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.

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Source data for enrichment analysis.

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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.

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Colocalization quantification source data.

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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).

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

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CXCL10 ELISA raw data.

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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).

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Relative abundances of MHC-I peptides determined by SureQuant.

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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).

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   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

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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

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