Modulation of host antigen presentation pathways by M.tuberculosis

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Modulation of host antigen presentation pathways by M.tuberculosis

M.tuberculosis employs various complex and highly evolved mechanisms to shift the delicate host-pathogen equilibrium in favour of the pathogen {15767567; 19563525; 20234378; 11244032}. Alteration of the host antigen presentation pathway is one of the main survival strategies used by M.tuberculosis to survive in the hostile milieu it encounters inside the host cells {19563525}. Since the T cell repertoire of the host does the central role in controlling M.tuberculosis infection, the pathogen has evolved mechanisms to modulate and interfere with the macrophage mediated antigen presentation to T cells leading to immune evasion {19302046}. Presentation and subsequent recognition of various loaded antigen presenting molecules especially through MHC ll molecules to CD4+ T-cell population induces cell activation and successive secretion of various cytokines leading to the enhancement of macrophage microbicidal functions and recruit pro-inflammatory leukocytes{16040149}(1)(1). IFN-γ and TNF-α are the important CD4+T cell derived cytokines that activate macrophages to produce NO and stimulate phago-lysosme biogenesis contributing to T-cell immunity to M.tuberculosis {20234378; 11179363; 7713566}. CD4+T cells was shown to kill M.tuberculosis infected targeted cells through the expression of Fas ligand and communicate with CD8+T cells and γδ T cells, which are important for the control of infection{19563525; 20234378; 11287139}. In addition, Srivastava et al recently showed that direct recognition of M.tuberculosis infected cells by CD4+T cells is required for the control of the infection in vivo{ 23817429}(2)(2). Considering the significant role of CD4+T cell activation in controlling M.tuberculosis infection, here we focus on various strategies used by M.tuberculosis to modulate MHC II mediated antigen presentation to T cells leading to immune subversion.

Macrophages and other antigens presenting cells alert and activate T cell repertoire to the presence of pathogens through recognition, ingestion and subsequent presentation of antigens. Antigen presentation of intracellular pathogen like M.tuberculosis is complex and it involves a series of highly organized events including the identification, uptake and delivery of the pathogen to the pertinent intracellular compartments followed by the enzymatic processing and loading of the processed antigen to antigen presenting molecules{ 6228574; 12776207}. The loaded antigen presenting molecule then gets exported to the cell surface where it presents to the appropriate T cell subsets {19563525}. Many points of this highly integrated pathway are interfered by M.tuberculosis to prevent its antigens from being presented efficiently {15767567}. Monocytes and macrophages infected with M.tuberculosis exhibit defective antigen processing and presentation ability {9794422; 10805975; 8039918; 23928171} and it is attributed to the multiple highly evolved mycobacterial mechanisms to gain the survival advantage over the host. By using a mathematical model of antigen presentation in macrophages, Stewart et al showed that modulating multiple cellular processes may serve as an optimal strategy for M.tuberculosis to maintain continuous inhibition of antigen presentation {15767567}. (3). In order to combat with these M.tuberculosis imposed modulation of antigen presentation strategies, the host is known to counteract by transferring soluble, unprocessed antigens to naive dendritic cells for effective presentation {24922576}.

Mechanisms of subversion of antigen presentation by M.tuberculosis

A. Modulation of MHC II expression

MHC II restricted CD4+T cells play central role in the containment of M.tuberculosis infection in humans and experimental animals, and several studies have reported that M.tuberculosis infected macrophages have diminished MHC II expression, decreased antigen presentation and altered CD4+T cell priming{19302046; 10934223; 11157048; 25000593 }. It is generally observed that direct modulation of MHC II expression by M.tuberculosis is more effective at inhibiting antigen presentation even though it shows a delay of at least 10 hrs than targeting other mechanisms involved in the presentation pathway because these mechanisms may be attenuated under certain conditions {15767567}. Considering the importance of MHC II pathway in controlling the infection , M.tuberculosis target and subvert this pathway specifically by various means to ensure its effective intracellular containment and subsequent survival.

Viable M.tuberculosis is not absolutely essential for MHC II inhibition, which can be achieved by the addition of M.tuberculosis lysate to macrophages {20234378}. Several mycobacterial lipoproteins like LpqH{11441098} LprG{15294983}, and LprA{16785538} were found to be the key components that inhibited MHC II expression. These lipoproteins are TLR2 agonists and excessive or prolonged TLR2 signalling induced by these M.tuberculosis lipoproteins results in the inhibition of MHC II molecules. Among these, the highly characterized LpqH (Rv 3763) codes for a major triacylated exported cell wall associated lipoprotein, 19 kDa lipoprotein antigen and it is reported to suppress the transcription of MHC II Transcriptional Transactivator (CIITA) through the modulation of another transcription factor C/EBP {19563525; 12816996}. It has already been shown that LpqH is shed from live intracellular mycobacteria {11123323}, making it available for the continuous stimulation of TLR2 and the resulting chronic TLR2 stimulation adds to prolonged MHC II inhibition and antigen presentation. In addition, mycobactrial cell wall glycolipids like LAM, LM and PIM also act as TLR agonists {20234378; 24983458} leading to chronic TLR signalling. In this context, an in vitromodel of antigen presentation study conducted by Pearl et al showed that ligands for TLR-9, 7, 4 and 2 increased the potential of APCs to present antigen-85B of BCG to CD4 T cells, which correlated with an increase in MHC-II expression through IL-10 down-regulation { 24384074 }.

Since naive CD4+T cells are mainly activated by dendritic cells, expression of high level of MHC II and other co-stimulatory molecules on these cells are essential for the efficient antigen presentation and T cell activation {12888794}. Recently, it was reported that M. tuberculosisinterferes with DC antigen presentation in a serine hydrolase, Hip1 dependent manner leading to reduced MHC expression, which was reversed by the infection with hip1 mutant {24659689}. Furthermore, the infection of macrophages with recombinant BCG expressing Ag85B-ESAT6 fusion construct was shown to be able to induce higher HLA-DR expression and thus improved antigen presentation leading to enhanced immune response { 20417300}.

B. Modulation of antigen processing and trafficking

M.tuberculosis affects the processing and subsequent trafficking of MHC II molecules mainly by interfering with the proteases present in the phagosomal compartment and by alkalinisation of intracellular components {16040149}. Degradation of the invariant chain (Ii) and removal of class II-associated invariant chain peptide (CLIP) depend on the activity of these proteases and it is necessary for the proper antigen loading to MHC II molecules prior to antigen presentation {12776207}. Cathepsins are cysteine proteases essential for the processing of Ii and Cathepsin S (CatS) is a prominent member of cathepsin family mediating the late steps of Ii cleavage {10562280}. Our lab had previously shown that infection of THP-1 differentiated macrophages with mycobacterium caused reduction of Cat S activity and gene expression through the induction of IL-10{16210638}. The reduction of Cat S activity was associated with reduction in the export of mature; peptide loaded MHC II molecules but increased the export of immature MHC II molecules associated with invariant chain to the macrophage surface. Restoration of Cat S expression by the addition of anti-IL-10 antibodies increased the export of mature MHC molecules to the infected cell surface. Moreover, other study conducted in our lab showed that restoration of Cat S activity by the infection of macrophages with human Cat S secreting BCG (rBCG-hcs) restored Cat S activity and improved the capacity of BCG infected macrophages to stimulate CD4+ T cells {17911599}. Other related study performed in our lab indicated that intra-phagosomal alkalinization by pathogenic mycobacteria expressing urease imparts significantly to the intracellular retention of class II dimers{15213164}. Urease encoded by mycobacterial ureC gene, which hydrolyzes urea into ammonia and carbon dioxide causes alkalinisation and defects in antigen processing and loading. More recently, it was shown that the superior protection exhibited by BCG ΔureC::hly vaccine, a urease deficient BCG strain equipped with the membrane-perforating listeriolysin (Hly) ofListeria monocytogenes is due to higher number of central memory CD4 T cells{ 24943726; 16110326; 24778634}. Due to the occurrence of Hly mediated perforation of the host cell phagosomal membrane, this strain induced the release of mycobacterial antigens into the cytosol of the host macrophage and thus enhanced cross-priming. Furthermore, BCG ΔureC::hly strain modified to co-express human interleukin-7 (hIL-7) or hIL-18 was shown to provide significantly better protection that wild type BCG in murine M.tuberculosis challenge model {24236077}.

An earlier study conducted in our lab pointed out that infection of macrophages with M.tuberculosis inhibits maturation of class II heterodimer during post-golgi transport to the cell surface and results in the intra-cellular accumulation of class II molecules{9794422}. In this study, we have shown that MHC II mRNA levels were unaltered in infected cells and at the same time, Noss et al. {10805975} demonstrated that MHC II mRNA levels decreased in infected cells. In this context, Stewart et al {15767567} performed a comparative analysis of these contrasting observations and pointed out that different experimental conditions used might have caused these contrasting observations.

Implication of mycobacterial phagolysome fusion in antigen presentation

Phagolysosome biogenesis block was one of the most important virulence traits described for M.tuberculosis {11483990}. The phagolysosome biogenesis block not only helps M.tuberculosis to avoid exposure to lysosomal hydrolases but also modulates the proteolytic processing and subsequent loading of processed peptides to MHC II molecules. Even though M.tuberculosis uses multiple ways to inhibit phago-lysosome fusion, failure to incorporate proton pump {8303277} and the retention of coronin 1 or TACO on phagosomes contribute significantly to the inhibitory effect {10338208}. It is widely accepted that phagolysosome biogenesis is crucial for the normal processing and presentation of antigens to evoke adaptive immunity and in this context, we have shown that lipoamide dehydrogenase (LpdC) of M.tuberculosis mediates the retention of coronin 1 on BCG vacuoles leading to phagolysosome fusion arrest {17652161}. We have also demonstrated that PtpA, a low-molecular weight tyrosine phosphatase, of M.tuberculosis interacts with VPS33B, a regulator of membrane fusion leading to the inhibition of phagosome-lysosome fusion and subsequent antigen presentation{18474358}. In addition, Johansen et al later showed that infection of murine bone marrow-derived dendritic cells with a mycobacterial mutant deficient in zmp1 gene which codes for zinc metalloprotease 1 resulted in increased presentation of MHC II restricted antigens {21471301}. Zinc metalloprotease 1 is known to interfere with inflammasome activation leading to defective phagosome maturation and subsequent antigen presentation. By altering the intra-phagosomal milieu from its designed function, M.tuberculosis may delay or prevent the effective antigen presentation to C4+T cell subsets.

Autophagy enhances antigen presentation

Autophagy is a homeostatic and inducible process whereby components of cytoplasm, including organelles and intracellular pathogens are sequestered in an autophagosome and delivered to lysosome for degradation {15607973} and these resulting peptides become potential source for antigen presentation and cross-priming of T cells {18305538; 20116986}. Cytoplasmic and nuclear antigens are reported to be delivered to MHC II molecules for presentation to CD4+Tcells through autophagy and in addition, autophagy has also been linked to MHC I mediated antigen cross-presentation {19265109; 21438870}.

Activation of autophagy by IFNγ is a host anti-mycobacterial defence mechanism that results in phagosome maturation and an increase in its acidification leading to M. tuberculosis killing {15607973; 16874070}. Autophagy is promoted by vitamin D3 and TLR4 via Toll–IL-1 receptor domain-containing adaptor-inducing IFNβ signalling leading to enhanced mycobacterial killing {15607973; 24754048; 17658277}. In an elegant study, Sudha et al showed that induction of autopahgy can be used to eliminate intra-cellular M.tuberculosis {16888103}. It has also been reported that M.tuberculosis H37Rv impairs autophagy at the step of autophagosome-lysosome fusion through ESX-1 mediated mechanism {22885411}. BCG vaccine efficacy was shown to be enhanced by augmenting autophagy mediated antigen presentation {19252503}. Rapamycin treatment was shown to overcome the autophagic block induced by M.tuberculosis through increased IL-12 expression leading to enhanced Th1 response. Moreover, IL-1β promoter polymorphism has been linked to susceptibility to tuberculosis infection {17854273} and it is reported that IL-1β promotes autophagosomal maturation and subsequent antigen presentation on M.tubercuosis infected macrophages {22921120; 23790398; 24032597}. It is widely accepted that autophagy contributes to vaccine stimulation of protective immunity through enhancing antigen presentation to T cells {25003089}. It was also reported that the incorporation of autophagy inducing element, the kinase defective mTOR (mTOR-KD)plasmid and Ag85B in separate mycobacterial plasmids into DNA vaccine elicited higher antibody responses as well as production of IFN-γ and IL-2 in the spleen and thus higher protective immune response {23228812}. Altogether, these studies clearly demonstrate the strong connection between autophagy induction and subsequent antigen presentation leading to better protective immune response against M.tuberculosis.

Apoptosis and antigen presentation

Apoptosis is a tightly regulated form of cell death where the cytoplasmic contents of the dying cells are confined within the membrane bound structures called apoptotic bodies, that express the so called eat me signal, phosphatidyl serine on their surface leading to the recognition and subsequent removal by the professional phagocytes through efferocytosis { 19259342; 21307848}. In addition to its role in intracellular pathogen removal by eliminating the favourable intra-cellular niche for replication, apoptosis is also reported to facilitate efficient antigen cross presentation leading to effective immune response against the pathogen {12872166}.

M.tuberculosis has evolved various mechanisms to actively block macrophage apoptosis { 21307848; 18651813; 23118880; 20676146} and in combination with various host cell subversion strategies, it helps the pathogen to gain a solid survival advantage over the host. It has already been reported that apoptosis of M.tuberculosis infected macrophages caused reduced viability of intra-cellular mycobacteria {23118880; 9605147} thus shifting the host-pathogen equilibrium in favour of the host. To facilitate its spread into neighbouring cells, M.tuberculosis turns the death mode of the infected macrophage to necrotic fate while avoiding the induction of the apoptotic mode(6). Professional phagocytes recognize and engulf the membrane bound apoptotic bodies through a process called efferocytosis and this binding is known to induce anti-inflammatory cytokine synthesis, thereby limiting the tissue damage {19259342; 16474428}. Efferocytosis enables the host cells to overcome the phagolysosome biogenesis block exerted by M.tuberculosis leading to mycobacterial killing. In addition to helping in eliminating the favourable intra-cellular environment for pathogen replication, apoptosis also facilitates mycobacterial antigen cross presentation leading to effective immune response through the detour pathway {19259342}. Here, the uninfected antigen presenting cells engulf these extracellular vesicles and cross-present mycobacterial antigens through MHC I pathway leading to effective CD8+T cell response. The detour pathway is also shown to facilitate mycobacterial lipid antigen presentation through CD1 molecules {19563525; 16413927}.

Individual M.tuberculosis genes that are reported to be implicated in blocking apoptosis have been identified, such as secA2 (Rv1821), which codes for a component of a secretion system that direct the transport of superoxide dismutase (SodA) out of the bacterial cell and nuoG (Rv3151), which encodes a subunit of NADH dehydrogenase type I complex in the mycobacterial membrane, and deletion of these genes is reported to result in a pro-apoptotic phenotype {17671656; 17658950}. Upon phagocytosis, M.tuberculosis encounters a hazardous intracellular environment that is created by variety of host factors including reactive oxygen and nitrogen intermediates {22773641}. Secreted SodA enables the pathogen to detoxify the reactive oxygen intermediates, which is known to induce host cell apoptosis if induced in sufficiently high level, there by adding the survival advantage. Earlier, Hinchey et al showed that inactivation of secA2 gene caused impaired superoxide dismutase secretion leading to enhanced apoptosis and in addition, it also showed to increase antigen specific CD8+T cell priming in vivo {17671656}. Similarly, deletion of nuoG gene was also shown to impair the ability of M.tuberculosis to inhibit macrophage apoptosis and this mutant exhibited significantly reduced virulence in mice {17658950}. Thus, the obstruction of macrophage apoptosis by M.tuberculosis allows the pathogen to subvert or delay the antigen presentation cascade leading to the establishment of a firm survival advantage over the host.

Table: Different strategies used to improve mycobacterial antigen presentation



Outcome of



Strategy used



Reduced Cats


Defective MHC II


Use of anti-IL-10 antibodies and using rBCG to secrete Cat S

{16210638; 17911599}


Urease production

Defective MHC II


Disruption of urease activity



Urease prodution

Defective MHC II


Disruption of urease activity and co-expression of IL7 & 18 in Hly backrgound

{24943726; 16110326; 24236077}


MARCH ligase activity

MHC II downregulation

Addition of TLR 7/9 ligands

{ 24384074}


Autophagy inhibition

Defective antigen presentation

Rapamycin treatment and encapsulated plasmids encoding Ag85B & mTOR

{ 23228812}


Serine hydrolase activity

Defective expression of MHC II and other co-stimulatory molecules

Disruption of serine hydrolase Hip1

{ 24659689}


Zinc metalloprotease


Interferes with inflammasome activation and defective antigen presentation

Deletion of zmp1 gene

{ 21471301}


HLA-DR expression

Defective HLA-DR expression

Modification of BCG to express Ag85B-ESAT6 fusion protein

{ 20417300}