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. 2011 Apr 15;186(8):4744-50.
doi: 10.4049/jimmunol.1003254. Epub 2011 Mar 14.

Endogenous MHC-related protein 1 is transiently expressed on the plasma membrane in a conformation that activates mucosal-associated invariant T cells

Wei-Jen Chua  1 , Sojung Kim, Nancy Myers, Shouxiong Huang, Lawrence Yu, Daved H Fremont, Michael S Diamond, Ted H Hansen
Affiliations

Endogenous MHC-related protein 1 is transiently expressed on the plasma membrane in a conformation that activates mucosal-associated invariant T cells

Wei-Jen Chua et al. J Immunol. .

Abstract

The development of mucosal-associated invariant T (MAIT) cells is dependent upon the class Ib molecule MHC-related protein 1 (MR1), commensal bacteria, and a thymus. Furthermore, recent studies have implicated MR1 presentation to MAIT cells in bacteria recognition, although the mechanism remains undefined. Surprisingly, however, surface expression of MR1 has been difficult to detect serologically, despite ubiquitous detection of MR1 transcripts and intracellular protein. In this article, we define a unique mAb capable of stabilizing endogenous mouse MR1 at the cell surface, resulting in enhanced mouse MAIT cell activation. Our results demonstrated that under basal conditions, endogenous MR1 transiently visits the cell surface, thus reconciling the aforementioned serologic and functional studies. Furthermore, using this approach, double-positive thymocytes, macrophages, and dendritic cells were identified as potential APCs for MAIT cell development and activation. Based on this pattern of MR1 expression, it is intriguing to speculate that constitutive expression of MR1 may be detrimental for maintenance of immune homeostasis in the gut and/or detection of pathogenic bacteria in mucosal tissues.

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Conflict of interest statement

Disclosures

The authors have no financial conflicts of interest.

Figures

FIGURE 1
FIGURE 1
B cells express a low level of endogenous MR1 at the cell surface. Different B cell lineages were stained by anti-MR1 mAb 8F2.F9 and analyzed by flow cytometry. Surface staining (upper panels) of endogenous MR1 is low, whereas intracellular (total) staining (lower panels) of endogenous MR1 is abundant in pre B, myeloma, and B1 B cell lines. By contrast, the A20 cell line does not express endogenous MR1. In the graphs (representing one of five independent experiments), the solid line indicates MR1 staining and the dotted line indicates the background of PE–anti-mouse IgG1 secondary Ab.
FIGURE 2
FIGURE 2
MAIT cell hybridoma activation is blocked by mAb 8F2.F9 but enhanced by mAb 8H9.D11 in the coculture with B cells. The MAIT cell hybridoma 6C2 was cocultured with B cell lines in the absence or presence of anti-MR1 mAbs 8F2.F9 and 8H9.D11. The IL-2 production of MAIT cell hybridomas in the coculture with CH27.mMR1 (A) or various B cell lines (B) is shown for three independent experiments. Experiments were performed in triplicate for each group. Error bars represent SD.
FIGURE 3
FIGURE 3
Blocking and enhancing mAbs to MR1 do not compete for binding to MR1. CH27.mMR1 were stained with 8F2.F9-AF647 in the absence or presence of various unlabeled competing mAbs and analyzed by flow cytometry. 8F2.F9-AF647 staining (MFI) at different concentrations of competing mAbs is shown as the percentage of staining without a competing mAb group. Data represent the results from one of three independent experiments.
FIGURE 4
FIGURE 4
Epitope mapping revealed that the enhancing mAb recognizes a putative hinge region in the α2 domain of MR1. A, Anti-MR1 mAbs 8F2.F9 and 8H9.D11 were tested for recognition of WT3 cells expressing wild-type mMR1 or mutants with single amino acid substitutions (mouse to human residues in α1 and α2 domains of mMR1). Anti-MR1 mAb 12.2 was used as a staining control for each mMR1 construct. The staining of anti-MR1 mAbs was visualized by PE–anti-mouse Ig secondary Ab followed by flow cytometry analysis. The plot shows the mean MFI (± SD) from three independent experiments. B, An mMR1-threading model was described previously (7). The mouse residues that were mutated to human residues in α1 and α2 domains of MR1 are labeled. The epitope residues of the enhancing mAb are labeled in red.
FIGURE 5
FIGURE 5
The enhancing mAb traps MR1 at the cell surface. CH27. mMR1 were incubated with medium alone, mouse IgG1 isotype control, 8F2.F9, or 8H9.D11 for 2 h or overnight before staining with 8F2.F9-AF647 and analysis by flow cytometry. A, Staining of CH27.mMR1 from the different overnight incubations from one of three experiments. B, 8F2. F9-AF647 staining of CH27.mMR1 from the 2-h or overnight incubations is shown as the percentage of the staining of the medium control in A. The plot shows the mean percentage (± SD) from three independent experiments.
FIGURE 6
FIGURE 6
Thymocytes, macrophages, and DCs express endogenous MR1 and activate MAIT cell hybridomas in the presence of the enhancing mAb. Thymocytes from wild-type (WT) and MR1 KO mice were used as APCs in the coculture with the MAIT cell hybridoma 6C2 in the absence or presence of anti-MR1 mAb 8H9.D11. A, The IL-2 production of 6C2 was measured by ELISA. The plot represents data from one of three independent experiments that were performed using one mouse for each genotype and set up in triplicate. B, Thymocytes were stained for surface expression of CD4 and CD8, followed by intracellular staining of mAb 8F2.F9 for MR1. In the graph (left panel), the blue line indicates the staining in WT thymocytes; the black line indicates the background in MR1 KO thymocytes. Right panel, MR1+ WT thymocytes were mostly CD4+CD8+. FACS analysis represents staining results of one mouse for each genotype in one representative experiment. C, Total cells (RBCs lysed), macrophages, and DCs from the spleen of WT mice were used as APCs to activate 6C2 in the presence of mAb 8H9.D11 using the same method described in A.
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