ALBERT

Description: Key Points CMV reactivation fundamentally resets posttransplant CD8 reconstitution, resulting in massive expansion of CMV-specific CD8 Tem. CMV reactivation is associated with defects in the underlying TCRβ immune repertoire.

Description: Key PointsThe transcriptional networks controlling breakthrough acute GVHD can be mapped, and correlate closely with clinical disease. Breakthrough acute GVHD is transcriptionally controlled by T-cell persistence, inflammation, and Th/Tc17 skewing.

Description: Abstract 4765 Introduction: Therapy for acute chest syndrome (ACS) in our hospital includes IV hydration, pain control, antibiotic therapy, incentive spirometry, and an inhaled bronchodilator in patients who are wheezing or who have a history of asthma. Patients also receive red blood cells as either a simple transfusion (ST) or an exchange transfusion using automated erythrocytapheresis (RCE). Here we present clinical and laboratory data from patients with ACS who were treated with RCE and compare these results to a contemporaneous control group of patients with ACS who were treated with ST. Methods: This was a retrospective study of all patients who presented to Arkansas Children's Hospital from 1996 through 2010 for the treatment of newly diagnosed ACS. Episodes of ACS were separated into a control group who were treated with ST and a RCE group. Patients were referred to the apheresis service by the treating hematologist for RCE when they presented with severe ACS and impending respiratory failure (n=20) or when they failed to improve, or deteriorated, following ST (n=29). Summary statistics were described as mean ± standard deviation for continuous data and frequency and percentage for categorical data. Because there were patients with multiple episodes, repeated measures analyses were performed to evaluate the clinical differences between the RCE and ST episodes. Results: We identified a total of 81 patients who were diagnosed with a total of 119 episodes of ACS. The average age at presentation was 8.5 years (range 5 months to 20 years), 64% were male, and 78% had SS disease. Twelve patients (accounting for 11 episodes each in the RCE and ST groups) had a previous history of asthma and 23 patients had multiple episodes of ACS (median 2, range 2–6). Episodes treated with RCE were significantly more likely to present with tachypnea, retractions, nasal flaring, and decreased air movement but episodes treated with ST were significantly more likely to present with cough and wheezing, even though a previous history of asthma was not more common in the ST group than in the RCE group. The RCE group spent significantly more time on oxygen, eight episodes required BiPAP, and 3 episodes required mechanical ventilation but no ST episodes required either BiPAP or mechanical ventilation. The median WBC at diagnosis was significantly higher in the RCE group but there were no significant differences in hemoglobin level or platelet count between the groups. As expected, blood product utilization was significantly higher in the RCE group. There were no procedural or catheter-related complications in the RCE group. Mean length of stay was not significantly longer in the RCE group but total hospital costs were significantly higher as expected in the RCE group due to the expense of RCE and the need for ICU care, BiPAP, and mechanical ventilation. There were no deaths from ACS during the study period. Additional variables that were studied but did not differ significantly between the groups included fever, sites of pain, vomiting, grunting, accessory muscle use, crackles, decreased breath sounds, color, oxygen saturation, chest radiograph findings, initial hemoglobin and platelet count, and post-treatment blood counts. Discussion: We found that children treated with RCE were sicker than those treated with ST, as determined by tachypnea, retractions, nasal flaring, decreased air movement, duration of oxygen therapy, need for BiPAP, and need for mechanical ventilation. We also found that children with ACS who present with cough and wheezing are significantly less likely to require RCE. We conclude that RCE is an effective therapy for sicker patients with ACS but that further work is required to develop a predictive scoring system to discriminate prospectively between children with ACS who will benefit from RCE and those who can be treated successfully with ST. Disclosures: No relevant conflicts of interest to declare.

Description: Graft versus host disease (GvHD) is the most common complication of hematopoietic stem cell transplant (HCT). However, our understanding of the molecular pathways that cause this disease remains incomplete, leading to inadequate targeted strategies for prevention and treatment. To address this, we determined the gene expression profile of non-human primate (NHP) T cells during active and partially controlled acute GvHD (aGvHD), in order to accomplish two goals: 1) uncover important genetic drivers of aGvHD and 2) identify novel, targetable pathways for optimal aGvHD prevention. Utilizing microarray technology, we measured the gene expression profiles of flow cytometrically purified CD3+ T cells from NHP recipients of MHC partially-matched HCT in three treatment cohorts resulting in increasing degrees of survival: 1) no immunoprophylaxis (No Rx, MST = 7.5); 2) sirolimus monotherapy (MST = 17) tacrolimus-methotrexate (Tac-Mtx) dual prophylaxis (MST = 49). Arrays were performed on T cells purified on Day +14 post-transplant (unless terminal analysis occurred earlier due to severe disease). This comparison allowed us to determine the impact of both mTOR and calcineurin inhibition on the molecular pathways dysregulated during GvHD, and to determine which genes and pathways remained dysregulated despite prophylaxis. Pathways identified by this strategy may contain new therapeutic targets unaffected by current immunoprophylactic approaches. We found that the transcriptional profile of donor T cells from HCT recipients with unprophylaxed GvHD was characterized by significant perturbation of pathways regulating T cell proliferation, effector function and cytokine synthesis (Figure 1a). By identifying pathways unaffected by sirolimus or tac-mtx therapy (Figure 1b), we discovered multiple potentially druggable targets not previously implicated in the pathophysiology of aGvHD. These targets prominently included the hedgehog and the aurora kinase A pathways. Utilizing a murine aGvHD model, we demonstrated that pharmacologic inhibition of these pathways could mitigate disease and improve survival (Figure 2a,b). These data provide the first identification of the T cell transcriptome of primate acute GvHD and the hedgehog and aurora kinase A pathways as novel potential targets for prevention of this disease. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: We have previously shown that blocking CD28:CD80/86 costimulation with CTLA4-Ig can be a successful strategy to inhibit GVHD. However, since CTLA4-Ig targets CD80/86, there remain concerns of off-target, immune activating effects, given that it can inhibit both positive signaling through CD28 and negative signaling through CTLA4. In order to specifically target CD28, we have developed a humanized anti-CD28 monovalent Fab antibody (FR104) and have tested its ability to protect against GVHD. Methods: NHP underwent MHC-mismatched transplantation after myeloablative radiation-based pre-transplant conditioning. They were transplanted with GCSF-mobilized PBSCs (4.2x10^8 +/- 1.1x10^8 TNC/kg and 1.9 x 10^7 +/- 0.5 x 10^7 CD3+ T cells/kg. GVHD prophylaxis consisted of either monotherapy with FR104 (5mg/kg given weekly) or with combination FR104 and rapamycin (rapamycin trough 5-15 ng/ml). These were compared to three control groups: no GVHD prophylaxis, rapamycin monotherapy and autologous HSCT. Clinical and histologic GVHD was monitored using our NHP grading scale, and longitudinal immunologic analysis was performed. Treatment was continued as tolerated with planned terminal analysis at 35 days in all survivors. Results: Untreated controls (n = 5) developed rapid, severe multi-organ GVHD (MST=7 days). Rapamycin monotherapy (n=6) partially protected against GVHD (MST =14 days). Monotherapy with FR104 showed a significant prolongation in survival compared to untreated animals (MST = 21 days, p=0.02 n = 3), with breakthrough GVHD (liver, skin predominant) ultimately occurring. In contrast to all other groups, combination therapy with FR104 and rapamycin (n=4) resulted in striking protection from GVHD with all animals reaching planned terminal analysis with minimal clinical disease (p=0.01 vs. untreated controls, p=0.01 vs. rapamycin monotherapy, Fig. 1). We have documented a major role for both T cell proliferation (Ki-67) and Granzyme B (Gran-B) overexpression in GVHD, with untreated controls demonstrating rampant CD8+ proliferation (81% + 5.4% Ki-67+ at day 7 vs. 4% pre-transplant) and Gran-B overexpression (81% + 2.7% Gran-Bvery high at day 7 vs. 0.5% + 0.5% pre-transplant). Monotherapy with either rapamycin or FR104 partially controlled proliferation (53% + 23% and 30% + 21.7%, respectively) and Gran-B overexpression (21% + 8.7% and 11% + 5%, respectively) on day 7. Combination therapy with FR104 + rapamycin resulted in synergistic control of both proliferation and Gran-B overexpression (0.6% + 0.2% Ki67+ and 2.3% + 1.3% Gran-B overexpression on Day 7), reverting the CD8+ activation phenotype to that observed in auto-HSCT controls (5.6% + 2.1% Ki67+, 9% + 3.2% Gran-B overexpression, Fig. 2). This reversion to the auto-HSCT phenotype was maintained until the terminal analysis, and the control of Day 7 proliferation and Gran-B overexpression correlated closely with GVHD-free survival. The control of CD28-mediated T cell activation in FR104-treated recipients resulted in significant preservation of the naïve CD8+ T cell (CD8 Tn) phenotype in these animals. Thus, in untreated controls and rapamycin monotherapy groups, the CD8 Tn population virtually disappeared post-transplant (2.1% + 0.7% and 6.6% + 1.5% at day 7, respectively compared to 33.6%+ 4.1% pre-transplant), consistent with widespread allo-stimulation and costimulation. In contrast, FR104 monotherapy resulted in partial preservation of CD8 Tn (24.3% + 2.8% at Day 7) and FR104 + rapamycin combination therapy promoted nearly complete preservation of CD8 Tn (48.3% + 0.9% at day +7), with maintenance of this naïve population for the duration of therapy (Fig. 3). Discussion: These results show, for the first time, that specific blockade of CD28 can inhibit NHP GVHD, and when coupled with rapamycin it can effectively control both the clinical and immunologic sequelae of this disease. CD28 blockade with FR104 + rapamycin significantly inhibited both CD8+ T cell proliferation and Granzyme B overexpression, and lead to robust preservation of the naïve T cell phenotype, consistent with efficient inhibition of allo-activation. Our results suggest that targeting the CD28:CD80/86 costimulation axis by selective CD28 blockade may be effective in inhibiting the T cell activation associated with GVHD, and is deserving of clinical evaluation for safety and efficacy in patients undergoing HCT. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Vanhove: Effimune: Research Funding.

Description: Although acute graft-versus-host-disease (AGVHD) is one of the major causes of non-relapse mortality after hematopoietic stem cell transplant (HCT), we are still unable to predict which patients will develop the most severe form of this disease, or which molecular pathways are dysregulated in the T cells that cause disease. Thus, understanding the molecular features of AGVHD is a critical unmet need. To address this, we have performed a companion mechanistic study as a part of our completed Phase 2 trial of abatacept, a CD28:CD80/86 costimulation blockade agent, for severe AGVHD prevention (Clinicaltrials.gov # NCT01743131, 'ABA2'). ABA2 has demonstrated significant improvement in AGVHD in patients prophylaxed with abatacept in addition to calcineurin inhibition (CNI) + Methotrexate (MTX) compared to controls receiving CNI/MTX alone. To begin to uncover mechanisms responsible for the control of AGVHD with abatacept, and given that CD4+ T cells have been consistently documented to be the main therapeutic target of this drug, we interrogated the transcriptome of CD4+ T cells reconstituting in patients prophylaxed with abatacept compared to CNI/MTX. To perform this analysis, we flow cytometrically sorted CD4+ T cells on Days 21-28 post-transplant from all patients on ABA2, as well as a cohort of 12 untransplanted healthy controls, and subsequently performed mRNA-sequencing on these cells. Weighted Gene Correlation Network Analysis (WGCNA) was performed on the top 6000 most variant transcripts from the resulting sequencing data. Hierarchical clustering of the WGCNA co-expression matrix enabled the identification of self-assembling modules (SAMs) that met a threshold of coexpression (Figure 1A). For the ABA2 dataset, we considered the following variables in the WGCNA model: patient cohort (7/8 patients, 8/8 patients, healthy controls), +/- prophylaxis with abatacept, CMV reactivation, EBV reactivation, Grade of GVHD (0-4), relapse, non-relapse mortality, and all-cause mortality. The WGCNA clustering analysis resulted in the identification of 4 discrete SAMs, which were highly correlated with clinical variable metamodules. This analysis revealed a strong positive correlation of a 476-gene SAM (the Turquoise module) in patients prophylaxed with CNI/MTX + placebo and anti-correlation of this module in patients prophylaxed with CNI/MTX + abatacept, as demonstrated in both the WGCNA heatmap and through Gene Set Enrichment Analysis (Figure 1 A-B). These opposing correlations suggested that interrogation of this module would reveal mechanistic correlates with standard prophylaxis that were decoupled by abatacept. Pathway analysis using the Reactome database (Figure 1C) revealed the turquoise SAM to be dominated by four types of pathways: (1) Those that define canonical cell-cycle pathways (2) Those involved in T cell metabolism (3) Those involved in apoptosis and (4) Those involved in T cell activation, consistent with upregulation of these transcripts in placebo versus abatacept patients. In addition to being highly correlated with patients receiving placebo, the expression of a subset of the transcripts in the Turquoise module were also directly correlated with the severity of AGVHD in these patients. Thus, linear regression analysis of the 476 transcripts in this module identified a subset of 93 genes for which transcript expression level was increased both in placebo compared to abatacept, and for which expression level also positively correlated with Grade of AGVHD. As with the Turquoise module as a whole, this subset of genes also formed a highly correlated network, linking transcripts involved in T cell proliferation, apoptosis, activation, metabolism as well as the T cell checkpoint (Figure 1D). This analysis represents the first comprehensive interrogation of the transcriptomic correlates of AGVHD. It identifies a novel set of transcripts which positively associate with the severity of AGVHD, and which costimulation blockade with abatacept down-regulates and de-couples from AGVHD severity. These results suggest a profound reprograming of T cell activation with abatacept that is correlated with the control of AGVHD. Disclosures Qayed: Bristol-Myers Squibb: Honoraria. Langston:Astellas Pharma: Other: Research Support; Incyte: Other: Research Support; Jazz Pharmaceuticals: Other: Research Support; Chimerix: Other: Research Support; Takeda: Other: Research Support; Kadmon Corporation: Other: Research Support; Novartis: Other: Research Support; Bristol Myers Squibb: Other: Research Support. Blazar:Fate Therapeutics, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; Abbvie Inc: Research Funding; Leukemia and Lymphoma Society: Research Funding; Childrens' Cancer Research Fund: Research Funding; KidsFirst Fund: Research Funding; Tmunity: Other: Co-Founder; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees. Kean:HiFiBio: Consultancy; BlueBirdBio: Research Funding; Gilead: Research Funding; Regeneron: Research Funding; EMDSerono: Consultancy; FortySeven: Consultancy; Magenta: Research Funding; Kymab: Consultancy; Jazz: Research Funding; Bristol Meyers Squibb: Patents & Royalties, Research Funding. OffLabel Disclosure: Abatacept: Approved for Rheumatoid Arthritis; used in this trial for prevention of GVHD.

Description: While novel therapeutics for graft versus host disease (GvHD) are desperately needed, new prevention and treatment strategies for this disease have been extremely slow to reach the clinic. One of the major barriers has been the historic lack of a powerful translational system capable of both (1) interrogating the clinical efficacy of novel approaches and (2) discovering the underlying mechanisms of GvHD prevention. Our lab has addressed this unmet need with the development of a non-human primate (NHP) model of GvHD, and the first therapeutic approach developed with the NHP model, costimulation blockade with CTLA4-Ig, is currently in clinical trials for GvHD prevention (Clinical Trials.gov #NCT01743131). While the first-in-disease trials of GvHD prevention add CTLA4-Ig to standard tacrolimus/methotrexate, previous work has suggested that mTOR inhibition with sirolimus is more pro-tolerogenic than calcineurin inhibition when paired with costimulation blockade. We have now addressed the possibility of pairing CTLA4-Ig with sirolimus to prevent GvHD in the NHP model, and have interrogated the outcomes using a systems-based approach. Our new experiments provide strong clinical, immunologic and trancriptomic evidence for potent synergy when CTLA4-Ig and sirolimus are combined for GvHD prevention. In this study, we determined the impact of the following treatment groups on GvHD after high-risk haplo-identical HSCT using T cell-replete peripheral blood stem cell transplantation: 1) no therapy (n=4) 2) CTLA4-Ig monotherapy (using the second generation CTLA4-Ig formulation, belatacept, n=3) 3) sirolimus monotherapy (n=4) 4) combination belatacept and sirolimus (n=3). To determine the relative impact of each therapeutic approach, we monitored clinical GvHD, GvHD-free survival, and flow cytometric signs of immune activation post-transplant. Moreover, to create a comprehensive molecular map of their impact on GvHD, we performed transcriptomic analysis, on CD3+/CD20- T cells that were purified on day +14 post-transplant and analyzed using the Affymetrix microarray platform. The synergistic impact of combined belatacept +sirolimus was evidenced through all analyses techniques: Thus, GvHD-free survival with belatacept + sirolimus was prolonged compared to all other groups (MST belatacept + sirolimus = 33d, p 〈 0.02 compared to MST for untreated controls (7.5d), belatacept monotherapy (9d, p 〈 0.03) and sirolimus monotherapy (12d) (Figure 1a). GvHD clinical scores mirrored the clinical survival (Figure 1b). In addition, canonical flow cytometric signs of CD8+ T cell activation (proliferation, measured by Ki-67, and excessive cytotoxicity measured by granzyme B overexpression) also mirrored the clinical synergy we measured with combined belatacept +sirolimus compared to all other groups (Figure 1c). Comparing the expression profile of T cells during acute GvHD in the four cohorts allowed examination of treatment synergy at an unprecedented level of molecular detail. Unsupervised analysis of transcriptomic signatures as a whole revealed clustering of principal component projections from belatacept + sirolimus to be strikingly similar to a large comparative healthy control cohort (n=28), underscoring the high degree of control alloreactivity with this regimen. Moreover, the comparison of differentially expressed genes from animals receiving belatacept + sirolimus revealed significant divergence from monotherapy with either belatacept or sirolimus, again underscoring the profound control of allo-activation that was observed with belatacept + sirolimus (Figure 2a). The mirror-image analysis provided additional support or this synergy, with transcriptional analysis of T cells from belatacept + sirolimus-treated animals most dissimilar from the transcriptome of unprophylaxed GvHD (Figure 2b). Those genes for which expression was significantly normalized by combination therapy showed pathway enrichment prominently in T cell effector function (prominently including granzyme signaling – Figure 2c), cytokine networks (prominently IL2, IL12 and IL-18 – Figure 2d), as well as in proliferation and cell cycle pathways (Figure 2e). These data reveal a treatment synergy between T cell costimulation blockade with CTLA-4-Ig and mTOR inhibition and suggest that this combination of therapy will be useful for acute GvHD immunoprophylaxis in humans. Disclosures No relevant conflicts of interest to declare.

Description: Allogeneic hematopoietic cell transplantation (HCT) may be curative for patients with marrow and immune disorders, but graft-vs-host-disease (aGVHD) and infections cause significant morbidity and non-relapse mortality. We have conducted a multicenter, double blind, placebo-controlled phase II trial of costimulation blockade with abatacept (Aba) combined with standard GVHD prophylaxis with a calcineurin inhibitor and methotrexate (CNI + MTX) following HLA matched unrelated donor transplant (n=142). In order to assess the effects of Aba on immune reconstitution, and to assess whether this reconstitution is influenced during CMV reactivation, we longitudinally evaluated post-transplant whole blood samples with multiparameter flow cytometry using markers for CD3, CD4, CD8, CD197 and CD45RA to measure reconstitution of CD4 and CD8 T cell populations and their respective memory subsets over time. Results: We observe that post-transplant CMV reactivation induces a marked expansion of CD8 effector memory (EM) cells, which is similar in magnitude for Aba vs placebo patients. We found that development of moderate (gr 2-4) or severe (gr 3-4) GVHD was not associated with an increased frequency of CMV reactivation, but patients with moderate GVHD showed a blunted expansion of CD8 EM cells compared to those without GVHD, and CD8 EM expansion was essentially absent among CMV reactivating patients with severe aGVHD. Clinical correlates will be presented. Conclusions: Our results suggest that adding abatacept to CNI/MTX does not materially affect reconstitution of T cell immunity in the presence or absence of CMV reactivation, but aGVHD remains a major driver of compromised immune recovery after HCT. Disclosures Watkins: Bristol Myers Squibb: Research Funding. Qayed:Mesoblast: Consultancy; Novartis: Consultancy. Horan:Bristol Myers Squib: Honoraria, Research Funding. Kean:gilead: Research Funding; bluebird bio: Research Funding; fortyseven: Consultancy; magenta: Research Funding; regeneron: Research Funding; hifibio: Consultancy; kymab: Consultancy; Bristol Meyers Squibb: Research Funding; novartis: Consultancy. Langston:Kadmon Corporation: Research Funding; Bristol Myers Squib: Research Funding; Incyte: Research Funding; Chimerix: Research Funding; Takeda: Research Funding; Astellas Pharmaceuticals: Research Funding; Jazz Pharmaceuticals: Research Funding.