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International Immunology Advance Access originally published online on March 15, 2007
International Immunology 2007 19(4):567-579; doi:10.1093/intimm/dxm022
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Comprehensive phenotypic analysis of the gut intra-epithelial lymphocyte compartment: perturbations induced by acute reovirus 1/L infection of the gastrointestinal tract

Mantej S. Bharhani, Jasvir S. Grewal, Richard Peppler, Candace Enockson, Lucille London and Steven D. London

Department of Microbiology and Immunology, Medical University of South Carolina, PO Box 250504, 173 Ashley Avenue, Charleston, SC 29425, USA

Correspondence to: S. D. London; E-mail: londonsd{at}musc.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Intestinal intra-epithelial lymphocytes (IELs) form a highly specialized lymphoid compartment. IELs consist primarily of T cells that are dispersed as single cells within the epithelial cell layer that surrounds the intestinal lumen. These lymphocytes along with lamina propria lymphocytes are considered to play an important role in the regulation of immune responses. IELs are heterogeneous with regard to phenotype, and they contain sub-populations with diverse functions. In our most recent study, we found that intra-duodenal inoculation of mice with reovirus serotype 1/strain Lang (reovirus 1/L) induced expression of both germinal center and T cell antigen and CD11c on IELs suggesting these cells to be the recently stimulated cells in gut mucosal tissue. We also demonstrated that IELs from these mice when cultured in vitro in the presence of reovirus 1/L-pulsed antigen-presenting cells generated reovirus 1/L-specific MHC-restricted CTL whose function was mediated utilizing perforin, Fas-FasL and TRAIL mechanisms. This present study provides a comprehensive analysis of the diverse subsets of IELs, which function with other mucosal cells to provide a strong, protective immunity in a highly regulated fashion inside the microenvironment of the intestinal epithelium. We demonstrated that the IEL population contains both thymus-dependent (TD) and thymus-independent (TI) lymphocytes in mice and that a complex phenotype is present when sub-populations are analyzed for TCR, Thy-1, CD4, CD8 and B220 expression in a comprehensive manner. In reovirus 1/L-inoculated mice, we found a decrease in the TI population and an increase in the TD population characterized by significant alterations in various sub-populations. This increase was largely due to an increase in CD4+, CD8+ and CD4/CD8 double-positive sub-populations of TD IELs. Intracellular cytokine analysis demonstrated induction of IFN-{gamma} and an increase in effector/cytotoxic CD8 and CD4 cells after reovirus 1/L infection. These results suggest that TD IELs may play an important role in the clearance of reovirus 1/L infection from gut.

Keywords: cytokines, IEL, LPL, mucosa, TCR{alpha}ß, TCR{gamma}{delta}


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Intra-epithelial lymphocytes (IELs) are the first cells to encounter pathogens that have invaded a host via the intestinal, respiratory or genitourinary tract epithelium. Intestinal IELs are found as single cells dispersed between the epithelial cells, and based on immunohistology, it has been estimated that the IELs of the mouse intestine alone amount to almost 50% of the total T cell number in all lymphoid organs (1–3). However, IELs are a phenotypically diverse population and are predominantly T cells (CD3+) with majority of them being CD8+ at most sites. Both TCR{alpha}ß+ and TCR{gamma}{delta}+ cells are found within IEL populations (2–4). There is increasing evidence utilizing mouse models that two populations of lymphocytes, which belong to different lineages, populate the gut epithelium. One IEL population [thymus dependent (TD)] is the progeny of precursors, which differentiate in the thymus and migrate after antigenic stimulation from the Peyer's patches (PP) to the gut epithelium via the hemolymphatic circuit (3,5–7). The second IEL population differentiates extrathymically. Although some of this population's precursors may arise and expand in the thymus (8), differentiation most likely occurs in the gut wall, and this population is termed thymus independent (TI) (3, 5, 9). Intestinal IELs can be subdivided into two major sub-populations based on the TCR and co-receptor type they express (3, 4). They consist of the TD IELs, which express TCR{alpha}ß together with CD4 or CD8{alpha}ß as TCR co-receptors, and the TI IELs subset, which can be either TCR{alpha}ß+ or TCR{gamma}{delta}+ (3, 5, 9). TI TCR{gamma}{delta}+ IELs express CD8{alpha}{alpha}, whereas TI TCR{alpha}ß+ IELs express either forms of CD8 (CD8{alpha}ß or CD8{alpha}{alpha}) (10). Both Thy-1+ and Thy-1– cells are found in the IEL population of normal mice, however, the TD population (TCR{alpha}ß+) is Thy-1+ and the TI populations (TCR{gamma}{delta}+ and TCR{alpha}ß+) are considered Thy-1– (11).

Since IELs are such a diverse population, there has been considerable interest in examining the changes in the proportions of different sub-populations of IELs after mucosal infection and defining the role of these sub-populations in regulating the mucosal immune response. The objective of this study was to first provide a comprehensive analysis of the range of IEL sub-populations present in conventional mice and then to evaluate changes in the phenotypic heterogeneity and function of IELs upon acute, gut mucosal stimulation. In this study, reovirus serotype 1/strain Lang (reovirus 1/L) was utilized, since gastrointestinal infection with reovirus 1/L in mice elicits cell-mediated immune responses in the gut as evidenced by the appearance of CD8+ reovirus 1/L-specific precursor CTL among PP lymphocytes and IELs (12–14). However, information is limited regarding the infection-associated changes in distinct sub-populations of IELs. Recently, we characterized the expression of activation markers and effector mechanisms involved in reovirus 1/L-specific cytotoxicity after intra-duodenal (i.d.) priming of mice with reovirus 1/L (15). We demonstrated that the majority of IELs expressed the phenotype TCR{alpha}ß+Thy-1+CD8+, which was similar to those found in the lamina propria and PP and these cells were cytotoxic and used perforin, Fas-FasL and TRAIL pathways for their cytotoxic effect (15). This prompted us to evaluate changes within the different sub-populations of the small intestine IELs after i.d. inoculation of mice with reovirus 1/L to pinpoint which particular cell types among the IEL sub-populations plays a role in this infection. Here, we demonstrate that after reovirus 1/L inoculation TCR{alpha}ß+Thy-1+CD4+, TCR{alpha}ß+Thy-1+CD8+ and TCR{alpha}ß+Thy-1+CD4+CD8+ populations were increased significantly at days 7 and 10 after inoculation, which returned to normal levels by day 14 coinciding with the course of infection in these mice. Further, intracellular cytokine analysis demonstrated the production of IFN-{gamma} and an increase in effector/cytotoxic CD8 and CD4 cells after reovirus 1/L infection. To our knowledge, this is the first study which describes in detail the changes in various sub-populations of IELs after a viral infection.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals and virus
Four-week-old female Balb/cJ mice were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Six- to eight-week old mice were used in all experiments. Mice were maintained under specific pathogen-free conditions in a BL-2 facility and provided sterile food and water ad libitum. All animal manipulations were performed in class II biological safety cabinets. Virally primed mice were kept physically isolated from all experimental and stock mice.

Reovirus 1/L was originally obtained from W. Joklik (Duke University School of Medicine, Durham, NC, USA). Third passage, gradient-purified stocks were titered by limiting dilution on L cell monolayers and used in these experiments (16).

Antibodies
The following mAbs were used in this study: anti-TCR{alpha}ß (clone H57-597), anti-TCR{gamma}{delta} (clone GL3) and anti-CD4 (clone CT-CD4) were purchased from Caltag (San Francisco, CA, USA). Anti-Thy-1.2 (anti-CD90, clone 53-2.1), anti-CD8{alpha} (clone 53-6.7), anti-CD8ß (clone 53.5.8), anti-B220 (clone RA3-6B2), anti-CD69 (clone H1.2F3), anti-CD16/CD32 (clone 2.4G2), anti-IL-4 (clone 11B11), anti-IL-5 (clone TRFK5), anti-IL-6 (clone MP5-20F3) and anti-IFN-{gamma} (clone XMG1.2) were purchased from PharMingen (San Diego, CA, USA). All antibodies were conjugated with FITC, PE, allophycocyanin (APC), PerCp or biotin. Streptavidin-conjugated PE, APC and PerCp (PharMingen) were used as second-step reagents to identify biotinylated primary antibodies. Each antibody was titrated to determine the optimal staining concentration for maximal signal.

Immunization
Mice were anesthetized with a 0.15-cc intra-peritoneal dose of 20% ketamine (Vetalar 100 mg cc–1; Fort Dodge Laboratories Inc., Ford Dodge, IA, USA) and 2.0% acepromazine maleate (PromAce 10 mg cc–1; Ayerest Laboratories, New York, NY, USA) and then inoculated i.d. with 3 x 107 Plaque-forming units (PFU) of reovirus 1/L in 50 µl of injectable grade 0.9% NaCl. Control mice were inoculated as above with the same volume of 0.9% NaCl.

Isolation of IEL and lamina propria lymphocytes
IEL and lamina propria lymphocytes (LPLs) were isolated from mice with some modifications of a protocol described previously (17). In brief, the intestines from the duodenum to the ileocaecal junction were removed and flushed with Ca2+-, Mg2+-free HBSS (CMF). PP and mesentery were removed and the intestines were opened longitudinally and cut into small pieces. The pieces were stirred three times for 30 min in CMF containing 10 mM HEPES, 25 mM NaHCO3, 2% fetal bovine serum (FBS), 1 mM EDTA and 1 mM dithiothreitol at 37°C. The eluted cells from the first two incubations were collected, passed through 74-µ nylon mesh to partially purify the IELs and kept at 37°C in CO2 incubator for 45–60 min to facilitate subsequent separation of IELs. The IELs were separated from epithelial cells by centrifugation through a discontinuous 44/67.5% Percoll (Pharmacia) gradient at 600 x g for 20 min. IELs were harvested from the interface between the 44 and 67.5% Percoll layers. For the isolation of LPLs, the EDTA treated intestinal pieces were washed and then digested for 90 min with RPMI-1640 containing 5% FBS, 10 mM HEPES, 25 mM NaHCO3, 100 U of Collagenase type II (Sigma) per milliliter, 0.5 mg of Dispase (Boehringer Mannheim) per milliliter and 100 U of DNAse (Boehringer Mannheim) per milliliter at 37°C. LPLs were then purified through a discontinuous 40/67.5% Percoll gradient centrifuged at 600 x g for 20 min. LPLs were harvested from the interface between the 40 and 67.5% Percoll layers.

Multicolor immunofluorescence staining for surface markers
IELs and LPLs (0.5 x 106–1 x 106 per sample) in HBSS–5% FBS containing 0.02% sodium azide were pre-incubated for 20 min with anti-CD16/CD32 (Fc block) to block Fc receptors to prevent non-specific binding. The cells were then incubated with appropriately titered mAbs for 30 min on ice. These antibodies were either directly labeled with FITC, APC, PE and PerCP or were biotinylated. In the case of biotinylated antibodies, streptavidin-conjugated PE, APC or PerCP was used as second-step reagents. After staining, the cells were washed with HBSS and fixed in 2% paraformaldehyde for 20 min at room temperature. Cells were analyzed for cell-surface marker expression using a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using CellQuest software (BD Biosciences).

Intracellular cytokine staining
IELs or LPLs (2 x 106 ml–1) were placed in a 24-well plate (2 ml per well) and stimulated with phorbol myristate acetate (PMA) (50 ng ml–1; Sigma) and ionomycin (500 ng ml–1; Sigma) in the presence of brefeldin A (10 µg ml–1; Sigma) for 4 h at 37°C in a CO2 incubator. Cultured cells were collected, incubated for 5 min at 37°C with DNAse (3 mg ml–1) to prevent clumping of the cells. Dead cells were removed by centrifugation at 600 x g for 20 min at 20°C over a 3 ml Ficoll-Paque cushion (Amersham). Viable cells (1.5 x 106 per sample) were suspended in HBSS–5% FBS with sodium azide and surface stained as detailed above. After surface staining, the cells were washed twice with HBSS without FBS and sodium azide and fixed with 4% Paraformaldehyde. Fixed cells were washed with HBSS and then permeabilized with 0.5% saponin in HBSS–5% FBS for 30 min at room temperature in the dark. Cells were then stained for 30 min with mAb diluted in 0.5% saponin at room temperature. After washing, the cells were suspended in HBSS and analyzed using a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using CellQuest software (BD Biosciences).

Reovirus 1/L-specific T lymphocytes
To generate antigen-specific effector cells, IELs were cultured in the presence of reovirus 1/L-infected (multiplicity of infection of 10), irradiated peritoneal exudate cells (PECs) for 16 h in RPMI–10% FBS as previously described (12). During the last 5 h of stimulation, brefeldin A (10 µg ml–1) was added to the culture. Cells were then harvested and stained for cell-surface markers and intracellular IFN-{gamma} expression as described above. Antigen-specific T cells were defined as the sub-population expressing both the very early activation marker, CD69 and IFN-{gamma} as recently described (18).

Statistical analysis
Data were analyzed for statistical significance using one-way analysis of variance. Data were considered significant at a P value of <0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Because IELs are such a diverse population, there has been considerable interest in examining the changes in the proportions of different sub-populations of IELs after mucosal infection and defining the role of these sub-populations in regulating the mucosal immune response. In this study, Balb/c mice were inoculated i.d. with reovirus 1/L (infected mice) or saline (control mice) and were sacrificed on days 3, 7, 10 and 14 after inoculation. The intestinal IELs were collected and the phenotype of the cells was analyzed by multicolor flow cytometry. Our results demonstrate that although the total number of IELs recovered after reovirus 1/L inoculation was not altered significantly (except on day 7 after inoculation), there were significant changes in the phenotype of various sub-populations of IELs as compared with control, saline-inoculated mice (Table 1).


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  		Table 1. Changes in the absolute cell number of IEL sub-populations after reovirus 1/L inoculation

 
Reovirus 1/L inoculation alters the expression pattern of TCR sub-populations in the IEL compartment
Two major populations of T lymphocytes exist in the gut mucosal epithelial compartment based on the expression of either TCR{gamma}{delta} or TCR{alpha}ß. Figure 1(A) demonstrates the percentage of IELs and Table 1 shows the absolute number of IELs that express TCR{gamma}{delta} or TCR{alpha}ß after i.d. inoculation with reovirus 1/L over a 14-day time course. For comparison, the percentage of IELs that express TCR{gamma}{delta} or TCR{alpha}ß in control, saline-inoculated mice are also shown. IELs obtained from control, saline-inoculated mice were 54.42 ± 4.38% TCR{gamma}{delta}+ and 18 ± 1.43% TCR{alpha}ß+ (Fig. 1A, Table 1). However, following reovirus 1/L inoculation, there was a significant decrease in the percentage of TCR{gamma}{delta}+ IELs on day 3 (48.5 ± 1.1%) and day 7 (46.7 ± 2.1%) after inoculation. The percentage of TCR{gamma}{delta}+ IELs returned to levels comparable to the control on days 10 and 14 after inoculation (Fig. 1A, Table 1). However, while the percentage of the TCR{gamma}{delta}+ IELs decreased, there was no significant change in the absolute number, suggesting that the decrease in the percentage of TCR{gamma}{delta}+ IELs may be due to an increase in another IEL population (Table 1). In contrast to the decrease in TCR{gamma}{delta}+ IELs, the percentage of TCR{alpha}ß+ IELs was significantly increased after reovirus 1/L inoculation, with 31.9 ± 1.5% TCR{alpha}ß+ lymphocytes on day 7 after inoculation and 28.3 ± 7.2% on day 10 after inoculation (Fig. 1A, Table 1). The percentage of TCR{alpha}ß+ IELs returned to levels comparable to control, saline-inoculated levels by day 14 after inoculation.


Figure 1
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  		Fig. 1. Reovirus 1/L inoculation alters the expression pattern of TCR sub-populations in the IEL compartment. Control (saline) and reovirus 1/L (i.d. with 3 x 107 PFU) -inoculated mice were sacrificed on days 3, 7, 10 and 14. The results shown for reovirus 1/L-inoculated mice are the mean percentage ± SD (three independent experiments with four mice each). The results shown for the control group are the average of control group values on days 3, 7, 10 and 14. (A) Expression of TCR{gamma}{delta}, TCR{alpha}ß, Thy-1, CD8, CD4 and CD4/CD8 IEL sub-populations after reovirus 1/L inoculation. The results demonstrate the changes in the percentage of TCR{gamma}{delta} (dotted gray bar), TCR{alpha}ß (dotted black bar), Thy-1 (dotted white bar), CD8 (hatched bar), CD4 (open bar) and CD4/CD8 (closed bar) IELs. (B) Phenotypic changes in the Thy-1+ IEL population after reovirus 1/L inoculation. The results demonstrate the changes in the percentage of TCR{gamma}{delta}–/TCR{alpha}ß–/Thy-1+ (hatched bar), TCR{gamma}{delta}+/Thy-1+ (dotted white bar) and TCR{alpha}ß+/Thy-1+ (dotted black bar) IEL. *P < 0.05 and **P < 0.01 as compared with control group.

 
Reovirus 1/L inoculation induced the expression of Thy-1, CD8 and CD4/CD8 in the IEL compartment
Thy-1 antigen expression is a marker of TD T lymphocytes. IELs obtained from control, saline-inoculated mice were 14.4 ± 2.48% Thy-1+ (Fig. 1A). Following reovirus 1/L inoculation, there was a significant increase in the percentage of Thy-1-expressing IELs which peaked on day 7 (32.5 ± 3.0%), remained elevated on day 10 (21.3 ± 1.9%) and approached levels comparable to control, saline-inoculated values by day 14 (17.5 ± 5.0%) after reovirus 1/L inoculation (Fig. 1A).

Further analysis demonstrated that Thy-1 was present on the TCR{gamma}{delta}+, TCR{alpha}ß+ and TCR{gamma}{delta}–/TCR{alpha}ß– IELs (Fig. 1B, Table 1). In control, saline-inoculated mice, the TCR{gamma}{delta}–/TCR{alpha}ß–/Thy-1+, TCR{gamma}{delta}+/Thy-1+ and TCR{alpha}ß+/Thy-1+ populations comprised an average 7.09 ± 0.93%, 2.56 ± 0.52% and 4.96 ± 1.08% of IELs, respectively (Fig. 1B). After reovirus 1/L inoculation, the percentage and absolute number of TCR{alpha}ß+/Thy-1+ IELs was significantly increased (P < 0.01) on day 7 (21.3 ± 2.7%) and day 10 (13.36 ± 1.78%) (Fig. 1B, Table 1). While there was a slight increase in the TCR{gamma}{delta}+/Thy-1+ population after reovirus 1/L inoculation, this increase was not significant as compared with the control, saline-inoculated group. In addition, although in the control, saline-inoculated mice there was a larger percentage of TCR{gamma}{delta}–/TCR{alpha}ß–/Thy-1+ IELs than either TCR{gamma}{delta}+/Thy-1+ or TCR{alpha}ß+/Thy-1+ IELs, the percentage of the TCR{gamma}{delta}–/TCR{alpha}ß–/Thy-1+ population did not significantly change at any time point following reovirus 1/L inoculation.

The IEL population in both control, saline-inoculated and reovirus 1/L-inoculated mice is also comprised of CD8+, CD4+ and CD4/CD8 double-positive (CD4+/CD8+) lymphocytes (Fig. 1A). The percentage of CD8+, CD4+ and CD4+/CD8+ lymphocytes in control, saline-inoculated mice was 62.3 ± 5.05%, 4.88 ± 1.11% and 2.52 ± 0.4%, respectively (Fig. 1A). In reovirus 1/L-inoculated mice, the percentage of CD8+ cells decreased significantly (53.34 ± 3.29%, P < 0.05) on day 3 after inoculation and then increased on day 7 after inoculation (66.37 ± 6.03%). However, this increase of CD8+ cells was not significant until day 10 after inoculation, when 71.37 ± 4.42% of the IELs were CD8+ (P < 0.05). The percentage of CD4+ lymphocytes in reovirus 1/L-inoculated mice increased significantly on day 3 after inoculation (7.64 ± 0.89%, P < 0.05). In addition, the percentage of CD4+/CD8+ cells also increased significantly (P < 0.05) after inoculation, from control levels of 2.52 ± 0.4% to 4.7 ± 1.31% on day 7 and to 3.6 ± 0.49% on day 10 after inoculation.

Reovirus 1/L inoculation induced the expression of TCR{alpha}ß+/CD8+ sub-population in the IEL compartment
Because the CD8+ lymphocyte population comprises a large proportion of the IEL population, both in control, saline-inoculated and reovirus 1/L-inoculated mice, the CD8+ population was further analyzed for changes in TCR{gamma}{delta} and TCR{alpha}ß expression following reovirus 1/L inoculation (Fig. 2). In control, saline-inoculated mice, the percentage of TCR{gamma}{delta}+/CD8+ IELs was 47.27 ± 4.48% while the percentage of TCR{alpha}ß+/CD8+ was 13.29 ± 2.51%. However, in reovirus 1/L-inoculated mice the percentage of TCR{gamma}{delta}+/CD8+ IELs was significantly decreased on day 3 (42.79 ± 3.1%, P < 0.05) and on day 7 (39 ± 2.5%, P < 0.01). In addition, in reovirus 1/L-inoculated mice the percentage of TCR{alpha}ß+/CD8+ IEL population increased significantly on day 7 (28.78 ± 6.45%, P < 0.05) and on day 10 (26.29 ± 5.55%, P < 0.05) after inoculation.


Figure 2
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  		Fig. 2. Phenotypic changes in the TCR{gamma}{delta}+/CD8+ and TCR{alpha}ß+/CD8+ IEL populations after reovirus 1/L inoculation. Control (saline) and reovirus 1/L (i.d. with 3 x 107 PFU) -inoculated mice were sacrificed on days 3, 7, 10 and 14. The results demonstrate the changes in the percentage of TCR{gamma}{delta}+/CD8+ and TCR{alpha}ß+/CD8+ IEL population over time. The results shown for reovirus 1/L-inoculated mice are the mean percentage ± SD (three independent experiments with four mice each). The results shown for control, saline-inoculated mice are the average of control group values on days 3, 7, 10 and 14. The results demonstrate the changes in the percentage of TCR{gamma}{delta}+/CD8+ (dotted grey bar) and TCR{alpha}ß+/CD8+ (dotted black bar) IELs. *P < 0.05 and **P < 0.01 compared with control group.

 
Reovirus 1/L inoculation altered the expression pattern of Thy-1+ and Thy-1– TCR sub-populations in the IEL compartment
The above results demonstrated significant changes in TCR{alpha}ß+, TCR{gamma}{delta}+ and Thy-1+ populations in the IELs following reovirus 1/L infection, and these changes warranted further investigation. Therefore, TCR{alpha}ß+/Thy-1+, TCR{alpha}ß+/Thy-1–, TCR{gamma}{delta}+/Thy-1+ and TCR{gamma}{delta}+/Thy-1– sub-populations were gated and analyzed for CD4 and CD8 expression.

CD4+, CD8+ and CD4+/CD8+ sub-populations of TCR{alpha}ß+/Thy-1+ IELs
Since we demonstrated an increase in the TCR{alpha}ß+/Thy-1+ IEL population after reovirus 1/L inoculation, we further investigated the expression of CD4 and CD8 on this sub-population by gating on the TCR{alpha}ß+/Thy-1+ IEL population. In control, saline-inoculated mice, the percentage of TCR{alpha}ß+/Thy-1+ IELs-co-expressing CD4 and CD8 was 4.5 ± 0.8%, the percentage expressing CD4 was 50.3 ± 3.05% and the percentage expressing CD8 was 31.42 ± 4.3% (Fig. 3A). However, in reovirus 1/L-inoculated mice, while the percentage of TCR{alpha}ß+/Thy-1+ IELs-expressing CD4 was significantly increased on day 3 (64.0 ± 1.7%, P < 0.01), the percentage of TCR{alpha}ß+/Thy-1+ IELs-expressing CD4 was significantly decreased on day 7 (27.0 ± 2.2%, P < 0.01), day 10 (27.4 ± 2.9%, P < 0.01) and day 14 (35.2 ± 3.2%, P < 0.05) as compared with control, saline-inoculated mice. In contrast to the CD4+ sub-population, the percentage of TCR{alpha}ß+/Thy-1+ IELs-expressing CD8 was significantly decreased on day 3 (24.4 ± 2.0%, P < 0.05). However, after day 3, this sub-population significantly increased (P < 0.01) throughout the study period to 58.9 ± 3.5%, 53.4 ± 2.7% and 51.0 ± 2.8% on days 7, 10 and 14, respectively. The percentage of TCR{alpha}ß+/Thy-1+ IELs-co-expressing CD4 and CD8 also increased significantly to 6.9 ± 0.5% (P < 0.05) on day 7 and to 12.2 ± 0.6% (P < 0.01) on day 10 after inoculation (Fig. 3A).


Figure 3
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  		Fig. 3. Reovirus 1/L inoculation altered the expression pattern of Thy-1+ and Thy-1– TCR sub-populations in the IEL compartment. Control (saline) and reovirus 1/L (i.d. with 3 x 107 PFU) -inoculated mice were sacrificed on days 3, 7, 10 and 14. The TCR{alpha}ß+/Thy-1+ population was gated and then analyzed for (A) CD4 and CD8 expression or (B) CD8{alpha}{alpha}, CD8{alpha}ß and CD8/B220 expression. The results shown for reovirus 1/L-inoculated mice are the mean percentage ± SD (three independent experiments with four mice each). The results shown for control, saline-inoculated mice are the average of control group values on days 3, 7, 10 and 14. (A) Phenotypic changes in the CD4+ and CD8+ sub-populations of the TCR{alpha}ß+/Thy-1+ IEL population after reovirus 1/L inoculation. TCR{alpha}ß+/Thy-1+/CD4+/CD8+ (hatched bar), TCR{alpha}ß+/Thy-1+/CD4+ (dotted white bar) and TCR{alpha}ß+/Thy-1+/CD8+ (dotted black bar) IELs. (B) Phenotypic changes in the CD8+ sub-population of the TCR{alpha}ß+/Thy-1+ IEL population after reovirus 1/L inoculation. TCR{alpha}ß+/Thy-1+/CD8{alpha}{alpha}+ (dotted white bar), TCR{alpha}ß+/Thy-1+/CD8{alpha}ß+ (dotted black bar), TCR{alpha}ß+/Thy-1+/CD8+/B220– (open bar) and TCR{alpha}ß+/Thy-1+/CD8+/B220+ (closed bar) IELs. *P < 0.05 and **P < 0.01 compared with control group.

 
CD8{alpha}{alpha}+ and CD8{alpha}ß+ sub-populations of TCR{alpha}ß+/Thy-1+ and TCR{alpha}ß+/Thy-1– IELs
Since we demonstrated an increase in the TCR{alpha}ß+/Thy-1+/CD8+ IEL population after reovirus 1/L inoculation, we further investigated the CD8+ subtype composition by gating on either the TCR{alpha}ß+/Thy-1+ (Fig. 3B) or TCR{alpha}ß+/Thy-1– IELs (Table 2) and analyzing for the expression of CD8{alpha}{alpha}, CD8{alpha}ß and CD8/B220. In control, saline-inoculated mice, the percentage of TCR{alpha}ß+/Thy-1+/CD8{alpha}{alpha}+ IELs was 14.35 ± 2.69% and the percentage of TCR{alpha}ß+/Thy-1+/CD8{alpha}ß+ IELs was 33.58 ± 4.59% (Fig. 3B). However, in reovirus 1/L-inoculated mice, the percentage of the CD8{alpha}{alpha}+ sub-population significantly decreased on day 3 (10.32 ± 0.58%, P < 0.01) and returned to control levels by day 7 (11.54 ± 4.23%) after inoculation as compared with control, saline-inoculated mice (Fig. 3B). However, by day 10, the CD8{alpha}{alpha}+ sub-population increased significantly to 26.3 ± 4.3% (P < 0.05) (Fig. 3B). In comparison, after reovirus 1/L inoculation, the CD8{alpha}ß+ sub-population also decreased significantly on day 3 of infection (22.31 ± 7.4%, P < 0.05), but then increased significantly throughout the study period (62.3 ± 12%, P < 0.01; 47.28 ± 6.0%, P < 0.05 and 59.49 ± 4.78%, P < 0.01, on days 7, 10 and 14, respectively).


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  		Table 2. Phenotypic changes in CD8{alpha}{alpha}+, CD8{alpha}ß+, CD8+/B220– and CD8+/B220+ sub-populations of TCR{alpha}ß+/Thy-1– IELs after inoculation with reovirus 1/L

 
The TCR{alpha}ß+/Thy-1+ IELs were also analyzed for CD8+/B220– and CD8+/B220+ sub-populations (Fig. 3B). In control, saline-inoculated mice, the percentage of TCR{alpha}ß+/Thy-1+/CD8+/B220– was 30.12 ± 6.83% and the percentage of TCR{alpha}ß+/Thy-1+/CD8+/B220+ was 14.34 ± 5.52%. In reovirus 1/L-inoculated mice, the percentage of the TCR{alpha}ß+/Thy-1+/CD8+/B220– sub-population increased significantly on day 7 (49.44 ± 9.67%, P < 0.05), day 10 (51.23 ± 9.53%, P < 0.05) and day 14 (53.98 ± 8.4%, P < 0.05) after inoculation. TCR{alpha}ß+/Thy-1+/CD8+/B220+ sub-population also increased on day 7 (21.51 ± 7.3%), day 10 (23.98 ± 5.05%) and day 14 (18.53 ± 2.6%) after inoculation. However, this increase was only significant on day 10 (P < 0.05).

When TCR{alpha}ß+/Thy-1– IELs were analyzed for CD8 variant expression, 80.84 ± 2.87% of the TCR{alpha}ß+/Thy-1– IELs were CD8{alpha}{alpha}+ and 10.27 ± 2.37% were CD8{alpha}ß+ in control, saline-inoculated mice (Table 2). These percentages did not change significantly following reovirus 1/L infection, with the exception of a significant reduction (73.62 ± 2.22%, P < 0.05) in the CD8{alpha}{alpha}+ sub-population on day 3 (Table 2). Similarly, percentages of CD8+/B220– (8.78 ± 3.12%) and CD8+/B220+ (82.13 ± 6.05%) sub-populations in the TCR{alpha}ß+/Thy-1– IELs in control, saline-inoculated mice did not change significantly following reovirus 1/L infection except on day 10 when the CD8+/B220+ sub-population increased significantly (89.86 ± 0.88%, P < 0.05).

CD8 and B220 sub-populations of TCR{gamma}{delta}+/Thy-1+ and TCR{gamma}{delta} /Thy-1– IELs
Table 3 shows the phenotypic changes in TCR{gamma}{delta}+/Thy-1+ and TCR{gamma}{delta}+/Thy-1– IELs. In control, saline-inoculated mice, the TCR{gamma}{delta}+ IELs were >90% CD8+ with little to no expression of CD4 (data not shown). Therefore, we investigated the expression of B220 on the CD8+ sub-population of both the TCR{gamma}{delta}+/Thy-1+ and TCR{gamma}{delta}+/Thy-1– IELs (Table 3). In control, saline-inoculated mice, the percentage of TCR{gamma}{delta}+/Thy-1–/CD8+/B220– and TCR{gamma}{delta}+/ Thy-1–/CD8+/B220+ were 1.26 ± 0.63% and 91.46 ± 1.74%, respectively. After reovirus 1/L inoculation, there was no significant change in the percentage of either the CD8+/B220+ or CD8+/B220– sub-population at any time point (Table 3). However, we did observe significant changes in the phenotype of the TCR{gamma}{delta}+/Thy-1+ IEL population after reovirus 1/L inoculation. In control, saline-inoculated mice, the percentage of TCR{gamma}{delta}+/Thy-1+/CD8+/B220– and TCR{gamma}{delta}+/Thy-1+/CD8+/B220+ were 10.84 ± 6.26% and 25.0 ± 4.73%, respectively. However, after reovirus 1/L inoculation, the CD8+/B220+ sub-population of TCR{gamma}{delta}+/Thy-1+ IELs significantly decreased (16.77 ± 3.33%, P < 0.05) on day 3 and then significantly increased on both day 7 (45.03 ± 11.4% P < 0.05) and day 10 (61.57 ± 17.3% P < 0.05).


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  		Table 3. Phenotypic changes in CD8+/B220– and CD8+/B220+ sub-populations of TCR{gamma}{delta}+/Thy-1+ and TCR{gamma}{delta}+/Thy-1– IELs after inoculation with reovirus 1/L

 
The reovirus 1/L-induced increase in the CD4/CD8 double-positive IEL population was due to an increase in the CD4+/CD8{alpha}ß+ sub-population
In Fig. 1, we demonstrated a significant increase in the percentage of CD4+/CD8+ IELs on days 7 and 10 after reovirus 1/L inoculation. Therefore, to evaluate the {alpha}{alpha} homodimer versus the {alpha}ß heterodimer chain of CD8 within this CD4+/CD8+ population, the CD4+/CD8{alpha}+ sub-population was gated and analyzed for the expression of CD8ß. In addition, the expression of TCR{alpha}ß was also investigated on this gated population (Fig. 4). In control, saline-inoculated mice, the percentage of the CD4+/CD8{alpha}ß+ population ranged from 30 to 42% and out of these between 13 and 18% were TCR{alpha}ß+. However, as shown in Fig. 4 (lower panels), there was a significant increase in the percentage of CD4+/CD8{alpha}ß+ IELs (37–73%) following infection with reovirus 1/L and these IELs were predominately TCR{alpha}ß+ (32–65%). Although this increase was observed through day 14 of infection, the maximum increase was seen 7 days after inoculation, with 73% of the CD4+/CD8+ IELs expressing the {alpha}/ß heterodimer and 65% also being TCR{alpha}ß+ (Fig. 4). Therefore, after infection, there was an almost 2-fold increase in the CD4+/CD8+ populations which can be accounted for in the CD4+/CD8{alpha}ß+ sub-population.


Figure 4
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  		Fig. 4. Phenotypic changes in the CD4+/CD8+ IEL sub-population after reovirus 1/L inoculation. Control (saline) and reovirus 1/L (i.d. with 3 x 107 PFU) -inoculated mice were sacrificed on days 7, 10 and 14. The CD4+/CD8{alpha}+ IEL sub-population was first gated to exclude all other cells, and then analyzed for TCR{alpha}ß and CD8ß expression. The upper panels show control, saline-inoculated mice and the lower panels show reovirus 1/L-inoculated mice. The values shown in the right panels are the percentage of the CD4+/CD8+ IEL sub-population expressing the {alpha}ß heterodimeric chains of the CD8 molecule and the TCR{alpha}ß. The results shown represent one of the three independent experiments (four mice per experiment) demonstrating similar results.

 
Reovirus 1/L infection induces a TH1-type response in both the IEL and LPL populations
The capacity for cytokine production by the IELs of control, saline-inoculated and reovirus 1/L-inoculated mice was also examined. For cytokine analysis, we focused on day 7 after infection, since our results demonstrated maximum phenotypic changes in the IEL population on that day. Cytokine production was assessed by flow cytometry after stimulation with PMA and ionomycin in the presence of brefeldin A, facilitating cytokine visualization by intracellular flow cytometric cytokine analysis. We evaluated IFN-{gamma} and IL-4 expression as representative of a TH1 or TH2 profile, respectively. The results, shown in Table 4, are expressed in terms of the number of cytokine-positive lymphocytes per 106 cells and the frequency of cytokine-producing lymphocytes. The total number of IFN-{gamma}-positive IELs was significantly increased after reovirus 1/L infection, with a frequency of 1/23 ± 5 on day 7 after infection compared with control frequencies of 1/74 ± 14 (Table 4). No significant change in the total number of IELs-expressing IL-4 was observed after reovirus 1/L inoculation (Table 4).


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  		Table 4. Cytokine production by IELs after reovirus 1/L inoculation

 
Our results demonstrated a significant induction of IFN-{gamma} expression when cultures were stimulated with PMA and ionomycin. Therefore, to evaluate whether the same increase in IELs-expressing IFN-{gamma} was observed after antigen-specific re-stimulation in vitro, we examined cytokine expression from IELs that were stimulated in vitro with reovirus 1/L-infected PEC (Table 4). As was observed after culture with PMA and ionomycin, there was a significant increase in the frequency of IFN-{gamma}-expressing IELs from a frequency of 1/77.5 in control, saline-inoculated mice to a frequency of 1/31.6 on day 7 after reovirus 1/L inoculation after in vitro re-stimulation with reovirus 1/L-infected PEC. According to a recent study (18), which applied intracellular IFN-{gamma} expression to calculate effector CTL in spleen cells, the antigen-stimulated IFN-{gamma}-producing T cells that co-expressed CD69 (a very early activation marker) were determined to be effector CTL. We applied this method to our IEL population and evaluated co-expression of CD69 and IFN-{gamma} as a marker for the presence of effector cells. While it is known that the majority of IELs in control, uninfected mice express CD69, we demonstrated that these CD69+ cells do not significantly express IFN-{gamma} (Fig. 5, top). However, in reovirus 1/L-infected mice, there was a significant increase in the percentage of CD69+/CD8+ (67% infected mice versus 19% control mice) or CD69+/CD4+ IELs (79% infected mice versus 21% control mice) that were IFN-{gamma} producing (Fig. 5, bottom). These CD4+ or CD8+ IFN-{gamma}-producing cells, therefore, fit the profile of effector cells induced in the intra-epithelial compartment following reovirus 1/L infection (18).


Figure 5
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  		Fig. 5. Phenotype of IEL-expressing IFN-{gamma} after reovirus 1/L inoculation. Control (saline) and reovirus 1/L (i.d. with 3 x 107 PFU) -inoculated mice were sacrificed on day 7. IELs were then surface stained with anti-CD8, -CD4 and -CD69 mAbs followed by intracellular staining with an anti-IFN-{gamma} mAb. The CD8+/CD69+ and CD4+/CD69+ populations were then gated and analyzed for IFN-{gamma} expression. The values shown in the left panels are the percentage of IELs co-expressing either CD4 or CD8 and CD69 (gated population). The percentage of the gated population expressing IFN-{gamma} is shown in the histogram on the right. The results shown represent one of the two independent experiments (four mice per experiment) demonstrating similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Intestinal IELs form a highly specialized lymphoid compartment and along with LPLs are considered to play an important role in the regulation of immune responses. IELs are a phenotypically diverse population with many reported sub-populations observed under various conditions. In this study, we investigated the IEL population of control (saline inoculated) and reovirus 1/L-inoculated Balb/c mice by multiparameter flow cytometry for phenotype and cytokine production. The objective was to perform a comprehensive analysis of the phenotypic heterogeneity and function of IELs upon acute, gut mucosal viral stimulation. To our knowledge, this is the first comprehensive analysis of the multiple IEL sub-populations reported in the literature in conventional as well acutely stimulated mice. In accordance with published studies, we demonstrated that the IEL population contains both TD and TI lymphocytes in conventional mice and that a complex phenotype is present when sub-populations are analyzed for TCR, Thy-1, CD4, CD8 and B220 expression in a comprehensive manner. In reovirus 1/L-inoculated mice, we found a decrease in the TI population and an increase in the TD population characterized by significant alterations in various sub-populations. In addition, intracellular cytokine analysis demonstrated induction of IFN-{gamma} and an increase in effector/cytotoxic CD8 and CD4 cells after reovirus 1/L infection. These results suggest that TD IELs play an important role in the clearance of reovirus 1/L infection, possibly through their cytolytic activity. These results are important in understanding the role of distinct sub-populations of IELs in the context of an acute gut mucosal infection.

Figure 6 provides a comprehensive summary of the phenotypic heterogeneity of IELs upon acute, gut mucosal stimulation with reovirus 1/L as compared with conventional mice. In these studies, we did not observe any significant differences between IEL sub-populations in normal mice kept under specific pathogen-free conditions and mice injected i.d. with saline and kept under specific pathogen-free conditions (data not shown). Therefore, control, saline-inoculated mice used in these studies are considered the phenotypic equivalent of normal mice. After reovirus 1/L infection, TCR{alpha}ß+, TCR{gamma}{delta}+, Thy-1+, Thy-1–, CD4+, CD8+ and CD4+/CD8+ populations of the IELs were significantly altered. Following reovirus 1/L infection, the TCR{alpha}ß+ population of the IELs increased while the TCR{gamma}{delta}+ population of the IELs decreased. TCR{alpha}ß+ IELs were either Thy-1+ or Thy-1–, but the increase in the TCR{alpha}ß+ population can be accounted for among the Thy-1+ sub-population. These TCR{alpha}ß+/Thy-1+ IELs are comprised of CD4+, CD8+ and CD4+/CD8+ sub-populations. The CD8+ lymphocytes expressed both dimer chain configurations (CD8{alpha}{alpha} and CD8{alpha}ß). Additionally, the CD8+ sub-population contained B220+ and B220– sub-populations. The B220+ IELs are thought to be activated/memory cells, which are activated in situ (19). The percentage of Thy-1+/B220+ IELs increased after reovirus 1/L infection suggesting that this increased B220+ IEL population is the activated population of IELs, which are activated in the epithelial site after reovirus 1/L infection.


Figure 6
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  		Fig. 6. Summary of the phenotypic changes in distinct sub-populations of IELs after reovirus 1/L infection. The values shown in thin-bordered box, dashed bordered box, thick-bordered box and double-bordered box and are of control groups on days 3, 7, 10 and 14, respectively. The values shown in shaded thin-bordered box, shaded dashed bordered box, shaded thick-bordered box and shaded double-bordered box are of reovirus 1/L-infected groups on days 3, 7, 10 and 14, respectively. Up arrow shows an increase and down arrow shows a decrease in expression as compared with control group. *P < 0.05 and **P < 0.01 as compared with control group. The values shown are the percentage of the total IELs.

 
In contrast to the TCR{alpha}ß+/Thy-1+ population, the TCR{alpha}ß+/Thy-1– population, which in control, saline-inoculated mice is larger than the TCR{alpha}ß+/Thy-1+ IEL population did not show any significant overall expansion following reovirus 1/L infection. The only significant change between control, saline-inoculated and reovirus 1/L-inoculated mice was within the CD8+ population of TCR{alpha}ß+/Thy-1– IELs, which decreased slightly during the first week of infection. Concerning the TCR{gamma}{delta}+ IEL population, it contained both Thy-1– and Thy-1+ sub-populations. Overall, the TCR{gamma}{delta}+ IELs decreased following infection, and this loss was due to a decrease in the Thy-1–/CD8+/B220+ sub-population. Currently available information about TCR{gamma}{delta}+ cells during the course of a viral infection is rudimentary. Given the dynamics of the TCR{gamma}{delta}+ population following infection, it is possible that TCR{gamma}{delta}+ IELs play a housekeeping role during the resolution phase of some inflammatory responses. In support of this concept, the state of intestinal bacterial colonization has been shown to affect the phenotypic characteristics of T cells in the intestinal immune system (20, 21).

During the early phase of reovirus 1/L infection (day 3), only the CD4+ population of the IELs increased, but later in the infection, CD8+ and CD4+/CD8+ populations also increased along with the CD4+ population. Little is known about the role of CD4+/CD8+ IELs in the mucosal immune response. One study demonstrated that CD4+/CD8+ IELs, which are primarily CD8{alpha}{alpha}, express a TH2-type cytokine profile, which suggests an anti-inflammatory role (22, 23). Therefore, these cells may have a unique immune regulatory function at the epithelial site. In support of a role for these cells in immune regulation, CD4+/CD8+ lymphocytes were observed in the draining lymph nodes following intra-nasal inoculation of reovirus 1/L and these cells respond via proliferation to reovirus 1/L-pulsed antigen-presenting cells in vitro (24). A recent study demonstrated that in contrast to CD8{alpha}{alpha}+ T cells, which are located mainly within the gut epithelium, CD4+ and CD8{alpha}ß+ T cells are present in the gut epithelium and lamina propria and are the progeny of common precursors that have proliferated under the same antigenic stimulation (25–27). Since CD4+ T cells comprise a larger proportion of the LPL population than CD8+ T cells, and the adhesive capacity of CD4+ cells to villus microvessels is greater than that of CD4– cells (28), the increase in CD4+ T cells in the intra-epithelial compartment 3 days after infection could be due to the migration of CD4+ T cells from the lamina propria to the epithelium. During the later phase of reovirus 1/L infection (days 7 and 10), the CD8+ IELs increased along with CD4+ IELs. This is likely due to the migration of primed PP CD8+ and CD4+ T cells to the intra-epithelial compartment through the lamina propria and the proliferation of CD8{alpha}ß+ IELs. In this regard, we have recently demonstrated that reovirus 1/L-primed PP cells adoptively transferred to normal or reovirus 1/L-inoculated mice, preferentially migrated to the intra-epithelial compartment of reovirus 1/L-infected mice as compared with normal mice (13). The literature also supports the hypothesis that the antigenic stimulation and proliferation of IEL precursors occur mainly in the PP followed by their rapid emigration to the intra-epithelial compartment and lamina propria through the mesenteric lymph nodes, thoracic duct and blood circulation (25–27). However, it is also possible that IELs, LPLs and PP lymphocytes upon meeting their cognate antigens in situ may undergo further stimulation and divide. Therefore, we suggest that the observed increase in TCR{alpha}ß+/Thy-1+ IELs following reovirus 1/L infection is due to migration of primed PP T cells to the epithelial site. However, the possibility of proliferation in situ of TCR{alpha}ß+/Thy-1+ IELs cannot be ruled out.

The decrease in reovirus 1/L-induced CD8+ IELs to control levels 14 days after infection might be due to depletion of unwanted T cells (CD8{alpha}{alpha} and CD8{alpha}ß) via apoptotic death after the clearance of the viral infection. In this study, the CD8{alpha}{alpha}+ T cells were decreased after reovirus 1/L infection, and we suspect this decrease was the cause of the significant decrease in the total number of CD8+ T cells on day 3 after reovirus 1/L infection. The decrease in the CD8+/B220+ sub-population of TCR{gamma}{delta}+/Thy-1+ IELs (CD8{alpha}{alpha}+/B220+) in our study also demonstrates indirectly that the decrease in the CD8{alpha}{alpha}+ sub-population of TCR{gamma}{delta}+ IELs also contributes to the decrease in total CD8+ IELs during the early stage of infection. There are several possible causes for this depletion of the CD8{alpha}{alpha}+ sub-population of IELs. Direct viral cytotoxicity may be playing a role, or some CD8{alpha}{alpha}+ T cells may also migrate to the periphery during reovirus 1/L infection. The proportion of CD8{alpha}{alpha}+ T cells has been shown to increase in the peripheral blood during chronic infection of immunodeficiency virus in cats (29). Previous studies have also suggested that CD8{alpha}{alpha}+ IELs were an immature extrathymic T cell subset that may differentiate through a CD4+/CD8{alpha}{alpha}+ stage into mature CD4+ and CD8{alpha}ß+ T cells in intestinal epithelium (30, 31). If this is the case, the increased CD4+/CD8{alpha}ß+ sub-population of TCR{alpha}ß+/Thy-1+ IELs after reovirus 1/L infection may be the intermediate stage of maturation of extrathymic immature CD4+/CD8{alpha}{alpha}+ T cells to mature CD4+ and CD8{alpha}ß+ IELs, a process which may require antigenic stimulation like the reovirus 1/L stimulation in this study. The CD4+/CD8{alpha}{alpha}+ cells may also be intermediate in the extrathymic differentiation pathway for local generation of resident CD8{alpha}{alpha}+ IELs (32). Interestingly, while a vast majority of TCR{gamma}{delta}+ IELs expresses CD8{alpha}{alpha} but not CD8{alpha}ß (which are considered to develop in intestinal epithelial layers independently of a functional thymus) (5, 10, 11), we demonstrated a small but significant CD8{alpha}ß population of the TCR{gamma}{delta}+ IELs (data not shown). This confirmed a recent study that demonstrated this functionally active population in athymic nu/nu mice (33). However, we did not see any significant change in the percentage of TCR{gamma}{delta}+/CD8{alpha}ß+ IELs after reovirus 1/L infection.

Gut mucosal infection with reovirus results in the presence of infectious virions in the intestine for up to 10 days after infection, with the titer decreasing 10-fold during this time period (34). In addition, reovirus 1/L infection results in the generation of MHC-restricted, reovirus 1/L-specific, cytolytic TCR{alpha}ß+/Thy-1+ IELs (12). Here, we demonstrate that the increase in the TCR{alpha}ß+/Thy-1+ IELs occurs early after infection and correlates with viral clearance and suggests that the TCR{alpha}ß+/Thy-1+ IELs may be playing an important role in the clearance of reovirus infection. This role could be executed through cytolytic activity or cytokine production. In these studies, infection with reovirus 1/L decreased the CD8{alpha}{alpha}+ IEL sub-population and increased CD8{alpha}ß+ and CD4+ IEL sub-populations. CD8+ cells provide protection against viruses by their cytolytic activity and locally generated TCR{alpha}ß+/CD8{alpha}{alpha}+ IELs exert only minimal virus-specific cytotoxicity, while maximum specific killing of virus is mediated by thymically derived TCR{alpha}ß+/CD8{alpha}ß+ IELs (13, 35). Therefore, the increase in CD8{alpha}ß+ IELs in response to reovirus 1/L infection could contribute to the CTL responses in the intestinal mucosa.

In the absence of deliberate stimulation, the cytokine profile of IELs fails to display normal polarization towards either TH1 or TH2 cytokine production despite the fact that they are morphologically in an active state. In the resting state, IELs produce IFN-{gamma} and IL-5 (36), however, after activation the majority of IELs adopts a TH1 cytokine profile particularly with respect to inflammatory cytokines (11). In terms of cytokine production, reovirus 1/L infection has been shown to induce a TH1 response in IELs (37), which includes anti-viral cytokines such as IFN-{gamma} and tumor necrosis factor-{alpha} (38). The present study extends these results and demonstrates that the increase in the CD4+ and CD8+ (TCR{alpha}ß+/Thy-1+) IEL sub-populations correlated with IFN-{gamma} induction by these cells over time. In addition, our results of antigen-driven intracellular IFN-{gamma} production, an assay that is used to measure potency of CD4+ and CD8+ IELs after reovirus 1/L infection, also demonstrated the induction of both effector/cytotoxic CD4+ and CD8+ IELs after reovirus 1/L infection. A recent report demonstrated that IELs recovered from reovirus 3/D-infected animals are primarily CD4+/CD43+/ICOS+ and secrete IFN-{gamma} in response to reovirus 3/D infection and potentially represent an activated virus-specific response (38, 39). Taken together with our findings that demonstrate activation of both CD4+ and CD8+ cells, these data support the proposal that both CD4+ and CD8+ viral-specific IEL sub-populations may play a role in the clearance of reovirus.

In conclusion, reovirus 1/L infection was found to alter the phenotypic profiles of the various IEL sub-populations. The results demonstrated increases in CD4+, CD8{alpha}ß+ and CD4+/CD8{alpha}ß+ populations of TCR{alpha}ß+/Thy-1+ IELs and a decrease in TCR{gamma}{delta}+/Thy-1–/CD8{alpha}{alpha}+ or TCR{alpha}ß+/Thy-1–/CD8{alpha}{alpha}+ IELs. The increase in CD4+ and CD8{alpha}ß cells and decrease in CD8{alpha}{alpha} cells may partially account for the altered T cell homeostasis in the intestinal epithelium following reovirus 1/L infection. Further, our results demonstrated that the potential of CD8+ and CD4+ IELs to produce IFN-{gamma} was up-regulated during reovirus 1/L infection. Taken together, the phenotype of the IELs after reovirus 1/L infection suggests that the protection against reovirus 1/L is most likely due to the activity of thymus-derived T cells that enter the intestine following antigenic stimulation. The increase in TCR{alpha}ß+ IELs after reovirus 1/L infection demonstrates that TCR{alpha}ß+ IELs play an important role in the immune response against reovirus 1/L.


   Acknowledgements
 
The authors would like to thank the Medical University of South Carolina (MUSC) and the Hollings Cancer Center for their support of the MUSC Analytical Flow Cytometry Facility. This work was supported by National Institutes of Health (NIH)/National Institute of Dental Craniofacial Research (NIDCR) grant number 5R01 DE12781 (S.D.L.) and NIH grant number CO6 RR015455 (MUSC).


   Abbreviations
 
APC, allophycocyanin
CMF, Ca2+-, Mg2+-free HBSS
FBS, fetal bovine serum
i.d., intra-duodenal
IEL, intra-epithelial lymphocyte
LPL, lamina propria lymphocyte
MUSC, Medical University of South Carolina
NIH, National Institutes Health
PEC, peritoneal exudate cell
PMA, phorbol myristate acetate
PP, Peyer's patche
reovirus 1/L, reovirus serotype 1/strain Lang
TD, thymus dependent
TI, thymus independent

   Notes
 
Transmitting editor: G. Trinchieri

Received 16 August 2006, accepted 31 January 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Rocha B, Vassalli P, Guy-Grand D. (1991) The V beta repertoire of mouse gut homodimeric alpha CD8+ intraepithelial T cell receptor alpha/beta+ lymphocytes reveals a major extrathymic pathway of T cell differentiation. J. Exp. Med. 173:483.[Abstract/Free Full Text]
  2. Hayday A, Theodoridis E, Ramsburg E, Shires J. (2001) Intraepithelial lymphocytes: exploring the Third Way in immunology. Nat. Immunol. 2:997.[CrossRef][Web of Science][Medline]
  3. Cheroutre H. (2004) Starting at the beginning: new perspectives on the biology of mucosal T cells. Annu. Rev. Immunol. 22:217.[CrossRef][Web of Science][Medline]
  4. Cheroutre H. (2005) IELs: enforcing law and order in the court of the intestinal epithelium. Immunol. Rev. 206:114.[CrossRef][Web of Science][Medline]
  5. Guy-Grand D, Cerf-Bensussan N, Malissen B, Malassis-seris M, Briottet C, Vassalli P. (1991) Two gut intraepithelial CD8+ lymphocyte populations with different T cell receptors: a role for the gut epithelium in T cell differentiation. J. Exp. Med. 173:471.[Abstract/Free Full Text]
  6. Rocha B, Vassalli P, Guy-Grand D. (1994) Thymic and extrathymic origins of gut intraepithelial lymphocyte populations in mice. J. Exp. Med. 180:681.[Abstract/Free Full Text]
  7. Guy-Grand D, Azogui O, Celli S, et al. (2003) Extrathymic T cell lymphopoiesis: ontogeny and contribution to gut intraepithelial lymphocytes in athymic and euthymic mice. J. Exp. Med. 197:333.[Abstract/Free Full Text]
  8. Lefrancois L and Olson S. (1994) A novel pathway of thymus-directed T lymphocyte maturation. J. Immunol. 153:987.[Abstract]
  9. Lin T, Matsuzaki G, Yoshida H, et al. (1994) CD3–CD8+ intestinal intraepithelial lymphocytes (IEL) and the extrathymic development of IEL. Eur. J. Immunol. 24:1080.[Web of Science][Medline]
  10. Lefrancois L. (1991) Intraepithelial lymphocytes of intestinal mucosa: curiouser and curiouser. Semin. Immunol. 3:99.[Medline]
  11. Beagley KW and Husband AJ. (1998) Intraepithelial lymphocytes: origins, distribution, and function. Crit. Rev. Immunol. 18:237.[Web of Science][Medline]
  12. London SD, Cebra JJ, Rubin DH. (1989) Intraepithelial lymphocytes contain virus-specific cytotoxic T cell precursors after gut mucosal immunization with reovirus serotype 1/Lang. Reg. Immunol. 2:98.[Medline]
  13. Cuff CF, Cebra CK, Rubin DH, Cebra JJ. (1993) Developmental relationship between cytotoxic {alpha}ß T cell receptor-positive intraepithelial lymphocytes and Peyer's patches lymphocytes. Eur. J. Immunol. 23:1333.[Web of Science][Medline]
  14. London SD, Rubin DH, Cebra JJ. (1987) Gut mucosal immunization with reovirus serotype 1/L stimulates viral specific cytotoxic T cell precursors as well as IgA memory cells in Peyer's patches. J. Exp. Med. 165:830.[Abstract/Free Full Text]
  15. Bharhani M, Grewal JS, Pilgrim MJ, et al. (2005) Reovirus serotype 1/strain Lang-stimulated activation of antigen-specific T lymphocytes in Peyer's patches and distal gut-mucosal sites: activation status and cytotoxic mechanisms. J. Immunol. 174:3580.[Abstract/Free Full Text]
  16. Rubin DH, Kornstein MJ, Anderson AO. (1985) Reovirus serotype 1 intestinal infection: a novel replicative cycle with ileal disease. J. Virol. 53:391.[Abstract/Free Full Text]
  17. Lefrancois L. (1994) Isolation of mouse small intestinal intraepithelial lymphocytes. In Coligan JE, Kruisbeek AM, Margulies DH, Shevak EM, Strober W (Eds.). Current Protocols in Immunology(Wiley, New York) pp. 191.
  18. Kumaraguru U and Rouse BT. (2000) Application of intracellular gamma interferon assay to recalculate the potency of CD8+ T-cell responses to herpes simplex virus. J. Virol. 74:5709.[Abstract/Free Full Text]
  19. Lefrancois L and Puddington L. (2001) Basic aspects of intraepithelial lymphocyte immunobiology. In Ogra PL, Mastecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR (Eds.). Mucosal Immunology 2nd edn (Academic press, San Diego) pp. 413.
  20. Bandeira A, Mota-Santos T, Itohara S, Hiuersser C, Tonegawa S, Coutinho A. (1990) Localization of {gamma}{delta} T-cells to the intestinal epithelium is independent of normal microbial colonization. J. Exp. Med. 172:239.[Abstract/Free Full Text]
  21. Lillehoj HS and Bacon LD. (1991) Increase of intestinal intraepithelial lymphocytes expressing CD8 antigen following challenge infection with Eimeria acervulina. Avian Dis. 35:294.[CrossRef][Web of Science][Medline]
  22. Fujihashi K, Yamamoto M, McGhee JR, Kiyono H. (1993) Alpha beta T cell receptor-positive intraepithelial lymphocytes with CD4+, CD8– and CD4+, CD8+ phenotypes from orally immunized mice provide Th2-like function for B cell responses. J. Immunol. 151:6681.[Abstract]
  23. Das G, Augustine MM, Das J, Bottomly K, Ray P, Ray A. (2003) An important regulatory role for CD4+CD8 alpha alpha T cells in the intestinal epithelial layer in the prevention of inflammatory bowel disease. Proc. Natl Acad. Sci. USA 100:5324.[Abstract/Free Full Text]
  24. Periwal SB and Cebra JJ. (1999) Respiratory mucosal immunization with reovirus serotype 1/L stimulates virus-specific humoral and cellular immune responses, including double-positive (CD4(+)/CD8(+)) T cells. J. Virol. 73:7633.[Abstract/Free Full Text]
  25. Arstila T, Arstila TP, Calbo S, et al. (2000) Identical T cell clones are located within the mouse gut epithelium and lamina propria and circulate in the thoracic duct lymph. J. Exp. Med. 191:823.[Abstract/Free Full Text]
  26. Kim SK, Reed DS, Heath WR, Carbone F, Lefrancois L. (1997) Activation and migration of CD8 T cells in the intestinal mucosa. J. Immunol. 159:4295.[Abstract]
  27. Tamura A, Soga H, Yaguchi K, et al. (2003) Distribution of two types of lymphocytes (intraepithelial and lamina-propria-associated) in the murine small intestine. Cell Tissue Res. 313:47.[CrossRef][Web of Science][Medline]
  28. Koseki S, Miura S, Fujimori H, et al. (2001) In situ demonstration of intraepithelial lymphocyte adhesion to villus microvessels of the small intestine. Int. Immunol. 13:1165.[Abstract/Free Full Text]
  29. Shimojima M, Miyazawa T, Kohmoto M, et al. (1998) Expansion of CD8alpha+beta– cells in cats infected with feline immunodeficiency virus. J. Gen. Virol. 79:91.[Abstract]
  30. Guy-Grand D and Vassalli P. (1993) Gut intraepithelial T lymphocytes. Curr. Opin. Immunol. 5:247.[CrossRef][Web of Science][Medline]
  31. Guy-Grand D and Vassalli P. (2002) Gut intraepithelial lymphocyte development. Curr. Opin. Immunol. 14:255.[CrossRef][Web of Science][Medline]
  32. Rozing J and de Geus B. (1995) Changes in the intestinal lymphoid compartment throughout life: implications for the local generation of intestinal T cells. Int. Rev. Immunol. 12:13.[Medline]
  33. Emoto Y, Emoto M, Miyamoto M, Yoshozawa I, Kaufmann SHE. (2004) Functionally active CD8{alpha}ß+TCR{gamma}{delta} intestinal epithelial lymphocytes in athymic nu/nu mice. Int. Immunol. 16:111.[Abstract/Free Full Text]
  34. Fan JY, Boyce CS, Cuff CF. (1998) T-helper 1 and T-helper 2 cytokine responses in gut associated lymphoid tissue following reovirus infection. Cell. Immunol. 188:55.[CrossRef][Web of Science][Medline]
  35. Muller S, Buhler-Jungo M, Mueller C. (2000) Intestinal intraepithelial lymphocytes exert potent protective cytotoxic activity during an acute virus infection. J. Immunol. 164:1986.[Abstract/Free Full Text]
  36. Taguchi T, Aicher WK, Fujihasgi K, et al. (1991) Novel function for intraepithelial lymphocytes: murine CD3+, {gamma}{delta}TCR T cells produce IFN-{gamma} and IL-5. J. Immunol. 147:3736.[Abstract]
  37. Kagi D and Hengartner H. (1996) Different roles for cytotoxic T cell in the control of infections with cytopathic versus noncytopathic viruses. Curr. Opin. Immunol. 8:472.[CrossRef][Web of Science][Medline]
  38. Montufar-Solis D, Garza T, Teng BB, Klein JR. (2006) Upregulation of ICOS on CD43+ CD4+ murine small intestinal intraepithelial lymphocytes during acute reovirus infection. Biochem. Biophys. Res. Commun. 342:782.[CrossRef][Web of Science][Medline]
  39. Wang HC, Montufar-Solis D, Teng BB, Klein JR. (2004) Maximum immunobioactivity of murine small intestinal intraepithelial lymphocytes resides in a subpopulation of CD43+ T cells. J. Immunol. 173:6294.[Abstract/Free Full Text]

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