Projects and summary of Major Achievements
A. Mechanisms of antigen presentation by cells infected with intracellular parastic protozoan, Toxoplasma gondii ( T.gondii)
The studies of immune responses of humans and mice to T.gondii have emphasized the nature of the antigen-presentation process. We have demonstrated vacuolar fusion with T.gondii membranes, providing a means through which T.gondii proteins gain access to the cytosolic compartment and thus can be processed for presentation in association with class I MHC molecules.
B. Analysis of T cell epitopes bound to MHC class I of T.gondii-infected cells
We have further extended this work to the examination of peptides derived from a T.gondii-infected cells. Naturally processed peptides derived from T.gondii were acid extracted from T.gondii-infected cells and detected by cytotoxic T lymphocytes ( CTL) dereived from peripheral blood lymphocytes of a patient with chronic toxoplasmosis. The CTL lines were obtained by weekly in vitro stimulation with a T.gondii-infected human B cell lymphoma line, ARH, which shares HLA-A2 with the patient. The lytic activity of these CTL lines against T.gondii-infected ARH and ARH pulsed with fraction 29 of a reverse-phase high performance liquid chromatography ( HPLC) extract from ARH was inhibited by an anti-HLA-A, B, C monoclonal antibody (mAb) and an anti-HLA-A2 mAb. Anti-HLA-DR mAb failed to block the lytic activity. Thus, the presentation of peptides by T.gondii-infected cells for CTL is mediated by HLA-A2 molecuiles.The amino acid sequence of the HLA-A2-bound peptide in fraction 29 was in part consistent with the predicted algorithm of HLA-A2-binding peptide motifs.
C. Roles of HSP70 in antigen presentation
HLA-DR-restricted CD4+ cytotoxic T lymphocytes (CTL) lines specific for T.gondii-infected melanoma cells have been established from peripheral blood lymphocytes (PBL) of a patient with chronic toxoplasmosis. The role of heat shock cognate protein (HSC) 71 in antigen (Ag) processing and presentation of T.gondii-infected melanoma cells to these CD4+ CTL lines was investigated. Human melanoma cell line, P36, pulsed with T.gondii-infected P36 cell-derived HSC71 was lysed by a T.gondii-specific CD4+ CTL line, Tx-HSC-1. The Tx-HSC-1 also killed T.gondii-infected P36 cells. The lytic activity of Tx-HSC-1 against P36 cells pulsed with T.gondii-infected P36 cell-derived HSC71 was inhibited by monoclonal antibodies (mAbs) against HSC71. Anti-human leukocyte antigen (HLA)-DR mAb also partially blocked the lytic activity whereas anti-HLA-A,B,C mAb did not block the lytic activity. In addition, flow cytometric analysis with these specific mAbs against HSC71 showed HSC71 to be expressed on the cell surface of T.gondii-infected as well as uninfected P36 cells. These data indicate that HSC71 molecules are expressed on human melanoma cell line, P36, and that HSC71 may play a potential role in Ag presentation and processing of T.gondii-infected P36 cells to CD4+ CTL.
D. Congenital toxoplasmosis in Japan
T.gondii infection is widespread among man and warm blooded animals including birds even in economically established countries such as theUnited States, European countries andJapan. Its prevalence varies widely with location and age of host . Seropositiveness of anti-T.gondii antibody inJapan is estimated as following formula; age x (0.1 - 1) %, i.e. 3-30% at 30 years old. One of the most severe form of toxoplasmosis is congenital toxoplasmosis. However, the mass screening of congenital toxoplasmosis with prenatal serodiagnosis in pregnant women has been believed meaningless or even hazardous for pregnant women because sero-positive results in prenatal examinations merely make them anxious inJapan Thus, detailed epidemiological records of congenital toxoplasmosis have not been maintained and researche has been neglected.
Under such a socio-medical atmosphere, we experienced a typical congenital toxoplasmosis case manifesting the triad of chorioretinitis, intracranial calcification, and hydrocephalus. The patient was definitively diagnosed by detection of T.gondii protozoa in the patient's spinal fluid atNagasaki Japan, in 1994. Since then, a higher frequency of congenital toxoplasmosis comparing with that of formerly believed has been speculated. We assumed that the difficulty to diagnose congenital toxoplasmosis was attributed to several factors including the lack of appropriate system of diagnosis. We have established quantitative competitive( QC)- PCR targeting T.gondii-specific SAG1 to overcome the difficulty of detecting intracellulary infective T.gondii. T.gondii-specific SAG1 gene( 759bp cDNA) has been cloned into pET-21b(+) vector, and a154 bp truncated SAG1-pET21(+) was used as the competitor in quntitative competitive(QC)-PCR. Genomic DNA was prepared from placenta, amniotic fluid, liquor, cord blood and peripheral blood. Since then, we have experienced 21 cases of symptomatic congenital toxoplasmosis untill the beginning of 1998. Therefore, actual incidence of congenital toxoplasmosis in Japan can be estimated relative to those of the United States( 40/530,000 newborns), Denmark ( 1/6,355 newborns) and European countries( 45/ 10,000 pregnancies).
In conclusion, surveillance for seroconversion in pregnant women should be mandated as an examination of congenital toxoplasmosis. Educational efforts to improve medical doctor's and pregnant women's understanding of the disease should be required inJapan.
E. Gene Vaccine
To develop a vaccine by augmenting the protective cellular immunity against T.gondii infection, T.gondii SAG1 gene-transfectants were established by using RMA.S, a murine TAP-deficient lymphoma line, as host APC. Immunization with the SAG1-transfected RMA.S induced a CD8+ CTL line specific for not only SAG1-transfected RMA.S but also T.gondii-infected RMA.S, and elicited protective effects against acute T.gondii-infection in C57BL/6 mice.
F. Virulency of T.gondii and the effector mechanisms of anti-T.gondii immunity by CTL
Virulency of T.gondii is correlated with the antigen presenting ability of T.gondii-infected cells, and down-regulated MHC class II expression of T.gondii-infected cells was up-regulated by IFN-gamma . IFN-gamma or CTL specific for T.gondii-infected cells did not kill free T.gondii tachyzoites directly. When free tachyzoites and T.gondii in the host cells were exposed to acidic condition, destruction of the outer cell membrane and nuclear membrane of T.gondii and degradation of SAG1 gene were observed. Thus, the final stage of killing of T.gondii may be mediated by other cell types such as macrophages than CTL.
G. Melanocytes as a professional antigen presentiong cell controling the pathogenicity of toxoplasmosis and Vogt-Koyanagi-Harada disease (VKH)
We have already reported that T.gondii-infected cell-specific CD4+CTL were induced by T.gondii-infected melanoma cells, while T.gondii-infected cell-specific CD8+CTL were induced by T.gondii-infected non-melanocytic cells. Similar immune responses were observed in patients with VKH in which self melanocytes are speculated as a target cell of autoimmunity. Melanoma-specific Tc1 CTL is exsistent in patients with toxoplasmosis and those with VKH. These data suggest that melanocytes may play a role in pathogenicity of toxoplasmosis and VKH by inducing Tc1.
H. Other parasites
Although the most important efforts have centered on T.gondii, we have also worked in other parasites and parasitic diseases such as amoebiasis, filariasis, anisakiasi, angiostrongylosis, malaria, schistosomiasis, strongyloidiasis, dirofilariasis, toxocariasis, paragonimiasis ( Paragonimus westermanii, Paragonimus miyazakii, Paragonimus yokogawaii), taeniasis, echinococcosis, etc.