The immunogenicity of HLA-A2-restricted T-cell epitopes in the S protein of the Severe acute respiratory syndrome coronavirus (SARS-CoV) and of human coronavirus strain 229e (HCoV-229e) was analyzed for the elicitation of a T-cell immune response in donors who had fully recovered from SARS-CoV infection. We employed online database analysis to compare the differences in the amino acid sequences of the homologous T epitopes of HCoV-229e and SARS-CoV. The identified T-cell epitope peptides were synthesized, and their binding affinities for HLA-A2 were validated and compared in the T2 cell system. The immunogenicity of all these peptides was assessed by using T cells obtained from donors who had fully recovered from SARS-CoV infection and from healthy donors with no history of SARS-CoV infection. HLA-A2 typing by indirect immunofluorescent antibody staining showed that 51.6% of SARS-CoV-infected patients were HLA-A2 positive. Online database analysis and the T2 cell binding test disclosed that the number of HLA-A2-restricted immunogenic epitopes of the S protein of SARS-CoV was decreased or even lost in comparison with the homologous sequences of the S protein of HCoV-229e. Among the peptides used in the study, the affinity of peptides from HCoV-229e (H77 and H881) and peptides from SARS-CoV (S978 and S1203) for binding to HLA-A2 was higher than that of other sequences. The gamma interferon (IFN-γ) release Elispot assay revealed that only SARS-CoV-specific peptides S1203 and S978 induced a high frequency of IFN-γ-secreting T-cell response in HLA-A2⁺donors who had fully recovered from SARS-CoV infection; such a T-cell epitope-specific response was not observed in HLA-A2⁺healthy donors or in HLA-A2⁻donors who had been infected with SARS-CoV after full recovery. Thus, T-cell epitopes S1203 and S978 are immunogenic and elicit an overt specific T-cell response in HLA-A2⁺SARS-CoV-infected patients.

Using bioinformatics, we have identified a novel tumor-specific gene BJ-TSA-9, which has been validated by Northern blot analysis and reverse transcription-polymerase chain reaction (RT-PCR). BJ-TSA-9 mRNA was expressed in 52.5% (21 of 40) of human lung cancer tissues and was especially higher in lung adenocarcinoma (68.8%). To explore the potential application of BJ-TSA-9 for the detection of circulating cancer cells in lung cancer patients, nested RT-PCR was performed. The overall positive detection rate was 34.3% (24 of 70) in peripheral blood mononuclear cells (PBMCs) of patients with various types of lung cancers and was 53.6% (15 of 28) in PBMCs of lung adenocarcinoma patients. In combination with the detection of two known marker genes SCC and LUNX, the detection rate was increased to 81.4%. A follow-up study was performed in 37 patients after surgical removal of tumor mass. Among nine patients with persistent detection of two to three tumor marker transcripts in PBMCs, six patients had recurrence/metastasis. In contrast, 28 patients with transient detection of one tumor marker or without detection of any tumor marker were all in remission. Thus, BJ-TSA-9 may serve as a marker for lung cancer diagnosis and as a marker, in combination with two other tumor markers, for the prediction of the recurrence and prognosis of lung cancer patients.

To identify tumour and tumour-associated antigens in patients with hepatocellular carcinoma (HCC) one may find potential diagnostic markers and immunotherapeutic targets. In the current study, 30 distinct antigens reactive with serum IgG from HCC patients were identified by serological analysis of cDNA expression libraries (SEREX). The mRNA expression patterns of 14 of these 30 antigens were altered in cancer as further revealed by cDNA microarray, with upregulation for nine and downregulation for five antigens. One of the upregulated antigens was cancer-testis (CT) antigen (CAGE), which had been previously reported to be expressed exclusively in normal gametogenic tissues and aberrantly expressed in a variety of cancer cells. In our study, CAGE mRNA was expressed in 39.4% of HCC patients, 73.3% of patients with gastric cancer and 30.8% of patients with colorectal cancer. Antibodies against CAGE protein were detected in approximately 5.1% of the sera from HCC patients, 8.3% of that from gastric cancer patients and 7.3% of that from colorectal cancer patients. The relative high incidence of CAGE in cancer cells makes it a potential target for vaccine design. Another antigen of great interest is transgelin 2. The overexpression of transgelin 2 mRNA in a large per cent (69%) of HCC points to its potential as a diagnostic marker for HCC.

The members of MAGE gene family are highly expressed in human hepatocellular carcinoma (HCC). In the present study, we tested the tumour-specific MAGE-1 and MAGE-3 transcripts in the peripheral blood of HCC patients by nested RT–PCR to detect the circulating tumour cells and evaluate their potential clinical implication. Of 30 HCC patients, the positive rate of MAGE-1 and MAGE-3 transcripts was 43.3% (13 out of 30) and 33.3% (10 out of 30) in PBMC samples, whilst the positive rate was 70% (21 out of 30) and 53.3% (16 out of 30) in the resected HCC tissue samples, respectively. The positivity for at least one MAGE gene transcript was 63.3% (19 out of 30) in PBMC samples of HCC patients and 83.3% (25 out of 30) in the resected HCC tissue samples. MAGE-1 and/or MAGE-3 mRNA were not detected in the PBMC of those patients from whom the resected HCC tissues were MAGE-1 or MAGE-3 mRNA negative, nor in the 25 PBMC samples from healthy donors. The detection of MAGE transcripts in PBMC was correlated with the advanced stages and tumour size of the HCC, being 82.4% (14 out of 17) in tumour stages III and IVa, 56.6% (five out of nine) in stage II, and null (nought out of four) in stage I. The serum α-FP in 33.3% (10 out of 30) of HCC patients was normal or slightly elevated (<40 ng ml⁻¹). However, six of these 10 patients (α-FP <40 ng ml⁻¹) were MAGE-1 and /or MAGE-3 mRNA positive in their PBMC. The follow-up survey of MAGE mRNA in PBMC was performed in 12 patients. Seven patients with persistent MAGE-1 and/or MAGE-3 mRNA positive or from negative turned to positive died because of metastasis and/or recurrence. In striking contrast, all four patients with MAGE-1 and/or MAGE-3 mRNA from positive turned to negative and one patient with persistent MAGE-3 transcript negative are alive after last test. Collectively, detection of MAGE transcripts with follow-up survey in PBMC is a feasible and reliable assay for the early prediction of the relapse and prognosis of the HCC patients.

The newly generated single-positive (SP) thymocytes undergo further maturation in the thymic medulla before their emigration to the periphery. The present study was undertaken to validate a developmental program we proposed for CD4SP medullary thymocytes and to explore the mechanisms regulating this process. During mouse ontogeny, the emergence of different subsets of CD4SP thymocytes followed a strict temporal order from SP1 to SP4. Parallel to the transition in surface phenotype, a steady increase in function was observed. As further evidence, purified SP1 cells were able to sequentially give rise to SP2, SP3, and SP4 cells in intrathymic adoptive transfer and in culture. Notably, the development of CD4SP cells in the medulla seemed to be critically dependent on a functionally intact medullary epithelial cell compartment becauseRelbandAiredeficiency were found to cause severe blockage at the transition from SP3 to SP4. Taken together, this work establishes an ontogenetically and functionally relevant maturation program for CD4SP thymocytes. Precise dissection of this program should facilitate further inquiry into the molecular mechanisms governing normal thymocyte development and its disturbance in pathological conditions.

In search for genes associated with hepatocellular carcinoma (HCC) by cDNA microarray, we found that the transcription of TSPY, ‘testis-specific protein Y-encoded’, was upregulated in HCC. Investigation of a broad spectrum of normal and malignant tissues by RT–PCR revealed the TSPY transcript selectively expressed in normal testis, different histological types of human neoplastic tissues, and tumour cell lines. The expression of TSPY in cancer cells was further confirmed by in situ hybridisation. Indirect immunofluorescence microscopy analysis showed that TSPY was localised mainly in the cytoplasm of transiently transfected cells. Testis-specific protein Y-encoded was detected in 50% (16 of 32) of well- and moderately differentiated HCC patients, in 16% (four of 25) of poorly differentiated HCC patients, and in 5% (one of 19) of renal cell cancer patients. A serological survey revealed that 6.6% (seven of 106) HCC patients had anti-TSPY antibody response, demonstrating the immunogenicity of TSPY in humans. In conclusion, these data suggest that TSPY is a novel cancer/testis (CT) antigen and may be a potential candidate in vaccine strategy for immunotherapy in HCC patients.