Gene expression of dysfunctional CD8+ T cells from the tumor microenvironment. Mus musculus

Although the presence of tumor-infiltrating lymphocytes (TILs) indicates an endogenous anti-tumor response, immune regulatory pathways can subvert the effector phase and enable tumor escape. Negative regulatory pathways include extrinsic suppression mechanisms but also a T cell-intrinsic dysfunctional state. A more detailed study has been hampered by a lack of cell surface markers defining dysfunctional TILs, and it is clear that PD-1 alone is not sufficient. Recently, we identified the transcription factor Egr2 as a critical component in controlling the anergic state in vitro. In the current study we show that the Egr2-driven cell surface proteins, LAG-3 and 4-1BB, identify dysfunctional tumor antigen-specific CD8+ TIL. Co-expression of 4-1BB and LAG-3 was seen on a majority of CD8+ TIL but not in lymphoid organs. Functional analysis revealed defective IL-2 and TNF-α production yet retained expression of IFN-γ and Treg-recruiting chemokines. Transcriptional and phenotypic characterization revealed co-expression of multiple additional co-stimulatory and co-inhibitory receptors. Administration of anti-LAG-3 plus anti-4-1BB mAbs was therapeutic against tumors in vivo, which correlated with a reversal of TIL dysfunction and restored effector phenotype. Our results indicate that co-expression of LAG-3 and 4-1BB characterize dysfunctional T cells within tumors, and that targeting these receptors has therapeutic utility. Overall design: 10 female mice purchased from Taconic farms were engrafted on both flanks with 2 million B16.SIY.dsRed tumor cells. After 14 days tumors were pooled, dissociated through a 50μm filter and washed with PBS 3-4 times. TILs were further enriched by layering Ficoll-Hypaque beneath the cell suspension followed by centrifugation without breaks for 30 min at 400 x g. The buffy-layer was isolated and washed twice with PBS before staining. Cells were sorted into RLT (QIAGEN) buffer and directly transferred to dry ice. RNA was isolated using the RNAeasy Micro RNA isolation Kit (QIAGEN) and submitted for QC and subsequent microarray analysis at the University of Chicago Genomics Core.