Tumor-infiltrating CD4+ T cells in patients with gastric cancer
- Long Yuan†1,
- Benling Xu†2,
- Peng Yuan1,
- Jinxue Zhou3,
- Peng Qin2,
- Lu Han2,
- Guangyu Chen2,
- Zhenlei Wang1,
- Zengci Run1,
- Peng Zhao1 and
- Quanli Gao1, 2Email author
© The Author(s) 2017
Received: 19 October 2017
Accepted: 27 November 2017
Published: 2 December 2017
T lymphocytes play an indispensably important role in clearing virus and tumor antigen. There is little knowledge about impacts of inhibitory molecules with cytokine on tumor-infiltrating CD4+ T-cells in the presence of gastric cancer (GC). This study investigated the distribution of tumor-infiltrating T-cells subset and the differentiation as well as inhibitory phenotype of T-cells from blood and tissues of GC patients.
Materials and methods
Patients with GC diagnosed on the basis of pre-operative staging and laparotomy findings were approached for enrollment between 2014 and 2015 at the Affiliated Cancer Hospital of Zhengzhou University, China. Phenotypic analysis based on isolation of tumor-infiltrating lymphocytes and intracellular IFN-γ staining assay is conducted. Statistical analysis is performed to show significance.
The results showed that the percentage of CD4+ T-cells among CD3+ cells in tumors was significantly higher than that in the matched paraneoplastic tissue. CD4+ CD25high CD127low regulatory T-cells (Tregs), PD-1+, Tim-3+, and PD-1+ Tim-3+ cells were up-regulated on tumor infiltrating T-cells from patients with GC compared to their expressions on corresponding peripheral blood and peritumoral T-cells. Blockades of PD-1+ and Tim-3+ were effective in restoring tumor infiltrating T-cells’ production of interferon-gamma (IFN-γ). Combined PD-1+ and Tim-3+ inhibition had a synergistic effect on IFN-γ secretion by CD4+ T-cells.
The results suggested that the composition, inhibitors, and location of the immune infiltrate should be considered when evaluating antitumor immunotherapy. A new insight into the mechanisms underlying T cell dysfunction is provided.
Gastric cancer (GC), one of the leading causes for worldwide mortality, is considered as the second most common cancer in China [1, 2]. Although the clinical management for GC is diverse, the prognosis of GC remains poor . The mechanism of clinical management is supposed to include increasing regulatory T-cells (Tregs), losing tumor antigen expression, and enhancing tumor expression of inhibitory ligand [4–7]. Reports proved that the depletion of Tregs could not enhance the efficacy of primary therapies in some cancers, which suggests that T cells may work in synergy with other mechanisms to suppress anti-tumor immunity [8–10].
Investigations showed that T-cell dysfunction in chronic virus infection and human tumor growth was related to the up-regulation of inhibitory molecules such as programmed death 1 (PD-1), T-cell immunoglobulin, and mucin-domain-containing molecule 3 (Tim-3) [11–14]. It provides a significant scheme for cancer treatment that invalidates these inhibitory pathways to resume exhausted T-cells. However, only 19.5% of GC patients respond to PD-1 inhibition .
Currently, there is little detail of T-cell subsets resident within gastric cancer tissues or the expression patterns in this microenvironment corresponding to those observed in paraneoplastic tissue or in the peripheral blood of GC patients. Moreover, the role of PD-1+, Tim-3+, and Tregs in the development and maintenance of tumor-infiltrating T-cell dysfunction in GC patients need to be investigated. Thus, it is crucial to discover the complex mechanisms for inducing T-cell dysfunction in GC patients by exploring the subset composition and functional properties of tumor-infiltrating T-cells.
In this study, we investigated the distribution of T-cells subset, the differentiation and inhibitory phenotype of T-cells from blood and tissues of GC patients. The results demonstrated that the T-cell subsets resident within GC is different from that in paraneoplastic tissue and blood, and the increase in Tim-3+ PD-1+ CD4+ T-cells in tumor tissues was found to correlate with the clinical cancer stage and Tregs. These data as well as the corresponding results and the methods provide a new immunotherapeutic approach for the clinical management of GC.
Materials and methods
Characteristics of patients
Total patients (n)
Median age in years (range)
AJCC stage (2010)
Weight loss (%)
Fresh venous blood was collected from patients or healthy donors (HDs) with EDTA-coated vacutainer tubes. Specific anti-CD3-PE-Cy7 (BioLegend, San Diego, CA, USA) or anti-CD3-FITC (BioLegend, San Diego, CA, USA), anti-CD25-PE (BD Pharmingen™, San Diego, California, USA), anti-CD127-APC (BioLegend, San Diego, CA, USA), anti-CD4-FITC (BioLegend, San Diego, CA, USA), anti-CD8-APC (BioLegend, San Diego, CA, USA), anti-CD45RA-FITC (BioLegend, San Diego, CA, USA), anti-CCR7-PE-Cy7 (BioLegend, San Diego, CA, USA), anti-CD4-PE (BD Biosciences, Oxnard, CA), anti-PD-1-FITC (BioLegend, San Diego, CA, USA), anti-CD4-PerCP-cy5.5 (BD Biosciences, Oxnard, CA), anti-Tim-3-PE (BD Pharmingen™, San Diego, California, USA), and anti-IFN-γ-APC (eBioscience, San Diego, CA, USA) were purchased for use. The viability of the cells was assessed by a violet amine reactive dye (Invitrogen, Carlsbad, CA).
Briefly, 50 μL of blood mixed with 5 μL of each antibody was incubated on ice for 20 min in the dark. Then, 2 mL of 1× lysis buffer (BD Biosciences, Oxnard, CA) was added to each sample and incubated at room temperature for 15 min. Samples were washed with FACS buffer (5% BSA in PBS, 0.09% sodium azide), and the pellets were resuspended in 300 μL of FACS buffer. Flow cytometry was performed on a BD FACS Aria II flow cytometer, and was analyzed with FlowJo software (TreeStar Inc., Ashland, OR, USA). The values were background-corrected by control sample or fluorescence minus one (FMO). Cells without surface makers were used as control samples.
Isolation of TILs
The fresh human tumor samples and matched paraneoplastic tissues from patients with GC were cut into pieces (3 ~ 5 mm3) and were treated with 1 μg/mL of collagenase (Sigma-Aldrich, St. Louis, MO, USA), 25 μg/mL of DNase (Sigma-Aldrich, St. Louis, MO, USA), and 2% fetal bovine serum in PBS at 37 °C for 1 to 1.5 h. The tissue homogenates were filtered by a 70-μm cell strainer (Falcon; BD Biosciences, Oxnard, CA) before density centrifugation. Density centrifugation was performed using Percoll density gradient, and cells at the interface between 40 and 80% discontinuous Percoll gradient were collected. The leukocyte’s viability was evaluated by trypan blue exclusion.
Intracellular IFN-γ staining assay
To evaluate the effect of the blockade of PD-1/PD-L1/2 and Tim-3/Tim-3-L pathways on the induction of IFN-γ, TILs and T-cells in noncancerous tissues were pre-incubation with blocking antibodies for 1 h, and stimulated with 5 μg/mL of anti-CD3 (eBioscience, San Diego, CA, USA) for 6 h . After incubation for another 2 h, 10 μg/mL of Brefeldin A (Sigma- Aldrich, St. Louis, MO, USA) was added to the culture medium. Cells were then stained with antibodies anti-CD3-PE-Cy7 and anti-CD4-PerCp-Cy5.5/anti-CD8-PerCp-Cy5.5, and marked with mAb against IFN-γ-APC (eBioscience, San Diego, CA, USA). Five hundred thousand events were recorded during flow cytometric analysis.
Statistical analysis was performed by GraphPad Prism 5.0 (GraphPad Software, US). Mann–Whitney test was used to assess the differences between the study groups. A pair wised t test was applied to compare the expression of the inhibitory molecules in cancer, noncancerous tissues, and blood. p < 0.05 was considered to be statistically significant.
T-cell subsets in GC
Tumor-infiltrating T-cell differentiation
The balance of T-cell differentiation plays an important role in successful immune controls. Therefore, T-cell differentiation in GC patients was analyzed according to the expressions of CCR7 and CD45RA. Both the tumor-infiltrating CD4+ and CD8+ T-cells were dominated by the effector memory phenotype (TEM, CCR7−CD45RA−, 55.52% for CD4+, 43.11% for CD8+) in GC. On CD4+ T-cells, there are 32.67% of central memory T-cells (TCM, CCR7+CD45RA−), 6.34% of terminally differentiated T-cells (Teff, CCR7−CD45RA+) and 5.72% of naive (Tnaive, CCR7+CD45RA+, 5.72%) T-cells (Additional file 1: Figure S1), while on CD8+ T-cells, there are 28.5% of Teff (28.5%), 14.57% of TCM (14.57%) and 13.81% of Tnaive (13.81%) T-cells (Additional file 1: Figure S1). TEM among CD4+ and CD8+ T-cells in tumor tissues was more frequent than TCM and Tnaïve T-cells in tumor tissues. On CD4+ T-cells, TEM was more frequent than Teff in tumor tissues, but on CD8+ T-cell, there was no difference between TEM and Teff. T-cells in paraneoplastic tissue displayed similar compositions compared to TILs. Overall, patterns of CD4+ and CD8+ T-cells differentiation revealed no differences between tumor and paraneoplastic tissue. It is very interesting that the frequencies of TEM and TCM on CD4+ and CD8+ T-cells in blood were significantly lower than that in tumors, while the frequencies of Teff and Tnaïve among CD4+ and CD8+ T-cells in blood were significantly higher than those in tumors and paraneoplastic tissues (Additional file 1: Figure S1). In addition, the frequencies of TEM and TCM on CD4+ T-cells had no significant difference between blood and paraneoplastic tissue, but the frequencies of TEM on CD8+ T-cells in paraneoplastic tissues were significantly higher than those in blood (37.16% vs. 9.89%, p = 0.002). T cell differentiation in HDs is similar to that in HDs (data not shown).
Tumor-infiltrating CD4+ CD25high CD127low T regulatory cells increase in GC patients
Elevated Tim-3 and PD-1 expression on tumor-infiltrating T lymphocytes in GC patients
The difference between the expression of Tim-3 and PD-1 in blood and tissues were also explored. Compared with the matched blood, the percentage of Tim-3+ cells in TILs were significantly increased (16.88% vs. 5.52%, p < 0.0001, Fig. 3e for CD4+; 21.96% vs. 4.56%, p < 0.0001, Additional file 2: Figure S2E for CD8+). PD-1+ was also expressed at a higher level on TILs (44.7% vs. 28.05%, p < 0.0001, Fig. 3f for CD4+; 56% vs. 27.1%, p < 0.0001, Additional file 2: Figure S2F for CD8+). Similarly, both the percentages of Tim-3+ and PD-1+ cells in paraneoplastic tissue were significantly lower than those in TILs (Tim-3+: 8.83% vs. 16.88%, p < 0.0001 for CD4+, 6.22% vs. 21.96%, p < 0.0001 for CD8+; PD-1+: 33.24% vs. 44.7%, p < 0.0001 for CD4+, 45.96% vs. 56%, p < 0.0001 for CD8+). Furthermore, PD-1+ Tim-3+ cells among T-cells were significantly higher in tumor tissues than those in blood (15.47% vs. 3.17%, p < 0.0001, Fig. 3g for CD4+; 18.2% vs. 1.45%, p < 0.0001, Additional file 2: Figure S2G for CD8+) and paraneoplastic tissues (15.47% vs. 6.64%, p < 0.0001, Fig. 3g for CD4+; 18.2% vs. 3.87%, p < 0.0001, Additional file 2: Figure S2G for CD8+). Percentages of PD-1+ and PD-1+ Tim-3+ in T-cells of paraneoplastic tissues were all significantly higher than those in blood for both CD4+ and CD8+ T-cells.
Ex vivo blockade of PD-1/PD-L1/2 and Tim-3/Tim-3-L pathways enhances the tumor-infiltrating T-cell IFN-γ production
Correlation of Tim-3+ PD-1+ CD4+/CD8+ T-cells with clinicopathological features
It has been reported that T lymphocytes played a critical role in controlling and eliminating cancer [3, 17, 18]. Researches revealed that tumor antigen would activate cytotoxic CD8+ T-cells which was enhanced by specific helper CD4+ T-cells [19, 20]. In our study, the GC tumors have higher percentages of CD4+ T-cells and a lower frequency of CD8+ T-cells compared with that in paraneoplastic tissues or blood, which indicated an increased CD4+/CD8+ ratio in tumors. In addition, the CD4+/CD8+ ratio in peripheral blood was significantly higher than that in matched paraneoplastic tissue. Our results showed that the T-cell subset distribution in patients with GC was different for varied tissues. The comparison is essential to the rational design for novel immuno-therapeutics strategy against GC. Previous reports showed that CD8+ T-cells prevailed over CD4+ T-cells in the tumor lesions derived from neuroblastoma patients, which meant that the CD4+/CD8+ ratio in tumors was lower than that in peripheral blood . However, these results were quite different from ours. In addition, the percentage of CD4+ T-cells was decreased in livers when compared with the blood in inflammatory liver injuries and viral infections . According to these studies, we conclude that different molecular mechanisms may cause the skewing of the CD4+/CD8+ T-cell ratio and the reason is not clear for the changes of CD4+ T-cells in GC patients, which needs further clinical investigations.
Previous studies have reported that there were increased frequency of effector and memory T-cells among virus-specific CD4+ T-cells during acute-resolving and chronic viral infections [18, 19]. In our research, the frequency of effector and memory T-cells among TILs and paraneoplastic T-cells elevated comparing with that of circulating T-cells, which was coincided with those in neuroblastoma patients. Fridman et al. found that the presence of CD8+ memory T-cells was associated with a favorable prognosis . However, the relationship between the presence of CD4+ memory T-cells and prognosis in patients with GC has yet to be elucidated, and it will be explored in further researches.
In addition, regulatory T-cells can regulate antigens and suppress immunity to cancer . However, the role of Tregs in anticancer immunity is more controversial. In some cancers such as ovarian carcinoma and HPV-associated patients, high numbers of tumor-infiltrating Tregs have shown poor outcomes [23, 24]. However, in colorectal cancer and lymphomas, Tregs have been reported as a positive prognostic factor [25, 26]. These findings implied that the role of Tregs might vary according to the type and etiology of the cancer. In GC, Choi et al. demonstrated that high level of Tregs among tumor-infiltrating CD4+ T-cells were favorable, but they only analyzed the Tregs in tumor tissue and peripheral blood from a healthy control . On the contrary, Hennequin et al. showed that low infiltration of Tregs were associated with better relapse-free survival in patients with localized gastric cancer . Moreover, there is still no definitive markers that used to define Treg. Instead, CD127low expression, which is known to be highly enriched in regulatory CD4+ CD25+ T-cells, might be the most specific marker that is known and used so far [27–29]. Therefore CD4+ CD25high CD127low was used to assess Tregs in CD4+ T-cells of GC patients in our study. We discovered an elevated level of Tregs in peripheral blood from patients with GC when compared with those from HDs. Notably, the percentage of CD25high CD127low T regulatory cells among TILs were significantly higher than their counterparts in peripheral blood and paraneoplastic tissue, while the Tregs in peripheral blood has no significant difference as compared with that in Nils, which indicated that the GC milieu favors the accumulation of immunosuppressive Tregs at the tumor site.
Recent studies suggested that cells express only PD-1+ indeed retain Ag responsiveness in tumors, while only co-expression of PD-1+ and Tim-3+ identifies the most profoundly hypo-functional T-cells [14–16], and the blockade of them rejuvenates tumor-infiltrating CD8+ T-cells function in cancer patients. In present study, we compared the expression of PD-1+ and Tim-3+ on CD4+/CD8+ T-cells in blood circulation, tumor, and paraneoplastic tissues from patients with GC, and further tested the changes of IFN-γ production on T-cells after in vitro blockade of PD-1+ and/or Tim-3+ pathways. Our results showed that the frequencies of Tim-3+, PD-1+, and PD-1+ Tim-3+ cells among CD4+/CD8+ cells in circulation were significantly higher in GC patients than that in HDs. These data were consistent with previous reports of increased PD-1+ and Tim-3+ expression on T-cells in GC, which suggested that PD-1+ and Tim-3+ may be involved in immune evasion in GC patients [30–33]. In addition, PD-1+ and Tim-3+ expression has been described in gastric patients with Helicobacter pylori (H. pylori) infection, which is a major cause of gastric cancer [34, 35]. Studies showed that the removal of H. pylori infection could theoretically decrease the number of cases by 89% [36, 37]. Therefore, the interaction among H. pylori infection, the expression of PD-1+ and Tim-3+, and GC needs to be further explored.
The hierarchical co-regulation of multiple negative regulatory pathways on CD4+ and CD8+ T-cells ;
The complex interactions between the inhibitory pathways during long-term in vitro conditions; and
The blocking Tim-3/galectin-9 interactions complementary to PD-1+ pathway inhibition .
According to abovementioned results, combination therapies of the immune checkpoint inhibitors with other targeted agents were alternative for GC patients.
One novel finding was that the percentages of Tim-3+ PD-1+ CD4+/CD8+ among TILs were associated with Tregs and clinical stages, which suggested that Tregs may induce the expression of PD-1+ and Tim-3+. A significant difference was observed between patients with advanced tumors and patients with stage I/II, which indicated that the exhausted T-cells may stimulate tumor progression.
In conclusion, to the best of our knowledge, this is the first study to characterize the subset composition and functional properties of multiple inhibitory molecules on TILs and the correlation of Tim-3+ PD-1+ CD4+/CD8+ T-cells in tumors with clinic opathological features and Treg in GC patients. Our findings provide new insights into the mechanisms underlying T-cell dysfunction and allow for future immunotherapeutic strategies in GC.
QG conceived the study. LY, BX, and PY performed experiments. JZ, PQ, LH, and GC conduct the analysis. ZW, ZR, PZ, and QG wrote the manuscript. All authors read and approved the final manuscripts.
The authors declare that they have no competing interests.
Availability of data and materials
Consent for publication
Ethics approval and consent to participate
The Institute Ethical Committee of the Affiliated Cancer Hospital of Zhengzhou University approved this study. Written informed consents were obtained from all patients.
This study is supported by the National Key Technology R&D Program (No. 2015BAI12B12) and National Clinical Research Center for Cancer, National Natural Science Foundation of China (Grant Number 81502648), Henan Medical Science and Technique Foundation (Grant Number 201401016), Zhengzhou Medical Science and Technique Foundation (Grant Number 153PKJGG061), Henan Students Studying Abroad Research Foundation (Grant Number 2015-5) and Henan Provincial Scientific and technological project (Grant Number 162102310046).
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