The effects of trastuzumab on the CD4+CD25+FoxP3+ and CD4+IL17A+ T-cell axis in patients with breast cancer

Abstract

In addition to the direct targeting effects on HER2-positive cells, trastuzumab may have a therapeutic role modulating the activity of the cellular immune system in patients with breast cancer. To investigate this further, the balance of T-regulatory (T(reg)), Th17, natural killer (NK) and NK T (NKT) cells before, during and after trastuzumab therapy was investigated. Sequential frequencies of circulating T(reg) cells, Th17 cells, NK and NKT cells were measured in peripheral blood of breast cancer patients and normal controls throughout therapy. Individuals with breast cancer had significantly higher T(reg) frequencies of peripheral blood compared with healthy controls (9.2 or 8.6 vs 6%; P<0.05), and no significant differences in T(reg) frequencies were observed between HER2-positive and HER2-negative individuals. The number of Th17 cells was lowest in HER2-positive patients compared with both healthy controls and HER2-negative patients (0.31 vs 0.75% or 0.84%; P=0.01). There appeared to be an inverse relationship between T(reg) and Th17 frequencies in metastatic breast cancer (MBC) with T(reg) levels significantly reduced during treatment with trastuzumab (P=0.04), whereas Th17 frequencies were concomitantly increased (P=0.04). This study supports earlier data that T(reg) cells are present at higher frequencies in breast cancer patients compared with healthy individuals. For the first time, we show that HER2-positive individuals with breast carcinomas have reduced numbers of circulating Th17 cells, which appear, in turn to have an inverse relationship with T(reg) frequency in MBC. The change in balance of the T(reg) : Th17 ratio appears to characterise the cancer state, and furthermore, is disrupted by trastuzumab therapy.

Figure 1

T_reg frequency is increased in breast cancer patients compared with healthy individuals. (A) A representative T_reg FACS plot showing CD4+FoxP3+ cells. By gating on the CD4+FoxP3+ cells, it was shown that they were also CD25 expressing (shown in histogram; key to histogram: isotype control (dotted grey line), CD4+FoxP3- cells (grey line), CD4+FoxP3+ cells (black line)). (B) Comparison of T_reg frequencies in healthy individuals (black), HER2− breast cancer patients (grey) and HER2+ breast cancer patients, either before or during treatment with trastuzumab (white). Shown is the frequency of T_reg cells as a percentage of total CD4+ cells. Error bars represent±s.e. P-values < 0.05, as measured by the Mann–Whitney U-test, were considered significant.

Figure 2

The number of circulatory Th17 cells is decreased in HER2+ breast cancer patients. The frequency of Th17 cells in HER2− breast cancer patients (grey) did not differ from that measured in healthy individuals (black). HER2+ breast cancer patients (white) had significantly lower Th17 frequency compared with both healthy individuals and HER2− patients, and the frequency of Th17s increased during trastuzumab therapy. Th17 frequency was measured as the frequency of CD4+IL17A+ T cells as a percentage of total CD4+ cells. Error bars represent±s.e. P-values < 0.05, as measured by the Mann–Whitney U-test, were considered significant.

Figure 3

The T_reg/Th17 bias is highest in HER2+ breast cancer. The T_reg : Th17 cell ratio in healthy individuals (black), HER2− breast cancer patients (grey) and HER2+ breast cancer patients treated with trastuzumab (white). The number of T_reg cells to Th17s is significantly higher in HER2+ breast cancer patients compared with healthy individuals and HER2− breast cancer patients, with a slight decrease during trastuzumab treatment. The number of T_reg cells to every Th17 cell is shown. P-values <0.05, as measured by the Mann–Whitney U-test, were considered significant.

Figure 4

The effect of breast cancer stage and trastuzumab on T_reg and Th17 frequencies. The frequency of T_reg cells and Th17s was measured pre- (white), during (grey) and post (black)-trastuzumab therapy in early breast cancer (EBC) and metastatic breast cancer (MBC) patients. The third histogram shows the relative frequencies seen in healthy and HER2− controls. (A) T_reg cells (B) Th17s. Trastuzumab therapy in MBC leads to a statistically significant decrease in the frequency of T_reg cells, and conversely, a statistically significant increase in Th17s. Error bars represent±s.e. P-values < 0.05, as measured by the Mann–Whitney U-test, were considered significant.

Figure 5

The T_reg/Th17 balance. The ratio of T_reg : Th17 cells was measured pre- (white), during (grey), and post (black)-trastuzumab therapy in early breast cancer (EBC) and metastatic breast cancer (MBC) patients. The third histogram shows the relative ratios seen in healthy and HER2− controls. The number of T_reg cells to every Th17 cell is shown. Error bars represent ±s.e. P-values <0.05, as measured by the Mann–Whitney U-test, were considered significant.