T CELL DIVERSITY AND FUNCTIONAL BIOLOGY

A. Andakyan¹, S. Burruss², L. Hong¹, X. Shen³, S. Romanov², F. Gao³, M. Fishbein⁴, N. Semiletova^1
1Surgery, UCLA Medical School, Los Angeles/UNITED STATES OF AMERICA, ²Surgery, UCLA Medical Schoold, Los Angeles/UNITED STATES OF AMERICA, ³Surgery, Dumont-UCLA Transplant Ctr, Los Angeles/CA/UNITED STATES OF AMERICA, ⁴, UCLA Medical School, Los Angeles/UNITED STATES OF AMERICA

Body: Despite advances in donor organ preservation and immunosuppressive therapies allograft chronic rejection stillrepresents the major cause of graft failure. The participation of mast cells in cardiac rejection remains controversial. Role of CD4CD25 mast cells in development of cardiac chronic rejection inconnection with emerging T regulatory cells (T-regs) was studied. Methods: Cardiac hosts were adoptively transferred with different subtypes of T-regs purified from tolerant (Tol-T-regs) andchronically rejecting (CR-T-regs) cardiac recipients. Results: ACI recipients of WF hearts treated with unfractioned CR-T-regs (10x10⁶) or with Tol-T-regs demonstrated stablesurvival of transplanted organ ( MST >120 days; n=8 & 17, respectively.). Histology after 100 days post-Tx revealed that recipients treated with CR-T-regs had transplant vascular disease(50-70% of vessel affected) accompanied with the strong mast cells infiltration (26.4 cells per field). Further analysis showed that these grafts were mostly CD3 negative but CD4, CD25 and FoxP3positive. On the contrary, recipients treated with Tol-T-regs had only 14.2 mast cells per field and post-Tx morphological changes were found in 30% of total blood vessels. These grafts were CD3negative also. However, strong CD4 and CD25 infiltration was not accompanied with the presence of FoxP3 positive cells. The hearts had only 6.4 of such cells per field that was in 5 times less thenin CR-T-regs treated group. Analysis of mRNA concentration for the surface chemokine (C-C motif) receptor 3 (CCR3) in the allograft tissues demonstrated that Tol-T-reg therapy sufficiently reducedexpression of CCR3 in transplanted hearts in compare to CR-T-reg treated grafts. CD8, CD4 and CD4CD25 syngeneic CR- and Tol-T-regs showed strong regulatory characteristic. All cardiac recipientstreated with these T-regs had indefinite prolongation of graft survival time. However, only the CD4⁺ Tol-T-reg reduced chronic rejection (NI=19+5, 21+6% of vessels) ascompared to CD8⁺ T-reg (NI=40+9 in 50+15% of vessels). The cytokine profile indicated a dominant IL-10 response in CD4 T-reg treated recipients. Alloantibody analysis showedupregulation of IL-4 and IL-12 dependent IgG2c immunoglobulins. Unlike in experiments with unfractioned T-regs, in studies with T-reg subtype transfers we did not find correlation of mast cellsinfiltration with the level of transplant chronic rejection. All groups appeared similar for the level of intragraft positive mast cells though they where on different stages of transplant vesseldisease and fibrosis. Treatment with CD8⁺ Tol-T-regs down-regulated CCR3 expression in the transplanted hearts compared to CD4⁺ Tol-T-regs. In all other groups CCR3 expressionshowed strong correlation with the level of infiltrated mast cells. Conclusion: Hosts of long-term surviving grafts generate T-reg cells of different regulatory characteristics in correlationwith the level of immune tolerance. True tolerant recipients can generate real T-regs that can ameliorate development of transplant vascular disease and generate uniqueCD4⁺CD25⁺FoxP3^-mast cells in the secondary hosts.

Disclosure: All authors have declared no conflicts of interest.