Identification and functional validation of MHC class I epitopes in the tumor-associated antigen 5T4

The cancer vaccine TroVax, modified vaccinia Ankara encoding the tumor-associated antigen 5T4, has been tested in phase I and II studies in colorectal cancer patients. Monitoring of 5T4-specific immune responses in patients receiving TroVax is critical since it could inform future refinements to the therapeutic or provide a surrogate marker of clinical efficacy. Tumor-specific cytotoxic T lymphocyte (CTL) are considered to be a key component of an effective anti-cancer immune response. Though numerous techniques have been employed to identify CTL epitopes, many are labor intensive, of variable reliability or biased toward common alleles such as human leukocyte antigen (HLA)-A2. A new high-throughput technique, iTopia, enables peptides to be evaluated on the basis of their physical binding properties for HLA alleles. This technique has been utilized to rapidly screen a panel of overlapping peptides, spanning the length of 5T4. Initially, peptides which bound to four class I alleles (A*0101, A*0201, A*0301 and B*0702) were identified and their physical binding characteristics assessed further by analysis of relative affinity and complex stability. 46 putative CTL epitopes have been identified which bind to at least one of the four HLA alleles. Using PBMCs from patients vaccinated with TroVax, we have used the interferon gamma (IFN gamma) ELISpot assay to validate one predicted A1 and two A2 epitopes. Conclusion: iTopia represents a rapid and high-throughput technique to identify CTL epitopes.     

Priming of T cells by exogenous antigen cross-presented on MHC class I molecules

Cross-presentation is the process whereby bone-marrow-derived antigen-presenting cells acquire, process and present exogenous antigen as peptides bound to MHC class I molecules to CD8(+) T cells. Professional antigen-presenting cells acquire cell-associated antigen predominantly in the form of protein, then process and present antigenic peptides on their surface MHC class I molecules via several mechanisms and efficiently cross-prime na?ve CD8(+) T cells in vivo. This pathway is of considerable interest because it has an important role in the immune surveillance of tissues for pathogens and cancers.     

Structure of an autoimmune T cell receptor complexed with class II peptide-MHC: insights into MHC bias and antigen specificity

T cell receptor crossreactivity with different peptide ligands and biased recognition of MHC are coupled features of antigen recognition that are necessary for the T cell's diverse functional repertoire. In the crystal structure between an autoreactive, EAE T cell clone 172.10 and myelin basic protein (1-11) presented by class II MHC I-Au, recognition of the MHC is dominated by the Vbeta domain of the TCR, which interacts with the MHC alpha chain in a manner suggestive of a germline-encoded TCR/MHC "anchor point." Strikingly, there are few specific contacts between the TCR CDR3 loops and the MBP peptide. We also find that over 1,000,000 different peptides derived from combinatorial libraries can activate 172.10, yet the TCR strongly prefers the native MBP contact residues. We suggest that while TCR scanning of pMHC may be degenerate due to the TCR germline bias for MHC, recognition of structurally distinct agonist peptides is not indicative of TCR promiscuity, but rather highly specific alternative solutions to TCR engagement.     

Comparison of gamma-interferon and alloantigen-induced T cell factors in the induction of MHC class II antigen expression and in the modulation of the immunogenicity of lymphocyte-free rat bone marrow

In a rat model, we compared the effects of various soluble products released by T cells, such as the unspecifically acting gamma-interferon or the newly detected alloantigen-induced factors that specifically act on nonlymphoid, hemopoietic bone marrow cells. We found two types of reactivity patterns with regard to the induction of MHC class II antigen expression on these cells. The very same patterns could be demonstrated when we investigated the modulation of their stimulatory capacity, i.e., their immunogenicity in a T proliferation assay. These findings are discussed in relation to the increasing incidence of immunologically mediated graft rejections in clinical bone marrow transplantation following T cell purging.     

Immunohistochemical characterization of infiltrating mononuclear cells in the rat heart with experimental autoimmune giant cell myocarditis.

The pathogenesis of giant cell myocarditis remains unclear. Subsets of inflammatory infiltrating cells may reflect the pathogenesis and etiology of the disease. Therefore, we examined subsets of infiltrating mononuclear cells in the heart of the rat with experimental giant cell myocarditis. Lewis rats were immunized with cardiac myosin in Freund's complete adjuvant (FCA). Severe myocarditis characterized by congestive heart failure and multinucleated giant cells were elicited. The lesions were composed of predominant mononuclear cells, polymorphonuclear neutrophils and fragments of degenerated myocardial fibres. The subsets of infiltrating mononuclear cells were investigated using MoAbs against rat CD4+ T cell (W3/25), CD8+ T cell (CX8), B cell (OX33) and macrophage (OX42). By serial examination, bound immunoglobulin could only be found on degenerated myocardial fibres. In this model, most infiltrating mononuclear cells were composed of macrophages and CD4+ T cells. The frequencies of macrophages and CD4+ T cells were 73.7% and 13.8%, respectively. CD8+ T cells were scarce and B cells were rare in the lesions. The frequencies of CD8+ T cells and B cells were 4.5% and 0.4%, respectively. The dominance of macrophages and CD4+ T cells was the constant finding among the sites of the lesions and throughout the course of the disease. These characteristic subsets of infiltrating cells were in contrast to those of murine viral myocarditis which were mainly composed of natural killer (NK) cells and CD8+ T cells. Clarifying the subsets of infiltrating cells in myocarditis may contribute to differential diagnosis of myocarditis between viral and autoimmune types. From this study, the pathogenesis of experimental autoimmune giant cell myocarditis seemed to be closely related to CD4+ T cells and macrophages.     

Dendritic cell/macrophage precursors capture exogenous antigen for MHC class I presentation by dendritic cells

Presentation of MHC class I antigens by professional antigen-presenting cells (APC) is an important pathway in priming cytotoxic T lymphocyte responses in vivo. This study sought to identify the nature of the professional APC responsible for indirect class I presentation by examining a special feature of professional APC, namely their ability to process exogenous forms of antigen for class I presentation. Incubation of highly purified bone marrow-derived precursor cells with chicken ovalbumin (OVA) led to the efficient presentation of the major class I-restricted OVA determinant by mature dendritic cells (DC), but not by macrophages (Mϕ) derived from the precursor population. DC as well as macrophages were, however, able to mediate class II presentation of OVA, suggesting that macrophages were deficient in class I processing but not in capturing exogenous OVA. The majority of mature DC, i.e. over 80 %, generated from the precursor cells pulsed with OVA, presented the class I OVA epitope. Upon maturation, class I presentation of OVA by DC was greatly reduced, suggesting that class I processing of exogenous antigen is modulated during DC maturation in a manner similar to class II antigen processing. This study shows that bone marrow-derived DC/Mϕ progenitors capture exogenous antigen for class I presentation, and that cells of the DC lineage can be functionally distinguished from cells of the macrophage lineage based on their ability to process exogenous antigen for class I presentation.     

MHC class I-mediated exogenous antigen presentation by exosomes secreted from immature and mature bone marrow derived dendritic cells

Exosomes are 50-90 nm vesicles with antigen presenting ability carrying major histocompatibility complex (MHC) class I, class II, abundant co-stimulatory molecules and some tetraspan proteins. Although dendritic cells (DCs) are one of the professional antigen presenting cells capable of presenting exogenous antigens in MHC class I-mediated antigen specific manner (cross-presentation), the cross-presentation ability by exosomes from immature or mature DCs are unknown. Here we show that exosomes released from ovalbumin (OVA) protein-pulsed bone marrow derived dendritic cells (BM-DCs) weakly present the peptide determinants to OVA specific MHC class I-restricted CD8(+) T cell hybridomas. The exosomes secreted by OVA(257-264) peptide- or OVA protein-pulsed mature BM-DCs activated OVA specific MHC class I-restricted T cell hybridomas more efficiently than those from immature BM-DCs. Transporters associated with antigen processing (TAP) deficient mice-derived BM-DCs were also used to examine whether functional TAP activity was required for cross-presentation by exosomes. The exosomes obtained from OVA(257-264) peptide-pulsed BM-DCs derived from TAP(-/-) mice showed a significant antigen presenting ability to OVA specific MHC class I-restricted T cell hybridomas. Altogether, our data indicate that BM-DCs secrete exosomes with weak cross-presentation ability.     

Tracking antigen-specific CD8+ T cells in the rat using MHC class I multimers

Studies of the quantitative and qualitative aspects of anti-microbial, anti-tumoral or autoreactive immune responses have been greatly facilitated by the possibility to stain antigen-specific CD8(+) T cells using fluorescently labeled multimeric major histocompatibility complex (MHC) class I/peptide complexes. So far, this technology has been developed for human and mouse, but not yet in the rat. Here, we describe the generation of the first rat MHC multimer. We produced a rat RT1(l) Pro5 MHC Pentamer combined with the immunodominant peptide for Borna disease virus (BDV), in order to study the characteristics of the antiviral CD8(+) T cell response. BDV is an RNA virus that can cause persistent infections of the central nervous system (CNS), often associated with prominent brain inflammation. In adult Lewis rats, of the RT1(l) MHC haplotype, BDV infection leads to severe immune-mediated neurological symptoms. The pathogenic role of the immune response is due primarily to antiviral CD8(+) T cells, many of them being specific for an immunodominant epitope located in the BDV nucleoprotein (N(230-238)). Ex vivo flow cytometry analyses revealed that 3 to 12% of CD8(+) T cells found in the brains of BDV-infected rats stained positively with the BDV-Pentamer. Interestingly, the frequency of Pentamer-positive cells increased up to 3.3 fold after a short resting period in culture. Virus-specific CD8(+) T cells were mainly detected in the brain and were virtually undetectable in peripheral lymphoid organs. This novel rat Pro5 MHC Pentamer represents an attractive tool for the detection, isolation and characterization of antigen-specific CD8(+) T cell responses in the rat.     

Expression of inhibitory receptors for MHC class I molecules on T cells

Inhibitory receptors (IRs) specific for MHC class I molecules and originally described on natural killer (NK) cells are also expressed on a fraction of peripheral T cells. The presence of these receptors on T cells is poorly understood. In this review, the different antigen specificities described to date for IR+ T cells and the expression pattern of these receptors on T cells are analyzed. This analysis indicates that the population of T cells defined by IR expression is heterogeneous and that different IRs (or families of IRs) may play different roles in T-cell biology.     

CD8+ T cells drive autoimmune hematopoietic stem cell dysfunction and bone marrow failure

Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction, resulting in varying degrees of hypoplasia and blood pancytopenia, and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4⁺ T cells in driving BM failure has been clearly established in several models. However, animal model data demonstrating a functional role for CD8⁺ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8⁺ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8⁺ T cells in BM failure and a beneficial role for Foxp3⁺ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice, which have a deficit in functional Tregs, develop spontaneous BM failure. Furthermore, we demonstrate a critical role for CD8⁺ T cells in the development of BM failure, which is dependent on the cytokine, IFNγ. CD8⁺ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells, resulting in anemia. Adoptive transfer experiments demonstrate that CD8⁺ T cells dramatically expedite disease progression and promote CD4⁺ T cell accumulation in the BM. Thus, BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNγ-producing CD8⁺ T cell (Tc1) response.     

Purification and characterization of human bone marrow lymphocyte subpopulations: evidence for the existence of an alloreactive immature T cell.

This study was conducted in order to clarify further the role of bone marrow lymphocyte (BML) subsets in graft-versus-host disease (GVHD) after clinical BM transplantation. Human BML separated by velocity sedimentation through a continuous sucrose gradient were found to consist of 38% T, 21% B and 41% null cells. T cells were purified from BML or peripheral blood lymphocytes (PBL) by rosette formation with sheep red blood cells (SRBC) and subsequent gradient centrifugation. Mitogenic stimulation of BML, PBL and the respective T cell subsets revealed the well-known reactivity of mature T lymphocytes, whereas non-T lymphocytes comprising T cell precursors showed only a weak response. In allogeneic stimulation kinetics, however, non-T-BML disclosed a delayed but marked responsiveness as compared to BML and T-BML suggesting that a T cell precursor matures under culture conditions. This cell type could be involved in GVHD in addition to mature T cells after clinical bone marrow transplantation.     

Quantification and characterization of myosin peptide-specific CD4+ T cells in autoimmune myocarditis

Characterization of autoantigen-specific CD4+ T cells at the single cell level is crucial for understanding the immunopathological mechanisms underlying autoimmune diseases. Cardiac myosin heavy chain (myhca) is the major autoantigen associated with autoimmune myocarditis both in humans and in experimental autoimmune myocarditis (EAM) in mice. In the current study, we evaluated two methods for the enumeration and phenotypic characterization of myhca-specific CD4+ T cells during the course of EAM. Both enzyme-linked immunospot (ELISPOT) and cytokine flow cytometry (CFC) assays were suitable for the detection and characterization of myhca-specific Th cells during acute myocardial inflammation and the late healing phase of the disease. Cytokine production of myhca-specific Th cells was restricted to interferon-gamma (IFNgamma). Only trace amounts of the Th2 cytokines IL-4 and IL-5 could be detected. Concomitant surface marker analysis in the CFC assay revealed the prototypical effector phenotype of myhca-specific Th1 cells during the acute phase of the disease. Taken together, the combination of both methods appears to be most appropriate for a comprehensive ex vivo single cell analysis of Th cells in heart-specific autoimmune disorders.     

Regulation of T cell function by NK cell receptors for classical MHC class I molecules

Inhibitory receptors for MHC class I molecules were initially characterised on NK cells. Human and mouse NK cell receptors (NKRs) are also expressed on T cells, predominantly on a subset of memory-phenotype CD8(+) T cells. This review focuses on the precise determination of interactions between NKRs and MHC class I, as well as on the unexpected in vivo function of NKRs on T cells.     

Solid-phase binding of class I and II MHC proteins: immunoassay and T cell recognition

Detergent-solubilized, affinity-purified class I and II MHC antigens can be immobilized on plastic by a simple detergent dilution procedure. The bound antigens retain allogeneic serological determinants and can be precisely quantitated by ELISA. The ability to quantitate immobilized antigen greatly facilitates purification by affinity chromatography. It is shown that differential elution can be used to highly enrich H-2Kd and Dd antigens from a single monoclonal antibody column which binds both. Binding of membrane proteins (class I, class II and plasma membrane protein) to plastic could be distinguished from binding of non-membrane proteins (bovine serum albumin and immunoglobulin) in competition studies and by comparison of their susceptibilities to inhibition by detergent. These contrasting properties suggested that MHC proteins may bind via their exposed hydrophobic regions and thus be oriented on the plastic surface. This was supported by the demonstration that immobilized class I is effectively recognized by alloantigen-specific cloned CTL to trigger the antigen-dependent degranulation response. Direct immobilization of MHC antigens, and probably other membrane proteins, provides an effective approach to the study of T cell recognition and triggering by physiological ligands.     

Similar structural requirements for T cell and antibody recognition of an MHC class I:peptide complex

We have generated a monoclonal antibody (Fab 13.4.1) using the phage display technique, which recognizes the murine MHC class I molecule, K^k, in complex with the influenza virus derived peptide, HA255-262, but not in complex with other peptides. Using BIAcore analysis the kinetics of the antibody was used to calculate the K_D to be about 10⁻⁹ M, which is typical of an antibody, and several orders of magnitude higher than the corresponding value for T cell receptors.

Given the specificity and affinity of Fab13.4.1 we expected it to inhibit K^k restricted and HA255-262 specific T cell hybridomas, but not K^k restricted T cells with different specificity, which indeed was found to be the case. Thus, Fab13.4.1, recognizes a K^k:HA255-262 dependent epitope which is related to the epitope recognized by the specific T cells. A more detailed analysis of the structural requirements for T cell and antibody recognition of the K^k:HA255-262 complex revealed that they use similar contacts points to interact with this MHC class I:peptide complex.     

Mycobacterium leprae 65hsp antigen expressed from a retroviral vector in a macrophage cell line is presented to T cells in association with MHC class II in addition to MHC class I.

Mycobacterium leprae lives free in the cytoplasm in infected macrophages. To test if an M. leprae antigen released into the cytoplasm would associate with major histocompatibility complex (MHC) class II we introduced the gene encoding the 65 kDa heat-shock protein (ML65hsp) into a retroviral shuttle vector (pZIPNeoSV(X)) and transfected the murine macrophage cell line J774G8. S1 nuclease mapping and Western blot analysis of the transfected cell line (CJ11) showed that specific messenger RNA and ML65hsp antigen were stably expressed. Presence of antigen at the cell surface was demonstrated by flow cytometric analysis with specific monoclonal antibodies (mAb). Antigen-specific T lymphocytes were stimulated by CJ11 cells to proliferate and release interleukins (IL-2 and IL-3). These responses were blocked by mAbs specific for either MHC class II or for the mycobacterial antigen. The endogenous antigen was also recognised by MHC class I-dependent cytotoxic T cells; cytotoxicity was inhibited by mAbs against either MHC class I molecules or ML65hsp. Thus, production of ML65hsp within the host cytoplasm resulted in association of the antigen with both MHC class I and MHC class II antigen-presenting structures and evoked both lymphocyte proliferation and cytotoxicity towards the antigen-presenting cell. These findings may be relevant to the development of recombinant subunit vaccines against intracellular pathogens.     

Thymic epithelial cells: antigen presenting cells that regulate T cell repertoire and tolerance development

During thymocyte development bone marrow-derived precursors in the thymus undergo a series of differentiation steps to produce self-tolerant, mature T lymphocytes. The thymus contains two functionally distinct anatomical compartments, consisting of a centrally located medulla surrounded by the thymic cortex. These compartments in turn are comprised of two major cellular components: (1) the T lymphoid compartment of developing thymocytes and (2) the thymic stroma consisting mainly of thymic epithelial cells (TECs). These epithelial cells are further separated into cortical and medullary TECs (cTECs and mTECs) based on their localization within the thymic cortex or medulla respectively. Reciprocal interactions between thymocytes and epithelial cells are required for the development of both cellular components into a functional thymic organ. Thymocytes provide trophic factors for the development of a complex three-dimensional epithelial cell network, while epithelial cells regulate T cell development through expression and presentation of self-antigens on major histocompatibility molecules. Our work focuses on how thymic epithelial cells regulate T cell development and function and on elucidating the mechanisms of thymic epithelial cell differentiation. Here we review current knowledge and provide our own insight into the development, differentiation and antigen presenting properties of TECs. We focus specifically on how mTECs regulate T cell repertoire selection and central tolerance.