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High Cytotoxic and Specific Migratory Potencies of Senescent CD8⁺CD57⁺ Cells in HIV-Infected and Uninfected Individuals¹

Yannick Le Priol^*, Denis Puthier, Cédric Lécureuil^*, Christophe Combadière^*, Patrice Debré^*, Catherine Nguyen and Béhazine Combadière^2,*

^* Institut National de la Santé et de la Recherche Médicale Unité 543, Université Pierre et Marie Curie Paris 6, Hôpital Pitié-Salpêtrière, Paris, France; and Institut National de la Santé et de la Recherche Médicale ERM206/TAGC, Université d’Aix-Marseille II, Parc Scientifique de Luminy, Marseille, France

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
CD8⁺CD57⁺ T lymphocytes, present at low levels in the peripheral blood of healthy individuals expand during HIV infection and remain elevated during chronic infection. Their role in the immune response remains unclear. We performed a large-scale gene array analysis (3158 genes) to characterize them and, interestingly, found no distinction in the transcriptional profiles of CD8⁺CD57⁺ T lymphocytes from HIV-infected and uninfected subjects. In both groups, these cells showed specificity for multiple Ags and produced large amounts of IFN- and TNF-. The transcriptional profiles of CD8⁺CD57⁺ and CD8⁺CD57^– cells, however, differed substantially. We propose that CD8⁺CD57⁺ cells were Ag-driven effector cells with very high cytotoxic effector potential including perforin, granzymes, and granulysin, regardless of HIV status. At both the messenger and protein levels, they expressed more adhesion molecules and fewer chemokine receptors (CCR7 and CXCR4) than CD8⁺CD57^– cells but expressed preferentially CX3CR1. The lower expression level of genes involved in cell cycle regulation showed limited proliferation capacities of CD8⁺CD57⁺ even in response to TCR and IL-2, IL-7, and IL-15 stimulation. CD8⁺CD57⁺ T cells from both HIV and uninfected subjects maintain effective cytotoxic potentials but are destined to migrate to nonlymphoid tissues without further cycling.

	Introduction

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Human immunodeficiency virus infection affects T lymphocyte homeostasis substantially, depleting CD4 T cells and modifies the ability of CD8 T cells to differentiate, migrate, and exert their immune functions (1, 2, 3). During chronic HIV infection the persistence of HIV-specific Ags drives CD8 T cell differentiation and defines the characteristics of the new CD8⁺ T cells. In addition, cytokine and other effector molecules affect the functioning of the cells and their role in the control of disease progression (4, 5, 6, 7). The CD8 T cell count and the proportion of CD8 T cells increase at seroconversion as they do in CMV or EBV infections (8, 9). But, it seems that the quality, rather than quantity, of the CD8⁺ T cell response is essential for effective immune control of infections. Our group has previously described (10, 11, 12, 13) differentiated T lymphocytes expressing CD57 markers, their number increases in HIV infection and correlates with disease progression. One hypothesis is the expansion and accumulation of these cells may result from persistent antigenic stimulation. From the first description of HNK-1 (CD57 marker) on large granular lymphocytes with cytotoxic activities (14, 15) until today, CD8⁺CD57⁺ T cells were intensely studied but remain largely undefined. Although antigenic stimulation drives their proliferation, it is still unclear whether their properties differ according to the level of Ag in vivo, i.e., for example, between HIV and uninfected (UI)³ subjects.

In UI individuals, the CD57 marker is expressed by 5–15% of PBMCs (16). The CD8⁺CD57⁺ T cell subset expands during chronic activation of the immune system, for example, viral infections (17, 18, 19, 20), after bone marrow transplantation (21), and with rheumatoid arthritis (22). These cells are a particular interest in HIV and CMV infection. Their potency as cytotoxic effectors is partially described by our group (13). Recent studies (23) report that HIV-specific CD8⁺CD57⁺ cells, defined as late-stage differentiated lymphocytes with short telomeres and a history of more cell divisions, lack proliferative ability and apoptosis sensitivity. In addition, CD8⁺CD57⁺ cells release a lectin-binding soluble factor that can inhibit Ag-specific and nonspecific cell cytotoxicity (11). We also recently demonstrated that late-stage differentiated CD8⁺CCR7^–CD45RA⁺ lymphocytes express mostly CX3CR1 chemokine receptors and belong to the cytotoxic lineage (perforin⁺, CD27^–, and CD28^–) (24). These findings suggest a differential tissue distribution of these effector/memory cells. Thus, HIV infection not only affects the differentiation and functional capacities of antiviral CD8 cells, but also leads to a new balance between the various CD8 T cell subsets and between lymphoid and nonlymphoid tissues. The ability of CD8⁺CD57⁺ cells to migrate also requires further investigation.

To improve our understanding of the role of CD8⁺CD57⁺ cells in healthy individuals and in HIV infection, we analyzed the transcriptional profiles of CD8⁺CD57⁺ cells in UI and HIV subjects and compared them to those of CD8⁺CD57^– cells.

	Materials and Methods

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Patients

DNA arrays analyzed PBMC samples from 12 healthy HIV^– (UI) volunteer donors (age range, 25–55 years) from the Etablissement Français du Sang and 120 HIV⁺ patients (age range, 19–73 years) were followed in the Infectious Disease Unit at Pitié-Salpêtrière Hospital. A pool of cells for UI groups contained four to five individuals. Because the quantity of blood per HIV patient was limited, we separately pooled the RNA isolated from the blood of HIV subjects to obtain at least 2 µg of total RNA per pool. Patients and volunteers provided informed consent in accordance with French ethical requirements. Table I summarizes the characteristics of each pool from HIV patients: mean age, CD8 count, CD4 count, CD4:CD8 ratio, and viral load. One pool contained blood from 9 to 49 patients as indicated. The percentage of patients with undetectable viral load was as follows: 93% in pool 1, 69 ± 2% in HIV pool 2, pool 3, pool 5, and 44% in pool 4. The Kruskal-Wallis test confirmed a difference of viremia between groups. This was confirmed using the Wilcoxon rank sum test that identified a significant difference between viral load of HIV pool 1 and HIV pool 4 group.

CD8⁺CD57⁺ and CD8⁺CD57^– cells were purified from PBMCs of UI and HIV subjects with the CD8⁺ T cell isolation kit used according to the manufacturer’s instructions (Miltenyi Biotec). Cells were then stained by an anti-CD57-FITC Ab (Immunotech), followed by anti-FITC magnetic beads (Miltenyi Biotec) according to the manufacturer’s instructions to separate CD8⁺CD57⁺ and CD8⁺CD57^– cells. Cell viability was calculated after beads purification by trypan blue staining and was >98% pure. The final purity for each fraction was calculated by flow cytometric analysis and was as follows for CD8⁺CD57⁺ and CD8⁺CD57^– fractions, respectively: HIV 1, 93 and 90%; HIV 2, 85 and 85%; HIV 3, 85 and 90%; HIV 4, 92 and 85%; HIV 5, 90 and 90%; UI 1, 95 and 95%; UI 2, 93 and 90%; UI 3, 85% CD8⁺CD57⁺, 90% CD8⁺CD57^–; UI 4, 72 and 88%; and UI 5, 85 and 85%. Cellular analysis of each fraction revealed mostly contamination of the CD8⁺CD57^– fraction with CD57⁺ cells and contamination of CD8⁺CD57⁺ fraction with CD8⁺ cells. The percentage of CD4⁺ T cells, monocytes (CD14), NK cells (CD16 and CD56), and B cells (CD19) was <3%.

Microarray

All data are MIAME compliant (accession no. A-MEXP-50 and E-MEXP-393; www.ebi.ac.uk/miamexpress). The feasibility, reproducibility, and sensitivity of spotting procedures have been previously described (25, 26, 27). Nylon microarrays were prepared as described using 3277 IMAGE cDNA clones (3158 nonredundant) coding for genes involved in the immunological process and ubiquitous biological function (26).

Total RNA was isolated from samples with the RNeasy Mini kit (Qiagen) and included DNase treatment according to the manufacturer’s instructions. Complex probes were prepared as described from 2 µg of total RNA and [³³P]dCTP labeled and then hybridized (26).

All samples were used for hybridization, i.e., 10 CD57⁺ samples and 10 CD57^– samples. However, a total of four samples showed low signal, close to membrane background. These samples were then excluded from the final analysis that included eight CD57^– samples and eight CD57⁺ samples.

The microarray used for this study contained 3552 elements encompassing 3277 IMAGE clones and controls (3158 nonredundant clones). After hybridization, images were quantified using the ArrayGauge software (Fujifilm Life Science). All images were carefully inspected, and spots with overestimated intensities due to neighborhood effects were flagged. Data were then processed using the R statistical and programming software (www.R-project.org) and array elements with at least one flagged value were excluded (n = 320).

Before normalization, a representative microarray was used to define the rank T of a threshold value above which a gene could be considered as detected. Based on this criterion, the 600 highest values of all of the microarrays were considered as being over this threshold. Next, quantile normalization was applied to the data set (ranks are not modified using this procedure). A filter was then used to minimize artifacts: a gene was kept for subsequent analysis whether the ranks across all of the samples of at least one of the class samples (CD57⁺/HIV^–; CD57^–/HIV^–; CD57⁺/HIV⁺; and CD57^–/HIV⁺) were always above T. This led to a list of 329 IMAGE clones (280 nonredundant genes). After log 2 transformation data were centered relative to the median for each gene and each array and used for classification algorithm.

We then classified the genes and samples by unsupervised hierarchic clustering, using the average linkage method and uncentered Pearson correlation coefficient as the distance metric (Cluster and Tree Viev software; www.microarrays.org/software) (28).

Discrimination between samples was studied using a two-sided unpaired t test. In addition, fold changes (FC) were also calculated FC = µ1– µ2, where µ1 and µ2 denote the means of the expression levels (in log scale) of a gene g in samples from classes 1 and 2, respectively. Genes were considered as differentially expressed, whether they met both criteria: t test p < 0.05 and FC > log₂ (1.5) (i.e.; FC > 1.5 in linear scale).

Flow cytometric analysis

Cell surface Ags were characterized on heparin-collected blood samples with a standard staining method that used the following mAbs: CX3CR1-FITC (MBL), CD8-allophycocyanin (BD Biosciences), or PE cyanin 5.5 (PE-Cy5.5; Caltag Laboratories); or PE (Immunotech); CD57-PE, or FITC, and CD127-PE were obtained from Immunotech; and CD11a-allophycocyanin, CD54-allophycocyanin, CCR5-PE, CXCR4-PE, CCR7-PE, CD122-PE, and CD25-allophycocyanin were purchased from BD Biosciences; granzyme A-FITC, -PE, granzyme B-allophycocyanin (all BD Biosciences), or phosphorylated c-jun-PE (p-c-jun; Santa Cruz Biotechnology). Direct and intracellular staining were performed on samples as previously described (24).

Annexin V staining involved cells stained in 1x PBS with CD8-PE (Immunotech) and CD57-FITC (BD Pharmingen), washed in annexin V binding buffer, and then stained with annexin V-allophycocyanin (BD Biosciences) in annexin V binding buffer following the manufacturer’s recommendations. One thousand CD8⁺CD57⁺ cells were acquired on the flow cytometer (FACSCalibur; BD Biosciences) and analyzed with CellQuest software (BD Biosciences).

For seven-color fluorescence analysis, cells were stained for membrane markers using CD8 Pacific blue, CD57-FITC (Immunotech), CCR7-allophycocyanin, CD3-peridin chlorophyll protein cyanin 5.5 (PerCP cyanin 5.5), CD27-PE (BD Biosciences), and CD45RA-ECD (Beckman Coulter); followed by intracellular staining for IFN- production. Cells were gated on CD3⁺CD8⁺CD57⁺ or CD3⁺CD8⁺CD57^–. At least 1,500,000 events were acquired and analyzed on an LSRII flow cytometer (BD Biosciences).

Cell function assays

Functional analyses of CD8⁺ T cells used purified CD8 positively selected lymphocytes (Miltenyi Biotec) from UI and HIV patients. Purity and viability were both >95%. Purified CD8 cells (1,000,000 cells/ml) were placed in R-10%: RPMI 1640 with 10% FCS, 2 mM glutamine, 10 mM sodium pyruvate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 µM nonessential amino acids (Invitrogen Life Technologies), and were or were not stimulated with 5 µg/ml anti-CD3 (UCHT1; Immunotech) or HIV-p17 overlapping 15-mer, covering the whole protein. Cells were then incubated overnight at 37°C. Brefeldin A (5 µg/ml) was added for 4 additional hours (Sigma-Aldrich). Cells were harvested for flow cytometric staining. CD8-PE (Immunotech) and CD57-FITC (BD Bioscience) membrane staining took place 18 h after TCR stimulation. Additional intracellular staining used cells that were fixed, permeabilized, and stained with Abs directed against IFN--allophycocyanin or TNF-- allophycocyanin (BD Biosciences) for 20 min at room temperature and washed three times in 1x PBS before flow cytometer analysis. Cytokine production analysis considered at least 5000 CD8⁺CD57⁺ events. In all data analyzing, expression of cytokines or cell markers are gated on CD8^highCD57⁺ compared with CD8^highCD57^– lymphocytes.

Data analysis used Prism 2.01 (GraphPad software). Statistical analyses used unpaired sample t tests for means. Statistical significance was set at p < 0.05.

Tetramer staining

The MHC class I tetramers used in this study were purchased from Beckman Coulter. The HIV-1-specific tetramers were HLA-A*201 complexed to the EBV- and CMV-specific tetramers were HLA-A*201 complexed to the peptides BMLF1-GLCTLVAML and pp65-NLVPMVATV, respectively. We analyzed whole-blood samples for Ag-specific T cells by flow cytometry. After RBC lysis, cells were washed once with 1x PBS containing 2% FCS and stained for 30 min with PE tetramer and for 15 min with mAbs CD8-allophycocyanin and CD57-FITC. They were then washed once and fixed with 1x PBS-1% paraformaldehyde. In all, 100,000 cells from a viable light scatter gate were acquired on the flow cytometer (FACSCalibur) and analyzed with CellQuest software (both BD Biosciences).

Proliferation analysis

Purified cells isolated from peripheral blood of HIV and UI subjects were labeled with seminaphthorhodafluor dye (SNARF-1; Molecular Probes) and stimulated or not for 36 h with 5 µg/ml anti-CD3 (UCHT1; Immunotech). Cells were then placed under various conditions in R-10%: IL-2 (5 U/ml; Boehringer Mannheim), IL-7 (10 ng/ml), and IL-15 (10 ng/ml) (both R&D Systems). Ten thousand CD8⁺ events were acquired from a viable light scatter gate for analyses with CellQuest software. SNARF⁺ cells were analyzed after gating on CD8^highCD57⁺ or CD8^highCD57^– cells. The analysis was performed using CellQuest software (BD Biosciences).

	Results

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Similar transcriptional profiles of CD8⁺CD57⁺ cells in UI and HIV subjects

We examined CD8⁺CD57⁺ gene expression patterns for HIV and UI subjects to compare them to the global transcriptional pattern of CD8⁺CD57^– cells (3158 nonredundant cDNA) (26). Four categories of samples were considered: CD8⁺CD57⁺ and CD8⁺CD57^– cells from UI donors or HIV patients. Gene and sample classifications obtained by unsupervised hierarchic clustering were applied, regardless of the CD57 or HIV status. Analysis of the unsupervised hierarchic clustering on 329 IMAGE clones (see Materials and Methods) showed no grouping of samples from UI or HIV subjects (Fig. 1). We obtained, instead, a drastic clustering of CD8⁺CD57⁺ cell samples and another of CD8⁺CD57^– cell samples, regardless of their HIV status, with all but one sample clustered in each group (Fig. 1). In addition, even though significant variation of viremia between HIV pool 1 and HIV pool 4 was observed, we did not find any effect on the sample clustering within the CD8⁺CD57⁺ samples.

View larger version (51K): [in this window] [in a new window]   FIGURE 1. Similar transcriptional profiles of CD8+CD57+ cells in UI and HIV subjects. Hierarchic clustering of 16 nylon arrays hybridized with CD8+CD57+ and CD8+CD57– T cell cDNA samples from UI and HIV-infected individuals (columns) vs 329 genes (of 3277 spotted cDNA clones) expressed at significant levels (rows). Genes considered to be expressed in either CD8+CD57+ or CD8+CD57– samples were included in the clustering analysis. The final analyses included eight CD8+CD57+ samples (three pools of UI subjects and five pools of HIV patients) and eight CD8+CD57– samples (four pools of UI subjects and four pools of HIV patients). A pseudocolor representation of gene expression is shown according to the scale at the bottom. Red, Expression levels more than median; green, levels less than the median.

These results show that the main differences were observed between CD8⁺CD57⁺ and CD8⁺CD57^– samples and that the transcriptional profile of CD8⁺CD57⁺ cells was similar between UI and HIV subjects.

Distinct gene array patterns in CD8⁺CD57⁺ and CD8⁺CD57^– cells

Because the unsupervised hierarchic clustering revealed significant differences between the CD8⁺CD57⁺ and CD8⁺CD57^– samples, we conducted a supervised analysis that considered these two groups of samples, regardless of HIV status. The criterion for identifying significant differences in gene expression between CD8⁺CD57⁺ and CD8⁺CD57^– samples was by calculating both p values after t test analysis, followed by FC calculation. Genes expression were considered significantly different when the t test p < 0.05, then FC = µ1 – µ2 >log₂ (1.5). Unsupervised hierarchic clustering classified differentially expressed genes and samples as shown in Fig. 2 using a color-scale representation. We found two distinct patterns: genes up-regulated in CD8⁺CD57⁺ but not in CD8⁺CD57^– cells (upper array) and genes down-regulated in this comparison (lower array; Fig. 2). Of 329 valid genes, we counted 61 up- or down-regulated. This result suggests that CD8⁺CD57⁺ cells are very different from CD8⁺CD57^– cells.

View larger version (55K): [in this window] [in a new window]   FIGURE 2. Distinct gene array patterns in CD8+CD57+ and CD8+CD57– cells. Hierarchic clustering of CD8+CD57+ and CD8+CD57– cell samples from HIV patients and UI individuals on the identified genes is reported. A pseudocolor representation of gene expression is shown according to the scale at the bottom. Red, Expression levels more than median; green, levels less than the median. Gene symbols are labeled on the right.

We found that three genes were highly up-regulated in CD8⁺CD57⁺ compared with CD8⁺CD57^– samples (FC > 2; p < 0.0001). These genes belonged to the cytotoxicity category (Fig. 2 and Table II). These genes clustered together and coded for compounds of azurophilic granules, which are a feature of effector cells. Two coded specifically for membrane-perturbed proteins, perforin (FC = 2.0) and granulysin (FC = 2.8), the third for the serine protease granzyme B (FC = 2.7). We also found significant up-regulation of another member of this group, granzyme M (FC = 0.9; p < 0.05). To confirm the transcriptional signature of the CD8⁺CD57⁺ T cell subset at the protein level, flow cytometric analysis compared both subsets in UI subjects (Table III). CD8⁺CD57⁺ cells expressed high levels of granzyme A (94 ± 7%) and granzyme B (91 ± 5%). Much lower percentages of these molecules were found in CD8⁺CD57^– cells (34 ± 18% granzyme A⁺ and 13 ± 10% granzyme B⁺).

Formation of the cytotoxic synapse depends on the actin cytoskeleton to move molecules in and of the forming synapse. It is thus interesting to note that the mRNA level of molecules with actin-bundling activity is modulated in both directions by CD8⁺CD57⁺ cells: actinin 1 (FC = –1.2; p < 0.0001), L-plastin (FC = 0.7; p < 0.001), and sodium/hydrogen exchanger regulator SLC9A3R1 (FC = +0.9; p < 0.001; Table II).

Many of the up-regulated genes included in cell-cell interactions are known to be involved in Ag-specific interactions with other cells of the immune system (Table II); these include the HLA-A (FC = 1.0; p < 0.05), HLA-C (FC = 1.0; p < 0.01), HLA-DPA1 (FC = 0.6; p < 0.05), and HLA-DPB1 (FC = 0.8; p < 0.001). The expression level of the gene encoding for the killer cell lectin-like receptor subfamily D member 1 is also higher in CD8⁺CD57⁺ than in CD8⁺CD57^– cells (FC = 1.1, p < 0.001). This gene may help recognize MHC class I HLA-E molecules.

These molecules may contribute to the tight seal that directs the cytolytic molecules to the target cell, thereby, avoiding damage to bystander cells or tissues. CD8⁺CD57⁺ cells are thus fully competent to interact effectively with target cells and deliver cytotoxic molecules to them.

Limited chemokine receptor expression on CD8⁺CD57⁺ cells

The capacity for migration and adhesion of CD8⁺CD57⁺ cells remains unknown. Gene array analysis showed significantly lower levels of homing chemokine receptor CCR7 (FC = –1.7; p < 0.001) on CD8⁺CD57⁺ cells (Table II). This decrease compromises the migration of these cells into lymph nodes. We studied the expression of several chemokine receptors on CD8⁺CD57⁺ cells to characterize their migration capacity in more detail (Table III). The membrane CCR7 expression level was consistent with the low level of CCR7 transcripts observed in the CD8⁺CD57⁺ but not the CD8⁺CD57^– T cell subset. Although 65 ± 9% of CD8⁺CD57^– cells expressed surface CCR7, very few CD8⁺CD57⁺ cells did so (4 ± 2%). The percentage of CXCR4⁺ cells was also significantly lower in CD8⁺CD57⁺ (10 ± 7%) than CD8⁺CD57^– cells (48 ± 13%; p < 0.001), again consistent with the transcript level (Tables II and III). CCR5 expression, however, remained similar in both cell populations (nonsignificant p value). CD8⁺CD57⁺ cells expressed higher levels of CX3CR1 (88 ± 7%) than CD8⁺CD57^– cells did (12 ± 5%; p < 0.001; Table III). In conclusion, the absence of CCR7 and CXCR4 on CD8⁺CD57⁺ cells and their high expression of CX3CR1 suggest that the latter receptor dictates the migratory pattern of these cells.

Intact cell adhesion potencies of CD8⁺CD57⁺ cells

Examining the level of adhesion molecules, we observed significantly more integrin ₇ (FC = 0.6; p < 0.05), _L (FC = 1.3; p < 0.0001), and ₂ (FC = 1.0, p < 0.01) subunits in CD8⁺CD57⁺ than in CD8⁺CD57^– cells (Table II). We also examined the level of expression of other genes encoding for molecules involved in interaction with components of the cell environment and up-regulated in CD8⁺CD57⁺ compared with CD8⁺CD57^– cells: the Fc fragment of IgG low-affinity IIIb receptor (FC = 1.8; p < 0.0001) and CD99 Ag (FC = 1.1; p < 0.001). The observation that all nine of the molecules involved in cell-cell interaction were up-regulated in CD8⁺CD57⁺ cells supports the hypothesis we tested, that CD8⁺CD57⁺ cells may be able to communicate and actively interact with their environment.

CD8⁺CD57⁺ T cell homeostasis

Close inspection of the group of genes related to the cell cycle and differentially expressed by CD8⁺CD57⁺ and CD8⁺CD57^– cells suggests that the former have limited proliferation abilities. Most genes differentially regulated in our samples controlled the G₁-S phase transition (e.g., Max dimerization protein 4: FC = 0.6; p < 0.01) (29). Modulation of several transcription and regulator factors could influence the proliferation potency of CD8⁺CD57⁺ cells. We note that FOXO1A (FC = –0.8; p < 0.001), and MLLT3 (FC = –0.6; p < 0.05) were down-regulated in CD8⁺CD57⁺ cells. Gene arrays revealed down-regulated gene expression of genes involved in T cell activation and survival such as the AP-1 subunits fos (FC = –1.1; p < 0.01) and jun B (FC = –1.4; p < 0.01) in CD8⁺CD57⁺ cells (Table II). Because c-jun is a part of the AP-1 subunit and phosphorylation is a mechanism that regulates transcription factor activity, we compared the percentage of CD8⁺CD57⁺ and CD8⁺CD57^– cells expressing the phosphorylated form of c-jun (p-c-jun). Only 61 ± 11% of CD8⁺CD57⁺ cells did so, although this form was detectable in almost all CD8⁺CD57^– cells (95 ± 6%; p < 0.001; Table III). Differentially expressed genes include those coding for proteins able to bind calcium, such as granulin (FC = –0.8; p < 0.05) and members of the S100 family (S100A12 (FC = –1.5; p < 0.05), S100A8 (FC = –1.9; p < 0.01), and S100A9 (FC = –2.1; p < 0.01). Calcium plays an important role in the proliferation and apoptotic death of immune cells. Taken together, our data indicate a lower level of cell survival in CD8⁺CD57⁺ cells.

Genes involved in the cell cycle constitute the transcriptional signature of CD8⁺CD57⁺ cells and supposedly their resistance and sensitivity to cell death (Table II). Gene array analysis also revealed transcript level modulations of the two death receptor subunits TNFRSF1B (FC = 0.8; p < 0.05) and TNFRSF25 (FC = –1.0; p < 0.05) between CD8⁺CD57⁺ and CD8⁺CD57^– cells.

We thus used annexin V, which binds to phosphatidyl serine, to identify preapoptotic CD8 cells. After in vitro TCR stimulation, substantially more CD8⁺CD57⁺ cells (2.9 ± 0.2%) than CD8⁺CD57^– cells (0.8 ± 0.2%; p < 0.001) reacted to the apoptotic marker annexin V (Table III). However, ex vivo we found no significant difference in the percentage of annexin V⁺ cells in CD8⁺CD57⁺ and CD8⁺CD57^– cells (data not shown).

In conclusion, CD8⁺CD57⁺ cells appear to be effector/memory cells with limited proliferative and survival potencies but with high cell-cell interaction and migratory capacities.

Ag specificity and effector functions in CD8⁺CD57⁺ cells of UI and HIV individuals

Microarray data showed that CD8⁺CD57⁺ are effector T cells. To complete this analysis, Ag specificity and effector function were further assessed. To estimate the proportion and diversity of Ag-specific CD8⁺CD57⁺ cells in both groups, we used HLA-A2 tetramers to evaluate their viral Ag specificity (EBV or CMV) in several HLA-A2 patients. CD8⁺CD57⁺ T cell subset included epitopes for CMV (pp65-NLVPMVATV) and EBV (BMLF1-GLCTLVAML) in UI individuals as did their counterpart CD57^– (Fig. 3A). The number of Ag-specific cells in the CD57⁺ subset than in CD57^– subset seems to behave similarly in HIV⁺ compared with HIV^– individuals, suggesting a close relationship.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. Ag diversity but similar effector functions in CD8+CD57+ cells of UI and HIV individuals. A, Whole-blood samples from UI and HIV were studied for antigenic specificity. Fluorescent coupled HLA-A2/peptide tetramer complexes for different viral epitopes EBV-BMLF1-GLCTLVAML and CMV-pp65-NLVPMVATV) were used. B, Purified CD8 T cells isolated from peripheral blood were stimulated overnight with anti-CD3 (5 µg/ml). Brefeldin A (5 µg/ml) was added 4 h before cells were harvested for flow cytometric analysis. Intracellular IFN- and TNF- accumulation 18 h after TCR stimulation were measured in CD8+CD57+ and CD8+CD57– cell populations. Results are represented as percentage of IFN-+ cells gated either CD8+CD57+ cells or CD8+CD57– cells.

To investigate the effector/memory phenotype of these cells in response to Ag, we examined by seven-color analysis the expression of CCR7, CD45RA, and CD27 on IFN--producing CD3⁺CD8⁺CD57⁺ cells (Fig. 3C, representative of three HIV⁺ cells individuals). We found that IFN-⁺CD8⁺CD57⁺ cells display effector/memory phenotype as defined by CD27^–CCR7^–CD45RA^+/–. IFN--producing CD3⁺CD8⁺CD57^– cells are CD27^–CCR7^–CD45RA^+/–.

In conclusion, CD8⁺CD57⁺ cells in both UI and HIV subjects are Ag-driven effector/memory cells with potential multiple Ag specificity.

Expression of cytokine receptors on CD8⁺CD57⁺ cells and effects of several cytokines on in vitro cell proliferation

Gene array analyses revealed significant up-regulation (FC = 0.6; p < 0.01) of the expression level of the transcript coding for the IL-2R -chain in CD8⁺CD57⁺ cells (Table II). The IL-7R transcript level was lower (FC = –1.1; p < 0.05) in CD8⁺CD57⁺ cells (compared with CD8⁺CD57^– cells). We thus evaluated the level of IL receptors using the common -chain subunit such as IL-2R, IL-7R, and IL-15R and known to be involved in the proliferation and survival of end-stage differentiated CD8 cells: IL-2R- (CD25), IL-2R- (CD122), and IL-7R (CD127) on CD8⁺CD57⁺ and CD8⁺CD57^– cells. We found that CD8⁺CD57⁺ cells had a significantly higher percentage of CD122^low positive cells (13 ± 14% vs 2 ± 2%; p = 0.028). The low percentage of CD127⁺ cells was an especially notable feature of the CD8⁺CD57⁺ T cell subset compared with CD8⁺CD57^– cells (14 ± 6%, vs 94 ± 3%; p < 0.001). In healthy donors, neither cell population expressed CD25 (data not shown). Despite an elevated level of the transcript for the IL-2R -chain, the membrane expression level of the three subunits, CD25, CD122, and CD127, did not rise. These results suggest a defect in the surface expression of IL-2R (absence of IL-2R-), IL-7R, and IL-15R (low IL-2R-) receptors on CD8⁺CD57⁺ cells.

These three cytokines bind -chain receptors and are known to be involved in survival and proliferation of end-stage differentiated CD8⁺ lymphocytes. To verify the capacity of CD8⁺CD57⁺ cells to proliferate in response to cytokine stimulation, we investigated the effect of these three cytokines on the in vitro proliferation capacities of CD8⁺CD57⁺ cells after TCR stimulation. Purified CD8 cells were labeled with the DNA-binding molecule SNARF-1 and stimulated for 36 h with anti-CD3 Ab. The cytokines were then added. We analyzed the SNARF-1 profiles of CD8⁺CD57⁺ and CD8⁺CD57^– cells at day 5 (Fig. 4). Each panel of Fig. 4 reports the percentage of undivided cells, cells with one or two divisions, and cells with at least three divisions for combinations of these cytokines. As expected, the proliferation capacity of CD8⁺CD57^– cells was high: in all cytokine conditions, up to 90% of cells had divided >3 times (IL-2, 94%; IL-7, 93%; IL-15, 93%; and all three cytokines, 91%). Thus, CD8⁺CD57^– cells proliferated after in vitro CD3 stimulation and IL-2, IL-7, and IL-15 helped to increase their proliferation rate. CD8⁺CD57⁺ cells, however, were less able to proliferate after in vitro CD3 stimulation (5% cells vs 78% for CD8⁺CD57⁺ cells). The addition of IL-2 or IL-15 slightly increased the number of CD8⁺CD57⁺ T cell divisions (IL-2, 17%, and IL-15, 11%), but IL-7 did not affect them in accordance to low level gene expression of its receptor as observed after microarray analysis. The profile of CD8⁺CD57⁺ cells cultivated in the presence of the three cytokines was similar to those of CD8⁺CD57⁺ cells cultivated in the presence of IL-2 or IL-15 alone and indicated no synergy between these cytokines. Similar results were observed in samples from HIV-infected subjects. Thus, CD8⁺CD57⁺ cells proliferate poorly in response to TCR or cytokine stimuli.

View larger version (36K): [in this window] [in a new window]   FIGURE 4. Expression of cytokine receptors on CD8+CD57+ cells and effects of several cytokines on in vitro cell proliferation. Effects of several cytokines on the in vitro proliferation of CD8+CD57+ cells. SNARF-1-labeled T cell proliferation profiles were assessed in the presence or absence of several cytokines after anti-CD3 stimulation. Purified CD8 cells isolated from peripheral blood of healthy donors were labeled with SNARF-1 and stimulated for 36 h with anti-CD3 (5 µg/ml). Cells were then placed for 3 days under different cytokine conditions: IL-2 (5 U/ml), IL-7 (10 ng/ml), and IL-15 (10 ng/ml). SNARF+ cells are gated on either CD8highCD57+ cells or CD8highCD57– lymphocytes. These results are representative of five experiments performed on cells from both healthy donors and HIV patients. The percentage of cells that did not proliferate, cells with one or two rounds of division, and cells with at least three rounds of division are reported.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

One aim for this study was to obtain a signature of the CD8⁺CD57⁺ T cell subset and thus enable more accurate assessment of its potential role. The comparison of CD8⁺CD57⁺ T and CD8⁺CD57^– T cell samples by both the hierarchic clustering analysis and differential analysis-based t test and FC calculation showed that CD8⁺CD57⁺ cells constitute a specific CD8 T cell subset. A limited number of genes (3158) and low amount of available materials for this study could lead to missing genes from the final analysis. In this study, we documented the principal transcriptional traits of CD8⁺CD57⁺ cells and found that this T cell subset: 1) may have a direct antiviral role via the lysis of virus-infected cells; 2) is composed of end-stage differentiated cytotoxic lymphocytes that have limited survival and proliferative capacities and are prone to apoptosis; 3) can communicate and act together with components of its environment; and 4) has the power to migrate to nonlymphoid tissues. One of the genes most overexpressed in CD8⁺CD57⁺ (vs CD8⁺CD57^–) cells codes for granulysin. This protein is present in the cytotoxic granules of CTL and NK cells and is a critical effector of the antimicrobial activity of CTLs against intracellular pathogens (37, 38). Granulysin is also a chemoattractant and a proinflammatory activator that actively recruits immune cells to inflammation sites (39), and its presence shows that the antiviral role of CD8⁺CD57⁺ cells involves mechanisms other than direct lysis of HIV-infected cells.

Phenotypic migratory markers such as CCR7 discriminate T cell subsets in the peripheral blood (40, 41). Changes in chemokine receptor expression were directly correlated with the activation state of the cells (40, 42). Other chemokine receptor expressions also tend to be modulated during differentiation. CX3CR1, for example, has been described (43) as a phenotypic marker of the CD4 and CD8 cytotoxic lineage. In this study, CD8⁺CD57⁺ cells appear not to express CCR7 or CXCR4 but rather to express mainly CX3CR1. Down-modulation of chemokine receptors on CD8⁺CD57⁺ lymphocytes suggests that CX3CR1 continues to be the sole homing chemokine receptor expressed by them. These results thus raise the question of where these cells migrate. One hypothesis is that in the absence of CCR7 and in the presence of CX3CR1, viral Ag-specific CD8 cells are inappropriately redirected to tissues, whereas virus replication persists in lymphoid tissues. This is consistent with the tissue infiltration of CD8 T cells frequently observed in the lungs of HIV patients (44, 45), which can culminate in a condition known as diffuse infiltrative CD8 lymphocytosis syndrome (46), and with our previous finding (12) of CD8⁺CD57⁺ cells infiltrating the lungs of HIV-infected individuals. This finding suggests that CD8⁺CD57⁺CX3CR1⁺ cells migrate to this tissue (10, 12). Moreover, the up-regulation of CX3CL1 in astrocytes from patients with AIDS dementia suggests that CD8⁺CD57⁺CX3CR1⁺ effector cells are recruited into the brain of HIV-infected patients (47, 48). CX3CL1 is also described in gut-associated lymphoid tissues and is particularly abundant in the lamina propria of HIV-infected patients and possibly in the T cell zone of lymph nodes of these patients, sites of high viral replication. Plasmacytoid dendritic cells (DC) are thought to be the principal source of CX3CL1 production in the lymphoid compartment (49). In the mouse model, FKN is expressed by DC in lymph node T cell areas (50). In human and murine models, CX3CL1 can be expressed by DC in epidermis and secondary lymphoid organs (51). These observations add new insight into the potential interaction of CD8⁺CD57⁺CX3CR1⁺ with different population of DC in tissues and lymphoid organs. However, CD8⁺CD57⁺ cells did not express CD62 ligand, which along with CCR7 are required for migration to lymphoid organs. These findings, in combination with the increased expression of several adhesion molecules, may have an important effect on the tissue distribution of these cells in HIV patients.

Although the limited survival and proliferative capacities of CD8⁺CD57⁺ cells have been previously reported (13, 23), we proposed here the first potent molecular explanations of these defects. Their limited power of proliferation may partially explain the ineffectiveness of host defenses against HIV (13, 23). Among the genes identified as differentially expressed by CD8⁺CD57⁺, three genes code for members of the S100 family of calcium-binding proteins involved in a variety of intracellular activities, including cell proliferation and differentiation. They also play a role in the dynamics of cytoskeleton constituents and in the structural organization of membranes. Suggesting functional consequences for the lower levels of S100A8 and S100A9 mRNA observed in CD8⁺CD57⁺ (vs CD8⁺CD57^–) cells is risky, because these may form a noncovalent heterodimer protein complex called calprotectin, which antagonizes the monomer functions (52). It is, therefore, necessary to further investigate the level of protein expression in CD8⁺CD57⁺ cells. We have, however, shown that IL-2 and IL-15, but not IL-7, increase the in vitro proliferation capacities of CD8⁺CD57⁺ cells slightly after TCR stimulation. The unresponsiveness to IL-7 correlates with the absence of IL-7R gene expression found on the microarray analysis. This observation was also consistent with results about the effect of IL-2 and IL-15 on HIV-specific CD8⁺ cells (53) and qualifies the absence of proliferation of CD8⁺CD57⁺ T after staphylococcal enterotoxin B or HIV stimulation in the presence of high concentrations of IL-15 reported by others (23). Our cDNA array experiments highlighted a decrease in the concentration of the AP-1 transcription factor and modulation of several genes controlling the G₁-S phase transition. AP-1 proteins, principally those belonging to the Jun group, may control cell life and death through their ability to regulate the expression and function of such cell cycle regulators as cyclin D1, myc, p53, p21, p19, and p16 (54).

The transcriptional signature of the CD8⁺CD57⁺ cells provides hypotheses about mechanisms involved in apoptosis and lack of proliferation as observed by others (23, 55, 56, 57, 58, 59) in different pathologies and tissues. Confirmation and further exploration of these possible mechanisms should help us to propose new molecules to boost survival, proliferation, and antiviral capacity of CD8⁺CD57⁺ cells, especially in HIV infection.

	Acknowledgments

 
We thank Prof. Brigitte Autran and Drs. G. Carcelain, P. Deterre, and F. Boutboul for their support and advice and all members of the Plate-forme Post Génomique de la Pitié Salpêtrière (Pitié-Salpétrière Hospital, Paris, France) and Marseille-Nice Genome Facilities (Marseille, France) for technical assistance and microarrays development.

	Disclosures

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The authors have no financial conflict of interest.

	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 Agence Nationale de Recherches sur le Syndrome d’immunodéficience acquise (SIDA) Y.L.P. was a recipient of a fellowship from the Agence Nationale de Recherche sur le SIDA.

² Address correspondence and reprint requests to Dr. Béhazine Combadière, Université Pierre et Marie Curie Paris 6, 91 bd de l’hôpital, 75013 Paris, France. E-mail address: combadie{at}ccr.jussieu.fr

³ Abbreviations used in this paper: UI, uninfected; FC, fold change; DC, dendritic cell; SNARF, seminaphthorhodafluor; p-c-jun, phosphorylated c-jun.

Received for publication December 13, 2005. Accepted for publication July 15, 2006.

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