JAK2/mTOR Inhibition Fails to Prevent Acute GVHD Despite Reduced Th1/Th17 cells: Final Phase II Trial Results.

Our phase I graft-versus-host disease (GVHD) prevention trial of JAK2 inhibitor, pacritinib, (recommended phase II dose: 100mg po BID day 0 to +70) plus sirolimus and tacrolimus (PAC/SIR/TAC) demonstrated the regimen was safe and free of pan-JAK myelosuppression after allogeneic hematopoietic cell transplantation (alloHCT). PAC inhibits IL-6 receptor activity and pathogenic Th1/Th17 differentiation in preclinical models and the phase I trial. Herein we report on our completed phase II trial of PAC/SIR/TAC after 8/8-HLA matched alloHCT. This single-arm phase II trial (NCT02891603) was powered to determine if PAC/SIR/TAC suppressed %pSTAT3+ CD4+ T cells at day +21 (primary endpoint: %pSTAT3+ CD4+ T cells ≤ 35%) and estimated grade II-IV acute GVHD by day +100. The impact of PAC/SIR/TAC on T cell subsets, CD28 (pS6 and pH3ser10), and IL-2 receptor (pSTAT5) signal transduction was also evaluated. Eligible patients (n=28) received alloHCT for hematologic malignancies or myeloproliferative neoplasms. Reduced or myeloablative intensity conditioning was permitted. PAC/SIR/TAC met the primary endpoint, reducing %pSTAT3+ CD4+ T cells to 9.62% at day +21. Th1/Th17 cells were decreased at day +21, increasing the ratio of Tregs to Th1 and Th17 cells with PAC/SIR/TAC at RP2D PAC compared to dose level 1 PAC. The cumulative incidence of grade II-IV acute GVHD by day +100 with PAC/SIR/TAC was similar to historic SIR/TAC values (46 v 43%). While PAC/SIR/TAC suppressed pSTAT3 and Th1/Th17 cells, the regimen did not improve acute GVHD prevention.

Amphiregulin, ST2, and REG3α biomarker risk algorithms as predictors of nonrelapse mortality in patients with acute GVHD.

ABSTRACT: Graft-versus-host disease (GVHD) is a major cause of nonrelapse mortality (NRM) after allogeneic hematopoietic cell transplantation. Algorithms containing either the gastrointestinal (GI) GVHD biomarker amphiregulin (AREG) or a combination of 2 GI GVHD biomarkers (suppressor of tumorigenicity-2 [ST2] + regenerating family member 3 alpha [REG3α]) when measured at GVHD diagnosis are validated predictors of NRM risk but have never been assessed in the same patients using identical statistical methods. We measured the serum concentrations of ST2, REG3α, and AREG by enzyme-linked immunosorbent assay at the time of GVHD diagnosis in 715 patients divided by the date of transplantation into training (2004-2015) and validation (2015-2017) cohorts. The training cohort (n = 341) was used to develop algorithms for predicting the probability of 12-month NRM that contained all possible combinations of 1 to 3 biomarkers and a threshold corresponding to the concordance probability was used to stratify patients for the risk of NRM. Algorithms were compared with each other based on several metrics, including the area under the receiver operating characteristics curve, proportion of patients correctly classified, sensitivity, and specificity using only the validation cohort (n = 374). All algorithms were strong discriminators of 12-month NRM, whether or not patients were systemically treated (n = 321). An algorithm containing only ST2 + REG3α had the highest area under the receiver operating characteristics curve (0.757), correctly classified the most patients (75%), and more accurately risk-stratified those who developed Minnesota standard-risk GVHD and for patients who received posttransplant cyclophosphamide-based prophylaxis. An algorithm containing only AREG more accurately risk-stratified patients with Minnesota high-risk GVHD. Combining ST2, REG3α, and AREG into a single algorithm did not improve performance.

Bispecific CD33/CD123 targeted chimeric antigen receptor T cells for the treatment of acute myeloid leukemia.

CD33 and CD123 are expressed on the surface of human acute myeloid leukemia blasts and other noncancerous tissues such as hematopoietic stem cells. On-target off-tumor toxicities may limit chimeric antigen receptor T cell therapies that target both CD33 and CD123. To overcome this limitation, we developed bispecific human CD33/CD123 chimeric antigen receptor (CAR) T cells with an "AND" logic gate. We produced novel CD33 and CD123 scFvs from monoclonal antibodies that bound CD33 and CD123 and activated T cells. Screening of CD33 and CD123 CAR T cells for cytotoxicity, cytokine production, and proliferation was performed, and we selected scFvs for CD33/CD123 bispecific CARs. The bispecific CARs split 4-1BB co-stimulation on one scFv and CD3ζ on the other. In vitro testing of cytokine secretion and cytotoxicity resulted in selecting bispecific CAR 1 construct for in vivo analysis. The CD33/CD123 bispecific CAR T cells were able to control acute myeloid leukemia (AML) in a xenograft AML mouse model similar to monospecific CD33 and CD123 CAR T cells while showing no on-target off-tumor effects. Based on our findings, human CD33/CD123 bispecific CAR T cells are a promising cell-based approach to prevent AML and support clinical investigation.

Phase II Study of Myeloablative 7-8/8-Matched Allotransplantation with Post-Transplantation Cyclophosphamide, Tacrolimus, and Mycophenolate Mofetil.

Graft-versus-host disease (GVHD) is the major toxicity of allogeneic hematopoietic cell transplantation (HCT). We hypothesized that a GVHD prophylaxis regimen of post-transplantation cyclophosphamide (PTCy), tacrolimus (Tac), and mycophenolate mofetil (MMF) would be associated with incidences of acute and chronic GVHD in patients receiving a matched or single antigen mismatched HCT. This Phase II study was conducted at the University of Minnesota using a myeloablative regimen of either total body irradiation (TBI) at a total dose of 1320 cGy, administered in 165-cGy fractions, twice daily from day -4 to day -1, or busulfan (Bu) 3.2 mg/kg daily (cumulative area under the curve, 19,000 to 21,000 µmol/min/L) plus fludarabine (Flu) 40 mg/m2 once daily on days -5 to -2, followed by a GVHD prophylaxis regimen of PTCy 50 mg/kg on days +3 and +4, Tac, and MMF beginning on day +5. The primary endpoint was the cumulative incidence of chronic GVHD necessitating systemic immunosuppression (IST) at 1 year post-transplantation. Between March 2018 and May 2022, we enrolled 125 pediatric and adult patients, with a median follow-up of 813 days. The incidence of chronic GVHD necessitating systemic IST at 1 year was 5.5%. The rate of grade II-IV acute GVHD was 17.1%, and that of grade III-IV acute GVHD was 5.5%. Two-year overall survival was 73.7%, and 2-year graft-versus-host disease-free, relapse-free survival was 52.2%. The 2-year cumulative incidence of nonrelapse mortality was 10.2%, and the rate of relapse was 39.1%. There was no statistically significant difference in survival outcomes between recipients of matched donor transplants versus recipients of 7/8 matched donor transplants. Our data show that myeloablative HCT with PTCy/Tac/MMF results in an extremely low incidence of severe acute and chronic GVHD in well-matched allogeneic HCT.

Phase II Study of Myeloablative 8/8- or 7/8-Matched Allotransplantation with Post-Transplant Cyclophosphamide, Tacrolimus, and Mycophenolate Mofetil: Marked Reduction in GVHD Risk Without Increased Relapse Risk Compared to Historical Cyclosporine/Methotrexate.

Introduction: Graft-versus host disease (GVHD) is a major limitation to the success of allogeneic hematopoietic cell transplant (HCT). We hypothesized that the GVHD prophylaxis regimen of post-transplant cyclophosphamide (PTCy), tacrolimus (Tac) and mycophenolate mofetil (MMF) would reduce the incidence of GVHD in patients receiving a matched or single antigen mismatched HCT without an increase in risk of malignant relapse. Methods: This is a phase II study conducted at the University of Minnesota using a myeloablative regimen of either: (A) total body irradiation (TBI, total dose 1320 cGy, administered in 165 cGy fractions, twice a day from days -4 to -1) or (B) Busulfan 3.2mg/kg daily (cumulative AUC 19,000 - 21,000 µmol/min/L) plus fludarabine 160mg/m2 days -5 to -2, followed by a GVHD prophylaxis regimen of PTCy (50mg/kg days +3 and +4), Tac and MMF (beginning day +5). The primary endpoint is cumulative incidence of chronic GVHD requiring systemic immunosuppression at 1-year post-transplant. We compared results to our previous myeloablative protocol for matched donors utilizing cyclosporine/methotrexate (CSA/MTX) GVHD prophylaxis. Results: From March 2018 - June 2022, we enrolled and treated 125 pediatric and adult patients with a median follow up of 472 days. Grade II-IV acute GVHD occurred in 16% (95% confidence interval (CI): 9-23%); Grade III-IV acute GVHD was 4% (CI: 0-8%). No patients experienced grade IV GVHD, and there were no deaths due to GVHD before day 100. Only 3 developed chronic GVHD requiring immune suppression, (4%, CI: 0-8%). Two-year overall survival (OS) was 80% (CI: 69-87%), and (graft-versus-host disease-free, relapse-free survival) GRFS 57% (CI: 45-67%), both higher than historical CSA/MTX. The incidence of grade II-IV aGVHD, cGVHD, and NRM were all lower with PTCy/Tac/MMF compared to historical CSA/MTX. One-quarter (25%) experienced relapse (CI: 15-36%) similar to historical CSA/MTX. There was no statistically significant difference in survival outcomes between recipients of matched versus 7/8 donors. Conclusion: Myeloablative HCT with PTCy/Tac/MMF results in extremely low incidence of severe acute or chronic GVHD, the primary endpoint of this clinical trial. Relapse risk is not increased compared to our historical CSA/MTX cohort.

Human Effectors of Acute and Chronic GVHD Overexpress CD83 and Predict Mortality.

PURPOSE: Acute and chronic GVHD remain major causes of transplant-related morbidity and mortality (TRM) after allogeneic hematopoietic cell transplantation (alloHCT). We have shown CD83 chimeric antigen receptor (CAR) T cells prevent GVHD and kill myeloid leukemia cell lines. In this pilot study, we investigate CD83 expression on GVHD effector cells, correlate these discoveries with clinical outcomes, and evaluate critical therapeutic implications for transplant recipients. EXPERIMENTAL DESIGN: CD83 expression was evaluated among circulating CD4+ T cells, B-cell subsets, T follicular helper (Tfh) cells, and monocytes from patients with/without acute or chronic GVHD (n = 48 for each group), respectively. CD83 expression was correlated with survival, TRM, and relapse after alloHCT. Differential effects of GVHD therapies on CD83 expression was determined. RESULTS: CD83 overexpression on CD4+ T cells correlates with reduced survival and increased TRM. Increased CD83+ B cells and Tfh cells, but not monocytes, are associated with poor posttransplant survival. CD83 CAR T eliminate autoreactive CD83+ B cells isolated from patients with chronic GVHD, without B-cell aplasia as observed with CD19 CAR T. We demonstrate robust CD83 antigen density on human acute myeloid leukemia (AML), and confirm potent antileukemic activity of CD83 CAR T in vivo, without observed myeloablation. CONCLUSIONS: CD83 is a promising diagnostic marker of GVHD and warrants further investigation as a therapeutic target of both GVHD and AML relapse after alloHCT.

Predictors and outcomes of flares in chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic cell transplantation (HCT) requires prolonged immunosuppressive therapy (IST), often requiring slow tapering with patients experiencing cGVHD flares and treatment failure. In 145 adult recipients developing cGVHD after matched sibling or umbilical cord blood donor HCT from 2010 to 2018, 2-year cumulative incidence of flares after cGVHD diagnosis was estimated at 60% (95% CI, 51-70%), with median time-to-first flare of 188 days (range, 16-751). Of 88 patients experiencing a flare, 32 (36%) had multiple flares (range, 2-4). First flare treatment consisted of an increase in prednisone dose in 77 patients (88%), plus topical therapy in 8 (9%) or another systemic IST in 43 patients (49%). Higher flare risk was associated with quiescent type of cGVHD at onset (HR 1.8; 95% CI: 1.1-2.7; p = 0.04). Patients without a flare required a shorter duration of IST and were more likely to achieve a durable discontinuation of systemic IST (86% vs. 31% for ≥6 consecutive months). Flares were associated with protective effect on relapse (HR 0.2, 95% CI: 0.1-0.3), however not with worsened 2-year NRM or OS. Flares of cGVHD identify a group needing better approaches to limit the duration of IST and thus the morbidity of cGVHD.

High cutaneous amphiregulin expression predicts fatal acute graft-versus-host disease.

BACKGROUND: Amphiregulin (AREG) is increased in circulation in acute graft-versus-host disease (aGVHD) and is associated with poor steroid response and lower survival. The expression of AREG in aGVHD target organs and its association with clinical outcomes are unknown. METHODS: We performed AREG immunohistochemical staining on skin specimens from 67 patients with aGVHD between the years 2010 and 2015. Two blinded reviewers assessed AREG expression and scored specimens with a semiquantitative scale ranging from 0 (absent) to 4 (most intense). RESULTS: Median AREG score of aGVHD cases was 3. Sixteen of 67 (23.9%) aGVHD cases had an AREG >3. High skin AREG expression (>3 vs. ≤3) was associated with increased overall clinical grade of aGVHD (52.9% vs. 33.4% clinical grade III-IV, p = 0.02), reduced 3-year overall survival (OS; 13% vs. 61%, p < 0.01), and increased 3-year non-relapse mortality (NRM; 56% vs. 20%, p = 0.05). CONCLUSION: High skin AREG immunohistochemical expression is associated with high clinical grade aGVHD, poor OS, and increased NRM.

Association of CD34 Cell Dose with 5-Year Overall Survival after Peripheral Blood Allogeneic Hematopoietic Cell Transplantation in Adults with Hematologic Malignancies.

Higher CD34 cell dose is associated with improved engraftment after peripheral blood allogeneic hematopoietic stem cell transplantation (alloHCT) but also may increase the risk of long-term complications, such as graft-versus-host disease (GVHD). Prior studies examining the relationship between CD34 cell dose and long-term survival outcomes have yielded conflicting results. In this study, we sought to clarify the prognostic impact of CD34 cell dose by examining a large contemporary cohort of patients undergoing alloHCT with a matched sibling peripheral blood stem cell (PBSC) donor. We retrospectively examined the impact of CD34 cell dose on overall survival (OS), neutrophil engraftment, platelet engraftment, treatment-related mortality, relapse, acute GVHD grade II-IV and III-IV, and chronic GVHD in 377 consecutive patients undergoing alloHCT with a PBSC graft source from a matched sibling donor at the University of Minnesota between 2002 and 2015. The patients were classified into 3 groups based on the tertile (T) of CD34 cell dose received: T1, <5 × 106 cells/kg; T2, 5 to 7.5 × 106 cells/kg; and T3, ≥7.5 × 106 cells/kg. Multivariable analysis demonstrated that high CD34 cell dose was associated with superior 5-year OS (hazard ratio [HR], 0.57; P = .01) and more rapid platelet engraftment (HR, 1.70; P < .01). Higher CD34 cell dose also was associated with improved absolute neutrophil count engraftment (T2: HR, 1.54; T3: HR, 1.52; P < .01). There was no association between CD34 cell dose and TRM or relapse at 5 years. Although higher CD34 cell dose was not associated with acute GVHD grade II-IV, it was associated with chronic GVHD (T2: HR, 1.68; T3: HR, 1.50; P = .04). Our data indicate that higher CD34 cell dose (>7.5 × 106/kg) is associated with superior OS at 5 years and improved engraftment but carries an increased risk of chronic GVHD. These data support a target CD34 cell dose goal of 7.5 × 106/kg for sibling PBSC graft donors.

Dual JAK2/Aurora kinase A inhibition prevents human skin graft rejection by allo-inactivation and ILC2-mediated tissue repair.

Prevention of allograft rejection often requires lifelong immune suppression, risking broad impairment of host immunity. Nonselective inhibition of host T cell function increases recipient risk of opportunistic infections and secondary malignancies. Here we demonstrate that AJI-100, a dual inhibitor of JAK2 and Aurora kinase A, ameliorates skin graft rejection by human T cells and provides durable allo-inactivation. AJI-100 significantly reduces the frequency of skin-homing CLA+ donor T cells, limiting allograft invasion and tissue destruction by T effectors. AJI-100 also suppresses pathogenic Th1 and Th17 cells in the spleen yet spares beneficial regulatory T cells. We show dual JAK2/Aurora kinase A blockade enhances human type 2 innate lymphoid cell (ILC2) responses, which are capable of tissue repair. ILC2 differentiation mediated by GATA3 requires STAT5 phosphorylation (pSTAT5) but is opposed by STAT3. Further, we demonstrate that Aurora kinase A activation correlates with low pSTAT5 in ILC2s. Importantly, AJI-100 maintains pSTAT5 levels in ILC2s by blocking Aurora kinase A and reduces interference by STAT3. Therefore, combined JAK2/Aurora kinase A inhibition is an innovative strategy to merge immune suppression with tissue repair after transplantation.

Objective and subjective physical function in allogeneic hematopoietic stem cell transplant recipients.

We conducted a prospective study of adult allogeneic hematopoietic cell transplantation (HCT) recipients to assess pre- and post-HCT physical function. Baseline measurements included a wrist actigraphy, a 6 min walk test (6MWT), an international physical activity questionnaire (IPAQ), and a Functional Assessment of Cancer Therapy-Bone Marrow Transplant (FACT-BMT) as well as serial post-HCT assessments of 6MWT, IPAQ, and FACT-BMT. Forty-seven patients were evaluable for functionality assessments, with a median follow-up of 54.5 months for surviving recipients. No patients demonstrated vigorous or very vigorous activity at any time during monitoring by wrist actigraphy; patients spent a median of 6 h daily sedentary. Self-reported activity via the IPAQ showed 36%, 43%, and 21% of subjects reporting light, moderate, and vigorous activity prior to HCT, respectively. Post-HCT 6MWTs on day +30 demonstrated the greatest association with subsequent survival and non-relapse mortality. A decline in 6MWT distance over time also demonstrated worsened overall survival. This study shows the feasibility of fitness assessments and the ability to risk stratify for subsequent mortality, particularly using the 6MWT on the day +30 single time point assessment and change scores from baseline to day +30 post HCT. These pilot findings suggest important targets for future study.

High incidence of thromboembolism in patients with chronic GVHD: association with severity of GVHD and donor-recipient ABO blood group.

Chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic cell transplantation (HCT) is associated with systemic inflammation and endothelial dysfunction, increasing risk for thromboembolic events (TEE). In 145 adult recipients who developed cGVHD after a matched sibling or umbilical cord blood donor HCT from 2010 to 2018, 32(22%) developed at least 1 TEE event, and 14(10%) developed 2 TEE events. The 5-year cumulative incidence of TEE was 22% (95% CI, 15-29%) with a median time from cGVHD to TEE of 234 days (range, 12-2050). Median time to the development of LE DVT or PE was 107 (range, 12-1925) compared to 450 days (range, 158-1300) for UE DVT. Cumulative incidence of TEE was 9% (95% CI, 0-20%), 17% (95% CI, 9-25%), and 38% (95% CI, 22-55%) in those with mild, moderate, and severe GVHD, respectively. Higher risk for TEE was associated with cGVHD severity (hazard ratio [HR] 4.9, [95% CI, 1.1-22.0]; p = 0.03), non-O-donor to recipient ABO match compared to O-donor to O-recipient match (HR 2.7, [95% CI, 1.0-7.5]; p = 0.053), and personal history of coronary artery disease (HR 2.4, [95% CI, 1.1-5.3]; p = 0.03). TEE was not associated with 2-year non-relapse mortality or 5-year overall survival.

Pacritinib Combined with Sirolimus and Low-Dose Tacrolimus for GVHD Prevention after Allogeneic Hematopoietic Cell Transplantation: Preclinical and Phase I Trial Results.

PURPOSE: In this first-in-human, phase I, GVHD prevention trial (NCT02891603), we combine pacritinib (PAC), a JAK2 inhibitor, with sirolimus to concurrently reduce T-cell costimulation via mTOR and IL6 activity. We evaluate the safety of pacritinib when administered with sirolimus plus low-dose tacrolimus (PAC/SIR/TAC) after allogeneic hematopoietic cell transplantation. PATIENTS AND METHODS: The preclinical efficacy and immune modulation of PAC/SIR were investigated in xenogeneic GVHD. Our phase I trial followed a 3+3 dose-escalation design, including dose level 1 (pacritinib 100 mg daily), level 2 (pacritinib 100 mg twice daily), and level 3 (pacritinib 200 mg twice daily). The primary endpoint was to identify the lowest biologically active and safe dose of pacritinib with SIR/TAC (n = 12). Acute GVHD was scored through day +100. Allografts included 8/8 HLA-matched related or unrelated donor peripheral blood stem cells. RESULTS: In mice, we show that dual JAK2/mTOR inhibition significantly reduces xenogeneic GVHD and increases peripheral regulatory T cell (Treg) potency as well as Treg induction from conventional CD4+ T cells. Pacritinib 100 mg twice a day was identified as the minimum biologically active and safe dose for further study. JAK2/mTOR inhibition suppresses pathogenic Th1 and Th17 cells, spares Tregs and antileukemia effector cells, and exhibits preliminary activity in preventing GVHD. PAC/SIR/TAC preserves donor cytomegalovirus (CMV) immunity and permits timely engraftment without cytopenias. CONCLUSIONS: We demonstrate that PAC/SIR/TAC is safe and preliminarily limits acute GVHD, preserves donor CMV immunity, and permits timely engraftment. The efficacy of PAC/SIR/TAC will be tested in our ongoing phase II GVHD prevention trial.

Outcomes of chronic graft-versus-host disease following matched sibling donor versus umbilical cord blood transplant.

We compared chronic graft-versus-host disease (cGvHD) following umbilical cord blood (UCBT) and matched sibling donor peripheral blood transplant (MSD). 145 patients (2010-2017) with cGvHD after MSD (n = 104) and UCBT (n = 41) were included. Prior acute GvHD was less frequent in MSD (55% vs. 85%; p = 0.01). Severe cGvHD (32% vs. 15%, p = 0.01) and de-novo onset (45% vs. 15%, p < 0.01) were more frequent following MSD. Liver was more frequently involved in MSD recipients (38% vs. 6%); and GI in UCBT (33% vs. 63%), both p < 0.01. Overall response (CR + PR) was similar between both cohorts. 2-year CR was higher in UCBT (14% vs 33%, p = 0.02). Karnofsky score (KPS) ≥ 90 at cGvHD diagnosis was associated with higher odds of response (95%CI: 1.42-10, p < 0.01). The cumulative incidence of durable discontinuation of immune-suppressive therapy, failure-free survival (FFS) and NRM at 2-years were similar between cohorts. KPS < 90 (95%CI: 3.1-24.9, p < 0.01) and platelets <100 × 10e9/L (95%CI: 1.25-10, p = 0.01) were associated with higher risk of NRM. UCBT patients were more likely to have a prior acute GvHD, less severe cGvHD and more likely to attain CR. Despite differences, both cohorts had similar NRM and FFS. High-risk groups, including those with platelets <100 × 10e9/L and KPS < 90, need careful monitoring and intensified therapy.

Current Concepts and Advances in Graft-Versus-Host Disease Immunology.

Worldwide, each year over 30,000 patients undergo an allogeneic hema-topoietic stem cell transplantation with the intent to cure high-risk hematologic malignancy, immunodeficiency, metabolic disease, or a life-threatening bone marrow failure syndrome. Despite substantial advances in donor selection and conditioning regimens and greater availability of allograft sources, transplant recipients still endure the morbidity and mortality of graft-versus-host disease (GVHD). Herein, we identify key aspects of acute and chronic GVHD pathophysiology, including host/donor cell effectors, gut dysbiosis, immune system and cytokine imbalance, and the interface between inflammation and tissue fibrosis. In particular, we also summarize the translational application of this heightened understanding of immune dysregulation in the design of novel therapies to prevent and treat GVHD.

Sole Upfront Therapy with Beclomethasone and Budesonide for Upper Gastrointestinal Acute Graft-versus-Host Disease.

Systemic glucocorticoids remain the standard treatment for gastrointestinal (GI) acute graft-versus-host disease (aGVHD) despite their toxicity and incomplete efficacy. Controlled trials have tested poorly absorbable steroids as adjuncts with systemic glucocorticoids, but only small case series have reported treatment with poorly absorbed beclomethasone dipropionate (BDP) and budesonide (BUD) alone. Our team has adopted the practice of administering BDP or BDP+BUD without systemic glucocorticoids as first-line therapy for isolated upper GI (UGI) aGVHD. We report results in 76 patients treated with BDP alone and in 81 patients treated with BDP+BUD, with allocation by physician choice. Almost all patients received peripheral blood stem cells (92%) from a fully HLA-matched related or unrelated donor (80%) after myeloablative conditioning (76%) for acute leukemia (49%), myelodysplastic syndrome (17%), non-Hodgkin lymphoma (14%), or another hematopoietic disorders (20%). After 28 days of treatment with BDP, 46% of the patients had a complete response (CR) and 10% had a partial response (PR); after 200 days, 61 (80%) patients were alive, 34% maintained a CR, and 3% maintained a PR, whereas 53% required additional immunosuppression (IS). After 28 days of treatment with BDP+BUD, 67% had a CR and 10% a PR; after 200 days, 74 (91%) patients were alive, 46% maintained a CR, and 2% maintained a PR, whereas 43% required additional IS. Among the entire cohort of 157 patients, 66 (42%) were treated successfully without systemic glucocorticoids. This study reports the efficacy of poorly absorbable steroids alone for patients with isolated UGI aGVHD. Prospective trials should test for the potential advantages of BDP and BUD use over systemic glucocorticoids.

Human CD83-targeted chimeric antigen receptor T cells prevent and treat graft-versus-host disease.

Graft-versus-host disease (GVHD) remains an important cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). For decades, GVHD prophylaxis has included calcineurin inhibitors, despite their incomplete efficacy and impairment of graft-versus-leukemia (GVL). Distinct from pharmacologic immune suppression, we have developed what we believe is a novel, human CD83-targeted chimeric antigen receptor (CAR) T cell for GVHD prevention. CD83 is expressed on allo-activated conventional CD4+ T cells (Tconvs) and proinflammatory dendritic cells (DCs), which are both implicated in GVHD pathogenesis. Human CD83 CAR T cells eradicate pathogenic CD83+ target cells, substantially increase the ratio of regulatory T cells (Tregs) to allo-activated Tconvs, and provide durable prevention of xenogeneic GVHD. CD83 CAR T cells are also capable of treating xenogeneic GVHD. We show that human acute myeloid leukemia (AML) expresses CD83 and that myeloid leukemia cell lines are readily killed by CD83 CAR T cells. Human CD83 CAR T cells are a promising cell-based approach to preventing 2 critical complications of allo-HCT - GVHD and relapse. Thus, the use of human CD83 CAR T cells for GVHD prevention and treatment, as well as for targeting CD83+ AML, warrants clinical investigation.

Facilitating resolution of life-threatening acute GVHD with human chorionic gonadotropin and epidermal growth factor.

Acute graft-versus-host disease (aGVHD) is a potentially fatal complication of allogeneic hematopoietic cell transplantation that fails to improve with intense immunosuppression in some patients. We hypothesized that urinary-derived human chorionic gonadotropin (uhCG) could help facilitate resolution of life-threatening aGVHD when added as supportive care via 2 potential mechanisms: immunomodulation (akin to its role in pregnancy) and supplementation of epidermal growth factor (EGF; to aid in epithelial repair). In a phase 1 study, 26 participants received subcutaneous injections of uhCG in addition to standard immunosuppression (13 receiving initial therapy for high-risk aGVHD [according to the Minnesota criteria] and 13 receiving second-line therapy). Participants underwent serial blood testing for biomarkers of hormone response, immune modulation, and aGVHD activity on study. uhCG was well tolerated, with no dose-limiting toxicities. Sixty-two percent of patients in the high-risk cohort and 54% of patients in the second-line cohort had a complete response at study day 28. Plasma EGF was elevated sixfold (from 4 to 24 pg/mL; P = .02) at 6 hours postdose in the high-risk cohort, in contrast to no peak in plasma EGF in the more severe second-line cohort. After 1 week of uhCG, patients reported a twofold increase in the regulatory T cell to conventional T-cell ratio, suggesting immune modulation despite high-dose steroids. Responding patients reported significantly lower plasma amphiregulin and higher plasma butyrate levels at study completion, suggesting improvement in mucosal damage over time. uhCG is a novel, safe, supportive therapy, proceeding to phase 2 testing at 2000 units/m2 in high-risk aGVHD. This study was registered at www.clinicaltrials.gov as #NCT02525029.

Metabolic reprogramming augments potency of human pSTAT3-inhibited iTregs to suppress alloreactivity.

Immunosuppressive donor Tregs can prevent graft-versus-host disease (GVHD) or solid-organ allograft rejection. We previously demonstrated that inhibiting STAT3 phosphorylation (pSTAT3) augments FOXP3 expression, stabilizing induced Tregs (iTregs). Here we report that human pSTAT3-inhibited iTregs prevent human skin graft rejection and xenogeneic GVHD yet spare donor antileukemia immunity. pSTAT3-inhibited iTregs express increased levels of skin-homing cutaneous lymphocyte-associated antigen, immunosuppressive GARP and PD-1, and IL-9 that supports tolerizing mast cells. Further, pSTAT3-inhibited iTregs significantly reduced alloreactive conventional T cells, Th1, and Th17 cells implicated in GVHD and tissue rejection and impaired infiltration by pathogenic Th2 cells. Mechanistically, pSTAT3 inhibition of iTregs provoked a shift in metabolism from oxidative phosphorylation (OxPhos) to glycolysis and reduced electron transport chain activity. Strikingly, cotreatment with coenzyme Q10 restored OxPhos in pSTAT3-inhibited iTregs and augmented their suppressive potency. These findings support the rationale for clinically testing the safety and efficacy of metabolically tuned, human pSTAT3-inhibited iTregs to control alloreactive T cells.

Stress responses, M2 macrophages, and a distinct microbial signature in fatal intestinal acute graft-versus-host disease.

Steroid-refractory intestinal acute graft-versus-host disease (aGVHD) is a frequently fatal condition with little known about mechanisms driving failed steroid responses in gut mucosa. To uncover novel molecular insights in steroid-refractory aGVHD, we compared gene expression profiles of rectosigmoid biopsies from patients at diagnosis of clinical stage 3-4 lower intestinal aGVHD (N=22), to repeat biopsies when the patients became steroid refractory (N=22), and normal controls (N=10). We also performed single gene analyses of factors associated with tolerance (programmed death ligand-1 [PDL1], indoleamine 2,3 dioxygenase [IDO1], and T cell immunoreceptor with Ig and ITIM domains [TIGIT]) and found that significantly higher expression levels of these aGVHD inhibitory genes (PDL1, IDO1, TIGIT) at aGVHD onset became decreased in the steroid-refractory state. We examined genes triggered by microbial ligands to stimulate gut repair, amphiregulin (AREG) and the aryl hydrocarbon receptor (AhR), and found that both AREG and AhR gene expression levels were increased at aGVHD onset and remained elevated in steroid-refractory aGVHD. We also identified higher expression levels of metallothioneines, metal-binding enzymes induced in stress responses, and M2 macrophage genes in steroid-refractory aGVHD. We observed no differences in T-cell subsets between onset and steroid-refractory aGVHD. Patients with a rapidly fatal course showed greater DNA damage and a distinct microbial signature at aGVHD onset, whereas patients with more prolonged survival exhibited a gene expression profile consistent with activation of Smoothened. Our results extend the paradigm beyond T cell-centric therapies for steroid-refractory GI aGVHD and highlight new mechanisms for therapeutic exploration.