Genki Kimura

Vγ1+ γδ T cells play protective roles at an early phase of murine cytomegalovirus infection through production of interferon-γ

Cytomegalovirus (CMV) causes severe opportunistic infection in immunocompromised hosts. The importance of conventional αβ T cells in protection against CMV infection has been well documented. However, the role of the second T‐cell population (which express the γδ T‐cell receptor) in CMV infection is not known. In the present study, we analysed the function and protective role of γδ T cells in a murine cytomegalovirus (MCMV) infection model. After intraperitoneal infection with MCMV, the number of γδ T cells increased in the liver and peritoneal cavity from day 3, and reached a peak on day 5. The γδ T cells showed an activated T‐cell phenotype and predominantly expressed Vγ1, which is known to be expressed by heat‐shock protein 65 (hsp 65)‐specific γδ T cells. Analysis of cytokine expression demonstrated that the MCMV‐induced γδ T cells expressed interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) but not interleukin‐4 (IL‐4), implying their participation in the cell‐mediated immune response against MCMV. Depletion of γδ T cells by anti‐T‐cell receptor (TCR) γδ monoclonal antibody (mAb) treatment resulted in significant increase of virus titre and decrease of IFN‐γ in the liver on day 3 after MCMV infection, which further supports the importance of γδ T cells in early protection against infection. Finally, the MCMV‐induced γδ T cells produced IFN‐γin vitro in response to hsp 65. Our results suggest that γδ T cells participate in early protection against MCMV infection through recognition of hsp 65 and production of IFN‐γ.

Oral administration of psk can improve the impaired anti-tumor CD4+ T-cell response in gut-associated lymphoid tissue (GALT) of specific-pathogen-free mice

We investigated both the effect and the mechanism of oral (p.o.) administration of PSK, a protein‐bound polysaccharide derived from Bosidiomycetes, on the anti‐tumor T‐cell response in gut‐associated lymphoid tissue (GALT). The p.o. administration of PSK significantly suppressed the growth of colon 26 carcinoma (C‐26) inoculated into the subserosal space of the cecum (i.c.), and augmented the tumor‐neutralizing activity of the draining mesenteric lymph node (LN) cells. PSK treatment also significantly decreased the levels of immunosuppressive factors such as plasma transforming growth factor (TGF)‐β in the i.c. C‐26‐inoculated mice. We also evaluated the improving effect of PSK on the anti‐tumor T‐cell response in GALT by utilizing B7‐transfected P815 mastocytoma (B7/P815). The PSK treatment promoted the rejection of i.c.‐inoculated B7/P815 and restored the CD4+ T‐cell‐dependent proliferative response of the draining mesenteric LN cells against in vitro restimulation. Furthermore, the treatment also decreased the TGF‐β production but increased the IFN‐γ production of these cells. The p.o. administration of PSK, however, showed no effect in the CD8+ T‐cell‐dependent cytolytic activity of the draining mesenteric LN cells after in vitro restimulation. Overall, these results indicate that the p.o. administration of PSK can improve the impaired anti‐tumor CD4+ T‐cell response in GALT, mainly through a suppression of TGF‐β production and a restoration of IFNγ‐production. Int. J. Cancer, 70:362–372, 1997.

Effects of inhibitors of the cytoplasmic structures and functions on the early phase of infection of cultured cells with simian virus 40

To obtain information about cytoplasmic structures and functions involving the entry of simian virus 40 virions into cells, we examined whether the inhibitors that affect the functions and/or structure of lysosomes, cell membrane, and cytoskeletons inhibit expression of nuclear T antigen in the SV40-inoculated rat 3Y1 and monkey CV-1 cells. Chloroquine, methylamine, and butylamine did not inhibit T-antigen expression, suggesting that lysosomal acidification is not required for establishment of infection. Cytochalasin B had no effect, suggesting that microfilaments are not involved. Monensin, colcemid, and amantadine each inhibited T-antigen expression at doses causing no obvious cytotoxicity. Maximal inhibition was seen when these inhibitors were added to the cultures within 1 hr (monensin), within 4 hr (colcemid), or within 12 hr (amantadine) after virion adsorption to the cell surface. When the inhibitor was present in the virus-inoculated cultures for 24 hr and then removed, nuclear T antigen began to be expressed at 4 hr (monensin), 9 hr (colcemid), or 1 hr (amantadine) after removal of the inhibitors. Results of SDS-PAGE analysis of immunoprecipitated radiolabeled proteins of infected cells revealed that amantadine inhibited synthesis of large and small T antigens as well as general protein synthesis. Inhibition by colcemid may be due to disruption of microtubules, because other microtubule-disrupting agents (colchicine, vinblastine, nocodazole, and podophyllotoxin) also inhibited appearance of nuclear T antigen but lumicolchicine and taxol did not. Electron microscopy revealed that, in the presence of colcemid, although the adsorbed virions were readily internalized to form pinosomes, vectorial movement of the pinosomes to the nucleus appeared to be inhibited. Results of electron microscopy also suggest that inhibition by monensin may occur mainly in internalization of adsorbed virions and that the inhibition is leaky such that the early steps of infection proceed slowly in the presence of monensin. We conclude that monensin, colcemid, and amantadine interfere with mutually different early events of SV40 infection.

Depression of acquired resistance against herpes simplex virus infection in neonatally thymectomized mice

An increasing number of reports have been appearing on the depression of acquired immunity by neonatal thymectomy. Thymeetomy in mice soon after birth is associated with the impairment of such immunologic activities as to produce serum antibody, to develop delayed hypersensitivity and to reject homograft [reviewed by Miller et al. (1), and Good and Papermaster (2)]. We have studied the effect of neonatal thymectomy on the development of protective immunity in various viral and bacterial infections of mice (3, 4, 5, 6). Previous report has dealt with the increased susceptibility of neonatally thymectomized mice to herpes simplex infection (4). This paper describes the marked depression of acquired resistance against herpes simplex infection in neonatally thymectomized mice. A half of each littermates of colony-bred CF1 mice were thymectomized before 16 hours after birth. Remaining half of the littermates were either sham-thymectomized or left intact. The completeness of the operation was examined by autopsies at the end of each experiment. For the immunization strain 1019C] of herpes simplex virus (HSV) was used. This is an avirulent HSV which was kindly given by l~rof. K. Yoshino of Yokohama City University School of Medicine and has been maintained by passage in Vero cell cultures, an established strain derived from African green monkey kidney cells. One-tenth ml of strain 1019C1 of HSV having a titer of 105.5 TCIDs0/0.1 ml was inoculated intraperitoneally at 14, 17 and 20 days of age. Three days after the last

Concomitant immunity against tumor development is enhanced by the oral administration of a kampo medicine, Hochu-ekki-to (TJ-41 : Bu-Zhong-Yi-Qi-Tang)

The oral administration of a kampo herbal medicine, Hochu-ekki-to (TJ-41: Bu-Zhong-Yi-Qi-Tang) using a water-supplying bottle resulted in a slight but significant inhibition of Meth A growth. The oral administration of TJ-41 with gastric gavage significantly enhanced the specific antitumor activity against Meth A at rechallenge on day 9. In a tumor-neutralizing assay, the tumor draining LN cells of the TJ-41 administered mice showed an antitumor activity against Meth A. In a cytolytic assay, the anti-Meth A specific cytolytic T lymphocyte activity was not detected in the spleen cells of the Meth A bearing and TJ-41 administered mice. The oral administration of TJ-41 enhanced the natural killer (NK) activity of the spleen cells in naive mice but could not improve the decreased NK activity of spleen cells from the tumor bearing mice. In a cytostatic assay, the peritoneal exudate cells from the Meth A bearing and TJ-41 administered mice showed a significantly higher amount of cytostatic activity against Meth A than that from either Meth A bearing or TJ-41 administered mice. These results indicate that the oral administration of TJ-41 into the tumor bearing mice may thus be able to enhance concomitant antitumor immunity through the augmentation of the cytostatic activity.

Genetic analysis of control of proliferation in fibroblastic cells in culture. I. Isolation and characterization of mutants temperature-sensitive for proliferation or survival of untransformed diploid rat cell line 3Y1

Mutants temperature sensitive for proliferation or survival were isolated from an untransformed diploid clone of fibroblastic rat cells (3Y1), according to an isolation protocol that selected for mutants defective at 38.5‡C (selection temperature) in undergoing the transition from quiescent to proliferating state while maintaining viability at 38.5‡ C. Of the 108 temperature-sensitive clones isolated, 32 were examined for survival in sparse cultures at 39.8‡ C (nonpermissive temperature) and classified into four classes. Results of temperature shift-up experiments suggest that functions defective in 11 of the 32 mutants are necessary not only for changing from the quiescent to proliferating state but also for maintenance of the proliferating state. Of the 32 mutants, 17 were assigned to eight complementation groups. Results of the physiological characterization of the representative mutants of each of the eight complementation groups are presented.

Role of Macrophages in Acute Murine Cytomegalovirus Infection

It has been recognized that macrophages play an important role in controlling virus infection in experimental animal models. To evaluate the role of macrophages in acute murine cytomegalovirus infection, macrophages in the spleen and the liver were eliminated by an intravenous injection of liposomes containing a cytolytic agent, dichloromethylene diphosphonate. The depletion of macrophages led to a significant increase of virus titer in the spleen and lungs in both susceptible BALB/c and resistant C57BL/6 mice during the first three days after intravenous infection. In the spleen, the increase of virus titer in macrophage‐depleted BALB/c mice was much greater than that in NK cell‐depleted mice. These results suggest that macrophages contribute to protection mainly by the mechanisms which are independent of NK cells during the first three days after infection. The increase of virus titer in macrophage‐depleted C57BL/6 mice was as great as that in NK cell‐depleted mice because of the high contribution of NK cells to protection in C57BL/6 mice. In the liver in both strains of mice, the effects of macrophage depletion on virus titer were not as much as those in the spleen and lungs. Furthermore, the local depletion of peritoneal macrophages resulted in a great increase of virus titer in the spleen at three days after intraperitoneal infection. We conclude that macrophages greatly contribute to decreasing the virus load in some organs possibly through either or both intrinsic and extrinsic mechanisms in the early phase of primary infection with murine cytomegalovirus.

Heat shock proteins and the antitumor T cell response.

Heat shock proteins (HSP) have been shown to participate in the antitumor T cell response. First, HSP play a crucial role in the intracellular pathway for antigen processing where HSP can make complexes with a broad spectrum of cellular proteins and peptides through their chaperone functions. In this pathway, macrophages are required for processing the chaperoned peptides to make stable molecules with the major histocompatibility complex (MHC) class I molecules, even when HSP-peptide complexes are exogenously administered. Through this pathway, vaccination with HSP-peptide complexes is thus able to elicit the response of CD8+ T cells specific for the chaperoned peptides. These findings suggest an essential role of HSP in ‘cross-priming’ and their usefulness for antitumor vaccination with tumor peptides. Second, HSP have been suggested to be expressed on the cell surface by transformation and, in addition, to function as antigen-presenting molecules for double negative T cells. Third, HSP derived from tumor cells have reportedly been recognized by T cells with either T cell receptor (TCR)-αβ or TCR-γδ. These lines of evidence therefore indicate that HSP may be potentially promising target molecules for antitumor T cell immunotherapy.

Protective effects of Hochu-ekki-to, a Chinese traditional herbal medicine against murine cytomegalovirus infection

The innate immunity against murine cytomegalovirus (MCMV) at the early phase of infection is mediated by NK cells and macrophages. We studied the effects of hochu-ekki-to (HET), a traditional Chinese herbal medicine, on the regulation of innate immunity mediated by NK cells and macrophages. We found the oral administration of HET to increase both the number of leukocytes in the spleen and liver and the splenic NK cell cytotoxicity associated with the increased induction of serum IFN-alpha/beta after an MCMV infection but it had no effect on liver NK cells. However, no differences were found in the serum IL-12, IFN-gamma, TNF-alpha and nitric oxide (NO) production in the culture of macrophages between the HET- and PBS-treated mice on day 2 after MCMV infection. In addition, HET-treated splenic and peritoneal macrophages were found to show a higher intrinsic resistance against in vitro MCMV infection than that of PBS-treated mice. Therefore, the HET-induced effects on NK cells and macrophages selectively reduced the viral load in the spleen but not in the liver at an early phase of MCMV infection. HET may thus be useful in the treatment of human cytomegalovirus infection which commonly occurs in HIV-infected AIDS patients.

Genetic analysis of control of proliferation in fibroblastic cells in culture. II. Alteration in proliferative and survival phenotypes in a set of temperature-sensitive mutants of rat 3Y1 cells after infection or transformation with simian virus 40

Mutants of rat 3Y1 fibroblasts, temperature sensitive for proliferation or survival and which represent each of eight complementation groups, were examined to determine whether cells made quiescent at confluence at 33.8‡C (permissive temperature) can be stimulated to enter S phase at 39.8‡C (nonpermissive temperature) by 20% serum or by infection with simian virus 40 (SV40). Three mutants with a short survival at 39.8‡ C did not enter S phase at 39.8‡ C under either condition. The remaining five entered S at 39.8‡C by infection with SV40. However, only one of these five entered S in response to high serum. After transformation with SV40, three mutants accumulating at 39.8‡C with a predominantly 2n (G1) DNA content did not proliferate, there was a rapid and extensive cell death, and the cells had a DNA content similar to that seen in randomly proliferating populations. The other two mutants, accumulating at 39.8‡ C with a predominantly 2n or 2n.4n DNA content, proliferated at this temperature after transformation with SV40. These results clearly indicate that SV40 interacts closely with cellular ts lesions related to control of proliferation and cell survival.