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Mucosal-associated invariant T (MAIT) cells express a semi-invariant T cell receptor (TCR) that detects microbial metabolites presented by the nonpolymorphic major histocompatibility complex (MHC)-like molecule MR1. The highly conserved nature of MR1 in conjunction with biased MAIT TCRα chain usage is widely thought to indicate limited ligand presentation and discrimination within a pattern-like recognition system. Here, we evaluated the TCR repertoire of MAIT cells responsive to three classes of microbes. Substantial diversity and heterogeneity were apparent across the functional MAIT cell repertoire as a whole, especially for TCRβ chain sequences. Moreover, different pathogen-specific responses were characterized by distinct TCR usage, both between and within individuals, suggesting that MAIT cell adaptation was a direct consequence of exposure to various exogenous MR1-restricted epitopes. In line with this interpretation, MAIT cell clones with distinct TCRs responded differentially to a riboflavin metabolite. These results suggest that MAIT cells can discriminate between pathogen-derived ligands in a clonotype-dependent manner, providing a basis for adaptive memory via recruitment of specific repertoires shaped by microbial exposure., (© 2014 Gold et al.)

Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) α-chain, TRAV1-2-TRAJ33, and are activated by vitamin B metabolites bound by the major histocompatibility complex (MHC)-related class I-like molecule, MR1. Understanding MAIT cell biology has been restrained by the lack of reagents to specifically identify and characterize these cells. Furthermore, the use of surrogate markers may misrepresent the MAIT cell population. We show that modified human MR1 tetramers loaded with the potent MAIT cell ligand, reduced 6-hydroxymethyl-8-D-ribityllumazine (rRL-6-CH₂OH), specifically detect all human MAIT cells. Tetramer(+) MAIT subsets were predominantly CD8(+) or CD4(-)CD8(-), although a small subset of CD4(+) MAIT cells was also detected. Notably, most human CD8(+) MAIT cells were CD8α(+)CD8β(-/lo), implying predominant expression of CD8αα homodimers. Tetramer-sorted MAIT cells displayed a T(H)1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1-rRL-6-CH₂OH tetramers detected CD4(+), CD4(-)CD8(-) and CD8(+) MAIT cells in Vα19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-β repertoire, and although the majority of human MAIT cells expressed TRAV1-2-TRAJ33, some expressed TRAJ12 or TRAJ20 genes in conjunction with TRAV1-2. Accordingly, MR1 tetramers allow precise phenotypic characterization of human and mouse MAIT cells and revealed unanticipated TCR heterogeneity in this population.

Mucosal-associated invariant T (MAIT) cells express a semiinvariant αβ T cell receptor (TCR) that binds MHC class I-like molecule (MR1). However, the molecular basis for MAIT TCR recognition by MR1 is unknown. In this study, we present the crystal structure of a human Vα7.2Jα33-Vβ2 MAIT TCR. Mutagenesis revealed highly conserved requirements for the MAIT TCR-MR1 interaction across different human MAIT TCRs stimulated by distinct microbial sources. Individual residues within the MAIT TCR β chain were dispensable for the interaction with MR1, whereas the invariant MAIT TCR α chain controlled specificity through a small number of residues, which are conserved across species and located within the Vα-Jα regions. Mutagenesis of MR1 showed that only two residues, which were centrally positioned and on opposing sides of the antigen-binding cleft of MR1, were essential for MAIT cell activation. The mutagenesis data are consistent with a centrally located MAIT TCR-MR1 docking that was dominated by the α chain of the MAIT TCR. This candidate docking mode contrasts with that of the NKT TCR-CD1d-antigen interaction, in which both the α and β chain of the NKT TCR is required for ligation above the F'-pocket of CD1d.

Ubiquitin (Ub) modification of proteins plays a prominent role in the regulation of multiple cell processes, including endoplasmic reticulum-associated degradation (ERAD). Until recently, ubiquitination of substrates was thought to occur only via isopeptide bonds, typically to lysine residues. Several recent studies suggest that Ub can also be coupled to nonlysine residues by ester/thiolester bonds; however, the molecular basis for these novel modifications remains elusive. To probe the mechanism and importance of nonlysine ubiquitination, we have studied the viral ligase murine K3 (mK3), which facilitates the polyubiquitination of hydroxylated amino acids serine/threonine on its ERAD substrate. In this paper, we identify Ube2j2 as the primary cellular E2 recruited by the mK3 ligase, and this E2-E3 pair is capable of conjugating Ub on lysine or serine residues of substrates. However, surprisingly, Ube2j2-mK3 preferentially promotes ubiquitination of hydroxylated amino acids via ester bonds even when lysine residues are present on wild-type substrates, thus establishing physiological relevance of this novel ubiquitination strategy.

Like CD1d-restricted iNKT cells, mucosal-associated invariant T cells (MAITs) are "innate" T cells that express a canonical TCRalpha chain, have a memory phenotype, and rapidly secrete cytokines upon TCR ligation. Unlike iNKT cells, MAIT cells require the class Ib molecule MHC-related protein I (MR1), B cells, and gut flora for development and/or expansion, and they preferentially reside in the gut lamina propria. Evidence strongly suggests that MAIT cell activation is ligand-dependent, but the nature of MR1 ligand is unknown. In this study, we define a mechanism of endogenous antigen presentation by MR1 to MAIT cells. MAIT cell activation was dependent neither on a proteasome-processed ligand nor on the chaperoning by the MHC class I peptide loading complex. However, MAIT cell activation was enhanced by overexpression of MHC class II chaperones Ii and DM and was strikingly diminished by silencing endogenous Ii. Furthermore, inhibiting the acidification of the endocytic compartments reduced MR1 surface expression and ablated MAIT cell activation. The importance of the late endosome for MR1 antigen presentation was further corroborated by the localization of MR1 molecules in the multivesicular endosomes. These findings demonstrate that MR1 traffics through endocytic compartments, thereby allowing MAIT cells to sample both endocytosed and endogenous antigens.

The mechanism by which substrates for endoplasmic reticulum-associated degradation are retrotranslocated to the cytosol remains largely unknown, although ubiquitination is known to play a key role. The mouse gamma-herpesvirus protein mK3 is a viral RING-CH-type E3 ligase that specifically targets nascent major histocompatibility complex I heavy chain (HC) for degradation, thus blocking the immune detection of virus-infected cells. To address the question of how HC is retrotranslocated and what role mK3 ligase plays in this action, we investigated ubiquitin conjugation sites on HC using mutagenesis and biochemistry approaches. In total, our data demonstrate that mK3-mediated ubiquitination can occur via serine, threonine, or lysine residues on the HC tail, each of which is sufficient to induce the rapid degradation of HC. Given that mK3 has numerous cellular and viral homologues, it will be of considerable interest to determine the pervasiveness of this novel mechanism of ubiquitination.

To characterize the process of class I assembly and maturation, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of intracellular and surface Ld molecules can be detected in an alternative conformation designated Ldalt1. Nascent Ldalt molecules are non-peptide ligand associated and are weakly associated with beta 2-microglobulin (beta 2m). Unexpectedly, when monoclonal antibodies were added directly to the lysis buffer, significant amounts of Ldalt/beta 2m heterodimer were detected, suggesting that beta 2m association is not necessarily sufficient to induce Ld conformation. By contrast, addition of peptide to cell lysates rapidly induced the folding of beta 2m-associated Ldalt to conformed Ld. Furthermore, the time course and dynamics of this conversion correlated precisely with peptide binding to Ld. The precursor-product relationship of Ldalt and conformed Ld was also visualized in vivo by pulse-chase analysis of BALB/c splenocytes. To investigate the factors that regulate intracellular transport of class I molecules, expression of Ld was studied in the peptide transport-deficient cell line, RMA.S-Ld, and in beta 2m-/- splenocytes. In contrast to wild-type cell lines, both Ldalt and conformed Ld are poorly expressed at the cell surface of RMA.S-Ld and beta 2m-/- splenocytes. Therefore, surface expression of Ldalt is dependent upon the concomitant expression of conformed Ld molecules. To determine whether surface Ldalt molecules can result from melting of conformed Ld molecules, surface Ld molecules were loaded with several different known Ld peptide ligands. Complexes of Ld with different ligands were found to have dramatically disparate surface half-lives. Importantly, the Ld peptide complexes that turned over the most rapidly resulted in the most gain in surface Ldalt, implying that peptide dissociation can induce the accumulation of nonconformed Ld heavy chains at the cell surface.

The preferential usage of certain T cell receptor (TCR) V beta genes has been well established in several major histocompatibility complex (MHC)-restricted immune responses. However, V beta usage among allogeneic responses remains unclear. Because recent findings of ours and others indicate that V beta 8 predominates in certain Ld-restricted, peptide-specific responses, we examined the V beta 8 usage in allogeneic responses to Ld. To selectively recognize the Ld molecule, cells from BALB/c-H-2dm2 (dm2), the Ld-loss mutant mouse, were stimulated in vitro or in vivo with wild-type BALB/c cells. We report here that after the intraperitoneal administration of the anti-V beta 8 monoclonal antibody (mAb) F23.1, peripheral V beta 8 T cells were depleted from dm2 mice. This in vivo depletion abrogated the ability of dm2 splenocytes to mount a primary response to Ld molecules. This abrogation was specific, since the response of V beta 8-depleted dm2 cells to Kb/Db antigens was the same as that of control nondepleted dm2 cells. Furthermore, in vivo depletion of V beta 8 cells was found to cause a dramatic prolongation of Ld-disparate skin grafts (mean survival time [MST] 22.1 +/- 2.1 vs. 10.3 +/- 1.1 d for saline-treated controls, or 10.9 +/- 1.7 d for controls treated with mAb KJ23 to V beta 17). By contrast, V beta 8 depletion had no effect on recipients grafted with haplotype-mismatched skin or single Dk-locus-disparate skin. These findings demonstrate that V beta 8+ T cells predominate in allogeneic response to Ld but not other alloantigens. The effect of V beta 8 depletion was found to be even more dramatic on recipients grafted with Ld-disparate vascularized heart transplants (MST > 100 vs. 8.6 +/- 0.5 d for controls). In total, these findings establish the efficacy of using mAb to the V beta gene family to specifically and significantly enhance the survival of allografts. The implications of detecting V beta 8 usage in both alloreactive or MHC-restricted TCR responses to the same class I molecule are discussed.

To determine the mechanism and structural consequences of peptide binding to class I molecules, we have studied the Ld molecule of the mouse. Previous studies have shown that a significant proportion of surface and intracellular Ld molecules can be detected in an alternative conformation designated Ldalt. Ldalt molecules are non-ligand associated and show weak if any beta 2-microglobulin (beta 2m) association. We report here that Ld molecules have a relatively rapid surface turnover compared with other class I molecules and that exogenous peptide dramatically prolongs Ld surface half-life. By contrast, Ldalt molecules are stably expressed on the surface and their half-life is unaffected by exogenous peptide. To study the surface interaction of peptide with Ld, live cells were incubated with iodinated peptides and Ld molecules were precipitated from cells precoated with monoclonal antibody before lysis. Using this assay, peptide binding to surface Ld molecules was found not to depend upon exchange with exogenous beta 2m, but did correlate with the level of beta 2m association. To study the intracellular interaction of peptide with Ld, cell lysates were used. In cell lysates, peptide was found to convert Ldalt molecules to properly folded Ld. This peptide-induced folding was almost complete at earlier but not later time points in pulse-chase analyses. Furthermore, conversion of Ldalt to Ld was found to affect almost exclusively immature (Endo Hs) class I molecules. Thus intrinsic properties of immature Ldalt molecules or their associated chaperonins are maintained in cell lysates that allow them to undergo de novo folding in vitro. These combined results demonstrate that immature Ldalt molecules are precursors awaiting constituents such as peptide and beta 2m that influence folding, whereas surface Ldalt molecules appear refractory to association with peptide, beta 2m, and consequent folding.

We have taken advantage of some unique properties of H-2Ld to investigate the determinant density requirements for cytotoxic T lymphocyte (CTL) priming versus effector function and to correlate the determinant density requirements with CD8 dependency. In a previous study (Lie, W.-R., N. B. Myers, J. Gorka, R. J. Rubocki, J. M. Connolly, and T. H. Hansen. 1990. Nature [Lond.]. 344:439), we demonstrated that culturing normal cells with peptides known to be restricted by H-2Ld led to a two- to fourfold increase in surface Ld expression. In the present study, we demonstrate the generation of Ld-restricted, peptide-specific in vitro primary CTL by culturing spleen cells with murine cytomegalovirus or tum- peptide at concentrations previously shown to result in maximum induction of Ld expression. Target cells can be sensitized for recognition by these CTL with lower dose of peptide than are required for the primary sensitization. This demonstrates differences in the determinant density requirements for priming versus effector function. The in vitro primary CTL generated with peptide can weakly lyse target cells that express the determinant endogenously, and CTL lines and clones capable of strong lysis of endogenous expressors are easily obtained. In both cases, target cells treated with exogenous peptide are lysed better than target cells expressing antigen endogenously. This suggested that there are differences in the determinant density of peptide-fed versus endogenous targets. This interpretation was substantiated when it was observed that the level of lysis of target cells expressing endogenous determinants correlated inversely with the amount of peptide required to sensitize targets for recognition by various tum- -specific CTL clones. Furthermore, simultaneous titration of both the peptide used to treat target cells and the antibody to CD8 revealed that the various CTL clones analyzed displayed widely disparate CD8 dependencies. In each case, the CD8 dependency correlated inversely with the determinant density requirement. Therefore, CD8 dependency of CTL is relative, but shows an absolute and quantitative correlation with their dependency on determinant density. These findings suggest that under physiologic conditions, where only low determinant densities are likely to be encountered, all CTL clones will show at least partial CD8 dependency.

To better understand the biological implications of the association of ligand with major histocompatibility complex class I molecules, we have studied the Ld molecule of the mouse. The culturing of various nonselected cell lines with three different known Ld peptide ligands resulted in a two- to fourfold specific increase in surface Ld expression as detected by 10 of 11 different monoclonal antibodies (mAbs) recognizing Ld epitopes. These findings suggest that Ld molecules are not saturated with endogenous peptide ligands and thus have accessible binding sites. Exploiting this feature of Ld we demonstrate that the physical association of Ld with ligand is exquisitely specific, indicating that they function in determinant selection. In addition, a non-peptide-bound antigenic variant of Ld was specifically detected with an exceptional mAb designated 64-3-7. In comparison with other Ld molecules, 64-3-7+ Ld molecules are not peptide ligand inducible, are more susceptible to proteolysis, lack beta 2 microglobulin association, and display a slower rate of oligosaccharide maturation. In spite of their deficiencies, the non-ligand-associated 64-3-7 Ld molecules were detected on the surface of all cell types tested; however, they appear not to be recognized by alloreactive cytotoxic T lymphocytes.

To resolve issues regarding the evolution of D region class I MHC genes and their relationship to other class I-encoding regions of the mouse, as well as man, we characterized the class I genes from the Dq region of the B10.AKM mouse strain. The Dq region was selected because it was known to express multiple gene products, yet two of the products previously characterized have structural features in common with the Ld molecule. Since DNA hybridization data defined similarities between the Dd and Dq regions, we used low-copy genomic or oligonucleotide probes derived from the Dd region of BALB/c (H-2d) to screen a B10.AKM cosmid library. Cosmid clones containing Dq, D2q, D3q, D4q, Lq, and Q1q genes have been isolated and aligned with the corresponding genes of the BALB/c MHC, thus demonstrating a similar gene organization. The two classical transplantation genes, Dq and Lq were found to be strikingly similar to each other such that exons 1-3 of Dq and Lq, are approximately 97% homologous, and exons 4-8 are identical. Furthermore, the implied amino acid sequences of both Lq and Dq molecules show considerable homology to Ld, particularly in regions presumed to be involved in ligand binding. These comparisons suggest not only that the Dq and Lq genes arose from the duplication of an Ld-like progenitor, but also that there is a selective advantage for the maintenance of an Ld-like structure. In addition, the 5' portion of the D4q gene was sequenced and found to have a 13-bp deletion and a 4-bp insertion within the alpha 2 exon. These result in a frame shift that creates a premature termination codon and potential polyadenylation site, respectively. Thus, D4q does not encode a typical class I molecule. Sequence comparisons suggest that the D4q gene did not arise from a duplication event involving an Ld-like gene such as Dq and Lq. Interestingly, the D4q molecule, if produced, would have amino acid residues in common with K and/or Q molecules that differ from those observed in D/L molecules. These findings, in conjunction with hybridization data, provide evidence that the D2, D3, and D4 genes were derived from Q genes by an unequal crossover event. Additional hybridization data using low-copy D region probes suggest that several different D region gene organizations exist among mice of different haplotypes. These and other recent molecular studies provide multiple examples of expansion and contraction of the class I genes in the D region.(ABSTRACT TRUNCATED AT 400 WORDS)

A significant within-strain correlation has been demonstrated between the levels of Ss and hemolytic complement (C) activity in two Ss-high strains. Mouse serum specifically depleted of Ss by absorption with F(ab')2 fragments of anti-Ss had negligible C activity. In control experiments, Ss-specific antigen-antibody complexes formed with F(ab')2 fragments did not fix rabbit C, and bovine serum albumin-specific antigen-antibody complexes formed with F(ab')2 fragments did not fix mouse C. Therefore the removal of C activity by anti-Ss [F(ab')2] was apparently not due to C fixation. These results suggest that the Ss protein is a necessary component of the C system.

The involvement of the different domains of the MHC class I molecule in CTL recognition was investigated. mAbs specific for the alpha 1/alpha 2 domains of H-2Ld interfered with both the primary and secondary generation and effector function of in vitro Ld-specific CTL. mAbs specific for the alpha 3 domain of H-2Ld interfered with the generation and function of primary in vitro Ld-specific CTL; however, there was no effect on the in vitro generation of secondary CTL and only partial inhibition of their function. In vivo treatment with graft-specific antibodies to both the alpha 3 domain and the alpha 1/alpha 2 domains together resulted in a dramatic enhancement of Ld- or Dd-disparate skin grafts, whereas the individual mAbs showed minimal effects. This suggested that the class I alpha 3 domain is recognized by alloreactive CTL. Several approaches were undertaken to examine whether recognition of the alpha 3 domain determinants is mediated by the Lyt-2 molecule. When mAbs specific for the alpha 3 domain of either H-2Ld or H-2Dd were used in vivo and in vitro, the resulting CTL population was not inhibited by antibody to the alpha 3 domain and was only partially inhibited by antibody to Lyt-2. We therefore observed a correlation between the effects of antibody to the class I alpha 3 domain of the target molecule and antibody to the Lyt-2 molecule on the CTL. To further test the relationship between CTL recognition of the alpha 3 domain and the involvement of Lyt-2, we used a cell expressing a mutation in the alpha 3 domain of the Dd molecule. The mutation resulted in a single amino acid substitution of glu to lys at residue 227 of the alpha 3 domain. Consistent with an earlier report, cells expressing the mutant Dd lys molecule were not lysed by CTL from a primary stimulation against the wild-type Dd glu molecule. However, this same cell line was killed by the Lyt-2-independent secondary Dd-specific CTL generated in the presence of antibody to the alpha 3 domain in vivo and in vitro. Furthermore, cells expressing the mutant Dd lys molecule failed to stimulate a primary response. In conclusion, several independent lines of evidence indicate that residues in the alpha 3 domain of the class I molecule are involved in recognition by the Lyt-2 molecule, and that Lyt-2-mediated recognition can be specifically blocked using mAb to determinants in the alpha 3 domain.

The H-2L locus is closely linked to H-2D and codes for antigenic specificities present on a 45,000 mol wt glycoprotein that is distinct from the molecule which bears the D region private specificity. It was found that BALB/c-H-2db mice, which lack detectable cell-surface H-2L gene products, were able to generate influenza- and vaccinia-immune cytotoxic T cells which lyse D region-compatible target cells, although they have been reported to be incapable of making a similar response to ectromelia virus (7). Thus, the lack of H-2L antigenic specificities does not produce a general loss of responsiveness for other viruses even when a highly cross-reactive pox virus (vaccinia) was studied. Antisera-blocking experiments utilizing sera specific for either L or D molecules indicated that BALB/c mice generate influenza virus-immune cytotoxic T-cell subsets which independently recognize H-2L and H-2D gene products in association with viral antigens. These results are the first indication that products of the H-2L locus can operate analogously to H-2K/D gene products in virus-immune T-cell recognition.

Sequential precipitations of soluble BALB/c antigen with antisera detecting private and public H-2 specificities indicated three distinct classes of molecules of 45,000 mol wt. However, only two of these classes of molecules were detectable in antigen from the loss mutant, BALB/c-H-2db. The class of molecules, detectable in the wild-type strain but missing in the mutant, does not bear private specificities but does react with an antiserum detecting H-2 public specificities. Absorption in mutant mice of the antiserum to public specificities, left antibodies specific for the antigen detectable in BALB/c but not BALB/c-H-2db. Genetic mapping studies using this specific antiserum indicated that the antigenic loss of this mutant is in a gene which maps in or close to the H-2D region, separable from the H-2K, S, G, Qa-2, and Tla regions.

Two phenomena appear to distinguish the D region class I genes from those in the K region in the murine MHC: (a) haplotype disparity in the number of expressed D region class I molecules has been observed; and (b) clines of closely related D region class I molecules among and within mice of different H-2 haplotypes can be defined. Both of these observations have been based on serological and peptide mapping analyses of these molecules. Recent reports using molecular biological approaches have corroborated these findings. Since the mouse strain B10.AKM expresses multiple D region class I antigens, all of which are closely related to the prototypic Ld molecule, we investigated the Dq region of B10.AKM using molecular approaches. Three D region class I genes were isolated from genomic B10.AKM bacteriophage and cosmid libraries. Based on alignment of those genes with the BALB/c D region class I genes by analogous restriction endonuclease sites and by hybridization of one of those genes with a D4d gene-derived oligonucleotide probe, we have designated these genes as Dq, Lq, and D4q. As determined by DNA-mediated gene transfer to mouse L cells followed by serological analyses, the Dq and Lq genes encode previously characterized Dq region class I antigens. The nucleic acid sequence comparisons of the Dq and Lq genes demonstrated a higher level of homology with the Ld and Db genes than with other D region class I genes. In addition, CTL stimulated with a Dq, Lq, or Ld gene transfectant showed strong crossreactions with the other transfectants as targets, suggesting that the products of these genes are also functionally related. Thus, these studies suggest that the L molecule represents a prototypic structure shared by several D region gene products, and furthermore, the duplication of an Ld-like progenitor gene resulted in two Dq region class I genes, Dq and Lq. Unexpectedly, the sequences determined for the Dq and Lq genes are nearly identical to the sequences of two genes, A166 and A149, respectively, which were reported to encode the tumor-specific antigens; these novel class I genes were isolated from an H-2k fibrosarcoma, 1591. This raises the distinct possibility that these purported tumor-specific class I genes were introduced into this tumor by contamination.

Anti-H-2 reagents have been tested on solubilized spleen cell preparations in combinations expected to be specific for D region products. Two different types of molecules were detected. One showed the expected reactivity with both antisera to private and antisera to public specificities. However, an additional molecule was detected which reacted only with antisera to public specificities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration patterns indicated that both products have a similar molecular size of approximately 45,000 daltons. The data therefore present chemical evidence for the existence of a third H-2-associated gene product of 45,000 mol wt in addition to the classical H-2K and H-2D antigens.