Immunopeptidomics reveals determinants of Mycobacterium tuberculosis antigen presentation on MHC class I

  1. Owen Leddy
  2. Forest M White
  3. Bryan D Bryson

  • Curated by eLife

    eLife logo

    eLife assessment

    This landmark study uses compelling approaches such as quantitative and screening mass spectrometry to identify peptides from tuberculosis bacteria that are presented by macrophages infected with this pathogen. The authors provide convincing evidence that the presentation of these antigens depends on a specialist bacterial secretion system. The study will be of interest to infectious disease specialists and of particular value for future vaccine development.

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

CD8+ T cell recognition of Mycobacterium tuberculosis ( Mtb )-specific peptides presented on major histocompatibility complex class I (MHC-I) contributes to immunity to tuberculosis (TB), but the principles that govern presentation of Mtb antigens on MHC-I are incompletely understood. In this study, mass spectrometry (MS) analysis of the MHC-I repertoire of Mtb -infected primary human macrophages reveals that substrates of Mtb ’s type VII secretion systems (T7SS) are overrepresented among Mtb -derived peptides presented on MHC-I. Quantitative, targeted MS shows that ESX-1 activity is required for presentation of Mtb peptides derived from both ESX-1 substrates and ESX-5 substrates on MHC-I, consistent with a model in which proteins secreted by multiple T7SSs access a cytosolic antigen processing pathway via ESX-1-mediated phagosome permeabilization. Chemical inhibition of proteasome activity, lysosomal acidification, or cysteine cathepsin activity did not block presentation of Mtb antigens on MHC-I, suggesting involvement of other proteolytic pathways or redundancy among multiple pathways. Our study identifies Mtb antigens presented on MHC-I that could serve as targets for TB vaccines, and reveals how the activity of multiple T7SSs interacts to contribute to presentation of Mtb antigens on MHC-I.

Article activity feed

  1. Version published to 10.7554/elife.84070 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    By performing immunopeptidomics of macrophages infected with virulent M. tuberculosis, the authors were able to appropriately address whether Mtb proteins are able to enter the MHC-I antigen processing pathway. Their interrogation provides convincing evidence that substrates of Mtb's type VII secretion systems (T7SS) are a significant contributor to the Mtb-derived peptides presented on MHC-I. Compelling data are provided to demonstrate that ESX-1 activity is required for the MHC-1 presentation of these newly identified peptides.

    Strength

    Employing a virulent strain of Mtb for infection of human monocyte-derived macrophages to identify Mtb proteins that access the MHC-I antigen processing pathways and the associated mechanisms.

    Weakness

    The immunogenicity of at least some of the identified peptides should have been evaluated.

    Although obtaining T cells from a cohort of TB-exposed patients was not within the scope of this study, we are also eager to assess the immunogenicity of the epitopes we identified in future work. In addition to the references we made in our initial submission to prior work showing that many of the proteins from which the epitopes we identified derive elicit T cell responses in Mtb-exposed humans, we’ve added references to prior studies that show that a few of the specific epitopes we identified are immunogenic, providing at least a preliminary indication that MHC-I peptides identified by MS can be immunogenic T cell epitopes (lines 420-423): “Individual peptides we identified by MS have also been previously shown to be recognized by human T cells, including EsxJ24-34 (Grotzke et al., 2010; Lewinsohn et al., 2013) and EsxA28-36 (Tully et al., 2005), providing a proof of concept that particular epitopes identified by MS can be immunogenic.”

  3. eLife

    eLife assessment

    This landmark study uses compelling approaches such as quantitative and screening mass spectrometry to identify peptides from tuberculosis bacteria that are presented by macrophages infected with this pathogen. The authors provide convincing evidence that the presentation of these antigens depends on a specialist bacterial secretion system. The study will be of interest to infectious disease specialists and of particular value for future vaccine development.

  4. eLife

    Reviewer #1 (Public Review):

    By performing immunopeptidomics of macrophages infected with virulent M. tuberculosis, the authors were able to appropriately address whether Mtb proteins are able to enter the MHC-I antigen processing pathway. Their interrogation provides convincing evidence that substrates of Mtb's type VII secretion systems (T7SS) are a significant contributor to the Mtb-derived peptides presented on MHC-I. Compelling data are provided to demonstrate that ESX-1 activity is required for the MHC-1 presentation of these newly identified peptides.

    Strength:

    Employing a virulent strain of Mtb for infection of human monocyte-derived macrophages to identify Mtb proteins that access the MHC-I antigen processing pathways and the associated mechanisms.

    Weakness:

    The immunogenicity of at least some of the identified peptides should have been evaluated.

  5. eLife

    Reviewer #2 (Public Review):

    In this study, the authors were seeking to determine the major antigens presented by the MHC-1 complex during the infection of human macrophages with virulent M. tuberculosis. Major strengths include rigorous and well-controlled experiments. The careful identification of mycobacterial peptides in the context of host peptides was impressive and well done. The results generally support the conclusions drawn in the study. Overall, the study is well-presented and rigorous and adds new knowledge to the field. This study provides new information regarding the mycobacterial peptides that are presented to the immune system via the MHC pathway, and the role of alternate secretion systems and known peptide processing pathways during M. tuberculosis infection of human macrophages. Importantly, adapting a protocol to identify MHC antigens in the BSL-3 pathogen will be of use to several fields. However, the study could be further strengthened by improving the discussion of prior work in the ESX and MHC fields to strengthen the context of this work and clarify its contribution to the field, as well as considering potential weaknesses of the study.

  6. eLife

    Reviewer #3 (Public Review):

    Mtb antigens were traditionally discovered through crude direct methods such as immune-blotting of Mycobacterium tuberculosis (Mtb) culture filtrate (or whole cell lysate), or indirectly through T cell / APC stimulation experiments. The manuscript addresses the critical question of which Mycobacterium tuberculosis (Mtb) antigens are presented in peptide form on the surface of macrophages that are actually infected with Mtb. The identification of such antigens is particularly important for defining targets for TB vaccine design since CD8 T cells are an important component of the adaptive immune response to Mtb and macrophages are the most important phagocyte target of Mtb infection. The authors directly isolate MHC-I molecules from human monocyte-derived macrophages, elute MHC-I bound peptides from several HLA types, and screen for sequences found among Mtb antigens, which they find to represent only 0.1% of all peptides screened. The authors make the interesting observation that the majority of peptides identified (13 of 16) correspond to antigens secreted by the unique Type-7 Secretion System (T7SS) of Mtb. Another strength is the experiments to determine whether these T7SS substrates preferentially gain access to the cytosol for MHC-I loading via phagosome permeabilization by identifying the colocalization of Mtb with markers of phagosomal membrane damage (rather than MHC-I). The authors used quantitative mass spec to quantify and compare the expression of two peptides presented on HLA-A*02:01 and -B*57:01, demonstrating similar expression after infection with H37Rv, but that infection with an Esx1-deficient Mtb mutant did not lead to the presentation of either peptide even though one of these peptides was part of a separate Esx locus. Although only two peptides were assessed and compared using quantitative mass spec, these data imply that Esx1 was required for the presentation of the antigens from which both peptides were derived. While the exact mechanism of antigen processing for HLA-I presentation is still unclear for the EsxA and EsxJKPW peptides, the authors tested several pathways including inhibitors of proteasome activity, cysteine cathepsin activity, and lysosomal acidification. In future follow-up studies, it would also be useful to know whether the pulldown of a broader selection of HLA-I alleles would yield the same peptides/classes of peptides vs. a broader repertoire. The conclusions of this paper are well-supported by the data. This rigorous analysis of peptides presented on macrophages in the context of Mtb infection will establish a precedent for use of these techniques to discover additional antigens and will inform vaccine development efforts.

  7. Version published to 10.1101/2022.08.30.505882v1 on bioRxiv