Brian C. Betts

Race/ethnicity affects the probability of finding an HLA-A, -B, -C and -DRB1 allele-matched unrelated donor and likelihood of subsequent transplant utilization

Factors relevant to finding a suitable unrelated donor and barriers to effective transplant utilization are incompletely understood. Among a consecutive series of unrelated searches (n=531), an 8/8 HLA-A, -B, -C and -DRB1-matched unrelated donor was available for 289 (54%) patients, 7/8 for 159 (30%) and no donor for 83 (16%). Patients of Caucasian race (P<0.0001) were more likely to find a donor. Younger age (P=0.01), Caucasian race (P=0.03), lower CIBMTR (Center for International Blood and Marrow Transplantation Research) risk (P=0.005), and 8/8 HLA matching (P=0.005) were associated with higher odds of reaching hematopoietic cell transplantation (HCT). In a univariate analysis of OS, finding a donor was associated with hazard ratio (HR) of 0.85 (95% CI 0.63–1.2), P=0.31. Karnofsky performance status (KPS) accounted for interaction between having a donor and survival. Patients with KPS 90–100 and a donor had significantly reduced hazard for death (HR 0.59, 95% CI 0.38–0.90, P=0.02). These data provide estimates of the probability to find an unrelated donor in the era of high-resolution HLA typing, and identify potentially modifiable barriers to reaching HCT. Further efforts are needed to enhance effective donor identification and transplant utilization, particularly in non-Caucasian ethnic groups.

The global severity of chronic graft-versus-host disease, determined by National Institutes of Health consensus criteria, is associated with overall survival and non-relapse mortality

Background The 2005 National Institutes of Health Consensus Development Conference on chronic graft-versus-host disease proposed major changes in the classification and grading of severity of chronic graft-versus-host disease. Design and Methods We aimed to study the association of the proposed chronic graft-versus-host disease classification and global severity with transplantation outcomes among a consecutive series of patients who received pharmacokinetically-targeted doses of intravenous busulfan and fludarabine conditioning followed by transplantation of allogeneic peripheral blood stem cells. Results From a total cohort (n = 242) of patients surviving more than 100 days after hematopoietic stem cell transplantation, 181 (75% of those at risk) had some manifestations of graft-versus-host disease after day 100. Of these, at onset 13 (7%) had late acute graft-versus-host disease, 62 (34%) had classic chronic graft-versus-host disease, and 106 (59%) had the overlap subtype of chronic graft-versus-host disease. The global severity of the chronic graft-versus-host disease was mild in 25% of cases, moderate in 46%, and severe in 29%. Multivariable modeling demonstrated the independent association of global severity of chronic graft-versus-host disease with overall survival (moderate/severe versus mild; HR 2.9, 95% CI 1.8–4.7, P<0.0001) and non-relapse mortality (moderate versus mild; HR 3.86, 95% CI 1.17–12.73, P=0.03, and severe versus mild (HR 10.06, 95% CI 3.07–32.97, P<0.001). The type of onset of progressive chronic graft-versus-host disease and the platelet count at the time of diagnosis of the disease were significantly associated with overall survival. The occurrence and severity of chronic graft-versus-host disease was also significantly associated with primary disease relapse. Conclusions Patients with moderate to severe chronic graft-versus-host disease, as determined by National Institutes of Health Consensus criteria, have an inferior overall survival and worse non-relapse mortality. Clinical and research advances are needed to improve the outcomes of affected patients.

Human regulatory T cells against minor histocompatibility antigens: ex vivo expansion for prevention of graft-versus-host disease

Alloreactive donor T cells against host minor histocompatibility antigens (mHAs) cause graft-versus-host disease (GVHD) after marrow transplantation from HLA-identical siblings. We sought to identify and expand regulatory CD4 T cells (Tregs) specific for human mHAs in numbers and potency adequate for clinical testing. Purified Tregs from normal donors were stimulated by dendritic cells (DCs) from their HLA-matched siblings in the presence of interleukin 2, interleukin 15, and rapamycin. Male-specific Treg clones against H-Y antigens DBY, UTY, or DFFRY-2 suppressed conventional CD4 T cell (Tconv) response to the specific antigen. In the blood of 16 donors, we found a 24-fold (range, 8-fold to 39-fold) excess Tconvs over Tregs reactive against sibling mHAs. We expanded mHA-specific Tregs from 4 blood samples and 4 leukaphereses by 155- to 405-fold. Cultured Tregs produced allospecific suppression, maintained demethylation of the Treg-specific Foxp3 gene promoter, Foxp3 expression, and transforming growth factor β production. The rare CD4 T conv and CD8 T cells in the end product were anergic. This is the first report of detection and expansion of potent mHA-specific Tregs from HLA-matched siblings in sufficient numbers for application in human transplant trials.

CD4+ T cell STAT3 phosphorylation precedes acute GVHD, and subsequent Th17 tissue invasion correlates with GVHD severity and therapeutic response

Th17 cells contribute to severe GVHD in murine bone marrow transplantation. Targeted deletion of the RORγt transcription factor or blockade of the JAK2‐STAT3 axis suppresses IL‐17 production and alloreactivity by Th17 cells. Here, we show that pSTAT3 Y705 is increased significantly in CD4+ T cells among human recipients of allogeneic HCT before the onset of Grade II–IV acute GVHD. Examination of target‐organ tissues at the time of GVHD diagnosis indicates that the amount of RORγt + Th17 cells is significantly higher in severe GVHD. Greater accumulation of tissue‐resident Th17 cells also correlates with the use of MTX‐ compared with Rapa‐based GVHD prophylaxis, as well as a poor therapeutic response to glucocorticoids. RORγt is optimally suppressed by concurrent neutralization of TORC1 with Rapa and inhibition of STAT3 activation with S3I‐201, supporting that mTOR‐ and STAT3‐dependent pathways converge upon RORγt gene expression. Rapa‐resistant T cell proliferation can be totally inhibited by STAT3 blockade during initial allosensitization. We conclude that STAT3 signaling and resultant Th17 tissue accumulation are closely associated with acute GVHD onset, severity, and treatment outcome. Future studies are needed to validate the association of STAT3 activity in acute GVHD. Novel GVHD prevention strategies that incorporate dual STAT3 and mTOR inhibition merit investigation.

Targeting Aurora kinase A and JAK2 prevents GVHD while maintaining Treg and antitumor CTL function

Inhibiting Aurora kinase A and JAK2 signal transduction pathways permits the differentiation of potent regulatory T cells while neutralizing alloreactive T cells and preventing graft-versus-host disease without impairing antitumor responses. Guiding T cells to stem GVHD Donor T cell–mediated graft-versus-host disease (GVHD) is a serious complication of stem cell transplantation, but complete inhibition of T cell activation might leave the patient susceptible to leukemia relapse. Betts et al. targeted two kinases involved in T cell costimulation and cytokine-responsiveness to blunt T cell responses. Dual targeting drove human CD4 T cells to become potently suppressive T regulatory cells instead of TH17 effector cells and also prevented GVHD in a humanized mouse model. CD8 T cells were capable of activation after a tumor challenge, indicating that patients receiving this treatment might be able to mount antileukemia responses. Graft-versus-host disease (GVHD) is a leading cause of nonrelapse mortality after allogeneic hematopoietic cell transplantation. T cell costimulation by CD28 contributes to GVHD, but prevention is incomplete when targeting CD28, downstream mammalian target of rapamycin (mTOR), or Aurora A. Likewise, interleukin-6 (IL-6)–mediated Janus kinase 2 (JAK2) signaling promotes alloreactivity, yet JAK2 inhibition does not eliminate GVHD. We provide evidence that blocking Aurora A and JAK2 in human T cells is synergistic in vitro, prevents xenogeneic GVHD, and maintains antitumor responses by cytotoxic T lymphocytes (CTLs). Aurora A/JAK2 inhibition is immunosuppressive but permits the differentiation of inducible regulatory T cells (iTregs) that are hyperfunctional and CD39 bright and efficiently scavenge adenosine triphosphate (ATP). Increased iTreg potency is primarily a function of Aurora A blockade, whereas JAK2 inhibition suppresses T helper 17 (TH17) differentiation. Inhibiting either Aurora A or JAK2 significantly suppresses TH1 T cells. However, CTL generated in vivo retains tumor-specific killing despite Aurora A/JAK2 blockade. Thus, inhibiting CD28 and IL-6 signal transduction pathways in donor T cells can increase the Treg/Tconv ratio, prevent GVHD, and preserve antitumor CTL.

CD8+ Tregs promote GVHD prevention and overcome the impaired GVL effect mediated by CD4+ Tregs in mice

ABSTRACT Adoptive natural regulatory T cell (nTreg) therapy has improved the outcome for patients suffering from graft-versus-host disease (GVHD) following allogeneic hematopoietic cell transplantation (Allo-HCT). However, fear of broad immune suppression and subsequent dampening of beneficial graft-versus-leukemia (GVL) responses remains a challenge. To address this concern, we generated alloreactive induced Tregs (iTregs) from resting CD4+ or CD8+ T cells and tested their ability to suppress GVH and maintain GVL responses. We utilized major mismatched and haploidentical murine models of HCT with host-derived lymphoma or leukemia cell lines to evaluate GVH and GVL responses simultaneously. Alloreactive CD4+ iTregs were effective in preventing GVHD, but abrogated the GVL effect against aggressive leukemia. Alloreactive CD8+ iTregs moderately attenuated GVHD while sparing the GVL effect. Hence, we reasoned that using a combination of CD4+ and CD8+ iTregs could achieve the optimal goal of Allo-HCT. Indeed, the combinational therapy was superior to CD4+ or CD8+ iTreg singular therapy in GVHD control; importantly, the combinational therapy maintained GVL responses. Cellular analysis uncovered potent suppression of both CD4+ and CD8+ effector T cells by the combinational therapy that resulted in effective prevention of GVHD, which could not be achieved by either singular therapy. Gene expression profiles revealed alloreactive CD8+ iTregs possess elevated expression of multiple cytolytic molecules compared to CD4+ iTregs, which likely contributes to GVL preservation. Our study uncovers unique differences between alloreactive CD4+ and CD8+ iTregs that can be harnessed to create an optimal iTreg therapy for GVHD prevention with maintained GVL responses.

STAT5 polarization promotes iTregs and suppresses human T-cell alloresponses while preserving CTL capacity

Alloreactivity negatively influences outcomes of organ transplantation or HCT from allogeneic donors. Standard pharmacologic immune suppression impairs T‐cell function and jeopardizes the beneficial reconstitution of Tregs. Murine transplantation models have shown that STAT3 is highly expressed in alloreactive T cells and may be therapeutically targeted. The influence and effects of STAT3 neutralization in human alloreactivity, however, remain to be elucidated. In this study, S3I‐201, a selective small‐molecule inhibitor of STAT3, suppressed human DC‐allosensitized T‐cell proliferation and abrogated Th17 responses. STAT3 blockade significantly enhanced the expansion of potent iTregs and permitted CD8+ cytolytic effector function. Mechanistically, S3I‐201 polarized the ratio of STAT phosphorylation in favor of STAT5 over STAT3 and also achieved a significant degree of Foxp3 demethylation among the iTregs. Conversely, selective impairment of STAT5 phosphorylation with CAS 285986‐31‐4 markedly reduced iTregs. STAT3 represents a relevant target for achieving control over human alloresponses, where its suppression facilitates STAT5‐mediated iTreg growth and function.

Pharmacokinetically-targeted BU and fludarabine as conditioning before allogeneic hematopoietic cell transplantation for adults with ALL in first remission

Allogeneic hematopoietic cell transplantation offers improved survival in patients with ALL, but with regimens containing TBI, the nonrelapse mortality is 20–40%. Efforts to lessen transplant toxicities by reducing conditioning regimen intensity have led to increased relapse risk. Therefore, there is a need for less toxic regimens that maintain an anti-leukemia effect. We report here a retrospective review of 65 patients with ALL in first remission receiving grafts from allogeneic donors after fludarabine 40 mg/m2/day for 4 days and i.v. BU targeted to a median daily area under the concentration–time curve below 6000 μmoles min/L. At 2 years after transplantation, OS was 65% (95% confidence interval (CI): 52–77%), relapse-free survival was 61% (95% CI: 48–73%), cumulative incidence of relapse was 26% (95% CI: 17–39%) and cumulative incidence of nonrelapse mortality was 14% (95% CI: 8–26%). Age over 35 years, Ph chromosome positivity and minimal residual disease at transplant did not adversely affect outcomes. Pharmacokinetically targeted BU and fludarabine can provide intensive pre-transplant conditioning for adults with ALL in first remission, with promising relapse-free and OS rates.

Human Dendritic Cells Mitigate NK-Cell Dysfunction Mediated by Nonselective JAK1/2 Blockade

Broad JAK inhibitors can improve graft-versus-host disease caused by stem cell transplants, but they reduce NK-cell numbers and activity. Selective inhibitors of JAK2, in concert with moDCs or Langerhans cells, preserve STAT5 signaling and NK-cell proliferation and function. Janus kinase (JAK) inhibitors have achieved positive responses in myeloproliferative neoplasms, but at the expense of decreased natural killer (NK) cell numbers and compromised function. Selective JAK2 inhibition may also have a role in preventing and treating graft-versus-host disease after allogeneic hematopoietic stem cell transplantation. Although JAK inhibitors can impair monocyte-derived dendritic cell (moDC) activation and function and suppress effector T-cell responses, the effects on NK cells and the relevant mechanisms remain undefined. Using common γc cytokines and distinct human dendritic cell (DC) subtypes, we compared the effects of a JAK2-specific (TG101348) with a less selective JAK1/2 (ruxolitinib) inhibitor on NK-cell activation and function. Ruxolitinib treatment completely blocked IL2, IL15, and DC-mediated STAT5 phosphorylation, along with the capacity of NK cells to secrete IFNγ or lyse NK cell–sensitive targets. Only NK-cell proliferation stimulated by moDCs resisted ruxolitinib treatment. In contrast, TG101348 treatment of stimulated NK cells resulted in far less functional compromise. TG101348 completely inhibited only soluble IL15-mediated STAT5 phosphorylation, which Langerhans-type DCs (LCs), presenting membrane-bound IL15 in trans, could salvage. These results demonstrate that ruxolitinibs nonselective inhibition of JAK1/2 results in profound NK-cell dysfunction by blocking downstream pSTAT5, hence providing a persuasive rationale for the development of selective JAK2 inhibitors for immunotherapeutic applications. Cancer Immunol Res; 5(1); 52–60. ©2016 AACR.

Prolonged sirolimus administration after allogeneic hematopoietic cell transplantation is associated with decreased risk for moderate-severe chronic graft-versus-host disease.

Effective pharmacological strategies employed in allogeneic hematopoietic cell transplantation should prevent serious chronic graft-versus-host disease and facilitate donor-recipient immune tolerance. Based on demonstrated pro-tolerogenic activity, sirolimus (rapamycin) is an agent with promise to achieve these goals. In a long-term follow-up analysis of a randomized phase II trial comparing sirolimus/tacrolimus versus methotrexate/tacrolimus for graft-versus-host disease prevention in matched sibling or unrelated donor transplant, we examined the impact of prolonged sirolimus administration (≥ 1 year post-transplant). Median follow-up time for surviving patients at time of this analysis was 41 months (range 27–60) for sirolimus/tacrolimus and 49 months (range 29–63) for methotrexate/tacrolimus. Sirolimus/tacrolimus patients had significantly lower National Institutes of Health Consensus moderate-severe chronic graft-versus-host disease (34% vs. 65%; P=0.004) and late acute graft-versus-host disease (20% vs. 43%; P=0.04). While sirolimus/tacrolimus patients had lower prednisone exposure and earlier discontinuation of tacrolimus (median time to tacrolimus discontinuation 368 days vs. 821 days; P=0.002), there was no significant difference in complete immune suppression discontinuation (60-month estimate: 43% vs. 31%; P=0.78). Prolonged sirolimus administration represents a viable approach to mitigate risk for moderate-severe chronic and late acute graft-versus-host disease. Further study of determinants of successful immune suppression discontinuation is needed.