Cytotoxic T cells and human herpes virus infections.
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Amino Acid Sequence↗
Biomedical subjects
L K Borysiewicz
Publications and source records attributed to L K Borysiewicz.
Down-regulation of integrin alpha 1/beta 1 expression and association with cell rounding in human cytomegalovirus-infected fibroblasts.
Human cytomegalovirus (HCMV) causes a c.p.e. characterized by rounding of the infected cell. Since interactions with the extracellular matrix may be involved in the cell rounding, we have analysed the expression of integrins, which are the main cell surface receptors involved in cell-substrate adhesion and spreading. By FACS analysis, a selective decrease in cell surface expression of alpha 1/beta 1 integrin was observed in HCMV-infected fibroblasts. This decrease coincides with cell rounding. Immunoprecipitation studies and FACS analysis of permeabilized cells have further demonstrated that total levels of this integrin are decreased in infected cells, suggesting that the reduction in cell surface alpha 1/beta 1 integrin is not due to a defect in transport to the surface. Furthermore, we have ruled out the possibility that the observed decrease in alpha 1/beta 1 expression is caused by a cytokine released from the infected cells by showing that the reduction is abolished by inactivating the HCMV with u.v. irradiation, and that conditioned medium from HCMV-infected cells has no effect on expression of alpha 1/beta 1 integrin in uninfected cells. Concomitant with the reduction in alpha 1/beta 1 levels, the HCMV-infected fibroblasts show a reduced ability to adhere to laminin and collagen IV. Taken together the data indicate that de novo synthesis of HCMV protein(s) causes a decreased assembly/expression of alpha 1/beta 1 integrin, coincident with the well characterized morphological alterations of the infected cell.
Cell Line↗
Human cytomegalovirus-infected cells have unstable assembly of major histocompatibility complex class I complexes and are resistant to lysis by cytotoxic T lymphocytes.
Viruses which cause persistence in the naturally infected host are predicted to have evolved immune evasion mechanisms. Human cytomegalovirus (HCMV) causes significant morbidity and mortality in immunocompromised patients yet persists without clinical manifestations in seropositive individuals who have normal immune function. We report that HCMV infection in vitro impairs major histocompatibility complex class I (MHC-I) assembly accompanied by resistance to killing by cytotoxic CD8+ T lymphocytes. Pulse-chase metabolic labelling experiments show that MHC-I complexes continue to be assembled by both uninfected and HCMV-infected cells. However, MHC-I molecules are unstable in HCMV-infected cells and are rapidly broken down. Endoglycosidase H treatment of immunoprecipitates indicates that the breakdown of MHC-I complexes in HCMV-infected cells occurs primarily in a pre-Golgi compartment. Interference with normal MHC-I assembly and expression, if relevant in vivo, may have implications for the restriction of the diversity of the CD8+ cytotoxic T lymphocyte repertoire directed against HCMV antigens and may be an important mechanism of viral persistence.
CD8 Antigens↗
Hereditary C1q deficiency and systemic lupus erythematosus.
We describe a 27-year-old women with systemic lupus erythematosus, C1q deficiency and cytomegalovirus retinitis. She suffered from severe SLE, with cutaneous and CNS involvement, and died of CNS disease aged 28. Review of 29 other published cases of C1q deficiency shows that SLE in these patients is often severe (five with CNS disease, ten with glomerulonephritis). The results of autoantibody studies in this and another patient with C1q deficiency and SLE are presented--both patients had autoantibodies to the extractable nuclear antigens, Sm, RNP and Ro, and one patient had high titres of antibodies to dsDNA. One of the patients had previously been treated with fresh frozen plasma, and antibodies to C1q were present in his serum. Homozygous C1q deficiency is associated with a very high prevalence of severe SLE with the full panoply of autoantibodies characteristic of this disease.
Adult↗
Subsets of CD8+, CD57+ cells in normal, healthy individuals: correlations with human cytomegalovirus (HCMV) carrier status, phenotypic and functional analyses.
Two different subsets of CD8+, CD57+ cells have been defined, one expressing high levels (CD8high+(CD57+)), the other expressing low levels of surface CD8 (CD8low+(CD57+)). Increased numbers of CD8high+(CD57+) cells correlated with previous HCMV infection. By three-colour fluorescence analysis, the CD8high+(CD57+) population expressed T cell markers such as CD3 and CD5, and most were alpha beta T cell receptor (alpha beta TCR)-positive. A significant proportion also expressed CD71 (transferrin receptor) and MHC class I, although little if any CD25 (IL-2R-p55). Some (> or = 40%) co-expressed CD45RA and CD45RO. The CD8low+(CD57+) population expressed classical natural killer (NK) cell markers--CD2, CD16 and CD56. The two subsets were also functionally distinct; CD8high+(CD57+) cells suppressed pokeweed mitogen (PWM)-driven, but not phytohaemagglutinin (PHA)-driven proliferation and immunoglobulin production; CD8low+(CD57+) cells exhibited NK cytotoxic activity which was not increased by interferon-alpha (IFN-alpha). Supernatant from cultured CD8high+(CD57+) cells suppressed PWM-driven immunoglobulin production, but not proliferation, and this effect was abrogated by physical separation with tissue culture inserts. Thus, a T cell subset expressing activation and memory T cell markers with direct non-specific suppressor activity was present in peripheral blood mononuclear cells (PBMC) of healthy subjects with asymptomatic HCMV infection.
Antigens, CD↗
Analysis of the human T-cell response to picornaviruses: identification of T-cell epitopes close to B-cell epitopes in poliovirus.
Little is known about the nature and specificity of T-cell-mediated responses to picornaviruses in humans. In this study, the nature of the T-cell response to seven picornaviruses, including polioviruses, coxsackieviruses B3 and B4, human rhinovirus 14, and encephalomyocarditis virus, was determined. Twenty-nine individuals responded to poliovirus type 3, coxsackievirus B3, and encephalomyocarditis virus by proliferation of T cells, and from such cultures, 130 virus-specific T-cell lines were established. T-cell lines generated in response to encephalomyocarditis virus were exclusively strain specific. However, the majority of T-cell lines established in response to viruses, other than encephalomyocarditis virus, were cross-reactive to each other. Their cross-reactivity was confirmed in 2 of the 30 picornavirus-specific clonally derived T-cell lines from two subjects, but the majority of these lines were serotype specific. T-cell epitopes adjacent to each of the B-cell antigenic sites in VP1 of poliovirus type 3 were identified. The response to the region adjacent to B-cell antigenic site 1 (residues 97 to 114) was dominant between individuals. The localization of this major CD4 T-cell epitope may permit the construction of chimeric viruses utilizing the natural picornavirus T-cell response to augment production of antibody specific for inserted sequences.
Amino Acid Sequence↗
Serum folate and chronic fatigue syndrome.
We assayed serum folate levels of 60 patients with chronic fatigue syndrome (CFS) and found that 50% had values below 3.0 micrograms/l. Some patients with CFS are deficient in folic acid.
Adolescent↗
Heterogeneity amongst natural killer cells revealed by limiting dilution culture; selectivity against virus-infected and tumour cell targets.
Previous studies have suggested that natural killer (NK) cells exhibit heterogeneous cytotoxicity towards different tumour cell targets. No studies have set out to determine whether different NK populations have relative selectivity for virus-infected cells. The aims of this study were to determine if this was the case for short-term clones, and whether there were differences in relative selectivity for particular target cells between clones with NK activity but with different surface phenotypes. Cells from different starting populations [whole peripheral blood lymphocytes (PBL), E-rosette positive or negative, CD16+ or CD3- cells] were grown in limiting dilution culture (LDC) with interleukin-2 (IL-2). The precursor frequency (NK-p) of cells proliferating and exhibiting NK activity towards various virus-infected or uninfected fibroblasts or tumour cell targets was determined by split-well analysis of the LDC. The relative NK-p were similar for different individuals, but were much lower for virus-infected fibroblasts than a tumour cell target. The pattern of cytotoxicity of 757 short-term clones, identified from the LDC, against four to five tumour and virus-infected target cells were analysed. We conclude that there was selective lysis of virus-infected cells by a proportion of NK clones which were predominantly PBL-derived (mainly CD3+). Twenty-six per cent of E(+)-derived clones lysed Molt4 cells only in the absence of phytohaemagglutinin (PHA), and a proportion of PBL- or E(+)-derived clones (up to 44%) lysed uninfected or virus-infected fibroblasts but not Molt4+PHA. Thus, under hese conditions lectin-induced cytotoxicity does not detect total potential cytotoxicity.
Adult↗
Lymphocyte culture from routine endomyocardial biopsies: lack of clinical or pathological correlations.
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Biopsy↗
Cytomegalovirus infection in heart-lung transplant recipients: risk factors, clinical associations, and response to treatment.
The risk factors, clinical associations, and response to treatment of cytomegalovirus (CMV) pneumonia and infection were studied in 65 recipients of heart-lung transplantation. There were 29 episodes of CMV pneumonia in 22 patients. In 80% (20/25) of episodes of CMV pneumonia treated with intravenous ganciclovir, the histologic changes resolved and the patient survived. Among seronegative recipients, a seropositive donor was a significant risk factor for CMV pneumonia and infection in the first 90 days after heart-lung transplantation (P = .004 and .002, respectively). Among seropositive recipients, there was no additional risk associated with a sero-positive donor. Rates of CMV pneumonia and infection were significantly increased when treatment with augmented immunosuppression had been given in the preceding 30 days (P less than .001). A significant association was found between CMV pneumonia or infection and pulmonary bacterial infections occurring 30 days before or after such an episode (P less than .001).
Adolescent↗
Infection and reactivation with cytomegalovirus strains in lung transplant recipients.
Cytomegalovirus pneumonia is a major cause of morbidity and death following lung transplantation (LT) (1). The case fatality rate is highest in the CMV-seronegative recipients (R-) of organs from seropositive donors (D+), which suggests that transmission of CMV may occur with the graft (1), but in seropositive recipients (R+) the comparative importance of reactivation of endogenous virus and reinfection with donor virus is poorly understood.
Cytomegalovirus↗
Human cytotoxic T cell responses to vaccinia virus vaccination.
Recombinant vaccinia viruses have been proposed as live virus vectors in man to generate both specific humoral and cellular immune responses. The generation of such responses following revaccination was followed in five normal volunteers. All five showed elevated (35- to 6500-fold) vaccinia virus-specific IgG by 14 days post-vaccination, following uncomplicated vaccination by dermal scarification. Four of the five had elevated total serum immunoglobulins with increased spontaneous and pokeweed mitogen-induced immunoglobulin production. Major histocompatibility complex class I-restricted vaccinia virus-specific cytotoxic T lymphocytes (CTLs) were not detected either directly, from peripheral blood mononuclear cells, or following secondary in vitro stimulation, even by limiting dilution analysis. These studies would suggest that vaccination with recombinant vaccinia viruses may provide an insufficient stimulus to the generation of CTLs in vivo.
Adult↗
Monocytes are a major site of persistence of human cytomegalovirus in peripheral blood mononuclear cells.
We have used the nested polymerase chain reaction (PCR) combined with fluorescence-activated cell sorting to define sites of latency of human cytomegalovirus (HCMV) in the peripheral blood of healthy subjects. Peripheral blood mononuclear (PBM) cells were separated into T cell or non-T cell populations and monocytes, and were then analysed by PCR for the presence of HCMV DNA. In five of six seropositive subjects, HCMV was found predominantly in the non-T cell population. Further analysis suggested that the virus was present in adherent cells and CD14+ cells. In three of nine seronegative subjects we could demonstrate HCMV DNA, which we do not believe was due to contamination, reproducibly by PCR. In one of these seronegative subjects, HCMV DNA was present predominantly in the non-T cell fraction of PBM cells. No HCMV DNA was detectable in the remaining six seronegative subjects. We conclude that, within the PBM cells of normal asymptomatic seropositive and some seronegative subjects, HCMV is present predominantly in the monocyte fraction. In addition, the detection of HCMV sequences in seronegative subjects may indicate that infection with HCMV is more widespread than conventional seroepidemiology suggests.
Antibodies, Viral↗
Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein.
Cell-mediated immunity is important in maintaining the virus-host equilibrium in persistent human cytomegalovirus (HCMV) infection. The HCMV 72-kDa major immediate early 1 protein (IE1) is a target for CD8+ cytotoxic T cells in humans, as is the equivalent 89-kDa protein in mouse. Less is known about responses against this protein by CD4+ T cells, which may be important as direct effector cells or helper cells for antibody and CD8+ responses. Proliferative-T-cell responses to HCMV IE1 were studied in normal seropositive subjects. Peripheral blood mononuclear cells from 85% of seropositive subjects proliferated in response to HCMV from infected fibroblasts, and of these, 73% responded to recombinant baculovirus IE1. Responding cells were predominantly CD3+ CD4+. IE1 antigen preparations, including baculovirus recombinant protein, transfected rat cell nuclei, and synthetic peptides, induced IE1-specific T-cell lines which cross-reacted between the preparations. The fine specificity of these IE1-specific T-cell lines was studied by using overlapping synthetic peptides encompassing the entire sequence of the IE1 protein. The regions of the IE1 molecule recognized were identified and these varied between individuals, possibly reflecting differences in major histocompatibility complex (MHC) class II haplotype. In one subject, the peptide specificities of proliferative and MHC class I-restricted cytotoxic determinants on IE1 were spatially distinct. Thus, no single immunodominant T-cell determinant within HCMV IE1 was identified, suggesting that multiple peptides or a region of the 72-kDa IE1 protein would be required to induce specific T-cell responses in humans.
Amino Acid Sequence↗
A report--chronic fatigue syndrome: guidelines for research.
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Fatigue Syndrome, Chronic↗
Pathogenesis of human cytomegalovirus disease and the kidney.
Uncertainty still surrounds many aspects of the pathogenesis of human cytomegalovirus (HCMV) infection. During asymptomatic infection HCMV is probably present in a small fraction of peripheral blood mononuclear cells (most likely monocytes); in active infection HCMV is detectable in a wider range of cells. In vitro experiments suggest that cellular control of transcription of HCMV immediate early (IE) gene expression is one key determinant of HCMV reactivation. The cytotoxic T cell response to IE and other virus gene products is probably important in subsequently limiting dissemination of HCMV infection. The basis for any association between active HCMV disease and allograft rejection is unclear, although homologies between cellular and viral proteins suggest possible mechanisms. Although HCMV may be associated with tubulointerstitial disease, there is no unequivocally established role for HCMV in the pathogenesis of human glomerular disease.
Animals↗
Automation of limiting dilution cytotoxicity assays.
Limiting dilution analysis is a valuable technique for the quantitation and clonal analysis of immunocompetent cells. However, manual processing of the large number of samples necessary for satisfactory statistical analysis is laborious, and consequently results in inaccuracies reflected as increased standard errors. We describe the application of an automated robotic liquid handling tool to process samples in limiting dilution cytotoxicity assays. Our studies have shown that automated liquid handling is more accurate than manual methods, and that errors are limited. This results in savings of both time and resources. Furthermore, the process may be adapted for the safe, remote handling of sterile cell cultures, and human pathogens.
Automation↗
Functional analysis of intercellular adhesion molecule-1-expressing human thyroid cells.
We have tested the potential role of thyroid cell intercellular adhesion molecule-1 (ICAM-1) expression by in vitro assays of cell clustering and cytotoxicity. Increased ICAM-1 appeared within 24 h of thyroid cell stimulation with cytokines and was not inhibited by the antithyroid drug methimazole. Autologous and allogeneic lymphocyte-thyroid cell cluster formation, assessed by flow cytometry, was reduced by about one-third in the presence of a monoclonal antibody against ICAM-1, regardless of whether thyroid cells were expressing basal levels of ICAM-1 or had been stimulated with interferon-gamma. The cytotoxicity produced by interleukin 2-stimulated allogeneic lymphocytes was not consistently inhibited by anti-ICAM-1 antibody, but phytohemagglutinin-stimulated lymphocytes showed a reduction of 23%-28% in cytotoxicity against untreated or interferon-gamma stimulated thyroid cells when the anti-ICAM-1 monoclonal antibody was present. Finally, thyroid cells could be infected by rhinovirus, confirming the presence of fully functional ligand. These results show that ICAM-1 expression by thyroid cells may enhance immune cell recognition and play some role in cytotoxicity, features which could be important in the initiation or perpetuation of autoimmune thyroiditis.
Cell Adhesion↗