Heterogeneous features of T-cell exhaustion and activation of hepatocellular carcinoma-infiltrating $CD8^+$ T cells

Abstract

T-cell exhaustion, or an impaired capacity to secrete cytokines and proliferate with overexpression of immune checkpoint receptors, occurs during chronic viral infections but has also been observed in tumors, including hepatocellular carcinomas (HCCs). T-cell exhaustion is also closely interconnected to T-cell activation, given that T-cell exhaustion arises due to excessive and prolonged T-cell activation to prevent immunopathology. I investigated features of exhaustion and activation in $CD8^+$ T cells isolated from HCC specimens. I obtained tumor specimens, along with adjacent non-tumor tissues and blood samples, from HCC patients who underwent surgical resection. Lymphocytes were isolated and used for multi-color flow cytometry, RNA-sequencing and in vitro functional restoration assays. $PD-1^{high}$, $PD-1^{int}$ and $PD-1^{neg}$ tumor-infiltrating $CD8^+$ T cells ($CD8^+$ TILs) had distinct gene expression profiles. $PD-1^{high}$ $CD8^+$ TILs expressed higher levels of genes that regulate T-cell exhaustion than $PD-1^{int}$ CD$8^+$ TILs. $PD-1^{high}$ $CD8^+$ TILs expressed TIM-3 and LAG-3, and a low proportion was $TCF-1^+$, T-bethigh/Eomeslow, and $CD127^+$. Differences in proportions of $PD-1^{high}$ $CD8^+$ TILs led to the identification of 2 subgroups of HCCs

A subgroup of HCC patients with enrichment of $PD-1^{high}$ $CD8^+$ TILs was associated with more aggressive biologic tumor features and severe T-cell dysfunction, and expressed multiple immune checkpoint receptors on CD8 TILs that were effectively reinvigorated by combined blockade of PD-1 along with TIM-3 or LAG-3. TIGIT-expressing $CD8^+$ TILs showed features of more progressed T-cell exhaustion when these cells were negative for CD226. I next examined the expression of co-stimulatory receptors on $CD8^+$ TILs to investigate features of T-cell activation. Among the examined co-stimulatory receptors, 4-1BB was most prominently expressed on $CD8^+$ TILs. 4-1BB expression was almost exclusively detected on $CD8^+$ T cells in the tumor—especially on $PD-1^{high}$ cells and not $PD-1^{int}$ and PD-1neg cells. Compared to $PD-1^{int}$ and $PD-1^{high}$ $4-1BB^{neg}$ $CD8^+$ TILs, $4-1BB^{pos}$$PD-1^{high}$ $CD8^+$ TILs exhibited higher levels of tumor reactivity and T-cell activation markers and significant enrichment for T-cell activation gene signatures. Per-patient analysis revealed positive correlations between percentages of $4-1BB^{pos}$ cells among $CD8^+$ TILs and levels of parameters of tumor reactivity and T-cell activation. Among highly exhausted $PD-1^{high}$ $CD8^+$ TILs, $4-1BB^{pos}$ cells harbored higher proportions of cells with proliferative and reinvigoration potential. 4-1BB agonistic antibodies enhanced the function of $CD8^+$ TILs and further enhanced the anti-PD-1-mediated reinvigoration of $CD8^+$ TILs, especially in cases showing high levels of T-cell activation. In conclusion, $CD8^+$ TILs in HCC patients exhibited heterogeneous features of T-cell exhaustion and activation determined by differential expression levels of PD-1 and 4-1BB. Based on the heterogeneity of $CD8^+$ TILs, individualized immunotherapeutic strategies such as combined checkpoint blockade or co-stimulation may provide better therapeutic outcomes.