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Journal of Virology, January 2007, p. 430-433, Vol. 81, No. 1
0022-538X/07/$08.00+0     doi:10.1128/JVI.01567-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

NKG2C⁺ NK Cells Are Enriched in AIDS Patients with Advanced-Stage Kaposi's Sarcoma

Martin R. Goodier,^1* C. M. Mela,¹ A. Steel,² B. Gazzard,² M. Bower,³ and F. Gotch¹

Department of Immunology, Imperial College London,¹ Directorate of HIV-GUM,² Clinical Oncology Unit, Chelsea and Westminster Hospital, London, United Kingdom³

Received 19 September 2006/ Accepted 4 October 2006

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Kaposi's sarcoma (KS) is an AIDS-defining condition in individuals with human immunodeficiency virus type 1 infection. We investigated the phenotype and function of the NKG2C⁺ NK cell population in individuals with AIDS and Kaposi's sarcoma. The staging of AIDS KS patients according to the AIDS Clinical Trial Group criteria revealed that patients with the S1 disease stage have a significantly higher proportion of NKG2C⁺ cells than those with the S0 disease stage. NKG2C⁺ cells from S1-stage patients are highly enriched for the expression of KIR3DL1, are depleted of NKp46, and respond poorly to major histocompatibility complex class I-positive target cells. These data demonstrate a link between NK cell phenotype and function and disease prognosis in AIDS.

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Kaposi's sarcoma (KS) is caused by infection with Kaposi's sarcoma herpesvirus (KSHV) and is an AIDS-defining illness in individuals with human immunodeficiency virus type 1 (HIV-1) infection.

Natural killer cells have been shown to target KSHV-infected cell lines and this activity is recovered in people with HIV and AIDS (PWHA) who resolve their KS lesions during antiretroviral therapy (22). However, the absolute numbers of NK cells do not appear to influence the relative risk of KS in PWHA, indicating that other factors control KSHV infection and the acquisition of KS lesions (23).

Recognition of infected target cells by NK cells is regulated by receptors for major histocompatibility complex class I (MHC-I) and pathogen-derived molecules (3, 14). Killer cell immunoglobulin-like receptor (KIR) family members, which are expressed on NK cells, influence the risk of progression to AIDS in combination with HLA-B alleles (15, 16). However, the proportion of NK cells expressing inhibitory KIR increases and those expressing activating natural cytotoxicity receptors (NCR) are less abundant in PWHA (1, 5, 6, 17).

We have shown that NK cells from PWHA are enriched for a subset bearing the C-type, lectin-like receptor NKG2C (20). The NKG2C receptor mediates NK cell responses to a limited set of ligands in association with HLA-E, including a human cytomegalovirus (HCMV) peptide, and to HCMV-infected fibroblasts (9, 10). However, NK cells bearing NKG2C predominantly express inhibitory KIR and have low levels of activating NCR, which may compromise their abilities to mount cytolytic responses (8, 20).

As NKG2C⁺ NK cells have a predominantly inhibitory phenotype, the dominance of this subset could have an impact on disease prognosis in AIDS patients, including those with KS. We used the AIDS Clinical Trial Group (ACTG) clinical staging system, which is applied to all AIDS KS patients upon diagnosis, to investigate the relationship between the NK cell phenotype and function and disease prognosis in HIV-1 infection.

Patients were recruited from the Chelsea and Westminster Hospital clinics with ethical approval and written informed consent. Samples were obtained from 21 PWHA with KS (mean age, 40 years [range, 23 to 72]; median CD4 count, 293 [range, 6 to 841]), 44 PWHA without KS lesions (mean age, 45 years [range, 28 to 63]; median CD4 count, 392 [range, 119 to 941]), and 9 uninfected healthy controls (mean age, 41 years [range, 27 to 58]). All patients had received antiretroviral therapy. KS patient staging was documented using the ACTG tumor, immune system, and systemic illness staging system (13). Peripheral blood mononuclear cells were prepared from venous blood by standard Ficoll-Hypaque separation. Cells were used directly for phenotypic analysis or cultured for degranulation assays (20).

We used flow cytometry to investigate whether the proportion of NKG2C⁺ cells was further increased in HIV-1-infected individuals with KS. Peripheral blood mononuclear cells from PWHA with and without KS and from healthy control individuals were analyzed for C-type lectin receptor expression after gating on CD3^– CD14^– CD19^– CD94⁺ cells as described previously (20). This method defines all of the NKG2A and NKG2C⁺ NK cells in the peripheral blood, including those present within the CD56^– NK cell population, which is increased in HIV-1 infection (18). The proportion of NKG2C-positive cells was significantly higher in both KS-negative and KS-positive PWHA (median, 50.3 and 46.5%, respectively; range, 0.1 to 96.1% and 1.9 to 85.4%, respectively) than that in healthy controls (median, 12.1%; range, 6.3 to 21%) (Fig. 1). Compared to that observed in the NKG2A⁺ cell population which dominates in healthy individuals, NKG2C⁺ NK cells from PWHA with KS or without KS had almost identical expression levels of NK receptors (Table 1). A higher proportion of NKG2C⁺ cells in both groups of patients expressed KIR and a lower proportion expressed NCR (Table 1).

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FIG. 1. Proportion of cells expressing C-type, lectin-like receptors in PWHA with and without KS and uninfected healthy controls. The proportion of cells expressing NKG2A and NKG2C was estimated after gating on CD94+ NK cells as described in the text. Shaded boxes represent NKG2A+ cells, and white boxes represent NKG2C+ cells. Boxes show medians and interquartile ranges; bars represent 10th and 90th percentiles.

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TABLE 1. Distribution of activating and inhibitory receptors on the NKG2A and NKG2C subsetsa

The staging of KS patients according to the ACTG criteria is regarded as an indicator of KS disease outcome. Patients with T0-, I0-, and S0-stage diseases have confined lesions, CD4 T-cell counts above 200, and good performance status without opportunistic infections, respectively (13). Individuals with T1 (disseminated KS)-, I1 (low CD4 T-cell counts)-, and S1 (opportunistic infections and/or poor performance status)-stage diseases have worse disease outcomes (13). We investigated the relationship between KS disease stage and the proportion of NKG2C⁺ cells in our AIDS KS patients. We saw no correlation between CD4⁺ T-cell count (I0 versus I1, n = 13 and n = 8, respectively), HIV-1 plasma RNA load or tumor stage (T0 versus T1, n = 11 and n = 10, respectively), and the proportion of NKG2C⁺ NK cells in our cohort of AIDS KS patients. However, we observed a significantly higher proportion of NKG2C⁺ cells in patients with S1-stage disease (n = 9; median, 67.8%; range, 34.5 to 85.4%) than in those with S0-stage disease (n = 12; median, 36.7%; range, 1.9 to 60.1%), irrespective of T and I stages (Fig. 2A). A reduction in the proportion of NKG2C⁺ cells expressing NKp30 and NKp46 and a significant reduction in the mean fluorescence intensity for NKp46 was observed in NKG2C⁺ NK cells from S1-stage AIDS KS patients (Fig. 2B and C). A significant increase in the proportion of cells expressing KIR3DL1 was also observed in S1-stage patients compared to that in S0-stage patients (Fig. 2B). Interestingly, there was no difference in NCR or KIR expression on NKG2A⁺ cells between S0- and S1-stage KS patients, indicating that phenotypic differences were occurring preferentially in the NKG2C⁺ cell population (data not shown).

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FIG. 2. Differences in NK cell receptor expression according to KS disease stage. (A) The proportion of NKG2A (shaded boxes)- and NKG2C (white boxes)-expressing cells in PWHA and KS with S0 (unhatched boxes) and S1 (hatched boxes) disease stages. (B) Proportion of individuals expressing activating NCR and inhibitory KIR receptors in gated NKG2C-positive cells from S0 (unhatched boxes)- and S1 (hatched boxes)-stage individuals. (C) Differences in the mean fluorescence intensity for NCR expression between S0 (unhatched boxes)-stage and S1 (hatched boxes)-stage individuals. Boxes show medians and interquartile ranges; bars represent 10th and 90th percentiles.

The low levels of NCR, in particular, NKp46, in S1 disease status, combined with the reciprocal increase in KIR3DL1 expression, could affect the ability of NKG2C⁺ cells to target MHC-I-positive cells infected with viruses or bacteria and tumor cells.

The ability of NKG2C⁺ cells to degranulate in response to the KSHV-infected primary effusion B-cell lymphoma cell line BCBL-1 (MHC-I positive) and K562 cells (MHC-I-negative) were therefore compared in S0- and S1-stage AIDS KS patients. NK cell subset degranulation assays were performed by the detection of CD107a as described previously (2). Peripheral blood mononuclear cells were cultured overnight with recombinant interleukin-15, which has previously been shown to amplify the activation and degranulation in cells from both HIV-1-infected and hepatitis C virus-infected individuals (7, 19). NKG2C⁺ cells from S1-stage patients had reduced degranulation in response to BCBL1 cells compared to that in S0-stage patients, as indicated by a lower proportion of CD107a-expressing cells (Fig. 3A, B, and D). No difference was observed between S0- and S1-stage patients in their responses to BCBL-1 targets after gating on NKG2C^– cells (Fig. 3D) and degranulation of either NKG2C⁺ or NKG2C^– cells in response to K562 cells (Fig. 3D). Although only a small proportion of NK cells from healthy control individuals expressed NKG2C, these mounted substantial degranulation responses against K562 cells and BCBL-1 cell lines (Fig. 3C). Similarly to S0-stage patients, a higher proportion of NKG2C⁺ cells responded to BCBL-1 cells in healthy individuals than in S1-stage patients, but these responses were similar to those of NKG2C^– cells (for healthy controls, NKG2C⁺, median, 42.2% [range, 25 to 56%]; NKG2C^–, median, 31.2% [range, 25.7 to 42%]). Both NKG2C⁺ and NKG2C^– cells from healthy control individuals made stronger degranulation responses to K562 than to BCBL-1 cells, as observed for PWHA, indicating that inhibitory receptors for MHC-I may be restricting the responses (for healthy controls, NKG2C⁺, median, 73.2% [range 73 to 83%]; NKG2C^–, median, 53.8% [range, 52 to 63%]). These results indicate that the NK cells from PWHA and KS with poor prognostic scores are enriched for NKG2C⁺ cells, which have reduced responses to MHC-I-positive target cells.

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FIG. 3. Poor degranulation of NKG2C+ cells in AIDS KS individuals with S1-stage disease. Representative plots for degranulation responses against BCBL1 cells (upper panel) and K562 cells (lower panel) from AIDS KS patients with (A) S0-stage and (B) S1-stage disease. Regions show gating for NKG2C+ and NKG2C– cells in S0- and S1-stage patients. (C) Representative plots from healthy control donors are shown. (D) Degranulation responses from NKG2C+ and NKG2C– cells from S0-stage patients (n = 11, unhatched boxes) and S1-stage patients (n = 9, hatched boxes). Boxes show medians and interquartile ranges; bars represent 10th and 90th percentiles.

The comparable phenotype of NK cells in PWHA to those in PWHA and KS implies that, as reported for NK cell numbers (23), susceptibility to KS lesions is not influenced by NK cell receptor expression. However, the enrichment of NKG2C⁺ cells in HIV-1-positive individuals with poor disease prognosis, as shown here for a subset of AIDS KS patients, implies that a functional deficit in these cells may impact disease outcome. We were able to assess the relationship between disease prognoses for PWHA and KS as these patients are ascribed defined prognostic scores upon diagnosis, and it may be that high levels of NKG2C⁺ cells also impact disease outcome in PWHA without KS. PWHA and poor disease prognoses for have experienced greater exposure to or a higher burden of agents such as CMV, which stimulates the proliferation of NKG2C⁺ cells via HLA-E (11) and is associated with disease progression in HIV-1 infection (4, 21). All of the PWHA with KS studied here tested positive for CMV immunoglobulin G (data not shown), indicating that CMV status is not the sole determining factor in driving NKG2C⁺ cells in these patients. It will be interesting to assess whether HLA-E and classical HLA-A and -B alleles, which influence NK and T-cell responses to CMV, also influence disease stage in KS patients (12). It therefore may be that disease outcome in AIDS KS patients is influenced indirectly by infections other than KSHV which influence immune competence. It is likely that both the proportion of NKG2C⁺ cells and the changes in inhibitory and activating NK receptors on this subset impact NK cell function in PWHA. Detailed analysis of the functional responses of NKG2C⁺ cells from PWHA may lead to therapeutic interventions which recover NK cell function and reverse the dominance of this subset in individuals with advanced disease.

 
We thank C. Boshoff, D. Bourmpoulia, and C. Burton for provision of materials, helpful discussions, and appraisal of the manuscript.

This work was supported by the St. Stephen's AIDS Trust and by a Medical Research Council UK program grant awarded to F. M. Gotch and C. Boshoff.

 
* Corresponding author. Mailing address: Department of Immunology, Imperial College London, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH, United Kingdom. Phone: (44) 208 746 5987. Fax: (44) 208 746 5997. E-mail: m.goodier{at}imperial.ac.uk.

Published ahead of print on 11 October 2006.

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Journal of Virology, January 2007, p. 430-433, Vol. 81, No. 1
0022-538X/07/$08.00+0     doi:10.1128/JVI.01567-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Goodier, M. R. Articles by Gotch, F. Search for Related Content PubMed PubMed Citation Articles by Goodier, M. R. Articles by Gotch, F. Pubmed/NCBI databases Medline Plus Health Information AIDS Kaposi's Sarcoma