The programed death-1 (PD-1) receptor is a member of the CD28 superfamily that negatively regulates T cell responses to antigen stimulation and enhances tolerance, triggered by interaction with its ligands PD-L1 and PD-L2 (52, 53). PD-1 is expressed on Tregs, natural killer (NK) cells, follicular T and B cells, and APCs (53). PD-1 has recently emerged as an important co-inhibitory molecule and “immune checkpoint” in a number of chronic intracellular infections, including TB, and overexpression of PD-1, cytotoxic T-lymphocyte antigen (CTLA-4) receptor, and TIM3 has been associated with T-cell exhaustion (54–56). In human TB infection, PD-1 activity induces Tregs and immunomosuppressive cytokines such as interleukin (IL)-10 and TGF-β (57, 58). HIV–TB coinfection is associated with increased expression of PD-1 on Mtb-specific cytokine-secreting CD4+ cell subsets (59).

Checkpoint blockade, i.e., removal of inhibitory coreceptor initiated signaling, is a strategy used successfully in cancer treatment for vaccine potentiation. Monoclonal antibodies (mAbs) that modulate inhibitor signaling by CTLA-4 and PD-1, e.g., ipilimumab, pembrolizumab, and nivolumab, are now in wide clinical use for several cancers (Table 1) (60). Combination of checkpoint blockade with PD-1 and CTLA-4 inhibition enhances response rates in patients with minimal responses to single checkpoint blockade with lesser toxicity compared to previous immunotherapies, e.g., IL-2 (60, 61). PD-1 blockade with the cancer vaccine TEGVAX in mice showed regression of established tumors, providing a rationale using vaccines combined with immune checkpoint blockade (62).

The overall role of PD-1 blockade in TB remains a complicated issue. PD-1-deficient mice have shown dramatically reduced survival compared with wild-type mice post-Mtb infection. Increased levels of pro-inflammatory cytokines and uncontrolled bacterial proliferation were seen in the lungs of these mice (63). These observations indicate that control of Mtb infection requires a carefully balanced immune response that contains the infection without causing pathology (64).

Interestingly, the specific role of PD1–L2 is currently of interest as a therapy for malaria. Blockade of PD1–L1 binding resulted in unfavorable outcome in a malaria mouse model, but blockade of PD1–L2 did not, suggesting different functions of the two PD-1 ligand functions in malarial infections (65). Administration of soluble multimeric PD-L2 outcompeted PD-L1 for PD-1 binding and improved CD4+ T cell responses in mice with lethal malaria, dramatically improving survival (66). The results also suggest that PD-L2 not only competes with PD-L1 but also has a stimulatory action via interaction with a different coreceptor.

Additional studies are needed to further investigate these issues that might allow tuning of TB vaccine responses to be more effective. Energy supply insufficiency due to cell metabolic alterations directed by PD-1 signaling leads to exhaustion and begins early in CD8+ T cell responses in chronic lymphocytic choriomeningitis virus infection (67). Downregulation of the glycolytic and peroxisome proliferator-activated receptor c coactivator 1α (PGC-1a) pathways leads to reduced mitochondrial energy availability. Additionally, mTOR signaling drives anabolic metabolism of effector T cells, exacerbating energy deficiency, further contributing to cell exhaustion. The reversion of these changes in a subset of exhausted CD8+ T cells by anti-PD-1 therapy suggests that targeted agents to directly inhibit these metabolic changes should be explored (67). The nuclear factor of activated T cells (NFAT) proteins is also primary in the establishment of T cell exhaustion in mouse models of tumor growth and bacterial infection (68). During persistent antigen stimulation, NFAT promotes T cell exhaustion by binding directly to regulatory regions of PD-1 and TIM3 (68). Targeting NFAT isoforms or their associated signaling molecules is being actively pursued in cancer therapy (69).

The various checkpoint co-inhibitors may need to be combined with T-cell co-stimulators (or stimulators could be used alone), for effective CD4+ Th1 and CD8+ T-cell development and subsequent sustained T-cell memory responses (70). CD134 (OX40), a TNF-α receptor superfamily member, is expressed transiently on newly activated T cells and NK cells (71, 72). CD134–CD134 ligand interactions are crucial for the expansion of effector T cells and generation of memory T cells (73). OX40 agonist mAb administration results in a Th1-cytokine-driven response enhancing IL-2 production by effector T cells and preventing the expression of CTLA-4, FoxP3, and IL-10 (71, 74). Compared with BCG vaccination alone, OX40 ligation with BCG vaccination in a murine model provided enhanced protection against aerosol and intravenous Mtb challenge with NK cells playing a crucial role (71). In cancer therapy, the beneficial results of combination immunotherapy targeting both costimulatory and co-inhibitory molecules (combined OX40/PD-1 or OX40/CTLA-4 mAb therapy) dramatically improved survival in the prostate, ovarian carcinoma, and sarcoma models (70, 75). The adjunct potential of OX40 agonists, in conjunction with CTLA–4 mAb, has been shown for infection by the intracellular bacterium, Leishmania donovani (76). The role of activation of stimulatory coreceptors to improve vaccine responses deserves further investigation.

Th1-type immunity is downregulated by a subset of immunosuppressive CD4+ T cells expressing CD25 and the transcription factor FoxP3, referred to as Tregs (77–79). As excessive inflammation related to highly activated populations of CD4+ T cells can cause tissue damage and exacerbate TB disease, a critical balance of regulatory and effector T-cell responses govern the outcome of infection (80, 81). Tregs suppress both T cells and APCs, thus enhancing persistent infection (79, 82–84). Expansion of Tregs in active TB has been observed both at organ-specific sites and in blood (80, 85, 86). Multiple studies suggest Tregs delay the arrival of potential effector T cells into the pulmonary lymph nodes during early infection, play a role in reactivation of latent infection, and have a positive correlation with bacterial burden and extent of active disease (80, 81, 87, 88). Contrasting results have been observed in the TB treatment phase, with some studies reporting declining levels of Tregs and others showing a transient increase (86, 89, 90).

Myeloid-derived suppressor cells are another group of regulatory cells controlling TB inflammation. MDSCs are a diverse population of monocyte, granulocyte, and DC precursors showing features of immune suppressors (91). Similar to macrophages, polarized MDSC lineages can be distinguished as M1 and M2 cells (92). Their numbers are expanded significantly in cancer and in pleural effusions and blood of TB patients (93, 94). MDSCs have been shown to become infected by Mtb, accumulate within the inflamed lung, interact with granuloma-residing cells, and decrease in number after TB chemotherapy (95, 96).

Studies of Treg suppression agents in combination with TB vaccination have yielded unclear results. A heterologous BCG/DNA-heat shock protein (HSP) 65 vaccination regimen was protective, and the effect was associated with lower numbers of Tregs in murine lungs (97). Concurrent inhibition of Th2 cells and Tregs by using the small molecule inhibitors Suplatast tosylate and D4476, respectively, during BCG vaccination improved vaccine efficacy in mice, by enhancing Mtb clearance, induction of superior Th1 responses, and long-lasting protective central memory T cell responses (98). However, reduction of Tregs using anti-CD25 antibody prior to BCG vaccination and an experimental ESAT-6 subunit TB vaccine administered concurrently with IL-28B that downregulates Tregs did not enhance the protective effect in mice models, suggesting a need to refine Treg inactivation strategies (79, 99).

Table 1 lists several other agents being tested for their regulatory cell modulation properties including damage-associated molecular patterns (DAMPs), inducible indoleamine 2,3-dioxygenase (IDO), TK, phosphodiesterase-5 (PDE5) and fatty acid oxidation (FAO), and arginase inhibitors.

Damage-associated molecular patterns (also known as alarmins) are released by dying or stressed cells and include HSPs, S100, and high mobility group box (HMGB1) proteins (111). Expansion of both Treg and MDSCs in cancer involves the participation of DAMPs (112). Interestingly, HMGB1 was evaluated as an adjuvant for a TB subunit HMGB1–ESAT-6 fusion protein vaccine in a mouse model. Poly-functional CD4 T cell-mediated immune response generated by the fusion protein vaccination correlated with protection against subsequent Mtb challenge (113). Tasquinimod, an S100A9 inhibitor currently in phase III trials for prostate carcinoma, decreased accumulation and activity of MDSCs with enhanced CD8+ responses in different combination immunotherapeutic strategies including a tumor vaccine in murine cancer models (114, 115). The potential use of DAMPs and DAMP inhibitors in TB vaccination strategies needs to be resolved.

Indoleamine 2,3-dioxygenase blocks T cell proliferation and modulates immune responses to Mtb (116–118). Increased IDO expression is strongly associated with poor outcome in many cancers and is emerging as new target for potentiation of cancer vaccines (119, 120). Similarly, reduced immunogenicity seen following MVA85A vaccination in South Africans compared to UK subjects has been attributed to increased baseline IDO activity (118). Use of IDO inhibitor drugs like epacadostat that significantly decrease Treg proliferation and increase CTL activity in vitro provide rationale for improving vaccine responses by combining with IDO inhibitors (121). A first-in-human, phase I clinical study, combining ipilimumab (anti CTLA-4) with a single-epitope peptide vaccine targeting IDO showed detectable T-cell responses in a subset of metastatic melanoma patients (120). New strategies targeting the IDO enzyme in cancer, either through vaccine components directed against IDO epitopes or silencing the IDO gene using small hairpin RNA plasmids, are some of the strategies with potential application in the context of TB vaccination (118, 122).

Tyrosine kinase inhibitors can also reduce the Tregs and MDSCs (123, 124), probably through multiple mechanisms, including cKIT inhibition. Sunitinib, currently used in cancer therapy, depletes MDSCs and synergized with a cancer vaccine to enhance antigen-specific immune responses and tumor eradication in mice (125). Similarly, dasatinib exhibits modest single-agent clinical efficacy but showed superior efficacy associated with reduced levels of MDSC and Treg populations in a combination treatment regimen with a DC vaccine candidate in a murine melanoma model (126). Imatinib, used as an approved therapeutic for chronic myelogenous leukemia, reduced bacterial load and associated pathology in mice infected with Mtb and acted in a synergistic manner with anti-TB drugs (47). Several mechanisms are possible for this effect, including inhibition of suppressor cells, and imatinib is being considered for study as a vaccine adjunct for infections.

Adenosine monophosophate-activated protein kinase (AMPK) is an energy sensor kinase that regulates cellular energy homeostasis and acts as a negative regulator of inflammation (127–129). AMPK-dependent control of T cell metabolism (as an immunometabolic checkpoint) is a crucial contributing factor to determine T cell development and effector responses. AMPK also has an essential role in memory T-cell differentiation by regulating the metabolic switch from primarily aerobic glycolysis to oxidative phosphorylation of lipids (129, 130). During Mtb infection, AMPK activation enhances autophagy, leading to phagosomal maturation and improved antimicrobial response (107, 129, 131).

The antidiabetic drug metformin (MET) is an AMPK activator that inhibits intracellular Mtb growth, restricts disease immunopathology, and induces expansion of Mtb-specific effector and memory T cells (Table 1) (128). Additionally, MET-treated diabetic patients with LTBI have increased numbers of Mtb-specific T cells (128). Although issues of dosage and incorporation into current TB treatments remain, MET is under consideration for evaluation as an adjunctive agent for TB treatment (132). Therapeutic cancer vaccination combined with MET improved tumor-infiltrating lymphocytes’ multifunctionality and protection from apoptosis and exhaustion (133). Additionally, MET is able to block M2-like macrophage polarization (partially through AMPK) in cancer. The Notch signaling pathway is a potentially important mechanism in this polarization (134, 135).

Silent information regulator 2 proteins or sirtuins (Sirts) are seven NAD-dependent protein deacetylases involved in stress adaption, cell survival, aging, and apoptosis (142–144). Sirts, specifically Sirt1, Sirt2, Sirt6, and Sirt7, are also emerging as important players in epigenetic changes (145). Sirt1 activates the key autophagy proteins ATG5, ATG7, ATG8/LC3, and Sirt1 and Sirt2 have anti-inflammatory roles, such as inhibition of NF-kB signaling and NLRP3 inflammasome activity (146–149). Sirt1 has recently emerged as a key regulator in immune regulation modulating both innate and adaptive responses.

Sirt1 inhibitors (Table 1) are able to block virus infections including HCMV, influenza A virus, and adenovirus Ad5 (150). In a tumor microenvironment, Sirt1 deficiency impacted immune suppression by switching MDSCs to a pro-inflammatory M1 phenotype through HIF-1α-dependent glycolysis (92). This suggests that Sirt1 inhibitors should be considered for evaluation as vaccine adjuncts. Conversely, the enzymatic activity of Sirt1 can be enhanced by small molecule activators including the natural polyphenol, resveratrol, and synthetic Sirt1 activator compounds (151). In respiratory syncytial virus infection, Sirt1 promotes an effective antiviral response through DC cytokine secretion and autophagy-mediated processes, providing an option for a novel vaccination strategy, such as a Sirt1-activating viral vaccine (152).

Sirtuins have multiple effects on immune regulation based on integration of immune cell metabolic and immune function regulation including potentially modulating T cell exhaustion. Sirt1 activates PGC-1a and negatively regulates NFAT, mTOR, and HIF-1a activity (149, 153, 154). This effect could reduce metabolic cell stresses and improve the effectiveness of PD-1 blockade therapy. Overall, the role of Sirt family in Mtb infection and its potential combined use with vaccination requires further exploration.

The canonical Wnt/β-catenin pathway plays an important role in the regulation of various cellular processes, including cell differentiation, apoptosis, polarity, motility, embryonic development, adult tissue homeostasis, and carcinogenesis (155–158). Infection with BCG activates Wnt/β-catenin signaling, resulting in the expression of a host of genetic signatures important for subsequent regulatory responses, including cyclooxygenase (COX)-2, suppressor of cytokine signaling-3 (SOCS-3), and Jagged1 (ligand for Notch1 signaling) (159–162). Additionally, wnt/β-catenin signaling in DCs induces production of various anti-inflammatory cytokines that induce Treg cells and suppress TH1/Th17 responses (163).

The wnt/β-catenin pathway has been suggested as a therapeutic target for adjunct treatments along with vaccination in the ovarian tumor microenvironment (164). Agents for targeting the wnt/β-catenin pathway include flavonoids, monensin, and resveratrol, and the use of antibodies against the Notch receptor or ligand (Table 1) (165). Additionally, telomere protection enzymes, the tankyrase (Tnks) subfamily of PARPs, control transcriptional response to secreted Wnt signaling molecules. Tnks inhibitors are currently being developed as therapeutic agents for targeting Wnt-related cancers (166). Despite recent reports of studies implicating Wnt signaling pathway in the development and progression of TB, the utilization of potential signaling inhibitors as effective adjuncts in TB vaccination remains to be explored.

Notch signaling regulates various developmental stages of T and B cells and T cell activation and differentiation (167). This highly conserved pathway also integrates further inputs from other signaling pathways. This cross talk includes upregulation of ligands or receptors (e.g., Wnt regulating Notch ligand levels) and co-regulation of shared target genes [e.g., Hes1 and Gsk3β regulation by Notch, Wnt, and sonic hedgehog (SHH)] (168, 169). Notch signaling directly controls PD-1 transcription in activated CD8+ T cells, as blocking Notch signaling leads to the inhibition of PD-1 expression (170). BCG infection of murine macrophages induced upregulation of Notch1 expression. Activation of Notch1 signaling was demonstrated in granulomatous lesions in brains of humans with TB meningitis in comparison to healthy individuals (167, 171). Several notch inhibitors, mainly gamma-secretase inhibitors, are now in clinical trials, e.g., RO4929097 (Table 1) (172).

Sonic hedgehog, a pleotropic member of the hedgehog family of signaling molecules, plays a key role in cellular homeostasis and development (173). Aberrant activation of the HH signaling pathway has been implicated in the pathogenesis of several types of cancer (174). SHH signaling constitutes one of the networks that macrophages use to tailor differential immune responses to infections with pathogenic mycobacteria. In addition to BCG triggering a robust activation of SHH signaling in macrophages, SHH signaling is heightened in humans with PTB and TBM (175). Additionally, TNFα-driven SHH signaling downregulates M. bovis-specific TLR2 responses through micro RNA-31 and -150 targeting MyD88 (175). These changes lead to modulation of a battery of genes that regulate various functions of macrophages genes, including SOCS-3, COX-2, MMP-9, and M1 and M2 genes and emphasize a novel role for SHH signaling in host immune responses to mycobacterial infections. Vismodegib and sonidegib are small molecule inhibitors targeting the SHH pathway in cancer (Table 1) (174, 176).

The binding of Wnt ligands secreted from various cell types to transmembrane receptors of the Frizzled family activates a signaling cascade resulting in the inhibition of a negative regulator of β-catenin levels, glycogen synthase kinase-3β (GSK3β) (177). GSK3β plays a pivotal role in regulating many cellular functions, including cell survival and apoptosis (165, 178–180). M. bovis BCG induces phosphorylation of GSK3 by the PI3K–Akt signaling pathway, switching the transcriptional activity to an anti-inflammatory CREB-driven cytokine response instead of the pro-inflammatory NF-κB transcription (181, 182). GSK3β is an upstream kinase regulating PD-1 transcription, and the use of GSK3β inhibitors in vivo downregulates PD-1 and enhances CTL clearance of viral infections (183). Several inhibitors of GSK3β are in various stages of development, e.g., tideglusib (Table 1) (184). Additionally, in a DC-based tumor vaccination murine model, inhibition of GSK3β by siRNA enhanced CTL activity via suppression of IDO expression (185).