WT CD4 Research Articles

P2RX7 enhances tumor control by CD8+ T cells in adoptive cell therapy

Abstract Expression of the purinergic receptor P2RX7 by CD8+ T cells promotes generation of memory populations following acute infections and is critical for responses to chronic infection. Despite these findings, recent studies have indicated that P2RX7 may limit the efficacy of anti-tumor responses. We show that P2RX7 is beneficial for optimal melanoma control in a mouse CD8+ T cell adoptive transfer model. Tumor specific P2rx7−/− CD8+ T cells exhibited impaired mitochondrial maintenance and function, but do not display signs of overt exhaustion or poor survival early in the anti-tumor response. However, as tumor burden increases the frequency P2RX7 deficient CD8+ T cells progressively declines relative to WT CD8+ T cells within the tumor. This was associated with worsening mitochondrial function and increased expression of some markers of T cell exhaustion among P2rx7−/− CD8+ T cells. Extending these studies, we found that transient in vitro stimulation of P2RX7 using the ATP analog BzATP led to enhanced B16 tumor control by CD8+ T cells. The improved ability of BzATP stimulated CD8+ T cells to prolong survival of tumor bearing mice was accompanied by enhanced mitochondrial function of tumor specific CD8+ T cells. These findings are in keeping with the concept that extracellular ATP (eATP) sensing by P2RX7 on CD8+ T cells is required for their ability to efficiently eliminate tumors by promoting mitochondrial fitness and indicates the potential for P2RX7 stimulation as a novel therapeutic treatment to enhance tumor immunotherapy. Supported by grants F30 award from the NIH (1F30CA250321-01)

The role of the Thrombin/PAR axis in modulating CD8+ T cell anti-tumor immunity

Abstract CD8+ tumor infiltrating lymphocytes (TIL) are critical for tumor clearance following immune checkpoint inhibitor (ICI) therapy. Thrombin, the central hemostatic protease, is activated in the tumor microenvironment and CD8+ T cells express the thrombin-cleavable protease activated receptors (PAR-1, -2, -3, -4). Recent studies showed that thrombin can enhance CD8+ T cell effector functions, but the role of thrombin in ICI is unknown. We hypothesized that thrombin enhances CD8+ TIL anti-tumor effector functions (αTEF) during ICI therapy. Consistent with this hypothesis, we found that reducing circulating prothrombin to ~10% of normal completely abrogated the efficacy of PD-1 blockade therapy to reduce tumor growth, reduced CD8+ TIL infiltration into tumor tissue, and limited αTEF. Mechanistic in vitro studies showed that thrombin stimulation of isolated murine or human CD8+ T cells resulted in a dose-dependent increase in survival, proliferation and cytokine production (activation profile). Surprisingly, when we stimulated CD8+ T cells lacking PAR-1 or PAR-2, we observed an increase in their activation profile relative to WT CD8+T cells in the absence of thrombin, which was further enhanced with thrombin. Moreover, stimulation of PAR-2 deficient CD8+ T cells with a specific PAR-1 activating peptide resulted in an increased activation profile compared to WT CD8+ T cells stimulated with the same peptide. Together these data suggest that 1) thrombin promotes adaptive tumor immunity in the context of ICI therapy, 2) thrombin promotes αTEF through PAR-1 dependent and independent mechanisms and 3) PAR-2 suppresses CD8+ T cell functions independently of thrombin and limits the capacity of PAR-1 activation to enhance effector functions. Supported by grants from NIH T32 (5T32AI118697-04)

TLR2/TLR4-Enhanced TIPE2 Expression Is Involved in Post-Hemorrhagic Shock Mesenteric Lymph-Induced Activation of CD4+T Cells.

PurposePost hemorrhagic shock mesenteric lymph (PHSML) return contributes to CD4+ T cell dysfunction, which leads to immune dysfunction and uncontrolled inflammatory response. Tumor necrosis factor α induced protein 8 like-2 (TIPE2) is one of the essential proteins to maintain the immune homeostasis. This study investigated the role of TIPE2 in regulation of CD4+ T lymphocyte function in interaction of PHSML and TLR2/TLR4.MethodsThe splenic CD4+ T cells were isolated from various mice (WT, TLR2-/-, TLR4-/-) by immunomagnetic beads, and stimulated with PHSML, normal lymphatic fluid (NML), respectively. Application of TIPE2-carrying interfering fragments of lentivirus were transfected to WT, TLR4-/-, and TLR2-/- CD4+ T cells, respectively. After interference of TIPE2, they were stimulated with PHSML and NML for the examinations of TIPE2, TLR2, and TLR4 mRNA expressions, proliferation, activation molecules on surface, and cytokine secretion function.ResultsPHSML stimulation significantly upregulated TIPE2, TLR2, and TLR4 mRNA expressions, decreased proliferation, CD25 expression, and IFN-γ secretion, and increased the secretion ability of IL-4 in WT CD4+ T cells. TIPE2 silencing enhanced proliferative capacity, upregulated CD25 expression, and increased IFNγ secretion in CD4+ T cells. PHSML stimulated TLR2-/-CD4+ T or TLR4-/-CD4+ T cells of which TIPE2 were silenced. TLR2 or TLR4 knockout attenuated PHSML-induced CD4+ T cells dysfunction; PHSML stimulation of silent TIPE2-expressing TLR2-/-CD4+ T or TLR4-/-CD4+ T revealed that the coexistence of low TIPE2 expression with lack of TLR2 or TLR4 eliminated this beneficial effect.ConclusionTIPE2 improves the PHSML-mediated CD4+T cells dysfunction by regulating TLR2/TLR4 pathway, providing a new intervention target following hemorrhagic shock-induced immune dysfunction.

TRPM2 Is Not Required for T-Cell Activation and Differentiation.

Antigen recognition by the T-cell receptor induces a cytosolic Ca2+ signal that is crucial for T-cell function. The Ca2+ channel TRPM2 (transient receptor potential cation channel subfamily M member 2) has been shown to facilitate influx of extracellular Ca2+ through the plasma membrane of T cells. Therefore, it was suggested that TRPM2 is involved in T-cell activation and differentiation. However, these results are largely derived from in vitro studies using T-cell lines and non-physiologic means of TRPM2 activation. Thus, the relevance of TRPM2-mediated Ca2+ signaling in T cells remains unclear. Here, we use TRPM2-deficient mice to investigate the function of TRPM2 in T-cell activation and differentiation. In response to TCR stimulation in vitro, Trpm2 -/- and WT CD4+ and CD8+ T cells similarly upregulated the early activation markers NUR77, IRF4, and CD69. We also observed regular proliferation of Trpm2 -/- CD8+ T cells and unimpaired differentiation of CD4+ T cells into Th1, Th17, and Treg cells under specific polarizing conditions. In vivo, Trpm2 -/- and WT CD8+ T cells showed equal specific responses to Listeria monocytogenes after infection of WT and Trpm2 -/- mice and after transfer of WT and Trpm2 -/- CD8+ T cells into infected recipients. CD4+ T-cell responses were investigated in the model of anti-CD3 mAb-induced intestinal inflammation, which allows analysis of Th1, Th17, Treg, and Tr1-cell differentiation. Here again, we detected similar responses of WT and Trpm2 -/- CD4+ T cells. In conclusion, our results argue against a major function of TRPM2 in T-cell activation and differentiation.

Identification of the Mouse T Cell ADP-Ribosylome Uncovers ARTC2.2 Mediated Regulation of CD73 by ADP-Ribosylation.

Mouse T cells express the ecto-ADP-ribosyltransferase ARTC2.2, which can transfer the ADP-ribose group of extracellular nicotinamide adenine dinucleotide (NAD+) to arginine residues of various cell surface proteins thereby influencing their function. Several targets of ARTC2.2, such as P2X7, CD8a and CD25 have been identified, however a comprehensive mouse T cell surface ADP-ribosylome analysis is currently missing. Using the Af1521 macrodomain-based enrichment of ADP-ribosylated peptides and mass spectrometry, we identified 93 ADP-ribsoylated peptides corresponding to 67 distinct T cell proteins, including known targets such as CD8a and CD25 but also previously unknown targets such as CD73. We evaluated the impact of ADP-ribosylation on the capability of CD73 to generate adenosine from adenosine monophosphate. Our results show that extracellular NAD+ reduces the enzymatic activity of CD73 HEK cells co-transfected with CD73/ARTC2.2. Importantly, NAD+ significantly reduced CD73 activity on WT CD8 T cells compared to ARTC2ko CD8 T cells or WT CD8 T cells treated with an ARTC2.2-blocking nanobody. Our study provides a comprehensive list of T cell membrane proteins that serve as targets for ADP-ribosylation by ARTC2.2 and whose function may be therefore affected by ADP-ribosylation.

Nrf2 Is a Critical Mediator of CD4 T Cell-Induced Acute Graft-Versus-Host Disease

Recent studies have shown that, in contrast to typical proliferating lymphocytes, alloreactive T (allo-T) cells can be specifically killed by apoptosis through compounds that lead to generation of intracellular reactive oxygen species. Nuclear factor erythroid-derived 2-like 2 (Nrf2) is an ubiquitously expressed transcription factor well-known for its role in regulating the cellular redox pathway, but little is known about its involvement in T-cell biology. In this study, we examined the contribution of Nrf2 to allo-T cells in the context of an allogeneic hematopoietic cell transplant (allo-HCT).To study the role of Nrf2 in T cell function, we first assessed the expression pattern of Nrf2 in activated T cells. We found that total cellular and nuclear Nrf2 expression in T cells were significantly increased 24 hours after in vitro α-CD3/CD28 stimulation (p<0.001), as well as in vivo in donor allo-T cells on day 14 in a well-established MHC-disparate murine allo-HSC model (C57BL/6→BALB/c) (p<0.05) (Fig 1). We next examined the effects of Nrf2 deficiency on T cell alloreactivity by transplanting WT or Nrf2-/- donor T cells in MHC-disparate (C57BL/6→BALB/c), as well as MHC-matched, minor antigen mismatched (C57BL/6→LP) murine models. In both models, allo-HCT recipients of Nrf2-/- donor T cells had significantly less acute graft-versus-host disease (GVHD)-induced mortality (p<0.01), morbidity (p<0.001), and intestinal pathology (p<0.05) (Fig 2a). To test if Nrf2 differentially regulates CD4+ versus CD8+ T cell function, we mixed and matched WT versus Nrf2-/- CD4+ and CD8+ T cells for transplant. Compared to allo-HCT recipients of donor WT CD4+: WT CD8+ T cells, we observed similar GVHD mortality in allo-HCT recipients of donor WT CD4+: Nrf2-/- CD8+ T cells, while we saw reduced GVHD mortality in allo-HCT recipients of donor Nrf2-/- CD4+: WT CD8+ T cells (p<0.01) (Fig 2b). These data support the notion that Nrf2 predominately regulates donor CD4+ and is dispensable for CD8+ T cells during allo-activation and the development of GVHD.We assessed if Nrf2 could affect polarization of donor CD4 T cells after allo-HCT, and found a two-fold increase in the proportion and absolute numbers of CD25+Foxp3+ regulatory T cells (Tregs) in the spleens of donor Nrf2-/- T cell allo-HCT recipients on day 7 post transplant (p<0.001) (Fig 3a). We next examined if Nrf2-deficiency results in greater induction of Tregs (iTregs) or stabilization of natural Tregs (nTregs). While we found an equal fraction of WT and Nrf2-/- naïve CD4+ T cells induced towards Tregs in vitro, we saw a significantly greater percentage of donor Nrf2-/- Tregs expressing Helios, a marker for thymus-derived nTregs, compared to donor WT Tregs in allo-HCT recipients on day 7 (p<0.01) (Fig 3b). Recent studies have suggested that Helios is required to stabilize the expression of FoxP3, and thus, the abundance and inhibitory activity of Tregs in inflammatory settings. In keeping with this notion, we discovered an increase in the cellular FoxP3 protein expression within donor Nrf2-/- Tregs in allo-HCT recipients (p<0.01) (Fig 3c). To further elucidate the biological significance of Nrf2 function on nTregs, we removed CD4+CD25+ putative Tregs from the donor inoculum and transplanted only conventional T cells (Fig 3d). We found that the survival advantage with donor Nrf2-/- T cells was abolished when Tregs were depleted from the donor graft, suggesting that Nrf2 expression in donor T cells negatively regulates nTregs during allo-activation, most likely through downregulation of Helios and FoxP3 expression.Finally, to determine if Nrf2 was critical for T-cell mediated GVT activities, we challenged allo-HCT recipients with A20 (B-cell lymphoma) cells. Using bioluminescent imaging, we found that Nrf2-/- allo-T cells were equally efficient in clearing tumor burdens, and along with their mitigated GVHD activities, resulting in a significantly improved survival outcome.Taken together, we identified a novel role of Nrf2 in CD4+ T cells in the context of allo-HCT. Nrf2 negatively regulated CD4+ nTreg through Helios downregulation during GVHD, but appeared to be dispensable in CD8+ mediated antitumor responses, further highlighting the possibility of separating GVT from GVHD activities. Our findings suggest that targeted manipulation of Nrf2 activities in allo-T cells may offer a useful clinical strategy to mitigate GVHD while preserving GVT responses. [Display omitted] DisclosuresJenq:Seres: Research Funding. van den Brink:PureTech Health: Consultancy; Seres: Research Funding; Jazz Pharmaceuticals: Consultancy; Therakos Institute: Other: Speaking engagement.

NKG2D Negatively Regulates CD4 T Cells in Allo-Response in Mice

NKG2D is a C-type lectin-like receptor which mainly expresses on mouse NK, activated CD8 and some γδ T cells. NKG2D is also found to express on human and mouse CD4 T cells, but the role of NKG2D in CD4 T cells is essentially unknown. We asked what subsets of CD4 T cells could express NKG2D; we activated and polarized CD4 T cells into Th0, Th1,Th2, Th17 and Foxp3+ Tregs in vitro and measured NKG2D expression on those subsets. We found that only a proportion of Th1 cells expressed NKG2D, whereas little or no NKG2D was expressed on other subsets of T cells. In a mixed lymphocyte reaction (MLR) assay, NKG2D was induced on a subset of CD4 T cells after stimulation with allogeneic antigen presenting cells (APCs) (Figure 1A). NKG2D+CD4+ T cells expressed higher levels of LAG-3, CD49b, CD80, FasL and PD-L1 (Figure 1B) and secreted more IL-10 and IFN-γ than NKG2D- CD4+ T cells (Figure 1C).Previous reports by others showed that NKG2D on NK and activated CD8 T cells contributing to graft versus host disease (GVHD) pathogenesis have been well-established, but the expression and function of NKG2D on CD4 T cells in GVHD development are unknown.To address these questions, we utilized a CD4-driven, MHC-mismatched aGVHD model of allogeneic bone marrow transplantation (allo-BMT), C57BL/6 to BALB/c. We monitored the development of NKG2D+CD4+ T cells in allogeneic recipients. We observed that NKG2D was continuously induced on CD4 T cells during the whole aGVHD process - gradually increased, peaked on day 14 and then slowly decreased after allo-BMT. By comparing the severity of aGVHD in recipients transferred with WT versus NKG2D-/- CD4 T cells. We observed that recipients transferred with NKG2D-/- CD4 T cells showed more severe GVHD demonstrated by a higher lethality (Figure 1D) and more severe diarrhea compared to WT counterpart. Mechanistically, NKG2D-/- CD4 T cells had a higher capacity of migrating to gut and liver than WT CD4 T cells reflected by the higher expression of α4β7 and CXCR3. Furthermore, NKG2D+CD4+ T cells secreted less proinflammatory cytokine IL-17 and more anti-inflammatory cytokine IL-10. Strikingly, NKG2D expression coincided with T-bet, but excluded RORγt or Foxp3 expression of CD4 T cells. Given NKG2D+CD4+ T cells expressed regulatory markers and secreted IL-10 and IFN-γ; we hypothesized that NKG2D+CD4+ T cells may serve as suppressive cells (e.g. Tr1 cells). To test this hypothesis, we sorted NKG2D+CD4+ and NKG2D-CD4+ T cells from recipients of allogeneic BMT, and performed in vitro suppressive assay. We observed that NKG2D+CD4+ T cells had a higher suppressive activity than NKG2D-CD4+ T cells. (Figure 1E)Taken together, the current work demonstrates that NKG2D may serve as a negative regulator on CD4 T cells, and NKG2D+CD4+ subset can function as a regulatory cell subset in the development of GVHD after allo-BMT. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting JAK2 Kinase Promotes Foxp3 Stability and Function of CD8iTregs in Prevention of Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Although donor T cells (Teff) have a beneficial effect on graft-versus-leukemia (GVL), they cause mortality and morbidity due to GHVD. Regulatory T cells (Tregs; CD4+Foxp3+) have been demonstrated to efficiently prevent GHVD by suppressing Teff proliferation. In addition to CD4+Foxp3+ Tregs, CD8+Foxp3+induced Tregs (CD8 iTregs) have also been identified not only inhibit Teff function, but also possess the GVL activity. However, instability of CD8 iTregs is an important barrier for GVHD clinical applications. Previous studies have revealed that Janus kinase2 (JAK2) signal transduction is an essential mediator of immune response. JAK2 activation is a positive regulator of Th1 and Th17 differentiation whereas JAK2 acts as a negative regulator in Tregs as its activation leads to Foxp3 instability.In this current study, we attempted to promote Foxp3 stability in CD8 iTregs by targeting JAK2 kinase using JAK2 conditional knockout mice (JAK2fl/flCD4Cre). The enhanced stability of CD8 iTregs could be a potential Treg-based therapy to prevent GVHD onset while maintaining GVL activity. In the generation of alloreactive iTregs in vitro, we found that a significant higher frequency of Foxp3+ cells could be induced from JAK2-/-CD8 T cells than WT CD8 T cells (Fig. 1A). We also observed that JAK2-/- CD8 iTregs exhibited higher levels of canonical Treg markers, Neuropilin-1 (NRP1), which is known to express on natural Tregs (nTregs) and has been proposed to associate with Treg stability (Fig. 1B). We initially examined in vitro stability of JAK2-/- CD8 iTregs in the presence of proinflammatory cytokines. Strikingly, JAK2-/-CD8 iTregs retained much higher Foxp3 than WT CD8 iTregs. Thus, we further investigated the function of JAK2-/- CD8 iTregs in preventing GVHD after allo-HSCT. Upon iTreg transfer into MHC-mismatched C57BL/6 (B6) to BALB/c model, JAK2-/- CD8 iTregs could significantly alleviate GVHD with a marked reduction of recipient's mortality (Fig. 1C).To understand the immune mechanisms of JAK2-/- CD8 iTregs in vivo, spleen, liver and mesenteric lymph nodes from recipient mice received JAK2-/- CD8 iTregs were analyzed on day 7 and 14 after allo-HSCT. JAK2-/- CD8 iTregs (Ly5.2+) remarkably suppressed Teff (Ly5.1+) expansion and maintain higher Foxp3 expression compared to WT counterparts (Fig. 1D). Consistently, lower levels of inflammatory cytokines (IFNγ, TNFα, IL-17 and IL-6) were detected from serum of mice received JAK2-/- CD8 iTregs than those of WT iTregs. For translational application, we evaluated the systemic effect of specific JAK2 kinase inhibitor (Pacritinib; PAC) on CD8 iTregs using a GVL model (B6 to BDF1). Mice transplanted with WT CD8iTregs plus P815 mastocytoma and then treated with Pacritinib could significantly prolong the survival and exhibited the GVL activity (Fig. 1E). In conclusion, our studies provide a strong rationale and means to stabilize CD8 iTregs by targeting JAK2 kinase, and stabilizedCD8 iTregs have a therapeutic potential for controlling GVHD in clinic. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The purinergic receptor P2RX7 promotes the accumulation of effector CD4+ T cells in the lung parenchyma during hypervirulent mycobacterial infection

Abstract CD4+ T cells are crucial to control pulmonary tuberculosis, but also favor uncontrolled inflammation in severe disease. Effective CD4+ T cell response to tuberculosis is related to tissue location: Lung-resident CD4+ T cells display higher effector function in comparison to circulating cells. Among the factors favoring T cell residency in non-lymphoid tissues, extracellular ATP sensing via P2RX7 is crucial for the generation of virus-specific resident memory CD8+ T cells. Yet, its role in CD4+ T cell non-lymphoid tissue residency has not been defined. Moreover, mycobacterial infection induces high extracellular ATP levels due to lung tissue damage, which is increased in response to hypervirulent strains. Here, we used P2rx7 ablation models to test the role of P2RX7 in CD4+T cell function in tuberculosis. In response to the hypervirulent Mycobacterium bovis MP287/03 strain, lung-resident WT CD4+ T cells express higher P2RX7 levels compared to circulating CD4+ T cells. Compared to WT mice, P2RX7-deficient (P2rx7−/−) mice display reduced numbers of antigen-specific (I-AbESAT-64-17) and IFN-γ-producing CD4+ T cells in the lung parenchyma. Using cell adoptive transfers, while recipient CD4-deficient mice transferred with CD4+ WT T cells are protected, mice that received CD4+P2rx7−/− T cells develop severe tuberculosis. Mechanistically, CD4+P2rx7−/− T cells have poorer accumulation and IFN-γ production in the lung parenchyma; moreover, they show reduced proliferation in situ. This defect in proliferation is exclusively observed in the lung, with no differences observed in draining lymph nodes. Together, our results show that P2RX7 promote the accumulation of protective CD4+ T cells in the lung during severe tuberculosis.

RNF5-deficiency inhibits CD8+ T cell exhaustion in chronic antigen stimulation

Abstract Functional T cell exhaustion develops with persistent antigen exposure during chronic viral infection and cancer. Greater insights into T cell exhaustion are needed to promote immunity to viruses and tumors. RNF5 (RING-finger protein 5) is an E3 ubiquitin ligase that functions in protein degradation. We previously found enhanced anti-tumor responses in RNF5−/− mice, which was coupled with greater recovery and function of CD8+ T cell in melanoma tumors. Tumor-infiltrating RNF5−/− CD8+ T cells expressed high levels of inhibitory receptors (IR), yet greater functionality compared to WT CD8+ T cells, suggesting greater activation. To further assess RNF5-dependent regulation of CD8+ T cell exhaustion, we used the chronic LCMV Cl13 infection that has defined states of progressive T cell exhaustion. Virus-specific CD8+ T cells were significantly increased in RNF5−/− mice leading to greater numbers of cytokine producers, which expressed high levels of IRs. They also expressed higher levels of TOX, a transcriptional regulator of T cell exhaustion and persistence. With low dose infection, RNF5−/− mice displayed decreased morbidity and CD8+ T cells acquired a highly functional memory phenotype rather than exhaustion. To identify T cell intrinsic role(s) of RNF5, we analyzed anti-viral responses in bone marrow chimeras lacking RNF5 only in T cells and used an in vitro T cell exhaustion assay. RNF5−/− CD8+ T cells also expressed high levels of IRs and TOX, as did the in vitro exhausted T cells yet importantly displayed enhanced cytokine production compared to WT cells. These data reveal a T cell-intrinsic role of RNF5 in promoting exhaustion in settings of chronic antigen stimulation.

CD73 Ectonucleotidase Restrains CD8+ T Cell Metabolic Fitness and Anti-tumoral Activity.

CD39 and CD73 are ectoenzymes that dephosphorylate ATP into its metabolites; ADP, AMP, and adenosine, and thus are considered instrumental in the development of immunosuppressive microenvironments. We have previously shown that within the CD8+ T cell population, naïve and memory cells express the CD73 ectonucleotidase, while terminally differentiated effector cells are devoid of this enzyme. This evidence suggests that adenosine might exert an autocrine effect on CD8+ T cells during T cell differentiation. To study the possible role of CD73 and adenosine during this process, we compared the expression of the adenosinergic signaling components, the phenotype, and the functional properties between CD73-deficient and WT CD8+ T cells. Upon activation, we observed an upregulation of CD73 expression in CD8+ T cells along with an upregulation of the adenosine A2A receptor. Interestingly, when we differentiated CD8+ T cells to Tc1 cells in vitro, we observed that these cells produce adenosine and that CD73-deficient cells present a higher cytotoxic potential evidenced by an increase in IFN-γ, TNF-α, and granzyme B production. Moreover, CD73-deficient cells presented a increased glucose uptake and higher mitochondrial respiration, indicating that this ectonucleotidase restrict the mitochondrial capacity in CD8+ T cells. In agreement, when adoptively transferred, antigen-specific CD73-deficient CD8+ T cells were more effective in reducing the tumor burden in B16.OVA melanoma-bearing mice and presented lower levels of exhaustion markers than wild type cells. All these data suggest an autocrine effect of CD73-mediated adenosine production, limiting differentiation and cytotoxic T cells’ metabolic fitness.

CARD11 Mutation Induces Oligoclonal Expansion of T-Cells, and Accelerates ATL Development in Combination with HBZ

Adult T-cell leukemia/lymphoma (ATL) is an aggressive peripheral T-cell lymphoma that develops in about 5% of human T-cell leukemia/lymphoma virus 1 (HTLV-1) carriers. In addition to viral oncogenes, namely tax and HTLV-1 bZIP factor (HBZ), gene mutations, highly enriched for T-cell receptor (TCR)-NF-kB signaling, should be involved in the development of ATL. Among gene mutations, mutation in CARD11, a cytoplasmic scaffolding protein required for TCR-induced NF-kB activation, was detected in 24% of ATL patients. Here we generated a mouse model for conditional expression of a human ATL-derived CARD11(E626K) gain-of-function mutant, and demonstrated CARD11 activation induced oligoclonal expansion of T-cell and infiltration to many organs. We also showed that expression of HBZ accelerated mutant CARD11-induced lymphoproliferative diseases. We introduced a human Card11(E626K) into the mouse genome at the ROSA26 locus. After crossing with CD4-Cre Tg, CARD11(E626K)CD4-Cre mice was obtained. In CD4+ cells from CARD11(E626K)CD4-Cre, the amount of cleaved BCL-10 and NF-kBp65 increased compared with those in WT CD4+cells, confirming the activation of NF-kB. About half of CARD11(E626K)CD4-Cre mice died on or after 6 months after birth. At 6 months, leukocytosis was observed in CARD11(E626K)CD4-Cre, and accordingly the number of CD4+ cells cells was about 1.43 times greater than those in WT mice. The most affected organ in CARD11(E626K)CD4-Cre mice was lung. Alveolar septum was thickened by infiltrated cells at 6 months, and worsened subsequently. CD3+ T-cell accumulated around capillary blood vessels, and had high proliferation indices (&amp;gt;50%), as assayed by Ki-67 staining. CARD11(E626K)CD4-Cre mice developed lymphadenopathy (4/8 mice (50%) at 6M and 4/6 mice (66.7%) at 12M). Normal lymph node (LN) architecture was barely preserved, and medullary sinus was expanded with CD3+ T-cell. Some of them were positive for FoxP3, and had moderate proliferation indices (25%-50%). Among CD4+ T-cell, the proportion of naive T-cell (Tn) decreased, and that of effector/memory T-cell (Tem) and regulatory T-cell (Treg) increased compared to WT LNs. The proportion of Treg in CD4+ T-cell from LN of 6M- and 12M-old CARD11(E626K)CD4-Cre mice was 25% and 40%, respectively, which values were much larger than the normal range as 10-15%. We next examined the clonality of CD4+ cells in spleen and swollen LNs from CARD11(E626K)CD4-Cre mice. The clonality of the TCR repertoires of 20 individual Vb gene famines from Vb1-20 was assessed by a PCR. The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 5 splenic CD4+ cells, and 1 of 2 LN CD4+ cells from CARD11(E626K)CD4-Cre mice. To assess the effect of HBZ constant expression on CARD11(E626K)CD4-Cre mice, we generated HBZ Tg, in which HBZ cDNA was expressed under the CD4 promoter. Similar to the previous report (by Satou et al.), our HBZ Tg showed increased number of Tem, destroyed architecture of lung such as thickened alveolar septum by infiltrated cells and decreased alveolar space by edema, and the lymphadenopathy after 12M (66.7%). We then crossed CARD11(E626K)CD4-Creand HBZ Tg, and obtained the compound mice. The compound mice caused more aggressive lymphoproliferative diseases compared with CARD11(E626K)CD4-Cre mice. Most of compound mice died within 8M. At 6M, architecture of lung, kidney, spleen, and LN was destroyed. In lung, alveolar space of lung was scarcely observed caused of T-cell invasion. Alveolar septum was thickened with infiltrated cells, and CD3+ cells accumulated around capillary blood vessel. Some of them were positive for FoxP3, and indicated moderate proliferation indices (25-50%). T-cell invasion was also observed in kidney. Lymphadenopathy was detected in 6 of 9 (66.7%) with completely destroyed architecture, increment of the proportion of Fox3+ cells, and moderate proliferation indices (25-50%). The clonality assay using the TCR repertoires exhibited an oligoclonal pattern in 4 of 4 splenic CD4+ cells, and 2 of 2 LNs CD4+ cells from compound mice. These results suggest that CARD11 mutant-induced NFkB activation and constant HBZ expression may have similar effects, such as T-cell infiltration into organs and LN adenopathy, and that the simultaneous occurrence of both may have additive effects. Disclosures Sugiyama: Chordia Therapeutics, Japan.: Current Employment. Morishita:Chordia Therapeutics Inc.: Current Employment, Current equity holder in private company. Shimoda:Perseus Proteomics: Research Funding; PharmaEssentia Japan: Research Funding; AbbVie Inc.: Research Funding; Astellas Pharma: Research Funding; Merck &amp; Co.: Research Funding; CHUGAI PHARMACEUTICAL CO., LTD.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Pfizer Inc.: Research Funding; Otsuka Pharmaceutical: Research Funding; Asahi Kasei Medical: Research Funding; Japanese Society of Hematology: Research Funding; The Shinnihon Foundation of Advanced Medical Treatment Research: Research Funding; Celgene: Honoraria; Shire plc: Honoraria; Novartis: Honoraria, Research Funding; Takeda Pharmaceutical Company: Honoraria; Bristol-Myers Squibb: Honoraria.

Lack of the E3 Ubiquitin Ligase March1 Affects CD8 T Cell Fate and Exacerbates Insulin Resistance in Obese Mice.

Obesity is a major risk factor for the development of insulin resistance and type 2 diabetes. However, the mechanisms that trigger the underlying adipose tissues inflammation are not completely understood. Here, we show that the E3 ubiquitin ligase March1 controls the phenotypic and functional properties of CD8+ T cells in mice white adipose tissue. In a diet-induced obesity model, mice lacking March1 [March1 knockout (KO)] show increased insulin resistance compared to their WT counterparts. Also, in obese March1 KO mice, the proportions of effector/memory (Tem) and resident/memory (Trm) CD8+ T cells were higher in the visceral adipose tissue, but not in the spleen. The effect of March1 on insulin resistance and on the phenotype of adipose tissue CD8+ T cells was independent of major histocompatibility complex class II ubiquitination. Interestingly, we adoptively transferred either WT or March1 KO splenic CD8+ T cells into obese WT chimeras that had been reconstituted with Rag1-deficient bone marrow. We observed an enrichment of Tem and Trm cells and exacerbated insulin resistance in mice that received March1 KO CD8 T cells. Mechanistically, we found that March1 deficiency alters the metabolic activity of CD8+ T cells. Our results provide additional evidence of the involvement of CD8+ T cells in adipose tissue inflammation and suggest that March1 controls the metabolic reprogramming of these cells.

Abstract A10: Constitutive HIF activity enhances T-cell antitumor immunity and memory T-cell formation in the murine adoptive cell therapy model

Abstract Purpose: To determine if unrestrained hypoxia inducible factor (HIF) activity in CD8 T cells improves antitumor response. Methods: CD8 T cells were isolated from von Hippel Lindau (VHL)-deficient (VHL KO) mice, which have unrepressed HIF1 and HIF2 activity, as well as from VHL/HIF1/HIF2 triple-deficient (TKO) and WT mice. Following in vitro activation, T cells were transferred to melanoma (B16) tumor-bearing mice and monitored for tumor progression and survival. In other studies, activated VHL KO and TKO CD8 cells were mixed with WT CD8 cells and transferred to B16 or adenocarcinoma (MC38) tumor-bearing mice; after 7-10 days, donor T cells were analyzed from tumors, spleens, and draining lymph nodes (LN). Results: Single transfer of VHL KO CD8 T cells to B16 tumor-bearing mice resulted in delayed tumor growth and prolonged survival compared to transfer of CD8 T cells from TKO or WT donors. Co-transfer of VHL KO cells with WT cells into B16 tumor-bearing mice resulted in superior accumulation of VHL KO cells in all tissues including tumors. In contrast, mixed transfer of TKO cells with WT cells to B16 tumor-bearing mice resulted in poorer accumulation of TKO cells in all tissues including tumors. VHL KO tumor-infiltrating lymphocytes (TILs) had superior expression of tissue resident memory (TRM) phenotype markers CD103 and CD69 compared to WT and TKO TILs. Similarly in the MC38 tumor model, VHL KO cells showed improved accumulation in all tissues compared to WT cells. Conclusions: Here we demonstrate that VHL deletion in CD8 T cells results in superior efficacy in delaying tumor growth and improving recipient survival, greater accumulation in multiple solid tumor models, and increased development of the TRM phenotype. Further, TKO recipients demonstrated increased tumor growth, reduced survival, reduced TIL numbers, and absence of TRM phenotype following adoptive transfer, confirming the HIF dependency of the VHL-deletion phenotype. Taken together, these data demonstrate that T cells with constitutive HIF activity provide a more robust antitumor response. Therefore, manipulation of HIF regulation could offer new tools for enhanced T-cell efficacy in adoptive cellular therapy for solid tumors. Citation Format: Colette R. Lauhan, Deborah Witherden, Ilkka Liikanan, Ananda Goldrath. Constitutive HIF activity enhances T-cell antitumor immunity and memory T-cell formation in the murine adoptive cell therapy model [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A10.

Understanding the role of hnRNPA1 on T cell differentiation and function in the Gut

Abstract Regulatory T cells (Treg) play a critical role in preventing autoimmunity diseases and maintaining tolerance in various tissues, particularly the gut. Peripheral derived Tregs (pTregs) are especially crucial in gut homeostasis and tolerance to commensal and food antigens. We have previously shown that knockdown of the RNA-binding protein, heterogeneous nuclear ribonucleoprotein (hnRNP) A1, reduces in vitro pTreg induction upon low TCR stimulation. In addition, we have shown that, under conditions of low TCR stimulation, in which pTreg are induced, hnRNP A1 is phosphorylated by Akt. In order to explore the impact of hnRNP A1 phosphorylation by Akt on pTreg induction in vivo we have generated a mutant mouse model, hnRNPA1-S199 in which the known Akt phosphorylation site on hnRNP A1 is mutated to Alanine. Extensive characterization of immune cells in this mutant mouse revealed no abnormalities in the number or frequency of major immune cell types at steady state. However, we identified abnormalities in the number of Treg in the mesenteric lymph nodes (mLN) in mutant mice, prompting further investigation. Using the Cytex Aurora flow cytometer we developed a 19 color T cell panel to characterize T cells in the mLN, Peyer’s patches and lamina propria. Detailed characterization of the pTreg and tTreg populations in the gut of WT and mutant mice will determine whether the abnormalities observed in the mLN are reflected in different intestinal areas. In addition, we are examining the induction of pTreg using an adoptive transfer model of OT-II TCR transgenic mutant and WT CD4 T cells followed by oral OVA administration. Further studies will focus on the mechanisms by which hnRNP A1 impacts pTreg induction.

YAP is a novel regulator of CD4+ and CD8+ T cells during activation, differentiation, and anti-tumor immunity

Abstract Advanced understanding of T cell regulation has fueled cancer immunotherapy, but durable responses remain confined to a minority of patients. Poor responses can be linked to either suppressed T cell function in tumors or “cold” tumors that exclude immune cells. Identifying novel T cell regulators involved in these processes is a high priority. Our studies identified the protein YAP as a novel regulator of T cells. YAP mRNA and protein were rapidly expressed after T cell activation in mouse CD4+ and CD8+ T cells. T cell-specific Yap knockout (YapKO) generated CD4+ and CD8+ T cells that express elevated levels of activation markers (CD25, CD44, CD69) compared to WT cells. YapKO increased the sensitivity of T cells to anti-CD3 dose, and increased CD4+ T cell differentiation efficiency under Th1, Th2, Th17, and Treg skewing conditions. YapKO CD8+ T cells expanded &amp;gt;20-fold more than WT when cultured in engineered hydrogel matrices optimized for T cell expansions and adoptive transfer. Syngeneic tumors grown in T cell YapKO mice had impaired growth and increased immune infiltration compared to tumors in WT hosts. Co-adoptive transfer of YapKO and WT CD8+ T cells showed only YapKO cells infiltrated B16F10 tumors. RNAseq of CD4+ and CD8+ T cells from tumors and draining lymph nodes showed gene expression changes including upregulation of T cell receptor and cytokine signaling pathways in YapKO hosts. T cells from tumor-draining lymph nodes were not significantly different between YapKO and WT. YapKO gene signatures correlated with immune infiltration and survival in multiple human cancers in TCGA. Our data demonstrated YAP is a novel regulator of diverse T cell functions and may be a useful target for enhancing responses to cancer immunotherapy.

The Role of PPARd in Alloreactive CD8 T cell Metabolism

Abstract Graft-versus-host disease (GVHD) is the most common cause of non-relapse mortality following allogeneic hematopoietic stem cell transplantation. Using a pre-clinical model of GVHD, previous work has found that CD8 T cells recovered on day 7 post-transplant upregulate the transcription factor, peroxisome proliferator activated receptor delta (PPARd). While PPARd has been shown to be a key regulator of fatty acid oxidation (FAO) in other tissues, it’s role in T cells is not well studied. We hypothesize that PPARd drives FAO in alloreactive CD8 T cells and that CD8 T cells lacking PPARd will be unable to oxidize fat and thus unable to cause GVHD. To investigate this hypothesis, ex vivo FAO was measured in day 7 WT versus PPARd KO CD8 T cells by quantitating conversion of 3H-palmitate to 3H2O. Production of 3H2O decreased by 50% in KO CD8 T cells, suggesting an inability to fully oxidize fat. To understand the mechanism underlying this decrease, we performed RNA sequencing and identified thirty genes that were differentially expressed in WT versus KO CD8s including 8 genes related to FAO. Strikingly, despite a decreased ability to oxidize fat, PPARd KO CD8 T cells were recovered in equal numbers on day 7 post transplantation, suggesting that CD8 T cells lacking PPARd adopt alternative metabolic pathways to generate sufficient energy for in vivo proliferation and activation. Ultimately, our analyses indicate that PPARd is required for FAO in alloreactive CD8 T cells, but this process is dispensable for short term proliferation and/or survival of CD8 T cells during GVHD. Future directions will identify compensatory metabolic pathways utilized by PPARd KO T cells through a combination of in vitro nutrient drop out experiments and metabolomic flux analyses.

Role of Granzyme B in mucosal immune responses

Abstract Granzyme B is a serine protease initially described in cell-mediated cytotoxicity. Recently, granzyme B has been found to be produced by non-cytotoxic cells, and elevated levels of granzyme B or cells expressing it have been implicated in many disease states. To investigate the role of granzyme B in mucosal immune responses, we utilized two models of intestinal inflammation: infection with Citrobacter rodentium, a colon-specific bacterium that induces intestinal inflammation in mice; and a T cell transfer model of colitis. Following infection, C. rodentium colonization was observed in the colon of both wt and grzB−/− animals at 14d post-infection. While wt mice displayed no overt signs of infection, severe weight loss as well as disease-associated changes in appearance and colon pathology were observed in grzB−/− mice at 10–14d post-infection. Similarly, adoptive transfer of grzB−/− CD4+CD45RBhi naïve effector T cells into rag2−/− recipients led to severe weight loss and disease-associated changes in appearance by 21d post-transfer, while rag2−/− recipients of wt CD4+CD45RBhi T cells were relatively unaffected at this early time point. Interestingly, disease in recipients of grzB−/− T cells corresponded with increased IL-17+ CD4+ T cells in the mesenteric lymph nodes and colon lamina propria relative to recipients that received wt T cells. These data suggest that granzyme B functions in a protective manner during intestinal inflammation, possibly playing a role in T cell differentiation by holding generation of IL-17-producing T cells in check.

The T-box transcription factors T-bet and Eomes repress protective Th17 CD4 T cell responses against Influenza A Virus

Abstract Promoting strong CD4 cell immunity is an attractive strategy to generate a universal Influenza A Virus (IAV) vaccine. Though most CD4 cell responses against IAV are classified as Th1, Th17 and cytotoxic CD4 cells (ThCTL) may also contribute to viral clearance. Interestingly, we find the transcription factor T-bet (Tbx21), the ‘master regulator’ of Th1 differentiation, to be dispensable for protective CD4 responses against IAV. Surprisingly, Tbx21−/− cells develop Th17 characteristics but retain Th1 functionality, highlighted by strong IFN-γ production. While the transcription factor Eomesodermin (Eomes) can compensate for loss of T-bet to promote antiviral CD8 responses, its roles in CD4 cells is still unclear. Here we analyze Eomes−/−, Tbx21−/− and Tbx21−/−Eomes−/− CD4 cell responses against IAV using an adoptive transfer model to determine the extent to which Eomes regulates antiviral CD4 functions. Eomes−/− cells mirrored the prototypical Th1 responses of WT CD4 cells. Strikingly, Tbx21−/−Eomes−/− cells exhibited near complete loss of both Th1 responses and ThCTL function. In contrast, Tbx21−/− Eomes−/− developed stronger Th17 attributes than Tbx21−/− cells. We assessed protection provided by transfer of in vitro Th17-primed WT, Eomes−/−, Tbx21−/− and Tbx21−/−Eomes−/− cells to unprimed WT mice infected with lethal IAV. While WT and Eomes−/− effectors gained Th1 characteristics in vivo, Tbx21−/−Eomes−/− cells strengthened their Th17 phenotype. Remarkably, all of the Th17 effectors were equally protective. Our observations reinforce the concept that Th17 cells can provide strong protection against IAV, independently of Th1 and ThCTL responses, and suggest Th17 cells use unique pathways to do so.

Enhanced anti-tumor response upon deficiency of Msn kinase MINK1 in mice

Abstract Misshapen (Msn)/NIK-related kinase 1 (MINK1) is a serine/threonine kinase belongs to the germinal center kinase (GCK) family. Previous studies indicated the correlation of MINK1 with tumor cell senescence and Th17 differentiation. Its dual role in modulating both tumor growth and T cell function indicated MINK1 a possible intervention target in tumor immunotherapy. We have utilized the MINK1 deficient mice to explore the possible role of MINK1 in the anti-tumor immune response. By using the subcutaneous tumor-xenograft model, we have found that MINK1 deficiency in mice resulted in slower tumor growth. We have also accumulated evidence in adoptive transfer and CD8+ T cells depletion experiments to show that the genetic ablation of MINK1 suppressed tumor growth primarily via CD8+ T cells without affecting other immune cells. Along with which, MINK1 deficient CD8+ T cells are superior in both tumor infiltration and the expression of effector markers such as TNF-α, IFN-γ, and Granzyme-B when compared to WT CD8+ T cells. Lower expression of exhaustion markers such as PD-1, TIM-3, and TIGIT were also observed in MINK1 KO CD8+ T cells. We are currently exploring the possible mechanisms of how MINK1 regulated CD8+ T cell function, and examine the possibility to target MINK1 in experimental tumor immunotherapy.