Mucosal-Associated Invariant T (MAIT) cells are highly activated in duodenal tissue of humans with Vibrio cholerae O1 infection: A preliminary report.

Mucosal-associated invariant T (MAIT) cells are unconventional T lymphocytes with a semi-conserved TCRα, activated by the presentation of vitamin B metabolites by the MHC-I related protein, MR1, and with diverse innate and adaptive effector functions. The role of MAIT cells in acute intestinal infections, especially at the mucosal level, is not well known. Here, we analyzed the presence and phenotype of MAIT cells in duodenal biopsies and paired peripheral blood samples, in patients during and after culture-confirmed Vibrio cholerae O1 infection. Immunohistochemical staining of duodenal biopsies from cholera patients (n = 5, median age 32 years, range 26-44, 1 female) identified MAIT cells in the lamina propria of the crypts, but not the villi. By flow cytometry (n = 10, median age 31 years, range 23-36, 1 female), we showed that duodenal MAIT cells are more activated than peripheral MAIT cells (p < 0.01 across time points), although there were no significant differences between duodenal MAIT cells at day 2 and day 30. We found fecal markers of intestinal permeability and inflammation to be correlated with the loss of duodenal (but not peripheral) MAIT cells, and single-cell sequencing revealed differing T cell receptor usage between the duodenal and peripheral blood MAIT cells. In this preliminary report limited by a small sample size, we show that MAIT cells are present in the lamina propria of the duodenum during V. cholerae infection, and more activated than those in the blood. Future work into the trafficking and tissue-resident function of MAIT cells is warranted.

Introduction

Mucosal-associated invariant T (MAIT) cells are a recently identified non-conventional T cell subset. They express an invariant T cell receptor (TCR) Vα chain (Vα7.2 or TRAV1-2 in humans) and a variable but restricted number of TCRβ chains. MAIT cells are found in mucosal tissues and associated organs, including the liver, lung, mesenteric lymph nodes, and intestinal epithelium [1]. In human peripheral blood, MAIT cells constitute approximately 1–10% of total T lymphocytes [2], and they account for up to 40% of T cells in the liver [3]. In the human intestine, they are located in both the lamina propria and as part of the intraepithelial lymphocyte compartment [4]. The antigen for MAIT cells has been identified as belonging to a class of transitory intermediates of the riboflavin synthesis pathway [5], which are produced by many, but not all, bacteria and yeast. These vitamin B metabolites are presented on the surface of MR1 [6], the non-polymorphic MHC class I related protein. MAIT cells are capable of releasing IFN-γ, TNF, and IL-17 in response to stimulation, and they also possess cytotoxic activity [7, 8], killing infected cells via granzyme B and perforin.

Cholera is a life-threatening diarrheal disease caused primarily by Vibrio cholerae O1, responsible for close to 3 million cases and 100,000 deaths annually in endemic countries alone [9]. The mechanisms of protection against cholera are not well understood, although we have previously shown that in patients hospitalized with severe cholera, both adaptive and innate immune responses are induced. We have demonstrated increases in circulating V. cholerae antigen-specific antibodies, as well as antigen-specific memory B and memory T cell responses in both children and adults after cholera infection [10, 11]. We have also shown, through endoscopically-obtained duodenal biopsies, that there is an increase in cells of the innate immune system and their mediators during acute cholera [12, 13].

We have previously reported that circulating MAIT cells are activated during V. cholerae O1 infection, and that in children, but not adults, their circulating numbers are significantly decreased by day 7 after infection and onward [14]. We also demonstrated an association between circulating MAIT cells and class-switched antibody responses against V. cholerae lipopolysaccharide (LPS). Despite their abundance in the intestinal mucosa, little is known regarding the activity of MAIT cells in mucosal tissue during acute enteric infection. Thus, our objective was to characterize MAIT cells in the intestinal mucosa during V. cholerae infection.

Methods

Ethics

This study was approved by the Ethical Review and Research Review Committees of the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b), and the Institutional Review Boards of Massachusetts General Hospital and the University of Utah.

Study population and sample collection

We enrolled patients admitted to the Dhaka Hospital of the icddr,b who had acute watery diarrhea and positive stool cultures for V. cholerae O1. We recruited patients who had no underlying medical conditions and had an otherwise normal physical examination and baseline laboratory parameters. All patients were rehydrated and provided antibiotics per hospital protocol (a minimum of 18–24 hours prior to enrollment) and were hemodynamically stable at the time of the procedure. After written informed consent, we performed esophagogastroduodenoscopy (EGD) on day 2 (acute illness phase), day 30 (convalescent phase), and day 180 (late convalescent phase) following admission. Using standard forceps, we obtained approximately six duodenal pinch biopsies (from second part of duodenum) of approximately 1 mm3 in diameter from each patient at each time point. We also obtained a stool sample from each patient at day 2 and venous blood samples at days 2, 7, 30 and 180. To characterize MAIT cell presence and activity in the duodenum during cholera, we obtained duodenal biopsies from a total of 15 cholera patients, on 10 of whom we performed flow cytometric (FC) analysis and 5 of whom we performed immunohistochemical (IHC) analysis (S1 Table). The median age of the FC group was 31 (range 23 to 36) years, and the median age of the IHC group was 30 (range 26–44) years. Only one female was recruited for each group. All patients mounted at least a 16-fold rise in vibriocidal response by day 7 of illness. These patients were from two larger cohorts named “SEGD” and “PIC”, both of which had paired biopsy and blood specimens. However, histology was performed from “PIC” patients only and flow cytometry was done in “SEGD” patients only (S1 Table).

Phenotyping of MAIT cells by flow cytometry

From venous blood samples, we isolated peripheral blood mononuclear cells (PBMCs) by differential centrifugation on Ficoll-Isopaque (Pharmacia, Piscataway, NJ). We stored plasma at -80°C for use in immunological assays as detailed below. From duodenal biopsy samples, we isolated lamina propria lymphocytes (LPLs) by incubation in 1 mM EDTA and 1 mM dithiothreitol (DTT) followed by filtering through a nylon cell strainer and treatment with collagenase and DNase, as we have previously described [15]. We washed and stained the freshly isolated PBMCs and LPLs with an established fluorochrome-conjugated antibody panel designed for MAIT cell isolation. Antibodies (S2 Table) were purchased from BioLegend (San Diego, CA), BD Biosciences (San Jose, CA), or Life Technologies (Carlsbad, CA): TCR Vα7.2-PE (Clone: 3C10, Cat# 351706, Biolegend), CD3-PE-Texas Red (Clone: 7D6, Cat# MHCD0317, Life technologies), CD4-Amcyan (Clone: SK3, Cat# 339187, BD), CD8-FITC (Clone: RPA-T8, Cat# 561948, BD Pharmingen), CD161-APC (Clone: DX12, Cat# 550968, BD Pharmingen), CD38-PE-Cy7 (Clone: HIT2, Cat# 303516, Biolegend), CD69-PerCP-Cy5.5 (Clone: FN50, Cat# 560738, BD Pharmingen), and DAPI (Cat# 564907, BD Pharmingen). After 45 minutes incubation at 4°C, we analyzed at least 105 lymphocytes on a FACSAria III flow cytometer (BD Biosciences, San Jose, CA) and analyzed data using FlowJo 10 software (TreeStar Inc, Ashland, OR). We used Cytometer Setup & Tracking beads (BD Biosciences) to check for inter-day variability, and Fluorescence Minus One (FMO) controls. We defined MAIT cells as live (DAPI−) CD3+CD161hiVα7.2+ cells, expressed as a percentage of total CD3+ lymphocytes, and used CD38 and CD69 as markers of cell activation.

Immunohistochemistry

In a separate set of patients, we embedded cryosections from duodenal biopsies in Tissue Tek OCT compound (Sakura USA, Torrance, CA) and used a Leica CM3000 Cryostat (Leica Instruments GmbH, Nussloch, Germany) to cut 5 μm sections, picked up on poly-L-lysine coated slides, and air-dried. In line with immunohistochemical methods from reports available at time of the experiments [16, 17], we stained the sections with primary antibodies against CD3, IL-18Rα, and Vα7.2, followed by corresponding secondary antibodies conjugated to: AF555, AF488, and Cy5. Antibodies were obtained from Dako (Carpinteria, CA), BioLegend, or LifeTechnologies. We counterstained with DAPI to visualize cell nuclei. We used the Nuance Multispectral Imaging system (CRI, Woburn, MA) to visualize and captured images with a digital camera. We analyzed the images with ImageJ software (US National Institutes of Health, Bethesda, MD). We defined MAIT cells as CD3+IL-18Rα+Vα7.2+ cells, as described previously [16]. For each patient, a single unblinded operator, using ImageJ software, enumerated both MAIT and CD3+ cells from a single biopsy section at each time point from paired (available at both day 2 and day 30) samples.

Vibriocidal and plasma antibody levels

To examine the vibriocidal antibody response to the two serotypes of V. cholerae O1 (Ogawa and Inaba), we performed the vibriocidal assay as previously described [18]. We quantified LPS (prepared from V. cholerae O1 as previously described [18])-specific IgA, IgG, and IgM antibody responses in plasma using a kinetic ELISA method [19].

Markers of intestinal inflammation and permeability

In a subset of patients from the flow cytometry cohort whom we had data from both day 2 and day 30, we performed ELISA to determine the concentration of myeloperoxidase (MPO; Alpco, Salem, NH) and alpha anti-trypsin (AAT; ImmuChrom GmBH, NC) from stool samples obtained at the time of admission (day 0), at dilution factors of 1:200 and 1:400, respectively.

Single Cell TCR sequencing

MAIT cells from PBMCs and LPLs from one donor, at acute stages of infection (days 2 and 7) and from a second donor at a convalescent stage of infection (day 180), were single cell sorted using the Aria II cell sorter (BD Biosciences) directly into One Step RT-PCR reaction mix (NEB) loaded in MicroAmp Optical 96-well reaction plates (Applied Biosystems). MAIT cells were defined as live (DAPI−) CD3+CD4−CD161hiVα7.2+ cells. Following reverse transcription and preamplification reaction, a series of three nested PCR’s were run using primers for TCR sequence and gene expression as described earlier [20]. To separate reads from every well in every plate according to specified barcodes we processed and demultiplexed raw sequencing data using a custom software pipeline described in [20]. The data were analyzed using the R package as described [20].

Statistics

We used the Wilcoxon signed-ranked test for comparisons of frequency and activation of MAIT cells between different study days (Fig 1). We used one-way ANOVA with Tukey’s multiple comparison test for comparison of two or more groups (Fig 2). We log transformed MPO and AAT values and used Spearman’s correlation to determine their association with changes in LPL MAIT cells (Fig 3). All P values were two-tailed, with a value of <0.05 considered the threshold for statistical significance. We performed analyses using STATA version 13.1 (StataCorp, College Station, TX), and GraphPad Prism version 6.0 (GraphPad Software, Inc., La Jolla, CA).

Fig 1

MAIT cell localization and frequency in LP by Immunohistochemistry.

Cryosections of duodenal biopsies obtained from five cholera patients (n = 5) were stained with antibodies as mentioned in methods and imaged using Leica CM3000 Cryostat. (A) Representative images of duodenal biopsies at day 2 and day 30, shows CD3+ T cells (red arrows), IL-18Rα+ T cells (green arrows), Vα 7.2+ cells (magenta arrows), and the merged image shows MAIT cells (yellow arrows indicating CD3+ IL-18Rα+ Vα7.2+ cells). (B) Frequency of MAIT cells, as a % of total CD3+ cells in the duodenum at day 2 and day 30 post infection (p.i.), and (C) Frequency of CD3+ cells, as % of total cells in the lamina propria area at day 2 and day 30 p.i. Statistical significance of the difference between day 2 and day 30 was determined using the Wilcoxon signed-ranked test.

Fig 2

MAIT cell frequency and activation in PBMCs and LPLs by flow cytometric analysis.

Lamina propria lymphocytes (LPLs) and PBMCs were isolated from patients. (A) Frequency of MAIT cells (live (DAPI−) CD3+CD161hiVα7.2+ cells), as % of total CD3+ lymphocytes, and (B) Frequency of CD3+ T cells, as of total lymphocyte population, in LPLs at day 2 (n = 10) and day 30 (n = 8) p.i., and PBMCs, at day 2 (n = 10), day 7 (n = 10), and day 30 (n = 9) p.i. (C) Frequency of CD38+ cells, and (D) frequency of CD69+ cells, gated on MAIT cells in LPLs at day 2 (n = 9) and day 30 (n = 7) p.i., and PBMCs, at day 2 (n = 9), day 7 (n = 9), and day 30 (n = 8) p.i. Horizontal bar shows median values in graphs. (E) Gating strategy used to identify MAIT cells and representative histograms showing % CD38+ and % CD69+ cells gated on MAIT cells in LPL and PBMCs. Statistical significance of difference between groups was determined using one-way ANOVA with Tukey’s post hoc testing. * denotes p ≤ 0.05, ** denotes p ≤ 0.01, and *** denotes p < 0.001.

Fig 3

MAIT cell number and correlation with fecal markers of intestinal permeability (myeloperoxidase (MPO)) and inflammation (alpha-1-1antitrypsin (AAT)).

Stool markers of intestinal permeability and inflammation were measured using ELISA, values were log transformed and the correlation with LPL MAIT cell number was determined using Spearman’s correlation. (A-B) shows the correlation of LPL MAITs with MPO (A) and with AAT (B), (n = 6). (C-D) shows the correlation of PBMC MAITs with MPO (C) and with AAT (D), (n = 7). [Δ%MAIT = day30—day2%MAIT].

Results

MAIT cells are found predominantly in the lamina propria of the duodenum, and are more abundant during acute infection than at convalescence

We performed immunohistochemistry from frozen sections of paired duodenal biopsies from five cholera patients. We identified MAIT cells as CD3+IL-18Rα+Vα7.2+ cells (Fig 1A). The majority of MAIT cells were identified in the lamina propria of the crypt, with no cells identified in the villi. By this technique, we found that the frequency of MAIT cells, as a % of total CD3+ cells, was statistically non-significantly higher at day 2 of infection compared to day 30 (p = 0.06, Fig 1B), though this was the smallest achievable p-value with only 5 paired observations. In contrast, we demonstrated that the occurrence of CD3+ cells, as % of total cells in the lamina propria, did not change between acute and convalescent stages of infection (Fig 1C).

Compared to peripheral blood MAIT cells, duodenal MAIT cells at both acute and convalescent stages are more activated, but present in similar frequencies

We performed flow cytometric analysis on LPLs isolated from duodenal biopsies in 10 patients, of which only 8 completed 30 days of follow-up. We found that during all phases of cholera, MAIT cells were present in the duodenal lamina propria at frequencies similar to those found in the periphery (Fig 2A). We demonstrated that the occurrence of CD3+ cells, as % of total cells in the lamina propria at the convalescent stage, is lower than in the periphery (Fig 2B). Using CD38+ as a marker of activation, we found that at both days 2 and 30, duodenal MAIT cells were significantly more activated than peripheral MAIT cells. At day 2, a mean of 60% of all duodenal MAITs were CD38+, compared to 15% of all peripheral MAIT cells (95% CI of difference 17–75, P = 0.0005); similarly, at day 30, a mean of 59% of duodenal MAIT cells were activated, compared to 21% of peripheral MAIT cells (95% CI of difference 6–70, P < 0.01) (Fig 2C). We also found that at day 30, percentage frequencies of CD69+ duodenal MAIT cells were significantly higher than peripheral MAIT cells (P < 0.05) (Fig 2D). We found no significant differences in CD38+ MAIT cells and CD69+ MAIT cells between duodenal MAIT cells at day 2 and day 30. Fig 2E shows the gating strategy for MAIT cells and representative flow cytometry plots of CD38+ and CD69+ MAIT cells in the lamina propria and the periphery.

Increased intestinal permeability and inflammation are associated with loss of duodenal (but not peripheral) MAIT cells

Given the known cytotoxic capacity of MAIT cells and their activation in inflammatory bowel disease [16, 21], we examined whether baseline intestinal inflammation was associated with changes in MAIT cell frequency observed in flow cytometry. We measured two common fecal markers of intestinal permeability (myeloperoxidase (MPO) and inflammation (alpha-1-1antitrypsin (AAT)) in six of the eight cholera patients from whom we had paired days 2 and 30 LPL MAIT cell data from the flow cytometry cohort, and seven of the eight from whom we had paired PBMC data. We found a high level of correlation between the markers and the loss of duodenal MAIT cells from day 2 to 30 (r = 0.90, p = 0.03 for MPO; r = 1.00, P = 0.003 for AAT; Fig 3A and 3B). We did not find any correlation between these markers and changes in the frequency of peripheral MAIT cells (r = -0.04, p = 0.96 for MPO; r = -0.50, P = 0.27 for AAT; Fig 3C and 3D).

Single-cell TCR analysis of duodenal MAIT cells reveals a different TCR usage than that of MAIT cells in peripheral blood during acute infection

Few studies have reported TCR usage of MAIT cells in tissues [22-24], with minimal data on paired αβ TCR usage. To understand how MAIT TCR repertoire is affected in LPLs and PBMCs during cholera, we utilized paired TCR-phenotype single-cell Illumina sequencing as previously described [20], on paired tissue-blood samples from two patients. The αβ TCR usage are shown as heatmaps in (Fig 4A–4D). When we compared TCR usage from PBMC (46 cells were sequenced at day 2 and 7) and LPL (46 cells were sequenced at day 2) samples that were available from the single patient examined during acute infection, we found differences in usage of TRAJ, TRBV, and TRBJ between the duodenal LPLs and circulating PBMCs (Fig 4A–4C). We noted that MAIT clones observed in PBMCs obtained at day 7 differed from those observed at day 2. When analyzing based on TCR β usage alone, in concurrence with previous studies [22-25], we also found that MAIT cells in PBMCs obtained at day 2 and day 7 expressed TRBV20 and TRBV6 (Fig 4C). Unfortunately, despite many unpaired TCRα and TCRβ sequences available from the acute day 2 LPL sample (Fig 4A–4C), there was only one paired TCRαβ clone sequenced (S3 Table), and thus comparisons between LPL and PBMC clonality could not be made based on paired TCR reads. On the other hand, numerous LPL-paired TCR reads were successfully sequenced for the convalescent stage (day 180) donor, for whom we sequenced 89 PBMC and 91 LPL cells and found overlapping MAIT TCR usage (Fig 4D and S3 Table), with the majority of PBMC and LPL MAIT cells expressing TRAJ33 (Fig 4A), TRBV7-2, TRBV20-1 or TRBV30 (Fig 4B), and TRBJ2-2 (Fig 4C).

Fig 4

Different distribution of MAIT TCR in LPL compared to PBMCs.

MAIT cells were sorted from LPL and PBMCs from two (n = 2) patients at the acute and convalescent stage of illness and TCR usage was analyzed at the single-cell level using Illumina MiSeq sequencing. (A) Paired MAIT TCRαβ usage in each patient is shown as a heat map with hierarchical clustering performed using Euclidean distance. (B, C, and D) TRAJ, TRBV and TRBJ usage in LPL and PBMCs is shown as heat map. (E) The length distribution of MAIT CDR3β sequence in LPL and PBMCs is shown as a bar graph. The x axis shows length distribution of amino acids and the y axis shows percentage frequency of CDR3β sequence found with that amino acid length.

MAIT TCR β-chain repertoire diversity resides within the complementarity determining region (CDR) 3β loop [26-28] and to determine if MAIT TCR usage differ at the CDR3 level, we investigated the length distribution of amino acids in the CDR3β region of PBMCs and LPLs. We observed that the percentage frequency of CDR3β sequences with 14 nucleotides was highest, and relatively higher in LPLs compared to PBMCs. In contrast, the percentage of CDR3β sequences with 15 nucleotides was relatively higher in PBMCs compared to LPL samples (Fig 4E). Overall, differential patterns of MAIT TCR usage was observed in the duodenum compared with peripheral blood during acute and convalescent infection.

Discussion

V. cholerae infection is caused by the ingestion of bacteria, followed by colonization of the small intestine where cholera toxin is elaborated, resulting in chloride ion secretion and secretory diarrhea [29]. MAIT cells are innate-like lymphocytes known to provide immediate effector functions in response to infections in human tissues [8, 30]. Although studies have described MAIT cells in the human intestinal mucosa [4, 31, 32], there is limited data available on MAIT cells in the intestinal mucosa during an acute intestinal infection. In this study, we performed endoscopy and obtained duodenal biopsies in a cohort of patients with culture-confirmed severe dehydrating V. cholerae O1 infection. We showed that during acute human cholera, MAIT cells in the duodenal mucosa are present at frequencies similar to that seen in the peripheral circulation. Using immunohistochemistry and multispectral imaging, we demonstrated that the vast majority of MAIT cells are in the lamina propria, predominantly in the crypt, and rarely in the villi. This is consistent with a previous report using MR1 tetramer staining of healthy human jejunal tissue, showing that MAIT cells reside predominantly near the base of the villi [4].

We have previously shown, in endoscopically-obtained biopsies from cholera patients, that during acute disease, there is an upregulation of innate responses, including infiltration of neutrophils, degranulation of mast cells, and expression of pro-inflammatory cytokines [13, 33]. Using immunohistochemistry and flow cytometry, we did not find any statistically significant differences in MAIT cell frequencies between acute infection compared to convalescence.

We have previously shown that in adults with cholera, peripheral MAIT cells are highly activated at day 7 following infection, and that MAIT cell frequencies remain stable for up to 90 days following infection [14]. Reports of MAIT cell kinetics in the intestinal mucosa are lacking, although studies have shown that MAIT cells are present and active in the gastric mucosa during H. pylori infection [34], at decreased frequencies in duodenal lamina propria in celiac disease patients compared to healthy controls [31], and decreased in the colon in chronic HIV infection [35, 36]. It was recently reported that MAIT cells are decreased in the circulation and accumulate in the inflamed mucosa of patients with inflammatory bowel diseases, where they display increased cytokine secretion capacities [16, 32, 37, 38]. In addition to this, a decrease in circulating MAIT cell frequencies has been reported previously in studies of live S. Typhi [39, 40] and live-attenuated Shigella dysenteriae 1 vaccine [7], suggesting that circulating MAIT cells may decrease in frequency in the blood as they move to locally inflamed and infected tissues. In this study, we showed that during cholera, an acute bacterial enteric infection, MAIT cells in the duodenum are activated at levels significantly higher than that in the peripheral blood, based on CD38 expression. Taken together, MAIT cells are present in the lamina propria of the duodenum during cholera, and more activated than those in the blood. We hypothesize that they play an important role in the innate immune response to cholera.

Studies in humans with celiac disease, characterized by increased small bowel permeability, have shown an association between intestinal pathology and loss of intestinal MAIT cells [31]. Similarly, studies of ileal biopsies from patients with inflammatory bowel disease showed an accumulation of MAIT cells in inflamed compared to healthy tissue [16]. We hypothesized that compromised gut barrier function would increase MAIT cell exposure to microbes, resulting in MAIT cell activation and cell death. Thus, we examined two common fecal markers of intestinal inflammation (MPO) and permeability (AAT) [41], and found a high heterogeneity among cholera patients. Notably, we showed that levels of these markers were highly correlated with the loss of duodenal, but not peripheral, MAIT cells. While these findings suggest that MAIT cell loss is associated with intestinal inflammation and permeability, we cannot make conclusions regarding causality or pathogenesis.

Recent studies suggest that variability in MAIT TCR affects microbial ligand discrimination, activation, and phenotype [40, 42, 43]. It has been shown that MAIT cells undergo clonal expansion after Salmonella enterica serovar Paratyphi A infection [40]. In our TCR analysis of two donors, we observed that during acute infection (P1) there was a different distribution of MAIT clones in LPLs compared to PBMCs. However, in the convalescent stage (P2), there was overlapping utilization of TCR usage. We hypothesize from these observations that during acute infection, the intestinal compartment may have more layers of functional and phenotypic heterogeneity of MAIT cells than seen in the peripheral blood. The low sample size and lack of paired blood and LPL samples significantly limit our TCR analyses, and thus further studies would be needed to confirm our observations regarding the TCR usage between LPL and blood MAITs, including the significance and generalizability of the TRAJ34 clones isolated.

Our study had a number of limitations. First, we were not able to assess cytokine secretion of MAIT cells during acute and convalescent stages of infection and further investigation of MAIT cell functionality in cholera is warranted. Second, our identification of MAIT cells was based on Vα7.2 antibody, as the MR1-tetramer was not available at the time that this study was conducted (2012–2014). Thus, our results are subject to lack of sensitivity to detect activated MAIT cells, given their potential for down-regulation of CD161 and TCRα chain [44], and may also include a small portion of non-MR1-restricted T cells. Thirdly, our study lacks duodenal biopsy data from healthy (non-cholera) participants, and thus comparisons of MAIT cell activation and frequency were only available between acute and convalescent stages of cholera. Fourthly, our conclusions regarding the immunohistochemical findings are limited by the small sample size and an unblinded operator, and our findings need to be confirmed in larger studies. Lastly, due to ethical and clinical limitations, our sampling of the duodenal mucosa was done only at days 2 and 30. It is likely that MAIT cells, given their innate-like nature in the mucosa, are recruited, activated and release effector molecules early in the course of infection, and that the days examined in our study do not adequately capture the granularity of the kinetics of the MAIT cell response [45-47]. Further studies with more frequent and prolonged sampling would help with examining the kinetics of MAIT cells during V. cholerae infection.

In conclusion, in this preliminary study limited by small sample size, we have shown that MAIT cells are present in the lamina propria of the duodenum and are highly activated (CD38+) compared to peripheral blood during human cholera infection. We hypothesize that the high day 2 MAIT frequency and activation in the duodenum seen in this study reflects the clonal expansion of MAIT cells during the early stages of cholera, though further studies into the functional cytotoxic abilities of duodenal MAIT cells to inactivate V. cholerae or their roles in innate and adaptive immune responses are needed.

Demographics and vibriocidal antibody titers (to the two V. cholerae O1 serotypes, Ogawa and Inaba) of study subjects. M = male; F = female; D = day.

(DOCX)

Click here for additional data file.

Detailed information for fluorochrome markers used.

(DOCX)

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Number of paired TCRαβ clones found in each sample.

PBMC = peripheral blood mononuclear cells; LPL = lamina propria lymphocyte; P1 = Patient 1; P2 = Patient 2; d = day. N = number of times each paired TCRαβ was found.

(DOCX)

Click here for additional data file.

16 Jul 2021

Dear Dr. Leung,

Thank you very much for submitting your manuscript "Mucosal-Associated Invariant T (MAIT) Cells are Highly Activated in Duodenal Tissue of Humans with Vibrio cholerae O1 Infection" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

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Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: Please see "Summary and General Comments" section

Reviewer #2: The population is not fully described, healthy controls are lacking and the sample sizes are small in some analyses. See full comments below.

Reviewer #3: The objectives are reasonably outlined, and the study design appropriate.

The population is well described and also appropriate for the study. The main worry here is the population size, as the inter-individual variation is large. The number of study patients is low, which makes it hard to make solid conclusions.

It is unclear from the text which statistical methods have actually been used in Fig. 1 and Fig. 4, the M&M section states Wilcoxon and the figure legends t-test.

No ethical concerns.

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Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: Please see "Summary and General Comments" section

Reviewer #2: There are unexplained inconsistencies between the data presented in different figures.

See full comments below.

Reviewer #3: The analysis is according to plan.

There are some issues with regard to result presentation. The IF staining in Fig. 1A is hard to interpret. Please provide a close up of the indicated MAIT cells, to show how they can be distinguished from the background. Please also show the DAPI stain so that individual cells can be identified.

There is also a lack of FMO controls in Fig. 2, which would be needed to judge the cut off for positive CD38 and CD39 staining.

Furthermore, the labeling of the axes in the dot plots in Fig. 2 and of the samples in Fig. 5A-D is very hard to read and clarity can be improved.

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Please see "Summary and General Comments" section

Reviewer #2: Conclusions are not always fully supported. See full comments below.

Reviewer #3: The low patient number makes it hard to draw solid conclusions. This limitation in mentioned, but not elaborated. The conflicting results from IHC and flow cytometry and how data can be used to advance understanding of the topic are not really discussed either.

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: Several small modifications. See fully comments (under minor comments heading) below.

Reviewer #3: 1. The first three references are quite old, and there are more recent reviews on the topic that could be used to introduce the reader to the topic of MAIT cells.

2. The first paragraph of the result section would be better suited in the materials and methods. Please also define VE blood group antigens in Suppl. Table 1.

3. The M&M sections states that Wilcoxon was used to compare values before and after infection, but in the legends to Fig. 1 and 4 it say paired t-test. Please clarify.

4. In Fig. 1C, only 1-4% of lamina propria lymphocytes are CD3+. This is surprisingly low, has there maybe been a mistake in the labeling of the y-axis?

5. In the text (line 193 and 216) it says that 7 patients were available for biopsies day 30, but in the corresponding plot in Fig. 2A, 8 individuals are shown. Please clarify.

6. In M&M, it is stated that cells were sorted from one donor for TCR analysis (line 146) while in the result section, data from two individuals are presented (line 237). Please clarify. The second patient appears to be biopsied at day 180, which is not mentioned in the M&M. Furthermore, how were these patients selected?

7. Reference 32 and 33 are the same.

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: The study by Bhuiyan et al. characterized MAIT cells present in duodenal biopsies and their paired peripheral blood samples in patients with culture-confirmed Vibrio cholerae O1 infection. The manuscript is sound, interesting, and presents novel findings reported straightforwardly.

A few suggestions are below:

1. Please transfer the paragraph containing the demographics from the “Results” (lines 171-177) to the “methods” section (i.e., “Study population and sample collection” section) of the manuscript.

2. Please explain, in the manuscript, the rationale for identifying MAIT cells using different markers (e.g., CD161 for blood and IL-18Rα for duodenal biopsies). The readers may be confused by a choice that is not explained.

3. Figure 1. Panel A would benefit from a figure with higher resolution and magnification.

4. Please confirm that day 2 and 30 biopsies were collected from the same area of the duodenum and not randomly. Add a statement clarifying this issue into the manuscript. Also, discuss further the discrepancy between duodenum MAIT cell frequencies obtained by immunochemistry (Fig. 1B) and those obtained by flow cytometry (Fig. 2A). Is it a sampling issue? Or experiment artifact? Collagenase might have impacted the expression of MAIT specific markers... Further immunochemistry stainings are necessary to clarify this issue.

5. References 38 and 39 are the same.

6. The discussion lacks publications showing MAIT cell kinetics (e.g., PMID: 23898209, PMID: 32811991, PMID: 28428786). Given that the authors dwell on the effects of MAIT cells in the duodenal biopsies and blood, it would perhaps be helpful if, at the end of the discussion, they committed to a statement on the pattern of the MAIT cell kinetics and its implications on cholera pathology.

7. Since no kinetic changes occurred in either blood or duodenum, please discuss the need for extra time points to establish the impact of MAIT cells on cholera infection

8. Material & Methods. There is no statement about blood collection at days 7 and 180, biopsies at day 180, or integrin β7 staining. If an oversight, please fix it.

9. Fix typo. “Results” section states, “The majority of MAIT cells were identified in the lamina propria, with no cells identified in the villous and crypt epithelia (lines 183 & 184), but the discussion describes, “we demonstrated that the vast majority of MAIT cells are in the lamina propria, predominantly in the crypts, and rarely in the epithelia.”( lines 271-273). In the discussion, do you imply beneath the crypts?

Reviewer #2: Summary

MAIT cells are known to be activated by diverse microbes and several mouse studies have shown their role in protecting against bacterial infection, but also in driving pathology in chronic infection. Studies of MAIT cells in humans during infection, particularly at mucosal sites, are currently limited.

Here, Bhuiyan and colleagues assess MAIT cell numbers in acute and convalescent Vibrio cholerae infection in duodenal biopsies and bloods from a small cohort of patients. The topic is interesting and the study would potentially add important information to the field. However, much of the data presented seems preliminary (for example the clonal expansion observed in one patient). He study lacks healthy control samples (which could easily be included for PBMCs if not biopsies). There is a relatively small number of patient used, and only small subsets of these analysed for the data shown in different figures (5 patients for some).

There are also inconsistencies between data from flow cytometry and histology that are not explained, and these then also affect downstream analyses.

Detailed comments below offer suggestions to improve the study.

Major comments

1. The histology images shown in Fig 1A is of low resolution (even in downloaded .tif file). Some of the arrows for Va7.2 staining do not appear to be pointing to cells. It is hard to assess from the histology presented if statements about location in the LP are correct (line 183).

2. How was the quantitation done in Fig 1B and C? i.e. how many sections, from how many biopsies were enumerated for each patient? Was the operator blinded? What is meant by “paired duodenal biopsies” in line 181?

3. How do the authors explain the inconsistency in terms of %MAITs of CD3+ T cells in duodenal samples between data shown in Fig 1B vs Fig 2A and Fig 1C vs 2B? In Fig. 1B, authors claimed that frequency of MAIT cells was significantly higher at D2 compare to D30. However, no difference was observed in Fig 2A.

4. It is a worry that the authors claim to show MAIT cell frequencies increase in acute infection vs convalescence (line 282) based on histology data, but that they “were unable to replicate this finding using flow cytometric analysis…”. This is cherry-picking since they could equally state there was no difference based on the flow data. Given that neither assessment used MR1 tetramers, which would precisely detect MAIT cells, can the authors be sure that the markers used do not change expression during activation leading to loss of signal and apparent decrease in MAIT cells?

5. In flow cytometry data, MAIT cells are defined as CD3+CD4−CD161hiVα7.2+ cells. However, some MAIT cells express CD4. It would be interesting to look at CD4 (and CD8) coreceptor expression if this data is available.

6. What is the percentage of MAIT cells among CD3+ cell in cholera-free duodenal tissue? Without such data, it is hard to claim that “MAIT cell frequencies are significantly increased during acute infection compared to convalescence”.

7. For Fig. 2C and D, duodenal MAIT cells highly express CD38 and CD69 at both acute and convalescent stages of cholera. Is this an intrinsic property of duodenal (or tissue) MAIT cells or induced by bacterial infection? In particular, CD69 expression is known to be high in tissues compared to PBMCs. In order to claim that duodenal MAIT cells in cholera infection are activated based on these markers (line 137), it is necessary to examine CD38 and CD69 expression on MAIT cells from uninfected duodenal samples.

8. For Fig. 2E, isotype or FMO control should be shown for CD69 and CD38 staining

9. For Fig. 4, FACS plots showing β7 expression on peripheral MAIT and non-MAIT cells should be included. Β7 data from healthy control is also required.

10. For Fig. 5A, is the dominant MAIT cell clone in the LPL a result of selective proliferation? Or preferential tissue homing? Again, uninfected controls would be welcomed here.

Minor comments

11. Line 60. “The ligand for MAIT cells…” This should be “antigen” and there is more than one described.

12. TNFa should be TNF consistent with current naming convention.

13. The methods are light on detail and insufficient for others to repeat. For example, antibodies should be listed with clone name, catalogue numbers and concentrations used for staining.

14. The patient information should be more detailed. For example, Inaba and Ogawa are only mentioned in Supp Table 1 and not explained. Are these patients part of a larger study? What are SEGD and PIC in the patient numbers? Which patient samples were used for FACS vs histology? The antibiotics given should be described along with the timing relative to biopsies (line 94). Is the use of these antibiotics known or anticipated to affect the MAIT cell response?

15. The labels on the gating strategy and % on histograms in fig 2E are impossible to read.

16. Data on peripheral MAIT cells (which the authors state did not correlate with Stool markers) should be shown in Fig 3.

17. Why was the B7 integrin expression only examined at day 7 (Fig 4), and why only on 4 patients? This figure should also show the expression for healthy controls. By itself this data is not meaningful to assess the gut homing capacity of MAIT cells in cholera and should be expanded or removed.

18.Labels on Fig 5 are hard to read, particularly the different samples.

19. Line 241 should state that the dominant clone was found in one of the two patients examined. The text also states that 46 clones were found in PBMC and LPL, but this data is not shown. Fig 1A should state (or in a table) how many times each sequence was identified. Similarly for convalescent clone data (line 248).

20. In fig 5B, the majority of clones from patient 1 express TRAJ34. This seems unusual for MAIT cells, although has been described in an “atypical”, TRAV1-2- MAIT cells (Gherardin et al. Immunity 2016, Koay et al. N. Comm 2019) and in one clone expanded from HIV infected individuals after stimulation with E. coli (Trivedi et al., ImunoHorizons 2021). Can the authors comment on why this may be and whether this is likely to be a generalisable phenomenon?

21. The data in Fig 5E doesn’t make sense. Frequencies should add up to 100 (line 254-259 in text).

22. Line 275: [35] is the wrong reference.

Reviewer #3: In this study, Bhuiyan et al investigate MAIT cell frequencies, phenotype and TCR rearrangements during acute and convalescent cholera infection. The authors should be commended for using intestinal material and paired data from the same individuals, as these tell much more than only circulating cells or animal models. However, it also creates problems such as limited tissue availability restricting the analyses that can be performed. It may also be hard to convince acutely ill patients to perform endoscopy for research purposes. This is probably to cause of one of the major limitations of the study, the low number of individuals included in the different analyses. Another major weakness is the unclear identification of MAIT cells in all the flow cytometry analyses.

Major points:

1. The use of Va7.2 and CD161 to identify mucosal MAIT cells is somewhat problematic, as there are other populations of CD161-expressing T cells in the intestinal mucosa. The authors would need to confirm their findings using MR1 tetramers and CD161 to identify MAIT cells. The MR1 tetramers are now available from the NIH tetramer facility since several years.

Furthermore, MAIT cells are said to be defined as CD3+CD4- cells expressing the MAIT TCR and CD161 (line 150). However, in the representative staining shown in Fig. 2, all T cells are included when the MAIT gate is set. Likewise, the manuscript consistently gives the number of MAIT cells per CD3+ cells, not CD8+ or CD3+CD4-, and this creates some confusion as to which gates were actually used. As Th17 cells and Treg can express CD161, some of them (expressing the Va7.2 as part of another non-MAIT TCR) may also have been included in the analyses.

2. The IF staining in Fig. 1A is hard to interpret. Please provide a close up of the indicated MAIT cells, to show how they can be distinguished from the background. Please also show the DAPI stain so that individual cells can be identified.

3. CD69 is a good activation marker for circulating T cells. In tissues however, CD69 also marks tissue-resident memory T cells, and is thus less suitable. Therefore, the authors may need to modify their statements on activation status in intestinal MAIT cells.

Please show the FMO controls for CD38 and CD69 in the intestinal samples in Fig. 2E.

4. There are clear differences with regard to changes in MAIT cell frequencies during the course of disease between IHC and flow cytometry. This should be discussed.

Furthermore, the authors’ previous finding of highly activated circulating MAIT cells on day 7 (ref 14) was not confirmed in the present study. This might also deserve some comments in the discussion.

The authors conclude that “MAIT cells are highly activated and present in the lamina propria of the duodenum during cholera”. However, the same can be said about the convalescent stage, as there is actually no difference between day 2 and day 30. A more correct statement would be that lamina propria MAIT cells are more activated than those in the blood.

There are several studies on MAIT cell activity following Salmonella infection or vaccination (for example Howson et al, Nature Comm 2018 and Salerno-Goncalves et al Front Immunol 2017) that could also be included in the discussion.

5. In Fig. 3, why not include the MAIT cell frequencies obtained from the IHC data? These are the patients that actually did show a change in MAIT cell frequencies during infection.

6. To evaluate the data presented in Fig. 4 properly, it would be better to show also the results from day 2 and day 30.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Marianne Quiding Järbrink

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22 Sep 2021

Submitted filename: pNTD 21-00600 Response to reviewers.docx

Click here for additional data file.

12 Oct 2021

Dear Dr. Leung,

Thank you very much for submitting your manuscript "Mucosal-Associated Invariant T (MAIT) Cells are Highly Activated in Duodenal Tissue of Humans with Vibrio cholerae O1 Infection" for consideration at PLOS Neglected Tropical Diseases. As with all papers reviewed by the journal, your manuscript was reviewed by members of the editorial board and by several independent reviewers. In light of the reviews (below this email), we would like to invite the resubmission of a significantly-revised version that takes into account the reviewers' comments.

Given the sample size in this study, the preliminary nature of the data it is recommended to add "a preliminary report" to the title. In addition, more details on the sample size, age and sex of the patients should be added to the abstract and summary.

In the conclusions (both abstract / discussion) please highlight the small sample size. Please also moderate the conclusions, (especially when some results were not statistically significant at p<0.05) and highlight the preliminary nature of the report.

Figures legend – please enter the number of samples tested in each group/time points. Figure 2: When using ANOVA please check if the underling assumptions of ANOVA were fulfilled, otherwise please use non-parametric test.

We cannot make any decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is also likely to be sent to reviewers for further evaluation.

When you are ready to resubmit, please upload the following:

[1] A letter containing a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

[2] Two versions of the revised manuscript: one with either highlights or tracked changes denoting where the text has been changed; the other a clean version (uploaded as the manuscript file).

Important additional instructions are given below your reviewer comments.

Please prepare and submit your revised manuscript within 60 days. If you anticipate any delay, please let us know the expected resubmission date by replying to this email. Please note that revised manuscripts received after the 60-day due date may require evaluation and peer review similar to newly submitted manuscripts.

Thank you again for your submission. We hope that our editorial process has been constructive so far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Khitam Muhsen

Guest Editor

PLOS Neglected Tropical Diseases

Alfredo Torres

Deputy Editor

PLOS Neglected Tropical Diseases

***********************

Given the sample size in this study, the preliminary nature of the data it is recommended to add "a preliminary report" to the title. In addition, more details on the sample size, age and sex of the patients should be added to the abstract and summary.

In the conclusions (both abstract / discussion) please highlight the small sample size. Please also moderate the conclusions, (especially when some results were not statistically significant at p<0.05) and highlight the preliminary nature of the report.

Figures legend – please enter the number of samples tested in each group/time points. Figure 2: When using ANOVA please check if the underling assumptions of ANOVA were fulfilled, otherwise please use non-parametric test.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: (No Response)

Reviewer #2: small sample size, but noting these samples are difficult to obtain and this is highlighted in the manuscript.

Reviewer #3: The weaknesses of the study have now been better spelled out in the manuscript.

--------------------

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: (No Response)

Reviewer #2: (No Response)

Reviewer #3: The inconsistencies in the result section have been clarified in the revised version of the manuscript. However, immunofluorescence pictures are not of an acceptable quality.

--------------------

Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: (No Response)

Reviewer #2: see comments below

Reviewer #3: These issues are adequately addressed in the revised manuscript.

--------------------

Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: (No Response)

Reviewer #2: see comments below

Reviewer #3: (No Response)

--------------------

Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: All the points raised by this reviewer have been addressed satisfactorily.

Reviewer #2: The authors have revised their manuscript addressing most comments resulting in an improved manuscript. They have removed some less convincing data (previous figure 4). Some issues remain as listed below.

Previous comment. 1. The histology images shown in Fig 1A is of low resolution (even in downloaded.tif file). Some of the arrows for Va7.2 staining do not appear to be pointing to cells. It is hard to assess from the histology presented if statements about location in the LP are correct (line 183).

Response: Thank you for your suggestion. Unfortunately, this is the highest resolution and magnification we were able to obtain. These images were obtained and saved with this resolution in 2013, and we are unable to improve the resolution of the saved images.

Remaining issues: the response does not address the main issues. It is still difficult to determine the validity of data presented in B and C when some arrows do not appear to be pointing to cells. Crypt and villi structures are hard to see. Noting the difficulty in sourcing these samples the authors could at least list this as a limitation and ensure their conclusions based on these results are appropriately phrased.

Previous comment. 2. How was the quantitation done in Fig 1B and C? i.e. how many sections, from how many biopsies were enumerated for each patient? Was the operator blinded? What is meant by “paired duodenal biopsies” in line 181?

Response: We have added to the Methods section (lines 145-147), “For each patient, a single operator, using ImageJ software, enumerated both MAIT and CD3+ cells from a single biopsy section at each time point from paired (available at both day 2 and day 30) samples.”

Remaining issues: The response does not fully address the previous comment. Was the operator blinded to the time points for each biopsy?

What were the absolute numbers counted? Were more biopsies available? What was the variation in numbers between patients? Drawing conclusions from such small numbers of biopsies is problematic in this reviewer’s opinion, particularly if the operator was not blinded to the grouping.

Previous comment. 3. How do the authors explain the inconsistency in terms of %MAITs of CD3+ T cells in duodenal samples between data shown in Fig 1B vs Fig 2A and Fig 1C vs 2B? In Fig. 1B, authors claimed that frequency of MAIT cells was significantly higher at D2 compare to D30. However, no difference was observed in Fig 2A.

Response: To address the discrepancy between flow cytometry and immunohistochemistry, we have now added to the discussion (line 291-298) as follows, “A potential explanation for this discrepancy include differences in sample preparation for flow cytometry and immunohistochemistry, as embedding and fixation of some antigenic epitopes could be lost during the processing and preparation of specimens, and collagenase treatment used to prepare samples for flow cytometry can also impact expression of surface markers [36, 37]. Furthermore, there is a lower detection threshold of flow cytometry compared with immunohistochemistry, whereby dim antigen expression may be easily distinguished from background. Such discrepancies between flow cytometric and immunohistochemical results have been reported by others [38-40].”

Remaining issues: It is unclear how these factors would differentially affect the results at day 2 and day 30.

Additionally, the histology differences are no longer reported as significant (p = 0.06) as the authors have changed the statistical test used, and thus claims of differences between the two time points should be avoided.

Previous comment. 4. It is a worry that the authors claim to show MAIT cell frequencies increase in acute infection vs convalescence (line 282) based on histology data, but that they “were unable to replicate this finding using flow cytometric analysis...”. This is cherry-picking since they could equally state there was no difference based on the flow data. Given that neither assessment used MR1 tetramers, which would precisely detect MAIT cells, can the authors be sure that the markers used do not change expression during activation leading to loss of signal and apparent decrease in MAIT cells?

Response: Unfortunately, during the period of this study (2012-2014), the MR1-tetramer was not available. Thus, we initially identified MAIT as CD3+CD4−CD161hiVα7.2+ for flow cytometry as we have used in our earlier publication (Ref 14). For immunohistochemistry, we used the staining markers for MAIT cells published previously by other groups (PMID 21084709, 24450998).

We have now added this statement to the Methods section (line 136-138): “In line with immunohistochemical methods from reports available at time of the experiments [16, 17],” To be more consistent with immunohistochemistry methods, we have revised our identification of MAIT cells by flow to not include CD4 gating (i.e. MAITs are CD3+CD161hiVα7.2+ cells), and we have made this changed in Methods section (line 129). We have now highlighted the discrepancy between flow cytometry and immunohistochemistry findings in the Discussion, as quoted in response to the question above.

Remaining issues: The flow cytometry data does not appear to be changed (Fig 2) to reflect the change in gating. As above, claims of “differences” should be avoided when not significant.

Previous comment. 5. In flow cytometry data, MAIT cells are defined as CD3+CD4−CD161hiVα7.2+ cells. However, some MAIT cells express CD4. It would be interesting to look at CD4 (and CD8) coreceptor expression if this data is available.

Response: Thank you for pointing this out. We have examined the flow cytometry data with and without CD4 involved in the gating strategy and this did not change any of our findings (no differences between day 2 and day 30). To address comments above and also posed by other reviewers, to be more consistent with MAIT identification by immunohistochemistry, we have changed the definition of MAIT cells to not include CD4 (as noted in statement above). Unfortunately, at the time of this study, we did not stain cells with CD8 antibody.

Remaining issues: OK. But data in figures does not appear to be changed.

Previous comment. 9. For Fig. 4, FACS plots showing β7 expression on peripheral MAIT and non-MAIT cells should be included. Β7 data from healthy control is also required.

Response: Given the lack of integrin β7 data from controls, and the limited information gained, we have decided to remove these data from our manuscript.

Remaining issues (minor): Integrin B7 Ab still appears in methods (line 123).

Previous comment. 21. The data in Fig 5E doesn’t make sense. Frequencies should add up to 100 (line 254- 259 in text).

Response: We apologize for this confusing figure. The Y axis is the absolute number of times (not percentage) of CDR3b with a particular amino acid length. This has been corrected in results (line 263 is updated to number instead of frequency), the y-axis title, and the figure legend (line 429) has been modified to, “x-axis shows length distribution of amino acids and y axis shows number of times CDR3β sequence was found with that amino acid length.”

Remaining issues: The figure now makes sense. However, if the authors wish to compare LPL to PBMC CDR3b length (line 263-) they need to consider the percentage of sequences rather than the numbers with each length. It seems the numbers in LPL at d2 are lower than PBMC due to the total number of sequences rather than a different pattern of CDR3b length. In fact, according to the results in Fig 5E (new 4E), a similar pattern of CDR3β sequence seems to be observed in MAIT cells from LPL and PBMC, with most frequent 14 nucleotide length.

Reviewer #3: The revised manuscript is much improved, and many inconsistencies have been addressed or clarified.

However, the resolution of the imunoflourescence pictures is still very poor, and they can’t be used to verify the conclusions made. The authors argue that the experiments were performed in 2013, and that the quality can’t be improved. If so, I would suggest that another set of biopsies are collected and evaluated using equipment that will yield pictures of sufficient quality to confirm the conclusions made previously.

Likewise, it is argued that as the experiments were performed before the MR1 tetramers became available, the authors can’t confirm the MR1 reactivity of the cells identified as MAIT cells. I would suggest a round of new FACS analyses using biopsies from cholera-infected or convalescent patients to confirm that the CD161+Va7.2+ cells detected in the duodenal mucosa are actually MR1-reactive.

--------------------

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at figures@plos.org.

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Reproducibility:

To enhance the reproducibility of your results, we recommend that you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option to publish peer-reviewed clinical study protocols. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

17 Mar 2022

Submitted filename: pNTD 21-00600R1 Response to reviewers.pdf

Click here for additional data file.

11 Apr 2022

Dear Dr. Leung,

We are pleased to inform you that your manuscript 'Mucosal-Associated Invariant T (MAIT) Cells are Highly Activated in Duodenal Tissue of Humans withVibrio cholerae O1 Infection: A Preliminary Report' has been provisionally accepted for publication in PLOS Neglected Tropical Diseases.

Before your manuscript can be formally accepted you will need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests.

Please note that your manuscript will not be scheduled for publication until you have made the required changes, so a swift response is appreciated.

IMPORTANT: The editorial review process is now complete. PLOS will only permit corrections to spelling, formatting or significant scientific errors from this point onwards. Requests for major changes, or any which affect the scientific understanding of your work, will cause delays to the publication date of your manuscript.

Should you, your institution's press office or the journal office choose to press release your paper, you will automatically be opted out of early publication. We ask that you notify us now if you or your institution is planning to press release the article. All press must be co-ordinated with PLOS.

Thank you again for supporting Open Access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Khitam Muhsen

Guest Editor

PLOS Neglected Tropical Diseases

Alfredo Torres

Deputy Editor

PLOS Neglected Tropical Diseases

***********************************************************

The authors addressed well all comments raised by the reviewers and editors.

Reviewer's Responses to Questions

Key Review Criteria Required for Acceptance?

As you describe the new analyses required for acceptance, please consider the following:

Methods

-Are the objectives of the study clearly articulated with a clear testable hypothesis stated?

-Is the study design appropriate to address the stated objectives?

-Is the population clearly described and appropriate for the hypothesis being tested?

-Is the sample size sufficient to ensure adequate power to address the hypothesis being tested?

-Were correct statistical analysis used to support conclusions?

-Are there concerns about ethical or regulatory requirements being met?

Reviewer #1: The relatively small sample size is an overall weakness, but it relates to the difficulties in obtaining human tissues (e.g., ethical, and logistical issues). The small sample size might have precluded the authors from rejecting the null hypothesis, and this shortcoming is now clearly explained in the discussion section of the revised version. The readers will now be able to judge the weaknesses and shortcomings of the present work, and the number of citations received by this manuscript, if published, will demonstrate its importance.

Reviewer #2: (No Response)

**********

Results

-Does the analysis presented match the analysis plan?

-Are the results clearly and completely presented?

-Are the figures (Tables, Images) of sufficient quality for clarity?

Reviewer #1: It has been a “tour de force” catching up on the many typos and inconsistencies in this manuscript. It is also a wonder how many of these issues could escape the attention of a large group of co-authors. Nevertheless, the manuscript is sound, interesting, and presents novel findings, fulfilling the PLOS NTD mission of publishing findings that improve the understanding of diseases affecting developing countries using a relevant human tissue model.

Reviewer #2: (No Response)

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Conclusions

-Are the conclusions supported by the data presented?

-Are the limitations of analysis clearly described?

-Do the authors discuss how these data can be helpful to advance our understanding of the topic under study?

-Is public health relevance addressed?

Reviewer #1: Although all the points raised by this reviewer have been addressed satisfactorily since the first revision, all the new changes/additions clearly add to the contribution of this paper.

Reviewer #2: (No Response)

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Editorial and Data Presentation Modifications?

Use this section for editorial suggestions as well as relatively minor modifications of existing data that would enhance clarity. If the only modifications needed are minor and/or editorial, you may wish to recommend “Minor Revision” or “Accept”.

Reviewer #1: I recommend an acceptance, though it would be great to have Figure 1A removed or cropped to better visualize the findings.

Reviewer #2: (No Response)

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Summary and General Comments

Use this section to provide overall comments, discuss strengths/weaknesses of the study, novelty, significance, general execution and scholarship. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. If requesting major revision, please articulate the new experiments that are needed.

Reviewer #1: Although all the points raised by this reviewer have been addressed satisfactorily since the first revision, all the new changes/additions clearly add to the contribution of this paper.

Reviewer #2: No further comments.

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Reviewer #1: No

Reviewer #2: No

30 Apr 2022

Dear Dr. Leung,

We are delighted to inform you that your manuscript, "Mucosal-Associated Invariant T (MAIT) Cells are Highly Activated in Duodenal Tissue of Humans withVibrio cholerae O1 Infection: A Preliminary Report," has been formally accepted for publication in PLOS Neglected Tropical Diseases.

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Thank you again for supporting open-access publishing; we are looking forward to publishing your work in PLOS Neglected Tropical Diseases.

Best regards,

Shaden Kamhawi

co-Editor-in-Chief

PLOS Neglected Tropical Diseases

Paul Brindley

co-Editor-in-Chief

PLOS Neglected Tropical Diseases