key: cord-0757669-45jc7gmd
authors: Zhong, Chen; Song, Zongchang; Li, Min
title: Gastric cancer patients display a distinctive population of IFNg(+)IL10(+) double positive CD8 T cells, which persists longer during prolonged activation
date: 2019-09-15
journal: Exp Cell Res
DOI: 10.1016/j.yexcr.2019.06.032
sha: 1cf210a0d7ab5720ae83bad2276f73299bd89a78
doc_id: 757669
cord_uid: 45jc7gmd

IL10 is generally regarded as a broad-spectrum regulatory cytokine. However, the role of IL10 in CD8 T cells remains controversial. In this study, we investigated the characteristics of endogenous IL10 by CD8 T cells in gastric cancer (GC) patients. Using intracellular staining, we found that in both GC patients and healthy controls, the majority of IL10-expressing CD8 T cells also presented concurrent IFNg expression. Interestingly, the frequency of IFNg(+)IL10(+) CD8 T cells was significantly higher in GC patients than in healthy controls, while the frequency of IFNg(+)IL10(-) CD8 T cells was significantly lower in GC patients than in healthy controls. Compared to the IFNg(-)IL10(-) CD8 T cells, both IFNg(+)IL10(-) and IFNg(+)IL10(+) CD8 T cells presented significantly higher expression of activation/inhibitory markers. Interestingly, the IFNg(+)IL10(+) cells presented lower PD1 and TIM3 and higher KLRG1 than the IFNg(+)IL10(-) CD8 T cells. Remarkably, the IFNg(+)IL10(+) CD8 T cells, but not the IFNg(+)IL10(-) CD8 T cells, were highly enriched in the CD45RO(+)CXCR5(+) subset. Prolonged activation resulted in significant enrichment of IFNg(+)IL10(+) CD8 T cells over time. Interestingly, compared to the CD45RO(+)CXCR5(-) CD8 T cells, the CD45RO(+)CXCR5(+) CD8 T cells presented stronger proliferation capacity at later stages of stimulation, and higher viability throughout the stimulation process. Overall, our investigation demonstrated that GC patients were enriched with a distinctive population of IFNg(+)IL10(+) double positive CD8 T cells, which resembled T follicular cytotoxic cells and could persist longer during prolonged activation.

Gastric cancer (GC) is one of the most common causes of cancerrelated deaths worldwide [1] , and its development is strongly linked to Helicobacter pylori (H. pylori) infection. However, only a tiny fraction of all H. pylori-infected individuals eventually develop GC [2] . The majority of infected cases remain asymptomatic through life. It is now understood that the chronic inflammatory response initiated by H. pylori critically contributes to GC pathogenesis [3] . Typically starting from the antral region that incites an inflammatory response, this H. pylori-mediated inflammation can gradually spread to the upper stomach over time and cause increasingly severe mucosal atrophy, intestinal metaplasia, and malignant transformation at the gastric lining [4, 5] .

Many branches of the immune system are involved in this process. H. pylori could directly elevate the expression of spermine oxidase in gastric epithelial cells and macrophages, and induce DNA damage through the production of reactive oxygen species [6, 7] . H. pylori and high salt diet also synergistically increase the production of several proinflammatory cytokines, such as IL1, IL6, and TNF, which possess angiogenic and pro-metastatic roles in tumor [8, 9] . Adaptive immune responses involving B cells and T cells likely also contributed to GC initiation. Mice infected with Helicobacter spp. showed symptoms of human GC patients, but not when B cells and T cells were absent [10, 11] . Further evidence demonstrated that Th1-mediated IFNg responses contributed to the gastric atrophy in mice infected with Helicobacter spp. [12, 13] .

On GC suppression once the tumor is established. Meta-analysis of tumorinfiltrating lymphocytes (TILs) in GC patients indicated that the frequency of tumor-infiltrating CD3 + T cells, especially the CD8 + TILs, was positively associated with overall survival [14] , while high expression of Foxp3, a transcription factor for regulatory T cells, in TILs was associated with reduced survival. High tumor-infiltrating CD3 + , CD8 + , and CD45RO + T cell frequency in addition was associated with lower lymph node metastasis and reduced recurrence following surgical resection [15, 16] . IL10 is a pleiotropic cytokine with multiple protective roles during inflammation. IL10 is shown to suppress innate and adaptive immune responses, mediate tissue repair, and maintain epithelium integrity [17] . In addition, IL10 can directly dampen the signal transduction of CD8 T cells, thus increasing the threshold required for antigen recognition and activation [18] . Interestingly, polymorphisms that increase IL10 production are associated with elevated GC risk [19] [20] [21] , which is contrary to the hypothesis that IL10 may prevent inflammation-induced disruption of the epithelium. Also, there is evidence that IL10 can promote the activation and expansion of tumor-resident CD8 T cells in murine tumor studies [22, 23] , which is in contrast with the common notion that IL10 inhibited CD8 T cell responses.

In this study, we investigated the endogenous production of IL10 by CD8 + T cells in GC patients. Interestingly, we found that CD8 T cells that produced cytokines could be separated into two distinctive subsets, the IFNg + IL10single positive type and the IFNg + IL10 + double positive type, the latter being more enriched in GC patients. These two subsets were not only different in cytokine production, but also different in the expression of surface markers, exhaustion markers, and proliferation and survival capacity.

This study included 26 patients with primary gastric adenocarcinoma, including 14 stage II and 12 stage III patients as defined by the American Joint Committee on Cancer [24] . All the patients were H. pylori infected. A total of 26 age-and sex-matched healthy volunteers were recruited as controls. The demographic data of patients and controls are presented at Table 1. The exclusion criteria were age outside of the 18 to 65 years range, familial history of GC, previous or ongoing chemotherapy and radiation therapy, autoimmune conditions, and other malignancies. All patients and controls donated 50 to 100 mL of peripheral blood samples, from which peripheral blood mononuclear cells (PBMCs) were harvested by Ficoll density gradient centrifugation. The diagnosis, sample collection, and experimentation were performed at the No. 960 Hospital, following approval by the Ethics Committee of No. 960 Hospital. All participants provided written informed consent.

CD8 T cells were isolated from total PBMCs via negative magnetic selection using EasySep Human CD8 + T Cell Enrichment Kit (Stemcell). Purity, assessed by staining of CD3 + CD8 + cells, was consistently above 97%. The CD8 T cells were then incubated in RPMI 1640 supplemented with L-glutamine, penicillin-streptomycin, and 10% fetal bovine serum (all from Gibco). For stimulation, Human T activator (CD3/CD28) beads (Gibco) were added at 2 beads per cell, and IL2 (Gibco) was supplemented at 50U/mL. The medium and IL2 were replaced every 3 days. At time points specified in the experiments, centrifugation was performed at 300 g for 5 min to separate the cells and the supernatant.

Violet Dead Cell Stain (Invitrogen) and different combinations of surface anti-human staining antibodies (BioLegend), including CD3, CD8, PD1, TIM3, KLRG1, CXCR5, and CD45RO, were added for 30 min on ice. The cells were then washed twice by centrifugation at 300g for 5 min, and permeabilized using CytoFix/CytoPerm (BD) for 15 min on ice. After washing, intracellular staining antibodies (BioLegend), including IFNg and IL10, were added for 30 min on ice in Perm Wash Buffer (BD). Samples were washed twice and acquired in BD LSR instrument. For each experiment, 5 × 10 4 lymphocytes at minimum were collected.

IFNg and IL10 concentration was measured using respective Human ELISA Kits (Invitrogen) according to the manufacturer's instructions. Standard curves were established using pure cytokine samples included in the kits.

CD8 T cells after magnetic negative selection were incubated with anti-human CD45RO and CXCR5 for 30 min on ice. The CD45RO + CXCR5 + CD8 T cells and CD45RO + CXCR5 -CD8 T cells were sorted using FACSAria instrument (BD). Following stimulation, cells were plated at 1 × 10 4 cells per well in a 96-well plate, and were incubated with tritiated thymidine (0.1 μCi/mL; Amersham Biosciences) for 6 hours, and the radioactivity was counted in a liquid scintillation counter.

Data were given as scatterplots or mean ± SD. Distribution was examined using D'Agostino-Pearson test. Differences between groups were examined using tests specified in the figure legend. Two-tailed p < 0.05 was considered significant.

We recruited 26 GC patients and 26 healthy controls. Circulating CD8 T cells were isolated from total PBMCs using magnetic sorting. The isolated CD8 T cells were then treated with anti-CD3/CD28 and IL2 to stimulate cytokine production. Using intracellular staining, we identified IFNg-expressing and IL10-expressing CD8 T cells in these subjects (Fig. 1A) . The frequency of IFNg-expressing IL10-nonexpressing (IFNg + IL10 -) CD8 T cells was at 12.9 ± 0.5% (mean ± SEM) in healthy controls and significantly lower at 8.7 ± 0.4% in GC patients (Fig. 1B) . The frequency of IFNg-expressing and IL10-expressing (IFNg + IL10 + ) CD8 T cells, on the other hand, was at 1.2 ± 0.1% in healthy controls and markedly higher at 3.8 ± 0.3% in GC patients (Fig. 1C) . The frequency of total IFNg-expressing (IFNg + IL10plus IFNg + IL10 + ) CD8 T cells was at 14.2 ± 0.5% in healthy controls and slightly lower at 12.5 ± 0.4% in GC patients (Fig. 1D) . Overall, these data demonstrated that GC patients presented an upregulation of the 

To characterize the IFNg/IL10-double positive CD8 T cell population in GC patients, we investigated the surface marker expression of these cells. First, the expression of inhibitory markers, including PD-1, Tim-3, and KLRG1, were examined by flow cytometry (Fig. 2A) . Compared to the CD8 T cells that did not express IFNg or IL10 (IFNg -IL10 -), the IFNg + IL10 -CD8 T cells presented significantly higher PD1 and TIM3 expression ( Fig. 2B and C) . Interestingly, the IFNg + IL10 + CD8 T cells presented significantly higher PD1 and TIM3 expression than IFNg -IL10cells and significantly lower PD1 and TIM3 expression than IFNg + IL10cells. The expression of KLRG1 was also higher in the IFNg + IL10 -CD8 T cells than in the IFNg -IL10 -CD8 T cells (Fig. 2D) . The IFNg + IL10 + CD8 T cells presented slightly higher KLRG1 than the IFNg + IL10 -CD8 T cells.

The PD1, TIM3, and KLRG1 expression pattern by IFNg + IL10 + CD8 T cells strongly resembled the expression pattern by CXCR5 + CD8 + T follicular cytotoxic (Tfc) cells, previously characterized in chronic LCMV infection [25] . Hence, the expression of CD45RO and CXCR5 were examined ( Fig. 3A and C) . On average, more than half of IFNg -IL10 -CD8 T cells were CD45RO-negative (Fig. 3B) . The IFNg + IL10and the IFNg + IL10 + CD8 T cells, on the other hand, were predominantly CD45RO-positive, and the frequency of CD45RO + cells in IFNg + IL10 + CD8 T cells was significantly higher than that in IFNg + IL10 -CD8 T cells (Fig. 3B) . The frequency of CXCR5-positive was lower than 5.0% in IFNg -IL10 -CD8 T cells from 26 GC patients (Fig. 3D) . In IFNg + IL10 -CD8 T cells, however, 16.4% on average were CXCR5-positive. Remarkably, in IFNg + IL10 + CD8 T cells, 80.4% cells on average were CXCR5-positive.

Subsequently, we sorted total CD8 T cells into CD45RO + CXCR5 + CD8 T cells and CD45RO + CXCR5 -CD8 T cells. Each subset was then treated in vitro with anti-CD3/CD28 and IL-2 for 24 hours. The IFNg and IL10 production was measured using a multiplex assay. The CD45RO + CXCR5 + CD8 T cells and the CD45RO + CXCR5 -CD8 T cells displayed markedly different capacity in IL10 production, with the former expressing high levels of IL10, while the latter expressed only minimally detectable IL10 (Fig. 4A) . Both the CD45RO + CXCR5 + CD8 T cells and the CD45RO + CXCR5 -CD8 T cells readily expressed IFNg, with the CD45RO + CXCR5 + CD8 T cells being more potent (Fig. 4B) .

Together, these data demonstrated that the IFNg + IL10 + CD8 T cells displayed lower PD1 and TIM3 than the IFNg + IL10 -CD8 T cells, and could be enriched in the CD45RO + CXCR5 + CD8 T cell fraction.

IFNg + IL10 + CD8 T cells were enriched during prolonged stimulation.

To examine the dynamics of IFNg and IL10 expression following extended stimulation, the CD8 T cells were sorted from PBMCs and stimulated with anti-CD3/CD28 and IL-2 for a total of 12 days, and the frequencies of IFNg + IL10and IFNg + IL10 + CD8 T cells were examined on day 1, day 3, day 6, day 9, and day 12 of stimulation (Fig. 5A) . The frequency of IFNg + IL10 -CD8 T cells increased from day 1 to day 6, then decreased from day 6 to day 12. The frequency of IFNg + IL10 -CD8 T cells, on the other hand, was increasing continuously until day 12. From day 1 to day 6, the frequency of IFNg + IL10cells was significantly (A) IL10 production by CD8 + CD45RO + CXCR5 + and CD8 + CD45RO + CXCR5 -T cells following 24-h stimulation using anti-CD3/CD28 beads and IL2. (B) IFNg production by CD8 + CD45RO + CXCR5 + and CD8 + CD45RO + CXCR5 -T cells following 24-h stimulation using anti-CD3/CD28 beads and IL2. Student's t-test. **p < 0.01. ***p < 0.001. C. Zhong, et al. Experimental Cell Research 382 (2019) 111487 higher than the frequency of IFNg + IL10 + cells. On day 9 or day 12, however, no significant differences were observed.

In the supernatant, we detected the cytokine concentration in the supernatant harvested at each time point. IL10 concentration presented an increasing trend, and compared to the concentration at day 1, the concentrations at day 6 to day 12 were significantly higher (Fig. 5B) . IFNg, on the other hand, increased initially and decreased after day 6. Compared to the IFNg concentration on day 1, the IFNg concentrations were higher on day 3 and day 6, comparable on day 9, and lower on day 12 (Fig. 5C) .

CD45RO + CXCR5 + CD8 T cells presented better proliferation and lower death than CD45RO + CXCR5 -CD8 T cells.

To assess the proliferation capacity of IFNg + IL10and IFNg + IL10 + CD8 T cells, we took advantage of the observation that IFNg + IL10 + CD8 T cells were enriched in CD45RO + CXCR5 + cells. The total CD8 T cells were sorted into CD45RO + CXCR5 + and CD45RO + CXCR5subsets. Each subset was then stimulated with anti-CD3/CD28 and IL2 in vitro, and the proliferation capacity was examined using thymidine incorporation assay (Fig. 6A) . The proliferation capacity of CD45RO + CXCR5 -CD8 T cells was significantly higher than that of Fig. 6 . Proliferation and viability of CD45RO + CXCR5 + and CD45RO + CXCR5 -CD8 T cells. CD8 + CD45RO + CXCR5 + and CD8 + CD45RO + CXCR5 -T cells were stimulated using anti-CD3/CD28 beads and IL2. Every 3 days, a portion of the cells were harvested to determine proliferation capacity and viability. (A) Radioactive thymidine incorporation of CD8 + CD45RO + CXCR5 + and CD8 + CD45RO + CXCR5 -T cells after 6-h pulsing with tritiated thymidine. (B) Viability of CD8 + CD45RO + CXCR5 + and CD8 + CD45RO + CXCR5 -T cells, measured using Violet Dead Cell Stain. (A and B) Two-way ANOVA followed by Sidak's multiple comparisons. *p < 0.05. **p < 0.01. ***p < 0.001. C. Zhong, et al. Experimental Cell Research 382 (2019) 111487 CD45RO + CXCR5 + CD8 T cells on day 3 and day 6, but quickly declined on day 9 and day 12, during which the proliferation capacity of CD45RO + CXCR5 + CD8 T cells continued to increase and exceeded that of CD45RO + CXCR5 -CD8 T cells. The viability of CD45RO + CXCR5 + and CD45RO + CXCR5 -CD8 T cells was then examined using flow cytometry. Throughout the course of stimulation, the CD45RO + CXCR5 + CD8 T cells presented higher viability than the CD45RO + CXCR5 -CD8 T cells (Fig. 6B ).

IL10 is generally regarded as a broad-spectrum regulatory cytokine that dampens inflammatory responses, including those mediated by CD8 T cells [17, 18] . In GC patients, IL10 expression level is positively associated with the density of microvessels and negatively associated with CD8 T cell infiltration in the tumor [26] . However, the appearance of IL10 is often accompanied by inhibitory immune cells, such as regulatory B cells, regulatory dendritic cells, M2-polarized macrophages, and tumor-associated macrophages [27] [28] [29] . Hence, some of the IL10mediated suppressive effects, previously described in other studies, might have required the coexistence of other inhibitory molecules and cell types. Indeed, IL10 in IL10 receptor (IL10R)-expressing CD8 T cells has been shown to strongly induce the phosphorylation of STAT3 and STAT1 and promote the in situ proliferation of tumor-resident CD8 T cells [22, 23] . In addition, in a coronavirus-infected murine encephalitis model, CD8 T cells that produced IL10 displayed higher cytokine and chemokine expression and stronger cytolytic activity [30] .

In this study, we investigated CD8 T cells that expressed IL10 endogenously. In both healthy controls and GC patients, CD8 T cells that expressed IL10 could be observed following anti-CD3/CD28 and IL2 stimulation. Interestingly, IL10-single positive cells were rare, and the majority of IL10-expressing cells also presented concurrent IFNg expression. GC patients had more IFNg/IL10-double positive CD8 T cells than healthy controls, while healthy controls had more IFNg-single positive CD8 T cells than GC patients, which seemed to suggest that the IFNg/IL10-double positive CD8 T cell population arose due to disease. We later found that prolonged stimulation alone could result in higher levels of IFNg/IL10-double positive cells and lower levels of IFNg-single positive cells. Hence, it is possible that CD8 T cells in GC patients are continuously being challenged with tumor antigens and/or H. pylori antigens, resulting in prolonged activation and higher frequencies of IFNg/IL10-double positive cells. In the future, the antigen specificity of IFNg/IL10-double positive CD8 T cells should be investigated.

A major limitation of this study is that we have not conclusively demonstrated the function of IFNg/IL10-double positive CD8 T cells in gastric cancer patients. We found that certain features of the IFNg/IL10double positive CD8 T cell subset, including lower PD1, lower TIM3, and higher KLRG1, resembled Tfc cells [25] . Indeed, we found that the IFNg/IL10-double positive cells were highly enriched in CD45RO + CXCR5 + CD8 T cells, while IFNg-single positive cells were enriched in CD45RO + CXCR5 -CD8 T cells. This feature would help us demonstrate the role of IFNg/IL10-double positive CD8 T cells in animal models using deletions and adoptive transfers. In past research, Tfc cells were shown to present stronger proliferation in response to PD1 blockade in exhausted CD8 T cells [31] , suggesting that they might be candidates of antitumor immunotherapy. However, regulatory functions also have been demonstrated in IL10-expressing CD8 T cells. For example, IL4 and IL12-induced IL10-expressing CD8 T cells was shown to inhibit graft-versus-host disease in vivo, and suppressed naive and effector T cell activation and IgG/IgE production [32] . Interestingly, CD8 T cell-mediated IL10 was shown to resolve chemotherapy-induced neuropathic pain [33] . Hence, IL10 expression might serve multiple roles in CD8 T cells, and the function of IFNg/IL10-double positive CD8 T cells should be further investigated using animal models.

None.

Global cancer incidence and mortality rates and trends -an update

Helicobacter pylori evolution and phenotypic diversification in a changing host

Aboul-Soud, From inflammation to gastric cancer: role of Helicobacter pylori

Development of gastric cancer and its prevention

Helicobacter pylori and gastrointestinal tract adenocarcinomas

Spermine oxidation induced by Helicobacter pylori results in apoptosis and DNA damage: implications for gastric carcinogenesis

Induction of polyamine oxidase 1 by Helicobacter pylori causes macrophage apoptosis by hydrogen peroxide release and mitochondrial membrane depolarization

Effect of NaCl and Helicobacter pylori vacuolating cytotoxin on cytokine expression and viability

NF-κBinduced NOX1 activation promotes gastric tumorigenesis through the expansion of SOX2-positive epithelial cells

The role of T cell subsets and cytokines in the pathogenesis of Helicobacter pylori gastritis in mice

Cellular immune responses are essential for the development of Helicobacter felis-associated gastric pathology

Helicobacter pylori-induced mucosal inflammation is Th1 mediated and exacerbated in IL-4, but not IFN-gamma, gene-deficient mice

Interferon gamma induction of gastric mucous neck cell hypertrophy

Prognostic role of tumor-infiltrating lymphocytes in gastric cancer: a systematic review and meta-analysis

Clinical impact of tumor-infiltrating CD45RO + memory T cells on human gastric cancer

Clinical significance of tumorinfiltrating lymphocytes for gastric cancer in the era of immunology

Regulation and functions of the IL-10 family of cytokines in inflammation and disease

Interleukin-10 directly inhibits CD8 + T cell function by enhancing N-glycan branching to decrease antigen sensitivity

Effects of interleukin-10 polymorphisms, helicobacter pylori infection, and smoking on the risk of noncardia gastric cancer

Effects of interleukin-10 gene polymorphism on the development of gastric cancer C

Japanese subjects

Interindividual variations in constitutive interleukin-10 messenger RNA and protein levels and their association with genetic polymorphisms

Interleukin-10 promotes the maintenance of antitumor CD8(+) T-cell effector function in situ

IL-10 directly activates and expands tumor-resident CD8+ T cells without De Novo infiltration from secondary lymphoid organs

Trotti, AJCC Cancer Staging Manual

Follicular CXCR5-expressing CD8(+) T cells curtail chronic viral infection

Interleukin-10 expression significantly correlates with minor CD8 + T-cell infiltration and high microvessel density in patients with gastric cancer

Regulatory B cells: origin, phenotype, and function

Innate and adaptive immune cells in the tumor microenvironment

Regulatory activity of autocrine IL-10 on dendritic cell functions

Highly activated cytotoxic CD8 T cells express protective IL-10 at the peak of coronavirus-induced encephalitis

Defining CD8+ T cells that provide the proliferative burst after PD-1 therapy

Cytokine-induced IL-10-secreting CD8 T cells represent a phenotypically distinct suppressor T-cell lineage

CD8+ T cells and endogenous IL-10 are required for resolution of chemotherapyinduced neuropathic pain

This study was supported by Science and Technology Program of Huzhou City (2018GY26).

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yexcr.2019.06.032.