Role of CYBR, a cytohesin binder and regulator scaffold protein, in cell-mediated immune response in vivo

Cybr (Cytohesin binder and regulator) is an adaptor protein involved in the assembly and recruitment of protein complexes associated with intracellular trafficking and signaling. Cybr has attracted attention as a potential key contributor to molecular mechanisms governing cells of the immune system due to its exclusive expression in cells of hematopoietic origin. Cybr interacts with members of the ADP ribosylation factor (ARF)-activating cytohesin family, mainly cytohesin-1, and it is involved in the cytohesin-1-mediated adhesion of LFA-1 to ICAM-1. Cybr expression is highly and rapidly responsive to, and regulated by, many cytokines and other soluble effectors of the immune system suggesting a potential functional role in the vesicle formation, endocytic trafficking, regulation of TCR signalling and regulation of dendritic cell (DC)/T cell interaction during the antigen presentation. To characterize the in vivo physiological role of this molecule, a Cybr-deficient mouse strain was created. Cybr deletion does not profoundly affect the development of the immune system, but Cybr-KO mice display a reduced or delayed capacity to respond to different stimuli and in stress conditions. This project aimed at investigating the biological function of Cybr in cell-mediated immune response to tumors induced by the retroviral complex constituted by the Moloney murine sarcoma virus/Moloney murine leukemia virus (M-MSV/MuLV, hereafter indicated as M-MSV). Intramuscular injection of M-MSV in immunocompetent C57BL/6 (B6) mice causes sarcomas that spontaneously regress because of a strong immune reaction primarily mediated by cytotoxic T lymphocytes (CTL) specific for viral antigens. Conversely, Cybr-deficient mice injected with M-MSV developed larger tumors than B6 mice, which additionally regressed with a slower kinetics. To disclose the biological bases of this behavior, M-MSV-injected Cybr-deficient and wild type mice were characterized for the lymphocyte phenotype and function in tumors, lymph nodes and spleens at the peak of tumor growth (day 11 to 15). We found a reduced number of CD4+ and CD8+ T cells, as well as of antigen-specific CTL, in the tumor infiltrating cells (TIL) of Cybr-deficient mice. However, this defect was recovered after a short temporal shift. Similarly, a three-day delay was also reported in the onset of lytic activity in Cybr-deficient respect to wild type CTLs. On the contrary, wild type or Cybr-deficient memory T cells from tumor regressor mice did not show any difference in terms of lytic activity. Overall, these data indicate that Cybr deficiency has a significant impact on the activation of naive T cells and expansion of primed T cells, but do not clarify whether Cybr mostly influence priming and/or cell adhesion or trafficking and migration of immune system cells. To address this issue, we transferred naive Cybr-KO or wild type GFP T cells in tumor-bearing RAG2-/- γc-/- mice, that lack T, B and NK cells and do not spontaneously regress M-MSV-induced tumors. Despite T cell transfer, tumors continued to growth indicating that transferred naive T cells were not able to mount a fully effective immune response in this setting. This was probably due to a suboptimal recruitment and priming phase in lymph nodes, that were found to be hypoplastic. To provide a mechanistic insight, reconstitution of nu/nu athymic B6 mice with T cell-depleted bone marrow from either wild type or Cybr-KO mice, followed by adoptive transfer of naive or memory T cells from Cybr-KO/GFP or B6/GFP animals, will provide the appropriate experimental set up to assess the role of Cybr in the APC or T cell compartments. Taken together, outlined results indicate that Cybr deficiency has a significant impact on antigen-specific immune response, but further studies have to be performed to fully dissect the role played by this molecule in the priming phase and in the delayed onset of lytic activity