Level of PD-1 expression on CD8+ T cells influence prognosis and respond to PD-1 therapy in a murine model
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* Benjamin A Kansy
* Raghvendra M Srivastava
* Hyun-Bae Jie
* Gulidanna Shayan
* Yu Lei
* Jing Li
* William Gooding
* Sven Brandau
* Stephan Lang
* Nicole Schmitt
* Gordon J Freeman
* David A Clump
* Robert L Ferris

*   Squamous Cell Carcinoma
*   Good Clinical Outcome
*   Neck Squamous Cell Carcinoma
*   Tumor Infiltrate Lymphocyte
*   Optimal Tumor

Meeting abstracts

## Introduction

Prognosis varies dramatically in head and neck squamous cell carcinoma (HNSCC) based on HPV status. In HPV+ patients, programmed death (PD)-1 expression has been linked to a better clinical outcome. We hypothesized that extent of PD-1 expression may differentially impact T cell phenotype, patient prognosis and response to anti-PD-1 immunotherapy in a murine HPV+ cancer model.

## Material and methods

Freshly isolated tumor infiltrating lymphocytes (TIL) from HNSCC patients were stained by flow cytometry for expression level of PD-1 expression (PD-1high vs. PD-1low), granzyme B, or IFN-y secretion by ELISPOT. The prognostic impact of PD-1high vs. PD-1low T cells was determined in a cohort of HNSCC patients (n=56, median follow up=19 mo). In a murine HNSCC model, PD-1high and PD-1low fractions were compared from CD3+ CD8+ PD-1+ cells and analyzed according to different treatment groups (untreated, anti-PD-1 mAb, radiotherapy and 3 different anti-PD-1/radiotherapy combinations).

## Results

CTLA-4 and PD-1 were significantly upregulated on both HPV+ and HPV- HNSCC patients' TIL, whereas PD-1+ CD8+ cells were significantly enriched in TIL from HPV+ patients (p=0.006). Interestingly, PD-1high cells represented a more dysfunctional phenotype, with severely compromised IFN-γ secretion (phigh CD8+ TIL were more likely to be HPV- and had a worse disease free survival (HR = 2.25; 95% CI = 1.46 – 3.15; p < .0001), while high fractions of PD-1low T cells were associated with better clinical outcome (HR= 0.19, 95% CI = .07 - .49, p = .0006), and were seen preferentially in HPV+ HNSCC patients. In the murine HNSCC model, anti-PD-1 mAb plus radiotherapy resulted in optimal tumor elimination, which was associated with an elimination of PD-1high CD8+ T cells and – most importantly – increase in PD-1low/intermed T cell frequencies (p < 0.5).

## Discussion

Consideration of different PD-1 expression levels on TIL segregates PD-1 expression as a marker of activated, competent tumor reactive T cells on the one hand (low/intermed expression), and as a marker of exhausted, dysfunctional cells in the tumor microenvironment on the other hand (high expression). These results emphasize the crucial role of differential PD-1 expression levels on HNSCC patients' effector T cells for prognosis and as a potential novel biomarker for anti-PD-1/PD-L1 based immunotherapy.

*   © The Author(s). 2015

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ([http://creativecommons.org/licenses/by/4.0](http://creativecommons.org/licenses/by/4.0)), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ([http://creativecommons.org/publicdomain/zero/1.0/](http://creativecommons.org/publicdomain/zero/1.0/)) applies to the data made available in this article, unless otherwise stated.