*Purpose: Allospecific memory T cells pose a threat to long-term allograft survival since they are somewhat resistant to immunosuppression. The objective of this study is to induce long-term allograft survival by suppressing the generation of donor-specific memory T cells using short-term treatment with a biological agent, such as a novel protein ESAT-6, which is originally identified as a T cell Ag in the culture filtrate of M. tuberculosis. Previous studies have demonstrated that ESAT-6 promotes the growth and pathogenesis of M. tuberculosis while weakening host immunity.

*Methods: In this study, B6 mice were transplanted with Balb/C islets and treated with ESTA-6 (50ug on days 0, 3, 6 and 10) and/or low doses of CTLA4-Ig (0.1 mg on days 0, 2, 4, and 6). To track and enumerate allospecific memory T cells via FACS analysis post-transplantation, purified Thy1.1+CD8+CD44- T cells were injected into B6 mice before grafting. Moreover, transcription factors EOMES and T-Bet, which control memory cell formation, were also determined via intracellular staining and FACS. One-way MLRs were set up to determine the effects of ESAT-6 on T cell proliferation in vitro.

*Results: We found that ESAT-6 significantly extended islet allograft survival (MST = 30 vs. 14 days, P<0.05). Importantly, treatments with both CTLA4-Ig and ESAT-6 further prolonged the allograft survival (MST = 88 vs. 30 days), with 50% of recipients achieving long-term allograft survival (>100 days). ESAT-6 also ameliorated cellular infiltration in islet allografts, as determined by H&E staining. Further, it reduced central memory CD8+ T cell numbers compared to control group 6 weeks after transplantation (Thy1.1+CD8+CD44^highCD62L^high: 1.1±0.2 vs. 2.4±0.3, x10000 /LN, P<0.05 and 4.3±0.5 vs. 8.2±1.1, x100000/spleen, P<0.05) while ESAT-6 plus CTLA4-Ig further reduced CD8+ central memory cell numbers compared to control group (0.7±0.1 vs. 2.4±0.3 /LN, P<0.01 and 3.0±0.4 vs. 8.2±1.1 /spleen, P<0.01). Interestingly, ESAT-6 only slightly lowered Thy1.1+ CD8+CD44^highCD62L^low effector memory numbers in both spleens and allografts (data not shown). Moreover, ESAT-6 significantly decreased EOMES expression in Thy1.1+CD8+ cells (MFI: 685±63 vs. 996±92, P<0.05) while slightly increasing their T-Bet expression. However, ESAT-6 did not alter CD4+FoxP3+ Treg frequency in recipient mice. Finally, ESAT-6 alone also inhibited T cell proliferation in vitro (CPM: 1.5±0.1 vs. 2.4±0.2, P<0.05).

*Conclusions: ESAT-6 inhibits allospecific central memory CD8+ T cell generation and islet allograft rejection. It also promotes the induction of long-term islet allograft survival. Even more mechanistic studies are currently underway. Thus, ESAT-6 could be potentially used to inhibit human allograft rejection in the future.

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