Quiescence of Memory CD8+ T Cells Is Mediated by Regulatory T Cells through Inhibitory Receptor CTLA-4
ABSTRACT: Immune memory cells are poised to rapidly expand and elaborate effector functions upon reinfection. However, despite heightened readiness to respond, memory cells exist in a functionally quiescent state. The paradigm is that memory cells remain inactive due to lack of TCR stimuli. Here we report a unique role of Tregs in orchestrating memory quiescence by inhibiting effector and proliferation programs through CTLA-4. Loss of Tregs resulted in activation of genome-wide transcriptional programs characteristic of potent effectors, and both developing and established memory quickly reverted to a terminally differentiated (KLRG-1hi/IL-7R±lo/GzmBhi) phenotype, with compromised metabolic fitness, longevity, polyfunctionality and protective efficacy. CTLA-4, an inhibitory receptor overexpressed on Tregs, functionally replaced Tregs in trans to rescue Treg-less memory defects and restore homeostasis of secondary mediators as well. These studies present CD28-CTLA-4-CD80/CD86 axis as a novel target to potentially accelerate vaccine-induced immunity and improve T-cell memory quality in current cancer immunotherapies proposing transient Treg-depletion. We used microarray analysis to detail the global programming of gene expression in LCMV GP33-specific CD8 T cells differentiated in the presence or absence of regulatory T cells Differentiation of memory CD8 T cells entails a progressive transition of highly activated effector program to a quiescent memory program. A key question in the field is to understand the factors that aid in the differentiation of memory cells from effector cells. It is a generally accepted paradigm that effector cells transition to a memory state by default after antigen clearance, since TCR stimuli is the key driver of effector programs in CD8 T cells. We hypothesized that the effector to memory transition of CD8 T cells involves active immunological brakes through regulatory T cells (Tregs) that allow the highly activated effector cells to convert into quiescent memory cells. To address this hypothesis, we used FoxP3-DTR mice to deplete Tregs during the window following antigen clearance, during which the effector CD8 T cells convert to long-lived memory cells. To get a deeper understanding of the global transcriptome of CD8 T cells as they transition from an effector to a memory state, we isolated and arrayed the antigen-specific CD8 T cells at day 16 post-infection that have experienced the transitional environment with and without the presence of Tregs.
Project description:During acute viral infections, naïve CD8+ T cells differentiate into effector CD8+ T cells and, after viral control, into memory CD8+ T cells. Memory CD8+ T cells are highly functional, proliferate rapidly upon reinfection and persist long-term without antigen. In contrast, during chronic infections, CD8+ T cells become “exhausted” and have poor effector function, express multiple inhibitory receptors, possess low proliferative capacity, and cannot persist without antigen. To compare the development of functional memory T cells with poorly functional exhausted T cells, we generated longitudinal transcriptional profiles for each. Naive CD44Lo CD8+ T cells were isolated and sorted from uninfected C57BL/6 mice and H2-Db GP33-specific CD8+ T cells were sorted using MHC-I tetramers at d6, 8, 15, and 30 p.i. with either LCMV Arm or LCMV clone 13. RNA from these CD8+ T cells was processed, amplified, labeled, and hybridized to Affymetrix GeneChip MoGene 1.0 st microarrays
Project description:During acute viral infections, naïve CD8+ T cells differentiate into effector CD8+ T cells and, after viral control, into memory CD8+ T cells. Memory CD8+ T cells are highly functional, proliferate rapidly upon reinfection and persist long-term without antigen. In contrast, during chronic infections, CD8+ T cells become “exhausted” and have poor effector function, express multiple inhibitory receptors, possess low proliferative capacity, and cannot persist without antigen. Exhuasted CD8+ T cells can be further segregated by their expression of the inhibitory cell surface receptor PD-1. We performed transcriptional profiling on both PD-1 High and PD-1 Intermediate H2-Db GP33-specific CD8+ T cells. H2-Db GP33-specific CD8+ T cells were sorted from C57BL/6 mice 30 days p.i. with LCMV clone 13. These cells were then segregated by their expression of the inhibitory cell surface receptor PD-1 into PD-1 High and PD-1 Intermediate subpopulations. We performed transcriptional profiling on these subpopulations.
Project description:Regulatory T cells play important roles in cancer development and progression by limiting the generation of innate and adaptive anti-tumor immunity. We hypothesized that in addition to natural CD4(+)CD25(+) regulatory T cells (Tregs) and myeloid-derived suppressor cells, tumor Ag-specific Tregs interfere with the detection of anti-tumor immunity after immunotherapy. Using samples from prostate cancer patients immunized with a DNA vaccine encoding prostatic acid phosphatase (PAP) and a trans-vivo delayed-type hypersensitivity (tvDTH) assay, we found that the detection of PAP-specific effector responses after immunization was prevented by the activity of PAP-specific regulatory cells. These regulatory cells were CD8(+)CTLA-4(+), and their suppression was relieved by blockade of CTLA-4, but not IL-10 or TGF-?. Moreover, Ag-specific CD8(+) Tregs were detected prior to immunization in the absence of PAP-specific effector responses. These PAP-specific CD8(+)CTLA-4(+) suppressor T cells expressed IL-35, which was decreased after blockade of CTLA-4, and inhibition of either CTLA-4 or IL-35 reversed PAP-specific suppression of tvDTH response. PAP-specific CD8(+)CTLA-4(+) T cells also suppressed T cell proliferation in an IL-35-dependent, contact-independent fashion. Taken together, these findings suggest a novel population of CD8(+)CTLA-4(+) IL-35-secreting tumor Ag-specific Tregs arise spontaneously in some prostate cancer patients, persist during immunization, and can prevent the detection of Ag-specific effector responses by an IL-35-dependent mechanism.
Project description:Tumor progression is facilitated by regulatory T cells (Treg) and restricted by effector T cells. In this study, we document parallel regulation of CD8(+) T cells and Foxp3(+) Tregs by programmed death-1 (PD-1, PDCD1). In addition, we identify an additional role of CTL antigen-4 (CTLA-4) inhibitory receptor in further promoting dysfunction of CD8(+) T effector cells in tumor models (CT26 colon carcinoma and ID8-VEGF ovarian carcinoma). Two thirds of CD8(+) tumor-infiltrating lymphocytes (TIL) expressed PD-1, whereas one third to half of CD8(+) TIL coexpressed PD-1 and CTLA-4. Double-positive (PD-1(+)CTLA-4(+)) CD8(+) TIL had characteristics of more severe dysfunction than single-positive (PD-1(+) or CTLA-4(+)) TIL, including an inability to proliferate and secrete effector cytokines. Blockade of both PD-1 and CTLA-4 resulted in reversal of CD8(+) TIL dysfunction and led to tumor rejection in two thirds of mice. Double blockade was associated with increased proliferation of antigen-specific effector CD8(+) and CD4(+) T cells, antigen-specific cytokine release, inhibition of suppressive functions of Tregs, and upregulation of key signaling molecules critical for T-cell function. When used in combination with GVAX vaccination (consisting of granulocyte macrophage colony-stimulating factor-expressing irradiated tumor cells), inhibitory pathway blockade induced rejection of CT26 tumors in 100% of mice and ID8-VEGF tumors in 75% of mice. Our study indicates that PD-1 signaling in tumors is required for both suppressing effector T cells and maintaining tumor Tregs, and that PD-1/PD-L1 pathway (CD274) blockade augments tumor inhibition by increasing effector T-cell activity, thereby attenuating Treg suppression.
Project description:Acquisition of effector properties is a key step in the generation of cytotoxic T lymphocytes (CTLs). Here we show that inflammatory signals regulate Dicer expression in CTL, and that deletion or depletion of Dicer in mouse or human activated CD8+ T cells causes upregulation of perforin, granzyme and effector cytokines. Genome-wide analysis of miRNA changes induced by exposure of differentiating CTLs to IL-2 and inflammatory signals identifies miR-139 and miR-150 as components of a miRNA network that controls perforin, eomesodermin (Eomes) and IL-2Ra expression in differentiating CTLs and whose activity is modulated by IL-2, inflammation and antigenic stimulation. Overall our data show that strong IL-2R and inflammatory signals act through Dicer and miRNAs to control the cytolytic program and other aspects of effector CTL differentiation. Comparison of control and Dicer knock-out CTLs differentiated in vitro; Comparison of wild type CTLs differentiated in vitro with or without inflammatory stimuli; Comparison of effector and memory precursor CTLs isolated from mice infected with LCMV-Armstrong
Project description:CTLA-4, an Ig superfamily molecule with homology to CD28, is one of the most potent negative regulators of T-cell responses. In vivo blockade of CTLA-4 exacerbates autoimmunity, enhances tumor-specific T-cell responses, and may inhibit the induction of T-cell anergy. Clinical trials of CTLA-4-blocking antibodies to augment T-cell responses to malignant melanoma are at an advanced stage; however, little is known about the effects of CTLA-4 blockade on memory CD8(+) T-cell responses and the formation and maintenance of long-term CD8(+) T-cell memory. In our studies, we show that during in vivo memory CD8(+) T-cell responses to Listeria monocytogenes infection, CTLA-4 blockade enhances bacterial clearance and increases memory CD8(+) T-cell expansion. This is followed by an accumulation of memory cells that are capable of producing the effector cytokines IFN-? and TNF-?. We also demonstrate that in a vaccination setting, blocking CTLA-4 during CD8(+) T-cell priming leads to increased expansion and maintenance of antigen-specific memory CD8(+) T cells without adversely affecting the overall T-cell repertoire. This leads to an increase in memory cell effector function and improved protective immunity against further bacterial challenges. These results indicate that transient blockade of CTLA-4 enhances memory CD8(+) T-cell responses and support the possible use of CTLA-4-blocking antibodies during vaccination to augment memory formation and maintenance.
Project description: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-?, 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.
Project description:The ability of some tumors to exclude effector T cells represents a major challenge to immunotherapy. T cell exclusion is particularly evident in pancreatic ductal adenocarcinoma (PDAC), a disease where blockade of the immune checkpoint molecule CTLA-4 has not produced significant clinical activity. In PDAC, effector T cells are often scarce within tumor tissue and confined to peritumoral lymph nodes and lymphoid aggregates. We hypothesized that CTLA-4 blockade, despite a lack of clinical efficacy seen thus far in PDAC, might still alter T cell immunobiology, which would have therapeutic implications. Using clinically relevant genetic models of PDAC, we found that regulatory T cells (Tregs), which constitutively express CTLA-4, accumulate early during tumor development but are largely confined to peritumoral lymph nodes during disease progression. Tregs were observed to regulate CD4+, but not CD8+, T cell infiltration into tumors through a CTLA-4/CD80 dependent mechanism. Disrupting CTLA-4 interaction with CD80 was sufficient to induce CD4 T cell infiltration into tumors. These data have important implications for T cell immunotherapy in PDAC and demonstrate a novel role for CTLA-4/CD80 interactions in regulating T cell exclusion. In addition, our findings suggest distinct mechanisms govern CD4+ and CD8+ T cell infiltration in PDAC.
Project description:In addition to CD4(+) regulatory T cells (Tregs), CD8(+) suppressor T cells are emerging as an important subset of regulatory T cells. Diverse populations of CD8(+) T cells with suppressive activities have been described. Among them, a small population of CD8(+)CD25(+)FOXP3(+) T cells is found both in mice and humans. In contrast to thymic-derived CD4(+)CD25(+)FOXP3(+) Tregs, their origin and their role in the pathophysiology of autoimmune diseases (AIDs) are less understood. We report here the number, phenotype, and function of CD8(+) Tregs cells in mice and humans, at the steady state and in response to low-dose interleukin-2 (IL-2). CD8(+) Tregs represent approximately 0.4 and 0.1% of peripheral blood T cells in healthy humans and mice, respectively. In mice, their frequencies are quite similar in lymph nodes (LNs) and the spleen, but two to threefold higher in Peyer patches and mesenteric LNs. CD8(+) Tregs express low levels of CD127. CD8(+) Tregs express more activation or proliferation markers such as CTLA-4, ICOS, and Ki-67 than other CD8(+) T cells. In vitro, they suppress effector T cell proliferation as well as or even better than CD4(+) Tregs. Owing to constitutive expression of CD25, CD8(+) Tregs are 20- to 40-fold more sensitive to in vitro IL-2 stimulation than CD8(+) effector T cells, but 2-4 times less than CD4(+) Tregs. Nevertheless, low-dose IL-2 dramatically expands and activates CD8(+) Tregs even more than CD4(+) Tregs, in mice and humans. Further studies are warranted to fully appreciate the clinical relevance of CD8(+) Tregs in AIDs and the efficacy of IL-2 treatment.
Project description:HLA-B*35Px is associated with HIV-1 disease rapid progression to AIDS. However, the mechanism(s) underlying this deleterious effect of this HLA allele on HIV-1 infection outcome has not fully understood. CD8+ T cells play a crucial role to control the viral replication but impaired CD8+ T cells represent a major hallmark of HIV-1 infection. Here, we examined the effector functions of CD8+ T cells restricted by HLA-B*35Px (HLA-B*35:03 and HLA-B*35:02), HLA-B*27/B57 and non-HLA-B*27/B57 (e.g. HLA-A*01, A*02, A*03, A*11, A*24, A*26, B*40, B*08, B*38, B*44). CD8+ T cells restricted by HLA-B*35Px exhibited an impaired phenotype compared with those restricted by HLA-B*27/B57 and even non-HLA-B*27/B57. CD8+ T cells restricted by non-HLA-B*27/B57 when encountered their cognate epitopes upregulated TIM-3 and thus became suppressed by regulatory T cells (Tregs) via TIM-3: Galectin-9 (Gal-9). Strikingly, CD8+ T cells restricted by HLA-B*35Px expressed fewer TIM-3 and therefore did not get suppressed by Tregs, which was similar to CD8+ T cells restricted by HLA-B*27/B57. Instead, CD8+ T cells restricted by HLA-B*35Px upon recognition of their cognate epitopes upregulated CTLA-4. The transcriptional and impaired phenotype (e.g. poor effector functions) of HIV-specific CD8+ T cells restricted by HLA-B*35 was related to persistent CTLA-4, elevated Eomes and blimp-1 but poor T-bet expression. As such, anti-CTLA-4 antibody, Ipilimumab, reversed the impaired proliferative capacity of antigen-specific CD8+ T cells restricted by HLA-B*35Px but not others. This study supports the concept that CD8+ T resistance to Tregs-mediated suppression is related to allele restriction rather than the epitope specificity. Our results aid to explain a novel mechanism for the inability of HIV-specific CD8+ T cells restricted by HLA-B*35Px to control viral replication.