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. 1999 Apr 19;189(8):1265-74.
doi: 10.1084/jem.189.8.1265.

The transmembrane sequence of human histocompatibility leukocyte antigen (HLA)-C as a determinant in inhibition of a subset of natural killer cells

D M Davis  1 , O Mandelboim, I Luque, E Baba, J Boyson, J L Strominger
Affiliations

The transmembrane sequence of human histocompatibility leukocyte antigen (HLA)-C as a determinant in inhibition of a subset of natural killer cells

D M Davis et al. J Exp Med. .

Abstract

Molecular interactions with the extracellular domains of class I major histocompatibility complex proteins are major determinants of immune recognition that have been extensively studied both physically and biochemically. However, no immunological function has yet been placed on the transmembrane or cytoplasmic amino acid sequences of these proteins despite strict conservation of unique features within each class I major histocompatibility complex locus. Here we report that lysis by a subset of natural killer (NK) cells inhibited by target cell expression of human histocompatibility leukocyte antigen (HLA)-Cw6 or -Cw7 was not inhibited by expression of chimeric proteins consisting of the extracellular domains of HLA-C and the COOH-terminal portion of HLA-G. Assays using transfectants expressing a variety of HLA-Cw6 mutants identified the transmembrane sequence and, in particular, cysteine at position 309 as necessary for inhibition of 68% (25/37) of NK cell lines and 23% (33/145) of NK clones tested. Moreover, these NK clones inhibited by target cell expression of HLA-Cw6 and dependent upon the transmembrane sequence were found not to express or to only dimly express NK inhibitory receptors (NKIR1) that are EB6/HP3E4-positive. Furthermore, assays using monoclonal antibody blocking suggest that an NK receptor other than NKIR1 or CD94 is responsible for recognition dependent upon the transmembrane sequence of HLA-Cw6.

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Figures

Figure 1
Figure 1
Alignment of amino acid sequences of HLA-Cw6, -Cw7, -G, -A2, and the chimera HLA-Cw6/TM G used in the current study. The location of the Bsu36I restriction site used to make the chimeras HLA-Cw6/G end and HLA-Cw7/G end is marked. Residues underlined were those mutated into stop codons in HLA-Cw6, and the cysteine at position 309 that was mutated into tryptophan is marked bold and underlined. The region of HLA-Cw6 altered to make HLA-Cw6/TM G is shaded.
Figure 2
Figure 2
The COOH-terminal portion of HLA-C is a determinant for inhibition of NK cell cytotoxicity. NK cell lines 986 (A) and I (B), selected by their susceptibility to inhibition by the expression of HLA-C proteins in 721.221 cells, were assayed for their capacity to be inhibited by expression of HLA-Cw6/G end and HLA-Cw7/G end at various E/T cell ratios. The data set shown is representative of three independent experiments using two or three independently transfected target cell lines for each class I MHC protein variant, except HLA-Cw7 and -G, for which only one transfectant was used.
Figure 3
Figure 3
The cytoplasmic tail of HLA-Cw6 is not a determinant in inhibition of NK cell–mediated lysis. NK cell lines 268 (A) and 264 (B), selected by their susceptibility to inhibition by target cell expression of HLA-Cw6, were assayed for their capacity to be inhibited by target cell expression of HLA-Cw6 with truncations placed along various positions in the cytoplasmic tail. Assays were performed at various E/T cell ratios shown. Data sets shown are representative of three independent experiments for each NK line. Another independently made transfectant for HLA-Cw6/315 stop behaved similarly. A transfectant expressing HLA-Cw6 with a stop codon placed at position 332 behaved similarly to other truncated -Cw6 transfectants.
Figure 4
Figure 4
The transmembrane sequence, and particularly cysteine at position 309, controls the capacity of HLA-Cw6 to inhibit lysis by some NK cell lines. Various arbitrarily numbered NK lines selected by their susceptibility to inhibition by target cell expression of HLA-Cw6 were assayed for their capacity to be inhibited by target cell expression of HLA-Cw6 with the transmembrane sequence replaced by that of HLA-G (HLA-Cw6/TM G) or the cysteine at position 309 replaced by tryptophan (HLA-Cw6/C309W). Lysis was assayed at the E/T ratio of ∼3:1, and at least two independent experiments were performed for each NK line. Two other independently made transfectants of HLA-Cw6/TM G gave similar results. The transfectant expressing HLA-Cw6/G end behaved similarly to that expressing -Cw6/TM G or -Cw6/C309W with all NK lines tested (not shown).
Figure 5
Figure 5
NK clones inhibited by target cell expression of HLA-Cw6 differ in their sensitivity to the transmembrane sequence of the protective class I MHC protein. NK clone 4 (A), NK clone 2 (B), and NK clone 82 (C), selected by their susceptibility to be inhibited by target cell expression of HLA-Cw6, were assayed for lysis of HLA-Cw6/TM G and -Cw6/C309W transfectants at the various E/T cell ratios shown. Data sets shown are representative of two independent experiments performed. Another independently made HLA-Cw6/TM G transfectant gave similar results (not shown). The transfectant expressing HLA-Cw6/G end behaved similarly to that expressing -Cw6/TM G or -Cw6/C309W (not shown).
Figure 6
Figure 6
Staining NK clones with mAbs to NK receptors reveals distinct phenotypes between clones that are sensitive to the transmembrane sequence of HLA-Cw6 and those that are not. Histograms of fluorescence intensity of NK clones stained with EB6 (thick gray line), HP3E4 (dotted line), and no first antibody (thin black line), followed by FITC- labeled goat anti–mouse Ig secondary antibody.
Figure 7
Figure 7
NK clones inhibited by target cell expression of HLA-Cw6 differing in their sensitivity to the transmembrane sequence of the protective class I MHC protein also differ in their sensitivity to blocking by mAb HP3E4. NK clone 15 (A) and NK clone X1 (B), selected by their susceptibility to inhibition by target cell expression of HLA-Cw6, were assayed for lysis of HLA-Cw6/TM G and -Cw6/C309W transfectants at the E/T cell ratio of 3:1 in the presence and absence of mAb HP3E4 (anti-NKIR1), as ascites diluted 1:1,000, and 20 μg/ml anti-CD94 mAb. Isotype control mAb TEPC183 (IgM ascites control for HP3E4) and GL183 (IgG1 control for CD94) had no effect on the NK cell lysis of any of the target transfectants (not shown). Data sets shown are representative of two independent experiments performed. Several other NK lines and clones behaved similarly to those shown. Inhibition of a minor subset of NK lines and clones by target cell expression of HLA-Cw6 could be blocked by anti-CD94 mAb with no correlation to whether or not each particular NK line or clone could be inhibited by target cell expression of HLA-Cw6/TM G or -Cw6/C309W (not shown).
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