Search

Posttranslational modifications can enhance immunogenicity of self-proteins. In several conditions, including hypertension, systemic lupus erythematosus, and heart failure, isolevuglandins (IsoLGs) are formed by lipid peroxidation and covalently bond with protein lysine residues. Here, we show that the murine class I major histocompatibility complex (MHC-I) variant H-2Db uniquely presents isoLG-modified peptides and developed a computational pipeline that identifies structural features for MHC-I accommodation of such peptides. We identified isoLG-adducted peptides from renal proteins, including sodium glucose transporter 2, cadherin 16, Kelch domain-containing protein 7A, and solute carrier family 23, that are recognized by CD8+ T cells in tissues of hypertensive mice, induce T cell proliferation in vitro, and prime hypertension after adoptive transfer. Finally, we find patterns of isoLG-adducted antigen restriction in class I human leukocyte antigens that are similar to those in murine analogs. Thus, we have used a combined computational and experimental approach to define likely antigenic peptides in hypertension.

The UV-mediated peptide exchange has enabled the generation of multiple different MHC multimer specificities in parallel, surpassing tedious individual refolding of MHC molecules with peptide ligands. Murine models are acknowledged as an effective tool for preclinical research to advance our understanding of immunological mechanisms, with the potential translatability of key learnings from mouse models to the clinic. The common inbred mouse strain BALB/c is frequently used in immunological research. However, for the BALB/c histocompatibility (H)-2 alleles availability of conditional ligand has been limited. To overcome this challenge, we design and experimentally validate conditional ligands restricted to murine MHC class I alleles H2D d and H2K d . In addition, we demonstrate the ability of the three H2 d molecules and two additional C57BL/6 H2 b molecules folded in-house with conditional ligands to generate fluorescently labeled peptide-H2 tetramers that allow staining of antigen-specific CD8+ T cells in splenocyte samples. Finally, we generate large peptide-H-2 multimer libraries with a DNA-barcode labeling system for high-throughput interrogation of CD8+ T cell specificity in murine splenocyte samples. Consequently, the described techniques will contribute to our understanding of the antigen-specific CD8+ T cell repertoire in murine preclinical models of various diseases., (© 2024. The Author(s).)

Fucosylation is involved in a wide range of biological processes from cellular adhesion to immune regulation. Although the upregulation of fucosylated glycans was reported in diseased corneas, its implication in ocular surface disorders remains largely unknown. In this study, we analyzed the expression of a fucosylated glycan on the ocular surface in two mouse models of dry eye disease (DED), the NOD.B10.H2 b mouse model and the environmental desiccating stress model. We furthermore investigated the effects of aberrant fucosylation inhibition on the ocular surface and DED. Results demonstrated that the level of type 2 H antigen, an α(1,2)-fucosylated glycan, was highly increased in the cornea and conjunctiva both in NOD.B10.H2 b mice and in BALB/c mice subjected to desiccating stress. Inhibition of α(1,2)-fucosylation by 2-deoxy-D-galactose (2-D-gal) reduced corneal epithelial defects and increased tear production in both DED models. Moreover, 2-D-gal treatment suppressed the levels of inflammatory cytokines in the ocular surface and the percentages of IFN-γ + CD4 + cells in draining lymph nodes, whereas it did not affect the number of conjunctival goblet cells, the MUC5AC level or the meibomian gland area. Together, the findings indicate that aberrant fucosylation underlies the pathogenesis of DED and may be a novel target for DED therapy.

Background: Major histocompatibility complex class I (MHCI) proteins present antigenic peptides for immune surveillance and play critical roles in nervous system development and plasticity. Most MHCI are transmembrane proteins. The extracellular domain of MHCI interacts with immunoreceptors, peptides, and co-receptors to mediate immune signaling. While the cytoplasmic domain also plays important roles in endocytic trafficking, cross-presentation of extracellularly derived antigens, and CTL priming, the molecular mediators of cytoplasmic signaling by MHCI remain largely unknown., Results: Here we show that the cytoplasmic domain of MHCI contains putative protein-protein interaction domains known as PDZ (PSD95/disc large/zonula occludens-1) ligands. PDZ ligands are motifs that bind to PDZ domains to organize and mediate signaling at cell-cell contacts. PDZ ligands are short, degenerate motifs, and are therefore difficult to identify via sequence homology alone, but several lines of evidence suggest that putative PDZ ligand motifs in MHCI are under positive selective pressure. Putative PDZ ligands are found in all of the 99 MHCI proteins examined from diverse species, and are enriched in the cytoplasmic domain, where PDZ interactions occur. Both the position of the PDZ ligand and the class of ligand motif are conserved across species, as well as among genes within a species. Non-synonymous substitutions, when they occur, frequently preserve the motif. Of the many specific possible PDZ ligand motifs, a handful are strikingly and selectively overrepresented in MHCI's cytoplasmic domain, but not elsewhere in the same proteins. Putative PDZ ligands in MHCI encompass conserved serine and tyrosine residues that are targets of phosphorylation, a post-translational modification that can regulate PDZ interactions. Finally, proof-of-principle in vitro interaction assays demonstrate that the cytoplasmic domains of particular MHCI proteins can bind directly and specifically to PDZ1 and PDZ4&5 of MAGI-1, and identify a conserved PDZ ligand motif in the classical MHCI H2-K that is required for this interaction., Conclusions: These results identify cryptic protein interaction motifs in the cytoplasmic domain of MHCI. In so doing, they suggest that the cytoplasmic domain of MHCI could participate in previously unsuspected PDZ mediated protein-protein interactions at neuronal as well as immunological synapses.

Current strategies to improve the current BCG vaccine attempt to over-express genes encoding specific M. tuberculosis (Mtb) antigens and/or regulators of antigen presentation function, which indeed have the potential to reshape BCG in many ways. However, these approaches often face serious difficulties, in particular the efficiency and stability of gene expression via nucleic acid complementation and safety concerns associated with the introduction of exogenous DNA. As an alternative, we developed a novel non-genetic approach for rapid and efficient display of exogenous proteins on bacterial cell surface. The technology involves expression of proteins of interest in fusion with a mutant version of monomeric avidin that has the feature of reversible binding to biotin. Fusion proteins are then used to decorate the surface of biotinylated BCG. Surface coating of BCG with recombinant proteins was highly reproducible and stable. It also resisted to the freeze-drying shock routinely used in manufacturing conventional BCG. Modifications of BCG surface did not affect its growth in culture media neither its survival within the host cell. Macrophages phagocytized coated BCG bacteria, which efficiently delivered their surface cargo of avidin fusion proteins to MHC class I and class II antigen presentation compartments. Thereafter, chimeric proteins corresponding to a surrogate antigen derived from ovalbumin and the Mtb specific ESAT6 antigen were generated and tested for immunogenicity in vaccinated mice. We found that BCG displaying ovalbumin antigen induces an immune response with a magnitude similar to that induced by BCG genetically expressing the same surrogate antigen. We also found that BCG decorated with Mtb specific antigen ESAT6 successfully induces the expansion of specific T cell responses. This novel technology, therefore, represents a practical and effective alternative to DNA-based gene expression for upgrading the current BCG vaccine.

Background: In mammalian cells, the quality control (QC) of properly folded proteins is monitored in the early secretory pathway, particularly in the endoplasmic reticulum (ER). Several proteins, including our protein of interest, major histocompatibility complex class I (MHC class I), can bypass the first line of ER-QC and reside in post-ER compartments in an unfolded form. Such forms entail both monomeric and dimeric structures that are devoid of peptides and thus cannot fulfill the immunological function of antigen presentation at the cell surface. MHC class I structures become mature and properly folded once loaded with the appropriate peptides in the framework of the peptide loading complex (PLC). Despite the flood of information on the diverse trafficking behavior of different MHC class I alleles, there is still controversy on the actual trajectory followed by improperly folded murine MHC class I alleles, namely H-2Kb. In this study, we employ an in vitro rapamycin trapping assay, live cell imaging, and a biochemical COPII budding approach to further investigate the trafficking of H-2Kb beyond the level of the ER., Results: We confirm the egress of H-2Kb in an unfolded form to a post-ER compartment from where they can cycle back to the ER. Deciphering the exact identity of the post-ER compartment by laser scanning microscopy did not only point to the existence of the ERGIC and cis-Golgi compartments as residency areas for unfolded proteins, but also to the involvement of an addional compartment, that lies in close proximity and possesses high resemblance to the aforementioned compartments. Interestingly, we were capable of showing using the same rapamycin trapping assay that H-2Kb can undergo a potential maturation event during their cycling; this is attained upon addition of peptides and trapping of accumulated post-ER molecules at the cell surface., Conclusions: Our findings deepen the understanding of H-2Kb trafficking outside the ER and pave the way to decipher the role and the trafficking of certain PLC chaperones, such as tapasin, throughout H-2K(b) post-ER QC. Finally, we demonstrate the plausible usage of the rapamycin assay to assess the trafficking of defected proteins especially in diseases and under therapeutic studies.

Ly49 receptors can be peptide selective in their recognition of MHC-I-peptide complexes, affording them a level of discrimination beyond detecting the presence or absence of specific MHC-I allele products. Despite this ability, little is understood regarding the properties that enable some peptides, when bound to MHC-I molecules, to support Ly49 recognition, but not others. Using RMA-S target cells expressing MHC-I molecules loaded with individual peptides and effector cells expressing the ectodomain of the inhibitory Ly49C receptor, we found that two adjacent amino acid residues, P2 and P3, both buried in the peptide binding groove of H-2Kb, determine mouse Ly49C specificity. If both are aliphatic residues, this is supportive. Whereas, small amino acids at P2 and aromatic amino acids at the P3 auxiliary anchor residue are detrimental to Ly49C recognition. These results resemble those with a rat Ly49 where the identity of a peptide anchor residue determines recognition, suggesting that dependence on specific peptide residues buried in the MHC-I peptide-binding groove may be fundamental to Ly49 peptide selectivity and recognition.

Background: Social deficit is one of the core symptoms of neuropsychiatric diseases, in which immune genes play an important role. Although a few immune genes have been shown to regulate social and emotional behaviors, how immune gene network(s) may jointly regulate sociability has not been investigated so far., Methods: To decipher the potential immune-mediated mechanisms underlying social behavior, we first studied the brain microarray data of eight inbred mouse strains with known variations in social behavior and retrieved the differentially expressed immune genes. We then made a protein-protein interaction analysis of them to find the major networks and explored the potential association of these genes with the behavior and brain morphology in the mouse phenome database. To validate the expression and function of the candidate immune genes, we selected the C57BL/6 J and DBA/2 J strains among the eight inbred strains, compared their social behaviors in resident-intruder and 3-chambered social tests and the mRNA levels of these genes, and analyzed the correlations of these genes with the social behaviors., Results: A group of immune genes were differentially expressed in the brains of these mouse strains. The representative C57BL/6 J and DBA/2 J strains displayed significant differences in social behaviors, DBA/2 J mice being less active in social dominance and social interaction than C57BL/6 J mice. The mRNA levels of H2-d1 in the prefrontal cortex, hippocampus, and hypothalamus and C1qb in the hippocampus of the DBA/2 J strain were significantly down-regulated as compared to those in the C57BL/6 J strain. In contrast, Polr3b in the hippocampus and Tnfsf13b in the prefrontal cortex of the DBA/2 J strain were up-regulated. Furthermore, C1qb, Cx3cl1, H2-d1, H2-k1, Polr3b, and Tnfsf13b were predicted to be associated with various behavioral and brain morphological features across the eight inbred strains. Importantly, the C1qb mRNA level was confirmed to be significantly correlated with the sociability in DBA/2 J but not in C57BL/6 J mice., Conclusions: Our study provided evidence on the association of immune gene network(s) with the brain development and behavior in animals and revealed neurobiological functions of novel brain immune genes that may contribute to social deficiency in animal models of neuropsychiatric disorders.

Type 1 diabetes is characterized by T-cell-mediated destruction of the insulin-producing β cells in pancreatic islets. A number of islet antigens recognized by CD8 T cells that contribute to disease pathogenesis in non-obese diabetic (NOD) mice have been identified; however, the antigenic specificities of the majority of the islet-infiltrating cells have yet to be determined. The primary goal of the current study was to identify candidate antigens based on the level and specificity of expression of their genes in mouse islets and in the mouse β cell line MIN6. Peptides derived from the candidates were selected based on their predicted ability to bind H-2K(d) and were examined for recognition by islet-infiltrating T cells from NOD mice. Several proteins, including those encoded by Abcc8, Atp2a2, Pcsk2, Peg3 and Scg2, were validated as antigens in this way. Interestingly, islet-infiltrating T cells were also found to recognize peptides derived from proglucagon, whose expression in pancreatic islets is associated with α cells, which are not usually implicated in type 1 diabetes pathogenesis. However, type 1 diabetes patients have been reported to have serum autoantibodies to glucagon, and NOD mouse studies have shown a decrease in α cell mass during disease pathogenesis. Our finding of islet-infiltrating glucagon-specific T cells is consistent with these reports and suggests the possibility of α cell involvement in development and progression of disease., (© 2014 John Wiley & Sons Ltd.)

The intracellular trafficking of major histocompatibility complex class I (MHC-I) proteins is directed by three quality control mechanisms that test for their structural integrity, which is correlated to the binding of high-affinity antigenic peptide ligands. To investigate which molecular features of MHC-I these quality control mechanisms detect, we have followed the hypothesis that suboptimally loaded MHC-I molecules are characterized by their conformational mobility in the F-pocket region of the peptide-binding site. We have created a novel variant of an MHC-I protein, K(b)-Y84C, in which two α-helices in this region are linked by a disulfide bond that mimics the conformational and dynamic effects of bound high-affinity peptide. K(b)-Y84C shows a remarkable increase in the binding affinity to its light chain, beta-2 microglobulin (β2m), and bypasses all three cellular quality control steps. Our data demonstrate (1) that coupling between peptide and β2m binding to the MHC-I heavy chain is mediated by conformational dynamics; (2) that the folded conformation of MHC-I, supported by β2m, plays a decisive role in passing the ER-to-cell-surface transport quality controls; and (3) that β2m association is also tested by the cell surface quality control that leads to MHC-I endocytosis., (© 2014. Published by The Company of Biologists Ltd.)

The formation of precise connections between retina and lateral geniculate nucleus (LGN) involves the activity-dependent elimination of some synapses, with strengthening and retention of others. Here we show that the major histocompatibility complex (MHC) class I molecule H2-D(b) is necessary and sufficient for synapse elimination in the retinogeniculate system. In mice lacking both H2-K(b) and H2-D(b) (K(b)D(b)(-/-)), despite intact retinal activity and basal synaptic transmission, the developmentally regulated decrease in functional convergence of retinal ganglion cell synaptic inputs to LGN neurons fails and eye-specific layers do not form. Neuronal expression of just H2-D(b) in K(b)D(b)(-/-) mice rescues both synapse elimination and eye-specific segregation despite a compromised immune system. When patterns of stimulation mimicking endogenous retinal waves are used to probe synaptic learning rules at retinogeniculate synapses, long-term potentiation (LTP) is intact but long-term depression (LTD) is impaired in K(b)D(b)(-/-) mice. This change is due to an increase in Ca(2+)-permeable AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors. Restoring H2-D(b) to K(b)D(b)(-/-) neurons renders AMPA receptors Ca(2+) impermeable and rescues LTD. These observations reveal an MHC-class-I-mediated link between developmental synapse pruning and balanced synaptic learning rules enabling both LTD and LTP, and demonstrate a direct requirement for H2-D(b) in functional and structural synapse pruning in CNS neurons.

The paramyxovirus pneumonia virus of mice (PVM) is a rodent model of human respiratory syncytial virus (hRSV) pathogenesis. Here we characterized the PVM-specific CD8(+) T-cell repertoire in susceptible C57BL/6 mice. In total, 15 PVM-specific CD8(+) T-cell epitopes restricted by H-2D(b) and/or H-2K(b) were identified. These data open the door for using widely profiled, genetically manipulated C57BL/6 mice to study the contribution of epitope-specific CD8(+) T cells to PVM pathogenesis.

The human adenovirus E3/19K protein is a type I transmembrane glycoprotein of the endoplasmic reticulum (ER) that abrogates cell surface transport of major histocompatibility complex class I (MHC-I) and MHC-I-related chain A and B (MICA/B) molecules. Previous data suggested that E3/19K comprises two functional modules: a luminal domain for interaction with MHC-I and MICA/B molecules and a dilysine motif in the cytoplasmic tail that confers retrieval from the Golgi apparatus back to the ER. This study was prompted by the unexpected phenotype of an E3/19K molecule that was largely retained intracellularly despite having a mutated ER retrieval motif. To identify additional structural determinants responsible for ER localization, chimeric molecules were generated containing the luminal E3/19K domain and the cytoplasmic and/or transmembrane domain (TMD) of the cell surface protein MHC-I K(d). These chimeras were analyzed for transport, cell surface expression, and impact on MHC-I and MICA/B downregulation. As with the retrieval mutant, replacement of the cytoplasmic tail of E3/19K allowed only limited transport of the chimera to the cell surface. Efficient cell surface expression was achieved only by additionally replacing the TMD of E3/19K with that of MHC-I, suggesting that the E3/19K TMD may confer static ER retention. This was verified by ER retention of an MHC-I K(d) molecule with the TMD replaced by that of E3/19K. Thus, we have identified the E3/19K TMD as a novel functional element that mediates static ER retention, thereby increasing the concentration of E3/19K in the ER. Remarkably, the ER retrieval signal alone, without the E3/19K TMD, did not mediate efficient HLA downregulation, even in the context of infection. This suggests that the TMD is required together with the ER retrieval function to ensure efficient ER localization and transport inhibition of MHC-I and MICA/B molecules.

Major Histocompatibility Complex (MHC), peptide and T-Cell Receptor (TCR) play an essential role of adaptive immune responses. Many prediction servers are available for identification of peptides that bind to MHC class I molecules but often lack detailed interacting residues for analysing MHC-peptide-TCR interaction mechanisms. This study considers both the interface similarity and the interacting force for identifying binding models. Our model, considering both the MHC-peptide and the peptide-TCR interfaces, is able to provide visualisation and the biological insights of binding models. We believe that our model is useful for the development of peptide-based vaccines.

Macrophages are critically involved in the interaction between T. crassiceps and the murine host immune system. Also, a strong gender-associated susceptibility to murine cysticercosis has been reported. Here, we examined the sex-associated expression of molecules MHC-II, CD80, CD86, PD-L1, and PD-L2 on peritoneal F4/80(hi) macrophages of BALB/c mice infected with Taenia crassiceps. Peritoneal macrophages from both sexes of mice were exposed to T. crassiceps total extract (TcEx). BALB/c Females mice recruit higher number of macrophages to the peritoneum. Macrophages from infected animals show increased expression of PDL2 and CD80 that was dependent from the sex of the host. These findings suggest that macrophage recruitment at early time points during T. crassiceps infection is a possible mechanism that underlies the differential sex-associated susceptibility displayed by the mouse gender.

Upon antigen recognition, T cells form either static (synapses) or migratory (kinapses) contacts with antigen-presenting cells. Addressing whether synapses and kinapses result in distinct T cell receptor (TCR) signals has been hampered by the inability to simultaneously assess T cell phenotype and behavior. Here, we introduced dynamic in situ cytometry (DISC), a combination of intravital multiphoton imaging and flow cytometry-like phenotypic analysis. Taking advantage of CD62L shedding as a marker of early TCR signaling, we examined how T cells sense TCR ligands of varying affinities in vivo. We uncovered three modes of antigen recognition: synapses with the strongest TCR signals, kinapses with robust signaling, and kinapses with weak signaling. As illustrated here, the DISC approach should provide unique opportunities to link immune cell behavior to phenotype and function in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

To study the CD8(+) T cell response against a mouse γ-herpes virus, we generated K(b)-MHV-68-ORF8(604-612)RAG(-/-) CD8(+) T cell receptor transnuclear (TN) mice as a source of virus-specific CD8(+) T cells. K(b)-ORF8-Tet(+) CD8(+) T cells, expanded in the course of a resolving MHV-68 infection, served as a source of nucleus donors. Various in vivo and ex vivo assay criteria demonstrated the fine specificity and functionality of TN cells. TN cells proliferated extensively in response to viral infection, helped control viral burden, and exhibited a phenotype similar to that of endogenous K(b)-ORF8-Tet(+) cells. When compared to OT-1 cells, TN cells displayed distinct properties in response to lymphopenia and cognate antigen stimulation, which may be attributable to the affinity of the TCR expressed by the TN cells. The availability of MHV-68-specific CD8(+) TCR TN mice provides a new tool for investigating aspects of host-pathogen interactions unique to γ-herpes viruses., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Prostate cancer (PCa) is the most commonly diagnosed type of cancer in men in western industrialized countries. As a public health burden, the need for the invention of new cost-saving PCa immunotherapies is apparent. In this study, we present a DNA vaccine encoding for the prostate-specific antigen prostatic acid phosphatase (PAP) linked to the J-domain and the SV40 enhancer sequence. The PAP DNA vaccine induced a strong PAP-specific cellular immune response after electroporation (EP)-based delivery in C57BL/6 mice. Splenocytes from mice immunized with PAP recognized the naturally processed PAP epitopes, indicating that vaccination with the PAP-J gene broke its self-tolerance against PAP. Remarkably, DNA vaccination with PAP-J inhibited tumor growth in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse model that closely resembled human PCa. Therefore, this study highlights a novel cancer immunotherapy approach with the potential to control PCa in clinical settings.

Natural killer (NK) cells can mediate the rejection of bone marrow allografts and exist as subsets based on expression of inhibitory/activating receptors that can bind MHC. In vitro data have shown that NK subsets bearing Ly49 receptors for self-MHC class I have intrinsically higher effector function, supporting the hypothesis that NK cells undergo a host MHC-dependent functional education. These subsets also play a role in bone marrow cell (BMC) allograft rejection. Thus far, little in vivo evidence for this preferential licensing across mouse strains with different MHC haplotypes has been shown. We assessed the intrinsic response potential of the different Ly49(+) subsets in BMC rejection by using β2-microglobulin deficient (β2m(-/-)) mice as donors. Using congenic and allogeneic mice as recipients and depleting the different Ly49 subsets, we found that NK subsets bearing Ly49s, which bind "self-MHC" were found to be the dominant subset responsible for β2m(-/-) BMC rejection. This provides in vivo evidence for host MHC class I-dependent functional education. Interestingly, all H2(d) strain mice regardless of background were able to resist significantly greater amounts of β2m(-/-), but not wild-type BMC than H2(b) mice, providing evidence that the rheostat hypothesis regarding Ly49 affinities for MHC and NK-cell function impacts BMC rejection capability.

Structural diversity in the peptide binding sites of the redundant classical MHC antigen presenting molecules is strongly selected in humans and mice. Although the encoded antigen presenting molecules overlap in antigen presenting function, differences in polymorphism at the MHC I A, B and C loci in humans and higher primates indicate these loci are not functionally equivalent. The structural basis of these differences is not known. We hypothesize that classical class I loci differ in their ability to direct effective immunity against intracellular pathogens. Using a picornavirus infection model and chimeric H-2 transgenes, we examined locus specific functional determinants distinguishing the ability of class I sister genes to direct effective anti viral immunity. Whereas, parental FVB and transgenic FVB mice expressing the H-2K(b) gene are highly susceptible to persisting Theiler's virus infection within the CNS and subsequent demyelination, mice expressing the D(b) transgene clear the virus and are protected from demyelination. Remarkably, animals expressing a chimeric transgene, comprised primarily of K(b) but encoding the peptide binding domain of D(b), develop a robust anti viral CTL response yet fail to clear virus and develop significant demyelination. Differences in expression of the chimeric K(b)α1α2D(b) gene (low) and D(b) (high) in the CNS of infected mice mirror expression levels of their endogenous H-2(q) counterparts in FVB mice. These findings demonstrate that locus specific elements other than those specifying peptide binding and T cell receptor interaction can determine ability to clear virus infection. This finding provides a basis for understanding locus-specific differences in MHC polymorphism, characterized best in human populations.

Susceptibility to respiratory syncytial virus (RSV) infection in mice is genetically determined. While RSV causes little pathology in C57BL/6 mice, pulmonary inflammation and weight loss occur in BALB/c mice. Using major histocompatibility complex (MHC)-congenic mice, we observed that the H-2(d) allele can partially transfer disease susceptibility to C57BL/6 mice. This was not explained by altered viral elimination or differences in the magnitude of the overall virus-specific cytotoxic T lymphocyte (CTL) response. However, H-2(d) mice showed a more focused response, with 70% of virus-specific CTL representing Vβ8.2(+) CTL directed against the immunodominant epitope M2-1 82, while in H-2(b) mice only 20% of antiviral CTL were Vβ9(+) CTL specific for the immunodominant epitope M187. The immunodominant H-2(d)-restricted CTL lysed target cells less efficiently than the immunodominant H-2(b) CTL, probably contributing to prolonged CTL stimulation and cytokine-mediated immunopathology. Accordingly, reduction of dominance of the M2-1 82-specific CTL population by introduction of an M187 response in the F1 generation of a C57BL/6N × C57BL/6-H-2(d) mating (C57BL/6-H-2(dxb) mice) attenuated disease. Moreover, disease in H-2(d) mice was less pronounced after infection with an RSV mutant failing to activate M2-1 82-specific CTL or after depletion of Vβ8.2(+) cells. These data illustrate how the MHC-determined diversity and functional avidity of CTL responses contribute to disease susceptibility after viral infection.

Background: Leishmania parasites have been reported to interfere and even subvert their host immune responses to enhance their chances of survival and proliferation. Experimental Leishmania infection in mice has been widely used in the identification of specific parasite virulence factors involved in the interaction with the host immune system. Cysteine-proteinase B (CPB) is an important virulence factor in parasites from the Leishmania (Leishmania) mexicana complex: it inhibits lymphocytes Th1 and/or promotes Th2 responses either through proteolytic activity or through epitopes derived from its COOH-terminal extension. In the present study we analyzed the effects of Leishmania (Leishmania) amazonensis CPB COOH-terminal extension-derived peptides on cell cultures from murine strains with distinct levels of susceptibility to infection: BALB/c, highly susceptible, and CBA, mildly resistant., Results: Predicted epitopes, obtained by in silico mapping, displayed the ability to induce cell proliferation and expression of cytokines related to Th1 and Th2 responses. Furthermore, we applied in silico simulations to investigate how the MHC/epitopes interactions could be related to the immunomodulatory effects on cytokines, finding evidence that specific interaction patterns can be related to in vitro activities., Conclusions: Based on our results, we consider that some peptides from the CPB COOH-terminal extension may influence host immune responses in the murine infection, thus helping Leishmania survival.

Major histocompatibility complex class I (MHCI) and MHCII proteins differ in structure and sequence. To understand how T cell receptors (TCRs) can use the same set of variable regions to bind both proteins, we have presented a comparison of a single TCR bound to both MHCI and MHCII ligands. The TCR adopts similar orientations on both ligands with TCR amino acids thought to be evolutionarily conserved for MHC interaction occupying similar positions on the MHCI and MHCII helices. However, the TCR antigen-binding loops use different conformations when interacting with each ligand. Most importantly, we observed alternate TCR core conformations. When bound to MHCI, but not MHCII, Vα disengages from the Jα β strand, switching Vα's position relative to Vβ. In several other structures, either Vα or Vβ undergoes this same modification. Thus, both TCR V-domains can switch among alternate conformations, perhaps extending their ability to react with different MHC-peptide ligands., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Human MUC1 is over-expressed in human adenocarcinomas and has been used as a target for immunotherapy studies. The 9-mer MUC1-9 peptide has been identified as one of the peptides which binds to murine MHC class I H-2K(b). The structure of MUC1-9 in complex with H-2K(b) has been modeled and simulated with classical molecular dynamics, based on the x-ray structure of the SEV9 peptide/H-2K(b) complex. Two independent trajectories with the solvated complex (10 ns in length) were produced. Approximately 12 hydrogen bonds were identified during both trajectories to contribute to peptide/MHC complex, as well as 1-2 water mediated hydrogen bonds. Stability of the complex was also confirmed by buried surface area analysis, although the corresponding values were about 20% lower than those of the original x-ray structure. Interestingly, a bulged conformation of the peptide's central region, partially characterized as a β-turn, was found exposed form the binding groove. In addition, P1 and P9 residues remained bound in the A and F binding pockets, even though there was a suggestion that P9 was more flexible. The complex lacked numerous water mediated hydrogen bonds that were present in the reference peptide x-ray structure. Moreover, local displacements of residues Asp4, Thr5 and Pro9 resulted in loss of some key interactions with the MHC molecule. This might explain the reduced affinity of the MUC1-9 peptide, relatively to SEV9, for the MHC class I H-2K(b).

After an infection, cytotoxic T lymphocyte precursors proliferate and become effector cells by recognizing foreign peptides in the groove of major histocompatibility complex (MHC) class I molecules expressed by antigen-presenting cells (APCs). Professional APCs specialized for T-cell activation acquire viral antigen either by becoming infected themselves (direct presentation) or by phagocytosis of infected cells, followed by transfer of antigen to the cytosol, processing and MHC class I loading in a process referred to as cross-presentation. An alternative way, referred to as 'cross-dressing', by which an uninfected APC could present antigen was postulated to be by the transfer of preformed peptide-MHC complexes from the surface of an infected cell to the APC without the need of further processing. Here we show that this mechanism exists and boosts the antiviral response of mouse memory CD8(+) T cells. A number of publications have demonstrated sharing of peptide-loaded MHC molecules in vitro. Our in vitro experiments demonstrate that cross-dressing APCs do not acquire peptide-MHC complexes in the form of exosomes released by donor cells. Rather, the APCs and donor cells have to contact each other for the transfer to occur. After a viral infection, we could isolate cross-dressed APCs able to present viral antigen in vitro. Furthermore, using the diphtheria toxin system to selectively eliminate APCs that could only acquire viral peptide-MHC complexes by cross-dressing, we show that such presentation can promote the expansion of resting memory T cells. Notably, naive T cells were excluded from taking part in the response. Cross-dressing is a mechanism of antigen presentation used by dendritic cells that may have a significant role in activating previously primed CD8(+) T cells.

The idiotypes of B cell lymphomas represent tumor-specific antigens. T cell responses induced by idiotype vaccination in vivo are directed predominantly against CDR peptides, whereas in vitro T cells also recognize framework-derived epitopes. To investigate the mechanisms regulating the specificity of idiotype-specific T cells, BALB/c or B10.D2 mice were immunized with mature dendritic cells loaded with H-2K(d)-restricted peptides from influenza hemagglutinin, or from shared (J region) or unique (CDR3) structures of the A20 lymphoma idiotype. Antigen-specific T cells were induced in vivo by the CDR3 and influenza epitopes, but not by the J peptide. Gene expression profiling of splenic regulatory T cells revealed vaccination-induced Treg activation and proliferation. Treg activity involved J epitope-dependent IL-10 secretion and functional suppression of peptide-specific effector T cells. Vaccination-induced in vivo proliferation of transgenic hemagglutinin-specific T cells was suppressed by co-immunization with the J peptide and was restored in CD25-depleted animals. In conclusion, Treg induced by a shared idiotype epitope can systemically suppress T cell responses against idiotype-derived and immunodominant foreign epitopes in vivo. The results imply that tumor vaccines should avoid epitopes expressed by normal cells in the draining lymph node to achieve optimal anti-tumor efficacy.

The T-cell receptor (TCR) interacts with peptide-major histocompatibility complexes (pMHC) to discriminate pathogens from self-antigens and trigger adaptive immune responses. Direct physical contact is required between the T cell and the antigen-presenting cell for cross-junctional binding where the TCR and pMHC are anchored on two-dimensional (2D) membranes of the apposing cells. Despite their 2D nature, TCR-pMHC binding kinetics have only been analysed three-dimensionally (3D) with a varying degree of correlation with the T-cell responsiveness. Here we use two mechanical assays to show high 2D affinities between a TCR and its antigenic pMHC driven by rapid on-rates. Compared to their 3D counterparts, 2D affinities and on-rates of the TCR for a panel of pMHC ligands possess far broader dynamic ranges that match that of their corresponding T-cell responses. The best 3D predictor of response is the off-rate, with agonist pMHC dissociating the slowest. In contrast, 2D off-rates are up to 8,300-fold faster, with the agonist pMHC dissociating the fastest. Our 2D data suggest rapid antigen sampling by T cells and serial engagement of a few agonist pMHCs by TCRs in a large self pMHC background. Thus, the cellular environment amplifies the intrinsic TCR-pMHC binding to generate broad affinities and rapid kinetics that determine T-cell responsiveness.

High avidity for antigen and diversity of T cell receptor (TCR) repertoire are essential for effective immunity against cancer. We have previously created a transgenic mouse strain with increased TCR avidity in a diverse T cell population. In this report, we show that strong alloreactive responses of transgenic T cells against targets with low MHC class I expression can be used for effective adoptive transfer of tumor immunity in vivo. Alloreactive transgenic T cells could be an effective therapeutic approach counteracting tumor evasion of the immune system.

Residues within processed protein fragments bound to major histocompatibility complex class I (MHC-I) glycoproteins have been considered to function as a series of "independent pegs" that either anchor the peptide (p) to the MHC-I and/or interact with the spectrum of alphabeta-T-cell receptors (TCRs) specific for the pMHC-I epitope in question. Mining of the extensive pMHC-I structural database established that many self- and viral peptides show extensive and direct interresidue interactions, an unexpected finding that has led us to the idea of "constrained" peptides. Mutational analysis of two constrained peptides (the HLA B44 restricted self-peptide (B44DPalpha-EEFGRAFSF) and an H2-D(b) restricted influenza peptide (D(b)PA, SSLENFRAYV) demonstrated that the conformation of the prominently exposed arginine in both peptides was governed by interactions with MHC-I-orientated flanking residues from the peptide itself. Using reverse genetics in a murine influenza model, we revealed that mutation of an MHC-I-orientated residue (SSLENFRAYV --> SSLENARAYV) within the constrained PA peptide resulted in a diminished cytotoxic T lymphocyte (CTL) response and the recruitment of a limited pMHC-I specific TCR repertoire. Interactions between individual peptide positions can thus impose fine control on the conformation of pMHC-I epitopes, whereas the perturbation of such constraints can lead to a previously unappreciated mechanism of viral escape.

Type 1 diabetes (T1D) is an autoimmune disease characterized by T cell-mediated destruction of insulin-producing pancreatic beta cells. In both humans and the non-obese diabetic (NOD) mouse model of T1D, class II MHC alleles are the primary determinant of disease susceptibility. However, class I MHC genes also influence risk. These findings are consistent with the requirement for both CD4(+) and CD8(+) T cells in the pathogenesis of T1D. Although a large body of work has permitted the identification of multiple mechanisms to explain the diabetes-protective effect of particular class II MHC alleles, studies examining the protective influence of class I alleles are lacking. Here, we explored this question by performing biochemical and structural analyses of the murine class I MHC molecule H-2K(wm7), which exerts a diabetes-protective effect in NOD mice. We have found that H-2K(wm7) molecules are predominantly occupied by the single self-peptide VNDIFERI, derived from the ubiquitous protein histone H2B. This unexpected finding suggests that the inability of H-2K(wm7) to support T1D development could be due, at least in part, to the failure of peptides from critical beta-cell antigens to adequately compete for binding and be presented to T cells. Predominant presentation of a single peptide would also be expected to influence T-cell selection, potentially leading to a reduced ability to select a diabetogenic CD8(+) T-cell repertoire. The report that one of the predominant peptides bound by T1D-protective HLA-A*31 is histone derived suggests the potential translation of our findings to human diabetes-protective class I MHC molecules.

Objective: In lethally irradiated bone marrow chimeras, part of the reconstituted T-cell compartment is derived from the irradiated host, but the detailed origin and functional activity of host-derived T cells has not been thoroughly analyzed. Herein, we determine the origin and function of radioresistant host-derived T cells., Materials and Methods: Lethally irradiated thymectomized or nonthymectomized C57BL/6 host mice were reconstituted with syngeneic bone marrow, itself incapable of generating T cells. Using fetal thymic organ cultures, bulk and limiting dilution assays on OP9-DL1 stromal cells, unambiguous cohorts of thymus-derived and peripheral T-cell-derived T cells were phenotypically characterized by flow cytometry and functionally characterized by their ability to participate in a T-cell-dependent antibody response., Results: Both thymus-derived and peripheral T-cell-derived host T cells are functional and can reconstitute 35% of the normal T-cell pool. By comparing thymectomized vs nonthymectomized hosts, host-derived T cells were shown to comprise a major (70%) subpopulation of de novo generated, thymus-derived, polyclonal, naïve cells, and a minor subpopulation of surviving, peripheral, oligoclonal, memory-like cells. Unlike euthymic recipients, mice whose T cells were derived from surviving peripheral T cells were frequently incapable of mounting a T-cell-dependent antibody response. Host-derived thymocytes regenerated in an interleukin-7-dependent fashion from conventional DN2 thymocytes and their differentiation recapitulated normal thymic ontogeny., Conclusion: We characterized, for the first time, functional radioresistant DN2-phenotype thymic T-cell precursors, the T-cell progeny of which might provide a first line of defense against infections during the lymphopenic phase post-bone marrow transplantation., (Copyright 2010 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Calreticulin is a lectin chaperone of the endoplasmic reticulum (ER). In calreticulin-deficient cells, major histocompatibility complex (MHC) class I molecules travel to the cell surface in association with a sub-optimal peptide load. Here, we show that calreticulin exits the ER to accumulate in the ER-Golgi intermediate compartment (ERGIC) and the cis-Golgi, together with sub-optimally loaded class I molecules. Calreticulin that lacks its C-terminal KDEL retrieval sequence assembles with the peptide-loading complex but neither retrieves sub-optimally loaded class I molecules from the cis-Golgi to the ER, nor supports optimal peptide loading. Our study, to the best of our knowledge, demonstrates for the first time a functional role of intracellular transport in the optimal loading of MHC class I molecules with antigenic peptide.

We previously showed that introduction of transporter associated with antigen processing (TAP) 1 into TAP-negative CMT.64, a major histocompatibility complex class I (MHC-I) down-regulated mouse lung carcinoma cell line, enhanced T-cell immunity against TAP-deficient tumour cells. Here, we have addressed two questions: (1) whether such immunity can be further augmented by co-expression of TAP1 with B7.1 or H-2K(b) genes, and (2) which T-cell priming mechanism (tumour direct priming or dendritic cell cross-priming) plays the major role in inducing an immune response against TAP-deficient tumours. We introduced the B7.1 or H-2K(b) gene into TAP1-expressing CMT.64 cells and determined which gene co-expressed with TAP1 was able to provide greater protective immunity against TAP-deficient tumour cells. Our results show that immunization of mice with B7.1 and TAP1 co-expressing but not H-2K(b) and TAP1 co-expressing CMT.64 cells dramatically augments T-cell-mediated immunity, as shown by an increase in survival of mice inoculated with live CMT.64 cells. In addition, our results suggest that induction of T-cell-mediated immunity against TAP-deficient tumour cells could be mainly through tumour direct priming rather than dendritic cell cross-priming as they show that T cells generated by tumour cell-lysate-loaded dendritic cells recognized TAP-deficient tumour cells much less than TAP-proficient tumour cells. These data suggest that direct priming by TAP1 and B7.1 co-expressing tumour cells is potentially a major mechanism to facilitate immune responses against TAP-deficient tumour cells.

Major histocompatibility complex (MHC) class I molecules have fundamental functions in the immune system. Recent studies have suggested that these molecules may also have non-immune functions in the nervous system, in particular related to synaptic function and plasticity. Because adult motoneurons express mRNAs for MHC class I molecules, we have examined their subcellular expression pattern in vivo and their role for the synaptic connectivity of these neurons. We observed immunoreactivity for classical MHC class I (Ia) protein in motoneuron somata, but the predominant expression was found in axons and presynaptically at neuromuscular junctions (NMJs). Peripheral nerve lesion induced a strong increase of motoneuron MHC class Ia (H2-K(b)/D(b)) mRNA, indicating a role for MHC class Ia molecules during regeneration. Accordingly, there was an accumulation of MHC class Ia proteins at the cut ends and in growth cones of motor axons after lesion. In K(b-/-)D(b-/-) mice (lacking MHC class Ia molecules), the time course for recovery of grip ability in reinnervated muscles was significantly delayed. Muscles from K(b-/-)D(b-/-) mice displayed an increased density and a disturbed distribution of NMJs and fewer terminal Schwann cells/NMJ compared with wild-type mice. A population of Schwann cells in sciatic nerves expressed the paired Ig receptor B, which binds to MHC class I molecules. These results provide the first evidence that neuronal MHC class Ia molecules are present in motor axons, that they are important for organization of NMJs and motor recovery after nerve lesion, and that their actions may be mediated via Schwann cells.

Background: A drawback of cancer gene therapy is the failure of toxic gene introduction into a proportion of the tumor cells, resulting in re-progression of the disease. Cancer cell-specificity of the gene introduction has also been problematic., Materials and Methods: Previously defined promoter/enhancer DNA of Q5 tumor antigen gene was used for the purpose of transcriptional targeting. A replication defective adenovirus carrying H-2D(d) cDNA placed downstream of the Q5 promoter/enhancer (Ad/Q5-H-2D(d)) was constructed and administered systemically to C57Bl/6 (H-2(b)) mice bearing pre-established hepatic metastasis of the syngeneic M5076 tumor., Results: 5 of the 10 mice showed complete regression of the tumor. Combination of the therapy with low dose cyclophosphamide (CY) administration augmented the therapeutic effect. The effect was brought about by tumor cell-specific allograft rejection reactions and by tumor antigen-specific reactions induced as "bystander effect" by the former reactions., Conclusion: The results provide an experimental basis of a highly efficient novel strategy of cancer gene therapy.

Background: Viral infection and neoplastic transformation trigger endoplasmic reticulum (ER) stress. Thus, a large proportion of the cells that must be recognized by the immune system are stressed cells. Cells respond to ER stress by launching the unfolded protein response (UPR). The UPR regulates the two key processes that control major histocompatibility complex class I (MHC I)-peptide presentation: protein synthesis and degradation. We therefore asked whether and how the UPR impinges on MHC I-peptide presentation., Results: We evaluated the impact of the UPR on global MHC I expression and on presentation of the H2Kb-associated SIINFEKL peptide. EL4 cells stably transfected with vectors coding hen egg lysozyme (HEL)-SIINFEKL protein variants were stressed with palmitate or exposed to glucose deprivation. UPR decreased surface expression of MHC I but did not affect MHC I mRNA level nor the total amount of intracellular MHC I proteins. Impaired MHC I-peptide presentation was due mainly to reduced supply of peptides owing to an inhibition of overall protein synthesis. Consequently, generation of H2Kb-SIINFEKL complexes was curtailed during ER stress, illustrating how generation of MHC I peptide ligands is tightly coupled to ongoing protein synthesis. Notably, the UPR-induced decline of MHC I-peptide presentation was more severe when the protein source of peptides was localized in the cytosol than in the ER. This difference was not due to changes in the translation rates of the precursor proteins but to increased stability of the cytosolic protein during ER stress., Conclusion: Our results demonstrate that ER stress impairs MHC I-peptide presentation, and that it differentially regulates expression of ER- vs. cytosol-derived peptides. Furthermore, this work illustrates how ER stress, a typical feature of infected and malignant cells, can impinge on cues for adaptive immune recognition.

Activation of CD4(+) T cells helps establish and sustain other immune responses. We have previously shown that responses against a broad set of nine CD4(+) T-cell epitopes were present in the setting of lymphocytic choriomeningitis virus (LCMV) Armstrong infection in the context of H-2(d). This is quite disparate to the H-2(b) setting, where only two epitopes have been identified. We were interested in determining whether a broad set of responses was unique to H-2(d) or whether additional CD4(+) T-cell epitopes could be identified in the setting of the H-2(b) background. To pursue this question, we infected C57BL/6 mice with LCMV Armstrong and determined the repertoire of CD4(+) T-cell responses using overlapping 15-mer peptides corresponding to the LCMV Armstrong sequence. We confirmed positive responses by intracellular cytokine staining and major histocompatibility complex (MHC)-peptide binding assays. A broad repertoire of responses was identified, consisting of six epitopes. These epitopes originate from the nucleoprotein (NP) and glycoprotein (GP). Out of the six newly identified CD4(+) epitopes, four of them also stimulate CD8(+) T cells in a statistically significant manner. Furthermore, we assessed these CD4(+) T-cell responses during the memory phase of LCMV Armstrong infection and after infection with a chronic strain of LCMV and determined that a subset of the responses could be detected under these different conditions. This is the first example of a broad repertoire of shared epitopes between CD4(+) and CD8(+) T cells in the context of viral infection. These findings demonstrate that immunodominance is a complex phenomenon in the context of helper responses.

Developmental exposure to noninherited maternal antigens (NIMA) exerts a tolerizing or sensitizing influence on clinical transplantation in humans and experimental animals. The aim of this study was to determine if strain and gender differences influence the NIMA effect. Six different mouse strain backcross matings of F(1) females with homozygous males ('NIMA backcross') and corresponding control breedings of F1 males with homozygous females were performed. H-2 homozygous offspring underwent heterotopic heart transplantation from fully allogeneic donors expressing noninherited H-2 antigens. A NIMA tolerizing effect on heart allograft outcome was found in three of six breeding models. In all three cases, the tolerizing antigens were from an H-2(d+) strain. The tolerogenic effect was greatest in male as compared with female recipients. Offspring from the three breeding models in which no tolerance was seen, appeared to be sensitized based on poorer graft survival, or enhanced T- or B-cell responses to the noninherited H-2(b or k) antigens. Significantly higher percentages of maternal antigen(+) cells were found in the peripheral blood of tolerant versus nontolerant strains of backcross mice prior to transplant. Our findings imply that transplants are predisposed to tolerance or rejection due to recipient developmental history and immunogenetic background.

Regulation of dendritic cell (DC) function is critical for maintaining self-tolerance and preventing autoimmunity. The dendritic cell-specific transmembrane protein (DC-STAMP) plays a key role in cell-cell fusion of osteoclasts and foreign body giant cells, but though originally identified in DCs, its specific roles there remain undefined. Here, we report that aged DC-STAMP-deficient mice display several systemic autoimmune symptoms such as spontaneous lymphoproliferation, splenomegaly associated with infiltration of T cells in several organs and increased serum anti-double-stranded DNA antibody production. Although a lack of DC-STAMP did not inhibit DC differentiation or proliferation, antigen presentation activity of DC-STAMP-deficient DCs was significantly up-regulated in both class I and II pathways through increased phagocytotic activity compared with wild-type DCs, an activity likely leading to autoimmunity. Our results indicate that DC-STAMP is required for proper regulation of DC activity and maintenance of immune self-tolerance.

We investigated whether the immunogenicity of antigens delivered by recombinant E1-deleted adenovirus (Ad) is impaired by the concomitant priming of specific immunity to protein antigens of the vector. A comparative evaluation of the immunogenicity of hepatitis B surface antigen (HBsAg or S) or ovalbumin (OVA) was carried out in mice injected with either the antigen-encoding Ad vector or a corresponding plasmid DNA vaccine. Recombinant Ad, but not the plasmid DNA vaccine, induced long lasting, specific CD8 T-cell immunity to immunodominant epitopes of the two antigens. In contrast, the HBsAg-encoding pCI/S DNA, but not the Ad/S vaccine, was shown to prime CD8 T-cell responses to subdominant HBsAg epitopes. Ad/S-primed CD8 T-cell responses to immunodominant epitopes of vector-encoded or capsid-delivered Ad proteins apparently suppressed CD8 T-cell priming to subdominant HBsAg epitopes. In B-cell-deficient mice, the established, Ad-specific T-cell immunity induced by vaccination with an irrelevant Ad vector impaired the priming of HBsAg-specific CD8 T-cell responses by Ad/S. It is clear, therefore, that a T-cell immunity specific for Ad proteins (either delivered with the Ad capsid or transcribed from the Ad genome) is efficiently primed by vaccination with Ad vectors, and can limit the immunogenicity (particularly of subdominant epitopes) of Ad vector-encoded transgenic antigens.

Antigen processing of respiratory syncytial virus (RSV) fusion (F) protein epitopes F85-93 and F249-258 presented to cytotoxic T-lymphocytes (CTLs) by the murine major histocompatibility complex (MHC) class I molecule Kd was studied in different viral contexts. Epitope F85-93 was presented through a classical endogenous pathway dependent on the transporters associated with antigen processing (TAP) when the F protein was expressed from either RSV or recombinant vaccinia virus (rVACV). At least in cells infected with rVACV encoding either natural or cytosolic F protein, the proteasome was required for epitope processing. In cells infected with rVACV encoding the natural F protein, an additional endogenous TAP-independent presentation pathway was found for F85-93. In contrast, epitope F249-258 was presented only through TAP-independent pathways, but presentation was brefeldin A sensitive when the F protein was expressed from RSV, or mostly resistant when expressed from rVACV. Therefore, antigen-processing pathways with different mechanisms and subcellular localizations are accessible to individual epitopes presented by the same MHC class I molecule and processed from the same protein but in different viral contexts. This underscores both the diversity of pathways available and the influence of virus infection on presentation of epitopes to CTLs.

Objective: Autoimmune diabetes in the nonobese diabetic (NOD) mouse model results from a breakdown of T-cell tolerance caused by impaired tolerogenic dendritic cell development and regulatory T-cell (Treg) differentiation. Re-establishment of the Treg pool has been shown to confer T-cell tolerance and protection against diabetes. Here, we have investigated whether murine thymic stromal lymphopoietin (TSLP) re-established tolerogenic function of dendritic cells and induced differentiation and/or expansion of Tregs in NOD mice and protection against diabetes., Research Design and Methods: We examined the phenotype of TSLP-conditioned bone marrow dendritic cells (TSLP-DCs) of NOD mice and their functions to induce noninflammatory Th2 response and differentiation of Tregs. The functional relevance of TSLP and TSLP-DCs to development of diabetes was also tested., Results: Our results showed that bone marrow dendritic cells of NOD mice cultured in the presence of TSLP acquired signatures of tolerogenic dendritic cells, such as an absence of production of pro-inflammatory cytokines and a decreased expression of dendritic cell costimulatory molecules (CD80, CD86, and major histocompatibility complex class II) compared with LPS-treated dendritic cells. Furthermore, TSLP-DCs promoted noninflammatory Th2 response and induced the conversion of naïve T-cells into functional CD4(+)CD25(+)Foxp3(+) Tregs. We further showed that subcutaneous injections of TSLP for 6 days or a single intravenous injection of TSLP-DCs protected NOD mice against diabetes., Conclusions: Our study demonstrates that TSLP re-established a tolerogenic immune response in NOD mice and protects from diabetes, suggesting that TSLP may have a therapeutic potential for the treatment of type 1 diabetes.

The identification of CTL epitopes from tumor antigens is very important for the development of peptide-based, cancer-specific immunotherapy. Heparanase is broadly expressed in various advanced tumors and can serve as a universal tumor-associated antigen. Although several epitopes of heparanase antigen are known in humans, the corresponding knowledge in mice is still rather limited. The present study was designed to predict and identify the CTL epitopes in the mouse heparanase protein. For this purpose, H-2K(b)-restricted CTL epitopes were identified by using the following four-step procedure: (a) a computer-based epitope prediction from the amino acid sequence of mouse heparanase, (b) a peptide-binding assay to determine the affinity of the predicted epitopes with the H-2K(b) molecule, (c) the testing of the induction of CTLs toward various carcinoma cells expressing heparanase antigens and H-2K(b), and (d) the induction of immunoprotection and immunotherapy in vivo. The results showed that, of the tested peptides, effectors induced by peptides of mouse heparanase at residue positions 398 to 405 (LSLLFKKL; mHpa398) and 519 to 526 (FSYGFFVI; mHpa519) lysed three kinds of carcinoma cells expressing both heparanase and H-2K(b) (B16 melanoma cells, EL-4 lymphoma cells, and Lewis lung cancer cells). In vivo experiments indicated that mHpa398 and mHpa519 peptides offered the possibility of not only immunizing against tumors but also treating tumor-bearing hosts successfully. Our results suggest that the mHpa398 and mHpa519 peptides are novel H-2K(b)-restricted CTL epitopes capable of inducing heparanase-specific CTLs in vitro and in vivo. These epitopes may serve as valuable tools for the preclinical evaluation of vaccination strategies.

Immune-independent diabetes often occurs via pancreatic beta cell dysfunction. However, the role of the tumour suppressor p53 that regulates cellular life and death in multiple tissues, in pancreatic cell death and diabetes has not been clarified. We have therefore utilized an established mouse model for diabetes in which the MHC class I antigen is overexpressed in pancreatic beta cells under the rat insulin promoter, to investigate the role of p53. We show that pancreatic beta cell death, as determined by TUNEL staining, is elevated in transgenic mice compared to wild-type mice. However, there was no increase in immuno-reactivity towards anti-p53 antibodies in the pancreas of transgenic mice over the course of diabetes formation and beta cell death, suggesting that p53 may not be involved in these processes. Interestingly, p53 expression was also not induced in pancreas upon gamma-irradiation, which resulted in a massive increase in the number of TUNEL-positive cells, suggesting that the p53 pathway may not be causally involved in pancreatic cell death. To further confirm these findings, we generated MHC class I transgenic mice lacking p53 expression. Absence of p53 did not result in any significant changes in pancreatic morphology or affect cell death levels. Importantly, p53 absence did not rescue the diabetic phenotype of the transgenic mice. The results therefore demonstrate that p53 may not be causally involved in pancreatic beta cell death, and suggests that the classical cell death pathway dependent on p53 may not be operating in pancreatic beta cells.

How the fetus escapes rejection by the maternal immune system remains one of the major unsolved questions in transplantation immunology. Using a system to visualize both CD4+ and CD8+ T cell responses during pregnancy in mice, we find that maternal T cells become aware of the fetal allograft exclusively through "indirect" antigen presentation, meaning that T cell engagement requires the uptake and processing of fetal/placental antigen by maternal APCs. This reliance on a relatively minor allorecognition pathway removes a major threat to fetal survival, since it avoids engaging the large number of T cells that typically drive acute transplant rejection through their ability to directly interact with foreign MHC molecules. Furthermore, CD8+ T cells that indirectly recognize fetal/placental antigen undergo clonal deletion without priming for cytotoxic effector function and cannot induce antigen-specific fetal demise even when artificially activated. Antigen presentation commenced only at mid-gestation, in association with the endovascular invasion of placental trophoblasts and the hematogenous release of placental debris. Our results suggest that limited pathways of antigen presentation, in conjunction with tandem mechanisms of immune evasion, contribute to the unique immunological status of the fetus. The pronounced degree of T cell ignorance of the fetus also has implications for the pathophysiology of immune-mediated early pregnancy loss.

alphabeta T cell receptors (TCRs) can crossreact with both self- and foreign- major histocompatibility complex (MHC) proteins in an enigmatic phenomenon termed alloreactivity. Here we present the 2.35 A structure of the 2C TCR complexed with its foreign ligand H-2L(d)-QL9. Surprisingly, we find that this TCR utilizes a different strategy to engage the foreign pMHC in comparison to the manner in which it recognizes a self ligand H-2K(b)-dEV8. 2C engages both shared and polymorphic residues on L(d) and K(b), as well as the unrelated QL9 and dEV8 peptide antigens, in unique pair-wise contacts, resulting in greater structural complementarity with the L(d)-QL9 complex. In the structure of an engineered, high-affinity 2C TCR variant bound to H-2L(d)-QL9, the "wild-type" TCR-MHC binding orientation persists despite modified TCR-CDR3alpha interactions with peptide. Thus, a single TCR recognizes two globally similar, but distinct ligands by divergent mechanisms, indicating that receptor-ligand crossreactivity can occur in the absence of molecular mimicry.

In the course of viral infection, the immune system exploits only a fraction of the available CTL repertoire and focuses on a few of a myriad of potentially antigenic peptides. This phenomenon, known as immunodominance, depends on a number of factors, including antigen processing and transport, MHC binding, competition for antigen-presenting cells, availability of the CD8 T cell repertoire and other mechanisms that function largely by restricting the immune response. Here we elucidate a novel mechanism that increases the immunodominance of the epitope rather by enhancing the immune response. Using a peptide-specific MHC-restricted mAb and functional assays of CTL activation, we show that T cells with high avidity for the immunodominant, H-2D(d) restricted, P18-I10 epitope expand rapidly following immunization, and this expansion in turn determines the level of the P18-I10 epitope immunodominance. This proliferation has little dependence on the number of MHC-peptide complexes. Since most self-reactive T cells of high avidity are depleted in the thymus, the selection of immunodominant epitopes based on the expansion of high-avidity T cells in the periphery reduces the potential for autoimmunity.

Cytotoxic T lymphocytes (CTLs) are critical for control of respiratory syncytial virus (RSV) infection in humans and mice. To investigate cellular immune responses to infection, it is important to identify major histocompatibility complex (MHC) class I-restricted CTL epitopes. In this study, we identified a new RSV-specific, H-2K(d)-restricted subdominant epitope in the M2 protein, M2(127-135) (amino acids 127 to 135). This finding allowed us to study the frequency of T lymphocytes responding to two H-2K(d)-presented epitopes in the same protein following RSV infection by enzyme-linked immunospot (ELISPOT) and intracellular cytokine assays for both lymphoid and nonlymphoid tissues. For the subdominant epitope, we identified an optimal nine-amino-acid peptide, VYNTVISYI, which contained an H-2K(d) consensus sequence with Y at position 2 and I at position 9. In addition, an MHC class I stabilization assay using TAP-2-deficient RMA-S cells transfected with K(d) or L(d) indicated that the epitope was presented by K(d). The ratios of T lymphocytes during the peak CTL response to RSV infection that were specific for M2(82-90) (dominant) to T lymphocytes specific for M2(127-135) (subdominant) were approximately 3:1 in the spleen and 10:1 in the lung. These ratios were observed consistently in primary or secondary infection by the ELISPOT assay and in secondary infection by MHC/peptide tetramer staining. The number of antigen-specific T lymphocytes dropped in the 6 weeks after infection; however, the proportions of T lymphocytes specific for the immunodominant and subdominant epitopes were maintained to a remarkable degree in a tissue-specific manner. These studies will facilitate investigation of the regulation of immunodominance of RSV-specific CTL epitopes.

NK cells are key effectors of innate immunity and host survival during cytomegalovirus (CMV) infection. Innate murine CMV (MCMV) resistance in MA/My mice requires Ly49H/m157-independent H-2k-linked NK cell control. Here we show that replacement of MA/My H-2k with C57L H-2b susceptibility genes led to a remarkable loss of innate virus immunity, though NK gamma interferon was induced in H-2b and H-2k strains shortly after infection. Thus, H-2b genes expressed in C57L or MA/My.L-H2b are sufficient in alerting NK cells to intrusion but fail to support NK restraint of viral infection. In addition, novel H-2 recombinant strains were produced and utilized in a further refinement of a critical genetic interval controlling innate H-2k-linked MCMV resistance. Importantly, this analysis excluded the gene interval from Kk class I through class II. The responsible gene(s) therefore resides in an interval spanning Dk class Ia and more-distal major histocompatibility complex (MHC) nonclassical class Ib genes. Recently, the NK activation receptor Ly49P and MHC class I Dk proteins were genetically implicated in MCMV resistance, in part because Ly49P-expressing reporter T cells could specifically bind Dk molecules on MCMV-infected mouse embryonic fibroblasts (MEFs). However, as we found that H-2k innate resistance differs in the C57L or MA/My backgrounds and because MCMV very efficiently downregulates H-2k class I proteins in L929 cells and primary MEFs shortly after infection, a Ly49P/Dk model should not fully explain H-2k-linked MCMV resistance.