Expression of PD-1 on CD4+ Tumor-Infiltrating Lymphocytes in Tumor Microenvironment Associated with Pathological Characteristics of Breast Cancer

Objective This study aimed to investigate the correlation of CD4+/PD-1+ or CD4+/PD-1− tumor-infiltrating lymphocytes with pathological characteristics in breast cancer patients. Methods A cross-sectional study consecutively recruited 133 patients with invasive ductal breast cancer. The expression of CD4, programmed cell death protein 1 (PD-1), CK7, CK20, E-cadherin, or Ki-67 was detected by immunohistochemistry. The associations between CD4+/PD-1+ or CD4+/PD-1− tumor-infiltrating lymphocytes and pathological characteristics were evaluated. Results Elderly patients intended to have a lower level of CD4+/PD-1− tumor-infiltrating lymphocytes (p < 0.05). Patients with positive E-cadherin expression had higher median cell counts of CD4+/PD-1− tumor-infiltrating lymphocytes than patients with negative E-cadherin expression (30/HPF versus 10/HPF, p < 0.05). Counts of CD4+/PD-1+ tumor-infiltrating lymphocytes had a significant correlation with Ki-67 index that the correlation coefficient was 0.29 (p = 0.001). Positive CK20 expression was related to a higher level of CD4+/PD-1− tumor-infiltrating lymphocytes than negative CK20 expression (73/HPF versus 30/HPF, p < 0.05). Conclusion CD4+/PD-1+ or CD4+/PD-1− tumor-infiltrating lymphocytes showed diverse association with pathological features of breast cancer. CD4+/PD-1+ tumor-infiltrating lymphocytes had a significant relationship with Ki-67 expression whereas CD4+/PD-1− tumor-infiltrating lymphocytes had a significant relationship with E-cadherin expression. Further studies are warranted to explore the immunomodulatory effects of phenotypes of CD4+ T cell subsets in breast cancer.


Introduction
Breast cancer (BC) is the most common female cancer worldwide with 1.67 million new cases (25% of all incident cancer cases) in 2012 [1], and the mortality rate was higher in underdeveloped countries than in developed countries [1]. Recently, tumor-infiltrating lymphocytes (TILs) were reported to be involved in BC development, prognosis, and immunotherapy efficacy.
TILs are mainly composed of different subtypes of T cells, which play an important role in antitumor immunity. A high proportion of TILs predicted a favorable prognosis of BC patients [2]. CD4 + T lymphocytes are helper T cells, and high counts of intratumoral CD4 + lymphocytes conferred a better BC survival [3]. Programmed cell death protein 1 (PD-1), as an immune checkpoint expressed on T cells, binds to programmed cell death ligand 1 (PD-L1) on the surface of cancer cells and suppresses antitumor functions of T lymphocytes. So the expression of PD-1 indicated exhausted function of lymphocytes and a high level of PD-1 + TILs correlated with a worse survival of BC [4]. According to PD-1 expression, CD4 + lymphocytes can be further classified into two subgroups, subsets of CD4 + /PD-1 + and CD4 + /PD-1 − T lymphocytes. The immune function of CD4 + T cells was affected by PD-1 expression. The immune function of CD4 + /PD-1 − TILs was less exhausted than that of CD4 + /PD-1 + TILs [5]. Among the HIV-infected patients, CD4 + /PD-1 + T lymphocytes played an important role in the HIV persistence and immunomodulatory microenvironment [6,7]. However, very few studies were focused on exhausted CD4 T cells in BC patients. Cytokeratins 7 (CK7) and 20 (CK20) were related to the pathological features of BC [8]. E-cadherin (E-Cad) and Ki-67 showed a significant relationship with BC prognosis [9]. These biomarkers were tested routinely in BC pathological diagnosis. We aimed to investigate the correlation of exhausted status of CD4+ helper T cells with pathological characteristics among BC patients. formaldehyde, embedded by paraffin, and stained by hematoxylin and eosin. Histopathologic feature was determined using serial 4 μm thickness sections derived from each specimen. Nottingham modification of the Bloom-Richardson system was used to classify histological grading of BC.
TILs were counted in randomly selected five different high-power fields (HPF) to obtain an average number in IHC sections by two pathologists. PD-1 was expressed in the cytoplasm of lymphocytes with the color of brown, CK7 and CK20 were expressed in the cytoplasm of BC cells with the color of brown, Ki-67 was expressed in nucleus of BC cells with color of brown, E-Cad was expressed on cytomembrane of BC cells with the color of brown, and CD4 was expressed on the cytomembrane of lymphocytes with the color of red. Double staining of CD4/PD-1 showed red cytomembrane and brown cytoplasm of lymphocytes. We counted the PD-1-or CD4-positive cells in 100 lymphocytes and calculated the expression rate. We counted PD-1positive cells among 100 CD4 + lymphocytes and calculated the proportion. Ki-67 index was estimated among 100 BC cells. Ki-67 index > 1% was defined as positive expression.

Statistical Analysis.
All analyses were conducted with SPSS software (version 17.0). Median and interquartile range (IQR) were used to describe TIL counts. Age was transformed in categorical scale by median of 55. The comparisons of TIL phenotypes were processed by Wilcoxon tests between age, nerve invasion, vascular invasion, and axillary lymph node involvement groups. The association of TIL phenotypes with histological grade was estimated by Spearman correlation tests. Wilcoxon tests were used to estimate the difference of TIL phenotypes between positive and negative expression of CK7, CK20, and E-Cad. Spearman correlation test was used to measure the relationship between Ki-67 index and cell counts of TIL phenotypes. All analyses were two sided, and a significant level was 0.05.

Discussion
Despite the development in therapies for BC, there is a gap in clinical efficacy between Chinese and US patients. The immune system is critically involved in tumor surveillance and development. Immunotherapy is a highly attractive alternative approach to treat patients with advanced BC [10].
An increased number of TILs was related to a better clinical outcome of cancer patients [11]. The dynamic change and dysfunction of TIL subsets in the immunosuppressive microenvironment contribute to the pathogenesis of cancer. A better understanding of TILs may facilitate the development of effective strategies for immunotherapy. An effective antitumor immune response requires both CD4 + and CD8 + T lymphocytes [12,13]. CD8 + T lymphocytes always play a critical role in antitumor immunity. The function of CD4 + T lymphocytes in cancer has recently been extensively studied in both animal models and patients. CD4 + T lymphocytes typically recognize peptides with the size of 12-16 AA, presented by MHC class II molecules [14]. These lymphocytes are important for the initiation and maintenance of adaptive immune responses. The subsets of CD4 + T lymphocytes had diverse antitumor immunity. The change of CD4 + T lymphocytes from Th1 to Treg and Th17 cells affected the immune functions [15].
Thrombocytopenia patients and chronic lymphocytic leukemia patients had a higher level (in both percentage and absolute number) of CD4 + /PD1 + lymphocytes than of    healthy controls [16][17][18][19]. The particular subsets of CD4 + lymphocytes had reduced proliferation ability and produced less amounts of IL-1, TNF-α, IL-10, and IFN-γ [17]. CD4 + /PD-1 + T lymphocytes were found to be in close contact with PD-L1 + chronic lymphocytic leukemia cells [19] and the activated PD-1/PD-L1 axis led to decreased production of IL-4 from CD4 + T lymphocytes [19]. High level of CD4 + /PD1 + lymphocytes, reduced cytokine secretion, dysfunctional proliferation, and increased apoptosis were also observed among sarcoidosis patents [20,21], but blocking PD-1 pathway in CD4 + T cells could reverse the proliferative capacity [21]. The expression of PD-1 on CD4 + lymphocytes indicated exhausted function [20,21]. CD4 + /PD1 + TILs were dysfunctional in the presence of PD-L1 among patients with head and neck squamous cell carcinomas [22] and glioblastoma multiforme (GBM) [5]. Though the immune function of CD4 + /PD-1 + T cells was exhausted in tumor microenvironment, CD4 + /PD-1 − effector TILs were more metabolically active and proliferative and enriched in immune costimulation gene sets than CD4 + /PD-1 + effector TILs from GBM [5]. PD-1 expression on CD4 + T cells contributed to the dysfunction of T cells. Compared with healthy controls, the expression of PD-1 on peripheral CD4+ T cells was decreased among psoriatic and rheumatoid arthritis (RA) patients [23,24]. High expression of CD4 + /PD-1 + T cells was related to a reduced activity score of RA [24]. This phenomenon reflected a negative regulation of immune response by CD4 + /PD-1 + lymphocytes in the pathogenesis of psoriasis and RA.
Elderly BC patients had more favorable prognostic factors than younger ones, such as higher grading score and hormone receptor expression; however, their survival was not as good as expected [25]. Hormone receptor positivity was increased from 60% in patients aged 30-35 to 85% in patients aged 80-85 [26]. About 60-70% of all BC patients were diagnosed at early stages; however, their prognosis varies greatly [27]. The variation in immune system of elderly patients accounted for the difference to some extent. In our study, the median of CD4 + TILs was 40/HPF in elderly BC patients, lower than in younger patients (56/HPF). Age was significantly associated with the number of CD4 + /PD1 − TILs. The median of CD4 + /PD1 − TILs was 32/HPF among cases equal to or younger than 55 and 25/HPF among those older than 55, implying the degradation of immune function among elderly patients.
The E-Cad, an important member of cell adhesion molecule family, is encoded by the cadherin gene located on chromosome 16q22.1. The E-Cad mediates molecular   recognition and binding, tumor infiltration, and metastasis [28]. Low expression of E-Cad was related to a poorer recurrence-free survival of invasive ductal BC [29]. Our study revealed that E-Cad expression was significantly associated with the number of CD4 + /PD-1 − TILs. BC cells expressing more E-Cad recruited more functional CD4 + TILs in tumor microenvironment.
Ki-67 is a cell cycle-dependent nuclear antigen and illustrates the proliferative activity of cells [30,31]. Expression rate of Ki-67 was regarded as a proliferation index in cancer cells [32]. We identified a correlation coefficient of 0.29 between Ki-67 index and the count of CD4 + /PD-1 + T cells.
A small sample size was one limitation of our study. In addition, we did not analyze intratumoral and stromal TILs separately. Not using flow cytometry to detect TILs phenotypes was another flaw.
The number of CD4 + /PD-1 + TILs was significantly related to Ki-67 expression, while the level of CD4 + /PD-1 − TILs significantly correlated with E-Cad expression. PD-1 expression on CD4 + TILs indicated dysfunctional immune microenvironment. Further studies are warranted to explore the immunotherapy for BC patients.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Disclosure
The supporting organizations had no role in the study design, data collection, analysis, and interpretation.