A strategy for uncovering germline variants altering anti-tumor CD8 T cell response

The treatment of malignant melanoma is being revolutionised by the deployment of T cell-augmenting immunotherapies (Waldman et al., 2020). However, only a subset of patients respond positively to these treatments (Larkin et al., 2015). Current evidence suggests that patients with a pre-existing CD8 T cell response in the tumor microenvironment (TME) benefit more from T cell augmenting immunotherapy (Erdag et al., 2012; Daud et al., 2016). It is speculated that genetic differences among human individuals may contribute to certain aspects of the anti-tumor CD8 T cell response in the TME (Kent et al., 2021). However, major technological challenges associated with the functional characterization of human genetic variation linked to tumor antigen-specific CD8 T cell responses have hindered progress in addressing these speculations. Here we suggest a convenient strategy for uncovering human genetic variants linked to tumor antigen-specific CD8 T cell responses in metastatic melanoma.

Single nucleotide variations (SNVs) are the most common type of genetic variation found in the general population, with an average estimate of about 4–5 million SNVs found in a single person's genome. SNVs may be rare and therefore unique to a few individuals (minor allele frequency, MAF < 0.01) or common and thus found in many individuals (MAF > 0.01). Coding region SNVs (cSNVs), which occur in the evolutionarily conserved phosphotyrosine (pTyr)-based signaling motifs (pTyr-motifs) of membrane-embedded or associated proteins, are highly likely to affect proximal signaling pathways. Hereafter, such membrane-proximal pTyr-motifs altering cSNVs are referred to as pTyr-SNVs. The T cell receptor (TCR) complex's pTyr-motifs act as mediators of the TCR-induced signal transduction process determining whether T cells respond positively or negatively to tumor antigen recognition. While the TCR lacks inherent signal-transducing activity, as it possesses no catalytic kinase or phosphatase domains, it forms a complex with the co-receptors CD3ɣ, CD3δ, CD3ε, and CD3ζ, which partake in the signaling cascade. In addition to the subunits of the TCR complex, non-TCR transmembrane proteins also contain pTyr-motifs in the cytoplasmic segments proximal to cell membranes. These include transmembrane proteins that belong to the class of co-stimulatory and inhibitory molecules (such as CD28, CD152/cytotoxic T-lymphocyte-associated protein 4 (CTLA4), CD278/inducible T-cell costimulator (ICOS), and CD279/programmed cell death protein 1 (PD-1) among others) and co-receptors (such as CD8a and CD8b). The engagement of the TCR/CD3 complex, co-stimulatory molecules, and co-receptors generates distinct membrane-proximal protein tyrosine phosphorylation events. In such events, the amplitude of proximal signaling is controlled in part by the most well-known pTyr-motifs, namely the immunoreceptor tyrosine-based activation motif (ITAM) and the immunoreceptor tyrosine-based inhibition motif (ITIM). Lesser-known pTyr-motifs located in the membrane-proximal segments or the non-catalytic cytoplasmic tails of membrane-associated protein molecules, such as the protein kinase C theta (PKCθ)-docking motif (p)YxxM, the RAS guanyl-releasing protein 1 (RASGRP1)-docking motif P(p)YAP, the growth factor receptor-bound protein 2 (GRB2)-docking motif (p)YxN, and the signal transducer and activator of transcription 3 (STAT3)-recruiting YxxQ motif (SRM), also play a role in modulating TCR-induced proximal signaling.

Upon antigen binding and co-receptor engagement, pre-activated lymphocyte cell-specific protein-tyrosine kinase (LCK) is recruited from the cytosol to the TCR complex. This recruitment of LCK results in the phosphorylation of ITAM contained in CD3 subunits. The CD3γ, CD3δ, and CD3ε subunits each contains a single ITAM, whereas CD3ζ contains three tandem ITAMs, yielding a total of ten ITAMs per TCR complex. Phosphorylation of tyrosines in the ITAM by LCK then leads to the recruitment of several non-receptor protein tyrosine kinases, and a cascade of downstream phosphate transfer signaling pathways is initiated, leading to the production of the second messengers diacylglycerol (DAG) and inositol (1,4,5)-trisphosphate (IP3). This triggers an increase in intracellular calcium and the activation of distal signaling events such as activation and nuclear translocation of several key transcription factors, including activator protein 1 (AP-1), nuclear factor- κB (NF- κB) and nuclear factor of activated T cells (NFAT), which together with non-TCR associated co-stimulatory receptor and cytokine receptor signals, orchestrate multiple T cell responses, such as proliferation, migration, cytokine production, and effector cytotoxic functions.

In addition to the TCR complex and co-receptor protein molecules, the primary structure of T cell co-stimulatory and co-inhibitory molecules, including the immune checkpoint molecules, can determine various aspects of immune regulation and proximal signaling mechanisms (He and Xu, 2020). For example, a mutation of Y191 within the pY(191)xxM motif in CD28, results in the complete loss of phosphatidylinositol 3-kinase (PI3–K) binding (Prasad et al., 1994); Y378F mutation within the ITIM in CD5, ablates Src homology region 2 domain-containing phosphatase-1 (SHP-1) binding (J et al., 1999); a single mutation of Y248F in the TxY(248)xxI motif renders PD-1 non-functional, resulting in significantly high levels of phosphorylated Ak strain transforming RAC-alpha serine/threonine-protein kinase (AKT), activation and expansion of T cells (Chemnitz et al., 2004). Similarly, increased IL-4 production and the exacerbation of autoimmune diseases by effector T cells were found in CTLA4 Y201V knock-in mice, which carry a mutation that disrupts the membrane-proximal YxxM motif in CTLA4 (Stumpf et al., 2014). Recently, in multiple T cell augmenting immunotherapy studies, artificial mutations altering the proximal signaling tyrosine-based sequence motifs were shown to improve the therapeutic outcomes of chimeric antigen receptor (CAR)-T cell therapies (Kagoya et al., 2018; Feucht et al., 2019; Guedan et al., 2020; Meng et al., 2020). Taken together, TCR-induced proximal signaling is a crucial determinant of tumor antigen-specific T cell responses⁠, and mutations in critical segments of proximal signaling proteins modulate TCR signaling outcomes.

In light of these studies, we sought to devise a convenient experimental strategy to facilitate the identification and biological characterization of rare pTyr-SNVs in the human genome that are linked to TCR-induced proximal signaling pathways (Fig. S1). Here we present a strategy that comprises an in vitro experimental setup, faithfully recapitulating tumor antigen-reactive CD8 T cell responses combined with an efficient T cell engineering method that allows the expression and characterization of pTyr-SNVs in tumor antigen-specific CD8 T cells.

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