This study included 128 individuals admitted to Dongyang People’s Hospital between September 2021 and August 2022. Based on the pathological analysis of breast tissue sections, all patients were divided into two groups: those with invasive breast cancer and those with benign tumors. Patients with ductal carcinoma in situ and tumors that were premalignant were not included in the study. Participants had to satisfy the following inclusion criteria (1): no other primary tumors (2); no adjuvant chemoradiotherapy prior to preoperative blood sample collection (3); no apparent signs of infection; and (4) no systemic diseases such as autoimmune and blood diseases. In addition, based on the inclusion criteria, patients with invasive breast cancer who underwent radical mastectomy or modified radical mastectomy without significant postoperative complications were included in the postoperative group and followed up for 2–3 weeks after surgery.

This study was approved by the Ethics Committee of Dongyang People’s Hospital (approval no.: 2021-YX-091). All patients enrolled in the study signed informed consent.

Preoperative fresh whole blood samples (2 mL) were collected in ethylenediaminetetraacetic acid anticoagulation tubes to study the characteristics of circulating CD4⁺Th cell subsets in patients with breast cancer. For patients with invasive breast cancer who underwent postoperative follow-up, 2 mL of fasting whole blood was collected again at 2–3 weeks after surgery to evaluate the dynamic changes in CD4⁺Th cell subsets upon surgery. Samples were examined within 24 h of collection.

To detect immune cell surface antigens, 10-Color flow cytometry was used. In brief, the standard assay procedure was as follows (1): 100 µL of whole blood was mixed thoroughly with pre-mixed antibody (CD45RA-fluorescein isothiocyanate, clone ALB11, Beckman Coulter; CD4-phycoerythrin, clone 13B8.2, Beckman Coulter; CD28-phycoerythrin-Texas, clone CD28.2, Beckman Coulter; PD-1-phycoerythrin-cyanin 5.5, clone PD1.3, Beckman Coulter; CD27-phycoerythrin-cyanin 7, clone 1A4CD27, Beckman Coulter; CCR7-allophycocyanin, clone G043H7, BioLegend; CD8-allophycocyanin-Alexa Fluor 700, clone B9.11, Beckman Coulter; CD3-allophycocyanin-Alexa Fluor 750, clone UCHT1, Beckman Coulter; CD57-pacific blue, clone NC1, Beckman Coulter; CD45-Krome Orange, clone J.33, Beckman Coulter) and left to stand for 15 min (2); commercial red blood cell lysate (OptiLyse C Lysing Solution, Beckman Coulter) was added for complete lysis of red blood cells (3); phosphate buffer solution was added for washing, the supernatant was removed after centrifugation, and then phosphate buffer solution was added for resuspension; and (4) flow cytometry results were analyzed using the accompanying software (version 2.0, Beckman Coulter). The gating strategy of CD4⁺Th subsets is shown in Supplementary Figure 1 .

CD3⁺CD4⁺CD8^-T cells were defined as CD4⁺Th. CD4⁺Th was divided into the following subgroups according to its effector memory differentiation status: CD4⁺ naive T cells (Tn) (CD45RA⁺CCR7⁺CD28⁺CD27⁺), CD4⁺ central memory T cells (Tcm) (CD45RA⁻CCR7⁺CD28⁺CD27^+/−), and CD4⁺ effector memory T cells (Tem) (CD45RA⁻CCR7^-CD28^+/−CD27^+/−). The proportion and absolute number of CD4⁺PD-1⁺T cells represent the exhaustion state of CD4⁺Th cells, and those of CD4⁺CD57⁺T cells represent the senescence state of CD4⁺Th cells.

The following criteria were used to determine positive immunohistochemistry: estrogen receptor (ER)/progesterone receptor (PR) positivity, the proportion of tumor nuclear stained ≥ 25% and/or intensity of staining ≥ 1+; C-erbB-2 positivity, immunohistochemical staining intensity ≥ 3+ and/or positive fluorescence in situ hybridization.

All statistical analyses of this study were conducted using IBM SPSS Statistics software (version 23.0). Categorical variables are presented as quantities (percentages). According to normal or non-normal distribution, continuous variables are presented as mean ± standard deviation or median (interquartile range). The differences between groups were compared using t-test for normally distributed variables and Wilcoxon rank-sum test for non-normally distributed variables. Postoperative dynamic changes in CD4⁺Th subsets were assessed using the paired-sample rank-sum test.

As shown in Table 2 , compared with that in patients with benign tumors, the proportion of CD4⁺Tn cells in patients with breast cancer decreased (mean: 27.9 vs. 33.5, P = 0.011), while the absolute number and proportion of CD4⁺CD57⁺T and CD4⁺PD-1⁺T cells significantly increased (P < 0.05).

In patients with positive and negative ER expression, the median CD4⁺PD-1⁺T (% of CD4⁺Th) population was 38.6% and 33.9%, respectively (P = 0.029, Figure 1A ). Similarly, patients with positive PR expression had higher levels of CD4⁺PD-1⁺T (% of CD4⁺Th) than those with negative PR expression (median: 38.6% vs. 34.10%, P = 0.032, Figure 1B ). No relationship existed between the proportion or absolute number of CD4⁺Th subsets and C-erbB-2 expression ( Figure 1C ).

Table 3 shows the dynamic changes in circulating CD4⁺Th cells before and after surgery. After tumor resection, the proportion and absolute number of CD4⁺Tcm decreased significantly compared with the preoperative values ( Figures 2A, B ). Moreover, the proportion and absolute number of CD4⁺Tem significantly increased after tumor resection ( Figures 2C, D ). Notably, tumor resection caused significant changes in the proportion and absolute number of CD4⁺CD57⁺T and CD4⁺PD-1⁺T, both of which showed a significant decrease ( Figures 2E, H ).

We observed that the proportion of CD4⁺Tn cells in breast cancer patients decreased. A similar distribution was previously found for head and neck cancer (20). The decrease in Tn, a newly formed reserve cell, suggests that the immune system’s reserves of CD4⁺T cells are depleted after prolonged activation by cancer antigens.

Antigen-specific CD4 persisted in Tcm and Tem cells. The functional expression of these two subsets is apparently different from one another, with the latter directly influencing patient prognoses. Tada et al. showed that low CD4⁺Tem level is a poor prognostic factor in patients with colorectal cancer (21). The current study found that the proportion and absolute number of CD4⁺Tcm cells decreased and that of CD4⁺Tem cells increased after surgery. This phenomenon of postoperative immune activation has also been reported in pancreatic cancer (22). For patients without distant metastasis, the decreased tumor burden can reverse the immunosuppressive status and improve patient prognosis.

PD-1 (CD279) is commonly used to assess T cell exhaustion as it negatively regulates T cell proliferation and cytokine production, resulting in dysregulation of host immunity (23). Zhu et al. found that CD4⁺PD-1⁺T cells in the peripheral blood of patients with thyroid cancer had a higher count than those of patients with nodular goiter (24). Furthermore, Rosenblatt et al. demonstrated a significant increase in PD-1 expression in circulating CD4⁺Th cell populations in patients with active myeloma (25). In our study, we found that the proportion and absolute number of CD4⁺PD-1⁺T cells in patients with breast cancer were higher than those in patients with benign tumors, and the proportion of CD4⁺PD-1⁺T was related to clinicopathology.

Numerous studies have shown that PD-1 expression is closely related to treatment response in patients with cancer (26, 27). However, some studies have reported that the prognostic value of PD-1 expression is uncertain because of the presence of high tumor heterogeneity, which is affected by the choice of detection method and antibody (28). At present, PD-1 detection is mostly done through the tumor tissue samples’ immunohistochemical analysis, which is not convenient for follow-up monitoring (29). In this study, circulating CD4⁺PD-1⁺T cells were detected with flow cytometry, and it was found that surgical resection of the tumor could reverse the immune exhaustion status of patients with breast cancer.

In the occurrence and development of breast cancer, in addition to T cell exhaustion, T cell senescence also produces malignant positive feedback. T cell exhaustion and senescence have similar manifestations but completely different origins. In contrast to exhaustion, which is controlled by extrinsic immunomodulatory mechanisms, immunosenescence is controlled in nature by the intrinsic stress response of immune cells (30). Studies have shown that CD57 is the most relevant marker of T cell senescence because the proliferation ability of CD57-expressing T cells is severely impaired in cancer (31). Shiraki et al. demonstrated that the percentage of CD4⁺CD57⁺T cells in the peripheral blood of patients with hepatitis C virus-associated hepatocellular carcinoma increased with tumor progression (32). Similar results can be seen in other malignancies, such as oral squamous cell carcinoma (33) and gastric cancer (34). In this study, the proportion and number of circulating CD4⁺CD57⁺ T cells in patients with breast cancer increased, and immunosenescence occurred.

Immunosenescence has long been considered irreversible. However, Beausejour et al. showed that immunosenescence may not necessarily be permanent (35). Moreover, Fornara et al. demonstrated a sustained decrease in the proportion of CD4⁺CD57⁺ T cells after surgery in patients who were long-term glioblastoma survivors (36). This study found that patients without distant metastasis showed a decreased CD4⁺CD57⁺ T cell proportion and absolute number after surgery, thus reversing immunosenescence.

This study describes the characteristics of circulating CD4⁺Th cells in patients with breast cancer and demonstrates for the first time that surgical treatment of breast cancer creates a new balance between immune suppression and immune stress in patients. However, this study has some limitations. First, prognostic information in patients with breast cancer cannot be obtained directly from this study. Therefore, more research is required to determine whether dynamic changes in CD4⁺Th influence the survival rate of patients. Additionally, the limited sample size from a single center limits the generalizability of results, requiring further validation.

In conclusion, this study found significant changes in circulating CD4⁺Th subsets in patients with breast cancer, which were related to clinicopathology. In addition, complete surgical resection of tumors can benefit patients, which can create a new balance between immunosuppression and immune activation, and reverse the state of immune exhaustion and immunosenescence.