Distinct Pro‐Inflammatory Species‐Specific Transcriptional Changes in Human T Cells Following Pig Xenogeneic Stimulation

Conventional T cell-directed immunosuppression is the mainstay of standard-of-care therapy to prevent graft rejection in clinical organ transplantation. However, it remains ineffective in preventing experimental and clinical organ xenograft rejection. Here, we explored the impact of allogeneic versus xenogeneic antigen stimulation on human T cell responses and gene profile. A comparable proliferative human T cell response was observed in vitro following stimulation with either human or pig cells. Yet, elevated High mobility group box-1 (HMGB1) levels were following xenogeneic but not allogeneic stimulation, suggesting a pro-inflammatory response. Next, human peripheral blood mononuclear cells (PBMC) were cultured with allogeneic human, "concordant" xenogeneic monkey, or "discordant" xenogeneic pig, intact cells, or cell lysates. Flow-sorted CD3⁺T cells were analyzed for gene expression using NanoString. A distinct pro-inflammatory gene profile was observed in human CD3⁺T cells following co-culture with discordant xenogeneic pig cells, but not concordant xenogeneic monkey cells or allogeneic human cells. Uniquely, stimulation with pig cells induced the expression of the transcription factor NCF4, which promotes inflammasome activation. Pig cell lysate, but not intact pig cells, induced high expression of the DNA-binding cytokine interleukin-26 gene. Collectively, these observations highlight the impact of xenogeneic stimulation of human T cells in pig xenograft recipients and concomitant inflammatory responses, which may contribute to immunosuppression-resistant xenograft rejection. Finally, the impact of genetic engineering of donor pigs on human T cell transcriptomic gene profile is yet to be determined.