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The Lymphopenic Mouse in Immunology: From Patron to Pariah
Abstract
A recent surge of interest in the behavior of T and B cells in lymphopenic model systems has resurrected a certain cynicism about the validity of using such models to answer important immunological questions. Here we discuss this skepticism in a broader historical context.
... 91 Therefore, autoimmunity resulting from Treg depletion in normal mice might not be due to abrogation of continuous suppression of some self-reactive T cells but rather from systemic homeostatic T cell expansion and activation associated with severe lymphopenia. 92,93 This suggests that conclusions regarding the role of Tregs using Foxp3 DTR mice should be taken with caution. However, it is important to note that in Foxp3 DTR mice tolerant of an allogeneic kidney, no tissue damage was detected in the native kidney and no signs of systemic autoimmunity were observed upon DT injections. ...
... Indeed, infusion of T cells into leukopenic animals is associated with systemic autoimmunity caused by homeostatic polyclonal expansion of pro-inflammatory T cells displaying an activated/memory effector phenotype. 92,93 Also, anti-CD25 Ab-depletion seemed to abolish self-tolerance in very young (<3 weeks) mice but not in adults. 100 This is a relevant issue since there is now plenty of evidence showing that ablation of Tregs in adult mice has no or little effect upon autoimmunity. ...
... Both a lymphopenic environment and a Treg cell deficiency are known to elicit autoimmune disorders in T cell-deficient mice infused with naive T cells. 92,93,100 Consequently, conclusions made from experiments involving a recipient thymectomy can be misleading. ...
Achieving host immune tolerance of allogeneic transplants represents the ultimate challenge in clinical transplantation. It has become clear that different cells and mechanisms participate in acquisition versus maintenance of allograft tolerance. Indeed, manipulations which prevent tolerance induction often fail to abrogate tolerance once it has been established. Hence, elucidation of the immunological mechanisms underlying maintenance of T cell tolerance to alloantigens is essential for the development of novel interventions that preserve a robust and long lasting state of allograft tolerance that relies on T cell deletion in addition to intra‐graft suppression of inflammatory immune responses. In this review, we discuss some essential elements of the mechanisms involved in the maintenance of naturally occurring or experimentally induced allograft tolerance, including the newly described role of antigen cross‐dressing mediated by extracellular vesicles.
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... The term "homeostatic proliferation" was originally coined to describe the slow T cell expansion observed when naive TCRtransgenic T cells were adoptively transferred into lymphopenic recipients, such as congenitally T cell-deficient RAG Ϫ/Ϫ mice or normal mice rendered lymphopenic by irradiation (2)(3)(4). Because donor TCR-transgenic T cells were RAG deficient, it ensured that these T cells were monoclonal, expressing a single ␣ TCR, and verified that this slow proliferation occurred in the absence of agonist peptides. ...
... Therefore, these functional impairments displayed by chronically activated T cells may contribute to the failure to clear virus. Programmed death-1 (PD-1), 4 a negative regulator of activated T cells (10), is strongly up-regulated on exhausted viralspecific CD8 T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection in mice and HIV infection in humans (11,12). Because blockade of PD-1 interaction with its ligand PD-L1 can restore function in exhausted CD8 T cells (11), it makes the case that PD-1 is not simply indicative of an exhausted state but also plays a key role in its maintenance. ...
... Homeostatic mechanisms that function to regulate peripheral T cell numbers may be basis for the association between autoimmunity and T cell lymphopenia observed in both animals and humans (4,25). Our studies of the homeostatic mechanisms operating in RasGRP1 Ϫ/Ϫ mice suggest that both self-and foreign-Ags could be driving T cell proliferation (Fig. 3). ...
The Ras-guanyl nucleotide exchange factor RasGRP1 is an important link between TCR-mediated signaling and the activation of Ras and its downstream effectors. RasGRP1 is especially critical for the survival and differentiation of developing thymocytes whereas negative selection of thymocytes bearing an autoreactive TCR appears to be RasGRP1 independent. Despite apparently normal central tolerance, RasGRP1−/− mice spontaneously acquire an acutely activated and proliferating CD4 T cell population that exhibits characteristics of T cell exhaustion, including strong expression of programmed cell death-1. To elucidate the basis for RasGRP1−/− CD4 T cell immune activation, we initiated a series of adoptive transfer experiments. Remarkably, the copious amounts of cytokines and self-Ags present in hosts made lymphopenic through irradiation failed to induce the majority of RasGRP1−/− CD4 T cells to enter cell cycle. However, their infusion into either congenitally T cell- or T/B cell-deficient recipients resulted in robust proliferation and L-selectin down-regulation. These findings imply that the activation and proliferation of RasGRP1−/− CD4 T cells may be dependent on their residence in a chronically immunocompromised environment. Accordingly, bacterial and viral challenge experiments revealed that RasGRP1−/− mice possess a weakened immune system, exhibiting a T cell-autonomous defect in generating pathogen-specific T cells and delayed pathogen clearance. Collectively, our study suggests that chronic T cell immunodeficiency in RasGRP1−/− mice may be responsible for CD4 T cell activation, proliferation, and exhaustion.
... Later on, in 1953, T cells became increasingly recognized as the major driver of anti-tumor immunity. Indeed, the first adoptive cell therapies in mice models, through transplantation of lymph nodes from mice inoculated with lymphosarcomas to healthy mice, also demonstrated successful transfer of antitumor activity [4,5]. Extensive characterization of the transferred lymphocyte populations lead to the identification of cytotoxic CD8 + T lymphocytes. ...
T lymphocytes are the major drivers of antitumor immunity. The recent clinical success of adoptive T cell therapies and immune checkpoint inhibitors has demonstrated the strength of modulating T cell function in fighting cancer. Nonetheless, a significant fraction of patients remain unresponsive largely due to the immunosuppressive tumor environment that blunts T cell activity. Small interfering RNAs (siRNAs) offer the potential to sequence-specifically silence the expression of negative regulator genes in T cells in a transient manner, thereby releasing the block on anti-tumor responses. Despite the current focus on small molecule- and antibody-based immune checkpoint inhibitors as well as T cell-directed delivery of mRNA and genome editing machinery, the application of siRNA involves important clinical advantages. The recent surge of adoptive cell therapies and development of new and potent delivery approaches has enabled efficient siRNA delivery to T cells both ex vivo and in vivo. As such, siRNA molecules have a newfound potential to improve the proliferation, survival, tumor infiltration and potency of T cells in cancer immunotherapy. In this review, we briefly discuss the extracellular and intracellular delivery hurdles associated with siRNA therapy, in particular with regard to T cell targeting. We provide a timely and comprehensive overview of current and emerging delivery technologies used for siRNA transfection, discussing their strengths and weaknesses from a clinical as well as a manufacturing point-of-view. Finally, we critically review the current status and new potential avenues for modulating T cell function in cancer immunotherapy using siRNA.
... Lymphopenia is known to exaggerate immune responses, especially after transfer of specific T cells (40,41). Lymphopenic proliferation and the resulting excessive immune responses are inhibited by Tregs (42). ...
The immune system in tolerance maintains cell-diversity without responding to self-antigens. Foxp3-expressing CD25+CD4+ regulatory T cells (Tregs) inhibit T cell activation through various molecular mechanisms. However, several key questions are still not resolved, including how Tregs control the immune response based on their self-skewed T cell receptor repertoire and how Tregs avoid impeding relevant immunity against pathogens. Here, we show that Tregs promote the proliferation of conventional T cells in the presence of excessive co-stimulation when murine T cells are stimulated in vitro with allogeneic antigen-presenting cells (APCs). Antigen-specific Tregs increase the number of cells interacting with dendritic cells (DCs) by increasing the number of viable DCs and the expression of adhesion molecules on DCs. Theoretical simulations and mathematical models representing the dynamics of T-APC interaction and T cell numbers in a lymph node indicate that Tregs reduce the dissociation probability of T cells from APCs and increase the new association. These functions contribute to tolerance by enhancing the interaction of low-affinity T cells with APCs. Supporting the theoretical analyses, we found that reducing the T cell numbers in mice increases the ratio of specific T cells among CD4+ T cells after immunization and effectively induces autoimmune diabetes in NOD mice. Thus, as a critical function, antigen-specific Tregs stabilize the immune state, irrespective of it being tolerant or responsive, by augmenting T-APC interaction. We propose a novel regulation model in which stable tolerance with large heterogeneous populations proceeds to a specific immune response through a transient state with few populations.
... Furthermore, the survival of the BCR-stimulated T2-B cells in the imb-1 mice was strictly dependent on the BAFF signaling system whereas non-stimulated naïve T1-B and T2-B cells could survive in these mice for some time without BAFF. We are aware of the fact that imb-1 mice are lymphopenic where the few generated B cells do not compete for limiting growth factors as is probably the case for newly generated B cells in wild-type mice (39,65,90,91). A survival of auto-reactive T cell clones in the thymus has also been observed in a lymphopenic mouse model (52). ...
Developing B cells undergo defined maturation steps in the bone marrow and in the spleen. The timing and the factors that control these differentiation steps are not fully understood. By targeting the B cell-restricted mb-1 locus to generate an mb-1 allele that expresses a tamoxifen inducible Cre and another allele in which mb-1 expression can be controlled by Cre, we have established a mouse model with an inducible B cell compartment. With these mice, we studied in detail the kinetics of B cell development and the consequence of BCR activation at a defined B cell maturation stage. Contrary to expectations, transitional 1-B cells exposed to anti-IgM reagents in vivo did not die but instead developed into transitional 2 (T2)-B cells with upregulated Bcl-2 expression. We show, however, that these T2-B cells had an increased dependency on the B cell survival factor B cell activating factor when compared to non-stimulated B cells. Overall, our findings indicate that the inducible mb-1 mouse strain represents a useful model, which allows studying the signals that control the selection of B cells in greater detail.
... TBI is often used interchangeably with antibody depletion experiments or gene-knockout mice to study immune cell reconstitution, but different methods of lymphodepletion cannot be assumed to be immunologically equivalent (55). The induction of homeostatic expansion of the T cell compartment after TBI preconditioning cannot be viewed as merely expanding to fill empty space (56). Rather, TBI induces a complex set of events, which includes the liberation of endogenous LPS capable of triggering TLR4 signals that enhance the efficacy of adoptively transferred T cells. ...
Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8 + T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8 + T cells in mice genetically deficient in all lym-phocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed com-mensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflam-matory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using poly-myxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand–containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8 + T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.
... However, a seemingly simple solution to this problem is to thwart the host antidonor immune response by using host animals that lack T cells due either to genetic ablation of key factors in T cell generation or using acute depletion measures such as irradiation, anti-T cell antibody treatment, or use of chemotherapeutic drugs. As has been discussed ( Singh & Schwartz, 2006), this use of lymphopenic animal models has a long and distinguished history in immunology and has other appealing features in addition to preventing donor cell rejection-such as substantially better adoptive transfer efficiency in lymphopenic hosts, in which there is more "space" for those donor cells. At face value, this approach performed nicely and yielded data supporting the idea that encounter with specific self-peptide/MHC molecules was critical for naïve T cell survival: various approaches showed that naïve CD4+ T cells transferred into Class II MHC-deficient (MHC-II −/− ) lymphopenic hosts were present at notably lower numbers than cells transferred into wild-type lymphopenic hosts ( Brocker, 1997;Kirberg, Berns, & von Boehmer, 1997;Takeda, Rodewald, Arakawa, Bluethmann, & Shimizu, 1996), and similar findings were reported for CD8+ T cells transferred into normal versus MHC-I −/− Tdepleted hosts ( Markiewicz et al., 1998;Nesic & Vukmanovic, 1998). ...
Memory T cells are usually considered to be a feature of a successful immune response against a foreign antigen, and such cells can mediate potent immunity. However, in mice, alternative pathways have been described, through which naïve T cells can acquire the characteristics and functions of memory T cells without encountering specific foreign antigen or the typical signals required for conventional T cell differentiation. Such cells reflect a response to the internal rather the external environment, and hence such cells are called innate memory T cells. In this review, we describe how innate memory subsets were identified, the signals that induce their generation and their functional properties and potential role in the normal immune response. The existence of innate memory T cells in mice raises questions about whether parallel populations exist in humans, and we discuss the evidence for such populations during human T cell development and differentiation.
© 2015 Elsevier Inc. All rights reserved.
... Though the validity of genetically lymphopenic mice as physiological models has been called into question, an enormous amount of research on complex aspects of T cell homeostasis has been enabled (95). Autoimmunity involves defects in both branches of the immune response: activation and regulation. ...
... To eliminate any possible contribution from the BM in the homeostatic control and life span of 19 + 45R lo cell subset, we performed adoptive transfer experiments into Rag2g 2/2 hosts, taking advantage of the fact that a lymphopenic environment maximizes the homeostatic cell proliferation in vivo under physiological conditions (33). Splenic 19 + 45R lo , 19 + 45R + , and peritoneal B1 cells were purified by double sorting from C57BL/6.Ly5.1 mice prior to their adoptive transfer to Rag2g 2/2 recipients (Fig. 4A). ...
The diversity in antibody repertoire relies on different B cell populations working efficiently to fulfil distinct specific functions. We recently described an innate-like CD19(+)CD45R(-/lo) (19(+)45R(lo)) cell population in postnatal unstimulated adult mice, a heterogeneous population containing cells expressing immunoglobulin M (IgM) and others behaving as differentiated mature B lymphocytes (intracytoplasmic IgG1, AID(+), Blimp-1(+)RAG2(-)). In the present study, we characterized the Ig repertoire expressed by splenic 19(+)45R(lo) cells, assuming that they would bear a restricted repertoire biased for germline rearrangements and low mutation rates similar to other innate-like cells. Sequences from 19(+)45R(lo) cells displayed a variety of V, D and J regions, and the analysis of the CDR-H3 region revealed an intermediate overall CDR-H3 length and moderate hydrophobicity. Both IgM and switched sequences of PD15 19(+)45R(lo) cells had shorter CDR-H3 region and fewer non-template N nucleotides than adult sequences, as expected for profiles that correspond to an immature phenotype. Regarding the mutation rate in the VH regions, IgG1 sequences already carried a high rate of replacement mutations at PD15, which increased further in the sequences obtained from adult mice. Moreover, statistical models suggest that a proportion of the switched sequences in adult 19(+)45R(lo) cells had experienced antigen selection, unlike other innate-like B cell compartments. © 2014 S. Karger AG, Basel.
... A major role for T cell lymphopenia has been implicated in breaking peripheral T cell tolerance in both young and adult mice [13,14]. Transient acute lymphopenia can be induced experimentally by the administration of drugs such as cyclophosphamide and cyclosporine [15,16] or repetitive low dose irradiation [17,18]. ...
Regulatory T (Treg) cells enforce T cell homeostasis and maintain peripheral T cell tolerance. Here we report a previously unappreciated phenomenon of acute T cell lymphopenia in secondary lymphoid organs and non-lymphoid tissues triggered by Treg cell depletion that precedes the expansion of self-reactive T cells. Lymphopenia affects both neonates and adults indicating a dominant role of Treg cells in maintaining peripheral T cell numbers regardless of the developmental stage. The lymphopenia was neither triggered by caspase-dependent apoptosis nor macrophage-mediated clearance of T cells, nor diminished survival of naïve or recently activated T cells due to paucity of IL-7. It is possible that transient lymphopenia associated with congenital or acute Treg cell deficiency may contribute to the development of T cell mediated autoimmune disorders.
... To eliminate any possible contribution from the BM in the homeostatic control and life span of 19 + 45R lo cell subset, we performed adoptive transfer experiments into Rag2g 2/2 hosts, taking advantage of the fact that a lymphopenic environment maximizes the homeostatic cell proliferation in vivo under physiological conditions (33). Splenic 19 + 45R lo , 19 + 45R + , and peritoneal B1 cells were purified by double sorting from C57BL/6.Ly5.1 mice prior to their adoptive transfer to Rag2g 2/2 recipients (Fig. 4A). ...
In the adult spleen, CD19⁺CD45R(-/lo) (19⁺45R(lo)) lymphocytes of embryonic origin exist as a distinct population to that of the conventional B cell lineage. These cells display a plasmablast phenotype, and they spontaneously secrete IgG1 and IgA, whereas the bone marrow population of 19⁺45R(lo) cells contains B1 progenitors. In this study, we show that 19⁺45R(lo) cells are also present in Peyer's patches and in the spleen throughout the life span of wild-type mice, beginning at postnatal day 7. Although this population is heterogeneous, the surface phenotype of most of these cells distinguishes them from follicular, transitional, marginal zone, and B1 cells. In CBA/CaHN mice, few 19⁺45R(lo) cells were detected at postnatal day 7, and none was observed in the adult spleen. Splenic 19⁺45R(lo) cells exhibited homeostatic BrdU uptake in vivo and actively transcribed cell cycle genes. When transferred to immunodeficient RAG2⁻/⁻γchain⁻/⁻ recipient mice, 19⁺45R(lo) cells survived and differentiated into IgG1- and IgA-plasma cells. Moreover, in vitro stimulation of splenic 19⁺45R(lo) cells with LPS, CpG, BAFF/IL4, and CD40/IL4 induced cell proliferation, IgG1/IgA secretion and the release of IL-10, suggesting a potential immunoregulatory role for this subset of innate-like B cells.
... Thus, a primary role for PD-1 is in the control of homeostatic activation, a process most strong in RTE cells as revealed during states of lymphopenia [56]. This perspective suggests a model whereby the relatively mild autoimmunity seen in PD-1 À/À mice may be explained by a partial loss in the ability to control homeostatic activation; the restricted lymphoid environment present in the neonatal period, when T cell homeostasis is established, prevents a complete loss of control. ...
Lymphopenia driven T cell activation is associated with autoimmunity. That lymphopenia does not always lead to autoimmunity suggests that control mechanisms may exist. We assessed the importance of the co-inhibitory receptor programmed death-1 (PD-1) in the control of lymphopenia-driven autoimmunity in newly generated T cells vs. established peripheral T cells and in thymic selection. PD-1 was not required for negative selection in the thymus or for maintenance of self tolerance following transfer of established PD-1⁻/⁻ peripheral T cells to a lymphopenic host. In contrast, PD-1 was essential for systemic self tolerance in newly generated T cells under lymphopenic conditions, as PD-1⁻/⁻ recent thymic emigrants (RTE), generated after transfer of PD-1⁻/⁻ hematopoietic stem cell (HSC) precursors or thymocytes into lymphopenic adult Rag⁻/⁻ recipients, induced a rapidly lethal multi-organ inflammatory disease. Disease could be blocked by using lymph node deficient recipients, indicating that lymphopenia driven PD-1⁻/⁻ T cell activation required access to sufficient lymph node stroma. These data suggested that PD-1⁻/⁻ mice themselves might be substantially protected from autoimmunity because their T cell repertoire is first generated early in life, a period naturally deficient in lymph node stroma. Consistent with this idea, neonatal Rag⁻/⁻ recipients of PD-1⁻/⁻ HSC were resistant to disease. Thus, a critical role of PD-1 resides in the control of RTE in lymphopenia. The data suggest that PD-1 and a paucity of lymphoid stroma cooperate to control autoimmunity in newly generated T cells. Clinical therapies for autoimmune disease employing lymphoablation and hematopoietic stem cell transplantation will need to take into account functional polymorphisms in the PD-1 pathway, if the treatment is to ameliorate rather than exacerbate autoimmunity.
... Because of the paucity of lymphocytes in these animals, signaling by various cytokines, self-peptide/MHC complexes, or foreign antigens may be atypical due to reduced T cell competition (Bourgeois and Stockinger, 2006). Mice such as those lacking components of the IL-7 signaling pathway may also have abnormal lymphoid tissues, impacting lymphocyte trafficking (Kondrack et al., 2003;Singh and Schwartz, 2006). Finally, IL-7 treatment increases thymic output, making it difficult to distinguish recent thymic emigrants (RTE) from preexisting (proliferating) naïve T-cells (Swainson et al., 2007;Sportes et al., 2008). ...
Interleukin-7 (IL-7) increases lymphocyte numbers, a critical feature of immune reconstitution, through mechanisms that are still poorly understood. Part of the problem is that IL-7 is produced in limited amounts by non-lymphoid cells, making in vivo studies of the cytokine's activity a challenge. To overcome this, we developed an in vitro system by which lymphocytes from secondary immune organs could be cultured to produce IL-7 responsive cells. Using this method, we showed that CD8(hi)CD44(hi) T cells accumulate in culture with IL-7 from a population of lymph node or splenic cells. These results were validated when a similar lymphocyte subset was found in mice expressing a constitutively active form of STAT5b, a key transducer of IL-7 signals. Interestingly, IL-7-expanded cells also up regulated the activation marker, CD69. The IL-7-derived CD44(hi)CD69(hi) cells were not generated from naïve cells, but expanded from an existing population, since culture in IL-7 of naïve lymphocytes from OT-1/Rag1(-/-) mice did not produce CD44(hi)CD69(hi) cells. Using the in vitro culture system to study lymphocytes from mice deficient in the apoptotic protein, BIM, we were able to attribute the expansion of CD8(hi)CD44(hi)CD69(hi) T cells to the proliferative and not survival activity of IL-7. The in vitro culture system provides an important new methodology to examine the activities of this essential as well as immunotherapeutic cytokine.
... TCR  levels were reduced in the small number of BCL11B-defi cient peripheral T lymphocytes ( Fig. 3 C ), suggesting that they are immature. In addition, there was a major reduction in the percentage of naive CD62L high CD44 low CD4 + and CD8 + T lymphocyte populations, whereas the CD44 high populations, comprising activated and memory-like T cells, were increased ( Fig. 3 D ), results which suggest a homeostatic deregulation in CD4 + and CD8 + T lymphocyte populations, which is often observed in lymphopenic mice ( 26 ). The lymph nodes and spleens of BCL11B F/F CD4cre mice presented a marked increase in percentage and absolute numbers of B220 + lymphocytes ( Table I and Table II ). ...
Transcriptional control of gene expression in double-positive (DP) thymocytes remains poorly understood. We show that the transcription factor BCL11B plays a critical role in DP thymocytes by controlling positive selection of both CD4 and CD8 lineages. BCL11B-deficient DP thymocytes rearrange T cell receptor (TCR) alpha; however, they display impaired proximal TCR signaling and attenuated extracellular signal-regulated kinase phosphorylation and calcium flux, which are all required for initiation of positive selection. Further, provision of transgenic TCRs did not improve positive selection of BCL11B-deficient DP thymocytes. BCL11B-deficient DP thymocytes have altered expression of genes with a role in positive selection, TCR signaling, and other signaling pathways intersecting the TCR, which may account for the defect. BCL11B-deficient DP thymocytes also presented increased susceptibility to spontaneous apoptosis associated with high levels of cleaved caspase-3 and an altered balance of proapoptotic/prosurvival factors. This latter susceptibility was manifested even in the absence of TCR signaling and was only partially rescued by provision of the BCL2 transgene, indicating that control of DP thymocyte survival by BCL11B is nonredundant and, at least in part, independent of BCL2 prosurvival factors.
... In our transfer experiments using nonlymphopenic hosts, we observed minimal proliferation for up to 7 d after transfer (unpublished data). This fi nding was in accordance with published data, indicating that lymphopenia is the major stimulus for B cell homeostatic proliferation (HP) ( 29 ). Thus, we used recipient B celldefi cient μ MT mice to compare the capacity of diff erent B cell subsets to respond to signals inducing HP. ...
We have characterized a distinct, late transitional B cell subset, CD21(int) transitional 2 (T2) B cells. In contrast to early transitional B cells, CD21(int) T2 B cells exhibit augmented responses to a range of potential microenvironmental stimuli. Adoptive transfer studies demonstrate that this subset is an immediate precursor of both follicular mature and marginal zone (MZ) B cells. In vivo, a large percentage of CD21(int) T2 B cells has entered the cell cycle, and the cycling subpopulation exhibits further augmentation in mitogenic responses and B cell-activating factor of the TNF family (BAFF) receptor expression. Consistent with these features, CD21(int) T2 cells exhibit preferential responses to BAFF-facilitated homeostatic signals in vivo. In addition, we demonstrate that M167 B cell receptor (BCR) idiotypic-specific B cells are first selected within the cycling CD21(int) T2 population, ultimately leading to preferential enrichment of these cells within the MZ B cell compartment. These data, in association with the coordinate role for BAFF and microenvironmental cues in determining the mature BCR repertoire, imply that this subset functions as a unique selection point in peripheral B cell development.
... The behavior of T lymphocytes under lymphopenic conditions has been studied mainly by adoptive cell transfers into irradiated or genetically lymphopenic mice (7,36). The spontaneous lymphopenic mouse model described here avoids ex vivo handling of T cells, which has been shown to change the expression level of 200 genes (13). ...
Naive T lymphocytes acquire a phenotype similar to Ag-experienced memory T cells as a result of proliferation under lymphopenic conditions. Such "memory-like" T (T(ML)) cells constitute a large fraction of the peripheral T cell pool in patients recovering from T cell ablative therapies, HIV patients under highly active antiretroviral therapy, and in the elderly population. To generate a model that allows characterization of T(ML) cells without adoptive transfer, irradiation, or thymectomy, we developed genetically modified mice that express diphtheria toxin A under control of a loxP-flanked stop cassette (R-DTA mice). Crossing these mice to CD4Cre mice resulted in efficient ablation of CD4 single-positive thymocytes, whereas double-positive and CD8 single-positive thymocytes were only partially affected. In the periphery the pool of naive (CD44(low)CD62L(high)) T cells was depleted. However, some T cells were resistant to Cre activity, escaped deletion in the thymus, and underwent lymphopenia-induced proliferation resulting in a pool of T(ML) cells that was similar in size and turnover to the pool of CD44(high)CD62L(low) "memory phenotype" T cells in control mice. CD4Cre/R-DTA mice remained lymphopenic despite the large available immunological "space" and normal Ag-induced T cell proliferation. CD4Cre/R-DTA mice showed a biased TCR repertoire indicating oligoclonal T cell expansion. Infection with the helminth Nippostrongylus brasiliensis resulted in diminished effector cell recruitment and impaired worm expulsion, demonstrating that T(ML) cells are not sufficient to mediate an effective immune response.
This overview of the immunology of hematopoietic cell transplantation (HCT) is focused on the individual outcomes of engraftment, graft-versus-host disease, control of malignancy, tolerance, and immune reconstitution, but these remain highly interconnected as different aspects of a single overall process. For this reason, the evaluation of interventions designed to influence one particular immunologic outcome of HCT requires equivalent scrutiny of other immunologic outcomes. Historically, clinical HCT has evolved from the biomedical knowledge and understanding gained from animal studies. Murine models have the advantages of well-defined immunogenetics and a wealth of reagents and methods for dissecting the cellular and humoral mechanisms involved in HCT immunobiology, while canine and non-human-primate models more closely mimic the immunologic and medical challenges of HCT in a large outbred species. The wide range of experimental manipulations afforded by animal models has allowed rapid progress in elucidating principles that improve the biomedical understanding of HCT immunology in all of its interconnected complexity.
The hyperacute rejection mediated by preexisting antibodies is a major impediment to the success of transplants across allogeneic and xenogeneic barriers. We report a new mouse model that allows us to not only monitor the sensitization of B cells mediating the hyperacute response but also validate therapeutic strategies for tolerizing them.
The new model system uses 5C.C7,RAG2 T-cell receptor transgenic T cells and B10.S(9R),CD3[Latin Small Letter Open E] hosts for adoptive transfer experiments.
In the allogeneic hosts, transgenic T cells expanded briefly before being chronically deleted. Once the deletion was initiated, a second graft of donor cells was used to assess a hyperacute response. The rapid rejection of the second cohort correlated with the appearance of donor-specific antibodies in the serum. Interestingly, chronically stimulated T cells were relatively resistant to hyperacute rejection, suggesting an explanation for the slower rejection kinetics of the first cohort even as the second cohort of identical donor cells was being hyperacutely rejected. Finally, we could tolerize the potential for a hyperacute response, by pretreating recipients with a single infusion of naive donor B cells before the first T-cell transfer. This treatment not only abrogated the development of a hyperacute response but also allowed the primary graft to survive in vivo for extended periods.
Inflammatory bowel diseases (IBD) are thought to be caused by a complex interaction of genetic, immunological, and environmental factors. Why is it that once an IBD develops it lasts a long time? Considering this simple question, we propose that coliotogenic memory CD4+T cells that remember the prototype of the disease in each patient are formed in IBD at the onset, and, perceiving them as "benign T-cell leukemia"-like lifelong memory CD4+T cells that hematogenously spread throughout the body, we thus propose that systemic circulating colitogenic memory CD4+T cells would be an ideal target for the treatment of IBD. Accordingly, selective depletion of colitogenic memory CD4+T cells by leukocytapheresis and blockade of circulation of colitogenic memory CD4+T cells by a newly developed immunosuppressant, FTY720, may be associated with dramatic efficacy and a marked reduction of inflammatory cytokines produced by activated leucocytes. We here describe the immunological pathogenesis focusing on the generation of circulating colitogenic memory CD4+T cells and the possible logics of leukocytapheresis and FTY720 for the treatment of IBD.
Ag persistence during high-titer chronic viral infections induces CD8 T cell dysfunction and lack of Ag-independent CD8 T cell memory formation. However, we have a poor understanding of the generation and maintenance of CD8 T cell memory during asymptomatic persistent viral infections, particularly gamma-herpesvirus infections. In this study, we demonstrate that the continuous presence of cognate Ag in the host is not required for the maintenance of CD8 T cell memory during a persistent gamma-herpesvirus infection. Importantly, the Ag-independent CD8 T cell memory that is maintained during gamma-herpesvirus persistence has the capacity to survive long-term under homeostatic conditions and to mount a protective recall response to a secondary encounter with the pathogen. These data highlight the ability of the immune system to maintain a population of protective memory CD8 T cells with capacity for long-term Ag-independent survival in the presence of systemic virus persistence.
Ex vivo-expanded tumor-infiltrating lymphocytes infused into lymphodepleted recipients has clear antitumor efficacy. More practical sources of such antitumor lymphocytes would broaden the application of this approach. Previously, we described an in situ vaccination combining chemotherapy with intratumoral injection of CpG-enriched oligonucleotides, which induced T-cell immunity against established lymphoma. An ongoing clinical trial of this maneuver has demonstrated clinical responses in lymphoma patients. Here, we use this vaccine maneuver to generate immune cells for transfer into irradiated, syngeneic recipients. Transferred tumor-specific T-effector (T(eff)) cells preferentially expanded, increasing the T(eff)/T-regulatory (T(reg)) ratio in these "immunotransplantation" recipients and curing large and metastatic tumors. Donor T cells were necessary for tumor protection, and CD8 T-cell immune responses were enhanced by posttransplantation booster vaccination. Hematopoietic stem cell transplantation is a standard therapy for lymphoma. Therefore, in situ tumor vaccination followed by immunotransplantation of harvested tumor-specific T cells could be directly tested in clinical trials to treat otherwise resistant malignancies.
Lymphodepletion with total body irradiation (TBI) increases the efficacy of adoptively transferred tumor-specific CD8(+) T cells by depleting inhibitory lymphocytes and increasing homeostatic cytokine levels. We found that TBI augmented the function of adoptively transferred CD8(+) T cells in mice genetically deficient in all lymphocytes, indicating the existence of another TBI mechanism of action. Additional investigation revealed commensal gut microflora in the mesenteric lymph nodes and elevated LPS levels in the sera of irradiated mice. These findings correlated with increased dendritic cell activation and heightened levels of systemic inflammatory cytokines. Reduction of host microflora using antibiotics, neutralization of serum LPS using polymyxin B, or removal of LPS signaling components using mice genetically deficient in CD14 and TLR4 reduced the beneficial effects of TBI on tumor regression. Conversely, administration of microbial ligand-containing serum or ultrapure LPS from irradiated animals to nonirradiated antibody-lymphodepleted mice enhanced CD8(+) T cell activation and improved tumor regression. Administration of ultrapure LPS to irradiated animals further enhanced the number and function of the adoptively transferred cells, leading to long-term cure of mice with large B16F10 tumors and enhanced autoimmune vitiligo. Thus, disruption of the homeostatic balance between the host and microbes can enhance cell-based tumor immunotherapy.
IgG antipolysaccharide (PS) and antiprotein responses to Streptococcus pneumoniae (Pn) are both CD4(+) T cell dependent. However, the primary IgG anti-PS response terminates more quickly, uses a shorter period of T cell help, fails to generate memory, and is more dependent on membrane Ig (mIg) signaling. We thus determined whether this limited anti-PS response to Pn reflected a greater propensity of PS-specific B cells to undergo apoptosis. We used mice that constitutively expressed the antiapoptotic protein Bcl-x(L) or Bcl-2 as a B cell-specific transgene. Both transgenic (Tg) mice exhibited increased absolute numbers of splenic B-1 and peritoneal B-1b and B-2 cells, subsets implicated in anti-PS responses, but not in marginal zone B (MZB) cells. Both Tg mouse strains elicited, in an apparently Fas-independent manner, a more prolonged and higher peak primary IgM and IgG anti-PS, but not antiprotein, response to Pn, but without PS-specific memory. A similar effect was not observed using purified PS or pneumococcal conjugate vaccine. In vitro, both splenic MZB and follicular Tg B cells synthesized DNA at markedly higher levels than their wild-type counterparts, following mIg cross-linking. This was associated with increased clonal expansion and decreased apoptosis. Using Lsc(-/-) mice, the Pn-induced IgG response specific for the capsular PS was found to be almost entirely dependent on MZB cells. Collectively, these data suggest that apoptosis may limit mIg-dependent clonal expansion of PS-specific B cells during a primary immune response to an intact bacterium, as well as decrease the pool of PS-responding B cell subsets.
Natural CD4(+)CD25(+) regulatory T cells (nTreg) have been shown to control graft-versus-host disease after hematopoietic stem cell transplantation (HSCT). Herein, we considered the possibility that the beneficial action of nTreg upon immune reconstitution in lymphopenic hosts involves dampening of the inflammatory response induced by bacterial products. We first observed that transfer of syngeneic CD4(+)CD25(-) T cells in RAG-deficient mice dramatically enhanced release of inflammatory cytokines and associated pathology upon endotoxin injection. Interferon (IFN)-gamma produced by T cells undergoing homeostatic proliferation was shown to be involved in the endotoxin hyperresponsiveness induced by CD4(+) T cell reconstitution. Co-transfer of CD4(+)CD25(+) nTreg with CD4(+)CD25(-) T cells inhibited the expansion of IFN-gamma-producing T cells and reduced endotoxin responses in RAG(-/-) mice. We conclude that (1) CD4(+) T cell reconstitution sensitizes lymphopenic hosts to endotoxin-induced pathology and (2) nTreg prevent this process by limiting the emergence of IFN-gamma-producing cells.
The transfer of transplantation immunity by lymph node cells has been the subject of investigation. Transplantable tumors have been used to provoke and to measure transplantation immunity. Cells from the lymph nodes draining a tumor homograft were transferred as mince or in suspension into the peritoneum of a secondary host to confer immunity. These cells could confer immunity while the immunizing graft was undergoing breakdown during the primary, and also during the more rapid secondary, response. Cells from other nodes and from the spleen, and also whole blood or serum failed in these experiments to transfer immunity. In one combination of tumor and host, serum from immunized donors enhanced tumor growth.
Evidence has been presented favoring the hypothesis that the lymph node cells were immunologically activated before transfer, and that they conferred immunity by continuing to function in their host. Immunization by tumor cells transferred along with the cells of the nodes could not account for the failure of lymph node transferred into susceptible animals to give rise to tumors; nor for the failure of tumor cells to give rise to immunity as rapidly as transferred lymph node cells. Freezing and thawing of the transferred cells prevented transfer of immunity. Cells from donors immunized against an isoantigen failed to confer immunity on hosts which carried that isoantigen, offering evidence of absorption of antibody. The duration of immunity transferred within an inbred strain was shorter than actively induced immunity, but longer than could have been expected of passively transferred immunity. After transfer of cells into foreign hosts, immunity declined more rapidly, as if the transferred cells were destroyed by the homograft reaction of the host.
The possibility that cells of the host were activated has also been discussed. A brief review showed that similar problems are raised in other systems of transfer of immunity by cells.
In this essay we suggest that the primary goal of the cells of the immune system is to ensure their own growth and survival. In adults, in steady-state conditions, the number and distribution of lymphocyte populations is under homeostatic control. New lymphocytes that are continuously produced in primary and secondary lymphoid organs must compete with resident cells for survival. We discuss recent findings supporting lymphocyte survival as a continuous active process and implicating cognate receptor engagement as fundamental survival signals for both T and B lymphocytes. The conflict of survival interests between different cell types gives rise to a pattern of interactions that mimics the behavior of complex ecological systems. In their flight for survival and in response to competition, lymphocytes use different survival signals within different ecological niches during cell differentiation. This is the case for T and B lymphocytes and also for naive and memory/activated T and B cells. We discuss how niche differentiation allows the co-existence of different cell types and guarantees both repertoire diversity and efficient immune responses.
The developmental requirements for immunological memory, a central feature of adaptive immune responses, is largely obscure. We show that as naive CD8 T cells undergo homeostasis-driven proliferation in lymphopenic mice in the absence of overt antigenic stimulation, they progressively acquire phenotypic and functional characteristics of antigen-induced memory CD8 T cells. Thus, the homeostasis-induced memory CD8 T cells express typical memory cell markers, lyse target cells directly in vitro and in vivo, respond to lower doses of antigen than naive cells, and secrete interferon gamma faster upon restimulation. Like antigen-induced memory T cell differentiation, the homeostasis-driven process requires T cell proliferation and, initially, the presence of appropriate restricting major histocompatibility complexes, but it differs by occurring without effector cell formation and without requiring interleukin 2 or costimulation via CD28. These findings define repetitive cell division plus T cell receptor ligation as the basic requirements for naive to memory T cell differentiation.
T cell receptor (TCR) signaling triggered by recognition of self-major histocompatibility complex (MHC) ligands has been proposed to maintain the viability of naïve T cells and to provoke their proliferation in T cell-deficient hosts. Consistent with this, the partially phosphorylated state of TCR zeta chains in naïve CD4+ and CD8+ T cells in vivo was found to be actively maintained by TCR interactions with specific peptide-containing MHC molecules. TCR ligand-dependent phosphorylation of TCR zeta was lost within one day of cell transfer into MHC-deficient hosts, yet the survival of transferred CD4+ lymphocytes was the same in recipients with or without MHC class II expression for one month. Thus, despite clear evidence for TCR signaling in nonactivated naïve T cells, these data argue against the concept that such signaling plays a predominant role in determining lymphocyte lifespan.
Adult naive T cells, which are at rest in normal conditions, proliferate strongly when transferred to lymphopenic hosts. In neonates, the first mature thymocytes to migrate to the periphery reach a compartment devoid of preexisting T cells. We have extensively analyzed the proliferation rate and phenotype of peripheral T cells from normal C57BL/6 and T cell antigen receptor transgenic mice as a function of age. We show that, like adult naive T cells transferred to lymphopenic mice, neonatal naive T cells proliferate strongly. By using bone-marrow transfer and thymic-graft models, we demonstrate that the proliferation of the first thymic emigrants reaching the periphery requires T cell antigen receptor-self-peptide/self-MHC interactions and is regulated by the size of the peripheral T cell pool.
Previous work has shown that memory-phenotype CD44(hi) CD8(+) cells are controlled by a cytokine, interleukin (IL)-15. However, the dependency of CD44(hi) CD8(+) cells on IL-15 is partial rather than complete. Here, evidence is presented that CD44(hi) CD8(+) cells comprise a mixed population of IL-15-dependent and IL-15-independent cells. The major subset of CD122(hi) CD44(hi) CD8(+) cells is heavily dependent on IL-15 by three different parameters, namely (1) "bystander" proliferation induced via IFN-induced stimulation of the innate immune system, (2) normal "background" proliferation, and (3) T cell survival; IL-15 dependency is most extreme for the Ly49(+) subset of CD122(hi) CD44(hi) CD8(+) cells. In contrast to CD122(hi) cells, the CD122(lo) subset of CD44(hi) CD8(+) cells is IL-15 independent; likewise, being CD122(lo), CD44(hi) CD4(+) cells are IL-15 independent. Thus, subsets of memory-phenotype T cells differ radically in their sensitivity to IL-15.
Cells of the popliteal lymph node were teased 3 days after the injection of Shigella paradysenteriae into the hind foot pads of rabbits. These cells were transferred to normal and x-irradiated recipients. It was noted that the serum titers of dysentery agglutinins in irradiated recipients were higher than in normal recipients. This was represented both in a higher peak: titer and a tendency to remain higher for a longer period than in normal animals. Recipients were x-irradiated within 1 hour after receiving cells of the lymph node prepared as indicated above. The serum titers of these recipients were markedly reduced in comparison with those of non-irradiated control animals. If the irradiation of the recipients followed the transfer of cells by a day, however, this difference was much smaller and in the case of a 2 day interval after the transfer of the lymph node cells the irradiation appeared to have no effect on the resulting serum titer.
The exact role of major histocompatibility complex (MHC) molecules in the peripheral survival of naive T cells is controversial, as some studies have suggested that they are critically required whereas others have suggested that they are not. Here we controlled for some of the features that differed among the earlier studies, and analyzed both the survival and expansion of naive CD4+ T cells transferred into MHC syngeneic, allogeneic, or MHC negative environments. We found that naive T cells transferred into MHC negative or allogeneic environments often fail to survive because of rejection and/or competition by natural killer (NK) cells, rather than failure to recognize a particular MHC allele. In the absence of NK cells, naive CD4+ T cells survived equally well regardless of the MHC type of the host. There was, however, an MHC requirement for extensive space-induced "homeostatic" expansion. Although the first few divisions occurred in the absence of MHC molecules, the cells did not continue to divide or transit to a CD44hi phenotype. Surprisingly, this MHC requirement could be satisfied by alleles other than the restricting haplotype. Therefore, space-induced expansion and survival are two different phenomena displaying different MHC requirements. Memory CD4+ T cells, whose survival and expansion showed no requirements for MHC molecules at all, dampened the space-induced expansion of naive cells, showing that the two populations are not independent in their requirements for peripheral niches.
Despite the ease of inhibiting immune responses by blockade of T-cell costimulation in naive rodent models, it is difficult to suppress those responses in animals with memory cells. Studies demonstrating the importance of alloreactive T-cell deletion during tolerance induction have promoted use of peritransplant T-cell-depleting therapies in clinical trials. But potentially complicating wide-scale, nonspecific T-cell depletion is the finding that extensive T-cell proliferation can occur under conditions of lymphopenia. This process, termed homeostatic proliferation, may induce acquisition of functional memory T cells. Here, using clinically relevant mouse models of peripheral T-cell depletion, we show that residual nondepleted T cells undergo substantial homeostatic expansion. In this setting, costimulatory blockade neither significantly suppresses homeostatic proliferation nor prevents allograft rejection. In addition, T cells that have completed homeostatic proliferation show dominant resistance to tolerance when adoptively transferred into wild-type recipients, consistent with known properties of memory cells in vivo. These findings establish the importance of homeostatic proliferation in clinically relevant settings, demonstrate the barrier that homeostatic proliferation can present to the induction of transplantation tolerance, and have important implications for transplantation protocols that use partial or complete peripheral T-cell depletion.
Naïve T cells proliferate independently of cognate antigen when introduced into lymphopenic hosts. Lymphopenia-induced proliferation depends on low-affinity MHC/self-peptide complexes and on IL-7. To elucidate the intracellular signals mediating this proliferation, we analyzed changes in gene expression in naïve CD8⁺ T cells at different times after their transfer into a lymphopenic environment. The genes induced in response to lymphopenia were largely an attenuated subset of those turned up by full antigenic stimulation, including genes related to cell cycling, whereas excluding genes specifically associated with effector activity. After the initial phase of proliferation in an empty compartment, the naïve T cells adopted a stable pattern of gene expression similar to that of antigen-experienced memory cells. Thus, T cells proliferating in lymphopenic hosts do not exhibit a unique gene-expression profile, instead relying on “traditional” signals for this antigen-independent proliferation; this process ultimately results in differentiation to “authentic” memory cells.
• gene-expression profiling
• memory
• T cell homeostasis
• lymphopenia
Overlapping roles have been ascribed for T cell anergy, clonal deletion, and regulation in the maintenance of peripheral immunological tolerance. A measurement of the individual and additive impacts of each of these processes on systemic tolerance is often lacking. In this report we have used adoptive transfer strategies to tease out the unique contribution of T cell intrinsic receptor calibration (adaptation) in the maintenance of tolerance to a systemic self-antigen. Adoptively transferred naïve T cells stably calibrated their responsiveness to a persistent self-antigen in both lymphopenic and T cell-replete hosts. In the former, this state was not accompanied by deletion or suppression, allowing us to examine the unique contribution of adaptation to systemic tolerance. Surprisingly, adapting T cells could chronically help antigen-expressing B cells, leading to polyclonal hypergammaglobulinemia and pathology, in the form of mild arthritis. The helper activity mediated by CD40L and cytokines was evident even if the B cells were introduced after extended adaptation of the T cells. In contrast, in the T cell-replete host, neither arthritis nor autoantibodies were induced. The containment of systemic pathology required host T cell-mediated extrinsic regulatory mechanisms to synergize with the cell intrinsic adaptation process. These extrinsic mechanisms prevented the effector differentiation of the autoreactive T cells and reduced their precursor frequency, in vivo.
Cells of the popliteal lymph node were teased 3 days after the injection of Shigella paradysenteriae into the hind foot pads of rabbits. These cells were transferred to normal and x-irradiated recipients. It was noted that the serum titers of dysentery agglutinins in irradiated recipients were higher than in normal recipients. This was represented both in a higher peak: titer and a tendency to remain higher for a longer period than in normal animals.
Recipients were x-irradiated within 1 hour after receiving cells of the lymph node prepared as indicated above. The serum titers of these recipients were markedly reduced in comparison with those of non-irradiated control animals. If the irradiation of the recipients followed the transfer of cells by a day, however, this difference was much smaller and in the case of a 2 day interval after the transfer of the lymph node cells the irradiation appeared to have no effect on the resulting serum titer.
Cells of the popliteal lymph node were teased 3 days after the injection of Shigella paradysenteriae into the hind foot pads of rabbits. These cells were transferred to normal and x-irradiated recipients. It was noted that the serum titers of dysentery agglutinins in irradiated recipients were higher than in normal recipients. This was represented both in a higher peak: titer and a tendency to remain higher for a longer period than in normal animals. Recipients were x-irradiated within 1 hour after receiving cells of the lymph node prepared as indicated above. The serum titers of these recipients were markedly reduced in comparison with those of non-irradiated control animals. If the irradiation of the recipients followed the transfer of cells by a day, however, this difference was much smaller and in the case of a 2 day interval after the transfer of the lymph node cells the irradiation appeared to have no effect on the resulting serum titer.
The adoptive secondary response of mice to conjugates of NIP (4-hydroxy-5-iodo-3-nitro-phenacetyl-) and DNP (2,4-dinitrophenyl-) is here used to elucidate the mechanism of cellular cooperation. The framework into which the experiments fit can be formulated as follows. Priming immunization raises a crop not only of specific antibody-forming-cell-precursors (AFCP) but also of specific helper cells. Upon secondary stimulation the helper cells serve a role as handlers or concentrators of antigen, thus enabling AFCP which would otherwise be incapable of reacting to initiate antibody synthesis. In this act of cooperation both cells recognise antigen; in the system examined here the helpers recognise carrier determinants and the AFCP recognise either the hapten or other carrier determinants. The first aim of the experiments was to raise populations of helpers and AFCP of distinguishable specificity. Mice were primed with NIP-Ovalbumin (OA) mixed with chicken γ-globulin (CGG) and bovine serum albumin (BSA); in comparison with controls primed with unmixed NIP-OA, their cells after transfer were relatively more sensitive to secondary stimulation with NIP-CGG or NIP-BSA and similar findings were obtained in cross-checks of these carriers, For reasons which are not entirely clear, non-transferred cells did not show the same effect. In further experiments cells primed with one conjugate (e. g. NIP-OA) were mixed with cells primed with another protein (e. g. BSA), transferred and challenged with the hapten conjugated to the second protein (i. e. NIP-BSA). In comparison with controls lacking the protein-primed cells, the mixture regularly showed greater sensitivity to stimulation. NIP and DNP were tested in many of the possible combinations with BSA, OA and CGG with the same result. The mixture system was used in the further analysis. Tests with allotype-marked protein-primed cells showed that these cells did not participate in the production of the anti-hapten antibody and could therefore properly be regarded as helpers. Tests of specificity showed that physical union of the hapten and carrier were required: cells primed with BSA, for example would not help NIP-OA-primed cells to make a response to NIP-HSA even when stimulated at the same time with BSA. Transfer of less than one-tenth of the spleen gives a maximum helper effect, whereas AFCP activity continues to rise as larger numbers of cells are transferred. Helper cells are therefore normally present in excess. Helper activity is more resistant than AFCP activity to irradiation, drugs and semi-allogeneic cell transfer across an H-2 barrier. This suggests that helper cells play a relatively passive role in the immune response. Several observations indicate that helper cells are thymus-derived mediators of cellular immunity. Passively transferred antibody did not substitute for helper cells. After immunization helper activity developed faster than AFCP activity. Spleen cells obtained from lethally-irradiated, thymocyte-repopulated, immunized donors provided help. Cells from the thymus-derived fraction of thymus/marrow chimeras also appear to provide help. Thus, the hapten-carrier cooperative response maps onto the well-established synergy of thymus and marrow in the response to foreign erythrocytes.
Homeostatic proliferation of naive T cells transferred to T cell-deficient syngeneic mice is driven by low-affinity self-MHC/peptide ligands and the cytokine IL-7. In addition to homeostatic proliferation, a subset of naive T cells undergoes massive proliferation in chronically immunodeficient hosts, but not in irradiated normal hosts. Such rapid T cell proliferation occurs largely independent of homeostatic factors, because it was apparent in the absence of IL-7 and in T cell-sufficient hosts devoid of functional T cell immunity. Strikingly, immunodeficient mice raised under germfree conditions supported only slow homeostatic proliferation, but not the marked T cell proliferation observed in conventionally raised immunodeficient mice. Thus, polyclonal naive T cell expansion in T cell-deficient hosts can be driven predominantly by either self-Ags or foreign Ags depending on the host’s previous state of T cell immunocompetency.
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.
Suspensions containing normal thymus, spleen, or marrow cells were injected into irradiated syngeneic mice which were subsequently given antigen. Normal spleen cells produced discrete areas of antibody production in recipient spleens, and the number of areas was proportional to the number of donor cells. Mice receiving both marrow and thymus cells produced more centers of hemolytic activity in their spleens than mice receiving cells of either type alone. Normal and immunized thymus cells produced little or no hemolytic activity, and normal marrow was also inactive.
Approximately 10% of peripheral CD4+ cells and less than 1% of CD8+ cells in normal unimmunized adult mice express the IL-2 receptor alpha-chain (CD25) molecules. When CD4+ cell suspensions prepared from BALB/c nu/+ mice lymph nodes and spleens were depleted of CD25+ cells by specific mAb and C, and then inoculated into BALB/c athymic nude (nu/nu) mice, all recipients spontaneously developed histologically and serologically evident autoimmune diseases (such as thyroiditis, gastritis, insulitis, sialoadenitis, adrenalitis, oophoritis, glomerulonephritis, and polyarthritis); some mice also developed graft-vs-host-like wasting disease. Reconstitution of CD4+CD25+ cells within a limited period after transfer of CD4+CD25- cells prevented these autoimmune developments in a dose-dependent fashion, whereas the reconstitution several days later, or inoculation of an equivalent dose of CD8+ cells, was far less efficient for the prevention. When nu/nu mice were transplanted with allogeneic skins or immunized with xenogeneic proteins at the time of CD25- cell inoculation, they showed significantly heightened immune responses to the skins or proteins, and reconstitution of CD4+CD25+ cells normalized the responses. Taken together, these results indicate that CD4+CD25+ cells contribute to maintaining self-tolerance by down-regulating immune response to self and non-self Ags in an Ag-nonspecific manner, presumably at the T cell activation stage; elimination/reduction of CD4+CD25+ cells relieves this general suppression, thereby not only enhancing immune responses to non-self Ags, but also eliciting autoimmune responses to certain self-Ags. Abnormality of this T cell-mediated mechanism of peripheral tolerance can be a possible cause of various autoimmune diseases.
Ionizing radiation can functionally alter the immune system and break self-tolerance. High dose (42.5 Gy), fractionated (2.5 Gy 17 times) total lymphoid irradiation (TLI) on mice caused various organ-specific autoimmune diseases, such as gastritis, thyroiditis, and orchitis, depending on the radiation dosages, the extent of lymphoid irradiation, and the genetic background of the mouse strains. Radiation-induced tissue damage is not the primary cause of the autoimmune disease because irradiation of the target organs alone failed to elicit the autoimmunity and shielding of the organs from irradiation was unable to prevent it. In contrast, irradiation of both the thymus and the peripheral lymphoid organs/tissues was required for efficient induction of autoimmune disease by TLI. TLI eliminated the majority of mature thymocytes and the peripheral T cells for 1 mo, and inoculation of spleen cell, thymocyte, or bone marrow cell suspensions (prepared from syngeneic nonirradiated mice) within 2 wk after TLI effectively prevented the autoimmune development. Depletion of T cells from the inocula abrogated the preventive activity. CD4+ T cells mediated the autoimmune prevention but CD8+ T cells did not. CD4+ T cells also appeared to mediate the TLI-induced autoimmune disease because CD4+ T cells from disease-bearing TLI mice adoptively transferred the autoimmune disease to syngeneic naive mice. Taken together, these results indicate that high dose, fractionated ionizing radiation on the lymphoid organs/tissues can cause autoimmune disease by affecting the T cell immune system, rather than the target self-Ags, presumably by altering T cell-dependent control of self-reactive T cells.
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.
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.
Positive selection to self-MHC/peptide complexes has long been viewed as a device for skewing the T cell repertoire toward recognition of foreign peptides presented by self-MHC molecules. Here, we provide evidence for an alternative possibility, namely, that the self-peptides controlling positive selection in the thymus serve to maintain the longevity of mature T cells in the periphery. Surprisingly, when total T cell numbers are reduced, these self-ligands become overtly stimulatory and cause naive T cells to proliferate and undergo homeostatic expansion.
TCR-self peptide:MHC interactions play a critical role in thymic positive selection, yet relatively little is known of their function in the periphery. It has been suggested that continued contact with selecting MHC molecules is necessary for long-term peripheral maintenance of naive T cells. More recent studies have also demonstrated a role for specific self peptide:MHC complexes in the homeostatic expansion of naive T cells in lymphopenic mice. Our examination of these processes revealed that, whereas self class II MHC molecules do have a modest effect on long-term survival of individual CD4+ T cells, interactions with specific TCR ligands are not required for peripheral naive CD4+ T cell maintenance. In contrast, selective engagement of TCRs by self-peptide:MHC complexes does promote proliferation of CD4+ T cells under severe lymphopenic conditions, and this division is associated with an activation marker phenotype that is different from that induced by antigenic stimulation. Importantly, however, the ability of naive T cells to divide in response to homeostatic stimuli does not appear to be stringently dependent on TCR-self peptide:MHC interactions. Therefore, these results show that the factors regulating survival and homeostatic expansion of naive T cells in the periphery are not identical. In addition, we provide evidence for a novel form of T cell proliferation that can occur independently of TCR signaling and suggest that this reflects another mechanism regulating homeostatic T cell expansion.
Efficient peptide presentation by professional APC to naive and effector CD4 T cells in vitro is limited to the first 1-2 days of culture, but is nonetheless optimum for effector expansion and cytokine production. In fact, prolonging Ag presentation leads to high levels of T cell death, decreased effector expansion, and decreased cytokine production by recovered effectors. Despite the absence of Ag presentation beyond day 2, T cell division continues at a constant rate throughout the 4-day culture. The Ag-independent later stage depends on the presence of IL-2, and we conclude optimum effector generation depends on an initial 2 days of TCR stimulation followed by an additional 2 days of Ag-independent, cytokine driven T cell expansion and differentiation.
The in vivo T cell response to persistent Ag contains a hyporesponsive phase following an initial expansion and subsequent partial deletion of the responding cells. The mechanism(s) responsible for this tolerance process is poorly understood. In this study, we describe a new paired transgenic model (TCR and Ag), which within 7-14 days produces 20-40 million hyporesponsive T cells. This state is characterized by an 85-95% reduction in all cytokine production, an impairment of re-expression of CD25 and CD69, and a desensitization of the proliferative response to Ag. TCR levels were normal, and in vivo mixing experiments showed no evidence for active suppression. The hyporesponsiveness partially dissipated without proliferation when the cells were transferred into a non-Ag-bearing host. If the second host expressed Ag, the T cells initially regained responsiveness, but then slowly entered an even deeper state of tolerance characterized by an additional 7- to 10-fold lowering of cytokine production and a greater desensitization of proliferation. Surprisingly, this readaptation took place with the same level of Ag presentation, suggesting that other parameters can influence the tolerance threshold. Both the readjustment in sensitivity and the reversal without Ag convincingly demonstrate for the first time a truly adaptive tolerance process in CD4+ T cells in vivo.
Thymectomy of BALB/c mice on day 3 of life results in the development of autoimmune gastritis (AIG) due to the absence of CD4(+)CD25(+) regulatory T cells. However, depletion of CD4(+)CD25(+) T cells by treatment with anti-CD25 rarely resulted in AIG. Depletion was efficient, as transfer of splenocytes from depleted mice induced AIG in nu/nu mice. One explanation for this result is that CD4(+)CD25(-) T cells upon transfer to nude recipients undergo lymphopenia-induced proliferation, providing a signal for T cell activation. Cotransfer of CD25(+) T cells did not inhibit initial proliferation but did suppress AIG. Surprisingly, immunization with the AIG target Ag, H/K ATPase, in IFA failed to induce disease in normal animals but induced severe AIG in CD25-depleted mice. These results demonstrate that second signals (nonspecific proliferation, TCR activation, or inflammation) are needed for induction of autoimmunity in the absence of CD25(+) regulatory T cells.
T cells expand without intentional antigen stimulation when transferred into adult lymphopenic environments. In this study, we show that the physiologic lymphopenic environment existing in neonatal mice also supports CD4 T cell proliferation. Strikingly, naive CD4 T cells that proliferate within neonates acquire the phenotypic and functional characteristics of memory cells. Such proliferation is inhibited by the presence of both memory and naive CD4 T cells, is enhanced by 3-day thymectomy, is independent of IL-7, and requires a class II MHC-TCR interaction and a CD28-mediated signal. CD44(bright) CD4 T cells in neonates have a wide repertoire as judged by the distribution of Vbeta expression. Thus, lymphopenia-induced T cell proliferation is a physiologic process that occurs during the early postnatal period.
CD4+CD25+ regulatory T cells are essential for the active suppression of autoimmunity. Here we report that the forkhead transcription factor Foxp3 is specifically expressed in CD4+CD25+ regulatory T cells and is required for their development. The lethal autoimmune syndrome observed in Foxp3-mutant scurfy mice and Foxp3-null mice results from a CD4+CD25+ regulatory T cell deficiency and not from a cell-intrinsic defect of CD4+CD25- T cells. CD4+CD25+ regulatory T cells rescue disease development and preferentially expand when transferred into neonatal Foxp3-deficient mice. Furthermore, ectopic expression of Foxp3 confers suppressor function on peripheral CD4+CD25- T cells. Thus, Foxp3 is a critical regulator of CD4+CD25+ regulatory T cell development and function.
The secretion of IL-2 is a critical and early landmark in the activation program of CD4(+) T cells in vitro, but the lack of sensitive assays has limited its application for studying T cell activation in vivo. Using a mouse cytokine capture assay we were able to detect the rapid secretion of IL-2 after an in vivo stimulus by 1-2 h in naive T cells and as early as 30 min in memory T cells. Maximal secretion was achieved within 1-2 h for memory cells or 6-8 h for naive T cells. Surprisingly IL-2 production terminated quickly in vivo and secretion was undetectable by 20-24 h in either cell type. We further demonstrated that this short duration of secretion can be influenced by cellular competition between Ag-specific CD4(+) T cells. The consequences of competition were mimicked by reducing the strength of the antigenic stimulus. These data argue that early competition between T cells influences both the eventual frequency of IL-2 producers in the population and also the duration of their secretion, potentially by altering the strength or duration of the stimulus available to each T cell.
Homeostatic proliferation of naive T cells transferred to T cell-deficient syngeneic mice is driven by low-affinity self-MHC/peptide ligands and the cytokine IL-7. In addition to homeostatic proliferation, a subset of naive T cells undergoes massive proliferation in chronically immunodeficient hosts, but not in irradiated normal hosts. Such rapid T cell proliferation occurs largely independent of homeostatic factors, because it was apparent in the absence of IL-7 and in T cell-sufficient hosts devoid of functional T cell immunity. Strikingly, immunodeficient mice raised under germfree conditions supported only slow homeostatic proliferation, but not the marked T cell proliferation observed in conventionally raised immunodeficient mice. Thus, polyclonal naive T cell expansion in T cell-deficient hosts can be driven predominantly by either self-Ags or foreign Ags depending on the host's previous state of T cell immunocompetency.
The adoptive secondary response of mice to conjugates of NIP (4‐hydroxy‐5‐iodo‐3‐nitro‐phenacetyl‐) and DNP (2,4‐dinitrophenyl‐) is here used to elucidate the mechanism of cellular cooperation. The framework into which the experiments fit can be formulated as follows. Priming immunization raises a crop not only of specific antibody‐forming‐cell‐precursors (AFCP) but also of specific helper cells. Upon secondary stimulation the helper cells serve a role as handlers or concentrators of antigen, thus enabling AFCP which would otherwise be incapable of reacting to initiate antibody synthesis. In this act of cooperation both cells recognise antigen; in the system examined here the helpers recognise carrier determinants and the AFCP recognise either the hapten or other carrier determinants.
The first aim of the experiments was to raise populations of helpers and AFCP of distinguishable specificity. Mice were primed with NIP‐Ovalbumin (OA) mixed with chicken γ‐globulin (CGG) and bovine serum albumin (BSA); in comparison with controls primed with unmixed NIP‐OA, their cells after transfer were relatively more sensitive to secondary stimulation with NIP‐CGG or NIP‐BSA and similar findings were obtained in cross‐checks of these carriers. For reasons which are not entirely clear, non‐transferred cells did not show the same effect. In further experiments cells primed with one conjugate ( e. g. NIP‐OA) were mixed with cells primed with another protein ( e. g. BSA), transferred and challenged with the hapten conjugated to the second protein ( i. e. NIP‐BSA). In comparison with controls lacking the protein‐primed cells, the mixture regularly showed greater sensitivity to stimulation. NIP and DNP were tested in many of the possible combinations with BSA, OA and CGG with the same result. The mixture system was used in the further analysis.
Tests with allotype‐marked protein‐primed cells showed that these cells did not participate in the production of the anti‐hapten antibody and could therefore properly be regarded as helpers. Tests of specificity showed that physical union of the hapten and carrier were required: cells primed with BSA, for example, would not help NIP‐OA‐primed cells to make a response to NIP‐HSA even when stimulated at the same time with BSA. Transfer of less than one‐tenth of the spleen gives a maximum helper effect, whereas AFCP activity continues to rise as larger numbers of cells are transferred. Helper cells are therefore normally present in excess.
Helper activity is more resistant than AFCP activity to irradiation, drugs and semi‐allogeneic cell transfer across an H‐2 barrier. This suggests that helper cells play a relatively passive role in the immune response.
Several observations indicate that helper cells are thymus‐derived mediators of cellular immunity. Passively transferred antibody did not substitute for helper cells. After immunization helper activity developed faster than AFCP activity. Spleen cells obtained from lethally‐irradiated, thymocyte‐repopulated, immunized donors provided help. Cells from the thymus‐derived fraction of thymus/marrow chimeras also appear to provide help.
Thus, the hapten‐carrier cooperative response maps onto the well‐established synergy of thymus and marrow in the response to foreign erythrocytes.
The overall size and the composition of the mature T cell pool are regulated by homeostatic mechanisms. Recent work has revealed that homeostatic signals are received from contact with two members of the common gamma chain family of cytokines, IL-7 and IL-15, and from self-MHC/peptide ligands. In essence, homeostasis of naïve T cells is regulated by IL-7 and self-MHC/peptide ligands and homeostasis of memory CD8 cells is controlled by IL-7 and IL-15. All of these signals also appear to be important to a varying degree for homeostasis of memory CD4 cells, but the details are less well understood than for other cell type.
Immunity to a plethora of microbes depends on a diverse repertoire of naïve lymphocytes and the production of long-lived memory
cells. We present evidence here that low clonal abundance in a polyclonal repertoire favors the survival and activation of
naïve CD4+ T cells as well as the survival of their memory cell progeny. The inverse relation between clonal frequency and survival
suggests that intraclonal competition could help maintain an optimally diverse repertoire of T cells and an optimal environment
for the generation of long-lived memory cells.
The survival of mature resting B cells in the periphery depends on signaling from the B-cell receptor (BCR) and the B-cell activating factor of the TNF family receptor (BAFF-R). Engagement of both receptors promotes NF-kappa B activity, which contributes to B-cell survival through different pathways. BCR signaling leads to activation of the inhibitor of NF-kappa B kinase (IKK) complex via Carma1, Bcl10 and MALT1, whereas BAFF-R engagement promotes processing of NF-kappa B2 protein p100, which is dependent on NF-kappa B-inducing kinase (NIK) and IKK alpha. Proximal signaling intermediates are potentially common to both pathways. We suggest that BCR and BAFF-R survival signaling are mutually dependent. In addition, we propose that BAFF-R signaling enhances the expression of survival genes through direct chromatin modifications in NF-kappa B target gene promoters.
Contribution a l'etude de l'origine des anticorps typhiques
- Deutsch
Deutsch, L. (1899). Contribution a l'etude de l'origine des anticorps
typhiques. Ann. Inst. Pasteur (Paris) 13, 689-727.
- W C Kieper
- A Troy
- J T Burghardt
- C Ramsey
- J Y Lee
- H Q Jiang
- W Dummer
- H Shen
- J J Cebra
Kieper, W.C., Troy, A., Burghardt, J.T., Ramsey, C., Lee, J.Y., Jiang,
H.Q., Dummer, W., Shen, H., Cebra, J.J., and Surh, C.D. (2005).
The impact of T cell intrinsic antigen adaptation on peripheral immune tolerance
- N J Singh
- C Chen
- R H Schwartz
Singh, N.J., Chen, C., and Schwartz, R.H. (2006). The impact of T cell
intrinsic antigen adaptation on peripheral immune tolerance. PLoS
Biol. 4, e430 10.1371/journal.pbio.0040340.