Tumor-associated macrophages (TAM) represent an abundant population, and in many tumors they outnumber other immune cells [12]. Although the majority of TAM are found in the invasive margin of the tumor, we can often find also elevated densities within the tumor core [13]. TAMs exhibit an extremely plastic phenotype and function, and two main subtypes have been described: M1 TAM (induced by Toll-like receptor ligands [e.g., lipopolysaccharide and IFN-γ]) which preferentially express pro-inflammatory cytokines and inducible nitric oxide synthase and M2 TAM (induced by IL-4 or IL-13) which express arginase 1, CD206, CD163, IL-4R, TGF-β1, and PDGF [12]. Some works suggest that while M1 TAM potentiate the antitumoral Th1 response and antagonize the suppressive activities of regulatory immune cells, M2 promote angiogenesis, tumor growth, and metastasis [13].

Natural killer cells are cytotoxic effector lymphocytes of the innate immune system whose primary function is to help control infections and tumors [14]. Two major mechanisms of recognition of tumor cells by this population have been described: they can recognize cells which have downregulated major histocompatibility complex class I expression (an immunotolerance phenomenon widely described in many cancer types), or they can bind to stress-induced ligands expressed on tumor cells (e.g., MICA or MICB, which bind to NKG2D expressed on the NK cell) [14].

The main function of dendritic cells (DC) is to establish a bridge between the innate and adaptive immune response. Under physiological circumstances, DC engulf and process nonself-antigens, and when they are exposed to danger or activation signals, they become activated and travel to secondary lymphoid structures in lymph nodes where they prime naïve B or T cells [15]. The DC phenotype is rather plastic, and they can produce a wide range of pro-inflammatory or immunosuppressive cytokines, as well as expressing a large series of activating or inhibition receptors, depending of the environment where they are embedded. The secondary lymphoid organs are protected environments and often provide an ideal milieu to promote a DC phenotype that effectively activates the adaptive immune response [16].

In many cancer types, tumor cells produce molecules that induce pro-inflammatory or tolerogenic DC and block their maturation at different stages. Often, intratumor DCs exhibit an immature and inhibitory phenotype [17]. Interestingly, in recent years, several works have described the presence of tertiary lymphoid structures (TLS) in the invasive margin of many cancer types [18], where in theory the DCs are protected from tumor-produced inhibitory substances and from where they can effectively prime the antitumor immune response [19].

TLS are highly organized lymphoid aggregates that develop in inflammatory pathologies. In cancer, TLS often develop in the invasive margin of the tumors and/or in the stroma and resemble those arising in other chronic infectious or autoimmune diseases [19]. Figure 2.2A illustrates TLS found in clear cell renal cell cancer (ccRCC). Characteristically, TLS exhibit an organization similar to secondary lymphoid organs, including a T cell zone (Fig. 2.2Aa) and a B cell follicular zone (Fig. 2.2Ab), and are often surrounded by high endothelial venules [20]. B cells in TLS form germinal centers; they undergo active proliferative machinery and somatic hypermutation [19]. T cells have a CD62L⁺/CD45RO⁺ central memory or a naïve phenotype, and some can be found in contact with mature DC which expresses the DC-Lamp marker (Fig. 2.2Aa) or at the periphery of B cell follicles (Fig. 2.2Ac) [20]. Follicular dendritic cells are also detected forming a network where immune complexes can form and be presented for selection of the high affinity B cells. Plasma cells that produce antibodies are located at the vicinity of TLS [21].

Primary tumors and metastases contain TLS at variable densities, depending on the tumor type and on the patient. As discussed below, it is assumed that TLS reflect the ongoing immune reaction within tumors. They allow the presentation of tumor antigens by mature dendritic cells to T cells leading to the differentiation of CD4⁺ Th1 cells as reflected by the expression of the T-bet marker and the T-B cell cooperation for B cell differentiation into plasma cells. All of these events can thus occur locally, within the tumor bed. To what extent TLS bypass the need of secondary lymphoid organs to mount or control the antitumor immune reaction remains an open issue.

CD4+ T-helper cells are divided into different subtypes, including Th1, Th2, Th17, Tfh, and Treg; each subpopulation accomplishes specific roles in the antitumor immune response. Overall, a Th1-oriented response antagonizes the tumor growth and is often associated with good clinical outcome [22]. In fact, Th1-oriented cells potentiate in situ the antitumor function of cytotoxic T cells, through the production of several cytokines including IL-2 and IFN-γ. Tfh cells interact with B cells in TLS, helping antibody production.

The role of other subpopulations of tumor-infiltrating CD4⁺ T cells (Th2, Th17, and Treg) is less well understood but is often associated with poor prognosis in different tumors [22]. Many studies suggest that Treg in cancer can dampen the antitumor immune response by two main mechanisms: (1) production of inhibitory cytokines (e.g., IL-10, TGF-β, and IL-35) and (2) suppression of DC development and maturation [23].

CD8⁺ T cells exert a very import function in the antitumor immune response, as they are responsible of tumor cell recognition and elimination. Due to their genome instability, tumor cells often express mutant proteins at their surface. Many of these are neoantigens that can induce a tumor-specific immune response. The primed CD8⁺ T cells are in charge of the tumor cells recognition and lysis, by mechanisms well described in the literature including the release of cytotoxic granules [24]. Interestingly, in the majority of tumors, infiltrating cytotoxic T cells express inhibitory receptors (e.g., PD-1, Tim-3, and Lag-3), whose function under physiological situations is to contract the immune response upon biding to their ligands. Many tumor cells in fact can take advantage of this inhibitory mechanism and in fact express a wide arrange of ligands (e.g., PD-L1, PD-L2) that help them escape for the T cell attack [25].

In inflammatory settings other than cancer, B cells enhance T cell responses by producing antibodies and stimulatory cytokines and chemokines, serving as local antigen presenting cells and organizing the formation of TLS that sustain the immune response. In cancer, B cell can exert all of these functions and overall have an antitumor effect. In addition, recent evidence suggests they can also play an immunomodulatory role through the production of IL-10, among other cytokines [26].

Chemokines ensure the local migration of these different cell types and cytokines allow their cooperation. In addition, many tumors are surrounded by a stroma containing an extracellular matrix composed of fibroblasts that form collagen fibers and produce enzymes––such as metalloproteases––that facilitate local invasion within tissues and ultimately the release of tumor cells that egress to the circulation and migrate in other tissues.

A direct consequence of these processes is that the tumor microenvironment is a tissue-dependent organized structure in which immune cells are common denominators. Figure 2.2B illustrates the presence of CD8⁺ T cells in the tumoral zone of clear cell renal cell cancer. A closer look into the organization of the immune microenvironment reveals that cells are not evenly distributed in the tumor area. Lymphocytes (T and B cells) are more abundant in the tissue border area called the invasive margin than in the center of the tumor [13]. They can be found dispersed or within aggregates, forming TLS in the invasive margin and/or in the stroma [18, 27]. Most of the T and B cells have a memory phenotype, CD8⁺ T, CD4⁺ Treg, Th1, Th2, Th17, and B cells being detected at variable densities, whereas naïve T cells and CD4⁺ Tfh are exclusively present within TLS. NK cells are detected in the tumor stroma. Some T cells are found in close contact with tumor cells in the center of the tumor. Myeloid cells such as macrophages, myeloid-derived suppressor cells, mast cells, and neutrophils are present at high densities, both in the invasive margin and the center of the tumor. Figure 2.2C illustrates the high density of CD163⁺ M2-oriented macrophages near the invasive margin of renal cell cancer. Immature dendritic cells are present at low densities, dispersed in the whole tumor area whereas mature dendritic cells are usually found within the TLS, in close contact with T cells (Fig. 2.2Aa). Importantly the immune composition of the tumor microenvironment evolves with the stages of tumor progression in a tumor-dependent manner. Thus, T cells are more numerous at the early stages of the disease in colorectal cancers and at their late stages in renal cell cancers [17, 28]. The density of B cells increases with tumor stage in colorectal cancers, as does that of the myeloid cells such as neutrophils, mast cells, immature dendritic cells, and macrophages. Thus, the tumor microenvironment is a complex structure, forming a tumor-dependent “immune landscape” that evolves during tumor progression.

The positive or negative role of B cells in antitumor immunity has been discussed for many years, mainly supported by mice studies. As compared to T cells, few clinical studies reported the prognostic role of intratumoral B cells. The majority of clinical studies have demonstrated that a high density of B cells within TME is associated with better prognosis including breast cancer [43], NSCLC [21], head and neck cancer [44], ovarian cancer [45], metastatic colorectal cancer [46], biliary tract cancer [47], and primary cutaneous melanoma [48]. Several nonexclusive mechanisms could explain the positive role of B cells in the antitumor immune response, some being antibody dependent by their capacity to trigger complement and antibody-dependent cell cytotoxicity (CDC and ADCC) or to form immune complexes able to activate DCs and others by acting as APC for CD4 [49] and CD8⁺ T cell immune responses [50]. Indeed, it has been shown that B cells play a major role during initial priming and expansion of CD4⁺ T cells [51], are able to cross-present antigens to CD8⁺ T cells [52], and can promote cytotoxic T lymphocyte survival and proliferation [53].

On the opposite, few clinical studies reported a pro-tumoral role of B cells within the TME [54, 55]. B cells may play a pro-tumor function by the maintenance of a chronic inflammation [56], by the promotion of neoangiogenesis [57], and/or by the direct inhibition of cytotoxic T cell responses [55]. Moreover, a subpopulation of immunoregulatory B cells called “Bregs” has been described and has been shown to favor the differentiation and the recruitment of Tregs, thus amplifying the immunosuppressive environment [58].

Beyond the density of B cells, an increasing number of studies reported that the spatial localization of these cells have an impact on patient’s outcome. In particular the density of B cell follicles characteristic of TLS is positively associated with outcomes. M.C. Dieu-Nojean and col. showed that an increase in B cell density within the TLS is associated with prolonged survival in NSCLC patients [21]. Similar results were reported in CRC [59] and oral squamous carcinoma [60].

Tumor-associated macrophages (TAM) are a major component of the TME, found both at the tumor core and the invasive margin. The prognostic value of TAM seems to be dependent of the tumor type. Increased density of TAMs is associated with a good prognosis in CRC [61], HCC [62], prostate [63], and cervical cancer [64]. At the opposite an increased TAM density is associated with poor prognosis in endometrial [65], gastric [66], urothelial [67], HCC [68], melanoma [69], breast [70], ovarian [71], bladder [67], NSCLC [72], and primary CRC tumors [13]. These discrepancies might be explained by the plasticity of these cells since we know that they can switch from a pro-tumoral function (M2) to an antitumoral function (M1) and vice versa [12]. M2 TAMs are associated with a shorter survival and M1 TAMs with a longer survival [22]. Unfortunately, there are no specific or consensual markers to define M1/M2 TAMs. Most of the studies used CD11c or NOS2 for M1 TAMs and CD163, CD204, or CD206 for M2 TAMs, but the use of these markers is still debated.

Tumors contain another heterogeneous subset of cells of myeloid origin, the myeloid-derived suppressor cells (MDSC). Such cells have an immature phenotype and exert profound immunosuppressive activities. Specific and robust tools are still needed for their identification in the human TME.

The most broadly used way to quantify tumor-infiltrating immune cells is to detect the protein expression of specific markers either by IHC or IF. These techniques have been improved in the last decade, allowing to detect multiple proteins (multiplex IHC or IF) and to quantify cells automatically. Nevertheless, they remain expensive and difficult to standardize across laboratories, and available antibodies could lack sensitivity or specificity to accurately detect some of immune cell populations.

Efforts have been made to use transcriptome to estimate the composition of the TME. Nevertheless, variability in the signal has limited its applicability until recently. New methods such as CIBERSORT [13] or MCP-counter [73] aim at providing very precise quantitative information about the cell content of heterogeneous samples. Using MCP-counter, we estimated the abundance of immune cells, fibroblasts, and endothelial cell infiltrates, in transcriptomes of 25 different cancers (n = 19,000). The results showed the relative heterogeneity of the cellular composition of the tumor microenvironment in different cancers and confirmed that the inferred density of CD8⁺ or cytotoxic T cells correlated with favorable prognosis in most cancer types [73] (Fig. 2.3).