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

A M Heeren,M C G Bleeker,C H Mom,A A Gassama,N Pocorni,G G Kenter,J Rotman,E S Jordanova,A G M Stam,T D De Gruijl,S Samuels,H J M A A Zijlmans

doi:10.1186/s40425-019-0526-z

Abstract

BackgroundCervical 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.MethodsTo 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).ResultsOur 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.ConclusionThese 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.

Highlights

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
In view of these low response rates on systemic immune checkpoint blockade in the metastatic setting, and since CxCa mainly spreads to adjoining tissues and pelvic lymph nodes before hematogenous spread occurs, we hypothesize that locally applied low-dose immunotherapy in earlier stages of the disease may more effectively achieve tumor control while lowering the risk of immune-related side effects associated with systemic administration of checkpoint inhibitors [18, 19]
Immunophenotyping of T-cell subsets in cervical cancer (CxCa) tumor-draining lymph nodes (TDLN) and primary tumors (PT) and expression of immune checkpoints We assessed the frequencies of various T-cell subsets in single-cell suspensions derived from 27 cervical TDLN and 10 PT

Summary

Introduction

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. Based on a reported overall response rate of 14.3%, of which 2.6% represented complete responses, pembrolizumab was recently approved by the FDA for PD-1 blockade in recurrent or advanced CxCa [16, 17] In view of these low response rates on systemic immune checkpoint blockade in the metastatic setting, and since CxCa mainly spreads to adjoining tissues and pelvic lymph nodes before hematogenous spread occurs, we hypothesize that locally applied low-dose immunotherapy in earlier stages of the disease may more effectively achieve tumor control while lowering the risk of immune-related side effects associated with systemic administration of checkpoint inhibitors [18, 19]

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