CAR-T and GvHD: Have We Engineered GvHD Out of Allogeneic CAR‑T Therapy?

Development of a Protective Cellular Therapy to Prevent Graft Versus Host Disease Post Allogenic Hematopoietic Stem Cell Transplant 2331939

Graft-versus-host disease (GVHD) remains a primary cause of morbidity and mortality post-allogeneic hematopoietic stem cell transplantation (allo-HSCT), driven by donor T cells’ dysregulated inflammation against host tissues. This limits survival and underscores the need for therapies that curb GVHD while preserving graft-versus-tumor (GVT) effects. Innate-like T cell subsets, including MAIT, NKT, and Vγ2 γδ T cells correlate with milder GVHD. These share the BTB-ZF transcription factor PLZF (Zbtb16), a master regulator of innate-like programming. Building on our finding that ectopic PLZF imparts innate-like traits to conventional αβ T cells, we tested if PLZF-overexpressing donor T cells suppress GVHD in vivo. In a B6→BALB/c acute GVHD model, lethally irradiated BALB/c recipients received bone marrow plus purified CD4+/CD8+ T cells from C57BL/6 donors, transduced with retroviral PLZF or empty vector (EV) control (n = 15/group). Mice were monitored daily for survival, clinical scores, and weight loss. In mouse models of acute GVHD (B6→BALB/c), adoptive transfer of retrovirally transduced, PLZF-overexpressing conventional T cells markedly delayed disease onset and enhanced survival relative to empty vector-transduced controls. Mechanistically, PLZF-overexpressing conventional T cell promoted expansion of regulatory T cell (Treg) populations, thereby driving a phenotypic shift toward innate-like suppression of alloimmunity. These results affirm PLZF overexpression as a viable, programmable approach for GVHD prophylaxis, poised for clinical advancement. Through innate-like T cell reprogramming, this strategy holds promise for revolutionizing allo-HSCT by decoupling immune tolerance from antitumor immunity. Current investigations are evaluating PLZF-overexpressing conventional T cell preservation of graft versus tumor (GVT) activity against leukemia and extending these findings to humanized models. NJ PHORCE Transplantation Immunology (TRAN)

Chronic xenogeneic GVHD after CAR T cell therapy limits long-term safety assessment of CAR effects using NSG mouse model.

The clinical successes of chimeric antigen receptor (CAR) T cells represent a major shift in immunotherapy. However, there is also increasing emphasis on potential long-term effects of CAR T cell therapy, especially using preclinical xenogeneic models. It has been previously demonstrated that only naïve, and not memory, peripheral blood human T cells can mediate a rapid and acute xenogeneic graft-versus-host disease (xenoGVHD). Here, we demonstrate that simply by altering the donor T cells in the process of generating CAR T cells, in which they are all memory phenotype, that the xenoGVHD outcome was markedly altered. Following tumor clearance with CAR T cell administration, we observed a significantly delayed (up to 200 days post-transfer with some donors) occurrence of lethal xenoGVHD, marked by profound scleroderma and multi-organ pathology consistent with chronic, not acute, GVHD. Notably, this novel chronic xenoGVHD occurred in the absence of B cells, which are classically associated with mediating chronic GVHD pathology. TCR-repertoire constriction during disease and the lack of disease using MHCI/II double-knockout NSG recipient mice confirmed the observed pathology was xenoGVHD and mediated by human-TCR:murine-MHC interactions. Interestingly, despite the consistent expansion of CAR-positive T cells during early tumor-clearance, a later emergence of CAR-negative populations during xenoGVHD also resulted. Our findings highlight xenoGVHD as a problem that makes long-term assessment of CAR T efficacy or toxicity highly problematic in xenograft models due to the artefact of xenoreactivity which is not representative of autologous clinical usage, and the profound effect that T cell alterations have in GVHD pathophysiology.

Lymphoid Tissue-Activated Donor T cells (LADTs) for Improved Donor Lymphocyte Infusion (DLI) against Blood Cancers 2250463

Donor lymphocyte infusion (DLI) is a key strategy for treating relapses after HLA-mismatched allogeneic hematopoietic stem cell transplantation (allo-HSCT) for blood cancers. In DLI, T cell alloreactivity drives both the highly beneficial graft vs leukemia (GvL) effect and the potentially fatal graft vs host diseases (GvHDs), which severely limits DLI’s application and efficacy. Here, we propose to separate GvL and GvHD by engineering Lymphoid Tissue-Activated Donor T cells (LADTs) that display alloreactivity only in lymphoid tissues where cancer cells accumulate but not in organs involved in GvHD including the skin, liver and guts. To generate LADTs, PBMCs were first used to expand alloreactive T cells from an HLA-mismatched donor. Then, T cell alloreactivity was abrogated by knocking out CD3ε using CRISPR/Cas9, which blocks cell surface expression of the TCR/CD3 complex. Finally, the T cells were engrafted with a B cell-sensing mechanism that includes a CD19-specific synthetic Notch receptor (synNotch) and an expression cassette that drives the expression of an exogenous copy of CD3ε in response to synNotch engagement. This mechanism enables LADTs to restore alloreactivity only in lymphoid organs where B cells are abundant but not in other organs where B cells are scarce. We have established a process of generating LADTs with consistent yields. LADTs bearing CD19-specific synNotch receptors restored surface expression of TCR/CD3 in response to CD19+ cells and showed strong cytotoxicity against HLA+ U266 myeloma cells but not against HLA- ones. After removing CD19+ cells, LADTs gradually lost TCR/CD3 cell surface expression. Our study demonstrated the feasibility of LADT generation and the proof-of-concept of using LADTs for improved DLI. This cellular immunotherapy approach does not depend on cancer-specific biomarkers thus may be applied to a wide range of hematopoietic malignancies. Nemours Foundation Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Universal CAR-T cell therapy: mechanisms and evasion strategies of bidirectional immune rejection.

This review systematically delineates the mechanisms underlying bidirectional immune rejection and comprehensively summarizes state-of-the-art mitigation strategies and highlights the development of alternative, inherently hypoimmunogenic cell sources as a fundamental approach to circumventing these immunological barriers.

[Novel therapeutic strategies to separate GVHD from GVL based on GVHD pathophysiology and mechanisms of leukemia relapse].

Crosstalk Between Donor T Cells and Recipient IL-33+ Target Tissue Stroma Controls Alloimmune Responses Causing Graft-versus-Host Disease 2260079

Graft-versus-host disease (GVHD) remains a major complication of allogeneic hematopoietic cell transplantation (AlloHCT). Conditioning with chemotherapy and radiation enables donor engraftment but causes tissue injury that upregulates the alarmin interleukin-33 (IL-33) in fibroblastic reticular cells (FRCs) in secondary lymphoid organs (SLO) and tissue stromal cells. While mechanisms releasing IL-33 from nuclear sequestration remain elusive, free IL-33 directly activates donor CD4+ T cells to promote Th1 differentiation and expansion. Early IL-33 blockade limits GVHD initiation, yet the role of local IL-33 in sustaining alloimmunity remains unclear. We used preclinical models to define how IL-33 signaling to donor T cells within SLO versus the gastrointestinal tract (GIT) stroma controls active GVHD and graft-versus-lymphoma (GVL) responses. BALB/c recipients received B6 T cell—depleted bone marrow, A20 lymphomas, and T cells from donors with inducible ST2 (IL-33R) deletion to control IL-33 signaling during GVHD and GVL. To identify stromal functions, B6 recipients lacking IL-33 in FRCs (CCL19-Cre×Il33fl/fl) or gut epithelium (Vil-Cre×Il33fl/fl) were transplanted with BALB/c T cells and bone marrow. T cell—dependent IL-33 release was tested using GzmB⁻/⁻, St2+/+, and St2⁻/⁻ T cells. Late ST2 deletion resolved GVHD without compromising GVL. IL-33 signaling increased Foxp3 levels and reduced T-bet expression in donor CD4+ T cells, while sustaining TCF-1. Unexpectedly, loss of FRC-derived IL-33 exacerbated GVHD, whereas gut epithelial IL-33 deletion was protective. IL-33 induced GzmB, and GzmB⁻/⁻ T cells displayed impaired activation and expansion. Early SLO-derived IL-33 establishes effector programming that influences later tissue responses. IL-33+ stroma—T-cell crosstalk programs donor CD4+ T-cell fate and shapes local alloimmunity during GVHD. IL-33—stroma feedback is a key signal to alloreactive T cells during GVHD. NIH/NIAID F30AI147437, NIH T32 CA082084, NIH/NHLBI R01HL122489, NIH/NIAID R21AI121981, NIH/NIAID R56AI13927 Transplantation Immunology (TRAN)