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The Journal of Immunology, 1999, 163: 2983-2989.
Copyright © 1999 by The American Association of Immunologists

A Dominant Role for the Thymus and MHC Genes in Determining the Peripheral CD4/CD8 T Cell Ratio in the Rat1

Jan G. M. C. Damoiseaux2,*, Bastien Cautain{dagger}, Isabelle Bernard{dagger}, Magali Mas{dagger}, Peter J. C. van Breda Vriesman*, Philippe Druet{dagger}, Gilbert Fournié{dagger} and Abdelhadi Saoudi{dagger}

* Department of Internal Medicine, Section Immunology, University Maastricht, Maastricht, The Netherlands, and {dagger} Institut National de la Santé et de la Recherche Médicale U28, Hôpital Purpan, Place du Dr Baylac, Toulouse, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During their development, immature CD4CD8 double positive thymocytes become committed to either the CD4 or CD8 lineage. The final size of the peripheral CD4 and CD8 T cell compartments depends on thymic output and on the differential survival and proliferation of the respective T cell subsets in the periphery. Our results reveal that the development of the distinct peripheral CD4/CD8 T cell ratio between Lewis and Brown Norway rats originates in the thymus and, as shown by the use of radiation bone marrow chimeras, is determined by selection on radio-resistant stromal cells. Furthermore, this difference is strictly correlated with the MHC haplotype and is the result of a reduction in the absolute number of CD8 T cells in Brown Norway rats. These data suggest that the distinct CD4/CD8 T cell ratio between these two rat strains is the consequence of differential interactions of the TCR/CD8 coreceptor complex with the respective MHC class I haplotypes during selection in the thymus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The majority of peripheral T cells fall into two main phenotypic categories: CD4 and CD8 T cells. In normal adults, the relative proportions of these two cell types seem to be finely controlled. It is well documented that there is an intraspecies difference in peripheral CD4/CD8 ratios in mice and humans and that the relative size of the peripheral CD4 and CD8 T cell compartments is genetically determined (1, 2). T cell development involves an obligatory interaction of the TCR on immature thymocytes with MHC-peptide complexes in the thymus. If the overall avidity of this interaction is relatively low, the corresponding thymocytes are stimulated to proceed along the maturation pathway (positive selection); if, however, the overall avidity is high, the thymocytes undergo cell death (negative selection) (3, 4, 5). Positive selection has been reported to take place predominantly in the cortex and is promoted by thymic epithelial cells, whereas negative selection occurs in the cortico-medullary junction and is mediated by bone marrow-derived dendritic cells (6). However, there is also evidence that negative selection is mediated by radio-resistant components of the thymus (7). CD4 and CD8 molecules act as coreceptors for recognition of class II and class I MHC Ags, respectively. Commitment to the CD4 or CD8 lineage is explained by the instructive model, which postulates that signaling through the coreceptor determines the fate of the double positive (DP)3 precursor cell, or alternatively, by the stochastic model, in which TCR engagement by DP thymocytes triggers random loss of one coreceptor (8, 9, 10).

After further maturation in the thymic medulla, the thymocytes enter the periphery as recent thymic emigrants (RTE) and replenish the peripheral T cell pool. The thymic output is independent of the size of the peripheral T cell pool (11). The peripheral regulation of the CD4/CD8 ratio may not only depend on the thymic output but also on the relative survival and expansion in the periphery of the respective T cell subsets. Indeed, it has been shown that, like for selection events in the thymus, for T cell survival and expansion in the periphery, an interaction with MHC is differentially required. MHC class I is required for peripheral survival of CD8 thymic emigrants (12), whereas MHC class II is not required for survival of newly generated CD4 T cells but is necessary to maintain the size of the peripheral T cell pool for extended periods (13).

Like humans and mice, rat strains, and in particular Lewis (LEW) and Brown Norway (BN) rats, differ in their peripheral CD4/CD8 T cell ratio (14). In this study, we analyzed the origin of the distinct CD4/CD8 ratio observed in LEW and BN rats. We have analyzed the presence of the respective subsets in different peripheral lymphoid compartments as well as in the thymus, and we have examined the role of MHC and non-MHC genes by using the respective MHC-congenic rat strains as well as (LEW x BN)F2 hybrids. Our results reveal that the development of the distinct peripheral CD4/CD8 T cell ratio between both strains originates in the thymus and, as shown by the use of radiation bone marrow chimeras, is determined during selection on radio-resistant stromal cells. Furthermore, this difference is strictly correlated with the MHC haplotype and is the result of a reduction in the absolute number of CD8 T cells in BN rats.


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

Specific pathogen-free LEW (RT1l), BN (RT1n), LEW-1N (RT1n), and BN-1L (RT1l) rats were obtained from the Central Animal Facility of University of Maastricht (Maastricht, The Netherlands). The LEW-1N and BN-1L strains were originally purchased from the Zentralinstitut für Versuchstierzucht (Hannover, Germany) and used as a breeding nucleus. (LEW x BN)F1 rats were obtained from Centre d’Elevage R. Janvier (Le Genest St. Isle, France) and were intercrossed to obtain (LEW x BN)F2 rats. Rats were male or female and 6–10 wk of age at the start of the experiment; the rats in individual experiments were of the same sex and the same age. All procedures were in accordance with national regulations on animal experiments.

Radiation bone marrow chimeras

Rats were given 8.5 Gy x-irradiation using an IBL (Paris, France) 437C 135Cesium irradiation machine or in case of BN bone marrow recipients, rats were given 10.2 Gy x-irradiation using a Philips (Hamburg, Germany) MU15F/225kV Röntgen irradiation machine 1 day before bone marrow transplantation. Recipient rats were given between 6 x 107 and 2 x 108 viable nucleated syngeneic bone marrow cells i.v. into the penis vein. At 6 wk postengraftment, the peripheral blood, lymph nodes, and spleen of the animals were analyzed for the CD4/CD8 T cell ratio.

Monoclonal Abs

The conjugated mouse anti-rat mAbs used for flow cytometry, FITC-conjugated OX-35 (CD4), PE-conjugated OX-8 (CD8{alpha}), R73 (TCR{alpha}ß), and OX-7 (Thy-1), are commercially available (PharMingen, San Diego, CA). The hybridomas producing mAb R73 (TCR{alpha}ß) (15) and 341 (CD8{alpha}ß heterodimers) (16) were kindly provided by Prof. Th. Hünig (Würzburg, Germany); the hybridomas producing mAb W3/25 (CD4) (17), OX-3 (RT1-Bl haplotype) (18), OX-8 (CD8{alpha}) (19), and OX-12 ({kappa}-light chain) (20), were kindly provided by Dr. D. Mason (Oxford, United Kingdom); the mAb 42-3-7 (RT1-An haplotype) and 163–7F3 (RT1-Al haplotype) were kindly provided by Dr. H. Kunz (Pittsburgh, PA); and the mAb GY15/19 (RT1-Dn haplotype) (21) was kindly provided by Dr. C. Boitard (Paris, France). The mAbs were differentially FITC- or biotin-conjugated using standard protocols.

Cell suspensions

The thymus, spleen, and lymph nodes (cervical and mesenteric) were teased apart, passed through a 100-µm mesh nylon gauze, and collected in balanced salt solution supplemented with 2% heat-inactivated FCS. The cells were washed twice by centrifugation (316 x g for 10 min at 4°C), resuspended in PBS containing 0.5% w/v BSA (PBS-BSA; Sigma, St. Louis, MO). Nucleated cells were counted in Türk solution with a Bürker haemocytometer (Emergo, Landsmeer, The Netherlands), while viability was assessed by trypan blue exclusion.

Heparinized blood was collected via the retro-orbital vein plexus. The PBL number was determined in Türk solution with a Bürker haemocytometer. The erythrocytes in the buffy coats of the peripheral blood and in the spleen cell suspensions were lysed with NH4Cl buffer (0.155 M NH4Cl, 0.01 M KHCO3, and 0.1 mM EDTA, pH 7.4), and next the cells were washed twice and resuspended in PBS-BSA. PBLs were enumerated and viability assessed as described above.

Flow cytometry

Single-cell suspensions were cell surface labeled, and two- or three-color immunofluorescence analysis was conducted, as described previously (22). Cells (5 x 105/sample) were centrifuged in a 96-well microtiter plate (236 x g for 3 min at 4°C) and resuspended in PBS containing 0.5% BSA, 10 mM NaN3, and a mixture of conjugated mAb. The biotin-conjugated mAb were stained in a second step with streptavidin-CyChrome (PharMingen). The phenotype was determined on a FACS-Calibur (Becton Dickinson, San Jose, CA) using the CellQuest software (Becton Dickinson) package for acquisition and analysis. The CD4/CD8 ratio in the periphery was calculated by gating on TCR{alpha}ß+ cells; in the thymus only TCR{alpha}ßhigh cells were included in the analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Distinct CD4/CD8 T cell ratios in LEW and BN rats originate in the thymus

It has been reported previously that the CD4/CD8 T cell ratio in the peripheral blood of LEW and BN rats is different, i.e., 3.4 (±0.3) and 14.1 (±2.8), respectively (14). The low CD4/CD8 T cell ratio observed in LEW rats is more typical of the norm than the high BN ratio because the ratio of dark August, piebald virol glaxo, and spontaneous hypertensive rats varies between 4 and 6 (unpublished results). In this study, we examined the CD4/CD8 T cell ratio in different lymphoid tissues, including the thymus, to determine whether this ratio is similar in all lymphoid compartments and whether it is established in the primary or secondary lymphoid organs. As shown in Fig. 1Go, the CD4/CD8 T cell ratio in the lymph nodes and peripheral blood was 3.8 ± 0.3 and 3.5 ± 0.4, respectively, in the LEW rat vs 12.3 ± 0.6 and 11.8 ± 0.7 in the BN rat. However, the CD4/CD8 T cell ratio in the spleen was reduced by one-half in both the LEW rat (1.9 ± 0.1) and the BN rat (5.1 ± 0.4). In the thymus, the CD4/CD8 ratio was determined by the relative presence of the CD4+CD8-TCR{alpha}ßhigh and the CD4-CD8+TCR{alpha}ßhigh thymocytes. The CD4/CD8 ratio in LEW thymus was 3.6 ± 0.1, whereas the ratio was 9.5 ± 0.7 in the BN thymus (Fig. 1Go). The results reveal that the difference in the peripheral CD4/CD8 T cell ratio between LEW and BN rats originates in the thymus. These data were supported by analysis of RTE in the peripheral blood. In the rat, it has been shown that T cells that have left the thymus recently, i.e., within ~7 days, express Thy-1 (23, 24). Indeed, the CD4/CD8 ratio within the Thy-1+ T cell population in the peripheral blood appeared to be similar to the CD4/CD8 ratio within the total peripheral T cell population of LEW and BN rats, i.e., 3.2 ± 0.2 and 9.0 ± 0.3, respectively.



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  		FIGURE 1. The peripheral CD4/CD8 T cell ratio originates in the thymus and is different between LEW and BN rats. Thymocytes, PBLs, lymph node cells, and splenocytes from naive LEW and BN rats were incubated with anti-TCR{alpha}ß, anti-CD4, and anti-CD8{alpha} mAbs and analyzed by flow cytometry. The results are depicted as contour plots of CD4 vs CD8 expression and represent TCR{alpha}ßhigh thymocytes or TCR{alpha}ß-positive T cells in peripheral blood, lymph nodes, and spleen. Numbers in the upper right quadrant represent the mean (±SD) CD4/CD8 T cell ratio of four animals of each strain. The data are of one of two representative experiments for the thymus and peripheral blood and one of four in case of the lymph nodes and spleen.

 
The difference in CD4/CD8 T cell ratio is not the result of CD8 expression by activated CD4 T cells

It has been shown in rats that, although CD8 T cells in the peripheral lymphoid organs almost exclusively express the CD8{alpha}ß heterodimer, activated CD4 T cells express the CD8{alpha}{alpha} homodimer (16). Because the CD4/CD8 T cell ratio in our study was calculated on the basis of CD8{alpha} expression on T cells using mAb OX-8, the observed difference in the CD4/CD8 T cell ratio between LEW and BN rats may be the result of an increased activation of the CD4 T cell compartment in LEW rats. To exclude this possibility, we determined the differential expression of CD8{alpha} and CD8{alpha}ß on peripheral blood T cells of LEW and BN rats. Fig. 2Go shows that the vast majority of CD8{alpha}+ T cells expressed the CD8{alpha}ß heterodimer in both rat strains. This finding implies that the CD4/CD8 T cell ratios observed in our study were hardly affected by the presence of activated CD4 T cells.



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  		FIGURE 2. The difference in CD4/CD8 T cell ratio is not the result of CD8{alpha} expression by activated CD4 T cells. PBLs from naive LEW and BN rats were incubated with a combination of anti-TCR{alpha}ß, anti-CD8{alpha}, and anti-CD8{alpha}ß mAbs and analyzed by flow cytometry. The results are depicted as contour plots of CD8{alpha} vs CD8{alpha}ß expression and represent TCR{alpha}ß-positive T cells. The plots shown represent a pool of four rats for each strain.

 
The distinct CD4/CD8 ratio in LEW and BN rats results from a quantitative difference in the CD8 T cell compartment

The relatively high CD4/CD8 ratio in BN rats could be the result of increased numbers of CD4 T cells or decreased numbers of CD8 T cells. To discriminate between these two possibilities, we calculated the absolute amount of CD4 and CD8 T cells in primary and secondary lymphoid organs. Fig. 3Go shows that the number of CD4 T cells did not differ between LEW and BN rats. However, the number of CD8 T cells was clearly decreased in all lymphoid tissues of BN rats as compared with LEW rats. This result indicates that the difference in CD4/CD8 T cell ratio is the result of a difference in the absolute amount of CD8 T cells. Furthermore, this deficient generation of CD8 T cell numbers in BN rats originates in the thymus.



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  		FIGURE 3. The absolute amount of CD8, but not CD4, T cells in the thymus and peripheral lymphoid organs differs between LEW and BN rats. Thymocytes, PBLs, lymph node cells, and splenocytes from naive LEW and BN rats were incubated with anti-TCR{alpha}ß, anti-CD4, and anti-CD8{alpha} mAbs and analyzed by flow cytometry. Leukocyte numbers of the complete thymus, 1 ml of peripheral blood, pooled mesenteric and cervical lymph nodes, and complete spleen were counted in Türk solution with a Bürker haemocytometer. Absolute numbers of CD4 and CD8 T cells of LEW (open bars) and BN (filled bars) rats were determined. The results are represented in bar diagrams as the mean (±SD) of the absolute amount of both CD4 and CD8 T cell populations of four animals of each rat strain. The data are of one of two representative experiments for the thymus and peripheral blood and one of four in case of the lymph nodes and spleen.

 
The CD4/CD8 T cell ratio is dependent on the MHC haplotype

To examine the role of MHC and non-MHC genes in determining the observed difference in CD4/CD8 T cell ratio between LEW and BN rats two independent systems were used. First we analyzed the CD4/CD8 T cell ratios in the peripheral blood of 48 (LEW x BN)F2 hybrids by flow cytometry for the combined expression of MHC haplotype and the expression of CD4 or CD8 T cells. It appeared that the CD4/CD8 T cell ratios in these hybrids were strictly correlated with the MHC haplotype, i.e., the homozygous LEW RT1l haplotype resulted in CD4/CD8 T cell ratios of 5.0 ± 1.2, whereas the homozygous BN RT1n haplotype revealed a ratio of 12.5 ± 2.0 (Fig. 4Go). The heterozygous situation resulted in an intermediate phenotype (8.6 ± 1.5).



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  		FIGURE 4. The CD4/CD8 T cell ratio in (LEW x BN)F2 hybrids is strictly correlated with MHC-haplotype. (LEW x BN)F2 hybrids (n = 48) were characterized first for the expression of LEW and/or BN MHC by incubation of the PBLs with MHC-haplotype-specific mAbs. Second, the CD4/CD8 T cell ratio in the peripheral blood was determined by incubating the leukocytes with anti-TCR{alpha}ß, anti-CD4 and anti-CD8{alpha} mAbs and analyzed by flow cytometry. The results are represented as the mean (±SD) CD4/CD8 T cell ratio of the homozygous BN haplotype (n/n; 11 rats), the heterozygous LEW/BN haplotype (n/l; 22 rats) and the homozygous LEW haplotype (l/l; 15 rats).

 
In a second system, we analyzed the CD4/CD8 T cell ratio in the primary and secondary lymphoid tissues of the respective MHC congenic strains: LEW-1N and BN-1L. First, we confirmed that these two rat strains were congenic for both MHC class I and MHC class II molecules by flow cytometric analysis of the MHC expression on lymph node cells using haplotype-specific mAbs. Indeed, LEW and BN-1L expressed the RT1l haplotype, whereas both BN and LEW-1N expressed the RT1n haplotype (data not shown). Analysis of the CD4/CD8 T cell ratio in the primary and secondary lymphoid organs showed that this ratio in BN-1L rats resembled the one in LEW rats, whereas the ratio in LEW-1N rats was similar to the one in BN rats (Fig. 5Go). These data confirm that the difference in peripheral CD4/CD8 T cell ratio between LEW and BN rats is dependent on the MHC haplotype. Furthermore, the analysis of the CD4/CD8 ratio in the thymus (Fig. 5Go) and within the RTE in the peripheral blood (Fig. 6Go) were in agreement with the dominant role of the thymus in the establishment of this ratio. Also in the MHC-congenic rat strains, the CD4/CD8 T cell ratio in the spleen was two times as low as compared with the other lymphoid compartments (Fig. 5Go).



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  		FIGURE 5. The CD4/CD8 T cell ratio in LEW, BN, and their respective MHC congenic rats is correlated with MHC-haplotype. Thymocytes, PBLs, lymph node cells and splenocytes from naive LEW, BN and their respective MHC congenic rats (LEW-1N and BN-1L) were incubated with anti-TCR{alpha}ß, anti-CD4 and anti-CD8{alpha} mAbs and analyzed by flow cytometry. The results represent the CD4/CD8 ratios determined within TCR{alpha}ßhigh thymocytes or TCR{alpha}ß-positive T cells in peripheral blood, lymph nodes, and spleen. The data are represented as the mean (±SD) CD4/CD8 T cell ratio of four animals of each rat strain. The data are one of two representative experiments for the thymus and peripheral blood and one of four in the case of the lymph nodes and spleen.

 


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  		FIGURE 6. The CD4/CD8 ratio within the recent thymic emigrants confirms the thymic origin of the peripheral CD4/CD8 T cell ratio. PBLs from naive LEW, BN, and their respective MHC congenic rats (LEW-1N and BN-1L) were incubated with a combination of anti-TCR{alpha}ß, anti-CD4, and anti-Thy-1 mAbs or a combination of anti-TCR{alpha}ß, anti-CD8{alpha}, and anti-Thy-1 mAbs and analyzed by flow cytometry. The results represent the CD4/CD8 ratios determined within the Thy-1+ and TCR{alpha}ß+ T cells (recent thymic emigrants). The data are represented as the mean (±SD) CD4/CD8 ratio of four animals of each rat strain.

 
The difference in CD4/CD8 ratio is determined by the nonhematopoietic thymic microenvironment

Because the difference in the peripheral CD4/CD8 T cell ratio between LEW and BN rats originates in the thymus, the difference may be the result of factors intrinsic to the thymus itself or to the hematopoietic precursor cells. Radiation bone marrow chimeras were produced to distinguish first between thymocyte-intrinsic factors and variations in the thymic microenvironment and second between selection on thymic epithelial cells and selection on hematopoietic stromal cells. Both BN and LEW recipients were reconstituted with bone marrow cells from LEW, BN, or (LEW x BN)F1 rats. The allelic difference in MHC class I haplotype between LEW and BN rats was used to distinguish T cells derived from the donor-type precursor cells. Fig. 7Go shows that the CD4/CD8 T cell ratio in the peripheral blood of BN recipients of LEW, BN, or (LEW x BN)F1 bone marrow was 8.4 ± 1.4, 11.2 ± 0.3, and 8.4 ± 1.1, respectively, whereas the ratio in LEW recipients of LEW, BN, or (LEW x BN)F1 bone marrow was 3.4 ± 0.2, 5.2 ± 0.4, and 3.8 ± 0.6, respectively. Similar results were obtained when lymph node was analyzed; analysis of the CD4/CD8 T cell ratio in the spleen revealed a similar reduction as observed in naive animals (Fig. 7Go). These data imply that the difference in CD4/CD8 T cell ratio does not depend on the origin of the hematopoietic cells and therefore excludes the involvement of thymocyte intrinsic factors and selection on hematopoietic stromal cells. However, our results indicate the involvement of selection on thymic epithelial cells in determining the difference in CD4/CD8 T cell ratio between LEW and BN rats.



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  		FIGURE 7. The distinct CD4/CD8 ratio is determined by the nonhematopoietic thymic microenvironment. PBLs, lymph node cells, and splenocytes from BN recipients of LEW (LEW -> BN), BN (BN -> BN), or (LEW x BN)F1 (F1 -> BN) bone marrow and LEW recipients of LEW (LEW -> LEW), BN (BN -> LEW), or (LEW x BN)F1 (F1 -> LEW) bone marrow were incubated with a combination of anti-TCR{alpha}ß, anti-CD4, and anti-donor-type MHC class I mAbs or with a combination of anti-TCR{alpha}ß, anti-CD8{alpha}, and anti-donor-type MHC class I mAbs and analyzed by flow cytometry 6 wk postengraftment. The results represent the CD4/CD8 ratios determined within TCR{alpha}ß-positive T cells of donor origin. The data are represented as the mean (±SD) CD4/CD8 T cell ratio of three to six animals of each combination.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have examined the role of the thymus and of MHC genes in the establishment of the peripheral CD4/CD8 T cell ratio in naive rats. Here we have used LEW, BN and their respective MHC congenic rats, (LEW x BN)F2 hybrids, and radiation bone marrow chimeras. Our results show that the difference in the CD4/CD8 T cell ratio between LEW and BN rats is determined in the thymus during selection on radio-resistant stromal cells and is MHC haplotype dependent. Furthermore, the variation in the CD4/CD8 T cell ratio is determined by a difference in the absolute amount of CD8, but not CD4 T cells.

The prominent role of the thymus is demonstrated by analysis of the CD4/CD8 ratio within the mature, single positive thymocytes and within the population of RTE in the peripheral blood. Both ratios were similar to the peripheral CD4/CD8 T cell ratio in LEW, BN, and their respective MHC congenic rats. In agreement with our data, it has been shown in mice that the thymus plays a crucial role in the establishment of the peripheral CD4/CD8 T cell ratio (25). However, there is a clear discrepancy between these two species. First, in the mouse the CD4/CD8 ratio in the thymus, as well as in the population of RTE, is ~2-fold higher as compared with the peripheral ratio indicating separate thymic and peripheral regulation of the CD4/CD8 ratio (25, 26). Second, the mouse also differs from the rat with respect to the role of the MHC haplotype in determining the peripheral CD4/CD8 T cell ratio (25). In the mouse, the ratio develops independently of the MHC haplotype, whereas our analysis of the MHC congenic rats, in combination with the (LEW x BN)F2 hybrids, reveals that in the rat the MHC genes play a dominant role in determining the peripheral CD4/CD8 T cell ratio. The discrepancy in the role of MHC in the development of CD4/CD8 T cell ratios may be the result of differences in the signaling mechanisms in the mouse and the rat. It has been shown that DP thymocytes from both species, when stimulated in vitro, respond with opposite lineage commitment, i.e., mouse thymocytes develop to CD4 single positive cells, whereas rat thymocytes develop to CD8 single positive cells. This opposite lineage decision may be explained by the thymic expression of a truncated CD8 {alpha}-chain unable to bind p56lck in mice but not in rats (27).

Our results on the thymic origin of the difference in the CD4/CD8 T cell ratio between LEW and BN rats indicates a role for selection events in the thymus. Independent of thymocyte development according to the instruction or stochastic model, both positive and negative selection require a proper interaction of the TCR and MHC molecules, which is further supported by the coreceptors CD4 and CD8 (3, 4, 5). Our study, using the radiation bone marrow chimeras, shows that the distinct CD4/CD8 T cell ratio in LEW and BN rats is controlled by factors that are extrinsic to the radio-sensitive hematopoietic cells but are intrinsic to the radio-resistant stromal cells. Because the radio-resistant stromal cells are not only involved in positive selection, but also in negative selection (7), our results can be explained by both selection events. The low amount of CD8 T cells in BN rats may be the result of defective positive selection or increased negative selection.

Recently, it has been shown that the difference in the level of expression of MHC class I proteins on thymic epithelial cells and dendritic cells influences the decision of immature thymocytes between positive and negative selection (28). Interestingly, LEW and BN rats differ in the composition of the MHC complex: the BN MHC haplotype encodes not one, as in case of LEW MHC, but two class Ia RT1-A molecules (29). This result implies that the amount of MHC class I molecules on the surface of the thymic stromal cells will be higher in BN rats than in LEW rats. Consequently, the BN thymic epithelial cells will be able to negatively select a larger fraction of MHC class I-restricted cells. Furthermore, it is expected that (LEW x BN)F1 rats may express an intermediate amount of MHC class I molecules. This finding will explain the intermediate CD4/CD8 T cell ratio in (LEW x BN)F2 rats with the mixed haplotype (Fig. 4Go).

Besides possible quantitative differences in the level of MHC class I expression on thymic epithelial cells between LEW and BN rats, also qualitative differences in the MHC class I molecules may explain the distinct CD4/CD8 T cell ratios. Because MHC class I proteins ligate with TCR and CD8 molecules, both interactions may be involved. First, CD8 is known to interact with a nonpolymorphic site of the {alpha}3 domain of MHC class I (RT1–A) (30), and this domain plays an important part in both positive and negative selection (31). An exposed loop involving residues 223–229 appears to be the major contact site of the {alpha}3 domain with the CD8 {alpha}-chain in human (30). Furthermore, HLA-Aw68.1 and HLA-Aw68.2, which do not bind CD8, have a valine residue at position 245 whereas all other HLA-A, -B, -C have alanine (32). Amino acid sequence alignment of the {alpha}3 domain of LEW and BN MHC class I revealed that the residues 223–229 and 245 are similar (33). Second, the array of peptides to be presented and therefore the TCR repertoire to be positively selected may be affected by the MHC polymorphism and/or by a polymorphism in the TAP. Indeed, it has been shown that LEW and BN MHC RT1-A are associated with distinct allelic forms of TAP, TAP-A, and TAP-B, respectively (29). Because the TAP isotype present in BN rats has a rather restricted peptide-transport specificity, this may result in less efficient positive selection of MHC class I-restricted CD8 T cells. Furthermore, this predicts that the TCR repertoire between LEW and BN CD8 T cells is different and this hypothesis will be a topic of our further investigation.

Besides the difference in CD4/CD8 T cell ratio between LEW and BN rats, these two rat strains also differ in their susceptibility to develop Th1- or Th2-mediated autoimmune diseases. LEW rats are susceptible to Th1-mediated autoimmune diseases like experimental allergic encephalomyelitis, experimental autoimmune uveoretinitis, and cyclosporin A-induced autoimmunity, whereas BN rats are resistant (34, 35, 36). In contrast, BN rats, but not LEW rats, are highly susceptible to Th2-mediated autoimmune disease like mercury disease and gold-salt induced autoimmunity (36, 37). Recently it has been shown that, like CD4 T cells, CD8 T cells are functionally heterogeneous as a result of their different profile of cytokine production (38, 39, 40). Furthermore, CD8 T cells have been considered to be involved in the down-regulation of Th2-mediated immune responses (41, 42, 43, 44). The susceptibility of BN rats to Th2-mediated autoimmune diseases is correlated with an obvious defect to generate a substantial amount of peripheral CD8 T cells. This finding suggests that BN rats may have defective development of one of the CD8 T cell subpopulations, i.e., Tc1. In agreement with this hypothesis, it has been shown that BN rats have a defect in cytotoxic T cell responses (45).

Altogether, our study on the unraveling of the mechanism responsible for the difference in the amount of CD8 T cells between LEW and BN rats directs for a dominant role of selection on radio-resistant stromal cells in the thymus and for the MHC haplotype. Differential interactions between MHC class I haplotype and CD8/TCR may result in the selection of different CD8 T cell repertoires as well as in an altered balance in the CD8 T cell subsets.


   Acknowledgments
 
We thank H. van der Heijden, M.-J. van de Gaar (Department of Immunology, University Maastricht, Maastricht, The Netherlands), M. Calise (Institut Fédératif de Recherche 30, Toulouse, France) for excellent technical assistance, and Dr. J. van Meerwijk for helpful discussions.


   Footnotes
 
1 This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM), the Cardiovascular Research Institute Maastricht, and by a grant from the Association pour la Recherche sur la Sclerose en Plaques, the Association Française contre les Myopathies, the Conseil Régional de la Région Midi-Pyrénées (RECH:97001931), and a collaborative grant from the Dutch Organization for Scientific Research and INSERM. A.S. is supported by the Centre National de la Recherche Scientifique and B.C. by the Ministère de l’Éducation Nationale, de la Recherche et de la Technologie. Back

2 Address correspondence and reprint requests to Dr. Jan G. M. C. Damoiseaux, Department of Internal Medicine, Section Immunology, University Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands. E-mail address: Back

3 Abbreviations used in this paper: DP, double positive; BN, Brown Norway; LEW, Lewis; RTE, recent thymic emigrants. Back

Received for publication December 14, 1998. Accepted for publication June 25, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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