A NOVEL BISPECIFIC CD8 TREG MODULATOR TARGETING CYTOLYTIC CD8 REGULATORY T CELLS REDUCES PATHOGENIC CD4 T CELLS AND INFLAMMATION IN TRANSLATIONAL MODELS OF INTESTINAL AUTOIMMUNE AND INFLAMMATORY DISEASE

Abstract

Abstract INTRODUCTION We have characterized a novel CD8 Treg network in autoimmune patient peripheral blood and tissues, in which a subset of cytolytic CD8 Treg eliminate pathogenic CD4 T cells, reducing inflammation and ameliorating disease in response to pathogenic CD4 T cell activation. We hypothesize that the CD8 Treg network is dysfunctional in patients with Celiac and Crohn’s disease, allowing pathogenic CD4 T cell expansion, the initiation of a cascade of inflammatory pathological consequences and the perpetuation of tissue destructive inflammation in Celiac disease and IBD. Here we demonstrate the use of bispecific CD8 Treg modulators that may have the potential to restore the elimination of pathogenic CD4 T cells in disease. METHODS CD8 Treg were cultured with autologous antigen presenting cells pulsed with gliadin peptides, and either primary autologous CD4 T cells or a target gliadin-responsive T cell line. Novel bispecific CD8 Treg Modulators were designed and evaluated for targeted binding by Octet and flow cytometry. The functional impact of CD8 Treg modulators was evaluated using flow cytometry, Incucyte, and supernatant analysis in PBMCs isolated from Celiac and Crohn’s disease patients. CD8 Treg Modulators were also tested using celiac patient duodenal tissue biopsies and in xenograft animal models of acute GVHD. RESULTS Activation of human CD8 Treg resulted in rapid cytolysis of pathogenic CD4 T cells as one mechanism of action. Upon the addition of CD8 Treg modulators, activation, and prevalence of CD8 Tregs increased along with the increase of degranulation and lytic molecules, and the death of antigen activated CD4 T cells. Following gliadin exposure of intestinal organoid cultures, in the presence of CD8 Treg modulators, CD8 Treg prevalence increased, coincident with the reduction of activated CD4 T cell and a decrease of epithelial cell death. The administration of CD8 Treg modulators in a humanized mouse model of acute graft versus host disease resulted in delayed onset and decreased severity of disease (based on clinical score and tissue pathology), and reduction of pro-inflammatory serum cytokines. CONCLUSION Our results indicate that CD8 Treg modulators activate CD8 Tregs in vitro, in vivo, and in human tissue resulting in decreased inflammation and amelioration of disease. Our data suggest that this network can be targeted by immune-modulating biologics to suppress pathogenic T cells to reduce disease severity & delay onset, as well as mitigate severity & frequency of flares in patients with Celiac disease and IBD. Figure 1. CD8 regulatory T cells (CD8 Treg) fail to eliminate pathogenic CD4 T cells in autoimmune disorders. CD8 Treg are responsible for eliminating pathogenic CD4 T cells when they become activated in response to an antigenic trigger. When successful, pathogenic CD4 T cells are eliminated, preventing their expansion and downstream inflammatory consequences, including plasmablast activation and autoantibody secretion, production of pro-inflammatory cytokines and chemokines, recruitment of inflammatory immune cells and bystander T cell activation. In autoimmunity, this surveillance process fails, resulting in inflammation and tissue damage. Figure 2. Mozart’s CD8 Treg Modulators delay disease onset and reduce severity of disease in xenograft models of acute GVHD. CD8 Treg, characterized by expression of inhibitory KIR checkpoint proteins, can prevent disease when mobilized (Figure 2A). NSG mice were injected once a week for 4 weeks (ending day 21) with 0.2mg/kg CD8 Treg modulators. Bispecific antibody like molecules directed against KIR and CD8 induce CD8 Treg mobilization in vivo resulting in reduced pathology, proinflammatory serum cytokines, and disease severity (Figure 2B). CD8 Treg modulators also delayed disease onset and enhanced survival (Figure 2C).