The neglected brothers come of age: B cells and Cancer

Abstract

The opposing roles of innate and adaptive immune cells in suppressing or supporting cancer initiation, progression, metastasis and response to therapy has been long debated. The mechanisms by which different monocyte and T cell subtypes affect and modulate cancer have been extensively studied. However, the role of B cells and their subtypes have remained elusive, perhaps partially due to their heterogeneity and range of actions. B cells can produce a variety of cytokines and present tumor-derived antigens to T cells in combination with co-stimulatory or inhibitory ligands based on their phenotype. Unlike most T cells, B cells can be activated by innate immune stimuli, such as endotoxin. Furthermore, the isotype and specificity of the antibodies produced by plasma cells regulate distinct immune responses, including opsonization, antibody-mediated cellular cytotoxicity (ADCC) and complement activation. B cells are shaped by the tumor environment (TME), with the capability to regulate the TME in return. In this review, we will describe the mechanisms of B cell action, including cytokine production, antigen presentation, ADCC, opsonization, complement activation and how they affect tumor development and response to immunotherapy. We will also discuss how B cell fate within the TME is affected by tumor stroma, microbiome and metabolism.

1. Introduction and a short history of “Bursa of Fabricus” cells

The first description of what we know as B cells today was in 1890 with the discovery of circulating antitoxins in immunity to diphtheria and tetanus by Emil von Behring and Shibasaburo Kitasato [1,2], while working at Berlin Institute of Infectious Diseases (Robert Koch institute). Later in 1900, another alumnus of the Koch’s institute, Paul Ehrlich hypothesized that an immune cell bearing many different antibody receptors could be stimulated by binding an antigen and subsequently produce and release more of the receptor type complementary to that antigen, what we know now as immunoglobulins (Igs) [3]. Ig-producing and secreting plasma cells [4,5], that are derived from B lymphocytes [6,7]. When Paul Ehrlich was describing his side-chain theory of antibody formation, explaining that the producers of circulating antitoxins were cells with pre-formed antibody receptors [8], he also suggested that these molecules, if reactive to tumors, could play a key role in cancer therapy [9].

The first clear evidence that B cells develop within a distinct organ was provided in 1956, when Bruce Glick removed the “Bursa” of Fabricius, a hindgut lymphoid organ, from newborn chicks, which resulted in depletion of antibody production [10]. The cells were called “Bursa of Fabricus” cells or “B” cells. Finally, in the decade between 1960-1970, the separate development of functionally linked lineages of lymphocytes known as B cells and T cells was accepted as a fundamental organizing principle of the adaptive immune system in all vertebrates by Jacques Miller and Max D. Cooper who discovered the thymus, as the tissue in which “T” cells develop and the “Bursa of Fabricus” in which “B” cells develop, respectively [2,11–14]. Since then, immunologists have defined many different sublineages of the clonally diverse B and T cells and described how they interact with each other and with the innate immune system to regulate infections, cancer and the development of autoimmune and inflammatory diseases. In this review, we will give a brief summary of B cell development, their different sublineages, and how their interactions with different arms of the innate and adaptive immune systems suppress or support cancer.

2. B cell development, antibody structure and diversity

In mammals, B cells originate from the fetal liver and bone marrow [15–17]. Lymphocytes can initiate specific immune responses against antigens by generating a nearly infinite diversity of antigen receptors. This is achieved largely by a somatic recombination process known as variable, diversity, and joining V(D)J recombination. Through V(D)J recombination, the variable region of antigen receptor genes is assembled from component germline V, D, and J gene segments. There are seven different loci that are rearranged to generate the antigen receptors of T and B lymphocytes. These include the immunoglobulin (Ig) heavy chain locus (IgH) and the Ig light chain (IgL) loci (Igκ and Igλ), which encode the antigen receptor (B cell receptor, BCR) and secreted antibodies (immunoglobulins, Ig). The rearrangement of these loci is tightly controlled in a lineage-, stage-, and allele specific manner. During B cell maturation, sequential intrinsic genetic DNA sequences are rearranged in the heavy and light chain immunoglobulin loci [18]. Cells at the pro-B stage of development initiate V(D)J gene segment recombination, using recombination activating gene 1/2 (RAG1/2) endonucleases to cleave the V, D and J segments, which are then joined by non-homologous end-joining (NHEJ) to form V(D)J exons [19]. Assembly of the heavy chain locus (IgH) precedes that of the light chains loci (IgL) [18]. The rearrangements of the IgH locus itself are sequential, with DH to JH joining occurring on both alleles prior to VH to DHJH segment rearrangement. The productive assembly of the VH-DHJH variable gene regions indirectly induces progression to the next stage of B cell differentiation. In this stage, IgL chains are assembled with Igκ rearrangements generally preceding those of Igλ, generating immature B cells that express μ plus IgL chains as surface IgM [18,20]. The random insertion and deletion of nucleotides at the junction sites of the V, (D) and J gene segments create fingerprint-like sequences of the junctional regions of Ig. These sequences are specific to each lymphocyte. Surface IgM⁺IgD⁺ B cells migrate to peripheral lymphoid tissues (spleen and lymph nodes) where they engage in antigen-dependent responses including class switch recombination (CSR) and somatic hypermutations (SHM) (Figure 1). Usually, mature naïve “follicular” B cells reside in the B cell follicles (Fo B cells) of the spleen and lymph nodes and become activated upon exposure to antigen (Figure 1). Activated FO B cells interact with follicular dendritic cells (FDC) and antigen specific follicular T helper cells (T_FH) and form the germinal center (GC) inside the B cell follicle, which has two regions, the dark (DZ) and light zone (LZ). The follicle is the site of affinity maturation, proliferation, and plasma cell and memory cell differentiation. CSR results in isotype switching and was thought to take place in the GC. However, new data suggest that CSR predominantly takes place before either GC or extrafollicular B cell differentiation, at the stage of the initial B cell:T cell interaction [21]. CSR is an intrachromosomal DNA rearrangement of the immunoglobulin (Ig) heavy-chain locus, which results in the deletion of μ–heavy-chain (IgM) constant region gene segment and its replacement by one of the other downstream constant region gene segments, which include γ (IgG), ε (IgE), and α (IgA) [22–24]. As a result, mature B cells express antibodies of the IgA, IgG, or IgE classes that differ in effector functions without altering their antigen specificity. Every isotype switching event also involves exposure to a particular cytokine; IL-4 regulates the IgE CSR, TGFβ induces the IgA CSR, and IFNγ usually results in IgG CSR (Figure 2). CSR and SHM are both regulated by activation-induced cytidine deaminase (AID, encoded by Aicda). Other nucleases and transcription factors involved in these processes are uracil-DNA glycosylase (UNG), apurinic-apyrimidinic endonuclease 1 (APE1), BCL6, IRF4, NF-κB, BLIMP1 and MYC [25]. The antigen-selected evolution of cumulative somatic mutations in V region genes (somatic hypermutations, SHM) is the underlaying mechanism for affinity maturation, a process that occurs primarily in the DZ of the germinal center and is needed for the generation of high affinity antibodies [2,26,27]. Moreover, in tissue-tertiary lymphoid structures (TLS), which are ectopic lymphoid organs that develop in non-lymphoid tissues at sites of chronic inflammation including tumors, antigen-specific T and B cells can undergo terminal differentiation into effector cells in the T- and B-cell-rich areas, respectively. Tumors contain variable proportions and type of TLS [28,29]. The proportion of TLS varies according to tumor types from around 10 %–20 % in HCC, and present in up to more than 80 % of tumors such as in CRC and lung squamous cell carcinoma primary tumors. In organized TLS, germinal center B cells establish close interactions with FDCs and T_FH in the course of differentiation into memory B cells and PCs (Figure 1B) [29,30]. Activated B cells, plasma cells, and the antibodies produced and secreted by plasma cells can migrate to sites of inflammation and infection to regulate innate and adaptive immune responses through a wide variety of mechanisms. The cellular functions of B cells and plasma cells include: a) Secretion of soluble factor e.g. cytokines; b) Expression of stimulatory and inhibitory ligands e.g. PD-L1, CD80, CD86; c) Priming of CD4⁺ and CD8⁺ T cells through MHC-I and MHC-II expression. The humoral functions of secretory immunoglobulins include: a) Neutralization, opsonization, and antibody-dependent phagocytosis; b) Antibody-dependent cellular cytotoxicity (ADCC); c) Complement activation (Figure 3). In the following section, we will discuss each of these functions in the context of cancer, and how the TME shapes the B cell response and vice versa.

Figure 1.

A) The development and differentiation of B cells. B cells develop in the bone marrow. During development, early B-cells similarly rearrange both of their IgH alleles before proceeding to rearrange their IgL alleles. V(D)J recombination in bone marrow pro-B cells first assembles IgH V(D)J exons leading to μ chain expression. Individual B cells express either Igκ or Igλ. Pre-pro-B cells associate with CXCL12^hi reticular cells, whereas pro-B cells move towards IL-7-expressing cells like MSC and stroma cells. B cells expressing cell-surface IgM exit the bone marrow and enter the blood to reach the spleen or lymph nodes, where they mature into peripheral mature B cells. In secondary lymphoid tissues, the germinal center is built within the B cell follicles upon infection or immunization. The GC is divided into two distinct compartments. The dark zone (DZ) contains a network of CXCL12-producing reticular cells and is the site of GC B cell proliferation and somatic hypermutation (SHM). B cells follow a CXCL13 gradient to enter the light zone (LZ) through expression of CXCR5. In the LZ, B cells capture antigen presented on follicular dendritic cells (FDCs) which they internalize, process and subsequently present to T follicular helper (TFH) cells in order to undergo selection. This process is regulated by T follicular regulatory (TFr) cells which are also present in the LZ. Cytokines and soluble factors secreted by TFH define isotype CSR. Upon receiving survival signals from TFH cells, B cells re-enter the DZ for further rounds of proliferation and SHM after which they exit the GC as memory B cells or high-affinity antibody-secreting plasma cells. Long-lived plasma cells reside in the bone marrow in direct contact to bone marrow stromal cells. B) The composition and function of tertiary lymphoid structures in cancer. TLS are formed in inflamed sites upon contact between lymphoid tissue organizer IL7⁺ stromal cell and a lymphoid tissue inducer cell (LTi) in a CXCL13 rich environment. Tissue resident monocytic cells, TH17 or B cells can function as LTi. CCL21 and CXCL12 participate in lymphocytes recruitment, while CXCL13 and CCL19, together with adhesion molecules, control the structural organization of the forming TLS. The infiltrating immune cells form aggregates with a T cell rich zone where mature DC present MHC-Cl II peptides to CD4⁺T cells, promoting their activation, proliferation and differentiation into T effector cells, including T_FH expressing the CXCL13 receptor, CXCR5. The T_FH migrate to the CXCL13-rich B cell zone where they induce the activation, proliferation and differentiation of antigen-specific B cells into plasmablasts or their migration into follicles to form GCs where B cells undergo CSR. TLS include a series of heterogenous structures from lymphoid aggregates of T and B cells to structures including follicles of B cells (disorganized to organized). TLS range from the early loose lymphoid aggregates (disorganized, E-TLS) without FDCs to primary follicle-like TLS (PFL-TLS) containing a network of CD21⁺ FDCs and secondary follicle-like TLS (SFL-TLS) containing a GC including a network of CD21⁺CD23⁺ FDCs, as described for secondary lymphoid tissues above. The activation and differentiation of T cells within TLS allows for modulation of their effector function in the TME. In SFL-TLS, CD20⁺B cells are located within and at the periphery of GCs, similar to GCs in LN. Analysis of the BCR repertoire in follicles, compared with peripheral B cells, reveals clonal expansion, suggesting the development of an antigen-driven B-cell response occurring within TLS-GC. B cells that successfully capture antigen and receive CD40-mediated help from T_FH survive and exit the GC as long-lived plasma cells or memory B cells. A fraction of the affinity matured B cells also undergo AID-dependent CSR that regulates isotype switching. Recent work suggests that this process could occur prior to differentiation of B cells into GC B cells or plasmablasts, rather than within GC as previously suggested. The question, if and at which stage of tumor-infiltrating TLSs maturation CSR appears, remains unknown.

Figure 2. Development of immunosuppressive or immunostimulating plasma cells.

T follicular helper cell (T_FH) differentiation and context-dependent T_FH heterogeneity regulate B cell CSR and isotypes. Following activation of naïve CD4⁺ T cells, TH cells proliferate and undergo fate decisions in response to cytokines and other differentiating factors. T_FH and B cell differentiation and isotypes are influenced by IL-6 and IL-21, and dependent on CD40 and ICOS signaling for expression of the transcription factor Bcl6. Cytokines including IL-12, IL-4, IL-1β, and many others, direct (A) TH1 and IgG; (B) TH2, IgG and IgE, (C) TH17, mucosal IgM and IgA and (D) regulatory cell differentiation. The microenvironment-dependent cytokines influence T_FH and B cell differentiation, lineages, and isotypes, which define their fate to be pro- or anti-tumorigenic.

Figure 3. The multiple functions of B cells and their contributions to cancer.

The functions of B cells and plasma cells include: A) Secretion of soluble factor and expression of ligands, opsonization; B) Interaction with microbiome; C) Antibody-dependent cellular cytotoxicity (ADCC); D) Priming of CD4₊ and CD8₊ T cells through MHC-I and MHC-II expression; E) Complement system activation; F) Induction of tertiary lymphoid structure (TLS) formation.

3. The multiple functions of B cells and their contributions to cancer

Several studies suggest that B cell presence and functionality is an important prognostic factor in cancer [31–42]. Plasma cells are present in many cancers, with the ability to produce large amounts of antibodies and cytokines [43,44]. Compared to T cells which are often scattered within tumors and sometimes in close contact with tumor cells, most B lymphocytes localize together forming clusters with varying size, cellular composition and maturation degree with T cells to from anything between a loose aggregate juxtaposing a T cell cluster in early (E)-aggregate (E-TLS) to denser organized primary follicle-like aggregates (PFL)- and secondary follicle-like (SFL)-TLS. Of the tumor-infiltrating immune cells, up to 3-30% are B and plasma cells based on cancer type, time point and the methods used, which is very similar compared to tumor-infiltrating-T cell numbers [37,40,41,45]. Immunoglobulins, dominantly IgG and IgA, specific for more than 2,000 antigens, which are often overexpressed by cancer cells, have been detected in sera of many cancer patients [46–49]. B cells and plasma cells are found scattered at tumor margins, close to cancer-associated fibroblasts (CAFs), in tumor-associated aggregates, and in unorganized clusters or organized tertiary lymphoid structures (TLS) [29,35,42,43,50]. However, tumor-infiltrating B and plasma cells have both tumor-promoting and -suppressing characteristics. This depends on their phenotype, their antibody isotype and production, the tumor type, TME, and their localization [51–53,53–56] (Figure 2,3). The Cancer Genome Atlas (TCGA) database analysis revealed that a high expression of B cell and plasma cell signature gene correlated with increased overall survival in patients with melanoma, lung adenocarcinoma, pancreatic adenocarcinoma and head and neck squamous cell carcinoma. By contrast, high levels of expression of these genes correlated with poorer clinical outcomes in patients with glioblastoma and clear cell renal cell carcinoma. High levels of immunoglobulin expression in tumors is also associated with increased survival in patients with melanoma, lung adenocarcinoma and head and neck squamous cell carcinoma, but it is associated with poor prognosis in patients with glioblastoma and renal cell carcinoma [51–53,57] (Table 1).

Table 1.

Infiltration and prognostic impact of B cells, plasma cells, serum immunoglobulins and TLS in human cancers

In particular, the cardinal role of B cells in regulation of CD8⁺ and CD4⁺ T cell responses ranges from their antitumorigenic function, including induction of cytotoxic CD8⁺ T cell (CTL) and CD4⁺ T helper cell (TH1) activation and effector function to their pro-tumorigenic function including suppression of CTLs and TH1 responses [30,32,35,42,43,58–62]. B cells and humoral immunity can also regulate anti-tumor immunity through opsonization, antibody-mediated cellular cytotoxicity, or activation of the complement system components C5a or C3a, which seem to either activate or suppress anti-tumor immunity in a context-dependent manner [62–66]. Below we will discuss different B and plasma cell effector functions and their contributions in cancer (Table 1).

3.1. Expression of soluble factors and ligands

The capability of B cells and particularly plasma cells to produce cytokines and soluble factors has long been underestimated [37,65,67–70]. In prostate cancer, newly recruited B cells promote aggressive hormone-refractory tumors by producing the proinflammatory cytokine lymphotoxin [71]. Lymphotoxin in combination with CXCL13, a chemoattractant for B lymphocytes, produced by T_FH cells and fibroblasts can support lymphangiogenesis and metastasis [50,72,73]. On the other hand, The CXCL13 signature was also associated with favorable prognosis in colorectal cancer and melanoma [30,41]. In this regards, the induction of lymphangiogenesis and tertiary follicle structures supported T cell priming and improved responses to immunotherapy [30,59,74]. Recently, B cells including heterogenous populations called “regulatory B cells (Bregs)” and “immunosuppressive plasma cells (ISPC)” were shown to attenuate the development of autoimmune disease [65,68,75,76] and antitumor immunity by expressing IL-10, PD-L1, FASL, TGF-β, IL-35 and Tim-1 [42,43,66,75,75,77–79,79,80]. Of note, Tim-1, a phosphatidylserine receptor expressed on B cells, induces IL-10 production by sensing apoptotic cells [43,79]. TIM-1⁺ B cells have recently been shown to express a number of co-inhibitory checkpoint receptors including TIGIT, Ebi3 (a subunit of IL-27), CD39, CD73 and PD-L1. Tim-1⁺ B cells also highly express a set of checkpoint receptors including CTLA4, Lag3, PD-1, and Tim-3 that have been associated with CD8⁺ T cell exhaustion/dysfunction and Treg differentiation [79,81,82]. Inversely, B cells, particularly IgG⁺ cells, can express factors that activate T cells and have tumor suppressive effects like Granzyme B, TRAIL, IFNγ and IL-12 [83]. B cells can also secrete the cytokine IL-6, a cytokine with a well described pro-tumorigenic role [84,85].

3.2. Plasma cells, isotypes, immunoglobulin

As stated above, the detection of antibodies, largely IgG and IgA, specific for tumor-associated antigens in both serum and TME confirmed the existence of humoral immune responses to tumors ([47,86–89]. Antibodies of different isotypes usually operate in distinct tissues and have different effector functions. CSR affecting the BCR cytoplasmic tail alters BCR functionality and each subclass (isotype) of secreted antibody can influence B cell differentiation, survival and function by stimulating a varied immune response [90]. Antibody functionality is also determined by the constant region (Fc) that can bind to Fc receptors expressed by various cells including macrophages and dendritic cells (DC) [91]. Factors and cytokines secreted by CD4⁺ T helper cells in combination with co-stimulatory ligands (CD40/CD40L) and cognate cell contact selectively direct B cells to switch to specific antibody classes and subclasses (Figure 1–3).

Recently, a meta-analysis, including data from 11-14 studies with approximately 2,000 untreated cancer cases and 1,000 control subjects, evaluated the association of serum immunoglobulin classes with solid cancer and addressed the question if the immune escape of tumors is accompanied by dysregulated systemic immunoglobulin class-switching. Significantly higher serum IgA levels in patients with solid malignancies compared with healthy individuals was observed, and this further increased in patients with advanced cancer, indicating an association of IgA class-switching with solid cancer diagnosis and confirming its immunosuppressive and pro-tumorigenic role [92]. IgA serum level has also been linked to older age, male gender, metabolic syndrome, and other well-known factors (e.g. obesity, alcohol, smoking) associated with immune dysregulation, cancer risk and unfavorable prognosis [92,93]. In this meta-analysis, no association between total serum IgG levels and solid cancer was found [92]. IgG accounts for 75% of serum immunoglobulins and consists of 4 different subclasses (IgG1–IgG4), which have been shown to differ in their inflammatory and ADCC abilities. Accordingly, a lower IgG1/total IgG ratio has been reported in gynecological [94], colorectal [95], head and neck [96], and breast cancer [97], confirming its anti-tumorigenic capability. On the other hand, a raised serum IgG4/total IgG ratio has been reported in hepatocellular carcinoma [98] and melanoma, [99] and has been associated with unfavorable disease prognosis.

Murine IgG2a and IgG2b with their human homologs IgG1 and IgG3 usually contribute to a proinflammatory Type I immunity against intracellular pathogens in concert with TH1 and ILC1 cells and bind to proinflammatory Fc receptors, FcγR1 and FcγRIV, [100–102]. In this regard, IgG2a⁺ antibodies failed to eliminate tumors in a FcγRIV^−/− mouse model [100]. These isotypes can effectively induce ADCC and activate the pro-inflammatory complement system [103,104], or produce Granzyme B, TRAIL and IL-12 [83]. Therefore they are associated with anti-tumor immunity and correlate with longer survival of cancer patients [38,51,53,105,106].

Murine IgG1, its human analog IgG4, and IgE usually contribute to Type II-like immunity. Murine IgG1 (hIgG4) can bind also to the inhibitory FcγRIIB with high affinity and fail to activate the pro-inflammatory complement pathway [104]. In this regard, IgG4 was shown to lack anti-tumor function and correlated with poor prognosis and induction of chronic inflammation probably by activation of tumor-associated macrophages and TAM2-like cells [89,99,107–111].

Binding of IgE to FcεRI can support the secretion of Type II cytokines like IL-4 and IL-13 by mast cells. These cytokines have a wide range of functions in tumors, being mostly pro-tumorigenic, including polarization of TH cells toward a TH2 phenotype, that lacks a strong-antitumor immunity [37,51].

Type III immunity is usually associated with protection of mucosal sites from extracellular pathogens by TH17 cells and IgA⁺ plasma cells. Secreted IgA⁺ antibodies usually bind to Polymeric immunoglobulin receptor (PIgR) to be transported through the epithelial layer [112–116]. The T cell-independent IgA CSR depends on retinoic acid and TGF-β, however TNF and inducible nitric oxide synthase (iNOS) can also attribute to their development in combination with BAFF and APRIL [113,117,118]. Intestinal secretory IgAs bind to PlgR in the mucosal layer and regulate the microbiome, which is mediated by dimeric secretory IgA that neutralizes antigens and prevents microbial adhesion to epithelial cells. IgA promotes bacterial symbiosis through protective opsonization, modification of their metabolism and epitope expression [119], which may induce immune tolerance and the maintenance of gut microbiome diversity [120]. Moreover, gut and lung microbiota can augment IgA class-switching through antigen presentation by CD103⁺ dendritic cells (DCs) and induction of TGFβ and IL10 [121]. Gut and skin residual CD103⁺ DCs are also able to induce T-cell anergy and Treg expansion by expressing aldehyde dehydrogenase and indoleamine 2,3-dioxygenase [122]. Tissue-specific immunoregulation may determine the ability of different tumors to regulate the IgA-microbiota axis in order to facilitate immune escape. A possible role of mucosal IgA in tumor immunity appears from its dynamic relationship with environmental factors, such as diet and the microbiota. Dysbiosis and changes in microbiome diversity was recently shown to influence multiple aspects of antitumor immunity, from altering the risk of developing cancer to regulating responses to immunotherapy [123–126]. In this regard, the role of mucosal IgA needs to be elucidated. IgA can also suppress tumor development in particular tumor types (e.g. colon cancer) for which a correlation between gut microbial homoeostasis and tumor growth has been established [42,127,128].

IgA and IgM can also contribute to a regulatory immunosuppressive response (Type IV, Figure 2) [42,43,75,76,112,116–118,129]. Tregs and regulatory innate lymphoid cells (ILCregs) express both IL-10 and TGF-β and support the development of immunosuppressive plasma cells (ISPC) in a T cell-dependent or -independent manner. We found a subpopulation of ISPCs that produces IgA, PD-L1, and IL-10 and strongly inhibit CTL activation in prostate cancer–bearing mice treated with the immunogenic chemotherapeutic agent oxaliplatin or in non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) [42,43]. Elimination of ISPCs, whose development depends on TGF-β signaling, strongly enhances the immunogenic response to low-dose oxaliplatin, resulting in tumor rejection [42,43]. Additional important features of IgA include that it does not activate the complement system and can mediate the regulatory effects of its main inducer, TGFβ [130,131]. In particular, monomeric IgA exerts inhibitory effects on many immune cell subsets via activation of FcαRI receptors [132,133] and induction of IL10 production [134], as well as by regulating proinflammatory cytokines [135]. Moreover, MDSCs were found to induce the differentiation of B cells into IgA⁺ plasma cells [136]. Recent high-throughput human studies involving B cell repertoire analysis confirmed that high proportions of IgA are associated with elevated cancer risk and poor prognosis in different tumor types, including melanoma, lung adenocarcinoma, bladder cancer, acute myeloid leukemia and HCC [38,51,53,137–139].

3.3. Antibody-mediated cellular cytotoxicity (ADCC)

B cells and humoral immunity have also been described to regulate anti-tumor immunity through antibody-mediated cellular cytotoxicity [140,141]. ADCC is an adaptive immune response largely mediated by NK cells through CD16 (FcγRIII) or FcγRIIC, which bind the Fc portion of IgG antibodies and trigger the lysis of targeted cells by expression of cytokines like IFNγ. Moreover, the positive correlation of IgG antibodies with good prognosis, and response to immunotherapy has been attributed partially to their ADCC capability [30,37,38,51,59]. Many therapeutic antibodies used clinically such as Rituximab (anti-CD20), cetuximab (anti-EGFR) and anti-GD2 attack cancer cells also through an ADCC dependent-mechanism [140–142]. Nevertheless, although tumor-associated Immunoglobulins were detected in the sera of many cancer patients [46,47,143], antibody-mediated cancer cell killing is usually impaired. Notably, antibody effector functions that are mediated by Fcγ receptors are also compromised during persistent infections, an effect attributed to formation of antigen/antibody immune complexes (ICs), suggesting that high concentrations of preexisting ICs can limit the effectiveness of antibody therapy in human cancer [144].

3.4. Interaction with the Complement system

The complement system can regulate B cell differentiation and also be regulated by the humoral immune system. Effective formation of GC requires the presence of T cells and a source of antigen which is retained on the surface of FDC via CD21 (complement receptor 2; CR2), CD35 (complement receptor 1; CR1) and FcR [145,146]. Moreover, antibodies can evoke complement-dependent cytotoxicity (CDC) that culminates in tumor cell elimination and regulate anti-tumor immunity by activating complement system components C5a or C3a [147,148], which can also suppress anti-tumor immunity in a context dependent manner [62,149]. However, the complement system can modulate the TME through a B cell-independent mechanism as well [146].

3.5. Antigen presentation

In addition to their role as antibody producing and secreting cells, B cells can function as professional antigen-presenting cells (APCs) for CD4⁺ T cells by expressing cell-surface major histocompatibility complex class II (MHCII) molecules with bound peptides that serve as α/β T cell receptor (TCR) ligands. MHCII restricted antigen presentation by B cells can play an important role in shaping the immune response, as shown for thymic B cells [150–152]. B cells can capture antigen and uptake to internalize the antigen:BCR complexes with MHCII in peptide-loading compartments, thus generating MHCII:peptide complexes followed by exocytic transport to present these complexes on their surface [150,153–157]. B cells also express co-stimulatory molecules, like CD40, CD80 and CD86. B cells’ ability to act as APC both in LN and TLS can explain the positive outcome of intratumoral B cell and T cell, in particular CD4⁺ T helper cells, cooperation in TLS during immunotherapy (Table 1) [29,30,35,59,60,83,158–160]. B cells can also capture immune complexes by a complement receptor–dependent mechanism from macrophages and transport the complexes to follicular dendritic cells, and later to T zone, which can enhance GC response and T cell activation [161]. However, much less is known about MHCI restricted antigen cross-presentation by B cells to prime CD8⁺ T cells.

Moreover, B cells can also indirectly regulate the priming of T cells through immune complexes (antigen-antibody complexes) harboring IgG-bound tumor antigens, that bind to FcγR expressed by dendritic cells and macrophages, thereby supporting phagocytosis and antigen cross-presentation and T cell priming [162–165].

4. Role of the tumor microenvironment in shaping the B cell repertoire

Regulation of hematopoietic stem cells (HSC) and B cell progenitors self-renewal and differentiation is thought to depend on their microenvironment, also called the “bone marrow” (BM) stem-cell niche [166,167]. Normal hematopoiesis requires a complex and reciprocal interaction between the BM (or fetal liver) microenvironment and HSC/ B cell progenitors [167]. The BM stem cell niche is composed of specialized cells like osteoblasts, responsible for osteogenesis (bone growth) and controlling HSC numbers and stromal cells including sinusoidal endothelial cells, supporting proliferation, differentiation, and transendothelial migration of HSC. B-cells are generated from HSC and develop in the BM. Afterwards mature B-cells migrate into the blood and reach peripheral lymphoid tissues. The development of B-cell precursors through various stages requires a coordinated interaction with BM stromal cells producing different factors in the BM niche, as well as the expression of specific transcription factors, such as Ikaros, E2A, and PAX5. In 1982, Whitlock and Witte described the growth of a B-cell precursor on BM-derived stromal cells in vitro [168]. For B-cell development several microenvironmental components have been identified, such as CXC-chemokine ligand 12 (CXCL12), FLT3 ligand, interleukin 7 (IL-7) and stem-cell factor (SCF) [167,169–171]. Different subtypes of BM stromal cells produce these factors [172,173]. Inflammatory responses in the TME are often accompanied by induction of fibrosis and recruitment of stromal cells like tumor-associated fibroblasts (CAF) and macrophages (TAM). CAFs are responsible for deposition of collagen and various extracellular matrix (ECM) components in the tumor microenvironment, where they stimulate cancer cell proliferation and angiogenesis [173,174]. CAFs and TAMs also have a critical immune function as they produce numerous cytokines and chemokines, including osteopontin (OPN), CXCL1, IL-6, CXCL12, TGF-β, IL-7, IL-1β, TN, iNOS, CCL-5, stromal-derived factor-1α (SDF-1α), and CXCL13, that are decisive for B cell differentiation, CSR, survival and effector functions, as discussed above [50,73,170,174–179]. The proinflammatory properties of TAMs and CAFs are enhanced by mediators secreted by resident immune cells [180], such as IL-1, which regulates and can be regulated by Fc receptors [181]. Activated CAFs and TAMs in the TME produce TGF-β, which inhibits NK cell and CTL activation, induces Treg and supports the IgA CSR [42,43,50,115,182,183]. CAF-derived CXCL13 mediates recruitment of B cells and lymphotoxin expression, which can lead to development of prostate and pancreatic cancers [50,71,184], but can also regulate TLS development and activation of anti-tumor responses [30,59,72,185,186]. Stromal cells and CAFs also secrete IL-6 and orchestrate B cell survival and lymphoid tissue growth and responses [187]. Most importantly, circulating TFH cells were found in tumors and tumor-infiltrating TLS that can regulate the B cell isotype via CSR by producing cytokines and soluble factors like IL-2 [188–192]. However, most tumor-infiltrating T_H/T_FH cells have a regulatory TrFH (IL-10, TGF-β), TH17 or TH2 (IL-4, IL-5) phenotype, which supports the development of immunosuppressive and anti-inflammatory isotypes [193]. IgA⁺ ISPC are derived from naïve B cells that are recruited into the TME by CAF-generated CXCL13 and CXCL12 [43,50]. Upon encounter of BCR-specific antigens, that could be bacterial-, food-, or tumor-derived, and further exposure to TGF-β and other cytokines, including IL-21 from circulatory TFH cells, lymphotoxin β (LTβ), IL-33, and IL-10, naïve IgM⁺ B cells undergo CSR to IgA⁺ plasma cells [42,43,113]. These anti-inflammatory and immunosuppressive effects are in line with the well-established homeostatic and regulatory role of IgA⁺ plasmocytes in mucosal immunity [112,117,194]. In this regard, an immune response coordinated by a pro-inflammatory environment and available immunogenic tumor antigens leads to the generation of enormous plasma cell clones producing high-affinity, tumor-specific IgG1 antibodies, while an immunosuppressive environment leads to B cells switching to IgA with the potential for immunosuppression.

5. Host metabolism and microbiome shape the cancer B cell repertoire

Obesity, aging and alcohol are fundamental supporters of cancer development [161], acting through induction of chronic inflammation, barrier disruption and microbiota dysbiosis [42,195–198]. Hypernutrition, alcohol consumption and aging also regulate the magnitude of B cell activity and effector function, although the underlying mechanisms remain poorly understood and have been attributed to metabolic reprograming, impairment of TFH and changes in cytokine gene expression [42,195,199–203]. Moreover, alcohol consumption, obesity and aging correlate with changes in IgA levels and diversity [204–207], which may further contribute to tumor development [42]. Mucosal or systemic microbiota exposures also shape the B cell repertoire, although its effects differs for each isotype; such as IgG and IgA. Therefore, the regulation of B cells by the microbiome and vice versa can affect the response to immunotherapy [123,208]. The development of B cells, including CSR, SHM and proliferation depends on sophisticated metabolic programing that includes anaerobic glycolysis, oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle and nucleotide biosynthesis. Recently, it was shown that GC B cells rely primarily on fatty acid oxidation (FAO) for proliferation [200,209]. However, it remains unknown how the B cell response is affected in cancer patients with metabolic disorders, and how or if the dysregulation of the B cell response contributes to cancer development and response to therapy.

6. Summary and Future perspective

In summary, recent data have established the crucial and important roles of B cells, plasma cells and antibodies in tumor development, progression and response to therapy. A sustained and long-term protective immune response to pathogens and tumors requires the organization and orchestration of cellular and humoral adaptive immune cells in concert with innate immune cells. A proinflammatory Type I response seems to be the most protective response that inhibits tumor development, supports tumor-rejection, and responds to immunotherapy, which usually includes CTLs, TH1, IgG1⁺ antibodies and TLS. However, the mechanisms by which different B cell types manifest their immunosuppressive and immunosupportive effects remain poorly understood. Future work needs to be done to systematically characterize tumor-infiltrating B cells and the transcription factors that regulate their development and antigen specificity.