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The Journal of Immunology, 2007, 178: 2104-2112.
Copyright © 2007 by The American Association of Immunologists, Inc.

CD18 Is Required for Intestinal T Cell Responses at Multiple Immune Checkpoints1

Marissa Marski, Alice L. Ye and Clara Abraham2

Section of Gastroenterology, Department of Medicine, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The intestinal immune response to oral Ags involves a complex multistep process. The requirements for optimal intestinal T cell responses in this process are unclear. LFA-1 plays a critical role in peripheral T cell trafficking and activation, however, its role in intestinal immune responses has not been precisely defined. To dissect the role of LFA-1 in intestinal immune responses, we used a system that allows for segregation of T cell migration and activation through the adoptive transfer of LFA-1-deficient (CD18–/–) CD4+ T cells from DO11.10 TCR transgenic mice into wild-type BALB/c mice. We find that wild-type mice adoptively transferred with CD18–/– DO11.10 CD4+ T cells demonstrate decreases in the numbers of Ag-specific T cells in the intestinal lamina propria after oral Ag administration. We also find that in addition to its role in trafficking to intestinal secondary lymphoid organs, LFA-1 is required for optimal CD4+ T cell proliferation in vivo upon oral Ag immunization. Furthermore, CD18–/– DO11.10 CD4+ T cells primed in the intestinal secondary lymphoid organs demonstrate defects in up-regulation of the intestinal-specific trafficking molecules, {alpha}4beta7 and CCR9. Interestingly, the defect in trafficking of CD18–/– DO11.10 CD4+ T cells to the intestinal lamina propria persists even under conditions of equivalent activation and intestinal-tropic differentiation, implicating a role for CD18 in the trafficking of activated T cells into intestinal tissues independent of the earlier defects in the intestinal immune response. This argues for a complex role for CD18 in the early priming checkpoints and ultimately in the trafficking of T cells to the intestinal tissues during an intestinal immune response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Infiltration of effector T cells into the intestine is essential for physiological responses to oral Ags, a critical component of immune responses in intestinal infections and intestinal inflammatory diseases. T cell function in the intestine requires passage through specific intestinal immune checkpoints. These include trafficking of naive T cells to intestinal secondary lymphoid organs, such as mesenteric lymph nodes (MLN)3 and Peyer’s patches (PP), T cell proliferation in these organs in response to oral Ag, up-regulation of trafficking molecules, and ultimately trafficking into intestinal tissues. The acquisition of expression of the integrin, {alpha}4beta7, and the chemokine receptor, CCR9, is critical for T cell trafficking from the secondary lymphoid structures into the intestinal tissues (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13). Previous studies have shown that {alpha}4beta7 contributes to rolling and adhesion of lymph node cells within both PP and lamina propria high endothelial venules (14, 15).

In addition to {alpha}4beta7, LFA-1 ({alpha}Lbeta2 or CD11a/CD18) also plays a role in intestinal T cell trafficking. The requirement for multiple integrins in mediating intestinal T cell migration and function reflects the complexity of the intestinal immune response. For example, whereas trafficking to MLN does not require {alpha}4beta7 (3, 5), LFA-1 is critical to MLN migration as well to PP migration (16, 17). In PP migration, {alpha}4beta7 functions as a bridge to LFA-1-dependent adhesion (15). Furthermore, despite the contribution of {alpha}4beta7 to trafficking and accumulation of T cells in specific intestinal lymphoid organs, it has been found not to play a role in T cell activation (18), whereas we hypothesize that LFA-1 plays a crucial role in intestinal T cell activation and differentiation in response to oral Ags. CD18-deficient mice have decreased numbers of T cells in intestinal tissues (19). Moreover, murine colitis is severely attenuated when LFA-1:ICAM interactions are blocked or absent before onset of disease (20, 21). This may, in part, reflect the demonstrated roles of LFA-1 in arrest on PP high endothelial venules (15), and trafficking to PP and MLN (16, 17). However, the intestinal immune checkpoints subsequent to PP and MLN entry that are affected by LFA-1:ICAM blockade are not known.

Thus, although LFA-1 is clearly important to intestinal immune function, its contributions to specific T cell functions in the intestine are not known. For example, functional absence of CD18 (e.g., CD18-deficient mice or blocking Abs) results in blockade of all beta2 integrin family members on many different hemopoietic cell subsets (e.g., NK cells, dendritic cells (DC), macrophages, B cells) that may contribute to altered T cell accumulation in intestinal tissues. In addition, although studies have demonstrated that LFA-1 participates in T cell trafficking to PP and MLN (16, 17), they have not addressed whether LFA-1 on T cells is necessary for T cell activation and acquisition of intestinal tropism in response to intestinal Ags once T cells have entered these intestinal secondary lymphoid structures. Furthermore, it is not known whether LFA-1 on T cells is directly required for subsequent migration to the lamina propria where T cells may exert effector functions. In fact, the role for LFA-1 in entry into PP and MLN may fully account for any role LFA-1 may have in effective accumulation of T cells in intestinal tissues.

LFA-1 is known to play a critical role in trafficking of naive T cells to peripheral secondary lymphoid organs, and in peripheral T cell activation (16, 17, 22, 23, 24, 25, 26, 27, 28, 29, 30). However, the requirement for LFA-1 in T cell responses in the intestinal immune system is largely undefined. The modulatory role of LFA-1 may have distinct features in the intestinal immune system when compared with immune responses that take place in peripheral lymph nodes for several reasons. T cells in the intestinal MLN have a decreased requirement for LFA-1 for initial entry (17), are exposed to higher levels of bacterial Ags, have an overall more activated status in comparison with that of T cells in the peripheral lymph nodes, and are activated by APCs with features distinct from those in the peripheral lymph nodes. As such, the role of LFA-1 in T cell activation and differentiation with the unique DC of the PP and MLN has not been examined. Thus, the role of LFA-1 in immune checkpoints subsequent to PP and MLN entry has not been defined.

Understanding the role for LFA-1 in intestinal immune checkpoints is critical to understanding its contributions to uncontrolled T cell activation and entry into intestinal tissues in diseases such as inflammatory bowel disease. To determine the role of LFA-1 in T cell immune responses in the intestine, we dissected its contribution at specific checkpoints of the immune response in vivo through the adoptive transfer of CD18–/– DO11.10 CD4+ T cells into wild-type (WT) recipients. We found that in addition to the defect in trafficking to the MLN and PP that had been reported previously, CD18–/– DO11.10 CD4+ T cells that have successfully entered those sites have additional defects in proliferation in these lymphoid organs in response to oral Ag and in differentiation to T cells expressing intestinal trafficking molecules. Furthermore, we show that CD18 has a role independent of its effects on T cell activation and intestinal-tropic differentiation in contributing to the optimal trafficking of activated T cells into the intestinal lamina propria. Taken together, these findings highlight the complex and broad involvement of CD18 at multiple levels in intestinal T cell immune responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

CD18–/– mice were generously provided by Dr. A. Beaudet (Baylor College of Medicine, Houston, TX) (29). CD18–/– mice were backcrossed onto the BALB/c background (nine generations) and then to DO11.10 TCR-transgenic mice. BALB/c mice were obtained from The Jackson Laboratory. BALB/c Thy1.1-congenic mice were generously provided by Dr. Alexander Khoruts (University of Minnesota, Minneapolis, MN). T cells were phenotyped through staining of peripheral blood cells with a biotinylated mAb, KJ1-26 (Caltag Laboratories), directed against the TCR clonotype expressed by DO11.10 T cells and by anti-CD18 (BD Pharmingen). Mice were maintained on autoclaved food and irradiated water in a specific pathogen-free facility with filtered air according to National Institutes of Health guidelines.

Abs and staining reagents

The following Abs and secondary reagents used for flow cytometry were purchased from BD Pharmingen and/or eBioscience: PE-, FITC-, biotin-, APC-Cy7-, allophycocyanin-labeled anti-CD4; PE-, biotin-, and FITC-labeled anti-CD18, PE-labeled anti-{alpha}4beta7; PE-Cy7 Thy1.2- and CyChrome-labeled streptavidin. The following Abs and secondary reagents were purchased from Caltag: PE-, PE-Cy5, and biotin-labeled KJ1-26. A goat polyclonal Ab raised to aa 10–37 of murine CCR9 was purchased from Abcam, and PE- and APC-labeled anti-goat secondary reagent were purchased from Jackson ImmunoResearch Laboratories.

Intestinal cell isolation

Lamina propria lymphocytes (LPL) were isolated essentially as described in Ref. 31 . In brief, PP were removed from the small bowel (SB). SBs and colons were cut longitudinally and then into ~1-mm pieces. Colonic or small bowel pieces were washed thoroughly (ice-cold PBS, 5% FCS) and then digested (PBS, 5 mM EDTA, 5% FCS) at 37°C for 1 h in a rotating incubator to remove the epithelial cells. The supernatants containing epithelial cells and intraepithelial lymphocytes were discarded, and intestines were washed twice in ice-cold PBS to remove the residual EDTA. The remaining tissue was then incubated for 2 h at 37°C in a rotating incubator in collagenase buffer consisting of RPMI 1640 supplemented with 10% FCS, 200 U/ml collagenase VIII (Sigma-Aldrich), 2 mM glutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, 10 mM HEPES, 50 µM 2-ME, essential amino acids, 0.5 µg/ml Fungizone, and 50 U/ml penicillin-50 µg/ml streptomycin. The cells were then filtered through a 40-µm pore size filter (Falcon) and washed twice in medium without collagenase.

In vivo T cell activation

Spleen-derived CD4+ T cells were isolated using CD4+ microbeads according to manufacturer’s instructions (Miltenyi Biotec). The efficacy of the CD4+ T cell purification was monitored by flow cytometry and was routinely >93% pure. Freshly isolated CD18+/– or CD18–/– DO11.10 CD4+ T cells were stained with 1.7 µM CFSE (Molecular Probes) and adoptively transferred by i.v. injection (2.5 x 106 of each per mouse) into WT BALB/c mice or WT Thy1.1 BALB/c mice. Twenty-four hours later, mice were immunized by oral gavage with 50 mg of chicken OVA (Sigma-Aldrich). Spleen, MLN, PP, and, where indicated, LPL, were harvested at the indicated days after immunization. Cells were stained with anti-CD4 and KJ1-26, along with Thy1.2 and/or CD18 where indicated, to assess activation (per CFSE dilution) and analyzed on a FACSCalibur or FACSCanto (BD Biosciences). In some experiments, the trafficking molecules {alpha}4beta7 and CCR9 were also assessed.

In vitro T cell proliferation

For in vitro stimulation, 5.0 x 104 freshly isolated spleen-derived CD18+/– or CD18–/– DO11.10 CD4+ T cells were incubated with PP or MLN CD11c-isolated cells (microbeads from Miltenyi) from WT mice injected s.c. with Flt3L-secreting melanoma tumors (generously provided by Dr. Ulrich von Andrian, Harvard Medical School, Boston, MA) (9, 11). T cell activation was conducted at the indicated DC:T cell ratios and with various concentrations of chicken OVA323–339 in a 96-well round-bottom plate. [3H]Thymidine was added to the cultures during the last 18 h of a 72-h assay.

In vitro T cell cultures

Freshly isolated spleen-derived CD18+/– or CD18–/– CD4+ T cells were stimulated with plate-bound anti-CD3 (4 µg/ml) and anti-CD28 (4 µg/ml) in the presence of 10 nM retinoic acid (Sigma-Aldrich). In other experiments, freshly isolated spleen-derived CD18+/– or CD18–/– DO11.10 CD4+ T cells were stimulated in the presence of 2 µg/ml OVA peptide at a 1:1 ratio (unless indicated otherwise) with PP or MLN-derived CD11c+ DC (Miltenyi) from WT mice injected s.c. with Flt3L-secreting melanoma cells. Cells were maintained in DMEM (Invitrogen Life Technologies) supplemented with 10% FCS, 2 mM glutamine, 0.1 mM nonessential amino acids, 20 mM HEPES, 100 U/ml penicillin-100 µg/ml streptomycin, and 50 µM 2-ME. Under both culture conditions, 10 U/ml recombinant human IL-2 was supplemented on day 3 after stimulation.

T cell homing

In vitro generated CD4+ T cells were harvested by Ficoll purification 5–6 days after stimulation. Freshly isolated or in vitro generated CD18+/– and CD18–/– CD4+ T cells were differentially stained with either 1.5 µM CFSE or 2.5 µM Cell Tracker Orange (CTO) (Molecular Probes). CD18+/– and CD18–/– CD4+ T cells were adoptively cotransferred at a 1:1 ratio (15–20 x 106 T cells each per mouse). Sixteen hours after transfer, PP, MLN, spleen, and, where indicated, LPL were harvested. Cells were stained for CD4+, and the homing index was calculated as transferred WT CD18+/– CD4+ T cells/CD18–/– CD4+ T cells adjusted for the initial input ratio.

Statistical analyses

Statistical comparisons of homing index between treated groups were assessed using a t test comparing experimental homing index vs homing index = 1. Cell accumulation between treated groups was assessed using a one-tailed Student t test. p < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD18 on CD4+ T cells is required for optimal DO11.10 CD4+ T cell accumulation in the intestinal lamina propria after oral Ag

To dissect the role of CD18 in CD4+ T cell immune responses within the intestinal lymphoid system, we first sought to determine whether there was a defect in the number of Ag-responsive CD4+ T cells in the intestinal lamina propria after activation through oral Ag. CD18 is expressed on hematopoietic cells (22), such that CD18–/– mice have a deficiency of CD18 on non-T cell subsets that may contribute to abnormal intestinal lymphoid organ immune responses. Therefore, we sought to investigate the role of CD18 specifically on CD4+ T cells through the adoptive transfer of TCR-transgenic CD4+ T cells. We used the adoptive transfer of CD18–/– mice x DO11.10 T cells (encode an OVA-specific, IAd-restricted TCR) into WT mice, such that only the Ag-specific T cells migrating and responding to immunization are CD18 deficient. We isolated CD4+ T cells from the spleen, given that the majority of T cells in the spleen have not differentiated into tissue-specific T cells and CD4+ T cells in CD18–/– mice do not have a trafficking defect to the spleen (17). These CD18–/– CD4+ T cells are deficient only in LFA-1, given that it is the only beta2 integrin expressed on peripheral CD4+ T cells (data not shown). CD18+/– mice are used as littermate controls for CD18–/– mice because both CD18+/+ and CD18+/– mice have identical cell surface expression of CD18 by flow cytometry. CD18+/– and CD18–/– CD4+ D011.10 T cells were adoptively cotransferred into WT Thy1.1 BALB/c mice; 24 h later, the mice were immunized with oral OVA. Absolute numbers of migrated CD4+ DO11.10 T cells to the intestinal lamina were determined 5 days after immunization to allow adequate time for trafficking to effector tissues. The number of CD18–/– DO11.10 CD4+ T cells in intestinal secondary lymphoid organs, MLN and PP, as well as in the small and large intestinal lamina propria, is decreased compared with CD18+/– CD4+ T cells (Fig. 1). Similar findings are observed at an earlier time point, day 4, after immunization (data not shown). Therefore, there is a defect in the total number of accumulated CD18–/– DO11.10 CD4+ T cells in the intestinal lamina propria after an oral immune response.


Figure 1
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  		FIGURE 1. Accumulation of CD18–/– DO11.10 CD4+ T cells in the SB and large bowel (LB) lamina propria is impaired after oral immunization. Freshly isolated splenic DO11.10 CD4+ T cells from Thy1.2 CD18+/– and CD18–/– mice were adoptively cotransferred into Thy1.1 WT BALB/c mice. Twenty-four hours later, the mice were orally gavaged with 50 mg of OVA Ag; 5 days after immunization, MLN, PP, LB LPL and SB LPL were harvested and the number of DO11.10 (KJ1-26+) CD4+ T cells was quantified (percent of DO11.10 CD4+ Thy1.2+ times the number of MLN, PP, LB LPL, or SB LPL cells) + SEM (n = 4 mice). Similar results were obtained when tissues were harvested day 4 after immunization. *, p < 0.05; **, p < 0.01.

 
CD18 is essential for trafficking of CD4+ T cells to secondary intestinal lymphoid organs

The defect in accumulation of CD18–/– DO11.10 CD4+ T cells in the intestinal lamina propria may be due to defects at various checkpoints in the immune response. The first step in the intestinal immune response requires the proper trafficking of Ag-specific T cells to the intestinal secondary lymphoid organs, including the MLN and PP. LFA-1 has been previously shown to play a role in trafficking to MLN and PP (16, 17). To confirm this role in cotransfer homing experiments using DO11.10 T cells, we determined the absolute numbers of migrated CD4+ DO11.10 T cells to the MLN and PP 16 h after adoptive cotransfer into WT BALB/c mice. The 16-h time point allows for distribution of T cells to the lymphoid organs. Migration of CD18–/– DO11.10 CD4+ T cells to both the MLN and PP is decreased compared with CD18+/– CD4+ T cells (Fig. 2). As controls, we also assessed trafficking to peripheral secondary lymphoid organs. As expected, trafficking of CD18–/– CD4+ T cells is decreased to the PLN, whereas it is increased to the spleen, compared with CD18+/– DO11.10 CD4+ T cells (Fig. 2) (17). The increased trafficking to the spleen is consistent with the splenic hypercellularity of CD4+ T cells in CD18-deficient mice under homeostatic conditions (16) and likely secondary to a redistribution of the CD4+ T cell population due to the inability to properly traffic to lymph nodes.


Figure 2
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  		FIGURE 2. Migration of CD18–/– DO11.10 CD4+ T cells to secondary intestinal lymphoid organs is impaired. Freshly isolated splenic DO11.10 CD4+ T cells from CD18+/– or CD18–/– mice were differentially stained with CFSE or CTO and adoptively cotransferred to WT BALB/c mice. Sixteen hours later, MLN, PP, PLN, and spleen were harvested and the percentage of CD18+/– and CD18–/– within each organ was determined. A, Representative FACS plots gated on CD4+ T cells indicating the percent of transferred cells in each organ; graph of the homing index (calculated as transferred WT CD18+/– CD4+ T cells/CD18–/– CD4+ T cells adjusted for the initial input ratio + SEM) (n = 4 mice) (B). Data represent at least two independent experiments. Similar results were obtained when either CD18+/– or CD18–/– T cells were stained with CFSE or CTO. ***, p < 0.001; {dagger}, p < 0.0001.

 
CD18 is critical for T cell proliferation in intestinal immune responses

In the context of the migration defect of CD18–/– CD4+ T cells to intestinal secondary lymphoid organs, we examined the activation of those CD4+ DO11.10 T cells that had successfully trafficked to the MLN of WT mice on exposure to oral Ag. Cell divisions, as indicated by CFSE dilution, 3 days after oral immunization were used as a measure of T cell activation. Upon cotransfer with CD18+/– DO11.10 CD4+ T cells into WT BALB/c mice, CD18–/– DO11.10 CD4+ T cells in MLN entered into cell division, but 25–50% less CD18–/– DO11.10 CD4+ T cells in MLN were able to progress to later cell divisions as compared with CD18+/– DO11.10 CD4+ T cells (Fig. 3A, cotransfer). The defect in CD18–/– DO11.10 CD4+ T cell activation in the MLN persisted at day 6 after immunization, indicating that the defect is not simply a delayed kinetic response (data not shown). We use cotransfer of CD18+/– and CD18–/– DO11.10 T cells as a means of more accurately comparing the responses of these T cells within the mice. However, it is possible that in the absence of competition from CD18+/– DO11.10 CD4+ T cells during T cell priming, the observed severe CD18–/– DO11.10 CD4+ T cell activation defect may significantly improve. Therefore, we adoptively transferred CD18+/– and CD18–/– DO11.10 CD4+ T cells into separate WT BALB/c mice and assessed priming in the MLN after oral OVA immunization. The activation defect in CD18–/– DO11.10 CD4+ T cells in MLN persisted despite lack of competition from CD18+/– DO11.10 CD4+ T cells (Fig. 3A, separate transfer). As expected, the total number of accumulated CD18–/– DO11.10 CD4+ T cells in the MLN after oral immunization under both cotransfer and separate transfer conditions was also decreased relative to CD18+/– controls (Fig. 3B). Therefore, CD18–/– DO11.10 CD4+ T cells demonstrate a persistent defect in both migration to the MLN and proliferation in the MLN in vivo upon oral immunization.


Figure 3
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  		FIGURE 3. CD18–/– DO11.10 CD4+ T cells have an activation defect upon oral OVA immunization. Freshly isolated spleen DO11.10 CD4+ T cells from CD18+/– or CD18–/– mice were labeled with CFSE and either adoptively cotransferred or separately transferred to WT BALB/c mice. Mice were orally gavaged with OVA 24 h later; 3 days after that, CFSE dilution (cell divisions) of DO11.10 CD4+ T cells (A), and the number of DO11.10 CD4+ T cells in MLN was measured (B; percent of DO11.10 CD4+ T cells times the number of MLN cells). The percent of CD18+/– and CD18–/– T cells in the MLN in the cotransferred conditions is indicated in the FACS plots. The percents of cells in the later generations of divisions are indicated. Compared with CD18+/– T cells, 25–50% less CD18–/– DO11.10 CD4+ T cells progressed into the later cell divisions in MLN on days 3–4. Data are representative of three to four mice per condition, six independent experiments on the cotransferred experiments, and five independent experiments in the separate transfers. *, p < 0.05.

 
CD18–/– DO11.10 CD4+ T cells demonstrate a defect in acquisition of intestinal-specific trafficking molecules after oral immunization

In light of the defect in proliferation observed in CD18–/– DO11.10 CD4+ T cells in MLN on administration of oral Ag, we questioned whether CD18–/– T cells are defective in T cell differentiation on activation with oral Ag. Specifically, we assessed whether CD18-deficient CD4+ T cells were defective in the up-regulation of the intestinal-specific trafficking molecules, {alpha}4beta7 and CCR9. The expression of these intestinal trafficking molecules is essential for proper trafficking of T cells to the intestinal tissues where they can then participate in the effector immune response. WT mice were adoptively cotransferred with CFSE-labeled CD18+/– and CD18–/– DO11.10 CD4+ T cells and orally immunized 24 h later with OVA. From 3 to 4 days after immunization, CD18–/– DO11.10 CD4+ T cells in both the MLN and PP demonstrate a defect in the percentage of T cells up-regulating {alpha}4beta7 and CCR9 (Fig. 4A). A defect in proliferation of CD18–/– DO11.10 CD4+ T cells is now observed in the PP as well as the MLN (Fig. 4A). The ability to up-regulate {alpha}4beta7 and CCR9 is acquired in the intestinal secondary lymphoid organs as T cells progress through cell division, such that it is possible that the differentiation defect observed in CD18–/– DO11.10 CD4+ T cells during oral OVA immunization is due to the defect in cell proliferation in the MLN and PP, and/or that there is an additional independent role for CD18 in differentiation of T cells expressing intestinal trafficking molecules. To examine these possibilities, we quantitated the level of {alpha}4beta7 and CCR9 expression on CD18–/– T cells at each cell division. We find that surface levels of these intestinal-specific trafficking molecules are equivalent between CD18+/– and CD18–/– T cells at a given cell cycle (Fig. 4B). Therefore, CD18–/– DO11.10 CD4+ T cells demonstrate a defect in the acquisition of intestinal trafficking molecules during oral immunization associated with their proliferation defect.


Figure 4
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  		FIGURE 4. CD18–/– DO11.10 CD4+ T cells demonstrate defects in acquisition of intestinal tropism in the MLN and PP. Freshly isolated spleen DO11.10 CD4+ T cells from CD18+/– and CD18–/– mice were labeled with CFSE and adoptively cotransferred at a 1:1 ratio into WT Thy1.1 BALB/c mice. Mice were orally gavaged with OVA 24 h later, and 3–4 days after that CFSE dilution (cell divisions) of DO11.10 CD4+ T cells vs {alpha}4beta7 or CCR9 in separate experiments (A) and the percent of DO11.10 CD4+ T cells + SEM (n = 4 mice) in MLN and PP expressing either {alpha}4beta7 or CCR9 correlated to the number of cell divisions (B) was measured. Data are representative of at least three independent experiments for each of the trafficking molecules.

 
CD18 is required for activation and up-regulation of the intestinal-tropic phenotype on T cells in vitro

One possibility for the defect in activation and differentiation of CD18–/– CD4+ DO11.10 T cells upon oral OVA immunization is that the CD18–/– T cell trafficking defect to the MLN and PP at the onset of the immune response may result in an insufficient critical T cell mass to allow for appropriate proliferation and differentiation. As a result, the defect in CD18–/– T cell proliferation and acquisition of intestinal tropism would be due to the initial migration effect rather than a direct defect in CD18–/– T cell activation and differentiation upon interaction with DC. In light of the critical role of DC from MLN and PP in T cell activation and differentiation, we first assessed whether the defect in CD18–/– DO11.10 CD4+ T cell proliferation was due to a defect in activation by DC from these secondary lymphoid organs. We used isolated WT MLN DC and PP DC to activate CD18+/– or CD18–/– DO11.10 CD4+ T cells in vitro. CD18–/– DO11.10 CD4+ T cells demonstrate an activation defect over a broad range of Ag doses at physiological DC:T cell ratios of 1:20 with both MLN DC (Fig. 5A) and PP DC (Fig. 5B). This activation defect persists even when the ratio of DC:T cells increases to 1:10, and finally 1:1, a much higher ratio than is observed in vivo in lymphoid structures. Therefore, CD18–/– DO11.10 CD4+ T cells have a defect in activation in vitro upon stimulation with either MLN or PP DC.


Figure 5
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  		FIGURE 5. CD18–/– DO11.10 CD4+ T cells have a defect in activation upon Ag presentation by either MLN or PP DC in vitro. MLN-derived DC (A) or PP-derived DC (B) were cocultured in separate experiments with splenocyte CD4+ T cells purified from CD18+/– or CD18–/– DO11.10 TCR-transgenic mice at a 1:20, 1:10 or 1:1 DC:T cell ratio in the presence of increasing concentrations of OVA peptide. Thymidine incorporation was measured during the last 18 h of a 72-h assay. Data are representative of at least three independent experiments.

 
As we next sought to use these differentiated T cells in experiments in vivo to assess the capacity of MLN DC and PP DC-instructed CD18–/– T cells to properly traffic, we expanded CD18+/– and CD18–/– DO11.10 CD4+ T cells over 5–6 days using both MLN and PP DC. We selected a moderately high dose of Ag at 2 µg/ml OVA peptide. We further used a 1:1 ratio of DC:T cells, similar to that used in previous reports (9, 11), to maximize T cell expansion for use in homing experiments in vivo. The in vitro stimulation of CD18–/– DO11.10 CD4+ T cells with MLN and PP DC at a 1:1 ratio creates much more favorable conditions for CD18–/– DO11.10 CD4+ T cell activation/expansion than would be present in vivo. We next sought to determine whether CD18–/– T cells stimulated under these conditions have defects in differentiation into intestinal-specific trafficking T cells expressing {alpha}4beta7 and CCR9 (9, 10, 11, 12, 13). We found that upon activation with either MLN or PP DC, a decreased percent of CD18–/– DO11.10 CD4+ T cells up-regulate {alpha}4beta7 and CCR9 (Fig. 6). Of those CD18–/– DO11.10 CD4+ T cells successfully up-regulating {alpha}4beta7 and CCR9, they did not increase expression to the same degree as did CD18+/– T cells (Fig. 6). Therefore, despite highly favorable activation conditions, CD18–/– DO11.10 CD4+ T cells have defects in differentiation into T cells expressing intestinal trafficking molecules upon stimulation with either MLN or PP DC.


Figure 6
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  		FIGURE 6. CD18–/– DO11.10 CD4+ T cells have a defect in acquisition of intestinal trafficking molecules upon Ag presentation by either MLN or PP DC in vitro. MLN-derived DC or PP-derived DC were cocultured with splenocyte CD4+ T cells purified from CD18+/– or CD18–/– DO11.10 TCR-transgenic mice at a 1:1 DC:T cell ratio in the presence of 2 µg/ml OVA peptide. Five days later, the expression of {alpha}4beta7 and CCR9 was assessed. Dotted line, naive DO11.10 CD4+ T cells; thick solid line, activated CD18+/– DO11.10 CD4+ T cells; thin solid line, activated CD18–/– DO11.10 CD4+ T cells.

 
MLN and PP DC-instructed CD18–/– DO11.10 CD4+ T cells demonstrate defects in intestinal homing

T cells activated by either MLN or PP DC demonstrate enhanced trafficking to the intestine in comparison to T cells activated by peripheral-derived DC (9, 10, 11, 12, 13). The decreased number of CD18–/– DO11.10 CD4+ T cells in the intestinal lamina propria after oral Ag may be due to the decreased number T cells in the MLN and PP available for trafficking to the intestine after the immune response, due to 1) initial migration defects and 2) expansion defects, as well as 3) defects in differentiation into intestinal homing cells and 4) an independent role for CD18 in the final phase of entry into the lamina propria. We now know that CD18–/– DO11.10 CD4+ T cells are defective in migration, proliferation, and differentiation into intestinal trafficking cells in vivo. We next sought to determine whether CD18–/– DO11.10 CD4+ T cells activated by either MLN or PP DC have defects in trafficking to the intestine. To eliminate the defects in available numbers of CD18–/– T cells in the secondary lymphoid structures that are observed in vivo, we transferred equal numbers of CD18–/– DO11.10 CD4+ T cells activated in vitro by either MLN or PP DC according to the conditions described in Fig. 6. On cotransfer of CD18+/– and CD18–/– DO11.10 CD4+ T cells, CD18–/– T cells activated by either PP (Fig. 7, A and B) or MLN (Fig. 7C) DC have defects in homing to secondary intestinal lymphoid organs and intestinal tissues, including MLN, PP, and large and SB lamina propria. The defect in intestinal-specific trafficking is more severe in CD18–/– T cells activated by PP DC. As an internal control, activated CD18–/– DO11.10 CD4+ T cells home well to the spleen, a secondary lymphoid organ which is not dependent upon CD18 for optimal trafficking (Fig. 7). Therefore, despite transfer of equal numbers of MLN or PP DC-instructed CD18–/– T cells, CD18–/– DO11.10 CD4+ T cells demonstrate defects in homing to the intestinal lymphoid system.


Figure 7
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  		FIGURE 7. Trafficking of CD18–/– DO11.10 CD4+ T cells instructed by either MLN or PP DC to the intestinal lymphoid system is impaired. Freshly isolated splenic DO11.10 CD4+ T cells from CD18+/– or CD18–/– mice were activated in vitro with 2 µg/ml OVA peptide at a 1:1 ratio with either PP or MLN DC. Five to six days after activation, CD18+/– and CD18–/– T cells were differentially stained with CFSE or CTO and adoptively cotransferred to WT BALB/c mice. Sixteen hours later, MLN, PP, LB, and SB LPL and spleen were harvested, and the percent of CD18+/– and CD18–/– within each organ was determined. A, Representative FACS plots gated on CD4+ T cells indicating the percent of PP DC-instructed T cells in each organ; B, PP DC-instructed T cell and MLN DC-instructed (C) T cell graphs of the homing index which was calculated as transferred WT CD18+/– CD4+ T cells/CD18–/– CD4+ T cells adjusted for the initial input ratio + SEM. Data are an average of two independent experiments with n = 3 mice per experiment. Similar results were obtained when either CD18+/– or CD18–/– T cells were stained with CFSE or CTO. *, p < 0.05; **, p < 0.01; {dagger}, p < 0.0001.

 
CD18–/– DO11.10 CD4+ T cells demonstrate a severe defect in homing to intestinal tissues despite expressing optimized intestinal trafficking molecules

The defect in intestinal trafficking exhibited by MLN or PP DC-instructed CD18–/– DO11.10 CD4+ T cells may be due to the decreased expression of intestinal-specific trafficking molecules on these T cells. Alternatively, CD18 may directly contribute to trafficking of activated CD4+ T cells into the intestine, independent of its role in the up-regulation of intestinal trafficking molecules during T cell activation. To investigate this latter possibility, we examined whether enforced expression of intestinal trafficking molecules on activated CD18–/– CD4+ T cells could overcome their defect in migration to intestinal tissues. Therefore, we activated CD18+/– and CD18–/– CD4+ T cells with plate-bound anti-CD3 and anti-CD28 in the presence of retinoic acid, which instructs T cells to up-regulate intestinal trafficking molecules and results in significantly improved homing to the intestine (32). We find that CD18–/– T cells activated under these conditions proliferate equivalently to CD18+/– T cells (Fig. 8, A and B), similar to previous reports using anti-CD3 stimulation (33). We further find that CD18–/– CD4+ T cells up-regulate {alpha}4beta7 and CCR9 to an equivalent level as is observed on CD18+/– T cells (Fig. 8C). Despite equivalent activation and intestinal-tropic differentiation of CD18–/– CD4+ T cells in vitro, these T cells continue to demonstrate a defect in homing to intestinal tissues (Fig. 8, D and E). Therefore, CD18 can contribute to optimal intestinal trafficking independent of its contributions to T cell proliferation and differentiation in the secondary lymphoid structures of the intestine.


Figure 8
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  		FIGURE 8. Trafficking of CD18–/– DO11.10 CD4+ T cells to the intestinal lymphoid system is defective despite optimal expression of intestinal trafficking molecules. Freshly isolated splenic CD4+ T cells from CD18+/– or CD18–/– mice were activated in vitro with anti-CD3/anti-CD28 in the presence of 10 nM retinoic acid. Cell proliferation was assessed by thymidine incorporation during the last 18 h of a 72 h assay (A) and CFSE dilution on days 2 and 3 after activation (B). Five to six days after activation, cell surface expression of {alpha}4beta7 and CCR9 was assessed (C; dotted line, naive CD4+ T cells; thick solid line, activated CD18+/– CD4+ T cells; thin solid line, activated CD18–/– CD4+ T cells). CD18+/– and CD18–/– T cells were then differentially stained with CFSE or CTO and adoptively cotransferred to WT BALB/c mice. Sixteen hours later, MLN, PP, LB, and SB LPL and spleen were harvested, and the percent of CD18+/– and CD18–/– within each organ was determined. D, Representative FACS plots gated on CD4+ T cells indicating the percent of transferred cells in each organ; E, graph of the homing index which was calculated as transferred WT CD18+/– CD4+ T cells/CD18–/– CD4+ T cells adjusted for the initial input ratio + SEM. Data are an average of six independent experiments with n = 3–4 mice per experiment. Similar results were obtained when either CD18+/– or CD18–/– T cells were stained with CFSE or CTO. {dagger}, p < 0.0001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we have defined the specific checkpoints of the intestinal immune response to which CD18 contributes. We find that CD18 is required for optimal CD4+ T cell accumulation in the intestinal lamina propria after oral Ag immunization. This is consistent with the decreased number of CD4+ T cells in the intestinal lamina propria of mice partially deficient in CD18 (34). We have identified that the defect in this final phase of CD4+ T cell accumulation is due to defects at multiple points in the intestinal immune response. In addition to the role that CD18 plays in migration of CD4+ T cells to MLN and PP, it is required for optimal proliferation of CD4+ DO11.10 T cells in these secondary intestinal lymphoid structures upon oral Ag administration. This defect in CD4+ T cell priming is associated with a defect in up-regulation of the intestinal trafficking molecules, {alpha}4beta7 and CCR9. Finally, CD18 plays a role in the trafficking of intestinal-tropic CD4+ T cells to the lamina propria independent of its role in activation and intestinal-tropic differentiation.

We have taken advantage of the ability to follow Ag-responsive CD4+ T cells to dissect the contribution of CD18 at each phase of the intestinal immune response. The defect in accumulation of Ag-specific CD18–/– CD4+ T cells in the intestinal lamina propria after oral Ag administration reflects a compounding of multiple defects in the immune response. Although we had previously identified a role for CD18 in T cell priming in vivo in the peripheral secondary lymphoid organs, the intestinal secondary lymphoid organs have different dependencies on trafficking molecules, have distinct DC-mediating T cell activation, have a larger percentage of activated T cells, and are continually exposed to intestinal bacterial Ags, such that the requirements for optimal T cell activation in these lymphoid structures may well have different requirements. Nevertheless, we find that optimal T cell activation in both the MLN and PP requires CD18. Considerations of mechanisms accounting for the CD18–/–CD4+ T cell activation defect may include both CD18’s adhesive and costimulatory functions (16, 17, 22, 23, 24, 25, 26, 27, 28, 29, 30).

Associated with the defect in CD18–/– DO11.10 CD4+ T cell progression through cell cycle in the MLN and PP upon oral Ag administration is a defect in the up-regulation of intestinal trafficking molecules generally acquired upon cell cycle progression in the MLN and PP. CD18 does not appear to have an independent contribution to up-regulation of {alpha}4beta7 and CCR9 beyond its role in optimizing T cell proliferation. When we examine CD18–/– DO11.10 CD4+ T cells for the up-regulation of {alpha}4beta7 and CCR9 on activation with MLN and PP DC in vitro, we see a defect in up-regulation despite highly favorable conditions for T cell activation at high 1:1 ratio DC:T cells and moderately high Ag dose. The defect in trafficking to the intestinal lymphoid system of MLN and PP DC-instructed CD18–/– T cells would likely be greater if the T cells were activated in vitro under lower DC:T cell ratios that are more consistent with those observed in vivo. The defect that is observed in the trafficking of MLN and PP DC-instructed CD18–/– DO11.10 CD4+ T cells to intestinal tissues likely reflects a combination of defects in up-regulation of intestinal-specific trafficking molecules, the deficiency of CD18, as well as possible defects in up-regulation of other adhesion molecules that may contribute to intestinal tissue trafficking. The identification of an independent role for CD18 in the trafficking of fully activated intestinal-tropic CD4+ T cells indicates that although intestinal-specific trafficking molecules are important for trafficking to intestinal tissues, they are insufficient for optimal trafficking in the absence of CD18. This is of particular relevance to diseases involving an existent population of activated, pathogenic CD4+ T cells that have been instructed to traffic to the intestine.

The infiltration of effector T cells into specific tissues is a critical feature of inflammatory disease. As a result, targeting both effector T cell activation and trafficking is increasingly used in the treatment of inflammatory diseases, such as inflammatory bowel disease, psoriasis, and multiple sclerosis. However, the outcomes of these interventions have been mixed. For example, one large study of blockade of {alpha}4 (thereby blocking {alpha}4beta7 and {alpha}4beta1) in patients with Crohn’s disease was of benefit in the maintenance phase of disease, although not in induction of remission in early phases of disease when there is significant ongoing T cell activation and populations of inflammatory effector T cells (35). This highlights the necessity of understanding the timing and role of various trafficking molecules in contributing to an immune process. The interaction of LFA-1 with its ligands (e.g., ICAM-1, ICAM-2, ICAM-3, and juntional adhesion molecule family members; Ref. 22) has been targeted in therapy of inflammatory diseases in both animal models and human disease, including inflammatory bowel disease (20, 36, 37, 38, 39, 40, 41, 42, 43, 44). As such, although LFA-1 has a role in trafficking to a number of lymphoid and nonlymphoid organs, in some models of intestinal inflammation, blockade of LFA-1 is, in fact, more effective than blockade of the more intestinal-specific integrins {alpha}4 and/or beta7 (45). This may be due to contributions to intestinal immune responses by LFA-1 that are not observed with {alpha}4beta7, such as trafficking to MLN or T cell activation, or to the need to block multiple molecules simultaneously due to redundancy in certain functions, such as trafficking. Therefore, although LFA-1 contributes to peripheral T cell functions, it also contributes to essential intestinal immune functions. Future interventions will require selecting appropriate technologies to mediate inhibition, optimizing the timing for intervention, determining the cooperation with other molecules in mediating intestinal immune responses, as well as carefully selecting the specific surface receptor/ligand to be targeted. As we have identified that CD18 can contribute to optimal DO11.10 CD4+ T cell responses at each phase of the intestinal immune response (migration, activation, and differentiation into intestinal-tropic T cells in the MLN and PP and trafficking into intestinal lamina propria), it has the potential to be an effective target at multiple time points in an intestinal inflammatory process.


   Acknowledgments
 
We thank Drs. Maria-Luisa Alegre, Judy H. Cho, Ellen Chuang, Yang-Xin Fu, and Jerrold R. Turner, for critical reading of the manuscript, and Drs. Arthur Beaudet, Alexander Khoruts, and Ulrich Von Andrian, for providing reagents.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 the Crohn’s and Colitis Foundation of America, National Institutes of Health Grant DK02905 (to C.A.), and University of Chicago Digestive Disease Center Grant DK42086. Back

2 Address correspondence and reprint requests to Dr. Clara Abraham, Department of Medicine, University of Chicago, 5841 South Maryland Avenue, MC 6084, Chicago, IL 60637. E-mail address: cabraham{at}medicine.bsd.uchicago.edu Back

3 Abbreviations used in this paper: MLN, mesenteric lymph nodes; WT, wild type; DO11.10 mice, OVA-specific TCR-transgenic mice; PP, Peyer’s patches; PLN, peripheral lymph nodes; LPL, lamina propria lymphocytes; DC, dendritic cells; CTO, cell tracker orange; LB, large bowel; SB, small bowel. Back

Received for publication August 30, 2006. Accepted for publication November 30, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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