Method of storage impacts graft immune cell populations in intestinal transplantation

Abstract

Intestinal transplantation (IT) is the only treatment for intestinal failure patients who cannot tolerate parenteral nutrition. Although cold storage (CS) is the gold standard for intestinal allograft preservation, normothermic machine perfusion (NMP) has improved transplantation success in other organs. Allograft immune modulation is thought to contribute to this success. The objective of this study was to assess the impact of NMP on intestinal immune cell populations as it relates to ultimate allograft and recipient health. We hypothesized that NMP would stabilize overall immune cell populations while reducing pro-inflammatory cohorts, thus improving IT success. Porcine intestines were stored for 6H at 4°C (CS-T6, n=6) or perfused at 34°C (NMP-T6, n=8) and transplanted. Samples were collected following intestinal procurement (T0), storage (T6), 1-hour post-transplant (T1PT), and euthanasia (T48). Flow cytometric profiling and quantification of intraepithelial (IEL) and lamina propria (LP) lymphocytes and NK cells were performed at T0, T6, and T48. Immunofluorescence (IF) identified CD3+ T-cells at T1PT. One-way ANOVA analyzed cell counts with significance set at p< 0.05. Flow cytometry revealed that NMP preserved CD3+ IELs in the jejunum at T6 (63.74% vs 32.79%, p< 0.0001), and in the ileum at T6 (51.31% vs 42.77%, p< 0.0001) and at T48 (57.73% vs 34.21%, p<0.0001). Immunofluorescent cell quantification confirmed this preservation of CD3+ IEL at T1PT in the crypts of the jejunum (4.9% vs 2.1 %, p< 0.05) and ileum (6.9% vs 2.8%, p< 0.001) compared to CS. However, upon further subpopulation analyses using flow cytometry, ileal IEL T-cells were reduced throughout NMP compared to CS (T6 28.7% vs 44.0%; T48 16.65% vs 22.37%; p< 0.0001). NK cells were also decreased in ileal and jejunal LP at NMP-T6 compared to T0 (3.6% vs 5.6%, p< 0.01; 2.4% vs 4.3% p< 0.05). NMP reduces NK cells, a cell type known to be associated with rejection. Interestingly, NMP preserved the overall CD3+ T cell populations in the jejunum and ileum at T6, T1PT, and T48. The subpopulation of IELs, however, were reduced in the ileum following NMP. The role of these populations, specifically in IT, has yet to be determined. Further characterization of T-cell subpopulations and dynamics is needed. Complimentary evaluations of the microbiome and proteomic responses to IT NMP are ongoing. Understanding immune cell dynamics will improve IT outcomes for patients requiring this critical, lifesaving surgery. U.S. Department of Defense PR181265; NIH K01OD010199 SERCA, T32OD011130-15. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.