A Critical Role of Host-Derived Semaphorin-4A for Regulating T Cell Immune Responses in Acute Graft Versus Host Disease

Abstract Semaphorins, originally identified as repulsive axon-guidance factors that participate in neuronal development. Several semaphorins are involved in various phases of immune responses, as has been shown recently. Semaphorin4A (Sema4A), a class IV transmembrane semaphorin, is preferentially expressed in dendritic cells (DCs) and Th1 cells. Previous studies suggested that Sema4A is involved in Ag-specific T cell priming, and in Th1 cell and Th17 cell differentiation. Additionally, Sema4A is required for the stability and function of Tregs. However, the role of Sema4A in alloimmune responses remains to be elucidated. In this study, we examined the contribution of Sema4A to T cell immune responses in the context of allogeneic hematopoietic stem cell transplantation (allo-HCT). To test the role of Sema4A in T cell proliferation, we performed in vitro T cell proliferation assay using dendritic cells harvested from wild-type or Sema4A-KO mice. Conventional CD4+T cells cocultured with DCs harvested from wild-type and Sema4A-KO mice proliferated equally in the presence of anti-CD3 antibody. In contrast, anti-CD3-induced proliferation of Tregs cocultured with DCs harvested from Sema4A-KO mice was attenuated compared to Tregs cocultured with DCs harvested from wild-type mice, suggesting that Sema4A is required for maximum proliferation of Tregs. We next investigated the effects of Sema4A deficiency in acute GVHD. We employed a murine acute GVHD model (B6→BALB/c) and monitored every day for survival. Body weight was assessed 2-3 times per week. First, to investigate the role of host Sema4A, lethally irradiated wild-type and Sema4A-KO mice were injected with T cell-depleted bone marrow cells and T cells isolated from wild-type B6. Sema4A-KO host mice developed significantly higher mortality compared to wild-type host mice (p<0.001) (Figure). On day 7 after allo-HCT, the proportion of Tregs in the spleen were significantly decreased in Sema4A-KO host mice compared to wild-type host mice (p<0.05). Both donor-derived preexisting natural Tregs and inducible Tregs in Sema4A-KO host mice were significantly decreased compared to wild-type host mice, although relatively less in inducible Tregs. We investigated the production of proinflammatory cytokines (IFN-g, IL-17, IL-4 and IL-13) of T cells in the spleen by intracellular cytokine staining. The proportion of IL-17+CD4+ T cells in Sema4A-KO host was slightly but not significantly decreased compared to wild-type host, whereas similar proportion of IFN-g+CD4+T cells was observed in Sema4A-KO and wild-type host mice. In contrast, the proportion of IL-4+CD4+ T cells in Sema4A-KO host was significantly increased in Sema4A-KO host mice compared to wild-type host mice, suggesting Sema4A deficiency skewed cytokine polarization of T cells after allo-HCT. Subsequently, we used Sema4A-KO and wild-type B6 mice donor to investigate the role of donor-derived Sema4A. Host mice transplanted from Sema4A-KO donor developed comparable mortality with host mice transplanted with wild-type donor, suggesting that donor-derived Sema4A does not play an important role in controlling graft versus host reactions in this model. Together, these results suggest that Sema4A contributes to Treg maintenance and the regulation of T cell responses after allo-HCT, which may have clinical implications for the novel approach in the treatment and protection of GVHD. Figure. Figure. Disclosures Ito: Bristol-Myers Squibb: Honoraria, Research Funding; Takeda: Honoraria; Novartis Pharma: Honoraria; Pfizer: Honoraria; Mundipharma: Honoraria; Celgene: Honoraria.

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Role of IL-2 secreted by PADRE-specific CD4+ T cells in enhancing E7-specific CD8+ T-cell immune responses

Gene Therapy ◽

10.1038/sj.gt.3303102 ◽

2008 ◽

Vol 15 (9) ◽

pp. 677-687 ◽

Cited By ~ 20

Author(s):

D Kim ◽

A Monie ◽

L He ◽

Y-C Tsai ◽

C-F Hung ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Cd4 T Cells ◽

Cd8 T Cell ◽

T Cell Immune Responses

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Erratum: Role of IL-2 secreted by PADRE-specific CD4+ T cells in enhancing E7-specific CD8+ T-cell immune responses

Gene Therapy ◽

10.1038/gt.2008.63 ◽

2008 ◽

Vol 15 (9) ◽

pp. 702-702

Author(s):

D Kim ◽

A Monie ◽

L He ◽

Y-C Tsai ◽

C-F Hung ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Cd4 T Cells ◽

Cd8 T Cell ◽

T Cell Immune Responses

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Su.67. Monocytes Modulate T-Cell Immune Responses in a Pge2-Cox-2 Dependent Manner and Induce Foxp3+-Cd4+- T-Cells

Clinical Immunology ◽

10.1016/j.clim.2006.04.494 ◽

2006 ◽

Vol 119 ◽

pp. S183

Author(s):

Sheraz Yaqub ◽

Tone Bryn ◽

Milada Mahic ◽

Einar Aandahl ◽

Kjetil Tasken

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Cd4 T Cells ◽

Dependent Manner ◽

T Cell Immune Responses ◽

Cox 2

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Promoting Regulation Via the Inhibition of DNAM-1 After Transplantation

Blood ◽

10.1182/blood.v120.21.338.338 ◽

2012 ◽

Vol 120 (21) ◽

pp. 338-338

Author(s):

Motoko Koyama ◽

Rachel D Kuns ◽

Stuart D Olver ◽

Katie E Lineburg ◽

Mary Lor ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

Steady State ◽

T Cell ◽

Median Survival ◽

Cd4 T Cells ◽

Cell Expansion ◽

Acute Gvhd ◽

Cd4 T Cell

Abstract Abstract 338 Graft-versus-host disease (GVHD) is the major limitation of allogeneic hematopoietic bone marrow transplantation (BMT). Donor T cells play pivotal roles in GVHD and graft-versus-leukemia (GVL) effects and following BMT all T cell fractions, including regulatory T cells (Treg) express the DNAX accessory molecule-1 (DNAM-1, CD226) and T cell Immunoglobulin and ITIM domain (TIGIT) molecule. DNAM-1 is a co-stimulatory and adhesion molecule, expressed mainly by NK cells and CD8+ T cells at steady state to promote adhesion to ligand (CD155, CD112)–expressing targets and enhance cytolysis. TIGIT is a regulatory ligand expressed predominantly by Treg as steady state which competes for CD155 binding, We have analyzed the role of this pathway in GVHD and GVL. Lethally irradiated C3H/Hej (H-2k) mice were injected with bone marrow cells and T cells from MHC disparate wild-type (wt) or DNAM-1–/– C57Bl6 (H-2b) mice. Recipients of DNAM-1–/– grafts were protected from GVHD (survival 67% vs. 7%, P < .0001). We also confirmed the role of DNAM-1 in GVHD in a MHC-matched BMT model (B6 → BALB/B (H-2b)) where GVHD is directed to multiple minor histocompatibility antigens. Next we examined the donor populations expressing DNAM-1 which mediate this effect. DNAM-1 had little impact on acute GVHD severity in the B6 → bm1 BMT model where GVHD is directed against an isolated MHC class I mismatch and is CD8-dependent. In contrast, recipients of wt bone marrow and DNAM-1–/– CD4 T cells survived long-term (compared to recipients of wt CD4 T cells, survival 81% vs. 25%, P = .003) in the B6 → B6C3F1 BMT model, confirming the protection from GVHD is CD4-dependent. Donor CD4 T cell expansion and effector function (Th1 and Th17), and CD8 T cell expansion and cytotoxic function were equivalent in recipients of wt and DNAM-1–/– grafts. However the percentage and number of Treg were significantly increased in recipients of DNAM-1–/– grafts compared to those of wt grafts. The depletion of Treg from donor grafts eliminated the protection from GVHD seen in the absence of DNAM-1 signalling (median survival 16 days vs. 15.5 days, P = 0.53). Adoptive transfer experiments using FACS-sorted Treg were undertaken to compare the relative ability of B6.WT and B6.DNAM-1–/– Treg to suppress GVHD. The majority of recipients of DNAM-1–/– Treg survived beyond day 50 (median survival; day 56), demonstrating a superior ability to suppress acute GVHD relative to wt Treg where the median survival was day 36 (survival 47% vs. 0%, P = .001). These data demonstrate that donor DNAM-1 expression promotes GVHD in a CD4+ T cell-dependent manner via the inhibition of donor Foxp3+ Treg. Finally, the absence of donor DNAM-1 did not influence leukemia-specific mortality in multiple GVL models, regardless of whether the tumor expressed CD155 or not. Thus we demonstrate that the DNAM-1 pathway promotes GVHD, putatively due to competition with TIGIT on Treg, thereby inhibiting regulatory function. This provides support for therapeutic DNAM-1 inhibition to promote tolerance not only after transplant but also in relevant inflammatory based diseases characterized by T cell activation. Disclosures: No relevant conflicts of interest to declare.

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β2- Adrenergic Signaling Regulates Graft Versus Host Disease after Allogenic Transplantation While Preserving Graft Versus Leukemia Effect

Blood ◽

10.1182/blood-2019-123139 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1915-1915 ◽

Cited By ~ 1

Author(s):

Hemn Mohammadpour ◽

Joseph L. Sarow ◽

George L. Chen ◽

Cameron R. MacDonald ◽

Umesh Sharma ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Adrenergic Receptor ◽

Graft Versus Host ◽

Graft Versus Leukemia ◽

Donor T Cells ◽

Β2 Adrenergic Receptor ◽

Ifn Γ

β2 adrenergic receptor signaling is a key regulator of various immune cells, including T cells; however, its role in T cell function in the context of graft versus host disease (GvHD) is poorly understood. We previously showed that housing mice at thermoneutral temperature (TT; 30°C), which reduces systemic adrenergic stress, increased the incidence and severity of GvHD after allogeneic hematopoietic cell transplant (allo-HCT) compared to mice housed at standard temperature (ST; 22°C) which exerts a mild but chronic adrenergic stress (Leigh et al J Immunol 2015). The increased incidence and severity of GvHD in TT mice can be reversed by the administration of a β2-adrenergic receptor (β2-AR) agonist, suggesting an important role of epinephrine and norepinephrine in allo-HCT outcome (Leigh et al., J. Immunol 2015; Mohammadpour et al J Immunol 2018). We investigated the mechanisms and downstream events of β2-AR signaling in donor T cells after allo-HCT by using β2-AR knockout (β2-AR-/-) mice and commercially available β2-AR agonists. The main goal here was to explore whether signaling through β2-AR in donor T cells could control GvHD incidence and severity without minimizing the graft-versus leukemia (GvL) effect. We utilized both a major MHC-mismatch C57B6 (H-2kb) into BALB/c (H-2kd) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2kb) into C3H/SW (H-2kb) model. Recipient BALB/c and C3H/SW WT mice were lethally irradiated with 850 and 1100 cGy respectively and injected by tail vein with T cell depleted bone marrow (TCD-BM) alone (3 ×106) or TCD-BM and splenic T cells derived from allogeneic WT or β2-AR-/- B6 donors (0.7 × 106 T cells in B6 → BALB/c and 1.5 × 106 in B6 → C3H/SW). We found that donor T cells express β2-AR after allo-HCT and that β2-AR expression on WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, and increased survival compared to mice receiving β2-AR-/- donor T cells (Figure 1A). Histopathologic examination showed that β2-AR-/- T cells induced more damage in the small and large intestine. To explore further the mechanism(s) by which β2-AR signaling controls the severity of GvHD, we used NanoString analysis and discovered that β2-AR-/- T cells have the Th1 phenotype with an increase in Tbx21, Ifng, Irf8 and Emoes genes, while WT CD4+ T cells had higher levels of Th2 and Treg associated genes, including Foxp3, Ptgs5, Tgfb2, Il10, Il21 and Il22. We also observed a significant increase in the inflammatory cytokines IFN-γ and IL-17 in β2-AR-/- CD4+ T cells from the spleen and liver on days 7 and 14 after allo-HCT as compared to WT T cells (Figure 1B), while the expression of IL-10 was significantly higher in WT T cells compared to β2-AR-/- T cells (P< 0.01). We next sought to determine whether GvL may be affected by use of long acting β2-AR agonist (Bambuterol) to control GvHD. Bambuterol was administered daily at a dose of 1mg/kg from day 0. We observed that Bambuterol controlled the severity and mortality of GvHD after allo-HCT in both major and minor mismatch mouse models, as evidenced by reduced weight loss and an improved clinical score and survival rate in mice receiving Bambuterol compared to vehicle (P<0.001). We showed that treatment increased the expression of IL-10 and decreased the expression of IFN-γ and IL-17 in CD4+ T cells. Interestingly, we found that β2-AR agonist treatment significantly increased the generation of myeloid derived suppressor cells (MDSCs) from WT BM without any effect on β2-AR-/- BM both in vitro and in vivo, suggesting an important role of β2-AR signaling in the generation of MDSCs. To investigate the effect of Bambuterol on GvL, the A20 lymphoma cell line was injected 4 hours before allo-HCT. Using two different doses of T cells (0.5 × 106 and 0.2 × 106) in B6 → BALB/c model, we found that Bambuterol preserved GvL by inducing CD44+ CD62L- NKG2D+ effector cells and CD44+ CD62L+ central memory cells. Since β2-AR agonists can affect cardiac function, we measured heart rate (HR) and blood pressure (BP) using a tail-cuff. There was no difference in BP and HR at day 21 and 28 after allo-HCT between mice receiving Bambuterol compared to mice receiving vehicle. In conclusion, these data reveal how β-AR signaling can influence donor T cell differentiation and function in murine GvHD models without decreasing GvL effect pointing to the feasibility of manipulation of β2-AR signaling to ameliorate clinical GvHD. Disclosures No relevant conflicts of interest to declare.

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CD25+CD4+ Regulatory T Cells from the Peripheral Blood of Asymptomatic HIV-infected Individuals Regulate CD4+ and CD8+ HIV-specific T Cell Immune Responses In Vitro and Are Associated with Favorable Clinical Markers of Disease Status

Journal of Experimental Medicine ◽

10.1084/jem.20032069 ◽

2004 ◽

Vol 200 (3) ◽

pp. 331-343 ◽

Cited By ~ 338

Author(s):

Audrey L. Kinter ◽

Margaret Hennessey ◽

Alicia Bell ◽

Sarah Kern ◽

Yin Lin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Immune Responses ◽

Cd4 T Cells ◽

Cellular Proliferation ◽

Cell Contact ◽

Hiv Disease ◽

T Cell Immune Responses

Human immunodeficiency virus (HIV) disease is associated with loss of CD4+ T cells, chronic immune activation, and progressive immune dysfunction. HIV-specific responses, particularly those of CD4+ T cells, become impaired early after infection, before the loss of responses directed against other antigens; the basis for this diminution has not been elucidated fully. The potential role of CD25+CD4+ regulatory T cells (T reg cells), previously shown to inhibit immune responses directed against numerous pathogens, as suppressors of HIV-specific T cell responses was investigated. In the majority of healthy HIV-infected individuals, CD25+CD4+ T cells significantly suppressed cellular proliferation and cytokine production by CD4+ and CD8+ T cells in response to HIV antigens/peptides in vitro; these effects were cell contact dependent and IL-10 and TGF-β independent. Individuals with strong HIV-specific CD25+ T reg cell function in vitro had significantly lower levels of plasma viremia and higher CD4+: CD8+ T cell ratios than did those individuals in whom this activity could not be detected. These in vitro data suggest that CD25+CD4+ T reg cells may contribute to the diminution of HIV-specific T cell immune responses in vivo in the early stages of HIV disease.

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Critical Role of T Cells in PF4/Heparin Antibody Production

Blood ◽

10.1182/blood.v124.21.1554.1554 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1554-1554

Author(s):

Yongwei Zheng ◽

Mei Yu ◽

Anand Padmanabhan ◽

Richard H. Aster ◽

Renren Wen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

B Cell ◽

Antibody Production ◽

Cd4 T Cells ◽

Wild Type ◽

Specific Antibodies ◽

Deficient Mice

Abstract Heparin-induced thrombocytopenia (HIT) is an antibody-mediated disorder that can cause arterial or venous thrombosis/thromboembolism, and platelet factor 4 (PF4)/ heparin-reactive antibodies are essential to the pathogenesis of HIT. Our recent studies have demonstrated that marginal zone (MZ) B cells play a major role in production of PF4/heparin-specific antibodies. However, the role of T cells in production of these pathogenic antibodies is not clear. Here we showed that PF4/heparin complex-induced production of PF4/heparin-specific antibodies was markedly impaired in mice, in which CD4 T cells were depleted by administration of GK1.5 anti-CD4 monoclonal antibody. As expected, the CD4 T cell-depleted mice responded normally to T cell-independent antigen TNP-Ficoll but not T cell-dependent antigen NP-CGG, in agreement with the lack of CD4 T cells in these GK1.5-treated mice. Further, following adoptive transfer of a mixture of wild-type splenic B cells and splenocytes from B cell-deficient μMT mice, T and B cell-deficient Rag1 knockout mice responded to PF4/heparin complex challenge to produce PF4/heparin-specific antibodies. In contrast, Rag1-deficient mice that received a mixture of wild-type splenic B cells and splenocytes from Rag1-deficient mice barely produced PF4/heparin-specific antibodies upon PF4/heparin complex challenge. These data suggest that T cells are required for production of PF4/heparin-specific antibodies. Consistent with this concept, mice with B cells lacking CD40 molecule, a B cell costimulatory molecule that helps T cell-dependent B cell responses, displayed a marked reduction of PF4/heparin-specific antibody production following PF4/heparin complex challenge. Also as expected, mice with CD40-deficient B cells were able to respond to T cell-independent antigen TNP-Ficoll but not T cell-dependent antigen NP-CGG, consistent with the lack of T-cell help in these mice. Taken together, these findings demonstrate that T cells play an essential role in production of PF4/heparin-specific antibodies by MZ B cells. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2018-99-118783 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 61-61 ◽

Cited By ~ 2

Author(s):

Melissa D Docampo ◽

Christoph K. Stein-Thoeringer ◽

Amina Lazrak ◽

Marina D Burgos da Silva ◽

Justin Cross ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Regulatory T Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Effector Memory ◽

Knock Out ◽

Graft Versus Host

Abstract INTRODUCTION: The 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 RESULTS: Using 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. CONCLUSIONS: We 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. Disclosures No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2018-99-110807 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 62-62

Author(s):

Clint Piper ◽

Vivan Zhou ◽

Brian T. Edelson ◽

Reshma Taneja ◽

William R. Drobyski

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Myeloid Cell ◽

Cd4 T Cell ◽

Graft Versus Host ◽

Gm Csf ◽

Rag 1 ◽

Csf Production

Abstract 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. Disclosures No relevant conflicts of interest to declare.

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MHC Class II on Ccl19+ Reticular Cells Mitigates Graft-Versus-Host Disease Via Maintenance of Regulatory T Cells

Blood ◽

10.1182/blood-2020-139374 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 23-23

Author(s):

Muhammad Haroon Shaikh ◽

Juan Gamboa Vargas ◽

Josefina Peña Mosca ◽

Duc Dung Le ◽

Hermann Einsele ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class Ii ◽

Cd4 T Cells ◽

Research Funding ◽

Class Ii ◽

Graft Versus Host ◽

Alloreactive T Cells ◽

Reticular Cells

Allogeneic T cell priming is considered as an essential event determining the outcome of allogeneic hematopoietic stem cell transplantation (allo-HCT), ideally triggering anti-leukemic responses (GvL effect) or, at worst, causing life-threatening acute graft-versus-host disease (aGvHD). During aGvHD initiation, alloreactive T cells are activated by host antigen presenting cells (APCs), rapidly expand and subsequently exert tissue damage. Recently, it was discovered that in absence of host hematopoietic APCs, aGvHD cannot be prevented, suggesting a crucial role of non-hematopoietic APCs for priming alloreactive T cells (Toubai et al., Blood 2012, Li et al., J Immunol. 2012, Koyama et al., Nat Med 2012). However, the exact location and identity of host non-hematopoietic APCs triggering alloreactive T cell responses remains controversial and needs to be proven in vivo. Fibroblastic reticular cells (FRCs) have shown to provide the crucial delta-like notch ligand to alloreactive T cells (Chung et al., JCI 2017) in aGvHD, therefore we investigated the role of FRCs MHC class II in aGvHD and their potential role as non-hematopoietic APCs in MHC class II dependent manner. In vitro cultured FRCs cell line as well as FRCs from lethally irradiated mice upregulate MHCII and co-stimulatory molecules. Moreover, FACS sorted FRCs (CD45-CD24-CD31-gp38+) were able to process DQ-OVA via MHC class II machinery, indicating that FRCs have the potential to activate CD4+ T cells. Employing allo-HCT mouse models in combination with flow cytometry and advanced microscopy techniques, we explored early alloreactive T cells activation initially in a myeloablatively conditioned MHC major mismatch allo-HCT setting (FVB/NàC57Bl/6). We generated MHCIIΔCcl19 mice with a Ccl19-intrinsic deletion of MHC class II on all Ccl19 expressing reticular lineage cells by crossing mice with floxed H2-Ab1 gene (H2-Ab1fl) with a mouse expressing Cre recombinase under the control of the Ccl19 promoter (Ccl19Cre). On day+3 after allo-HCT, CD4+ T cells activation (CD44 and CD25 expression) and proliferation (Ki67 expression and CFSE dilution) did not differ in the MHCIIΔCcl19 mice from H2-Ab1fl wildtype littermates. To further elucidate FRCs MHC class II in aGvHD milieu, we utilized iFABP-tOVA transgenic model in which OVA is expressed by intestinal epithelial cells as well as ectopically by FRCs of the lymphoid organs. OT-II cells transferred from RagΔ background mice failed to proliferate in the mLNs of lethally irradiated iFABP-tOVA, whereas excessive proliferation was observed in CD11c.DOG mice (where OVA is presented by CD11c-expressing cells). Taken together these results indicate that MHCII on FRCs does not play a role in direct antigen presentation and CD4+ T cell activation. Next, we asked whether MHCII on FRCs influences alloreactivity of CD4+ T cells in the symptomatic phase of aGvHD. Indeed, in MHCIIΔCcl19 mice, CD4+ T cells expressed higher levels of effector molecules: CD44 and CD127 as well as the proliferation marker Ki67 on day +30 of allo-HCT. Furthermore, the proportion of donor CD90.1+CD4+FoxP3+ regulatory T cells (Tregs) were reduced in MHCIIΔCcl19 mice as compared to H2-Ab1fl wild-type littermates. This led to overall poor survival of MHCIIΔCcl19 mice by day+60 after allo-HCT. At this time point in MHCIIΔCcl19 mice CD4+ T cells displayed higher levels of CD44, CD127 and Ki67 and down-regulated PD-1 and Lag3. To further elucidate the effect of FRCs MHC class II on CD4+FoxP3+ donor Tregs, we transplanted CD90.1+CD4+CD25hi Tregs, TCD BM from FVB mice along with naïve luc+ CD90.1+CD4+ T cells from FVB.L2G85 mice. Tregs protected against aGvHD in H2-Ab1fl littermate controls whereas Tregs could not protect MHCIIΔCcl19 recipients rendering them susceptible to aGvHD and to poor overall survival. Conclusively, these results indicate for the first time that MHC class II on FRCs assists to maintain donor Tregs in the SLOs after allo-HCT. Conclusively, we propose a model in which FRCs promote T cell alloreactivity by providing notch ligands (Chung et al., JCI 2017) in the initiation phase and mitigate aGvHD by maintenance of Tregs via MHC class II in the aGvHD-effector phase. Disclosures Einsele: Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Takeda: Consultancy, Honoraria, Speakers Bureau; Sanofi: Consultancy, Honoraria, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Research Funding, Speakers Bureau.

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