Gartlan, Kate H.

Abstract Allogeneic stem cell transplantation (alloSCT) is a highly effective, curative therapy for leukemia yet does not provide a survival benefit above autologous SCT in patients with multiple myeloma (MM). To explore this, we developed preclinical models of SCT using C57Bl/6 recipient mice and either C57Bl/6 (ASCT) or C3H.SW (alloSCT) donor grafts. Importantly, these models recapitulated the clinical setting whereby alloSCT provided superior outcomes, compared to ASCT, in recipients bearing MLL-AF9-driven acute myeloid leukemia (AML) but not in those bearing Vk*MYC-MM. Interestingly, we found that MM-specific, T cell-mediated immunity was generated after ASCT, which failed due to MM-induced T cell exhaustion. MM relapse after ASCT could be prevented by TIGIT or PD-1 targeted immune checkpoint inhibition or via depletion of suppressive CSF1-R+ myeloid cells. Conversely, after alloSCT, T cell exhaustion was driven principally by alloantigen and CD8 T cells expressed high levels of PD-1, TIGIT and TIM-3. Furthermore, Vk*MYC myeloma exploited this alloantigen-driven T cell exhaustion via expression of high levels of PD-L1 and CD155 (the cognate ligands for PD-1 and TIGIT), which were expressed minimally on MLL-AF9 AML. To exploit alloSCT in MM, we used post-transplant cyclophosphamide (PT-Cy) to delete high affinity alloreactive T cells that generate the exhausted donor T cell pool. Subsequent administration of CD137 agonists enhanced T cell activation and cytolytic activity within bone marrow, without exacerbating GVHD. Thus, PT-Cy provides a platform for optimizing immunotherapy after alloSCT.

Abstract Multiple myeloma (MM) is a largely incurable hematological malignancy characterized by clonal expansion of plasma cells in bone marrow (BM). Autologous stem cell transplantation (ASCT) remains a standard treatment for MM, allows intensive cytoreduction and induces inflammation in the context of lymphodepletion. We hypothesized that SCT may overcome myeloma-induced immune defects to restore immune-equilibrium. To address this, we developed a model of ASCT for MM using Vk*MYC myeloma. Surprisingly, we demonstrate the induction of T cell-dependent MM control after SCT as recipients of syngeneic BM grafts containing T cells had prolonged survival and reduced tumor burden compared to BM alone. This anti-myeloma activity was independent of γδ T, natural killer (NK) and NK T cells and was dependent on CD4+and CD8+ T cells. MM-primed T cells, harvested from SCT recipients with controlled MM, protected against relapse in secondary recipients and were Vk*MYC clone-specific. Recipients of MM-primed T cells had increased central memory CD8+ T cells and transfer of MM-primed CD8+ T cells was sufficient to prevent relapse. Furthermore, TCR sequencing of CD8+ T cells from mice with controlled MM revealed a higher clonotype overlap relative to MM-free SCT recipients, consistent with MM-specific T cells. Donor IFNγ secretion and signaling was critical to protective immunity and CD8+ T cells from mice with relapsed MM had impaired IFNγ production compared to those with controlled disease. Importantly, IFNγ production was profoundly augmented by CD137 agonist treatment such that majority of mice controlled disease. These data provide new insights into the mechanisms of action of SCT in myeloma and suggest the use of CD137 agonists to prevent relapse.

Abstract Autologous stem cell transplantation (ASCT) remains an important consolidation treatment for multiple myeloma (MM) patients, even in the era of novel agents. The prolongation of plateau-phase induced by ASCT is generally attributed to intensive cytoreduction. However, ASCT generates inflammation and profound lymphodepletion, which may result in hitherto unexpected immunological effects. To investigate potential immunological contributions to myeloma control after ASCT, we developed preclinical models of transplantation for MM using Vk*MYC myeloma that generates bony lytic lesions, a serum M band and marrow plasmacytosis that are hallmarks of clinical disease. Myeloma-bearing B6 recipients underwent myeloablative conditioning and were transplanted with naïve B6 bone marrow (BM) grafts with or without T cells from donors that were myeloma-naïve (SCT) or had low M bands at the time of harvest to mimic ASCT. Surprisingly, we demonstrate the broad induction of T cell-dependent myeloma control with enhanced median survival in recipients of grafts containing T cells compared to T cell depleted (TCD) BM alone (SCT= 91 days and ASCT > 100 days post-transplant vs TCD BM alone= 44 days; p<0.0001). Myeloma was most efficiently controlled when recipients were transplanted with memory T cells (CD44+) from autologous grafts (median survival: ASCT-CD44+ T cells >90 days post-transplant vs. CD44─ T cells = 50 days; p = 0.0006). Importantly, T cells adoptively transferred from recipients surviving > 120 days (MM-primed) protected secondary recipients compared to T cells from naïve donors (median survival: MM-primed > 120 days post-transplant vs 65 days naïve T cells; p = 0.0003). Furthermore, MM-primed CD8 T cells were restricted in TCR repertoire and provided protection in a myeloma clone-specific fashion, indicative of a tumor-specific T cell response. Despite this immune-mediated control of myeloma after SCT, progression still occurred in the majority of recipients. We phenotyped CD8+ T cells from the BM of MM-relapsed, MM-controlled and MM-free (that had never seen myeloma) mice 8 weeks after SCT. Expression of the inhibitory receptors, programmed cell death protein 1 (PD-1) and T cell immunoreceptor with Ig and ITIM domains (TIGIT) on BM CD8+ T-cells strongly correlated with myeloma cell number (r = 0.729, p<0.0001 and r = 0.796, p<0.0001 respectively). Additionally, the co-stimulatory/adhesion receptor CD226 (DNAM-1) was markedly downregulated as myeloma progressed (r = - 0.865, p<0.0001), as was interferon-γ secretion (r = - 0.76, p = 0.0022). t-SNE analysis confirmed an irreversible exhaustion signature at myeloma progression, characterized by the absence of DNAM-1 and co-expression of PD-1, TIM-3, TIGIT together with CD101 and CD38. Immune-checkpoint inhibition (CPI) early post-SCT, using antibodies against PD-1 or TIGIT facilitated long-term myeloma control (median survival in both treatment arms > 120 days post-SCT vs. 60 and 68 days respectively; p <0.05). Furthermore, TIGIT blockade limited CD8+ T cell exhaustion, increased CD107a and IFNγ secretion and expanded a memory CD8+ T cell population in the BM. Genetic deletion of either IFNγ or the IFNγ receptor from the donor graft resulted in dramatic myeloma progression after SCT. Consequently, treatment with a CD137 (4-IBB) agonist early after SCT profoundly augmented CD8+IFNγ+GranzymeB+ T-cell expansion in the BM, such that majority of treated animals eliminated myeloma and survived long-term. These data provide insights into an unappreciated mechanism of action of ASCT whereby myeloma immune-equilibrium is established and suggest that combination with immunotherapeutic strategies is a rational approach to generate long term disease control. Disclosures Smyth: Bristol Myers Squibb: Other: Research agreement; Tizona Therapeutics: Research Funding.