Eomesodermin Regulates The Early Activation Of Alloreactive CD4 T Cells and Is Critical For Both Gvh and GVL Responses

Abstract Despite increasing insights into its immunobiology, graft vs host disease (GVHD) remains a major obstacle for successful allogeneic hematopoietic stem/progenitor cell transplantation (allo-HCT). Separation of GVHD from graft vs. leukemia/lymphoma (GVL) responses also remains an elusive goal for allo-HSCT. Efforts to delineate the transcriptional networks regulating T cell differentiation post-HCT have suggested that multiple transcription factors may be involved in the regulation of alloreactive helper T (Th) cells and GVHD. However, conflicting data have emerged regarding the role of Th1 and Th17 pathways, and it remains unclear which transcription factors mediate the early activation of alloreactive T cells necessary for subsequent GVHD development. The T-box transcription factor eomesodermin (Eomes) cooperates with T-bet to regulate CD8 T cell cytotoxic function, IFNy production, and memory cell formation. Recently, a role for Eomes in CD4 Th cell polarization has been described as well. In order to evaluate the role of Eomes in T cell function in the context of allo-HCT, we used a MHC-disparate mouse model (C57BL/6 into BALB/c) with T cell depleted donor bone marrow (TCD-BM) and wild-type (WT) or Eomes knock out (KO) donor T cells. Recipients were conditioned with lethal total body irradiation. Eomes deficiency in donor T cells led to a significant reduction in GVHD mortality (Fig 1, p<.001), morbidity (p<.001), and intestinal pathology (p<.05, colon). Notably, Eomes KO T cells exerted significantly less GVHD mortality than T-bet KO T cells (Fig 1, p<.001). Given the reduced gastrointestinal (GI) GVHD observed with Eomes KO T cells, we next analyzed the expression of homing molecules important for T cell migration to the GI tract. Consistent with reduced GI GVHD, we detected reduced expression of α4β7 integrin on Eomes KO donor CD8 T cells one week post-HCT. We also observed an increase in the proportion and absolute numbers of Foxp3+ regulatory T cells, as well as a decrease in expression of T-bet in mesenteric lymph nodes (MLNs). Moreover, we found decreased production of IFNy by Eomes KO donor CD4 T cells two weeks (spleen and MLN, p<.001) and three weeks (spleen, p<.01) post-HCT without a comcomitant increase in IL-17. We also found increased IL-4 production by Eomes KO CD4 T cells two weeks post-HCT (MLN, p<.05), indicating a shift from Th1 to Th2 polarization in the absence of Eomes. Strikingly, one of the greatest differences we observed between WT and Eomes KO donor T cells was impaired early activation of CD4 T cells; Eomes deficiency was associated with reduced proliferation (p<.001), reduced expression of CD25 (p<.001, spleen; p<.001, MLN), and increased expression of CD62L (p<.01, spleen; p<.001, MLN) in CD4 T cells within the first 72 hours post-HCT (Fig 2). In order to determine if Eomes was important for T cell-mediated GVL responses, we performed allo-HCT in the presence of A20 lymphoma cells. Despite the reduction in GVHD mortality as described above, A20 tumor challenge led to increased mortality in recipients of Eomes KO T cells, indicating that Eomes was also critical for effective GVL function. Given the importance of Eomes in early alloactivation of CD4 T cells, we evaluated if the impaired GVL function was due to an intrinsic CD8 defect or lack of CD4 help. B6 TCD-BM was transplanted into BALB/c recipients along with either WT or Eomes KO CD4 or CD8 T cells. Eomes deficiency in both CD4 and CD8 T cells again led to significant mortality, but HCT with Eomes KO CD4 T cells and WT CD8 T cells led to the greatest survival due to less GVHD and intact GVL (Fig 3), suggesting that Eomes is essential for intrinsic CD8 function during GVL, but not for CD4 help. In summary, we identified distinct requirements for Eomes in CD4 versus CD8 T cells in the context of allo-HCT. Eomes regulated multiple aspects of CD4 T cell function following allo-HCT, including early activation, cytokine production, and gut trafficking. The multifacted functions of Eomes in CD4 T cells likely explain its requirement for GVHD. In contrast, Eomes deficiency in CD8 T cells led to impaired GVL, consistent with its established importance for cytotoxic CD8 T cell differentiation. To our knowledge, this is one of the first descriptions of a transcription factor necessary for effective GVL capacity. Our results suggest that selective manipulation of Eomes function in T cell subsets may be useful for both limiting GVHD and enhancing GVL. Disclosures: No relevant conflicts of interest to declare.

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Role of PD-1 during effector CD8 T cell differentiation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1718217115 ◽

2018 ◽

Vol 115 (18) ◽

pp. 4749-4754 ◽

Cited By ~ 92

Author(s):

Eunseon Ahn ◽

Koichi Araki ◽

Masao Hashimoto ◽

Weiyan Li ◽

James L. Riley ◽

...

Keyword(s):

T Cells ◽

Cell Differentiation ◽

T Cell ◽

Viral Infection ◽

Cd8 T Cells ◽

Cell Epitope ◽

Cd8 T Cell ◽

T Cell Differentiation ◽

Lcmv Infection

PD-1 (programmed cell death-1) is the central inhibitory receptor regulating CD8 T cell exhaustion during chronic viral infection and cancer. Interestingly, PD-1 is also expressed transiently by activated CD8 T cells during acute viral infection, but the role of PD-1 in modulating T cell effector differentiation and function is not well defined. To address this question, we examined the expression kinetics and role of PD-1 during acute lymphocytic choriomeningitis virus (LCMV) infection of mice. PD-1 was rapidly up-regulated in vivo upon activation of naive virus-specific CD8 T cells within 24 h after LCMV infection and in less than 4 h after peptide injection, well before any cell division had occurred. This rapid PD-1 expression by CD8 T cells was driven predominantly by antigen receptor signaling since infection with a LCMV strain with a mutation in the CD8 T cell epitope did not result in the increase of PD-1 on antigen-specific CD8 T cells. Blockade of the PD-1 pathway using anti–PD-L1 or anti–PD-1 antibodies during the early phase of acute LCMV infection increased mTOR signaling and granzyme B expression in virus-specific CD8 T cells and resulted in faster clearance of the infection. These results show that PD-1 plays an inhibitory role during the naive-to-effector CD8 T cell transition and that the PD-1 pathway can also be modulated at this stage of T cell differentiation. These findings have implications for developing therapeutic vaccination strategies in combination with PD-1 blockade.

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Identification of a Discrete Subpopulation of T-Cells Over-Expressing HDAC11 with a TH17 Phenotype

Blood ◽

10.1182/blood.v116.21.588.588 ◽

2010 ◽

Vol 116 (21) ◽

pp. 588-588

Author(s):

Karrune Woan ◽

Fengdong Cheng ◽

Hongwei Wang ◽

Jennifer Rock-Klotz ◽

Zi Wang ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd4 T Cells ◽

Cell Function ◽

Conflicts Of Interest ◽

Negative Regulator ◽

Egfp Expression ◽

In Vivo Models

Abstract Abstract 588 We recently defined a novel role of histone deacetylase 11 (HDAC11), the newest member of the HDAC family, as a negative regulator of IL-10 gene transcription in antigen-presenting cells (APCs).1 To better understand the role of HDAC11 gene expression in immune cells in vivo, we have utilized a BAC (Bacterial artificial chromosome) transgenic mouse in which the EGFP reporter gene was inserted downstream of the HDAC11 promoter region but immediately upstream of the HDAC11 coding sequence (TgHDAC11-EGFP mice).2 In the steady-state, macrophages and B-cells isolated from spleen of TgHDAC11-EGFP mice express low levels of HDAC11 as evidenced by a slight shift in EGFP fluorescence from background. In sharp contrast, we identified a discrete population (11.9%) of T-cells over-expressing HDAC11 as demonstrated both by flow cytometry for EGFP and by qRT-PCR for HDAC11, a majority of which were CD4+ T-cells. Sorting of this EGFP+, CD4+ T-cell population confirmed that the increased EGFP expression correlated with an increased HDAC11mRNA expression. Reminiscent of our prior data in APCs, the increased expression of HDAC11 in T-cells was also inversely correlated with IL-10mRNA expression. Further analyses revealed that in the absence of any stimulation or T-cell polarizing conditions, this EGFP positive population expressed significantly elevated levels of ROR-γt and IL-17 mRNA, markers specific for the TH17 subpopulation. Polarization of wild type CD4+ T-cells into functional TH17 cells was associated with reduction of HDAC11 expression, suggesting a potential role for HDAC11 in regulating T-cell function and/or activation, in particular within the TH17 subset. Further support for this regulatory role of HDAC11 has been provided by our additional findings that T-cells devoid of HDAC11 are indeed hyper-reactive in vitro and in in vivo models. 1. Villagra A, et al. Nat Immunol. 2009 Jan;10(1):92-100. 2. Gong S, et al. Nature. 2003 Oct 30;425(6961):917-25. Disclosures: No relevant conflicts of interest to declare.

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Combined Deficiency of p50 and cRel in CD4+ T Cells Reveals an Essential Requirement for Nuclear Factor κB in Regulating Mature T Cell Survival and In Vivo Function

Journal of Experimental Medicine ◽

10.1084/jem.20021610 ◽

2003 ◽

Vol 197 (7) ◽

pp. 861-874 ◽

Cited By ~ 94

Author(s):

Ye Zheng ◽

Monika Vig ◽

Jesse Lyons ◽

Luk Van Parijs ◽

Amer A. Beg

Keyword(s):

T Cells ◽

T Cell ◽

Cell Survival ◽

Cd4 T Cells ◽

Cell Function ◽

Nuclear Factor ◽

T Cell Function ◽

T Cell Survival

Signaling pathways involved in regulating T cell proliferation and survival are not well understood. Here we have investigated a possible role of the nuclear factor (NF)-κB pathway in regulating mature T cell function by using CD4+ T cells from p50−/− cRel−/− mice, which exhibit virtually no inducible κB site binding activity. Studies with these mice indicate an essential role of T cell receptor (TCR)-induced NF-κB in regulating interleukin (IL)-2 expression, cell cycle entry, and survival of T cells. Our results further indicate that NF-κB regulates TCR-induced expression of antiapoptotic Bcl-2 family members. Strikingly, retroviral transduction of CD4+ T cells with the NF-κB–inducing IκB kinase β showed that NF-κB activation is not only necessary but also sufficient for T cell survival. In contrast, our results indicate a lack of involvement of NF-κB in both IL-2 and Akt-induced survival pathways. In vivo, p50−/− cRel−/− mice showed impaired superantigen-induced T cell responses as well as decreased numbers of effector/memory and regulatory CD4+ T cells. These findings provide the first demonstration of a role for NF-κB proteins in regulating T cell function in vivo and establish a critically important function of NF-κB in TCR-induced regulation of survival.

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Organ- and Disease-Stage-Specific Regulation of Toxoplasma gondii-Specific CD8-T-Cell Responses by CD4 T Cells

Infection and Immunity ◽

10.1128/iai.00098-06 ◽

2006 ◽

Vol 74 (10) ◽

pp. 5790-5801 ◽

Cited By ~ 26

Author(s):

Sonja Lütjen ◽

Sabine Soltek ◽

Simona Virna ◽

Martina Deckert ◽

Dirk Schlüter

Keyword(s):

T Cells ◽

T Cell ◽

Toxoplasma Gondii ◽

Cd8 T Cells ◽

Functional Activity ◽

Cd4 T Cells ◽

Cd8 T Cell ◽

Cd4 T Cell ◽

Ifn Γ

ABSTRACT Toxoplasma gondii induces a persistent central nervous system infection, which may be lethally reactivated in AIDS patients with low CD4 T-cell numbers. To analyze the role of CD4 T cells for the regulation of parasite-specific CD8 T cells, mice were infected with transgenic T. gondii expressing the CD8 T-cell antigen β-galactosidase (β-Gal). Depletion of CD4 T cells prior to infection did not affect frequencies of β-Gal876-884-specific (consisting of residues 876 to 884 of β-Gal) CD8 T cells but resulted in a pronounced reduction of intracerebral β-Gal-specific gamma interferon (IFN-γ)-producing and cytolytic CD8 T cells. After cessation of anti-CD4 treatment a normal T. gondii-specific CD4 T-cell response developed, but IFN-γ production of intracerebral β-Gal-specific CD8 T cells remained impaired. The important supportive role of CD4 T cells for the optimal functional activity of intracerebral CD8 T cells was also observed in mice that had been depleted of CD4 T cells during chronic toxoplasmosis. Reinfection of chronically infected mice that had been depleted of CD4 T cells during either the acute or chronic stage of infection resulted in an enhanced proliferation of β-Gal-specific IFN-γ-producing splenic CD8 T cells. However, reinfection of chronically infected mice that had been depleted of CD4 T cells in the acute stage of infection did not reverse the impaired IFN-γ production of intracerebral CD8 T cells. Collectively, these findings illustrate that CD4 T cells are not required for the induction and maintenance of parasite-specific CD8 T cells but, depending on the stage of infection, the infected organ and parasite challenge infection regulate the functional activity of intracerebral CD8 T cells.

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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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The Role of β-Catenin in Th1 Immune Response against Tuberculosis and Profiles of Expression in Patients with Pulmonary Tuberculosis

Journal of Immunology Research ◽

10.1155/2021/6625855 ◽

2021 ◽

Vol 2021 ◽

pp. 1-15

Author(s):

Kunlong Xiong ◽

Jinxia Niu ◽

Ruijuan Zheng ◽

Zhonghua Liu ◽

Yanzheng Song ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Mononuclear Cells ◽

Cd4 T Cell ◽

Cell Frequency ◽

Tnf Α ◽

Ifn Γ

β-Catenin is a key molecule of canonical Wnt/β-catenin pathway. Its roles and expression profiles in T cells of tuberculosis (TB) remain unclear. The aim of this study was to explore the role of β-catenin in CD4+ T cells and its expression characteristics in patients with pulmonary tuberculosis (PTB). In this study, CD4+ T cell-specific β-catenin conditional knockout mice (β-CAT-cKO mice) were aerosol infected with Mycobacteria tuberculosis (Mtb) H37RV with wild-type mice as controls. Four weeks after infection, the mRNA expression of IFN-γ, TNF-α, and TCF-7 in the lungs of mice was measured. CD4, CD8, β-catenin, IFN-γ, and TNF-α in mononuclear cells from the lungs and spleens were measured by flow cytometry, and the pathological changes of lungs were also observed. Patients with PTB were enrolled, with blood samples collected and PBMCs isolated. The expressions of β-catenin, IFN-γ, TNF-α, and PD-1 in CD4+ and CD8+ T cells were measured by flow cytometry. Results showed a decreased frequency of and reduced IFN-γ/TNF-α mRNA expression and secretion by CD4+ T cells in the lungs of infected β-CAT-cKO mice compared with infected wild-type controls, and only slightly more inflammatory changes were observed in the lungs. β-catenin expressions in CD4+ and CD8+ T cells were significantly decreased in blood cells of patients with severe PTB compared with those in mild PTB. The stimulation of peripheral blood mononuclear cells (PBMCs) with lithium chloride (LiCl), a stimulant of β-catenin, resulted in the increase in CD4+ T cell frequency, as well as their secretion of IFN-γ and TNF-α. β-Catenin demonstrated a moderately positive correlation with PD-1 in CD4+ T cells. β-Catenin along with PD-1 and IFN-γ in CD4+ T cells had a high correlation with those in CD8+ T cells. In conclusion, β-catenin may be involved in the regulation of Th1 response and CD4+ T cell frequency in TB.

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Peri-Transplant Steroids Enhance GVL and Attenuate GVHD By Redistributing Alloreactive Donor T Cells from the Gut into the Bone Marrow

Blood ◽

10.1182/blood-2019-129341 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3210-3210

Author(s):

Takayuki Inouye ◽

Motoko Koyama ◽

Ensbey Kathleen ◽

Nicholas Greene ◽

Luke Samson ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Cell Expansion ◽

Effector Memory ◽

Gi Tract ◽

Donor T Cells ◽

T Cell Expansion

Leukemia relapse represents a failure of graft-versus-leukemia (GVL) and remains the major limitation of allogeneic stem cell/bone marrow transplantation (BMT). Graft-versus-host disease (GVHD) within the gastrointestinal (GI) tract is the principal determinant of transplant-related mortality and is initiated by a network of alloantigen presentation by professional and non-professional APC that prime donor T cells in the GI tract and related lymphoid structures. Since GVL and lethal GVHD are mediated by donor T cells at spatially distinct sites; bone marrow (BM) and the GI tract respectively, we sought tractable approaches to spatially separate alloreactive responses at these two locations. The administration of high dose steroids in the peri-transplant period is permissive of T cell replete HLA-haploidentical BMT and significant GVL effects (Ogawa H, et al. BBMT. 2006). We utilized murine haploidentical BMT models (B6D2F1 → B6C3F1, B6 → B6D2F1) with recipient background MLL/AF9 primary acute myeloid leukemia (AML), with or without dexamethasone (Dex) administration (5 mg/kg/day i.p., days -1 to +5). Dex-treatment improved transplant survival (from 25% to 68% at day 100, P=0.0012) with significant reductions in GVHD histopathology specifically in the colon (histopathology scores 8.7±1.0 vs 4.6±0.8, P< 0.05), despite excellent leukemia control. To understand this paradox, we analyzed the kinetics of donor T cell expansion after BMT. In the mesenteric lymph node (mLN), Dex treatment significantly suppressed the expansion of both CD4 and CD8 T cells (3.3±0.3 x 105 vs 1.4±0.3 x 105, P< 0.001 and 4.2±0.4 x 105 vs 2.1±0.4 x 105, P< 0.01 respectively) and the activation of CD4 T cells (CD25 MFI: 2021±146 vs 1056±102, P< 0.01). In contrast, donor effector/memory CD44+ CD8 T cells were expanded in the BM of Dex treated recipients (1.9±0.3 x 105 vs 3.1±0.4 x 105, P< 0.05) that demonstrated high per cell cytolytic activity against leukemia (specific lysis: 65±2.4 % vs 62±2.6 % in untreated vs Dex-treated, P> 0.05). Surprisingly, there was no difference in proliferation (cell tracking dye dilution: 63±5.5 % vs 57±5.5 % in untreated vs Dex-treated, P> 0.05) or apoptosis (caspase-3: 6.6±0.4 % vs 6.1±0.6 %, caspase-8: 20±1.6 % vs 17±3.3 % in untreated vs Dex-treated, respectively, P> 0.05) of CD4 T cells in the mLN between the two groups. We undertook experiments with luciferase expressing T cells and noted that Dex-treatment preferentially inhibited T cell accumulation in the GI tract, but not marrow after BMT. Thus, it appeared that Dex treatment preferentially re-distributed donor T cells from the GI tract to the bone marrow. We next determined if Dex exerted effects via direct signaling to the donor T cell. We thus transplanted glucocorticoid receptor (GR)-deficient or intact T cells (GRfl/fl lck-Cre mice). Dex-treatment reduced donor CD4 T cell expansion in the mLN independent of their expression of the GR (untreated vs Dex-treated: 2.8±0.6 x 105 vs 1.2±0.3 x 105, lckCREGRfl/fl and 2.4±0.3 x 105 vs 1.4±0.4 x 105, GRfl/fl littermates, P< 0.05 both groups). Thus steroid effects were mediated indirectly, putatively via effects on recipient alloantigen presentation. There was a marked reduction in recipient dendritic cells (DC) and macrophages expressing the Ea peptide within MHC class II in the GI tract of Dex-treated recipients (terminal Ileum YAe+ DC number 896±93 vs 356±40, P< 0.01, YAe+ macrophage number 1035±136 vs 355±97, P< 0.01). In conjunction with this, expression of the gut homing integrin a4b7 expression was reduced in CD4 T cells from Dex treated recipient mLN (25±1.6 % vs 17±1.7 %, P< 0.01), while the marrow homing integrin VLA-4 (a4b1) was increased (a4: 62±2.2 % vs 75±1.6 %, P< 0.001, b1: 52±2.5 % vs 61±1.6 %, P< 0.05) in donor CD8 T cells from Dex treated recipient BM. Finally, Dex treatment enhanced GVL against a second primary AML (BCR/ABL-NUP98/HOXA9) relative to untreated recipients and those receiving post-transplant cyclophosphamide (PT-Cy) (relapse rate: 0% vs 40% vs 100% at day 35 in Dex vs untreated vs PT-Cy, PT-Cy vs Dex-treated, P< 0.0001; untreated vs Dex-treated, P=0.029). These data suggest a potential therapeutic strategy to modulate antigen presentation in the GI tract and consequent integrin imprinting that minimizes GVHD lethality whilst enhancing GVL within BM. Disclosures No relevant conflicts of interest to declare.

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Definition of a Diagnostic Window Prior to the Onset of Clinically Apparent Acute Graft-Versus-Host Disease.

Blood ◽

10.1182/blood.v116.21.3746.3746 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3746-3746

Author(s):

Carina A Bäuerlein ◽

Simone S Riedel ◽

Brede Christian ◽

Ana-Laura Jordán Garrote ◽

Agnes Birner ◽

...

Keyword(s):

T Cells ◽

Cell Migration ◽

T Cell ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Critical Time ◽

Time Points ◽

T Cell Migration ◽

Donor T Cells

Abstract Abstract 3746 Acute graft-versus-host disease (aGvHD) is an immune syndrome after allogeneic hematopoietic cell transplantation (allo-HCT) caused by alloreactive donor T cells that attack the gastrointestinal tract, liver and skin. Thus, early T cell migration patterns to these organs could provide first cues for the onset of aGvHD. Hence, a unique surface marker profile of donor T cells at early time points after allo-HCT may be an indicator for patients at risk of aGVHD. Therefore, we analyzed the course of donor T cell activation, proliferation and homing in a clinical relevant murine MHC minor mismatch (miHAg) allo-HCT model to define critical time points and marker profiles for the detection of alloreactive T cells. Luciferase-labeled C57Bl/6 (H-2b) T cells plus bone marrow cells were transplanted into conditioned (8 Gy) MHC major mismatched Balb/c (H-2d) or miHAg Balb/b (H-2b) recipients. Donor T cell migration was visualized by in vivo bioluminescence imaging (BLI) and cells were characterized by multiparameter flow cytometry for 30 consecutive days after allo-HCT. GVHD scoring was performed by histopathology. Donor T cells proliferated exclusively in secondary lymphoid organs until day+3 (initiation phase) before migrating via the peripheral blood into target organs (effector phase). This occured in both models, MHC major mismatch and miHAg allo-HCT, which resulted in hyper-acute (starting at day+6) or acute GVHD (starting at day+21), respectively. In the hyper-acute scenario one wave of T cell migration starting at day+4 sufficed to cause lethal aGVHD. We detected a 4000-fold increase in CD4 and a 1500-fold increase in CD8 donor T cell numbers in the peripheral blood between day+3 and day+6 in this model. In contrast, in the more clinical relevant miHAg allo-HCT model we found 3 waves of T cell migration with peaks at days +6, +11 and +15 after allo-HCT. In the peripheral blood CD4 T cells increased 20-fold, CD8 T cells 50-fold between day+3 and day+6, but more than 40-fold (CD4) and 400-fold (CD8) between day+3 and day+11. After the third peak on day+15 a period followed when we could only detect very few migrating donor T cells in the peripheral blood before aGvHD became clinically apparent on day+21. Next, we asked whether we could identify alloreactive T cells by testing a large panel of surface markers at the defined migration peaks. Indeed, allogeneic T cells upregulated certain homing receptors at these peaks (e.g. at day+11: α4β7 integrin: 27% of CD4 T cells, 3.4×104/ml, 60% of CD8 T cells, 1.6×105/ml; P-selectin ligand: 28% of CD4 T cells, 3.5×104/ml, 35% of CD8 T cells, 9.1×104/ml). In contrast, syngeneic transplanted mice only showed a constant low expression level of those receptors (e.g. at day+11: α4β7 integrin: 20% of CD4 T cells, 9.6×103/ml, 5% of CD8 T cells, 3.1×103/ml; P-selectin ligand: 17% of CD4 T cells, 8.5×103/ml, 10% of CD8 T cells, 6.6×103/ml). However, other markers such as CD44 could be found on more than 80% of all donor T cells in allogeneic or syngeneic recipients. Our results in this clinical relevant mouse model show accelerating waves of T cell migration consistent with an enhancing feedback loop model of aGvHD pathogenesis. The homing receptor expression profile of donor T cells correlated with critical migration waves and clearly differed between mice with or without aGvHD. The assessment of critical time points frame a diagnostic window for a potential predictive test based on the dynamic change of the T cell homing receptor profile after allo-HCT. This preclinical study now awaits to be evaluated in patients undergoing allo-HCT. Disclosures: No relevant conflicts of interest to declare.

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Abrogation of Donor T Cell IL-21 Signaling Leads to Tissue-Specific Modulation of Immunity and Separation of Gvhd From GVL

Blood ◽

10.1182/blood.v116.21.729.729 ◽

2010 ◽

Vol 116 (21) ◽

pp. 729-729

Author(s):

Alan M. Hanash ◽

Lucy W. Kappel ◽

Nury L. Yim ◽

Rebecca A. Nejat ◽

Gabrielle L. Goldberg ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Lymph Nodes ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Inflammatory Cytokine ◽

Wild Type ◽

Mesenteric Lymph Nodes ◽

Mesenteric Lymph ◽

Donor T Cells

Abstract Abstract 729 Allogeneic hematopoietic transplantation is frequently the only curative therapy available to patients with hematopoietic malignancies, however transplant success continues to be limited by complications including graft vs. host disease (GVHD) and disease relapse. Separation of GVHD from graft vs. leukemia/lymphoma (GVL) responses continues to be a major goal of experimental and clinical transplantation, and better understanding of T cell immunobiology may lead to novel strategies to accomplish this goal. Interleukin 21 (IL-21) is a pro-inflammatory cytokine produced by Th17 helper T cells, and abrogation of IL-21 signaling has recently been demonstrated to reduce GVHD while retaining GVL. However, the mechanisms by which IL-21 may lead to a separation of GVHD and GVL are incompletely understood. In order to characterize the effect of IL-21 on GVH and GVL T cell responses, we compared wild type and IL-21 receptor knockout (IL-21R KO) donor T cells in a C57BL/6 into BALB/c murine MHC-mismatched bone marrow transplant (BMT) model. Lethally irradiated BMT recipients of IL-21R KO T cells demonstrated decreased GVHD-related morbidity (p<.05) and mortality (p<.01), and decreased histopathologic evidence of GVHD within the small intestine (p<.05). While this reduction in IL-21R KO T cell-mediated GVHD was associated with increased donor regulatory T cells two to three weeks post-BMT (p<.001), IL-21 signaling in both donor CD4 and donor CD8 T cells was found to contribute to GVHD mortality (p<.01 for CD4, p<.05 for CD8). Analysis of IL-21R expression by wild type T cells demonstrated receptor upregulation upon polyclonal activation in vitro and upon alloactivation in vivo (p<.01). However, this IL-21R upregulation was not required for in vivo alloactivation, as IL-21R KO and wild type donor T cells demonstrated equivalently greater proliferation in allogeneic vs. syngeneic recipients (p<.001), equivalent upregulation of CD25 (p<.001), and equivalent downregulation of CD62L (p<.01 for CD8 T cells). Despite this equivalent alloactivation, IL-21R KO T cells demonstrated decreased infiltration within the small intestine (p<.05), decreased infiltration in mesenteric lymph nodes (p<.05 for CD8 T cells, p<.001 for CD4 T cells), and decreased inflammatory cytokine-producing CD4 T cells within mesenteric lymph nodes (p<.01 for IFN-g, p<.001 for TNF-a, Figure 1A). Consistent with this, transplanted IL-21R KO donor T cells demonstrated decreased expression of a4b7 integrin (LPAM, p<.05), a molecule known to be involved in homing of GVHD-mediating donor T cells to the gut. However, in contrast to the reduced inflammatory cytokine-producing CD4 T cells observed in mesenteric lymph nodes, IL-21R KO helper T cell cytokine production was maintained in spleen (Figure 1B) and peripheral lymph nodes, and IL-21R KO T cells were able to protect recipient mice from lethality due to A20 lymphoma (p<.001). In summary, abrogation of IL-21 signaling in donor T cells leads to tissue-specific modulation of immunity, such that gastrointestinal GVHD is reduced, but peripheral T cell function and GVL capacity are retained. Targeting IL-21 for therapeutic intervention is an exciting strategy to separate GVHD from GVL, and this novel approach should be considered for clinical investigation to improve transplant outcomes and prevent malignant relapse. Disclosures: No relevant conflicts of interest to declare.

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Transient NKG2D Blockade Attenuates Graft-Versus-Host Disease While Preserving Graft-Versus-Leukemia Effects

Blood ◽

10.1182/blood.v122.21.3242.3242 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3242-3242 ◽

Cited By ~ 1

Author(s):

Mobin Karimi ◽

Theresa M Leichner ◽

Atsushi Satake ◽

David Raulet ◽

Taku Kambayashi

Keyword(s):

T Cells ◽

T Cell ◽

Mouse Model ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Cd8 T Cell ◽

Allogeneic Hsct ◽

Isotype Control ◽

Isotype Control Antibody

Abstract In allogeneic hematopoietic stem cell transplantation (HSCT), identification of mechanisms to control GVHD yet maintain GVL responses is of critical importance. One key effector cell that mediates both GVHD and GVL is the CD8+ T cell, which expands in response to T cell receptor (TCR) stimulation by allogeneic MHC class I molecules during allogeneic HSCT. In addition, co-stimulatory molecules facilitate the TCR-mediated activation process and the effector function of CD8+ T cells. Recent data suggest that NKG2D may play a co-stimulatory role in activation and in augmenting anti-tumor cytotoxic responses of CD8+ T cells. NKG2D is an NK cell-associated receptor that is also expressed on all human CD8+ T cells and on activated/memory mouse CD8+ T cells. NKG2D recognizes a diverse array of MHC-related ligands that are expressed by many tumors and induced on cells under stress such as myeloablative conditioning during HSCT. As the role of NKG2D in allogeneic HSCT is unknown, we hereby investigated the role of NKG2D on CD8+ T cells in a mouse model of GVHD and GVL. Our results show that a large fraction (40-50%) of mouse CD8+ T cells inducibly express NKG2D upon activation by allogeneic MHC in vitro and in vivo. To test the role of NKG2D in GVHD pathogenesis, we employed a major MHC-mismatched mouse model of GVHD involving the transplantation of C57BL/6-derived CD8+ T cells and bone marrow (BM) into lethally irradiated Balb/c mice (B6→Balb/c). Using 3 different approaches to block NKG2D on CD8+ T cells (shRNA-mediated silencing, germline NKG2D deficiency, and antibody blockade), we found that weight loss, clinical score, and survival were significantly improved in transplanted mice with NKG2D blockade. The attenuation in GVHD correlated with a significant reduction in TNFα and IFNγ production, cytotoxicity, and proliferation (BrdU incorporation) by CD8+ T cells. Although CD4+ T cells did not express NKG2D, a protective effect of NKG2D blockade was still observed in GVHD induced by a mixture of CD8+ and CD4+ T cells, albeit to a lesser extent. We next tested the effects of NKG2D on CD8+ T cell-mediated GVL. To this end, irradiated Balb/c mice were transplanted with C57BL/6-derived CD8+ T cells and BM, challenged intravenously with luciferase-positive A20 leukemia cells, and followed by total body imaging of luciferase-expressing cells. Given that NKG2D ligands are constitutively expressed on many tumor cells and plays an important role in their eradication, we predicted that continuous NKG2D blockade would inhibit GVL effects. However, as NKGD ligands are upregulated only transiently on stressed normal tissue, we reasoned that transient NKG2D blockade might be sufficient to attenuate GVHD and allow CD8+ T cells to regain their GVL function. To test this hypothesis, we compared the effect of anti-NKG2D antibody as continuous treatment or as 5-day transient treatment to mice receiving isotype control antibody. As expected, mice that received isotype control antibody cleared the A20 cells but developed severe GVHD. Continuous anti-NKG2D antibody-mediated blockade improved GVHD but also blunted the GVL response leading to increased A20 growth. In contrast, a large proportion of mice transiently treated with anti-NKG2D antibody cleared the A20 cells, while maintaining the attenuated GVHD state. Together, these data support a positive role of NKG2D on CD8+ T cells in mediating GVHD and GVL. Given the transient nature of NKG2D ligand upregulation on stressed tissues, a window of opportunity may exist where transient NKG2D blockade could provide a novel therapeutic strategy for treatment of acute GVHD while preserving the GVL function of CD8+ T cells after allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

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