in CD45-Deficient Mice T Cells + Lymphocytes, NK Cells, and NK 1.1 ...

Development of Intestinal Intraepithelial Lymphocytes, NK Cells, and NK 1.1ⴙ T Cells in CD45-Deficient Mice1 Steven M. Martin,* Indira K. Mehta,† Wayne M. Yokoyama,†‡ Matthew L. Thomas,*‡ and Robin G. Lorenz2*† The transmembrane protein tyrosine phosphatase CD45 is differentially required for the development and function of B, T, and NK cells, with mice partially deficient for CD45 having a significant inhibition of T cell, but not NK or B cell, development. CD45-mediated signaling has also been implicated in the development of intrathymic, but not extrathymic, intestinal intraepithelial T lymphocytes (iIELs) in the CD45ex6ⴚ/ⴚ mouse. As NK1.1ⴙ CD3ⴙ (NK-T) cells can also develop through extrathymic pathways, we have investigated the role of CD45 in NK-T cell development. In mice with a complete absence of CD45 expression (CD45ex9ⴚ/ⴚ) the NK-T cell population was maintained in the iIEL compartment, but not in the spleen. Functionally, CD45deficient NK-T cells were unable to secrete IL-4 in response to TCR-mediated signals, a phenotype similar to that of CD45deficient iIELs, in which in vitro cytokine production was dramatically reduced. Using the CD45ex9ⴚ/ⴚ mouse strain, we have also demonstrated that only one distinct population of NK-T cells (CD8ⴙ) appears to develop normally in the absence of CD45. Interestingly, although an increase in cytotoxic NK cells is seen in the absence of CD45, these NK calls are functionally unable to secrete IFN-␥. In the absence of CD45, a significant population of extrathymically derived CD8␣␣ⴙ iIELs is also maintained. These results demonstrate that in contrast to conventional T cells, CD45 is not required during the development of CD8ⴙ NK-T cells, NK cells, or CD8␣␣ⴙ iIELs, but is essential for TCR-mediated function and cytokine production. The Journal of Immunology, 2001, 166: 6066 – 6073.

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onventional T cell subsets depend upon the thymus for their development; however, other lineages, such as CD8␣␣⫹ intestinal intraepithelial lymphocytes (iIELs),3 ⫹ CD8␣␤ NK1.1⫹ CD3⫹ T cells, and NK cells develop normally in athymic (nude) mice (1–7). The development of these lineages appears to be controlled by IFN regulatory factor 1 and its regulation of IL-15 gene expression (8 –12). The iIEL compartment contains a rich and phenotypically diverse T lymphocyte population whose size, by some estimates, rivals that of the pool of T lymphocytes in the spleen (13). All iIEL express the ␣IEL (␣E)␤7 integrin that binds E-cadherin and mediates adherence to epithelial cells (14 –16). The vast majority of the iIEL T lymphocyte population expresses the CD8␣-chain; however, only ⬃20% of these T lymphocytes express the CD8␤-chain (17). CD8␣␤ heterodimer expression is characteristic of thymus-derived CD8⫹ T lymphocytes, while CD8␣␣ homodimer expression has been associated with extrathymically derived T lymphocytes in nude mice and in

Departments of *Pathology and Immunology and †Medicine and ‡Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110 Received for publication April 17, 2000. Accepted for publication March 12, 2001. 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 The Charles E. Culpeper Foundation and Washington University Digestive Diseases Research Core Center Grant P30DK52574. S.M.M. is supported by National Institutes of Health Training Grant T32AI07163. R.G.L. is a Charles E. Culpeper Foundation Medical Scholar. M.L.T. was an investigator with Howard Hughes Medical Institute. W.M.Y. is an investigator with Howard Hughes Medical Institute. 2 Address correspondence and reprint requests to Dr. Robin G. Lorenz, Department of Pathology and Immunology (Box 8118), Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail address: [email protected] 3 Abbreviations used in this paper: iIEL, intestinal intraepithelial lymphocytes; CD45ex6⫺/⫺, CD45 exon 6 deficient; CD45ex9⫺/⫺, CD45 exon 9 deficient; NK-T cell, NK1.1⫹ CD3⫹ cell; LAKs, IL-2-activated NK cells.

Copyright © 2001 by The American Association of Immunologists

thymectomy models (18 –20). Both TCR␣␤⫹ and TCR␥␦⫹ T lymphocytes in the iIEL can express the CD8␣␣ homodimer, whereas only TCR␣␤⫹ T lymphocytes express the CD8␣␤ heterodimer (13). Thus, TCR␥␦⫹ and a portion of the TCR␣␤⫹ iIEL population are considered to be of extrathymic origin. The site of extrathymic iIEL maturation has not been clearly established, although there is evidence that the intestine is involved (21–25). Functionally, iIEL are known to make Th1 cytokines (IL-2 and IFN-␥) (26). NK cells represent a population of lymphocytes that can lyse tumor cells that may lack MHC class I expression. NK cells share multiple surface molecules with T cells and may be generated from common precursors; however, functional NK cells can develop in the absence of a thymus (27). Additionally, there is a subset of lymphocytes that share receptor structures common to both T cell and NK cell lineages, the NK1.1⫹ CD3⫹ cell (NK-T cell) (3, 5, 28). This lymphocyte population coexpresses the ␣␤ TCR and the NK surface receptor NKR-P1 (NK1.1 in the C57BL/6 mouse strain). There are two phenotypically and functionally distinct subsets of NK-T cells described in most tissues. One population is CD4⫹ or CD4⫺CD8⫺ (double negative) and is CD1d and thymus dependent, while the CD8⫹ population is CD1d and thymus independent. Most CD1d- and thymus-dependent NK-T cells use one of only three TCR V␤ domains (V␤8.2, V2␤7, or V␤2) and a single V␣ domain (V␣14) (5, 28, 29). In addition to this restricted TCR repertoire, CD1d- and thymus-dependent NK-T cells have the other unusual property of rapidly releasing IL-4 within 1 h of TCR engagement. In contrast, CD8⫹ NK-T cells express heterogeneous TCR, do not produce IL-4 rapidly (7), and can develop extrathymically from fetal liver precursors (3, 7). The transmembrane protein tyrosine phosphatase CD45 (leukocyte common Ag, Ly-5) has been shown to be critically important for thymic T lymphocyte development (30, 31). CD45 is expressed on all nucleated cells of hemopoietic origin, including B, T, and NK cells (32). Multiple isoforms of CD45 exist, ranging in size 0022-1767/01/$02.00

The Journal of Immunology ?from 180 to 220 kDa. The physiological significance of the multiple isoforms remains unclear; however, a switch to smaller isoforms on T lymphocytes has been correlated with a memory phenotype in humans, and the basal biochemical association of CD45 with the TCR complex is regulated in part by CD45 isoform expression (33, 34). The observed heterogeneity is due to alternative splicing of three variable exons of CD45, exons 4, 5, and 6. These exons contain multiple O-linked glycosylation sites, allowing for the large mass difference between isoforms (32). Three different strains of CD45 mutant mice have been produced, one in which the variable exon 6 was replaced by the neomycin cassette (CD45 exon 6 deficient, CD45ex6⫺/⫺), one in which exon 12 was replaced, and another in which exon 9 was replaced (CD45 exon 9 deficient, CD45ex9⫺/⫺) (30, 31, 35). In CD45ex6⫺/⫺ mice, CD45 expression can be detected at low levels in spleen and lymph nodes. The CD45ex9⫺/⫺ mouse strain lacks a constant exon of CD45; thus, no CD45 expression can be detected in these mice. These three CD45-deficient mouse strains have been found to have similar phenotypes. Maturation of T lymphocytes is primarily blocked at the CD4 CD8 double-positive stage in the thymus of CD45-deficient mice. Few CD4⫹ TCR␣␤⫹ lymphocytes can be found in the spleen and lymph nodes of either mouse strain. It has recently been reported that in the CD45ex6⫺/⫺ mouse strain, CD45 expression is required for intrathymic, but not extrathymic, iIEL development (36). Interestingly, there is also an increase in NK cells in the CD45ex6⫺/⫺ mouse strain (37). As NK cells can also develop in the absence of a thymus, these findings suggest that CD45 expression is not required for the development of any lymphocyte subset that may be dependent on an extrathymic process. To test this hypothesis, we investigated the role of CD45 in the development of NK-T cells. In addition, as the CD45ex6⫺/⫺ mouse strain has a significant low level expression of CD45, we decided to evaluate what role, if any, this low level CD45 expression had on CD8␣␣⫹ iIEL, NK, and NK-T cell development through evaluation of the CD45ex9⫺/⫺ mouse strain. The results of this study showed that CD45 is not essential for the development of lymphocyte subsets of extrathymic origin, but is critical for the development of full functional capacity.

Materials and Methods Mice CD45ex6⫺/⫺ mice have been previously described and were maintained in a specific pathogen-free animal facility (30). CD45ex9⫺/⫺ mice (C57BL/ 6-OlaHsd-Ptprctm1) were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in a specific pathogen-free facility (31). All deficient mice used were backcrossed at least seven generations to the C57BL/6 background; therefore, age-matched C57BL/6 mice (Taconic Farms, Germantown, NY) were used as wild-type controls. All mice, unless otherwise indicated, were 10 –14 wk of age.

Cell preparation Single-cell suspensions of splenic lymphocytes were prepared by mechanical dissociation and lysing of contaminating erythrocytes with ACK lysing buffer (BioWhittaker, Walkersville, MD). To prepare iIELs, intestines from sacrificed mice were removed, and visible Peyer’s patches were discarded. Lengthwise incisions in the intestine were made, and the intestines were washed three to five times or until the wash was clear using ice-cold PBS (BioWhittaker). Intestines were then cut into 1-cm pieces and placed on a rotating platform at 100 rpm for 30 min at 37°C in RPMI 1640 (BioWhittaker) containing 2% FBS (HyClone, Logan, UT), 50 ␮g/ml gentamicin (Life Technologies, Gaithersburg, MD), 2 mM Glutamax I (Life Technologies), 1 mM sodium pyruvate (BioWhittaker), 50 U/ml penicillin-50 ␮g/ml streptomycin (Life Technologies), 50 ␮M 2-ME (Fisher Scientific, Pittsburgh, PA), 100 ␮M nonessential amino acids (BioWhittaker), 0.075% (w/v) sodium bicarbonate (BioWhittaker), and 10 mM HEPES (BioWhit-

6067 taker) (R2 medium). The intestinal pieces were then shaken vigorously (30 s, four times), and iIELs were enriched by passage through a siliconized glass-wool column (38).

Abs and cell lines The following Abs were purchased from PharMingen (San Diego, CA): biotinylated anti-␣IEL integrin chain, clone 2E7; purified anti-CD3, clone 145-2C11; FITC anti-CD4, clone GK1.5; FITC anti-CD8␣, clone 53-6.7; FITC anti-CD8␤, clone 53-5.8; FITC anti-CD45, clone 30F11.1; purified and FITC anti-TCR␤, clone H57-597; purified anti-TCR␥␦, clone GL4, FITC anti-TCR␥␦, clone GL3; biotinylated anti-NK-1.1; isotype controls hamster IgG group 1, ␬, clone A19-3 and hamster IgG group 2, ␭, clone Ha4/8; and PE-conjugated streptavidin. Biotinylated anti-Thy1.2 (CD90.2), clone 5a-8 was purchased from Caltag (Burlingame, CA), and FITC-conjugated goat anti-Armenian hamster IgG was purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The IL-2/IL-4dependent cell line CTLL was used in bioassays for cytokine production by iIEL (39).

Flow cytometric analysis Cells were resuspended at a concentration of 107 cells/ml in flow buffer (1⫻ PBS/0.05% BSA; Fisher). Aliquots (100 ␮l) were incubated with 1 ␮g of Fc Block (PharMingen) for 15 min on ice, and the primary (biotinylated) Ab was added and incubated for 30 min on ice. Cells were washed twice with flow buffer and resuspended in 100 ␮l with the appropriate secondary Ab, streptavidin conjugate, or directly conjugated Ab. Cells were incubated for 30 min on ice and washed twice with flow buffer. Analysis was performed on a FACStar (Becton Dickinson, San Jose, CA) using CellQuest software (Becton Dickinson). Ten thousand gated cells were analyzed per sample.

In vitro functional assays To measure IL-2 production, 2 ⫻ 105 iIEL/well were incubated on immobilized anti-CD3, anti-TCR␣␤, anti-TCR␥␦, or control Ab in a 96-well tissue culture plate (no. 3799; Costar, Cambridge, MA) for 18 h at 37°C in RPMI 1640 containing 10% FCS (HyClone), 2 mM Glutamax I, 50 U/ml penicillin-50 ␮g/ml streptomycin, 50 ␮M 2-ME, and 100 ␮M nonessential amino acids (R10 medium). The number of iIELs was analyzed by hemocytometer counting of viable lymphocytes as determined by morphological criteria and trypan blue exclusion. The percentage of iIEL in each intestinal preparation ranged from 1.9 to 17.3%. One hundred microliters of culture supernatant was harvested from each well and added to wells of a 96-well culture plate (Costar no. 3595) containing 100 ␮l of RPMI 1640 with 10% FBS, 2 mM Glutamax I, 50 ␮g/ml gentamicin (R10), and 2.5 ⫻ 104 CTLL/ml. The assay plate was incubated for 18 h at 37°C and then pulsed with 1 ␮Ci of [3H]thymidine (NEN, Boston, MA) for 6 – 8 h. Assays were harvested on a Skatron Micro 96 harvester (Molecular Dynamics, Sunnyvale, CA). To analyze splenic NK-T cell function, groups of two mice were given 2 ␮g of anti-CD3 (2C11) by i.v. injection. The spleens were removed after 90 min, and 1 ⫻ 107 splenocytes/ml were cultured in R10 for 2 h. For the analysis of iIEL NK-T cell function, iIELs were isolated as described above, and 1 ⫻ 107 cells were cultured for 4 h at 37°C in 5% CO2 in the presence of 100 ␮g/ml anti-CD3 (2C11) in R10. IL-4 levels were measured in culture supernatants using a mouse IL-4 ELISA kit (PharMingen). IL-2-activated NK cell (LAK) preparations were performed as previously described (40). Briefly, splenocyte suspensions were made in HBSS and 10% FCS. After RBC lysis, washed cells were incubated in a nylonwool column for 45 min at 37°C. Nonadherent cells were cultured in RPMI 1640 with 10% FCS, Glutamax I, penicillin/streptomycin, 2-ME, and 800 U/ml of rIL-2 (Chiron, Emeryville, CA) at 4 ⫻ 106/ml. On day 3 adherent cells were removed with Versene (Life Technologies) and washed. The cells were then incubated in 1/1 dilutions of culture supernatants GK1.5 (anti-CD4) and YTS169.4 (anti-CD8␣) Abs and a 1/15 dilution of Low Tox M Rabbit Complement (Cederlane Laboratories, Hornby, Ontario, Canada) for 45 min at 37°C. After washing, the remaining cells were expanded for 2– 4 days in 800 U/ml of rIL-2 at 4 ⫻ 105 cells/ml. Two assays of in vitro NK cell activity were performed. Natural killing assays were performed as previously described (40). Briefly, tumor targets were radiolabeled with 51Cr (25 ␮Ci/106 cells) for 90 min in the absence of FCS. Effector cells were added to 96-well round-bottom plates at densities sufficient to achieve the indicated E:T cell ratios. Radiolabeled targets were added (104/well) and incubated for 4 h at 37°C. One hundred and fifty microliters of supernatant was harvested and assayed for 51Cr release. Specific lysis was determined as follows: % specific lysis ⫽ 100 ⫻ (exp ⫺ spont)/(max ⫺ spont). In vitro IFN-␥ release by NK cells was performed as previously described (41). Briefly, 96-well flat-bottom plates were coated with goat anti-

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iIEL AND NK-T CELL DEVELOPMENT IN CD45-DEFICIENT MICE

mouse F(ab⬘)2 (Jackson ImmunoResearch) and then mouse anti-NK1.1 at the indicated concentrations. NK cell populations were incubated for 4 h at 37°C at 2 ⫻ 105 cells/well. IFN-␥ was measured in culture supernatants using a mouse IFN-␥ ELISA kit (PharMingen).

Results NK cells are increased, while NK-T cells are decreased, in spleens of CD45⫺/⫺ mice Through the use of flow cytometry, we examined whether NK and NK-T cells could develop in CD45-deficient mice. An increase in NK cells was previously described in CD45ex6⫺/⫺ mice; however, as these mice express low levels of CD45, we wanted to evaluate the numbers of NK cells in the completely CD45-deficient CD45ex9⫺/⫺ mouse. As shown in Table I, there was a 2-fold increase in the percentage of NK cells in the spleens of CD45ex9⫺/⫺ mice. This translates into a significant (3.5-fold) increase in the total number of splenic NK cells. This substantial change in NK cells is in contrast to the dramatic decrease in NK-T cell percentages and numbers in the spleens of CD45-deficient mice. As shown in Table I, CD3⫹ NK1.1⫹ cells were at the limit of reliable detection in CD45ex9⫺/⫺ mice (⬎10-fold decreased). This decrease precluded the identification of NK-T cell subpopulations in the spleens of CD45ex9⫺/⫺ mice. Intestinal IEL are present in CD45ex9⫺/⫺ mice Enriched iIELs were stained for ␣IEL expression after harvest, and ␣IEL-positive cells were detected by flow cytometry. In C57BL/6 mice, 12.3 ⫾ 1.5% of the total intestinal preparation (iIEL and enterocytes) was positive for ␣IEL, whereas in CD45ex6⫺/⫺ and CD45ex9⫺/⫺ mice only 3.9 ⫾ 0.8 and 2.6 ⫾ 0.6%, respectively, of the intestinal preparation were ␣IEL positive. Based on these percentages and the total number of cells (epithelial and iIEL) in the preparations, the absolute number of iIELs in the respective populations was calculated (Table II). A decrease in the absolute number of iIELs was observed in both CD45ex6⫺/⫺ and CD45ex9⫺/⫺ mice (Table II and Fig. 1A). Phenotype of iIEL in CD45ex6⫺/⫺ mice Because of the presence of a significant, albeit reduced, population of iIEL in the CD45ex6⫺/⫺ and CD45ex9⫺/⫺ mouse strains, further phenotypic characterization was performed. To normalize for number of iIEL, lymphocytes were stained for ␣IEL and the marker of interest. Thus, all values are the percentage iIEL (␣IEL-positive cells) positive for the second marker, allowing for comparison between C57BL/6 and deficient mice. All iIEL from C57BL/6 mice expressed CD45, while only 25% of iIEL from the CD45ex6⫺/⫺ mice express CD45 (Fig. 1, B–D).

This percentage is higher than the low, but detectable, levels of expression in other lymphoid compartments of CD45ex6⫺/⫺ mice (CD45ex6⫺/⫺ splenocytes, 3– 4% CD45⫹). It should also be noted that the intensity of CD45 expression on iIELs was slightly decreased (mean fluorescence intensity: C57BL/6, 1299; CD45ex6⫺/⫺, 568). iIEL from the CD45ex9⫺/⫺ strain did not express CD45 (Fig. 1, B and E), thus confirming the complete absence of CD45 protein expression in this mutant strain. With respect to CD8 expression, 79% of the iIEL in the CD45ex6⫺/⫺ mice and 77.5% of the iIEL in the CD45ex9⫺/⫺ mice expressed the CD8␣ chain compared with 86% in the C57BL/6 control (Table III and Fig. 2A). However, while 12.9% of the CD45ex6⫺/⫺ iIEL and 15% of the C57BL/6 iIEL expressed the CD8␤ chain, only 4.5% of iIELs in an age-matched CD45ex9⫺/⫺ mouse were CD8␤⫹ (Fig. 2B and Table III). To determine whether there was attenuation of TCR␣␤ or TCR␥␦ populations, iIEL were costained for ␣IEL and either TCR␣␤ or TCR␥␦. Both T lymphocyte populations were present in the CD45ex6⫺/⫺ and CD45ex9⫺/⫺ animals, although with slightly decreased percentages compared with C57BL/6 (Table III). The majority of CD45positive cells in the CD45ex6⫺/⫺ iIELs (94%) are TCR␣␤ positive (Fig. 3A). As previously described, there was an increase in CD4positive iIELs in the CD45ex6⫺/⫺ mouse; however, this elevation was not seen in the CD45ex9⫺/⫺ iIELs, indicating a decrease in CD4-positive iIELs in the complete absence of CD45. The percentage of NK and NK-T cells is increased in iIELs in CD45-deficient mice To examine whether part of the retained iIELs in CD45-deficient mice were NK and/or NK-T cells, we analyzed the dual expression of NK1.1 and CD3 on iIELs by flow cytometry. As shown in Fig. 4A, C57BL/6 mice have a minor population (mean ⫾ SE, 7.8 ⫾ 0.8) of NK1.1⫹CD3⫹ cells in the iIEL population. This population of NK-T cells is significantly elevated in the CD45ex6⫺/⫺ (12.5 ⫾ 4.6%) and CD45ex9⫺/⫺ (12.3 ⫾ 1.4%) iIEL populations (Table I and Fig. 4). In addition, although the normal iIEL population has almost no measurable NK cells (1.8 ⫾ 0.6%), the iIEL populations from CD45-deficient mice have a significantly increased percentage of NK cells (Table I and Fig. 4; CD45ex6⫺/⫺, 5.9 ⫾ 2.4%; CD45ex9⫺/⫺, 8.8 ⫾ 2.5%). This increase in the percentage of NK cells in the iIEL population results in an increase in the total number of NK cells in the iIEL population that is similar to the fold increase in the total number of NK cells in the spleen. As two clearly distinct populations of NK-T cells have been described, we used flow cytometric analysis to analyze whether there was selective survival of these subpopulations in CD45ex9⫺/⫺ iIELs. In the C57BL/6 spleen, the majority of NK-T

Table I. The development of NK and NK-T cells in the spleen and iIEL of CD45ex9-deficient micea Spleen

iIEL

Mice

C57BL/6

CD45ex9⫺/⫺

C57BL/6

CD45ex9⫺/⫺

Total cell No. (⫻106) % NK1.1⫹ CD3⫺ No. NK cells (⫻105) % NK1.1⫹ CD3⫹ No. NK-T (⫻105) % NK1.1⫹ CD4⫹ % NK1.1⫹ CD8␣⫹

81 ⫾ 3 4.3 ⫾ 0.3 35 ⫾ 3 2.1 ⫾ 0.3 17 ⫾ 3 0.7 ⫾ 0.2 0.2 ⫾ 0.1

141 ⫾ 10 8.8 ⫾ 1.1 121 ⫾ 9ⴱ .09 ⫾ .01ⴱ 1.2 ⫾ 0.1ⴱ BDLb BDL

3.9 ⫾ 0.7 1.8 ⫾ 0.7 0.7 ⫾ 0.1 7.8 ⫾ 0.8 3.0 ⫾ 0.1 0.7 ⫾ 0.1 7.0 ⫾ 1.3

1.6 ⫾ 0.4 8.8 ⫾ 2.5ⴱ 1.4 ⫾ 0.1 12.3 ⫾ 1.4ⴱ 2.0 ⫾ 0.1ⴱ 1.1 ⫾ 0.3 14.4 ⫾ 1.6ⴱ

a Total spleen cells and iIELs were isolated, counted, and stained with hamster anti-CD3 and biotinylated NK1.1 mAb, followed by FITC anti-hamster and streptavidin-PE or biotinylated NK1.1 mAb, followed by streptavidin-PE and FITC anti-CD4 or CD8␣. Percentages of cells in whole spleen were analyzed by flow cytometry, and numbers of each population were calculated (mean ⫾ SEM). Results are means of four separate experiments (n ⫽ 6, C57BL/6; n ⫽ 3, CD45ex9⫺/⫺). ⴱ, p ⬍ 0.05 for C57BL/6 vs CD45ex9⫺/⫺ mice. b BDL, Below detection limit.

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Table II. Number of iIELs in intestines of CD45-deficient micea

Mice

C57BL/6 CD45ex6⫺/⫺ CD45ex9⫺/⫺

Total No. of Intestinal Cells (⫻107)

␣IEL⫹ (%)

Total No. of iIELs (⫻106)

CD45⫹ ␣IEL⫹ (%)

3.2 ⫾ 0.4 3.6 ⫾ 0.5 6.5 ⫾ 1.0

12.3 ⫾ 1.5 3.9 ⫾ 0.8 2.6 ⫾ 0.5

3.9 ⫾ 0.7 1.3 ⫾ 0.2ⴱ 1.6 ⫾ 0.4

99.5 ⫾ 0.2 24.8 ⫾ 7.1ⴱ 0.1 ⫾ 0.1ⴱ

a Total intestinal cells were isolated as described in Materials and Methods, counted and stained with biotinylated ␣IEL mAb, followed by FITC anti-CD45 and streptavidin-PE. Percentages of cells in isolated intestinal preparations were analyzed by flow cytometry, and numbers of each population were calculated (mean ⫾ SEM). Results are means of four separate experiments (n ⫽ 6, C57BL/6; n ⫽ 5, CD45ex6⫺/⫺; n ⫽ 3, CD45ex9⫺/⫺). ⴱ, p ⬍ 0.05 for CD45ex9⫺/⫺ or CD45ex6⫺/⫺ vs C57BL/6 mice.

cells were CD4⫹ or CD4⫺CD8⫺, in agreement with previous reports (Table I) (7, 42). Surprisingly, however, as is shown in Table I and Fig. 4, almost all NK-T cells in the iIEL compartment were CD8⫹. When these results were compared with the subpopulations of NK-T cells in the CD45ex9⫺/⫺ iIEL compartments, there was a retention of the CD8⫹ NK-T cell population (Table I). There was no clearly defined population of CD4⫹ or CD4⫺CD8⫺ NK-T cells in the iIEL compartment (Fig. 4). Functional activity of CD45-deficient iIELs, NK-T cells, and NK cells To test the functional capability of the remaining iIEL in the CD45ex6⫺/⫺ and CD45ex9⫺/⫺ mice, the production of IL-2 in response to immobilized anti-TCR complex Abs was performed. Proliferation of the IL-2-dependent CTLL cell line was measured. Although incubation of C57BL/6 iIEL with anti-CD3, antiTCR␣␤ and anti-TCR␥␦ Abs resulted in significant IL-2 production (Fig. 3B and data not shown), minimal IL-2 production was activated by Ab-mediated TCR stimulation in CD45ex6⫺/⫺ or CD45ex9⫺/⫺ iIEL (Fig. 3B). Therefore, the reduced number of iIELs in CD45-deficient strains is coupled with a significant impairment of iIEL function measured in vitro. As shown above, the total number of splenic T cells bearing the NK-T cell phenotype by FACS analysis was decreased ⬎10-fold in CD45-deficient mice. To determine whether there were cells capable of NK-T cell function in the absence of cells expressing the typical NK-T cell phenotype profile, we assessed IL-4 production after i.v. administration of anti-CD3 mAb (Fig. 5A). A complete loss of NK-T cell function was demonstrated in both the CD45ex6⫺/⫺ and CD45ex9⫺/⫺ strains. When iIEL NK-T cell function was analyzed in a similar fashion, low amounts of IL-4 production could be measured in the C57BL/6 population, while no cytokine production could be measured in the CD45ex9⫺/⫺ population (Fig. 5B). Therefore, although these mouse strains demonstrate that iIEL NK-T cells can develop in the absence of CD45 cell surface expression, these NK-T cells are unable to perform the function of rapid IL-4 release after anti-CD3 stimulation. We also used the CD45ex9⫺/⫺ mouse strain to address whether NK cells in the complete absence of CD45 are fully functional. In agreement with the results from the CD45ex6⫺/⫺ mouse, which express CD45 at low levels, NK cells isolated from CD45ex9⫺/⫺ spleenocytes have normal cytotoxic activities, as assessed by NK lysis of YAC-1 targets (Fig. 5C). However, when antiNK1.1-induced IFN-␥ production by LAKs was assessed, there was a dramatic reduction in the amount of cytokine produced by CD45ex9⫺/⫺ LAKs (Fig. 5D). This decreased cytokine production was also noted for TNF-␣ (data not shown).

Discussion The transmembrane protein tyrosine phosphatase CD45 has been shown to be critically important for the development of two pop-

ulations of lymphocytes, the NK cell and thymically derived T lymphocytes (30, 31, 37, 43). In CD45ex6⫺/⫺ spleens there is a marked increase in the number of NK cells with normal cytotoxic activity. As NK cell development is influenced by T cell cytokines such as IL-2, the presence of a small population of mature T cells in CD45ex6⫺/⫺ mice may have an influence on the maintenance of the NK cell population in these mutant mice. Therefore, the completely CD45-deficient CD45ex9⫺/⫺ mice were tested for the presence of NK cells in the spleen. Strikingly, the number of NK cells was even more dramatically increased in the spleens of CD45ex9⫺/⫺ mice. The total number of NK cells was increased 4-fold over that in wild-type C57BL/6 mice, indicating that the NK cell population can develop independently of CD45. There are several potential explanations for this expansion of NK cells in the absence of CD45. First, as the cellular composition of the spleen in

FIGURE 1. Presence of iIEL in CD45-deficient mice. Intestinal cells were stained for CD45 and the ␣IEL integrin chain. A, The total number of iIELs was decreased in CD45-deficient mice, as determined by the percentage of total epithelial and lymphoid cells positive for the ␣IEL integrin chain by flow cytometric analysis (see also Table II). The level of CD45 expression on ␣IEL⫹ lymphocytes is shown for wild-type and CD45-deficient mouse strains (B). Note the widely varying levels of expression in the CD45ex6⫺/⫺ strain. Histograms show the level of CD45 expression in C57BL/6 mice (C), CD45ex6⫺/⫺ mice (D), and CD45ex9⫺/⫺ mice (E). Note that the surface expression of CD45 as well as the number of cells positive for CD45 are reduced in the CD45ex6⫺/⫺ mouse strain. Results are representative of three experiments.

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iIEL AND NK-T CELL DEVELOPMENT IN CD45-DEFICIENT MICE Table III. Characterization of iIEL in CD45-deficient Micea Mouse

C57BL/6 CD45ex6⫺/⫺ CD45ex9⫺/⫺

CD8␣⫹

CD8␤⫹

CD4⫹

TCR␣␤⫹

TCR␥␦⫹

86.0 ⫾ 0.1 79.0 ⫾ 4.4 77.5 ⫾ 2.0ⴱ

16.4 ⫾ 0.7 12.9 ⫾ 6.5 4.5 ⫾ 2.6ⴱ

7.6 ⫾ 0.6 12.3 ⫾ 3.0 4.0 ⫾ 0.1ⴱ

46.1 ⫾ 5.4 44.8 ⫾ 9.4 52.9 ⫾ 6.3

47.1 ⫾ 6.1 40.8 ⫾ 5.3 22.3 ⫾ 6.6

a iIEL were harvested and stained as in Figs. 1 and 2. iIEL are defined as aIEL-expressing cells. The percentages of iIEL that are CD4⫹, CD8␣⫹, CD8␤⫹, TCR␣␤⫹, and TCR␥␦⫹ are shown. Results are representative of at least three experiments. ⴱ, p ⬍ 0.05 for CD45ex9⫺/⫺ or CD45ex6⫺/⫺ vs C57BL/6 mice.

the absence of CD45 is dramatically different from that of a normal spleen, it is possible that the expansion of NK cells is being driven by an external stimulus. A second possibility is that the CD45 phosphatase and the intracellular signals it regulates are directly involved in the maintenance of NK cell homeostasis. This role may be similar to the role that CD45 plays in the down-regulation of kinase activity of Src family members during integrin-mediated adhesion in macrophages (44). In agreement with the reported functional capacity of NK cells in the CD45ex6⫺/⫺ mouse, NK cells from the spleens of CD45ex9⫺/⫺ mice maintain cytotoxic activity. However, in addition to cytotoxic activity, NK cells produce a variety of cytokines, including IFN-␥, TNF-␣, and GM-CSF, in response to cross-linking of NK cell activation receptors, such as NK1.1 (41, 45). As it is not currently known whether cytokine production is stimulated by precisely the same pathway that activates NK cell cytotoxicity, it was important to also investigate this aspect of NK cell function. In contrast to the normal levels of cytotoxicity seen with NK cells from the CD45ex9⫺/⫺ mice, there was a dramatic loss of the capacity of NK cells to produce cytokines in response to NK1.1 cross-linking. Although the NK cell activation receptor(s) involved in YAC-1 killing is still unknown, this result implicates CD45 in either the pathway necessary to induce NK cell cytokine production or in the development of this functional capacity. Most importantly, it implies that the pathways that activate NK cell cytotoxicity and NK cell cytokine production are divergent.

The NK-T cell has recently been classified as a lymphocyte subset that shares features common with both NK cells and conventional T cells (3, 7, 28). As the NK cell population is increased in both strains of CD45-deficient mice, the presence or absence of NK-T cells was investigated. In CD45ex9⫺/⫺ mice, the total number of splenic NK-T cells was significantly decreased, while a significant increase in NK-T cells was seen in the iIEL population. Our finding that the subpopulation of NK-T cells that is retained in the iIEL compartment in the absence of CD45 is a CD8⫹ NK-T cell supports the hypothesis that this subpopulation can develop in a thymus-independent manner. Its maintenance in CD45-deficient mice is consistent with the maintenance of other extrathymically derived populations. The functional potential of these NK-T cells has been investigated by in vivo triggering of their TCR-CD3 complex with mAb. Wild-type, C57BL/6 splenic NK-T cells can pro-

FIGURE 3. Functional capacity of CD45-deficient iIELs. A, CD45ex6⫺/⫺ iIEL used for functional studies were stained for CD45 expression and either TCR␣␤ (left) or TCR␥␦ (right). The majority of CD45⫹ iIELs are TCR␣␤⫹. B, C57BL/6 iIEL stimulated by anti-CD3 in vitro produce IL-2 (f) in a dosedependent fashion. Decreased or absent IL-2 production was observed in CD45ex6⫺/⫺ (‚) and CD45ex9⫺/⫺ (E) iIELs.

FIGURE 2. CD8␣␣ and CD8␣␤ iIEL in CD45-deficient mice. A, CD8␣ expression on iIEL is maintained in both CD45-deficient mouse strains as assessed by cell surface expression. B, CD8␤ expression is reduced in both CD45-deficient strains, compared with expression on C57BL/6 wild-type iIEL. Results are representative of three experiments. The iIEL are defined as ␣IEL-positive cells.

duce large amounts of IL-4 in vitro after short term in vivo stimulation with anti-CD3; however, this capacity for IL-4 secretion was completely lost in the CD45-deficient NK-T cells. This lack of rapid cytokine secretion after anti-CD3 stimulation of NK-T cells

The Journal of Immunology

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FIGURE 4. NK-T and NK cells in iIELs of CD45-deficient mice. NK-T cells were identified by flow cytometric analysis of NK1.1 and CD3. Wildtype C57BL/6 iIELs have a very small population of CD3⫹NK1.1⫹ NK-T cells. This subpopulation is increased in the CD45ex9⫺/⫺ strain, as is the NK cell population (CD3⫺NK1.1⫹). NK-T cells in the iIEL compartment predominantly express the CD8␣-chain, and not CD4, in both wild-type C57BL/6 and CD45ex9⫺/⫺ strains.

has been previously described for the CD8⫹ NK-T cell subpopulation, which is the predominant population of NK-T cells in the iIEL compartment (7). Recent evidence has suggested that CD45 is not required for the extrathymic development of T lymphocytes; however, these studies used the CD45ex6⫺/⫺ mouse strain in which a subset of peripheral T cells still expresses low levels of CD45 (36). Through the use of an alternative mouse strain engineered to have a complete absence of CD45 surface expression, we have now demonstrated that it is only the extrathymically derived CD8␣␣⫹ iIEL population that is maintained in the complete absence of CD45 expression. Both classical CD8␣␤⫹ and CD4⫹ iIELs are significantly reduced in the CD45ex9⫺/⫺ mouse intestine. This is in contrast to the previously reported increase in CD4⫹ iIELs in the CD45ex6⫺/⫺ mouse strain and demonstrates that the low level expression of surface CD45 in CD45ex6⫺/⫺ mouse iIELs has a measurable effect on the overall iIEL population. Specifically, the majority of this CD45⫹ population is CD4⫹TCR␣␤⫹, a population that is drastically reduced in the CD45ex9⫺/⫺ mouse (36). Because the precise location of extrathymic iIEL maturation has not been determined, we have not been able to identify which isoform of CD45 is used in the maturation of these CD4⫹TCR␣␤⫹ iIEL. However, previous authors have reported that iIELs from wild-type C57BL/6 mice express significant amounts of two-exon, single-exon, and zero-variable exon forms of CD45 (46). The two-

FIGURE 5. Functional capacity of CD45ex9⫺/⫺ NK-T and NK cells. A, Splenic CD45ex6⫺/⫺ and CD45ex9⫺/⫺ NK-T cells produce no measurable IL-4 in vitro after in vivo anti-CD3 stimulation. The numbers of lymphocytes were equivalent in all cultures as determined by morphologic criteria. B, CD45ex9⫺/⫺ NK-T cells from the iIEL compartment were also unable to produce cytokines after anti-CD3 stimulation. C, CD45ex9⫺/⫺ NK cells (‚) maintain specific killing activity, albeit decreased compared with that of C57BL/6 controls (f), against YAC-1 target cells. D, IFN-␥ production by C57BL/6 LAKs (left) after NK1.1 stimulation at the indicated doses. CD45ex9⫺/⫺ LAKs did not produce IFN-␥ after NK1.1 stimulation.

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iIEL AND NK-T CELL DEVELOPMENT IN CD45-DEFICIENT MICE

exon form was a mixture of exons 4 and 5 and exons 5 and 6, while the single-exon form solely used exon 5. This exon usage is in contrast to the exon usage seen in the thymus, which has been reported to progress from the zero variable exon form in immature thymocytes (CD4⫹ CD8⫹) to single- or double-exon usage, which predominantly expresses exons 5 and 6 (47). This differential usage of CD45 isoforms could explain the presence of iIELs in the CD45ex6⫺/⫺ mice, as they still have the potential to express exons 4 and 5. It should be noted that the pool of TCR-expressing lymphocytes in the spleens of both strains of CD45-deficient mice was comparable in these experiments (data not shown). Both the CD45ex6⫺/⫺ and CD45ex9⫺/⫺ mouse strains have reduced numbers of iIELs. This is in agreement with the decrease in total numbers of iIELs previously reported in older CD45ex6⫺/⫺ mice (36). Although the iIEL of CD45ex6⫺/⫺ mice have preferentially maintained the CD8␣␣⫹ population, a minor population of classic (CD8␣␤⫹) iIEL can be detected. However, not all the CD8␤⫹ are Thy-1⫹, an indication that they also may not be thymically derived (data not shown). This is in contrast to the CD45ex9⫺/⫺, which develop minimal CD8␣␤⫹ iIELs (Table III). Recent observations in the IL-2/15R␤ chain-deficient mouse revealed the lack of TCR␣␤⫹CD8␣␣⫹ iIEL (10). Taken together, these data suggest CD8␣␣⫹ iIEL development is dependent on the expression and function of the IL-2/15R␤ chain, but not CD45. In summary, our findings have revealed that CD45 is not required for the development or maintenance of CD8⫹ NK-T cells, NK cells, or CD8␣␣ iIELs. These three populations have several common features. First, in contrast to mainstream T cells, all three of these populations have the capacity to use a thymic-independent developmental pathway (1–5). Second, the maturation pathway of these lymphocyte subsets is severely impaired in IFN regulatory factor 1-, IL-15-, and IL-15R␣-deficient mice (9, 11, 12). Third, all three populations constitutively express the IL-2R␤ chain/15R ␤-chain and proliferate in response to IL-15 (8, 48, 49). IL-15 has been shown to use the IL-2R␤ chain in lymphocytes (49 –51), and IL-15 mRNA is abundant in the intestine of mice (R. D. Newberry, S. M. Martin, and R. G. Lorenz, unpublished observation). The development of these three unconventional lymphocyte subsets in the absence of CD45 allows a hypothesis regarding their development, i.e., that none of these subsets requires signaling through the TCR for their initial development and maturation. This is in contrast to the decreased functional capacity of CD8␣␣ iIELs and NK-T cells in CD45-deficient mice, which rely on triggering of the TCR-CD3 complex for cytokine secretion.

Acknowledgments We thank Kimberly Goodwin for technical assistance, Parveen Chand for help with flow cytometry, and Laurie Shornick, Andrew Chan, and Herbert Virgin for discussions and critical reading of this manuscript. This work is dedicated to the memory of Matthew L. Thomas (1953–1999).

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