Behbehani, Gregory K.

Abstract High‐dimensional mass cytometry data potentially enable a comprehensive characterization of immune cells. In order to positively affect clinical trials and translational clinical research, this advanced technology needs to demonstrate a high reproducibility of results across multiple sites for both peripheral blood mononuclear cells (PBMC) and whole blood preparations. A dry 30‐marker broad immunophenotyping panel and customized automated analysis software were recently engineered and are commercially available as the Fluidigm® Maxpar® Direct™ Immune Profiling Assay™. In this study, seven sites received whole blood and six sites received PBMC samples from single donors over a 2‐week interval. Each site labeled replicate samples and acquired data on Helios™ instruments using an assay‐specific acquisition template. All acquired sample files were then automatically analyzed by Maxpar Pathsetter™ software. A cleanup step eliminated debris, dead cells, aggregates, and normalization beads. The second step automatically enumerated 37 immune cell populations and performed label intensity assessments on all 30 markers. The inter‐site reproducibility of the 37 quantified cell populations had consistent population frequencies, with an average %CV of 14.4% for whole blood and 17.7% for PBMC. The dry reagent coupled with automated data analysis is not only convenient but also provides a high degree of reproducibility within and among multiple test sites resulting in a comprehensive yet practical solution for deep immune phenotyping.

Introduction. Our previous flow cytometry-based comparison of HIV(+) and HIV(-) autologous hematopoietic stem cell transplant (auto-HSCT) recipient immunomes at 56, 180 and 365 days post-transplant to each other and to healthy controls (HCs) showed that both sets of auto-HSCT recipient immunomes approached HCs over time, but retained significant differences. HIV(+), but not HIV(-), auto-AHCT recipients retained pro-inflammatory features consistent with chronic HIV infection. Here, we report the results of a quantitative and functional analysis of immune reconstitution in HIV(+) patients treated with allogeneic hematopoietic stem cell transplant (allo-HSCT), in comparison with HIV(+) auto-HSCT recipients and HCs. Methods. Blood samples were collected for analysis at days 56, 180 and 365 post-transplant from HIV(+) transplant recipients and at 1 time point from HCs. Whole blood analysis was performed by five-color flow cytometry across 100 immune marker combinations. Comparisons were made between HIV(+) allo-HSCT recipients (n=17, acute myeloid leukemia, acute lymphocytic leukemia, myelodysplastic syndrome, Hodgkin and non-Hodgkin lymphoma that received myeloablative or reduced intensity conditioning on the BMT-CTN-0903/AMC-080 trial), HIV(+) auto-HSCT recipients (n=36, aggressive B cell non-Hodgkin lymphoma or Hodgkin lymphoma that received myeloablative conditioning on the BMT-CTN-0803/AMC-071 trial) and 71 HCs. Unsupervised principal component analysis (PCA) examined differences in immune cell proportions, identified by flow cytometry across 100 cell subsets at each time point. Wilcoxon rank-sum tests compared median absolute counts and median proportions of cell subsets. An independent feature importance score analysis (FIS) identified contributions of immune cell populations expressing specific immune marker combinations to the differences between HIV(+) auto-HSCT recipients, HIV(+) allo-HSCT recipients and HCs. Functional responsiveness of HIV(+) allo-HSCT recipients' T cells to stimulation with CD3- and CD28-directed antibodies, NK cells to stimulation with IL-12 and IL-18 and monocytes to stimulation with lipopolysaccharide (LPS) was assessed in a preliminary mass cytometry on peripheral blood mononuclear cells isolated at the same time points (n=2) and compared to HCs (n=2). Results. PCA showed that immunomes of HIV(+) allo-HSCT recipients and HIV(+) auto-HSCT recipients clustered together with each other, but away from HCs at all time points throughout the post-transplant year. FIS identified: 1) 13 cell subsets that defined the difference between HIV(+) allo-HSCT recipients (all visits) and HCs, and 2) 11 immune cell subsets that defined the difference between HIV(+) auto-HSCT recipients (all visits) and HCs; in both of these comparisons, activated CD3+/HLA-DR+ T cells had the greatest impact on the difference between HIV(+) and HC immunomes. At 1 year, both HIV(+) transplant recipient cohorts had higher absolute numbers of activated T cells, effector T cells and CD8+ T cells than HCs (Wilcoxon rank-sum test, p<0.0031). HIV(+) autologous and allogeneic HSCT recipients also had lower numbers of CD4+ T cells, naïve T cells and activated NK cells compared to HCs (p<0.0031). FIS also identified 20 immune cell subsets that defined the difference between HIV(+) autologous and allogeneic HSCT recipients immunomes at 1 year, with CD8+/CD27- effector T cell subset exerting the highest impact on the difference. Preliminary functional mass cytometry analysis of 2 HIV(+) allo-HSCT recipients and 2 HCs showed that: 1) IFNʏ production by CD8+ T cells was increased above that of HCs at all time points. 2) Expanded populations of CD4+/T-bet+ cytotoxic cells expressing granzyme B and perforin, and CD8+ cytotoxic T cells expressing granzyme B and perforin, persisted in HIV(+) allo-HSCT recipients at all time points, but not in HCs. 3) NK cells retained an ability to produce IFNʏ in response to stimulation with IL-12 and IL-18 in HIV(+) allo-SCT recipients. 4) Monocytes showed an enhanced production of TNFα in response to stimulation with LPS in HIV(+) allo-HSCT recipients compared to HCs at 1 year post-HSCT. Conclusion. Chronic HIV infection confers the pro-inflammatory immune features on the phenotypic and functional profiling of the T lymphocyte immunome of stem cell transplant recipients, irrespective of allogeneic or autologous stem cell donor source. Disclosures Devine: Bristol Myers: Other: Grant for monitoring support & travel support; Kiadis Pharma: Other: Protocol development (via institution); Magenta Therapeutics: Other: Travel support for advisory board; My employer (National Marrow Donor Program) has equity interest in Magenta. Noy:Janssen: Consultancy; Medscape: Honoraria; Prime Oncology: Honoraria; NIH: Research Funding; Pharamcyclics: Research Funding; Raphael Pharma: Research Funding. Popat:Bayer: Research Funding; Incyte: Research Funding; Jazz: Consultancy. Hofmeister:Celgene: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; Nektar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Imbrium: Membership on an entity's Board of Directors or advisory committees; Oncopeptides: Membership on an entity's Board of Directors or advisory committees. Navarro:Atara Biotherapeutics: Employment, Equity Ownership. Behbehani:Fluidigm corporation: Other: Travel funding. Lozanski:Boehringer Ingelheim: Research Funding; Beckman Coulter: Research Funding; Stemline Therapeutics Inc.: Research Funding; Genentec: Research Funding. Baiocchi:Prelude: Consultancy.

Significance In this work we report on the previously uncharacterized clinical and biological role for EGFL7 in acute myeloid leukemia (AML). Patients with increased EGFL7 mRNA expression had lower complete remission rates and shorter overall and event-free survival, demonstrating the clinical relevance of EGFL7 expression in cytogenetically normal AML. Our results show that AML blasts are able to synthesize and secrete EGFL7 protein, promoting autocrine blast cell growth. Inhibition of EGFL7 results in decreased proliferation and induces apoptosis of AML cells. Taken together, our data provide the rationale for targeting EGFL7 using blocking antibodies as a therapy for patients with AML.