Genes and Immunity (2002) 3, 144–150  2002 Nature Publishing Group All rights reserved 1466-4879/02 $25.00 www.nature.com/gene Linkage analysis of variations in CD4:CD8 subsets between C57BL/6 and DBA/2

C Myrick1,4, R DiGuisto2, J DeWolfe2, E Bowen1,3, J Kappler2, P Marrack2 and EK Wakeland1,3 1Center for Mammalian Genetics, College of Medicine, University of Florida, Gainesville, FL 32610, USA; 2Department of Immunology, Howard Hughes Medical Institute and National Jewish Med. and Res. Centre, Denver, CO 80206, USA; 3Center for Immunology, University of Texas Southwestern Medical Center, Dallas, TX 75235, USA

The ratio of CD4 T cells to CD8 T cells (CD4:CD8 ratio) varies over two-fold between C57BL/6 and DBA/2 mice for both T cell precursors in the and mature T cells in the periphery. Correlation analysis of the CD4:CD8 ratio in thymic precursors vs peripheral T cells in F2 and backcross mice found that thymic precursor ratios are inherited independently from those in the periphery, indicating that the CD4:CD8 ratios in these populations are affected by distinct genetic mechanisms. A genome scan of progeny in the phenotypic extremes identified three quantitative trait loci (QTLs). Trmq1 (for T cell ratio modifier QTL 1) was detected in the telomeric end of c6 (peak marker D6Mit15 at 74 cM) and had a maximum LOD score of 4.6. Trmq2, in the telomeric half of c2, peaked at D2MIT483 and had a maximum LOD score of 3.41. Both of these QTLs impacted the CD4:CD8 ratios in peripheral T cells and had no impact on variation in this ratio among thymic precursors. However, heterozygosity for the H2 complex was suggestively associated (LOD score of 2.43) with increases in CD4 T cells among T cell precursors in the thymus. All of these QTLs were affected by epistatic interactions, indicating that additional modifiers in the B6 and DBA/2 genomes modulate this phenotype. Genes and Immunity (2002) 3, 144–150. DOI: 10.1038/sj/gene/6363819

Keywords: T cells; CD4 T cell subsets; CD8 T cell subsets; genome scan; quantitative trait loci; epistasis

Introduction The correlation between the ratio of CD4 to CD8 T cells in the thymus and periphery is not always good, how- Most normal mice and human beings contain more CD4 ever. For example, depletion of peripheral CD4+ T cells T cells than CD8 T cells in their thymuses and peripheral does not lead to increased production of CD4+ cells by lymphoid tissues. However, numerous factors affect the thymus.17 Therefore it is likely that genes other than these numbers. For example, the CD4:CD8 ratio falls as those thought to affect T cell maturation; for example, 1–4 animals age. Infection, particularly with viruses, tends genes that act exclusively on mature T cells; may also to increase disproportionately the numbers of mature T affect the ratio of CD4:CD8 T cells in the periphery. 5–8 cells bearing CD8. Autoimmunity, diet and even As an initial step in the identification of such genes, we exposure to sunshine have been reported to affect the studied the CD4:CD8 ratios of F2 and backcross progeny 9–11 ratio. generated by crossing C57B1/6 and DBA/2, two strains Although environmental and other factors may affect that differ markedly in their thymus and peripheral the numbers of CD4 to CD8 T cells in an individual, it is CD4:CD8 ratios. C57B1/6 mice have a comparatively low 12–14 clear that the ratio is also under genetic control. Since ratio of CD4 to CD8 T cells, whereas the ratio is high the maturation of CD4 and CD8 T cells is controlled by in DBA/2 mice. Our results indicate that, in this strain ␣␤ interactions of their T cell receptors with class II and combination, the ratios in thymus were not well corre- class I major histocompatibility complex (MHC) lated with those in lymph nodes. In the lymph nodes, the and peptides in the thymus, one would predict that CD4 ratios were only slightly affected by genes mapping to to CD8 ratios would be affected by TCR and MHC genes. MHC or TCR␣ or TCR␤. Instead, the peripheral CD4:CD8 Indeed there is evidence that in certain mouse strain com- ratios were controlled by at least two genes mapping to 14,15 binations, this is the case. Other proteins expressed in mouse chromosomes 6 and 2. The inheritance of this the thymus, such as Notch, may also affect the balance phenotype appears to be quite complex and to be subject 16 between CD4 and CD8 T cells. to significant epistatic interactions.

Correspondence: E Wakeland, Center for Immunology, University of Results Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235–9093, USA. E-mail: Edward.WakelandȰemail.swmed.edu 4 Inheritance pattern of CD4:CD8 ratios in F2 and Present address: Emory Vaccine Center, Emory University School backcross progeny of Medicine, Atlanta, GA 30329, USA + + This work was supported by NIH Grants Al-17966, Al-17134, Al- To measure accurately the ratio of CD4 to CD8 T cells 18785, and Al-22295. in individual mice, cells isolated from the thymus, lymph Received 16 April 2001; revised and accepted 5 October 2001 nodes, and spleen were stained with anti-␣␤TCR, anti- Linkage analysis in CD4:CD8 T cell subsets C Myrick et al 145 CD4 and anti-CD8. Flow cytometry was then used to presented for the lymph node and thymus in Figures 1b determine the ratio of the numbers of ␣␤TCR+ T cells and 1c. The phenotype was distributed normally in both bearing CD4 to those bearing CD8. Since CD4:CD8 ratio F2 and BC mice, suggesting that the ratios in both organs varies with age,1–4 the ratios in these mice were all meas- are inherited in a quantitative fashion and are potentially ured between 4 to 8 weeks of age. The CD4:CD8 ratios affected by several quantitative trait loci (QTL) that vary for B6, DBA/2, (B6 × DBA.2)F1, (B6 × DBA/2)F2, and (B6 between the parental strains. Not unexpectedly, the ratios × DBA/2)F1 × DBA/2 testcrosses are presented for of BC mice tended to be higher than those of F2 animals. lymph node T cells in Figure 1a. As observed previously, In addition, the ratios for both F2 and BC mice almost B6 mice have a lower ratio of CD4 to CD8 T cells than completely encompassed parental values. A few F2 mice DBA/2 mice, and this was true in all organs tested (data had ratios that were even lower than those of their low, for thymus and spleen not shown). The data in Figure 1a C57BI/6 grandparents, however, suggesting that this illustrate the magnitude of the genetic variance in phenotype may be affected by epistatic interactions CD4:CD8 ratio between C57BL/6 and DBA/2 that was between loci in the two genomes. detected in our colony and demonstrate that the low ratio of B6 mice is dominant in F1 crosses between these CD4:CD8 ratios in thymus and lymph nodes strains. segregate independently We generated 304 intercross (F2) and 261 F1 × DBA/2 As an initial step towards performing a genome scan to backcross (BC) animals to study the genetic basis for this identify the locations of QTLs affecting CD4:CD8 ratios, phenotypic variance. The distribution of CD4:CD8 ratios we identified progeny in the phenotypic extremes of the among individual mice in these testcross populations is F2 and BC testcrosses. Previous theoretical and empirical studies have demonstrated that genotypic analyses of progeny from the phenotypic extremes in a segregating population provide the highest information content for linkage analysis.18 To our surprise, we found that pro- geny in the phenotypic extremes for CD4:CD8 ratios in LN samples were generally not the same as those based on a similar analysis of CD4:CD8 ratios in the thymus. For example, of the 40 F2 animals with the 20 highest or 20 lowest lymph node ratios, only four were also in these extremes in an analysis of the thymus. In contrast, the extremes in spleen and lymph node samples were very similar. This result suggested that CD4:CD8 in the thy- mus was segregating independently from this same phenotype in peripheral organs. To assess this possibility more formally, we tested the correlation of phenotypic segregation in both crosses for all three organs. As shown in Figure 2, the spleen and lymph node ratios were well correlated, while the thymus ratios did not correlate well with either peripheral organ (spleen vs thymus data not shown). The correlations of log CD4:CD8 ratios measured in the thymus and periphery (0.33 for thymus and lymph nodes, and 0.34 for thymus and spleen) were significantly smaller than the correlation of 0.63 between log ratios in the lymph node and spleen (z =−5.1, P  0.0001). Similar results were obtained in an analysis of the BC values. These results indicated that the ratios of CD4 to CD8 in the organ in which most ␣␤TCR+ T cells are generated, the thymus, do not necessarily predict ratios in the per- iphery, an observation that has been reported before.17 Consequently the CD4:CD8 values in the thymus and periphery are almost certainly controlled by different genes, at least in the strain combination studied here.

Linkage analysis of loci impacting CD4:CD8 ratios in the periphery Since CD4:CD8 ratios in the thymus segregated indepen- dently from this ratio in peripheral organs, it was neces- sary to identify testcross progeny in the phenotypic extremes for each organ separately. The spleen and lymph node values correlated well. Consequently, we Figure 1 Phenotypes of parental strains and crosses. (a) Lymph selected animals in the phenotypic extremes of the lymph node CD4:CD8 ratios from 4 to 8 week old parental mice and node values, since the phenotypic values were con- crosses. Distribution of lymph node (b) and thymic (c) CD4:CD8 ratios are normal. Bars undermeath graphs represent the pheno- sidered to be experimentally more robust (due to the typic extremes from which mice were selected for genotyping of higher concentration of T cells in lymph nodes vs spleen). BC (white bars) and F2 (black bars) testcrosses. The phenotypic distribution and mice selected for geno-

Genes and Immunity Linkage analysis in CD4:CD8 T cell subsets C Myrick et al 146 1), mapped to the telomeric end of c6 (peak marker D6Mit15 at 74 cM) and was found in the F2 cross (Figure 3b). Analysis of this locus with MAPMAKER.QTL indi- cated a QTL between D6MIT198 and D6MIT15 with a maximum LOD score of 4.6. As shown in Table 1, pro- geny with one or two DBA/2 alleles had higher average lymph node CD4:CD8 ratios than B6 allele homozygotes, consistent with the dominant inheritance of a DBA/2 allele mediating higher CD4:CD8 levels. This result sug- gests that the expression of this allele is impacted by epis- tatic interactions, given that (B6 × DBA/2)F1 animals express low CD4:CD8 ratios. As expected, Trmq1 was not detected in the BC analysis, because all these animals would have at least one copy of the DBA/2 allele. The second gene affecting lymph node ratios was found by analysis of BC mice. This gene, Trmq2, is located in a telomeric segment of c2. As shown in Figure 4, MAP- MAKER-QTL analysis of this region identified a QTL with a maximum LOD score of 3.41 within the interval delineated by D2MIT61 and D2MIT17. Animals hetero- zygous at this locus had a higher CD4:CD8 than animals homozygous for the DBA/2 allele indicating that the B6 allele of this gene mediates higher CD4:CD8 levels (Table 1). Given that B6 mice express a low CD4:CD8 ratio, these results indicate that Trmq2 is phenotypically suppressed by another gene or genes within the B6 background, again, indicative of strong epistatic interactions affecting the expression of this phenotype. Trmq2 was also seen weakly in the analyses of F2 mice, giving a peak LOD score near 2.0 (Figure 3a). Other immunologically important genes such as H2 on c17, TCR␣ on c14, and TCR␤ on c6 (Figures 3 and 4) were not associated with lymph node CD4:CD8 in these crosses. Trmq1 and Trmq2 were not significantly associated with CD4:CD8 ratios in the thymus. Similarly, TCR␣ and Figure 2 Lack of correlation between CD4:CD8 ratios in periphery TCR␤ showed no association with the thymic CD4:CD8 and thymus. Scatter plots of the CD4:CD8 values obtained in indi- (Figures 3 and 4). However, the H2 region on c17 was vidual backcross progeny from (a) T cell populations of thymus vs lymph node; and (b) T cell populations of spleen vs lymph node. associated with the ratio in BC animals at a suggestive The correlations of log CD4:CD8 ratios measured in the thymus level with a maximum LOD score of 2.43 (Figure 4d). and periphery (0.33 for thymus and lymph nodes, and 0.34 for thy- Analysis of this region with MAPMAKER-QTL identified mus and spleen) were significantly smaller than the correlation of a QTL locus within the genomic segment delineated by 0.63 between log ratios in the thymus and spleen (z =−5.1, P  D17MIT34 and D17MIT10. Heterozygous animals had a 0.0001). higher average ratio compared to animals homozygous for the DBA/2 allele (Table 1). This finding may reflect typic analysis among the F2 and BC testcross progeny for the increased numbers of MHC class II molecules that the lymph node phenotype and thymus phenotype are are expressed in B6 × DBA/2 heterozygotes, which will presented in Figure 1b and 1c. express heterodimeric subunit pairs of the A molecule, The genomes of these animals were scanned as pre- and E␤bE␣d molecule that is not found in either par- viously described19,20 using 84 microsatellite markers dis- ental strain. tributed on all 19 autosomes to provide coverage of Ͼ95% of the genome to within 15 cM of a tested polymor- Confirmation of Trmq1 phism. Linkage groups were assembled using MAP- To confirm the linkage of Trmq1 with CD4:CD8 ratios and MAKER-EXP and the locations of QTLs were sought for to assess the potency of this locus as a candidate for con- each of the phenotypes using both Bymarker and MAP- genic dissection, additional testcross animals. were bred MAKER-QTL as previously described.21 This analysis and the Trmq1 allele they were likely to contain was revealed significant or suggestive linkage for three auto- determined by typing them for a haplotype of markers somal loci. Figures 3 and 4 present the results of these spanning the Trmq1 interval on chromosome 6. These ani- scans for the relevant chromosomes together with results mals were then sacrificed and their lymph node CD4:CD8 for chromosomes 14 (TCR␣) and 6 (TCR␤), which have ratios were measured. As we had previously found, ani- been implicated in control of CD4:CD8 ratio by pre- mals with one or two copies of the DBA/2 allele at vious scans.14 D6Mit15 had a higher average CD4:CD8 ratio than B6 Two genomic segments significantly associated with homozygotes at this locus. This was confirmed by a two- CD4:CD8 ratios in peripheral organs were identified, sample T test, P Ͻ 0.002 (Table 2). based on criteria established by Lander and Kruglyak.22 Since Trmq1 is a dominant DBA/2 gene, the relation- The first, designated Trmq1 (for T cell ratio modifier QTL ship between Trmq1 allele and CD4:CD8 could also be

Genes and Immunity Linkage analysis in CD4:CD8 T cell subsets C Myrick et al 147

Figure 3 Linkage associations detected in a genome scan of (B6 × DBA/2)F2 progeny. LOD score values for CD4:CD8 ratio generated with MapMaker/QTL are plotted on relevant chromosomes from centromere (left) to telomere (right). Solid line at LOD = 4.3 depicts significant association and dashed line at 2.8 depicts suggestive thresholds (from Lander and Kruglyak22). Shown below each plot are polymorphic microsatellite markers used to scan each chromosome and their distances from the centromere. The 95% confidence interval for Trmq1 (b) is shown as a darkened arrow. TCR␣, TCR␤, and H2 are shown relative to markers typed. All other chromosomes showed no suggestive or significant associations with CD4:CD8 ratios. tested in the opposite BC (F1 × B6). Such animals were predominantly influenced by distinct genetic and mol- therefore bred, typed for D6MIT15 allele and their lymph ecular mechanisms, an idea which was confirmed by the node CD4:CD8 ratios were determined. The association observed differences in linkage results. In this regard, the of Trmq1 with CD4:CD8 ratios was less robust in this independent segregation of the CD4:CD8 ratio of thymic testcross, although the trend was in the appropriate precursors in comparison to that of mature T cells in the direction (P Ͻ 0.05, Table 2). This may reflect the weaker periphery indicates that significant compensatory adjust- impact of a single allele DBA/2 allele on the phenotype ments in the sizes of these T cell subsets are made as when bred into the B6 genome. Taken together, these these subsets expand and populate the peripheral lym- data confirm the detection of Trmq1 as a locus impacting phatics. CD4:CD8 ratios in the C57BL/6 and DBA/2 combination. Several previous reports have analyzed the genetic fac- tors that control CD4:CD8 ratios in mice. Sim et al14 Discussion showed that the ratios of these cells are affected in the thymus by genes mapping to the TCR␣ locus. Several These findings provide some interesting insights into recent publications have shown that the size of the CD8+ processes regulating the homeostasis of CD4 and CD8 T population in the thymus can be increased by increased cell subsets in mice. A comparison of these subsets in B6 expression of proteins thought to improve and DBA/2 mice clearly indicates a strong genetic survival such as notch23 and bcl-2.24 In a similar vein, component influences their prevalence in both thymic T overexpression of –1orB7–2 decreases the CD4:CD8 cell precursor populations and in mature T cells in the ratio, both in the thymus and periphery and conversely, periphery. The poor correlation of the CD4:CD8 ratios the CD4:CD8 ratio is increased in B7–1/B7–2 double between these two populations suggests that they are knock-out mice.25 The authors of this study show that the

Genes and Immunity Linkage analysis in CD4:CD8 T cell subsets C Myrick et al 148

Figure 4 Linkage associations detected in a genome scan of (DBA/2 × B6)F1 × DBA/2 progeny. LOD score values for CD4:CD8 ratio generated with MapMaker/QTL are plotted on relevant chromosomes from centromere (left) to telomere (right). Solid line at LOD = 3.3 depicts significant and dashed line at 1.9 depicts suggestive thresholds (from Lander and Kruglyak22). The 95% confidence interval for Trmq2 (a) is shown as a darkened bar. All other chromosomes showed no suggestive or significant associations with CD4:CD8 ratios.

effects of B7 require CD28 on the T cells involved and phenotypes mediated by these allelic intervals is purely suggest that the effects might be mediated through CD28 conjectural at this stage. In our experience, serious pos- induction of Bcl-2 or Bcl-xl. In this regard, it is interesting itional candidate testing should not be initiated in inter- that Bcl-xl maps to , although to a region vals that are larger than about 2 cM. that is somewhat distal to that identified in the mapping Finally, we have used a simple confirmation test to described here. validate the impact of Trmq1 on CD4:CD8 ratios. This test Consistent with the detection of a strong genetic was performed on test progeny produced at the component in the phenotypic variance between B6 and University of Florida, while the original mapping study DBA/2, linkage analyses detected three robust QTLs via was performed with mice produced at the University of a genome scan of less than 100 progeny derived from the Colorado. Although the CD4:CD8 ratio values detected phenotypic extremes in two testcrosses. The positions of differed somewhat in the two colonies, essentially the the two named loci, Trmq1 and Trmq2, are presented in same results were obtained. Figure 5. As with all QTLs, the support intervals ident- This confirmation test also allows an initial assessment ifying their positions are large in the initial stage of analy- of the feasibility of utilizing congenic dissection to further sis and, as a result, a variety of genes of immunologic characterize Trmq1. Since the production of congenic import are located within the 95% support intervals of strains is a time-consuming and costly enterprise, data these two QTLs. The relevance of these genes to the supporting the potential value of this approach for the

Genes and Immunity Linkage analysis in CD4:CD8 T cell subsets C Myrick et al 149 Table 1 Mode of inheritance of Trmq1 and Trmq2

Cross Organ Locus CM Mean ± s.d. of LN CD4:CD8 LOD Inheritance of ‘High Allele’ BB BD DD

F2 LN D6Mit135 63 1.5 ± 1.0 3.0 ± 1.2 3.1 ± 1.3 1.2 D6Mit198 67 1.5 + 1.0 3.1 + 1.0 3.3 + 1.3 4.1 DBA/2 dominant D6Mit15 74 1.5 ± 1.0 2.9 ± 1.1 3.5 ± 1.2 4.6 BC LN D2Mit37 45 3.6 ± 1.5 2.6 ± 1.3 1.3 D2Mit483 53 3.8 ± 1.4 2.4 ± 1.2 3.4 D2Mit207 60 3.8 ± 1.4 2.4 ± 1.2 3.4 B6 dominant D2Mit17 69 3.8 ± 1.5 2.4 ± 1.1 3.0 D2Mit21 80 3.6 ± 1.5 2.5 ± 1.3 1.7 Thy D17Mit34 18 8.8 ± 4.1 5.4 ± 3.9 1.6 D17Mit10 24 8.8 ± 4.3 4.8 ± 3.2 2.3 B6 dominant D17Mit20 30 8.5 ± 4.4 5.2 ± 3.5 1.4

Significant LOD scores are in bold.

Table 2 Genotype-phenotype assessment confirms Trmq1 were used for analysis. were gated using forward and side scatter and 105 lymphocytes were coun- Cross Mean LN CD4:CD8 ± s.d. (n) P ted. TCR+ staining was used to create the second gate. From the TCR+ population, CD4+ and CD8+ cells were BB BD DD gated. The percentage of CD4+ cells was divided by the percentage of CD8+ cells to determine the CD4:CD8 ratio. (B × D) F2 1.3 ± 0.1 (8) 1.5 ± 0.2 (7) 1.6 ± 0.2 (8) 0.002* (B × D) × B 1.5 ± 0.3 (14) 1.6 ± 0.2 (15) — 0.05 Genetic mapping and statistical analysis *B6 homozygotes compared to DBA/2 homozygotes. DNA was extracted from liver for genotyping. SSCP loci were selected from microsatellite loci identified as being polymorphic (http:www-genome.wi.mit.edu) between analysis of a specific phenotype early in the process is of C57Bl/6 and DBA/2 and primers were obtained from significant valuable. Our results with Trmq1 support the Research Genetics (Huntsville, AL, USA). Marker pos- feasibility of producing an appropriate congenic strain as itions were obtained from The Jackson Laboratory Mouse a strategy to characterize its individual phenotype. Genome Database (http://www.informatics.jax.org). Finally, the testcross progeny groups produced via geno- Standard PCR was performed at experimentally determ- type-phenotype assessment could also be used for a com- ined optimal annealing temperature for each primer pair. parison of gene expression profiles in the affected target PCR products were visualized with EtBr on 5% agarose tissue. This might provide interesting preliminary infor- gels. Data were analyzed using MapMaker/QTL, mation about the molecular mechanisms mediating the Bymarker, and two-sample t-tests. Criteria for declaring a significant linkage association in a mapping cross fol- phenotype, especially if powerful positional candidates 22 were present within the relevant intervals. lowed guidelines suggested by Lander and Krugylak.

Materials and methods Linkage confirmation test Lymph nodes (inguinal) from 4 to 8 week old animals were used to make single cell suspensions, and 1.5 × 106 Mice cells were triple-stained with CD3-biotin, CD4-FITC, C57Bl/6 and DBA/2 were obtained from The Jackson CD8␣-PE antibodies obtained from Pharmingen (San Laboratory (Bar Harbor, ME, USA). Animals used in the Diego, CA, USA). Non-specific staining was blocked with mapping crosses were bred and maintained in SPF 10% horse serum diluted in FACS buffer (5% horse conditions at the National Jewish Center, Denver, CO. serum, 1% NaAzide in PBS) for 20 min. Directly conju- Animals for the genotype-phenotype assessment experi- gated antibodies were added to blocking buffer for ments were housed in a conventional colony at the Uni- 30 min on ice followed by three washes in FACS buffer. versity of Florida, Gainesville, FL. Staining was repeated with avidin-quantum red. After the last wash, cells were fixed with 1% formaldehyde (in CD4:CD8 ratios PBS) and analysis was performed on FACSort (Becton- Thymus, spleen, and the combined inguinal, axillary, and Dickinson). Analysis of stained cells was carried out as brachial lymph nodes from 4 to 8 week old animals were × 6 above, but CD3 staining instead of TCR staining was used to make single cell suspensions. Cells (1 10 ) were used to create the second gate. suspended in normal mouse serum and 2.4G2 solution for FcR and non-specific blocking. TCR-biotin was added to blocking solution for 30 min. After three washes, Acknowledgements CD8␣-FITC, CD4-PE, and strep-avidin cytochrome were incubated with cells for 30 min followed by three washes. The authors would like to thank Ms Kushnir and Gajen- FACScan and FACS Calibur (Becton-Dickinson, USA) dra Tulsian for their help with animal care.

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