- Original Articles
- Open Access
Oligoclonality in the Human CD8+ T Cell Repertoire in Normal Subjects and Monozygotic Twins: Implications for Studies of Infectious and Autoimmune Diseases
© Molecular Medicine 1995
- Published: 1 September 1995
We have previously demonstrated CD8+ T cell clonal dominance using a PCR assay for the CDR3 length of T cell receptors belonging to a limited number of TCRBV segments/families. In this study, we have modified this approach in order to analyze more comprehensively the frequency of oligoclonality in the CD8+ T cell subset in 25 known TCRBV segments/families. In order to assess the relative roles of genes and environment in the shaping of a clonally restricted CD8+ T cell repertoire, we have analyzed clonal dominance in the CD8+ T cell population of monozygotic twins, related siblings, and adoptees.
Materials and Methods
Oligoclonality was assessed in the CD8+ T cell subsets using a multiplex PCR approach to assay for CDR3 length variation across 25 different TCRBV segments/families. Specific criteria for oligoclonality were established, and confirmed by direct sequence analysis of the PCR products. This assay was used to investigate the CD8+ T cell repertoire of 56 normal subjects, as well as six sets of monozygotic (MZ) twins.
Seventy-two percent of normal subjects (n = 56) had evidence of oligoclonality in the CD8+ T cell subset, using well-defined criteria. Although MZ twins frequently displayed CD8+ T cell clonal dominance, the overall pattern of oligoclonality was very diverse within each twin pair. However, we occasionally observed dominant CD8+ T cell clones that were highly similar in sequence in both members of some twin pairs. Not a single example of such similarity was observed in normal controls or siblings.
Oligoclonality of circulating CD8+ T cells is a characteristic feature of the human immune system; both host genetic factors and environment shape the pattern of oligoclonality in this T cell subset. The high frequency of this phenomenon in normal subjects provides a background with which to evaluate CD8+ T cell oligoclonality in the setting of infection or autoimmune disease. Further phenotypic and functional characterization of these clonally expanded T cells should provide insight into normal immune homeostasis.
The majority of peripheral T cells recognize peptide/MHC complexes by means of a clonotypic α /β T cell receptor (1). The potential repertoire of these clonotypic receptors is very large, in major part due to the somatic diversification which takes place during thymocyte development (2). Therefore, extreme clonality within the peripheral T cell compartment has generally been viewed as due to malignant or premalignant transformation of such cells. However, recent studies have shown that quite restricted clonotypes may arise in the course of some antigen specific responses (3, 4) and that even in the absence of malignancy or an obvious antigenic challenge, oligoclonal dominance and/or expansion can occasionally be observed in the peripheral CD8+ T cell compartment (5–8).
The T cell receptor repertoire has been studied in a wide variety of human diseases including autoimmune (7, 9, 10), infectious (11), and neoplastic (12) disorders. In some instances, oligoclonality has been found in blood or at sites of inflammation. In order to interpret these studies, it is necessary to characterize the normal T cell receptor repertoire with regard to the frequency of oligoclonality. We have previously demonstrated CD8+ T cell clonal dominance in normal individuals using a polymerase chain reaction (PCR) assay for the CDR3 length of T cell receptors for a limited number of TCRBV segment/families (8). We have now modified this approach in order to analyze more comprehensively the frequency of oligoclonality in the CD8+ T cell population. The results show that oligoclonality is an extremely frequent and characteristic feature of the CD8+ T cell repertoire in normal subjects. Studies of monozygotic twin pairs indicate a large role for environment in the generation of this oligoclonality, a finding consistent with our previous observation that CD8+ clonal dominance is enriched in the memory (CD45RO+) cell compartment (8). Therefore, this feature of the CD8+ T cell repertoire probably reflects previous or ongoing immune recognition events; the ubiquitous nature of CD8+ oligoclonality must be taken into account when interpreting T cell repertoire studies in the setting of human disease.
Immunomagnetic Separation of CD4+ and CD8+ T Cells
Peripheral blood mononuclear cells (PBMC) were obtained from the peripheral blood of the study subjects, by centrifugation over Ficoll-Hypaque (Lymphoprep; Nyegaard, Oslo, Norway). The cells were washed twice in Dulbecco’s modified Eagle’s medium (DMEM)-1% FCS and resuspended in DMEM-10% FCS at a concentration of 10 to 20 × 106 cells/ml. Positive selection for CD8+ cells was carried out by incubating the PBMC with anti-CD8 immunomagnetic beads (DYNAL, Great Neck, NY, U.S.A.) for 30 min at 4°C on a rotating shaker, as recommended by the manufacturer. The unbound cells were then incubated with anti-CD4 immunomagnetic beads for selection of CD4+ T cells. The cells bound to the beads were placed directly into RNAzol (Biotecx, Houston, TX, U.S.A.) for isolation of total RNA.
Multiplex PCR Assay for CDR3 Length of TCRBV Chains
Sequence of the primers used
Specific combinations of 2 or 3 Vβ (BV)-specific forward primers (20 pmoles each) were used for each multiplex PCR reaction. Each reaction also contained 20 pmoles of a reverse primer specific for the C region (CB-R), out of which 3 pmoles were end labeled with 32P using T4 kinase (GIBCO-BRL). The BV primer combinations were selected on the basis of the location of each V specific primer with respect to the end of the V region. The different reaction sets used were as follows: A: TCRBV1,18,23; B: BV2,4,8; C: BV3,13S1; D: BV5S2,5S1; E: BV6,20; F: BV7,22; G: BV9,16; H: BV11,12; I: BV15,13S2; J: BV14,17; K: BV19,24; and L: BV10,21. Figure 1 represents the strategy used in a multiplex PCR assay using a combination of upstream TCRBV1, 18, and 23 specific primers and a common radiolabeled CB-R primer.
A total of 10 µ1 of the cDNA was used for each PCR reaction. Conditions for the PCR on the cycle sequencer (model 9600; Perkin-Elmer Corp., Norwalk, CT, U.S.A.) were as follows: denaturation at 94°C for 30 sec, annealing at 55°C for 30 sec and extension at 72°C for 1 min. PCR buffer conditions were Tris HCL 10 mM pH 8.3, MgCl2 2 mM, 50 mM KCL, with 20 pmoles of each primer in a 100-µ1 reaction volume. After 35 cycles, an additional extension at 72°C for 10 min was carried out. Five microliters of the amplified products were loaded on a standard 6% Polyacrylamide sequencing gel. Bands (spaced three base pairs apart) were visualized after overnight exposure to Kodak AR film. The radioactive bands were also analyzed using a phosphorimager (model Phosphorlmmager SF; Molecular Dynamics, Sunnyvale, CA, U.S.A.).
Direct Sequencing of the PCR Amplified Products
In selected samples, the cDNA was reamplified using a single TCRBV segment primer and an unlabeled CB-R/BJ-R primer. The amplified product was used as a substrate for DNA sequencing using the CB-R/BJ-R primer, following purification over Magic Prep DNA purification columns (Promega, Madison, WI, U.S.A.). Direct sequencing of the PCR products was carried out with fluorescent dideoxy terminators and analyzed on an Applied Biosystems Model 373A Automated Sequenator (Foster City, CA, U.S.A.). The sequences were further confirmed using specific BJ-R primers (Table 1) for sequencing the same PCR products.
In samples where direct sequencing was not possible, the PCR amplified products were cloned using the TA cloning system (Novagen, Madison, WI, U.S.A.). Plasmid DNA was extracted from the resulting recombinant clones using the Magic Miniprep DNA purification system (Promega, Madison, WI, U.S.A.). The plasmid DNA was sequenced as described above using the T7 promoter primer.
Results of direct sequencing of PCR products to establish criteria for oligoclonality by CDR3 length analysis
Single Clonal Sequencea
Counts in Dominant Band
CB-R or BJ-R (%)
1 nt. Position (%)
2 nt. Positions (%)
3 nt. Positions (%)
>3 nt. Positions (%)
≧50% (n = 34)
45–50% (n = 15)
Four families of healthy related siblings with two to five members in each, with ages ranging between 18 and 40 years participated in this study. A pair of healthy adoptees of ages 19 and 20 years who were raised together for 19 years were also included in this study.
Multiplex PCR Assay for Oligoclonality of the TCRBV Chain
Criteria for Clonal Dominance by CDR3 Length Analysis
Restriction in CDR3 length within a particular BV segment is visualized as a dominant band depending on its intensity with respect to the rest of the bands within the TCRBV specific PCR product. The radioactive gels were scanned using a phosphorimager and specific criteria were used to define a band as “dominant”-namely, that >50% of the counts within a V segment/family are contained within the dominant band. The clonality of these dominant bands was established by direct sequencing of the PCR product with the CB-R primer. As shown in Table 2, 16/34 instances (47%), in which the counts within the dominant band were 50% or greater, a single readable sequence was obtained using the CB-R primer. Sequencing with a specific BJ-R primer (selected on the basis of the first CB-R primed sequence) yielded a cleanly readable sequence in an additional 35% of cases. Thus, in fully, 82% of the cases in which the 50% criteria for dominance were met, direct sequencing of the PCR product yielded a single dominant sequence. If we reduced the stringency of our criteria for “dominance” to 45–50% of the counts contained in a single band, direct sequencing with the CB-R primer resulted in a clear readable sequence in only 1 out of 15 cases (6%). However, sequencing with a specific BJ-R primer worked in an additional nine cases.
In the remaining cases (18%) where the 50% criteria for dominance were met, some ambiguity of sequence was observed at one or more nucleotide positions, as shown in Table 2. Thus, for example, 9% of the sequences in this group had discrepancies at more than three nucleotide positions, although even in these instances an underlying group of related sequences was obviously present rather than a mixed random sequence. A similar situation is observed when a 45–50% criteria is used, although a larger number of sequences with ambiguous nucleotides were present. Therefore, we have preliminarily settled on the 50% criteria as an reflection of oligoclonality; this almost certainly underestimates the frequency of this phenomenon.
Occasionally, two or more apparently “dominant” bands are appreciated on visual inspection of the gel (for example, see Fig. 4, twin 5K). Direct sequencing of such a PCR product results in a mixed sequence. This problem can be resolved by molecular cloning of the PCR products followed by sequencing of the plasmid clones. This situation was infrequent; except as noted, these instances are not included in the data reported below.
Clonality within the CD8 TCR Repertoire of Normal Individuals
The multiplex PCR assay for CDR3 length was used to assess the CD8+ T cell receptor repertoire in 46 unrelated normal individuals. Clonal dominance was observed in all 25 TCRBV segments/families studied, although with varying frequencies as represented in Fig. 3. Overall, a higher frequency (>10% of subjects) of CD8+ T cell clonality involving TCRBV5S1, -8, -10, -14 and -15 was observed while relatively few instances (<2% of subjects) of clonality involving TCRBV3, -4, -9, -13S2, -17, -22, and -23 were detected. The typical pattern of the radioactive bands spaced three base pairs apart following separation through a sequencing gel was not observed in TCRBV19 PCR products. This is consistent with a nonfunctional gene segment (M. A. Robinson, personal communication) and therefore, we eliminated TCRBV19 from our subsequent analysis. Oligoclonality was occasionally observed in CD8+ T cells bearing the TCRBV10 segment. However, the band patterns were often heterogeneous; clear separation of bands 3 bp apart, was not observed in all the individuals tested, perhaps suggesting
MZ Twins Frequently Display Different Patterns of Clonal Dominance in the CD8+ T Cell Population
MZ twin sets used for study and the corresponding TCRBV segments in which CD8 + oligoclonality was observed
CD8 Oligoclonality in TCRBV
9, 11, 15, 16
1, 5S1, 8, 11, 17, 24
5S2, 7, 12, 13S2, 16
8, 13S2, 19
Infrequent Occurrence of Similar Dominant Sequences in MZ Twin Pairs
Sequence Analysis of TCRBV Chains in MZ Twin Pairs
Nucleotide and amino acid sequences of the CDR3 regions of TCRBV chains which are dominant in both members of twin sets 2, 5, and 6
T T S G K acg act agc ggg aaa
T T S G K acg act agc ggg aaa
D P G T L G I gac cca ggg act ctg ggt atc
D P G T L A gac ccc ggg acc tta gct
D P G Q L A I gat ccg gga cag ctc gct atc
F G D M ttt ggg gat atg
F G D M ttc ggc gac atg
In the case of twin set 5, direct sequencing of the BV2 PCR products from both members with CB-R primer yielded a mixed sequence probably due to the presence of more than one dominant band within the BV2 PCR product of 5K, and the fact that <50% of the counts were contained in the dominant band of 5J (Fig. 4). Therefore, the BV2 PCR products from both members of twin set 5 were molecularly cloned using the TA cloning system. In twin member 5K, three out of three molecular clones containing TCRs with a CDR3 length of 12 aa had the same sequence, “DPGTLGI”. This was also observed following direct sequencing of the TCRBV2-specific PCR product with the BJ1S2 primer. In twin member 5J, two out of three clones containing TCRs with a CDR3 length of 12 aa had the same sequence, “DPGTLA”, while the third clone contained a very similar TCRBV2 sequence, “DPGQLAI”. The “DPGTLA” sequence was confirmed by direct sequencing of the BV2 PCR products using the appropriate BJ sequencing primer (BJ1S2). Thus, the TCR clones from both members of this twin set shared a common sequence of “DPGTL” in the first five residues of the N-D-N region. The codons for this stretch of amino acids were not identical (Table 4).
PCR Analysis with V-J Primer Pairs to Detect Underlying Clonal Dominance in Twin Pairs
Siblings Display Diverse Patterns of CD8+ T Cell Oligoclonality
In order to investigate the relative roles of genetic and nongenetic factors in the shaping of the CD8+ T cell repertoire, normal healthy related siblings and adoptees were chosen as a control population with which to compare the results on MZ twins. Related siblings from four families were tested for shared TCRs within their oligoclonal CD8+ T cell populations. Fifteen instances of clonal dominance involving 10 different TCRBV families were observed in these 12 individuals. However, there were no instances of shared clonal dominance observed between any related sibling pair. Oligoclonality was also tested in a pair of unrelated siblings (i.e., adoptees who had lived together for 19 years). Clonal dominance involving TCRBV10 was seen in a single instance in one adopted sibling and was not present in the other adoptee. Therefore, no apparent similarities were observed between the CD8+ T cell receptor repertoires in individuals who have shared similar environments during their childhood.
In this study we have established that prominent oligoclonality within the CD8+ T cell population is an extremely common and characteristic feature of the human immune system. Previous reports from this (8) and other laboratories (5, 6) have indicated that this phenomenon is occasionally observed in normal individuals. However, we have now comprehensively assessed the presence of oligoclonality in a population of 60 individuals and have shown that the majority of subjects exhibit evidence of oligoclonality within the CD8+ T cell repertoire.
In order to study T cell populations for the presence of oligoclonal dominance, we have modified previously described assays for the CDR3 length of T cell receptor β chain (8, 17, 18) to allow for the analysis of multiple TCRBV families within a single PCR reaction. In this manner, we were able to assess the presence of oligoclonality in 26 TCRBV segments/families using only 12 PCR reactions. In addition, we have developed criteria for identifying oligoclonality based upon direct sequence analysis of these PCR products. As shown in Table 2, we have found that when a band of one length contains >50% of the counts within a given V segment amplification group, then direct sequencing of the PCR product almost always (82% of the time) reveals a single dominant sequence. If a less stringent criterion is used for clonal dominance (45–50% of the counts in the dominant band), the success rate of finding an unambiguous dominant sequence using CB-R as the sequencing primer is substantially lower (Table 2). Thus we have settled on the 50% value as a reasonable starting point for estimating the frequency of clonal dominance, although it clearly underestimates the phenomenon overall.
Having established preliminary criteria for detecting oligoclonality within T cell populations, we determined the frequency of this phenomenon in a group of unrelated normal subjects (n = 46). When CD4+ T cells were examined, not a single instance of clonal dominance was detected in 12 individuals, consistent with our previous observations (8). In contrast, 72% (33/46) of subjects had evidence of clonal dominance involving at least one V segment family in the CD8+ T cell subset. The distribution of oligoclonality among the different V segment families is shown in Fig. 3, and reveals that this phenomenon can involve all the V segments/families under study. On average, 1.5 instances of clonal dominance per individual are found in the CD8+ T cells of the normal population. A weak correlation was observed between the age of the subjects and the number of CD8+ T cell clones detected, although subjects in their teens also displayed four to five CD8 clones per individual.
In our previous, more limited analysis (8), we observed the appearance of clonally dominant CD8+ T cells after immunization and established that these cells are markedly enriched in the CD45RO+ subset. Thus, although there appears to be a considerable influence of environment on the appearance of these cells, we wondered whether host genetic factors might also control the specificity of these clones. We therefore compared the oligoclonality of the CD8+ T cell repertoire in six sets of monozygotic twins. Most of the twin pairs exhibited very diverse patterns of clonality in the CD8+ compartment. Furthermore, reanalysis of some twin pairs with selected V-J combinations to search for any “hidden” similarities resulted in the detection of just one shared clonality out of 10 instances.
However, in three different MZ twins, we found highly similar or identical TCRB receptors in both members of the twin pair (see Fig. 4 and Table 4). In twin pair 6, identical amino acid sequences were encoded by different nucleotide sequences, strongly indicating a role for selection in the generation of these clonally dominant receptors. In order to control for the possible effects of a shared environment in generating this phenomenon, we searched for evidence of shared patterns of oligoclonality among the siblings of four families and in one pair of genetically unrelated adoptees raised together from the first few months of life. We did not observe any instance of similar clonality among these 14 subjects, nor among any of our normal control population. Although the number of control observations is small, the data suggest that host genetic factors do play a role in the patterns of oligoclonality we have observed in the CD8+ T cell population. A larger study of unrelated adoptees raised together from birth will be required to definitively establish this fact.
Two of the twin pairs we studied were discordant for an autoimmune disease. Although there were differences in the patterns of CD8+ oligoclonality in these twin pairs, the high frequency of differences among normal MZ twins emphasizes the difficulty of interpreting such data. Indeed, recent studies on antigen-specific T cell responses in normal MZ twins suggest a highly variable pattern of immune responses can occur in such individuals (19). Thus, it is risky to ascribe T cell repertoire differences in twins as necessarily being due to the presence of disease in one member of the twin pair.
These results indicated that there exists a large amount of diversity in the patterns of CD8+ T cell oligoclonality, with complex genetic and environmental contributions to this phenomenon. The function of these oligoclonal cell populations is obviously of some interest. We have recently found that, in normal individuals, oligoclonal expansion of these cells is frequently enriched within the CD57+ subset of CD8+ T cells (20), a population which appears to have regulatory properties (21). Interestingly, recent work by Behar et al. indicates that in patients with rheumatoid arthritis, clonally expanded CD8+ T cell populations can exhibit autologous reactivity (22). In the setting of rheumatoid arthritis, CD8+ oligoclonality may preferentially involve particular V segment families, including TCRAV12 (7) and TCRBV3 (Ref. 23 and R. Hingorani et al., submitted). These findings suggest that further characterization of this cell population may provide insight into normal immune homeostasis and contribute to an understanding of variations in disease activity in patients with autoimmune diseases.
This work was supported by grants to PKG from the National Multiple Sclerosis Foundation and the National Institutes of Health (NIH) (AI33454). JS is supported by NIH Grants AI22005 and AR40982 as well as a grant from the Juvenile Diabetes Foundation. JM and RH are recipients of posdoctoral fellowship awards from the American Heart Association. Generous support was also provided to PKG by Mr. and Mrs. James C. Dudley and the Guilford Fund. We thank Velia Trejo for the analysis of androgen receptor polymorphism in MZ twin pairs. Colleen Millan for expert assistance with DNA sequencing, and Dr. Charles Christian for access to MZ twin pairs.
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