CD4-CD8- Double Negative Regulatory T Cell Based Therapy Induces Donor-Specific Tolerance in Mouse Composite Tissue Allotransplantation Model

Cheng-Hung Lin¹, Timothy Ng², Dong Zhang², Wensheng Zhang², Gerald Brandacher¹, W.P. Andrew Lee¹, Xin Xiao Zheng².

¹Department of Plastic and Reconstructive Surgery, Johns Hopkins University School of Medicine, MD, USA; ²Division of Plastic and Reconstructive Surgery, University of Pittsburgh Medical Center, PA, USA.

Background: Composite tissue allotransplantation (CTA) can restore appearance, function and dignity to the deformed patient. But CTA requires high-dosed immunosuppressive drugs, which can cause severe side effects. Regulatory cell-based therapy may wean patients off immunosuppressants. In our previous report, ex vivo CD4⁺ T-cells converted CD4^-CD8^- double negative regulatory T cells (DN Tregs), which regulate allo- and auto- immune responses, significantly prolong islet allograft survival. In this study, we explore the DN Treg-based therapy in mouse CTA model.

Materials and Methods: We tested the DN Treg-based therapy (5 x 10⁶ cells) with a short course of low-dosed immunosuppressant, rapamycin (0.6 mg/kg/day for 28 days), in an MHC completely mismatched mouse heterotopic CTA model (DBA/2 to C57BL/6). Antilymphocyte serum (ALS) and/or IL-2/Fc were also used in the experimental groups in conjunction with DN Tregs and rapamycin.

Results: DN Tregs and rapamycin had a synergistic effect on decreasing both CD4⁺ and CD8⁺ T cell proliferation and increasing apoptosis detected by significantly induced Annexin V staining for both CD4⁺ and CD8⁺ T cells in vivo. In mouse CTA model, the single transfer of DN Tregs plus 28-day IL-2/Fc and low-dosed rapamycin treatment significantly prolonged hindlimb allograft survival in comparison with untreated group (MST 46 days vs. 10 days, p=0.0026) and IL-2/Fc plus rapamycin treated group (MST 46 days vs. 32 days, p=0.0064). Moreover, the addition of ALS to DN Tregs, rapamycin and IL-2/Fc treatment could achieve permanent engraftment in mouse CTA model (> 180 days). Macrochimerism was detected 30 days after hindlimb osteomyocutaneous flap transplantation. Moreover, significant increase of CD4⁺Foxp3⁺ Tregs was found over the period of 3 months post transplantation. Donor specific tolerance was reflected in vivo by survivors accepting donor while rejecting third-party skin grafts.

Conclusion: Using ex vivo CD4⁺ T-cells converted DN Tregs is a highly potent and antigen specific induction method. DN Tregs prevent acute rejection and induce donor-specific hindlimb osteomyocutaneous flap tolerance. DN Tregs-based therapy may represent a novel strategy to prevent rejection and induce tolerance in CTA.