Differential CD4/CD8 subset-specific expression of highly homologous rat Tcrb-V8 family members suggests a role of CDR2 and/or CDR4 (HV4) in MHC class-specific thymic selection

Thomas Herrmann, Kathrin Hofmann, Nora E. Torres Nagel, Anne Asmuß, Thomas Hünig and Kurt Wonigeit1

Institut für Virologie und Immunobiologie, Julius-Maximillians-Universität Würzburg, Versbacherstrasse 7, 97078 Würzburg, Germany
1 Klinik für Abdominal- und Transplantationschirurgie, Medizinische Hochschule Hannover, 30625 Hannover, Germany

Correspondence to: T. Herrmann fax: +49-931-201-2243, e-mail: herrmann-t{at}vim.uni-wuerzburg.de


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Different rat Tcrb haplotypes express either TCR ß variable segment (Tcrb-V) 8.2l or 8.4a. Both V segments bind the mAb R78 but differ by one conservative substitution (L14V) and clusters of two and four substitutions in the complementarity-determining region (CDR) 2 and CDR4 [hypervariable loop 4 (HV4)]. Independently of MHC alleles numbers of R78+CD4+ cells are lower in Tcrb-V8.2l-expressing than in Tcrb-V8.4a-expressing strains. Expression of R78+ TCR during T cell development, analysis of backcross populations and generation of a Tcrb congenic strain [LEW.TCRB(AS)] define two mechanisms how Tcrb haplotypes affect the frequency of R78+ cells, one acting prior to thymic selection leading to up to 2-fold higher frequency of Tcrb-V8.4a versus Tcrb-V8.2l in unselected thymocytes and another occurring between the TCRlow and the CD4/CD8 single-positive stage. The latter leads to a 50% reduction of frequency of Tcrb-V8.4a CD8+ cells but not CD4+ cells and does not affect either subset of Tcrb-V8.2l cells. A comparison of rat classical class I MHC (RT1.A) sequences and current models of TCR–MHC–peptide interaction suggests that this reduction in frequency of Tcrb-V8.4a CD8 cells may be a consequence of differential selection of Tcrb-V8.2l versus Tcrb-V8.4a TCR by differential binding of CDR2ß to highly conserved areas of C-terminal parts of the {alpha} helices of class I MHC molecules.

Keywords: complementarity-determining region, MHC, polymorphism, superantigens, TCR, T cell repertoire, V segments


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
{alpha}ßTCR of theoretically up to 1015 different specificities can be generated by recombination of V{alpha}/J{alpha} and Vß/Dß/Jß segments, insertion of N and P nucleotides, and combination of different TCR {alpha} and ß chains (1). These randomly generated specificities are subject to selection during thymocyte development. This selection generates a repertoire of TCR preferentially binding to complexes of foreign peptides and self MHC molecules (24). The binding of the TCR and its co-receptors CD4 and CD8 to intrathymically expressed complexes of MHC and peptides stops further rearrangement of TCR genes and determines thymic selection. The differentiation (positive selection) to mature T cells requires TCR–MHC–peptide interaction of appropriate strength (either affinity or avidity) while lack of such binding results in `death by negligence or ignorance'. Thymocytes with potentially autoreactive TCR of high affinity for self molecules are deleted by induction of apoptosis (negative selection) (35). At the level of thymocyte populations, selection by MHC starts with cortical thymocytes expressing low levels of TCR but high levels of both CD4 and CD8, and ends with medullary thymocytes which express either CD4 or CD8 and high levels of TCR. Consequently, differences in V segment usage between these immature (TCRlow) and mature CD4+ or CD8+ TCRhigh thymocytes or CD4+ and CD8+ peripheral lymphocytes can be expected to be a result of thymic selection and to reflect strength of binding of TCR comprising these V segments to their respective intrathymic ligands. These ligands can be either MHC–peptide complexes or endogenous superantigens (sAg) (3,4).

Effects of MHC on V segment usage of peripheral CD4+ and CD8+ cells have been described for mice, rats and humans, and support a concept of an `intrinsic' affinity of germline-encoded V{alpha} and Vß segments for MHC as its natural ligand. Bias in V segment usage can be determined by MHC class or MHC isotypes, such as in the case of preferential expression of V{alpha}3.1. and V{alpha}3.2 in mouse CD4+ and CD8+ (6) cells or of Vß6+CD4+ mouse cells selected by H2E (7) but can also be specific for alleles of certain MHC molecules as in the case of positive selection of rat Tcra-V8.2+CD8+cells by RT1.Af (8). Endogenous sAg shape thymic generation of the Tcrb-V repertoire as it has been demonstrated for mtv-encoded sAg (mtv-sAg) in mice (9) but the recent finding of a human retroviral sAg (10) increases the probability of such a mechanism acting in other species as well. The TCR–sAg interaction of developing thymocytes usually leads to negative selection of CD4+ and CD8+ cells but this deletion is sometimes more efficient for CD4+ than for CD8+ cells and even positive selection by mtv-sAg may occur (reviewed in 9,11).

At the molecular level many functional as well as an increasing number of crystallographic studies give the following picture of TCR–ligand interaction (1217). Germline encoded V{alpha} and Vß segments bear complementarity-determining regions (CDR) 1 and 2, and V{alpha}J{alpha} and VßJßDß transitions form the CDR3 which bind to the peptide–MHC complex. CDR3ß, CDR1ß and all CDR of the TCR {alpha} chain contact the MHC molecule. Binding to peptide occurs also by all CDR of the {alpha} chain, and by CDR1 and CDR3 of the ß chain. Contacts of CDR2 of the ß chain with the MHC or peptide have not been found in the first TCR-A6–HLA-A2–peptide crystal (13) but binding to the {alpha}1 helix of the MHC molecule occurs in other TCR–class I MHC complexes (14,16,17). Binding to sAg involves the CDR1 and CDR2 (18,19) and the hypervariable loop 4 (HV4) of the ß chain, also referred to as CDR4 (2022).

So far the most detailed information on TCR regions involved in Tcr-V-specific positive selection stem from investigation of an opposite subset preference of V{alpha}3.2 and V{alpha}3.1 bearing mouse cells (23). Both V segments differ in their CDR1 and CDR2 and analysis of mice expressing mutated V{alpha}3 segments mapped these areas as being important for MHC class-specific positive selection. In rat, the importance of germline-encoded V{alpha} segments for MHC restriction is supported by studies on RT1f-driven overselection and sequence analysis of Tcra-V8.2+TCR (8, 24) used in a RT1.Af-directed alloresponse.

Here we describe genetic factors controlling the frequency of expression by CD4+ and CD8+ cells of alleles of two highly homologous Tcrb-V segments. These V segments (Tcrb-V8.2l and Tcrb-V8.4a) are characterized as follows. Both bind to the same mAb (R78) but are expressed by different Tcrb haplotypes. Tcrb-V8.2l is expressed by R78+ cells of Tcrbl strains (e.g. LEW, WF, BN and BH) and responds to the sAg staphylococcal enterotoxin B (SEB). Tcrb-V8.4a is also found in R78+ cells but is SEB unresponsive and is part of the Tcrb1 haplotype (e.g. DA and F344) and of related variant haplotypes (Tcrbav1 and Tcrbav2) as described in this paper. The regions controlling the differential SEB response of the R78+ cells have been mapped to clusters of two and four substitutions in the CDR2 and CDR4 (HV4) respectively (25,26). Here we define mechanisms which control the frequency and CD4/CD8-specific expression of Tcrb-V8.2l and Tcrb-V8.4a by analysis of expression of R78+ TCR at different stages of T cell development and backcross studies. We propose that the structural differences in the CDR2 and/or CDR4 of both V segments contribute to differential maturation of MHC class I- and class II-restricted cells.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Animals
Young adult female AUG, AGUS, BDII, DA, F344, PVG and WF rats were purchased from Harlan Winkelmann (Borchem, Germany). LEW rats were obtained from our own animal facilities at the Institut für Virologie und Immunbiologie, Würzburg (breeding pairs were from the Zentralinstitut für Versuchstierkunde, Hannover, Germany and Charles River Deutschland, Sulzfeld, Germany). AS, BH, LEW.1U and LEW.1A rats were from the animal facilities of the Medical School Hannover, Hannover. LEW.TCRB(AS) were generated by serial backcross onto LEW background for six generations and subsequent intercross. Offspring was selected for high numbers of R78+ CD4 cells. The determination of the Tcrbav2 haplotype is described in the Results section of this paper (Fig. 2Go).



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Fig. 2. PCR-RFLP and Southern blot determine Tcrb-C1 alleles and Tcrb haplotypes. (A) Parts of the 3'-untranslated region of Tcrb-C1 were amplified by PCR and MspI-digested fragments were analyzed on a 1% agarose gel. Names of analyzed rat strains are indicated. (B) EcoRI-digested genomic DNA of indicated rat strains was separated on an 0.8% agarose gel and probed with Tcrb-C. For the left gel mol. wt markers run in parallel are shown. F344 and DA belong to the Tcrba haplotype, AGUS represents a variant of this haplotype (Tcrbav1). The Tcrb haplotype of AS and LEW.TCRB(AS) represents another variant of Tcrba designated Tcrbav2..

 
Antibodies
mAb of the following specificities were used: R73; rat {alpha}ßTCR (27), R78; rat Tcrb-V8.2 (28), G101; rat Tcrb-V10 (28), His 42; rat Tcrb-V16 (28, 29), G177; rat Tcra-V8.2, W3/25; rat CD4 (30), OX8 rat CD8{alpha} (30), 3.4.1; rat CD8{alpha}ß (31), RT1.Al; 5F3A (32) and isotype-matched control antibodies. Purification by Protein A–Sepharose and biotinylation and FITC labeling followed standard procedures. Phycoerythrin (PE) conjugates (anti-CD4) and some preparations of FITC- and biotin-labeled mAb were obtained from PharMingen (San Diego, CA). Polyclonal antibodies used were donkey anti-mouse Ig (DaMIg; Dako Diagnostika, Hamburg, Germany)–PE, sheep anti-mouse Ig and normal mouse Ig (Sigma, Deisenhofen, Germany).

Immunofluorescence and flow cytometry
Lymphocytes were characterized by two- or three-color immunofluorescence and flow cytometry. Briefly, cells were stained indirectly using a primary mouse mAb and PE-labeled secondary DaMIg, followed by another primary mouse mAb labeled with FITC. For a third color biotin-labeled mAb were added and revealed by streptavidin 670 (Life Technologies, Eggerstein, Germany). In some cases PE-conjugated primary mAb were taken. To avoid capture of primary mAb by PE-labeled secondary DaMIg, normal mouse Ig was used in excess to block potential free binding capacity on DaMIg. Samples were analyzed on a FACScan flow cytometer (Becton Dickinson, Sunnyvale, CA) with the use of Lysys II or CellQuest software. Light scatter gates were set to include all viable nucleated cells.

Cells
Suspensions of lymph node and thymocytes cells were prepared as previously described (33). Peripheral blood was hemolysed for flow cytometry, for culture experiments peripheral blood leukocytes were obtained by taking heparinized blood from the tail vein and subsequent purification with Ficoll-Hypaque (Pharmacia, Freiburg, Germany) according to the manufacturer's instructions. P3/2 cells are murine DAP-3 fibroblasts stably expressing DR1 (34) and kindly provided by Dr R.-P. Sekaly (Clinical Research Institute, Montreal, Canada). R78+ T cell hybridomas were generated from concanavalin A-stimulated lymph node cells (25).

Cell culture, SEB and stimulation by mAb R78
Cell culture was performed at 37°C in 5% CO2 and humidified atmosphere in RPMI 1640, supplemented as described (33). SEB stimulation and analysis of stimulated cells was performed as previously described (25). P3/2 cells were seeded at 5x104 cells/cm2 in culture flasks and treated for 45 min with mitomycin C (50 µg/ml; Sigma) in situ 18h later. Free mitomycin C was removed by three washes with BSS/0.1% BSA and 106 lymph node cells/ml complete RPMI plus 200U/ml IL-2 and SEB (supernatant of recombinant SEB, 1:100) were added. Stimulation with SEB lasted 3–4 days, subsequently cells were resuspended in fresh culture medium supplemented with 200 U IL-2/ml and analyzed 1 day later by immunofluorescence and flow cytometry. In a typical experiment 50–80% of resulting cells are activated by the criteria of blastoid transformation as determined by light scatter characteristics and frequency of an irrelevant Tcrb-V segment (Tcrb-V16 stained by mAb HIS42) was reduced by a minimum 50% (25). Tcrb-V and CD4 expression of blast cells was analyzed by immunofluorescence and flow cytometry as described (25).

Nucleic acid isolation
RNA was obtained by homogenization of the cells in guanidine thiocyanate and pelleting the RNA through a CsCl cushion according to (35). Genomic DNA was isolated from tail, ear or liver by digestion in 700 µl TENS buffer (50 mM Tris, 0.1 M Na–EDTA, 0.1 M NaCl and 1% SDS) with 30 µl Proteinase K (Sigma; 20 mg/ml, 56°C, over night) followed by a digestion with 35 µl RNase A (10 mg/ml; Sigma) for 60 min at 37°C. After mixing 250 µl of saturated 6 M NaCl were added, the probe centrifuged (7 min, 14,000 r.p.m.) and the supernatant recovered. The DNA was precipitated with 1 volume isopropanol and recovered by coiling, washed with 70% ethanol, and resolved in TE buffer (1 mM Tris and 0.1 mM EDTA).

cDNA synthesis, PCR, Tcrb-C1 typing and sequencing
Single-stranded cDNA was synthesized by using Superscript II (Gibco/BLR, Eggenstein, Germany), RNasin (Promega, Feinbiochemikalien, Heidelberg, Germany) and oligo(dT)15 (Promega) according to the manufacturer's instructions. Subsequently, PCR was performed on a Perkin Elmer Thermocycler 480 following the Boehringer Mannheim PCR protocol (Boehringer Mannheim, Mannheim, Germany) modified to achieve a final MgCl2 concentration of 1.5 mM. PCR consisted of 30 cycles of 94°C (1 min), primer specific annealing temperature (1 min) and 72°C (1 min plus 1 s elongation for each successive cycle). After completing the cycles the probes were exposed to 72°C for 10 min. For amplifying Tcrb-V8.2/8.4 from cDNA of R78+ T cell hybridomas Tcrb-V8.2-specific primer (5'-GGT GAC ATT AAA AGG AGG AAA GGT G) and internal Tcrb-C primer (5'-TGT TTG TCT GCG ATC TCT GC) were used at 60°C annealing temperature.

The 3'-untranslated region from genomic DNA was amplified with Tcrb-C1 sense (5'-AGTGCTCTGGGGGGACAGCAGC) and antisense primer (5'-CTACTCCACAATGGCTGCAGCG). All PCR products were purified by QIAquick Spin PCR purification kit (Quiagen, Hilden, Germany). For Tcrb-C1 typing purified PCR products were digested by MspI. Sequencing was either performed as described (25) by dideoxynucleotide termination method using a T7 sequencing kit (Pharmacia) and TdT to extend unspecifically terminated strands or with the ABI PRISM dye terminator cycle sequencing ready reaction kit (Perkin Elmer) and an ABI 310 analyser.

Southern blot analysis
Genomic DNA was digested with EcoRI and separated on a 0.8% agarose gel running a 1 kb ladder (Stratagene, Heidelberg, Germany) in parallel. Transfer was done by alkali blotting onto Hybond-N+ membrane (Amersham-Buchler, Braunschweig, Germany) according to manufacturer's instructions. The Tcrb-C probe was isolated by NotI–BamHI digestion from a Tcrb-C comprising pCRScript plasmid kindly provided by G. Giegerich, Neurology Department, University of Wuerzburg. Labeling was performed with Multiprime DNA labeling system with [32P]dCTP (Amersham-Buchler). Free label was removed by G-50 Sephadex. Hybridization was performed with QuickHyb Hybridization solution (Stratagene, Heidelberg, Germany) with 30 min prehybridization and 2 h hybridization at 68°C. Subsequently the membrane was washed 3–4 times with 0.2xSSC and 0.1% SDS and analyzed by autoradiography.

Generation of R78+ cells and RNase protection assay with Tcrb-V8.2/8.4 specific probes
R78+ T cells were enriched and stimulated with immobilized mAb R78 by panning exactly as described in (26). Nearly all cells were blasts, and >90% were R78+ as analyzed by immunofluorescence and flow cytometry. RNA was tested for expression of Tcrb-V8.2/8.4 genes with an RNase protection assay using Tcrb-V8.2l-, Tcrb-V8.2a- and Tcrb-V8.4a/l-specific probes, and a Tcrb-C probe for calibration.


    Results
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 Abstract
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 Methods
 Results
 Discussion
 References
 
Frequency of R78+CD4+ cells correlates with allelic differences of the Tcrb but not of MHC restriction elements
Peripheral CD4 and CD8 lymph node cells of various rat inbred strains were analyzed by two-color immunoflow cytometry for the frequency of expression of the R78 epitope. Figure 1Go shows pronounced strain-specific variation of frequency of R78+CD4+ cells and to some extent of R78+CD8+ cells. These differences occur among strains sharing identical class I and class II restriction elements, suggesting independence of the MHC (shown in Fig. 1Go for three different MHC haplotypes).



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Fig. 1. Increased frequency of R78+(CD4+) cells correlates with Tcrb irrespective of MHC type. R78+CD4+ or CD8+ cells of rat strains with indicated alleles of MHC loci RT1A and RT1B/D were analyzed by two-color flow cytometry of lymph node cells. Values represent the frequency of R78+ cells of {alpha}ßTCR+CD4+ and CD8+ cells respectively. An asterisk indicates strains sharing the Tcrb-C1 allele of LEW. Error bars give SD of analysis of three to five animals.

 
A possible explanation for the variation observed could be a strain-specific structural variation among Tcrb-V used by R78+ cells. Such a variation is well documented for R78+ cells of LEW and DA (26). R78+ cells of LEW bear Tcrb-V8.2l TCR and respond to SEB while R78+ cells of DA express the SEB unresponsive Tcrb-V8.4a TCR. This strain-specific difference in the SEB response extends to other strains (25,36), and correlates with a polymorphism revealed by RNase protection (25) on assay with Tcrb-V8.2-specific probes and the Tcrb haplotype (Tcrbl for SEB responders: LEW, WF, PVG and BN; Tcrba for SEB non-responders: DA and F344) (22,32). Therefore we used differences in the SEB response of Tcrb-V8.2l and Tcrb-V8.4a TCR as a first read out for a possible variation of the R78+ Tcrb-V segments among the strains studied. The SEB response was determined by measuring the frequency of R78+ cells among CD4+ blast cells after 4 days co-culture of peripheral lymph node cells with recombinant SEB presented by HLA-DR1 transfected mouse fibroblasts. The use of these cells as antigen-presenting cells (APC) diminishes possible effects of allelic differences between rat class II MHC on SEB presentation and allows direct comparison of the T cell response of rats of various RT1 haplotypes (25). The results summarized in table 1Go define strains with SEB-responsive R78+ cells (LEW, WF and BH) with a 4- to 8-fold increase of frequency of R78+CD4+ cells and strains which lack such a response, which leads to reduced frequencies of R78+CD4+ blasts due to activation of R78 T cells (DA, F344, AS, AGUS and BDII). Interestingly, the frequency of R78+CD4+ cells in the starting population was lower in responder than in non-responder strains (Fig. 1Go) suggesting that structural differences between R78+ V segments might control both the frequency of R78+ cells in the naive repertoire as well as their sAg response.


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Table 1. R78+ cells and Tcrb genes
 
R78+ cells of strains carrying Tcrba and related variant Tcrb haplotypes express Tcrb-V8.4a
Direct evidence for the expression of different R78-reactive Tcrb-V segments in sAg responder and non-responder strains was obtained by generating R78+ T cell hybridomas of AS, AGUS and BDII rats and analyzing their Tcrb-V. Two hybridomas per strain were tested. Tcrb-V segments were amplified by RT-PCR and directly sequenced. All three strains express the Tcrb-V8.4a sequence previously described for DA and F344 (25). Thus in agreement with previous findings for DA and F344 (25) Tcrb-V8.4a is expressed in R78+ cells of strains with SEB-unresponsive R78+ cells (DA, F344, AS, AGUS and BDII), in contrast to SEB-responding R78+ cells of LEW and WF which express Tcrb-V8.2l (25,26). In case of AS rats the exclusive expression of Tcrb-V8.4a by R78+ cells was also tested by RNase protection assays using RNA isolated from a polyclonal population of activated R78+ cells to protect Tcrb-V8.2l-, Tcrb-V8.2a- and Tcrb-V8.4a-specific probes. The picture obtained was essentially the same as previously found for R78+ DA cells (26), i.e. protection of the Tcrb-V8.4a probe but no protection of the Tcrb-V8.2 probes (data not shown).

In order to investigate whether the differential expression of Tcrb-V8 family members correlates with Tcrb haplotypes, all strains were tested for a MspI restriction fragment length polymorphism (RFLP) in the 3'-untranslated region of the Tcrb-C1 gene. Figure 2(A)Go shows that all SEB non-responder strains share this restriction site, which is missing in SEB responder strains. Southern blot analysis of EcoRI-digested genomic DNA with a Tcrb-C probe allowed further differentiation of Tcrb alleles (Fig. 2bGo). Consistent with previous reports (3739) a 4.1 kb fragment was identified in LEW. Based on the Tcrb-C1 allele (38) (Fig. 2aGo) and response of R78+ cells to SEB LEW, WF and BH rats carry the Tcrbl haplotype. EcoRI fragments of 3.4 kb were identified for DA, F344 and AGUS (37,38,40). The latter shares a number of other RFLP with DA and F344 but differs by a PvuII RFLP (39 and data not shown) and represents together with AUG and LER a variant of Tcrba named Tcrbav (39,41). Southern blot analysis of BDII was not performed but based on the presence of the Tcrb-C1 allele and the missing SEB response it can be expected to carry a Tcrb haplotype more related to Tcrba than to Tcrbl. A 5.1 kb EcoRI RFLP of AS and LEW.TCRB(AS) has not been described previously, and their Tcrb allele is provisionally named Tcrbav2. Tcrbav2 is more similar to Tcrba(v1) than to Tcrbl by the criteria of Tcrb-V8.4a expression and presence of the Tcrb-C1 MspI site. The results of Tcrb typing, Tcrb-V8.4a expression and SEB response are summarized in Table 1Go.

Expression of Tcrb-V8.2l and Tcrb-V8.4a at different stages of T cell differentiation
Three Tcrb haplotypes were analyzed in greater detail [Tcrbl: LEW, Tcrba: DA, F344, Tcrbav2 AS, LEW.TCRB(AS)] in order to test parameters leading to differential subset preference of Tcrb-V8.2l versus Tcrb-V8.4a. Animals of the Tcrbl haplotype (LEW) express Tcrb-V8.2l in their R78+ cells while those of the Tcrba (DA and F344) and Tcrbav2 haplotype [AS and LEW.TCRB(AS)] express Tcrb-V8.4a. The developmental level at which control of the frequency of R78+ cells takes place was determined by analysis of R78+ cell frequency among TCRlow cells (mainly unselected thymocytes), CD4/CD8 single-positive thymocytes (mainly selected thymocytes) and peripheral T cells. Accordingly, proportions of mature R78+ thymocytes and R78+ peripheral CD4+ and CD8+ T cells were determined by three-color analysis using R78-, CD4-and CD8{alpha}-specific mAb as shown in Fig. 3Go. The proportion of immature R78+ TCRlow thymocytes was determined by two-color flow cytometry as exemplified in the same figure. In all cases the proportion of R78+ cells given were normalized against the overall frequency of the respective R73+ ({alpha}ßTCR+) cells.



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Fig. 3. Determination of the proportion of TCR+ cells which are R78+ during different stages of thymocyte development. Values given in Fig. 4Go were calculated from the type of analysis depicted in this figure. Proportion of TCR+ immature thymocytes expressing R78+TCR was determined by two-color flow cytometry using biotinylated R78 (Fl3) and FITC-labeled {alpha}ßTCR mAb R73 (Fl1) from numbers of cells found in the left gate of the upper left window. Frequency of R78+CD4+ or CD8+ thymocytes was determined by three-color flow cytometry from populations gated on R5 and R6. Values were normalized as percentage with total number of TCR+ within this gate cells. Frequency of CD4+ cells carrying the TCR was always >96%, but varied for CD8{alpha}+ cells between 50 and 75%. The data shown are obtained with LEW thymocytes.

 
The analysis of animals representing the three Tcrb haplotypes showed a high degree of reproducibility between individual animals of the same haplotype. Furthermore only small differences between the frequency of R78+ cells among single-positive CD4+ and CD8+ thymocytes compared to CD4+ and CD8+ cells isolated from peripheral lymphoid organs were found. The Tcrb haplotypes showed distinct patterns of expression of R78+ cells during T cell development. Preselection frequency of R78+ cells was lowest in Tcrb-V8.2l-expressing LEW rats (4.5–5%). Numbers were higher in Tcrb-V8.4a-expressing Tcrb haplotypes but still varied between these Tcrb haplotypes. AS and LEW.TCRB(AS) (Trcbav2) had a proportion of 10–11% R78+ immature TCR+ thymocytes, compared to 6.5–7% expressed by DA and F344 (both Tcrba). The patterns of expression during different stages of thymocyte maturation allow us to separate the tested Tcrb haplotypes in two groups. One comprises the Tcrb-V8.2l-expressing LEW rats with similar frequencies of R78+ TCRlow cells, CD4+ thymocytes, CD8+ thymocytes, CD4+ peripheral T cells and CD8+ peripheral T cells. The second group comprising the Tcrb-V8.4a-expressing haplotypes (Tcrba and Tcrbav2) shows a reduction of the frequency of R78+CD8+ thymocytes and of R78+CD8+ peripheral T cells by ~50% compared to R78+ TCRlow cells and CD4+ thymocytes and peripheral CD4+ T cells.

These results suggest two mechanisms of intrathymic control of the frequency of R78+ cells, which both may be under control of the Tcrb. The first mechanism acts prior to thymic selection. It determines the total number of R78+ cells which enter thymic maturation and differs between single Tcrb haplotypes. The second mechanism correlates with expression of Tcrb-V8.2l by R78+ cells of Tcrbl rats (LEW) and of Tcrb-V8.4a by R78+ cells of Tcrba (DA and F344) and Tcrbav2 [AS and LEW(TCRB.AS)] rats. This mechanism affects cells between the TCRlow and the single-positive CD4+ and CD8+ stage of thymic maturation, and acts differentially on CD4+ and CD8+ cells. As discussed below, it is likely to reflect the structural differences between Tcrb-V8.2l and Tcrb-V8.4a.

Co-segregation of the controlling element for R78+ cell numbers and Tcrb haplotype in backcross populations
Thus far the hypothesis that the Tcrb haplotype has a major effect on the frequency of R78 cells and their distribution within the CD4 and CD8 compartment rests on two findings. (i) The association of R78+ cell numbers and Tcrb haplotype in a panel of inbred strains (Figs 1 and 2GoGo), and (ii) the results obtained for the newly derived strain LEW.TCRB(AS) (Figs 2 and 3GoGo). This strain has been generated by selecting animals with high R78+CD4+ T cell numbers in blood for further breeding during the backcrossing phase. A breeding scheme led to the transfer of the Tcrbav2 haplotype of AS onto a LEW background as shown by PCR and Southern blot analysis of the fully established strain (Fig. 2Go). It is unlikely that this strain differs from the LEW background in any other genetic region relevant for thymic selection except the Tcrb locus and closely adjacent regions. The name LEW.TCRB(AS) was assigned following standard nomenclature for congenic strains.

A third approach to demonstrate formally the decisive role of Tcrb haplotype for controlling the size and CD4/CD8 composition of the R78+ population in the T cell repertoire was the analysis of segregating populations. In this experiment effects on the CD4/CD8 ratio should be apparent for backcrosses of Tcrb haplotypes expressing different R78+ Tcrb-V segments such as the Tcrb-V8.2l- and Tcrb-V8.4a-expressing haplotypes Tcrbl and Tcrbav2. No effects on the CD4/CD8 ratio are expected for backcrosses of Tcrb haplotypes which express the same Tcrb-V segment in their R78+ cells (e.g. Tcrb-V8.4a in Tcrba and Tcrbav2 strains). Both possibilities were tested with backcrosses of LEW (Tcrbl), AS (Tcrbav2) and DA (Tcrba) (Fig. 5Go and Table 2Go).




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Fig. 5. Segregation analysis in two backcross populations. Panel a (left) LEWx(LEWxAS) animals. Panel b (right) (ASxDA)xDA animals. Results are presented separately for Tcrb homozygous and Tcrb heterozygous animals. For comparison the respective data of the parental strains and appropriate F1 hybrids are also given. Frequency of R78+CD4/CD8 cells was determined by two-color flow cytometry of lymph node cells (A) and mononuclear blood cells (B), and was normalized against frequency of {alpha}ßTCR+CD4+ or {alpha}ßTCR+CD8+ cells. av2/l and l/l designate Tcrb alleles in the LEWx(LEWxAS) backcross. a/a and av2/a designate the Tcrb alleles of the (ASxDA)xDA backcross. CD4/CD8 subset preferences of R78+ cells were calculated from the values of the upper graph and are given as proportion of R78+ cells of total CD8+ T cells. (55)

 

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Table 2. Analysis of backcrosses for correlation of frequency of R78+CD4+ and R78+CD8+ cells with alleles of Tcrb, Tcra and RT1
 
Figure 5(A)Go and Table 2Go show the results for 20 LEWx(LEWxAS) backcross rats and clearly demonstrate that animals typing positive for Tcrbav2 (typing was done by the MspI RFLP of Tcrb-C1 shown in Fig. 2Go) have higher numbers of R78+ cells in the CD4 and CD8 compartment. In agreement with the results obtained for the AS strain the difference is much stronger in the CD4 than in the CD8 compartment, leading to a significantly lower population of CD8 cells in the total R78+ population (38.0 ± 2% for the Tcrbav2/l animals versus 46.3 ± 2% for the Tcrbl/l animals). The results are very similar to those observed in F1(LEWxAS) hybrids and the parental LEW strain respectively (Fig. 5aGo).

The results in Fig. 3Go suggest that the different frequencies of R78+(CD4+) cells observed in different Tcrb-V8.4a-expressing strains (Tcrba: DA, F344: 7–8%, Tcrbav2 and Tcrbav1: AS and AGUS: 11–13%) are mainly due to Tcrb controlled determination of the number of R78+ cells before thymic selection starts. Such a control would be expected to lead to a co-segregation of the Tcrb haplotype and R78+ cell frequency without effect on the CD4/CD8 ratio. This hypothesis was tested in a population of 28 (ASxDA)xDA backcross animals which was typed for RT1, Tcra, and Tcrb haplotypes. The Tcrb haplotypes were determined by the EcoRI RFLP shown in Fig. 2Go. Tcra alleles were tested by staining with the mAb G177 detecting an epitope of Tcra-V8.2 present in LEW and AS rats but not in DA rats (8). RT1 alleles were typed with the RT1.Al-specific mAb 5F3A. The MHC and Tcra haplotype had no significant effect on the numbers of R78+ cells in the animals of this population (Table 2Go). This was in clear contrast to the Tcrb haplotype. Heterozygous a/av2 had a moderately but significantly higher number of R78+(CD4+) cells than homozygous a/a animals (Table 2Go). Again the numbers found for the two sets of hybrids closely resembled those found for F1 and parental DA-animals (Fig. 5Go), strongly suggesting control exclusively by the Tcrb haplotype. Between both groups no significant differences in CD4/CD8 subset bias were found (Tcrba/a, %CD8: 33.8 ± 3.3%; Tcrba/av2, 35.6 ± 5.2%). This further supports the hypothesis that the Tcrb controled differences in the frequency of R78+ cells among Tcrb-V8.4a-expressing strains appear at the preselection level of repertoire generation.

In summary, backcross analysis of Tcrb-V8.2l- or Tcrb-V8.4a-expressing LEW and AS rats demonstrates control by the Tcrb haplotype of the total frequency of R78+ cells as well as of strain-specific differences in CD4/CD8 subset preference of R78+ cells. In contrast, the backcross of DA and AS strains which differ in RT1, Tcra and Tcrb but which both express Tcrb-V8.4a reveals only a correlation between Tcrb haplotypes and the total frequency of R78+ cells but not with the CD4/CD8 ratio within this population.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Germline-encoded V segments contribute differentially to the affinity of TCR for certain MHC classes, isotypes or alleles. These differences lead to varying efficiencies in positive or sometimes also negative selection, which finally result in distinct CD4/CD8 subset preferences for given V segments. Recently, mutational analysis of mouse V{alpha} segments allowed to map CDR1{alpha} and CDR2{alpha} as being important for MHC class-specific selection (6). In this study we compared the highly homologous V segments Tcrb-V8.2l and Tcrb-V8.4a which both are recognized by the mAb R78 but differ in their reactivity for the sAg SEB as a consequence of amino acid substitutions in the CDR2 and CDR4 (HV4), and are expressed by rats of distinct Tcrb haplotypes; Tcrb-V8.2l by Tcrbl and Tcrb-V8.4a by Tcrba and related haplotypes. Both V segments were analyzed with respect to expression during thymocyte maturation and CD4/CD8 subset distribution, in order to test possible effects of structural differences between both V segments on MHC class specific selection.

The frequency of expression of R78+ Tcrb-V8.2l or Tcrb-V8.4a peripheral T cells was determined in different rat inbred strains and effects of Tcrb haplotype on the frequency of R78+ cells were found. Animals of two known (Tcrba/ Tcrbav1) and one new (Tcrbav2) Tcrb-V8.4a-expressing Tcrb haplotypes and of the Tcrb-V8.2l-expressing Tcrbl haplotype were compared. Tcrbl strains have a relatively low frequency of ~5% R78+ cells and show only a weak CD4 bias of R78+ cells, in contrast to three closely related Tcrb-V8.4a-expressing Tcrb haplotypes (Tcrba/Tcrbav1/Tcrbav2). These express higher numbers of (CD4+)R78+ cells than Tcrbl strains, suggesting effects of the Tcrb haplotypes on R78+ cell frequency and CD4/CD8 ratio of R78+ cells. A slight increase in frequency (5.5–6.5%) of R78+CD4+ was found in RT1u strains WF and LEW.1U but is likely to be a consequence of the known preferential MHC-specific `overselection' of Tcrb-V8.2l CD4+ cells by RT1.Bu/Du (8).

The first mechanism by which the Tcrb locus affects the frequency of expression of R78+ TCR acts before thymic selection. It becomes visible as different proportions of R78+ TCR+ cells in essentially unselected TCRlowCD4+CD8+ thymocytes of different Tcrb haplotypes and by examination of the R78+ cells in backcross studies of Tcrb-V8.4a-expressing Tcrb haplotypes, which varied in frequency of R78+ cells but showed no significant differences in the CD4/CD8 ratio. This variation in V segment usage of the unselected thymocytes results most likely from differences in frequencies of recombination of the respective V segments. One simple mechanism to increase probability of usage of a V segment is the reduction of the number of available V segments as a consequence of deletions in the Tcrb locus as it has been described for mice (42). This mechanism can be excluded by genetic analysis for the well-defined Tcrba and Tcrbl haplotypes (4345), and is also unlikely to apply for AS, AGUS and BDII, since they use other V segments such as Tcrb-V8.5 (or 8.3), 10 and 16 with similar frequencies as RT1-matched Tcrbl or Tcrba strains (8 and N. E. Torres Nagel et al., data not shown). Additionally, immature thymocytes of AS and LEW.TCRB(AS) rats show a slight decrease of Tcrb-V10+ T cells compared with other Tcrb haplotypes and not an increase which would be expected with a reduced number of Tcrb-V segments available for recombination (data not shown). An alternative explanation for different frequencies of V segment usage would be a polymorphism of the recognition signal sequences or of the spacer regions of the V segments as it has been proposed to explain different frequencies of expression of human BV3S2 alleles (46,47). This possibility could be tested in future experiments by determination of the sequences adjacent to Tcrb-V8.4a/Tcrb-V8.2l.

The relative frequency of expression by CD4+ and CD8+ cells differs for Tcrb-V8.4a and Tcrb-V8.2l TCR as can be concluded from co-segregation of CD4 subset bias and Tcrb-V8.4a-expressing haplotype. Analysis of R78+ TCR expression of immature TCRlow thymocytes, mature CD4+ or CD8+ thymocytes and peripheral T cells explains this bias as a consequence of inefficient maturation of Tcrb-V8.4a CD8+ thymocytes compared to Tcrb-V8.4a CD4+ or to Tcrb-V8.2l thymocytes, which could be due to either partial negative selection or to inefficient positive selection by class I MHC. Independently of the type of selection acting, the observed reduction of frequency of R78+CD8+ cells in Tcrba and related haplotypes points towards a differential interaction of Tcrb-V8.2l and Tcrb-V8.4a comprising TCR with class I MHC, since effects of allelic differences in Tcrb-J segments are not expected to act in the observed V-specific manner. A comparison of the primary structure of the V segments allows us to define candidate areas which may be important for MHC class-specific selection, especially since both V segments differ only by 7 amino acids, i.e. one conservative substitution (L14V) and clusters of two substitutions in the CDR2 and four substitutions in the CDR4 (HV4) (26). The L14V substitution is unlikely to play a role in thymic selection taken its distance from the TCR–MHC–peptide interface and the functional similarity of Tcrb-V8.2 alleles, which vary also by the L14V substitution but not in SEB recognition (26) or preferential usage in the RT1.Bl-restricted response to myelin basic protein (41). More likely to be important are the differences between Tcrb-V8.2l and Tcrb-V8.4a in CDR2 and/or CDR4 (HV4).

We see three possibilities how these differences could influence maturation of CD8+ thymocytes. The least likely one would be differential reactivity of both V segments for an endogenous sAg with the consequence of negative selection of Tcrb-V8.4aCD8+ but not CD4+ cells. So far no evidence for endogenous sAg or Mls-like loci exists for the rat (44), and more importantly negative selection by sAg usually affects both CD4+ and CD8+ cells (48) or predominantly CD4+ cells (49) but not CD8+ cells only. A second possibility would be interference of CDR2 or CDR4 with interaction of the TCR and its CD4 or CD8 co-receptors. Such a possibility cannot be ruled out, but preliminary reports on sites for co-receptor TCR interaction suggest only the possibility of interaction of TCR V{alpha} but not of the TCR Vß with CD4 or CD8 (50). We favor the third possibility, i.e. different strength of binding of the CDR2ß to class I MHC, to explain differential selection by class I MHC. Although CDR2ß contacts have not been reported for the TCR-A6–HLA-A2–peptide complex (13) they occur in other TCR–class I MHC complexes (14,16,17) where they interact with the central and/or the C-terminal part of the {alpha}1 helix of class I MHC. In addition to the TCR–MHC interaction found in the crystals, functional studies suggest the possibility of a 180° turn of the TCR with respect to the MHC and binding of the CDR2ß to the C-terminal part of the {alpha}2 helix (23). In both cases contacts of the CDR2ß with the MHC could result in differential binding to class I MHC of Tcrb-V8.2l versus Tcrb-V8.4a TCR. The lack of effects of RT1.A polymorphisms on Tcrb-V8.2l versus Tcrb-V8.4a CD8+ thymocyte frequencies is in line with conservation or identity of RT1.A alleles in these areas. Most residues of the C-terminal half of the {alpha} helices which are not covered by the peptide and thus provide possible contact points for CDR2ß (51) are identical ({alpha}1 helix: positions Q72, R75, V76, R79, R82 and G83; {alpha}2 helix: E161, G162, E166, R170 and E173) for tested RT1.A alleles [a, l and u (5254)]. Similar contacts of CDR2ß could also be envisaged for the {alpha}1 helix of class II MHC and may contribute to preferential selection by class II MHC such as in the case of enhanced positive selection of mouse Vß6 by H2E molecules (7). The proposed contribution of CDR2ß to class I MHC restriction and a not yet excluded involvement of the CDR4 (HV4) in Vß skewing can be tested by generation and analysis of animals expressing TCR with mutated Tcrb-V8 segments analogous to experiments performed for the CDR1{alpha} and CDR2{alpha} (6).

In conclusion it has been shown that a set of highly homologous Tcrb-V8 family members differ remarkably in their performance during intrathymic selection. These effects are found in the presence of different MHC haplotypes and thus appear not to depend on polymorphic MHC determinants. It is suggested that the relevant elements of different Tcrb-V8 members are structural differences of the CDR2ß and/or the CDR4 (HV4).



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Fig. 4. Expression of R78+ TCR during different stages of T cell development. Rat strains of different Tcrb haplotypes were analyzed as shown in Fig. 3Go. Tested rat strains and their Tcrb haplotypes are given in the figure. Analysis of lymph node cells was also performed by three-color flow cytometry as shown in Fig. 3Go for CD4+/CD8+ thymocytes. L.AS stands for LEW.TCRB(AS)

 

    Acknowledgments
 
This work is supported by Deutsche Forschungsgemeinschaft He 2346/1-2 + 1-3 and SFB 265. We thank Petra Buss and Astrid Dinkel for excellent technical assistance


    Abbreviations
 
CDRcomplementarity determining region
DaMdonkey anti-mouse
HV4hypervariable region 4
RT1rat MHC
sAgsuperantigen
SEBstaphylococcus enterotoxin B
RFLPrestriction fragment length polymorphism
TcraTCR {alpha} chain locus
TcrbTCR ß chain locus
Tcrb-VTCR ß chain variable segment
Tcrb-Vgene encoding Tcrb-V

    Notes
 
Transmitting editor: H. R. MacDonald

Received 14 August 1998, accepted 17 November 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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