ABSTRACT
Immunochemical studies (sequential immunoprecipitation followed by SDS-PAGE and two-dimensional gel electrophoresis) have shown that the monoclonal antibody (MoAb) 26.163 reacts with HLA-DR, DQ, and DP antigens. Testing with isolated alpha and beta chains of HLA class II antigens and immunoblot analysis also demonstrated that the determinant defined by the MoAb 26.163 is localized on beta chains and does not require their association with alpha chains for its expression. The MoAb 26.163 appears to be the first example of a monoclonal antibody with specificity for the gene products of HLA-DR, DQ, and DP loci.
Subject(s)
Antibodies, Monoclonal , Epitopes/analysis , HLA-D Antigens/genetics , HLA-DP Antigens/genetics , HLA-DQ Antigens/genetics , HLA-DR Antigens/genetics , Antibodies, Monoclonal/classification , Chromosome Mapping , Collodion , Electrophoresis, Polyacrylamide Gel , Epitopes/genetics , Humans , Immunoglobulin alpha-Chains/genetics , Paper , Precipitin Tests/methodsABSTRACT
A panel of five murine monoclonal antibodies to canine T-lymphocytes were produced. Antibodies 4.78, 12.125 and 8.358 reacted with approximately 18%, 39% and 60% peripheral blood lymphocytes, respectively. Two color flow cytometric analysis showed that lymphocytes expressing 1.140, 4.78, 8.53 and 12.125 were subsets of lymphocytes expressing 8.358. The lymphocytes expressing 8.358 were negative for surface immunoglobulin. The subsets defined by 1.140, 4.78 or 8.53, 12.125 were mutually exclusive and together account for most cells expressing 8.358 in the peripheral blood, spleen, and lymph node. In the thymus, approximately 47% cells were positive for both 1.140/4.78 and 8.53/12.125. SDS-PAGE analysis of radiolabelled thymus cell lysates demonstrated that antibodies 1.140 and 4.78 immunoprecipitated a 32,35 kd heterodimer under reducing conditions and 12.125 immunoprecipitated a single 56 kd chain under reducing and non-reducing conditions. Antibodies 8.53/12.125 and 1.140/4.78 react with canine lymphocyte populations that occur in proportions similar to lymphocytes expressing CD4 and CD8 like molecules in several primate and non-primate species. The molecules recognized by 12.125 and 1.140/4.78 were similar in size and subunit composition to human CD4 and CD8.
Subject(s)
Antibodies, Monoclonal/immunology , Dogs/immunology , T-Lymphocyte Subsets/immunology , Animals , Antibodies, Monoclonal/biosynthesis , Antigens, Surface/immunology , Bone Marrow/immunology , CD4-Positive T-Lymphocytes/immunology , Electrophoresis, Polyacrylamide Gel , Flow Cytometry , Immunoglobulin Isotypes/immunology , Lymphoid Tissue/immunology , Mice , Mice, Inbred BALB C , T-Lymphocytes, Regulatory/immunologyABSTRACT
An IgG1 mouse monoclonal antibody, designated 1.646, is described which recognizes a cytoplasmic antigen of equine mononuclear phagocytes. Indirect fluorescent antibody staining of peripheral blood leukocytes reveals a granular cytoplasmic staining, predominantly in adherent blood mononuclear cells. Indirect fluorescent antibody staining is positive for alveolar and peritoneal macrophages. In some horses, a few neutrophils are also stained. In equine tissue samples stained by immunohistochemistry, the distribution of positive cells is consistent with the distribution of tissue macrophages. The most intense and reliable staining occurs with splenic and lymph node macrophages. Hepatic Kupffer cells also stain with antibody 1.646, although the intensity of that staining is somewhat variable between horses. A granular pattern of staining typical of lipofuscin deposition is also seen in liver sections. There is also pale staining of some biliary and renal tubular epithelium. Equine erythrocytes, platelets and lymphocytes are not recognized by this antibody, and neither are monocyte/macrophages of human, canine or feline origin. Antibody 1.646 recognizes two proteins (150 and 30 kDa) of equine monocyte-derived macrophages when assayed by Western immunoblot. Because of the distribution of staining (tissue mononuclear phagocytes, lipofuscin-containing storage granules, biliary and renal tubular epithelium, and some neutrophils) we hypothesize that antibody 1.646 recognizes a cytoplasmic antigen that is closely associated with lysosomal membranes.
Subject(s)
Antibodies, Monoclonal/biosynthesis , Horses/immunology , Leukocytes, Mononuclear/immunology , Phagocytes/immunology , Animals , Antibodies, Monoclonal/immunology , Antibodies, Viral/biosynthesis , Antibody Specificity , Antigens, Viral/immunology , Blotting, Western/veterinary , Equine Infectious Anemia/etiology , Equine Infectious Anemia/immunology , Fluorescent Antibody Technique/veterinary , Hybridomas , Immunoglobulin G/biosynthesis , Immunoglobulin G/immunology , Liver/immunology , Lung/immunology , Lymph Nodes/immunology , Macrophages/immunology , Mice , Spleen/immunologyABSTRACT
We describe the development of three monoclonal antibodies to feline T lymphocytes. Antibody 1.572 stains 93% of feline thymocytes, 49% of lymph node, and 65% of spleen lymphocytes. Two-color analysis shows 1.572 does not stain Ig-bearing cells, and 1.572-positive lymphocytes plus Ig-positive lymphocytes make up approximately 90% of peripheral blood lymphocytes (PBL), suggesting that 1.572 is a pan-T cell marker. The other two monoclonal antibodies, 3.357 and CAT30A, stain a smaller population of thymocytes (59%) of which 40% express both antigens. The 3.357 antigen is found on 23% of lymph node and 47% of spleen lymphocytes, while the CAT30A antigen is found on 29% of lymph node and 19% of spleen lymphocytes. Two-color analysis shows that 3.357 and CAT30A stain mutually exclusive subpopulations of 1.572-positive cells. Using thymocytes as an antigen source, antibody 3.357 precipitated a molecule of 66,000 molecular weight (Mw) under nonreducing conditions and a heterodimer of 32,000 and 34,000 under reducing conditions, suggesting that 3.357 recognizes the feline CD8 homologue. Antibody CAT30A precipitated a molecule of 55,000 Mw under both reducing and nonreducing conditions, which suggests it recognizes the feline CD4 homologue. Analysis of PBL profiles of 35 normal cats using the three monoclonal antibodies indicates that the distribution of feline PBL subpopulations is similar to man, including the CAT30A:3.357 ratio (1.74), which is identical to reported CD4:CD8 ratios in man. Based on these data, the feline CD4 and CD8 homologues are similar to those reported in other species.
Subject(s)
Antibodies, Monoclonal/immunology , Cats/immunology , T-Lymphocyte Subsets/immunology , Animals , Antibodies, Monoclonal/isolation & purification , Antigens, Surface/immunology , Electrophoresis, Polyacrylamide Gel , Epitopes/immunology , Flow Cytometry , Hybridomas/immunology , Lymph Nodes/cytology , Lymph Nodes/immunology , Mice , Precipitin Tests , Specific Pathogen-Free Organisms , Spleen/cytology , Spleen/immunology , Thymus Gland/cytology , Thymus Gland/immunologyABSTRACT
The age-related changes in numbers and proportions of peripheral blood lymphocyte subsets (pan-T, CD4, CD8, Ig, and null) were evaluated by two-color flow cytometry in 16 feline fetuses (56-58 days gestational age) and 21 kittens from birth through 8 weeks of age. Populations of pan-T+, CD4+, and CD8+ cells increased in total numbers as a function of increases in total lymphocyte numbers while proportions of these subsets remained relatively static. In contrast, both the total number and proportion of Ig+ cells increased from birth to 4 weeks of age, after which there were essentially no changes. Null (pan-T-, Ig-) cells were highest during late gestation and declined steadily thereafter to become a minimal component of the peripheral lymphocyte subsets. Compared with normal adult values, CD4/CD8 ratios were high throughout the 8 week study period. These results illustrate that the neonatal cat blood lymphocyte profile undergoes maturational changes and emphasize the importance of evaluating age-matched controls in studies of conditions that may alter feline lymphocyte subsets.
Subject(s)
Aging/immunology , Animals, Newborn/growth & development , Animals, Newborn/immunology , Embryonic and Fetal Development/immunology , Lymphocyte Subsets/physiology , Animals , CD4-CD8 Ratio/veterinary , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/physiology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/physiology , Cats , Female , Lymphocyte Subsets/cytology , PregnancyABSTRACT
One hundred ninety six dogs with spontaneously occurring lymphoproliferative disorders were immunophenotyped. Dogs with lymphoma (175) were determined to be derived from B-cells in 134/175 (76%), T-cells in 38/175 (22%) and 3/175 (2%) were null cells (non-reactive with any canine-specific lymphocyte antibody). Dogs with T-cell lymphomas were at significantly higher risk of relapse and early death compared with B-cell lineage lymphoma following therapy (52 vs. 160 days; p < 0.001 and 153 vs. 330 days; p < 0.001, respectively). Hypercalcemia was associated only with CD4+ lymphomas. A nonimmunoglobulin B-cell marker (B5), expressed in 95% of nonneoplastic lymphocytes, was expressed at a reduced level in 63% (64/104) of dogs with B-cell lymphoma. Dogs with lymphoma in which the B5 antigen was expressed below normal levels experienced shorter progression free survival (125 vs. 202 days; p < 0.05) and overall survival times (203 vs. 385 days; p < 0.05) than dogs with B-cell lymphoma in which the B5 antigen was expressed normally. Chronic lymphocytic leukemia in dogs was primarily associated with a CD8+ phenotype (8/12) and acute lymphoblastic leukemia was determined to be of either null cell (4/9) or T-cell (3/9) phenotype. Although canine and human non-Hodgkin's lymphoma are phenotypically similar, canine leukemia is phenotypically distinct from human leukemia. The development of canine-specific probes has facilitated a priori assessment of treatment outcome in dogs with lymphoma and may in the future contribute to the comparative understanding of leukemo- and lymphoma-genesis in these species.
Subject(s)
Antigens, Surface/analysis , B-Lymphocytes/chemistry , Leukemia, Experimental/immunology , Lymphoma/immunology , Animals , Biomarkers , Dogs , Immunophenotyping , Leukemia, Experimental/mortality , Lymphoma/mortality , Prospective Studies , Survival AnalysisABSTRACT
The temporal appearance of 4 viral proteins was detected in bluetongue virus (BTV)-infected Vero cells by indirect immunofluorescence staining with monoclonal antibodies (MAb) to BTV structural proteins VP2 and VP7 and nonstructural proteins NS1 and NS2. Bluetongue viral proteins were detected at distinct intervals after inoculation of Vero cells; VP7 was first detected 3 hours after inoculation, NS1 and NS2 at 5 hours after inoculation, whereas VP2 was not detected until 8 hours after inoculation. Patterns of fluorescence varied with the fixative used, but each MAb induced a distinct pattern of fluorescence in infected cells. Flow cytometry, which was used with each of the 4 MAb, proved to be an accurate and sensitive method of detecting BTV-infected P3 mouse myeloma cells. The temporal appearance of each viral protein in BTV-infected P3 cells was similar to that detected in BTV-infected Vero cells. Advantages of flow cytometry over conventional immunofluorescence staining to detect BTV-infected cells included: (1) enumeration of the proportion of infected cells in a population; (2) further characterization of infected cells, including estimates of their viability; and (3) computer-assisted storage and analysis of data obtained.
Subject(s)
Antibodies, Monoclonal , Bluetongue virus , Reoviridae , Viral Proteins/analysis , Animals , Cell Separation , Flow Cytometry/methods , Fluorescent Antibody Technique , Time Factors , Vero Cells , Viral Proteins/biosynthesis , Viral Structural Proteins/analysis , Viral Structural Proteins/biosynthesisABSTRACT
An overview of monoclonal antibody technology and some examples of its relevance to veterinary medicine are presented in this article. A technical description of the generation of immune spleen cells and hybridization is included. Feline leukemia, canine parvovirus, and their respective diseases are included as examples of cases in which monoclonal antibodies can be applied in the diagnosis and characterization of these diseases and their etiologic agent.
Subject(s)
Antibodies, Monoclonal , Virus Diseases/veterinary , Animals , Cat Diseases/diagnosis , Cats , Dog Diseases/diagnosis , Dogs , Enzyme-Linked Immunosorbent Assay/veterinary , Hybridomas , Leukemia/diagnosis , Leukemia/veterinary , Leukemia Virus, Feline/immunology , Leukemia Virus, Feline/isolation & purification , Mice , Parvoviridae/immunology , Parvoviridae Infections/diagnosis , Parvoviridae Infections/veterinary , Virus Diseases/diagnosisABSTRACT
Flow cytometric analysis of the ploidy of normal and neoplastic hepatocyte nuclei obtained from adult woodchucks, a model of human hepadnavirus-induced hepatocellular carcinoma, was performed. All 36 samples of nuclei from non-neoplastic liver from woodchuck hepatitis virus-infected or uninfected liver were diploid, indicating that age-related nuclear polyploidization does not occur in this species, unlike other rodents. Individual or multiple hepatic neoplasms were obtained from each of 14 woodchuck hepatitis virus-infected woodchucks. Nineteen samples of hepatocellular carcinoma and eight adenomas were examined. Aneuploid nuclei were detected in 10 of the hepatocellular carcinomas and three of the adenoma samples. Similar DNA indexes, ranging from 1.11 to 1.22, were found in 7 of the 10 aneuploid HCCs and all 3 aneuploid adenomas. Nine of the 19 hepatocellular carcinoma samples and 5 of the 8 adenomas were diploid. Four of the diploid hepatocellular carcinomas had increased proportions of tetraploid nuclei. The presence of aneuploid nuclei was not related to histological appearance of the neoplasms or serum gamma-glutamyltranspeptidase levels. Because none of the hepatocellular carcinomas metastasized, the presence of aneuploidy could not be related to biological behavior. We determined the proportion of uninucleate and binucleate hepatocytes in hepatocellular carcinoma and nonneoplastic liver. Approximately 7% of hepatocytes were binucleate in nonneoplastic liver from woodchuck hepatitis virus-infected and uninfected liver. Only 2% of malignant hepatocytes were binucleate. The results of this study indicate that aneuploidy is a common change in hepatic neoplasms from woodchuck hepatitis virus-infected woodchucks.
Subject(s)
DNA, Neoplasm/analysis , DNA/analysis , Hepatitis B Virus, Woodchuck , Hepatitis B/metabolism , Liver Neoplasms/chemistry , Liver/chemistry , Ploidies , Adenoma, Liver Cell/chemistry , Adenoma, Liver Cell/etiology , Adenoma, Liver Cell/pathology , Aneuploidy , Animals , Carcinoma, Hepatocellular/chemistry , Carcinoma, Hepatocellular/etiology , Carcinoma, Hepatocellular/pathology , Cell Nucleus/chemistry , Cell Nucleus/pathology , Diploidy , Flow Cytometry , Hepatitis B/complications , Hepatitis B/pathology , Liver/cytology , Liver/pathology , Liver Neoplasms/etiology , Liver Neoplasms/pathology , MarmotaABSTRACT
Feline immunodeficiency virus (FIV) infection in the cat is similar to human immunodeficiency virus type 1 infection in causing a selective reduction in CD4+ cell numbers, leading to inversion of the CD4+/CD8+ ratio. To determine whether FIV, similar to human immunodeficiency virus type 1, has a tropism for CD4+ cells, we examined the in vitro and in vivo susceptibilities of feline lymphocyte subpopulations to FIV infection. Infection of interleukin-2-dependent CD4+ or CD8+ lymphocyte cultures with the NCSU1 isolate of FIV (FIV-NCSU1) resulted in syncytium formation, cell death, and Mg(2+)-dependent reverse transcriptase (RT) activity in both cases. Monoclonal antibodies to feline lymphocyte subsets were used to sort peripheral blood mononuclear cells from FIV-infected cats into highly (> 95%) purified CD4+ cell, CD8+ cell, immunoglobulin-positive (Ig+) cell, and monocyte subpopulations. The mononuclear cell subpopulations were analyzed for FIV provirus by polymerase chain reaction and Southern blot analysis and for virus expression by RT activity. All 16 cats infected with FIV-NCSU1 demonstrated FIV provirus in CD4+ cell-, CD8+ cell-, and Ig+ cell-enriched lymphocyte populations. Southern blot detection of amplified gag gene sequences and limiting-cell-dilution polymerase chain reaction analysis indicated that Ig+ cells carried a higher FIV provirus burden in chronically (> or = 1-year) infected cats than either CD4+ or CD8+ cells. In contrast, CD4+ cells carried the greatest provirus burden in acutely (2- to 4-week) infected cats. FIV provirus was detected in monocytes from only 1 of 10 cats with asymptomatic infection. Addition of culture supernatants from enriched CD4+, CD8+, and Ig+ cells from FIV-infected cats to an FIV-susceptible CD4+ lymphocyte culture resulted in syncytium formation, cell death, and RT activity. Infection of Ig+ cells is not unique to FIV-NCSU1, as lymphocyte subpopulations from other cats with natural infections and cats infected with the Petaluma or Mount Airy isolate of FIV demonstrated a similar distribution of FIV provirus and RT activity. These data suggest that FIV possesses a broad tropism for peripheral blood mononuclear cells and that an Ig+ cell may serve as a major reservoir for the virus in chronically infected cats.
Subject(s)
Immunodeficiency Virus, Feline/physiology , Lymphocyte Subsets/microbiology , Proviruses/physiology , T-Lymphocyte Subsets/microbiology , Animals , Antibodies, Monoclonal , Base Sequence , Blotting, Southern , CD4-CD8 Ratio , Cats , DNA, Viral/analysis , Flow Cytometry , Genes, gag , Immunodeficiency Virus, Feline/genetics , Immunodeficiency Virus, Feline/immunology , Lymphocyte Subsets/immunology , Magnesium/pharmacology , Molecular Sequence Data , Oligodeoxyribonucleotides , Polymerase Chain Reaction , Proviruses/genetics , Proviruses/immunology , RNA-Directed DNA Polymerase/analysis , RNA-Directed DNA Polymerase/metabolism , T-Lymphocyte Subsets/immunologyABSTRACT
We describe studies of a human leukocyte antigen, termed Leu-13, that is defined by a mouse monoclonal antibody that reacts with T and B lymphocytes. Optimal staining of T cells requires a much higher concentration of alpha Leu-13 (greater than or equal to 200 micrograms/ml) than alpha Leu-4 (6 micrograms/ml). However, the fluorescence intensity of T cells labeled with alpha Leu-13, washed and kept at 4 degrees C for different periods, does not diminish more rapidly than the fluorescence of T cells stained with alpha Leu-4 and treated the same way. These results indicate that the novel binding pattern of alpha Leu-13 is not due to weak affinity. Immunoprecipitation and SDS-PAGE analysis indicates that alpha Leu-13 precipitates a major 16-kilodalton (16kd) band and a minor 26kd component from Nonidet P-40-solubilized membrane of normal T cells that are surface-labeled with 125I. The similar sizes of the 16kd to 26kd (Leu-13) and 22kd to 28kd (Leu-4) molecules prompted us to compare the capacity of alpha Leu-13 and alpha Leu-4 to trigger T cell functions, and to co-modulate and co-precipitate the T cell antigen receptor. Unlike alpha Leu-4, alpha Leu-13 does not trigger T cell proliferation or augment NK activity, but inhibits the mitogenic effect of alpha Leu-4/T3 antibodies. alpha Leu-13 also induces T cells to clump into large aggregates after 16 hr of culture at 37 degrees C, but not at 4 degrees C. T cell aggregation is triggered by concentrations of alpha Leu-13 as low as 12.5 ng/ml. At this concentration, alpha Leu-13 is not detectable on the surface of T cells by indirect immunofluorescence analysis in a FACS. On leukemic T cells, alpha Leu-4 was found to co-modulate and co-precipitate a 38kd to 44kd dimer having apparent idiotypic determinants, whereas alpha Leu-13 did not. The possible significance of these findings to immune regulation by T cells is discussed.
Subject(s)
Antibodies, Monoclonal/physiology , Antigens, Surface/immunology , Leukocytes/immunology , T-Lymphocytes/physiology , Animals , Antibodies, Monoclonal/immunology , Antigens, Surface/isolation & purification , Binding Sites, Antibody , Binding, Competitive , Cell Aggregation , Humans , Leukocytes/classification , Leukocytes/metabolism , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Molecular Weight , Precipitin Tests , Sezary Syndrome/immunology , T-Lymphocytes/immunologyABSTRACT
Cultures of adherent and non-adherent bovine blood mononuclear cells were infected with bluetongue virus (BTV) serotype 10. Production of BTV proteins in mononuclear cell cultures was detected by immune precipitation of viral proteins from [35S]methionine-labelled extracts of these cells, by immunofluorescence staining of cells using monoclonal antibodies (MAbs) to BTV proteins VP7 and NS2, and by flow cytometry with MAbs to VP2, VP7, NS1 and NS2. BTV-infected cells were most numerous in cultures of adherent mononuclear cells; infected cells were initially identified as monocytes on the basis of their morphology, and size and scatter characteristics as determined by analysis with a fluorescence-activated cell sorter (FACS). The majority of adherent mononuclear cells with these scatter characteristics were confirmed to be monocytes by FACS analysis with a MAb specific for bovine monocytes. Identification of BTV-infected adherent mononuclear cells as monocytes was further established by double immunofluorescent labelling, as infected adherent cells reacted with the MAb specific for bovine monocytes, and with another MAb specific for class II antigen. Infection of adherent mononuclear cells was also confirmed by transmission electron microscopy, as BTV virions and tubules were present in lysates of cultures of BTV-infected adherent mononuclear cells and within the cytoplasm of adherent cells. In contrast, BTV proteins were detected in few cells identified as lymphocytes on the basis of their scatter characteristics, and mean fluorescence of such cells was considerably less than that of BTV-infected monocytes. Viraemia persisted until 35 days after inoculation of a colostrum-deprived calf inoculated with BTV. Virus was isolated from blood mononuclear cells at 1 week after infection of the calf, but not thereafter. BTV infection of blood mononuclear cells was demonstrated until 9 days after inoculation by indirect immunofluorescence staining of mononuclear cells. In contrast, virus was consistently isolated from erythrocyte-enriched preparations throughout viraemia in titres comparable to those in whole blood. These results indicate that although bovine monocytes are readily infected in vitro with this strain of BTV serotype 10, infection of blood monocytes is unlikely to be responsible for the prolonged viraemia that consistently occurs in BTV-infected cattle.
Subject(s)
Bluetongue virus/growth & development , Monocytes/microbiology , Reoviridae/growth & development , Animals , Antibodies, Monoclonal , Antibody Specificity , Autoradiography , Bluetongue/blood , Bluetongue/microbiology , Bluetongue virus/immunology , Bluetongue virus/ultrastructure , Cattle , Cells, Cultured , Flow Cytometry , Fluorescent Antibody Technique , Monocytes/analysis , Monocytes/ultrastructure , Precipitin TestsABSTRACT
The acute stage of feline immunodeficiency virus (FIV) infection is characterized by a CD8+ anti-FIV response that parallels the appearance of a CD8+ subpopulation with reduced expression of the beta chain (CD8 alpha + beta lo). The relationship between the CD8 alpha + beta lo phenotype and CD8+ anti-FIV activity was examined. Flow cytometric analysis of peripheral blood mononuclear cells with anti-CD8 beta chain monoclonal antibody 117 revealed that the CD8 alpha + beta lo phenotype expanded throughout the asymptomatic infection, constituting 80%-90% of the CD8 beta + cells in long-term-infected cats. Purified CD8 alpha + beta hi and CD8 alpha + beta lo subpopulations were analyzed for anti-FIV activity in an acute infection assay. Anti-FIV activity resided principally in the CD8 alpha + beta lo population and was demonstrated in acute FIV infections, as well as in long-term asymptomatic infections. These data suggest that a unique CD8 alpha + beta lo anti-FIV phenotype arises early in infection and may play a major role in eliminating virus and maintaining the asymptomatic infection.
Subject(s)
CD8 Antigens/biosynthesis , CD8-Positive T-Lymphocytes/immunology , Feline Acquired Immunodeficiency Syndrome/immunology , Immunity, Cellular , Acute Disease , Animals , Antibody Specificity , CD8 Antigens/immunology , Carrier State , Cats , Down-Regulation , Flow Cytometry , Immunodeficiency Virus, Feline/isolation & purification , PhenotypeABSTRACT
The acute stage of feline immunodeficiency virus (FIV) infection is characterized by the appearance of a major CD8 subpopulation with reduced expression of the CD8 beta chain (CD8alpha+betalo). CD8 antiviral activity was subsequently shown to be mediated by the CD8alpha+betalo phenotype, which is the dominant CD8 phenotype in long-term infected cats. Two- and three-color flow cytometric analysis demonstrated that the CD8alpha+betalo subset is L-selectin negative (CD62L-) and has increased expression of CD44, CD49d, and CD18, consistent with an activation phenotype. The CD8alpha+betaloCD62L- cells but not the CD8alpha+betahiCD62L+ cells demonstrated strong antiviral activity in the FIV acute-infection assay. The progressive expansion of the CD8alpha+betaloCD62L- effector subset cells in FIV-infected cats parallels that seen in human immunodeficiency virus (HIV)-infected patients, suggesting that failure in homeostatic mechanisms regulating lymphocyte activation or trafficking (or both) may be a consequence of both HIV and FIV infections.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Feline Acquired Immunodeficiency Syndrome/immunology , Immunodeficiency Virus, Feline/immunology , L-Selectin/analysis , T-Lymphocyte Subsets/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cats , Immunophenotyping , Integrins/metabolism , Leukocytes, Mononuclear/immunology , Lymph Nodes/immunologyABSTRACT
A full-length feline immunodeficiency virus NCSU1 (FIV-NCSU1) genome (JSY3) was cloned directly from FIV-NCSU1-infected feline CD4+ lymphocyte (FCD4E) genomic DNA and identified by PCR amplification with 5' long terminal repeat, gag, env, and 3' long terminal repeat primer sets. Supernatant from FCD4E cells cocultured with JSY3-transfected Crandell feline kidney (CrFK) cells was used as an inoculum. Cell-free JSY3 virus was cytopathogenic for FCD4E lymphocytes but did not infect CrFK cells in vitro. To determine in vivo infectivity and pathogenesis, six young adult specific-pathogen-free cats were inoculated with cell-free JSY3 virus. Provirus was detected at 2 weeks postinfection (p.i.) and was still detectable at 25 weeks p.i. as determined by gag region PCR-Southern blot analysis of peripheral blood mononuclear cell lysates. Infectious virus was recovered from peripheral blood mononuclear cells at 6 and 25 weeks p.i., and an antibody response to FIV was detected by 4 weeks. In the acute phase of infection, JSY3 provirus was found only in the CD4+ lymphocyte subset; however, by 14 weeks p.i., the greatest provirus burden was detected in B lymphocytes. All six cats were panlymphopenic at 2 weeks p.i., CD4+/CD8+ ratios were inverted by 6 weeks p.i., and five of the six cats developed lymphadenopathy by 10 weeks p.i. To determine if the JSY3 molecular clone caused immunodeficiency similar to that of the parental wild-type FIV-NCSU1, the cats were challenged with the low-virulence ME49 strain of Toxoplasma gondii at 29 weeks p.i. Five of six cats developed clinical signs consistent with generalized toxoplasmosis, and three of six cats developed acute respiratory distress and required euthanasia. Histopathologic examination of the severely affected cats revealed generalized inflammatory reactions and the presence of T. gondii tachyzoites in multiple tissues. None of the six age- and sex-matched specific-pathogen-free cats inoculated with only T. gondii developed clinical disease. Our results suggest that the pathogenesis of the molecularly cloned NCSU1 JSY3 is similar to that of wild-type FIV-NCSU1.