Initiation of V(D)J Recombination by Dβ-Associated Recombination Signal Sequences: A Critical Control Point in TCRβ Gene Assembly

T cell receptor (TCR) β gene assembly by V(D)J recombination proceeds via successive Dβ-to-Jβ and Vβ-to-DJβ rearrangements. This two-step process is enforced by a constraint, termed beyond (B)12/23, which prohibits direct Vβ-to-Jβ rearrangements. However the B12/23 restriction does not explain the order of TCRβ assembly for which the regulation remains an unresolved issue. The initiation of V(D)J recombination consists of the introduction of single-strand DNA nicks at recombination signal sequences (RSSs) containing a 12 base-pairs spacer. An RSS containing a 23 base-pairs spacer is then captured to form a 12/23 RSSs synapse leading to coupled DNA cleavage. Herein, we probed RSS nicks at the TCRβ locus and found that nicks were only detectable at Dβ-associated RSSs. This pattern implies that Dβ 12RSS and, unexpectedly, Dβ 23RSS initiate V(D)J recombination and capture their respective Vβ or Jβ RSS partner. Using both in vitro and in vivo assays, we further demonstrate that the Dβ1 23RSS impedes cleavage at the adjacent Dβ1 12RSS and consequently Vβ-to-Dβ1 rearrangement first requires the Dβ1 23RSS excision. Altogether, our results provide the molecular explanation to the B12/23 constraint and also uncover a ‘Dβ1 23RSS-mediated’ restriction operating beyond chromatin accessibility, which directs Dβ1 ordered rearrangements

RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement

V(D)J recombination of TCR loci is regulated by chromatin accessibility to RAG1/2 proteins, rendering RAG1/2 targeting a potentially important regulator of lymphoid differentiation. We show that within the human TCR-α/δ locus

Single-cell profiling identifies pre-existing CD19-negative subclones in a B-ALL patient with CD19-negative relapse after CAR-T therapy

International audienceChimeric antigen receptor T cell (CAR-T) targeting the CD19 antigen represents an innovative therapeutic approach to improve the outcome of relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL). Yet, despite a high initial remission rate, CART therapy ultimately fails for some patients. Notably, around half of relapsing patients develop CD19 negative (CD19 neg) BALL allowing leukemic cells to evade CD19-targeted therapy. Herein, we investigate leukemic cells of a relapsing BALL patient, at two-time points: before (T1) and after (T2) anti-CD19 CART treatment. We show that at T2, the BALL relapse is CD19 negative due to the expression of a non-functional CD19 transcript retaining intron 2. Then, using single-cell RNA sequencing (scRNAseq) approach, we demonstrate that CD19 neg leukemic cells were present before CART cell therapy and thus that the relapse results from the selection of these rare CD19 neg BALL clones. In conclusion, our study shows that scRNAseq profiling can reveal pre-existing CD19 neg subclones, raising the possibility to assess the risk of targeted therapy failure

Isolation and enrichment of mouse splenic T cells for ex vivo and in vivo T cell receptor stimulation assays

International audienceSpecific antigen recognition by T cell receptor (TCR) activates TCR signaling pathway, leading to T cell proliferation and differentiation into effector and memory cells. Herein, we describe protocols for TCR stimulation assays, including procedures for the isolation and enrichment of mouse splenic T cells for ex vivo TCR stimulation with anti-CD3/CD28 antibodies, and the use of ovalbumin-OT-II mouse model for in vivo TCR stimulation. We applied this protocol to show that MYC protein is essential for T cell proliferation and differentiation

Single‐cell transcriptomics uncovers an instructive T‐cell receptor role in adult γδ T‐cell lineage commitment

International audienceAfter entering the adult thymus, bipotent T-cell progenitors give rise to ab or cd T cells. To determine whether the cd T-cell receptor (TCR) has an instructive role in cd T-cell lineage commitment or only "confirms" a pre-established cd Τ-cell lineage state, we exploited mice lacking expression of LAT, an adaptor required for cd TCR signaling. Although these mice showed a T-cell development block at the CD4 À CD8 À double-negative third (DN3) stage, 0.3% of their DN3 cells expressed intermediate levels of cd TCR (further referred to as cd int) at their surface. Single-cell transcriptomics of LAT-deficient DN3 cd int cells demonstrated no sign of commitment to the cd T-cell lineage, apart from cd TCR expression. Although the lack of LAT is thought to tightly block DN3 cell development, we unexpectedly found that 25% of LAT-deficient DN3 cd int cells were actively proliferating and progressed up to the DN4 stage. However, even those cells failed to turn on the transcriptional program associated with the cd T-cell lineage. Therefore, the cd TCR-LAT signaling axis builds upon a cd T-cell uncommitted lineage state to fully instruct adult cd T-cell lineage specification

Multiplexed single-cell RNA-sequencing of mouse thymic and splenic samples

International audienceMultiplexed single-cell RNA-sequencing (scRNA-seq) enables investigating several biological samples in one scRNA-seq experiment. Here, we use antibodies tagged with a hashtag oligonucleotide (HTO) to label each sample, and 103 Genomics technology to analyze singlecell gene expression. Advantages of sample multiplexing are to reduce the cost of scRNA-seq assay and to avoid batch effect. It may also facilitate cell-doublet removal and the merging of several scRNA-seq assays. Herein, we apply multiplexed scRNA-seq to investigate mouse thymocytes and splenic T lymphocytes development

Calcium Signaling Is Impaired in PTEN-Deficient T Cell Acute Lymphoblastic Leukemia

International audiencePTEN (Phosphatase and TENsin homolog) is a well-known tumor suppressor involved in numerous types of cancer, including T-cell acute lymphoblastic leukemia (T-ALL). In human, loss-of-function mutations of PTEN are correlated to mature T-ALL expressing a T-cell receptor (TCR) at their cell surface. In accordance with human T-ALL, inactivation of Pten gene in mouse thymocytes induces TCRαβ + T-ALL development. Herein, we explored the functional interaction between TCRαβ signaling and PTEN. First, we performed single-cell RNA sequencing (scRNAseq) of PTEN-deficient and PTEN-proficient thymocytes. Bioinformatic analysis of our scRNAseq data showed that pathological Pten del thymocytes express, as expected, Myc transcript, whereas inference of pathway activity revealed that these Pten del thymocytes display a lower calcium pathway activity score compared to their physiological counterparts. We confirmed this result using ex vivo calcium flux assay and showed that upon TCR activation tumor Pten del blasts were unable to release calcium ions (Ca 2+ ) from the endoplasmic reticulum to the cytosol. In order to understand such phenomena, we constructed a mathematical model centered on the mechanisms controlling the calcium flux, integrating TCR signal strength and PTEN interactions. This qualitative model displays a dynamical behavior coherent with the dynamics reported in the literature, it also predicts that PTEN affects positively IP3 (inositol 1,4,5-trisphosphate) receptors (ITPR). Hence, we analyzed Itpr expression and unraveled that ITPR proteins levels are reduced in PTEN-deficient tumor cells compared to physiological and leukemic PTEN-proficient cells. However, calcium flux and ITPR proteins expression are not defective in non-leukemic PTEN-deficient T cells indicating that beyond PTEN loss an additional alteration is required. Altogether, our study shows that ITPR/Calcium flux is a part of the oncogenic landscape shaped by PTEN loss and pinpoints a putative role of PTEN in the regulation of ITPR proteins in thymocytes, which remains to be characterized

MYC deficiency impairs the development of effector/memory T lymphocytes

International audienceIn the thymus, T cell progenitors differentiate in order to generate naive T lymphocytes which migrate in the periphery where they will fulfill their function in the adaptive immune response. During thymopoiesis, genomic alterations in thymocytes can promote leukemia development. Among recurrent alteration is PTEN inactivation, which is associated to MYC overexpression. Herein, we used conditional Pten and Myc knockout mouse models and single-cell RNA-sequencing approach, to investigate the impact of MYC loss on physio-pathological development of PTEN-proficient or PTEN-deficient T lymphocytes. First, our results confirm that MYC is mandatory for PTEN loss-mediated leukemogenesis, while it is not required for terminal steps of thymopoiesis. In contrast, we uncovered that Myc ablation in CD4+CD8+ thymocytes disrupts T lymphocytes homeostasis in the spleen, notably by drastically reducing the number of MYC-deficient effector/memory T cells. Collectively, our data show that besides naive T cells proliferation, MYC is essential for effector/memory differentiation

Diagnostic challenge in a case of ambiguous lineage acute leukemia with PICALM::MLLT10 rearrangement

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