The incidence and mortality rates of esophageal cancer are ranked seventh and sixth in the world, respectively (1). More than 90% of esophageal cancer is esophageal squamous cell carcinoma (ESCC). Patients with ESCC are typically diagnosed at the locally advanced stage and concurrent chemoradiotherapy (CCRT) is the standard treatment (2).

As anti-PD-1 antibodies were first used to treat drug-resistant solid tumors in 2006 (3), immune checkpoint blockades have recently been successfully applied in various cancers (4–7). Several studies including researches on glioma, implanted breast and colorectal carcinomas, and non-small cell lung cancer have supported the combination of radiotherapy and immunotherapy (8–11). From July 2017 to January 2018, we firstly conducted a phase 1b clinical trial (NCT03222440) of radiotherapy concurrently combined with an anti-PD-1 antibody, camrelizumab, as first-line treatment in patients with locally advanced ESCC who were intolerant to or refused CCRT. This combination therapy had manageable toxicity and preliminary antitumor efficacy for locally advanced ESCC (12).

The occurrence and development of tumors are closely related to antitumor immune responses. Peripheral blood lymphocytes (PBLs) can directly reflect a patient’s systemic immune status. To date, a few studies focused on PBLs have preliminarily confirmed that the changes of lymphocyte subsets before and after treatment have predictive significance for treatment response (13–15). For example, increased CD8⁺CD28⁺ T cells predict better early response to stereotactic ablative radiotherapy in non-small cell lung cancer and high frequency of PD-1⁺TIGIT⁺CD8⁺ T cells predict better response to anti-PD-1 therapy in melanoma and Merkel cell carcinoma.

CD8⁺ T cells are the main effector T cells that play a vital role in antitumor immunity. Based on CD45RA and CD62L expression, CD8⁺ T cells are divided into four differentiation subtypes: naïve T cells (CD45RA⁺CD62L⁺), central memory T cells (Tcm, CD45RA^-CD62L⁺), effector memory T cells (Tem, CD45RA^-CD62L^-), and CD45RA⁺ effector memory T cells (TEMRA, CD45RA⁺CD62L^-) (16). Molecular markers can characterize the state of T cells. PD-1 is a checkpoint molecule which regulates immunocytes status (17). Eomes and T-bet are two members of the T-box transcription factor family (18). Both of them regulate T-cell effector functions, including IFN-γ production (19–21). IFN-γ is a crucial cytokine for innate and adaptive immunity and contributes to the antitumor immune response through its immunostimulatory and immunomodulatory effects (22). CD38 and HLA-DR are markers of T cell activation (23, 24). Detecting the expression of these molecule markers could indicate repressed or activated state of T cells.

At present, there are few reports on feature changes of CD8⁺ T cells in the peripheral blood of patients with ESCC under treatment. We previously reported the changes of main T-cell subsets, CD4⁺ and CD8⁺ T cells, under radiotherapy plus immunotherapy (12). In the present study, by further monitoring the expression levels of molecular markers and differentiation subsets in the peripheral CD8⁺ T cells of patients with locally advanced ESCC before and during radiotherapy plus immunotherapy or CCRT, we attempted to determine the dynamic changes of activation, function and differentiation status of peripheral CD8⁺ T cells and compare the similarities and differences between the two cohorts. This may provide evidence for the understanding of systematic immune status of patients undergoing treatment and contribute to the treatment of patients with locally advanced ESCC.

Our study revealed that PD-1⁺CD8⁺ T cells exhibited higher functional and activated characteristics and had more memory properties than PD-1^-CD8⁺ T cells in the two treatment groups. Besides, both RT combined with immunotherapy and CCRT treatment activated PD-1⁺ and PD-1^- CD8⁺ T cells, while the two treatments had different effects on the differentiation state of PD-1⁺ and PD-1^- CD8⁺ T cells.

Both RT plus camrelizumab and CCRT treatments increased the expression levels of CD38 and HLA-DR in peripheral CD8⁺ T cells suggesting that both treatments activate CD8⁺ T cells. Besides, we found effective binding of anti-PD-1 antibody camrelizumab with PD-1 on CD8⁺ T cells during RT plus camrelizumab treatment. Anti-PD-1 antibody directly competed with PD-L1 on tumor cells to bind PD-1 on immune cells, thereby reducing the inhibitory effects of tumor cells on immune cells. This might be one of the reasons for the significantly better prognosis of radiotherapy combined with immunotherapy than CCRT in our preliminary survival analysis.

We found that functional molecules IFN-γ, as well as activated molecules CD38 and HLA-DR, were all higher in PD-1⁺CD8⁺ T cells than those in PD-1^-CD8⁺ T cells in two groups both before and during treatment. These results indicated that PD-1⁺CD8⁺ T cells might have higher activity and stronger immune function than PD-1^-CD8⁺ T cells. It was consistent with the reports that adoptive transfer of PD-1⁺ T cells to tumor-bearing mice resulted in tumor control, in contrast to the adoptive transfer of PD-1^- T cells (25, 26). Additionally, the ability of PD-1⁺CD8⁺ T cells maintained stable, regardless RT plus camrelizumab or CCRT administrated.

In addition, PD-1⁺CD8⁺ T cells consisted of less naïve but more memory subsets compared with PD-1^-CD8⁺ T cells before treatment in both groups. It has been reported that expression of PD-1 identified a neoantigen-specific anti-tumor T cell response in peripheral CD8⁺ T cells (27). Neoantigen burdened by tumor cells stimulated and developed activation and differentiation of T cells resulting in more memory participants in the T-cell pool. The change of T-cell differentiation state was regularly accompanied by the change of activation and function characteristics. These evidences were consistent with our findings of higher proportions of memory subsets and stronger activation in PD-1⁺CD8⁺ T cells. Whether PD-1⁺CD8⁺ T cells contain more neoantigen-specific T cells with increased activation in ESCC deserved further exploration.

When dynamically monitoring the differentiation status of peripheral CD8⁺ T cells, Tcm subset increased in PD-1⁺CD8⁺ T cells and Tem subset increased in PD-1^-CD8⁺ T cells after RT plus camrelizumab treatment, while only Tcm subset in PD-1^-CD8⁺ T cells elevated after CCRT treatment. Since Tcm and Tem had different capabilities and life cycles responding to immune induction (28–30), our results suggested that RT plus camrelizumab and CCRT treatment might have partial distinct effects on the activation of memory T cells. The increase of different memory subsets in PD-1⁺ and PD-1^- CD8⁺ T cells might be related to the different expression of transcriptional factors in different memory subsets. It was demonstrated that T-bet, Blimp1, ID2, and STAT4 were associated with Tem subset, while Eomes, TCF1, BCL-6, ID3, and STAT3 were associated with Tcm subset (28). Our data showed that PD1⁺CD8⁺ T cells had higher expression level of Eomes than PD1^-CD8⁺ T cells in RT plus camrelizumab group. This might be one of the reasons why Tcm subset increased in PD-1⁺CD8⁺ T cells rather than in PD-1^-CD8⁺ T cells after RT plus camrelizumab treatment.

It was reported that PD-1^high CD8⁺ T cells predicted response to PD-1 blockade and correlated with increased overall survival and 80% of patients with clinical benefit exhibited PD-1⁺ CD8⁺ T-cell responses to PD-1 targeted immunotherapy in non-small cell lung cancer (31, 32). And tumor-specific CD8⁺ T cells were enriched among PD-1⁺ cells (27, 33). So our results that RT plus camrelizumab treatment increased PD-1⁺CD8⁺ Tcm while CCRT had no influence on any PD-1⁺CD8⁺ subset suggested that RT plus camrelizumab treatment might exhibit stronger CD8⁺ T-cell response than CCRT. This might also be one of the reasons for the better prognosis of radiotherapy combined with immunotherapy than CCRT in our preliminary survival analysis.

Radiotherapy promoted the release of tumor neoantigen, consequently activating neoantigen presentation and inducing specific anti-tumor immune response in local tumor microenvironment (34). Our present results from ESCC patients revealed that besides the local anti-tumor microenvironment, the systematic anti-tumor immune was also greatly promoted by these treatments.

For patients with locally advanced ESCC who were intolerable or refused surgery, CCRT is the standard treatment. However, the survival for these patients was still poor after CCRT. Recently, accumulated evidences have shown the important role of tumor-infiltrating immune cells in treatment outcome in ESCC (35, 36). The results from phase 3 clinical trials revealed the promising anti-tumor effect of anti-PD-1 antibody in advanced ESCC patients (37, 38). It is great interesting to find out whether combining chemoradiotherapy or radiotherapy with immunotherapy might increase the survival of ESCC patients. As a result, we laughed the first phase 1b clinical trial (No. NCT03222440) in 2017 to evaluate the toxicity and preliminary anti-tumor efficiency of radiotherapy plus camrelizumab in patients with locally advanced ESCC who were intolerable or refused surgery as well as CCRT. In the present study, we originally reported the systematic immune traits, including the activated, functional and differentiation status, under RT plus camrelizumab treatment and compared these findings with what we found in CCRT group. Our results would indicate the underling anti-tumor mechanisms under radiotherapy combined with immunotherapy.

Nevertheless, there were obvious limitations in this study. Because of the characteristics of the phase 1b clinical trial combing RT and camrelizumab, only 19 patients were finally included in the study. And the peripheral blood was also collected from patients receiving CCRT during the same period of the phase 1b study. The sample size was insufficient, and therefore our results were preliminary at present. We would continue to carry out relevant studies and collect more cases for analysis to verify our findings. In addition, we would take further studies on the underlying mechanisms of the different effects of the two treatments on the immune status of ESCC patients.

This study identified the systematic immune status of patients undergoing treatment. The effects of RT concurrently combined with immunotherapy and CCRT on the expression levels of molecular markers of peripheral CD8⁺ T cells and on the characteristics and differentiation state of PD-1⁺ and PD-1^- CD8⁺ T-cell subsets were both similar and different. The two treatments had similar activation effects on peripheral CD8⁺ T cells with different PD-1 properties but had different effects on their differentiation status. In general, RT concurrently combined with immunotherapy attained better prognosis than CCRT, which had the potential to be an ideal option for patients with locally advanced ESCC. Our results provided potential clues to the reasons underlying the difference in prognosis of the two treatments. Indeed, our results are still preliminary and further researches are needed to verify our findings and clarify the underlying mechanisms.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

The studies involving human participants were reviewed and approved by the Ethical Committee of Tianjin Medical University Cancer Institute and Hospital. The patients/participants provided their written informed consent to participate in this study.

CY, QP, PW and WZ contributed to the study conception and design. ZG, XM, YangL, XW, DH, TZ, and XC contributed to samples preparation and data collection. Data analysis was performed by HW and YangL. The first draft of the manuscript was written by HW, YangL, CY and WZ, and polished by JZ. All authors read and approved the manuscript.

This work was supported by the National Natural Science Foundation of China [grant number 82073348, 81602565 and 81872462], Chinese National Key Research and Development Project [2018YFC1315601] and Tianjin Key Medical Discipline (Specialty) Construction Project [TJYXZDXK-009A].

Author JZ is employed by Jiangsu Hengrui Pharmaceuticals Co., Ltd.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.