A structural basis for LCMV immune evasion: subversion of H-2D(b) and H-2K(b) presentation of gp33 revealed by comparative crystal structure.Analyses

LCMV infection of H-2(b) mice generates a CD8(+) CTL response mainly directed toward three immunodominant epitopes. One of these, gp33, is presented by both H-2D(b) and H-2K(b) MHC class I molecules. The virus can escape immune recognition in the context of both these MHC class I molecules through single mutations of the peptide. In order to understand the underlying structural mechanism, we determined the crystal structures of both complexes. The structures reveal that the peptide is presented in two diametrically opposed manners by H-2D(b) and H-2K(b), with residues used as anchor positions in one MHC class I molecule interacting with the TCR in the other. Importantly, the peptide's N-terminal residue p1K protrudes from the binding cleft in H-2K(b). We present structural evidence that explains the functional consequences of single mutations found in escape variants.     

Steric recognition of T-cell receptor contact residues is required to map mutant epitopes by immunoinformatical programmes

MHC class I-restricted CD8 T-lymphocyte epitopes comprise anchor motifs, T-cell receptor (TCR) contact residues and the peptide backbone. Serial variant epitopes with substitution of amino acids at either anchor motifs or TCR contact residues have been synthesized for specific interferon-γ responses to clarify the TCR recognition mechanism as well as to assess the epitope prediction capacity of immunoinformatical programmes. CD8 T lymphocytes recognise the steric configuration of functional groups at the TCR contact side chain with a parallel observation that peptide backbones of various epitopes adapt to the conserved conformation upon binding to the same MHC class I molecule. Variant epitopes with amino acid substitutions at the TCR contact site are not recognised by specific CD8 T lymphocytes without compromising their binding capacity to MHC class I molecules, which demonstrates two discrete antigen presentation events for the binding of peptides to MHC class I molecules and for TCR recognition. The predicted outcome of immunoinformatical programmes is not consistent with the results of epitope identification by laboratory experiments in the absence of information on the interaction with TCR contact residues. Immunoinformatical programmes based on the binding affinity to MHC class I molecules are not sufficient for the accurate prediction of CD8 T-lymphocyte epitopes. The predictive capacity is further improved to distinguish mutant epitopes from the non-mutated epitopes if the peptide-TCR interface is integrated into the computing simulation programme.     

Role of alpha3 domain of class I MHC molecules in the activation of high- and low-avidity CD8+ CTLs

CD8 can serve as a co-receptor or accessory molecule on the surface of CTL. As a co-receptor, CD8 can bind to the alpha3 domain of the same MHC class I molecules as the TCR to facilitate TCR signaling. To evaluate the role of the MHC class I molecule alpha3 domain in the activation of CD8(+) CTL, we have produced a soluble 227 mutant of H-2D(d), with a point mutation in the alpha3 domain (Glu227 --> Lys). 227 mutant class I-peptide complexes were not able to effectively activate H-2D(d)-restricted CD8 T cells in vitro, as measured by IFN-gamma production by an epitope-specific CD8(+) CTL line. However, the 227 mutant class I-peptide complexes in the presence of another MHC class I molecule (H-2K(b)) (that cannot present the peptide) with a normal alpha3 domain can induce the activation of CD8(+) CTL. Therefore, in order to activate CD8(+) CTL, the alpha3 domain of MHC class I does not have to be located on the same molecule with the alpha1 and alpha2 domains of MHC class I. A low-avidity CD8(+) CTL line was significantly less sensitive to stimulation by the 227 mutant class I-peptide complexes in the presence of the H-2K(b) molecule. Thus, low-avidity CTL may not be able to take advantage of the interaction between CD8 and the alpha3 domain of non-presenting class I MHC molecules, perhaps because of a shorter dwell time for the TCR-MHC interaction.     

Evaluation of T-cell responses to peptides and lipopeptides with MHC class I binding motifs derived from the amino acid sequence of the 19-kDa lipoprotein of Mycobacterium tuberculosis

Cytotoxic T-lymphocyte (CTL) epitopes on the 19-kDa lipoprotein from Mycobacterium tuberculosis were identified by the use of lipopeptides and their cytokine profile studied. Selection of candidate CTL epitopes was based on synthetic peptides derived from the amino acid sequence of the 19-kDa lipoprotein showing major histocompatibility complex class I (MHC-I) binding motifs (H-2D(b) and H-2L(d)). Their ability to up-regulate and stabilize MHC-I molecules on the mouse lymphoma cell line RMA-S was studied. Similar studies were performed with peptides, in which the anchor amino acid of the H-2D(b) MHC-I motif was replaced by alanine. Three out of five peptides with H-2D(b) or H-2L(d) binding motifs and their corresponding lipopeptides as well, up-regulated and stabilized the H-2D(b) molecules on RMA-S cells. Replacement of the anchor amino acid residues of the H-2D(b) MHC-I motif by alanine revealed that the anchor amino acid asparagine at position 5, contributed more to binding of peptide to H-2D(b) molecules than leucine at position 11. The closely related lipopeptides LP19c and LP19d, in combination with incomplete Freund's adjuvant (IFA), induced CTL responses in C57BL/6 (H-2(b)) mice. These CTLs could recognize the naturally processed antigen, i.e. the 19-kDa antigen protein produced and processed by the EX-19 cell line. The capacity of the various lipopeptides to induce CTL correlated well with the ability of the (lipo)peptide to up-regulate and to stabilize H-2D(b) molecules. Lipopeptide LP19c primed spleen cells showed a T helper type one profile after in vitro stimulation with P19c and P19d 19 kDa peptides. The approach to characterize presumptive 19-kDa CTL epitopes might lead to selection of promising CTL epitopes, which can be applied in the development of subunit tuberculosis vaccines.     

Exact prediction of a natural T cell epitope.

T lymphocytes recognize their antigen as peptides associated with major histocompatibility complex (MHC) molecules. Peptides naturally presented by MHC class I molecules are uniform in length and have a specific motif, both defined by the respective MHC allele (Falk, K. et al. Nature 1991. 351:290). These allele-specific motifs should allow exact prediction of natural T cell epitopes. H-2Kb-restricted epitopes, for example, have a length of eight amino acid residues and conserved anchor residues at positions 5 and 8. According to this information, we predicted the natural Kb-restricted epitope of ovalbumin, thought to be contained in the 19-mer IINFEKLTEWTSSNVMEER, to be SIINFEKL. Here we show that this prediction is correct. Thus, exact prediction of natural T cell epitopes is possible.     

Dendritic cells infected with Mycobacterium bovis bacillus Calmette Guerin activate CD8(+) T cells with specificity for a novel mycobacterial epitope

Although CD4(+) T cells are essential for protective immunity against Mycobacterium tuberculosis infection, recent reports indicate that CD8(+) T cells may also play a critical role in the control of this infection. However, the epitope specificity and the mechanisms of activation of mycobacteria-reactive CD8(+) T cells are poorly characterized. In order to study the CD8(+) T cell responses to the model mycobacterial antigen, MPT64, we used recombinant vaccinia virus expressing MPT64 (VVWR-64) and a panel of MPT64-derived peptides to establish that the peptide MPT64(190-198) contains an H-2D(b)-restricted CD8(+) T cell epitope. A cytotoxic T lymphocyte response to this peptide could be demonstrated in M. bovis bacillus Calmette Guerin (BCG)-infected mice following repeated in vitro stimulation. When bone marrow-derived dendritic cells (DC) were infected with BCG, the expression of MHC class I molecules by DC was up-regulated in parallel with MHC class II and B7-2, whereas CD1d expression level was not modified. Moreover, BCG-infected DC activated MPT64(190-198)-specific CD8(+) T cells to secrete IFN-gamma, although with a lower efficacy than VVWR-64-infected DC. The production of IFN-gamma by MPT64(190-198)-specific CD8(+) T cells was inhibited by antibodies to MHC class I, but not to CD1d. These data suggest that mycobacteria-specific CD8(+) T cells are primed during infection. Therefore, anti-mycobacterial vaccine strategies targeting the activation of specific CD8(+) T cells by DC may have improved protective efficacy.     

The CD8alphabeta co-receptor on double-positive thymocytes binds with differing affinities to the products of distinct class I MHC loci

The CD8 co-receptor is essential for TCR-dependent immune recognition and T cell development involving peptides bound to MHC class I (MHCI) molecules. The dominant interaction of CD8 alpha alpha and alpha beta co-receptors is with the alpha3 domain of an MHCI molecule. Whether this interaction is different for the products of various MHCI loci is currently unknown. Here we examine the interaction between H-2K(b) and H-2D(b), the two MHCI molecules in the C57BL / 6 mouse, and CD8 using H-2K(b) and H-2D(b) tetramers. The MHCI molecules bind to the CD8alpha beta co-receptor on double-positive thymocytes with different avidities (H-2K(b) > D(b)). The differences are linked to their respective alpha3 domains. Hence, an H-2D(b)K(b) tetramer comprising D(b)alpha1--alpha2 and K(b)alpha3 domains shows more binding than H-2D(b). We also quantitated the monomeric affinities of CD8alpha alpha and CD8alpha beta for H-2K(b) and H-2D(b). The H-2K(b) interaction with CD8alpha alpha and CD8alpha beta is stronger than that of H-2D(b). Given that T cell repertoire selection of DP thymocytes is a function of both TCR-pMHCI and CD8alpha beta-pMHCI avidities, these differences may explain the dominant role of H-2K(b) as compared to H-2D(b) in CD8 T cell development of C57BL / 6 mice. The influence of allelic and non-allelic alpha3 polymorphisms on thymic selection processes are discussed.     

MUC1 peptide epitopes associated with five different H-2 class I molecules

We have previously described the induction of murine CD8⁺ major histocompatibility complex (MHC) class I-restricted cytotoxic T cells (CTL) recognizing the 20-amino acid repeat region of the human mucin 1 (MUC1) variable number of tandem repeats region (VNTR), a mucin greatly increased in expression in breast cancer and proposed as a target for immunotherapy. In that study, CTL could detect MUC1 peptides associated with the MHC of all nine strains examined, and we now report the different epitopes presented by five different MHC class I molecules. The epitopes were defined in CTL assays using peptide-pulsed phytohemagglutinin blasts or MHC class I-transfected L cells as targets; in addition, peptide binding assays and T cell proliferation studies were performed. Within the 20-amino acid VNTR, nine potential epitopes could be defined. The epitopes for the four MHC class I molecules [K^b (three epitopes), D^d, L^d and K^k] were closely related, all containing the amino acids PDTRPAP. For D^b, three epitopes were identified, all containing APGSTAP. Most of the epitopes did not contain a consensus motif for the particular MHC class I allele, and bound with low ‘affinity’, compared with known high-affinity peptides. CD8⁺ T cell proliferation also occurred to the same MHC class I-presented epitopes. Finally, when conventional anchor residues were introduced into the peptides, peptide binding increased, whereas CTL recognition was either retained (K^b) or lost (D^b) depending on the epitope.     

Selective absence of CD8+ TCRalpha beta+ intestinal epithelial cells in transgenic mice expressing beta2-microglobulin-associated ligands exclusively on thymic cortical epithelium

Whereas interactions between the TCRalpha beta and self MHC:peptide complexes are clearly required for positive selection of mature CD4(+) and CD8(+) T cells during intrathymic development, the role of self or foreign ligands in maintaining the peripheral T cell repertoire is still controversial. In this report we have utilized keratin 14-beta2-microglobulin (K14-beta2m)-transgenic mice expressing beta2m-associated ligands exclusively on thymic cortical epithelial cells to address the possible influence of TCR:ligand interactions in peripheral CD8(+) T cell homeostasis. Our data indicate that CD8(+) T cells in peripheral lymphoid tissues are present in normal numbers in the absence of self MHC class I:peptide ligands. Surprisingly, however, steady state homeostasis of CD8(+) T cells in the intestinal epithelium is severely affected by the absence of beta2m-associated ligands. Indeed TCRalpha beta(+) IEL subsets expressing CD8alpha beta or CD8alpha alpha are both dramatically reduced in K14-beta2m mice, suggesting that the development, survival or expansion of CD8(+) IEL depends upon interaction of the TCR with MHC class I:peptide or other beta2m-associated ligands elsewhere than on thymic cortical epithelium. Collectively, our data reveal an unexpected difference in the regulation of CD8(+) T cell homeostasis by beta2m-associated ligands in the intestine as compared to peripheral lymphoid organs.     

MHC class I-associated peptides identified from normal platelets and from individuals with idiopathic thrombocytopenic purpura

Major histocompatibility complex (MHC) class I molecules bind and display peptide antigens on the cell surface. CD8(+) T lymphocytes recognize peptides in association with class I proteins to initiate a cytotoxic immune response. To understand the specificity of such immune responses and to facilitate the development of therapies for disease, it is important to identify MHC-presented peptides. In this study, platelets, easily obtainable and often associated with immune-mediated disease, were selected to identify MHC class I-associated peptides. MHC-associated peptides presented on platelets of normal individuals and individuals with idiopathic thrombocytopenic purpura (ITP) were characterized. ITP is characterized by the premature immune destruction of platelets. It is associated with the production of antiplatelet autoantibodies, most often targeting platelet membrane GPIIb/IIIa or GPIb/IX. In addition to characterizing five fully and several partially sequenced peptides from platelets, the peptide GPRGA(L/I)S(L/I)(L/I) was identified from four of the five ITP patients. The anchor motif of this peptide correlates with the presence of the HLA-B7 allele. A BLAST search identified this peptide as GPIb (4-12). In conclusion, platelets from normal and ITP individuals can present peptides from general cellular proteins and platelet specific proteins, such as GPIb, to the immune system via MHC class I.     

Antigen-specific cytotoxic T lymphocytes protect against lethal West Nile virus encephalitis

Infection with West Nile virus (WNV) causes fatal encephalitis in immunocompromised animals. Previous studies in mice have established that T cell protection is required for clearance of WNV infection from tissues and preventing viral persistence. The current study assessed whether specific WNV peptide epitopes could elicit a cytotoxic T lymphocyte (CTL) response capable of protecting against virus infection. Hidden Markov model analysis was used to identify WNV-encoded peptides that bound the MHC class I proteins K(b) or D(b). Of the 35 peptides predicted to bind MHC class I molecules, one immunodominant CTL recognition peptide was identified in each of the envelope and non-structural protein 4B genes. Addition of these but not control peptides to CD8(+) T cells from WNV-infected mice induced IFN-gamma production. CTL clones that were generated ex vivo lysed peptide-pulsed or WNV-infected target cells in an antigen-specific manner. Finally, adoptive transfer of a mixture of envelope- and non-structural protein 4B-specific CTL to recipient mice protected against lethal WNV challenge. Based on this, we conclude that CTL responses against immundominant WNV epitopes confer protective immunity and thus should be targets for inclusion in new vaccines.     

Augmented induction of CD8+ cytotoxic T-cell response and antitumour resistance by T helper type 1-inducing peptide

The effector CD8(+) T cells recognize major histocompatibility complex (MHC) class I binding altered self-peptides expressed in tumour cells. Although the requirement for CD4(+) T helper type 1 (Th1) cells in regulating CD8(+) T cells has been documented, their target epitopes and functional impact in antitumour responses remain unclear. We examined whether a potent immunogenic peptide of Mycobacterium tuberculosis eliciting Th1 immunity contributes to the generation of CD8(+) T cells and to protective antitumour immune responses to unrelated tumour-specific antigens. Peptide-25, a major Th epitope of Ag85B from M. tuberculosis preferentially induced CD4(+) Th1 cells in C57BL/6 mice and had an augmenting effect on Th1 generation for coimmunized unrelated antigenic peptides. Coimmunization of mice with Peptide-25 and ovalbumin (OVA) or Peptide-25 and B16 melanoma peptide [tyrosinase-related protein-2 (TRP-2)] for MHC class I led to a profound increase in CD8(+) T cells specific for OVA and TRP-2 peptides, respectively. This heightened response depended on Peptide-25-specific CD4(+) T cells and interferon-gamma-producing T cells. In tumour protection assays, immunization with Peptide-25 and OVA resulted in the enhancement of CD8(+) cytotoxic cell generation specific for OVA and the growth inhibition of EL-4 thymoma expressing OVA peptide leading to the tumour rejection. These phenomena were not achieved by immunization with OVA alone. Peptide-25-reactive Th1 cells counteractivated dendritic cells in the presence of Peptide-25 leading them to activate and present OVA peptide to CD8(+) cytotoxic T cells.     

Multiple proteases process viral antigens for presentation by MHC class I molecules to CD8(+) T lymphocytes

Recognition by CD8(+) cytotoxic T lymphocytes of any intracellular viral protein requires its initial cytosolic proteolytic processing, the translocation of processed peptides to the endoplasmic reticulum via the transporters associated with antigen processing, and their binding to nascent major histocompatibility complex (MHC) class I molecules that then present the antigenic peptides at the infected cell surface. From initial assumptions that the multicatalytic and ubiquitous proteasome is the only protease capable of fully generating peptide ligands for MHC class I molecules, the last few years have seen the identification of a number of alternative proteases that contribute to endogenous antigen processing. Trimming by non-proteasomal proteases of precursor peptides produced by proteasomes is now a well-established fact. In addition, proteases that can process antigens in a fully proteasome-independent fashion have also been identified. The final level of presentation of many viral epitopes is probably the result of interplay between different proteolytic activities. This expands the number of tissues and physiological and pathological situations compatible with antigen presentation, as well as the universe of pathogen-derived sequences available for recognition by CD8(+) T lymphocytes.     

Lysis of interferon-gamma activated Schwann cell by cross-reactive CD8+ alpha/beta T cells with specificity for the mycobacterial 65 kd heat shock protein.

Heat shock protein (hsp) 65 is a major T cell antigen of Mycobacterium leprae. The hsp 65 of M. leprae is nearly identical in M. bovis/M. tuberculosis (greater than 95% protein sequence homology) and surprisingly similar in man (65% protein sequence homology). Recently, we had provided evidence in a murine model that CD8+ T cells recognize and lyse Schwann cells presenting M. leprae antigen in the context of major histocompatibility (MHC) class I gene products. Because murine Schwann cells are class I negative, antigen presentation requires prior stimulation with interferon-gamma (IFN-gamma). CD8+ T cells were activated against tryptic fragments of mycobacterial hsp 65. These T cells recognized epitopes of hsp 65 which had been generated through the cytoplasmic class I processing pathway. They were also capable of lysing Schwann cells which had been activated by IFN-gamma and not primed with nominal hsp 65 peptides. In contrast, T cells activated against tryptic ova peptides only lysed Schwann cells which had been both stimulated with IFN-gamma and primed with ova peptides. Evidence is presented that class I (H-2D) restricted, CD8+ alpha/beta T lymphocytes with specificity for the mycobacterial hsp 65 recognize IFN-gamma-stimulated Schwann cells probably because they are specific for a(n) epitope(s) shared by the bacterial hsp and a host cognate. Activation of autoreactive T cells with specificity to shared epitopes could contribute to nerve damage in tuberculoid leprosy which is characterized by low to absent M. leprae in Schwann cells.     

Transient expression of bacterial gene fragments in eukaryotic cells: implications for CD8(+) T cell epitope analysis

CD8(+) T cells are potent effectors of acquired immunity against some viruses and intracellular bacterial pathogens. Antigens recognized by CD8(+) T cells are small, 8-9 amino acid peptides derived from proteins produced by the pathogen. These peptides are presented by MHC class I molecules on the surface of the infected cell. When characterizing the CD8(+) T cell response to a bacterial or viral pathogen, it is often necessary to express an antigenic protein in a eukaryotic host cell that is capable of processing and presenting peptide epitopes to antigen-specific CD8(+) T cells. We describe a system designed to transiently express bacterial polypeptides and MHC class I molecules in eukaryotic cells. Recognition of these peptide-MHC complexes stimulates TNF production by antigen-specific CD8(+) T cell lines. This system should be useful for analysis of CD8(+) T cell epitope-containing bacterial gene fragments when expression of the entire bacterial protein is detrimental to the eukaryotic cell, or when overexpression of the bacterial gene is detrimental to the bacterial cloning strain. Furthermore, this system can be used for the rapid mapping of CD8(+) T cell epitopes within a protein.     

Specificity, magnitude, and kinetics of MOG-specific CD8+ T cell responses during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) has traditionally been thought to be almost exclusively mediated by CD4(+) effector T cells. Here, we provide evidence for the existence of mouse CD8(+) T cells that are specific for an epitope of the myelin oligodendrocyte glycoprotein (MOG). Using a panel of truncated MOG peptides, we have identified the minimal epitope recognized by these T cells as MOG 37-46. This peptide, while possessing relatively low affinity for H-2D(b), efficiently stimulates IFN-gamma production from MOG-specific CD8(+) T cell lines in vitro and induces EAE in vivo. To further characterize the magnitude and kinetics of expansion of the MOG-specific CD8(+) T cell population in vivo, we used MOG 37-50/H-2D(b) MHC tetramers to visualize MOG-specific CD8(+) effectors in the peripheral lymphoid organs and central nervous system during the course of EAE induction and progression. Our results identify MOG-specific CD8(+) T cells in the central nervous system prior to and after the onset of disease, suggesting that CD8(+) T cells are a possible target for therapeutic intervention during EAE.     

Idiotypic T cells specific for Morbillivirus nucleocapsid protein process and present their TCR to cognate anti-idiotypic CD8+ T cells

CD8(+) T cells are activated by the presentation of antigenic peptide through MHC class I molecules. Newly synthesized proteins formed as defective ribosomal products (DRiPs) can act as a major source of antigenic peptides for MHC class I presentation pathway. Majority of these peptides are generated from the intracellular degradation of self antigens. In the present study, we have shown that newly synthesized T cell receptor (TCR) beta chains formed as DRiPs in T cells are ubiquitinated and degraded by the proteasomes. These TCR-DRiPs are processed and presented by activated T cells to cognate anti-idiotypic CD8(+) T cells. Presentation of TCR idiopeptide (peptide derived from the variable region of idiotypic TCR) by activated T cells leads to Bcl-2 expression and cytokine secretion by anti-idiotypic CD8(+) T cells. Presentation of intracellular antigen by T cells may have important implications in immunoregulation, control of lymphotropic virus infection and autoimmune diseases.     

Identification of broadly recognized, T helper 1 lymphocyte epitopes in an equine lentivirus

Equine infectious anaemia virus (EIAV) is a horse lentivirus causing lifelong, persistent infection. During acute infection, CD8(+) cytotoxic T lymphocytes (CTL) are probably involved in terminating plasma viraemia. However, only a few EIAV CTL epitopes, restricted to fewer horse major histocompatibility complex (MHC) class I alleles, are known. As interferon-gamma (IFN-gamma)-secreting CD4(+), T helper 1 (Th1) lymphocytes promote CTL activity and help maintain memory CTL, identifying broadly recognized EIAV Th1 epitopes would contribute significantly to vaccine strategies seeking to promote strong CTL responses among horses with varying class I haplotypes. To this end, peripheral blood mononuclear cells (PBMC) from 10 MHC disparate, EIAV-infected horses were tested in T-lymphocyte proliferation assays for recognition of peptides from the Gag p26 capsid region and a portion of Pol. Both regions are highly conserved among EIAV isolates, and this Pol region is 51-63% homologous to other lentiviral Pol proteins. Seven of 10 horses recognized peptide Gag 221-245, and peptides Gag 242-261 and Pol 323-344 were recognized by five and four horses, respectively. Furthermore, the Gag peptides were recognized by two additional horses after resolving their initial plasma viraemia, indicating that these two peptides can be immunodominant early in infection. Gag peptide-responsive PBMC produced only IFN-gamma, indicating a Th1 response, while Pol 323-344-responsive PBMC produced IFN-gamma both with and without interleukin-4. PBMC from uninfected horses failed to either proliferate or secrete cytokines in response to peptide stimulation. Finally, CD4(+) T lymphocytes were required for proliferation responses, as shown by assays using CD4- versus CD8-depleted PBMC.