Cord Blood Regulatory T Cells Do Not Impair Anti-Myeloma Effect of Conventional T Cells

Abstract

Introduction Regulatory T cells (Tregs) are potent immunosuppressors and, specifically, cord blood (CB) Tregs are emerging as a promising treatment for graft vs. host disease as well as immune mediated bone marrow failure disorders. We and others have shown that when compared to peripheral blood (PB), CB Tregs have a higher expression of Helios, a marker of thymic Tregs; exert superior suppression on proliferating conventional T cells (Tcons) and do not secrete inflammatory IL-17 or express RORүt under stressful conditions. Such a stable suppressor profile coupled with their ready availability as an off-the-shelf product positions CB Tregs to be a very attractive therapeutic agent. Multiple myeloma, a plasma cell clonal malignancy, has been incurable and required more effective and safer therapy. Objectives We hypothesized that adoptive therapy with CB Tregs might not interfere with the anti-myeloma activity and relieve the adverse effects related to the excess immune response during the immune therapy. Methods NOD-SCID IL2rγnull (NSG) mice were used to first establish the myeloma tumor model, where mice were injected intravenously via tail vein with 3 × 106 GFP-Firefly luciferase labeled MM.1S cells. For generating CB Tregs, Treg cell enrichment was performed from the whole CB unit using CD25+ magnetic beads followed by continuous culture in the presence of CD3/28 T-cell activator beads, and human recombinant interleukin-2 for 14 days. To assess the CB Tregs impact on anti-myeloma effect by Tcons, the mice inoculated MM.1S cells were injected with 5 × 106 PB CD3+Tcons on day 14 with or without 10 × 106 ex-vivo expanded CB Tregs on day 16, 23, and 30. CB Tregs alone arm was also set up to assess the CB Tregs impact on myeloma progression. The progression of MM.1S cells was monitored by in vivo bioluminescence imaging (BLI). Results The expanded CB Tregs showed a consistent phenotype of CD4+25+127- FoxP3high and more than 80% suppression of the proliferating CD4+ Tcons in vitro. Tcons with or without CB Tregs regressed the myeloma cells in the mice while CB Tregs alone did not have any impact. Notably, the relapse was slightly delayed in the Tcons with CB Tregs recipients compared to the Tcons alone (Figure A). Although CB Tregs recipients died due to graft vs host disease eventually, the survival was not inferior to that in the Tcons alone (Figure B). Upon euthanasia, myeloma cells were barely detectable in the both arms of Tcons with or without CB Tregs compared to large tumor burden in the control arm. Of note, extramedullary tumor was seen in the Tcons alone arm (Figure C). Conclusions Our results demonstrate that ex-vivo expanded CB Tregs do not impair, rather might enhance, the anti-myeloma activity of Tcons in a xenogenic model. These data provide additional insight of the Tregs new role in the tumor environment and may contribute to the development of the novel immunotherapies in the future.