T cells, including CD4+ and CD8+ cells, function as immune effectors to induce adaptive immunity. After activation, CD8+ T lymphocytes differentiate into cytotoxic cells, and CD4+ T cells originate three subpopulations of T helper (Th) cells: type 1 Th (Th1), type 2 Th (Th2), and type 17 Th (Th17) cells. CD8 lymphocytes destroy tumor cells and CD4+ subpopulations can produce pro- or antitumor responses [48,49]. CD4+ Th-1 cells release proinflammatory cytokines, such as TNF-α, IL-2, and INF-γ, inducing a potent antitumor response. Further, they promote the antitumor activity of macrophages, including NK and T cells [49,50]. CD4+ Th-2 cells secrete cytokines that have been reported to play a role in tumor growth and metastasis, such as IL-4, IL-5, and IL-13 [51]. However, they also release IL-10, which has both pro- and antitumor properties [52]. Additionally, CD4+ Th-17 cells can induce tumor growth after being activated by TGF-β, IL-6, or IL-23 [53].

In HER2-positive breast cancer, a variety of studies have evaluated the significance of T cell subpopulations on the onset and progression of the disease. In a cohort of 1334 primary invasive breast cancer patients with long-term follow-up, CD8+ T lymphocyte density was assessed. The results showed a significant association between a higher number of CD8+ lymphocytes and a better clinical outcome, regardless of other clinical parameters, including HER2 status [54]. Moreover, elevated CD8+ and low Forkhead box protein 3 positive (FOXP3+) T-cell infiltrates have been identified to serve as an independent predictor of improved OS and RFS after treatment with neoadjuvant chemotherapy in patients with HER2-positive and HER2-negative breast cancer [55]. In addition, breast carcinomas with a higher number of CD8+ T cells have shown a greater benefit from treatment with trastuzumab [56]. Recently, prognostic subsets of T cells have been identified in breast tumors by an automated tool to determine optimal cluster numbers in single-cell RNA sequencing data. Consistent with previous results, CD8+ subsets and Tregs were associated with improved survival in breast cancer patients, and a significant correlation between CD4+ naive T cell expression and OS was identified in breast cancer subtypes HER2-positive and TN [57]. Lately, patients with early HER2-positive breast cancer treated with anti-HER2 therapies, lapatinib plus trastuzumab, in the absence of chemotherapy, have shown a correlation between TIL subsets and pCR rate. The results showed higher pCR rates in patients with CD4+, CD8+, high CD20+ stromal TILs, and high CD20+ intratumoral TILs who had been treated with anti-HER2 therapies [58]. In addition, Datta and colleagues evaluated the CD4+ Th1 immune response in patients with primary invasive HER2-positive breast cancer treated with trastuzumab plus chemotherapy. The results showed that patients who achieved pCR after receiving neoadjuvant therapy had a greater Th1 CD4+ response [59].

Treg cells were described in 1995 and comprise a distinct group of CD4+ T lymphocytes with immunosuppressive properties [60]. Treg cell activity has been related to poor immunological response, and it has been proposed that they could represent a critical mechanism for immune evasion by tumors, including breast cancers. Treg cells are characterized by the expression of FOXP3, a transcription factor that participates in the differentiation, development, maintenance, and function of the Treg cell population [61]. The suppression of the immune response by Treg cells is produced by different mechanisms. These include the inhibition of activated T cells through interaction with the CTLA-4 protein that inhibits the costimulatory molecule of CD28 T cells, as well as the release of cytokines that promote the death of tumor T cells, such as TGF-β, IL-10, and IL-35 [62].

The role of Treg cells in HER2-positive breast cancer has been evaluated in several studies. In a cohort of 3992 breast cancer patients, Treg TILs were assessed by immunohistochemistry. They identified an improved survival in HER2-positive/ER (estrogen receptor)-negative patients with high Treg TIL infiltrates and coexistent CD8+ T-cells. Nevertheless, high Treg TIL quantities without CD8+ T cells were not associated with better survival in ER-positive breast cancer patients [63]. Based on the studies to date, further research is needed to clarify the role of Treg cells in tumoral immune response and their interaction with other cell populations present in the tumor [64]. Thus, future studies on their prognostic role in patient samples are necessary to finally determine the clinical importance of Treg cell assessment.

In contrast to the high attention given to T lymphocytes in tumor progression, the role of B lymphocytes within the tumor microenvironment remains underexplored. In addition to their function of secretion of antibodies and cytokines, which trigger a humoral antitumor response, B cells are able to recognize antigens, regulate antigen processing and presentation, and promote and modulate innate and T cell immunity [65]. This contributes to a plethora of functions in the tumor microenvironment, sometimes contradictory between promotion and regression [66]. Finally, increasing evidence has revealed that high B cell levels are a positive prognostic factor in breast cancer. In fact, tumor-infiltrating B cell densities have been shown to increase in breast tumor tissue compared with normal breast tissue. Moreover, tumor-infiltrating B cells have also been associated with global TILs, CD4+ and CD8+ T cells, higher tumor grade and proliferation, and HR negativity [67]. In a cohort of biopsies from invasive breast cancer patients obtained before neoadjuvant chemotherapy treatment, the expression of CD3, CD8, and CD20 markers in stromal TIL infiltrates was evaluated. Interestingly, they identified CD20 as the most sensitive and specific marker predicting pCR after chemotherapy treatment. Specifically, high CD20 expression was also described to predict pCR in those cases with the HER2-positive subtype [68]. In HER2-positive breast cancer, high levels of B lymphocytes has been correlated with a higher proportion of patients achieving pCR, following lapatinib and trastuzumab treatment without chemotherapy [58]. In contrast, another study showed higher levels of tumor-infiltrating B cells in the pretreatment biopsies of breast cancer patients. Following neoadjuvant treatment with anthracycline plus taxane-based therapy or with trastuzumab, a statistically significant decrease in B cell counts in tumor samples was reported [69]. Therefore, additional research is required to determine the clinical relevance of this immune population in breast cancer due to considerable controversy regarding the prognostic impact of this subpopulation of immune cells.

NK cells are the main players of innate immunity. Although they are cytotoxic effector cells, they also are involved in the modulation of immune reactions through the secretion of cytokines and chemokines. They are identified by their surface markers CD56 and CD16 and can be subdivided into different populations according to their relative expression [70]. Since many studies have demonstrated their cytotoxicity against tumoral cells, NK cells are recognized as crucial agents of immunosurveillance and elimination phases during cancer immunoediting [71]. However, their specific role in breast tumor development, as well as in therapy response, remains still unclear.

A study performed with early breast cancer patients showed the association between the increased expression of molecules involved in the activation of NK cells (such as NK cell receptors) and the increase in RFS [72]. In patients with locally advanced disease, high levels of NK cells were found to be significantly related with an increase in pCR rates. Further, the reduction in NK-mediated cytotoxicity was significantly associated with a poorer response to neoadjuvant chemotherapy [73]. Particularly in patients with HER2-positive breast cancer that had been treated with anti-HER2 agents, an association between increased NK cell levels and pCR was observed [74]. Moreover, in vivo studies using HER2-positive breast cancer mouse models have shown that NK cell depletion abolished the activity of anti-HER2 monoclonal antibodies [75,76]. In patients with early breast cancer, HER2-specific antibodies have been shown to trigger NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) [77,78]. New therapeutic approaches aiming to enhance NK cell activation by anti-HER2 agents are currently being evaluated [79], including the systemic treatment with recombinant cytokines [80], ADAM inhibitors [81], and the delivering of Toll-like receptor (TLR) ligands to the tumor site [82].

It has been suggested that the efficacy of HER2-blocking agents could be affected by NK cell differentiation [83]. Interestingly, circulating CD57+ NK cell numbers have been found to be associated with the acquisition of resistance to anti-HER2 therapies [83]. Therefore, CD57+ NK cell determination could emerge as a useful biomarker for improving clinical management of these sets of patients.

Many immune cells are key players in the tumor microenvironment. However, tumor-associated macrophages (TAMs) have gained special attention in the last decade. In addition to their ability to phagocytize cancer cells [84], they can also recruit other immune cells, as well as present antigens to T cells [85]. Macrophages are commonly grouped into M1 and M2 macrophages, which are two polarized groups discriminated by different functions and cell surface markers. However, both M1 and M2 tumor-infiltrating macrophages are generally identified by the CD68 marker [84,85]. Over the years, several mechanisms by which TAMs can modulate tumor microenvironment have been described, including immunosuppressive actions through programmed death-ligand 1 (PD-L1) expression and the promotion of tumor growth, invasion, and angiogenesis [86,87,88]. M1 macrophages undergo classical macrophage activation, which means they are stimulated by INF-γ and TLR ligands. The M1 phenotype is characterized by expression of CD80 and CD86 (costimulatory molecules) and the secretion of cytokines with proinflammatory properties (such as IL-12 and IL-23). They are commonly associated with Th1 cytotoxic responses that promote tumor destruction [89,90]. M2 macrophages have an alternative activation, stimulated by IL-4/IL-13. They are characterized by CD163, CD204, and CD206 markers, the secretion of IL-10, and the expression of EGF and VEGF. They have been typically associated with immunosuppressive and protumorigenic effects because of their contribution to the activation of Th2 immune responses [90,91,92]. As a result of the accumulating evidence enhancing their role in tumor development, TAMs have emerged as a promising therapeutic target in breast cancer research [93,94]. However, their role is not fully understood, and their relationship with therapy efficacy and patient outcome remains unclear.

Since elevated TAM (CD68+) infiltration in breast stroma has been negatively correlated with clinic-pathological features, such as tumor size, HR status, histologic grade, and age [95,96], it has been disclosed as a prognosis factor in breast cancer patients [97]. Of note, it could be difficult to deepen the interpretation of these results because of the fact that CD68, despite being a pan-macrophage marker, may be expressed in other immune populations [98].

Studies in early breast cancer patients have shown that high numbers of CD163+ M2-macrophages significantly correlate with short RFS and OS rates [99,100]. In this set of patients, high CD163+ infiltration was strongly associated with unfavorable prognosis factors, such as proliferation, poor tumor differentiation, and ER negativity, supporting the importance of TAM polarization into M1 and M2 phenotypes in breast cancer disease [99]. In human invasive breast cancer samples, a high density of TAMs was found to be significantly correlated with the reduction of RFS and OS. Further, TAM infiltration was reported as an independent prognostic factor [101]. T-cell immunoglobulin and mucin domain-3 (TIM-3) is an immune checkpoint molecule that may be expressed in T cells. Interestingly, TIM-3 may represent a potential target to overcome resistance to programmed cell death protein 1 (PD-1) blockade therapies [102]. In HER2-positive patients with metastatic disease (with brain metastases), high densities of TIM-3+ CD163+ macrophages have recently been associated with worse OS of patients [103]. Interestingly, TAM polarization and recruitment into the tumor microenvironment have been associated, not only with poor clinical outcomes in patients with breast tumors [100,104,105], but also with the hampering of anti-HER2-specific agents’ activity [106]. As mentioned previously, M2 macrophage phenotypes have been highly associated with tumoral progression. Importantly, in mouse models, TAM depletion has been shown to improve the therapeutic effects of the anti-HER2 antibody trastuzumab [107]. In addition, the study also highlights the relevance of the repolarization of M2 TAMs into M1 phenotypes after treatment with IL-21, as it has also been reported to enhance the therapeutic effect of trastuzumab [107]. Although these findings present a promising approach to address resistance, further research will be needed to clarify the potential value of TAM repolarization in clinical practice.