Molecular Pathways Responsible for Adaptive Resistance to Single-Checkpoint Inhibition May Reveal Biomarkers and Immunotherapy Targets in Multiple Myeloma Patients

Immune checkpoints blocking antibodies represent a promising strategy to harness anti-tumor immune responses and improve the clinical outcome of patients with hematological malignancies. Early studies based on single-agent PD-1 blockade have fallen short of clinical expectations in multiple myeloma patients. Thus, several strategies are under consideration to implement and optimize the clinical efficacy of immune checkpoint blockade in multiple myeloma patients. We have focused on Vγ9Vδ2 T cells because we believe that these cells are very sensitive tools to assess the immune competence of multiple myeloma (MM) patients in the tumor microenvironment. By interrogating the bone marrow (BM) Vγ9Vδ2 T-cell immune reactivity to phosphoantigens, we have revealed a very early and unexpected tumor microenvironment commitment to immune suppression which is already detectable in monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted in clinical remission after autologous stem cell transplantation. Multiple cell subsets (myeloma cells, myeloid-derived suppressor cells, regulatory T cells, bone marrow stromal cells) are involved in Vγ9Vδ2 T-cell inhibition via several immune suppressive mechanisms including the immune checkpoint circuity.

We have shown that there is a multifaceted immune checkpoint expression in the tumor microenvironment which is dependent on the clinical stage and cannot be neutralized with single-agent PD-1 blockade. To test our hypothesis that adaptive resistance to PD-1 blockade in MM patients may be due to the compensatory up-regulation of other immune checkpoints, we analyzed the expression of other inhibitory receptors (TIM-3, LAG-3, TIGIT and CTLA-4) in PB and BM Vg9Vd2 T cells from healthy donors and MM patients and observed that also TIM-3 beyond PD-1 is significantly upregulated in BM Vγ9Vδ2 T cells from MM patients at diagnosis. Unlike healthy immune cells, the attempt to activate functionally exhausted immune cells can further compromise their anti-tumor functions. pAg stimulation of PD-1+ and TIM-3+ BM Vγ9Vδ2 T cells further increases their inhibitory receptor expression. Interestingly, TIM-3 up-regulation is even more pronounced than PD-1 up-regulation in BM Vγ9Vδ2 T cells and it is specular to Vγ9Vδ2 T -cell proliferation, confirming that any inappropriate attempt to activate these cells leads further worsen their immune competence status. Our observations about the compensatory up-regulation of multiple inhibitory receptors (PD-1 and TIM-3) on BM Vγ9Vδ2 T cells and the further up-regulation of TIM-3 after anti-PD1 treatment suggested us to assess the combination of both immune checkpoint inhibitors (anti-PD1 and anti-TIM3) to overcome the PD-1 single blocking resistance and recover anti-myeloma reactivity. TIM-3 blockade alone was able to partially recover pAg-induced Vγ9Vδ2 T-cell proliferation but the best recovery was obtained when pAg stimulation is carried out in the presence of concurrent PD-1 and TIM-3 blockade. To investigate the molecular mechanisms behind the up-regulation of multiple immune checkpoints responsible for PD-1-mediated hyporesponsiveness and the effector cells exhaustion, we analyzed the main PD-1-mediated (PI3K/AKT) and TIM-3-mediated (JAK/STAT) signal transduction pathways. After pAg stimulation, purified Vγ9Vδ2 T cells from peripheral blood (PB) CTRL, PB MM and BM MM has been tested by western blot analysis for the expression of several intracellular proteins: Ras, phospho-ERK1/2, ERK1/2, phospho-Akt, Akt, phospho- JAK1, JAK1, phospho-STAT1, STAT1, phospho-STAT3, STAT3. Vγ9Vδ2 T cells isolated from BM MM and PB MM showed a remarkable down-modulation of pAKT, a key player of PD-1-mediated signal transduction, and pJAK-1 and pSTAT1, both involved in TIM-3-mediated signaling pathway. Morever, we observed a significantly reduced expression of the transcription factor T-bet, the downstream target gene of pSTAT1, selectively in Vγ9Vδ2 T cells isolated from the tumor site, suggesting that low T-bet expression is a further hallmark of BM Vγ9Vδ2 T cells exhaustion.

Altogether, our findings allow to identify potential molecular mechanisms responsible for the poor efficacy of single immune checkpoint blockade in the tumor microenvironment and to design rational combination therapeutic strategy to be applied to the different clinical settings.