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Kinetics of gene expression induced in the presence or absence of CTLA4-Ig in TCR-activated CD4+ T cells. Naïve CD4+ T cells were activated with αCD3+APCs in the presence or absence of CTLA4-Ig for 0, 3, 7, and 14 hours. CD4 cells were purified and subjected to RNA preparation and RNA-Seq analysis. Data shown are K-mean clustering of genes differentially expressed during activation compared to 0 hours with p value <0.05 and RPKM>1 in at least one condition. Selected gene ontology pathways overrepresented in each cluster are shown to the right together with the corresponding p-value.
Source publication
During activation, T cells integrate multiple signals from APCs and cytokine milieu. The blockade of these signals can have clinical benefits as exemplified by CTLA4-Ig, which blocks interaction of B7 co-stimulatory molecules on APCs with CD28 on T cells. Variants of CTLA4-Ig, abatacept and belatacept are FDA approved as immunosuppressive agents in...
Contexts in source publication
Context 1
... better understand the mechanism of CTLA4-Ig action during T cell activation, we analyzed the kinetics of gene expression in human CD4 cells stimulated by αCD3 and APCs in the pres- ence or absence of CTLA4-Ig (Fig 3). In both conditions, CD4 cells upregulated a large group of genes targeted by TCR signals (cluster IV), confirming intact activity of TCR downstream pathways. ...
Context 2
... at early time points, only naïve cells activated by αCD3 in the presence of co-stimulation were able to upregulate genes encod- ing cytokines and cytokines receptors, such as IL2, CSF2, LTA, IL2RA, IL15RA, and IL12RB2, and transcription factors (TFs) that are important in immune response-BATF, IRFs, MYB, BHLHE40, REL, and TBX21 (cluster II). CD4 cells activated in the presence of CTLA4-Ig had a delayed response with lower levels of induction of these genes at later time points (Fig 3, cluster II effector [E] vs. anergic [A]). Similarly, genes downregulated following T cell activation were reduced slower in the presence of CTLA4-Ig treatment (cluster III, 3 hours). ...
Citations
... Belatacept (cytotoxic T lymphocyte-associated antigen 4 [CTLA4]-Ig; LEA29Y; Bristol Myers Squibb) is a human fusion protein combining the extracellular portion of CTLA-4 that has been mutated to confer greater binding avidity to CD80 and CD86 and the constant region fragment of human IgG1. CTLA-4 binds to surface costimulatory ligands of antigen-presenting cells (APCs) and in a lesser extent on T cells, and thus, prevents their interaction with CD28, thereby blocking T cell activation 7,8 . ...
The humoral response mediated by alloantibodies directed against donor HLA molecules (DSAs) is one of the main causes of graft loss in kidney transplantation. Understanding the pathophysiology leading to humoral kidney rejection as the development of therapeutic tools is therefore a main objective in the field of solid organ transplantation and necessitate adapted experimental models. Among the immunosuppressive agents used in renal transplantation, belatacept, a fusion protein targeting T costimulatory molecules has shown its ability to prevent more efficiently the secretion of DSA by different mechanisms including a direct action on plasma cells but also on B lymphocytes and follicular helper T lymphocytes (Tfh) cooperation. This cellular cooperation occurs within germinal centers (GC), the seat of B lymphocytes differentiation. Here, we aimed to develop a dedicated mouse model in which human GC would be functional to study the effect of belatacept on GC formation and the ability of B lymphocytes to secrete immunoglobulin. We next demonstrate that belatacept inhibits the formation of these GCs, by inhibiting the frequency of Tfh and B lymphocytes. This alters the B maturation and therefore the generation of plasma cells and consequently, immunoglobulin secretion.
... Our previous studies highlighted the efficacy of CTLA4-Ig treatment to reduce the in vitro pro-inflammatory cytokine production (IL-6, TNFα, IL-1β) as well as the CD86 expression in cultured synovial macrophages, and this effect seems to be mediated by the upregulation of the NF-kB inhibitor IkB-a [36][37][38]. Moreover, Rochman et al. demonstrated the effect of abatacept in reducing the gene expression of NF-kB and AP-1 transcription factors along with the remarkable proliferation decrease of T regulatory cells [55]. Additionally, Lorenzetti et al. reported a dose-dependent decrease of CD80 and CD86 induced by the treatment with abatacept in B cells [56]. ...
Abstract Background In rheumatoid arthritis (RA), macrophages play an important role in modulating the immunoinflammatory response through their polarisation into “classically” (M1) or “alternatively activated” (M2) phenotypes. In RA, CTLA4-Ig (abatacept) reduces the inflammatory activity of macrophages by interacting with the costimulatory molecule CD86. The study aimed to investigate the efficacy of CTLA4-Ig treatment to induce an M2 phenotype both in M1-polarised monocyte-derived macrophages (MDMs) obtained from healthy subjects (HS) and in cultured MDMs obtained from active RA patients. Methods Cultured MDMs were obtained from peripheral blood mononuclear cells of 7 active RA patients and from 10 HS after stimulation with phorbol myristate acetate (5 ng/mL) for 24 h. HS-MDMs were then stimulated with lipopolysaccharide (LPS, 1 mg/mL) for 4 h to induce M1-MDMs. M1-MDMs and RA-MDMs were treated with CTLA4-Ig (100 μM and 500 μM) for 3, 12, 24, and 48 h. The gene expression of CD80, CD86, and TLR4 (M1 markers); CD163, CD204, and CD206 (surface M2 markers); and MerTK (functional M2 marker) was evaluated by qRT-PCR. The protein synthesis of surface M2 markers was investigated by Western blotting. The statistical analysis was performed by the Wilcoxon t-test. Results In LPS-induced HS-M1-MDMs, CTLA4-Ig 100 μM and 500 μM significantly downregulated the gene expression of M1 markers (3 h p
... The activation of naive T cells requires antigen recognition, but also an interaction of B7 with the CD28 receptor. CD28 in the only costimulatory receptor constitutively expressed on naive T cells; therefore, its presence is fundamental for the initiation and maintenance of T-cell-mediated immune responses [16,17]. In chronic-inflammation situations such as chronic kidney disease and diabetes mellitus (DM), the transformation of receptor repertoire on T cells, including the loss of the CD28 molecule, results in a cell type of which the phenotype and activities resemble NK cells, called the NK-like T cell [2,10,11,18,19]. ...
Background:
End-stage renal disease (ESRD) is associated with alterations in T-cell immunity, including increased CD28null and reduced regulatory T cells (Tregs). However, whether immune disturbances are due to ESRD or primary disease is not yet clear. As diabetes mellitus is the leading cause of ESRD, we evaluated its impact on the immune profile of ESRD patients.
Methods:
CD28null, Tregs, and natural killer cells were initially analyzed by flow cytometry in 30 predialysis ESRD patients due to diabetes (DM), 30 non-DM (NDM), and 25 healthy controls. Measurements were repeated after 6 months on hemodialysis (HD) or peritoneal dialysis (CAPD).
Results:
The percentage of CD4 + CD28null cells, CD8 + CD28null cells, and Tregs showed significant differences in DM, NDM, and controls; mean rank 33.71 vs. 25.68 vs. 18.88, p = 0.006, 37.79 vs. 28.82 vs. 17.08, p = 0.008, and 20.79 vs. 26.12 vs. 41.33, p = 0.001, respectively. DM vs. NDM had increased CD4 + CD28null and CD8 + CD28null cells, 11.5% (1.5%-24%) vs. 4.1% (0-42.3%), p = 0.02 and 61.3% (24%-76%) vs. 43% (5.7%-85%), p = 0.04, respectively. After 6 months on HD but not CAPD, DM showed a significant further increase in CD4 + CD28null cells, from 30 (14-100) to 52.7 (15-203), p = 0.02; and CD8 + CD28null cells, from 137 (56-275) to 266 (103-456), p = 0.01.
Conclusions:
Diabetes mellitus affects T-cell subtypes even at predialysis stage, though changes become more prominent after commencement on HD.
... In support of this hypothesis, one study has described a CCR7 + CD45RA + autoreactive Melan-A-specific CD8 + T cell population in healthy people that were suppressed, via regulatory T cells, in their ability to both proliferate and produce cytokines in response to TCR ligation (50). Other studies have also associated the naïve marker of CR45RA with anergic T cells (51). Further studies are needed to better understand the dynamics of T cell populations in the marrow of patients with AML. ...
Significance
Acute myeloid leukemia (AML) is the most common acute leukemia in adults, with a 5-y survival of 29%. Immunotherapy is based on the premise that tumors suppress the immune system. We investigated the status of T cell immunity in AML at the time of diagnosis. We found a significant association between T cell percentage in the bone marrow and overall survival in newly diagnosed AML patients. When we evaluated the T cells from the bone marrow of patients with AML, one-third displayed profound functional impairment. Most of these compromised T cells, however, could be rescued using checkpoint inhibitors. Our results support the development of immune checkpoint therapy to combat this deadly disease.
... The activation is accomplished through simultaneous stimulation of the TCR and costimulatory receptors such as CD28. Downstream NFATs, AP-1 (a heterodimer of FOS and JUN proteins), and NF-κB are activated via Ca 2+calcineurin, MAPK, and PI3K/PKC pathways (Fathman and Lineberry, 2007;Crabtree and Olson, 2002;Jain et al., 1994;Rochman et al., 2015). Concurrently with activation signals, differentiation signals provided by the cytokine milieu lead to the activation of JAK-STAT pathways, induction of lineage-specific transcription factors (TFs), and eventually lineage-specific cytokine gene expression . ...
... To better understand the role of AP-1-induced chromatin remodeling in the greater context of the immune response, we next performed T cell activation in the absence of CD28 costimulation ( Fig. 6 A). Previous studies have shown that the lack of co-stimulation reduces activation of AP-1 and eventually leads to the induction of anergy (Macián et al., 2002;Kriegel et al., 2009;Rochman et al., 2015). Indeed, cells not receiving CD28 co-stimulation (anergy) showed dramatic reduction of nuclear translocation of AP-1 and, to a smaller degree, NF-κB p50, whereas nuclear levels of NFATs and NF-κB p65 were reduced only slightly (Fig. S5 A). ...
... It has long been known that activation of T cells requires two signals. Work from the Rao laboratory and others has shown that insufficient induction of AP-1 in the absence of costimulation fails to induce Il2 gene expression and eventually leads to anergy (Macián et al., 2002;Kriegel et al., 2009;Rochman et al., 2015). However, murine cFOS knockout T cells demonstrated normal response to activation, suggesting either that cFOS is not necessary (Jain et al., 1994) or that the other FOS family members, such as FOSB, FRA-1, and FRA-2, could substitute for cFOS (Fleischmann et al., 2000;Gruda et al., 1996). ...
Activation of T cells is dependent on the organized and timely opening and closing of chromatin. Herein, we identify AP-1 as the transcription factor that directs most of this remodeling. Chromatin accessibility profiling showed quick opening of closed chromatin in naive T cells within 5 h of activation. These newly opened regions were strongly enriched for the AP-1 motif, and indeed, ChIP-seq demonstrated AP-1 binding at >70% of them. Broad inhibition of AP-1 activity prevented chromatin opening at AP-1 sites and reduced the expression of nearby genes. Similarly, induction of anergy in the absence of co-stimulation during activation was associated with reduced induction of AP-1 and a failure of proper chromatin remodeling. The translational relevance of these findings was highlighted by the substantial overlap of AP-1–dependent elements with risk loci for multiple immune diseases, including multiple sclerosis, inflammatory bowel disease, and allergic disease. Our findings define AP-1 as the key link between T cell activation and chromatin remodeling.
... The activation is accomplished through simultaneous stimulation of the T cell receptor (TCR) and co-stimulatory receptors such as CD28. Downstream NFATs, AP-1 (a heterodimer of FOS and JUN proteins), and NFB are activated via Ca 2+ -calcineurin, MAP kinase and PI3K/PKC pathways (Fathman and Lineberry, 2007;Crabtree and Olson, 2002;Jain et al., 1994;Rochman et al., 2015). Concurrently with activation signals, differentiation signals provided by the cytokine milieu lead to activation of JAK-STAT pathways, induction of lineagespecific transcription factors (TFs), and eventually lineage-specific cytokine gene expression . ...
... In order to better understand the role of AP-1-induced chromatin remodeling in the greater context of the immune response, we next performed T cell activation in the absence of CD28 co-stimulation ( Fig. 6 A). Previous studies have shown that the lack of co-stimulation reduces activation of AP-1 and eventually leads to the induction of anergy (Macian et al., 2002;Kriegel et al., 2009;Rochman et al., 2015). Indeed, cells not receiving CD28 co-stimulation (Anergy) showed dramatic reduction of nuclear translocation of AP-1 and to a smaller degree NFB p50, whereas nuclear levels of NFATs and NFB p65 were reduced only slightly (Fig. S6 A). ...
... It has long been known that activation of T cells requires two signals. Work from the Rao laboratory and others has shown that insufficient induction of AP-1 in the absence of costimulation fails to induce Il2 gene expression and eventually leads to anergy (Macian et al., 2002;Kriegel et al., 2009;Rochman et al., 2015). However, murine cFOS knock-out T cells demonstrated normal response to activation, suggesting that either cFOS was not necessary (Jain et al., 1994) or that the other FOS family members, such as FOSB, FRA-1, and FRA-2, could substitute for cFOS (Fleischmann et al., 2000;Gruda et al., 1996). ...
Activation of T cells is dependent on organized and timely opening and closing of chromatin. Herein, we identify AP-1 as the transcription factor that directs most of this remodeling. Chromatin accessibility profiling showed quick opening of closed chromatin in naive T cells within 5 hours of activation. These newly open regions were strongly enriched for the AP-1 motif, and indeed, ChIP-seq demonstrated AP-1 binding at more than 70% of them. Broad inhibition of AP-1 activity prevented chromatin opening at AP-1 sites and reduced expression of nearby genes. Similarly, induction of anergy in the absence of co-stimulation during activation, was associated with reduced induction of AP-1 and a failure of proper chromatin remodeling. The translational relevance of these findings was highlighted by the substantial overlap of AP-1 dependent elements with risk loci for multiple immune diseases, including multiple sclerosis, inflammatory bowel disease and allergic disease. Our findings define AP-1 as the key link between T cell activation and chromatin remodeling.
... Abatacept has been shown to be safe and effective in oligo-and polyarticular JIA (49,(89)(90)(91)(92), adult DM/polymyositis (34,53), a case report of steroid-sparing abatacept in complex JDM (93) and a trial in JDM is underway (27). A reduction of the T cell activation state is the main reported effect of abatacept (90,(94)(95)(96). Surprisingly, the majority of studies found abatacept decreases the frequency of Tregs (90,95,(97)(98)(99), with some studies showing an increase in function (99). ...
Regulatory T cells (Tregs) are believed to be dysfunctional in autoimmunity. Juvenile idiopathic arthritis (JIA) and juvenile dermatomyositis (JDM) result from a loss of normal immune regulation in specific tissues such as joints or muscle and skin, respectively. Here, we discuss recent findings in regard to Treg biology in oligo-/polyarticular JIA and JDM, as well as what we can learn about Treg-related disease mechanism, treatment and biomarkers in JIA/JDM from studies of other diseases. We explore the potential use of Treg immunoregulatory markers and gene signatures as biomarkers for disease course and/or treatment success. Further, we discuss how Tregs are affected by several treatment strategies already employed in the therapy of JIA and JDM and by alternative immunotherapies such as anti-cytokine or co-receptor targeting. Finally, we review recent successes in using Tregs as a treatment target with low-dose IL-2 or cellular immunotherapy. Thus, this mini review will highlight our current understanding and identify open questions in regard to Treg biology, and how recent findings may advance biomarkers and new therapies for JIA and JDM.
... The patient had been on belatacept for 20 months, resulting in a long-term lack of CD28-mediated costimulation. Belatacept, which can provide significantly better renal function and both patient and renal graft survival than calcineurin inhibitor-based immunosuppression [8], provokes T-cell anergy in vivo [9]. The CD4 T-cell count recovery observed in our patient was not associated with improved functionality in terms of cytokine secretion, proliferation or cytotoxicity. ...
Progressive multifocal leukoencephalopathy (PML) is a deadly demyelinating disease due to CNS replication of the human polyomavirus JC-virus (JCV) in immunosuppressed patients. The only effective therapeutic approach is to restore anti-JCV T-cell responses. Here, we describe a case of rapidly fatal PML with JCV T-cell anergy in a renal transplant patient treated with CTLA4-Ig (belatacept, a CD28-B7 costimulation blocker and T-cell anergy inducer). T-cell anergy could not be reversed despite several therapeutic approaches. PML secondary to biotherapy-induced T-cell anergy may thus represent a subset of PML with major resistance to anti-JCV immune recovery.
... In this setting, ct-CD45-Ig (hence referred to as ct-CD45) was used at saturating conditions (Supporting Information Fig. 3 and [5]). CTLA4-Ig has been described to inhibit the T-cell antigen-presenting cell interaction, but does not act directly on T cells [7,8], thus serving as negative control in this system. ...
The cytoplasmic tail of CD45 (ct-CD45) is proteolytically cleaved and released upon activation of human phagocytes. It acts on T cells as an inhibitory, cytokine-like factor in vitro. Here we show, that ct-CD45 is abundant in human peripheral blood plasma from healthy adults compared with plasma derived from umbilical cord blood and plasma from patients with rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Plasma depleted of ct-CD45 enhanced T-cell proliferation, while addition of exogenous ct-CD45 protein inhibited proliferation and reduced cytokine production of human T lymphocytes in response to TCR signaling. Inhibition of T-cell proliferation by ct-CD45 was overcome by co-stimulation via CD28. T-cell activation in the presence of ct-CD45 was associated with an upregulation of the quiescence factors Schlafen family member 12 (SLFN12) and Krueppel-like factor 2 (KLF2) as well as of the cyclin-dependent kinase (CDK) inhibitor p27kip1. In contrast, positive regulators of the cell cycle such as cyclin D2 and D3 as well as CDK2 and CDK4 were found to be downregulated in response to ct-CD45. In summary, we demonstrate that ct-CD45 is present in human plasma and sets the threshold of T-cell activation. This article is protected by copyright. All rights reserved.
... In vitro assays using naive human CD4 + T cells show that CTLA4Ig blocks activation of transcription factors known to be preferentially activated by CD28 signals, including cJUN and NF-kB. CD28 blockade by CTLA4Ig induces hyporesponsiveness but did not induce a Treg cell phenotype (Rochman et al., 2015). However, artificial in vitro systems are unlikely to reflect the complex mechanism of action that CTLA4Ig uses to have effects on both effector and Treg cells. ...
Ligation of the CD28 receptor on T cells provides a critical second signal alongside T cell receptor (TCR) ligation for naive T cell activation. Here, we discuss the expression, structure, and biochemistry of CD28 and its ligands. CD28 signals play a key role in many T cell processes, including cytoskeletal remodeling, production of cytokines, survival, and differentiation. CD28 ligation leads to unique epigenetic, transcriptional, and post-translational changes in T cells that cannot be recapitulated by TCR ligation alone. We discuss the function of CD28 and its ligands in both effector and regulatory T cells. CD28 is critical for regulatory T cell survival and the maintenance of immune homeostasis. We outline the roles that CD28 and its family members play in human disease and we review the clinical efficacy of drugs that block CD28 ligands. Despite the centrality of CD28 and its family members and ligands to immune function, many aspects of CD28 biology remain unclear. Translation of a basic understanding of CD28 function into immunomodulatory therapeutics has been uneven, with both successes and failures. Such real-world results might stem from multiple factors, including complex receptor-ligand interactions among CD28 family members, differences between the mouse and human CD28 families, and cell-type specific roles of CD28 family members.
