Regulatory T Cell Depletion during Stem Cell Mobilization Enhances Anti-Myeloma Immunity after Experimental Autologous Stem Cell Transplantation

Autologous stem cell transplantation (ASCT) with G-CSF/plerixafor mobilized peripheral blood stem cells (PBSC) remains the standard of care for newly diagnosed eligible multiple myeloma (MM) patients and prolongs progression-free survival. However, most patients eventually progress and require additional therapy. We have previously demonstrated that experimental ASCT can enhance myeloma-specific immunity and limits disease progression characterized by immunological escape. Here we explored the ability of stem cell mobilization (SCM) to mobilize putative myeloma-specific T cells from the bone marrow (BM) and concurrent effects of regulatory T cell depletion (Treg), hypothesizing that this may represent an opportunity to optimize myeloma-specific immunity prior to ASCT. In myeloma patients we noted increased proportions of subsets of effector memory CD8 T cells, including a CX3CR1/granzyme B (GzmB)+ subset in PBSC grafts relative to BM (P<0.01). TIGIT+ Treg were also significantly expanded in PBSC (P <0.01). To study the relevance of these effects, we transferred congenically marked BM T cells into syngeneic mice. We noted that BM-derived effector (CD44+) CD8 cells preferentially homed back to BM (vs. spleen/blood), consistent with T cell residency at this site. SCM subsequently promoted the migration of these resident effector CD8 T cells from BM (4-fold reduction, P <0.001) to blood (5-fold expansion, P <0.001), confirming that PBSC contain BM resident T cells. BM Foxp3+ Treg were also reduced in BM and increased in blood after SCM. Mobilized Treg were characterized by an immunosuppressive phenotype with high levels of CD73, Nrp-1, CTLA4 and TIGIT, and this represents a possible suppressive pathway invoked by SCM. We thus examined the role of BM Treg in limiting myeloma-specific immunity during SCM with Foxp3-GFP-DTR mice. Depletion of Treg with diphtheria toxin (DT) during SCM promoted the expansion of antigen experienced CD8 T cells that secreted IFNg, TNF and GzmB. We next hypothesized that expansion of these polyfunctional cells would enhance myeloma-specific immunity after ASCT. To test this, we treated Vk*MYC bearing Foxp3-GFP-DTR donor mice with DT (vs. saline) 1 week after tumor injection followed by SCM. CD8 T cells from mobilized grafts were transplanted with BM and CD4 T cells from naïve B6 into lethally irradiated Vk*MYC bearing recipients (Figure 1A). Recipients transplanted with CD8 T cells from Treg depleted donors had dramatically improved tumor control (Figure 1B) after ASCT compared to recipients of cells from Treg intact donors (survival 100 days post-ASCT; 100% for DT group vs 13% for saline group, P <0.0001). Rechallenge of mice surviving over 100 days after ASCT confirmed MM clone-specific immunity with complete elimination of the MM seen at initial ASCT, but not an unrelated clone. We next studied clinically relevant approaches to mimic this scenario. Administration of TIGIT, CTLA4, CD25 or LAG3 antibodies resulted in inadequate Treg depletion in BM to recapitulate this effect. In contrast, a synthetic IL-2/IL-15 mimetic NL-201 that selectively binds the beta and gamma chains of IL-2 receptor but not the high affinity IL-2Ra (CD25) reduced BM Treg (45.9% vs 18.6%, P <0.05) and expanded polyfunctional effector cells expressing IFNg+TNF+GzmB+ in SCM grafts. MM-bearing mice transplanted with NL-201 mobilized CD8 T cells were also protected against relapse after ASCT (survival 100 days post-ASCT; 87.5% for NL-201 group vs 25% for saline group, P <0.05). This protection was characterized by expansion of antigen experienced (CD38+) and cytokine secreting (TNF, IFNg) memory (CD44+) CD8 T cells in the BM 3 weeks after ASCT. BM CD8 T cells and Treg are efficiently mobilized after G-CSF/plerixafor. Depletion of Treg in donors during SCM dramatically increases myeloma specific T cells in stem cell grafts and enhances myeloma-specific immunity after ASCT. Stem cell mobilization in the presence of the synthetic IL-2/15 mimetic NL-201 is a promising clinically tractable approach to recapitulate these effects. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal