Background: Patients with heterozygous signal transducer and activator of transcription 1 (STAT1) gain-of-function (GOF) pathogenic variants exhibit an array of clinical phenotypes including susceptibility to multiple infections, autoimmunity, and cancer predisposition. Previous studies to characterize the pathways involved and explore therapeutic interventions have been constrained by the technology to perform in-depth immunophenotyping. Mass cytometry has allowed us to perform extensive immune profiling of patients with inborn errors of immunity (IEI) to help gain a better understanding of disease pathology. STAT1 gain-of-function (GOF) mutations have demonstrated higher levels of phosphorylated STAT1 in response to type I and II interferons, but the response to other cytokines is less understood. Using advanced cytometry, we demonstrate a unique pattern of STAT1 phosphorylation in response to IL-6 stimulation in T-cell subsets and this differential pattern may play a role in T-cell differentiation and memory in STAT1 GOF patients. Cases: We report two patients with heterozygous STAT1 GOF mutations in the coiled-coil domain. For both patients, the clinical phenotype was largely consistent with other STAT1 GOF patients, one (P1, c.800C>T; p.ala267Val) presented with secondary HLH due to histoplasmosis, and the second (P2, c.866A>G; p.Tyr289Cys) presented with presumed vaccine strain varicella zoster virus (VZV) meningitis and subsequent history of recurrent herpes simplex virus (HSV) skin lesions. Patient peripheral blood mononuclear cells (PBMCs) were evaluated by fluorescence flow cytometry and cytometry by time of flight (CyTOF) as previously described (Roussel et al. J Leukoc Biol. 2017) to evaluate the impact of STAT1 GOF mutations on T-cell immunophenotype and cytokine signaling. Results: Utilizing cytometric data, we were able to identify similar patterns of T cell distribution on t-distributed stochastic neighbor embedding (t-SNE) plots for both patients with STAT1 GOF that were distinct compared to healthy controls (Fig 1a). In the T-cell compartment, both patients had decreased Th17 and Treg populations and an increased Th1/Th2 ratio compared to healthy donor (Fig 1b). In response to stimulation with IFNg or IL-6, there were also clear patterns with the two patients compared to healthy controls. Levels of p-STAT1 and p-STAT3 were assessed in STAT1 GOF and health donor PBMCs at several times points between 15 and 120 minutes, after stimulation with either IFNg or IL-6. Using fluorescence flow, we found that IL-6 stimulation led to greater than anticipated p-STAT1 response at all timepoints compared to a much more muted response to IFNg. The cell subsets highlighted in the t-SNE after IL-6 stimulation differ from the cell subsets that respond to IFNg stimulation in patients and healthy control (Fig 2a), suggesting that distinct cell populations are driving the response to IL-6. By evaluating these IL-6 responsive subsets in comparison with healthy control by CyTOF, we identified an exaggerated p-STAT1 response to IL-6 in the memory T-cell populations in P2 (Fig 2b). Conclusions: These two unique clinical presentations demonstrate that with similar mutations in the coiled-coil domain of STAT1, yet largely differing clinical presentations, the immune profiling patterns of the patients compared to healthy controls can drive further work on disease characterization and therapeutic interventions. This is relevant for this patient cohort, as treatment recommendations for STAT1 GOF are not well established. Long-term outcomes with JAK inhibition are lacking and transplant survival rates to date have been very poor compared to other immune diseases including familial HLH. With identification of IL-6 signaling playing a potential role in T-cell maturation, further studies will need to be performed to determine if IL-6 modulation could be used as a treatment modality. We conclude that performing deep immunophenotyping of patients with inborn errors of immunity (IEIs) such as STAT1 GOF can point to new disease mechanisms of human immunity and inflammation and lead to improved understanding of the role of cytokine and cellular signaling in normal hematopoiesis and cellular maturation. Figure 1 Figure 1. Disclosures Rathmell: Sitryx: Consultancy, Current equity holder in publicly-traded company, Research Funding; Caribou: Consultancy, Current equity holder in publicly-traded company, Current holder of stock options in a privately-held company; Nirogy: Consultancy, Current holder of stock options in a privately-held company; Merck: Speakers Bureau; Pfizer: Speakers Bureau; Mitobridge: Consultancy; Incyte: Research Funding; Calithera: Research Funding; Tempest: Research Funding.

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Introduction The BMT-CTN 1204 study for Hemophagocytic Syndromes or Selected Primary Immune Deficiencies (NCT01998633) (RICHI) was a single arm study testing safety and efficacy of reduced intensity conditioning (RIC) with alemtuzumab (1mg/kg), fludarabine (150 mg/m2) and melphalan (140 mg/m2). Survival was favorable compared to historical studies, but patients experienced high rates of mixed chimerism (MC) and ultimate secondary graft failure (GF). Mechanisms for GF are not known. Expansion of recipient T cells and interferon-gamma pathway activation have been proposed as drivers for GF. However, high peri-transplant alemtuzumab levels have been associated with higher risk of MC and eventual secondary GF, suggesting an inverse relationship between GF and immune activation in the context of RIC. In order to elucidate mechanisms of GF for patients on the RICHI study, we systematically evaluated cytokine patterns and alemtuzumab levels and their association with durable engraftment. Methods Serial blood samples were collected, processed, and stored for consenting patients at day -14 (window: day -28 to -14), day -7 (+/- 1 day), day -1 (+/- 1), day +1 (+1 to +3), day +14 (+/- 2), day +28 (+/- 2), day +42 (+/- 3), day +70 (+/- 10), and day +100 (+/- 10). Alemtuzumab levels were measured using a flow cytometric assay as previously described. Comprehensive cytokine analysis was performed for over 100 analytes using the MagPix platform. Primary GF was defined as donor chimerism <5% prior to day +42. Secondary GF was defined as donor chimerism <5% after initial engraftment and/or requirement of donor lymphocyte infusion (DLI) or second HCT (with or without conditioning) to manage MC or graft loss. Mixed chimerism (MC) was defined as donor chimerism <95% on at least one occasion. Results Thirty-three patients were included in this study with HLH (n=25), CAEBV (n=3), CGD (n=2), HIGM (n=2), and IPEX (n=1). All patients received bone marrow grafts and 27 (82%) patients had unrelated donors. Twenty-one grafts were 8/8 or 6/6 HLA-matched (64%) and 12 grafts were 7/8 HLA-matched (36%). Among all patients, 1 patient (3%) developed primary GF, 22 (67%) developed mixed chimerism (MC), and 11 patients (33%) developed secondary GF. Survival with sustained engraftment without DLI or second HCT was 40.0%. We first evaluated peripheral blood levels of 100+ cytokines. Analysis revealed significant differences between patients with and without GF as shown in Figure 1A. Notably, on day +14 and +28, patients with secondary GF had significantly lower CXCL9 levels than those without GF. We then estimated the cumulative incidence (CI) of secondary GF among patients with CXCL9 levels above and below the day +14 median level of 2394pg/mL. The CI of secondary GF in patients with a day +14 CXCL9 level ≤2394pg/mL was 73.6% vs 0% in patients with a level >2394pg/mL (p=0.002). The CI of secondary GF in patients with a day +28 CXCL9 level ≤2867pg/mL (day +28 median) was 64.3%, vs 0% in patients with levels >2867pg/mL (p=0.004). We then sought to correlate CXCL9 levels with alemtuzumab exposure, as high alemtuzumab levels would result in more efficient T cell depletion of donor grafts that could lead to lower CXCL9 levels. Indeed, CXCL9 levels inversely correlated with day 0 alemtuzumab levels. Patients with day 0 alemtuzumab levels >0.32µg/mL had lower CXCL9 levels compared to patients with levels ≤0.32µg/mL (Figure 1B). Finally, we examined the impact of alemtuzumab levels on MC and secondary GF. Patients with day 0 alemtuzumab levels ≤0.32µg/mL had a lower CI of MC compared to patients with levels >0.32µg/mL, 14.3% vs 90.9%, respectively (p=0.03). The CI of secondary GF was 0% in patients with day 0 alemtuzumab levels ≤0.32µg/mL compared to 54.3% in patients with levels >0.32µg/mL (p=0.08). Conclusions This study demonstrates a strong relationship between alemtuzumab levels and durable engraftment. Further, interferon gamma activity, as reflected by CXCL9, inversely correlates with peri-transplant alemtuzumab levels in this prospective cohort treated with RIC. Our findings support the paradigm that higher alemtuzumab levels drive efficient T cell depletion of the stem cell product which increases the risk of MC and secondary GF, suggesting that donor T cells promote engraftment via a graft versus hematopoiesis function. Precision alemtuzumab dosing strategies may offer an opportunity to improve outcomes for patients who undergo RIC HCT. Figure 1 Figure 1. Disclosures Pulsipher: Adaptive: Research Funding; Equillium: Membership on an entity's Board of Directors or advisory committees; Jasper Therapeutics: Honoraria. Bollard: Neximmune: Current equity holder in publicly-traded company; Catamaran Bio: Membership on an entity's Board of Directors or advisory committees; Cabaletta Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Cellectis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Repertoire Immune Medicines: Current equity holder in publicly-traded company; ROCHE: Consultancy, Honoraria; SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kean: Regeneron: Research Funding; Bristol Myers Squibb: Patents & Royalties: From clinical trial data, Research Funding; Bluebird Bio: Research Funding; Gilead: Research Funding; Vertex: Consultancy; Novartis: Consultancy; EMD Serono: Consultancy. Jordan: Sobi: Consultancy. Allen: Sobi: Consultancy. OffLabel Disclosure: Alemtuzumab, humanized monoclonal antibody against CD52, used as part of allogeneic HCT conditioning