Highlights d CD34 + human thymic progenitors present a spectrum of specification and commitment states d Earliest progenitors are CD7and exhibit stem-cell-like and T-primed transcriptomes d Loss of B-potential precedes that of myeloid and NK potentials during T-commitment d A CD34 + subpopulation of cells is primed for the plasmacytoid dendritic lineage

Pediatric oncology patients are at risk for poor outcomes with respiratory viral infections. Outcome data for COVID‐19 in children and young adults with cancer are needed; data are sparse for obese/overweight and adolescent and young adult subgroups. We conducted a single center cohort study of COVID‐19 outcomes in patients younger than 25 years with cancer. Candidate hospitalization risk factors were analyzed via univariable and multivariable analyses. Eighty‐seven patients with cancer and COVID‐19 were identified. Most were Hispanic/Latinx (n = 63, 72%). Forty‐two (48%) were overweight/obese. Anticancer therapy included chemotherapy only (n = 64, 74%), chimeric antigen receptor T‐cells (CAR‐T, n = 7), hematopoietic stem cell transplantation (HSCT, n = 12), or CAR‐T and HSCT (n = 4). There was no COVID‐19 related mortality. Twenty‐six patients (30%) required COVID‐19 related hospitalization; 4 required multiple hospitalizations. Nine (10%) had severe/critical infection; 6 needed intensive care. COVID‐19 resulted in anticancer therapy delays in 22 (34%) of 64 patients on active therapy (median delay = 14 days). Factors associated with hospitalization included steroids within 2 weeks prior to infection, lymphopenia, previous significant non‐COVID infection, and low COVID‐19 PCR cycle threshold value. CAR‐T recipients with B‐cell aplasia tended to have severe/critical infection (3 of 7 patients). A COVID‐19 antibody response was detected in 14 of 32 patients (44%). A substantial proportion of COVID‐19 infected children and young adults with cancer require inpatient management; morbidity may be high in B‐cell immunodeficiency. However, a majority of patients can be taken through chemotherapy without prolonged therapy delays. Viral load is a potential outcome predictor in COVID‐19 in pediatric cancer.

The initial stages of thymopoiesis are characterized by the induction of T-lineage genes (specification) and the repression of alternative lineage genes (commitment). Multilineage (Thy1 and Thy2) and committed (Thy3) populations representing successive differentiation stages have been defined within CD34+ progenitor cells in the human thymus by the expression of CD7 and CD1a. However, due to intra-population heterogeneity and unstudied transitional states between populations, our understanding of the transcriptional programs that launch human thymopoiesis remains incomplete. Also, the interpretation of species specific gene expression profiles seen in these populations is confounded by species related differences in progenitor immunophenotypes. To our knowledge, whole transcriptome profiles of human thymic CD34+ progenitors at single cell resolution have not been reported. To resolve the transcriptional landscape of the initial stages of thymopoiesis, we performed single cell RNA-Seq (sRNA-Seq) of FACS sorted unfractionated human thymic CD34+CD4-CD8- cells from 3 donors using 10X (1 thymus, 5000 cells), Indrop (1 thymus, 2000 cells), or BD genomics (1 thymus, 192 index sorted cells) sequencing Averaged sRNA-Seq expression profiles recapitulated previous bulk RNASeq data validating our sRNA-Seq pipeline. Transcriptomes of index sorted cells mapped appropriately to those predicted to be Thy1,Thy2, or Thy3 cells from CD34, CD7, and CD1a RNA levels in the 10x and indrops data indicating the validity of identifying Thy1-3 cells by RNA levels. Seurat clustering showed that CD34+ thymocytes consist of a continuum of cell states rather than discrete populations. Nine cell states marked by gradual changes in CD2, CD44, CD7, CD1A, and CD34 RNA levels were seen: state 1 (Thy1), states 2-6 (Thy2), and states 7-9 (Thy3). Thy2 and Thy3 cells spanned several previously undescribed states with distinct expression profiles. Single cells co-expressing stem and T-lineage genes were seen in state 1 indicating an early onset of T-lineage priming that occurs prior to downregulation of stem cell genes. Within Thy2 cells, high expression of stem and alternative lineage genes was mostly restricted to states 2 and 3, which represent novel Thy2 subpopulations. State 2 cells co-expressed T-cell and high levels of innate immune genes, particularly IRF8. State 3 cells showed low CD2 expression and co-expressed T-cell and stem, myeloid, B-cell, and/ or NK genes. These results indicate concomitant priming of multiple lineage transcriptional programs rather than just the presence of unilineage gene program expressing subpopulations in the earliest thymic progenitor cells. In preliminary experiments, FACS sorted CD2 low Thy2 cells generated T-cells as well as a higher output of alternative lineage cells than CD2 high Thy2 cells. States 7-9 showed committed profiles with upregulated T-cell genes and silenced stem and alternative lineage genes. While state 9 (late Thy3) showed high RAG1 expression and cell cycle arrest, most Thy3 cells (states 7 and 8) were cycling indicating that commitment, which precedes T-cell receptor rearrangement, is not associated with proliferation arrest. Monocle pseudotime analysis finely resolved expression profiles along the differentiation trajectory within Thy1-3 populations: e.g. genes repressed earlier (e.g. MEIS1) vs later (e.g. DTX1). BCL11B, a key T-cell commitment gene was one of the earliest transcription factors (TF) to be induced during human thymopoiesis, and upregulation of TCF7 and BCL11B occurred concurrently. In contrast, in mice induction of Bcl11b occurs later after Tcf7 is fully upregulated. Thus, the initial stages of thymopoiesis are characterized by a continuum of multilineage transcriptional priming followed by a gradual transition to a T-lineage restricted gene program, a process similar to that recently described for lineage commitment of bone marrow hematopoietic progenitors but not previously reported in the thymus. SRNA-Seq yielded expression profiles along numerous data-points on the differentiation trajectory within heterogenous populations, providing high transcriptional resolution not possible with bulk data. We found differences in key TF expression profiles that could imply species specific regulatory mechanisms. Our data are a resource for elucidating transcriptional networks in T-cell development and T-cell leukemias. Disclosures No relevant conflicts of interest to declare.

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Summary Single cell RNA sequencing of human thymic cells is dependent on isolation of highly pure and viable cell populations. This protocol describes the isolation of CD34 + progenitor and more differentiated CD34 – fractions from post-natal thymic tissue to study thymopoiesis. CD34 + cells represent <1% of thymic cells, so this protocol uses magnetic- followed by fluorescence-activated cell separation to isolate highly enriched CD34 + cells. For complete details on the use and execution of this protocol, please refer to Le et al. (2020) .

Backgroud: Despite advances in therapy and improved survival, relapsed and refractory B-cell precursor acute lymphoblastic leukemia (r/r BCP-ALL) in pediatric and adult patients still remains a problem. Chimeric antigen receptor T cells against CD19 (CD19 CAR T) show promising results in patients with r/r BCP-ALL. However, relapse of the disease still occurs with appreciable frequency even with this novel therapy. As a significant number of relapses post-CAR T lack surface CD19 expression, further CD19-directed therapy is not an option for these cases. Hypothesis: Sometimes despite CAR T engraftment and establishment of B-cell aplasia, relapse still occurs. We hypothesized that, similarly to cell adhesion mediated chemotherapeutic drug resistance (CAM-DR), cell adhesion mediated CAR T-cell resistance (CAM-CART-R) can contribute to relapse of ALL. Results: To test our hypothesis, primary ALL cells were treated with CD19 CAR T cells either with murine calvaria-derived bone marrow stromal cells, OP9, or cultured only with media in short term cultures. We observed B-ALL cells treated with CD19 CAR T on OP9 has 10-20% higher viability compared to B-ALL and CD19 CAR T co-culture in medium alone, supporting the notion of CAM-CART-R. We also determined that soluble factors in OP9 primed medium may contribute to CAM-CART-R. However, the direct stromal contact mediated significant protection again CAR T induced apoptosis of B-ALL cells. To determine the molecular mechanisms underlying the survival promoting effects of stromal cells on CD19-, these cells were starved in serum-free media for 4hours and then treated with PI3Kδ inhibitor CAL-101 or DMSO and co-cultured with OP9 cells for 1 hour. We found that p-Akt is upregulated by stromal contact in CD19-negative B-ALL cells post-CAR T therapy and that PI3Kδ inhibition using can downregulate p-Akt in CD19-negative B-ALL patients. Critically, we investigated whether CD19 CAR T cells were functional under these conditions. For this purpose, we determined if stromal contact of ALL cells or stromal contact of CAR T cells changes the intracellular cytokine milieu of CD19 CAR T cells and found that intracellular IL-6, TNF- α and IFN-γ were reduced upon stromal contact supporting our hypothesis of a role of stromal cells in CAM-CART-R. We also determined that immune checkpoints molecules on T cells are unaffected by OP9 cells. Despite the reduction of cytokine level in T cells upon co-culture with B-ALL cells on OP9, PD-1, TIM-3 and LAG3 expression on CD19 CAR T cells after 2 days of co-culture was not altered as determined by flow cytometry. Resistance of ALL cells to CD19 CART cells was not mediated through checkpoint inhibition, since the PD-1/PD-L1 inhibitor Nivolumab failed to enhance ALL killing. Phenotypic profiling of thirteen cases of primary ALL relapse post-CD19 CAR T cell therapy showed high expression of adhesion molecules including integrin α4. Phenotypic analysis also revealed high expression of integrins is retained in primary ALL cells after CD19 knockout in one case. To explore possible solutions to overcome CAM-CART-R, we examined a strategy of blocking specifically integrin α4. We have previously shown that blocking integrin α4 can de-adhere CD19-negative B-ALL relapse post-CAR T cell therapy from their respective counter-ligands in vitro and that these cells can benefit from integrin blocking therapy in vivo. We have now confirmed this in NSG mice injected with CD19-negative B-ALL cells from a patient with post-CAR T cell relapse. Mice were treated intraperitoneally (n=6/group) with total immunoglobulin (Ig) control or humanized anti-human integrin α4 antibody Natalizumab (NZM). As a result, Natalizumab monotherapy significantly prolonged survival of leukemic mice compared to control Ig group (66 days (Ig) vs 85 days (NZM) p<0.005). Further combination treatments with chemotherapy are in progress. Conclusion: In summary, our data indicate that similarly to CAM-DR, CAM-CART-R can occur resulting in relapse of ALL. Targeting adhesion molecules may be a new approach to treat or prevent relapse following CD19 CAR T cell therapy for . Disclosures Ahmed: CellMedica: Other: Royalties; Celgene: Other: Royalties; Adaptimmune: Membership on an entity's Board of Directors or advisory committees. Babak:Simurx. Inc: Other: Founder . Pulsipher:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Other: Education for employees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Amgen: Other: Lecture; Bellicum: Consultancy; Miltenyi: Research Funding; Medac: Honoraria. Wayne:AbbVie: Consultancy; Kite, a Gilead Company: Consultancy, Research Funding; Servier: Consultancy; Spectrum Pharmaceuticals: Consultancy, Research Funding. Abdel-Azim:Adaptive: Research Funding.