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478 Calcineurin Inhibitors Inhibit Tolerance Induction By Suppressing Terminal Differentiation of Donor Exhausted T Cells after Allogeneic SCT

Program: Oral and Poster Abstracts
Type: Oral
Session: 701. Experimental Transplantation: Basic and Translational: Novel Mechanisms of Acute and Chronic Graft-versus-Host Disease
Hematology Disease Topics & Pathways:
Research, Biological therapies, Translational Research, GVHD, Checkpoint Inhibitor, Diseases, Immune Disorders, immune mechanism, Therapies, immunology, Biological Processes, Transplantation
Sunday, December 11, 2022: 10:15 AM

Hajime Senjo, M.D.1, Daigo Hashimoto, MD1, Shinpei Kubota, M.D., Ph.D.2*, Yuki Tanaka, Ph.D.2*, Shinpei Harada3*, Kazuki Yoneda, M.D.1*, Zixuan Zhang1*, Xuanzhong Chen1*, Ryo Kikuchi, M.D.1*, Yuta Hasegawa, M.D., Ph.D.1*, Hiroyuki Ohigashi, M.D., Ph.D.1*, Takahide Ara, M.D., Ph.D.1*, Yoshinori Hasegawa, Ph.D.4*, Masaaki Murakami, V.M.D., Ph.D.2* and Takanori Teshima, M.D., Ph.D.1

1Department of Hematology, Hokkaido University Faculty of Medicine, Sapporo, Japan
2Molecular Psychoneuroimmunology, Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan
3Department of Hematology, Hokkaido University Faculty of Medicine, Sapporo City, Japan
4Department of Applied Genomics, Kazusa DNA Research Institute, Chiba, Japan

[Introduction] Calcineurin inhibitors (CNIs) reduce acute GVHD by inhibiting NFAT signaling in donor T cells but fail to prevent chronic GVHD (cGVHD). We have previously shown that donor alloreactive T cells differentiated into exhausted T cells with severely impaired alloreactivity after mouse allogeneic hematopoietic stem cell transplantation (allo-SCT) (Asakura S: JCI 2010). Given the critical role of NFAT signaling in T-cell exhaustion, we explored the role of CNIs in donor T-cell exhaustion and tolerance induction after allo-SCT using mouse SCT models with host alloantigen (allo-Ag)-specific 2C TCR-transgenic (2C) T cells and single cell RNA sequencing (scRNAseq) of donor T cells. [Methods] B6D2F1 recipients (H-2b/d) were lethally irradiated and transplanted with 1 x 106 2C T cells (CD8.1+) plus 1 x 106 wild type T cells (WT, CD45.1+) of B6 background (H-2b) together with T-cell-depleted bone marrow cells (TCD-BM) from WT B6 (CD45.2+) on day 0. In some experiments, recipients were p.o. injected with 25 mg/kg cyclosporin (CSP) daily on days 0-14. [Results] Flowcytometric analysis (FCM) showed 2C CD8+ T cells differentiated into terminally exhausted T cells (Tex) expressing multiple co-inhibitory receptors such as PD-1 and TIGIT on day 14 after allo-SCT, that led to unresponsiveness of donor T cells to PD-1 blockade. Next, we performed scRNAseq of donor T cells harvested from CSP- or vehicle-treated recipients on day 14 after allo-SCT. UMAP analysis identified 4 clusters of CD4+ T cells (C10-C13) and 9 clusters of CD8+ T cells (C1-C9), and all of them expressed Tox and Pdcd1, indicating that all donor T cells entered differentiation pathway to exhaustion. CSP remarkably increased C10 (CD4+) and C1 (CD8+) characterized by lower expression of Tox and Pdcd1 and higher expression of Ly6c2 compared to other clusters (Fig 1). FCM confirmed CSP significantly expanded PD-1+TIGIT-, and TOXlow cells in 2C and WT CD8+ and WT CD4+ donor T cells on day 14, and these cells persisted at least 2 wks after cessation of CSP. After adoptively transferred into irradiated secondary B6D2F1 recipients, Ly6C+ T cells purified from CSP-treated recipients were differentiated into Ly6C- Tex, while Ly6C- T cells remained to be Ly6C-, indicating that CSP-induced Ly6C+ donor T cells are precursors of Tex (pTex) like cells. We named these cells as pTex-CNI, based on lower TCF-1 expression compared to TCF-1high canonical pTex. We found that, among various agents known to suppress GVHD or Tex differentiation, only CSP and ibrutinib induced ToxlowPD-1+TIGIT-Ly6C+ pTex-CNI, suggesting that NFAT inhibition is critical for pTex-CNI induction. Importantly, only Ly6C+ donor T cells, but not Ly6C- cells, induced cGVHD after adoptive transfer, suggesting that pTex-CNI play a critical role in pathophysiology of cGVHD (Fig 2). pTex-CNI demonstrated marked proliferative response and differentiation toward Tex upon PD-1 blockade as canonical pTex did in tumor and viral infection models in the previous reports (Sade-Feldman M: Cell 2018, Hudson WH: Immunity 2019). Administration of PD-L1 mAbs from day 14 enhanced OXPHOS of donor T cells and eradicated host-type leukemia cells only in CSP-treated recipients. Post-transplant cyclophosphamide (PTCy) is widely used in clinical allo-SCT, and CNIs are started either before SCT or after PTCy typically on day 5. We found that pTex-CNI were induced only when CSP was added to PTCy from day -1, but not from day 5, suggesting that NFAT inhibition early after allo-SCT is critical for induction of pTex-CNI. Finally, we confirmed that T cells from the patients who underwent HLA-matched PBSCT with tacrolimus (TAC)-based GVHD prophylaxis started from day -1 expressed significantly lower levels of PD-1, TIGIT, and TOX on day 28 compared to those from patients underwent haploidentical PBSCT with PTCy-based GVHD prophylaxis combined with TAC started from day 5. [Conclusion] Our study for the first time demonstrated that CNI inhibits terminal differentiation of donor exhausted T cells after allo-SCT. In mouse models, pTex-CNI contributed to development of cGVHD and PD-1 blockade-induced anti-tumor response after allo-SCT. The timing of CNI initiation in PTCy-based allo-SCT could determine the persistence of donor T cell alloreactivity after allo-SCT, that could impact on the strategies of cessation of immunosuppressants and treatment of relapsed diseases. These should be tested in the future clinical studies.

Disclosures: Teshima: Astellas: Research Funding; Eisai: Research Funding; Chugai: Research Funding; Bristol-Myers Squibb: Honoraria; Asahi Kasei Pharma: Research Funding; Merck Sharp & Dohme: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Other: Manuscript preparation; Fuji Pharma: Research Funding; NIPPON SHINYAKU: Research Funding; Kyowa Kirin: Honoraria, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Sanofi: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: Manuscript preparation; Luca Science Inc.: Research Funding; Sumitomo Pharma: Research Funding; ONO: Research Funding; SHIONOGI: Research Funding; Priothera SAS: Research Funding; Otsuka: Research Funding; AbbVie: Honoraria; Celgene: Honoraria; Meiji Seika Pharma: Other: non-financial support; DAIICHI SANKYO: Other: non-financial support ; AstraZeneca: Other: non-financial support ; Roche Diagnostics: Other: non-financial support .

*signifies non-member of ASH