Wei-Feng Chen

Large Scale Identification of Human Hepatocellular Carcinoma-Associated Antigens by Autoantibodies

Autoantibodies are often detected in hepatocellular carcinoma (HCC), and these responses may represent recognition of tumor Ags that are associated with transformation events. The identities of these Ags, however, are less well known. Using serological analysis of recombinant cDNA expression libraries (SEREX) from four HCC patients, we identified 55 independent cDNA sequences potentially encoding HCC tumor Ags. Of these genes, 15 are novel. Two such proteins, HCA587 and HCA661, were predominantly detected in testis, but not in other normal tissues, except for a weak expression in normal pancreas. In addition to HCC, these two Ags can be found in cancers of other histological types. Therefore, they can be categorized as cancer-testis (CT) Ags. Two other Ags (HCA519 and HCA90) were highly overexpressed in HCC and also expressed in cancer cell lines of lung, prostate, and pancreas, but not in the respective normal tissues. Four other Ags were identified to be expressed in particular types of cancer cell lines (HCA520 in an ovarian cancer cell line, HCA59 and HCA67 in a colon cancer cell line, HCA58 in colon and ovarian cancer cell lines), but not in the normal tissue counterpart(s). In addition, abundant expression of complement inactivation factors was found in HCC. These results indicate a broad range expression of autoantigens in HCC patients. Our findings open an avenue for the study of autoantigens in the transformation, metastasis, and immune evasion in HCC.

Identification of genes differentially expressed in human hepatocellular carcinoma by a modified suppression subtractive hybridization method

To identify differentially expressed genes in human HCC in China, we applied a modified SSH method for cDNA subtraction. Such modification has made the method more effective for subtraction. We have obtained 36 and 24 differentially expressed cDNA fragments after modified SSH from 4 paired samples of human HCC and non‐HCC tissues, respectively. Reverse Northern blotting analysis was performed to further identify the genes differentially expressed in the HCC and non‐HCC tissue samples. There were 25 genes really overexpressed in HCC, and their corresponding encoding molecules may reflect the events of cell accelerated metabolism, proliferation, angiogenesis, anti‐apoptosis, tumorigenesis (TLH107, TFH9) and the potential for metastasis. Of the 25 genes overexpressed in HCC, 5 were novel and their full‐length cDNAs were cloned. These 5 novel genes are functionally associated with the occurrence and development of HCC according to the Database analysis. In the paired non‐HCC tissues, there were 15 genes lowly or not expressed in HCC, and their encoding proteins function as tumor suppressors (TFA3, TFA11), acute‐phase reactive proteins, and the blood plasma proteins that are mainly or exclusively synthesized in the liver. The distinct profiles of the differentially expressed genes in HCC and the paired non‐HCC tissues have partially reflected the genetic alterations during HCC tumorigenesis. The novel HCC‐specific gene TLH6 and the CT antigen encoding gene TLH107 may have diagnostic and therapeutic potentials in HCC and/or other solid cancers.

The Spontaneous CD8+ T-Cell Response to HLA-A2-Restricted NY-ESO-1b Peptide in Hepatocellular Carcinoma Patients

Purpose: Hepatocellular carcinoma (HCC) can express various cancer-testis antigens including NY-ESO-1, members of the SSX family, members of the MAGE family, SCP-1, and CTP11. Immunotherapy directed against these antigens is a potential alternative treatment for HCC. To date, it remains unclear whether HCC patients have spontaneous immune responses to these tumor antigens. The objectives of this study were to measure immune responses to NY-ESO-1, a promising cancer vaccine candidate, in HCC patients using the HLA-A2–restricted NY-ESO-1b peptide (p157-165) to measure cellular responses and whole protein to measure antibody responses. Experimental Design: In HLA-A2+ patients with NY-ESO-1+ HCC, we analyzed T-cell antigen-dependent interferon (IFN)-γ and/or Granzyme B release by enzyme-linked immunospot (ELISPOT) assay and IFN-γ–producing intracellular cytokine flow cytometry (CytoSpot). As an assay independent of T-cell function, we performed tetramer staining. Antibodies to whole NY-ESO-1 were assayed by enzyme-linked immunosorbent assay. Results: The frequency of specific CD8+ T-cell responses to NY-ESO-1b in 28 NY-ESO-1 mRNA+HLA-A2+ HCC patients was 35.7% (10 of 28). The average magnitude of effector CD8+ T cells was 0.3% (89 ± 59 per 2.5 × 104 CD8+ cells) and 1.2% as measured by IFN-γ release ELISPOT and CytoSpot assays, respectively. These in vitro induced NY-ESO-1b–specific CD8+ T cells can also recognize HepG2 cells transfected with pcDNA3.1-NY-ESO-1 in both IFN-γ and Granzyme B ELISPOT assays. Frequencies of NY-ESO-1b–specific T cells in several patients were confirmed by tetramer staining. Nonfunctional tetramer+CD8+ T cells were also present. The CD8+ T-cell response was apparently increased in patients with late-stage HCC. A discordance between antibody and CD8+ T-cell responses in HCC patients was observed. Conclusions: The elevated frequency of specific CD8+ T-cell responses to NY-ESO-1b in NY-ESO-1 mRNA+HLA-A2+ HCC patients suggests that NY-ESO-1 is appropriate for use in the immunotherapy of HCC patients.

Developmental pathway of CD4+CD8− medullary thymocytes during mouse ontogeny and its defect in Aire−/− mice

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 because Relb and Aire deficiency 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.

PLAC1 is a tumor-specific antigen capable of eliciting spontaneous antibody responses in human cancer patients

Immunoselection and tumor evasion constitutes one of the major obstacles in cancer immunotherapy. A potential solution to this problem is the development of polyvalent vaccines, and the identification of more tumor‐specific antigens is a prerequisite for the development of cancer vaccines. To identify novel tumor‐specific antigens, suppression subtractive hybridization (SSH) was performed to isolate genes differentially expressed in human hepatocellular cancer (HCC) tissues. PLAC1 (PLACenta‐specific 1) was one of the genes identified highly expressed in HCC tissues but not in paired noncancerous tissues. Further analyses revealed its expression in several other types of cancer tissues as well as tumor cell lines, but not in normal tissues except for placenta. Among HCC samples tested, 32% (22/69) showed PLAC1 mRNA expression while the protein was detected in 23.3% (7/30). A serological survey revealed that 3.8% (4/101) of HCC patients had anti‐PLAC1 antibody response, suggesting the immunogenicity of PLAC1 in HCC patients. PLAC1 represents a new class of tumor associated antigen with restricted expression in placenta and cancer tissues, that may serve as a target for cancer vaccination.

The immunosuppressive tumor microenvironment in hepatocellular carcinoma.

Increasing evidence indicates the immunosuppressive nature of the local environment in tumor. The present study was focused on analyzing the immune status within hepatocellular carcinoma. In contrast to the increasing number of CD4+ T cells, CD8+, CD3−CD56+, CD3+CD56+, and γδT cells were all found to be under-represented in tumor infiltrating lymphocytes. Notably, the relative abundance of CD3+CD56+ cells appeared to be correlated with patient survival. Functional analysis demonstrated that CD4+ cells in the tumor tended to produce more IL-10 but less IFN-γ, whereas CD8+ cells showed impaired capacity for the production of both IFN-γ and perforin. Consistent with previous reports, we observed a significant increase of Foxp3+ cells in the tumor tissue. Intriguingly, although over 90% of CD4+CD25high cells were found to be Foxp3+, the majority of Foxp3+ cells were identified in the CD4+CD25medium and CD4+CD25− subsets. In support of its role as a negative regulator, CD4+CD25high cells suppressed the proliferation of CD4+CD25− cells isolated from the same tissues in an APC dependent manner. In conclusion, the tumor microenvironment of hepatocellular carcinoma is featured by the presence of multiple immunosuppressive factors.

Characterization of the in vivo dynamics of medullary CD4+CD8- thymocyte development.

Our previous studies have defined a differentiation program followed by the newly generated single-positive (SP) thymocytes before their emigration to the periphery. In the present study, we further characterize the development of CD4SP cells in the thymic medulla using mainly intrathymic adoptive transfer assays. By analyzing the differentiation kinetics of the donor cells, which were shown to home correctly to the medullary region following adoptive transfer, we established the precursor–progeny relationship among the four subsets of CD4SP thymocytes (SP1–SP4) and demonstrated that the progression from SP1 to SP4 was unidirectional and largely synchronized. Notably, while the phenotypic maturation from SP1 to SP4 was achieved in 2–3 days, a small fraction of donor cells could be retained in the thymus for a longer period, during which they further matured in function. BrdU incorporation indicated that cell expansion occurred at multiple stages except SP1. Nevertheless, CFSE labeling revealed that only a limited number of cells actually divided during their stay in the medulla. As to the thymic emigration, there was a clear bias toward cells with increasing maturity, but no distinction was found between dividing and nondividing thymocytes. Collectively, these data not only provide solid evidence for a highly ordered differentiation program for CD4SP thymocytes, but they also illustrate several important features associated with the developmental process.

HCA587 antigen expression in normal tissues and cancers: correlation with tumor differentiation in hepatocellular carcinoma.

The HCA587 gene, identified by serological analysis of recombinant cDNA expression library (SEREX) from a hepatocellular carcinoma (HCC) patient, encodes a new member of cancer-testis antigens. HCA587 mRNA expression in normal tissues and cancers has been previously reported. To estimate its immunogenicity to induce immune response, it is essential to analyze HCA587 expression at the protein level. In this study anti-HCA587 polyclonal antibody, termed “TC-1,” was generated, and the expression of HCA587 protein was assessed by immunohistochemical staining in a panel of normal and tumor tissue sections. No HCA587 protein was shown in normal tissues except germ cells in testis and Purkinji cells in cerebellum. In HCC specimens the HCA587 protein was expressed in 37.1% (26 of 70) samples. The expressed protein was either located in the cytoplasm or nucleus depending on the individual samples. More importantly, there appears to be correlation between the tumor differentiation of HCC and HCA587 protein expression, ie, the lower differentiation, the higher percentage of protein expression. Coincidentally, seroreactivity showed that the Ab specific to recombinant HCA587 protein was detected only in the sera of three patients with poorly differentiated HCCs. HCA587 antigen was also expressed in different proportions in melanoma, lymphoma, pancreatic cancer, and lung cancer.

Human TFDP3, a novel DP protein, inhibits DNA binding and transactivation by E2F

The two known DP proteins, TFDP1 and -2, bind E2Fs to form heterodimers essential for high affinity DNA binding and efficient transcriptional activation/repression. Here we report the identification of a new member of the DP family, human TFDP3. Despite the high degree of sequence similarity, TFDP3 is apparently distinct from TFDP1 in function. Although TFDP3 retained the capacity to bind to E2F proteins, the resulting heterodimers failed to interact with the E2F consensus sequence. In contrast to the stimulatory effect of TFDP1, TFDP3 inhibited E2F-mediated transcriptional activation. Consistent with this observation, we found that ectopic expression of TFDP3 impaired cell cycle progression from G1 to S phase instead of facilitating such a transition as TFDP1 does. Sequence substitution analysis indicated that the DNA binding domain of TFDP3 was primarily responsible for the lack of DNA binding ability of E2F-TFDP3 heterodimers and the inhibition of E2F-mediated transcriptional activation. Fine mapping further revealed four amino acids in this region, which were critical for the functional conversion from activation by TFDP1 to suppression by TFDP3. In conclusion, these studies identify a new DP protein and a novel mechanism whereby E2F function is regulated.

Specific CD8 + T cell responses to HLA-A2 restricted MAGE-A3 p271–279 peptide in hepatocellular carcinoma patients without vaccination

The MAGE-A3 protein, one of the promising tumor antigens for immunotherapy, is highly expressed in human hepatocellular carcinoma (HCC). In this study, we estimated the specific CD8+ T cell immune response to MAGE-A3 p271–279 peptide (M3271) in the peripheral blood of HCC patients without antigen vaccination in order to evaluate its immunotherapeutic potential in these patients. After expansion in vitro, the functional IFN-γ producing M3271 specific CD8+ T cells were detected in 30.8% (8/26) of HLA-A2+MAGE-A3+ HCC patients. The effector CD8+ T cells could release cytotoxic molecules of granzyme B and perforin after restimulation with natural HLA-A2+MAGE-A3+ HCC cell lines in the samples tested. The functional supertype of HLA-A2 in the presentation of HLA-A*0201 restricted M3271 peptide has been identified in the Chinese HCC patients of Han ethnicity, that widely expanded the applicability of this tumor peptide vaccine in Chinese HCC patients. Thus, the functionally detectable pre-existence of M3271-specific CD8+ T cells in HCC patients makes M3271 a potential target for immunotherapy in these patients. The responsive CD8+ T cells to both NY-ESO-1 and MAGE-A3 antigens provide a rationale for the application of a bivalent vaccine in HCC patients with tumors expressing both antigens.