Ihor S. Misko

A case report: Immune responses and clinical course of the first human use of granulocyte/macrophage-colony-stimulating-factor-transduced autologous melanoma cells for immunotherapy

Abstract The first use of granulocyte/macrophage-colony-stimulating-factor-transduced, lethally irradiated, autologous melanoma cells as a therapeutic vaccine in a patient with rapidly progressive, widely disseminated malignant melanoma resulted in the generation of a novel antitumour immune response associated with partial, albeit temporary, clinical benefit. An initially negative reaction to non-transduced, autologous melanoma cells was converted to a delayed-type hypersensitivity (DTH) reaction of increasing magnitude following successive vaccinations. While intradermal vaccine sites showed prominent dendritic cell accrual, DTH sites revealed a striking influx of eosinophils in addition to activated/memory T lymphocytes and macrophages, recalling the histology of challenge tumour cell rejection in immune mice. Cytotoxic T lymphocytes (CTL) reactive with autologous melanoma cells were detectable at high frequency after vaccination, not only in limiting-dilution analysis, but also in bulk culture without added cytokines. Clonal analysis of CTL showed a conversion from a purely CD8+ response to a high proportion of CD4+ clones following vaccination. A prominent acute-phase response manifested by a five- to tenfold increase in C-reactive protein was observed, as was a systemic eosinophilia. Vaccination resulted in the regression of axillary lymphatic metastases, stabilisation of pulmonary metastases, and a dramatic, reversible increase in cerebral oedema associated with multiple central nervous system metastases; however, lesions in the adrenal glands, pancreas and spleen proved refractory. The antitumour effects and immune response were not detectable 2 months following the last vaccination. Irradiation of the extensive cerebral metastases resulted in rapid deterioration and death of the patient.

A functional link for major TCR expansions in healthy adults caused by persistent Epstein-Barr virus infection.

Dramatic clonal expansions of unknown functional significance have been documented in the T cell receptor (TCR) alpha beta peripheral blood repertoires of apparently healthy adults. In this study, we provide evidence that persistent infection with the ubiquitous Epstein-Barr virus (EBV) causes major distortions within the memory repertoire of healthy virus carriers. Using complementarity determining region 3 (CDR3) length analysis to measure repertoire diversity, dominant expansions that dramatically skewed the entire TCRBV6 blood repertoire towards oligoclonality were enriched in the CD8(+)CD45RO+CD45RA- subset of HLA B8(+) healthy virus carriers. Evidence of phenotypic heterogeneity between individuals was also observed for these expansions based on their variable coexpression of CD45RO and CD45RA. TCR junctional region sequencing revealed that these expansions were clonal and that they represented commonly selected HLA B8-restricted memory cytotoxic T cells that recognize the immunodominant latent EBV epitope, FLRGRAYGL. Furthermore, the functional identity of these virus-specific CD8(+) T cells was confirmed by their FLRGRAYGL-specific cytotoxicity. Therefore, the functional significance of dramatic clonal expansions in healthy adults can be linked in some cases to virus-specific CD8(+) T cells that play an essential role in immunosurveillance. This first identified link for expansions in the circulation of healthy adults strongly implies that restricted-memory TCR responses to environmental antigens play a pivotal role in expansion development, which should have an important impact on studies interpreting TCR expansion patterns in health and disease.

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.

Dominant Cytotoxic T Lymphocyte Response to the Immediate-Early Trans-Activator Protein, BZLF1, in Persistent Type A or B Epstein-Barr Virus Infection

Five healthy human leukocyte antigen-B8 (HLA-B8)-positive virus carriers were studied to investigate the CD8+ cytotoxic T lymphocyte (CTL) response to an HLA-B8-restricted peptide, RAKFKQLLQ, located in the Epstein-Barr virus (EBV) immediate-early trans-activator protein, BZLF1. Of the 5 virus carriers, 4 were infected with type A and 1 with type B EBV. Using limiting-dilution analysis of peripheral blood mononuclear cells, a high RAKFKQLLQ-specific CTL precursor frequency was demonstrated after specific peptide or autologous lymphoblastoid cell line stimulation in both type A and type B EBV carriers. The RAKFKQLLQ-specific CTL precursor frequencies in all 5 persons were at least as dominant as those observed with two other EBV-associated, HLA-B8-restricted latent epitopes, FLRGRAYGL and QAKWRLQTL. These findings show that healthy virus carriers maintain a high frequency of BZLF1-specific memory T cells, potentially to control virus spread from lytically infected cells.

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.

The ecology and pathology of Epstein-Barr virus

Epstein‐Barr virus achieves its ubiquitous and uniform epidemiological distribution by a dual strategy of latency to guarantee lifelong persistence and intermittent replication to guarantee transmission. These two functions appear to dictate residence in different cell types: latency in B lymphocytes and replication in epithelial cells. Both of these cell compartments are potential sites for FBV‐associated malignancies.

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.

Failure of Epstein‐Barr virus‐specific cytotoxic T lymphocytes to lyse B cells transformed with the B95‐8 strain is mapped to an epitope that associates with the HLA‐B8 antigen

There are two types, A and B, of Epstein‐Barr virus (EBV) and B95‐8 represents the common type A laboratory strain. Herein, we show in a family study that paternal EBV‐specific cytotoxic T lymphocytes (CTL) generated in short‐term cultures following stimulation with the autologous B95‐8‐transformed lymphoblastoid cell line (LCL) or B cells freshly infected with the B95‐8 isolate did not lyse haploidentical B95‐8 LCL expressing the HLA‐A1‐B8,‐DR3 paternal haplotype. In contrast, the haploidentical B95‐8 LCL expressing the HLA‐A11,‐B51,‐DR7 paternal haplotype was strongly lysed. Moreover, paternal CTL generated in response to stimulation with the B95‐8 LCL expressing the haploidentical HLA‐A1,‐B8, ‐DR3 paternal haplotype included an allogeneic response against the maternal haplotype but no EBV‐specific response as shown by the poor lysis of the autologous LCL target cells. However, stimulation with the haploidentical HLA‐A11,‐B51, ‐DR7 paternal haplotype resulted in the generation of both an allogeneic and an EBV‐specific response. CTL clones were generated from two HLA‐B8+ donors in response to stimulation with the autologous type A LCL transformed with wildtype EBV. The clones were cross‐reactive for an immunodominant B95‐8‐associalcd peptide epitope that interacted with the HLA‐B8 allele but failed to lyse B95‐8‐transformed LCL largets unless the targets were pre‐coated with the exogenous peptide. A CTL clone that was initially stimulated with the autologous BL74 LCL lysed the spontaneous autologous LCL and spontaneous LCL from an HLA‐B8+ donor, but failed to lyse the B95‐8 LCL from that donor. The observed haplotype preference can be explained in terms of sequence variation between the B95‐8 and the corresponding wildtype epitope. Our findings may help to clarify the role of EBV in the pathogenesis of primary Sjögrens syndrome which is closely associated with HLA‐B8.

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.

Resistance to etoposide‐induced apoptosis in a Burkitt's lymphoma cell line

Burkitts lymphoma cells that vary in their phenotypic characteristics show significantly different degrees of susceptibility to radiation‐induced apoptosis. Propensity to undergo apoptosis is reflected in the degradation of substrates such as DNA‐dependent protein kinase but the status of bcl‐2, c‐myc and p53 has been uninformative. In this study, we have focused on 2 Epstein‐Barr virus (EBV)‐associated Burkitts cell lines, one (WW2) susceptible and the other (BL29) resistant to etoposide‐induced apoptosis. Differences in expression of BHRF1, an EBV gene that is homologous to the Bcl‐2 proto‐oncogene and known to inhibit apoptosis, or changes in apoptosis inhibitory proteins (IAPs), did not appear to account for the difference in susceptibility in the 2 cell lines. Cytoplasmic extracts from etoposide‐treated WW2 cells caused apoptotic changes in nuclei isolated from either BL29 or WW2 cells, whereas extracts from BL29 cells failed to do so. In addition, extracts from etoposide‐treated WW2 cells degraded the catalytic subunit of DNA‐dependent protein kinase (DNA‐PKcs), an important indicator of apoptosis, but this protein was resistant to degradation by BL29 extracts. It appears likely that caspase 3 (CPP32) is involved in this degradation since it was activated only in the apoptosis susceptible cells and the pattern of cleavage of DNA‐PKcs was similar to that reported previously with recombinant caspase 3. As observed previously, addition of caspase 3 to nuclei failed to induce morphological changes indicative of apoptosis, but addition of caspase 3 to nuclei in the presence of extract from the resistant cells led to apoptotic changes. We conclude that resistance to apoptosis in BL29 cells is due to a failure of etoposide to activate upstream effectors of caspase activity. Int. J. Cancer 77:755–762, 1998.