Towards a vaccine against rheumatic fever.

Rheumatic fever (RF) is an autoimmune disease which affects more than 20 million children in developing countries. It is triggered by Streptococcus pyogenes throat infection in untreated susceptible individuals. Carditis, the most serious manifestation of the disease, leads to severe and permanent valvular lesions, causing chronic rheumatic heart disease (RHD). We have been studying the mechanisms leading to pathological autoimmunity in RF/RHD for the last 15 years. Our studies allowed us a better understanding of the cellular and molecular pathogenesis of RHD, paving the way for the development of a safe vaccine for a post-infection autoimmune disease. We have focused on the search for protective T and B cell epitopes by testing 620 human blood samples against overlapping peptides spanning 99 residues of the C-terminal portion of the M protein, differing by one amino acid residue. We identified T and B cell epitopes with 22 and 25 amino acid residues, respectively. Although these epitopes were from different regions of the C-terminal portion of the M protein, they showed an identical core of 16 amino acid residues. Antibodies against the B cell epitope inhibited bacterial invasion/adhesion in vitro. Our results strongly indicated that the selected T and B cell epitopes could potentially be protective against S. pyogenes.

Cellular autoreactivity against heat shock protein 60 in renal transplant patients: peripheral and graft-infiltrating responses.

Autoreactivity to heat shock protein 60 (Hsp60) has been implicated in the pathogenesis and regulation of chronic inflammation, especially in autoimmune diseases. In transplantation, there is a lack of information regarding the cytokine profile and specificity of cells that recognize self-Hsp60 as well as the kinetics of autoreactivity following transplantation. We studied the cellular reactivity of peripheral and graft-infiltrating lymphocytes against Hsp60 in renal transplant patients. Cytokine production induced by this protein in peripheral blood mononuclear cells indicated a predominance of interleukin (IL)-10 during the late post-transplantation period, mainly in response to intermediate and C-terminal peptides. Patients with chronic rejection presented reactivity to Hsp60 with a higher IL-10/interferon (IFN)-gamma ratio compared to long-term clinically stable patients. Graft-infiltrating T cell lines, cocultured with antigen-presenting cells, preferentially produced IL-10 after Hsp60 stimulation. These results suggest that, besides its proinflammatory activity, autoreactivity to Hsp60 in transplantation may also have a regulatory role.

Rheumatic fever: how S. pyogenes-primed peripheral T cells trigger heart valve lesions.

The pathogenesis of rheumatic fever (RF) is related to autoimmune humoral and cellular responses against human tissues triggered by Streptococcus pyogenes. CD4(+) T cells are the ultimate effectors of chronic heart lesions in rheumatic heart disease (RHD). Heart-infiltrating CD4(+) T cell clones are able to recognize heart tissue and streptococcal antigens by molecular mimicry. The streptococcal M5(81-103) region, an immunodominant region, was recognized by both intralesional and peripheral T cell clones (62% and 38%, respectively). Peripheral T lymphocytes from Brazilian patients with severe RHD preferentially recognized the M5(81-96) peptide, in the context of HLA-DR7(+) and DR53(+) molecules. HLA-DR7 seems to be related to the development of multiple valvular lesions in RHD patients from different countries. In addition, the fact that peripheral and intralesional T cells recognized the M5(81-103) region points to this region as one of the streptococcal triggers of autoimmune reactions in RHD. T cell repertoire analysis from peripheral and intralesional T cell lines derived from RHD patients showed several oligoclonal expansions of BV families. Major expansions were found in the heart lesions, suggesting that such T cell populations preferentially migrate from the periphery to the heart. Some cross-reactive intralesional T cell clones displayed the same T cell receptor (TCR) BVBJ and CDR3 sequences, showing a degenerate pattern of antigen recognition. Heart tissue-infiltrating cells from myocardium and valvular tissue produced TNF-alpha, IFN-gamma, IL-10, and IL-4, whereas few cells from valvular tissue produced IL-4, showing that the lack of regulation in the valves could be responsible for the permanent and progressive valvular lesions.

How an autoimmune reaction triggered by molecular mimicry between streptococcal M protein and cardiac tissue proteins leads to heart lesions in rheumatic heart disease.

Molecular mimicry between microbial antigens and host tissue is suggested as a mechanism for post-infectious autoimmune disease. In the present work we describe the autoimmune reactions of two severe rheumatic heart disease (RHD) patients, through an analysis of heart-infiltrating T-cell repertoire, antigen recognition, and cytokine production induced by specific antigens. T-cell clones derived from oligoclonally expanded T cells in the heart cross-recognized M5 peptides, heart tissue-derived proteins, and myosin peptides. We show, using binding affinity assays, that an immunodominant streptococcal peptide (M5(81-96)) is capable of binding to the HLA-DR53 molecule. The same peptide was recognized by an infiltrating T-cell clone from a patient carrying HLA-DR15, DR7, and DR53 molecules. This suggests that this peptide is probably presented to T cells in the context of the HLA-DR53 molecule. Cross-reactive heart-infiltrating T cells activated by the M5 protein and its peptides and by heart tissue-derived proteins produced predominantly inflammatory cytokines. Interleukin (IL)-4 was produced in small amounts by mitral valve intralesional T-cell lines and clones. Altogether, these results suggest that mimicry between streptococcal antigens and heart-tissue proteins, combined with high inflammatory cytokine and low IL-4 production, leads to the development of autoimmune reactions and cardiac tissue damage in RHD patients.

T-cell reactivity against streptococcal antigens in the periphery mirrors reactivity of heart-infiltrating T lymphocytes in rheumatic heart disease patients.

T-cell molecular mimicry between streptococcal and heart proteins has been proposed as the triggering factor leading to autoimmunity in rheumatic heart disease (RHD). We searched for immunodominant T-cell M5 epitopes among RHD patients with defined clinical outcomes and compared the T-cell reactivities of peripheral blood and intralesional T cells from patients with severe RHD. The role of HLA class II molecules in the presentation of M5 peptides was also evaluated. We studied the T-cell reactivity against M5 peptides and heart proteins on peripheral blood mononuclear cells (PBMC) from 74 RHD patients grouped according to the severity of disease, along with intralesional and peripheral T-cell clones from RHD patients. Peptides encompassing residues 1 to 25, 81 to 103, 125 to 139, and 163 to 177 were more frequently recognized by PBMC from RHD patients than by those from controls. The M5 peptide encompassing residues 81 to 96 [M5(81-96) peptide] was most frequently recognized by PBMC from HLA-DR7+ DR53+ patients with severe RHD, and 46.9% (15 of 32) and 43% (3 of 7) of heart-infiltrating and PBMC-derived peptide-reactive T-cell clones, respectively, recognized the M5(81-103) region. Heart proteins were recognized more frequently by PBMC from patients with severe RHD than by those from patients with mild RHD. The similar pattern of T-cell reactivity found with both peripheral blood and heart-infiltrating T cells is consistent with the migration of M-protein-sensitized T cells to the heart tissue. Conversely, the presence of heart-reactive T cells in the PBMC of patients with severe RHD also suggests a spillover of sensitized T cells from the heart lesion.

Molecular evidence for antigen-driven immune responses in cardiac lesions of rheumatic heart disease patients.

Rheumatic heart disease (RHD) is a sequel of post-streptococcal throat infection. Molecular mimicry between streptococcal and heart components has been proposed as the triggering factor of the disease, and CD4(+) T cells have been found predominantly at pathological sites in the heart of RHD patients. These infiltrating T cells are able to recognize streptococcal M protein peptides, involving mainly 1-25, 81-103 and 163-177 N-terminal amino acids residues. In the present work we focused on the TCR beta chain family (TCR BV) usage and the degree of clonality assessed by beta chain complementarity-determining region (CDR)-3 length analysis. We have shown that in chronic RHD patients, TCR BV usage in peripheral blood mononuclear cells (PBMC) paired with heart-infiltrating T cell lines (HIL) is not suggestive of a superantigen effect. Oligoclonal T cell expansions were more frequently observed in HIL than in PBMC. Some major BV expansions were shared between the mitral valve (Miv) and left atrium (LA) T cell lines, but an in-depth analysis of BJ segments usage in these shared expansions as well as nucleotide sequencing of the CDR3 regions suggested that different antigenic peptides could be predominantly recognized in the Miv and the myocardium. Since different antigenic proteins probably are constitutively represented in myocardium and valvular tissue, these findings could suggest a differential epitope recognition at the two lesional heart sites after a common initial bacterial challenge.