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I S Misko

Publications and source records attributed to I S Misko.

At least 37 records · Page 2Linked to original sources
PubMed1991

Composite response of naive T cells to stimulation with the autologous lymphoblastoid cell line is mediated by CD4 cytotoxic T cell clones and includes an Epstein-Barr virus-specific component.

We have approached the challenge of generating a primary T cell response to Epstein-Barr virus (EBV) in vitro by stimulating naive T cells with the autologous EBV-transformed lymphoblastoid cell line (LCL), a rich source of EBV-associated cytotoxic T lymphocyte (CTL) epitopes. Responsive T cells from three EBV-seronegative donors were cloned in agarose, phenotyped for T cell markers by flow cytometry, and their cytotoxic properties analyzed in the 51Cr release assay. Most clones (greater than 95%) expressed the CD4 phenotype and 59% of these clones showed cytotoxic properties. The dominant CTL response was specific for FCS-associated epitopes presented by FCS-grown autologous LCL target cells and was restricted by class II HLA antigens. Other clonal components included: (i) an EBV-specific response by HLA-restricted CD4 CTL clones that did not discriminate between A- and B-type EBV transformants; (ii) an EBV-specific response by an HLA-restricted CD4 CTL clone that discriminated between A- and B-type transformants, and (iii) a nonspecific cytotoxic response by CD3+,4+,8-, CD3+,4-,8-, and CD3-,4-,8- clones that were broadly allotypic or restricted to the lysis of K562 target cells. The EBV-specific CTL clones did not lyse the autologous EBV-negative B or T cell blasts and their specificity patterns of lysis were supported by the cold target competition data. These studies highlight the role of CD4 CTL in the establishment in vitro of a primary immune response to a human virus.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Cytotoxic T lymphocyte discrimination between type A Epstein-Barr virus transformants is mapped to an immunodominant epitope in EBNA 3.

An immunodominant Epstein-Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) epitope, represented by peptide 68, has been mapped to the EBV nuclear antigen, EBNA 3. The epitope is recognized by class I-restricted CTLs through HLA-B8 and is functionally active on type A but not type B lymphoblastoid cell lines (LCLs). Herein we show that peptide 68 is not expressed as a functional CTL epitope by type A LCLs infected with an EBV B95-8 isolate. CTLs from cultures stimulated with autologous type A IARC-BL74 or QIMR-WIL LCLs lysed autologous cells stimulated with phytohaemagglutinin (PHA blasts) and coated with exogenous peptide 68. No peptide 68-specific CTLs were generated in cultures stimulated with autologous type A B95-8 or type B AG876 LCLs. However, the B95-8 LCL coated with peptide 68 was effective in the induction of a peptide-specific CTL response. A peptide 68-specific CTL clone failed to lyse the B95-8 LCL, type B AG876 LCL and PHA blasts, although such targets were lysed when coated with peptide 68.

Amino Acid Sequence↗
PubMed1991

Oligopeptide induction of a secondary cytotoxic T-cell response to Epstein-Barr virus in vitro.

Three Epstein-Barr virus (EBV) nuclear antigen (EBNA)-encoded oligopeptide epitopes have been mapped, each capable of acting as a recognition determinant for class I-restricted lysis by CD8+ cytotoxic T lymphocytes (CTL). This report shows that each peptide, when presented on an appropriate autologous antigen-presenting cell (APC), also stimulates EBV-specific memory T cells present in peripheral blood mononuclear cell (PBMC) populations to develop in vitro into peptide-specific CTL. These CTL specifically lysed autologous EBV-infected lymphoblastoid cell lines (LCL) and peptide-sensitized uninfected targets. Identical viral oligopeptides could therefore function as recognition determinants for both the induction and commission of class I-restricted specific cytotoxicity. A model system is described in which autologous phytohaemagglutinin (PHA) blasts present exogenous peptide during the stimulation phase. The magnitude of the peptide-specific CTL response was dependent on the concentration of peptide added to the APC and specific lysis was inhibited by anti-class I monoclonal antibody (MoAb) but not anti-class II MoAb. Cultures depleted of CD8+ T cells by cell separation with immunomagnetic beads prior to stimulation invariably failed to generate a peptide-specific CTL response. However, the effect of CD4 depletion on CTL activity was equivocal and indicated that a need for CD4+ T cells as accessory helper cells may depend on the efficiency of the APC to elaborate their own help. This model has advantages in the analysis of events involved in the development of CTL activity in vitro.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Expression of Epstein-Barr virus nuclear antigens in anti-IgM-stimulated B cells following recombinant vaccinia infection and their recognition by human cytotoxic T cells.

Cytotoxic T lymphocytes (CTL) recognizing Epstein-Barr virus (EBV) nuclear antigens (EBNA) are an important host defence mechanism in restricting the proliferation of EBV-infected B cells. Previously, B-type lymphoblastoid cell lines (LCL) infected with vaccinia recombinants encoding for the EBNA proteins have been used to identify A-type-specific CTL epitopes. However, to localize the CTL epitopes encoded by both A- and B-type transformants, B-type LCL are an inappropriate host for vaccinia. In the present study, an alternative host cell for vaccinia infection is described. Initial studies demonstrated that anti-IgM (mu-chain specific)-stimulated human B cells allowed vaccinia virus to replicate more efficiently than either phytohaemagglutinin-stimulated lymphocytes (PHA blasts) or CTL and expressed EBNA proteins following recombinant vaccinia infection. Furthermore, the presentation and recognition of target epitopes expressed on vaccinia-infected anti-mu-stimulated B cell blasts were comparable to that on similarly infected LCL. Anti-mu-stimulated B cells were used to define the CTL epitopes recognized by a panel of CTL clones from an EBV-immune donor. Using recombinant vaccinia-infected anti-mu-stimulated B cells, the CTL response from this donor was mapped to the EBNA6 protein. Most importantly, in vitro stimulation of unfractionated mononuclear cells with vaccinia-infected anti-mu B cells activated a memory CTL response. Based on the vaccinia results, screening of peptides from EBNA6 localized the epitope for the majority of the EBNA6-specific CTL clones to the sequence EENLLDFVRFM, apparently in association with HLA-B44. This work clearly demonstrates that anti-mu-stimulated B cells not only provide an efficient model for localizing the CTL epitope(s) but also raises the possibility of reactivating a memory T-cell response to any gene product expressed by recombinant vaccinia.

Antigens, Viral↗
PubMed1990

Lymphokine-activated killer (LAK) cells discriminate between Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cells.

We have generated in vitro lymphokine-activated killer (LAK) cells from healthy donors by stimulating their mononuclear leukocytes with recombinant interleukin-2 (rIL-2) (100 U/ml). After 6 days in culture, the lytic properties of the LAK cells were analyzed in the 51Cr-release assay by utilizing a target panel of 6 paired lines consisting of an Epstein-Barr virus (EBV)-positive Burkitt's lymphoma (BL) cell line and an EBV-transformed lymphoblastoid cell line (LCL) from the same donor, the Raji BL line and the natural killer (NK) cell-sensitive K562 line. The patterns of lysis showed that the LAK cells discriminated between two categories of BL cell lines. Group I/II BL tumor cells which expressed the common acute lymphoblastic leukemia antigen (CALLA), the BL-associated glycolipid antigen (BLA) and phenotypically resembled biopsy cells were strongly lysed whereas group III BL cells which had assumed an LCL-like phenotype during culture and lacked the CALLA and BLA surface markers were only poorly lysed. The LCL targets were generally resistant to lysis but the K562 cell line was particularly sensitive. The outcome of cell depletion and monoclonal antibody (MAb) studies indicated that the LAK cell populations were phenotypically and functionally heterogeneous and consisted of at least 2 subpopulations of effector cells; a tumor-specific component and an NK-cell-mediated component.

Animals↗
PubMed1990

Patterns of reactivity of Epstein-Barr virus-specific T cells in A-type donor cultures after reactivation with autologous A- or B-type transformants.

Regression endpoints were assessed in cultures from 11 Epstein-Barr (EB) virus A-type seropositive donors and 2 seronegative donors using A- and B-type EB virus preparations. In 9/11 of the seropositive donors, the resulting endpoints using A-type or B-type virus were similar and demonstrated a significant T cell response to both virus types. However, the regression endpoints for 2/11 seropositive donors were reproducibly higher with B-type virus compared with A-type virus, indicating a weak T cell response to the B-type virus compared with that to the A-type virus. Seronegative donor cultures showed no regression. The patterns of reactivity of bulk cultures of EB virus-specific cytotoxic T cells and T cell clones from selected seropositive donors were compared. Four of six donors showed evidence of a cytotoxic T cell response to A- and B-type autologous transformants while cytotoxic T cells from 2/6 donors (corresponding to those identified as lacking regression to B-type virus) lysed autologous targets infected with A-type but not B-type virus. The results show that while most A-type seropositive donors are capable of mounting a T cell response to A- and B-type virus, certain donors apparently lack B-type reactivity.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

An Epstein-Barr virus-specific cytotoxic T cell epitope in EBV nuclear antigen 3 (EBNA 3).

Epstein-Barr virus (EBV)-specific CTL clones were isolated that recognized A-type EBV transformants but not B-type transformants. These A-type-specific CTL clones (HLA B8 restricted) were used to screen peptides derived from the EBV nuclear antigens (EBNAs) 2, 3, 4, and 6 as potential CTL epitopes. Of the 76 peptides screened, one sequence from EBNA 3 (residues 329-353) was recognized by A-type-specific CTL clones after absorption onto target cells (either autologous B-type transformants or PHA blasts). This report is the first description of an EBV target epitope recognized by specific CTL clones.

Amino Acid Sequence↗
PubMed1989

Expression of Epstein-Barr virus nuclear antigens 3, 4, and 6 are altered in cell lines containing B-type virus.

A high proportion of HIV-positive sera were found to react with 130- and 180-kDa antigens which were present in the Jijoye cell line. The majority of the HIV-positive sera which detected these antigens also contained antibodies to Epstein-Barr virus (EBV) nuclear antigen 2B (EBNA2B) suggesting a relationship between B-type EBV strains and the expression of the 130K/180K antigens. Cell lines were established by infection of B lymphocytes with different A- and B-type strains of EBV. Incubation of these lines with sera from individuals infected with either A-type or B-type EBV strains demonstrated that the 130K and 180K antigens were only expressed by cell lines containing B-type virus. Sera from individuals infected with A-type EBV did not react with the 180K antigen in any cell lines but could detect EBNAs 3, 4, and 6 antigens in the A-type cell lines. Restriction enzyme analysis of the BamHI E region of the EBV genome revealed marked differences between the A and B types of the virus. These results demonstrate that expression of antigens encoded from the BamHI E region of EBV (EBNAs 3, 4, and 6) are altered in cell lines transformed by B-type strains of EBV.

Antigens, Viral↗
PubMed1989

Interleukin-2 receptors in infectious mononucleosis.

The majority of peripheral blood mononuclear cells (PBMC) from acute-phase infectious mononucleosis (IM) patients express high levels of T10 (CD38) and HLA-DR surface antigens, which are markers characteristic of activated T cells. However, Tac antigen (p55) expression on these cells was not detectable by flow cytometric immunofluorescence, and only a low level of specific interleukin-2 (IL-2) binding was found by Scatchard analysis. These results suggest that IL-2 receptors (IL-2R) are lost or down-regulated on activated T cells in acute IM. Since a large proportion of T cells die during the first 24 h of in vitro culture in the absence of exogenous IL-2, the data implicate a physiological role for the observed low levels of IL-2R on T cells.

Humans↗
PubMed1989

T lymphocytes in infectious mononucleosis; effect of IL-2 on the outgrowth of Epstein-Barr virus-infected cells.

The addition of interleukin-2 (IL-2) to lymphocyte cultures from acute infectious mononucleosis (IM) donors dramatically increased the incidence of regression in such cultures and resulted in the emergence of an IL-2 dependent, CD3 Epstein-Barr virus nuclear antigen (EBNA)-negative cell population. Corresponding cultures seeded in the absence of IL-2 rarely regressed and were quickly dominated by IL-2 independent, CD3-negative, EBNA-positive cells. Lymphocyte cultures from Epstein-Barr virus (EBV) seropositive donors showed enhanced regression in the presence of IL-2 but failed to regress after the removal of the E-rosetting population. Cultures from EBV-seronegative donors showed no evidence of regression in the presence or absence of IL-2. E-rosetting cells isolated from cultures from acute IM donors that had been cultured in the presence of IL-2 lysed autologous and allogeneic lymphoblastoid cell lines.

Antibodies, Viral↗
PubMed1988

Cytotoxic T-cell clones discriminate between A- and B-type Epstein-Barr virus transformants.

Epstein-Barr virus (EBV) is the aetiological agent of infectious mononucleosis and is associated with Burkitt's lymphoma and nasopharyngeal carcinoma. The virus is harboured for life in all previously infected individuals and is apparently controlled by a population of EBV-specific memory T lymphocytes, specifically activated to recognize the functionally defined lymphocyte-detected membrane antigen. Two types (A and B) of EBV have been identified that show DNA sequence divergence within the BamH1 WYH region of the genome encoding the transformation-associated antigen, Epstein-Barr nuclear antigen 2 (EBNA 2) (ref. 4). To define the function of EBNA 2 in T-cell recognition, we have compared the ability of EBV-specific cytotoxic T-cell clones to distinguish between autologous B lymphocytes transformed by A- or B-type virus. We have now isolated both CD4 and CD8 cytotoxic T-cell clones that recognize autologous A-type but not B-type transformed lymphoblastoid cell lines, thus providing the first evidence that EBV-specific T-cell recognition can be mediated by EBNA 2. As this antigen is not expressed in Burkitt's lymphoma, this finding explains the failure of EBV-specific T-cell surveillance to eliminate the tumour.

B-Lymphocytes↗
PubMed1984

HLA-DR-antigen-associated restriction of EBV-specific cytotoxic T-cell colonies.

EBV-specific cytotoxic T cells can be generated in vitro in a secondary response. Several previous studies with bulk cultures provided evidence that cytotoxicity was restricted by HLA-A,B-related antigens. In the present family study, the EBV-specific cytotoxic T-cell response of a normal EBV-seropositive donor was analysed in detail by T-cell colony formation. Peripheral blood mononuclear cells were stimulated by the gamma-irradiated autologous lymphoblastoid cell line (LCL) and 3 days later seeded into agarose. Colonies were harvested, amplified by addition of interleukin-2 (IL-2), and analysed for T-cell markers and specificity in 51Cr-release assays. Twenty-two colonies were studied: all colonies were OKT3+, five were predominantly OKT4+, 9 were OKT8+ and 8 were mixtures. As expected from previous work, the OKT8+ colonies were cytotoxic for the autologous LCL target and cytotoxicity was blocked by monoclonal antibody (W6/32) to the nonpolymorphic determinants of HLA-A,B,C antigens. Significantly, the OKT4+ colonies tested also showed specific cytotoxicity, but lysis of the autologous LCL was blocked by the monoclonal antibody (OKlal) to the non-polymorphic determinants of HLA-DR antigens. Two interesting patterns of specificity were seen in cytotoxicity tests on sibling LCL targets. In one pattern, targets bearing the A11, B5, DR7 haplotype were lysed, while those bearing the A1, B8, DR3 were not, indicating haplotype preference. In the other pattern, there was lysis of the autologous cell line but not of the sibling targets. These results including HLA-DR-associated restriction, haplotype preference and strict self-preference, further illustrate the complexity of the EBV-cytotoxic T-cell response.

Adult↗
PubMed1984

Factors influencing the human cytotoxic T cell response to autologous lymphoblastoid cell lines in vitro.

Epstein-Barr virus (EBV)- and fetal calf serum (FCS)-specific cytotoxic human T cells can be generated in vitro, and have been shown to be HLA-antigen restricted. In the present work, peripheral blood mononuclear cells were stimulated with the gamma-irradiated autologous lymphoblastoid cell line (LCL) grown previously in FCS, human serum, or without serum. The induction and generation of cytotoxic T cells was carried out exclusively in culture medium containing autologous serum. With EBV-seronegative responders, FCS-grown stimulator LCL generated a FCS-specific cytotoxic T cell response. AB serum-grown LCL generated only a weak response, except at a high stimulatory dose, where the response tended to be essentially nonspecific. EBV-seropositive responders, in contrast, gave a typical secondary EBV-specific response regardless of the serum in which the LCL had been grown previously; no FCS response was detected. Dose-response and cold target inhibition studies confirmed these results. EBV immunity obviously plays a major role in the T cell response to the autologous LCL, which can no longer be viewed simply as a form of autologous mixed leucocyte reaction.

ABO Blood-Group System↗
PubMed1982

Evidence for the involvement of HLA-DR antigens in restricted cytotoxicity by fetal calf serum-specific human T cells.

Fetal calf serum (FCS) generated at least two distinct populations of human cytotoxic cells in vitro. One population expressed natural killer (NK) cell-like activity and lysed K562 and HSB-2 targets more effectively than autologous or allogeneic lymphoblastoid cell lines (LCLs). The other population contained FCS-specific cytotoxic T cells which preferentially lysed the autologous LCLs and showed minimal lysis of K562. E-rosette separation and cold target competition experiments clearly established that NK cells were not involved in the self-reactive lysis. Moreover, the lytic activity of the E-rosetted T cells was reduced by up to 95% when autologous target cells were grown in human AB serum rather than FCS, showing that FCS-associated determinants on targets were essential in the cytolytic phase. Autologous LCLs grown in FCS were also considerably stronger competitors than human serum-grown LCLs. The consistent self-preferred lysis suggested that HLA antigen-related restriction was involved, but the patterns of lysis did not implicate HLA-A or B antigens, and monoclonal antibody (W6/32) to an A, B, and C monomorphic determinant failed to block FCS-specific lysis. In contrast, monoclonal antibody (DA.2) to a monomorphic determinant of DR effectively blocked FCS-specific lysis. Cytotoxicity tests with a small panel of DR-typed donors indicated that strong cross-reactions were invariably associated with sharing of DR antigens, particularly DR2, and to a lesser but significant extent DR7. Although DR antigen sharing did not always result in lysis of allogeneic targets, the overall evidence strongly suggests that FCS-specific T-cell cytotoxicity in humans is restricted by products encoded by or associated with the DR genes.

Animals↗
PubMed1981

Generation in vitro of EBV-induced specific cytotoxic T cells in autologous serum avoids complications due to self-preferred foetal calf serum-specific T-cell cytotoxicity.

A previous report has established that in cultures of human mononuclear leukocytes, foetal calf serum (FCS) is capable of generating high levels of T cells preferentially cytotoxic for the autologous lymphoblastoid cell line (LCL). The present study compared the capacity of Epstein-Barr virus (EBV) to generate cytotoxic T cells in cultures of mononuclear cells grown in FCS in this system. Five EBV-seropositive and three seronegative donors were used and cultures were harvested at 14 days. With cultures from seropositive donors, whether grown in FCS or in autologous serum, EBV infection generated T cells cytotoxic for the autologous LCL; the response in uninfected control cultures was markedly lower. With seronegative donor cultures grown in FCS, there was virtually no difference in the capacity of T cells generated in infected or uninfected cultures to lyse the autologous LCL. Moreover, cells from seronegative donors cultured in human serum gave no detectable lysis of autologous LCL in either infected or uninfected cultures, clearly showing the absence of a response to EBV. This evidence shows that it is possible to distinguish the generation of specific cytotoxic T cells by FCS from generation by EBV, and with certain donors the apparently EBV-induced response may actually include a significant component induced by FCS in the medium. The cytotoxicity patterns of EBV-induced and FCS-induced T cells for autologous and allogeneic LCL targets showed a degree of parallelism, stressing the need for caution in interpretation of data obtained from cultures using FCS.

Animals↗