Yik Y. L. Yu

Human Mucosal Associated Invariant T Cells Detect Bacterially Infected Cells

A first indication of the biological role of mucosal associated invariant T (MAIT) cells reveals that this discrete T cell subset is broadly reactive to bacterial infection. In particular MAIT cells recognize Mycobacterium tuberculosis-infected lung airway epithelial cells via the most evolutionarily conserved major histocompatibility molecule.

MR1-restricted MAIT cells display ligand discrimination and pathogen selectivity through distinct T cell receptor usage

MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, and the TCR repertoire is distinct within individuals, indicating that the MAIT cell repertoire is shaped by prior microbial exposure.

Cutting Edge: Single-Chain Trimers of MHC Class I Molecules Form Stable Structures That Potently Stimulate Antigen-Specific T Cells and B Cells

We report in this work the expression and characterization of class I molecules expressed as single-chain trimers consisting of an antigenic peptide-spacer-β2-microglobulin-spacer H chain. Our results indicate that these single-chain constructs assemble efficiently, maintain their covalent structure, and are unusually stable at the cell surface. Consequently, these constructs are at least 1000-fold less accessible to exogenous peptide than class I molecules loaded with endogenous peptides, and they are potent simulators of peptide-specific CTL and Abs. Our combined findings suggest that single-chain trimers may have applications as DNA vaccines against virus infection or tumors.

Biochemical Features of the MHC-Related Protein 1 Consistent with an Immunological Function

MHC-related protein (MR)1 is an MHC class I-related molecule encoded on chromosome 1 that is highly conserved among mammals and is more closely related to classical class I molecules than are other nonclassical class I family members. In this report, we show for the first time that both mouse and human MR1 molecules can associate with the peptide-loading complex and can be detected at low levels at the surface of transfected cells. We also report the production of recombinant human MR1 molecules in insect cells using highly supplemented media and provide evidence that the MR1 H chain can assume a folded conformation and is stoichiometrically associated with β2-microglobulin, similar to class I molecules. Cumulatively, these findings demonstrate that surface expression of MR1 is possible but may be limited by a specific ligand or associated molecule.

Allorecognition by murine natural killer cells: lysis of T-lymphoblasts and rejection of bone-marrow grafts

Summary: Natural killer (NK) cells of inbred mice reject allogeneic bone‐marrow cells, and NK cells of F1 hybrid mice can reject parental bone‐marrow cells (hybrid resistance). In some cases these patterns of rejection can be mimicked in vitro by utilizing IL‐2 cultured NK effector cells and allogeneic or parental T‐lymphoblasts as target cells. Lysis of allogeneic parental targets in vitro can be explained on the basis of the missing self hypothesis. Subsets of NK cells that bear non‐overlapping MHC class I inhibitory receptors belonging to the Ly49 family lyse allogeneic targets because they do not express self class I molectiles of the NK cell donor. Parental strain targets are lysed because they do not express all of the self class I antigens of the Fl hybrid, and hence fail to deliver inhibitory signals to all subsets of Fl NK cells. The expression of Ly49 receptors on NK cells is regulated by liost MHC to ensure maximal sensitivity to alterations in self class I molecules and to prevent autoreactivity. In many instances, however, the rejection of allogeneic bone marrow cells in vivo cannot be readily explained by the missing self hypothesis. In these instances, it appears that rejection is initiated by class 1 MHC receptors on NK ceils Out recognize allogeneic class I molecules as non‐self, and activate rather than inhibit NK cell function.

Kb, Kd, and Ld Molecules Share Common Tapasin Dependencies as Determined Using a Novel Epitope Tag

The endoplasmic reticulum protein tapasin is considered to be a class I-dedicated chaperone because it facilitates peptide loading by proposed mechanisms such as peptide editing, endoplasmic reticulum retention of nonpeptide-bound molecules, and/or localizing class I near the peptide source. Nonetheless, the primary functions of tapasin remain controversial as do the relative dependencies of different class I molecules on tapasin for optimal peptide loading and surface expression. Tapasin dependencies have been addressed in previous studies by transfecting different class I alleles into tapasin-deficient LCL721.220 cells and then monitoring surface expression and Ag presentation to T cells. Indeed, by these criteria, class I alleles have disparate tapasin-dependencies. In this study, we report a novel and more direct method of comparing tapasin dependency by monitoring the ratio of folded vs open forms of the different mouse class I heavy chains, Ld, Kd, and Kb. Furthermore, we determine the amount of de novo heavy chain synthesis required to attain comparable expression in the presence vs absence of tapasin. Our findings show that tapasin dramatically improves peptide loading of all three of these mouse molecules.

Role of murine NK cells and their receptors in hybrid resistance

Hybrid resistance refers to the rejection of parental strain bone marrow cells by natural killer cells of mice that are F1 hybrids derived from two inbred parental strains. This pattern of rejection is not seen in solid organ transplants. Progress in understanding this exception to the laws of transplantation genetics has occurred with the recent discovery of negative signaling receptors for MHC class I molecules. In the last year the discovery of natural killer cell subsets with non-overlapping inhibitory receptors for parental class I molecules has provided an explanation for hybrid resistance. In some instances, however, positive rather than negative signaling seems to be the basis for rejection of allogeneic as well as parental marrow cell grafts.

Association of ERp57 with Mouse MHC Class I Molecules Is Tapasin Dependent and Mimics That of Calreticulin and not Calnexin

Before peptide binding in the endoplasmic reticulum, the class I heavy (H) chain-β2-microglobulin complexes are detected in association with TAP and two chaperones, TPN and CRT. Recent studies have shown that the thiol-dependent reductase, ERp57, is also present in this peptide-loading complex. However, it remains controversial whether the association of ERp57 with MHC class I molecules precedes their combined association with the peptide-loading complex or whether ERp57 only associates with class I molecules in the presence of TPN. Resolution of this controversy could help determine the role of ERp57 in class I folding and/or assembly. To define the mouse class I H chain structures involved in interaction with ERp57, we tested chaperone association of Ld mutations at residues 134 and 227/229 (previously implicated in TAP association), residues 86/88 (which ablate an N-linked glycan), and residue 101 (which disrupts a disulfide bond). The association of ERp57 with each of these mutant H chains showed a complete concordance with CRT, TAP, and TPN but not with calnexin. Furthermore, ERp57 failed to associate with H chain in TPN-deficient .220 cells. These combined data demonstrate that, during the assembly of the peptide-loading complex, the association of ERp57 with mouse class I is TPN dependent and parallels that of CRT and not calnexin.

Tapasin enhances peptide-induced expression of H2-M3 molecules, but is not required for the retention of open conformers

H2-M3 is a class Ib MHC molecule that binds a highly restricted pool of peptides, resulting in its intracellular retention under normal conditions. However, addition of exogenous M3 ligands induces its escape from the endoplasmic reticulum (ER) and, ultimately, its expression at the cell surface. These features of M3 make it a powerful and novel model system to study the potentially interrelated functions of the ER-resident class I chaperone tapasin. The functions ascribed to tapasin include: 1) ER retention of peptide-empty class I molecules, 2) TAP stabilization resulting in increased peptide transport, 3) direct facilitation of peptide binding by class I, and 4) peptide editing. We report in this study that M3 is associated with the peptide-loading complex and that incubation of live cells with M3 ligands dramatically decreased this association. Furthermore, high levels of open conformers of M3 were efficiently retained intracellularly in tapasin-deficient cells, and addition of exogenous M3 ligands resulted in substantial surface induction that was enhanced by coexpression of either membrane-bound or soluble tapasin. Thus, in the case of M3, tapasin directly facilitates intracellular peptide binding, but is not required for intracellular retention of open conformers. As an alternative approach to define unique aspects of M3 biosynthesis, M3 was expressed in human cell lines that lack an M3 ortholog, but support expression of murine class Ia molecules. Unexpectedly, peptide-induced surface expression of M3 was observed in only one of two cell lines. These results demonstrate that M3 expression is dependent on a unique factor compared with class Ia molecules.

Induction of hematopoietic and endothelial cell program orchestrated by ETS transcription factor ER71/ETV2.

The ETS factor ETV2 (aka ER71) is essential for the generation of the blood and vascular system, as ETV2 deficiency leads to a complete block in blood and endothelial cell formation and embryonic lethality in the mouse. However, the ETV2‐mediated gene regulatory network and signaling governing hematopoietic and endothelial cell development are poorly understood. Here, we map ETV2 global binding sites and carry out in vitro differentiation of embryonic stem cells, and germ line and conditional knockout mouse studies to uncover mechanisms involved in the hemangiogenic fate commitment from mesoderm. We show that ETV2 binds to enhancers that specify hematopoietic and endothelial cell lineages. We find that the hemangiogenic progenitor population in the developing embryo can be identified as FLK1highPDGFRα−. Notably, these hemangiogenic progenitors are exclusively sensitive to ETV2‐dependent FLK1 signaling. Importantly, ETV2 turns on other Ets genes, thereby establishing an ETS hierarchy. Consequently, the hematopoietic and endothelial cell program initiated by ETV2 is maintained partly by other ETS factors through an ETS switching mechanism. These findings highlight the critical role that transient ETV2 expression plays in the regulation of hematopoietic and endothelial cell lineage specification and stability.