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Background: Checkpoint inhibition is a potent strategy to reinvigorate T cells. However, aCTLA-4 or aPD-1 monotherapy has not been effective for the majority of patients, resulting in the exploration of combinatorial approaches to improve treatment efficacy. One such target is LAG-3, which is upregulated on T cells that have experienced repeated antigen exposure, such as in the tumor microenvironment (TME), and is associated with reduced T cell effector function. In addition, high LAG-3 expression on regulatory T cells (Tregs) has been reported for patients with varying cancer types, providing an additional rationale for targeting LAG-3 with the aim of reducing immune suppression within the TME. We hypothesized that the combination of aPD-1 and aLAG-3 would synergize to promote tumor regression and increase survival via a reduction in tumor-induced immune suppression and enhanced CD8+ T cell effector function.
Methods: CT26 (colon carcinoma) tumor-bearing BALB/c mice received aPD-1 and/or aLAG-3 (200 μg/dose; ip) 3x/week on days 7, 10, and 13 post-tumor implant. Tumor growth (area) was assessed 2-3x/week and mice were sacrificed when tumors exceeded 150 mm2. In additional cohorts, tumors were harvested 7 days post-treatment (d17) and tumor- infiltrating lymphocytes (TIL) were analyzed by flow cytometry. Responders to combined aPD-1/aLAG3 therapy were designated as those exhibiting decreased tumor size on the day of harvest (d17) compared to maximum tumor growth post-implant.
Results: Combined aPD-1/aLAG-3 immunotherapy significantly improved the survival of CT26 tumor-bearing mice compared to monotherapy (p<0.05). Further analysis revealed that aPD-1/aLAG-3 therapy significantly increased the percentage of CD8+ TIL compared to aPD-1 (p<0.01) or aLAG-3 (p<0.05) alone. Additionally, we observed increased effector function in CD8+ TIL from aPD-1/aLAG-3-treated mice, as evidenced by increased cytotoxicity (granzyme A; p<0.05) and cytokine production (TNF-a; p<0.05 and IFN-g; p<0.01). Interestingly, responders to aPD-1/aLAG-3 therapy were enriched for CD8+ TIL with higher cytolytic activity and effector cytokine production, which correlated with a reduction in PD-1 MFI amongst the PD-1+/CD8+ TIL. Lastly, aPD-1/aLAG-3 treatment significantly increased the frequency of effector CD4+ T cells (Teff) compared to FoxP3+CD4+ Tregs (p<0.05).
Conclusions: In summary, these data suggest that aPD-1/aLAG-3 immunotherapy increased recruitment of CD8+ TIL exhibiting enhanced effector function, increased CD4+ Teff/Treg ratios, which likely mitigated Treg-mediated immune suppression. Together, these positive immunological changes led to a more immune stimulatory TME capable of supporting tumor regression and significantly improved tumor-free survival.
Cancer, immunotherapy, checkpoint blockade, PD-1, LAG-3, tumor infiltrating lymphocytes, TIL, T cells, tumor microenvironment, cancer immunity, flow cytometry
Sturgill, Elizabeth; Mick, Courtney; Jenkins, David; Kaufmann, Johanna; and Redmond, William L., "Combined anti-PD-1 and anti-LAG-3 checkpoint blockade enhances CD8+ TIL effector function while reducing Tregs leading to reduced immune suppression and improved overall survival" (2018). Society for Immunotherapy of Cancer 2018 Annual Meeting Posters. 17.