GvHD After Haploidentical Transplant: A Novel, MHC-Defined Rhesus Macaque Model Identifies CD28-Negative CD8+ T Cells as a Reservoir of Breakthrough T Cell Proliferation During Costimulation Blockade and Sirolimus-Based Immunosuppression

Abstract 2550

We have developed a novel, MHC-defined rhesus macaque model of total body irradiation-based haploidentical hematopoietic stem cell transplantation. This model has permitted us, for the first time, to perform a rigorous study of the cellular and molecular basis of uncontrolled primate GvHD, and to evaluate the efficacy of a novel, clinically-relevant T cell costimulation blockade-based immunosuppressive regimen to control this disease.

We have found that after unprophylaxed haploidentical transplant, severe GvHD developed, which was characterized by rapid clinical decline, and widespread T-cell infiltration and organ damage, with histopathologic evidence of disease in the lungs, the liver, and the GI tract. Mechanistic analysis revealed activation as well as possible counter-regulation, with rapid, CD8-predominant T-cell expansion and accumulation of both CD8+ and CD4+ granzyme B+ effector cells as well as FoxP3^pos/CD27^high/CD25^pos/CD127^low CD4+ T-cells. In addition, CD8+ cells downregulated CD127 and BCl-2 and upregulated Ki-67, consistent with a highly activated, proliferative profile. A cytokine storm also occurred, with GvHD-specific secretion of IL-1Ra, IL-18, and CCL4.

The combination of CD40/CD28 costimulation blockade (using a monoclonal antibody against CD40 and the CTLA4Ig fusion protein) and mTOR inhibition with sirolimus (Costimulation Blockade and Sirolimus, “CoBS”) resulted in striking protection against GvHD. Thus, at the 30-day primary end-point, CoBS-treated recipients demonstrated 100% survival compared to no survival in untreated recipients. Long-term analysis revealed that CoBS treatment resulted in mean survival increasing from 11.6 to 62 days (p<0.01) with significant blunting of both T-cell expansion and activation. However, some CoBS-treated animals did eventually develop GvHD, with both clinical and histopathologic evidence of smoldering disease. We used multiplexed cytokine-secretion analysis as well as multicolor flow cytometry to determine the origins of the reservoir of the CoBS-resistant breakthrough immune activation. We found that this reservoir included the secretion of IFNγ, IL-2, MCP-1 and IL12/23. In addition, animals developing breakthrough GvHD demonstrated CoBS-resistant proliferation of CD28-negative, CD8+ T-cells, and in vitro analysis confirmed that allo-proliferation of these CD28-negative T cells was resistant to immunosuppression with CTLA4Ig, which targets the CD28/B7 T cell costimulation pathway.

These results demonstrate the utility of this novel primate model to provide mechanistic insights into the molecular and cellular basis of GvHD as well as to allow a rigorous evaluation of novel, clinically-relevant immunosuppression strategies. Our results with CoBS suggest that while significant disease control was accomplished, the CD28-negative CD8+ T cell compartment was relatively resistant to CD28/CD40 costimulation blockade and sirolimus, and that adjuvant treatments targeting this subpopulation may be needed for full disease control, especially after high-risk, T cell replete haploidentical transplantation.