Thymocytes from mice immunized against an allograft render bone-marrow cells specifically cytotoxic.

Thymocytes from C57BL mice immunized with the DBA/2 lymphoma L5178Y exert in vitro an immunologically specific cytotoxic action against the target cells in the presence of bone-marrow cells. Neither the nonimmune bone marrow nor the immune thymocytes are by themselves cytotoxic. The cells in the bone marrow which take part in the cytotoxic action adhere to glass and are sensitive to anti-macrophage serum. These bone-marrow cells can also be rendered specifically cytotoxic by exposure to the supernatant obtained from a culture of immune thymocytes with the specific target cells. The thymocytes before they are confronted with the specific target cells are very radiosensitive; however, on coming into contact with the target cells, an immunologically specific increase in RNA synthesis occurs and thereafter the thymocytes' capacity to render bone-marrow cells cytotoxic is relatively radioresistant. Two classes of immune lymphocytes occur in mice immunized with allogeneic cells, those that are capable of killing target cells directly and those that produce a factor capable of rendering macrophages (or monocytes) specifically cytotoxic. In the thymus of immune animals only the latter are found while both categories are present in the spleen and lymph nodes of immune animals.

Thymus-derived lymphocytes produce an immunologically specific macrophage-arming factor.

Spleen cells from mice immunized with an allogeneic tumor when cultured with the specific tumor cells release into the supernatant a specific macrophage-arming factor(s) (SMAF) which binds nonspecifically to macrophages from both mice and rats and renders these cytotoxic to the specific tumor cells. SMAF also binds in an immunologically specific way to the target cells. SMAF-treated target cells grow normally in the absence of macrophages but are killed in the presence of normal macrophages. Thymus-derived cells are necessary for the production of SMAF since (a) after treatment with anti-theta serum immune spleen cells fail to release SMAF; (b) spleen cells from immunized T cell-deprived mice (thymectomized as adults followed by whole body irradiation and restored with bone marrow) fail to produce SMAF on stimulation with the specific target cells. While SMAF has the properties of a cytophilic antibody, it does not belong to one of the established classes of immunoglobulin since high activity is found after column separation in a fraction having a molecular weight between 50,000-60,000 daltons.

Retrovirus-induced feline pure red cell aplasia. Hematopoietic progenitors are infected with feline leukemia virus and erythroid burst-forming cells are uniquely sensitive to heterologous complement.

Feline leukemia virus subgroup C/Sarma (FeLV-C) induces pure red cell aplasia (PRCA) in cats. Just before the onset of anemia, erythroid colony-forming cells (CFU-E) become undetectable in marrow culture, yet normal frequencies of erythroid burst-forming cells (BFU-E)- and granulocyte-macrophage colony-forming cells (CFU-GM) persist. To determine if erythroid progenitors were uniquely infected with retrovirus, marrow mononuclear cells from cats viremic with FeLV-C were labeled with monoclonal antibodies to gp70 and then analyzed with a fluorescence-activated cell sorter. Both erythroid and granulocyte-macrophage progenitors were among cells sorting positively, suggesting that infection of BFU-E alone did not result in PRCA. The results were confirmed by complement (C') lysis studies using baby rabbit or guinea pig sera as sources of C'. These studies also suggested that BFU-E from cats with PRCA were unusually sensitive to C' alone, without the addition of antibody. In further studies, we demonstrated that C' activation was via the classical pathway and that C' sensitivity was unique to BFU-E and not a property of CFU-E, CFU-GM, or progenitors that were capable of giving rise to BFU-E in suspension culture. As BFU-E from cats viremic with FeLV-A/Glasgow-1 or the Rickard strain of feline leukemia virus were not sensitive to C', this finding may relate to the pathogenesis of feline PRCA. We hypothesize that, in cats viremic with FeLV-C, the abnormal C' sensitivity of BFU-E leads to the absence of CFU-E and anemia.

Detection of complement-dependent lytic antibodies in sera from feline leukemia virus-infected cats by the chromium-51 release assay.

Sera from feline leukemia virus-infected cats lysed FL74 cells in the presence of an appropriate complement source; lysis was detected with the use of the 51 Cr release method. Serum complement from both rabbits and guinea pigs mediated lysis. Partial correlation of results was obtained from preliminary comparisons of lysis and immunofluorescence tests with the use of FL74 cells as targets.

Protection of cats against progressive fibrosarcomas and persistent leukemia virus infection by vaccination with feline leukemia cells.

Young cats (3-6 mo old) were challenged with oncogenic Snyder-Theilen feline sarcoma virus (FeSV) after vaccination with live or killed FL74 cat lymphoma cells. Compared with controls immunized with normal cat fibroblasts, the FL74-vaccinated cats exhibited increased resistance to FeSV-induced progressive primary and disseminated secondary tumors. Maximum protection was achieved by vaccination with live FL74 cells or with a low dose of freeze-thawed cells, but tumor cells inactivated by glutaraldehyde or paraformaldehyde were also effective. Infectious helper feline leukemia virus (FeLV) was detected in the blood of all cats after FeSV challenge, but the duration and magnitude of this viremia were reduced in animals that had been previously vaccinated with live, freeze-thawed, or paraformaldehyde-fixed cells. Although immunized cats were resistant to FeSV-induced tumors and FeLV viremia, no evidence was obtained to suggest that vaccination with dead cells induced detectable circulating antibody prior to challenge with oncogenic virus. After FeSV challenge, complement-dependent antibody to feline oncornavirus-associated cell membrane antigen (CDA-FOCMA) appeared at high titer in cats that were destined either to survive tumor-free or to develop small, localized, and eventually regressing tumors. Cats immunized with live FL74 cells developed CDA-FOCMA prior to challenge, and antibody appeared in these cats following an episode of transient FeLV viremia induced by virus replicating from the injected tumor cells. Therefore, apparently, a state of transient or persistent FeLV viremia regularly preceded detection of CDA-FOCMA activity. Several individually derived feline lymphoma cell lines were used as targets for CDA-FOCMA, and the results suggested that lytic activity is directed to multiple antigen determinants expressed differently by individual feline lymphomas.

Comparison of feline leukemia virus-infected and normal cat T-cell lines in interleukin 2-conditioned medium.

Following mitogen stimulation (phytohemagglutinin or concanavalin A), feline lymphocytes were maintained in medium containing feline interleukin 2. Lymphocyte lines derived from the blood of feline leukemia virus-infected cats contained cells 50 to 90% of which expressed virus antigens, but lymphocyte lines derived from uninfected cats remained virus free. Leukemia virus-positive and -negative lymphocyte lines retained total dependence upon the presence of interleukin 2 for finite life spans of 20 to 40 cell divisions. Cell-doubling times and surface properties (membrane immunoglobulin negative, guinea pig red cell rosette positive) of all T-cell lines were similar. All cat lymphocyte lines rapidly developed strong but nonspecific cytotoxic effects against a variety of established cat target lines, including normal and leukemia virus-infected fibroblasts and virus-producing lymphomas. Attempts to infect virus-negative lymphocytes with leukemia virus in vitro produced lines containing 1 to 4% of infected cells; subsequently, this level of infection remained constant. Within observation limits, the characteristics of feline leukemia virus-infected and normal cat T-cells were similar. Leukemia virus infection did not predispose target lymphocytes to exhibit properties in vitro that might be associated with preneoplastic change, such as rapid or infinite cell division or development of independence from interleukin 2 regulation.

Natural feline leukemia virus infection and the immune response of cats of different ages.

Forty-two kittens and 28 adult cats were placed as tracers in leukemia cluster environments in contact with resident cats, 30% of which were persistently infected with feline leukemia virus (FeLV). After 7 months exposure, FeLV viremia had been detected in 71% of the tracer kittens, although only 55% of these remained persistently infected; in the same period, 11% of tracer adults became infected, but by 2 years the figure reached 43%. Mean latent periods before detection of viremia were 3.4 +/- 1.8 (S.D.) and 13.0 +/- 5.9 months for kittens and adults, respectively. First detection of FeLV infection was accompanied by a sharp although transient drop in peripheral white blood cell numbers, and infection onset triggered the humoral immune response which was comprised of separate antibodies with virus-neutralizing and tumor lysis activities. High titers of virus-neutralizing antibody appeared in transiently viremic cats immediately following elimination of viremia; this antibody was rarely detected in cats that remained persistently viremic. Lytic complement-dependent antibody to feline oncornavirus-associated cell membrane antigen appeared in most cats 1 to 2 weeks after FeLV infection was first detected, and subsequently high titers of this antibody remained in both transiently and persistently infected cats. If the rate of FeLV infection was summarized by using viremia and/or antibody appearance, then 95% of the kittens became infected within 1 year and 61% of the adults within 2 years. Adult cats are, therefore, susceptible to FeLV infection following long-term natural exposure, and their apparent resistance cannot be attributed to a protective humoral immune response that developed immediately after exposure commenced.

Presence of fucolipid antigens with mono- and dimeric X determinant (Lex) in the circulating immune complexes of patients with adenocarcinoma.

A series of fucosylated glycosphingolipids with the Lewisx (Lex) determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) have been shown to accumulate in human adenocarcinomas. Lex glycolipids were eluted from Protein A-silica columns over which plasma from patients with adenocarcinoma had previously been perfused. The fact that Protein A has strong affinity for IgG and IgG-immune complexes suggested that the Lex antigens isolated from Protein A eluates were complexed with IgG. Lewisx antigen eluted from Protein A columns banded in the immune complex-enriched region (below IgG) of neutral sucrose density gradients. A modified Raji cell assay and an anticomplement C1q enzyme-linked immunosorbent assay were also used for measurement of Lex antigen associated with C3- and C1q-CIC, respectively. Following affinity purification of Lex-IgG complexes and subsequent dissociation of these immune complexes, human antibodies were isolated which reacted with purified glycosphingolipids containing Lex. Levels of Lex-IgG complexes were found to be 2- to 5-fold higher in eluates of Protein A-silica columns perfused with plasma from adenocarcinoma patients compared to eluates from columns perfused with plasma from healthy individuals and patients with other cancers. These assays may prove to be of diagnostic and/or prognostic significance in adenocarcinoma.

Feline immunodeficiency virus and feline leukemia virus infections and their relationships to lymphoid malignancies in cats: a retrospective study (1968-1988).

Sera from 353 cats with naturally occurring feline leukemia virus (FeLV) infection were collected in Boston, Los Angeles, New York City, and Seattle between 1968 and 1988. These sera were retrospectively assayed by enzyme-linked immunosorbent assay for antibodies to feline immunodeficiency virus (FIV). Fifty-one (14.4%) of the FeLV-positive sera had antibodies to FIV, indicating dual oncovirus and lentivirus infections. FIV infections were confirmed by Western blot analysis, antibodies against the 15 and 27 kDa proteins being used as definitive markers. FIV infection was diagnosed in one cat sampled in 1968 and in eight other cats sampled before 1975 in New York City. Illnesses exhibited by coinfected cats were similar to those of cats infected with FeLV only. Two unrelated cats with multicentric fibrosarcomas were found to be simultaneously infected with FIV, FeLV, and feline sarcoma virus. FIV was less contagious than FeLV in 73 cats residing in an exposure household between 1977 and 1980 as determined by evaluation of sera collected sequentially. In this household, 15 resident cats became FeLV infected whereas no cats contracted FIV infection. Comparison of serologic results from 53 cats with leukemia/lymphoma and matched controls confirmed a strong correlation between FeLV viremia and leukemia/lymphoma. A significant correlation between FIV infection and lymphoproliferative malignancies was also found independent of FeLV infection.

Complement origin determines lytic activity of antibodies to nucleated target cells. Comparison of common complement sources.

The effects of complement from different species of animals were measured in a variety of antibody assays by using 51Cr-labelled target cells. Sheep antibodies were measured in samples of lymph and serum obtained from animals immunised with allogeneic lymphocytes, transplanted with allogeneic kidney grafts, or immunised with mouse tumour cells. Mouse (C57BL) antibodies were measured after immunisation with allogeneic tumour (P815) or after transplantation of allogeneic thyroid grafts (BALB/c). Different species of complement gave quantitative and sometimes qualitative differences when used to assay the same samples of antibody. In all systems tested, rabbit complement caused lysis of target cells at low antibody concentration when guinea pig and rat complements gave negative results, in some antibody-target cell combinations sheep complement was as effective as rabbit complement in mediating lysis. The different complement sources showed no selective lytic affinity for either IgM or IgG1 antibody subclasses purified from immune sheep lymph. Lysis of P815 target cells occurred more quickly when mediated by rabbit or sheep complement than when mediated by guinea pig or rat complement. Complement-dependent lysis sometimes occurred in systems where antibody, target cells and complement were obtained from the same species.

Rejection of allogeneic tumor cells growing in mouse cerebrospinal fluid. Functional analysis of the inflammatory process.

Mice injected intracerebrally (i.c.) with mastocytoma cells reject the tumor, which grows rapidly in cerebrospinal fluid (CSF), within 7 or 8 days. The characteristics of the inflammatory exudate have been examined by several different, functional criteria. Potent cytotoxic T-lymphocyte populations are readily demonstrated in CSF, and both phagocytic and non-phagocytic cells capable of mediating antibody-dependent cell-mediated cytotoxicity are also present. However, no tumor-specific antibody was detected in CSF of mice that had recently rejected the tumor, using sensitive complement-dependent and lymphocyte-dependent cytotoxic assay systems. The process of cell-mediated tumor rejection does not obviously compromise the blood-CSF barrier to immunoglobulins.

Retroviral vector-transduced cells expressing the core polyprotein induce feline immunodeficiency virus-specific cytotoxic T-lymphocytes from infected cats.

The core polyprotein of feline immunodeficiency virus (FIV) was expressed in primary feline T-lymphocytes using a retroviral vector. These cells were used as antigen-presenting stimulator cells (APSC) for the in vitro induction of cytotoxic T-lymphocytes (CTL) from feline peripheral blood mononuclear cells (PBMC). CTL from 4 cats chronically infected with the Petaluma strain of FIV specifically lysed autologous FIV-infected targets in an MHC-restricted manner. The CD8 phenotype of more than 70% of the induced effector cells (97% for cells from one cat) was consistent with MHC class I-restricted cytotoxicity. In addition, it was possible to detect low levels of core polyprotein-specific lysis from effector cells of two of the FIV-infected cats. When observed, the level of lysis, measured as a percentage of specific 111In release, was lower for the transgenic gag-expressing targets than for FIV-infected targets. The difference in killing may reflect the low level of core CTL were not detected in either PBMC stimulated with cells transduced by a retroviral vector without the FIV gag sequence or PBMC from an uninfected cat stimulated with autologous transgenic APSC. The detection of FIV-specific CTL from infected cats following stimulation with transgenic APSC suggests a role for retroviral vectors in determining CTL specific for individual lentiviral proteins in protective immunity.

FIV infection of IL-2-dependent and -independent feline lymphocyte lines: host cells range distinctions and specific cytokine upregulation.

We have analyzed the ability of three molecular clones of feline immunodeficiency virus (FIV) and an ex vivo variant to infect nine distinct specific-pathogen-free feline cell lines in tissue culture. The purpose of these studies was to elucidate mechanisms by which host cells regulate the level of virus infection and expression and to assess host cell cytokine responses to virus infection. Cells used for the analyzes included four IL-2-dependent continuous T-cell lines (104-C1, 104-C7, MCH5-4 and DB FeTs) which arose from long-term passage, followed by limiting dilution cloning of peripheral blood mononuclear cells (PBMCs); two IL-2-independent T-cell lines (104-C1DL and MCH5-4DL) which originated from two of the IL-2-dependent lines, 104-C1 and MCH5-4; respectively; Crandell feline kidney cells (CrFK); G355-5 brain-derived glial cells; and the T-cell lymphoma line, 3201. Cells were infected with FIV-PPR, FIV-34TF10, FIV 34TF10orf2rep, and a variant arising from FIV-PPR during ex vivo passage on 104-C1DL cells, termed FIV-PPRglial. Infection of the IL-2-dependent T-cell line, 104-C1, by FIV-PPR resulted in the specific and distinct upregulation of cytokine expression. In particular, these cells doubled their expression of the pleiotropic cytokines, interleukin-4 and interleukin-12 after FIV infection. Interferon-gamma production also increased after infection with FIV whereas, TNFalpha expression remained constant. Also, a marked upregulation of MHC class II expression was noted post infection of MCH5-4 and 104-C1 cells with FIV-PPR. Similar results were obtained after infection with FIV-34TF10orf2rep, indicating that the upregulation of cytokine expression is not an isolate-specific phenomenon. Changes in cytokine and class II expression are similar to various reports for the in vivo cytokine alterations in FIV, SIV and HIV infections. The ex vivo infection of these cell lines offers amanipulable system to examine the mechanism(s) by which lentiviruses alter cytokine expression.

Feline immunodeficiency virus (FIV)-associated lymphoma: a potential role for immune dysfunction in tumourigenesis.

To determine the potential role of immune dysfunction in feline immunodeficiency virus (FIV)-associated lymphomagenesis, we present the results of immunological monitoring during the chronic phase of experimental FIV infection in two cats which subsequently developed lymphoma. In one cat, C1, cell-mediated immunity was depressed throughout the monitoring period but particularly from 125-200 weeks post-infection (pi), when this cat demonstrated profoundly impaired lymphocyte blastogenesis and markedly increased interleukin-1 (IL-1) production compared to age-matched, uninfected control cats. Lymphocyte function in the other cat, C2, was preserved to a greater degree. Alterations in the levels of immunoglobulin isotypes M, A and G in CD4+-, CD8+- and CD21+-lymphocyte sub-sets were demonstrated in both cats. Southern blot analysis revealed the presence of integrated FIV-provirus in tumour DNA from C2 but not C1 indicating a possible direct role for the virus in the former case only. In this study we have characterised, for the first time, the FIV-induced immune dysfunction in cats which developed lymphoma, demonstrating potential indirect mechanisms of tumourigenesis.

Interaction of acute feline herpesvirus-1 and chronic feline immunodeficiency virus infections in experimentally infected specific pathogen free cats.

Cats with or without chronic feline immunodeficiency virus (FIV) infection were exposed to feline herpesvirus, type 1 (FHV-1). FIV infected cats became sicker than non-FIV infected cats and required more supportive treatment. However, there were no differences in the length of their illness or in the levels and duration of FHV-1 shedding. FHV-1 infection caused a transient neutrophilia at Day 7 with a rapid return to preinfection levels. The neutrophilia coincided with a transient lymphopenia that was accompanied by a decline in both CD4+ and CD8+ T-lymphocytes. A brief decrease in the CD4+/CD8+ T-lymphocyte ratio occurred at Day 14 in both FIV infected and non-infected cats. This decrease was mainly the result of an absolute and transient increase in CD8+ T-lymphocytes. CD4+ and CD8+ T-lymphocyte numbers and CD4+/CD8+ T-lymphocyte ratios returned to baseline within 4-8 weeks in both FIV infected and non-infected cats. FIV infected cats produced less FHV-1 neutralizing antibodies during the first 3 weeks of infection than non-FIV infected animals. The IgM FHV-1 antibody response was depressed in FIV infected cats whereas the IgG antibody response was unaffected. FHV-1 infection evoked a comparable transient loss of lymphocyte blastogenic responses to concanavalin A and pokeweed mitogen in both FIV infected and non-infected cats. However, response to pokeweed mitogen took longer to return to normal in FIV infected animals. Lymphocytes from FIV infected cats had a greater and more sustained proliferative response to FHV-1 antigen than non-FIV infected cats. The ongoing IgG antibody response to FIV was not affected by FHV-1 infection.

Fatal enteritis associated with coronavirus infection in cats.

This report describes five cases of naturally occurring feline coronavirus enteritis. The affected animals, aged 2 months to 7 years, had a clinical history of intestinal symptoms, including diarrhoea or vomiting, or both. They exhibited variable histological changes in the epithelium of the small intestine, ranging from degeneration of single cells and detachment of groups of cells from the villous tips to regenerative processes of the crypt epithelia. Post-mortem diagnosis was based on the immunohistochemical demonstration of coronavirus antigen within intestinal epithelial cells and on the electron microscopical demonstration of coronavirus particles in the faeces. In addition, one animal was immunohistochemically positive for antigens of feline leukaemia virus (FeLV) and exhibited intestinal changes consistent with FeLV-associated enteritis. Two cats were tested serologically for feline immunodeficiency antibodies, with negative results. The findings indicate that natural coronavirus infection is a potential cause of severe enteritis in juvenile and adult cats.

Variable expression of major histocompatibility complex class II in the domestic cat.

This paper describes the characterisation of six independently produced monoclonal antibodies (mAbs) specific for non-polymorphic determinants of feline major histocompatibility complex (MHC) class II. One mAb is an anti-sheep class II which cross-reacts with the cat and five have been produced in response to immunisation with purified feline immunodeficiency virus. Despite their independent source all the mAbs have identical reactivities, immunoprecipitating two complex groups of polypeptides of M(r) 33 to 36.000 (MHC class II alpha chains) and M(r) 28 to 31,000 (MHC class II beta chains). Immunoblot analysis showed them to be beta chain-specific. One and two-dimensional electrophoresis revealed the complexity of feline class II mass and charge and implied the expression of multiple class II loci in the cat. Furthermore, it was demonstrated that distinct cell populations expressed a distinct range of class II variants. This suggesting either the differential expression or the distinct post-translational modification of lymphocytes from different sites. The mAbs have also been used for the detailed examination of the cellular distribution and tissue localisation of MHC class II in the cat.

Severe neutropenia associated with griseofulvin therapy in cats with feline immunodeficiency virus infection.

Griseofulvin administration was associated with the development of absolute neutropenia in six of seven (86%) cats with feline immunodeficiency virus (FIV) infection. The neutropenia was severe (less than 400 neutrophils/microliter) in four of the six affected cats, and one cat died from sepsis. Neutrophil counts returned to baseline values within 15 days after drug withdrawal in all surviving cats. No symptoms or hematologic abnormalities were observed in four normal (FIV-seronegative) cats treated with the same lot of griseofulvin at equivalent doses. Neutropenia recurred in two of two FIV-seropositive cats upon griseofulvin rechallenge. Cats with FIV infections appear to be at increased risk for griseofulvin-associated neutropenia. This phenomenon may be analogous to the increased frequency of antibiotic-induced neutropenias observed in humans infected with the human immunodeficiency virus.

Complement "specificity" and interchangeability: measurement of hemolytic complement levels and use of the complement-fixation test with sera from common domesticated animals.

The results from studies to measure lytic complement (C') in sera of different animal species were reviewed. The traditional system, using sheep red blood cells (RBC) and rabbit antibody, was confirmed as the most sensitive to measure C' levels in man, monkey, dog, guinea pig, and rat serums. Sera C' from horse, cow, and sheep were found to be best assayed using rabbit RBC, whereas C' from goat, cat, and rabbit were best assayed with human RBC. Antibodies and C' from the same species usually mediated lysis of foreign RBC, but this lysis occurred more readily with some RBC targets than with others and may be associated with the presence of natural antibodies in the test sera. The effects of the species origin of a C' source in immunologic reactions in vitro and in vivo are discussed.

Increased sensitivity of canine sarcoma cells to lysis with alloantiserum and complement after pretreatment of cells with doxorubicin HC1.

The 51Cr release assay was used to detect lysis of canine sarcoma cells incubated in vitro with alloantiserum and complement. Preincubation of the target cells with the anti-cancer agent doxorubicin-HCl increased the sensitivity of the cells to antibody-mediated lysis. The effect was dependent upon drug dose and was not reproduced by treatment with methotrexate, cytarabine, or dactinomycin. The observation suggests a possible ancillary mode of action for doxorubicin-HCl on neoplastic cells.