T-Cell Exhaustion Determined by PD-1 Expression on Tumor-Infiltrating CD8 T-Cells in Hepatocellular Carcinoma

( Shin Hwang ),( Hyung-don Kim ),( Su-hyung Park ),( Gi-won Song ),( Seongyeol Park ),( Min Kyung Jung ),( Kyung Hwan Kimv ),( Sukyeong Eo ),( Deok-bog Moon ),( Young Seok Ju ),( Eui-cheol Shin ) 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: Phenotypic and functional characteristics of tumor-infiltrating CD8 T-cells (CD8 TILs) has not been well studied in the context of heterogeneity of exhausted T-cells and patient stratification in hepatocellular carcinoma (HCC). Methods: We identified distinct T-cell exhaustion profiles determined by PD-1 expression levels on CD8 TILs from 48 HCC patients undergoing surgical resection by multi-color flow cytometry and RNA sequencing. Results: CD8 TILs from HCC patients significantly overexpressed immune checkpoint receptors such as PD-1, TIM-3 and LAG-3. CD8 TILs could be further stratified into PD-1^hi, PD-1^int and PD-1^neg subpopulations, and TIM-3 and/or LAG-3 were exclusively expressed on PD-1^hi, not PD-1^int, subpopulation. RNA expression profiles were distinct among PD-1^hi, PD-1^int and PD-1^neg CD8 TILs and a gene module of T-cell exhaustion was significantly enriched in PD-1^hi CD8 TILs. HCC patients could be classified into two distinct subgroups based on the proportion of PD-1^hi CD8 TILs; High PD-1 expressers and Low PD-1 expressers. High PD-1 expressers had more severely dysfunctional CD8 TILs with exhaustion profiles that were observed in PD-1^hi CD8 TILs, compared to Low PD-1 expressers. High PD-1 expressers were associated with aggressive biologic tumor features. Combination blockades of Tim-3 or LAG-3 in addition to PD-1 blockade further enhanced proliferative responses of CD8 TILs from High PD-1 expressers. Conclusions: CD8 TILs displayed heterogeneous exhaustion profiles determined by differential PD-1 expression levels in HCC patients. A certain subgroup of HCC patients expressing multiple immune checkpoint receptors appears to be eligibly indicated for immune checkpoint inhibitors for effective reinvigoration of anti-tumor immunity.

Heterogeneous Exhaustion Status of Tumor-Infiltrating CD8 T Cells Determines Distinct Subgroups of Hepatocellular Carcinoma Patients

( Hyung-don Kim ),( Gi-won Song ),( Seongyeol Park ),( Min Kyung Jung ),( Kijong Yi ),( Min Hwan Kim ),( Hyo Jeong Kang ),( Chang-hoon Yoo ),( Kyung Hwan Kim ),( Sukyeong Eo ),( Deok-bog Moon ),( Seun 대한간학회 2018 춘·추계 학술대회 (KASL) Vol.2018 No.1

Aims: 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). We investigated features of exhaustion in CD8⁺ T cells isolated from HCC specimens. Methods: We obtained HCC specimens, along with adjacent non-tumor tissues and blood samples, from 79 patients who underwent surgical resection at Asan Medical Center (in Seoul, Korea) from April 2016 through December 2017. Intrahepatic lymphocytes and tumor-infiltrating T cells were analyzed by flow cytometry. Tumor-infiltrating CD8⁺ T cells were sorted by flow cytometry into populations based on expression level of programmed cell death 1 (PDCD1 or PD1): PD1-high, PD1-intermediate, and PD1-negative. Sorted cells were analyzed by RNA-seq. Proliferation and production of interferon gamma (IFNG) and tumor necrosis factor (TNF) by CD8⁺ T cells were measured in response to anti-CD3 and antibodies against immune checkpoint receptors including PD1, hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3), lymphocyte activating 3 (LAG3), or isotype control. Tumor-associated antigen-specific CD8⁺ T cells were identified using HLA-A^*0201 dextramers. Results: PD1-high, PD1-intermediate, and PD1-negative CD8+ T cells from HCCs had distinct gene expression profiles. PD1-high cells expressed higher levels of genes that regulate T-cell exhaustion than PD1-intermediate cells. PD1-high cells expressed TIM3 and LAG3, and a low proportion was TCF1⁺, TBET^high/ eomesodermin^low, and CD127+. PD1-high cells produced the lowest amounts of IFNG and TNF upon anti-CD3 stimulation. Differences in proportions of PD1-high CD8⁺ T cells led to the identification of 2 subgroups of HCCs; HCCs with a larger proportion of PD1-high cells were more aggressive than HCCs with a smaller proportion. Incubation of CD8⁺ T cells from HCCs with high proportions of PD1-high cells with antibodies against PD1 and TIM3 or LAG3 further restored proliferation and production of IFNG and TNF in response to anti-CD3. Conclusions: We found HCC specimens to contain CD8⁺ T cells that express different levels of PD1. HCCs with high proportions of PD1-high CD8⁺ T cells express TIM3 and/or LAG3 and produce low levels of IFNG and TNF in response to anti-CD3. Incubation of these cells with antibodies against PD1 and TIM3 or LAG3 further restore proliferation and production of cytokines; HCCs with high proportions of PD1-high CD8⁺ T cells might be more susceptible to combined immune checkpoint blockade-based therapies.

Association Between Expression Level of PD1 by Tumor-Infiltrating CD8⁺ T Cells and Features of Hepatocellular Carcinoma

Kim, Hyung-Don,Song, Gi-Won,Park, Seongyeol,Jung, Min Kyung,Kim, Min Hwan,Kang, Hyo Jeong,Yoo, Changhoon,Yi, Kijong,Kim, Kyung Hwan,Eo, Sukyeong,Moon, Deok-Bog,Hong, Seung-Mo,Ju, Young Seok,Shin, Eui- Elsevier 2018 Gastroenterology Vol.155 No.6

<P><B>Background & Aims</B></P> <P>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). We investigated features of exhaustion in CD8<SUP>+</SUP> T cells isolated from HCC specimens.</P> <P><B>Methods</B></P> <P>We obtained HCC specimens, along with adjacent nontumor tissues and blood samples, from 90 patients who underwent surgical resection at Asan Medical Center (Seoul, Korea) from April 2016 through April 2018. Intrahepatic lymphocytes and tumor-infiltrating T cells were analyzed by flow cytometry. Tumor-infiltrating CD8<SUP>+</SUP> T cells were sorted by flow cytometry into populations based on expression level of programmed cell death 1 (PDCD1 or PD1): PD1-high, PD1-intermediate, and PD1-negative. Sorted cells were analyzed by RNA sequencing. Proliferation and production of interferon gamma (IFNG) and tumor necrosis factor (TNF) by CD8<SUP>+</SUP> T cells were measured in response to anti-CD3 and antibodies against immune checkpoint receptors including PD1, hepatitis A virus cellular receptor 2 (HAVCR2 or TIM3), lymphocyte activating 3 (LAG3), or isotype control. Tumor-associated antigen-specific CD8<SUP>+</SUP> T cells were identified using HLA-A*0201 dextramers. PDL1 expression on tumor tissue was assessed by immunohistochemistry.</P> <P><B>Results</B></P> <P>PD1-high, PD1-intermediate, and PD1-negative CD8<SUP>+</SUP> T cells from HCCs had distinct gene expression profiles. PD1-high cells expressed higher levels of genes that regulate T-cell exhaustion than PD1-intermediate cells. PD1-high cells expressed TIM3 and LAG3, and low proportions of TCF1<SUP>+</SUP>, TBET<SUP>high</SUP>/eomesodermin<SUP>low</SUP>, and CD127<SUP>+</SUP>. PD1-high cells produced the lowest amounts of IFNG and TNF upon anti-CD3 stimulation. Differences in the PD1 expression patterns of CD8<SUP>+</SUP> T cells led to the identification of 2 subgroups of HCCs: HCCs with a discrete population of PD1-high cells were more aggressive than HCCs without a discrete population of PD1-high cells. HCCs with a discrete population of PD1-high cells had higher levels of predictive biomarkers of response to anti-PD1 therapy. Incubation of CD8<SUP>+</SUP> T cells from HCCs with a discrete population of PD1-high cells with antibodies against PD1 and TIM3 or LAG3 further restored proliferation and production of IFNG and TNF in response to anti-CD3.</P> <P><B>Conclusions</B></P> <P>We found HCC specimens to contain CD8<SUP>+</SUP> T cells that express different levels of PD1. HCCs with a discrete population of PD1-high CD8<SUP>+</SUP> T cells express TIM3 and/or LAG3 and produce low levels of IFNG and TNF in response to anti-CD3. Incubation of these cells with antibodies against PD1 and TIM3 or LAG3 further restore proliferation and production of cytokines; HCCs with a discrete population of PD1-high CD8<SUP>+</SUP> T cells might be more susceptible to combined immune checkpoint blockade–based therapies.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>