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The Journal of Immunology, 2007, 179: 221-228.
Copyright © 2007 by The American Association of Immunologists, Inc.
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A New Subset of Human Naive CD8+ T Cells Defined by Low Expression of IL-7R{alpha}1

Nuno L. Alves2,3,*, Ester M. M. van Leeuwen2,4,*,{dagger}, Ester B. M. Remmerswaal*, Nienke Vrisekoop{ddagger}, Kiki Tesselaar{ddagger}, Eddy Roosnek§, Ineke J. M. ten Berge*,{dagger} and René A. W. van Lier5,*

* Department of Experimental Immunology, {dagger} Department of Internal Medicine, Division of Nephrology, Academic Medical Center, Amsterdam, The Netherlands, {ddagger} Department of Immunology, University Medical Center Utrecht, Utrecht, The Netherlands; and § Department of Internal Medicine, Division of Hematology, Geneva University Hospital, Geneva, Switzerland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Concomitant with an increased number of memory-type cells, the amount of naive T cells steadily declines with age. Although the regulatory mechanisms behind this conversion are not fully understood, the suggestion is that both alterations in thymic output and homeostatic signals mold the naive T cell pool. In this study, we identify a new subset of circulating CD27highCD45RAhigh CD8+ T cells characterized by low IL-7R{alpha} message and protein expression. Analysis of TCR repertoire and TCR excision circle content together with ex vivo recovery of IL-7R{alpha} expression indicated that these cells should be placed into the naive T cell pool. Compared with conventional IL-7R{alpha}high naive T cells, this subset displayed significantly lower levels of CD28 and higher levels of HLA-DR. Proliferative responses to anti-CD3/CD28 mAbs were indistinguishable from conventional naive T cells, but the responsiveness to IL-7 was limited. Strikingly, IL-7R{alpha}low naive T cells were particularly increased in circumstances of naive CD8+ T cells shortage, as in the elderly, in patients early after hemopoietic stem cell transplantation, and in HIV-infected individuals. As common {gamma} chain cytokines induce rapid down-regulation of IL-7R{alpha}, we propose that this new subset of naive T cells may encompass cells that have recently received homeostatic signals.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Throughout life, the maintenance of a functional diverse pool of T cells is necessary to preserve immunocompetence. T cell numbers are fairly constant despite the input of naive T cells from the thymus, their maintenance in the periphery, the expansion and waning of specific clonotypes caused by multiple pathogen challenges, and the formation and turnover of memory cells (1). Hence, the size of the peripheral T cell pool is under tight regulatory mechanisms.

Several studies have shown that cytokines belonging to the common {gamma} chain ({gamma}c)6 cytokine receptor family, especially IL-7 and IL-15, are pivotal for T cell homeostasis (2, 3, 4). In mice, ablation of IL-7 and IL-15, or their respective specific receptor components IL-7R{alpha}- and IL-15R{alpha}-chain, causes an overt defect in the maintenance of naive and memory CD8+ T cells, respectively (5, 6, 7, 8). These different cytokine requirements exhibited by distinct T cell subsets may be conditioned by the expression of cytokine receptor subunits. Naive CD8+ T cells express both the {alpha} chain and {gamma} chain of the IL-7R complex (9, 10), and IL-7R{alpha} expression has been used together with several other cell surface markers to typify naive T cells (10, 11, 12, 13).

The number, percentage, and diversity of naive T cells declines with age (14, 15). Besides conversion to the memory pool after Ag challenge, this finding indicates that during their lifespan naive T cells may be exposed to selective pressures through the availability of homeostatic cytokines (e.g., IL-7, IL-15). Because IL-7R{alpha} levels are dynamically regulated by homeostatic cytokines and Ag (10, 13, 16, 17, 18), we hypothesized that its expression in naive T cells could be affected under circumstances of altered composition of the T cell pool.

In this study, we analyzed IL-7R{alpha} expression in naive CD8+ T cells. We identified a subset of circulating naive CD8+ T cells (CD27highCD45highIL-7R{alpha}low) particularly increased in the elderly, in patients early after bone marrow transplantation, in HIV infection, and in those whose percentage inversely correlated with the frequency of total naive CD8+ T cells. Apart from IL-7R{alpha}, this subset had phenotypic and functional traits that placed it into the naive T cell pool. Our data suggest that IL-7R{alpha}low naive T cells may constitute a pool of circulating naive cells that have recently exited from cytokine-rich niches such as secondary lymphoid organs.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents and mAbs

Monoclonal Abs FITC-conjugated TCR-{alpha}beta, PE-conjugated CD4, PerCP-Cy5.5-conjugated CD3, allophycocyanin-conjugated CD3, PerCP-Cy5.5-conjugated CD8, biotin-conjugated CD122, biotin-conjugated CD132, and PerCP-Cy5.5-conjugated streptavidin were purchased from BD Biosciences. PE-Cy7-conjugated CCR7, allophycocyanin-Cy7-conjugated CD8, and PE-Cy7-conjugated CD28 were obtained from eBioscience. Allophycocyanin-conjugated CD45RA was obtained from BD Pharmingen. FITC- and biotin-conjugated CD27 (clone 3A12) were obtained from Sanquin. Biotin-conjugated polyclonal anti-IL-15R{alpha} was obtained from R&D Systems. PE-conjugated anti-IL-7R{alpha} (CD127) was purchased from Immunotech. Anti-human CD8 microbeads (MACS) were purchased from Miltenyi Biotec. Cells were stained with indicated mAbs and were analyzed on a FACSCalibur (BD Biosciences). Six-color FACS analysis was performed on the LSRII FACS (BD Biosciences).

Healthy individuals and patients

Peripheral blood was drawn from 43 healthy individuals, ranging in age from 18 to 83 years. Nenonatal blood was obtained from umbilical cord blood.

Thymectomized patients

Three patients (two male and one female) who were studied had been thymectomized at least 5 years previously because of myasthenia gravis. They were age 35, 39, and 40 years, respectively, and their drug therapy consisted of prednisone, azathioprine, and pyridostigmine (Mestinon).

HIV-infected patients

Cryopreserved peripheral blood samples from 14 antiretroviral HIV-1-infected individuals were analyzed. The samples were collected as baseline samples in a study concerning long-term immune reconstitution during highly active antiretroviral therapy. Median CD4 count of the HIV-1-infected patients was 150 cells/µl (range 20–490 cells/µl).

Patients after hemopoietic stem cell transplantation (HSCT)

All four patients in the study transplanted for leukemia received grafts from their HLA identical sibling after classical conditioning with total body irradiation and cyclophosphamide. No posttransplantation complications that would need a durable increase of the immunosuppressive regimen occurred and all patients remained in complete remission during the period of the study.

The study was approved by the Medical Ethical Committee, and written informed consent was obtained from all participants.

Cell preparation and sorting

Human PBMCs, from healthy donors or patients, and umbilical cord blood mononuclear cells were isolated by Ficoll-Isopaque density gradient centrifugation (Pharma; Nycomed). For cell sorting, CD8+ T cells were purified from total PBMC by positive selection using the MACS system, as previously described (9). Purified CD8+ T cells were stained with FITC-conjugated CD27, PE-conjugated IL-7R{alpha}, PerCP-Cy5.5-conjugated CD8, and allophycocyanin-conjugated CD45RA and sorted in CD8highCD27highCD45RAhighIL-7R{alpha}low and CD8highCD27highCD45RAhighIL-7R{alpha}high cells (BD Biosciences). The purity of the sorted populations was assessed by FACS analysis and it was >95%.

CFSE labeling

Purified T cells were pelleted and resuspended in PBS at a final concentration of 5–10 x 106 cells/ml. Next, cells were labeled in 0.5 µM (final concentration) of CFSE (Molecular Probes) in PBS for 10 min at 37°C. Cells were washed and subsequently resuspended in IMDM supplemented with L-Glutamine and 25 mM HEPES (BioWhittaker) containing 10% human pool serum (BioWhittaker), 100 ng/ml streptomycin (Invitrogen Life Technologies), 10 U/ml penicillin (Yamanouchi Pharma), and 3.57 x 10–4 (%v/v) 2-ME (Merck) as culture medium.

Cell culture

Cells were cultured in culture medium for the indicated time points at 37°C in 5% CO2 atmosphere. For cytokine activation, cells were cultured in the presence or absence of IL-7 (Strahtmann). For combined TCR-CD28 activation, cells were cultured with plate-bound anti-CD3 mAbs (1.0 µg/ml, clone 16A9; Sanquin) in the presence or absence of soluble anti-CD28 mAbs (5 µg/ml, clone 15E8; Sanquin).

Real-time PCR

Total RNA was extracted from freshly isolated cells using GenElute Mammalian total RNA Miniprep kit (Sigma-Aldrich). Oligo(dT)-primed cDNA was synthesized using avian myeloblastosis virus reverse transcriptase (Roche Molecular Biochemicals). From these cDNA pools, specific targets were amplified by PCR performed with LightCycler FastStart DNA Master SYBR Green I from Roche, using the sense and antisense IL-7R{alpha} primers 5'-TCGCAGCACTCACTGACC-3' and 5'-CGGGAAGGAGCCAATGAC-3', the LEF-1 primers 5'-GCTTTATCCAGGCTGGTCTGCAA-3' and 5'-GACCTGTACCTGATGCAGATTCCT-3', and 18 S primers 5'-GGACAACAAGCTCCGTGAAGA-3' and 5'-CAGAAGTGACGCAGCCCTCTA-3', respectively. The results were normalized to 18 S rRNA.

Reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) procedure and analysis

The apoptotic gene expression profile measured by RT-MLPA was performed as previously described (19). In brief, MLPA probes consist of two oligonucleotides that anneal to adjacent sites on a target sequence and are then ligated by a heat stable ligase. RNA was first reverse transcribed using a gene-specific probe mix, and the resulting cDNA was annealed overnight at 60°C to the MLPA probes and covalently linked by Ligase-65 at 54°C (MRC-Holland). Ligation products were amplified and fluorescently labeled by PCR. Samples were analyzed on an ABI 3100 capillary sequencer (Applied Biosystems), and data were analyzed with Genescan and Genotyper (Applied Biosystems), and further analyzed with Microsoft Excel spreadsheet software. The sum of all peak data was set at 100% to correct for fluctuations in total signal between samples, and individual peaks were calculated relative to the 100% value.

Vbeta repertoire and CDR3 length distribution analysis

cDNA was subjected to Vbeta PCR as previously described (20). As constant beta primers, the FAM-labeled primers (5'-CAGGCACACCAGCAGTGTGGC and 5'-AGATCTCTGCTTCTGATGGCTC; Invitrogen Life Technologies) were used. Vbeta PCR products were visualized on a 1% agarose gel. For the spectratyping, aliquots of positive samples were mixed with Genescan-500 ROX size standards and run on an ABI 3100 capillary sequencer (Applied Biosystems) in Genescan mode. Data were analyzed with Genescan software packages (Applied Biosystems).

TCR excision circle (TREC) analysis

DNA of the sorted cell subsets was isolated using the QIAamp blood kit (Qiagen). Signal joint TREC analysis numbers and the amount of input DNA were quantified using real-time PCR as previously described (21). The number of C{alpha} constant regions was measured to determine the amount of DNA. The signal joint TREC copies and the amount of C{alpha} were determined using a dilution series of a standard sample, which was included in each PCR experiment. From the average TREC content, as measured per microgram of DNA, the TREC content per cell was calculated by assuming that 1 µg of DNA corresponds to 150,000 cells.

Statistical analysis

The two-tailed Mann-Whitney U test was used for analysis of differences between groups. For correlations, the spearman nonparametric correlation test was used. Values for p < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Identification of a subset of IL-7R{alpha}low cells with naive CD8+ T cell traits

We characterized the expression of IL-7R{alpha} on human CD8 T cell subsets in the peripheral blood of individuals of different ages (Fig. 1), using CD27 and CD45RA as cell surface markers (11). As expected (9, 10, 11), in neonatal blood the majority of CD8 T cells possessed a naive phenotype (CD27highCD45RAhigh), were included in the IL-7R{alpha}high gate, and were hardly found in IL-7R{alpha}low fraction. A minor population of IL-7R{alpha}low cells was occasionally found with an effector phenotype (CD27–CD45RAhigh) (Fig. 1B, top row). In healthy adults, IL-7R{alpha}high cells included CD27highCD45RAhigh naive cells and CD27highCD45RAlow memory cells. Conversely, IL-7R{alpha}low cells were predominantly CD27–CD45RAhigh, although a small fraction was present in CD27highCD45RAlow and unexpectedly also in the putatively naive CD27highCD45RAhigh gate (9.1 ± 6.0%) (Fig. 1B, bottom row). The latter observation pointed to the existence of a novel T cell subset and subsequent experiments were designed to clarify its origin.


Figure 1
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  		FIGURE 1. Presence of IL-7R{alpha}low cells within naive CD27highCD45RAhigh CD8+ T cell gate. A, Representative example of IL-7R{alpha} subset discrimination within CD8+ lymphocyte gate (adult healthy donor). B, IL-7R{alpha}low cells are found in the naive CD8 T cell gate in peripheral blood of adult healthy donors. Umbilical cord blood mononuclear cells from neonatal (top row) and PBMCs from adult (bottom row) healthy individuals (n = 43) were stained with indicated markers followed by FACS analysis. T cell subset distribution, based on CD27 and CD45RA, in total CD8high gate is depicted (left). CD8 T cell subsets found within IL-7R{alpha}low (middle) and IL-7R{alpha}high (right) gate are represented. Numbers shown indicate percentage of CD27highCD45RAhigh cells found within IL-7R{alpha}low and IL-7R{alpha}high gate.

 
CD27highCD45RAhighIL-7R{alpha}low cells are naive CD8+ T cells

Analysis of the expression of TCR-{alpha}beta complex showed that CD27highCD45RAhighIL-7R{alpha}low cells were typical mature {alpha}beta CD8+ T cells (Fig. 2A). To test whether this subset consisted of naive cells or Ag-experienced cells that had reverted their phenotype from RO to RA expression, we performed a comparative analysis on the TCR Vbeta repertoire and replicative history by quantifying their content of TREC (21). As depicted in Fig. 2B, the different TCR Vbeta chains were equally represented in both sorted, CD27highCD45RAhighIL-7R{alpha}low, and CD27highCD45RAhighIL-7R{alpha}high CD8+ T cell populations. Close inspection of the CDR3 length distribution within Vbeta family members showed that IL-7R{alpha}low cells possessed a TCR repertoire as diverse as their IL-7R{alpha}high counterparts (Fig. 2C). Importantly, analysis of TREC content, as a marker for replicative history, showed that IL-7R{alpha}low cells were undistinguishable from conventional naive cells. In contrast, memory-type CD8+ T cells (CD27highCD45RAlow) did show the expected dilution of TRECs caused by extensive proliferation (Fig. 2D). Together, these combined findings strongly indicate that the CD27highCD45RAhighIL-7R{alpha}low cells constitute a subset of genuine naive CD8+ T cells.


Figure 2
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  		FIGURE 2. CD27high CD45RAhighIL-7R{alpha}low cells possess an identical broader TCR repertoire and TREC content as conventional naive CD8+ T cells. A, Representative staining demonstrating that CD8highCD27highCD45RAhighIL-7R{alpha}low cells are {alpha}beta T cells. CD8highCD27highCD45RAhighIL-7R{alpha}low and CD8highCD27highCD45RAhighIL-7R{alpha}high cells were sorted from adult healthy donors. B, Diverse Vbeta repertoire of CD27highCD45RAhighIL-7R{alpha}low cells. Analysis was done on cDNA obtained from RNA of sorted populations. The bands on gel were retrieved after PCR for the different Vbeta families mentioned on top of the pictures. Representative experiments (n = 3) from one donor. C, Spectratyping of the Vbeta families found in both populations. Each peak represents CDR3 regions with a certain length spaced by three nucleotides. Numbers indicate the corresponding Vbeta family, with the different cell populations shown. D, Identical TREC content in both populations. TREC content was analyzed in both CD27highCD45RAhighIL-7R{alpha}low/high populations. Total naive (nonfractionated) and memory CD8 T cells (CD27highCD45RAlow) are shown as internal control. Data shown are the mean of at least three independent sorts.

 
IL-7R{alpha}low cells bear an unconventional naive cell surface phenotype

For simplicity, this study refer to CD27highCD45RAhighIL-7R{alpha}low and CD27highCD45RAhighIL-7R{alpha}high cells as IL-7R{alpha}low or IL-7R{alpha}high naive cells, respectively. Conventional naive CD8+ T cells can be discriminated by the simultaneous expression of an array of distinct cell surface markers such as CD27, CD45RA, CD28, CCR7, and IL-7R{alpha} (9, 11, 12). We characterized IL-7R{alpha}low naive cells phenotypically and compared them with the conventional IL-7R{alpha}high naive CD8+ T cells. IL-7R{alpha}low cells significantly expressed higher levels of HLA-DR and lower levels of CD28, whereas the expression of CCR7 tended to be lower (Fig. 3A, top). Analysis of the expression of the IL-15R components in both subsets showed equivalent levels of {gamma}c (CD132) and IL-15R{alpha}-chain, but suggested an augmented expression of IL-2/15 beta-chain (CD122) on IL-7R{alpha}low naive cells (Fig. 3A, bottom). Confirming previous findings (9, 10, 22), in vitro culture in the presence of IL-15 in the absence or presence of IL-7 yielded a similar phenotype in naive CD8+ T cells (Fig. 3B). Hence, IL-7R{alpha}low naive cells bear combined traits of naive and {gamma}c cytokine-activated T cells.


Figure 3
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  		FIGURE 3. IL-7R{alpha}low cells display an altered naive T cell phenotype. A, IL-7R{alpha}low naive CD8+ T cells (Figure 3) were characterized phenotypically for the indicated markers by flow cytometry using the BD LSRII analyzer that enables the detection of up to 8 fluorescent colors. IL-7R{alpha}high naive CD8+ T cells were used as comparison ({blacksquare}). The mean fluorescence intensity (MFI) for a panel of different markers is represented. Mean MFI and SD of n = 3–6 donors. Values for p represent the statistical analysis for differences within groups. B, Purified naive CD8+ T cells, obtained from umbilical cord blood, were stimulated in the presence of combined IL-7 and IL-15 (10 ng/ml each). After 4 days the cells were stained for the indicated cell surface markers, followed by FACS analysis. Ex vivo (thin line histogram) and cytokine-activated cells (thick line histogram) are shown. Data are from one representative experiment of two conducted.

 
IL-7R{alpha}low naive cells have an adequate proliferative response to combined TCR-CD28 activation but limited responsiveness to IL-7

We next assessed whether IL-7R{alpha}low naive cells were able to respond to distinct mitogenic stimuli. Both IL-7R{alpha}low and IL-7R{alpha}high naive populations were sorted, labeled with CFSE, and stimulated with combined CD3/CD28 mAbs or IL-7. As shown in Fig. 4, the response to CD3/CD28 mAbs was undistinguishable between both subsets. In contrast, IL-7R{alpha}low cells displayed a limited proliferative response to IL-7 compared with IL-7R{alpha}high counterparts. Thus, our data showed that IL-7R{alpha}low naive cells are capable of expanding to stimulation through TCR and importantly show that these cells are not anergic following TCR-CD3 engagement.


Figure 4
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  		FIGURE 4. Proliferative response of IL-7R{alpha}low naive cells to combined TCR-CD28 or {gamma}c cytokine IL-7. Purified CD8+ T cells from adult healthy donors were stained with Abs to CD8, CD27, CD45RA, and IL-7R{alpha}. Sorted IL-7R{alpha}low and IL-7R{alpha}high naive CD8+ T cells were labeled with CFSE and stimulated for 3 days with CD3/CD28 mAbs or for 6 days with IL-7 (10 ng/ml). Cell division was measured by FACS analysis. Data represent one of two experiments conducted.

 
IL-7R{alpha}low naive cells bear a partial molecular signature of IL-7 signaling

The reduced expression of IL-7R{alpha} in naive CD8+ T cells exposed to IL-7 (10) has recently been demonstrated to be regulated at the transcriptional level (13). Furthermore, it has been shown that IL-7R{alpha} signals inhibit the expression of the transcription factor LEF-1 (23) and have prosurvival effects in T cells through up-regulation of Bcl-2 and Mcl-1 (3, 24). Based on the data, we considered that the IL-7R{alpha}low naive cell subset could define a pool of circulating naive cells that had recently been in contact with IL-7 or other {gamma}c cytokines. Analysis of mRNA levels in sorted cells showed that concordantly with low IL-7R{alpha} expression detected at protein level, IL-7R{alpha}low naive cells exhibited significant lower levels of IL-7R{alpha} mRNA as compared with IL-7R{alpha}high counterparts, whereas mRNA levels for LEF-1 tended to be diminished (Fig. 5A). To broaden the analysis of putative targets of IL-7R{alpha} signals, we examined the levels of Bcl-2 family members by RT-MLPA (19). Comparison of the apoptotic gene expression profiles showed neither difference in Bcl-2 and Mcl-1 levels nor in other Bcl-2 family proteins included in the assay (Fig. 5B and data not shown). Undetectable levels of survivin (25) indicated that IL-7R{alpha}low naive cells are not cycling (data not shown).


Figure 5
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  		FIGURE 5. IL-7R{alpha}low naive cells display a partial molecular signature for IL-7 signals. IL-7R{alpha}low (Figure 5) and IL-7R{alpha}high ({blacksquare}) naive CD8+ T cells were sorted, and mRNA was isolated. A, The expression of IL-7R{alpha} and LEF-1 was compared by real-time PCR. The mean and SD of six (IL-7R{alpha}) or three (LEF-1) donors is shown. B, Bcl-2 and Mcl-1 levels were measured by RT-MLPA, and the relative expression for each was calculated as described in Materials and Methods. Data represent average of three experiments performed. Values for p represent the statistical analysis for differences within groups. C, IL-7R{alpha} expression is recovered in IL-7R{alpha}low naive CD8+ T cells. i, IL-7R{alpha} expression was analyzed in sorted populations following overnight (O/N) culture in plain medium. Purity after sort (left) and overnight culture are shown (right). After sort, day 0 cultured (thin line histogram), overnight cultured cells labeled with IL-7R{alpha} mAb (bold line histogram), and unlabeled overnight cultured cells (dashed line histogram) are depicted. A representative experiment of n = 3 is shown. ii, Total viability in the cultures. Viability was assessed by FACS analysis based on viable gating. Mean and SD of three experiments is represented.

 
Additionally, we investigated whether IL-7R{alpha} protein expression in IL-7R{alpha}low cells was shut down permanently. Overnight incubation of sorted populations in plain culture medium showed that IL-7R{alpha}high naive cells increased IL-7R{alpha} expression. Importantly, under the same conditions IL-7R{alpha}low cells also rapidly re-expressed IL-7R{alpha} to a similar level as ex vivo IL-7R{alpha}high cells (Fig. 5C, i). To exclude the possibility that selection of cells expressing higher levels of IL-7R{alpha} during overnight cultures might explain this finding, cell death was measured. The viability of both populations remained above 80% although IL-7R{alpha}low cells appeared to be more susceptible to cell death (Fig. 5C, ii). These data suggest that a change in environmental cues can rapidly increase in IL-7R{alpha} mRNA levels in naive IL-7R{alpha}low CD8+ T cells.

Increased frequency of IL-7R{alpha}low naive cells in individuals with low abundance of naive CD8+ T cells

Next, we considered the possibility that the frequency of IL-7R{alpha}low naive cells could be altered in circumstances in which normal T cell homeostasis is disturbed, either in physiological situations (elderly) or pathological condition (HSCT or HIV infection). Analysis on the frequency of IL-7R{alpha}low cells found within the naive CD8+ T cell gate, in neonates and several individuals ranging in age from 18 to 86 years, revealed that this population was nearly undetectable in neonates, constant in adult life, and particularly augmented in the elderly (Fig. 6A). Their frequency remained unaltered in thymectomized patients who possessed normal T cell numbers. Their frequency was also significantly augmented both in HIV-infected patients and early after HSCT (Fig. 6B).


Figure 6
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  		FIGURE 6. Frequency of IL-7R{alpha}low naive cells inversely correlates with the abundance of naive CD8+ T cells. Total PBMCs were isolated, stained with Abs to CD8, CD27, CD45RA, and IL-7R{alpha} and subjected to FACS analysis. A, IL-7R{alpha}low naive CD8+ T cells are increased in the elderly. The percentage of IL-7R{alpha}low cells found within the naive CD8+ gate is represented vs age. Measurements are obtained from 43 healthy donors. B, Frequency of IL-7R{alpha}low found in the naive CD8 gate in peripheral blood of thymectomized (T{phi}) (n = 3 patients), HIV-infected (n = 12 patients), and HSCT (n = 4 patients) is shown. HSCT patients were followed early (3–5 mo), intermediate (10–13 mo), and late (≥2 years) after transplantation. Patient ages vary between ages 28 and 45 years. Age-matched healthy individuals are represented (left). *, p < 0.05 significant difference in patient groups. C, Relation between the percentages of IL-7R{alpha}low cells found within the naive gate and the frequency of total naive cells for CD8 and CD4 subsets. i, IL-7R{alpha}low naive CD8 T cells in healthy individuals, HSCT transplanted, and HIV-infected patients. In HSTC results, square, diamond, and triangle symbols represent early, intermediate, and late time points. ii, All individual groups are included (healthy, T{phi}, HSCT, and HIV). Measurements are obtained from 62 healthy donors and patients. Results represent total data in the naive CD8 (left) or CD4 (right) T cell subsets.

 
The frequency of IL-7R{alpha}low naive cells in patients after HSCT decreased over time (Fig. 6B, right end). Because this decrease occurred concomitantly with the recovery and restoration of naive T cell pool (26), we reasoned that the frequency of IL-7R{alpha}low naive cells could be related with the abundance of naive CD8+ T cells. Indeed, the presence of this population inversely correlated with the frequency of total naive CD8+ T cells in both healthy and patients groups (Fig. 6C, i) and thus seemed to be unrelated with pathological conditions. This correlation became even more clear when healthy individuals and patients were plotted together (Fig. 6C, ii, left). In the CD4+ T cell subset a similar subset of IL-7R{alpha}low cells could be found but their frequency was substantially lower than in the CD8+ T cell compartment (Fig. 6C, ii, right). Comparable to the CD8+ T cells subset, also CD4+ IL-7R{alpha}low cells appeared to be most prominent in individuals with low levels of naive cells.

Overall, our data strongly suggest that the emergence of IL-7R{alpha}low naive cells in the circulation occurs in circumstances of naive T cell scarcity.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The decline in the number of naive T cells with the subsequent reduction in the diversity of the Ag-receptor repertoire is one of the most prominent features of aging of the immune system. Naive CD8+ T cells constitute one arm of the adaptive immune system equipped with a variety of Ag-recognizing receptors. Thus, to preserve full immunocompetence it is pivotal to maintain a diverse and functional pool of naive lymphocytes. Several phenotypic markers, including IL-7R{alpha}, have been used to characterize naive cells. In this study, we have identified a new subset of circulating naive CD8+ T cells typified by a low expression of IL-7R{alpha}. This population was absent in neonatal blood, present at low frequency in healthy adults, and particularly increased under conditions of low frequency of naive CD8+ T cells. Phenotypically this subset displayed combined traits of conventional and {gamma}c cytokine-activated naive T cells.

IL-7R{alpha} has been shown to be down-regulated by both cytokines and Ag stimulation (10, 16, 17, 23). From this perspective, the origin of IL-7R{alpha}lowCD27highCD45RAhigh cells, Ag- or cytokine-induced, became an obvious question. Analysis of TREC levels showed that these cells had not undergone extensive proliferation in vivo as they had a similar replicative history as typical naive cells, contrasting the successive rounds of divisions undergone by memory-type cells (Fig. 2). The combined analysis of TREC and diversity of TCR repertoire (Fig. 2) strongly indicated that the IL-7R{alpha}lowCD27highCD45RAhighCD8high population consisted of naive T cells. This subset appeared to be distinct from both the intermediate effector cells previously described by Rufer et al. (27) and IL-7R{alpha}low effector/memory cells found to be increased in the elderly (28). The latter are far differentiated cells, characterized by lack of CD27 and CD28, and therefore different from IL-7R{alpha}low naive cells described in our study (CD27highCD28dim). The existence of this population was not previously noted by Kim et al. (28). A possible explanation may be the use of different markers to define naive cells, i.e., CCR7 (28) vs CD27 (our manuscript). The finding that the levels of CCR7 tend to differ in IL-7R{alpha}low and IL-7R{alpha}high naive cells indeed supports this explanation (Fig. 3). In addition, low expression of IL-7R{alpha} has been recently proposed to be used as a marker for regulatory CD4 T cells, which have been defined as anergic to TCR-CD28 stimulation (29, 30). Yet, as IL-7R{alpha}low naive CD8 T cells exhibited a normal response to TCR-CD28 mAb as conventional naive T cells (Fig. 4), this subset presumably does not contain regulatory CD8+ T cells.

A number of findings indicate that the population of IL-7R{alpha}low naive cells represents a circulating pool of naive cells that has recently received composite signals in {gamma}c cytokine-rich microenvironments. First, IL-7 and IL-15 have been shown to induce distinct features in naive T cells in vitro. Although IL-7 maintains a naive phenotype except for down-regulating IL-7R{alpha}, IL-15 induces a primed phenotype (9, 10, 22, 31). Interestingly, naive CD8+ T cells cultured in the presence of both cytokines also acquired an intermediate phenotype (10) (Fig. 3B) that resembled in many ways the unconventional naive cells identified in this study (CD27highCD45highIL-7R{alpha}lowCD28lowHLA-DRhigh) (Fig. 3). Second, IL-7R{alpha} and IL-2/15Rbeta-chain expression have been shown to be decreased and augmented, respectively, following in vitro culture with IL-7 and IL-15 (9, 13). Ex vivo analysis of the expression of different components of cytokine receptors, IL-7R{alpha} and IL-2/15Rbeta (Figs. 1 and 3), revealed similar changes. IL-7 and IL-15 stimulate T cell survival by enhancing the expression of prosurvival members of the Bcl-2 family, such as Bcl-2 and Mcl-1 (3, 24). However, no differences in the expression of different members of the Bcl-2 family, including Bcl-2, were found when IL-7R{alpha}low and IL-7R{alpha}high naive cells were compared (Fig. 5B). The similarities in Bcl-2 levels between both subsets of naive cells could be explained by the transient transcriptional effect provoked by IL-7 in vitro (our unpublished data). IL-7R{alpha} and LEF-1 have been shown to be transcriptionally down-regulated by {gamma}c cytokines, particularly IL-7 (13, 23). Consistently, we found reduced levels of mRNA for IL-7R{alpha} and LEF-1 in IL-7R{alpha}low naive cells (Fig. 5A). IL-7R{alpha} protein was rapidly recovered following ex vivo culture (Fig. 5C), suggesting only a temporary reduced IL-7R{alpha} expression. Taken together, our data suggest that IL-7R{alpha}low naive cells display a partial signature for {gamma}c-signaled cells. It is worthy to note that the maintenance of memory T cells depends also on IL-15 and IL-7 (4), and thus similar mechanism may exist in the control of memory homeostasis. In fact, we found that IL-7R{alpha} expressions is absent on some memory CD8 T cells of healthy individuals (16) (Fig. 1). Moreover, its expression is diminished on memory CD8 T cells early after HSTC and it is partly increased on this subset at later time points concomitantly with restoration of normal T cell numbers (data not shown). Importantly, as IL-7R{alpha} expression is also down-regulated by Ag (16, 17), the inexistence of markers that enable distinguishing Ag- from cytokine-mediated activation precludes to discern with certainty the origin of IL-7R{alpha}low memory cells.

Following emigration from the thymus, naive cells possess limited access to different niches in the organism, moving from blood to secondary lymphoid organs and returning to the circulation via lymphatic vessels. The peripheral maintenance of naive CD8+ T cells is dependent on IL-7 and IL-15 working in conjunction with TCR signaling received from contact with self-MHC-peptide complexes (4). In contrast to IL-7 that is constitutively expressed, IL-15 seems to be particularly produced under stress responses (2, 32). We therefore hypothesized that the IL-7R{alpha}low naive population could be expanded in pathological conditions in which deregulated IL-15 levels have been reported (i.e., rheumatoid arthritis and inflammatory bowel disease) (32). However, its frequency remained normal in the peripheral blood of patients with rheumatoid arthritis and inflammatory bowel disease, compared with healthy individuals (data not shown). Possibly, the localized expression of IL-15 together with the limited accessibility of naive T cells into the inflamed microenvironment may explain our observations.

The frequency of IL-7R{alpha}low naive cells was, however, increased in circumstances in which the amount of naive T cells declined, as in the elderly, early after bone marrow transplantation, or in HIV infection (Fig. 6). In agreement with these findings, it has recently been shown both in humans and mice that IL-7R{alpha} is down-regulated on CD8 T cells during lymphopenic conditions (33, 34, 35). Noteworthy, IL-7R{alpha}low cells are predominantly found in the CD8 but not CD4 T cell subset. Despite that both subsets are able to respond to IL-7, the responsiveness of naive CD4 T cells to IL-15 is limited compared with that of their CD8 counterparts (9, 31, 36). These inherent distinct responses may be related to the predominant presence of this subset within CD8 T cells. The emergence of IL-7R{alpha}low naive CD8+ T cells in situations of low abundance of naive T cells may be a mere reflection of increased IL-7 and/or IL-15 bioavailability per cell. Supposing that cytokine levels are constant and limited under normal circumstances (37), a decrease in naive cell numbers would render them more accessible to IL-7 and IL-15 and therefore impact IL-7R{alpha} expression. In fact, augmented IL-7 levels have been detected in lymphopenic conditions caused by HIV infection or after bone marrow transplantation (38, 39). Alternatively, it may well be that the production of these {gamma}c cytokines increases when naive T cells drop (40). Understanding the regulation of these pivotal homeostatic factors in normal and altered conditions is far from complete and constitutes an important challenge in the future. In light with our findings, measuring IL-7R{alpha}low naive cells in situations of T cell lymphopenia might provide insights into the repopulation potential of the naive CD8+ T cell compartment.


   Acknowledgments
 
We thank B. Hooibrink (Department of Cell Biology and Histology, Academical Medical Center, Amsterdam, The Netherlands) for cell sorting, A. Jaspers for technical help, and Drs. E. Eldering, M. Nolte, and P. Baars for helpful discussion and critical reading of the manuscript.


   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 study was funded by a VICI Project grant and Research Grant 912-04-032 both from the Netherlands Organization for Scientific Research (to R.A.W.v.L.). Back

2 N.L.A. and E.M.M.v.L. contributed equally to this work. Back

3 Current address: Unite des Cytokines et Developpment Lymphoide, Institut Pasteur, Paris, France. Back

4 Current address: Department of Immunology, The Scripps Research Institute, La Jolla, CA 92092. Back

5 Address correspondence and reprint requests to Dr. René A. W. van Lier, Academic Medical Center, Laboratory for Experimental Immunology, K0-146, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail address: r.vanlier{at}amc.uva.nl Back

6 Abbreviations used in this paper: {gamma}c, common {gamma} chain; TREC, TCR excision circle; HSCT, hemopoietic stem cell transplantation; RT-MLPA, reverse transcriptase multiplex ligation-dependent probe amplification. Back

Received for publication March 23, 2007. Accepted for publication April 18, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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