Research ArticleExperimental Studies
Open Access

Peripheral CD4+ T Cells Predict T Cell Immunity in Lung Tissues of Non-small Cell Lung Cancer Patients

MARI TONE, TOMOMI ISONO, YOKO YAMAMOTO, YOSHITO TAKEDA, YASUSHI SHINTANI, ATSUSHI KUMANOGOH, HISASHI WADA and KOTA IWAHORI
Anticancer Research March 2025, 45 (3) 909-920; DOI: https://doi.org/10.21873/anticanres.17478

Abstract

Background/Aim: Previous investigations showed that non-small cell lung cancer (NSCLC) patients with a high percentage of a peripheral CD4+ T cell subset were more likely to have severe immune-related adverse events (irAEs) due to anti-PD-1 therapy. The present study investigated the relationship between a peripheral CD4+ T cell subset and T cell immunity in the non-tumor lung tissues of patients with NSCLC to clarify the rationale of predictive biomarkers for anti-PD-1-related pneumonitis.

Patients and Methods: We analyzed the T cell profiles and functions in peripheral blood and non-tumor lung tissues surgically resected from patients with NSCLC.

Results: In patients with NSCLC with a high percentage of the peripheral CD4+ T cell subset (CD45RA+CD25+CD4+ T cells), non-tumor lung tissues had a high percentage of PD-1+CD4+ T cells and a low percentage of PD-1+ effector regulatory T (Treg) cells. The percentage of PD-1+ effector Treg cells negatively correlated with IFNγ and TNFα production by CD4+ T cells in the lung tissues of patients with NSCLC.

Conclusion: Patients with NSCLC with a high percentage of the peripheral CD4+ T cell subset are at an increased risk of anti-PD-1-related pneumonitis, which activates PD-1+CD4+ T cells in the absence of the suppressive activity of effector Treg cells in lung tissues.

Keywords:

Introduction

Immune checkpoint inhibitors (ICIs) benefit a limited population of patients with non-small cell lung cancer (NSCLC); however, some patients develop severe immune-related adverse events (irAEs). Therefore, the need for predictive biomarkers of the treatment efficacy and severe irAEs of ICIs is increasing. Regarding treatment efficacy, PD-L1 staining of tumor tissues has been developed as companion diagnostics to anti-PD-1 therapy (1, 2). However, no predictive biomarkers of irAEs are currently approved for ICI treatments. Patients with NSCLC who developed irAEs from anti-PD-1 therapy achieved better outcomes than those without irAEs (3-8). However, a significant difference in overall survival was observed between mild and severe irAEs (9). Previous studies on treatment efficacy and irAEs focused on CD4+ T cell subsets (10-16). The CD4 T helper (Th) 1 subset was identified as a prominent component of anti-tumor immunity. IFNg released by Th1 cells has been associated with the development of irAEs (17). In addition to Th1 cells, regulatory T cells (Treg cells) play a critical role in irAEs (18, 19). Bronchoalveolar lavage fluid samples from patients with ICI-related pneumonitis showed the down-regulated expression of PD-1 and CTLA-4 in Treg cells, suggesting an attenuated suppressive phenotype (20). In the analysis of peripheral blood before and after anti-PD-1 therapy, patients with severe irAEs exhibited a significantly lower-fold increase in the frequency of effector Treg cells (21). We also reported that patients with NSCLC with a high percentage of a peripheral CD4+ T cell subset (CD45RA+ CD25+CD4+ T cells) were more likely to develop severe irAEs (22).

ICI-related pneumonitis is one of the most critical adverse events among irAEs. Patients with NSCLC with interstitial lung disease (ILD) are at an increased risk of ICI-related pneumonitis (23-28). We previously showed that T cells were active participants and balanced in part by Treg cells in the lung tissues of patients with NSCLC with stable ILD (29).

These findings on T cells in peripheral blood and lung tissues indicate a relationship with irAEs. Therefore, we herein investigated the peripheral CD4+ T cell subset and their T cell immunity in the non-tumor lung tissues of patients with NSCLC to clarify the rationale of predictive biomarkers for ICI-related pneumonitis.

Patients and Methods

Patient selection and data collection. The study protocol was approved by the Institutional Ethics Committee of Osaka University Hospital (IRB number 13266). Written informed consent was obtained from participants before their inclusion in the study. Surgery was performed on patients between May 2015 and September 2020. Source data comprised patients with NSCLC recruited from Osaka University Hospital. All specimens were collected from excess clinical samples obtained from patients on whom the surgical resection of primary lung tumors was performed. Any additional surgical sampling beyond the required surgery was not conducted. Demographic and clinical data were obtained before surgery. Patients were followed up for 60 months for a time-to-event analysis, in which recurrence and death were considered to be events for overall survival. The present study was conducted according to the principles of the Declaration of Helsinki.

Sample preparation. Peripheral blood and non-tumor lung specimens were obtained from patients with NSCLC during surgical resection of primary lung neoplasms. Non-tumor lung samples were collected at a maximum distance from the tumor site.

Lung tissues were minced in a 6-cm dish and enzymatically dissociated into a single-cell suspension using a Tumor Dissociation Kit for humans (Miltenyi Biotec, Bergisch Gladbach, Germany) and a gentleMACS Dissociator (Miltenyi Biotec) following the manufacturer’s protocols. The resulting cell suspension was filtered through a 70-μm nylon cell strainer (BD Biosciences, Franklin Lakes, NJ, USA) to remove red blood cells using BD Pharm Lyse (BD Biosciences). Subsequently, dead cells and debris were eliminated by centrifugation in an isodensity Percoll solution (GE Healthcare, Piscataway, NJ, USA) prior to flow cytometry analysis. The remaining cells were cryopreserved in liquid nitrogen for subsequent in vitro stimulation for intracellular cytokine staining.

White blood cells were isolated from peripheral blood using BD Pharm Lyse (BD Biosciences) red blood cell lysis buffer and were then analyzed using flow cytometry.

Flow cytometry analysis. A flow cytometric analysis was performed on BD LSRFortessa X-20 (Becton Dickinson, Franklin Lakes, NJ, USA) with FACSDiva software (Becton Dickinson). Surface and intracellular marker staining was conducted after the FcR block using Human TruStain FcX Fc Receptor blocking solution (BioLegend, San Diego, CA, USA). Cells were incubated with the Zombie NIR Fixable Viability Kit (BioLegend).

The following antibodies were used for surface marker staining: FITC anti-human CD45RA (clone HI100) (Bio Legend), PE anti-human CD25 (clone BC96) (BioLegend), BV510 anti-human CD8a (clone RPA-T8) (BioLegend), BV605 anti-human CD103 (clone Ber-ACT8) (BioLegend), BV711 anti-human CD4 (clone OKT4) (BioLegend), APC anti-Tim-3 (clone F38-2E2) (BioLegend), Alexa Fluor 700 anti-human CD3 (clone UCHT1) (BioLegend), BV785 anti-human CD45 (clone HI30) (BioLegend), and PE-Cy7 anti-human PD-1 (clone EH12.1) (BD Bioscience). Mouse IgG1Embedded Image isotype control (clone MOPC-21) (BioLegend) was used as the isotype control.

In the intracellular staining of Foxp3, the following antibodies were used for cell surface staining: PE-CF594 anti-human CD4 (clone RPA-T4) (Becton Dickinson), Alexa Fluor 700 anti-human CD3 (clone SK7) (BioLegend), FITC anti-human CD45RA (clone HI100) (BioLegend), Brilliant Violet 785 anti-human CD45 (clone HI30) (BioLegend), Brilliant Violet 711 anti-human CD8a (clone RPA-T8) (BioLegend), and Brilliant Violet 421 anti-human PD-1 (clone EH12.2H7) (BioLegend). Mouse IgG1Embedded Image isotype control (clone MOPC-21) (BioLegend) was used as the isotype control. After cell surface staining, cell fixation and perforation were performed with the Fox P3/Transcription Factor Staining Buffer Set (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Intracellular FoxP3 staining was conducted using APC anti-human FoxP3 (clone PCH101) (Invitrogen).

In the intracellular cytokine staining analysis, Alexa Fluor 700 anti-human CD3 (clone UCHT1) (BioLegend), PE anti-human CD4 (clone SK3) (BioLegend), and APC anti-human CD8a (clone RPA-T8) (BioLegend) antibodies were used for cell surface staining. Cell fixation and perforation were then performed with the Transcription Factor Buffer Set (Becton Dickinson) according to the manufacturer’s protocol. Intracellular staining was conducted using the following antibodies: Brilliant Violet 421 anti-human IFNγ (clone 4S.B3) (BioLegend), PE-Cyanine7 anti-human TNF-α (clone MAb11) (eBioscience, Waltham, MA, USA), and PerCP-eFluor710 anti-human IL-2 (clone MQ1-17H12) (Invitrogen). Mouse IgG1Embedded Image isotype control (clone MOPC-21) (BioLegend) was used as the isotype control.

In vitro stimulation for intracellular cytokine staining. Frozen cells were rapidly thawed in a 37°C water bath and immediately used for the phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation. Between 5.0×105 and 1.0×106 thawed cells were suspended in 1 ml of AIM V™ Medium (Thermo Fisher, Waltham, MA, USA) and then added to 24-well plates. Samples were stimulated with 50 ng/ml PMA (Sigma-Aldrich, St. Louis, MO, USA), 1 μM ionomycin (Sigma-Aldrich), and a 1:1,500 dilution of Protein Transport Inhibitor BD GolgiPlug™ (BD Biosciences) at 37°C, 5% CO2, for 4 h. A 1:1,500 dilution of Protein Transport Inhibitor was added to wells containing negative control samples (without any stimulation). Harvested cells were washed and stained with antibodies as described in the “Flow cytometry analysis” section.

Statistical analysis. A two-tailed Student’s t-test was used to examine the significance of differences between samples. Relationships between paired data were analyzed using Pearson’s correlation coefficient. In the survival analysis, the starting point was the day of surgery. The Kaplan–Meier method was used to analyze OS with differences between groups being calculated using the Log-rank test. A p-value <0.05 was considered to be significant. These analyses were performed using JMP software (SAS Institute, Inc., Cary, NC, USA).

Results

Patient characteristics. We previously demonstrated that patients with advanced NSCLC with a high percentage of a peripheral CD4+ T cell subset (CD45RA+CD25+CD4+ T cells) (≥6% of CD4+ T cells) prior to anti-PD-1 therapy were more likely to develop severe irAEs, including ICI-related pneumonitis, which led to the discontinuation of anti-PD-1 therapy (Figure 1A) (22). However, the rationale for the relationship between the peripheral CD4+ T cell subset and T cell immunity in non-tumor lung tissue has not yet been clarified. In the present study, we investigated the relationship between peripheral CD4+ T cell subset and T cell immunity in the non-tumor lung tissues of patients with NSCLC eligible for the surgical resection of primary lung tumors.

Figure 1.
Figure 1.

The peripheral CD4+ T cell subset correlated with severe immune-related adverse events (irAEs) due to anti-PD-1 therapy in patients with non-small cell lung cancer (NSCLC). (A) Two representative samples (a flow cytometry analysis of peripheral blood in patients with advanced NSCLC with non-severe irAEs and ICI-related pneumonitis after anti-PD-1 therapy) showing CD45RA and CD25 positivity after gating on CD4+CD3+ PBMCs. (B) Kaplan–Meier curves for overall survival after surgery were compared between NSCLC patients with a high percentage of the peripheral CD4+ T cell subset (the ratio of CD45RA+CD25+ in CD4+ T cells >6%, n=49) and a low percentage of the peripheral CD4+ T cell subset (the ratio of CD45RA+CD25+ in CD4+ T cells <6%, n=44) at the surgical resection of primary lung tumors.

Patient characteristics are shown in Table I. We divided the study population into two groups: a high peripheral CD4+ T cell group (the percentage of CD45RA+CD25+CD4+ T cells ≥6% of CD4+ T cells) and a low peripheral CD4+ T cell group (the percentage of CD45RA+CD25+CD4+ T cells <6% of CD4+ T cells). No significant differences were observed in age, sex, smoking status, or histology between the two groups. The Kaplan–Meier analysis showed that overall survival was slightly longer in the high peripheral CD4+ T cell subset than in the low peripheral CD4+ T cell subset (p= 0.1648) (Figure 1B).

View this table:
Table I.

Baseline characteristics of the study population.

Relationship between peripheral CD4+ T cell subset and T cell profile in lung tissues. To clarify the relationship between peripheral CD4+ T cell subset and T cell profile in non-tumor lung tissues, we analyzed T cells in the peripheral blood and non-tumor lung tissues of patients with NSCLC using flow cytometry.

We examined immune checkpoint molecules (PD-1 and Tim-3) in CD4+ and CD8+ T cells in lung tissues and found that the ratio of PD-1+ in CD4+ T cells in lung tissues was higher in the high peripheral CD4+ T cell subset than in the low peripheral CD4+ T cell subset (p=0.0092) (Figure 2A). Although there were fewer Tim-3+ T cells than PD-1+ T cells in lung tissues, the ratio of Tim-3+ in CD8+ T cells in lung tissues was higher in the high peripheral CD4+ T cell subset than in the low peripheral CD4+ T cell subset (p=0.0247) (Figure 2A). We then analyzed CD103, a marker of tissue-resident memory T cells, in CD4+ and CD8+ T cells in lung tissues and found no significant difference in the ratio of CD103+ T cells in lung tissues between the high and low peripheral CD4+ T cell subsets (Figure 2B).

Figure 2.
Figure 2.

T cell profiles in lung tissues of non-small cell lung cancer (NSCLC) patients with high and low peripheral CD4+ T cell subsets. (A) The ratios of immune checkpoint molecule (PD-1 and Tim-3)-expressing cells in the CD4+ and CD8+ T cells of lung tissues were analyzed using flow cytometry between NSCLC patients with high peripheral CD4+ T cell subset (n=48) and low peripheral CD4+ T cell subset (n=41). Data represent the mean±standard error of the mean (SEM). (B) The ratios of CD103-expressing cells in the CD4+ and CD8+ T cells of lung tissues were analyzed using flow cytometry between NSCLC patients with high peripheral CD4+ T cell subset (n=47) and low peripheral CD4+ T cell subset (n=41). Data represent the mean±SEM.

We also investigated CD45RA-Foxp3hi Treg cells (effector Treg cells), CD4+Foxp3− conventional T cells (Tconv cells), and PD-1 expression in these cells. In the low peripheral CD4+ T cell subset, the ratio of PD-1+ in effector Treg cells was higher than that in Tconv cells in lung tissues (p=0.001) (Figure 3A). However, in the high peripheral CD4+ T cell subset, no significant difference was observed in the ratio of PD-1+ cells between effector Tregs and Tconv cells in lung tissues (Figure 3A). We also found that patients with a high percentage of peripheral CD4+ T cell subset had a low percentage of effector Treg and PD-1+ effector Treg cells in non-tumor lung tissues (Figure 3B).

Figure 3.
Figure 3.

Correlation between the peripheral CD4+ T cell subset and the balance between effector and regulatory T cells in lung tissues of patients with non-small cell lung cancer (NSCLC). (A) The ratio of PD-1+ cells in effector Treg (CD45RA-Foxp3hi CD4+ T) cells and Tconv (CD4+Foxp3-conventional T) cells in the lung tissues of NSCLC patients with high peripheral CD4+ T cell subset (right, n=31) and low peripheral CD4+ T cell subset (left, n=30). (B) The ratios of peripheral CD45RA+CD25+ in CD4+ T cells were analyzed for correlations with the ratios of effector Treg (left, n=68) and PD-1+ effector Treg (right, n=61) cells in CD4+ T cells in the lung tissues of patients with NSCLC. Each dot represents one patient.

These results indicated that patients with NSCLC with a high percentage of peripheral CD4+ T cell subset had a high ratio of PD-1+ in CD4+ T cells and a low ratio of PD-1+ effector Treg cells in lung tissues.

Functional analysis of T cells in lung tissues. Based on the results of the T cell profile, we examined the relationship between the T cell profile and function in the lung tissues of patients with NSCLC. T cell function in lung tissues was examined by analyzing cytokine production from T cells after the stimulation with PMA/ionomycin. The ratio of PD-1+ in CD4+ T cells did not correlate with cytokine production by CD4+ T cells in lung tissues (Figure 4A). However, the ratio of PD-1+ effector Treg in CD4+ T cells negatively correlated with IFNγ and TNFα production by CD4+ T cells in lung tissues (Figure 4B).

Figure 4.
Figure 4.

Correlations between cytokine production and T cell subsets in lung tissues of patients with non-small cell lung cancer (NSCLC). (A) The ratios of cytokine (IFNγ, TNFα, and IL-2)-producing cells in CD4+ T cells were analyzed for correlations with the ratios of PD-1+ cells in the CD4+ T cells of lung tissues. Each dot represents one patient (n=39). (B) The ratios of cytokine (IFNγ, TNFα, and IL-2)-producing cells in CD4+ T cells were analyzed for correlations with the ratios of PD-1+ effector Treg cells (CD45RA-Foxp3hi) in the CD4+ T cells of lung tissues. Each dot represents one patient (n=38).

Based on these results, patients with NSCLC with a high percentage of peripheral CD4+ T cell subset lack PD-1+ effector Treg cells in lung tissues, which is related to the inhibition of IFNγ and TNFα production.

These findings in the present study indicated that the peripheral CD4+ T cell subset was correlated with the PD-1+ CD4+ and effector Treg cells in lung tissue, suggesting the peripheral CD4+ T cell subset as a predictive biomarker for anti-PD-1-related pneumonitis.

Discussion

We herein examined the relationship between the peripheral CD4+ T cell subset (CD45RA+CD25+CD4+ T cells) and T cell immunity in the non-tumor lung tissues of patients with NSCLC. We found that patients with NSCLC with a high percentage of the peripheral CD4+ T cell subset had a high ratio of PD-1+ in CD4+ T cells and a low ratio of PD-1+ effector Treg cells in lung tissues, which was related to the inhibition of IFNγ and TNFα production. These results indicated that patients with NSCLC with a high percentage of the peripheral CD4+ T cell subset were at an increased risk of anti-PD-1-related pneumonitis, which activates PD-1+CD4+ T cells in the absence of the suppressive activity of effector Treg cells. These results support our previous finding of the peripheral CD4+ T cell subset as a predictive biomarker of severe irAEs.

Regarding the role of PD-1 in Treg cells, the anti-PD-1 antibody has been shown to bind to PD-1 on Treg cells and enhance the suppressive activity of these cells (30-33). An analysis of tumor-infiltrating lymphocytes from the gastric tumors of patients who developed rapid tumor growth after anti-PD-1 therapy indicated a higher frequency of proliferating Tregs in the post-treatment tumor micro-environment than in patients who did not experience rapid tumor growth (31). Based on these findings, our results indicated that patients with NSCLC with a high percentage of the peripheral CD4+ T cell subset lacked the suppressive activity of Treg cells in their lung tissues after anti-PD-1 therapy because of a low ratio of PD-1+ effector Treg cells.

There have been reports concerning the predictive value of the CD4+ T cell subset in relation to the efficacy of immune checkpoint inhibitors (34, 35). We previously demonstrated that patients with NSCLC with a high percentage of the peripheral CD4+ T cell subset were more likely to develop severe irAEs (22). CD45RA+CD25+CD4+ T cells include naïve Treg cells expressing CD45RA as a marker of naïve T cells (36, 37). However, CD45RA+ CD25+CD4+ T cells were considered to consist of not only naïve Treg cells but also activated CD4+ T cells expressing CD25 as an activation marker. In our previous study, the majority of CD45RA+CD25+CD4+ T cells in patients with severe irAEs did not express Foxp3, which is a specific marker for Treg cells (22). Since ICI-related pneumonitis reportedly results from the balance between effector and Treg cells in the lungs (20), peripheral CD45RA+CD25+ CD4+ T cells are considered to reflect the interaction of these cells in lung tissues.

There are several limitations that need to be addressed. In the present study, we examined the relationship between peripheral CD4+ T cell subset and T cell immunity in lung tissues surgically resected from patients with NSCLC. However, the relationship between T cells in the peripheral blood and lung tissues of patients with NSCLC with anti-PD-1-related pneumonitis remains unclear because of the difficulties associated with obtaining these clinical samples. Furthermore, the antigen specificity of T cells in peripheral and lung tissues was not clarified. PD-1+CD4+ T cells in the lung tissue of patients with NSCLC with a high percentage of the peripheral CD4+ T cell subset may include T cells specific for autoantigens on alveolar epithelial cells.

Conclusion

In conclusion, the peripheral CD4+ T cell subset correlated with PD-1+CD4+ T cells and PD-1+ effector Treg cells in non-tumor lung tissues, indicating a relationship with anti-PD-1-related pneumonitis in patients with NSCLC. Further studies are needed to establish a rationale for predictive biomarkers of anti-PD-1-related pneumonitis for patients with NSCLC.

Acknowledgements

The Authors would like to thank Medical English Service (Kyoto, Japan) for editing the manuscript.

Footnotes

  • Authors’ Contributions

    KI: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Data Curation, Writing – Original Draft, Writing – Review & Editing, Visualization. TI: Investigation, Data Curation, Formal analysis, Writing – Review & Editing. YY: Investigation, Data Curation, Formal analysis, Writing – Review & Editing. MT: Data Curation, Writing – Review & Editing. YT: Supervision, Writing – Review & Editing. YS: Supervision, Writing – Review & Editing. AK: Supervision, Writing – Review & Editing. HW: Supervision, Writing – Review & Editing.

  • Conflicts of Interest

    The Authors have no conflicts of interest to declare in relation to this study.

  • Funding

    This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI (21K08153 and 24K11315 to KI) and a research grant from the Japan Agency for Medical Research and Development (AMED) (24ama221340h9901 to KI).

  • Received January 14, 2025.
  • Revision received January 27, 2025.
  • Accepted January 29, 2025.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Peripheral CD4+ T Cells Predict T Cell Immunity in Lung Tissues of Non-small Cell Lung Cancer Patients
MARI TONE, TOMOMI ISONO, YOKO YAMAMOTO, YOSHITO TAKEDA, YASUSHI SHINTANI, ATSUSHI KUMANOGOH, HISASHI WADA, KOTA IWAHORI
Anticancer Research Mar 2025, 45 (3) 909-920; DOI: 10.21873/anticanres.17478
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