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Gamma delta T-lymphocytes and anti-heat shock protein reactivity in bovine leukaemia virus infected cattle
Abstract
Bovine leukemia virus (BLV) induces a chronic infection in cattle that may result in persistent lymphocytosis (PL) and, sometimes, enzootic bovine leukosis. The cellular and humoral immune responses of the host following infection have been extensively investigated but little is known about the involvement of gamma delta T-cells in BLV pathogenesis. The affluence of these cells in cattle, and particularly in the peripheral blood of young ruminants, may suggest a particular role for them in defense mechanisms. In this study we have examined circulating gamma delta lymphocytes that express workshop clusters 1 (WC1) and 2 (WC2). In healthy cattle the WC1 cell count tends to decrease with age and adult cattle blood has statistically lower numbers (19.0 +/- 6.6%) than that of young animals (40.1 +/- 7.2%). However, in the blood of BLV-seropositive adult cattle and mainly in BLV+ PL+ animals the population of WC1 cells is elevated compared with uninfected animals (P < 0.007). Likewise, the WC2 cells count is increased (P < 0.01) in BLV+PL+. Furthermore, we have investigated whether BLV infection up-regulates the expression of heat shock proteins (HSP) which in turn could augment the humoral response. Anti-HSP70 activity was examined in the sera of 34 BLV-infected cattle and 40 healthy controls by ELISA. Significantly higher activities (P < 0.001) were observed in BLV-infected cattle.
... Anti-HSP70 activity was examined in the sera of 34 BLV-infected cattle and 40 healthy controls by ELISA. Significantly higher activities (p<0.001) were observed in BLVinfected animals [60]. Several studies have proposed the implication of HSP in prion diseases. ...
Heat shock proteins (HSPs) are the molecular chaperones, that are not only expressed during the normal growth process of cell cycle consecutively, but also get induced in cells during various stress conditions produced by cellular insult, environmental changes, temperature, infections, tumors etc. According to their molecular weight and functions, HSPs are divided into five major families. HSP90, HSP70, HSP60 and HSP100 are the most studied members of the family. Experimental studies have proved that overexpression and/or inhibition of HSPs play an important role in maintaining the tolerance and cell viability under above-described stress conditions. HSP90 is found to be a promising candidate for the diagnosis, prognosis and treatment of cancer. Similarly, HSP70, HSP60 and small HSPs experimentally and clinically have potential for the treatment of neurodegenerative disease, ischemia, cell death, autoimmunity, graft rejection, etc. In a way, exploring, the cytoprotective and immunoregulatory role of HSPs can open a new avenue for the drug discovery and treatment of critical diseases.
... In another study on influenza virus, it was shown that HSP 70 interacts with the ribonucleoprotein complex and inhibits its nuclear export leading to the inhibition of virus production [21]. We and other reported the presence of natural auto anti-HSP antibodies in the sera of normal and retrovirus infected human and animals [22][23][24][25] but little is known concerning their role. ...
Adult T cell leukemia is a fatal malignant transformation caused by the human T-cell lymphoptropic virus type I (HTLV-I). HTLV-I is only associated with the development of this disease in a small percentage of infected individuals. Using two rabbit transformed T-cell lines; RH/K30 (asymptomatic) and RH/K34 (leukemogenic), we have investigated the expression of heat shock proteins (HSP) 90 and 70 and the role of anti-HSPs antibodies on virus production. HSPs surface expression was higher on RH/K34 than RH/K30 cells. Heat treatment of cells increased the expression of HSPs proteins and virus production; HSPs augmentation was stabilized after 12 h and virus production reached a maximum between 8 h–12 h then returned to normal level after 24 h of culture. Incubation of cells only with rabbit anti-HSP 70 antibodies prevented virus production specifically in the leukemogenic cell line. The results indicate a relationship between HSP 70 and virus production.
A single and rapid method to obtain an antigenic fraction of excretory-secretory antigens (ESAs) from Fasciola hepatica suitable for serodiagnosis of fascioliasis is reported. The procedure consists in the negative selection of F. hepatica ESAs by hydroxyapatite (HA) chromatography (HAC; fraction HAC-NR) followed by antigen precipitation with 50% ammonium sulphate (AS) and subsequent recovery by means of a Millex-GV or equivalent filter (Fi-SOLE fraction). Tested in indirect ELISA, the Fi-SOLE antigens detected natural infections by F. hepatica with 100% sensitivity and 98.9% specificity in sheep, and 97.7% sensitivity and 97.7% specificity in cattle, as determined by ROC analysis. The SDS-PAGE and proteomic nano-UHPLC-Tims-QTOF MS/MS analysis of fractions showed that the relative abundance of L-cathepsins and fragments thereof was 57% in fraction HAC-NR and 93.8% in fraction Fi-SOLE. The second most abundant proteins in fraction HAC-NR were fatty-acid binding proteins (11.9%). In contrast, free heme, and heme:MF6p/FhHDM-1 complexes remained strongly bond to the HA particles during HAC. Interestingly, phosphorylcholine (PC)-bearing antigens, which are a frequent source of cross-reactivity, were detected with an anti-PC mAb (BH8) in ESAs and fraction HAC-NR but were almost absent in fraction Fi-SOLE.
A single and rapid method to obtain an antigenic fraction of excretory-secretory antigens (ESAs) from Fasciola hepatica ESAs suitable for serodiagnosis of fascioliasis is reported. The procedure consists in the negative selection of F. hepatica ESAs by hydroxyapatite (HA) chromatography (HAC; fraction HAC-NR) followed by antigen precipitation with 50% ammonium sulphate (AS) and subsequent recovery by means of a Millex-GV or equivalent filter (Fi-SOLE fraction). Tested in indirect ELISA, the Fi-SOLE antigens detected natural infections by F. hepatica with 100% sensitivity and 98.9% specificity in sheep, and 97.7% sensitivity and 97.7% specificity in cattle, as determined by ROC analysis. The SDS-PAGE and proteomic nano-UHPLC-Tims-QTOF MS/MS analysis of fractions showed that the relative abundance of L-cathepsins and fragments thereof was 57% in fraction HAC-NR and 93.8% in fraction Fi-SOLE. The second most abundant proteins in fraction HAC-NR were fatty-acid binding proteins (11.9%). In contrast, free heme, and heme:MF6p/FhHDM-1 complexes remained strongly bond to the HA particles during HAC. Interestingly, phosphorylcholine (PC)-bearing antigens, which are a frequent source of cross-reactivity, were detected with an anti-PC mAb (BH8) in ESAs and fraction HAC-NR but were almost absent in fraction Fi-SOLE.
The study aimed to explore the serum levels of HSP70 and identify its possible association with serum cortisol, thyroid hormones, and acute-phase protein concentrations in cattle naturally infected with foot-and-mouth disease (FMD) virus. After the FMD outbreak in an organized dairy cattle farm in India, blood samples were obtained from clinically infected (n = 40) and apparently healthy (n = 30) animals. Samples were processed and tested by an in-house DIVA assay for confirmation of FMD infection. Serum was analyzed for HSP70, cortisol, T4, T3, haptoglobin, and serum amyloid A by enzyme-linked immunosorbent assay (ELISA). HSP70 concentrations were significantly higher in the serum of clinically infected cattle (p < 0.01) than the healthy group. To the best of our knowledge, this is the first report describing the elevated serum levels of HSP70 under infectious diseases of bovines. Cortisol (p < 0.05), haptoglobin (p < 0.001), and serum amyloid A (p < 0.05) concentrations also markedly increased in the diseased animals; however, no differences (p > 0.05) were found in T4 and T3 levels between healthy and infected cattle. Elevated HSP70 concentration correlated positively with high cortisol (p < 0.05) and haptoglobin (p < 0.001) levels suggesting an essential link between these acute events during clinical infectious phase of FMD.
Bovine leukemia virus (BLV) causes enzootic bovine leukosis (EBL). The BLV genome encodes Tax protein, a transcriptional activator of viral gene expression that binds to the BLV long terminal repeat (LTR). Heat shock factor 1 (HSF1) is a known regulator of the heat shock response proteins, including heat shock proteins. In the present study, the BLV LTR was investigated for interaction of heat shock element (HSE) with HSF1 and the viral Tax protein. It could be confirmed that a functional HSE is well conserved in different BLV strains. The LTR transcriptional activity, as measured by luciferase reporter assay, was upregulated by bovine HSF1 - without Tax expression - in feline CC81 cells. The HSF1 activated LTR transcription by binding to the HSE. LTR-activation was lost upon HSE removal from the LTR and upon expression of a mutant HSF1 lacking the DNA-binding domain. We conclude that BLV LTR is activated to a basal level by host transcriptional factor HSF1, but without Tax protein involvement.
The research covered 66 Black-and-White breed heifers originating from one herd. Tests were performed in the 3rd, 4th, 5th and 7th month of life of the heifers. Diagnosis of enzootic bovine leukosis was confirmed using the PCR test and ELISA. The profile of peripheral blood lymphocytes was determined with the use of immunoflourescence method with monoclonal antibodies. The results suggest an incomplete expression of the immunological function of the γ-lactalbumin gene product in heifers in their first months of life. In conclusion, the results obtained indicate that the natural BLV infection and the age of heifers and interaction between these factors influence the population number of CD 19+ B lymphocytes.
The article presents the results of research done to date on the variability of expression of superficial markers on leukocytes in cattle infected with bovine leukemia virus (BLV). Studies carried out so far show that these changes mainly concern B lymphocytes, and, to a lesser degree monocytes, macrophages and T lymphocytes. The article also describes the role of flow cytometry as a quick and precise method for assessing immunophenotype cells in the process of leukemia in cattle.
Immune responses were examined in cattle between 3-5 years after experimental inoculation with bovine immunodeficiency virus (BIG) and/or bovine leukemia virus (BLV). Lymphocyte proliferative responses to Con A or to allogeneic lymphocytes with foreign major histocompatibility complex molecules (allo MHC) were determined by 3H-thymidine incorporation assays. Antigen-specific antibody and lymphocyte proliferative responses were measured following vaccination with tetanus toxoid (TT) and bovine herpes virus-1 (BHV-1). Lymphocytes from BIV-infected cattle had significantly (p<0.05) reduced proliferative responses to Con A, but responses to allo-MHC and TT did not differ from those of uninfected controls. BIV infection also had little effect on TT-specific antibody responses in vivo. In contrast, BLV-infected cattle had significantly increased secondary antibody responses to vaccination with TT, as well as enhancement of antibody responses to BHV-1. Co-infection with BIV did not alter the BLV effect, suggesting a lack of significant interaction between the two viruses in vivo. Numbers of circulating mononuclear cells were also higher in BLV-infected cattle, which was attributable to increases in both T and B cell numbers. Unstimulated lymphocytes from BLV-infected cattle had significantly increased spontaneous uptake of 3H-thymidine in vitro. When differences in counts per minute were analyzed, lymphocytes from BLV-infected cattle had slightly increased proliferative responses to Con A, but no consistent alternations in responsiveness to allo-MHC, TT, or BHV-1. The observed increase in antibody responses to non-BLV antigens suggests that at least in clinically asymptomatic cattle, BLV infection may cause a non-specific B cell activation.
An experimental model of chronic infection with bovine leukemia virus (BLV) was established in young calves within a relatively short time. In the sera of all infected calves, precipitating antibodies were detected within 5 weeks after infection but upon disease progression pattern of cellular profiles varied. Three calves exhibited transient lymphocytosis 3-5 weeks after infection, two became persistent lymphocytotic (PL+) by that time and one stayed non-lymphocytotic (PL-) for 11 weeks and became PL+ after 4.5 months. Eventually all infected calves became PL+ by the end of the experiment, 6-12 months after infection. Increase of total counts of peripheral blood mononuclear cells (PBMC) related to polyclonal expansion of B-cells. The latter was assessed in all infected calves where the expansion of CD5-bearing cells (B+ CD5+) correlated with increase or decrease of total PBMC counts. Other cell populations such as CD4 and CD8 were also affected. Percentages decreased by 5 weeks after experimental infection to about half their original values though actual cell numbers stayed relatively stable. The experimental model we established compared well with field cases of naturally BLV-infected cattle and thus permitted the investigation of the disease at early stages of infection.
Understanding the immunological function of bovine gammadelta T cells is essential for evaluating their role in the response to infectious agents and for determining the potential of targeting this population with vaccines. This study examined the age dependent changes of circulating CD2(+) and CD2(-) gammadelta T cells as well as differences in the expression of activation markers between these two populations. Changes in activation marker expression following vaccination with Vira Shield 5 are also discussed. CD62L was expressed on all CD2(-) gammadelta T cells but only a subset of CD2(+) gammadelta T cells and following vaccination there was a significant decrease in the percentage of CD2(-)/CD62L(+) gammadelta T cells but not CD2(+)/CD62L(+) gammadelta T cells. Both CD2(-) and CD2(+) gammadelta T cells consistently expressed high levels of CD44. The majority of both CD2(-) and CD2(+) gammadelta T cells also expressed CD45R, however, more of the CD2(-) cells were CD45R(neg/lo). Following vaccination there was a significant decrease in the percentage of CD2(-) and CD2(+) gammadelta T cells that expressed CD44 and CD45R. These data indicate significant differences in activation expression on CD2(-) and CD2(+) gammadelta T cells, which adds to the growing evidence that there may be functional as well as phenotypic differences between these two populations of bovine gammadelta T cells.
We report the complete 8714-nucleotide sequence of the integrated bovine leukemia virus genome and deduce the following genomic organization: 5' LTR-gag-pol-env-pXBL-3' LTR, where LTR represents a long terminal repeat and pXBL represents a region containing unidentified open reading frames. This genomic structure is similar to that of human T-cell leukemia virus. The LTR contains a putative splice donor site in the R region. The gag gene encodes a precursor protein with the form NH2-p15-p24-p12-COOH. The NH2- and COOH-terminal regions of the pol product show stronger homologies with those of avian, rather than murine, type C retrovirus, and its structure is identical to that of avian virus. The env gene encodes a surface glycoprotein (gp51) and a transmembrane protein (gp30). In contrast to the pol product, the gp30 shows stronger sequence homology with a murine, rather than avian homologue, indicating the chimeric nature of the bovine leukemia virus genome. Comparisons of the best conserved pol sequences and overall genomic organizations between several major oncoviruses allow us to propose that bovine leukemia and human T-cell leukemia viruses constitute a group, designated as type "E," of Oncovirinae.
Eight human T cell leukemia virus type I (HTLV-I)-infected cell lines were derived in vitro from peripheral blood mononuclear
cells of 8 rabbits. Each rabbit was then inoculated with its own HTLV-I-transformed cells, after which all but 1 rabbit had
anti-heat-shock protein (hsp) antibodies in sera. Cell line RH/K34, which failed to raise a response to hsp70, caused lethal
leukemia when >2 00D7; 108 live cells were injected into unrelated outbred rabbits. Rabbits injected with cell-free virus isolated from RH/K34 cells
produced anti-hsp70 antibodies and became infected but developed no fatal disease. ELISA inhibition and flowcytometry analyses
indicated that hsp molecules are expressed on the surface of RH/K34 and RH/K30, a nonlethal HTLV-I cell line used for comparison;
surface hsp expression does not occur normally. Two proteins of ∼72 and 93 kDa were detected by Western blot in extracts of
RH/K30 cells. Presence of anti-hsp70 antibodies correlated with resistance to lethal doses of live RH/K34 cells, suggesting
that hsp immunity may influence the outcome of RH/K34 pathogenicity.
We have investigated the induction of heat shock proteins (HSPs) in mice infected with vaccinia virus. Vaccinia virus replicates to high levels in the ovaries of infected mice and causes a significant inhibition of host cell DNA, RNA, and protein synthesis. Many HSPs are constitutively expressed in murine ovarian tissue at low levels, consistent with their obligatory role in normal physiological events. In contrast with these events, HSP expression was augmented in virus-infected mouse ovaries 6 days postinfection. In particular, there was a dramatic increase in the expression of a protein identified as the inducible 72-kDa HSP. Analysis of cellular mRNA confirmed this protein to be the major mouse inducible HSP70 and demonstrated its presence within virus-infected cells. Hence, we have demonstrated the expression of stress proteins during poxvirus infection in vivo.
After a review of earlier work, an improved technique for the preparation of the antigen used in the agar gel immuno-diffusion test is described.
Recently emerging evidence indicates that the heat shock proteins (HSPs) gp96, hsp90, and hsp70 associate with antigenic peptides derived from cellular proteins. This evidence forms the basis of the following two hypotheses: 1) that HSPs constitute a relay line in which the peptides, after generation in the cytosol by the action of proteases, are transferred from one HSP to another, until they are finally accepted by MHC class I molecules in the endoplasmic reticulum, and 2) that the binding of peptides by HSPs constitutes a key step in the priming of cytotoxic T lymphocytes (CTLs) in vivo. The following chain of events is suggested: HSPs are released from virus-infected cells or tumor cells in vivo during lysis of cells during infection or by the action of antibodies or nonspecific effectors. The HSPs, which are now complexed with antigenic peptides derived from the cognate cells, are taken up by macrophage or other specialized antigen-presenting cells, possibly by a receptor-mediated mechanism. The HSP-borne peptide is then routed to the endogenous presentation pathway in the antigen-presenting cell and is displayed in the context of that cell's MHC class I, where it is finally recognized by the precursor CTLs. Thus it is suggested that, as with antigen presentation by MHC class II molecules, presentation by MHC class I molecules is also carried out primarily by the host antigen-presenting cells. This mechanism explains the phenomenon of cross-priming and has implications for the development of immunological strategies against cancer and infectious diseases.
Investigations were conducted to determine the mechanisms that account for differences in the responses of BoCD4+ lymphocytes from mammary gland secretions (MGS) in healthy cows and in cows with Staphylococcus aureus infection. The proliferative response to lectins and S. aureus antigens of mammary gland lymphocytes from healthy, S. aureus immunized cows was less than the response of peripheral blood lymphocytes. The lower responses of mammary gland lymphocytes were attributable both to less efficient antigen presentation by mammary gland antigen-presenting cells (APC) than by peripheral blood APC, and to lower responsiveness of mammary gland lymphocytes to lectins and antigen.In addition, the proliferative response of infected mammary gland lymphocytes was less than the response of uninfected mammary gland lymphocytes. This difference resulted from decreased proliferation of BoCD4+ lymphocytes in infected MGS. Flow cytometric analysis revealed that infected MGS contained increased numbers of BoCD8+ cells which coexpressed an activation molecule, ACT2, relative to BoCD8+ cells from uninfected MGS. Removal of BoCD8+, ACT2+ lymphocytes resulted in increased antigen responsiveness by lymphocytes from infected mammary glands. Also, when purified BoCD4+ lymphocytes were stimulated with antigen in the presence of varying numbers of ACT2+, BoCD8+ lymphocytes, antigen responsiveness was decreased in a dose-related manner.These data demonstrate that hyporesponsiveness of mammary gland lymphocytes to lectins and S. aureus antigen is, in part, mediated by activated BoCD8+ lymphocytes and suggest that this population enhances persistent intramammary infection by S. aureus.
Peripheral blood T-cell subsets were obtained from an experimentally sensitized bovine and from nine bovines naturally infected with Mycobacterium paratuberculosis and tested for their ability to respond to antigen. It was determined that following antigen challenge, proliferative responses followed a biphasic pattern: an initial CD4+ proliferative response, a period of anergy, and a final response governed by gamma/delta T lymphocytes. The anergic phase was characterized by a dramatic drop in peripheral blood CD4+ cells; the nature of the non-responsiveness could not be determined. The anergic phase was followed by increased proliferative responses of non-MHC restricted gamma/delta T lymphocytes. Although CD4+ cells had the ability to proliferate in response to M. paratuberculosis antigens in the absence of gamma/delta T cells, antigen-primed CD4+ lymphocytes failed to incorporate [3H]-thymidine in the presence of gamma/delta T cells and M. paratuberculosis antigen. It was concluded that M. paratuberculosis-specific gamma/delta T lymphocytes have immunoregulatory function and exhibit cytotoxic activity against antigen-primed CD4+ helper cells. The data suggest that the inability of effector cell populations to prevent intracellular proliferation of M. paratuberculosis may be a result of cytotoxic killing of the T helper lymphocyte population required for macrophage activation.
The study of immunity to infectious agents has proved valuable for analysing the role of T-cell subsets. This was true when Zinkernagel and Doherty demonstrated MHC restriction using viral and bacterial systems, and was re-emphasized at a recent meeting, where data were presented on the role of T cells and cytokines in viral, bacterial, protozoan and helminth infections. This report summarizes some of the highlights.
The 14 mAbs representing workshop cluster 1 recognise a 215/300 kDa antigen expressed on a subpopulation of lymphocytes which express low levels of CD5 but are negative for other B and T cell markers defined by workshop antibodies. Separate studies with cDNA probes for bovine CD3 and T cell receptor indicate that these lymphocytes are gamma/delta T cells. It is of note that the different mAbs react with varying proportions of this cell population, suggesting that the antigen undergoes considerable post-translational modification. A further two mAbs, designated workshop cluster 2, react with a 37/47 kDa heterodimeric molecule expressed in a subpopulation of the WC1+ cells and on an additional small population of T lymphocytes. The cell populations recognised by the two mAbs are different although they overlap in some animals. It is suggested that these mAbs may be specific for T cell receptor molecules.
The lymphoid systems of sheep and cattle contain a large number of gamma delta T cells, in striking contrast to the lymphoid systems of humans and mice. In neonatal animals particularly, these cells comprise the predominant fraction of T cells in the blood. Here Wayne Hein and Charles Mackay discuss what is currently known about the ontogeny, phenotype, tissue distribution and function of gamma delta T cells in ruminants. There are a number of interesting molecular features that characterize ruminant gamma delta T cells, but these do not entirely explain the high frequency of use of the gamma delta T-cell receptor in these animals. Studies on sheep, cattle or other animals that preferentially use gamma delta T cells should provide insights into the biological significance of the existence of two distinct forms of the T-cell receptor.
Most T cells recognize antigen through the T-cell antigen receptor (TCR)alpha beta-CD3 complex on the T-cell surface. A small percentage of T cells, however, do not express alpha beta but a second type of TCR complex designated gamma delta (ref. 2). Unlike alpha beta+ lymphocytes, gamma delta+ lymphocytes do not generally express CD4 or CD8 molecules, and the nature of antigen recognition by these cells is unknown. To study antigen recognition by gamma delta+ lymphocytes we raised a gamma delta+ alpha beta- -CD4-CD8- line from an individual immune to PPD (purified protein derivative). This line showed a specific proliferative response to PPD and to a recombinant mycobacterial heat-shock protein (HSP) of relative molecular mass 65,000 (65K). The gamma delta+ line was shown to exhibit a major response to HSP in the presence of autologous antigen-presenting cells (APCs). Minor responses occurred, however, with APCs matched for some HLA class I or II antigens, whereas no response occurred with HLA-mismatched APCs. These findings, therefore, document the requirement of HSP-reactive gamma delta+ lymphocytes for histocompatible APCs.
The objective of the present study was to find out whether BLV infection, known to cause immunodisturbances in cows, might also bring about decreased productivity, reproductive rate and a shorter life span. More than 100 pairs of dairy cows, and a whole population of 3000 milch cows, were studied for this report. The findings revealed that a BLV-positive cow had a shorter life span than both its seronegative counterpart and the entire milch cow population. It also produced a total of 3.5% less milk and had a mean of 48 more days open than did the BLV-negative cow. The differences in survival rate were highly significant, while, at a level of 5%, those of productivity and reproductive rate were not. The implications of these findings are discussed. A highly significant correlation was also shown between BLV infection and the persistence of Trichophyton verrucosum infection in cows. The presented data indicate that BLV infection might affect the immune system of a cow to such a degree that it ceases to be productive enough to be kept within a herd. Thus it is usually culled before any severe symptoms of disease emerge.
Bovine leukemia virus (BLV) replication in B lymphocytes from experimentally infected sheep depends on three conditions. First, viral production is detected only when the infected animals exhibit blood lymphocytosis. Second, it requires in vitro cultivation but is never observed in vivo. Third, enhancement of virus expression after phytohemaglutinin (PHA) or concavalin A stimulation is observed in lymphocyte cultures for all infected animals, whereas specifically B cell mitogens are inefficient. The PHA effect is linked to the presence of T lymphocytes. In addition, the conditioned medium prepared from PHA-stimulated normal lymphocytes greatly enhances BLV production. Altogether, these results establish that T lymphocytes, through a lymphokine production, are important and may be essential for BLV growth in B lymphocytes.
Bovine leukaemia virus (BLV) is the aetiological agent of a chronic lymphatic leukaemia/lymphoma in cows, sheep and goats. Infection without neoplastic transformation has also been demonstrated in pigs, rhesus monkeys, chimpanzees and rabbits and observed in capybaras and water buffaloes. Structurally and functionally, BLV is a relative of human T lymphotropic viruses 1 and 2 (HTLV-I and HTLV-II) since all three viruses show clear-cut sequence homologies. The pathology of the BLV-induced disease, most notably the absence of chronic viraemia, a long latency period and lack of preferred proviral integration sites in tumours, is similar to that of adult T-cell leukaemia/lymphoma induced by HTLV-I. The most striking feature of the three naturally transmitted leukaemia viruses is the X region located between the env gene and the long terminal repeat (LTR) sequence. The X region contains several overlapping long open reading frames, one of which, designated XBL-1, encodes a trans-activator function capable of increasing the level of gene expression directed by BLV-LTR and is most probably involved in genetic instability of BLV-infected cells of the B-cell lineage. The 'genetic instability' may put the cell into a state of fragility, ready to move along a number of stages towards full malignancy. Little is known about these events and their causes and we present some theoretical possibilities. BLV infection has a worldwide distribution. In temperate climates the virus spreads mostly through iatrogenic transfer of infected lymphocytes. In warm climates and in areas heavily populated by haematophagous insects, there are indications of insect-borne propagation of the virus.
Peripheral blood mononuclear cells (PBMCs) from bovine leukaemia virus (BLV)-seronegative cattle and from BLV-seropositive cows either with normal haematological values or persistent lymphocytosis were tested for their proliferative response to BLV antigens. Cells from only BLV-infected cattle with normal lymphocyte counts were stimulated to a detectable level by the fetal lamb kidney cell supernatant containing BLV antigens. Proliferation assays performed with the purified major core protein p24 indicated that this protein has to be processed through a chloroquine-sensitive compartment before being recognized by CD4+ T lymphocytes. Forty-one 15-mer overlapping peptides spanning the entire p24 sequence were synthesized and analysed for their stimulating potential. It appeared that two regions included T cell epitopes recognized by PBMCs from three of five animals tested. These regions were represented by amino acids 31 to 55 (PGSQVWIQTLRLAILQADPTPADLE) and 141 to 165 (AESYVEFVNRLQISLADNLPDGVPK). The possible implication of this cell-mediated immune response in BLV pathogenesis and vaccine development is discussed.
The effects of bovine leukosis virus (BLV) on the phenotypic and functional characteristics of peripheral blood mononuclear cells were investigated. Whole blood differentials showed that persistently lymphocytotic (BLV+PL) dairy cattle had more lymphocytes and fewer neutrophils than the aleukemic seropositive (BLV+AL) or seronegative (BLV-) animals. Flow cytometric analyses of peripheral blood mononuclear cells indicated that the BLV+PL animals had more B lymphocytes, with a concomitant decrease in CD2 positive cells when compared with the BLV- group. Mononuclear cells from the BLV+AL animals also had fewer CD2 positive cells, but no differences in B lymphocytes were observed when compared with BLV- cattle. Peripheral blood mononuclear cells were used in blastogenesis assays to assess the functional ability of lymphocytes. Lymphocytes from BLV+PL animals had lower proliferative responses to concanavalin A and pokeweed mitogen when compared with cells from the BLV- or BLV+AL groups. The level of spontaneous blastogenesis in the absence of mitogenic stimulation was high for lymphocytes obtained from BLV+AL cattle. Cultures of lymphocytes obtained from BLV+PL animals produced greater amounts of interleukin-2 (IL-2) than BLV+AL and BLV- groups, although no differences were observed in the expression of IL-2 receptors. The development of uncontrolled lymphocytosis in BLV-infected cattle may result from an altered responsiveness to IL-2-regulated B-lymphocyte proliferation.
Although gamma delta T cells have been postulated to act as a surveillance mechanism that eliminates transformed or otherwise damaged cells, little is known about tumor recognition by gamma delta T cells, including the Ags that are recognized and the molecules that present them. Previously, we described human gamma delta CTL that recognize the autologous B cell lymphoma. Here we report that these gamma delta CTL lyse heterologous cells transfected with the tumor Ig lambda chain gene. Furthermore, the lambda chain is recognized as processed peptide in an Id-specific manner. T cell recognition does not involve classical MHC molecules, but it could be blocked by Abs directed against the heat shock protein grp75. These findings show a specific gamma delta T cell response to a highly polymorphic Ag such as tumor Id and implicate heat shock protein as a molecule required for recognition.
Bovine leukemia virus (BLV) is a retrovirus similar to the human T-cell leukemia virus (HTLV). Most BLV infected animals (70%) develop a B-cell lymphoproliferative syndrome with altered productive traits and 1 to 5% die with B-cell lymphosarcomas. Although BLV infection is world-wide, western European countries have almost eradicated it by slaughtering the seropositive animals. BLV infection remains endemic in many countries including the United States and prophylactic strategies involving recombinant vaccine vectors, genetically modified BLV and transgenic animals resistant to the infection are under study.
A panel of monoclonal antibodies to bovine leukocyte differentiation molecules was used to evaluate peripheral blood and splenic lymphocytes from cattle of various ages. The major population of peripheral blood lymphocytes from neonatal calves was gamma delta T-cell receptor (TCR1) positive, as determined by TCR1-N12 expression. TCR1-N12+ lymphocytes were decreased in number in older calves, and were lowest in adult cattle. The major subpopulation of TCR1-N12+ cells from peripheral blood coexpressed WC1, but not BoCD2. A small subpopulation of peripheral blood TCR1-N12+ cells from cattle of all ages coexpressed BoCD2, but not WC1. The TCR1-N12+ BoCD2+ lymphocytes made up the largest TCR1-N12+ lymphocyte subpopulation in spleens of both calves and adults. The TCR1-N12+WC1+ splenic lymphocytes were present as a small population. The data indicate that two subpopulations of TCR1+ lymphocytes are present in cattle of all ages. These two subpopulations are differentially distributed between blood and spleen, with TCR1-N12+WC1+ lymphocytes predominating in blood, and TCR1-N12+BoCD2+ cells predominating in spleen.
We previously investigated a novel heat-inducible transformation-associated cell surface Ag that is expressed on the activated H-ras oncogene-transformed rat fibrosarcoma W31, but not its parental nontransformed fibroblast WFB. This Ag was detected by mAb 067. Herein, we characterized the molecular nature of the Ag by using anti-heat shock protein (HSP) mAb. The accumulated data indicated that the cell surface expression of Ag was clearly enhanced by several stressors, such as TNF, L-azetidine-2-carboxylic acid, and sodium arsenite. The immunoprecipitate made with mAb 067 and W31 cell lysates reacted with anti-rat 70 kDa heat shock cognate (HSC) mAb, TG5E, indicating that 067-defined Ag may be a rat 70 kDa HSC. Because this Ag seemed to be one of the transformation-associated Ag of WFB, we further studied whether it could play an important role in the host's anti-tumor immunity. Peripheral T cells of rats primed with live BCG showed cytotoxicity to W31 but not to WFB. Because the possibility existed that HSP may interact with certain populations of T cells, we focused on the reactivity of CD4-CD8- double negative T (DNT) cells against 067-defined molecule. DNT cells from spleen and PBL of live BCG-primed rats showed the cytotoxicity against W31 cells. This cytotoxicity was completely blocked by mAb 067 and anti-CD3 mAb. However, it was not blocked by mAb R48B1 and 109, which detect the MHC class I nonpolymorphic determinant and a target molecule of the cytolysis by poly I:C-induced NK cells, respectively. Furthermore, brefeldin A was able to block the cytotoxicity against W31 targets by DNT cells, but not by NK cells. These data suggest that 70 kDa HSC may be a tumor Ag and may act as a presenting molecule perhaps complexed with cellular peptides to certain DNT cells.
Heat shock protein transfer peptides during antigen processing and CTL priming
- Srivastava