Tolerance Of CD8 Research Articles

CD8+ T cell tolerance: It doesn’t translate

The mechanisms that make and break CD8+ Tcell tolerance to self-antigens remain unclear. In this issue of Immunity, Van Der Byl etal. show that tolerant CD8+ Tcells rapidly adopt an epigenetically and transcriptionally distinct cell state and exhibit impaired protein translation. Breaking tolerance requires both inflammation and increased antigen exposure to augment MYC expression and restore translation.

Tumor-associated CD8+T cell tolerance induced by erythroid progenitor cells.

CD8+T cell tolerance plays an important role in tumor escape. Recent studies have shown that CD45+ erythroid progenitor cells (CD45+EPCs) generated through splenic extramedullary erythropoiesis suppress tumor immunity. However, the mechanism underlying how CD45+EPCs mediate CD8+T cell tolerance remains incompletely understood and requires further research. In this study, the antigen-processing abilities of CD45+EPCs was verified through both in vitro and in vivo experiments. We have used the method of co-culture in vitro and adoptive transfer experiments in vivo to explore the effects of CD45+EPCs on CD8+T cell tolerance. RNA-sequencing analysis and blocking experiments were used to evaluate the role of ROS in the CD45+EPC mediated tolerance of CD8+T cells. Finally, we incorporated uric acid into the adoptive transfer experiments to rescue the CD45+EPC mediated tumor-promoting effect. We found that CD45+EPCs take up soluble proteins, present antigenic epitopes on their surface, and induce antigen-specific CD8+T cell anergy. In addition, we found that CD45+EPC directly nitrates tyrosine within the TCR/CD8 complex via the production of reactive oxygen species and peroxynitrite, preventing CD8+ T cells from responding to their specific peptide antigens. Furthermore, uric acid treatment effectively abolished the immunosuppressive effects of CD45+EPCs during CD8+T cell adoptive transfer, thereby enhancing the anti-tumor efficacy. These results demonstrated that CD8+T cell tolerance in tumor-bearing mice is induced by CD45+EPCs. The results of this study have direct implications for tumor immunotherapy.

A multiomics analysis-assisted deep learning model identifies a macrophage-oriented module as a potential therapeutic target in colorectal cancer

Colorectal cancer (CRC) is a common malignancy involving multiple cellular components. The CRC tumor microenvironment (TME) has been characterized well at single-cell resolution. However, a spatial interaction map of the CRC TME is still elusive. Here, we integrate multiomics analyses and establish a spatial interaction map to improve the prognosis, prediction, and therapeutic development for CRC. We construct a CRC immune module (CCIM) that comprises FOLR2+ macrophages, exhausted CD8+ Tcells, tolerant CD8+ Tcells, exhausted CD4+ Tcells, and regulatory Tcells. Multiplex immunohistochemistry is performed to depict the CCIM. Based on this, we utilize advanced deep learning technology to establish a spatial interaction map and predict chemotherapy response. CCIM-Net is constructed, which demonstrates good predictive performance for chemotherapy response in both the training and testing cohorts. Lastly, targeting FOLR2+ macrophage therapeutics is used to disrupt the immunosuppressive CCIM and enhance the chemotherapy response invivo.

Novel PD-L1/BTLA Checkpoint Axis Exploited for Bacterial Immune Escape by Restraining CD8+ T Cell-Initiated Adaptive Immunity in Zebrafish.

Programmed death-ligand 1/programmed cell death 1 (PD-L1/PD-1) is one of the most important immune checkpoints in humans and other mammalian species. However, the occurrence of the PD-L1/PD-1 checkpoint in evolutionarily ancient vertebrates remains elusive because of the absence of a PD-1 homolog before its appearance in tetrapods. In this article, we identified, to our knowledge, a novel PD-L1/B and T lymphocyte attenuator (BTLA) checkpoint in zebrafish by using an Edwardsiella tarda-induced bacterial infection model. Results showed that zebrafish (Danio rerio) PD-L1 (DrPD-L1) and BTLA (DrBTLA) were differentially upregulated on MHC class II+ macrophages (Mϕs) and CD8+ T cells in response to E. tarda infection. DrPD-L1 has a strong ability to interact with DrBTLA, as shown by the high affinity (KD = 5.68 nM) between DrPD-L1/DrBTLA proteins. Functionally, the breakdown of DrPD-L1/DrBTLA interaction significantly increased the cytotoxicity of CD8+BTLA+ T cells to E. tarda-infected PD-L1+ Mϕ cells and reduced the immune escape of E. tarda from the target Mϕ cells, thereby enhancing the antibacterial immunity of zebrafish against E. tarda infection. Similarly, the engagement of DrPD-L1 by soluble DrBTLA protein diminished the tolerization of CD8+ T cells to E. tarda infection. By contrast, DrBTLA engagement by a soluble DrPD-L1 protein drives aberrant CD8+ T cell responses. These results were finally corroborated in a DrPD-L1-deficient (PD-L1-/-) zebrafish model. This study highlighted a primordial PD-L1/BTLA coinhibitory axis that regulates CD8+ T cell activation in teleost fish and may act as an alternative to the PD-L1/PD-1 axis in mammals. It also revealed a previously unrecognized strategy for E. tarda immune evasion by inducing CD8+ T cell tolerance to target Mϕ cells through eliciting the PD-L1/BTLA checkpoint pathway.

The effect of tolerogenic PLG nanoparticles on transgenic CD8+ T cells

Abstract Antigen-loaded, biodegradable PLG nanoparticles have demonstrated tolerance induction in models of autoimmune disease. Here we report on the effect of these particles on transgenic CD8+ T cells. Wild-type mice received transgenic cells, were treated with nanoparticles, and then had their spleens and/or pancreases removed for flow cytometric analysis. Relative to controls, mice treated with these particles exhibited a larger percentage of transgenic CD8+ T cells in spleens, suggesting that a sequestration of autoreactive T cells prevents their trafficking to inflamed tissues. Furthermore, nanoparticle treatment led to a higher percentage of cells with surface markers indicative of a regulatory phenotype. These results imply that altered trafficking and an expansion of regulatory T cells are two mechanisms by which PLG nanoparticles induce tolerance in CD8+ T cell - mediated autoimmune disease. Supported by a grant from the JDRF

Abstract 1376: Dysfunctional and tolerant CD8 T cell persistence in premalignant lesions

Abstract T cells have the potential to carry out potent anti-tumor responses. Most studies examining T cell-cancer interactions have been directed toward late-stage tumors, but much less is known about how T cells respond to premalignant lesions. Using a clinically relevant liver cancer mouse model (Albumin-Stop-Large T Antigen; AST) in which the SV40 Large T Antigen (TAG) serves as oncogene and tumor neoantigen, we previously demonstrated that TAG-specific CD8 T cells (TCR-TAG) rapidly become dysfunctional (unable to produce cytokines and cytolytic molecules) after entering premalignant liver lesions, although they persist long-term. We hypothesized that peripheral tolerance mechanisms, which prevent autoimmunity against self-antigens, drive T cell dysfunction in premalignant lesions. To test this, we compared CD8 T cell (TCR-GAG) responses to a liver self-antigen (GAG) to TCR-TAG responses to the TAG neoantigen in premalignant hepatocytes. We found that liver-reactive TCR-GAG were unable to produce cytokines, similar to neoantigen-specific TCR-TAG; however, in contrast, they failed to persist. To test whether oncogenic transformation of hepatocytes promoted T cell persistence, we crossed Alb-GAG and AST mice to generate a model in which GAG and TAG was expressed in premalignant hepatocytes (ASTxAlb-GAG). We found that both TCR-GAG and TCR-TAG transferred into ASTxAlb-GAG mice bearing premalignant lesions driven by the TAG oncogene rapidly lost the ability to produce cytokines; however, only TCR-TAG persisted while TCR-GAG quickly disappeared. To understand why the TCR-GAG failed to persist, we assessed proliferation and cell cycling after T cells encountered their cognate antigen. While both TCR-GAG and TCR-TAG were rapidly activated and began proliferating, TCR-GAG exited cell cycle and began decreasing in number after four cell divisions, while TCR-TAG continued to divide and increase in number. Antigen amount and localization may regulate T cell proliferation and persistence. We found much higher levels of TAG-encoding versus GAG-encoding mRNA in the liver, suggesting that higher antigen expression may be required for T cell persistence. T cell responses can be directed against both shared tumor self-antigens and neoantigens. By dissecting the determinants of CD8 T cell function and persistence in response to self and non-self-antigens, these studies could inform strategies to boost anti-tumor CD8 T cell responses in cancer patients. Citation Format: Jessica J. Roetman, Megan M. Erwin, Minna K. Apostolova, Mary Philip. Dysfunctional and tolerant CD8 T cell persistence in premalignant lesions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1376.

CD4+ CTLs Act as a Key Effector Population for Allograft Rejection of MSCs in a Donor MHC-II Dependent Manner in Injured Liver.

Mesenchymal stromal/stem cells (MSCs) have been considered an attractive source of cytotherapy due to their promising effects on treating various diseases. Allogeneic MSCs (allo-MSCs) are extensively used in clinical trials due to their convenient preparation and credible performance. Traditionally, allo-MSCs are considered immunoprivileged with minimal immunogenicity and potent immunomodulatory capacity. However, growing evidence has suggested that allo-MSCs also induce immune response and cause rejection after transplantation, but the underlying cellular and molecular mechanisms remain to be elucidated. Here, we demonstrated that allografted MSCs upregulated MHC-II upon stimulation of IFN-γ in hepatic inflammatory environment by using mouse model of CCl4-induced liver injury. MHC-II upregulation enhanced the immunogenicity of allo-MSCs, leading to the activation of alloreactive T cells and rejection of allo-MSCs. However, MHC-II deficiency impaired the allogenic reactivity, thereby rescuing the loss of allo-MSCs. Mechanistically, CD4+ cytotoxic T lymphocytes (CTLs), rather than CD8+ CTLs, acted as the major effector for allo-MSC rejection. Under liver injury condition, the transplanted allo-MSCs upregulated CD80 and PD-L1, and CD8+ CTLs highly expressed CTLA-4 and PD-1, thereby inducing immune tolerance of CD8+ T cells to allo-MSCs. On the contrary, CD4+ CTLs minimally expressed CTLA-4 and PD-1; thus, they remain cytotoxic to allo-MSCs. Consequently, transplantation of MHC-II-deficient allo-MSCs substantially promoted their therapeutic effects in treating liver injury. This study revealed a novel mechanism of MSC allograft rejection mediated by CD4+ CTLs in injured liver, which provided new strategies for improving clinical performance of allo-MSCs in benefiting hepatic injury repair.

The PD-1 Checkpoint Receptor Maintains Tolerance of Self-Reactive CD8 T Cell in Skin

The PD-1 Checkpoint Receptor Maintains Tolerance of Self-Reactive CD8 T Cell in Skin

Inhibition of autoimmune enteritis in IFABP-Ova mice by LCMV infection

Abstract Epidemiological data indicate that infections play a role in preventing autoimmunity. Thus, the decline in the incidences of infectious diseases and the increase in the frequency of autoimmune diseases also known as the hygiene hypothesis suggest that there might be a link between the two phenomena. In this study using the iFABP-Ova transgenic mouse model, that expresses ovalbumin in the small intestine, we showed that prior LCMV infection dampened self-reactive effector CD8 T cell responses and protected mice from enteric autoimmunity. LCMV specific memory CD8 T cells, generated after infection, were not required for this protection as elimination of these cells using a depleting antibody still protected the mice from enteric autoimmunity. Moreover, we found that infection induced suppression is specific to self antigen reactive CD8 T cells without affecting foreign antigen specific CD8 T cell responses. Lastly, we found that a similar suppression of self-reactive CD8 T cells occurs in iFABP-Ova mice when tolerant CD8 T cells are previously present. Thus there might be some similarities in the mechanism of suppression of self-reactive CD8 T cells by viral infections and tolerance generation. Taken together these data demonstrate that prior viral infections or tolerant CD8 T cells have the ability to suppress self-reactive autoimmune responses and provide protective effects without compromising immunity to foreign pathogens.

Inventories of naive and tolerant mouse CD4 T cell repertoires reveal a hierarchy of deleted and diverted T cell receptors

Deletion or Treg cell differentiation are alternative fates of autoreactive MHCII-restricted thymocytes. How these different modes of tolerance determine the size and composition of polyclonal cohorts of autoreactive T cells with shared specificity is poorly understood. We addressed how tolerance to a naturally expressed autoantigen of the central nervous system shapes the CD4 T cell repertoire. Specific cells in the tolerant peripheral repertoire either were Foxp3+ or displayed anergy hallmarks and, surprisingly, were at least as frequent as in the nontolerant repertoire. Despite this apparent lack of deletional tolerance, repertoire inventories uncovered that some T cell receptors (TCRs) were lost from the CD4 T cell pool, whereas others mediated Treg cell differentiation. The antigen responsiveness of these TCRs supported an affinity model of central tolerance. Importantly, the contribution of different diverter TCRs to the nascent thymic Treg cell population reflected their antigen reactivity rather than their frequency among precursors. This reveals a multilayered TCR hierarchy in CD4 T cell tolerance that separates deleted and diverted TCRs and assures that the Treg cell compartment is filled with cells of maximal permissive antigen reactivity.

Reprogramming responsiveness to checkpoint blockade in dysfunctional CD8 T cells

Established T cell dysfunction is a barrier to antitumor responses, and checkpoint blockade presumably reverses this. Many patients fail to respond to treatment and/or develop autoimmune adverse events. The underlying reason for T cell responsiveness remains elusive. Here, we show that susceptibility to checkpoint blockade is dependent on the activation status of T cells. Newly activated self-specific CD8 T cells respond to checkpoint blockade and cause autoimmunity, which is mitigated by inhibiting the mechanistic target of rapamycin. However, once tolerance is established, self-specific CD8 T cells display a gene signature comparable to tumor-specific CD8 T cells in a fixed state of dysfunction. Tolerant self-specific CD8 T cells do not respond to single or combinatorial dosing of anti-CTLA4, anti-PD-L1, anti-PD-1, anti-LAG-3, and/or anti-TIM-3. Despite this, T cell responsiveness can be induced by vaccination with cognate antigen, which alters the previously fixed transcriptional signature and increases antigen-sensing machinery. Antigenic reeducation of tolerant T cells synergizes with checkpoint blockade to generate functional CD8 T cells, which eliminate tumors without concomitant autoimmunity and are transcriptionally distinct from classic effector T cells. These data demonstrate that responses to checkpoint blockade are dependent on the activation state of a T cell and show that checkpoint blockade-insensitive CD8 T cells can be induced to respond to checkpoint blockade with robust antigenic stimulation to participate in tumor control.

Forced expression of IL-7R promotes CD8 T cell cytotoxicity to self antigen.

Cross-presentation of apoptotic cell associated antigens by immature dendritic cells prevents the activation of self reactive CD8 T cells. Tolerized self reactive CD8 T cells down-regulate IL-7R expression on their surface. Whether over-expression of IL-7R can reverse their fate and function has not been examined. In this paper, we showed forced expression of IL-7R in OT-I T cells by a transgene enhanced CD8 T cell mediated diabetes in the RIP-mOVA model. Although IL-7R Tg (transgenic) did not completely reverse the deletion of OT-I T cells, it provided a significant survival advantage over w.t OT-I T cells. Furthermore, IL7R Tg OT-I T cells isolated from diabetic pancreata displayed increased production of IFN-γ, higher expression of T-bet, and increased externalization of CD107a. We also found that immature DCs containing apoptotic cells expressed high levels of PDL-1 on their surface. Although IL-7R Tg did not change PD1 expression on activated OT-I cells in vivo, the transgene enabled a significantly lower number of OT-I T cells to induce diabetes in the absence of PDL-1. Our results demonstrated that forced expression of IL-7R not only improved the functionality of tolerized CD8 T cells, it also acted in synergy with PDL-1 deficiency to further promote CD8 T cell cytotoxicity to self antigens.

P32: A NOVEL IMMUNOTHERAPEUTIC VACCINE TO MODULATE CYTOKINE RESPONSES OF ANTIGEN‐SPECIFIC CD4+ T CELLS IN PEANUT ALLERGY

Peanut allergy, a potentially life-threatening condition, has emerged as a major public health concern in developed countries. There are currently no approved treatment options, with dietary avoidance being the only recommended strategy for prevention of fatal anaphylactic reactions. We propose the use of a novel, proprietary Sementis Copenhagen Vector (SCV)-based peanut hypoallergenic vaccine (SCV-PHAV) to induce sustained unresponsiveness and immune tolerance to peanuts, an objective that eludes current immunotherapy modalities in allergy. We established an ex vivo human dendritic cells (DCs): CD4+ T cell culture system to investigate the capacity of SCV-PHAV instructed dendritic cells (DCs) to facilitate the production of a tolerant Th1-biased peanut-specific CD4+ T cell response. Briefly, monocyte-derived DCs and CD4+ T cells were isolated from the blood of peanut allergic patients. Peanut-pulsed and SCV-PHAV vaccinated DCs were incubated with CD4+ T cells for 2 weeks. At the end of the incubation period, cultures were labelled with a proliferation dye, re-stimulated with peanut extract and the cytokine profiles of proliferating antigen-specific T cells was examined by flow cytometry. A significantly higher Th1/Th2 ratio was detected in SCV-PHAV vaccinated cultures compared to peanut-pulsed cultures (n = 5). A consistent increase was observed in separate ex vivo DC:CD4 T cultures set up 6-months apart from the same patient, confirming both assay reproducibility and vaccine efficacy. In addition, we were able to demonstrate an antigen-specific Th1 bias following long-term cultures (5–6 weeks) in the presence of peanut antigens. The SCV-PHAV vaccine can modulate the cytokine signatures of peanut-reactive CD4+ T cells from an allergic, Th2-biased to a tolerant, Th1-biased profile.

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. III. Interleukin-5 (IL-5) promotes survival of alloantigen-specific CD4+ T regulatory cells

CD4+T cells mediate antigen-specific allograft tolerance, but die in culture without activated lymphocyte derived cytokines. Supplementation of the media with cytokine rich supernatant, from ConA activated spleen cells, preserves the capacity of tolerant cells to transfer tolerance and suppress rejection. rIL-2 or rIL-4 alone are insufficient to maintain these cells, however. We observed that activation of naïve CD4+CD25+FOXP3+Treg with alloantigen and the Th2 cytokine rIL-4 induces them to express interleukin-5 specific receptor alpha (IL-5Rα) suggesting that IL-5, a Th2 cytokine that is produced later in the immune response may promote tolerance mediating Treg.This study examined if recombinant IL-5(rIL-5) promoted survival of tolerant CD4+, especially CD4+CD25+T cells. CD4+T cells, from DA rats tolerant to fully allogeneic PVG heart allografts surviving over 100days without on-going immunosuppression, were cultured with PVG alloantigen and rIL-5. The ability of these cells to adoptively transfer tolerance to specific-donor allograft and suppress normal CD4+T cell mediated rejection in adoptive DA hosts was examined. Tolerant CD4+CD25+T cells' response to rIL-5 and expression of IL-5Rα was also assessed.rIL-5 was sufficient to promote transplant tolerance mediating CD4+T cells' survival in culture with specific-donor alloantigen. Tolerant CD4+T cells cultured with rIL-5 retained the capacity to transfer alloantigen-specific tolerance and inhibited naïve CD4+T cells' capacity to effect specific-donor graft rejection. rIL-5 promoted tolerant CD4+CD25+T cells' proliferation in vitro when stimulated with specific-donor but not third-party stimulator cells. Tolerant CD4+CD25+T cells expressed IL-5Rα.This study demonstrated that IL-5 promoted the survival of alloantigen-specific CD4+CD25+T cells that mediate transplant tolerance.

Cytokines affecting CD4+ T regulatory cells in transplant tolerance. II. Interferon gamma (IFN-γ) promotes survival of alloantigen-specific CD4+ T regulatory cells

CD4+T cells that transfer alloantigen-specific transplant tolerance are short lived in culture unless stimulated with specific-donor alloantigen and lymphocyte derived cytokines. Here, we examined if IFN-γ maintained survival of tolerance transferring CD4+T cells.Alloantigen-specific transplant tolerance was induced in DA rats with heterotopic adult PVG heart allografts by a short course of immunosuppression and these grafts functioned for >100days with no further immunosuppression. In previous studies, we found the CD4+T cells from tolerant rats that transfer tolerance to an irradiated DA host grafted with a PVG heart, lose their tolerance transferring ability after 3days of culture, either with or without donor alloantigen, and effect rejection of specific-donor grafts. If cultures with specific-donor alloantigen are supplemented by supernatant from ConA activated lymphocytes the tolerance transferring cells survive, suggesting these cells depend on cytokines for their survival. In this study, we found addition of rIFN-γ to MLC with specific-donor alloantigen maintained the capacity of tolerant CD4+T cells to transfer alloantigen-specific tolerance and their ability to suppress PVG allograft rejection mediated by co-administered naïve CD4+T cells. IFN-γ suppressed the in vitro proliferation of tolerant CD4+T cells. Tolerant CD4+CD25+T cells did not proliferate in MLC to PVG stimulator cells with no cytokine added, but did when IFN-γ was present. IFN-γ did not alter proliferation of tolerant CD4+CD25+T cells to third-party Lewis. Tolerant CD4+CD25+T cells' expression of IFN-γ receptor (IFNGR) was maintained in culture when IFN-γ was present. This study suggested that IFN-γ maintained tolerance mediating alloantigen-specific CD4+CD25+T cells.

Utilizing self-specific CD8 T cells for the treatment of cancer

Abstract CD8 T cells are potent effectors that monitor the body for infections as well as malignancies. CD8 T cell infiltration into tumors is correlated with better prognoses in many cancers. However, the tumor microenvironment is often toleragenic, which treatments like checkpoint inhibitors seek to overcome. Such treatments have been met with some success, but their benefits remain elusive for a subset of patients. Using a strict model of tolerance to intestinal self-antigen, we show that newly activated CD8 T cells respond best to checkpoint blockade therapy, while CD8 T cells in a toleragenic environment, even for a short time, do not respond. We have developed a therapeutic vaccination strategy that generates a targeted CD8 T cell response with renewed susceptibility to checkpoint blockade. Using heterologous prime boost vaccination model, we have refined tolerance reversal to a subset of self-antigen specific CD8 T cells, and eliminated the need for broadly immunostimulatory treatments. This model has been used to reverse tolerance in CD8 T cells specific for engineered antigens in the small intestine as well as naturally occurring self-antigens in the skin. The expanded populations of skin-specific CD8 T cells can be utilized for the treatment of melanoma. These melanocyte-specific CD8 T cells are functional and generate tissue resident memory (Trm) populations throughout the body, including the skin. Additionally, melanocyte-specific CD8 T cells can cooperate with neo-antigen specific CD8 T cells to aid in tumor clearance. By investigating tolerance reversal in self-specific CD8 T cells we have uncovered ways to improve upon current anti-cancer treatments and developed potentially translatable anti-cancer vaccine strategies.

Visualizing antigen-specific CD8 T cell dynamics in the small intestine

Abstract The small intestine (SI) represents the largest mucosal barrier between the environment and the host. As such, the SI limits both normal and pathogenic microbe invasion and must balance immune tolerance and activation. CD8ab T cells constitute a dominant lymphocyte population in the SI and contribute to both of these processes but detailed investigation into their in situ behavior in response to various stimuli is lacking. We visualized Ag-specific CD8 T cell migration in the SI using two-photon microscopy to understand CD8 T cell immunosurveillance. We found that pathogen-specific CD8 T cells are initially slowed early after infection but regain motility directly after Ag clearance. At a memory time point, CD8 T cells exhibited slower migration concurrent with acquisition of a resident memory phenotype. Current studies are focused on determining what is required for CD8 T cell motility throughout the course of infection. Additionally, as tolerance reversal and expansion of self-specific CD8 T cells has recently become a focus of cancer therapy, we characterized in situ behavior of self-specific CD8 T cells. Using a mouse expressing a model Ag in the SI as a self-protein, we observed self-specific CD8 T cells before and after tolerance reversal. Tolerant CD8 T cells migrate significantly faster than pathogen-specific cells at acute time points after infection. Contrastingly, ex-tolerant CD8 T cells were comparable to pathogen-specific CD8 T cells indicating that indeed their in situ scanning behavior had been altered upon tolerance reversal. These observations have provided valuable insight into CD8 T cell motility within the SI and can inform targeted treatment options and vaccines to ensure effective immunosurveillance of this tissue.

Rescue of Tolerant CD8+ T Cells during Cancer Immunotherapy with IL2:Antibody Complexes

Interleukin-2 (IL2) was among the earliest reagents used for cancer immunotherapy due to its ability to support the survival and function of tumor-reactive T cells. However, treatment with IL2 is accompanied by off-target toxicity and low response rates in patients. In mouse models, these issues are largely overcome when IL2 is administered as a cytokine/antibody complex (IL2c). The complex has a longer serum half-life and can be designed for preferential cytokine delivery to specific cells of interest. Early studies showed IL2c could boost antitumor immunity in mice by activating tumor-reactive CD8+ T cells. But such functional T cells are often limited in the tumor microenvironment, where instead unresponsive tolerant T cells are eventually eliminated by apoptosis, representing a major obstacle to the success of cancer immunotherapy. We found that IL2c treatment rescued tumor-specific CD8+ T cells from a state of established tolerance, providing effective immunotherapy in tumor-bearing mice. Expression of the transcription factor T-bet was necessary to drive intratumoral IFNγ production and effector activity by T cells rescued with IL2c. Furthermore, IL2c promoted T-bet expression in human CD4+ and CD8+ T cells in humanized tumor-bearing mice, but also increased the frequency of Foxp3+ regulatory T cells. Our study reveals a novel role for IL2c as a powerful immunotherapeutic reagent capable of reversing tolerance in tumor-reactive T cells, and provides the first evidence that IL2c influences human T cells in vivo, highlighting the translational potential to modulate human antitumor immune responses. Cancer Immunol Res; 4(12); 1016-26. ©2016 AACR.

FOXO3 is differentially required for CD8+ T-cell death during tolerance versus immunity.

Peripheral tolerance mechanisms limit autoimmunity by constitutively eliminating self-reactive CD8+ T cells from the periphery in a process called deletion. Previous work has demonstrated that this deletion process is mediated by BIM-dependent apoptotic death due to transcriptional induction of the Bim gene. Currently, the transcriptional pathways responsible for Bim induction during peripheral deletion remain unclear. We speculated that the transcriptional regulator FOXO3 may induce BIM-dependent death during peripheral deletion, as it has been implicated in Bim induction and cell death during effector CD8+ T-cell differentiation. Despite observing less Akt-dependent inactivation of FOXO transcription factors in tolerised cells relative to effector cells, we demonstrate that FOXO3-deficient CD8+ T cells induce Bim and die normally during peripheral deletion. These data thus demonstrate that BIM-dependent death during CD8+ T-cell deletion is FOXO3 independent. Furthermore, these data provide the first evidence that the pathways responsible for Bim induction and cell death during effector differentiation versus tolerance of CD8+ T cells are molecularly distinct.

Tracking of TCR-Transgenic T Cells Reveals That Multiple Mechanisms Maintain Cardiac Transplant Tolerance in Mice.

Solid organ transplantation tolerance can be achieved following select transient immunosuppressive regimens that result in long-lasting restraint of alloimmunity without affecting responses to other antigens. Transplantation tolerance has been observed in animal models following costimulation or coreceptor blockade therapies, and in a subset of patients through induction protocols that include donor bone marrow transplantation, or following withdrawal of immunosuppression. Previous data from our lab and others have shown that proinflammatory interventions that successfully prevent the induction of transplantation tolerance in mice often fail to break tolerance once it has been stably established. This suggests that established tolerance acquires resilience to proinflammatory insults, and prompted us to investigate the mechanisms that maintain a stable state of robust tolerance. Our results demonstrate that only a triple intervention of depleting CD25+ regulatory T cells (Tregs), blocking programmed death ligand-1 (PD-L1) signals, and transferring low numbers of alloreactive T cells was sufficient to break established tolerance. We infer from these observations that Tregs and PD-1/PD-L1 signals cooperate to preserve a low alloreactive T cell frequency to maintain tolerance. Thus, therapeutic protocols designed to induce multiple parallel mechanisms of peripheral tolerance may be necessary to achieve robust transplantation tolerance capable of maintaining one allograft for life in the clinic.