HLA-G Transference From Multipotent Mesenchymal Stromal Cells to Activated T-Lymphocytes.

Abstract 3674

Poster Board III-610

HLA-G is a nonclassic human leukocyte antigen which is characterized by its limited variability, its highly tissue-specific expression and for its very distinct immunological role. Instead of triggering immune responses, HLA-G is exclusively inhibitory, suppressing immune cell functions. For instance, HLA-G is expressed by cells in sites considered immunologically privileged, such as the fetal cytotrophoblast, at the fetal–maternal interface, were it acts protecting the fetal tissue from the mother's immune response. HLA-G is also pathologically expressed in a diverse set of tumors, acting as an immunescape mechanism. Trough splicing mechanisms, HLA-G can be expressed as a membrane-bound or as a soluble isoform. Interestingly, the transference of membrane fragments between cells, a process called trogocytosis, can spread HLA-G inhibitory function beyond the reach of HLA-G-expressing cells. For instance, upon trogocytosis, effector CD4+ T cells stop proliferating, stop responding to stimulation, and behave as regulatory T cells. Recently, mesenchymal stromal cells (MSC) were shown to secrete soluble HLA-G, adding to the existing set of secreted molecules, by which MSC can modulate cells of the immune system. Despite the importance of secreted factors, cell-to-cell contacts have an important role in the immunological effects exerted by MSC. Based in these facts, we hypothesized that one of the mechanisms used by MSC to immunomodulate T cells, could involve trogocytosis mediated HLA-G transference. To test this, CD3+ T cell were immunomagnetically selected from peripheral blood mononuclear cells (PBMC) and pre-activated for 72hs using anti-CD2/CD3/CD28 beads. Activated T-cells were then incubated for 30 min, alone or with MSC. After this period, cells were recovered and the transfer of membrane bound molecules from MSC to T-cells was analyzed by flow cytometry (3 experiments) using antibodies against HLA-G. Confocal microscopy was carried in an additional experiment, using antibodies against HLA-G, CD140B (a specific MSC marker) and CD3. DAPI was used for nuclear staining. Flow cytometry analysis revealed that activated CD3+ T-cells did not express HLA-G, while MSC expressed HLA-G intracytoplasmatic. After 30 min of co-incubation, membrane bound HLA-G was detected in a variable percentage of CD3+ cells, ranging from 3 to up to 16%, characterizing the transfer of HLA-G from MSC to CD3+ T-cells. Confocal microscopy revealed that activated T-cells cultured alone did not stained for HLA-G or CD140B, while, about 12% of the CD3+ cells stained for membrane HLA-G, following the 30 min incubation with MSC. Additionally, CD140B was also transferred from MSC to CD3+ T-cells. These data are the first demonstration of trogocytosis mediated transfer of HLA-G from MSC to activated T lymphocytes. Given the significance of HLA-G expression and trogocytosis in normal and pathological situations, this newly described immunological mechanism should be considered in the development and application of MSC-based therapies. Supported by CNPq and FAPESP.