Loss of the GVL Effect By Distinct Expression of Migration Markers As a Mechanism of Immune Escape in Adult T Cell Leukemia/Lymphoma (ATLL), a Malignant Counterpart of Regulatory T Cells

Adult T-cell leukemia/lymphoma (ATLL) is highly aggressive CD4+ T cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1). Normal cellular counterpart of ATL is considered to be CD4+ regulatory T cells (Treg) and therefore ATL cell and Treg cell show similar phenotypic characteristics including the expression of CD25, Foxp3 and CCR4. Treg is immunosuppressive cells which contribute the maintenance of peripheral tolerance. Previous studies suggested that murine Treg show the distinct characteristics in cell-migration after activation, however, the expression of migration-related markers after activation on human Treg has not been well studied. In this study, we here examined the expression pattern of surface and intracellular markers including activation/migration receptors on Tregs from healthy individuals and Tregs and ATLL cells from 7 patients before and after allogeneic hematopoietic stem cell transplantation (HSCT). Peripheral blood samples were obtained before and at 2, 4, 6, 8, and 12 weeks after HSCT and thereafter every 3 months. Tregs were defined as CD4+CD7+CADM1-CD25+CD127- and ATLL cells were defined as CD4+CD7-CADM1+, and these were compared with CD4+ conventional T cells (Tcons). Gated Tregs and ATLL cells were examined for expression of CD45RA, CD62L, CCR4, CCR7, Ki-67, BCL2, PD-1 and PD-L1. Plasma TARC/CCL17 and MDC/CCL22, endogenous ligands of CCR4, were also measured by ELISA. First, we analyzed the expression of chemokine receptors of Treg from healthy donors. Our data showed that CD45RA+ naïve-type Treg express high level of CCR7 and little expression of CCR4 while CD45RA- activated/memory-phenotype Treg express low level of CCR7 and high level of CCR4. To elucidate the alteration of chemokine receptor expressions just after cell-activation in each Treg subpopulation, we sorted out 4 different CD4+ T cell subsets (CD45RA+ Tcon, CD45RA- Tcon, CD45RA+ Treg and CD45RA- Treg) from healthy donors, cultured with anti-CD3/CD28 antibodies and then measured the proliferation and the migration markers by CFSE-based assay. Tcons promptly decreased CCR7 after cell-division, in contrast, Tregs increased the expression of CCR7, suggesting Treg appears to have distinct migration attribute in which Treg may migrate to lymph node from periphery after activation. Next, we examined Tregs and ATLL cells from patients who received HSCT. 3 patients received mogamulizumab (Mog), a CCR4 monoclonal antibody, before HSCT and showed significantly lower residual ATL at HSCT as compared to 5 patients without pre-HSCT use of Mog (0.3% vs 15.8% of CD4+, P<0.05). Patients with pre-SCT Mog failed early expansion of Treg (3.0% vs 14.6% of CD4+ at week4, P<0.05) and the delayed recovery was prolonged over 12 months. 3 patients developed relapse. PD-L1 on ATL cells was negative at HSCT in these patients but was strongly expressed at relapse. The expression of CD25 was low at the early stage of relapse, which was later upregulated. Interestingly, relapsed ATLLs involved CD45RA+CD62L- or CD45RA-CD62L- subset with high CCR7 and Ki-67 and the slightly decreased CCR4, those are very similar to normal Treg after activation. TARC and MDC were highly elevated just after the treatment but was the elevation was transient. Our data demonstrated that Tregs and ATLL cells showed the similar expression of migration markers on activation and it is clearly different from conventional T cells. ATLL cells at relapse after HSCT showed the decreased skin-oriented CCR4 and the increased lymph node-oriented CCR7, suggesting activated ATLL cells might escape from the immunological surveillance by GVL which is actively exerted in GVHD-target tissues including skin. Our data might provide important information to develop efficient HSCT for ATLL.