After the diagnosis of BC was confirmed by pathological examination, 52 untreated patients with BC (mean age = 48.9±1.55 years) were recruited into the study. According to the pathology reports, 23 out of 52 LNs were involved (44.23%). Most patients were in stage II (29/52, 55.77%), and in most, the tumor type was invasive ductal carcinoma (IDC, 41/50, 82.0%). The main clinical and pathological characteristics of the patients are summarized in Table 1.
Frequency of CD8+ memory T cell subsets in tumor-draining lymph nodes
The average frequency of different memory T cell subtypes in the CD8+ lymphocyte population along with mean expression of CD95 on the surface of these cells are reported in Table 2. As shown, 8.43±0.49 of the lymphocytes in TDLNs of patients with BC were CD8-positive. In this group, more than 47% (47.65±2.66%) of the cells expressed CD45RO, a marker of the memory T cell phenotype.
Memory CD8+ T cell subsets in patients with different clinical and pathological characteristics
In the next step, we investigated the association of memory CD8+ subsets and naive CD8+ cells with different clinical and pathological parameters. Statistical analysis showed that the percentage of CD95+CD8+ and CD45ROlow CD8+ cells was significantly higher in involved lymph nodes comparing to tumor-free ones (P=0.036 and P=0.048, respectively). The percentage of CD45RO+CD8+ cells was also higher in patients with larger tumors (T2 vs. T1, P=0.035). While the frequency of CD8+ lymphocytes was significantly lower in patients of N1 (with 1–3 involved nodes) and N2 (with 3–9 involved nodes) compared to patients with free nodes (P=0.004 and P=0.025, respectively).
CD8+ TCM cells
The total frequency of TCM with the CD8+CCR7+CD45RO+CD95+ phenotype was 33.84±2.16 in draining lymph nodes of patients with BC. We also investigated two different subsets with low and high CD45RO expression (Table 2). Our analysis showed that the frequency of total TCM cells and the subset with low CD45RO expression (CD45ROlow TCM) was significantly greater in involved nodes compared to tumor-free ones (P=0.024 and P=0.017, respectively; Fig. 2a). Among patients in different pathological stages, CD95 expression (based on MFI) on the surface of TCM overall, and the CD45ROhi TCM and CD45ROlow TCM subsets, was higher only in patients with stage II compared to those with stage I disease (P=0.004, P=0.015 and P=0.001, respectively). In addition, the expression of CD95 on TCM was also higher in TDLNs of patients with moderately differentiated tumor cells (grade II) compared with those with well-differentiated tumors (grade I, P=0.019). Regarding the number of involved lymph nodes, CD95 expression on TCM, CD45ROhi and CD45ROlow TCM subsets was notably higher in patients with N1 disease compared to the node-free group (P<0.0001, P=0.002 and P<0.001, respectively). Mean fluorescence intensity for CD95 on TCM cells was also greater in the N3 group (P=0.037) compared to the N0 group. Furthermore, the percentage of CD45ROhi TCM cells was also greater in patients with larger tumor sizes (T2 vs. T1; P=0.038).
CD8+ TEM cells
Approximately 9% of CD8+ cells (9.24±0.78%) in draining LNs of patients with BC had the effector memory phenotype (CD8+CCR7-CD45RO+CD95+). Analysis of CD95 expression on TEM cells in patients with different clinical and pathological characteristics indicated higher expression of this molecule in N1 patients compared to node-free patients (P=0.020).
CD8+ TSCM cells
The frequency of the CD8+CCR7+CD45RO-CD95+ phenotype, considered here to
reflect TSCM cells, was 9.40±1.37%. Although the frequencies of these cells did not differ significantly among patients with different clinical and pathological characteristics, mean expression of CD95 (based on MFI) on the surface of TSCM showed an increase in patients with stage II disease (P=0.012) compared to those in stage I. The expression of CD95 on these cells was also greater in TDLNs of N1 patients compared to node-free patients (N0, P=0.003).
Naive CD8+ lymphocytes
In addition to memory CD8+ lymphocytes, we also determined the percentage of lymphocytes with the naive phenotype (CCR7+CD45RO-CD95-) in TDLNs. The percentage of naive CD8+ T cells was significantly lower in tumor-involved lymph nodes compared to tumor-free ones (P=0.022, Fig. 2a).
Correlations among frequencies of different CD8+ lymphocyte subsets
We also investigated the correlations among different subsets, and between subsets and patients’ ages, with the Spearman correlation test (Fig. 2b). The results showed that the percentage of naive cells had a strong negative correlation with TCM (P<0.001, R=−0.736), their CD45ROhi (P<0.001, R=−0.773) and CD45ROlow (P<0.001, R=−0.682) subsets, and TEM cells (P<0.001, R=−0.645). The percentage of CD45ROlow TCM cells had a positive correlation with CD45ROhi TCM (P<0.001, R=0.650) and TSCM (P=0.035, R=0.294) subsets, and the percentage of CD45ROhi TCM cells correlated strongly with TEM cells (P=0.001, R=0.433). We also observed a positive association between age and the frequency of CD45RO+CD8+ (P=0.042, R=0.283), TCM (P=0.022, R=0.318) and TSCM (P=0.049, R=0.275) subsets. Conversely, a negative correlation was observed between age and the frequency of TN (P=0.025, R=−0.0310).