*Purpose: Exosomes are nanoparticles (30-200nm) released by many cell types. They carry nucleic acid and proteins that may reflect conditional changes in their tissue counterparts. They may play a role in modulating antigen presentation, cytokine production and cell proliferation. Previously we reported the biomarker potential of donor tissue specific exosomes for noninvasive monitoring of rejection. We propose that allograft rejection also results in time-specific changes in T cell specific exosomes. We studied this in a mouse model of full major histocompatibility mismatch heart transplantation.

*Methods: Full major histocompatibility mismatch heterotopic heart transplantation (BALB/c [H2-K^d] into C57BL/6 [H2-K^b]) was performed (n=42) and mice were sacrificed for plasma exosome analysis at time points: pre-transplant, days 1, 2, 4, 7, 11, and 30. Exosomes were isolated by size exclusion chromatography/ ultracentrifugation. T cell exosomes were purified by affinity antibody coupled bead (NHS-CD3ε) isolation and applied for downstream cargo analysis. Microarray analysis of T cell specific exosome RNA cargo was performed. BALB/c hearts were transplanted into immunodeficient (C57BL/6 Prkdc^SCID) recipients (n=26) to validate recipient origin.

*Results: Exosomes expressed bona fide exosome specific markers, but H-2k^d protein was detected only in BALB-C mouse plasma exosomes (Fig. 1A). T cell specific exosomes (CD3+) were quantified on nanoparticle detector, showing increased signals post-transplants peaking at day 7 (Fig. 1B). T cell exosomes were not detected in immunodeficient recipients, suggesting minimal to no contribution from passenger leukocytes. T cell specific exosomes showed increased protein expression of CD4, TCR, FOXP3, INF-γ, and SERCA2 (Fig. 1C). RT-PCR analysis showed enriched expression of mRNAs for CD3ε, TCR, CXCL-10, cytokine INF-γ, IL-2, and FOXP3 in T cell exosomes (Fig. 1D). Analysis of T cell exosome mRNA and microRNA cargoes demonstrated significant association with immunologic and hematologic pathways.

*Conclusions: Acute rejection leads to time specific changes in circulating T cell specific exosomes. Upregulation of protein and RNA markers implicated in T cell mediated alloreactivity and rejection suggest that T cell exosomes may play a role in immunologic processes. T cell specific exosome profiling may serve as a biomarker of cell mediated rejection.

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