Efficacy of PD-1 blockade in cervical cancer is related to a CD8+FoxP3+CD25+ T-cell subset with operational effector functions despite high immune checkpoint levels

AM Heeren, J Rotman, A.G.M. Stam, Noëlle Pocorni, Awa Gassama, S Samuels, Maaike C. G. Bleeker, CH Mom, H.J.M.A.A. Zijlmans, GG Kenter, ES Jordanova, TD de Gruijl

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Abstract

Background: Cervical cancer (CxCa) is mainly a locally invading disease that metastasizes to loco-regional lymph node basins before involving distant organs in more advanced stages. Local immune potentiation of tumor-draining lymph nodes (TDLN) may thus protect against tumor progression. Methods: To identify therapeutic targets for local immune modulation, multi-parameter flow cytometric T-cell profiling of primary cervical tumors (PT) and TDLN (n = 37) was performed. The in-vitro effect of PD-1 blockade on T-cell reactivity to HPV16 E6 oncoproteins was determined in cultures of TDLN and PT single cell suspensions (n = 19). Also, intracellular cytokine staining (ICS) upon anti-CD3 stimulation was performed in metastatic TDLN (LN+) and PT (n = 7), as well as multiplexed immunofluorescence histochemistry staining (n = 8). Results: Our data revealed elevated rates of activated regulatory T cells (aTregs) and of central or effector memory CD8 + T cells in metastatic TDLN (LN+) as compared to tumor-free TDLN (LN-), and equally high or even higher rates of these subsets in PT. Both memory subsets co-expressed multiple immune checkpoints. PD-1 blockade significantly enhanced detectable E6-specific T-cell responses in 4/5 HPV16+ LN+ and in 1/5 HPV16+ PT. Whereas aTreg rates were higher in anti-PD-1 non-responders, in responders elevated levels of CD8 +FoxP3 +CD25 + T cells were observed, which correlated with the efficacy of PD-1 blockade (P = 0.018). This subset was characterized by an early effector memory phenotype with particularly high levels of co-expressed PD-1, CTLA-4, TIM-3 and LAG-3 checkpoints, but, rather than exhausted, was shown upon polyclonal activation to produce higher levels of Granzyme-B and effector cytokines as compared to its CD8 +FoxP3 - counterparts. Conclusion: These observations support local PD-(L)1 blockade to interrupt loco-regional immune suppression in CxCa and control metastatic spread to TDLN. Furthermore, our data identify CD8 +FoxP3 +CD25 + T cells as therapeutic targets, which may also serve as predictive biomarker for PD-(L)1 checkpoint blockade.

Original languageEnglish
Article number43
JournalJournal for Immunotherapy of Cancer
Volume7
Issue number1
DOIs
Publication statusPublished - 12 Feb 2019

Cite this

@article{c18559e9ae754d2c8eacf0cb6ec45b68,
title = "Efficacy of PD-1 blockade in cervical cancer is related to a CD8+FoxP3+CD25+ T-cell subset with operational effector functions despite high immune checkpoint levels",
abstract = "Background: Cervical cancer (CxCa) is mainly a locally invading disease that metastasizes to loco-regional lymph node basins before involving distant organs in more advanced stages. Local immune potentiation of tumor-draining lymph nodes (TDLN) may thus protect against tumor progression. Methods: To identify therapeutic targets for local immune modulation, multi-parameter flow cytometric T-cell profiling of primary cervical tumors (PT) and TDLN (n = 37) was performed. The in-vitro effect of PD-1 blockade on T-cell reactivity to HPV16 E6 oncoproteins was determined in cultures of TDLN and PT single cell suspensions (n = 19). Also, intracellular cytokine staining (ICS) upon anti-CD3 stimulation was performed in metastatic TDLN (LN+) and PT (n = 7), as well as multiplexed immunofluorescence histochemistry staining (n = 8). Results: Our data revealed elevated rates of activated regulatory T cells (aTregs) and of central or effector memory CD8 + T cells in metastatic TDLN (LN+) as compared to tumor-free TDLN (LN-), and equally high or even higher rates of these subsets in PT. Both memory subsets co-expressed multiple immune checkpoints. PD-1 blockade significantly enhanced detectable E6-specific T-cell responses in 4/5 HPV16+ LN+ and in 1/5 HPV16+ PT. Whereas aTreg rates were higher in anti-PD-1 non-responders, in responders elevated levels of CD8 +FoxP3 +CD25 + T cells were observed, which correlated with the efficacy of PD-1 blockade (P = 0.018). This subset was characterized by an early effector memory phenotype with particularly high levels of co-expressed PD-1, CTLA-4, TIM-3 and LAG-3 checkpoints, but, rather than exhausted, was shown upon polyclonal activation to produce higher levels of Granzyme-B and effector cytokines as compared to its CD8 +FoxP3 - counterparts. Conclusion: These observations support local PD-(L)1 blockade to interrupt loco-regional immune suppression in CxCa and control metastatic spread to TDLN. Furthermore, our data identify CD8 +FoxP3 +CD25 + T cells as therapeutic targets, which may also serve as predictive biomarker for PD-(L)1 checkpoint blockade.",
keywords = "Cervical cancer, FoxP3, HPV16 E6, Lymph nodes, PD-1 blockade, T cells",
author = "AM Heeren and J Rotman and A.G.M. Stam and No{\"e}lle Pocorni and Awa Gassama and S Samuels and Bleeker, {Maaike C. G.} and CH Mom and H.J.M.A.A. Zijlmans and GG Kenter and ES Jordanova and {de Gruijl}, TD",
year = "2019",
month = "2",
day = "12",
doi = "https://doi.org/10.1186/s40425-019-0526-z",
language = "English",
volume = "7",
journal = "Journal for Immunotherapy of Cancer",
issn = "2051-1426",
publisher = "BioMed Central",
number = "1",

}

TY - JOUR

T1 - Efficacy of PD-1 blockade in cervical cancer is related to a CD8+FoxP3+CD25+ T-cell subset with operational effector functions despite high immune checkpoint levels

AU - Heeren, AM

AU - Rotman, J

AU - Stam, A.G.M.

AU - Pocorni, Noëlle

AU - Gassama, Awa

AU - Samuels, S

AU - Bleeker, Maaike C. G.

AU - Mom, CH

AU - Zijlmans, H.J.M.A.A.

AU - Kenter, GG

AU - Jordanova, ES

AU - de Gruijl, TD

PY - 2019/2/12

Y1 - 2019/2/12

N2 - Background: Cervical cancer (CxCa) is mainly a locally invading disease that metastasizes to loco-regional lymph node basins before involving distant organs in more advanced stages. Local immune potentiation of tumor-draining lymph nodes (TDLN) may thus protect against tumor progression. Methods: To identify therapeutic targets for local immune modulation, multi-parameter flow cytometric T-cell profiling of primary cervical tumors (PT) and TDLN (n = 37) was performed. The in-vitro effect of PD-1 blockade on T-cell reactivity to HPV16 E6 oncoproteins was determined in cultures of TDLN and PT single cell suspensions (n = 19). Also, intracellular cytokine staining (ICS) upon anti-CD3 stimulation was performed in metastatic TDLN (LN+) and PT (n = 7), as well as multiplexed immunofluorescence histochemistry staining (n = 8). Results: Our data revealed elevated rates of activated regulatory T cells (aTregs) and of central or effector memory CD8 + T cells in metastatic TDLN (LN+) as compared to tumor-free TDLN (LN-), and equally high or even higher rates of these subsets in PT. Both memory subsets co-expressed multiple immune checkpoints. PD-1 blockade significantly enhanced detectable E6-specific T-cell responses in 4/5 HPV16+ LN+ and in 1/5 HPV16+ PT. Whereas aTreg rates were higher in anti-PD-1 non-responders, in responders elevated levels of CD8 +FoxP3 +CD25 + T cells were observed, which correlated with the efficacy of PD-1 blockade (P = 0.018). This subset was characterized by an early effector memory phenotype with particularly high levels of co-expressed PD-1, CTLA-4, TIM-3 and LAG-3 checkpoints, but, rather than exhausted, was shown upon polyclonal activation to produce higher levels of Granzyme-B and effector cytokines as compared to its CD8 +FoxP3 - counterparts. Conclusion: These observations support local PD-(L)1 blockade to interrupt loco-regional immune suppression in CxCa and control metastatic spread to TDLN. Furthermore, our data identify CD8 +FoxP3 +CD25 + T cells as therapeutic targets, which may also serve as predictive biomarker for PD-(L)1 checkpoint blockade.

AB - Background: Cervical cancer (CxCa) is mainly a locally invading disease that metastasizes to loco-regional lymph node basins before involving distant organs in more advanced stages. Local immune potentiation of tumor-draining lymph nodes (TDLN) may thus protect against tumor progression. Methods: To identify therapeutic targets for local immune modulation, multi-parameter flow cytometric T-cell profiling of primary cervical tumors (PT) and TDLN (n = 37) was performed. The in-vitro effect of PD-1 blockade on T-cell reactivity to HPV16 E6 oncoproteins was determined in cultures of TDLN and PT single cell suspensions (n = 19). Also, intracellular cytokine staining (ICS) upon anti-CD3 stimulation was performed in metastatic TDLN (LN+) and PT (n = 7), as well as multiplexed immunofluorescence histochemistry staining (n = 8). Results: Our data revealed elevated rates of activated regulatory T cells (aTregs) and of central or effector memory CD8 + T cells in metastatic TDLN (LN+) as compared to tumor-free TDLN (LN-), and equally high or even higher rates of these subsets in PT. Both memory subsets co-expressed multiple immune checkpoints. PD-1 blockade significantly enhanced detectable E6-specific T-cell responses in 4/5 HPV16+ LN+ and in 1/5 HPV16+ PT. Whereas aTreg rates were higher in anti-PD-1 non-responders, in responders elevated levels of CD8 +FoxP3 +CD25 + T cells were observed, which correlated with the efficacy of PD-1 blockade (P = 0.018). This subset was characterized by an early effector memory phenotype with particularly high levels of co-expressed PD-1, CTLA-4, TIM-3 and LAG-3 checkpoints, but, rather than exhausted, was shown upon polyclonal activation to produce higher levels of Granzyme-B and effector cytokines as compared to its CD8 +FoxP3 - counterparts. Conclusion: These observations support local PD-(L)1 blockade to interrupt loco-regional immune suppression in CxCa and control metastatic spread to TDLN. Furthermore, our data identify CD8 +FoxP3 +CD25 + T cells as therapeutic targets, which may also serve as predictive biomarker for PD-(L)1 checkpoint blockade.

KW - Cervical cancer

KW - FoxP3

KW - HPV16 E6

KW - Lymph nodes

KW - PD-1 blockade

KW - T cells

UR - http://www.scopus.com/inward/record.url?scp=85061491625&partnerID=8YFLogxK

U2 - https://doi.org/10.1186/s40425-019-0526-z

DO - https://doi.org/10.1186/s40425-019-0526-z

M3 - Article

VL - 7

JO - Journal for Immunotherapy of Cancer

JF - Journal for Immunotherapy of Cancer

SN - 2051-1426

IS - 1

M1 - 43

ER -