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Differential NKp30 Inducibility in Chimpanzee NK Cells and Conserved NK Cell Phenotype and Function in Long-Term HIV-1-Infected Animals¹

Erik Rutjens^2,*, Stefania Mazza^2,,, Roberto Biassoni, Gerrit Koopman^*, Luana Radic, Manuela Fogli^3,#, Paola Costa^,, Maria Cristina Mingari^¶,||, Lorenzo Moretta^,,, Jonathan Heeney^* and Andrea De Maria^4,,||,#

^* Biomedical Primate Research Centre, Rijswijk, The Netherlands; Centro di Eccellenza per la Ricerca Biomedica, Genoa, Italy; Dipartimento di Medicina Sperimentale, Università di Genova, Genoa, Italy; Istituto Scientifico Giannina Gaslini, Genoa, Italy; ^¶ Dipartimento di Oncologia Biologia e Ginecologia, Università di Genova, Genoa, Italy; ^|| Istituto Nazionale per la Ricerca sul Cancro e Centro di Biotechnologie Avanzate, Genoa Italy; and ^# Dipartimento di Medicina Interna, Università di Genova, Genoa, Italy

	Abstract

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HIV-1 infection in chimpanzees, the closest human relative, rarely leads to disease progression. NK cells contribute to the shaping of adaptive immune responses in humans and show perturbed phenotype and function during HIV-1 infection. In this study, we provide full phenotypic, molecular, and functional characterization for triggering molecules (NKp46, NKp30 NKp80, and NKG2D) on Pan troglodytes NK cells. We demonstrate that, in this AIDS-resistant species, relevant differences to human NK cells involve NKp80 and particularly NKp30, which is primarily involved in NK-dendritic cell interactions. Resting peripheral chimpanzee NK cells have low or absent NKp30 molecule expression due to posttranscriptional regulation and increase its levels upon in vitro activation. Following long-standing HIV-1 infection, peripheral NK cells in chimpanzees have conserved triggering receptor expression and display moderate phenotypic and functional decreases only once activated and cultured in vitro. These data suggest that one of the keys to successful lentivirus control may reside in part in a different regulation of NK cell-triggering receptor expression.

	Introduction

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Chimpanzees (Pan troglodytes), the closest living relatives of humans (1), are the reservoirs of SIVcpz, the lentivirus most closely related to HIV-1 groups M (pandemic in humans) and N (2). Unlike other primate species, chimpanzees are able to support a persistent infection of specific isolates of HIV-1. Contrary to HIV-1-infected humans, however, they are relatively resistant to the development of AIDS (3).

Longitudinal follow-up of chimpanzees experimentally or naturally infected with CCR5-tropic HIV-1 strains or with SIVcpz maintain high plasma virus loads, (4) and normal CD4 T cell levels and have remained asymptomatic (3) with one notable exception (5). They maintain an intact lymphoid microenvironment (6) rich in dendritic cells (DCs)⁵ driving effective, low-level, Ag-specific CD4⁺ and CD8⁺ T cell responses (7, 8, 9, 10) without extensive lymphocyte activation (11, 12, 13).

Contrary to chimpanzees, only a minority of humans (<2%) develop an unusually benign clinical course of HIV-1 infection, defined long-term nonprogressive (LTNP) disease (14, 15, 16). LTNP patients respond to long-term HIV infection in a similar way as chimpanzees with respect to the breadth of a HIV-specific CD8⁺ T cell repertoire and the persistence of virus-specific CD4⁺ T cell specificity (17, 18, 19, 20, 21). These findings are in sharp contrast with the majority of HIV-1-infected humans, where virus replication is only temporarily controlled by a vigorous CD8⁺ CTL increase during acute infection (22) followed by progressive loss of HIV-1-specific CD4⁺ cell responses with T cell activation (23, 24, 25), impaired CTL function (17, 26, 27, 28), and loss and dysfunction of DC populations (29).

Although a variety of both host- and virus-related factors have been described in chimpanzees (2, 4, 5, 6, 10), none of them alone allows us to fully reconcile the benign course of the infection with a unique correlate that could help explain the breadth and continuous HIV-specific CD8+ CTL response.

Following encounters with virus-infected or tumor-transformed cells, NK cells provide a first line of defense by Ag-independent recognition of specific triggering ligands that develop through cytotoxicity and by the release of cytokines and chemokines that, in turn, can activate or recruit multiple cell types (30, 31, 32, 33). Molecules that are predominantly responsible for delivering activating signals leading to peripheral NK cell triggering are the natural cytotoxicity receptors (NCRs) and NKG2D, whereas other surface molecules, including NKp80, NTB-A, 2B4, and the DNAX accessory molecule (DNAM), display a costimulatory effect upon the recognition of their respective ligands on target cells (31, 33, 34). NK cells also express a set of MHC-specific receptors that mediate inhibitory signals to the cell through target structure dephosphorylation, thereby overriding any existing activation event and providing an "off" signal that prevents unnecessary activation or autoreactivity (31, 32, 34, 35).

Recently, accumulating evidence of the relevance of NK cell and DC cross-talk at sites of primary inflammation as well as in secondary lymphoid tissues has been recognized as a tool involved in modulating and editing the intensity and quality of adaptive immune responses (30, 36, 37, 38, 39).

Derangement of NK cell numbers and function has been detected since the early epidemic of HIV-1 infection in humans (40), occurring soon after seroconversion (41). Decreased NK cell cytolytic function is determined by a reduction of NKp46, NKp30, and NKp44 NCRs (42), particularly in a subset of NK cells that are significantly activated (43). In addition, these cells may have reduced CD56 surface expression and reduced cytokine production (44). They also have been recently shown to predominantly express triggering NKG2C (CD159c) molecules in association with CD94 (NKG2C/CD94) with a reduced expression of HLA-E-specific inhibitory NKG2A (CD159a)/CD94 molecules (45) that is possibly associated with CMV latency (46).

With the exception of preserved CC chemokine production and Ab-dependent cellular cytotoxicity (ADCC) cytolytic function in infected chimpanzees (11, 12), little information is available regarding chimpanzee NK cells. We therefore decided to study the molecular phenotypic and functional characteristics of chimpanzee triggering receptors to investigate the hypothesis that potentially different patterns of NCR expression or function may accompany and potentially associate with the lack of disease progression and efficient long-term containment of virally determined or immune-mediated damage in chimpanzees.

	Materials and Methods

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Animal subjects and human donors

PBMC were obtained from adult healthy chimpanzees housed at the Biomedical Primate Research Centre, Rijswijk, the Netherlands. Animals (subspecies P. troglodytes verus) were housed in groups according to the International Guidelines for Nonhuman Primate Care. PBMC were isolated using density gradient centrifugation (lymphocyte separation medium) and cryopreserved. Human PBMC were similarly obtained and cryopreserved from six healthy donors. Stored surplus samples were available for six HIV-infected and eight uninfected chimpanzees. HIV-1-infected animals had a mean age of 30 years with a mean of 19.6 years of infection, negative plasma viremia, and the presence of proviral DNA in PBMC. All animals were in good health and had no sign of immunodeficiency. A comparable group of uninfected chimpanzees were studied.

Monoclonal Abs and immunofluorescence analysis

The following panel of anti-human mAbs was used: KD1 (IgG2a anti-CD16); GPR165 (IgG2a anti-CD56); AZ20 (IgG1 anti-NKp30); 7A6 (IgG1 anti-NKp30); F252 (IgM anti-NKp30); BAB281 (IgG1 anti-NKp46); KL247 (IgM anti-NKp46); KS38 (IgM anti-NKp44); Z231 (IgG1 anti-NKp44); MA152 (IgG1 anti-NKp80); and BAT221 or ON72 (IgG1 anti-NKG2D). In addition, human SK7 (CD3) FITC/allophycocyanin, human 3G8PE (CD16), human SK1 (CD8) PerCP/FITC (BD Biosciences), and IgG1 (BioSource International) were purchased. FITC- and PE-conjugated anti-isotype goat anti-mouse second reagent Abs were purchased from Southern Biotechnology.

The reactivity of mAbs with PBMC populations was assessed by indirect immunofluorescence and flow cytofluorometric analysis as described earlier (13). All samples were analyzed on a flow cytofluorometer (FACSort; BD Biosciences). Data were analyzed using CellQuest software (BD Biosciences). Cells were gated by forward and side scatter parameters based on low scatter and small size. Results are expressed as the logarithm of green/red fluorescence intensity (arbitrary units) vs the number of events. For each analysis 10,000 events were counted.

Cell cultures

Cryopreserved human and chimpanzee PBMC were thawed and washed in full medium. PBMC were enriched for NK cells using the MACS NK isolation kit II from Miltenyi Biotec. After immunomagnetic selection, cells were cultured on irradiated (5000 rad) feeder cells in the presence of rIL-2 at 100 U/ml (Proleukin; Chiron) (27, 47). The culture medium used was RPMI 1640 supplemented with 10% FCS, L-glutamine (2 mM/L), and 1% antibiotic mixture (penicillin/streptomycin).

Cytotoxicity assay

A series of FcR⁺ and FcR^– target cells were used in the various cytolytic assays. The P815 murine mastocytoma (FcR⁺) and the FO1 human melanoma (FcR^–) cell lines were used as targets. NK cell-enriched populations were tested for cytolytic activity in a 4-h ⁵¹Cr release assay as previously described (13), either in the absence or the presence of various mAbs. The concentration of the various mAbs was 10 µg/ml for the masking experiments and 0.5 µg/ml for the redirected killing experiments. The E:T ratios are indicated in the text. Murine and human cell lines were used to warrant comparability of results due to their availability and their efficient performance in macaques also (47).

Statistical analysis

Box plot and nonparametric tests were performed using the StatView 4.2 program (Abacus Concepts).

Cytokine assay

IL-2-activated NK cells were harvested and resuspended in complete medium without IL-2 for at least 6 h at 37°C; the cells were subsequently incubated in PMA/ionomycin (2 h) followed by the addition of GolgiPlug (BD Pharmingen) overnight, permeabilization/fixation according to Cytofix/Cytoperm protocol, and subsequent staining for IFN- and TNF-. Cells were fixed overnight with 2% paraformaldehyde and analyzed on a cytofluorometer (FACSort; BD Biosciences).

Isolation of P. troglodytes NKp46, NKp30, NKp80 and NKG2D cDNA by RT-PCR

Total RNA was extracted from 5 x 10⁵ frozen cell pellets using RNAClean (TIB MOLBIOL) from either resting or IL-2-cultured NK cells. First-strand cDNA oligo(dT)-primed synthesis was performed using a RevertAid H Minus cDNA synthesis kit following the manufacturer’s instructions (Fermentas International). RT-PCR amplification was performed with the following primers: NKp46 (1014-bp amplicon), 5'-GAATCTGAGCGATGTCTTCC-3' (NKp46 ATG II; up) and 5'-TCCGTGGGTCCAACACAG-3' (NKp46 open reading frame; reverse); NKp30 (789-bp fragment), 5'-ACCCAGACCTCACTGCT-3' (1C7; forward) and 5'-TATTGGGTGAATGACAGTGTTC-3' (7A6/36.37; reverse); NKp80 (859-bp product): 5'-ACTCACATTGAAGATGCAAGATG-3' (M; up) and 5'-GCTAGACCAGTGTCGATGATGG-3' (M; down); and NKG2D (691-bp PCR fragment), 5'-AGTATTTGATGGGGTGGATTC-3' (H. sapiens/P. troglodytes NKG2D; up) and 5'-TTCCTGGCTTTTATTGAGATG-3' (H. sapiens/P. troglodytes NKG2D; down).

Amplifications were performed after completely denaturing the template for 10 min at 94°C and then 40 cycles at 94°C for 30 s and 55°C for 45 s followed by a 7-min extension at 72°C using Taq recombinant DNA polymerase (Invitrogen Life Technologies). All of the amplification products, which contained the complete open reading frame, were subcloned into pcDNA3.1/V5-His-TOPO vector (Invitrogen Life Technologies) and checked for their DNA sequences using the BigDye terminator cycle sequencing kit and a 3100 Applied Biosystems automatic sequencer.

Analysis of NKp30 mRNA transcription in resting and IL-2-cultured NK cells

The presence of a specific transcript for NKp30 in resting or activated NK cells of both chimpanzee and human origin was compared with the level of transcription of -actin mRNA. The same amount of RNA was analyzed for specific amplification of short amplicons by RT-PCR, and the amplified products, obtained using the same PCR condition mentioned above, have been evaluated after electrophoresis on an ethidium bromide-stained 1.5% agarose gel. To this end, the primer sequences have been designed on different exons to be cross-reactive with both chimpanzee and human sequences: NKp30 (139-bp product), 5'-GACATGGCCTGGATGCTG-3' (1C7 open reading frame 2; up) and CTTGGCTGGCATTGAAGG (1C7; down); and -actin (249-bp amplicon), 5'-ACTCCATCATGAAGTGTGACG-3' (-actin; up) and 5'-CATACTCCTGCTTGCTGATCC-3' (-actin; down).

	Results

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Flow cytometric characteristics of peripheral blood NK cells in chimpanzees

To verify the possibility of defining comparable subsets of NK cells in chimpanzee and human donors, PBMC were thawed and immediately analyzed for CD16, CD56, and CD3 expression. Cytofluorometric analysis revealed that within chimpanzee CD3^– PBMC, two populations with different CD16 surface molecule densities could be identified. Both CD16^dim and CD16^bright cells were lacking CD3 expression, contrary to what was observed in human CD16⁺ PBMC (Fig. 1). In addition, a higher proportion of CD3⁺CD16⁺ PBMC could be detected in chimpanzees compared with human samples (15.44 ± 2.36 vs 8.48 ± 3.64%, respectively; p < 0.01), largely due to an increased representation of CD3^–CD16^dim cells. Exploratory sample analysis also showed that the CD3^–CD56⁺ population in chimpanzees was indistinct because of the poor expression of CD56 as opposed to standard human donor samples (Fig. 1). Therefore, CD16⁺CD3^– PBMC were operatively considered to represent chimpanzee NK cells.

View larger version (41K): [in this window] [in a new window]   FIGURE 1. Expression of CD16 and CD56 on human and chimpanzee NK cells. Cytofluorometric density plots show the expression of CD3, CD56, and CD16 of one representative healthy volunteer donor (bottom row) and one chimpanzee (top row). The experiments are representative of four performed on each species.

Previous work has shown that mAbs specific for human NCRs (NKp46 and NKp30) (46) and other triggering receptors (NKG2D and NKp80) (47) also react with homologous molecules on macaque NK cells. We therefore first studied whether mAbs directed against these four triggering receptors could be used to identify chimpanzee NK cells for the purpose of subsequently studying their reactivity pattern and functional characteristics. We first analyzed by three-color fluorescence analysis the reactivity of anti-NKp30, anti-NKp46, anti-NKp80, and anti-NKG2D mAbs on CD3^–CD16⁺ PBMC from eight uninfected chimpanzees. Cytofluorometric analysis performed by gating on CD3^–CD16⁺ cells confirmed the reactivity of anti-NKp46, anti-NKp80, and anti-NKG2D mAbs with chimpanzee NK cells (Fig. 2A). Unexpectedly, however, we could not find significant reactivity of anti-NKp30 mAbs on resting peripheral NK cells by cytofluorometric analysis in five of eight animals (Fig. 2A), and we found low level expression in the other three animals. This finding of absent to very low NKp30 expression (median 4.8%) could be reproduced in all of the animals studied as shown by box plot analysis of the whole cohort (Fig. 2B). Similar results were obtained when three different NKp30-specific mAbs of different isotypes were used. These findings differ to a considerable extent with what is usually observed on resting human peripheral NK cells from healthy donors, where NKp30 is constitutively expressed on the majority of resting peripheral NK cells (50–85% of NK cells) (Fig. 2).

View larger version (18K): [in this window] [in a new window]   FIGURE 2. Expression of NK cell receptors on fresh human and chimpanzee NK cells. A, Representative cytofluorometric analysis of human and chimpanzee NK cells. NK cells were gated according to lymphocyte forward and side scatter pattern and three-color flow cytometry was performed. Binding of the indicated mAbs is shown on CD3–CD16+ cells. Green fluorescence histograms indicate the reactivity of the anti-NKp46, NKp30, NKp80, and NKG2D Abs in the gated NK population from individuals representative of the two species. B, Box-plot analysis for the proportion of NK cells stained with the indicated mAbs as determined by three-color cytofluorometry. Lines represent median values for the group, boxes show the 25th and 75th percentiles, and bars show SD. Analysis was performed on six human donors and six chimpanzees. *, p = 0.027; #, p = 0.0033. C, Box-plot analysis of mean fluorescence intensity patterns for the given mAbs after three-color cytofluorometric analysis.

In addition to the unexpected pattern of reactivity of anti-NKp30 mAbs, chimpanzee NK cells also stained to a reduced level with anti-NKp80 and to a lesser degree with anti-NKp46 as well, whereas comparable levels of anti-NKG2D binding were observed (Fig. 2, A and B).

Analysis of mean fluorescence intensity (MFI) as a measure of surface molecule density revealed that it was only slightly lower in chimpanzee compared with human NK cells, suggesting that the observed differences were due to differences in the proportion of cells expressing the molecules of interest rather that to a decreased molecule density on chimpanzee NK cells (Fig. 2C). Similar to what is observed in human peripheral blood, no staining for anti-NKp44 mAbs was detected in any of the animals, and further analysis was limited to the above mentioned triggering receptors (31, 33, 42, 44).

Taken together, these results show the presence of mAb reactivity with chimpanzee CD3^–CD16⁺ peripheral NK cells for three of the triggering receptors and also show that compared with human peripheral NK cells major differences are present, particularly for the molecule recognized by anti-NKp30.

Cloning of cDNA encoding P. troglodytes NKP30, NKP46, NKP80, and NKG2D triggering receptors

Basic local alignment search tool (BLAST) and Clustal analyses were performed using information regarding cDNA sequences coding for NK receptors in different primate species (47, 48), and data were available in the European Molecular Biology Laboratory/National Center for Biotechnology Information database for selecting primers able to cross-amplify the corresponding cDNA expressed in NK cells of chimpanzee origin.

In this study we isolated and characterized different cDNAs encoding the NKp46 (NCR1; clone no. 7 (GenBank accession no.AM110137) and clone no.3 (GenBank accession no.AM110136)), NKp30 (NCR3; GenBank accession no.AJ516006), NKp80 (Klrf1; GenBank accession no.AM087960), and NKG2D (Klrk1; GenBank accession no.AM113545) receptors. The two NKP46 isolated forms code for proteins of 304 and 291 aa, respectively. The shorter form is an alternatively spliced product that completely lacks exon 2, which is known to encode a portion of the signal peptide. An identical alternatively spliced NKp46 form, lacking exon 2, has been characterized in humans and is probably the result of a mistakenly spliced DNA due to the short length of introns 1 and 2 of 77 and 188 bp, respectively.

P. troglodytes NKp46 (AM110137) shares a 98% amino acid identity compared with the human homologue and displays five residue differences; four of them are located in the extracellular portion, whereas the other is intracytoplasmatic (Table I). Among the extracellular amino acid differences, two substitutions map proximal to the transmembrane (TM) portion (amino acid sequence nos. 26 and 24, respectively) and the other two are present in a -strand and a loop as described by the crystal structure of human NKp46 (Protein Data Bank code 1OLL) (49). The location of these substitutions hardly modify the receptor’s three-dimensional structure and are unlikely to significantly modify mAb reactivity. Additional comparison of NKp46 sequences from chimpanzee and Macaca mulatta (47 , 48) revealed an overall amino acid identity of 86% with 30 residues changed only in the extracellular portion. A similar comparison of human with macaque protein sequences (47, 48) results in a different composition in 31 aa residues (Fig. 3).

View larger version (19K): [in this window] [in a new window]   FIGURE 3. Graphical representation of primate NK cell-triggering receptor amino acid pairways sequence homology. Numbers indicate different residues between the receptors of the indicated species, Hs, H. sapiens; Pt, P. troglodytes; Mm, M. mulatta (rhesus); Mf, Macaca fascicularis (cynomolgus); Pp, Pongo pygmaeus. Asterisks (*) indicate that an evaluation of sequence alignments has been performed for the extracellular portion only.

The chimpanzee NKp80 sequence encodes a 231-aa protein sharing 98% identity when compared with human homologues. Only four amino acids are different from those in human NKp80, and among them two map in the extracytoplasmic region while one difference each is found in the TM and intracytoplasmic regions (Table I). A comparison of the chimpanzee and human extracellular NKp80 displays 16 residue differences when compared with previously determined macaque sequences (47 , 48) (Fig. 3).

P. troglodytes NKG2D displays two residue substitutions when compared with the human sequence (99% identities for 216 aa) (Table I). A comparison of either the chimpanzee or the human NKG2D sequence to that of macaques reveals a one-amino acid difference in the extracellular region. Similarly, P. troglodytes and Rhesus macaque NKG2D are characterized by 95% overall protein identities (10 residues) and display a single amino acid substitution in the extracellular region (Fig. 3). These analyses indicate that extracellular NKG2D regions are highly conserved in primates while the TM and intracytoplasmic regions appear to be less closely related, although the polar amino acid arginine in the TM region is conserved among all the primate species analyzed.

These data demonstrate a very high degree of sequence conservation between NK receptors in chimpanzee and humans, especially considering the extracellular region of the analyzed receptors. These results were in line with expectations, considering that NK triggering receptor sequences derived from macaques have slightly lower degrees of homology but can be still positively recognized in vivo and in transfection experiments (47, 48).

Phenotypic characterization of purified, in vitro activated chimpanzee NK cells

The extremely high triggering molecule sequence homology and the finding of NKp30 mRNA transcription even with low or absent surface molecule expression led us to next study whether the P. troglodytes triggering receptor function is conserved compared with human homologues and whether NKp30 expression would be up-regulated on chimpanzee NK cells once they are activated in vitro.

To this purpose, chimpanzee and human purified peripheral NK cells were activated in vitro in the presence of human rIL-2 and irradiated feeder cells and were analyzed after 15 days of in vitro culture for the expression of NKp46, NKp30, NKp80, and NKG2D. Similarly, NK cell populations were derived in vitro also for human healthy volunteers. To avoid interindividual differences due to minor proportions of contaminant CD3⁺ cells, three-color fluorescence analysis was performed to analyze only CD3^– cells.

All of the triggering receptors were expressed on activated NK cells (Fig. 4A). NKp46 and NKG2D expression on chimpanzee NK cells was not different to that of human NK cells, whereas NKp30 and NKp80 were expressed on a lower proportion of cells (p = 0.0283 and p = 0.0163 respectively; Mann-Whitney U test). MFI analysis also showed that the density of the expressed molecules was comparable on human and chimpanzee cells as far as NKp46, NKp80, and NKG2D are concerned, while cells expressing NKp30 have a considerably (50%) lower median molecule number per cell in chimpanzee (43 vs 27 MFI, p < 0.05; Mann-Whitney U test) (Fig. 4B). In agreement with previous results obtained in macaques, NKp44 reactivity was absent on chimpanzee activated NK cells in vitro (three animals), whereas it was detectable on human NK cell populations (47).

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Expression of triggering receptors on purified chimpanzee NK cells cultured in vitro in the presence of rIL-2. A, Box-plot analysis of the proportion of in vitro activated NK cells derived from human donors (open boxes) and chimpanzees (shaded boxes) expressing the indicated triggering receptors. B, Box-plot analysis for mean fluorescence intensity staining of in vitro activated NK cells derived from human donors (open boxes) and chimpanzees (shaded boxes). C, Comparison of the expression of NKp30 on resting (open boxes) and activated (shaded boxes) NK cells from human donors and chimpanzees. *, p < 0.05.

Analysis of the other triggering receptors on in vitro activated NK cells also showed that the proportion of cells expressing NKp46 and NKG2D was comparable to that of human NK cells, whereas significantly decreased expression was observed for those expressing NKp80 (Fig. 4, A and B) when compared with both chimpanzee resting purified and human activated NK cell populations. The presence of different functional characteristics for different triggering receptors, as is the case for NKp80 and NKp30, argues against their generalized inducibility in chimpanzee NK cells and further supports the novel finding of a unique NKp30 inducibility in P. troglodytes.

Functional analysis of in vitro activated chimpanzee NK cells

Given the constraints and limitations in obtaining freshly separated chimpanzee peripheral NK cells, functional experiments could be performed exclusively on in vitro activated NK cell cultures derived from cryopreserved samples from both species.

When in vitro cultured NK cell populations were assayed in a redirected killing assay using P815 cells and appropriate mAbs, it was evident that NKp30 or NKp46 triggering induced target cell killing (Fig. 5A) in agreement with their expression by cytofluorometric analysis. Compared with human NK cell cultures, decreased cytolytic activity was detected for NKp30- and NKp80-mediated killing in agreement with the lower levels of expression of these triggering receptors. Differences in cytolytic activity were more blunted or absent for receptors that are expressed at comparable levels, (e.g., NKp46 and NKG2D).

View larger version (18K): [in this window] [in a new window]   FIGURE 5. Cytolytic activity of NK cell populations derived in vitro. A, NK cell effectors were assayed in a 4-h 51Cr release assay against P815 cells at 5:1 E:T cell ratios either in the absence (control) or the presence of anti-CD16, anti-NKp46, anti-NKp30, anti-NKp80, anti-NKG2D, or a combination of anti-NKp30 plus anti-NKp80 mAbs (IgG). The increase in 51Cr release over baseline (irrelevant mAb) reflects the ability of a given mAb to trigger the lytic machinery of the cells. Human donors (open bars), chimpanzees (shaded bars). All experiments were performed using Effector dilution curves in triplicate. Bars represent mean values from four donor/chimpanzee NK cultures (±SD). B, Tumor cell killing assay and mAb-mediated masking of triggering receptors. NK cell populations from human donors (open bars) and chimpanzees (shaded bars) were assayed for cytolytic activity against FO1 melanoma cells either in the absence or the presence of mAbs masking the receptors (µ isotype). E:T cell ratios were 5:1. The decrease in the 51Cr release value over baseline (none) reflects the ability of the mAbs to mask the interaction of specific activating molecules on the effector with its ligand(s) on the target cell. The bars labeled "all mAbs" represent anti-NKp46 + anti-NKp30 plus anti-NKp80 plus anti-NKG2D. Bars represent mean values from four donor/chimpanzee NK cultures (±SD). C, Cytokine production in NK cells stimulated with PMA/ionomycin. Density plots showing double staining for IFN- and TNF- before and after overnight stimulation with PMA/ionomycin. Experiments are representative of three performed.

Next, mAb-mediated masking experiments (Fig. 5B) were performed to confirm triggering receptor activity and specificity. Using FO1 cells as targets to explore NK cell cytolytic function, it could be verified that both human and chimpanzee NK cell populations actively killed these targets. When all of the triggering receptors were masked by a mixture of mAbs specific for the presently explored triggering receptors (NKp30, NKp46, NKG2D, and NKp80), lysis was reduced almost entirely using both human and chimpanzee NK cells.

Finally, we assessed cytokine production by chimpanzee NK cell populations using cytofluorometric detection of intracellular IFN- and TNF- production. Activated NK cells were exposed to PMA/ionomycin followed by four-color cytofluorometric analysis using mAbs specific for CD3, CD16, TNF-, and IFN-. Simultaneous expression of TNF- and IFN- could be detected in >70% of the CD3^–CD16⁺ NK cells in chimpanzees at a level comparable to that in human cells in all samples analyzed (Fig. 5C).

These experiments reveal that NK cell function in P. troglodytes is comparable to that in Homo sapiens, as individual triggering receptors can be specifically recognized by mAbs and are functional upon cross-linking. Functional comparison with human NK cell populations shows similar cytokine production capability with slight reductions in cytotoxic function proportional to the decreased expression of some triggering molecules (notably NKp30 and NKp80).

Analysis of NKp30 mRNA expression in chimpanzee NK cells

The extremely reduced or absent expression of NKp30 on resting NK cells from P. troglodytes presently described is a characteristic unique to chimpanzees that is not shared with H. sapiens. Together with its de novo expression on in vitro cultured NK cell populations it provides evidence for its inducibility, because the event of an in vitro expansion of a minority of NKp30⁺NKp46⁺ NK cells from a predominant peripheral NKp30^null/lowNKp46⁺ population is unlikely. The mechanism(s) underlying this regulation could be either transcriptional or posttranscriptional; we therefore studied resting peripheral NKp30 transcripts compared with activated NK cell cultures where this molecule is expressed and functional.

Screening for NKp30 mRNA by PCR amplification of total cDNA showed that NKp30 mRNA transcription is present in resting peripheral NK cells in both P. troglodytes and H. sapiens. Upon NK cell activation in vitro in the presence of rIL-2, the transcription of NKp30 mRNA increased in both species (Fig. 6A). To verify the qualitative results obtained by gel electrophoresis analysis, we further studied transcript inducibility by using a quantitative PCR approach with real-time PCR. These analyses confirmed an increased transcript expression in activated NK cells compared with resting peripheral purified NK cells in both chimpanzee and human samples (Fig. 6B).

View larger version (53K): [in this window] [in a new window]   FIGURE 6. Analysis of NKp30 mRNA expression in resting (r) and activated (a) purified human and chimpanzee NK cells. A, PCR products of resting and activated NK cells of a chimpanzee show that NKp30 mRNA is present in both populations, similar to NKp46 mRNA. B, Quantitative real-time PCR analysis of NKp30 mRNA transcripts in resting and activated NK cells. Bars represent the fold increase in NKp30 (open bars) and NKp46 (shaded bars) mRNA molecules over resting conditions after in vitro activation of purified NK cells (±SD). Tick marks represent one unit of fold increase.

Analysis of NK cell expression and function in HIV-infected chimpanzees

There is no specific information deriving from patients with LTNP disease to evaluate their NK cell function and phenotype. The opportunity to examine the closest primate relative to humans that invariably controls HIV-1 infection was therefore taken to explore NK cell phenotype and function.

An analysis of cryopreserved aliquots of stored PBMC from infected chimpanzees (four animals) was undertaken using the same conditions described for experiments on NK cells from uninfected chimpanzees. Cytofluorometric analysis of peripheral NK cells in HIV-1-infected animals showed that, similar to uninfected chimpanzees, a low density of NKp30 molecules was expressed in a negligible proportion of NK cells (Fig. 7, A and B). Surface expression of the other triggering receptors followed a pattern comparable to that of NK cells from uninfected chimpanzees (Fig. 7B). Also, an analysis of surface density, as determined by MFI, showed that there was a moderate decrease in NKp30 and NKp46 surface density on NK cells from infected animals without reaching statistical significance (not shown). Thus, contrary to what is known to occur in viremic HIV-1-infected patients, no relevant perturbation of NKp30 or NKp46 is present on NK cells derived from HIV-1-infected chimpanzees.

View larger version (28K): [in this window] [in a new window]   FIGURE 7. Phenotypic analysis of NK cells in HIV-1-infected chimpanzees. A, Cytofluorometric analysis of uninfected and HIV-1-infected chimpanzee NK cells. NK cells were gated according to lymphocyte forward and side scatter pattern, and three-color flow cytometry was performed. Green fluorescence histograms indicate the reactivity of the anti-NKp46, anti-NKp30, anti-NKp80, and anti-NKG2D Abs on CD3–CD16+ cells. Data are representative for six individuals in each of the two conditions (uninfected and infected). B, Expression of triggering receptors on peripheral NK cells of uninfected (open bars) and HIV-1-infected (shaded bars) chimpanzees. Bars represent mean proportion of cells (±SD) expressing a given molecule for all the animals studied. C, Expression of triggering receptors on in vitro activated NK cells in uninfected (open bars) and HIV-1-infected (shaded bars) chimpanzees. Bars represent mean proportion of cells (±SD) expressing a given molecule for all the animals studied. D, Cytolytic activity of NK cell populations derived in vitro from uninfected and infected chimpanzees. NK cell effectors were assayed in a 4-h 51Cr release assay against P815 cells at 5:1 E:T cell ratios either in the presence of an irrelevant mAb or the presence of anti-NKp46, anti-NKp30, and anti-NKG2D. Human donors (open bars), chimpanzees (shaded bars). All experiments were performed using effector dilution curves in triplicate. Bars represent mean values from one representative experiment of three performed (±SD). Lysis in the presence of control mAbs was subtracted in all cases.

Thus, peripheral NK cells in HIV-1-infected chimpanzees present only moderate differences in triggering receptor expression compared with uninfected animals, and a relatively conserved NK cell function and phenotype occurs after HIV-1 infection in chimpanzees.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In this study we provide for the first time a complete molecular, phenotypic, and functional characterization of a set of triggering receptors (including NCRs) in uninfected and HIV-1-infected chimpanzees. Among the receptors studied, NKp30 has been repeatedly shown to be involved in NK-DC cross-talk, contributing to DC-induced activation of NK cells and, at the same time, to killing of immature DC by NK cells (30).

Initial phenotypic characterization of peripheral NK cells in chimpanzees suggested the possibility of different distributions of natural cytotoxicity receptors compared with that in humans. In fact, mAbs recognizing human NKp30 triggering receptors failed to react with chimpanzee peripheral resting NK cells, whereas this was not the case for other triggering receptors, including NKp46.

Molecular analysis of chimpanzee mRNA through cDNA amplification and sequencing confirmed extremely high sequence homology for all of the triggering receptors considered here. Compared with previous data in macaques (47, 48), receptor homology increased considerably in chimpanzees, reaching an extremely high level of similarity to human molecules as expected by the relative positions in evolutional proximity by the three species. In particular, sequences of chimpanzee NKp30 had a single amino acid substitution in the extracellular domain, indicating that a poor or absent recognition by three different mAbs specific for NKp30 molecules was unlikely. This was further confirmed by the identification of NKp30 molecules on chimpanzee NK cells after activation, indicating that this major triggering receptor has a largely inducible phenotype in chimpanzees. This inducibility is unique, as previous reports of NKp30 phenotypic, functional, and molecular characterization in macaques and in humans, where HIV-1 infection is predominantly progressive, did not show a similar behavior (31, 32, 33, 46, 47).

The finding of constitutive NKp30 mRNA transcription and its increased transcription upon cell activation with the same order of magnitude as human NKp30, human NKp46, and chimpanzee NKp46 indicates that, in addition to promoter-induced increased transcription in activated NK cells, NKp30 expression on resting chimpanzee NK cells may be regulated at a posttranscriptional level. Possible explanations for the simultaneous presence of transcribed mRNA without surface molecule expression (51, 52) may involve mRNA stabilization (52) or regulation of ribosomal entry (53) by as yet undescribed factors.

The specific interactions between NKp30 and its ligand(s) expressed on DCs leads to NK cell activation, immature DC maturation, and immature DC killing by NK cells in humans (37, 54). Little or nothing is known as of the nature of the interaction between these cells in P. troglodytes. The choice of the triggering receptors presently analyzed was initially suggested by their expression also on macaque cells, where 2B4, DNAM, and NTB-A cannot be detected by available mAbs (47, 48). Based on recent evidences for an additional relevance for DNAM in the interaction between NK cells and DCs (56), further studies are needed to include and extend the analysis of these coreceptors on chimpanzee NK cells, which could not be addressed here due to the unavailability of fresh cell samples in abidance with the present continental regulations. With these limitations, the present study shows a high sequence similarity for all the four triggering receptors that were analyzed and a similar function of chimpanzee NKp30 as compared with its human homologue. This finding could lead to the suggestion that, in the presence of similar rules of engagement for NK cells and DCs in chimpanzees and humans, decreased or absent NKp30 expression on NK cells could potentially contribute to decreased DC maturation, decreased NK cell activation, and, at least in part, to the observed broad but low level HIV-1-specific CD8⁺ CTL responses in infected chimpanzees with very limited levels of peripheral cell activation (7, 8, 9, 10, 30, 36, 37).

It is most likely that the relative resistance of chimpanzees to AIDS is complex and multifactorial as recently reviewed (2). However, the single most important underlying theme that distinguishes chimpanzees from AIDS-susceptible species is the preservation of APC function and sustained adaptability of responses even in the presence of high virus loads. Direct proof in this respect still needs to be obtained, requiring a comprehensive investigation of DC subsets involving comparative studies of matching DC and NK populations from cohorts of infected and noninfected humans and chimpanzees.

In addition to the relevant aspects of chimpanzee NKp30 regulation and expression, a particular behavior was noted in the present analysis for NKp80. Its cross-linking has been shown to increase NCR-mediated target cell lysis in humans (50), and its lower level of expression in chimpanzee NK cells when associated with extremely low or absent NKp30 expression may result in a reduced editing potential of resting NK cells on DC-mediated adaptive responses (55). Whether and how these characteristics are associated with the unique ability to control virus replication and immune damage by chimpanzees needs future attention using appropriate samples and extending the analysis to other relevant coreceptors such as DNAM (56).

In addition to uninfected chimpanzees, we also extended this study to the expression of the major NCRs on chronically infected chimpanzees who have long-standing nonprogressing asymptomatic infection. Different from humans, chimpanzee peripheral NK cells have unchanged expression of triggering receptors compared with uninfected animals without evidence of overt functional defects, in line with their conserved adaptive immune function (8, 9, 11).

Relevant interest has been raised recently concerning the relative contribution of innate immune responses in the outcome of early events on the evolution of chronic viral replication. Immunogenetic evidence links the prevalence of a weak inhibitory interaction of KIR2DL3 and HLA-Cw3 in patients with successful clearance of acute hepatitis C virus infection (57). The presence of activating KIR2DS1 in human papillomavirus (HPV)-infected patients is linked to chronic infection and progression to cervical cancer (58). Similarly, HIV-1-infected patients have been shown to experience only slowly progressive disease when expressing KIR3DS1 and HLA-Bw4 (59). These observations suggest that efficient defenses against different viruses might benefit from different natural immune responses. The predominance of active NK cell responses could lead to more efficient clearance of acute hepatitis C virus infection (57) or to improved control on chronically replicating HIV-1 (59). In contrast, other viral infections (i.e., HPV) do not progress to overt disease when there is a predominant inhibitory interaction between NK and target cells (58). Along this line, our finding of low or absent NKp30 expression in AIDS-resistant chimpanzees may, at least in part, correspond to mechanisms observed for HPV-associated disease progression and could additionally integrate and complement the recently described relevant expression of ITIM-linked inhibitory CD33-related sialic acid binding Ig-like lectins (Siglecs) on chimpanzee leukocytes, leading to the dampening and control of adaptive immune responses (60). Reduced NK-mediated inflammation, bystander killing, and particularly the dampening of the reciprocal interaction-activation with DCs could result in the specific but not vehement adaptive immune responses that accompany HIV-1 control in this species.

	Acknowledgments

 
We appreciate the assistance of B. Verstrepen, H. Niphuis, S. Hoffman and S. Canu.

	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 in part by grants from the National Institutes of Health (POI AI48225), Istituto Superiore di Sanità (Programma Nazionale AIDS 40F.55, Italian Concerted Action for AIDS Vaccine, Accordi di Collaborazione Scientifica 40D61, 45D/1.13, 45F12), Ministero della Salute (RF 2002/149), Associazione Italiana per la Ricerca sul Cancro, and Ministero dell’Istruzione dell’Università e della Ricerca.

² E.R. and S.M. equally contributed to this work.

³ Current address: National Institute of Allergy and Infectious Diseases, Bethesda, MD 20892.

⁴ Address correspondence and reprint requests to Dr. Andrea De Maria, Department of Internal Medicine, University of Genoa, Largo R. Benzi 10, 16132 Genoa, Italy. E-mail address: de-maria{at}unige.it

⁵ Abbreviations used in this paper: DC, dendritic cell; DNAM, DNAX accessory molecule; HPV, human papillomavirus; LTNP, long-term nonprogressive; MFI, mean fluorescence intensity; NCR, natural cytotoxicity receptor; TM, transmembrane.

Received for publication August 2, 2006. Accepted for publication November 8, 2006.

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