*Purpose: A diverse microbiome is important in the generation, maintenance, and function of the immune system. Specific bacterial communities may have a relationship with organ rejection, infectious complications, and other clinical events. Understanding these relationships would prove valuable for prevention of complications, serve as biomarkers for high-risk patients, and may hold clues to establish tolerance. Utilization of a germ-free mouse model enables investigation of known microbial compositions to evaluate for differences in immune response.

*Methods: Splenocytes from male B6 12-14-week-old germ-free (GF), conventionalized (ConvD: colonized with a mixture of healthy human donors), or conventionally-raised (ConvR) mice were analyzed by flow cytometry for lymphocyte composition and mixed-lymphocyte reaction (MLR) for alloresponse. Potential human subjects for fecal microbial transfer to GF mice were identified by an internal kidney registry between ages 18-70, no prior other organ transplant, and no active infections. A stool sample was collected from patients who were highly-sensitized (CPRA>80), non-sensitized (CPRA<20), or post-kidney transplant +/- rejection). Bacterial composition will be identified by 16S rRNA sequencing and analyzed for differences in operational taxonomic units (OTUs). Samples will be selected for colonization of GF mice for transplant assays.

*Results: Preliminary lymphocyte analysis showed compared to ConvD mice, GF mice had a higher % B220 cells (70.5 +/- 0.7 vs 67.7 +/-.78, p=0.04) and % CD8 of T cells (43.8 +/-1 vs 40 +/-0.4, p=0.02), fewer CD4 of T cells ( 51.5 +/1.1 vs 55.6 +/- 0.46, p=0.03), and no significant difference in CD4 CD25 T cells (p=0.15), memory CD4 T cells (p=0.12), or memory CD8 T cells (p=0.06). Following a 5 day MLR, CD8 T cell proliferation by Ki-67 staining was greater in GF than ConvD mice (72% vs 60%, p<0.05.) however there was no significant difference of Ki-67 staining of CD4 T cells. Forty-five stool samples were collected from all subgroups and controls, and 16S sequencing data is forthcoming.

*Conclusions: Germ-free mice colonized with human stool provides a translational model to investigate the influence of microbial communities on transplant immunology. Preliminary studies show mice with a diverse healthy flora have an altered lymphocyte profile and a dampened alloresponse compared to germ-free controls. Future studies using samples selected by bacterial profile or clinical relevance to sensitization or rejection will inform the role of the microbiome in transplant immunology.

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