Bruce R. Blazar’s research while affiliated with University of Minnesota and other places

Pathogenic antibodies produced by alloreactive B cells mediate antibody-mediated rejection after kidney transplantation, but the mechanisms remain poorly understood. Follicular regulatory T (Tfr) cells modulate follicular helper T cell-mediated B cell responses, but the functions of Tfr in controlling alloreactive antibody are unknown. Here we study the developmental signals and functions of Tfr cells in mouse allogeneic kidney transplantation models, and show that costimulatory blockade alters the development of Tfr cells disproportionately by decreasing germinal center (GC)-like Tfr cells but increasing follicular-like Tfr cells. Functionally, global Tfr cell deletion results in accelerated graft rejection and increases in donor-specific B cells in both draining lymph nodes and kidney allografts. Mechanistically, Tfr cell deletion increases GC B cell expression of pro-inflammatory cytokines such as IL-15, while neutralization of IL-15 compensates for the loss of Tfr cells and prolongs the survival of mice receiving kidney transplants. Together our preclinical mouse data demonstrate how Tfr restrains kidney allograft rejection by limiting alloreactive B cell responses.

Chronic graft-versus-host disease (cGVHD) remains the leading cause of non-relapse morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Effective therapeutics agents targeting dysregulated cytokines including IL-17 and CSF-1 have been defined in preclinical models of cGVHD, and efficacy in subsequent clinical trials has led to their recent FDA approval. Despite this, these agents are effective in only a subset of patients, expensive, difficult to access outside the US, and used in a trial-and-error fashion. The ability to readily discern druggable, dysregulated immunity in these patients is desperately needed to facilitate the selection of appropriate treatment and to potentially identify high-risk individuals for preemptive therapy. We used single cell sequencing-based approaches in our informative preclinical cGVHD models to "reverse engineer" temporal IL-17 and CSF-1 signatures in mouse blood that could be used to interrogate patients. We defined distinct, non-intuitive IL-17 and CSF-1 signatures in mouse blood monocytes that could be identified in relevant monocytes within 70% of patients at diagnosis of cGVHD and in half of patients at day +100 post-HCT who subsequently developed cGVHD. These signatures can now be evaluated prospectively in clinical studies to help delineate potential responder and non-responders to relevant therapeutics targeting these pathways.

Chronic graft-versus-host disease (cGVHD) is an immune-mediated, heterogeneous, multiorgan complication affecting allogeneic hematopoietic cell transplantation recipients, leading to increased morbidity, mortality, and decline in health-related quality-of-life. Advances in understanding the complex disease pathophysiology, and collaborative efforts lead by the National Institutes of Health to standardize criteria for clinical trials, led to bench-to-bedside efforts resulting in the development of 4 US Food and Drug Administration-approved agents for the treatment steroids-refractory cGVHD since 2017. Despite the remarkable advances in the field of hematopoietic cell transplantation in prevention of cGVHD, and more treatment options, the outcome of patients with moderate-severe cGVHD remains suboptimal. Essential to successful cGVHD management is to recognize the disease at early stages before the onset of irreversible damage, allowing for personalized multidisciplinary specialized interventions that include pharmacologic therapies and additional supportive care measures. The aim of this review is to summarize key areas of active clinical research and new developments in cGVHD therapeutic approaches, with focus on (1) preemptive therapy, (2) upfront therapy beyond corticosteroids, (3) treatment refractory cGVHD novel agents, role of combination therapies, and organ-specific approaches, and (4) challenges, gaps, and future directions.

The success of allogeneic hematopoietic stem cell transplantation (allo-HSCT) can be limited by graft-versus-host disease (GVHD). T-cell activation is a key factor in GVHD progression. Costimulatory signals can be counterbalanced by co-inhibitory signals such as the checkpoint molecule VISTA (V-domain Ig-containing suppressor of T-cell activation)/PD-1H that restrains activation and maintains donor T-cell quiescence. A single dose of anti-VISTA mAb prevents acute GVHD lethality in multiple models. Naïve donor T-cells express moderate VISTA levels, which transiently increase in allo-HSCT recipients in association with TCR signaling, leading to heightened susceptibility to anti-VISTA mAb-mediated depletion, in contrast to donor T-cells transferred to syngeneic recipients. Anti-VISTA mAb donor T-cell depletion was compatible with rapamycin but incompatible with peri-transplant tacrolimus GVHD prophylaxis. Targeting VISTA exclusively on host cells or donor CD8+ T-cells was not protective against GVHD lethality. Instead, anti-VISTA mAb mediated deletion of alloreactive donor T-cells depended on targeting a third (non-T) cell type. Further mechanistic studies indicated that donor T-cells concurrently exposed to anti-VISTA mAb in vivo but not preincubated in vitro before adoptive T-cell transfer were eliminated via FcR-mediated phagocytosis. In a lymphoma challenge model, a graft-versus-lymphoma effect was fully retained when anti-human VISTA mAb exclusively targeted donor CD4+ T-cells and delayed but mostly retained when unseparated donor T-cells were infused. In a xenogeneic GVHD model, anti-human VISTA mAb reduced donor T-cell expansion, VISTA T-cell expression levels, and recipient lethality. Together, these data support a novel clinical translational pathway in which acute GVHD lethality can be mitigated without negating the GVL effect.

Cytotoxic attack particles released by CTLs and NK cells include diverse phospholipid membrane and glycoprotein encapsulated entities that contribute to target cell killing. Supramolecular attack particles (SMAPs) are one type of particle characterized by a cytotoxic core enriched in granzymes and perforin surrounded by a proteinaceous shell including thrombospondin (TSP)-1. TSP-4 was also detected in bulk analysis of SMAPs released by CTLs; however, it has not been investigated whether TSP-4 contributes to distinct SMAP types or the same SMAP type as TSP-1 and, if in the same type of SMAP, whether TSP-4 and TSP-1 cooperate or compete. Here, we observed that TSP-4 expression increased upon CD8 ⁺ T cell activation while, surprisingly, TSP-1 was down-regulated. Correlative Light and Electron Microscopy and Stimulated Emission Depletion microscopy localized TSP-4 and TSP-1 in SMAP-containing multicore granules. Superresolution dSTORM revealed that TSP-4 and TSP-1 are usually enriched in the same SMAPs while particles with single-positive shells are rare. Retention Using Selective Hooks assays showed that TSP-4 localizes to the lytic granules faster than TSP-1 and promotes its accumulation therein. TSP-4 contributed to direct CTL-mediated killing, as previously shown for TSP-1. TSP-4 and TSP-1 were both required for latent SMAP-mediated cell killing, in which released SMAPs kill targets after removal of the CTLs. Of note, we found that chronic lymphocytic leukemia (CLL) cell culture supernatants suppressed expression of TSP-4 in CTL and latent SMAP-mediated killing. These results identify TSP-4 as a functionally important component of SMAPs and suggest that SMAPs may be targeted for immune suppression by CLL.

Chronic graft-versus-host disease (cGVHD) is the leading cause of morbidity and non-relapse associated mortality following allogeneic hematopoietic cell transplantation (aHSCT). Treating steroid resistant/refractory cGVHD remains challenging. Epigenetic regulators can have global transcriptional effects that control donor T-cell responses. We previously showed that inhibiting histone lysine motifs by chromatin-modifying enzymes can ameliorate murine cGVHD. Targeting donor T-cell DNA methyltransferases reduce acute GVHD. Here, we sought to investigate the DNA demethylase Tet (ten-eleven translocase) methylcytosine dioxygenases 2 (Tet2) and Tet3 in T follicular helper cell (TFH) dependent cGVHD. In a clinically relevant model of cGVHD that recapitulates pulmonary fibrosis from bronchiolitis obliterans, recipients of Tet2 deleted donor T-cells did not have improved pulmonary function tests in contrast to the markedly improved pulmonary function in Tet3 deleted donor T-cells. Tet3 deleted donor T-cells did not impair TFH-dependent germinal center (GC) formation. Unexpectedly, TET3 deficiency resulted in elevated GATA3 expression in and IL-4 production by TFH cells. TET3 deficient TFH cells supported GC B-cell immunoglobulin (Ig) class switching to nonpathogenic IgG1 but not pathogenic IgG2c allowing mice to escape cGVHD pulmonary fibrosis. Elevated GATA3 expression and disruption of IgG2c class switching was recapitulated in an in-vitro human GC culture system. These studies provide new insights into the function of Tet3 in TFH driven Ig class switching and suggest a new approach to mitigate cGVHD.

Tissue-specific T cell immune responses play a critical role in maintaining organ health but can also drive immune pathology during both autoimmunity and alloimmunity. The mechanisms controlling intratissue T cell programming remain unclear. Here, we leveraged a nonhuman primate model of acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation to probe the biological underpinnings of tissue-specific alloimmune disease using a comprehensive systems immunology approach including multiparameter flow cytometry, population-based transcriptional profiling, and multiplexed single-cell RNA sequencing and TCR sequencing. Transcriptional profiling revealed substantial biological differences between T cells infiltrating the lung and liver during aGVHD. These included enrichment for transcriptional pathways controlling extracellular matrix remodeling and chemotaxis in the lung and enrichment for transcriptional pathways linked to nucleic acid metabolism and proliferation in the liver. Single-cell RNA sequencing and TCR sequencing substantiated divergent organ-specific transcriptional programing of tissue-infiltrating T cells, which was linked to clonal expansion, with expanded clones progressively enriched for C-X3-C motif chemokine receptor 1 ( CX3CR1 )–expressing CD8 effector T cells in the lung and eomesodermin ( EOMES )–expressing CD8 effector-memory T cells in the liver. This divergent evolution of T cells was maintained even for T cells sharing the same TCRs, indicating its independence from antigen specificity. Together, these results provide insights into the role that tissue microenvironment–derived signals play in local T cell transcriptional programming during alloimmune-mediated clonal expansion and suggest potential opportunities to develop tissue-specific therapeutics to curtail pathogenic immunity after transplant.

Background: GVHD remains a major cause of non-relapse mortality (NRM) after hematopoietic cell transplant (HCT). Abatacept, a costimulatory blockade agent targeting the CD28:CD80/86 pathway, was FDA-approved in 2021 for GVHD prophylaxis in patients undergoing an 8/8 HLA-matched unrelated donor (MUD), or 7/8-HLA mismatched unrelated donor (MMUD) HCT for a hematologic malignancy, in part based on the ABA2 trial (NCT01743131). Here, we present 5-yr follow-up data for ABA2. Methods: ABA2 patient eligibility criteria (described in detail in PMID: 33449816) included patients ≥ 6 yr with heme malignancies undergoing MUD or MMUD HCT after intensive conditioning. MUD patients were enrolled on the randomized, double-blind placebo-controlled arm, with patients randomized to either 4 doses of abatacept (10mg/kg/dose on Day -1, +5, +14 +28) or placebo. MMUD patients all received 4 doses of abatacept. 185 patients were enrolled: 69 MUD-placebo, 73 MUD-abatacept, 43 MMUD-abatacept. GVHD prophylaxis also included a calcineurin inhibitor (CNI, continued through Day +100 followed by a wean) and methotrexate (MTX, 15mg/m2 on day +1 and 10mg/m2 on Day +3, +6, +11). Both bone marrow and PBSC grafts were used. All patient data were independently monitored through 5 yr by the NMDP CRO. Here we report cumulative incidence (CI) data at 2-, 3- and 5-yr for overall survival (OS), relapse, relapse-free survival (RFS), non-relapse mortality (NRM), systemic therapy (ST)-requiring chronic GVHD (cGVHD), and grade 3-4 acute GVHD (aGVHD)-free/ST-requiring cGVHD-free/relapse-free survival (GRFS). Results: The median follow-up was 53.8 months, 56.1 months and 60 months for the MUD-placebo, MUD-abatacept, and MMUD-abatacept cohorts, respectively. The 2-, 3- and 5-yr OS was 65.2%, 62.3% and 54.3% for MUD-placebo, 75.2%, 68.0% and 61.0% for MUD-abatacept, and 78.8%, 74.0% and 71.7% for MMUD-abatacept cohorts, respectively. The 2-, 3- and 5-yr CI of relapse was 25.7%, 27.5% and 27.5% for MUD-placebo, 21.7%, 25.0% and 25.0% for MUD-abatacept, and 9.7%, 9.7% and 12.6% for MMUD-abatacept cohorts, respectively. The 2-, 3- and 5-yr CI of NRM was 18.1%, 20.0%, 28.7% for MUD-placebo, 14.4%, 18.1% and 22.5% for MUD-abatacept, and 15.0%, 20.2% and 20.2% for MMUD-abatacept cohorts, respectively. The 2-, 3- and 5-yr CI of RFS was 60.9%, 58.0% and 51.7% for MUD-placebo, 67.0%, 61.4% and 58.1% for MUD-abatacept, and 76.7%, 72.1% and 69.8% for MMUD-abatacept cohorts, respectively. The 1-yr CI of grade 3-4 acute GVHD was 14.8% for MUD-placebo, 8.4% for MUD-abatacept, and 2.3% for MMUD-abatacept cohorts, respectively. The 2-, 3- and 5-yr CI of ST-requiring chronic GVHD was 49.6%, 52.0% and 52.0% for MUD-placebo, 54.1%, 58.5% and 58.5% for MUD-abatacept, and 72.5%, 75.2% and 78.0% for MMUD-abatacept cohorts, respectively. The 2-, 3- and 5-yr cumulative incidence of GRFS was 27.5%, 26.1%, and 26.1% for MUD-placebo, 27.4%, 21.9% and 21.9% for MUD-abatacept, and 23.3%, 20.9% and 16.3% for MMUD-abatacept cohorts, respectively. Conclusions: Here we demonstrate that the central outcomes previously reported for ABA2 at 2 yr post-HCT (PMID: 33449816) were durable at 3- and 5-yr, with relatively stable OS, relapse, NRM, cGVHD and RFS. These data are important given new CIBMTR comparisons demonstrating that, for both MUD and MMUD recipients at 2 yr, abatacept/CNI/MTX is superior for OS and RFS compared to both CNI/MTX and CNI/MTX/ATG, and is similar to post-transplant cyclophosphamide (PT-Cy, Blood 2024, PMID:39028876). Moreover, the 3-yr outcome data for MMUD patients presented here for ABA2 also appear similar to the recently reported 3-yr outcomes for myeloablative conditioning (MAC) and PT-Cy for the MMUD-15 trial (PMID: 36584941, 5-yr outcomes not yet reported for MMUD-15). Most notable is the fact that despite increased cGVHD in ABA2 compared to MMUD-15, the GRFS is similar in both trials (20.9% GRFS at 3 yr for ABA2 versus 16.9% for PT-Cy after MAC HCT). These similar composite endpoints may be driven by lower apparent relapse rates in ABA2 (9.7% at 3-yr for ABA2 and 50.5% at 3-yr for MAC PT-Cy in MMUD-15). Together, these data lead to two key conclusions: (1) That the addition of abatacept continues to abrogate historic disparities in OS and RFS for HLA-mismatched vs HLA-matched HCT (extending our previous comparison (PMID:34753172) to 5-yrs post-HCT). (2) That a head-to-head randomized trial of abatacept- versus PT-Cy-based prophylaxis is warranted.