Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a leading cause of infectious disease mortality worldwide, causing approximately 10 million new cases of active TB disease and 1.5 million deaths per year (World Health Organization 2022, 2021). Currently, the only clinically licensed vaccine to prevent TB is Bacille Calmette-Guerin (BCG), which protects children against disseminated Mtb infection (Roy et al., 2014), but provides limited and highly variable protection against pulmonary TB in adults (Fine, 1995). More effective vaccines against TB are therefore needed, but identifying Mtb antigens capable of eliciting protective immunity remains challenging.

Multiple convergent lines of evidence from experiments in mouse and non-human primate models of TB show that CD8+ T cells can contribute to immune control of Mtb infection (Chen et al., 2009; Flynn et al., 1992; Woodworth et al., 2008b), but the antigenic targets of protective CD8+ T cell immunity to Mtb infection have not been conclusively defined. In murine models, CD8+ T cells specific for some immunodominant Mtb antigens poorly recognize Mtb-infected macrophages (Yang et al., 2018), implying that infected macrophages may not present all Mtb antigens that elicit cytokine-producing CD8+ T cell responses (Kamath et al., 2004; Lewinsohn et al., 2017). These results suggest a need to directly identify which Mtb antigens are presented on MHC-I by infected phagocytes (Flynn et al., 2011).

It is currently unknown which Mtb proteins are able to enter MHC-I antigen processing pathways in macrophages infected with virulent Mtb. Whereas some bacterial species are lysed following phagocytosis and expose their internal cell contents to antigen processing pathways (Shen et al., 1998), a high proportion of virulent Mtb remains intact and viable in macrophages (Armstrong and Hart, 1975; Lee et al., 2008; Lewis et al., 2003), leading us to hypothesize that only a subset of Mtb proteins may be accessible for processing and presentation on MHC-I. Here, we use MS-based identification of peptides bound to MHC-I (immunopeptidomics) to directly identify Mtb-derived peptides presented on MHC-I in primary human macrophages infected with virulent Mtb H37Rv, revealing potential targets for CD8+ T cell-mediated immunity. Additionally, we use targeted MS to quantify changes in the presentation of Mtb peptides resulting from genetic perturbations to Mtb and chemical perturbations to the host cell, allowing us to probe host and bacterial determinants of antigen presentation on MHC-I in Mtb infection.

To identify Mtb antigens presented on MHC-I, we infected primary human monocyte-derived macrophages with Mtb H37Rv, isolated MHC-I by immunoprecipitation 72 hours post-infection, purified the associated peptides, and identified peptides by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). We selected the 72 hour time point over shorter time points to allow sufficient time for antigen processing and presentation on MHC-I. Past experience suggested that choosing a longer time point would have resulted in a high rate of cell death. For an initial set of three analyses, we adapted a previously described protocol (Stopfer et al., 2020; Protocol 1 – see Methods) for use in a biosafety level 3 (BL3) setting. We developed a further optimized protocol (Protocol 2 – see Methods) that we used for 3 additional analyses (Figure 1A). The class I human leukocyte antigen (HLA) genotypes of the 6 donors analyzed were largely distinct from one another (Table 1), enabling us to identify peptides associated with a variety of HLA alleles.

Figure 1 with 8 supplements see all

Download asset Open asset

Figure 1—source data 1

Source data for enrichment analysis.

https://cdn.elifesciences.org/articles/84070/elife-84070-fig1-data1-v2.zip

Download elife-84070-fig1-data1-v2.zip

We identified thousands of peptides for each primary cell donor, with a length distribution typical of MHC-I peptides (Figure 1—figure supplement 1A) and retention times that correlated well with hydrophobicity (Figure 1—figure supplement 1B). Unsupervised clustering of identified peptides using GibbsCluster 2.0 (Andreatta et al., 2017) revealed groups corresponding to the known peptide sequence binding motifs of class I HLA alleles expressed by each donor (Figure 1—figure supplement 2), confirming the specificity of our pulldowns. Mtb-derived MHC-I peptides detected in each pulldown were predicted to bind at least one class I HLA allele expressed by the donor (Figure 1—figure supplement 3). Mtb peptides made up less than 0.1% of MHC-I peptides identified, and this proportion was not discernibly increased by pre-treating macrophages with IFN-γ or treating with cycloheximide to inhibit host protein synthesis (Figure 1B).

Putative Mtb peptides that passed manual inspection of MS/MS spectra and extracted ion chromatograms (see Methods) were further validated using internal standard parallel reaction monitoring (IS-PRM, also known as SureQuant) (Gallien et al., 2015; Stopfer et al., 2021a; Figure 1—figure supplement 4). Putative Mtb peptides that had MS/MS spectra that closely matched that of a synthetic stable isotope-labeled (SIL) standard, co-eluted with the SIL standard, and were not detected in mock-infected control samples by SureQuant were considered correctly identified, authentic Mtb peptides (Figure 1—figure supplement 5). 77.85% of MASCOT identifications of putative Mtb peptides were rejected after manual data inspection, and of the remaining candidates a further 51.51% were rejected following analysis by SureQuant (Table 2), highlighting the need for rigorous validation using best practices (Fritsche et al., 2021) when identifying pathogen-derived peptides among an MHC repertoire dominated by host-derived peptides.

Of the 16 Mtb-derived MHC-I peptides we identified, 13 (81.25%) derived from proteins secreted via type VII secretion systems (T7SS) (Figure 1C). The Mtb genome encodes five of these protein export machines (designated ESX-1, 2, 3, 4, and 5; Abdallah et al., 2007), and we identified MHC-I peptides derived from proteins known to be secreted by three of these systems [ESX-1 (Guinn et al., 2004; Millington et al., 2011; Stanley et al., 2003), ESX-3 (Siegrist et al., 2009; Tufariello et al., 2016), and ESX-5 (Abdallah et al., 2006; Daleke et al., 2012; Ekiert and Cox, 2014; Shah et al., 2015)]. For several T7SS substrates, we identified multiple peptides from the same protein, and/or identified the same peptide across multiple donors. These antigens included EsxA, PPE20, EspC, and PE35, as well as sequences conserved among the four nearly identical members of the EsxJ family of proteins (EsxJ, EsxK, EsxP, and EsxW – referred to here as EsxJKPW). T7SS substrates were significantly overrepresented in the MHC-I repertoire relative to the whole Mtb proteome, both at the peptide level (p<10^–10; binomial test with Bonferroni correction) and at the protein level (p<10^–7; hypergeometric test with Bonferroni correction; Figure 1D). Proteins without identifiable secretion signals were significantly underrepresented among Mtb-derived MHC-I peptides (p<10^–8; binomial test with Bonferroni correction) and source proteins (p<10^–6; hypergeometric test with Bonferroni correction). While some of the Mtb antigens we identified are highly abundant compared to the rest of the Mtb proteome (e.g. EsxB), the abundances of other T7SS substrates are near the mean (Figure 1—figure supplement 1C; Schubert et al., 2015), suggesting that the overrepresentation of T7SS substrates is not solely due to greater abundance relative to the rest of the Mtb proteome. These results suggest that T7SS substrates may preferentially gain access to MHC-I antigen processing pathways.

To nominate possible mechanisms by which Mtb antigens might be processed and loaded onto MHC-I, we used confocal microscopy to examine the intracellular fate of Mtb in primary human macrophages. We hypothesized that Mtb antigens could either (1) be processed by endolysosomal proteases and loaded onto MHC-I in Mtb-containing compartments, or (2) gain access to cytosolic antigen processing pathways via permeabilization of the phagosome membrane (Grotzke et al., 2010; Grotzke et al., 2009; van der Wel et al., 2007; Figure 2A). In a subset of macrophages at both early and late timepoints, a subset of Mtb co-localized with Galectin-3, a marker of phagosomal membrane permeabilization (Chauhan et al., 2016; Watson et al., 2012; Figure 2B–C; Figure 2—figure supplement 1A). A subset of Mtb also co-localized with P62, an autophagy adaptor protein that recognizes cytosol-exposed bacteria (Watson et al., 2012; Zheng et al., 2009; Figure 2B–C; Figure 2—figure supplement 1B). Since bacteria become ubiquitinated upon exposure to the cytosol and recruitment of autophagy adaptor proteins to the Mtb phagosome has previously been associated with damage to the phagosomal membrane (Watson et al., 2012), these results suggest that Mtb gains access to the host cytosol in primary human macrophages, as has previously been shown in murine macrophages (Mittal et al., 2018) and in cell lines (Beckwith et al., 2020). A subset of Mtb-containing phagosomes co-localized with the late endosome and lysosome marker LAMP-1 (Figure 2B–C; Figure 2—figure supplement 1C), suggesting access to endolysosomal proteases, but Mtb-containing phagosomes did not co-localize with MHC-I itself (Figure 2B–C; Figure 2—figure supplement 1D), suggesting that Mtb antigens were unlikely to be loaded onto MHC-I in the Mtb-containing phagosome. These results suggested that access to the host cell cytosol represented a likely route for processing and presentation of Mtb antigens. An Mtb strain deficient in the activity of the ESX-1 secretion system (eccCa1:Tn) exhibited reduced co-localization with Galectin-3 and P62 (Figure 2D–F). Our results are consistent with prior studies demonstrating that ESX-1 activity is required for Mtb to damage the phagosome membrane (Augenstreich et al., 2017; Simeone et al., 2012; Watson et al., 2012) and show that this phenomenon occurs in our primary human macrophage infection model.

Figure 2 with 1 supplement see all

Download asset Open asset

Figure 2—source data 1

Single-channel images.

https://cdn.elifesciences.org/articles/84070/elife-84070-fig2-data1-v2.zip

Download elife-84070-fig2-data1-v2.zip

Figure 2—source data 2

Colocalization quantification source data.

https://cdn.elifesciences.org/articles/84070/elife-84070-fig2-data2-v2.zip

Download elife-84070-fig2-data2-v2.zip

Our microscopy results led us to hypothesize that ESX-1-mediated phagosomal membrane damage might be required for Mtb antigens to access MHC-I antigen processing pathways (Figure 3A). If this were the case, ESX-1 activity would be required for presentation not only of peptides derived from ESX-1 substrates (e.g. EsxA_28-36 – sequence LLDEGKQSL), but also peptides derived from substrates of other T7SSs (e.g. EsxJKPW_24-34 – sequence QTVEDEARRMW – which is derived from ESX-5 substrates that do not require ESX-1 for secretion; Champion et al., 2006). To test this hypothesis, we turned to quantitative targeted MS (SureQuant) to quantify changes in the presentation of EsxA_28-36 and EsxJKPW_24-34 across multiple experimental conditions (Figure 3B). We used primary macrophages from donors expressing HLA-A*02:01 and HLA-B*57:01 for these experiments to ensure presentation of the target peptides. Because the other Mtb epitopes detected in our untargeted MS experiments are not expected to bind these HLA alleles, we only targeted EsxA_28-36 and EsxJKPW_24-34. While this targeted approach is limited in the number of epitopes it can detect, it enables reliable and accurate quantification of peptides across experimental conditions with low sample input. Because methionine residues of peptides can oxidize during sample handling, we targeted both the oxidized and non-oxidized form of EsxJKPW_24-34 where possible. In addition to the SIL synthetic trigger peptides required for SureQuant, we also spiked pre-formed heavy isotope labeled peptide-MHC complexes (hipMHCs) into the lysates prior to immunoprecipitation to provide an internal standard that can be used to normalize out technical variation, which improves the accuracy of label-free quantification (Stopfer et al., 2020).

Figure 3 with 1 supplement see all

Download asset Open asset

Figure 3—source data 1

Relative abundances of Mtb-derived MHC-I peptides determined by SureQuant.

https://cdn.elifesciences.org/articles/84070/elife-84070-fig3-data1-v2.zip

Download elife-84070-fig3-data1-v2.zip

Figure 3—source data 2

CXCL10 ELISA raw data.

https://cdn.elifesciences.org/articles/84070/elife-84070-fig3-data2-v2.zip

Download elife-84070-fig3-data2-v2.zip

Targeted MS analysis revealed that macrophages infected with ESX-1-deficient Mtb (eccCa1:Tn) did not present either EsxA_28-36 or EsxJKPW_24-34 on MHC-I (Figure 3c). We assessed alternative explanations for this difference in presentation of EsxA_28-36 and EsxJKPW_24-34 and observed no difference in Mtb outgrowth (Figure 3D), the proportion of cells infected (Figure 3—figure supplement 1A–B), or total MHC-I surface expression (Figure 3—figure supplement 1C–D) between macrophages infected with wild-type or ESX-1-deficient Mtb. These results show that ESX-1 activity is required for presentation of EsxA_28-36 and EsxJKPW_24-34 on MHC-I in macrophages.

To determine whether the absence of EsxA_28-36 and EsxJKPW_24-34 in the MHC-I repertoire of macrophages infected with ESX-1-deficient Mtb could be attributed to a loss of type I interferon signaling (Stanley et al., 2007), we added exogenous IFN-β to macrophages infected with ESX-1-deficient Mtb and again quantified presentation of EsxA_28-36 and EsxJKPW_24-34 by SureQuant. Addition of exogenous IFN-β restored a type I interferon response as measured by production of CXCL10 (Figure 3E), but did not rescue presentation of EsxA_28-36 or EsxJKPW_24-34 on MHC-I (Figure 3F). These results show that presentation of EsxA_28-36 or EsxJKPW_24-34 on MHC-I is dependent on ESX-1 activity but independent of downstream type I interferon signaling, consistent with a model in which ESX-1-mediated phagosomal damage enables Mtb antigens to access MHC-I antigen processing pathways.

Many MHC-I peptides are proteolytically processed by the proteasome (Kloetzel, 2001), while others are processed by endosomal or lysosomal proteases (Grotzke et al., 2017; Shen et al., 2004). To determine whether these mechanisms contribute to presentation of Mtb peptides on MHC-I, we treated HLA-A*02:01+, HLA-B*57:01+ macrophages with inhibitors of proteasome activity (MG-132), cysteine cathepsin activity (E64d), and lysosomal acidification (bafilomycin; Figure 4A). We quantified presentation of EsxA_28-36 and EsxJKPW_24-34 on MHC-I along with five host-derived HLA-A*02:01-binding peptides identified in previous studies (Stopfer et al., 2021b) that could be reliably detected in the MHC-I repertoire of macrophages. We began drug treatment of the macrophages prior to infection with Mtb and limited the duration of infection to 24 hr so that the cells could be treated with drug for the full duration of the infection without excessive cytotoxicity (Figure 4B). Treatment with MG-132 inhibited proteasome activity, as measured by accumulation of proteins modified with K48-linked polyubiquitin (Figure 4—figure supplement 1A–B). Treatment with E64d inhibited cathepsin B activity, as measured by a fluorometric assay (Figure 4—figure supplement 1C). Treatment with bafilomycin inhibited lysosomal acidification, as measured by lysotracker staining (Figure 4—figure supplement 1D–E). All three drugs exhibited minimal cytotoxicity in macrophages at the doses used in our immunopeptidomic experiments (Figure 4—figure supplement 2), did not inhibit phagocytosis of Mtb or bacterial outgrowth (Figure 4—figure supplement 3), and did not have a significant effect on surface MHC-I levels (Figure 4—figure supplement 3).

Figure 4 with 4 supplements see all

Download asset Open asset

Figure 4—source data 1

Relative abundances of MHC-I peptides determined by SureQuant.

https://cdn.elifesciences.org/articles/84070/elife-84070-fig4-data1-v2.zip

Download elife-84070-fig4-data1-v2.zip

Treatment with MG-132 reduced presentation of MHC-I peptides derived from cytosolic or nuclear host proteins, but not peptides derived from Mtb proteins or secreted or endomembrane compartment localized host proteins (Figure 4C). Treatment with E64d decreased presentation of peptides derived from endosomal or secreted proteins and had a range of effects on peptides derived from cytosolic or nuclear proteins, but did not decrease presentation of Mtb peptides (Figure 4C). Treatment with bafilomycin broadly increased presentation of target MHC-I peptides (Figure 4C). This increase was statistically significant for EsxA_28-36 (Figure 4D; p<0.05, one-way ANOVA with Dunnett’s multiple comparisons test), though not for EsxJKPW_24-34 (Figure 4E). Cell surface levels of HLA-DR and HLA-DQ were not significantly affected by bafilomycin or E64d treatment (Figure 4—figure supplement 4), suggesting that E64d and bafilomycin did not substantially stall MHC-II antigen presentation and their effects of on MHC-I presentation could not be explained as indirect effects of modulating antigen entry into the MHC-II antigen processing pathway. Our results suggest that processing of Mtb peptides for presentation on MHC-I relies on antigen processing proteases other than the proteasome, cysteine cathepsins, or other acidification-dependent lysosomal proteases, and/or that multiple redundant pathways contribute to processing of Mtb peptides for presentation on MHC-I.

Our analysis of the MHC-I repertoire of primary human macrophages infected with Mtb revealed that T7SS substrates are a prominent source of Mtb peptides presented on MHC-I. Our findings contrast with those of previous studies of the immunopeptidome of macrophage cell lines infected with BCG (Bettencourt et al., 2020) and the avirulent strain Mtb H37Ra (Flyer et al., 2002), in which the mycobacterial peptides identified included twin arginine translocation (Tat) pathway substates, Sec pathway secretion substrates, membrane-associated proteins, and cytosolic proteins, with no apparent enrichment for T7SS substrates. Given that we showed ESX-1 activity contributes to presentation of Mtb antigens in infection with virulent Mtb H37Rv, the fact that BCG and Mtb H37Ra are both deficient in ESX-1 activity (Frigui et al., 2008; Gordon et al., 1999; Guinn et al., 2004; Lewis et al., 2003; Pym et al., 2002) may in part explain this difference in the mycobacterial peptides presented on MHC-I. The absence of ESX-1 in BCG therefore could limit the ability of BCG to prime effective T cell responses against Mtb, not only because of the absence of antigens encoded in the ESX-1 locus but also because of altered or reduced presentation of other antigens (Pym et al., 2003).

Our findings contrast with previous work arguing that ESX-1 activity is dispensable for presentation of Mtb antigens on MHC-I (Lewinsohn et al., 2006; Woodworth et al., 2008a). ESX-1 activity is not required for Mtb-infected human monocyte-derived dendritic cells to present an epitope derived from TB8.4 (a Sec pathway substrate) to cognate T cells (Lewinsohn et al., 2006). While ESX-1 activity is required for priming of CD8+ T cell responses specific for EsxB in vivo in mice, it is dispensable for priming of CD8+ T cell responses specific for antigens exported via other secretion systems (such as TB8.4 and EsxH) (Woodworth et al., 2008b). While these prior results suggest that the requirements for presentation of Mtb antigens on MHC-I may vary among Mtb antigens and/or vary among antigen presenting cell types, our results show that ESX-1 activity is essential for the presentation of certain Mtb antigens in infected human macrophages, beyond ESX-1 substrates alone. Whereas in axenic culture, secretion of ESX-5 substrates is independent of ESX-1 function (Champion et al., 2006; Shah and Briken, 2016), the fact that presentation of peptides derived from an ESX-5 substrate on MHC-I requires ESX-1 activity supports the hypothesis that the localization of secreted Mtb proteins within a host cell may depend on the activity of multiple T7SSs. This dependence on multiple T7SSs has previously been shown for other ESX-5 substrates such as CpnT (Izquierdo Lafuente et al., 2021), and a functional interdependence between ESX-1 and ESX-3 (which respectively damage the phagosome membrane and prevent host membrane repair) has previously been proposed (Mittal et al., 2018). Our results suggest that the interdependence of Mtb T7SSs in an intracellular context influences the availability of Mtb antigens for processing and presentation on MHC-I.

Prior studies have shown that Mtb antigens must access the cytosol to be presented on MHC-I in human monocyte-derived dendritic cells (Grotzke et al., 2010; Grotzke et al., 2009; Lewinsohn et al., 2006), and our results are consistent with a model in which ESX-1-mediated phagosomal membrane damage enables Mtb antigens to access cytosolic antigen processing and presentation pathways. However, further evidence will be needed to establish whether phagosomal membrane damage alone is sufficient to enable presentation of Mtb antigens on MHC-I in the absence of other ESX-1 functions. Given that our quantitative MS experiments only targeted two Mtb-derived epitopes, further experiments will also be needed to determine whether these results extend to all Mtb T7SS substrates or all Mtb MHC-I antigens, or only a subset.

In this study, we used strain H37Rv for the sake of consistency with other prior studies on antigen presentation on MHC-I and phagosome membrane damage in Mtb-infection, but antigen presentation may vary among Mtb isolates. Mtb isolates have been shown to differ in their level of ESX-1 activity (Solans et al., 2014), as well as carrying mutations in Esx-family proteins and other T7SS substrates (Saelens et al., 2022; Uplekar et al., 2011). Further studies across multiple isolates may therefore reveal differences in antigen presentation that could be relevant for the design of broadly protective vaccines.

We showed that presentation of two Mtb-derived peptides on MHC-I was independent of the activity of proteases commonly associated with antigen processing (the proteasome and cysteine cathepsins), suggesting an alternate processing mechanism. Other proteases have previously been proposed to contribute to proteolytic processing of antigens for presentation on MHCs, including tripeptidyl peptidase II (Geier et al., 1999; Guil et al., 2006; Lázaro et al., 2015; York et al., 2006), nardilysin (Kessler et al., 2011), thimet oligopeptidase (Kessler et al., 2011), metalloproteinases (Lorente et al., 2012), and serine cathepsins such as Cathepsin G that are not inhibited by E64d (Burster et al., 2010). Some of these or other enzymes may be responsible for proteolytic processing of EsxA_28-36 and EsxJKPW_24-34, or may have a redundant role that can compensate for inhibition of conventional antigen processing pathways. Further work will be needed to determine which proteolytic pathways contribute to presentation of Mtb T7SS substrates on MHC-I.

In addition to T7SS substrates, we also identified peptides derived from a Sec pathway substrate (TB8.4), and from uncharacterized hypothetical proteins (Rv1211, Rv3196A). Rv1211 and Rv3196A have both previously been detected by MS in culture filtrates of Mtb (Bell et al., 2012), as has the Mycobacterium marinum ortholog of Rv3196A (MMAR_1367; Cronin et al., 2022), suggesting that either or both of these proteins could be secreted despite lacking readily identifiable secretion signals. TB8.4, Rv1211, and Rv3196A are all low-molecular-weight proteins, which we speculate could make it easier for them to translocate through pores in a permeabilized phagosomal membrane. Further investigation of the localization of these proteins may help us better understand how and to what extent small secreted Mtb proteins other than known T7SS substrates can contribute to the MHC-I repertoire.

Some of the Mtb MHC-I peptides we identified derive from proteins known or suspected to be associated with the mycobacterial outer membrane (Abdallah et al., 2006; Sampson et al., 2001). These include PPE51 (Wang et al., 2020), PPE20 (Boradia et al., 2022), PPE60 (Su et al., 2018), EspC (Champion et al., 2009; Lou et al., 2017), and PE13 [the co-transcribed binding partner (Chen et al., 2017) of the putative outer membrane protein PPE18 (Nair et al., 2009)]. While some of these proteins can also be found in soluble form in culture filtrates (Tufariello et al., 2016), others may be stably associated with the mycobacterial outer membrane (Wang et al., 2020). The mechanism by which antigens associated with the outer membrane are processed and presented could differ from that of soluble secreted proteins.

Prior work shows that at least some of the MHC-I antigens we identified as potential vaccine targets are immunogenic in humans. Lewinsohn et al., 2017 showed that in humans with prior exposure to Mtb, EsxA, EsxB, PPE51, and EsxJKPW are sources of immunodominant CD8+ T cell antigens, while other antigens identified in our study elicited intermediate responses (PE13, PE35, PPE60, PPE19), and one elicited no detectable response (PPE20). In a separate study, CD8+ T cells (as well as CD4+ T cells) recognized multiple epitopes derived from EspC (Millington et al., 2011). Individual peptides we identified by MS have also been previously shown to be recognized by human T cells, including EsxJ_24-34 (Grotzke et al., 2010; Lewinsohn et al., 2013) and EsxA_28-36 (Tully et al., 2005), providing a proof of concept that particular epitopes identified by MS can be immunogenic. Antigens like PPE20 that did not elicit measurable CD8+ T cell responses in the context of natural infection should not necessarily be ruled out as vaccine targets, as a lack of CD8+ T cell recognition in the setting of natural infection does not necessarily imply that these antigens could not be immunogenic. Bioinformatic studies have also shown that predicted class I HLA-binding motifs are statistically underrepresented in several Esx-family protein sequences, suggesting that these proteins may be under selective pressure to escape recognition by CD8+ T cells (Maman et al., 2011). This finding provides further support for the idea that T cell recognition of T7SS substrates presented on MHC-I is functionally relevant to protective immunity against Mtb infection.

We did not detect any peptides derived from several antigens previously shown to stimulate CD8+ T cell responses in Mtb infection, including responses restricted to HLA alleles represented in our DDA MS analyses [for example, EsxH and FbpB (Axelsson-Robertson et al., 2015)]. This could be due to any of several biological and/or technical reasons. Given that the Mtb-derived MHC-I peptides we detected were enriched for T7SS substrates but not for substrates of the Sec or Tat secretion pathways, it is possible that the subcellular localization of antigens secreted via these pathways is not optimal for efficient presentation on MHC-I in macrophages. In this case, other types of APCs that differ in their antigen processing and presentation capabilities could still present peptides from these antigens to prime the previously observed CD8+ T cell responses. The same could also be true of certain T7SS substrate antigens (such as EsxH) if T7SS substrates vary in their subcellular localization or if presentation of certain epitopes requires antigen processing pathways not active in macrophages. In future studies, isolating MHC peptides from multiple myeloid cell types may help determine whether heterogeneity in antigen presentation among cell types contributes to apparent discrepancies between the Mtb-specific CD8+ T cell repertoire and the macrophage pMHC repertoire. Some peptides may also have been missed because they were lost to adsorption during sample handling, because they were below the limit of detection due to low abundance or ionization efficiency, or because they co-eluted with other, more abundant peptides and were therefore not prioritized for acquisition in DDA analyses. In future work, pMHC internal standards added prior to immunoprecipitation may enable accurate estimates of the limit of MS detection for specific peptides, to determine whether technical factors led to a lack of detection. Our results do not rule out presentation of additional categories of antigens besides those we detected.

Our results identify several Mtb proteins that are accessible for presentation on MHC-I in Mtb-infected human macrophages, suggesting that these could serve as targets for subunit vaccines designed to elicit CD8+ T cell-mediated protection against TB. By showing that T7SS substrates are overrepresented in the MHC-I repertoire of macrophages and revealing mechanistic determinants of Mtb antigen processing, our results have the potential to guide future screens to identify additional vaccine targets. Our findings advance the field’s understanding of antigen presentation in TB and suggest novel potential targets for TB vaccine development.

The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2022) partner repository with the dataset identifiers PXD037837 (DDA data) and PXD037843 (SureQuant data). Microscopy data analysis scripts are available on GitHub at https://github.com/oleddy/local_correlation_analysis (copy archived at Leddy, 2023).

1. 1. Bryson BD

   (2023) PRIDE

   ID PXD037837. DDA data.

   https://www.ebi.ac.uk/pride/archive/projects/PXD037837
2. 1. Bryson BD

   (2023) PRIDE

   ID PXD037843. SureQuant data.

   https://www.ebi.ac.uk/pride/archive/projects/PXD037843

1. 1. Abdallah AM
   2. Verboom T
   3. Hannes F
   4. Safi M
   5. Strong M
   6. Eisenberg D
   7. Musters RJP
   8. Vandenbroucke-Grauls CMJE
   9. Appelmelk BJ
   10. Luirink J
   11. Bitter W

   (2006) A specific secretion system mediates PPE41 transport in pathogenic mycobacteria

   Molecular Microbiology 62:667–679.

   https://doi.org/10.1111/j.1365-2958.2006.05409.x

   • PubMed
   • Google Scholar
2. 1. Abdallah AM
   2. Gey van Pittius NC
   3. Champion PAD
   4. Cox J
   5. Luirink J
   6. Vandenbroucke-Grauls CMJE
   7. Appelmelk BJ
   8. Bitter W

   (2007) Type VII secretion -- mycobacteria show the way

   Nature Reviews. Microbiology 5:883–891.

   https://doi.org/10.1038/nrmicro1773

   • PubMed
   • Google Scholar
3. 1. Andreatta M
   2. Alvarez B
   3. Nielsen M

   (2017) GibbsCluster: unsupervised clustering and alignment of peptide sequences

   Nucleic Acids Research 45:W458–W463.

   https://doi.org/10.1093/nar/gkx248

   • PubMed
   • Google Scholar
4. 1. Armstrong JA
   2. Hart PD

   (1975) Phagosome-Lysosome interactions in cultured macrophages infected with virulent tubercle bacilli: reversal of the usual nonfusion pattern and observations on bacterial survival

   The Journal of Experimental Medicine 142:1–16.

   https://doi.org/10.1084/jem.142.1.1

   • PubMed
   • Google Scholar
5. 1. Augenstreich J
   2. Arbues A
   3. Simeone R
   4. Haanappel E
   5. Wegener A
   6. Sayes F
   7. Le Chevalier F
   8. Chalut C
   9. Malaga W
   10. Guilhot C
   11. Brosch R
   12. Astarie-Dequeker C

   (2017) Esx-1 and phthiocerol dimycocerosates of Mycobacterium tuberculosis act in concert to cause phagosomal rupture and host cell apoptosis

   Cellular Microbiology 19:e12726.

   https://doi.org/10.1111/cmi.12726

   • PubMed
   • Google Scholar
6. 1. Axelsson-Robertson R
   2. Ju JH
   3. Kim HY
   4. Zumla A
   5. Maeurer M

   (2015) Mycobacterium tuberculosis-specific and MHC class I-restricted CD8+ T-cells exhibit a stem cell precursor-like phenotype in patients with active pulmonary tuberculosis

   International Journal of Infectious Diseases 32:13–22.

   https://doi.org/10.1016/j.ijid.2014.12.017

   • Google Scholar
7. 1. Beckwith KS
   2. Beckwith MS
   3. Ullmann S
   4. Sætra RS
   5. Kim H
   6. Marstad A
   7. Åsberg SE
   8. Strand TA
   9. Haug M
   10. Niederweis M
   11. Stenmark HA
   12. Flo TH

   (2020) Plasma membrane damage causes NLRP3 activation and pyroptosis during Mycobacterium tuberculosis infection

   Nature Communications 11:2270.

   https://doi.org/10.1038/s41467-020-16143-6

   • PubMed
   • Google Scholar
8. 1. Bell C
   2. Smith GT
   3. Sweredoski MJ
   4. Hess S

   (2012) Characterization of the Mycobacterium tuberculosis proteome by liquid chromatography mass spectrometry-based proteomics techniques: a comprehensive resource for tuberculosis research

   Journal of Proteome Research 11:119–130.

   https://doi.org/10.1021/pr2007939

   • PubMed
   • Google Scholar
9. 1. Bettencourt P
   2. Müller J
   3. Nicastri A
   4. Cantillon D
   5. Madhavan M
   6. Charles PD
   7. Fotso CB
   8. Wittenberg R
   9. Bull N
   10. Pinpathomrat N
   11. Waddell SJ
   12. Stylianou E
   13. Hill AVS
   14. Ternette N
   15. McShane H

   (2020) Identification of antigens presented by MHC for vaccines against tuberculosis

   NPJ Vaccines 5:2.

   https://doi.org/10.1038/s41541-019-0148-y

   • PubMed
   • Google Scholar
10. Preprint

    1. Boradia V
    2. Frando A
    3. Grundner C

    (2022) Calcium Transport by the Mycobacterium Tuberculosis PE15/PPE20 Proteins

    bioRxiv.

    https://doi.org/10.1101/2022.05.30.494056

    • Google Scholar
11. 1. Burster T
    2. Macmillan H
    3. Hou T
    4. Boehm BO
    5. Mellins ED

    (2010) Cathepsin G: roles in antigen presentation and beyond

    Molecular Immunology 47:658–665.

    https://doi.org/10.1016/j.molimm.2009.10.003

    • PubMed
    • Google Scholar
12. 1. Champion PAD
    2. Stanley SA
    3. Champion MM
    4. Brown EJ
    5. Cox JS

    (2006) C-Terminal signal sequence promotes virulence factor secretion in Mycobacterium tuberculosis

    Science 313:1632–1636.

    https://doi.org/10.1126/science.1131167

    • PubMed
    • Google Scholar
13. 1. Champion PAD
    2. Champion MM
    3. Manzanillo P
    4. Cox JS

    (2009) Esx-1 secreted virulence factors are recognized by multiple cytosolic AAA ATPases in pathogenic mycobacteria

    Molecular Microbiology 73:950–962.

    https://doi.org/10.1111/j.1365-2958.2009.06821.x

    • PubMed
    • Google Scholar
14. 1. Chauhan S
    2. Kumar S
    3. Jain A
    4. Ponpuak M
    5. Mudd MH
    6. Kimura T
    7. Choi SW
    8. Peters R
    9. Mandell M
    10. Bruun JA
    11. Johansen T
    12. Deretic V

    (2016) TRIMs and galectins globally cooperate and TRIM16 and galectin-3 co-direct autophagy in endomembrane damage homeostasis

    Developmental Cell 39:13–27.

    https://doi.org/10.1016/j.devcel.2016.08.003

    • PubMed
    • Google Scholar
15. 1. Chen CY
    2. Huang D
    3. Wang RC
    4. Shen L
    5. Zeng G
    6. Yao S
    7. Shen Y
    8. Halliday L
    9. Fortman J
    10. McAllister M
    11. Estep J
    12. Hunt R
    13. Vasconcelos D
    14. Du G
    15. Porcelli SA
    16. Larsen MH
    17. Jacobs WR
    18. Haynes BF
    19. Letvin NL
    20. Chen ZW

    (2009) A critical role for CD8 T cells in a nonhuman primate model of tuberculosis

    PLOS Pathogens 5:e1000392.

    https://doi.org/10.1371/journal.ppat.1000392

    • PubMed
    • Google Scholar
16. 1. Chen X
    2. Cheng H-F
    3. Zhou J
    4. Chan C-Y
    5. Lau K-F
    6. Tsui SK-W
    7. Au SW-N

    (2017) Structural basis of the PE-PPE protein interaction in Mycobacterium tuberculosis

    The Journal of Biological Chemistry 292:16880–16890.

    https://doi.org/10.1074/jbc.M117.802645

    • PubMed
    • Google Scholar
17. 1. Creasy BM
    2. Hartmann CB
    3. White FKH
    4. McCoy KL

    (2007) New assay using fluorogenic substrates and immunofluorescence staining to measure cysteine cathepsin activity in live cell subpopulations

    Cytometry. Part A 71:114–123.

    https://doi.org/10.1002/cyto.a.20365

    • PubMed
    • Google Scholar
18. 1. Cronin RM
    2. Ferrell MJ
    3. Cahir CW
    4. Champion MM
    5. Champion PA

    (2022) Proteo-genetic analysis reveals clear hierarchy of ESX-1 secretion in Mycobacterium marinum

    PNAS 119:e2123100119.

    https://doi.org/10.1073/pnas.2123100119

    • PubMed
    • Google Scholar
19. 1. Daleke MH
    2. van der Woude AD
    3. Parret AHA
    4. Ummels R
    5. de Groot AM
    6. Watson D
    7. Piersma SR
    8. Jiménez CR
    9. Luirink J
    10. Bitter W
    11. Houben ENG

    (2012) Specific chaperones for the type VII protein secretion pathway

    The Journal of Biological Chemistry 287:31939–31947.

    https://doi.org/10.1074/jbc.M112.397596

    • PubMed
    • Google Scholar
20. 1. Ekiert DC
    2. Cox JS

    (2014) Structure of a PE–PPE–espg complex from Mycobacterium tuberculosis reveals molecular specificity of Esx protein secretion

    PNAS 111:14758–14763.

    https://doi.org/10.1073/pnas.1409345111

    • Google Scholar
21. 1. Fine PEM

    (1995) Variation in protection by BCG: implications of and for heterologous immunity

    The Lancet 346:1339–1345.

    https://doi.org/10.1016/S0140-6736(95)92348-9

    • Google Scholar
22. 1. Flyer DC
    2. Ramakrishna V
    3. Miller C
    4. Myers H
    5. McDaniel M
    6. Root K
    7. Flournoy C
    8. Engelhard VH
    9. Canaday DH
    10. Marto JA
    11. Ross MM
    12. Hunt DF
    13. Shabanowitz J
    14. White FM

    (2002) Identification by mass spectrometry of CD8 (+) -T-cell Mycobacterium tuberculosis epitopes within the rv0341 gene product

    Infection and Immunity 70:2926–2932.

    https://doi.org/10.1128/IAI.70.6.2926-2932.2002

    • PubMed
    • Google Scholar
23. 1. Flynn JL
    2. Goldstein MM
    3. Triebold KJ
    4. Koller B
    5. Bloom BR

    (1992) Major histocompatibility complex class I-restricted T cells are required for resistance to Mycobacterium tuberculosis infection

    PNAS 89:12013–12017.

    https://doi.org/10.1073/pnas.89.24.12013

    • PubMed
    • Google Scholar
24. 1. Flynn JL
    2. Chan J
    3. Lin PL

    (2011) Macrophages and control of granulomatous inflammation in tuberculosis

    Mucosal Immunology 4:271–278.

    https://doi.org/10.1038/mi.2011.14

    • PubMed
    • Google Scholar
25. 1. Frigui W
    2. Bottai D
    3. Majlessi L
    4. Monot M
    5. Josselin E
    6. Brodin P
    7. Garnier T
    8. Gicquel B
    9. Martin C
    10. Leclerc C
    11. Cole ST
    12. Brosch R

    (2008) Control of M. tuberculosis ESAT-6 secretion and specific T cell recognition by PhoP

    PLOS Pathogens 4:e33.

    https://doi.org/10.1371/journal.ppat.0040033

    • PubMed
    • Google Scholar
26. 1. Fritsche J
    2. Kowalewski DJ
    3. Backert L
    4. Gwinner F
    5. Dorner S
    6. Priemer M
    7. Tsou CC
    8. Hoffgaard F
    9. Römer M
    10. Schuster H
    11. Schoor O
    12. Weinschenk T

    (2021) Pitfalls in HLA ligandomics-how to catch a Li (E) gand

    Molecular & Cellular Proteomics 20:100110.

    https://doi.org/10.1016/j.mcpro.2021.100110

    • PubMed
    • Google Scholar
27. 1. Gallien S
    2. Kim SY
    3. Domon B

    (2015) Large-Scale targeted proteomics using internal standard triggered-parallel reaction monitoring (IS-PRM)

    Molecular & Cellular Proteomics 14:1630–1644.

    https://doi.org/10.1074/mcp.O114.043968

    • PubMed
    • Google Scholar
28. 1. Geier E
    2. Pfeifer G
    3. Wilm M
    4. Lucchiari-Hartz M
    5. Baumeister W
    6. Eichmann K
    7. Niedermann G

    (1999) A giant protease with potential to substitute for some functions of the proteasome

    Science 283:978–981.

    https://doi.org/10.1126/science.283.5404.978

    • PubMed
    • Google Scholar
29. 1. Gordon SV
    2. Brosch R
    3. Billault A
    4. Garnier T
    5. Eiglmeier K
    6. Cole ST

    (1999) Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays

    Molecular Microbiology 32:643–655.

    https://doi.org/10.1046/j.1365-2958.1999.01383.x

    • PubMed
    • Google Scholar
30. 1. Grotzke JE
    2. Harriff MJ
    3. Siler AC
    4. Nolt D
    5. Delepine J
    6. Lewinsohn DA
    7. Lewinsohn DM

    (2009) The Mycobacterium tuberculosis phagosome is a HLA-I processing competent organelle

    PLOS Pathogens 5:e1000374.

    https://doi.org/10.1371/journal.ppat.1000374

    • PubMed
    • Google Scholar
31. 1. Grotzke JE
    2. Siler AC
    3. Lewinsohn DA
    4. Lewinsohn DM

    (2010) Secreted immunodominant Mycobacterium tuberculosis antigens are processed by the cytosolic pathway

    Journal of Immunology 185:4336–4343.

    https://doi.org/10.4049/jimmunol.1000801

    • PubMed
    • Google Scholar
32. 1. Grotzke JE
    2. Sengupta D
    3. Lu Q
    4. Cresswell P

    (2017) The ongoing SAGA of the mechanism (S) of MHC class I-restricted cross-presentation

    Current Opinion in Immunology 46:89–96.

    https://doi.org/10.1016/j.coi.2017.03.015

    • PubMed
    • Google Scholar
33. 1. Guil S
    2. Rodríguez-Castro M
    3. Aguilar F
    4. Villasevil EM
    5. Antón LC
    6. Del Val M

    (2006) Need for tripeptidyl-peptidase II in major histocompatibility complex class I viral antigen processing when proteasomes are detrimental

    Journal of Biological Chemistry 281:39925–39934.

    https://doi.org/10.1074/jbc.M608522200

    • Google Scholar
34. 1. Guinn KM
    2. Hickey MJ
    3. Mathur SK
    4. Zakel KL
    5. Grotzke JE
    6. Lewinsohn DM
    7. Smith S
    8. Sherman DR

    (2004) Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis: RD1 genes affect secretion and virulence of M. tuberculosis

    Molecular Microbiology 51:359–370.

    https://doi.org/10.1046/j.1365-2958.2003.03844.x

    • Google Scholar
35. Preprint

    1. Hallgren J
    2. Tsirigos KD
    3. Pedersen MD
    4. Almagro Armenteros JJ
    5. Marcatili P
    6. Nielsen H
    7. Krogh A
    8. Winther O

    (2022) DeepTMHMM Predicts Alpha and Beta Transmembrane Proteins Using Deep Neural Networks

    bioRxiv.

    https://doi.org/10.1101/2022.04.08.487609

    • Google Scholar
36. 1. Hulkower KI
    2. Butler CC
    3. Linebaugh BE
    4. Klaus JL
    5. Keppler D
    6. Giranda VL
    7. Sloane BF

    (2000) Fluorescent microplate assay for cancer cell-associated cathepsin B

    European Journal of Biochemistry 267:4165–4170.

    https://doi.org/10.1046/j.1432-1327.2000.01458.x

    • Google Scholar
37. 1. Izquierdo Lafuente B
    2. Ummels R
    3. Kuijl C
    4. Bitter W
    5. Speer A

    (2021) Mycobacterium tuberculosis toxin cpnt is an ESX-5 substrate and requires three type VII secretion systems for intracellular secretion

    MBio 12:e02983-20.

    https://doi.org/10.1128/mBio.02983-20

    • PubMed
    • Google Scholar
38. 1. Kamath AB
    2. Woodworth J
    3. Xiong X
    4. Taylor C
    5. Weng Y
    6. Behar SM

    (2004) Cytolytic CD8+ T cells recognizing CFP10 are recruited to the lung after Mycobacterium tuberculosis infection

    The Journal of Experimental Medicine 200:1479–1489.

    https://doi.org/10.1084/jem.20041690

    • PubMed
    • Google Scholar
39. 1. Kessler JH
    2. Khan S
    3. Seifert U
    4. Le Gall S
    5. Chow KM
    6. Paschen A
    7. Bres-Vloemans SA
    8. de Ru A
    9. van Montfoort N
    10. Franken KLMC
    11. Benckhuijsen WE
    12. Brooks JM
    13. van Hall T
    14. Ray K
    15. Mulder A
    16. Doxiadis IIN
    17. van Swieten PF
    18. Overkleeft HS
    19. Prat A
    20. Tomkinson B
    21. Neefjes J
    22. Kloetzel PM
    23. Rodgers DW
    24. Hersh LB
    25. Drijfhout JW
    26. van Veelen PA
    27. Ossendorp F
    28. Melief CJM

    (2011) Antigen processing by nardilysin and thimet oligopeptidase generates cytotoxic T cell epitopes

    Nature Immunology 12:45–53.

    https://doi.org/10.1038/ni.1974

    • PubMed
    • Google Scholar
40. 1. Kloetzel PM

    (2001) Antigen processing by the proteasome

    Nature Reviews. Molecular Cell Biology 2:179–187.

    https://doi.org/10.1038/35056572

    • PubMed
    • Google Scholar
41. 1. Lázaro S
    2. Gamarra D
    3. Del Val M

    (2015) Proteolytic enzymes involved in MHC class I antigen processing: a guerrilla army that partners with the proteasome

    Molecular Immunology 68:72–76.

    https://doi.org/10.1016/j.molimm.2015.04.014

    • PubMed
    • Google Scholar
42. Software

    1. Leddy O

    (2023) Local correlation analysis, version swh:1:rev:da390142bcc385d4a3bd12275241291a626ad426

    Software Heritage.

    https://archive.softwareheritage.org/swh:1:dir:39f77f0cbe8587a47955ba70bba162152bb07cb9;origin=https://github.com/oleddy/local_correlation_analysis;visit=swh:1:snp:bae11d47b63a1480bb3b697148891b588a693c0e;anchor=swh:1:rev:da390142bcc385d4a3bd12275241291a626ad426
43. 1. Lee JS
    2. Krause R
    3. Schreiber J
    4. Mollenkopf H-J
    5. Kowall J
    6. Stein R
    7. Jeon B-Y
    8. Kwak J-Y
    9. Song M-K
    10. Patron JP
    11. Jorg S
    12. Roh K
    13. Cho S-N
    14. Kaufmann SHE

    (2008) Mutation in the transcriptional regulator PhoP contributes to avirulence of Mycobacterium tuberculosis H37Ra strain

    Cell Host & Microbe 3:97–103.

    https://doi.org/10.1016/j.chom.2008.01.002

    • PubMed
    • Google Scholar
44. 1. Lewinsohn D.M.
    2. Grotzke JE
    3. Heinzel AS
    4. Zhu L
    5. Ovendale PJ
    6. Johnson M
    7. Alderson MR

    (2006) Secreted proteins from Mycobacterium tuberculosis gain access to the cytosolic MHC class-I antigen-processing pathway

    Journal of Immunology 177:437–442.

    https://doi.org/10.4049/jimmunol.177.1.437

    • PubMed
    • Google Scholar
45. 1. Lewinsohn DM
    2. Swarbrick GM
    3. Cansler ME
    4. Null MD
    5. Rajaraman V
    6. Frieder MM
    7. Sherman DR
    8. McWeeney S
    9. Lewinsohn DA
    10. Shams H

    (2013) Human Mycobacterium tuberculosis CD8 T cell antigens/epitopes identified by a proteomic peptide library

    PLOS ONE 8:e67016.

    https://doi.org/10.1371/journal.pone.0067016

    • Google Scholar
46. 1. Lewinsohn DA
    2. Swarbrick GM
    3. Park B
    4. Cansler ME
    5. Null MD
    6. Toren KG
    7. Baseke J
    8. Zalwango S
    9. Mayanja-Kizza H
    10. Malone LL
    11. Nyendak M
    12. Wu G
    13. Guinn K
    14. McWeeney S
    15. Mori T
    16. Chervenak KA
    17. Sherman DR
    18. Boom WH
    19. Lewinsohn DM

    (2017) Comprehensive definition of human immunodominant CD8 antigens in tuberculosis

    NPJ Vaccines 2:8.

    https://doi.org/10.1038/s41541-017-0008-6

    • PubMed
    • Google Scholar
47. 1. Lewis KN
    2. Liao R
    3. Guinn KM
    4. Hickey MJ
    5. Smith S
    6. Behr MA
    7. Sherman DR

    (2003) Deletion of RD1 from Mycobacterium tuberculosis mimics Bacille Calmette-Guérin attenuation

    The Journal of Infectious Diseases 187:117–123.

    https://doi.org/10.1086/345862

    • PubMed
    • Google Scholar
48. 1. Lorente E
    2. García R
    3. Mir C
    4. Barriga A
    5. Lemonnier FA
    6. Ramos M
    7. López D

    (2012) Role of metalloproteases in vaccinia virus epitope processing for transporter associated with antigen processing (TAP) -independent human leukocyte antigen (HLA) -B7 class I antigen presentation

    Journal of Biological Chemistry 287:9990–10000.

    https://doi.org/10.1074/jbc.M111.314856

    • Google Scholar
49. 1. Lou Y
    2. Rybniker J
    3. Sala C
    4. Cole ST

    (2017) EspC forms a filamentous structure in the cell envelope of Mycobacterium tuberculosis and impacts ESX-1 secretion

    Molecular Microbiology 103:26–38.

    https://doi.org/10.1111/mmi.13575

    • PubMed
    • Google Scholar
50. 1. Maman Y
    2. Nir-Paz R
    3. Louzoun Y

    (2011) Bacteria modulate the CD8+ T cell epitope repertoire of host cytosol-exposed proteins to manipulate the host immune response

    PLOS Computational Biology 7:e1002220.

    https://doi.org/10.1371/journal.pcbi.1002220

    • PubMed
    • Google Scholar
51. 1. Millington KA
    2. Fortune SM
    3. Low J
    4. Garces A
    5. Hingley-Wilson SM
    6. Wickremasinghe M
    7. Kon OM
    8. Lalvani A

    (2011) Rv3615c is a highly immunodominant RD1 (region of difference 1) -dependent secreted antigen specific for Mycobacterium tuberculosis infection

    PNAS 108:5730–5735.

    https://doi.org/10.1073/pnas.1015153108

    • PubMed
    • Google Scholar
52. 1. Mittal E
    2. Skowyra ML
    3. Uwase G
    4. Tinaztepe E
    5. Mehra A
    6. Köster S
    7. Hanson PI
    8. Philips JA

    (2018) Mycobacterium tuberculosis type VII secretion system effectors differentially impact the ESCRT endomembrane damage response

    MBio 9:e01765-18.

    https://doi.org/10.1128/mBio.01765-18

    • PubMed
    • Google Scholar
53. 1. Nair S
    2. Ramaswamy PA
    3. Ghosh S
    4. Joshi DC
    5. Pathak N
    6. Siddiqui I
    7. Sharma P
    8. Hasnain SE
    9. Mande SC
    10. Mukhopadhyay S

    (2009) The PPE18 of Mycobacterium tuberculosis interacts with TLR2 and activates IL-10 induction in macrophage

    The Journal of Immunology 183:6269–6281.

    https://doi.org/10.4049/jimmunol.0901367

    • Google Scholar
54. 1. Perez-Riverol Y
    2. Bai J
    3. Bandla C
    4. García-Seisdedos D
    5. Hewapathirana S
    6. Kamatchinathan S
    7. Kundu DJ
    8. Prakash A
    9. Frericks-Zipper A
    10. Eisenacher M
    11. Walzer M
    12. Wang S
    13. Brazma A
    14. Vizcaíno JA

    (2022) The pride database resources in 2022: a hub for mass spectrometry-based proteomics evidences

    Nucleic Acids Research 50:D543–D552.

    https://doi.org/10.1093/nar/gkab1038

    • PubMed
    • Google Scholar
55. 1. Pym AS
    2. Brodin P
    3. Brosch R
    4. Huerre M
    5. Cole ST

    (2002) Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti

    Molecular Microbiology 46:709–717.

    https://doi.org/10.1046/j.1365-2958.2002.03237.x

    • PubMed
    • Google Scholar
56. 1. Pym AS
    2. Brodin P
    3. Majlessi L
    4. Brosch R
    5. Demangel C
    6. Williams A
    7. Griffiths KE
    8. Marchal G
    9. Leclerc C
    10. Cole ST

    (2003) Recombinant BCG exporting ESAT-6 confers enhanced protection against tuberculosis

    Nature Medicine 9:533–539.

    https://doi.org/10.1038/nm859

    • PubMed
    • Google Scholar
57. 1. Reynisson B
    2. Alvarez B
    3. Paul S
    4. Peters B
    5. Nielsen M

    (2020) NetMHCpan-4.1 and netmhciipan-4.0: improved predictions of MHC antigen presentation by concurrent motif deconvolution and integration of MS MHC eluted ligand data

    Nucleic Acids Research 48:W449–W454.

    https://doi.org/10.1093/nar/gkaa379

    • PubMed
    • Google Scholar
58. 1. Roy A
    2. Eisenhut M
    3. Harris RJ
    4. Rodrigues LC
    5. Sridhar S
    6. Habermann S
    7. Snell L
    8. Mangtani P
    9. Adetifa I
    10. Lalvani A
    11. Abubakar I

    (2014) Effect of BCG vaccination against Mycobacterium tuberculosis infection in children: systematic review and meta-analysis

    BMJ 349:g4643.

    https://doi.org/10.1136/bmj.g4643

    • PubMed
    • Google Scholar
59. 1. Saelens JW
    2. Sweeney MI
    3. Viswanathan G
    4. Xet-Mull AM
    5. Jurcic Smith KL
    6. Sisk DM
    7. Hu DD
    8. Cronin RM
    9. Hughes EJ
    10. Brewer WJ
    11. Coers J
    12. Champion MM
    13. Champion PA
    14. Lowe CB
    15. Smith CM
    16. Lee S
    17. Stout JE
    18. Tobin DM

    (2022) An ancestral mycobacterial effector promotes dissemination of infection

    Cell 185:4507–4525.

    https://doi.org/10.1016/j.cell.2022.10.019

    • PubMed
    • Google Scholar
60. 1. Sampson SL
    2. Lukey P
    3. Warren RM
    4. van Helden PD
    5. Richardson M
    6. Everett MJ

    (2001) Expression, characterization and subcellular localization of the Mycobacterium tuberculosis PPE gene Rv1917c

    Tuberculosis 81:305–317.

    https://doi.org/10.1054/tube.2001.0304

    • Google Scholar
61. 1. Schubert OT
    2. Ludwig C
    3. Kogadeeva M
    4. Zimmermann M
    5. Rosenberger G
    6. Gengenbacher M
    7. Gillet LC
    8. Collins BC
    9. Röst HL
    10. Kaufmann SHE
    11. Sauer U
    12. Aebersold R

    (2015) Absolute proteome composition and dynamics during dormancy and resuscitation of Mycobacterium tuberculosis

    Cell Host & Microbe 18:96–108.

    https://doi.org/10.1016/j.chom.2015.06.001

    • PubMed
    • Google Scholar
62. 1. Shah S
    2. Cannon JR
    3. Fenselau C
    4. Briken V

    (2015) A duplicated ESAT-6 region of ESX-5 is involved in protein export and virulence of mycobacteria

    Infection and Immunity 83:4349–4361.

    https://doi.org/10.1128/IAI.00827-15

    • PubMed
    • Google Scholar
63. 1. Shah S
    2. Briken V

    (2016) Modular organization of the ESX-5 secretion system in Mycobacterium tuberculosis

    Frontiers in Cellular and Infection Microbiology 6:49.

    https://doi.org/10.3389/fcimb.2016.00049

    • PubMed
    • Google Scholar
64. 1. Shen H
    2. Miller JF
    3. Fan X
    4. Kolwyck D
    5. Ahmed R
    6. Harty JT

    (1998) Compartmentalization of bacterial antigens: differential effects on priming of CD8 T cells and protective immunity

    Cell 92:535–545.

    https://doi.org/10.1016/s0092-8674(00)80946-0

    • PubMed
    • Google Scholar
65. 1. Shen L
    2. Sigal LJ
    3. Boes M
    4. Rock KL

    (2004) Important role of cathepsin S in generating peptides for TAP-independent MHC class I crosspresentation in vivo

    Immunity 21:155–165.

    https://doi.org/10.1016/j.immuni.2004.07.004

    • PubMed
    • Google Scholar
66. 1. Siegrist MS
    2. Unnikrishnan M
    3. McConnell MJ
    4. Borowsky M
    5. Cheng TY
    6. Siddiqi N
    7. Fortune SM
    8. Moody DB
    9. Rubin EJ

    (2009) Mycobacterial ESX-3 is required for mycobactin-mediated iron acquisition

    PNAS 106:18792–18797.

    https://doi.org/10.1073/pnas.0900589106

    • PubMed
    • Google Scholar
67. 1. Simeone R
    2. Bobard A
    3. Lippmann J
    4. Bitter W
    5. Majlessi L
    6. Brosch R
    7. Enninga J

    (2012) Phagosomal rupture by Mycobacterium tuberculosis results in toxicity and host cell death

    PLOS Pathogens 8:e1002507.

    https://doi.org/10.1371/journal.ppat.1002507

    • PubMed
    • Google Scholar
68. 1. Solans L
    2. Aguiló N
    3. Samper S
    4. Pawlik A
    5. Frigui W
    6. Martín C
    7. Brosch R
    8. Gonzalo-Asensio J

    (2014) A specific polymorphism in Mycobacterium tuberculosis H37Rv causes differential ESAT-6 expression and identifies whib6 as a novel ESX-1 component

    Infection and Immunity 82:3446–3456.

    https://doi.org/10.1128/IAI.01824-14

    • PubMed
    • Google Scholar
69. 1. Stanley SA
    2. Raghavan S
    3. Hwang WW
    4. Cox JS

    (2003) Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system

    PNAS 100:13001–13006.

    https://doi.org/10.1073/pnas.2235593100

    • PubMed
    • Google Scholar
70. 1. Stanley SA
    2. Johndrow JE
    3. Manzanillo P
    4. Cox JS

    (2007) The type I IFN response to infection with Mycobacterium tuberculosis requires ESX-1-mediated secretion and contributes to pathogenesis

    Journal of Immunology 178:3143–3152.

    https://doi.org/10.4049/jimmunol.178.5.3143

    • PubMed
    • Google Scholar
71. 1. Stopfer LE
    2. Mesfin JM
    3. Joughin BA
    4. Lauffenburger DA
    5. White FM

    (2020) Multiplexed relative and absolute quantitative immunopeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition

    Nature Communications 11:2760.

    https://doi.org/10.1038/s41467-020-16588-9

    • PubMed
    • Google Scholar
72. 1. Stopfer LE
    2. Flower CT
    3. Gajadhar AS
    4. Patel B
    5. Gallien S
    6. Lopez-Ferrer D
    7. White FM

    (2021a) High-Density, targeted monitoring of tyrosine phosphorylation reveals activated signaling networks in human tumors

    Cancer Research 81:2495–2509.

    https://doi.org/10.1158/0008-5472.CAN-20-3804

    • PubMed
    • Google Scholar
73. 1. Stopfer LE
    2. Gajadhar AS
    3. Patel B
    4. Gallien S
    5. Frederick DT
    6. Boland GM
    7. Sullivan RJ
    8. White FM

    (2021b) Absolute quantification of tumor antigens using embedded MHC-I isotopologue calibrants

    PNAS 118:e2111173118.

    https://doi.org/10.1073/pnas.2111173118

    • PubMed
    • Google Scholar
74. 1. Su H
    2. Zhang Z
    3. Liu Z
    4. Peng B
    5. Kong C
    6. Wang H
    7. Zhang Z
    8. Xu Y

    (2018) Mycobacterium tuberculosis PPE60 antigen drives Th1/Th17 responses via Toll-like receptor 2–dependent maturation of dendritic cells

    Journal of Biological Chemistry 293:10287–10302.

    https://doi.org/10.1074/jbc.RA118.001696

    • Google Scholar
75. 1. Teufel F
    2. Almagro Armenteros JJ
    3. Johansen AR
    4. Gíslason MH
    5. Pihl SI
    6. Tsirigos KD
    7. Winther O
    8. Brunak S
    9. von Heijne G
    10. Nielsen H

    (2022) SignalP 6.0 predicts all five types of signal peptides using protein language models

    Nature Biotechnology 40:1023–1025.

    https://doi.org/10.1038/s41587-021-01156-3

    • PubMed
    • Google Scholar
76. 1. Tufariello JM
    2. Chapman JR
    3. Kerantzas CA
    4. Wong KW
    5. Vilchèze C
    6. Jones CM
    7. Cole LE
    8. Tinaztepe E
    9. Thompson V
    10. Fenyö D
    11. Niederweis M
    12. Ueberheide B
    13. Philips JA
    14. Jacobs WR

    (2016) Separable roles for Mycobacterium tuberculosis ESX-3 effectors in iron acquisition and virulence

    PNAS 113:E348–E357.

    https://doi.org/10.1073/pnas.1523321113

    • PubMed
    • Google Scholar
77. 1. Tully G
    2. Kortsik C
    3. Höhn H
    4. Zehbe I
    5. Hitzler WE
    6. Neukirch C
    7. Freitag K
    8. Kayser K
    9. Maeurer MJ

    (2005) Highly focused T cell responses in latent human pulmonary Mycobacterium tuberculosis infection

    Journal of Immunology 174:2174–2184.

    https://doi.org/10.4049/jimmunol.174.4.2174

    • PubMed
    • Google Scholar
78. 1. Uplekar S
    2. Heym B
    3. Friocourt V
    4. Rougemont J
    5. Cole ST
    6. Flynn JL

    (2011) Comparative genomics of Esx genes from clinical isolates of Mycobacterium tuberculosis provides evidence for gene conversion and epitope variation

    Infection and Immunity 79:4042–4049.

    https://doi.org/10.1128/IAI.05344-11

    • Google Scholar
79. 1. van der Wel N
    2. Hava D
    3. Houben D
    4. Fluitsma D
    5. van Zon M
    6. Pierson J
    7. Brenner M
    8. Peters PJ

    (2007) M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells

    Cell 129:1287–1298.

    https://doi.org/10.1016/j.cell.2007.05.059

    • PubMed
    • Google Scholar
80. 1. Wang Q
    2. Boshoff HIM
    3. Harrison JR
    4. Ray PC
    5. Green SR
    6. Wyatt PG
    7. Barry CE III

    (2020) PE/PPE proteins mediate nutrient transport across the outer membrane of Mycobacterium tuberculosis

    Science 367:1147–1151.

    https://doi.org/10.1126/science.aav5912

    • Google Scholar
81. 1. Watson RO
    2. Manzanillo PS
    3. Cox JS

    (2012) Extracellular M. tuberculosis DNA targets bacteria for autophagy by activating the host DNA-sensing pathway

    Cell 150:803–815.

    https://doi.org/10.1016/j.cell.2012.06.040

    • PubMed
    • Google Scholar
82. 1. Woodworth JS
    2. Fortune SM
    3. Behar SM

    (2008a) Bacterial protein secretion is required for priming of CD8+ T cells specific for the Mycobacterium tuberculosis antigen CFP10

    Infection and Immunity 76:4199–4205.

    https://doi.org/10.1128/IAI.00307-08

    • PubMed
    • Google Scholar
83. 1. Woodworth JS
    2. Wu Y
    3. Behar SM

    (2008b) Mycobacterium tuberculosis-specific CD8+ T cells require perforin to kill target cells and provide protection in vivo

    Journal of Immunology 181:8595–8603.

    https://doi.org/10.4049/jimmunol.181.12.8595

    • PubMed
    • Google Scholar
84. Software

    1. World Health Organization 2022

    (2021)

    Global tuberculosis report, version 1.1.1

    World Health Organization.
85. 1. Yang JD
    2. Mott D
    3. Sutiwisesak R
    4. Lu YJ
    5. Raso F
    6. Stowell B
    7. Babunovic GH
    8. Lee J
    9. Carpenter SM
    10. Way SS
    11. Fortune SM
    12. Behar SM

    (2018) Mycobacterium tuberculosis-specific CD4+ and CD8+ T cells differ in their capacity to recognize infected macrophages

    PLOS Pathogens 14:e1007060.

    https://doi.org/10.1371/journal.ppat.1007060

    • PubMed
    • Google Scholar
86. 1. York IA
    2. Bhutani N
    3. Zendzian S
    4. Goldberg AL
    5. Rock KL

    (2006) Tripeptidyl peptidase II is the major peptidase needed to TRIM long antigenic precursors, but is not required for most MHC class I antigen presentation

    Journal of Immunology 177:1434–1443.

    https://doi.org/10.4049/jimmunol.177.3.1434

    • PubMed
    • Google Scholar
87. 1. Zheng YT
    2. Shahnazari S
    3. Brech A
    4. Lamark T
    5. Johansen T
    6. Brumell JH

    (2009) The adaptor protein p62/SQSTM1 targets invading bacteria to the autophagy pathway

    Journal of Immunology 183:5909–5916.

    https://doi.org/10.4049/jimmunol.0900441

    • PubMed
    • Google Scholar

Author details

1. Owen Leddy

   1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States
   2. Ragon Institute of Massachusetts General Hospital, Harvard, and MIT, Cambridge, United States
   3. Koch Institute for Integrative Cancer Research, Cambridge, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3437-1956

2. Forest M White

   1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States
   2. Koch Institute for Integrative Cancer Research, Cambridge, United States
   3. Center for Precision Cancer Medicine, Cambridge, United States

   Contribution

   Conceptualization, Data curation, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Contributed equally with

   Bryan D Bryson

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1545-1651

3. Bryan D Bryson

   1. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States
   2. Ragon Institute of Massachusetts General Hospital, Harvard, and MIT, Cambridge, United States

   Contribution

   Conceptualization, Data curation, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Contributed equally with

   Forest M White

   For correspondence

   bryand@mit.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1716-6712

• 3,115

  views

• 427

  downloads

• 15

  citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.