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The Balance Between CD45RC^high and CD45RC^low CD4 T Cells in Rats Is Intrinsic to Bone Marrow-Derived Cells and Is Genetically Controlled¹

Jean-Francois Subra^2,*, Bastien Cautain^2,*, Emmanuel Xystrakis^*, Magali Mas^*, Dominique Lagrange^*, Harry van der Heijden, Marie-Jose van de Gaar, Philippe Druet^*, Gilbert J. Fournié^*, Abdelhadi Saoudi^3,* and Jan Damoiseaux

^* Institut National de la Santé et de la Recherche Médicale, Unité 28, Institut Fédératif de Recherche 30, Hôpital Purpan and Université Paul Sabatier, Toulouse, France; and Department of Immunology, University Maastricht, Maastricht, The Netherlands

	Abstract

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The level of CD45RC expression differentiates rat CD4 T cells in two subpopulations, CD45RC^high and CD45RC^low, that have different cytokine profiles and functions. Interestingly, Lewis (LEW) and Brown Norway (BN) rats, two strains that differ in their ability to mount type 1 and type 2 immune responses and in their susceptibility to autoimmune diseases, exhibit distinct CD45RC^high/CD45RC^low CD4 T cell ratios. The CD45RC^high subpopulation predominates in LEW rats, and the CD45RC^low subpopulation in BN rats. In this study, we found that the antiinflammatory cytokines, IL-4, IL-10, and IL-13, are exclusively produced by the CD45RC^low CD4 T cells. Using bone marrow chimeras, we showed that the difference in the CD45RC^high/CD45RC^low CD4 T cell ratio between naive LEW and BN rats is intrinsic to hemopoietic cells. Furthermore, a genome-wide search for loci controlling the balance between T cell subpopulations was conducted in a (LEW x BN) F₂ intercross. Genome scanning identified one quantitative trait locus on chromosome 9 (17 centiMorgan (cM); log of the odds ratio (LOD) score 3.9). In addition, two regions on chromosomes 10 (28 cM; LOD score 3.1) and 20 (40 cM; LOD ratio score 3) that contain, respectively, a cytokine gene cluster and the MHC region were suggestive for linkage. Interestingly, overlapping regions on these chromosomes have been implicated in the susceptibility to various immune-mediated disorders. The identification and functional characterization of genes in these regions controlling the CD45RC^high/CD45RC^low Th cell subpopulations may shed light on key regulatory mechanisms of pathogenic immune responses.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is well established that CD4 T cells can be divided in several subsets as based on the expression of the leukocyte common Ag CD45 (1). This molecule exists at the leukocyte cell surface in a number of different isoforms generated by differential splicing of mRNA transcribed from three different exons, A, B, and C, that encode amino acid sequences of the extracellular NH₂ terminus of the molecule. In the rat, the mAb MRC-OX22 binds to the high m.w. isoform CD45RC and defines subpopulations of CD4 T cells with different cytokine profiles and functions (2, 3, 4, 5, 6). Following mitogenic stimulation in the presence of accessory cells, CD45RC^high CD4 T cells produce IL-2 and IFN-, but express little amounts of IL-4 mRNA, whereas CD45RC^low CD4 T cells produce less IL-2 and IFN- and express high amounts of IL-4 mRNA (2). The production of IL-10 and IL-13 by these subpopulations has not yet been determined. Functional studies in vivo have shown that the CD45RC^high CD4 T cell subset mediates alloreactivity in lethal graft-vs-host reactions or in the popliteal lymph node assay, but provides little or no B cell help for secondary Ab responses (7). Reciprocal activities are found for the CD45RC^low CD4 T cell subset. Further work has shown that neither subset is homogeneous (5, 8, 9, 10) and that the balance between the CD45RC^high and CD45RC^low subpopulations is influenced by the Ags in the periphery. Indeed, upon activation, the naive CD45RC^high population becomes CD45RC^low and the CD45RC^low activated memory population may revert to a CD45RC^high resting memory population depending on the persistence of Ag (9, 11).

Several experimental systems have revealed a functional interregulation between the CD45RC^high and CD45RC^low CD4 T cell subsets. In nude rats, the adoptive transfer of CD45RC^high CD4 T cells from congenic euthymic donors induces a fatal wasting disease, while cotransfer of both subpopulations, or of the CD45RC^low cells alone, has no effect (4). It has been shown that lymphopenia-induced diabetes in PVG RT.1^u rats or thyroiditis in PVG RT.1^c rats can also be prevented by adoptive transfer of CD45RC^low but not of CD45RC^high CD4 T cells (5, 12). In the thyroiditis model, it has been found that IL-4 and TGF- are involved in the immunoregulation mediated by the CD45RC^low CD4 T cell subpopulation (12). Taken together, these data show that the CD45RC^high CD4 T cells contain T cells with a pathogenic potential, while the CD45RC^low CD4 T cells exhibit a regulatory function, and that the latter population has a dominant effect in regulating the former. However, in the mercuric chloride-induced disease, a protective immunoregulatory role has been ascribed to the CD45RC^high CD4 T cells. Indeed, upon in vivo depletion of the CD45RC^high CD4 T cells, HgCl₂-injected animals develop more severe Th2-dependent tissue injury than controls (13).

Brown Norway (BN)⁴ and Lewis (LEW) rats are known to be entirely different with respect to the polarization of their immune responses as well as in their susceptibility to induce autoimmune diseases (14, 15). LEW rats are highly susceptible to develop Th1-mediated experimental allergic encephalomyelitis and cyclosporin A-induced autoimmunity, whereas BN rats are resistant (16, 17, 18). On the other hand, BN rats are highly susceptible to Th2-mediated immunological disorders as induced upon chronic injections of nontoxic doses of HgCl₂ or gold salts (14, 19), while LEW rats are resistant. Interestingly, although there is no direct evidence that the CD45RC^high/CD45RC^low ratio within the CD4 T cell compartment is involved in the susceptibility to develop autoimmunity, we and others did show that LEW rats have a preponderance of CD45RC^high T cells, while in BN rats the CD45RC^low CD4 T cells predominate (20, 21).

In the present study, we examined the factors that influence the balance between the Th subsets in rats as defined by the expression of CD45RC. We first showed that, upon stimulation in an APC-independent system, the antiinflammatory cytokines IL-4, IL-10, and IL-13 are almost exclusively produced by the CD45RC^low subpopulation of CD4 T cells. Next, by using bone marrow chimeras, we showed that the difference in CD45RC^high/CD45RC^low ratio between LEW and BN rats is intrinsic to hemopoietic cells. Finally, a genome-wide search for quantitative trait loci (QTL) in an F₂ intercross between LEW and BN rats identified three regions in chromosomes 9, 10, and 20 that could control the CD45RC^high/CD45RC^low CD4 T cell balance.

	Materials and Methods

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Rats

Specific pathogen-free LEW (RT1^l) and BN (RT1ⁿ) rats were obtained either from Center d’Elevage R. Janvier (Le Genest St. Isle, France) or from the Central Animal Facility of University Maastricht. The (LEW x BN) F₁ rats were obtained from Center d’Elevage R. Janvier and were intercrossed to obtain 104 (LEW x BN) F₂ rats. Rats were anesthetized and bled, as previously described (22). To study the Th2 response (IgE) in these F₂ rats, they were s.c. injected with aurothiopropanol sulfonate (Atps; Allochrysine; Laboratory Solvay Pharma, Suresnes, France), at 20 mg/kg body weight three times per week for 5 wk, as described (22). For making bone marrow chimeras, the (LEW x BN) F₁ were obtained from Charles River (Someren, The Netherlands). Rats were male or female and 8–10 wk of age at the start of the experiment. All procedures were in accordance with national regulations on animal experiments.

Antibodies

The mAbs used in this study were as follows: W3/25 (anti-rat CD4) (23), OX6 (anti-rat MHC class II) (24), OX8 (anti-rat CD8) (25), OX12 (anti-rat -light chain) (26), OX21 (anti-human C3b inactivator) (27), OX22 (anti-rat CD45RC) (7), R73 (anti-rat TCR) (28), V65 (anti-rat TCR) (29), JJ319 (anti-rat CD28) (30), and 10.78 (anti-rat NKR-P1) (31). The hybridomas OX6, OX8, OX12, OX21, OX22, and W3/25 were kindly provided by Dr. D. Mason (Oxford, U.K.). The hybridomas 10.78, JJ319, V65, and R73 were kindly provided by Dr. Th. Hünig (Würzburg, Germany). The conjugated mouse anti-rat mAbs used for flow cytometry, FITC-conjugated OX22, FITC-conjugated R73, and biotinylated W3/25 were prepared in our own laboratory according to standard protocols. PE-conjugated R73, OX35 (anti-rat CD4), and OX39 (anti-rat CD25) are commercially available (PharMingen, San Diego, CA). The biotinylated mAb 42-3-7 (RT1-Aⁿ haplotype) and 163-7F3 (RT1-A^l haplotype) were kindly provided by Dr. H. Kunz (Pittsburgh, PA).

Cell suspensions and flow cytometry

Heparinized blood was collected via the retroorbital vein plexus. The erythrocytes in the buffy coats of the peripheral blood were lysed with NH₄Cl buffer (0.155 M NH₄Cl, 0.01 M KHCO₃, 0.1 mM EDTA, pH 7.4), and next the cells were washed twice and resuspended in PBS-BSA. The spleen and lymph nodes (cervical and mesenteric) were teased apart and passed through a 100-µm mesh nylon gauze and collected in balanced salt solution supplemented with 2% heat-inactivated FCS. The cells were washed twice by centrifugation (316 x g, 10 min, 4°C), resuspended in PBS containing 0.5% w/v BSA (PBS-BSA; Sigma, St. Louis, MO). In case of spleen cell suspensions, the erythrocytes were lysed as described above. Nucleated cells were counted in Türk solution with a Bürker Haemocytometer, while viability was assessed by trypan blue exclusion.

Single cell suspensions were cell surface labeled, and three-color immunofluorescence analysis was conducted. Briefly, cells (5 x 10⁵/sample) were centrifuged in a 96-well microtiter plate (236 x g, 3 min, 4°C) and resuspended in PBS-BSA containing 10 mM NaN₃ and a cocktail of FITC-labeled, PE-labeled, and biotinylated mAbs. The biotin-conjugated mAb were stained in a second step with streptavidin-CyChrome (PharMingen). Data were collected on 10,000 cells, as determined by forward and side light scatter intensity on a XL Coulter (Coultronics, Margency, France) or a FACSCalibur (Becton Dickinson, San Jose, CA) cytometer using the Cell Quest software package (Becton Dickinson) for analysis. Unless stated otherwise, the CD45RC expression was analyzed by gating on TCR and CD4 double-positive lymphocytes.

Isolation of CD4 T cells

Rat T cells were negatively selected from lymph node and spleen cells using anti-mouse IgG magnetic microbeads (Dynal, Oslo, Norway). Briefly, cells were washed and incubated 30 min on ice with a cocktail of the following mAbs: OX6, OX8, OX12, 10.78, and V65. After washing and incubation with anti-mouse IgG-coupled microbeads under agitation, CD4 T cells were purified by magnetic depletion. The purity of the negatively selected cells was controlled by flow cytometric analysis using triple staining with OX8-FITC, R73-PE, and biotinylated W3/25, as well as by using rabbit anti-mouse IgG-FITC. The purity was between 95 and 98%. CD45RC^high and CD45RC^low CD4 T cell subpopulations were obtained by cell fractionation using Elite coulter cell sorter (Coultronics), following labeling CD4 T cells with FITC-conjugated OX22. Purity of sorted populations was always 96–99%.

T cell stimulation

The culture medium was RPMI 1640 (Life Technologies, Cergy pontoise, France) containing 10% FCS, 1% pyruvate, 1% nonessential amino acids, 1% L-glutamine, 1% penicillin-streptomycin, and 2 x 10^-5 M 2-ME. For stimulation in the absence of APC, 10⁵ T cells were incubated with plate-bound TCR mAb (R73) and soluble CD28 mAb (JJ319), as already described (30). Proliferation was measured by the degree of [³H]thymidine uptake during the last 18 h of a 72-h culture period, and results were expressed as mean cpm of triplicate cultures. At various times throughout the culture (24 or 36, 48 or 60, and 72 h), supernatants were removed and stored at -20°C for cytokine determination; cells were harvested following 24- or 36-h stimulation and RNA purified for analysis of lymphokine gene expression by RT-PCR.

Cytokine assays

IFN-, IL-2, and IL-10 protein in the supernatant were measured by specific ELISA. Ninety-six-well plates were coated overnight at 4°C with 5 µg/ml of an anti-rat IFN- mAb (DB1) (32), 1 µg/ml of rabbit anti-rat IL-2 Ab (PharMingen), or 1 µg/ml of an anti-rat IL-10 mAb (A5-7; PharMingen). Serial dilutions of tissue culture supernatant (100 µl/well), followed by biotinylated DB12, an anti-rat IFN- mAb (32); biotinylated A38-3 (PharMingen), an anti-rat IL-2 mAb; or biotinylated A5-6 (PharMingen), an anti-rat IL-10 mAb, were sequentially incubated for 2 h at room temperature, separated by three washes. The bound biotinylated Abs were revealed by additional 60-min incubation with alkaline phosphatase-conjugated streptavidin (Jackson ImmunoResearch Laboratories, Avondale, PA). The assay was developed by adding the enzyme substrate 4-nitrophenylphosphate disodium (Sigma) at 1 mg/ml in diethanolamine buffer, pH 9.6, for 90 min at room temperature. The absorbance was measured at 405 nm using automated microplate ELISA reader (Emax; Molecular Devices, Sunnyvale, CA). For IFN-, values were expressed as U/ml derived from a standard curve constructed using rat rIFN-. This cytokine and anti-rat IFN- mAbs were a gift from Dr. P. Van der Meide (TNO, Rijswijk, The Netherlands). For IL-2, values were expressed as U/ml derived from a standard curve constructed using rat rIL-2 (PharMingen). For IL-10, values were expressed as pg/ml derived from a standard curve constructed using rat rIL-10 (PharMingen). TNF- protein in the supernatant was measured by biological assay using a mouse fibroblast line (L929) sensitive to TNF-mediated cytotoxicity. Briefly, 3 x 10⁴ cells/well were added to a flat-bottom plate in 100-µl vol and allowed to adhere overnight. An equal volume of serial dilutions of supernatant with 0.1 µg/well of actinomycin D was added to the cells. Cell viability was determined 18 h later by addition of crystal violet. After 10 min of incubation, the plates were washed and 150 µl of 95% ethanol was added in each well. Absorbance was measured in a microplate reader with a 570-nm filter. The results were expressed as pg/ml derived from a standard curve constructed using rat rTNF- (PharMingen). IL-4 and IL-13 mRNA was detected by RT-PCR, as has been already published (33). Following amplification, 5 µl of the amplified product were then separated by electrophoresis on 2% agarose minigels and visualized by ethidium bromide staining. Photographs of gels were digitized, and densitometric analysis of the bands was performed by using the Gel Analyst program (GreyStone; ICONIX, Courtaboeuf, France). Results were expressed in arbitrary units and represent the ratio of the intensity of the band for cytokine to the intensity of the band for GAPDH x 100.

Radiation bone marrow chimeras

Rats were given 10.2 Gy total body irradiation using a Philips µ15F/225kV Röntgen irradiation machine (Hamburg, Germany) 1 day before bone marrow transplantation. Recipient rats were given 10⁸ viable nucleated bone marrow cells i.v. into a tail vein. At 10 wk postengraftment, the peripheral blood of the animals was analyzed for the state of activation (CD25) on donor-derived T cells. Donor or recipient origin of the T cells was determined by inclusion of the RT1-A haplotype-specific mAb. At 12 and 14 wk postengraftment, the peripheral blood of the animals was analyzed for the expression of CD45RC within the CD4 T cells of donor origin. At week 14, spleen and lymph nodes were also included in the analysis.

Genome scan

Genomic DNA from the F₂ rats was prepared from tail tips using the High Pure PCR Template Preparation Kit from Boehringer Mannheim (Meylan, France) according to the manufacturer’s procedure. DNA was stored at -20°C in distilled water. Genotypes were determined by PCR amplification of DNA fragments known to contain simple sequence length polymorphism between BN and LEW. When available, microsatellite marker loci were chosen at approximately 20- to 30-cM intervals based on the genetic map of the rat (http://www.genome.wi.mit.edu, fttp://well.ox.ac.uk/pub/genetics/ratmap) (34, 35). A list of the markers used in this study is available on request. Primers were obtained from Genset (Paris, France). PCR amplifications were performed in GeneAmp PCR Systems 9600 and 9700 (PE Applied Biosystems, Foster City, CA) in 15-µl vol using 50 ng of genomic DNA, 0.4–0.6 U of Taq DNA polymerase (AmpliTaq Gold from Perkin-Elmer, Norwalk, CT), 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl₂, 0.2 mM of each dNTP, 0.5 µM of each primer, 90 µg/ml Cresol Red, and 13% sucrose. After an initial step at 95°C for 10 min, 39 cycles of 95°C for 20 s, 52°C or 60°C for 30 s, and 72°C for 30 s were used. The final elongation time at 72°C was 10 min. The PCR products were size fractionated on 4% agarose (MetaPhor, TEBU, Le Perray-en-Yvelines, France) and visualized under UV light after staining with 1 µg/ml ethidium bromide.

Linkage maps were estimated using the Kosambi map function within the MAPMAKER/EXP 3.0b computer package, and quantitative trait linkage analysis was performed by interval mapping using MAPMAKER/QTL 1.0b (36) under the assumptions of a free genetic model. A total of 98.6% of the rat genetic map was scanned to within 30 cM of a marker. The further uncovered distances were respectively of 32, 41, and 40 cM for chromosomes 5, 16, and X. LOD score thresholds to achieve the genome-wide significance levels of 5% (significant linkage) were, as proposed by Lander and Kruglyak (37), 4.3 when no model of gene action was specified ("free" genetics), 3.4 when gene action was restricted to recessive or dominant models, and 3.3 when gene action was restricted to an additive model. For suggestive linkage, log of the odds ratio (LOD) score values were, respectively, 2.8 ("free" genetics), 2 (recessive or dominant), and 1.9 (additive). When a peak LOD score (36) was statistically significant or suggestive, we genotyped all F₂ individuals with additional markers when possible, to increase the marker density in the region of the chromosome over which the LOD score drops by 2 from its peak value.

Normality of the distributions was assessed using Kolmogorof Smirnov’s test after logarithmic transformation of the data. One-way ANOVA was also done using the SPSS 8.0.1F statistical package. After establishing the equality between the variances (Levene’s test), Tukey’s post hoc test was performed to choose the model of inheritance.

ELISA for quantitation of serum IgE concentrations and study of correlation

The Atps-injected 104 (LEW x BN) F₂ rats were bled once per week, and their sera were frozen at -20°C. The total serum IgE concentration was measured by specific ELISA, as already described (22). The Spearman’s rank test was used to detect a correlation between the IgE response to Atps injections and the CD45RC^high/CD45RC^low CD4 T cell ratios using the SPSS 8.0.1F package.

	Results

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Differential cytokine production by CD45RC^high and CD45RC^low CD4 T cells

CD4 T cells were purified from LEW spleen and lymph nodes using negative selection. Next the cells were fractionated into CD4 CD45RC^high (95% purity) and CD4 CD45RC^low (97% purity) fractions by cell sorting, as indicated in Fig. 1. Upon stimulation with bound anti-TCR and soluble anti-CD28, both T cell subpopulations proliferated equally well (Fig. 2A), but produced different cytokine profiles. The analysis of the antiinflammatory cytokines, IL-4, IL-10, and IL-13, revealed that these cytokines were mainly produced by the CD45RC^low CD4 T cell subset (Fig. 2, B–D). The inflammatory cytokines, IL-2, TNF-, and IFN-, were produced by both subsets, but the CD45RC^high population produced more IL-2 and TNF- (Fig. 2, E–F), while the CD45RC^low population produced more IFN- (Fig. 2G). Similar results were obtained when BN CD4 T cell subsets were tested (data not shown). These results show that upon stimulation in the absence of APC, the inflammatory cytokines are produced by both T cell subpopulations, while the antiinflammatory cytokines are almost exclusively produced by the CD45RC^low CD4 T cell subset.

View larger version (31K): [in this window] [in a new window]   FIGURE 1. Fractionation of CD45RChigh and CD45RClow CD4 T cell subpopulations. To purify CD4 T cells, spleen and lymph node cells from naive LEW rats were depleted of CD8+ and TCR T cells, B cells, monocytes, and NK cells using a standard technique of negative selection, as described in Materials and Methods. The cells were double color labeled with mAb to CD4 (W3/25) and to CD8 (OX8) before (dot plot on the left) and after purification (dot plot on the right). The obtained purified CD4 T cells were further fractionated by cell sorting on the basis of expression of CD45RC using FITC-conjugated OX22. The gates used to purified CD45RChigh and CD45RClow subpopulations are indicated in the histogram in the middle of this figure. The values represent the percentage of each cell population.

View larger version (27K): [in this window] [in a new window]   FIGURE 2. Differential cytokines production by CD45RChigh and CD45RClow CD4 T cells. The purified CD45RClow () and CD45RChigh () T cells from LEW rats were stimulated with plate-bound anti-TCR supplemented (0.2 µg) or not (0 µg) with soluble anti-CD28. A, Proliferation was assessed with an 18-h [3H]thymidine pulse added after 48-h stimulation, and results were expressed as cpm. Tissue culture supernatants collected 24, 48, and 72 h after stimulation with anti-TCR and anti-CD28 were assayed for IL-10 (B), IL-2 (E), and IFN- (G) protein using capture ELISA and for TNF- (F) using biological assay. In C and D, the figures show, respectively, the IL-4 and IL-13 mRNA expression following stimulation for 24 h. Results are expressed in arbitrary units that represent the ratio between the intensity of the band for IL-4 or IL-13 and the housekeeping gene GADPH. The stimulation with anti-TCR only did not induce the production of these cytokines. The data shown represent the quantification made in a pool of triplicate culture for each time point and are representative of three independent experiments.

It has been reported previously that the CD45RC^high/CD45RC^low ratio within the CD4 T cell compartment differs between rat strains (20, 21, 38). Indeed, upon analysis of peripheral blood, the CD45RC^high/CD45RC^low ratios within the CD4 T cell compartment were 2.2 (±0.3) in LEW rats and 0.9 (±0.1) in BN rats (Fig. 3). (LEW x BN) F₁ rats exhibited an intermediate phenotype, resulting in a CD45RC^high/CD45RC^low ratio of 1.2 ± 0.1. To examine whether the distinct CD45RC^high/CD45RC^low ratio within the CD4 T cell compartment between LEW and BN rats is intrinsic to hemopoietic cells or is determined in the periphery, we generated bone marrow chimeras. Since activation of T cells results in down-modulation of CD45RC molecules, we first analyzed the state of T cell activation within the donor-type T cells in our chimeras by flow cytometry using anti-CD25 mAb and anti-MHC class I haplotype-specific mAbs. In all chimeras, the percentage of activated donor-type T cells was less than 9% (data not shown). We did also analyze the state of T cell chimerism using anti-TCR mAb and anti-MHC class I haplotype-specific mAbs and showed that the chimerism in F₁ recipients of BN and LEW bone marrow cells was 75% and 87%, respectively, while the chimerism of BN and LEW recipients of F₁ bone morrow cells was 96% and 92%, respectively (data not shown). The analysis of CD45RC expression by donor-type peripheral blood CD4 T cells revealed that the difference in CD45RC^high/CD45RC^low CD4 T cell ratio between naive LEW and BN rats is intrinsic to the hemopoietic cells. Indeed, F₁ recipients of BN and LEW bone marrow cells exhibited a clear difference in the CD45RC^high/CD45RC^low ratio (Fig. 4A). On the other hand, BN, LEW, and F₁ recipients of F₁ bone marrow cells had similar ratios (Fig. 4B). These results indicate that the balance between CD45RC^high and CD45RC^low CD4 T cell subpopulations is genetically determined in bone marrow-derived cells and that the nonhemopoietic peripheral factors do not contribute to this balance.

View larger version (18K): [in this window] [in a new window]   FIGURE 3. LEW and BN rats exhibit different percentage of CD45RClow and CD45RChigh CD4 T cell subpopulations. Peripheral blood leukocytes from naive LEW, BN, and (LEW x BN) F1 rats were incubated with anti-TCR (R73), anti-CD4 (W3/25), and anti-CD45RC (OX22) mAbs and analyzed by flow cytometry, as indicated in Materials and Methods. The results represent histograms of the CD45RC expression by CD4 T cells. The data are of one randomly chosen rat among four in each rat strain. The value on the top of each histogram represents the mean (±SD) of CD45RC CD4 T cell subpopulations of four rats per group.

View larger version (27K): [in this window] [in a new window]   FIGURE 4. The distinct CD45RChigh/CD45RClow ratio between naive LEW and BN rats is intrinsic to hemopoietic cells. Peripheral blood leukocytes from (LEW x BN) F1 recipients of BN (BNF1), LEW (LEWF1), or (LEW x BN) F1 (F1F1) bone marrow (A) and LEW (F1LEW), BN (F1LEW), and F1 (F1LEW) recipients of (LEW x BN) F1 bone marrow (B) were incubated with a combination of anti-CD45RC, anti-CD4, and anti-donor type MHC class I mAbs, and analyzed by flow cytometry 14 wk postengraftment. The results represent the CD45RChigh/CD45RClow ratios determined within CD4-positive T cells of donor origin. The data are represented as the mean (±SD) CD45RChigh/CD45RClow T cell ratio of five to six animals of each combination. Open circle represents the result of individual rats.

The expression of the CD45RC marker on peripheral CD4⁺ T cells was studied in 104 (LEW x BN) F₂ rats, and a genome-wide search for loci controlling this expression was conducted using a panel of polymorphic microsatellite markers covering 99% of the rat genome within 30 cM of a marker. Whenever a QTL was found or suggested using the MAPMAKER software (36), ANOVA with Tukey’s post hoc test was performed for the marker at the peak of the QTL. The mode of inheritance was defined as additive, dominant, or recessive according to the significance of differences in the mean values of the trait between rats that were homozygous BN, heterozygous BN/LEW, or homozygous LEW. We identified one significant locus on chromosome 9 and two suggestive loci on chromosomes 10 and 20. Results are shown in Table I and Fig. 5. These three QTLs account for 40% of the genetic variance of the percentage of the CD4 CD45RC^high population. The size of the QTLs, as defined by the two-LOD support intervals, are respectively 17 cM on chromosome 9, 28 cM on chromosome 10, and 40 cM on chromosome 20. Fig. 6 shows this trait according to the genotypes of the marker located at the peak of the QTLs. For the three QTLs, the BN genotype was associated with a low percentage of the CD4 CD45RC^high population. For the expression of this phenotype, the mode of gene action appears to fit with a BN recessive (or LEW dominant) mode for the QTL found on chromosome 9 and with a BN dominant (or LEW recessive) mode for the QTLs found on chromosomes 10 and 20.

View larger version (29K): [in this window] [in a new window]   FIGURE 5. Diagrammatic representation of chromosomes and QTLs location on chromosomes 9, 10, and 20. Genetic maps were constructed using data collected on the progeny of the 104 (LEW x BN) F2 hybrid rats investigated in this study. On the right of each chromosome are indicated the markers and the distance in centiMorgans between two consecutive markers, as calculated by MAPMARKER/EXP 3.0 using the kosambi mapping function. The positions of the centromeres are shown as closed circles. Black rectangles located in the chromosome indicate the QTLs for the percentage of the CD45RChigh CD4 T cell population. Vertical bars on the left of each chromosome show the position of various QTLs that have been previously described in these regions. Atps1, Atps2, and Atps3 are loci for susceptibility to develop IgE response in rats injected with Atps (19 22 ). Eae4 and eae-x are loci for susceptibility to EAE (18 46 ). The locus for EAE susceptibility described on chromosome 10 has not yet been named and is therefore indicated Eae-x. The cytokine gene cluster indicated in chromosome 10 contains the IL-4, IL-5, IL-3, IFN-regulatory factor-1 (47 ). MHC on top of chromosome 20 expends on a 3-megabase region (48 ). In all cases, the sizes and locations of the QTLs are defined by their two-LOD support intervals.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. Percentage of CD4 CD45RChigh T cells according to the genotypes of 104 (LEW x BN) F2 hybrid rats for the microsatellite marker located at the peak of QTLs found on chromosomes 9 (D9Cbr1), 10 (D10Wox13), and 20 (D20Wox5). Location of markers on the genetic map of the rat is shown in Fig. 5. Symbols represent the mean values, and vertical bars indicate the SEM. The statistical significance of differences found between groups is indicated above horizontal lines.

To establish a possible link between the CD45RC^high/CD45RC^low ratio within the CD4 T cell compartment and the distinct functional characteristics of LEW and BN rats, we examined in the 104 F₂ rats whether the high percentage of CD4 CD45RC^high is correlated with a low IgE response upon treatment with gold salts. The results in Table II show that a negative correlation exists between the percentage of CD45RC^high CD4 T cells and the IgE response on days 7, 28, and 35 after the first gold salts injection. The strongest correlation is found with the serum IgE concentration on day 7 (p = 0.001). A correlation also exists with the mean IgE response from day 0 to day 35.

	Discussion

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In our study, we found that the CD45RC^low subpopulation produced both pro- and antiinflammatory cytokines, upon stimulation with anti-TCR and anti-CD28. Whether these cytokines are produced by the same cells or by different T cell subpopulations is not known. There is a large body of evidence that there is functional and phenotypical heterogeneity not only within the CD45RC^low CD4 T cells, but also within the CD45RC^high CD4 T cell subset (5, 6, 10, 38). Therefore, the expression of the CD45RC isoform cannot be used to unambiguously identify a unique differentiation state. Concerning the CD45RC^low CD4 T cell subpopulation, there are at least three distinct subsets: 1) recent thymic emigrants that can be identified by the expression of Thy-1; 2) activated, Ag-primed T cells that neither express RT6 nor Thy-1; and 3) Th2-like memory cells that have been involved in the regulation of autoimmunity and are also called regulatory T cells. This cell subset depends on the continuous presence of Ag and has been shown to express RT6 but not Thy-1. With respect to the CD45RC^high subpopulation, at least two different subsets can be distinguished: 1) naive T cells that express RT6 but not Thy-1, and 2) Th1-like memory cells that express neither RT6 nor Thy-1. Whether these Th1-like memory cells are identical to the memory cells that have been reported to be independent on persisting Ag is not yet clarified. Taken together, the maturation and the level of CD45RC expression by peripheral CD4 T cells are dependent on Ag encounter and Ag persistence in the periphery (9, 11). The establishment of the CD45RC^high/CD45RC^low CD4 T cell ratio may therefore involve all the actors that are implicated in the generation of immune responses. These actors include bone marrow-derived hemopoietic cells such as APC and T cells, but also nonhemopoietic factors such as Ag and mediators that are involved in the interaction between the neuroendocrine and immune systems. In the present study, we showed that the CD45RC^high/CD45RC^low CD4 T ratio is genetically determined and is intrinsic to bone marrow-derived cells. Indeed, the difference in CD45RC^high/CD45RC^low CD4 T cell ratio between naive LEW and BN rats is dependent on bone marrow donor type, but not on the genetic make-up of the recipient, as shown by bone marrow chimeras.

We found that, upon stimulation in an APC-independent system, the CD45RC^low CD4 T cell subpopulation produced more IL-4, IL-10, IL-13, but less IL-2 and TNF- than CD45RC^high CD4 T cell subpopulation. However, one unexpected but repeatable finding was the higher IFN- production by the CD45RC^low CD4 T cell subpopulation. This result contrasts with the finding that, following mitogenic stimulation in the presence of accessory cells, the highest amounts of IFN- protein were produced by the CD45RC^high CD4 T cell subpopulation (2, 41). However, a discrepancy between IFN- mRNA expression and protein production by CD45RC^low CD4 T cells has been observed in one of these studies (2). This result indicates that the CD45RC^low CD4 T cells are not defective in IFN- production but, in contrast to CD45RC^high population, this population might regulate translation or secretion of the IFN- protein in a manner that would prevent high levels of release (2). Our results are in agreement with this hypothesis and suggest that the interaction between CD45RC^low CD4 T cells and accessory cells may mediate this regulation. In addition, it is tempting to speculate that accessory cells mighthave a differential effect in controlling IFN- production by CD45RC^high and CD45RC^low CD4 T cells via the differential expression of some regulatory molecules on these CD4 T cell subpopulations. This supposition is currently under exploration. In agreement with this hypothesis, it has been shown more recently that CD45RB^low, but not CD45RB^high, CD4 T cells express the negative regulator of T cell activation CTLA-4 (42). Furthermore, the blockade of CTLA-4/B7 interaction in vivo by anti-CTLA-4 Ab was reported to result in the exacerbation of classical Th1-mediated autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE) and diabetes (43, 44, 45).

Genome-wide search linkage analysis of the CD45RC^high/CD45RC^low CD4 T cell ratio in (LEW x BN) F₂ rats identified three regions localized on chromosomes 9, 10, and 20. The regions on chromosome 10 and 20 were only supportive for linkage to the ratio. The locus found on chromosome 20 includes the MHC region that is associated with the development of various immune-mediated disorders in rat, mouse, and human. However, the QTL found in this study expand on 40 cM, and it is possible that the gene(s) that controls the CD45RC T cell subsets ratio is (are) located outside the 3 cM of the MHC region. Interestingly, overlapping regions on rat chromosomes 9 and 10 and on some homologous regions to rat chromosome 10 in man and mouse are involved in susceptibility and/or severity to various immune-mediated disorders (Table III, Fig. 5). These data suggest that the CD45RC^high/CD45RC^low CD4 T cell ratio could be related to clinically distinct forms of immune-mediated disorders, including autoimmune diseases.

	Acknowledgments

 
We thank I. Bernard (Institut National de la Santé et de la Recherche Médicale (Unité 28) and G. Cassar (Institut Fédératif de Recherche 30, Toulouse, France) for excellent technical assistance; M. Calise, S. Apolinaire-Pilipenko, and P. Aregui (Institut Fedératif de Recherche 30, Toulouse, France) for taking care of the animal house; and Marie-Odile Christen (Solvay Pharma Laboratory) for her support and interest in our work.

	Footnotes

 
¹ This work was supported by Institut National de la Santé et de la Recherche Médicale, the Université Paul Sabatier, the Conseil Général de Région Midi-Pyrénées (Contract 97001931), the Ministère de l’Aménagement du Territoire et de l’Environnement, the Cardiovascular Research Institute Maastricht, by grants from Association pour la Recherche sur la Sclérose en Plaques and Association Française contre les myopathies, the Solvay Pharma Laboratory, and a collaborative grant from the Dutch Organization for Scientific Research (NWO) and Institut National de la Santé et de la Recherche Médicale. J.-F.S. is supported by Société de Néphrologie, J.-F.S. and E.X. by Fondation pour la Recherche Médicale, and M.M. by Association de Recherche sur la Polyarthrite Rhumatoïde. A.S. is supported by Center National de la Recherche Scientifique, and B.C. by Ministère de l’Éducation Nationale, de la Recherche et de la Technologie.

² J.-F.S. and B.C. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Abdelhadi Saoudi, Institut National de la Santé et de la Recherche Médicale, Unité 28, Hôpital Purpan, place du Dr Baylac, 31059 Toulouse Cedex, France.

⁴ Abbreviations used in this paper: BN, Brown Norway; Atps, aurothiopropanol sulfonate; EAE, experimental autoimmune encephalomyelitis; LEW, Lewis; LOD, log of the odds ratio; QTL, quantitative trait locus; cM, centiMorgan.

Received for publication August 25, 2000. Accepted for publication December 12, 2000.

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