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. 2014 Jun 19;5:294.
doi: 10.3389/fimmu.2014.00294. eCollection 2014.

Characterization and Functional Properties of Gastric Tissue-Resident Memory T Cells From Children, Adults, and the Elderly

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Free PMC article

Characterization and Functional Properties of Gastric Tissue-Resident Memory T Cells From Children, Adults, and the Elderly

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

Abstract

T cells are the main orchestrators of protective immunity in the stomach; however, limited information on the presence and function of the gastric T subsets is available mainly due to the difficulty in recovering high numbers of viable cells from human gastric biopsies. To overcome this shortcoming we optimized a cell isolation method that yielded high numbers of viable lamina propria mononuclear cells (LPMC) from gastric biopsies. Classic memory T subsets were identified in gastric LPMC and compared to peripheral blood mononuclear cells (PBMC) obtained from children, adults, and the elderly using an optimized 14 color flow cytometry panel. A dominant effector memory T (TEM) phenotype was observed in gastric LPMC CD4(+) and CD8(+) T cells in all age groups. We then evaluated whether these cells represented a population of gastric tissue-resident memory T (TRM) cells by assessing expression of CD103 and CD69. The vast majority of gastric LPMC CD8(+) T cells either co-expressed CD103/CD69 (>70%) or expressed CD103 alone (~20%). Gastric LPMC CD4(+) T cells also either co-expressed CD103/CD69 (>35%) or expressed at least one of these markers. Thus, gastric LPMC CD8(+) and CD4(+) T cells had the characteristics of TRM cells. Gastric CD8(+) and CD4(+) TRM cells produced multiple cytokines (IFN-γ, IL-2, TNF-α, IL-17A, MIP-1β) and up-regulated CD107a upon stimulation. However, marked differences were observed in their cytokine and multi-cytokine profiles when compared to their PBMC TEM counterparts. Furthermore, gastric CD8(+) TRM and CD4(+) TRM cells demonstrated differences in the frequency, susceptibility to activation, and cytokine/multi-cytokine production profiles among the age groups. Most notably, children's gastric TRM cells responded differently to stimuli than gastric TRM cells from adults or the elderly. In conclusion, we demonstrate the presence of gastric TRM, which exhibit diverse functional characteristics in children, adults, and the elderly.

Keywords: LPMC; gastric tissue-resident/memory T cells; multifunctionality; stomach.

Figures

Figure 1
Figure 1
Methodological diagram for the isolation of gastric LPMC. A detailed description of the optimized procedure for isolation of LPMC is found in the Section “Materials and Methods.”
Figure 2
Figure 2
Cell yields from gastric biopsies (LPMC) from different age groups. (A) Comparison of biopsy weight and viable cell yields obtained from children, adults, and the elderly. Trypan blue exclusion was used to enumerate live viable cells. (B) Cell yields were expressed as total viable cells per milligram of tissue and compared between the three age groups. (C) Correlation between the age of volunteers and cell yields obtained from biopsies as continuous variables. Results were analyzed using Spearman’s correlation (n = 57); *p < 0.05.
Figure 3
Figure 3
Characterization of memory T (TM) cells in gastric LPMC and PBMC from children, adults, and the elderly. (A) Representative scatter plots of the gating strategy used to characterize T cell subsets in LPMC and PBMC: naïve (CD62L+CD45RA+), central memory (TCM, CD62L+CD45RA), effector memory (TEM, CD62LCD45RA), and effector memory expressing CD45RA (TEMRA, CD62LCD45RA+). Data shown are from a 12 year-old child. (B) Cumulative data showing the median % and range of CD3, CD4, CD8, TEM populations in gastric LPMC and PBMC from all three age groups. (C) Identification of tissue-resident memory T (TRM) cells in gastric LPMC. Representative plots showing expression of TRM cells as defined by the concomitant expression of CD103 and CD69 markers on CD8+ and CD4+ T cells in gastric LPMC and PBMC. (D) Cumulative data (n = 10) showing the percentage of gastric TRM cells among CD8+ and CD4+ T cells from LPMC and PBMC (***p < 0.0005).
Figure 4
Figure 4
Characterization of integrin α4β7 expression on memory T (TRM and TEM) cells in gastric LPMC and PBMC. (A) Representative scatter plots showing integrin α4β7 expression on CD8+ and CD4+ gastric LPMC and PBMC (data shown are from a 12-year-old child). (B) Cumulative data (n = 14) showing the percentage of CD8+ and CD4+ TRM (LPMC) as well as CD8+ and CD4+ TEM (PBMC) cells expressing integrin α4β7. Significant differences are denoted as follows: **p < 0.005; ***p < 0.0005. (C) Cumulative data comparing the percentage of CD8+ or CD4+ TRM subsets (LPMC) expressing integrin α4β7 by age group. Closed and open symbols represent CD8+ and CD4+ TRM cells, respectively: adults (Ad); children (Ch); elderly (El).
Figure 5
Figure 5
Activation of CD8+ and CD4+ tissue-resident memory T (TRM) cells in gastric LPMC. Representative plot of the activation of gastric LPMC CD8+ (A) or CD4+ (B) TRM (CD62LCD45RACD69+ CD103+) by two stimulants: (1) staphylococcal enterotoxin B (SEB; 10 μg/ml) and (2) anti-CD3/CD28 beads (α-CD3 α-CD28) to produce IL-2, IFN-γ, MIP-1β, TNF-α, IL-17A, and up-regulation of the expression of CD107a. Cells left unstimulated were used as negative control (Media, C-). Cumulative data comparing baseline activation levels of gastric LPMC CD8+ (C) and CD4+ (D) TRM (white portion of the bar) to PBMC (black portion of the bar). In (C,D) significant differences between TEM and TRM are indicated with asterisks on top of each bar; *p < 0.05; **p < 0.005; ***p < 0.0005.
Figure 6
Figure 6
Multifunctional gastric LPMC CD8+ TRM and PBMC CD8+ TEM responses to SEB stimulation in adults, children, and the elderly. Multifunctionality was determined by simultaneous detection of two or more functions performed by CD8+ TRM (LPMC) or CD8+ TEM (PBMC). Six functions were evaluated: production of five cytokines/chemokines (IFN-γ, TNF-α, IL-2, IL-17A, MIP-1β) and expression of CD107a in response to SEB stimulation. (A) Scatter plot showing the six predominant function patterns in LPMC CD8+ TRM and (B) in PBMC CD8+ TEM cells from adults (red circles, n = 9), children (black squares, n = 7), and the elderly (blue triangles, n = 8). Multifunctionality was analyzed using the FCOM feature of WinList. Significant differences between age groups were denoted by asterisks (*p < 0.05). Black dotted boxes indicate the same multiple cytokine-producing cells in LPMC and PBMC CD8+ T subsets.
Figure 7
Figure 7
Multifunctional gastric LPMC CD4+ TRM and PBMC CD4+ TEM responses to SEB stimulation in adults, children, and the elderly. Multifunctionality was determined by simultaneous detection of two or more functions performed by CD4+ TRM (LPMC) or CD4+ TEM (PBMC). Six functions were evaluated: production of five cytokines/chemokines (IFN-γ, TNF-α, IL-2, IL-17A, MIP-1β) and expression of CD107a in response to SEB stimulation. (A) Scatter plot showing the six predominant function patterns in LPMC CD4+ TRM and (B) in PBMC CD4+ TEM cells from adults (red circles, n = 9), children (black squares, n = 7), and the elderly (blue triangles, n = 8). Multifunctionality was analyzed using the FCOM feature of WinList. Significant differences between age groups were denoted by asterisks (*p < 0.05). Black dotted boxes indicate the same multiple cytokine-producing cells in LPMC and PBMC CD4+ T subsets.

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