The majority of H2-M3 is retained intracellularly in a peptide-receptive state and traffics to the cell surface in the presence of N-formylated peptides.

We used a new monoclonal antibody (mAb 130) to analyze the intracellular trafficking and surface expression of H2-M3, the major histocompatibility complex class Ib molecule that presents N-formylated peptides to cytotoxic T cells. M3 surface expression is undetectable in most cell types due to the paucity of endogenous antigen. M3 is induced on the cell surface by addition of high-affinity N-formylated peptides from mitochondria and listeria. Peptide-induced M3 expression is most efficient on antigen presenting cells. Basal and inducible expression of M3 is transporter associated with antigen processing (TAP)-dependent, distinguishing M3 from the class Ib molecules TL and CD1. Unlike the expression of class Ia molecules and a previously described M3/L(d) chimera, surface expression of M3 cannot be rescued by lowered temperature, suggesting that the alpha3 domain and transmembrane region of M3 may control trafficking. Pulse-chase analysis and use of trafficking inhibitors revealed a pool of empty M3 in the endoplasmic reticulum or early Golgi apparatus. Addition of exogenous peptide allows maturation with kinetics matching those of D(d). The lack of endogenous N-formylated peptide allows discovery of novel pathogen-derived peptides in normal antigen presenting cells. The nonpolymorphic nature of M3 and its ability to present bacterial antigens rapidly and dominantly make it an attractive target for peptide vaccination strategies.

Induction of M3-restricted cytotoxic T lymphocyte responses by N-formylated peptides derived from Mycobacterium tuberculosis.

Major histocompatibility complex (MHC) class I-restricted CD8(+) T cells play a critical role in the protective immunity against Mycobacterium tuberculosis (Mtb). However, only a few Mtb peptides recognized by MHC class Ia-restricted CD8(+) T cells have been identified. Information on epitopes recognized by class Ib-restricted T cells is even more limited. M3 is an MHC class Ib molecule that preferentially presents N-formylated peptides to CD8(+) T cells. Because bacteria initiate protein synthesis with N-formyl methionine, the unique binding specificity of M3 makes it especially suitable for presenting these particular bacterial epitopes. We have scanned the full sequence of the Mtb genome for NH2-terminal peptides that share features with other M3-binding peptides. Synthetic peptides corresponding to these sequences were tested for their ability to bind to M3 in an immunofluorescence-based peptide-binding assay. Four of the N-formylated Mtb peptides were able to elicit cytotoxic T lymphocytes (CTLs) from mice immunized with peptide-coated splenocytes. The Mtb peptide-specific, M3-restricted CTLs lysed the Mtb-infected macrophages effectively, suggesting that these N-formylated Mtb peptides are presented as the naturally processed epitopes by Mtb-infected cells. Furthermore, T cells from Mtb-infected lungs, spleen, and lymph nodes responded to N-formylated Mtb peptides in an M3-restricted manner. Taken together, our data suggest that M3-restricted T cells may participate in the immune response to Mtb.

The selection of M3-restricted T cells is dependent on M3 expression and presentation of N-formylated peptides in the thymus.

The major histocompatibility complex (MHC) class Ib molecule H2-M3 binds N-formylated peptides from mitochondria and bacteria. To explore the role of M3 expression and peptide supply in positive and negative selection, we generated transgenic mice expressing an M3-restricted TCR-alpha/beta from a CD8(+) T cell hybridoma (D7) specific for a listerial peptide (LemA). Development of M3-restricted transgenic T cells is impaired in both beta2-microglobulin-deficient and transporter associated with antigen processing (TAP)-deficient mice, but is not diminished by changes in the H-2 haplotype. Maturation of M3/LemA-specific CD8(+) single positive cells in fetal thymic organ culture was sensitive to M3 expression levels as determined by antibody blocking and use of the castaneus mutant allele of M3. Positive selection was rescued in TAP(-/-) lobes by nonagonist mitochondrial and bacterial peptides, whereas LemA and a partial agonist variant caused negative selection. Thus, M3-restricted CD8(+) T cells are positively and negatively selected by M3, with no contribution from the more abundant class Ia molecules. These results demonstrate that class Ib molecules can function in thymic education like class Ia molecules, despite limited ligand diversity and low levels of expression.

Tissue distribution, regulation and intracellular localization of murine CD1 molecules.

CD1 molecules are MHC-unlinked class Ib molecules consisting of classical (human CD 1a-c) and non-classical subsets (human CD1d and murine CD1). The characterization of non-classical subsets of CD1 is limited due to the lack of reagents. In this study, we have generated two new anti-mouse CD1 monoclonal antibodies, 3H3 and 5C6, by immunization of hamsters with purified CD1 protein. These antibodies recognize CD1-transfected cells and have no reactivity to cells isolated from CD1-/- mice. Both antibodies precipitate the 52 kDa heavy chain and 12 kDa beta2m from thymocytes and splenocytes by radio-immunoprecipitation. Deglycosylation of CD1 reduces molecular mass of the heavy chain by 7.5 kDa, which can be detected by 3H3 but not 5C6. 3H3 and 5C6 detect surface CD1 expression on cells from the thymus, spleen, lymph node and bone marrow, but not on intestinal epithelial cells. Developmentally, CD1 is expressed on thymocytes prior to TCR rearrangement and remains constant throughout thymic development. CD1 is expressed early in the fetal liver (day 14) and remains expressed in hepatocytes postnatally. These data support evidence of a role for CD1 in the selection and/or expansion of NK1- T cells of both thymic origin and extrathymic origin. Unlike classical class I molecules, murine CD1 levels are not affected by IFN-gamma, but like human CD1b can be up-regulated by IL-4 and GM-CSF although only moderately. Similar to human CD1b, murine CD1 is found by immunofluorescence microscopy on the cell surface, and in various intracellular vesicles, including early and late endosomes. Localization in endocytic compartments indicates that murine CD1 may be capable of binding endocytosed antigens.

Development of the Taiwanese Depression Questionnaire.

BACKGROUND: Previous studies have shown that different cultures have various modes for emotional expression which suggest that Western scales have certain limitations. The purpose of this study was to develop a culturally relevant depression screening questionnaire: the "Taiwanese Depression Questionnaire" (TDQ) which can be used to conduct epidemiological surveys in Taiwanse society. METHODS: Based on the results of previous studies and related references, a draft of the TDQ was constructed. After several research group discussions, the first version of a 20-item draft was developed. By purposive sampling, 48 patients, from depressive disorder and anxiety disorder groups of Chang Gung Memorial Hospital's psychiatric clinics, self-rated the draft to test the wording, sequences, and its concurrent validity. After item analysis, a meeting of experts, and draft modification, an 18-item second version of the draft was established. Afterwards, 107 community subjects, recruited from Jenwu Township of Kaohsiung County, were sampled for the receiver operating characteristic (ROC) curve analysis, conventional validity index, and internal consistency of reliability. RESULTS: The results demonstrate that Cronbach's alpha coefficient (0.90), concurrent validity, and the area under the ROC curves (0.92) are all quite satisfactory. The 18-item TDQ had a sensitivity of 0.89 and a specificity of 0.92 at a cutoff score of 19. CONCLUSIONS: The TDQ is a culturally relevant questionnaire, which is adaptable for screening depressive people in the local communities.

Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice.

The MHC class lb molecule, CD1, has been conserved throughout mammalian evolution. To assess the function of CD1 in lymphocyte development, we generated mice with targeted disruption of the CD1.1 and CD1.2 genes. CD1-deficient mice have normal numbers of CD4+ and CD8+ T cells but marked reduction in NK1.1-bearing T cells, particularly those with a canonical gene rearrangement of V alpha14-J alpha281. CD1-deficient mice are unable to generate a rapid IL-4 response following systemic T cell activation but can generate effective antigen-specific Th2 responses. Thus, CD1 is required for the development of a specialized subset of T lymphocytes with a monomorphic antigen receptor. The rapid effector cytokine secretion of these T cells suggests that CD1 educates adaptive immune cells to subserve functions of innate immunity.

Clinical characteristics of outpatients at a psycho-oncology clinic in a radiation oncology department.

BACKGROUND: Psychosocial care in cancer medicine has become increasingly important. The first psychiatric consultation-liaison (C-L) outpatient clinic in Taiwan was established in a department of radiation oncology in a medical center in Oct. 1998. METHODS: From October 1998 through January 2000, 121 patients were referred for psychosocial evaluation. Six referred patients were excluded because of cerebral complications of malignant disease or coincidental psychiatric disorder. The remaining 115 patients were referred because of psychological problems related to their malignant disease. These patients were divided into 'depression related disorder' and 'anxiety related disorder' groups according to psychiatric standard diagnoses. The records of these referred patients were retrospectively analyzed based on the psychiatric diagnoses. RESULTS: The outpatient utilization rate for psychiatric consultation escalated from 0% to 5.92% after the C-L clinic was established. Patients with nasopharynx, breast, and head and neck cancer had higher referral, rates (over 10%) than patients with other types of cancer. There were significant differences in major subjective psychiatric problems and psychological reactions between patients with psychiatric diagnosis and those without psychiatric diagnosis. Cancer patients who were diagnosed with 'depression related disorder' visited this clinic more times than those with 'anxiety related disorder'. The former received antidepressant drugs more frequently than the latter. There were also more multiple visits in the former group than the latter group. CONCLUSION: Locating a C-L clinic in a radiation oncology department improves access to psychiatric evaluation, early detection and continuous intervention can then be offered to cancer patients with anxiety or depression, especially those with 'depression related disorder'.