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Short Communication |
expression in CD8
+
– T cells of feline immunodeficiency virus-infected cats
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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+
– and fCD8+low cells in peripheral blood. To investigate these cells in more detail, an anti-fCD3
mAb, termed NZM1, was generated, which recognizes the extracellular epitope of the fCD3 molecule. The anti-fCD3 mAb proved to be more suitable for identifying feline T cells than the anti-fCD5 one, which has been used as a pan-T-cell reagent in cats, because of the presence of fCD5+fCD3– cells among lymphocytes. Although the fCD8+– and fCD8+low cells in the FIV-infected cats expressed fCD3, a subset of fCD8+– cells expressed fCD3 antigen at a lower level than the T cells whose phenotype was fCD4+, or fCD8+low. The lower expression of fCD3 may be associated with the immune status of fCD8+– T cells.
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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TOPABSTRACTMAIN TEXTREFERENCES |
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). The CD8 antigen consists of two polypeptides, CD8 and CD8, and exists as a heterodimer (CD8) or a homodimer (CD8). In humans, T-cell receptor (TCR) T cells express the CD8 heterodimer, and TCR
T cells and natural killer cells express the CD8 homodimer (Moebius et al., 1991). Feline immunodeficiency virus (FIV) infection in cats has been studied extensively as an animal model for the persistent infections and pathogenesis caused by HIV (for a review see Miyazawa, 2002). Previously, we found that feline CD (fCD)8+– and fCD8+low cells increased in number in the peripheral blood of FIV-infected cats (Shimojima et al., 1998a). These subsets were reported to play roles in the suppression of FIV replication (Bucci et al., 1998; Flynn et al., 2002; Gebhard et al., 1999; Shimojima et al., 2004). The induction of similar subpopulations was also confirmed in human diseases, such as HIV infection (Schmitz et al., 1998). However, it remains unknown whether the fCD8+– and fCD8+low cells are T cells or natural killer cells. The phenotypic characterization of fCD8+– and fCD8+low cells in FIV-infected cats is difficult due to a lack of monoclonal antibodies (mAbs) against appropriate surface markers.
Cells of the T-cell lineage bear a TCR–CD3 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 TCR–CD3 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 1–5). These findings indicate that the mAb NZM1 is directed against the fCD3 molecule.
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with NZM1 also induced T-cell proliferation as demonstrated with anti-CD3 mAbs of other species (Leo et al., 1987; Tsoukas et al., 1985; Yang et al., 1996). Feline PBMCs (2x105) separated from heparinized whole blood of a specific-pathogen-free (SPF) cat were suspended in 100 µl RPMI 1640 medium containing fetal calf serum (10 %, v/v) and antibiotics, and plated in a well of a 96-well flat-bottomed microculture plate. The PBMCs were cultured in the presence of the anti-fCD4 mAb [4D9 (IgG1); Shimojima et al., 1997], anti-fCD8 [12A3 (IgG2a); Shimojima et al., 1998b] or NZM1 (final dilution, ascites 1 : 103, 1 : 104 or 1 : 105) for 72 h at 37 °C in a humidified atmosphere of 5 % CO2 in air. The proliferation of PBMCs was measured by MTT assay (Mosmann, 1983). The cells proliferated to a greater extent when cultured with NZM1 than with 4D9 or 12A3 (P<0·005, n=3; data not shown). We considered that NZM1 recognizes the extracellular epitope of fCD3, as it could stain feline PBMCs without permeabilization in the immunofluorescence analysis and induce the proliferation of feline PBMCs in the co-cultivation experiments.
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 8–10 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 (fCD5–PE), 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 fCD3high and fCD3low subsets (Fig. 2d), which indicates that fCD8+– cells in FIV-infected cats consist of fCD3high and fCD3low subsets (Shimojima et al., 1998a; Stievano et al., 2003).
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–FITC and either fCD4–PE (Fel7; Ackley et al., 1990), fCD8–PE (FT2; Klotz & Cooper, 1986), fCD8 (2D7; Shimojima et al., 1998b), or the mixture of fCD4–PE, fCD8 and fCD8–PE. A secondary rat anti-mouse IgG2a antibody conjugated with PE (Zymed Laboratories) was used for the detection of fCD8. The uninfected and FIV-infected groups gave distinctive patterns, and representative results are shown in Fig. 3. Most of the fCD4+ and fCD8+ cells were fCD3+. The fCD3+ cell population consisted of fCD4+ (46·3±2·4 %; Fig. 3a), fCD8+ (41·9±2·3 %; Fig. 3b) and fCD4– fCD8–– (9·3±0·6 %; Fig. 3d) cells in the SPF cats (n=3). Most of the fCD3low cells in the FIV-infected cats were fCD5low fCD4– fCD8low– (Fig. 3d–g). In addition, fCD8low cells whose population expanded in FIV-infected cats also expressed fCD3 (Fig. 3c, g).
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+– and fCD8+low cells in the FIV-infected cats expressed fCD3, hence these subsets are T cells. It is still unknown at present whether fCD8+–, fCD8+low and fCD3+ fCD4– fCD8–– cells are T cells, as no reagent specific for the feline TCR - or -chain is available. We also found a lower level of expression of the fCD8 molecule in fCD8+– subsets. A decreased expression of CD8 is reported in CD3+ cells but not natural killer cells in HIV-infected individuals (Ginaldi et al., 1997). Downregulation of fCD8 expression may contribute to the progressive reduction of fCD8+ cell function in FIV-infected cats. Several factors may be involved in the change of fCD3 expression in FIV infection. In general, the CD3low T cell is a recently antigen-activated or memory cell. It is reported that both activated and non-activated T cells from HIV-positive patients express less CD3 than those from control subjects (Ginaldi et al., 1997). As CD3 plays an important role in signalling of TCR/CD3, fCD3low cells might raise the activation threshold and contribute to the lack of effective immune surveillance. There is a continuous loss of naïve CD4 and CD8 T cells and expansion of memory cells in HIV-infected patients (Bass et al., 1992). As the majority of fCD8+– cells show an increase in fCD11a expression, one of the activation antigens (Shimojima et al., 2003) and CD8+– memory T cells descend directly from clonally expanded CD8++ T cells (Konno et al., 2002), we speculate that fCD3lowfCD8+– T cells consist of activated memory subsets. Hohdatsu et al. (2003) reported controversial anti-FIV activities of fCD8+– and fCD8+low subsets. Not all fCD8+– and fCD8+low cells, but some with enough fCD3 expression, may have strong anti-FIV activity.
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.
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