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IL-7 mediates the homeostasis of naive and memory CD8 T cells in vivo
Abstract
The naïve and memory T lymphocyte pools are maintained through poorly understood homeostatic mechanisms that may include signaling via cytokine receptors. We show that interleukin-7 (IL-7) plays multiple roles in regulating homeostasis of CD8+ T cells. We found that IL-7 was required for homeostatic expansion of naïve CD8+ and CD4+ T cells in lymphopenic hosts and for CD8+ T cell survival in normal hosts. In contrast, IL-7 was not necessary for growth of CD8+ T cells in response to a virus infection but was critical for generating T cell memory. Up-regulation of Bcl-2 in the absence of IL-7 signaling was impaired after activation in vivo. Homeostatic proliferation of memory cells was also partially dependent on IL-7. These results point to IL-7 as a pivotal cytokine in T cell homeostasis.
... Cascades of Toll-like receptors (TLRs) and pathways regulating the response to SARS-CoV-2 infection show progressive activation in c1 and c6. Importantly, the activation pattern in c1 after the second vaccine dose indicated a proliferation signature, reprogramming of metabolism and the memory T cell preservation (TXNIP, GATA3, and IL7R) (Fig. 2c, d) [33][34][35][36] . ...
... With this approach, we found 179 genes whose expressions were significantly modulated by effectorness and vaccination acting jointly due to their cross-reactive effect (Supplementary Table 1). Among them, we found genes with a high impact on the effector response such as IFNG, TNF, and XCL2, and several genes crucial for durable T CM phenotype preservation including IL7R, TCF7, GATA3, CXCR3, and EOMES 34,35,[40][41][42] . Profiling the expression of these genes over different time points revealed their progressive correlation with the vaccine stimulations ( Fig. 4). ...
T cells provide rapid cellular immunity against pathogens. Here, we conducted matched single-cell RNA-sequencing and γδ-TCR-sequencing to delineate the molecular changes in γδ T cells during a longitudinal study following mRNA SARS-CoV-2 vaccination. While the first dose of vaccine primes Vδ2 T cells, it is the second administration that significantly boosts their immune response. Specifically, the second vaccination uncovers memory features of Vδ2 T cells, shaped by the induction of AP-1 family transcription factors and characterized by a convergent central memory signature, clonal expansion, and an enhanced effector potential. This temporally distinct effector response of Vδ2 T cells was also confirmed in vitro upon stimulation with SARS-CoV-2 spike-peptides. Indeed, the second challenge triggers a significantly higher production of IFNγ by Vδ2 T cells. Collectively, our findings suggest that mRNA SARS-CoV-2 vaccination might benefit from the establishment of long-lasting central memory Vδ2 T cells to confer protection against SARS-CoV-2 infection.
... To identify such a factor, we first examined expression levels of IL-7 and IL-15, which are required for memory T cell maintenance [4][5][6][7] . No difference was observed in Il7 and Il15 mRNA levels in spleens of MHCII −/− and WT mice ( Supplementary Fig. 2a). ...
The mechanisms by which the number of memory CD8 T cells is stably maintained remains incompletely understood. It has been postulated that maintaining them requires help from CD4 T cells, because adoptively transferred memory CD8 T cells persist poorly in MHC class II (MHCII)-deficient mice. Here we show that chronic interferon-γ signals, not CD4 T cell-deficiency, are responsible for their attrition in MHCII-deficient environments. Excess IFN-γ is produced primarily by endogenous colonic CD8 T cells in MHCII-deficient mice. IFN-γ neutralization restores the number of memory CD8 T cells in MHCII-deficient mice, whereas repeated IFN-γ administration or transduction of a gain-of-function STAT1 mutant reduces their number in wild-type mice. CD127high memory cells proliferate actively in response to IFN-γ signals, but are more susceptible to attrition than CD127low terminally differentiated effector memory cells. Furthermore, single-cell RNA-sequencing of memory CD8 T cells reveals proliferating cells that resemble short-lived, terminal effector cells and documents global downregulation of gene signatures of long-lived memory cells in MHCII-deficient environments. We propose that chronic IFN-γ signals deplete memory CD8 T cells by compromising their long-term survival and by diverting self-renewing CD127high cells toward terminal differentiation.
... 74,75 IL-7 modulates homeostasis of CD8 + T N and T M subsets. 76 The IL-7/IL-15 combination triggers in vitro expansion of CAR-T cells with higher T SCM subtypes. 77 Interestingly, some studies have indicated that IL-15 alone is much better than the IL-7/IL-15 combination in CAR-T culture for antitumor activity. ...
Treatment of multiple myeloma (MM) has evolved remarkably over the past few decades. Autologous stem cell transplantation, as well as proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies, has substantially improved the prognosis of patients with MM. Novel therapies, including chimeric antigen receptor-T cells, bispecific T-cell engagers, antibody-drug conjugates, histone deacetylase inhibitors, and nuclear export inhibitors, have provided more options. However, MM remains incurable. T cells are the principal weapons of antitumor immunity, but T cells display a broad spectrum of dysfunctional states during MM. The promising clinical results of T-cell-directed immunotherapies emphasize the significance of enhancing T-cell function in antimyeloma treatment. This review summarizes the potential effects of these antimyeloma agents on T-cell function and discusses possible optimized strategies for MM management by boosting T-cell immunity.
... Treatment with the cytokine IL-7 can induce the maturation and differentiation of thymic cells into TNs, enhancing the diversity of the TCR [152]. Schluns et al. demonstrated that IL-7 is necessary for the steady-state expansion of immature CD4 + T-cells, and IL-7 treatment restored thymic generation in IL-7-deficient mice [153]. Additionally, IL-22 may contribute to thymic regeneration in mice. ...
With the increasing proportion of the aging population, neurodegenerative diseases have become one of the major health issues in society. Neurodegenerative diseases (NDs), including multiple sclerosis (MS), Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS), are characterized by progressive neurodegeneration associated with aging, leading to a gradual decline in cognitive, emotional, and motor functions in patients. The process of aging is a normal physiological process in human life and is accompanied by the aging of the immune system, which is known as immunosenescence. T-cells are an important part of the immune system, and their senescence is the main feature of immunosenescence. The appearance of senescent T-cells has been shown to potentially lead to chronic inflammation and tissue damage, with some studies indicating a direct link between T-cell senescence, inflammation, and neuronal damage. The role of these subsets with different functions in NDs is still under debate. A growing body of evidence suggests that in people with a ND, there is a prevalence of CD4+ T-cell subsets exhibiting characteristics that are linked to senescence. This underscores the significance of CD4+ T-cells in NDs. In this review, we summarize the classification and function of CD4+ T-cell subpopulations, the characteristics of CD4+ T-cell senescence, the potential roles of these cells in animal models and human studies of NDs, and therapeutic strategies targeting CD4+ T-cell senescence.
... While substantial evidence shows that DCs and TRM cells such as Th2/Tc2, Th22/Tc22, and Th17/Tc17 are present in large numbers in lesions after the subsidence of inflammation in AD, their specific mechanisms in the recurrence of AD require further investigation (179). Moreover, TRM can persist in skin tissues for months to years and is primarily regulated by the local microenvironment (IL-7, IL-15, and transforming growth factor-β) generated by KCs and fibroblasts (180,181). To sum up, the interaction between TRM cells and the skin barrier plays a key role in the recurrence of psoriasis and AD (Supplementary Figure S2). ...
As chronic inflammatory conditions driven by immune dysregulation are influenced by genetics and environment factors, psoriasis and atopic dermatitis (AD) have traditionally been considered to be distinct diseases characterized by different T cell responses. Psoriasis, associated with type 17 helper T (Th17)-mediated inflammation, presents as well-defined scaly plaques with minimal pruritus. AD, primarily linked to Th2-mediated inflammation, presents with poorly defined erythema, dry skin, and intense itching. However, psoriasis and AD may overlap or transition into one another spontaneously, independent of biological agent usage. Emerging evidence suggests that defects in skin barrier-related molecules interact with the polarization of T cells, which forms a skin barrier-inflammatory loop with them. This loop contributes to the chronicity of the primary disease or the transition between psoriasis and AD. This review aimed to elucidate the mechanisms underlying skin barrier defects in driving the overlap between psoriasis and AD. In this review, the importance of repairing the skin barrier was underscored, and the significance of tailoring biologic treatments based on individual immune status instead of solely adhering to the treatment guidelines for AD or psoriasis was emphasized.
... However, while CD127 expression prior to HSCT was expected to match the mother's CD127 levels, we were surprised to see a severe reduction in the expression both in CD4 and CD8 T cells of P2 pre-HSCT (Fig. 2C). Because the expression of CD127 is high in resting T cells and is lost in activated or exhausted T cells [19][20][21][22][23][24][25][26], we hypothesized that maternal T cells that are engrafted in P2's blood acquired an activated/exhausted state due to continuous alloantigen exposure. HLA class I/II staining was increased on CD4 and CD8 T cells in P2 compared to healthy donors, confirming the activated status of these cells, which explains the loss of CD127 on T cells in P2 (Fig. 2D). ...
Purpose
The interleukin-7 receptor (IL-7R) is primarily expressed on lymphoid cells and plays a crucial role in the development, proliferation, and survival of T cells. Autosomal recessive mutations that disrupt IL-7Rα chain expression give rise to a severe combined immunodeficiency (SCID), which is characterized by lymphopenia and a T⁻B⁺NK⁺ phenotype. The objective here was to diagnose two siblings displaying the T⁻B⁺NK⁺ SCID phenotype as initial clinical genetic testing did not detect any variants in known SCID genes.
Methods
Whole genome sequencing (WGS) was utilized to identify potential variants causing the SCID phenotype. Splicing prediction tools were employed to assess the deleterious impact of the mutation. Polymerase Chain Reaction (PCR), Sanger sequencing, flow cytometry, and ELISA were then used to validate the pathogenicity of the detected mutation.
Results
We discovered a novel homozygous synonymous mutation in the IL7R gene. Our functional studies indicate that this variant is pathogenic, causing exon 6, which encodes the transmembrane domain, to be preferentially spliced out.
Conclusion
In this study, we identified a novel rare synonymous mutation causing a loss of IL-7Rα expression at the cellular membrane. This case demonstrates the value of reanalyzing genetic data based on the clinical phenotype and highlights the significance of functional studies in determining the pathogenicity of genetic variants.
... Moreover, IL7 has been shown to play an important role in the development and preservation of memory immune cell function. Previous work has demonstrated that IL7 can induce naive T-cells to differentiate into a memory phenotype [20,21], and promote the proliferation and activity of effector T-cells against cancer [22][23][24]. In addition, suppressive regulatory T-cells (Tregs) have been shown to express relatively low levels of the IL7 receptor (IL7R) [25]. ...
Clinically approved head and neck squamous cell carcinoma (HNSCC) immunotherapies manipulate the immune checkpoint blockade (ICB) axis but have had limited success outside of recurrent/metastatic disease. Interleukin-7 (IL7) has been shown to be essential for effector T-cell survival, activation, and proliferation. Here, we show that IL7 in combination with radiotherapy (RT) is effective in activating CD8 + T-cells for reducing tumor growth. Our studies were conducted using both human papillomavirus related and unrelated orthotopic HNSCC murine models. Immune populations from the tumor, draining lymph nodes, and blood were compared between treatment groups and controls using flow cytometry, proteomics, immunofluorescence staining, and RNA sequencing. Treatment with RT and IL7 (RT + IL7) resulted in significant tumor growth reduction, high CD8 T-cell tumor infiltration, and increased proliferation of T-cell progenitors in the bone marrow. IL7 also expanded a memory-like subpopulation of CD8 T-cells. These results indicate that IL7 in combination with RT can serve as an effective immunotherapy strategy outside of the conventional ICB axis to drive the antitumor activity of CD8 T-cells.
... involved in T cell egress from peripheral tissues, thus helping TRM cells to remain in the tissue for a long time.39,40 IL-7 and IL-15 also promote the survival and proliferation of TRM cells.[41][42][43] IL-33 is produced by epithelial cells in response to tissue damage, and it promotes the survival and activation of TRM cells through the ST2 receptor.44 ...
Tissue‐resident memory T cells (TRM) are a specialized subset of T cells that reside in tissues and provide long‐term protective immunity against pathogens that enter the body through that specific tissue. TRM cells have specific phenotype and reside preferentially in barrier tissues. Recent studies have revealed that TRM cells are the main target of immune checkpoint inhibitor immunotherapy since their role in cancer immunosurveillance. Furthermore, TRM cells also play a crucial part in pathogenesis of immune‐related adverse events (irAEs). Here, we provide a concise review of biological characteristics of TRM cells, and the major advances and recent findings regarding their involvement in immune checkpoint inhibitor immunotherapy and the corresponding irAEs.
... However, the differential sensitivity to JAK3i may relate to varying levels of IL-7 receptor (IL-7R) expression. While high IL-7R levels are required for the survival and proliferation of naive and memory T cells, T cell receptor signaling downregulates IL-7R expression in activated effector T cells (45,46). It means that T H 2 cells are not dependent on the downstream signaling pathways of IL-7R for their survival and functions, which explains why JAK3i did not affect them. ...
Steroids are the standard treatment for allergic airway inflammation in asthma, but steroid-refractory asthma poses a challenge. Group 2 innate lymphoid cells (ILC2s), such as T helper 2 (T H 2) cells, produce key asthma-related type 2 cytokines. Recent insights from mouse and human studies indicate a potential connection between ILC2s and steroid-resistant asthma. Here, we highlight that lung ILC2s, rather than T H 2 cells, can develop steroid resistance, allowing them to persist and maintain their disease-driving activity even during steroid treatment. The emergence of multipotent IL-5 ⁺ IL-13 ⁺ IL-17A ⁺ ILC2s is associated with steroid-resistant ILC2s. The Janus kinase 3 (JAK3)/signal transducer and activator of transcription (STAT) 3, 5, and 6 pathways contribute to the acquisition of steroid-resistant ILC2s. The JAK3 inhibitor reduces ILC2 survival, proliferation, and cytokine production in vitro and ameliorates ILC2-driven Alternaria -induced asthma. Furthermore, combining a JAK3 inhibitor with steroids results in the inhibition of steroid-resistant asthma. These findings suggest a potential therapeutic approach for addressing this challenging condition in chronic asthma.
... IL-7 is a cytokine produced primarily by the stromal cells of lymphoid tissues. IL-7 is required for normal T cell development and homeostasis of naive T cell populations [16,17]. CAR-T cells expanded in the presence of IL-7 or engineered to constitutively secrete transgenic IL-7 have been shown to have enhanced antitumor potential [18,19]. ...
CAR-T cell therapy has emerged as a breakthrough therapy for the treatment of relapsed and refractory hematologic malignancies. However, insufficient CAR-T cell expansion and persistence is a leading cause of treatment failure. Exogenous or transgenic cytokines have great potential to enhance CAR-T cell potency but pose the risk of exacerbating toxicities. Here we present a chemical-genetic system for spatiotemporal control of cytokine function gated by the off-patent anti-cancer molecular glue degrader drug lenalidomide and its analogs. When co-delivered with a CAR, a membrane-bound, lenalidomide-degradable IL-7 fusion protein enforced a clinically favorable T cell phenotype, enhanced antigen-dependent proliferative capacity, and enhanced in vivo tumor control. Furthermore, cyclical pharmacologic combined control of CAR and cytokine abundance enabled the deployment of highly active, IL-7-augmented CAR-T cells in a dual model of antitumor potency and T cell hyperproliferation.
... IL7 is a promising candidate for enhancing the antitumor function of CAR T cells. Its signaling in T cells promotes their survival, proliferation, and the formation of memory T cells [66][67][68][69]. IL7 has shown to be less toxic at certain doses compared to other cytokines like IL2, IL12, and IL15 in clinical trials [70][71][72][73]. ...
Simple Summary
Glioblastoma (GBM) is a highly aggressive brain tumor with low survival rates and limited treatment options. This review explores the potential of chimeric antigen receptor (CAR) T-cell immunotherapy as a promising approach for treating GBM. While CAR T-cell therapy has shown success in blood malignancies, it faces challenges when applied to brain tumors, such as tumor heterogeneity and the inhibitory tumor microenvironment. The review discusses existing research and future prospects for CAR T-cell immunotherapy in glioblastoma, highlighting the need for innovative strategies to improve outcomes for patients. Overall, this review provides insights into the current state and future directions of immunotherapy for GBM.
Abstract
Glioblastoma (GBM), the most common primary brain tumor in adults, is characterized by low survival rates and a grim prognosis. Current treatment modalities, including extensive surgical resection, chemotherapy, and radiation therapy, often yield limited success due to the brain’s sensitivity, leading to significant side effects. Exciting advancements in immunotherapy have recently shown promise in treating various types of tumors, raising hopes for improved outcomes in brain tumor patients. One promising immunotherapy approach is chimeric antigen receptor (CAR) T-cell therapy, which recognizes surface proteins on targeted tumor cells and redirects cytotoxicity towards specific targets. This review aims to discuss the existing research and future prospects for CAR T-cell immunotherapy in treating glioblastoma.
... How memory T cells are generated remains unclear and may vary in different effector cell types. A small fraction of CD8 + T cells in lymphocytic choriomeningitis virus (LCMV) or Listeria monocytogenes infection acquired memory precursor characteristics with increased expression of IL-7 receptor subunit-α (IL-7Rα), CD27, and B cell lymphoma 2 (BCL-2), and decreased expression of killer cell lectin-like receptor G1 (KLRG1), distinct from the KLRG1 hi IL7Rα low CD27 hi BCL-2 hi terminal effector cells (Kaech et al., 2003;Joshi et al., 2007;Joshi and Kaech, 2008;Sarkar et al., 2008;Schluns et al., 2000). Memory CD8 + T cells in circulation have been classified as Tcm or Tem cells. ...
T follicular helper (Tfh) cells, essential for germinal center reactions, are not identical, with different phenotypes reported. Whether, when, and how they generate memory cells is still poorly understood. Here, through single-cell RNA-sequencing analysis of CXCR5⁺Bcl6⁺ Tfh cells generated under different conditions, we discovered, in addition to PD-1hi effector Tfh cells, a CD62L⁺PD1low subpopulation. CD62L-expressing Tfh cells developed independently from PD-1⁺ cells and not in direct contact with B cells. More importantly, CD62L⁺ Tfh cells expressed memory- and stemness-associated genes, and with better superior long-term survival, they readily generated PD-1hi cells in the recall response. Finally, KLF2 and IL7R, also highly expressed by CD62L⁺ Tfh cells, were required to regulate their development. Our work thus demonstrates a novel Tfh memory-like cell subpopulation, which may benefit our understanding of immune responses and diseases.
... Cytokine receptors Il12rb1, Il15r, Il2rb, and Il2rg were increased in DS CD8 T-cells, consistent with an effector phenotype (Supplemental Table 1). CD8 T-cells from DS mice showed evidence of terminal differentiation, with high expression of effector transcripts like Eomes, Tgfb1, IFNg, Gzmk, and Il10 and low expression of Il7r and Tcf7 (TCF-1) [63][64][65] . Despite the absence of experimental antigen, the most differentially upregulated transcripts in DS CD8 T-cells were exhaustion-associated inhibitory receptors like Pdcd1 (PD-1), Havcr2 (Tim-3), Ctla4, Entpd1 (CD39), and Tigit ( Figure 4B). ...
Hemophagocytic Lymphohistiocytosis (HLH) and Macrophage Activation Syndrome (MAS) are life-threatening hyperinflammatory syndromes. Familial HLH is caused by genetic impairment of granule-mediated cytotoxicity (e.g., perforin-deficiency). MAS is linked to excess activity of the inflammasome-activated cytokine IL-18. Though individually tolerated, mice with dual susceptibility (Prf1-/-Il18tg; DS) succumb to spontaneous, lethal hyperinflammation. We hypothesized that understanding how these susceptibility factors synergize would uncover key pathomechanisms in the activation, function, and persistence of hyperactivated CD8 T-cells. In IL-18 transgenic (Il18tg) mice, IL-18 effects on CD8 T-cells drove MAS following a viral (LCMV), but not innate (TLR9), trigger. In vitro, CD8 T-cells also required T-cell receptor (TCR) stimulation to fully respond to IL-18. IL-18 induced, but perforin deficiency impaired, immunoregulatory Restimulation-Induced Cell Death (RICD). Paralleling hyperinflammation, dual susceptibility mice displayed massive post-thymic oligoclonal CD8 T-cell hyperactivation in their spleens, livers, and bone marrow as early as 3 weeks. These cells increased proliferation and IFNg production contrasted with increased expression of receptors and transcription factors associated with exhaustion. Broad-spectrum antibiotics and anti-retrovirals failed to ameliorate disease. Attempting to genetically "fix" TCR antigen-specificity instead demonstrated the persistence of spontaneous HLH and hyperactivation chiefly on T-cells that had evaded TCR fixation. Thus, drivers of HLH may preferentially act on CD8 T-cells: IL-18 amplifies activation and demand for RICD, whereas perforin supplies critical immunoregulation. Together, these factors promote a terminal CD8 T-cell activation state combining features of exhaustion and effector function. Therefore, susceptibility to hyperinflammation may converge on a unique, unrelenting, and antigen-dependent state of CD8 T-cell hyperactivation.
... Consistent with the upregulation of a pro-survival gene network ( Fig. 1 and Supplementary Fig. 3c), expression of the let-7 transgene improved CTL viability during cytokine withdrawal in vitro 12 (Fig. 3a). Following contraction, cytokines such as IL−15 and IL-7 promote memory T cell differentiation and survival, and can be used to generate memory CD8 T cells in vitro 45,49,67,68 . As expected, WT CTLs upregulated the expression of Tcf7, Sell, Ccr7, and CD127 only in the presence of IL-15, whereas let-7Tg CTLs displayed an enhanced memory phenotype regardless of the use of IL-2 or IL-15 (Fig. 3b). ...
The success of the CD8 T cell-mediated immune response against infections and tumors depends on the formation of a long-lived memory pool, and the protection of effector cells from exhaustion. The advent of checkpoint blockade therapy has significantly improved anti-tumor therapeutic outcomes by reversing CD8 T cell exhaustion, but fails to generate effector cells with memory potential. Here, using in vivo mouse models, we show that let-7 miRNAs determine CD8 T cell fate, where maintenance of let-7 expression during early cell activation results in memory CD8 T cell formation and tumor clearance. Conversely, let-7-deficiency promotes the generation of a terminal effector population that becomes vulnerable to exhaustion and cell death in immunosuppressive environments and fails to reject tumors. Mechanistically, let-7 restrains metabolic changes that occur during T cell activation through the inhibition of the PI3K/AKT/mTOR signaling pathway and production of reactive oxygen species, potent drivers of terminal differentiation and exhaustion. Thus, our results reveal a role for let-7 in the time-sensitive support of memory formation and the protection of effector cells from exhaustion. Overall, our data suggest a strategy in developing next-generation immunotherapies by preserving the multipotency of effector cells rather than enhancing the efficacy of differentiation.
... (i) Stromal cell-immune cell contact-dependent PI3K and APRIL induces NF-kB signaling and prevents mitochondrial and ER stress of memory plasma cells [140]; (ii) Stromal cell CD80/CD86 expression provides CD28 stimulation in BM-resident plasma cells, leading to sustained antibody responses [141]; (iii) Stromal cells providing superior bio-availability of IL-15 cause upregulation of glucocortioid-induced TNF receptor (GITR) on CD8 MTCs [142]; (iv) Stromal-cell-derived Il-7 mediates homeostasis of naïve and memory CD8 T cells in vivo [143]; (v) Stromal-cell-expressed VCAM-1 holds immune cells in the niche and maintains plasma cell longevity [144]. ...
Bone marrow is known as the site of hematopoiesis. What is not being described in textbooks of immunology is the fact that bone marrow is not only a generative, but also an antigen-responsive, immune organ. It is also a major storage site for antigen-specific memory B and T cells. That bone marrow is a priming site for T cell responses to blood borne antigens was discovered exactly 20 years ago. This review celebrates this important discovery. The review provides a number of examples of medical relevance of bone marrow as a central immune system, including cancer, microbial infections, autoimmune reactions, and bone marrow transplantation. Bone marrow mesenchymal stem cell-derived stromal cells provide distinct bone marrow niches for stem cells and immune cells. By transmitting anti-inflammatory dampening effects, facilitating wound healing and tissue regeneration mesenchymal stem cells contribute to homeostasis of bone and other tissues. Based on the evidence presented, the review proposes that bone marrow is a multifunctional and protective immune system. In an analogy to the central nervous system, it is suggested that bone marrow be designated as the central immune system.
... Relative to other clusters, cluster 1 showed increased expression of Tcf7 and Il7r, which are upregulated on naive T cells (Fig. 3B) (50)(51)(52). Cluster 1 cells also showed high expression of Txnip, whose expression has been negatively associated with T cell activation (53)(54)(55), and Cxcr3, which we previously showed is expressed on CD8+ T cells migrating into the skin in AA (26). Cluster 2 showed high expression of Ifng, one of the main T cell-derived cytokines in AA (3), and Cd69, a marker of T cell activation and tissue retention (56). ...
Alopecia areata (AA) is among the most prevalent autoimmune diseases, but the development of innovative therapeutic strategies has lagged due to an incomplete understanding of the immunological underpinnings of disease. Here, we performed single-cell RNA sequencing (scRNAseq) of skin-infiltrating immune cells from the graft-induced C3H/HeJ mouse model of AA, coupled with antibody-based depletion to interrogate the functional role of specific cell types in AA in vivo. Since AA is predominantly T cell-mediated, we focused on dissecting lymphocyte function in AA. Both our scRNAseq and functional studies established CD8+ T cells as the primary disease-driving cell type in AA. Only the depletion of CD8+ T cells, but not CD4+ T cells, NK, B, or γδ T cells, was sufficient to prevent and reverse AA. Selective depletion of regulatory T cells (Treg) showed that Treg are protective against AA in C3H/HeJ mice, suggesting that failure of Treg-mediated immunosuppression is not a major disease mechanism in AA. Focused analyses of CD8+ T cells revealed five subsets, whose heterogeneity is defined by an "effectorness gradient" of interrelated transcriptional states that culminate in increased effector function and tissue residency. scRNAseq of human AA skin showed that CD8+ T cells in human AA follow a similar trajectory, underscoring that shared mechanisms drive disease in both murine and human AA. Our study represents a comprehensive, systematic interrogation of lymphocyte heterogeneity in AA and uncovers a novel framework for AA-associated CD8+ T cells with implications for the design of future therapeutics.
... IL-6 rescues T-cells from apoptosis by inhibiting the downregulation of Bcl-2 in a dose dependent manner which promotes the survival of stimulated T-cells [60,61]. There are multiple pathways through which T cells can undergo cell death and deprivation of the key survival cytokines can result in increased death of T cells [62][63][64][65]. Inflammatory cytokines such as TNFα have been shown to promote cell death of T cells through TNF-R1 signaling [66]. ...
XIAP is an endogenous inhibitor of cell death and inactivating mutations of XIAP are responsible for X-linked lymphoproliferative disease (XLP-2) and primary immunodeficiency, but the mechanism(s) behind these contradictory outcomes have been unclear. We report that during infection of macrophages and dendritic cells with various intracellular bacteria, XIAP restricts cell death and secretion of IL-1β but promotes increased activation of NFκB and JNK which results in elevated secretion of IL-6 and IL-10. Poor secretion of IL-6 by Xiap-deficient antigen presenting cells leads to poor expansion of recently activated CD8 T cells during the priming phase of the response. On the other hand, Xiap-deficient CD8 T cells displayed increased proliferation and effector function during the priming phase but underwent enhanced contraction subsequently. Xiap-deficient CD8 T cells underwent skewed differentiation towards short lived effectors which resulted in poor generation of memory. Consequently Xiap-deficient CD8 T cells failed to provide effective control of bacterial infection during re-challenge. These results reveal the temporal impact of XIAP in promoting the fitness of activated CD8 T cells through cell extrinsic and intrinsic mechanisms and provide a mechanistic explanation of the phenotype observed in XLP-2 patients.
... Several cytokines of the γ− common, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, promote the effector response of CD8 + T cells, as well as the survival of long-lived memory cells [111]. For example, IL-2 promotes the proliferation and accumulation of memory CD8 + T cells [112]; IL-7 promotes the homeostasis of naïve and memory CD8 + T cells [113,114], while IL-15 and IL-21 promote the survival of HIV-specific CD8 + T cells, the production of IFN-γ, the expression of cytotoxic molecules, and control of HIV in vitro [115][116][117]. IL-15 also promotes the metabolic plasticity of HIV-specific CD8 + T cells, a characteristic of cells derived from elite controllers [117]. ...
Achieving a cure for HIV infection is a global priority. There is substantial evidence supporting a central role for CD8+ T cells in the natural control of HIV, suggesting the rationale that these cells may be exploited to achieve remission or cure of this infection. In this work, we review the major challenges for achieving an HIV cure, the models of HIV remission, and the mechanisms of HIV control mediated by CD8+ T cells. In addition, we discuss strategies based on this cell population that could be used in the search for an HIV cure. Finally, we analyze the current challenges and perspectives to translate this basic knowledge toward scalable HIV cure strategies.
... Interleukin-7 (IL-7) is a critical cytokine in the development and expansion of T cells. It is mainly secreted by thymic and bone marrow stromal cells and plays a significant role in health maintenance and disease prevention [8]. IL-7 signaling deficiency can result in severe immunodeficiency and, following antigen exposure, contributes to memory T cell development and long-term T cell survival [9]. ...
Interleukin-7 (IL-7) plays a vital role in the homeostasis of CD4+ and CD8+ T cells. Although IL-7 has been implicated in T helper (Th)1- and Th17-mediated autoinflammatory diseases, its role in Th2-type allergic disorders, such as atopic dermatitis (AD), remains unclear. Thus, to elucidate the effects of IL-7 deficiency on AD development, we generated IL-7-deficient AD-prone mice by backcrossing IL-7 knockout (KO) B6 mice onto the NC/Nga (NC) mouse strain, a model for human AD. As expected, IL-7 KO NC mice displayed defective development of conventional CD4+ and CD8+ T cells compared with wild type (WT) NC mice. However, IL-7 KO NC mice presented with enhanced AD clinical scores, IgE hyperproduction, and increased epidermal thickness compared with WT NC mice. Moreover, IL-7 deficiency decreased Th1, Th17, and IFN-γ-producing CD8+ T cells but increased Th2 cells in the spleen of NC mice, indicating that a reduced Th1/Th2 ratio correlates with severity of AD pathogenesis. Furthermore, significantly more basophils and mast cells infiltrated the skin lesions of IL-7 KO NC mice. Taken together, our findings suggest that IL-7 could be a useful therapeutic target for treating Th2-mediated skin inflammations, such as AD.
... For example, treatment with IL-2 in HIV-infected patients can significantly increase CD4 + T cell counts and enhance immune function (170). IL-7 is also crucial in T cell homeostasis as it maintains T cell survival, induces proliferation, and promotes de novo production (171,172). The IL-7 responsiveness is largely dependent on the presence or absence of the IL-7 receptor (IL-7R). ...
Highly active antiretroviral therapy (ART) can effectively inhibit virus replication and restore immune function in most people living with human immunodeficiency virus (HIV). However, an important proportion of patients fail to achieve a satisfactory increase in CD4⁺ T cell counts. This state is called incomplete immune reconstitution or immunological nonresponse (INR). Patients with INR have an increased risk of clinical progression and higher rates of mortality. Despite widespread attention to INR, the precise mechanisms remain unclear. In this review, we will discuss the alterations in the quantity and quality of CD4⁺ T as well as multiple immunocytes, changes in soluble molecules and cytokines, and their relationship with INR, aimed to provide cellular and molecular insights into incomplete immune reconstitution.
... Besides meeting metabolic requirements, the skin environment contains a plethora of locally residing antigen presenting cells producing chemokines and cytokines including IL-7, IL-15, and TGFβ for T RM maintenance. 92,93 Cross talk of T RM with antigen presenting cells and stromal cells is extensively reviewed elsewhere. 94 In steady state, the percentage of skin T RM proliferating within a week, detected as bromodeoxyuridine (BrDU)-uptaking cells, is below 5%. 1 This number is lower than that of T RM in other tissues, for example the brain parenchyma, which suggests superior survival capacity of individual skin T RM in a resting state due to adaptation to local nutrient availability. ...
The human skin is populated by a diverse pool of memory T cells, which can act rapidly in response to pathogens and cancer antigens. Tissue‐resident memory T cells (TRM) have been implicated in range of allergic, autoimmune and inflammatory skin diseases. Clonal expansion of cells with TRM properties is also known to contribute to cutaneous T‐cell lymphoma. Here, we review the heterogeneous phenotypes, transcriptional programs, and effector functions of skin TRM. We summarize recent studies on TRM formation, longevity, plasticity, and retrograde migration and contextualize the findings to skin TRM and their role in maintaining skin homeostasis and altered functions in skin disease.
... 105 IL-15 promoted mitochondrial biogenesis and expression of an enzyme that controls fatty acid oxidation. 105 Both examples illustrate how cytokines that have long been associated with memory T-cell survival [106][107][108][109][110] might mediate their effects through subset-specific modulation of T-cell metabolism. ...
Memory T cells form an essential part of immunological memory, which can last for years or even a lifetime. Much experimental work has shown that the individual cells that make up the memory T‐cell pool are in fact relatively short‐lived. Memory T cells isolated from the blood of humans, or the lymph nodes and spleen of mice, live about 5–10 fold shorter than naive T cells, and much shorter than the immunological memory they convey. The commonly accepted view is, therefore, that long‐term T‐cell memory is maintained dynamically rather than by long‐lived cells. This view is largely based on memory T cells in the circulation, identified using rather broad phenotypic markers, and on research in mice living in overly clean conditions. We wondered to what extent there may be heterogeneity in the dynamics and lifespans of memory T cells. We here review what is currently known about the dynamics of memory T cells in different memory subsets, locations in the body and conditions of microbial exposure, and discuss how this may be related to immunometabolism and how this knowledge can be used in various clinical settings.
Interleukin-7 (IL-7) is considered a critical regulator of memory CD8 ⁺ T cell homeostasis, but this is primarily based on analysis of circulating and not tissue-resident memory (T RM ) subsets. Furthermore, the cell-intrinsic requirement for IL-7 signaling during memory homeostasis has not been directly tested. Using inducible deletion, we found that Il7ra loss had only a modest effect on persistence of circulating memory and T RM subsets and that IL-7Rα was primarily required for normal basal proliferation. Loss of IL-15 signaling imposed heightened IL-7Rα dependence on memory CD8 ⁺ T cells, including T RM populations previously described as IL-15-independent. In the absence of IL-15 signaling, IL-7Rα was upregulated, and loss of IL-7Rα signaling reduced proliferation in response to IL-15, suggesting cross-regulation in memory CD8 ⁺ T cells. Thus, across subsets and tissues, IL-7 and IL-15 act in concert to support memory CD8 ⁺ T cells, conferring resilience to altered availability of either cytokine.
Highlights
Tissue-resident and circulating memory CD8 ⁺ T cells modestly decline after loss of IL-7Rα IL-7Rα is required for normal self-renewal of memory CD8 ⁺ T cells
Combined loss of IL-7 and IL-15 causes a profound defect across memory CD8 ⁺ T cell subsets Cross-regulation of IL-7 and IL-15 signaling occurs in memory CD8 ⁺ T cells
Abstract Figure
Reproductive cancers are malignancies that develop in the reproductive organs. One of the leading cancers affecting the male reproductive system on a global scale is prostate cancer (PCa). The negative consequences of PCa metastases endure and are severe, significantly affecting mortality and life quality for those who are affected. The association between inflammation and PCa has captured interest for a while. Inflammatory cells, cytokines, CXC chemokines, signaling pathways, and other elements make up the tumor microenvironment (TME), which is characterized by inflammation. Inflammatory cytokines and CXC chemokines are especially crucial for PCa development and prognosis. Cytokines (interleukins) and CXC chemokines such as IL-1, IL-6, IL-7, IL-17, TGF-β, TNF-α, CXCL1–CXCL6, and CXCL8–CXCL16 are thought to be responsible for the pleiotropic effects of PCa, which include inflammation, progression, angiogenesis, leukocyte infiltration in advanced PCa, and therapeutic resistance. The inflammatory cytokine and CXC chemokines systems are also promising candidates for PCa suppression and immunotherapy. Therefore, the purpose of this work is to provide insight on how the spectra of inflammatory cytokines and CXC chemokines evolve as PCa develops and spreads. We also discussed recent developments in our awareness of the diverse molecular signaling pathways of these circulating cytokines and CXC chemokines, as well as their associated receptors, which may one day serve as PCa-targeted therapies. Moreover, the current status and potential of theranostic PCa therapies based on cytokines, CXC chemokines, and CXC receptors (CXCRs) are examined.
T cells are an important component of adaptive immunity and protect the host from infectious diseases and cancers. However, uncontrolled T cell immunity may cause autoimmune disorders. In both situations, antigen-specific T cells undergo clonal expansion upon the engagement and activation of antigens. Cellular metabolism is reprogrammed to meet the increase in bioenergetic and biosynthetic demands associated with effector T cell expansion. Metabolites not only serve as building blocks or energy sources to fuel cell growth and expansion but also regulate a broad spectrum of cellular signals that instruct the differentiation of multiple T cell subsets. The realm of immunometabolism research is undergoing swift advancements. Encapsulating all the recent progress within this concise review in not possible. Instead, our objective is to provide a succinct introduction to this swiftly progressing research, concentrating on the metabolic intricacies of three pivotal nutrient classes—lipids, glucose, and amino acids—in T cells. We shed light on recent investigations elucidating the roles of these three groups of metabolites in mediating the metabolic and immune functions of T cells. Moreover, we delve into the prospect of “editing” metabolic pathways within T cells using pharmacological or genetic approaches, with the aim of synergizing this approach with existing immunotherapies and enhancing the efficacy of antitumor and antiinfection immune responses.
Interleukin-7 (IL-7) is a versatile cytokine that plays a crucial role in regulating the immune system’s homeostasis. It is involved in the development, proliferation, and differentiation of B and T cells, as well as being essential for the differentiation and survival of naïve T cells and the production and maintenance of memory T cells. Given its potent biological functions, IL-7 is considered to have the potential to be widely used in the field of anti-tumour immunotherapy. Notably, IL-7 can improve the tumour microenvironment by promoting the development of Th17 cells, which can in turn promote the recruitment of effector T cells and NK cells. In addition, IL-7 can also down-regulate the expression of tumour growth factor-β and inhibit immunosuppression to promote anti-tumour efficacy, suggesting potential clinical applications for anti-tumour immunotherapy. This review aims to discuss the origin of IL-7 and its receptor IL-7R, its anti-tumour mechanism, and the recent advances in the application of IL-7 in tumour therapy.
The 1858C>T allele of the tyrosine phosphatase PTPN22 is present in 5–10% of the North American population and is strongly associated with numerous autoimmune diseases. Although research has been done to define how this allele potentiates autoimmunity, the influence PTPN22 and its pro-autoimmune allele has in anti-viral immunity remains poorly defined. Here, we use single cell RNA-sequencing and functional studies to interrogate the impact of this pro-autoimmune allele on anti-viral immunity during Lymphocytic Choriomeningitis Virus clone 13 (LCMV-cl13) infection. Mice homozygous for this allele (PEP-619WW) clear the LCMV-cl13 virus whereas wildtype (PEP-WT) mice cannot. This is associated with enhanced anti-viral CD4 T cell responses and a more immunostimulatory CD8α ⁻ cDC phenotype. Adoptive transfer studies demonstrated that PEP-619WW enhanced anti-viral CD4 T cell function through virus-specific CD4 T cell intrinsic and extrinsic mechanisms. Taken together, our data show that the pro-autoimmune allele of Ptpn22 drives a beneficial anti-viral immune response thereby preventing what is normally a chronic virus infection.
Lymph nodes are crucial organs of the adaptive immune system, orchestrating T cell priming, activation and tolerance. T cell activity and function are highly regulated by lymph nodes, which have a unique structure harbouring distinct cells that work together to detect and respond to pathogen-derived antigens. Here we show that implanted patient-derived freeze-dried lymph nodes loaded with chimeric antigen receptor T cells improve delivery to solid tumours and inhibit tumour recurrence after surgery. Chimeric antigen receptor T cells can be effectively loaded into lyophilized lymph nodes, whose unaltered meshwork and cytokine and chemokine contents promote chimeric antigen receptor T cell viability and activation. In mouse models of cell-line-derived human cervical cancer and patient-derived pancreatic cancer, delivery of chimeric antigen receptor T cells targeting mesothelin via the freeze-dried lymph nodes is more effective in preventing tumour recurrence when compared to hydrogels containing T-cell-supporting cytokines. This tissue-mediated cell delivery strategy holds promise for controlled release of various cells and therapeutics with long-term activity and augmented function.
Adoptive cell therapy (ACT) is an immunotherapeutic approach that involves isolating T cells from a patient, culturing them ex vivo, then re-infusing the cells back into the patient. Although this strategy has shown remarkable efficacy in hematological malignancies, the solid-tumour microenvironment (TME) has presented serious challenges for therapy efficacy. Particularly, the TME has immunosuppressive signaling and presents a metabolically challenging environment that leads to T cell suppression. T cell metabolism is an expanding field of research with a focus on understanding its inherent link to T cell function. Here, we review the current model of T cell metabolism from naïve cells through effector and memory life stages, as well as updates to the model from recent literature. These models of metabolism have provided us with the tools and understanding to explore T cell metabolic and mitochondrial insufficiency in the TME. We discuss manipulations that can be made to these mitochondrial and metabolic pathways to enhance persistence of infused T cells, overcome the metabolically challenging TME and improve the efficacy of therapy in ACT models. Further understanding and investigation of the impact of metabolic pathways on T cell performance could contribute to improving therapy efficacy for patients.
The delicate balance of immune homeostasis is regulated by the interactions between cytokines and their cognate cell surface signaling receptors. There is intensive interest in harnessing cytokines as drugs for diseases such as cancer and autoimmune disorders. However, the multifarious and often contradictory activities of cytokines, coupled with their short serum half-lives, limit clinical performance and result in dangerous toxicities. There is thus growing emphasis on manipulating natural cytokines to enhance their selectivity, safety, and durability through various strategies. One strategy that has gained traction in recent years is the development of anticytokine Abs that not only extend the circulation half-life of cytokines but also specifically bias their immune activities through multilayered molecular mechanisms. Although Abs are notorious for their antagonistic activities, this review focuses on anticytokine Abs that selectively agonize the activity of the target protein. This approach has potential to help realize the clinical promise of cytokine-based therapies.
CAR-T cell therapy is revolutionizing the treatment of hematologic malignancies. However, there are still many challenges ahead before CAR-T cells can be used effectively to treat solid tumors and certain hematologic cancers, such as T-cell malignancies. Next-generation CAR-T cells containing further genetic modifications are being developed to overcome some of the current limitations of this therapy. In this regard, genome editing is being explored to knock out or knock in genes with the goal of enhancing CAR-T cell efficacy or increasing access. In this chapter, we describe in detail a protocol to knock out genes on CAR-T cells using CRISPR–Cas9 technology. Among various gene editing protocols, due to its simplicity, versatility, and reduced toxicity, we focused on the electroporation of ribonucleoprotein complexes containing the Cas9 protein together with sgRNA. All together, these protocols allow for the design of the knockout strategy, CAR-T cell expansion and genome editing, and analysis of knockout efficiency.
T cell-based adoptive cell therapy (ACT) has exhibited excellent antitumoral efficacy exemplified by the clinical breakthrough of chimeric antigen receptor therapy (CAR-T) in hematologic malignancies. It relies on the pool of functional T cells to retain the developmental potential to serially kill targeted cells. However, failure in the continuous supply and persistence of functional T cells has been recognized as a critical barrier to sustainable responses. Conferring stemness on infused T cells, yielding stem cell-like memory T cells (T SCM ) characterized by constant self-renewal and multilineage differentiation similar to pluripotent stem cells, is indeed necessary and promising for enhancing T cell function and sustaining antitumor immunity. Therefore, it is crucial to identify T SCM cell induction regulators and acquire more T SCM cells as resource cells during production and after infusion to improve antitumoral efficacy. Recently, four common cytokine receptor γ chain (γc) family cytokines, encompassing interleukin-2 (IL-2), IL-7, IL-15, and IL-21, have been widely used in the development of long-lived adoptively transferred T SCM in vitro. However, challenges, including their non-specific toxicities and off-target effects, have led to substantial efforts for the development of engineered versions to unleash their full potential in the induction and maintenance of T cell stemness in ACT. In this review, we summarize the roles of the four γc family cytokines in the orchestration of adoptively transferred T cell stemness, introduce their engineered versions that modulate T SCM cell formation and demonstrate the potential of their various combinations.
Cancer immunotherapy is moving toward combination regimens with agents of complementary mechanisms of action to achieve more frequent and robust efficacy. However, compared with single-agent therapies, combination immunotherapies are associated with increased overall toxicity because the very same mechanisms also work in concert to enhance systemic inflammation and promote off-tumor toxicity. Therefore, rational design of combination regimens that achieve improved antitumor control without exacerbated toxicity is a main objective in combination immunotherapy. Here, we show that the combination of engineered, tumor matrix-binding interleukin-7 (IL-7) and IL-12 achieves remarkable anticancer effects by activating complementary pathways without inducing any additive immunotoxicity. Mechanistically, engineered IL-12 provided effector properties to T cells, while IL-7 prevented their exhaustion and boosted memory formation as assessed by tumor rechallenge experiments. The dual combination also rendered checkpoint inhibitor (CPI)–resistant genetically engineered melanoma model responsive to CPI. Thus, our approach provides a framework of evaluation of rationally designed combinations in immuno-oncology and yields a promising therapy.
Objectives
Thymus implantation is a recently FDA‐approved therapy for congenital athymia. Patients receiving thymus implantation develop a functional but incomplete T cell compartment. Our objective was to develop a mouse model to study clinical thymus implantation in congenital athymia and to optimise implantation procedures to maximise T cell education and expansion of naïve T cells.
Methods
Using Foxn1 nu athymic mice as recipients, we tested MHC‐matched and ‐mismatched donor thymi that were implanted as fresh tissue or cultured to remove donor T cells. We first implanted thymus under the kidney capsule and then optimised intramuscular implantation. Using competitive adoptive transfer assays, we investigated whether the failure of newly developed T cells to expand into a complete T cell compartment was because of intrinsic deficits or whether there were deficits in engaging MHC molecules in the periphery. Finally, we tested whether recombinant IL‐7 would promote the expansion of host naïve T cells educated by the implanted thymus.
Results
We determined that thymus implants in Foxn1 nu athymic mice mimic many aspects of clinical thymus implants in patients with congenital athymia. When we implanted cultured, MHC‐mismatched donor thymus into Foxn1 nu athymic mice, mice developed a limited T cell compartment with notably underdeveloped naïve populations and overrepresented memory‐like T cells. Newly generated T cells were predominantly educated by MHC molecules expressed by the donor thymus, thus potentially undergoing another round of selection once in the peripheral circulation. Using competitive adoptive transfer assays, we compared expansion rates of T cells educated on donor thymus versus T cells educated during typical thymopoiesis in MHC‐matched and ‐mismatched environments. Once in the circulation, regardless of the MHC haplotypes, T cells educated on a donor thymus underwent abnormal expansion with initially more robust proliferation coupled with greater cell death, resembling IL‐7 independent spontaneous expansion. Treating implanted mice with recombinant interleukin (IL‐7) promoted homeostatic expansion that improved T cell development, expanded the T cell receptor repertoire, and normalised the naïve T cell compartment.
Conclusion
We conclude that implanting cultured thymus into the muscle of Foxn1 nu athymic mice is an appropriate system to study thymus implantation for congenital athymia and immunodeficiencies. T cells are educated by the donor thymus, yet naïve T cells have deficits in expansion. IL‐7 greatly improves T cell development after thymus implantation and may offer a novel strategy to improve outcomes of clinical thymus implantation.
A cooperated, powerful, but self-constrained immune reaction to a goal antigen is produced by the immune system's cells in conjunction with cytokines, which act as molecular messengers. Increased efforts to identify cytokines and use of their extensive signalling networks to create cancer treatments have coincided with the increased interest in using the resistant system to fight cancer during the past two decades. The main cytokines used in cancer immunotherapy will be reviewed, along with their fundamental biology and therapeutic uses, in this chapter. The chapter will also go through novel distribution strategies, biological agent sequences, new cytokines in diagnosing research and potential future directions for cytokine-based research in the future. Immune cell chemotaxis and the growth of lymphoid tissues are mediated by chemokines, which are chemotactic cytokines. Recent research has shown that chemokines and their corresponding receptors are essential for the growth of cancer and the inflammation that results from it. As a result of these discoveries, the chemokine system is now a current possible goal for cancer therapy. The chemokine system regulates the flow of immune cells into the tumor microenvironment, producing both pro- and anti-immunity and promoting or inhibiting tumor growth and angiogenesis, metastasis and proliferation. Growing evidences support the chemokine system's positive predictive importance in cancer patients. Depending on the kind of cancer, CCL10, CXCL2 and CX3CL1/CX3CR1 might be one of the two favourable or unfavourable prognostic variables; however, CCL14 and CXCL1 have strong predicting value. More chemokines with poor prognostic value include CXCL1, CXCL8 and CXCL12. Despite significant advancements in our comprehension of the complicated complexion of the system chemokine in tumor biology, our grasp of the chemokine system's diverse functions in many cancers is still restricted. Additional research is required to clarify the many functions of the system chemokine in the development of cancer and to confirm their potential utility in cancer prognosis.
Within the brain, a pro-inflammatory response is essential to prevent clinical disease due to Toxoplasma gondii reactivation. Infection in the immunocompromised leads to lethal Toxoplasmic encephalitis while in the immunocompetent, there is persistent low-grade inflammation which is devoid of clinical symptoms. This signifies that there is a well-balanced and regulated inflammatory response to T. gondii in the brain. T cells are the dominant immune cells that prevent clinical disease, and this is mediated through the secretion of effector molecules such as perforins and IFN-γ. The presence of cognate antigen, the expression of survival cytokines, and the alteration of the epigenetic landscape drive the development of memory T cells. However, specific extrinsic signals that promote the formation and maintenance of memory T cells within tissue are poorly understood. During chronic infection, there is an increase in extracellular glutamate that, due to its function as an excitatory neurotransmitter, is normally tightly controlled in the CNS. Here we demonstrate that CD8+ T cells from the T. gondii-infected brain parenchyma are enriched for metabotropic glutamate receptors (mGluR's). Characterization studies determined that mGluR+ expression by CD8+ T cells defines a distinct memory population at the transcriptional and protein level. Finally, using receptor antagonists and agonists we demonstrate mGluR signaling is required for optimal CD8+ T cell production of the effector cytokine IFNγ. This work suggests that glutamate is an important environmental signal of inflammation that promotes T cell function. Understanding glutamate's influence on T cells in the brain can provide insights into the mechanisms that govern protective immunity against CNS-infiltrating pathogens and neuroinflammation.
CD8+ iNKT cells are functionally different from other iNKT cells, and are enriched in human but not in mouse. To date, their developmental pathway and molecular basis for fate decision remain unclear. Here, we report enrichment of CD8+ iNKT cells in neonatal mice due to their rapider maturation kinetics than CD8- iNKT cells. Along developmental trajectories, CD8+ and CD8- iNKT cells separate at stage 0, following stage 0 DP iNKT cells, and differ in HIVEP3 expression. HIVEP3 is lowly expressed in stage 0 CD8+ iNKT cells and negatively controls their development, whereas it is highly expressed in stage 0 CD8- iNKT cells and positively controls their development. Despite no effect on IFN-γ, HIVEP3 inhibits granzyme B but promotes IL-4 production in CD8+ iNKT cells. Together, we reveal that, as a negative regulator for CD8+ iNKT fate decision, low expression of HIVEP3 in stage 0 CD8+ iNKT cells favors their development and Th1 biased cytokine responses as well as high cytotoxicity.
NK cells and CD8 T cells use cytotoxic molecules to kill virally infected and tumor cell targets. While perforin and granzyme B (GzmB) are the most commonly studied lytic molecules, less is known about granzyme K (GzmK). However, this granzyme has been recently associated with improved prognosis in solid tumors. In this study, we show that, in humans, GzmK is predominantly expressed by innate-like lymphocytes, as well as a newly identified population of GzmK+CD8+ non- mucosal-associated invariant T cells with innate-like characteristics. We found that GzmK+ T cells are KLRG1+EOMES+IL-7R+CD62L-Tcf7int, suggesting that they are central memory T and effector memory T cells. Furthermore, GzmK+ cells are absent/low in cord blood, suggesting that GzmK is upregulated with immune experience. Surprisingly, GzmK+ cells respond to cytokine stimuli alone, whereas TCR stimulation downregulates GzmK expression, coinciding with GzmB upregulation. GzmK+ cells have reduced IFN-γ production compared with GzmB+ cells in each T cell lineage. Collectively, this suggests that GzmK+ cells are not naive, and they may be an intermediate memory-like or preterminally differentiated population. GzmK+ cells are enriched in nonlymphoid tissues such as the liver and adipose. In colorectal cancer, GzmK+ cells are enriched in the tumor and can produce IFN-γ, but GzmK+ expression is mutually exclusive with IL-17a production. Thus, in humans, GzmK+ cells are innate memory-like cells that respond to cytokine stimulation alone and may be important effector cells in the tumor.
Cytokines contribute to T cell homeostasis at all stages of T cell existence. However, the particular cytokine involved varies as T cells progress from a naïve through an activated to a memory state. In many cases the important cytokines are members of the interleukin 2 subfamily of the short-chain type I cytokines. A case is made for the idea that the evolutionary divergence of the short-chain family allowed for concurrent divergence in leukocytes.
C57BL/6 mice genetically deficient in interleukin 15 (IL-15−/− mice) were generated by gene targeting. IL-15−/− mice displayed marked reductions in numbers of thymic and peripheral natural killer (NK) T cells, memory phenotype CD8+ T cells, and distinct subpopulations of intestinal intraepithelial lymphocytes (IELs). The reduction but not absence of these
populations in IL-15−/− mice likely reflects an important role for IL-15 for expansion and/or survival of these cells. IL-15−/− mice lacked NK cells, as assessed by both immunophenotyping and functional criteria, indicating an obligate role for IL-15
in the development and functional maturation of NK cells. Specific defects associated with IL-15 deficiency were reversed
by in vivo administration of exogenous IL-15. Despite their immunological defects, IL-15−/− mice remained healthy when maintained under specific pathogen-free conditions. However, IL-15−/− mice are likely to have compromised host defense responses to various pathogens, as they were unable to mount a protective
response to challenge with vaccinia virus. These data reveal critical roles for IL-15 in the development of specific lymphoid
lineages. Moreover, the ability to rescue lymphoid defects in IL-15−/− mice by IL-15 administration represents a powerful means by which to further elucidate the biological roles of this cytokine.
The c-kit ligand (KL; Steel factor, mast cell growth factor, stem cell factor) is a hematopoietic factor that has been shown to act as a potent cofactor for hematopoietic growth and differentiation in vitro. The in vivo effects of KL, however, have been variable. To study the hematopoietic role of KL in vivo, we evaluated KL gene expression in both normal mice and mice recovering from myelosuppressive radiation exposure using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique. In a single RNA sample, we found that the RT-PCR technique has high precision (co-efficient of variation, 15.7%). Amplifications of serial 1:2 dilutions of template RNA precisely correlated with starting RNA concentrations at 20 cycles or at 25 cycles, depending on the level of expression. Amplification of individual normal bone marrow and spleen cell RNA showed basal expression in all normal bone marrows but irregular expression in normal spleens. On day 2 after a sublethal 7.75-Gy (0.4 Gy/min) 60Co irradiation, splenic KL gene expression increased approximately 2.5-fold (P = .011), and bone marrow expression increased 15-fold (P = .004). During a 28-day postirradiation recovery period, KL expression increased in bone marrow on days 2 through 7. Splenic expression during the same period was more variable. In conclusion, the KL gene is invariably expressed in normal murine spleens. Postirradiation, recovering bone marrow and spleen both express increased levels of KL mRNA at day 2 and continue to express increased levels for several days postexposure. These data support a role for KL in the endogenous recovery of hematopoiesis after hypoplastic injury.
In vitro studies have defined an essential role for stromal cells in supporting B-cell development, including production of lymphopoietic cytokines. It has been suggested that stromal cells are equivalent to adventitial reticular cells in the marrow; however, evidence of reticular cells producing cytokines has been difficult to obtain. Staining of bone marrow (BM) sections with antibodies to interleukin-7 (IL-7) showed a reticular pattern, mimicking that obtained using antibodies to vascular cell adhesion molecule 1 (VCAM-1), a molecule present on both stromal cells in vitro and reticular cells. To more closely examine cytokine production within normal marrow, an immunomagnetic separation scheme was devised to directly enrich VCAM-1+ stromal cells. Twenty to thirty percent of cells isolated in the VCAM-1+ fraction shared characteristics with stromal cells from long term BM cultures, including cellular morphology and expression of alkaline phosphatase and alpha actin. These were termed "reticular stromal" cells. Immunohistochemical staining showed that virtually all of the latter cells possessed cytoplasmic IL-7 protein, and about half expressed stem cell factor. In contrast with cultured stromal cells, very few had detectable macrophage-colony-stimulating factor. These data constitute the first report of cytokine expression by marrow reticular cells in vivo. The implications of this data with respect to the existence of stromal cell subsets and their regulation of lymphopoiesis is discussed.
Interleukin 7 (IL-7) stimulates the proliferation of B cell progenitors, thymocytes, and mature T cells through an interaction with a high affinity receptor (IL-7R) belonging to the hematopoietin receptor superfamily. We have further addressed the role of IL-7 and its receptor during B and T cell development by generating mice genetically deficient in IL-7R. Mutant mice display a profound reduction in thymic and peripheral lymphoid cellularity. Analyses of lymphoid progenitor populations in IL-7R-deficient mice define precisely those developmental stages affected by the mutation and reveal a critical role for IL-7R during early lymphoid development. Significantly, these studies indicate that the phase of thymocyte expansion occurring before the onset of T cell receptor gene rearrangement is critically dependent upon, and mediated by the high affinity receptor for IL-7.
Prompt, cytokine-mediated restoration of hematopoiesis is a prerequisite for survival after irradiation. Therapy with biologic response modifiers (BRMs), such as LPS, 3D monophosphoryl lipid A (MPL), and synthetic trehalose dicorynomycolate (S-TDCM) presumably accelerates hematopoietic recovery after irradiation by enhancing expression of cytokines. However, the kinetics of the cytokine gene response to BRMs and/or irradiation are poorly defined. One hour after sublethal (7.0 Gy) 60Cobalt gamma irradiation, B6D2F1/J female mice received a single i.p. injection of LPS, MPL, S-TDCM, an extract from Serratia marcescens (Sm-BRM), or Tween 80 in saline (TS). Five hours later, a quantitative reverse transcription-PCR assay demonstrated marked splenic gene expression for IL-1 beta, IL-3, IL-6, and granulocyte-CSF (G-CSF). Enhanced gene expression for TNF-alpha, macrophage-CSF (M-CSF), and stem cell factor (SCF) was not detected. Injection of any BRM further enhanced cytokine gene expression and plasma levels of CSF activity within 24 h after irradiation and hastened bone marrow recovery. Mice injected with S-TDCM or Sm-BRM sustained expression of the IL-6 gene for at least 24 h after irradiation. Sm-BRM-treated mice exhibited greater gene expression for IL-1 beta, IL-3, TNF-alpha, and G-CSF at day 1 than any other BRM. When challenged with 2 LD50/30 of Klebsiella pneumoniae 4 days after irradiation, 100% of Sm-BRM-treated mice and 70% of S-TDCM-treated mice survived, whereas < or = 30% of mice treated with LPS, MPL, or TS survived. Thus, sublethal irradiation induces transient, splenic cytokine gene expression that can be differentially amplified and prolonged by BRMs. BRMs that sustained and/or enhanced irradiation-induced expression of specific cytokine genes improved survival after experimental infection.
On the basis of their surface markers, T lymphocytes are divided into subsets of "naive" and "memory cells". We have defined the interrelationship and relative life spans of naive and memory T cells by examining the surface markers on murine T cells incorporating bromodeoxyuridine, a DNA precursor, given in the drinking water. Three findings are reported. First, using a new method we show that the release of newly formed naive T cells from the unmanipulated thymus is very low (confirming the findings of others with surgical approaches). Second, in thymectomized mice, T cells with a naive phenotype remain in interphase for prolonged periods; however, some of these cells divide and retain (or regain) their "naive" markers. Third, most T cells with a memory phenotype divide rapidly, but others remain in interphase for many weeks. Collectively, the data indicate that long-lived T cells have multiple phenotypes and contain a mixture of memory cells, naive (virgin) cells, and memory cells masquerading as naive cells.
A monoclonal antibody, A7R34, that recognizes the high-affinity interleukin 7 receptor (IL-7Ra) and blocks the binding between IL-7 and IL-7Ra has been produced. Cell surface staining with A7R34 demonstrated that IL-7Ra is expressed in both B- and T-cell lineages. In the bone marrow, immature B-lineage cells that do not express surface IgM were IL-7Ra+. In the thymus, IL-7Ra was detected in CD4-8- T cells and also in CD4 or CD8 single-positive cells but not in CD4+8+ double-positive cells. In the peripheral lymphoid tissues, both CD4 and CD8 single-positive cells were the major cell types that express IL-7Ra. Addition of A7R34 to a long-term B-precursor-cell culture inhibited proliferation of the B-lineage cells, indicating that IL-7 is an absolute requirement for in vitro B-cell genesis. Consistent with this in vitro result, continuous injection of A7R34 into an adult mouse resulted in a decrease of B-precursor cells and also of thymocytes, whereas a considerable fraction of mature B and T cells in the peripheral tissues persisted over 2 weeks of the experiment. When A7R34 injection is started from day 14 of gestation, it is possible to produce mice that lack B cells. These results indicate that IL-7 is an essential molecule for generation of both B and T cells in murine bone marrow and thymus, respectively. Moreover, IL-7Ra would be the sole receptor system regulating these processes.
Mice lacking functional IL-7 or IL-7R alpha genes are severely deficient in developing thymocytes, T cells, and B cells. IL-7 and IL-7 receptor functions are believed to result in lymphoid cell proliferation and cell maturation, implying signal transduction pathways directly involved in mitogenesis and elaboration of developmentally specific new gene programs. Here, we show that enforced expression of the bcl-2 gene in T-lymphoid cells (by crossing in the Emu-bcl-2 transgene) in IL-7R alpha-deficient mice results in a significant restoration of thymic positive selection and T cell numbers and function. We propose cell survival signals to be the principal function of IL-7R engagement in thymic and T cell development.
Although much is known about the activation, proliferation, and function of CD4(+) T cells, little is known about how they survive as resting T cells in animals. Resting T cells have a half-life in animals of more than a week; however, when they are removed from animals and placed in tissue culture their half-life falls to approximately 24 h. In this paper, we show that the survival of resting T cells in vitro is promoted by two cytokines, interleukins 4 and 7 (IL-4, IL-7). They may do this in part by maintaining levels of survival-promoting proteins such as Bcl-2 in the cells, because the levels of Bcl-2 and Bcl-Xl in resting T cells fall rapidly after the cells are isolated from animals, and are maintained by culture in IL-4. Because the IL-4 receptor is known to signal through the JAK1 and JAK3/Stat6 pathway, we tested whether Stat6 was required for IL-4- dependent T cell survival. Surprisingly, we found that IL-4 rescued T cells from apoptosis in what appeared to be a Stat6-independent manner. These results demonstrate that the survival of resting T cells is an active process that can be affected by signals delivered by cytokines and also suggest that the IL-4 receptor on resting T cells may use a novel signaling pathway to facilitate T cell viability.
Thymic T cell development is controlled by T cell receptor (TCR)-major histocompatibility complex (MHC) interactions, whereas a further dependence of peripheral mature T cells on TCR-MHC contact has not been described so far. To study this question, CD4 T cell survival was surveyed in mice lacking MHC class II expression and in mice expressing MHC class II exclusively on dendritic cells. Since neither of these mice positively select CD4 T cells in the thymus, they were grafted with MHC class II-positive embryonic thymic tissue, which had been depleted of bone marrow derived cells. Although the thymus grafts in both hosts were repopulated with host origin thymocytes of identical phenotype and numbers, an accumulation of CD4+ T cells in peripheral lymphoid organs could only be observed in mice expressing MHC class II on dendritic cells, but not in mice that were completely MHC class II deficient. As assessed by histology, the accumulating peripheral CD4 T cells were found to be in close contact with MHC class II+ dendritic cells, suggesting that CD4 T cells need peripheral MHC class II expression for survival and that class II+ dendritic cells might play an important role for the longevity of CD4 T cells.
MHC molecules influence the fate of T lymphocytes at two important stages of their differentiation. Recognition of self peptide/MHC complexes in the thymus determines whether immature T cells should live and mature into immunocompetent T cells or whether they should die. In the periphery, recognition of Ags presented by MHC molecules induces T cell activation, proliferation, and differentiation into effector/memory T cells. We describe in this work a third role that MHC molecules play in T cell physiology. CD8+ thymic emigrants require presence of MHC class I molecules in the periphery to seed the peripheral lymphoid organs. Numbers of CD8+ T cells are reduced severely in both the thymus and the periphery of beta2-microglobulin-deficient (beta2m[-/-]) mice. When grafted with wild-type (beta2m[+/+]) thymic epithelium, immature beta2m(-/-) T cells that populate the graft develop into functional mature CD8+ cells. However, significant numbers of peripheral CD8+ cells in grafted beta2m(-/-) mice can be observed only after injection of MHC class I-expressing cells in the periphery. Thus, naive T cells in the periphery do not passively await antigenic stimulation, but actively engage in interactions with self MHC molecules that may promote their survival.
The thymus is essential for the initial seeding of T cells to the periphery, but its role in maintaining the adult T cell pool remains poorly defined. We investigated whether changes to the rate of T cell export could form part of the mechanism(s) controlling the homeostatic regulation of the size and composition of the peripheral T cell pool. Using neonatal thymi grafted under the kidney capsule, we found that irrespective of whether the pool was oversupplied (by thymic grafts) or undersupplied (due to neonatal thymectomy), the thymic export rate was constant from both the host and graft thymus, and the periphery remained constant in size. Recent thymic emigrants (RTE) were also tracked to determine the extent of their acceptance into the T cell pool of a normal mouse. As a population, RTE are phenotypically mature, but were distinct from resident T cells in the periphery, being released in a CD4/CD8 ratio approximately twice that of established peripheral T cells. This export ratio is similar to that of T cells in the mature thymic compartment, but soon after entry into the periphery, the ratio falls, indicating separate thymic and peripheral regulation of the CD4/CD8 ratio. RTE may also be preferentially incorporated into the periphery, causing displacement of resident T cells, thus maintaining the size of the peripheral pool. Although not vital for the maintenance of a functional T cell pool, the acceptance of RTE in a "full" peripheral pool would ensure that the T cell receptor repertoire is kept diverse and that the T cell population encompasses a broad range of naive as well as memory T cells.
Signals from the IL-7R are essential for normal thymocyte development. We isolated thymocytes from early developmental stages and observed that suspensions of pro-T1, -T2, and -T3 cells rapidly died in culture. Addition of IL-7 promoted their survival, but did not induce cell division. Pro-T4 cells did not undergo rapid cell death, and their survival was therefore independent of IL-7. Death in the absence of IL-7 showed the hallmarks of apoptosis, including DNA fragmentation and annexin V binding; however, caspase inhibitors blocked DNA fragmentation, but did not block cell death. The trophic effect of IL-7 was partially inhibited by blocking protein synthesis. The p53 pathway was not involved in this death pathway, since pro-T cells from p53-/- mice also underwent cell death in the absence of IL-7. The Fas/Fas ligand pathway was not involved in cell death, since Fas-deficient pro-T cells died normally in the absence of IL-7, anti-Fas Abs did not protect cells from death in the absence of IL-7, and Fas expression was undetectable on cells at these stages. The IL-7 trophic affect correlated with increased intracellular levels of Bcl-2 and decreased levels of Bax, whereas no Bcl-X(L), Bcl-w, or Bad was detectable. Thus, maintaining a favorable Bcl-2/Bax ratio may account for the trophic action of IL-7.
We compared peripheral and mucosal primary CD8 T cell responses to inflammatory and noninflammatory forms of antigen in a
T cell-adoptive transfer system. Immunization with the soluble antigen, ovalbumin (ova), administered i.p. or orally without
adjuvant, activated nonmucosal CD8 T cells but did not induce cytotoxic activity. However, after activation, the transferred
cells entered the intestinal mucosa and became potent antigen-specific killers. Thus, exogenous intact soluble protein entered
the major histocompatibility complex class I antigen presentation pathway and induced mucosal cytotoxic T lymphocytes. Moreover,
distinct costimulatory requirements for activation of peripheral versus mucosal T cells were noted in that the CD28 ligand,
B7-1, was critical for activated mucosal T cell generation but not for activation of peripheral CD8 T cells. The costimulator,
B7-2, was required for optimum activation of both populations. Infection with a new recombinant vesicular stomatitis virus
encoding ovalbumin induced lytic activity in mucosal as well as peripheral sites, demonstrating an adjuvant effect of inflammatory
mediators produced during virus infection. Generation of antiviral cytotoxic T lymphocytes was also costimulation-dependent.
The results indicated that induction of peripheral tolerance via antigen administration may not extend to mucosal sites because
of distinct costimulatory and inflammatory signals in the mucosa.
The adoptive transfer of TCR-transgenic T cells into syngeneic recipients allows characterization of individual T cells during in vivo immune responses. However, the proliferative behavior of individual T cells and its relationship to effector and memory function has been difficult to define. Here, we used a fluorescent dye to dissect and quantify T cell proliferative dynamics in vivo. We find that the average Ag-specific CD4+ T cell that undergoes division in vivo generates >20 daughter cells. TCR and CD28 signals cooperatively determine the degree of primary clonal expansion by increasing both the proportion of Ag-specific T cells that divide and the number of rounds of division the responding T cells undergo. Nonetheless, despite optimal signaling, up to one-third of Ag-specific cells fail to divide even though they show phenotypic evidence of Ag encounter. Surprisingly, however, transgenic T cells maturing on a RAG-2-/- background exhibit a responder frequency of 95-98% in vivo, suggesting that maximal proliferative potential requires either a naive phenotype or allelic exclusion at the TCRalpha locus. Finally, studies reveal division cycle-dependent expression of markers of T cell differentiation, such as CD44, CD45RB, and CD62L, and show also that expression of the cytokines IFN-gamma and IL-2 depends primarily on cell division rather than on receipt of costimulatory signals. These results provide a quantitative assessment of T cell proliferation in vivo and define the relationship between cell division and other parameters of the immune response including cytokine production, the availability of costimulation, and the capacity for memory.
We investigated the mechanism by which alpha/beta T cells expand upon transfer to T cell-deficient host mice by injecting carboxyfluorescein diacetate succinimidyl ester-labeled T cells into mice depleted of T cells by sublethal irradiation. We found that CD4+ T cells divided when transferred to irradiated hosts and that the division of more than half of these cells required class II expression. However, division of transferred CD4+ T cells did not occur in irradiated hosts that expressed class II molecules occupied solely by the peptide responsible for thymic selection, indicating that peptides distinct from those involved in thymic selection cause the division of CD4+ T cells in irradiated mice. These data establish that class II-bound peptides control the expansion of CD4+ T cells transferred to T cell-deficient hosts and suggest that the same peptides contribute to the maintenance of T cell numbers in normal mice.
In vitro studies have defined an essential role for stromal cells in supporting B-cell development, including production of lymphopoietic cytokines. It has been suggested that stromal cells are equivalent to adventitial reticular cells in the marrow; however, evidence of reticular cells producing cytokines has been difficult to obtain. Staining of bone marrow (BM) sections with antibodies to interleukin-7 (IL-7) showed a reticular pattern, mimicking that obtained using antibodies to vascular cell adhesion molecule 1 (VCAM-1), a molecule present on both stromal cells in vitro and reticular cells. To more closely examine cytokine production within normal marrow, an immunomagnetic separation scheme was devised to directly enrich VCAM-1+ stromal cells. Twenty to thirty percent of cells isolated in the VCAM-1+ fraction shared characteristics with stromal cells from long term BM cultures, including cellular morphology and expression of alkaline phosphatase and alpha actin. These were termed “reticular stromal” cells. Immunohistochemical staining showed that virtually all of the latter cells possessed cytoplasmic IL-7 protein, and about half expressed stem cell factor. In contrast with cultured stromal cells, very few had detectable macrophage-colony-stimulating factor. These data constitute the first report of cytokine expression by marrow reticular cells in vivo. The implications of this data with respect to the existence of stromal cell subsets and their regulation of lymphopoiesis is discussed.
We grafted fetal thymi from wild-type mice into immunodeficient RAG-2-/- or class II-/-RAG-2-/- (class II MHC-) recipients and followed the fate of naive CD4+ T cells derived from the grafts. In both types of recipients, newly generated CD4+ T cells proliferated to the same extent in the periphery and rapidly filled the empty T cell compartment. However, CD4+ T cells in class II- recipients gradually decreased in number over 6 months. These results show that interactions between the TCR and class II molecules are not required for newly generated CD4+ T cells to survive and proliferate, but are necessary to maintain the size of the peripheral T cell pool for extended periods.
Purpose: To investigate cytokine gene expression in the lung after single and fractionated doses of radiation, and to investigate the effect of steroids and the genetic background. Materials and methods: Expression of cytokine genes (mTNF-alpha, mIL-1alpha, mIL-1beta, mIL-2, mIL-3, mIL-4, mIL-5, mIL-6, mIFNgamma) in the lungs of C3H/HeJ and C57BL/6J mice was measured by RNase protection assay at different times after various doses of radiation. The effects of dexamethasone and fractionated radiation treatment on gene expression were also studied. Results: IL-1beta was the major cytokine induced in the lungs of C3H/HeJ mice within the first day after thoracic irradiation. Radiation doses as low as 1Gy were effective. Responses to 20Gy irradiation peaked within 4-8h and subsided by 24h. With the exception of IL-1alpha and TNF-alpha, the other cytokines that were investigated had undetectable pre-treatment mRNA levels and were not radiation inducible. Similar responses were seen in C57BL/6J mice, although TN...
Interleukin (IL)-7 is a potent stimulus for immature T and B cells and, to a lesser extent, mature T cells. We have inactivated the IL-7 gene in the mouse germline by using gene-targeting techniques to further understand the biology of IL-7. Mutant mice were highly lymphopenic in the peripheral blood and lymphoid organs. Bone marrow B lymphopoiesis was blocked at the transition from pro-B to pre-B cells. Thymic cellularity was reduced 20-fold, but retained normal distribution of CD4 and CD8. Splenic T cellularity was reduced 10-fold. Splenic B cells, also reduced in number, showed an abnormal population of immature B cells in adult animals. The remaining splenic populations of lymphocytes showed normal responsiveness to mitogenic stimuli. These data show that proper T and B cell development is dependent on IL-7. The IL-7-deficient mice are the first example of single cytokine-deficient mice that exhibit severe lymphoid abnormalities.
The requisite molecular interactions for CD8 T cell memory were determined by comparison of monoclonal naı̈ve and memory CD8+ T cells bearing the T cell receptor (TCR) for the HY antigen. Naı̈ve T cells required only the right major histocompatibility
complex (MHC) class I–restricting molecule to survive; to expand, they also needed antigen. In contrast, for survival, memory
cells did not require the restricting MHC allele, but needed only a nonspecific class I; for expansion the correct class I,
but not antigen, was required. Thus, maintenance of CD8 T cell memory still required TCR–MHC class I interactions, but memory
T cells may have a lower functional activation threshold that facilitates secondary responses.
Using a semi-quantitative polymerase chain reaction (PCR) technique we have examined the expression of a panel of cytokines during thymus development, localizing the expression to individual components of the thymic stroma and thymocytes at different maturational stages. The expression of interleukin (IL)-7, stem cell factor (SCF), IL-1α and granulocyte-monocyte-colony-stimulating factor (GM-CSF) mRNA was mapped to individual stromal cell types, while the expression of IL-1α and GM-CSF, along with interferon (IFN)-γ and IL-4 was detected in the lymphoid compartment of fetal day (Fd) 14 thymus. The expression of lymphoid-specific cytokine genes was selectively down-regulated in thymocytes undergoing maturation. CD3−/104+8+ cells, representing an intermediate stage of thymocyte maturation, were devoid of cytokine gene expression. Their CD3+ progeny, on the other hand, expressed IFN-γ mRNA, supporting the notion that positive selection of cells for further maturation induces the reexpression of some cytokine genes. The cytokine profiles of the various stromal components differed. Purified major histocompatibility complex class II+ cortical epithelial cells strongly expressed IL-7 and SCF, but only limited expression of IL-lα and GM-CSF could be detected. Fetal mesenchyme, on the other hand, expressed SCF, IL-lα and GM-CSF but not IL-7. The importance of these cytokine profiles in relation to T cell development is discussed.
Interleukin-7 (IL-7) has long been known as a potent growth factor in lymphocyte development. However, recent data obtained in vitro revealed additional functions for this cytokine, e.g. IL-7 influences the generation of cytotoxic T cells and NK cells, and in higher concentrations, activates monocytes in a manner similar to bacterial lipopolysaccharide. Furthermore, human tonsillar B cells are able to proliferate upon stimulation by IL-7 and cross-linking of the B cell receptor. Considering the latter role of IL-7 for B cell proliferation, we investigated which cells of the human immune system express IL-7 in vivo. mRNA expression of tonsillar cells was analyzed using the reverse transcription polymerase chain reaction, and protein expression was assessed by immunohistochemical staining of frozen sections. Among a variety of different immune cell types isolated from human tonsils, only follicular dendritic cells (FDC) expressed specific IL-7 products, whereas B and T cells were consistently negative. Immunohistochemical staining of tonsillar sections revealed the expression of immunoreactive IL-7 protein on FDC, and a pronounced expression could also be detected in oral mucosa and vascular endothelial cells. For the latter, we could also demonstrate mRNA expression in primary cultured cells. In light of the previous finding that IL-7 can act as a co-stimulus for the induction of proliferation in tonsillar B cells, these data suggest a role of IL-7 in the germinal center reaction. It is tempting to speculate that FDC may function as regulatory cells in B cell development in the tonsil, as epithelial nurse cells are thought to govern T cell development in the thymus.
We have monitored the expression of interleukin-7 (IL-7) in the developing embryonic mouse thymus by a combination of quantitative polymerase chain reaction (PCR) and immunofluorescence microscopy. A strong specific signal for IL-7 mRNA was detected by day 12 in the developing fetal thymus. IL-7 mRNA was found to be maximally expressed on day 15, and then decreased over the next 5 days. Immunofluorescence staining of fetal thymus sections using an anti-IL-7 antibody confirmed these PCR data. IL-7 protein expression was first detected at day 13 of development. At 14 days the intensity of the staining increased by a factor of three and stayed at this level over the next 4 days. The same anti-IL-7 antibody used for immunofluorescence, blocked the proliferation of fetal thymocytes in organotypic cultures. In addition, we detected mRNA coding for IL-2 and SCF (also known as the steel factor or KL) in embryonic thymocytes. The implications of these findings for early thymocyte growth are discussed.
The regulation of murine T cell proliferation by IL-7 was investigated. Highly purified resting splenic T cells were induced to proliferate in a short term assay by IL-7 in the presence of the comitogen, Con A. The proliferation of these resting T cells showed both IL-2-dependent and -independent components as determined by the susceptibility of the response to the blocking effects of anti-IL-2 mAb. Furthermore, IL-7 was found to augment the Con A-induced production of IL-2 and expression of IL-2R by resting splenic T cells. In contrast, Con A blasts and long term, Ag-dependent cloned T cells proliferated in response to IL-7 independently of any involvement of IL-2. Finally, differences were observed between IL-7 and IL-6 with regard to the regulation of T cell growth and activation. As with IL-7, IL-6 stimulated resting splenic T cells to proliferate in the presence of comitogen. However, in contrast to IL-7, IL-6 failed to stimulate the proliferation of Con A blasts or T cell clones and did not augment the Con A-induced expression of IL-2R on resting T cells.
Peripheral T lymphocytes are self-renewable cell populations since, when transferred into syngeneic T cell-deficient athymic mice, they expand in the absence of exogenous antigen stimulation. Quantification of the expansion potential of CD4+ cells by transfer of the same population into successive host mice shows that these cells are able to divide up to 56 times in vivo. Therefore, as a population, CD4+ cells can increase in size 8 x 10(5)-fold, an expansion potential of similar magnitude to that previously reported for colony-forming units. Injection of different numbers of T cells at different CD4/CD8 ratios is followed by T cell accumulation to a similar plateau in recipient nude mice. This indicates that peripheral T lymphocytes are tightly regulated by homeostatic mechanisms that control pool sizes and CD4/CD8 ratios, in a manner independent of the cell input into the peripheral compartment. This kinetic behavior of mature T cells permits the maintenance at the periphery of any T cell specificity previously selected in the thymus. The expansion capacity of peripheral T cells may also allow extensive modulation of peripheral T cell specificities, which would confer a major role to post-thymic selection of mature peripheral T cell repertoires.
Athymic nude rats (PVG.rnu/rnu) were injected at 6 to 10 wk of age with 1 to 200 million thoracic duct lymphocytes (TDL) containing 40 to 60% mature T cells. Thereafter TDL-injected nude recipients were monitored for evidence of T cell function for up to 2 yr. W3/25+ T helper (Th) cells in lymph nodes (LN) increased from 7% at 2 wk to 30% at 8 wk after TDL transfer. The percent of W3/25+ cells remained elevated for the life of the recipient (up to 2 yr), approximating normal levels. The total size of the recirculating pool expanded in TDL-injected nude rats to reach 2/3 the level of euthymic controls by 16 wk, an increase of 10-fold to 15-fold in W3/25+ cells. The expansion of the W3/25+ population was independent of initial TDL dose. With time spleen and LN acquired a normal histological appearance including the development of germinal centres and a marked increase in cellularity in T cell traffic areas. TDL-injected nude rats rejected skin allografts with near normal kinetics. In addition graft vs host (GVH) responsiveness, assessed by the popliteal LN assay, progressively increased reaching a level 9 mo to 1 yr after replacement that resembled the GVH activity in euthymic controls.
The percentage of haem-containing nucleated RBC precursor cells in marrows and spleens of W/Wv and Sl/Sld anaemic mice increased in response to hypoxia produced by reduced pressure, red-cell loss, or red-cell destruction by phenylhydrazine. The response in the spleens of W/Wv mice occurred several days later than the marrow response, in contrast to normal or Sl/Sld mice in which the marrow and spleen responses occurred simultaneously. These responses were also simultaneous in W/Wv mice whose anaemia had been cured by an injection of normal stem cells. After RBC destruction by phenylhydrazine the duration and degree of tissue hypoxia necessary to stimulate the Sl/Sld response varied widely among individual Sl/Sld mice. In Sl/Sld mice with anaemias alleviated by implants of intact normal spleens, most erythropoiesis was contained within these normal spleen grafts.
Polycythaemic W/Wv and Sl/Sld anaemic mice gave defective responses to erythropoietin; the defect was not caused by delayed erythroid maturation. Serum samples from anaemic mice responding to RBC destruction by phenylhydrazine contained high concentrations of erythropoietin in quantities inversely proportional to the donor's PCV (packed cell volume), but did not contain any factors, other than erythropoietin, to stimulate erythropoiesis in polycythaemic Sl/Sld anaemic mice.
Five rat monoclonal antibodies have been derived that express specificities for determinants present on the molecular complex bearing the Lyt 2 antigen. SDS-polyacrylamide gel electrophoresis of 125I-labeled polypeptides precipitated by each of these antibodies reveal 3 components (150,000, 75,000, and 33,000 daltons), and 2 components (44,000 and 33,000 daltons) when analyzed under nonreducing and reducing conditions, respectively. Two of these antibodies are IgG and are specific for the Lyt 2.2 determinant; the other 3 are IgM and react with determinants other than Lyt 2.2, which are nonpolymorphic. Each of the 5 antibodies can block the cytolytic activities of 5-day MLC cells or of cloned cytolytic T cells in the absence of C. Treatment of responding spleen cells with any of these antibodies and C inhibits the generation of cytolytic activity in MLC.
Mature T cells may be produced in the thymus, or by expansion in the periphery. While thymus output of virgin cells ensures repertoire diversity, peripheral expansion increases the size of rare clones, and thus the efficiency of immune responses. We studied the role of both phenomena in the generation of the CD8+ T cell pool using RAG-/- female mice expressing a transgenic T cell receptor specific for the male antigen; nude mice injected with peripheral T cells; and euthymic irradiated chimeras injected with bone marrow and mature T cells. Our results show that the total number of virgin and activated T cells, each constituting about half of the peripheral T cell pool, was regulated independently, revealing an efficient mechanism to maintain repertoire diversity while optimizing the immune response.
We have studied memory in T cell receptor (TCR) transgenic mice expressing a Db-restricted TCR specific for the male peptide (H-Y). CD8+ T cells from female TCR transgenic C57BL/6 (B6) mice were activated by transferring them into X-irradiated male (B6 x bm12)F1 hybrid recipients. Subsequently, they were highly purified by cell sorting and transferred for various lengths of time into female B6 nu/nu recipient mice. Other nu/nu recipient mice received highly purified naive T cells expressing the transgenic TCR. The functional potential of naive and "memory" T cells was analyzed by stimulation with male cells in vivo. The results show that memory cells can be derived from activated T cells and persist in the absence of antigen for at least 13 weeks. Naive and memory T cells differ in that memory T cells give a more vigorous and sustained response than naive T cells.
The interaction of mucosal lymphocytes and intestinal epithelial cells is thought to be important in regulating immune response in the intestinal mucosa, but conclusive evidence is limited. Here we demonstrate the expression of IL-7 mRNA in human intestinal mucosa by combined reverse transcription PCR and Southern blot hybridization. Immunohistochemistry and in situ hybridization confirm the presence of IL-7 in intestinal epithelial cells, especially in epithelial goblet cells. Moreover, IL-7 receptor expression in mucosal lymphocytes is demonstrated by immunohistochemistry and in situ hybridization, as well as by Southern blot and flow cytometric analysis of freshly isolated lamina propria lymphocytes. In contrast, IL-7 receptor could not be detected in the cell surface of freshly isolated PBLs. The functional activity of IL-7 receptor is demonstrated by the utility of recombinant IL-7 to stimulate the growth of lamina propria lymphocytes, and conversely inhibit CD3-dependent proliferation of these cells. In contrast, IL-7 caused no significant increase in DNA synthesis and cell numbers when added to PBLs. These findings suggest that human intestinal epithelial cells and epithelial goblet cells produce IL-7, and locally produced IL-7 may serve as a potent regulatory factor for intestinal mucosal lymphocytes.
The interleukin-2 receptor gamma chain (IL-2R gamma) is a necessary component of functional IL-2 receptors. IL-2R gamma mutations result in X-linked severe combined immunodeficiency (XSCID) in humans, a disease characterized by the presence of few or no T cells. In contrast, SCID patients with IL-2 deficiency and IL-2-deficient mice have normal numbers of T cells, suggesting that IL-2R gamma is part of more than one cytokine receptor. By using chemical cross-linking, IL-2R gamma was shown to be physically associated with the IL-7 receptor. The presence of IL-2R gamma augmented both IL-7 binding affinity and the efficiency of internalization of IL-7. These findings may help explain the defects of XSCID. Given its role in more than one cytokine receptor system, the common gamma chain (gamma c) is proposed as the designation for IL-2R gamma.
The gamma chain of the interleukin-2 (IL-2) receptor is an indispensable subunit for IL-2 binding and intracellular signal transduction. A monoclonal antibody to the gamma chain, TUGm2, inhibited IL-2 binding to the functional IL-2 receptors and also inhibited IL-4-induced cell growth and the high-affinity binding of IL-4 to the CTLL-2 mouse T cell line. Another monoclonal antibody, TUGm3, which reacted with the gamma chain cross-linked with IL-2, also immunoprecipitated the gamma chain when cross-linked with IL-4. These results suggest that the IL-2 receptor gamma chain is functionally involved in the IL-4 receptor complex.
We have used organ culture of fetal thymic lobes from T cell receptor (TCR) transgenic beta 2M(-/-) mice to study the role of peptides in positive selection. The TCR used was from a CD8+ T cell specific for ovalbumin 257-264 in the context of Kb. Several peptides with the ability to induce positive selection were identified. These peptide-selected thymocytes have the same phenotype as mature CD8+ T cells and can respond to antigen. Those peptides with the ability to induce positive selection were all variants of the antigenic peptide and were identified as TCR antagonist peptides for this receptor. One peptide tested, E1, induced positive selection on the beta 2M(-/-) background but negative selection on the beta 2M(+/-) background. These results show that the process of positive selection is exquisitely peptide specific and sensitive to extremely low ligand density and support the notion that low efficacy ligands mediate positive selection.
While early thymic T cell precursor populations and their maturational sequence have been recently identified, the signals driving differentiation are unknown. While cytokines may play an integral role in T cell development, various mouse models rendered genetically deficient for specific cytokines do not display abnormalities in T cell development. Recently, we have generated IL-7 -/- mice and reported that IL-7 plays a unique and nonredundant role in lymphopoiesis. These mice displayed a 10- to 20-fold reduction in the total number of T and B cells. Here, we show that IL-7 -/- mice display a sharp reduction in both the frequency and absolute number of adult thymic gamma delta T cells while retaining normal frequencies of alpha beta T cells. This defect in gamma delta T cell production extends to peripheral organs as IL-7 -/- mice are essentially devoid of splenic and intestinal intraepithelial gamma delta T cells. This aberrant phenotype was traced back to impaired fetal gamma delta T cell maturation. In the absence of IL-7, differentiation of immature V gamma 3 low-CD24+ fetal T cells to mature V gamma 3 high CD24- cells is inhibited. In contrast, NK cell maturation appears to be only mildly affected in the absence of IL-7. To further clarify the role of IL-7 in thymic development, detailed analysis of CD3-4-8- thymic precursors was performed. A partial inhibition in the differentiation of CD44+25+ pro-T cells into CD44-25+ pre-T cells was observed. Unexpectedly, the lack of IL-7 resulted in decreased expression of CD117 (c-kit) on both CD4 low and pro-T cells, suggesting that IL-7 may influence the expression of other cytokine receptors involved in early hemopoietic development. Together, these data clarify the developmental abnormalities during T cell development due to the absence of IL-7.
Mice genetically deficient in IL-7R(alpha) are highly lymphopenic in the peripheral lymphoid organs. The functional competence of T cells that have developed in the absence of an IL-7R signal was investigated. Three important observations were made using several in vitro activation regimens. First, stimulation of T cells from IL-7R -/- mice at limiting dilution with immobilized Abs to CD3, CD4 or CD8, and CD18 revealed a six- to sevenfold reduction in the frequency of clonogenic T cells compared with T cells from IL-7R +/+ mice. IL-7R -/- T cells were also significantly less responsive to alloantigen as well as to receptor-independent stimuli such as PMA and ionomycin. Furthermore, the average clone size of single IL-7R -/- T cells was 50% smaller than that of IL-7R +/+ T cells. These data suggest that the reduced clonogenicity was predominantly due to intrinsic deficiencies in the ability of IL-7R -/- T cells to proliferate upon stimulation. Second, analysis of the kinetics of cell growth of IL-7R -/- T cells revealed that a significant proportion of T cells failed to proliferate within the first 72 h of in vitro stimulation, with the majority undergoing programmed cell death. Third, both clonogenic IL-7 -/- T cells and IL-7R +/+ T cells showed a similar proliferative response in the presence of IL-2 and similar survival kinetics, indicating that a subpopulation of IL-7R -/- T cells is functionally mature. We propose that an absence of IL-7R signaling not only affects T cell development in the thymus, but also results in the accumulation of functionally inactive T cells in the periphery.
Expression of activation markers and proliferative status were measured in peripheral CD4+ and CD8+ T cells of various T cell receptor (TCR)-transgenic mice either before or after intentional antigenic stimulation. In the absence of intentional immunization, CD4+ T cells persisted as resting or partially activated and cycling cells depending on the specificity of their TCR. Similar results were obtained following transfer into T cell-deficient recipients, i.e. T cells that were not cycling in situ did not cycle after transfer, whereas cells that were proliferating in situ also cycled after transfer. Thus, the TCR of some cells in the absence of intentional antigenic stimulation may bind to some unidentified ligand that does not induce tolerance, but rather slow expansion. In a different sort of experiment, activated T cells that were derived from noncycling naive T cells by deliberate antigenic stimulation continued to cycle slowly even a long time after transfer into antigen-free recipients that did not induce proliferation of the naive cells. Thus, lymphokines or ligands that do not induce activation of naive T cells may be responsible for the maintenance of memory cells. Our experiments show that the latter does not depend on a second TCR expressed by the memory cells, since memory T cells from RAG-2(-/-) TCR-transgenic mice persisted to a similar extent.
Interleukin-7 (IL-7)-deficient mice exhibit an early defect in lymphopoiesis. We examined Bcl-2 expression and the cell cycle status of immature thymocyte subsets in these mice. In IL-7-deficient mice, developmental transition to a T cell-committed fate was accompanied by a striking loss of Bcl-2 protein expression and an increased relative proportion of cells in the G0/G1 stage of the cell cycle. Short-term culture of immature thymocytes with rIL-7 caused up-regulation of Bcl-2 protein and cell survival. These data specify a T cell lineage developmental transition point, prior to T cell antigen receptor rearrangement, where IL-7 signal transduction is linked to an anti-apoptosis mechanism and the cell cycle.
Mature single-positive (SP) T lymphocytes enter a “resting” state in which they are proliferatively quiescent and relatively
resistant to apoptosis. The molecular mechanisms regulating this quiescent phenotype were unknown. Here it was found that
the expression of a Kruppel-like zinc finger transcription factor, lung Kruppel-like factor (LKLF), is developmentally induced
during the maturation of SP quiescent T cells and rapidly extinguished after SP T cell activation. LKLF-deficient T cells
produced by gene targeting had a spontaneously activated phenotype and died in the spleen and lymph nodes from Fas ligand–induced
apoptosis. Thus, LKLF is required to program the quiescent state of SP T cells and to maintain their viability in the peripheral
lymphoid organs and blood.
The putative effects of interleukin (IL)-7, operating in the context of extracellular matrix (ECM), on the adhesion of human T cells were examined. Recombinant human, IL-7 was found to bind ECM or fibronectin (FN) with IC50 values of 10-100 nM. Nanogram amounts of both soluble and, especially, FN- or ECM-bound IL-7, which differentially affected the morphologies of FN-adherent T cells, induced the adhesion of resting CD4+ and CD8+ T cells in dose-dependent and beta 1 integrin-dependent manners. Under static and flow conditions, soluble IL-7 also induced the binding of unstimulated T cells to vascular cell adhesion molecule-1, suggesting that this cytokine can also modulate integrin binding to endothelial cell ligands. The effects of affinity modulation by IL-7 of FN-specific beta 1 integrins depend on the presence of soluble FN, which inhibited T cell adhesion to FN induced by FN-bound IL-7 or by an integrin-specific affinity-modulating monoclonal antibody, but not by soluble IL-7 or phorbol 12-myristate 13-acetate. These findings provide an example of a major ECM integrin ligand, FN, which is capable of modulating its adhesive interactions with specific immune cells by associating with and presenting a cytokine in a bio-active state.
B- and T-lymphocyte populations have an independent homeostatic regulation of resting (B and T) and activated (B) or memory (T) cell compartments. This organization may provide an efficient mechanism to ensure simultaneously a first natural barrier of protection against common pathogens, the maintenance of immunological T-cell memory and a reservoir of repertoire diversity capable of dealing with new antigenic challenges.
Interleukin-7 (IL-7) supports the proliferation of mature T lymphocytes, however, the cellular source of IL-7 for T lymphocyte activation has not been well established. We therefore investigated whether human peripheral blood dendritic cells (DC) produce IL-7 as a contribution towards T lymphocyte activation. Human CMRF-44+/CD14-/CD19- low density DC, purified after overnight tissue culture, contained IL-7 transcripts, detected by direct cell reverse transcription-polymerase chain reaction. Intracytoplasmic staining confirmed IL-7 protein in at least a subpopulation of cultured low density DC. In contrast, resting/immature DC, isolated directly by immunodepletion of lineage marker positive cells, contained no IL-7 mRNA. Thus, the expression of IL-7 by DC follows the pattern described previously for CD80, CD86 and CD40. However, tissue culture of purified resting/immature DC, in contrast to CD80, CD86 and CD40, failed to induce IL-7 transcripts. The functional importance of DC IL-7 expression was demonstrated in an allogeneic mixed leukocyte reaction (MLR). Neutralising mAb to IL-7 significantly inhibited T lymphocyte proliferation when low DC numbers were used, but at higher stimulator numbers, anti-IL-7 mAb failed to inhibit an allogeneic MLR. This suggests, that when DC are in excess, other co-stimulatory pathways can compensate for the lack of IL-7. Addition of IL-7 to a MLR caused a significant increase in the proliferative response stimulated by monocytes and B lymphocytes but not by DC. These data support the concept of an initial phase of antigen uptake by DC followed by the optimisation of DC co-stimulatory potential. The co-stimulatory repertoire expressed, including IL-7, may be regulated by exogenous stimuli, thereby ensuring DC flexibility in mounting a response appropriate to the environmental changes.
Proliferation of memory-phenotype (CD44hi) CD8+ cells induced by infectious agents can be mimicked by injection of type I interferon (IFN I) and by IFN I-inducing agents such as lipopolysaccharide and Poly I:C; such proliferation does not affect naive T cells and appears to be TCR independent. Since IFN I inhibits proliferation in vitro, IFN I-induced proliferation of CD8+ cells in vivo presumably occurs indirectly through production of secondary cytokines, e.g., interleukin-2 (IL-2) or IL-15. We show here that, unlike IL-2, IL-15 closely mimics the effects of IFN I in causing strong and selective stimulation of memory-phenotype CD44hi CD8+ (but not CD4+) cells in vivo; similar specificity applies to purified T cells in vitro and correlates with much higher expression of IL-2Rbeta on CD8+ cells than on CD4+ cells.
This study examines the influence of IL-7 on post-thymic CD4+ T cells using cord blood as a model system. Survival of naive cord blood T cells in the presence of IL-7 alone was significantly prolonged by up-regulating bcl-2, thereby preventing apoptosis while maintaining maximal cell viability. Cultures without IL-7 showed high rates of apoptosis resulting in 50% cell death by day 5 of culture. Upon phorbol 12-myristate 13-acetate + ionomycin stimulation, accumulation of cytoplasmic IL-2 was similar to that observed in freshly isolated cells, but no IL-4- or IFN-gamma-positive cells were detected. IL-7 maintained the naive T cells in a quiescent state expressing the CD45RA antigen. A significant finding was the loss of CD38 antigen expression on the naive cord blood T cells to levels similar to that observed on adult naive T cells. In contrast to the reduced proliferative response of fresh cord blood T cells to anti-CD2 + CD28 stimulation, the proliferative response of IL-7-treated cells was similar to that of adult naive T cells. This study shows that as well as maintaining the naive T cell pool by enhancing cell survival and up-regulating bcl-2 expression, IL-7 also functions as a maturation factor for post-thymic naive T cells.
The IL-15 receptor alpha subunit (IL-15Ralpha) mediates high-affinity binding of IL-15, a pleiotropic cytokine implicated in the development of innate immune cells. We have generated IL-15Ralpha null (IL-15Ralpha-/-) mice to understand the role of IL-15Ralpha in immune development and function. IL-15Ralpha-/- mice are markedly lymphopenic despite grossly normal T and B lymphocyte development. This lymphopenia is due to decreased proliferation and decreased homing of IL-15Ralpha-/- lymphocytes to peripheral lymph nodes. These mice are also deficient in natural killer cells, natural killer T cells, CD8+ T lymphocytes, and TCRgammadelta intraepithelial lymphocytes. In addition, memory phenotype CD8+ T cells are selectively reduced in number. Thus, IL-15Ralpha has pleiotropic roles in immune development and function, including the positive maintenance of lymphocyte homeostasis.
A novel cytokine from a thymic stromal cell line (thymic stromal lymphopoietin (TSLP)) promotes the development of B220+/IgM+ immature B cells when added to fetal liver cultures, long term bone marrow cultures, or bone marrow cells plated in semisolid medium. Because the activities of TSLP overlap with those of IL-7 in some in vitro assays, we compared the signaling mechanisms employed by TSLP and IL-7. Proliferation of a factor-dependent pre-B cell line (NAG8/7) in response to either TSLP or IL-7 was inhibited by anti-IL-7R alpha mAbs, suggesting that the functional TSLP receptor complex uses IL-7R alpha. In contrast, three different Abs to the common cytokine receptor gamma-chain had no effect on the response of these cells to TSLP, indicating that the functional TSLP receptor complex does not use the common cytokine receptor gamma-chain. Both cytokines induced activation of Stat5, but only IL-7 induced activation of the Janus family kinases Jak1 and Jak3. In fact, TSLP failed to activate any of the four known Janus family kinases, suggesting that Stat5 phosphorylation is mediated by a novel mechanism. Taken together, these data support the idea that TSLP can make unique contributions to B lymphopoiesis and indicate that it does so by mechanisms distinct from IL-7.
The diversity of naive CD4 T cells plays an important role in the adaptive immune response by ensuring the capability of responding to novel pathogens. In the past, it has been generally accepted that naive CD4 T cells are intrinsically long-lived; however, there have been studies suggesting some CD4 T cells are short-lived. In this report, we identify two populations of naive CD4 T cells: a long-lived population as well as a short-lived population. In addition, we identify two factors that contribute to the establishment of long-lived naive CD4 T cells. We confirm earlier findings that MHC class II interaction with the TCR on CD4 T cells is important for survival. Furthermore, we find that MHC class II alleles with the correct restriction element for Ag presentation mediate the peripheral survival of naive CD4 T cells more efficiently than other positively selecting alleles, regardless of the selecting MHC in the thymus. The second component contributing to the survival of naive CD4 T cells is contact with the cytokines IL-4 and IL-7. We find that the physiological levels of IL-4 and IL-7 serve to enhance the MHC class II-mediated survival of naive CD4 T cells in vivo.
In the absence of thymic emigration, the peripheral T cell pool is maintained by division of mature lymphocytes. We have examined the molecular interactions required for peripheral CD8+ T cell expansion in lymphopenic mice without conventional antigenic stimulation. Expansion of CD8+ T cells in lymphopenic hosts was found to be peptide specific. An antagonist peptide known to serve as a ligand for positive selection of these T cells promoted expansion; however, a control peptide that binds the same class I molecule did not. Surprisingly, the cells undergoing proliferation in lymphopenic hosts did not mature to cytotoxic effectors and displayed a partially activated surface phenotype. These data suggest that division of T cells in the periphery of lymphopenic hosts requires specific recognition of self-peptide/MHC complexes, similar to the signal for thymocyte maturation.