HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sim, B.-C. Articles by Gascoigne, N. R. J. Search for Related Content PubMed PubMed Citation Articles by Sim, B.-C. Articles by Gascoigne, N. R. J.

Reciprocal Expression in CD4 or CD8 Subsets of Different Members of the V11 Gene Family Correlates with Sequence Polymorphism¹

Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Previous staining studies with TCR V11-specific mAbs showed that V11.1/11.2 (AV11S1 and S2) expression was selectively favored in the CD4⁺ peripheral T cell population. As this phenomenon was essentially independent of the MHC haplotype, it was suggested that AV11S1 and S2 TCRs exert a preference for recognition of class II MHC molecules. The V segment of the TCR -chain is suggested to have a primary role in shaping the T cell repertoire due to selection for class I or II molecules acting through the complementarity determining regions (CDR) 1 and CDR2 residues. We have analyzed the repertoire of V11 family members expressed in C57BL/6 mice and have identified a new member of this family; AV11S8. We show that, whereas AV11S1 and S2 are more frequent in CD4⁺ cells, AV11S3 and S8 are more frequent in CD8⁺ cells. The sequences in the CDR1 and CDR2 correlate with differential expression in CD4⁺ or CD8⁺ cells, a phenomenon that is also observed in BALB/c mice. With no apparent restriction in TCR J usage or CDR3 length in C57BL/6, these findings support the idea of V-dependent T cell repertoire selection through preferential recognition of MHC class I or class II molecules.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The TCR has the potential for enormous diversity. This arises from somatic rearrangement of multiple gene segments encoding TCR variable (V), diversity (D), and joining (J) domains with additional variability contributed by the removal and/or template-independent addition of nucleotides 1 . However, this diversity is limited by the fact that the majority of thymocytes die as the repertoire is shaped by positive and negative selection forces during development. Thymocytes bearing TCRs that recognize self-peptides bound to MHC molecules with a sufficiently high overall avidity are clonally deleted or functionally inactivated 2 . In contrast, T cells recognizing MHC molecules with a lower but nevertheless critical overall avidity are positively selected for maturation 2, 3 . With few exceptions, mature ß T lymphocytes in the periphery are either CD8⁺ cytotoxic T cells restricted to class I MHC molecules or CD4⁺ Th cells restricted to MHC class II molecules. It is evident that TCR specificity and MHC recognition are important determinants that will influence the choice between CD4 or CD8 T cell lineage. The skewed expression of TCR-ß transgenes always correlates with the transgenic receptor’s specificity for either MHC class I or class II molecules 2 . CD4⁺ cells were absent in MHC class II-deficient mice, whereas thymuses of mice that do not express ß₂-microglobulin protein and hence lack MHC class I protein contain very few mature CD8⁺ thymocytes 2 .

Staining with two independently developed V11.1/11.2-specific mAbs showed that their epitopes were strongly biased to expression in the CD4⁺ subset 4, 5 . This selective influence was not very sensitive to differences in MHC alleles. In comparing numerous mouse strains, it was found that the percentage of V11.1/11.2-expressing T cells in the CD4⁺ subset was higher in I-E⁺ relative to I-E^- strains 4, 6 . This and the overall bias toward CD4⁺ cells led to the postulation that V11.1/11.2 TCR favored interaction with MHC class II molecules and that V usage in general can differentially influence the selection of T cells into the CD4⁺ or CD8⁺ subsets (Refs. 4 and 5 and reviewed in 7 . In support of this, all other TCR V elements analyzed to date, inclusive of members of the mouse V2 8 , V3 9, 10, 11 , and V8 12 families, as well as rat V8 13, 14 and human V12 15 , are also observed to be preferentially expressed by either CD4 or CD8 T cells irrespective of MHC haplotypes 7 . However, recent data on the relation of signal strength to selection on class I vs class II makes it unclear whether the effect is due to stronger or weaker recognition of one class of MHC molecules 16, 17 . We have recently demonstrated that genetic polymorphism in the number of CD4⁺ and CD8⁺ cells is tightly linked to polymorphism in the TCR -chain locus (Tcra)³ 18 . Taken together, these observations provide us with a new perspective on how TCR V regions can participate directly in the self-MHC recognition process that shapes the TCR repertoire. Consistent with this view is the idea that TCR appears to have an intrinsic ability to interact with either MHC class I or II molecules 19, 20 . We have used the TCR V3 family extensively to study the effect of V on T cell repertoire selection. It is evident from these findings that the skewing of individual V3 family members’ expression into the CD4 or CD8 subsets is due to selection acting through the complementarity determining regions (CDR) 1 and CDR2 residues for class I or II molecules 10, 11 . This observed effect depends primarily on the V domain, which is, on average, dominant over contributions from the ß-chains and the compositions of the CDR3/J 10, 11 .

Structures of TCR/MHC-peptide complexes in both mouse and human models show the germline encoded CDR1 and CDR2 of the TCR making significant contact with the MHC class I molecule 21, 22, 23 . In one case, while the CDR1 and CDR2 contact the class I molecule, there was little or no contribution from the CDR1ß and CDR2ß in this interaction 22 . In the other case, both - and ß-chain of a V3Vß8 TCR made significant contacts with class I using all three CDRs, but the CDR1 and CDR2 contacts included bonds between highly conserved residues of both TCR and class I 23 . Most significantly, V residues shown to be important in determining MHC-class restriction 10, 11 were found to be major interaction sites with conserved MHC class I residues 23 .

These findings have strong implications for the control of the T cell repertoire. Therefore, we decided to test whether polymorphism in the CDR1 and CDR2 regions in another V family is likely to result in differential selection of T cells. We show here that two of the four expressed TCR V11 family members in B6 mice are over-represented in the CD8⁺ T cell subset, with the other two members being biased to the CD4⁺ population. Variability in the CDR1 and CDR2 regions correlates with differential expression of the family members in the CD8 or CD4 peripheral T cell subsets. The CD4-skewed members, AV11S1 (V11.1) and AV11S2 (V11.2), share identical amino acid residues in the CDR1 and CDR2 segments, which differ significantly from those present in the CD8-skewed members, AV11S3 (V11.3) and the previously undescribed AV11S8. In line with this observation, the V11 family members bearing CDR1 composition similar to that of AV11S1 and S2 in BALB/c mice are also more frequent in the CD4⁺ T cell subset. BALB/c family members with CDR1 sequences similar to AV11S3 and S8 are more biased toward CD8⁺ cells. Analysis of TCR J usage in B6 mice indicate a broad and random distribution in both T cell populations, with the possible exception of the AV11S3 clones in the CD8 set. The CDR3 length is also not conserved and ranges from 6–11 amino acids with 8- and 9-amino acid-long CDR3s being the most frequent. Hence, studies on two different TCR V families yield parallel observations in that unique residues present in the CDR1 and CDR2 play a primary role in MHC class-selection. The array of V genes within various TCR V families, each having its own inherent preference for MHC class I or II molecules, will inevitably contribute to the overall shaping of the T cell repertoire.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

The C57BL/6J (B6) and BALB/cByJ (BALB/c) mice were obtained from The Scripps Research Institute vivarium, while the seventh generation C57BL/6J-Tcra^tm1Mom 24 TCR -chain knockout mice (Tcra^-/-) were from Jackson Laboratories (Bar Harbor, ME). All mice are cared for as specified by institutional guidelines.

Genomic Southern blot analysis

Genomic DNA was extracted from the liver of B6 mice and subjected to restriction endonuclease digestion according to standard procedure 25 . Digested DNA was electrophoresed through a 0.8% agarose gel and blotted onto -probe GT membrane (Bio-Rad, Hercules, CA). Hybridization with a 300-bp V11-specific probe was conducted in QuikHyb buffer (Stratagene, San Diego, CA) at 68°C. This probe, isolated from a cDNA subclone of ADV11S5 5 , includes the whole leader sequence and about 87% of the V region. The filter was washed in 3x SSC/0.1%SDS at 65°C with a final wash in 0.3x SSC/0.1%SDS. No cross-hybridization to other V families was observed.

RT-PCR and sequencing

Lymphocytes from spleens and lymph nodes were isolated from (B6 x Tcra^-/-)F₁ and (BALB/c x Tcra^-/-)F₁ mice and double-stained with anti-CD4-RED613 (clone H129.19; Life Technologies, Grand Island, NY) and anti-CD8-FITC (clone 53-6.7; PharMingen, San Diego, CA) Abs. The cells were then sorted into CD4⁺ and CD8⁺ T cell subsets. Extraction of total RNA 26 from both sorted populations of T cells was followed by the synthesis of first strand cDNA using the Superscript preamplification system (Life Technologies). Double-stranded cDNA was generated and amplified by nested PCR using Vent polymerase (New England Biolabs, Beverly, MA). In the first round of PCR, the 5' primer used was specific for the V11 leader sequence (5'-TTCCGAGCTCATGCAGAGGAACCTGGGAGCT-3') with the 3' primer complementary to the 3' end of the -chain C region (5'-TGTGATGCCACGTTGACCGAGAAAAGCTTT-3'). The second round of PCR used the 3' end of the leader sequence (5'-CTGTGGGTGCAGATTTGC-3') as 5' primer with an oligonucleotide complementary to the 5' end of the C region as the 3' primer (5'-CCCAGAACCTGCTGTGTACTCTAGACGG-3'). In CDR1 analysis of (BALB/c x Tcra^-/-)F₁ mice, the 3' primer was substituted for a stretch of highly conserved nucleotide sequences between CDR1 and CDR2 regions (5'-GAATTCCAGGGGCAGCCT-3') in the second nested PCR. Both 5' primers will anneal equally well to published V11 family members AV11S1 (Tcra^b), AV11S2 (Tcra^b), AV11S3 (Tcra^b), and AV11S4 (Tcra^a). No 5' sequences are available for ADV11S5 (Tcra^a, Tcra^d), AV11S6 (Tcra^d), and AV11S7 (Tcra^a), but these genes were cloned using PCR with identical primers 5 . Products from the second-round PCR reaction were isolated and cloned into pCR-script Amp SK(+) (Stratagene, San Diego, CA). Clones were randomly picked from each T cell subset and sequenced using the T7 and/or M13 reverse primers. The Protein and Nucleic Acids Core facility of The Scripps Research Institute uses the FS dye terminator cycle sequencing method in conjunction with the Applied Biosystems/Perkin-Elmer models 373x1 and 377 DNA sequencers. The nucleotide sequences we obtained were aligned to published V11 sequences 5, 27 with nomenclature for V and TCR J regions in accordance with Refs. 27 and 28, respectively.

PCR and sequencing of V11-specific genomic DNA

The number and sizes of V11-hybridizing DNA fragments from HindIII-digested genomic DNA were first determined from Southern blot analysis. Genomic DNA fragments corresponding to these V11-specific bands were isolated and independently amplified with a 5' primer annealing to the 5' end of the V11 leader sequence (5'-TTCCGAGCTCATGCAGAGGAACCTGGGAGCT-3') in conjunction with a 3' primer specific for the 3' portion of the V region (5'-GAGGACTCAGGCACTTACTTC 3'). The resultant PCR products were isolated, cloned into pCR-script Amp SK(+), and the nucleotide sequence of the V region determined. Nucleotide sequences obtained were aligned to the V11 sequences analyzed in this study (Fig. 1). Three clones were sequenced for each of the different sized genomic bands, with the exception of the 5.5-kb band, for which seven clones were analyzed.

View larger version (37K): [in this window] [in a new window]   FIGURE 1. Nucleotide (A) and deduced amino acid (B) sequences of expressed V11 family members. Sequence comparison of the V region of the four different V11 family members are shown. The amino acid numbering system used is in accordance with Ref. 27. Hence, the CDR1 comprises of seven amino acids and includes residues 24–31; the six amino acid block from residue 48–53 constitutes the CDR2 region. Both regions are underlined. The CDR3 region starts with residue 93, the third residue after the conserved cysteine at position 90. The CDR4 region stretches approximately from residue 67 to residue 71. Dashes indicate nucleotide sequence identity.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

Sequences of the expressed members of the V11 family in B6 mice

The two available Abs specific for the V11 family (1.F2 and RR8-1) do not differentiate between AV11S1 (V11.1) and AV11S2 (V11.2) and do not recognize other members of the family in B6 mice 4, 5 . RR8-1 also recognizes AV11S6 in the Tcra^d haplotype 5 . No Abs are available that detect other members of the V11 family. Analysis of published V11 sequences reveals substantial polymorphism, some of which is due to allelic polymorphism and some of which represents different members of the family. It is difficult to distinguish between these 5, 27, 29 . Due to the strong evidence we have acquired from the V3 family for the role of TCR CDR1 and CDR2 residues in distinguishing between MHC class I or II molecules in repertoire selection 10, 11 , we wish to confirm that this is indeed a general phenomenon and not a peculiarity of the V3 family. We have approached this by nucleotide sequencing. By means of RT-PCR on sorted populations of CD4⁺ and CD8⁺ cells, the V regions of expressed members of the V11 family were cloned and sequenced. B6 Tcra hemizygous mice (i.e., [B6 x Tcra^-/-]F₁) were used so as to eliminate artifacts from cells expressing two -chain mRNAs 30, 31 . A total of 22 different V11 clones were sequenced from CD4⁺ cells and 20 from the CD8⁺ cells. Only clones that differed in the CDR3 region were included in the analysis so as to ensure that they represented independent clones. Four different classes of nucleotide sequences were detected (Fig. 1). These are AV11S1 (known previously as V11.1^b), AV11S2 (V11.2^b), AV11S3 (V11.3^b), and AV11S8, which we describe here. AV11S1, AV11S2, and AV11S3 are established Tcra^b haplotype V11 family members, having been described from the B10.A strain 5, 32, 33, 34 . AV11S3 and AV11S8 are identical in amino acid sequence except at amino acid residue 44. They must be considered as two independent genes in B6, due to the presence of six other silent mutations dotted throughout the nucleotide sequences.

Composition of the CDR1 and CDR2 regions in repertoire selection

A pattern in the distribution of the different V11 family members’ representation in the CD4 and CD8 T cell subsets was observed among the randomly picked and sequenced -chain cDNAs. Both AV11S1 and AV11S2 were found to be more frequently expressed in the CD4⁺ than the CD8⁺ peripheral T cell subset (Table I). This finding is consistent with earlier FACS staining experiments with anti-AV11S1, S2 mAbs RR8-1, and 1.F2 4, 5 . The skewing of AV11S1 is much more marked than that of AV11S2, which is marginal (see Discussion). Conversely, AV11S8 is strongly skewed into the CD8⁺ set and not well represented in the CD4⁺ population. Expression of AV11S3 is also more frequent in the CD8⁺ cells, but the low frequency of expression does not allow us to state that it is as strongly skewed as AV11S8. However, this sequence was originally derived from a class I-restricted T cell 34 .

View this table: [in this window] [in a new window]   Table I. Distribution of TCR V11 family members into the CD4 and CD8 T cell subsets in B6 hemizygous mice

Southern analysis of V11 gene family in B6

A total of five V11-hybridizing DNA bands were observed in HindIII endonuclease-digested genomic liver DNA from the wild-type B6 mouse. Because four different members of the V11 family were found to be expressed, we decided to determine which of these correspond to the different bands on the Southern blot. The HindIII-digested DNA revealed fragments of the following sizes: 25 kb, 10 kb, 6.5 kb, 5.5 kb, and 4.2 kb (Fig. 2). A previous study on B10.A reported the presence of these fragments, but with the 6.5-kb band being indistinct 36 . To reconcile the ambiguity in restriction fragment lengths between B6 and B10.A (both of which are Tcra^b haplotype), and to determine which gene resides on which genomic band, we purified, cloned, and sequenced the respective V11-specific genomic fragments from B6. The 25-kb fragment has the AV11S3 nucleotide sequence, the 10-kb band has the nucleotide sequence of AV11S2, the 6.5-kb band corresponds to the AV11S3 nucleotide sequence, the 5.5-kb fragment corresponds to AV11S1, and the 4.2-kb fragment corresponds to the sequence of AV11S8. The sequences of the respective V11 family members are reported in Fig. 1. We sequenced seven clones derived from the 5.5-kb band because it shows stronger hybridization than other bands in the Southern blot, and we suspected that there could be more than one V11 gene present on a band of this size. Only one type of sequence, AV11S1, was found among these clones. Because these clones have identical sequences, including the L-V intron (data not shown), these are probably derived from the same gene. Another possibility is that there are two identical copies of the gene, resulting from a gene duplication, which both reside on a 5.5-kb fragment (either the same or a different fragment) of the Tcra. The AV11S3 nucleotide sequences yielded by the 25-kb and 6.5-kb fragments have identical leader, intron, and V region sequences (data not shown). Hence, the 25-kb fragment observed here is likely to be a remnant of a partial digestion. The very weak 6.5-kb band and the strong 25-kb band observed in B10.A could then be accounted for by an even less complete HindIII digest in the previous experiment 36 . However, we cannot rule out the presence of two identical copies of the AV11S3 gene. These data indicate the presence of four different V11 family members in the B6 (Tcra^b) strain of mice, although it is possible that there may be more than four genes.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Southern blot analysis of the Tcra V11 family in B6. Liver genomic DNA from B6 mice was digested with HindIII and probed with a V11 cDNA gene segment. The restriction fragment length polymorphism pattern obtained for the V11 family is shown with the molecular size markers indicated on the left. The nomenclature of the V11 gene present within the corresponding genomic band is shown on the right. The likely partial digest band is indicated (p).

Analysis of TCR J usage and CDR3 length distribution

Differential TCR J usage and CDR3 composition has been found in positive repertoire selection in comparison to allorecognition 13, 14 . Positive selection of rat V8.2 (AV8S2) on the class I molecule RT1^f showed no specific CDR3/J requirements. This same V element is preferentially used in alloresponses to RT1^f. In alloresponses, the CDR3/J composition of the AV8S2 genes was highly selected. This was attributed to the requirement for a stronger MHC interaction during allorecognition 14 . Positive selection requires a lower-affinity interaction between TCR and MHC than does activation of mature T cells 3 . Length distribution of CDR3 has been used as a measure for diversity 37 . Therefore, the role of TCR J usage and CDR3 length distribution in -chain-dependent T cell repertoire selection was analyzed.

Table II lists all TCR Js used, with the corresponding CDR3 lengths, by the various V11 cDNA clones in both T cell populations. A total of 24 different TCR J-segments were used by the 20 CD8-derived and 22 CD4-derived sequences. Only three TCR Js were used in both CD4s and CD8s, namely J12, J30, and J31. These Js, in addition to J9, were also the most frequently used. J31 was used five times, J12 was used four times, while J9 and J30 were each used thrice. J9 was used by all three AV11S3 clones in the CD8 set. The significance of this preferred J usage exclusively by AV11S3 is at present unclear due to the small sample size. With the exception of J9, no selective TCR J usage was observed, indicating that differential selection of V11 elements into the CD8 or CD4 subsets is compatible with a diverse set of J regions. The actual boundary of the CDR3 has been determined based on the mouse 2C TCR crystal structure 21 . It begins with residue 93, three residues after the conserved cysteine at position 90 and continues to the residue before the conserved phenylalanine in the FGXG J-region motif. In both T cell subsets, the CDR3 segment ranges from 6–11 amino acids in length with 8- and 9-amino acid-long CDR3s being the most common (Table II). When analyzed separately as CD4-skewed or CD8-skewed V11 members expressed in either T cell subsets, no significant difference in CDR3 length distribution was observed (data not shown). The rather broad range of CDR3 length used argues that selection of the V11 elements is not strongly dependent on CDR3 length. Indeed, it was previously found that positive repertoire selection had no effect on CDR3 length 37 .

View this table: [in this window] [in a new window]   Table II. TCR CDR3/J compositions in the CD4 and CD8 sets1

Representation of V11 sequences in CD4⁺ and CD8⁺ subsets in the Tcra^a haplotype

The above data imply that the overall TCR V skewing into the CD4 or CD8 subset is due mainly to the V segment and is not achieved by differential TCR J element usage or a restricted CDR3 sequence. The differences in the CDR1 and CDR2 regions observed in B6 are also evident in V11 genes from other Tcra haplotypes 5, 27, 29 . Therefore, we extended our study to include the Tcra^a haplotype by using (BALB/c x Tcra^-/-) hemizygous mice. The CDR1 region of expressed members of the V11 family were sequenced from sorted peripheral CD4 and CD8 T cell subsets as outlined above. Two classes of V11 CDR1 amino acid compositions were observed in BALB/c, one similar to AV11S1, S2 (most likely ADV11S5 and AV11S6) and the other similar to AV11S3, S8 (most likely AV11S4 and S7). The AV11S1, S2-related CDR1s, with the sequences FTTTMRS and FTTTTRS, constitute 71% of the CD4⁺ V11 repertoire vs 52% of the CD8⁺ repertoire in BALB/c. On the other hand, CDR1s, which resemble those of AV11S3, S8, (FSIAATT and FSIATTT), make up 48% of the CD8⁺ V11 repertoire, but only 29% of the CD4⁺ repertoire (Table III). However, it must be noted that the sequences likely representing AV11S7 are equally represented in CD4⁺ and CD8⁺ subsets, possibly due to the compensatory effect of other polymorphic residues (see Discussion). Therefore, as with the Tcra^b haplotype (B6), the composition of CDR1 and CDR2 regions in Tcra^a haplotype influences expression in the CD4 and CD8 subsets.

View this table: [in this window] [in a new window]   Table III. Differential CDR1 expression in CD4 and CD8 subsets of BALB/c mice1

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Single residues of either CDR1 and CDR2 can markedly affect selection. This was shown in the V3 family; when residues in a CD4-skewed member were mutated to the corresponding residue from the CD8-skewed member, the transgene expression was skewed to the CD8 cells 10 . It is noteworthy that the residues at position 51 in CDR2 in both V11 and V3 are the same in the CD4- and CD8-skewed members of these families: serine in AV11S1, S2, AV3S3, S5, and S9; proline in AV11S3, S8, AV3S2 (Ref. 11 and here). Residue 51 is an important contact point with MHC in the V3/K^b crystal structure 23 , suggesting that the presence of proline or serine in this position has a dramatic effect on MHC restriction. Overselection of -chains into the CD4 or CD8 T cell subsets seems to depend primarily on V expression, irrespective of CDR3/J composition. Such findings indicate that a considerable degree of specificity is imparted by V alone in interaction with MHC. Indeed, crystal structures of TCR in complex with MHC/peptide ligands, in both mouse and human models, have revealed significant contact points between the CDR1 and CDR2 and the MHC molecules 21, 22, 23, 35 . In both TCR/MHC-peptide structures, the TCR is oriented diagonally across the class I molecule. The CDR3 of - and ß-chains interacts with the central region of the bound peptide as well as with the -helices of the class I-molecule. The CDR2 lies mostly over the C-terminal end of the MHC 2-helix while CDR1 lies over both the N-terminal region of the 1-helix and N terminus of peptide. Conversely, the CDR1ß lies mostly over the N terminus of the 2-helix and CDR2ß over the C terminus of the 1-helix 21, 22, 23, 35 . In one case, all six CDRs contact the MHC-peptide ligand, but with particularly close interactions between residues in the CDR1 and CDR2 and conserved residues on class I -helices 23, 35 . In other cases, CDR1 and CDR2 both make strong interactions with class I -helix residues, but the corresponding Vß regions have very limited interactions 22 . This illustrates that the CDR1 and CDR2 have a primary role in binding to MHC. Potential TCR contact sites along the MHC class I and II -helices are remarkably conserved within the MHC class. Seven of nine and six of seven residues contacted by the TCR -chain in these structures are conserved among MHC class I molecules 22, 23, 35 . For MHC class II molecules, eight of the nine residues in the analogous positions are conserved among alleles. However, significant structural and amino acid differences exist between class I and II proteins in the regions where CDR1 and CDR2 bind 38 . For example, the CDR1 binding region is toward the N terminus of the 1-helix of class I. In class II molecules, the analogous region is a short ß-strand. These differences may ensure class-distinction. Hence, it is plausible that a V element can interact better with most alleles of either MHC class I or II 7 .

It was noted that AV11S1 exhibits a stronger CD4-skewing pattern than AV11S2 in B6. Evidence from our V3 study suggested that residues located on the fourth hypervariable region, between the D and E ß-strands of the V structure, may also affect the degree of skewing 11 . This "CDR4" comprises approximately residues 67–71. Of the five amino acid residues that differ between AV11S1 and AV11S2, two residues (67 and 71) lie in CDR4. Hence, although the compositions of CDR1 and CDR2 are major determinants for MHC class-specificity, it is possible that residues positioned outside canonical TCR CDR regions may have some influence in the strength of MHC class-selection. Of the two AV11S3- and S8-like sequences in BALB/c (most likely AV11S4 and S7), one is clearly skewed to expression in the CD8⁺ cells, whereas the other is represented equally in CD4⁺ and CD8⁺ cells. There are seven differences between the AV11S4 and S7 gene sequences, including one close to the N terminus (residue 2) plus the difference in the CDR1. The CDR4s are positioned over the C terminus of the 2-helix in four TCR/MHC-peptide structures 21, 22, 23, 35, 39 . In the structure of a human TCR bound to MHC class I, a CDR4 residue contacts the class I 2-helix and the N-terminal residue contacts the 1-helix 22 . In the mouse structure, the CDR4ß forms part of the interaction surface 23 . It has also been demonstrated that N-terminal residues on V can affect TCR engagement with MHC-peptide complex 40 . Thus, it is likely that differences in CDR4 sequences or N-terminal residues could affect MHC interactions directly, in some cases, by contact with the MHC, or potentially indirectly, by altering the conformation of CDR1 or CDR2.

It has been documented that TCR has an intrinsic ability to interact with MHC proteins 19, 20 . The mouse Tcra locus is large and, as a result of separate gene duplication events in different parts of the locus, each family has evolved to have numerous closely related V genes. Differences in number and organization of V-gene elements in turn allows us to distinguish between the five Tcra haplotypes on the basis of restriction fragment length polymorphisms 32, 41, 42, 43, 44, 45 . Our findings, together with the fact that concentration of within-family diversity is often clustered in the CDR1 and CDR2 regions 5, 7, 27, 46, 47, 48 , have strong implications as to how the CD4/CD8 T cell repertoire will be shaped 7 . Polymorphism between CD4/CD8 ratios is closely linked to Tcra haplotypes 18 . With each member being biased to recognize either class I or II protein as governed by their CDR1 and CDR2 sequences, and the conservation of skewed selection across different MHC haplotypes, within-family, and allelic polymorphism in the Tcra-V locus will have major consequences on the expressed T cell repertoire.

	Acknowledgments

 
We thank Robert Escalera for his early contributions to this project and Anna Zal and Shane Marine for technical assistance.

	Footnotes

 
¹ This work was supported by National Institutes of Health Grant GM48002. B.-C.S. is supported by a Special Fellowship from the Leukemia Society of America. This is publication number 11010-IMM from The Scripps Research Institute.

² Address correspondence and reprint requests to Dr. Nicholas R. J. Gascoigne, Department of Immunology, IMM1, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037. E-mail address:

³ Abbreviations used in this paper: Tcra, TCR -chain locus; CDR, complementarity determining region(s).

Received for publication February 25, 1998. Accepted for publication December 8, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Davis, M. M., P. J. Bjorkman. 1988. T-cell antigen receptor genes and T-cell recognition. Nature 334:395.[Medline]
2. Jameson, S. C., K. A. Hogquist, M. J. Bevan. 1995. Positive selection of thymocytes. Annu. Rev. Immunol. 13:93.[Medline]
3. Alam, S. M., P. J. Travers, J. L. Wung, W. Nasholds, S. Redpath, S. C. Jameson, N. R. J. Gascoigne. 1996. T cell receptor affinity and thymocyte positive selection. Nature 381:616.[Medline]
4. Jameson, S. C., J. Kaye, N. R. J. Gascoigne. 1990. A T cell receptor V region selectively expressed in CD4⁺ cells. J. Immunol. 145:1324.[Abstract]
5. Jameson, S. C., P. B. Nakajima, J. L. Brooks, W. Heath, O. Kanagawa, N. R. J. Gascoigne. 1991. The T cell receptor V11 gene family. Analysis of allelic sequence polymorphism and demonstration of J-region dependent recognition by allele-specific antibodies. J. Immunol. 147:3185.[Abstract]
6. Merkenschlager, M., C. Benoist, D. Mathis. 1994. MHC control of the naive TCR -chain repertoire. J. Immunol. 153:3005.[Abstract]
7. Sim, B.-C., D. Lo, N. R. J. Gascoigne. 1998. Preferential expression of TCR V regions in CD4/CD8 subsets: class discrimination or co-receptor recognition?. Immunol. Today 19:276.[Medline]
8. Pircher, H., N. Rebaï, M. Groettrup, C. Grégoire, D. E. Speiser, M. P. Happ, E. Palmer, R. M. Zinkernagel, H. Hengartner, B. Malissen. 1992. Preferential positive selection of V2⁺CD8⁺ T cells in mouse strains expressing both H-2^k and T cell receptor V^a haplotypes: determination with a V2-specific monoclonal antibody. Eur. J. Immunol. 22:399.[Medline]
9. Utsunomiya, Y., J. Bill, E. Palmer, K. Gollob, Y. Takagaki, O. Kanagawa. 1989. Analysis of a monoclonal rat antibody directed to the -chain variable region (V3) of the mouse T cell antigen receptor. J. Immunol. 143:2602.[Abstract]
10. Sim, B.-C., L. Zerva, M. I. Greene, N. R. J. Gascoigne. 1996. Control of MHC restriction by TCR V CDR1 and CDR2. Science 273:963.[Abstract]
11. Sim, B.-C., J. L. Wung, N. R. J. Gascoigne. 1998. Polymorphism within a TCRAV family influences the repertoire through class I/II restriction. J. Immunol. 160:1204.[Abstract/Free Full Text]
12. Tomonari, K.. 1992. Negative selection of Tcra-V8⁺ CD8⁺ T cells by MHC class I molecules. Immunogenetics 35:291.[Medline]
13. Torres-Nagel, N., T. Herrmann, G. Giegerich, K. Wonigeit, T. Hünig. 1994. Preferential TCR V usage in positive repertoire selection and alloreactivity of rat T-lymphocytes. Int. Immunol. 6:1367.[Abstract/Free Full Text]
14. Torres-Nagel, N., A. Deutschländer, T. Herrmann, B. Arden, T. Hünig. 1997. Control of TCR V-mediated positive repertoire selection and alloreactivity by differential J usage and CDR3 composition. Int. Immunol. 9:1441.[Abstract/Free Full Text]
15. DerSimonian, H., H. Band, M. B. Brenner. 1991. Increased frequency of T cell receptor V12.1 expression on CD8⁺ T cells: evidence that V participates in shaping the peripheral T cell repertoire. J. Exp. Med. 174:639.[Abstract/Free Full Text]
16. Matechak, E. O., N. Killeen, S. M. Hedrick, B. J. Fowlkes. 1996. MHC class II-specific T cells can develop in the CD8 lineage when CD4 is absent. Immunity 4:337.[Medline]
17. Itano, A., P. Salmon, D. Kioussis, M. Tolaini, P. Corbella, E. Robey. 1996. The cytoplasmic domain of CD4 promotes the development of CD4 lineage T cells. J. Exp. Med. 183:731.[Abstract/Free Full Text]
18. Sim, B.-C., N. Aftahi, C. Reilly, B. Bogen, R. H. Schwartz, N. R. J. Gascoigne, D. Lo. 1998. Thymic skewing of the CD4/CD8 ratio maps with the T cell receptor -chain locus. Curr. Biol. 8:701.[Medline]
19. 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]
20. Merkenschlager, M., D. Graf, M. Lovatt, U. Bommhardt, R. Zamoyska, A. G. Fisher. 1997. How many thymocytes audition for selection?. J. Exp. Med. 186:1149.[Abstract/Free Full Text]
21. Garcia, K. C., M. Degano, R. L. Stanfield, A. Brunmark, M. R. Jackson, P. A. Peterson, L. Teyton, I. A. Wilson. 1996. An ß T cell receptor structure at 2.5Å and its orientation in the TCR-MHC complex. Science 274:209.[Abstract/Free Full Text]
22. Garboczi, D. N., P. Ghosh, U. Utz, Q. R. Fan, W. E. Biddison, D. C. Wiley. 1996. Structure of the complex between human T-cell receptor, viral peptide and HLA-A2. Nature 384:134.[Medline]
23. Garcia, K. C., M. Degano, L. R. Pease, M. Huang, P. A. Peterson, L. Teyton, I. A. Wilson. 1998. Structural basis of plasticity in T cell receptor recognition of a self peptide-MHC antigen. Science 279:1166.[Abstract/Free Full Text]
24. Mombaerts, P., A. R. Clarke, M. A. Rudnicki, J. Iacomini, S. Itohara, J. Lafaille, L. Wang, Y. Ichikawa, R. Jaenisch, M. L. Hooper, S. Tonegawa. 1992. Mutations in T cell receptor genes and ß block thymocyte development at different stages. Nature 360:225.[Medline]
25. Sambrook, J., E. Fritsch, T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
26. Chomczynski, P., N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156.[Medline]
27. Arden, B., S. P. Clark, D. Kabelitz, T. W. Mak. 1995. Mouse T-cell receptor variable gene segment families. Immunogenetics 42:501.[Medline]
28. Koop, B. F., L. Rowen, K. Wang, C. L. Kuo, D. Seto, J. A. Lenstra, S. Howard, W. Shan, P. Deshpande, L. Hood. 1994. The human T-cell receptor TCRAC/TCRDC (C/C) region: organization, sequence, and evolution of 97.6 kb of DNA. Genomics 19:478.[Medline]
29. Osman, G. E., M. Toda, O. Kanagawa, L. E. Hood. 1993. Characterization of the T cell receptor repertoire causing collagen arthritis in mice. J. Exp. Med. 177:387.[Abstract/Free Full Text]
30. Malissen, M., J. Trucy, E. Jouvin-Marche, P.-A. Cazenave, R. Scollay, B. Malissen. 1992. Regulation of TCR and ß gene allelic exclusion during T-cell development. Immunol. Today 13:315.[Medline]
31. Alam, S. M., I. N. Crispe, N. R. J. Gascoigne. 1995. Allelic exclusion of mouse T cell receptor chains occurs at the time of thymocyte TCR up-regulation. Immunity 3:449.[Medline]
32. Becker, D. M., P. Patten, Y. Chien, T. Yokota, Z. Eshhar, M. Giedlin, N. R. J. Gascoigne, C. Goodnow, R. Wolf, K. Arai, M. M. Davis. 1985. Variability and repertoire size of T-cell receptor V gene segments. Nature 317:430.[Medline]
33. Fink, P. J., L. A. Matis, D. L. McElligott, M. A. Bookman, S. M. Hedrick. 1986. Correlations between T cell specificity and the structure of the antigen receptor. Nature 321:219.[Medline]
34. Malissen, M., J. Trucy, F. Letourneur, N. Rebaï, D. E. Dunn, F. W. Fitch, L. Hood, B. Malissen. 1988. A T cell clone expresses two T cell receptor genes but uses one ß heterodimer for allorecognition and self MHC-restricted antigen recognition. Cell 55:49.[Medline]
35. Ding, Y. H., K. J. Smith, D. N. Garboczi, U. Utz, W. E. Biddison, D. C. Wiley. 1998. Two human T cell receptors bind in a similar diagonal mode to the HLA-A2/Tax peptide complex using different TCR amino acids. Immunity 8:403.[Medline]
36. Nakajima, P. B., J. P. Di Vincenzo, S. C. Jameson, N. R. J. Gascoigne. 1992. Chromosome 14 in B10.A(18R) mice is recombinant and includes Tcra-V^a alleles. Immunogenetics 35:190.[Medline]
37. Rock, E. P., P. R. Sibbald, M. M. Davis, Y. Chien. 1994. CDR3 length in antigen-specific immune receptors. J. Exp. Med. 179:323.[Abstract/Free Full Text]
38. Stern, L. J., D. C. Wiley. 1994. Antigenic peptide binding by class I and class II histocompatibility antigens. Structure 2:245.[Medline]
39. Teng, M. K., A. Smolyar, A. G. D. Tse, J. H. Liu, J. Liu, R. E. Hussey, S. G. Nathenson, H. C. Chang, E. L. Reinherz, J. H. Wang. 1998. Identification of a common docking topology with substantial variation among different TCR-peptide-MHC complexes. Curr. Biol. 8:409.[Medline]
40. Seibel, J. L., N. Wilson, H. Kozono, P. Marrack, J. W. Kappler. 1997. Influence of the NH₂-terminal amino acid of the T cell receptor chain on major histocompatibility complex (MHC) class II plus peptide recognition. J. Exp. Med. 185:1919.[Abstract/Free Full Text]
41. Singer, P. A., R. J. McEvilly, R. S. Balderas, F. J. Dixon, A. N. Theofilopoulos. 1988. T-cell receptor -chain variable-region haplotypes of normal and autoimmune laboratory mouse strains. Proc. Natl. Acad. Sci. USA 85:7729.[Abstract/Free Full Text]
42. Klotz, J. L., R. K. Barth, G. L. Kiser, L. E. Hood, M. Kronenberg. 1989. Restriction fragment length polymorphisms of the mouse T-cell receptor gene families. Immunogenetics 29:191.[Medline]
43. Jouvin-Marche, E., M. Goncalves 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]
44. Jouvin-Marche, E., I. Hue, P. N. Marche, C. Liebe-Gris, J.-P. Marolleau, B. Malissen, P.-A. Cazenave, M. Malissen. 1990. Genomic organization of the mouse T cell receptor V family. EMBO J. 9:2141.[Medline]
45. Gascoigne, N. R. J.. 1995. Genomic organization of T cell receptor genes in the mouse. J. I. Bell, and M. J. Owen, and E. Simpson, eds. T cell receptors 288. Oxford University Press, Oxford.
46. Chou, H. S., M. A. Behlke, S. A. Godambe, J. H. Russell, C. G. Brooks, D. Y. Loh. 1986. T cell receptor genes in an alloreactive CTL clone: implications for rearrangement and germline diversity of variable gene segments. EMBO J. 5:2149.[Medline]
47. Wang, K., C.-L. Kuo, K.-C. Cheng, M.-K. Lee, B. Paeper, B. F. Koop, T.-J. Yoo, L. Hood. 1994. Structural analysis of the mouse T-cell receptor Tcra V2 subfamily. Immunogenetics 40:116.[Medline]
48. Gahéry-Ségard, H., E. Jouvin-Marche, A. Six, C. Gris-Liebe, M. Malissen, B. Malissen, P. A. Cazenave, P. N. Marche. 1996. Germline genomic structure of the B10.A mouse Tcra-V2 gene subfamily. Immunogenetics 44:298.[Medline]

This article has been cited by other articles:

				N. Torres-Nagel, B. Mehling, A.-F. LeRolle, E. Joly, and T. Hunig Genetic control of peripheral TCRAV usage by representation in the preselection repertoire and MHC allele-specific overselection Int. Immunol., January 1, 2001; 13(1): 63 - 73. [Abstract] [Full Text] [PDF]

				M. Correia-Neves, C. Waltzinger, J.-M. Wurtz, C. Benoist, and D. Mathis Amino Acids Specifying MHC Class Preference in TCR V{alpha}2 Regions J. Immunol., November 15, 1999; 163(10): 5471 - 5477. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sim, B.-C. Articles by Gascoigne, N. R. J. Search for Related Content PubMed PubMed Citation Articles by Sim, B.-C. Articles by Gascoigne, N. R. J.