Mycobacterium leprae-Specific, HLA Class II-Restricted Killing of Human Schwann Cells by CD4+ Th1 Cells: A Novel Immunopathogenic Mechanism of Nerve Damage in Leprosy

Peripheral nerve damage is a major complication of reversal (or type-1) reactions in leprosy. The pathogenesis of nerve damage remains largely unresolved, but detailed in situ analyses suggest that type-1 T cells play an important role. Mycobacterium leprae is known to have a remarkable tropism for Schwann cells of the peripheral nerve. Reversal reactions in leprosy are often accompanied by severe and irreversible nerve destruction and are associated with increased cellular immune reactivity against M. leprae. Thus, a likely immunopathogenic mechanism of Schwann cell and nerve damage in leprosy is that infected Schwann cells process and present Ags of M. leprae to Ag-specific, inflammatory type-1 T cells and that these T cells subsequently damage and lyse infected Schwann cells. Thus far it has been difficult to study this directly because of the inability to grow large numbers of human Schwann cells. We now have established long-term human Schwann cell cultures from sural nerves and show that human Schwann cells express MHC class I and II, ICAM-1, and CD80 surface molecules involved in Ag presentation. Human Schwann cells process and present M. leprae, as well as recombinant proteins and peptides to MHC class II-restricted CD4+ T cells, and are efficiently killed by these activated T cells. These findings elucidate a novel mechanism that is likely involved in the immunopathogenesis of nerve damage in leprosy. Acute reactional episodes are major complications in leprosy. Type-1 reversal reactions (RR)3 in particular can result in irreversible tissue damage and nerve destruction. Such reactions are characterized by strongly increased cellular immune responses in peripheral blood and lesions (1) accompanied by the abundant presence of local CD4+ T cells (1, 2, 3) and type-1 cytokines (2, 4, 5, 6). In tuberculoid and RR granulomas, cells that express serine esterase, a component of cytotoxic granules, colocalize with CD4+ CD45RO+ memory T cells (2), and analysis of Mycobacterium leprae-reactive T cells confirmed that these T cells indeed produce serine esterase in vitro (7). The induction of cytolytic CD4+ Th1-like cells during mycobacterial infections has been documented extensively (7, 8, 9, 10), further suggesting that cytotoxic Th1 cells may play a major role in the protection against and the immunopathology of mycobacterial infections. However, direct evidence for a pathogenic role of T cells in Schwann cell damage is lacking. Better insight into the immunopathogenesis of Schwann cell damage in leprosy is required, given the major impact of nerve damage in leprosy. M. leprae has a remarkable affinity for Schwann cells, the molecular basis of which has been elucidated recently: M. leprae binds specifically to the G domain of the extracellular matrix protein laminin-2, which ligates to α/β-dystroglycan receptor-complexes on myelinating Schwann cells (11, 12, 13). Thus, M. leprae exploits interactions between matrix- and cytoskeletal-linked glycoproteins to target and infect Schwann cells. The recent elucidation of this mechanism now provides novel opportunities to disrupt interactions between M. leprae, Schwann cells, and inflammatory T cells and is of potential value in the prevention or treatment of nerve damage. Previously, Steinhoff et al. showed in a mouse model that Schwann cells can be lysed by CD8+ T cells in an Ag-specific manner (14), suggesting that murine Schwann cells are susceptible to killing by CD8+ T cells. However, CD4+ T cells, which form the major cellular component of granulomatous leprosy lesions, were not examined. We now have established human Schwann cell cultures and analyzed their Ag-presenting capacity and their susceptibility to killing by M. leprae-reactive T cells in a human setting. Our data show that human Schwann cells process and present M. leprae to Ag-specific T cells and are subsequently killed during this event. We propose that this could be an important mechanism in nerve damage

Hypomethylating drugs convert HA-1-negative solid tumors into targets for stem cell-based immunotherapy

Clinical responses of solid tumors after allogeneic human leukocyte antigen-matched stem cell transplantation (SCT) often coincide with severe graft-versus-host disease (GVHD). Targeting minor histocompatibility antigens (mHags) with hematopoiesis- and cancer-restricted expression, for example, HA-1, may allow boosting the antitumor effect of allogeneic SCT without risking severe GVHD. The mHag HA-1 is aberrantly expressed in cancers of most entities. However, an estimated 30% to 40% of solid tumors do not express HA-1 (ie, are HA-1(neg)) and cannot be targeted by HA-1-specific immunotherapy. Here, we investigated the transcriptional regulation of HA-1 gene expression in cancer. We found that DNA hypermethylation in the HA-1 promoter region is closely associated with the absence of HA-1 gene expression in solid tumor cell lines. Moreover, we detected HA-1 promoter hypermethylation in primary cancers. The hypomethylating agent 5-aza-2'-deoxycytidine induced HA-1 expression only in HA-1(neg) tumor cells and sensitized them for recognition by HA-1-specific cytotoxic T lymphocytes. Contrarily, the histone deacetylation inhibitor trichostatin A induced HA-1 expression both in some HA-1(neg) tumor cell lines and in normal nonhematopoietic cells. Our data suggest that promoter hypermethylation contributes to the HA-1 gene regulation in tumors. Hypomethylating drugs might extend the safe applicability of HA-1 as an immunotherapeutic target on solid tumors after allogeneic SC

Identification and Characterization of the ESAT-6 Homologue of Mycobacterium leprae and T-Cell Cross-Reactivity with Mycobacterium tuberculosis

In this paper we describe identification and characterization of Mycobacterium leprae ESAT-6 (L-ESAT-6), the homologue of M. tuberculosis ESAT-6 (T-ESAT-6). T-ESAT-6 is expressed by all pathogenic strains belonging to the M. tuberculosis complex but is absent from virtually all other mycobacterial species, and it is a promising antigen for immunodiagnosis of tuberculosis (TB). Therefore, we analyzed whether L-ESAT-6 is a similarly powerful tool for the study of leprosy by examining T-cell responses against L-ESAT-6 in leprosy patients, TB patients, and exposed or nonexposed healthy controls from areas where leprosy and TB are endemic and areas where they are not endemic. L-ESAT-6 was recognized by T cells from leprosy patients, TB patients, individuals who had contact with TB patients, and healthy individuals from an area where TB and leprosy are endemic but not by T cells from individuals who were not exposed to M. tuberculosis and M. leprae. Moreover, leprosy patients who were not responsive to M. leprae failed to respond to L-ESAT-6. A very similar pattern was obtained with T-ESAT-6. These results show that L-ESAT-6 is a potent M. leprae antigen that stimulates T-cell-dependent gamma interferon production in a large proportion of individuals exposed to M. leprae. Moreover, our results suggest that there is significant cross-reactivity between T-ESAT-6 and L-ESAT-6, which has implications for the use of ESAT-6 as tool for diagnosis of leprosy and TB in areas where both diseases are endemic

Postgenomic Approach To Identify Novel Mycobacterium leprae Antigens with Potential To Improve Immunodiagnosis of Infection

Early detection of Mycobacterium leprae infection is considered an important component of strategies aiming at reducing transmission of infection, but currently available diagnostic tools often lack sufficient sensitivity and specificity to reach this goal. Recent comparative genomics have revealed the presence of 165 M. leprae genes with no homologue in M. tuberculosis. We selected 17 of these genes for further study. All 17 genes were found to be expressed at the mRNA level in M. leprae from infected mice and from a multibacillary leprosy patient. Additional comparative genomic analyses of all currently available mycobacterial genome databases confirmed 12 candidate genes to be unique to M. leprae, whereas 5 genes had homologues in mycobacteria other than M. tuberculosis. Evaluation of the immunogenicity of all 17 recombinant proteins in PBMC from 127 Brazilians showed that five antigens (ML0576, ML1989, ML1990, ML2283, and ML2567) induced significant gamma interferon levels in paucibacillary leprosy patients, reactional leprosy patients, and exposed healthy controls but not in most multibacillary leprosy patients, tuberculosis patients, or endemic controls. Importantly, among exposed healthy controls 71% had no detectable immunoglobulin M antibodies to the M. leprae-specific PGL-I but responded to one or more M. leprae antigen(s). Collectively, the M. leprae proteins identified are expressed at the transcriptome level and can efficiently activate T cells of M. leprae-exposed individuals. These proteins may provide new tools to develop tests for specific diagnosis of M. leprae infection and may enhance our understanding of leprosy and its transmission