scholarly journals Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade

2020 ◽  
Author(s):  
Douglas C. Palmer ◽  
Beau R. Webber ◽  
Yogin Patel ◽  
Matthew J. Johnson ◽  
Christine M. Kariya ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
High Efficiency ◽  
Tumor Regression ◽  
Physiological Role ◽  
Tumor Infiltrating Lymphocytes ◽  
Negative Regulator ◽  
Knock Out

AbstractWhile neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

scholarly journals Internal checkpoint regulates T cell neoantigen reactivity and susceptibility to PD1 blockade

2020 ◽  
Author(s):  
Douglas Palmer ◽  
Beau Webber ◽  
Yogin Patel ◽  
Matthew Johnson ◽  
Christine Kariya ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
High Efficiency ◽  
Tumor Regression ◽  
Physiological Role ◽  
Tumor Infiltrating Lymphocytes ◽  
Negative Regulator ◽  
Knock Out

Abstract While neoantigen-specific tumor infiltrating lymphocytes (TIL) can be derived from in antigen-expressing tumors, their adoptive transfer fails to consistently elicit durable tumor regression. There has been much focus on the role of activation/exhaustion markers such as PD1, CD39 and TOX in TIL senescence. We found these markers were inversely expressed to Cytokine-Induced SH2 protein (CISH), a negative regulator of TCR signaling and tumor immunity in mice. To evaluate the physiological role of CISH in human TIL we developed a high-efficiency CRIPSR-based method to knock out CISH in fully mature TIL. CISH KO resulted in increased T cell receptor (TCR) avidity, tumor cytolysis and neoantigen recognition. CISH expression in the tumor resections correlated with TIL inactivity against p53 hotspot mutations and CISH KO in TIL unmasked reactivity against these universal neoantigens. While CISH KO resulted in T cell hyperactivation and expansion it did not alter maturation, perhaps by preferential PLCγ-1 and not AKT inhibition. Lastly, CISH KO in T cells increased PD1 expression and the adoptive transfer of Cish KO T cells synergistically combines with PD1 antibody blockade resulting in durable tumor regression and survival in a preclinical animal model. These data offer new insights into the regulation of neoantigen recognition, expression of activation/exhaustion markers, and functional/maturation signals in tumor-specific T cells.

scholarly journals Spontaneous Intestinal Tumorigenesis inApc/Min+Mice Requires Altered T Cell Development with IL-17A

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Wook-Jin Chae ◽  
Alfred L. M. Bothwell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Gene Mutation ◽  
Adoptive Transfer ◽  
Tumor Regression ◽  
T Cell Development ◽  
Cell Development ◽  
Intestinal Tumorigenesis

The control of inflammatory diseases requires functional regulatory T cells (Tregs) with significant Gata-3 expression. Here we address the inhibitory role of Tregs on intestinal tumorigenesis in theApc/Min+mouse model that resembles human familial adenomatous polyposis (FAP).Apc/Min+mice had a markedly increased frequency of Foxp3+ Tregs and yet decreased Gata-3 expression in the lamina propria. To address the role of heterozygousApcgene mutation in Tregs, we generatedFoxp3-Cre,Apcflox/+mice. Tregs from these mice effectively inhibited tumorigenesis comparable to wild type Tregs after adoptive transfer intoApc/Min+mice, demonstrating that the heterozygousApcgene mutation in Tregs does not induce the loss of control over tumor microenvironment. Adoptive transfer of in vitro generatedApc/Min+iTregs (inducible Tregs) failed to inhibit intestinal tumorigenesis, suggesting that naïve CD4 T cells generated fromApc/Min+mice thymus were impaired. We also showed that adoptively transferred IL-17A-deficientApc/Min+Tregs inhibited tumor growth, suggesting that IL-17A was critical to impair the tumor regression function ofApc/Min+Tregs. Taken together, our results suggest that both T cell development in a functional thymus and IL-17A control the ability of Treg to inhibit intestinal tumorigenesis inApc/Min+mice.

333 Targeting the apical intracellular checkpoint CISH unleashes T cell neoantigen reactivity and effector program

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A359-A359
Author(s):  
Douglas Palmer ◽  
Beau Webber ◽  
Yogin Patel ◽  
Matthew Johnson ◽  
Christine Kariya ◽  
...  
Keyword(s):  
Clinical Trial ◽  
T Cells ◽  
T Cell ◽  
High Efficiency ◽  
Tumor Regression ◽  
Human Tumor ◽  
Tumor Infiltrating Lymphocytes ◽  
T Cell Therapy ◽  
Human T Cells

BackgroundNeoantigen-specific T cells isolated from tumors have shown promise clinically but fail to consistently elicit durable tumor regression. Expression of the intracellular checkpoint CISH is elevated in human tumor infiltrating lymphocytes (TIL) and has been shown to inhibit neoantigen reactivity in murine TIL.MethodsTo explore CISH function in human T cells we developed a CRISPR/Cas9-based strategy to knockout (KO) CISH in human T cells with high-efficiency (>90%) and without detectable off-target editing.ResultsCISH KO in peripheral blood T cells enhanced proliferation, cytokine polyfunctionality, and cytotoxicity in vitro. To determine if CISH KO similarly enhances TIL function, we developed a clinical-scale, GMP-compliant manufacturing process for CISH disruption in primary human TIL. In process validation runs we achieved CISH KO efficiencies >90% without detectable off-target editing while maintaining high viability and expansion. Compared to WT controls, CISH KO in patient-derived TIL demonstrated increased proliferation, T cell receptor (TCR) avidity, neoantigen recognition, and unmasked reactivity to common p53 mutations. Hyperactivation in CISH KO TIL did not increase differentiation, suggesting that CISH KO may uncouple activation and differentiation pathways. Single cell profiling identifies a pattern of CISH expression inverse to key regulators of activation, and CISH KO in human TIL increases PD1 expression. Adoptive transfer of Cish KO T cells synergistically combines with PD1 inhibition resulting in durable tumor regression in mice, highlighting orthogonal dual cell surface and intracellular checkpoint inhibition as a novel combinatorial approach for T cell immunotherapy.ConclusionsThese pre-clinical data offer new insight into neoantigen recognition and serve as the basis for a recently initiated human clinical trial at the University of Minnesota (NCT04426669) evaluating inhibition of the novel intracellular immune checkpoint CISH in a CRISPR-engineered, neoantigen-specific T cell therapy for solid tumors. Updates from the clinical trial will be highlighted.Trial RegistrationNCT04426669

scholarly journals Tumor masses support naive T cell infiltration, activation, and differentiation into effectors

2010 ◽  
Vol 207 (8) ◽  
pp. 1791-1804 ◽  
Author(s):  
Elizabeth D. Thompson ◽  
Hilda L. Enriquez ◽  
Yang-Xin Fu ◽  
Victor H. Engelhard
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Activation ◽  
Ex Vivo ◽  
Tumor Infiltrating Lymphocytes ◽  
T Cell Infiltration

Studies of T cell responses to tumors have focused on the draining lymph node (LN) as the site of activation. We examined the tumor mass as a potential site of activation after adoptive transfer of naive tumor-specific CD8 T cells. Activated CD8 T cells were present in tumors within 24 h of adoptive transfer and proliferation of these cells was also evident 4–5 d later in mice treated with FTY720 to prevent infiltration of cells activated in LNs. To confirm that activation of these T cells occurred in the tumor and not the tumor-draining LNs, we used mice lacking LNs. Activated and proliferating tumor-infiltrating lymphocytes were evident in these mice 24 h and 4 d after naive cell transfer. T cells activated within tumors acquired effector function that was evident both ex vivo and in vivo. Both cross-presenting antigen presenting cells within the tumor and tumor cells directly presenting antigen activated these functional CD8 effectors. We conclude that tumors support the infiltration, activation, and effector differentiation of naive CD8 T cells, despite the presence of immunosuppressive mechanisms. Thus, targeting of T cell activation to tumors may present a tool in the development of cancer immunotherapy.

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

Blood ◽  
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.

scholarly journals Tumor Regression and Autoimmunity after Reversal of a Functionally Tolerant State of Self-reactive CD8+ T Cells

2003 ◽  
Vol 198 (4) ◽  
pp. 569-580 ◽  
Author(s):  
Willem W. Overwijk ◽  
Marc R. Theoret ◽  
Steven E. Finkelstein ◽  
Deborah R. Surman ◽  
Laurina A. de Jong ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Tumor Antigen ◽  
Tumor Regression ◽  
Peptide Ligand ◽  
Altered Peptide Ligand ◽  
Large Numbers ◽  
Tolerant State

Many tumor-associated antigens are derived from nonmutated “self” proteins. T cells infiltrating tumor deposits recognize self-antigens presented by tumor cells and can be expanded in vivo with vaccination. These T cells exist in a functionally tolerant state, as they rarely result in tumor eradication. We found that tumor growth and lethality were unchanged in mice even after adoptive transfer of large numbers of T cells specific for an MHC class I–restricted epitope of the self/tumor antigen gp100. We sought to develop new strategies that would reverse the functionally tolerant state of self/tumor antigen-reactive T cells and enable the destruction of large (with products of perpendicular diameters of >50 mm2), subcutaneous, unmanipulated, poorly immunogenic B16 tumors that were established for up to 14 d before the start of treatment. We have defined three elements that are all strictly necessary to induce tumor regression in this model: (a) adoptive transfer of tumor-specific T cells; (b) T cell stimulation through antigen-specific vaccination with an altered peptide ligand, rather than the native self-peptide; and (c) coadministration of a T cell growth and activation factor. Cells, vaccination, or cyto-kine given alone or any two in combination were insufficient to induce tumor destruction. Autoimmune vitiligo was observed in mice cured of their disease. These findings illustrate that adoptive transfer of T cells and IL-2 can augment the function of a cancer vaccine. Furthermore, these data represent the first demonstration of complete cures of large, established, poorly immunogenic, unmanipulated solid tumors using T cells specific for a true self/tumor antigen and form the basis for a new approach to the treatment of patients with cancer.

Abstract 16615: Reconstitution of CD4 T Cells Causes Cardiac Fibrosis and Myocardial Dysfunction in T Cell Specific S1P Receptor 1 Deficient Diabetic Mice

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Chowdhury S Abdullah ◽  
Zhu-Qiu Jin
Keyword(s):  
T Cells ◽  
Body Weight ◽  
T Cell ◽  
Adoptive Transfer ◽  
Cd4 T Cells ◽  
Cardiac Fibrosis ◽  
Myocardial Dysfunction ◽  
Diabetic Mice ◽  
Knock Out ◽  
S1p Receptor

Involvement of T cells in fibrosis has been reported but modulation of their role in context of diabetic fibrogenesis has yet to be determined. Our previous studies indicated that T cell S1P receptor 1 (S1P1) genetic depletion ensues sustained lymphopenia in circulation and reduced fibrosis in streptozotocin-induced type 1 murine diabetic model. We hypothesized that adoptive transfer of T cells to T-cell S1P1 receptor knock-out (TS1P1KO) mice would abolish cardioprotection and antifibrotic effect as observed earlier. TS1P1KO and littermate wild-type (WT) mice were divided into vehicle and streptozotocin (STZ) (50 mg/kg body weight for five days, i.p.) groups. Naïve CD4 T cells were isolated by positive selection through MS column from CD4 magnetic microbeads-labeled WT mice splenocytes. Isolated CD4 T cells (purity >95%) were adoptively transferred (i.v.) into the mice of above groups at dose of one million cells. Body weight (g) and blood glucose level (mg/dl) were monitored. CD4 and CD8 T cells in blood were counted by flow cytometry. Heart histology was studied in H&E stained sections and pathological grading was given. Masson Trichrome stained heart sections were digitally imaged at 16x magnification and percent of fibrotic area was quantified by using NIH ImageJ. Cardiac contractility was measured in ex-vivo Langendorff’s heart perfusion system at the end of 11 weeks. TS1P1KO mice had ~90% reduced T cells (CD4 cells: 1.15±0.30% vs 25.06±0.64%, CD8 cells: 2.09±0.42% vs 14.72±0.38% in WT, **P<0.01, n=4-5) in blood. KO diabetic mice without adoptive transfer of CD4 T cells exhibited about 70% less fibrotic area (11.86±4.34% vs 46.48±8.06% in WT STZ, *P<0.05, n=7-9) and improved cardiac structure and function. Adoptively CD4 T cells recipient KO diabetic mice presented cardiac structural disorganization (histological score: 9.25±0.95 vs. 1.29±0.52 in KO STZ without transfer, *P<0.05, n=4-7) and increased myocardial fibrosis (37.11±3.22% vs. 11.86±4.34% in KO STZ without transfer, *P<0.05, n=4-7) with reduced cardiac contractile force compared with KO diabetic mice without CD4 T cells transfer. In conclusion, reconstitution of CD4 T cells increases cardiac fibrosis and attenuates cardiac function in lymphopenic T cell S1P1 knock-out diabetic mice.

CD24-Siglec-G Interaction Plays an Important in Reducing Experimental Graft-Versus-Host Disease (GVHD)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 453-453
Author(s):  
Tomomi Toubai ◽  
Rebecca Evers ◽  
Yaping Sun ◽  
Isao Tawara ◽  
Chen Liu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
Immune Activation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Negative Regulator ◽  
Clinical Severity ◽  
Allogeneic Bmt

Abstract Abstract 453 The role of host antigen presenting cells (APCs) on negatively regulating GVHD is not well understood. Members of the sialic acid binding Ig–like lectin-G (Siglec-G) is an immunoreceptor tyrosine-based inhibitory motifs (ITIM) or ITIM-like regions in its intracellular domain that negatively regulates immune activation induced by non-infectious damage associated molecules (DAMPs). Following conditioning for allogeneic BMT, several DAMPs are released which stimulate host APCs and enhance GVHD. But the role of negative regulators of DAMP associated immune activation, such as Siglecs, in regulating allo-reactivity is not known. We therefore utilized well defined clinically relevant murine models of allogeneic BMT to test the hypothesis that deficiency of a negative regulator of responses to DAMPs in the hosts, namely Siglec-G, will increase GVHD. B6 wild type (WT) and Siglec-G−/− animals were lethally irradiated (13Gy) and transplanted on day 0 with 5×106 bone marrow and 3×106 splenic CD90+T cells from either syngeneic WT-B6 or MHC mismatched BALB/c donors. The Siglec-G−/− animals showed significantly worse survival than the allo-WT animals (p=0.0045). The increased mortality was associated with an increase in GVHD specific clinical severity (p<0.05), donor T cell expansion (p<0.03), and serum levels of pro-inflammatory cytokines (TNFα, IFN-γ, p<0.05) on day +7 after BMT. We next evaluated whether this was because of Siglec-G deficiency only on the radiosensitive host APCs. To this end we generated [B6àB6Ly5.2] and [Siglec-G−/−àB6Ly5.2] BM chimeras and utilized them as recipients following lethal radiation. They were injected with 5×106 BM and 3×106 CD90+ T cells from either syngeneic WT B6 or allogeneic BALB/c donors. The allogeneic [Siglec-G−/−àB6Ly5.2] animals demonstrated significantly worse survival than the [B6àB6Ly5.2] animals (p<0.0001). We determined the converse, i.e. analyzed the role of Siglec-G on radio-resistant host APCs by generating [B6Ly5.2àB6] and [B6Ly5.2àSiglec-G−/−] BM chimeras and utilized them as recipients. [B6Ly5.2àSiglec-G−/−] chimeras demonstrated similar survival as [B6Ly5.2àB6] chimeras. These data collectively demonstrate that Siglec-G expression only on the host radiosensitive APCs is critical for protection from GVHD. To confirm the role of increased DAMPs in causing greater mortality we tested whether the intensity of conditioning affects the serum level of DAMPs (HMGB1, proinflammatory cytokines) and found that significantly greater levels of DAMPs were observed in the mice that received 13Gy than 8 Gy. Furthermore, consistent with the increased levels of DAMPs, Siglec-G−/− animals showed higher GVHD only after 13Gy radiation but not after 8Gy conditioning. Because responses to non-infectious DAMPs are regulated by Siglec-G through its interaction with CD24 we next hypothesized that enhanced CD24-Siglec-G interaction would mitigate GVHD following myeloablative conditioning. We first characterized stimulation of allogeneic T cell responses by Siglec-G−/− APCs. We utilized CD24−/− and WT BALB/c T cells as responders in an MLR with B6 and Siglec-G−/−stimulators. We found that Siglec-G−/− APCs expanded the CD24−/− T cells more than WT-B6 APCs. We next tested in vivo whether enhanced CD24-Siglec-G interaction would mitigate GVHD. We utilized a novel CD24 fusion protein (day-1, 100mg/mouse) and found that it decreased GVHD mortality only in the WT but not in the Siglec-G−/− animals. Together our data demonstrate a critical role for CD24-Siglec-G interactions in regulating GVHD and suggest that administration of the novel CD24 fusion protein may be an innovative strategy to mitigate GVHD. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hypertension in response to CD4+ T cells from reduced uterine perfusion pregnant rats is associated with activation of the endothelin-1 system

2012 ◽  
Vol 303 (2) ◽  
pp. R144-R149 ◽  
Author(s):  
Kedra Wallace ◽  
Sarah Novotny ◽  
Judith Heath ◽  
Janae Moseley ◽  
James N. Martin ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Rat Serum ◽  
Pregnant Rats ◽  
Endothelin 1 ◽  
Induced Hypertension ◽  
Uterine Perfusion ◽  
Placental Ischemia

We have shown that adoptive transfer of CD4+ T cells from placental ischemia (reduction in uteroplacental perfusion, RUPP) rats causes hypertension and elevated inflammatory cytokines during pregnancy. In this study we tested the hypothesis that adoptive transfer of RUPP CD4+ T cells was associated with endothelin-1 activation as a mechanism to increase blood pressure during pregnancy. CD4+ T cells from RUPP or normal pregnant (NP) rats were adoptively transferred into NP rats on gestational day 13. Mean arterial pressure (MAP) was analyzed on gestational day 19, and tissues were collected for endothelin-1 analysis. MAP increased in placental ischemic RUPP rats versus NP rats (124.1 ± 3 vs. 96.2 ± 3 mmHg; P = 0.0001) and increased in NP recipients of RUPP CD4+ T cells (117.8 ± 2 mmHg; P = 0.001 compared with NP). Adoptive transfer of RUPP CD4+ T cells increased placental preproendothelin-1 mRNA 2.1-fold compared with NP CD4+ T cell rats and 1.7-fold compared with NP. Endothelin-1 secretion from endothelial cells exposed to NP rat serum was 52.2 ± 1.9 pg·mg−1·ml−1, 77.5 ± 4.3 pg·mg−1·ml−1 with RUPP rat serum ( P = 0.0003); 47.2 ± .16 pg·mg−1·ml−1 with NP+NP CD4+ T cell serum, and 62.2 ± 2.1 pg·mg−1·ml−1 with NP+RUPP CD4+ T cell serum ( P = 0.002). To test the role of endothelin-1 in RUPP CD4+ T cell-induced hypertension, pregnant rats were treated with an endothelin A (ETA) receptor antagonist (ABT-627, 5 mg/kg) via drinking water. MAP was 92 ± 2 mmHg in NP+ETA blockade and 108 ± 3 mmHg in RUPP+ETA blockade; 95 ± 5 mmHg in NP+NP CD4+ T cells+ETA blockade and 102 ± 2 mmHg in NP+RUPP CD4+ T cells+ETA blockade. These data indicate the importance of endothelin-1 activation to cause hypertension via chronic exposure to activated CD4+ T cells in response to placental ischemia.

scholarly journals Stim1 Deletion Synthetically Rescues Ezh2-Null Effector T Cells and Alloimmunity

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4533-4533
Author(s):  
Ying Wang ◽  
Shan He ◽  
Yongnian Liu ◽  
Robert Hooper ◽  
Hongshuang Yu ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Effector T Cells ◽  
Conditional Knockout ◽  
Negative Regulator ◽  
Sequencing Analysis ◽  
Major Barrier ◽  
Donor T Cells

Abstract Graft-versus-host disease (GVHD) remains a major barrier for the success of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We have identified the central role of the histone methyltransferase Ezh2 in regulating allogeneic T-cell expansion, differentiation and function. Conditional loss of Ezh2 in donor T cells inhibits GVHD in mice due to the inability of alloreactive T cells to persist. However, the molecular mechanism by which Ezh2 deficiency causes alloreactive T cell death remains unknown. Here we demonstrate that genetic deletion of Stromal Interaction Molecule (Stim) 1, a dynamic endoplasmic reticulum Ca2+ sensor and regulator of Ca2+ signaling, rescues antigen-activated Ezh2-null (Ezh2-/-) T cells, leading to restored persistence of alloreactive effector T cells in mice and severe GVHD. Using RNA-sequencing analysis, we found Ezh2-deficiency led to the upregulation of multiple genes (e.g., Ifng, Prf1, Ccl5, Ccl4, Upp1 and Spp1) known to be regulated by Ca2+ signals through calcineurin (CN), the primary target of the immunosuppressant cyclosporine A (CsA). This reverse correlation between Ezh2 inhibition and CsA-treatment for gene expression suggests that Ezh2 may antagonize Ca2+ signaling in activated T cells. Calcium signaling assays revealed higher cytosolic Ca2+ uptake and more frequent Ca2+ oscillations in Ezh2-/- T cells. Moreover, Ezh2-/- T cells exhibited significantly increased polarization of Stim1 and Orai1 in the cellular membrane. These data reveal an unexpected role of Ezh2 as a negative regulator of Ca2+ entry, thereby serving as a 'brake' for Ca2+ signaling. Using the C57BL/6 (B6) into Balb/c mouse GVHD model, we found significantly fewer Ezh2-/- or Stim1-/- IFN-g-secreting effector T cells compared to the WT counterparts on day 8 or 14 post-transplantation. In contrast, deleting Stim1 from Ezh2-/- donor T cells rescued the cells in the spleen and liver, producing even more donor T cells and IFN-g-secreting effector T cells compared to WT T cells and inducing severe GVHD. We further examined the cell autonomous effect of Stim1 deletion on the rescue of Ezh2-/- T cells by mixing WT T cells (B6/SJL, CD45.1) with Ezh2- and/or Stim1- conditional knockout T cells (i.e., Ezh2-/-, Stim1-/- or Ezh2-/- x Stim1-/- B6 T cells (CD45.2)) at a ratio of 1:1 before transferring into the Balb/c mice. While loss of either Ezh2 or Stim1 led to lower frequency of IFN-g+IL-2+ effector T cells, combined deletion of both genes restored the frequency and number of IFN-g+IL-2+ effector T cells to that of WT T cells. Thus, Stim1-mediated Ca2+ signals are crucial for mediating cell death in alloantigen-driven Ezh2-/- effector T cells. To further determine whether the inhibition of CN-NFAT contributes to the rescue, we treated T cell receptor (TCR)-activated Ezh2-/- T cells with CsA or the calcium release-activated channel specific inhibitor BTP2, respectively, in vitro. While BTP2 dramatically improved the survival of IFN-g-producing effector T cells, CsA did not, suggesting the involvement of CN-NFAT-independent pathways. Ca2+ overload is known to impair mitochondrial function and cause massive cell death. As compared to TCR-activated WT T cells, activated Ezh2-/- T cells displayed significantly less ATP, lower mitochondrial membrane potential, enlarged mitochondrial mass, and decreased capacity to upregulate oxidative phosphorylation. Stim1 deletion largely reversed the metabolic defect in Ezh2-/- T cells, indicating the critical role of mitochondrial metabolism in rescuing these T cells. Considered together, our findings identify the remarkable coordination between Ezh2- and Stim1-regulated effector T cell persistence. As such, these investigations may lead to new approaches to inhibit GVHD, with broad implications to defining fundamental mechanisms of T cell differentiation for control of adaptive immunity, such as tumor immunity and autoimmunity. Disclosures Reshef: Incyte: Consultancy; Takeda Pharmaceuticals: Consultancy; Pfizer: Consultancy; Kite Pharma: Consultancy; Atara Biotherapeutics: Consultancy; Bristol-Myers Squibb: Consultancy.

Sign in / Sign up

Export Citation Format

Share Document