Emergence of Donor T Cells That Recognize Nonpolymorphic Antigens During Graft Versus Host Disease,

Abstract 4013

Chronic graft versus host disease (GVHD) is a major cause of morbidity and mortality in allogeneic stem cell transplant recipients and typically develops from antecedent acute GVHD. In contrast to acute GVHD which is characterized by a restricted set of organ involvement, chronic GVHD has more protean manifestations with much broader tissue involvement that can affect nearly all organ sites. Moreover, clinical manifestations in chronic GVHD often bear striking similarity to what is observed in autoimmune diseases. How GVHD evolves from an acute inflammatory syndrome in which donor T cells recognize polymorphic antigens expressed by host APCs to one that is characterized by autoimmune manifestations is not well understood. More specifically, it is not clear whether T cells that mediate autoimmune manifestations during chronic GVHD continue to respond to polymorphic antigens or whether T cells that recognize nonpolymorphic antigens emerge in these recipients. To address this issue, we performed a comprehensive analysis of the clonotypic T cell response in mice that developed autoimmune-mediated GVHD. To conduct these studies, lethally irradiated Balb/c [H-2^d] mice were transplanted with C57BL/6 [H-2^b] bone marrow and spleen cells to induce acute GVHD. At 19–21 days post transplantation, spleen cells or purified T cells from completely donor-engrafted mice were transferred into non irradiated syngeneic B6 Rag animals. In this model, animals develop pathological damage in the colon 60–70 days post transfer characterized by lamina propria inflammation, goblet cell depletion, and crypt cell destruction. This is attributed to the presence of autoreactive donor T cells in the original spleen cell inoculum that expand in syngeneic recipients due to loss of effective T cell regulation. To examine the clonotypic T cell response, we performed T cell receptor beta spectratyping on pathologically involved colonic tissue to identify over represented, skewed bands that were shared by replicate mice within the 21 Vβ families. These bands within a given Vβ family were sequenced to define the specific T cell clonotypes within colitic tissue. In the vast majority of families across multiple experiments, there were high frequency clonotypes that were present in all replicate mice and comprised 50–90% of all sequences. Notably, these shared clonotypes between replicate animals had the same CDR3 nucleotide sequence, indicating that they were the same T cell clones. The presence of high frequency clonotypes was also evidence that autoimmunity was characterized by antigen-driven expansion of a limited number of clones. During the progression from acute to chronic GVHD in humans, host APCs are eliminated and presentation of host peptides is by donor APCs through the indirect alloreactive pathway. To simulate this condition in our experimental model, we created chimeric animals by transplanting B6 Rag BM cells into lethally irradiated Balb/c Rag mice. Spleen cells from primary B6→Balb/c GVHD mice were then transferred into these fully donor-engrafted chimeric animals. Clonotypic analysis of T cells obtained from replicate mice with colitis revealed that dominant clonotypes were observed and comprised 65–80% of all sequences, similar to what was observed during conditions of autoimmunity. Given these similar results in models of autoimmunity and indirect alloimmunity, we then determined whether T cells that were capable of responding to B6 and Balb/c antigens when presented by B6 APCs could be identified. To address this question, the same pooled spleen cell suspension from primary (B6→Balb/c) GVHD mice was transferred into B6 Rag and chimeric (B6 Rag→Balb/c Rag) animals. We observed that T cell clonotypes with the same nucleotide sequences could be identified in pathologically involved colon tissue from both cohorts of mice indicating the emergence of T cells that were capable of recognizing both B6 and Balb/c antigens presented by B6 APCs. These results demonstrate that the loss of self tolerance which occurs during acute GVHD leads to the emergence of broadly reactive donor T cells that are capable of recognizing nonpolymorphic antigens that are shared between donor and host. These data also provide a mechanistic explanation for how autoimmunity develops as a consequence of GVHD and help explain the progression of tissue involvement that characterizes the transition from acute to chronic GVHD.