To elucidate the transcriptional landscape that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitors spanning the earliest stages of B and T lymphoid specification. Over 3000 novel long non-coding RNA genes (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage-specific and more lineage-specific than protein coding patterns. Protein-coding genes co-expressed with neighboring lncRNA genes were enriched for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships between the earliest progenitors in the human bone marrow and thymus.

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

Chimeric antigen receptor (CAR) T-cells targeting CD19 demonstrate remarkable efficacy in treating B-lineage acute lymphoblastic leukemia (BL-ALL), yet up to 39% of treated patients relapse with CD19(−) disease. We report that CD19(−) escape is associated with downregulation, but preservation, of targetable expression of CD20 and CD22. Accordingly, we reasoned that broadening the spectrum of CD19CAR T-cells to include both CD20 and CD22 would enable them to target CD19(−) escape BL-ALL while preserving their upfront efficacy. Supplementary informationThe online version of this article (https://

The initial stages of T-cell differentiation are characterized by a progressive commitment to the T-cell lineage, a process that involves the loss of alternative (myelo-erythroid, NK, B) lineage potentials. Aberrant differentiation during these stages can result in T-cell acute lymphoblastic leukemia (T-ALL). However, the mechanisms regulating the initial stages of human T-cell differentiation are obscure. Through loss of function studies we showed BCL11B, a transcription factor recurrently mutated T-ALL, is essential for T-lineage commitment, particularly the repression of NK and myeloid potentials and the induction of T-lineage genes, during the initial stages of human T-cell differentiation. In gain of function studies, BCL11B inhibited growth of and induced a T-lineage transcriptional program in T-ALL cells. We found previously unknown differentiation stage-specific DNA binding of BCL11B at multiple T-lineage genes; target genes showed BCL11B dependent expression, suggesting a transcriptional activator role for BCL11B at these genes. Transcriptional analyses revealed differences in the regulatory actions of BCL11B between human and murine thymopoiesis. Our studies show BCL11B is a key regulator of the initial stages of human T-cell differentiation and delineate the BCL11B transcriptional program, enabling the dissection of the underpinnings of normal T-cell differentiation and providing a resource for understanding dysregulations in T-ALL.

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Introduction The initial stages of thymopoiesis are characterized by a progressive commitment to the T-cell lineage, which involves the loss of alternative (myelo-erythroid, NK, B) lineage potentials. Dysregulated differentiation during these stages can result in T-cell acute lymphoblastic leukemia (T-ALL). While the regulation of murine T-lineage commitment has been investigated through studies of the early stages of thymopoiesis, very few studies of early human thymopoiesis exist. Hence, our understanding of the mechanisms underlying the initial stages of human thymopoiesis is incomplete. The transcription factor BCL11B is recurrently mutated in T-ALL. BCL11B is repressed in Early T-cell precursor ALL (ETP-ALL), a T-ALL subtype with a differentiation arrest at an early stage of thymopoiesis. However, the function of BCL11B in human thymopoiesis has not been defined. Furthermore, the DNA binding targets of BCL11B during thymopoiesis, and thereby its role in the regulation of T-lineage transcription networks, are largely undefined. We investigated the role of BCL11B in the initial stages of human thymopoiesis. Methods BCL11B expression in human bone marrow (BM) progenitors and thymic populations was analyzed by RNA-Seq and qPCR. CD34+ cord blood (CB) progenitors transduced with BCL11B knockdown (KD) or scrambled control shRNA lentivirus were co-cultured with OP9DLL1 stroma to assess the effect of BCL11B loss of function on the initial stages of thymopoiesis. The earliest stages of thymopoiesis are recapitulated in the OP9DLL1 system through the sequential appearance of multilineage CD7+CD1a- and T-lineage restricted CD7+CD1a+ T-cell precursors. The effect of KD on alternative lineage potentials was assessed by isolating CD7+CD1a- and CD7+CD1a+ T-cell precursors from KD and control OP9DLL1 co-cultures and then replating them in NK, B, myeloid and/or erythroid assays. T-ALL cells transduced with a BCL11B overexpression or control lentivirus were analyzed by RNA-Seq to determine the effect of BCL11B gain of function on T-cell differentiation at a transcriptional level. ChIP-Seq and RNA-Seq of CD34+ and CD34- cells from the human thymus were performed to define differentiation stage specific BCL11B DNA binding targets and their expression. Results BCL11B was not expressed in BM cells and first became detectable in CD34+ thymic progenitors. A progressive increase in expression was seen when comparing thymic populations at successive stages of T-lineage differentiation. Similar percentages of total CD7+ cells (sum of CD7+CD1a- and CD7+CD1a+ cells) were seen in OP9DLL1 co-cultures initiated with BCL11B KD and control CD34+ CB progenitors. However, KD CD34+ cells showed reduced differentiation into CD7+CD1a+ T-cell precursors (mean %CD7+CD1a+ cells seen in control co-cultures =30.88%, vs 17.85% in KD co-cultures, p<0.05, n=7 independent experiments, 4 shRNA sequences), indicating a differentiation arrest at the CD7+CD1a- stage. KD T-cell precursors showed increased myeloid and NK potentials (p<0.05, n=2 independent experiments). BCL11B overexpression rapidly induced a T-lineage transcriptional program in T-ALL cells (n=3 experiments), including the upregulation of several critical T-lineage genes. ChIP-Seq revealed distinct BCL11B DNA binding profiles in CD34+ (5032 binding sites) and CD34- (12849 binding sites) human thymic cells with an overall gain in binding with differentiation. Most sites were in gene promoter regions. Combined genome wide analysis of gene expression and BCL11B DNA binding targets during thymopoiesis revealed previously unknown differentiation stage-specific binding at multiple sites, which were enriched for critical T-lineage differentiation genes, and binding was associated with upregulation of these target genes (n=2 biological replicates). Conclusions BCL11B is essential for T-lineage differentiation and commitment through the repression of NK and myeloid potentials, during the initial stages of human thymopoiesis. BCL11B binds to multiple T-lineage genes in a stage specific manner and induces T-lineage genes, a BCL11B transcriptional program that has not been previously described in murine or human thymopoiesis. Our data strongly support the notion that BCL11B repression at least partly contributes to the aberrant differentiation in human ETP-ALL and provide a framework for understanding human thymopoiesis and T-ALL. Disclosures Crooks: Kite Pharma: Patents & Royalties: Kite Pharma holds an exclusive license to certain intellectual property, Research Funding.

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.

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) .