Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas

Guoying Zhou, Dave Sprengers, Patrick P.C. Boor, Michail Doukas, Hannah Schutz, Shanta Mancham, Alexander Pedroza-Gonzalez, Wojciech G. Polak, Jeroen de Jonge, Marcia Gaspersz, Haidong M Dong, Kris Thielemans, Qiuwei Pan, Jan N.M. IJzermans, Marco J. Bruno, Jaap Kwekkeboom

Research output: Contribution to journalArticle

70 Citations (Scopus)

Abstract

Background & Aims Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell–mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non–small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4+ and CD8+ T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions. Methods We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8+ and CD4+ T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. Results Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8+ and CD4+ T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8+ TIL, compared with other CD8+ TIL. Compared with TIL that did not express these inhibitory receptors, CD8+ and CD4+ TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8+ and CD4+ TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions. Conclusions The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer.

Original languageEnglish (US)
Pages (from-to)1107-1119.e10
JournalGastroenterology
Volume153
Issue number4
DOIs
StatePublished - Oct 1 2017

Fingerprint

Tumor-Infiltrating Lymphocytes
Hepatocellular Carcinoma
Lymphocytes
T-Lymphocytes
Antibodies
Neoplasm Antigens
Neoplasms
Cytotoxic T-Lymphocytes
Cell Death
Granzymes
Programmed Cell Death 1 Receptor
Liver
Cytokines
Ligands
CD4 Antigens
Proteins
Liver Neoplasms
Renal Cell Carcinoma
Non-Small Cell Lung Carcinoma
Dendritic Cells

Keywords

  • Galectin 9
  • GPC3
  • Immunotherapy
  • MAGEC2

ASJC Scopus subject areas

  • Gastroenterology

Cite this

Zhou, G., Sprengers, D., Boor, P. P. C., Doukas, M., Schutz, H., Mancham, S., ... Kwekkeboom, J. (2017). Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas. Gastroenterology, 153(4), 1107-1119.e10. https://doi.org/10.1053/j.gastro.2017.06.017

Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas. / Zhou, Guoying; Sprengers, Dave; Boor, Patrick P.C.; Doukas, Michail; Schutz, Hannah; Mancham, Shanta; Pedroza-Gonzalez, Alexander; Polak, Wojciech G.; de Jonge, Jeroen; Gaspersz, Marcia; Dong, Haidong M; Thielemans, Kris; Pan, Qiuwei; IJzermans, Jan N.M.; Bruno, Marco J.; Kwekkeboom, Jaap.

In: Gastroenterology, Vol. 153, No. 4, 01.10.2017, p. 1107-1119.e10.

Research output: Contribution to journalArticle

Zhou, G, Sprengers, D, Boor, PPC, Doukas, M, Schutz, H, Mancham, S, Pedroza-Gonzalez, A, Polak, WG, de Jonge, J, Gaspersz, M, Dong, HM, Thielemans, K, Pan, Q, IJzermans, JNM, Bruno, MJ & Kwekkeboom, J 2017, 'Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas', Gastroenterology, vol. 153, no. 4, pp. 1107-1119.e10. https://doi.org/10.1053/j.gastro.2017.06.017
Zhou, Guoying ; Sprengers, Dave ; Boor, Patrick P.C. ; Doukas, Michail ; Schutz, Hannah ; Mancham, Shanta ; Pedroza-Gonzalez, Alexander ; Polak, Wojciech G. ; de Jonge, Jeroen ; Gaspersz, Marcia ; Dong, Haidong M ; Thielemans, Kris ; Pan, Qiuwei ; IJzermans, Jan N.M. ; Bruno, Marco J. ; Kwekkeboom, Jaap. / Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas. In: Gastroenterology. 2017 ; Vol. 153, No. 4. pp. 1107-1119.e10.
@article{aac683f7741c4a5aa1be771dff10aedf,
title = "Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas",
abstract = "Background & Aims Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell–mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non–small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4+ and CD8+ T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions. Methods We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8+ and CD4+ T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. Results Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8+ and CD4+ T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8+ TIL, compared with other CD8+ TIL. Compared with TIL that did not express these inhibitory receptors, CD8+ and CD4+ TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8+ and CD4+ TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions. Conclusions The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer.",
keywords = "Galectin 9, GPC3, Immunotherapy, MAGEC2",
author = "Guoying Zhou and Dave Sprengers and Boor, {Patrick P.C.} and Michail Doukas and Hannah Schutz and Shanta Mancham and Alexander Pedroza-Gonzalez and Polak, {Wojciech G.} and {de Jonge}, Jeroen and Marcia Gaspersz and Dong, {Haidong M} and Kris Thielemans and Qiuwei Pan and IJzermans, {Jan N.M.} and Bruno, {Marco J.} and Jaap Kwekkeboom",
year = "2017",
month = "10",
day = "1",
doi = "10.1053/j.gastro.2017.06.017",
language = "English (US)",
volume = "153",
pages = "1107--1119.e10",
journal = "Gastroenterology",
issn = "0016-5085",
publisher = "W.B. Saunders Ltd",
number = "4",

}

TY - JOUR

T1 - Antibodies Against Immune Checkpoint Molecules Restore Functions of Tumor-Infiltrating T Cells in Hepatocellular Carcinomas

AU - Zhou, Guoying

AU - Sprengers, Dave

AU - Boor, Patrick P.C.

AU - Doukas, Michail

AU - Schutz, Hannah

AU - Mancham, Shanta

AU - Pedroza-Gonzalez, Alexander

AU - Polak, Wojciech G.

AU - de Jonge, Jeroen

AU - Gaspersz, Marcia

AU - Dong, Haidong M

AU - Thielemans, Kris

AU - Pan, Qiuwei

AU - IJzermans, Jan N.M.

AU - Bruno, Marco J.

AU - Kwekkeboom, Jaap

PY - 2017/10/1

Y1 - 2017/10/1

N2 - Background & Aims Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell–mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non–small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4+ and CD8+ T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions. Methods We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8+ and CD4+ T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. Results Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8+ and CD4+ T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8+ TIL, compared with other CD8+ TIL. Compared with TIL that did not express these inhibitory receptors, CD8+ and CD4+ TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8+ and CD4+ TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions. Conclusions The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer.

AB - Background & Aims Ligand binding to inhibitory receptors on immune cells, such as programmed cell death 1 (PD-1) and cytotoxic T-lymphocyte associated protein 4 (CTLA4), down-regulates the T-cell–mediated immune response (called immune checkpoints). Antibodies that block these receptors increase antitumor immunity in patients with melanoma, non–small-cell lung cancer, and renal cell cancer. Tumor-infiltrating CD4+ and CD8+ T cells in patients with hepatocellular carcinoma (HCC) have been found to be functionally compromised. We analyzed HCC samples from patients to determine if these inhibitory pathways prevent T-cell responses in HCCs and to find ways to restore their antitumor functions. Methods We collected HCC samples from 59 patients who underwent surgical resection from November 2013 through May 2017, along with tumor-free liver tissues (control tissues) and peripheral blood samples. We isolated tumor-infiltrating lymphocytes (TIL) and intra-hepatic lymphocytes. We used flow cytometry to quantify expression of the inhibitory receptors PD-1, hepatitis A virus cellular receptor 2 (TIM3), lymphocyte activating 3 (LAG3), and CTLA4 on CD8+ and CD4+ T cells from tumor, control tissue, and blood; we studied the effects of antibodies that block these pathways in T-cell activation assays. Results Expression of PD-1, TIM3, LAG3, and CTLA4 was significantly higher on CD8+ and CD4+ T cells isolated from HCC tissue than control tissue or blood. Dendritic cells, monocytes, and B cells in HCC tumors expressed ligands for these receptors. Expression of PD-1, TIM3, and LAG3 was higher on tumor-associated antigen (TAA)-specific CD8+ TIL, compared with other CD8+ TIL. Compared with TIL that did not express these inhibitory receptors, CD8+ and CD4+ TIL that did express these receptors had higher levels of markers of activation, but similar or decreased levels of granzyme B and effector cytokines. Antibodies against CD274 (PD-ligand1 [PD-L1]), TIM3, or LAG3 increased proliferation of CD8+ and CD4+ TIL and cytokine production in response to stimulation with polyclonal antigens or TAA. Importantly, combining antibody against PD-L1 with antibodies against TIM3, LAG3, or CTLA4 further increased TIL functions. Conclusions The immune checkpoint inhibitory molecules PD-1, TIM3, and LAG3 are up-regulated on TAA-specific T cells isolated from human HCC tissues, compared with T cells from tumor-free liver tissues or blood. Antibodies against PD-L1, TIM3, or LAG3 restore responses of HCC-derived T cells to tumor antigens, and combinations of the antibodies have additive effects. Strategies to block PD-L1, TIM3, and LAG3 might be developed for treatment of primary liver cancer.

KW - Galectin 9

KW - GPC3

KW - Immunotherapy

KW - MAGEC2

UR - http://www.scopus.com/inward/record.url?scp=85031662977&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85031662977&partnerID=8YFLogxK

U2 - 10.1053/j.gastro.2017.06.017

DO - 10.1053/j.gastro.2017.06.017

M3 - Article

C2 - 28648905

AN - SCOPUS:85031662977

VL - 153

SP - 1107-1119.e10

JO - Gastroenterology

JF - Gastroenterology

SN - 0016-5085

IS - 4

ER -