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Characterization of CD4+ and CD8+ T cells responses in the Mixed Lymphocyte Reaction by flow cytometry and single cell RNA sequencing

ABSTRACT: The Mixed Lymphocyte Reaction (MLR) consists in the allogenic co-culture of monocytes derived dendritic cells (MoDCs) with T cells from another donor. This in vitro assay is largely used for the assessment of immunotherapy compounds. Nevertheless, the phenotypic changes associated with lymphocyte responsiveness under MLR have never been thoroughly evaluated. Here, we used multiplex cytokine and chemokine assays, multiparametric flow cytometry and single cell RNA-sequencing to deeply characterize T cells activation and function in the context of CD4+- and CD8+-specific MLR kinetics. We showed that CD4+ and CD8+ T cells in MLR share common classical markers of response such as polyfunctionality, increased proliferation and CD25 expression but differ in their kinetics and amplitude of activation as well as their patterns of cytokines secretion and immune checkpoints expression. The analysis of immunoreactive KI67+CD25+ T cells identified PBK, LRR1 and MYO1G as new potential biomarkers of MLR response. Using cell-cell communication network inference and pathway analysis on single cell RNA-sequencing data, we also highlighted key components of the immunological synapse occurring between T cells and the stimulatory MoDCs together with downstream signaling pathways involved in CD4+ and CD8+ T cells activation. These results provide a deep understanding of the kinetics of the MLR assay for CD4+ or CD8+ T cells and may allow to identify new strategies for immunotherapy in cancer.

ORGANISM(S): Homo sapiens

PROVIDER: GSE242428 | GEO | 2024/01/05

REPOSITORIES: GEO

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Project description:Colorectal cancer (CRC) is one of the most common cancers in France (36,000 new cases / year) and nearly 16,000 people die each year from this disease. The lymph node involvement of the surgical specimen is today the main tool on which is based the adjuvant treatment decision after curative surgical resection. The study of new predictive factors to identify patients at risk for developing a local or metastatic recurrence is therefore a major challenge. It is now clear that the immune system plays a role in the control of tumor’s development, and it was shown that there was a correlation between the presence of a CD3+ T-lymphocyte infiltrate in colorectal cancers and patient survival. Preliminary studies suggest an important role of regulatory T-lymphocyte in the modulation of the antitumor immune response. The aim of our study is to follow a cohort of patients operated for colon cancer with curative intent to highlight the prognostic characteristics of the tumoral infiltrate by various lymphocyte populations (particularly T-lymphocytes but also B-lymphocytes and regulatory lymphocytes). It will be performed a preoperative analysis of blood circulating lymphocytes with antibodies specific for different cell populations (CD3, CD4, CD8, CD56, CD16, CD19, CD2) and stage of activation (CD25, CD69, HLA-DR ) or differentiation (CD24, CD38, CD27, CD103, CD62L, CCR7, CD45RA / RO, IgD). The presence of regulatory T-lymphocytes will also be analyzed. It will be performed on tumor sample a Tissue Microarrays for immunohistochemical study to determine the presence of different lymphocyte populations. We systematically study the markers CD68 (monocytes / macrophages), CD56 (NK cells), CD20 and CD79a (B cells / plasma cells), CD3 (T cells), CD8 (cytotoxic T), CD4 (helper T) FoxP3 (regulatory T), cytotoxicity of CD8 markers (Fas ligand, perforin and granzyme) and MHC I (antigen presentation) to explore the innate and adaptive immune responses. For each section, the different zones will be analyzed (center and invasive margin and healthy tissue). The main objective of the study is the influence of the tumor infiltration rate by CD3 + T cells on disease free survival at 2-years in patients with non-metastatic colon cancer resection. The secondary objective is to search a correlation between the rate of T-lymphocytes on preoperative blood sample and on tumor sample.

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Characterization of CD4+ and CD8+ T cells responses in the Mixed Lymphocyte Reaction by flow cytometry and single cell RNA sequencing

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