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.

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.

Pre-transplant and longitudinal changes in faecal microbiome characteristics are associated with subsequent development of chronic graft-versus-host disease.

The role of the gastrointestinal microbiome in predisposing to chronic graft-versus-host disease (cGVHD), an immune-mediated haematopoietic cell transplant (HCT) complication, is not well defined. We examined the relationship of the host faecal microbiome with subsequent cGVHD development by analysing baseline stool samples as well as post-HCT changes in microbiome composition and metabolite pathway analyses. We analysed pre-transplant baseline samples from 11 patients who subsequently developed cGVHD compared to 13 controls who did not develop acute GVHD or cGVHD at any time. We found a significant differential abundance of multiple taxa at baseline between cGVHD versus controls, including the Actinobacteria phylum and Clostridium genus. A subgroup analysis of longitudinal samples within each patient revealed a greater loss of alpha diversity from baseline to post-engraftment in patients who subsequently developed cGVHD. Metabolic pathways analysis revealed that two pathways associated with short-chain fatty acid metabolism were enriched in cGVHD patient microbiomes: ß-oxidation and acyl-CoA synthesis, and γ-aminobutyrate shunt. In contrast, a tryptophan catabolism pathway was enriched in controls. Our findings show a distinct pattern of baseline microbiome and metabolic capacity that may play a role in modulating alloreactivity in patients developing cGVHD. These findings support the therapeutic potential of microbiome manipulation for cGVHD prevention.

Amphiregulin as monitoring biomarker for life-threatening acute graft-versushost disease: secondary analysis of two prospective clinical trials.

Not available.

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.

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.

Targeting JAK2 reduces GVHD and xenograft rejection through regulation of T cell differentiation.

Janus kinase 2 (JAK2) signal transduction is a critical mediator of the immune response. JAK2 is implicated in the onset of graft-versus-host disease (GVHD), which is a significant cause of transplant-related mortality after allogeneic hematopoietic cell transplantation (allo-HCT). Transfer of JAK2-/- donor T cells to allogeneic recipients leads to attenuated GVHD yet maintains graft-versus-leukemia. Th1 differentiation among JAK2-/- T cells is significantly decreased compared with wild-type controls. Conversely, iTreg and Th2 polarization is significantly increased among JAK2-/- T cells. Pacritinib is a multikinase inhibitor with potent activity against JAK2. Pacritinib significantly reduces GVHD and xenogeneic skin graft rejection in distinct rodent models and maintains donor antitumor immunity. Moreover, pacritinib spares iTregs and polarizes Th2 responses as observed among JAK2-/- T cells. Collectively, these data clearly identify JAK2 as a therapeutic target to control donor alloreactivity and promote iTreg responses after allo-HCT or solid organ transplantation. As such, a phase I/II acute GVHD prevention trial combining pacritinib with standard immune suppression after allo-HCT is actively being investigated (https://clinicaltrials.gov/ct2/show/NCT02891603).

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.

Hypoalbuminemia at Day +90 Is Associated with Inferior Nonrelapse Mortality and Overall Survival in Allogeneic Hematopoietic Cell Transplantation Recipients: A Confirmatory Study.

Prognostic biomarkers in allogeneic hematopoietic cell transplantation (allo-HCT) are needed to improve risk assessment and help guide therapeutic and surveillance strategies to mitigate the risk of death from the procedure. We previously identified hypoalbuminemia at day +90 post-transplantation as an independent predictor of increased nonrelapse mortality (NRM) and inferior overall survival (OS) in patients with acute myelogenous leukemia and myelodysplastic syndrome who were treated with an allo-HCT. Here, we aim to confirm the prognostic significance of day +90 hypoalbuminemia in 783 patients, median age 52 years (range, 18 to 76), who received an allo-HCT for various hematologic malignancies and bone marrow failure syndromes. Multivariate analysis for NRM demonstrated a negative effect of low serum albumin levels (<3.0 versus 3.0 to 3.5 versus >3.5 g/dL) at day +90 post-transplantation (hazard ratios, 8.03 [95% CI, 3.59 to 17.97] versus 2.84 [95% CI, 1.59 to 5.08] versus reference; P < .0001). This was also the case for OS (hazard ratios, 6.86 [95% CI, 4.24 to 11.10] versus 1.52 [95% CI, 1.05 to 2.20] versus reference; P < .0001). Patients with hypoalbuminemia at day +90 post-transplantation are more likely to die from causes other than relapse, particularly infections. This large study confirms the ability of day +90 serum hypoalbuminemia to predict worse NRM and inferior OS. Presence of hypoalbuminemia at day +90 should drive a more rigorous real-time surveillance strategy considering the anticipated high-risk of NRM and poor survival in these patients. Future studies should consider incorporating day +90 serum albumin levels in prognostic models of NRM and OS.

Hypoalbuminaemia segregates different prognostic subgroups within the refined standard risk acute graft-versus-host disease score.

Hypoalbuminaemia has been previously described to predict worse non-relapse mortality (NRM) and inferior overall survival (OS) in allogeneic haematopoietic cell transplant (allo-HCT) recipients. Here, we evaluate the role of hypoalbuminaemia (<35 g/l) at time of onset of acute graft-versus-host disease (aGVHD) when incorporated into the refined aGVHD score. The study population consisted of 522 patients, median age 53 (18-75) years, who underwent an allo-HCT mostly for haematological malignancies. Standard risk (SR) aGVHD comprised 467 patients (89%) and the number of high risk (HR) cases was 55 (11%). Median follow-up for all surviving patients was 26 (3-55) months. Two-year OS was significantly better in patients with SR aGVHD with a serum albumin ≥35 g/l compared to SR with albumin <35 g/l [70% (95% CI = 64-76%) vs. 49% (95% CI = 42-56%), P < 0·0001]. Also, patients with SR aGVHD and a serum albumin level of ≥35 g/l had a significantly lower NRM at 1-year post-transplantation [6% (95% CI = 3-10%) vs. 25% (95% CI = 20-32%), P < 0·0001]. After our findings are validated in a large cohort of patients, we propose that hypoalbuminaemia should be incorporated into the refined aGVHD risk score to further its ability to predict outcomes within this group.

IL-2 promotes early Treg reconstitution after allogeneic hematopoietic cell transplantation.

Graft-versus-host disease (GvHD) remains a major cause of transplant-related mortality. Interleukin-2 (IL-2) plus sirolimus (SIR) synergistically reduces acute GvHD in rodents and promotes regulatory T cells. This phase II trial tested the hypothesis that IL-2 would facilitate STAT5 phosphorylation in donor T cells, expand regulatory T cells, and ameliorate GvHD. Between 16th April 2014 and 19th December 2015, 20 patients received IL-2 (200,000 IU/m2 thrice weekly, days 0 to +90) with SIR (5-14 ng/mL) and tacrolimus (TAC) (3-7 ng/mL) after HLA-matched related or unrelated allogeneic hematopoietic cell transplantation (HCT). The study was designed to capture an increase in regulatory T cells from 16.0% to more than 23.2% at day +30. IL-2/SIR/TAC significantly increased regulatory T cells at day +30 compared to our published data with SIR/TAC (23.8% vs. 16.0%, P=0.0016; 0.052 k/uL vs. 0.037 k/uL, P=0.0163), achieving the primary study end point. However, adding IL-2 to SIR/TAC led to a fall in regulatory T cells by day +90 and did not reduce acute or chronic GvHD. Patients who discontinued IL-2 before day +100 showed a suggested trend toward less grade II-IV acute GvHD (16.7% vs. 50%, P=0.1475). We surmise that the reported accumulation of IL-2 receptors in circulation over time may neutralize IL-2, lead to progressive loss of regulatory T cells, and offset its clinical efficacy. The amount of phospho-STAT3+ CD4+ T cells correlated with donor T-cell activation and acute GvHD incidence despite early T-cell STAT5 phosphorylation by IL-2. Optimizing IL-2 dosing and overcoming cytokine sequestration by soluble IL-2 receptor may sustain lasting regulatory T cells after transplantation. However, an approach to target STAT3 is needed to enhance GvHD prevention. (clinicaltrials.gov identifier: 01927120).

Ofatumumab in combination with glucocorticoids for primary therapy of chronic graft-versus-host disease: phase I trial results.

Standard primary therapy for chronic graft-versus-host disease (GVHD) is incompletely effective. Based on biologic insights implicating pathogenic B cells, we conducted a phase I trial examining the combination of standard (1 mg/kg/day prednisone) glucocorticoid therapy with ofatumumab, a humanized anti-CD20 monoclonal antibody, for primary chronic GVHD therapy. Patients ages ≥ 18 with National Institutes of Health Consensus moderate-to-severe chronic GVHD newly requiring 1 mg/kg/day prednisone were treated at 3 escalating dose levels (300 mg, 700 mg, and 1000 mg) of i.v. ofatumumab on days 1 and 14 of initial glucocorticoid therapy. Dose-limiting toxicity (DLT) was defined by grade 4 infusion reactions, related grade 4 constitutional symptoms, related grade ≥ 3 organ toxicities, or grade 4 neutropenia lasting > 14 days. A total of 12 patients (median age 54; range, 25 to 72) were treated (dose level 1: n = 3; level 2: n = 3; level 3: n = 6). At enrollment, overall chronic GVHD was moderate (n = 7) or severe (n = 5), with diverse organ involvement (skin: n = 8; mouth: n = 8; eye: n = 8; lung: n = 4; gastrointestinal: n = 3; liver: n = 5; genital: n = 2; joint/fascia: n = 5). Infusion of ofatumumab was well tolerated, and no DLT was observed. From the total number of adverse events (n = 29), possibly related adverse events (n = 4) included grade 1 fatigue, grade 1 transaminitis, and 2 infusion reactions (grades 2 and 3). Infectious complications were expected, and there were no cases of hepatitis B reactivation or progressive multifocal leukoencephalopathy. Ofatumumab in combination with prednisone is safe and a phase II examination of efficacy is ongoing.

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.

Human Langerhans cells use an IL-15R-α/IL-15/pSTAT5-dependent mechanism to break T-cell tolerance against the self-differentiation tumor antigen WT1.

Human CD34(+) progenitor-derived Langerhans-type dendritic cells (LCs) are more potent stimulators of T-cell immunity against tumor and viral antigens in vitro than are monocyte-derived DCs (moDCs). The exact mechanisms have remained elusive until now, however. LCs synthesize the highest amounts of IL-15R-α mRNA and protein, which binds IL-15 for presentation to responder lymphocytes, thereby signaling the phosphorylation of signal transducer and activator of transcription 5 (pSTAT5). LCs electroporated with Wilms tumor 1 (WT1) mRNA achieve sufficiently sustained presentation of antigenic peptides, which together with IL-15R-α/IL-15, break tolerance against WT1 by stimulating robust autologous, WT1-specific cytolytic T-lymphocytes (CTLs). These CTLs develop from healthy persons after only 7 days' stimulation without exogenous cytokines and lyse MHC-restricted tumor targets, which include primary WT1(+) leukemic blasts. In contrast, moDCs require exogenous rhuIL-15 to phosphorylate STAT5 and attain stimulatory capacity comparable to LCs. LCs therefore provide a more potent costimulatory cytokine milieu for T-cell activation than do moDCs, thus accounting for their superior stimulation of MHC-restricted Ag-specific CTLs without need for exogenous cytokines. These data support the use of mRNA-electroporated LCs, or moDCs supplemented with exogenous rhuIL-15, as vaccines for cancer immunotherapy to break tolerance against self-differentiation antigens shared by tumors.

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.

Anti-IL6-receptor-alpha (tocilizumab) does not inhibit human monocyte-derived dendritic cell maturation or alloreactive T-cell responses.

Significant comorbidites and lethality complicate GVHD and its treatment. Targeting the cytokine milieu may improve GVHD control; and IL6 is an attractive candidate, given its role in dendritic cell activation and T-cell differentiation. Tocilizumab is a humanized mAb to IL6-receptor-α (IL6R-α), which is Food and Drug Administration-approved for treatment of rheumatoid arthritis. Mouse transplant models have demonstrated that IL6 blockade also improves GVHD scores and survival. Definitive immunologic effects of IL6 inhibition have not emerged given inconsistent alterations in regulatory T cells (Tregs) and suppression of T-cell proliferation. Despite on-target suppression of IL6R-α signaling in human monocyte-derived dendritic cells (moDCs) and T cells, our data show no effect on moDC maturation/activation, alloreactive T-cell proliferation, Treg expansion, or allogeneic Th1/Th17 responses in vitro. These findings merit attention in any clinical trials of tocilizumab for GVHD prevention or treatment and provide a rationale for evaluating more specific inhibitors of downstream JAK2/STAT3 signaling as well.

Janus kinase-2 inhibition induces durable tolerance to alloantigen by human dendritic cell-stimulated T cells yet preserves immunity to recall antigen.

Janus kinase-2 (JAK2) conveys receptor-binding signals by several inflammatory cytokines, including IL-6, via phosphorylation of signal transducer and activator of transcription 3 (STAT3). We demonstrate that selective JAK2 inhibition by TG101348 during initial encounters between human T cells and allogeneic monocyte-derived dendritic cells induces durable, profound, and specific T-cell tolerance upon reexposure to the same alloantigens. Subsequent responses by nonalloreactive T cells to stimulation de novo by a pathogenic nominal antigen remain intact. TG101348 also suppresses primed T-cell responses when present only during alloantigen restimulation. TG101348 ablates IL-6/JAK2-mediated phosphorylation of STAT3, but has no off-target effects on IL-2 or IL-15/JAK3/pSTAT5-dependent signaling, which sustain the responses of regulatory T cells (Tregs) and other effector T cells. JAK2 inhibition preserves Treg numbers and thereby enhances the ratio of CD4(+) Tregs to CD8(+)CD25(+) effector T cells in favor of Tregs. JAK2 inhibition also reduces the production of IL-6 and TNF-α in allogeneic MLRs, impairing the activation of central and effector memory T cells as well as the expansion of responder Th1 and Th17 cells. While we have reported the limitations of isolated IL-6R-α inhibition on dendritic cell-stimulated alloreactivity, we demonstrate here that JAK2 represents a relevant biologic target for controlling GVHD or allograft rejection without broader immune impairment.