Hideo Nariuchi

Polarization of Naive CD4+ T Cells Toward the Th1 Subset by CTLA-4 Costimulation

In this study, we examined in vitro the role of CTLA-4 costimulation in the polarization of naive CD4+ T cells toward the Th1 subset. When CTLA-4 costimulation was blocked by the inclusion of anti-CTLA-4 Fab in cultures during priming of naive CD4+ T cells with anti-CD3 in the presence of splenic adherent cells, they were polarized toward the Th2 subset. Conversely, the engagement of CTLA-4 with immobilized anti-CTLA-4 or with CD80-P815 cells polarized naive CD4+ T cells costimulated with anti-CD3 and anti-CD28 toward the Th1 subset. The CTLA-4 costimulation during priming augmented TGF-β1 mRNA accumulation in naive CD4+ T cells, and the inclusion of anti-TGF-β in cultures for priming suppressed the effect of CTLA-4 costimulation on the Th1 polarization. The addition of low doses of TGF-β1 in cultures for priming of naive CD4+ T cells enhanced the production of Th1 cytokines upon secondary stimulation, although Th2 cytokine production was not affected by the doses of TGF-β1. The CTLA-4 costimulation was also shown to suppress IL-4 production of naive CD4+ T cells upon priming. These results indicate that the costimulation against CTLA-4 drives polarization of naive CD4+ T cells toward the Th1 subset independent of IL-12 through, at least in part, the enhancement of TGF-β1 production, and it also hampers Th2 subset differentiation by affecting IL-4 production of naive CD4+ T cells.

A Critical Role of Fc Receptor-Mediated Antibody-Dependent Phagocytosis in the Host Resistance to Blood-Stage Plasmodium berghei XAT Infection

Plasmodium berghei XAT is an irradiation-induced attenuated variant derived from the lethal strain P. berghei NK65, and its blood-stage parasites are spontaneously cleared in immune competent mice. In the present study, we studied the mechanism of host resistance to blood-stage malaria infection using P. berghei XAT. Infection enhanced Ab-dependent phagocytosis of PRBC by splenic macrophages in wild-type C57BL/6 mice. In contrast, FcR γ-chain knockout (FcRγ−/−) mice, which lack the ability to mediate Ab-dependent phagocytosis and Ab-dependent cell-mediated cytotoxicity through FcγRI, FcγRII, and FcγRIII, could not induce Ab-dependent phagocytic activity. These FcRγ−/− mice showed increased susceptibility to the P. berghei XAT infection, with eventually fatal results, although they produced comparable amounts of IFN-γ by spleen cells and anti-XAT Abs in serum. In addition, passive transfer of anti-XAT IgG obtained from wild-type mice that had recovered from infection into FcRγ−/− mice could not suppress the increase in parasitemia, and almost all of these mice died after marked parasitemia. In contrast, passive transfer of anti-XAT IgG into control wild-type mice inhibited the increase in parasitemia. IFN-γ−/− mice, which were highly susceptible to the P. berghei XAT infection, failed to induce Ab-dependent phagocytic activity and also showed reduced production of serum anti-XAT IgG2a isotype compared with control wild-type mice. These results suggest that FcR-mediated Ab-dependent phagocytosis, which is located downstream of IFN-γ production, is important as an effector mechanism to eliminate PRBC in blood-stage P. berghei XAT infection.

Impairment of signal transduction in T cells from old mice.

T cells from old mice showed impaired proliferative response to antigenic stimulation. To understand the mechanism underlying the age-related impairment of T cell functions, the signal transduction pathway was examined and compared between T cells from young and old mice, and between T cell clones established from a young and old mouse. The age-related changes in T cells were as follows: (1) reduction in the expression and the activation of protein tyrosine kinases associated with T cell receptor (TCR) after antigenic stimulation; (2) reduced phosphorylation of phospholipase C gamma 1 (PLC gamma 1); (3) reduced production of second messengers such as inositoltrisphosphate (IP3) and diacylglycerol (DAG); and (4) reduced influx of Ca2+ ion. Thus, a T cell clone established from an old mouse showed impaired proliferation by stimulation with anti-CD3 antibody, but was fully activated to the level of a T cell clone from a young mouse by stimulation with phorbol acetate myristate (PMA) plus ionomycin (INM). However, splenic T cells freshly prepared from old mice did not show full recovery by the same treatment. The results indicate that one major blockade in the signal transduction of T cells from old mice is present in the pathway just after TCR, but besides this, the blockade is also present in multiple sites down-stream, which can not be bypassed by stimulation with PMA plus INM.

Purification and partial amino acid sequence of rabbit tumor necrosis factor

Good production of tumor necrosis factor (TNF) in the rabbit was obtained using Propionibacterium acnes IID 912 as a priming agent and subsequent administration of lipopolysaccharide. The physicochemical characteristics of rabbit TNF were very similar to those of murine TNF. The molecular weight of rabbit TNF was 39,000 as estimated by gel filtration, and 18,000 by SDS‐PAGE. The isoelectric point was determined as ph 4.0 by isoelectric focusing. Rabbit TNF was stable within the ph range of 5.5 to 11.0, and was stable at 56°C for 8 hr. It was digested by trypsin, pancreatic protease and elastase, but was resistant to neuraminidase . The amino acid sequence of rabbit TNF was determined as Ser‐Ala‐Ser‐Arg‐Ala‐Leu‐ …. Monoclonal antibody against rabbit TNF completely inhibited both the in vivo and in vitro activity of rabbit TNF. However, this antibody could not inhibit the action of murine TNF. Antitumor activity of rabbit TNF was shown against murine and human cancer cells in vivo and in vitro, and rabbit TNF was also capable of distinguishing malignant cells from normal cells.

Expression and co-stimulatory function of B7-2 on murine CD4+ T cells.

Co‐stimulatory signals mediated by the interaction of B7‐1/B7‐2 with CD28 are important for the activation of CD4+ T cells stimulated with antigen on antigen‐presenting cells. There are controversies about the expression and function of B7‐1/B7‐2 on CD4+ T cells. The aim of this study was to analyze the expression of B7‐1/B7‐2 on naive and memory CD4+ T cells and the co‐stimulatory function in the activation of naive CD4+ T cells stimulated by TCR ligation. Present results indicate that memory CD4+ T cells express B7‐2 molecules on their surface, whereas naive CD4+ T cells do not. Neither memory nor naive CD4+ T cells expressed B7‐1 molecule on their surface, although B7‐1 mRNA was faintly expressed in memory T cells. B7‐2 molecules expressed on memory T cells co‐stimulated CD4+ naive T cells stimulated with plate‐coated anti‐CD3 to produce IL‐2. Naive CD4+ T cells were shown to express B7‐2 after co‐stimulation with B7‐2 and TCR ligation, because the naive T cells stimulated with anti‐CD3 and B7‐2CHO expressed B7‐2 on their surface, although it remained to be studied whether the co‐stimulation with B7‐2 directly induced B7‐2 expression on naive T cells. Our present results indicate that memory CD4+ T cells play some role in the activation of naive CD4+ T cells through the co‐stimulation with B7‐2 molecules.

Impairment in the Expression and Activity of Fyn During Differentiation of Naive CD4+ T Cells into the Th2 Subset

We previously showed that the amounts of Fyn protein in Th2 clones were approximately one-third to one-fifth of those in Th1 clones. In this study we examined the role of Fyn in the polarization of naive CD4+ T cells toward the Th2 subset using fyn−/− mice. The fyn−/− naive CD4+ T cells efficiently produced Th2 cytokines and polarized toward the Th2 subset even in the absence of IL-4 and IL-13. The expression of Fyn in wild-type CD4+ T cells decreased at a transcription level concomitant with polarization toward the Th2 subset. These results suggest that Fyn plays a role in the down-regulation of the differentiation of naive CD4+ T cells into the Th2 subset.

Interleukin-12-Dependent Mechanisms in the Clearance of Blood-Stage Murine Malaria Parasite Plasmodium berghei XAT, an Attenuated Variant of P. berghei NK65

The mechanism of development of host resistance to blood-stage malarial infection was studied by use of an irradiation-induced attenuated variant, Plasmodium berghei XAT, obtained from a lethal strain, P. berghei NK65. The infection enhanced mRNA expression of interleukin (IL)-12 p40 and also of interferon (IFN)-gamma, IL-4, IL-10, and cytokine-inducible nitric oxide synthase (iNOS) in spleen. Treatment of these mice with anti-IL-12 or anti-IFN-gamma led to the progression of parasitemia and fatal outcome. Anti-IL-12 treatment significantly reduced the secretion and mRNA expression of IFN-gamma and greatly diminished the augmentation of iNOS mRNA expression. In addition, recombinant IL-12 administration delayed the onset of parasitemia because of the enhanced IFN-gamma production. These results suggest that blood-stage P. berghei XAT infection induces IL-12 production, which is important for the development of host resistance via IFN-gamma production.

Eosinophilia, IgE production, and cytokine production by lung T cells in surface CD4-deficient mutant mice infected with Toxocara canis.

Mutant mice deficient in CD4+ T cells and their normal and heterozygous littermates were infected with Toxocara canis, and compared for eosinophilia, total and Toxocara‐specific immunoglobulin E (IgE) production, and in vitro cytokine production by lung cells. The numbers of eosinophils in the peripheral blood of normal and heterozygous mice peaked on days 10 and 21, although mutant mice showed eosinophilia with a peak on day 10. This indicates that the first peak on day 10 is CD4 independent and the second peak is CD4 dependent. Before infection, the levels of total IgE had no significant difference among the three groups of mice. Total and Toxocara‐specific IgE in all genotypes of mice increased after infection, and was the highest in normal mice and the lowest in mutant mice. In vitro production of interleukin (IL)‐5 and IL‐4 by total lung cells was the highest in normal mice and the lowest in mutant mice. CD4+ and CD4− CD8− T lymphocytes, but not CD8+ T lymphocytes produced IL‐5 and IL‐4 when incubated with anti‐CD3 monoclonal antibody (mAb) and lung‐adherent cells. These results indicated that IL‐5 and IL‐4 were produced mainly by CD4+ cells and partly by CD4− CD8− cells, but not by CD8+ cells. In addition, cytokine production by CD4+ cells was affected by the number of CD4 molecules on their surface.

A substrain of NZB mouse as an animal model of autoimmune inner ear disease

A substrain of an autoimmune-prone mouse, NZB/kl, was found to show spontaneous elevation of the auditory brainstem response (ABR) threshold with age. Morphological examination of the inner ear in NZB/kl mice with high ABR thresholds revealed pathological changes confined to the stria vascularis, including marked thickening of the capillary basement membrane which contained many foamy structures, and vacuolar degeneration of the intermediate cells. Circular or granular IgM deposits and some IgG deposits were found in the stria vascularis in the mice with high ABR thresholds, suggesting that deposits of immune complexes (mainly IgM antibodies) could cause strial damage that resulted in the ABR threshold elevation. Another substrain of NZB mice, NZB/san, showed lower levels of IgM immune complexes and anti-ss DNA antibodies, and did not develop either inner ear morphological changes or a high ABR threshold. NZB/kl mice may provide a useful animal model for studying the mechanism of autoimmune inner ear disease.

Decline of T cell-related immune functions in cancer patients and an attempt to restore them through infusion of activated autologous T cells

We developed a scoring system that can combine several immunological parameters and express the immune status of individuals as a simple numeral. T cell immune score was obtained by using 5T cell-related parameters: number of T cells, ratio of CD4(+)T cells to CD8(+)T cells, number of naïve T cells, ratio of naïve T cells to memory T cells, and T cell proliferative index (TCPI). TCPI was calculated by using number of T cells and their proliferative activity. We assessed T cell immune score in 103 patients with colorectal cancer and 51 healthy age-matched controls. The results were as follows: (1) T cell-immune score of patients in stages I-IV before surgery was significantly decreased as compared with controls. (2) The number of regulatory T cells in patients in stages I-IV gradually increased with disease progression. (3) T cell immune score was strongly suppressed after surgery, but were recovered to the initial level within a month. (4) Furthermore, restoration of immunological function was attempted in cancer patients by infusion of activated autologous T cells. The effectiveness was confirmed by an increase of TCPI in many cancer patients.