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The Journal of Immunology, 2002, 168: 3833-3838.
Copyright © 2002 by The American Association of Immunologists

The State of CD4+ T Cell Activation Is a Major Factor for Determining the Kinetics and Location of T Cell Responses to Oral Antigen1

Hae-ock Lee2,3, Cristine J. Cooper2,4, Jung-hee Choi, Ziad Alnadjim and Terrence A. Barrett5

Departments of Medicine and Microbiology-Immunology, Northwestern University, Chicago, IL 60611


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Current models suggest that inductive immune responses to enteric Ag are initiated in Peyer’s patches (PP) and mesenteric lymph nodes (MLN) followed by migration of activated, memory-like CD4+ T cells to extralymphoid sites in the intestinal lamina propria (LP). The resultant immune system contains both naive and activated T cells. To examine the differential responses of naive and memory-like T cells to oral Ag, bone marrow chimeras (BMC) were generated. Irradiated BALB/c hosts were reconstituted with a mix of DO11.10 x RAG-1-/- and BALB/c bone marrow. In unprimed DO11.10 and BMC models, LP and PP DO11.10 T cells responded to oral Ag with similar kinetics. Responses of activated, memory-like T cells to oral Ag were examined in thymectomized BMC 60 days after i.p. immunization with OVA peptide in Freund’s adjuvant (OVA323–339/CFA). Results indicate that i.p. OVA323–339/CFA generated a high proportion of memory-like CD45RBlow DO11.10 T cells in peripheral lymphoid (40%) and intestinal LP (70%) tissue. Previously activated DO11.10 T cells in the LP responded to oral Ag earlier and at 50% higher levels compared with memory CD4+ T cells localized to PP tissue. These data indicate that responses to oral Ag in antigenically naive animals are initiated in PP whereas in Ag-experienced animals LP T cells respond earlier and more vigorously than cells in PP. Taken together, these data suggest that previous activation alters the hierarchy of T cell responses to oral Ag by enhancing the efficiency of LP T cell activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A significant proportion of foreign Ag is encountered by the host immune system through the intestinal mucosa. Peyer’s patches (PP)6 are considered to be the primary site for entry and processing of oral Ag. Several lines of investigation suggest that luminal Ag is transferred across specialized epithelial cells, called M cells overlying PP (1), and passed to APC in the subepithelial dome, germinal center, and interfollicular regions (2, 3, 4, 5). However, PP and M cells do not appear to be required for oral Ag uptake, and M cells or epithelial cells are unlikely to directly present MHC class II-restricted Ags themselves (6, 7). Ag absorbed through the intestine is also delivered to T cells at sites outside the PP. Oral Ag can be detected in the serum within 1 h of ingestion (8, 9, 10), and circulating oral Ag activates T cells in the mesenteric lymph nodes (MLN) and spleen (11). Intestinal Ag trapped by migrating DC or other bone marrow-derived APC also may present enteric Ag in the draining MLN (7, 12). In comparison with Ag entry through PP, the pathway of Ag delivery to the lamina propria (LP) is less well characterized. There is considerable evidence that intestinal epithelial cells may present Ag directly to LP T cells beneath them (13); however, the kinetics for that presentation may be delayed compared with more traditional Ag-presenting populations (e.g., macrophages). It has been suggested that oral protein is digested by intestinal enzymes, yielding peptide Ag that may permeate through the epithelium and be presented by APC in the LP (14). Whether oral Ag is primarily delivered to T cells in intestinal LP via the systemic circulation or by direct absorption across the epithelial barrier is unclear.

The activation status of the host affects how oral Ag impacts systemic immune responses. In naive animals, oral Ag induces systemic tolerance; whereas in immunized animals, Ag feeding may enhance effector responses (15, 16, 17, 18, 19). Studies in naive animals indicate that Ag feeding induces systemic tolerance through induction of clonal anergy or active suppression depending on the Ag dose (20, 21, 22). In studies that have examined the effects of oral Ag in Ag-experienced animals, oral Ag generally failed to suppress immune responses. For example, Chung et al. (23) found that memory lymphocytes were refractory to oral tolerance, and studies by Peng et al. (17) indicated that in previously immunized mice, medium or low dose Ag feeding increased serum Ab levels. These findings are consistent with data in animal models of autoimmune disease where Ag feeding has exacerbated tissue destruction (24, 25). Taken together, these data suggest that the activation state of T cells affects the impact oral Ag has on systemic host immune responses.

Distinct combinations of naive and activated T cells populate tissue compartments of the gut-associated lymphoid tissue (26). CD4+ and CD8+ T cells in the intestinal LP are predominantly activated, memory-like effector cells (27, 28, 29, 30, 31, 32). We have previously found that Ag-experienced CD4+ T cells in the LP respond to lower levels of systemic Ag compared with naive cells in peripheral lymphoid tissue (33). PP and MLN CD4+ T cells are a mixture of naive and activated populations and have well-studied responses to oral Ag. Another possibility is that prior exposure to an Ag may alter the activation threshold of intestinal T cells as well as the relative contribution of transepithelial routes of Ag absorption. Studies from Berin et al. (34) and Yang et al. (35) suggest that previous sensitization may accelerate transepithelial transport of enteric Ag. We hypothesized that Ag experience and anatomical localization affects both the kinetics and intensity of T cell responses to oral Ag. This concept might alter notions of how oral Ag is presented to mucosal T cells. In naive mice, several studies suggest that PP and MLN are the predominant sites of T cell activation by oral Ag. We suspected that in immunized mice, responses to oral Ag may occur earlier and at higher levels in the LP compared with PP. To test this hypothesis, we used the DO11.10 Ag-specific TCR-transgenic mouse model (36). Mixed bone marrow chimeric mice (BMC) were generated by combining marrow from DO11.10 x RAG-1-/- and BALB/c mice. Resultant mice were populated by naive OVA-specific T cells throughout peripheral lymphoid tissues with relatively small but detectable numbers of cells in the intestinal LP. To examine responses to oral Ag in previously immunized mice, BMCs were thymectomized and sensitized with i.p.OVA323–339 peptide in CFA. In vivo responses to oral OVA were measured by assessing 5-bromo-2'-deoxyuridine (BrdU) uptake in CD4+ DO11.10 T cells. The results indicate that naive T cells respond to oral Ag in Peyer’s patches 12 h before MLN and splenic tissues. In previously sensitized BMC, T cell responses to oral Ag were initially detected in the intestinal LP followed by PP, MLN, and splenic tissues. Taken together, the findings suggest that naive T cell responses to oral Ag are initiated in PP tissues whereas responses of previously activated T cells occur in the LP followed by lower responses in the PP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

BALB/c mice were obtained from the National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD). D011.10 TCR-transgenic mice (a gift from Dr. Kenneth Murphy, Washington University, St. Louis, MO) were bred to RAG-1-/- mice on a BALB/c background (a gift from Dr. R. Coffman, DNAX, Palo Alto, CA) in facilities at the Lakeside Veteran’s Administration Medical Sciences Building and maintained under specific pathogen-free conditions. Previously, DO11.10 mice were backcrossed to BALB/c mice for 14 generations and to RAG-1-/- for 7 generations. DO11.10 x RAG-1-/- mice were screened by flow cytometry using transgenic TCR-specific mAb KJ1-26 (a gift from Dr. P. Marrack, National Jewish Medical and Research Center, Denver, CO) and B220 (BD PharMingen, San Diego, CA).

In vivo Ab treatment

MECA-367 (anti-mucosal addressin cell adhesion molecule (MAdCAM)) cell line was obtained through American Type Culture Collection (Manassas, VA). HERMES-1 rat isotype control hybridoma was obtained at the Developmental Studies Hybridoma Bank (University of Iowa, Ames, IA). MECA-367 and HERMES-1 hybridoma were grown in a bioreactor (Heraeus, Osterod, Germany) and Ab purified with the Gammabind plus protein G column (Amersham Pharmacia, Piscataway, NJ). Purified Abs were checked for endotoxin by the Limulus assay (Associates of Cape Cod, Falmouth, MA) and found to be <1 endotoxin U (EU)/ml. MECA 367 (0.2 mg) or control HERMES-1 Ab (0.2 mg) was given 2 h before OVA feeding.

Generation of BMC, thymectomy, and immunization

Bone marrow cells were collected from BALB/c and DO11.10 x RAG-1-/- mice and mixed at a 3:1 ratio. BALB/c mice were sublethally irradiated at 550 rad and injected i.v. with 107 marrow cells within 2 h of irradiation. BMC were kept for 8 wk before manipulations. Some BMC were adult thymectomized by suction as previously described (37).

To generate activated T cells, BMC were immunized i.p. with 150 µg of OVA323–339 (from Dr. D. G. Klapper, University of North Carolina, Chapel Hill, NC) in CFA. Mice were kept for 6–8 wk before use.

Cell isolation

Spleen and MLN were mechanically disassociated and RBC lysed. Cell suspensions were washed and stored in DMEM (Life Technologies, Gaithersburg, MD) with 5% FCS (5% DMEM) on ice until used. Small intestines were removed and flushed with cold PBS to remove fecal contents. Peyer’s patches were excised, mechanically dissociated, and stored in 5% DMEM. Intestines were opened longitudinally, minced into 5- to 10-mm pieces and washed extensively with cold PBS. Mucosal pieces were then digested at 37°C with 5 mM EDTA (Sigma-Aldrich, St. Louis, MO) and 10% newborn calf serum (Life Technologies) in PBS. After digestion with EDTA, mucosal pieces were washed with cold PBS, and the supernatants were discarded. The remaining tissue was then digested in a buffer containing 100 U/ml collagenase (Sigma-Aldrich), 25 mM HEPES (Life Technologies), 7 mM CaCl2 (Sigma-Aldrich), and 20% newborn calf serum (Life Technologies) in DMEM. After each 30-min interval, the cells released were centrifuged, washed, and stored in 10% DMEM on ice; and the mucosal pieces were replaced into the collagenase buffer. LP cells were kept on ice overnight and then diluted in 5% DMEM containing 0.3 mg/ml DTT (Life Technologies), and viable cells were isolated by centrifugation over Nycoprep 1.077 (Accurate Chemical, Westbury, NY). After centrifugation, cells were collected from the interface, washed, and pelleted.

Feeding and BrdU incorporation analysis

Mice were fed 250 mg chicken OVA using a 20-gauge feeding needle (Popper & Sons, New Hyde Park, NY). Twelve hours before sacrifice, mice were given 1 mg BrdU (Sigma-Aldrich) in 200 µl sterile PBS by i.p. injection with additional BrdU provided in the drinking water (1 mg/ml). The dose of Ag feeding was selected that induced consistent levels and kinetics of intestinal T cell BrdU incorporation for mice within experimental groups.

At sacrifice, lymphocytes were prepared as described. Cells were stained with anti-CD4-allophycocyanin (BD PharMingen) and KJ1-26.1-BIO. Biotinylated KJ1-26.1 was visualized with streptavidin-PE (BD PharMingen). Cells were fixed overnight in 1% paraformaldehyde (Polysciences, Warrington, PA) in PBS with 0.2% Tween 20 (Sigma-Aldrich). Samples were incubated with 50 Kunitz U of DNase I (Boehringer Mannheim, Indianapolis, IN) in magnesium-calcium DNase buffer (5 mM MgCl2, 5 mM CaCl2 in 1x PBS) for 1 h at 37°C. After a wash with FACS buffer containing 0.2% Tween 20, 10 µl of FITC-conjugated anti-BrdU (BD Biosciences, San Jose, CA) were added for 45 min at 4°C. Samples were washed with FACS buffer-Tween 20 and analyzed by flow cytometry. Events were collected and analyzed using BD Biosciences FACStation and CellQuest software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
T cell responses to oral Ag in D011.10 mice

The hierarchy of T cell responses to oral Ag was initially studied in unprimed, intact DO11.10 mice. Studies from our laboratory (26) and others (38) suggest that environmental Ags activate transgenic T cells in the intestinal LP and PP of DO11.10 mice through endogenous TCR. Activation of LP-transgenic T cells via endogenous TCR generates populations of effector CD4+ T cells that express surface and functional phenotypes typical of CD4+ LP T cells (27, 28, 29, 39). Expression of Ag-specific TCR by transgenic T cells allows us to examine the effects of antigenic stimulation on mixed populations of naive and activated T cells. To determine the location, degree, and kinetics of T cell activation, BrdU incorporation was assessed in control and OVA-fed mice. As shown in Fig. 1Go, <1% of transgenic T cells were BrdU+ in the spleen and MLN given control protein (swine gamma-globulin) by intragastric administration. Slightly higher BrdU incorporation was detected in the intestine (LP and PP) than in the spleen and MLN of control mice, possibly due to activation of dual TCR-bearing cells by environmental Ags (26). Oral Ag induced BrdU incorporation among transgenic cells 18 h after Ag feeding in both the LP (39% BrdU+) and PP (24% BrdU+) tissues. BrdU incorporation increased from 0.3 to 5.7% of transgenic cells at 24 h in the MLN and reached a maximum of 6% at 30 h in the spleen. Taken together, these studies suggest that oral Ag effectively activates intestinal T cells in both LP and PP compartments at time points before MLN and splenic tissues.



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  		FIGURE 1. Kinetics and location of DO11.10 T cell responses to OVA feeding. DO11.10 mice were fed 250 mg of OVA and pulsed with BrdU at 12-h intervals (Materials and Methods). Time points above the figure specify hours after OVA feeding and the starting point of BrdU pulse. Cells from the spleen (SPL), MLN, PP, and the intestinal LP were isolated and stained with anti-CD4, KJ1-26, and anti-BrdU Ab (Materials and Methods). CD4+ cells were gated and shown for KJ1-26 vs anti-BrdU staining profiles. For controls, swine gamma-globulin was fed instead of OVA. Numbers in the upper right quadrant indicate percent of BrdU+KJ1-26+ cells over total CD4+KJ1-26+ cells. The results shown are representative of those obtained from three independent experiments.

 
To address whether intestinal migration of activated transgenic T cells contributed to BrdU incorporation detected for LP and PP T cells, DO11.10 mice were treated with blocking mAb directed against MAdCAM. MAdCAM binding is required for migration of activated T cell blasts to the intestine (40). We have used anti-MAdCAM mAb to block 70 and 97% of the recruitment of DO11.10 T cells to the PP and LP of adoptive transfer mice sensitized with i.p. OVA323–339/CFA (C. J. Cooper et al., unpublished observations). Results in Fig. 2Go indicate that MAdCAM blockade did not affect the proportions of BrdU+-transgenic T cells detected in the LP or PP 7 days after Ag feeding. These findings suggest that intestinal migration does not significantly contribute to levels of BrdU incorporation detected in the LP and PP after Ag feeding.



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  		FIGURE 2. Intestinal migration does not significantly contribute to initial levels of BrdU incorporation detected in the LP and PP after Ag feeding. DO11.10 mice were fed 250 mg OVA and injected with control or anti-MAdCAM Ab. BrdU was administered during the experimental period. Cells from the spleen, MLN, LP, and PP were isolated 7 days later and stained with anti-CD4, KJ1-26, and anti-BrdU Ab. The results are shown as a means (percent BrdU+KJ1-26+ cells over total CD4+KJ1-26+ cells) ± SD from three experiments. i.g., intragastric.

 
Activation of naive DO11.10 T cells in mixed BMC

We previously reported that in DO11.10 x RAG-1-/- mice, naive T cells expressing transgenic TCR were present in the nonlymphoid LP compartment of the intestine (26). However, the absence of normal lymphoid structures limits the usefulness of DO11.10 x RAG-1-/- mice for examining T cell responses in vivo. To examine naive DO11.10 T cell responses in mice with relatively normal proportions of peripheral B and T cell populations, bone marrow from DO11.10 x RAG-1-/- and BALB/c donors were infused into irradiated BALB/c recipients. Mice were examined 8 wk later to allow full reconstitution of lymphoid and nonlymphoid compartments. Our analysis indicated that abundant numbers of DO11.10 T cells were present in peripheral lymphoid tissues of chimeric mice with low, yet detectable numbers of transgenic T cells in the LP (Fig. 4GoA). An examination of lymphocytes in chimeric mice indicate that >90% DO11.10 T cells from spleen, MLN, PP, and LP express a pattern of activation markers typical of naive cells (CD45RBhighCD69-) (Fig. 3Go).



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  		FIGURE 4. Kinetics and location of transgenic T cell responses to OVA feeding in naive and activated BMC mice. Naive and activated BMC mice were generated as in Fig. 3Go, fed 250 mg of OVA, and then pulsed with BrdU at 12-h intervals. Cells from the spleen, MLN, PP, and LP were isolated and stained with anti-CD4, KJ1-26, and anti-BrdU Ab as in Materials and Methods. A, CD4+ cells were gated and shown for KJ1-26 vs anti-BrdU Ab staining profiles at 24–36 h. Percent of upper left (KJ1-26+) and upper right (BrdU+KJ1-26+) quadrant is shown. B and C, Percent of BrdU+KJ1-26+ cells over total CD4+KJ1-26+ cells are shown in OVA-fed naive (B) and activated (C) BMC mice over a time course. The results shown are representative of those obtained from three independent experiments. FL1-H, Fluorescence.

 


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  		FIGURE 3. Surface phenotypes of transgenic T cells in the naive and activated BMCs. BMC were generated using BALB/c and DO11.10 x RAG-/- mice (Naive BMC). Eight weeks later, a group of mice were thymectomized and immunized with OVA323–339 peptide to generate activated/memory T cells (Activated BMC). After another 8 wk, cells from the spleen, MLN, PP, and LP were isolated and stained with fluorochrome-conjugated anti-CD4, KJ1-26, and anti-CD45RB (A) or anti-CD69 (B). Percentages of CD45RBlow (A) and CD69+ (B) KJ1-26+ T cells are shown. Data are representative of two independent experiments.

 
To examine the effects of oral Ag on naive T cells in the intestine and periphery, BrdU incorporation was assessed at intervals after OVA feeding. Data in Fig. 4GoB indicate that initial responses of naive DO11.10 T cells to oral Ag were detected in PP tissue 24 h after Ag feeding (31% BrdU+ DO11.10 T cells). Interestingly, an increased proportion of BrdU+ DO11.10 T cells were also observed in the LP at 24 h (44% BrdU+ DO11.10 T cells) with peak activation levels detected 36 h after Ag feeding (56% BrdU+ DO11.10 T cells). Nonetheless, the relative impact of oral Ag on naive T cells in this compartment may be minimal given that data in Fig. 4GoA indicate that the overall proportion of DO11.10 T cells in the LP of naive BMCs was relatively low (0.9% of CD4+ T cells). DO11.10 T cell responses to oral Ag were delayed in MLN and splenic tissues compared with intestinal tissues. Levels of BrdU+ DO11.10 T cells increased in the MLN and spleen of naive mice given BrdU 36 h after OVA feeding and peak levels were detected in the MLN in mice given BrdU 48 h after Ag feeding (38% BrdU+ DO11.10 T cells). Thus, the temporal sequence of T cell responses to oral Ag progressed from the intestine to more distant lymphoid sites.

To assess the hierarchy of T cell responses to oral Ag in previously immunized mice, mixed DO11.10 x RAG-1-/-:BALB/c BMCs were examined 60 days after sensitization with i.p. OVA323–339/CFA. In recent studies (C. J. Cooper and T. A. Barrett, unpublished observations), we demonstrated that i.p. OVA323–339/CFA induces migration of activated DO11.10 T cells in adoptive transfer mice. Mice were thymectomized 8 wk after reconstitution and before immunization to prevent recent thymic emigrants from diluting the pool of activated cells. Analysis of the surface phenotype expressed by T cells in sensitized mice (Fig. 3Go) indicated that >40% of DO11.10 cells in PP and MLN and >70% of DO11.10 T cells in LP compartments express low levels of CD45RB. Interestingly, DO11.10 cells in the peripheral lymphoid tissue did not express the early activation marker CD69 whereas >80% of LP cells were CD69+. The relatively high proportion of cells expressing CD69 in immunized mice was in contrast to LP cells in naive mixed BMC where the proportion of CD69+ DO11.10 T cells was <5%. These results indicate that in mice immunized 60 days earlier, DO11.10 T cells in peripheral lymphoid tissues returned to the resting state. However, in the LP, local factors helped to maintain the activated state of Ag-experienced LP T cells. Overall, these data suggest that Ag immunization in mixed BMC generates populations of Ag-experienced DO11.10 T cells localized to peripheral lymphoid and intestinal tissues.

To examine the effect of oral Ag on populations of previously activated cells, BrdU incorporation was assessed in OVA-fed mice 60 days after immunization with i.p. OVA323–339/CFA. Data in Fig. 4GoC indicate that within 12 h of OVA feeding, levels of BrdU incorporation were relatively low in peripheral and intestinal lymphoid (<2% BrdU+ DO11.10 cells) as well as LP (<4% BrdU+ DO11.10 cells) tissues. These levels were similar to those detected in swine gamma-globulin-fed controls (data not shown). The low levels of BrdU incorporation may have been due to bystander mechanisms of cell turnover or indicative of low levels of cross-reactivity to environmental Ags for DO11.10 x RAG-1-/- T cells. The earliest increase in BrdU uptake by DO11.10 T cells in OVA-fed mice was observed in the LP. Seventeen percent of DO11.10 T cells given BrdU 18 h after OVA feeding became BrdU+, whereas 43% of DO11.10 LP T cells incorporated BrdU from 24 to 36 h. Compared with LP responses, levels of BrdU incorporation in DO11.10 T cells in the PP, MLN, and spleen were lower and more delayed. In PP, MLN, and splenic tissues, the peak increase in BrdU incorporation was detected in mice given BrdU from 36 to 48 h (15, 30, and 15% BrdU+ DO11.10 T cells, respectively). Representative data from naive and activated BMC analyzed 24–36 h after OVA feeding are shown in Fig. 4GoA. The data show that OVA feeding induced BrdU incorporation for a greater proportion of DO11.10 T cells in the LP (45% BrdU+ DO11.10 cells) than in PP, MLN, or spleen (8–9% BrdU+ DO11.10 cells) of activated mice. In naive mice, the proportion of BrdU+ DO11.10 T cells is greater in LP than in PP tissue, but the proportion of transgenic of total CD4+ T cells is lower (0.9%) than that in PP (20.2%). Thus, the relatively high proportion of BrdU+ DO11.10 T may not directly correlate with the strength of immune response in vivo due to low precursor frequencies. CD4+ T cells in the LP of humans are normally composed of cells that express an activated memory-like phenotype (30). Thus, these data suggest that initial responses to oral Ag in previously sensitized individuals occur in the LP.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Incorporation of BrdU was used to examine the hierarchy of T cell responses to oral Ag. BrdU labeling has been used to assess both B and T cell responses in vivo (41, 42, 43, 44, 45, 46). In this study, labeling of Ag-specific T cells was assessed after continuous BrdU administration over a 12-h interval. In this approach (47), the fraction of BrdU+ cells detected at a given time point is indicative of 1) the input of cells into the compartment, 2) cell proliferation within the compartment, and 3) the loss of cells due to death and/or emigration from the compartment. Results of anti-MAdCAM mAb treatment herein (Fig. 2Go) suggest that relatively low levels of DO11.10 T cell migration into the intestine occur after Ag feeding. Chen et al. (48) reported that apoptosis of transgenic T cells in the intestine was not induced until 48 h after Ag feeding. In related studies, we found that peak levels of PP and LP T cell apoptosis were induced 48–72 h after Ag feeding without evidence for increased T cell apoptosis in the LP at 24 h when BrdU responses peaked (data not shown). Although it is difficult to assess the rate of cell emigration from the LP compartment, we have not detected cells outside the intestine that express the characteristic CD45RBlowL-selectin-CD44high{alpha}4{beta}7high surface phenotype associated with LP T cells. These results are consistent with other studies that suggest that LP T cells are long-lived residents of the intestine with little recirculation (49). Thus, the extent to which our assessment of T cell responses to Ag feeding was compromised by variables related to cell immigration, cell death, and/or cell emigration seems limited. We therefore propose that levels of BrdU labeling of DO11.10 T cells reported herein accurately reflect the kinetics and distribution of T cell responses induced by oral Ag.

Despite the presence of CD45RBhigh T cells in the Ag-experienced mice, responses occur with delayed kinetics compared with naive cells. Several possibilities may explain these data. First, it is possible that previously activated cells that down-regulated the CD45RB phenotype may have reverted to a CD45RBhigh status. The reversion of memory cells to express naive markers has been reported by Tough and Sprent (41) and others and is reviewed by Dutton et al. (50) and Sprent and Surh (51). The alternative (and not mutually exclusive) explanation is that more naive than memory cells survive after Ag sensitization. Given this possibility, PP cells in activated BMC should behave more like cells in naive mice. In related data, we found that PP responses in intact DO11.10 mice (that contain both naive and memory cells) respond with kinetics similar (early) to that of LP cells. These early responses are reminiscent of the early responses seen in naive BMC. Thus, naive cells can respond to Ag efficiently despite the presence of activated cells. The correlation of data between models suggest that DO11.10 PP T cells in activated BMC are likely a mixture of memory cells, naive (virgin) cells (never activated by Ag), and memory cells masquerading as naive cells as suggested by Sprent et al. (41). A better understanding of the distinct patterns of PP Ag presentation to naive and activated populations of T cells may be needed to understand the distinct kinetics of PP responses in intact DO11.10, naive, and activated BMC.

Our results suggest that oral Ag readily permeates intestinal mucosa and activates T cells in both PP and the LP. BrdU incorporation studies indicate that oral Ag is absorbed directly into subepithelial nonlymphoid LP as well as lymphoid PP tissue. Findings in PP are consistent with reports indicating that dendritic cells (DC), subepithelial dome, and interfollicular regions acquire enteric Ag for presentation to local T cells (3, 4, 52). These studies suggest that PP DC are divided into three subsets: myeloid (CD11b+CD11c+); lymphoid (CD11c+CD8+); and double-negative (CD11b-CD8-). Furthermore, DC subsets localize to distinct regions of the PP where they perform different roles in directing immune responses. The segregation of DC plus Ag to separate PP regions may explain the delayed kinetics of DO11.10 PP T cell responses to oral Ag in previously immunized BMC. Thus, DC migration to sites of Ag presentation may affect the kinetics of PP T cell responses to oral Ag.

Activation of LP T cells at relatively early time points suggest an efficient entry and presentation of Ag in this compartment. Previous studies suggest that oral protein is partially, if not completely, digested before crossing the mucosal lining. Hanson et al. (9) detected immunologically active peptide in serum within hours of intragastric administration. Thus, it is possible that oral protein enters mucosal compartments as peptide fragments that bind class II MHC molecules expressed by epithelial cells as well as LP APC. Studies from several groups indicate that intestinal epithelial cells express class II MHC and function as APC (53, 54, 55, 56). Studies of Ag presentation by enterocytes (56) suggest that the kinetics of Ag digestion and processing may be delayed (~18 h) compared with "professional" APC populations such as macrophages or DC that present peptide Ag within 8 h of protein uptake. However, if Ag crosses into subepithelial spaces as free peptide, it is possible that binding to class II MHC molecules occurs on basal surfaces exposed to underlying LP. Studies from Harper et al. (57) suggest that DC in the LP may present oral Ag to local T cells. These and other data support the notion that oral Ag permeates the epithelial barrier where it is presented to resident LP T cells by local APC (14).

Results indicate that presentation of oral Ag to activated T cells in the LP was highly efficient. In activated BMCs, LP T cell responses to oral Ag preceded PP, MLN, and splenic T cell responses (Fig. 4GoC). We suspect that the hierarchy of T cell responses in previously stimulated mice reflects the biologic importance of presenting Ag to memory-like effector T cells localized to the LP. Memory T cells respond to Ag more rapidly and at a lower threshold than do naive T cells (58). Thus, it is reasonable to postulate that memory-like T cells in the LP are conditioned to respond quickly in the course of mucosal immune responses to enteric Ag. Along with previous activation, our analysis of the surface phenotype expressed by LP T cells in activated BMC suggests that local factors help to maintain the activation state of cells in this compartment. Cells in the LP continued to express CD69 60 days after Ag administration. In contrast, T cells in peripheral lymphoid tissue were CD69-. We have considered at least two explanations for these results. First, it is possible that properties of mucosal APC operate to maintain Ag in the LP at levels that can activate local T cells. This possibility is difficult to address in models of prolonged Ag persistence. Secondly, soluble factors released from local immune cells may affect the activation state and CD69 expression of LP T cells. For example, IL-15 perpetuates activation of CD8+ T cells (59, 60). In eosinophils, IFN-{gamma} enhances CD69 expression (61). A similar IFN-{gamma}-induced molecular pathway may operate in the LP where levels of IFN-{gamma} are relatively high. Lastly, chemokine signaling in CD4+ T cells may enhance and maintain T cell activation in the LP. Studies with stromal cell-derived factor-1, RANTES, macrophage-inflammatory protein-1, macrophage-inflammatory protein-1{beta}, and monocyte-chemotactic protein-1 indicate that chemokines enhance CD25 expression, IL-2 production, and proliferation of anti-CD3-stimulated peripheral T cells (62). In studies by Bacon et al. (63), it was shown that high concentrations of RANTES stimulated T cells without anti-CD3 stimulation. Interestingly, the induction and maintenance of CD69 expression in the LP were detected in immunized but not in naive mice (Fig. 3Go). Thus, these data suggest that previous activation is required for LP T cell expression of CD69 that is maintained by local factors in the mucosal interstitial environment. Further delineation of these factors will help define the unique nature of mucosal T cell responses to oral Ag.


   Acknowledgments
 
We thank Dr. Jerry Trier (Harvard Medical School, Brigham and Women’s Hospital, Boston, MA) for intuitive and insightful discussion during the inception of this paper.


   Footnotes
 
1 This study was supported by grants from the National Institutes of Health (DK47073) and the Crohn’s and Colitis Foundation of America. Back

2 H.L. and C.J.C. contributed equally to this work. Back

3 Current address: Department of Ophthalmology, Washington University School of Medicine, St. Louis, MO 63110. Back

4 Current address: Department of Immunology, University of Washington, Seattle, WA 98195. Back

5 Address correspondence and reprint requests to Dr. Terrence A. Barrett, Department of Medicine, Division of Gastroenterology and Hepatology, Searle Building, Room 10-526, Northwestern University, 303 East Chicago Avenue, Chicago, IL 60611. E-mail address: tabarrett{at}northwestern.edu Back

6 Abbreviations used in this paper: PP, Peyer’s patch; BMC, bone marrow chimera; BrdU, bromodeoxyuridine; DC, dendritic cell; MAdCAM, mucosal addressin cell adhesion molecule; MLN, mesenteric lymph node; LP, lamina propria. Back

Received for publication September 27, 2001. Accepted for publication February 13, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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