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 TCR? 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 TCR? 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.
We used a newly generated T-cell receptor mimic monoclonal antibody (TCRm MAb) that recognizes a known nonself immunodominant peptide epitope from West Nile virus (WNV) NS4B protein to investigate epitope presentation after virus infection in C57BL/6 mice. Previous studies suggested that peptides of different length, either SSVWNATTAI (10-mer) or SSVWNATTA (9-mer) in complex with class I MHC antigen H-2D(b) , were immunodominant after WNV infection. Our data establish that both peptides are presented on the cell surface after WNV infection and that CD8(+) T cells can detect 10- and 9-mer length variants similarly. This result varies from the idea that a given T-cell receptor (TCR) prefers a single peptide length bound to its cognate class I MHC. In separate WNV infection studies with the TCRm MAb, we show that in vivo the 10-mer was presented on the surface of uninfected and infected CD8?(+) CD11c(+) dendritic cells, which suggests the use of direct and cross-presentation pathways. In contrast, CD11b(+) CD11c(-) cells bound the TCRm MAb only when they were infected. Our study demonstrates that TCR recognition of peptides is not limited to certain peptide lengths and that TCRm MAbs can be used to dissect the cell-type specific mechanisms of antigen presentation in vivo.
Mucosal-associated invariant T cells (MAIT cells) express a semi-invariant T cell receptor (TCR) ?-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-CH?OH), 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 CD8?(+)CD8?(-/lo), implying predominant expression of CD8?? homodimers. Tetramer-sorted MAIT cells displayed a T(H)1 cytokine phenotype upon antigen-specific activation. Similarly, mouse MR1-rRL-6-CH?OH tetramers detected CD4(+), CD4(-)CD8(-) and CD8(+) MAIT cells in V?19 transgenic mice. Both human and mouse MAIT cells expressed a broad TCR-? 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.
Aberrantly or excessively expressed proteins in the endoplasmic reticulum are identified by quality control mechanisms and dislocated to the cytosol for proteasome-mediated, ubiquitin-dependent degradation by a process termed endoplasmic reticulum-associated degradation (ERAD). In addition to its role in degradation, ubiquitination has also been implicated in substrate dislocation, although whether direct ubiquitin conjugation of ERAD substrates is required for dislocation has been difficult to ascertain. An obstacle in probing the mechanism of quality control-induced ERAD is the paucity of ERAD substrates being dislocated and detected at any given time. To obviate this problem, we report here the use of a sensitive biotinylation system to probe the dislocation of major histocompatibility complex I (MHCI) heavy chain substrates in the absence of immune evasion proteins. Using this assay system the dislocation of MHCI heavy chains was found not to require potential ubiquitin conjugation sites in the cytoplasmic tail or Lys residues in the ectodomain. By contrast, dislocation of MHCI heavy chains did require deubiquitinating enzyme activity and rapid proteasome-mediated degradation required Lys residues in MHCI heavy chain ectodomain. These combined findings support the model that the endoplasmic reticulum quality control-induced dislocation of MHCI heavy chains may not require direct ubiquitination/deubiquitination as is required for proteasome-mediated degradation post dislocation.
CD161(++) CD8(+) T cells represent a novel subset that is dominated in adult peripheral blood by mucosal-associated invariant T (MAIT) cells, as defined by the expression of a variable-? chain 7.2 (V?7.2)-J?33 TCR, and IL-18R?. Stimulation with IL-18+IL-12 is known to induce IFN-? by both NK cells and, to a more limited extent, T cells. Here, we show the CD161(++) CD8(+) T-cell population is the primary T-cell population triggered by this mechanism. Both CD161(++) V?7.2(+) and CD161(++) V?7.2(-) T-cell subsets responded to IL-12+IL-18 stimulation, demonstrating this response was not restricted to the MAIT cells, but to the CD161(++) phenotype. Bacteria and TLR agonists also indirectly triggered IFN-? expression via IL-12 and IL-18. These data show that CD161(++) T cells are the predominant T-cell population that responds directly to IL-12+IL-18 stimulation. Furthermore, our findings broaden the potential role of MAIT cells beyond bacterial responsiveness to potentially include viral infections and other inflammatory stimuli.
Attempts to generate robust anti-tumour cytotoxic T lymphocyte (CTL) responses using immunotherapy are frequently thwarted by exhaustion and anergy of CTL recruited to tumour. One strategy to overcome this is to retarget a population of virus-specific CTL to kill tumour cells. Here, we describe a proof-of-principle study using a bispecific conjugate designed to retarget ovalbumin (OVA)-specific CTL to kill tumour cells via CD20. A single-chain trimer (SCT) consisting of MHCI H-2K(b)/SIINFEKL peptide/beta 2 microglobulin/BirA was expressed in bacteria, refolded and chemically conjugated to one (1:1; F2) or two (2:1; F3) anti-hCD20 Fab fragments. In vitro, the [SCT × Fab] (F2 and F3) redirected SIINFEKL-specific OT-I CTL to kill CD20(+) target cells, and in the presence of CD20(+) target cells to provide crosslinking, they were also able to induce proliferation of OT-I cells. In vivo, activated OT-I CTL could be retargeted to kill [SCT × Fab]-coated B cells from hCD20 transgenic (hCD20 Tg) mice and also EL4 and B16 mouse tumour cells expressing human CD20 (hCD20). Importantly, in a hCD20 Tg mouse model, [SCT × Fab] administered systemically were able to retarget activated OT-I cells to deplete normal B cells, and their performance matched that of a bispecific antibody (BsAb) comprising anti-CD3 and anti-CD20. [SCT × Fab] were also active therapeutically in an EL4 tumour model. Furthermore, measurement of serum cytokine levels suggests that [SCT × Fab] are associated with a lower level of inflammatory cytokine release than the BsAb and so may be advantageous clinically in terms of reduced toxicity.
Mucosal-associated invariant T cells are a unique population of T cells that express a semi-invariant ?? TCR and are restricted by the MHC class I-related molecule MR1. MAIT cells recognize uncharacterized ligand(s) presented by MR1 through the cognate interaction between their TCR and MR1. To understand how the MAIT TCR recognizes MR1 at the surface of APCs cultured both with and without bacteria, we undertook extensive mutational analysis of both the MAIT TCR and MR1 molecule. We found differential contribution of particular amino acids to the MAIT TCR-MR1 interaction based upon the presence of bacteria, supporting the hypothesis that the structure of the MR1 molecules with the microbial-derived ligand(s) differs from the one with the endogenous ligand(s). Furthermore, we demonstrate that microbial-derived ligand(s) is resistant to proteinase K digestion and does not extract with common lipids, suggesting an unexpected class of antigen(s) might be recognized by this unique lymphocyte population.
Smallpox decimated humanity for thousands of years before being eradicated by vaccination, a success facilitated by the fact that humans are the only host of variola virus. In contrast, other orthopoxviruses such as cowpox virus can infect a variety of mammalian species, although its dominant reservoir appears to be rodents. This difference in host specificity suggests that cowpox may have developed promiscuous immune evasion strategies to facilitate zoonosis. Recent experiments have established that cowpox can disrupt MHCI antigen presentation during viral infection of both human and murine cells, a process enabled by two unique proteins, CPXV012 and CPXV203. While CPXV012 inhibits antigenic peptide transport from the cytosol to the ER, CPXV203 blocks MHCI trafficking to the cell surface by exploiting the KDEL-receptor recycling pathway. Our recent investigations of CPXV203 reveal that it binds a diverse array of classical and non-classical MHCI proteins with dramatically increased affinities at the lower pH of the Golgi relative to the ER, thereby providing mechanistic insight into how it works synergistically with KDEL receptors to block MHCI surface expression. The strategy used by cowpox to both limit peptide supply and disrupt trafficking of fully assembled MHCI acts as a dual-edged sword that effectively disables adaptive immune surveillance of infected cells.
Herpesviruses are thought to be highly genetically stable, and their use as vaccine vectors has been proposed. However, studies of the human gammaherpesvirus, Epstein-Barr virus, have found viral isolates containing mutations in HLA class I-restricted epitopes. Using murine gammaherpesvirus 68 expressing ovalbumin (OVA), we examined the stability of a gammaherpesvirus antigenic locus under strong CD8 T cell selection in vivo. OVA-specific CD8 T cells selected viral isolates containing mutations in the OVA locus but minimal alterations in other genomic regions. Thus, a CD8 T cell response to a gammaherpesvirus-expressed antigen that is not essential for replication or pathogenesis can result in selective mutation of that antigen in vivo. This finding may have relevance for the use of herpesvirus vectors for chronic antigen expression in vivo.
New DNA sequencing platforms have revolutionized human genome sequencing. The dramatic advances in genome sequencing technologies predict that the $1,000 genome will become a reality within the next few years. Applied to cancer, the availability of cancer genome sequences permits real-time decision-making with the potential to affect diagnosis, prognosis, and treatment, and has opened the door towards personalized medicine. A promising strategy is the identification of mutated tumor antigens, and the design of personalized cancer vaccines. Supporting this notion are preliminary analyses of the epitope landscape in breast cancer suggesting that individual tumors express significant numbers of novel antigens to the immune system that can be specifically targeted through cancer vaccines.
Post-translational modification by ubiquitination determines intracellular location and fate of numerous proteins, thus impacting a diverse array of physiologic functions. Past dogma has been that ubiquitin was only coupled to substrates by isopeptide bonds to internal lysine residues or less frequently peptide bonds to the N-terminus. Enigmatically, however, several proteins lacking lysines had been reported to retain ubiquitin-dependent fates. Resolution of this paradox was afforded by recent observations that ubiquitination of substrates can also occur on cysteine or serine and threonine residues by thio- or oxy-ester bond formation, respectively (collectively called esterification). Although chemically possible, these bonds were considered too labile to be of physiological relevance. In this review we discuss recent evidence for the ubiquitination of protein substrates by esterification and speculate on its mechanism and its physiological importance.
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.
Major histocompatibility complex (MHC) class I molecules can be engineered as single chain trimers (SCTs) that sequentially incorporate all three subunits of the fully assembled proteins, namely peptide, ?2 microglobulin, and heavy chain. SCTs have been made with many different MHC-peptide complexes and are used as novel diagnostic and therapeutic reagents, as well as probes for diverse biological questions. Here, we review the recent and diverse applications of SCTs. These applications include new approaches to enumerate disease-related T cells, DNA vaccines, eliciting responses to pre-assembled MHC-peptide complexes, and unique probes of lymphocyte development and activation. Future applications of SCTs will be driven by their further engineering and the ever-expanding identification of disease-related peptides using chemical, genetic and computational approaches.
Control of infection with Mycobacterium tuberculosis (Mtb) requires Th1-type immunity, of which CD8+ T cells play a unique role. High frequency Mtb-reactive CD8+ T cells are present in both Mtb-infected and uninfected humans. We show by limiting dilution analysis that nonclassically restricted CD8+ T cells are universally present, but predominate in Mtb-uninfected individuals. Interestingly, these Mtb-reactive cells expressed the Valpha7.2 T-cell receptor (TCR), were restricted by the nonclassical MHC (HLA-Ib) molecule MR1, and were activated in a transporter associated with antigen processing and presentation (TAP) independent manner. These properties are all characteristics of mucosal associated invariant T cells (MAIT), an "innate" T-cell population of previously unknown function. These MAIT cells also detect cells infected with other bacteria. Direct ex vivo analysis demonstrates that Mtb-reactive MAIT cells are decreased in peripheral blood mononuclear cells (PBMCs) from individuals with active tuberculosis, are enriched in human lung, and respond to Mtb-infected MR1-expressing lung epithelial cells. Overall, these findings suggest a generalized role for MAIT cells in the detection of bacterially infected cells, and potentially in the control of bacterial infection.
The generation of a robust CD8(+) T cell response is an ongoing challenge for the development of DNA vaccines. One problem encountered with classical DNA plasmid immunization is that peptides produced are noncovalently and transiently associated with MHC class I molecules and thus may not durably stimulate CD8(+) T cell responses. To address this and enhance the expression and presentation of the antigenic peptide/MHC complexes, we generated single-chain trimers (SCTs) composed of a single polypeptide chain with a linear composition of antigenic peptide, beta(2)-microglobulin, and H chain connected by flexible linkers. In this study, we test whether the preassembled nature of the SCT makes them effective for eliciting protective CD8(+) T cell responses against pathogens. A DNA plasmid was constructed encoding an SCT incorporating the human MHC class I molecule HLA-A2 and the immunodominant peptide SVG9 derived from the envelope protein of West Nile virus (WNV). HLA-A2 transgenic mice vaccinated with the DNA encoding the SVG9/HLA-A2 SCT generated a robust epitope-specific CD8(+) T cell response and showed enhanced survival rate and lower viral burden in the brain after lethal WNV challenge. Inclusion of a CD4(+) Th cell epitope within the SCT did not increase the frequency of SVG9-specific CD8(+) T cells, but did enhance protection against WNV challenge. Overall, these findings demonstrate that the SCT platform can induce protective CD8(+) T cell responses against lethal virus infection and may be paired with immunogens that elicit robust neutralizing Ab responses to generate vaccines that optimally activate all facets of adaptive immunity.
It is commonly believed that delivery of antigen into the class I antigen presentation pathway is a limiting factor in the clinical translation of DNA vaccines. This is of particular concern in the context of cancer vaccine development as many immunodominant peptides derived from self tumor antigens are not processed and presented efficiently. To address this limitation, we have engineered completely assembled peptide/MHC class I complexes whereby all three components (class I heavy chain, beta(2)m, and peptide) are attached by flexible linkers and expressed as a single polypeptide (single chain trimers or SCT). In this study, we tested the efficacy of progressive generations of SCT DNA vaccines engineered to (1) enhance peptide binding, (2) enhance interaction with the CD8 coreceptor, and/or (3) activate CD4(+) helper T cells. Disulfide trap SCT (dtSCT) have been engineered to improve peptide binding, with mutations designed to create a disulfide bond between the class I heavy chain and the peptide linker. dtSCT DNA vaccines dramatically enhance the immune response to model low affinity antigens as measured by ELISPOT analysis and tumor challenge. SCT engineered to enhance interaction with the CD8 coreceptor have a higher affinity for the TCR/CD8 complex, and are associated with more robust CD8(+) T cell responses following vaccination. Finally, SCT constructs that coexpress a universal helper epitope PADRE, dramatically enhance CD8(+) T cell responses. Taken together, our data demonstrate that dtSCT DNA vaccines coexpressing a universal CD4 epitope are highly effective in generating immune responses to poorly processed and presented cancer antigens.
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.
This unit describes a method for constructing a class I MHC molecule with a bound peptide as a single polypeptide chain, termed SCT, for single chain trimer. The component organization of the SCT appears to be widely applicable to different mouse or human MHC class I isotypes bound by different antigenic peptides. The enhanced peptide occupancy afforded by the SCT format makes these molecules effective reagents as DNA vaccines, multimeric staining reagents to enumerate CD8 T cells, and probes of lymphocyte biology.
Pathogen recognition by T cells is dependent on their exquisite specificity for self-major histocompatibility complex (MHC) molecules presenting a bound peptide. Although this specificity results from positive and negative selection of developing T cells in the thymus, the relative contribution of these two processes remains controversial. To address the relation between the selecting peptide-MHC complex and the specificity of mature T cells, we generated transgenic mice that express a single peptide-MHC class I complex. We demonstrate that positive selection of CD8 T cells in these mice results in an MHC-specific repertoire. Although selection on a single complex is peptide promiscuous, mature T cells are highly peptide specific. Thus, positive selection imparts MHC and peptide specificity on the peripheral CD8 T cell repertoire.
Downregulation of MHC class I on the cell surface is an immune evasion mechanism shared by many DNA viruses, including cowpox virus. Previously, a cowpox virus protein, CPXV203, was shown to downregulate MHC class I. Here we report that CPXV12 is the only other MHC class I-regulating protein of cowpox virus and that it uses a mechanism distinct from that of CPXV203. Whereas CPXV203 retains fully assembled MHC class I by exploiting the KDEL-mediated endoplasmic reticulum retention pathway, CPXV12 binds to the peptide-loading complex and inhibits peptide loading on MHC class I molecules. Viruses deleted of both CPXV12 and CPXV203 demonstrated attenuated virulence in a CD8 T cell-dependent manner. These data demonstrate that CPXV12 and CPXV203 proteins combine to ablate MHC class I expression and abrogate antiviral CD8 T cell responses.
The cell surface display of peptides by MHC class I molecules to lymphocytes provides the host with an important surveillance mechanism to protect against invading pathogens. However, in turn, viruses have evolved elegant strategies to inhibit various stages of the MHC class I antigen presentation pathway and prevent the display of viral peptides. This Review highlights how the elucidation of mechanisms of viral immune evasion is important for advancing our understanding of virus-host interactions and can further our knowledge of the MHC class I presentation pathway as well as other cellular pathways.
A plethora of ubiquitin ligases determine the intracellular location and fate of numerous proteins in a substrate-specific manner. However, the mechanisms for these functions are incompletely understood. Most ligases have structurally related RING domains that are critical for ligase activity including the recruitment of ubiquitin conjugating enzymes. Here we probe the function of the RING-CH domain of murine gamma-herpesvirus-68 ligase mK3 that functions as an immune evasin by targeting major histocompatibility complex (MHC) class I heavy chains for endoplasmic reticulum-associated degradation (ERAD). Interestingly, mK3 mediates ubiquitin conjugation via ester bonds to S or T residues in addition to conventional isopeptide linkages to K residues. To determine the mechanism of non-K ubiquitination of substrates, we introduced into an mK3 background the RING-CH domains of related viral and cellular MARCH (membrane associated RING-CH) ligases. We found that although a conserved W present in all viral RING-CH domains is critical for mK3 function, sequences outside the RING-CH domain determine whether and which non-lysine substrate residues can be ubiquitinated by mK3. Our findings support the model that viral ligases have evolved a highly effective strategy to optimally orient their RING domain with substrate allowing them to ubiquitinate non-K residues.
Several nonclassical major histocompatibilty antigens (class Ib molecules) have emerged as key players in the early immune response to pathogens or stress. Class Ib molecules activate subsets of T cells that mount effector responses before the adaptive immune system, and thus are called innate T cells. MR1 is a novel class Ib molecule with properties highly suggestive of its regulation of mucosal immunity. The Mr1 gene is evolutionarily conserved, is non-Mhc linked, and controls the development of mucosal-associated invariant T (MAIT) cells. MAIT cells preferentially reside in the gut, and their development is dependent on commensal microbiota. Although these properties suggest that MAIT cells function as innate T cells in the mucosa, this has been difficult to test, due to the (i) paucity of MAIT cells that display MR1-specific activation in vitro and (ii) lack of knowledge of whether or not MR1 presents antigen. Here we show that both mouse and human MAIT cells display a high level of cross-reactivity on mammalian MR1 orthologs, but with differences consistent with limited ligand discrimination. Furthermore, acid eluates from recombinant or cellular MR1 proteins enhance MAIT cell activation in an MR1-specific and cross-species manner. Our findings demonstrate that the presentation pathway of MR1 to MAIT cells is highly evolutionarily conserved.
Immunological synapse formation between T cells and target cells can affect the functional outcome of TCR ligation by a given MHC-peptide complex. Although synapse formation is usually induced by TCR signaling, it is not clear whether other factors can affect the efficiency of synapse formation. Here, we tested whether cytokines could influence synapse formation between murine CTLs and target cells. We found that IL-12 enhanced synapse formation, whereas TGFbeta decreased synapse formation. The enhanced synapse formation induced by IL-12 appeared to be functional, given that IL-12-treated cells could respond to weak peptides, including self-peptides, to which the T cells were normally unresponsive. These responses correlated with expression of functionally higher avidity LFA-1 on IL-12-treated CTLs. These findings have implications for the function of IL-12 in T cell-mediated autoimmunity.
HIV infection is associated with immune dysfunction, perturbation of immune-cell subsets and opportunistic infections. CD161++ CD8+ T cells are a tissue-infiltrating population that produce IL17A, IL22, IFN, and TNF?, cytokines important in mucosal immunity. In adults they dominantly express the semi-invariant TCR V?7.2, the canonical feature of mucosal associated invariant T (MAIT) cells and have been recently implicated in host defense against pathogens. We analyzed the frequency and function of CD161++ /MAIT cells in peripheral blood and tissue from patients with early stage or chronic-stage HIV infection. We show that the CD161++ /MAIT cell population is significantly decreased in early HIV infection and fails to recover despite otherwise successful treatment. We provide evidence that CD161++ /MAIT cells are not preferentially infected but may be depleted through diverse mechanisms including accumulation in tissues and activation-induced cell death. This loss may impact mucosal defense and could be important in susceptibility to specific opportunistic infections in HIV.
One of the hallmarks of viral immune evasion is the capacity to disrupt major histocompatibility complex class I (MHCI) antigen presentation to evade T-cell detection. Cowpox virus encoded protein CPXV203 blocks MHCI surface expression by exploiting the KDEL-receptor recycling pathway, and here we show that CPXV203 directly binds a wide array of fully assembled MHCI proteins, both classical and non-classical. Further, the stability of CPXV203/MHCI complexes is highly pH dependent, with dramatically increased affinities at the lower pH of the Golgi relative to the endoplasmic reticulum (ER). Crystallographic studies reveal that CPXV203 adopts a beta-sandwich fold similar to poxvirus chemokine binding proteins, and binds the same highly conserved MHCI determinants located under the peptide-binding platform that tapasin, CD8, and natural killer (NK)-receptors engage. Mutagenesis of the CPXV203/MHCI interface identified the importance of two CPXV203 His residues that confer low pH stabilization of the complex and are critical to ER retrieval of MHCI. These studies clarify mechanistically how CPXV203 coordinates with other cowpox proteins to thwart antigen presentation.
The mature conformation of major histocompatibility complex class I (MHC-I) proteins depends on the presence of bound peptides, permitting recognition at the cell surface by CD8(+) T lymphocytes. Newly synthesized MHC-I molecules in the endoplasmic reticulum are maintained in a peptide-receptive (PR) transition state by several chaperones until they are released concomitant with the loading of peptides. By determining the crystallographic structure of a region of an MHC-I molecule that is recognized by a unique monoclonal antibody and comparing this with docking and molecular dynamics simulations with the whole molecule, we demonstrate the movement of a hinged unit supporting the part of the binding groove that interacts with the amino terminal residues of the bound peptide. This unit contains a conserved 310 helix that flips from an exposed "open" position in the PR form to a "closed" position in the peptide-loaded (PL) mature molecule. These analyses indicate how this segment of the MHC-I molecule moves to help establish the A and B pockets critical for tight peptide binding and the stable structure required for antigen presentation and T cell recognition at the cell surface.
We assessed gene expression in tissue macrophages from various mouse organs. The diversity in gene expression among different populations of macrophages was considerable. Only a few hundred mRNA transcripts were selectively expressed by macrophages rather than dendritic cells, and many of these were not present in all macrophages. Nonetheless, well-characterized surface markers, including MerTK and Fc?R1 (CD64), along with a cluster of previously unidentified transcripts, were distinctly and universally associated with mature tissue macrophages. TCEF3, C/EBP-?, Bach1 and CREG-1 were among the transcriptional regulators predicted to regulate these core macrophage-associated genes. The mRNA encoding other transcription factors, such as Gata6, was associated with single macrophage populations. We further identified how these transcripts and the proteins they encode facilitated distinguishing macrophages from dendritic cells.
Activation of naïve cluster of differentiation (CD)8(+) cytotoxic T lymphocytes (CTLs) is a tightly regulated process, and specific dendritic cell (DC) subsets are typically required to activate naive CTLs. Potential pathways for antigen presentation leading to CD8(+) T-cell priming include direct presentation, cross-presentation, and cross-dressing. To distinguish between these pathways, we designed single-chain trimer (SCT) peptide-MHC class I complexes that can be recognized as intact molecules but cannot deliver antigen to MHC through conventional antigen processing. We demonstrate that cross-dressing is a robust pathway of antigen presentation following vaccination, capable of efficiently activating both naïve and memory CD8(+) T cells and requires CD8?(+)/CD103(+) DCs. Significantly, immune responses induced exclusively by cross-dressing were as strong as those induced exclusively through cross-presentation. Thus, cross-dressing is an important pathway of antigen presentation, with important implications for the study of CD8(+) T-cell responses to viral infection, tumors, and vaccines.
Mucosa-associated invariant T (MAIT) cells are a unique population of ?? T cells in mammals that reside preferentially in mucosal tissues and express an invariant V? paired with limited V? T-cell receptor (TCR) chains. Furthermore, MAIT cell development is dependent upon the expression of the evolutionarily conserved major histocompatibility complex (MHC) class Ib molecule MR1. Using in vitro assays, recent studies have shown that mouse and human MAIT cells are activated by antigen-presenting cells (APCs) infected with diverse microbes, including numerous bacterial strains and yeasts, but not viral pathogens. However, whether MAIT cells play an important, and perhaps unique, role in controlling microbial infection has remained unclear. To probe MAIT cell function, we show here that purified polyclonal MAIT cells potently inhibit intracellular bacterial growth of Mycobacterium bovis BCG in macrophages (M?) in coculture assays, and this inhibitory activity was dependent upon MAIT cell selection by MR1, secretion of gamma interferon (IFN-?), and an innate interleukin 12 (IL-12) signal from infected M?. Surprisingly, however, the cognate recognition of MR1 by MAIT cells on the infected M? was found to play only a minor role in MAIT cell effector function. We also report that MAIT cell-deficient mice had higher bacterial loads at early times after infection compared to wild-type (WT) mice, demonstrating that MAIT cells play a unique role among innate lymphocytes in protective immunity against bacterial infection.
MHC class I (MHC-I) proteins of the adaptive immune system require antigenic peptides for maintenance of mature conformation and immune function via specific recognition by MHC-I-restricted CD8(+) T lymphocytes. New MHC-I molecules in the endoplasmic reticulum are held by chaperones in a peptide-receptive (PR) transition state pending release by tightly binding peptides. In this study, we show, by crystallographic, docking, and molecular dynamics methods, dramatic movement of a hinged unit containing a conserved 3(10) helix that flips from an exposed "open" position in the PR transition state to a "closed" position with buried hydrophobic side chains in the peptide-loaded mature molecule. Crystallography of hinged unit residues 46-53 of murine H-2L(d) MHC-I H chain, complexed with mAb 64-3-7, demonstrates solvent exposure of these residues in the PR conformation. Docking and molecular dynamics predict how this segment moves to help form the A and B pockets crucial for the tight peptide binding needed for stability of the mature peptide-loaded conformation, chaperone dissociation, and Ag presentation.
Upon Ag exposure, most memory T cells undergo restimulation-induced cell death. In this article, we describe a novel synthetic agonist, an N-terminal extended decamer peptide expressed as a single-chain trimer, the amino-terminal extended peptide MHC class I single-chain trimer (AT-SCT), which preferentially promotes the growth of memory human CD8(+) T cells with minimal restimulation-induced cell death. Using CMV pp65 and melanoma gp100 Ags, we observe the in vitro numerical expansion of a clonally diverse polyfunctional population of Ag-specific CD8(+) T cells from healthy individuals and vaccinated melanoma patients, respectively. Memory CD8(+) T cells stimulated with AT-SCT presented on MHC class I/II-null cells show reduced cytokine production, slower kinetics of TCR downregulation, and decreased cell death compared with native nonamer MHC class I single-chain trimer (SCT)-activated T cells. However, both ERK phosphorylation and cell cycle kinetics are identical in AT-SCT- and SCT-activated T cells. Probing of SCT and AT-SCT peptide-MHC complexes using fluorochrome-conjugated TCR multimers suggests that nonamer- and decamer-linked peptides may be anchored differently to the HLA-A2 peptide-binding groove. Our findings demonstrate that modified peptide-MHC structures, such as AT-SCT, can be engineered as T cell agonists to promote the growth and expansion of memory human CD8(+) T cells.
Mucosal-associated invariant T (MAIT) cells express a semiinvariant ?? 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 V?7.2J?33-V?2 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 ? chain were dispensable for the interaction with MR1, whereas the invariant MAIT TCR ? chain controlled specificity through a small number of residues, which are conserved across species and located within the V?-J? 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 ? chain of the MAIT TCR. This candidate docking mode contrasts with that of the NKT TCR-CD1d-antigen interaction, in which both the ? and ? chain of the NKT TCR is required for ligation above the F-pocket of CD1d.
Viral immune invasion proteins are highly effective probes for studying physiological pathways. We report here the characterization of a new viral ubiquitin ligase pK3 expressed by rodent herpesvirus Peru (RHVP) that establishes acute and latent infection in laboratory mice. Our findings show that pK3 binds directly and specifically to class I major histocompatibility proteins (MHCI) in a transmembrane-dependent manner. This binding results in the rapid degradation of the pK3/MHCI complex by a mechanism dependent upon catalytically active pK3. Subsequently, the rapid degradation of pK3/MHCI secondarily causes the slow degradation of membrane bound components of the MHCI peptide loading complex, tapasin, and transporter associated with antigen processing (TAP). Interestingly, this secondary event occurs by cellular endoplasmic reticulum-associated degradation. Cumulatively, our findings show pK3 uses a unique mechanism of substrate detection and degradation compared with other viral or cellular E3 ligases. More importantly, our findings reveal that in the absence of nascent MHCI proteins in the endoplasmic reticulum, the transmembrane proteins TAP and tapasin that facilitate peptide binding to MHCI proteins are degraded by cellular quality control mechanisms.
To circumvent limitations of poor antigen presentation and immunogenicity of DNA vaccines that target induction of CD8(+) T cell immunity, we have generated single chain MHC I trimers (MHC I SCTs) composed of a single polypeptide chain with a linear composition of antigenic peptide, ?2-microglobulin, and heavy chain of a MHC class I molecule connected by flexible linkers. Because of its pre-assembled nature, the SCT presents enhanced expression and presentation of the antigenic peptide/MHC complexes at the cell surface. Furthermore, DNA vaccination with a plasmid DNA encoding an SCT incorporating an immunodominant viral epitope elicited protective CD8(+) T cell responses against lethal virus infection. To extend these findings, here we tested the efficacy of SCT DNA vaccines against bacterial infections. In a mouse infection model of Listeria monocytogenes, the SCT DNA vaccine encoding H-2K(d) and the immunodominant peptide LLO 91-99 generated functional primary and memory peptide-specific CD8(+) T cells that confer partial protection against L. monocytogenes infection. DNA immunization of K(d)/LLO(91-99) SCTs generated functional memory CD8(+) T cells independently of CD4(+) T cells, although the expression of cognate or non-cognate CD4(+) helper T cell epitopes further enhanced the protective efficacy of SCTs. Our study further demonstrates that the SCT serves as a potent platform for DNA vaccines against various infectious diseases.
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