Evidence that human CD8+CD45RA+CD27 cells are induced by antigen and evolve through extensive rounds of division

Dörte Hamann1, Stefan Kostense1, Katja C. Wolthers1, Sigrid A. Otto1, Paul A. Baars1,2, Frank Miedema1,3 and René A. W. van Lier1,2

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


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
We recently showed that circulating human CD8+ effector cells have a CD45RA+CD27 membrane phenotype. In itself this phenotype appeared to pose a paradox: CD45RA, a marker expressed by unprimed cells, combined with absence of CD27, characteristic for chronically stimulated T cells. To investigate whether differentiation towards the CD45RA+CD27 phenotype is dependent on antigenic stimulation and involves cellular division, TCR Vß usage and telomeric restriction fragment (TRF) length were analyzed within distinct peripheral blood CD8+ subsets. FACS analysis showed that the TCR Vß repertoire of CD8+CD45RA+CD27 cells differed significantly from that of unprimed CD8+CD45RA+CD27+ cells. Moreover, in two out of six individuals large expansions of particular Vß families were observed in the CD8+CD45RA+CD27 subset. CDR3 spectrotyping and single-strand confirmation analysis revealed that within the CD8+CD45RA+CD27 population most of the 22 tested Vß families were dominated by oligoclonal expansions. The mean TRF length was found to be 2.3 ± 1.0 kb shorter in the CD8+CD45RA+CD27 subset compared with the unprimed CD8+CD45RA+CD27+ population, but did not differ substantially from that of memory type, CD8+CD45RACD27+ T cells. These findings indicate that the CD8+CD45RA+CD27 cytotoxic effector population consists of antigen-induced, clonally expanded cells and confirm that the expression of CD45RA is not a strict marker of antigen non-experienced T cells.

Keywords: CD8, CD27, CD45RA, single-strand confirmation polymorphism, telomeric restriction fragment


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
The relationship between CD8+ effector and memory cells is not yet fully understood (1). Studies on this issue have been seriously hampered by the fact that cell surface markers that distinguish unprimed and primed cells are not useful in separating memory from effector cells (2). Recently, we have shown that in the human peripheral blood CD8+ T cell population next to unprimed or naive CD45RA+CD27+ cells, two distinct subsets can be defined with phenotypic and functional characteristics of primed cells: a memory type CD45RACD27+ population and a CD45RA+CD27 cytotoxic effector subset (3). The possibility to distinguish CD8+ effector from memory cells based on their membrane phenotype opens up new opportunities to address the question of the lineage relationship.

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.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
mAb
The mAb CLB-CD8/1, CLB-CD14/1, CLB-FcR gran1 (CD16), CLB-CD19/1, biotinylated CLB-CD27/1 and CLB-CD27/1–FITC were all produced at the CLB. Phycoerythrin (PE)-conjugated CD45RA mAb 2H4-RD1 was obtained from Coulter Immunology (Hialeah, FL). FITC-conjugated CD16 and TCR{alpha}ß mAb were from Becton Dickinson (San Jose, CA). TCR Vß mAb directed against Vß5.1, 5.2, 5.3, 6.1, 8.1 and 12.1 (all FITC conjugated) were purchased from T Cell Sciences (Cambridge, MA). TCR VßmAb recognizing Vß3.1, 13.6, 17, 18, 21.3 and 22.1 were from Immunotech (Marseilles, France).

Cell preparation
Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of healthy blood donors (20–40 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+TCR{alpha}ß+CD16 as determined by immunofluorescence analysis with directly labeled mAb. CD8+ cells were either used as a total population or were subsequently stained with CD45RA–PE and CD27–FITC, 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-mouse–FITC staining. After blocking free binding sites of the goat anti-mouse conjugate with 10% normal mouse serum, cells were stained with CD45RA–PE 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 streptavidin–Red 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 2–5x106 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 [{alpha}-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 {lambda}/HindIII.

Statistics
Correlation analysis was performed with Spearman rank correlation.


    Results and discussion
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 
Vß repertoire of CD8+ T cell subsets
The TCR repertoire of mature T cells is determined by negative and positive selection processes in the thymus, which in turn are dependent on the expression of particular MHC class I and II alleles. Next to this, it will be subject to changes imposed by antigenic challenge. Since naive or unprimed T cells by definition have not yet encountered antigen, their pattern of TCR Vß expression should reflect the genetically determined repertoire of an individual. A different pattern of TCR Vß expression in primed cells would imply that these cells have evolved by antigenic stimulation.

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. 1Go). 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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Fig. 1. Vß expression in CD8+ T cell subsets. Purified CD8+ cells were stained with Vß-specific mAb in combination with CD45RA and CD27 mAb and analyzed on a FACScan. Within each indicated subset at least 5000 events were analyzed. Shown are the percentages of Vß cells within the distinct CD8+ subsets of two representative healthy individuals. Either large expansions were found within the CD45RA+CD27 subset (A) or most of the measured Vß families were almost undetectable in this population (B).

 
TCR Vß expression differed substantially between CD8+ subsets (Fig. 1Go and Table 1Go). Whereas Vß family expression in the CD45RA+CD27+ population of all six healthy donors ranged between 0.3 and 7%, two individuals had large expansions of certain Vß subfamilies in both CD27 subsets (being either CD45RA+ or CD45RA ) ranging from 19 to 83% of one particular Vß chain (Fig. 1AGo and data not shown). Remarkably, two of the remaining four individuals showed almost undetectable expression of most of the measured V ß families in the CD45RA+CD27 population (Fig. 1BGo and data not shown). The latter suggests that these individuals had large expansions in Vß families that were not evaluated. Neither expansion nor deletion of certain Vß families were found in the CD45RACD27+ subset (Fig. 1A and BGo). Correlation analysis of Vß expression revealed that the greatest difference was observed between CD45RA+CD27 effector cells and the unprimed CD45RA+CD27+ subset (R = 0.40 ± 0.21). CD45RACD27 cells showed an intermediate correlation with unprimed cells (R = 0.60 ± 0.11). The highest concordance was found between the unprimed CD45RA+CD27+ population and the primed CD45RACD27+ subset (R = 0.70 ± 0.16).


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Table 1. Mean correlation values (R) between CD8+ and CD4+ T cell subsets for the TCR Vß chain expression
 
Both CDR3 spectrotyping and SSCP analysis was performed to investigate whether the observed differences in the Vß family usage between naive CD45RA+CD27+ and primed CD27 cells were related to clonal outgrowth. Due to the limited number of CD8+ cells available from buffy coats of blood donors, determination of Vß expression levels and CDR3 spectrotyping and SSCP analysis could not be done on the same samples, and the latter analyses were therefore performed on purified CD8+ populations from two additional individuals. In total, 22 TCR Vß (1,2,3,4,5,6,7,8,9,11,12,13,14,15,16,17,18,20,21,22,23and24) were analyzed for their CDR3 and SSCP pattern in all subsets. Since the results were similar for all families, representative data are shown for four families (Fig. 2Go). The CD45RA+CD27+ naive subset showed a relatively normal CDR3 size distribution, whereas all primed subsets expressed a skewed CDR3 size pattern ranging from moderate perturbations within the CD45RACD27+ memory cells to highly skewed profiles within both CD45RACD27 and CD45RA+CD27 populations. SSCP analyses confirmed that the disturbed patterns in the primed subsets were dominated by oligoclonal expansions that were again most obvious in both CD27 populations.



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Fig. 2. CDR3 spectrotyping and SSCP analysis of representative Vß families in CD8+ T cell subsets from two healthy blood donors. Purified CD8+ cells were sorted into the indicated populations to a minimum of 106 cells/subset. CDR3 were amplified by Vß-specific PCR. CDR3 sizes were analyzed on an automatic sequencer and CDR3 sequence diversity was assessed by SSCP profiles.

 
Our findings show that post-thymic antigen-dependent differentiation of unprimed CD8+ cells is accompanied by considerable changes of the {alpha}ß TCR repertoire which are most evident in the CD45RA+CD27 cytotoxic effector population.

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 1Go). 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 {alpha}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 4–14% 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 BGo). 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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Fig. 3. (A) Southern blot of telomeric DNA from CD45RA+CD27+, CD45RA+CD27 and CD45RACD27+ CD8+ T cell subsets from one representative donor. Genomic DNA was isolated from the purified subsets, digested, separated by electrophoresis, blotted and hybridized to the radiolabeled probe (TTAGGG)5. Autoradiography was performed overnight. The mol. wt marker is shown with molecular sizes in the right column. Calculation of the mean telomere length was performed using Phosphor-Imager software which determines the integrated signal of the area above the background. The mean value in kb was calculated using the mol. wt marker {lambda}/Hin dIII. (B) Mean telomere length of CD8+ T cell subsets. Individual donors are represented by distinct symbols.

 
To get information about the lineage relationship between CD45RACD27+ memory-type cells and CD45RA+CD27 effector cells, the mean TRF length of these two populations was compared. The average mean TRF length was not significantly different between CD45RA+CD27 effector cells (8.8 ± 2.7) and memory-type CD45RACD27+ cells (8.2 ± 1.9). Given the limited sensitivity of the technique and the variation observed between donors, a definite answer to the question whether CD45RA+CD27 effector cells have evolved from the CD45RACD27+ memory-type subset cannot be given.

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 TCR–antigen 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.


    Abbreviations
 
PBMCperipheral blood mononuclear cell
SSCPsingle-strand confirmation polymorphism
TRFtelomeric restriction fragment

    Notes
 
Transmitting editor: K. Okumura

Received 6 July 1998, accepted 10 March 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 

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