Functionally impaired follicular helper T cells induce regulatory B cells and CD14+ human leukocyte antigen-DR- cell differentiation in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) represents one of the most common and aggressive cancers worldwide, as it typically displays irreversible progression and poor prognosis. Interaction between programmed death 1 (PD-1) and its ligand, PD-L1, plays important roles in tumor immunology. Follicular helper T (Tfh) cells have characteristically high PD-1 expression; thus, in the present study, we investigated the role of circulating Tfh cells and their correlation with disease-free survival after tumor resection in NSCLC. We found significantly higher number of Tfh cells but lower serum interleukin (IL)-21 levels in NSCLC patients, especially in those with advanced stage (III and IV), indicating that the function of Tfh cells to produce IL-21 was impaired. Further analysis showed that the increase in Tfh cells was attributable to an expansion of the PD-1+ -Tfh2 and PD-1+ -Tfh17 subtypes. Functional analysis showed that Tfh cells from NSCLC patients induced the differentiation of regulatory B cells and CD14+ human leukocyte antigen (HLA)-DR- cells. Interestingly, the number of Tfh1 subtypes in NSCLC patients was negatively correlated with disease-free survival after tumor resection. In short, the high number and abnormal function of Tfh cells could cause further immunosuppression and lead to tumor development in NSCLC. Rescuing Tfh functions therefore represents a potential therapeutic strategy in NSCLC.

INTRODUCTION

Lung cancer is the leading cause of cancer‐related death worldwide.1 Non‐small cell lung cancer (NSCLC) accounts for approximately 75% of lung cancers, is usually diagnosed at advanced stages and has a 5‐year survival rate of <5%.2 Targeted therapy improves the survival rate for advanced NSCLC, although this only benefits a subset of patients.3 Studies have shown that the interaction between programmed death 1 (PD‐1) and its ligand, PD‐L1, inhibit immune responses in tumors. PD‐1/PD‐L1 checkpoint inhibitors show impressive antitumor activity in NSCLC.4, 5, 6, 7, 8 The recently defined follicular helper T (Tfh) cells express characteristically high PD‐1, which is a common mediator of immunosuppression in multiple cancers,9, 10 but it is not clear whether Tfh cells can also inhibit immune responses in NSCLC.

Follicular helper T cells coexpress high levels of surface C‐X‐C motif chemokine receptor 5 (CXCR5) and inducible costimulator (ICOS), with Bcl‐6 as their distinguishing transcription factor and interleukin (IL)‐21 as their characteristic cytokine.11, 12, 13 Both peripheral circulating and germinal center (GC) Tfh cells support B‐cell activation, expansion and differentiation into plasma cells and memory B cells in GC, thereby promoting B‐cell immunity.13, 14, 15 Human circulating Tfh cells are divided into three major functional subtypes (Tfh1, Tfh2, and Tfh17),16 with skewing to Tfh2 and Tfh17 subtypes observed in autoimmune disease and several tumors.17, 18

Recent studies have suggested that Tfh cells and IL‐21 regulate humoral immunity and cell‐mediated antiviral/tumor responses in some infectious diseases and tumors.19, 20 Breast cancer patients with higher Tfh cell infiltrates in the tumor show better survival over those with lower Tfh cell infiltration.21 However, we do not know whether changes in circulating Tfh cells in NSCLC are associated with tumor development, progression and, eventually, clinical prognosis.

In the present study, we aimed to investigate the levels of circulating Tfh cells in the peripheral blood of newly diagnosed NSCLC patients to explore their role in the pathogenesis of NSCLC and to show their predictive value for disease‐free survival.

MATERIALS AND METHODS

Patients

From June 2015 to March 2017, 57 newly diagnosed NSCLC patients (34 males and 23 females; median age 60.48 years; range 38 to 81 years) at Zhejiang Provincial People's Hospital before any systemic or local treatment was given were enrolled in the present study. Of all 57 NSCLC patients, 14, 12, 15, and 16 were classified into stages I, II, III and IV, respectively, based on criteria described in the seventh edition International Association for the Study of Lung Cancer (IASLC) TNM staging system. Detailed clinical characteristics of NSCLC patients are shown in Table 1. We also recruited 20 age‐matched healthy subjects (HS) from the physical examination department. The study was approved by the Zhejiang Provincial People's Hospital Review Board. All methods were carried out in accordance with the 1975 Declaration of Helsinki. All tumor specimens in the pathological analysis were obtained with informed consent with approval by the ethics committee of our hospital.

Table 1

Clinical characteristics of NSCLC patients and HS

	NSCLC	HS
Gender	57	20
Male	34	12
Female	23	8
Years (range)	60.48 ± 12.46	60.36 ± 12.07
>60	29	9
≤60	28	11
Histology
Adenocarcinoma	31	NA
Squamous	12	NA
Other types	14	NA
TNM stage
I	14	NA
II	12	NA
III	15	NA
IV	16	NA
PD‐L1, n
Positive	25	NA
Negative	32	NA
White blood cell (×109/L)	6.95 ± 2.31	6.00 ± 0.97
Lymphocyte (×109/L)	1.76 ± 0.52	2.21 ± 0.50

HS, healthy subjects; NA, not applicable; NSCLC, non‐small cell lung cancer; PD‐L1, programmed death ligand 1.

Cell isolation

Peripheral blood mononuclear cells were isolated from blood samples using a Ficoll‐Hypaque density gradient centrifugation method. Serum samples were obtained by centrifugation at 1500 g for 10 minutes and were immediately stored at −80°C. Serum IL‐21 was assessed using ELISA (eBioscience, San Diego, CA, USA).

CD4+CXCR5+ICOS+PD‐1+ Tfh cells, CD19+IgD+ naive B cells, and CD14+HLA‐DR− cells from six HS and six NSCLC patients were purified using a FACSAria III Aria cell sorter (Becton Dickinson, Sparks, MD, USA) based on the expression of CD4, CXCR5, ICOS, and PD‐1 or CD19 and IgD or CD14 and HLA‐DR. Cell purity was confirmed to be >95% by flow cytometry.

Flow cytometry analysis

The following cell surface antibodies were used: PerCP‐CD3 (clone SK7; BD Biosciences, San Diego, CA, USA), PC7‐CD4 (clone 13B8.2; Beckman Coulter, Marseille cedex, France), Alexa Fluor 488‐CXCR5 (clone RF8B2; BD Biosciences), APC‐ICOS (clone ISA‐3; BD Biosciences), PerCP‐cy7‐PD‐1 (clone H12.1; BD Biosciences), APC‐CXCR3 (clone IC6; BD Biosciences), PerCP‐cy5.5‐CCR6 (clone 11A9; BD Biosciences), FITC‐CD19 (clone J4.119; Beckman Coulter) and PE‐CD14 (clone RMO52; Beckman Coulter). After cells were incubated with cell surface antibodies for 30 minutes at 4°C in the dark, they were washed with PBS and then analyzed by flow cytometer. CD4+CXCR5+ICOS+PD‐1+ Tfh cells were identified based on ICOS and PD‐1 expression after cells were gated on CD3+CD4+CXCR5+ (Figure S1). Tfh subtypes were determined according to CXCR3 and CCR6 expression after cells were gated on CD3+CD4+CXCR5+ (Figure S1) and the PD‐1 expression of the three subtypes was further analyzed.

For the detection of intracellular cytokines following cell surface staining, cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences) and then stained using PE‐IL‐10 (clone JES5‐19F1; BD Biosciences) and PE‐cy7‐TGF‐β (clone TW4‐9E7; BD Biosciences) or Alexa Fluor 488‐TNF‐α (clone MAb11; BD Biosciences). Stained cells were then analyzed using a FACS Canto II flow cytometer and Diva software (Becton Dickinson). All staining was carried out according to the manufacturer's protocol.

Function analyses of Tfh cells

Isolated Tfh cells (1.5 × 104) were cultured either alone or 1:1 with purified CD19+IgD+ cells (1.5 × 104) in complete RPMI 1640 containing l‐glutamine, NaHCO3, 10% FCS and penicillin/streptomycin (100 U/mL) in 96‐well U‐bottom plates in the presence of 2 μg/mL Staphylococcal Exterotoxin B (SEB) for 72 hours, with PIB (phorbol‐12‐myristate‐13‐acetate + ionomycin + brefeldin A) added in the last 5 hours, as described elsewhere. Cells were then stained with FITC‐CD19, permeabilized, stained intracellularly with PE‐IL‐10 and PE‐cy7‐transforming growth factor beta (TGF‐β) and analyzed by flow cytometry. The supernatant was harvested for IL‐10 and TGF‐β detection.

Isolated Tfh cells (1.5 × 104) were cultured either alone or 1:1 with purified CD14+HLA‐DR− cells (1.5 × 104) in complete RPMI 1640 containing l‐glutamine, NaHCO3, 10% FCS and penicillin/streptomycin (100 U/mL) in 96‐well U‐bottom plates for 72 hours, with PIB added in the last 5 hours as described elsewhere. Cells were then stained with PE‐CD14, permeabilized, stained intracellularly with Alexa Fluor 488‐tumor necrosis factor (TNF)‐α and analyzed by flow cytometry. Supernatant TNF‐α levels were examined by ELISA.

Enzyme‐linked immunosorbent assay

Human IL‐21, IL‐10, TGF‐β and TNF‐α ELISA Ready‐Set‐Go Kits (eBioscience) were used to examine cytokine levels following instructions provided by the manufacturer.

Immunohistochemistry for PD‐L1

All measurements for PD‐L1 were obtained according to the immunohistochemistry (IHC) protocols provided by the manufacturers. All IHC results were checked independently by two pathologists. The cutoff for PD‐L1 expression on tumor cells (Dako, 22C3, Copenhagen, Denmark; approved by the FDA) was equal to or more than 50% staining.

Statistical analysis

Statistical analysis was carried out with GraphPad Prism 5.01 software (GraphPad Software Inc., San Diego, CA, USA). The statistical tests used for data analysis included the Mann‐Whitney U test and the Pearson test for correlation analysis. Quantitative data are presented as the mean values ± standard deviations (SD). Differences were considered to be statistically significant at values of P < .05 and P < .01.

RESULTS

Elevated numbers of Tfh cells and skewing to PD‐1+‐Tfh2 and PD‐1+‐Tfh17 subtypes in NSCLC patients

Compared to HS, a significantly higher frequency and number of circulating CD4+PD‐1+, Tfh cells were observed in NSCLC patients (Figure 1A,B), but there was no significant difference in the frequency and number of CD4+CXCR5+ cells between the two groups (Figure 1A,B). Because Tfh cells can be divided into Tfh1 (CXCR3+CCR6−), Tfh2 (CXCR3−CCR6−) and Tfh17 (CXCR3−CCR6+) subtypes (Figure 1C,D), we further compared the prevalence of these three subtypes in NSCLC patients. Tfh2 and Tfh17 subtypes were significantly elevated in NSCLC patients, whereas Tfh1 subtypes were decreased in NSCLC patients (Figure 1C,D).

Figure 1

Elevated follicular helper T (Tfh) cells and skew to Tfh1 and Tfh17 subtypes in non‐small cell lung cancer (NSCLC) patients. A, Frequency in CD4+ T cells and number of CD4+ CXCR5+, CD4+ PD‐1+ and Tfh cells in healthy subjects (HS) (n = 20) and NSCLC patients (n = 57) are shown in the top and bottom panels, respectively. B, Representative dot plots of CD4+ CXCR5+ (top panel), CD4+ PD‐1+ (middle panel), Tfh cells (bottom panel) from one HS and one NSCLC patient are shown. Numbers indicate frequency of cells among CD4+ T cells. C, Frequency in CD4+ T cells and number of Tfh1, Tfh2 and Tfh17 subtypes are shown in the top and bottom panels, respectively. D, Representative dot plots of Tfh1, Tfh2 and Tfh17 subtypes from one HS and one NSCLC patient are shown. E, Mean fluorescence intensity (MFI) of PD‐1+ in Tfh1, Tfh2 and Tfh17 subtypes is shown. F, Representative flow cytometric plots of PD‐1 in Tfh1, Tfh2 and Tfh17 subtypes from one HS and NSCLC patient are shown in the left and right panels, respectively. G, Frequency in CD4+ T cells and number of PD‐1 in Tfh1, Tfh2 and Tfh17 are shown in the left and right panels, respectively. H‐M, Differences in the number of CD4+ CXCR5+ (H), CD4+ PD‐1+ (I), Tfh (J), Tfh1 (K), Tfh2 (L) and Tfh17 subtypes (M) in NSCLC patients with different stages. CXCR5, C‐X‐C motif chemokine receptor 5; PD‐1, programmed death 1

We also investigated PD‐1 expression of three Tfh subtypes in HS and NSCLC patients and found higher mean fluorescence intensity (MFI) of PD‐1 in Tfh1 than in Tfh2 and Tfh17, and higher MFI of PD‐1 in Tfh1 in NSCLC patients than in HS (Figure 1E,F). However, we further compared PD‐1 expression of three Tfh subtypes in NSCLC patients with those in HS and found that the frequency and the number of PD‐1+‐Tfh2 and PD‐1+‐Tfh17 in NSCLC patients were higher than those in HS (Figure 1G). These data suggest that circulating Tfh cells, especially PD‐1+‐Tfh2 and PD‐1+‐Tfh17 subtypes, expand in NSCLC and indicate a potential involvement of Tfh cells in NSCLC.

Non‐small cell lung cancer patients can be classified into stage I, stage II, stage III and stage IV according to the TNM classification system, with severity increasing from stage I to stage IV. Thus, we further analyzed the numbers of circulating Tfh cells in NSCLC patients with different stages. Our data showed lower numbers of CD4+PD‐1+ (Figure 1I), Tfh cells (Figure 1J), Tfh2 (Figure 1L) and Tfh17 subtypes (Figure 1M) but a higher number of Tfh1 subtypes (Figure 1K) in NSCLC patients with early stage (I and II) relative to those with advanced stage (III and IV); however, there was no significant difference in the number of CD4+CXCR5+ cells in NSCLC with different stages (Figure 1H).

Decreased serum IL‐21 levels and its negative association with circulating Tfh cells in NSCLC patients

Interleukin‐21 is produced mainly by Tfh cells. Although the number of Tfh cells was elevated in NSCLC patients, in the present study, we observed decreased serum IL‐21 levels (Figure 2A), especially in those with advanced stage (III and IV; Figure 2B). Moreover, IL‐21 levels were significantly and negatively correlated with the number of circulating Tfh cells in NSCLC patients (Figure 2C). We further analyzed IL‐21 levels relative to Tfh subtypes and found that the number of Tfh17 subtypes was negatively correlated with IL‐21 levels (Figure 2F), whereas the number of Tfh1 and Tfh2 subtypes were not correlated with IL‐21 levels in NSCLC patients (Figure 2D,E). These results indicated that the function of Tfh cells to produce IL‐21 could be impaired in NSCLC patients.

Figure 2

Correlation of serum interleukin (IL)‐21 levels with circulating follicular helper T (Tfh) cells in non‐small cell lung cancer (NSCLC) patients. A, Serum IL‐21 levels were measured for healthy subjects (HS) (n = 20) and NSCLC patients (n = 57). B, IL‐21 levels in NSCLC patients with different stages. C‐F, Correlation of IL‐21 levels with the number of Tfh, Tfh1, Tfh2 and Tfh17 subtypes in NSCLC patients, respectively

Follicular helper T cells from NSCLC patients induce differentiation of regulatory B cells and CD14+HLA‐DR− cells

We know that interaction of PD‐1 and PD‐L1 inhibits immune responses in tumors. Our previous study found high numbers of IL‐10+ regulatory B cells and CD14+HLA‐DRlow/− myeloid‐derived suppressor cells (MDSC) in NSCLC patients22 and, thus, in the present study, we investigated the functional effect of Tfh cells on CD19+IgD+ naïve B cells and CD14+HLA‐DR− cells. Tfh cells were sorted from six HS and six NSCLC patients and were cocultured 1:1 with autologous CD19+IgD+ naïve B cells in the presence of SEB. After 72 hours, we observed a slightly higher frequency of IL‐10‐ and TGF‐β‐producing CD19+ B cells (Figure 3A‐C) and supernatant IL‐10 and TGF‐β levels (Figure 3D,E) when cocultured with Tfh cells compared to CD19+IgD+ cells alone from NSCLC patients.

Figure 3

Follicular helper T (Tfh) cells from non‐small cell lung cancer (NSCLC) patients induce differentiation of regulatory B cells and CD14+ HLA‐DR − cells. FACS‐sorted Tfh cells were cultured 1:1 with CD19+IgD+ naïve B cells or CD14+ HLA‐DR − monocytes from healthy subjects (HS) (n = 6) and NSCLC patients (n = 6). Intracellular levels of interleukin (IL)‐10 and transforming growth factor (TGF)‐β among CD19+ B cells and tumor necrosis factor (TNF)‐α among CD14+ monocytes were measured by flow cytometry. Supernatant cytokines were measured by ELISA. A‐B, Graphs show the frequency of CD19+ IL‐10+ and CD19+ TGF‐β+ before or after CD19+IgD+ naïve B cells cocultured with Tfh cells. C, Representative dot plots show the gating strategy and the frequency of live CD19+ B cells, CD19+ CD10+ and CD19+ TGF‐β+ from a single HS (top panel) and NSCLC patient (bottom panel). All the B cell subsets were analyzed after gating on live CD19+ cells. D‐E, Supernatant IL‐10 and TGF‐β levels from HS and NSCLC patients. F, Graphs show the frequency of CD14+ TNF‐α+ before and after CD14+ HLA‐DR − monocytes cocultured with Tfh cells. G, Representative dot plots show the gating strategy and the frequency of live CD14+ cells, CD14+ TNF‐α+ from a single HS (top panel) and NSCLC patient (bottom panel). CD14+ TNF‐α+ were analyzed after gating on live CD14+ cells. H, Supernatant TNF‐α levels from HS and NSCLC patients. HLA‐DR, human leukocyte antigen‐DR

Similarly, after coculture with Tfh cells from NSCLC patients, a higher, statistically significant frequency of TNF‐α‐producing CD14+HLA‐DR− cells (Figure 3F,G) and supernatant TNF‐α levels (Figure 3H) was observed when compared to CD14+HLA‐DR− cells alone. However, coculture with Tfh cells from HS had no significant effect on the cytokine production of CD19+IgD+ naïve B cells and CD14+HLA‐DR− cells (Figure 3).

High expression of PD‐L1 on tumor cells is significantly correlated with high numbers of circulating Tfh cells in NSCLC patients

Non‐small cell lung cancer is known to upregulate PD‐L1 expression, and high PD‐L1 expression is an independent predictor of poor prognosis.23, 24 In the present study, we found that 32 NSCLC samples (56.1%) stained negative for PD‐L1, and 25 NSCLC samples (43.9%) stained positive for PD‐L1 (Figure 4A,B). PD‐L1‐positive NSCLC tumor cells were significantly correlated with high numbers of CD4+PD‐1+ (Figure 4D) and Tfh cells (Figure 4E), but there was no significant correlation with CD4+CXCR5+ (Figure 4C) and clinicopathological factors (data not shown). As previously reported, we also found that NSCLC patients with PD‐L1‐negative tumor cells had longer disease‐free survival after tumor resection (Figure 4F).

Figure 4

Non‐small cell lung cancer (NSCLC) tumor cell programmed death ligand 1 (PD‐L1) expression and its association with circulating follicular helper T (Tfh) cells. A, Negative cell surface staining (brown) and B, positive PD‐L1 staining are shown for NSCLC tumor cells (20×). C‐E, Frequency and number of circulating CD4+ CXCR5+, CD4+ PD‐1+ and Tfh cells between patients with PD‐L1‐negative and ‐positive tumors are shown in the left and right panels, respectively. F, Disease‐free survival in patients with PD‐L1‐negative and ‐positive tumors. CXCR5, C‐X‐C motif chemokine receptor 5; PD‐1, programmed death 1

Negative correlation between Tfh1 subtypes with disease‐free survival

Thus, we determined whether Tfh cells with high PD‐1 expression were also predictive of survival time in NSCLC. We analyzed the correlation between Tfh cells and disease‐free survival after tumor resection. Interestingly, the number of Tfh1 subtypes was positively correlated with disease‐free survival of NSCLC patients (Figure 5D). However, no significant association was found between CD4+CXCR5+, CD4+PD‐1+, Tfh cells, Tfh2, or Tf17 subtypes and disease‐free survival of NSCLC patients (Figure 5A,B,C,E,F).

Figure 5

Correlation between follicular helper T (Tfh) cells and disease‐free survival. A‐F, Correlations between the number of CD4+ CXCR5+, CD4+ PD‐1+, Tfh cells, Tfh1, Tfh2, and Tfh17 subtypes with disease‐free survival

Circulating Tfh cells in NSCLC patients with different histological subtypes

Non‐small cell lung cancer comprises several subtypes based on histological examination in which adenocarcinoma and squamous cell carcinoma are the two major subtypes. We investigated the levels of circulating CD4+CXCR5+, CD4+PD‐1+ and Tfh cells in these NSCLC histological subtypes. Our data showed that the frequency and the number of circulating CD4+CXCR5+ and Tfh cells were higher in adenocarcinoma than in squamous cell carcinoma or in the other types, but the level of CD4+PD‐1+ was no different in the three histological subtypes (Figure 6). However, the level of circulating CD4+CXCR5+, CD4+PD‐1+ and Tfh cells was not significantly different between squamous cell carcinoma and other types.

Figure 6

Circulating follicular helper T (Tfh) cells in non‐small cell lung cancer patients with different histological subtypes. Frequency in CD4+ T cells and number of CD4+ CXCR5+, CD4+ PD‐1+, and Tfh cells are shown in the top and bottom panels, respectively. CXCR5, C‐X‐C motif chemokine receptor 5; PD‐1, programmed death 1

DISCUSSION

Follicular helper T cells represent a distinct CD4+ T subset that helps B cells in GC differentiate into long‐lived memory B and plasma cells.13, 14, 15 Studies in several tumors have shown that Tfh cells have quantitative or functional abnormalities.25, 26, 27 In the present study, we investigated circulating Tfh cells in newly diagnosed NSCLC patients and identified the expansion of circulating CD4+PD‐1+ and Tfh cells, especially in those with advanced stage (III and IV). We also observed a great skewing toward PD‐1+‐Tfh2 and PD‐1+‐Tfh17 subtypes in NSCLC. In addition, we found increased circulating CD4+PD‐1+ and Tfh cells in adenocarcinoma than in squamous cell carcinoma or other types, which suggested different histological subtypes of NSCLC might have different pathways of tumorigenesis.

Several studies have confirmed the antitumor role of IL‐21 in different tumors.28, 29 IL‐21 is the characteristic cytokine produced by Tfh cells. De Leur et al30 have shown that Tfh2 and Tfh17, but not Tfh1, could effectively induce naive B cells to produce immunoglobulins through secreting of IL‐21. Here, we found decreased IL‐21 levels and a negative correlation between IL‐21 and Tfh and Tfh17 cells, indicating that IL‐21 production of Tfh17 cells could be greatly impaired in NSCLC. GC and circulating Tfh cells can stimulate the production of antibodies through the expression of IL‐10 and IL‐21.31 Ma32 reported that Tfh cells from NSCLC patients were defective at helping naive B cells induce IgM, IgG, and IgA production. Tfh cells do not effectively produce IL‐21, which further does not effectively stimulate the production of antibodies. Overall, our data indicate that Tfh cells from NSCLC patients have antitumor potential but are functionally impaired.

To further clarify the role of Tfh cells in NSCLC, we analyzed the functional effect of Tfh cells on CD19+IgD+ naïve B cells and CD14+HLA‐DR− cells. Our results showed that Tfh cells from NSCLC patients promoted the release of TGF‐α and IL‐10 from CD19+ B cells and the release of TNF‐α from CD14+ cells. Increased PD‐1+‐Tfh2 and PD‐1+‐Tfh17 subtypes in NSCLC could inhibit immune response through interaction with PD‐L1. The high number and abnormal function of Tfh cells induce the differentiation of regulatory B cells and CD14+HLA‐DR− cells, which could cause further immunosuppression and lead to tumor development and even metastasis.

High PD‐L1 expression in NSCLC was an independent predictor of poor prognosis.33 We found that high expression of PD‐L1 in tumor cells is significantly correlated with a high number of circulating Tfh cells in NSCLC patients. NSCLC patients with PD‐L1‐negative tumor cells had longer disease‐free survival, as reported in other studies.23, 24 Gu‐Trantien et al found that when Tfh cell infiltration in breast cancer patients was greater, the prognosis of patients was better. Additionally, impaired function of Tfh cells is associated with hepatocellular carcinoma progression.34, 35 Thus, Tfh cells are likely to be associated with prognosis of tumor. Interestingly, in the present study, we found that the number of Tfh1 subtypes was negatively correlated with disease‐free survival after tumor resection, which may be related to immunosuppression induced by high PD‐1 expression in Tfh1 subtypes. However, our data were based on a relatively small number of patients. For this reason, we were unable to reliably assess the predictive utility of Tfh1 subtypes on NSCLC patient survival and will expand our samples and conduct a larger study.

Based on our results, it is possible that elevated Tfh cells cause further immunosuppression in NSCLC. Whether rescuing Tfh functions can induce better B‐cell responses and improve antitumor immunity in NSCLC is still unknown and will require further animal experiments and clinical trials for elucidation.

CONFLICTS OF INTEREST

Authors declare no conflicts of interest for this article.

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