T lymphocytes are able to recognize tumor antigens and control neoplasms. However, a series of functional alterations, such as immuno-senescence and exhaustion may affect an effective antitumor response. Multiple myeloma (MM) is a neoplasia characterized by an infiltration of neoplastic B cells in the bone marrow. The prevalent median age group in MM patients is 65 years old; therefore, patients are mainly experiencing an immunosenescent process that may potentially affect the functional status of T lymphocytes. Considering that autologous CAR-T cells (Chimeric Antigen Receptors) treatment for MM showed an overall rate of response of about 30%, we hypothesize that patients’ derived T lymphocytes may potentially present a dysfunctional profiling and generating inefficient CAR-T products. Thus, we aim to investigate the molecular landscape of T lymphocytes from patients with MM using multiparametric flow cytometry and functional assays.

Healthy donors (HD) (n = 20) and MM patients’ (n = 30) peripheral blood will be collected with written consentient agreement. T lymphocytes from both groups will be obtained by ficoll gradient and further evaluated according to distinct functional assays. Cells will be submitted to distinct protocols of activation such as: anti-CD3/CD28 microbeads; variable concentrations of IL-2 and IL-7 cytokines; and PMA/ionomicin stimulation. After stimulated, we will investigate surface molecules associated to exhaustion and senescence: CTLA-4 CD160, CD244, PD1, CD62L, TIM3and LAG3; transcription factors: GATA-3, FOXP3 and T-bet; and intracellular cytokine/factors production: perforin, granzyme-B, TNF-alpha and IFN-gamma using flow cytometry. In addition, migration assays will be performed by cultivating T cells in trans well system in the presence of CXCL9 and CXCL10 chemokines. Moreover, we will evaluate differences in the activity of enzymes related to cell metabolism: EC2.7.1.1, EC 1.1.1.49, EC 2.7, and EC 2.7.1.40. Results: Preliminary set-up experiments revealed that T cells from HD individuals respond to the distinct stimulators by up-regulating all markers of activation. In addition, T cells cultivated in the presence high dose of IL-2 for five days showed an important rate of mortality.

Experiments using T lymphocytes from MM patients are ongoing and will delineate the molecular and functional status of these cells in the course of the disease.