Genetic dissection of the effects of stimulatory and inhibitory IgG Fc receptors on murine lupus

Immune complex (IC)-mediated tissue inflammation is controlled by stimulatory and inhibitory IgG Fc receptors (FcgammaRs). Systemic lupus erythematosus is a prototype of IC-mediated autoimmune disease; thus, imbalance of these two types of FcgammaRs is probably involved in pathogenesis. However, how and to what extent each FcgammaR contributes to the disease remains unclear. In lupus-prone BXSB mice, while stimulatory FcgammaRs are intact, inhibitory FcgammaRIIB expression is impaired because of promoter region polymorphism. To dissect roles of stimulatory and inhibitory FcgammaRs, we established two gene-manipulated BXSB strains: one deficient in stimulatory FcgammaRs (BXSB.gamma(-/-)) and the other carrying wild-type Fcgr2b (BXSB.IIB(B6/B6)). The disease features were markedly suppressed in both mutant strains. Despite intact renal function, however, BXSB.gamma(-/-) had IC deposition in glomeruli associated with high-serum IgG anti-DNA Ab levels, in contrast to BXSB.IIB(B6/B6), which showed intact renal pathology and anti-DNA levels. Lymphocytes in BXSB.gamma(-/-) were activated, as in wild-type BXSB, but not in BXSB.IIB(B6/B6). Our results strongly suggest that both types of FcgammaRs in BXSB mice are differently involved in the process of disease progression, in which, while stimulatory FcgammaRs play roles in effecter phase of IC-mediated tissue inflammation, the BXSB-type impaired FcgammaRIIB promotes spontaneous activation of self-reactive lymphocytes and associated production of large amounts of autoantibodies and ICs

Commensal bacteria regulate thymic Aire expression.

Commensal bacteria in gastrointestinal tracts are reported to function as an environmental factor to regulate intestinal inflammation and immune responses. However, it remains largely unknown whether such bacterial function exerts any effect on other immune organs distant from the intestine. In this study, the influence of commensal bacteria in the thymus, where T cell lineages develop into mature type to form proper repertoires, was investigated using germ-free (GF) mice and Nod1-deficient mice lacking an intracellular recognition receptor for certain bacterial components, in which a commensal bacterial effect is predicted to be less. In both mice, there was no significant difference in the numbers and subset ratios of thymocytes. Interestingly, however, autoimmune regulator (Aire) expression in thymic epithelial cells (TECs), main components of the thymic microenvironment, was decreased in comparison to specific pathogen-free (SPF) mice and Nod1 wild-type (WT) mice, respectively. In vitro analysis using a fetal thymus organ culture (FTOC) system showed that Aire expression in TECs was increased in the presence of a bacterial component or a bacterial product. These results suggest that through their products, commensal bacteria have the potential to have some effect on epithelial cells of the thymus in tissues distant from the intestine where they are originally harbored

The Inhibitory Effects of Anti-ERC/Mesothelin Antibody 22A31 on Colorectal Adenocarcinoma Cells, within a Mouse Xenograft Model

The expression of Renal Carcinoma (ERC)/mesothelin is enhanced in a variety of cancers. ERC/mesothelin contributes to cancer progression by modulating cell signals that regulate proliferation and apoptosis. Based on such biological insights, ERC/mesothelin has become a molecular target for the treatment of mesothelioma, pancreatic cancer, and ovarian cancer. Recent studies revealed about 50–60% of colorectal adenocarcinomas also express ERC/mesothelin. Therefore, colorectal cancer can also be a potential target of the treatment using an anti-ERC/mesothelin antibody. We previously demonstrated an anti-tumor effect of anti-ERC antibody 22A31 against mesothelioma. In this study, we investigated the effect of 22A31 on a colorectal adenocarcinoma cell line, HCT116. The cells were xenografted into BALB/c nu/nu mice. All mice were randomly allocated to either an antibody treatment group with 22A31 or isotype-matched control IgG1κ. We compared the volume of subsequent tumors, and tumors were pathologically assessed by immunohistochemistry. Tumors treated with 22A31 were significantly smaller than those treated with IgG1κ and contained significantly fewer mitotic cells with Ki67 staining. We demonstrated that 22A31 exhibited a growth inhibitory property on HCT116. Our results implied that ERC/mesothelin-targeted therapy might be a promising treatment for colorectal cancer

Establishment of a Therapeutic Anti-Pan HLA-Class II Monoclonal Antibody That Directly Induces Lymphoma Cell Death via Large Pore Formation.

To develop a new therapeutic monoclonal Antibody (mAb) for Hodgkin lymphoma (HL), we immunized a BALB/c mouse with live HL cell lines, alternating between two HL cell lines. After hybridization, we screened the hybridoma clones by assessing direct cytotoxicity against a HL cell line not used for immunization. We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line. A newly established mouse anti-human mAb (4713) triggered cytoskeleton-dependent, but complement- and caspase-independent, cell death in HL cell lines, Burkitt lymphoma cell lines, and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice improved survival significantly. mAb 4713 was revealed to be a mouse anti-human pan-HLA class II mAb. Treatment with this mAb induced the formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pore formation. This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma

Lower Aire expression in TECs of GF mice.

<p>(A) Thymocytes obtained from SPF and GF mice were stained with antibodies against CD4 and CD8. Cell numbers and proportions of CD4/8 subsets were almost equal in the two mice. (B) After roughly enriching CD45⁻ cells by depleting CD45⁺ cells from whole thymic cells using AutoMACS, they were stained with anti-I-A^d-FITC, CD45-APC and UEA1-biotin and then Streptavidin-PE. Circulated areas in GF and SPF mice in left panels were isolated by FACS Aria to be CD45⁻I-A⁺ cells, and they were used for real-time PCR analysis. In separate experiments using 4 GF and SPF mice each, circulated areas were further analyzed for the proportions of UEA1⁺I-A⁺ cells in CD45⁻I-A⁺ cells. The average proportions for gated UEA1⁺I-A⁺ cells in CD45⁻I-A⁺ cells from independent experiments are shown in the right panel. (C) CD45⁻I-A⁺ cells isolated as TECs from SPF and GF mice were analyzed for Aire mRNA expression by real-time PCR. Relative quantification of gene expression is shown as mean of values from triplicate samples, and the lowest Aire expression level in GF was arbitrarily set at 1. The relative gene expression level is shown for individual TECs from 4 heads each of GF and SPF mice. *P<0.05. (D) For FACS analysis of Aire experiments, enriched CD45⁻depleted cells were first stained with CD45-APC and UEA1-biotin facilitated with Streptavidin-PE, and anti-I-A/I-E-Pacific Blue (to distinguish the anti-Aire reaction that was conjugated with Alexa Fluor 488). Then, for intracellular Aire staining, the cells were fixed as described in Materials and Methods and stained with Aire-A488 or control antibody. Representative FACS plots of Aire expression of SPF (black line) and GF (red line) mice are shown. Black and red dotted lines indicate isotype controls of SPF and GF mice, respectively. The mean fluorescence intensity (MFI) for Aire from three independent experiments is shown (right). *P<0.05.</p

SEB induces Aire expression through RANKL.

<p>(A) TECs were isolated from E17 BALB/c thymic lobes subjected to FTOC with or without SEB (10 µg/ml) for 3 days, and their gene expression was analyzed by real-time PCR. (B) RANKL expression in DP and 4SP cells isolated from FTOC was analyzed by real-time PCR. (C) In upper panels, thymocytes obtained from FTOC were stained with antibodies against CD4 and CD8, and analyzed by flow cytometry. The absolute cell numbers in three independent experiments were as follows. Total cell number, untreated: 1.1±0.5×10⁷, SEB: 1.9±0.4×10⁶; DP, untreated: 3.4±1.2×10⁵, SEB: 2.6±1.1×10⁴; 4SP, untreated: 5.0±3.0×10⁴, SEB: 1.1±0.3×10⁴. Percentages of DP and 4SP cells are shown. Lower panels show the flow cytometric profiles of DP and 4SP cells further stained with anti-RANKL or control antibody. MFI is indicated in the panels. (D) TECs isolated from 3-day FTOC with or without SEB in the presence or absence of anti-RANKL antibody were submitted to gene expression analysis by real-time PCR. In all panels of (A), (B) and (D), the untreated samples were arbitrarily set at 1 and data bars represent mean ± SD of at least three independent experiments. *P<0.05.</p

Lower Aire expression in TECs of Nod1^−/− mice.

<p>(A) Gene expression of TECs isolated from 5-wk-old wild type mice was examined by PCR. (B) From littermates, TECs were isolated individually from 7 heads of Nod1^+/+ (WT) mice and 11 heads of Nod1^−/− mice. mRNA expression was estimated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105904#pone-0105904-g001" target="_blank">Fig. 1C</a>. The lowest Aire or Ctsl expression level in Nod1^−/− mice was arbitrarily set at 1. ***P<0.001. (C) For FACS analysis, the Aire expression on TECs (UEA1⁺I-A⁺CD45⁻) in I-A⁺CD45⁻ cells of Nod1^+/+ and Nod1^−/− mice was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105904#pone-0105904-g001" target="_blank">Fig. 1D</a> legend. Representative FACS plots of Aire expression of Nod1^+/+ WT (black line) and Nod1^−/− (red line) mice are shown. Black and red dotted lines indicate isotype controls of Nod1^+/+ and Nod1^−/− mice, respectively. MFI for Aire from three independent experiments is shown (right). **P<0.01. (D) Expression of Aire-dependent (Spt1, Expi and S100a8) and -independent (Crp) genes in TECs from Nod1^+/+ (n = 5) and Nod1^−/− (n = 5) mice were analyzed by real-time PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105904#pone-0105904-g001" target="_blank">Fig. 1C</a>. *P<0.05, **P<0.01.</p

Cytotoxic effects of mAb4713 on lymphoma/leukemia cell lines.

<p>Cytotoxic effects of mAb4713 on lymphoma/leukemia cell lines.</p

Reactivity and cytotoxic activity of mAb 4713 against lymphoma cell lines.

<p>(A) Flow cytometry analysis of mAb 4713 reactivity analyzed by incubating different lymphoma cell lines with mAb 4713 (4°C; 2 h), followed by Alexa 488-conjugated rat anti-mouse Igs (green histogram). Cytotoxicity was analyzed based on propidium iodide (PI) staining of dead cells (red histogram). (B) Cytotoxic effects of mAb 4713 on IL-2-dependent or IL-2-independent ATL cell lines analyzed by PI staining after incubation with mAb 4713 (37°C; 1 or 2 h). Similar data were obtained in two independent experiments. (C) Kaplan–Meier survival analysis of mAb 4713-treated C.B-17/ICR-SCID mice bearing Raji lymphoma xenografts. The Raji-injected SCID mice (n = 5) were treated with mAb 4713 once (red line) or twice (brown line), or with control IgG once (green line). **p = 0.01 versus isotype control.</p