Donor T cell STAT3 deficiency enables tissue PD-L1-dependent prevention of graft-versus-host disease while preserving graft-versus-leukemia activity.

STAT3 deficiency (STAT3-/-) in donor T cells prevents graft-versus-host disease (GVHD), but the impact on graft-versus-leukemia (GVL) activity and mechanisms of GVHD prevention remains unclear. Here, using murine models of GVHD, we show that STAT3-/- donor T cells induced only mild reversible acute GVHD while preserving GVL effects against nonsusceptible acute lymphoblastic leukemia (ALL) cells in a donor T cell dose-dependent manner. GVHD prevention depended on programmed death ligand 1/programmed cell death protein 1 (PD-L1/PD-1) signaling. In GVHD target tissues, STAT3 deficiency amplified PD-L1/PD-1 inhibition of glutathione (GSH)/Myc pathways that regulate metabolic reprogramming in activated T cells, with decreased glycolytic and mitochondrial ATP production and increased mitochondrial ROS production and dysfunction, leading to tissue-specific deletion of host-reactive T cells and prevention of GVHD. Mitochondrial STAT3 deficiency alone did not reduce GSH expression or prevent GVHD. In lymphoid tissues, the lack of host-tissue PD-L1 interaction with PD-1 reduced the inhibition of the GSH/Myc pathway despite reduced GSH production caused by STAT3 deficiency and allowed donor T cell functions that mediate GVL activity. Therefore, STAT3 deficiency in donor T cells augments PD-1 signaling-mediated inhibition of GSH/Myc pathways and augments dysfunction of T cells in GVHD target tissues while sparing T cells in lymphoid tissues, leading to prevention of GVHD while preserving GVL effects.

Donor programmed cell death 1 ligand 1 is required for organ transplant tolerance in major histocompatibility complex-mismatched mixed chimeras although programmed cell death 1 ligand 1 and major histocompatibility complex class II are not required for inducing chimerism.

Induction of major histocompatibility complex (MHC) human leukocyte antigen (HLA)-mismatched mixed chimerism is a promising approach for organ transplantation tolerance; however, human leukocyte antigen-mismatched stable mixed chimerism has not been achieved in the clinic. Tolerogenic dendritic cell (DC) expression of MHC class II (MHC II) and programmed cell death 1 ligand 1 (PD-L1) is important for immune tolerance, but whether donor-MHC II or PD-L1 is required for the induction of stable MHC-mismatched mixed chimerism and transplant tolerance is unclear. Here, we show that a clinically applicable radiation-free regimen can establish stable MHC-mismatched mixed chimerism and organ transplant tolerance in murine models. Induction of MHC-mismatched mixed chimerism does not require donor cell expression of MHC II or PD-L1, but donor-type organ transplant tolerance in the mixed chimeras (MC) requires the donor hematopoietic cells and the organ transplants to express PD-L1. The PD-L1 expressed by donor hematopoietic cells and the programmed cell death 1 expressed by host cells augment host-type donor-reactive CD4+ and CD8+ T cell anergy/exhaustion and differentiation into peripheral regulatory T (pTreg) cells in association with the organ transplant tolerance in the MC. Conversely, host-type Treg cells augment the expansion of donor-type tolerogenic CD8+ DCs that express PD-L1. These results indicate that PD-L1 expressed by donor-type tolerogenic DCs and expansion of host-type pTreg cells in MHC-mismatched MCs play critical roles in mediating organ transplant tolerance.

Trafficking between clonally related peripheral T-helper cells and tissue-resident T-helper cells in chronic GVHD.

Chronic graft-versus-host disease (cGVHD) is an autoimmune-like syndrome. CXCR5-PD-1hi peripheral T-helper (Tph) cells have an important pathogenic role in autoimmune diseases, but the role of Tph cells in cGVHD remains unknown. We show that in patients with cGVHD, expansion of Tph cells among blood CD4+ T cells was associated with cGVHD severity. These cells augmented memory B-cell differentiation and production of immunoglobulin G via interleukin 21 (IL-21). Tph cell expansion was also observed in a murine model of cGVHD. This Tph cell expansion in the blood is associated with the expansion of pathogenic tissue-resident T-helper (Trh) cells that form lymphoid aggregates surrounded by collagen in graft-versus-host disease (GVHD) target tissues. Adoptive transfer experiments showed that Trh cells from GVHD target tissues give rise to Tph cells in the blood, and conversely, Tph cells from the blood give rise to Trh cells in GVHD target tissues. Tph cells in the blood and Trh cells in GVHD target tissues had highly overlapping T-cell receptor α and ß repertoires. Deficiency of IL-21R, B-cell lymphoma 6 (BCL6), or T-bet in donor T cells markedly reduced the proportions of Tph cells in the blood and Trh cells in GVHD target tissues and reduced T-B interaction in the lymphoid aggregates. These results indicate that clonally related pathogenic Tph cells and Trh cells traffic between the blood and cGVHD target tissues, and that IL-21R-BCL6 signaling and T-bet are required for the development and expansion of Tph and Trh cells in the pathogenesis of cGVHD.

Retention of Donor T Cells in Lymphohematopoietic Tissue and Augmentation of Tissue PD-L1 Protection for Prevention of GVHD While Preserving GVL Activity.

Allogeneic hematopoietic cell transplantation (Allo-HCT) is a curative therapy for hematological malignancies (i.e., leukemia and lymphoma) due to the graft-versus-leukemia (GVL) activity mediated by alloreactive T cells that can eliminate residual malignant cells and prevent relapse. However, the same alloreactive T cells can cause a serious side effect, known as graft-versus-host disease (GVHD). GVHD and GVL occur in distinct organ and tissues, with GVHD occurring in target organs (e.g., the gut, liver, lung, skin, etc.) and GVL in lympho-hematopoietic tissues where hematological cancer cells primarily reside. Currently used immunosuppressive drugs for the treatment of GVHD inhibit donor T cell activation and expansion, resulting in a decrease in both GVHD and GVL activity that is associated with cancer relapse. To prevent GVHD, it is important to allow full activation and expansion of alloreactive T cells in the lympho-hematopoietic tissues, as well as prevent donor T cells from migrating into the GVHD target tissues, and tolerize infiltrating T cells via protective mechanisms, such as PD-L1 interacting with PD-1, in the target tissues. In this review, we will summarize major approaches that prevent donor T cell migration into GVHD target tissues and approaches that augment tolerization of the infiltrating T cells in the GVHD target tissues while preserving strong GVL activity in the lympho-hematopoietic tissues.

Steroid-Refractory Gut Graft-Versus-Host Disease: What We Have Learned From Basic Immunology and Experimental Mouse Model.

Intestinal graft-versus-host disease (Gut-GVHD) is one of the major causes of mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). While systemic glucocorticoids (GCs) comprise the first-line treatment option, the response rate for GCs varies from 30% to 50%. The prognosis for patients with steroid-refractory acute Gut-GVHD (SR-Gut-aGVHD) remains dismal. The mechanisms underlying steroid resistance are unclear, and apart from ruxolitinib, there are no approved treatments for SR-Gut-aGVHD. In this review, we provide an overview of the current biological understanding of experimental SR-Gut-aGVHD pathogenesis, the advanced technology that can be applied to the human SR-Gut-aGVHD studies, and the potential novel therapeutic options for patients with SR-Gut-aGVHD.

Reversal of autoimmunity by mixed chimerism enables reactivation of ß cells and transdifferentiation of α cells in diabetic NOD mice.

Type 1 diabetes (T1D) results from the autoimmune destruction of ß cells, so cure of firmly established T1D requires both reversal of autoimmunity and restoration of ß cells. It is known that ß cell regeneration in nonautoimmune diabetic mice can come from differentiation of progenitors and/or transdifferentiation of α cells. However, the source of ß cell regeneration in autoimmune nonobese diabetic (NOD) mice remains unclear. Here, we show that, after reversal of autoimmunity by induction of haploidentical mixed chimerism, administration of gastrin plus epidermal growth factor augments ß cell regeneration and normalizes blood glucose in the firmly established diabetic NOD mice. Using transgenic NOD mice with inducible lineage-tracing markers for insulin-producing ß cells, Sox9+ ductal progenitors, Nestin+ mesenchymal stem cells, and glucagon-producing α cells, we have found that both reactivation of dysfunctional low-level insulin expression (insulinlo) ß cells and neogenesis contribute to the regeneration, with the latter predominantly coming from transdifferentiation of α cells. These results indicate that, after reversal of autoimmunity, reactivation of ß cells and transdifferentiation of α cells can provide sufficient new functional ß cells to reach euglycemia in firmly established T1D.

Haploidentical mixed chimerism cures autoimmunity in established type 1 diabetic mice.

Clinical trials are currently testing whether induction of haploidentical mixed chimerism (Haplo-MC) induces organ transplantation tolerance. Whether Haplo-MC can be used to treat established autoimmune diseases remains unknown. Here, we show that established autoimmunity in euthymic and adult-thymectomized NOD (H-2g7) mice was cured by induction of Haplo-MC under a non-myeloablative anti-thymocyte globulin-based conditioning regimen and infusion of CD4+ T cell-depleted hematopoietic graft from H-2b/g7 F1 donors that expressed autoimmune-resistant H-2b or from H-2s/g7 F1 donors that expressed autoimmune-susceptible H-2s. The cure was associated with enhanced thymic negative selection, increased thymic Treg (tTreg) production, and anergy or exhaustion of residual host-type autoreactive T cells in the periphery. The peripheral tolerance was accompanied by expansion of donor- and host-type CD62L-Helios+ tTregs as well as host-type Helios-Nrp1+ peripheral Tregs (pTregs) and PD-L1hi plasmacytoid DCs (pDCs). Depletion of donor- or host-type Tregs led to reduction of host-type PD-L1hi pDCs and recurrence of autoimmunity, whereas PD-L1 deficiency in host-type DCs led to reduction of host-type pDCs and Helios-Nrp1+ pTregs. Thus, induction of Haplo-MC reestablished both central and peripheral tolerance through mechanisms that depend on allo-MHC+ donor-type DCs, PD-L1hi host-type DCs, and the generation and persistence of donor- and host-type tTregs and pTregs.