TCR Stimulation Research Articles

Upregulation of SLAMF3 on human T cells is induced by palmitic acid through the STAT5-PI3K/Akt pathway and features the chronic inflammatory profiles of type 2 diabetes

Metabolic stress-induced low-grade chronic inflammation plays an important role in the development of insulin-resistance and type 2 diabetes (T2D). Emerging evidence highlights the importance of directly elucidating T-cell activation under the obesity-induced metabolic stress condition, as T cells primed under such conditions were found to acquire a unique phenotype and function. Herein, we found a significant upregulation of signaling lymphocytic activation molecule family member 3 (SLAMF3) expression on T cells from T2D patients compared to those of healthy controls. Importantly, SLAMF3 upregulation was associated with an increased ability to produce proinflammatory cytokines. Significantly increased SLAMF3 expression was seen in T2D patient T cells that produce IFN-γ or IL-17 upon short (4-h) stimulation, compared to non-cytokine-producing T cells. In line with this finding, SLAMF3high T cells were significantly more sensitive than SLAMF3low T cells to TCR stimulation with anti-CD3/CD28 antibodies. Furthermore, treatment with palmitic acid (PA) led to significant upregulation of SLAMF3 on human T cells primed by anti-CD3/CD28 antibodies and on Jurkat cells, a human T-cell line. RNA sequencing revealed strong activation of the PI3K/Akt signaling pathway in T cells that were primed with PA. Further mechanistic studies showed that inhibition of PI3K/Akt signaling, or its upstream mediator STAT5 can prevent PA-induced SLAMF3 upregulation on T cells. These results indicate that SLAMF3 upregulation is associated with T-cell activation and cytokine production in T2D patients, and suggest that elevated saturated fatty acids in T2D patients may induce SLAMF3 upregulation on T cells via activation of the STAT5-PI3K/Akt signaling pathway.

Thymic Development of a Unique Bone Marrow-Resident Innate-like T Cell Subset with a Potent Innate Immune Function.

Mainstream CD8+ and CD4+ T cells of αβ lineage are developed in the thymus through TCR-mediated selection in the context of MHC class I and MHC class II in association with self-peptides, respectively. In addition, minor αβT cells bearing invariant TCRs, NKT cells, and mucosal-associated invariant T cells are selected via MHC-like molecules, CD1d, and MR1 complexed with nonpeptide Ags, respectively, parts of which express neither CD4 nor CD8. In this study, we indicate that bone marrow (BM), but barely other lymphoid tissues, harbors CD4/CD8 double-negative αβT cells with an apparently diverse TCR repertoire at considerable proportions in healthy adult mice. The BM-resident double-negative αβT (BMDNT) cells are developed in the thymus in a Notch and IL-7-dependent manner but independently of known restriction elements, including MHC class I, MHC class II, CD1d, and MR1. These cells are sustained in BM throughout the adult stage with "homeostatic" proliferation via IL-1β derived from normal myeloid cells dominating the BM environment. Although BMDNT cells secrete a unique set of cytokines, including IL-17, GM-CSF, IL-3, and CCL chemokines on TCR stimulation, these T cells also express a series of NK receptors and exhibit a potent NK-like cytotoxic activity. Furthermore, BMDNT cells show robustly accelerated proliferation and activation following systemic administration of TLR ligands likely through the enhanced production of IL-1β by myeloid cells in situ. Our results suggest that αβT lineage cells that are developed in the thymus by default of TCR-mediated selection are maintained and differentiated to innate-like T cells in BM and may play a role in innate immunity in the hematopoietic environment.

Abstract 2378: Differential exhaustion on cytokine release (DECREASE) by ERY974, a novel T-cell-redirecting antibody targeting glypican-3: A new type of T-cell exhaustion

Abstract Background: T cell-redirecting antibody (TRAB), which bispecifically binds to CD3 and tumor antigen, is a key player in next-generation cancer immunotherapy (CIT). Because TRAB can redirect T cells regardless of their TCR specificity, it is expected to be efficacious in immune checkpoint inhibitor-resistant tumors. The CD19-targeting bispecific T cell engager, blinatumomab, has been used for the treatment of blood cancers, and CEA-TCB and IMCgp100 have shown promising clinical efficacy in solid tumors as well. ERY974 is another promising TRAB targeting glypican-3 (GPC3) and a phase 1 study is in progress (Ishiguro et al., Sci Transl Med 2017). On the other hand, cytokine release syndrome (CRS) has been recognized as a common side effect of TRAB. Thus, mitigation of CRS is an urgent issue. Intra-patient step-up dosing regimens have been incorporated in clinical trials to reduce cytokine release, but the mechanism behind this phenomenon remains elusive. In this study, we pre-clinically explored the phenomenon and its mechanism by using ERY974. Method & Results: GPC3-expressing human cancer cells were incubated with human PBMCs and ERY974. The incubated PBMCs were harvested and mixed with newly prepared GPC3-expressing cancer cells and higher doses of ERY974. The pre-treated PBMCs showed reduced cytokine production compared to that without pre-treatment, while maintaining the same level of T cell-dependent cellular cytotoxicity (TDCC). Comprehensive gene expression analysis of the pre-treated PBMCs was also conducted. A murine cancer cell line expressing human GPC3 was established and implanted into immune competent mice. A mouse surrogate version of ERY974 (mERY974) was first administered at a low dose expected to elicit weak anti-tumor activity, and mice were then treated with a higher dose of mERY974. Anti-tumor activity was similar regardless of the pre-treatment, but plasma cytokine levels were shown to be reduced in the pre-treated mice upon administration of the higher dose of mERY974. Conclusion: Cytokine production following the high-dose ERY974 treatment was mitigated by the low-dose pre-treatment in vitro and in vivo. Cytotoxic activity did not decrease in this setting, thus demonstrating that the pre-treatment selectively suppressed only cytokine production. It is well known that repeated stimulation of TCR leads to T-cell exhaustion. The phenomenon we observed here can also be characterized as a sort of exhaustion, but is quite unconventional. Thus, we propose a new type of exhaustion and suggest calling it ‘Differential exhaustion on cytokine release (DECREASE)’. Management of CRS is critical for the clinical use of TRAB. A detailed analysis of the mechanism of DECREASE currently in progress, is expected to contribute to the development of desired dosing regimens for this new type of CIT agent. Citation Format: Mayumi Hoshino, Yuji Sano, Yasuko Kinoshita, Yumiko Azuma, Toshiaki Tsunenari, Yoko Kayukawa, Mizuho Noguchi, Takahiro Ishiguro, Shohei Kishishita, Noriaki Sawada, Mika Endo, Junichi Nezu. Differential exhaustion on cytokine release (DECREASE) by ERY974, a novel T-cell-redirecting antibody targeting glypican-3: A new type of T-cell exhaustion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2378.

Abstract 3272: RTX-212, an allogeneic red cell therapeutic expressing 4-1BBL and IL-15TP, exhibits potent in vitro and in vivo activity and a favorable safety profile

Abstract Recombinant agonists that activate co-stimulatory and cytokine receptors have shown limited clinical activity perhaps due to 1) toxicity; 2) a need for coordinated activation of co-stimulatory and cytokine pathways; or 3) the failure of agonist antibodies to recapitulate signaling by endogenous ligands. To address these limitations, Rubius Therapeutics developed genetically engineered red cells with cell surface expression of both co-stimulatory and cytokine ligands, which present ligands in their native form through cell-cell contact to potently activate and expand both T and NK cells. On-target, off-tissue toxicity may be limited due their biodistribution, which is restricted to the vascular system. IL-15 is known to promote NK survival and CD8 T cell memory and 4-1BB agonists are known to promote T cell prolioferation and survival. Rubius Therapeutics has developed RTX-212, an allogeneic red cell therapeutic genetically engineered to co-express 4-1BBL and an IL-15/IL-15Rαfusion (IL-15TP). In vitro assays demonstrated that the combination of both ligands on RTX-212 expanded both memory CD8 T cells (2.5-fold) and NK cells (10-15-fold), in the absence of TCR stimulation. RTX-212 further induced dramatic proliferation of CD8 T cells and CD4 T cells in the presence of TCR stimulation with increased IFNγsecretion. In addition to the synergistic effects of 4-1BBL and IL-15TP, each molecule provided complementary functions that expanded the activity of RTX-212 beyond RTX-4-1BBL or RTX-IL-15TP alone. IL-15TP uniquely activated NK cytotoxicity and ADCC, while 4-1BBL uniquely stimulated CD4 and CD8 T cell proliferation and production of IFNγ. To evaluate in vivo immune responses, anti-tumor activity and safety, a mouse surrogate therapeutic, mRBC-212, was developed where recombinant Fc-IL-15-sushi and m4-1BBL were chemically conjugated to mouse red blood cells. This surrogate overcame the rapid clearance of human red cells in mice. Intravanous administration of mRBC-212 to C57Bl6 mice that received B16F10 melanoma cells IV, showed a 66% decrease in the number of lung metastases compared to control mice (p=0.0001) and was associated with a significant increase in NK cell infiltration into the lungs (p=0.02). In a CT26 tumor model, mRBC-212 treated mice exhibited 55% tumor growth inhibition, which was accompanied by a 1.7-fold increase in the tumor infiltration of proliferating and cytotoxic CD8 T cells. Mice treated with the highest feasible dose of mRBC-212 showed no change in serum transaminases, infiltration of CD8 T cells and macrophages to the liver or liver inflammation score compared to agonistic 4-1BB antibodies treated mice. Taken together, these data indicate that RTX-212 has the potential to be an effective therapy with an improved safety profile compared to 4-1BB agonist antibodies and IL-15 agonists, supporting its clinical development. Citation Format: Anne-Sophie Dugast, Shannon McArdel, Maegan Hoover, Enping Hong, Shannon Curtis Leonard, Arjun Bollampalli, Douglas C. McLaughlin, Jennifer Mellen, Torben Straight Nissen, Christopher L. Carpenter, Thomas J. Wickham, Sivan Elloul. RTX-212, an allogeneic red cell therapeutic expressing 4-1BBL and IL-15TP, exhibits potent in vitro and in vivo activity and a favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3272.

Frontline Science: Late CD27 stimulation promotes IL-7Rα transcriptional re-expression and memory T cell qualities in effector CD8+ T cells.

We previously demonstrated that CD27 co-stimulation during a primary CD8+ T-cell response was critical for the expression of IL-7Rα on acute effector CD8+ T cells, providing an essential element in the generation of CD8+ T-cell memory to infectious pathogens. IL-7 plays a critical role in the generation and maintenance of memory CD8+ T cells, and IL-7Rα has been regarded as a functional marker of long-lived memory precursor effector cells. While IL-7Rα is downregulated acutely upon TCR stimulation, the regulation of the emergence of IL-7Rα expressing cells around the peak of primary CD8+ responses is less clear. Re-expression could be a default outcome after withdrawal of TCR stimulation. Alternatively, specific stimuli could actively antagonize the downregulation or promote the recovery of IL-7Rα in Ag-activated CD8+ T cells. By utilizing agonistic mAb and transgenic models, here we show: (1) CD27 stimulation acts directly on CD8+ T cells to enhance IL-7Rα-expressing effectors; (2) CD27 stimulation neither alleviates the downregulation of IL-7Rα upon TCR signaling nor promotes the expansion/survival of IL-7Rα-expressing effectors, but facilitates IL-7Rα re-expression; (3) CD27 stimulation regulates Il7ra mRNA abundance but not protein distribution. Importantly, CD27 stimulation promotes not only IL-7Rα, but also the common γ chain of the receptor and the downstream signaling mediated by pSTAT5. Our results demonstrate a previously unappreciated role of CD27 stimulation as a positive regulator of IL-7Rα during CD8 T-cell responses, provide insights into the mechanistic basis by which CD27 stimulation influences CD8+ T-cell memory differentiation, and highlight the potential of targeting CD27-CD70 axis to enhance IL-7 signaling for antiviral/antitumor immunotherapy.

Akt and STAT5 mediate na\xefve human CD4+ T-cell early metabolic response to TCR stimulation

Metabolic pathways that regulate T-cell function show promise as therapeutic targets in diverse diseases. Here, we show that at rest cultured human effector memory and central memory CD4+ T-cells have elevated levels of glycolysis and oxidative phosphorylation (OXPHOS), in comparison to naïve T-cells. Despite having low resting metabolic rates, naive T-cells respond to TCR stimulation with robust and rapid increases in glycolysis and OXPHOS. This early metabolic switch requires Akt activity to support increased rates of glycolysis and STAT5 activity for amino acid biosynthesis and TCA cycle anaplerosis. Importantly, both STAT5 inhibition and disruption of TCA cycle anaplerosis are associated with reduced IL-2 production, demonstrating the functional importance of this early metabolic program. Our results define STAT5 as a key node in modulating the early metabolic program following activation in naive CD4+ T-cells and in turn provide greater understanding of how cellular metabolism shapes T-cell responses.

Investigating Epidermal T Cell Function by C-C Chemokine Receptor 6

Abstract The skin is the largest organ on the body and is responsible for providing protection from foreign pathogens. When skin tissue damage occurs, immune cells aid in wound repair. Epidermal T cells play a key role in maintaining skin homeostasis by their secretion of cytokines and chemokines to facilitate inflammatory responses following tissue damage. During obesity and type 2 diabetes, epidermal T cells exhibit reduced production of select cytokine and chemokine receptors one of which is C-C Chemokine receptor 6 (CCR6). The principle ligand for CCR6 is C-C Chemokine Ligand 20 (CCL20). Upon CCL20 binding, CCR6 induces lymphocyte migration however, little is known about the regulatory role CCR6 plays in cellular functions such as cytokine production and cell surface receptor expression. For this study, murine epidermal T cells were harvested and stimulated in the presence or absence of CCL20. Epidermal T cell production of cytokines and chemokines in response to CCL20 were characterized via Luminex assay. Consequently to being stimulated with CCL20 epidermal T cells upregulate IL-2R and downregulate their T cell receptor both of which signify cell activation. Additionally, CCL20 costimulates TCR stimulation to activate epidermal T cells. Using immunofluorescence microscopy, we show that CCR6 has a pivotal role in activation causing morphological changes to skin resident T cells. Future studies will investigate CCR6 activation and receptor expression of αβ and γδ in human skin.

IL-15 Activates Liver Sinusoidal Mucosal-associated Invariant T Cells to Exert TCR/MR1-independent Innate-like Cytotoxicity

Abstract Mucosal-associated invariant T (MAIT) cells are the most dominant population of innate-like T cells in the human liver and predominantly localized in liver sinusoids. MAIT cells can be activated by MR1-dependent TCR stimulation or cytokines such as IL-12 and IL-18. However, effects of IL-15 on MAIT cells have not been comprehensively studied, particularly in the aspect of their innate-like cytotoxic activity. In the present study, we examined effects of IL-15 on liver sinusoidal MAIT cells obtained from donor liver perfusates during liver transplantation. IL-15 activated MAIT cells to have a cytotoxic phenotype and induces cellular proliferation. Moreover, IL-15-activated liver sinusoidal MAIT cells exerted innate-like cytotoxicity that is independent of TCR and MR1. Increased target cell conjugation and NKG2D-dependent stimulation were responsible for innate-like cytotoxicity of IL-15-activated MAIT cells, and PI3K–mTOR pathway was critically required for IL-15-induced innate-like cytotoxicity. Furthermore, we observed an increased NKG2D expression in intrahepatic MAIT cells from patients with acute hepatitis A, in which liver injury is known to be mediated by IL-15-induced innate-like cytotoxicity of T cells (Immunity 2018, 48:161). In summary, our data demonstrate IL-15 activates liver sinusoidal MAIT cells to exert TCR/MR1-independent cytotoxicity, and suggest a potential role of liver sinusoidal MAIT cells in immune responses or immunopathogenesis in liver diseases.

Immune checkpoint inhibitors reverse T-cell functional suppression in the bone marrow of a subset of AML patients

Abstract Acute Myeloid Leukemia is a blood cancer with poor prognosis despite decades of therapeutic development. Immune checkpoint inhibitors (ICIs) have been shown to be effective in many cancers, but their utility in AML has not been clearly established. To better understand the immune landscape of the AML microenvironment, we performed immunophenotyping on fresh bone marrow cells from AML patients using mass-spec based flow cytometry. In addition, we performed functional assays to evaluate T-cell proliferative capacity and determine whether AML bone marrow T cells can be modified by ICI treatment. Sixty two bone marrow samples from patients with AML were analyzed. We performed T-cell stimulation assays to measure proliferation and cytokine production in the presence and absence of ICIs. Samples were sub-categorized as being proliferators or non-proliferators based on the percent of T-cell division in response to TCR stimulation. Phenotypic analysis showed proliferation status correlated with a specific T-cell profile. Additionally, within the non-proliferator group, 70% overcame immune suppression and responded to at least one of the ICIs tested. In conclusion, we show that T-cell dysfunction is present in a subset of patients with AML. Nonetheless, this suppression can be reversed by ICIs in many cases, supporting the use of ICIs in AML therapy.

Eomes transcription factor is required for the development and differentiation of invariant NKT cells

Eomes regulates the differentiation of CD8+ T cells into effector and memory phases. However, its role in invariant (i)NKT cells remains unknown. Here, we show the impact of Eomes on iNKT cells in the thymus and peripheral tissue using conditional knockout (Eomes-cKO) mice. In the thymus, CD1d-tetramer+CD24+CD44−NK1.1−CD69+stage 0 iNKT cells express higher levels of Eomes than the other iNKT stages. We also found that Eomes regulates NKT1 cell differentiation predominantly. Interestingly, the expression of Eomes in the steady state is low, but can be upregulated after TCR stimulation. We also showed epigenetic changes in the Eomes locus after activation. In addition, vaccination of C57BL/6, but not Eomes-cKO mice with iNKT ligand-loaded dendritic cells generated KLRG1+iNKT cells in lung, characterized as effector memory phenotype by transcriptome profiling. Thus, Eomes regulates not only the differentiation of NKT1 cells in the thymus, but also their differentiation into memory-like KLRG1+iNKT cells in the periphery.

Nef-mediated inhibition of NFAT following TCR stimulation differs between HIV-1 subtypes

Functional characterisation of different HIV-1 subtypes may improve understanding of viral pathogenesis and spread. Here, we evaluated the ability of 345 unique HIV-1 Nef clones representing subtypes A, B, C and D to inhibit NFAT signalling following TCR stimulation. The contribution of this Nef function to disease progression was also assessed in 211 additional Nef clones isolated from unique subtype C infected individuals in early or chronic infection. On average, subtype A and C Nef clones exhibited significantly lower ability to inhibit TCR-mediated NFAT signalling compared to subtype B and D Nef clones. While this observation corroborates accumulating evidence supporting relative attenuation of subtypes A and C that may paradoxically contribute to their increased global prevalence and spread, no significant correlations between Nef-mediated NFAT inhibition activity and clinical markers of HIV-1 infection were observed, indicating that the relationship between Nef function and pathogenesis is complex.

Inhibition of T cell activation and function by the adaptor protein CIN85.

T cell activation is initiated by signaling molecules downstream of the T cell receptor (TCR) that are organized by adaptor proteins. CIN85 (Cbl-interacting protein of 85 kDa) is one such adaptor protein. Here, we showed that CIN85 limited T cell responses to TCR stimulation. Compared to activated wild-type (WT) T cells, those that lacked CIN85 produced more IL-2 and exhibited greater proliferation. After stimulation of WT T cells with their cognate antigen, CIN85 was recruited to the TCR signaling complex. Early TCR signaling events, such as phosphorylation of ζ-chain-associated protein kinase 70 (Zap70), Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP76), and extracellular signal-regulated kinase (Erk), were enhanced in CIN85-deficient T cells. The inhibitory function of CIN85 required the SH3 and PR regions of the adaptor, which associated with the phosphatase suppressor of TCR signaling-2 (Sts-2) after TCR stimulation. Together, our data suggest that CIN85 is recruited to the TCR signaling complex and mediates inhibition of T cell activation through its association with Sts-2.

Abstract A202: Lysophosphatidic acid impedes the effector function of CD8+ T-cells through LPA5R

Abstract Immunotherapies have demonstrated the utility of targeting the immune system for the eradication of tumors, yet a majority of patients remain refractory to such treatments. Therefore, further advances in our understanding of T-cell activation and inhibitory signals are required to find alternative combinational strategies to treat cancers. Lysophosphatidic acid (LPA) is a bioactive lipid that has been characterized to promote tumor growth via distinct mechanisms and thus displays multiple “hallmarks” of tumorgenesis, including enhancing tumor-promoting inflammation, metastasis, and angiogenesis. Despite LPA receptors (LPARs) being expressed on all immune cells, the impact of LPA on immune cells remains unclear. Work from our lab has previously shown that LPA signals via LPAR5 expressed by CD8+ T-cells to suppress TCR-mediated intracellular calcium mobilization. Thus, we propose that aberrant expression of LPA by diverse tumors serves also to dampen adaptive immune responses, thereby creating an immune suppressive tumor microenvironment. Here, we demonstrate that CD8+ T-cells express three of the six known LPA receptors, and that LPA signaling through LPA5R on CD8+ T-cells has not only a profound inhibition of Ca2+ release after TCR stimulation, but also impedes ERK and Nur77 TCR induced signaling pathway. Importantly we now demonstrate that LPA signaling also decreases the cytolytic function of CD8+ T-cells by inhibiting granule exocytosis. Both adoptive transfer of Lpar5-/- CD8+ T-cells and implantation of tumors into Lpar5-/- hosts leads to greater control of tumor growth, underscoring an immunosuppressive signal mediated by LPA through LPA5R on CD8+ T-cells. Finally, human CD8+ T-cells also express the same LPARs as in the mouse and similarly fail to efficiently release Ca2+ upon TCR stimulation in the presence of LPA. Pharmaceutical inhibition of the human LPA5 receptor restores Ca2+ flux, suggesting a similar mechanism seen in mice. Thus, our data illuminate a novel lipid-receptor interaction that suppresses CD8+ T-cell function in both human and murine cells. Citation Format: Divij Mathew, Pamela Strauch, Roberta Pelanda, Raul Torres. Lysophosphatidic acid impedes the effector function of CD8+ T-cells through LPA5R [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A202.

Abstract A197: The HMG transcription factor TOX induces a transcriptional and epigenetic program of CD8+ T-cell exhaustion in chronic infection and cancer

Abstract The protective capacity of the adaptive immune system relies on efficient and coordinated transitions between cellular fates. Following initial activation by specific antigen, naïve CD8+ T-cells proliferate extensively and undergo a highly orchestrated program of molecular rewiring and differentiation into effector CD8+ T-cells (TEFF) that can mediate protection through cytotoxicity and production of inflammatory cytokines. If the infection or antigen is cleared, most of this TEFF pool dies, but a subset persists, undergoing additional differentiation to form a pool of long-lived, self-renewing memory T-cells (TMEM) capable of mounting rapid recall responses. In contrast, during chronic infections or cancer, when T-cell stimulation persists, this program of functional T-cell differentiation is diverted and T-cells fail to sustain robust effector functions, instead becoming exhausted. Exhausted CD8+ T-cells (TEX) may balance limited pathogen or tumor control while restraining damaging immunopathology, but the consequence of restrained functionality is disease persistence and possible progression. Though first described in mice infected with lymphocytic choriomeningitis virus (LCMV), it is now clear that T-cell exhaustion is a common feature of many chronic infections as well as a variety of cancers in both mice and humans. Indeed, TEX are highly therapeutically relevant since these cells are a major target of checkpoint blockade mediated immune reinvigoration in human cancer patients. T-cell exhaustion is characterized by the progressive decline in effector function including the hierarchical loss of inflammatory cytokine production (IL-2, TNFa, IFNg). TEX also sustain high co-expression of multiple inhibitory receptors (PD-1, LAG3, TIGIT, CD160, TIM-3, 2B4), have reduced glycolytic and oxidative phosphorylation capacity, and impaired proliferation and survival. Underlying these major differences in TEX compared to TEFF and TMEM, is a distinct transcriptional program highlighted by altered use of key transcription factors and altered transcriptional circuits. Moreover, recent epigenetic analyses revealed that TEX differ from TEFF and TMEM by ~6,000 open chromatin regions, similar to differences between other major hematopoietic lineages, suggesting that TEX are not simply a state of activation of TEFF or TMEM, but rather are a distinct immune lineage. Yet the mechanisms that initiate this TEX fate commitment and epigenetic and transcriptional programming have thus far remained poorly understood, and exhaustion-specific TFs or transcriptional programming activities have remained elusive. Here we identify the HMG-box protein TOX as a master regulator of the TEX lineage. We find that robust TOX expression is limited to chronic infections and cancers in both mice and humans. TOX is largely dispensable for TEFF and TMEM formation, but in the absence of TOX, TEX do not form. Instead, TOX-deficiency results in the accumulation and subsequent rapid deletion of TEFF cells. TOX is induced by calcineurin and, specifically, NFAT2 and then operates in a feed-forward loop to become calcineurin independent and is durably expressed at a high level in TEX, but not TEFF or TMEM. TOX interacts with histone modifying enzyme proteins including the HBO1 complex and the acetyl-transferase Kat7, providing a mechanism for TEX lineage-specific epigenetic changes. Thus, high and sustained induction of TOX causes lineage commitment to TEX by translating persisting TCR stimulation cues into a distinct TEX transcriptional and epigenetic developmental program. Citation Format: Omar Khan, Josephine R. Giles, Sierra McDonald, E. John Wherry. The HMG transcription factor TOX induces a transcriptional and epigenetic program of CD8+ T-cell exhaustion in chronic infection and cancer [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A197.

Abstract A215: Thymocyte selection-associated HMG box protein TOX is a master regulator of tumor-specific T-cell dysfunction

Abstract Tumor-specific CD8 T-cells in cancers enter a state of dysfunction characterized by the expression of inhibitory receptors and failure to produce effector cytokines and cytotoxic molecules. Here we identify the nuclear factor, Thymocyte selection-associated HMG box protein, TOX, as a master regulator of tumor-specific T-cell dysfunction. TOX is uniquely expressed in dysfunctional CD8 T-cells from mouse and human tumors but absent in functional T-cells. TOX expression is driven by continuous TCR stimulation and NFAT activity. Forced expression of TOX in functional effector T-cells was sufficient to induce a transcriptional program of dysfunction through the concerted expression of genes encoding numerous inhibitory receptors and dysfunction-associated transcription factors. Notably, TOX-deficient tumor-infiltrating T-cells did not upregulate inhibitory receptors such as PD1, LAG3, CD38, or CD39 and maintained high TCF1 expression. Surprisingly, despite their normal, “non-exhausted” phenotype, TOX-deficient T-cells failed to make effector cytokines, suggesting that loss of effector function in tumor-specific T-cells is uncoupled from inhibitory receptor expression. Furthermore, TOX-deficient T-cells failed to persist in tumors, ultimately undergoing activation-induced cell death. We propose that the TOX-induced transcriptional program of hyporesponsiveness is a physiologic negative feedback mechanism that prevents overstimulation; thus TOX is absolutely required for T-cell survival in the setting of chronic antigen stimulation as in cancers. Citation Format: Andrew C. Scott, Steven Camara, Peter Lauer, Alexandra Synder, Dmitriy Zamarin, Tyler Walther, Olivier Levy, Michael Glickman, Jonathan Kaye, Mary Philip, Andrea Schietinger. Thymocyte selection-associated HMG box protein TOX is a master regulator of tumor-specific T-cell dysfunction [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A215.

Upregulation of SLAMF3 on Human T cells Is Induced by Palmitic Acid Through the STAT5-PI3K/Akt Pathway and Features the Chronic Inflammatory Profiles of Type 2 Diabetes

Background: Metabolic stress-induced low-grade chronic inflammation plays an important role in the development of insulin-resistance and type 2 diabetes (T2D). Emerging evidence highlights the importance of directly elucidating T cell activation under the obesity-induced metabolic stress condition and the underline mechanisms, which remain elusive. Methods: T cells from T2D patients and health controls were analyzed for activation and cytokine production by flow cytometry. The effect of palmitic acid on T cells was assessed by in vitro assays. RNA sequencing and inhibitor blocking assays were used to determine the signaling pathways involved in palmitic acid-induced SLAMF3 upregulation on T cells. Findings: We found a significant upregulation of SLAMF3, a molecule regulates immune function through homophilic interactions, on T cells from T2D patients compared to those of healthy controls. Importantly, SLAMF3 upregulation was associated with an increased ability to produce proinflammatory cytokines including IFN-γ and IL-17. Additionally, SLAMF3high T cells were significantly more sensitive than SLAMF3low T cells to TCR stimulation with anti-CD3/CD28 antibodies. Palmitic acid was found to cause significant upregulation of SLAMF3 on primary human T cells as well as Jurkat cells. Further mechanistic studies showed that inhibition of PI3K/Akt signaling, or its upstream mediator STAT5 can prevent palmitic acid-induced SLAMF3 upregulation on T cells. Interpretation: These results indicate that SLAMF3 upregulation is associated with T cell activation and cytokine production in T2D patients, and suggest that elevated saturated fatty acids in T2D patients may induce SLAMF3 upregulation on T cells via activation of the STAT5-PI3K/Akt signaling pathway. Funding Statement: This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030303), Chinese MOST (2015CB964400 and 2017YFA0104402), NSFC (81570145, 81870091, 91642208, 91742107, and 81501279), Plan B for Bethune Project of Jilin University (2015210), an innovation capability project of Jilin Province Development and Reform Commission (2017C019), an open topic of Jilin Province Science and Technology Agency (20170623092TC-01 & 20180623083TC-01). Declaration of Interests: The author(s) declare no competing financial interests. Ethics Approval Statement: Protocols involved in the use of human samples were approved by the Institutional Review Board, and all of the experiments were performed in accordance with the protocols.

IL-27 and TCR Stimulation Promote T Cell Expression of Multiple Inhibitory Receptors.

Inhibitory receptors (IR) are a diverse group of cell surface molecules that modulate T cell activation, but there are gaps in our knowledge of the cell-extrinsic factors that regulate their expression. The present study found that in vivo overexpression of IL-27 in mice led to increased T cell expression of PD-L1, LAG-3, TIGIT, and TIM-3. In vitro, TCR stimulation alone promoted expression of multiple IRs, whereas IL-27 alone induced expression of PD-L1. However, the combination of intermediate TCR stimulation and IL-27 resulted in synergistic induction of LAG-3, CTLA-4, and TIGIT. In vivo, infection with Toxoplasma gondii resulted in parasite-specific effector T cells that expressed high levels of IR, and at local sites of infection where IL-27 production was highest, IL-27 was required for maximal effector cell expression of PD-L1, LAG-3, CTLA-4, and TIGIT. Together, these results affirm the critical role of TCR signals in the induction of IR expression but find that during infection, IL-27 promotes T cell expression of IR.

Safety and efficacy of Tet-regulated IL-12 expression in cancer-specific T cells

ABSTRACTWe explored whether engineering of T cell specificity and effector function improves immunotherapy of solid tumors. Although IL-12 can enhance cancer immunity, a strategy of safe IL-12 delivery without toxicity is currently lacking. We engineered T cells to express IL-12 controlled by the NFAT promoter responsive to TCR stimulation, or by the Tet-On promoter responsive to doxycycline. In vivo, NFAT-engineered T cells caused lethal toxicity, while Tet-engineered T cells were safe in the absence of doxycycline. Combining gene transfer of the melanoma-specific TRP2-TCR with Tet-IL-12 engineering revealed that temporal induction of IL-12 was essential to inhibit the growth of B16F10 melanoma tumors. Induced IL-12 increased the number of tumor-infiltrating T cells and also prevented the down-modulation of the TRP2-TCR and the associated up-regulation of the PD1 marker that was observed in the absence of IL-12. In addition, temporal induction of IL-12 expression also increased the number of plasmacytoid DC in the tumor micro-environment. We show that repeated induction of IL-12 can be used to enhance control of tumor growth without encountering systemic toxicity. The observation that TCR engineering combined with Tet-regulated IL-12 expression can achieve tumor immunity without the side effects that are usually associated with the in vivo use of IL-12 warrants translation of this concept into the clinic.

Immunomodulatory Effects of Pevonedistat, a NEDD8-Activating Enzyme (NAE) Inhibitor, in Chronic Lymphocytic Leukemia (CLL)

Introduction: T cells from patients with CLL and lymphoma show highly impaired immune synapse formation, cytotoxic function, and adhesion and migration capabilities. Recent advances in immunooncology led to the emergence of therapeutic agents that permit reversal of T-cell exhaustion in cancer. However, rational development of novel combination approaches in immunotherapy requires detailed understanding of how targeted therapies influence T-cell function.We have shown that pevonedistat (TAK-924), an investigational NAE inhibitor, abrogates NFκB activation in CLL cells. Pevonedistat forms a covalent adduct with NEDD8, a ubiquitin-like modifier, thereby disrupting its interaction with NAE. This leads to reduced activity of Cullin-RING ligases (CRLs), a group of ubiquitin ligases that require modification by NEDD8 for their function. Ultimately, a decrease in CRL activity leads to reduced ubiquitination and proteasomal degradation of CRL substrates, extending the half-life of these proteins, including inhibitor of NFκB (IκB). Moreover, NFκB is critical in T-cell function. However, limited data exist on the effects of targeting neddylation on T-cell response. Here, we demonstrate that targeting neddylation in vitro preserves T-cell functionality and may lead to favorable T-cell population shifts in CLL.Methods: Peripheral blood mononuclear cells were isolated from patients with CLL (n=50), and T cells were purified using Dynabeads. Pevonedistat was obtained from Millennium Pharmaceuticals, Inc., a wholly owned subsidiary of Takeda Pharmaceutical Company Limited (Cambridge, MA).Results: In vitro T-cell receptor (TCR; CD3/CD28) stimulation induced T-cell activation and proliferation. Continuous treatment of T cells with pevonedistat led to rapid (2 hour) disruption of cullin neddylation, followed by a significant reduction in activity of NFκB and NFAT as assessed by immunoblotting and immunofluorescence. Despite this reduction, CD4 and CD8 T cells continued to respond to TCR stimulation, with relative abundance of early markers of activation (CD40L, CD69). However, we observed reduced expression of CD25 and PD-1 at 72 hours. Continuous treatment with pevonedistat led to a dose-dependent decrease in IL-2 secretion and reduced proliferation of the CD4 T-cell subset (CFSE, Ki-67) but did not induce apoptosis. Unlike CLL cells, CD4 T cells did not undergo DNA re-replication and G2/M arrest in response to pevonedistat.We further analyzed T-cell subsets following TCR stimulation. Concurrent treatment with pevonedistat led to an increase in IFNγ-secreting CD4 T cells, whereas IL-4 production decreased, suggesting a shift toward the Th1 phenotype. Furthermore, we observed a robust decrease of the iTreg population, accompanied by downregulation of FoxP3 mRNA and protein within the CD4 T-cell subset, indicating that targeting neddylation may help to reverse the immunosuppressive phenotype in CLL.To mimic the in vivo pharmacokinetics of pevonedistat, we performed drug washouts where CLL-derived T cells were exposed to 2-hour pulse treatment with 1 µM pevonedistat prior to TCR stimulation. Under these conditions, cullin neddylation and NFκB activity began to recover by 8 hours, with near complete recovery by 24 hours. Moreover, pevonedistat did not disrupt allogeneic (OCI-LY19 cells) or autologous (CD40L-stimulated CLL cells) T-cell cytotoxicity. Meanwhile, CD8 T cells continued to produce perforin and granzyme B.Conclusions: Our data suggest that pharmacologic targeting of NAE preserves T-cell cytotoxic function and may enhance anti-tumor immunity in CLL. Combined with our earlier reports that targeting NAE kills CLL cells under lymph node-mimicking conditions, these data provide a strong rationale for continued investigation of pevonedistat in CLL and lymphoid malignancies. DisclosuresSpurgeon:Bristol Myers Squibb: Research Funding; Gilead Sciences, Inc.: Consultancy, Research Funding; Oncternal: Research Funding; Acerta: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Pharmacyclics: Consultancy, Research Funding; MEI Pharma: Consultancy. Berger:Takeda Pharmaceuticals International Co.: Employment. Danilov:Gilead Sciences: Consultancy, Research Funding; Astra Zeneca: Consultancy; Verastem: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Aptose Biosciences: Research Funding; Takeda Oncology: Research Funding; TG Therapeutics: Consultancy; Bayer Oncology: Consultancy, Research Funding.

Ex Vivo Evidence for the Combination of Immune Checkpoint Inhibition with TGF-β Blockade to Enhance Anti-Tumor T Cell Responses in Multiple Myeloma

Immunosuppressive milieu of multiple myeloma is associated with various cellular and non-cellular factors that foster immune escape leading to tumor progression. While immune checkpoint inhibitors have achieved significant clinical success in many types of solid tumors, recent clinical trials of immune checkpoint blockade performed in patients with multiple myeloma failed to demonstrate significant anti-tumor efficacy. To enhance the clinical efficacy of immune checkpoint blockade in multiple myeloma, elaborate characterization of tumor antigen-specific T cells is an essential prerequisite. In the present study, we investigated the immunophenotypic and functional characteristics of tumor antigen-specific T cells in patients with multiple myeloma. In addition, using direct ex vivo experimental techniques, we tried to examine how to manipulate the immunosuppressive microenvironment to maximize anti-myeloma responses of the tumor-specific T cells.We first tried to define and characterize CD8+ T cell population capable of specifically recognizing and reacting to myeloma cells in bone marrow of myeloma patients using MHC multimer technique. In selected patients, we could successfully define CD8+ T cell population specifically recognizing the HLA-A*0201-restricted epitopes (either “SLLMWITQC” or “LLLGIGILV”), included in myeloma tumor antigens NY-ESO-1 and HM1.24. The vast majority of myeloma-antigen specific CD8+ T cells expressed high level of PD-1 and also co-expressed other types T cell inhibitory receptors. More strikingly, PD-1+ myeloma-specific CD8+ T cells had a distinct pattern of transcriptional factor expression, high level of Eomes and low level of T-bet (EomeshiT-betlo), indicating that they were profoundly exhausted functionally and that a simple blockade of PD-1/PD-L1 binding might not be enough to reinvigorate their anti-myeloma activity.Consistent with the immunophenotypes of myeloma-specific CD8+ T cells, malignant plasma cells in bone marrow of myeloma patients, defined as CD14-CD19-CD138+CS1+CD56hi, also expressed PD-L1 abundantly, compared to normal plasma cells, suggesting that PD-1/PD-L1 axis plays a major role in making myeloma-recognizing T cells unresponsive to TCR stimulation. Interestingly, in addition to tumor cells, various types of immune cells comprising myeloma microenvironment also highly express PD-L1. Indeed, in response to ex vivo TCR stimulation with anti-CD3, CD8+ T cells from myeloma bone marrow showed lower proliferation and reduced production of anti-tumor effector cytokines (INF-γ and TNF-α), compared to marrow-infiltrating CD8+ T cells of diffuse large B cell lymphoma and Hodgkin lymphoma patients with extensive bone marrow involvement. However, even in the presence of anti-PD-1, myeloma-specific responses of marrow-infiltrating CD8+ T cells was only modestly improved in terms of proliferation and cytokine production.Next, we investigated whether blocking TGF-β signaling in combination with PD-1/PD-L1 axis blockade could restore the function of marrow-infiltrating CD8+ T cells of myeloma patients, since TGF-β produced by clonal plasma cells and bone marrow stromal cells is critical in immunosuppressive microenvironment of multiple myeloma. To this end, we found that combination of TGF-β signaling blockade (either anti-TGF-β1 or Galunisertib, a small molecule inhibitor of TGF-β receptor I) with anti-PD-1 significantly increased the frequencies of IFN-γ- and/or TNF-α-producing CD8+ T cells in response to ex vivo TCR stimulation, compared to a single PD-1 or a single TGF-β blockade. Likewise, myeloma antigen-specific proliferation of CD8+ T cells was significantly enhanced with addition of TGF-β signaling blockade.Taken together, although PD-1/PD-L1 axis acts as a major component of immunosuppressive milieu in multiple myeloma, the efficacy of PD-1 blockades in multiple myeloma might be hampered by complicated microenvironment consisting of T cell-intrinsic and -extrinsic factors. Our results provide an ex vivo evidence of incorporating TGF- β signaling blockade to immune checkpoint inhibition to enhance anti-tumor T cell responses in multiple myeloma DisclosuresNo relevant conflicts of interest to declare.