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. 2015 Sep 17;6:466.
doi: 10.3389/fimmu.2015.00466. eCollection 2015.

Mucosal-Associated Invariant T Cells in the Human Gastric Mucosa and Blood: Role in Helicobacter Pylori Infection

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Free PMC article

Mucosal-Associated Invariant T Cells in the Human Gastric Mucosa and Blood: Role in Helicobacter Pylori Infection

Jayaum S Booth et al. Front Immunol. .
Free PMC article

Abstract

Mucosal-associated invariant T (MAIT) cells represent a class of antimicrobial innate-like T cells that have been characterized in human blood, liver, lungs, and intestine. Here, we investigated, for the first time, the presence of MAIT cells in the stomach of children, adults, and the elderly undergoing routine endoscopy and assessed their reactivity to Helicobacter pylori (H. pylori - Hp), a major gastric pathogen. We observed that MAIT cells are present in the lamina propria compartment of the stomach and display a similar memory phenotype to blood MAIT cells. We then demonstrated that gastric and blood MAIT cells are able to recognize H. pylori. We found that CD8(+) and CD4(-)CD8(-) (double negative) MAIT cell subsets respond to H. pylori-infected macrophages stimulation in a MR-1 restrictive manner by producing cytokines (IFN-γ, TNF-α, IL-17A) and exhibiting cytotoxic activity. Interestingly, we observed that blood MAIT cell frequency in Hp(+ve) individuals was significantly lower than in Hp(-ve) individuals. However, gastric MAIT cell frequency was not significantly different between Hp(+ve) and Hp(-ve) individuals, demonstrating a dichotomy between blood and gastric tissues. Further, we observed that the majority of gastric MAIT cells (>80%) expressed tissue-resident markers (CD69(+) CD103(+)), which were only marginally present on PBMC MAIT cells (<3%), suggesting that gastric MAIT cells are readily available to respond quickly to pathogens. These results contribute important new information to the understanding of MAIT cells function on peripheral and mucosal tissues and its possible implications in the host response to H. pylori.

Keywords: H. pylori; age-related; cytotoxic; gastric MAIT; stomach.

Figures

Figure 1
Figure 1
MAIT cells are present in the human gastric mucosa and exhibit T effector memory phenotype. (A) Identification of MAIT cell subsets [CD8+, CD4+, and CD4CD8 (DN)] in PBMC and (B) in gastric LPMC in a representative individual as CD161hi TCR Vα7.2 (box). (C) Comparison of gastric (red dots; n = 27) and PBMC (black squares; n = 29) MAIT cell subsets. Significant differences between MAIT cell subsets in (i) PBMC [black asterisks (*)] and (ii) gastric LPMC [red asterisk (*)]. Blue asterisks (*) denote significant differences in MAIT cell subsets between PBMC and LPMC. Horizontal lines represent medians (red bars: gastric MAIT; black bars: PBMC MAIT). (D) Expression of CD45RA and CD62L to evaluate memory [TCM (CD62L+CD45RA), TEM (CD62LCD45RA), and TEMRA (CD62LCD45RA+)] and Tnaive (CD62L+CD45RA+) subpopulations in MAIT subsets from PBMC (n = 29) and (E) LPMC (n = 27). (F) Comparison of TEM MAIT cell subsets between PBMC and LPMC. *P < 0.05; **P < 0.005; ***P < 0.0005.
Figure 2
Figure 2
MAIT cells in blood (PBMC) and gastric LPMC in children, adults, and the elderly. (A) PBMC and (B) gastric LPMC obtained from children (n = 12), adults (n = 21), and the elderly (n = 10) were evaluated for the presence of MAIT cells. Significant differences among age groups are denoted by asterisks (*P < 0.05). (C) Correlation of CD8+ MAIT cells with age in PBMC (n = 46) and (D) in gastric LPMC (n = 43) was performed using Spearman’s correlation analysis. Horizontal green lines in (A,B) represent medians.
Figure 3
Figure 3
Responses of blood CD8+ MAIT cells from healthy adults to H. pylori-infected macrophages. (A) Representative volunteer showing the induction of cytokine production (IFN-γ, TNF-α, IL-17A) and up-regulation of CD107a expression by CD8+ MAIT cells following stimulation by H. pylori-infected differentiated THP-1 macrophages (Mϕ) at increasing effector to target (E:T) ratios (5:1, 10:1, 20:1, 50:1). (B) Cumulative data (n = 11) displaying the percentages of cytokine-producing and CD107a-expressing CD8+ MAIT cells following stimulation with H. pylori-infected Mϕ (E:T 5:1, 10:1, 20:1, 50:1), non-infected Mϕ, and effector cells alone. Shown are significant differences (*) between non-infected and infected targets. Significances were P < 0.0005 for CD107 and TNF-α and P < 0.005 for IFN-γ at all E:T ratios and P < 0.05 for IL-17A at 10:1, 20:1, and 50:1 E:T ratios. (C) Cumulative data (n = 11) showing cytokine production and expression of CD107a following stimulation with H. pylori-infected Mϕ at E:T (50:1) ratio, non-infected Mϕ, and effector cells alone. Horizontal black lines in (C) represent medians. Significant differences were determined between non-infected and H. pylori-infected Mϕ (*P < 0.05; **P < 0.005; ***P < 0.0005).
Figure 4
Figure 4
Responses of blood CD4CD8 (DN) MAIT subsets from healthy adults to H. pylori-infected macrophages. (A) Representative volunteer showing the induction of cytokine production (IFN-γ, TNF-α, IL-17A) and up-regulation of CD107a expression in DN MAIT cells following stimulation with media (alone), non-infected THP-1 macrophages (Mϕ), or H. pylori-infected THP-1 Mϕ (E:T – 50:1). (B) Cumulative data (n = 11) showing production of cytokines (IFN-γ, TNF-α, and IL-17A) and expression of CD107a by DN MAIT cells following stimulation with H. pylori-infected THP-1 Mϕ (E:T – 50:1). (C) CD69 up-regulation by CD3+CD161+TCR Vα7.2+ cells following H. pylori-infected THP-1 Mϕ stimulation (E:T – 50:1) and assessment of the level of cytokines production (IFN-γ, TNF-α, IL-17A) and CD107a expression in CD69+ MAIT cell subsets (CD8+ and CD4CD8) following stimulation with H. pylori-infected THP-1 Mϕ (E:T – 50:1) (n = 11). Horizontal black lines in (B,C) represent medians. Significant differences are denoted by asterisks (**P < 0.005).
Figure 5
Figure 5
Blood MAIT cells from healthy adults exhibit multifunctional abilities and are cytotoxic to H. pylori-infected macrophages. (A) Multifunctional activities of MAIT cells were determined by simultaneous detection of three intracellular cytokines (IFN-γ, TNF-α, and IL-17A) and expression of CD107a by CD8+ MAIT cells following stimulation with H. pylori-infected THP-1 Mϕ (E:T – 50:1). Scatter plot shows single-cytokine-producing and CD107-expressing cells and the six predominant multi-cytokine-producing/CD107-expressing patterns using PBMCs from healthy adult volunteers. Horizontal red lines indicate the median responses. Multifunctionality was analyzed using the FCOM feature of WinList. (B) H. pylori CTL responses by MAIT cells were measured at various effector:target ratios (50:1, 25:1, 12.5:1, 6.25:1) in non-infected (NI) and H. pylori-infected (INF) THP-1 Mϕ. Blocking of CTL responses was performed using anti-human MR-1 antibody (goat, polyclonal) (10 μg/ml) or goat IgG control (immunoglobulin control; IgC; 10 μg/ml). (C) H. pylori CTL responses by MAIT cells were measured at two effector:target ratios (50:1 and 25:1) in NI and INF THP-1 Mϕ. Blocking of CTL responses were performed using the anti-human MR-1 mAb (clone 26.5) (10 μg/ml) or a matched isotype control (IC, 10 μg/ml). Lines show the mean percentages of cytotoxicity at different E/T ratios from triplicate wells. The data are representative of four separate experiments.
Figure 6
Figure 6
MR1 restriction of responses by blood MAIT CD8+ and CD4CD8 DN subsets from healthy adults to H. pylori-infected primary autologous macrophages. Representative volunteer showing the induction of cytokine production (IFN-γ and TNF-α) and up-regulation of CD107a expression in (A) CD8+ and (B) DN MAIT cell subsets following stimulation with non-infected primary autologous macrophages (Mϕ); H. pylori-infected primary autologous Mϕ (E:T 5:1 ratio); H. pylori-infected primary autologous Mϕ (E:T – 5:1 ratio) + α-MR1 (26.5 monoclonal ab) (10 μg/ml); or H. pylori-infected primary autologous Mϕ (E:T – 5:1 ratio) + isotype control (10 μg/ml). Cumulative data (n = 5) showing MR1-restricted production of IFN-γ, TNF-α, and expression of CD107a by (C) CD8+ and (D) DN MAIT cells following stimulation with the four treatments described above. The % of responses compared to media control (% of media control) was calculated as (cytokine production/CD107 expression in cells stimulated with H. pylori-infected primary autologous macrophages containing either α-MR1 or IC)/(cytokine production/CD107 expression in cells stimulated with H. pylori-infected primary macrophages) × 100. Horizontal black lines in (B,D) represent medians. Significant differences are denoted by asterisks (*P < 0.05; **P < 0.005).
Figure 7
Figure 7
Responses of human gastric MAIT cells to H. pylori-infected macrophages. (A) Representative volunteer showing the induction of cytokine production (IFN-γ, TNF-α) and expression of CD107a by gastric CD8+ MAIT following stimulation by H. pylori-infected and non-infected THP-1 Mϕ. Shown are cumulative data (n = 8) of (B) IFN-γ production, (C) TNF-α production, and (D) expression of CD107a by CD8+ MAIT following stimulation by H. pylori-infected Mϕ and non-infected THP-1 Mϕ. Horizontal lines in (B–D) represent medians. Significant differences are denoted by **P < 0.005.
Figure 8
Figure 8
MAIT cells are lower in blood but not in the gastric mucosa of H. pylori-infected volunteers. Aggregate data of the percentages of MAIT cell subsets (CD8+ and CD4CD8) in H. pylori-negative (Hp−ve) (n = 20) and H. pylori-positive (Hp+ve) (n = 5) volunteers in (A) PBMC and (B) gastric LPMC. Hp−ve volunteers were age-matched to Hp+ve volunteers. (C) Representative example of tissue-resident T markers (CD69 and CD103) expression on CD8+ MAIT cells from PBMC and gastric LPMC. (D) Cumulative data (n = 6) comparing the expression of TR MAIT cells in PBMC and LPMC. Horizontal lines in (A,B,D) represent medians. Significant differences are denoted by asterisks (*P < 0.05, **P < 0.005).

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