EVALUATION OF T- REGULATORY CELLS IN BREAST CANCER

Ola Sayed Mohamed Ali 1 , * Maha A. El-Taweel 2 , Iman Attia Abdel El-Gawad 2 and Eman Ahmed Gouda 3 . 1. Professor of Biochemistry, Faculty of Pharmacy for girls, Al-Azhar University. 2. Associate professor of clinical pathology, National Cancer Institute, Cairo University. 3. B.Sc. of pharmaceutical sciences, Faculty of Pharmacy, Cairo University. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History


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CD4+CD25+ Treg cells were significantly higher in the de novo compared to the healthy controls and in the aftersurgery group compared to the healthy controls and benign breast groups (P<0.0001), while CD4+CD25hi were significantly higher in both de novo and after surgery patients as compared to healthy controls (P<0.0001) ( Table 2). A significant positive correlation was detected between CD4+CD25+ and CD4+CD25hi Treg cells in both de novo and benign patients' groups, while a significant negative correlation was detected between CD4+CD25and CD8+CD25+ Treg cells in the after-surgery group (Table 3).
CD4+CD25+ Treg cells showed significant negative correlation with age in both de novo and after surgery groups (P=0.038, r=-0.388 & P=0.012, r=-0.612 respectively), and significant positive correlation with CEA in the benign group (P=0.005, r=0.722). CD8+CD25+ Treg cells showed significant positive correlation with age in the aftersurgery group (P=0.048, r= 0.487) ( Table 4). CD4+CD25+ and CD4+CD25hi Treg cells were higher in premenopausal compared to postmenopausal de novo breast cancer patients (P =0.017 and 0.051 respectively). CD4+CD25hi Treg cells were significantly higher in the non-metastatic compared to the metastatic patients in the de novo group (P =0.041), while CD4+CD25hi Treg cells were higher in grade III compared to grade II patients in the after-surgery group (P= 0.011) (Data not shown)

Discussion: -
In Egypt, breast cancer ranked the first, constituting 17.50% of total malignancies in females (Meloni et al., 2006).
Although T cells represent the most important immunological response in tumor growth, they become suppressive CD4 + and CD8 + Treg cells after chronic stimulation and interactions with tumor cells, thus promoting rather than inhibiting cancer development and progression (Mokhtar et al., 2007).
In this study, CD4+ lymphocytes were predominant in peripheral blood of de novo breast cancer patients and healthy controls groups compared to the after-surgery group (P=0.04). Mozaffari et al. (2007) reported the same predominance of absolute number of CD4+ lymphocytes although it was statistically non-significant. Leong et al. (2006) showed that CD4+ lymphocyte % was the 2 nd predominant subset (after CD8+) in the tumor microenvironment. Different sample analyses and different sample sizes may be the contributing factors for this discrepancy.
Tumor cells commonly participate in the generation of Treg cells, which provides an explanation for the observation that elevated numbers of Treg cells have been found in many types of cancer. It appears that TGF-β, secreted by the tumor itself or tumor-stimulated myeloid cells, plays a central role in tumor-mediated development of Treg cells by converting naïve T cells into Treg cells (Perez et al., 2007).
As regardsCD4+CD25+ and CD4+CD25hi, they were significantly higher in the de novo and the after-surgery cancer patients compared to the healthy controls and the benign patients' group.
Our result is consistent withWang et al. (2011) who foundthe same result in their study on peripheral blood of de novo breast cancer patients.
In our study, CD4+CD25hi subset was higher in the after-surgery patients compared to the de novo patients (P=0.04) and this was in accordance with the finding of Mozaffari et al (2007).
CD8+CD25+Treg cells share phenotypic and functional features with CD4+CD25+ Treg cells (Cosmi et al., 2006).We detected that the benign patients group had the highest percentage of CD8+CD25+% Treg cells subset, followed by the after-surgery group. The de novo breast cancer patients and healthy controls group were almost the same, the differences were non-significant. . Mozaffari  A significant positive correlation was detected between the CD4+CD25+ % and the CD4+CD25hi % in the de novo breast cancerpatients group (P< 0.0001, r = 0.673) and in the benign group (P=0.002, r = 0.79), while there was a significant negative correlation between the CD4+CD25+ % and CD8+CD25+ % in the after-surgery patients group (P= 0.018, r= -0.583).
In agreement with our results, Zhu et al. (2015) and Leong et al. (2006) have demonstrated an insignificant negative correlation between the CD4+CD25+ cells and CD8+CD28+CD25+ obtained from TILs of breast cancer patients. They also found a significant positive correlation between CD4+CD25+ and CD8+CD28-subsets. In that study, CD8+CD28+CD25+ were defined as effector T cells and CD8+CD28-subset as Treg cells.
In our study, the observed negative relationship may be explained by the finding that not all CD8 + CD25+ population are Treg cells (Bisikirska et al., 2005).
CorrelatingCD4+CD25+ % and CD8+CD25+% with some of the prognostic factors of breast cancer revealed a significant negative correlation between the CD4+CD25+ % and the age in the de novo and after surgery groups. However, a significant positive correlation was found between CD8+CD25+% and age in the after-surgery group.
Similarly, Chui et al. (2004) reported that CD4+ Treg cells were higher in the blood of breast cancer patients < 65 years in age. Tsaknaridis et al. (2003) also demonstrated that the suppressive activity of human CD4+CD25+ T cells declines with age in his study done on normal subjects. In contrast, Leong et al. (2006) found a significant positive correlation with age (> 50 years old).
This diversity of results may be due to differences in sample size or ethnic diversity. found that CD4+CD25+FOXP3+ regulatory TILs are a poor prognostic indicator in ER+ breast cancer, but a favorable prognostic factor in the HER2+/ER-ve subtype. Leong et al. (2006) found a significant positive correlation between CD4+CD25-T subset of TILs in breast cancer patients and ER. Estrogen has been reported to play a role in regulating the activation of human T cells particularly CD4+ and CD8+ T cells. The importance of ER-α in regulating the immune system is further supported by an in vivo murine model, where a high frequency of immature double CD4+CD8+ thymocytes was found in ER-αnegative mice as compared to the ER-α positive mice (Erlandsson et al., 2001).

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This discrepancy between results may be attributed to the predominance of ER positivity in our studied population, different ethnic groups or menopausal status of the studied population.
In conclusion, some Treg cells as CD4+CD25+ cells are predominant in breast cancer patients, and show negative correlation with age and with CD8+CD25+ lymphocytes, which are predominant in the benign patients and after surgery patients.
Treg cells may play a role in modulation of effector T cell responses against breast tumors. Some Treg cells such as CD4+CD25+ cells maybe increased in cases of malignancy, while others, as CD8+CD25+ lymphocytes seem to play a protective role against malignancy. Further studies and larger sample sizes are needed to clarify the diagnostic and prognostic role of Treg cells in breast cancer progression. Treg can act as an immune-modulator in the therapeutic treatment of breast cancer patients weather alone or in combination with therapeutic vaccines and cytokines. a) b) (Fig 1) a):-Positive expression of CD4+CD25+ and CD4+CD25hi subsets gated from CD4+ lymphocytes in a de novo patient. b) CD8+CD25+ subsets histogram from CD3+ lymphocytes gate in an after-surgery patient.