The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van Meerwijk, J. P. M.
Right arrow Articles by MacDonald, H. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Meerwijk, J. P. M.
Right arrow Articles by MacDonald, H. R.
The Journal of Immunology, 1998, 160: 3649-3654.
Copyright © 1998 by The American Association of Immunologists

Thymic Lineage Commitment Rather Than Selection Causes Genetic Variations in Size of CD4 and CD8 Compartments

Joost P. M. van Meerwijk1, Teresa Bianchi, Samuel Marguerat2 and H. Robson MacDonald3

Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
During their development, immature CD4+CD8+ thymocytes become committed to either the CD4 or CD8 lineage. Subsequent complete maturation of CD4+ and CD8+ cells requires a molecular match of the expressed coreceptor and the MHC specificity of the TCR. The final size of the mature CD4+ and CD8+ thymic compartments is therefore determined by a combination of lineage commitment and TCR-mediated selection. In humans and mice, the relative size of CD4+ and CD8+ peripheral T cell compartments shows marked genetic variability. We show here that genetic variations in thymic lineage commitment, rather than TCR-mediated selection processes, are responsible for the distinct CD4/CD8 ratios observed in common inbred mouse strains. Genetic variations in the regulation of lineage commitment open new ways to analyze this process and to identify the molecules involved.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
The peripheral T lymphocyte repertoire is shaped by a variety of TCR-dependent and -independent mechanisms. In the thymus, only immature thymocytes expressing a TCR that is capable of recognition of self MHC/peptide complexes survive the process of positive selection (reviewed in Refs. 1 and 2). This mechanism is thought to select useful T lymphocytes from an immature pool of precursors with an estimated frequency of MHC reactivity of only 5% (3). Potentially autoreactive thymocytes are physically deleted by induction of apoptosis upon activation by thymic professional APCs (reviewed in Refs. 4 and 5). The latter process (negative selection) eliminates approximately half of the positively selectable thymocytes (6, 7). Finally, in peripheral lymphoid organs, T lymphocytes need to interact with MHC molecules to survive (8, 9, 10).

While complete maturation of thymocytes depends on a molecular match of the expressed coreceptor and MHC class specificity of the TCR, recent data on transgenic and mutant mice and on in vitro differentiation systems indicate that the initial CD4 vs CD8 lineage choice by CD4+CD8+ precursors probably is independent of the MHC class specificity of the TCR (reviewed in 1 . Lineage commitment appears, therefore, to be of a stochastic nature rather than instructed by TCR-MHC interactions.

The relative size of the peripheral CD4 and CD8 T cell compartments is known to be genetically determined in mice and man (11, 12, 13, 14). The contribution of TCR-mediated selection and/or lineage commitment to this genetic variation is unknown. We report here a detailed analysis of the mechanism(s) responsible for the distinct CD4/CD8 ratios observed in the commonly used laboratory mouse strains C57BL/6 and DBA/2. These strains were chosen because of the availability of relatively large numbers of C57BL/6 x DBA/2 recombinant inbred (BXD RI)4 strains as well as congenic strains expressing different MHC and TCR alleles. Our results indicate that the distinct relative sizes of the CD4 and CD8 compartments in these mice are determined by genetic variations in the process of thymic lineage commitment rather than by TCR-mediated positive or negative selection.


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

DBA/2, C57BL/6, C57BL/10, B10.D2, and (C57BL/6 x DBA/2)F1 mice were obtained from Harlan Sprague Dawley, Zeist, Netherlands. Recombinant inbred C57BL/6 x DBA/2 mice (BXD RI) were purchased from The Jackson Laboratory, Bar Harbor, ME. C57BL/6 Vßa and DBA/2 Vßa mice were provided by H. Bour and P. Brawand and originally obtained from Dr. A. Livingstone, Basel Institute for Immunology, Basel, Switzerland. These mice carry the TCR Vßa locus (containing a deletion of several Vß regions as well as point mutations in others (15, 16)) derived from C57L mice and were backcrossed for 15 generations.

Cytofluorometric analysis

Three- and four-color flow cytometric analyses of thymocytes were performed using the following mAbs: anti-TCRß-FITC (H57-597 (PharMingen, San Diego, CA); anti-CD8ß-FITC (H35-17.2) (17); anti-CD5.1-FITC (H11-86.1, PharMingen); anti-HSA/CD24-FITC (M1/69) (18); anti-CD69-FITC (H1.2F3) (19); anti-CD4-PE (H129.19, Boehringer Mannheim, Mannheim, Germany); anti-TCRß-PE (H57-597, PharMingen); anti-CD4-Red613 (H129.19, Life Technologies, Gaithersburg, MD); anti-CD8-Red613 (53-6.7, Life Technologies); anti-CD8-APC (53.6.7, PharMingen).

Cell cycle analysis was performed on electronically sorted CD4+CD8-TCRhigh and CD4-CD8+TCRhigh cells as well as on unpurified thymocytes. Cells (0.5–10 x 104) were permeabilized in 0.2% Nonidet P-40 and DNA stained with 50 µg/ml propidium iodide. During analysis, doublets were excluded using pulse processing.

Cytofluorometric analysis was performed using FACScan and FACStarPlus flow cytometers (Becton Dickinson, San Jose, CA).

Bone marrow chimeras

Mixed bone marrow chimeras were produced as follows: lethally irradiated (900 rad {gamma} irradiation, Cs137 source) DBA/2 and B10.D2 hosts were reconstituted the next day by i.v. injection of a mixture of 1 to 2 x 107 B10.D2 and DBA/2 bone marrow cells (1:1 ratio) that were T cell depleted using anti-Thy1 IgM AT83 (20) and complement (Saxon Europe, Suffolk, U.K.). Mice were kept on antibiotic containing water (0.2% Bactrim, Roche, Basel, Switzerland) until analysis at 6 wk postengraftment.

Analysis of BXD RI mice was performed using hemopoietic chimeras produced by reconstituting lethally irradiated (C57BL/6 x DBA/2)F1 hosts with T cell-depleted bone marrow derived from BXD RI (as well as control C57BL/6 and DBA/2) mice. Per bone marrow origin, three hosts were injected.

Analysis of recombinant inbred strains

Thymocytes from BXD RI hemopoietic chimeras were analyzed 6 wk after engraftment. Ratios of CD4+CD8-TCRhigh to CD4-CD8+TCRhigh thymocytes and their mean and SD were determined. Assignment of the observed CD4/CD8 ratio phenotype to that of DBA/2 or C57BL/6 was determined using Student’s t test. The allelic distributions of the TCR{alpha} and CD8{alpha} genes were retrieved from the Mouse Genome Database (MGD), Mouse Genome Informatics, The Jackson Laboratory (World Wide Web URL: http://www.informatics.jax.org, June, 1997).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Distinct CD4/CD8 ratios in C57BL/6 and DBA/2 mice originate in the thymus

Differences in the numeric ratios of CD4+ to CD8+ T lymphocytes in the lymph nodes of different mouse strains have been described previously (13, 14). We investigated whether the difference in CD4/CD8 ratio between C57BL/6 and DBA/2 mice is due to extrathymic or intrathymic mechanisms. As shown in Figure 1Go, the percentage of CD4+ cells among T lymphocytes in the blood of DBA/2 mice was slightly higher than that in C57BL/6 mice. In contrast, fewer CD8+ T cells were found among PBL of DBA/2 than of C57BL/6 mice. Accordingly, the ratio of CD4+ to CD8+ T lymphocytes was significantly (~2-fold) higher in DBA/2 mice than in C57BL/6 animals (Fig. 1Go). This difference in CD4/CD8 ratios could be due to peripheral and/or intrathymic mechanisms. To distinguish between these possibilities, we analyzed thymocytes from C57BL/6 and DBA/2 mice. Again, we observed a slightly increased percentage of mature CD4+CD8-TCRhigh cells in DBA/2 mice as compared with C57BL/6 animals, while CD4-CD8+TCRhigh thymocytes were present at a lower frequency (Fig. 1Go). The CD4/CD8 ratio was approximately 1.5-fold higher in DBA/2 as compared with C57BL/6 mice (Fig. 1Go). Therefore, the distinct CD4/CD8 ratios observed in peripheral T lymphocytes in these mouse strains originate in the thymus.



View larger version (38K):
[in this window]
[in a new window]
  		FIGURE 1. Distinct C57BL/6 and DBA/2 CD4/CD8 ratios in peripheral blood T lymphocytes originate in the thymus. Peripheral blood and thymocytes of C57BL/6 and DBA/2 mice were incubated with anti-TCR, anti-CD4, and anti-CD8 Abs, and percentages of mature CD4+ and CD8+ cells as well as CD4/CD8 ratios were determined by cytofluorometry. In the analysis of PBL, 3 animals were used per mouse strain, while the data for thymocytes are compiled from four experiments with a total of 13 mice per strain. The difference in thymocyte CD4/CD8 ratios between C57BL/6 and DBA/2 is highly significant (Student’s t test, p < <0.0001).

 
The difference in CD4/CD8 ratios is not due to proliferation or thymic retention

Differences in steady state representations of mature thymocyte populations may be caused by variations in the generation, thymic retention, or proliferation of those cells. To distinguish between these possibilities, we examined cell cycle status as well as phenotype of mature thymocytes using markers in which expression levels change during final maturation.

The mature CD4+ and CD8+ thymocyte populations are known to contain dividing cells (21, 22). We investigated whether this intrathymic proliferation could account for the difference in CD4/CD8 ratios observed in C57BL/6 and DBA/2 mice. Electronically sorted CD4+CD8-TCRhigh and CD4-CD8+TCRhigh thymocytes from the two mouse strains were detergent permeabilized and their DNA content analyzed by flow cytometry (Fig. 2GoA). The proportion of cycling mature CD4+ and CD8+ thymocytes was similar in C57BL/6 and DBA/2 mice. Therefore, differences in proliferation of mature thymocytes in C57BL/6 and DBA/2 mice cannot account for the differences in CD4/CD8 ratio.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 2. Differentiation rather than proliferation or retention of thymocytes causes variation in CD4/CD8 ratio. A, Total thymocytes and electronically sorted CD4+CD8- TCRhigh and CD4-CD8+TCRhigh cells (purity > 95%) were detergent permeabilized, and DNA content was assessed by propidium iodide staining and flow cytometry. Statistics indicate mean percentage (±SD, n = 5) of cells within depicted gates (S + G2/M). Thymocytes were labeled with anti-CD4, anti-CD8, anti-TCR, and anti-CD69 (B) or anti-HSA (C) Abs. CD69 and HSA fluorescence histograms of electronically gated CD4+CD8- TCRhigh and CD4-CD8+TCRhigh thymocytes are shown. Numbers indicate mean percentage (±SD, n = 5) of events within depicted CD69 or HSA gates among total, CD4+CD8- TCRhigh, and CD4-CD8+ TCRhigh thymocytes. Bar graphs indicate mean ratios (±SD) of percentage of CD69highCD4+CD8-TCRhigh to CD69highCD4-CD8+TCRhigh (B) and HSAhighCD4+ CD8-TCRhigh to HSAhigh CD4-CD8+TCRhigh thymocytes (C). CD4/CD8 ratios of DBA/2 cells are significantly higher than those of C57BL/6 thymocytes shown in B (p < <0.001) and C (p < <0.001).

 
CD69 is an early lymphocyte activation marker that is transiently up-regulated during thymic positive selection (23, 24, 25). CD69 expression is gradually extinguished on CD4+CD8-TCRhigh and CD4-CD8+TCRhigh thymocytes. No gross differences in the proportion of CD69low cells among mature CD4+ or CD8+ thymocytes in DBA/2 vs C57BL/6 mice were observed (Fig. 2GoB). Moreover, when considering only the least mature CD69high CD4+CD8-TCRhigh and CD4-CD8+TCRhigh cells, a relative difference in the CD4/CD8 ratios of C57BL/6 vs DBA/2 mice was found that was similar to that obtained using CD4, CD8, and TCR as criteria (Fig. 2GoB), suggesting that this difference is not caused by increased retention of mature thymocytes.

The expression of heat-stable Ag (HSA, CD24), a marker highly expressed on immature thymocytes, is also down-modulated during final maturation of CD4+CD8-TCRhigh and CD4-CD8+TCRhigh thymocytes (26, 27). Moreover, in contrast to the more mature HSAlow thymocytes, immature TCRhighHSAhigh cells are known to respond very poorly to mitogenic stimuli. The proportion of HSAlow cells among CD4+CD8-TCRhigh and CD4-CD8+TCRhigh thymocytes was indistinguishable in C57BL/6 vs DBA/2 mice (Fig. 2GoC). Again, the ratio of the least mature HSAhigh CD4+CD8-TCRhigh to CD4-CD8+TCRhigh cells was significantly higher in DBA/2 than in C57BL/6 mice (Fig. 2GoC), and the relative difference in the ratios between these mouse strains was similar to that obtained using thymocytes gated on CD4, CD8, and TCR only (Fig. 1Go). Collectively, these data indicate that the difference in the CD4/CD8 ratio between C57BL/6 and DBA/2 mice is not caused by increased retention or proliferation of mature thymocytes but rather by variations in the differentiation of those cells, i.e., thymocyte CD4/CD8 lineage choice and/or TCR-mediated selection processes.

Distinct CD4/CD8 ratios develop independently of MHC haplotype

The processes of thymic positive and negative selection depend on interactions between the clonotypic TCR and its MHC/peptide ligand. The different MHC haplotypes expressed by DBA/2 (H-2d) and C57BL/6 (H-2b) mice may therefore cause variations in positive and/or negative selection and may thus explain the different CD4/CD8 ratios. To investigate this possibility, we analyzed C57BL/10 congenic mice expressing products of the DBA/2-derived MHC locus (B10.D2). C57BL/10 (B10) mice are genetically very similar to C57BL/6 mice and had a thymic CD4/CD8 ratio similar to that of C57BL/6 animals (Figs. 1Go and 3Go). The CD4/CD8 ratio in B10.D2 mice was similar to that of B10 rather than DBA/2 animals. Therefore, the different CD4/CD8 ratios observed in DBA/2 and C57BL/10 mice are dictated by genetic background rather than by MHC haplotype.



View larger version (50K):
[in this window]
[in a new window]
  		FIGURE 3. MHC haplotype does not influence CD4/CD8 ratio. Thymocytes from C57BL/10, DBA/2, and B10.D2 mice were analyzed by flow cytometry using anti-TCR, anti-CD8, and anti-CD4 Abs. Percentages of CD4+CD8-TCRhigh and CD4-CD8+TCRhigh thymocytes and CD4/CD8 ratios were determined. Depicted are mean values ± SD (n = 5). The CD4/CD8 ratio in DBA/2 mice is significantly higher than in C57BL/10 (p < 0.001) and B10.D2 (p < 0.001) mice.

 
Difference in CD4/CD8 ratio is determined by thymocyte-intrinsic factors

Antigenic peptides and superantigens presented by MHC molecules are also known to be involved in thymic positive and negative selection processes (reviewed in Refs. 2, 28, and 29). Moreover, during development thymocytes interact with a variety of APC and thymic stromal cells (reviewed in 30 . Since both factors probably vary between the C57BL/6 and DBA/2 mouse strains, we sought to investigate their involvement in the generation of the different CD4/CD8 ratios.

Radiation bone marrow chimeras were produced in which B10.D2 and DBA/2 thymocytes developed in an identical microenvironment. Lethally irradiated B10.D2 and DBA/2 hosts were reconstituted with a mixture of DBA/2 and B10.D2 bone marrow cells at a ratio of 1:1. The allelic difference in CD5 between B10 and DBA/2 mice (31) was used to distinguish thymocytes derived from the two types of precursor cells (Fig. 4Go). The CD4/CD8 ratio of B10.D2 and DBA/2 thymocytes developing in DBA/2 hosts was similar to that of thymocytes from normal B10.D2 and DBA/2 mice, respectively (Fig. 4Go). Moreover, the relative difference in the CD4/CD8 ratios between B10.D2 and DBA/2 cells was also conserved in B10.D2 hosts, although individual CD4/CD8 ratio values were slightly higher (Fig. 4Go). Therefore, thymocyte-intrinsic factors rather than variations in thymic microenvironment clearly determine the difference in CD4/CD8 ratio between B10.D2 and DBA/2 mice.



View larger version (43K):
[in this window]
[in a new window]
  		FIGURE 4. Distinct CD4/CD8 ratios are determined by a thymocyte-intrinsic mechanism. Lethally irradiated B10.D2 and DBA/2 hosts were reconstituted with a mixture of bone marrow cells from the two strains at a ratio of 1:1. Six weeks later, thymocytes were analyzed by four-color flow cytometry using anti-CD5.1, anti-TCR, anti-CD4, and anti-CD8 Abs. CD4/CD8 contour plots of CD5.1- (B10.D2-derived) and CD5.1+ (DBA/2-derived) thymocytes are depicted with the CD4+CD8- and CD4-CD8+ gates used to analyze TCR expression (not shown). Numbers indicate percentages (±SD, n = 5) of CD4+CD8-TCRhigh and CD4-CD8+TCRhigh cells among CD5.1- or CD5.1+ thymocytes. Bar graphs depict CD4/CD8 ratios (±SD) calculated for each individual chimera. The CD4/CD8 ratio of B10.D2 thymocytes is significantly lower that that of DBA/2 cells in B10.D2 (p < 0.01) and DBA/2 (p < <0.001) hosts.

 
Difference in CD4/CD8 ratio is TCR{alpha}ß independent

Since the difference in CD4/CD8 ratio between C57BL/6 and DBA/2 mice seems to be determined by a thymocyte intrinsic mechanism(s), we subsequently analyzed surface molecules known to be involved in TCR-dependent clonotypic selection mechanisms. The major thymocyte surface molecule implicated in positive and negative selection is the clonotypic TCR{alpha}ß heterodimer. To our knowledge, no differences between the products of the TCRß loci of DBA/2 and C57BL/6 mice have been reported. The ß-chain-encoding segments of the two loci have not been completely sequenced, however, and differences may have remained undetected. To investigate whether the TCRß locus influences the development of different CD4/CD8 ratios in the two strains of mice, we analyzed DBA/2 and C57BL/6 congenic mice expressing the products of an identical, C57L-derived TCRß locus (Vßa) containing a deletion of several Vß segments as well as point mutations in others (15, 16). A similar difference in thymic CD4/CD8 ratios was observed in C57BL/6 Vßa vs DBA/2 Vßa and C57BL/6 Vßb vs DBA/2 mice Vßb (Fig. 5Go). Moreover, C57BL/6 Vßa and DBA/2 Vßa-derived thymocytes had CD4/CD8 ratios indistinguishable from those of C57BL/6 and DBA/2 mice (Fig. 5Go). Therefore, the difference in CD4/CD8 ratio between C57BL/6 and DBA/2 mice cannot be explained by variations in expressed TCR Vß segments. Interestingly, despite the very significant (approximately twofold) difference in the number of Vß segments encoded by the Vßa and Vßb alleles (16), similar CD4/CD8 ratios develop, reinforcing the concept that CD4/CD8 ratios develop in a selection independent manner.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 5. TCR Vß repertoire does not influence CD4/CD8 ratio. Thymocytes from C57BL/6 and DBA/2 congenic mice expressing TCR Vßa or Vßb alleles were incubated with anti-CD4, anti-CD8, and anti-TCR Abs and the ratio of CD4+CD8-TCRhigh to CD4-CD8+TCRhigh cells determined by flow cytometry. Graphs depict mean values ± SD (C57BL/6 Vßa/Vßb, n = 5; DBA/2 Vßa/Vßb, n = 3).

 
Inasmuch as allelic differences between the DBA/2 and C57BL/6 TCR C{alpha} and V{alpha} loci have been reported (32, 33), we also investigated whether this variation causes different CD4/CD8 ratios in C57BL/6 and DBA/2 mice using BXD RI strains. To synchronize thymocytes developing in the several BXD RI strains and to limit our analysis to thymocyte-intrinsic factors, we produced hemopoietic chimeras reconstituting (C57BL/6 x DBA/2)F1 hosts with BXD RI-derived hemopoietic cells. Chimeras with CD4/CD8 ratios that were assignable to the C57BL/6 or DBA/2 parental strains (11 of 22 total) are shown in Figure 6Go. The distribution of CD4/CD8 ratios among the BXD RI chimeric thymocytes did not correlate with that of the TCR{alpha} alleles. Therefore, the observed difference in CD4/CD8 ratio is not caused by allelic differences in the DBA/2 and C57BL/6 TCR{alpha} loci. Taken together, the data indicate that variations in the expressed TCR are not responsible for the development of different CD4/CD8 ratios in C57BL/6 and DBA/2 mice.



View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 6. TCR{alpha} and CD8{alpha} do not determine CD4/CD8 ratio. Lethally irradiated (C57BL/6 x DBA/2)F1 hosts were reconstituted with 1–2 x 107 bone marrow cells from C57BL/6 ("B"), DBA/2 ("D"), or BXD RI ("1", "6", etc.) donors. Six weeks later, thymocytes were analyzed by flow cytometry using anti-CD4, anti-CD8, and anti-TCR Abs. CD4+CD8-TCRhigh:CD4-CD8+TCRhigh ratios were determined for each series of three chimeras that received the same bone marrow; mean values (±SD) are depicted. The CD4/CD8 ratio of thymocytes from a given chimera was assigned to either C57BL/6 ("B") or DBA/2 ("D") using Student’s t test. The BXD RI strain distribution of allelic forms of TCR{alpha} and CD8{alpha} is included for comparison.

 
Variation in the CD4/CD8 ratio develops independently of CD4 and CD8

Coreceptors (CD4 and CD8) are known to influence positive and negative selection processes. Thus, differences in expression level of coreceptors change the balance between no selection and positive or negative selection (34, 35, 36, 37, 38). Since expression levels of coreceptors are similar in C57BL/6 and DBA/2 mice (data not shown), we analyzed whether allelic differences in the encoding genes might be responsible for the different thymic CD4/CD8 ratios. To our knowledge, no allelic differences in the products of the CD4 and CD8ß genes of these two mouse strains have been reported. However, DBA/2 and C57BL/6 mice express different CD8{alpha} alleles (CD8{alpha}.1 and CD8{alpha}.2, respectively) (31), and the distribution of the two alleles in the BXD RI strains is known. As shown in Figure 6Go, the distribution of the CD4/CD8 ratios among BXD RI thymocytes in hemopoietic chimeras does not correlate with that of the CD8{alpha} alleles. Moreover, BALB/c mice, which express the same CD8{alpha} and CD8ß alleles as C57BL/6 animals (31), have a CD4/CD8 ratio at least as high as that in DBA/2 mice (data not shown and Refs. 13 and 14). Therefore, the difference in CD4/CD8 ratios observed in C57BL/6 vs DBA/2 mice appears not to be due to differences in expression level or expressed alleles of CD4 and/or CD8.

Collectively, our data indicate that the distinct relative sizes of the CD4 and CD8 compartments observed in C57BL/6 and DBA/2 mice are not due to differences in positive and/or negative selection, since the involvement of TCR ligands, thymic microenvironment, and the TCR complex itself has been excluded. Indeed, large variations in expression of MHC and TCR genes do not significantly alter the CD4/CD8 ratio, indicating that it develops independently of thymic selection processes.

The distinct CD4/CD8 ratios observed in C57BL/6 and DBA/2 mice are most readily explained by the variation in the process of CD4 vs CD8 lineage commitment, although other possibilities cannot formally be excluded. Recently, extensive analysis of the mechanism underlying this process has revealed that the MHC class specificity of the TCR expressed by thymocytes probably does not determine their CD4 vs CD8 lineage choice (reviewed in 1 . Rather, the correlation between the MHC class specificity of the TCR and the lineage in which the thymocyte can successfully develop is proposed to be determined by TCR specificity-dependent selection mechanisms following lineage commitment. Our data indicating that a TCR selection-independent mechanism determines CD4/CD8 ratio in normal, unmanipulated mice supports current stochastic models of lineage commitment.

It has recently been suggested that the CD4/CD8 lineage choice may be determined by the activity of Notch1 (39). Although the exact distribution of the C57BL/6 and DBA/2 alleles of Notch1 in the BXD RI strains is not known, it has been reported that three recombinations occurred between Notch1 and Ass1 (40). Since the CD4/CD8 ratio differs in at least eight BXD RI strains from the inherited Ass1 allele (data not shown), the Notch1 molecule itself does not seem to determine CD4/CD8 ratio. Nevertheless, it remains possible that other molecules involved in Notch1 signaling (e.g., Numb (41) and Jagged (42)) are involved.

Analysis of thymocyte CD4/CD8 lineage commitment has been performed in vivo using mice with targeted mutations of several genes or expression of a variety of transgenes and in vitro using thymocyte differentiation assays (reviewed in 1 . Our data indicating genetic variations in CD4/CD8 lineage choice in common laboratory mouse strains open new avenues to analyzing this process and to identifying the molecules involved.


   Acknowledgments
 
We thank Dr. Alexandra Livingstone (Basel Institute for Immunology, Basel, Switzerland) for generously providing C57BL/6 Vßa and DBA/2 Vßa congenic mice. These mice were bred in our facilities and provided by Hélène Bour and Pierre Brawand. Pierre Zaech is acknowledged for expert flow cytometry.


   Footnotes
 
1 Current address: National Institute of Health and Medical Research (INSERM) U395, Purpan Hospital, and University "Paul Sabatier," Toulouse, France. Back

2 Current address: Department of Genetics and Microbiology, University of Geneva Medical School, Geneva, Switzerland. Back

3 Address correspondence and reprint requests to Dr. H. Robson MacDonald, Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne, 1066 Epalinges, Switzerland. Back

4 Abbreviations used in this paper: BXD RI, C57BL/6 x DBA/2 recombinant inbred strain; HSA, heat-stable Ag. Back

Received for publication October 27, 1997. Accepted for publication December 8, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Guidos, C. J.. 1996. Positive selection of CD4+ and CD8+ T cells. Curr. Opin. Immunol. 8:225.[Medline]
  2. Jameson, S. C., K. A. Hogquist, M. J. Bevan. 1995. Positive selection of thymocytes. Annu. Rev. Immunol. 13:93.[Medline]
  3. Zerrahn, J., W. Held, D. H. Raulet. 1997. The MHC reactivity of the T cell repertoire prior to positive and negative selection. Cell 88:627.[Medline]
  4. Nossal, G. J.. 1994. Negative selection of lymphocytes. Cell 76:229.[Medline]
  5. Sprent, J., S. R. Webb. 1995. Intrathymic and extrathymic clonal deletion of T cells. Curr. Opin. Immunol. 7:196.[Medline]
  6. Ignatowicz, L., J. Kappler, P. Marrack. 1996. The repertoire of T cells shaped by a single MHC/peptide ligand. Cell 84:521.[Medline]
  7. van Meerwijk, J. P. M., S. Marguerat, R. K. Lees, R. N. Germain, B. J. Fowlkes, H. R. MacDonald. 1997. Quantitative impact of thymic clonal deletion on the T cell repertoire. J. Exp. Med. 185:377.[Abstract/Free Full Text]
  8. Takeda, S., H. R. Rodewald, H. Arakawa, H. Bluethmann, T. Shimizu. 1996. MHC class II molecules are not required for survival of newly generated CD4+ T cells, but affect their long-term life span. Immunity 5:217.[Medline]
  9. Tanchot, C., F. A. Lemonnier, B. Perarnau, A. A. Freitas, B. Rocha. 1997. Differential requirements for survival and proliferation of CD8 naive or memory T cells. Science 276:2057.[Abstract/Free Full Text]
  10. Rooke, R., C. Waltzinger, C. Benoist, D. Mathis. 1997. Targeted complementation of MHC class II deficiency by intrathymic delivery of recombinant retroviruses. Immunity 7:123.[Medline]
  11. Amadori, A., R. Zamarchi, G. De Silvestro, G. Forza, G. Cavatton, G. A. Danieli, M. Clementi, L. Chieco-Bianchi. 1995. Genetic control of the CD4/CD8 T-cell ratio in humans. Nat. Med. 1:1279.[Medline]
  12. Amadori, A., R. Zamarchi, L. Chieco-Bianchi. 1996. CD4:CD8 ratio and HIV infection: the "tap-and-drain" hypothesis. Immunol. Today 17:414.[Medline]
  13. Kraal, G., I. L. Weissman, E. C. Butcher. 1983. Genetic control of T-cell subset representation in inbred mice. Immunogenetics 18:585.[Medline]
  14. Sprent, J., M. Schaefer. 1990. Antigen-presenting cells for CD8+ T cells. Immunol. Rev. 117:213.[Medline]
  15. Smith, L. R., A. Plaza, P. A. Singer, A. N. Theofilopoulos. 1990. Coding sequence polymorphisms among Vß T cell receptor genes. J. Immunol. 144:3234.[Abstract]
  16. Behlke, M. A., H. S. Chou, K. Huppi, D. Y. Loh. 1986. Murine T-cell receptor mutants with deletions of ß-chain variable region genes. Proc. Natl. Acad. Sci. USA 83:767.[Abstract/Free Full Text]
  17. Golstein, P., C. Goridis, A.-M. Schmitt-Verhulst, B. Hayot, A. Pierres, A. van Agthoven, Y. Kaufmann, Z. Eshhar, M. Pierres. 1982. Lymphoid cell surface interaction structures detected using cytolysis-inhibiting monoclonal antibodies. Immunol. Rev. 68:5.[Medline]
  18. Springer, T., G. Galfre, D. S. Secher, C. Milstein. 1978. Monoclonal xenogeneic antibodies to murine cell surface antigens: identification of novel leukocyte differentiation antigens. Eur. J. Immunol. 8:539.[Medline]
  19. Yokoyama, W. M., F. Koning, P. J. Kehn, G. M. Pereira, G. Stingl, J. E. Coligan, E. M. Shevach. 1988. Characterization of a cell surface-expressed disulfide-linked dimer involved in murine T cell activation. J. Immunol. 141:369.[Abstract]
  20. Sarmiento, M., M. R. Loken, F. W. Fitch. 1981. Structural differences in cell surface T25 polypeptides from thymocytes and cloned T cells. Hybridoma 1:13.[Medline]
  21. Ernst, B., C. D. Surh, J. Sprent. 1995. Thymic selection and cell division. J. Exp. Med. 182:961.[Abstract/Free Full Text]
  22. Huesmann, M., B. Scott, P. Kisielow, H. von Boehmer. 1991. Kinetics and efficacy of positive selection in the thymus of normal and T cell receptor transgenic mice. Cell 66:533.[Medline]
  23. Bendelac, A., P. Matzinger, R. A. Seder, W. E. Paul, R. H. Schwartz. 1992. Activation events during thymic selection. J. Exp. Med. 175:731.[Abstract/Free Full Text]
  24. Swat, W., M. Dessing, H. von Boehmer, P. Kisielow. 1993. CD69 expression during selection and maturation of CD4+8+ thymocytes. Eur. J. Immunol. 23:739.[Medline]
  25. van Meerwijk, J. P. M., R. N. Germain. 1993. Development of mature CD8+ thymocytes: selection rather than instruction?. Science 261:911.[Abstract/Free Full Text]
  26. Nikolic-Zugic, J., M. J. Bevan. 1990. Functional and phenotypic delineation of two subsets of CD4 single positive cells in the thymus. Int. Immunol. 2:135.[Abstract/Free Full Text]
  27. Ramsdell, F., M. Jenkins, Q. Dinh, B. J. Fowlkes. 1991. The majority of CD4+8- thymocytes are functionally immature. J. Immunol. 147:1779.[Abstract]
  28. Simpson, E., P. J. Dyson, A. M. Knight, P. J. Robinson, J. I. Elliott, D. M. Altmann. 1993. T-cell receptor repertoire selection by mouse mammary tumor viruses and MHC molecules. Immunol. Rev. 131:93.[Medline]
  29. Acha-Orbea, H., H. R. MacDonald. 1995. Superantigens of mouse mammary tumor virus. Annu. Rev. Immunol. 13:459.[Medline]
  30. Anderson, G., N. C. Moore, J. J. T. Owen, E. J. Jenkinson. 1996. Cellular interactions in thymocyte development. Annu. Rev. Immunol. 14:73.[Medline]
  31. III Morse, H. C., F. W. Shen, U. Hämmerling. 1987. Genetic nomenclature for loci controlling mouse lymphocyte antigens. Immunogenetics 25:71.[Medline]
  32. Dembic, Z., W. Bannwarth, B. A. Taylor, M. Steinmetz. 1985. The gene encoding the T-cell receptor {alpha}-chain maps close to the Np-2 locus on mouse chromosome 14. Nature 314:271.[Medline]
  33. Jouvin-Marche, E., M. G. Morgado, N. Trede, P. N. Marche, D. Couez, I. Hue, C. Gris, M. Malissen, P. A. Cazenave. 1989. Complexity, polymorphism, and recombination of mouse T-cell receptor gene families. Immunogenetics 30:99.[Medline]
  34. Lee, N. A., D. Y. Loh, E. Lacy. 1992. CD8 surface levels alter the fate of {alpha}/ß T cell receptor-expressing thymocytes in transgenic mice. J. Exp. Med. 175:1013.[Abstract/Free Full Text]
  35. Robey, E. A., F. Ramsdell, D. Kioussis, W. Sha, D. Loh, R. Axel, B. J. Fowlkes. 1992. The level of CD8 expression can determine the outcome of thymic selection. Cell 69:1089.[Medline]
  36. Casey Crooks, M. E., D. R. Littman. 1994. Disruption of T lymphocyte positive and negative selection in mice lacking the CD8 ß chain. Immunity 1:277.[Medline]
  37. Itano, A., D. Cado, F. K. Chan, E. Robey. 1994. A role for the cytoplasmic tail of the ß chain of CD8 in thymic selection. Immunity 1:287.[Medline]
  38. Davis, C. B., D. R. Littman. 1995. Disrupted development of thymocytes expressing a transgenic TCR upon CD4 overexpression. Int. Immunol. 7:1977.[Abstract/Free Full Text]
  39. Robey, E., D. Chang, A. Itano, D. Cado, H. Alexander, D. Lans, G. Weinmaster, P. Salmon. 1996. An activated form of Notch influences the choice between CD4 and CD8 T cell lineages. Cell 87:483.[Medline]
  40. Reaume, A. G., R. A. Conlon, R. Zirngibl, T. P. Yamaguchi, J. Rossant. 1992. Expression analysis of a Notch homologue in the mouse embryo. Dev. Biol. 154:377.[Medline]
  41. Zhong, W., J. N. Feder, M. M. Jiang, L. Y. Jan, Y. N. Jan. 1996. Asymmetric localization of a mammalian Numb homolog during mouse cortical neurogenesis. Neuron 17:43.[Medline]
  42. Lindsell, C. E., C. J. Shawber, J. Boulter, G. Weinmaster. 1995. Jagged: a mammalian ligand that activates Notch1. Cell 80:909.[Medline]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
V. Thomas-Vaslin, H. K. Altes, R. J. de Boer, and D. Klatzmann
Comprehensive Assessment and Mathematical Modeling of T Cell Population Dynamics and Homeostasis
J. Immunol., February 15, 2008; 180(4): 2240 - 2250.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. Shanker, N. Auphan-Anezin, P. Chomez, L. Giraudo, B. V. d. Eynde, and A.-M. Schmitt-Verhulst
Thymocyte-Intrinsic Genetic Factors Influence CD8 T Cell Lineage Commitment and Affect Selection of a Tumor-Reactive TCR
J. Immunol., April 15, 2004; 172(8): 5069 - 5077.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. Karlsson, X. Zhao, I. Lonskaya, M. Neptin, R. Holmdahl, and A. Andersson
Novel Quantitative Trait Loci Controlling Development of Experimental Autoimmune Encephalomyelitis and Proportion of Lymphocyte Subpopulations
J. Immunol., January 15, 2003; 170(2): 1019 - 1026.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. S. Sobel, L. Morel, R. Baert, C. Mohan, J. Schiffenbauer, and E. K. Wakeland
Genetic Dissection of Systemic Lupus Erythematosus Pathogenesis: Evidence for Functional Expression of Sle3/5 by Non-T Cells
J. Immunol., October 1, 2002; 169(7): 4025 - 4032.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
E. Jimenez, A. Vicente, R. Sacedon, J. J. Munoz, G. Weinmaster, A. G. Zapata, and A. Varas
Distinct Mechanisms Contribute to Generate and Change the CD4:CD8 Cell Ratio During Thymus Development: A Role for the Notch Ligand, Jagged1
J. Immunol., May 15, 2001; 166(10): 5898 - 5908.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
J. G. M. C. Damoiseaux, B. Cautain, I. Bernard, M. Mas, P. J. C. van Breda Vriesman, P. Druet, G. Fournie, and A. Saoudi
A Dominant Role for the Thymus and MHC Genes in Determining the Peripheral CD4/CD8 T Cell Ratio in the Rat
J. Immunol., September 15, 1999; 163(6): 2983 - 2989.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
R. D. McFarland, D. C. Douek, R. A. Koup, and L. J. Picker
Identification of a human recent thymic emigrant phenotype
PNAS, April 11, 2000; 97(8): 4215 - 4220.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by van Meerwijk, J. P. M.
Right arrow Articles by MacDonald, H. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by van Meerwijk, J. P. M.
Right arrow Articles by MacDonald, H. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS