Persistent Oligoclonal CD4dimCD8þT Cells inPeripheral Blood

Claude Lambert,* Cristina Iobagiu, and Christian GeninImmunology Laboratory, University Hospital of St. Etienne, St. Etienne, France

Background: Routine CD4/CD8 T-cell phenotyping may shows a small fraction of CD4dimCD8+ T cellswith a homogeneous appearance as described for lymphoproliferative syndromes or chronic infections.The aim of this study was to elucidate the significance of CD4dimCD8þ T cells and their degree ofdiversity.Methods: Phenotyping was performed in 272 samples from healthy donors, elderly patients, and immu-

nocompromised (human immunodeficiency virus or renal transplantation) patients.Results: The CD4dimCD8+ T cells had decreased fluorescence intensity for CD4 but not for CD8. The

frequency of patients with CD4dimCD8+ T cells (>20 cells/ll; 10.3% of patients with human immunodefi-ciency virus and 7.7% with renal transplantation) was not significantly different when compared withhealthy donors (9.7%). The CD4dimCD8+ T cells did not express the activation marker CD69. The CD8 ofCD4dimCD8+ T cells expressed the heterodimeric (ab) isoform. In 13 of 26 samples, the apparently highlyhomogeneous CD4dimCD8+ T cells expressed one predominant T-cell receptor Vb clonotype. These predo-minant clonotypes were widely distributed among patients: Vb 5.2, 17, 2, 3, 5.1, 13.1, 14, and 20.Conclusions: Whether these findings demonstrate an oligoclonal reaction to chronic inflammation or

an emerging lymphoproliferative disorder must be elucidated in a long-term longitudinal study. Byanalogy to monoclonal gammopathy, we propose to name this phenomenon ‘‘oligoclonal clonopathy ofundetermined significance.’’ ' 2005 Wiley-Liss, Inc.

Key terms: T-cell subsets; CD4dimCD8þ T cells; double-positive T cells; unconventional T cells

CD4 and CD8 are coreceptors involved in binding ofT-cell receptors (TCRs) to specific antigens presented byMCH class II or class I molecules. The two coreceptorsare transiently coexpressed by early thymocytes. How-ever, it is usually admitted that mature conventionalT cells express in a mutually exclusive manner only oneof them in relation to their helper (CD4þ) or cytotoxic(CD8þ) activity. Recent phenotyping techniques usingmultiparametric labeling have led to occasional observa-tions of a minor group of CD3þ T cells that coexpressboth coreceptors in peripheral blood (1–3). On theseunconventional T cells, each coreceptor is expressed atdifferent levels of fluorescence intensity in flow cytome-try. Sometimes they have a homogeneous appearance ondot plots, suggesting a restricted diversity. The most fre-quent phenotype, CD4brightCD8dimCD3þ, has beendescribed in healthy donors and different diseases (2–4).Its clinical significance is not elucidated, except in thecase of proliferative disorders (5,6). It has also beenobserved in animals (7). An oligoclonal expansion couldbe induced by a chronic stimulation (8–10), but anemerging lymphoproliferative disorder cannot beexcluded (11). An alternative CD4dimCD8þ phenotype is

even less known (11–13), and its immunologic or clini-cal significance remains to be clarified.CD4/CD8 lymphocyte phenotyping is the most fre-

quently performed analysis in flow cytometry for moni-toring cellular immunology. Although fundamentalimmunology has made tremendous progress in recentyears, partly with the help of technologic improvementsin cytometry, the routine applications remain mostly basic(CD4þ CD8þ T-cell counts) and simple, although muchinformation is available. In routine analysis of peripheralblood, an additional homogeneous CD4dimCD8þ T-cellsubset is occasionally observed that we cannot interpretfor clinical use. The aim of this study was to characterizethese unconventional T cells and to approach an under-standing of their immunologic significance. Our results

*Correspondence to: Dr. Claude Lambert, Immunology Laboratory,Hopital Bellevue, CHU St. Etienne, F 42055 St. Etienne Cedex 2,France. E-mail: claude.lambert@univ-st-etienne.frReceived 26 July 2004; Accepted 2 December 2004Published online 11 April 2005 Wiley InterScience (www.

interscience.wiley.com).DOI: 10.1002/cyto.b.20047

Cytometry Part B (Clinical Cytometry) 66B:10–17 (2005)

' 2005 Wiley-Liss, Inc.

argue in favor of considering more than the two conven-tional CD4þ or CD8þ T-cell subsets in clinical immunolo-gic studies.

MATERIALS AND METHODS

Lymphocyte analyses were performed in 272 bloodsamples taken during routine practice from 31 healthyblood donors (15 women and 16 men, 46.5 6 13.3 yearsold, age range 18–68 years), 93 patients with the humanimmunodeficiency virus (HIV; 28 women and 65 men,40.1 6 10.3 years old, age range 23–78 years), and 106renal transplantation patients (38 women and 68 men,52.8 6 13.7 years old, age range 20–82 years). All HIVpatients received highly active antiretroviral therapy. Wealso analyzed 21 volunteers older than 70 years whoresided in a medical institute (12 women and 9 men,82.2 6 5.6 years old, age range 73–90 years). Amongthese subjects, frequencies were evaluated in 31 healthydonors, 21 elderly patients, and unselected, consecutivepatients who had HIV (n ¼ 78) and renal transplantation(n ¼ 78; Table 1).

Lymphocytes were immunolabeled by using a combi-nation of anti-CD3, phycoerythrin (PE), and cyanin 5(PE-Cy5, clone UCHT1); anti–CD4-PE and Texas Red (PE-TR; clone SFCI12T4D11); anti–CD8a-PE (cloneSFCI21thyD3); and anti-CD45 and fluorescein isothiocya-nate (FITC; clone B3821F4A; all from TetraChrom, Beck-man-Coulter, Fullerton, CA, USA). TCR/CD8 isoformexpression was analyzed using anti-TCRa/b PE-Cy5(clone BMA031), anti-TCRg/d FITC (clone Immu510),anti-CD8a PE-TR (clone SFCI21thyD3), and anti-CD8b PE(clone 2ST8.5H7; all from Immunotech, Beckman-Coul-ter). In a few cases, results were confirmed with anti-CD4 FITC (clone M310, DakoCytomation, Copenhagen,Denmark) instead of anti-TCRg/d in the same combina-tion. Activated T cells were identified with anti–CD3-PE-Cy5, anti-CD8a PE-TR (clone SFCI21thyD3), anti-CD56-PE (clone N901; Beckman-Coulter), and anti-CD69 FITC(clone TP1.55.3; Immunotech). The activation status of

the CD8 subsets was further analyzed in 10 patients byusing anti-CD8a PE-TR , anti-CD8b PE, and anti-TCRa/blabeling combined with anti-CD25-FITC (clone ACT-1;DakoCytomation). CD69 and CD25 labelings were pre-viously validated in vitro in peripheral blood mononuclearcells from healthy donors stimulated with phytohemagglu-tinin (maximum expression at 24 and 48 h, respectively;data not shown). Antibody staining was always comparedwith matched isotype controls (same concentration, samemanufacturer).Immunolabeling procedures were performed in 100 ml

of ethylenediaminetetraacetic acid plus anticoagulatedfresh peripheral blood (�4 h after withdrawal), incu-bated with 10 to 20 ml of each antibody according to themanufacturer’s instructions, gently mixed, and incubatedfor 20 min at room temperature in the dark. Red bloodcells were then lysed, and remaining cells were fixedwith formaldehyde by using Immunoprep reagent on aT-Q prep machine (Beckman-Coulter). Samples werethen stored in the dark at room temperature and ana-lyzed within 1 h on a four-color EPICS XL flow cyt-ometer (Beckman-Coulter). Just before analysis, CD3/CD4/CD8/CD45-labeled samples were mixed with anequal volume (100 ml) of standardized fluorescent beads(Flowcount, Beckman-Coulter) for absolute quantifica-tion, according to the Beckman-Coulter quality assur-ance certified procedures. A minimum of 6,000lymphocyte events were analyzed. Lymphocytes wereselected on the basis of CD45/side scatter dot plot.T lymphocytes were selected by CD3 expression fromCD45-gated lymphocytes. CD4 and CD8 were thenanalyzed from CD3þ lymphocytes. When another para-meter was added, CD45 was suppressed and lympho-cytes were selected on forward versus side scatter dotplot.The absolute membrane density of the CD8 corecep-

tor was evaluated in five samples by indirect labelingwith the QuiFiKit (DakoCytomation) according to themanufacturer’s instructions. Briefly, the kit provides a

Table 1Comparative Analysis of CD4dimCD8þ T-Cell Subsets Analyzed in 208 Consecutive Samples*

Healthy donors HIVþ Kidney transplant Elderly

n 31 78 78 21Age (years) 46.5 6 13.3 40.3 6 10.2 52.3 6 14.1 82.2 6 5.3Women/men# 15/16 22/56 26/52 12/9CD4þ T cells/m1 952 6 309 511 6 288§ 790 6 402

y830 6 454

y

CD8þ T cells/m1 457 6 231 1,167 6 980§ 518 6 217 436 6 360gd T cells/m1 51 6 37.4 88 6 93.5§ 52 6 60.7 37 6 27%CD4dimCD8þ 0.2–4.7 0.1–3.8 0.1–6.1 0.1–2.5CD4dimCD8þ/m1 2–82 1–68 0–40 1–44>20 cells/m1 3 (9.7%) 8 (11%) 6 (8 %) 4 (19%)

*CD4dimCD8þ T cells are expressed as a percentage of CD3þ T cells or as absolute values from asingle-platform system (Flowcount, Bcckman-Coulter). An increase in CD4dimCD8þ T cells at morethan 20 cells/m1 was not associated with an immune deficiency status, especially CD4þ or CD8þ lym-phocytosis. An increased frequency of CD4dimCD8þ T cells with age is possible.y

P < 0.05 versus donors, paired t test.§P < 0.001 versus donors, paired t test.#P ¼ 0.038, chi-square test.

11CIRCULATING CD4dimCD8þ T CELLS

mixture of beads coated with four calibrated amounts ofa mouse monoclonal immunoglobulin G–mimickinglabeled cells. T cells from total fresh blood samples werelabeled with unconjugated immunoglobulin G anti-CD8a(clone SFCI21thyD3; Beckman-Coulter) at a saturatingconcentration. T cells and calibration beads were thenlabeled with an FITC-conjugated goat anti-mouse immu-noglobulin G. A standard curve of fluorescence intensitywas plotted as a function of the number of molecules oncalibrated beads and the number of anti-CD8 on T cellsthat could be interpolated from it.

The clonotypes of TCR Vb chain were identified byusing pairs of conjugated monoclonal antibodies to Vb1(clone BL37.2) PE and Vb2 (clone MPB2D5) FITC, Vb3(clone CH92) FITC and Vb4 (clone WJF24) PE, Vb5.1(clone IMMU157) FITC and Vb5.3 (clone 3D11) PE,Vb5.2 (clone 36213) FITC and Vb8 (8.1 and 8.2; clone56C5) PE, Vb7 (clone ZOE) FITC and Vb9 (clone FIN9)PE, Vb11 (clone C21) PE and Vb12 (clone VER2.32.1)FITC, Vb13.1 (clone IMMU222) PE and Vb13.6 (cloneJU-74) FITC, Vb14 (clone CAS1.1.3) PE and Vb16 (cloneTAMAYA 1.2) FITC, Vb17 (clone E17.5F3) FITC andVb18 (clone BA62) PE, Vb20 (clone ELL 1.4) FITC andVb22 (clone IMMU 546) PE, Vb21.3 (clone IG125) FITCand Vb23 (clone AF23) PE (all purchased from Immuno-tech). Each pair was combined with anti-CD8a PE-TR(clone SFCI21thyD3; Beckman-Coulter) and anti-CD4 PE-Cy5 (clone MT310; DakoCytomation).

Statistical analysis were performed with chi-square test(frequency distributions), paired or unpaired Student’st test (quantitative values between groups), and linearregressions (between quantitative data) with determina-tion coefficient (r2) by using Excel (Microsoft, Redmond,WA, USA) and Statistica (Statsoft Inc., Tulsa, OK, USA).Results are expressed as mean 6 standard deviation.

RESULTS

Evidence for a CD4dimCD8þ T-Cell Subset

In routine analysis of T cells, we occasionally observeda small fraction of CD3þ T cells expressing lower levelsof CD4 (Fig. 1a). These cells always coexpressed CD8 atthe same level as conventional CD8þCD4� T cells (meanfluorescence intensity [MFI] 24.8 6 5.4; Fig. 1b). Thisfraction was subsequently named CD4dimCD8þ T cells.The CD4 MFI on this fraction was clearly decreased (MFI10.1 6 3.5) compared with conventional CD4þCD8� Tcells (MFI 24.0 6 2.6; paired t test on 38 representativesamples: P < 0.0001). The flow cytometer was set sothat more than 99% of isotype controls had fluorescenceintensities lower than 1.0. The fluorescence intensity ofCD4 had a trimodal distribution (Fig. 1c) showing a clearadditional CD4dim peak between the usual CD4 brightand negative peaks. Back-gating showed that theseCD4dimCD8þ T cells were not distinguishable from lym-phocytes on forward/size scatter.

Frequency of CD4dimCD8þ T-Cell Subsets

CD4dimCD8þ T cells was frequently observed in immu-nocompromised patients (HIVþ or kidney transplant reci-pients) routinely analyzed. Because preliminaryobservations relied on visual dot plot selection, we sys-tematically numbered these cells by batch analysis of 78consecutive patients from each group. Levels ofCD4dimCD8þ T cells were widely distributed, rangingfrom 1 to 68 cells/ml (Table 1).To obtain reference values, we analyzed a cohort of 31

consecutive healthy blood donors. Surprisingly, weobserved a similar discrepancy of data, with values rangingfrom 0 to 90 (median 7.5) CD4dimCD8þ T cells/ml (repre-senting 0.1% to 6.1% of total T cells, median 0.6%). How-ever, we observed that the vast majority of samples hadvalues clustered below 20/ml CD4dimCD8þ T cells (Fig. 2).In the absence of clear normal values, we arbitrarily chosethis level as a reference threshold. A few (9.7%) healthydonors had a high increase of that subset (Table 1).With regard to patient data, we observed that

increased CD4dimCD8þ T cells (>20 cells/ml) were not

related to immunodeficiency, with the percentage beingnonsignificantly different in renal transplantation recipi-

FIG. 1. a: CD4/CD8 dot plots from a representative patient clearlyshow a double CD4þ T-cell population expressing high (MFI 22.7)or low (MFI 3.28) levels of CD4. b: Although CD4bright T cellsdid not express CD8, CD4dim T cells expressed high levels of CD8.c: CD4 fluorescence intensity has a trimodal distribution with oneunusual intermediate peak.

12 LAMBERT ET AL.

ents (n ¼ 6–7.9%) and HIVþ patients (n ¼ 8–11%) com-pared with healthy donors (nonsignificant).

Increased CD4dimCD8þ T cells were associated overallwith higher lymphocytosis of CD4þ (831 6 488, range245–1,962, compared with patients with low

CD4dimCD8þ: 644 6 360, range 9–1,936; P ¼ 0.014,t test) and CD8þ T cells (966 6 898, range 180–3,845,vs. 566 6 530, range 24–4,874; P < 0.0001, t test). How-ever, CD4dimCD8þ T-cell counts were not directly corre-

lated to levels of CD8þ or CD4þ T cells (not shown).The increase in CD4dimCD8þ T cells was not related

to gender (41.2% of women had high CD4dimCD8þ and31.7% of women had low CD4dimCD8þ) or age (median

44 in subjects 32–71 years old and median 46 in those18–82 years old, respectively). However, this does notrule out an age effect because we observed in theelderly cohort an increased frequency of CD4dimCD8þ Tcells that was almost twice as high as in healthy donors(4 of 21, 19%). However, the absolute number ofCD4dimCD8þ T cells did not correlate with patients’ages. This was not statistically significant (chi-squaretest), but the size of the cohort was too small to allowfor reliable conclusions.

CD4dimCD8þ T Cells Not Recently Activated

We found no significant expression in the activationmarker CD69 on CD4dimCD8þ and conventional CD8þ

fresh peripheral T cells (results not shown). Further, ahomogeneous dot plot of CD4dimCD8þ T cells was con-

stantly observed on repeated analysis over a period of afew years in several patients (results not shown).

TCR and CD8 Phenotype of CD4dimCD8 T Cells

After simultaneously analyzing CD4 with TCRab, CD8b,and CD8a in 14 samples, we observed that all CD4dimCD8þ

T cells expressed the TCRab and the vast majorityexpressed CD8a and CD8b chains (Fig. 3a–c). This sug-gests that, unlike CD4þCD8dim T cells, CD4dimCD8þ T cellsexpress the ab heterodimeric form of the CD8 corecep-tor and belong to the systemic, thymus-dependentimmune system. However, the CD4dim also appeared insome cases on CD8aa T cells (Fig. 3d). The increase inthe CD4dimCD8þ T-cell subset was not associated withan increase in mucosa-associated lymphoid subsets suchas gd T cells or CD4�CD8aa T cells.

FIG. 3. CD8a/CD8b expression. a: CD4 and CD8 were analyzed onTCRabþ T cells selected from lymphocytes gated on forward/side scat-ter parameters. b: Most CD8þTCRab cells expressed CD8a and CD8bchains. In most patients, CD4dim CD8þ expressed CD8a and CD8b(c: data from one representative patient) but CD8a alone was alsoobserved (d: data from another representative patient).

FIG. 2. CD4dimCD8þ T-cell subset distribution in consecutive sam-ples. The size of the subset was analyzed with CD45/CD3/CD4/CD8immunolabeling in fresh peripheral blood samples from 31 healthydonors, 78 HIV þ patients, 78 kidney transplant recipients, and 21elderly volunteers. A minority of samples (8–19%) in all four groupshad high levels of CD4dimCD8þ T cells (>20 cells/ml).

13CIRCULATING CD4dimCD8þ T CELLS

Diverse Repertoire of CD4dimCD8 T Cells

Because the observed homogeneity on dot plots maydemonstrate monoclonal expansion, we analyzed the dis-tribution of the TCR Vb clonotypes in 26 patients withunambiguous CD4dimCD8þ dot plots.

The TCR Vb clonotypes were widely distributed onconventional CD4þ T cells (Fig. 4a). Most of the clono-types were expressed on 0.25% to 5% of CD4 T cells. Afew clonotypes were expressed on more than 5% of CD4þ

T cells, especially Vb2, (expressed on 10–23% of CD4þTcells in 14 patients, 33%) but also Vb3, Vb5.1, and Vb17.

In contrast, the Vb clonotype distribution was muchmore restricted on CD8þ T cells (Fig. 4b); many Vb clo-notypes (Vb4, Vb5.2, Vb5.3, Vb11, Vb14 and Vb18)were undetectable (expressed on <0.25% of CD4�CD8þ

T cells) in most patients (48% to 98%). Only a few clono-types were expressed by more than 5% of CD8þ T cells:Vb2 (expressed on 5–13.2% of CD8þ T cells in 45% ofpatients), Vb3 (5.1–27.3% in 47% of patients), Vb17(5.2–16.3% in 35% of patients), and Vb1 (5.4–20.4% in27% of patients). Moreover, a few patients expressedone dominant Vb clonotype (10–39.3% of CD4�CD8þ Tcells), especially Vb3 (13 patients, 22%) and Vb8, 13.1,and 17 (four patients, 8% for each clonotype).

In contrast, 13 of 23 patients (56.5%) expressed onepredominant clonotype (40–94% of CD4dimCD8þ T cells).The same clonotype was never overexpressed (<10% ofT cells) in the other CD4þ or CD8þ T-cell subsets in thesame patient (Fig. 5). The predominant clonotypes werewidely diverse (Fig. 6): Vb5.2 and Vb17 (in two patientseach) and Vb2, Vb3, Vb5.1, Vb13.1, Vb14, and Vb20(one patient). When no dominant clonotype wasobserved, the sum of clonotypes was detected on only23% to 66% of cells, suggesting that another clonotypethat was not tested in that panel might have expanded.

DISCUSSION

Double-positive CD4þCD8þ T cells have beendescribed for several years but have never been properlycharacterized, and their immunologic and clinical signifi-cance remains unclear. They are more frequentlyobserved during viral infection and in this case areusually transient, disappearing within a few months (3).However, they have also been observed in blood donorswho are not infected with chronic virus infections (2).The existence of this cell subset is not questionable

when four-parameter labeling is used: lymphocytes wereelectronically selected by their physical (forward and

FIG. 4. Diversity of conventional T cells. Clonotypes of TCR Vb chain were analyzed on CD4þ or CD8þ peripheral blood T cells from 26 patients(different symbols) with unambiguous CD4dimCD8þ T-cell subsets. a: All clonotypes except Vb23 were expressed by CD4þ T cells and many clono-types were expressed by more than 5% of CD4þ T cells, especially Vb2, Vb3, Vb5.1, and Vb17. b: Conversely, Vb2, Vb3, and Vb17 were the mostfrequent clonotypes on CD8þ T cells, but many clonotypes were frequently not expressed (Vb4, Vb5.2, Vb5.3, Vb9, Vb11, Vb14, Vb18, and Vb23).

14 LAMBERT ET AL.

side scatter) properties and their level of CD45 expres-sion, which was clearly higher than on any other leuko-cytes. Double-positive T cells were then identified bytriple labeling with CD3, CD4, and CD8. Standardizedsettings of the flow cytometer and assurance quality vali-dation make the results highly reproducible. Double-posi-tive CD4þCD8þ T cells have to be distinguished fromthe occasional nonspecific double CD4/CD8 labeling,easily recognized, and usually decreased by washing ofcells. This is observed in patients who have had recentsevere infections. This phenomenon is not understoodand is rapidly reversible.

Double-positive labeling is well known on early thy-mocytes, which corresponds to a maturation stagebefore the thymocytes are engaged in a CD4þ or CD8þ

single-positive lineage. These early thymocytes are nor-mally not detected in peripheral blood, especially inadult patients whose thymus is not functional for severalyears, unless under drastic conditions (12).Unconventional T-cell populations are present in most

healthy donors but at very low levels, which are difficultto distinguish from electronic noise. However, in a fewsamples, they are significantly increased and becomeobvious on dot plots observation, as illustrated in

FIG. 5. Restricted diversity of unconventional T cells in one characteristic patient. CD4dimCD8þ T cells (black bars) expressed almost exclusively(83.7% of cells) one Vb clonotype (Vb3 in that case). In contrast, the Vb clonotypes were widely distributed on conventional CD4þ T cells (gray bars)and much less on CD8þ T cells (white bars).

FIG. 6. Clonotype distribution of CD4dimCD8þ T cells in all patients tested. One predominant clonotype was observed at high frequency (25–88%of CD4dimCD8þ T cells in 13 of 26 cases with unambiguous dot blots). Vb5.2 and Vb 17 were the most frequent among predominant clonotypes.Many clonotypes were not expressed, the same as on CD8þ T cells, suggesting that the CD4dimCD8þ T cells are derived from the CD8þ T-cell pool.

15CIRCULATING CD4dimCD8þ T CELLS

Figure 1. Because the information is hidden, there is nochance for clinicians to consider it and elucidate its sig-nificance. Most of the time, clinicians would be embar-rassed with that information because its clinicalsignificance is unclear.

They seem to be persistent and were observed onrepeated analyses in patients periodically tested overmonths or years of follow-up (our experience) (1). Itwas difficult to define reference values because the dotplot appearance was much influenced by lymphocytosis.We observed that up to 90% of healthy donors hadfewer than 20 CD4dimC8þ T cells per microliter. We con-sider it a reasonable reference number until its clinicalrelevance can be defined. The increase in this subsetwas sometimes highly significant, reaching three- or four-fold above the threshold level.

Moreover, in some cases, the dot plot had a veryhomogeneous distribution, suggesting that only arestricted number of specificities was concerned. Thishomogeneity was confirmed with TCR clonotyping inaccordance with a previous report (1) and most prob-ably corresponds to at least an oligoclonal expansion ofthese cells.

Because flow cytometers allow reproducible measure-ment of the level of fluorescence labeling, it becameobvious that double-positive T cells correspond to twodifferent phenotypes. One type expresses high levels ofCD4 and low levels of CD8 (2,3). We and others haveshown that these CD4þCD8dim T cells are most probablyderived from CD4þ T cells and express the homodimericaa isoform of CD8, suggesting that they are related tomucosal immunity. In the present study, we have demon-strated that the CD4dimCD8þ T cells express usual levelsof ab heterodimeric form of CD8, suggesting that, unlikeCD4þCD8dim T cells, the CD4dimCD8þ T cells were notderived from mucosa-associated lymphoid tissue.

There is sparse information available on the ontogenyof this phenotype. Because, unlike CD4þ T cells, the Vbrepertoire of the CD4dimCD8þ T cells is highly restricted,we believe they are closely related to conventionalCD8þ T cells. These T cells are probably not early thy-mocytes. Further, experimental data have shown thatCD4 expression may be peripherally reacquired by someCD8þ T cells under particular stimulation conditions(13,14). The same observation was made for CD8aareexpression on CD4þ T cells (9–15). However, in thecase of our observations, this coexpression is not a sim-ple transient activation artifact because the phenomenonis long-lasting and the CD4dimCD8þ T we observed werenot in activation status (not expressing CD69). Thus, weconclude that these CD4dimCD8þ T cells are an authen-tic subset of CD8þ T cells.

The functional advantage of these CD4dimCD8þ T cellsis not known and, unlike for CD4þCD8dim T cells (10),there is no report of a well-characterized CD4dimCD8þ

cell line to our knowledge. Do these CD8 T cells acquirehelper activity and the ability to interact with MHC classII molecules? These CD4dimCD8þ T cells have beenshown to have a preferential T-helper 2-like activity in

producing interleukin-4 and not interleukin-2 or inter-feron-g (16), unlike CD4þCD8dim T cells, which are pre-ferentially of type T-helper 1 (17). Further, like Ortolaniet al. (3), we did not observe expression of CD56 onthese CD4dimCD8þ T cells, as we occasionally did onCD4þCD8dim T cells, suggesting they do not possesscytotoxic activity (18). However, acquiring expression ofCD4 makes CD8 T cells sensitive to HIV infection (13),although we did not observe a particular decrease ofthese cells in HIVþ patients.What is the significance of such persistence? Does this

phenomenon only reflect a scar of a dominant ancientstimulation such as a viral infection (19)? Is thisrestricted stimulation still going on or do these particularcells have a persistent proliferative capacity? They havebeen associated with chronic immune disorders (4–20)and autoimmunity (17). The oligoclonality may also indi-cate a lymphoproliferative disorder, as described for theCD4þCD8dim phenotype (5,6). By analogy to monoclonaldysglobulinemia of undetermined significance, we con-sider it possible that some of these cases may progresstoward a real lymphoproliferative syndrome. We proposeto name this phenomenon ‘‘oligoclonal clonopathy ofundetermined significance.’’ Long-term follow-up of sucha cohort should allow a definition of predictive criteria.Meanwhile, we believe that patients with oligoclonal clo-nopathy of undetermined significance should be fol-lowed clinically.

ACKNOWLEDGMENTS

We thank Pascale Saby for skillful technical assistance.

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17CIRCULATING CD4dimCD8þ T CELLS