Pan-Cancer Proteomics Analysis Reveals Wiskott-Aldrich Syndrome Protein as a Potential Regulator of Programmed Death-Ligand 1.

The programmed death-ligand 1 (PD-L1) is a key mediator of immunosuppression in the tumor microenvironment. The expression of PD-L1 in cancer cells is useful for the clinical determination of an immune checkpoint blockade (ICB). However, the regulatory mechanism of the PD-L1 abundance remains incompletely understood. Here, we integrated the proteomics of 52 patients with solid tumors and examined immune cell infiltration to reveal PD-L1-related regulatory modules. Wiskott-Aldrich syndrome protein (WASP) was identified as a potential regulator of PD-L1 transcription. In two independent cohorts containing 164 cancer patients, WASP expression was significantly associated with PD-L1. High WASP expression contributed to immunosuppressive cell composition, including cells positive for immune checkpoints (PD1, CTLA4, TIGIT, and TIM3), FoxP3+ Treg cells, and CD163+ tumor-associated macrophages. Overexpression of WASP increased, whereas knockdown of WASP decreased the protein level of PD-L1 in cancer cells without alteration of PD-L1 protein stability. The WASP-mediated cell migration and invasion were markedly attenuated by the silence of PD-L1. Collectively, our data suggest that WASP is a potential regulator of PD-L1 and the WASP/PD-L1 axis is responsible for cell migration and an immunosuppressive microenvironment.

Differential induction of donor-reactive Foxp3+ regulatory T cell via blockade of CD154 vs CD40.

Recently published studies in both murine models and a meta-analysis of non-human primate renal transplant studies showed that anti-CD154 reagents conferred a significant survival advantage over CD40 blockers in both animal models and across multiple organs. Here we sought to compare the induction of donor-reactive forkhead box P3+-induced regulatory T cells (Foxp3+ iTreg) in mice treated with anti-CD154 versus anti-CD40 monoclonal antibodies (mAbs). Results indicated that while treatment with anti-CD154 mAb resulted in a significant increase in the frequency of donor-reactive CD4+ Foxp3+ iTreg following transplantation, treatment with anti-CD40 or Cd40 deficiency failed to recapitulate this result. Because we recently identified CD11b as an alternate receptor for CD154 during alloimmunity, we interrogated the role of CD154:CD11b interactions in the generation of Foxp3+ iTreg and found that blockade of CD11b in Cd40-/- recipients resulted in increased donor-reactive Foxp3+ iTreg as compared with CD40 deficiency alone. Mechanistically, CD154:CD11b inhibition decreased interleukin (IL)-1ß from CD11b+ and CD11c+ dendritic cells, and blockade of IL-1ß synergized with CD40 deficiency to promote Foxp3+ iTreg induction and prolong allograft survival. Taken together, these data provide a mechanistic basis for the observed inferiority of anti-CD40 blockers as compared with anti-CD154 mAb and illuminate an IL-1ß-dependent mechanism by which CD154:CD11b interactions prevent the generation of donor-reactive Foxp3+ iTreg during transplantation.

TIGIT agonism alleviates costimulation blockade-resistant rejection in a regulatory T cell-dependent manner.

Belatacept-based immunosuppression in kidney transplantation confers fewer off-target toxicities than calcineurin inhibitors but comes at a cost of increased incidence and severity of acute rejection, potentially due to its deleterious effect on both the number and function of Foxp3+ regulatory T cells (Tregs). TIGIT is a CD28 family coinhibitory receptor expressed on several subsets of immune cells including Tregs. We hypothesized that coinhibition through TIGIT signaling could function to ameliorate costimulation blockade-resistant rejection. The results demonstrate that treatment with an agonistic anti-TIGIT antibody, when combined with costimulation blockade by CTLA-4Ig, can prolong allograft survival in a murine skin graft model compared with CTLA-4Ig alone. Further, this prolongation of graft survival is accompanied by an increase in the frequency and number of graft-infiltrating Tregs and a concomitant reduction in the number of CD8+ T cells in the graft. Through the use of Treg-specific TIGIT conditional knockout animals, we demonstrated that the TIGIT-mediated reduction in the graft-infiltrating CD8+ T cell response is dependent on signaling of TIGIT on Foxp3+ Tregs. Our results highlight both the key functional role of TIGIT on Foxp3+ Tregs under conditions in which CTLA-4 is blocked and the therapeutic potential of TIGIT agonism to optimize costimulation blockade-based immunosuppression.

A five-protein prognostic signature with GBP2 functioning in immune cell infiltration of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is of poor clinical outcomes, and currently lacks reliable prognostic biomarkers. By analyzing the datasets of the Cancer Genome Atlas (TCGA) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC), we established a five-protein prognostic signature containing GBP2, HLA-DRA, ISG15, ISG20 and ITGAX. Our data indicate that this signature was closely correlated with advanced stage, higher pathological grade, and unfavorable survivals in patients with ccRCC. We further functionally characterized GBP2. Overexpression of GBP2 enhanced the phosphorylation of STAT2 and STAT3 to trigger JAK-STAT signaling and promote cell migration and invasion in ccRCC. Treatment of Ruxolitinib, a specific inhibitor of JAK/STAT, attenuated the GBP2-mediated phenotypes. Patients with high GBP2 expression were accompanied with more infiltration of immune cells positively stained with CD3, CD8, CD68, and immune checkpoint markers PD-1 and CTLA4, which was validated by Opal multiplex immunohistochemistry in ccRCC tissues. More CD8 + T cells and CD68 + macrophages were observed in patients expressing high GBP2. Taken together, a five-protein prognostic signature was constructed in our study. GBP2 has an oncogenic role via modulating JAK-STAT signaling and tumor immune infiltration, and thus may serve as a potential therapeutic target in ccRCC.

Selective CD28 blockade impacts T cell differentiation during homeostatic reconstitution following lymphodepletion.

Introduction: Costimulation blockade targeting the CD28 pathway provides improved long-term renal allograft survival compared to calcineurin inhibitors but may be limited as CTLA-4-Ig (abatacept, belatacept) blocks both CD28 costimulation and CTLA-4 coinhibition. Directly targeting CD28 while leaving CTLA-4 intact may provide a mechanistic advantage. Fc-silent non-crosslinking CD28 antagonizing domain antibodies (dAb) are currently in clinical trials for renal transplantation. Given the current standard of care in renal transplantation at most US centers, it is likely that lymphodepletion via thymoglobulin induction therapy could be used in patients treated with CD28 antagonists. Thus, we investigated the impact of T cell depletion (TCD) on T cell phenotype following homeostatic reconstitution in a murine model of skin transplantation treated with anti-CD28dAb. Methods: Skin from BALB/cJ donors was grafted onto C56BL/6 recipients which were treated with or without 0.2mg anti-CD4 and 10µg anti-CD8 one day prior to transplant and with or without 100µg anti-CD28dAb on days 0, 2, 4, 6, and weekly thereafter. Mice were euthanized six weeks post-transplant and lymphoid cells were analyzed by flow cytometry. Results: Anti-CD28dAb reversed lymphopenia-induced differentiation of memory CD4+ T cells in the spleen and lymph node compared to TCD alone. Mice treated with TCD+anti-CD28dAb exhibited significantly improved skin graft survival compared to anti-CD28dAb alone, which was also improved compared to no treatment. In addition, the expression of CD69 was reduced on CD4+ and CD8+ T cells in the spleen and lymph node from mice that received TCD+anti-CD28dAb compared to TCD alone. While a reduced frequency of CD4+FoxP3+ T cells was observed in anti-CD28dAb treated mice relative to untreated controls, this was balanced by an increased frequency of CD8+Foxp3+ T cells that was observed in the blood and kidney of mice given TCD+anti-CD28dAb compared to TCD alone. Discussion: These data demonstrate that CD28 signaling impacts the differentiation of both CD4+ and CD8+ T cells during homeostatic reconstitution following lymphodepletion, resulting in a shift towards fewer activated memory T cells and more CD8+FoxP3+ T cells, a profile that may underpin the observed prolongation in allograft survival.

Anti-TIGIT differentially affects sepsis survival in immunologically experienced versus previously naive hosts.

Mounting evidence suggests that the balance of T cell costimulatory and coinhibitory signals contributes to mortality during sepsis. Here, we identified a critical role of the coinhibitory molecule T cell Ig and ITIM domain (TIGIT) in regulating sepsis mortality. Because TIGIT is significantly upregulated on memory T cells, we developed a "memory mouse" model to study the role of TIGIT during sepsis in a more physiologically relevant context. Mice received sequential pathogen exposure and developed memory T cell frequencies, similar to those observed in adult humans, and were then subjected to sepsis induction via cecal ligation and puncture. Our results show that targeting the TIGIT pathway during sepsis is fundamentally different in previously naive versus memory mice, in that αTIGIT Ab had no effect on survival in previously naive septic mice but sharply worsened survival in memory septic mice. Mechanistically, αTIGIT increased apoptosis of memory T cells, decreased T cell function, and downregulated the costimulatory receptor DNAM on memory CD8+ T cells in memory septic mice, but not in previously naive septic mice. Additionally, αTIGIT diminished Helios expression in Tregs in memory but not previously naive septic mice. These data highlight fundamental differences in the pathophysiological impact of targeting TIGIT in immunologically experienced versus previously naive hosts during sepsis.

Memory T cell-mediated rejection is mitigated by FcγRIIB expression on CD8+ T cells.

Donor-reactive memory T cells generated via heterologous immunity represent a potent barrier to long-term graft survival following transplantation because of their increased precursor frequency, rapid effector function, altered trafficking patterns, and reduced reliance on costimulation signals for activation. Thus, the identification of pathways that control memory T cell survival and secondary recall potential may provide new opportunities for therapeutic intervention. Here, we discovered that donor-specific effector/memory CD8+ T cell populations generated via exposure to acute vs latent vs chronic infections contain differential frequencies of CD8+ T cells expressing the inhibitory Fc receptor FcγRIIB. Results indicated that frequencies of FcγRIIB-expressing CD8+ donor-reactive memory T cells inversely correlated with allograft rejection. Furthermore, adoptive T cell transfer of Fcgr2b-/- CD8+ T cells resulted in an accumulation of donor-specific CD8+ memory T cells and enhanced recall responses, indicating that FcγRIIB functions intrinsically to limit T cell CD8+ survival in vivo. Lastly, we show that deletion of FcγRIIB on donor-specific CD8+ memory T cells precipitated costimulation blockade-resistant rejection. These data therefore identify a novel cell-intrinsic inhibitory pathway that functions to limit the risk of memory T cell-mediated rejection following transplantation and suggest that therapeutic manipulation of this pathway could improve outcomes in sensitized patients.

CD28-Dependent CTLA-4 Expression Fine-Tunes the Activation of Human Th17 Cells.

Previous work has demonstrated that Th17 memory cells but not Th1 cells are resistant to CD28/CTLA-4 blockade with CTLA-4 Ig, leading us to investigate the individual roles of the CD28 and CTLA-4 cosignaling pathways on Th1 versus Th17 cells. We found that selective CD28 blockade with a domain antibody (dAb) inhibited Th1 cells but surprisingly augmented Th17 responses. CD28 agonism resulted in a profound increase in CTLA-4 expression in Th17 cells as compared with Th1 cells. Consistent with these findings, inhibition of the CD28 signaling protein AKT revealed that CTLA-4 expression on Th17 cells was more significantly reduced by AKT inhibition relative to CTLA-4 expression on Th17 cells. Finally, we found that FOXO1 and FOXO3 overexpression restrained high expression of CTLA-4 on Th17 cells but not Th1 cells. This study demonstrates that the heterogeneity of the CD4+ T cell compartment has implications for the immunomodulation of pathologic T cell responses.

CD11b is a novel alternate receptor for CD154 during alloimmunity.

Antagonism of the CD154/CD40 pathway is a highly effective means of inducing long-term graft survival in preclinical models. Using a fully allogeneic murine transplant model, we found that CD154 blockade was more effective in prolonging graft survival than was CD40 blockade, raising the possibility that CD154 binds a second receptor. To test this, we queried the impact of CD154 antagonism in the absence of CD40. Data indicated that anti-CD154 functioned to reduce graft-infiltrating CD8+ T cells in both WT and CD40-/- hosts. Because it has recently been reported that CD154 can ligate CD11b, we addressed the impact of blocking CD154-CD11b interactions during transplantation. We utilized a specific peptide antagonist that prevents CD154 binding of CD11b but has no effect on CD154-CD40 interactions. CD154:CD11b antagonism significantly increased the efficacy of anti-CD40 in prolonging allograft survival as compared to anti-CD40 plus control peptide. Mechanistically, CD154:CD11b antagonism functioned to reduce the frequency of graft-infiltrating CD8+ T cells and innate immune cells. These data therefore demonstrate that blocking CD154 interactions with both CD40 and CD11b is required for optimal inhibition of alloimmunity and provide an explanation for why CD40 blockers may be less efficacious than anti-CD154 reagents for the inhibition of allograft rejection.

2B4 Mediates Inhibition of CD8+ T Cell Responses via Attenuation of Glycolysis and Cell Division.

We recently showed that 2B4 expression on memory T cells in human renal transplant recipients was associated with reduced rates of rejection. To investigate whether 2B4 functionally underlies graft acceptance during transplantation, we established an experimental model in which 2B4 was retrogenically expressed on donor-reactive murine CD8+ T cells (2B4rg), which were then transferred into naive recipients prior to skin transplantation. We found that constitutive 2B4 expression resulted in significantly reduced accumulation of donor-reactive CD8+ T cells following transplantation and significantly prolonged graft survival following transplantation. This marked reduction in alloreactivity was due to reduced proliferation of CD8+ Thy1.1+ 2B4rg cells as compared with control cells, underpinned by extracellular flux analyses demonstrating that 2B4-deficient (2B4KO) CD8+ cells activated in vitro exhibited increased glycolytic capacity and upregulation of gene expression profiles consistent with enhanced glycolytic machinery as compared with wild type controls. Furthermore, 2B4KO CD8+ T cells primed in vivo exhibited significantly enhanced ex vivo uptake of a fluorescent glucose analogue. Finally, the proliferative advantage associated with 2B4 deficiency was only observed in the setting of glucose sufficiency; in glucose-poor conditions, 2B4KO CD8+ T cells lost their proliferative advantage. Together, these data indicate that 2B4 signals function to alter T cell glucose metabolism, thereby limiting the proliferation and accumulation of CD8+ T cells. Targeting 2B4 may therefore represent a novel therapeutic strategy to attenuate unwanted CD8+ T cell responses.

Selective CD28 blockade attenuates CTLA-4-dependent CD8+ memory T cell effector function and prolongs graft survival.

Memory T cells pose a significant problem to successful therapeutic control of unwanted immune responses during autoimmunity and transplantation, as they are differentially controlled by cosignaling receptors such as CD28 and CTLA-4. Treatment with abatacept and belatacept impede CD28 signaling by binding to CD80 and CD86, but they also have the unintended consequence of blocking the ligands for CTLA-4, a process that may inadvertently boost effector responses. Here, we show that a potentially novel anti-CD28 domain antibody (dAb) that selectively blocks CD28 but preserves CTLA-4 coinhibition confers improved allograft survival in sensitized recipients as compared with CTLA-4 Ig. However, both CTLA-4 Ig and anti-CD28 dAb similarly and significantly reduced the accumulation of donor-reactive CD8+ memory T cells, demonstrating that regulation of the expansion of CD8+ memory T cell populations is controlled in part by CD28 signals and is not significantly impacted by CTLA-4. In contrast, selective CD28 blockade was superior to CTLA-4 Ig in inhibiting IFN-γ, TNF, and IL-2 production by CD8+ memory T cells, which in turn resulted in reduced recruitment of innate CD11b+ monocytes into allografts. Importantly, this superiority was CTLA-4 dependent, demonstrating that effector function of CD8+ memory T cells is regulated by the balance of CD28 and CTLA-4 signaling.

Age-Associated B Cells Express a Diverse Repertoire of VH and Vκ Genes with Somatic Hypermutation.

The origin and nature of age-associated B cells (ABCs) in mice are poorly understood. In this article, we show that their emergence required MHC class II and CD40/CD40L interactions. Young donor B cells were adoptively transferred into congenic recipients and allowed to remain for 1 mo in the absence of external Ag. B cells expressing the T-bet transcription factor, a marker for ABCs, were generated after multiple cell divisions from C57BL/6 donors but not from MHC class II- or CD40-deficient donors. Furthermore, old CD154 (CD40L)-deficient mice did not accrue ABCs, confirming that they arise primarily through T-dependent interactions. To determine what Igs ABCs express, we sequenced VH and Vκ rearranged genes from unimmunized 22-mo-old C57BL/6 mice and showed that they had a heterogeneous repertoire, which was comparable to that seen in old follicular and marginal zone B cell subsets. However, in contrast to the follicular and marginal zone cells, ABCs displayed significant somatic hypermutation. The mutation frequency was lower than found in germinal center cells after deliberate immunization, suggesting that ABCs have undergone mild stimulation from endogenous Ags over time. These observations show that quiescent ABCs are Ag-experienced cells that accumulate during T cell-dependent responses to diverse Ags during the life of an individual.

Enhanced Requirement for TNFR2 in Graft Rejection Mediated by Low-Affinity Memory CD8+ T Cells during Heterologous Immunity.

The affinity of a TCR binding to peptide:MHC profoundly impacts the phenotype and function of effector and memory cell differentiation. Little is known about the effect of low-affinity priming on memory cell generation and function, which is particularly important in heterologous immunity, when microbe-specific T cells cross-react with allogeneic Ag and mediate graft rejection. We found that low-affinity-primed memory CD8(+) T cells produced high levels of TNF ex vivo in response to heterologous rechallenge compared with high-affinity-primed memory T cells. Low-affinity secondary effectors significantly upregulated TNFR2 on the cell surface and contained a higher frequency of TNFR2(hi) proliferating cells. Low-affinity-primed secondary effectors concurrently downregulated TNF production. Importantly, blockade of TNFR2 attenuated graft rejection in low- but not high-affinity-primed animals. These data establish a functional connection between TNF signaling and TCR-priming affinity and have implications for the immunomodulation of pathogenic T cell responses during transplantation.

Low-Affinity Memory CD8+ T Cells Mediate Robust Heterologous Immunity.

Heterologous immunity is recognized as a significant barrier to transplant tolerance. Whereas it has been established that pathogen-elicited memory T cells can have high or low affinity for cross-reactive allogeneic peptide-MHC, the role of TCR affinity during heterologous immunity has not been explored. We established a model with which to investigate the impact of TCR-priming affinity on memory T cell populations following a graft rechallenge. In contrast to high-affinity priming, low-affinity priming elicited fully differentiated memory T cells with a CD45RB(hi) status. High CD45RB status enabled robust secondary responses in vivo, as demonstrated by faster graft rejection kinetics and greater proliferative responses. CD45RB blockade prolonged graft survival in low affinity-primed mice, but not in high affinity-primed mice. Mechanistically, low affinity-primed memory CD8(+) T cells produced more IL-2 and significantly upregulated IL-2Rα expression during rechallenge. We found that CD45RB(hi) status was also a stable marker of priming affinity within polyclonal CD8(+) T cell populations. Following high-affinity rechallenge, low affinity-primed CD45RB(hi) cells became CD45RB(lo), demonstrating that CD45RB status acts as an affinity-based differentiation switch on CD8(+) T cells. Thus, these data establish a novel mechanism by which CD45 isoforms tune low affinity-primed memory CD8(+) T cells to become potent secondary effectors following heterologous rechallenge. These findings have direct implications for allogeneic heterologous immunity by demonstrating that despite a lower precursor frequency, low-affinity priming is sufficient to generate memory cells that mediate potent secondary responses against a cross-reactive graft challenge.

Retrogenic ICOS Expression Increases Differentiation of KLRG-1hiCD127loCD8+ T Cells during Listeria Infection and Diminishes Recall Responses.

Following T cell encounter with Ag, multiple signals are integrated to collectively induce distinct differentiation programs within Ag-specific CD8(+) T cell populations. Several factors contribute to these cell fate decisions, including the amount and duration of Ag, exposure to inflammatory cytokines, and degree of ligation of cosignaling molecules. The ICOS is not expressed on resting T cells but is rapidly upregulated upon encounter with Ag. However, the impact of ICOS signaling on programmed differentiation is not well understood. In this study, we therefore sought to determine the role of ICOS signaling on CD8(+) T cell programmed differentiation. Through the creation of novel ICOS retrogenic Ag-specific TCR-transgenic CD8(+) T cells, we interrogated the phenotype, functionality, and recall potential of CD8(+) T cells that receive early and sustained ICOS signaling during Ag exposure. Our results reveal that these ICOS signals critically impacted cell fate decisions of Ag-specific CD8(+) T cells, resulting in increased frequencies of KLRG-1(hi)CD127(lo) cells, altered BLIMP-1, T-bet, and eomesodermin expression, and increased cytolytic capacity as compared with empty vector controls. Interestingly, however, ICOS retrogenic CD8(+) T cells also preferentially homed to nonlymphoid organs and exhibited reduced multicytokine functionality and reduced ability to mount secondary recall responses upon challenge in vivo. In sum, our results suggest that an altered differentiation program is induced following early and sustained ICOS expression, resulting in the generation of more cytolyticly potent, terminally differentiated effectors that possess limited capacity for recall response.

Inhibition of Donor-Reactive CD8+ T Cell Responses by Selective CD28 Blockade Is Independent of Reduced ICOS Expression.

Programmed T cell differentiation is critically influenced by the complement of costimulatory and coinhibitory signals transmitted during initial antigen encounter. We previously showed that selective CD28 blockade with novel domain antibodies that leave CTLA-4-mediated coinhibitory signaling intact resulted in more profound attenuation of donor-reactive T cell responses and improved graft survival in a murine transplant model. Selective CD28 blockade was also associated with decreased ICOS expression on donor-reactive CD8+ T cell responses as compared to CTLA-4 Ig, but the functional importance of this reduced ICOS expression was not known. In this study, we created retrogenic donor-reactive CD8+ T cells that overexpress ICOS in order to determine whether reduced ICOS expression mechanistically underlies the increased efficacy of selective CD28 blockade in controlling graft-specific T cell responses as compared to conventional costimulation blockade with CTLA-4 Ig. Results indicated that the ability of selective CD28 blockade to blunt donor-reactive CD8+ T cell expansion following transplantation was independent of its ability to inhibit ICOS expression. Furthermore, we have previously published that 2B4 coinhibitory signals are functionally important for controlling graft-specific CD8+ T cell responses in mice treated with CD28 blockade. Here we used a co-adoptive transfer approach to determine that 2B4 coinhibitory signals on antigen-specific CD8+ T cells function in a cell-intrinsic manner to limit ICOS expression in the setting of selective CD28 blockade.

2B4 (CD244) induced by selective CD28 blockade functionally regulates allograft-specific CD8+ T cell responses.

Mounting evidence in models of both autoimmunity and chronic viral infection suggests that the outcome of T cell activation is critically impacted by the constellation of co-stimulatory and co-inhibitory receptors expressed on the cell surface. Here, we identified a critical role for the co-inhibitory SLAM family member 2B4 (CD244) in attenuating primary antigen-specific CD8(+) T cell responses in the presence of immune modulation with selective CD28 blockade. Our results reveal a specific up-regulation of 2B4 on antigen-specific CD8(+) T cells in animals in which CD28 signaling was blocked. However, 2B4 up-regulation was not observed in animals treated with CTLA-4 Ig (abatacept) or CD28 blockade in the presence of anti-CTLA-4 mAb. 2B4 up-regulation after CD28 blockade was functionally significant, as the inhibitory impact of CD28 blockade was diminished when antigen-specific CD8(+) T cells were deficient in 2B4. In contrast, 2B4 deficiency had no effect on CD8(+) T cell responses during unmodified rejection or in the presence of CTLA-4 Ig. We conclude that blockade of CD28 signals in the presence of preserved CTLA-4 signals results in the unique up-regulation of 2B4 on primary CD8(+) effectors, and that this 2B4 expression plays a critical functional role in controlling antigen-specific CD8(+) T cell responses.

Candida-elicited murine Th17 cells express high Ctla-4 compared with Th1 cells and are resistant to costimulation blockade.

Effector and memory T cells may cross-react with allogeneic Ags to mediate graft rejection. Whereas the costimulation properties of Th1 cells are well studied, relatively little is known about the costimulation requirements of microbe-elicited Th17 cells. The costimulation blocker CTLA-4 Ig has been ineffective in the treatment of several Th17-driven autoimmune diseases and is associated with severe acute rejection following renal transplantation, leading us to investigate whether Th17 cells play a role in CD28/CTLA-4 blockade-resistant alloreactivity. We established an Ag-specific model in which Th1 and Th17 cells were elicited via Mycobacterium tuberculosis and Candida albicans immunization, respectively. C. albicans immunization elicited a higher frequency of Th17 cells and conferred resistance to costimulation blockade following transplantation. Compared with the M. tuberculosis group, C. albicans-elicited Th17 cells contained a higher frequency of IL-17(+)IFN-γ(+) producers and a lower frequency of IL-10(+) and IL-10(+)IL-17(+) cells. Importantly, Th17 cells differentially regulated the CD28/CTLA-4 pathway, expressing similarly high CD28 but significantly greater amounts of CTLA-4 compared with Th1 cells. Ex vivo blockade experiments demonstrated that Th17 cells are more sensitive to CTLA-4 coinhibition and therefore less susceptible to CTLA-4 Ig. These novel insights into the differential regulation of CTLA-4 coinhibition on CD4(+) T cells have implications for the immunomodulation of pathologic T cell responses during transplantation and autoimmunity.

Inhibition of CD8+ T cell-derived CD40 signals is necessary but not sufficient for Foxp3+ induced regulatory T cell generation in vivo.

Current models of CD4(+) T cell help suggest a major role for CD154 binding to CD40 expressed on dendritic cells, with a lesser role for direct T:T interactions via CD40 expressed on CD8(+) T cells. However, the contribution of CD8(+) T cell-derived CD40 signals during the donor-reactive T cell response to a transplant has never been studied. In this study, we examined the graft-rejection kinetics and CD4(+) and CD8(+) donor-reactive T cell responses under conditions in which CD40 was genetically ablated only on APC, as well as under conditions in which CD40 was genetically ablated only on donor-reactive CD8(+) T cells. Our results revealed a significant role for CD8(+) T cell-expressed CD40 in the augmentation of donor-reactive CD8(+) T cell responses following transplantation and showed that CD40 expressed on CD8(+) T cells must be inhibited to allow conversion of CD4(+) T cells into induced regulatory T cells. Thus, this study identifies a major role for CD8(+) T cell-derived CD40 signals as a critical switch factor that both promotes optimal differentiation of cytokine-producing CD8(+) effector T cell responses and inhibits the differentiation of Ag-specific Foxp3(+) induced regulatory T cells in vivo.

CD40/CD154 blockade inhibits dendritic cell expression of inflammatory cytokines but not costimulatory molecules.

Blockade of the CD40/CD154 pathway remains one of the most effective means of promoting graft survival following transplantation. However, the effects of CD40/CD154 antagonism on dendritic cell (DC) phenotype and functionality following transplantation remain incompletely understood. To dissect the effects of CD154/CD40 blockade on DC activation in vivo, we generated hematopoietic chimeras in mice that expressed a surrogate minor Ag (OVA). Adoptive transfer of OVA-specific CD4(+) and CD8(+) T cells led to chimerism rejection, which was inhibited by treatment with CD154 blockade. Surprisingly, CD154 antagonism did not alter the expression of MHC and costimulatory molecules on CD11c(+) DCs compared with untreated controls. However, DCs isolated from anti-CD154-treated animals exhibited a significant reduction in inflammatory cytokine secretion. Combined blockade of inflammatory cytokines IL-6 and IL-12p40 attenuated the expansion of Ag-specific CD4(+) and CD8(+) T cells and transiently inhibited the rejection of OVA-expressing cells. These results suggest that a major effect of CD154 antagonism in vivo is an impairment in the provision of signal three during donor-reactive T cell programming, as opposed to an impact on the provision of signal two. We conclude that therapies designed to target inflammatory cytokines during donor-reactive T cell activation may be beneficial in attenuating these responses and prolonging graft survival.