The effect of blocking immune checkpoints LAG-3 and PD-1 on human invariant Natural Killer T cell function

Invariant Natural Killer T (iNKT) cells undergo immune exhaustion during chronic activation caused by cancer and viral infections, such as HIV. Exhaustion is marked by cell dysfunction and increased expression of immune checkpoint proteins programmed cell-death-1 (PD-1) and lymphocyte-activation-gene-3 (LAG-3). We hypothesize that blockade of PD-1 and/or LAG-3 will enhance iNKT cell function. Utilizing peripheral blood mononuclear cells from healthy donors, LAG-3 and PD-1 expression on iNKT cells was assessed using flow cytometry following in vitro stimulation with iNKT-specific stimulant α-galactosylceramide (n = 4). Efficacy of anti-LAG-3 and/or anti-PD-1 antibody blockades in enhancing iNKT cell function was assessed by determining proliferative capacity and IFN-γ production (n = 9). LAG-3 and PD-1 expression on iNKT cells peaked at Day 4 (98.8%; p ≤ 0.0001 and 98.8%; p = 0.005, respectively), followed by steep decrease by Day 10, coinciding with peak iNKT cell proliferation. In a 10-day blocking assay, both the anti-PD-1 alone and dual anti-PD-1 and anti-LAG-3 significantly increased iNKT proliferation (6 and 6.29 log2 fold-change respectively) compared to the no blockade control (ANOVA-p = 0.0005) with the dual blockade system being more effective (t-test-p = 0.013). This provides proof-of-concept for LAG-3 and PD-1 as immunotherapeutic targets to enhance human iNKT cell function, with the long-term goal of addressing immune exhaustion.