Figure 1.
Peptide-dependent tetramer staining of primary NK cells and CD8+ T cells from an unimmunized, uninfected rhesus macaque.
(A) To identify the tetramer-positive cells, whole blood was stained with Mamu-A1*00201 Gag71-79 GY9 followed by monoclonal antibodies to the indicated cell type-specific markers. (B) The frequency of tetramer-positive CD8+ T cells versus CD16+ NK cells was determined by gating sequentially on CD8 followed by CD3 (I) or CD16 (II). (C) To determine if tetramer staining is dependent on the peptide bound by Mamu-A1*00201, whole blood was stained with Mamu-A1*00201 tetramers folded with eight different SIV peptides, Gag71-79 GY9 (GSENLKSLY), Env788-795 RY8 (RTLLSRVY), Env317-325 KM9 (KTVLPVTIM), Nef248-256 LM9 (LTARGLLNM), Nef159-167 YY9 (YTSGPGIRY), Env296-304 RY9 (RTIISLNKY), Vif97-104 WY8 (WTDVTPNY), and Vif89-97 IW9 (ITWYSKNFW), followed by monoclonal antibodies to CD3, CD8 and CD16. After gating on CD8+ lymphocytes, the percentages of tetramer-positive CD3– versus CD3+ cells were determined.
Figure 2.
Amino acid sequence alignments for KIR alleles cloned from a rhesus macaque with a tetramer-positive NK cell population in peripheral blood.
The predicted amino acid sequences are shown for six Mamu-KIR3DL alleles (A), three Mamu-KIR3DS alleles (B), and two Mamu-KIR2DL04 alleles (C). Positions of amino acid identity with the consensus sequence shown at the top are indicated by a period and translational stop sites are indicated with an asterisk.
Figure 3.
Mamu-A1*00201 is a ligand for Mamu-KIR3DL05.
Jurkat cells were transfected with constructs expressing each of the six Mamu-KIR3DL (A), three Mamu-KIR3DS (B), and two Mamu-KIR2DL04 (C) alleles cloned from Mm 337-07, and stained with Gag71-79 GY9 and Nef159-167 YY9 tetramers. The KIRs were expressed from a bicistronic vector designed to introduce a common leader peptide followed by an HA tag at the N-terminus of the D0 domain, and to co-express eGFP from a downstream internal ribosomal entry site. Jurkat cells were electroporated with the KIR expression constructs and stained the following day with APC-conjugated Mamu-A1*00201 tetramers, followed by a PE-conjugated antibody to the HA tag. Tetramer versus HA staining was determined after gating on the eGFP+ cell population. Quadrant gates were set using empty vector-transfected controls stained with tetramer and antibody to the HA tag.
Figure 4.
Polymorphisms in the D0 and D1 domains of Mamu-KIR3DL05 modulate the avidity and peptide-selectivity of binding to Mamu-A1*00201.
(A) Jurkat cells were transfected with HA-tagged Mamu-KIR3DL05 expression constructs, and stained the next day with APC-conjugated Mamu-A1*00201 tetramers (Gag71-79 GY9, Env788-795 RY8, Nef159-167 YY9 or Vif89-97 IW9), followed by a PE-conjugated antibody to the HA tag. Quadrant gates were set using empty vector-transfected controls stained with tetramer and antibody to the HA tag. (B) An alignment comparing the predicted amino acid sequences of the D0, D1 and D2 domains for seven different Mamu-KIR3DL05 alleles. Positions of amino acid identity with the consensus sequence are indicated by a period. The shaded regions correspond to loops predicted to contact surfaces of the peptide-MHC class I complex [37]. The plus signs beneath the alignment indicate unique residues in the D1 domain of mmKIR3DL05x that coincide with, or are immediately adjacent to, predicted MHC class I-contact loops.
Table 1.
Relative binding of Mamu-A1*00201 tetramers folded with four different SIV peptides to seven allotypes of Mamu-KIR3DL05.
Figure 5.
Amino acid differences in the third MHC class I-contact loop of the D1 domain account for the preferential binding of mmKIR3DL05x to the Nef159-167 YY9 tetramer.
(A) Positions in the D1 domain of mmKIR3DL05x that differ from Mamu-KIR3DL05*008 are highlighted in a three-dimensional model of KIR3DL*015 bound to HLA-A*2402 [37]. The residues indicated in yellow are located in surface-exposed loops in close proximity to the bound peptide. The residues indicated in red represent differences in the D1 domain at sites that do not contribute directly to interactions with MHC class I ligands. The residues highlighted in magenta represent positions 77-83 of the α1 domain corresponding to the Bw6 of Mamu-A1*00201. (B) Jurkat cells were electroporated with constructs expressing recombinants of Mamu-KIR3DL05*008 and mmKIR3DL05x, for which residues of the second (L2) and third (L3) predicted MHC class I-contact loops in D1 were exchanged, and stained with APC-conjugated tetramer (Gag71-79 GY9 or Nef159-167 YY9) followed by a PE-conjugated antibody to the HA tag. Tetramer versus HA staining was determined after gating on the eGFP+ cell population and quadrant gates were set using control cells transfected with an empty vector.
Figure 6.
Mamu-A1*00201+ target cells suppress the degranulation of tetramer-positive NK cells.
(A) Primers specific for exon 5 of Mamu-KIR3DL05 were used to amplify a 156 bp sequence from genomic DNA. Primers specific for a conserved 300 bp region of Mamu-DRB were included as an internal control. PCR products were separated on a 1% agarose gel containing ethidium bromide and visualized by UV transillumination. (B) Peripheral blood from two Mamu-KIR3DL05− and eight Mamu-KIR3DL05+ rhesus macaques was stained with the Gag71-79 GY9 and monoclonal antibodies to CD3, CD8 and CD16. The percentages of tetramer-positive cells were determined for CD3-CD8+ versus CD3+CD8+ lymphocytes. Of the eight Mamu-KIR3DL05+ animals, four were Mamu-A1*00201+ (Mm 177-05, Mm 350-04, Mm R02020 and Mm R95117) and four were Mamu-A1*00201- (Mm 20-05, Mm 376-04, Mm RHAX18 and Mm R03035). With the exception of Mm AP78 and Mm AD73INF08, which were negative for Mamu-KIR3DL05, tetramer-positive NK cells were detected in peripheral blood for each of these animals. Mm AP78, Mm AD73INF08, Mm 177-05, and Mm RHAX18 were uninfected at the time of this analysis. Mm R02020 and Mm R95117 were infected with SIVsmmE660. Mm 350-04 and 376-04 were infected with SIVmac239Δnef. Mm 20-05 and Mm R03035 were infected with SIVmac239. The SIV-infected animals are indicated with an asterisk. (C) Freshly isolated PBMC from two Mamu-A1*00201+ and two Mamu-A1*00201- macaques were stimulated with parental 721.221 cells, or 721.221 cells expressing individual rhesus macaque MHC class I molecules. Mm 337-07 was uninfected at the time of this analysis. The cells were incubated overnight at a 5:1 PBMC to target cell ratio in the presence of a monoclonal antibody to CD107a. Following stimulation, the cells were stained with the Gag71-79 GY9 tetramer and antibodies to CD3, CD8, CD16 and NKG2A. After gating on CD3-NK2GA+ lymphocytes, the upregulation of CD107a on tetramer-positive versus tetramer-negative NK cells was determined.