HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xystrakis, E. Articles by Saoudi, A. Search for Related Content PubMed PubMed Citation Articles by Xystrakis, E. Articles by Saoudi, A. Pubmed/NCBI databases Gene Substance via MeSH

Functional and Genetic Analysis of Two CD8 T Cell Subsets Defined by the Level of CD45RC Expression in the Rat¹

Emmanuel Xystrakis^2,*, Pierre Cavailles^2,*, Anne S. Dejean^*, Bastien Cautain^3,*, Céline Colacios^*, Dominique Lagrange^*, Marie-Jose van de Gaar, Isabelle Bernard^*, Daniel Gonzalez-Dunia^*, Jan Damoiseaux, Gilbert J. Fournié^* and Abdelhadi Saoudi^4,*

^* Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 563, Institut Fédératif de Recherche (IFR) 30, Hôpital Purpan and Université Paul Sabatier, Toulouse, France; and Department of Immunology, University Maastricht, Maastricht, The Netherlands

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Differential cytokine production by T cells plays an important role in the outcome of the immune response. We show that the level of CD45RC expression differentiates rat CD8 T cells in two subpopulations, CD45RC^high and CD45RC^low, that have different cytokine profiles and functions. Upon in vitro stimulation, in an Ag-presenting cell-independent system, CD45RC^high CD8 T cells produce IL-2 and IFN- while CD45RC^low CD8 T cells produce IL-4, IL-10, and IL-13. In vitro, these subsets also exhibit different cytotoxic and suppressive functions. The CD45RC^high/CD45RC^low CD8 T cell ratio was determined in Lewis (LEW) and Brown-Norway (BN) rats. These two rat strains differ with respect to the Th1/Th2 polarization of their immune responses and to their susceptibility to develop distinct immune diseases. The CD45RC^high/CD45RC^low CD8 T cell ratio is higher in LEW than in BN rats, and this difference is dependent on hemopoietic cells. Linkage analysis in a F₂(LEW x BN) intercross identified two quantitative trait loci on chromosomes 9 and 20 controlling the CD45RC^high/CD45RC^low CD8 T cell ratio. This genetic control was confirmed in congenic rats. The region on chromosome 9 was narrowed down to a 1.2-cM interval that was found to also control the IgE response in a model of Th2-mediated disorder. Identification of genes that control the CD45RC^high/CD45RC^low CD8 T cell subsets in these regions could be of great interest for the understanding of the pathophysiology of immune-mediated diseases.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antibodies recognizing different isoforms of the leukocyte common Ag CD45 have revealed a phenotypic and functional heterogeneity in mature T cells. In the rat, the mAb MRC-OX22 binds to the high m.w. isoform CD45RC and defines two subpopulations of CD4 T cells with different cytokine profiles and functions (1, 2, 3). Polyclonal activation of the CD45RC^high and CD45RC^low CD4 T cell subsets in vitro showed that the CD45RC^high CD4 T cells synthesize predominantly IL-2 and IFN-, whereas CD45RC^low CD4 T cells synthesize IL-4, IL-10, and IL-13 (2, 4). Functional analysis of CD45RC^high and CD45RC^low CD4 T cells has demonstrated that important regulatory interactions occur between these subsets in vivo (3, 5). Recently, we showed that the balance between CD45RC^high and CD45RC^low CD4 T cells in rats is intrinsic to bone marrow-derived cells and that it is genetically controlled by regions on chromosomes (c) 9, 10, and 20 (4). These regions have been implicated in the susceptibility to various immune-mediated disorders (4, 6) indicating that the CD45RC^high/CD45RC^low ratio may contribute to susceptibility to immune-mediated diseases.

CD8 T cells play important roles, either as effector or regulatory T cells, in autoimmune diseases (7, 8), allergy (9), organ transplantation (10, 11), protection of the host against infectious diseases (12, 13), and cancer (14, 15). Recently, we showed that the difference in susceptibility between Lewis (LEW)⁵ and Brown-Norway (BN) rats to develop, respectively, type 1- and type 2-mediated immune disorders is associated with a qualitative difference in their CD8 T cell compartment (6, 16). BN CD8 T cells produce little IFN- and IL-2, but express high levels of IL-4, when compared with LEW CD8 T cells. It is known that CD8 T cells have the potential to produce a wide array of cytokines and distinct subsets of CD8 T cells have been described in mice (17, 18), rats (19), and humans (20). However, the level of CD45RC expression has never been addressed as a marker for functionally distinct CD8 T cell subsets.

In the present study, we demonstrate for the first time that CD45RC expression distinguishes two CD8 T cell subsets in the rat: CDR45RC^high and CD45RC^low. Upon in vitro stimulation in an APC-independent system, CD45RC^high CD8 T cells produce mainly IL-2 and IFN-, while the CD45RC^low CD8 T cells produce only IL-4, IL-10, and IL-13. We also show that CD45RC^low and CD45RC^high CD8 T cells exhibit different cytotoxic and regulatory functions in vitro. Furthermore, we demonstrate that the balance between CD45RC^high and CD45RC^low CD8 T cells is mainly determined by bone marrow-derived cells and is genetically controlled by two regions on chromosomes 9 (c9) and 20 (c20). Interestingly, the same region on c9 also appeared to regulate Th2-mediated disorder.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals

Male LEW and BN rats were obtained from Centre d’Elevage R. Janvier (Le Genest St. Isle, France) or from the Central Animal Facility of University Maastricht. (LEW x BN)F₁ rats were obtained from Centre d’Elevage R. Janvier and were intercrossed to obtain 104 (LEW x BN)F₂ rats. For the generation of bone marrow chimeras, male (LEW x BN)F₁ rats were obtained from Charles River Laboratories (Someren, The Netherlands). The MHC-congenic BN.1L and LEW.1N rats were obtained by the cross-intercross-backcross method and were backcrossed 20 times (unpublished observation, Dr. H. J. Hedrich, Medizinische Hochschule, Hannover, Germany). They were originally purchased from the Zentralinstitut für Versuchstierzucht (ZFV; Hannover, Germany) and used as a breeding nucleus in Maastricht since 1994. The genetic maps of these congenic lines were constructed using 16 polymorphic markers covering the chromosome c20 with a mean average spacing of 3 cM between markers. One hundred sixty markers covering 99% of the other autosomal chromosomes with an average spacing of 10 cM were used to control the genetic background of the congenic lines. In the BN.1L line the marker D4Wox19 showed a homozygous LEW genotype, defining a highest possible LEW interval of 23.3 cM on c4; in the LEW.1N line, D16Wox10 and D18Mit3 showed BN genotypes defining highest possible BN intervals of 3.4 cM on c16 and of 18.6 cM on c18, respectively. The BN congenic lines for LEW chromosome 9 were made in our own animal facilities (see below). To study the Th2 response in these congenic lines, rats were injected s.c. with aurothiopropanol sulfonate (Atps; Allochrysine; Laboratory Solvay Pharma, Suresnes, France), and their sera were tested for IgE production on day 7 after three injections of Atps (20 mg/kg body weight) as described (21). Rats were 8–10 wk of age at the start of the experiment. Breeding and experimental procedures were conducted in accordance with European guidelines.

Antibodies

The mAbs used for flow cytometry and for purification of T cell subpopulations were as follows: W3/25 (anti-rat CD4), OX6 (anti-rat MHC class II), OX8 (anti-rat CD8), OX12 (anti-rat L chain), OX21 (anti-human C3b inactivator), OX22 (anti-rat CD45RC), OX39 (anti-rat CD25), OX40, OX85 (anti-rat L selectin), R73 (anti-rat TCR), V65 (anti-rat TCR), JJ319 (anti-rat CD28), 341 (anti-rat CD8), and 3.2.3 (anti-rat NKR-P1). The Abs used for cytokine neutralization were 2G7 (which recognizes both active and latent forms of TGF1, TGF2, and TGF3) mAb and rabbit anti-rat IL-10 (kindly provided by Dr. J. Khalif, Pasteur Institut, Lille, France). The hybridomas OX6, OX7, OX8, OX12, OX21, OX22, OX39, OX40, OX85, and W3/25 were kindly provided by Dr. D. Mason (Medical Research Council Cellular Immunology Unit, Oxford, U.K.) and the hybridomas JJ319, V65, and R73 by Dr. T. Hünig (Virology and Immunology Institute, Würzburg, Germany). The conjugated mouse anti-rat mAbs used for flow cytometry, FITC-conjugated OX8, OX22, and R73 mAbs, and biotinylated W3/25, OX6, OX8, OX18, OX39, OX40, OX85, and JJ319 mAbs were prepared in our own laboratory according to standard protocols. PE-conjugated R73, 341, and OX39 are commercially available (BD 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. W. Kunz (Department of Pathology, University of Pittsburgh, Pittsburgh, PA).

Flow cytometry

Heparinized blood was collected via the retro-orbital vein plexus. The erythrocytes were lysed with NH₄Cl buffer (0.155 M NH₄Cl, 0.01 M KHCO₃, 0.1 mM EDTA, pH 7.4), and the cells were washed twice and resuspended in PBS-BSA. 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 mixture of FITC-labeled, PE-labeled, and biotinylated mAbs. The biotin-conjugated mAb were stained in a second step with streptavidin-CyChrome (BD Pharmingen). Data were collected on a XL Coulter (Coultronics, Margency, France) or a FACSCalibur (BD Biosciences, San Jose, CA) cytometer using the CellQuest software package (BD Biosciences) for analysis.

Isolation of CD45RC T cell subsets

Rat CD8 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 mixture of the following mAbs: OX6, W3/25, OX12, 3.2.3, and V65. After washing and incubating with anti-mouse IgG-coupled microbeads under agitation, CD8 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 CD45RC^high and CD45RC^low CD8 T cell subpopulations were obtained by positive and negative selection using magnetic beads (AutoMacs; Miltenyi Biotec, Bergisch Gladbach, Germany). Purified CD8 T cells were incubated with OX22-FITC and R73-PE mAbs and selected using anti-FITC and anti-PE magnetic beads (AutoMacs; Miltenyi Biotec). Purity of sorted populations was always >95%.

T cell stimulation and analysis of T cell proliferation

The culture medium was RPMI 1640 (Invitrogen Life Technologies, Cergy Pontoise, France) containing 10% FCS, 1% sodium pyruvate, 1% nonessential amino acids, 1% L-glutamine, 1% penicillin-streptomycin, and 2 x 10^–5 M 2-ME. For proliferation and cytokine profile studies, purified LEW CD45RC CD8 T cell subsets were polyclonally stimulated using anti-TCR and anti-CD28 mAbs or specifically stimulated with alloantigens in a mixed leukocyte reaction (MLR). For polyclonal activation, 2.5 x 10⁵ T cells were incubated with plate-bound anti-TCR mAb and soluble anti-CD28 mAb as already described (22). For MLR, stimulation was performed by incubating fractionated CD45RC CD8 T cell subsets (3 x 10⁵ cells/well) with 1.5 x 10⁵ T cell-depleted and -irradiated (2500 rad) splenocytes from semiallogeneic (LEW x BN)F₁ rats in the presence of rat IL-2 (10 U/ml). In coculture experiments, LEW CD8 CD45RC^high T cells (3 x 10⁵ cells/well) were stimulated with semiallogeneic T cell-depleted and irradiated F₁ spleen cells (1.5 x 10⁵ cells/well) and variable numbers of LEW CD8 CD45RC^low or CD8 CD45RC^high T cells. Proliferation was measured by [³H]thymidine uptake during the last 18 h of a 48 h (anti-TCR + anti-CD28) or a 96 h (for MLR) culture period. Results were expressed as mean cpm of triplicate cultures.

Cytokine analysis

At various times throughout the culture (36 and 60 h for anti-TCR stimulation, 72, 96, and 120 h for MLR) supernatants were removed and stored at –20°C for cytokine determination; cells were harvested and RNA purified for analysis of lymphokine gene expression by RT-PCR. We measured IFN-, IL-2, and IL-10 production in the supernatant by specific ELISA as described (16). Transcript levels of IL-4, IL-13, and hypoxanthine phosphoribosyltransferase (HPRT) were quantified using real-time quantitative PCR and SYBR green DNA Dye (ABI Prism 5700; PerkinElmer/Applied Biosystems, Foster City, CA) as described (16). Results were expressed as the intrasample ratio of IL-4 to HPRT mRNA copy numbers.

In vitro cytotoxic responses

Fractionated LEW CD45RC^high and CD45RC^low CD8 T cells were differentiated into effector cells by in vitro stimulation with allogeneic BN APC and exogenous rat IL-2 (10 U/ml) for a period of 4 days. The target cells were BN myelocytic leukemia (BNML) cells (23). Target cells were labeled with 0.1 mCi of ⁵¹Cr at 37°C for 2 h, washed three times with medium, and incubated with effector cells at various E:T ratios in a final volume of 200 µl/well. After 6 h of incubation, 50 µl of supernatant was collected and radioactivity was counted in a gamma counter. Percentages of specific ⁵¹Cr release were calculated according to the formula: 100 x (test release – spontaneous release)/(maximal release – spontaneous release), where test release, spontaneous release, and maximal release were, respectively, release in the presence of effector cells, in the presence of medium alone, and in the presence of 10% bleach.

Radiation bone marrow chimeras

Rats were given 10.2 Gy total body irradiation using a Philips MU15F/225kV Röntgen irradiation machine (Hamburg, Germany) 1 day before bone marrow transplantation. Recipient rats were given 10⁸ viable nucleated bone marrow cells i.v. At 10 wk postengraftment, the peripheral blood of the animals was analyzed for the origin of the T cells by using 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 CD8 T cells of donor origin. At week 14, spleen and lymph nodes were also included in the analysis.

Genotypic and linkage analyses, development of BN.LEWc9 congenic lines

Genotypic analyses and linkage maps were performed as previously described (21). Linkage analysis was performed with the MAPMAKER/QTL 1.1 computer program (Whitehead Institute, Cambridge, MA) (24). The initial genome screen was performed using 119 polymorphic microsatellite markers covering 99% of the rat genetic map with an average spacing of 14 cM between markers. Thereafter, additional markers on c9 and c20 were used to refine the quantitative trait loci (QTLs) found on these chromosomes. Congenic lines were developed by successive backcrosses on the BN genetic background as described (25) and subsequently maintained by brother-sister mating.

ELISA for quantitation of serum IgE concentrations

LEW, BN, and BN.LEWc9 rats were injected with Atps and bled once a week and their sera were frozen at –20°C. The total serum IgE concentration was measured by specific ELISA as described (21).

Statistical analysis

The analysis of the differences between groups of rats made according to their genotypes was performed using the Kruskall-Wallis H test and was subsequently confirmed by the Mann-Whitney test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
CD8 T cells can be divided into two subsets according to their level of CD45RC expression

In the present study, we show for the first time that CD8 T cells can be divided into two cell subsets according to the level of CD45RC expression. As shown in Fig. 1A, the analysis of peripheral blood CD8 T cells in LEW rats revealed that 20 ± 3% express low levels of CD45RC (CD45RC^low) while 80 ± 3% express high levels of CD45RC (CD45RC^high). Similar results were obtained in lymph node and spleen CD8 T cells (data not shown). These cell subsets can be highly purified (>95%) from lymph node and spleen cells by magnetic beads as described in Materials and Methods (Fig. 1B). Phenotypical analysis of CD45RC^high and CD45RC^low CD8 T cells from naive LEW rats, using several activation markers (CD28, L-selectin, OX40, MHC class I and II, and CD25), revealed that both subsets had similar expression levels of these molecules (data not shown).

View larger version (24K): [in this window] [in a new window]   FIGURE 1. The level of CD45RC expression distinguishes CD8 T cells into two subsets. A, Peripheral blood leukocytes from naive LEW rats were incubated with anti-TCR (R73-PE), anti-CD4 (W3/25-biot), or anti-CD8 (OX8-biot) and anti-CD45RC (OX22-FITC) mAbs and analyzed by flow cytometry, as indicated in Materials and Methods. The results represent histograms of CD45RC expression on gated CD4 (top panel) and CD8 (bottom panel) T cells. The values on the top of each histogram represent the mean of the percent of the CD45RClow T cell subset of four individual rats. B, To purify CD8 T cells, spleen and lymph node cells from naive LEW rats were depleted of CD4+ and TCR T cells, B cells, monocytes, and NK cells using a standard technique of negative selection as described in Materials and Methods. The obtained purified CD8 T cells were further fractionated by magnetic beads on the basis of expression of CD45RC using FITC-conjugated OX22. The values represent the purity of each cell population.

First, we showed that LEW CD45RC CD8 T cell subsets produce different cytokine profiles, upon in vitro activation using bound anti-TCR and soluble anti-CD28 mAbs (Fig. 2). Indeed, although both populations proliferated equally well, the CD45RC^high CD8 T cells produced predominantly IL-2 and IFN-, while the CD45RC^low CD8 T cells produced IL-10 and expressed IL-4 and IL-13 mRNA. Second, we assessed the cytotoxic capacity of LEW CD45RC^high and CD45RC^low CD8 T cell subsets after allogeneic stimulation, using irradiated T cell-depleted BN spleen cells as sources of alloantigens and exogenous rat IL-2. As shown in Fig. 3A, only CD45RC^high CD8 T cells were cytotoxic against BNML target cells. Finally, we evaluated, in coculture experiments, a possible interregulation between CD45RC^low and CD45RC^high CD8 T cell subsets after allogeneic stimulation. As shown in Fig. 3B, the LEW CD8 CD45RC^low population inhibited, in a dose-dependent fashion, the proliferation and IFN- production of LEW CD8 CD45RC^high T cells stimulated with semiallogeneic T cell-depleted spleen cells. In three independent experiments, a significant suppression was observed at a suppressor-responder ratio of 1:1 (the inhibition was 37 ± 22% for the proliferative response and 51 ± 24% for IFN- production). We also demonstrated that this suppression was not dependent on the production of IL-10 or TGF- because the neutralization of these cytokines did not affect the suppression mediated by the CD45RC^low CD8 T cells (data not shown). In contrast, in these coculture experiments, the CD45RC^high CD8 T cell subset did not suppress IL-10 production by the CD45RC^low CD8 T cells (data not shown). Together, these data demonstrate that freshly isolated CD45RC CD8 T cell subsets exhibit different cytokine profiles and functions.

View larger version (24K): [in this window] [in a new window]   FIGURE 2. CD45RChigh and CD45RClow CD8 T cells produce distinct cytokine profiles. The purified CD45RClow () and CD45RChigh () CD8 T cells from LEW rats were stimulated with plate bound anti-TCR supplemented (0.02, 0.2 µg/ml) or not with soluble anti-CD28 mAbs. In A, proliferation was assessed with an 18 h [3H]thymidine pulse added after 48 h of stimulation and results were expressed as cpm. Tissue culture supernatants collected 36 and 60 h after stimulation with anti-TCR and anti-CD28 were assayed for IL-2 (B), IFN- (C), and IL-10 (D) protein using capture ELISA. In E and F, the figures show, respectively, the IL-4 and IL-13 mRNA expression following stimulation for 36 h using quantitative RT-PCR. Results are expressed as the mean cytokine-HPRT ratio. Stimulation with anti-TCR only did not induce the production of these cytokines. The data shown are representative of three independent experiments.

View larger version (17K): [in this window] [in a new window]   FIGURE 3. CD45RChigh and CD45RClow CD8 T cells exhibit distinct functions. A, Purified LEW CD8 CD45RC subsets were differentiated into effector cells by in vitro stimulation with allogeneic BN splenocytes for 4 days in the presence of exogenous rat IL-2. These effector cells were tested for their ability to kill Cr-labeled BNML cells as described in Materials and Methods. B, Purified LEW CD45RChigh and CD45RClow CD8 T cells (3 x 105 cells/well) were stimulated in vitro in MLR using irradiated T cell-depleted allogeneic (LEW x BN)F1 spleen cells as stimulators (1.5 x 105 cells/well) in the presence of exogenous rat IL-2 (10 U/ml). In coculture experiments, LEW CD45RChigh CD8 T cells were stimulated as before in the presence of variable numbers of LEW CD45RClow CD8 T cells. The proliferation was assessed with an 18 h [3H]thymidine pulse added after 96 h of culture. Tissue culture supernatants collected 96 h after stimulation were assayed for IFN- protein levels using capture ELISA. The data shown represent the quantification made in triplicate cultures and are representative of three independent experiments.

Recently, we showed that the CD8 T cell compartment plays a dominant role in the difference of cytokine production by LEW and BN T cells (6, 16). Because CD45RC CD8 T cell subsets produce different cytokines, we compared the CD45RC expression profiles of LEW and BN CD8 T cells. Fig. 4A shows that while LEW CD8 T cells have a preponderance of the CD45RC^high subset (CD45RC^high/CD45RC^low ratio: 4.0 ± 0.7), BN CD8 T cells contain an equal number of CD45RC^high and CD45RC^low T cells (CD45RC^high/CD45RC^low ratio: 0.9 ± 0.1). (LEW x BN)F₁ rats exhibited an intermediate phenotype (Fig. 4A) resulting in a CD45RC^high/CD45RC^low ratio of 2.4 ± 0.3. As for LEW CD45RC CD8 T cell subsets, BN CD45RC^high CD8 T cells produced mainly IL-2 and IFN-, while BN CD45RC^low CD8 T cells produced IL-10 and expressed IL-4 and IL-13 mRNA (data not shown).

View larger version (25K): [in this window] [in a new window]   FIGURE 4. LEW and BN rats exhibit a distinct CD45RChigh/CD45RClow ratio that is mainly determined by the origin of the hemopoietic cells. In A, peripheral blood leukocytes from naive LEW, BN, and (LEW x BN)F1 rats were incubated with anti-TCR (R73-PE), anti-CD8 (OX8-biot), and anti-CD45RC (OX22-FITC) mAbs and analyzed by flow cytometry as indicated in Materials and Methods. The results represent histograms of CD45RC expression on gated CD8 T cells. The value on the top of each histogram represents the mean (±SD) of CD45RC CD8 T cell subpopulations of four rats per group. In B and C, peripheral blood leukocytes from (LEW x BN)F1 recipients of BN (BN F1) or LEW (LEW F1) bone marrow (B) and LEW (F1 LEW) and BN (F1 BN) recipients of (LEW x BN)F1 bone marrow (C) were incubated with a combination of anti-CD45RC (OX22-FITC), anti-CD8 (341-PE), and anti-donor type MHC class I (42-3-7-biot or 163-7F3-biot) mAbs and analyzed by flow cytometry 14 wk postengraftment. The results represent the CD45RChigh/CD45RClow ratios determined within CD8 T cells of donor origin. The data are represented as the mean CD45RChigh/CD45RClow T cell ratio of five to six animals of each combination. •, The results of individual rats.

To examine whether the distinct CD45RC^high/CD45RC^low ratio within the CD8 T cell compartment between LEW and BN rats is intrinsic to hemopoietic cells or is determined in the periphery, we generated bone marrow chimeras. We first analyzed 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 75.2 ± 2.4% and 87.0 ± 5.9%, respectively, while the chimerism of BN and LEW recipients of F₁ bone marrow cells was 96.3 ± 1.6% and 92.3 ± 1.7%, respectively. The analysis of CD45RC expression by donor-type peripheral blood CD8 T cells revealed that the difference in the CD45RC^high/CD45RC^low CD8 T cell ratio between LEW and BN rats is dependent on the origin of hemopoietic cells. Indeed, F₁ recipients of BN and LEW bone marrow cells exhibited a significant difference (p = 0.006) in the CD45RC^high/CD45RC^low ratio (Fig. 4B) while the ratios in BN and LEW recipients of F₁ bone marrow cells were not significantly different (Fig. 4C). Similar results were obtained when lymph node and spleen cells were analyzed in these bone marrow chimeras (data not shown). Altogether, these data demonstrate that the balance between CD45RC^high and CD45RC^low CD8 T cell subsets is mainly determined by the origin of the bone marrow-derived cells.

Localization of two regions on chromosomes 9 and 20 that control the CD45RC^high/CD45RC^low CD8 T cell ratio in LEW and BN rats

The genome wide search conducted with 119 markers in the (LEW x BN)F₂ rats identified two QTLs associated with the expression of the CD45RC marker on peripheral CD8 T cells on top of the acrocentric c9 between D9Wox24 and D9Wox21 and on top of the short arm of c20 between D20UW1 and D20Rat31. To confirm and refine the localization of these regions, linkage studies were performed with additional markers on c9 (total number 17) and c20 (total number 11). As shown in Table I, significant linkage was found for both QTLs. Together, these two QTLs account for 40% of the variance of the percentage of the CD8 CD45RC^low population. The size of the QTLs, as defined by the two-logarithm of odd ration (lod) support intervals, are, respectively, 13.1 cM on c9 and 12 cM on c20 (not shown). Fig. 5 shows the percentage of CD8 CD45RC^low T cells according to the genotypes of the marker located at the peaks of the QTLs. For the two QTLs, the BN genotype was associated with a high percentage of CD8 CD45RC^low T cells. However, the mode of gene action appears different for the two QTL (Table I and Fig. 5). For the QTL found on c9, the mode of inheritance was defined as recessive BN (Fig. 5A); on c20 it was defined as additive (Fig. 5B).

View larger version (16K): [in this window] [in a new window]   FIGURE 5. Percentage of CD8 CD45RClow T cells according to the genotypes of 104 (LEW x BN)F2 hybrids rats for the microsatellite marker located at the peak of QTLs found on chromosomes 9 and 20. A, D9Got8; B, D20Rat41. Location of markers on the genetic map of the rat can be seen in Figs. 6 (c20) and 7 (c9). ll: Rats with a homozygous LEW genotype (n = 24 and 26 for D9Got8 and D20Rat41, respectively); nl: rats with a heterozygous LEW/BN genotype (n = 55 and 54 for D9Got8 and D20Rat41, respectively); nn: rats with a homozygous BN genotype (n = 25 and 24 for D9Got8 and D20Rat41, respectively). Symbols represent the mean values and vertical bars indicate the SEM. The statistical significance of differences found between groups is indicated above horizontal lines (Mann and Whitney test).

The gene or group of genes controlling the CD45RC^high/CD45RC^low CD8 T cell ratio on chromosome 20 is localized within an 8.4 cM interval

Because the QTL found on the short arm of c20 included the MHC region, available BN and LEW lines congenic for the MHC region of the other strain (named, respectively, BN.1L and LEW.1N) were used to confirm and further refine its localization. The genetic map of these congenic lines is shown in Table II. The CD45RC^high/CD45RC^low T cell ratio of these congenic lines and a schematic representation of the genotypes are shown in Fig. 6. The CD45RC^high/CD45RC^low CD8 T cell ratios were similar in the BN parental strain and in its BN.1L congenic line thus indicating that the gene(s) that control(s) the trait is not located in the chromosomal segments of the MHC region from LEW origin introgressed into the BN genetic background. Therefore, the region telomeric to the D20Rat41 marker can be excluded from the QTL. By contrast, the CD45RC^high/CD45RC^low CD8 T cell ratio observed in the LEW.1N rats is significantly decreased (p = 0.001) compared with the ratio observed in the LEW parental strain. This observation not only confirms the presence in the QTL of a gene or a group of genes that controls this trait, but also allows its localization to be refined to a segment of at most 8.4 cM, with D20Rat41/D20Rat2 and D20Rat31/D20Got13 as telomeric and centromeric boundaries, respectively.

View larger version (26K): [in this window] [in a new window]   FIGURE 6. Studies of BN and LEW lines congenic for the MHC region of the other strain refine the localization of the genetic control of the CD45RChigh/CD45RClow CD8 T cell ratio on chromosome 20 to an interval of 8.4 cM including part of the centromeric MHC region. The upper portion of the figure shows individual values of the CD45RChigh/CD45RClow CD8 T cell ratio in congenic lines, and in the BN and LEW strains. BN.1L, Line of BN rat congenic for the MHC region from LEW strain; LEW.1N, line of LEW rat congenic for the MHC region from BN strain. The lower portion represents schematically the genetic map of the rats with, on the left, the boundary markers, and on the right, the cumulative genetic distances in centiMorgans (cM, Kosambi units) that are drawn to scale. The white and black colors indicate, respectively, the LEW and BN genotypes. The gray color indicates ambiguous genotypes (either BN or LEW), due to crossover breakpoints between markers. Note that the six markers shown on the figure are located on the p12 region of the short arm of the c20.

The gene or group of genes controlling the CD45RC^high/CD45RC^low CD8 T cell ratio on chromosome 9 is localized within an 1.2 cM interval that also controls the IgE response in a model of Th2-mediated disorder

Three lines of the BN rats, congenic for short segments of the centromeric end of c9 from LEW origin (BN.LEWc9-B, -E, and -F lines), were developed and investigated. The results concerning the CD45RC^high/CD45RC^low CD8 T cell ratio and the genotypes of the congenic lines are shown in Fig. 7. In two of the three congenic lines (BN.LEWc9-B and -F), the chromosomal region of LEW origin introgressed into the BN genomic background was responsible for a significant increase in the CD45RC^high/CD45RC^low ratio, compared with the BN parental strain (p = 0.001). By contrast, the T cell subset ratio of the BN.LEWc9-E congenic line was identical to that of the parental BN strain. This result allows the upper part of c9 between D9Wow24 and D9Got200 to be excluded from the locus. Thus, the genetic maps of the BN.LEWc9 congenic lines indicate that a gene or a group of genes localized within an 1.2 cM centromeric interval on c9, between D9Got200 and D9Got8, controls the CD45RC^high/CD45RC^low CD8 T cell ratio. We also analyzed in these congenic lines the IgE response after Atps treatment and showed a striking relationship between the IgE response and the CD45RC CD8 T cell subset ratio. Indeed, the two BN.LEWc9 congenic lines (B and F) that show an increase in the CD45RC^high/CD45RC^low CD8 T cell ratio also show a dramatic decrease in the IgE response to Atps compared with the results obtained in the parental BN strain or in the BN.LEWc9-E line that shows a T cell subset ratio identical to that of the BN strain (mean ± SD: BN.LEWc9-B, 154 ± 58 µg/ml, n = 7; BN.LEWc9-F, 151 ± 64 µg/ml, n = 11; BN, 1022 ± 320 µg/ml, n = 6; BN.LEWc9-E, 1033 ± 191 µg/ml, n = 10).

View larger version (28K): [in this window] [in a new window]   FIGURE 7. A 1.2-cM centromeric interval on chromosome 9 controls the CD45RChigh/CD45RClow CD8 T cell ratio. The upper portion of the figure shows individual values of the CD45RChigh/CD45RClow CD8 T cell ratio in BN.LEWc9-B, -F, and -E congenic lines, and in the BN and LEW strains. The lower portion represents schematically the genetic map of the rats with, on the left, the boundary markers, and, on the right, the cumulative genetic distances in centiMorgans (cM, Kosambi units) that are drawn to scale. The white, black, and gray colors indicate, respectively, the LEW, BN, and undetermined genotypes as indicated in Fig. 6. Note that c9 is an acrocentric chromosome.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

T cell heterogeneity may reflect different lineages or merely different stages of a linear differentiation pathway (26). Naive, effector, and memory T cells represent different stages in a linear developmental pathway and can be clearly distinguished by functional assays and also by cell surface markers (27, 28). It has been previously proposed that CD45 isoforms are able to distinguish naive and memory T cells. However, more recently, it has become clear that discrimination between naive and memory states based only on CD45 isoform expression, including CD45RC, is insufficient. Indeed, with time, memory T cells may revert to the expression of CD45 isoforms that previously defined naive cells (27, 29, 30). In this study, we can exclude that this marker defines activated effector cells. Indeed, both CD45RC CD8 T cell subsets have a morphology of small resting lymphocytes and do not express activation markers. In addition, we show that CD45RC CD8 T cell subsets produce distinct effector cytokines, indicating that these subsets may represent divergent developmental pathways.

Upon in vitro stimulation and depending on cytokines present during primary stimulation, naive CD8 T cells can differentiate into two subsets, Tc1 and Tc2, secreting different cytokine patterns (17, 18). Tc1 cells, are defined as CD8 T cells that secrete IFN-, but not IL-4, IL-5, or IL-10; Tc2 cells are CD8 T cells that secrete IL-4, IL-5, and IL-10, but not IFN-. There is substantial evidence that these CD8 T cell subsets also exist in vivo and that the polarized pattern of secreted cytokines may influence the nature of the resulting immune responses (13, 20, 31, 32, 33, 34, 35). According to the cytokine profile observed in CD45RC CD8 T cell subsets, the CD45RC^low cells are Tc2-like, while the CD45RC^high cells are Tc1-like. These results indicate that the level of CD45RC expression may be used as a phenotypic marker for Tc1 and Tc2 cells to study and monitor in vivo CD8 T cell-mediated immune responses. This is supported by our findings showing that 1) animals prone to develop type 2 immune responses exhibit a high proportion of Tc2-like cells as compared with animals that preferentially develop type 1 responses; 2) CD8 T cells play a dominant role in the difference between these two rat strains to produce type 1 and type 2 cytokines (16); and 3) the difference in the proportion of these CD8 T cells subsets is genetically controlled by the same regions that have been described to be involved in several immunopathological manifestations (6, 36).

The recent release of an annotated draft of the whole rat genome sequence (http://www.ensembl.com) allowed a better knowledge of the QTLs that control the CD45RC^high/low subsets of CD8 T lymphocytes. The QTL found in an 8.4-cM interval that covers 9.22 megabases of the p12 region on the short arm of c20 shows conserved synteny with parts of the human chromosome regions 6p21.31, 21q22.3, and 22q11.23 and of mouse c17 and c10. The centromeric portion of the QTL belongs to the centromeric region of the MHC. Studies in humans and rodents have established that the chromosomal region that includes the MHC genes plays a major role in controlling susceptibility to a wide range of diseases including autoimmune and allergic diseases (37, 38). Because the identified QTL contains probably >180 genes, the identification of candidate genes requires further genetic dissection to narrow down the locus. The centromeric portion of the chromosomal region identified on c9 is homologous to regions of mouse chromosome 17 and of human chromosome 19. However, it is not yet possible to establish precisely the physical map of the QTL on c9 by comparative genomics because we have found that its physical map, as published in the data banks, is not correct.

A previous study conducted in F₂ rats issued from a LEW x BN cross pointed to the presence of QTLs on c9, c10, and c20 that control the CD45RC expression in CD4 T lymphocytes (4), but the levels of significance for the presence of these loci were lower than those found in the present study. The QTLs found on c10 and c20 were only suggestive for linkage. However, taken together, this previous study and the present study strongly suggest that the QTLs identified on c9 and c20 are central to the CD45RC^high/CD45RC^low balance in both CD4 and CD8 T cells, and as such may contribute to the orientation of immune responses along different pathways. The role of the MHC region has been widely studied in several models of immune diseases in the BN and LEW rats (6). The locus found on c20 that includes part of the MHC region overlaps with Eae1 and Aiid1 (Atps-induced immunological disorder, formerly named Atps1) loci that control, respectively, the susceptibility to experimental autoimmune encephalomyelitis (EAE) (39) and to immunological disorders triggered by Atps. EAE is an experimental model of multiple sclerosis to which LEW and DA rats are susceptible and the BN rat is resistant (36). By contrast, the BN rats are susceptible and the LEW rats are resistant to the Th2-mediated disorders triggered by Atps. Interestingly, the locus found on c9 overlaps with Eae4 and Aiid3, two loci that also control, respectively, the susceptibility of DA rats to EAE (36) and of BN rats to the Atps-triggered disease (21). In the present study, we have found a striking relationship between the CD8 T cell subset ratios and the IgE response to Atps according to the genotypes of the three studied BN.LEWc9 congenic lines. Both traits were found to be under the control of one gene or set of genes located in a 1.2-cM interval. However, one cannot exclude that both traits are under separate genetic control. Alternatively, a single gene or set of genes that controls the balance between the two CD8 T cell and therefore the balance between Th1 and Th2 cytokines could indirectly exert some control on the Th1 or Th2 type of the immune response. In other words, allelic variants of genes at the identified chromosomal regions on c9 and c20 that control the expression on CD45RC on T cells could be implicated in the control of T cell polarization. Such genes could have pleiotropic effects, influencing the clustering of immune dysfunction in the BN/LEW models of immune-mediated diseases. This hypothesis fits with the observation that BN rats are prone to develop Th2-mediated diseases but resistant to the development of EAE, while the LEW rats are prone to develop Th1-mediated autoimmune diseases but are resistant to immunological disorders induced by gold salts (6).

In conclusion, our work shows that the level of CD45RC expression on rat CD8 T cells discriminates two cell subsets that produce different types of cytokines and exert different functions in vitro. Because CD8 T cells play an important role in several pathological manifestations including infectious diseases, cancer, autoimmune diseases, and allergic manifestations, the functional analysis of the CD45RC CD8 T cell subsets in these pathological manifestations is of great interest. A better understanding of the origin, regulation, and function of these CD8 T cell subsets may be important for the development of optimal immune intervention strategies.

	Acknowledgments

 
We thank Roland Liblau for critically reading the manuscript, Harry van der Heijden for technical assistance, and Maryline Calise, Sylvie Pilipenko-Appolinaire, Patrick Aregui, Marie-Andrée Daussion, Eliane Pélissou, Magali Toulouse, and Aline Tridon (Zootechnic Unit, Institut Fédératif de Recherche 30) for careful handling of the rats.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by INSERM, ZonMW (Netherlands Organization for Scientific Research)/INSERM, Association de la Recherche contre le Cancer, the Université Paul Sabatier, the Conseil Général de Région Midi-Pyrénées, the Génopole Toulouse Midi-Pyrénées, the Ministère de l’Aménagement du Territoire et de l’Environnement, the Ministère de l’Education Nationale, de la Recherche et de la Technologie, the Association de Recherche sur la Polyarthrite Rhumatoïde, the Etablissement français des Greffes, and the Ligue Contre le Cancer. A.S. is supported by "Centre National de la Recherche Scientifique"; E.X. is supported by INSERM and Fondation pour la Recherche Médicale. P.C., A.S.D., and C.C. are supported by grants from the Ministère de l’Education Nationale, de la Recherche et de la Technologie.

² E.X. and P.C. equally contributed to this work.

³ Current address: Department de Biologie du Developpement, Institut Jacques Monod, Centre National de la Recherche Scientifique et Universités Paris 6 et 7, 2, place Jussieu-Tour 43-3eme etage 75251 Paris Cedex 05 France.

⁴ Address correspondence and reprint requests to Dr. Abdelhadi Saoudi, INSERM Unité 563, Centre de Physiopathologie Toulouse Purpan Bat B, Hôpital Purpan, BP 3028, 31024 Toulouse Cedex 3, France. E-mail address: Abdelhadi.Saoudi{at}toulouse.inserm.fr

⁵ Abbreviations used in this paper: LEW, Lewis; BN, Brown-Norway; Atps, aurothiopropanol sulfonate; MLR, mixed leukocyte reaction; HPRT, hypoxanthine phosphoribosyltransferase; BNML, BN myelocytic leukemia cell; lod, logarithm of odd ration; QTL, quantitative trait locus; EAE, experimental autoimmune encephalomyelitis.

Received for publication January 26, 2004. Accepted for publication June 23, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Spickett, G. P., M. R. Brandon, D. W. Mason, A. F. Williams, G. R. Woollett. 1983. MRC OX-22, a monoclonal antibody that labels a new subset of T lymphocytes and reacts with the high molecular weight form of the leukocyte-common antigen. J. Exp. Med. 158:795.[Abstract/Free Full Text]
2. McKnight, A. J., A. N. Barclay, D. W. Mason. 1991. Molecular cloning of rat interleukin 4 cDNA and analysis of the cytokine repertoire of subsets of CD4⁺ T cells. Eur. J. Immunol. 21:1187.[Medline]
3. Powrie, F., D. Mason. 1990. OX-22^high CD4⁺ T cells induce wasting disease with multiple organ pathology: prevention by the OX-22low subset. J. Exp. Med. 172:1701.[Abstract/Free Full Text]
4. Subra, J. F., B. Cautain, E. Xystrakis, M. Mas, D. Lagrange, H. van der Heijden, M. J. van de Gaar, P. Druet, G. J. Fournie, A. Saoudi, J. Damoiseaux. 2001. The balance between CD45RC^high and CD45RC^low CD4 T cells in rats is intrinsic to bone marrow-derived cells and is genetically controlled. J. Immunol. 166:2944.[Abstract/Free Full Text]
5. Fowell, D., D. Mason. 1993. Evidence that the T cell repertoire of normal rats contains cells with the potential to cause diabetes: characterization of the CD4⁺ T cell subset that inhibits this autoimmune potential. J. Exp. Med. 177:627.[Abstract/Free Full Text]
6. Fournie, G., B. Cautain, E. Xystrakis, J. Damoiseaux, M. Mas, D. Lagrange, I. Bernard, J. F. Subra, L. Pelletier, P. Druet, A. Saoudi. 2001. Cellular and genetic factors involved in the difference between Brown-Norway and Lewis rats to develop respectively type-2 and type-1 immune-mediated diseases. Immunol. Rev. 184:145.[Medline]
7. Huseby, E. S., D. Liggitt, T. Brabb, B. Schnabel, C. Ohlen, J. Goverman. 2001. A pathogenic role for myelin-specific CD8⁺ T cells in a model for multiple sclerosis. J. Exp. Med. 194:669.[Abstract/Free Full Text]
8. Liblau, R. S., F. S. Wong, L. T. Mars, P. Santamaria. 2002. Autoreactive CD8 T cells in organ-specific autoimmunity: emerging targets for therapeutic intervention. Immunity 17:1.[Medline]
9. Kemeny, D. M., A. Noble, B. J. Holmes, D. Diaz-Sanchez. 1994. Immune regulation: a new role for the CD8⁺ T cell. Immunol. Today 15:107.[Medline]
10. Martin, P. J.. 1993. Donor CD8 cells prevent allogeneic graft rejection in mice: potential implications for marrow transplantation in humans. J. Exp. Med. 178:703.[Abstract/Free Full Text]
11. Fowler, D. H., J. Breglio, G. Nagel, M. Eckhaus, R. E. Gress. 1996. Allospecific CD8 Tc1 and Tc2 populations in GVL and GVHD. J. Immunol. 157:4811.[Abstract]
12. Van Lier, R. A., I. J. Ten Berge, L. E. Gamadia. 2003. Human CD8⁺ T-cell differentiation in response to viruses. Nat. Rev. Immunol. 3:931.[Medline]
13. Cerwenka, A., T. M. Morgan, A. Harmsen, R. W. Dutton. 1999. Migration kinetics and final destination of type1 and type 2 CD8 effector cells predict protection against pulmonary virus infection. J. Exp. Med. 189:423.[Abstract/Free Full Text]
14. Dobrzanski, M. J., J. B. Reome, R. W. Dutton. 1999. Therapeutic effect of tumor-reactive type 1 and type 2 CD8⁺ T cell subpopulations in established pulmonary metastases. J. Immunol. 162:6671.[Abstract/Free Full Text]
15. Valmori, D., C. Scheibenbogen, V. Dutoit, D. Nagorsen, A. M. Asemissen, V. Rubio-Godoy, D. Rimoldi, P. Guillaume, P. Romero, D. Schadendorf, et al 2002. Circulating tumor-reactive CD8⁺ T cells in melanoma patients contain a CD45RA⁺CCR7^– effector subset exerting ex vivo tumor-specific cytolytic activity. Cancer Res. 62:1743.[Abstract/Free Full Text]
16. Cautain, B., J. Damoiseaux, I. Bernard, E. Xystrakis, E. Fournie, P. van Breda Vriesman, P. Druet, A. Saoudi. 2002. The CD8 T cell compartment plays a dominant role in the deficiency of Brown-Norway rats to mount a proper type 1 immune response. J. Immunol. 168:162.[Abstract/Free Full Text]
17. Sad, S., R. Marcotte, T. R. Mosmann. 1995. Cytokine-induced differentiation of precursor mouse CD8⁺ T cells into cytotoxic CD8⁺ T cells secreting Th1 or Th2 cytokines. Immunity 2:271.[Medline]
18. Croft, M., L. Carter, S. L. Swain, R. W. Dutton. 1994. Generation of polarized antigen-specific CD8 effector populations: reciprocal action of interleukin (IL)-4 and IL-12 in promoting type 2 versus type 1 cytokine profiles. J. Exp. Med. 180:1715.[Abstract/Free Full Text]
19. Noble, A., P. A. Macary, D. M. Kemeny. 1995. IFN- and IL-4 regulate the growth and differentiation of CD8⁺ T cells into subpopulations with distinct cytokine profiles. J. Immunol. 155:2928.[Abstract]
20. Salgame, P., J. S. Abrams, C. Clayberger, H. Goldstein, J. Convit, R. L. Modlin, B. R. Bloom. 1991. Differing lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science 254:279.[Abstract/Free Full Text]
21. Mas, M., J. F. Subra, D. Lagrange, S. Pilipenko, D. Gauguier, P. Druet, G. J. Fournié. 2000. Rat chromosome 9 bears a major susceptibility locus for IgE response. Eur. J. Immunol. 30:1698.[Medline]
22. Tacke, M., G. Hanke, T. Hanke, T. Hunig. 1997. CD28-mediated induction of proliferation in resting T cells in vitro and in vivo without engagement of the T cell receptor: evidence for functionally distinct forms of CD28. Eur. J. Immunol. 27:239.[Medline]
23. Martens, A. C. M., D. W. Van Bekkum, A. Hagenbeek. 1990. The BN acute myelocytic leukemia (BNML) (a rat model for studying human acute myelocytic leukemia (AML)). Leukemia 4:241.[Medline]
24. Paterson, A. H., E. S. Lander, J. D. Hewitt, S. Peterson, S. E. Lincoln, S. D. Tanksley. 1988. Resolution of quantitative traits into Mendelian factors by using a complete linkage map of restriction fragment length polymorphisms. Nature 335:721.[Medline]
25. Mas, M., P. Cavailles, C. Colacios, J. F. Subra, D. Lagrange, M. Calise, M. O. Christen, P. Druet, L. Pelletier, D. Gauguier, G. J. Fournie. 2004. Studies of congenic lines in the Brown Norway rat model of Th2-mediated immunopathological disorders show that the aurothiopropanol sulfonate-induced immunological disorder (Aiid3) locus on chromosome 9 plays a major role compared to Aiid2 on chromosome 10. J. Immunol. 172:6354.[Abstract/Free Full Text]
26. Woodland, D. L., R. W. Dutton. 2003. Heterogeneity of CD4⁺ and CD8⁺ T cells. Curr. Opin. Immunol. 15:336.[Medline]
27. Tough, D. F.. 2003. Deciphering the relationship between central and effector memory CD8+ T cells. Trends Immunol. 24:404.[Medline]
28. Dutton, R. W., L. M. Bradley, S. L. Swain. 1998. T cell memory. Annu. Rev. Immunol. 16:201.[Medline]
29. Bell, E. B., S. Hayes, M. McDonagh, C. Bunce, C. Yang, S. M. Sparshott. 2001. Both CD45R^low and CD45R^high "revertant" CD4 memory T cells provide help for memory B cells. Eur. J. Immunol. 31:1685.[Medline]
30. Bell, E. B., S. M. Sparshott, C. Bunce. 1998. CD4⁺ T-cell memory, CD45R subsets and the persistence of antigen–a unifying concept. Immunol. Today 19:60.[Medline]
31. Hussell, T., C. J. Baldwin, A. O’Garra, P. J. M. Openshaw. 1997. CD8⁺ T cells control Th2-driven pathology during pulmonary respiratory syncytial virus infection. Eur. J. Immunol. 27:3341.[Medline]
32. Srikiatkhachorn, A., T. J. Braciale. 1997. Virus-specific CD8⁺ T lymphocytes downregulate T helper cell type 2 cytokine secretion and pulmonary eosinophilia during experimental murine respiratory syncytial virus infection. J. Exp. Med. 186:421.[Abstract/Free Full Text]
33. Maggi, E., M. G. Giudizi, R. Biagiotti, F. Annunziato, R. Manetti, M. P. Piccinni, P. Parronchi, S. Sampognaro, L. Giannarini, G. Zuccati, et al 1994. Th2-like CD8⁺ T cells showing B cell helper function and reduced cytolytic activity in human immunodeficiency virus type 1 infection. J. Exp. Med. 180:489.[Abstract/Free Full Text]
34. Kemeny, D. M., B. Vyas, M. Vukmanovic-Stejic, M. J. Thomas, A. Noble, L. C. Loh, B. J. O’Connor. 1999. CD8⁺ T cell subsets and chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care Med. 160:S33.[Abstract/Free Full Text]
35. Seder, R. A., G. G. Le Gros. 1995. The functional role of CD8⁺ T helper type 2 cells. J. Exp. Med. 181:5.[Free Full Text]
36. Dahlman, I., L. Jacobsson, A. Glaser, J. C. Lorentzen, M. Andersson, H. Luthman, T. Olsson. 1999. Genome-wide linkage analysis of chronic relapsing experimental autoimmune encephalomyelitis in the rat identifies a major susceptibility locus on chromosome 9. J. Immunol. 162:2581.[Abstract/Free Full Text]
37. Allcock, R. J., A. M. Martin, P. Price. 2000. The mouse as a model for the effects of MHC genes on human disease. Immunol. Today 21:328.[Medline]
38. Nepom, G. T.. 1989. MHC genes in HLA-associated disease. Curr. Opin. Immunol. 2:588.[Medline]
39. Bergsteinsdottir, K., H. T. Yang, U. Pettersson, R. Holmdahl. 2000. Evidence for common autoimmune disease genes controlling onset, severity, and chronicity based on experimental models for multiple sclerosis and rheumatoid arthritis. J. Immunol. 164:1564.[Abstract/Free Full Text]
40. Lander, E. S., P. Green, J. Abrahamson, A. Barlow, M. J. Daly, S. E. Lincoln, L. Newburg. 1987. MAPMAKER: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1:174.[Medline]
41. Lander, E., L. Kruglyak. 1995. Genetic dissection of complex traits: guidelines for interpreting and reporting linkage results. Nat. Genet. 11:241.[Medline]

This article has been cited by other articles:

				R. Dawes, S. Petrova, Z. Liu, D. Wraith, P. C. L. Beverley, and E. Z. Tchilian Combinations of CD45 Isoforms Are Crucial for Immune Function and Disease J. Immunol., March 15, 2006; 176(6): 3417 - 3425. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Xystrakis, E. Articles by Saoudi, A. Search for Related Content PubMed PubMed Citation Articles by Xystrakis, E. Articles by Saoudi, A. Pubmed/NCBI databases Gene Substance via MeSH