S121 SINGLE-CELL CHARACTERIZATION OF THE MULTIPLE MYELOMA (MM) IMMUNE MICROENVIRONMENT IDENTIFIES CD27- T CELLS AS POTENTIAL SOURCE OF TUMOR-REACTIVE LYMPHOCYTES
Authors: C. Botta, C. Perez, N. Puig, M.T. Cedena, L. Cordon, A. Zabaleta, L. Burgos, C. Maia, I. Goicoechea, S. Rodriguez, I. Rodriguez, S. Sarvide, D. Alignani, A. Vilas-Zornoza, E. Lorenzo-Vivas, L. Rosiñol, A. Oriol, M.-J. Blanchard, R. Rios, A. Sureda, R. Martinez, J. Martin, J. Bargay, J. de la Rubia, M. Rossi, P. Tagliaferri, P. Tassone, J. Merino, F. Prosper, A. Orfao, M.-V. Mateos, J.-J. Lahuerta, J. Bladé, J. San Miguel, B. Paiva
Journal: HemaSphere (2019)

Background: The broad use of immunomodulatory drugs (IMiDs) and the breakthrough of novel immunotherapies in MM urge the optimization of immune monitoring for tailored treatments based on better prediction of patients’ response. For instance, current T cells immune monitoring is of limited value because the phenotype of tumor-reactive T cells is uncertain. Aims: To characterize the MM immune microenvironment at the single-cell level and to identify clinically relevant subsets for effective immune monitoring. Methods: We used a semi-automated pipeline to unveil full cellular diversity based on unbiased clustering, in a large flow cytometry dataset of 86 newly-diagnosed MM patients enrolled in the PETHEMA/GEM2012MENOS65 clinical trial, including evaluation at diagnosis, after induction with bortezomib, lenalidomide, dexamethasone (VRD), autologous transplant and VRD consolidation. Immunophenotyping was performed using the first 8-color combination (CD19, CD27, CD38, CD45, CD56, CD81, CD117, CD138) of the next-generation flow (NGF) panel for MRD assessment. Results were then validated in additional 145 patients enrolled in the same trial. Deep characterization of T cells was performed using 18-color multidimensional flow cytometry (MFC) and combined single-cell (sc) RNA/TCR sequencing (10xGenomics). Results: Simultaneous analysis of the entire dataset (n = 333 files) unbiasedly identified 25 cell clusters (including 9 myeloid and 13 lymphocytes subsets) in the MM immune microenvironment. Afterwards, we correlated the cellular abundance of each cluster and specific cell ratios determined at all time points with laboratory parameters, the International Staging System (ISS), FISH cytogenetics and progression-free survival (PFS). 10 variables had significant impact in PFS and the ratio between CD27- vs CD27+ T cells emerged as an independent prognostic factor (HR:0.09, p = 0.03) together with the ISS in a Cox regression model. The 3-year PFS rates of patients with high vs low CD27-/CD27+ ratios were 94% vs 71% (p = 0.02), respectively; these findings being confirmed in the validation dataset. Thus, we observed in the entire cohort (n = 231) that a prognostic score including both the CD27-/CD27+ T cell ratio (HR:0.21, p = 0.013) and ISS (HR:1.41, p = 0.015) outperformed each factor alone (HR:0.06, p = 0.007). To gain further insight into the biological significance of the CD27-/CD27+ T cell ratio, we performed scRNA/TCRseq in 23,673 lymphocytes from 3 MM patients. Downstream analysis unveiled that CD27- T cells were predominantly CD8 and included senescent, effector and exhausted clusters. By contrast, CD27+ T cells were mainly CD4 and the remaining CD8 T cells had a predominant immune suppressive phenotype (ie. high GZMK, TIGIT, LAG3 and PD1 expression levels). Such T cell clustering was validated by 18-color MFC that confirmed higher reactivity for PD1 and TIGIT in CD27+ T cells. Simultaneous scTCRseq revealed a total of 30 different clonotypes. Interestingly, most clonotypes (25/30) where found in CD27- T cells and, using the VDJB database, the CDR3 sequences of clonotypic effector/exhausted CD27- T cells were predicted to recognize MM-related epitopes such as MLANA, HM1.24 (CD319), TKT, or IMP2. Summary/Conclusion: We show for the first time that potential MM-reactive T cells are CD27- and that their abundance in the immune microenvironment of newly-diagnosed MM patients is prognostic, possibly due to their reactivation after treatment with IMiDs and autologous transplant. Because NGF is broadly used, these results are readily applicable for effective T cell immune monitoring.