Generation in vitro of HLA-restricted EB virus-specific cytotoxic human T cells by autologous lymphoblastoid cell lines: the role of previous EB virus infection and foetal calf serum

The cytotoxic response of human lymphocytes to the autologous lymphoblastoid cell line (LCL) was investigated with five Epstein-Barr virus (EBV)-seropositive and four seronegative donors and an additional donor who seroconverted during the course of the study. LCL stimulation was carried out usually at a stimulator-to-responder cell ratio which varied from 1:200 to 1:20,000. Cytotoxic T cells from 7- and 14-day cultures were assayed on autologous and allogeneic LCL target cells and the K562 line, using the ⁵¹Cr release assay. Without exception, the cytotoxic T cells generated in 7- and 14-day LCL-stimulated cultures from seropositive donors were strongly lytic for the autologous LCL. With some donors, the cytotoxic response increased with increasing autologous LCL stimulation, while with others even the lowest level of stimulation induced a potent response. Overall, the cytotoxic T cells generated in cultures from seropositive donors showed a striking preferential lysis of the autologous LCL and cross-reactive killing of allogeneic LCL target cells matched for certain HLA-B antigens. Lysis of the K562 line, an indicator target for natural-killer-like activity, was consistently negligible. In comparison to the considerable self-preferred lysis obtained with T cells from LCL-stimulated cultures, the level of autologous killing by T cells generated in control unstimulated cultures was relatively low. The cytotoxicity patterns obtained with cytotoxic T cells generated in cultures from seronegative donors were markedly different from those obtained with seropositive donors. The level of autologous killing by T cells harvested from LCL-stimulated cultures was considerably lower than that obtained with seropositive donors and often did not increase above that seen in control cultures, except at the highest stimulatory dose. Moreover, preferential lysis of the autologous LCL target cells by T cells from LCL-stimulated cultures was essentially due to the foetal calf serum (FCS)-induced cytotoxicity and there was no obvious HLA-antigen-restricted killing of allogeneic target cells. Lysis of K562 target cells was generally more pronounced at the highest level of stimulation than that observed with seropositive donors. The distinctive cytotoxic T-cell response patterns observed with the seropositive and seronegative donor cultures were both observed with cultures from an individual donor who was tested before and after infectious mononucleosis. Our cytotoxicity data are interpreted as indicating that with LCL-stimulated cultures from seropositive donors the cytotoxic T-cell response to autologous LCL is predominantly directed against EBV-associated antigens and is HLA-antigen restricted. In contrast, with seronegative donor cultures, the cytotoxic T-cell response is primarily due to FCS-induced cytotoxicity, although a response against other LCL-associated antigens may be superimposed in cultures stimulated with high levels of autologous LCL.     

Enhancement of outgrowth of EB virus-transformed cells from normal human peripheral blood by a tumor promoter, TPA

Effect of 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on the establishment of lymphoblastoid cell lines (LCL) following spontaneous tranformation of B cells by Epstein-Barr virus (EBV) from peripheral lymphocytes of healthy EBV-seropositive adults was examined. In the lymphocyte culture with TPA (0.5 ng/ml), the frequency of establishment of LCL increased and the time required for appearance of transformation decreased. A larger number of EBV-positive cells in the peripheral blood were detected in the assay by co-cultures with cord blood lymphocytes in the presence of TPA than in the absence of it. The enhancement of LCL establishment by TPA was largely abolished in the culture with EBV neutralizing antibody-containing human umbilical cord serum. This suggests that the enhancement by TPA may be caused mostly by transformation of coresident B lymphocytes infected with active virus induced from the EBV genome-positive cells by TPA.     

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.     

Induction of epstein-barr virus by a new tumor promoter,teleocidin, compared to induction by TPA

The effect of teleocidin, a new, naturally occurring tumor promoter, on induction of Epstein-Barr virus (EBV), was compared with that of a known tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Early antigen (EA) and/or capsid antigen (VCA) of EBV was induced in the EBV genome-carrying cell lines C-6 and P3HR-I cells by teleocidin, its effect being maximal at a concentration of 12.5 ng/ml. The production of infectious EBV from P3HR-I cells was enhanced by teleocidin maximally at a concentration of 0.5 to 2.5 ng/ml. The outgrowth of EBV-transformed cells from peripheral lymphocytes of seropositive healthy donors was also enhanced by teleocidin at a concentration of 0.02 to 0.5 ng/ml. TPA tested simultaneously in all experiments exhibited the same activities as teleocidin, and was effective at similar concentrations. Teleocidin enhanced both EA and VCA synthesis in P3HR-I cells additively with n-butyrate, but not with TPA. This suggests that teleocidin and TPA have a common mechanism of action, although their chemical structures are different.     

Defective cell-mediated response to EBV-transformed B cells in a healthy individual with regular EBV antibody titers

We have analyzed the EBV-related immune parameters of a healthy EBV-seropositive individual (ST) who has regular antibody titers but defective inhibitory capacity toward the growth of autologous EBV-infected B cells. This in vitro function reflects the EBV-specific memory because it does not occur in experiments performed with cells of seronegative individuals. An analysis of events following in vitro EBV infection showed that lymphocytes of ST behaved in some tests in the same way as those collected from seronegative individuals. These parameters were: lack of gamma-IFN production 24 hr after EBV infection; low production of soluble factors that inhibit EBV-induced B-cell proliferation; lack of generation of LCL selective cytotoxicity after repeated stimulation with autologous LCL; and high proportion of EBNA-positive cells in 7-day-old EBV-infected cultures. On the other hand, cellular memory to the virus detected by the production of IL-2 24 hr after infection, and by the production of LIF upon exposure to EBV-encoded antigens, conformed with the results obtained with seropositive individuals. T-cell-mediated inhibition of EBV-induced B-cell growth in vitro has been regarded as a corollary of in vivo control of EBV-infected B cells. However, it is absent or has a low efficiency in certain disease categories which are not accompanied by risk of B-EBV growth. Our results with a healthy individual also indicate that several mechanisms contribute to a harmless life-long virus carrier state.     

Induction of Epstein-Barr virus-associated nuclear antigen during in vitro transformation of human lymphoid cells.

Human lymphoid cells isolated from the peripheral blood of adults, from cord blood, and from fetal liver, spleen, bone marrow, and thymus were cultivated with or without a cell-free preparation of Epstein-Barr virus (EBV) with demonstrated transforming activity. The cultures were examined for the EBV-associated nuclear antigen (EBNA) and for transfromation into permanent lymphoblastoid cell lines (LCL). EBNA, seen only in cultures that had received exogenous EBV, was detected between days 1 and 6 after addition of EBV, most frequently on day 3. EBNA-positive cells had a lymphoblastoid appearance. Transformation into established LCL became apparent between days 12 and 19. The addition of pokeweed mitogen to cultures containing EBV enhanced the development of EBNA, whereas phytohemagglutinin or concanavalin A had no such effect. Neither EBNA nor transfomration was observed in lymphoid cells from fetal thymus. In fetal spleen, bone marrow, and liver cells, EBV regularly induced EBNA and LCL transformation.     

Decreased calcium dependence of lymphoblastoid cell lines compared with Burkitt lymphoma cell lines

The extracellular calcium level required for proliferation was compared in B lymphoid cell lines from various sources by determining the calcium concentration at which long-term proliferation was inhibited by 50% (CaPD50). Fourteen Burkitt lymphoma (BL) lines had a mean CaPD50 of 44 +/- 28 microM whereas 45 lymphoblastoid cell lines (LCLs) obtained by in vitro transformation of B lymphocytes with Epstein-Barr virus (EBV) had a mean CaPD50 of 3.6 +/- 1.8 microM. This difference applied also to autologous BL lines and LCLs established from the same patient. The decreased calcium requirement of virally-transformed compared with tumour-derived cell lines therefore appears to be a universal phenomenon in mammalian cells. Within the BL group, no correlation was found between the calcium requirement for proliferation and presence or absence of the EBV genome. Arrest of BL lines and LCLs occurred in the G1 phase of the cell cycle and was readily reversed by addition of calcium to the medium. One anomalous LCL was found which showed a high CaPD50 (43 +/- 6 microM) and accumulated in both G1 and G2. These results, in combination with a previous study of EBV transformation in vitro, indicate that the calcium dependence of B lymphocytes generally decreases in the following order: normal cells greater than BL cells = early stage transformation greater than LCL. The 2 transformed phenotypes thus distinguished in human lymphoid cells may offer unique opportunities for defining the status and expression of EBV in vitro and in vivo.     

Long-term T-cell-mediated immunity to Epstein-Barr virus in man. III. Activation of cytotoxic T cells in virus-infected leukocyte cultures.

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.     

Effects of Phorbol Esters and Cytokines (Interleukin-2,-4, and -6) on the Proliferation and Surface Phenotype of Epstein-Barr Virus Immortalised Human B Lymphocytes

Epstein-Barr virus (EBV)-induced in vitro infection of peripheral blood mononuclear cells (PBMCs) leads to a polyclonal proliferation and immortalisation of B lymphocytes. In the present study we determined the effects of three different cytokines, interleukin-2 (IL-2), interleukin-4 (IL-4) and interleukin-6 (IL-6), and the tumour promoting phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on EBV-immortalised B lymphocytes. These factors have known activities on normal B cells. IL-4 and IL-6 increased significantly EBV-B cell proliferation after 3 and 5 days of culture, where IL-2 had no effect. The effect of IL-4 and IL-6 on EBV-B cells was abolished after pre-incubation with anti-IL-4 and anti-IL-6 neutralising antisera, respectively. TPA induced a dose dependent inhibition of proliferation both in serum free and 10% fetal calf serum (FCS) supplemented culture medium. Combinations of TPA and interleukins did not restore lymphoblastoid cell proliferation to background levels. All possible combinations of the three cytokines showed no synergistic or antagonistic effect on proliferation. TPA induced significant phenotypic changes of EBV immortalised B lymphocytes, by increasing IL-2 receptor (IL-2R) expression and decreasing CD20 and CD23 antigen expression. Other B cell differentiation antigens; HLA-DR, CD19, and transferrin receptor (CD71), did not demonstrate significant changes. A dose dependent inhibition of CD21 and increase in CD22 expression was observed in 2 out of 3 lymphoblastoid cell lines tested.     

Lysis of fresh human tumor cells by autologous peripheral blood lymphocytes and tumor-infiltrating lymphocytes activated by PSK

The protein-bound polysaccharide PSK was tested for the ability to induce in vitro autologous tumor killing (ATK) activity in human cancer patients. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) demonstrated various levels of cytotoxicity against autologous, freshly isolated tumor cells. When PBL and TIL were cultured overnight with PSK, ATK activity was induced in previously non-reactive cases and augmented in previously reactive samples. The PSK effect was observed with PSK concentrations of 10-100 micrograms/ml that could be obtained in the blood of cancer patients who received standard oral administration of PSK. The manifestation of PSK-induced ATK required active cell metabolism and RNA and protein syntheses, but not DNA synthesis of lymphocytes. PSK-induced enhancement of ATK was not abrogated by monoclonal antibodies (mAb) directed against interferon (IFN) alpha or IFN gamma. In addition, mAb that neutralized interleukin-2 (IL-2) or mAb reactive with alpha-chain or beta-chain of IL-2 receptors (IL-2R) had no effect on PSK-induced ATK activity. Supernatants from PSK-stimulated lymphocyte cultures did not induce ATK. Cell fractionation experiments revealed that CD3-CD16+ large granular lymphocytes (LGL) and/or CD3+CD16- T lymphocytes were responsible for both spontaneous and PSK-induced ATK. PSK-activated LGL, but not T lymphocytes expressed lysis of fresh allogeneic tumor cells. These results indicate that PSK activates PBL and TIL to exhibit ATK independently of IL-2/IL-2R systems.     

Epstein-Barr virus-induced transformation of human leukocytes after cell fractionation.

The efficiency of transformation of human lymphocytes after infection with Epstein-Barr virus (EBV) was determined in fractionated and non-fractionated preparations derived from 16 human cord blood samples and two blood samples from adult donors. The transformation efficiency of macrophage-depleted leukocytes was consistently lower as compared to non-fractionated leukocytes. Additional depletion of B-cells resulted in a further decrease. Reduction of T-cells, however, did not influence significantly the transformation rate. In non-fractionated leukocyte cultures, as well as in macrophage-depleted and B-cell enriched cultures, colonies of transformed cells were regularly observed within the first week of cultivation. All cell lines established after EBV-infection revealed membrane-bound immunoglobulin. Reconstruction of macrophages-depleted, B-cell enriched or B-cell depleted cultures with autologous macrophages resulted in an increase of the transfromation efficiency up to the values of non-fractionated leukocyte preparations. Addition of heterologous human embryonic lung fibroblasts resulted in a similar increase. The results support the interpretation that EBV transforms only those cells of the hematopoetic system which are derived from the bone-marrow entity. The transformation efficiency is considerably increased by co-cultivation of lymphocytes with macrophages and heterologous human fibroblasts which seem to excert a feeder-layer effect by enhancing survival of lymphocytes in vitro.     

SCID mouse model of Epstein-Barr virus-induced lymphomagenesis of immunodeficient humans.

Immunodeficient humans are at very high risk of developing Epstein-Barr virus (EBV)-induced lymphomagenesis. Severe combined immunodeficient mice (SCID) have been shown to develop lymphoproliferative disease (LPD) following transfer of peripheral blood leukocytes (PBL) with latent EBV. To study mechanisms of lymphomagenesis, we compared results of engraftment of PBL from normal donors and immunodeficient donors with X-linked lymphoproliferative disease (XLP). Graft-versus-host disease (GVHD) developed in 6 of 10 SCID mice 4 to 8 weeks following transfer of PBL from normal donors. In contrast, none of 9 mice engrafted with PBL from XLP patients with T-cell defects showed GVHD. LPD developed in mice regardless of the immune competence of the donors. The expression of EBV-encoded proteins, results of immunophenotyping, and karyotyping of the LPD lesions revealed lethal oligoclonal LPD owing to transfer of latent EBV in B cells in mice engrafted with PBL from seropositive donors. Polyclonal LDP developed in mice engrafted with PBL from seronegative patients which were infected with B95-8 virus 6 weeks after transfer of the cells. This model is useful for investigating mechanisms of EBV-induced LDP in immunodeficient patients.     

Human allogeneic melanoma-reactive T-helper lymphocyte clones: functional analysis of lymphocyte-melanoma interactions.

Lymphocyte clones were isolated from CD4+ peripheral-blood lymphocytes (PBL) of melanoma (Me) patient 9923 (HLA-DR7, DQw2, w6), co-cultured for 30 days with autologous accessory cells, allogeneic Me (Me 1811) (HLA-DR7, DQw1, w2), IL-1 beta (2 U/ml) and IL-2 (15 IU/ml). The 55 clones tested displayed a CD3+, CD4+, CD8-, T-cell receptor (TCR) alpha/beta+, gamma/delta- phenotype. Twenty clones were assayed for proliferation in the presence of Me 1811 and B-lymphoblastoid cell line (LCL) 1811, both expressing HLA-class-I and -II (DR7 and DQw2 shared with patient 9923), intercellular adhesion molecule-1 (ICAM-1) and lymphocyte-function-associated antigen-3 (LFA-3) molecules. Eight clones were found to be reactive to Me 1811 but not to LCL 1811. Specificity analysis of these 8 clones revealed that each of them proliferated only to Me 1811, not to other 14 Me and 12 different LCL, suggesting recognition of melanoma-associated antigen (MAA) expressed on the stimulating Me. One clone (103) was analyzed in more detail. A wider specificity analysis showed that it reacted to Me 1811 but not to 10 other Me expressing or not HLA-DR7, 5 normal melanocyte cultures (2 of them typing HLA-DR7-positive when exposed to interferon-gamma--IFN-gamma), 4 tumors other than Me and 20 different LCL. Clones did not show proliferation in the presence of autologous Me cells. Clone proliferation in response to Me 1811 was significantly inhibited by monoclonal antibodies (MAbs) directed to CD3, TCR alpha/beta, TCR beta chain V12, CD4 and HLA-DR. Moreover, following stimulation with Me 1811, clone 103 showed increased surface expression of CD25 (IL-2 receptor) and CD71 (transferrin receptor) and produced significant amounts of IL-2 and IFN-gamma. The supernatant taken from co-culture of clone 103 with Me 1811 augmented the cytotoxicity of PBL 9923 and other allogeneic PBL against K562 and Me 1811. Thus, the lymphocyte clone 103 is a CD4+ Th clone which uses its CD3/TCR alpha/beta complex to recognize an MAA in conjunction with HLA-DR7. Availability of this type of reagent may prove useful to identify and characterize MAA recognized by T lymphocytes.     

Sensitivity of different target cells to the killing action of peripheral lymphocytes stimulated by autologous lymphoblastoid cell lines

Peripheral lymphocytes obtained from two individuals with a previous history of infectious mononucleosis were exposed to mitomycin-treated cells of the autologous lymphoblastoid cell line (LCL) established during the acute phase of the disease. This resulted in a stimulation of DNA synthesis, comparable to or even exceeding a one-way MLC with allogeneic lymphocytes. The cytotoxic effect of the stimulated lymphocytes was tested by colony inhibition or ⁵¹Cr release, against a large LCL panel, including the autologous line and allogeneic lines from patients with Burkitt's lymphoma (BL), nasopharyngeal carcinoma (NPC), infectious mononucleosis (IM), leukemia, myeloma, or normal donors. While the majority of the lines were highly sensitive to the killing action, three were relatively resistant. The same pattern of sensitivity was obtained with effector cells stimulated by autologous LCL derived from IM or BL patients. The majority of the target LCLs had B-cell characteristics and carried the EBV genome, but three cell lines that were devoid of the EBV genome, were also sensitive. These lines included two lymphoid cell lines, one of them a T-cell line, and a myeloma line. Fresh peripheral lymphocytes from normal donors or acute IM patients, PHA-induced blasts and blast cells from a case of acute myeloid leukemia were resistant.     

Lysis of Fresh Human Tumor Cells by Autologous Peripheral Blood Lymphocytes and Tumor-infiltrating Lymphocytes Activated hy PSK

The protein-bound polysaccharide PSK was tested for the ability to induce in vitro autologous tumor killing (ATK) activity in human cancer patients. Peripheral blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) demonstrated various levels of cytotoxicity against autologous, freshly isolated tumor cells. When PBL and TIL were cultured overnight with PSK, ATK activity was induced in previously non-reactive cases and augmented in previously reactive samples. The PSK effect was observed with PSK concentrations of 10–100 μg/ml that could be obtained in the blood of cancer patients who received standard oral administration of PSK. The manifestation of PSK-induced ATK required active cell metabolism and RNA and protein syntheses, but not DNA synthesis of lymphocytes. PSK-induced enhancement of ATK was not abrogated by monoclonal antibodies (mAb) directed against interferon (IFN)α or IFNγ-. In addition, mAb that neutralized interleukin-2 (IL-2) or mAb reactive with α-chain or β -chain of IL-2 receptors (IL-2R) had no effect on PSK-induced ATK activity. Supernatants from PSK-stimulated lymphocyte cultures did not induce ATK. Cell fractiona-tion experiments revealed that CD3^-CD16⁺ large granular lymphocytes (LGL) and/or CD3⁺CD16^- T lymphocytes were responsible for both spontaneous and PSK-induced ATK. PSK-activated LGL, but not T lymphocytes expressed lysis of fresh allogeneic tumor cells. These results indicate that PSK activates PBL and TIL to exhibit ATK independently of IL-2/IL-2R systems.     

B cells from leukemic patients are relatively resistant to in-vitro EBV transformation

Samples of peripheral blood mononuclear cells (PBMC) from normal donors or from leukemic patients were used to obtain Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL). Whereas the rate of transformation was around 85% with normal donors, it was only around 20% with leukemic patients. However no explanation for this resistance of B cells from leukemic patients to in-vitro EBV transformation could be found. Indeed no correlation exists between this EBV resistance and the age, sex, diagnosis or chemotherapeutic regimen. Furthermore no correlation is apparent between the percentage of B2+ cells, which should have EBV receptors, and the successful EBV transformation.     

Enhanced susceptibility of adriamycin-treated human renal cell carcinoma cells to lysis by peripheral blood lymphocytes and tumor infiltrating lymphocytes

Previous studies indicated that the anticancer agent adriamycin (ADR) could induce activation of cytotoxic T lymphocytes (CTL) and natural killer cells. In this study, we investigated the effect of ADR on the susceptibility of human renal cell carcinoma (RCC) cells to lysis by peripheral blood lymphocytes (PBL) and tumor infiltrating lymphocytes (TIL). Treatment of human RCC cell line ACHN and freshly derived RCC cells with ADR at 1 microg/ml or more for 3 h significantly enhanced their susceptibility to lysis by PBL (P<0.05). This ADR-induced enhancement of susceptibility of RCC cells to lysis by PBL was also observed when freshly derived TIL were used as effector cells (P<0.05). ADR up-regulated the expression of leukocyte function-associated antigen-3 (LFA-3) and intercellular adhesion molecule-1 (ICAM-1), which are critical in the binding and killing of CTL against cancer cells. Of the five fresh RCC cell cultures treated with ADR, LFA-3 was increased in all and ICAM-1 was increased in three of them, respectively (P<0.05). Up-regulation of LFA-3 and ICAM-1 was also observed in ACHN cells treated with two derivatives of ADR, epirubicin and pirarubicin. ADR further significantly increased the bindings of PBL to RCC cells (P<0.05). These findings suggest that treatment of RCC patients with low doses of ADR may sensitize the RCC cells to killing by PBL and TIL and may be a novel immunotherapeutic modality for the treatment of drug-resistant and/or immune-resistant RCC. The inducing of LFA-3 and ICAM-1 by ADR may be involved in the enhancement of susceptibility of PBL and TIL-mediated cytolysis in human RCC cells.     

Verapamil preferentially potentiates in-vitro cytotoxicity of vincristine on malignant lymphoid cells.

Verapamil has been shown to overcome acquired drug resistance to vincristine in P388 leukemia both in vitro and in vivo. To study the selectivity of this action, the effect of addition of verapamil on the cytotoxicity of vincristine was studied using lymphocytes from eight patients with chronic lymphocytic leukemia (CLL), lymphoblasts from a T-acute lymphoblastic leukemia (T-ALL) cell line (GM 3639), and peripheral blood lymphocytes (PBL) from eight normal healthy volunteers. Using the differential staining cytotoxicity (DiSC) assay, we demonstrated that verapamil at 1 microM concentration potentiated the in-vitro cytotoxicity of vincristine on CLL and GM 3639 cells in concentrations of 0.04-0.25 micrograms/l. There was however, no enhancement of cytotoxicity noted against the control PBL. The data demonstrate that verapamil preferentially enhances the in-vitro cytotoxicity of vincristine on CLL and GM 3639 cells but no enhancement of cytotoxicity is seen against PBL.     

Establishment of human cytotoxic T-cell lines specific for Epstein-Barr virus-transformed autologous cells

EBV-specific cytotoxic T cells (Tc) induced in vitro have continuously proliferated in vitro for over 9 months. The long-term maintenance of the Tc growth was dependent on periodic supplementation of both irrediated EBV-transformed autologous lymphoblastoid cell line (LCL) cels and conditioned medium. The latter was derived from supernatants of human spleen-cell cultures stimulated with phytohemagglutinin. The cultured Tc maintained significant cytotoxic activity to autologous LCL cells but not to EBV-unrelated target cells, including K-562, B-, T-, null-cell lines and mitogen-stimulated antologous peripheral blood lymphocytes. Thus, established EBV-specific Tc lines from five different individuals always exhibited highly significant cytotoxicity against exhibited highly significant cytotoxicity against autologous LCL cells but not always against allogeneic LCL cells. Furthermore, restriction of the Tc to the autologous LCL was more pronounced after long-term culture than it was initially. This suggests that certain clones of Tc which arignificant cytotoxicity against exhibited highly significant cytotoxicity against autologous LCL cells but not always against allogeneic LCL cells. Furthermore, restriction of the Tc to the autologous LCL was more pronounced after long-term culture than it was initially. This suggests that certain clones of Tc which arignificant cytotoxicity against exhibited highly significant cytotoxicity against autologous LCL cells but not always against allogeneic LCL cells. Furthermore, restriction of the Tc to the autologous LCL was more pronounced after long-term culture than it was initially. This suggests that certain clones of Tc which are probably restricted to HLA are selectively established during long-term cultivation.     

Paired Epstein-Barr virus (EBV)-negative and EBV-converted Burkitt lymphoma lines: stimulatory capacity in allogeneic mixed lymphocyte cultures

Epstein-Barr virus (EBV)-negative Burkitt lymphoma lines (BLE-) and their in vitro EBV-converted sublines (BLEc), obtained by infection with the P3HRI and B95-8 strains of EBV, were compared for their capacity to induce T-lymphocyte proliferation in allogeneic mixed lymphocyte cultures (MLC). Regardless of the virus strain used for conversion, the BLEc lines induced a considerably stronger primary MLC response than their EBV-negative parentals. Only the BLEc lines were able to maintain T-lymphocyte proliferation in repeated stimulations. The low proliferative response observed in cultures stimulated with BLE- cells was not due to the generation of suppressor cells or to the release of inhibitory factors. The increased stimulatory capacity of BLEc lines was unrelated to changes in expression of MHC class-I and class-II antigen, or of B-cell activation markers, and was not due to the reactivation of EBV-specific memory T cells, since lymphocytes from EBV-seropositive and seronegative donors responded similarly. The results indicate that the capacity of BL cells to elicit cellular immune responses may be influenced by their EBV-carrying status.