*Purpose: Identifying new molecular targets based on our understanding of the biology of the allo-immune response in the allograft microenvironment is critical to develop more targeted therapies in transplantation. We recently showed in a high-throughput-analysis that CD74 gene-expression was highly enriched in urinary exosomes of kidney transplant recipients during rejection. Surprisingly, CD74, a molecule known as a chaperone for MHC-II molecule and the receptor for macrophage-inhibitory-factor (MIF) signaling, hasn’t been explored in allo-immunity.

*Methods: CRISPR-Cas9 technology was used to generate CD74-loxP and subsequently CD74KO mouse to study the role of CD74 in allo-immune response in a fully mismatched murine heart transplant model. We performed immunophenotyping of graft infiltrating cells as well as allograft draining lymph node and spleen in addition to ex-vivo functional assays. Moreover, we performed Tregs proliferation and suppression assays in-vitro.

*Results: Surprisingly, fully mismatched BALB/c heart transplants in CD74KO mice recipients resulted in indefinite allograft survival as compared to 7 days in WT controls (MST >100 vs 7 days, n=6/group, p=0.008). Interestingly, we observed a 4-fold increase in CD4 regulatory T cells (Treg) infiltrating the allograft of CD74KO recipients as compared to WT controls on day 7 post transplantation (7.9% vs 38.9%, p<0.0001). Depletion of Tregs using anti-CD25 antibody resulted in acute graft loss in CD74KO recipients (MST 19 days post-treatment, p=0.03), showing a critical role of CD4 Tregs in mediating allograft survival in this model. In addition, graft infiltrating Tregs showed significantly higher PD-1 frequency and higher Granzyme B expression by MFI as compared to controls (16.2% vs 52.9%, p=0.0003 and 2184 vs 3586, p=0.02 respectively). Significantly higher frequency of WT Tregs in the allograft and dLN expressed CD74 (mean 35% and 24% respectively), as compared to spleen (mean 2.9%). Interestingly, Tregs with highest expression of CD74 showed lower FoxP3 MFI compared to CD74lo Tregs (16295 vs 12948, p=0.01). In-vitro, Tregs isolated from CD74KO mice exhibited higher proliferative capacity and suppressive function compared to WT Tregs. Stimulated CD74KO Tregs in vitro maintained higher frequency of FoxP3 expression comparted to WT. We are performing RNA sequencing of CD74+ and CD74- negative Tregs in addition to ChIPSeq analysis to dissect the role of CD74 in controlling Tregs related genes. We generated CD74-loxP FoxP3-GFP-Cre mice which will help us to dissect the role of CD74 in Tregs.

*Conclusions: Our data shows that allo-immune activated Tregs express CD74 that plays a critical role in suppressing its homeostasis and stability. Targeting CD74 is a promising approach to induce tolerance. Further studies are required to fully explore role of this molecule in allo-immune response.

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