Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens

Reviewer #1 (Public review):

Summary:

The manuscript "Synaptotagmin 1 and Synaptotagmin 7 promote MR1-mediated presentation of Mycobacterium tuberculosis antigens", authored by Kim et al., showed that the calcium-sensing trafficking proteins Synaptotagmin (Syt) 1 and Syt7 specifically promote (are critical for) MAIT cell activation in response to Mtb-infected bronchial epithelial cell line BEAS-2B (Fig. 1) and monocyte-like cell line THP-1 (Figure 3) . This work also showed co-localization of Syt1 and Syt7 with Rab7a and Lamp1, but not with Rab5a (Figure 5). Loss of Syt1 and Syt7 resulted in a larger area of MR1 vesicles (Figure 6f) and an increased number of MR1 vesicles in close proximity to an Auxotrophic Mtb-containing vacuoles during infection (Figure 7ab). Moreover, flow organellometry was used to separate phagosomes from other subcellular fractions and identify enrichment of auxotrophic Mtb-containing vacuoles in fractions 42-50, which were enriched with Lamp1+ vacuoles or phagosomes (Figures 7e-f).

Strengths:

This work nicely associated Syt1 and Syt7 with late endocytic compartments and Mtb+ vacuoles. Gene editing of Syt1 and Syt7 loci of bronchial epithelial and monocyte-like cells supported Syt1 and Syt7 facilitated maintaining a normal level of antigen presentation for MAIT cell activation in Mtb infection. Imaging analyses further supported that Syt1 and Syt7 mutants enhanced the overlaps of MR1 with Mtb fluorescence, and the MR1 proximity with Mtb-infected vacuoles, suggesting that Syt1 and Syt7 proteins help antigen presentation in Mtb infection for MAIT activation.

Weaknesses:

Additional data are needed to support the conclusion, "identify a novel pathway in which Syt1 and Syt7 facilitate the translocation of MR1 from Mtb-containing vacuoles" and some pieces of other evidence may be seen by some to contradict this conclusion.

Reviewer #2 (Public review):

Summary:

The study demonstrates that calcium-sensing trafficking proteins Synaptotagmin (Syt) 1 and Syt7 are involved in the efficient presentation of mycobacterial antigens by MR1 during M. tuberculosis infection.

This is achieved by creating antigen-presenting cells in which the Syt1 and Syt7 genes are knocked out. These mutated cell lines show significantly reduced stimulation of MAIT cells, while their stimulation of HLA class I-restricted T cells remains unchanged. Syt1 and Syt7 co-localize in a late endo-lysosomal compartment where MR1 molecules are also located, near M. tuberculosis-containing vacuoles.

Strengths:

This work uncovers a new aspect of how mycobacterial antigens generated during infection are presented. The finding that Syt1 and Syt7 are relevant for final MR1 surface expression and presentation to MR1-restricted T cells is novel and adds valuable information to this process.

The experiments include all necessary controls and convincingly validate the role of Syt1 and Syt7.

Another key point is that these proteins are essential during infection, but they are not significant when an exogenous synthetic antigen is used in the experiments. This emphasizes the importance of studying infection as a physiological context for antigen presentation to MAIT cells.

An additional relevant aspect is that the study reveals the existence of different MR1 antigen presentation pathways, which differ from the endoplasmic reticulum or endosomal pathways that are typical for MHC-presented peptides.

Weaknesses:

The reduced MAIT cell response observed with Syt1 and Syt7-deficient cell lines is statistically significant but not completely abolished. This may suggest that only some MR1-loaded molecules depend on these two Syt proteins. Further research is needed to determine whether, during persistent M. tuberculosis infection, enough MR1-loaded molecules are produced and transported to the plasma membrane to sufficiently stimulate MAIT cells.

The study proposes that other Syt proteins might also play a role, as outlined by the authors. However, exploring potential redundant mechanisms that facilitate MR1 loading with antigens remains a challenging task.

Reviewer #3 (Public review):

Summary:

In the submitted manuscript, the authors investigate the role of Synaptotagmins (Syt1) and (Syt7) in MR1 presentation of MtB.

Strengths:

In the first series of experiments, the authors determined that knocking down Syt1 and Sy7 in antigen-presenting cells decreases IFN-γ production following cellular infection with Mtb. These experiments are well performed and controlled.

Weaknesses:

Next, they aim to mechanistically investigate how Syt1 and Syt7 affect MtB presentation. In particular, they focus on MR1, a non-classical MHC-I molecule known to present endogenous and exogenous metabolites, including MtB metabolites.

Results from these next series of experiments are less clear. Firstly, they show that knocking down Syt1 and Sy7 does not change MtB phagocytosis as well as MR1 ER-plasma membrane translocation. Based on this, they suggest that Syt1 and Syt7 may affect MR1 trafficking in endosomal compartments. However, neither subcellular compartment analysis nor flow organelleometry clearly establishes the role of Syt1 and Syt7 in MtB trafficking.

Altogether, the notion that Synaptotagmins facilitate MR1 interaction with Mtb-containing compartments and its vesicular transport was already known. As such, the manuscript should add additional insight on where/how the interaction occurs. The reviewer is left with the notion that Syt1 and Sy7 may affect MR1 presentation, facilitating the trafficking of MR1 vesicles from endosomal compartments to either the cell surface or other endosomal compartments. The analysis is observational and additional data or discussion could address what the insight gained beyond what is already known from the literature.