1 Department of Clinical Viro-Immunology and
2 Department of Immunobiology, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB) and Laboratory of Experimental and Clinical Immunology, Academic Medical Centre, 1066 CX Amsterdam, The Netherlands
3 Department of Human Retrovirology, Academic Medical Centre, University of Amsterdam,1066 CX Amsterdam, The Netherlands
Correspondence to: R. A. W. van Lier, Dept. Clinical Viro-Immunology, CLB, Plesmanlaan 125, 1066 CX Amsterdam, the Netherlands
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Abstract |
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Keywords: CD8, CD27, CD45RA, single-strand confirmation polymorphism, telomeric restriction fragment
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Introduction |
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T cells being either CD4+ or CD8+ irreversibly switch off CD27 expression when stimulated for prolonged periods (4,5). On the other hand, although rare CD8+ clones that can express CD45RA under certain conditions have been described (6), activation of unprimed CD8+ T cells results in the loss of CD45RA expression and gain of CD45RO expression (7 and D. Hamann, unpublished data). Therefore, until now it has been impossible to address the mechanism of the generation of CD8+CD45RA+CD27 effector T cells using in vitro culture systems. Since as mentioned above TCR-induced activation induces a rapid loss of the CD45RA expression from the surface of dividing T cells, the presence of the CD45RA antigen could imply that these cells have developed by differentiation from naive cells without cellular division. During this process the CD27 molecule is down-regulated but CD45RA expression is unaltered. Indeed, we have provided evidence that the differentiation of CD27+ cells into CD27 cells in the CD4+CD45RO+ memory compartment occurs without substantial cellular division (8). Alternatively, CD45RA+CD27 cells could have developed from CD45RA+CD27+ cells via CD45RACD27+ and CD45RACD27 stages, including proliferation and switch to CD45RO expression, down-regulation of CD27 expression, and re-expression of CD45RA.
Here we studied Vß usage and telomeric restriction fragment (TRF) length of distinct CD8+ cell populations to address the question whether the differentiation into CD8+CD45RA+CD27 effector T cells is dependent on antigen-specific stimulation and whether this process involves extensive cellular division.
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Methods |
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Cell preparation
Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy blood donors (2040 years of age) by Ficoll-Isopaque density centrifugation (Pharmacia, Uppsala, Sweden). CD8+ T cells were generated by positive enrichment using the MACS system (Miltenyi, Bergisch Gladbach, Germany) as previously described (3). Briefly, PBMC were stained with CD8 microbeads and enrichment was performed with BS columns (capacity: 108 cells) using the VarioMACS magnet according to the manufacturer's instructions. The resulting CD8+ T cells were >98% CD8+TCRß+CD16 as determined by immunofluorescence analysis with directly labeled mAb. CD8+ cells were either used as a total population or were subsequently stained with CD45RAPE and CD27FITC, and sorted into CD45RA+CD27+, CD45RA+CD27 and CD45RACD27+ populations (purity >98%) on a FACStar (Becton Dickinson, Mountain View, CA). CD4+ T cells were purified by incubating the PBMC with CD8, CD14, CD16 and CD19 mAb followed by negative depletion with Dynabeads-M450 (Dynal, Oslo, Norway). The purity of this population was >99%.
Analysis of TCR Vß expression with mAb
Triple-color immunofluorescence analysis was performed as previously described (8). Briefly, purified CD4+ or CD8+ T cells were first incubated with an unlabeled TCR V
ß-specific mAb followed by goat anti-mouseFITC staining. After blocking free binding sites of the goat anti-mouse conjugate with 10% normal mouse serum, cells were stained with CD45RAPE mAb and biotinylated CD27 mAb. Alternatively, cells were simultaneously incubated with a FITC-conjugated TCR Vß-specific mAb, PE-labeled CD45RA mAb and biotinylated CD27 mAb. The latter was subsequently detected with streptavidinRed 670 (Life Technologies, Gaithersburg, MD).
Evaluation of the TCR Vß repertoire with CDR3 spectrotyping and single-strand confirmation polymorphism (SSCP) analysis
CDR3 spectrotyping and SSCP analysis was performed as previously described (9). Purified CD8+ cells from two healthy blood donors were prepared as described above, and subsequently sorted into CD45RA+CD27+, CD45RACD27+, CD45RACD27 and CD45RA+CD27 populations to a minimum of 106 cells/subset. RNA was isolated and cDNA synthesized according to the manufacturer's protocol (Life Technologies). Primary PCR was performed with paired TCR Vß primers (10) and was adjusted for total TCR Vß cDNA yield per sample (11). For nested PCR (11), single TCR Vß primers were used, in combination with a TCR Cß primer, labeled with fluorescent 6FAM or HEX.
CDR3 pattern analysis.
PCR products were heated for 2 min at 92°C and run on a 5% polyacrylamide gel together with a TAMRA-labeled size standard. CDR3 peak patterns were visualized and analyzed using an ABI-377 DNA sequencer (Perkin Elmer, Foster City, CA).
SSCP analysis.
TCR Vß amplification products were denatured at 92°C for 2 min and run on a neutral 5% polyacrylamide + 5% glycerol gel. The gel was kept at 30°C to allow the single-strand products to fold according to their sequence during the run. An ABI-377 sequencer was used to analyze the sequence-dependent mobility profiles.
Determination of the TRF length
TRF length in CD8+ T cell subsets was analyzed by the Southern blot technique. DNA was isolated from 25x106 cells of each subset by the Qiagen Blood and Body Fluid Protocol according to the manufacturer's instructions (Qiagen, Hilden, Germany). Genomic DNA (5 µg) was digested with 40 U of HinfI and RsaI (Life Technologies), and completeness of digestion was monitored by gel electrophoresis. The digested DNA was electrophoresed on 0.6% agarose gels (50 mA, 24 h). Gels were then denatured in 0.25 N HCl and neutralized in 0.4 N NaOH/0.6 M NaCl, and blotted to Genescreen Plus (DuPont) in 0.5 N NaOH/1.5 M NaCl. Blots were washed twice in 2xSSC and cross-linked (Stratalinker; Stratagene). The telomeric probe (TTAGGG)5 was radiolabeled with [-32P]dCTP with terminal transferase (Boehringer Mannheim, Almere, The Netherlands). Hybridization was at 65°C in 0.5 M Na2HPO4/7% SDS (pH 7.2). Blots were washed with 3xSSC/0.5% SDS decreasing to 0.1xSSC/0.5% SDS (15 min at 65°C). Blots were exposed to Phosphor-Imager screens (Fuji, Kanegawa, Japan) for 4 h or overnight and mean telomere length was analyzed by Phosphor-Imager software (TINA; Raytest, Straubenhardt, Germany) which determines the integrated signal of the area above the background. The mean value in kb was calculated using the mol. wt marker
/HindIII.
Statistics
Correlation analysis was performed with Spearman rank correlation.
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Results and discussion |
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The expression of TCR Vß elements in distinct T cell subsets was determined with Vß-specific mAb and flow cytometric analysis. Antibodies directed against 12 different Vß subfamilies were used that cover ~30% of the repertoire in both the CD4+ and CD8+ subsets (see Fig. 1). As a measure of concordance of the Vß repertoire between two given subsets, correlation coefficients (Spearman correlation) were calculated (R = 1, being a perfect correlation and R = 0, being no correlation). Thus, changes in the TCR Vß repertoire due to antigenic selection in primed populations would result in a low correlation coefficient when compared to the unprimed cells.
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Interestingly, in contrast to CD8+ cells, within the CD4+ population overall a relative similarity in the Vß repertoires of different subsets was observed (mean R values >0.74) (Table 1). Primed CD45RACD27+ cells had an almost identical Vß usage (R = 0.92 ± 0.02) as unprimed CD45RA+CD27+ cells. These data demonstrate that the antigen-dependent development of CD45RACD27+ cells from CD45RA+CD27+ cells does not lead to obvious modulations of the Vß repertoire. This is in accordance with earlier studies that revealed a remarkable similarity in the Vß usage of circulating CD4+ T cells between monozygotic twins (12,13). However, the Vß usage of highly differentiated CD45RACD27 effector cells was less comparable with that of unprimed CD45RA+CD27+ cells (R = 0.74 ± 0.11). This lower concordance suggests that prolonged antigenic stimulation (4, 1200
5) eventually does induce alterations of the
ß TCR repertoire of CD4+ T cells. In contrast to the CD8+ population, CD45RA+CD27 cells are almost absent from the peripheral CD4+ population of healthy adults (<1%) (8) and could therefore not be analyzed.
In conclusion, post-thymic alterations of the TCR repertoire are detectable in the CD27 subsets within both the CD8+ and the CD4+ compartment, suggesting that CD27 cells have been selected in vivo through antigenic stimulation. In the CD8+ compartment, the CD27 subset can comprise up to 50% of the total population in apparently healthy individuals (3). Changes in the Vß repertoire will therefore become apparent if the total CD8+ population is analyzed. In line with this, differences in Vß expression of the entire CD8 subset have been reported in monozygotic twins that were especially marked where one individual had an underlying disease (13). In contrast, since CD45RACD27 cells comprise only 414% of the circulating CD4+ cells in healthy individuals (8) moderate changes in their Vß expression pattern will not become evident if the total CD4+ 1300 population is studied.
Determination of the mean telomere length of CD8+ T cell subsets
The question whether CD8+CD45RA+CD27 cell development is accompanied by extensive cellular division was addressed by determining the average TRF length of the distinct CD8+ subsets. Telomeres consist of several thousand repeats of hexameric sequences at the end of every chromosome (14). In somatic cells, the average telomere length shortens between 50 and 100 bp with each round of replication (15,16). Analysis of the telomere length thus allows an assessment of the replicative history of a cell population.
To investigate TRF length, purified CD8+ T cells from six healthy donors were sorted into CD45RA+CD27+, CD45RA+CD27 and CD45RACD27+ subsets and telomeric DNA was analyzed by Southern blot technique (Fig. 3A and B). Due to the low frequency of CD45RACD27 cells in the peripheral blood of healthy donors (mean 4 ± 3%) (3) this subset was not accessible for this type of analysis. In all six donors studied, mean TRF length of primed CD45RACD27 1400
+ cells was shorter than that of unprimed CD45RA+CD27+ cells (mean loss 1.8 ± 0.8 kb) which corresponds well to previous data showing a 1.4 kb difference between unprimed and primed CD4+ cells (17). The same was true for CD45RA+CD27 cytotoxic effector cells that had a 2.3 ± 1.0 kb shorter TRF length compared to CD45RA+CD27+ unprimed cells. This finding demonstrates that the cytotoxic effector population has evolved by extensive cellular division in vivo. Our data are in good agreement with the differences in TRF length in CD8+ T cell subsets as determined by a recently developed fluorescent in situ hybridization technique (18). Although cellular division induced by stimulation via the TCR appears to be accompanied by a down-regulation of the CD45RA isoform expression (19), CD4+CD45RA+ T cells have been shown to proliferate in vitro upon culture with a combination of cytokines without switching to CD45RO expression (2022). However, under these conditions CD4+CD45RA+ cells did not down-regulate CD27 expression (D. Hamann, unpublished observation). Moreover, this antigen-independent proliferation should not alter the TCR Vß repertoire of the cells. Thus, the finding of shortened TRF in combination with an altered Vß repertoire implies that effector cells do not directly differentiate from the unprimed CD45RA+CD27+ pool, but rather may acquire the CD45RA 1500
+ CD27 phenotype via transition through a CD45RA stage.
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Loss of CD27 appears to be an irreversible differentiation event (5), which is at variance with the linear differentiation model, that predicts that whereas most of the effectors die, some cells survive and constitute the memory pool (1). Rather, the finding that the mean TRF length of CD45RA+CD27 cells is in the same range as that of CD45RACD27+ cells fits with a model in which initial encounter of antigen by CD8+ cells leads to activation and clonal proliferation as a consequence of which cells loose CD45RA expression and reach a CD45RACD27+ 1600 stage. Dependent on the strength of the TCRantigen interaction together with the expression of certain co-stimulatory molecules on the antigen-presenting cell, cells from this population will stop proliferating and further differentiate into effector cells, whereas others will compose the memory pool (23). We have previously shown that CD8+CD45RA+CD27 cells share a number of phenotypic and functional characteristics with the CD8+CD28 population, including high expression of CD11b and CD57, cytotoxic activity, and poor proliferative potential (3). Interestingly, mean TRF length of CD8+CD28 has been demonstrated to be on average 1.4 kb shorter compared to CD8+CD28+ cells in healthy individuals (24). Shortened TRF have also been described in the CD8+CD28 population of HIV-1-infected individuals (25). From this it was concluded that CD28 cells may have reached a state of replicative senescence causally related with TRF loss (2426). The shortening in TRF length described in the CD8+CD28 population is comparable to that found in the CD45RA+CD27 cytotoxic effector population, suggesting that the low proliferative potential of CD45RA+CD27 effector cells could be a consequence of their relatively short TRF. However, our finding that CD45RA+ 1700 CD27 effector cells and CD45RACD27+ memory-type cells have a comparable low mean TRF length demonstrates that the poor proliferative capacity of the CD45RA+CD27 cells cannot simply be explained by a state of replicative senescence since memory type CD45RACD27+ cells extensively proliferate when stimulated in vitro (3).
Concluding remarks
The skewed Vß repertoire and the shortened telomeres support the idea that CD8+CD45RA+CD27 effector T cells are antigen-primed, clonally expanded cells. The combined data imply that CD8+CD45RA+CD27 cells do not directly differentiate from the unprimed CD8+CD45RA+CD27+ pool but rather develop via CD8+CD45RACD27+ and CD8+ 1800
CD45RACD27 stages. In our opinion re-expression of the CD45RA on the fully differentiated effector cells reflects the inability of these cells to become engaged in TCR-induced proliferation. Importantly, in accordance with recent data obtained in animal models (27), the data imply that one has to be cautious interpreting the expression of CD45RA on human T cells as a token of antigenic virginity.
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Abbreviations |
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PBMC | peripheral blood mononuclear cell |
SSCP | single-strand confirmation polymorphism |
TRF | telomeric restriction fragment |
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Notes |
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Received 6 July 1998, accepted 10 March 1999.
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References |
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