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}$$PD-1^{high}$ $CD8^+$ TILs exhibited higher levels of tumor reactivity and T-cell activation markers and significant enrichment for T-cell activation gene signatures. Per-patient analysis revealed positive correlations between percentages of $4-1BB^{pos}$ cells among $CD8^+$ TILs and levels of parameters of tumor reactivity and T-cell activation. Among highly exhausted $PD-1^{high}$ $CD8^+$ TILs, $4-1BB^{pos}$ cells harbored higher proportions of cells with proliferative and reinvigoration potential. 4-1BB agonistic antibodies enhanced the function of $CD8^+$ TILs and further enhanced the anti-PD-1-mediated reinvigoration of $CD8^+$ TILs, especially in cases showing high levels of T-cell activation. In conclusion, $CD8^+$ TILs in HCC patients exhibited heterogeneous features of T-cell exhaustion and activation determined by differential expression levels of PD-1 and 4-1BB. Based on the heterogeneity of $CD8^+$ TILs, individualized immunotherapeutic strategies such as combined checkpoint blockade or co-stimulation may provide better therapeutic outcomes.
T 세포 탈진은 T 세포에서 면역관문 수용체의 과발현과 동반된 기능 저하로 정의되며, 이는 만성 바이러스 감염에서 보고되었지만, 최근 간세포암을 포함한 악성 종양에서도 관찰되고 있으며 종양내 활성화와도 밀접하게 연관되어 있다. 이에 간세포암 환자의 종양-침투 $CD8^+$ T 세포의 T 세포 탈진 및 활성화의 특성을 연구하였다. 종양조직, 비종양 간조직, 말초 혈액에서 림프구를 분리하여 유세포 분석, RNA-seq과 T 세포 기능분석을 하였다. $PD-1^{high}$, $PD-1^{int}$ 그리고 PD-1neg 종양-침투 $CD8^+$ T 세포들은 특징적인 유전자 발현 양상을 보였고, $PD-1^{high}$ 세포들에서 T 세포 탈진의 전형적인 특징들이 관찰되었다. $PD-1^{high}$ 세포군의 존재 유무에 따라서 서로 다른 환자군을 제시하였고, 이들 세포군이 있는 환자에서 더 공격적인 종양의 생물학적인 특성이 관찰되었다. 이들의 종양-침투 $CD8^+$ T 세포에서만 다른 면역 관문 수용체인 TIM-3, LAG-3가 동시의 발현되었고, 이들의 기능은 복합 면역 관문 억제로 추가적으로 회복되었다. TIGIT을 발현하는 종양-침투 $CD8^+$ T 세포는 CD226 부재시에만 더 진행된 탈진의 특성을 보였다. 종양-침투 $CD8^+$ T 세포 에서 보조자극 수용체의 발현을 비교하였고, 4-1BB가 특징적으로 $PD-1^{high}$ 세포에 과발현됨을 확인하였다. $4-1BB^{pos}$$PD-1^{high}$ 종양-침투 $CD8^+$ T 세포들은 종양-반응성과 T 세포 활성화를 나타내는 지표와 T 세포 증식 및 재활성화와 관련된 지표도 더 높은 것을 확인하였다. 4-1BB의 항진 항체는 종양-침투 $CD8^+$ T 세포의 기능과 PD-1 차단으로 이한 T 세포 재활성화을 더욱 촉진하였고, 이는 T 세포의 활성화 정도가 높은 종양에서 더 특징적이었다. 결론적으로,간세포암 환자의 종양-침투 $CD8^+$ T 세포는 이질적인 T 세포 탈진 및 활성화 특징을 보이며, 이로 인한 개별적인 면역항암치료가 적용될 것으로 기대된다.
