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The Journal of Immunology, 2005, 175: 7889-7897.
Copyright © 2005 by The American Association of Immunologists

CD18 Is Required for Optimal Development and Function of CD4+CD25+ T Regulatory Cells1

Marissa Marski*, Sravanthi Kandula*, Jerrold R. Turner{dagger} and Clara Abraham2,*

* Department of Medicine and {dagger} Department of Pathology, University of Chicago, Chicago, IL 60637


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD4+CD25+ T regulatory (Treg) cells inhibit immunopathology and autoimmune disease in vivo. CD4+CD25+ Treg cells’ capacity to inhibit conventional T cells in vitro is dependent upon cell-cell contact; however, the cell surface molecules mediating this cell:cell contact have not yet been identified. LFA-1 (CD11a/CD18) is an adhesion molecule that plays an established role in T cell-mediated cell contact and in T cell activation. Although expressed at high levels on murine CD4+CD25+ Treg cells, the role of LFA-1 in these cells has not been defined previously. We hypothesized that LFA-1 may play a role in murine CD4+CD25+ Treg function. To evaluate this, we analyzed LFA-1-deficient (CD18–/–) CD4+CD25+ T cells. We show that CD18–/– mice demonstrate a propensity to autoimmunity. Absence of CD18 led to diminished CD4+CD25+ T cell numbers and affected both thymic and peripheral development of these cells. LFA-1-deficient CD4+ CD25+ T cells were deficient in mediating suppression in vitro and in mediating protection from colitis induced by the transfer of CD4+CD25– T cells into lymphopenic hosts. Therefore, we define a crucial role for CD18 in optimal CD4+CD25+ Treg development and function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Regulatory CD4+CD25+ T (Treg)3 cells are critical for inhibiting immunopathology in vivo. This has been demonstrated in models of colitis, gastritis, diabetes, experimental autoimmune encephalomyelitis, and fetal rejection (1, 2, 3). Moreover, either the number or function of these cells is reduced in mouse and human autoimmune diseases (4, 5, 6). CD4+CD25+ Treg cells inhibit conventional T cell proliferation in vitro and control T cell numbers and effector functions in vivo (7). CD4+CD25+ Treg cells develop in the thymus (8), and they specifically express the transcription factor FoxP3 (9, 10). There is also evidence that either peripheral development or expansion of CD25+ Treg cells occurs (11, 12, 13).

The mechanisms wherein CD4+CD25+ Treg cells mediate suppression of immune responses have not been fully elucidated. Many studies demonstrate a requirement for cell-cell contact (3). However, the molecules mediating this contact have not yet been identified. Potential candidate adhesion molecules expressed on CD4+CD25+ T cells include ICAM-1, P-selectin, CD18, {beta}7, CD103, and L-selectin (14), but ICAM-1 and P-selectin were found not to be required for CD25+-suppressive function in a model of experimental autoimmune encephalomyelitis (15), and {beta}7 integrins were recently found not to be required for CD25+-mediated protection from colitis (16). Although high expression of L-selectin and CD103 on CD4+CD25+ Treg cells identifies a more potent suppressive subset, a functional role for these adhesion molecules has not been defined (14, 17). Whether CD18 is required for the optimal function of murine CD4+CD25+ T cells, either in vitro or in vivo, is unknown. In addition, the role of CD18 in the development of CD4+CD25+ T cells has not been reported.

LFA-1 ({alpha}L/{beta}2 or CD11a/CD18) is a member of the {beta}2 integrin family (18). Integrins are a large family of heterodimeric membrane glycoproteins composed of noncovalently linked {alpha}- and {beta}-chains that are involved in cell-cell adhesion. LFA-1 is expressed on peripheral blood lymphocytes, neutrophils, monocytes, granulocytes, and NK cells, where it binds multiple ligands, including ICAM-1, ICAM-2, or ICAM-3 (18). LFA-1 is the only member of the {beta}2 integrin family to be expressed on peripheral CD4+ T cells, where it plays an established role in trafficking and activation (18, 19, 20, 21, 22). Consistent with this, LFA-1 is the only {beta}2 integrin family member expressed on CD4+CD25+ T cells (data not shown). The physiological importance of LFA-1 is manifest in those individuals lacking the {beta}2 subunit in a disease known as leukocyte adhesion deficiency (LAD). These patients are characterized by an inability to clear pathogens, recurrent infections, and, frequently, death at an early age (23, 24). Notably, there have been reports of inflammatory bowel disease in patients with LAD (25). CD11a–/– and CD18–/– mice share many of these characteristics, including decreased CD4+ and CD8+ T cells in peripheral lymph nodes (PLN) and in extralymphoid tissue sites (e.g., lung, liver) (19, 22), and in vivo T cell functional defects, such as in tumor rejection, delayed-type hypersensitivity, and infectious responses (26, 27, 28, 29). Although the inflammatory manifestations and premature death seen in CD18–/– mice have been presumed to be infectious in nature (30), it is of significance that CD18–/– mice demonstrate a persistence of the inflammatory skin lesions despite being housed in a germfree facility (31). Thus, an infectious etiology is excluded under these conditions and this invokes the possibility of a defect in regulatory immune homeostasis and development of autoimmune disease.

We hypothesized that LFA-1 might play a role in CD4+CD25+ Treg cell homeostasis. Using CD18–/– mice, we demonstrate that CD18 is necessary for the development of optimal numbers of both innate and peripherally induced CD4+CD25+ Treg cells, as well as for the suppressor function of these cells. Consistent with these defects is the development of autoantibodies, indicating a propensity to autoimmunity in the absence of CD18.


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

CD18–/– mice on a C57BL/6 background were generously provided by A. Beaudet (Baylor College of Medicine, Houston, TX) (28). CD18–/– mice were backcrossed onto the BALB/c background (nine generations) and in some cases onto DO11.10 TCR transgenic mice. BALB/c.RAG-1–/– and BALB/c mice were obtained from The Jackson Laboratory. 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 in microisolator cages with filtered air. Experiments were performed in agreement with our institutional animal care and use committee and according to the National Institutes of Health guidelines for animal use.

Abs and staining reagents

The following Abs and secondary reagents used for flow cytometry were purchased from BD Pharmingen: PE-, FITC-, biotin-, allophycocyanin-labeled anti-CD4, PE- and FITC-labeled anti-CD18 and -CD25, biotin-labeled CD62L, FITC-labeled CD44, PE-labeled CD69, and CyChrome-labeled streptavidin. FITC-labeled FoxP3 was purchased from eBioscience. The following Abs and secondary reagents were purchased from Caltag Laboratories: PE- and biotin-labeled KJ1–26. Alkaline phosphatase-conjugated IgG was purchased from Southern Biotechnology Associates.

Autoimmunity model

RAG-1–/– mice were adoptively transferred with 20 x 106 splenocytes from 5-wk-old CD18+/– or CD18–/– mice. Mice were monitored by serum anti-DNA Abs assessed at weeks 3, 6, and 8 posttransfer. At 8 wk, mice were sacrificed, and tissues were harvested. These tissues were fixed in neutral-buffered formalin and paraffin-embedded by standard methods. Well-oriented 3-µm sections were stained by H&E. These were examined by a gastrointestinal surgical pathologist (J.R.T.) blinded to the experimental treatment.

Measurement of anti-DNA Abs

Ninety-six-well plates (Costar) were coated with sperm DNA (50 µg/ml) (Sigma-Aldrich) in PBS and then blocked with PBS/5% FCS. Duplicates of four to five dilutions of serum (1/200, 1/400, 1/800, 1/1600, and 1/3200) were incubated in the wells at 37°C x 1 h. Plates were washed and incubated with IgG conjugated with alkaline phosphatase (Southern Biotechnology Associates). Plates were washed and incubated with phosphatase substrate (Sigma-Aldrich), and optical densities were determined at 405 {lambda}.

RNA preparation and real-time RT-PCR

CD4+CD25+ T cells were isolated by MO-FLO sorting (DakoCytomation) from the thymus and spleen of CD18+/– or CD18–/– mice. RNA was harvested using TRIzol (Invitrogen Life Technologies), and residual DNA was removed with DNaseI treatment (Promega). RNA was reverse transcribed to cDNA with SuperScript II as per manufacturer instructions (Invitrogen Life Technologies). The level of FoxP3 mRNA was determined by real-time PCR using Sybergreen (Molecular Probes) and Taqman Universal PCR Mastermix (Applied Biosystems) on the iCycler (Bio-Rad). GAPDH endogenous control was used to normalize RNA. Primers were as follows: FoxP3 forward: 5'-TCCCAGAGTTCTTCCACAAC-3', and reverse, 5'-TAAGGGTGGCATAGGTGAAA-3'; GAPDH forward, 5'-CATGGCCTTCCGTGTTCCTA-3', and GAPDH reverse, 5'-TGTCATCATACTTGGCAGGTTTCT-3'. {Delta}{Delta}CT for relative quantitation was calculated using CD4+CD25– T cells as the calibrator.

Induction of peripheral CD4+CD25+ T cells

Spleen-derived CD4+ T cells were isolated using CD4+ microbeads according to manufacturer instructions (Miltenyi Biotec). Freshly isolated (2.5 x 106) CD18+/– or CD18–/– RAG-1–/– DO11.10 CD4+ T cells were adoptively transferred by i.v. injection into BALB/c mice. Twenty-four hours later, the mice were immunized with 5 µg/ml chicken OVA peptide 323–339 by i.v. injection, and 8 days after immunization, spleen and PLN were harvested, and cells were stained with anti-CD4+, anti-KJ1–26, and anti-CD25+ Abs to assess induction of CD4+CD25+ T cells.

In vitro suppression of T cell activation

For assessment of CD18+/– or CD18–/– CD4+CD25+ T cell suppression in vitro of freshly isolated CD4+ T cells, spleen-derived CD4+ T cells were first isolated using CD4+ microbeads (Miltenyi Biotec), and then CD25– or CD25+ populations were further sorted using the MO-FLO sorter (DakoCytomation). In some cases, CD25+ T cells were additionally stained with CD62L for the isolation of CD25+CD62Lhigh T cells. A total of 5.0 x 104 CD4+ T cells was incubated with 1 µg/ml anti-CD3 (145-2C11) and 2.5 x 105 irradiated (3000 rad) splenocytes along with increasing concentrations of CD18+/– or CD18–/– CD4+CD25+ T cells in a 96-well flat-bottom plate. [3H]Thymidine was added to the cultures during the last 18 h of a 72-h assay.

CD4+CD25–-mediated colitis

Spleen-derived CD4+ T cells were first isolated using CD4+ microbeads (Miltenyi Biotec), and then CD25– or CD25+ populations were further sorted using the MO-FLO sorter (DakoCytomation). Colitis was induced through the transfer of CD18+/–CD4+CD25– T cells (5 x 105) into RAG-1–/– mice, which were placed on a tap water and regular food diet. Protection from colitis was examined through cotransfer of either CD18+/– or CD18–/–CD4+CD25+ T cells (1 x 105) (from mice 4–5 wk of age) with the CD4+CD25– T cells. Mice were monitored over 8–9 wk for weight loss. Mice were sacrificed at 8–9 wk, and colon specimens were fixed in neutral-buffered formalin, paraffin-embedded, sectioned, and stained by H&E. These were examined by a gastrointestinal surgical pathologist (J.R.T.) blinded to the experimental treatment, and activity was scored using a standard five-tier scale of inactive (score of 0), minimal activity (score of 1), mild activity (score of 2), moderate activity (score of 3), and severe activity (score of 4). Minimal activity was defined as an increase in lamina propria neutrophils, lymphocytes, and plasma cells without significant numbers of intraepithelial neutrophils. Mild activity included intraepithelial neutrophils, but no crypt abscesses. Moderate activity was defined by the presence of crypt abscesses. Severe activity included erosions and/or ulceration. Spleen and large bowel lamina propria lymphocytes (LPL) were harvested and examined for CD4+ T cells. LPL were isolated essentially as per Ref.32 . In brief, colons were cut longitudinally and then into ~1-mm pieces. Colonic pieces were washed thoroughly (ice-cold PBS, 5% FCS) and then digested (PBS, 5 mM EDTA, and 5% FCS) at 37°C x 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 collegenase 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, 40 µg/ml gentamicin, and 50 U/ml penicillin-50 µg/ml streptomycin. The cells were then filtered through a 40-µm filter (BD Biosciences) and washed twice in medium without collagenase.

Statistical analyses

Statistical comparisons of anti-DNA Abs, cell percentages and numbers, body weights, and pathology scores between treated groups were assessed using a one-tailed Student’s t test. Values of p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
CD18–/– splenocytes transferred into RAG-1–/– mice lead to intestinal inflammation

To evaluate whether defects in regulatory immune homeostasis might contribute to the inflammatory manifestations in CD18–/– mice, we investigated CD18–/– splenocytes in a general model of autoimmunity. Transfer of splenocytes into lymphopenic hosts can lead to selective proliferation of autoreactive T cells (33) and has been used as a means of assessing a propensity toward autoimmunity (34). Both CD18+/+ and CD18+/– mice have an identical cell surface expression of CD18 by flow cytometry, and CD18+/– mice are used as littermate controls for the CD18–/– mice. The activation markers on CD18–/– splenocytes are not increased (Fig. 1A). Therefore, we transferred equivalent numbers of CD18+/– or CD18–/– splenocytes into RAG-1–/– mice. RAG-1–/– mice transferred with CD18–/– splenocytes uniformly demonstrated an increase of ileal (Fig. 1B) and colonic (data not shown) disease. This was characterized by marked wall thickening and transmural inflammation (Fig. 1B). Epithelial regeneration, typified by frequent mitotic figures, and evidence of chronic injury, including increased numbers of Paneth cells, were also present.



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  		FIGURE 1. CD18–/– splenocyte transfer into RAG-1–/– mice results in a progressive increase in anti-DNA Abs and intestinal inflammation. A, Spleen from CD18+/– and CD18–/– mice were stained CD4, CD69, and CD44, and expression of CD69 and CD44 on gated CD4+ T cells is shown. A total of 20 x 106 CD18+/– (n = 6) or CD18–/– (n = 5) splenocytes was transferred into RAG-1–/– mice. B, Eight weeks after transfer, mice were sacrificed, and the ileum was harvested and stained by H&E. CD18+/– splenocyte-transferred mice demonstrate normal ileum (mucosa, scale bar = 100 µM) and crypts with Paneth cells (scale bar = 20 µM). CD18–/– splenocyte-transferred mice (magnifications are identical to CD18+/–-transferred mice) demonstrate ileal inflammatory disease with marked wall thickening and increased lamina propria immune cells, decreased goblet cells, and reactive epithelial changes. Examination of crypt epithelium shows increased mitotic activity and expansion of Paneth cells. The average number of Paneth cells increased from 1.1 ± 0.1 per crypt in CD18+/–-transferred mice to 5.5 ± 0.2 (p < 0.001) per crypt in CD18–/–-transferred mice. C, Serial dilutions of serum anti-DNA Abs were measured by ELISA at weeks 3, 6, and 8 after transfer. The data are represented as relative units = OD (± SEM). *, p < 0.05.

 
CD18–/– mice demonstrate high titers of anti-DNA Abs

The development of intestinal inflammation in RAG-1–/– mice transferred with CD18–/– splenocytes was unexpected in light of the known defects in CD18–/– hemopoietic cell trafficking and activation (19, 22, 28, 35). To further study whether these findings were consistent with autoimmunity, we assessed whether recipient mice have increased production of autoantibodies. RAG-1–/– mice transferred with CD18–/– splenocytes demonstrated a progressive increase in serum anti-DNA titers for at least 8 wk after splenocyte transfer (Fig. 1C). Similarly, CD18–/– mice had higher titers of anti-DNA Abs than did their CD18+/– littermate controls (Fig. 2).



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  		FIGURE 2. CD18–/– mice have elevated titers of anti-DNA Abs. Serial dilutions of serum from CD18+/– (n = 7) and CD18–/– (n = 9) mice were assessed for anti-DNA Abs by ELISA. The data are represented as relative units = OD (± SEM).

 
CD18–/– mice demonstrate defects in thymic and peripheral CD4+CD25+ T cell numbers

The development of intestinal inflammation and anti-DNA Abs in the transfer of CD18–/– splenocytes to RAG-1–/– mice could potentially be explained by increased autoreactive T cells and/or decreased numbers or function of regulatory populations. To evaluate the possibility of autoreactive T cells, we examined the V{beta} repertoire in CD18–/– mice as a means of assessing potential defects in thymic selection and T cell repertoire. The V{beta} repertoire in CD18–/– mice was equivalent to that of CD18+/– littermate controls, and the deletion of V{beta}5 and V{beta}8 was observed as expected in BALB/c mice (data not shown). Although other subtleties beyond V{beta} repertoire may be present, these data suggest the absence of increased autoreactive T cells.

Therefore, we next considered defects in regulatory T cell populations. The colonic and ileal inflammation induced by the transfer of CD18–/– splenocytes to RAG-1–/– mice had similarities to the disease observed in the CD4+CD45RBhigh transfer model into immunodeficient hosts. CD4+CD25+ T cells can rescue the intestinal inflammation observed in the CD4+CD45RBhigh model (36). Therefore, we examined whether there was a quantitative defect of CD4+CD25+ T cells in CD18–/– mice. Mice were examined between 4 and 8 wk of age, at which time there is no evidence of spontaneous inflammation of various organs by H&E staining (e.g., kidney, pancreas, liver, stomach, and small and large intestines). In comparison to CD18+/– mice, the percentage of CD4+CD25+ T cells (Fig. 3, A and C) is decreased in the mesenteric lymph node (MLN) and spleen, and the absolute number of CD4+CD25+ T cells (Fig. 3D) is decreased in the MLN and PLN of CD18–/– mice. As is observed in the CD11a–/– mice (19), the number of total CD4+ T cells in MLN and PLN of CD18–/– mice is decreased (Fig. 3B), accounting for the relative enhancement in the defect in CD18–/–CD4+CD25+ T cell numbers in these organs. Although the sum total CD4+ T cells in the periphery of CD18–/– mice is not decreased due to the redistribution of CD4+ T cells to the spleen, the total number of CD4+CD25+ T cells is decreased in these mice. As the most significant defect in CD4+CD25+ T cells was in the MLN, a lymphoid organ that contains increased populations of effector T cells that can also express CD25+, we assessed the FoxP3-expressing populations in the lymph nodes of CD18–/– mice. The defects in percentage and number of FoxP3-expressing CD4+ T cells in the MLN of CD18–/– mice (Fig. 3, E and F) parallel that observed with CD4+CD25+ T cells.



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  		FIGURE 3. CD18–/– mice have a decreased number of CD4+CD25+ T cells in their secondary lymphoid organs. PLN and MLN from CD18+/– and CD18–/– mice were harvested and stained for expression of CD25 and CD4 (A). Bar graphs indicate (B) the absolute number of CD4+ T cells and (C) the percentage and (D) absolute number (= % CD4+CD25+ T cells x total number of lymphoid cells) of CD25+CD4+ T cells in each lymphoid organ for CD18+/– (n = 23) and CD18–/– (n = 23) mice. PLN and MLN were assessed for CD4+FoxP3+ T cell populations by (E) the percentage (% CD4+FoxP3+ T cells) and (F) the absolute number (= % of CD4+FoxP3+ T cells x total number of lymphoid cells) ± SEM in each lymphoid organ for CD18+/– (n = 6) and CD18–/– (n = 6) mice. **, p < 0.01; ***, p < 0.001.

 
The decrease in peripheral CD4+CD25+ T cells in CD18–/– mice may be secondary to defects in either thymic or peripheral CD4+CD25+ T cell development/expansion. To discriminate between these mechanisms, we first assessed defects in thymic CD4+CD25+ T cells development. The percentage (Fig. 4A) and absolute number (Fig. 4B) of single positive (SP) CD4+CD25+ thymocytes was decreased in CD18–/– mice. Notably, the percentage of single and double positive thymic subsets was equivalent in CD18+/– and CD18–/– littermate controls (data not shown). Thus, together with the observed normal V{beta} repertoire, these data indicate that there is not a global defect in thymic selection. To determine whether the CD4+CD25+ thymocytes that do develop in CD18–/– mice activate appropriate transcriptional pathways present in regulatory T cells, we assessed FoxP3 mRNA levels on SP CD4+CD25+ thymocytes. CD18–/– SP CD4+CD25+ thymocytes expressed FoxP3 mRNA at levels equal to CD18+/– SP CD4+CD25+ thymocytes (Fig. 4C). We further find that the CD4+CD25+ T cells in peripheral spleen of CD18–/– mice expressed FoxP3 mRNA (Fig. 4D) and protein (Fig. 4, E and F) at levels equivalent to those of CD18+/–CD4+CD25+ T cells. Thus, SP CD4+CD25+ thymocytes, which do develop in CD18–/– mice, have appropriate levels of FoxP3, but the absence of CD18 limits efficient development of the number of SP CD4+CD25+ thymocytes.



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  		FIGURE 4. CD18–/– mice have a decreased number of SP CD4+CD25+ thymocytes. Thymocytes from CD18+/– (n = 25) or CD18–/– (n = 25) mice were assessed for single positive CD4+CD25+ populations by flow cytometry. The data are indicated as (A) a percentage and (B) an absolute number (% SP CD4+CD25+ thymocytes x number of thymocytes) ± SEM. C, SP CD4+CD25+ thymocytes from CD18+/– (n = 3) or CD18–/– (n = 3) mice or (D) CD4+CD25+ splenocytes from CD18+/– (n = 6) or CD18–/– (n = 6) mice were sorted by MO-FLO, and FoxP3 mRNA levels were assessed by real-time PCR. The fold increase in FoxP3 levels are calculated relative to CD4+CD25– cells, and samples are normalized to GAPDH. Splenocytes from CD18+/– (n = 6) or CD18–/– (n = 6) mice were assessed for intracellular FoxP3 levels by flow cytometry. The data are indicated as representative flow cytometry gated on CD4+ T cells for expression of CD25+ and FoxP3 (or isotype control) (E) and percentage of CD4+CD25+ T cells expressing FoxP3 ± SEM in comparison to CD4+CD25– T cells (F). *, p < 0.05; ***, p < 0.001.

 
CD18–/– mice demonstrate defects in development of peripheral CD4+CD25+ T cells to low-dose Ag

The number of CD4+CD25+ T cells increase in response to low doses of peripheral Ag (10, 11, 12, 13, 37). In particular, adoptively transferred DO11.10 RAG–/– CD4+ T cells develop an increased percent of CD4+CD25+ T cells in response to low-dose OVA peptide in the spleen and PLN, and these cells function as suppressor T cells (11). CD18+/– and CD18–/– DO11.10 RAG-1–/– mice have equivalent and low numbers of CD4+CD25+ splenocyte T cells (<1.0%) before activation. To assess whether CD18–/–CD4+ T cells can expand the CD4+CD25+ T cell population after immunization with low-dose Ag, we adoptively transferred CD18+/– or CD18–/–CD4+RAG-1–/– DO11.10 T cells into BALB/c mice. The mice were immunized with 5 µg/ml OVA peptide, and 8 days after immunization, spleen and PLN were harvested. Mice transferred with CD18–/–CD4+RAG-1–/– DO11.10 T cells developed a lower percentage of CD4+CD25+ T cells in the spleen and PLN upon low-dose OVA immunization in comparison to mice transferred with CD18+/– T cells (Fig. 5A). These results correlate to a decreased number of accumulated CD18–/–CD4+CD25+ T cells in the lymphoid organs (Fig. 5B). As we used DO11.10 CD4+ T cells (i.e., not on a RAG-1–/– background) in our previous experiments, we also assessed whether these cells demonstrated a similar defect. We found a defect in the development of CD4+CD25+ T cells (percentage and absolute number) under low-dose OVA immunization when using CD18–/–CD4+ DO11.10 T cells as well (data not shown).



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  		FIGURE 5. CD18–/–CD4+ DO11.10 RAG-1–/– T cells have a defect in the development of CD4+CD25+ peripheral T cells after low-dose OVA immunization. Freshly isolated RAG-1–/– DO11.10 CD4+ splenocyte T cells from CD18+/– or CD18–/– mice were adoptively transferred into WT BALB/c mice. Mice were immunized 24 h later with i.v. 5 µg of OVA peptide, and 8 days later, spleen and PLN were harvested, and (A) the percentage and (B) the absolute number (= % CD4+CD25+ T cells x total number of lymphoid cells) of DO11.10 CD4+CD25+ T cells were assessed. The data are represented as ± SEM (n = 3 mice). The data are representative of two independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 
CD18–/–CD4+CD25+ Treg cells demonstrate defects in mediating suppression in vitro

To investigate whether CD18 plays a role in the ability of CD4+CD25+ T cells to mediate suppressive function, we isolated CD4+CD25+ T cells from the spleen of CD18+/– or CD18–/– mice. In comparison to CD18+/–CD4+CD25+ T cells, CD18–/–CD4+CD25+ T cells were less efficient at mediating in vitro suppression of CD4+ T cell activation upon stimulation with anti-CD3 and irradiated wild-type (WT) splenocytes (Fig. 6A). The CD62Lhigh subfraction has been demonstrated to contain a greater degree of suppressor activity (17, 38). Therefore, we also assessed whether the defect persisted even when CD18–/–CD4+CD25+CD62Lhigh T cells were used as regulatory T cells and found a defect in the suppression of this subset as well (Fig. 6B). Thus, CD18–/–CD4+CD25+ T cells clearly demonstrate a defect in mediating suppression in vitro.



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  		FIGURE 6. CD18–/–CD4+CD25+ T cells have a defect in suppressor function in vitro. WT CD4+CD25– T cells were cocultured with 1 µg/ml anti-CD3 and irradiated splenocytes, along with an increasing number of either CD18+/– or CD18–/– (A) CD4+CD25+ or (B) CD4+CD25+CD62Lhigh T cells. Thymidine incorporation was measured during the last 18 h of a 72-h assay. The ratio of CD4+CD25– T cells to CD4+CD25+ T cells is indicated. The data are represented as counts per minute ± SEM. The data are representative of four and two independent experiments, respectively.

 
CD18–/–CD4+CD25+ T cells have in vivo suppressor function defects

To determine whether the defect in CD18–/–CD4+CD25+ T cell function was also present in vivo, we used the CD4+CD25– T cell transfer into RAG-1–/– mice model of colitis. The cotransfer of CD4+CD25+ T cells is able to prevent the colitis caused by the transfer of pathogenic T cells into lymphopenic mice (36). Therefore, we are able to investigate the role of CD18 specifically on the CD4+CD25+ T cell subset in vivo. Consistent with published results, the defect in weight gain observed by the transfer of CD18+/–CD4+CD25– T cells was attenuated upon the transfer of CD18+/–CD4+CD25+ T cells (Fig. 7A). Remarkably, CD18–/–CD4+CD25+ T cells were not able to mediate the same degree of protection in CD4+CD25–-transferred mice (Fig. 7A). The inability of CD18–/–CD4+CD25+ T cells to prevent disease was confirmed by histopathological examination. These mice exhibited chronic mucosal injury, as evidenced by crypt architectural distortion, surface ulceration, and transmural disease that included wall thickening and inflammatory infiltration of the mucosa and muscularis propria that was similar to mice receiving only CD4+CD25– T cells (Fig. 7B). In contrast, these changes were diminished significantly by cotransfer of CD18+/–CD4+CD25+ T cells. When disease activity present in histologic sections was assessed semiquantitatively, it was also apparent that cotransfer of CD18+/–CD4+CD25+ T cells diminished activity while cotransfer of CD18–/–CD4+CD25+ T cells did not (Fig. 7C). This was further corroborated by the accumulation of colonic CD4+ LPL (Fig. 7D) and splenic CD4+ T cells (Fig. 7E), which has been previously correlated with the severity of colitis (39). In light of the increased trafficking of CD18–/–CD4+ T cells to the spleen (22), we considered that the increased number of CD4+ T cells in the mice cotransferred with CD18–/–CD4+CD25+ T cells may be due to the trafficking of these cells to the spleen. However, in mice transferred with CD18+/–CD4+CD25– T cells + CD18–/–CD4+CD25+ T cells, >90% of the CD4+ T cells in the spleen and >95% CD4+ T cells in the colon were CD18+/– (data not shown). As a result, the expansion of CD4+ T cells in the spleen and the colon is due to the disease-inducing CD18+/–CD4+CD25–-transferred cells. Taken together, these results demonstrate that CD18–/–CD4+CD25+ T cells are unable to prevent CD4+CD25–-induced colitis.



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  		FIGURE 7. CD18–/–CD4+CD25+ T cells from CD18–/– demonstrate a defect in protection from WT CD4+CD25–-induced colitis after transfer into RAG-1–/– mice. A total of 5 x 105 CD18+/– CD+CD25– T cells were transferred into RAG-1–/– mice alone or with 1 x 105 CD18+/– or CD18–/–CD4+CD25+ T cells. A, Weekly weights were measured. Line labels indicate the cotransferred CD4+CD25+ T cells. B, Mice were sacrificed at 8–9 wk, and H&E staining of the colon specimens was performed. A low power exam of the mucosa demonstrates that CD18+/–CD25– T cells transferred alone show transmural inflammation, intestinal wall thickening, and crypt architectural distortion (scale bar = 200 µM). Higher power examination of the mucosal surface (scale bar = 50 µM) demonstrates intraepithelial neutrophils and sheets of overlying neutrophils, while examination of the muscularis propria (scale bar = 50 µM) reveals extensive inflammatory infiltration. Mice in which CD18+/–CD25– T cells were cotransferred with CD18+/–CD25+ T cells demonstrate amelioration of all aspects of disease (note that the intestinal wall is substantially thinner at the same magnification as the diseased colon), whereas cotransfer with CD18–/–CD25+ offered no significant protection. C, Pathology scores are indicated. Numbers of CD4+ T cells in (D) the LPL and (E) the spleen were assessed. Of note is that one mouse in the group transferred with CD18+/–CD25– T cells alone, and two mice in the group cotransferred with CD18+/–CD25– and CD18–/–CD25+CD4+ T cells died before completion of the study. These mice demonstrated weight loss. Data are represented as CD4+ T cell numbers ± SEM. n = 4–6 for each condition assessed. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The inflammatory manifestations observed in CD18-deficient mice have been attributed to the susceptibility of these mice to infection. However, CD18–/– mice demonstrate certain indications of defects in regulatory immune homeostasis, including that some inflammatory disease persists even in germfree facilities (31). Thus, we considered the hypothesis that CD18–/– mice might have defects in Treg function. We find that CD18 deficiency leads to an increased propensity toward autoimmunity. Our data clearly demonstrate that CD18 is required for the development of optimal numbers of innate thymic CD4+CD25+ T cells and induced peripheral CD4+CD25+ T cells. In addition, CD18 expression by CD4+CD25+ T cells is required for optimal suppressor function in vitro and is necessary for the in vivo CD4+CD25+ T cell-mediated protection of CD4+CD25–-induced colitis. The development of intestinal inflammation has also been described in humans with CD18 dysfunction in the disease LAD (25).

Our data show that CD18 is required for optimal numbers of CD4+CD25+ T cells in the thymus. The requirement for CD18 in CD4+CD25+ thymic development might be on various hematopoietically derived cells within the thymus. IL-2, CD28, B7, and CD40 contribute to thymic development of CD4+CD25+ Treg cells (4, 40, 41, 42, 43). CD4+CD25+ thymic development also depends on MHC class II (MHC II), and these cells have more efficient interactions with MHC II peptides (8, 40, 44, 45, 46). CD4+CD25+ thymocytes are thought to represent a population of thymocytes, which are selected for their higher affinity for MHC II:self-peptide. This may account for the specific decrease in the SP CD4+CD25+ thymocyte population, in the context of normal percentages of CD4+, CD8+, and CD4+CD8+ thymocytes (data not shown; Ref.28) in CD18–/– mice. Numerous studies have demonstrated that LFA-1 enhances the engagement of the TCR for its MHC II:peptide ligand, thereby allowing for a 10–100 times decrease in dose of MHC II:peptide Ag in the activation of mature T cells (20, 47, 48). The absence of CD18 likely reduces the thymocyte affinity/avidity for MHC:self-peptide, which may simply shift the threshold of selection in the total thymocyte population but result in a selective defect in the CD4+CD25+ T cell population. The equivalent FoxP3 expression on a per cell basis among those SP CD4+CD25+ thymocytes present argues that those cells selected are, in fact, regulatory cells.

Our data also demonstrate that CD18 is required for induction of optimal numbers of CD4+CD25+ T cells in the periphery to low-dose Ag. Peripheral expansion of CD4+CD25+ T cells has been shown to occur under various conditions, including upon immunization with low-dose peptide, upon oral Ag exposure, with lymphopenic proliferation, and upon exposure to TGF-{beta} (11, 37, 49, 50). This peripheral expansion may be due to proliferation of existing CD4+CD25+ T cells, as well as an education of CD4+CD25– T cells (51). Physiological maintenance of peripheral CD4+CD25+ T cells requires the presence of selecting self-peptide, B7-1/-2 and IL-2 (42, 52). Given the known defect in the activation and IL-2 production of CD18–/– DO11.10 CD4+ T cells upon peripheral immunization (22), possibilities for the defect we observe in the induction of peripheral CD18–/– DO11.10 CD4+CD25+ T cells include inadequate TCR activation and IL-2 production.

We show that CD18–/–CD4+CD25+ T cells have a clear defect in suppressor function in vitro. The development of CD18–/–CD4+CD25+ T cells into Treg cells appears to be intact given the equivalent expression of the essential transcription factor for regulatory function, FoxP3, in CD18+/– and CD18–/–CD4+CD25+ thymocytes and peripheral CD4+CD25+ T cells. However, we cannot completely exclude the possibility that the absence of CD18 results in an indirect effect on any number of possible molecules, which, in turn, contributes to defects in suppressor function. Attempts to selectively block LFA-1 on WT CD4+CD25+ T cells in the in vitro suppression assay and in vivo are confounded by the direct effects of blocking Abs on the proliferation of the responder CD4+CD25– T cells, as LFA-1:ICAM-1 interactions are required on this population for optimal proliferation (data not shown). The mechanisms through which CD4+CD25+ Treg cells mediate their functions are not completely understood. However, studies in human and murine CD4+CD25+ T cells have identified prevention of dendritic cell (DC) maturation, interactions through latent Ag gene of TGF-{beta}, lysis of autologous cells, and the induction of IDO in DC as putative mechanisms whereby these cells contribute to suppression of T cell activation (53, 54, 55, 56). A recent report describes the ability of human Treg cells to mediate autologous target cell death and that this process depends on CD18 (57). It has not yet been reported whether this process also occurs in murine systems. LFA-1 is expressed at high levels on CD4+CD25+ T cells (14). The deficiency of LFA-1 on CD4+CD25+ T cells may result in defective regulatory function through a variety of mechanisms, including a defect in: 1) the cell-cell contact required by CD4+CD25+ T cells to mediate their function; and 2) adequate engagement of the TCR on CD4+CD25+ T cells. The defect in cell-cell contact could result in suboptimal effects on target cells, such as DC, with a defect in the generation of suppressor DC (55) (i.e., induction of IDO, down-regulation of costimulatory molecules on DC) or in the interaction with surface TGF-{beta}. Regulatory activity of T cells has been shown to correlate with possession of high avidity for MHC:self-peptide complexes (28). LFA-1 on the cell surface of CD4+CD25+ T cells may be providing for enhanced avidity, leading to improved competition of CD4+CD25+ T cells for their ligand. In addition, as engagement of the TCR on CD4+CD25+ T cells is required for their function, a defect in this engagement in the absence of CD18 may result in a defect in the regulatory function of the Treg cells.

The disruption of LFA-1:ICAM-1 interactions in vivo affects a wide variety of cell:cell interactions such that the impact on a given process is ultimately an integration of these effects. In fact, given the importance of LFA-1:ICAM-1 interactions in the proper activation of effector T cells, inhibition of these interactions has resulted in the amelioration of a number of inflammatory diseases, including various Th1-mediated diseases and murine models of lupus (58, 59, 60, 61). A similar complexity exists in CD28–/– mice. CD28–/– plays a critical role for effector T cell responses, and CD28–/– mice demonstrate numerous defects in T cell activation, function, and amelioration of various inflammatory diseases (62). However, careful studies revealed a reduced number of CD4+CD25+ T cells with increased autoimmunity under specific circumstances, such as in the NOD background (4) and a failure of CD28–/–CD4+CD25+ thymocytes to demonstrate suppressor function (43). The defect in the ability of CD18–/–CD4+CD25+ T cells to protect from CD4+CD25–-mediated colitis may be due to mechanisms other than those addressed in vitro and unique to the in vivo conditions of this model. Upon CD4+CD25+ T cell transfer into RAG-1–/– mice, the cells undergo lymphopenic proliferation, and CD4+CD25+ T cells, which have undergone lymphopenic proliferation, develop enhanced regulatory function (63). LFA-1 may be playing a role in this process. Furthermore, LFA-1 is critical for optimal trafficking of CD4+ T cells to the PLN, MLN, and various peripheral tissues, including the intestine (19, 22, 64). The trafficking patterns of CD4+CD25+ T cells have not been clearly defined. There have been certain studies examining specific chemokine receptors and the capacity to migrate to various chemokines under particular conditions (14, 65). In addition, the expression of a host of different adhesion molecules have been identified on the surface of CD4+CD25+ Treg cells (14). However, the necessity of these trafficking molecules for trafficking to specific lymphoid organs and tissues under various conditions has not been fully defined.

In conclusion, these studies demonstrate that CD18 is required for the development of optimal numbers of thymic and peripheral CD4+CD25+ T cells, as well as the optimal function of murine CD4+CD25+ T cells in vitro and in vivo. The absence of CD18 on CD4+CD25+ T cells may lead to defects in immune homeostasis and contribute to the development of autoimmune diseases. In addition, in the use of blocking anti-CD18 or anti-LFA-1 Abs in clinical disease, one needs to consider potential consequences on CD4+CD25+ T cells.


   Acknowledgments
 
We thank Marisa Alegre, Yang-Xin Fu, and Alexander Khoruts for helpful discussions; Marisa Alegre, Judy Cho, Anita Chong, Ellen Chuang, Yang-Xin Fu, Thomas Gajewski, and Lisa Sevilla for critical reading of the manuscript; and Arthur Beaudet for providing reagents.


   Disclosures
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 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 National Institutes of Health Grants DK02905 (to C.A.) and DK61931 and DK68271 (to J.R.T.), University of Chicago Digestive Disease Center Grant DK42086, and the Crohn’s and Colitis Foundation of America and Louis Block Fund (to C.A.). Back

2 Address correspondence and reprint requests to Dr. Clara Abraham, Department of Medicine, Section of Gastroenterology, 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: Treg, T regulatory; LAD, leukocyte adhesion deficiency; PLN, peripheral lymph node; MLN, mesenteric lymph node; SP, single positive; WT, wild type; LPL, lamina propria lymphocyte; MHC II, MHC class II; DC, dendritic cell. Back

Received for publication June 15, 2005. Accepted for publication October 1, 2005.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
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J. D. Glawe, D. R. Patrick, M. Huang, C. D. Sharp, S. C. Barlow, and C. G. Kevil
Genetic Deficiency of Itgb2 or ItgaL Prevents Autoimmune Diabetes Through Distinctly Different Mechanisms in NOD/LtJ Mice
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D. Q. Tran, D. D. Glass, G. Uzel, D. A. Darnell, C. Spalding, S. M. Holland, and E. M. Shevach
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J. Clarencio, C. I. de Oliveira, C. Favali, O. Medina, A. Caldas, C. H. Costa, D. L. Costa, C. Brodskyn, A. Barral, and M. Barral-Netto
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Y. Onishi, Z. Fehervari, T. Yamaguchi, and S. Sakaguchi
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Y. Liang, C. Liu, J. Y. Djeu, B. Zhong, T. Peters, K. Scharffetter-Kochanek, C. Anasetti, and X.-Z. Yu
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X. Wu, R. Guo, Y. Wang, and P. N. Cunningham
The role of ICAM-1 in endotoxin-induced acute renal failure
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R. Castriconi, A. Dondero, C. Cantoni, M. Della Chiesa, C. Prato, M. Nanni, M. Fiorini, L. Notarangelo, S. Parolini, L. Moretta, et al.
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H. W. Lim, H. E. Broxmeyer, and C. H. Kim
Regulation of Trafficking Receptor Expression in Human Forkhead Box P3+ Regulatory T Cells
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K. Wing, Z. Fehervari, and S. Sakaguchi
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