Published papers

In cancer patients, sentinel lymph nodes (SLNs) are crucial in the induction of antitumor T cells. However, in many cases, SLNs and tumors appear to be in immunosuppressive condition through mechanisms yet to be elucidated. In this study, the role of tumor-derived TGF-β1 in the generation of immunosuppressive microenvironments of tumors and SLNs was investigated. Murine colorectal carcinoma CT26 transduced with TGF-β1 cDNA (CT26-TGF-β1) showed enhanced tumor growth compared with mock-transduced CT26 (CT26-Mock) when implanted in syngeneic Balb/c mice, even though CT26-TGF-β1 shows slower growth in vitro. This enhanced growth was not observed when implanted in immunodeficient mice, suggesting that TGF-β1 enhanced tumor growth by suppressing antitumor T-cell responses. Analysis of immune cells in CT26-TGF-β1-implanted mice revealed impairment of dendritic cells (DCs), decrease of CD8 T cells, and increase of MDSCs and Tregs in the tumors. Similarly, the SLNs of these mice showed an increase of MDSCs, Tregs, and PD-L1 DCs, and decrease of T-cell stimulatory activity of DCs accompanied by decreased CD80 expression and TNF-α production. In addition, induction of tumor antigen-specific T cells from SLNs of the CT26-TGF-β1-implanted mice was significantly reduced. These results demonstrate that overproduction of TGF-β1 is critical for the generation of immunosuppressive microenvironments in both tumors and SLNs, which may result in suppression of spontaneous antitumor CD8 T-cell responses. Therefore, TGF-β1 is an attractive target for restoration of immunosuppressive condition in cancer patients.

The American Joint Committee on Cancer/Union Internationale Contre le Cancer (AJCC/UICC) TNM staging system provides the most reliable guidelines for the routine prognostication and treatment of colorectal carcinoma. This traditional tumour staging summarizes data on tumour burden (T), the presence of cancer cells in draining and regional lymph nodes (N) and evidence for distant metastases (M). However, it is now recognized that the clinical outcome can vary significantly among patients within the same stage. The current classification provides limited prognostic information and does not predict response to therapy. Multiple ways to classify cancer and to distinguish different subtypes of colorectal cancer have been proposed, including morphology, cell origin, molecular pathways, mutation status and gene expression-based stratification. These parameters rely on tumour-cell characteristics. Extensive literature has investigated the host immune response against cancer and demonstrated the prognostic impact of the in situ immune cell infiltrate in tumours. A methodology named 'Immunoscore' has been defined to quantify the in situ immune infiltrate. In colorectal cancer, the Immunoscore may add to the significance of the current AJCC/UICC TNM classification, since it has been demonstrated to be a prognostic factor superior to the AJCC/UICC TNM classification. An international consortium has been initiated to validate and promote the Immunoscore in routine clinical settings. The results of this international consortium may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

Purpose and Methods. T cells play an important role in in vivo rejection of human melanoma. Human melanoma antigens recognized by autologous T cells were identified. These antigens are classified as tissue (melanocyte)- specific proteins, cancer-testis antigens (proteins expressed in normal testis and various cancers), tumor-specific peptides derived from mutations in tumor cells, and others. Results. A variety of mechanisms generating T cell epitopes on tumor cells were discovered. Various clinical observations, including tumor regression observed in adoptive transfer of gp 100-reactive T cells suggest that these identified melanoma peptides may function as tumor rejection antigens. Immunodominant common epitopes that could expand melanoma-reactive cytotoxic T lymphocytes (CTLs) in vitro were found in the MART-1 and gp 100 antigens. New immunization protocols - including immunization with peptides, recombinant viruses, plasmid DNAs, and dendritic cells pulsed with peptides as well as adoptive transfer of in vitro-generated CTLs by stimulation with antigenic peptides - were developed (phase I clinical trials have been performed in the Surgery Branch of the National Cancer Institute, Bethesda, MD, U.S.A.). Immunization with the gp100(209(210M)) peptide that was modified to have high HLA-A2 binding affinity, along with incomplete Freund's adjuvant and interleukin (IL)-2, resulted in a 42% response rate in patients with melanoma. Conclusion. These immunotherapies need further improvement due to the mechanisms of tumor escape from T cell responses.

Objective. To examine serial changes in serum anti-topoisomerase I (anti-topo I) antibody levels in patients with systemic sclerosis (SSc), as well as associations with clinical features and the in vivo activation status of circulating topo I-reactive T and B cells. Methods. Serum anti-topo I antibody levels were serially measured at different time points in 28 SSc patients who were positive for anti-topo I antibody at their first visit (range of followup 6-29 years). The patients were subgrouped according to the disappearance (group 1) or persistence (group 2) of anti-topo I antibody. Clinical findings as well as T and B cell responses to topo I were compared between these 2 groups. Results. Serum anti-topo I antibody disappeared during the period of followup in 6 patients (group 1), but persisted in 22 patients (group 2). Loss of anti-topo I antibody occurred within 10 years after the first visit and independently of treatment. Group I patients had less extensive skin and lung involvement and better survival rates than did group 2 patients. Complete loss of anti-topo I antibody followed a reduction in isotype expression and epitope reactivities. The kinetics of in vitro T cell proliferation induced by topo I were delayed and circulating topo I- reactive T Cells were less frequently detected in group I versus group 2 patients, suggesting that the disappearance of anti-topo I antibody was due to loss of activation of topo I-reactive T cells. In vitro production of anti-topo I antibody in peripheral blood mononuclear cell cultures in response to antigenic stimulation in both group 1 and group 2 patients indicated persistence of anti-topo I antibody-producing 'memory' B cells even after the loss of serum anti-topo I antibody. Conclusion. Our results indicate that there is a distinct subset of anti-topo I-positive SSc patients who lose anti-topo I antibody during the disease course and have a favorable outcome. In vivo production of anti-topo I autoantibody may require antigenic stimulation that activates topo I-reactive T and B cells.

Objective. To identify the T cells responsive to β2-glycoprotein I (β2GPI) that mediate antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). Methods. In vitro proliferative responses and anti-β2GPI antibody production induced by β2GPI were examined in peripheral blood mononuclear cell (PBMC) cultures from 12 APS patients, 13 systemic lupus erythematosus patients without APS, and 12 healthy donors. Results. Peripheral blood T cells from all subjects failed to respond to β2GPI in its native form. In contrast, reduced β2GPI was able to stimulate T cells not only from all 12 patients with anti-β2GPI antibodies, but also from 10 of 25 individuals without anti-β2GPI antibodies. The specificity of the responses to β2GPI was confirmed by activation of the reduced β2GPI-primed T cells by recombinant β2GPI in secondary cultures. Characterization of the T cell response induced by β2GPI revealed that the response was associated with the presence of the DR53-associated alleles, the responding T cells were CD4+ and restricted by HLA class II, and antigenic peptides were located in domains IV and/or V. Anti-β2GPI antibody production was induced specifically in anti-β2GPI antibody-positive patients, in PBMC cultures with reduced β2GPI. Anti-β2GPI antibodies produced in vitro recognized β2GPI immobilized with cardiolipin or β2GPI coated on 'high-binding' polystyrene plates. Conclusion. These results strongly suggest that CD4+ and HLA class II-restricted T cells responsive to β2GPI are involved in the production of antiphospholipid antibodies in APS patients.

We recently identified CD4+ T cells that are autoreactive to β2-glycoprotein I (β2GPI) and that promote antiphospholipid antibody production in patients with antiphospholipid syndrome (APS). In this study, T-cell receptor (TCR) β chains of β2GPI-reactive T cells were examined in 8 β2GPI-responders, including 5 patients with APS and 3 healthy subjects, using polymerase chain reaction and single-strand conformation polymorphism (PCRSSCP) analysis combined with in vitro stimulation of peripheral blood T cells with recombinant β2GPI. The TCR Vβ segments that expanded oligoclonally after stimulation with β2GPI varied among responders, but the Vβ7 and Vβ8 segments were commonly detected in 6 and 4 β2GPI-responders, respectively. Analysis of the complementarity-determining region 3 sequence of β2GPI-reactive T cells revealed limited diversity, and all Vβ7+ TCRs had an amino acid motif of TGxxN/Q or minor variations. The Vβ8+ TCRs had another motif, PxAxxD/E. Surprisingly, an identical Vβ7+ TCRβ chain was used by β2GPI-reactive T cells in 3 patients with APS. There was no apparent difference in the TCRβ usage between APS patients and healthy responders. Some of the Vβ7+ TCRs with the TGxxN/Q motif detected by PCR-SSCP analysis were also used by β2GPI-specific CD4+ T-cell clones responsive to an immunodominant epitope containing the major phospholipid-binding site. Depletion of Vβ7+ or Vβ8+ T cells from the peripheral blood mononuclear cell cultures significantly inhibited in vitro anti-β2GPI antibody production in response to β2GPI. Our results indicate preferential usage of TCRβ chains by β2GPI-reactive T cells. These TCRβ chains can be reasonable targets for TCR-based immunotherapy for patients with APS.

We have recently reported that in patients with chronic immune thrombocytopenic purpura (IMTP), circulating T and B cells that are responsive to gpIIb-IIIa can induce anti-platelet autoantibody production. In this study, the frequencies and activation status of gpIIb-IIIa-reactive T and B cells were evaluated in the peripheral blood and spleen obtained from nine IMTP patients undergoing splenectomy. There was no difference in gpIIb-IIIa-reactive T cell frequencies between peripheral blood and spleen (6.4 ± 2.6 vs 5.2 ± 2.4 per 105 T cells), as determined by limiting dilution analysis, but activated T cells responsive to gpIIb-IIIa showing accelerated proliferation kinetics and those expressing CD154 were more frequent in spleen than in peripheral blood. The frequencies of anti-gpIIb-IIIa Ab-producing B cells, as determined by ELISPOT assay, were also similar in peripheral blood and spleen (61.2 ± 24.0 vs 77.7 ± 45.3 per 105 B cells); however, an anti-gpIIb-IIIa Ab was spontaneously produced by splenocytes in vitro, but scarcely secreted by PBMCs. CD19-/surface Ig-/CD38+/CD138+ plasma cells secreting anti-gpIIb-IIIa Ab were exclusively detected in the spleen. In serial analysis, the frequencies of circulating gpIIb-IIIa-reactive T and B cells were decreased after splenectomy in patients with a complete response, but were unchanged in nonresponders. These findings indicate that an interaction between gpIIb-IIIa-reactive T and B cells inducing anti-platelet Ab production in IMTP patients occurs primarily in the spleen and that the significant number of gpIIb-IIIa-reactive T and B cells activated in the spleen are released into the circulation as memory cells.

Background: Deletions and point mutations of the p16 gene are detectable in more than 50% of ovarian cancer cells. In this study, we examined the effect of p16 gene transduction on the growth of ovarian cancer cells and on the effect of anti-cancer agents. Materials and Methods: p16-null human ovarian cancer cell lines, SKOV-3 and OVCAR-5, were used in this study. We transduced the full-length human p16 gene using recombinant adenovirus (AxCA-hp16). Results: The spontaneous growth of these cells was significantly inhibited by hp16 transduction. MTT assay revealed that AxCA-hp16 infection induced chemoresistance in both cell lines. Flow cytometric analysis revealed that only hp16-transduced SKOV-3, were arrested at the G1-phase for 3 days whereas those infected with AxCA-mock and OVCAR-5 infected with both recombinant viruses did not. Western blot analysis showed increased microtubule-associated proteins 4 (MAP4) in both cell lines. Conclusion: These results suggest that in SKOV-3 cells, G1-arrest induced by p16-transduction prevents paclitaxel- and vindesine- induced cell death, and in OVCAR-5 cells, the other unknown mechanisms play a role of chemoresistance.

Tumor cells arising from a particular tissue may exhibit the same gene expression patterns as their precursor cells. To test this proposition, we have analyzed the expression of a neural RNA-binding protein, Musashi1, in primary human central nervous system (CNS) tumors. In rodents, Musashi1 is expressed predominantly in proliferating multipotent neural precursor cells, but not in newly generated postmitotic neurons. The expression of Musashi1 is downregulated with the successive progression of neurogenesis. In normal adult human tissues, we detected low levels of Musashi1 expression in brain and testis by RT-PCR analysis. In an RNA panel of 32 cancer tissues and cell lines, elevated expression of Musashi1 was seen in all five malignant gliomas studied, in contrast to the slight expression seen in other tumor cells, including those in several melanomas and a prostate cancer. Western blot analysis showed strong Musashi1 expression in malignant gliomas compared with nonneoplastic brain tissue. Glioblastomas, the most malignant form of glioma, showed higher Musashi1 expression than less malignant gliomas by immunohistochemical analysis. Tumors with strong Musashi1 expression tended to have high proliferative activity. Thus, the expression of Musashi1 correlated with the grade of the malignancy and proliferative activity in gliomas. These results suggest that primary CNS tumors may share gene expression patterns with primitive, undifferentiated CNS cells and that Musashi1 may be a useful marker for the diagnosis of CNS tumors.