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The Journal of Immunology, 2002, 168: 162-170.
Copyright © 2002 by The American Association of Immunologists

The CD8 T Cell Compartment Plays a Dominant Role in the Deficiency of Brown-Norway Rats to Mount a Proper Type 1 Immune Response1

Bastien Cautain*, Jan Damoiseaux{dagger}, Isabelle Bernard*, Emmanuel Xystrakis*, Emmanuelle Fournié*, Peter van Breda Vriesman{dagger}, Philippe Druet* and Abdelhadi Saoudi2,*

* 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 {dagger} 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 regulating the nature of an immune response. In the rat, Brown-Norway (BN) and Lewis (LEW) strains differ markedly in their susceptibility to develop either type 1 or type 2-mediated autoimmune manifestations. BN rats are susceptible to type 2-dependent systemic autoimmunity, while LEW rats are resistant. Conversely, type 1-mediated, organ-specific autoimmune disease can be easily induced in LEW, but not in BN, rats. The mechanisms involved in the differential development of type 1 and type 2 immune responses by these two strains are still unknown. In the present study we analyzed the contributions of APC, CD4 and CD8 T cells, and MHC molecules in the difference between LEW and BN rats to develop a type 1 immune response. First, we show that the defect of BN T cells to produce type 1 cytokines in vitro does not require the presence of APC and, by using an APC-independent stimulation assay, we have localized the defect within the T cell compartment. Both CD4 and CD8 T cells are involved in the defect of BN rats to develop a type 1 immune response with a major contribution of the CD8 T cell compartment. This defect is associated with an increase in the type 2 cytokine IL-4 in both BN T cell populations, but neutralization of this cytokine does not restore this defect. Finally, by using MHC congenic rats, we show that the MHC haplotype is not involved in the defect of BN T cells to mount a proper type 1 cytokine response.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4 T cells play a central role in the induction and regulation of the immune response and have been shown to be phenotypically and functionally heterogeneous in rats (1), mice (2), and humans (3). In the rat, CD4 T cells can be subdivided into two major subsets based on their different lymphokine production patterns (1). Th1 cells, which produce IL-2 and IFN-{gamma}, preferentially induce the synthesis of Abs expressing the IgG2b isotype (4). By their production of IFN-{gamma} Th1 cells are responsible for directing cell-mediated immune responses leading to the eradication of intracellular pathogens (5). This cell subset may also cause immunopathology and organ-specific autoimmune disease if dysregulated (6). Conversely, Th2 cells produce IL-4 and help B cells to produce IgG1 and IgE (2, 5). This cell subset has been strongly implicated in atopy and allergic inflammation (5). Several studies showed that Th1 and Th2 cells can arise from the same T cell precursor. The development of these functionally distinct T cell subsets is influenced by many factors, such as cytokines that are present in the T cell microenvironment during Ag presentation and initiation of T cell responses (7, 8, 9). Aside from cytokines, it is now clear that the polarization of Th cells is also influenced by other factors, such as the type of APC (10), the genetic predisposition (11, 12) and the hormonal status of the host (13), the Ag ligand density (14, 15), the affinity of ligand-TCR interactions (16, 17), and the costimulatory signals (18). Recently, numerous studies have shown that the effector functions of CD8 T cells overlap those of CD4 T cells much more than previously anticipated (19, 20). Naive CD8 T cells can differentiate into at least two subsets with distinct cytokine patterns: T cytotoxic-1 cells secrete a Th1-like cytokine pattern, while T cytotoxic-2 cells produce Th2 cytokines (19, 20). Currently, it is customary to consider IFN-{gamma} to represent a typical type 1 cytokine, whereas the signature cytokine of the type 2 response is IL-4.

Brown-Norway (BN) and Lewis (LEW) rats are known to be two extremes with respect to their polarization of the immune response (21) as well as their susceptibility for experimental autoimmune diseases. LEW rats are susceptible to Th1-mediated autoimmune diseases such as experimental allergic encephalomyelitis, experimental autoimmune uveoretinitis, and cyclosporin A-induced autoimmunity, while BN rats are resistant (22, 23, 24). In contrast, BN rats, but not LEW rats, are highly susceptible to Th2-mediated autoimmune disease such as mercury disease and gold salt-induced autoimmunity (25, 26). Although these two rat strains have often been used in experimental models of autoimmune diseases to elucidate the pathophysiology and to identify genetic markers for these diseases (26, 27, 28), the reason for the difference in their cytokine response has not been studied.

In the present study we analyzed the contributions of APC, CD4, and CD8 T cells and MHC molecules in the difference between LEW and BN rats to develop a type 1 immune response. First, we show that the defect in BN T cells to produce type 1 cytokines in vitro does not require the presence of APC and is mainly localized in the T cell compartment, since LEW and BN T cells, upon stimulation with anti-TCR and anti-CD28, still exhibit distinct type 1 cytokine responses. Both CD4 and CD8 T cells are involved in the defect of BN rats to develop a type 1 immune response, with a major contribution of the CD8 T cell compartment. This defect is not mediated by the high IL-4 expression by BN T cells. Finally, upon comparison of LEW, BN, and their respective MHC congenic rats, we showed that MHC is not involved in the different type 1 cytokine profile of these rat strains.


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

Eight- to 10-wk-old LEW, BN, and (LEW x BN)F1 male rats were used in this study. These animals were obtained from Center d’Elevage R. Janvier (Le Genest St. Isle, France) and maintained in our animal house facility. LEW-1N and BN-1L rats were obtained from the Central Animal Facility of the University of Maastricht (Maastricht, The Netherlands). The BN-1L and LEW-1N rats were originally purchased from Zentralinstitut für Versuchstierzucht (ZFV, Hannover, Germany) and have been used as a breeding nucleus in Maastricht since 1994. The rats were made congenic by the cross-intercross-backcross method and have been backcrossed 20 times (Dr. H. J. Hedrich, Medizinische Hochschule, Hannover, Germany; unpublished observations). All procedures were performed in accordance with national regulations on animal experiments.

Antibodies

The mAbs used for flow cytometry and for purification of T cell subpopulations were as follows: W3/25 (anti-rat CD4) (29), OX6 (anti-rat MHC class II) (30), OX8 (anti-rat CD8) (31), OX12 (anti-rat {kappa} L chain) (32), OX21 (anti-human C3b inactivator) (33), OX81 (anti-rat IL-4) (34), R73 (anti-rat TCR{alpha}{beta}) (35), V65 (anti-rat TCR{gamma}{delta}) (36), JJ319 (anti-rat CD28) (37), and 10.78 (anti-rat NKR-P1) (38). The hybridomas OX6, OX8, OX12, OX21, OX81, and W3/25 were provided by Dr. D. Mason (Oxford, U.K.). The hybridomas 10.78, JJ319, V65, and R73 were provided by Dr. T. Hünig (Wurzburg, Germany).

Isolation of T cells, CD4 T cells, CD8 T cells, and APC

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 for 30 min on ice with a cocktail of the following mAbs: OX6, OX12, 10.78, and V65. After washing and incubation with anti-mouse IgG-coupled microbeads under agitation, T cells were purified by magnetic depletion. For the purification of CD4 and CD8 T cells a similar technique was applied with the addition of OX8 or W3/25 mAbs to the mixture, respectively. As a source of APC, T cell-depleted splenocytes were used. The splenocytes were incubated with R73 and V65 and negatively selected using the magnetic beads as described above. 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 using rabbit anti-mouse IgG-FITC.

Flow cytometry

For flow cytometry 5 x 105 cells/sample were centrifuged in a 96-well microtiter plate (236 x g, 5 min, 4°C) and resuspended in 50 µl PBS containing 1% FCS and the mAbs of choice. In case of conjugated primary Abs, the cells were triple stained for 30 min on ice with FITC-labeled, PE-labeled, and biotinylated mAbs. The biotin-conjugated mAbs were stained in a second step with streptavidin-CyChrome (BD PharMingen, San Diego, CA). When unlabeled primary mAb (in the case of CD28) was used, cells were consecutively stained with 1) the unlabeled primary mAb, 2) FITC-conjugated goat anti-mouse Fab, 3) biotin- and PE-conjugated secondary mAbs, and 4) streptavidin-CyChrome. After each incubation excess reagents were removed by extensive washing. Data were collected on 10,000 cells as determined by forward and size light scatter intensities on a XL Coulter cytometer (Coultronics, Margency, France) and analyzed using CellQuest software (BD Biosciences, Mountain View, CA).

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. Ag-specific stimulation was performed as previously described (21). The stimulation assay in the presence of APC was performed by incubating 105 T cells from LEW, BN, and (LEW x BN)F1 rats with 1 µg/ml Con A and several concentrations of irradiated (2500 rad), T cell-depleted splenocytes from LEW and BN rats. For stimulation in the absence of APC, 105 T cells were incubated with plate-bound TCR mAb (R73) and soluble CD28 mAb (JJ319) as previously described (37). When indicated, anti-rat IL-4 mAb (OX81), isotype-matched control mAb (OX21), or mouse rIL-12 (gift from Dr. Presky, Hoffmann-La Roche, Nutley, NJ) were added to the culture in the indicated concentration. 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 counts per minute of triplicate cultures. At various times throughout the culture, supernatants were removed and stored at -20°C for cytokine determination.

Cytokine assays

IFN-{gamma} and IL-2 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-{gamma} mAb (DB1) (39) or 1 µg/ml rabbit anti-rat IL-2 Ab (BD PharMingen). Serial dilutions of tissue culture supernatant (100 µl/well), followed by biotinylated DB12, an anti-rat IFN-{gamma} mAb (39), or biotinylated A38-3 (BD PharMingen), an anti-rat IL-2, were sequentially incubated for 2 h at room temperature, separated by three washes. The bound biotinylated Abs were revealed by an 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, St. Louis, MO) at 1 mg/ml in diethanolamine buffer, pH 9.6, for 90 min at room temperature. The absorbance was measured at 405 nm using an automated microplate ELISA reader (Emax, Molecular Devices, Sunnyvale, CA). For IFN-{gamma}, values were expressed as units per ml derived from a standard curve constructed using rat recombinant IFN-{gamma}. This cytokine and anti-rat IFN-{gamma} mAbs were gifts from Dr. P. van der Meide (TNO, Rijswijk, The Netherlands). For IL-2, values were expressed as units per ml derived from a standard curve constructed using rat recombinant IL-2 (BD PharMingen). The IL-4 protein in the supernatant was analyzed by ELISA and biological assay based on the effect of IL-4 on MHC class II up-regulation on B cells as described previously (34). For quantification of IL-4 mRNA, total RNA was isolated from stimulated T cells using the Promega isolation kit (Promega, Madison, WI). The cDNA was prepared as described previously (1). Transcript levels of IL-4 and hypoxanthine phosphoribosyltransferase (HPRT)3 were quantified using real-time quantitative PCR and SYBR Green DNA dye (ABI PRISM 5700, Perkin-Elmer Applied Biosystems, Foster City, CA). Primer sequences were as follows: IL-4, 5'-CGGTGAACTGAGGAAACTCTGTAG-3' (sense) and 5'-CACGGTGCAGCTTCTCAGTG-3' (antisense); and HPRT, 5'-TGTTGGATACAGGCCAGACTTTGT-3' (sense) and 5'-TCCACTTTCGCTGATGACACA-3' (anti-sense). Results were expressed as the intrasample ratio of IL-4 to HPRT mRNA copy numbers.

ELISA for serum IgE and IgG subclasses

For detection of total IgE and IgG subclasses in the sera from naive rats, a standard ELISA technique was applied. Briefly, microtiters plates (Falcon 3012, BD Labware, Oxnard, CA) were coated overnight at 4°C with 0.5 µg/ml mouse anti-rat {epsilon} (MARE-1), 0.5 µg/ml goat anti-rat IgG (provided by E. Druet, Toulouse, France), or 2 µg/ml mouse anti-rat {gamma}1 (MARG1-2) and {gamma}2b (MARG2b-3) mAbs (LO-IMEX, Brussels, Belgium). Bound IgE, IgG1, and IgG2b were revealed using peroxidase mouse anti-rat {kappa}{lambda}-chains (MARK-1 + MARL-15). For total IgG measurement, sera were incubated with peroxidase-conjugated goat anti-rat IgG Fc{gamma} (Jackson ImmunoResearch). The plates were washed and incubated with substrate 3,3'-5,5'-tetramethylbenzidine (Fluka Chemie, Buchs, Switzerland). The reaction was stopped by adding 50 µl/well H2SO42N, and absorbance was read at 450 nm using an automated microplate ELISA reader (Emax, Molecular Devices). Each serum was tested in duplicate and was assessed at four different dilutions.

Statistical analysis

Results are expressed as the mean ± SD, and overall differences between variables were evaluated by Mann-Whitney U test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BN rats have a defective type 1 immune response compared with LEW rats

There is a large body of evidence that BN rats have a defect that prevents them from mounting a proper type 1 immune response. Indeed, we have reported that even using a strong type 1 promoting adjuvant (CFA), immune lymph node cells from BN rats produce less IL-2 and IFN-{gamma}, but more IL-4, than immune lymph node cells from LEW rats (21). To determine whether such a distinction in cytokine profile between LEW and BN rats is already apparent in animals before immunization, we analyzed the type 1 (IgG2b) and type 2 (IgG1 and IgE)-associated Igs in sera derived from naive animals. The results revealed first that BN rats had significantly higher amounts of total IgE (Fig. 1GoA; p = 0.0001) and IgG (Fig. 1GoB; p = 0.0002) than LEW rats, and second that in BN rats there was a preponderance of the type 2-associated IgG1 subclass, while in LEW rats the type 1-associated IgG2b subclass predominated (Fig. 1GoC).



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  		FIGURE 1. Isotype and IgG subclass distribution in serum from naive BN and LEW rats. LEW and BN rats were bled at 10 wk of age, and total IgE (A), total IgG (B), IgG2b, and IgG1 were quantified by ELISA. The results of the IgG subclasses are presented as the IgG1/IgG2b ratio (C). The results are represented in bar diagrams as the mean of 8–12 animals of each rat strain from a pool of three independent experiments. **, p < 0.01.

 
The defect in the ability of BN rats to mount a proper type 1 cytokine response is localized within the T cell compartment

First, we examined the cytokine profile of purified T cells from LEW (95 ± 2% purity) and BN (93 ± 2% purity) rats stimulated with various amounts of syngeneic, irradiated, T cell-depleted splenocytes (<0.5% of contaminating T cells) and Con A. BN T cells proliferated slightly less (Fig. 2GoA) and produced significantly lower amounts of type 1 cytokines IL-2 (Fig. 2GoB) and IFN-{gamma} (Fig. 2GoC) than LEW T cells. It is well established that APC skew the cytokine response of T cells via cognate APC-T cell interactions and production of soluble factors (10, 40, 41, 42). To examine a possible involvement of APC in the distinct cytokine response of LEW and BN rats, we stimulated (LEW x BN)F1 T cells (98 ± 2% purity) with various amounts of T cell-depleted, irradiated splenocytes from BN and LEW rats in the presence of Con A. (LEW x BN)F1 T cells proliferated equally well upon stimulation with both types of APC (Fig. 2GoD) and produced similar amounts of IL-2 (Fig. 2GoE) and IFN-{gamma} (Fig. 2GoF). However, after 60 h of stimulation in the presence of BN APC the IL-2 production was slightly, but significantly, lower (p = 0.02) than that in the presence of LEW APC (data not shown). These data indicate that the APC are not involved in the defective IFN-{gamma} response of BN rats.



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  		FIGURE 2. APC are not involved in the defective IFN-{gamma} response of BN rats. Top panels, Purified T cells from LEW ({square}; four individual rats) and BN ({blacksquare}; four individual rats) rats were stimulated with Con A (1 µg/ml) in the presence of their syngeneic APC at several APC-T cell ratios. Bottom panels, Purified T cells from (LEW x BN)F1 rats were stimulated with Con A (1 µg/ml) in the presence of APC from LEW ({square}; four individual rats) and BN ({blacksquare}; four individual rats) rats at several APC-T cell ratios. Proliferation (A and D) was assessed with an 18-h [3H]thymidine pulse added after 48-h stimulation, and results are expressed as the mean [3H]thymidine incorporation with background proliferation subtracted ({Delta}cpm) ± SD. Tissue culture supernatants were assayed by capture ELISA for IL-2 (B and E) and IFN-{gamma} production (C and F) after 36 (IL-2) and 60 h (IFN-{gamma}) of stimulation. The results of one representative experiment of three are shown and are expressed as the mean ± SD of values obtained from four individual rats. *, p = 0.02.

 
To confirm that T cells are directly involved in the distinct type 1 cytokine production of LEW and BN rats, we purified T cells from both rat strains and stimulated them in an APC-independent system using anti-TCR and anti-CD28 mAbs. The purity of T cells was controlled by flow cytometry and was 95 ± 1% for BN T cells and 96 ± 1% for LEW T cells.

TCR and CD28 expression was similar on T cells from both rat strains, ruling out a role for these molecules in the differential cytokine responses (data not shown). In this APC-independent system, it first appeared that stimulation with anti-TCR Ab alone did not induce T cell proliferation or cytokine production, and second, that upon stimulation with anti-TCR and anti-CD28 mAbs, the peak of cytokine production was 24 and 48 h for IL-2 and IFN-{gamma}, respectively. Upon stimulation with anti-TCR and 0.2 µg/ml anti-CD28 mAb, BN T cells proliferated slightly less (Fig. 3GoA; p = 0.02)) and produced significantly lower amounts of IFN-{gamma} (Fig. 3GoC; p = 0.02) than LEW T cells. In this condition of stimulation, the production of IL-2 was not significantly different between T cells of both rat strains (Fig. 3GoB). When the T cells were stimulated with the lowest concentration of anti-CD28 mAb (0.02 µg/ml), BN and LEW T cells proliferated equally well (Fig. 3Go, A and D), but BN T cells produced significantly lower amounts of IL-2 (Fig. 3Go, B and E; p = 0.0001) and IFN-{gamma} (Fig. 3Go, C and F; p = 0.0003) than LEW T cells. Taken together, the difference in cytokine response between LEW and BN T cells is still apparent in an APC-independent system. This indicates that the distinct immune response of LEW and BN rats is mainly localized within the T cell compartment.



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  		FIGURE 3. The defect in the ability of BN rats to mount a type 1 cytokine response is localized in the T cell compartment. The top panels (A–C) show the results of one representative experiment of three. The purified T cells from LEW ({square}; four individual rats) and BN ({blacksquare}; four individual rats) rats were stimulated with plate-bound anti-TCR and two concentrations of soluble anti-CD28 mAb. A, Proliferation was assessed with an 18-h [3H]thymidine pulse added 48 h after stimulation, and results are expressed as the mean counts per minute ± SD. Tissue culture supernatants were assayed for IL-2 (B) and IFN-{gamma} (C) protein using capture ELISA. IL-2 and IFN-{gamma} production were assessed after 24- and 48-h stimulation, respectively. The bottom panels (D–F) show the pooled results obtained from three independent experiments; dots represent individual animals (12 rats in each group). The proliferation (D) and IL-2 (E) and IFN-{gamma} (F) production were assessed as described above using 0.02 µg/ml anti-CD28 mAb. To pool independent experiments, the results were normalized. In each experiment, the results were expressed as arbitrary units (AU) by considering the mean of all LEW and BN values as 100 AU. *, p < 0.05; **, p < 0.01.

 
Both CD4 and CD8 BN T cells are involved in the defect in mounting a proper type 1 cytokine response with a major participation of the CD8 T cell compartment

We showed recently that BN rats have approximately 3 times fewer CD8 T cells than LEW rats (43). Since the CD8 T cell population is recognized to be a major source of IFN-{gamma}, we examined whether the defect in BN T cells to produce IL-2 and IFN-{gamma} could be attributed to the distinct CD4/CD8 T cell ratio. For this purpose, we purified CD4 and CD8 T cells of both LEW and BN rats and stimulated them in the APC-independent system as described above. CD4 T cells purified from BN and LEW rats were, respectively, 97 ± 2 and 95 ± 2% pure. Upon stimulation with anti-TCR and 0.02 µg/ml anti-CD28 mAb, the LEW CD4 T cells proliferated better (Fig. 4Go, A and D; p = 0.02) and produced significantly more IL-2 (Fig. 4Go, B and E; p = 0.0002) and IFN-{gamma} (Fig. 4Go, C and F; p = 0.0003) than BN CD4 T cells. Upon stimulation with 0.2 µg/ml anti-CD28 mAb, the proliferative response (Fig. 4GoA) and IL-2 production (Fig. 4GoB) remained different, but the production of IFN-{gamma} by CD4 T cells was similar between both rat strains (Fig. 4GoC). CD8 T cells from LEW and BN were, respectively, 74 ± 2 and 88 ± 6% pure. The contaminating cells did not express TCR and therefore are unlikely to be stimulated in our system. As in the case of CD4 T cells, the CD8 T cells from LEW rats proliferated better (Fig. 5GoA; p = 0.02) and produced more IL-2 (Fig. 5GoB; p < 0.04) and IFN-{gamma} (Fig. 5GoC; p = 0.02) than BN CD8 T cells upon stimulation with both concentrations of anti-CD28. Taken together, these results show that both CD4 and CD8 T cells are involved in the defect of BN T cells to produce type 1 cytokines compared with LEW T cells and that CD8 T cells play a major role in this defect. Indeed, if one takes into account the absolute amount of CD8 T cells and the IFN-{gamma} production per cell, it appears that the LEW CD8 T cell compartment produces 20 times more IFN-{gamma} than the BN CD8 T cell compartment (Table IGo).



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  		FIGURE 4. BN CD4 T cells produce less type 1 cytokines than LEW CD4 T cells. The top panels (A–C) show the results of one representative experiment of three individual experiments. The purified CD4 T cells from LEW ({square}; four individual rats) and BN ({blacksquare}; four individual rats) rats were stimulated with plate-bound anti-TCR and two concentrations of soluble anti-CD28 mAb. The proliferative response (A) and IL-2 (B) and IFN-{gamma} (C) production were assessed as described in Fig. 3Go. The bottom panels (D–F) show the pooled results obtained from three independent experiments; the results are represented in bar diagrams as the mean of 12 rats in each group (dots represent individual animals). The proliferation (D) and IL-2 (E) and IFN-{gamma} (F) production were assessed as described in Fig. 3Go. *, p < 0.05; **, p < 0.01.

 


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  		FIGURE 5. CD8 T cells are involved in the defect of BN rats to mount a proper type 1 cytokine response. Purified CD8 T cells from LEW ({square}; four individual rats) and BN ({blacksquare}; four individual rats) rats were stimulated with plate-bound anti-TCR and two concentrations of soluble anti-CD28 mAb. A, Proliferation was assessed with an 18-h [3H]thymidine pulse added after 48-h stimulation, and results are expressed as the mean [3H]thymidine incorporation (counts per minute) ± SD. Tissue culture supernatants were assayed by capture ELISA for IL-2 (B) and IFN-{gamma} (C) production after 24- and 48-h stimulation, respectively. The figure shows the results of one representative experiment of three. *, p < 0.05.

 

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  		Table I. Contribution of the CD4 and CD8 T cell compartments to the difference in IFN-{gamma} production between LEW and BN T cells

 
The MHC haplotype is not involved in the difference of BN and LEW T cells to produce type 1 cytokines

To study the influence of MHC molecules in the different abilities of BN and LEW T cells to mount a type 1 immune response, we analyzed the proliferative response and cytokine production of purified T cells from MHC-congenic rats, LEW-1N and BN-1L. The purity of T cells was 87 ± 2 and 95 ± 1% for BN-1L and LEW-1N T cells, respectively. The results presented in Fig. 6Go show that while T cells from LEW-1N and BN-1L proliferated equally well in response to anti-TCR and anti-CD28 mAbs (Fig. 6GoA), they produced different amounts of IL-2 and IFN-{gamma}. BN-1L T cells produced significantly lower amounts of IL-2 (Fig. 6GoB; p = 0.02) and IFN-{gamma} (Fig. 6GoC; p = 0.02) compared with LEW-1N T cells. Furthermore, the comparison of LEW, BN, BN-1L and LEW-1N T cells shows that although T cells from these rat strains proliferated equally well (Fig. 6GoD), in terms of IL-2 and IFN-{gamma} production BN-1L T cells responded in a similar way as BN T cells, while LEW-1N T cells exhibited the same type 1 cytokine profile as LEW T cells (Fig. 6Go, E and F). Similar results were obtained with purified CD4 T cells from these four rat strains (data not shown). These results indicate that the MHC is not involved in the different type 1 cytokine profiles of LEW and BN T cells. For analysis of the cytokine profile of CD8 T cells we only compared LEW and BN-1L rats, since they have relatively high amounts of CD8 T cells compared with BN and LEW-1N rats (43). This enabled us to circumvent the problem of contamination of CD8 T cells as observed in BN rats. Indeed, the purity of CD8 T cells from BN-1L rats was 87 ± 2% and was comparable to the purity of CD8 T cells from LEW rats (91 ± 3%). Upon stimulation with the anti-TCR and anti-CD28 mAbs, CD8 T cells from LEW and BN-1L rats proliferated equally well (Fig. 7GoA), but BN-1L CD8 T cells produced significantly less IL-2 (Fig. 7GoB; p = 0.02) and IFN-{gamma} (Fig. 7GoC; p = 0.02) than LEW CD8 T cells. Again, CD8 T cells from both BN-1L and LEW rats produced much more IFN-{gamma} compared with a similar amount of CD4 T cells from the respective rat strains (data not shown). Together our results show that the MHC haplotype is not responsible for the difference in type 1 cytokine production between LEW and BN T cells.



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  		FIGURE 6. The difference between BN and LEW T cells in production of type 1 cytokines is not MHC dependent. The purified T cells from LEW-1N ({square}; four individual rats) and BN-1L ({blacksquare}; four individual rats) rats were stimulated with plate-bound anti-TCR and two concentrations of soluble anti-CD28 mAb. The proliferative response (A) and IL-2 (B) and IFN-{gamma} (C) production were assessed as described in Fig. 3Go. The bottom panels (D–F) show the pooled results obtained from three independent experiments with LEW and BN rats and from one experiment with MHC-congenic rats; the results are represented in a bar diagram as the mean of 4 or 12 rats in each group (dots represent individual animals). The proliferation (D) and IL-2 (E) and IFN-{gamma} (F) production were assessed as described above using 0.02 µg/ml anti-CD28. To compare independent experiments, the results were normalized as described in Fig. 3Go. *, p < 0.05; **, p < 0.01.

 


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  		FIGURE 7. The MHC haplotype is not involved in the difference between BN-1L and LEW CD8 T cells in the production of type 1 cytokines. Purified CD8 T cells from LEW ({square}; four individual rats) and BN-1L ({blacksquare}; four individual rats) rats were stimulated with plate-bound anti-TCR and two concentrations of soluble anti-CD28 mAb. The proliferative response (A) and IL-2 (B) and IFN-{gamma} (C) production were assayed as described in Fig. 3Go. The figure shows the results of one representative experiment of two individual experiments. *, p < 0.05.

 
Contributions of IL-4 and IL-12 to the defect in the ability of BN T cells to mount a proper type 1 cytokine response

The supernatants obtained in the previous experiments were tested for the presence of IL-4 using ELISA and a biological assay. This cytokine was undetectable whatever the stimulus, time course, and assay (data not shown). Therefore, we performed an additional experiment and analyzed IL-4 mRNA by quantitative RT-PCR in purified CD4 and CD8 T cells from LEW and BN rats. Upon stimulation with anti-TCR and anti-CD28 mAbs, BN CD4 (Fig. 8GoA) and CD8 (Fig. 8GoB) T cells exhibited high amounts of IL-4 mRNA compared with their LEW counterparts. Similar results were obtained when BN-1L CD4 and CD8 T cells were compared with their LEW counterparts (data not shown). IL-4 is associated with Th2-type immune responses and can either inhibit (44, 45) or, in some cases, promote Th1-type immune response (46, 47, 48, 49, 50). To analyze the contribution of this cytokine, we tested the effect of an anti-rat IL-4 mAb on IFN-{gamma} production by T cells from BN-1L and LEW rats that have the same CD4/CD8 T cell ratio. Fig. 8GoC shows that the addition of anti-rat IL-4 mAb, but not of the isotype control, inhibits IFN-{gamma} production by anti-TCR- and anti-CD28-stimulated BN-1L (96 ± 2% purity) and LEW (97 ± 2% purity) T cells. Similar results were obtained when these T cells were stimulated with syngeneic APC and Con A in the presence of anti-rat IL-4 mAb (Fig. 8GoD). These data suggest that the observed increase in IL-4 mRNA expression by BN-1L T cells is not responsible for their deficiency in IFN-{gamma} production. In contrast, IL-4 is necessary for IFN-{gamma} production by both LEW and BN-1L T cells. We also analyzed whether T cells of BN genetic background are defective in their response to IL-12, a key cytokine for the differentiation of T cells to produce type 1 cytokines. The addition of IL-12 significantly increased the amount of IFN-{gamma} produced by both LEW and BN-1L T cells stimulated with anti-TCR and anti-CD28 mAbs (Fig. 8GoC) or syngeneic APC and Con A (Fig. 8GoD), suggesting that these cells are not defective in their response to IL-12. However, BN-1L T cells still produce less IFN-{gamma} than LEW T cells stimulated under the same conditions (Fig. 8Go, C and D).



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  		FIGURE 8. Contributions of IL-4 and IL-12 to the defect in the ability of BN T cells to mount a proper type 1 cytokine response. Purified CD4 (A) and CD8 (B) T cells from LEW and BN rats were stimulated with plate-bound anti-TCR and soluble anti-CD28 mAb (0.2 µg/ml). IL-4 mRNA was assayed by quantitative RT-PCR after 24 h of stimulation. The results shown are expressed as the mean IL-4/HPRT ratio ± SD of values obtained from four individual rats in each group. Purified T cells from LEW ({square}; four individual rats) and BN-1L ({blacksquare}; four individual rats) rats were stimulated with anti-TCR and anti-CD28 mAb (0.02 µg/ml; C) or with syngeneic APC (T/APC ratio = 1) and Con A (1 µg/ml; D) in the presence of IL-12 (1000 and 100 pg/ml), anti-rat-IL-4 mAb (OX81, 10 µg/ml), or isotype control (OX21, 10 µg/ml). Tissue culture supernatants were assayed by capture ELISA for IFN-{gamma} production after 48 (C) and 72 (D) h of stimulation. The figure shows the results of one representative experiment of two. Cytokine productions by LEW and BN or BN-1L T cells stimulated under the same conditions were compared using Mann-Whitney U test. *, p < 0.05; **, p < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Differential cytokine production by T cells plays an important role in regulating the nature of an immune response and thereby the outcome of immunopathologic manifestations such as autoimmunity, allograft rejection, and control of tumors and infections (5, 6). In the rat, BN and LEW rats differ markedly in their susceptibility to develop either type 1- or type 2-mediated autoimmune manifestations. BN rats are susceptible to type 2-dependent systemic autoimmunity, while LEW rats are resistant. Conversely, type 1-mediated, organ-specific autoimmune disease can be easily induced in LEW, but not in BN, rats (22, 23, 25, 27). Furthermore, we showed recently that even using a strong type 1-promoting adjuvant, immune lymph node cells from BN rats are still defective in producing IFN-{gamma} (21). Although these two rat strains have often been used in experimental models of autoimmune diseases to elucidate the pathophysiology and to identify genetic markers for these diseases (26, 27, 28), the reason for the difference in their cytokine responses has not been studied. In the present paper we show that the defect of BN T cells to produce type 1 cytokines in vitro resides within the T cell and not the APC compartment. This defect is associated with an increase in the type 2 cytokine IL-4, but the neutralization of this cytokine in vitro does not restore, but, rather, augments, this defect. Furthermore, the analysis of the CD4 and CD8 compartment revealed that both CD4 and CD8 T cells are involved in the difference between LEW and BN rats, with a major contribution of the CD8 T cell compartment. Finally, by using MHC congenic rats, LEW-1N and BN-1L, it appeared that the distinct type 1 immune response between LEW and BN T cells is not influenced by the MHC haplotype.

It is well recognized that APC are important regulators of Th cell functions; in particular, they influence the cytokine profile of both naive and memory Th cells. Concerning IFN-{gamma} production, there is a large body of evidence that APC-derived factors, such as NO and PGE2, directly inhibit the production of this cytokine by T cells (41, 42, 51, 52). Besides these factors, costimulatory molecules and APC-derived cytokines have been reported to influence the cytokine profile of T cells (7, 9, 18). Preliminary data show that LEW and BN APC produce similar amounts of IL-12 p40 mRNA upon stimulation with Staphylococcus aureus Cowan, and IFN-{gamma} and express similar levels of the costimulatory molecules CD54, CD80, and CD86 (data not shown). In our present study we show that APC of BN and LEW origin do not differentially influence the IFN-{gamma} response of (LEW x BN)F1 T cells. These data indicate that APC-derived mediators are not directly involved in the defect of BN T cells to produce IFN-{gamma}. This was further supported by our results obtained in the APC-independent culture system using anti-TCR and anti-CD28 mAbs for stimulation. Indeed, BN T cells produced significantly less type 1 cytokines than LEW T cells.

Although MHC linkage to polarized Th1-type and Th2-type immune responses has been reported (53, 54), our results exclude such a role in the difference in type 1 cytokine production between LEW and BN T cells. Indeed, BN rats congenic for the LEW MHC (BN-1L rats) responded in a similar way as the BN rats, and LEW rats congenic for the BN MHC (LEW-1N rats) exhibited the same cytokine response as the LEW rats. In agreement with our study it has been shown that the MHC genes are not involved in the regulation of the type 1/type 2 cytokine balance and therefore do not play a dominant role in determining susceptibility to several experimental autoimmune diseases (11, 55, 56).

In mice it has also been demonstrated that non-MHC-linked genetic background controls the Th1/Th2 development, resulting in either resistance or susceptibility to pathogens such as Leishmania major (57). BALB/c strains produce a Th2 response to L. major and succumb to infection, whereas B10.D2 and C57BL/6 produce a Th1 response and are resistant (12). The comparison of the intrinsic tendencies of CD4 T cells with identical TCR and Ag specificity but distinct genetic background has shown that after Ag stimulation under neutral conditions, naive CD4 T cells from BALB/c and B10.D2 backgrounds preferentially differentiate toward Th2 and Th1 phenotypes, respectively (58). It has been proposed that this biased Th2 development depends on the rapid extinction of IL-12 responsiveness due to selective loss of the IL-12R {beta}2 subunit, which is required for IL-12 signaling and Th1 cell development (59). We showed that the addition of IL-12 in vitro enhanced the production of IFN-{gamma} by BN-1L T cells, but the amounts produced were still lower than those produced by LEW T cells stimulated under the same conditions (Fig. 8Go, C and D). Furthermore, by using intracytoplasmic staining for IFN-{gamma}, we showed that Ag-specific BN T cells respond to IL-12, but the percentage of IFN-{gamma}-producing T cells is still lower compared with LEW T cells (unpublished observations). Whether the defect in IFN-{gamma} production associated with the biased Th2 development observed in BN T cells is also dependent on selective loss of the IL-12R {beta}2 subunit has to be investigated.

IL-4 is antagonistic for many of the activities of IFN-{gamma}, since this cytokine suppresses the development of Th1 cells and directly inhibits the synthesis of IFN-{gamma} by T lymphocytes (8, 60). In the present study we showed that the defect of IFN-{gamma} production by BN CD4 and CD8 T cells is associated with increased expression of IL-4 mRNA. However, the neutralization of this cytokine, using anti-rat IL4 mAb, not only did not restore this defect, but suppressed IFN-{gamma} production by both LEW and BN T cells. These data suggest that the observed increase in IL-4 mRNA expression by BN T cells is not responsible for their deficiency in IFN-{gamma} production. In contrast, IL-4 is necessary for the synthesis of IFN-{gamma} by both LEW and BN T cells. In agreement with our data, there is a large body of evidence that IL-4 can help in Th1-type immune responses in rats and mice (46, 47, 48, 49, 50).

The comparison of the T cell compartment between LEW and BN rats revealed that there is a quantitative and a qualitative difference. Concerning the quantitative difference, BN rats have fewer T cells than LEW rats, and this is due to a defect in the CD8 T cell compartment. Indeed, BN rats have 3 times fewer CD8 T cells than LEW rats (43). With respect to the qualitative difference, stimulation of similar amounts of CD4 or CD8 T cells revealed that both T cells populations of BN rats produce significantly less type 1 and more type 2 cytokines than their LEW counterparts. When taking into account the absolute numbers of CD4 and CD8 T cells as well as the amount of IFN-{gamma} produced per cell, the LEW CD8 T cell compartment produces about twice as much IFN-{gamma} as the LEW CD4 T cell compartment. On the contrary, the BN CD8 T cell compartment produces only one-quarter the amount of IFN-{gamma} produced by the CD4 T cell compartment (Table IGo). Taken together, the LEW CD8 T cell compartment produces 20 times more IFN-{gamma} than the BN CD8 T cell compartment, indicating that this population plays a major role in the difference in type 1 cytokine production between LEW and BN rats. The defective IFN-{gamma} production by the BN CD8 T cell compartment may account for the susceptibility of this rat strain to develop type 2 immune responses. Indeed, CD8 T cells have been shown to down-regulate several Th2-mediated immune responses via the secretion of IFN-{gamma} (61, 62, 63, 64, 65). Whether depletion of CD8 T cells in LEW rats also results in a decreased type 1 immune response and corresponding immunopathologic manifestations upon challenge remains to be determined.


   Acknowledgments
 
We thank H. van der Heijden, M.-J. van de Gaar (Department of Immunology, University Maastricht, Maastricht, The Netherlands) for excellent technical assistance, P. Aregui and M. Calise (Institut Fédératif de Recherche 30, Toulouse, France) for taking care of the animal house, and Dr. P. Van der Meide (Netherlands Central Organization for Applied Scientific Research, Rijswijik, The Netherlands) for supplying anti-IFN-{gamma} Abs and IFN-{gamma}.


   Footnotes
 
1 This work was supported by Institut National de la Santé et de la Recherche Médicale, the Cardiovascular Research Institute Maastricht, grants from Association pour la Recherche sur la Sclérose en Plaques and Association Française contre les Myopathies, and a collaborative grant from the Dutch Organization for Scientific Research and Institut National de la Santé et de la Recherche Médicale. E.X. is supported by Fondation pour la Recherche Médicale and Institut National de la Santé et de la Recherche Médicale. A.S. is supported by Centre National de la Recherche Scientifique. B.C is supported by Ministère de l’Éducation Nationale, de la Recherche et de la Technologie and Fondation pour la Recherche Médicale. Back

2 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. E-mail address: abdelhadi.saoudi{at}purpan.inserm.fr Back

3 Abbreviation used in this paper: HPRT, hypoxanthine phosphoribosyltransferase. Back

Received for publication January 11, 2001. Accepted for publication October 29, 2001.


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 Introduction
 Materials and Methods
 Results
 Discussion
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