Tuning of NK-Specific HLA-C Expression by Alternative mRNA Splicing

Figures

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  Figure 1. Individuals with homozygous HLA-C alleles exhibit distinct patterns of alternative transcripts. (A) PCR amplification of cDNA from individuals homozygous for specific HLA-C alleles. NK cell cDNA from HLA-C homozygous donors (C*06/*06; C*03/*03; C*04/*04) was amplified with exon -1a forward and exon 8 reverse primers. The size of bands in the DNA marker lanes (kb) and the size range of major bands amplified from the cDNAs is indicated. HLA-C allele is indicated as (C*##) above each lane. (B) Sequencing analysis of PCR products. A summary of the exon structure of translatable mRNA isoforms detected is shown. All isoforms listed contain exons 1–8 comprising the complete open reading frame. The alternative 5′-UTR exons a, b, c, and 1 are numbered as in Li et al. (12). (C) Summary of the HLA-C alternative exons observed in this study. The relative genomic position, name, and size in bp is shown for each of the exons present in the NK-Pro transcripts listed in (B). The locations of the NK-Pro and the ATG initiation codon of the HLA-C gene are indicated.

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  Figure 2. Western blotting of constructs for HLA-C*03 and HLA-C*04 indicate variation in protein expression due to alternative splicing. HLA-C allele is indicated at left. The exons present in each splice variant are indicated as per the format a/b/c/1-. HLA-C band intensity was quantified and normalized against actin intensity utilizing ImageJ, shown at the right. The results shown are representative of three independent experiments performed.

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  Figure 3. Western blotting of HLA-C*03 constructs containing exon 1 splice forms of increasing length indicate inclusion of a long ORF reduces protein expression. (A) Schematic illustrating splice forms utilized for the Western blot panel. Exons are indicated in bold. ORF start and stop codons are indicated by black and red lines, respectively. ORF Length is shown as blue text. Kozak sequence strength is indicated by the red asterisks, where at least one Kozak sequence enhancing nucleotide is present. The red box indicates the ORF associated with lower expression of a1/b1/c3/15 and a3/16. (B) Western blot comparing HLA-C*03 constructs with increasing exon 1 length. Alternative exons present in each construct are indicated on the left. ImageJ normalized lane intensities are indicated on the right. The results shown are representative of three independent experiments performed.

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  Figure 4. HLA-C*06 is expressed at markedly higher levels than -C*03 and -C*04 alleles. The exon content of each HLA-C cDNA is listed and shown schematically on the left. The results of Western blotting of transfected JAR cells is shown in the panel on the right. ImageJ normalized lane intensities are indicated on the right. The results shown are representative of three independent experiments performed.

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  Figure 5. Western Blot comparing HLA-C*06 and HLA-C*12. The 5′-UTR structure of clones and a schematic representation of exon content for each structure is shown on the left. HLA-C allele is indicated as C*06 or C*12. The 5′-UTR is also indicated on the left, immediately adjacent to the Western blot. ImageJ actin normalized HLA-C intensity is indicated rightmost. The results shown are representative of three independent experiments performed.

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  Figure 6. HLA-C*03 and -C*06 expression is similar at low temperature. Growth temperature of JAR cells transfected with HLA-C expression constructs is indicated on the left. The transfected HLA-C*03 a1/b1/c2/1-3 cDNA is indicated as (C*03) and the a1/b2/c2/1-3 HLA-C*06 cDNA is indicated as (C*06), (–) represents cells transfected with a negative control vector. ImageJ actin-normalized HLA-C intensities are quantified and shown at right. Results are reflective of three independent experiments.

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  Figure 7. Expression levels of HLA-C alleles on peripheral blood NK cells are consistent with the expression level observed by Western blot analysis. The mean fluorescence intensity of HLA-C expression on peripheral blood NK cells is shown for donors that are homozygous for HLA-C*03 (*03/*03) or HLA-C*06 (*06/*06), and heterozygous individuals possessing HLA-C*03 and HLA-C*04 (*03/*04). Statistical significance (p-value < 0.05) is indicated by the asterisks (unpaired t-test). (A) HLA-C intensity for CD56bright NK cells. (B) HLA-C intensity for CD56dim NK cells. The *06/*06 individuals have a significantly higher level of HLA-C expression than either the *03/*03 or *03/*04 individuals (*03/*03 vs. *06/*06 p = 0.016, *03/*04 vs. *06/*06 p = 0.0146, unpaired t-test).