Characterisation of Human CD83 Expression on Immune Cells and Their Targeting with CD83 Antibodies to Prevent Graft Versus Host Disease in Allogeneic Haematopoietic Cell Transplantation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3081-3081
Author(s):  
Derek NJ Hart ◽  
Xinsheng Ju ◽  
Zehra Elgundi ◽  
Nirupama Verma ◽  
Pablo Silveira ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Cells ◽  
Cell Activation ◽  
Splice Variants ◽  
Scid Mice ◽  
Human Pbmc ◽  
Graft Versus Host ◽  
Equity Ownership

Abstract Introduction: CD83 is an important marker of activated dendritic cells (DC) but it is also expressed on other immune cells. Polyclonal anti-CD83 antibody depletes activated DC and prevents human peripheral blood mononuclear cell (PBMC) induced xenogeneic graft versus host disease (GVHD) in immunosuppressed SCID mice (J Exp Med 2009;206;387). We therefore generated a potential therapeutic human anti-CD83 mAb (3C12C), which had similar efficacy and T cell sparing effects in the same model (Leukemia 2015; in press). To investigate the specific immunosuppressive effect of 3C12C further, we undertook a comprehensive analysis of CD83 expression and its glycosylation pattern on various immune cell populations and tested the effect of 3C12C on T cell function using preclinical models, including a human CD83 (hCD83) knock in (KI) mouse. Methods: A panel of mouse and recombinant mAbs to hCD83 were used to analyse its expression by flow cytometry on resting and activated healthy donor PBMC. The expression of potential CD83 splice variants was examined by PCR. T cell expression was examined by flow cytometry and confocal microscopy after PHA, CD3/CD28 beads and allogeneic mixed leukocyte reaction (alloMLR) culture. Control human IgG1 (trastuzumab) and 3C12C mAbs were tested (0.125mg d-1) in a xenogeneic model of GVHD utilizing human PBMC transplanted into total body irradiation and anti-NK conditioned SCID mice. The genetically engineered hCD83 KI mouse was shown to be immune-competent and used to test the effect of 3C12C on LPS activated DC and T cells. Results: There were distinct CD83 splice variants (full length CD83, splicing variant CD83a, CD83b and CD83c) in different immune cells. CD83 glycosylation status also differed with high glycosylation required for surface expression on activated DC, whereas its expression on activated B cells and monocytes was resistant to de-glycosylation. Increases in CD83 expression on T cells occurred early with different kinetics, underlining the distinct signal pathway involved. The 3C12C mAb reduced T cell proliferation in the alloMLR but did not affect cytomegalovirus (CMV) or influenza (Flu) specific CD8+T cell numbers. Treatment with 3C12C prevented GVHD in human PBMC transplanted SCID mice, which otherwise developed histological GVHD between d8-13. Human DC were activated by d2 and expressed the CMRF-44 activation marker plus CD83, CD80 and CD86. Treatment with 3C12C mAb eliminated CD83+ CMRF44+ DC early post-transplant and reduced T cell activation. Further studies, established CMV and Flu specific T cells were retained and responded to antigen by IFNg production. Furthermore, Treg numbers were preserved. The 3C12C mAb depleted LPS activated DC in hCD83 KI mice in experiments performed prior to commencing transplant studies. Conclusion: These findings suggest that the potential therapeutic human anti-CD83 mAb induced significant immune suppression, by depletion of activated DC and consequential modulation of T cell activation. The reduction in allo/xeno activated T cells may result in part from a direct effect of anti-CD83 on early T cell responses. This apparently selective immunosuppressive effect preserves anti-viral T cell immunity and Treg pathways, suggesting that 3C12C merits further investigation as a novel agent for GVHD prophylaxis. Disclosures Hart: DendroCyte BioTech Pty Ltd: Equity Ownership. Clark:DendroCyte BioTech Pty Ltd: Equity Ownership.

T Cell Activation and Regulation in Graft-Versus-Host Disease: Integral Role of CD28, CTLA4 and GITR Splice Variants.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3054-3054
Author(s):  
Yuji Miura ◽  
Christopher J. Thoburn ◽  
Emilie C. Bright ◽  
Elizabeth C. Matsui ◽  
William H. Matsui ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Splice Variants ◽  
Treg Cells ◽  
Mrna Splice ◽  
Graft Versus Host

Abstract Graft-versus-host disease (GVHD) is a serious, life-threatening complication that occurs following allogeneic (allo) bone marrow transplantation (BMT). The use of non-specific immunosuppression or T cell depletion has reduced the incidence of GVHD but at the expense of increased rates of infection and leukemic relapse. Modulation of the major costimulatory pathway (CD28/CTLA4:B7) involved in T cell activation and regulation may lead to specific immune tolerance in the absence of global non-specific immunosuppression. The identification of mRNA splice variants encoding for soluble forms of CD28, CTLA4 and GITR suggests that costimulation of T cells is complex and is not limited to cell-cell contact. The present studies examined the hypothesis that the onset of GVHD and the re-establishment of immune tolerance correlate with the expression levels of these costimulatory molecules. mRNA transcript levels for the soluble (s) and full-length (fl; cell surface associated) variants assessed by quantitative PCR, were temporally examined in peripheral blood lymphocytes (PBLs) from patients undergoing alloBMT (n=38) or autologous (auto) BMT (n=39) with the induction of autoGVHD by cyclosporin A treatment post-transplant. Levels of s and fl CD28 mRNA transcripts in PBLs were significantly increased (>1.5 fold, P<0.05) in patients developing either allo or autoGVHD compared to patients who do not develop GVHD. s and flCTLA4 levels in patients at the onset of allo and autoGVHD were significantly decreased compared to healthy controls (n=22) (>2.3-fold, P<0.01). s and flCTLA4 expression in patients with autoGVHD was significantly decreased compared to patients without autoGVHD (>2.1-fold). sCTLA4 expression in patients with alloGVHD was significantly decreased than patients without alloGVHD. Interestingly, temporal analysis revealed that the levels for sCTLA4 paralleled the recovery from GVHD implicating an active process in the establishment of non-responsiveness. CD28, CTLA4 and GITR s and fl mRNA levels in CD4+CD25+ T regulatory (Treg) cells from allo and autoBMT patients were significantly increased (7-, 41- or 22-fold, P<0.01) compared to the CD4+CD25− subset. Additional studies attempted to identify the potential role of the sCTLA4 protein (encoded by the mRNA splice variant) on the regulation of the lymphocyte response mediated by Treg cells. Addition of the Treg cells to a mixed lymphocyte reaction suppressed the proliferative response of CD8+ T cells to alloantigens (75% suppression; >4 fold reduction of 3H-thymidine incorporation). However, pretreatment of the Treg subset with short interfering RNA (siRNA) to knockdown sCTLA4 gene (confirmed by quantitative PCR) significantly reduced the ability of these cells to suppress the response (minimal suppression was detected, 6%). In vitro siRNA studies also indicated that Treg cells with inhibited sCTLA4 expression were unable to suppress the response of IL-2-stimulated autoreactive CD8+ T cells. Taken together, the results indicate that increased expression of CTLA4 (soluble and cell-surface associated) and the “negative” signal delivered by this molecule to the T cell may regulate the development of GVHD and help to re-establish self tolerance after BMT. Defining the role of costimulation and the modulation of this pathway on immune recognition and regulation not only provides opportunities to enhance the re-establishment of tolerance but also may help to intensify anti-tumor immunotherapeutic strategies.

scholarly journals Role of Phosphodiesterase 7 (PDE7) in T Cell Activity. Effects of Selective PDE7 Inhibitors and Dual PDE4/7 Inhibitors on T Cell Functions

2020 ◽  
Vol 21 (17) ◽  
pp. 6118 ◽  
Author(s):  
Marianna Szczypka
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Splice Variants ◽  
Cell Activity ◽  
Cell Functions ◽  
Simultaneous Inhibition ◽  
And Function

Phosphodiesterase 7 (PDE7), a cAMP-specific PDE family, insensitive to rolipram, is present in many immune cells, including T lymphocytes. Two genes of PDE7 have been identified: PDE7A and PDE7B with three or four splice variants, respectively. Both PDE7A and PDE7B are expressed in T cells, and the predominant splice variant in these cells is PDE7A1. PDE7 is one of several PDE families that terminates biological functions of cAMP—a major regulating intracellular factor. However, the precise role of PDE7 in T cell activation and function is still ambiguous. Some authors reported its crucial role in T cell activation, while according to other studies PDE7 activity was not pivotal to T cells. Several studies showed that inhibition of PDE7 by its selective or dual PDE4/7 inhibitors suppresses T cell activity, and consequently T-mediated immune response. Taken together, it seems quite likely that simultaneous inhibition of PDE4 and PDE7 by dual PDE4/7 inhibitors or a combination of selective PDE4 and PDE7 remains the most interesting therapeutic target for the treatment of some immune-related disorders, such as autoimmune diseases, or selected respiratory diseases. An interesting direction of future studies could also be using a combination of selective PDE7 and PDE3 inhibitors.

scholarly journals Non-voltage-gated L-type Ca2+Channels in Human T Cells

2004 ◽  
Vol 279 (19) ◽  
pp. 19566-19573 ◽  
Author(s):  
Leanne Stokes ◽  
John Gordon ◽  
Gillian Grafton
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Splice Variants ◽  
Antigen Receptor ◽  
Dominant Form ◽  
Voltage Gated ◽  
Antigen Receptor Signaling ◽  
Human T Cells

In T lymphocytes, engagement of the antigen receptor leads to a biphasic Ca2+flux consisting of a mobilization of Ca2+from intracellular stores followed by a lower but sustained elevation that is dependent on extracellular Ca2+. The prolonged Ca2+flux is required for activation of transcription factors and for subsequent activation of the T cell. Ca2+influx requires as yet unidentified Ca2+channels, which potentially play a role in T cell activation. Here we present evidence that human T cells express a non-voltage-gated Ca2+channel related to L-type voltage-gated Ca2+channels. Drugs that block classical L-type channels inhibited the initial phase of the antigen receptor-induced Ca2+flux and could also inhibit the sustained phase of the Ca2+signal suggesting a role for the L-type Ca2+channel in antigen receptor signaling. T cells expressed transcripts for the α11.2 and α11.3 pore-forming subunits of L-type voltage-gated Ca2+channels and transcripts for all four known β-subunits including several potential new splice variants. Jurkat T leukemia cells expressed a small amount of full-length α11.2 protein but the dominant form was a truncated protein identical in size to a truncated α11.2 protein known to be expressed in B lymphocytes. They further expressed a truncated form of the α11.3 subunit and auxiliary β1- and β3-subunit proteins. Our data strongly suggest that functional but non-voltage-gated L-type Ca2+channels are expressed at the plasma membrane in T cells and play a role in the antigen receptor-mediated Ca2+flux in these cells.

Clinical Dosing Regimen of Selinexor Maintains Normal Immune Homeostasis and T Cell Effector Function in Mice: Implications for Combination with Immunotherapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2525-2525
Author(s):  
Paul M Tyler ◽  
Mariah M Servos ◽  
Boris Klebanov ◽  
Trinayan Kashyap ◽  
Sharon Shacham ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
T Cell Development ◽  
Immune Homeostasis ◽  
Dosing Regimen ◽  
Employment Equity ◽  
Equity Ownership

Abstract Selinexor (KPT-330) is a first in class nuclear transport inhibitor of exportin-1(XPO1) currently in advanced clinical trials to treat patients with solid and hematological malignancies. To determine how selinexor might impact anti-tumor immunity, we analyzed immune homeostasis in mice treated with high selinexor doses (15 mg/kg, three times a week: M, W, F) and found disruptions in T cell development, a progressive loss of CD8 T cells and increases in inflammatory monocytes. Antibody production in response to immunization was mostly normal. Precursor populations in bone marrow and thymus were unaffected by high doses of selinexor, suggesting that normal immune homeostasis could recover. We found that high dose of selinexor given once per week preserved nearly normal immune functioning, whereas a lower dose given 3 times per week (7.5 mg/kg, M, W, F) was not able to restore immune homeostasis. Both naïve and effector CD8 T cells cultured in vitro showed impaired activation in the presence of selinexor. These experiments suggest that XPO1 function is required for T cell development and function. We then determined the minimum concentration of selinexor required to block T cell activation, and showed that T cell inhibitory effects of selinexor occur at levels above 100nM, corresponding to the first 24 hours post-oral dosing of 10 mg/kg. In a model of implantable melanoma, we used selinexor treatment at the clinically relevant dosing regimen of 10 mg/kg with a 5-day drug holiday (M, W selinexor treatment). After two weeks of treatment, tumors were harvested and tumor infiltrating leukocyte (TIL) populations were analyzed. This treatment led to intratumoral IFNg+, granzyme B+ cytotoxic CD8 T cells that were comparable to vehicle treated mice. Overall, selinexor treatment leads to transient inhibition of T cell activation but the clinically relevant once and twice weekly dosing schedules that incorporate sufficient drug holidays allow for normal CD8 T cell functioning and development of anti-tumor immunity. These results provide additional support to the recommended selinexor phase 2 dosing regimen, as was determined recently (Razak et al. 2016). Disclosures Klebanov: Karyopharm Therapeutics: Employment, Equity Ownership. Kashyap:Karyopharm Therapeutics: Employment, Equity Ownership. Shacham:Karyopharm Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Landesman:Karyopharm Therapeutics: Employment, Equity Ownership. Dougan:Karyopharm Therapeutics: Consultancy. Dougan:Karyopharm Therapeutics: Consultancy.

scholarly journals Allogeneic T Cells Utilize Glycolysis As the Predominant Metabolic Pathway to Induce Acute Graft-Versus-Host Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2419-2419
Author(s):  
Hung Nguyen ◽  
Kelley MK Haarberg ◽  
Yongxia Wu ◽  
Jianing Fu ◽  
Jessica Lauren Heinrichs ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Aerobic Glycolysis ◽  
Cell Metabolism ◽  
Metabolic Process ◽  
Naive T Cells ◽  
Graft Versus Host ◽  
Naïve T Cells

Abstract Allogeneic hematopoietic cell transplantation (allo-HCT) is an effective therapy for hematologic malignancies through T cell-mediated graft-versus-leukemia (GVL) effects, but allogeneic T cells often lead to severe graft-versus-host disease (GVHD). Cell metabolism plays pivotal roles in T-cell activation, differentiation, and function. However, understanding of T cell-metabolism is still superficial, and even less is known how metabolism regulates T-cell response to alloantigens and GVHD induction after allo-HCT. In this study, using a high-throughput liquid-and gas-chromatography-based metabolic approach, we compared the metabolic process of allogeneic versus syngeneic T cells at day 4 (early preclinical stage), day 7 (preclinical stage), and day 14 (clinical stage) post bone marrow transplantation (BMT), with naïve T cells as additional controls. Over 180 metabolites were identified and quantified. T cells after being transferred into pre-conditioned recipients were undergoing metabolic reprogramming reflected by attenuated levels of metabolites involving anabolic pathways of lipids, amino acids, nucleotides and carbohydrates in allogeneic and syngeneic T cells compared to those in naïve T cells. In comparison with syngeneic T cells, allogeneic T cells exhibited increased oxidative stress, reflected by higher levels of eicosanoid, cyclooxygenase, and lipoxygenase-oxidized eicosanoids, and decreased levels of antioxidant compounds such as glutathione (GSH) and glutathione disulfide (GSSG). To obtain biomass for robust proliferation followed by alloantigen stimulation, allogeneic T cells further increased pentose phosphate and polyamine synthesis by day 7 post-BMT. We also observed that allogeneic T cells and syngeneic T cells expressed comparable levels of metabolites in fatty acid and glutamine oxidized in tricarboxylic acid (TCA) cycle, which was much lower than those of naïve T cells. Importantly, allogeneic T cells exhibited higher levels of metabolites in glycolysis as compared to syngeneic T cells regardless of time points. Consistently, using Seahorse approach, we also found that allogeneic T cells significantly increased aerobic glycolysis as compared to syngeneic T cells post-BMT, whereas oxidative phosphorylation was similar. Moreover, blocking glycolysis with 2-deoxyglucose remarkably inhibited donor T-cell proliferation, expansion and Th1 differentiation after allo-BMT. Thus, aerobic glycolysis rather than mitochondrial oxidative phosphorylation is the preferential metabolic process required for the optimal expansion and activation of allogeneic T cells. Given mechanistic target of rapamycin (mTOR) plays an essential role in controlling T-cell metabolism particularly in glycolysis, we hypothesized that targeting mTOR would prevent GVHD by inhibiting glycolytic metabolism. Using pharmacological and genetic approaches, we unequivocally demonstrated that mTOR, especially mTORC1, was essential for T-cell glycolytic activity and for GVHD induction. Mechanistically, mTORC1 promoted T-cell activation, expansion, Th1 differentiation, and migration into GVHD target organs, but inhibited the generation of induced T regulatory cells. In conclusion, the current work provides compelling evidence that allogeneic T cells utilize glycolysis as a predominant metabolic process after BMT. Furthermore, we validate glycolysis or its key regulator, such as mTORC1, to be a valid therapeutic target for the control of GVHD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Highly Selective Irreversible ITK Inhibitor Cpi-818 Reduces Acute Graft-Versus Host Disease

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3814-3814
Author(s):  
Sarah Lindner ◽  
Nicole Lee ◽  
Gabriel K Armijo ◽  
Marina D Burgos da Silva ◽  
Pamela S Herrera ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Serum Concentrations ◽  
Graft Versus Host ◽  
Advisory Boards ◽  
Group 2 ◽  
Privately Held

Abstract Background Acute graft-versus-host disease (aGVHD) remains a major limitation of allogeneic hematopoietic cell transplantation (allo-HCT) despite prophylactic immunosuppression. Not all patients who develop aGVHD respond to currently available treatment and thus there is a pressing need for novel prophylactic and therapeutic strategies. Interleukin-2-Inducible T-Cell Kinase (ITK) is a Tec-family, non-receptor tyrosine kinase expressed in T-cells, homologous with Bruton's tyrosine kinase (BTK) in B cells and is involved in proximal T-cell receptor (TCR) signaling, activating Phospholipase Cγ1 (PLCγ1) and initiating a signaling cascade that includes the NFAT, NF-κB, and MAPK pathways tuning T-cell activation, proliferation, and differentiation. Targeting ITK (ITK-/- in T cell graft and short-term exposure of donor graft to ITK inhibitor) has been shown to be associated with less GVHD in preclinical studies (Mammadli et al 2020, Kondo et al 2021). CPI-818 is a highly selective irreversible ITK inhibitor that covalently binds to cysteine 442 of ITK and abolishes kinase activity. In vitro assays demonstrate that CPI-818 inhibits phosphorylation of the ITK substrate PLC γ1 (Y783) and downstream signaling molecules ERK and S6, and blocks IL-2 secretion. CPI-818 reduced clinical and histological disease severity in a Th1-driven T cell adoptive transfer model of colitis (not published). CPI-818 is currently being evaluated in a Phase 1/1b study in patients with relapsed/refractory T-cell lymphoma (NCT03952078), and thus far appears well tolerated. Methods and Results We used 2 well established preclinical aGVHD models (MHC-disparate B6 → BALB/c and minor MHC-mismatched B6 → 129) to explore the effects of ITK inhibition by CPI-818. Recipient mice were treated with CPI-818-formulated (300 mg/kg/day) or control diet from day -7 to day 90 relative to allo-HCT. We found significantly improved survival (MHC-disparate: p<0.0001, n=20/group, 2 independent experiments, Figure A; minor mismatch: p=0.019, n=20/group, 2 independent experiments), in mice treated with CPI-818 compared with control. In the MHC-disparate model we observed reduced serum concentrations of IL-5 and IL-17a on day 7 after allo-HCT (IL-5: 6.9 vs 22.1 pg/mL, p=0.048; IL-17a: 2.7 vs 9.2 pg/mL, p=0.037, 10 mice/group, Figure B). When we analyzed T cells in the spleen, 3 days post transplant, we saw reduced proliferation of both CD4 and 8 T cells as measured by CFSE dilution (CD4: 73.0 vs 85.1%, p=0.002, CD8: 53.5 vs 65.1%, p=0.002, n=10/group, Figure C), accompanied by a reduction in CD4 T cell expression of CD25 (MFI CD25, 2034.5 vs 2693.5, p=0.037, 10 mice/group, Figure D). By day 7, we observed reduced T cell activation in the small and large intestine (p<0.02, n=10/group, Figure D). Conversely, CPI-818 treated mice had increased serum concentrations of anti-inflammatory IL-10 (Day 7, 10.1 vs 3.1 pg/mL, p=0.012, n=10/group, Figure B) and increased proportions of FoxP3+ T reg in mesenteric lymph nodes (6.2 vs 3.1%, p=0.002, n=10/group, Figure E) and both small and large intestines (small intestine: p=0.01, n=10/group; large intestine: p=0.004, Figure E) on day 7 after allo-HCT. In vitro, 1uM CPI-818 reduced proliferation of T cells exposed to LPS-activated allogeneic DCs compared with vehicle control (CD4: 73.0 vs 81.4%, p=0.008; CD8: 90.1 vs 93.9%, p=0.012; data from 3 independent experiments). This was also seen when T cells were stimulated with CD3/CD28 beads (CD4: 66.2 vs 81.3%, p=0.03; CD8: 67.3 vs 90.6, p=0.03; data from 2 independent experiments) and was not caused by reduced viability of T cells due to drug exposure. Conclusion These data in two clinically relevant GVHD models demonstrate that ITK inhibition has potential as a novel targeted approach to prevent aGVHD through a) the suppression of T cell activation and proliferation, b) decreased concentrations of pro-inflammatory cytokines and increased concentration of anti-inflammatory cytokines. CPI-818 is the most potent and selective ITK inhibitor reported to date and these data highlight its promise as a novel agent for the prevention of aGVHD. Figure 1 Figure 1. Disclosures Smith: Janssen: Consultancy, Honoraria. Perales: Celgene: Honoraria; Kite/Gilead: Honoraria, Other; MorphoSys: Honoraria; Merck: Honoraria; Incyte: Honoraria, Other; Cidara: Honoraria; Bristol-Myers Squibb: Honoraria; Omeros: Honoraria; Novartis: Honoraria, Other; Medigene: Honoraria; Karyopharm: Honoraria; Nektar Therapeutics: Honoraria, Other; Equilium: Honoraria; Takeda: Honoraria; Sellas Life Sciences: Honoraria; NexImmune: Honoraria; Miltenyi Biotec: Honoraria, Other; Servier: Honoraria. Buggy: Corvus Pharmaceuticals Inc: Consultancy, Current holder of individual stocks in a privately-held company. Janc: Corvus Pharmaceuticals Inc: Current Employment, Current holder of individual stocks in a privately-held company. van den Brink: Forty-Seven, Inc.: Honoraria; WindMILTherapeutics: Honoraria; Kite Pharmaceuticals: Other; Da Volterra: Other: has consulted, received honorarium from or participated in advisory boards; Notch Therapeutics: Honoraria; Pluto Therapeutics: Current holder of stock options in a privately-held company, Other: has consulted, received honorarium from or participated in advisory boards ; GlaskoSmithKline: Other: has consulted, received honorarium from or participated in advisory boards; Synthekine (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Priothera: Research Funding; Wolters Kluwer: Patents & Royalties; Pharmacyclics: Other; Rheos: Honoraria; Nektar Therapeutics: Honoraria; Ceramedix: Other: has consulted, received honorarium from or participated in advisory boards ; Novartis (Spouse): Other: has consulted, received honorarium from or participated in advisory boards; Lygenesis: Other: has consulted, received honorarium from or participated in advisory boards ; Frazier Healthcare Partners: Honoraria; DKMS (nonprofit): Other; Juno Therapeutics: Other; MagentaTherapeutics: Honoraria; Merck & Co, Inc: Honoraria; Amgen: Honoraria; Therakos: Honoraria; Jazz Pharmaceuticals: Honoraria; Seres: Other: Honorarium, Intellectual Property Rights, Research Fundingand Stock Options.

PI3K-Independent Signaling Pathways Contribute to ICOS-Mediated T-Cell Costimulation in Acute Graft-Versus-Host Disease in Mice.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3003-3003
Author(s):  
Jun Li ◽  
Julie Leconte ◽  
Kenrick Semple ◽  
Jessica Heinrichs ◽  
Claudio Anasetti ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Acute Gvhd ◽  
Calcium Flux ◽  
Pi3k Signaling ◽  
Graft Versus Host ◽  
Acute Graft

Abstract Abstract 3003 ICOS provides an important costimulation to promote T-cell activation and function. Using a knock-in mouse strain, termed ICOS-YF, in which the cytoplasmic tail of ICOS cannot activate phosphoinositide 3-kinase (PI3K), we have shown that ICOS-PI3K signaling axis is critical for the generation of follicular helper T cells. We also observed that, in both CD4+ and CD8+ T cells, ICOS could potentiate TCR-mediated calcium flux in a PI3K-independent manner in vitro. Although ICOS can potentiate TCR-mediated calcium flux independent of PI3K, its biological significance is unclear. To address this question, we studied the function of ICOS-YF T cells in comparison with ICOS wild-type (WT) and knock-out (KO) T cells in MHC-mismatched bone marrow transplantation (BMT) models. Severity of acute graft-versus-host disease (GVHD) was evaluated based on recipient survival, body weight change, and pathologic scores. Consistent with the data previously published by us and others, ICOS KO T cells had significantly reduced ability to cause acute GVHD as compared to WT T cells. We further observed that YF T cells were significantly more capable in causing GVHD than KO T cells, but less capable than WT T cells. Mechanically, the levels of serum TNFa and IFNg were similar in the recipients of YF or KO T cells, but significantly lower than those of WT T cells. However, on the per-cell basis, YF CD8+ T cells expressed similar levels of intracellular IFNg as WT T cells, but significantly higher than KO T cells. We further compared the ability of CD4+ or CD8+ T cells alone in the induction of acute GVHD, and found that CD4+ T cells from YF and KO mice were similarly impaired in their capacity to induce acute GVHD. In contrast, the pathogenic capacity of CD8+ T cells from YF mice was comparable to that of WT cells, whereas KO CD8+ T cells were significantly less pathogenic. These results suggest that although both CD4+ and CD8+ T cells depend on ICOS costimulation, the downstream signaling pathways they utilize are distinct: CD4+ T cells depend on ICOS-PI3K signaling whereas CD8+ T cells are more dependent on PI3K-independent pathways, probably calcium signaling. Taken together, our study reveals a complexity in ICOS signaling mechanisms in T cell activation and GVHD induction. Disclosures: No relevant conflicts of interest to declare.

Immunologic Effects of CCR5 Blockade in Graft-Versus-Host Disease Prophylaxis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 920-920 ◽  
Author(s):  
Ryan H Moy ◽  
Austin P Huffman ◽  
Lee P Richman ◽  
Lisa Crisalli ◽  
James A Hoxie ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Graft Versus Host Disease ◽  
Cell Activation ◽  
Ccr5 Expression ◽  
Allogeneic Hsct ◽  
Graft Versus Host ◽  
And Control ◽  
Ccr5 Blockade

Abstract Background: Acute graft-versus-host disease (GVHD) is a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Experimental models and clinical studies suggest that blocking lymphocyte trafficking with the chemokine receptor CCR5 antagonist maraviroc (MVC) ameliorates visceral GVHD. However, the effects of CCR5 blockade on immune reconstitution and activation in allogeneic HSCT recipients remain unknown. Methods: We previously reported a phase I/II study of 38 high-risk patients undergoing reduced-intensity allogeneic HSCT with standard GVHD prophylaxis (tacrolimus, methotrexate) combined with brief (33-day) MVC treatment. We compared the clinical and immunologic outcomes of these patients to a contemporary control cohort of 115 consecutive patients undergoing allogeneic HSCT with standard GVHD prophylaxis alone. Multivariate analysis was used to assess the effect of MVC treatment on the incidence of GVHD. Flow cytometry on day 30 and day 60 PBMC samples was used to monitor immune cell subsets, T cell activation markers and chemokine receptor expression. We also used a multiplex Luminex assay to quantify serum cytokine levels at day 30. Results: Since our initial report, longer follow up (median 37 months) now shows that patients who received MVC have reduced incidence rates of acute grade II-IV GVHD (adjusted hazard ratio [aHR]=0.42, 95% CI 0.21-0.84, p=0.015) and grade III-IV GVHD (aHR=0.43, 95% CI 0.17-1.09, p=0.075) compared to our control cohort (Figure 1). There was no difference in chronic GVHD between the groups. Early lymphocyte recovery was similar between MVC and control cohorts, with no significant difference in absolute lymphocyte, CD4 T cell and CD8 T cell counts at day 30. We observed increased CCR5 expression on CD4 T cells, CD8 T cells and B cells at day 30 in response to MVC treatment (Figure 2A-B). However, CCR5 expression on these cell types equalized between MVC and control patients by day 60. Day 30 MVC samples also demonstrated decreased percentages of CD38+ CD4 T cells and HLA-DR+ CD8 T cells (Figure 2C-D), an effect that also faded by day 60. These data suggest that inhibition of lymphocyte trafficking with CCR5 blockade dampens peripheral T cell activation. Notably, some patients developed acute GVHD despite MVC prophylaxis, and therefore we investigated differences between MVC responders and non-responders. Patients who developed GVHD had a higher percentage of activated CD38+ CD4 T cells in peripheral blood (Figure 3A) and an increased proportion of naive CD4 (36.5% vs 18.1% p=0.005) and CD8 (35.5% vs 11.9% p=0.0008) T cells, which has been associated with the development of GVHD. We also observed higher CCR5 expression on CD8 T cells from MVC non-responders (CCR5 MFI 8800 vs 6595, p=0.02). Intriguingly, while MVC responders and non-responders had similar serum concentrations of CCR5 ligands (MIP-1a, MIP-1b, Rantes), MVC non-responders had elevated levels of CXCR3 ligands (IP-10, MIG) (Figure 3B-C). Taken together, these data suggest that lymphocyte migration via CXCR3 may be a potential escape mechanism for GVHD initiation and T cell activation in the setting of CCR5 blockade. Conclusion: Brief CCR5 blockade in high-risk patients inhibits T cell activation and reduces the incidence of GVHD. MVC does not inhibit early lymphocyte recovery, which was similar in treated and control patients. This supports the hypothesis that MVC treatment would not impair graft-versus-leukemia activity. Our analysis of immunologic outcomes of MVC reveals a distinct immunologic effect for CCR5 blockade in allogeneic HSCT recipients and suggests a novel resistance mechanism. These studies bolster CCR5 antagonism as an effective strategy for GVHD prevention and provide support for extended MVC treatment duration and investigation into CXCR3 as a therapeutic target. Figure 1. Incidence of GVHD in control and MVC cohorts Figure 1. Incidence of GVHD in control and MVC cohorts Figure 2. MVC increases CCR5 expression and dampens T cell activation at day 30 *p<0.05; **p<0.01; ***p<0.001 Figure 2. MVC increases CCR5 expression and dampens T cell activation at day 30 *p<0.05; **p<0.01; ***p<0.001 Figure 3. Increased T cell activation and serum CXCR3 ligands in MVC non-responders *p<0.05; ***p<0.001 Figure 3. Increased T cell activation and serum CXCR3 ligands in MVC non-responders *p<0.05; ***p<0.001 Disclosures Off Label Use: Off label use of CCR5 antagonist maraviroc for the prevention of graft-versus-host disease. Vonderheide:Pfizer: Research Funding. Porter:Pfizer: Research Funding.

scholarly journals The Proteomic Landscape of Resting and Activated CD4+ T Cells Reveal Insights into Cell Differentiation and Function

2020 ◽  
Vol 22 (1) ◽  
pp. 275
Author(s):  
Yashwanth Subbannayya ◽  
Markus Haug ◽  
Sneha M. Pinto ◽  
Varshasnata Mohanty ◽  
Hany Zakaria Meås ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Immune Cells ◽  
Cd4 T Cells ◽  
Cell Activation ◽  
Cd4 T Cell ◽  
Proteomic Profiling ◽  
Altered Expression ◽  
Adaptive Immune Cells

CD4+ T cells (T helper cells) are cytokine-producing adaptive immune cells that activate or regulate the responses of various immune cells. The activation and functional status of CD4+ T cells is important for adequate responses to pathogen infections but has also been associated with auto-immune disorders and survival in several cancers. In the current study, we carried out a label-free high-resolution FTMS-based proteomic profiling of resting and T cell receptor-activated (72 h) primary human CD4+ T cells from peripheral blood of healthy donors as well as SUP-T1 cells. We identified 5237 proteins, of which significant alterations in the levels of 1119 proteins were observed between resting and activated CD4+ T cells. In addition to identifying several known T-cell activation-related processes altered expression of several stimulatory/inhibitory immune checkpoint markers between resting and activated CD4+ T cells were observed. Network analysis further revealed several known and novel regulatory hubs of CD4+ T cell activation, including IFNG, IRF1, FOXP3, AURKA, and RIOK2. Comparison of primary CD4+ T cell proteomic profiles with human lymphoblastic cell lines revealed a substantial overlap, while comparison with mouse CD+ T cell data suggested interspecies proteomic differences. The current dataset will serve as a valuable resource to the scientific community to compare and analyze the CD4+ proteome.

scholarly journals CD20-TCB, a Novel T-Cell-Engaging Bispecific Antibody, Induces T-Cell-Mediated Killing in Relapsed or Refractory Non-Hodgkin Lymphoma: Biomarker Results From a Phase I Dose-Escalation Trial

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5319-5319 ◽  
Author(s):  
Ann-Marie E Bröske ◽  
Ian James ◽  
Anton Belousov ◽  
Enrique Gomez ◽  
Marta Canamero ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Board Of Directors ◽  
T Cell Activation ◽  
Research Funding ◽  
Cell Activation ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Introduction: CD20-TCB (RG6026) is a novel T-cell-engaging bispecific (TCB) antibody with a '2:1' molecular format that comprises two fragment antigen binding regions that bind CD20 (on the surface of B cells) and one that binds CD3 (on the surface of T cells). CD20-TCB offers the potential for increased tumor antigen avidity, rapid T-cell activation, and enhanced tumor cell killing versus other bispecific formats. The safety, tolerability, pharmacokinetics, biomarkers, and antitumor activity of CD20-TCB are currently being investigated in a multicenter Phase I dose-escalation trial (NP30179; NCT03075696). We recently presented preliminary clinical data demonstrating promising clinical activity in relapsed or refractory (R/R) non-Hodgkin lymphoma (NHL) patients with indolent or aggressive disease (Dickinson et al. ICML 2019). Here, we present preliminary blood and tissue biomarker analyses to explore modes of action, support optimal biological dose selection, and identify potential outcome predictors. Methods: For biomarker analyses, we performed immune profiling of peripheral blood by flow cytometry, analyzed plasma cytokine levels by ELISA, and characterized baseline and on-treatment tumor biopsies by immunohistochemistry/immunofluorescence assays and RNA sequencing. Biomarker data were obtained from 122 patients dosed with 0.005-25mg CD20-TCB. Results: CD20-TCB infusion led to a rapid and transient reduction in T cells in the peripheral circulation (T-cell margination) in all patients. T-cell margination reached nadir 6 hours after the first CD20-TCB infusion, and showed a strong association with CD20-TCB dose and receptor occupancy (RO%; as determined by Djebli et al. ASH 2019). Interestingly, rebound of T cells 160 hours after the first CD20-TCB infusion was associated with response to treatment. Responding patients showed long-term T-cell activation after the first infusion of CD20-TCB at doses from 0.6mg and above. T-cell activation was demonstrated by 2-4-fold elevation of T-cell activation markers such as Ki67, HLA-DR, PD-1, ICOS, OX40, and 4-1BB, which was sustained up to Cycle 5 (105 days). Analysis of paired pre- and on-treatment tumor biopsies (n=6) obtained before and 2-3 weeks after the first dose of CD20-TCB showed evidence of T-cell-mediated tumor cell killing. Analysis of archival and pre-treatment tumor biopsies (n=80) revealed that clinical responses were achieved irrespective of the amount of tumor T-cell infiltration at baseline. In contrast, preliminary baseline bulk tumor RNA sequencing data (n=46) showed upregulation of gene signatures associated with cell proliferation/Myc and T-cell subsets (effector vs exhausted-like) in non-responding patients. Conclusions: In this study, we demonstrated the mode of action of CD20-TCB, a novel bispecific antibody with promising clinical activity in R/R NHL. We also demonstrated that biomarker data on T-cell activation can support dose finding in conjunction with pharmacokinetics. Additional analysis is ongoing to evaluate response predictors and better characterize the population that will benefit most from T-cell mediated therapies. Disclosures Bröske: Roche: Employment, Equity Ownership. James:A4P Consulting Ltd: Consultancy. Belousov:Roche: Employment. Gomez:F. Hoffmann-La Roche Ltd: Employment. Canamero:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Ooi:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Grabole:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Wilson:F. Hoffmann-La Roche Ltd: Employment. Korfi:F. Hoffmann-La Roche Ltd: Consultancy. Kratochwil:F. Hoffmann-La Roche Ltd: Employment. Morcos:Roche: Employment, Equity Ownership. Ferlini:Roche: Employment, Equity Ownership. Thomas:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Dimier:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Moore:F. Hoffmann-La Roche Ltd: Employment, Equity Ownership. Bacac:Roche: Employment, Equity Ownership, Patents & Royalties: Patents, including the one on CD20-TCB. Weisser:Pharma Research and Early Development Roche Innovation Center Munich: Employment, Equity Ownership, Patents & Royalties. Dickinson:Merck Sharpe and Dohme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: CD20-TCB (also known as RG6026, RO7082859) is a full-length, fully humanized, immunoglobulin G1 (IgG1), T-cell-engaging bispecific antibody with two fragment antigen binding (Fab) regions that bind to CD20 (on the surface of B cells) and one that binds to CD3 (on the surface of T cells) (2:1 format). The 2:1 molecular format of CD20-TCB, which incorporates bivalent binding to CD20 on B cells and monovalent binding to CD3 on T cells, redirects endogenous non-specific T cells to engage and eliminate malignant B cells. CD20-TCB is an investigational agent.

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