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Inborn errors of immunity that present with concomitant immunodeficiency and auto-inflammation are therapeutically challenging; furthermore, complexity is added when they are caused by mutations in genes that encode for proteins expressed beyond immune cells. The ubiquitin-proteasome system is the main intracellular proteolytic machinery and participates in most cellular processes by degrading ubiquitinated proteins. Mutations in proteasome subunits resulting in proteasome deficiency cause a severe auto-inflammatory disease characterized by chronic auto-inflammation neutrophilic dermatosis and fever, collectively referred to as Proteasome Associated Auto-inflammatory Syndromes (PRAAS). POMP is a chaperone for proteasome assembly and AD mutations in POMP cause a form of PRAAS with prominent immunodeficiency referred to as POMP-related auto-inflammation and immune dysregulation (PRAID) manifesting with recurrent, severe and opportunistic infections in addition to inflammatory features that are characteristic for all PRAAS disorders, most importantly early-onset neutrophilic dermatosis. JAK inhibitors partially control the disease in individuals with PRAAS, however life-threatening, recurrent and opportunistic infections in patients with POMP mutations limit immunosuppressive therapies and prompted consideration of hematopoietic stem cell transplant (HSCT). We describe successful HSCT in two patients with POMP deficiency. Despite POMP being ubiquitously expressed, the immunologic and auto-inflammatory phenotype were both ameliorated through HSCT which suggests that the clinical and immunological features of PRAID are predominantly derived from a proteasome defect in hematopoietic cells. To our knowledge, these are the first patients with a form of PRAAS cured by HSCT, opening new therapeutic possibilities for these diseases.

Adenosine deaminase (ADA) deficiency causes ∼13% of cases of severe combined immune deficiency (SCID). Treatments include enzyme replacement therapy (ERT), hematopoietic cell transplant (HCT), and gene therapy (GT). We evaluated 131 patients with ADA-SCID diagnosed between 1982 and 2017 who were enrolled in the Primary Immune Deficiency Treatment Consortium SCID studies. Baseline clinical, immunologic, genetic characteristics, and treatment outcomes were analyzed. First definitive cellular therapy (FDCT) included 56 receiving HCT without preceding ERT (HCT); 31 HCT preceded by ERT (ERT-HCT); and 33 GT preceded by ERT (ERT-GT). Five-year event-free survival (EFS, alive, no need for further ERT or cellular therapy) was 49.5% (HCT), 73% (ERT-HCT), and 75.3% (ERT-GT; P < .01). Overall survival (OS) at 5 years after FDCT was 72.5% (HCT), 79.6% (ERT-HCT), and 100% (ERT-GT; P = .01). Five-year OS was superior for patients undergoing HCT at

Hematopoietic stem cell transplant (HSCT) is a curative option for patients with high-risk acute lymphoblastic leukemia (ALL), but relapse remains a major cause of treatment failure. To prevent disease relapse, we prepared and infused donor-derived multiple leukemia antigen–specific T cells (mLSTs) targeting PRAME, WT1, and survivin, which are leukemia-associated antigens frequently expressed in B- and T-ALL. Our goal was to maximize the graft-versus-leukemia effect while minimizing the risk of graft-versus-host disease (GVHD). We administered mLSTs (dose range, 0.5 × 107 to 2 × 107 cells per square meter) to 11 patients with ALL (8 pediatric, 3 adult), and observed no dose-limiting toxicity, acute GVHD or cytokine release syndrome. Six of 8 evaluable patients remained in long-term complete remission (median: 46.5 months; range, 9-51). In these individuals we detected an increased frequency of tumor-reactive T cells shortly after infusion, with activity against both targeted and nontargeted, known tumor-associated antigens, indicative of in vivo antigen spreading. By contrast, this in vivo amplification was absent in the 2 patients who experienced relapse. In summary, infusion of donor-derived mLSTs after allogeneic HSCT is feasible and safe and may contribute to disease control, as evidenced by in vivo tumor-directed T-cell expansion. Thus, this approach represents a promising strategy for preventing relapse in patients with ALL.

Despite improvements, mortality after allogeneic hematopoietic cell transplantation (HCT) for nonmalignant diseases remains a significant problem. We evaluated whether pre-HCT conditions defined by the HCT Comorbidity Index (HCT-CI) predict probability of posttransplant survival. Using the Center for International Blood and Marrow Transplant Research database, we identified 4083 patients with nonmalignant diseases transplanted between 2007 and 2014. Primary outcome was overall survival (OS) using the Kaplan-Meier method. Hazard ratios (HRs) were estimated by multivariable Cox regression models. Increasing HCT-CI scores translated to decreased 2-year OS of 82.7%, 80.3%, 74%, and 55.8% for patients with HCT-CI scores of 0, 1 to 2, 3 to 4, and ≥5, respectively, regardless of conditioning intensity. HCT-CI scores of 1 to 2 did not differ relative to scores of 0 (HR, 1.12 [95% CI, 0.93-1.34]), but HCT-CI of 3 to 4 and ≥5 posed significantly greater risks of mortality (HR, 1.33 [95% CI, 1.09-1.63]; and HR, 2.31 [95% CI, 1.79-2.96], respectively). The effect of HCT-CI differed by disease indication. Patients with acquired aplastic anemia, primary immune deficiencies, and congenital bone marrow failure syndromes with scores ≥3 had increased risk of death after HCT. However, higher HCT-CI scores among hemoglobinopathy patients did not increase mortality risk. In conclusion, this is the largest study to date reporting on patients with nonmalignant diseases demonstrating HCT-CI scores ≥3 that had inferior survival after HCT, except for patients with hemoglobinopathies. Our findings suggest that using the HCT-CI score, in addition to disease-specific factors, could be useful when developing treatment plans for nonmalignant diseases.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a rare X-linked disorder caused by a loss of function mutation in the FOXP3 gene. It manifests early in infancy with clinical symptoms including autoimmune enteropathy, type 1 diabetes mellitus, and eczema. While aberrant FOXP3 expression has been associated with several types of cancer, little is known regarding the risk of cancer in patients with IPEX harboring the characteristic FOXP3 mutation. Here, we present a unique case of a primary signet ring gastric adenocarcinoma in a pediatric patient with IPEX syndrome.

Chronic granulomatous disease (CGD) is a primary immune deficiency due to defects in phagocyte respiratory burst leading to severe and life-threatening infections. Patients with CGD also suffer from disorders of inflammation and immune dysregulation including colitis and granulomatous lung disease, among others. Additionally, patients with CGD may be at increased risk of systemic inflammatory disorders such as hemophagocytic lymphohistiocytosis (HLH). The presentation of HLH often overlaps with symptoms of systemic inflammatory response syndrome (SIRS) or sepsis and therefore can be difficult to identify, especially in patients with a primary immune deficiency in which incidence of infection is increased. Thorough evaluation and empiric treatment for bacterial and fungal infections is necessary as HLH in CGD is almost always secondary to infection. Simultaneous treatment of infection with anti-microbials and inflammation with immunosuppression may be needed to blunt the hyperinflammatory response in secondary HLH. Herein, we present a series of X-linked CGD patients who developed HLH secondary to or with concurrent disseminated CGD-related infection. In two patients, CGD was a known diagnosis prior to development of HLH and in the other two CGD was diagnosed as part of the evaluation for HLH. Concurrent infection and HLH were fatal in three; one case was successfully treated, ultimately receiving hematopoietic stem cell transplantation. The current literature on presentation, diagnosis, and treatment of HLH in CGD is reviewed.

Primary Immune Regulatory Disorders (PIRD) are an expanding group of diseases caused by gene defects in several different immune pathways, such as regulatory T cell function. Patients with PIRD develop clinical manifestations associated with diminished and exaggerated immune responses. Management of these patients is complicated; oftentimes immunosuppressive therapies are insufficient, and patients may require hematopoietic cell transplant (HCT) for treatment. Analysis of HCT data in PIRD patients have previously focused on a single gene defect. This study surveyed transplanted patients with a phenotypic clinical picture consistent with PIRD treated in 33 Primary Immune Deficiency Treatment Consortium centers and European centers. Our data showed that PIRD patients often had immunodeficient and autoimmune features affecting multiple organ systems. Transplantation resulted in resolution of disease manifestations in more than half of the patients with an overall 5-years survival of 67%. This study, the first to encompass disorders across the PIRD spectrum, highlights the need for further research in PIRD management. ispartof: FRONTIERS IN IMMUNOLOGY vol:11 ispartof: location:Switzerland status: published

The Primary Immune Deficiency Treatment Consortium (PIDTC) performed a retrospective analysis of 662 patients with severe combined immunodeficiency (SCID) who received a hematopoietic cell transplantation (HCT) as first-line treatment between 1982 and 2012 in 33 North American institutions. Overall survival was higher after HCT from matched-sibling donors (MSDs). Among recipients of non-MSD HCT, multivariate analysis showed that the SCID genotype strongly influenced survival and immune reconstitution. Overall survival was similar for patients with RAG, IL2RG, or JAK3 defects and was significantly better compared with patients with ADA or DCLRE1C mutations. Patients with RAG or DCLRE1C mutations had poorer immune reconstitution than other genotypes. Although survival did not correlate with the type of conditioning regimen, recipients of reduced-intensity or myeloablative conditioning had a lower incidence of treatment failure and better T- and B-cell reconstitution, but a higher risk for graft-versus-host disease, compared with those receiving no conditioning or immunosuppression only. Infection-free status and younger age at HCT were associated with improved survival. Typical SCID, leaky SCID, and Omenn syndrome had similar outcomes. Landmark analysis identified CD4(+) and CD4(+)CD45RA(+) cell counts at 6 and 12 months post-HCT as biomarkers predictive of overall survival and long-term T-cell reconstitution. Our data emphasize the need for patient-tailored treatment strategies depending upon the underlying SCID genotype. The prognostic significance of CD4(+) cell counts as early as 6 months after HCT emphasizes the importance of close follow-up of immune reconstitution to identify patients who may need additional intervention to prevent poor long-term outcome.

Introduction: Transplant-associated thrombotic microangiopathy (TA-TMA) is an increasingly recognized hematologic complication after allogeneic hematopoietic cell transplantation (HCT). While few studies have reported germline association with rare variants in complement genes using targeted next generation sequencing (NGS) method, they were limited by small sample size (≤40 TMA cases) and lack of analysis of non-complement genes (PMIDs 26603840, 32131130). In the present study, we employed whole exome sequencing (WES) to assess rare variant contribution to the development of TMA in a hypothesis-driven pathway-specific approach. Methods: In the current case-control genetic association study conducted at Fred Hutchinson Cancer Research Center, we selected 100 patients with a diagnosis of TMA and pre-transplant DNA samples (case definition described previously in PMID 30940363, 33836868). We then performed incidence density sampling to randomly select 100 non-TMA controls after allogeneic HCT matching by age, sex, race, and year of HCT. WES (germline variant detection 40x) was conducted using Illumina NovaSeq. Sequence reads were mapped to hg38 reference genome followed by deduplication and base quality score recalibration. Joint-genotyping was performed to call single nucleotide polymorphism (SNPs) and insertion/deletion (indels) using the GATK v3.3 and Atlas2. Variants were filtered during quality control (QC) and variant quality score recalibration (VQSR) and annotated using ANNOVAR and Ensembl VEP. To optimize signal detection by reducing neutral background variation, we defined qualifying variants as those meeting all 3 criteria: 1) novel or rare variants with a minor allele frequency (MAF) We then focused on the exome profiles of 5 a priori selected genetic pathways: complement regulation (17 genes), VWF and coagulation (7 genes), VWF clearance (10 genes), ADAMTS13 mimics or interacting proteins (10 genes), and angiopoietin family and endothelial activation (7 genes). Pathway-based and gene-based collapsing association tests were performed using the Optimized Sequence Kernel Association (SKAT-O) test as an optimal test combining burden test and SKAT. Results: After joint variant calling, 91 TMA cases and 93 non-TMA controls passed all QC filters (Table 1). Among 1,485 variants detected in the 5 pathways after QC, 60 variants (73 total mutations) were considered as qualifying variants with MAF Conclusion: Contrary to the initial hypothesis, we did not observe pathogenic germline rare variants in the complement regulation pathway in patients with TA-TMA. Instead, we found a significant association in the VWF clearance pathway, particularly that of the LRP1 gene. In recent years, researchers have shown that VWF can bind to and activate complement proteins. Impaired VWF clearance could lead to the higher predisposition for complement activation observed in patients with TA-TMA. Future functional studies are needed to determine the impact of VWF clearance on the pathogenesis of the disease. Figure 1 Figure 1. Disclosures Sartain: Alexon Pharamaceuticals: Membership on an entity's Board of Directors or advisory committees. Lee: Incyte: Research Funding; Janssen: Other; Takeda: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; Kadmon: Research Funding; National Marrow Donor Program: Membership on an entity's Board of Directors or advisory committees; Syndax: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding.

Background: Hematopoietic stem cell transplant (HSCT) is a curative option for patients with high-risk Acute Lymphoblastic Leukemia (HR-ALL), but relapse remains a major cause of treatment failure. Strategies to enhance the graft-versus-leukemia (GVL) effect have been employed to prevent relapse, including modulating immune suppression post-HSCT to hasten immune reconstitution or with the use of donor lymphocyte infusions (DLIs). However, DLIs carry a significant risk of graft-versus-host disease (GVHD) due to the concurrent transfer of alloreactive T cells. To enhance the GVL effect while minimizing GVHD, we developed a protocol for the generation of ex vivo expanded, donor-derived T-cell lines targeting PRAME, WT1 and Survivin - tumor associated antigens that are frequently expressed in both B- and T-cell ALL. These multi-antigen-targeted T cells (multiTAAs) were adoptively transferred to pediatric and adult patients with HR-ALL who had undergone an allogeneic HSCT. Methods: Donor-derived multiTAA-specific T cells were generated by co-culturing PBMCs with autologous DCs loaded with pepmixes (15 mer peptides overlapping by 11 amino acids) spanning all 3 target antigens in the presence of a Th1-polarizing/pro-proliferative cytokine cocktail. Following 2-4 rounds of stimulation these multiTAA-specific T cells were infused to patients with ALL who had undergone an HSCT but remained at a high risk for disease relapse. Results: We have generated 15 clinical grade multiTAA-specific T cell lines comprising CD3+ T cells (mean 95.1±1.9%) with a mixture of CD4+ (mean 22.8±6.3%) and CD8+ (mean 52.5±5.3%) cells, which expressed central [CD45RO+/CD62L+: 13.5±2.8%] and effector memory markers [CD45RO+/CD62L-: 56.4±3.8%]. The expanded lines recognized the targeted antigens PRAME (range 0-370 SFC/2x10 5), WT1 (0-363 SFC/2x10 5), and Survivin (0-65 SFC/2x10 5) in an IFNg ELIspot. None of the lines reacted against non-malignant patient-derived cells (3.7±0.8% specific lysis; E: T 20:1) - a study release criterion indicating lack of alloreactivity. We have infused 11 HR-ALL patients (8 pediatric and 3 adult) with donor-derived multiTAA-specific T cells to prevent disease relapse (Table 1). Patients were administered with up to 4 infusions of cells at 3 escalating dose levels, ranging from 0.5 - 2x10 7 cells/m 2. Infusions were well tolerated with no dose-limiting toxicity, GVHD, cytokine release syndrome or other adverse events. Three patients were not evaluable per study criteria as they received >0.5mg/kg of steroids (2 patients received stress doses for septic shock and 1 for elevated liver enzymes presumed to be GVHD that was later ruled out) within 4 weeks of infusion and were replaced. Six of the 8 remaining patients infused remain in CR on long-term follow up at a median of 46.5 months post-infusion (range 9-51 months). In patients who remained in long term CR we detected an expansion of tumor-reactive T cells in their peripheral blood post-infusion against both targeted (WT1, Survivin, PRAME) and non-targeted antigens (SSX2, MAGE-A4, -A1, -A2B, -C1, MART1, AFP and NYESO1) reflecting epitope and antigen spreading, which correlated temporally (within 4 weeks) with multiTAA infusions. By contrast in the two patients who relapsed we saw no evidence of in vivo T cell amplification within the first 4 weeks after infusion. Conclusion: The preparation and infusion of donor-derived multiTAA-specific T cells to patients with B- and T-ALL post allogeneic HSCT is feasible, safe and as evidenced by in vivo tumor-directed T cell expansion and antigen spreading in patients, may contribute to disease control. This strategy may present a promising addition to current immunotherapeutic approaches for prophylaxis for leukemic relapse in HSCT recipients. Figure 1 Figure 1. Disclosures Vasileiou: Allovir: Consultancy. Tzannou: Gileas: Honoraria; Allovir: Current equity holder in publicly-traded company. Kuvalekar: Allovir: Consultancy. Watanabe: Allovir: Consultancy. Grilley: QB Regulatory Consulting: Other: Ownership, project management support, Research Funding; Marker: Consultancy, Other: Regulatory and project management support; Allovir: Current equity holder in publicly-traded company, Other: Leadership. Hill: Incyte: Membership on an entity's Board of Directors or advisory committees. Omer: Allovir: Research Funding. Gottschalk: Tessa Therapeutics: Consultancy; Immatics: Membership on an entity's Board of Directors or advisory committees; Other: Other: patents and patent applications in the field of cancer cell and gene therapy ; Tidal: Consultancy; Novartis: Consultancy; Catamaran Bio: Consultancy. Heslop: Gilead: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Kuur Therapeutics: Research Funding; GSK: Membership on an entity's Board of Directors or advisory committees; Allovir: Current equity holder in publicly-traded company; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Marker Therapeutics: Current equity holder in publicly-traded company; Fresh Wind Biotherapies: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees. Rooney: Allogene: Patents & Royalties; Bellicum: Patents & Royalties; Bluebird: Current equity holder in publicly-traded company; Allovir: Current equity holder in publicly-traded company; Alimera: Consultancy; Memgen: Consultancy; TScan Therapeutics: Consultancy; Takeda: Patents & Royalties; Marker: Current equity holder in publicly-traded company; Tessa: Consultancy, Other: Leadership, Research Funding. Vera: Allovir: Consultancy, Current equity holder in publicly-traded company, Other: Leadership, travel , accomodations, expenses, Patents & Royalties; Marker: Current Employment, Other: Travel, Accomodations, Expenses, Patents & Royalties, Research Funding. Leen: Allovir: Consultancy, Current equity holder in publicly-traded company; Marker: Current equity holder in publicly-traded company.

Purpose Improvement of cure rates for patients treated with allogeneic hematopoietic stem-cell transplantation (HSCT) will require efforts to decrease treatment-related mortality from severe viral infections. Adoptively transferred virus-specific T cells (VSTs) generated from eligible, third-party donors could provide broad antiviral protection to recipients of HSCT as an immediately available off-the-shelf product. Patient and Methods We generated a bank of VSTs that recognized five common viral pathogens: Epstein-Barr virus (EBV), adenovirus (AdV), cytomegalovirus (CMV), BK virus (BKV), and human herpesvirus 6 (HHV-6). The VSTs were administered to 38 patients with 45 infections in a phase II clinical trial. Results A single infusion produced a cumulative complete or partial response rate of 92% (95% CI, 78.1% to 98.3%) overall and the following rates by virus: 100% for BKV (n = 16), 94% for CMV (n = 17), 71% for AdV (n = 7), 100% for EBV (n = 2), and 67% for HHV-6 (n = 3). Clinical benefit was achieved in 31 patients treated for one infection and in seven patients treated for multiple coincident infections. Thirteen of 14 patients treated for BKV-associated hemorrhagic cystitis experienced complete resolution of gross hematuria by week 6. Infusions were safe, and only two occurrences of de novo graft-versus host disease (grade 1) were observed. VST tracking by epitope profiling revealed persistence of functional VSTs of third-party origin for up to 12 weeks. Conclusion The use of banked VSTs is a feasible, safe, and effective approach to treat severe and drug-refractory infections after HSCT, including infections from two viruses (BKV and HHV-6) that had never been targeted previously with an off-the-shelf product. Furthermore, the multispecificity of the VSTs ensures extensive antiviral coverage, which facilitates the treatment of patients with multiple infections.

Human metapneumovirus (hMPV) is a respiratory virus detected in ≥9% of allogeneic hematopoietic stem cell transplant (HSCT) recipients, in whom it can cause significant morbidity and mortality. Given the lack of effective antivirals, we investigated the potential for immunotherapeutic intervention, using adoptively transferred T cells. Thus, we characterized the cellular immune response to the virus and identified F, N, M2-1, M, and P as immunodominant target antigens. Reactive T cells were polyclonal (ie, they expressed CD4 and CD8), T-helper type 1 polarized, and polyfunctional (ie, they produced interferon γ, tumor necrosis factor α, granulocyte-macrophage colony-stimulating factor, and granzyme B), and they were able to kill autologous antigen-loaded targets. The detection of hMPV-specific T cells in HSCT recipients who endogenously controlled active infections support the clinical importance of T-cell immunity in mediating protective antiviral effects. Our results demonstrate the feasibility of developing an immunotherapy for immunocompromised patients with uncontrolled infections.

Cytomegalovirus (CMV) infections remain a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT), and standard antiviral therapies are associated with significant side effects and development of drug-resistant mutants. Adoptively transferred donor-derived CMV-specific T cells (CMVSTs) can provide an alternative treatment modality with few side effects but are not widely available due to their patient-specific nature. Here we report the establishment and use of a bank of CMVSTs derived from just 8 CMV-seropositive donors, with HLA types representing the diverse US population, as an “off-the-shelf” therapy to treat drug-refractory infections. To date, we have screened 29 patients for study participation and identified a suitable line, with ≥2 of 8 shared HLA antigens, for 28 (96.6%) patients with a median of 4 shared HLA antigens. Of these, 10 patients with persistent/refractory CMV infections or disease were eligible for treatment; a single infusion of cells produced 3 partial responses and 7 complete responses, for a cumulative response rate of 100% (95% confidence interval, 69.2-100) with no graft-versus-host disease, graft failure, or cytokine release syndrome. Potential wider use of the tested CMVSTs across transplant centers is made more feasible by our ability to produce sufficient material to generate cells for >2000 infusions from a single donor collection. Our data indicate that a “mini” bank of CMVSTs prepared from just 8 well-chosen third-party donors can supply the majority of patients with an appropriately matched line that produces safe and effective anti-CMV activity post-HSCT.

Wiskott-Aldrich syndrome (WAS) is an X-linked disease caused by mutations in the WAS gene, leading to thrombocytopenia, eczema, recurrent infections, autoimmune disease, and malignancy. Hematopoietic cell transplantation (HCT) is the primary curative approach, with the goal of correcting the underlying immunodeficiency and thrombocytopenia. HCT outcomes have improved over time, particularly for patients with HLA-matched sibling and unrelated donors. We report the outcomes of 129 patients with WAS who underwent HCT at 29 Primary Immune Deficiency Treatment Consortium centers from 2005 through 2015. Median age at HCT was 1.2 years. Most patients (65%) received myeloablative busulfan-based conditioning. With a median follow-up of 4.5 years, the 5-year overall survival (OS) was 91%. Superior 5-year OS was observed in patients 95%) vs low-level (5%-49%) donor myeloid engraftment. In summary, HCT outcomes for WAS have improved since 2005, compared with prior reports. HCT at a younger age continues to be associated with superior outcomes supporting the recommendation for early HCT. High-level donor myeloid engraftment is important for platelet reconstitution after either myeloablative or busulfan-containing reduced intensity conditioning. (This trial was registered at www.clinicaltrials.gov as #NCT02064933.)

Serious viral infections, due to delayed immune reconstitution, are a leading cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation (HSCT). Thus, many transplant centers prospectively track cellular immune recovery by evaluating absolute cell numbers and the phenotypic profile of reconstituting T cell subsets to identify individuals who are at highest risk of infection. Conventional assessments, however, fail to measure either the antigen specificity or functional capacity of reconstituting cells—both factors that correlate with endogenous antiviral protection. In this pilot study, we sought to address this limitation by prospectively investigating the tempo of endogenous immune reconstitution in a cohort of 23 pediatric HSCT patients using both quantitative (flow cytometry) and qualitative (IFNγ ELISpot) measures, which we correlated with either the presence or absence of infections associated with cytomegalovirus, adenovirus, Epstein-Barr virus, BK virus, human herpes virus 6, respiratory syncytial virus, parainfluenza, influenza, and human metapneumovirus. We present data spanning 12 months post-transplant demonstrating the influence of conditioning on immune recovery and highlighting the differential impact of active viral replication on the quantity and quality of reconstituting cells. Judicious use of standard (phenotypic) and novel (functional) monitoring strategies can help guide the clinical care and personalized management of allogenic HSCT recipients with infections.

IntroductionInflammatory bowel disease (IBD) affects approximately 1/3 of patients with chronic granulomatous disease (CGD). Comprehensive investigation of the effect of allogeneic hematopoietic cell transplantation (HCT) on CGD IBD and the impact of IBD on transplant outcomes is lacking.MethodsWe collected data retrospectively from 145 patients with CGD who had received allogeneic HCT at 26 Primary Immune Deficiency Treatment Consortium (PIDTC) centers between January 1, 2005 and June 30, 2016.ResultsForty-nine CGD patients with IBD and 96 patients without IBD underwent allogeneic HCT. Eighty-nine percent of patients with IBD and 93% of patients without IBD engrafted (p = 0.476). Upper gastrointestinal acute GVHD occurred in 8.5% of patients with IBD and 3.5% of patients without IBD (p = 0.246). Lower gastrointestinal acute GVHD occurred in 10.6% of patients with IBD and 11.8% of patients without IBD (p = 0.845). The cumulative incidence of acute GVHD grades II-IV was 30% (CI 17-43%) in patients with IBD and 20% (CI 12-29%) in patients without IBD (p = 0.09). Five-year overall survival was equivalent for patients with and without IBD: 80% [CI 66-89%] and 83% [CI 72-90%], respectively (p = 0.689). All 33 surviving evaluable patients with a history of IBD experienced resolution of IBD by 2years following allogeneic HCT.ConclusionsIn this cohort, allogeneic HCT was curative for CGD-associated IBD. IBD should not contraindicate HCT, as it does not lead to an increased risk of mortality. This study is registered at clinicaltrials.gov NCT02082353.

Adoptive transfer of virus-specific T cells (VSTs) has been shown to be safe and effective in stem cell transplant recipients. However, the lack of virus-experienced T cells in donor cord blood (CB) has prevented the development of ex vivo expanded donor-derived VSTs for recipients of this stem cell source. Here we evaluated the feasibility and safety of ex vivo expansion of CB T cells from the 20% fraction of the CB unit in pediatric patients receiving a single CB transplant (CBT). In 2 clinical trials conducted at 2 separate sites, we manufactured CB-derived multivirus-specific T cells (CB-VSTs) targeting Epstein-Barr virus (EBV), adenovirus, and cytomegalovirus (CMV) for 18 (86%) of 21 patients demonstrating feasibility. Manufacturing for 2 CB-VSTs failed to meet lot release because of insufficient cell recovery, and there was 1 sterility breach during separation of the frozen 20% fraction. Delayed engraftment was not observed in patients who received the remaining 80% fraction for the primary CBT. There was no grade 3 to 4 acute graft-versus-host disease (GVHD) associated with the infusion of CB-VSTs. None of the 7 patients who received CB-VSTs as prophylaxis developed end-organ disease from CMV, EBV, or adenovirus. In 7 patients receiving CB-VSTs for viral reactivation or infection, only 1 patient developed end-organ viral disease, which was in an immune privileged site (CMV retinitis) and occurred after steroid therapy for GVHD. Finally, we demonstrated the long-term persistence of adoptively transferred CB-VSTs using T-cell receptor-Vβ clonotype tracking, suggesting that CB-VSTs are a feasible addition to antiviral pharmacotherapy.

To test the feasibility of a single T-cell manipulation to eliminate alloreactivity while sparing antiviral and antitumor T cells, we infused 12 haploidentical hematopoietic stem cell transplant patients with increasing numbers of alloreplete haploidentical T cells expressing the inducible caspase 9 suicide gene (iC9-T cells). We determined whether the iC9-T cells produced immune reconstitution and if any resultant graft-versus-host disease (GVHD) could be controlled by administration of a chemical inducer of dimerization (CID; AP1903/Rimiducid). All patients receiving >10(4) alloreplete iC9-T lymphocytes per kilogram achieved rapid reconstitution of immune responses toward 5 major pathogenic viruses and concomitant control of active infections. Four patients received a single AP1903 dose. CID infusion eliminated 85% to 95% of circulating CD3(+)CD19(+) T cells within 30 minutes, with no recurrence of GVHD within 90 days. In one patient, symptoms and signs of GVHD-associated cytokine release syndrome (CRS-hyperpyrexia, high levels of proinflammatory cytokines, and rash) resolved within 2 hours of AP1903 infusion. One patient with varicella zoster virus meningitis and acute GVHD had iC9-T cells present in the cerebrospinal fluid, which were reduced by >90% after CID. Notably, virus-specific T cells recovered even after AP1903 administration and continued to protect against infection. Hence, alloreplete iC9-T cells can reconstitute immunity posttransplant and administration of CID can eliminate them from both peripheral blood and the central nervous system (CNS), leading to rapid resolution of GVHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes. This trial was registered at www.clinicaltrials.gov as #NCT01494103.

The HLH-2004 criteria are used to diagnose hemophagocytic lymphohistiocytosis (HLH), yet concern exists for their misapplication, resulting in suboptimal treatment of some patients. We sought to define the genomic spectrum and associated outcomes of a diverse cohort of children who met the HLH-2004 criteria. Genetic testing was performed clinically or through research-based whole-exome sequencing. Clinical metrics were analyzed with respect to genomic results. Of 122 subjects enrolled over the course of 17 years, 101 subjects received genetic testing. Biallelic familial HLH (fHLH) gene defects were identified in only 19 (19%) and correlated with presentation at younger than 1 year of age (P < .0001). Digenic fHLH variants were observed but lacked statistical support for disease association. In 28 (58%) of 48 subjects, research whole-exome sequencing analyses successfully identified likely molecular explanations, including underlying primary immunodeficiency diseases, dysregulated immune activation and proliferation disorders, and potentially novel genetic conditions. Two-thirds of patients identified by the HLH-2004 criteria had underlying etiologies for HLH, including genetic defects, autoimmunity, and malignancy. Overall survival was 45%, and increased mortality correlated with HLH triggered by infection or malignancy (P < .05). Differences in survival did not correlate with genetic profile or extent of therapy. HLH should be conceptualized as a phenotype of critical illness characterized by toxic activation of immune cells from different underlying mechanisms. In most patients with HLH, targeted sequencing of fHLH genes remains insufficient for identifying pathogenic mechanisms. Whole-exome sequencing, however, may identify specific therapeutic opportunities and affect hematopoietic stem cell transplantation options for these patients.

The Primary Immune Deficiency Treatment Consortium (PIDTC) is enrolling children with severe combined immunodeficiency (SCID) to a prospective natural history study. We analyzed patients treated with allogeneic hematopoietic cell transplantation (HCT) from 2010 to 2014, including 68 patients with typical SCID and 32 with leaky SCID, Omenn syndrome, or reticular dysgenesis. Most (59%) patients were diagnosed by newborn screening or family history. The 2-year overall survival was 90%, but was 95% for those who were infection-free at HCT vs 81% for those with active infection (P = .009). Other factors, including the diagnosis of typical vs leaky SCID/Omenn syndrome, diagnosis via family history or newborn screening, use of preparative chemotherapy, or the type of donor used, did not impact survival. Although 1-year post-HCT median CD4 counts and freedom from IV immunoglobulin were improved after the use of preparative chemotherapy, other immunologic reconstitution parameters were not affected, and the potential for late sequelae in extremely young infants requires additional evaluation. After a T-cell-replete graft, landmark analysis at day +100 post-HCT revealed that CD3 < 300 cells/μL, CD8 < 50 cells/μL, CD45RA < 10%, or a restricted Vβ T-cell receptor repertoire (

Summary In areas without newborn screening for severe combined immunodeficiency (SCID), disease-defining infections may lead to diagnosis, and in some cases, may not be identified prior to the first year of life. We describe a female infant who presented with disseminated vaccine-acquired varicella (VZV) and vaccine-acquired rubella infections at 13 months of age. Immunological evaluations demonstrated neutropenia, isolated CD4 lymphocytopenia, the presence of CD8+T cells, poor lymphocyte proliferation, hypergammaglobulinaemia and poor specific antibody production to VZV infection and routine immunizations. A combination of whole exome sequencing and custom-designed chromosomal microarray with exon coverage of primary immunodeficiency genes detected compound heterozygous mutations (one single nucleotide variant and one intragenic copy number variant involving one exon) within the IL7R gene. Mosaicism for wild-type allele (20–30%) was detected in pretransplant blood and buccal DNA and maternal engraftment (5–10%) demonstrated in pretransplant blood DNA. This may be responsible for the patient's unusual immunological phenotype compared to classical interleukin (IL)-7Rα deficiency. Disseminated VZV was controlled with anti-viral and immune-based therapy, and umbilical cord blood stem cell transplantation was successful. Retrospectively performed T cell receptor excision circle (TREC) analyses completed on neonatal Guthrie cards identified absent TREC. This case emphasizes the danger of live viral vaccination in severe combined immunodeficiency (SCID) patients and the importance of newborn screening to identify patients prior to high-risk exposures. It also illustrates the value of aggressive pathogen identification and treatment, the influence newborn screening can have on morbidity and mortality and the significant impact of newer genomic diagnostic tools in identifying the underlying genetic aetiology for SCID patients.

Human phosphoglucomutase 3 (PGM3) catalyzes the conversion of N-acetyl-glucosamine (GlcNAc)-6-phosphate into GlcNAc-1-phosphate during the synthesis of uridine diphosphate (UDP)-GlcNAc, a sugar nucleotide critical to multiple glycosylation pathways. We identified three unrelated children with recurrent infections, congenital leukopenia including neutropenia, B and T cell lymphopenia, and progression to bone marrow failure. Whole-exome sequencing demonstrated deleterious mutations in PGM3 in all three subjects, delineating their disease to be due to an unsuspected congenital disorder of glycosylation (CDG). Functional studies of the disease-associated PGM3 variants in E. coli cells demonstrated reduced PGM3 activity for all mutants tested. Two of the three children had skeletal anomalies resembling Desbuquois dysplasia: short stature, brachydactyly, dysmorphic facial features, and intellectual disability. However, these additional features were absent in the third child, showing the clinical variability of the disease. Two children received hematopoietic stem cell transplantation of cord blood and bone marrow from matched related donors; both had successful engraftment and correction of neutropenia and lymphopenia. We define PGM3-CDG as a treatable immunodeficiency, document the power of whole-exome sequencing in gene discoveries for rare disorders, and illustrate the utility of genomic analyses in studying combined and variable phenotypes.

Adoptive transfer of donor-derived T lymphocytes expressing a safety switch may promote immune reconstitution in patients undergoing haploidentical hematopoietic stem cell transplant (haplo-HSCT) without the risk for uncontrolled graft versus host disease (GvHD). Thus, patients who develop GvHD after infusion of allodepleted donor-derived T cells expressing an inducible human caspase 9 (iC9) had their disease effectively controlled by a single administration of a small-molecule drug (AP1903) that dimerizes and activates the iC9 transgene. We now report the long-term follow-up of 10 patients infused with such safety switch-modified T cells. We find long-term persistence of iC9-modified (iC9-T) T cells in vivo in the absence of emerging oligoclonality and a robust immunologic benefit, mediated initially by the infused cells themselves and subsequently by an apparently accelerated reconstitution of endogenous naive T lymphocytes. As a consequence, these patients have immediate and sustained protection from major pathogens, including cytomegalovirus, adenovirus, BK virus, and Epstein-Barr virus in the absence of acute or chronic GvHD, supporting the beneficial effects of this approach to immune reconstitution after haplo-HSCT. This study was registered at www.clinicaltrials.gov as #NCT00710892.

If hemophagocytic lymphohisticytosis (HLH)–causing mutations are found in a well child, should that patient receive a bone marrow transplant? In this issue of Blood, Lucchini et al have collected a unique subset of patients with biallelic mutations causing HLH and provide an important recommendation for the management of asymptomatic carriers (ACs) with the same mutation as affected siblings, index cases (ICs).1

Introduction: Hematopoietic stem cell transplantation (HSCT) for patients with hemophagocytic lymphohistiocytosis (HLH) following myeloablative conditioning regimens (MAC) is associated with high rates of non-relapse mortality. A previously reported prospective, phase 2 multi-center trial (RICHI) using using RIC strategy of fludarabine, melphalan and alemtuzumab (day -14) for HLH and primary immune deficiency syndromes (PIDS) demonstrated improved mortality rates but fewer than half the patients with HLH (41%) successfully engrafted without secondary graft failure, need for donor lymphocyte infusion (DLI) or second transplant. Incorporation of thiotepa during conditioning has been to shown to be safe and improve engraftment. We report the results of a retrospective analysis of nine consecutive patient treated with the inclusion of thiotepa into the RICHI backbone (RICHI+TT). Methods: Patients received a single additional dose of thiotepa 10mg/kg on day -3 added to the fludarabine/melphalan/alemtuzumab backbone (RICHI+TT) with the same graft-versus-host disease prophylaxis of methyprednisolone through day +28 and cyclosporine through day 180. To determine sustained engraftment, we used the same parameters the RICHI study defined as > 5 % donor chimerism without any intervention and alive at 1 year post-transplant. Results: Our cohort consisted of 8 males and 1 female with a median age of 7 years (range 1-18 years). Seven patients had HLH with proven pathogenic genetic mutations (biallelic PRF1 - 2, UNC13D - 2, STXBP1- 1, RAB27A-1, STAT3 gain of function-1), while the other 2 patients had HLH without identified pathogenic mutations (1- chronic active EBV, 1- juvenile idiopathic arthritis with refractory macrophage activation syndrome).The majority of patients received a bone marrow product (n = 8), one patient received a peripheral blood stem cell product; 6 patients received a graft from a matched related donor , two from a mismatched unrelated donor, and one from a matched unrelated donor. All patients engrafted at a median of 15 days post-transplant (8 patients at 100% donor chimera; 1 patient at 99% donor chimera at initial engraftment). Six of the 9 patients were evaluable to assess donor chimerism at 1 year as per study definitions with a median follow up of 875 days (range: 366 -1000 days). All 6 patients had > 5% donor chimerism and were alive at 1 year. Five of the 6 evaluable patients met criteria for sustained donor engraftment without need for intervention and all maintained 100% donor chimerism at last follow-up (Table 1). Only one of the six patients had evidence of falling donor chimerism; this stabilized at 40% donor chimerism after DLI. No patients had primary or secondary graft failure. Three patients were not evaluable for long-term assessment due to death prior to 1 year. Six of the 9 patients described here are alive and disease-free with stable long-term engraftment. The incorporation of Thiotepa to the RICHI backbone improved on previously reported sustained donor engraftment (Table 2). Conclusions: The RICHI+TT approach had better long-term donor engraftment with a decreased need for DLI or second transplant without increased rates of non-relapse mortality. Prospective studies are needed to determine the optimal treatment strategies for patients with HLH who require HSCT for cure. Disclosures Heslop: Cell Medica: Research Funding; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Marker Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Allovir: Equity Ownership; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees.

High risk or relapsed pediatric acute myeloid leukemia (AML) is potentially curable with allogeneic hematopoietic stem cell transplantation (HSCT). The use of a matched related donor (MRD) is the standard of care for these patients, but in the majority of patients a MRD is not available. Significant improvements have been made with supportive care practices, but there are limited outcome data for AML in pediatric patients transplanted in the contemporary era. We report retrospective results of HSCT in eighty-seven pediatric patients with high risk or relapsed AML performed at a single institution between 2008 and 2018. The median age at the time of HSCT was 8 years old (range: 0.6-20 years). More than 2/3rd of patients were in remission at time of HSCT with 43 patients (49%) transplanted in CR1 and 18 patients (21%) in CR2. Twenty six patients (30%) had active disease (defined as any evidence of disease) at the time of HSCT. Patients were transplanted using a matched related donor (MRD, n=21), a matched unrelated donor (MUD, n=25), a mismatched unrelated donor (MMUD, n=15), a haplo-identical donor (haplo, n=12) or an umbilical cord blood (UCB, n=14) graft. The majority of patients (78/87, 90%) received myeloablative conditioning while 9 patients (10%) received reduced intensity conditioning regimens. MRD and UCB graft recipients did not receive serotherapy. MUD, MMUD and haplo graft recipients (n=52) received serotherapy, the majority of whom received an alemtuzumab-based regimen, 42/52 (81%). Haplo-identical transplants were performed using CD34+ selected grafts. Three-year overall survival (OS) and disease free survival (DFS) for the entire cohort were 52% (95% CI: 41-62%) and 49% (95% CI: 38-59%) respectively. OS differed significantly according to disease status at time of transplant (p=0.018), with 3 year OS: 60%, (95% CI: 43-74%) for patients in CR1, 56%, (95% CI: 31-75%) for patients in CR2 and 34%, (95% CI: 17-52%) for patients with active disease. OS differed by donor type (p=0.058). OS was comparable for patients with MRD (71%, 95% CI: 46-86%) and 10/10 MUD (59%, 95% CI: 37-75%) (p=0.67). Patients with alternate donor sources had worse outcomes (MMUD 33%, 95% CI: 10-59%; UCB 43%, 95% CI: 18-66%; Haplo 33%, 95% CI: 10-59%) as compared to MRD (MMUD vs MRD, p=0.042, UCB vs MRD, p=0.046, haplo vs MRD p=0.017). There was a low incidence of Grade III-IV GVHD (8%, n=9) and extensive chronic GVHD (9%, n=7) likely secondary to the use of alemtuzumab-based conditioning regimens for MUD, MMUD and haplo donors. The 3-year non-relapse mortality was low at 11%, (95% CI: 5-19%). The primary cause of death was relapse, with a 3-year cumulative incidence of relapse of 40%, (95% CI: 30-50%). Patients with active disease at transplant had significantly higher cumulative incidence of relapse (sHR: 2.42, 95% CI: 1.18-4.95, p=0.016) compared to patients in remission. There was no difference in cumulative incidence of relapse between patients in CR1 and CR2 (p=0.598). In the contemporary era, outcomes after allogeneic HSCT are comparable for MRD and 10/10 MUD for pediatric AML. Myeloablative conditioning regimens that incorporated alemtuzumab were well tolerated with low rates of NRM and very low rates of acute and chronic GVHD. Relapse remains the major cause of treatment failure and future trials evaluating use of cellular therapies and targeted agents post-HSCT will be needed to improve current rates of relapse. Disclosures No relevant conflicts of interest to declare.

Adoptive transfer of virus-specific T cells can prevent and treat serious infections with Epstein-Barr virus (EBV), cytomegalovirus (CMV), and adenovirus (Adv) after allogeneic hematopoietic stem cell transplant. It has, however, proved difficult to make this approach widely available since infectious virus and viral vectors are required for T cell activation, followed by an intensive and prolonged culture period extending over several months. We now show that T cells targeting a range of viral antigens derived from EBV, CMV, and Adv can be reproducibly generated in a single culture over a 2-3-week period, using methods that exclude all viral components and employ a much-simplified culture technology. When administered to recipients of haploidentical (n = 5), matched unrelated (n = 3), mismatched unrelated (n = 1) or matched related (n = 1) transplants with active CMV (n = 3), Adv (n = 1), EBV (n = 2), EBV+Adv (n = 2) or CMV+Adv (n = 2) infections, the cells produced complete virological responses in 80%, including all patients with dual infections. In each case, a decrease in viral load correlated with an increase in the frequency of T cells directed against the infecting virus(es); both immediate and delayed toxicities were absent. This approach should increase both the feasibility and applicability of T cell therapy. The trial was registered at www.clinicaltrials.gov as NCT01070797.

Transplantation of bone marrow cells leads to engraftment of osteopoietic and hematopoietic progenitors. We sought to determine whether the recently described transient expansion of the host osteoblastic niche after marrow radioablation promotes engraftment of both osteopoietic and hematopoietic progenitor cells. Mice infused with marrow cells 24 hours after total body irradiation (TBI) demonstrated significantly greater osteopoietic and hematopoietic progenitor chimerism than did mice infused at 30 minutes or 6 hours. Irradiated mice with a lead shield over 1 hind limb showed greater hematopoietic chimerism in the irradiated limb than in the shielded limb at both the 6- and 24-hour intervals. By contrast, the osteopoietic chimerism was essentially equal in the 2 limbs at each of these intervals, although it significantly increased when cells were infused 24 hours compared with 6 hours after TBI. Similarly, the number of donor phenotypic long-term hematopoietic stem cells was equivalent in the irradiated and shielded limbs after each irradiation-to-infusion interval but was significantly increased at the 24-hour interval. Our findings indicate that a 24-hour delay in marrow cell infusion after TBI facilitates expansion of the endosteal osteoblastic niche, leading to enhanced osteopoietic and hematopoietic engraftment.

Summary Chronic granulomatous disease (CGD) patients are highly susceptible to invasive aspergillosis and might benefit from aspergillus-specific T cell immunotherapy, which has shown promise in treating those with known T cell defects such as haematopoietic stem cell transplant (HSCT) recipients. But whether such T cell defects contribute to increased risks for aspergillus infection in CGD is unclear. Hence, we set out to characterize the aspergillus-specific T cell response in CGD. In murine CGD models and in patients with CGD we showed that the CD4+ T cell responses to aspergillus were unimpaired: aspergillus-specific T cell frequencies were even elevated in CGD mice (P

Cellular therapies could play a role in cancer treatment and regenerative medicine if it were possible to quickly eliminate the infused cells in case of adverse events. We devised an inducible T-cell safety switch that is based on the fusion of human caspase 9 to a modified human FK-binding protein, allowing conditional dimerization. When exposed to a synthetic dimerizing drug, the inducible caspase 9 (iCasp9) becomes activated and leads to the rapid death of cells expressing this construct.We tested the activity of our safety switch by introducing the gene into donor T cells given to enhance immune reconstitution in recipients of haploidentical stem-cell transplants. Patients received AP1903, an otherwise bioinert small-molecule dimerizing drug, if graft-versus-host disease (GVHD) developed. We measured the effects of AP1903 on GVHD and on the function and persistence of the cells containing the iCasp9 safety switch.Five patients between the ages of 3 and 17 years who had undergone stem-cell transplantation for relapsed acute leukemia were treated with the genetically modified T cells. The cells were detected in peripheral blood from all five patients and increased in number over time, despite their constitutive transgene expression. A single dose of dimerizing drug, given to four patients in whom GVHD developed, eliminated more than 90% of the modified T cells within 30 minutes after administration and ended the GVHD without recurrence.The iCasp9 cell-suicide system may increase the safety of cellular therapies and expand their clinical applications. (Funded by the National Heart, Lung, and Blood Institute and the National Cancer Institute; ClinicalTrials.gov number, NCT00710892.).

Background: Autoimmune hemolytic anemia is a well-recognized complication of hematopoietic stem cell transplant (HSCT). While occurring in only ~2-6% of pediatric patients receiving HSCT, it is associated with significant morbidity and mortality of up to 50%. Post-transplant AIHA is poorly studied due to small patient numbers. Therefore, risk factors for the development of post-transplant AIHA are not well delineated and optimal treatment strategies are not known. Disease course is often refractory and multiply relapsing, with current treatment approaches remaining empiric. The most commonly utilized immunomodulatory therapies include glucocorticoids, intravenous immune globulin (IVIG), and rituximab; and potentially repeat bone marrow transplantation. Better understanding of the underlying immunobiology in post-transplant AIHA is imperative, as it may uncover the potential for newer, more effective targeted treatment options and better elucidate pre-disposing risk factors which could be modified for future HSCT patients, resulting in overall improvement in clinical outcomes among this group of high-risk patients. Objective: Through retrospective chart review of a large cohort of pediatric patients developing post-transplant AIHA, we aim to better describe the clinical characteristics, treatment responses, and long-term outcomes of patients with this rare and poorly-studied HSCT complication in order to highlight important areas for future research. Furthermore, we aim to describe correlating immunobiology in a sub-population of patients experiencing refractory AIHA post-HSCT to elucidate potential therapeutic targets and identify peri-transplant immune modulation modifications which may be beneficial in preventing this complication. Methods: We completed a retrospective chart review including patients aged 0 - 18 with post-transplant AIHA over a recent 6-year period (2012 - 2018) at a large pediatric transplant center. Potential patients were identified via query of the electronic medical record (EMR), and further stratified according to eligibility criteria for further full review. AIHA was defined as the presence of hemolytic anemia (as evidenced by at least 2 of the following parameters: elevated LDH, elevated bilirubin, low haptoglobin, reticulocytosis, or evidence of hemolysis on the peripheral smear) and positive Coombs or Direct Antiglobulin Test (DAT). Patients diagnosed with autoimmune hemolytic anemia prior to first HSCT or found to have an alternate etiology of hemolytic anemia were excluded. Eight patients from among this cohort of post-transplant AIHA patients were enrolled in a biology study with the aim of evaluating T cell recovery in this population as compared to that in matched post-transplant control patients in whom AIHA did not occur. T cell subsets, including sub-analysis of CD4+ T helper cells, CD8+ cytotoxic T cells, and FoxP3+CD25+ T regulatory cells, were measured at monthly time-points following enrollment. Both studies were conducted in accordance with institutional IRB-approved protocols. Results: Thirty-six pediatric patients with post-transplant AIHA were identified (median age 6.3 years). Median time to the onset of AIHA following HSCT was 188 days (range 37 - 563 days). Among patients treated for AIHA, 48% had documented disease resolution, while 52% did not achieve resolution of AIHA and had an average disease duration of 822 days. Mean duration of disease for all those achieving resolution of post-transplant AIHA was 472 days. Importantly, mortality among our cohort of post-transplant AIHA patients was 33%. Among the sub-population of patients in whom phenotypic immune reconstitution was monitored, we demonstrated a trend toward statistically significantly reduced T regulatory cell populations in post-transplant AIHA patients as compared to matched post-transplant control patients (Figure 1). Conclusions: Autoimmune hemolytic anemia following HSCT is characterized by a refractory and relapsing course and high associated morbidity and mortality rates with limited effective therapeutic options. Impaired immune reconstitution following HSCT, specifically delayed T regulatory cell re-population, may be implicated in the complex immunopathology of post-transplant AIHA development and persistence, thereby representing an important area for future research and potential therapeutic targeting. Disclosures No relevant conflicts of interest to declare.

Introduction: Matched related donor (MRD) hematopoietic cell transplant (HCT) is an accepted treatment for sickle cell disease (SCD) despite risk of graft-versus-host disease and graft rejection. Alemtuzumab is a potent lymphocytic medication that can reduce the risk of GVHD; however, it is associated with mixed donor chimerism (MDC) and graft loss when used with submyeloablative conditioning. We explored the use of myeloablative chemotherapy in conjunction with alemtuzumab in MRD HCT for SCD as a method to concurrently prevent GVHD and promote durable engraftment. Methods: We retrospectively reviewed engraftment, GVHD and survival for patients that underwent MRD HCT for SCD at Texas Children's Hospital between 2003-2017. All patients received busulfan dose adjusted to achieve target area under the curve of 800-1200μM per minute with doses administered intravenously every 6 hours for 4 days and cyclophosphamide 50mg/kg daily for 4 days. GVHD prophylaxis included intravenous alemtuzumab daily for 3-4 doses starting on day -5 (5-15kg=3mg, 15-30kg=5mg, >30kg= 10mg daily), methotrexate and a calcineurin inhibitor. Donor chimerism was evaluated via short tandem repeats (STR) or fluorescent-in-situ hybridization (FISH) of nucleated cells from the peripheral blood. If MDC was detected, follow-up chimerism studies were obtained every 1-4 weeks until stabilization on 2 or more consecutive evaluations. Persistent MDC was defined by the presence of recipient cells on 2 consecutive evaluations without return to full donor chimerism at last follow-up. Results: Thirty-eight consecutive patients underwent MRD transplant (3 non-sibling and 35 sibling) with a median age at transplant of 8.6 yrs (range: 2.9-18.4yr). Stem cell source consisted of bone marrow grafts for all patients. Two patients concurrently received cord blood from the same donor to augment cell dose. Neutrophil engraftment was achieved in all patients at a median time of 19 days (range: 13-24 days). Incidence of persistent MDC was 62.8% with a median last chimerism of 94% donor cells (range: 24-100%). Three of 22 patients (13.6%) with persistent MDC had Conclusions: Myeloablative conditioning was well tolerated in this patient population, and the addition of alemtuzumab minimized occurrence of severe GVHD. While MDC was observed, chimerism stabilized at >50% donor cells in most patients and no graft rejection or recurrence of SCD occurred with a median follow-up of 2.9 yrs. The use of this regimen may be a promising approach to achieve low rates of GVHD while maintaining low rates of transplant related complications for patients with SCD that can tolerate myeloablative chemotherapy. Disclosures No relevant conflicts of interest to declare.

Mesenchymal stromal cells (MSCs) have enormous potential for the regeneration of bone, cartilage, and other tissues derived from primitive mesoderm. Despite extensive research, there is still no single marker that reliably identifies MSCs within the bone marrow. Using immunocytochemistry and flow cytometry, we demonstrate here that the neural ganglioside GD2 is expressed by MSCs either newly isolated from bone marrow or expanded in tissue culture; this finding was supported by reverse transcriptase–polymerase chain reaction (RT-PCR) analysis showing expression of the mRNA for GD2 synthase, an essential enzyme for GD2 biosynthesis. GD2 was also expressed on MSCs isolated from adipose tissue, but not on foreskin fibroblasts. Importantly, MSCs were the only cells within normal marrow that expressed this marker. Thus, GD2 appears to be the first reported single surface marker that uniquely distinguishes MSCs from other marrow elements. GD2 may prove valuable to study MSC biology and for the preparation of MSCs for clinical applications.

Background Viral infections are a leading fatal complication for patients with primary immunodeficiencies (PIDs) who require hematopoietic stem cell transplantation (HSCT). Use of virus-specific T lymphocytes (VSTs) has been successful for the treatment and prevention of viral infections after HSCT for malignant and nonmalignant conditions. Here we describe the clinical use of VSTs in patients with PIDs at 4 centers. Objective We sought to evaluate the safety and efficacy of VSTs for treatment of viral infections in patients with PIDs. Methods Patients with PIDs who have received VST therapy on previous or current protocols were reviewed in aggregate. Clinical information, including transplantation details, viral infections, and use of antiviral and immunosuppressive pharmacotherapy, were evaluated. Data regarding VST production, infusions, and adverse reactions were compared. Results Thirty-six patients with 12 classes of PID diagnoses received 37 VST products before or after HSCT. Twenty-six (72%) patients had received a diagnosis of infection with cytomegalovirus, EBV, adenovirus, BK virus, and/or human herpesvirus 6. Two patients were treated before HSCT because of EBV-associated lymphoproliferative disease. Partial or complete responses against targeted viruses occurred in 81% of patients overall. Time to response varied from 2 weeks to 3 months (median, 28 days). Overall survival at 6 months after therapy was 80%. Four patients had graft-versus-host disease in the 45 days after VST infusion, which in most cases was therapy responsive. Conclusion VSTs derived from either stem cell donors or third-party donors are likely safe and effective for the treatment of viral infections in patients with PIDs.

Haploidentical hematopoietic stem cell transplantation (haplo-HSCT) is an effective therapeutic strategy for transplant candidates lacking an MHC-matched donor. Removal of T cells from the graft is required, however, to prevent lethal graft-versus-host disease (GvHD). Removal of all T cells increases the risk of graft rejection, relapse, and viral and other opportunistic infections. Several strategies have been used to overcome these barriers, but it is difficult to develop a single T-cell manipulation that both eliminates alloreactivity and spares T cells representing all the available anti-viral and anti-tumor specificities in the donor's peripheral blood. We determined whether alloreplete haploidentical T cells expressing the human caspase-9 (iC9) safety gene can produce immune reconstitution in patients undergoing haplo-HSCT, and whether any resultant GvHD from these alloreplete cells can be controlled by administration of a dimerizing drug (AP1903). After transplant, patients were infused with iC9-T cells using a dose-escalation schedule from 1×104 cells/kg to 5×106 cells/kg. We measured the subsequent immune reconstitution of iC9-T cell recipients. Subjects received dimerizing drug (AP1903) if they developed GvHD. Here we reported ten patients (median age, 13 years; range, 3 – 50 years) who received increasing numbers of alloreplete donor-T cells expressing iC9 (iC9-T). All patients receiving >104 alloreplete iC9 T-lymphocytes/kg engrafted with these cells. There was rapid reconstitution of immune responses toward EBV, CMV, HHV6, VZV and BK viruses, and concomitant control of any active infections. Four patients received a single dose of AP1903, and 85-95% of circulating Cd3+CD19+ T cells were eliminated within 30 minutes of dimerizer infusion, with no recurrence of GvHD within 90 days. In one of these patients with an associated cytokine release syndrome (CRS- hyperpyrexia, high levels of pro-inflammatory cytokines and rash), symptoms and signs of this disorder resolved within 2 hours of dimerizer infusion. One patient with VZV meningitis and acute GvHD had iC9-T cells present in the CSF which were reduced by >90% after dimerizing drug. Notably, virus-specific T cells recovered even after administration of AP1903 and continued to protect the patients against infection. Hence alloreplete iC9-T cells can reconstitute immunity post-transplant and can be eliminated both from peripheral blood and CNS by administration of dimerizing drug, leading to a rapid resolution of GvHD and CRS. The approach may therefore be useful for the rapid and effective treatment of toxicities associated with infusion of engineered T lymphocytes.

Adoptive transfer of cytomegalovirus (CMV)-specific T cells derived from adult seropositive donors can effectively restore antiviral immunity after transplantation. However, CMV-seronegative donors lack CMV-specific memory T cells, which restricts the availability of virus-specific T cells for immunoprophylaxis. We demonstrate the feasibility of deriving CMV-specific T cells from naïve cells for T cell therapy. Naïve T cells primed to recognize CMV were restricted to different, atypical epitopes than T cells derived from CMV-seropositive individuals; however, these two cell populations had similar avidities. CMV-seropositive individuals also had T cells recognizing these atypical epitopes, but these cells had a lower avidity than those derived from the seronegative subjects, which suggests that high-avidity T cells to these epitopes may be lost over time. Indeed, recipients of cord blood (CB) grafts who did not develop CMV were found by clonotypic analysis to have T cells recognizing atypical CMVpp65 epitopes. Therefore, we examined unmanipulated CB units and found that T cells with T cell receptors restricted by atypical epitopes were the most common, which may explain why these T cells expanded. When infused to recipients, naïve donor-derived virus-specific T cells that recognized atypical epitopes were associated with prolonged periods of CMV-free survival and complete remission. These data suggest that naïve-derived T cells from seronegative patients may be an additional source of cells for CMV immunoprophylaxis.

It remains difficult to treat the multiplicity of distinct viral infections that afflict immunocompromised patients. Adoptive transfer of virus-specific T cells (VSTs) can be safe and effective, but such cells have been complex to prepare and limited in antiviral range. We now demonstrate the feasibility and clinical utility of rapidly generated single-culture VSTs that recognize 12 immunogenic antigens from five viruses (Epstein-Barr virus, adenovirus, cytomegalovirus, BK virus, and human herpesvirus 6) that frequently cause disease in immunocompromised patients. When administered to 11 recipients of allogeneic transplants, 8 of whom had up to four active infections with the targeted viruses, these VSTs proved safe in all subjects and produced an overall 94% virological and clinical response rate that was sustained long-term.

Background The approach to the diagnosis of severe combined immunodeficiency disease (SCID) and related disorders varies among institutions and countries. Objectives The Primary Immune Deficiency Treatment Consortium attempted to develop a uniform set of criteria for diagnosing SCID and related disorders and has evaluated the results as part of a retrospective study of SCID in North America. Methods Clinical records from 2000 through 2009 at 27 centers in North America were collected on 332 children treated with hematopoietic stem cell transplantation (HCT), enzyme replacement therapy, or gene therapy for SCID and related disorders. Eligibility for inclusion in the study and classification into disease groups were established by using set criteria and applied by an expert review group. Results Two hundred eighty-five (86%) of the patients were determined to be eligible, and 47 (14%) were not eligible. Of the 285 eligible patients, 84% were classified as having typical SCID; 13% were classified as having leaky SCID, Omenn syndrome, or reticular dysgenesis; and 3% had a history of enzyme replacement or gene therapy. Detection of a genotype predicting an SCID phenotype was accepted for eligibility. Reasons for noneligibility were failure to demonstrate either impaired lymphocyte proliferation or maternal T-cell engraftment. Overall (n = 332) rates of testing were as follows: proliferation to PHA, 77%; maternal engraftment, 35%; and genotype, 79% (mutation identified in 62%). Conclusion Lack of complete laboratory evaluation of patients before HCT presents a significant barrier to definitive diagnosis of SCID and related disorders and prevented inclusion of subjects in our observational HCT study. This lesson is critical for patient care, as well as the design of future prospective treatment studies for such children because a well-defined and consistent study population is important for precision in outcomes analysis. © 2013 American Academy of Allergy, Asthma & Immunology.

Despite advances in antiviral drugs, Cytomegalovirus (CMV) infections remain a significant cause of morbidity and mortality in immunocompromised individuals. We have recently demonstrated in hematopoietic stem cell transplant (HSCT) recipients that adoptively-transferred virus-specific T cells, generated from healthy 3rd party donors and administered as an "ready to administer" product, can be curative, even in patients with drug-refractory CMV infections. However, broader implementation has been hindered by the postulated need for extensive panels of T cell lines representing a diverse HLA profile, as well as the complexities of large scale manufacturing for widespread clinical application. To address these potential issues, we have developed a decision tool that identified a short list of donors who provide HLA coverage for >90% of the stem cell transplant population. Furthermore, to generate banks of CMV-specific T cells from these donors, we have created a simple, robust, and linearly scalable manufacturing process. To determine whether these advances would enable the widespread application of "ready to administer" T cells, we generated CMV cell banks (Viralym-C™) from 9 healthy donors selected by our decision tool, and initiated a fixed-dose (2x107 cells/m2) Phase I clinical trial for the treatment of drug-refractory CMV infections in pediatric and adult HSCT recipients. To generate the Viralym-C™ banks, we stimulated donor peripheral blood mononuclear cells (PBMCs) with overlapping peptide libraries spanning the immunodominant CMV antigens pp65 and IE1. Cells were subsequently expanded in a G-Rex device, resulting in a mean fold expansion of 103±12. The lines were polyclonal, comprising both CD4+ (21.3±6.7%) and CD8+ (74.8±6.9%) T cells, and expressed central CD45RO+/CD62L+ (58.5±4.2%) and effector memory markers CD45RO+/CD62L- (35.3±12.2%). Furthermore, the lines generated were specific for the target antigens (IE1: 419±100; pp65 1070±31 SFC/2x105, n=9). To date, we have screened 12 patients for study participation, and from our bank of just 9 lines we have successfully identified a suitable line for all patients within 24 hours. Of these, 6 patients have been infused; 5 received a single infusion and 1 patient required 2 infusions for sustained benefit. There were no immediate infusion-related toxicities; and despite the HLA disparity between the Viralym-C lines and the patients infused, there were no cases of de novo or recurrent graft versus host disease (GvHD). One patient developed a transient fever a few hours post-infusion, which spontaneously resolved. Based on viral load, measured by quantitative PCR, or symptom resolution (in patients with disease), Viralym-C™ cells controlled active infections in all 5 evaluable patients; 4 patients had complete responses, and 1 patient had a partial response within 4 weeks of cell infusion. One patient with CMV retinitis had complete resolution of symptoms following Viralym-C™ infusion. In conclusion, our results demonstrate the feasibility, preliminary safety and efficacy of "ready to administer" Viralym-C™ cells that have been generated from a small panel of healthy, eligible CMV seropositive donors identified by our decision support tool. These data suggest that cost-effective, broadly applicable T cell anti-viral therapy may be feasible for patients following HSCT and potentially other conditions. Disclosures Tzannou: ViraCyte LLC: Consultancy. Leen:ViraCyte LLC: Equity Ownership, Patents & Royalties. Kakarla:ViraCyte LLC: Employment.