PD1 and LAG3 converge to limit polyfunctionality and systemic immunity

Abstract

Abstract Targeting PD1 has yielded clinical success across many tumor types, yet a significant proportion of patients remain unresponsive to treatment. Co-expression of PD1 and LAG3 inhibitory receptors (IRs) on CD8+ tumor-infiltrating T cells (TIL) is associated with an exhausted phenotype and dual blockade synergistically limits tumor growth, greater than targeting PD1 alone. The cellular and mechanistic basis for PD1/LAG3 synergy is unknown. We have generated conditional knockin mice that facilitates the CD8+ T cell-restricted deletion of Pdcd1 and/or Lag3 when crossed to E8ICre.GFP. These mice have also been crossed with a pMEL TCR transgenic mouse, congenically marked to allow intrinsic analysis of PD1 and/or LAG3-deficient CD8+ T cells, and controls, in a ‘quad’ co-adoptive transfer system. Pdcd1L/L and/or Lag3L/L-yfp E8ICre.GFP mice show reduced B16-F10 tumor growth with improved survival, compared to Lag3L/L-yfp E8ICre.GFP mice and controls. CD8+ TIL frequency is increased with loss of both IRs, as a result of enhanced proliferation. Although expression of TIM3, TIGIT and 2B4 IRs is maintained, CD8+ TIL isolated from Pdcd1L/LE8ICre.GFP and Pdcd1L/ Lag3L/L-yfp E8ICre.GFP mice show increased polyfunctionality, by IFNg, TNFa and GzmB release. This was confirmed to be largely driven by effector and chemoattractive secretions by analysis with a 28-plex single-cell cytokine response panel (Isoplexis). Overall, these data suggest that PD1 and LAG3 synergize to have a dominant effect on CD8+ TIL and limit antitumor immune effects, as intrinsic removal of both IRs results in reduced tumor growth that substantially impacts anti-tumor immunity. These results are encouraging for the development of co-targeting LAG3 and PD1 in the clinic.