The activation of T cells from a memory to an effector state depends on the integration of the positive signal from the T cell receptor upon binding with a cognate antigen and the negative signals from any of several inhibitory signaling receptors (ISRs) binding their cognate ligands on the antigenic cell’s surface. ISRs, typified by receptors like PD-1 and CTLA-4, downregulate TCR-mediated signal transduction and raise the threshold of immune activation, preventing the effector phase. In this study, Alan Frey identified protocadherin-18 (pcdh18) as a novel ISR expressed in memory T cells. The only known ligand to pcdh18 is itself, which functions in trans.

In a previous study by the Frey lab, pcdh18 was found to be expressed on dysfunctional tumor infiltrating lymphocytes (TILs) from MCA38 colon tumor-bearing mice. These TILs lacked lytic function against pcdh18-expressing MCA38 cells immediately after isolation, however, their function could be restored after brief culture, suggesting that the interaction with tumor cells was related to their dysfunction. In this new work, Frey used shRNA to knock down pcdh18 on tumor cells, and TILs from the tumors were functional immediately upon isolation. Similarly, in mice engineered to lack pcdh18 expression on T cells (pcdh18^-/-), tumor growth was significantly delayed compared to wild type mice, suggesting that pcdh18^-/- T cells were more effective against pcdh18-expressing MCA38 tumors than pcdh18⁺ T cells. Together, this data confirms Frey’s hypothesis that pcdh18 on tumor cells (acting as ligand) initiates the inhibitory function of pcdh18 on T cells (acting as receptor).

To determine why tumor growth was only delayed, and not entirely halted in pcdh18^-/- mice, Frey analyzed the gene expression of TILs isolated from these animals. While their gene expression profiles showed significant upregulation of immune function genes related to T cell activation, signaling, effector functions, and chemokine and cytokine receptors, they also showed increased expression of genes encoding other known negative regulators, including PD-1 and CTLA-4.

Since PD-1 is highly expressed on wild-type TILs, and further upregulated on TILs from pcdh18^-/- mice, Frey tested the addition of anti-PD-1 therapy in both wild type and pcdh18^-/- models. In wild type mice, anti-PD-1 delayed tumor growth, but only when administered at an early stage (day 5). In pcdh18^-/- mice, the addition of anti-PD-1 further inhibited tumor growth, even if treatment was delayed (to day 10). TILs from the combination group also produced slightly more IFNγ than those treated with PD-1 blockade alone. Depletion of CD8⁺ T cells abrogated the effect of anti-PD-1 in both mouse models, indicating their essential role in mediating the delay of tumor growth.

While genetically knocking out pcdh18 helped prove its role as an ISR, Frey developed and tested a murine anti-pcdh18 serum in anticipation of the development of a clinical therapy. As monotherapies, anti-PD-1 was still more effective than anti-pcdh18, which only slightly delayed tumor growth. Combining the two antibody therapies, however, was much more effective than the use of either as a single agent checkpoint blockade, suggesting anti-pcdh18 could be used to improve the efficacy of anti-PD-1 therapies.

In a translational study, Frey explored the expression of pcdh18 in human T cells. Immunohistochemistry of a normal human lymph node showed regions where low proportions (~5%) of both CD4⁺ and CD8⁺ cells expressed pcdh18. Using freshly isolated peripheral blood mononuclear cells and fluorescence-activated cell sorting, high pcdh18 expression was observed in CD4⁺ and CD8⁺ effector memory T cell populations, and conversion of central memory CD8⁺ T cells to an effector memory phenotype demonstrated upregulation of pcdh18 protein. Finally, in vitro activation of human CD8⁺ effector memory T cells coincided with an increase in pcdh18 expression and loss in viability; anti-pcdh18 sera rescued viability and restored INFγ expression.

The expression of pcdh18 in human memory T cells further supports its role as an ISR with regulatory control over the effector phase, opening the door to its potential as a clinically-relevant target for immunotherapy, especially in conjunction with anti-PD1 therapy.

by Lauren Hitchings