In vivo eradication of MLL/ENL leukemia cells by NK cells in the absence of adaptive immunity.

It remains unclear how the immune system affects leukemia development. To clarify the significance of the presence of immune systems in leukemia development, we transferred MLL/ENL leukemia cells into immune-competent or immune-deficient mice without any preconditioning including irradiation. The wild-type mice did not develop leukemia, whereas all the Rag2(-/-)γc(-/-) mice lacking both adaptive immune cells and natural killer (NK) cells developed leukemia, indicating that leukemia cells were immunologically rejected. Interestingly, leukemia cells were also rejected in 60% of the Rag2(-/-) mice that lacked adaptive immune cells but possessed NK cells, suggesting that NK cells play a substantial role in the rejection of leukemia. Moreover, engraftment of leukemia cells was enhanced by NK cell depletion in Rag2(-/-) recipients and inhibited by transfer of NK cells into Rag2(-/-)γc(-/-) recipients. Upregulation of NKG2D (NK group 2, member D) ligands in MLL/ENL leukemia cells caused elimination of leukemia cells by NK cells. Finally, we found that leukemia cells resistant to elimination by NK cells had been selected during leukemia development in Rag2(-/-) recipients. These results demonstrate that NK cells can eradicate MLL/ENL leukemia cells in vivo in the absence of adaptive immunity, thus suggesting that NK cells can play a potent role in immunosurveillance against leukemia.

Protection against colitis by CD100-dependent modulation of intraepithelial γδ T lymphocyte function.

Intraepithelial γδ T lymphocytes (γδ IEL) have important roles in repair of tissue damage at epithelial sites, such as skin and intestine. Molecules that orchestrate these γδ T-cell functions are not well defined. Recently, interaction of the semaphorin CD100 on skin γδ T cells with plexin B2 on keratinocytes was shown to be important for effective γδ T-cell function in the epidermis, which raised the possibility that CD100 may exert similar functions in the intestinal tract. In this study, we find that CD100 is expressed on all IEL, and plexin B2 is present on all epithelial cells of the mouse colon. Using the dextran sulfate sodium (DSS) mouse model of colitis, disease severity is significantly exacerbated in CD100-deficient (CD100(-/-)) mice, with increased colon ulceration and mucosal infiltration with inflammatory cells. The severe colitis in CD100(-/-) mice is attributable to the failure of the colon epithelium to mount a proliferative response to damage. Unlike wild-type γδ IEL, γδ IEL from CD100(-/-) mice fail to produce keratinocyte growth factor-1 (KGF-1) in response to DSS treatment. Administration of recombinant KGF-1 to CD100(-/-) animals ameliorates disease and reverses colitis susceptibility. These results demonstrate that CD100-mediated signals are critical for effective activation of γδ IEL to produce growth factors, including KGF-1, that are required for healing of the colon epithelium during colitis.

A retrospective analysis of 335 Japanese lung cancer patients who responded to initial gefitinib treatment.

BACKGROUND: Gefitinib treatment results in considerably better progression-free survival compared with that of platinum doublets in the first line treatment of nonsmall-cell lung cancer (NSCLC) carrying an activating epidermal growth factor receptor (EGFR) mutation. Some patients who respond to gefitinib have an overall survival (OS) of more than 5 years, whereas other initial responders do less well. Although there has been considerable effort made to elucidate the mechanisms of acquired resistance, there have only been a few studies that addressed the effect of clinical backgrounds and treatment histories on the survival of the patients who had responded to an EGFR-tyrosine kinase inhibitor (TKI). In this study, we especially focused on the clinical benefit of EGFR-TKI administration after progression. PATIENTS AND METHODS: We retrospectively analyzed consecutive patients with advanced NSCLC who were diagnosed before October 2010, treated with gefitinib after July 2002, and responded to it. The primary objective of this study was to evaluate how clinical backgrounds and treatment histories influence survival of the patients who respond to gefitinib. The secondary objectives were to evaluate the safety of long-term gefitinib use and to establish the optimal treatment sequence using a dynamic treatment regimen analysis (DTRA). RESULTS: A total of 335 patients were recruited. Twenty-eight (8.4%) patients survived more than 5 years. Sixty-five and 93 patients received gefitinib as rechallenge and beyond progressive disease (BPD), respectively. A statistically significant difference in OS was observed between the patients who underwent gefitinib rechallenge and those who did not rechallenge (median: 1272 days vs. 774 days; p < 0.001), a result supported by a DTRA. Patients treated with gefitinib BPD also showed a tendency of longer survival. CONCLUSIONS: Gefitinib rechallenge and BPD played a central role in long term survival of the patients who initially responded to gefitinib.

Retention of tocilizumab and anti-tumour necrosis factor drugs in the treatment of rheumatoid arthritis.

OBJECTIVES: The retention of the anti-rheumatic agent tocilizumab (TCZ) has not been well documented in patients with rheumatoid arthritis (RA). We conducted an observational study to compare the retention of TCZ and anti-tumour necrosis factor (TNF) drugs in the treatment of patients with RA. METHOD: We reviewed continuation rates and causes of discontinuation of biological agents (biologics) by assessing medical records of patients with RA who were administered biologics at our institute from September 1999 to April 2012, using the Osaka University Biologics for Rheumatic Diseases (BiRD) registry. RESULTS: A total of 401 patients were included. TCZ, infliximab (IFX), etanercept (ETN), and adalimumab (ADA) were administered to 97, 103, 143, and 58 patients, respectively. There were some differences between the baseline characteristics of the groups. The median duration (range) of TCZ, IFX, ETN, and ADA administration was 2.5 (0.1-12.6), 1.9 (0.0-7.7), 2.9 (0.0-11.3), and 1.3 (0.0-3.4) years, respectively. Continuation rates for TCZ and ETN were significantly higher than those for IFX and ADA. Multivariate analyses showed that discontinuation due to lack or loss of efficacy was significantly less common in the TCZ group than in the other groups. Discontinuation due to overall adverse events was not significantly different between treatment groups. CONCLUSION: TCZ and ETN show better retention than IFX or ADA in the treatment of RA.

CD138-negative clonogenic cells are plasma cells but not B cells in some multiple myeloma patients.

Clonogenic multiple myeloma (MM) cells reportedly lacked expression of plasma cell marker CD138. It was also shown that CD19(+) clonotypic B cells can serve as MM progenitor cells in some patients. However, it is unclear whether CD138-negative clonogenic MM plasma cells are identical to clonotypic CD19(+) B cells. We found that in vitro MM colony-forming cells were enriched in CD138(-)CD19(-)CD38(++) plasma cells, while CD19(+) B cells never formed MM colonies in 16 samples examined in this study. We next used the SCID-rab model, which enables engraftment of human MM in vivo. CD138(-)CD19(-)CD38(++) plasma cells engrafted in this model rapidly propagated MM in 3 out of 9 cases, while no engraftment of CD19(+) B cells was detected. In 4 out of 9 cases, CD138(+) plasma cells propagated MM, although more slowly than CD138(-) cells. Finally, we transplanted CD19(+) B cells from 13 MM patients into NOD/SCID IL2Rγc(-/-) mice, but MM did not develop. These results suggest that at least in some MM patients CD138-negative clonogenic cells are plasma cells rather than B cells, and that MM plasma cells including CD138(-) and CD138(+) cells have the potential to propagate MM clones in vivo in the absence of CD19(+) B cells.

Serum chemokine profile in patients with bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune inflammatory disease causing blister formation at the dermoepidermal junction. Cutaneous infiltration of activated CD4+ T cells and eosinophils is an early event in blister formation during the disease process, suggesting that the trafficking of circulating leucocytes through the sites of inflammation is crucial in the pathogenesis of the disease. While the accumulated evidence suggests that some cytokines are involved in the pathogenesis, there have been few reports about serum chemokine profiles in patients with BP. OBJECTIVES: To determine serum profiles of various chemokines and their clinical association in patients with BP. METHODS: Concentrations of 10 chemokines - interferon (IFN)-gamma-inducible protein-10 (IP-10), monokine induced by IFN-gamma (MIG), macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, eotaxin, monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3 and growth-regulated oncogene-alpha- were measured in serum samples from 38 patients with BP, 16 with pemphigus vulgaris (PV) and 17 normal controls using a sandwich immunoassay-based multiplex protein array system. RESULTS: While there was no significant increase in any serum chemokine levels in patients with PV, serum levels of IP-10 and MCP-1 were significantly increased in patients with BP compared with healthy controls. Furthermore, serum levels of IP-10, MIG, MCP-1 and eotaxin in patients with BP increased significantly with disease severity as determined by the area affected. CONCLUSIONS: These observations suggest that an elaborately orchestrated network of chemokines, especially MCP-1 and IP-10, contributes to the pathomechanism of BP.

Biological functions and signaling of a transmembrane semaphorin, CD100/Sema4D.

The semaphorin proteins were identified originally as axonal guidance factors functioning during neuronal development. In addition to this function, several semaphorins play diverse roles outside the nervous system. The class 4 semaphorin CD100/Sema4D, which utilizes plexin-B1 and CD72 as receptors, exerts important biological effects on a variety of cells, including the neuronal, epithelial and immune cells. Here, we review recent advances exploring the molecular mechanisms governing the biological functions of CD100/Sema4D.

The CD100-CD72 interaction: a novel mechanism of immune regulation.

CD100 is a 150 kDa transmembrane protein that belongs to the semaphorin family. Many members of the semaphorin family are known to play crucial roles in axon guidance, acting as chemorepulsive factors during neuronal development. CD100 is the first member of the semaphorin family for which crucial roles in the immune system have been identified. Although plexin-B1 has been shown to be the receptor for CD100 in nonlymphoid tissues, CD72 functions as its receptor in lymphoid tissues. CD100 plays a nonredundant role in the immune response by a unique mechanism that involves switching off the negative signals mediated by CD72.

Enhanced immune responses in transgenic mice expressing a truncated form of the lymphocyte semaphorin CD100.

CD100/Sema4D is a 150-kDa transmembrane protein that belongs to the semaphorin family. Binding of CD100 to CD72 enhances the immune response by turning off the negative signaling effects of CD72. To investigate the physiological functions of CD100 in vivo, we generated transgenic mice expressing a truncated form of CD100. A large amount of the soluble form of CD100 was detected in the sera of mice expressing a truncated form of CD100, although the amount of CD100 was only slightly elevated on the surface of B cells. In the mutant mice the development of conventional B and T cells appeared normal in terms of the surface marker phenotypes, while the number of CD5(+) B-1 cells in the peritoneal cavity increased in comparison with wild-type mice. In vitro proliferation and Ig production of B cells in response to CD40 stimulation were considerably enhanced in mice expressing a truncated form of CD100. Additionally, in vivo both Ab responses against T cell-dependent Ags and generation of Ag-specific T cells were enhanced. Furthermore, introduction of the CD100-transgene could restore in vitro B cell responses as well as in vivo Ab production against T cell-dependent Ag in CD100-deficient mice. Collectively, these results not only indicate that CD100 has an important role in the immune system, but also that the soluble form of CD100 released from the cell surface can exert functions in vivo.

Functional soluble CD100/Sema4D released from activated lymphocytes: possible role in normal and pathologic immune responses.

CD100/Sema4D is a 150-kd transmembrane protein that belongs to the semaphorin family. The interaction of CD100 with CD72 is critical for the immune system. In CD100-deficient mice, the production of specific antibodies against T-cell-dependent antigens is severely impaired, but not against T-cell-independent antigens. Here, a functional soluble CD100 protein (sCD100) released from activated lymphocytes is reported. sCD100 was detected in culture supernatants of activated lymphocytes. Either affinity-purified from supernatants of activated T-cells, or produced as a recombinant sCD100 protein consisting of the extracellular region of the mouse CD100 fused to the human IgG1 Fc (CD100-Fc), sCD100 significantly enhanced CD40-induced B-cell responses. Furthermore, sCD100 was detected either in sera of mice immunized with T-cell-dependent antigens, or in sera of MRL/lpr mice, but not in sera of mice immunized with T-cell-independent antigens. A significant correlation was observed between the level of sCD100 and the titer of autoantibodies in the serum of MRL/lpr mice. This study's findings suggest a potential role for sCD100 in immune responses, including production of antibody, and autoimmune diseases. (Blood. 2001;97:3498-3504)

Increased T cell autoreactivity in the absence of CD40-CD40 ligand interactions: a role of CD40 in regulatory T cell development.

Mutations in the CD40 ligand (CD40L) gene lead to X-linked immunodeficiency with hyper-IgM, which is often associated with autoimmune diseases. To determine the contribution of defective CD40-CD40L interactions to T cell autoreactivity, we reconstituted CD40-CD40L interactions by transferring T cells from CD40-deficient mice to syngenic athymic nude mice and assessed autoimmunity. T cells from CD40-deficient mice triggered autoimmune diseases accompanied with elevations of various autoantibodies, while those from wild-type mice did not. In CD40-deficient mice, the CD25(+) CD45RB(low) CD4(+) subpopulation which regulates T cell autoreactivity was markedly reduced. CD40-deficient APCs failed to induce T regulatory cells 1 producing high levels of an inhibitory cytokine, IL-10 in vitro. Furthermore, autoimmune development was inhibited when T cells from CD40-deficient mice were cotransferred with CD45RB(low) CD4(+) T cells from wild-type mice or with T regulatory cells 1 induced on CD40-expressing APCs. Collectively, our results indicate that CD40-CD40L interactions contribute to negative regulation of T cell autoreactivity and that defective interactions can lead to autoimmunity.

Identification of CD72 as a lymphocyte receptor for the class IV semaphorin CD100: a novel mechanism for regulating B cell signaling.

We have identified the lymphocyte semaphorin CD100/Sema4D as a CD40-inducible molecule by subtractive cDNA cloning. CD100 stimulation significantly enhanced the effects of CD40 on B cell responses. Administration of soluble CD100 markedly accelerated in vivo antigen-specific antibody responses. CD100 receptors with different binding affinities were detected on renal tubular cells (K(d) = approximately 1 x 10(-9)M) and lymphocytes (K(d) = approximately 3 x 10(-7)M). Expression cloning revealed that the CD100 receptor on lymphocytes is CD72, a negative regulator of B cell responsiveness. CD72 thus represents a novel class of semaphorin receptors. CD100 stimulation induced tyrosine dephosphorylation of CD72 and dissociation of SHP-1 from CD72. Our findings indicate that CD100 plays a critical role in immune responses by the novel mechanism of turning off negative signaling by CD72.

The class IV semaphorin CD100 plays nonredundant roles in the immune system: defective B and T cell activation in CD100-deficient mice.

The class IV semaphorin CD100/Sema4D differentially utilizes two distinct receptors: plexin-B1 in nonlymphoid tissues, such as brain and kidney, and CD72 in lymphoid tissues. We have generated CD100-deficient mice and demonstrated that they have functional defects in their immune system, without apparent abnormalities in other tissues. The number of CD5(+) B-1 cells was considerably decreased in the mutant mice, whereas conventional B cells and T cells appeared to develop normally. In vitro proliferative responses and immunoglobulin production were reduced in CD100-deficient B cells. The humoral immune response against a T cell-dependent antigen and in vivo priming of T cells were also defective in the mutant mice. These results demonstrate nonredundant and essential roles of CD100-CD72 interactions in the immune system.

gp130-Dependent signalling pathway is not enhanced in gp130 transgenic heart after LIF stimulation.

Activation of gp130 transduces a hypertrophic signal in the heart, but it is not clear whether signalling through gp130 is enhanced when gp130 is overexpressed in vivo. We generated gp130 transgenic mice (TG) and examined the activation of signalling pathways downstream of gp130 in the hearts. The tyrosine phosphorylation of gp130 was enhanced, the phosphorylation of STAT3 and ERK (extracellular signal regulated kinase) 1/2 was increased and induction of the beta-myosin heavy chain (MHC) gene was observed in TG hearts without significant phenotypic changes. Intravenous administration of leukaemia inhibitory factor (LIF) induced tyrosine phosphorylation of STAT3 and ERK 1/2 and expression of c-fos and beta-MHC mRNAs in wild-type littermates' (WT) hearts. However, enhancement of STAT3 and ERK 1/2 phosphorylation or augmented mRNA expressions was not observed in TG hearts after LIF stimulation. Next, STAT-induced STAT inhibitor (SSI) mRNA expression was examined. The expression of SSI-1, SSI-2, and SSI-3 mRNAs was significantly augmented in TG hearts after LIF stimulation. These results indicate that overexpressed gp130 does not always enhance downstream signals in the hearts and suggest that the SSI family plays a role in the regulation of the gp130-dependent signalling pathway in the hearts.

Impairment of antigen-specific antibody production in transgenic mice expressing a dominant-negative form of gp130.

gp130 is a common signal-transducing receptor component for the interleukin 6 family of cytokines functioning in, for example, immune, hematopoietic, and nervous systems. In this study, to investigate the physiological functions of gp130 and to determine the pathological consequences of impaired gp130 signals, we have generated transgenic mice expressing a cytoplasmically truncated form of mouse gp130. Expression of this form of gp130 in lymphocytes significantly suppressed interleukin 6-induced tyrosine phosphorylation of endogenous gp130 and a downstream signaling molecule, signal transducer and activator of transcription 3, indicating that this form has a dominant negative function. In spite of the impaired gp130 signals, the development of lymphocytes in the transgenic mice appeared normal in terms of surface marker phenotypes. These mice, however, exhibited severe defects in antigen-specific antibody production of most immunoglobulin isotypes other than IgM after immunization with 2,4-dinitrophenol-conjugated ovalbumin. These results demonstrate in vivo that gp130 is essential for antigen-specific antibody production.