Wild-type CD4 Research Articles

Helios Deficiency Predisposes the Differentiation of CD4+Foxp3- T Cells into Peripherally Derived Regulatory T Cells.

The transcription factor Helios is expressed in a large percentage of Foxp3+ regulatory T (Treg) cells and is required for the maintenance of their suppressive phenotype, as mice with a selective deficiency of Helios in Treg cells spontaneously develop autoimmunity. However, mice with a deficiency of Helios in all T cells do not exhibit autoimmunity, despite the defect in the suppressor function of their Treg cell population, suggesting that Helios also functions in non-Treg cells. Although Helios is expressed in a small subset of CD4+Foxp3- and CD8+ T cells and its expression is upregulated upon T cell activation, its function in non-Treg cells remains unknown. To examine the function of Helios in CD4+Foxp3- T cells, we transferred Helios-sufficient or -deficient naive CD4+Foxp3- TCR transgenic T cells to normal recipients and examined their capacity to respond to their cognate Ag. Surprisingly, Helios-deficient CD4+ T cells expanded and differentiated into Th1 or Th2 cytokine-producing effectors in a manner similar to wild-type TCR transgenic CD4+ T cells. However, the primed Helios-deficient cells failed to expand upon secondary challenge with Ag. The tolerant state of the Helios-deficient memory T cells was not cell-intrinsic but was due to a small population of Helios-deficient naive T cells that had differentiated into Ag-specific peripheral Treg cells that suppressed the recall response in an Ag-specific manner. These findings demonstrate that Helios plays a role in the determination of CD4+ T cell fate.

Abstract 936: Regulation of CD8+ T-cell function and antitumor activity by DGKα and DGKζ

Abstract Diacylglycerol kinases (DGKs) are a family of enzymes that metabolize the lipid diacylglycerol (DAG) to form phosphatidic acid. In T cells, two isoforms of DGK, DGKα and DGKζ, serve an important role in dampening signal transduction downstream of the T cell receptor (TCR), where they phosphorylate DAG generated by PLCγ1 after activation of proximal signaling molecules. DAG generated downstream of TCR binds to Protein Kinase C θ (PKCθ) and Ras Guanyl Releasing Protein 1 (RasGRP1), facilitating T cell activation. Elimination of either DGK isoform leads to increased generation of DAG and subsequent strengthened signaling downstream of RasGRP1 and PKCθ. Functionally, this results in formation of CD8+ T cells with enhanced cytokine production and proliferation after TCR stimulation. While mice or CD8+ T cells deficient in DGKα or DGKζ are both known to demonstrate enhanced immune responses to tumor, there has not been, to date, a direct comparison of the anti-tumor activity between mice lacking the two isoforms. We sought to determine which isoform of DGK more intensely attenuated anti-tumor immunity in order to identify which would be a better target for immune-based cancer therapies. We inoculated C57BL/6 mice with a variety of syngeneic tumor cell lines - MC38 colon carcinoma, B16F1 melanoma, and C1498 leukemia - and directly compared survival and tumor growth between mice deficient in DGKα or DGKζ in the presence or absence of anti-PD1 treatment. We found that DGKζ-/- mice more efficiently suppressed growth of subcutaneously implanted tumor cells when compared with DGKα-/- mice across the three model systems and further observed that the combination of DGKζ-deficiency and anti-PD1 were additive in tumor control. Additionally, relative to wildtype CD8+ T cells, we found that DGKζ-/- CD8+ T cells demonstrated qualitatively enhanced mRNA changes as assessed by RNA-seq analysis of cells after TCR stimulation. Pathway analyses from RNA-seq similarly indicated enhanced quantitative changes in DGKζ-/- T cells relative to DGKα-/- T cells, findings confirmed with in vitro biochemical and functional assays. Lastly, using CRISPR-based approaches to eliminate DGKα or DGKζ, we observed similar biochemical changes in human T cells when compared with T cells from gene-deficient mice. Our data, employing genetic approaches to modulate DGKα or DGKζ in both human and mouse models, suggests that DGKζ is a dominant regulator of TCR driven T cell activation and is consistent with the observed superior anti-tumor immune response. Citation Format: Susan Wee, Junchen Gu, Cindy Wang, Carolyn Cao, Sandra Holzhauer, Heshani Desilva, Erin Wesley, Susan Tsai, Douglas Evans, Matthew Riese. Regulation of CD8+ T-cell function and antitumor activity by DGKα and DGKζ [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 936.

MicroRNA-155 Controls T Helper Cell Activation During Viral Infection.

MicroRNA (miR) 155 has been implicated in the regulation of innate and adaptive immunity as well as autoimmune processes. Importantly, it has been shown to regulate several antiviral responses, but its contribution to the immune response against cytopathic viruses such as vesicular stomatitis virus (VSV) infections is not known. Using transgenic/recombinant VSV expressing ovalbumin, we show that miR-155 is crucially involved in regulating the T helper cell response against this virus. Our experiments indicate that miR-155 in CD4+ T cells controls their activation, proliferation, and cytokine production in vitro and in vivo upon immunization with OVA as well as during VSV viral infection. Using intravital multiphoton microscopy we analyzed the interaction of antigen presenting cells (APCs) and T cells after OVA immunization and found impaired complex formation when using miR-155 deficient CD4+ T cells compared to wildtype CD4+ T cells ex vivo. In contrast, miR-155 was dispensable for the maturation of myeloid APCs and for their T cell stimulatory capacity. Our data provide the first evidence that miR-155 is required for efficient CD4+ T cell activation during anti-viral defense by allowing robust APC-T cell interaction required for activation and cytokine production of virus specific T cells.

Critical Role for TCR Signal Strength and MHC Specificity in ThPOK-Induced CD4 Helper Lineage Choice.

Sustained TCR signaling is critical for ThPOK induction in MHC class II (MHCII)-signaled thymocytes leading to the CD4 helper lineage commitment. ThPOK suppresses the cytotoxic program in the signaled thymocytes and is shown to be necessary and sufficient for the CD4 helper lineage choice. Accordingly, loss and gain of ThPOK function redirects MHCII- and MHC class I (MHCI)-signaled thymocytes into the CD8 cytotoxic and CD4 helper lineage, respectively. However, the impact of a defined ThPOK level on the CD4 helper lineage choice of MHCII- and MHCI-specific thymocytes and the role of TCR signaling in this process is not evaluated. Equally, it is not clear if suppression of the cytotoxic program by ThPOK is sufficient in redirecting MHCI-restricted thymocytes into the CD4 helper lineage. In this study, we have investigated CD8 to CD4 helper lineage redirection in three independent ThPOK overexpressing transgenic mouse lines. Our analysis shows that one of the transgenic lines, despite overexpressing ThPOK compared with wild-type CD4 mature T cells and compromising cytotoxic program, failed to redirect all MHCI-signaled thymocytes into the CD4 helper lineage, resulting in the continued presence of CD8+ mature T cells and the generation of a large number of double negative mature T cells. Critically, the same ThPOK transgene completely restored the CD4 helper lineage commitment of MHCII-specific Thpok -/- thymocytes. Importantly, augmenting TCR signaling significantly enhanced the ThPOK-mediated CD4 helper lineage choice of MHCI-specific thymocytes but was still substantially less efficient than that of MHCII-specific thymocytes expressing the same amount of ThPOK. Together, these data suggest that the ThPOK-induced CD4 helper lineage commitment is strongly influenced by TCR signal strength and MHC specificity of developing thymocytes.

ITK signaling differentiates GVT and GVHD after allogeneic bone marrow transplantation by regulating IRF-4, JAK/STAT and Eomesodermin expression

Abstract Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective therapy for patients with hematologic malignancies. Donor T cells contained within the graft prevent tumor recurrence by exhibiting graft-versus-tumor (GVT) effects but also cause graft-versus-host disease (GVHD); therefore, novel treatment strategies are needed to maintain GVT while suppressing GVHD. We investigated the role of TCR-mediated ITK activation in mediating GVT vs. GVHD effects after allo-HSCT. We discovered that T cells from ITK−/−mice display a CD62Lhi CD122+ CD44hi Innate Lymphoid Cell-Like (ILCL) functional phenotype, and higher Eomes but not T bet expression compared to WT. We showed that T cells from ITK−/−mice reduced IRF-4, JAK1 and JAK2 and STAT3. T cells from ITK−/− mice displayed reduced cytokine production but showed preserved cytotoxicity after allo-HSCT. These cells also showed defective upregulation of the chemokine receptors CX3CR3, CXCR1, and CXCR6, which correlated with their reduced migration into GVHD target organs. The defective migration of ITK deficient T cells into GVHD target organs contributed to separation of GVHD and GVT effects, since ITK deficeint T cells cleared intravenously injected but not subcutaneously injected tumor cells in allo-HSCT mice. Moreover, pharmacological ITK inhibition attenuated GVHD and preserved GVT function by wild type CD8+ T cells. Together, our data suggest that ITK inhibition could be used as therapy after allo-HSCT to reduce GVHD while preserving the beneficial GVT effects by donor T cells

Intrinsic Alteration of Differentiation and Glycolysis in T Lymphocytes Immediately After TCR Activation Through PSGL-1 Signaling

Abstract P-selectin glycoprotein ligand-1 (PSGL-1) is an adhesion molecule expressed on the surface of naïve, effector, and memory CD4+ and CD8+ T cells. We identified PSGL-1 to be an immune checkpoint inhibitor as antibody-mediated ligation of PSGL-1 promotes T cell exhaustion and deletion of PSGL-1 prevents chronic viral infection and inhibits tumor. To investigate the role of PSGL-1 signaling in the development of T cell responses, we assessed the differentiation state, expansion capacity, and glycolytic profile of PSGL-1-deficient T cells. PSGL-1+/− OT-II CD4+ T cells demonstrated increased skewing towards IL-17A+ T cells compared to wild-type OT-II CD4+ T cells under TH17 conditions, and reduced IFNg+ cells under both TH0 and TH1 conditions. In a B16-OVA tumor model, we observed increased IL-13+ CD4+ T cells among the intratumoral T cell compartment in PSGL-1−/− mice. In vitro activation with a sub-optimal dose of αCD3 of PSGL-1−/− OT-II CD4+ T cells demonstrated increased expansion and expression of CD25 compared to wild-type OT-II CD4+ T cells. Further, adoptively transferred in vitro-activated PSGL-1−/− CD4+ T cells demonstrated greater expansion in vivo upon adoptive transfer into RAG−/− host mice. Using the Seahorse glycolysis stress test, we identified that both CD4+ and CD8+ PSGL-1−/− T cells demonstrate increased glycolysis after 72 hours of in vitro activation compared to wild-type T cells. Further, in situ activation of PSGL-1−/− CD8+ T cells demonstrates that at both sub-optimal and optimal levels of αCD3 stimulation, PSGL-1−/− CD8+T cells have increased glycolysis and increased glycolytic capacity. Taken together, these data show that PSGL-1 signaling has an intrinsic and immediate role in the development of T cell responses.

STAT1 restrains peripheral naive CD8+ T cell responsiveness to homeostatic cytokines by regulating mTORC1 signaling

Abstract Biotechnology and Research Institute for Agriculture and Life Sciences, Seoul National University, South Korea In a steady-state, naïve CD8+ T cells are kept to be quiescent in response to homeostatic cues such as self-ligands and cytokines while preserving a robust responsiveness to foreign antigens. Although such a steady-state quiescence involves multiple regulators, an exact nature of the regulators and underlying mechanisms are still incompletely understood. Here we showed that the signal transducer and activator of transcription 1 (STAT1), a key transcription factor downstream of type I and II interferon receptor signaling, plays a cell-intrinsic role in regulating naïve CD8+ T cell responsiveness to tonic homeostatic cytokines. Thus, mice lacking STAT1 led to abnormalities in the composition and number of naïve and effector/memory phenotype among CD8+ T cells. The observed alterations were not due to dysregulation in thymic development but rather resulted from enhanced proliferative capacity of peripheral mature STAT1-deficient naïve CD8+ T cells in a cell-autonomous manner. In vitro analyses revealed that two key cytokines, IL-7 and type I interferon, serve as a major driver to induce abnormal proliferation of STAT1-deficient but not wild-type CD8+ T cells depending on a pathway that is sensitive to rapamycin, indicating a role of mTORC1 signaling. Based on these findings, we suggest that, under steady-state, naïve CD8+ T cells that are continuously exposed to homeostatic cytokines actively restrain their responsiveness to the “Goldilocks” levels – that is, not too strong to induce activation/proliferation and not too weak to lose a survival ability – presumably via blocking mTORC1 pathway, therefore maintaining a functional state of quiescence before encountering a cognate foreign antigen.

CD4 cell-derived IL-2 synergizes with influenza infection to exacerbate lung inflammation

Abstract Defining the most penetrating correlates of protective memory T cells is key for designing improved vaccines and T cell therapies. Here, we evaluate how interleukin (IL-2) production by CD4 memory cells, a widely held indicator of their protective potential, impacts recall responses against murine influenza A virus (IAV). Unexpectedly, we show that IL-2-deficient memory cells are more effective on a per cell basis at combating IAV than wild-type memory cells marked by strong IL-2 production. Improved outcomes orchestrated by IL-2-deficient cells include reduced weight loss and improved respiratory function that correlate with reduced levels of a broad array of inflammatory factors in the infected lung. Blocking CD70-CD27 signals that reduce T cell IL-2 production tempers the inflammation induced by wild-type memory CD4 cells and improves the outcome of IAV infection in vaccinated mice. Using a reductionist model, we show that IL-2 drives rapid and extremely potent lung inflammation involving NK cells and neutrophils, which can synergize with sublethal IAV infection to promote acute morbidity and mortality. These results suggest that IL-2 production is dispensable for optimal protective CD4 T cell recall responses and that the lung environment is particularly sensitive to IL-2-induced inflammation.

Lymphocyte Activation Gene-3 Maintains Mitochondrial and Metabolic Quiescence in Naive CD4+ T Cells

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed by CD4+ Tcells and tempers their homeostatic expansion. Because CD4+ Tcell proliferation is tightly coupled to bioenergetics, we investigate the role of LAG-3 in modulating naive CD4+ Tcell metabolism. LAG-3 deficiency enhances the metabolic profile of naive CD4+ Tcells by elevating levels of mitochondrial biogenesis. Invivo, LAG-3 blockade partially restores expansion and the metabolic phenotype of wild-type CD4+ Tcells to levels of Lag3-/- CD4+ Tcells, solidifying that LAG-3 controls these processes. Lag3-/- CD4+ Tcells also demonstrate greater signal transducer and activator of transcription 5 (STAT5) activation, enabling resistance to interleukin-7 (IL-7) deprivation. These results implicate this pathway as a target of LAG-3-mediated inhibition. Additionally, enhancement of STAT5 activation, as a result of LAG-3 deficiency, contributes to greater activation potential inthese cells. These results identify an additional mode of regulation elicited by LAG-3 in controlling CD4+ Tcell responses.

Histone H3K27 Demethylase Negatively Controls the Memory Formation of Antigen-Stimulated CD8+ T Cells.

Although the methylation status of histone H3K27 plays a critical role in CD4+ T cell differentiation and its function, the role of Utx histone H3K27 demethylase in the CD8+ T cell-dependent immune response remains unclear. We therefore generated T cell-specific Utx flox/flox Cd4-Cre Tg (Utx KO) mice to determine the role of Utx in CD8+ T cells. Wild-type (WT) and Utx KO mice were infected with Listeria monocytogenes expressing OVA to analyze the immune response of Ag-specific CD8+ T cells. There was no significant difference in the number of Ag-specific CD8+ T cells upon primary infection between WT and Utx KO mice. However, Utx deficiency resulted in more Ag-specific CD8+ T cells upon secondary infection. Adoptive transfer of Utx KO CD8+ T cells resulted in a larger number of memory cells in the primary response than in WT. We observed a decreased gene expression of effector-associated transcription factors, including Prdm1 encoding Blimp1, in Utx KO CD8+ T cells. We confirmed that the trimethylation level of histone H3K27 in the Prdm1 gene loci in the Utx KO cells was higher than in the WT cells. The treatment of CD8+ T cells with Utx-cofactor α-ketoglutarate hampered the memory formation, whereas Utx inhibitor GSK-J4 enhanced the memory formation in WT CD8+ T cells. These data suggest that Utx negatively controls the memory formation of Ag-stimulated CD8+ T cells by epigenetically regulating the gene expression. Based on these findings, we identified a critical link between Utx and the differentiation of Ag-stimulated CD8+ T cells.

Mouse PVRIG Has CD8+ T Cell-Specific Coinhibitory Functions and Dampens Antitumor Immunity.

A limitation to antitumor immunity is the dysfunction of T cells in the tumor microenvironment, in part due to upregulation of coinhibitory receptors such as PD-1. Here, we describe that poliovirus receptor-related immunoglobulin domain protein (PVRIG) acts as a coinhibitory receptor in mice. Murine PVRIG interacted weakly with poliovirus receptor (PVR) but bound poliovirus receptor-like 2 (PVRL2) strongly, making the latter its principal ligand. As in humans, murine NK and NKT cells constitutively expressed PVRIG. However, when compared with humans, less PVRIG transcript and surface protein was detected in murine CD8+ T cells ex vivo However, activated CD8+ T cells upregulated PVRIG expression. In the mouse tumor microenvironment, infiltrating CD8+ T cells expressed PVRIG whereas its ligand, PVRL2, was detected predominantly on myeloid cells and tumor cells, mirroring the expression pattern in human tumors. PVRIG-deficient mouse CD8+ T cells mounted a stronger antigen-specific effector response compared with wild-type CD8+ T cells during acute Listeria monocytogenes infection. Furthermore, enhanced CD8+ T-cell effector function inhibited tumor growth in PVRIG-/- mice compared with wild-type mice and PD-L1 blockade conferred a synergistic antitumor response in PVRIG-/- mice. Therapeutic intervention with antagonistic anti-PVRIG in combination with anti-PD-L1 reduced tumor growth. Taken together, our results suggest PVRIG is an inducible checkpoint receptor and that targeting PVRIG-PVRL2 interactions results in increased CD8+ T-cell function and reduced tumor growth.See related article on p. 257.

Relative Frequencies of Alloantigen-Specific Helper CD4 T Cells and B Cells Determine Mode of Antibody-Mediated Allograft Rejection.

Humoral alloimmunity is now recognized as a major determinant of transplant outcome. MHC glycoprotein is considered a typical T-dependent antigen, but the nature of the T cell alloresponse that underpins alloantibody generation remains poorly understood. Here, we examine how the relative frequencies of alloantigen-specific B cells and helper CD4 T cells influence the humoral alloimmune response and how this relates to antibody-mediated rejection (AMR). An MHC-mismatched murine model of cardiac AMR was developed, in which T cell help for alloantibody responses in T cell deficient (Tcrbd−/−) C57BL/6 recipients against donor H-2Kd MHC class I alloantigen was provided by adoptively transferred “TCR75” CD4 T cells that recognize processed H-2Kd allopeptide via the indirect-pathway. Transfer of large numbers (5 × 105) of TCR75 CD4 T cells was associated with rapid development of robust class-switched anti-H-2Kd humoral alloimmunity and BALB/c heart grafts were rejected promptly (MST 9 days). Grafts were not rejected in T and B cell deficient Rag2−/− recipients that were reconstituted with TCR75 CD4 T cells or in control (non-reconstituted) Tcrbd−/− recipients, suggesting that the transferred TCR75 CD4 T cells were mediating graft rejection principally by providing help for effector alloantibody responses. In support, acutely rejecting BALB/c heart grafts exhibited hallmark features of acute AMR, with widespread complement C4d deposition, whereas cellular rejection was not evident. In addition, passive transfer of immune serum from rejecting mice to Rag2−/− recipients resulted in eventual BALB/c heart allograft rejection (MST 20 days). Despite being long-lived, the alloantibody responses observed at rejection of the BALB/c heart grafts were predominantly generated by extrafollicular foci: splenic germinal center (GC) activity had not yet developed; IgG secreting cells were confined to the splenic red pulp and bridging channels; and, most convincingly, rapid graft rejection still occurred when recipients were reconstituted with similar numbers of Sh2d1a−/− TCR75 CD4 T cells that are genetically incapable of providing T follicular helper cell function for generating GC alloimmunity. Similarly, alloantibody responses generated in Tcrbd−/− recipients reconstituted with smaller number of wild-type TCR75 CD4 T cells (103), although long-lasting, did not have a discernible extrafollicular component, and grafts were rejected much more slowly (MST 50 days). By modeling antibody responses to Hen Egg Lysozyme protein, we confirm that a high ratio of antigen-specific helper T cells to B cells favors development of the extrafollicular response, whereas GC activity is favored by a relatively high ratio of B cells. In summary, a relative abundance of helper CD4 T cells favors development of strong extrafollicular alloantibody responses that mediate acute humoral rejection, without requirement for GC activity.This work is composed of two parts, of which this is Part I. Please read also Part II: Chhabra et al., 2019.

Duox1 Regulates Primary B Cell Function under the Influence of IL-4 through BCR-Mediated Generation of Hydrogen Peroxide.

Engagement of the BCR with Ags triggers signaling pathways for commitment of B lymphocyte responses that can be regulated, in part, by reactive oxygen species. To investigate the functional relevance of reactive oxygen species produced in primary B cells, we focused on the role of the hydrogen peroxide generator Duox1 in stimulated splenic B cells under the influence of the TH2 cytokine IL-4. We found that H2O2 production in wild type (WT) and Nox2-deficient CD19+ B cells was boosted concomitantly with enhanced expression of Duox1 following costimulation with BCR agonists together with IL-4, whereas stimulated Duox1-/- cells showed attenuated H2O2 release. We examined whether Duox1-derived H2O2 contributes to proliferative activity and Ig isotype production in CD19+ cells upon BCR stimulation. Duox1-/- CD19+ B cells showed normal responses of Ig production but a higher rate of proliferation than WT or Nox2-deficient cells. Furthermore, we demonstrated that the H2O2 scavenger catalase mimics the effect of Duox1 deficiency by enhancing proliferation of WT CD19+ B cells in vitro. Results from immunized mice reflected the in vitro observations: T cell-independent Ag induced increased B cell expansion in germinal centers from Duox1-/- mice relative to WT and Nox2-/- mice, whereas immunization with T cell-dependent or -independent Ag elicited normal Ig isotype secretion in the Duox1 mutant mice. These observations, obtained both by in vitro and in vivo approaches, strongly suggest that Duox1-derived hydrogen peroxide negatively regulates proliferative activity but not Ig isotype production in primary splenic CD19+ B cells.

Immune regulation of neuronal injury and repair by observing T cell activation

Background and Hypothesis: 
 It is unknown how the immune system maintains the majority of facial motoneuron (FMN) survival after axotomy. IL-10 cytokine is necessary for FMN survival and CD4+ T cells are activated and play a critical role in survival, but do not produce IL-10. It was proposed that the source of IL-10 resides in the CNS; however, it is possible that antigen presenting cells (APC) produce IL-10 which activate CD4+ T cells to a neuroprotective phenotype. The regulation of IL-10 receptors (IL-10R) in immunodeficient compared to wild-type (WT) mice in the facial nucleus was studied in this experiment, as well as the possibility of the PNS producing IL-10. 
 Experimental Design or Project Methods: 
 To study APC’s role in motoneuron survival, we transferred WT whole splenocytes into global IL-10 knock out (KO) mice prior to axotomy. To study IL-10R gene expression, immunodeficient RAG-2 KO mice received WT or IL-10R-/- CD4+ T cells prior to axotomy. 
 Results: 
 qPCR revealed that WT mice upregulate IL-10R after axotomy, whereas RAG-2 KO mice had decreased expression comparatively. RAG-2 mice who received WT CD4+ T cells transfer restored IL-10R comparable to WT values.IL-10R was rescued in RAG-2 mice after the adoptive transfer of WT CD4+T cells. When IL-10R-/- CD4+ cells were transferred into RAG-2 mice, IL-10R values were restored; however, these T cells were unable to rescue FMN survival. 
 Conclusion and Potential Impact: 
 If WT whole splenocytes transferred into global IL-10 KO mice rescue FMN survival, it implies that APC play a role in producing IL-10. If they cannot mediate rescue, then peripheral IL-10 is unlikely sufficient for FMN survival. CD4+ T cells regulate central IL-10R response and must respond to IL-10 to mediate FMN survival. The transfer of whole splenocytes provides APCs capable of producing IL-10 and CD4+ T cells capable of responding to IL-10.

Expression of the Butyrate/Niacin Receptor, GPR109a on T Cells Plays an Important Role in a Mouse Model of Graft Versus Host Disease

INTRODUCTIONThe intestinal microbiota is essential for the fermentation of fibers into the short-chain fatty acids (SCFA) butyrate, acetate and propionate. SCFA can bind to G-protein-coupled receptors GPR41, GPR43 and GPR109a to activate downstream anti-inflammatory signaling pathways. In colitis or graft versus host disease (GVHD), GPR43 signaling has been reported as an important regulator of intestinal homeostasis by increasing the pool of regulatory T cells. In contrast to GPR43, which binds preferentially propionate and acetate, GPR109a is the major receptor for butyrate. We and others have demonstrated that butyrate can ameliorate gastrointestinal injury during GVHD through enterocyte protection. Therefore, we hypothesized that GPR109a plays an important role in the pathophysiology of intestinal GVHD, focusing specifically on alloreactive T cells. METHODS AND RESULTSUsing mouse models of GVHD, we examined the role of the butyrate/niacin receptor, GPR109a in allogeneic hematopoietic cell transplantation (allo-HCT). First, we studied whether a genetic knock-out (KO) of GPR109a in transplant recipient mice affected GVHD, but GPR109a-KO recipient mice did not exhibit increased mortality from GVHD compared to wild type (WT) mice. We next investigated the role of GPR109a in the donor compartment by transplanting either BM or T cells from WT or GPR109a-KO mice into major MHC mismatched BALB/c host mice. Mice transplanted with B6 BM, with T cells from a GPR109a-KO mouse into BALB/c hosts displayed a lower incidence of lethal GVHD (Fig. 1A). To determine whether the attenuation of GVHD is intrinsic to GPR109a-KO T cells, we established BM chimeras and performed a secondary transplant by transplanting B6 BM + (B6 à Ly5.1) or (GPR109a à Ly5.1) T cells into BALB/c hosts. We observed the same improvement in survival in mice that received GPR109a-KO T cells. This indicates an intrinsic role for GPR109a specifically in the donor hematopoietic compartment.Having identified a T-cell specific requirement for GPR109a we next examined expression of GPR109a on WT T cells in vitro at baseline and following stimulation with CD3/28 and found GPR109a significantly upregulated on both CD4+ and CD8+ T cells after 72 hours of stimulation (Fig 1B). At steady state in vivo, we observed the same numbers and percentages of splenic effector memory, central memory, and naïve CD4+ T cells as well as regulatory T cells in WT B6 mice and GPR109a-KO mice, suggesting normal T cell development in the knockout mice. In an in vitro mixed lymphocyte reaction (MLR), GPR109a-KO CD4+ T cells become activated, proliferate, polarize and secrete cytokine (specifically IFNg) to the same level as WT CD4+ T cells, suggesting normal functional capacity. However, after allo-HCT in mice we observed significantly fewer CD4+ and CD8+ T cells, and specifically fewer effector memory CD4+ T cells (Fig. C), in the small and large intestines of mice that received GPR109a-KO T cells at day 7 post transplant. In contrast, we found significantly more regulatory T cells in the intestines (Fig. 1D) and the spleen of GPR1091-KO T cell recipients, while numbers and percentages of polarized Th1 and Th17 T cells were similar between the two groups. We further 16S rRNA sequenced the gut microbiota of mice at day 7 after transplant and observed an increased relative abundance of bacteria from the genus Clostridium (Fig. 1D) along with an increased concentration of cecal butyrate as measured by GC-MS (Fig. 1E). In a preliminary graft versus tumor (GVT) experiment, we found that mice that received A20 tumor cells and GPR109a-KO T cells exhibited increased survival compared to mice that received A20 tumor cells and WT T cells. These preliminary findings suggest that GPR109a-KO T cells maintain their graft versus tumor response while causing less GVHD, and exclude a defective functional capacity. CONCLUSIONSWe report a novel role of the butyrate/niacin receptor GPR109a on donor T cells in allo-HCT as a genetic knock-out on T cells attenuates lethal GVHD. As these T cells are tested as functionally intact, we propose that the reduction in overall T cells of KO T cell recipients may underlie the attenuation in GVHD. Furthermore, such a reduction in allograft-induced gut injury is accompanied by maintenance of the gut commensal Clostridium and butyrate production, which is known to protect the intestinal epithelium and increases the regulatory T cell pool. DisclosuresNo relevant conflicts of interest to declare.

A Bhlhe40/GM-CSF Axis Potentiates Gastrointestinal Tract Inflammation during Acute Graft Versus Host Disease

Damage to the gastrointestinal tract is the major cause of morbidity during acute graft-versus-host disease (GVHD). While T cells are the proximate drivers of GVHD, disease induction and amplification rely on crosstalk between the innate and adaptive arms of the immune system. The cellular and cytokine networks which mediate this interplay, however, are not well understood. We previously identified a colitogenic CD4+ IL-23R+ CD11c+ T cell population that possesses an innate-like gene expression signature, indicating that these cells appear to be positioned at the interface of the innate and adaptive arms of the immune system. Notably, we also observed that these cells had increased expression of bhlhe40 which is a transcription factor that has been shown to regulate the production of GM-CSF. Given the well characterized ability of GM-CSF to activate myeloid cell populations in autoimmunity, we sought to define the role of this transcription factor and cytokine as a potential bridge between innate and adaptive immunity in GVHD. Using a well-defined murine GVHD model [C57BL/6 (H-2b)→Balb/c (H-2d)], we observed that mice transplanted with Rag-1-/- bone marrow (BM) and CD4+ bhlhe40-/- T cells were completely protected from GVHD, whereas animals transplanted with Rag-1-/- BM and wild type (WT) CD4+ T cells uniformly developed lethal disease. Further analysis revealed that CD4+ bhlhe40-/- T cells produced less GM-CSF and more IL-10 than their WT counterparts, and had preferentially less pathological damage in the colon. To examine the specific role of GM-CSF, we employed the same GVHD model along with a corresponding syngeneic control (B6→B6.PL). We observed robust GM-CSF production in allogeneic, but not syngeneic, recipients in all GVHD target tissues, but most prominently in the colon. This was largely attributable to donor-derived CD4+ T cells, as there was little GM-CSF produced by CD8+ T cells. Notably, whereas the vast majority (~80%) of these cells in the lung and liver also produced IFN-γ, ~50% of GM-CSF-expressing CD4+ T cells in the colon only produced GM-CSF, suggesting that these cells might represent a separate CD4+ T cell lineage. In that regard, antibody blockade of IL-6, IL-23 and IL-27 had no effect on the frequency of CD4+ GM-CSF+ T cells, indicating that the development of these cells was not regulated by cytokines affecting TH1 and TH17 differentiation. To define the functional significance of donor T cell-derived GM-CSF, recipients were transplanted with BM or BM plus splenocytes from WT or GM-CSF-/- animals. Recipients of GM-CSF-/- grafts had significantly increased survival when compared to WT controls. Furthermore, histological analysis demonstrated a significant reduction in pathology in the colon of animals that received GM-CSF-/- grafts, as well as a decrease in infiltrating TH1 cells, whereas there was no difference in pathological damage in the lung or liver. A similar outcome was observed in complementary experiments in which recipient animals that were treated with an anti-GM-CSF antibody had significantly increased survival compared to mice treated with an isotype control antibody. To confirm a role for GM-CSF signaling in CD4+ T cells, Balb/c recipients were transplanted with Rag-1-/- BM alone or together with purified CD4+ T cells from WT or GM-CSF-/- mice. Mice that received CD4+ GM-CSF-/- T cells had a significant increase in survival compared to those that received WT CD4+ T cells, confirming a proinflammatory role for GM-CSF production by donor CD4+ T cells. Given that GM-CSF acts on a diverse subset of innate immune cells, we then examined which myeloid cell subsets were responsive to GM-CSF two weeks post-transplantation when donor APCs have repopulated the APC compartment. Using established markers for macrophages, neutrophils, and dendritic cells, we observed no difference in the number of donor macrophages or neutrophils between groups. However, there was a significant reduction in dendritic cells (DCs) in the colon of mice receiving CD4+ GM-CSF-/- T cells, and donor-derived DCs were virtually absent from the mesenteric lymph nodes, indicating that GM-CSF facilitates the accumulation of DCs in the GI tract and associated lymphoid tissue during GVHD. Collectively, these studies demonstrate that a CD4+ T cell-intrinsic bhlhe40/GM-CSF axis potentiates gastrointestinal inflammation during GVHD by promoting inflammatory cytokine production and DC recruitment. DisclosuresNo relevant conflicts of interest to declare.

Critical Role of Histone Demethylase Jmjd3 in T Cell Migration

Jmjd3, a histone H3K27 demethylase, is known to play a critical role in macrophage and T cell differentiation, but its role in T cell migration and T cell memory maintenance remains largely unknown. In this study, we show that Jmjd3 deficiency resulted in multiple alterations in T cell migration. Jmjd3 deletion limits CD4+ T cells egress out of the thymus, leading to thymic T-cell accumulation and peripheral lymphoid organ T-cell reduction. Gene profiling analysis of wild-type and Jmjd3-deficient CD4+ T cells identified altered expression of Jmjd3 target genes that correlated with changes in H3K27 and/or H3K4 trimethylation in promoters and gene body regions. The expression of the Jmjd3 target gene Pdlim4 is also regulated by Klf2, which regulates T-cell migration. Thus, our findings identify a novel Jmjd3 target gene, Pdlim4, in CD4+ T cell migration and provide insight into the molecular mechanisms by which Jmjd3 regulates T-cell migration. DisclosuresNo relevant conflicts of interest to declare.

Peroxisome Proliferator Activated Receptor-Delta Controls Fat Oxidation in Alloreactive T Cells

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is a curative treatment for high-risk leukemia and multiple non-malignant hematologic disorders. However, the routine use of alloHSCT remains limited by acute graft-versus-host disease (GVHD), where activated donor T cells attack and destroy host tissues in the skin, gastrointestinal tract, and liver. We have previously shown that GVHD-causing T cells increase fat oxidation compared to both syngeneic and naive T cells. To explore this adaptation mechanistically, we studied the role of the transcription factor Peroxisome Proliferator Activated Receptor delta (PPAR-δ) in alloreactive donor T cells during the initiation of GVHD. By day 7 post-transplant, alloreactive T cells up-regulated PPAR-δ >5-fold compared to pre-transplant naive T cells (p<0.0001, Figure 1A). Furthermore, PPAR-δ was necessary for maximally severe GVHD, as major-MHC mismatched B6xDBA2 F1 mice receiving donor T cells deficient in exon 4 of PPAR-δ (PPAR-δ KO) survived longer than mice receiving wildtype (WT) T cells (p<0.007, Figure 1B).We next investigated the mechanism underlying this observed decrease in GVHD severity. As a transcription factor, PPAR-δ controls expression of multiple genes involved in fat transport and oxidation. To determine its role in alloreactive cells, RNA was collected from CD4 and CD8 T cells on day 7 post-transplant and levels of 8 known PPAR-δ targets quantitated by RT-PCR. These 8 targets were selected from a longer list of genes known to be up-regulated in alloreactive cells. Transcript levels of both carnitine palmitoyl transferase-1a (CPT-1a) and CD36 decreased in PPAR-δ KO CD8 T cells (Figure 2A), with decreases in CD36 protein levels confirmed by immunoblot (Figure 2B). Interestingly, changes in CPT-1a and CD36 did not occur in PPAR-δ KO CD4 T cells. To assess the functional consequence of these changes, day 7 WT versus PPAR-δ KO CD8 T cells were plated with 3H-palmitate and fat oxidation measured ex vivo. Consistent with a decrease in expression of genes involved in fat transport and mitochondrial fat import, fat oxidation decreased by >75% in PPAR-δ KO CD8 cells (Figure 2C). However, despite these decreases, the number of PPAR-δ KO CD8 T cells recovered on day 7 post-transplant was equivalent to WT T cells (Figure 3A, left panel). In contrast, PPAR-δ KO CD4 T cell numbers decreased by 30% on day 7, despite equivalent levels of CD36 and CPT1a (Figure 3A, right panel).Finally, we addressed whether pharmacologic inhibition of PPAR-δ might also effectively mitigate GVHD. Administration of the PPAR-δ inhibitor GSK3787 on days 3-6 post-transplant substantially decreased the number of donor T cell recovered on day 7 (Figure 3B), with PPAR-δ impairment corroborated by a decrease in CPT1a gene transcription. However, instead of improving recipient health, GSK3787 treatment instead worsened weight loss and increased rates of post-transplant morbidity and mortality. From these data, we conclude that PPAR-δ is necessary in alloreactive T cells to cause maximally severe GVHD and that mechanistically, an absence of PPAR-δ impairs fat oxidation in CD8 T cells without impacting CD8 T cell numbers. In contrast, PPAR-δ deficiency decreases the number of CD4 T cells post-transplant, but does so without impacting CPT1a or CD36 levels, highlighting clear differences in metabolic reprogramming between CD4 and CD8 alloreactive cells. Finally, our data suggest that systemic inhibition of PPAR-δ post-transplant is not feasible given a sharp increase in toxicity. Future work will elucidate the mechanism of PPAR-δ in CD4 T cells, define the additional metabolic adaptations of CD8 cells which lack PPAR-δ, and determine if similar changes occur in human T cells. Together, these studies will test whether cellular inhibition of PPAR-δ represents a clinically-relevant, future therapy for GVHD. DisclosuresNo relevant conflicts of interest to declare.

During acute graft versus host disease CD28 deletion in donor CD8+ , but not CD4+ , Tcells maintain antileukemia responses in mice.

Donor lymphocyte infusions together with allogeneic hematopoietic stem cell transplantation are routinely used as second-line treatment for hematological malignancies. Mature Tcells in the graft crucially mediate a graft versus leukemia (GvL) response, but also attack healthy tissues in the recipient leading to potentially life-threatening acute graft versus host disease. Using inducible CD28 knockout C57BL/6 mice as T-cell donors, we have now assessed whether CD28 costimulation of donor CD4+ and/ or CD8+ Tcells is required for an efficient GvL effect after allogeneic T-cell transplantation into BALB/c recipients. Our results show that CD28 costimulation of donor CD8+ cytotoxic, but not CD4+ helper, Tcells was dispensable for curing mice from the BCL-1 lymphoma. Therefore, donor lymphocyte infusion treated lymphoma-bearing BALB/c recipient mice showed enhanced long-term survival when receiving CD28-deficient as compared to wild-type donor CD8+ Tcells together with wild-type conventional and regulatory CD4+ Tcells. The same was observed when donor CD8+ and conventional and regulatory CD4+ Tcells were CD28 deficient. Our data, thus, suggest that systemic CD28 blockade, for example, with the drug FR104 might also reduce acute graft versus host disease in patients after allogeneic hematopoietic stem cell transplantation, while maintaining the protective GvL response.

Loss of G\u03b1q impairs regulatory B-cell function

BackgroundRecent studies have shown a crucial role of Gαq in immune regulation, but how Gαq modulates regulatory B-cell (Breg) function is still unclear. We address this here.MethodsCD19+IL-10+ Bregs of wild-type (WT) and Gnaq−/− mice were analyzed by flow cytometry after stimulation by lipopolysaccharide. The WT and Gnaq−/− Bregs were isolated and cocultured with WT CD4+CD25− T cells in the presence of T-activator, and the proliferation of T cells and differentiation of regulatory T cells (Tregs) were analyzed by flow cytometry. We used inhibitors of PI3 kinase (PI3K), extracellular regulated protein kinases 1/2 (Erk1/2), and p38 mitogen-activated protein kinase (p38 MAPK) to detect the pathways involved in the regulation of Gαq on Breg differentiation, which were confirmed by western blot analysis. Furthermore, the expression level of Gαq was assessed by quantitative real-time PCR in peripheral blood mononuclear cells (PBMCs) from healthy controls and rheumatoid arthritis patients. The frequency of CD19+CD24hiCD38hi B cells in PBMCs was detected by flow cytometry, and the association of the Gαq mRNA expression level and the frequency of CD19+CD24hiCD38hi B cells was analyzed by Spearman test.ResultsThe differentiation of CD19+IL-10+ Bregs was inhibited in the Gnaq−/− mice. In addition, Gαq depletion showed an impaired suppressive function of Bregs on T-cell proliferation, which might be due to the decreased Treg expansion. Mechanically, our data demonstrated that the PI3K, Erk1/2, and p38 MAPK signaling pathways were required for regulation of Gαq on Bregs, and blockage of these signaling pathways impaired Breg differentiation. Consistent with our previous studies, we also found a decreased frequency of CD19+CD24hiCD38hi Bregs in rheumatoid arthritis patients. As expected, a significantly positive correlation was investigated between CD19+CD24hiCD38hi Bregs with Gαq mRNA expression.ConclusionsOur results indicate that Gαq plays a critical role in the differentiation and immunosuppression of Bregs, and it may provide a new therapeutic target for autoimmune diseases.