Development of a Novel Method for in Vitro Analysis of CD8 Thymocyte Selection and Maturation,

Abstract 3235

T cell development relies on the interaction between the T-cell receptor (TCR) on thymocytes and the self-major histocompatibility complex (MHC) expressed on thymic epithelial cells in the thymus. This process, called positive selection, rescues developing thymocytes from cell death while leading to their differentiation into mature T cells. Since it is believed that the proper development of CD8 T cells requires an intact thymus, several groups studied their development using fetal or reaggregation thymus organ cultures in vitro. Unfortunately, these models were shown to be cumbersome requiring a complicated set-up while generating limited cellular yield. Thus, we sought of developing a novel in vitro system using bone marrow-derived stromal cells to support CD8 T cell development and maturation in vitro. We selected the OTI system as a working model due to the availability of previously identified positively selecting peptides. Non-selected T-cell-committed double-positive (DP) OTI thymocytes (CD4⁺CD8⁺CD69⁻) were first fractionated based on the surface expression intensity of both TCR and CD5. These 3 subsets designated as TCR^loCD5^lo (DP1), TCR^intCD5^hi (DP2), and TCR^hiCD5^int (DP3) express different levels of ZAP70. Following fractionation, the DP subsets were co-cultured with bone marrow-derived stromal cells presenting OTI-selecting peptides. In the absence of cytokines, no CD8⁺ OTI cell development occurred in vitro. When repeated in the presence of γc-cytokines (IL2, IL4, IL7, IL9, IL15 and IL21) only rIL4 and rIL7 were able to induce CD8 T cell development. Supplementing the co-culture system with rIL4 led to the generation of 50–60% single-positive (SP) CD8 T cells only from the DP3 fraction whereas rIL7 induced the development of a minor fraction of CD8 T cells from DP2s (3–4%) and a major population from DP3 (50–76%). Furthermore, we found that rIL4 treatment triggers the development of 2 distinct populations of SP OTI cells (based on their CD8 expression intensity) which we termed CD8^int and CD8^hi. When analyzed by flow-cytometry, ex vivo generated CD8^int, but not CD8^hi, expressed high levels of CD69, PD-L1 and CD44. In contrast, SP CD8 T cells developed in the presence of rIL7 did not upregulate these markers. Since IL7 promotes survival and proliferation of TCR-triggered DPs while IL4 affects their differentiation, we admixed both cytokines during the co-culture and found a dominant rIL4 effect: the phenotype of SP CD8 T cells was similar to that induced by rIL4 alone. Taken together, our findings demonstrate that some DP thymocytes are efficiently selected in our system by OTI-specific positively selecting peptides. Notably, the addition of rIL7 leads to the development and maturation of classic CD8 T cells whereas rIL4 induces both classic and innate CD8 T cells.

This work was supported by grant a from CIHR.