ORIGINAL RESEARCH ARTICLE
published: 19 June 2014
doi: 10.3389/fimmu.2014.00294
Characterization and functional properties of gastric
tissue-resident memory T cells from children, adults,
and the elderly
Jayaum S. Booth 1,2 † , Franklin R. Toapanta 1,3 † , Rosangela Salerno-Goncalves 1,2 , Seema Patil 3,4 ,
Howard A. Kader 2 , Anca M. Safta 2 , Steven J. Czinn 2 , Bruce D. Greenwald 3,4 and Marcelo B. Sztein 1,2,3 *
1
2
3
4
Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, MD, USA
Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD, USA
Department of Medicine, University of Maryland School of Medicine, Baltimore, MD, USA
Division of Gastroenterology and Hepatology, University of Maryland School of Medicine, Baltimore, MD, USA
Edited by:
Eric Cox, Ghent University, Belgium
Reviewed by:
Diane Bimczok, University of Alabama
at Birmingham, USA
Charles Kelly, King’s College London,
UK
*Correspondence:
Marcelo B. Sztein, Center for Vaccine
Development, University of Maryland
School of Medicine, 685 West
Baltimore Street, HSF-1 Room 480,
Baltimore, MD 21201, USA
e-mail: msztein@medicine.
umaryland.edu
†
Jayaum S. Booth and Franklin R.
Toapanta have contributed equally to
this work.
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 PBMCTEM counterparts. Furthermore, gastric CD8+ TRM and CD4+ TRM cells
demonstrated differences in the frequency, susceptibility to activation, and cytokine/multicytokine 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, stomach, gastric tissue-resident/memory T cells, multifunctionality
INTRODUCTION
In human , peripheral blood memory T (TM ) cells are commonly
grouped into two major subsets based on their functional status
and expression of defined homing receptors (e.g., CD62L, CCR7,
and CD45RA) (1): central memory T (TCM ) and effector memory
T (TEM ) cells. While TCM cells express the lymph node-targeting
molecules CD62L and CCR7, TEM cells largely lack these receptors,
and typically express defined homing molecules that endows them
with the ability to migrate to peripheral non-lymphoid tissues (1,
2). Recently, a novel population of T cells known as tissue-resident
memory CD8+ T (TRM ) cells has been described. These TRM cells
have the ability to remain for long periods of time in peripheral
tissues (e.g., intestinal and vaginal mucosa, skin, brain, and salivary
glands) after pathogenic clearance. These cells have been shown
to be antigen-specific, express markers of CD8+ TEM cells, and
their survival appears to be antigen-independent (3–6). Studies in
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epithelial and neuronal tissues have shown that TRM are characterized by their expression of high levels of CD103 (the α-chain of the
integrin αEβ7) and CD69 (a surface molecule typically found on
recently activated T cells) (7, 8). While initially described in mice,
these cells have been recently also identified in human tissues (9).
The stomach’s primary function is to digest food. With its low
pH environment, the stomach has a secondary function in limiting the number of microorganisms that enter the intestinal tract.
However, some microorganisms such as Helicobacter pylori (H.
pylori) can cause significant pathogenesis and have a niche in this
harsh environment. Various immune cells have been identified in
stomach biopsies obtained during esophagogastroduodenoscopy
(EGD) procedures. Immune populations described in gastric lamina propria mononuclear cells (LPMC) include γδT cells (10),
CD13+ macrophages (Mφ) (M1 and M2) (11), dendritic cells
(DC) (12), natural killer (NK) (13), NK-T (14), neutrophils, B
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Booth et al.
cells (15), and T (CD4+ and CD8+ ) cells (14, 16, 17). Various
studies have demonstrated that intestinal immune cells (innate
and adaptive) are phenotypically and functionally different from
their systemic counterparts. For example, intestinal Mφ is more
phagocytic and bactericidal but secrete less pro-inflammatory
cytokines than their peripheral blood counterparts (18). Additionally, the CD4+ /CD8+ ratio is inverted (~1:3) in gastric LPMC
(from healthy volunteers) compared to peripheral blood mononuclear cells (PBMC) (~3:1) (16, 19, 20). Despite these observations,
very little is known about the different T cell subsets present in
the gastric mucosa of healthy humans. For example, it is currently
unknown whether TCM and TEM cells have similar percentage
distribution in the gastric mucosa as in peripheral blood. Furthermore, despite that the acidic environment of the stomach provides
a different environment than the one found in the mucosa of the
small and large intestines, the gastric mucosa is part of the digestive
tract and therefore has the potential to harbor TRM cells. However, the presence of TRM cells and their ability to exhibit effector
functions (e.g., cytokine production and cytotoxicity) is currently
unknown. In addition, no study has assessed the frequency of these
cells in the gastric mucosa and differences in various age groups
(children, adults, and the elderly). In the present study, after optimizing an isolation method for LPMC from gastric biopsies, we
characterized in depth the memory CD4+ and CD8+ T cell subsets
in gastric LPMC of healthy human volunteers. We demonstrated
that the most abundant CD8+ T cell population in the stomach
(>80%) was TRM (CD62L− , CD45RA− , CD103+ , and CD69+ ).
These cells were able to produce various cytokines (either single or multiple cytokines simultaneously) when stimulated with
mitogens and demonstrated differences in the strength and quality of the responses among different age groups (adults, children,
and the elderly). These findings were only partially mirrored by
gastric CD4+ TRM cells, since only ~35% of the cells showed coexpression of CD103 and CD69. However, these cells were also
able to produce cytokines and showed differences among the age
groups evaluated. These novel findings suggest that TRM might
play a key role in protection from gastric infections and offer new
insights into age differences in gastric immunity.
Age-associated responses in gastric TRM
antrum mucosa was either normal (n = 12) or exhibited mild
inflammation (n = 45). No concurrent GI disease/disorders or
other illnesses that may affect the GI tract were present. Additionally, all volunteers were confirmed to be H. pylori negative as determined by culture and rapid Urease test (CLO test).Tissue samples
collected during EGD were transported to the laboratory facilities
in a tube containing RPMI 1640 (Gibco, Carlsbad, CA, USA) with
antibiotics/antifungal (Penicillin/Streptomycin/Amphotericin B;
Gibco) and processed immediately after collection as shown in
Figure 1 and Figure S1 in Supplementary Material. We first compared two methods the isolation of gastric LPMC: (i) a conventional method (CM) and (ii) bullet blender (BB) method. The
CM method consisted of three steps: (a) removal of intraepithelial
lymphocytes (IEL) [HBSS + EDTA (1 mM)], (b) digestion of the
resulting tissues (collagenase D/DNase I), and (c) disaggregation
of the tissues (by teasing of the tissues between the frosty ends
of two microscope glass slides). The BB method also consisted
of three steps. The first two steps were similar to the CM whist
the last step consisted of homogenizing the gastric biopsy tissues
using a BB (Next Advance, Averill Park, NY, USA) (Figure 1). To
perform these methods, media was removed from the biopsies
by using a 70-µm cell strainer (BD Falcon, Franklin Lakes, NJ,
USA) and dried through the filter using sterile gauze. The tissue
was then transferred to a pre-weighted 1.5 ml centrifuge tube and
the net weight measured. Biopsies were then rapidly transferred
to a 50 ml conical tube containing 10 ml of HBSS without CaCl2 ,
MgCl2 , MgSO4 (Gibco) with antibiotics/antifungal mix (Gibco)
and EDTA (1 mM) and incubated at 37°C for 30 min while shaking. The tissues were washed with 10 ml of HBSS buffer (with
CaCl2 , MgCl2 ) (Gibco) without EDTA and incubated for 10 min
at room temperature (RT) while shaking. The tissues were then
enzymatically digested either in six well plates (CM method) or
1.5 ml sterile screw-top polypropylene microcentrifuge tubes (BB
method) containing 1 ml of digestion solution. Tubes used for
the BB method also contained two stainless steel beads (3.2 mm
MATERIALS AND METHODS
VOLUNTEERS AND ISOLATION OF PERIPHERAL BLOOD MONONUCLEAR
CELLS
Volunteers were recruited from the Baltimore–Washington area
and University of Maryland, Baltimore campus. Written informed
consent was obtained and all procedures were approved by the
University of Maryland, Baltimore IRB. Immediately after blood
draws, PBMC were isolated by density gradient centrifugation
and used freshly for stimulation and characterization. Blood and
gastric biopsies were collected at the same time. A total of 57
volunteers (aged 7–85 years) were evaluated.
ISOLATION OF LAMINA PROPRIA MONONUCLEAR CELLS
Gastric biopsies were collected from volunteers (7–85 years-old)
referred for outpatient diagnostic upper endoscopy (EGD) at the
University of Maryland Medical Center. The indications for EGD
included abdominal pain, heartburn, GERD, dysphagia, and acute
gastritis. Diagnostic pathology reports showed that the stomach’s
Frontiers in Immunology | Mucosal Immunity
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.”
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Booth et al.
diameter; Next Advance Inc., Averill, NY, USA). The enzymatic
digestion solution consisted of 1 ml of RPMI containing 10 µl
of fetal bovine serum (FBS) (Gemini Bioproducts, West Sacramento, CA, USA), 10 µl antibiotics/antifungal mix (Gibco), 10 µl
of 2.5 M CaCl2 , 10 µl of Collagenase D (100 µg/ml; Roche, Indianapolis, IN, USA), and 1 µl DNase I (10 µg/ml; Affymetrix,
Cleveland, OH, USA). The biopsies (20 mg maximum per tube)
were digested for 45 min at 37°C with intermittent pipetting (CM
method) or shaking (BB method). Following 45 min incubation, the tissues were disaggregated using the frosty ends of glass
slides (CM method). In the case of the BB method, following
the 45 min digestion the tube was placed in a BB homogenizer
(Next Advance Inc., NY, USA) and the tissue homogenized for
30 s (speed 1). Tissues were further incubated for 15 min (37°C).
After the second digestion by either method, cells were collected
in a 50 ml tube through a 70 µm cell strainer and centrifuged
at 1400 rpm. Cells were then washed and re-suspended in complete RPMI [Heat inactivated FBS (10%), l-glutamine (2 mM),
non-essential Amino acids (1×) (Gibco 11140), HEPES buffer
(10 mM) (Gibco 15630-080), Sodium pyruvate (2.5 mM) (Lonza
13-155E), Penicillin/streptomycin (100 U/ml–100 µg/ml) (Sigma
P0781), Gentamicin (50 µg/ml) (Gibco 15750-060)], and counted
using Kova Glasstic Slides (Hycor Biomedical, CA, USA). Cells
were either stained immediately for immunophenotyping by flow
cytometry or stimulated with mitogens overnight before staining
(see below). To evaluate whether the enzymatic digestion resulted
in loss of surface receptors, PBMC were either treated or untreated
with the same digestion mix and processed as detailed above
and assessed for surface markers (Figures S1C,D in Supplementary Material). To determine the effect of collagenase D in the
digestion mix on surface marker expression the above procedure
was followed using an enzymatic mix in which collagenase D was
replaced with dispase (1 µg/ml) (Figure S1C in Supplementary
Material).
FLOW CYTOMETRY PROCEDURES AND STAINING
Ex vivo stimulation
Freshly isolated cells from PBMC and gastric biopsies (LPMC)
were re-suspended in complete media and stimulated with
medium, staphylococcal enterotoxin B (SEB) (10 µg/ml; Sigma)
or Dynabeads Human T activator CD3/CD28 (4 × 104 beads/ml)
(Invitrogen Dynal, Oslo, Norway). For each treatment, 1 × 105
LPMC and 1 × 106 PBMC were cultured in 200 µl and 1 ml total
volumes, respectively, and incubated at 37°C in 5% CO2 . In some
experiments, cells were stained with CD107a-FITC at the time of
stimulation. After 2 h, GolgiStop (Monensin, BD) and GolgiPlug
(Brefeldin A, BD) were added at concentrations of 0.5 µl/ml and
cultures continued overnight at 37°C in 5% CO2 .
Surface and intracellular staining
Following stimulation, PBMC and LPMC were plated in 96well V-bottom plates for staining. Cells were washed twice with
phosphate buffered saline (PBS) and stained for live/dead discrimination using Invitrogen LIVE/DEAD fixable yellow dead
cell stain kit (YEVID) (Invitrogen, Carlsbad, CA, USA). Blocking of Fc receptors was performed using human immunoglobulin
(3 µg/ml; Sigma) and was followed by surface staining, performed
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Age-associated responses in gastric TRM
as previously described (21). Briefly, cells were stained with fluorescently labeled monoclonal antibodies (mAbs) directed to
CD14-BV570 (clone M5E2, Biolegend, San Diego, CA, USA)
and CD19-BV570 (clone HIB19, Biolegend), CD3-BV650 (clone
OKT3, Biolegend), CD4-PE-Cy5 (clone RPA-T4, BD), CD8PerCP-Cy5.5 (clone SK-1, Becton–Dickinson, BD), CD45RAbiotin (clone HI100, BD), integrin α4β7-Alexa Fluor 647 (clone
Act-1, Leukosite, Cambridge, MA, USA), and CD62L-Alexa Fluor
780 (clone DREG-5, eBioscience, San Diego, CA, USA) at 4°C
for 30 min. Staining with streptavidin-QDot800 (Invitrogen) was
performed for panels that included biotin-conjugated mAbs for
30 min at 4°C. The cells were then fixed and permeabilized using IC
fixation and permeabilization buffers (eBioscience) according to
the manufacturer’s recommendations. Intracellular staining with
mAbs to IL-17A-BV421 (clone BL168, Biolegend), IL-2-BV605
(clone MQ1-17H12, Biolegend), IFN-γ-PE-Cy7 (clone B27, BD),
TNF-α-Alexa 700 (clone MAb11, BD), MIP-1β-PE (clone 24006,
R&D Systems, Minneapolis, MN, USA), and CD69-ECD (clone
TP1.55.3, Beckman Coulter, Danvers, MA, USA) was performed at
4°C overnight. After staining, cells were fixed in 1% paraformaldehyde and stored at 4°C until data collection. Data were collected
using a customized LSRII flow cytometer (BD) and then analyzed
using WinList version 7 (Verity Software House, Topsham, ME,
USA) software package. Graphs were generated using GraphPad
Prism version 5.03 (GraphPad Software, San Diego, CA, USA).
In experiments designed to characterize TRM cells, the staining
panels were modified as follows. LPMC and PBMC were stained
with mAbs directed to CD103-Alexa Fluor 488 (clone Ber-ACT8,
Biolegend), CD14-BV570 (clone M5E2, Biolegend, San Diego,
CA, USA), CD13-Pacific Orange (clone WM-15 eBioscience,
San Diego, CA, USA conjugated to Pacific Orange in-house),
CD19-BV570 (clone HIB19, Biolegend), CD3-BV650 (OKT3,
Biolegend), CD4-PE-Cy5 (clone RPA-T4, BD), CD8-PerCP-Cy5.5
(clone SK-1, BD), CD45RA-biotin (clone HI100, BD), integrin
α4β7-Alexa Fluor 647 (clone Act-1, Leukosite, Cambridge, MA,
USA), and CD62L-Alexa Fluor 780 (clone DREG-5, eBioscience,
San Diego, CA, USA) and intracellularly with mAbs to IL-17ABV421 (clone BL168, Biolegend), IL-2-BV605 (clone MQ1-17H12,
Biolegend), IFN-γ-PE-Cy7 (clone B27, BD), TNF-α-Alexa 700
(clone MAb11, BD), MIP-1β-PE (clone 24006, R&D systems, Minneapolis, MN, USA), and CD69-ECD (clone TP1.55.3, Beckman
Coulter, Danvers, MA, USA).
FCOM ANALYSIS FOR MULTIFUNCTIONALITY
FCOM (Verity Software House, Topsham, ME, USA) is an analytical tool that is used to classify events based on combinations of
selected gates. FCOM reduces multiparameter data to a series of
multiple acquisition gates, one for every possible combination.
FCOM was employed to determine the subsets of CD4+ and
CD8+ producing multiple cytokines and/or expressing CD107a
expression (i.e., multifunctionality).
STATISTICAL ANALYSIS
Data were analyzed using GRAPHPAD PRISM™ 5.03 statistical
software (Graphpad, San Diego, CA, USA). Statistical differences
in median values between two groups were determined using
Mann–Whitney tests. Statistical differences between multiple
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Booth et al.
groups (more than two) were determined by Kruskal–Wallis tests
and the Dunn’s post-test was used to compare selected group pairs.
Values of *p < 0.05, **p < 0.005, ***p < 0.0005 were considered
significant.
RESULTS
GASTRIC LPMC ISOLATION AND CELL YIELDS FROM CHILDREN,
ADULTS, AND ELDERLY VOLUNTEERS
Several methodologies to isolate gastric leukocytes from human
stomach biopsies have been reported; however, there is a lack of
consensus in the type of digestion enzymes to use, their concentration, digestion periods and whether or not to use mechanical
dissociation techniques. Therefore, we optimized a protocol for
isolation of gastric LPMC. We first compared two methods: (i)
a conventional method (CM) and (ii) a blender method (BB)
(described in detail in Section “Materials and Methods”). In the
BB method, we optimized the homogenization step regarding the
speed, time, and number of beads needed for a gentle dissociation
of the cells from the gastric tissues. We found that homogenizing the tissue for 30 s at a speed of 1 and using 2 beads (stainless
steel; 3.2 mm diameter) resulted in optimal cell yields (Figure S1A
in Supplementary Material). This optimized BB method yielded
superior cell numbers (1.1 × 104 /mg of tissue) from human biopsies compared to the CM method (0.6 × 104 /mg of tissue) (Figure
S1B in Supplementary Material). Two digestion enzymes (collagenase D and dispase) were then compared by substituting each
one using the optimized BB method. We observed that collagenase D treatment resulted in better cell yields and cell surface
marker preservation than dispase, which had a marked effect on
the expression of cell surface markers as shown by lower MFI
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
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Age-associated responses in gastric TRM
for CD4+ and CD8+ T cells in both PBMC and LPMC isolated
cells (Figure S1C in Supplementary Material). We further evaluated the effect of collagenase D on tissues by using PBMC treated
in similar fashion as biopsies with the BB method. The results
showed no significant differences in the expression levels of CD3,
CD4, CD8, CD45RA, CD62L, and integrin α4β7 surface markers
between PBMC treated with or without collagenase D (Figure S1D
in Supplementary Material).
Stomach biopsies (antrum) obtained from H. pylori negative
(CLO test negative) adult (18–64 years), children (7–17 years), and
elderly (65–85 years) volunteers were processed as described in
Section“Materials and Methods”and Figure 1. Gastric LPMC were
isolated and enumerated from five biopsy samples from each adult
and elderly volunteer and three biopsy samples from each child
(Figure 2A). The viable cell yields in biopsies from adult and the
elderly ranged from 230,000 to 2,300,000 (median 634,000) and
240,000 to 1,300,000 (median 605,000) cells, respectively; whereas
in children’s biopsies cell yields ranged from 320,000 to 734,000
(median: 492,000) cells (Figure 2A). The total viable cell yields
in the children group was significantly lower (p < 0.05) than in
the adult group. However, the weight of biopsies varied between
age groups as samples from children were significantly (p < 0.05)
smaller in size and weight (8.8–33.4 mg) than samples from adults
(20.5–76 mg) and the elderly (31.7–51.9 mg) (Figure 2A). To
compare the viable cell yields among age groups we calculated
the number of viable cells per milligram of tissue. The results
showed that the cell yields obtained from biopsies of children
(13,000–49,000 viable cells/mg of tissue, median: 21,000) were
significantly higher (p < 0.05) than those obtained from biopsies of the elderly (6000–49,000, median: 15,500) (Figures 2A,B).
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.
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Finally, using Pearson’s regression analysis, we observed a significant inverse correlation (r = −0.3, p = 0.021) between the number
of isolated cells per milligram of tissue and age (Figure 2C).
CHARACTERIZATION OF T CELL SUBSETS IN LPMC AND PBMC
Human gastric T lymphocytes have been shown to display a
Th1 type response (IFN-γ, TNF-α, and IL-2 secretion) toward
pathogens, such as H. pylori (16). However, the TM subset(s) that
secrete(s) these cytokines and differences among the various age
groups have not been explored. To address these shortcomings, the
presence of T cells (CD3+ CD13− CD19− ) in LPMC and PBMC
was assessed (Figure 3A). The frequency of these cells was significantly lower in LPMC than in PBMC from adult volunteers
(median: 13.7 vs. 74.7%; p < 0.0005) (Figures 3A,B). Similar findings were observed in children and the elderly (Figure 3B). When
CD3+ T cells were divided into CD4+ (CD3+ CD4+ ) and CD8+
(CD3+ CD8+ ) T cells, the latter were more abundant in LPMC
than in PBMC (Figures 3A,B). Therefore, there was an inversion
in the CD4/CD8 ratio in LPMC (~1:3) compared to PBMC (~3:1).
These results confirmed and extended those reported by others
(16, 19, 20) by showing that these differences were observed in
all age groups (Figure 3B). When CD4+ T cells in LPMC of children and the elderly were compared to adults, children showed
a significantly higher frequency of these cells (Figure 3B). In
contrast, PBMC from all three groups expressed similar levels of
CD3+ , CD4+ , and CD8+ T cells (Figure 3B). Next, we evaluated
the presence of TM cells in LPMC and PBMC using CD62L and
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 , CD62L− CD45RA− ), and effector memory expressing
CD45RA (TEMRA , CD62L− CD45RA+ ). Data shown are from a 12 year-old child.
(B) Cumulative data showing the median % and range of CD3, CD4, CD8, TEM
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Age-associated responses in gastric TRM
CD45RA markers. These markers define four different subsets:
(i) T central memory (TCM ) (CD62L+ CD45RA− ), (ii) T naïve
(Tnaive ) (CD62L+ CD45RA+ ), (iii) T TEM (CD62L− CD45RA− ),
and (iv) CD45RA positive T TEMRA (CD62L− CD45RA+ ) (22).
Interestingly, the vast majority of adult CD8+ and CD4+ T cells in
LPMC showed a TEM phenotype (>70%) (CD62L− , CD45RA− )
(Figures 3A,B). This phenotype was also dominant in children and
the elderly (Figure 3B). In contrast, the classic memory (TCM , TEM ,
and TEMRA ) and naïve subsets defined by CD62L and CD45RA
were identified in PBMC (Figures 3A,B). The finding that LPMC
CD4+ and CD8+ T cells showed a dominant TEM phenotype
suggested that these cells could represent a population of gastric tissue-resident memory T (TRM ) cells. The hallmark of TRM
is the surface expression of high levels of CD103 and CD69 (4, 6–
8); therefore, we evaluated expression of these markers in LPMC
and PBMC. We observed that most CD8+ T cells in LPMC coexpressed CD103 and CD69 (mean: 81.1%; median: 80.5%), while
a much lower percentage of CD4+ T cells in LPMC co-expressed
these markers (mean: 35%; median: 28.8%) (Figures 3C,D). Similar results were observed in all the volunteers tested (Figure 3D).
On the other hand, CD103 and CD69 were virtually absent from
CD4+ and CD8+ T cells in PBMC. Further analysis revealed that
CD8+ T cells in LPMC either co-expressed CD103 and CD69
(>70%) or expressed CD103 alone (~20%); therefore, more than
90% of these cells expressed at least one marker that defined them
as TRM cells (Figure 3C). Similarly, even though only ~30% of
CD4+ T cells in LPMC co-expressed CD103 and CD69, expression
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).
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of CD69 or CD103 alone was found in ~35 and ~10% of these cells,
respectively. In sum, the majority of the CD4+ and CD8+ T cells
in LPMC expressed molecules compatible with those reported for
TRM cells. In contrast, the expression of CD103 and CD69 was virtually absent from PBMC CD4+ and CD8+ T cells (Figures 3C,D).
Expression of the homing marker integrin α4β7 was assessed in
PBMC and LPMC in the subsets showing the TEM phenotype (TRM
in LPMC and TEM in PBMC) (Figure 4A). Gastric CD8+ TRM
and CD4+ TRM showed a significantly lower level of expression of
integrin α4β7 (p < 0.05) compared to CD8+ TEM and CD4+ TEM
(Figures 4A,B). Interestingly, there were no significant differences
in integrin α4β7 expression among gastric LPMC CD8+ TRM or
CD4+ TRM between adult, children, and the elderly (Figure 4C).
MITOGEN ACTIVATION OF LPMC AND PBMC
We next examined whether isolated gastric LPMC CD8+ or CD4+
TRM cells were functionally active by exploring whether they
responded to mitogen stimulation by producing cytokines and upregulating the expression of CD107a, a marker of degranulation
associated with cytotoxic activity (23). Furthermore, we explored
whether there were any differences in the responses between the
different age groups. Gastric LPMC and PBMC were stimulated
with various T cell stimulants including: (i) SEB (superantigen)
and (ii) α-CD3/CD28 coated beads (TCR stimulation). As negative control, cells were incubated in media alone. The concomitant
production of multiple cytokines (IFN-γ, TNF-α, IL-2, IL-17A,
and MIP-1β) and up-regulation of CD107a were determined in
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
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Age-associated responses in gastric TRM
LPMC (Figures 5A,B) and PBMC in all three age groups. Upregulation of CD69 as a cell activation marker was only considered
for PBMC since in LPMC (TRM ) this marker is highly expressed
regardless of stimulation (Figures 3C,D). In PBMC, the main TM
subsets responding to the stimulations were CD8+ and CD4+
TEM and TEMRA and these results were consistent with previous
reports from our group, as well as others (6, 24). The percentages of gastric CD8+ TRM and CD4+ TRM producing cytokines
and up-regulating CD107a following stimulation were compared
to CD8+ TEM and CD4+ TEM (PBMC) and the results in all age
groups are summarized in Tables 1–4.
Interestingly, control media gastric CD8+ and CD4+ TRM cells
from adult volunteers showed higher percentages of cells producing cytokines and up-regulating expression of CD107a than media
only CD8+ TEM and CD4+ TEM (PBMC) cells (Figures 5C,D).
This difference in baseline cytokine production was statistically
significant (p < 0.05) only in CD8+ T cells (Figures 5C,D).
Of note, in children, while higher CD8+ TRM cells producing cytokines and up-regulating the expression of CD107a were
observed, no statistically significant differences were found compared to CD8+ TEM cells in media controls (Table 1). In the elderly
group, a percentage of cells producing significant higher baseline levels of IL-2 and MIP-1β in gastric CD8+ TRM cells were
identified. Concerning the other cytokines investigated, as well
as CD107a, although a similar trend to the other age groups was
observed, the differences did not reach statistical significance, likely
due to high subject-to-subject variability (Table 1). Additionally,
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).
June 2014 | Volume 5 | Article 294 | 6
Booth et al.
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 (CD62L− CD45RA− CD69+
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
the diagnostic pathology reports allowed us to explore whether the
higher baseline cytokines levels of CD8+ and CD4+ TRM cells were
due to extrinsic factors causing inflammation of the antral mucosa.
Volunteers were classified as having either normal or mildly
inflamed (mild diffuse erythema, mild diffuse inflammation, reactive changes) antral mucosa and the baseline cytokines levels were
compared between these two groups (Figure S2 in Supplementary
Material). There were no differences between the normal and“mild
inflammation” groups for any of the cytokines/chemokines (IL-2,
IFNγ, MIP-1β, TNFα, and IL-17A) and CD107a at baseline in
CD8+ TRM cells (Figure S2 in Supplementary Material). Although
CD4+ TRM cells showed somewhat increased baseline cytokines
levels (IL-2, IFNγ, TNFα, and IL-17A) in the “mild inflammation”
group, they were not statistically different than those observed in
the normal group (Figure S2 in Supplementary Material).
Gastric CD8+ TRM and CD4+ TRM cells from adult volunteers
stimulated with mitogens (SEB and anti-CD3/CD28) produced
most of the assessed cytokines at higher levels than media control
cells (Figures 5A,B; Tables 1–4). In general, higher percentages
were observed in gastric CD8+ TRM and CD4+ TRM cells producing cytokines and expressing CD107a than in CD8+ TEM and
CD4+ TEM cells. In some instances, the cytokine production differences were statistically significant (Tables 1 and 2). Overall,
results were similar in all age groups (Tables 1 and 2).
We next compared cytokine production by adults, children,
and the elderly in both PBMC and LPMC populations (results
are summarized in Table 3). CD8+ TEM cells from children did
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Age-associated responses in gastric TRM
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.
not show statistically significant differences when compared to
adults for any of the cytokines evaluated or CD107a expression. In
contrast the elderly group demonstrated significantly higher number of CD8+ TEM cells (p < 0.05) producing MIP-1β at baseline
levels (compared to adults) and this cytokine was identified in a
higher percentage of cells following stimulation (Table 3). Other
cytokines observed in a higher percentage of cells following stimulation in the elderly were TNF-α (anti-CD3/CD28) and CD107a
expression (SEB and anti-CD3/CD28) (Table 3). In children, a significantly higher percentage of CD8+ TRM cells (LPMC) expressed
CD107a at baseline than in adults (Table 3). Neither children nor
the elderly showed differences in the percentage of CD4+ TEM
cells (PBMC) producing cytokines compared to adults. On the
other hand, in children, at baseline, the percentage of CD4+ TRM
cells (LPMC) producing IFN-γ and TNF-α was higher than in
adults. However, no differences were noted following stimulation
(Table 3). In the elderly, the percentage of CD4+ TRM cells producing TNF-α, at basal levels were also significantly higher than
in adults. Moreover, the percentage of CD4+ TRM cells producing
significantly higher levels of IL-2 (anti-CD3/CD28), IFN-γ (antiCD3/CD28), and TNF-α (SEB and anti-CD3/CD28) following
stimulation was also enhanced (Table 3).
We also compared the percentage of cells producing cytokines
following SEB and anti-CD3/CD28 stimulation to the control cells
in all age groups. The results are summarized in Table 4. SEB and
anti-CD3/CD28 beads efficiently induced CD8+ TEM and CD4+
TEM cells from adults and the elderly to produce cytokines and
June 2014 | Volume 5 | Article 294 | 7
Booth et al.
Age-associated responses in gastric TRM
Table 1 | Comparison of PBMC CD8+ TEM and gastric LPMC CD8+ TRM cells activation responses in adult, children, and the elderly.
Cytokine
IL-2
INF-γ
MIP-1β
TNF-α
IL-17A
CD107a
Stimulant
Adult
Children
Elderly
CD8+ TEM
CD8+ TRM
CD8+ TEM
CD8+ TRM
CD8+ TEM
CD8+ TRM
Median
% (Range)
Median
% (Range)
Median
% (Range)
Media
0.1 (0–2)
1.2 (0–2)a,**
0.3 (0–1)
1.0 (0–2)
0.1 (0–1)
1.7 (0–3)**
SEB
1.7 (0–17)
1.6 (0–42)
0.3 (0–2)
1.5 (1–2)
1.8 (1–13)
3.9 (1–22)
α-CD3/CD28
2.0 (0–12)
1.7 (0–11)
0.3 (0–1)
1.0 (0–3)
1.4 (1–9)
4.0 (1–12)
Media
0.2 (0–6)
0.4 (0–3)
1.6 (1–3)
0.6 (0–2)
1.7 (0–5)
SEB
8.6 (0–37)
15.7 (8–27)
1.3 (0–3)*
4.3 (1–19)
10.3 (6–17)
6.3 (2–57)
11.5 (2–28)
α-CD3/CD28
3.1 (0–19)
13.8 (3–30)**
2.7 (1–6)
12.3 (6–23)*
6.5 (1–15)
20.9 (10–28)*
4.8 (1–10)*
Media
0.2 (0–6)
1.9 (0–8)**
0.5 (0–4)
0.6 (0–6)
1.0 (0–4)
SEB
5.0 (0–20)
15.6 (5–31)*
4.4 (0–7)
8.6 (3–15)
15.5 (1–55)
17.8 (3–30)
α-CD3/CD28
2.3 (0–20)
16.7 (3–49)**
1.4 (0–14)
8.9 (2–42)
14.6 (1–39)
33.2 (8–55)
Media
0.2 (0–3)
1.4 (0–4)*
0.9 (0–3)
1.9 (1–3)
0.9 (0–2)
0.7 (0–4)
SEB
2.6 (0–32)
9.1 (4–22)
2.6 (2–21)
3.4 (1–15)
9.4 (1–49)
7.6 (1–17)
α-CD3/CD28
2.9 (0–13)
7.8 (2–15)*
1.9 (1–5)
8.5 (4–11)
9.9 (1–21)
9.7 (1–13)
Media
0.1 (0–2)
0.7 (0–2)*
0.5 (0–1)
1.1 (0–2)
0.1 (0–1)
0.8 (0–2)
SEB
1.1 (0–4)
1.1 (0–7)
0.3 (0–2)
2.2 (0–9)
0.2 (0–1)
0.4 (0–10)
α-CD3/CD28
2.0 (0–5)
1.0 (0–3)
0.2 (0–1)
1.9 (0–2)*
0.2 (0–1)
0.9 (0–5)**
Media
0.1 (0–4)
1.4 (0–3)**
0.8 (0–4)
3.4 (2–4)
1.1 (0–4)
2.6 (1–5)
SEB
5.2 (0–21)
12.9 (7–40)*
4.6 (3–21)
9.8 (7–23)
14.2 (5–45)
12.9 (3–26)
α-CD3/CD28
1.3 (0–12)
10.8 (2–18)**
3.0 (2–7)
16.6 (7–28)**
12.7 (1–21)
12.3 (10–19)
Gastric LPMC CD8+ TRM responses (IL-2, IFN-γ, MIP-1β, TNF-α, IL-17A, and CD107a) to two stimulants (SEB and α-CD3/CD28 beads) were compared to PBMC CD8+
TEM responses obtained from adults, children, and the elderly. Significant differences are shown in highlighted colors as determined by Mann–Whitney tests. Adults:
n = 10; children: n = 10; elderly: n = 10.
a
Light green color = significant increase in the frequency of CD8+ TRM (LPMC) compared to CD8+ TEM (PBMC); *p < 0.05; **p < 0.005.
CD107a expression compared to media control (Table 4). Interestingly, even though both stimulants induced CD8+ TEM cells
to produce cytokines in children (Table 1), the results were not
statistically significant as compared to media (Table 4), and only
CD107a up-regulation was significantly induced by SEB in children. SEB and anti-CD3/CD28 beads were unable to stimulate
CD8+ TRM cells to produce IL-17A in any of the three age groups
and IL-2 was significantly induced only in the elderly (Table 4).
CD8+ TRM cells were efficiently induced to produce IFN-γ and
CD107a expression in all three age groups by both SEB and antiCD3/CD28 beads (p < 0.05). Furthermore, MIP-1β was efficiently
induced in adults and the elderly, but not in children by both
stimulants. TNF-α was also significantly induced in all three age
groups but only by anti-CD3/CD28 beads. Neither SEB nor antiCD3/CD28 beads were able to increase the percentage of CD4+
TRM cells producing IL-2, MIP-1β, and expressing CD107a in children and IL-17A in adults. However, at least one of these stimulants
was able to stimulate CD4+ TRM to produce IL-2, IFN-γ, MIP-1β,
TNF-α, and CD107a in adults and the elderly (Table 4).
MULTIFUNCTIONAL GASTRIC CD8+ AND CD4+ TRM/EM
CD4+ and CD8+ T cells that produce two or more cytokines
simultaneously (multifunctional) have enhanced functionality
and are more likely to correlate with protection from disease when
Frontiers in Immunology | Mucosal Immunity
compared to single cytokine-producing cells (25–28). The induction of multifunctional cells in the human gastric mucosa has not
yet been reported and whether these cells play a role in the development or resolution of pathogenesis remains unknown. Thus,
we investigated whether CD4+ and CD8+ TRM (LPMC) obtained
from the three age groups had multifunctional properties following SEB stimulation. All possible combinations (64 in total) for five
cytokines (IFN-γ, TNF-α, IL-2, IL-17A, MIP-1β) and expression of
CD107a were analyzed in multidimensional space using the WinList FCOM function. Similar analyses were performed in CD4+
TEM and CD8+ TEM (PBMC) populations. The results demonstrated that stimulation elicits multifunctional responses in gastric
CD4+ TRM and CD8+ TRM cells. Similarly, CD4+ TEM and CD8+
TEM cells demonstrated multifunctionality, which is consistent
with previous results from our group as well as others (Figures 6
and 7) (6, 24, 25, 29). For simplicity, shown are only the six highest
expressing multifunctional CD8+ TRM and CD8+ TEM cell groups
(Figures 6A,B). Double, triple, quadruple, and quintuple cytokine
secreting cells CD8+ TRM and CD8+ TEM were found in all age
groups albeit at different percentages. Interestingly, of the six highest multifunctional groups in gastric CD8+ TRM cells, four were
also found in CD8+ TEM cells (dotted boxes) (Figures 6A,B). We
then compared the magnitude of multifunctional T cells between
the age groups and identified some differences. A significantly
June 2014 | Volume 5 | Article 294 | 8
Booth et al.
Age-associated responses in gastric TRM
Table 2 | Comparison of PBMC CD4+ TEM and gastric CD4+ TRM cells activation responses in adult, children, and the elderly.
Cytokine
IL-2
INF-γ
MIP-1β
TNF-α
IL-17A
CD107a
Stimulant
Adult
Children
Elderly
CD4+ TEM
CD4+ TRM
CD4+ TEM
CD4+ TRM
CD4+ TEM
CD4+ TRM
Median
% (Range)
Median
% (Range)
Median
% (Range)
Media
0.3 (0–3)
0.5 (0–2)
0.5 (0–2)
1.3 (0–3)
SEB
2.5 (0–60)
3.4 (0–35)
1.5 (1–5)
0 (0–6)
α-CD3/CD28
2.2 (0–38)
8.9 (0–21)
1.2 (0–6)
Media
0.2 (0–2)
0.3 (0–2)
SEB
3.0 (0–15)
7.0 (0–11)
α-CD3/CD28
0.6 (0–5)
5.6 (5–22)a, ***
0.3 (0–4)
1.6 (0–9)
14.8 (1–31)
8.7 (0–32)
8.8 (0–20)
7.9 (3–27)
21.4 (5–32)
0.7 (0–3)
4.8 (0–9)
0.6 (0–3)
5.8 (2–9)
3.7 (0–25)
6.2 (3–18)
3.6 (1–19)
4.1 (1–9)
10.9 (2–25)
3.8 (1–14)
14.1 (2–27)
0.0 (0–5)
Media
0.2 (0–1)
1.5 (0–8)
0.3 (0–2)
4.7 (0–6)
0.7 (0–1)
1.3 (0–8)
SEB
1.5 (0–13)
4.8 ( − 33)
0.8 (0–5)
11.4 (0–35)
1.5 (1–3)
13.5 (1–21)*
α-CD3/CD28
0.8 (0–9)
9.2 (1–38)**
0.3 (0–3)
15.0 (0–31)
1.2 (0–3)
0.0.3 (7–34)***
Media
0.1 (0–3)
2.3 (0–6)
1.2 (0–6)
4.2 (1–6)
2.8 (0–5)
1.2 (0–6)
SEB
0.9 (0–55)
10.8 (2–30)
15.7 (5–21)
6.3 (2–14)
41.1 (1–53)
9.9 (0–21)b, *
α-CD3/CD28
0.6 (24)
5.8 (14–42)***
12.2 (5–25)
24.6 (8–60)
23.7 (2–31)
27.6 (4–33)
0.0 (0–3)
Media
0.2 (0–2)
0.9 (0–3)
0.3 (0–3)
1.4 (0–3)
0.3 (0–1)
SEB
2.1 (1–16)
2.4 (0–12)
3.1 (0–7)
6.3 (2–25)
1.5 (1–3)
2.1 (1–9)
α-C03/CD28
3.8 (0–14)
4.8 (0–12)
1.6 (1–5)
14.1 (3–19)*
0.9 (0–2)
2.9 (0–14)
Media
0.2 (0–1)
0.2 (0–3)
0.3 (0–1)
1.3 (0–5)
0.2 (0–2)
0.0 (0–1)
SEB
1.5 (1–7)
4.0 (0–11)
1.2 (0–3)
5.7 (2–19)*
1.6 (1–3)
2.2 (0–11)
α-C03/C028
0.7 (0–3)
5.9 (0–8)*
0.7 (0–2)
6.3 (2–12)*
1.4 (0–3)
3.5 (0–12)
Gastric LPMC CD4+ TRM responses (IL-2, IFN-γ, MIP-1β, TNF-α, IL-17A, and CD107a) to two stimulants (SEB and anti-CD3/CD28) were compared to PBMC CD4+ TEM
responses obtained from adults, children, and the elderly. Significant differences are shown in highlighted colors as determined by Mann–Whitney tests. Adults:
n = 10; children: n = 10; elderly: n = 10.
a
Light green color = significant increase in the frequency of CD4+ TRM (LPMC) compared to CD4+ TEM (PBMC).
b
Light blue color = significant decrease in the frequency of CD4+ TRM (LPMC) compared to CD4+ TEM (PBMC); *p < 0.05; **p < 0.005; ***p < 0.0005.
higher percentage of CD8+ TRM cells from the elderly contained
double (CD107a+ MIP-1β+ ) cytokine-producing cells than adults
and children (Figure 6A). Similarly, quintuple (CD107a+ IFN-γ+
TNF-α+ IL-2+ MIP-1β+ ) cytokine-producing cells in the elderly
were significantly more abundant than in adults (Figure 6A). In
peripheral blood, differences between the age groups within the
multi-cytokine-producing sets were also noted. The percentage
of double (TNF-α+ IL-2+ ) and triple (IFN-γ+ TNF-α+ IL-2+ ,
as well as CD107a+ IFN-γ+ MIP-1β+ ) CD8+ TEM cells in the
elderly group was significantly higher than in children (Figure 6B).
Furthermore, the percentage of triple (CD107a+ IFN-γ+ MIP1β+ ) CD8+ TEM cells was higher in the elderly than in adults
(Figure 6B).
We also assessed multifunctionality in CD4+ TRM and CD4+
TEM cells (Figure 7). Both of these cell populations have the potential to become multifunctional and showed differences in the age
groups within various multifunctional sets. Of the six highest multifunctional populations in gastric CD4+ TRM cells (LPMC), only
two were also found in CD4+ TEM cells (PBMC) (dotted boxes)
(Figures 7A,B). Double and quadruple cytokine-producing cells
were observed in gastric CD4+ TRM cells. The percentage of dual
producer cells (TNF-α+ IL-2+ and IL-2+ MIP-1β+ ) in the elderly
was significantly higher than in children (Figure 7A). Moreover,
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the percentage of gastric CD4+ TRM cells producing IL-2 and MIP1β in elderly volunteers was also higher than in adults (Figure 7A).
Interestingly, the percentage of dual producer CD4+ TRM cells
consisting of IL-17 (CD107a+ IL-17A+ and TNF-α+ IL-17A+ )
in children was significantly higher than adults and the elderly
(Figure 7A). Similarly, PBMC CD4+ TEM stimulated with SEB
display activated cells that contained double, triple, and quadruple
cytokine-producing cells (Figure 7B). In the elderly, a significantly
higher percentage of CD4+ TEM cells produced double (IFNγ+ TNF-α+ or TNF-α+ IL-2+ ), triple (CD107a+ IL-2+ TNF-α+
and IFN-γ+ TNF-α+ IL-2+ ), and quadruple (IFN-γ+ TNF-α+
IL-2+ MIP-1β+ ) cytokines than children and adult volunteers
(Figure 7B).
DISCUSSION
Recent reports have described the presence of TRM cells (CD8+ )
in mucosal surfaces (4–6, 9). These cells, originally described in
the mouse model, were very recently identified in the lungs of
humans (9), along with CD4+ TRM cells, which have been less
well characterized. In the stomach mucosa, several immune cells
have been described and although different methodologies for isolation of mononuclear cells from biopsies have been reported, the
optimal conditions remain largely undefined. In this manuscript,
June 2014 | Volume 5 | Article 294 | 9
Booth et al.
Age-associated responses in gastric TRM
Table 3 | Summary of gastric LPMC TRM cells and PBMC TEM (CD4+ and CD8+ ) responses between children and the elderly to adults.
Cytokine
Stim
PBMC
LPMC
PBMC
LPMC
CD8+ TEM
CDS+ TRM
CD4+ TEM
CD4+ TRM
Cha
IL-2
Elb
Ch
El
Ch
El
Ch
El
Med
SEB
α-CD3/CD28c
IFN-γ
*
Med
*
SEB
*
α-CD3/CD28
MIP-1β
TNF-α
Med
*,d
SEB
*
α-CD3/CD28
*
Med
*
SEB
α-CD3/CD28
IL-17A
*
**
*
**
Med
SEB
α-CD3/CD28
CD107a
Med
*
SEB
*
α-CD3/CD28
**
LPMC CD8+ and CD4+ TRM and PBMC TEM responses (IL-2, IFN-γ, MIP-1β, TNF-α, IL-17A, and CD107a) to stimulation by two mitogens (SEB and anti-CD3/CD28) were
determined and compared between children (Ch) or elderly (El) to adults (children vs. adult and elderly vs. adult). Significant differences are shown in highlighted
colors as determined by Mann–Whitney test. Adults: n = 8; children: n = 5; elderly: n = 7. Empty (white) cells indicate non-significant differences between children or
elderly vs. adults as determined by Mann–Whitney tests.
a
Children; b Elderly; c Anti-CD3/CD28 beads; d Light green color = significant in cytokine production compared to *p < 0.05; *p < 0.005.
we report an optimized method for the isolation of human gastric leukocytes from stomach biopsies and, using this method, the
identification of CD8+ TRM and CD4+ TRM cells. Moreover, we
explored the ability of these cells to produce cytokines following
stimulation with various mitogens, as well as demonstrated the
multifunctional nature of these responses. Finally, we investigated
whether there are differences in the quality and magnitude of the
responses in various age groups.
Our cell isolation protocol (LPMC) from gastric biopsies
involved the removal of epithelial cells and a mild enzymatic
digestion step (13) that was combined with a mild mechanical
disruption step using stainless steel beads (BB). This additional
step allowed for maximum dislodgement of cells with minimal
damage and provided a more consistent and uniform homogenization, decreasing variation between samples and generating higher
viable cell yields. Compared to previously published reports, our
method, at a minimum doubled the number of viable LPMC isolated from gastric biopsies (12, 30). However, given that biopsies
size and weight varies considerably during sampling and that in
most studies the biopsy weights have not been reported, the real
efficiency of the methods cannot be directly compared. Cell yields
expressed as total number of viable cells per milligram of tissue in
“dried” biopsies would be optimal to enable this assessment across
Frontiers in Immunology | Mucosal Immunity
studies. Interestingly, the number of cells per milligram of tissue in
children was higher than in the elderly (Figure 2B). These results
were similar to those reported by Bontems et al., who suggested
that there was higher cellularity in children than in adults; however, no statistical differences were reported in that study (17).
The data from elderly volunteers allowed us to extend the time
frame of evaluation and confirmed that as the age of the volunteers increased, the number of mononuclear cells isolated in the
stomach decreased.
Consistent with previous reports, the frequency of CD3+ cells
was lower as a percentage of total LPMC cells in the gastric mucosa
than in PBMC (19, 31). Additionally, CD4+ and CD8+ T cells
from gastric LPMC were found at similar frequencies as reported
by others (14, 16) and the vast majority of gastric CD8+ and
CD4+ T cells showed a TEM phenotype (CD62L− , CD45RA− ).
These results provide evidence that the optimized cell isolation
method described in the present manuscript did not result in cell
subset selection bias. Therefore, these cells appeared to be of the
newly defined tissue-TRM cells in human intestinal tissues (32).
This presumption was confirmed by investigating the expression
of hallmark receptors for these cells, including CD103 and CD69,
which were expressed by T cells isolated from gastric tissues. Interestingly, differences were noted between CD8+ TRM and CD4+
June 2014 | Volume 5 | Article 294 | 10
Booth et al.
Age-associated responses in gastric TRM
Table 4 | Summary of TRM and TEM responses to stimulation in children, elderly, and adults.
Cytokine
IL-2
PBMC
CD8+ TEM
LPMC
CD8+ TRM
PBMC
CD4+ TEM
LPMC
CD48+ TRM
SEB
α3/28a
SEB
α3/28
SEB
α3/28
SEB
α3/28
**,e
**
**
*
**
Eld
***
**
*
*
**
*
*
Ad
***
**
***
***
**
*
*
**
**
**
*
**
Age
Adb
Chc
IFN-γ
Ch
MIP-1β
*
El
**
**
*
**
**
**
Ad
**
*
**
***
**
*
El
**
**
*
**
***
*
*
**
Ad
***
**
***
***
*
*
**
***
**
*
*
**
*
**
**
**
**
**
*
***
***
*
**
*
**
*
Ch
TNF-α
Ch
IL-17A
El
**
**
Ad
*
*
Ch
CD107a
El
*
Ad
***
Ch
*
El
***
***
**
***
**
**
**
**
***
**
*
**
*
*
**
Gastric LPMC CD8+ and CD4+ TRM and PBMC TEM responses (IL-2, IFN-γ, MIP-1β, TNF-α, IL-17A, and CD107a) to stimulation by two mitogens (SEB and anti-CD3/CD28)
were determined and compared to media stimulation (negative control) in adults (Ad), children (Ch), and the elderly (El). Significant differences are shown in highlighted
colors as determined by Mann–Whitney tests. Adults: n = 8; children: n = 5; elderly: n = 7. Empty (white) cells indicate non-significant differences between stimulated
and non-stimulated (media) cultures as determined by Mann–Whitney tests.
a
Anti-CD3/CD28 beads; b Adults; c Children; d Elderly; e Light green color = significant increase in cytokine production compared to media stimulation, *p < 0.05;
**p < 0.005; ***p < 0.0005.
TRM cells in gastric tissues. For example, the large majority of
CD8+ TRM cells co-expressed CD103 and CD69, whilst only a
small proportion expressed CD103 alone. In contrast, only ~35%
of CD4+ TRM cells co-expressed CD103 and CD69. Therefore,
CD8+ TRM and CD4+ TRM in the human gastric lamina propria
exhibited a differential expression pattern of molecules reported to
define TRM in other human mucosal tissues (7, 8). These observations suggest that CD4+ TRM cells are a more heterogeneous and
complex population than CD8+ TRM , possibly composed by various subsets. Future studies are necessary to address this important
question. Of note, CD4+ and CD8+ TRM were present in children,
adult, and the elderly at similar frequencies.
Migration of immune cells from peripheral blood to gut tissues is driven by the expression of tissue-specific homing receptors
such as integrin α4β7 and CCR9 (33, 34). Since gastric TRM cells
are expected to permanently reside in this tissue, we reasoned that
up-regulation of CD103, which binds to E-cadherin and allows
homing at the mucosal level, will result in down-regulation of integrin α4β7. Consistent with this, CD8+ TRM and CD4+ TRM cells
showed a significant down-regulation of integrin α4β7 compared
to its expression levels in CD8+ TEM and CD4+ TEM (PBMC).
These results are consistent with data reported in the mouse model
in tissues isolated from the small intestine (32, 35). Similar results
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were seen in adults, children, and the elderly. Whether CCR9 is
also down-regulated in CD8+ and CD4+ TRM cells remains to be
explored. The fact that we were able to identify CD8+ TRM and
CD4+ TRM cells in LPMC, which contain cells from the lamina
propria, suggests that TRM cells are either constantly mobilizing
between the epithelial and lamina propria layers of the stomach or
reside mainly in the lamina propria layer. In an attempt to address
this question, we assessed the IEL fraction in some volunteers
and identified mainly CD8+ T cells, most of which co-expressed
CD103 and CD69 (data not shown). CD4+ T cells were also identified in the IEL fraction, but at such low frequencies that we
were unable to ascertain their levels of expression of CD103 and
CD69 (data not shown). It is reasonable to speculate that TRM cells
mainly reside in the lamina propria and once they migrate to the
epithelium (i.e., becoming part of the traditional IEL subset) are
unable to re-enter the lamina propria. In the latter scenario, TRM
cells in the lamina propria will constantly supply cells that migrate
to the epithelial layer. This would require a change in expression
of homing markers and would suggest that, in addition to CD103,
other receptor(s) yet to be identified is(are) involved in the homing of these cells from the lamina propria to the epithelium. At this
time our data are unable to determine which of these hypotheses
is correct. However, it is likely that TRM cells shuttle between these
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Booth et al.
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.
two compartments working as sentinel cells and when a specific
antigen is encountered, these cells are rapidly activated, producing
cytokines and acquiring CTL activity.
CD8+ TRM cells have been described in mice as well as humans,
and reactivity of these cells to antigens derived from pathogens
has been demonstrated (4–6, 9). We identified CD8+ TRM and
CD4+ TRM cell in the stomach of volunteers confirmed to be
H. pylori negative (as determined by CLO test) (36). While H.
pylori is well recognized for its role in the development of gastritis, peptic ulcer, and adenocarcinoma, it does not affect the
composition of the gastric community (37). The gastric microbiota has been shown to contain a diverse community of 128
phylotypes (37), which could provide the underlying T cells with
antigen(s) to regulate their development. It can be speculated that
unidentified infectious agents or the gastric microbiota, through
conserved epitopes that resemble those of pathogens, play a role
in the development of TRM cells (38). Whichever the event(s)
that triggers their development, it appears that they occur at a
young age, since even the youngest children evaluated in our
Frontiers in Immunology | Mucosal Immunity
Age-associated responses in gastric TRM
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.
studies (i.e., 7-year-old) showed the presence of these unique
cells. Future experiments designed to address these questions
will include the investigation of the role of the microbiota in
the development of TRM cells and a comparison of the cytokine
production by CD4+ TRM and CD8+ TRM cells from H. pylori positive and healthy volunteers following stimulation with H. pylori
antigens.
Gastric CD8+ TRM and CD4+ TRM cells obtained from biopsies
of children, adults, and the elderly were responsive to SEB and antiCD3/CD28 beads stimulations by secreting Th1 cytokines (IL-2,
IFN-γ, TNF-α, IL-17A, MIP-1β) and up-regulating the cytotoxicity marker CD107a. These results confirm and extend studies in
which gastric CD4+ T cells obtained from healthy adults (H. pylori
negative) secreted Th1 cytokines (IFN-γ and TNF-α) when stimulated with PMA/Ionomycin (16, 31). Interestingly, CD8+ TRM cells
appeared to produce cytokines constitutively; a higher percentage
of TRM cells cultured in media only showed cytokine production
compared to CD8+ TEM cells. Similar results, albeit not statistically
significant, were identified in CD4+ TRM cells. These observations
June 2014 | Volume 5 | Article 294 | 12
Booth et al.
suggest that TRM cells are more prone to activation and possibly
have a lower antigenic threshold for stimulation than their peripheral blood counterparts. However, it is important to consider that
while the volunteers were H. pylori negative, they were referred
for EGD due to the presence of clinical symptoms (e.g., dysphagia,
heartburn, GERD, etc.). Therefore, to determine if gastric TRM
cells were activated in response to an inflammatory environment
resulting from the underlying clinical condition(s), we stratified
the baseline cytokine levels based on the pathology findings from
each volunteer (normal and “mild inflammation”) (Figure S2 in
Supplementary Material). Neither CD8+ TRM nor CD4+ TRM cells
showed statistically significant differences between the normal and
mild inflammation groups. These results support the idea that TRM
cells show a persistent activation state in “normal volunteers.” An
alternative explanation for the persistent activation state of TRM
cells could involve the role of the gastric microbiota. Thus, future
studies should be directed to explore this and other alternative
explanations. Whether the higher percentage of cells producing
cytokines and up-regulating CD107a spontaneously have a deleterious effect at the gastric mucosal level or that this enhanced
inflammatory environment benefits the host by limiting colonization with pathogens remains to be explored. Overall, CD8+ TRM
cells from adult, children, and the elderly responded to the stimuli
and the cytokine production was higher compared to PBMC, but
more evident in adults and elderly than in children.
There is little information on the induction of local immune
responses in the gastric mucosa from children (39, 40). Few studies have evaluated the cytokine responses in the gastric mucosa of
this age group and the results are contradictory (17, 41, 42). One
study found that lower levels of IFN-γ were produced in culture
supernatants of gastric mucosa tissues from children compared to
adults, but no differences in TNF-α, IL-2, or IL-10 were detected
regardless of their H. pylori status (17). On the other hand, a recent
study showed that in H. pylori infected children, the gastric concentration of IL-1α and TNF-α were significantly higher than that
in infected adults whereas IL-2, IL-12p70, and IFN-γ were lower in
infected children than in infected adults (42). Of note, differences
in cytokine profiles were observed between infected and uninfected individuals in both age groups (42). Epidemiological studies
have also suggested that unlike adults, children rarely develop peptic ulcers or gastric atrophy (43–45). This suggests that children
may display a unique immunological milieu that limits gastric
mucosal damage. In our study, even though the phenotype and
abundance of gastric CD8+ TRM and CD4+ TRM in children were
similar to those of adults and the elderly, their responses were different. CD4+ TRM and CD8+ TRM cells from children responded
only moderately to the mitogenic stimulations and secreted lower
amounts of cytokines than their adult and the elderly counterparts. Our results are consistent, and markedly extend, previous
studies demonstrated that gastric T cells from children are less
responsive to stimulation than adults. Moreover, our results provide novel information on cells isolated from elderly subjects.
Additionally, it has been shown that Th1 and Th17 responses in
children are down-regulated, resulting in reduced gastritis due to
H. pylori infections (46). This observation contrasts with that of
adults, in whom H. pylori infections usually result in significant
inflammation. Furthermore, in children who are positive for H.
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Age-associated responses in gastric TRM
pylori, the levels of regulatory T cells (Tregs ) and IL-10 secreting
cells in the gastric mucosa are higher than in H. pylori infected
adults (46, 47). Therefore, these observations suggest that in children the regulatory mechanisms at the gastric level are more active
than in adults and this could contribute to the limited reactivity
identified in CD4+ and CD8+ TRM cells. Future studies involving
the investigation of the presence and functional properties of Tregs
in the gastric mucosa of children as compared to adults/elderly
will shed light into this important question.
Multifunctionality analysis following SEB stimulation confirmed that TRM are multifunctional and also reinforced the idea
that age is a significant factor. Interestingly, various multi-cytokine
production patterns demonstrated that in the elderly a higher
percentage of cells produced multiple cytokines than in children,
suggesting that elderly cells are more reactive to stimulation. This
data further confirmed and extend the observations in this manuscript that cells from children are less susceptible to activation.
Of note, there were a few instances in which cells from children
produced more cytokines than those isolated from the elderly
(e.g., CD4+ TRM dual producers CD107a and IL-17A, as well as
TNF-α and IL-17A). This reinforces the indication that cytokine
production is age-related.
In summary, we developed a consistent method for isolation of
immune cells from the gastric biopsies that increased cell yields
and allowed the identification of CD8+ TRM and CD4+ TRM cells
in children, adults, and the elderly. We demonstrated that these
cells were functional and responsive to various categories of stimulants. Finally, we show that gastric cells of children respond differently to stimuli than adults and the elderly in terms of cytokines
and multi-cytokine production suggesting that unique regulatory
mechanisms are operative in the children’s gastric mucosa.
ACKNOWLEDGMENTS
We are indebted to the volunteers who allowed us to perform this
study. We thank Onyinye Erondu, Robin Barnes, and the staff from
the Recruiting Section of Center for Vaccine Development for their
help in collecting gastric biopsies and blood specimens and Ms.
Regina Harley and Catherine Storrer for excellent technical assistance. This work was supported, in part, by NIAID U19 AI082655
(CCHI) to Marcelo B. Sztein.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at http://journal.frontiersin.org/Journal/10.3389/fimmu.
2014.00294/abstract
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Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 18 April 2014; accepted: 05 June 2014; published online: 19 June 2014.
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Age-associated responses in gastric TRM
Citation: Booth JS, Toapanta FR, Salerno-Goncalves R, Patil S, Kader HA, Safta AM,
Czinn SJ, Greenwald BD and Sztein MB (2014) Characterization and functional properties of gastric tissue-resident memory T cells from children, adults, and the elderly.
Front. Immunol. 5:294. doi: 10.3389/fimmu.2014.00294
This article was submitted to Mucosal Immunity, a section of the journal Frontiers in
Immunology.
Copyright © 2014 Booth, Toapanta, Salerno-Goncalves, Patil, Kader, Safta, Czinn,
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