Expression of the mouse MHC class Ib H2-T11 gene product, a paralog of H2-T23 (Qa-1) with shared peptide-binding specificity

Abstract

The mouse MHC class Ib gene H2-T11 is 95% identical at the DNA level to H2-T23, which encodes Qa-1, one of the most studied MHC class Ib molecules. H2-T11 mRNA was observed to be expressed widely in tissues of C57BL/6 mice, with the highest levels in thymus. To circumvent the availability of a specific mAb, cells were transduced with cDNA encoding T11 with a substituted α3 domain. Hybrid T11D3 protein was expressed at high levels similar to control T23D3 molecules on the surface of both TAP(+) and TAP(-) cells. Soluble T11D3 was generated by folding in vitro with Qa-1 determinant modifier, the dominant peptide presented by Qa-1. The circular dichroism spectrum of this protein was similar to that of other MHC class I molecules, and it was observed to bind labeled Qa-1 determinant modifier peptide with rapid kinetics. By contrast to the Qa-1 control, T11 tetramers did not react with cells expressing CD94/NKG2A, supporting the conclusion that T11 cannot replace Qa-1 as a ligand for NK cell inhibitory receptors. T11 also failed to substitute for Qa-1 in the presentation of insulin to a Qa-1-restricted T cell hybridoma. Despite divergent function, T11 was observed to share peptide-loading specificity with Qa-1. Direct analysis by tandem mass spectrometry of peptides eluted from T11D3 and T23D3 isolated from Hela cells demonstrated a diversity of peptides with a clear motif that was shared between the two molecules. Thus, T11 is a paralog of T23 encoding an MHC class Ib molecule that shares peptide-binding specificity with Qa-1 but differs in function.

Fig. 1

Alignment of amino acid sequences of Qa-1^b, Qa-1^a and T11 ectodomains. The amino acid sequences of Qa-1^b, Qa-1^a and T11 ectodomains were aligned, using CLUSTALW, SDSC Biology WorkBench 3.2 (http://workbench.sdsc.edu). Alpha 1, 2 and 3 domains are boxed. Consensus key: * fully conserved;: strongly conserved; weakly conserved.

Fig. 2

Expression of T11 gene. (A) PCR amplification of T11 and T23 genes from the wild type C57BL/6 (B6) and Qa-1^b−/− (KO) mouse genomic DNA. The T11 amplicon size was 628bp and the T23 amplicon size was 677bp. (B) Reverse-transcriptional PCR amplification of T11 and T23 transcripts from B6 mouse spleen and thymus. The amplicon sizes are 415 bp and 438 bp for T11 and T23 respectively. RT: reverse transcriptase. (C) Quantitative-PCR to determine the T11 transcription level in different tissues. The total RNA was extracted from Qa-1^b−/− mice. The reference gene was RNA polymerase 2A (POLR2A). The ratio of T11 to POLR2A was calculated according to the Pfaffl method. The qPCR was done in triplicates (n=3) and the experiment was repeated twice with similar results. One of them was shown. (D) Quantitative-PCR to determine the T11 transcription level in different cell subpopulations of thymus and spleen. The total RNA was extracted from thymus and spleen of Qa-1^b−/− mice. The qPCR was done as above, at least in triplicates. (E) Qa-1/T23 and T11 expression after stimulation. Splenocytes of B6 or Qa-1^b−/− mice were stimulated with plate-bound anti-CD3/28 Ab. Surface expression of Qa-1 on B6 splenocytes was determined by FACS. The total RNA was extracted from unstimulated or stimulated B6 or Qa-1^b−/− splenocytes. The qPCR for T23 used RNA from B6 or for T11 used RNA from Qa-1^b−/− splenocytes, respectively. The qPCR was done as same as above, in triplicates (n= 3) and the experiment was repeated twice with similar results. One of surface staining of Qa-1was shown.

Fig. 3

Expression of hybrid T11D3 and T23D3 molecules and test of the function of hybrid molecule expressing cells as Ag presenting cells. (A) FACS analysis of hybrid T11D3 and T23D3 on the surface of Hela and T2 cells. Transduced Hela cells were stained with 28-14-8s (α-D^b α3) and T2 cells were stained with 28-14-8s and 6A8 (α-Qa-1^b α2), respectively, shown as marked (red lines). The staining of isotype control Ab is blue. (B) Antigen presentation assay to test capability of the hybrid MHC-Ib molecule expressing cells to present insulin to 6C5 T hybridoma cells specific to bovine insulin (bINS). The assay was set up in triplicates (n=3) and the experiment was repeated twice with similar results. One of them was shown.

Fig. 4

In vitro folding of the hybrid T11D3 and T23D3 molecules. (A) S300 spectrum of the folding products. Arrows indicate the correct folding product peak. Top panel: black line, T23D3-β2m folding, gray line, T23D3-β2m-Qdm folding; bottom panel: T11D3-β2m folding, black line, T23D3-β2m folding, gray line, T11D3-β2m-Qdm folding. (B) SDS-PAGE analysis of the purified folding products. The MHC heavy chain is ~33kD and the β2m light chain is −14kD. (C) Eu-based immunoassay to examine the folding products. Folding MHC monomer was captured by the anti-β2m mAb and detected by biotinylated 28-14-8s and 6A8 (b-28-14-8s and b-6A8). The assay was set up in triplicates (n=3) and the experiment was repeated twice with similar results. One of them was shown. (D) In vitro folded MHC monomers were analyzed using far-UV circular dichroism. Each spectrum curve was the average of three independent scans, and a representative one was shown. (E) Thermal denaturation curves were generated from CD signals recorded at 222nm and the temperature was increased at 2°C interval from 25°C to 79°C. Each curve presented the average of three independent experiments.

Fig. 5

Qdm-binding capability of T11D3. (A) Eu-based immunoassay to test the ability of folded T11 and T23 monomers of binding Qdm peptide. Folded MHC Ib monomers were captured on plates by the coated the anti-b2m mAb and incubated with the biotin-labeled peptides at room temperature overnight. The assay was set up in triplicates (n=3) and the experiment was repeated twice with similar results. One of them was shown. (B) and (C) Fluorescence polarization (FP) assay. Folded MHC Ib monomers were incubated with Alexfluro488 labeled Qdm peptides and the FP signal was recorded every 60 seconds at 37°C or 25°C, respectively. The experiments were repeated twice (37°C) and three times (25°C) with similar results. One of each was shown.

Fig. 6

MHC Ib-Qdm tetramer staining of lymphocytes. B6 spleen and liver lymphocytes were stained with surface markers for B cells (B220), T cells (CD3ε), NK cells (NKp46) and the hybrid T23D3-Qdm or T11D3-Qdm tetramers. APC labeled streptavidin was used as a negative control. The lymphocytes were gated out for FACS analysis. The staining was repeated twice with similar results and one of each was shown.

Fig. 7

Peptide elution from Hela-T11D3 and Hela-T23D3 cells. (A) Length distribution of the peptides eluted from Hela-T11D3 and Hela-T23D3 cells. (B) The unique peptides from the T23 eluted and T11 eluted peptide pool were analyzed. Numbers of 8mer, 9mer and 10mer peptides were shown in the Venn diagram. (C) Sequence logo of the total eluted unique peptides from T23 and (D) T11. Each column represents one amino acid position in the peptide. Amino acids with different properties were labeled with different colors.