Crucial Contributions by T Lymphocytes (Effector, Regulatory, and Checkpoint Inhibitor) and Cytokines (TH1, TH2, and TH17) to a Pathological Complete Response Induced by Neoadjuvant Chemotherapy in Women with Breast Cancer

Figure 3

FOXP3⁺ (a, b) and CTLA-4⁺ (c, d) Tregs in the sections of LLABCs, using IHC staining, at 400x magnification. Briefly, heat-mediated antigen retrieval was performed using citrate buffer, pH 6 (20 mins). The sections were then incubated with MAbs to FOXP3 (Abcam, ab20034) at a concentration of 20 μg/mL for 30 mins at RT and MAbs to CTLA-4 (Santa Cruz Bio, sc-376016) at a 1 : 300 dilution for 30 mins at RT. Polymeric HRP-linker antibody conjugate was used as secondary antibody. DAB chromogen was used to visualize the staining. The sections were counterstained with haematoxylin. (a, c) Low level of FOXP3⁺, CTLA-4⁺ Treg infiltration; (b, d) high level of FOXP3⁺ and CTLA-4⁺ Treg infiltration. The average number of brown nuclear-stained (FOXP3), membrane-stained (CTLA-4) cells, regardless of intensity, in contact with tumour cells or within tumour cell nests (Itu: intratumoural) and in the interstitial stroma (Str: stromal) per HPF was counted.

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