We have continued to focus our studies on 1) the development of a model that may provide insights into mechanisms involved in autoimmune reactions in skin and in the maintainance of tolerance to epidermally-derived proteins, 2) a mouse model in which a cytokine storm occurs, 3) determining the role of CD8+ T cells in tumor progression when mice are experienceing a graft vs. host-like diseases (GvHD), 4) determining the early molecular events that occur in situ in this GvHD model, and 5) determining the potential role of Janus kinase inhibitors, like tofacitinib, as well as histadine decarboxylase inhibitors like Vorinostat, and azole compounds like ketaconazole in the inhibition of the GvHD effector functions of T cells in this model. The major project that we are pursuing involves the characterization of a model of skin autoimmunity and peripheral immunological tolerance induction. We have developed transgenic mice that have a K14-ovalbumin (K-14 OVA) encoding gene. Some of the mice express the OVA on the epithelial cell surfaces (K14-mOVA) while others express soluble OVA (K14-sOVA). We are studying these mice and have also crossed these mice with those that have a TCR transgene that recognizes ovalbumin in association with H-2b (OT-I). These mice have the TCR for ovalbumin and express ovalbumin in the epidermis but have no apparent disease. We are also using the K-14 OVA mice as targets for immunological reactions in the skin. When T cells from OT-I mice (that have a T cell receptor for the OVA peptide and MHC class I H-2b antigens) are injected into either the single Tg mice or the double Tg mice their role in causing inflammatory skin lesions has been assessed. We have found that the OT-I CD8+ T cells induce a GvHD-like disease in the single Tg (k14-mOVA) mice....this is characterized by swelling of the feet from days 6-14 and development of redness and scaling of the skin within 7 days. The mice lose weight and die between weeks 2 and 3. The mice probably die because of a mucositis (tongue and esophagus)-they are unable to eat properly. In sharp contrast, the double Tg (K14-mOVA X OT-I) mice, despite their expressing OVA in the skin, are unaffected when the OT-I CD8+ T cells are injected into them. One of the aims of our current studies is to better understand why there is tolerance in these double Tg mice. We have identified IL-15 as a critical molecule that is involved in the GvHD reactions. From these studies, using microarray technology, we have identified a transcription factor, IRF8, that is a potentially important molecule in the CD8 OT-I cells that induce disease. We have functionally characterized this transcription factor and found that IRF8 integrates the TCR/co-stimulation and gamma-c-cytokine signaling pathways and mediates the transition of na?ve CD8 T cells to effector cells. We have thus identified IRF8 as one of the molecular regulators of CD8 T cell differentiation. We have also found that the PD-1/PDL-1/2 system plays a role in controlling disease (and in maintaining tolerance) in certain of these mice and we have been able to break this tolerance by inhibiting or knocking out PD-i from the transferred OT-I cells. These OT-IPD-IKO cells will induce substantial GVHD in these otherwise tolerant mice. On the other hand, we have found that HDAC inhibitors will block GVHD as well as the elicitation phase of contact hypersensitivity if given by gavage to mice. We have also continued our studies of K14-OVA mice that produce soluble OVA (K14-sOVA). When OT-I cells are injected into these mice, almost all mice die after 5-7 days. and this is due to an acute mucositis and esophagitis. We are currently studying the mechanism by which this destruction takes place. We found that K14-sOVA crossed with OT-I mice die within 15-21 days of birth. The cause of death is probably due autoreactivity of OT-I cells that have not been deleted by the thymus. Recent studies have shown that we can obviate death by injecting the relevant peptide (SIINFEKL) on days 5 and 9 to these double transgenic mice. Thus, while all of these double transgenic mice die within 21 days if PBS is injected on days 5 and 9 of birth, about 80% survive if 100 micrograms of peptide is injected on days 5 and 9. The mechanism by which these peptides protect the double Tg mice continues to be studied- findings to date suggest that there is a depletion of CD8 cells in these mice and these CD8 T cells express many fewer CD8 molecules than do the double Tg mice that die. There is also downregulation of the V alpha chain of the TCR on these CD8+ T cells. Furthermore, CD8 T cells from healthy (treated) mice were anergic and could not be activated by exogenous IL-2. A block in IL-2/IL-7 signaling via the STAT5 pathway provided the basis for low surface expression of the CD8 coreceptor and failure of IL-2 to break CD8 T cell anergy. Thus, the soluble TCR ligand triggered multiple tolerance mechanisms in these sOVA/OT-I mice, making this treatment approach a potential paradigm for modulating human autoimmune diseases. These same SIINFEKL peptides obviate the intense inflammatory reaction (total obliteration of ears) that occurs when trangenic sOVA mice are crossed with OT-I mice. We have also embarked on studies that test the role of OT-I cells in mOVA Tg mice that are carrying OVA-expressing tumors and are then adoptively transferred with OT-I cells. Preliminary studies indicate that the OT-I cells obliterate the tumor and/or growth is markedly attenuated. Preliminary studies of the in situ events that occur in the GvHD in mice expressing mOVA have demonstrated that there is upregulation of CXCL9 and CXCL10 chemokines in the skin and draining lymph nodes of mice 3 days after onset of the GvHD reaction that occurs after adoptive transfer of OT-I cells. CXCR3 is also upregulated in the OT-I cells that are seen in the draining lymph nodes. Finally, we have had some success in developing a mouse model of cytokine storm that closely resembles what happens in human beings. This model involves the injection of the SIINFEKL peptide into OT-I mice that have a TCR that recognizes this peptide in association with class I MHC molecules. We are embarking on studies to understand the mechanisms underlying this reaction and to develop agents that can block the reaction.
