HLA Class I Subtype-Dependent Expansion of KIR3DS1+ and KIR3DL1+ NK Cells during Acute Human Immunodeficiency Virus Type 1 Infection

Galit Alter; Suzannah Rihn; Katharine Walter; Anne Nolting; Maureen Martin; Eric S Rosenberg; Jeffrey S Miller; Mary Carrington; Marcus Altfeld

doi:10.1128/JVI.00256-09

. 2009 Apr 22;83(13):6798–6805. doi: 10.1128/JVI.00256-09

HLA Class I Subtype-Dependent Expansion of KIR3DS1⁺ and KIR3DL1⁺ NK Cells during Acute Human Immunodeficiency Virus Type 1 Infection ^▿

Galit Alter ^1,^*, Suzannah Rihn ¹, Katharine Walter ¹, Anne Nolting ¹, Maureen Martin ^2,^†, Eric S Rosenberg ¹, Jeffrey S Miller ³, Mary Carrington ^2,^†, Marcus Altfeld ¹

PMCID: PMC2698561 PMID: 19386717

Abstract

NK cells are critical in the early containment of viral infections. Epidemiological and functional studies have shown an important role of NK cells expressing specific killer immunoglobulin-like receptors (KIRs) in the control of human immunodeficiency virus type 1 (HIV-1) infection, but little is known about the mechanisms that determine the expansion of these antiviral NK cell populations during acute HIV-1 infection. Here we demonstrate that NK cells expressing the activating receptor KIR3DS1⁺ and, to a lesser extent, the inhibitory receptor KIR3DL1⁺ specifically expand in acute HIV-1 infection in the presence of HLA-B Bw480I, the putative HLA class I ligand for KIR3DL1/3DS1. These data demonstrate for the first time the HLA class I subtype-dependent expansion of specific KIR⁺ NK cells during an acute viral infection in humans.

NK cells are cytotoxic effector cells that play a vital role in the innate immune response to viral infections (9, 12, 33). The critical role of NK cells in acute viral infections has been best characterized in acute murine cytomegalovirus (MCMV) infection (14, 28). While several murine lab strains are resistant to MCMV infection, others are highly susceptible. Resistance to MCMV infection was mapped to a gene encoding an activating NK cell receptor, Ly49H, which has been shown to be critical in the early recognition and control of MCMV infection via the direct recognition of a viral product (M157) expressed on infected cells (28). Remarkably, MCMV-infected mice exhibit a dramatic expansion of NK cells during acute infection, but this expansion is restricted to the specific accumulation of Ly49H⁺ NK cells (16). Data from these studies suggest that the antiviral activity of the Ly49H⁺ NK cells is linked to their ability to expand early in infection, prior to the development of adaptive antiviral immunity.

While the critical role of Ly49H⁺ NK cells in MCMV infection has been well established, very little is known about the clonal composition of NK cells that expand in human viral infections, and the NK cell receptors that mediate their antiviral activity. Unlike T cells and B cells, the specificity of NK cells is not determined by a single NK cell receptor (8); rather, NK cells express an array of activating and inhibitory receptors that regulate their activity. While the expression of these receptors is stochastic, the random combinations of different receptors on the surface of a given NK cell clone determine its ability to respond to a specific target cell (26, 27). It has been suggested that individual NK cell populations expressing a specific array of receptors may respond differentially to diverse viral infections (7). This has been further supported by epidemiological studies associating the expression of individual activating or inhibitory NK cell receptors in combination with their HLA class I ligands with better or worse disease outcomes in viral infections such as hepatitis C virus (22), human immunodeficiency virus (HIV) (29, 30), human papillomavirus (11), and CMV (7). The functional basis for this protective immunity mediated by NK cells in human viral infections remains largely unknown.

Similar to MCMV infection, highly functional NK cells expand rapidly in acute HIV-1 infection, prior to the induction of adaptive immune responses (2). One particular activating killer immunoglobulin-like NK cell receptor (KIR3DS1), in combination with its putative ligand, an HLA-B allele with isoleucine at position 80 (HLA-B Bw480I), has been shown to be associated with slower HIV-1 disease progression (29). We have recently shown that KIR3DS1⁺ NK cells can effectively suppress HIV-1 replication in HLA-B Bw480I⁺ target cells in vitro (1). Furthermore, a subset of inhibitory alleles from the same locus, KIR3DL1, that show high cell surface expression levels have similarly been associated with slower disease progression toward AIDS in the presence of their ligand, HLA-B Bw480I (30). These data suggest that both KIR3DS1⁺ and KIR3DL1⁺ NK cells may play a critical role in the control of natural HIV-1 infection, depending on the interaction with their ligand on infected cells (4). However, the mechanisms underlying their protective role are not understood.

Given the critical role of NK cells in acute viral infections and the described expansion of NK cells overall during acute HIV-1 infection (16), we assessed clonal NK cell expansions during acute HIV-1 infection by quantitative PCR and flow cytometric analysis. Here we report an HLA class I subtype-dependent specific expansion of KIR3DS1⁺ and KIR3DL1⁺ NK cells during acute HIV-1 infection. These data demonstrate for the first time the impact of the HLA class I ligands on clonal NK cell expansions during an acute human viral infection.

MATERIALS AND METHODS

Study subjects.

A total of 64 subjects were recruited for this study. Thirty-one subjects were identified during primary HIV-1 infection, either prior to the development of any detectable antibodies in a p24^gag enzyme-linked immunosorbent assay (acute infection, n = 14) or at a time when they had detectable antibody responses against p24 (enzyme-linked immunosorbent assay positive) but less than three bands in an HIV-1 Western blot (early infection, n = 17). Seventeen of these subjects identified during primary HIV-1 infection expressed both KIR3DS1 and KIR3DL1 (no subjects encoded for KIR3DL1*004, and 14/17 had at least one high/intermediate allele) (21, 37). Longitudinal samples were obtained over the course of their first year of untreated infection for 12 subjects that coexpressed HLA-B Bw480I or were homozygous for HLA-B Bw6. Subjects in primary HIV-1 infection had a an average viral load of 1,730,170 copies of viral RNA per ml plasma (range, 1,270 to 9,620,000 copies per ml) and an average CD4 T-cell count of 559 CD4⁺ T cells per μl of blood (range, 290 to 981 cells per μl). Fourteen subjects with chronic untreated HIV-1 infection were recruited with an average viral load of 73,290 copies of viral RNA per ml plasma (range, 8,100 to 750,000) and an average CD4 count of 346 CD4⁺ T cells/μl (range, 23 to 620 cells per μl). Finally, 19 HIV-1-uninfected subjects were enrolled as a control population. The KIR and HLA genotypes for all study participants are listed in Table 1. The Massachusetts General Hospital Institutional Review Board approved the study, and each subject gave written informed consent for participation in the study.

TABLE 1.

HLA and KIR genotypes

Patient group	Genotype
	HLA						KIR
	HLA-A	HLA-A	HLA-B	HLA-B	HLA-C	HLA-C	2DL1	2DL2	2DL3	2DL4	2DL5	2DS1	2DS2	2DS3	2DS4	2DS5	3DL1	3DS1
Primary	0201	0101	1801	5101	0701	1505	1	0	1	1	1	1	0	0	197	1	1	1
infection	2301	0301	5301	5801	0401	0701	1	1	1	1	1	1	1	1	197	0	1	1
	1101	0201	5101	5301	0401	1402	1	0	1	1	1	1	1	0	197	1	1	1
	0101	7400	0801	5201	1202	0701	1	0	1	1	1	0	0	0	197, 219	1	1	1
	0201	0211	5701	4802	1203	0401	1	1	1	1	1	1	1	0	197	1	1	1
	0301	2402	1402	0702	1203	0802	1	1	1	1	1	1	1	1	197	0	1	1
	0201	0201	0801	1501	0401	0301	1	0	1	1	1	1	0	0	219	1	1	1
	0301	0301	0702	3503	0702	0802	1	1	1	1	1	1	1	0	219	1	1	1
	0301	2601	1402	1501	0702	0303	1	1	0	1	1	0	1	1	219	0	1	1
	0101	0101	1517	3501	0701	0701	1	1	1	1	1	1	1	0	197	1	1	1
	0201	3201	1501	1517	0303	0401	1	1	1	1	1	1	1	0	197	1	1	1
	0201	3001	1501	3906	0701	1502	1	1	1	1	1	1	1	0	197	1	1	1
	0101	0201	1302	4402	0501	0602	1	1	1	1	0	0	1	0	197	0	1	1
	0101	0201	0801	3701	0602	0701	1	1	1	1	1	1	1	1	197	0	1	1
	0101	0301	0801	4901	0701	0702	1	1	1	1	0	0	1	0	197	0	1	1
	0301	0301	4001	2705	0501	0602	1	1	0	1	1	0	1	1	197	0	1	1
	0201	2902	1501	4403	0304	1601	1	0	1	1	0	0	0	0	197, 219	0	1	1
	3201	3201	4402	3906	0501	1502	1	0	1	1	0	0	0	0	197	0	1	0
	0201	0201	2705	3501	0401	0702	1	1	1	1	0	0	1	0	197, 219	0	1	0
	0201	0301	3501	4402	0401	0501	1	0	1	1	0	0	0	0		0	1	0
	1101	0201	3501	4402	0501	0401	1	0	1	1	0	0	0	0	197	0	1	0
	0101	3201	4402	0801	0501	0701	1	0	1	1	0	0	0	0	219	0		0
	1101	0301	2705	3801	0202	1203	0	1	0	1	0	0	1	0	219	0	1	0
	0201	0101	3701	4403	1601	0602	1	0	1	1	0	0	0	0	197, 219	0	1	0
	6801	0201	4402	4403	0401	0501	1	1	1	1	0	0	1	0	197, 219	0	1	0
	0201	0101	0801	4402	0501	0710	1	0	1	1	0	0	0	0	197	0	1	0
	2601	6802	1801	4402	0701	0302	1	1	1	1	0	0	1	0	219	0	1	0
	2601	0201	3801	4403	0401	1203	1	1	1	1	1	0	1	1	197, 219	0	1	0
	2902	1101	0801	4402	0401	0401	1	0	1	1	0	0	0	0	197, 219	0	1	0
	0202	0201	3501	4001	0302	1502	1	1	1	1	1	0	1	1	197	0	1	0
	2501	0201	1801	1801	1203	0702	1	1	1	1	0	0	1	0	197, 219	0	1	0
Chronic	2501	2902	1801	4501	0602	1203	1	0	1	1	1	1	0	0	197	1	1	1
	2902	3101	4001	4403	0304	1601	1	0	1	1	1	1	0	0	197	1	1	1
	0101	0217	1540	5703	0303	0701	1	1	0	1	1	0	1	1	0	1	0	1
	2301	2902	1503	5101	0210	1502	1	1	0	1	1	1	1	1	0	1	0	1
	1101	3201	3501	4402	0401	0401	1	0	1	1	1	1	0	0	197	1	1	1
	0201	6802	0702	5301	0401	0702	1	0	1	1	1	1	1	0	0	1	0	1
	2902	2902	1501	4403	0303	1601	1	1	1	1	1	1	1	0		1	1	1
	0101	2601	2705	5601	0102	0102	1	1	1	1	0	0	1	0	197	0	1	0
	0301	3002	1801	0801	0501	0702	1	1	1	1	0	0	1	0	197	0	1	0
	0101	0201	2705	3701	0602	1203	1	0	1	1	0	0	0	0	197, 219	0	1	0
	0101	0201	4403	5301	0401	1601	1	0	1	1	0	0	0	0	197	0	1	0
	0101	0301	0801	1801	0701	0701	1	1	1	1	1	0	1	1	197	0	1	0
	3002	3301	4501	5801	0701	1601	1	1	1	1	0	0	1	0	219	0	1	0
	0101	2902	0702	5701	0701	0702	1	1	1	1	1	0	1	1	197	0	1	0
Negative	0301	2601	4402	5801	0302	0501	1	1	1	1	1	1	1	1	197	0	1	1
controls	2301	3202	3905	4402	0501	0702	1	0	1	1	1	1	0	0	197	1	1	1
	0206	2402	2705	3906	0202	0702	1	1	1	1	1	1	1	0	219	1	1	1
	0201	6601	3501	4102	0401	1700	1	0	1	1	1	1	0	0	0	1	0	1
	0201	3002	0702	1801	0501	1203	1	1	0	1	1	1	1	1	197	0	1	1
	0101	2402	0801	1801	0701	1203	1	1	1	1	1	1	1	0	219	1	1	1
	0301	2601	3801	5201	0304	1203	1	0	1	1	1	1	1	0	197	1	1	1
	2301	6801	4101	4101	0801	1700	1	1	0	1	1	1	1	1	197	1	1	1
	1101	2402	0702	4001	1203	1505	1	0	1	1	1	1	0	1	0	1	0	1
	0101	3002	0801	4201	0701	1505	1	0	1	1	1	1	0	0	219	1	1	1
	0201	2402	2702	3801	0202	1203	1	0	1	1	0	0	0	0	197, 219	0	1	0
	0301	6802	1402	1402	0802	0802	0	1	0	1	1	1	1	0	197	1	1	0
	2402	2402	4001	4403	0304	0701	1	0	1	1	0	0	0	0	219	0	1	0
	0201	0201	1302	5201	0602	1202	1	0	1	1	0	0	0	0	197, 219	0	1	0
	0301	3201	3801	4402	0501	1203	1	1	1	1	1	0	1	1	197	0	1	0
	0201	2402	0702	3501	0401	0702	1	1	1	1	0	0	1	0	197, 219	0	1	0
	0101	0205	0801	3801	0701	1203	1	1	1	1	0	0	1	0	197, 219	0	1	0
	0201	3001	1801	4201	0501	1700	1	1	0	1	1	0	1	1	197	1	1	0
	0201	3201	1302	5801	0602	0701	1	1	1	1	0	0	1	0	197, 219	0	1	0

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Quantitative reverse transcription-PCR KIR mRNA expression.

NK cell populations were isolated from peripheral blood mononuclear cells (PBMCs) by high-speed cell sorting using a fluorescence-activated cell sorter (BD FACSAria). For these cell-sorting experiments, PBMCs were labeled with anti-CD3-phycoerythrin-Cy5.5 (anti-CD3-PE-Cy5.5), anti-CD56-PE-Cy7, anti-CD16-allophycocyanin-Cy7 (anti-CD16-APC-Cy7), anti-CD14-PE-Cy5, and anti-CD19-PE-Cy5 antibodies. Gates were set to only include CD3⁻ CD14⁻ CD19⁻ CD56^+/− CD16^+/− NK cells, while all CD3⁺ CD14⁺ CD19⁺ cells were excluded. The average purity of sorted NK cell populations was 98.8% (range, 96.4 to 99.5%). Sorted NK cells were collected directly in RNA stabilizing buffer (RLT; Qiagen) and stored at −80°C. RNA was prepared using the RNeasy kit (Qiagen) and then used to prepare cDNA using the Superscript III kit (Invitrogen). The level of transcription of all KIRs was measured by quantitative PCR with SYBR green (Stratagene) as described by Cooley et al. (13). To ensure specificity, dissociation curves were analyzed upon each run. The relative expression of KIR mRNA was normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in sorted NK cell RNA preparations. The levels of KIR transcripts were then expressed as 2^50-cycle number above threshold.

Quantification of KIR3DS1 and KIR3DL1 expression by flow cytometry.

The frequency of KIR3DS1⁺ and KIR3DL1⁺ NK cells was quantified by flow cytometry using the combination of DX9 and z29 antibodies as described previously (10, 32, 36). While z27 (Beckman Coulter) is able to bind both the highly homologous extracellular regions of KIR3DL1 and KIR3DS1, the DX9 antibody (BD Biosciences) only binds to KIR3DL1. Thus, by using the combination of the two antibodies it is possible to distinguish the proportion of NK cells that express KIR3DL1 (DX9⁺ z27⁺) or KIR3DS1 (DX9⁻ z27⁺) or that are double negative (DX9⁻ z27⁻). PBMCs were stained with DX9-fluorescein isothiocyanate (BD Biosciences), z27-PE (Beckman Coulter), CD3-Pacific Blue, CD56-PE-Cy7, CD16-APC-Cy7, CD14-PE-Cy5, and CD19-PE-Cy5 (Becton Dickinson, San Jose, CA). Following gating on CD3⁻ CD14⁻ CD19⁻ CD56^+/− CD16^+/− cells, gates were set to separate z27^hi DX9^hi (KIR3DL1⁺), z27^lo DX9⁻ (KIR3DS1⁺), or z27⁻ DX9⁻ (KIR3DL1⁻/KIR3DS1⁻) NK cells. A minimum of 2 × 10⁵ cells were acquired on an LSRII, and the data were analyzed with Flow Jo.

Statistical analysis.

To test for differences in the mean between several populations, an analysis of variance with Tukey's correction was employed for all comparisons that were below P < 0.05. P values of <0.05 were considered significant.

RESULTS

Preferential expansion of KIR⁺ NK cells during acute HIV-1 infection.

The human Ly49 analogues, KIRs, are expressed stochastically on the surface of NK cells and are critical for monitoring alterations in major histocompatibility complex expression during viral infection, as shown in the KIR gene cluster in the NCBI database (M. Carrington and P. Norman, 2003). While acute HIV-1 infection is associated with a dramatic expansion of cytolytic NK cells (2, 3), it is uncertain whether this expansion occurs nonspecifically or specifically in KIR-expressing NK cells. Due to the high degree of homology between KIR molecules, commercially available antibodies are promiscuous and recognize groups of similar KIRs, and therefore they do not allow for the separate identification of KIRs of the same family. We therefore assessed the overall change in KIR⁺ NK cells by flow cytometry using a pool of antibodies (CD158a, CD158b, CD158e, and NKB1) that recognize KIR2DL1/2DL2/2DL3/3DL1/2DS1/2DS2. There was a dramatic expansion of KIR⁺ CD56^dim CD16⁺ NK cells in acute HIV-1 infection compared to uninfected controls (Fig. 1A and B; P = 0.01). We confirmed that chronic HIV-1 infection was also associated with a slight increase in KIR⁺ NK cells compared to HIV-negative controls (18, 19, 31), albeit at lower levels than those observed in acute infection. Thus, there was a significant early expansion of KIR⁺ CD56^dim CD16⁺ NK cells during acute HIV-1 infection.

FIG. 1. — Expansion of KIR⁺ NK cells in acute HIV-1 infection. (A) Primary flow panels demonstrate the increase of CD3⁻ CD56^dim KIR⁺ (CD158a, CD158b, CD158e, and NKB1⁺) NK cells in acute infection (left) compared to the level in a patient with chronic infection (middle) or an uninfected control (right). (B) The dot plot shows the overall increase of total KIR⁺ NK cells in patients with acute and chronic HIV-1 infection compared to uninfected controls (HIV neg).

Elevated mRNA levels of activating KIRs in acute HIV-1 infection and inhibitory KIRs in chronic HIV-1 infection.

To further resolve whether the KIR⁺ NK cell expansion identified in acute infection was related to elevated activating or inhibitory KIR expression at different stages of HIV-1 infection, we adapted a quantitative panel of quantitative PCR primers (13, 18) to comprehensively characterize changes in specific KIRs in sorted bulk NK cells from all 64 subjects (Table 1). Overall KIR transcript levels were significantly higher in patients with chronic HIV-1 infection than those in both acutely infected individuals and uninfected controls (Fig. 2A; P < 0.01), while there was only a trend toward higher overall KIR transcript levels in acutely infected individuals compared to negative controls. Furthermore, when the KIRs were split into activating (short cytoplasmic tail) or inhibitory (long cytoplasmic tail) receptors, the levels of inhibitory KIR transcripts were significantly higher in chronic HIV-1 infection (Fig. 2B; P < 0.05 for both comparisons), while the activating KIRs were elevated in acutely infected individuals (Fig. 2B; P < 0.05 for both comparisons) compared to uninfected controls. These data demonstrate differential expansion of transcript levels of activating and inhibitory KIRs in NK cells at different stages of HIV-1 infection.

FIG. 2. — Preferential increase in KIR3DS1 transcript levels in acute HIV-1 infection. We analyzed changes in KIR transcription in purified bulk CD3⁻ CD56^+/− CD16^+/− NK cells by quantitative SYBR green PCR. There was an overall increase in KIR transcripts in bulk NK cells in both chronic and acutely infected individuals compared to HIV-negative (HIV−) controls (A). This increase was associated with elevated inhibitory KIR mRNA levels in chronic HIV infection and activating KIR mRNA levels in acute infection (B). Elevated KIR3DS1 transcript levels dominated in primary HIV-1 infection (C). N, HIV negative; A, acute HIV infection; C, chronic HIV infection; *, P < 0.05.

Significant expansion of KIR3DS1⁺ and KIR3DL1⁺ NK cells in the presence of HLA-B Bw480I during acute HIV-1 infection.

Acute MCMV infection is associated with a dramatic nonspecific expansion of NK cells, followed by a specific expansion of a single population of Ly49H-bearing NK cells that are critically involved in the early containment and eventual clearance of the infection (16). We were therefore interested to assess whether similarly a specific population of NK cells expanded preferentially in HIV-1 infection. Deconvolution of transcript levels for each of the 12 KIRs revealed a significant expansion of KIR2DL1 mRNA levels in chronic HIV-1 infection (Fig. 2C; P < 0.05 for both comparisons), likely as a homeostatic mechanism to increase the activation threshold of these cytolytic cells, and elevated expression of three of the six activating KIRs in acute HIV-1 infection (Fig. 2C), similar to the nonspecific expansion observed in lymphocytic choriomeningitis virus. However, among the three activating KIRs that were expressed at elevated levels, mean differences in KIR3DS1 in acute infection were highest compared to the other two groups (Fig. 2C; P < 0.05 for both comparisons). These data suggest that acute HIV-1 infection is associated with the preferential increase of KIR3DS1 mRNA transcript levels.

Despite the fact that we observed a general increase in the level of KIR3DS1 transcripts, there was a significant heterogeneity in mRNA levels ranging from 2.7 × 10⁴ to 1.5 × 10⁹ (Fig. 2C) in subjects expressing both KIR3DS1 and KIR3DL1. Similarly, we also observed a similar wide range of KIR3DL1 transcriptional levels ranging from 2 × 10² to 1.6 × 10⁷ (Fig. 2C). We therefore performed several analyses to identify the underlying factors accounting for the observed heterogeneity in KIR3DS1 and KIR3DL1 mRNA expression levels during acute HIV-1 infection. No correlation was observed between KIR3DS1/L1 mRNA expression levels and viral load or CD4⁺ T-cell count (data not shown), suggesting that other factors likely account for differences in the level of mRNA transcripts in this population of 17 KIR3DS1⁺/3DL1⁺ individuals identified during acute HIV-1 infection.

Epidemiological data suggest that KIR3DS1 and some alleles of KIR3DL1 in conjunction with HLA-B Bw480I are associated with slower HIV-1 disease progression (29, 30). To determine whether the presence or absence of HLA-B Bw480I has an impact on the level of KIR3DS1/L1 mRNA expression during acute HIV-1 infection, we compared transcript levels among acutely infected subjects that either possessed the ligand/putative ligand HLA-B Bw480I or expressed only HLA-B Bw6, a family of HLA class I molecules that does not interact with KIR. KIR3DS1 transcript levels in acute infection were 14-fold higher in subjects that encoded at least one copy of HLA-B Bw480I than those in subjects who possessed two copies of HLA-B Bw6 (Fig. 3A; P = 0.02). KIR3DS1 levels were also 32-fold higher in HLA-B Bw480I⁺ individuals with acute HIV-1 infection than those in HLA-B Bw480I⁺ HIV-negative controls (P = 0.01). Furthermore, there was a trend toward increased levels of KIR3DL1 transcript levels in acutely infected subjects that encoded for at least one copy of HLA-B Bw480I compared to HLA-B Bw6⁺ individuals (KIR3DL1 mRNA levels were sixfold higher in HLA-B Bw480I⁺ subjects [Fig. 3A; P = 0.07), and KIR3DL1 transcript levels were fivefold higher in HLA-B Bw480I⁺ with acute infection to HLA-B Bw480I⁺ HIV-1-negative controls. These data suggest that the presence of HLA-B Bw480I was critical in driving the early transcriptional activation of KIR3DS1 and, to a lesser extent, KIR3DL1 during acute HIV-1 infection.

FIG. 3. — Early and persistent elevation in KIR3DS1 and KIR3DL1 transcripts in the presence of HLA-B Bw480I. To determine whether the presence of the putative ligand HLA-B Bw480I had a role in modulating the expression of KIR3DS1 and KIR3DL1 transcripts, we compared transcript levels for these two receptors at the first visit during primary infection and after 1 year of untreated HIV-1 infection compared to those in HIV-negative individuals. All subjects were KIR3DS1/3DL1⁺ and expressed at least one copy of HLA-B Bw480I or two copies of HLA-B Bw6. The dot plots show that the mRNA levels of KIR3DS1 (left) and KIR3DL1 (right) transcripts were elevated in subjects coexpressing HLA-B Bw480I compared to those that only expressed HLA-B Bw6 during primary infection and that the transcript levels stayed elevated over the course of the first year of infection (A). The kinetics of KIR3DS1 (left) or KIR3DL1 (right) transcript decay are depicted on the line graph (B), demonstrating the rapid loss of both receptor transcripts in subjects that lack the coexpression of HLA-B Bw480I.

HLA class I-dependent persistence of KIR3DS1⁺ and KIR3DL1⁺ NK cells.

We next monitored longitudinal changes in KIR3DS1 and KIR3DL1 mRNA transcripts over the course of the first year of untreated infection in individuals identified during acute HIV-1 infection. Following 1 year of infection, the level of KIR3DS1 and KIR3DL1 transcripts declined. Subjects that expressed at least one copy of HLA-B Bw480I, however, maintained 14-fold-higher KIR3DS1 mRNA levels than subjects that only expressed Bw6 (Fig. 3A and B; P = 0.04). Similarly the level of KIR3DL1 transcripts was significantly higher in HLA-B Bw480I subjects than that in HLA-B Bw6⁺ subjects (Fig. 3A and B; P = 0.002). Furthermore, the level of KIR3DS1 and KIR3DL1 transcripts declined below those observed in HIV-negative controls in subjects that were HLA-B Bw6 homozygous, whereas transcript levels for these receptors remained above those observed in uninfected controls in the presence of HLA-B Bw480I (Fig. 3A). Overall, these data show that the mRNA expression levels of KIR3DS1 and KIR3DL1 during acute and early HIV-1 infection are significantly affected by the presence or absence of the ligand/putative ligand HLA-B Bw480I for these KIRs.

To address whether the above data demonstrating a significant impact of HLA-B Bw480I on mRNA transcript levels of KIR3DS1 and KIR3DL1 in NK cells also translated into a higher frequency of NK cells expressing the respective KIRs, we used a combination of two commercially available antibodies against KIR (z27 and DX9) that have recently been shown to allow for the quantification of KIR3DS1⁺ (z27⁺ DX9⁻) and KIR3DL1⁺ (z27⁺ DX9⁺) NK cells by flow cytometry (Fig. 4A) (10, 32, 36). This flow-based approach allowed us to ensure that the increased expression of these two KIRs was not simply attributable to differences in NK cell subset redistribution (3). We therefore quantified the proportion of KIR3DS1⁺ and KIR3DL1⁺ NK cells over the course of the first year of HIV-1 infection, using the same time points used for the quantification of KIR transcripts. KIR3DS1⁺ and KIR3DL1⁺ NK cells were elevated in acute HIV-1 infection compared to uninfected controls (Fig. 4B; P < 0.05 for both comparisons). Furthermore, the levels of KIR3DS1 and KIR3DL1 were significantly higher in subjects that expressed at least one copy of HLA-B Bw480I than those in subjects expressing HLA-B Bw6 (Fig. 4B, P = 0.03 and P = 0.05, respectively). While the proportions of the KIR3DS1⁺ and KIR3DL1⁺ NK cells declined during the first year of infection, KIR3DS1⁺ and KIR3DL1⁺ NK cells remained higher in subjects expressing HLA-B Bw480I than those in HLA-B Bw6⁺ subjects (Fig. 4C; P = 0.02 and P = 0.002, respectively) and persisted at higher levels than those observed in HLA-B Bw480I⁺ HIV-negative controls (Fig. 4B; P = 0.03 and P = 0.04, respectively). These flow cytometric data reflect results similar to those observed at the transcript levels, demonstrating that KIR3DS1⁺ and KIR3DL1⁺ NK cells expand and persist preferentially during primary HIV-1 infection in the presence of their putative ligand.

FIG. 4. — Early and persistent expansion of KIR3DS1⁺ and KIR3DL1⁺ NK cells in the presence of HLA-B Bw480I. To confirm that the changes observed on the transcriptional level correlated with differences on the protein level, we compared the proportions of KIR3DS1⁺ and KIR3DL1⁺ NK cells by flow cytometry in the same subjects described above, at the first visit during primary infection and after 1 year of untreated infection, compared to the level in HIV-uninfected controls. The flow plot depicts the gating strategy utilized to quantify the proportion of KIR3DS1⁺ (z27⁺ DX9⁻) and KIR3DL1⁺ (z27⁺ DX9⁺) NK cells (A). There was a clear elevation in KIR3DS1⁺ and KIR3DL1⁺ NK cells in uninfected controls in HLA-B Bw480I⁺ subjects, but not Bw6⁺ subjects (B), and this difference was amplified during primary infection and remained elevated over the levels during the first year of infection (B and C).

DISCUSSION

Acute viral infections are typically characterized by a rapid expansion of NK cells. In MCMV infection, this expansion is preferentially restricted to a population of NK cells expressing the activating receptor Ly49H⁺, which recognizes a ligand encoded by the virus (5). While a dramatic expansion of total bulk NK cells occurs during acute HIV-1 infection (2), little is known about the specific expansion of individual NK cell populations during this early window of infection. The delineation of clonal NK cell expansion in human viral infections is seriously hampered by the limitations of commercially available antibodies that only recognize half of the KIRs and do not differentiate between activating and inhibitory KIRs. We therefore adapted a quantitative RT-PCR approach (13) to monitor changes in transcriptional expression of KIR mRNAs in NK cells, and demonstrate that acute HIV-1 infection is marked by a rapid nonspecific increase in activating KIR transcription, an upregulation of KIR2DL1 transcripts in chronic HIV-1 infection, and a clonal NK cell redistribution that is strongly regulated by the presence of HLA class I ligands.

HIV-1 peak viremia declines following acute infection, and the level of early containment of HIV replication correlates with the rate of disease progression (20). While CD8⁺ T cells have been implicated in this containment of viral replication (25, 35), several additional factors may contribute to the initial decline of viremia. One proposed factor is the rapid loss of CD4⁺ T cells, in particular in gut-associated lymphoid tissue, resulting from HIV-1 infection, limiting the number of available target cells (34). Furthermore, the decline in HIV-1 replication and the resolution of acute infection symptoms frequently precede the development of significant virus-specific B- and T-cell responses, strongly suggesting an important role of the innate immune response in this early containment of infection while the adaptive immune response is still developing. Epidemiological evidence suggests that individuals that coexpress KIR3DS1 or some alleles of KIR3DL1 in conjunction with their putative HLA class I ligand (HLA-B Bw480I) exhibit significantly lower viral replication and slower progression toward AIDS (29, 30); however, the underlying mechanism(s) for the protective effect of these KIR/HLA compound genotypes remains to be defined.

Previous studies have shown that NK cells expand in numbers during the first days to weeks of acute HIV-1 infection (2); however, it is uncertain whether specific populations of antiviral NK cells expand during this early window. Using a quantitative RT-PCR approach (13), we studied changes in transcriptional levels of individual KIR mRNAs and KIR⁺ NK cell populations over the course of HIV-1 infection. Similar to acute MCMV infection (16), we demonstrate that acute HIV-1 infection is also associated with a nonspecific expansion of activating KIR (KIR2DS1, KIR2DS2, and KIR3DS1)-expressing NK cells. However, following this nonspecific expansion, a specific accumulation of protective KIR3DS1- and KIR3DL1-expressing NK cells appears to occur, but only in the presence of their putative ligand, HLA-Bw480I. Taken together, these data along with previously published work by Martin et al. (29, 30) suggest that part of the protective activity of these KIR/HLA compound genotypes may be the result of a proliferative signal elicited by HLA-B Bw480I to KIR3DS1 and KIR3DL1⁺ NK cells, allowing them to expand sufficiently to potentially help contain early viral replication in acute HIV-1 infection.

What might account for the activation and expansion of KIR3DS1⁺ and KIR3DL1⁺ NK cells in acute HIV-1 infection? The coexpression of KIR and its HLA class I ligand has been shown to result in a preferential accumulation of NK cells bearing the binding KIR in healthy donors compared to individuals that do not express the KIR ligand (37). Here we show that HIV-1 infection exaggerates these differences in such a way that the proportion of NK cells expressing KIR3DS1 and KIR3DL1 is significantly higher in subjects that coexpress the putative HLA class I ligand family for these receptors. Inhibitory KIR ligands have recently been shown to play a critical role in modulating NK cell function during NK cell development (6, 17, 23). Engagement of an inhibitory KIR by its ligand during NK cell development serves as a critical checkpoint that results in the generation of highly functional NK cells that are easily inhibited by self HLA class I molecules expressed on normal cells in the periphery (6, 17, 23). Given this intimate interaction between the NK cell receptor and its ligand during early NK cell development, it is plausible that HLA-B Bw480I may deliver critical signals to KIR3DL1⁺ NK cells allowing them to develop into highly functional killers that are able to respond aggressively to target cells that do not express their respective self ligands at a sufficient level. This model supports epidemiological data demonstrating that KIR3DL1 allele subtypes encoding for KIR3DL1 receptors expressed at higher levels on the surface of NK cells are highly protective against HIV-1 disease progression when expressed in conjunction with their ligand HLA-B Bw480I (30). KIR3DL1 molecules expressed at high levels may deliver stronger licensing signals to the NK cell during development when engaged with HLA-B Bw480I compared to KIR3DL1 molecules expressed at lower levels, resulting in more potent NK cell populations (4). This is further supported by recent data suggesting a dose-dependent effect of ligand on KIR-mediated NK cell licensing, as increased expression levels of inhibitory NK cell receptor ligands correlate with elevated NK cell effector functions (24). Thus, overall the presence of HLA-B Bw480I may deliver increased licensing signals through inhibitory KIR3DL1 to NK cells, resulting in higher activation of these NK cells in response to target cells expressing no or reduced levels of HLA class I. Additionally we observed a slight increase in the level of both KIR3DS1 and KIR3DL1 transcripts and cell numbers even in HIV-uninfected controls that possessed HLA-B Bw480I. These results suggest that, even in the absence of infection, the presence of HLA-B Bw480I may have an impact on the persistence of populations bearing these receptors in the peripheral circulation, albeit at significantly lower levels than those observed in acute HIV-1 infection.

While NK cell licensing provides a model to explain the preferential expansion of KIR3DL1⁺ NK cells in HLA-B Bw480I⁺ subjects, thus far licensing appears to function through inhibitory KIR only. Thus, a different mechanism might account for the observed changes in KIR3DS1⁺ NK cells following exposure to HIV-1. Previously we have shown that KIR3DS1⁺ NK cells become activated by CD4⁺ T cells expressing HLA-B Bw480I when they become infected with HIV, resulting in potent inhibition of viral replication (1). These data suggest that KIR3DS1 may sense HIV infection specifically through HLA-B Bw480I. While the precise ligand for KIR3DS1 has not been identified, epidemiological and functional data suggest a crucial role of HLA-B Bw480I molecules in modulating the function of KIR3DS1⁺ NK cells during HIV-1 infection (1, 30). In addition, here we observed an early expansion of KIR3S1⁺ NK cells in the presence of HLA-B Bw480I, suggesting that in addition to delivering a potent antiviral signal to KIR3DS1⁺ NK cells, HLA-B Bw480I may also provide a proliferative signal during acute HIV-1 infection to drive the expansion of this potentially antiviral subset of NK cells. Despite the fact that there is no concrete evidence thus far to demonstrate a physical interaction between KIR3DS1 and HLA-B Bw480I, an activating NK cell receptor in mice, which appears to be involved in the control of MCMV infection, was recently shown to analogously interact with its major histocompatibility complex class I ligand H-2D^k only in collaboration with another yet-undefined protein (15). Thus, it is equally possible that the interaction between KIR3DS1 and HLA-B Bw480I, which may present specific HIV-1-derived epitopes or epitopes derived from self stress proteins, might require an additional cellular protein expressed during HIV-1 infection.

Overall we demonstrate the HLA-B Bw480I-dependent expansion of KIR3DS1⁺ and KIR3DL1⁺ NK cells in acute HIV-1 infection. These results demonstrate for the first time a ligand-specific expansion of KIR⁺ NK cell populations in an acute human viral infection. The early accumulation of highly activated NK cells may provide a potent first-line defense allowing for the initial control of acute HIV-1 replication while adaptive immune responses are still developing.

Acknowledgments

This work was supported by NIH grants R01-AI067031 and PO1-AI074415. The project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under contract N01-C0-12400. CHAVI (Center for HIV/AIDS Vaccine Immunology) provided support for the development of the quantitative RT-PCR. We thank the Philip T. and Susan Ragon Foundation for support.

The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Footnotes

^▿

Published ahead of print on 22 April 2009.

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[r1] 1.Alter, G., M. P. Martin, N. Teigen, W. H. Carr, T. J. Suscovich, A. Schneidewind, H. Streeck, M. Waring, A. Meier, C. Brander, J. D. Lifson, T. M. Allen, M. Carrington, and M. Altfeld. 2007. Differential natural killer cell-mediated inhibition of HIV-1 replication based on distinct KIR/HLA subtypes. J. Exp. Med. 2043027-3036. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r2] 2.Alter, G., N. Teigen, R. Ahern, H. Streeck, A. Meier, E. S. Rosenberg, and M. Altfeld. 2007. Evolution of innate and adaptive effector cell functions during acute HIV-1 infection. J. Infect. Dis. 1951452-1460. [DOI] [PubMed] [Google Scholar]

[r3] 3.Alter, G., N. Teigen, B. T. Davis, M. M. Addo, T. J. Suscovich, M. T. Waring, H. Streeck, M. N. Johnston, K. D. Staller, M. T. Zaman, X. G. Yu, M. Lichterfeld, N. Basgoz, E. S. Rosenberg, and M. Altfeld. 2005. Sequential deregulation of NK cell subset distribution and function starting in acute HIV-1 infection. Blood 1063366-3369. [DOI] [PubMed] [Google Scholar]

[r4] 4.Altfeld, M., and P. Goulder. 2007. ‘Unleashed’ natural killers hinder HIV. Nat. Genet. 39708-710. [DOI] [PubMed] [Google Scholar]

[r5] 5.Andrews, D. M., A. A. Scalzo, W. M. Yokoyama, M. J. Smyth, and M. A. Degli-Esposti. 2003. Functional interactions between dendritic cells and NK cells during viral infection. Nat. Immunol. 4175-181. [DOI] [PubMed] [Google Scholar]

[r6] 6.Anfossi, N., P. Andre, S. Guia, C. S. Falk, S. Roetynck, C. A. Stewart, V. Breso, C. Frassati, D. Reviron, D. Middleton, F. Romagne, S. Ugolini, and E. Vivier. 2006. Human NK cell education by inhibitory receptors for MHC class I. Immunity 25331-342. [DOI] [PubMed] [Google Scholar]

[r7] 7.Bashirova, A. A., M. P. Martin, D. W. McVicar, and M. Carrington. 2006. The killer immunoglobulin-like receptor gene cluster: tuning the genome for defense. Annu. Rev. Genomics Hum. Genet. 7277-300. [DOI] [PubMed] [Google Scholar]

[r8] 8.Biassoni, R., C. Cantoni, D. Pende, S. Sivori, S. Parolini, M. Vitale, C. Bottino, and A. Moretta. 2001. Human natural killer cell receptors and co-receptors. Immunol. Rev. 181203-214. [DOI] [PubMed] [Google Scholar]

[r9] 9.Biron, C. A. 1999. Initial and innate responses to viral infections—pattern setting in immunity or disease. Curr. Opin. Microbiol. 2374-381. [DOI] [PubMed] [Google Scholar]

[r10] 10.Carr, W. H., D. B. Rosen, H. Arase, D. F. Nixon, J. Michaelsson, and L. L. Lanier. 2007. Cutting edge: KIR3DS1, a gene implicated in resistance to progression to AIDS, encodes a DAP12-associated receptor expressed on NK cells that triggers NK cell activation. J. Immunol. 178647-651. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r11] 11.Carrington, M., S. Wang, M. P. Martin, X. Gao, M. Schiffman, J. Cheng, R. Herrero, A. C. Rodriguez, R. Kurman, R. Mortel, P. Schwartz, A. Glass, and A. Hildesheim. 2005. Hierarchy of resistance to cervical neoplasia mediated by combinations of killer immunoglobulin-like receptor and human leukocyte antigen loci. J. Exp. Med. 2011069-1075. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

HLA Class I Subtype-Dependent Expansion of KIR3DS1⁺ and KIR3DL1⁺ NK Cells during Acute Human Immunodeficiency Virus Type 1 Infection ^▿

Galit Alter

Suzannah Rihn

Katharine Walter

Anne Nolting

Maureen Martin

Eric S Rosenberg

Jeffrey S Miller

Mary Carrington

Marcus Altfeld

Abstract