T cell receptor β‐chain repertoire analysis reveals the association between neoantigens and tumour‐infiltrating lymphocytes in multifocal papillary thyroid carcinoma

To explore whether a few nonsynonymous somatic mutations could induce activation and proliferation of neoantigen‐specific tumour‐infiltrating lymphocytes (TILs) in tumours with low mutation rates, we analysed a patient with multifocal papillary thyroid carcinoma (seven noncontiguous cancer foci) to investigate the relationship between neoantigens and TILs. These seven foci had a few or no nonsynonymous somatic mutations; moreover, multiple loci had similar or different spectra of mutations. We used high‐throughput sequencing of the rearranged genes in T cell receptor β‐chain (TCRβ) to reveal the basic characteristics of T cells in seven tumour foci and matched adjacent normal tissue. We found that in multifocal papillary thyroid carcinoma the number of nonsynonymous somatic mutations was positively associated with oligoclonal TCRβ repertoire, and tumour foci with similar spectra of mutations had higher overlap of TCRβ repertoire. In conclusion, the number of nonsynonymous somatic mutations is small in tumours with low mutation rates but these mutations still play an important role in activating neoantigen‐specific TILs.     

T cell receptor β‐chain repertoire analysis of tumor‐infiltrating lymphocytes in pancreatic cancer

Pancreatic cancer is lethal due to lack of perceptible symptoms and effective treatment methods. Immunotherapy may provide promising therapeutic choices for malignant tumors like pancreatic cancer. Tumor‐infiltrating lymphocytes (TIL) in tumor mesenchyme could recognize peptide antigens presented on the surface of tumor cells. The present study aimed to test the relationship between the T cell receptor (TCR) β repertoire of the tumor and peripheral blood, and also to investigate the intra‐tumor spatial heterogeneity of the TCR β repertoire in pancreatic cancer. To the best of our knowledge, this is the first study to evaluate the clonal composition of TCR β repertoire in TIL across the spatial extent of pancreatic cancer. In this study, we studied 5 patients who were diagnosed with primary pancreatic cancer. Ultra‐deep sequencing was used to assess the rearrangement of the TCR β‐chain (TCR β) gene. HE staining and immunohistochemistry of CD3, CD4, CD8 and HLA class I were used to show histopathology and immune conditions macroscopically. TIL repertoire showed that different regions of the same tumor showed a greater number of repertoire overlaps between each other than between peripheral blood, which suggested that T cell clones in pancreatic cancer might be quite different from those in peripheral blood. In contrast, intra‐tumoral TCR β repertoires were spatially homogeneous between different regions of a single tumor tissue. Based on these results, we speculated that the cellular adaptive immune response in pancreatic cancer was spatially homogeneous; this may pave the way for immunotherapy for the treatment of pancreatic cancer patients.     

Doxorubicin sensitizes human tumor cells to NK cell‐ and T‐cell‐mediated killing by augmented TRAIL receptor signaling

Doxorubicin (DOX) is an anthracycline antibiotic that is widely used to treat different types of malignancy. In this study, it was studied whether DOX could be used to render tumor cells susceptible to apoptosis by NK and T cells. Pretreatment with subapoptotic doses of DOX sensitized tumor cell lines of various histotypes to both NK and T cells resulting in a 3.7 to 32.7% increase in lysis (2.5 mean fold increase, p < 0.0001) and a 2.9 to 14.2% increase in lysis (3.0 mean‐fold increase, p < 0.05), respectively. The sensitizing effect of the drug was primarily dependent on the tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL)/TRAIL‐receptor signaling, but not on Fas‐ligand, perforin, NKG2D or DNAM‐1. The central role of the TRAIL signaling pathway was further supported by an increased expression of TRAIL‐R2 on DOX‐treated tumor cells and by downregulation of cellular FLICE inhibitory protein, the inhibitors of death receptor‐mediated apoptosis. Compared to untreated cells, pretreatment of tumor cells with DOX showed increased processing and activation of caspase‐8 on coculture with NK or T cells. The significance of this treatment strategy was confirmed using a xenogeneic tumor‐bearing mouse model. Tumor progression was delayed in mice that received either NK cells (p < 0.05) or T cells (p < 0.0001) following DOX treatment compared to mice receiving either cell type alone. Moreover, combined infusion of both NK and T cells following DOX treatment not only delayed tumor progression but also significantly improved the long‐term survival (p < 0.01). Based on these findings, it was proposed that DOX can be used to improve the efficacy of adoptive cell therapy in patients with cancer.     

Expression of activating natural killer‐cell receptors is a hallmark of the innate‐like T‐cell neoplasm in peripheral T‐cell lymphomas

Peripheral T‐ or natural killer (NK)‐cell lymphomas are rare and difficult‐to‐recognize diseases. It remains arduous to distinguish between NK cell‐ and cytotoxic T‐lymphocyte‐derived lymphomas through routine histological evaluation. To clarify the cells of origin, we focused on NK‐cell receptors and examined the expression using immunohistochemistry in 22 cases with T‐ and NK‐cell neoplasms comprising angioimmunoblastic T‐cell lymphoma, anaplastic lymphoma kinase (ALK)‐positive and ‐negative anaplastic large‐cell lymphomas, extranodal NK/T‐cell lymphoma, nasal type, monomorphic epitheliotropic intestinal T‐cell lymphoma, aggressive NK‐cell leukemia, and other peripheral T‐cell lymphomas. Inhibitory receptor leukocyte immunoglobulin‐like receptor subfamily B member 1 (LILRB1) was detected in 14 (64%) cases, whereas activating receptors DNAM1, NKp46, and NKG2D were expressed in 7 (32%), 9 (41%), and 5 (23%) cases, respectively. Although LILRB1 was detected regardless of the disease entity, the activating NK‐cell receptors were expressed predominantly in TIA‐1‐positive neoplasms (DNAM1, 49%; NKp46, 69%; and NKG2D, 38%). In addition, NKp46 and NKG2D were detected only in NK‐cell neoplasms and cytotoxic T‐lymphocyte‐derived lymphomas including monomorphic epitheliotropic intestinal T‐cell lymphoma. One Epstein‐Barr virus‐harboring cytotoxic T‐lymphocyte‐derived lymphoma mimicking extranodal NK/T‐cell lymphoma, nasal type lacked these NK‐cell receptors, indicating different cell origin from NK and innate‐like T cells. Furthermore, NKG2D expression showed a negative impact on survival among the 22 examined cases, which mainly received the standard chemotherapy regimen (log‐rank test, P = .024). We propose that the presence of activating NK‐cell receptors may provide new insights into understanding peripheral T‐cell lymphomas and characterizing them as innate‐like T‐cell neoplasm.     

An adaptive immune response driven by mature,antigen‐experienced T and B cells within the microenvironment of oral squamous cell carcinoma

Lymphocyte infiltrates have been observed in the microenvironment of oral cancer; however, little is known about whether the immune response of the lymphocyte infiltrate affects tumor biology. For a deeper understanding of the role of the infiltrating‐lymphocytes in oral squamous cell carcinoma (OSCC), we characterized the lymphocyte infiltrate repertoires and defined their features. Immunohistochemistry revealed considerable T and B cell infiltrates and lymphoid follicles with germinal center‐like structures within the tumor microenvironment. Flow cytometry demonstrated that populations of antigen‐experienced CD4+ and CD8+ cells were present, as well as an enrichment of regulatory T cells; and T cells expressing programmed death‐1 (PD‐1) and T cell Ig and mucin protein‐3 (Tim‐3), indicative of exhaustion, within the tumor microenvironment. Characterization of tumor‐infiltrating B cells revealed clear evidence of antigen exposure, in that the cardinal features of an antigen‐driven B cell response were present, including somatic mutation, clonal expansion, intraclonal variation and isotype switching. Collectively, our results point to an adaptive immune response occurring within the OSCC microenvironment, which may be sustained by the expression of specific antigens in the tumor.     

Clinical development of anti‐CD19 chimeric antigen receptor T‐cell therapy for B‐cell non‐Hodgkin lymphoma

B‐cell non‐Hodgkin lymphoma (B‐NHL) is the most frequent hematological malignancy. Although refined chemotherapy regimens and several new therapeutics including rituximab, a chimeric anti‐CD20 monoclonal antibody, have improved its prognosis in recent decades, there are still a substantial number of patients with chemorefractory B‐NHL. Anti‐CD19 chimeric antigen receptor (CAR) T‐cell therapy is expected to be an effective adoptive cell treatment and has the potential to overcome the chemorefractoriness of B‐cell leukemia and lymphoma. Recently, several clinical trials have shown remarkable efficacy of anti‐CD19 CAR T‐cell therapy, not only in B‐acute lymphoblastic leukemia but also in B‐NHL. Nonetheless, there are several challenges to overcome before introduction into clinical practice, such as: (i) further refinement of the manufacturing process, (ii) further improvement of efficacy, (iii) finding the optimal infusion cell dose, (iv) optimization of lymphocyte‐depleting chemotherapy, (v) identification of the best CAR structure, and (vi) optimization of toxicity management including cytokine release syndrome, neurologic toxicity, and on‐target off‐tumor toxicity. Several ways to solve these problems are currently under study. In this review, we describe the updated clinical data regarding anti‐CD19 CAR T‐cell therapy, with a focus on B‐NHL, and discuss the clinical implications and perspectives of CAR T‐cell therapy.     

Toll‐like receptor‐4 signaling in mantle cell lymphoma

BACKGROUND:

Mantle cell lymphoma (MCL) is an incurable B‐cell malignancy, and patients with this disease have the poorest prognosis among all patients with B‐cell lymphomas. The signaling pathways that trigger MCL escape from immune surveillance are unclear. Because Toll‐like receptors (TLRs) initiate innate and adaptive immune responses against invading pathogens, the authors investigated the impact of TLR signaling in MCL cells.

METHODS:

TLR expression was examined in MCL cell lines and in primary tumors. The examination focused on TLR4 and its ligand lipopolysaccharide (LPS) on MCL cells and their function on MCL proliferation and immune evasion.

RESULTS:

MCL cells expressed multiple TLRs, and TLR4 was among the highest expressed molecules. The activation of TLR4 signaling in MCL cells by LPS induced MCL proliferation and up‐regulated the secretion of cytokines like interleukin‐6 (IL‐6), IL‐10, and vascular endothelial growth factor (VEGF). LPS‐pretreated MCL cells inhibited the proliferation and cytolytic activity of T cells by secreted IL‐10 and VEGF, and neutralizing antibodies against these cytokines restored their functions. Similar results were observed in TLR4‐positive/myeloid differentiation 88 (MyD88)‐positive primary lymphoma cells but not in TLR4‐positive/MyD88‐negative primary lymphoma cells from patients with MCL. Knockdown of TLR4 on MCL cells abrogated the effect of LPS on MCL cells in term of cell growth or secretion of the cytokines and evasion of the immune system.

CONCLUSIONS:

The current results indicated that TLR4 signaling triggers a cascade that leads to MCL growth and evasion from immune surveillance. Thus, TLR4 signaling molecules may be novel therapeutic targets in patients with MCL. Cancer 2013. © 2012 American Cancer Society.     

Spatiotemporal homogeneity and distinctness of the T‐cell receptor β‐chain repertoires in Epstein–Barr virus‐associated primary and metastatic nasopharyngeal carcinomas

Nasopharyngeal carcinoma (NPC) is an Epstein–Barr virus (EBV)‐associated lymphoepithelioma. The aim of this study was to characterize the homogeneity and distinctness of the T‐cell repertoires within and between primary and metastatic NPCs. We used ultra‐deep sequencing of the hypervariably rearranged antigen‐binding CDR3 regions of T‐cell receptor beta (TCR_beta) to comprehensively profile the T‐cell repertoires in NPC patients receiving definitive chemoradiotherapy with long‐term follow‐up. We observed not only various spatially heterogeneous patient‐specific TCR_beta clone compositions that changed with time but also several commonly enriched TCR_beta subclones that were constantly shared between primary NPCs in the head and neck regions, locally recurrent tumors after treatment and later‐developed distant metastatic tumors in the liver, lung and bone. Comparison of the overlap frequency of the T‐cell clonality between TCR_beta repertoires enabled us to calculate the pairwise genetic distance between primary NPCs of different patients and different sites of metastatic or recurrent NPCs. The constructed NPC phylogeny clearly differentiated the low‐risk patients without relapse from the high‐risk patients with distant metastasis after chemoradiotherapy. In contrast to the rather low frequency of nonsilent somatic mutations in NPC cells, the degrees of similarity and divergence of NPC‐infiltrating lymphocyte TCR_beta repertoires among different patients showed prognostication. Moreover, the persistent presence of commonly NPC‐shared in‐frame TCR_beta CDR3 gene sequences spatiotemporally identified in the NPC‐infiltrating lymphocytes within varied EBV‐positive NPCs and their metastases suggest the existence of frequently shared epitopes of neoantigens virally or nonvirally displayed on cancer cells, thereby providing opportunities for the development of precisely tumor‐targeted immunotherapy for distant metastasis.     

Combined chemoradiotherapy and programmed cell death‐ligand 1 blockade leads to changes in the circulating T‐cell receptor repertoire of patients with non‐small‐cell lung cancer

Combined chemoradiotherapy (CRT) and programmed cell death‐ligand 1 (PD‐L1) blockade is a new care standard for unresectable stage III non‐small‐cell lung cancer (NSCLC). Although this consolidation therapy has improved the overall survival of patients with NSCLC, the synergistic action mechanisms of CRT and immunotherapy on T cells remain unclear. In addition, there is a paucity of reliable biomarkers to predict clinical responses to therapy. In this study, we analyzed T‐cell receptor (TCR) sequences in the peripheral blood of five patients with NSCLC. T‐cell receptor analysis was undertaken before treatment, after CRT, and after PD‐L1 blockade. Notably, we observed the expansion and alteration of the dominant T‐cell clonotypes in all cases with a complete response. In contrast, neither expansion nor alteration of the TCR repertoire was observed in cases with progressive disease. T cell expansion was initiated after CRT and was further enhanced after PD‐L1 blockade. Our findings suggest the systemic effect of CRT on circulating T cells in addition to the curative effect on limited tumor sites. Dynamic changes in circulating T‐cell clonotypes could have a prognostic significance for combined CRT and PD‐L1 blockade.     

Cadmium‐coordinated supramolecule suppresses tumor growth of T‐cell leukemia in mice

Cadmium is a toxic pollutant with occupational and environmental significance, due to its diverse toxic effects. Supramolecules that conjugate and decontaminate toxic metals have potential for use in treatment of cadmium intoxication. In addition, metal‐coordinating ability has been postulated to contribute to the cytotoxic effects of anti‐tumor agents such as cisplatin or bleomycin. Thiacalixarenes, cyclic oligomers of p‐alkylphenol bridged by sulfur atoms, are supramolecules known to have potent coordinating ability to metal ions. In this study, we show that cadmium‐coordinated thiacalix[4]arene tetrasulfate (TC4ATS‐Cd) exhibits an anti‐proliferative effect against T‐cell leukemia cells. Cadmium exhibited cytotoxicity with IC₅₀ values ranging from 36 to 129 μM against epithelia‐derived cancer cell lines, while TC4ATS‐Cd elicited no significant cytotoxicity (IC₅₀ > 947 μM). However, a number of T‐cell leukemia cell lines exhibited marked sensitivity to TC4ATS‐Cd. In Jurkat cells, toxicity of TC4ATS‐Cd occurred with an IC₅₀ of 6.9 μM, which is comparable to that of 6.5 μM observed for cadmium alone. TC4ATS‐Cd induced apoptotic cell death through activation of caspase‐3 in Jurkat cells. In a xenograft model, TC4ATS‐Cd (13 mg/kg) treatment significantly suppressed the tumor growth of Jurkat cells in mice. In addition, TC4ATS‐Cd‐treated mice exhibited significantly less cadmium accumulation in liver and kidney compared to equimolar cadmium‐treated mice. These results suggest that cadmium‐coordinated supramolecules may have therapeutic potential for treatment of T‐cell leukemia.     

Characterization of tumor infiltrating lymphocytes in paired primary and metastatic renal cell carcinoma specimens

Renal cell carcinoma (RCC) is one of the most chemo- and radio-resistant malignancies, with poor associated patient survival if the disease metastasizes. With recent advances in immunotherapy, particularly with PD-1/PD-L1 blockade, outcomes are improving, but a substantial subset of patients does not respond to the new agents. Identifying such patients and improving the therapeutic ratio has been a challenge, although much effort has been made to study PD-1/PD-L1 status in pre-treatment tumor. However, tumor infiltrating lymphocyte (TIL) content might also be predictive of response, and our goal was to characterize TIL content and PD-L1 expression in RCC tumors from various anatomic sites. Utilizing a quantitative immunofluorescence technique, TIL subsets were examined in matched primary and metastatic specimens. In metastatic specimens, we found an association between low CD8+ to Foxp3+ T-cell ratios and high levels of PD-L1. High PD-L1-expressing metastases were also found to be associated with tumors that were high in both CD4+ and Foxp3+ T-cell content. Taken together these results provide the basis for combining agents that target the PD-1/PD-L1 pathway with agonist of immune activation, particularly in treating RCC metastases with unfavorable tumor characteristics and microenvironment. In addition, CD8+ TIL density and CD8:Foxp3 T-cell ratio were higher in primary than metastatic specimens, supporting the need to assess distant sites for predictive biomarkers when treating disseminated disease.     

Development of a T‐cell receptor multimer with high avidity for detecting a naturally presented tumor‐associated antigen on osteosarcoma cells

For efficacy of peptide vaccination immunotherapy for patients with cancer, endogenous expression of the target peptide/human leukocyte antigen (HLA) on cancer cells is required. However, it is difficult to evaluate the expression status of a peptide/HLA complex because of the lack of a soluble T‐cell receptor (TCR) that reacts with tumor‐associated antigen (TAA) with high avidity. In the present study, we developed two soluble TCR‐multimers that were each directed to TAA, survivin‐2B (SVN‐2B) and PBF in the context of HLA‐A24 (SVN‐2B TCR‐multimer and PBF TCR‐multimer, respectively), from CTL clones that were established from peptide‐vaccinated patients. Both TCR multimers could recognize cognate peptide‐pulsed antigen‐presenting cells, C1R‐A24 cells, in a CD8‐independent method. Moreover, the PBF TCR‐multimer successfully recognized a PBF peptide naturally presented on HLA‐A24⁺PBF⁺ osteosarcoma cells. Taken together, the results indicated that a TCR‐multimer might be useful for detection of a TAA‐derived peptide presented by HLA in patients receiving immunotherapy.     

TCR repertoire intratumor heterogeneity of CD4+ and CD8+ T cells in centers and margins of localized lung adenocarcinomas

Intratumor heterogeneity (ITH) of T cell receptor (TCR) repertoire in different T-cell subsets and locations in lung adenocarcinomas was unclear. Here, we investigated percentages and TCR repertoire of freshly isolated CD4⁺ and CD8⁺ tumor infiltrating lymphocytes (TILs) in tumor centers and margins by flow cytometry on 80 tumor samples from 20 patients and high-throughput TCR sequencing on 27 and 25 samples of CD4⁺ and CD8⁺ TILs from seven patients. Our results demonstrated that amount and TCR repertoire diversity of CD4⁺ TILs were significantly higher than those of CD8⁺ TILs and moreover substantial ITH regarding amount and TCR repertoire of CD4⁺ and CD8⁺ TILs were observed. Additionally, ITH of CD4/CD8 T-cell ratio and CD8⁺ TIL repertoire across center regions was lower than that across margin regions. The amount and TCR repertoire ITH of CD4⁺ and CD8⁺ TILs and mean clonality of CD8⁺ TILs in tumor centers were associated with relapse. Our study provides insights into amount and TCR repertoire ITH of CD4⁺ and CD8⁺ TILs in tumor centers and margins as well as corresponding association with prognosis in lung adenocarcinoma patients, suggesting potential clinical significance of TCR repertoire.     

Immune‐checkpoint molecules on regulatory T‐cells as a potential therapeutic target in head and neck squamous cell cancers

Immune‐checkpoint inhibitors improve the survival of head and neck squamous cell carcinoma (HNSCC) patients. Although recent studies have demonstrated that the tumor immune microenvironment (TIME) has critical roles in immunotherapy, the precise mechanisms involved are unclear. Therefore, further investigations of TIME are required for the improvement of immunotherapy. The frequency of effector regulatory T‐cells (eTregs) and the expression of immune‐checkpoint molecules (ICM) on eTregs and conventional T‐cells (Tconvs) both in peripheral blood lymphocytes (PBL) and tumor‐infiltrating lymphocytes (TIL) from HNSCC patients were analyzed by flow cytometry and their distributions were evaluated by multi‐color immunofluorescence microscopy. High frequency eTreg infiltration into HNSCC tissues was observed and high expressions of CD25, FOXP3, stimulatory‐ICM (4‐1BB, ICOS, OX40 and GITR) and inhibitory‐ICM (programmed cell death‐1 [PD‐1] and cytotoxic T‐lymphocyte‐associated protein‐4 [CTLA‐4]) were found on invasive eTregs. In contrast, the expression of stimulatory‐ICM on Tconvs was low and the expression of inhibitory‐ICM was high. In addition, ICM‐ligands (programmed cell death‐1 [PD‐L1], galectin‐9 and CEACAM‐1) were frequently expressed on cancer cells. PD‐L1 and galectin‐9 were also expressed on macrophages. PD‐1⁺ T‐cells interacted with PD‐L1⁺ cancer cells or PD‐L1⁺ macrophages. This suggested that in TIL, eTregs are highly activated, but Tconvs are exhausted or inactivated by eTregs and immune‐checkpoint systems, and ICM and eTregs are strongly involved in the creation of an immunosuppressive environment in HNSCC tissues. These suggested eTreg targeting drugs are expected to be a combination partner with immune‐checkpoint inhibitors that will improve immunotherapy of HNSCC.     

Long term presence of a single predominant tyrosinase‐specific T‐cell clone associated with disease control in a patient with metastatic melanoma

In an earlier study, we described a patient who developed an anti‐tyrosinase T‐cell response leading to long‐term tumor control. Here we analyzed this response with regard to T‐cell receptor (TCR) Vβ family usage and clonality in order to further elucidate the nature of the T cell response in this patient. For identification of expanded specific cytotoxic T‐cell (CTL) clones, tetramer enrichment of tyrosinase reactive T‐cells was followed by comparative quantitative reverse transcribed PCR (qRT PCR) quantification of all TCR Vβ‐families and sequencing of family Vβ4 elevated in the enriched fraction. The predominant specific clone was quantified by clonotypic qRT PCR in multiple samples from blood, bone marrow, and tumor tissue. FACS analyses with staining of TYR.A2 and TCR Vβ4 were performed. Epitope specific enrichment revealed an isolated increase of Vβ−family 4. FACS analysis showed a shift of specific CTLs to Vβ‐family 4 during tumor regression with a maximum of 80% of all TYR.A2 specific cells belonging to this family. Sequencing revealed a single predominant clone against polyclonal background coding for identical CDR3 loops. The predominant clone was highly expressed in bone marrow and tumor tissue, and was detectable in blood over a period of ten years. Considering the results of previous studies showing a specific effector phenotype in blood and a specific memory compartment in bone marrow of this patient, this data implicate the predominant clone featured all attributes of a sufficient CTL response including homing capacity and memory formation resulting in long term clonal persistence and tumor control.     

Programmed death‐ligand 1 is prognostic factor in esophageal squamous cell carcinoma and is associated with epidermal growth factor receptor

Programmed death‐ligand 1 (PD‐L1) expression either indicates immune inhibitory status or concurrent immune response. Although the relationship between PD‐L1 and clinical outcomes has been studied widely in recent years, its role in prognosis of esophageal squamous cell carcinoma (ESCC) remains unclear. Here, we assessed the significance of PD‐L1 in ESCC and its association with epidermal growth factor receptor (EGFR) and radiation response. We found that PD‐L1 was present both on the surface of tumor cells and tumor‐infiltrating immune cells. Patients with tumor‐infiltrating immune cell PD‐L1 expression had better survival. PD‐L1 expression on immune cells was an independent prognostic factor for patients with ESCC. PD‐L1 expression either on tumor‐infiltrating immune cells or tumor cells was negatively associated with EGFR expression. EGFR/PD‐L1 pairs could separate the survival between EGFR low/PD‐L1 positive and EGFR high/PD‐L1 negative groups. In ESCC cell lines with EGFR high expression, PD‐L1 expression was induced significantly when EGFR signaling was activated by radiation and was dramatically inhibited by an EGFR tyrosine kinase inhibitor. In conclusion, tumor‐infiltrating immune cell PD‐L1 expression is an independent prognostic factor for ESCC, and the association between EGFR and PD‐L1 is vital to determining survival. It is important to consider radiotherapy‐induced imbalance of pro‐tumor and anti‐tumor immune response. A combination of radiotherapy and PD‐L1‐targeted therapy could be a promising therapeutic strategy for ESCC patients.