Differences in cellular immunity between humans and chimpanzees in relation to their relative resistance to aids
Rutjens, E.D.I.
Citation
Rutjens, E. D. I. (2011, February 3). Differences in cellular immunity between humans and chimpanzees in relation to their relative resistance to aids. Retrieved from
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CD8 + NK cells are predominant in chimpanzees and characterized by high NCR expression and cytokine production and are preserved in chronic HIV-1 infection
European journal of immunology, 2010, 40(5):1440-50
Abstract
HIV-1 infection in humans results in an early and progressive NK cell dysfunction and an accumulation of an ‘anergic’ CD56-, CD16+ NK subset, which is
characterised by a low NCR expression and low cytokine producing capacity. In contrast to humans, chimpanzee NK cells do not display a distinguishable CD56bright and CD56dim subset, but as shown here could be subdivided into functionally
different CD8+ and CD8- subsets. The CD8+ NK cells expressed significantly higher levels of triggering receptors including NKp46, and upon in vitro activation,
produced more IFN-, TNF- and CD107 than their CD8- counterparts. In addition, chimpanzee CD8- NK cells had relatively high levels of HLA-DR expression,
suggestive of an activated state. Killing inhibitory receptors (KIR) were expressed only at low levels, however, upon in vitro stimulation they were up-regulated on CD8+ but not on CD8- NK cells and were functionally capable of inhibiting NKp30 triggered killing. In contrast to HIV-1 infected humans, infected chimpanzees
maintained their dominant CD8+ NK cell population, with high expression of NCRs.
Introduction
Following encounters with virus-infected or neoplastic cells, NK cells provide a first line of defence by antigen-independent recognition of specific triggering ligands, resulting in cytotoxic activity and/or release of cytokines and chemokines [1-3]. Molecules that are predominantly responsible for delivering activating signals leading to NK cell triggering are the natural cytotoxicity receptors (NCR) and NKG2D, while other surface molecules including NKp80, NTB-A, 2B4, and DNAM provide a co-stimulatory effect upon recognition of their respective ligands on target cells [1, 4, 5]. NK cells express a set of MHC specific receptors that mediate inhibitory signals to the cell through target structure dephosphorylation, thereby overriding an inappropriate activation event [1, 3, 5, 6]. The finding of NCRs, inhibitory receptors and cytokines in combination with CD16 and CD56 has provided evidence for two distinct circulating NK cell populations in humans. These include a CD56dimCD16+ subset with strong expression of NCRs and strong killing capacity, predominant killing inhibitory receptor (KIR) expression and slow IFN
response, which function as peripheral effectors and a CD56brightCD16+ [7, 8] subset with lower NCR expression, high IFN expression, predominant NKG2A+ phenotype as inhibitory receptor. In particular conditions, either pathological or in vitro, other NK cell phenotypes have been described, including a so called “exhausted” subset that is CD56- CD16+ and is deficient for NCRs and cytokine expression [9, 10], “helper” NK cells that express CD83 and CCR7 upon culture with IL-18 [11], and modulatory NK cells [12].
HIV-1 infection in humans has a profound impact on NK responses, ultimately resulting in major changes in NK populations and function. Decreased NK cell cytolytic function is observed in association with a reduced expression of NCRs (i.e.; NKp46, NKp30, NKp44) [13-16]. This change is ultimately exemplified by a shift in relative NK populations with an evolving predominance of CD56-CD16+NCRlowHLADR+CD69+
“exhausted” cells in chronic HIV-1 infected humans [9, 10, 13, 14] and leads to impaired killing of iDC with altered DC editing [17].
Chimpanzees (Pan troglodytes) are genetically and evolutionary most closely related to humans and are the only identified reservoir of HIV-1 group “M” and “N” related lentiviruses, designated as SIVcpz [18, 19]. Remarkably, when chimpanzees are exposed to human adapted variants (HIV-1 group M) of this virus the vast majority (more than 99%) control infection and remain asymptomatic, suggesting adaptation of virus and host
defence mechanisms [20-22]. Chimpanzees elicit effective antigen specific CD4+ and CD8+ T-cell responses to HIV-1 [23-25] in the presence of an intact lymphoid microenvironment [26].
NK cells have been reported to be present at elevated levels in chimpanzees relative to humans [27, 28]. Strikingly, over 50% of all NK cells in chimpanzees express the CD8
chain [28]. In contrast, humans possess a small population of CD8+ NK cells and these have been reported to display enhanced survival during target cell killing, mediated by homotypic cross interaction between CD8 chains expressed on neighbouring NK cells [29]. This important feature in combination with previously reported differences in expression of CD56 and NKp30 [30] has lead us to further characterize and compare NK cell subsets in HIV-1 infected and uninfected chimpanzees with particular focus on the CD8+ NK population which declines early in infected humans. Here we demonstrate that in contrast to humans, CD8+ NK cells in chimpanzees express and maintain high levels of killing enhancing receptors (NCR) and vigorously upregulate CD107 and IFN
expression upon stimulation. Importantly, despite chronic HIV-1 infection in chimpanzees both CD8+ NK cell phenotype as well as balance of different NK sub- populations is preserved.
Materials and Methods
Animal subjects and human donors
Peripheral blood mononuclear cells (PBMC) were obtained from adult healthy chimpanzees (P.troglodytes) housed at the Biomedical primate research centre in Rijswijk, the Netherlands. Animals were housed in groups according to the European and international guidelines for non-human primate care. PBMC were isolated using density gradient centrifugation (LSM) and subsequently frozen in FCS with 10% DMSO and stored at -135C. Cryopreserved samples were used from a cohort of 9 infected chimpanzees, of which 8 had been experimentally infected with HIV-1, either in 1984 (n=2), 1991 (n=5) or 1996 (n=1). One animal carried the SIVcpzANT virus. Six HIV-1 infected human donors were used for detailed comparative studies.
Monoclonal antibodies and immuno-fluorescence analysis
The following panel of anti-human mAbs was used in these studies: anti-CD16: KD1 (IgG2a); anti-CD56: GPR165 (IgG2a), 123C3 (IgG1) (Sanbio, Uden, Netherlands) ; anti- NKp30: AZ20 (IgG1); anti-NKp46: BAB281 (IgG1), KL247 (IgM); anti-NKp80: MA152 (IgG1); anti-NKG2D: BAT221 or ON72 (IgG1); anti-NKp44: KS38 (IgM), Z231 (IgG1);
anti-P58.1: 11PB6 (IgG1); anti-P58.2: GL183 (IgG2A); anti-NTBA: MA127 (IgG1); anti HLA-DR: D1-12 (IgG2A); anti Granzyme B: GB11 (IgG1, kindly provided by Dr. C.E.
Hack, Sanquin, Amsterdam, The Netherlands). In addition commercial preparations of anti-human CD3: SK7 FITC/APC or SP34.2 Al700; CD16: 3G8 PE/PE-Cy7; CD8: SK1 PERCP/FITC/APC-Cy7 ; HLA-DR: L243 PerCP; CD94 HP-3D9 FITC (all from Becton Dickinson, San Jose); CD14: RM052 ECD (Beckman Coulter); CD20: B9E9 ECD (Beckman Coulter) were used. Fluorescein isothiocyanate- (FITC) and phycoerithrine (PE) conjugated isotype specific Goat anti-Mouse secondary antibodies were purchased from Southern Biotechnology (Birmingham, AL).
Binding of mAbs with PBMC populations was assessed by indirect immuno-fluorescence and flow cytometric analysis as described earlier [13, 14]. Analysis of the CD56 expressing subset of CD3-CD16- cells was performed by using a combination of CD94FITC, NKp30PE, NKp44PE, NKp46PE, CD14ECD, CD20ECD, HLA-DRPerCP, CD16PE-Cy7, CD56APC, CD3Al700, CD8APC-Cy7. All samples were analyzed by flow cytometry and data were processed using Cellquest and Diva software (Becton Dickinson, Mountainview, CA). Results are expressed as the proportion of the cells expressing a given surface antigen as compared to a negative control consisting of cells stained with irrelevant mAbs, or as mean fluorescence intensity channel.
Cell cultures
Cryopreserved Human and Chimpanzee peripheral blood mononuclear cells (PBMC) were thawed and washed in RPMI 1640 supplemented with 10% foetal calf serum, l- glutamine (2mM/l) and 1% antibiotic mixture (penicillin 5mg/ml, streptomycin 5mg/ml stock solution). PBMC were enriched for NK cells by negative selection against CD3, CD4, CD14, CD19, CD34 using MACS NK isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). After immunomagnetic selection, cells were cultured in the presence of r-IL-2 100U/ml (Proleukin, Chiron Corp., Emeryville, CA). Irradiated (50
Gy) feeder cells, consisting of a mixture of human PBMC and 221G cell-line, were added to support the IL-2 driven outgrowth of the NK cells .
Cytotoxicity Assays
The FcR-positive P815 murine mastocytoma target cells were used in the various functional cytolytic assays. NK cell-enriched populations were tested for cytolytic activity in a 4-hr 51Cr-release assay as previously described [13], either in the absence or in the presence of various mAbs. The concentration of the various mAbs was 0.5 µg/ml for the redirected killing experiments. The E/T ratios are indicated for each experiment.
Cytokine Assay
IL-2 activated NK cells were harvested and re-suspended in complete medium without IL-2 for at least 2 hours at 37C, subsequently, cells were incubated with either PMA (20 ng/ml) plus ionomycin (1 g/ml) or co cultured with K562 target cells for 2 hours, then followed by the addition of Golgiplug (Becton Dickinson). CD107a was present during this culture period. After overnight incubation, cells were permeabilized and fixed according to the Becton Dickinson Cytofix/Cytoperm protocol and subsequently stained for IFN and TNF (Becton Dickinson). Cells were fixed overnight with 2% para- formaldehyde and all samples were analyzed by flow cytometry FACSort & FACSaria, Beckton Dickinson).
Isolation of complete ORF of different Pan troglodytes KIR and NKG2A cDNAs by RT- PCR
Complete chimpanzee 5’ cDNA end KIR sequences were obtained by RACE (Roche, Mannheim, Germany) using the following primers: Pt-KIR-SP1 (5’-gaacgtgcaggtgtctgga- 3’), Pt-KIR-SP2 (5’-gatagggggagtgagtaac-3’) and Pt-KIR-SP3 (5’-tttcctgtggacgccctca-3’).
The nested PCR were performed at 94°C/2’ followed by 30 cycles 94°C/15”, 58°C/30”, 72°C/40” and a 72°C/7’final elongation step. Amplicons were subcloned into pcDNA2.1 TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by capillary electrophoresis (Applied Biosystems Europe, Belgium).
Primers designed on the above sequence and in conserved region of human KIR gene were used to amplify by RT-PCR the complete ORF of Pt-KIR: Pt-KIR orf frw (5’- accggcagcaccatgtc-3’) and Pt-KIR orf dw (5’-gtcatcctgcaatgttggtc-3’). Reaction was performed at 94°C/2’ followed by 30 cycles at 94°C/15”, 58°C/30”, 72°C/40” and a 72°C/7’ extension step (Expand High Fidelity PCR system; Roche, Mannheim, Germany).
The 1349 bp amplicon containing the complete ORF of P.tro KIR3DL4 (AM400232) were cloned into pcDNA2.1-TOPO vector, checked for DNA sequence and subsequently subcloned into the BamHI/XhoI sites of pRB1 expression vector.
The Pt-NKG2A was amplified using the NKG2A frw (5’- acactgcagagatggataacc-3’) and the NKG2A rev (5’-aaaatgagcccgacacaaatg-3’) to obtain a 907bp amplicon containing the 702bp NKG2A complete ORF (AM400232). This cDNA has been subcloned into the pcDNA3.1 (Invitrogen, Carlsbad, CA) eukaryotic expression vector.
Transient transfections to confirm P.t KIR and NKG2A specificity
Transient transfections were performed with HEK293T cells as described [33], and analyzed by indirect immuno-fluorescence and cytometric analysis using 11pb6 mAb (anti H.sap KIR2DL1; IgG1) followed by a PE-conjugated anti-mouse reagent. Control transfections using Homo sapiens KIR2DL1 (cl.47.11) and Homo sapiens KIR2DL3 (cl.6), were used as positive or negative samples, respectively.
For the analysis of NKG2A surface expression Homo sapiens or Pan troglodytes NKG2A cDNA constructs were co-transfected with Homo sapiens CD94-encoding expression vectors, since NKG2A is known to be expressed on the cell surface only in the presence of CD94 molecules [39]. Cytometric analysis of CD94/NKG2A transfectants were performed using mAbs Z199 and Z270, specific for human NKG2A molecules, the P25 mAb specific for both human NKG2A and human NKG2C receptors and, as control, the anti-human CD94 XA185 mAb.
Results
Phenotypic characterisation of chimpanzee NK cell subpopulations
Chimpanzee PBMC from normal healthy animals were thawed and analysed for CD3, CD16, CD56 and CD8 expression. NK cell populations were analysed by selecting CD3 negative cells within an extended lymphocyte gate as to incorporate the large granular cells. As reported previously [30], similar to humans, a large proportion of these CD3 negative cells expressed CD16.
Figure 1. Definition of NK cell populations in peripheral blood in humans versus chimpanzees. Expression of CD16 versus CD8, CD56, granzyme B and CD94 was evaluated in PBMC by first selecting cells within an extended lymphocyte gate, incorporating the large granular cells, and subsequent gating on the CD3 negative population. Dot plots show the distribution of CD16 versus CD8, CD56, Granzyme B or CD94, on the CD3 negative cells on human PBMC (top row) and chimpanzee PBMC (middle row). The subpopulation of CD3-CD16-CD56+ cells was first analysed for expression of CD14,20 and HLA-DR (bottom row). Subsequently, the CD14, CD20, HLA-DR negative cells were analysed for expression of CD94 versus a combination of NKp30,NKp44,NKp46 NCRs.
However, in contrast to the uniform CD16 bright population in humans, in chimpanzees a CD16 bright and CD16 dim population could be discerned, CD56 expression was largely absent and a dominant CD8+ subpopulation was seen [28, 30]. As shown in figure 1, the two CD16 subsets were partly CD8 positive and to a large extend CD94 positive.
Strikingly, the CD16 bright cells did not express granzyme B (figure 1) or granzyme A (not shown), while these markers were present on CD16 dim cells. Of additional note is the presence of a CD3-CD16-CD56+ population. Further analysis showed that these cells are largely negative for CD14, CD20 and HLA-DR and have a high expression of CD94 and NCRs (figure 1). Because these cells only form a minor subset (0.68 ± 0.46 % of the lymphogate) only phenotypic analysis could be performed and subsequent studies focused on, comparative receptor expression on the CD3-CD16+CD8+ versus the CD3-CD16+CD8- subset. As shown in figure 2 the cytotoxicity receptors NKp46 and NKG2D as well as the co-activatory receptors NKp80 and NTB-A were all expressed at higher levels in the CD8+ subset. As previously shown, NKp30 was negative on freshly isolated chimpanzee NK cells, in contrast to human NK cells [30]. Since reduced NCR expression has previously been noted in activated, HLA-DR positive, human NK cells, its expression was analyzed on chimpanzee NK cells [13]. As shown in figure 2, relatively high percentages of HLA-DR positive cells were found in the CD8- subset, while only limited expression was seen on CD8+ NK cells.
Figure 2. Expression of cytotoxicity receptors and HLA-DR on chimpanzee NK cells.
Expression of activating receptors and HLA-DR is shown as percentage positive cells in the CD8+ (black bar) versus the CD8- (white bar) NK population (lymphogate selected, CD3 negative CD16 positive peripheral blood cells) in healthy control chimpanzees. Average expression +/- standard deviation from 3 individual animals is shown. Asterisks show statistically significant differences between CD8+ versus CD8- cells as calculated by students t test (P<0.05).
0,00 10,00 20,00 30,00 40,00 50,00 60,00 70,00 80,00 90,00
NKp46 NKp30 NKp80 NKG2D NTB-A HLA-DR
% positive cells
HIV- CD8+
HIV- CD8-
*
* * *
NKG2A and the KIR receptors p58.1, p58.2, p70 were only expressed at modest intensity, with a Mean Fluorescence Intensity of 2.9 ± 1.1; 2.4 ± 1.6; 1.5 ± 1.5 and 2.8 ± 0.7 respectively. Overall, based on this phenotypic analysis, human homologes of the CD16+CD56dim population could not be identified in uninfected chimpanzees.
Chimpanzee CD8- NK cells seem to represent an activated subpopulation with decreased expression of activatory receptors including NKp46/NKp80/NKG2D.
Phenotypic and functional analysis of in vitro activated chimpanzee NK subpopulations
In order to study whether expression of CD8 defined two separate NK cell subsets or represented the same NK cell type at different stages of cell activation/differentiation, a phenotypic stability study was performed using CD3/CD4/CD14/CD19/CD34 depleted lymphocytes cultured in the presence of IL-2 and irradiated feeder cells. Upon culture, the relative composition of these cells with regard to CD8 expression remained unaltered.
However, upregulation of CD56 expression and a slight reduction of CD16 fluorescence intensity were detected in a subset of the CD8 positive cells (figure 3). Subsequently, we studied the expression of NCRs and inhibitory receptors on these same activated CD8+ and CD8- NK subsets. Although there was substantial animal-to-animal variation, the data presented in table 1 illustrate that for each of the 3 chimpanzees tested, the expression of all activating NK cell receptors was markedly higher in CD8+ than in the CD8- NK cells.
Hence, in vitro stimulation per se was not capable of inducing high levels of expression of these markers in CD8- cells. Interestingly, the expression of NKp30, which could not be detected on fresh chimpanzee NK cells, was upregulated following stimulation in CD8+ cells of all chimpanzees. Reactivity to mAbs specific for KIRs and NKG2A remained low on CD8- NK cells, but was increased on the CD8+ NK subset (table 1).
Since the culture conditions and the limited amount of available cryopreserved samples prevented us from performing further functional experiments on enriched CD8+ or CD8- NK cell fractions derived in vitro, we proceeded to study the overall cytotoxic activity of these populations by performing redirected killing assays using P815 cells loaded with antibodies specifically recognising each specific receptor. P815 murine mastocytoma cells express FcR, and are commonly used to explore "Redirected Killing" also known as
"reverse ADCC" employing mouse mAbs. In this assay mAbs bind to the triggering
molecule (e.g. NCRs on chimpanzee NK cells) they specifically recognize via the Fab moiety, while the mAb Fc is bound by FcR on P815 cells.
Figure 3. Phenotypic characterization of in vitro activated NK cells. First CD3 negative cells were selected for this analysis. Plots show expression of CD16 versus CD8 or CD56, and CD56 versus CD8 in the CD3 negative compartment of IL-2 activated NK cells grown on feeder cells (14 day culture).
In contrast to observations in human NK cell populations, relevant basal cytolytic activity was observed in chimpanzee NK cells directed against ligand specific P815 targets at relatively low effector/target cell ratios. As shown in figure 4, both spontaneous lysis as well as lysis induced through NKp30 ligation was markedly reduced by mAb directed against human p58.1, NKG2A and p70. These experiments show for the first time that molecules specifically recognized by mAbs raised against human KIRs and NKG2A are functional and that their cross-linking determines inhibition of chimpanzee NK cell activity.
Table I: Relative expression of NK receptors in CD8+ versus CD8- subpopulations of NK cells in HIV negative healthy chimpanzees after in vitro stimulation for 14 days with IL-2 and heterologous target cells (percentage positive cells is shown).
NKP46 NKP30 NKP80 NKG2D NTB-A NKG2A P58.1 P70 P58.2 HLA-DR
CD8+ 82.6 83.0 51.3 73.6 36.8 12.8 19.6 5.4 9.7 59.3
CD8- 44.7 42.6 20.8 34.2 10.1 0.2 1,8 0.0 0.1 41.7
CD8+ 15.7 11.0 35.1 44.0 35.6 3.9 7.2 2.8 3.0 12.0
CD8- 5.8 2.7 0.7 12.9 8.0 0.1 6.5 0.0 0.1 14.8
CD8+ 34.2 33.1 32.3 46.1 40.9 17.4 21.3 15.6 13.3 3.3
CD8- 7.3 13.3 2.7 12.3 6.1 0.5 1.0 0.0 0.4 4.0
Chimp 1 HIV-
Chimp 2 HIV- Chimp 3
HIV-
Molecular characterization of chimpanzee MHC class I inhibitory receptors.
While the mAbs used in this analysis were all selected on basis of binding to specific human NK receptors, sequence analysis of chimpanzee NKp46, NKp30, NKp80, NKG2D cDNA revealed a striking degree of sequence conservation, especially in the extracellular binding region of the receptors, making this interspecies comparison possible [30].
However, much less information was available with regard to the inhibitory receptors. In order to verify that the mAb used by us are cross reactive against chimpanzee NK cell receptors and to identify which inhibitory receptors are specifically recognized, we next focused our attention to the isolation of cDNAs specifically reacting with the corresponding mAbs. To this end, RNA was extracted from selected purified NK cell populations displaying 11pb6 mAb (anti-human KIR2DL1/DS1) surface reactivity. Since at that time all the chimpanzee KIR sequences available in databases were missing the complete open reading frame (ORF), including the ATG start-codon critical for the translation and fundamental to code for signal peptide, we cloned the complete cDNA.
FACS analysis on transfected HEK293T cells showed that P.tro-KIR3DL4 (AM400232) was 11pb6 mAb reactive (Figure 5, panel A).
Figure 4. Killing inhibitory function of receptors expressed on in vitro activated NK cells.
Inhibition of cytolytic activity against P815 murine mastocytoma cells mediated by mAb directed against p58.1, NKG2A, p70 in the absence (spontaneous NK mediated lysis (left half of the graph)) or presence of triggering NKp30 mAb (right half of the graph). Lysis is depicted as percentage relative to experimental wells where no inhibitory antibody was added. Negative values represent a reduction of background lysis of the target cells by ligation of inhibitory receptors. A representative example out of 3 separate experiments is shown.
-15 -10 -5 0 5 10 15 20 25
P58.1 NKG2A P70 NKP30+P58.1 NKP30+NKG2A NKP30+P70 NKp30
relative % lysis
These findings confirmed the presence of functional MHC-class I inhibitory receptors in the chimpanzee NK cell populations and their direct involvement as demonstrated by inhibition of cytotoxicity experiments using the anti-p58.1 11bp6 mAb (Figure 4).
The analysis of the Pt-KIR3DL4 amino acid sequence revealed that all the residues involved in defining C2-specificity (“group 2” HLA class I-specificity, i.e. HLA-Cw2, - Cw4, -Cw6) typical of KIR2DL1/p58.1 receptors were conserved both in human KIR2DL1 and Pt-KIR3DL4, as recently also shown by others [31]. In particular, M44, directly involved in the hydrogen bond interaction with the “group-2” HLA-C alleles in humans and the other polymorphic residues involved in the HLA-C binding site (M33, N46, T48, R68 and T70) may also contribute to a similar interaction of KIR3DL4 with chimpanzee MHC alleles.
Within the same context, mAb reactivity also demonstrated functional inhibition of chimpanzee NK cells in the presence of mAb Z270 that is specific for human NKG2A and mediates functional NK cell inhibition upon binding to its ligand. After cDNA isolation and sequence analysis, cDNA expression constructs using chimpanzee NKG2A cDNA (AM502818) were prepared. Since NKG2A cannot be expressed on the cell surface in the absence of CD94, the constructs were also co-transfected with CD94 cDNA (GU256361) and analyzed by indirect immuno-fluorescence using the mAbs specific for human NKG2A or CD94 including those that had been used for functional assays (Z270, Z199, P25, XA185). As shown in figure 5 panel B, all the mAbs confirmed the same pattern of reactivity against human and chimpanzee derived sequences.
Hence, these results show that mAbs specific for human KIR2DL1 recognize a homologous inhibitory receptor in chimpanzees (KIR3DL4) and support the specific nature of the functional effects seen in cytotoxicity assays. Similarly, human NKG2A- specific mAbs recognize a functional homologue in chimpanzees.
Functional differences between CD8+ and CD8- NK cells.
The two main functional properties of NK cells in humans are the production of cytokines and cytolysis of cells that have down-modulated MHC-I. Both of these functions were studied in chimpanzee NK cells activated by PMA/ionomycin or via co-culture with K562 target cells using multi-parameter FACS analysis. K562 cells (human erythroleukemia) are commonly used to provide a generic estimate of the cytolytic activity of resting NK
cells (or of IL-2 activated T and NK cell killing activity) and are usually resistant to lysis by resting T cells. Detection of CD107a was used as a read out of lytic capacity.
A
B
Figure 5. Surface expression of human and chimpanzee KIR and NKG2A in transiently co-transfected cells.
Panel A: FACS analysis of HEK293T cells transiently transfected either with human KIR2DL1/U24076 (left), chimpanzee (P. tro) KIR3DL4/AM400232 (middle) or human KIR2DL2/U24074 (right panel) cDNA constructs and stained with the 11pb6 mAb (originally selected for human KIR2DL1 reactivity).
Panel B: FACS analysis of HEK293T cells transiently co-transfected with human CD94 and either human (H. sap) NKG2A or chimpanzee (P. tro.) NKG2A (AM502818), showing staining of the Z199, Z270 and P25 mAbs (in humans Z199 and Z270 are specific for NKG2A, while P25 recognizes both NKG2A and NKG2C). The CD94-specific XA185 mAb was used as transfection control. Red lines represent reactivity to the indicated mAb. Results are expressed as logarithm of red fluorescence intensity (arbitrary units) versus number of events. For each analysis 104 events have been acquired.
It is present in cytotoxic granules of NK cells as well as cytotoxic T-lymphocytes and is transiently expressed on the cell surface during degranulation [32]. Previous experiments have shown induction of IFN and TNF in chimpanzee NK cells upon PMA/ionomycin stimulation [30]. As shown in figure 6, the induction of CD107a as well as TNF and IFN- was largely restricted to the CD8+ subset, implying full functional capacity of these cells, while CD8- NK cells had a much lower expression. Co-cultivation of chimpanzee NK cells with the K562 NK target line resulted in the induction of both cytokine expression and degranulation, which again was a predominant characteristic in the CD8+ subset (figure 6). From multiple experiments it can be concluded that in agreement with
H.sap
P.tro
Z199 Z270 P25 XA185
H.sap
P.tro
Z199 Z270 P25 XA185
their phenotypic expression of triggering receptors (figure 2), CD8+ cells are functionally armed. Alternatively, the CD8- NK subset represented a relatively functionally non- responsive population. As this was detected following maximal PMA/ionomycin stimulation, a stimulus far beyond physiological norms of a dampened triggering receptor expression, this NK subset in chimpanzees may represent their exhausted or anergic population.
Figure 6. Cytokine production capacity and induction of CD107 expression in chimpanzee CD8+ versus CD8- NK cells. (A) Gating strategy. Analysis was performed by selecting cells within an extended lymphocyte gate that were CD3 negative. Subsequently, the expression of different cytokines or CD107 was plotted against CD8. Shown is expression of TNF (B), IFN (C), CD107 (D) in CD8+ (black bars) and CD8- (open bars) on IL-2 activated NK cell cultures after K562 target cell or PMA/ionomycin stimulation.
TNF and IFN are measured by intracellular cytokine staining, while CD107 is detected via incubation with anti CD107 mAb during stimulation. Averages with +/- standard deviation of 3 separate individual chimpanzees is shown. Asterisks represent statistical significant differences between CD8+ and CD8- cell by students t test (P<0,05).
NK subset composition and phenotype in HIV-1 infected chimpanzees.
Given the changes in NK subset composition that are observed in HIV-1 infected humans, we undertook to study the phenotype of NK cell subsets in a cohort of 9 HIV-1 infected chimpanzees. Relative to the group of 13 non-infected animals there was a modest A
B
Figure 7. NK cell number and phenotype in HIV-1 infected chimpanzees.
A) Absolute number of NK cells (CD3-CD16+) (left graph) and percentage of NK cells that express CD8 (right graph) in non-infected (black dot, n=13) versus HIV-1 infected (black square, n=9) chimpanzees is shown. The mean is indicated by a horizontal bar. Statistical evaluation was performed by two tailed students t test B) Expression of activating receptors and HLA-DR is shown as percentage of positive cells in the CD8+ (black bar) versus the CD8- (white bar) NK population (lymphogate selected, CD3 negative CD16 positive peripheral blood cells) in infected chimpanzees. Average expression +/- standard deviation from 3 individual experiments is shown. Asterisks show statistical significant differences between CD8+ versus CD8- cell as calculated by students t test (P<0.05).
0 10 20 30 40 50 60 70 80 90 100
NKp46 NKp30 NKp80 NKG2D NTB-A HLA-DR
% positive cells
HIV+ CD8+
HIV+ CD8-
*
*
*
*
*
comparison with uninfected animals (p<0.01), which correlated with CD4 count (p=0.015 Spearman correlation test), the relative subset composition of the CD8+ versus the CD8- NK subset remained unaltered (figure 7a).
NK cells in HIV-1 infected chimpanzees are known to express NCRs and co-activatory receptors [30]. As shown in figure 7b, the NCRs and co-activatory receptors were found predominantly expressed in the CD8+ NK cell subset, similar to the uninfected chimpanzees (figure 2). In addition the level of expression of these receptors was found to be similar in uninfected as compared to HIV-1 infected animals (figure 2, 7). Inhibitory receptors were only detected at low intensity with an MFI of 5.2 ± 5.4; 6.4 ± 3.7; 1.3 ± 0.9 and 4.4 ± 2.0 for NKG2A, p58.1, p58.2, p70 respectively, which is similar to the low expression seen in uninfected chimpanzees. As shown in table 2, NK cells in HIV-1 infected chimpanzees were responsive to IL-2 stimulation as were the uninfected chimpanzees. Upregulation of NCRs as well as expression of KIRs and NKG2A was observed, again with predominant expression of NCRs in the CD8+ subset. The subset of CD3-CD16-CD56+ expressing cells described in figure 1 was found to be present in HIV- 1 infected chimpanzees at a similar frequency (0.88 ± 0.77 % of the lymphogate) and had similar phenotypic characteristics (not shown). In conclusion, despite a two-fold decrease in absolute NK cell numbers, HIV infected chimpanzees maintain their CD8+ NK subset, with unaltered NCR expression.
Table II: Relative expression of NK receptors in CD8+ versus CD8- subpopulations of NK cells in HIV-1 infected chimpanzees after in vitro stimulation for 14 days with IL-2 and heterologous target cells (percentage positive cells is shown).
NKP46 NKP30 NKP80 NKG2D NTB-A NKG2A P58.1 P70 P58.2 HLA-DR
CD8+ 25.0 7.2 78.2 26.8 42.4 3.8 3.8 4.2 2.3 13.3
CD8- 3.5 1.9 3.9 3.2 6.1 1.1 1.7 1.3 0.7 86.2
CD8+ 6.8 4.6 12.8 51.2 55.2 9.2 6.6 11.2 12.3 26.5
CD8- 0.0 3.6 0.5 11.4 7.6 0.0 1.0 0.0 0.0 34.7
CD8+ 57.3 53.8 53.2 80.0 31.1 19.1 34.0 18.6 15.3 50.3
CD8- 13.6 12.3 9.0 29.6 1.7 0.2 5.8 1.4 0.6 30.1
Chimp 2 HIV+
Chimp 3 HIV+
Chimp 1 HIV+
Discussion
Underlying HIV-1 infection in humans is a progressive decay of immune function, characterized by a loss of T and B-cell and antigen presenting cell function. Also striking are changes in NK cell phenotype and function with a progressive accumulation of NKp30dullNKp46dull, CD56-/+CD16+HLADR+ cells [9, 13, 14], that have low cytolytic as well as cytokine producing capacity [10].
Previous observations in chimpanzees reported the dominant expression of CD8 on approximately 50% or more of the NK cells (defined as CD3 negative, CD16 positive lymphocytes) [27, 28] and characterized activatory NK cell receptors expressed on their surface [30]. In contrast to humans, chimpanzee NK cells cannot be easily defined by CD56 expression, but rather by subsets of CD3-CD8 expression. The consequences of low CD56 expression are at present still unclear and probably reflect a different regulation of this molecule. The observed up-regulation after IL-2 stimulation and our conformation with a second mAb make a simple lack of cross reactivity unlikely. We demonstrate that a high proportion of this specie’s CD8+ NK cells express functional triggering receptors (NKp46, NTB-A, NKp80, NKG2D). In contrast, there was consistently a much lower expression of triggering receptors in the CD8 negative subset.
In agreement with this high NCR expression, CD8+ NK cells also showed high granzyme B expression and upregulation of CD107 surface expression, following stimulation, which is indicative of lytic capacity. The same CD8+ subset was also capable of producing high levels of cytokines following stimulation. Importantly, human NK cells show different functional programs in the two main peripheral NK cell populations (cytotoxicity and cytokine production in CD56dimCD16+ and CD56brightCD16+/-, respectively) [7, 8]. The present work, indicates that these two programs (cytotoxicity and cytokine production) are both predominantly confined to CD8+CD16+ NK cells in chimpanzees, underscoring a difference in innate immune function in chimpanzees in addition to the patterns of NKp30 expression/function [30] and of NKp44 induction [33].
Recently six inhibitory receptors were described in chimpanzees, which recognized mutually exclusive C2 and C1 epitopes of MHC-I and induced specific killing of MHC transfected target cells [31]. We observed NK inhibitory receptors such as NKG2A or p58.1 (CD158a), to be expressed at low levels in this species on resting NK cells.
Molecular characterization and transfection experiments of the NKG2A receptor in
chimpanzees confirmed cross reactivity and specificity of mAbs recognizing the human homologue. Similarly, mAbs directed against human CD85e (p70) or CD158b (p58.2) displayed low-level reactivity. This finding may reflect low expression of NKp30 [30]
maintaining a balanced functionality of “resting” NK cells.
The fact that these inhibitory receptors were upregulated during in vitro culture of NK cells is somewhat reminiscent of our recent demonstration of NKp30 inducibility in this species [30]. The de novo KIR expression on activated NK cells could possibly serve to more tightly regulate their killing capacity and thus provide an important feed back mechanism for dampening the innate/adaptive immune responses in situations of persistent antigen (with decreased T and NK cell activation). This may also facilitate fine editing of adaptive immune responses through NK-DC interactions in situations of evolving antigens [34-36].
The small population of CD8-CD16+ NK cells was also characterized by a relatively high HLA-DR expression. Recently, in HIV-1 infected humans an HLA-DR positive subset was described with reduced expression of NCRs, which phenotypically may be most similar to these chimpanzee CD8-CD16+ NK cells [13, 14]. It is tempting to speculate that this minor chimpanzee CD8- subset, which also have reduced expression of CD107a, IFN and TNF upon stimulation with PMA/ionomycin as well as K562 target cells, resembles the so-called “exhausted” CD56-CD16+ NK cell subset that is greatly increased in HIV-1 infected humans. However, the fact that this subset is present to similar levels in both uninfected and infected chimpanzees rather reflects a constitutive difference of NK cell regulation in the two species. Indeed, while the absolute number of NK cells slightly diminished in HIV-infected chimpanzees, the relative composition of the CD8+ and CD8- subsets remained unaltered as did their pattern of activation and inhibitory marker expression. The data concur with earlier findings regarding preservation of NK cell mediated lytic capacity against K562 target cells in HIV infected chimpanzees [37].
In view of the increased mortality and AIDS like immunopathology that was recently reported in wild chimpanzees infected with the naturally acquired SIVcpz [38], it would be interesting to understand the impact that SIVcpz infection has on the NK cell populations in this species.
In conclusion, in depth analysis of chimpanzee NK cell phenotype and function reveals relevant differences in subset composition from human studies. The CD16+CD8- NKp46dullHLA-DR+ subset present in uninfected and in chronically infected chimpanzees
is likely a constitutive property of NK cell subset composition in this species, contrary to humans. More importantly, the functionally competent CD8+DR- NK cell subset remains intact with high NCR expression despite persistent lentiviral replication and may exemplify differences in NK cell homeostasis relevant for the resistance to activatory effects of HIV-1 on the immune system.
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