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The Journal of Immunology, 1999, 163: 301-311.
Copyright © 1999 by The American Association of Immunologists

Invariant or Highly Conserved TCR {alpha} Are Expressed on Double-Negative (CD3+CD4-CD8-) and CD8+ T Cells

Mei Han, Lisa Harrison, Patricia Kehn, Kimberly Stevenson, Jeffrey Currier and Mary Ann Robinson1

Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, Twinbrook II Facility, National Institutes of Health, Rockville, MD 20852


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TCR {alpha} (TCRA) expression was examined in RNA samples from PBMC and isolated populations of CD4+, CD8+, and DN T cells from 15 healthy individuals. The expressed TCR repertoire was surveyed using spectratype analysis, a technique that displays the distribution of complementarity determining region 3 (CDR3) lengths for each TCRAV gene family. The results revealed the presence of unusual populations of double-negative (DN; CD4-CD8-CD3+) T cells that express invariant or conserved TCRAV4A, AV7, AV19, and AV24 chains. Each of the conserved TCRA families was over-represented in >70% of the individuals studied, and all individuals expressed at least one of the over-represented TCRAV families. Over-represented conserved AV4A or AV7 sequences were also present in CD8+ T cells from most donors. The extent of TCRA sequence conservation is unparalleled. TCRAV4A, AV19, and AV24 sequences were invariant, although AV4A and AV19 transcripts contained N region additions. TCRAV24 transcripts derived from the direct juxtaposition of V and J gene segments. TCRAV7 sequences showed some diversity in two amino acids encoded at junctions of V and J gene segments. Although derivation of DN T cells with conserved TCRA chains is puzzling, the wide-spread expression of these unusual cells suggests an important function.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
T lymphocytes recognize Ag as peptides presented in the context of MHC molecules with the fine specificity of the T cell defined by the TCR (1). The majority of T lymphocytes express a TCR composed of an {alpha}- and a ß-chain. Crystal structures of {alpha}ßTCR, MHC, and peptide complexes have revealed contact between residues in MHC peptide and both the {alpha}- and ß-chains of the TCR (2, 3, 4). The region of the TCR that is primarily involved in Ag-MHC contact is the complementarity determining region 3 (CDR3),2 which is the most variable region of both {alpha}- and ß-chains. The TCRß (TCRB) repertoire of healthy individuals and of individuals with certain diseases has been investigated (5, 6, 7, 8), whereas the expressed TCR{alpha} (TCRA) repertoire has not been extensively characterized. The TCRA gene complex spans 1072 kb on chromosome 14. The TCRA gene complex consists of 42 variable (V) gene segments that are grouped into 32 families based upon >75% identity at the nucleotide level (9). The TCRAV gene segments recombine with 1 of 61 TCRA joining (J) gene segments encoded in a 33-kb region located 5' of the {alpha} constant region (TCRAC) gene segment (10). Diversity in the TCRA CDR3 derives from different combinations of V and J gene segments and from N region nucleotides that are added somatically.

In the present report, TCRA expression of healthy individuals was investigated by using spectratype and sequence analyses. Spectratype analysis is a technique that displays the array of CDR3 lengths associated with each TCRAV family (5, 6, 7, 8). Mean distributions of CDR3 lengths for the 32 TCRA families were established by analyses of PBMC, CD4+, CD8+, and DN T cells from 15 healthy adult individuals. Gaussian distributions of CDR3 lengths were observed for CD4+ T cells, and deviations from Gaussian distributions were observed increasingly in PBMC, CD8+, and DN T cells, respectively. Similarities were observed in the size of a skewed peak in AV4A, AV7, AV19, AV24, and AV31 families. Sequence analyses revealed invariant or conserved sequences in AV4A, AV7, AV19, and AV24 that were present in multiple unrelated individuals. In addition, the same AV4A and AV7 sequences were over-represented in the CD8+ T cell subpopulations.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
TCRAV PCR primer design

The complete sequence of the TCRA gene complex has been entered in GenBank (accession no. AE000521). Sequences of 42 TCRAV genes were aligned using MAP (multiple sequence alignment program). TCRAV genes were grouped into 32 subfamilies based on the standard of 75% or greater nucleotide identity. PCR primers were designed to amplify all members of each VA family, but to contain sufficient mismatches with other TCRAV families to minimize the possibility of amplifying other TCRAV genes. The 34 AV primers used for PCR amplification in these experiments are listed in Fig. 1Go.



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  		FIGURE 1. Sequences of the oligonucleotide primers for 32 TCRAV gene families used in this study for PCR amplification and primer-extension reactions. The length in base pairs from the beginning of the oligonucleotide to the 5' end of the CDR3 coding region is shown for each AV region primer.

 
Definition of TCRA CDR3 region

Conserved sequences at the 3' end of TCRAV (Y-L/F-C-A-{square}) and at the 5' end of TCRAJ gene segments (F-G-{square}-G-T) serve as markers in the definition of the CDR3 region. The {square} corresponds to any amino acid. The CDR3 region includes amino acids beginning with the third amino acid after the invariant C residue in all TCRAV genes (Y-L/F-C-A-{square}-1) and spans to the amino acid immediately preceding the TCRAJ motif (2-F-G-{square}-G-T).

PBMC isolation and T cell separation

Peripheral blood (50–100 ml) was taken from 15 healthy adult individuals by venipuncture, and PBMC were subsequently isolated by centrifugation of the blood over lymphocyte separation medium, a ficoll derivative (Organon Technika, Durham, NC). The PBMC were enumerated and an aliquot of 5 x 106 cells was kept on ice (4°C) as the unfractionated PBMC population. The remaining cells were incubated with anti-CD14- and anti-CD19-coated magnetic beads (Miltenyi Biotec, Sunnyvale, CA) at 4°C for 15 min in a volume of 100 µl/107 cells. After incubation and one wash, the cell-bead mixture was applied to a MACS AS separation column attached to a SuperMACS (Miltenyi Biotec) separation magnet to deplete the population of B lymphocytes and monocytes. The T lymphocyte enriched population was then incubated with anti-CD8 (Leu-2A) MACS MicroBeads (Miltenyi Biotec) and passed through a MACS AS column to collect CD8- cells, and CD8+ T cells were eluted from the magnetic beads according to the manufacturer’s instructions. The CD8- cell population was then incubated with anti-CD4 (Leu-3A) (Miltenyi Biotec) and passed through a MACS AS column to collect CD4-CD8- (DN) cells, and CD4+ T cells were eluted. The CD8+- and CD4+-enriched and the DN populations were then washed, enumerated, and kept for subsequent analysis. Following the same procedure, certain DN samples were separated into CD56- and CD56+ populations using anti-CD56 (NCAM 16.2) MicroBeads (Miltenyi Biotec).

RNA extraction and cDNA synthesis

Total RNA was prepared from PBMC and T cell subsets using the RNeasy Total RNA Minikit (Qiagen, Santa Clarita, CA). Total RNA (up to 20 µg) was eluted from each column and used as the template for first strand cDNA synthesis. The reaction primer oligo(dT)(12, 13, 14, 15, 16, 17, 18) (2.5 µg) was annealed to the eluted RNA by incubation at 70°C for 10 min. The reaction mixture was brought up to a total volume of 100 µl (50 mM Tris-HCl, 75 mM KCl, 5 mM MgCl2, 10 mM DTT, and 500 mM each dNTP), and cDNA synthesis was completed by primer extension with SuperScript II RNase H- reverse transcriptase (500 U/reaction) (Life Technologies, Gaithersburg, MD) at 42°C for 2 h. The cDNA was diluted 1:5 with DNase-free H2O and stored at -80°C before use as the template for PCR amplification.

TCR spectratype analysis

PCR amplification was performed in a total reaction volume of 50 µl (10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2, and 200 mM each dNTP), using AmpliTaq Gold DNA polymerase (1 U/reaction) (PE-Applied Biosystems, Foster City, CA). Reactions were primed with the TCRAC region-specific oligonucleotide oTCA4 and each of the TCRAV region specific oligonucleotides (1 mM final concentration) and submitted to 35 cycles of amplification. Reaction conditions consisted of an initial incubation at 95°C for 12 min (to activate Amplitaq Gold), followed by template denaturation at 95°C for 30 s, primer annealing at 60°C for 30 s, and primer extension at 72°C for 90 s. Five microliters of each PCR product was then used as template for a single-stranded primer-extension reaction using the same reaction mix as for PCR amplification in a total volume of 20 µl. This reaction was primed with a nested TCRAC region fluorescently labeled oligonucleotide oTCRAC (6-FAM fluorophore; Life Technologies). Primer extension was performed using AmpliTaq DNA polymerase (0.5 U/reaction) (PE-Applied Biosystems) and 3 cycles of template denaturation at 95°C for 2 min, primer-annealing at 60°C for 2 min, and primer-extension at 72°C for 10 min. Fluorescent products were analyzed by separation on a 4% acrylamide, 6 M urea sequencing gel using an ABI-377 DNA sequencer (PE-Applied Biosystems). The gel-loading mix consisted of 2 µl of primer-extension reaction product, 2 µl of formamide, 0.5 µl of loading dye (25 mM EDTA, 50 mg/ml blue dextran), and 0.5 µl of GeneScan-500 TAMRA dye-labeled size standards (PE-Applied Biosystems). This mixture was denatured at 95°C for 2 min, and 2 µl was loaded onto the gel and run for 2 h. DNA products of the appropriate lengths were analyzed using GeneScan software version 2.1 (PE-Applied Biosystems), and areas under each peak were used for subsequent analysis.

Flow cytometric analysis

PBMC from healthy individuals were isolated by ficoll density gradient centrifugation and washed three times in PBS. Cell purity after magnetic bead separation was determined by staining with FITC-conjugated anti-CD4 or CD8, PE-conjugated anti-CD3 or CD56, and PerCP conjugated anti-CD8, CD4, or CD56 (Becton Dickinson, Mountain View, CA) for 30 min on ice. Stained cells were washed twice in PBS containing 1% FCS and 0.01% sodium azide before analysis on a Becton Dickinson FACScan. Lymphocytes were gated on forward versus side scatter, and 10,000 events were collected. Analysis was performed using the CellQuest software, and markers were based on unstained controls. Other Abs used for staining, but for which data are not shown, include anti-VA24 (Immunotech, Westbrook, ME) and FITC-conjugated anti-TCR{alpha}ß, TCR{gamma}{delta}, CD16, CD44, and goat anti-mouse Ig (Becton Dickinson).

Quantitative analysis

A formal mathematical analysis of the data was performed to permit an objective and sensitive determination of the magnitude of perturbation or skewing of a given CDR3 profile from a Gaussian distribution. After analysis using GeneScan software, data from each CDR3 profile were imported into a spreadsheet and translated into a probability distribution, P (i) = Ai/({Sigma}iAi) (11). The area under the curve (Ai) of each CDR3 peak (i), corresponding to a discrete amino acid length, was expressed as fraction of the total area under the profile ({Sigma}iAi). To provide a baseline for the calculation of generalized hamming distances (HD) for each profile, the average probability distribution was calculated for each AVj family using the CD4+ profiles obtained from 15 individuals, Pj (i) = {Sigma}j[Pj(i)]/15. This was performed for each AV family, since some families consistently showed a larger number of possible CDR3 lengths than others (ranges from 7 to 11), and some families have different mean CDR3 lengths. A normal distribution was then fit to the average distribution profiles and used to represent the theoretical unperturbed repertoire for each AVj family. The generalized HD was then used to calculate the degree of skewing or perturbation of any given profile from the predicted normal distribution. The extent of perturbation or skewing at each TCR length, Di = Pi (observed) - Pi (expected) was calculated for each profile. The HD for each profile was then calculated using the formula: HD = 100[{Sigma}i D (i)]/2, where [{Sigma}i D (i)] is the sum of the absolute differences of the distances between two probability distributions at each TCR length. For a profile showing no perturbation from normal, the value for HD = 0%, while two completely discordant profiles will have an HD = 100%, and two randomly drawn profiles should have an HD = 50%. The HD-scoring method was used, as it is a more robust test for biological significance than the {chi}2 test.

TCRA sequencing

DNA sequence analyses were performed on selected samples by ligation of 0.5–1 ng of PCR product with pCR2.1 vector (TA cloning kit; Invitrogen, Carlsbad, CA) at 16°C overnight. OneShot competent cells (Invitrogen) were used for the transformation. After PCR screening, positive colonies were selected and expanded. Plasmids were purified, inserts were sequenced using an ABI 377 DNA sequencer, and the sequence data were analyzed using Factura software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Spectratype analyses of peripheral T lymphocytes

The extent of diversity in the human TCRA repertoire was examined by spectratype analysis, a technique that provides a quantitative assessment of the array of CDR3 lengths present in transcribed TCRA chains. It was possible to design primers that detect the members of all 32 functional TCRAV families (Fig. 1Go) from the sequence of the complete TCRA gene complex available in GenBank. For both AV1 and AV4 families, two primers were necessary because of sequence variation among subfamily members.

A complete TCRA spectratype analysis of the PBMC from a healthy adult individual is shown in Fig. 2Go. Profiles of the 32 TCRAV families show distributions of CDR3 lengths that differ by 3 bp and correspond to in-frame TCRA transcripts. The length of the CDR3 region can be deduced from the lengths of the PCR products when compared with that derived from a TCRA chain clone with a known sequence. In this experiment, TCRA CDR3 lengths ranged from 3 aa to 19 aa. For most of the TCRAV families, the distribution of CDR3 lengths appeared as a Gaussian distribution by visual examination. However, there were certain notable exceptions. The TCRAV profiles for the individual shown in Fig. 2Go showed single dominant peaks in the spectratype profiles of AV4A, AV7, AV29, and AV30 and skewed profiles for AV3, AV8, AV12, AV24, AV26, AV27, and AV31. Skewed TCRB spectratype profiles have been observed in peripheral CD8+ T cells of healthy individuals and have been shown to represent clonal or oligoclonal expansions of the TCRBV family within the CD8+ T cell subset (12, 13, 14, 15, 16). It was next asked whether skewed distribution of CDR3 lengths within TCRAV families reflected a clonal or oligoclonal expansion within a single T cell subset.



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  		FIGURE 2. Representative TCRAV spectratype profiles derived from the PCR amplification of cDNA from PBMC samples from one donor. The x-axis of each plot corresponds to size in nucleotides; peaks are separated by 3 bases to correspond to in-frame transcripts. The y-axis corresponds to fluorescence intensity and is proportional to the frequency of transcripts present in that peak.

 
Spectratype analyses of T cell subsets

PBMC were depleted of B cells and monocytes using a combination of anti-CD14- and anti-CD19-coated magnetic beads. CD8+ and CD4+ T cells were isolated sequentially by positive selection using anti-CD8- then anti-CD4-coated magnetic beads. The remaining cells comprised the CD4-8- or DN population. The isolated cell populations were tested for purity by flow cytometry using anti-CD3, anti-CD4, and anti-CD8; the average purity was 97% for the CD4+, 86% for the CD8+, and 94% for the DN populations.

Spectratype analysis was performed on PBMC, and isolated populations of CD4+, CD8+, and DN T cells from 15 unrelated healthy adult individuals. A total of 2040 spectratype profiles were produced, and each was compared with a normal distribution. A formal quantitative assessment designated HD was applied to give a numerical value for the degree to which a profile deviates from an idealized normal distribution. For example, a profile with an HD score of 0% shows total concordance with a normal distribution, whereas a profile with an HD score of 100% shows complete discordance. The HD scores for each TCRAV family in the 15 individuals are shown by a dot in Fig. 3Go with representative profiles for the ranges of HD scores shown at the top of the figure.



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  		FIGURE 3. Summary of TCRA chain HD from PBMC, CD4+, CD8+, and DN cell populations of 15 healthy adult donors. The x-axis represents the HD score in percent, while each closed circle represents a single AV family for each donor. Numbers along the y-axis represent individual donors. Above are shown five CDR3 spectratype profiles representing the patterns that can be observed for the indicated HD values.

 
The CD4+ populations have profiles that most closely approximate normal distributions of CDR3 lengths, thus the HD scores are lower than those for the other subpopulations of T cells. To establish an operational definition of skewed TCRA CDR3 profiles, the 95% confidence interval for the mean of all TCRAV families in the CD4+ subpopulation from all 15 individuals was used. Thus, in the present report, HD values of >19.2% are considered to be skewed. Using this definition, the mean HD and percentage of skewed profiles observed for all TCRAV families in the PBMC, CD4+, CD8+, and DN T cell populations are shown in Table IGo.


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  		Table I. Skewing in T cell subpopulations

 
Skewed CDR3 length distributions were observed in only 3.9% of the TCRAV samples in the CD4+ cell populations from the 15 individuals. The mean HD was 8.8%, indicating that the majority of TCRAV families showed normal distributions of CDR3 lengths in the CD4+ subpopulations. In contrast, 48.8% of the TCRAV samples were skewed in the CD8+ population with the mean HD equal to 24.1%. Therefore, similar to what was observed for TCRB, the CD8+ cell populations showed significant skewing of TCRA CDR3 length distributions. The most skewed subpopulation was the DN subset. The mean HD was high (37%), and 82.6% of the DN samples were skewed. The PBMC samples being comprised of the above subpopulations (CD4+, CD8+, and DN) showed an intermediate level of skewing with a mean HD of 14.9% and 22.6% of the samples having skewed distributions of CDR3 lengths. Skewed peaks observed in the PBMC samples corresponded to skewed peaks present in at least one of the subpopulations.

Similarities in spectratype profiles of DN T cells among multiple individuals

Examination of the mean profiles for each TCRAV family, combining data from all 15 individuals studied, revealed similarities in spectratype profiles of DN T cells. Skewing in the mean profile would reflect that the same CDR3 length was over-represented in multiple individuals. The mean spectratype profiles of five TCRAV families showed non-Gaussian distributions in DN populations with one CDR3 length represented at frequencies between 1.8 and 4.3 times greater than expected for a normal distribution (Fig. 4Go).



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  		FIGURE 4. Mean profiles for TCRAV families that show skewed distributions with one CDR3 length over-represented. The length of the CDR3 region is shown on the x-axis, and the mean frequency of transcripts is shown on the y-axis. Data from all of the 15 individuals studied are combined.

 
TCRAV4A transcripts with a CDR3 length of 9 aa were present in the mean spectratype profile 3.2 times more frequently than expected. A total of 13 of the 15 individual donors studied showed over-representation of TCRAV4A transcripts that correspond to a CDR3 length of 9 aa. TCRAV7 with a CDR3 of 8 aa was found at 4.3 times the expected frequency and over-represented in 14 individuals. AV19 with a CDR3 of 9 aa was found 1.8 times more frequently and over-represented in 12 individuals. TCRAV24 with a CDR3 length of 11 aa was present 2.1 times more frequently than expected and over-represented in 11 of the 15 donors studied. AV31 with 9 aa was found at 2.1 times the expected frequency and over-represented in 9 individuals (Table IIGo).


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  		Table II. Overrepresented TCRA in DN and CD8+ T cells

 
Comparison of spectratype profiles from the four cell populations, PBMC, CD4+, CD8+, and DN cells, reveals the extent of skewing in the DN population. A different donor was selected to represent each TCRAV family; the profiles shown are typical of individuals that present a skewed profile for the designated TCRAV family. Samples from DN cells showed single dominant peaks for AV4A, AV7, AV19, AV24, and AV31, which is in marked contrast to the normal distributions for each of these families from CD4+ populations (Fig. 5Go).



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  		FIGURE 5. Representative spectratype profiles of PBMC, CD4+, CD8+, and DN cell populations for TCRAV families showing over-representation of a CDR3 length. The size of over-represented CDR3 lengths is indicated in PBMC panel and indicated by the dashed line in all profiles. Profiles of all T cell subsets for each TCRAV family are from a single donor; different representative donors are shown for each TCRAV family.

 
There are additional TCRAV families that show the over-representation of one CDR3 length in DN T cells; however, the extent of over-representation was less and fewer individuals had the over-represented peak. A single-sized CDR3 length was over-represented in TCRAV3, AV9, AV15, AV27, and AV30 families, but was present in only 3–7 individuals. These TCRAV families have not been investigated in further depth in the present report.

Similarities in spectratype profiles of CD8+ cells among multiple individuals

Fig. 5Go also reveals skewed profiles for TCRAV4A and AV7 in the CD8+ subpopulation for the individuals shown. The size of the peak in the CD8+ T cells is identical to that present in the DN subpopulation. The mean CD8+ spectratype profiles for TCRAV4A and AV7 families were examined to determine whether skewed profiles were observed in multiple individuals. The mean profiles of AV4A and AV7 families for CD8+ cells showed non-Gaussian distributions of CDR3 lengths. TCRAV4A transcripts with a CDR3 length of 9 aa were present 2.4 times more frequently than expected and were over-represented in 10 of the 13 individuals, who also showed this length over-represented in their DN cells. TCRAV7 transcripts corresponding to a CDR3 of 8 aa were over-represented (3.5 times) in all of 14 individuals with that length CDR3 over-represented in their DN population (Table IIGo). The TCRAV4A and AV7 peaks were also evident in the PBMC (Fig. 5Go). One individual showed over-representation of same-sized AV24 peak in both DN and CD8+ cells (11 aa), and one individual showed over-representation of same sized AV19 peak in both DN and CD8+ cells (9 aa). Skewing observed in the remaining TCRAV families was variable among individuals in CDR3 length and occurrence.

Stability of TCRA spectratype profiles over time

Full TCRA spectratype profiles were determined on seven individuals on at least two different occasions with blood samples taken 3 mo to 2 yr apart. The patterns of expression of the four conserved TCRAV families were consistent over time. Over-representation of TCRAV4A, AV7, AV19, and AV24 was consistently maintained within the same T cell subsets in all individuals. Individuals who were negative for the over-representation of a TCRAV family remained negative. Two individuals with TCRAV4A over-represented in DN but not CD8+ T cells maintained that pattern over a 2-yr period. Thus, the expression of conserved TCRAV4A, AV7, AV19, and AV24 families is stable in healthy adult individuals.

Sequence analysis of TCRA transcripts with over-represented CDR3 lengths

Spectratype analysis identifies CDR3 lengths within a TCRA family that are over-represented, but does not reveal the extent of diversity in the transcripts having that CDR3 length. Analyses were performed to determine the sequences of transcripts in the five TCRAV families that showed an over-represented CDR3 length. Samples from four to eight individuals with the over-represented CDR3 length in the DN populations and samples from one to seven individuals with the over-represented CDR3 length in CD8+ cells were cloned and sequences were determined. Absolutely conserved or predominant sequences were observed for AV4A, AV7, AV19, and AV24 (Fig. 6Go), whereas AV31 sequences were unique to each individual.



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  		FIGURE 6. Frequency of conserved CDR3 sequences in TCRAV4A, AV7, AV19, and AV24 genes from CD8+ and DN cell populations. The TCRAV genes, the conserved sequences, and the TCRAJ gene segments utilized are listed. Frequency lists the number of clones that were identical to the conserved sequence over the total number of clones sequenced. {square} indicates that multiple amino acids were observed at this position.

 
Sequences of TCRAV4A transcripts were determined for clones derived from the DN population of eight donors and from the CD8+ T cells of five donors. Identical AV4A DNA sequences were observed in 66/76 clones from DN cells and in 34/57 clones from CD8+ T cells. All clones utilize TCRAV4S1 and AJ29 gene segments. The invariant sequence was found in the DN and CD8+ subpopulations of all individuals studied.

The presence of conserved TCRAV7 in the DN T cells has been reported (17), but their presence in the CD8+ population was not noted in this study. A total of 177 TCRAV7 clones from DN and CD8+ T cell populations derived from 7 donors were sequenced, and 139 clones had a CDR3 of 8 aa. Of the clones with an 8-aa CDR3 length, 123 clones utilized TCRAJ33. Diversity was observed in the two amino acids encoded at the junction of V and J gene segments (marked by a {square} in Fig. 6Go). A valine residue was the most frequently observed amino acid in the first variable position (92/123), which, according to the numbering system used here, would be considered part of the V gene segment. Methionine was the most frequently observed residue in the second variable amino acid position (54/123); Val and Met were present together in 38/123 clones. There were 16 clones with an 8-aa CDR3 that utilized TCRAJ gene segments other than the conserved TCRAJ33. A Val residue was present in the first variable position in all 16 clones, and 4 clones had both Val and Met residues at corresponding positions in the CDR3. Each individual expressed numerous different sequences in DN and in CD8+ cells; many sequences were present in both cell populations from a single individual.

A total of 30 of 57 clones derived from the DN cell populations of 8 individuals had an invariant TCRAV19 sequence with a CDR3 of 9 aa using AJ48. In addition, one of these individuals showed over-representation of transcripts corresponding to a CDR3 of 9 aa in the CD8+ population, and 8/22 clones from the CD8+ cells of this individual had the invariant TCRAV19 sequence.

The reported invariant TCRAV24 sequence using AJ 18 with a CDR3 of 11 aa (17, 18) was observed in 18 of 35 clones analyzed derived from DN T cells from 4 individuals. In addition, one of these individuals also showed over-representation of transcripts corresponding to a CDR3 length of 11 aa in the CD8+ cell population. Ten clones derived from CD8+ cells were sequenced; none showed the invariant sequence.

Sequences were determined for 54 TCRAV31 clones with a 9-aa CDR3 length derived from six individuals. Comparison of sequences of clones from a single individual showed that all clones were identical. Comparisons of sequences between individuals revealed no sequence similarities. Thus, TCRAV31 transcripts were conserved in CDR3 length, but not in sequence.

Origin of conserved TCRA chains

Transcripts from AV4A, AV7, AV19, and AV24 families had conserved TCRAJ genes and CDR3 sequences. Examination of the origin of the conserved sequences by comparison of the transcripts with germline sequences may reveal features that favor these specific rearrangement events. With availability of the sequence of the full TCRA complex, it was possible to determine the origin of nucleotides that encode the CDR3 region. Alignments of cDNA and genomic sequences are shown in Fig. 7Go.



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  		FIGURE 7. Origin of conserved TCRAV sequences. The top line in each box lists the nucleotides and deduced amino acid sequences for each of the over-represented TCRAV transcripts. Below are the germline TCRAV and TCRAJ gene segments. Junctions of V and J gene segments are outlined. In TCRAV7, the outlined V and M residues were observed most frequently at those positions.

 
The nucleotide sequences of TCRAV4A, AV19, and AV24 transcripts were invariant among all individuals, but only AV24 transcripts could be the product of the direct juxtaposition of AV (AV24) and AJ (AJ18) gene segments. TCRAV4A and AV19 contained N region additions, and, despite redundancy in the codons for the amino acid present, all individuals used the same nucleotide sequence.

TCRAV4A transcripts have a CDR3 region of 9 aa with the sequence R-D-V-G-N-T-P-L-V. The R-D residues are encoded by the AV4A gene segment and the G-N-T-P-L-V residues by the TCRAJ29 gene segment. The central valine residue is the result of N region additions; however, the last nucleotide of the codon may derive from AJ29. The invariant TCRAV19 sequence had a 9-aa CDR3 with the sequence K-N-F-G-N-E-K-L-T. Most of the CDR3 is encoded by the TCRAJ48 gene segment with the leading lysine residue encoded by N nucleotides. It is possible for the first residue of the lysine codon to derive from the AV19 gene segment. Genomic sequences of the TCRAV4A and TCRAV19 gene segments were confirmed in five of the donors in this study.

In contrast, limited diversity is present in TCRAV7 clones using the TCRAJ33 gene segment with a CDR3 of 8 aa. There are two TCRAV7 genes; however, most of the conserved AV7 clones (118/122) utilized the AV7.2 gene segment rather than the AV7.1 gene segment. Diversity in the conserved TCRAV7 transcripts is found in the two residues that border the junction of V and J gene segments. According to the definition of the CDR3 used here, the first variable residue lies in the V region. The germline sequences in part account for the predominance of valine and methionine residues in the variable positions. Codons for valine and methionine residues are present in the AV7 and AJ33 gene segments, respectively. Some diversity arose from variation in the direct joining of V to J gene segments, and additional diversity arose from N region nucleotides. For example, there is 4-fold redundancy in valine codons, and all four codons were found in the clones.

The distribution of conserved TCRAV into CD56+ and CD56- DN T cells

The invariant TCRAV24 has been reported to be present on DN T cells that express NK markers (19, 20, 21). The representation of the other conserved TCRAV families in DN NK+ and NK- populations was investigated. Isolated DN cell populations were separated into NK+ and NK- populations using anti-CD56 magnetic beads, and the resulting NK- populations (CD56-depleted) were >98% pure. Spectratype profiles for AV4A, AV7, AV19, and AV24 are shown in Fig. 8Go. Three of the four individuals studied, donors 2, 5, and 8, expressed all four of the over-represented TCRAV families, whereas donor 6 expressed all but the conserved TCRAV19 sequence.



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  		FIGURE 8. Spectratype profiles of DN/CD56+ and DN/CD56- cell populations from four donors. The size of over-represented CDR3 lengths is indicated by the dashed line in all profiles. The donors expressed all of the over-represented sequences, with the exception of donor 6, who did not express the over-represented AV19 sequence.

 
The conserved TCRAV were present in both DN CD56+ and DN CD56- cell populations. TCRAV4A with a CDR3 length of 9 aa were present in both DN CD56+ and DN CD56- cells. TCRAV4A with other CDR3 lengths were present in both subpopulations, but more predominant in the DN CD56- cells. Conserved TCRAV7 transcripts were present in both DN CD56+ and DN CD56- cell populations. TCRAV19 transcripts with 9 aa CDR3 regions were more prominent in the DN CD56- than in the DN CD56+ cell populations. TCRAV24 transcripts with 11 aa CDR3 lengths were present at approximately equivalent levels in both DN CD56+ and CD56- subpopulations, which was confirmed by FACScan analyses of isolated populations using an Ab to TCRAV24 (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Diversity in the TCR repertoire of circulating peripheral T cells is characterized by a normal distribution of CDR3 lengths within the majority of TCRAV families. Skewing in the distribution of CDR3 lengths for a TCRAV family is sometimes observed in CD8+ and/or DN T cells, but skewing is rarely observed in CD4+ T cells. Comparison of spectratype profiles from multiple unrelated individuals generally reveals patterns of skewed CDR3 lengths specific for each individual. Thus, the observation that conserved TCRAV chains are present in multiple unrelated individuals is unusual and suggests that these cells have an important function.

The T cells with conserved TCRA are puzzling in several ways. First, identical conserved TCRA sequences are found in multiple unrelated individuals, but none of the conserved sequences were detected in all individuals studied. Second, conserved TCRA chains are found on populations of T cells that lack both CD4 and CD8 coreceptor molecules, and, in some individuals, the same conserved TCRA sequences are also found on CD8+ cells. Third, the level of TCRA sequence conservation is extensive in spite of N region additions. Fourth, TCRA chains undergo rearrangement only after a functional TCRB chain has been expressed, yet analyses of TCRB expression have not identified parallel conserved TCRB chains.

Expression of conserved TCRAV sequences in 15 unrelated healthy individuals

The conserved TCRA sequences were found in multiple unrelated individuals from diverse ethnic backgrounds. None of the conserved sequences were observed to be present in all of the individuals studied, although all individuals showed over-representation of at least one of the conserved TCRAV families. Expression of conserved TCRA sequences was stable over time. The same conserved sequences were present, and no additional sequences were detected in an individual at intervals of up to 2 yr. These observations suggest that T cells with conserved TCRA recognize a common Ag that is presented by molecules with limited polymorphism. These data further suggest that the common Ag is persistently present in multiple individuals in the population.

Several studies report that DN T cells with uncharacterized TCR recognize a variety of nonprotein lipid or glycolipid Ags when presented by CD1 molecules (22, 23, 24, 25, 26, 27, 28). Populations of DN T cells with the conserved TCRAV24 have been well characterized in terms of the glycolipid recognized, the cytokines produced in response to Ag, and the participation of costimulatory molecules (17, 18, 19, 20, 21, 29, 30, 31). CD1d has been established to be the Ag-presenting molecule for the invariant TCRAV24-expressing DN T cells.

Expression of conserved TCRAV sequences in T cell subsets

The conserved TCRAV4A, AV7, and AV19 sequences differ from the invariant TCRAV24 in T cell subset localization. TCRAV24 is found on DN and CD4+, but never on CD8+ T cell (19, 20). TCRAV4A and AV7 sequences are present in DN and often in CD8+ T cells. The conserved TCRAV19 sequence was found in the CD8+ population of one of the individuals with it highly represented in their DN population.

A recent study reports that CD8+ T cells can also recognize nonprotein lipids from Mycobacterium tuberculosis in the context of CD1a or CD1c (32). Thus, it is possible that T cells bearing the conserved TCRAV4A, AV7, and AV19 may recognize a nonprotein, lipid, or glycolipid Ag in the context of a highly conserved nonconventional class I-like molecule.

The presence of conserved TCRAV4A and AV7 sequences in CD8+ and DN T cells suggests that these peripheral DN T cells with conserved TCRA chains derive from differentiated CD8+ T cells. Furthermore, CD8+ T cells have been reported to convert to a DN phenotype upon stimulation by specific Ag (33) or a combination of cytokines, PMA, and ionomycin (34). Modulation of coreceptor molecules may not be limited to CD8. The conserved TCRA sequences were found in both CD56+ and CD56- DN subsets, which may be because of CD56 modulation.

Junctional diversity in conserved TCRAV sequences

TCRAV4A, AV7, and AV19 are also distinguished from TCRAV24 by the presence of N region nucleotide additions. TCRAV24 can be the result of a direct joining of TCRAV and TCRAJ gene segments. TCRAV4A and AV19 sequences include N region residues, and, even with the addition of nongermline encoded residues, cDNA sequences from all individuals were identical. Conservation of amino acid sequence suggests selection based upon specificity, but conservation at the mRNA level is puzzling. The possibility that genomic organization would favor certain specific rearrangement events was investigated by examination of the relative positions of the gene segments involved in the conserved sequences within the TCRA gene complex.

The pairs of V and J gene segments found together are not in proximity to one another in the genome, as shown schematically in Fig. 9Go. TCRAV7S1 and AV7S2 are located at the 5' end of the cluster of V gene segments in the TCRAV gene complex, whereas TCRAV19 is located at the 3' end. The TCRAJ gene segments used by the conserved transcripts are dispersed across the AJ region and numbered consecutively 3' to 5' (10). All rearrangement events involve elements that span considerable distance in the TCRA gene complex. In addition, the heptamer and nonomer recombination signal sequences for the paired AV and AJ gene segments were examined. No striking similarities were evident to suggest preferential joining among recombination signal sequences.



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  		FIGURE 9. Schematic representation of the TCRA gene complex showing the positions of the TCRAV and TCRAJ gene segments involved in constructing the conserved TCRA sequences. Lines above and below mark the gene segments juxtaposed in the rearrangement of conserved TCRA sequences.

 
The level of diversity observed in TCRAV7 sequences is very similar to that observed in extensive studies of TCRB from CD8+ T cell clones that recognize a single peptide presented by a single MHC molecule (35, 36, 37, 38, 39) or CD8+ T cells selected by binding of MHC tetramers and Ag (40, 41, 42). In each of these studies, only one TCRBV gene was used with a few BJ genes. The CDR3 lengths of TCR were identical and contained conserved amino acid motifs. These similarities suggest a relationship between the conserved AV7 sequences and CD8+ T cells with a defined specificity. However, the donors expressing the conserved AV7 sequence were from diverse ethnic backgrounds and did not express a common MHC class I Ag.

TCRBV expression with conserved TCRAV

Expression of TCRB has been studied in considerably greater depth than TCRA expression, and conserved sequences in peripheral blood samples from multiple individuals have not been observed. TCRB spectratype profiles of PBMC, and isolated populations of CD4+, CD8+, and DN T cells were examined in parallel with the present studies. No over-represented TCRB chains that corresponded to the over-represented TCRA were observed. Studies of TCRAV24 using a mAb have identified several TCRBV families associated with the conserved {alpha}-chain. Even though certain TCRBV families seem to be associated with the invariant TCRAV24, there is significant diversity in CDR3 lengths and sequences (17, 19, 20). It is also possible that several TCRBV families are used to form the receptor with conserved TCRAV4A, AV7, and AV19 chains. However, any heterogeneity in the TCRB chain paired with a conserved TCRA chain presents an enigma because the TCRB chain undergoes rearrangement before the TCRA chain during development in the thymus.

Origin of peripheral DN T cells

DN T cells represent the earliest stage of differentiation in the thymus. In the mature thymus, DN T cells comprise ~5% of total thymocytes and represent at least three discrete populations. One population expresses TCR {gamma}{delta}, another population expresses TCR{alpha}ß, and the remainder are committed to the {alpha}ß lineage but have yet to rearrange their TCR. Upon expression of a functional TCRB chain, along with the pre-T cell {alpha}-chain, both CD4 and CD8 coreceptor molecules are also expressed on the cell surface (43). The presence of CD4 and CD8 is critical to the positive and negative selective forces that shape the peripheral TCR repertoire.

In the periphery, DN T cells (CD3+ CD4-8-) are principally TCR{gamma}{delta}-positive, and those with TCR{alpha}ß represent only 0.1–2% of the PBMC in healthy individuals. The populations of peripheral DN T cells with conserved TCRA chains may derive from the population of thymic DN T cells; they may derive from CD4+ or CD8+ T cells that have modulated the expression of coreceptor molecules; or, alternatively, they may develop in a thymus-independent manner. Preliminary spectratype results from human thymic tissue did not reveal the over-representation of conserved TCRAV4A, AV7, AV19, and AV24 sequences (unpublished observations). Therefore, if the conserved DN T cells are derived from DN T cells in the thymus, skewing of the conserved populations occurs after these cells have left the thymus, presumably because of stimulation and expansion by Ag.

The widespread presence of DN T cells with conserved TCRA chains in the population indicates that these cells represent effectors of important immune responses. Furthermore, these data suggest that the Ag-presenting molecules must be highly conserved in the human population. DN T cells with conserved TCRA are unusual cells in terms of the expression of TCR and coreceptor molecules. The extent of conservation of TCRA sequences is unparalleled, and the mechanisms used in the generation of diversity may be unconventional. DN T cells have been recently described to recognize nonprotein lipids or glycolipid Ags (22, 23, 24, 25, 26, 27, 28, 29, 30, 31), but the functions of DN T cells with conserved TCRAV4A, AV7, and AV19 remain to be determined.


   Acknowledgments
 
We thank Drs. A. A. Ansari, V. M. Hirsch, and T. J. Kindt for comments on the manuscript. We also thank Ms. N. Cogan for secretarial assistance.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. Mary Ann Robinson, Twinbrook II Facility, 12441 Parklawn Drive, Rockville, MD 20852. E-mail address: Back

2 Abbreviations used in this paper: CDR3, complementarity determining region 3; DN, double-negative T cells; TCRB, TCR ß; TCRA, TCR {alpha}; TCRAV, TCRA variable region; HD, Hamming distance; aa, amino acid. Back

Received for publication March 19, 1999. Accepted for publication April 22, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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