Biomedical subjects
Publications and source records attributed to D J Moss.
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Unfractionated mononuclear (UM) cells and T cells freshly prepared from the blood of adult donors were co-cultivated in microtest plate wells with progressively lower numbers of cells from the autologous EB-virus-transformed B-cell line. The fresh cells present in co-cultures from EB virus antibody-negative (seronegative) donors regularly facilitated autologous cell line outgrowth, monitored after 4 weeks, whereas outgrowth was markedly inhibited in the corresponding co-cultures from seropositive donors. Larger-scale co-cultures, set up at a ratio of 80-100 fresh UM cells to one autologous virus-transformed B cell, were harvested after 8 to 12 days and the T-cell subpopulation was examined for cytotoxicity both by growth inhibition and by chromium release assays. Cytotoxic T cells were generated exclusively in seropositive donor co-cultures and were strongly active against the autologous virus-transformed cell line without affecting either autologous uninfected B cells or any of a range of EB virus genome-negative target cell lines chosen as sensitive indicators of non-specific cytotoxicity. Recognition of allogeneic EB-virus-transformed cells was restricted to those whose HLA-A and/or B and/or B and/or C antigen expression matched that of the effector cells themselves;; moreover target cell lysis was specifically inhibited in the presence of monoclonal antibodies binding to these HLA antigens. The results indicate that EB-virus-specific HLA-restricted memory T cells, present in the blood of previously-infected individuals, can be reactivated in vitro using the established autologous virus-transformed cell line as a stimulus. THe reactivated cytotoxic cells appear to recognize a virus-induced lymphocyte-detected membrane antigen, LYD-MA, analogous to that first invoked to explain the cytotoxic response to primary EB virus infection observed during infectious monoucleosis.
The experiments show that the phenomenon of regression, seen exclusively in Epstein-Barr (EB) virus-infected cultures of mononuclear cells from EB virus antibody-positive donors, is mediated by cytotoxic T cells reactivated in vitro and specifically recognizing an EB virus-induced lymphocyte-detected membrane antigen LYDMA. Thus, effector T cells from regressing cultures kill autologous EB virus-transformed cells but not autologous pokeweed mitogen-stimulates lymphoblasts nor any of a range of EB virus genome-negative human hemopoietic cell lines (K562, HSB2, BJAB, EB4) particularly sensitive to nonspecific natural killer-like activities. Moreover, these reactivated effector cells exhibit classical HLA restriction of target cell recognition; in a survey of 14 effector cell donors, preferential lysis of the autologous virus-transformed line was a consistent feature, while the relative degree of lysis of allogeneic lines was in general directly related to the number of HLA-A and B antigens shared between effector and target cells. The pattern of reactivity shown by effector T cell preparations from any one donor was strikingly reproducible, and the results from a number of donors revealed differences between particular HLA-A and B antigens with respect to the level of EB virus-specific killing which was associated with sharing through these determinants.
Monoclonal antibodies specifically binding common determinants on all HLA-A, B anc C antigen molecules blocked the lysis of EB virus-transformed target cells by EB virus-specific cytotoxic T cells reactivated in vitro. Blocking was mediated through binding of the antibodies to the target rather than to the effector cells and was maximal (75 to 85% inhibition of lysis) at saturating antibody concentrations. A similar blocking effect was also shown by a monoclonal antibody binding to beta 2-microglobulin, a molecule physically associated with HLA-A, B and C antigens on the target cell surface, but not by a monoclonal antibody binding to the non-HLA-associated leukocyte-common antigen. Saturating concentrations of monoclonal antibodies specific for common determinants on all HLA-DRw antigen molecules either had no effect at all upon EB virus-specific T cell cytotoxicity or caused a slight, but nonspecific, inhibition. The results demonstrate unequivocally that HLA-A, B and C antigens on the target cell surface are indeed the polymorphic elements which impose genetic restriction upon EB virus-specific cytotoxic T cell function.
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Under appropriate culture conditions, EB virus infection of lymphocytes from seropositive donors leads to regression of transformation, and this was shown previously to be due to activation in a secondary immune response to T lymphocytes inhibitory for the autologous lymphoblastoid cell line. Regression can be quantified by determining the number of cells required for its expression. To investigate the development of memory T cells with EB-virus specificity in the primary infection, a comparison was made of the capacity for regression of lymphocytes from 16 cases of infectious mononucleosis (IM) and 13 normal donors. With 9 normal seropositive donors a mean lymphocyte concentration of 4.6 X 10(5)/ml was required to achieve 50% regression. In contrast, with 8 cases of IM tested within 1 week of onset, a much higher mean lymphocyte concentration (3.7 X 10(6)/ml) was necessary. Six of these IM cases, and another not tested in the first week, were tested on several occasions between 5 and 23 weeks after onset, and showed a slight reduction in the mean cell concentration required for regression (1.5 X 10(6)/ml). Six additional were tested 23--83 weeks after onset by which time the cell concentration required for 50% regression (mean = 4.5 X 10(5)/ml) had reached the level shown by normal seropositive donors. Regression did not occur with lymphocytes from seronegative donors, even at the highest cell concentration. Recombination cultures of T-cell-depleted and T-cell-enriched lymphocyte populations from 3 IM cases in ratios of 1:7 to 7:1 showed that the failure of regression in acute IM was not due simply to lack of sufficient numbers of T cells. The results indicate that EB-virus-specific memory T-cell activity as detected by the regression test is absent in the acute phase of IM, becomes evident at low levels 5--23 weeks after onset, and reaches a maximum after about 6 months.
The specificity of cytotoxic T cells generated in Epstein-Barr virus (EBV)-infected lymphocyte cultures was investigated, using a 51Cr release assay. Potent cytotoxic T cells with preferred specificity directed to antigens expressed on autologous lymphoblastoid cell line (LCL) target cells were present in 14-day cultures of lymphocytes from EBV-seropositive donors and not from seronegative donors. Moreover, the cytotoxic patterns obtained with a panel of HLA antigen-related and unrelated LCL target cells, supported by unlabeled target inhibition tests, strongly indicate that T cell cytotoxicity to EBV is restricted to HLA antigens.
Regression of EB-virus-induced transformation occurs exclusively in cultures of leukocytes from seropositive donors. Studies have shown that the strength of regression could be assayed in terms of the proportion of T cells which must be added to the autologous EB virus-infected T-cell-depleted population in order to establish regression in the culture. The in vitro regression phenomenon was strongly T-cell-dependent but did not require the presence of either monocytes or, as a potential antigenic stimulus, the residual viral envelope material on the surface of virus-infected B cells. The T-cell-depleted population from seropositive donors sometimes transformed (7/60 cultures) without the experimental addition of virus. Regression appeared to be independent of cytotoxic mechanisms involving anti-viral antibodies and not to be mediated by soluble factors released into culture medium.
Experiments have been conducted to determine the role played by immune T cells in the regression of EB-virus-induced transformation which is exclusively seen in leukocyte cultures from sero-positive donors. Kinetic studies suggest that, in virus-infected cultures from such donors, a population of T cells proliferates within the first 2 weeks apparently in response to the appearance of virus-infected B cells. This proliferation continues to some extent during the period of regression. Nonspecific induction of T-cell proliferation by PHA did not induce regression in virus-infected cultures from seronegative donors and acutally prevented the regression in seropositive donor cultures. T cells harvested from seropositive donor cultures 11-14 days post infection were generally much more inhibitory to the growth of the autologous EB-virus-transformed cell line than were T cells either freshly prepared from whole blood or harvested from corresponding uninfected cultures; this inhibitory activity was either absent or much diminished when assayed against allogeneic target cell lines. The results suggest that virus-specific memory T cells capable of mounting a cytotoxic response when properly challenged in vitro.
A series of 10 cell lines established from human malignant melanomas is described. The morphology varied but, in general, was epithelioid. Five produced visible melanin in culture but others produced melanin precursors. All lines were aneuploid and each had a distinctive human karyotype. One line appeared not to have the same sex as the patient but its karyotype was distinct from that of other lines.
Peripheral blood mononuclear cells from donors of known serological status with respect to EB virus were exposed to the virus in vitro and then cultured at various cell concentrations. All cultures from nine seronegative adult and 12 foetal donors gave rise to cell lines following subculture 4 weeks post infection. In contrast, seropositive donor cultures seeded at the higher cell concentrations developed foci of proliferating EBNA-positive cells within the first 1--2 weeks but thereafter regressed completely and subcultures made after 4 weeks never gave rise to cell lines. Out of 18 seropositive donors tested, 15 showed regression in all cultures seeded at 10(6) cells/ml and above, and with the other three donors a proportion of replicate cultures regressed. T-cell depletion and reconstitution experiments showed that the effect was absolutely dependent upon the presence in the cultures of T cells from these seropositive donors. The results strongly suggest that the regression phenomenon is an in vitro expression of long-term T-cell-mediated immunity to EB virus which the large majority, if not all, infected individuals possess.
Non-adherent lymphocyte populations prepared from donors with previous EBV infection transformed in the usual way in response to EBV. However, transformation was completely inhibited in the presence of adult but not fetal human fibroblasts. Such inhibition was associated with lymphocytes from donors with prior EBV infection, but not from those without, and there appears to be an immunological basis for inhibition. Inhibition was associated with production of EBNA and was reversible. Addition of adult fibroblasts to non-adherent lymphocytes up to 2-4 days after infection with EBV also resulted in inhibition of transformation. These results are interpreted as indicating that EBV transformation involves two recognizable phases, with an immunological block between EBNA production and cell proliferation.
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Transformation of a special population of non-adherent human lymphocytes by EB virus (EBV) is reversibly inhibited by co-cultivation on adult human fibroblasts. Neither fluid from adult fibroblast cultures nor extracts of fibroblasts inhibited such transformation, and the growth of already transformed lymphocytes was not inhibited on adult fibroblasts. The EBV-associated nuclear antigen (EBNA) was detectable in inhibited cultures, with a maximum level of about 15% at 14 days after which it decreased gradually. Reversal of inhibition at 28 days, by either addition of phytohaemagglutinin or by removal of the lymphocytes from the adult fibroblasts, resulted in a prompt increase in the percentage of EBNA-positive cells and typical lymphoblastoid outgrowth. Transformation of EBV-infected non-adherent lymphocytes could be inhibited by the addition of adult fibroblasts up to 2-4 days after infection. The results indicate that, in the EBV-infection of non-adherent lymphocytes on adult fibroblasts, a block resulting in inhibition of transformation occurs between the production of EBNA and the onset of autonomous proliferation of the infected lymphocytes.
A nuclear antigen, apparently the Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA), was detected by anticomplement immunofluorescence (ACIF) tests in adult peripheral blood leukocytes infected with the QIMR-WIL strain of EBV. EBNA was not detectable at 24 h but appeared in about 11% of the cells by 3 days, and by 5 days up to 64% of the cells were positive. Proliferation of EBNA-positive cells at this stage was confirmed by autoradiography. There was a good correlation between the concentration of virus and the number of EBNA-positive cells in the first 5-7 days. The subsequent course of events was found to be influenced by the initial cell concentration and the time of subculture. EBNA production was delayed in cells infected with higher dilutions of virus but subsequently appeared in a high proportion of cells. Indirect immunofluorescence failed to detect viral capsid antigen (VCA) or early antigen (EA) by 10 days. The results show that EBV infection was abortive and that the critical events of viral transformation occurred within the first few days.
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