Hideto Tamura

Tumor-associated B7-H1 promotes T-cell apoptosis: a potential mechanism of immune evasion.

B7-H1, a recently described member of the B7 family of costimulatory molecules, is thought to be involved in the regulation of cellular and humoral immune responses through the PD-1 receptor on activated T and B cells. We report here that, except for cells of the macrophage lineage, normal human tissues do not express B7-H1. In contrast, B7-H1 is abundant in human carcinomas of lung, ovary and colon and in melanomas. The pro-inflammatory cytokine interferon-γ upregulates B7-H1 on the surface of tumor cell lines. Cancer cell–associated B7-H1 increases apoptosis of antigen-specific human T-cell clones in vitro, and the apoptotic effect of B7-H1 is mediated largely by one or more receptors other than PD-1. In addition, expression of B7-H1 on mouse P815 tumor increases apoptosis of activated tumor-reactive T cells and promotes the growth of highly immunogenic B7-1+ tumors in vivo. These findings have implications for the design of T cell–based cancer immunotherapy.

B7-H4, a Molecule of the B7 Family, Negatively Regulates T Cell Immunity

We identify a B7 family molecule, B7-H4, by protein sequence analysis and comparative molecular modeling. While B7-H4 mRNA is widely distributed in mouse and human peripheral tissues, cell surface expression of B7-H4 protein is limited and shows an inducible pattern on hematopoietic cells. Putative receptor of B7-H4 can be upregulated on activated T cells. By arresting cell cycle, B7-H4 ligation of T cells has a profound inhibitory effect on the growth, cytokine secretion, and development of cytotoxicity. Administration of B7-H4Ig into mice impairs antigen-specific T cell responses whereas blockade of endogenous B7-H4 by specific monoclonal antibody promotes T cell responses. B7-H4 thus may participate in negative regulation of cell-mediated immunity in peripheral tissues.

Costimulating aberrant T cell responses by B7-H1 autoantibodies in rheumatoid arthritis

A pathogenic hallmark of rheumatoid arthritis (RA) is persistent activation of self-reactive CD4(+) T cells. The cause of this aberrant activity remains elusive. We report here detection of autoantibodies against B7-H1, a recently described member of the B7 family, in 29% of patients with RA versus 4% of healthy donors. High-level expression of cell surface B7-H1 are found on activated human CD4(+), CD8(+), and CD45RO(+) T cells. Immobilized autoantibodies to B7-H1 are capable of costimulating the proliferation of CD4(+) T cells in vitro, and the presence of these autoantibodies correlates with active disease status. Using immobilized B7-H1 mAbs and programmed death 1Ig, we demonstrate that engagement of B7-H1 on CD4(+) T cells costimulates proliferation and secretion of IL-10, and subsequently leads to programmed cell death, accompanied with upregulated expression of TNF-related apoptosis-inducing ligand and activation of caspase-3. Taken together with our previous findings, these data indicate a bidirectional signaling role of B7-H1 in T cell costimulation and apoptosis and implicate B7-H1 autoantibodies as contributing to the progression of RA by inducing aberrant T cell responses.

Diagnostic utility of flow cytometry in low-grade myelodysplastic syndromes: a prospective validation study.

The diagnosis of myelodysplastic syndromes is not always straightforward when patients lack specific diagnostic markers, such as blast excess, karyotype abnormality, and ringed sideroblasts. This article proposes a flow cytometry protocol that can be used in the diagnostic work-up of low-grade myelodysplastic syndrome patients who lack specific diagnostic markers. See related perspective article on page 1041. Background The diagnosis of myelodysplastic syndromes is not always straightforward when patients lack specific diagnostic markers, such as blast excess, karyotype abnormality, and ringed sideroblasts. Design and Methods We designed a flow cytometry protocol applicable in many laboratories and verified its diagnostic utility in patients without those diagnostic markers. The cardinal parameters, analyzable from one cell aliquot, were myeloblasts (%), B-cell progenitors (%), myeloblast CD45 expression, and channel number of side scatter where the maximum number of granulocytes occurs. The adjunctive parameters were CD11b, CD15, and CD56 expression (%) on myeloblasts. Marrow samples from 106 control patients with cytopenia and 134 low-grade myelodysplastic syndromes patients, including 81 lacking both ringed sideroblasts and cytogenetic aberrations, were prospectively analyzed in Japan and Italy. Results Data outside the predetermined reference range in 2 or more parameters (multiple abnormalities) were common in myelodysplastic syndromes patients. In those lacking ringed sideroblasts and cytogenetic aberrations, multiple abnormalities were observed in 8/26 Japanese (30.8%) and 37/55 Italians (67.3%) when the cardinal parameters alone were considered, and in 17/26 Japanese (65.4%) and 42/47 Italians (89.4%) when all parameters were taken into account. Multiple abnormalities were rare in controls. When data from all parameters were used, the diagnostic sensitivities were 65% and 89%, specificities were 98% and 90%, and likelihood ratios were 28.1 and 8.5 for the Japanese and Italian cohorts, respectively. Conclusions This protocol can be used in the diagnostic work-up of low-grade myelodysplastic syndromes patients who lack specific diagnostic markers, although further improvement in diagnostic power is desirable.

Marrow stromal cells induce B7-H1 expression on myeloma cells, generating aggressive characteristics in multiple myeloma.

Tumor-associated B7-H1 molecules inhibit antitumor immunity in some malignancies. We found that B7-H1 expression on patient myeloma cells and human myeloma cell lines (HMCLs) was upregulated by cultivating the cells with autologous stromal cells and the human stromal cell line HS-5. Among major cytokines produced by HS-5 cells, interleukin (IL)-6-induced B7-H1 expression on HMCLs. Moreover, HS-5 cell-mediated B7-H1 expression was downregulated by inhibiting IL-6. B7-H1+ HMCLs were more proliferative and less susceptible to antimyeloma chemotherapy compared with B7-H1− HMCLs. Moreover, the former cells showed higher levels of Bcl-2 and FasL expression than the latter. Finally, B7-H1 molecules on HMCLs induced T-cell apoptosis and anergy of tumor-specific T cells. Consistent with these in vitro observations, patients whose myeloma cells expressed high levels of B7-H1 had higher myeloma cell percentages in the bone marrow (BM) and higher serum lactate dehydrogenase levels compared with other myeloma patients. In addition, B7-H1 expression levels were often upregulated after myeloma patients relapsed or became refractory to therapy. Our data indicate that the BM microenvironment upregulates B7-H1 expression on myeloma cells, which links to the two biological actions of inducing T-cell downregulation and enhancing aggressive myeloma-cell characteristics. Modulating the B7-H1 pathway may be worthwhile in myeloma.

Blockade of LIGHT/LTβ and CD40 signaling induces allospecific T cell anergy, preventing graft-versus-host disease

Previous studies have shown that blockade of LIGHT, a T cell costimulatory molecule belonging to the TNF superfamily, by soluble lymphotoxin β receptor–Ig (LTβR-Ig) inhibits the cytotoxic T lymphocyte (CTL) response to host antigenic disparities and ameliorates lethal graft-versus-host disease (GVHD) in a B6 to BDF1 mouse model. Here, we demonstrate that infusion of an mAb against CD40 ligand (CD40L) further increases the efficacy of LTβR-Ig, leading to complete prevention of GVHD. We further demonstrate that alloantigen-specific CTLs become anergic upon rapid expansion, and persist in the tolerized mice as a result of costimulatory blockade. Transfer of anergic CTLs to secondary F1 mice fails to induce GVHD despite the fact that anergic CTLs can be stimulated to proliferate in vitro by antigens and cytokines. Our study provides a potential new approach for the prevention of lethal GVHD.

Multicenter validation of a reproducible flow cytometric score for the diagnosis of low-grade myelodysplastic syndromes: results of a European LeukemiaNET study

Background The current World Health Organization classification of myelodysplastic syndromes is based morphological evaluation of bone marrow dysplasia. In clinical practice, the reproducibility of the recognition of dysplasia is usually poor especially in cases that lack specific markers such as ring sideroblasts and clonal cytogenetic abnormalities. Design and Methods We aimed to develop and validate a flow cytometric score for the diagnosis of myelodysplastic syndrome. Four reproducible parameters were analyzed: CD34+ myeloblast-related and B-progenitor-related cluster size (defined by CD45 expression and side scatter characteristics CD34+ marrow cells), myeloblast CD45 expression and granulocyte side scatter value. The study comprised a “learning cohort” (n=538) to define the score and a “validation cohort” (n=259) to confirm its diagnostic value. Results With respect to non-clonal cytopenias, patients with myelodysplastic syndrome had increased myeloblast-related cluster size, decreased B-progenitor-related cluster size, aberrant CD45 expression and reduced granulocyte side scatter (P<0.001). To define the flow cytometric score, these four parameters were combined in a regression model and the weight for each variable was estimated based on coefficients from that model. In the learning cohort a correct diagnosis of myelodysplastic syndrome was formulated in 198/281 cases (sensitivity 70%), while 18 false-positive results were noted among 257 controls (specificity 93%). Sixty-five percent of patients without specific markers of dysplasia (ring sideroblasts and clonal cytogenetic abnormalities) were correctly classified. A high value of the flow cytometric score was associated with multilineage dysplasia (P=0.001), transfusion dependency (P=0.02), and poor-risk cytogenetics (P=0.04). The sensitivity and specificity in the validation cohort (69% and 92%, respectively) were comparable to those in the learning cohort. The likelihood ratio of the flow cytometric score was 10. Conclusions A flow cytometric score may help to establish the diagnosis of myelodysplastic syndrome, especially when morphology and cytogenetics are indeterminate.

Expression of functional B7-H2 and B7.2 costimulatory molecules and their prognostic implications in de novo acute myeloid leukemia.

Purpose: The B7 family molecules have been shown to regulate immune responses in both positive and negative fashions. Their roles in the progression of human cancers, however, are not well established. The aim of this study was to examine whether leukemic cells of acute myeloid leukemia express functional B7 family molecules and, if so, whether such expression has any clinical significance. Experimental Design: The expression of four B7 family molecules, B7.1, B7.2, B7-H1, and B7-H2, on leukemic cells from acute myeloid leukemia patients was analyzed by flow cytometry. The function of the expressed molecules was examined by the allogeneic mixed lymphocyte-leukemic cell reaction, and their relationship to the clinical data and survival was analyzed. Results: Although B7.1 and B7-H1 expressions were rare, the cells from a substantial number of acute myeloid leukemia cases expressed B7.2 and B7-H2 molecules [mean percentages of B7.2- and B7-H2-positive cells were 28.9% (n = 58) and 15.3% (n = 59), respectively]. Patients in whom >25% of leukemic cells expressed B7-H2 had significantly shorter survival, and this B7-H2 positivity had the strongest prognostic value when B7-H2 and other prognostic factors were analyzed together by multivariate analysis (P = 0.0108). Furthermore, B7.2 expression was associated with hyperleukocytosis (P = 0.026). Consistent with this finding, acute myeloid leukemia cells expressing B7.2 and B7-H2 induced allogeneic CD4+ T cells to proliferate and secrete interleukin-4 and interleukin-10 in vitro, effects that were partially blocked by antibodies against B7.2 and B7-H2. Conclusions: Our results indicate the expression of functional B7.2 and B7-H2 molecules, and these molecules may facilitate progression of acute myeloid leukemia.

Interferon-γ and tumor necrosis factor-α induce an immunoinhibitory molecule, B7-H1, via nuclear factor κB activation in blasts in myelodysplastic syndromes

During disease progression in myelodysplastic syndromes (MDS), clonal blasts gain a more aggressive nature, whereas nonclonal immune cells become less efficient via an unknown mechanism. Using MDS cell lines and patient samples, we showed that the expression of an immunoinhibitory molecule, B7-H1 (CD274), was induced by interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) on MDS blasts. This induction was associated with the activation of nuclear factor-kappaB (NF-kappaB) and nearly completely blocked by an NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). B7-H1(+) MDS blasts had greater intrinsic proliferative capacity than B7-H1(-) MDS blasts when examined in various assays. Furthermore, B7-H1(+) blasts suppressed T-cell proliferation and induced T-cell apoptosis in allogeneic cocultures. When fresh bone marrow samples from patients were examined, blasts from high-risk MDS patients expressed B7-H1 molecules more often compared with those from low-risk MDS patients. Moreover, MDS T cells often overexpressed programmed cell death 1 (PD-1) molecules that transmit an inhibitory signal from B7-H1 molecules. Taken together, these findings provide new insight into MDS pathophysiology. IFNgamma and TNFalpha activate NF-kappaB that in turn induces B7-H1 expression on MDS blasts. B7-H1(+) MDS blasts have an intrinsic proliferative advantage and induce T-cell suppression, which may be associated with disease progression in MDS.

Interferon-gamma and tumor necrosis factor-alpha induce an immunoinhibitory molecule, B7-H1, via nuclear factor-kappaB activation in blasts in myelodysplastic syndromes.

During disease progression in myelodysplastic syndromes (MDS), clonal blasts gain a more aggressive nature, whereas nonclonal immune cells become less efficient via an unknown mechanism. Using MDS cell lines and patient samples, we showed that the expression of an immunoinhibitory molecule, B7-H1 (CD274), was induced by interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha) on MDS blasts. This induction was associated with the activation of nuclear factor-kappaB (NF-kappaB) and nearly completely blocked by an NF-kappaB inhibitor, pyrrolidine dithiocarbamate (PDTC). B7-H1(+) MDS blasts had greater intrinsic proliferative capacity than B7-H1(-) MDS blasts when examined in various assays. Furthermore, B7-H1(+) blasts suppressed T-cell proliferation and induced T-cell apoptosis in allogeneic cocultures. When fresh bone marrow samples from patients were examined, blasts from high-risk MDS patients expressed B7-H1 molecules more often compared with those from low-risk MDS patients. Moreover, MDS T cells often overexpressed programmed cell death 1 (PD-1) molecules that transmit an inhibitory signal from B7-H1 molecules. Taken together, these findings provide new insight into MDS pathophysiology. IFNgamma and TNFalpha activate NF-kappaB that in turn induces B7-H1 expression on MDS blasts. B7-H1(+) MDS blasts have an intrinsic proliferative advantage and induce T-cell suppression, which may be associated with disease progression in MDS.