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

PTEN (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 TCRαß+ T-ALL development. Herein, we explored the functional interaction between TCRαß 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 Ptendel thymocytes express, as expected, Myc transcript, whereas inference of pathway activity revealed that these Ptendel 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 Ptendel blasts were unable to release calcium ions (Ca2+) 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.

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

Specific 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. For complete details on the use and execution of this protocol, please refer to Nozais et al. (2021).

MYC deficiency impairs the development of effector/memory T lymphocytes.

In 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.

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

Multiplexed 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 (Ab-HTO) to label each sample, and 10× Genomics technology to analyze single-cell 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. For complete details on the use and execution of this protocol, please refer to Nozais et al. (2021).

A Quantitative Tri-fluorescent Yeast Two-hybrid System: From Flow Cytometry to In cellula Affinities.

We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on a small but diverse set of PPIs (eleven protein families from six organisms) with different affinities; the dissociation constants range from 117 pm to 17 µm After only two hours of reaction, expression of the reporter can be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflect the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.