T. H. M. Ottenhoff

Mycobacterium leprae-specific protein antigens defined by cloned human helper T cells

Leprosy displays a remarkable spectrum of symptoms correlating with the T-cell-mediated immune reactivity of the host against the causative organism, Mycobacterium leprae1. At one pole of this spectrum are lepromatous leprosy patients showing a M. leprae-specific T-cell unresponsiveness2; at the other are tuberculoid leprosy patients displaying both acquired immunity and delayed-type hypersensitivity against M. leprae which are thought to be conferred by helper T (Th) cells1,3–5. Because well-defined M. leprae antigens are crucial for the prevention and control of leprosy1,6,7, we have cloned M. leprae- reactive T cells (TLC) of the helper phenotype from a tuberculoid leprosy patient. As reported here, these TLC show an unexpected diversity in the recognition of M. leprae and related mycobacteria, which is different from that exhibited by monoclonal antibodies8,9. Half of these TLC are completely or almost M. leprae-specific, whereas the other half are cross-reactive with most or all other mycobacteria. A M. leprae protein of relative molecular mass (Mr) 36,000 (36K) defined by a M. leprae-specific monoclonal antibody7,8,10 stimulates 4 out of 6 TLC tested. Each of these TLC recognizes a different antigenic determinant, one of which is M. leprae-specific. The previous paper describes other M. leprae-specific T-cell clones half of which recognize an epitope on a M. leprae protein of Mr 18 K.

Epitope Specificity of Anti–Heat Shock Protein 65/60 Serum Antibodies in Atherosclerosis

Levels of specific antibodies (Ab) against mycobacterial and human heat shock protein (hsp) 65/60 are increased in the sera of patients with atherosclerotic lesions and have been demonstrated to be capable of mediating endothelial cytotoxicity. To clarify the antigen epitopes recognized by these serum Abs, Ab binding to hsp65 deletion mutants (Dms), as well as to overlapping 15-mer and 8-mer hsp65 peptides, was assessed. Western blotting of hsp65 Dms indicated the presence of at least one epitope between amino acid (aa) residues 171 and 276, recognized by both high-titer sera and affinity-purified anti-hsp65/60 Ab. Fluorescence immunoassays using 53 15-mer peptides and Pin ELISA using 526 7-mer peptides demonstrated three distinct, conserved sequences with high affinity to high-titer sera and purified anti-hsp65/60 Ab. Two N-terminal sequences, aa 97-109 and aa 179-187, and one C-terminal sequence, aa 504-512, were identified. These three epitopes recognized by anti-hsp65/60 Ab may serve as autoantigens in certain circumstances in vivo. This phenomenon could contribute to the initiation of atherosclerosis by an autoimmune reaction.

Immunological Crossreactivity of the Mycobacterium leprae CFP‐10 with its Homologue in Mycobacterium tuberculosis

Mycobacterium tuberculosis culture filtrate protein‐10 (CFP‐10) (Rv3874) is considered a promising antigen for the immunodiagnosis of tuberculosis (TB) together with early secreted antigens of M. tuberculosis (ESAT‐6). Both ESAT‐6 and CFP‐10 are encoded by the RD1 region that is deleted from all tested M. bovis bacille Calmette–Guérin (BCG) strains but present in M. leprae, M. tuberculosis, M. bovis, M. kansasii, M. africanum and M. marinum. In this study, the homologue of CFP‐10 in M. leprae (ML0050) is identified and characterized. Interferon‐γ production in response to this homologue by T cells from leprosy patients, TB patients and unexposed controls shows that CFP‐10 of M. leprae is a potent antigen that crossreacts with CFP‐10 of M. tuberculosis at the T‐cell level. This crossreactivity has implications for the use of CFP‐10 of these mycobacterial species as diagnostic tool in areas endemic for both the diseases.

Molecular and immunological analysis of a fibronectin‐binding protein antigen secreted by Mycobacterium leprae

By screening a Mycobacterium lepraeλgt11 genomic DNA library with leprosy‐patient sera we have previously identified 50 recombinant clones that expressed novel M. leprae antigens (Sathish et al., 1990). In this study, we show by DNA sequencing and immunoblot analysis that three of these clones express a M. leprae homologue of the fibronectin‐binding antigen 85 complex of mycobacteria. The complete gene was characterized and it encodes a 327‐amino‐acid polypeptide, consisting of a consensus signal sequence of 38 amino acids followed by a mature protein of 289 amino acids. This is the first sequence of a member of the M. leprae antigen 85 complex, and Southern blotting analysis indicated the presence of multiple genes of the 85 complex in the genome of M. leprae. The amino acid sequence displays 75–85% sequence identity with components of the antigen 85 complex from M. tuberculosis, M. bovis BCG and M. kansasii. Furthermore, antibodies to the antigen 85 complex of M. tuberculosis and M. bovis BCG reacted with two fusion proteins containing the amino acid regions 55–266 and 265–327 of the M. leprae protein. The M. leprae 30/31 kDa protein induces strong humoral and cellular responses, as judged by Western blot analysis with patient sera and proliferation of T cells derived from healthy individuals and leprosy patients. Amino acid regions 55–266 and 265–327 both were shown to bind to fibronectin, indicating the presence of at least two fibronectin‐binding sites on the M. leprae protein. These data indicate that this 30/31 kDa protein is not only important in the immune response against M. leprae, but may also have a biological role in the interaction of this bacillus with the human host.

Regulation of mycobacterial heat-shock protein-reactive T cells by HLA class II molecules: lessons from leprosy.

Major hislocompatibility (MHC) class II molecules are heterodimeric cell surface glycoproteins that are expressed by immunocompetenl cells. MHC class II molecules present processed peptide fragments to T-lymphocyte receptors, and thus can restrict and regulate T-cell responses against specific antigens. Class II molecules are highly polymorphic. This polymorphism is responsible for the genetically controlled differences in T cell-dependent responses and presumably also for genetic differences in disease susceptibility. A human disease where (HLA) class II genes play an important role is leprosy, the chronic infectious disease caused by Mycobacterium leprae. Protective immunity against M. leprae and other mycobacterial species is dependent on antigen-specific helper T lymphocytes. This is elegantly illustrated by the leprosy spectrum where strong Tlympbocyte reactivity to M. leprae is seen in the tuberculoid or high responder form of the disease as well as in healthy exposed individuals, but low or no Tcell responsiveness in the disseminated lepromatous form (Bloom & Godal 1983). HLA class Il-linked genes control the type of leprosy that develops upon infection as well as the type of cell-mediated immunity against M. leprae and related mycobacteria in skin testing (reviewed by Ottenhoff & de Vries 1987). Since leprosy type and skin test responsiveness both strongly correlate with M. lepraespecific T-cell reactivity, HLA class II gene products might regulate these responses by restricting and regulating T-helper cell activity against M. leprae

Mannose receptor mediated uptake of antigens strongly enhances HLA-class II restricted antigen presentation by cultured dendritic cells.

Dendritic cells (DCs) use macropinocytosis and mannose receptor mediated endocytosis for the uptake of exogenous antigens. Here we show that the endocytosis of the mannose receptor and mannosylated antigen is distinct from that of a non-mannosylated antigen. Shortly after internalization, however, both mannosylated and non-mannosylated antigen are found in an MIIC like compartment. The mannose receptor itself does not reach this compartment, and probably releases its ligand in an earlier endosomal structure. Finally, we found that mannosylation of peptides strongly enhanced their potency to stimulate HLA class II-restricted peptide-specific T cell clones. Our results indicate that mannosylation of antigen leads to selective targeting and subsequent superior presentation by DCs which may be useful for vaccine design.

Salivary Anti-hsp65 Antibodies as a Diagnostic Marker for Gingivitis and a Possible Link to Atherosclerosis

Levels of specific salivary IgA antibodies against mycobacterial heat shock protein (hsp) 65 are significantly increased in patients with gingivitis when compared to clinically healthy subjects. The process of identifying the hsp65 epitopes recognized by the salivary antibodies, binding to overlapping 15-mer-hsp65 peptides, was assessed. Time-resolved fluorescence immunoassays using 15-mer overlapping peptides spanning the whole hsp65 molecule revealed six distinct sequences recognized by anti-hsp65 IgA antibodies. Due to the high degree of sequence homology between mycobacterial hsp65, cognates of the hsp60 family of oral bacterial flora and human hsp60, these six epitopes may serve as cross-reactive autoantigens in certain circumstances in vivo and could incite an autoimmune response that contributes to the initiation of gingivitis.

CLIP binds to HLA class II using methionine-based, allele-dependent motifs as well as allele-independent supermotifs

The invariant chain (Ii) region that interacts with class II and inhibits premature peptide binding has been mapped to amino acids 82-107, known as CLIP. It is unclear whether CLIP binds directly to the class II peptide binding groove and thus competitively blocks binding of other peptides, or whether it binds to conserved class II sites and indirectly inhibits peptide binding by inducing conformational changes in class II. Here we show evidence that strongly suggests that CLIP binds within the peptide binding groove, as CLIP binds to various HLA-DR alleles using allele-dependent as well as allele-independent, methionine-based binding motifs. First, a core sequence of 12 amino acids was identified within CLIP which is required for optimal binding to DR1, DR2, DR3(17) and DR7. This sequence is composed of CLIP p88-99 (SKMRMATPLLMQ). By substitution analysis, all three methionine residues appeared to control CLIP binding to HLA-DR. However, whereas M90 controlled binding to all four alleles, M92 and M98 were of different importance for the various alleles: M92 is involved in CLIP binding to DR1 and DR3(17) but not to DR2 or DR7, and M98 controls CLIP binding to DR2, DR3(17) and DR7 but not DR1. Also, CLIP competes with known immunogenic peptides for class II binding in a manner indistinguishable from regular, class II binding competitor peptides. Finally, the dissociation rates of CLIP-class II complexed are similar to those of antigenic peptide-class II complexes. Thus, CLIP most likely binds to the class II peptide binding groove, since most allelic class II differences are clustered here. CLIP uses unconventional methionine anchor residues representing an allele-independent supermotif (M90) as well as allele-dependent motifs (M92 and M98).

Flexibility in T‐cell receptor ligand repertoires depends on MHC and T‐cell receptor clonotype

T‐cell receptors (TCR) recognize peptides complexed to self‐major histocompatibility complex (MHC) molecules. Recognition of peptide/MHC ligands by the TCR is highly peptide specific. However, certain TCRs can also recognize sequence‐related and ‐unrelated (‘mimicry’) epitopes presented by homologous MHC molecules. Using two human, human leucocyte antigen‐DR1 (HLA‐DR1)‐restricted T‐cell clones specific for HA p307–319, we identified several diverse combinations of peptide‐MHC complexes that are functionally equivalent in their ability to trigger T‐cell stimulation. These findings demonstrate that a single TCR can productively interact with different peptides complexed to self‐ as well as non‐self‐MHC molecules. This extended reactivity is human leucocyte antigen (HLA) allele and TCR clonotype dependent, as the peptide repertoire recognized depends on the presenting HLA‐DR molecule and varies among different TCRs that both recognize the HA p307–319/DR1 complex. Importantly, certain peptide analogues can completely change the HLA‐restriction pattern of the TCR: T‐cell recognition of the wild‐type peptide that was absent in the context of a non‐self HLA‐DR molecule, was restored by complementing substitutions in altered peptide ligands, that could not be presented by the original restriction element. This mechanism may play an important role in allorecognition.

Bronchoalveolar lavage cells from sarcoidosis patients and healthy controls can efficiently present antigens

Objectives. The interaction between antigen‐presenting cells (APC) and T lymphocytes, that recognize the antigen‐HLA complex using its T cell‐receptor for antigen, is of crucial importance for a subsequent specific immune response. In patients with pulmonary sarcoidosis, the local antigen‐ presenting capacity in the lungs has been suggested to be abnormally enhanced, and implicated in the immunopathogenesis of the disease. This study was aimed at increasing the understanding of the capacity to present antigens by APC in the lung compartment.