Squamous cell carcinomas escape immune surveillance via inducing chronic activation and exhaustion of CD8+ T Cells co-expressing PD-1 and LAG-3 inhibitory receptors

Abstract

Squamous cell carcinoma (SCC) is the second commonest type of skin cancer. Moreover, about 90% of head and neck cancers are SCCs. SCCs develop at a significantly higher rate under chronic immunosuppressive conditions, implicating a role of immune surveillance in controlling SCCs. It remains largely unknown how SCCs evade immune recognition. Here, we established a mouse model by injecting tumor cells derived from primary SCCs harboring KrasG12D mutation and Smad4 deletion into wild-type (wt) or CD8−/− recipients. We found comparable tumor growth between wt and CD8−/− recipients, indicating a complete escape of CD8+ T cell-mediated anti-tumor responses by these SCCs. Mechanistically, CD8+ T cells apparently were not defective in infiltrating tumors given their relatively increased percentage among tumor infiltrating lymphocytes (TILs). CD8+ TILs exhibited phenotypes of chronic activation and exhaustion, including overexpression of activation markers, co-expression of programmed cell death 1 (PD-1) and lymphocyte activation gene-3 (LAG-3), as well as TCRβ downregulation. Among CD4+ TILs, T regulatory cells (Tregs) were preferentially expanded. Contradictory to prior findings in melanoma, Treg expansion was independent of CD8+ T cells in our SCC model. Unexpectedly, CD8+ T cells were required for promoting NK cell infiltration within SCCs. Furthermore, we uncovered AKT-dependent lymphocyte-induced PD-L1 upregulation on SCCs, which was contributed greatly by combinatorial effects of CD8+ T and NK cells. Lastly, dual blockade of PD-1 and LAG-3 inhibited the tumor growth of SCCs. Thus, our findings identify novel immune evasion mechanisms of SCCs and suggest that immunosuppressive mechanisms operate in a cancer-type specific and context-dependent manner.

Functional systemic CD 4 immunity is required for clinical responses to PD ‐L1/ PD ‐1 blockade therapy

“…An increasing number of studies are linking PD‐1/LAG‐3 co‐expression in T cells to resistance to anti‐PD‐L1/PD‐1 therapies (Mishra et al , ; Huang et al , ; Williams et al , ; Johnson et al , ). Our study prompts the clinical evaluation of patients with systemic CD4 T‐cell dysfunctionality by PD‐1/LAG‐3 dual‐blockade strategies.…”

Lessons learned from SMAD4 loss in squamous cell carcinomas

“…K15.Kras G12D .Smad4 −/− SCC cells are metastatic in mouse models . K15.Kras G12D .Smad4 −/− SCC tumor transplants develop secondary tumors, despite the infiltration of CD8+ T cell into the primary tumor microenvironment . However, K15.Kras G12D .Smad4 −/− SCC primary tumor growth was unaffected by the presence of CD8+ cells, suggesting that immune evasion or T cell exhaustion.…”

“…Several immune checkpoints have been implicated in preventing cytotoxic T cell killing of tumor cells including programmed cell death 1 (PD‐1), PD‐ligand 1 (PD‐L1), and lymphocyte activation gene‐3 (LAG‐3) . The K15.Kras G12D .Smad4 −/− SCC tumors were infiltrated with CD8+ T cells that were PD‐1+ and LAG‐3+, indicating that these CD8+ cells are dysfunctional for antitumor immunity . Furthermore, a large percent of the CD8+ T cells downregulated T cell antigen receptor ß chain, an indication of suppressed CD8+ T cells .…”

“…The K15.Kras G12D .Smad4 −/− SCC tumors were infiltrated with CD8+ T cells that were PD‐1+ and LAG‐3+, indicating that these CD8+ cells are dysfunctional for antitumor immunity . Furthermore, a large percent of the CD8+ T cells downregulated T cell antigen receptor ß chain, an indication of suppressed CD8+ T cells . Isolated K15.Kras G12D .Smad4 −/− SCC tumor cells also expressed higher PD‐L1 in response to infiltrating CD8+ T cells and K15.Kras G12D .Smad4 −/− SCC tumor cells isolated from CD8‐null mice had reduced PD‐L1 expression, which further support an immune‐evasion phenotype by these Smad4‐null tumors.…”

“…Alternatively, immunotherapies may be effective against SMAD4 deficient SCCs and there are several FDA approved checkpoint inhibitors that target PD‐1 or PD‐L1. For instance, K15.Kras G12D .Smad4 −/− SCC tumor growth was suppressed by dual‐blockade of immune checkpoint inhibitors, anti‐PD‐1 and anti‐LAG‐3 . Immunotherapies also have the potential to prime the host immune system to recognize tumor neoantigens associated with genomic instability and help prevent future tumor reoccurrence.…”