scholarly journals Comprehensive Analysis of Chemokines in Host Organs and Their Corresponding Receptors on Donor T-Cells in Xenogeneic Gvhd Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5676-5676
Author(s):  
Yasufumi Kawasaki ◽  
Kazuya Sato ◽  
Hirofumi Nakano ◽  
Kiyomi Mashima ◽  
Daisuke Minakata ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Chemokine Receptors ◽  
Research Funding ◽  
Cell Homing ◽  
Human T Cell ◽  
Human T Cells ◽  
Donor T Cells ◽  
T Cell Homing

Abstract Background After hematopoietic stem cell transplantation, donor T-cells home to secondary lymphoid organs and recognize alloantigens within MHC molecules presented by host APCs. Following activation, donor T-cells acquire effector functions and then migrate into host organs along the chemokine gradients. Animal models targeting chemokine signals for prevention or treatment of GVHD have shown promising results; however, there have been significant inconsistencies among studies probably due to differences in species and conditioning regimens. The aim of this study is to evaluate the role of chemokines and their receptors, CCR5 (receptor of CCL3-5) and CXCR3 (receptor of CXCL9-10), in human T-cell homing and the development of GVHD using xenogeneic GVHD mouse model. Methods NOG mice received 250cGy of total body irradiation (TBI) if not otherwise specified, and were subsequently injected intravenously with human pan T-cells. All mice developed severe GVHD and died within 2 weeks, while the mice that received TBI only survived without any symptoms of GVHD. Peripheral blood was collected from mice at a certain interval for chemokine measurement. To assess the expression of chemokine receptors and genes associated with T-cell homing, cells were harvested from GVHD target organs of mice at day 9. For CCR5 blockage, mice were treated with 31 mg/kg maraviroc once daily by oral gavage after transplantation. Results Extensive infiltration of human T-cells and tissue destruction were observed in lungs and liver, but less severely in colon of GVHD mice. Consistent with this, quantitative real-time PCR analysis for five chemokine-related genes detected up-regulation of murine CXCL9 and CXCL10 in lungs, CCL4 in lungs and liver, but no up-regulation in colon. Similarly, the multiplex analysis of nine chemokines in plasma showed a marked increase in murine CCL4, CXCL9, and CXCL10 in GVHD mice. These observations suggest that the increased expression of CCL4, CXCL9, and CXCL10 on individual organs and following their systemic release play a critical role in the homing of allogeneic T-cells. Quantitative real-time PCR analysis of 84 genes associated with chemokines and chemokine receptors in human T-cells obtained from GVHD target organs revealed down-regulation of 36 genes, most of which are critical for T-cell homing into lymph nodes, such as CCL21 (-6.73-fold) and its receptor, CCR7 (-51.6-fold), and up-regulation of 16 genes such as CCL3 (225.5-fold), CCL4 (25.2-fold), CCR1 (11.4-fold), CCR5 (3.94-fold), and CXCL10 (2.88-fold). Focusing on chemokine receptors on human T-cells, flow cytometric analysis showed significantly higher expression of CCR5 on CD4+ and CD8+ T-cells, and CXCR3 on CD4+ T-cells in GVHD mice, whereas CXCR3 on CD8+ T-cells was strongly expressed even in resting state. Tissue damages were less apparent in GVHD mice that received human T-cells only compared with irradiated GVHD mice. Consistent with this, not only a total number but also the proliferation rate of human T-cells was decreased in non-irradiated GVHD mice. Also, non-irradiated GVHD mice showed significantly decreased plasma CCL4 and CXCL10 levels in plasma, and lower expression of CCR5 on CD4+ and CD8+ T-cells, and CXCR3 on CD4+ T-cells. The same was observed, to a significantly greater extent, in MHC class I/II deficient mice, suggesting that recognition of host MHC molecules by T-cells are critical for both host and donor chemokine signals. Taken together, TBI promotes host chemokine secretion and chemokine receptor expression on donor T-cells, leading to faster recruitment of donor T-cells into host organs and their proliferation. Contrary to the previous reports, CCR5 inhibitor treatment failed to attenuate GVHD and to improve the survival of mice. Although none of chemokine ligands but CCL4 was up-regulated on the liver, the number of infiltrated T-cells and tissue destruction were almost equivalent compared to the control. These observations indicate that compensatory chemokine pathways involving alternative receptors for CCL3-5, such as CCR1 and CCR2 on effector T-cells may overcome CCR5 blockage. Conclusion This study firstly provides a comprehensive picture of human T-cell homing through CCR5 and CXCR3 signaling in xenogeneic GVHD models. Our data supports the development of novel preventive and therapeutic strategies targeting chemokine signaling for GVHD. Disclosures Fujiwara: Shire: Consultancy; Pfizer: Consultancy; Chugai: Consultancy; Kirin: Consultancy; Kyowa-Hakko: Consultancy; Astellas: Consultancy. Ohmine:Kyowa Hakko Kirin: Speakers Bureau; Takara Bio: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Celgene Corporation: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Alexion Pharmaceuticals: Speakers Bureau; Ono Pharmaceutical: Consultancy. Muroi:Japanese Red Cross Society: Speakers Bureau; Dickinson and Company: Speakers Bureau; Becton: Speakers Bureau; JCR: Speakers Bureau. Kanda:Taisho-Toyama: Research Funding; Ono: Consultancy, Honoraria, Research Funding; Asahi-Kasei: Research Funding; Takeda: Consultancy, Honoraria, Research Funding; Sanofi: Research Funding; Tanabe-Mitsubishi: Research Funding; CSL Behring: Research Funding; Dainippon-Sumitomo: Consultancy, Honoraria, Research Funding; Shionogi: Consultancy, Honoraria, Research Funding; Novartis: Research Funding; Kyowa-Hakko Kirin: Consultancy, Honoraria, Research Funding; Astellas: Consultancy, Honoraria, Research Funding; Eisai: Consultancy, Honoraria, Research Funding; Otsuka: Research Funding; MSD: Research Funding; Chugai: Consultancy, Honoraria, Research Funding; Taiho: Research Funding; Nippon-Shinyaku: Research Funding; Pfizer: Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Mochida: Consultancy, Honoraria; Alexion: Consultancy, Honoraria; Takara-bio: Consultancy, Honoraria.

scholarly journals NLRP6 in Donor T Cells Separately Regulates CD4 and CD8 Mediated Graft-Versus-Host Disease in Experimental Murine BMT

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1926-1926
Author(s):  
Masahiro Suto ◽  
Eri Matsuki ◽  
Erika Sekiguchi ◽  
Hiroya Tamaki ◽  
Isao Tawara ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Research Funding ◽  
Pharmaceutical Research ◽  
T Cell Responses ◽  
Inflammasome Activation ◽  
Activated T Cells ◽  
Cell Responses ◽  
Donor T Cells

NLRP6 (NOD-like receptor family pyrin domain containing 6) is an important inflammasome component and is highly expressed in intestinal epithelial and in immune cells. NLRP6 mediated inflammasome activation plays a critical role in response to intestinal infection and preventing dysbiosis of gut microbiota through the secretion of IL-18 and mucus. However, we recently found that NLRP6 plays a pathogenic role in GVHD that is independent of microbial dysbiosis, which is in contrast to its well-appreciated microbiome-dependent protective role in intestinal colitis and tumorigenesis. Interestingly, we also found that activated T cells increased NLRP6 expression, but the T cell autonomousrole of NLRP6 in regulating T cell responses is unknown. Because NLRP6 is an important regulator of GVH responses, we tested the hypothesis that NLRP6 deficiency in donor T cells would ameliorate GVHD. To test our hypothesis, we first performed adetailed phenotypic analysis of various T cell subsets and activation markers in naïve NLRP6-/-and wild-type (WT) B6 animals and found a similar distribution of naïve, memory, effector and regulatory T cells. In order to examine whether the absence of NLRP6 in donors affects GVHD, WT-BALB/canimals were lethally irradiated (700cGy) and transplanted on day 0 with 5x106bone marrow and 1.0x106 splenic CD90+T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or NLRP6-/-animals. Contrary to our hypothesis, the recipients receiving donor T cells from NLRP6-/-animals showed a significantly worse survival compared to allogeneic WT-B6 animals (p<0.05). GVHD mortality and severity were also increased in an MHC mismatched B6 into B10.BR model, and in an MHC mismatched haploidentical B6 into F1model (p<0.05). In contrast, GVHD severity and mortality were similar in an MHC matched multiple minor antigen mismatched B6 into C3H.sw model. We hypothesized that GVHD severity and mortality was similar in the B6 into C3H.sw model because NLRP6 regulates CD4+ and CD8+ T cell responses, differently. In order to test this, we transplanted C3H.sw recipients as above except we infused either 1x106CD4+ or CD8+ T cells from B6-WT or NLRP6-/-animals. GVHD severity and mortality (P<0.05) were enhanced only when NLRP6-/-CD4+ T cells transplanted. We confirmed enhanced GVHD mortality and severity mediated by donor NLRP6-/-CD4+ T cells in a second MHC-mismatched GVHD model, B6 into BALB/c (p<0.05). To explore how NLRP6 effects T cell responses independent ofinflammasome activation, we tested naïve T cell proliferation in vitro after allogeneic or non-specific TCR stimulation by anti-CD3 and CD28 antibody and found that NLRP6-/-CD4+ but not CD8+T cells proliferated more than WT-B6 CD4+ or CD8+ T cells, respectively, following either stimulus. Furthermore, allogeneicNLRP6-/-T cells also caused greater mortality compared to WT allogenic T cells in a non-irradiated B6 into F1 model, which lacks inflammasome activation associated with conditioning induced DAMPs and PAMPs. Microarray analysis of activated T cells from NLRP6-/-animals showed higher expression of IL-2 and IFN-γ than WT B6 T cells, and we observed no effect of NLRP6 in a Treg suppression assay. These data suggest that NLRP6 regulates CD4+ T cell- mediated immune responses and that NLRP6 in donor T cells is critical for controlling CD4+ T cell mediated GVHD. The effect of NLRP6 on T cell mediated GVL is currently under investigation. Disclosures Tawara: Kyowa Hakko Kirin: Honoraria, Research Funding; Ono Pharmaceutical: Research Funding; Astellas Pharma: Research Funding. Ishizawa:Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; Novartis: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau.

Cytokine Production Signatures Are Intrinsically Associated with Human T Cell Maturation Stages.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1337-1337
Author(s):  
Krishna V. Komanduri ◽  
Tae Kon Kim ◽  
Eric D. Wieder ◽  
Lisa S. St. John
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cytokine Production ◽  
Memory T Cells ◽  
Cell Maturation ◽  
Human T Cell ◽  
Human T Cells ◽  
Maturation Stages ◽  
T Cell Maturation

Abstract Our recent published studies have suggested that impaired immune reconstitution after allogeneic stem cell transplantation is associated with a greater proportion of circulating late memory T cells, defined phenotypically. To characterize the relationship between immunophenotypic markers of T cell maturation and functional attributes of T cells, we optimized an 8-color, 10-parameter cytokine flow cytometry (CFC) approach and studied T cells from healthy donors. T cells were exposed to stimuli that both bypass (PMA:Ionomycin, P:I) and signal through the T cell receptor (Staph enterotoxin B, SEB; and CMV pp65 peptide pools) and stained with CD45RA and CD27 to demarcate naïve (N, CD45RA+CD27+), and three progressively mature memory subsets: M1 (CD45RA−CD27+), M2 (CD45RA−CD27−), M3 (CD45RA+CD27−) CD4+ and CD8+ T cells. We assessed the 15 possible combinations of cells producing IL-2, IFNγ, TNFα, and MIP1β alone or in combination within maturation subsets. When we initially studied the production of individual cytokines, we found that the bulk of IL-2 production was produced by activated N and M1 cells in both CD4 and CD8 lineages. In contrast, IFNγ and MIP1β were produced by later maturation stages (M2 and M3) of CD4+ and CD8+ T cells. In contrast to the polarized production of individual cytokines at the extremes of the maturation spectrum, early and middle memory cells (M1 and M2) cells produced heterogeneous combinations of cytokines (e.g, IL-2+IFNγ+ and TNFα+MIP-1β+ cells). We also found that IL-2/IFNγ co-producing cells, shown to be particularly important for the control of chronic viral pathogens, exist mainly in the M1 and M2 stages, and not the M3 stage. The above results were consistent with both P:I and SEB stimulation, and across several healthy subjects tested. Our cross-sectional results were confirmed by in vitro differentiation experiments, wherein we sorted naive (CD45RA+CD27+) CD4+ and CD8+ T cells and demonstrated that their function evolved as expected following stimulation with PHA and IL-2, which resulted in differentiation into M1 and M2 cells in culture. Finally, we stimulated PBMC from healthy CMV-seropositive donors with a CMV pp65 peptide mixtures and examined maturation and cytokine production. Consistent with prior observations, most CMV-specific T cells were M2 and M3 cells. Surprisingly, the most abundant functional subsets consisted of cells producing either MIP1β alone or MIP1β and other cytokines. Consistent with our results following polyclonal stimulation, we found that IL-2/IFNγ co-producing CMV specific T cells existed in M1 and M2, but not in the M3 stage. These results demonstrate that: Functional cytokine signature is strongly associated with T cell maturation stage; Nearly all IL-2 production occurs in N, M1 and M2 cells; M3 cells produce little IL-2, but substantial amounts of MIP1β; IL-2/IFNγ co-production is rare in M3 cells, but exist in M1 and M2 cells, perhaps suggesting why late stage skewing of memory T cells may lead to functional T cell impairment in vivo; and that MIP1β is the most abundant cytokine produced by CMV-specific T cells. Overall, our results demonstrate that phenotypically defined maturation stages in both CD4+ and CD8+ T cell lineages are strongly associated with functional signatures irrespective of stimulus type, and that multidimensional analyses of human T cells may be beneficial when assessing human T cells in the clinical setting.

scholarly journals Galectin-3 Signaling in Donor T Cells Regulates Acute Graft Versus Host Disease (aGvHD) after Allogenic Transplantation

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2765-2765
Author(s):  
Hemn Mohammadpour ◽  
Takemasa Tsuji ◽  
Cameron R. MacDonald ◽  
Joseph L. Sarow ◽  
Jingxin Qiu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Cd4 T Cells ◽  
Research Funding ◽  
T Cell Proliferation ◽  
Advisory Committees ◽  
Human T Cells ◽  
Donor T Cells

Abstract Galectin-3 (Gal-3) is a unique member of the galectin family of lectins. Gal-3 possesses immune-regulatory functions depending on the immune cell and the immunologic situation. There are no studies that specifically delineate the role of Gal-3 in the setting of acute GvHD but mounting research suggests that dysregulation of pathways involving the galectin family may contribute to the pathogenesis of other immune disorders. Gal-3 is expressed by many types of immune cells, including T-cells. It suppresses signaling downstream of the TCR, decreases effector T-cell cytokine production, but increases the development and differentiation of memory T cells, myeloid cells, and macrophages. We investigated the mechanisms and downstream events of Gal-3 signaling in donor T cells after Allo-HCT, using Gal-3 knockout (Gal-3 -/-) mice. We further studied the effect of Gal-3 in controlling aGvHD incidence and severity while preserving the Graft-versus Leukemia (GvL) effect by overexpressing Gal-3 in human T cells. We utilized both a major MHC-mismatch (C57B/6 (H-2 b) into BALB/c (H-2 k) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2 b) into C3H/SW (H-2 b) model. Lethally irradiated recipient BALB/c and C3H/SW WT animals were injected with T cell depleted bone marrow alone (3 ×10 6) or with splenic T cells derived from allogeneic WT or Gal-3 -/- B6 donors (0.7 × 10 6 T cells in B6 → BALB/c and 1.5 × 10 6 in B6 → C3H/SW). We found that donor T cells express Gal-3 after Allo-HCT and that Gal-3 expression in WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, decreased clinical GvHD scores, and longer survival when compared to mice receiving Gal-3 -/- donor T cells (Figure 1A). We studied the mechanisms by which Gal-3 signaling controls the severity of aGvHD. Using flow cytometry analysis, we determined that Gal-3 plays a critical role in T cell proliferation and exhaustion. Gal-3 -/- T cells have a cytotoxic T phenotype with increased IFN-ℽ and GM-CSF production in T cells from the spleen and liver tissues on days 7 and 14 after Allo-HCT when compared to WT T cells (Figure 1B). There was a significant increase in T cell proliferation in Gal-3 -/- CD4 +T cells with a significantly higher level of IFN- ℽ mediated activation induced cell death (AICD) when compared to WT T cells. Gal-3 expression in T cells significantly increased the expression of exhaustion markers evidenced by a higher percentage of Slamf6 + Tim-3 + in WT T cells when compared to Gal-3 -/- T cells (Figure 1B). Gal-3 induced T cell exhaustion by through overactivation of NFAT signaling (data not shown). We sought to determine whether overexpression of Gal-3 in human T cells could control GvHD without affecting GVL. Gal-3 was overexpressed in human T cells using retrovirus containing Gal-3, vector alone and control T cells: Gal-3 T cells (T RV-Gal-3), GFP T cells (T RV-GFP) and control T cells were injected in irradiated NSG-HLA-A2 mice. All human cells expressed HLA-A2. Gal-3 overexpression in T cells effectively controlled the severity and mortality of GvHD after Allo-HCT in this humanized murine model of GvHD, evidenced by decreased body weight loss and decreased GvHD clinical scores in recipients transplanted with Gal-3 T cells when compared to control or GFP T cells (Figure 1C). Gal-3 overexpression did not impair the GvL effect when T cells cultured with Raji and THP-1 cell lines in vitro (data not shown). Gal-3 overexpression in T cells increased the frequencies of exhausted CD4 + T cells, and central memory CD4 + T cells while decreasing the percentage of effector CD4 T cell and INF-ℽ + CD4 + T cells. Clinical GI colon biopsies from patients undergoing allo-HCT were evaluated for Gal-3 expression in T cells using the multi-color Vectra 3 Automated Quantitative Pathology Imaging System. T cells in the colon biopsies expressed Gal-3. There was a significant correlation between Gal-3 MFI in CD4+ T cells, and GI histopathology score when analyzing Gal-3 intensity on Gal-3-expressing T cells. The Gal-3 MFI in CD4+ T cells was significantly lower in biopsies with higher colon GI histopathology scores (III-IV) compared to with lower colon GI histopathology scores I-II. In conclusion, these data reveal how Gal-3 can influence donor T cell proliferation and function in preclinical aGvHD models and point to the feasibility of manipulation of Gal-3 signaling to ameliorate aGvHD in the clinical setting. Figure 1 Figure 1. Disclosures Blazar: Rheos Medicines: Research Funding; Carisma Therapeutics, Inc: Research Funding; Equilibre Pharmaceuticals Corp: Research Funding; Tmunity Therapeutics: Other: Co-founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. McCarthy: Magenta Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bluebird: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Juno: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees.

scholarly journals The Absence of NLRP6 in Donor T Cells Exacerbates Gvhd

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2766-2766
Author(s):  
Masahiro Suto ◽  
Eri Matsuki ◽  
Masahiro Miyata ◽  
Erika Sekiguchi ◽  
Hiroya Tamaki ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Research Funding ◽  
Cell Responses ◽  
Donor T Cells ◽  
Induced Apoptosis

Abstract The Nlrp6 (NOD-like receptor family pyrin domain containing 6) inflammasome is important for intestinal epithelial cell innate immune responses and for maintaining gut homeostasis by preventing microbial dysbiosis. Contrary to its role in epithelial cell inflammasome-mediated responses, we recently showed that Nlrp6 in gut epithelial cells exacerbates GVHD in a manner independent of the inflammasome or gut microbiota. However, donor allogeneic T cells are also critical for GVHD development, yet, the function of Nlrp6 in allogeneic T cells is unknown. We hypothesized that Nlrp6 deficient donor T cells would ameliorate experimental GVHD. To test our hypothesis, WT-BALB/crecipients were lethally irradiated and transplanted on day 0 with 5x10 6 bone marrow and 1.0x10 6 splenic CD90 +T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or Nlrp6 -/- donors. Contrary to our hypothesis, the survival of allogeneic recipients of Nlrp6 -/- donor T cells was significantly worse than those receiving WT-B6 T cells (p&lt;0.05). Nlrp6 -/- donor T cells also caused greater GVHD mortality and morbidity in an MHC mismatched haploidentical B6 into B6D2F1 model (p&lt;0.05) and an MHC mismatched B10.BR into B6 model. Similar results were obtained using B6 into BALB/c and B6 into B6D2F1 models performed at the University of Michigan, suggesting our results were not unique to local environmental factors. By contrast, GVHD severity and mortality were similar in an MHC matched multiple minor antigen mismatched B6 into C3H.sw model. Because the B6 into C3H.sw model is largely driven by CD8+ T cells whereas the previous models are mediated by both CD4+ and CD8+ T cells, we examined whether Nlrp6 separately regulates CD4+ and CD8+ T cell-mediated GVHD. In order to test this, we transplanted C3H.sw recipients as above except we infused either 1x10 6 CD4+ or CD8+ T cells from B6-WT or Nlrp6 -/- animals. GVHD severity and mortality (P&lt;0.05) were enhanced only when Nlrp6 -/- CD4+ T cells were transplanted. These data suggested that Nlrp6 regulates allogeneic T cell responses in a subset-specific manner. To explore how Nlrp6 regulates intrinsic responses in donor T cell subsets, we tested naïve T cell proliferation in vitro after allogeneic or non-specific TCR stimulation. Consistent with the lack of increased GVHD induced by CD8+ Nlrp6 -/- donor T cells in the B6 into C3H.sw model, Nlrp6 -/- CD4+ but not CD8+ T cells proliferated more than WT-B6 CD4+ or CD8+ T cells, respectively, when stimulated with either anti-CD3/CD28 antibodies or lethally irradiated allogeneic antigen presenting cells in a mixed lymphocyte reaction. In addition, activation-induced apoptosis was decreased in Nlrp6 -/- CD4+ T cells compared to WT T cells. Importantly, Treg suppressive function was not altered in Nlrp6 -/- T cells. Therefore, increased proliferative responses and resistance to activation-induced apoptosis may have contributed to the enhanced GVHD caused by Nlrp6 -/- donor T cells. Increased Th1 and Th17 polarization is associated with worse GVHD. Because only CD4+ Nlrp6 -/- T cells enhanced GVHD, we tested whether Nlrp6 influenced T helper cell differentiation into Th1, Th17, and Th2 subsets. Consistent with our in vivo data, Th1 in vitro differentiation was enhanced in Nlrp6 -/- CD4+ T cells. To determine the molecular signaling events altered by Nlrp6 deficiency, we tested various T cell activation signaling pathways and found that phosphorylation of ZAP-70 was increased in Nlrp6 -/- T cells. These data suggested that Nlrp6 in donor T cells may regulate allo-immune responses via ZAP-70 pathway. GVH and graft-versus-tumor (GVT) responses are intricately linked. Because CD8+ responses were not affected by Nlrp6 deficiency, we hypothesized that GVT responses would be unaltered in Nlrp6 -/- donor T cells. Indeed, Nlrp6 -/- T cells showed equivalent in vivo GVL responses to MLL-AF4 leukemia cells as WT-T cells. Hence Nlrp6in donor T cells is not required for GVT responses. Altogether our data suggested that Nlrp6 negatively-regulates allogeneic donor CD4+ T cell responses, possibly via negative regulation of ZAP-70 signaling, resulting in mitigation of GVHD and maintenance of robust GVT responses. Disclosures Ishizawa: AbbVie: Research Funding; Eisai: Honoraria; Chugai: Honoraria; Ono: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Bayer: Research Funding; Bristol Myers Squibb: Speakers Bureau; Pfizer: Research Funding; Kyowa Kirin: Consultancy; SymBio: Honoraria, Research Funding; Otsuka: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Sanofi: Research Funding; IQVIA: Research Funding.

scholarly journals Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the C-type lectin Mincle

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Josephine F. Reijneveld ◽  
Mira Holzheimer ◽  
David C. Young ◽  
Kattya Lopez ◽  
Sara Suliman ◽  
...  
Keyword(s):  
T Cells ◽  
Fatty Acid ◽  
Immune System ◽  
T Cell ◽  
Cross Reactivity ◽  
Lipid Binding ◽  
Human Immune System ◽  
Human T Cell ◽  
Human T Cells ◽  
Pure Compounds

AbstractThe cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

scholarly journals Human CD8+ T-cells Recognizing Peptides from Mycobacterium tuberculosis (Mtb) Presented by HLA-E Have an Unorthodox Th2-like, Multifunctional, Mtb Inhibitory Phenotype and Represent a Novel Human T-cell Subset

2015 ◽  
Vol 11 (3) ◽  
pp. e1004671 ◽  
Author(s):  
Krista E. van Meijgaarden ◽  
Mariëlle C. Haks ◽  
Nadia Caccamo ◽  
Francesco Dieli ◽  
Tom H. M. Ottenhoff ◽  
...  
Keyword(s):  
T Cells ◽  
Mycobacterium Tuberculosis ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Subset ◽  
T Cell Subset ◽  
Human T Cell

Generation of primary antigen-specific human cytotoxic T lymphocytes in human/mouse radiation chimera

Blood ◽  
1996 ◽  
Vol 88 (2) ◽  
pp. 721-730 ◽  
Author(s):  
H Segall ◽  
I Lubin ◽  
H Marcus ◽  
A Canaan ◽  
Y Reisner
Keyword(s):  
T Cells ◽  
T Cell ◽  
Human Lymphocytes ◽  
Scid Mice ◽  
Human Antibody ◽  
Adoptive Therapy ◽  
Activation Markers ◽  
Human T Cell ◽  
Human T Cells

Severe combined immunodeficient (SCID) mice are increasingly used as hosts for the adoptive transfer of human lymphocytes. Human antibody responses can be obtained in these xenogeneic chimeras, but information about the functionality of the human T cells in SCID mice is limited and controversial. Studies using human peripheral blood lymphocytes (PBL) injected intraperitoneally (IP) into SCID mice (hu-PBL-SCID mice) have shown that human T cells from these chimeras are anergic and have a defective signaling via the T-cell receptor. In addition, their antigenic repertoire is limited to xenoreactive clones. In the present study, we tested the functionality of human T cell in a recently described chimeric model. In this system, BALB/c mice are conditioned by irradiation and then transplanted with SCID bone marrow, followed by IP injection of human PBL. Our experiments demonstrated that human T cells, recovered from these hu-PBL-BALB mice within 1 month posttransplant, proliferated and expressed activation markers upon stimulation with anti-CD3 monoclonal antibody. A vigorous antiallogeneic human cytotoxic T-lymphocyte (CTL) response could be generated in these mice by immunizing them with irradiated allogeneic cells. Moreover, anti-human immunodeficiency virus type 1 (HIV-1) Net- specific human CTLs could be generated in vivo from naive lymphocytes by immunization of mouse-human chimeras with a recombinant vaccinia-nef virus. This model may be used to evaluate potential immunomodulatory drugs or cytokines, and could provide a relevant model for testing HIV vaccines, for production of antiviral T-cell clones for adoptive therapy, and for studying human T-cell responses in vivo.

scholarly journals Characterization of Changes in the T-Cell Receptor Repertoire in Patients with Acute Myeloid Leukemia with Durable Remission Following Allogeneic Stem Cell Transplant

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5186-5186
Author(s):  
Ronald M. Paranal ◽  
Hagop M. Kantarjian ◽  
Alexandre Reuben ◽  
Celine Kerros ◽  
Priya Koppikar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
Cell Receptor ◽  
T Cell Repertoire ◽  
Cell Repertoire ◽  
Donor T Cells ◽  
Durable Remission

Introduction: Allogeneic hematopoietic stem-cell transplantation (HSCT) is curative for many patients with advanced hematologic cancers, including adverse-risk acute myeloid leukemia (AML). This is principally through the induction of a graft-versus-leukemia (GVL) immune effect, mediated by donor T-cells. The incredible diversity and specificity of T-cells is due to rearrangement between V, D, and J regions and the random insertion/deletion of nucleotides, taking place in the hypervariable complementarity determining region 3 (CD3) of the T-cell receptor (TCR). Massively parallel sequencing of CDR3 allows for a detailed understanding of the T-cell repertoire, an area relatively unexplored in AML. Therefore, we sought out to characterize the T-cell repertoire in AML before and after HSCT, specifically for those with a durable remission. Methods: We identified 45 bone marrow biopsy samples, paired pre- and post-HSCT, from 14 patients with AML in remission for > 2 years as of last follow-up. We next performed immunosequencing of the TCRβ repertoire (Adaptive Biotechnologies). DNA was amplified in a bias-controlled multiplex PCR, resulting in amplification of rearranged VDJ segments, followed by high-throughput sequencing. Resultant sequences were collapsed and filtered in order to identify and quantitate the absolute abundance of each unique TCRβ CDR3 region. We next employed various metrics to characterize changes in the TCR repertoire: (1) clonality (range: 0-1; values closer to 1 indicate a more oligoclonal repertoire), it accounts for both the number of unique clonotypes and the extent to which a few clonotypes dominate the repertoire; (2) richness with a higher number indicating a more diverse repertoire with more unique rearrangements); (3) overlap (range: 0-1; with 1 being an identical T-cell repertoire). All calculations were done using the ImmunoSeq Analyzer software. Results: The median age of patients included in this cohort was 58 years (range: 31-69). Six patient (43%) had a matched related donor, and 8 (57%) had a matched unrelated donor. Baseline characteristics are summarized in Figure 1A. Six samples were excluded from further analysis due to quality. TCR richness did not differ comparing pre- and post-HSCT, with a median number pre-HSCT of 3566 unique sequences (range: 1282-22509) vs 3720 (range: 1540-12879) post-HSCT (P = 0.7). In order to assess whether there was expansion of certain T-cell clones following HSCT, we employed several metrics and all were indicative of an increase in clonality (Figure 2B). Productive clonality, a measure of reactivity, was significantly higher in post-transplant samples (0.09 vs 0.02, P = 0.003). This is a measure that would predict expansion of sequences likely to produce functional TCRs. The Maximum Productive Frequency Index was higher post-HSCT indicating that the increase in clonality was driven by the top clone (most prevalent per sample). Similarly for the Simpson's Dominance index, another marker of clonality which was higher post-HSCT (0.01 vs 0.0009, P = 0.04). In order to determine whether this clonal expansion was driven by TCR clones shared among patients, we compared the degree of overlap in unique sequences among pre and post-HSCT samples. We found there was very little overlap between samples in the pre and the post-transplant setting and no change in the Morisita and Jaccard Overlap Indices. Conclusions: In conclusion, we show in this analysis an increase in clonality of T-cells following HSCT in patients with AML. This is likely related to the GVL effect after recognition of leukemia antigens by donor T cells and subsequent expansion of these T-cells. These expanded T-cell clonotypes were unlikely to be shared by patients in this cohort, likely reflecting the variety of antigens leading to the GVL effect. This could have direct implications on TCR-mediated immune-therapies given the likely need for a personalized, patient-specific design for these therapies. Figure 1 Disclosures Kantarjian: BMS: Research Funding; Novartis: Research Funding; AbbVie: Honoraria, Research Funding; Jazz Pharma: Research Funding; Astex: Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Agios: Honoraria, Research Funding; Daiichi-Sankyo: Research Funding; Takeda: Honoraria; Amgen: Honoraria, Research Funding; Cyclacel: Research Funding; Ariad: Research Funding; Pfizer: Honoraria, Research Funding. Short:Takeda Oncology: Consultancy, Research Funding; AstraZeneca: Consultancy; Amgen: Honoraria. Cortes:Takeda: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Sun Pharma: Research Funding; BiolineRx: Consultancy; Novartis: Consultancy, Honoraria, Research Funding; Astellas Pharma: Consultancy, Honoraria, Research Funding; Merus: Consultancy, Honoraria, Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Biopath Holdings: Consultancy, Honoraria; Daiichi Sankyo: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding. Jabbour:Cyclacel LTD: Research Funding; Pfizer: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding. Molldrem:M. D. Anderson & Astellas Pharma: Other: Royalties.

scholarly journals Phase 2 Study of Combination of Cytarabine, Idarubicin, and Nivolumab for Initial Therapy of Patients with Newly Diagnosed Acute Myeloid Leukemia

Blood ◽  
2017 ◽  
Vol 130 (Suppl_1) ◽  
pp. 815-815
Author(s):  
Farhad Ravandi ◽  
Naval Daver ◽  
Guillermo Garcia-Manero ◽  
Christopher B Benton ◽  
Philip A Thompson ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Myeloid Leukemia ◽  
Research Funding ◽  
T Cell Subsets ◽  
Newly Diagnosed ◽  
Effector Cells ◽  
T Effector Cells ◽  
Grade 3

Abstract Background: Blocking PD-1/PD-L1 pathways enhances anti-leukemia responses by enabling T-cells in murine models of AML (Zhang et al, Blood 2009). PD-1 positive CD8 T-cells are increased in bone marrow (BM) of pts with AML (Daver et al, AACR 2016). PD1 inhibition has shown activity in AML (Berger et al, Clin Cancer Res 2008). We hypothesized that addition of nivolumab to an induction regimen of ara-C and idarubicin may prolong relapse-free survival (RFS) and overall survival (OS); this study was designed to determine the feasibility of this combination. Methods: Pts with newly diagnosed acute myeloid leukemia (by WHO criteria; ≥20% blasts) and high risk MDS (≥10% blasts) were eligible to participate if they were 18-65 yrs of age and had adequate performance status (ECOG ≤3) and organ function (LVEF ≥ 50%; creatinine ≤ 1.5 g mg/dL, bilirubin ≤ 1.5 mg/dL and transaminases ≤ 2.5 times upper limit of normal). Treatment included 1 or 2 induction cycles of ara-C 1.5 g/m2 over 24 hours (days 1-4) and Idarubicin 12 mg/m2 (days 1-3). Nivolumab 3 mg/kg was started on day 24 ± 2 days and was continued every 2 weeks for up to a year. For pts achieving complete response (CR) or CR with incomplete count recovery (CRi) up to 5 consolidation cycles of attenuated dose ara-C and idarubicin was administered at approximately monthly intervals. Eligible pts received an allogeneic stem cell transplant (alloSCT) at any time during the consolidation or thereafter. Results: 3 pts with relapsed AML were treated at a run-in phase with a dose of nivolumab 1 mg/kg without specific drug-related toxicity. Subsequently, 32 pts (median age 53 yrs; range, 26-65) were treated as above including 30 with AML (24 de novo AML, 2 therapy-related AML, 3 secondary AML and 1 therapy-related secondary AML) and 2 high risk MDS. Pre-treatment genetic risk by ELN criteria was 11 adverse, 16 intermediate, and 5 favorable, including 2 FLT3 -ITD mutated, 5 NPM1 mutated, and 7 TP53 mutated. All 32 pts were evaluable for response and 23 (72%) achieved CR/CRi (19 CR, 4 CRi). The 4-week and 8 week mortality was 6% and 6%. The median number of doses of nivolumab received was 6 (range, 0-13); one pt did not receive nivolumab due to insurance issues. 9 pts underwent an alloSCT. After a median follow-up of 8.3 mths (range, 1.5-17.0) the median RFS among the responding pts has not been reached (range, 0.1 - 15.8 mths) and the median OS has not been reached (range 0.5-17.0 mths). Grade 3/4 immune mediated toxicities have been observed in 5 pts and include rash, pancreatitis, and colitis. Other grade 3/4 toxicities thought to be potentially related to nivolumab include cholecystitis in one pt. 9 pts proceeded to an alloSCT. Donor source was matched related in 2, matched unrelated in 6 and haplo-identical in 1 pt. Conditioning regimen was Fludarabine plus busulfan-based in 8, and fludarabine plus melphalan in 1 pt. 4 pts developed graft versus host disease (GVHD)(grade I/II in 3, grade III/IV in 1), which responded to treatment in 3. Multicolor flow-cytometry studies are conducted by the Immunotherapy Platform on baseline (prior to first dose of nivolumab) and on-treatment BM aspirate and peripheral blood to assess the T-cell repertoire and expression of co-stimulatory receptors and ligands on T-cell subsets and leukemic blasts, respectively. The baseline BM was evaluated on 23 of the 32 evaluable pts, including 18 responders and 5 non-responders. Pts who achieved a CR/CRi had a trend of higher frequency of live CD3+ total T cell infiltrate as compared to non-responders in the baseline BM aspirates (Fig 1A). We evaluated expression of immune markers on T cell subsets: CD4 T effector cells [Teff]: CD3+CD4+CD127lo/+Foxp3-, CD4 T regulatory cells [Treg]: CD3+CD4+CD127-Foxp3+, and CD8 T cells. At baseline, BM of non-responders had significantly higher percentage of CD4 T effector cells co-expressing the inhibitory markers PD1 and TIM3 (p&lt;0.05) and a trend towards higher percentage of CD4 T effector cells co-expressing PD1 and LAG3 compared to responders (Fig 1B). Co-expression of TIM3 or LAG3 on PD1+ T cells have been shown to be associated with an exhausted immune phenotype in AML (Zhou et al., Blood 2011). Conclusion: Addition of nivolumab to ara-C and anthracycline induction chemotherapy is feasible and safe in younger pts with AML. Among the pts proceeding to alloSCT the risk of GVHD is not significantly increased. Figure 1 Figure 1. Disclosures Daver: Pfizer Inc.: Consultancy, Research Funding; Otsuka America Pharmaceutical, Inc.: Consultancy; Sunesis Pharmaceuticals, Inc.: Consultancy, Research Funding; Novartis Pharmaceuticals Corporation: Consultancy; Bristol-Myers Squibb Company: Consultancy, Research Funding; Kiromic: Research Funding; Karyopharm: Consultancy, Research Funding; Jazz: Consultancy; Immunogen: Research Funding; Daiichi-Sankyo: Research Funding; Incyte Corporation: Honoraria, Research Funding. Thompson: Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jabbour: Bristol-Myers Squibb: Consultancy. Takahashi: Symbio Pharmaceuticals: Consultancy. DiNardo: Novartis: Honoraria, Research Funding; Daiichi-Sankyo: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Celgene: Honoraria, Research Funding. Sharma: Jounce: Consultancy, Other: stock, Patents & Royalties: Patent licensed to Jounce; Astellas: Consultancy; EMD Serono: Consultancy; Amgen: Consultancy; Astra Zeneca: Consultancy; GSK: Consultancy; Consetellation: Other: stock; Evelo: Consultancy, Other: stock; Neon: Consultancy, Other: stock; Kite Pharma: Consultancy, Other: stock; BMS: Consultancy. Cortes: BMS: Consultancy, Research Funding; Sun Pharma: Research Funding; Novartis Pharmaceuticals Corporation: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Teva: Research Funding; ImmunoGen: Consultancy, Research Funding; ARIAD: Consultancy, Research Funding. Kantarjian: Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Novartis: Research Funding; Bristol-Meyers Squibb: Research Funding; Pfizer: Research Funding.

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