T-Bet Is Critical for the Development of Acute Graft-Versus-Host Disease Through Controlling T Cell Differentiation and Function

Abstract Abstract 452 Background: Allogeneic hematopoietic cell transplantation (HCT) offers great promise for the treatment of hematologic malignancies. However, HCT benefits are frequently offset by graft-versus-host disease (GVHD). Donor T cells can differentiate into Th1 or Th17 subset that contribute to GVHD. The T-box transcription factor T-bet is important for promoting the differentiation of naïve CD4+T cells into Th1 phenotype, while simultaneously inhibiting Th2 and Th17 lineage commitment. Published data indicate that donor T cells deficient for IFN-γ induce exacerbated GVHD. In contrast, our recent study showed that T cells deficient for T-bet were impaired in the induction of GVHD. Given T-bet is a master regulator for the differentiation into Th1 cells that produce IFN-γ, the underlining mechanisms accounted for the distinct outcomes caused by T-bet- versus IFN-γ-deficient donor T cells are not clear. Method: We evaluated the roles of T-bet and IFN-γ in acute GVHD induced by naïve CD4+ T cells or polarized Th17 cells using murine allogeneic bone marrow transplantation (allo-BMT) model. WT, T-bet knockout (KO) and IFN-γ KO mice on C57BL/6 (B6) background were used as donors, and lethally irradiated BALB/c mice were used as recipients. Pathologic analysis and serum cytokine detection were done 14 days after adoptive transfer of WT, T-bet–/–, and IFN-γ–/– CD4+ T cells. Using microarray technology, gene expression profile on donor T cells was analyzed 7 days after adoptive transfer by sorting donor-derived CD4+ T cells from the recipients of WT, T-bet–/– or IFN-γ–/– CD4+ T cells. Results: We compared the ability of WT, T-bet–/–, and IFN-γ–/– CD4 T cells in the induction of acute GVHD. In the comparison with WT cells, IFN-γ–/– CD4 T cells caused similar or even more severe GVHD as expected. In sharp contrast, T-bet–/– CD4 T cells induced much ameliorated GVHD, as significantly higher survival and less body weight loss were observed in the recipients of T-bet–/–T cells. Pathology study on GVHD target organs showed that recipients of T-bet–/– donor T cells had markedly reduced T cell infiltration and tissue damage in liver, gut, and skin, when compared with those of WT or IFN-γ–/– T cells. Reduced GVHD in the recipients of T-bet–/– T cells was consistent with significantly lower levels of pathogenic cytokines IFN-γ, TNF-α, and IL-2 but higher IL-10 (anti-inflammatory), IL-6 (Th17 related) and IL-4 (Th2 related) in serum as compared with those in the recipients of WT T cells. Mechanistic studies in vitro revealed that T-bet–/– CD4 T cells expressed significantly lower levels of IFN-γ, CXCR3 (Th1 specific chemokine receptor) and CD122 (T cell activation marker), but higher levels of IL-17 (Th17 cytokine) and CCR6 (Th17 specific chemokine receptor) compared with WT CD4 T cells, indicating that T-bet–/– T cells impaired in differentiating into Th1 cells and instead into Th17 cells. Given Th17 subset only is capable of causing GVHD and T-bet–/– T cells are prone to Th17-differentiation, we assessed the role of T-bet or IFN-γ in the development of GVHD by comparing the pathogenicity of in vitro polarized WT, T-bet–/– and IFN-γ–/– Th17 cells. While IFN-γ–/– Th17 cells had a comparable ability to cause GVHD compared with WT Th17 cells, T-bet–/– Th17 cells had reduced pathogenicity, and caused ameliorated GVHD. Furthermore, microarray analysis identified genes that are regulated by T-bet but independent of IFN-γ, including Cxcr3, Ccr5, Ccl3, Ccl4, Klrc1, Klrd1, Nkg7 and Pdcd1, which may explain the compromised ability of T-bet−/− not IFN-γ–/–T cells in the induction of GVHD. Conclusions: We conclude that T-bet is required for Th1 differentiation and optimal function of Th17 cells, and it can also control T cell infiltration into GVHD target organs by regulating chemokines and their receptors. Thus, failure in Th1 generation, migration and reduced activity of polarized Th17 cells are likely accounted for impaired ability of T-bet−/− CD4 T cells in the induction of acute GVHD. The current study suggests that targeting T-bet or regulating its downstream effectors independent of IFN-γ may be a promising strategy to control GVHD after allogeneic HCT in clinic. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

In Allogeneic Transplant Recipients Th17 Cells Originate From Donor Stem Cells and Reside In Target Organs of Chronic Graft-Versus-Host Disease

Blood ◽

10.1182/blood.v122.21.5425.5425 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5425-5425

Author(s):

Antonia MS Mueller ◽

Mareike Florek ◽

Natascha J Kuepper ◽

Jessica Poyser ◽

Judith A. Shizuru

Keyword(s):

T Cells ◽

T Cell ◽

Th17 Cells ◽

Acute Gvhd ◽

Chronic Gvhd ◽

Cell Infiltration ◽

Host Disease ◽

T Cell Infiltration ◽

Target Organs ◽

Donor T Cells

Abstract Graft-vs-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation (HCT). Acute GVHD results from activated donor T cells that infiltrate and damage target organs, producing an inflammatory state. In contrast, the pathophysiology of chronic graft-vs-host disease (cGVHD) remains poorly understood. cGVHD can follow acute GVHD or emerge de novo (>d+100). The clinical picture varies and manifestations can resemble autoimmune disorders. Because IL-17 has emerged as a principal cytokine involved in autoimmunity, Th17 cells have attracted much attention in the transplant community. While IFNg-producing Th1 cells appear to drive acute GVHD, the role of Th17 cells in the pathophysiology of GVHD has not been fully clarified. Here, we used an established minor-antigen disparate mouse model of acute and chronic GVHD to examine the emergence of IL-17+CD4+ Th17 cells post-HCT. Lethally irradiated BALB.B mice received pure hematopoietic stem cells (HSC; cKIT+Thy1.1loSca1+Lin-) or HSC plus splenic T cells from C57BL/6 donors (HSC: GFP; TC: CD45.1+). At several time points lymphoid and GVHD target organs were analyzed for donor T cell infiltration and T cell IL-17 expression. In this model recipients of HSC + T cells developed acute GVHD with intestinal involvement (diarrhea, weight loss) and a mortality of ∼30%, while mice given pure HSC remained healthy. Survivors stabilized around d45, but developed clinically evident chronic GVHD after 6-12 mo manifested by sclerodermatous skin excoriations and liver fibrosis/cirrhosis. Donor T cell infiltration of tissues (spleen, lymph nodes (LN), liver, intestines) was high at 2 and 4 wks post-HCT, but there was no detectable IL-17 production by CD4 cells during acute GVHD. The degree of donor T cell infiltration decreased (as acute GVHD improved) in these tissues. However, at 2 mo post-HCT higher percentages CD4+IL-17+ cells were observed, first in intestines and mesenteric LN, followed by liver and skin. At all time points post-HCT proportions of Th17 cells were higher in HCT recipients (of HSC +/- T cells) as compared to normal wild-type (WT) tissues. To summarize, our key findings are: (i) In our model acute GVHD was driven by adoptively transferred mature (CD4+) T cells that acquired a Th1 phenotype, whereas IL-17 producing donor cells were not detectable during this period. IFNg and T-bet are negative regulators of RORgT, the master regulator of Th17. Thus, this observation is consistent with the idea that in the presence of donor Th1 cells the development of Th17 cells is suppressed. (ii) The effect of Th1-related suppression of Th17 persisted beyond the acute phase: recipients of T-cell replete grafts that survived acute GVHD but later developed chronic GVHD did not demonstrate increased CD4+IL-17+ cells. In these mice, organ-infiltrating donor T cells were primarily adoptively transferred T cells, supporting the postulation that no plasticity exists between committed Th1 and Th17 cells. (iii) Signs of chronic GVHD were observed in animals that had not suffered from severe acute GVHD. In particular, in groups without acute GVHD we observed CD4+IL-17+ cells starting at 2 mo, peaking around 6 mo and which stabilized >1 yr post-HCT. In spleen and peripheral LN of these mice only low levels of CD4+IL-17+ cells were detectable, but their proportion was high in GVHD target organs (liver, intestines, skin). The susceptibility of organs appeared to change post-HCT with high proportions of CD4+IL-17+ cells in the intestines at 2 mo post-HCT that decreased over time. In contrast, CD4+IL-17+ cells in the liver increased later in the time course (Figures). (iv) A centrally important observation was that CD4+IL-17+ cells primarily originated from donor HSC, even in recipients of mature donor T cells. Likewise, recipients of pure HSC showed increasing proportions of Th17 cells over time, and could also manifest signs of cGVHD. From our model we conclude that IL-17 does not contribute to acute inflammatory GVHD. However, IL-17 can be involved in an alternative pathophysiologic mode of chronic GVHD development in the absence of acute inflammation. Since CD4+IL-17+ cells derive from donor HSC and undergo maturation in the host this form of GVHD is delayed, and the emergence and activity of these cells appears to constitute a true autoimmune phenomenon. Our novel hypothesis may explain parts of the complex and obscure pathophysiology of chronic GVHD. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

In Vitro Polarized Th17 CD4+ T Cells Home Primarily to Sites of Initial Antigen Encounter

Blood ◽

10.1182/blood.v112.11.2561.2561 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2561-2561

Author(s):

Joseph H. Chewning ◽

Weiwei Zhang ◽

Trenton Schoeb ◽

Casey Weaver

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Th17 Cells ◽

Cd4 T Cell ◽

T Helper ◽

Facs Analysis ◽

Ifn Γ

Abstract The Th1 and Th2 lineages of CD4+ T helper cells are essential for control of host infection. Both lineages respond to antigenic stimulation with distinct effector functions and cytokine profiles. Differential homing patterns permit localization within specific tissue sites where these cells interact with other immune cells to promote the immune response. Variability in T helper lineage homing is due, in part, to differing chemokine receptor expression patterns. This laboratory and others recently described another CD4+ T helper lineage, Th17. Following stimulation, Th17 cells also produce a unique cytokine profile, including interleukin (IL)-17, IL-21, and IL-22. The Th17 lineage has now been implicated in the pathogenesis of several human autoimmune diseases, including psoriasis and inflammatory bowel disease, and appears to be critical for the inflammation of both the skin and gastrointestinal tract, respectively, seen in these diseases. It is not well understood whether Th17 cells arise within the inflammatory milieu in these tissues, or whether these cells possess a distinct homing pattern. We have performed studies using in vitro polarized Th17 cells for the study of tissue homing patterns in vivo. Experiments were performed using the well-described HLA Class II-disparate C57BL/6 (B6) to B6.C-H-2bm12 (bm12) model. Previous studies have established CD4+ T cell-dependent inflammation in this model. Naïve CD4+ T cells from B6 mice were polarized to the Th17 lineage in vitro using standard techniques, including IL-6 and TGF-β. FACS analysis of the Th17 cells prior to adoptive transfer revealed IL-17-positive staining in >60% cells and IFN-γ-positivity in <10%. Th17 or Th2-polarized control cells (1 × 106) were transferred into lethally irradiated bm12 mice (or syngeneic B6 control mice). Mice receiving Th17 cells demonstrated weight gain in the initial weeks compared to Th2 control recipients, but less than B6 syngeneic recipients. The Th17 recipients appeared less active, however, and most mice in this group eventually became moribund, requiring euthanasia. Complete necropsy was performed on mice from each group at intervals following transfer. Tissue analysis in the Th17 recipients revealed marked inflammation within the lungs, skin, liver, and gastrointestinal tract. Syngeneic B6 recipients of Th17 cells also demonstrated a similar tissue pattern, but with markedly reduced inflammation. Tissues from the bm12 Th2-polarized cell control mice, as well as T cell depleted marrow alone recipients did not demonstrate significant inflammation. Additional time course experiments revealed the initial target organs affected as the lungs and stomach, with subsequent involvement of other affected organs. FACS analysis of recipient hematopoietic tissues, using CD45.1 isotype distinction, revealed Th17 cell proliferation within the bm12 allogeneic recipients compared to the B6 syngeneic recipient mice (25–35% total cells of donor origin compared to 2–8%, respectively). CD4+ T cell counts performed on recipient spleens confirmed increased proliferation of Th17 cells within the allogeneic recipient compared to Th2 allogeneic and Th17 syngeneic controls (108 total donor-derived cells compared to 106 and 107, respectively). Cytokine analysis was performed by FACS on CD4+ T cells harvested from tissues. In contrast to pre-transfer analysis, the transferred CD4+ T cells harvested from allogeneic bm12 recipients secreted increased amounts of IFN-γ (12–33%) concomitant with a decrease in IL-17 production. Our studies demonstrate that Th17 CD4+ T cells are able to home to mucosal sites of early antigen encounter, in both the allogeneic and syngeneic setting. This pattern is consistent with the known role of IL-17 in innate immune response to infection. In the setting of chronic T cell stimulation, we also observed that Th17 cells can transition to a Th1-like, IFN-γ-producing CD4+ T cell. The skin, lungs, and GI tract are important sites of initial antigen encounter, and understanding the CD4+ Th17 T cell homing and proliferation patterns could have important implications in understanding both innate and adaptive immune responses to acute infection. Ongoing studies are underway to identify the role of specific chemokine receptors responsible for Th17 homing.

Download Full-text

Absence of Inducible Costimulator (ICOS) on Alloreactive T Cells Reduces Graft-versus-Host-Disease While Leaving Graft-Versus-Tumor Activity Intact.

Blood ◽

10.1182/blood.v104.11.592.592 ◽

2004 ◽

Vol 104 (11) ◽

pp. 592-592

Author(s):

Vanessa M. Hubbard ◽

Jeffrey M. Eng ◽

Kartono H. Tjoe ◽

Teresa Ramirez-Montagut ◽

Stephanie J. Muriglan ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class I ◽

Graft Versus Host ◽

Cell Numbers ◽

Alloreactive T Cells ◽

Inducible Costimulator ◽

Target Organs ◽

Donor T Cells ◽

Ifn Γ

Abstract Inducible costimulator (ICOS) is a member of the B7 family that is expressed on activated and memory T cells and is involved in the regulation of TH1 and TH2 effector cytokine production after CD3/TCR stimulation. Studies with ICOS inhibition or ICOS−/− recipients have demonstrated prolonged allograft survival after heart or liver transplantation in animal models. To study the role of ICOS expression on alloreactive T cells in graft-versus-host disease (GVHD), we used allogeneic MHC class I and II disparate hematopoietic stem cell transplantation (allo-HSCT) models. We first analyzed the expression of ICOS by transferring CFSE-labeled donor T cells into irradiated allogeneic recipients and observed an increased expression of ICOS on alloreactive T cells compared to non-alloreactive T cells. We then studied B6-ICOS−/− alloreactive T cells and found intact proliferation in vivo (as determined by adoptive transfer of CFSE labeled T cells and donor T cell numbers in the spleen of allo-HSCT recipients), intact cytotoxicity, intact up regulation of activation markers, but decreased IFN-γ production in vitro. We then performed GVHD experiments in two models with full MHC class I and II disparity and observed significantly less GVHD morbidity and mortality in recipients of ICOS−/− donor T cells. Furthermore, histopathological analysis demonstrated less GVHD in all target organs (skin, liver, small bowel and large bowel) of recipients of ICOS−/− splenic T cells compared to recipients of wild type T cells. We harvested target organs (spleen, thymus, liver and gut) on days 7, 14, and 21 to examine donor T cell content (naïve and activated T cells) and found no significant difference in the total T cell numbers and subpopulations. Interestingly, in GVHD/graft-versus-tumor (GVT) experiments, ICOS−/− donor T cells displayed intact GVT activity, while their GVH activity was diminished. We then tested the levels of IFN-γ in the sera of mice undergoing GVHD and observed decreased serum levels in recipients of B6-ICOS−/− T cells. In conclusion, alloreactive ICOS−/− donor T cells display less GVHD morbidity and mortality due to decreased IFN-γ production, while proliferation, infiltration and GVT activity remain intact. These data suggests that strategies to inhibit ICOS could be useful for the prevention and/or treatment of GVHD in recipients of an allo-HSCT.

Download Full-text

Adoptive T Cell Therapy of Human CMV Infection in Immunocompromised Hosts: In Vitro Generation of CMV-Specific T Cells from Naïve Precursors

Blood ◽

10.1182/blood.v112.11.206.206 ◽

2008 ◽

Vol 112 (11) ◽

pp. 206-206 ◽

Cited By ~ 1

Author(s):

Sonja Schmucker ◽

Mario Assenmacher ◽

Jurgen Schmitz ◽

Anne Richter

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Peptide Pool ◽

Adoptive T Cell Therapy ◽

T Cell Therapy ◽

A Cell ◽

Ifn Γ

Abstract Adoptive transfer of virus-specific T cells is a promising therapy for the treatment of infections in immunocompromised patients. Virus-specific T cells can readily be obtained from antigen-experienced, but not naïve donors. In this study we describe a cell culture system for the in vitro generation of CMV-specific T cells from naive T cells derived from CMV-seronegative donors. We isolated naïve T cells by magnetic depletion of non-T cells, CD25+ regulatory T cells, and CD45RO+ effector and memory T cells from peripheral blood mononuclear cells (PBMC) of CMV-seronegative donors. These naïve T cells were co-cultured with autologous mature monocyte-derived DC (MoDC) loaded with a pool of overlapping peptides from the CMV protein pp65. CD3-depleted autologous PBMC were used as feeder cells and CD28 antibody, IL-2, IL-7, and IL-15 were added to the culture. Already only 9–13 days after starting the priming culture, frequencies of 0.0024% and 0.009% pp65495–503/A2-tetramer+ cells among CD8+ T cells were found for 2 HLA-A2+ blood donors. In contrast pp65495–503/A2-tetramer+ T cells were not detectable when naive T cells were cultured with unpulsed MoDC. Tetramers are suitable tools for the identification of antigen-specific T cells but are restricted to single epitopes of mainly CD8+ T cells. To analyze primed CD4+ T cells as well as CD8+ T cells having specificities other than for the peptide pp65495–503, we looked for upregulation of the activation marker CD137 after a second stimulation and found increased frequencies of CD137+ CD4+ T cells as well as CD137+ CD8+ T cells in the pp65-primed cell cultures only when restimulated with the peptide pool of pp65. Because IFN-γ is important for the control of CMV infection, we studied the capability of the in vitro primed pp65-specific CD4+ and CD8+ T cells to produce this cytokine. Restimulation of the T cells with pp65 peptide pool induced IFN-γ secretion in up to 3.9% of the CD8+ T cells and up to 3.8% of the CD4+ T cells in each of six donors tested. No specific IFN-γ production was detected after restimulation with an irrelevant IE-1 peptide pool. As expected the frequency of pp65-specific T cells in the priming cultures is low. For generation of T cell lines, we magnetically enrich pp65- specific T cells according to their IFN-γ secretion using the cytokine secretion assay technology. After further cultivation for 2 weeks the antigen-specificity of the expanded T cells was again evaluated. Only if restimulated with the pp65 peptide pool 56.6% of the CD4+ T cells showed upregulated expression of the activation marker CD154 (CD40L). Cytokine analysis of the cells revealed IFN-γ production in 40.2% of the CD4+ T cells, of which 36% co-expressed IL-2, indicating the functionality of the in vitro primed and expanded T cells. In conclusion, we established a cell culture system for in vitro priming of CMV-specific CD4+ and CD8+ T cells derived from peripheral blood of donors not infected by CMV. This should extend the application of adoptive T cell therapy to patients for whom immune donors are not available.

Download Full-text

IL-17A-Expressing CD4+ T Cells Must Acquire the Expression of T-Bet and IFN-γto Destroy Tumor Cells In Vivo.

Blood ◽

10.1182/blood.v114.22.468.468 ◽

2009 ◽

Vol 114 (22) ◽

pp. 468-468

Author(s):

Pawel Muranski ◽

Sid P Kerkar ◽

Zachary A Borman ◽

Robert Reger ◽

Luis Sanchez-Perez ◽

...

Keyword(s):

T Cells ◽

Treatment Efficacy ◽

Cd4 T Cells ◽

Th17 Cells ◽

End Effector ◽

Polarized Cells ◽

Ifn Γ

Abstract Abstract 468 We have recently demonstrated that Th17-polarized TCR transgenic CD4+ T cells specific for TRP-1 melanoma antigen are superior to Th1-polarized cells in mediating effective anti-tumor responses against advanced disease after adoptive transfer. The therapeutic activity of Th17-skewed cells is critically dependent on their ability to secrete IFN-γ, suggesting that the Th17 subset might evolve in vivo. However, the developmental program of Th17-polarized cells in vivo remains substantially un- elucidated. We developed a novel TCR-transduction technique that enabled us to rapidly confer specificity for a cognate antigen upon any population of T cells, regardless of its genetic background, its previous polarization history or its state of differentiation. Using adoptive transfers into tumor-bearing hosts, we were able to study the functionality of these genetically-engineered T cells in vivo. In vitro, CD4+ T cells cultured in type 17 conditions acquired end-effector phenotype (CD62Llow, CD45RBlow), but proliferated slower than cells grown in type 1 condition. Thus, we hypothesized that Th17-polarized cells might represent a less mature, more central-memory like subset. This notion was supported by their ability to secrete high quantities of IL-2 and higher expression of IL-7 receptor. In contrast, Th1-polarized cells upon in vitro re-stimulation upregulated PRDM1 that encodes BLIMP1, a molecule associated with the end-effector senescent phenotype. Moreover, Th1-skewed cells overexpressed caspase 3 and were prone to activation-induced cell death as measured by annexin V assay, while type 17 cells were resistant to apoptosis, and robustly expanded in secondary cultures. Using the TCR gene transfer technique we tested the treatment outcomes when Th17-polarized cells deficient for IL-17A were used. In contrast to wild-type (WT)-derived Th17 cells that effectively eradicated established tumors, we observed significant impairment of treatment with IL-17A-deficent cells. Similarly, we observed reduction in treatment efficacy when CCR6-deficient Th17 cells were transferred. CCR6 is a receptor for CCL20, a chemokine highly induced Th17 cells and thought to contribute to the trafficking of those cells to the site of inflammation. In both cases however, the addition of exogenous vaccination and IL-2 significantly improved treatment efficacy. Thus, we concluded that Th17-associated factors play the role in the anti-cancer activity of type 17 cells. To address the question whether plasticity of Th17-skewed effectors is important for their function upon ACT, we treated animals with TCR-transduced Th17-skewed cells derived from IFN-γ-deficient CD4+ cells as well as from t-bet-deficient mice, which are not able to develop type 1 responses. In contrast to WT-derived Th17 effectors, IFN-γ-deficient cells did not show any anti-tumor activity, while t-bet-deficient Th17 cells were able to mediate only minimal delay in tumor growth, suggesting that indeed the capacity to acquire Th1-like properties is essential for the anti-tumor function of Th17-skewed lymphocytes. Overall, here we demonstrate that TCR gene engineered Th17-polarized cells can efficiently treat advanced tumor. The high activity of in vitro-generated anti-tumor Th17 cells relies on the contribution of type 17-associated characteristics, including both the secretion of inflammatory factors IL-17A and CCL20, as well as the superior capacity to survive and expand upon the secondary stimulation. Importantly however, type 1-defining t-bet-mediated plasticity in the lineage commitment is required for the full therapeutic effect, underscoring the dualistic nature of Th17-skewed cells. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Hyperactivable NFAT1 Ameliorates Autoimmune Encephalitis In Vivo.

Blood ◽

10.1182/blood.v114.22.711.711 ◽

2009 ◽

Vol 114 (22) ◽

pp. 711-711

Author(s):

Srimoyee Ghosh ◽

Sergei B Koralov ◽

Irena Stevanovic ◽

Mark S Sundrud ◽

Yoshiteru Sasaki ◽

...

Keyword(s):

T Cells ◽

Transgenic Mice ◽

Cd4 T Cells ◽

Th17 Cells ◽

Autoimmune Encephalitis ◽

Cell Types ◽

Wild Type ◽

Ifn Γ

Abstract Abstract 711 Naïve CD4 T cells differentiate into diverse effector and regulatory subsets to coordinate the adaptive immune response. TH1 and TH2 effector subsets produce IFN-γ and IL-4, respectively, whereas proinflammatory TH17 cells are key regulators of autoimmune inflammation, characteristically produce IL-17 and IL-22 and differentiate in the presence of inflammatory cytokines like IL-6 and IL-21 together with TGF-β. Naive T cells can also differentiate into tissue-protective induced T regulatory (iTreg) cells. NFAT proteins are highly phosphorylated and reside in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. In this study, we investigated the role of the Ca/NFAT signaling pathway in regulating T cell differentiation and the development of autoimmune diseases. We generated transgenic mice conditionally expressing a hyperactivable version of NFAT1 (AV-NFAT1) from the ROSA26 locus. To restrict AV-NFAT1 expression to the T cell compartment, ROSA26-AV-NFAT1 transgenic mice were bred to CD4-Cre transgenic mice. Naïve CD4 T cells freshly isolated from AV mice produced significantly less IL-2 but increased amounts of the inhibitory cytokine IL-10. To investigate the role of NFAT1 in the generation of TH1, TH2, Tregand TH17 cells, the respective cell types were generated from CD4 T cells of AV mice by in vitro differentiation. T cells from AV-NFAT1 mice exhibited a dysregulation of cytokine expression, producing more IFN-γ and less IL-4. While the numbers of CD4+CD25+ “natural” Treg cells in peripheral lymphoid organs and their in vitro suppressive functions were slightly decreased in AV mice, iTreg generation from CD4+CD25- T cells of AV mice as compared to wild type cells was markedly enhanced. Moreover, TH17 cells generated in vitro from CD4 T cells of AV mice in the presence of IL-6, IL-21 and TGF-β exhibited dramatically increased expression of both IL-10 and IL-17 as compared to wild type controls. To investigate putative NFAT binding sites in the IL-10 and IL-17 gene loci, we performed chromatin immunoprecipitation experiments. We show that NFAT1 can bind at the IL-17 locus at 3 out of 9 CNS regions which are accessible specifically during TH17 but not during TH1 and TH2 differentiation. Furthermore, we provide evidence that NFAT1 binds one CNS region in the IL10-locus in TH17 cells. To verify our observations in vivo, we induced experimental autoimmune encephalitis (EAE) in AV mice and wild type controls with the immunodominant myelin antigen MOG33-55 emulsified in complete Freund‘s adjuvant. While wild type animals showed a normal course of disease with development of tail and hind limb paralysis after approximately 10 days, AV mice showed a markedly weaker disease phenotype with less severe degrees of paralysis and accelerated kinetics of remission. Moreover at the peak of the response, there were fewer CD4+CD25- but more CD4+CD25+ T cells in the CNS of AV animals compared to wild type controls. Surprisingly, these cells produced significantly more IL-2, IL-17 and IFN-γ upon restimulation, even though they displayed decreased disease. In summary, our data provide strong evidence that NFAT1 contributes to the regulation of IL-10 and IL-17 expression in TH17 cells and show that increasing NFAT1 activity can ameliorate autoimmune encephalitis. This could occur in part through upregulation of IL-10 expression as observed in vitro, but is also likely to reflect increased infiltration of regulatory T cells into the CNS as well as increased conversion of conventional T cells into Foxp3+ regulatory T cells within the CNS. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Absence of Coronin 1B in Donor T Cells Diminishes Acute Gvhd By Impairing T Cell Accumulation in Secondary Lymphoid Tissue

Blood ◽

10.1182/blood.v126.23.1880.1880 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1880-1880

Author(s):

Trisha Dant ◽

Danny Bruce ◽

Leshara Fulton ◽

Michelle West ◽

Niko Foger ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Lymphoid Tissue ◽

Acute Gvhd ◽

Wild Type ◽

Target Organs ◽

Donor Cells ◽

Donor T Cells

Abstract Allogeneic stem cell transplant is a standard treatment for patients with high-risk and relapsed myeloid and lymphoid malignancies. However, donor T cells from the stem cell graft mediate graft-versus-host disease (GVHD), which is a common cause of morbidity and mortality for transplant recipients. Our group and others have shown that migration of donor T cells into secondary lymphoid tissue (SLT) and subsequent migration to target organs is critical to the pathogenesis of acute GVHD. The Coronin family of proteins consists of actin-binding proteins, which regulate filament formation by interacting with the Arp2/3 complex. Coronin 1B, a ubiquitously expressed member of the Coronin family, is required for lamellipodial protrusion and effective cell migration. Previous work has not evaluated a role for this protein in the function of T lymphocytes or during acute GVHD. To evaluate the effect of Coronin 1B in acute GVHD pathogenesis, we transplanted B6 T cell depleted bone marrow cells with wild type or Coronin 1B-/- T cells to lethally irradiated B6D2 and BALB/c recipient mice and evaluated clinical score of GVHD and overall survival. B6D2 recipients of Coronin 1B-/- T cells demonstrated 100% survival (Figure 1A. p< .001 as determined by Log-rank (Mantel-Cox) test) and significantly decreased clinical scores after transplant. This was confirmed with improvement in survival in BALB/c recipients of Coronin 1B-/- T cells. Additionally, Coronin 1B-/- T cells were capable of eliminating P815 tumor cells, indicating that loss of Coronin 1B does not inhibit graft-versus-tumor activity. By day 12 post- transplant, all mice receiving bone marrow alone developed tumor compared to none of the mice receiving Coronin 1B-/- T Cells. However, protection was not complete as 40% of Coronin 1B-/- T cell recipients developed tumor by day 23. To determine the effect of Coronin 1B on T cell migration during GVHD, B6D2 recipients were given GFP-expressing wild type or Coronin 1B-/- T cells along with T cell depleted bone marrow. Lymphoid tissue and target organs were harvested and analyzed by flow cytometry or GFP ELISA. We observed decreased accumulation of Coronin 1B-/- CD4+ (Figure 1B. p< .01 as determined by Student's t -test) and CD8+ T cells in the inguinal lymph node, mesenteric lymph node, and the spleen 4 days after transplant with no difference in accumulation in lymphoid tissue on days 7 and 14 after transplant. Additionally, we found decreased accumulation of Coronin 1B-/- donor T cells in the lung, colon and spleen 14 days after transplant (Figure 1C. p< .05 by Student's t -test). We also quantified the amount of cytokine in target organs by ELISA, and observed a decrease in IFN-γ and TNF-α in the colon 14 days after transplant. Our data demonstrate that Coronin 1B-/- T cells elicit reduced GVHD compared to wild type T cells. This was correlated with decreased accumulation of Coronin 1B-/- T cells in SLT early after transplant. These data indicate that targeting the migration of T cells to SLT is a viable approach to prevent acute GVHD. Figure 1. (A) Kaplan Meier curve comparing B6D2 recipients of Coronin 1B-/- T cells and wild type (WT) T Cells. (B) Decreased accumulation of Coronin 1B-/- T Cells 4 Days after transplant. For panels (B) and (C) black bars indicate recipients of WT T cells while red bars indicate recipients of Coronin 1B-/- T cells. Inguinal lymph nodes (ILN) were pooled from n=5 mice from each group. Spleens were analyzed individually. GFP expressing donor cells were analyzed by flow cytometry. Representative image of two experiments. (C) Coronin 1B-/- T cells express decreased accumulation in the lung, colon and spleen 14 days after transplant. Target organs were analyzed by GFP ELISA to detect GFP+ Donor Cells (n=5 in each group). Figure 1. (A) Kaplan Meier curve comparing B6D2 recipients of Coronin 1B-/- T cells and wild type (WT) T Cells. (B) Decreased accumulation of Coronin 1B-/- T Cells 4 Days after transplant. For panels (B) and (C) black bars indicate recipients of WT T cells while red bars indicate recipients of Coronin 1B-/- T cells. Inguinal lymph nodes (ILN) were pooled from n=5 mice from each group. Spleens were analyzed individually. GFP expressing donor cells were analyzed by flow cytometry. Representative image of two experiments. (C) Coronin 1B-/- T cells express decreased accumulation in the lung, colon and spleen 14 days after transplant. Target organs were analyzed by GFP ELISA to detect GFP+ Donor Cells (n=5 in each group). Disclosures No relevant conflicts of interest to declare.

Download Full-text

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.

Download Full-text

Critical and Opposing Effects of T Cell-Derived TNFα and Interferon-γ on IL-17 Induction Assessed in Vitro and in Vivo Following Allogeneic Bone Marrow Transplantation

Blood ◽

10.1182/blood.v112.11.3482.3482 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3482-3482

Author(s):

Minghui Li ◽

Kai Sun ◽

Mark Hubbard ◽

Doug Redelman ◽

Angela Panoskaltsis-Mortari ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Th17 Cells ◽

Allogeneic Bone Marrow Transplantation ◽

Allogeneic Bone ◽

Allogeneic Bmt

Abstract IL-17-producing CD4 T cells (Th17) are a recently identified T helper subset that plays a role in mediating host defense to extracellular bacteria infections and is involved in the pathogenesis of many autoimmune diseases. In vitro induction of IL-17 in murine CD4+ T cells has been shown to be dependent on the presence of the proinflammatory cytokines TGF-β and IL-6 whereas IFNγ can suppress the development of Th17 cells. In the current study, we examined the roles of TNFα and IFNγ on IL-17 production by purified T cells in vitro and in vivo after allogeneic bone marrow transplantation (BMT). We present findings that expression of TNFα by the T cell itself is necessary for optimal development of Th17 under in vitro polarizing conditions. A novel role for T cell-derived TNFα in Th17 induction was observed when in vitro polarization of Tnf−/−CD4+ T cells resulted in marked reductions in IL-17+CD4+ T cells compared to Tnf+/+CD4+ T cells. In marked contrast, T cell-derived IFNγ markedly inhibited Th17 development as more IL-17+CD4+ T cells were found in Ifnγ−/−CD4+ T cells than in Ifnγ+/+CD4+ T cells, and of particular interest was the dramatic increase in IL-17+CD8+ cells from Ifnγ−/− mice. To determine if T cell-derived TNFα or IFNγ can regulate Th17 development in vivo we examined the differentiation of alloreactive donor T cells following allogeneic BMT. We have found that donor-derived Th17 cells can be found in lymphoid tissues and GVHD-affected organs after allogeneic BMT. However, transfer of Tnf−/− CD4+ T cells after allogeneic BMT resulted in marked reductions in Th17 cells in the spleen (18×103 vs 7×103, P<0.05). In agreement with the in vitro data and in contrast to what was observed with transfer of Tnf−/− CD4+ T cells, transfer of donor Ifnγ−/− T cells resulted in marked increases in not only IL-17+CD4+ but also IL-17+CD8+ T cells infiltrating the liver (7×103 vs 14×103, P<0.05; 4×104 vs 12.5×104, P<0.05). These results suggest that the donor T cell-derived TNFα and IFNγ opposingly regulate IL-17 induction of both CD4+ and CD8+ T cells in vitro and after allogeneic BMT which correlates with GVHD pathology.

Download Full-text

Donor T Cells Intrinsic Responses to Damps Regulated By Siglec-G-CD24 Axis Mitigate Gvhd but Maintain GVL in Experimental BMT Model

Blood ◽

10.1182/blood.v126.23.229.229 ◽

2015 ◽

Vol 126 (23) ◽

pp. 229-229 ◽

Cited By ~ 1

Author(s):

Tomomi Toubai ◽

Corinne Rossi ◽

Katherine Oravecz-Wilson ◽

Nathan Mathewson ◽

Cynthia Zajac ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

T Cell Responses ◽

Innate Immune ◽

T Cell Subsets ◽

Activation Markers ◽

Cell Responses ◽

Target Organs ◽

Donor T Cells

Abstract Innate immune receptors like pattern recognition receptors (PRRs) including toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD) like-receptors (NLR) on immune cells play an important role in initiating inflammatory responses to damage- and pathogen- associated molecular patterns (DAMPs and PAMPs) expressed on invading pathogens or released from damaged cells. Although it is well known that DAMPs directly modulate innate immune functions, it is less clear whether DAMPs directly regulate T cell intrinsic function. Members of the sialic acid binding Ig-like lectin (Siglec) family have immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM-like regions in their intracellular domain that negatively regulate immune activation induced by DAMPs. Our previous data suggested that the Siglec- G-CD24 interaction in host APCs plays an important role in the negative regulation of graft-versus host (GVH) responses. However, the T cell autonomous role of Siglec-G in the regulation of T cell responses is not known. Because Siglecs are important negative regulators of immune responses, we tested the hypothesis that the deficiency of Siglec-G in donor T cells would enhance GVHD. To test our hypothesis, we first examined detailed phenotypic analysis of various T cell subsets and activation markers in naïve Siglec-G-/- and wild-type (WT) B6 animals and found similar distribution of naïve, memory, effector and regulatory T cells. In order to examine whether the absence of Siglec-G in donors affects GVHD, WT-BALB/cmice were lethally irradiated (850cGy) and transplanted on day 0 with 5x106 bone marrow and 0.5x106 splenic CD90+ T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or Siglec-G-/- animals. The recipients receiving donor T cells from Siglec-G-/- animals showed a significantly worse survival compared to allogeneic WT-B6 animals (p<0.05). This increased mortality was also associated with more severe GVHD damage in target organs and a higher expansion of activated CD69+, IFN-r+, and IL-17A+ donor T cells in the spleen and target organs. Enhanced GVHD mortality and severity was also observed in MHC mismatched haploidentical matched B6 in to F1models (p<0.05). To explore the mechanism, we tested whether Siglec-G deficiency alters the naïve T cell responses in vitro after allogeneic or non-specific TCR stimulation in the absence of exogenous DAMPs. Interestingly Siglec-G-/- T cells showed similar proliferation in vitro, when compared to WT B6 T cells. In addition, Siglec-G-/- Tregs are equally suppressive in suppression assay and Siglec-G-/- T cells showed severe GVHD even Tregs are depleted in allo-BMT. However, Siglec-G-/- T cells showed a higher proliferation after direct TCR stimulation (CD3/CD28) with addition of DAMP (HMGB-1) when compared to WT T cells in vitro, suggesting direct T cell intrinsic effects. Consistent with this result, allogeneic Siglec-G-/- T cells caused similar mortality compared to WT controls in non-irradiated B6 into F1 model due to the absence of DAMPs from conditioning. To test the critical cellular mechanisms, we examined the function of endogenous Siglec-G ligand, CD24. We utilized BALB/c CD24-/- animals as hosts in same BMT model and found that CD24-/- animals showed an enhanced GVHD mortality and severity when compared to WT animals (p<0.05). To enhance Siglec-G-CD24 axis, we utilized a novel CD24 fusion protein (CD24Fc) in same BMT model and found that CD24 Fc ameliorated GVHD severity and mortality in not only allogeneic WT-B6 animals (p<0.05) but also CD24-/- animals (p<0.05). Next we explored DAMPs regulation by Siglec-G-CD24 axis in GVL. We utilized the same model of CD24Fc treatment but added P815 at the same time of allo-BMT and found that CD24Fc treated animals showed equivalent GVL to non-treated animals, suggesting that regulation of DAMPs with CD24Fc mitigates GVHD with maintaining GVL effect. Collectively our data suggested that the expression of both Siglec-G on donor T cells and CD24 on hosts is critical for controlling GVHD in the context of DAMPs released from conditioning, and represents a novel strategy that CD24Fc can mitigates GVHD with maintaining GVL. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures No relevant conflicts of interest to declare.

Download Full-text