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The Journal of Immunology, 2004, 173: 6294-6302.
Copyright © 2004 by The American Association of Immunologists

Maximum Immunobioactivity of Murine Small Intestinal Intraepithelial Lymphocytes Resides in a Subpopulation of CD43+ T Cells1

Heuy-Ching Wang*, Dina Montufar-Solis*, Ba-Bie Teng{dagger} and John R. Klein2,*

* Department of Diagnostic Sciences, Dental Branch, and {dagger} Research Center for Human Genetics, Institute of Molecular Medicine, University of Texas Health Science Center, Houston, TX 77030


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
CD43 has been linked to many function-associated T cell activities. Using mAbs that recognize two different CD43 determinants, we show that, although mouse small intestinal intraepithelial lymphocytes (IELs) expressed the CD43 core molecule reactive with mAb R2/60, only about one-half of the total IELs—including some but not all of the TCR{alpha}{beta} and TCR{gamma}{delta} cells—expressed the CD43 S7– reactive determinant. CD43 S7+ IELs secreted more IL-2, IL-4, IL-10, IL-17, and IFN-{gamma} following anti-CD3 stimulation, and were >4-fold more cytotoxic in fresh isolates and >16-fold more cytotoxic after anti-CD3 stimulation, than S7– IELs. S7+ but not S7– IELs from the ileum of IL-10–/– mice spontaneously produced IFN-{gamma}. In vivo BrdU uptake by IELs in non-Ag-primed mice was greatest in the S7+ population, indicating that significantly more S7+ IELs than S7– IELs undergo cell expansion under normal homeostatic conditions. DNA microarray analyses showed that S7+ IELs expressed higher levels of genes associated with activated T cells, whereas S7– IELs expressed genes used in the regulation of NK cells. These findings define two functionally distinct populations of IELs based on CD43 expression independent of TCR class, and they identify a subset of IELs that may serve as a target to better control intestinal inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Murine small intestinal intraepithelial lymphocytes (IELs)3 exist in a novel state of activation. This can be seen, for example, by the fact that freshly isolated IELs without deliberate in vitro activation have cytotoxic activity (1, 2, 3, 4, 5), yet they do not undergo proliferation or secrete T cell cytokines until after a TCR/CD3-mediated signal has been received (4, 5, 6). Within 12–24 h of CD3-mediated activation, however, IELs secrete IFN-{gamma} more quickly and to higher levels than CD3-stimulated lymph node T cells (5). Consistent with that, murine IELs express CD69 in vivo and ex vivo (5, 7), although they lack other markers commonly associated with activated T cells, including Ly6C and CD25 (5). Further evidence for an unconventional activation state of IELs is supported by studies using DNA microarray analyses of {gamma}{delta} IELs in Yersinia pseudotuberculosis-infected and uninfected mice (8), and by serial analyses of gene expression of {alpha}{beta} and {gamma}{delta} IELs (9). Of interest, in both studies, IELs expressed genes associated with T cell activation while concomitantly expressing genes expressed during the deactivation of T cells. However, IELs are phenotypically and functionally heterogeneous. Thus, analyses of cells based on TCR{alpha}{beta} or TCR{gamma}{delta} differences alone could easily reflect functionally different subsets, or cells at different stages of differentiation, within those groups.

CD43 is a ubiquitous but enigmatic molecule on thymocytes, peripheral T cells, and B cells, and is also found on early myeloid and lymphoid bone marrow hemopoietic precursors (10, 11, 12, 13). CD43 has been functionally linked to a variety of T cell activities, including coactivation and enhanced cytotoxicity of CD8+ T cells (14, 15, 16). Moreover, CD43 is known to exert positive or negative regulatory effects depending upon when CD43 stimulation occurs during the immune response (17, 18, 19). In lymphocytic choriomeningitis virus-infected CD43–/– mice, for example, CD8+ effector T cells were generated with the same efficiency as in CD43+/+ mice, but were less capable of expansion of naive Ag-specific T cells, had a slight delay in effector cell trafficking into the CNS, and displayed a significantly retarded contraction phase of the immune response after infection (19). Additionally, high levels of CD43 expression on CD4+ T cells have been shown to curtail activation-induced cell death (20). Interestingly, T cell proliferation and activation in CD43-deficient mice is elevated rather than depressed (17, 18), as would be expected from the costimulatory effects in normal mice. Taken together, these findings point to dynamic regulatory effects of CD43 depending upon the circumstances of the immune response and the type of T cells involved.

Previous studies from our laboratory examined the expression of CD43 on murine IELs from normal mice and from mice with graft-vs-host disease (21). That study revealed the surprising finding that the pattern of CD43 expression differed for IELs compared with peripheral T cells in that, although all murine IELs were reactive with mAb R2/60, an Ab that reacts with the CD43 core molecule, only about one-half of the IELs expressed the neuraminidase-sensitive CD43 determinant recognized by mAb S7. By comparison, all peripheral T cells expressed the S7 determinant (21). In an effort to understand that disparity for IELs, we have conducted studies using populations of IELs differentiated by S7 expression. As reported here, there were major differences among IELs such that S7+ cells 1) synthesized significantly more IL-2, IL-4, IL-10, IL-17, and IFN-{gamma}, 2) were more cytolytic in redirected cytotoxicity assays, 3) had markedly greater in vivo proliferation, and 4) displayed major differences in lymphocyte gene activation profiles than S7– IELs. These findings thus identify populations of IELs that can be applied to studies aimed at more precisely understanding IELs according to their state of activation, and they may have value for therapeutically targeting specific IEL subsets under conditions of nonphysiological inflammation or autoimmunity.


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

Adult female BALB/c and C57BL/6 mice were purchased from Harlan Sprague Dawley (Houston, TX). IL-10–/– (B6.129P2-Il10tm1Cgn/J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were used in accord with the University of Texas Animal Welfare Guidelines.

Isolation and purification of IELs

Isolation of small intestine IELs was done as previously described (4, 5). Briefly, after tissue digestion with 2 mM DTT and 5 mM EDTA, IELs were separated from epithelial cells by centrifugation over a 40/70% Percoll gradient. For IEL fractionation into S7+ and S7– populations, IELs were stained by direct labeling at 4°C with PE-anti-CD43 (S7) and sorted using a Vantage SE Turbo high-speed cell sorter (BD Biosciences, Mountain View, CA). Cell sorting was done by first selecting the IEL population according to parameters of forward- and right-angle scatter, which we have previously determined to include >95% CD3+ cells, and second, by gating onto the S7+ and S7– subsets.

Abs and flow cytometry

Abs used in this study were as follows: purified NA/LE anti-CD3 (145-2C11); FITC-anti-TCR{beta} (H57-597); FITC-anti-TCR{delta} (GL3); CyChrome-anti-CD8{alpha} (53-6.7); biotynylated-anti-CD8{beta} (53-5.8); purified, PE-, FITC-, and biotin-anti-CD43 (S7); FITC-CD44 (IM7); purified NA/LE anti-hamster Ab (G94-56); streptavidin-allophycocyanin; strepavidin-CyChrome; streptavidin-PE; FITC-anti-BrdU; GolgiStop (monensin) (BD Pharmingen, San Diego, CA); FITC-anti-IL-4 (BVD6-24G2); FITC-anti-IL-10 (JES5-16E3); PE-anti-IFN-{gamma} (XMG1.2); FITC control (eBRG1); PE control (eBRG1); PE-anti-CD94 (18d3); PE-anti-NK (Ly-49F/I/C/H) (eBioscience, San Diego, CA). Anti-CD43 mAb R2/60 was biotinylated in our laboratory. Alkaline phosphatase anti-rat IgG was purchased from Southern Biotechnology Associates (Birmingham, AL). Flow cytometric analyses were done on a FACSCalibur flow cytometer using CellQuest software (BD Biosciences). Intracellular staining was done using IELs cultured in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS (Sigma-Aldrich) for 24 h with or without anti-CD3 stimulation as previously described (4). Monensin (1 µl/ml) was added for the last 5 h of culture. Intracellular staining of IELs from 14-mo-old IL-10–/– mice was done using cells recovered from the lower one-third of the small intestine, followed by in vitro culture in RPMI 1640 containing 10% FBS and 1 µl/ml monensin for 5 h to allow for intracellular accumulation of cytokines. Cells were collected and stained for CD43 S7, IFN-{gamma}, and IL-4, or IL-10.

Enzyme-linked immunoassays and redirected cell-mediated cytotoxicity assay

Commercial cytokine assay kits for IL-2, IL-4, IL-10, IL-17, and IFN-{gamma} were purchased from eBioscience. S7+ and S7– IEL populations were obtained by flow cytometric cells sorting. In vitro anti-CD3 stimulation of IELs was done by coating plates overnight at 4°C with 10 µg/ml anti-hamster Ig in PBS. Wells were washed, and 1 µg/ml anti-CD3 mAb was added in PBS for 2 h at 37°C followed by 20-min blocking with 50% newborn calf serum. Cell-free supernatants were collected after 24 h and assayed for the presence of cytokines. Redirected cell-mediated assays were done as previously reported (4, 5) using S7+ and S7– IELs purified by flow cytometric cell sorting. For determining the effects of anti-CD3 stimulation on the cytotoxic activity of S7– and S7+ IELs, cell-sorted populations were cultured for 48 h in plates coated with anti-CD3 mAb (4). Viable cells were collected and assayed in the redirected cytotoxicity assay.

Western blot

A total of 107 whole unfractionated IELs, or IELs depleted of S7+ cells by MACS (autoMACS; Miltenyi Biotec, Auburn, CA), were solubilized in 50 µl of detergent buffer consisting of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM NaF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin, and 1% Nonidet P-40 (Sigma-Aldrich). A volume of 12.5 µl of cell lysates was mixed with 2x sample buffer (Bio-Rad, Hercules, CA) containing 5% 2-ME (Sigma-Aldrich) and boiled for 5 min. A volume of 15 µl of sample was added to precast 7.5% SDS-glycine cells (Bio-Rad). After electrophoresis, proteins were transferred to PVDF membranes (Bio-Rad). Membranes were blocked overnight with 5% nonfat dry milk in PBS, washed, and reacted for 2 h with 1 µg/ml purified S7 mAb, and washed and reacted for 2 h with 1/2000 dilution of alkaline phosphatase anti-rat IgG. Membranes were washed, developed using an ECL protocol (ICN Pharmaceuticals, Costa Mesa, CA), and exposed to Kodak (Rochester, NY) x-ray photographic film.

In vivo BrdU labeling

Mice were injected i.p. twice 4 h apart with 1 mg of BrdU (Sigma-Aldrich) suspended in PBS. Twenty-four hours after the last injection, IELs were recovered and stained for intracellular BrdU uptake using FITC-anti-BrdU Ab with PE-anti-CD43 Ab.

DNA microarrays

IELs, enriched for S7+ and S7– cells by high-speed cell sorting, were washed in PBS and lysed with buffer provided in the RNeasy mini-kit 50 (Qiagen, Valencia, CA). Three independent samples were used for S7– gene chip analyses, and two independent samples were used for S7+ gene chip analyses. Each sample consisted of cell-sorted IELs from three to four mice. RNAs were extracted and stored at –80°C until a sufficient amount was obtained to yield 8 µg for each sample. cDNAs were prepared from samples. The first-strand cDNA was primed with a T7-(dT)24 primer at 70°C and catalyzed by SuperScript II RT (Invitrogen Life Technologies, Carlsbad, CA) at 42°C. The second-strand cDNA was synthesized by nick translational replacement in the presence of Escherichia coli DNA polymerase, E. coli RNase H, and E. coli DNA ligase (Invitrogen Life Technologies). To synthesize the biotin-labeled cRNA, the dsDNA was used as template for in vitro transcription with Enzo Bioarray High Yield RNA Transcription Labeling kit (Affymetrix, Santa Clara, CA). Fragmentation of the labeled cRNA was done at 95°C before the hybridization mixture was generated; the hybridization mixture was made by adding 10 µg of biotin-labeled cRNA from each sample along with Eukaryotic Hybridization Controls and Control Oligonucleotide B2 (Affymetrix). The mixture was then hybridized to GeneChip Murine Genome U74Av2 (Affymetrix). Chips were washed and stained with streptavidin-PE (Molecular Probes, Eugene, OR). Signals were amplified by hybridizing with biotin-labeled anti-streptavidin-PE (Vector Laboratories, Burlingame, CA) and restained with streptavidin-PE (Molecular Probes). The arrays were scanned with GeneArray Scanner and visualized with Microarray Suite, version 5.0 (Affymetrix). Gene expression levels were averaged for the three S7– samples and for the two S7+ samples for determination of fold-change values and p values. Statistical analysis was done using Data Mining Tool, version 3 (Affymetrix); statistical significance was defined as ≥2-fold increase with a p value of ≤0.05, similar to that previously reported (22). Absolute call values for gene expression levels were determined for the five samples using Gene Expression MAS 5.0 software (Affymetrix), using default values provided by the manufacturer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
CD8+ IELs express the CD43 core molecule but differentially express a murine CD43 S7 isoform

Studies from our laboratory demonstrated that the CD43 determinant recognized by the S7 mAb is expressed on 85–90% of all CD8+ peripheral T cells in mice (16). However, although all IELs expressed CD43 as seen by reactivity with mAb R2/60 (Fig. 1), the S7 isoform was differentially expressed on small intestinal IELs. Thus, the S7 determinant was expressed on 99.2% of the TCR{alpha}{beta}+CD8{beta}+ IELs (Fig. 2, A and B), and on 80.7% of the TCR{alpha}{beta}+CD8{alpha}+ IELs (A and C). A total of 19.3% of the TCR{alpha}{beta}+CD8{alpha}+ cells were S7– cells (Fig. 2, A and C). In contrast, 42.4% of the TCR{gamma}{delta}+CD8a+ IELs were S7+ cells (Fig. 2, D and F), and 57.6% were S7– cells (D and F). The overall distribution of S7 expression on CD8{alpha} and CD8{beta} IELs is shown in Fig. 2, G–I.



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  		FIGURE 1. Nearly all murine small intestinal IELs express the CD43 core molecule recognized by mAb R2/60. Data are representative of four independent experiments.

 


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  		FIGURE 2. The CD43 S7 Ag is differentially expressed on murine IELs. A–C, The CD43 S7 Ag is expressed on nearly all TCR{alpha}{beta}+CD8{beta}+ IELs, and most but not all TCR{alpha}{beta}+CD8{alpha}+ IELs. D–F, TCR{gamma}{delta}+CD8{alpha}+ IELs consist of both S7+ and S7– subsets. G–I, Three-color staining demonstrating that CD8{beta}+ IELs are S7+ cells, whereas the S7 determinant is differentially expressed on CD8{alpha}+ IELs. Data are representative of five independent experiments.

 
The fact that not all IELs express the S7 Ag by flow cytometry did not formally rule out the possibility that the S7 determinant was expressed on S7– cells, because the lack of staining could be due to a failure of mAb S7 to react with some IELs. To address this, Western blotting was done using the S7 mAb with cell lysates from whole IELs (i.e., cells that included the S7+ population), and IELs from which the S7+ subset had been removed by negative MACS sorting. As seen in Fig. 3, the S7 mAb was nonreactive by Western blotting for cell lysates from S7-depleted IELs, whereas lysates from unfractionated IELs identified a 114- to 118-kDa component characteristic of that recognized by mAb S7 (21), thereby confirming the lack of expression of the S7 determinant on CD43 S7– IELs.



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  		FIGURE 3. CD43 S7– IELs are nonreactive with S7 mAb by Western blotting. Cell lysates from equivalent numbers of whole IELs and IELs depleted of S7+ cells by MACS sorting were electrophoresed and blotted with mAb CD43 S7 as described in Materials and Methods. The lack of reactivity of the S7 mAb against S7-depleted cell lysates confirms that the S7 determinant is not expressed on those cells. Data are representative of two independent experiments.

 
Following anti-CD3 activation, S7+ IELs secrete higher levels of cytokines, and both S7+ and S7– populations contain Th1, Th2, and Th0 cells

To determine how S7+ and S7– IELs differ functionally, purified S7+ and S7– IELs were assayed for their ability to secrete five IEL cytokines that are prominently involved in IEL regulation and/or activation (IL-2, IL-4, IL-10, IL-17, and IFN-{gamma}). After isolation and purification by Percoll gradient centrifugation, S7+ and S7– IELs were enriched to >98% purity by high-speed flow cytometric cell sorting. Equal numbers of each population were cultured for 24 h in 24-well tissue culture plates coated with anti-hamster Ig plus hamster anti-CD3 mAb or control mAb. Cell-free supernatants were collected and screened for IL-2, IL-4, IL-10, IL-17, and IFN-{gamma} activities by enzyme immunoassay. Findings from these experiments were notable in that S7+ cells produced significantly higher levels of all five cytokines compared with S7– IELs (Fig. 4), indicating that there was a major difference in the levels of cytokine production by S7+ IELs following anti-CD3 stimulation.



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  		FIGURE 4. CD43 S7+ IELs produce significantly greater levels of IL-2, IL-4, IL-10, IL-17, and IFN-{gamma} than S7– IELs. IELs were enriched into S7+ and S7– populations by flow cytometric cells sorting. Purified cells were stimulated with immobilized anti-CD3 mAb for 24 h; cell-free supernatants were collected and assayed for cytokine activity using enzyme-linked immunoassays. Data are mean values of two independent experiments; bars indicate range of values for both experiments.

 
Because S7+ and S7– IELs secreted both Th1 (IL-2 and IFN-{gamma}) and Th2 (IL-4 and IL-10) cytokines, albeit at quantitatively different levels, it was of interest to determine the extent to which activated IELs were Th1 or Th2 cells, or whether they were Th0 cells that produced both Th1 and Th2 cytokines (23, 24). IELs were cultured for 24 h with or without anti-CD3 stimulation. Cells were collected and stained for three-color flow cytometric analysis based on expression of S7, intracellular IFN-{gamma}, and intracellular IL-4 or IL-10. After anti-CD3 stimulation, the majority of cytokine-secreting cells in both the S7+ and S7– groups were Th1 cells that produced IFN-{gamma} but not IL-4, although proportionally more Th1 cells were present in the S7+ population (Fig. 5A). Additionally, a small proportion of the S7+ and S7– cells were Th2 cells that produced IL-4 only following anti-CD3 stimulation (Fig. 5A). Interestingly, some cells in both the S7+ and S7– populations were Th0 cells that produced IFN-{gamma} and IL-4; this was particularly true for the S7+ cells (Fig. 5A). The pattern for IL-10 staining reflected that observed for IL-4 in that most IL-10-producing S7+ cells also produced IFN-{gamma} (Fig. 5B). Cells cultured without anti-CD3 stimulation did not synthesize IFN-{gamma}, IL-4, or IL-10 (data not shown).



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  		FIGURE 5. Anti-CD3 stimulation of IELs results in Th1, Th2, and Th0 profiles. A and B, IELs were cultured for 24 h with plate-bound anti-CD3 mAb and stained for three-color flow cytometric analysis for surface CD43 S7, intracellular IFN-{gamma}, and intracellular IL-4 or IL-10. In both the S7+ and S7– populations, the majority of IELs were Th1 cells that synthesized IFN-{gamma}. IL-4+ and IL-10+ cells were present among both the S7+ and S7– populations, although proportionally more S7+ cells were IL-4+ or IL-10+ cells. Note the presence of Th0 cells, particularly among the S7+ population. Cursor settings are positioned according to staining with FITC-Ig and PE-Ig control Abs. Data are representative of four independent experiments for each group.

 
CD43 S7+ but not S7– IELs in IL-10–/– mice are the primary source of IFN-{gamma}

Studies involving anti-CD3 stimulation of IELs were relevant for understanding the process of activation as it would occur in response to Ag involving a TCR-mediated signal. It was of interest, however, to also examine the responses of S7+ and S7– IELs during pathophysiological conditions of chronic intestinal inflammation. Because most adult IL-10–/– mice >15 wk of age develop intestinal inflammation in the colon, ileum, and jejunum (25), small intestinal IELs from the ileum of 14-mo-old IL-10–/– mice with rectal prolapse were isolated and cultured for 5 h without anti-CD3 stimulation in RPMI 1640 supplemented with 10% FBS plus monensin to permit the accumulation of intracellular cytokines generated in situ during the inflammatory process. Cells were harvested and stained for CD43 S7 expression and intracellular IFN-{gamma} and IL-4. As seen in Fig. 6A, IELs from the ileum of normal C57BL/6 mice cultured without anti-CD3 stimulation produced neither IFN-{gamma} nor IL-4 (B). In contrast, a significant proportion of S7+ ileal IELs from IL-10–/– mice without anti-CD3 stimulation spontaneously produced IFN-{gamma} (Fig. 6B). Moreover, IFN-{gamma}-producing cells by S7– IELs was <10% of that of S7+ IELs (Fig. 6B). In neither the S7+ nor the S7– IEL subset was there a significant number of IL-4-producing cells (Fig. 6B). These findings make an important point in that they indicate that the basic relationship between S7+ IELs and activation observed for CD3-stimulated IELs also held true in a model of intestinal inflammation induced by cytokine dysregulation.



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  		FIGURE 6. IELs from the ileum of IL-10–/– mice with rectal prolapse spontaneously produce IFN-{gamma} predominantly within the S7+ population. A, Ileal IELs from normal C57BL/6 mice cultured for 5 h without anti-CD3 stimulation fail to synthesize IFN-{gamma} or IL-4. B, Ileal IELs from a 14-mo-old IL-10–/– mice with rectal prolapse cultured for 5 h without anti-CD3 stimulation synthesized IFN-{gamma} in the S7+ population but only minimally in the S7– population, indicating that the S7+ subset is the likely source of proinflammatory cytokines in IL-10–/– with active intestinal inflammation. Data are representative of three IL-10–/– mice.

 
CD43 S7+ cells have higher cytolytic activity and in vivo proliferation than S7– IELs

Small intestinal IELs are known to express cytolytic activity in freshly isolated preparation without overt activation; this can be demonstrated using anti-CD3 or anti-TCR Abs to bridge effector cells to target cells (1, 2, 3, 4, 5). Freshly isolated IELs were enriched by high-speed cell sorting into S7+ and S7– populations and tested for lytic activity in the redirected assay. As seen in Fig. 7A, there were ~4-fold higher levels of cytolytic activity of S7+ cells compared with an equivalent number of S7– cells.



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  		FIGURE 7. CD43 S7+ IELs are more cytotoxic than S7– IELs. A, IELs from normal mice were purified into S7+ and S7– populations by flow cytometric cell sorting, and assayed against P815 target cells in the presence of anti-CD3 mAb. S7+ IELs expressed ~4-fold greater cytotoxic activity than S7– IELs. B, After 48 h of anti-CD3 stimulation, there is ~2-fold increase in the lytic activity of S7– IELs compared with fresh S7– IELs (A); however, there is ~16-fold increase in the lytic activity of the S7+ cell population, indicating that S7+ IELs are more cytotoxic than S7– IELs both before and after CD3-mediated activation. C, A greater proportion of CD43 S7+ IELs undergo in vivo than CD43 S7– IELs in normal nonprimed mice. Twenty-four hours after in vivo BrdU injection, IELs were isolated and stained for intracellular BrdU with anti-CD43 S7 staining. Data are representative of two independent experiments.

 
To determine how CD3-mediated activation might affect the cytotoxic activity of S7– and S7+ IELs, S7– and S7+ cells were purified by flow cytometric cell sorting and were cultured in plates containing immobilized anti-CD3 mAb. After 48 h, cells were collected and assayed for lytic activity in the redirected cytotoxicity assay. Although the lytic activity of both the S7– and the S7+ population increased over that of fresh IELs, there were quantitative differences in the increase in lytic activity of the S7– vs the S7+ cells. Whereas the cytolytic activity of S7– IELs increased by ~2-fold, the activity of the S7+ IELs increased by ~16-fold over that of fresh IELs (Fig. 7, A and B). Additionally, the cytotoxic activity of the S7+ IELs was at least 16-fold greater than that of the S7– IELs in CD3-stimulated cultures (Fig. 7B).

Murine IELs have been shown to undergo cell proliferation in vivo as demonstrated by BrdU incorporation (26). Based on the above findings of heightened cytolytic activity and cytokine secretion by S7+ IELs, we sought to determine whether S7 expression correlated with the in vivo proliferation capacity of IELs. Normal mice were injected twice with BrdU as described in Materials and Methods. Twenty-four hours after the last injection, animals were sacrificed, IELs were recovered, and cells were stained for intracellular BrdU in conjunction with CD43 S7 expression. As seen in Fig. 7C, significantly more CD43 S7+ than S7– IELs incorporated BrdU in vivo, thus indicating that S7+ cells are an actively dividing cell population in vivo, either from a homeostatic proliferation process or possibly due to Ag-driven activation in vivo.

Gene array studies point to significant differences in the functional roles of S7+ and S7– IELs

To more extensively examine the differences in IELs based on CD43 S7 expression, DNA microarray analyses were done using cell-sorted S7+ and S7– IELs. Findings from these studies reinforced the observations that functional differences exist between S7+ and S7– IELs. By three criteria (fold increase; value of p < 0.05; and absolute call for gene expression), S7+ IELs expressed significantly higher levels of genes for a number of functionally relevant genes. The most notable of these were the CD6, CD44, and CD97 cell surface activation markers; CCR2, CXCR3, and MIP-{beta} gene expression; and the cathepsin L protease gene (Table I). S7– IELs also displayed several prominent features. Perhaps the most significant of these was the strong association between S7– cells and the expression of genes for receptors linked to NK cell activation/inhibition (Ly49E-GE, Ly49G.2, CD94/NKG2, Ly49H, and Ly49C) (Table II). It should be noted that, although the majority of the genes with increased gene expression for the S7– also were expressed by S7+ cells (absolute call), gene expression for those nonetheless occurred at significantly higher levels than for S7+ cells (fold increase, and value of p < 0.05). Although this likely points to a real difference between those populations at a functional level, it similarly reinforces the tendency of S7+ IELs to have more broad-ranging effector activities than S7– cells.


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  		Table I. Results of gene array analyses of CD43 S7+ and S7– IEL: genes expressed higher in CD43 S7+ IELs

 

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  		Table II. Results of gene array analyses of CD43 S7+ and S7– IEL: genes expressed higher in CD43 S7– IELs

 
To confirm at the protein level the gene expression patterns observed in Tables I and II, freshly isolated IELs were stained for expression of CD43 S7, CD44, Ly-49F/I/C/H, and CD94. Consistent with the gene array findings, CD43 S7+ cells expressed CD44 at high levels, whereas CD44 was differentially expressed on S7– IELs. Conversely, more than twice as many CD43 S7– IELs expressed the NK markers, Ly-49F/I/C/H and CD94, than S7+ IELs (Fig. 8).



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  		FIGURE 8. To confirm the validity of gene expression patterns observed in Tables I and II, freshly isolated IELs were stained according to S7 expression and CD44, Ly-49F/I/C/H, and CD94. Note that nearly all CD43 S7+ cells were CD44+ cells, whereas CD44 was differentially expressed on S7– IELs. Conversely, a greater proportion of CD43 S7– IELs expressed Ly-49F/I/C/H and CD94 than CD43 S7+ IELs, thus confirming the basic pattern observed from the gene array studies. Data are representative of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The distribution of IELs throughout the intestinal epithelium suggests that they play an important strategic role in the protection against the entry and dissemination of foreign Ag. Yet the extensive phenotypic and functional complexities of the IELs have made it difficult to fully appreciate their involvement in local immunity. The findings described in this study offer new insight into IEL biology by defining functionally distinct populations of cells that differ according to the expression of a CD43 glycoform. The presence of the CD43 S7 Ag on IELs correlated with enhanced activity of three fundamental properties of activated CD8+ T cells, namely, CD3-mediated cytotoxicity, cell proliferation, and CD3-induced cytokine secretion. Interestingly, although S7+ IELs produced significantly more cytokines than S7– cells for all of the cytokines tested, the wide spectrum of the immunobiological effects mediated by those cytokines suggests that additional functional heterogeneity also may exist within the S7+ population. Additional studies can now focus on defining those subpopulations in more detail.

A particularly important aspect of this study concerns the information derived from the gene array analyses, which revealed major differences in S7+ and S7– cells that, in some cases, could not have been easily predicted using other approaches. The presence of high levels of CD44, CD97, and CD6 gene expression in S7+ IELs parallels the stronger effector responses of those cells as seen by their in vitro cytotoxic and cytokine responses (Figs. 4 and 7). In studies of whole unfractionated IELs, CD44 spans a range of expression from low to high, with CD44high IELs most likely constituting recently activated cells as predicted by the up-regulation of CD44 shortly after anti-CD3 stimulation (5). The gene array findings regarding CD44 thus further underscore the notion that most S7+ IELs are highly activated cells. Similarly, greater gene expression of CD97 suggests that S7+ IELs are activated T cells, possibly with enhanced cell adhesion (27), that would augment the effector activity of the S7+ population. Perhaps the most striking aspect of these data, however, was the enormously higher level of CD6 gene expression (45-fold) in S7+ vs S7– IELs. Although CD6 has been linked to T cell activation (28), expression of the CD6 gene also has been shown to be down-regulated on activated tumor-specific T cells (29). Moreover, a role for CD6 during intrathymic positive selection has recently been reported (30), thus raising the possibility that CD6 expression on S7+ IELs may be emblematic of lineage-associated differences between S7+ and S7– IELs.

The higher levels of gene expression of CCR2, CXCR3, and MIP-1{beta} in CD43 S7+ IELs are significant for several reasons. CCR2 is expressed on activated T cells (31), and is increased in IL-10–/– mice with colitis (32). Moreover, CCR2–/– mice have less intestinal adhesion and ulceration than wild-type animals following dextran sodium sulfate exposure (33). CXCR3 expression is elevated in Rag-2–/– mice with colonic inflammation following transfer of CD45RBhigh cells (32), and CXCR3 appears to play a role in lymphocyte trafficking and localization during inflammation (34). MIP-1{beta} is elevated in patients with ulcerative colitis and Crohn’s disease (35). Taken together, those findings are in line with likelihood that S7+ cells may be prone to participate in the inflammatory response within the intestinal mucosa.

The most significant feature of the gene array studies for the S7– population was the expression of genes for receptors associated with NK cell activation/inhibition. Although the majority of those genes (Ly49E-GE, Ly49G.2, CD94/NKG2, and Ly49C) have NK inhibitory activities (36, 37), the Ly49H receptor has been shown to activate NK cells (38, 39, 40, 41, 42). On human IELs, the NKG2D receptor for the MHC class I chain-related epithelium-expressed stress-induced molecules (MIC-A and MIC-B) is constitutively expressed at low levels, and is up-regulated following anti-CD3 stimulation or upon culture with IL-15 (43), resulting in enhanced NKG2D-mediated cytotoxicity and heightened IFN-{gamma} production (43). Clearly, differences in the expression of NK receptor molecules could reflect discrete NK subsets present among the S7– IEL population. However, regulation of NK activity could occur at the level of ligand expression, particularly if different NK receptors are coexpressed on the same cell; this would be true for the Ly49H NK activating receptor, which binds to the m157 protein of the mouse CMV (44), for example. More important in the context of the present study, however, the association of NK receptors on S7– IELs implies that those IELs differ significantly from S7+ IELs in the mechanisms by which they recognize Ag.

The observation that the CD43 S7 marker is expressed to varying degrees across both TCR{alpha}{beta} and TCR{gamma}{delta} cells, coupled with the differences in the functional responses of S7+ and S7– cells, lends credence to the notion that many similar aspects of the IEL immune response are distributed across subsets independent of TCR expression (45). Moreover, in addition to the functional differences between S7+ and S7– IELs, the possibility that the CD43 S7 Ag may have significance as a lineage-related marker of IELs should not be discounted. Note that the majority (though not all) of TCR{gamma}{delta} IELs were CD43 S7– cells, whereas the majority (though not all) of the TCR{alpha}{beta} IEL were S7+ cells. This is in line with the purported classification of IELs according to thymus-independent (TCR{alpha}{beta} CD8{alpha}{alpha} and TCR{gamma}{delta} CD8{alpha}{alpha}), and thymus-dependent (TCR{alpha}{beta} CD8{alpha}{beta}) lineages (46). Although extensive experimental work involving bone marrow reconstitution of athymic mice will be needed to define the relationship between S7 expression and IEL lineage derivation, if true, analyses of IELs by S7 expression would provide a valuable tool for penetrating into the abstruse lineage/function relationship of IELs.

By all criteria (cytokine production following anti-CD3 stimulation and in IL-10–/– mice, cytotoxicity in fresh IELs and following anti-CD3 stimulation, and gene array studies), CD43 S7+ IELs have the potential to be the most potent effector cells, but also to be the most destructive within the local intestinal environment. Thus, CD43 may have considerable usefulness for further dissecting regulatory and effector properties of IELs, and for therapeutic approaches aimed at controlling hyperactivated IELs involved in intestinal immunopathology. This could occur by selective elimination or inactivation of IELs expressing the CD43 S7 glycoform through Ab immunotherapy. In cases of chronic intestinal inflammation, this may be particularly important when coupled with therapeutic approaches targeted to specific inflammatory molecules (47, 48). Similar combined therapies aimed at multiple entities have been shown to have beneficial synergistic effects in situations such as with the transplantation of allograft islets in type 1 diabetes (49).


   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 in part by National Institutes of Health Grant DK35566. Back

2 Address correspondence and reprint requests to Dr. John R. Klein, Department of Diagnostic Sciences, Room 3.094F, Dental Branch, University of Texas Health Science Center at Houston, 6516 M. D. Anderson Boulevard, Houston, TX 77030. E-mail address: john.r.klein{at}uth.tmc.edu Back

3 Abbreviation used in this paper: IEL, intraepithelial lymphocyte. Back

Received for publication September 11, 2003. Accepted for publication September 3, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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