Intraduodenal priming of mice with reovirus serotype 1/strain Lang (reovirus 1/L) stimulates gut lymphocytes and generates precursor and effector CTLs. Our earlier studies demonstrated that germinal center and T cell Ag (GCT) is a marker which identifies reovirus 1/L-specific precursor CTL and effector CTL in Peyer’s patches (PP) of reovirus 1/L-inoculated mice. In this study, we characterized the expression of the activation markers, GCT and CD11c, on reovirus 1/L-stimulated gut lymphocytes and the effector mechanisms involved in reovirus 1/L-specific cytotoxicity. We found that intraduodenal reovirus 1/L inoculation of mice induced the expression of both GCT and CD11c on PP lymphocytes (PPL), intraepithelial lymphocytes (IEL), and lamina propria lymphocytes (LPL), and these activated cells expressed Fas ligand (FasL). The majority of the GCT+CD11c+ IEL and LPL expressed a phenotype, TCRαβ+Thy-1+CD8+ similar to that expressed on reovirus 1/L-stimulated PPL. However, splenic lymphocytes expressed GCT but not CD11c after stimulation with reovirus 1/L. Perforin, Fas-FasL, and TRAIL pathways were found to be involved in PPL, IEL, and LPL cytotoxic activity against reovirus 1/L-infected targets. In PPL, perforin and Fas-FasL pathways were more effective than TRAIL. In IEL, all three cytotoxic mechanisms were equally as effective. However, LPL prefer Fas-FasL and TRAIL over perforin. Further, we demonstrated the preferential migration of GCT+ PPL to the intraepithelial compartment and the lamina propria. These results suggest that GCT and CD11c can be used as activation markers for gut lymphocytes and CD11c can also be used to differentiate between activated gut and systemic lymphocytes.

The gut mucosal immune system consists of two functionally distinct types of tissue: 1) inductive sites, consisting primarily of Peyer’s patches (PP)4 and mesenteric lymph nodes; and, 2) effector sites, comprising two major sites, the lamina propria (LP) and the epithelium-associated cells, intraepithelial lymphocytes (IEL) (1, 2). Unlike the majority of nonintestinal lymphocytes, many LP lymphocytes (LPL) and IEL exhibit phenotypic and functional characteristics of activated cells (3, 4). The LP lymphocyte population is comprised of CD4 and CD8 T cells and of B cells and plasma cells secreting primarily IgA (5). However, lymphocytes in the intraepithelial compartment are phenotypically diverse and are comprised of two major CD8+ subsets based on TCR expression: γδ or αβ (3). Although it was believed that the development of CD8 αβ (TCR-αβ IEL) was thymus-dependent and that the development of CD8αα IEL (TCR-γδ and -αβ) was thymus-independent, it is now clear that most IEL require some form of thymic influence for complete maturation and expression of their surface molecules (6, 7). Thus, it is important to determine the mechanisms by which IEL are selected and subsequently respond to Ag. However, the activated nature of resident intestinal T cells makes it difficult to study primary T cell activation in the mucosa.

Intraduodenal (i.d.) inoculation of reovirus serotype 1/strain Lang (reovirus 1/L) has proven to be an effective model for studying mucosal T cells and their properties. Our previous studies using this model have demonstrated that i.d. inoculation of reovirus 1/L leads to the generation of MHC-restricted reovirus 1/L-specific precursor (p) CTL in PP and the intraepithelial compartment as well as IgA memory B cells in PP (8, 9). We also have reported that both reovirus 1/L-specific pCTL and effector (e) CTL, generated by in vitro restimulation of PP lymphocytes (PPL) with reovirus 1/L, express the germinal center and T cell Ag (GCT) (10). This Ag is characterized by a mAb that was originally found to bind to germinal center (GC) B cells present in the spleens of Ag-stimulated mice and also a subpopulation of CD8+ cells (11). The expression of GCT on eCTL and pCTL indicates that GCT may provide a means to detect pCTL or eCTL in all gut mucosal tissues after their stimulation in the PP (10). CD11c has also been demonstrated to be a hallmark of T cell activation in vivo and an indicator of ongoing Ag-specific T cell activation in the intestinal epithelium (3). Therefore, CD11c may prove to be a useful marker for identifying activated gut mucosal lymphocytes in our model of gut mucosal reovirus 1/L infection. It has been known for some time that activated T cells traffic to the intestine, although the precise subsets and molecular mechanisms involved in this process are not fully understood (12). Although reovirus 1/L infection leads to the generation of MHC-restricted virus-specific TCRαβ IEL (9), and this response parallels the induction of a response in PP, it is unclear whether IEL are primed in PP, in situ, or elsewhere (13). In addition, whether IELs can be activated in situ via Ag presentation by intestinal epithelial cells or whether IELs are activated outside of the epithelium followed by migration into the mucosa is unknown.

Virus-specific pCTL have been isolated from PP and IEL after i.d. priming with reovirus 1/L (8, 9). However, the mechanisms by which these cells mediate the killing of virally infected target cells are still poorly understood. Recent studies of CTLs have defined two major pathways of contact-dependent cytotoxicity in vitro (14, 15). The first pathway involves granule exocytosis and pore formation in the target cell membrane through extracellular Ca2+-dependent polymerization of perforin. The perforin/granzyme pathway of cytotoxicity is the principal modus operandi for CD8+ T cells. The second pathway is mediated by T cell Fas ligand (FasL) engagement of Fas expressed by target cells. Use of the Fas-FasL pathway of target cell lysis is thought to be favored by CD4+ T cells (16). However, human virus-specific CD4+ T cells can also use the perforin/granzyme pathway of cytotoxicity (17). Recent work has revealed that T cell induction of target cell apoptosis may involve other receptor-mediated pathways, the most notable of which is TRAIL (18).

The objective of this work is to gain further understanding of the cellular and molecular interactions, which occur within the gut, that result in protective mucosal immunity. In this report, we demonstrate that GCT and CD11c are expressed by recently activated gut lymphocytes. We characterized the pathways of cytotoxicity used by PPL, LPL, and IEL and found the involvement of perforin, Fas-FasL, and TRAIL, although to a different extent in each of these populations. We also provide evidence that it is the recently stimulated (GCT+) PPL population which preferentially migrates to the intraepithelial compartment and LP following enteric virus infection.

Four-week-old female BALB/cJ mice were purchased from The Jackson Laboratory. Six- to 8-wk-old mice were used in all experiments. Mice were maintained under specific pathogen-free conditions 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 other experimental and stock mice.

Reovirus 1/L was originally obtained from Dr. W. Joklik (Duke University School of Medicine, Durham, NC). Third passage, gradient-purified stocks were titered by limiting dilution on L cell monolayers (19).

The following mAbs were used in this study: anti-TCR-αβ (clone H57-597), anti-TCR-γδ (clone GL3), and anti-CD4 (clone CT-CD4) were purchased from Caltag Laboratories. Anti-Thy-1.2 (clone 53-2.1), anti-CD8α (clone 53-6.7), anti-CD8β (clone 53.5.8), anti-B220 (clone RA3-6B2), anti-CD16/CD32 (Fc block, clone 2.4G2), anti-CD11c (clone HL3), anti-FasL (clone MFL3), and anti-hamster IgG mixture (clones G70-204 and G94-56) were purchased from BD Pharmingen. Anti-GCT (clone 1024CD3.5) was a gift from Dr. F. M. Platt (Oxford University, Oxford, U.K.). Abs were conjugated with FITC, PE, allophycocyanin, PerCP, or biotin. Streptavidin-conjugated PE, allophycocyanin, and PerCP (BD Pharmingen) were used as second-step reagents to identify biotinylated primary Abs. Each Ab was titrated to determine the optimal staining concentration for maximum signal.

Mice were anesthetized with a 0.15 cc i.p. dose of 20% ketamine (Vetalar 100 mg/cc; Fort Dodge Laboratories) and 2.0% acepromazine maleate (PromAce 10 mg/cc; Ayerest Laboratories) and then inoculated i.d. with 3 × 107 PFU of reovirus 1/L in 50 μl of 0.9% NaCl (8). Control mice were inoculated as above with the same volume of 0.9% NaCl.

IEL and LPL were isolated from mice with some modifications of a protocol described previously (20). In brief, the intestines from the duodenum to the ileocecal junction were removed and flushed with Ca2+, Mg2+-free HBSS (CMF). PPs 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% FBS, 1 mM EDTA, and 1 mM DTT at 37°C. The eluted cells from the first two incubations were collected, passed through 74 μm nylon mesh to partially purify the IEL and kept at 37°C in CO2 incubator for 45–60 min. IEL were subsequently separated from epithelial cells by centrifugation through 44/67.5% Percoll (Pharmacia) gradient at 600 × g for 20 min. IEL were harvested from the interface between the 44% and 67.5% Percoll layers. For the isolation of LPL, 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/ml Collagenase type II (Sigma-Aldrich), 0.5 mg/ml Dispase II (Boehringer Mannheim), and 100 U/ml DNase I (Boehringer Mannheim) at 37°C. LPL were then purified through a 40/67.5% Percoll gradient centrifuged at 600 × g for 20 min. PPs were isolated and digested with collagenase and dispase, as described above. Splenocytes were isolated by pressing the spleen between the two frosted slides. RBCs were removed by lysis with 0.84% ammonium chloride.

Single cell suspensions (0.5–1 × 106/sample) in HBSS-5% FBS containing 0.02% sodium azide were preincubated for 20 min with anti-CD16/32 to block FcRs (FcγIII A/B) to prevent nonspecific binding. The cells were then incubated with the relevant appropriately titered mAbs for 30 min on ice. These Abs were either directly labeled with FITC, allophycocyanin, PE, and PerCP, or were biotinylated. In the case of biotinylated Abs, streptavidin-conjugated PE, allophycocyanin, or PerCP were used as second step reagents. After washing with HBSS without phenol red and FBS, the cells were fixed in 2% paraformaldehyde for 20 min at room temperature and analyzed for cell surface marker expression using a FACSCalibur flow cytometer (BD Biosciences). Data were analyzed using CellQuest software (BD Biosciences).

PPL, IEL, and LPL were harvested and RNA was extracted by the chloroform/isopropanol method using TRI-reagent (Sigma-Aldrich). Using a one-step RT-PCR kit (Qiagen), RNA (500 ng) was first reverse-transcribed, and then PCR-amplified. Amplification was performed in a DNA thermal cycler (PerkinElmer/Cetus) set at 1 min each at 94°C, 58°C and 72°C for 30 cycles for FasL, perforin, and β-actin followed by an extension at 72°C for 10 min. Amplification for TRAIL was for 28 cycles at 92°C (30 s), 57°C (30 s), and 72°C (2 min). After amplification, the PCR product was electrophoresed on a 1.5% agarose gel and visualized by ethidium bromide staining under UV illumination. The specific primers for FasL (sense 5′-CTG GAA TGG GAA GAC ACA TA-3′ and antisense 5′-AAA GGT CTT AGA TTC CTC AA-3′), perforin (sense 5′-CAC AAG TTC GTG CCA GGT GTA-3′ and antisense 5′-GCA TGC TCT GTG GAG CTG TTA-3′), TRAIL (sense 5′-TCA CCA ACG AGA TGA AGC AGC-3′ and antisense 5′-CTC ACC TTG TCC TTT GAG ACC-3), and β-actin (sense 5′-TTG TAA CCA ACT GGG ACG ATA TGG-3′ and antisense 5′-GAT CTT GAT CTT CAT GGT GCT AGG-3′) were purchased from Integrated DNA Technologies. Amplified products for β-actin, TRAIL, perforin, and FasL were 760, 513, 491, and 214 bp, respectively. For semiquantitative RT-PCR analysis, band intensities on scanned gels were analyzed using specific bands of the housekeeping gene β-actin transcripts as a reference using the public domain NIH Image program developed at the National Institutes of Health.

To generate Ag-specific CTL effector cells, gut lymphocytes (PPL, LPL, and IEL) isolated as described above from enterically primed mice were cultured with some modifications (8). In brief, lymphocytes were cultured in 96-well microtiter plates at a density of 1 × 106 cells/ml in 200 μl of RPMI 1640 medium supplemented with 5 × 10−5 M 2-ME, 2 mM l-glutamine, 10% FCS, gentamicin, penicillin, streptomycin, and nystatin. Thioglycolate-elicited peritoneal exudate stimulator cells (PEC) pulsed with reovirus 1/L at a multiplicity of infection (MOI) of 2 for 1 h were used as a source of APCs. PEC (5 × 104) were added to each well containing lymphocytes. For PPL and LPL, cultures were harvested on day 5. In the case of IEL, cultures were supplemented with rIL-2 (100 pg/ml) and IL-15 (10 ng/ml) (BioSource International) on day 1 and harvested the following day. For all populations, live cells were enriched at the interface of a 44/67.5% Percoll gradient before analyzing their cytotoxic activity.

Cytolytic activity was measured by a lactate dehydrogenase (LDH) release assay (Cytotoxicity Detection kit; Roche Diagnostics) from PPL, IEL, and LPL isolated on day 10 post-reovirus 1/L inoculation (peak time point for FasL expression, see Fig. 7). Effector cells, as prepared above, were added in triplicate to U-bottom wells of 96-well microtiter plate and serial dilutions were made at the indicated E:T ratios. Reovirus 1/L-infected (MOI = 10) or noninfected KD2SV target cells were then added to each well. The plates were then incubated at 37°C. After 4 h of incubation, the plates were centrifuged at 250 × g for 10 min. Cell-free supernatants (50 μl) from each well were collected and transferred to a 96-well flat-bottom microtiter plate. Fifty microliters of freshly prepared reaction mixture was added to each well and incubated at room temperature for 10–30 min in the dark. The released LDH activity was measured at OD490 with a reference wavelength of 650 nm. Maximum LDH release was determined by incubation of target cells in 100 μl of 2% Triton X-100 and spontaneous LDH release was determined by addition of medium only. The percentage of specific LDH released was calculated as percent-specific cytotoxicity = (experimental release − effector spontaneous release − target spontaneous release)/(maximum target release − spontaneous target release). Results are expressed as the mean percent-specific cytotoxicity ± SD.

FIGURE 7.

Expression of FasL on PPL, IEL, and LPL. The results show induction of FasL on GCT+ CD11c+ (A) PPLs, (B) IEL, and (C) LPL at various time points post-reovirus 1/L infection. The values shown in the panels are the percentage of GCT+CD11c+ cells expressing FasL. The results shown represent one of three experiments demonstrating similar results.

FIGURE 7.

Expression of FasL on PPL, IEL, and LPL. The results show induction of FasL on GCT+ CD11c+ (A) PPLs, (B) IEL, and (C) LPL at various time points post-reovirus 1/L infection. The values shown in the panels are the percentage of GCT+CD11c+ cells expressing FasL. The results shown represent one of three experiments demonstrating similar results.

Close modal

To evaluate the role of perforin in CTL-mediated cytolysis, effector cells were pretreated with Concanamycin A (CMA; Sigma-Aldrich) for 2 h at a final concentration of 100 nM to inactivate perforin. Similarly, anti-FasL mAb (10 μg/ml; BD Pharmingen) was used to inhibit Fas-FasL mediated lysis and anti-TRAIL mAb (10 μg/ml; R & D Systems) was used to inhibit TRAIL-mediated cytotoxicity. Appropriate isotype matched control mAbs were used in all experiments and no significant effect on the cytotoxic activity was observed (data not shown).

BALB/cJ mice were inoculated i.d. with reovirus 1/L as described, and PPLs were isolated after 3 days. Cells were labeled with the fluorescent dye, CFSE (Molecular Probes). Briefly, cells were suspended in HBSS without serum containing 5 μM CFSE for 30 min at 37°C for staining and for an additional 20 min in HBSS containing 10% FBS to stop the reaction. The labeled cells were washed twice with HBSS before transfer via tail vein injection into recipient mice. One day after cell transfer (1 × 107 cells/mouse), the recipient mice were inoculated i.d. with 3 × 107 PFU of reovirus 1/L (infected group) or saline (control group) and sacrificed 3 days after cell transfer. The IEL, LPL, and PPL were isolated, stained for GCT, and analyzed for CFSE+ and GCT+ cells as described above.

The ANOVA method was used to analyze the data. A p value of <0.05 was considered to indicate significance.

To determine whether a GCT+ lymphocyte population developed in gut mucosal inductive and effector sites following reovirus 1/L inoculation, BALB/cJ mice were inoculated i.d. with 3 × 107 PFU of reovirus 1/L. PPL, IEL, and LPL were obtained and analyzed for the expression of the GCT Ag by flow cytometry on days 3, 7, and 10 postinoculation. In these studies, B cells in the PPL and LPL were identified by expression of the cell surface molecule, B220. We verified that 100% of the B220+ LPL and PPL also expressed the CD19 B cell marker, thus demonstrating the phenotype of the B cell populations in the these gut-mucosal compartments (data not shown). We found that in control, saline-inoculated mice, a very small population of PPL, IEL, and LPL express GCT (2–6%). However, a single i.d. application of reovirus 1/L resulted in a marked increase in the proportion of GCT expressing PPL, IEL, and LPL (10 - 26%) (Figs. 1,A, 2,A, and 3 A).

FIGURE 1.

Induction of GCT on PPL. A, The results show percent expression (bars) and absolute numbers (lines) of GCT+ cells at various time points post-reovirus 1/L infection. Control (□, ▴) and infected (▧, •) mice were inoculated i.d. with either saline or reovirus 1/L (3 × 107 PFU), respectively. The values shown are the mean ± SD of three independent experiments using four mice per time point. ∗ and # represent p < 0.05 compared with the control group. B, Expression of GCT on B220+ PPL on day 3 and expression of GCT on B220+, CD4+, and CD8+ PPL on day 7 post-reovirus 1/L infection. The values shown in the panels are the percentage of PPL expressing GCT alone (upper left) or coexpressing GCT and the indicted surface Ag (upper right). The results shown represent one of the three independent experiments demonstrating similar results.

FIGURE 1.

Induction of GCT on PPL. A, The results show percent expression (bars) and absolute numbers (lines) of GCT+ cells at various time points post-reovirus 1/L infection. Control (□, ▴) and infected (▧, •) mice were inoculated i.d. with either saline or reovirus 1/L (3 × 107 PFU), respectively. The values shown are the mean ± SD of three independent experiments using four mice per time point. ∗ and # represent p < 0.05 compared with the control group. B, Expression of GCT on B220+ PPL on day 3 and expression of GCT on B220+, CD4+, and CD8+ PPL on day 7 post-reovirus 1/L infection. The values shown in the panels are the percentage of PPL expressing GCT alone (upper left) or coexpressing GCT and the indicted surface Ag (upper right). The results shown represent one of the three independent experiments demonstrating similar results.

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FIGURE 2.

Induction of GCT on IEL. A, The results show percent expression (bars) and absolute numbers (lines) of GCT+ cells at various time points post-reovirus 1/L infection. Control (□, ▴) and infected (▧, •) mice were inoculated i.d. with either saline or reovirus 1/L (3 × 107 PFU), respectively. The values shown are the mean ± SD of three experiments using four mice per time point. ∗ and # represent p < 0.05 compared with the control group. B, Expression of GCT on different subpopulations of IEL on day 7 post-reovirus 1/L infection. The values shown in the panels are the percentage of IEL expressing GCT alone (upper left) or coexpressing GCT and the indicted surface Ag (upper right). The results shown represent one of the three experiments demonstrating similar results. C, Expression of GCT on subpopulations of CD8+ IEL on day 7 post-reovirus 1/L infection. The CD8α+GCT+ cells were gated to exclude all other cells and further analyzed for CD8β and CD4 expression. The values shown in the panels are the percentage of either CD8β+ or CD4+ IEL expressing GCT alone (upper left) or coexpressing GCT (upper right). The results shown represent one of the three experiments demonstrating similar results.

FIGURE 2.

Induction of GCT on IEL. A, The results show percent expression (bars) and absolute numbers (lines) of GCT+ cells at various time points post-reovirus 1/L infection. Control (□, ▴) and infected (▧, •) mice were inoculated i.d. with either saline or reovirus 1/L (3 × 107 PFU), respectively. The values shown are the mean ± SD of three experiments using four mice per time point. ∗ and # represent p < 0.05 compared with the control group. B, Expression of GCT on different subpopulations of IEL on day 7 post-reovirus 1/L infection. The values shown in the panels are the percentage of IEL expressing GCT alone (upper left) or coexpressing GCT and the indicted surface Ag (upper right). The results shown represent one of the three experiments demonstrating similar results. C, Expression of GCT on subpopulations of CD8+ IEL on day 7 post-reovirus 1/L infection. The CD8α+GCT+ cells were gated to exclude all other cells and further analyzed for CD8β and CD4 expression. The values shown in the panels are the percentage of either CD8β+ or CD4+ IEL expressing GCT alone (upper left) or coexpressing GCT (upper right). The results shown represent one of the three experiments demonstrating similar results.

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FIGURE 3.

Induction of GCT on LPL. A, The results show percent expression (bars) and absolute numbers (lines) of GCT+ cells at various time points post-reovirus 1/L infection. Control (□, ▴) and infected (▧, •) mice were inoculated i.d. with either saline or reovirus 1/L (3 × 107 PFU), respectively. The values shown are the mean ± SD of three experiments using four mice per time point. ∗ and # represent p < 0.05 compared with the control group. B, Expression of GCT on subpopulations of LPL on day 7 post-reovirus 1/L infection. The values shown in the panels are the percentage of LPL expressing GCT alone (upper left) or coexpressing GCT and the indicted surface Ag (upper right). The results shown represent one of three experiments demonstrating similar results.

FIGURE 3.

Induction of GCT on LPL. A, The results show percent expression (bars) and absolute numbers (lines) of GCT+ cells at various time points post-reovirus 1/L infection. Control (□, ▴) and infected (▧, •) mice were inoculated i.d. with either saline or reovirus 1/L (3 × 107 PFU), respectively. The values shown are the mean ± SD of three experiments using four mice per time point. ∗ and # represent p < 0.05 compared with the control group. B, Expression of GCT on subpopulations of LPL on day 7 post-reovirus 1/L infection. The values shown in the panels are the percentage of LPL expressing GCT alone (upper left) or coexpressing GCT and the indicted surface Ag (upper right). The results shown represent one of three experiments demonstrating similar results.

Close modal

The expression and absolute number of GCT+ cells in PP of reovirus 1/L-inoculated mice was significantly higher than controls at all time points investigated (Fig. 1,A). GCT was strongly induced on PPL (24.3 ± 1.6% GCT+) following i.d. reovirus 1/L inoculation peaking at day 3 (Fig. 1,A). Although the relative proportions of B220+, CD4+, and CD8+ PPL did not change significantly postinoculation (Table I), on day 3 postinoculation, B220+GCT+ cells were 21.4%, and this population decreased to 2.7% by day 7 after inoculation (Fig. 1,B and Table I). Both GCT+CD8+ and GCT+CD4+ cells were present in the PP following inoculation with reovirus 1/L, and ∼50% of the CD8+ cells coexpressed GCT (Fig. 1,B and Table I).

Table I.

GCT expression on PPL subpopulationsa

Day 3Day 7
Total B220+B220+ GCT+Total CD4+CD4+ GCT+Total CD8+CD8+ GCT+Total B220+B220+ GCT+Total CD4+CD4+ GCT+Total CD8+CD8+ GCT+
Control 75.0 ± 3.60 1.1 ± 0.37 16.6 ± 1.52 0.2 ± 0.20 4.5 ± 0.61 0.9 ± 0.11 72.0 ± 3.60 1.2 ± 0.47 16.6 ± 0.57 0.3 ± 0.20 3.6 ± 0.57 1.1 ± 0.17 
Infected 67.3 ± 3.88 21.7 ± 1.77b 19.6 ± 0.57 1.9 ± 0.20b 6.0 ± 1.00 2.1 ± 0.50b 69.0 ± 1.73 3.2 ± 0.40b 19.6 ± 1.52 2.2 ± 0.26b 6.0 ± 1.00b 3.1 ± 0.65b 
Day 3Day 7
Total B220+B220+ GCT+Total CD4+CD4+ GCT+Total CD8+CD8+ GCT+Total B220+B220+ GCT+Total CD4+CD4+ GCT+Total CD8+CD8+ GCT+
Control 75.0 ± 3.60 1.1 ± 0.37 16.6 ± 1.52 0.2 ± 0.20 4.5 ± 0.61 0.9 ± 0.11 72.0 ± 3.60 1.2 ± 0.47 16.6 ± 0.57 0.3 ± 0.20 3.6 ± 0.57 1.1 ± 0.17 
Infected 67.3 ± 3.88 21.7 ± 1.77b 19.6 ± 0.57 1.9 ± 0.20b 6.0 ± 1.00 2.1 ± 0.50b 69.0 ± 1.73 3.2 ± 0.40b 19.6 ± 1.52 2.2 ± 0.26b 6.0 ± 1.00b 3.1 ± 0.65b 
a

Control and infected mice were inoculated i.d. with either saline or reovirus I/L (3 × 107 PFU), respectively. The results show the percent of PPLs expressing B220, CD4, or CD8 and the percent expression of GCT on these subpopulations. The results shown (from days 3 and 7 post-reovirus I/L infection) are the mean ± SD of three independent experiments using four mice per time point.

b

, p < 0.05 compared with the control group.

GCT expression was also induced in the IEL population following reovirus 1/L inoculation. In contrast to PPL where GCT expression peaked on day 3, both the absolute number and percent expression of GCT in IEL peaked on day 7, with 13.6 ± 1.6% GCT+ lymphocytes compared with 3.6 ± 0.5% in saline-inoculated controls (Fig. 2,A). IEL are typically a complex population, and our detailed analysis of different subpopulations of IEL in BALB/cJ mice after i.d. inoculation with reovirus 1/L demonstrates an increase in the percentage of TCRαβ+, Thy-1+, CD4+, CD8+, and CD4+/CD8+ cells5 (Fig. 2,B and Table II). Therefore, the induction of GCT expression was analyzed on these distinct IEL subpopulations. In control mice, the TCRγδ+ cells do not express GCT but a small percentage (0.8–1.5%) of TCRαβ+, Thy-1+, CD8+, and CD4+ cells express GCT (Fig. 2,B and Table II). However, after reovirus 1/L inoculation, the expression of GCT was induced on TCRαβ+ (7.3%), Thy-1+ (9.8%), and CD8+ (9.0%) cells. Because the CD8+ cells in the epithelial site are either CD4/8+ or CD4+/8+ and are further subdivided by the expression pattern of CD8 (αα homodimer chain of CD8 molecule or αβ heterodimer chain of the CD8 molecule), these CD8+ IEL subpopulations were further investigated for GCT expression (Fig. 2,C). For this, the GCT+CD8α+ cells were gated to exclude all other cells and further analyzed for GCT+CD8β+ and GCT+CD4+ cells. We found that in control mice 75% of the GCT+CD8+ IEL were CD8αβ+ and 38% of the GCT+CD8+ IEL were CD4+ (Fig. 2,C). However, after reovirus 1/L inoculation, the percentage of the GCT+CD8αβ+ cells increased to 88% and the percentage of the GCT+CD4+ IEL decreased to 17% (Fig. 2 C).

Table II.

GCT expression on IEL subpopulationsa

Total TCRγδ+TCRγδ+ GCT+Total TCRαβ+TCRαβ+ GCT+Total Thy-1+Thy-1+ GCT+Total CD8+CD8+ GCT+Total CD4+CD4+ GCT+
Control 55.6 ± 3.27 0.2 ± 0.08 18.4 ± 1.07 1.1 ± 0.3 15.3 ± 2.03 0.5 ± 0.33 62.2 ± 3.68 1.83 ± 0.55 4.5 ± 0.63 0.4 ± 0.18 
Infected 45.1 ± 1.2b 0.4 ± 0.34 30.6 ± 4.04b 9.1 ± 1.35b 28.4 ± 2.92b 10.4 ± 1.15b 68.3 ± 2.97 9.2 ± 1.3b 8.8 ± 1.96 1.13 ± 0.51 
Total TCRγδ+TCRγδ+ GCT+Total TCRαβ+TCRαβ+ GCT+Total Thy-1+Thy-1+ GCT+Total CD8+CD8+ GCT+Total CD4+CD4+ GCT+
Control 55.6 ± 3.27 0.2 ± 0.08 18.4 ± 1.07 1.1 ± 0.3 15.3 ± 2.03 0.5 ± 0.33 62.2 ± 3.68 1.83 ± 0.55 4.5 ± 0.63 0.4 ± 0.18 
Infected 45.1 ± 1.2b 0.4 ± 0.34 30.6 ± 4.04b 9.1 ± 1.35b 28.4 ± 2.92b 10.4 ± 1.15b 68.3 ± 2.97 9.2 ± 1.3b 8.8 ± 1.96 1.13 ± 0.51 
a

Control and infected mice were inoculated i.d. with either saline or reovirus I/L (3 × 107 PFU), respectively. The results show the percent of IELs expressing TCRγδ, TCRαβ, Thy-1, CD8, or CD4 and the percent expression of GCT on these subpopulations. The results shown (from day 7 post-reovirus I/L infection) are the mean ± SD of three independent experiments using four mice per time point.

b

, p < 0.05 compared with the control group.

Like the IEL, a small proportion of the LPL in control mice also expressed GCT (4.86 ± 0.3%) (Fig. 3,A). The percentage and absolute number of GCT+ LPL also increased significantly on days 3 (8.36 ± 1.5%), 7 (10.27 ± 0.9%), and 10 (8.8 ± 1.1%) post-reovirus 1/L inoculation (Fig. 3,A). In control mice, a small proportion of B220+ (1.3%), CD4+ (2.0%), and CD8+ (0.5%) LPL express GCT (Fig. 3,B and Table III). However, after reovirus 1/L inoculation the percentage of GCT+B220+ (3.1%) and GCT+CD8+ (6.9%) LPL increased significantly (Fig. 3,B and Table III). Thus, GCT expression identifies unique subpopulations within all compartments of the gut-mucosal immune system after the i.d. inoculation of reovirus 1/L.

Table III.

GCT expression on LPL subpopulationsa

Total B220+B220+GCT+Total CD4+CD4+GCT+Total CD8+CD8+GCT+
Control 30.3 ± 3.15 1.0 ± 0.25 23.6 ± 2.5 1.3 ± 0.65 4.5 ± 1.04 1.4 ± 0.40 
Infected 31.1 ± 4.54 2.4 ± 0.65b 31.0 ± 8.18 1.5 ± 0.5 9.1 ± 1.87b 6.9 ± 1.90b 
Total B220+B220+GCT+Total CD4+CD4+GCT+Total CD8+CD8+GCT+
Control 30.3 ± 3.15 1.0 ± 0.25 23.6 ± 2.5 1.3 ± 0.65 4.5 ± 1.04 1.4 ± 0.40 
Infected 31.1 ± 4.54 2.4 ± 0.65b 31.0 ± 8.18 1.5 ± 0.5 9.1 ± 1.87b 6.9 ± 1.90b 
a

Control and infected mice were inoculated i.d. with either saline or reovirus I/L (3 × 107 PFU), respectively. The results show the percent of LPLs expressing B220, CD4, or CD8 and the percent expression of GCT on these subpopulations. The results shown (from day 7 post-reovirus I/L infection) are the mean ± SD of three independent experiments using four mice per time point.

b

, p < 0.05 compared with the control group.

CD11c has also been shown to be an indicator of ongoing Ag-specific T cell activation in the intestinal epithelium (3). Therefore, we investigated the induction of CD11c as an activation marker on IEL along with LPL and PPL after i.d. inoculation with reovirus 1/L. As shown in Fig. 4,A, 15–20% of IEL isolated from control mice expressed CD11c and this percentage as well as the absolute number increased significantly to 41.7 ± 3.5% and 43 ± 4% on days 7 and 10, respectively, following reovirus 1/L inoculation. In control mice, CD11c was found on all IEL subpopulations investigated; αEβ7+ (18%), TCRγδ+ (12%), TCRαβ+ (2%), and Thy-1+ (5%) (Fig. 4,B and Table IV). After reovirus 1/L inoculation, there was a significant induction of CD11c on the homing receptor αEβ7+ (32%), TCRαβ+ (19%), and Thy-1+ (21%) populations, but not on the TCRγδ+ population (Fig. 4,B and Table IV). Because GCT and CD11c expression may be indicators of ongoing Ag-specific activation in the gut-mucosal lymphoid compartments, we investigated the coexpression of GCT and CD11c on IEL, PPL, LPL, and splenic lymphocytes following i.d. reovirus 1/L inoculation. Unlike the IEL population in control mice (17.7%), a very small percentage of the LPL (4.8%) and PPL (4.1%) expressed CD11c (Fig. 4,C). However, following reovirus 1/L inoculation, the percentage of CD11c+ lymphocytes increased significantly to a peak of 7.96% in the LPL on day 7, and to 26.5% in the PP on day 3 (Fig. 4,C). Interestingly, the entire GCT+ population of IEL (10.0%), LPL (7.9%), and PPL (19.7%) are CD11c+. However, in contrast, splenic lymphocytes do not express CD11c after either i.d. (Fig. 4,C) or i.p. (data not shown) inoculation with reovirus 1/L, even though there is a marked up-regulation of GCT expression in splenic lymphocytes after reovirus 1/L inoculation (16% in control vs 30% in reovirus 1/L inoculation) (Fig. 4 C).

FIGURE 4.

Induction of CD11c on IEL. A, The results show percent expression (bars) and absolute numbers (lines) of CD11c+ cells at various time points post-reovirus 1/L infection. Control (□, ▴) and infected (▧, •) mice were inoculated i.d. with either saline or reovirus 1/L (3 × 107 PFU), respectively. The values shown are the mean ± SD of three experiments using four mice per time point. ∗ and # represent p < 0.05 compared with the control group. B, Expression of CD11c on different subpopulations of IEL on day 7 post-reovirus 1/L inoculation. The values shown in the panels are the percentage of IEL expressing CD11c alone (upper left) or coexpressing CD11c and the indicated cell surface Ag (upper right). C, Coexpression of CD11c and GCT on IEL, LPL, PPL, and splenic lymphocytes (SPL). The values shown in the panels are the percentage of cells expressing GCT alone (upper left), CD11c alone (lower right), or coexpressing CD11c and GCT. The results shown are from day 7 (IEL, LPL, and SPL) or day 3 (PPL) post-reovirus 1/L infection when maximum expression of GCT and CD11c is observed. The results shown represent one of three experiments demonstrating similar results.

FIGURE 4.

Induction of CD11c on IEL. A, The results show percent expression (bars) and absolute numbers (lines) of CD11c+ cells at various time points post-reovirus 1/L infection. Control (□, ▴) and infected (▧, •) mice were inoculated i.d. with either saline or reovirus 1/L (3 × 107 PFU), respectively. The values shown are the mean ± SD of three experiments using four mice per time point. ∗ and # represent p < 0.05 compared with the control group. B, Expression of CD11c on different subpopulations of IEL on day 7 post-reovirus 1/L inoculation. The values shown in the panels are the percentage of IEL expressing CD11c alone (upper left) or coexpressing CD11c and the indicated cell surface Ag (upper right). C, Coexpression of CD11c and GCT on IEL, LPL, PPL, and splenic lymphocytes (SPL). The values shown in the panels are the percentage of cells expressing GCT alone (upper left), CD11c alone (lower right), or coexpressing CD11c and GCT. The results shown are from day 7 (IEL, LPL, and SPL) or day 3 (PPL) post-reovirus 1/L infection when maximum expression of GCT and CD11c is observed. The results shown represent one of three experiments demonstrating similar results.

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Table IV.

CD11c expression on IEL subpopulationsa

Total αEβ7+αEβ7+ CD11c+Total TCRγδ+TCRγδ+ CD11c+Total TCRαβ+TCRαβ+ CD11c+Total Thy-1+Thy-1+ CD11c+
Control 78.7 ± 3.89 15.1 ± 2.57 55.6 ± 3.27 10.9 ± 0.64 18.4 ± 1.07 2.1 ± 0.12 15.3 ± 2.03 5.1 ± 0.66 
Infected 77.5 ± 5.37 30.7 ± 2.47b 45.1 ± 3.99b 13.9 ± 1.96 30.6 ± 4.04b 14.3 ± 2.17b 28.4 ± 2.92b 11.2 ± 1.16b 
Total αEβ7+αEβ7+ CD11c+Total TCRγδ+TCRγδ+ CD11c+Total TCRαβ+TCRαβ+ CD11c+Total Thy-1+Thy-1+ CD11c+
Control 78.7 ± 3.89 15.1 ± 2.57 55.6 ± 3.27 10.9 ± 0.64 18.4 ± 1.07 2.1 ± 0.12 15.3 ± 2.03 5.1 ± 0.66 
Infected 77.5 ± 5.37 30.7 ± 2.47b 45.1 ± 3.99b 13.9 ± 1.96 30.6 ± 4.04b 14.3 ± 2.17b 28.4 ± 2.92b 11.2 ± 1.16b 
a

Control and infected mice were inoculated i.d. with either saline or reovirus I/L (3 × 107 PFU), respectively. The results show the percent of IEL expressing αEβ7, TCRγδ, TCRαβ, or Thy-1 and the percent expression of CD11c on these subpopulations. The results shown (from day 7 post-reovirus I/L infection) are the mean ± SD of three independent experiments using four mice per time point.

b

, p < 0.05 compared with the control group.

Because we have previously demonstrated that PPL, LPL, and IEL are capable of producing CTL activity following reovirus 1/L inoculation (8, 9), we next investigated the possible involvement of FasL, perforin, and TRAIL in inducing the death of virus-infected target cells. Accordingly, the induction of specific mRNAs was assayed in PPL, IEL, and LPL by RT-PCR. Fig. 5 demonstrates that at 10 days postinoculation with reovirus 1/L, mRNA expression for FasL and perforin is evident in all three populations studied (PPL, IEL, LPL). The ratios of perforin/β-actin or FasL/β-actin mRNA were significantly higher (p < 0.05) in infected IEL and LPL populations when compared with the respective controls (Fig. 5,B). However, in the case of PPL, while there was a slight increase in the perforin mRNA expression in the infected PPL population, this increase was not significant (p > 0.05). In contrast, mRNA expression for TRAIL as well as the ratio of TRAIL/β-actin mRNA were significantly increased only in the LPL population (Fig. 5).

FIGURE 5.

Induction of perforin, FasL, and TRAIL mRNA in reovirus 1/L-infected mice. A, PPL, IEL, and LPL were harvested on day 10 post-reovirus 1/L infection and RNA was analyzed for perforin, FasL, and TRAIL transcripts by RT-PCR. Lane 1, control PPL; lane 2, infected PPL; lane 3, control IEL; lane 4, infected IEL; lane 5, control LPL; and lane 6, infected LPL. The results shown represent one of three experiments demonstrating similar results. B, Semiquantitative analysis of perforin, FasL, and TRAIL mRNA expression from control (□) and infected samples (▨). Values are the average ratio of perforin:β-actin, FasL:β-actin, and TRAIL:β-actin mRNA from three independent experiments. ∗, p < 0.05 compared with control group.

FIGURE 5.

Induction of perforin, FasL, and TRAIL mRNA in reovirus 1/L-infected mice. A, PPL, IEL, and LPL were harvested on day 10 post-reovirus 1/L infection and RNA was analyzed for perforin, FasL, and TRAIL transcripts by RT-PCR. Lane 1, control PPL; lane 2, infected PPL; lane 3, control IEL; lane 4, infected IEL; lane 5, control LPL; and lane 6, infected LPL. The results shown represent one of three experiments demonstrating similar results. B, Semiquantitative analysis of perforin, FasL, and TRAIL mRNA expression from control (□) and infected samples (▨). Values are the average ratio of perforin:β-actin, FasL:β-actin, and TRAIL:β-actin mRNA from three independent experiments. ∗, p < 0.05 compared with control group.

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To examine the pathway of cytotoxicity used by various gut-mucosal lymphoid compartments in reovirus 1/L-inoculated mice, CTL assays were performed in the presence of specific inhibitors of either the Fas/FasL (anti-FasL Ab), perforin (CMA), or TRAIL (anti-TRAIL Ab) cytotoxic pathways before culture with virally infected target cells. PPL isolated on day 10 post-reovirus 1/L inoculation showed high cytolytic activity against reovirus 1/L-infected target cells as compared with uninfected target cells (Fig. 6,A). The cytolytic activity significantly decreased in the presence of anti-FasL Ab, CMA, or anti-TRAIL Ab (Fig. 6,A). However, pretreatment with anti-TRAIL Ab treatment was the least effective in decreasing cytolytic activity at E:T ratios of less than or equal to 5:1 (Fig. 6,A). Similar to the PPL population, the IEL and LPL populations isolated on day 10 post-reovirus 1/L inoculation also showed high cytolytic activity against reovirus 1/L-infected target cells as compared with uninfected target cells (Fig. 6,B). In the IEL population, the cytolytic activity was decreased to a similar extent in the presence of either anti-FasL Ab, CMA, or anti-TRAIL Ab (Fig. 6,B). In the LPL population, CMA was the least effective in decreasing cytolytic activity of LPL at all E:T ratios. Cytolytic activity was also decreased in the presence of anti-FasL or anti-TRAIL Ab (Fig. 6 C). However, treatment with anti-TRAIL Ab completely inhibited LPL-mediated cytolytic activity at all E:T cell ratios. Thus, our data demonstrate that there is a differential use of various cytolytic pathways by PPL, IEL, and LPL, with the Fas-FasL pathway being strongly used in all populations.

FIGURE 6.

Reovirus 1/L-specific cytotoxicity from various gut-mucosal lymphoid populations. (A) PPL, (B) IEL, and (C) LPL were obtained 10 days postinfection with reovirus 1/L (3 × 107 PFU). After in vitro generation of cytotoxic cells, PPL, IEL, and LPL were incubated with either reovirus 1/L-infected (▧) or uninfected KD2SV target cells. Cytotoxicity was measured after a 4-h incubation. No significant lysis was observed in the presence of uninfected target cells. Where indicated, the effector cells were treated with CMA (▦), anti-FasL (▤), or anti-TRAIL (□) before culture with target cells. The results shown represent one of three experiments demonstrating similar results. The values shown are the mean ± SD of triplicate wells from one representative experiment. ∗, p < 0.05 and # p < 0.01 compared with untreated effector + infected target cells (▧).

FIGURE 6.

Reovirus 1/L-specific cytotoxicity from various gut-mucosal lymphoid populations. (A) PPL, (B) IEL, and (C) LPL were obtained 10 days postinfection with reovirus 1/L (3 × 107 PFU). After in vitro generation of cytotoxic cells, PPL, IEL, and LPL were incubated with either reovirus 1/L-infected (▧) or uninfected KD2SV target cells. Cytotoxicity was measured after a 4-h incubation. No significant lysis was observed in the presence of uninfected target cells. Where indicated, the effector cells were treated with CMA (▦), anti-FasL (▤), or anti-TRAIL (□) before culture with target cells. The results shown represent one of three experiments demonstrating similar results. The values shown are the mean ± SD of triplicate wells from one representative experiment. ∗, p < 0.05 and # p < 0.01 compared with untreated effector + infected target cells (▧).

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Because the Fas/FasL pathway appears to play an important role in the cytolytic mechanism used by reovirus 1/L-specific mucosal effector cells, we next investigated the expression of FasL on activated cells. For these studies, we focused on the GCT+CD11c+ lymphocyte population, because we have demonstrated that this is the phenotype of activated cells obtained from gut-mucosal lymphoid tissues after reovirus 1/L infection. Fig. 7 details FasL expression on the GCT+CD11c+ lymphocytes obtained from PPL, IEL, and LPL on days 3, 7, and 10 post-reovirus 1/L inoculation. Although on day 3 postinoculation, only 0–5% of GCT+CD11c+ cells in any of the populations express FasL, the percentage of GCT+CD11c+ cells expressing FasL increased to between 4 and 14% by day 7. Peak FasL expression occurred on day 10 with 99% of the PPL, 83% of the IEL, and 77% of the LPL GCT+CD11c+ populations expressing FasL (Fig. 7). Clearly, the activated cells (GCT+CD11c+), with their high level of FasL expression, have the potential to play an important role in the clearance of reovirus 1/L infection, through the Fas/FasL-mediated pathway of cytolytic activity.

In this and previous studies (10), we have demonstrated that reovirus 1/L-stimulated gut-mucosal lymphocytes obtained from the PP, intestinal epithelium, and LP express the GCT activation marker. Maximal GCT expression is observed on PPL on day 3 post-reovirus 1/L inoculation, whereas GCT expression is observed later in the time course in other gut mucosal sites (day 7 in LPL and day 10 in IEL). In addition, the entire GCT+ population of IEL, LPL, and PPL was CD11c+ (Fig. 4,C), whereas splenic lymphocytes, which also expressed GCT after reovirus 1/L infection, do not express CD11c (Fig. 4 C). These results suggest that the GCT+CD11c+ IEL and LPL may be derived from the GCT+CD11c+ PPL that migrated to the gut LP and intestinal epithelium. To investigate this hypothesis, we isolated the PPL from BALB/cJ mice 3 days after immunization with reovirus 1/L when PPL showed maximum GCT expression, labeled these cells with CFSE, and transferred them i.v. into recipient BALB/cJ mice. Twenty-four hours later, the recipient mice were inoculated i.d. with either saline or reovirus 1/L. The recipient mice were sacrificed 3 days after donor cell transfer.

Fig. 8,A demonstrates the CSFE labeling of PPL before adoptive transfer. Fig. 8,B demonstrates the location of CSFE+-transferred PPL in the PP, LP, intestinal epithelium, and spleen of either saline-inoculated (control) or reovirus 1/L-inoculated mice. Table V tabulates the fold increase (percent CSFE in reovirus 1/L-inoculated recipients/percent CSFE in saline control recipients) of the localization of CSFE+-transferred donor PPL in the indicated tissues. CFSE-stained donor PPL were detected in all tissues tested. In all cases, higher numbers of CSFE-stained cells were observed in the reovirus 1/L-inoculated recipient mice, with the spleen having the highest fold increase in CSFE-stained cells (7.00), followed by the intestinal epithelium (3.87), the PP (2.17), and the lamina propria (1.75) (Table V). However, the subpopulation of CFSE+ donor PPL which express GCT were preferentially enriched in gut-mucosal tissues as compared with the spleen (intestinal epithelium >30, lamina propria 8.67, PP 4.92 vs 2.00 for the spleen; Table V and Fig. 8,B). A reverse pattern was observed for the CFSE+ donor PPL that did not express GCT. GCTCFSE-stained donor PPL were enriched in the spleen as compared with the gut-mucosal tissues (9.00 for the spleen vs intestinal epithelium 1.80, lamina propria 1.16, PP 1.47; Table V). The fold difference in GCT+CSFE+ lymphocytes as compared with total CSFE+ lymphocytes was greatest in gut-mucosal tissues (2.27 to >7.75) vs the spleen (0.29). Alternatively, the fold difference in GCTCSFE+ lymphocytes as compared with total CSFE+ lymphocytes was greatest in the spleen (1.29) vs gut-mucosal tissues (0.68 to 0.47). These results suggest that the GCT+CFSE+ donor PPL preferentially migrate to effector mucosal sites (gut lamina propria and intestinal epithelium) as opposed to those that migrate to the spleen which are overwhelmingly GCT.

FIGURE 8.

Adoptively transferred PPL migrate to the intestinal mucosa. PPL were isolated from donor mice 3 days post-reovirus 1/L infection. These cells were then stained with CFSE, and transferred (1 × 107/mouse) i.v. into recipient mice. The recipient mice were inoculated i.d. with 3 × 107 PFU of reovirus 1/L (infected group) or with saline (control group) one day after cell transfer. A, Flow cytometric profile of CFSE-labeled PPLs. Thin line shows CFSE-negative cells and bold line shows CFSE-positive donor cells. B, GCT+CFSE-labeled cells in PP, LP, and intraepithelial compartments in uninfected or reovirus 1/L-infected mice 3 days after donor cell transfer. The values shown in the panels are the percentage of cells stained with CFSE (lower right) or stained with CFSE and expressing GCT (upper right). The results shown represent one of two experiments demonstrating similar results.

FIGURE 8.

Adoptively transferred PPL migrate to the intestinal mucosa. PPL were isolated from donor mice 3 days post-reovirus 1/L infection. These cells were then stained with CFSE, and transferred (1 × 107/mouse) i.v. into recipient mice. The recipient mice were inoculated i.d. with 3 × 107 PFU of reovirus 1/L (infected group) or with saline (control group) one day after cell transfer. A, Flow cytometric profile of CFSE-labeled PPLs. Thin line shows CFSE-negative cells and bold line shows CFSE-positive donor cells. B, GCT+CFSE-labeled cells in PP, LP, and intraepithelial compartments in uninfected or reovirus 1/L-infected mice 3 days after donor cell transfer. The values shown in the panels are the percentage of cells stained with CFSE (lower right) or stained with CFSE and expressing GCT (upper right). The results shown represent one of two experiments demonstrating similar results.

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Table V.

Migration of CSFE-labeled PPL

TissueTotal CFSEaGCT+CFSE+GCTCFSE+
PPL 2.17 4.92b 2.27c 1.47d 0.68e 
LPL 1.75 8.67b 4.95c 1.16d 0.66e 
IEL 3.87 >30b >7.75c 1.80d 0.47e 
Spleen 7.00 2.00b 0.29c 9.00d 1.29e 
TissueTotal CFSEaGCT+CFSE+GCTCFSE+
PPL 2.17 4.92b 2.27c 1.47d 0.68e 
LPL 1.75 8.67b 4.95c 1.16d 0.66e 
IEL 3.87 >30b >7.75c 1.80d 0.47e 
Spleen 7.00 2.00b 0.29c 9.00d 1.29e 
a

Ratio of CSFE+ lymphocytes in reovirus 1/L-inoculated recipient mice/CSFE+ lymphocytes in saline-inoculated control recipient mice.

b

Ratio of GCT+CSFE+ lymphocytes in reovirus 1/L-inoculated recipient mice/GCT+CSFE+ lymphocytes in saline-inoculated control recipient mice.

c

Fold difference GCT+CSFE+ lymphocytes as compared to total CSFE+ lymphocytes (ratio b/ratio a).

d

Ratio of GCTCSFE+ lymphocytes in reovirus 1/L-inoculated recipient mice/GCTCSFE+ lymphocytes in saline-inoculated control recipient mice.

e

Fold difference GCTCSFE+ lymphocytes as compared to total CSFE+ lymphocytes (ratio d/ratio a).

The gut-mucosal immune system exists in a chronically activated state in non-germfree, conventional animals. Because of the background of activated lymphocytes in the various gut-mucosal populations (PP, LP, and gut epithelium), it is difficult to dissect newly activated populations that are responding to new antigenic stimulation. In this paper, we have used gut-mucosal infection with reovirus 1/L to dissect recently stimulated lymphocyte populations after acute antigenic stimulation. We have previously reported that GCT is a marker, which identifies reovirus 1/L-specific pCTL and eCTL in PP (10). Additionally, we have also reported that IEL from mice i.d. inoculated with reovirus 1/L and cultured in vitro in the presence of reovirus 1/L-pulsed APCs generated reovirus 1/L-specific MHC-restricted CTL (9). Whether the IEL compartment and LP contain a population of GCT+ lymphocytes, which function as pCTL and eCTL remains to be determined. Therefore, the present study was undertaken to investigate the activation status of IEL, LPL, and PPL in reovirus 1/L-inoculated BALB/cJ mice with regards to the expression of GCT. In addition, CD11c has been demonstrated to be an activation marker that is chronically expressed in the lymphocyte population of the intestinal epithelium (3). Therefore, we hypothesized that the expression of the GCT and CD11c activation markers may provide a means to identify recently stimulated cells in gut-mucosal tissue and may be used to delineate the developmental relationship between PPLs, IELs, and LPL. In this report, we demonstrate a unique subpopulation of activated, CD11c+ GCT+ gut-mucosal lymphocytes in the PP, intestinal epithelium, and LP which originates in the PP and populates the gut-epithelium and LP after acute gut-mucosal reovirus 1/L stimulation.

In the mouse, CD11c has previously been demonstrated to be expressed in vivo only by splenic dendritic cells (21). However, the expression of CD11c on IEL, following graft-vs-host disease, a hallmark of T cell activation in vivo, and an indicator of ongoing Ag-specific T cell activation has also been shown (3). Our results demonstrate for the first time the expression of CD11c on PPL and LPL following reovirus 1/L infection, and the entire GCT+ lymphocyte population in the PP, the intraepithelial compartment, and the LP was CD11c+. However, CD11c+ lymphocytes, particularly in the intraepithelial compartment where the cells remain in continuous contact with gut flora, are not all GCT+. In addition, this GCT+CD11c+ population is not observed in the spleen of conventional or reovirus 1/L-inoculated mice. Therefore, we hypothesize that the GCT+CD11c+ populations in PPL, IEL and LPL, but not the GCTCD11c+ population, contain the recently activated Ag-specific population of lymphocytes.

Previous studies have shown that the TCRαβ+ population of IEL differentiates in the thymus and migrates after antigenic stimulation from the PPs to the gut epithelium via a hemolymphatic circuit (22, 23). Because a developmental relationship between PPL and IEL has been previously demonstrated (6), and our results demonstrated maximum GCT expression in PP before maximal expression in the IEL and LPL, we hypothesized that GCT+CD11c+ lymphocytes appearing in the intestinal epithelium and LP originate from the GCT+CD11c+ PPL. Our data clearly demonstrated the enrichment of GCT+ CSFE-labeled PPL in gut-mucosal tissues as compared with the GCT CSFE-labeled population. Because the GCT+ PPL have been shown to be the activated population which expresses the CD11c activation marker, these results support the conclusion that activated lymphocytes which originate in the PP in response to reovirus 1/L infection preferentially migrate to effector mucosal sites like the LP and intraepithelial compartments. These results are in contrast to those recently reported that activated CD8 T cells migrated pervasively to a number of lymphoid and nonlymphoid tissues regardless of the site of activation (24). The difference with our study is that we looked at the migration of lymphocytes in the inductive compartment (PP) of the gut-mucosal immune system and then compared the migration pattern of specific subpopulations defined by GCT expression. Although the ratio of migration of CSFE-labeled PPL was the highest in the spleen, this migration pattern was accounted for by the GCT CSFE population as compared with the GCT+ CSFE population which was greatly enriched in the gut, with the highest migration to the LP compartment.

Although the CD8+ IELs contain complex subpopulations such as CD8αα+, CD8αβ+, TCRγδ+, TCRαβ+, Thy-1+ and Thy-1, we demonstrated that the GCT Ag was induced only on TCRαβ+Thy-1+CD8αβ+ IELs. This is the same phenotype of the CD8+ lymphocytes found in PP and LP. However, because CD8αβ+ cells are present in spleen and are also typically TCRαβ+ Thy-1+, it is theoretically possible that the GCT+ TCRαβ+ Thy-1+ CD8αβ+ cells present in the intraepithelial compartment or LP may be spleen cells which have migrated to these sites. However, this is not likely, considering that while there was no expression of CD11c on splenic lymphocytes after i.d. or i.p. inoculation with reovirus 1/L, the entire GCT+ population in PP, the LP, and the intraepithelial compartment was CD11c+. Considering this phenotypic evidence, it is possible that GCT+CD11c+ lymphocytes from the PP, which may be considered as recently primed cells after i.d. inoculation with reovirus 1/L, migrate to LP and intraepithelial compartments to act there as effector cells. This is supported by a study demonstrating that severe combined immunodeficient mice infected orally with reovirus and given GCT+CD8+ lymphocytes clear the virus, whereas infected recipients that received naive lymphocytes or B cells do not clear the virus (25). Therefore, it is clear that GCT expression on mucosal lymphocytes indicates activated T lymphocytes which may play an important role in the clearance of viral infections. Like CD11b, which is associated with activated CD8+ T cells that preferentially recirculate to sites of inflammation in vivo (26), CD11c may also be involved in the homing of activated cells from the PP to the LP and the intraepithelial compartment.

We have previously demonstrated reovirus 1/L-specific CTLs in the PP and gut epithelium after reovirus 1/L inoculation (8, 9). In this report, we demonstrate for the first time reovirus 1/L-specific CTL activity in the LP. Two major pathways for cell-mediated cytotoxicity have been described. One pathway is mediated by exocytosis of perforin and granzymes. This pathway is Ca2+-dependent and is important in the clearance of virus-infected cells (16). A second pathway is Ca2+-independent and involves the interaction between FasL expression on the effector cell and Fas expression on the target cell (16, 27). An additional pathway for cell-mediated cytotoxicity is apoptosis induction mediated by TRAIL. These pathways can be involved in antitumor and antiviral activity (27). In this report, we demonstrated reovirus 1/L-specific CTL activity from all three gut-mucosal populations studied (PPL, IEL, and LPL). These results are consistent with previous studies which demonstrated that intraepithelial T cells are cytotoxic (28, 29) and that LPL are also cytotoxic, although to a lesser degree (29, 30). We further demonstrated the involvement of perforin, Fas/FasL, and the TRAIL pathways for PPL, IEL, and LPL cytotoxicity. In the case of PPL, the perforin and Fas/FasL pathways were predominant because the cytotoxic activity was completely inhibited at E:T ratios of 5 or lower (p < 0.01), whereas TRAIL was less effective especially at lower E:T ratios. In IEL, all three pathways were equally as effective, whereas for LPL, the Fas/FasL and TRAIL pathways were predominant. TRAIL-induced apoptosis appears to be most prominent in the case of LPL because it completely inhibited cytotoxicity at all E:T ratios analyzed. In support of this observation, TRAIL mRNA was significantly induced only in the LPL population. To our knowledge, this is the first finding of a functional role for TRAIL-mediated cytotoxicity in gut-mucosal populations. This finding is consistent with a previously published report demonstrating that human IEL and LPL express TRAIL mRNA, although the functional significance of TRAIL expression was not determined (31). However, the expression of TRAIL in both normal and diseased intestinal mucosal is an area of active interest (32). Because the Fas/FasL pathway was common to all gut mucosal populations studied, we analyzed these populations for FasL expression. We found increased FasL mRNA expression on all the three populations. These results are consistent with earlier studies in humans and mice, which demonstrated that IEL and LPL populations used the Fas/FasL pathway (30, 31). However, we further demonstrated by flow cytometry that FasL is induced on the newly activated GCT+CD11c+ lymphocytes. The duration of the increase in FasL expression on GCT+ cells in our study correlates with the known time course of clearance of reovirus from the intestine (33) suggesting an important role for these cells in viral clearance.

Because the gut-mucosal immune system exists in a chronically activated state in non-germfree, conventional animals, it is difficult to gain an understanding of the cellular and molecular interactions, which occur within the gut, that result in protective mucosal immunity. Further, because of the background of activated lymphocytes in the various gut-mucosal populations (PP, LP, and gut epithelium), it is difficult to dissect newly activated populations that are responding to new antigenic stimulation. In this report, we demonstrated that the GCT and CD11c surface Ag markers are expressed by recently activated PPLs as well as by lymphocytes in gut-mucosal tissues including the intraepithelial compartment and LP. This is in contrast to lymphocytes obtained from the spleen after i.d. reovirus 1/L inoculation where the GCT-expressing population did not coexpress the CD11c marker. Therefore, this unique population (GCT+, CD11c+) identifies recently stimulated lymphocyte populations in the gut-mucosal immune system but not in systemic sites such as the spleen. In addition, we further demonstrated that this population first appears in the PP, and subsequently, preferentially migrates to the intraepithelial compartment and LP following enteric virus infection suggesting that lymphocytes activated in PP migrate to and populate the intraepithelial compartment and lamina propria. Thus, GCT and CD11c can be used as activation markers for gut lymphocytes and CD11c can also be used to differentiate between activated gut and activated systemic lymphocytes. These results, coupled with our increased understanding of the cytolytic mechanisms used in the various gut-mucosal lymphoid compartments, will allow further detailed analysis of Ag-specific immunity to viral infections in the context of mucosal vs systemic immunity.

The authors have no financial conflict of interest.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

This work was supported by National Institutes of Health/National Institute of Dental and Craniofacial Research Grant Number 5R01 DE12781 (to S.D.L.).

4

Abbreviations used in this paper: PP, Peyer’s patch; LP, lamina propria; IEL, intraepithelial lymphocyte; LPL, LP lymphocyte; PPL, PP lymphocyte; i.d., intraduodenal; reovirus 1/L, reovirus serotype 1/strain Lang; pCTL, precursor CTL; eCTL, effector CTL; GC, germinal center; GCT, GC and T cell Ag; FasL, Fas ligand; CMF, Ca2+, Mg2+-free HBSS; PEC, peritoneal exudate stimulator cell; MOI, multiplicity of infection; LDH, lactate dehydrogenase; CMA, Concanamycin A.

5

M. S. Bharhani, J. S. Grewal, R. Peppler, C. Enockson, L. London, and S. D. London. Comprehensive phenotypic analysis of the gut intraepithelial lymphocyte compartment: perturbations induced by acute reovirus 1/L infection of the gastrointestinal tract. Submitted for publication.

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