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Long-Term Stable Expanded Human CD4⁺ T Cell Clones Specific for Human Cytomegalovirus Are Distributed in Both CD45RA^high and CD45RO^high Populations¹

Department of Medicine, University of Cambridge Clinical School, Cambridge, United Kingdom

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
T cells play an important role in the control of human CMV (HCMV) infection. Peripheral blood CD4⁺ T cell proliferative responses to the HCMV lower tegument protein pp65 have been detected in most healthy HCMV carriers. To analyze the clonal composition of the CD4⁺ T cell response against HCMV pp65, we characterized three MHC class II-restricted peptide epitopes within pp65 in virus carriers. In limiting dilution analysis, we observed high frequencies of pp65 peptide-specific CD4⁺ T cells, many of which expressed peptide-specific cytotoxicity in addition to IFN- secretion. We analyzed the clonal composition of CD4⁺ T cells specific for defined HCMV peptides by generating multiple independent peptide-specific CD4⁺ clones and sequencing the TCR -chain. In a given carrier, most of the CD4⁺ clones specific for a defined pp65 peptide had identical TCR nucleotide sequences. We used clonotype oligonucleotide probing to quantify the size of individual peptide-specific CD4⁺ clones in whole PBMC and in purified subpopulations of CD45RA^highCD45RO^low and CD45RA^lowCD45RO^high cells. Individual CD4⁺ T cell clones could be large (0.3–1.5% of all CD4⁺ T cells in PBMC) and were stable over time. Cells of a single clone were distributed in both the CD45RA^high and CD45RO^high subpopulations. In one carrier, the virus-specific clone was especially abundant in the small CD28^–CD45RA^high CD4⁺ T cell subpopulation. Our study demonstrates marked clonal expansion and phenotypic heterogeneity within daughter cells of a single virus-specific CD4⁺ T cell clone, which resembles that seen in the CD8⁺ T cell response against HCMV pp65.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human CMV (HCMV)³ is a betaherpesvirus that infects 60–90% of individuals depending on the population studied. Following primary HCMV infection the virus persists lifelong in a latent state in cells of the myeloid lineage, with intermittent viral reactivation and shedding from mucosal surfaces, and containment by the host immune response (1). In immunocompromised individuals, uncontrolled HCMV reactivation can cause serious disease. There is much evidence from animal models (2) and from studies of immunosuppressed humans (3) that virus-specific CD8⁺ T cells have an important role in protection against CMV disease. In healthy long-term HCMV carriers, there are high frequencies of circulating HCMV-specific CD8⁺ T cells, many of which recognize specific peptides within either the viral tegument protein pp65 or the immediate early protein IE72 (4, 5). In a given carrier, most of the CD8⁺ T cells specific for a given viral peptide comprise a few individual CD8⁺ T cell clones that have undergone large stable clonal expansions in vivo (4, 6). Using oligonucleotide probing specific for the hypervariable TCR -chain sequence of each immunodominant CD8⁺ clone, we found that individual virus-specific CD8⁺ clones can be markedly heterogeneous in their expression of various cell surface proteins including the costimulatory molecule CD28 (7, 8) and CD45RA and CD45RO isoforms of the leukocyte common Ag (9).

There is increasing evidence that HCMV-specific CD4⁺ T cells are also an important correlate of protection against HCMV disease (10, 11, 12, 13). In healthy HCMV carriers, typically 1–2% of all circulating CD4⁺ T cells are specific for HCMV (14, 15). Many of these virus-specific CD4⁺ T cells recognize pp65, and in a given carrier typically focus on one or a few epitopes within pp65 (5, 16). In part because fewer peptide binding motifs are known for MHC class II alleles than for MHC class I alleles (17), relatively few MHC class II-restricted epitopes within pp65 have been identified (5, 18, 19), and the MHC restriction of these epitopes has not always been formally defined. In a limited number of healthy HCMV carriers using a combination of intracellular cytokine detection by flow cytometry, spectrotype analysis, and TCR sequencing, it has been shown that the CD4⁺ T cell response to whole HCMV is oligoclonal. Bitmansour et al. (20, 21) demonstrated striking focusing of TCR V usage among CD4⁺ T cells stimulated with whole HCMV Ag, and showed that these V expansions were composed of a limited number of clonotypes; the same clonotypes were identified when CD4⁺ T cells were stimulated with individual immunodominant pp65 peptides. Studies of the surface phenotype of HCMV-specific CD4⁺ T cells have been hampered by the lack of MHC class II tetramers complexed with HCMV peptides. Existing techniques to identify HCMV-specific CD4⁺ T cells involve the staining of HCMV-stimulated PBMC with two or three different fluorochrome-linked mAbs. One such study has demonstrated that CD4⁺ T cells responding to stimulation by whole HCMV are enriched within CD45RO^high, CD27^–, CD62 ligand (CD62L^–) and CCR7^– subpopulations (14). Phenotypic analysis at a clonal level has not been performed.

The high m.w. isoform of leukocyte common Ag CD45RA was previously thought to identify naive T cells, whereas the low m.w. isoform CD45RO identified Ag-experienced T cells (22, 23). Analysis of virus-specific CD8⁺ T cells in carriers of HCMV and EBV has demonstrated that Ag-experienced cells are distributed in both the CD45RO^high and CD45RA^high populations (9, 24, 25). There is evidence to suggest that the same is true of Ag-experienced virus-specific CD4⁺ T cells (26, 27, 28).

In this study in HCMV carriers we characterized CD4⁺ T cell epitopes within HCMV pp65, and formally defined the MHC class II restriction of these peptides. We found that in a given HCMV carrier, the majority of independently derived CD4⁺ T cell clones specific for a defined pp65 peptide had identical TCR nucleotide sequences. We used oligonucleotide clonotype probing to demonstrate that these defined clonotypes were present in purified subpopulations of CD45RA^highCD45RO^low and CD45RA^lowCD45RO^high CD4⁺ T cells. In one individual we showed that a pp65 peptide-specific CD4⁺ clone was particularly enriched within the CD28^–CD45RA^high subpopulation. Using both limiting dilution analysis (LDA) and molecular clonotype analysis we demonstrated that the size of individual peptide-specific CD4⁺ clonotypes was stable over a period of years and accounted for 0.3–1.5% of all CD4⁺ T cells.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Donors and tissue typing

One HIV seronegative laboratory donor and two HIV-infected subjects were studied. All donors were HCMV seropositive. In these subjects we previously identified strong CD8⁺ T cell responses against allele-specific peptides of HCMV pp65 (6). The MHC class II tissue type of each donor was determined by the tissue typing laboratory (Addenbrooke’s Hospital, Cambridge, U.K.).

Peptides of the lower matrix protein pp65

A panel of 20 aa long (20 mer) peptides overlapping by 10 aa that spanned the whole of pp65 was constructed as previously described (Affiniti Research Products, Exeter, U.K.) (4). Initial screening for peptide-specific CD4⁺ T cells was performed using pools of peptides containing three to five consecutive 20 mer peptides. To define more closely the epitope within an individual 20 mer peptide, we also used 14 or 15 aa long peptides previously described (4). All peptides were dissolved in RPMI 1640 at 200 µg/ml and frozen in small aliquots at –70°C.

Preparation of defined subsets of CD4⁺ cells for LDA and oligonucleotide probing

PBMC were prepared from fresh heparinized venous blood samples by Ficoll-Hypaque (Lymphoprep; Nyegaard, Oslo, Norway) density gradient centrifugation. CD16⁺ NK cells were depleted using anti-CD16 IgM (Leu-11b; BD Biosciences, Oxford, U.K.) and complement (4). CD8⁺ cells were then depleted using anti-CD8-conjugated MACS microbeads according to manufacturer’s instructions (Miltenyi Biotec, Dreiech, Germany); enrichment for CD4⁺ cells gave improved efficiency of subsequent cell sorting, and improved sensitivity for clonotype detection. For HIV-infected subjects, CD16^–CD8^– cells were further separated into CD45RA-depleted and CD45RO-depleted subpopulations populations using anti-CD45RA FITC or anti-CD45RO FITC and anti-FITC MACS microbeads (Miltenyi Biotec). For healthy HCMV carrier 009 who was HIV negative, sorting of PBMC into subpopulations for clonotype probing was performed using a FACSVantage cell sorter. mAbs used for sorting were anti-CD45RA Tricolor, anti-CD28 PE, and anti-CD4 FITC. The purity of lymphocyte subpopulations obtained FACSVantage or MACS microbeads was always >98%. To determine the proportion of CD4⁺ T cells that possessed the relevant TCR V segment within each sorted population, three- or four-color flow cytometry was performed on aliquots of PBMC. Abs used were TCR V-specific mAbs anti-V4 PE, anti-V5.3 PE, anti-V12 FITC; anti-CD4 PerCP or anti-CD4 allophycocyanin; anti-CD45RA FITC or PE; anti-CD45RO FITC or PE; and anti-CD28 Tricolor (Beckman-Coulter/Immunotech, Fullerton, CA).

Functional analysis of peptide-specific T cells

Aliquots of CD16^–CD8^– responder cells were used to identify MHC class II-restricted pp65 epitopes. We set up replicate microcultures of CD16^–CD8^– responder cells (20,000–40,000 cells per well), and stimulated each replicate (n = 3–6) with irradiated autologous PBMC pulsed with 1 of 11 pools of overlapping pp65 peptides, in RPMI 1640 supplemented with 10% human AB serum and recombinant human IL-2 5 IU/ml (Medical Research Council AIDS Reagent Project, Potters Bar, U.K.). After 14 days each set of replicates was assayed for cytotoxicity against radiolabeled autologous lymphoblastoid cell lines pulsed with the pool of peptides that had been used for stimulation or unpulsed in 4 h ⁵¹Cr release cytotoxicity assays. Where three of three or six of six replicates demonstrated pool-specific cytotoxicity, we retested pool-specific responder CD4⁺ T cells against aliquots of target cells pulsed with each individual peptide of the relevant pool.

The methodology of LDA used was as previously described (6). Replicate microcultures (n = 27) of CD16^– responder cells were set up over an appropriate range of dilutions. APCs were autologous irradiated peptide-pulsed PBMC, in medium supplemented with 10% FCS (Myoclone; Invitrogen Life Technologies, Grand Island, NY), 10% human AB serum and human recombinant IL-2 5 IU/ml. On day 14, the cells in each individual well were divided into three aliquots that were assayed for cytotoxicity against peptide-pulsed autologous lymphoblastoid cell lines, unpulsed autologous, or peptide-pulsed MHC mismatched lymphoblastoid cell lines in 4-h ⁵¹Cr release cytotoxicity assays. In some experiments, supernatants from individual LDA microcultures were harvested before cytotoxicity assay and the supernatants assayed for IFN- by ELISA. In additional experiments autologous lymphoblastoid cell lines infected with recombinant vaccinia virus expressing HCMV pp65 or expressing an irrelevant protein were used as target cells as previously described (6). The method of calculation of precursor frequency was as previously described (4).

Intracellular cytokine assay

IFN- intracellular cytokine staining was performed as described by Hoffmeister et al. (29). Briefly, PBMC were incubated for 2 h with the peptide of choice, an irrelevant peptide (negative control), or staphylococcal enterotoxin B (positive control). Cells were incubated for a further 6 h in the presence of brefeldin A to block secretion of IFN-. Cells were washed, stained with anti-CD8 Tricolor or anti-CD4 allophycocyanin, permeablized using a fix and permeablization kit (Caltag Laboratories, Burlingame, CA) then stained with anti-IFN- PE (Caltag Laboratories). Cells were fixed by resuspension in PBS containing 2% paraformaldehyde then analyzed by flow cytometry.

Generation of single cell clones and clonally derived T cell microcultures

We identified individual LDA microcultures that showed strong peptide-specific cytotoxicity on the master plate at or below the responder cell dilution where <50% of replicate microcultures showed peptide-specific killing (at these low dilutions, individual LDA microcultures contained single peptide-specific clones) (6). Residual cells from selected microcultures were subcultured at 0.5 cells/well to generate formal single cell clones using restimulation with PHA-P and irradiated allogeneic PBMC (6). All clones were CD4⁺CD8^– by flow cytometry. In addition, for other selected microcultures we expanded the residual cells by mitogen restimulation to generate multiple independent clonally derived T cell cultures. All clones and cultures were retested and confirmed to show strong peptide-specific cytotoxicity, before mRNA extraction and cDNA synthesis as described (6).

TCR -chain and -chain PCR amplification and sequencing

To determine TCR - and -chain V region usage of T cell clones, PCR amplification and sequencing was performed using a panel of TCR V primers and a C primer or a panel of TCR V primers and a C primer as described (6). The -chain hypervariable CDR3 region is as defined by Kabat et al. (30), starting at position 95 of V and finishing at the phenylalanine of the motif FGXG within J.

Quantitative clonotypic analysis

From the TCR -chain hypervariable sequence of each immunodominant peptide-specific CD4⁺ T cell clone, we designed a complementary 15–20 mer oligonucleotide probe. Such probes are highly specific for individual T cell clonotypes (6, 9). mRNA was extracted from purified populations of cells, reverse transcribed into cDNA and amplified using TCR V-specific PCR primers as previously described (6). A positive control sample from the original defined biological T cell clone and a negative control sample from the pooled PBMC of four HCMV seronegative donors were amplified simultaneously in duplicate using the same primers. Each PCR product was separated on an agarose gel and blotted onto a zeta-probe nylon filter (Bio-Rad, Hercules, CA). After washing and prehybridization, the filter was incubated overnight with [-³²P] end-labeled clonotypic probe in hybridization buffer. After washing, the amount of probe that had bound to each sample on the filter was quantitated using an Instant Imager (Beckman Coulter). The filter was stripped by soaking in 0.4 M NaOH, washed and then rehybridized with a TCR -chain constant region probe that detects all amplified TCR sequences (9). In each experiment we corrected for any differences in radioactive labeling of the clonotypic and constant region probes by measuring the radioactivity of each probe bound to the positive control sample as a reference standard (TCR -chain sequences amplified from the in vitro derived T cell clone contain the unique hypervariable region and constant region in equal amounts). In each population of CD4⁺ T cells studied, we calculated the relative abundance of the clonotype sequence as a proportion of all amplified TCR sequences of the same V family as: (test sample cpm (probed with clonotypic probe)/test sample cpm (probed with constant probe) x 100)/positive control cpm (probed with clonotypic probe)/positive control cpm (probed with constant probe)).

In each population of CD4⁺ T cells studied, we quantified the proportion of cells that possessed the relevant TCR V segment by flow cytometry using TCR V-specific mAbs (earlier described). The absolute clone size within CD4⁺ cells of each subpopulation was then estimated from the proportion of clonotype sequence within all amplified sequences multiplied by the proportion of the CD4⁺ T cells that stained with the V-specific mAb within each subpopulation.

The V4 family comprises V4.1 and V4.2. For clonotype 4A, we used a V4 family specific primer and V4 family specific Ab. The V5 family comprises V5.1–V5.7—for clonotype 4C we used a V5.3-specific primer, and an Ab that is completely specific for V5.3. The V12 family comprises V12.1, V12.2, and V12.3—an Ab completely specific for V12.3 is not available. For clonotype 4B we used a V12.3 specific primer and we used an anti-V12 family specific Ab. For this clonotype the estimated clone size was 2–20 times higher than the corresponding frequency estimates from LDA (see Fig. 4); we may have overestimated the size of clonotype 4B.

View larger version (26K): [in this window] [in a new window]   FIGURE 4. Longitudinal analysis of clone size of defined CD4+ clonotypes and peptide-specific CTLp frequency. A, HCMV carrier 009. Top, At five time points over 3 years the proportion of clonotype 4C within all V5.3 sequences in PBMC (Table I) was stable. Bottom, The clone size expressed as cells of the clone/106 CD4+ T cells () was also stable over 3.5 years. The frequency of CD4+ CTLp specific to aa 367–380 () was high and maintained at a similar level at two time points 8 mo apart. B, HIV-infected subject H0043 with (top) HIV viral load (copies/ml plasma) and CD4+ T cell count (per microliter of whole blood), over the period of study during which antiretroviral therapy commenced. The proportion of clonotype 4B sequence (middle) within all V12.3 sequences in PBMC was stable over 4 years. The clone size of clonotype 4B (bottom) and the frequency of CD4+ CTLp specific for aa 259–273 were maintained at a high level over 3.5 years.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

For each of three HCMV-seropositive individuals (donors 009, H0043, and H0049), we identified an immunodominant pp65 peptide recognized by CD4⁺ T cells (data for donor H0049 shown in Fig. 1A and Table I), and for donor 009 we identified a 14 mer containing the minimal epitope (Fig. 1B and Table I).

View larger version (8K): [in this window] [in a new window]   FIGURE 1. Identification of immunodominant CD4+ T cell epitopes within pp65. A, For donor H0049 we retested pool B1-specific responder cells against target cells incubated with five individual peptides that constituted pool B1 and against the first peptide of pool B2. There was strong killing of peptide aa 41–60, with no evidence of killing of peptide aa 31–50 or aa 51–70. B, For donor 009, peptide aa 361–380 was recognized by pool B8-specific responder cells. Peptide aa 367–380 contains the minimal epitope required for recognition by pool B8-specific CD4+ T cells.

View this table: [in this window] [in a new window]   Table I. Multiple independent CD4+ T cell clones specific for a defined HCMV pp65 peptide have identical TCR -chain nucleotide sequences

View larger version (16K): [in this window] [in a new window]   FIGURE 2. The dose-response curve for two pp65 peptide epitopes. Using clonally derived effector cells from donor 009 specific for aa 367–380 at an E:T ratio of 5:1 (maximum specific lysis 68%) or a bulk CTL line from donor H0043 specific for aa 41–60 (maximum specific lysis 58%) at a E:T ratio of 40:1 we showed that the peptide concentration needed for half maximum sensitization of target cells was >10-fold lower for aa 367–380 compared with aa 41–60.

In previous studies, the MHC class II allele restricting individual CD4⁺ T cell epitopes within pp65 has been deduced rather than formally defined by analyzing which MHC class II molecules are shared by different donors that recognize the same epitope. In this study we were able directly to demonstrate the MHC class II molecule restricting the three peptides previously described by testing peptide-specific CD4⁺ T cell clones against autologous and partially matched target cells.

In donor 009, peptide aa 367–380 is restricted by HLA-DR11 (Fig. 3A); peptides overlapping with this epitope were recognized by CD4⁺ T cells from a total of eight DR11⁺ HCMV carriers (5, 18). In donor H0049, peptide aa 41–60 is restricted by HLA-DQ6 (data not shown). One previous study identified three healthy HCMV carriers who recognized aa 41–55 and shared both HLA-DQ6 and HLA-DR15 (5). In donor H0043, peptide aa 259–273 is restricted by HLA-DR13 (data not shown).

View larger version (17K): [in this window] [in a new window]   FIGURE 3. (A) Peptide aa 367–380 is presented by HLA-DR11. A panel of partial MHC class II-matched EBV-transformed lymphoblastoid cell lines was used to identify the MHC class II allele that presents aa 367–380 to donor 009 aa 367–380-specific effector CD4+ T cells (E:T is 10:1). There was strong killing of peptide-pulsed autologous cells and cells matched at HLA-DR11, but no killing of target cells matched at other class I or class II alleles but mismatched at HLA-DR11. B, In split well analysis, almost all individual microcultures that showed strong MHC-restricted peptide-specific killing also showed IFN- secretion. Replicate LDA cultures initially contained 1250 CD16–CD8– responder cells from donor 009 and were stimulated with peptide aa 367–380 pulsed irradiated PBMC.

Using split well analysis in LDA, for each microculture we simultaneously analyzed IFN- secretion into the supernatant (quantified by ELISA) and peptide-specific cytotoxicity. At limiting dilution, almost all individual microcultures that showed strong MHC-restricted peptide-specific killing also showed IFN- secretion; a small number of microcultures that showed IFN- secretion showed no detectable killing. In donor 009, the frequency of CD4⁺ CTLp specific to aa 367–380 as assessed by cytotoxicity was 460/10⁶ CD4⁺ T cells; in the same LDA, the frequency of aa 367–380 specific CD4⁺ T cells as estimated by IFN- secretion was 1010/10⁶ CD4⁺ T cells (Fig. 3B). In this donor we used cytoplasmic IFN- detection to confirm our estimate of the frequency of CD4⁺ T cells specific to aa 367–380 at a further time point: 1280/10⁶ CD4⁺ T cells. In the same experiment, the frequency of CD4⁺ T cells specific to an irrelevant peptide was 110/10⁶ CD4⁺ T cells.

We measured high frequencies of CD4⁺ CTLp specific for pp65 peptides in LDA. In donor H0049 at one time point, the frequency of CD4⁺ CTLp specific to aa 41–60 was 620/10⁶ CD4⁺ T cells. In donor 009, the frequency of CD4⁺ CTLp specific for aa 367–380 was stably maintained at 420–430/10⁶ CD4⁺ T cells at two time points 8 mo apart (Fig. 4A). In donor H0043, the frequency of CD4⁺ CTLp specific for aa 259–273 varied between 250-5660/10⁶ CD4⁺ T cells at nine time points over 3.5 years (Fig. 4B). In all experiments the frequency of CD4⁺ CTLp specific for control unpulsed target was <15/10⁶ CD4⁺ T cells. At two time points in donor H0043 in split well analysis we simultaneously quantitated the frequency of CD4⁺ CTLp specific for targets pulsed with pp65 aa 259–273 and for targets infected with recombinant vaccinia virus either expressing pp65 or a control protein. At both time points, we measured high frequencies of aa 259–273 specific CD4⁺ CTLp (250–570/10⁶ CD4⁺ T cells), however at both time points the frequency of CTLp specific for vaccinia pp65 was very low, and identical with that of the negative control vaccinia (12–17/10⁶ CD4⁺ T cells). This may reflect the fact that when Ag is expressed in the cytoplasm by vaccinia virus, delivery to the MHC class II pathway for recognition by CD4⁺ T cells is inefficient.

The clonal composition of memory CD4⁺ T cells specific for pp65 epitopes is highly focused

We determined how many different T cell clones contribute to the population of CD4⁺ T cells specific for an individual pp65 peptide. We generated multiple independent peptide-specific formal single cell clones, determined the TCR -chain V region usage of each set of clones using a panel of TCR V primers and a C primer, and sequenced each TCR -chain (Table I). In each of the three donors studied, the clonal composition of the memory CD4⁺ T cell response to a given pp65 peptide was highly focused; for each donor, one predominant TCR -chain nucleotide sequence was detected in multiple independently derived formal T cell clones specific for a given peptide. In subject H0049, eight of eight independent aa 41–60 specific CD4⁺ T cell clones had the same V4⁺ TCR -chain nucleotide sequence, demonstrating that this clone accounted for the large majority of the aa 41–60-specific memory T cells in this donor. In donor H0043, six of seven independent aa 259–273-specific T cell clones derived at two time points had the same V12.3⁺ TCR -chain nucleotide sequence. In donor 009, three of four independent aa 367–380-specific T cell clones derived at two time points had the same TCR V5.3⁺ TCR -chain nucleotide sequence. We further determined the TCR -chain V region usage of these three clones using a panel of TCR V primers and a C primer. By sequencing each TCR -chain we showed that all three clones also had identical V24⁺ TCR -chain nucleotide sequences (data not shown). These results closely resemble our previous observation of clonal focusing of CD8⁺ CTL specific for peptides of pp65 in healthy HCMV carriers (6). In our study we have formally demonstrated that these clonal expansions are indeed specific to individual viral peptides.

Cells of pp65-specific CD4⁺ clonotypes are distributed in both CD45RA^highCD45RO^low and CD45RA^lowCD45RO^high CD4⁺ subpopulations

We examined the distribution of individual clonotypes in the CD45RA^high and CD45RO^high subpopulations of CD4⁺ T cells using quantitative oligonucleotide clonotype probing. Peptide-specific clonotypes often accounted for a very high percentage of TCR sequences in cells of the same V family (Table II and Fig. 5). For clonotypes 4B and 4C, cells of the clone were distributed in both the CD45RA^high and CD45RO^high subpopulations (Table II and Fig. 5). In donor H0043 the distribution of clonotype 4B between CD45RA^high and CD45RO^high subpopulations was similar at two time points 8 mo apart (Table II and Fig. 5). In donor 009, in addition we confirmed that clonotype 4C was relatively evenly distributed between CD45RA^high and CD45RO^high subpopulations by using a different oligonucleotide probe for the same clone based on the hypervariable region of the TCR -chain (Table II). Clonotype 4A from donor H0049 was almost entirely partitioned in the CD45RO^high population; the proportion of clonotype 4A sequence in CD45RA^high CD4⁺ cells was almost at the limit of detection of the assay as determined by background binding of the clonotype specific probe (Table II).

View this table: [in this window] [in a new window]   Table II. Proportion of clonotype sequence within all sequences of the same V family and clone size if individual clonotypes in different CD4+ subpopulations determined by oligonucleotide probing

View larger version (83K): [in this window] [in a new window]   FIGURE 5. Cells of an expanded pp65-specific CD4+ T cell clone are present in both CD45RAhigh and CD45ROhigh populations: from the TCR -chain of a V12.3+ pp65 aa 259–273-specific CD4+ T cell clone of donor H0043, we designed an oligonucleotide probe based on the nucleotide sequence of the hypervariable region and the ends of the adjoining V and J regions. mRNA was extracted from subpopulations of CD4+ PBMC, and cDNA was synthesized and amplified using a V12.3-specific primer and a C-specific primer. A positive control sample of cDNA of the biological CD4+ clone and a negative control sample from pooled PBMC of four HCMV-negative healthy donors was also amplified at the same time using the same primers. PCR products (I) were blotted onto a filter, which was first probed using the labeled clonotypic probe. By stripping the filter and reprobing (II) with a conserved constant region-specific probe that detects all amplified TCRs, it was possible to calculate the relative abundance of clonotype sequence as a proportion of all TCR sequences of the V12.3 family.

From the relative abundance of clonotype within cells of the same TCR V family, and the proportion of V⁺ cells in the CD4⁺ population determined by flow cytometry, the number of cells of the clone in 10⁶ CD4⁺ T cells of each phenotype could be estimated (Table II). Individual clones that recognized pp65 peptides could be very large (up to 13,000 per 10⁶ CD4⁺ T cells, namely 1.3% of all CD4⁺ T cells in PBMC). For clonotypes 4A and 4B, the clone size in 10⁶ cells of each subpopulation was 14 times greater in the CD45RO^high subpopulation compared with the CD45RA^high subpopulation. Clonotype 4C was relatively more enriched in the CD45RA^high subpopulation.

Clonotype 4C is enriched within CD28^–CD45RA^high cells

To address which subpopulation of CD45RA^high cells may contain Ag-experienced virus-specific cells, we studied the distribution of clonotype 4C from donor 009 in three sorted subpopulations of CD4⁺ PBMC defined by CD28 and CD45RA expression (Fig. 6). For the CD4⁺ population as a whole, 45% of cells were CD28⁺CD45RA^high, 38% were CD28⁺CD45RA^low, and only 0.7% were CD28^–CD45RA^high cells; the remaining cells were CD28⁺CD45RA^int. For V5.3⁺ CD4⁺ cells, 34% were CD28⁺CD45RA^high, 36% were CD28⁺CD45RA^low, and 3.6% were CD28^–CD45RA^high cells. Clonotype 4C was particularly enriched in this small CD28^–CD45RA^high population, accounting for 82% of V5.3⁺ sequences in CD28^–CD45RA^high cells. Clonotype 4C was detectable at low level within CD28⁺CD45RA^high cells (Fig. 6). The purity of the sorted CD28⁺CD45RA^high population was 99.3%. The proportion of CD28^–CD45RA^high cells contaminating this sorted CD28⁺CD45RA^high population (the subset containing the majority of clonotype sequence) was 0.00% (0 of a total of 11,181 sorted cells). The clone size in each of these CD4⁺ populations were: CD28^–CD45RA^high population 44,600/10⁶ cells; CD28⁺CD45RA^low population 530/10⁶ cells; and CD28⁺CD45RA^high population 130/10⁶ cells. The difference between the percentage of clonotype 4C detected in sorted CD28⁺CD45RA^high cells and the binding of the clonotype probe to the negative control was small, and this small difference may not be significant. To define precisely the truly naive population of CD4⁺ or CD8⁺ T cells, sorting of cells using more than three cell surface markers may be necessary (31); cells sorted using two markers such as CD28⁺CD45RA^high might therefore contain low frequencies of virus-specific clonotypes.

View larger version (38K): [in this window] [in a new window]   FIGURE 6. Cells of an expanded pp65-specific CD4+ T cell clone are enriched in CD28–CD45RAhigh cells. From the TCR -chain of a V5.3+ pp65 aa 367–380-specific CD4+ T cell clone of donor 009, we designed a clonotypic probe as described in Fig. 5. We used this probe to determine the relative abundance of clonotype sequence as a proportion of all TCR sequences of the V5.3 family in subpopulations of CD4+ PBMC (illustrated in FACS plot). Clonotype 4C was particularly abundant in CD28–CD45RAhigh cells.

In all donors we found that the relative abundance of clonotype sequence within cells of the same V family was stable over time. In donor H0049 clonotype 4A accounted for 22–24% of all V4⁺ sequences at two time points 4 mo apart (Table II). In donors 009 and H0043 the abundance of clonotype 4B and 4C sequences within V⁺ cells was stable at multiple time points over 3.5 years (Fig. 4). In these two donors, by correcting for the proportion of V5.3⁺ and V12⁺ cells in the CD4⁺ population we found that the size of clonotypes 4B and 4C remained stable over time (Fig. 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
To characterize the HCMV-specific CD4⁺ T cell response, we identified MHC class II-restricted pp65 epitopes in three HCMV carriers. In a given HCMV carrier, the majority of independently derived CD4⁺ T cell clones specific for a defined pp65 epitope had identical TCR -chain nucleotide sequences. By using oligonucleotide clonotype probing, we demonstrate for the first time that these clonotypes were present in both CD45RA^highCD45RO^low and CD45RA^lowCD45RO^high CD4⁺ T cell subpopulations and further found that in one individual a pp65 peptide-specific CD4⁺ clone was particularly enriched within the CD28^–CD45RA^high subpopulation. The size of individual peptide-specific CD4⁺ clonotypes as quantitated by LDA and clonotype probing was stable over 3–4 years and accounted for 0.3–1.5% of all CD4⁺ T cells.

The CD4⁺ T cell clones that we studied were selected both for growth in vitro and for peptide-specific cytotoxicity. It is possible that the composition of peptide-specific CD4⁺ T cell clones in vivo may be more heterogeneous than suggested by our in vitro analysis because some peptide-specific clones may show reduced in vitro growth and/or have lower TCR affinity for specific peptide/MHC. It is also possible that additional noncytotoxic clones are present, however using split well LDA we found only a small number of microcultures that showed IFN- secretion but no detectable killing; in general almost all microcultures that showed IFN- secretion also showed strong MHC-restricted peptide-specific killing. Measurements of CD4⁺ CTLp frequency using a cytotoxicity readout provides a minimum estimate of the frequency of peptide-specific CD4⁺ T cells.

Bitmansour et al. (20, 21) observed a similar degree of clonal focusing of IFN--expressing CD4⁺ T cells following stimulation either with whole HCMV Ag or with individual immunodominant peptides. There was striking focusing of TCR V usage among peptide-stimulated CD4⁺ T cells; further, each V expansion was oligoclonal upon spectrotyping and TCR sequencing. Identical dominant clonotypes were seen when CD4⁺ T cells were stimulated with whole HCMV Ag suggesting that the degree of clonal focusing observed was independent of the method of T cell activation in vitro.

In all three subjects, we found high frequencies of peripheral blood CD4⁺ T cells specific for pp65 peptides as assessed by LDA and by clonotype probing. Other studies have demonstrated comparable high frequencies of HCMV-specific CD4⁺ T cells. By stimulating PBMC with peptide pools or HCMV viral lysate and quantitating total HCMV-specific CD4⁺ T cells using intracellular IFN- cytokine staining, a median of 1–2% of all circulating CD4⁺ T cells have been found to be specific for HCMV (14, 15), and up to 0.2% of all CD4⁺ T cells specific for pp65 (5).

In our subjects, the clone size of an individual peptide-specific T cell clone was consistently greater than the precursor frequency of CD4⁺ T cells specific to the same peptide, which indicates that not all of the T cell clones detected by the clonotype probing technique proliferated efficiently in LDA culture or were cytotoxic. For clonotypes 4A, 4B, and 4C the estimated cloning efficiency (the LDA response as a proportion of peptide-specific CD4⁺ T cell clone size) was 4–47%. Bitmansour et al. (21) demonstrated that in short-term in vitro assays, the threshold for activation varied across T cells of the same CD4⁺ clone, which may be one reason in our assays that cloning efficiency was substantially <100%. In our previous studies of HCMV and HIV-specific CD8⁺ CTL we also found that in many instances the clone size of an individual peptide-specific CTL clone was greater than the frequency of CTLp specific to the same peptide (7, 9). For CD8⁺ CTL, it is recognized that the functional response requiring clonal expansion in vitro in LDA may underestimate the absolute number of Ag-specific cells as determined by peptide/MHC class I tetramer staining (32). In general we found that assessing pp65-specific CD8⁺ T cell frequencies by staining with peptide tetramers gave a three times higher frequency than LDA (33).

The size of individual CD4⁺ T cell clones specific for pp65 peptides could be very large and was maintained at a stable level over three to four years. Because HCMV is a persistent viruses characterized by sustained or intermittent viral reactivation and Ag expression, repeated exposure of virus-specific memory CD4⁺ T cells to viral Ag in vivo over time may lead to selective expansion and maintenance of large Ag-experienced T cell clones, possibly those that express certain high affinity TCR (34). For CD8⁺ T cells, we have observed comparably large stable clonal expansions of pp65-specific CD8⁺ T cells (7, 8). Other persistent virus infections have also been associated with large clonal expansions of peptide-specific CD8⁺ T cells including EBV (35) and HIV-1 (36). By contrast to HCMV, the magnitude of the CD8⁺ T cell response to a nonpersistent virus, influenza, is 10- to 100-fold lower (37), and diminishes after resolution of acute infection (38).

CD8⁺ CTL specific for HCMV are distributed between CD45RO^high and CD45RA^high CD8⁺ cells, and we have provided direct evidence that in primary HCMV infection defined virus-specific CD8⁺ T cell clonotypes can revert from CD45RO^high to CD45RA^high (9). Previous phenotypic studies of CD45RA^high CD4⁺ T cells suggested the presence of both naive and Ag-experienced cells within this population, but the specificity of the Ag-experienced cells was not determined (31). In this study we show for the first time large clone sizes of the same pp65-specific CD4⁺ T cell clonotype in both the CD45RO^high and in the CD45RA^high CD4⁺ cells of long-term virus carriers. Clonotype 4A was predominantly CD45RO^high. The clone size of clonotype 4B was substantially greater in the CD45RO population compared with the CD45RA population (17,400/10⁶ CD45RO^high cells compared with 2000/10⁶ CD45RA^high cells). Clonotype 4C showed the reverse: 200/10⁶ CD45RO^high cells compared with 1400/10⁶ CD45RA^high cells. Within CD45RA^high cells clonotype 4C was particularly enriched within the small CD28^–CD45RA^high subpopulation; in the same donor we previously demonstrated a large expanded pp65-specific CD8⁺ T cell clone that was also CD28^–CD45RA^high (39). Sester et al. (14) and Amyes et al. (26) stimulated CD4⁺ T cells with whole HCMV Ag and identified HCMV-specific T cells by intracellular cytokine staining. The majority of these T cells were CD45RO^high, although some individuals had a significant proportion of CD45RA^high IFN- expressing CD4⁺ T cells. The Ag experienced CD45RA^high population may have stringent requirements for in vitro activation. In previously vaccinated subjects, the in vitro proliferative response of CD45RA^high CD4⁺ cells to tetanus toxoid was very weak (40), but was significantly enhanced by the addition of anti-CD28 Ab (27).

The generation of phenotypic diversity within the clonal progeny of a single virus-specific T cell may be an instructive process as a result of differences in the activation state of APCs to which the naive cell and later daughter cells are exposed (41, 42), and differences in the cytokine milieu during their activation and differentiation (43). An alternative possibility is that activation of a single CD4⁺ T cell might give rise to daughter cells of different phenotypes by a stochastic process. In either case, the diversity of phenotypes may be modified by subsequent selection of those daughter cells whose activation state and/or homing pathway are best suited to control the virus at a given site (44). The extent to which CD4⁺ T cells undergo conversion from CD45RO^high to CD45RA^high either in vitro or in vivo has been much debated (45). Conversion from CD45RO^high to CD45RA^high may be influenced by characteristics of an Ag, including the cell type and tissue location in which Ag is expressed. Different Ags of a single virus can influence the differentiation of responding T cells. Both CD4⁺ and CD8⁺ T cells specific for EBV latent Ags are predominantly CD45RO^high, whereas cells specific for EBV lytic Ags are present in both CD45RO^high and CD45RA^high subpopulations (25, 26).

From our work in an albeit limited number of individuals, we conclude that the CD4⁺ T cell response against HCMV pp65 comprises expanded individual clones that are stably maintained over time with marked phenotypic heterogeneity among cells of a single clone. These properties closely resemble those of the CD8⁺ T cell response against HCMV pp65.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by Medical Research Council Program Grant G9202171 (to J.G.P.S.) and a Medical Research Council Cooperative Group grant. A.J.C. is a Lister Institute Research Fellow.

² Address correspondence and reprint requests to Dr. Andrew J. Carmichael, Department of Medicine, University of Cambridge Clinical School, Hills Road, Cambridge CB2 2QQ, U.K. E-mail address: ac71{at}medschl.cam.ac.uk

³ Abbreviations used in this paper: HCMV, human CMV; LDA, limiting dilution analysis; CTLp, CTL precursor.

Received for publication May 11, 2004. Accepted for publication August 12, 2004.

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