Mycobacterium tuberculosis (Mtb), the causative agent of pulmonary tuberculosis (TB), remains the most important pathogen worldwide in terms of accumulated mortality. The World Health Organization has estimated that 10 million new cases of TB and 1.421 million deaths caused by Mtb occurred in 2018 (1). The convergence of the Mtb and human immunodeficiency virus (HIV) epidemics, as well as the lack of new vaccines capable of conferring significant protection against TB have limited the control of this global health treat. Failure to create an effective vaccine for TB has been largely due to an incomplete understanding of the immune mechanisms associated with protective immunity against Mtb. In fact, for many years, the TB vaccine field has established the paradigm that CD4+ T memory cell responses mediated by IFN-γ are the chief immune mechanism which controls the spread of Mtb within the infected lung (2, 3). Despite its relevance, this mechanism has erroneously been considered the sole correlate of protection in TB (4). Moreover, recent findings have raised uncertainty about the protective capacity of IFN-γ-mediated CD4+ T cell memory against Mtb. For instance, T cell epitopes have been demonstrated to be well-conserved in Mtb, suggesting that the pathogen may take advantage of its recognition by T cells (5). Furthermore, recent TB vaccine candidates targeting IFN-γ-mediated T cell functions have failed to provide improved effectiveness compared to the Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine (6). Finally, IFN-γ has shown a poor predictive value in discriminating between subjects receiving BCG vaccination that will receive protection from those that will develop active TB (7). The discussion of the protective capacity of T cell memory responses against Mtb is beyond the scope of the present review, but further evidence has been extensively revised and analyzed by other researchers (8).

Hence, the TB vaccination field would benefit from the exploration of novel correlates of protection and the development of new strategies to disrupt the natural immune responses induced by Mtb to ensure its survival. Recently, some authors have proposed that this goal could be achieved through two complementary approaches: 1) inducing immune memory responses lacking or being strong enough to overcome the characteristics of the natural anti-Mtb immune responses that are beneficial for the pathogen, but with minimal risk of immunopathology, or 2) triggering very early protective responses that prevent the establishment of evasive mechanisms used by Mtb to manipulate the innate immune response (9). A growing body of evidence suggests that these approaches could be achieved by targeting immune cell populations other than T cells (10–13). In particular, it has been increasingly accepted that B cells actively participate in anti-Mtb immunity, either as secondary actors providing support and shaping the quality of T cell-memory responses, or as protagonists mediating direct effector functions against Mtb (14). Similarly, different subpopulations of innate immune cells that possess a previously unrecognized capacity to mount secondary “memory-like” responses are equally capable of limiting Mtb growth (11, 15). Therefore, in this review we summarize the memory responses of innate immune cells and B cells against Mtb and analyze how their functions may constitute novel correlates of protection that can be potentially harnessed for TB vaccine development.

As mentioned before, the study of the mechanisms underlying immunity to Mtb infection has focused on immunological memory mediated by adaptive immune cells, mainly CD4+ T helper lymphocytes. However, human studies have shown that up to a quarter of the individuals that are in close contact with active TB patients remain clear of the infection (16). These individuals test negatively in the purified protein derivative (PPD) skin test and IFN-γ release assays (IGRAs) (16, 17), which are two indirect readouts of adaptive responses against immune-dominant Mtb antigens. A positive BCG vaccination history has been associated with this state of immune protection (16). This suggests that in such “resistant” close-contacts, innate immune responses potentiated by BCG vaccination are sufficient to restraint Mtb infection.

Notably, recent investigations have uncovered the role of “trained immunity” in the pathogenesis of different infectious diseases. This concept describes an enhanced capacity of innate leukocytes to respond to secondary challenges by the same or by unrelated microorganisms after an initial antigenic exposure (18). The first hints of trained immunity induced by mycobacterial components were observed in mice treated with muramyl dipeptide (19), Freund's complete adjuvant (20), and BCG/PPD (21). Mycobacterial component preparations conferred protection against Klebsiella pneumoniae, foot and mouth disease virus (FMDV), and Candida albicans infection, respectively. Interestingly, protection from FMDV was independent of neutralizing antibodies (20), whereas control of systemic candidiasis occurred before the development of delayed-type hypersensitivity (21). Similarly, BCG exposure was shown to limit Schistosoma mansoni infestation in nude mice, suggesting a role for trained immunity in BCG-induced cross-protection (22). In humans, BCG vaccination conferred heterologous immunity against lower respiratory tract pathogens (23, 24), yellow fever virus (25), and neonatal sepsis (24). Furthermore, BCG vaccination was demonstrated to induce protection against intestinal parasites (26), even in individuals infected with HIV, suggesting that innate immune cells mediate such cross-protection.

Epigenetic changes that facilitate the expression of inflammatory genes and generate metabolic reprogramming are responsible for inducing trained immunity against mycobacterial and other microbial components. In these epigenetic changes, long non-coding RNAs participate in the formation of chromatin loops that bring several genes into a close spatial relationship, making these genetic regions more accessible to the enzymatic complexes responsible for epigenetic priming. Three cellular subsets appear to be involved in the trained immune response against Mtb: monocyte-macrophages, myeloid cell precursors, and innate lymphoid cells (ILCs), these latter including natural killer (NK) cells (18).

The role of B cells in the defense against pathogens greatly relies on their capacity to generate immune memory, providing the host with a durable reservoir of antigen-specific antibodies, as well as a pool of long-lasting antibody-producing cells that can re-expand in case of a secondary challenge. During a primary response to an antigen, naïve B cells may transform into effector B cells and subsequently experience class switching, recombination, and affinity maturation within germinal centers (GC), with the support of follicular T helper (Tfh) cells. This GC reaction is a common pathway that gives rise to memory B cells (MBCs) and plasmablasts. MBCs secrete null amounts of antibodies but in case of a re-encounter with the same pathogen, they can generate plasmablasts within hours. Plasmablasts are B cells capable of secreting reduced amounts of antibodies and more importantly, are precursors of plasma cells (PCs), short-lived cellular units that use their protein machinery for the production and secretion of massive amounts of antigen-specific antibodies. PCs are unable to proliferate without antigenic stimulation; however, a selected minority of PCs can generate anti-apoptotic processes and migrate to the bone marrow, thus becoming long-lived PCs (LLPCs, also termed memory PCs). These LLPCs survive without antigenic stimulation and continuously liberate antigen-specific antibodies to the circulation (108, 109).

For many years, the role of antibody-mediated B cell memory responses in TB has remained controversial (110). However, several studies have demonstrated the importance of this response in combatting this disease. In fact, elevated serum titers of PPD-specific antibodies correlate with protection against TB in high-risk individuals and are a better indicator of LTBI than the skin test reaction (111). Such antibodies induce the stimulation of PBMCs in vitro, suggesting that after biding to its specific antigen, these immunoglobulins can trigger effector responses of different immune cells. In line with these findings, it has been recently described that the antibodies from LTBI subjects have an increased capacity to bind to the FcγRIII and trigger the effector activities of NK cells and macrophages (10). This functional difference is associated with distinctive patterns of glycosylation of the Fab domain of antibodies from LTBI patients compared to individuals with active TB (10). Functional humoral responses have also been linked to the development of sterilizing immunity in individuals resistant to TB. In such resistant subjects an IFNγ-independent response mediated by CD4+CD40L/CD154+ T cells induces T-follicular B cell help and the production of several non-Th subset-specific cytokines critical for B cell activation (112). Furthermore, in a recent study, the humoral response of this human population was characterized by an IgG1-dominant state specific to ESAT6/ CFP10, LAM and PPD. Conversely LTBI individuals displayed a diversified IgG subclass response (112). Additionally, antibodies from individuals with LTBI can restrict Mtb intracellular growth in a more successful fashion compared with antibodies from patients with active TB (113). Whether antibodies contribute to the “resistant” phenotype observed in some individuals remains an undetermined matter at this moment, however it is currently known that post-vaccination serum favors Mtb phagocytosis by macrophages, enhances phagolysosome fusion and inhibits intracellular Mtb growth (113). Furthermore, pooled IgG from LTBI individuals may eradicate intracellular bacilli through a process that likely involves the inflammasome (114). These findings suggest that antibody-mediated B-cell memory responses play a role in the defense against Mtb infection and may be targeted to induce protective immunity through vaccination. Unfortunately, the dynamics of the antibody mediated immune responses (AMIR) during TB progression remain insufficiently characterized. Nonetheless, as MBCs are not only located in lymphoid organs but can also be found in peripheral blood, some studies have examined the kinetics of circulating MBC populations in patients with distinct forms of TB.

Some preliminary findings suggest that the proportion of distinct MBC subsets may predict the clinical status of Mtb-infected patients (Figure 2), arguing in favor of a role of B cell memory in TB (108, 115–119). For instance, MBC, plasmablasts, and PCs subsets are significantly enriched in the circulation of patients with active TB, and their proportions diminish after the conclusion of anti-TB treatment, suggesting that these B cells subsets undergo a contraction phase after antigen clearance (119). Indeed, the maintenance of MBCs in circulation is dependent on the presence of Mtb antigens and can be eliminated after 12 weeks of antibiotic treatment (116), but serum IgG levels remain augmented even after a 6 month course of antibiotic therapy in TB patients (119). Thus, during active TB, the presence of the pathogen may trigger the generation of high-affinity antibody-mediated memory responses to Mtb antigens. Plasmablasts can readily induce class-switched and cytokine-producing PCs upon antigen exposure or re-exposure if they are derived from naïve B cells or antigen-experienced MBCs, respectively (14, 119). These responses may reduce Mtb burden in active TB patients and curb their disease outcome, as well as maintain a LTBI status. In fact, it was demonstrated that PCs derived from peripheral blood cells of LTBI subjects produce significant amounts of antibodies and IL-17 when exposed to Mtb antigens (115). Thus, the dual presence of MBCs and plasmablasts in the peripheral blood has been proposed as a marker of resolving active disease and LTBI. On the other hand, the presence of activated antigen-specific plasmablasts but not MBCs in the peripheral circulation may reflect the initial stages of active TB and the lack of both MBCs and plasmablasts in circulation may indicate uncontrolled infection (118). Particularly, non-switched IgD+ MBCs have been found reduced in distinct tissue compartments during advanced active TB (120, 121). Other studies have found that higher proportions of circulating MBCs but not plasmablasts in peripheral blood along with a prominent serum antibody-mediated memory response is indicative of a healthy condition after sterilizing immunity following Mtb infection (118). This coincides with the observation that healthy subjects who have resided in TB-endemic areas have greater frequencies of peripheral blood MBCs and antibody-mediated responses compared to subjects from other world areas (122). Therefore, although MBCs might contract after Mtb control, they remain preconditioned to mount secondary responses in case of reinfection.

The role and dynamics of LLPCs and marginal zone (MZ) B cells during TB is still under investigation. Recently, it was observed that BCG vaccination can elicit the generation of PPD-specific LLPCs in humans (122). Additionally, another study found that LLPCs are important contributors of cytokine production during LTBI (115). MBCs and LLPCs generate the AMIR through the production of IL-21 and the subsequent induction of a B cell maturation loop involving the formation of PCs (108, 116). Preliminary evidence suggests that this response is crucial for controlling Mtb infection (108, 116). The role of MZ B cells during TB has not been adequately characterized and inconsistencies have arisen between mouse and human studies (121). These cells are normally activated by T cell independent antigens and have innate-like B cell memory characteristics. MZ B cells contribute to the AMIR through the production of IgM and IgG3 antibodies (117). Interestingly, a pilot study indicated that MZ B cells are present in the peripheral circulation during active TB at significantly lower levels compared to healthy controls (116).

Beyond antibody production, B cells play a crucial role in regulating innate and adaptive immune responses to infectious agents even in disease states dominated by T lymphocytes, as is the case of TB. B cells impact cellular adaptive immunity and are necessary for the adequate activation and maturation of memory T cells (123–125). This regulation is achieved through direct interactions between B and T cells that occur during both primary and secondary antigen responses. Such interactions are varied and include antigen presentation, co-stimulatory signaling, cytokine priming and antibody-mediated effector functions, as shown in Figure 3. In fact, B cell-deficient mice and human patients receiving B cell depletion therapy generally present alterations in their CD4+ T cell and CD8+ T cell repertoires (126). However, B cells exert contrasting effects on T cell responses. For instance, B1 and B2 cell subsets are capable of producing IFN-γ, TNF-α, IL-12 or IL-2, IL-13, and IL-4, thus inducing Th1 or Th2 immune responses, respectively. Furthermore, B-cells also display different regulatory phenotypes capable of generating Th10 responses via IL-10, TGF-β, IL-35, FasL, or PD-L1. Besides T cell activation, B cells also regulate T cell proliferation and contraction during acute immune responses and participate in the maintenance and reactivation of T cell memory responses via antibody-independent mechanisms (127). Most of these B cell functions have not been adequately characterized during TB, although it is currently known that B cells can get infected by Mtb via micropinocytosis (128), and B cells can produce cytokines in response to infection with Mtb in vivo (129).

In addition, B cells are also crucial for the induction and maturation of antigen-presenting environments (APEs) and APCs (127). In TB, B cells participate in the formation of granulomas and play a pivotal role in the development of TLNs (Figure 3), two APEs that are critical for Mtb control (123, 124). As such, B cell-deficient mice infected with Mtb display disrupted lung granuloma and TLN formation (34). Moreover, the production of cytokines and antibodies by B cells contributes to set up activation thresholds for macrophages, DCs and other non-professional APCs within and outside APEs during TB (123), which subsequently will determine the quality and dynamics of effector and memory T cell responses. In this regard, during active pulmonary TB, humans, mice, and non-human primates develop TLNs in the surroundings of granulomas. These TLNs assume the structure and function of GCs containing B cells with different maturation profiles, Tfh-like CXCR5+ cells, and follicular DCs (34, 130). Additionally, pulmonary TLNs facilitate the interaction between these and other immune cells and become the anatomical foci that possess the highest levels of cellular proliferation during active TB. The presence and organization of these TLNs is associated with immune protection and LTBI, and their absence or disorganization may lead to uncontrolled progression of active TB (34).

Importantly, the possibility that Mtb might manipulate B cell and B cell memory responses exists, as B cells are susceptible to multiple effects of the bacteria including their direct infection via micropinocytosis (128). For instance, tissue-like memory B-cells (TLMs) are increased in the blood of patients with active TB and reduced in healthy controls (Figure 2). TLMs constitute a population of MBCs, also known as exhausted B-cells, that express low levels of CD21 and CD27 and increased levels of inhibitory receptors, having a diminished capacity to respond to antigenic stimuli. The presence of these cells in the circulation is associated with adverse clinical outcomes in chronic viral infections and is likely associated with active TB as well (128). Additionally, a population of B CD27-/IgD- memory cells has been shown to be increased in the pleural fluid of patients with tuberculous pleurisy as compared to healthy subjects. In these patients, pleural B cells interfered with the protective production of IFN-γ by NK cells and T lymphocytes (121). Collectively, these data indicate that targeting B cell functions for vaccination purposes in TB must be conducted carefully to avoid possible detrimental effects to the host.

Humoral responses characterized by the production of high-affinity neutralizing antibodies have been the main target for the development of vaccines that confer protective immunity against several bacterial pathogens (108). Currently, the exception to the rule seems to be TB, as most of the TB vaccine candidates that are being evaluated in clinical trials are designed to exploit cell-mediated immunity rather than humoral immunity (131). Despite this, the evidence reviewed here suggests that both arms of the adaptive immune response should be evaluated in vaccine-development programs. Induction strategies should attempt to provide the host with a pool of antigen-specific MBCs capable of producing high-affinity neutralizing and polyfunctional antibodies which can potentiate macrophage and NK cell effector functions. Antibody-mediated activation of innate immunity may contribute to prevent the establishment of an immune microenvironment manipulated by Mtb. Vaccines targeting B cell responses can also promote the cooperation between B and T cells necessary for efficient T cell memory responses. For example, in a preclinical mouse model, the adoptive transfer of B cells that had internalized the naked plasmid pcDNA3 encoding the Mycobacterium leprae 65-kDa heat shock protein into B cell deficient mice resulted in the establishment of strong CD8+ T cell memory responses that conferred protection against Mtb challenge (132). Furthermore, novel ways to promote the formation of protective TLNs within the lungs deserves further investigation. Finally, the fact that a robust MBC response can be induced in the adjacent mesothelium upon primary mucosal Mtb exposure advocates in favor of using novel routes of vaccine administration (119, 121).

On the other hand, the memory-like properties of innate immune cells must also prompt us to discover novel ways to potentiate protective long-lasting anti-TB innate immune mechanisms. These strategies must aim to provide the host with a pool of primed long-lived innate cells with enhanced capacity to directly respond to the invading pathogen and with an increased adjuvant activity to support the correct development of effective T-cell memory responses. For instance, targeting trained immunity in myeloid cells may provide the host with metabolically and epigenetically reprogramed phagocytes ready to act before the contact with Mtb. Furthermore, memory-like AMs could initiate the early formation of protective granulomas around the sites of Mtb exposure, while the enhancement of ILCs activity could promote the establishment of an early immunological microenvironment advantageous for the host. Finally, Mtb-induced memory-like NK cells may serve as innate effector cells with increased cytokine production and cytotoxic activity. Targeting innate immunity in TB vaccination strategies may benefit a significant proportion of patients co-infected with Mtb and HIV by conferring them with T-cell independent protective immunity. HIV/AIDS remains the leading comorbidity in TB, and according to the 2019 WHO TB report, a third of HIV-related deaths were due to TB (1).

In conclusion, we must think outside the box and look for novel protective immune responses against Mtb beyond T cell memory responses. Future investigations in the TB field warrant the study of mechanisms implicated in trained immunity and B cell memory, as well as the discovery of routes and adjuvants for TB vaccine administration that may potentiate these and other overlooked protective immune responses against Mtb infection (131, 133).

JC-P and LW designed the study and drafted the manuscript. EY, JZ, and RH-P contributed in the writing process of the manuscript and revised it for intellectual content. All the authors revised and approved the final version of the manuscript.