MHC class II restricted recognition of FMDV peptides by bovine T cells.

A putative synthetic vaccine for foot-and-mouth disease (FMDV15) has proved less successful in a host species, cattle, than predicted by results in a small-animal model. Possible reasons for this include non-recognition by T cells influenced by major histocompatibility complex (MHC)-linked immune response gene control. It is now possible to type for human leucocyte antigen (HLA) DR-like bovine MHC (BoLA) class II polymorphisms with a one-dimensional isoelectric focusing (IEF) technique. Using this method 14 unrelated cattle were selected with eight different BoLA class II IEF types. After immunization with FMDV15, 13 cattle generated a T-cell response to FMDV15. However, the fine specificity and magnitude of the response was related to BoLA class II type. The non-response by one animal and low response by two other animals were associated with two of the BoLA class II types. Response to the region 149-158 was immunodominant and animals which did not respond to this region had low responses to the whole peptide. Using FMDV-specific T-cell lines five BoLA class II types associated with responder animals were able to present FMDV15 in an MHC class II-restricted fashion, indicating that this peptide is capable of binding to different MHC class II molecules and may account for the broad response observed. The restriction patterns of the lines indicated that the IEF method does not distinguish all functional polymorphisms. At least two of the IEF-defined types could each be split into two distinct specificities and revealed that the three sets of animals with identical IEF types in fact expressed distinct restriction elements.     

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.     

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.     

Mixed-haplotype MHC class II molecules select functional CD4+ T cells

MHC class II molecules are formed from polymorphic alpha and beta chains. While pairing of chains is most efficient within class II isotypes and haplotypes, limited pairing and surface expression of mixed-haplotype and -isotype class II molecules is common. The function of such molecules in antigen presentation has been established by the unique restriction of responses in F1 mice. However, it has not been established whether mixed class II molecules are able to mediate selection of functional T cells and how the reduced avidity of the TCR/MHC interaction influences the repertoire. In this report we have addressed these issues through the production of mice expressing solely mixed-haplotype class II molecules. The mixed class II molecules promote selection of a small CD4+ T cell repertoire with modified TCR use. The selected CD4+ T cells are functional in vivo and in vitro.     

Antigen-independent acquisition of MHC class II molecules by human T lymphocytes

We report here that human T lymphocytes have the capacity of acquiring large amounts of MHC class II molecules from various types of antigen-presenting cells (APC) in an antigen-independent manner. The transfer of MHC class II molecules from APC to T cell required direct cell-to-cell contact and appeared to involve the interaction of numerous adhesion molecules between these cells. Depletion of cholesterol from the plasma membrane reduced the amount of MHC class II transferred onto the T cells. Most significantly, the newly acquired MHC class II molecules were capable of efficiently presenting antigen to T helper cells. These results suggest that T cells are able to interact with other T cells to regulate immune responses by presenting MHC peptide complexes that have been snatched away from nearby APC.     

Self-peptides with intermediate capacity to bind and stabilize MHC class I molecules may be immunogenic

Thirty self-peptides were selected on the basis of their predicted binding to H-2b molecules. The binding of peptides was ascertained experimentally by biochemical (KD measurements) and cellular [major histocompatibility complex class I (MHC-I) stabilization] assays. A weak, but significant, correlation between KD measurements and MHC-I stabilization was observed. Mice (n = 99) were immunized with individual peptides. Twenty-eight peptides were found to induce peptide-specific cytotoxic activity, and a total of 84 mice developed significant cytotoxic T lymphocyte (CTL) responses after immunization. Only one of the 21 mice immunized with high-affinity peptides developed a peptide-specific CTL response of 29 lytic units per 106 splenocytes, whereas 11 of the 42 mice immunized with intermediate-affinity peptides developed peptide-specific CTL responses at this level (P < 0.05). These observations suggest the absence of tolerance towards most MHC-I-restricted self-peptides and that strong antiself immunity can be generated preferentially towards self-peptides with an intermediate affinity for MHC-I. These data should be considered in the design of tumour vaccines based on MHC-I-binding self-peptides.     

Inhibition of T cell activation by blockade of MHC class II molecules

Autoimmune diseases result from the activation of self-reactive T cells induced by autoantigens or by foreign antigens cross-reactive with an autoantigen. A striking characteristic of autoimmune diseases is the increased frequency of certain HLA alleles in affected individuals. Moreover, as demonstrated for example in rheumatoid arthritis and insulin-dependent diabetes mellitus, class II alleles positively associated with autoimmune diseases share amino acid residues in the hypervariable HLA regions involved in peptide binding. Therefore, it is likely that disease-associated HLA class II molecules have the capacity to bind the autoantigen and present it to T cells, thereby inducing and maintaining, under appropriate conditions, the autoimmune disease. The data reviewed here demonstrate MHC-selective inhibition of antigen-induced T cell responses in vivo by parenterally administered soluble, MHC-binding peptide competitors, under conditions in which the competitor is not immunogenic. This suggests the feasibility of a therapeutic approach based on MHC blockade in the treatment of HLA-linked autoimmune diseases.     

Identification of MHC class II restricted T-cell-mediated reactivity against MHC class I binding Mycobacterium tuberculosis peptides

Major histocompatibility complex (MHC) class I restricted cytotoxic T lymphocytes (CTL) are known to play an important role in the control of Mycobacterium tuberculosis infection so identification of CTL epitopes from M. tuberculosis is of importance for the development of effective peptide-based vaccines. In the present work, bioinformatics technology was employed to predict binding motifs of 9mer peptides derived from M. tuberculosis for the 12 HLA-I supertypes. Subsequently, the predicted peptides were synthesized and assayed for binding to HLA-I molecules in a biochemically based system. The antigenicity of a total of 157 peptides with measured affinity for HLA-I molecules of K(D) ≤ 500 nM were evaluated using peripheral blood T cells from strongly purified protein derivative reactive healthy donors. Of the 157 peptides, eight peptides (5%) were found to induce T-cell responses. As judged from blocking with HLA class I and II subtype antibodies in the ELISPOT assay culture, none of the eight antigenic peptides induced HLA class I restricted CD8(+) T-cell responses. Instead all responses were blocked by pan-HLA class II and anti-HLA-DR antibodies. In addition, CD4(+) T-cell depletion before the 10 days of expansion, resulted in total loss of reactivity in the ELISPOT culture for most peptide specificities. FACS analyses with intracellular interferon-γ staining of T cells expanded in the presence of M. tuberculosis peptides confirmed that the responsive cells were indeed CD4(+). In conclusion, T-cell immunity against HLA-I binding 9mer M. tuberculosis-derived peptides might in many cases turn out to be mediated by CD4(+) T cells and restricted by HLA-II molecules. The use of 9mer peptides recognized by both CD8(+) and CD4(+) T cells might be of importance for the development of future M. tuberculosis peptide-based vaccines.     

Cytotoxic T lymphocyte recognition of hybrid MHC class I molecules

We have utilized a group of MHC class I genes produced by in vitro recombination between Dp and Dd to study recognition of MHC class I molecules by cytolytic T cells (CTLs). Both polyclonal allo-specific and H-2-restricted CTLs require that alpha 1 and alpha 2 of the target class I molecule be derived from the same haplotype for efficient killing. By using T-cell lines we showed that within the bulk population there must exist a fraction of T cells which can recognize epitopes in alpha 1 or alpha 2. Critical residues for T-cell recognition have been identified using these chimeric genes.     

Endogenous antigen presentation by MHC class II molecules

T cell recognition of antigen requires that a complex form between peptides derived from the protein antigen and cell surface glycoproteins encoded by genes within the major histocompatibility complex (MHC). MHC class II molecules present both extracellular (exogenous) and internally synthesized (endogenous) antigens to the CD4 T cell subset of lymphocytes. The mechanisms of endogenous antigen presentation are the subject of this review. Isolation and amino acid sequencing of peptides bound to the class II molecule indicate that a very high proportion (70–90%) of the total peptides presented by the class II molecule are in fact derived from the pool of proteins that are synthetized within the antigen-presenting cell (APC). This type of sequence information as well as the study of model antigens has indicated that proteins expressed in a diversity of intracellular sites, including the cell surface, endoplasmic reticulum and cytosol can gain access to the class II molecule, albeit with different efficiencies. The main questions that remain to be answered are the intracellular trafficking patterns that allow colocalization of internally synthesized antigens with the class II molecule, the site(s) within the cell where peptide: class II molecule complex formation can take place and whether presentation of ‘foreign’ as well as ‘self’ antigens takes place by mechanisms that vary from one cell type to another or that vary with the metabolic state of the APC. If such variability exists, is would imply that the array of peptides displayed by class II molecules at the cell surface has similar variability, a possibility that would impact on self tolerance and autoimmunity.     

Sheep MHC class II molecules. I. Immunochemical characterization

The physicochemical features, biosynthesis and glycosylation of sheep class II molecules were investigated using a panel of seven monoclonal antibodies. The class II molecules recognized by different monoclonal antibodies could be differentiated using SDS-PAGE. Two monoclonal antibodies, SBU.II 42-20 and 49-1, reacted with dissociated sheep class II molecules and recognized epitopes on class II alpha and beta polypeptides, respectively. The structure of the sheep class II heterodimer differed from that of mouse and man in that it was unstable in the presence of 1% SDS at 20 degrees and, following reduction, sheep beta polypeptides displayed a marked increase in MW, resulting in the apparent co-migration of reduced alpha and beta polypeptides on SDS-PAGE. This phenomenon was not seen using sheep class II molecules synthesized in the presence of tunicamycin. Pulse-chase analyses of biosynthetically labelled sheep class II molecules suggested the rapid association and glycosylation of sheep class II alpha and beta polypeptides during synthesis. Both alpha and beta polypeptides of sheep class II molecules carried N-linked oligosaccharides of MW 6,000 and 3,000, respectively. However, unlike human class II oligosaccharides, these were exclusively of the complex or sialylated type.     

Phosphorylation of class I but not class II MHC molecules by membrane-localized protein kinase C

Membranes were isolated from B cells stimulated with phorbol 12-myristate 13-acetate (PMA) for a time sufficient to allow maximal redistribution and activation of protein kinase C (PKC). Exposure of such membranes to a short incubation with [γ-³²P]ATP resulted in the detection of at least nine unique or hyperphosphorylated membrane proteins by SDS-PAGE and autoradiography. The appearance of these phosphoproteins was blocked by pretreatment of the membranes with H-7 or sangivamycin, two selective inhibitors of PKC. In addition, membranes purified from B cells treated with an inactive phorbol ester or stimulated with dibutyryl cAMP failed to exhibit a pattern of new phosphoproteins. These results are consistent with the involvement of PKC in the phosphorylation of the proteins. These phosphoproteins are also candidates for proteins whose functions are modified as a consequence of early signal delivery to resting B cells following membrane immunoglobulin occupancy. This system was utilized to identify the heavy chain of MHC class I molecules as one of the membrane proteins phosphorylated by PKC. The MHC class II molecules were not phosphorylated in membranes isolated from PMA-treated normal B cells or from PMA-treated B cells which had previously been exposed to IL-4. These results indicate that class I, but not class II, MHC molecules are phosphorylated by PKC. It is possible that such a modification of cell surface class I molecules may be involved during the process of signal transduction leading to B cell activation.     

Mutations of class II MHC molecules

It remains unclear how the tertiary interaction of T-cell receptor, la molecule and foreign antigen results in the extensive diversity of the helper T cell repertoire. Here Laurie Glimcher and Irwin Griffith focus on what has been learned about the relationship between structure and function of the la molecule from the use of mouse strains with mutations in the genes coding for these glycoproteins.     

Vesicles bearing MHC class II molecules mediate transfer of antigen from antigen-presenting cells to CD4+ T cells

Previous studies have provided evidence that myelin basic protein (MBP)-specific rat T cells acquire antigen via transfer of preformed peptide/MHC class II complexes from splenic antigen-presenting cells (APC). The purpose of the present study was to determine how T cells acquire peptide/MHC class II complexes from APC in vitro. Our results show that a MHC class II+ T cell line, R1-trans, released MHC class II-bearing vesicles that directly stimulated MBP-specific CD4+ T cells. Vesicles expressing complexes of MHC class II and MBP were also specifically cytotoxic to MBP-specific T cells. Surviving T cells acquired MHC class II/antigen complexes from these vesicles by a mechanism that did not require protein synthesis but depended on specific TCR interactions with peptide/self MHC complexes. Furthermore, MBP/MHC class II-bearing vesicles enabled T cells to present MBP to other T cell responders. These studies provide evidence that APC release vesicles expressing preformed peptide/MHC class II complexes that interact with clonotypic TCR, allowing MHC class II acquisition by T cells. Vesicular transport of antigen/MHC class II complexes from professional APC to T cells may represent an important mechanism of communication among cells of the immune system.     

Peptides derived from the whole sequence of BCR-ABL bind to several class I molecules allowing specific induction of human cytotoxic T lymphocytes

Chronic myeloid leukemia (CML) is characterized cytogenetically by a t(9;22) translocation which generates a hybrid bcr-abl gene, encoding a p210^bcr-abl fusion protein. The induction in vitro of leukemia-specific T cells reactive with p210^bcr-abl is a strategy developed for an immunological therapeutic approach in CML. Peptides from the junction region of this chimeric protein have been considered as potential targets for a cytotoxic response against leukemic cells. However, only a few peptides encompassing the two p210^bcr-abl breakpoints have been shown to bind to the most common HLA class I molecules, which limits the number of patients who could benefit from this approach. We assume that the presence of chimeric BCR-ABL protein in leukemic cells may affect processing and delivery of peptides, possibly giving rise to new epitopes at the cell surface. We selected 162 peptides from the whole sequence of this protein, including 14 peptides of the b2a2 and b3a2 junctions, which had an anchor motif for a common HLA class I molecule. We tested their ability to bind to eight HLA class I molecules (HLA-A1, -A2, -A3, -A11, -B7, -B8, -B27, -B44). We identified 48 peptides from outside the junction region, with intermediate or strong binding capacities to these HLA class I molecules contrasting with only six junction peptides with a moderate binding capacity to HLA-A3/A11, -B8, or -B44 molecules. Moreover, cytotoxic T lymphocyte lines specific for various peptides outside the junction were generated from peripheral blood mononuclear cells of HLA-A2 or -B7 healthy donors and from one CML patient. These results contribute to evaluation of immunity to the BCR-ABL chimeric protein. Further studies are required to investigate whether such epitopes are correctly processed and presented by leukemic cells.     

Mls allo-determinants are recognized in an MHC class II antigen-dependent but unrestricted fashion by a discrete set of T cells

Blocking studies carried out with anti-H-2 class II antigen antibodies show that H-2 class II molecules are intimately involved in the recognition of Mlsa determinants by unprimed, specifically responsive T cells. The blocking of the anti-Mlsa response by these antibodies were not due to inhibition of IL-1 production by H-2 class II antigen positive macrophages. A strain analysis indicates that the response to Mlsa is regulated by H-2-linked genes and that this effect is exerted at the level of the stimulator cells; however, the response to Mls epitopes does not appear to be H-2 restricted. Finally, the ability of spleen cells from Mls-incompatible mice to induce a state of Mls-specific, clonal deletion type tolerance by neonatal injection, shows that Mls determinants exist in qualitatively allelic forms and suggests that these determinants are recognized by specific clones of T cells.