1 Department of Veterinary Microbiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2 Host and Defense, PRESTO, Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
3 Laboratory of Veterinary Public Health, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
Correspondence
Takayuki Miyazawa
takavet{at}obihiro.ac.jp
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ABSTRACT |
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Present address: Department of Virology II, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama-shi, Tokyo 208-0011, Japan.
Present address: Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo 108-8639, Japan.
Present address: Department of Pathobiology, College of Veterinary Medicine, University of Florida, Gainesville, FL 32611, USA.
||Present address: Department of Biological Chemistry, UC Davis School of Medicine, UC Davis Cancer Center, Sacramento, CA 95817, USA.
¶Present address: Food Safety Commission, The Cabinet Office, Japanese Government, Nagata-cho, Chiyoda-ku, Tokyo, Japan.
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MAIN TEXT |
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Cells of the T-cell lineage bear a TCRCD3 complex consisting of variable or
TCR chains associated with invariant CD3 chains of
,
,
and
(Ashwell & Klausner, 1990
). The CD3
chain appears to be the most immunogenic and exposed part of CD3, as anti-human CD3 mAbs are predominantly directed to epitopes of the CD3
subunit (Transy et al., 1989
). Only completely assembled TCRCD3 complex can be expressed on the T-cell surface (Clevers et al., 1988
). Therefore, mAbs for CD3
have exquisite specificity for T cells and are widely used to identify T cells in both humans (Reinherz et al., 1979
) and mice (Leo et al., 1987
). To investigate feline T cells, Joling et al. (1996)
reported that an anti-human CD3
polyclonal antibody, prepared from rabbits immunized with peptides of the cytoplasmic domain of human CD3
, cross-reacted with feline CD3
and could be used for immunohistochemical studies in cats. However, this antibody was inconvenient as the permeabilization of cells is necessary for flow cytometric analysis. Instead of a specific anti-fCD3 mAb, f43 mAb, which recognizes the feline homologue of the CD5 antigen, has been used as a pan-T-cell reagent in cats (Ackley & Cooper, 1992
). However, the CD5 molecule is also expressed on a subset of B cells in humans, rabbits and mice (Caligaris-Cappio et al., 1982
; Manohar et al., 1982
; Raman & Knight, 1992
), therefore f43 mAb appears to be inappropriate for the detection of feline T cells. In order to solve this problem, we prepared a mAb termed NZM1 that detects the fCD3
antigen in immunoblotting and flow cytometric analyses, and characterized the fCD8
+
and fCD8
+
low cells in FIV-infected cats.
Hybridomas were generated from BALB/c mice immunized with insect cells (Sf9 cells) infected with the recombinant baculovirus rAcfCD3, which carries cDNA encoding the fCD3
molecule (Nishimura et al., 1998
). A positive hybridoma designated NZM1 (IgG3) was selected based on the reactivity with a T-lymphoblastoid cell line, MYA-1 cells (Miyazawa et al., 1989b
), by an indirect immunofluorescence assay using a fluorescein isothiocyanate (FITC)-conjugated secondary antibody. The specificity of NZM1 was confirmed by the immunoblotting analysis using Sf9 cells infected with rAcfCD3
and feline peripheral blood mononuclear cells (PBMCs) as antigens (Fig. 1
). As a control, a rabbit polyclonal antibody against the cytoplasmic region of human CD3
(Dako A/S) was used. Secondary antibodies conjugated with horseradish peroxidase were used to detect positive signals as described previously (Miyazawa et al., 1989a
). NZM1 recognized several bands of about 25 kDa in Sf9 cells infected with rAcfCD3
(Fig. 1
, lane 8) but not in mock-infected cells (Fig. 1
, lane 6) or cells infected with the control baculovirus (Fig. 1
, lane 7). NZM1 was confirmed to react with a 25 kDa molecule of MYA-1 cells (Fig. 1
, lane 9) and feline PBMCs (Fig. 1
, lane 10), which was identical to the molecule recognized by the anti-human CD3
polyclonal antibody (Fig. 1
, lanes 15). These findings indicate that the mAb NZM1 is directed against the fCD3
molecule.
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Two cats infected with each of the FIV TM1 (cat 103) and TM2 (cat 104) strains for 11 years (Miyazawa et al., 1989a) and one infected with the Petaluma strain for 2 years (cat 115) were used in the flow cytometric analysis. Three adult SPF cats aged 810 years (cats 102, 201 and 202) were used as uninfected controls. All cats were clinically healthy. PBMCs were suspended in a sorter buffer (PBS containing 3 % fetal calf serum and 0·05 % sodium azide) and centrifuged at 800 r.p.m. to remove platelets. The mAb NZM1 was labelled with FITC (fCD3
FITC) according to a standard procedure. PBMCs were washed twice in the cold sorter buffer and incubated with fCD3
FITC. After washing with the sorter buffer, stained cells were analysed after gating for lymphocytes based on light (forward and side) scatters using a flow cytometer FACScan with CELLQUEST software (Becton Dickinson). The different subpopulations were expressed as percentages of the total lymphocyte population. The uninfected and FIV-infected groups gave distinctive patterns of fCD3
expression, and representative results are shown in Fig. 2
. In FIV-uninfected SPF cats, the fCD3
molecule was expressed on 57·2±9·5 % (n=3) of peripheral lymphocytes (Fig. 2a
). On the other hand, two subsets of fCD3+ cells, fCD3high (33·1±16·5 %, n=3) and fCD3low (20·7±9·3 %, n=3), were detected in the FIV-infected cats (Fig. 2c
). As the fCD5 antigen has been considered a pan-T-cell molecule in cats, PBMCs were labelled with fCD3
FITC and phycoerythrin (PE)-conjugated anti-fCD5 mAb (fCD5PE), f43 (Ackley & Cooper, 1992
) and analysed by flow cytometry (Fig. 2b, d
). Although most of the fCD5 cells expressed the fCD3
molecule, there was a substantial number of fCD5+fCD3
cells in FIV-uninfected SPF cats (2·0±1·7 %, n=3; Fig. 2b
). So anti-fCD5 mAb appears to be unsuitable for the detection of feline T cells. The expression of fCD5 antigen on feline B cells has not been characterized in detail, and it is unknown whether this subset corresponds to CD5+ B cells in humans and mice. It should also be noted that the fCD5low population consisted of fCD3
high and fCD3
low subsets (Fig. 2d
), which indicates that fCD8
+
cells in FIV-infected cats consist of fCD3
high and fCD3
low subsets (Shimojima et al., 1998a
; Stievano et al., 2003
).
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Trimble & Lieberman (1998) reported the expansion of CD3
subsets in a substantial fraction of CD8+ T cells in HIV-infected patients. They classified the CD8+ cells into the subpopulations CD8+CD3
and CD8+CD3
+. They did not mention the fluorescent intensity of the CD3
molecule on CD3+ cells, and concluded that the downregulation of CD3
expression is independent of other TCR/CD3 components. A decrease in CD3
mRNA levels was also reported in T cells from AIDS patients (Geertsma et al., 1999
), but that of CD3
mRNA levels has not yet been discussed. Although downregulation of CD3 expression on CD4+ and CD8+ cells is reported in HIV-infected patients, its relationship with CD3
expression is unclear (Ginaldi et al., 1997
). In the fCD3 complex, fCD3
is the only molecule whose cDNA has been identified, and NZM1 is the first mAb specific to the fCD3 component. Therefore it is not known at present whether the fCD3
downregulation involves a decrease of other feline TCR/CD3 components, including fCD3
. If the downregulation of fCD3
in the fCD8+ cells of FIV-infected cats correlates with disease progression, as does that of CD3
in HIV infection (Geertsma et al., 1999
), the measurement of fCD3
expression may contribute to our understanding of the immune status of FIV-infected cats.
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ACKNOWLEDGEMENTS |
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Received 10 March 2004;
accepted 11 May 2004.