Nongnit Lewin

Bcl-2, Bax and p53 expression in B-CLL in relation to in vitro survival and clinical progression.

Our previous data have shown that isolated leukemic cells from progressive chronic lymphocytic leukemia (B‐CLL) patients respond to growth stimulation in vitro and express high levels of p53, immunoreactive with the configuration‐specific antibody PAb 240. We have now analyzed the in vitro survival of B‐CLL cells in relation to Bcl‐2, Baxα and p53 expression and compared this with the clinical progression of the disease. Leukemic cells from patients with progressive disease demonstrated higher in vitro survival, compared with non‐progressive B‐CLL and normal B cells. All cells were sensitive to treatment with a combination of glucocorticoid and cAMP. Bcl‐2 protein levels were not related to clinical progression, as measured by flow cytometry. Competitive PCR showed that Bcl‐2 mRNA was over‐expressed in most of the B‐CLL samples and that p53 mRNA expression was similar between B‐CLL groups and normal values and thus not related to clinical progression. However, since Baxα expression was lower in progressive than in non‐progressive patients, the Bcl‐2/Baxα ratio at the mRNA level was significantly higher in the progressive group. Our data suggest that the Bcl‐2/Baxα ratio is important for the regulation of B‐CLL cell survival, that p53 over‐expression in progressive B‐CLL is the result of post‐transcriptional modifications and that a directed PKA activation may potentiate the cytolytic effect of glucocorticoids in vivo.

Autologous T lymphocytes may specifically recognize leukaemic B cells in patients with chronic lymphocytic leukaemia

This study analysed a naturally occurring specific cellular immunity against tumour cells in chronic lymphocytic leukaemia (CLL) patients. Five out of eight patients had blood T lymphocytes able to recognize spontaneously and specifically the autologous tumour B cells (proliferation assay). In these five patients, detection of cytokines by real‐time reverse transcription polymerase chain reaction (RT‐PCR) revealed that granulocyte–macrophage colony‐stimulating factor (GM‐CSF) was the most abundant cytokine gene expressed by the T cells that recognized the autologous tumour B cells. Other activated cytokine genes were γ‐interferon (IFN), interleukin (IL)‐2 and tumour necrosis factor (TNF)‐α, but not IL‐4. This profile suggests a type 1 anti‐B‐CLL T‐cell response. CD80 and CD54 were relatively downregulated on the native tumour B cells compared with control normal B cells. Upregulation of CD80 on the leukaemic cells was mandatory for the induction of such a specific T‐cell response. CD80 and CD54 monoclonal antibodies inhibited the specific T‐cell DNA synthesis proliferation. The proliferative T‐cell response was either MHC class I or class II restricted (inhibition by monoclonal antibodies). The specific cytokine gene expression could be found in isolated CD4, as well as CD8, T‐cell subsets. This study demonstrated the presence of a potential natural specific CD4, as well as a CD8 type 1 T‐cell immunity against the leukaemic CLL tumour B cells in CLL. A further detailed analysis of the spontaneous anti‐CLL T‐cell immunity is warranted that may facilitate the development of effective anti‐tumour vaccines in CLL.

Intracellular T cell cytokines in patients with B cell chronic lymphocytic leukaemia (B‐CLL)

Abstract: Analysis of cytokine production is a tool to functionally characterise T cells. In this study, spontaneous and polyclonal activation induced cytokine production in T cells were assessed by flow cytometry in patients with B‐CLL. Patients with progressive disease had a significantly increased number of T cells spontaneously producing IL‐2, IL‐4 and GM‐CSF as compared to healthy donors and patients with non‐progressive CLL, which was not the case for TNF‐α and IFN‐γ producing T cells. However, no difference in the frequency of T cells producing these cytokines was seen comparing patients with non‐progressive disease to control donors. Polyclonal activation of B‐CLL T cells in vitro induced an increased proportion of T cells producing these five cytokines in patients as well as in control donors, indicating that T cells in CLL patients might have a relatively well preserved functional capacity. However, the increase in GM‐CSF, TNF‐α and IL‐4 producing T cells was more marked in CLL patients than in controls. Furthermore, following activation, a higher frequency of cytokine‐producing T cells was noted in patients with progressive disease as compared to those with non‐progressive disease. The augmented number of cytokine‐producing T cells in CLL may indicate an up‐regulated capability of T cells to secrete cytokines, especially in patients with progressive CLL. The increased production of the T cell derived cytokines GM‐CSF, TNF‐α, IL‐4 and IL‐2 is interesting, as these cytokines have previously been shown to support growth of B‐CLL leukaemic cells in vitro and as T cells might specifically recognise the autologous leukaemic B cells in vivo. The findings may suggest a role for T cells in the pathogenesis of B‐CLL.

B-CLL cells with unusual properties

In studies concerning the interaction of B‐CLL cells and Epstein‐Barr virus (EBV), we encountered one patient whose cells had several unusual properties. In addition to the B‐cell markers, the CLL cells expressed the exclusive T‐cell markers CD3 and CD8 and carried a translocation t(18,22)(q21;q11), involving the bcl‐2 and Igλ loci. The patient represents the 4th reported CLL case with this translocation. The CLL cells could be infected and immortalized by the indigenous and by the prototype B958 virus in vitro. The T‐cell markers were not detectable on the established lines. In all experiments the immortalized lines originated from the CLL cells. Their preferential emergence over virus‐infected normal B cells may be coupled to the high expression of the bcl‐2 gene due to the translocation. In spite of the sensitivity of CLL cells to EBV infection in vitro, no EBNA‐positive cells were detected in the ex vivo population. In vitro, we could generate cytotoxic function in T‐lymphocyte cultures which acted on autologous EBV‐infected CLL cells. Therefore we assume that if such cells emerged in vivo they were eliminated by the T‐cell response.

EBV Infection of B-CLL Cells in vitro Potentiates Their Allostimulatory Capacity if Accompanied by Acquisition of the Activated Phenotype

Epstein‐Barr virus (EBV)‐carrying immortalized lymphoblastoid cell lines (LCLs) stimulate autologous T lymphocytes in vitro. This T‐cell response is independent of the EBV‐specific cellular memory because it also occurs in experiments with cells of seronegative individuals. The question can be posed whether the T‐cell‐stimulatory potential of the LCL is coupled to its immortalized state. B‐CLL cells were exploited to study this question because the majority of clones, represented by different patients, can be infected with EBV but they rarely become immortalized. We have investigated the phenotypic changes and the T‐cell‐stimulatory capacity of EBV‐infected B‐CLL cells. One aliquot of CLL cells was infected with EBV, another was activated with a mixture of Staphylococcus aureus (SAC), IL‐2 and the supernatant from the T‐cell hybridoma MP6 (activation mixture, AcMx) and the third aliquot received both treatments. In accordance with the individual features of the clonal populations represented by each patient, the immunophenotypic changes imposed by these treatments differed. With the samples of 3 patients the allo‐stimulatory potential showed the following ranking order: EBV and AcMx‐treated cells > AcMx‐treated > EBV‐infected. An analysis of several activation‐related surface markers and adhesion molecules on the cells did not reveal any association between their expression and the EBV‐imposed potentiation of allostimulatory capacity. These results may be extrapolated to EBV‐genome‐carrying normal B cells, suggesting that they can persist in vivo only as long as they have the resting phenotype. Once they are activated, these cells may be recognized and eliminated by T lymphocytes.

EBV-Activation of Human B-Lymphocytes

The Epstein-Barr virus (EBV) is a human herpes virus originally discovered in Burkitt lymphoma cell lines. The virus was found to specifically infect a sub-population of B-lymphocytes. In vitro infected B-cells are transformed into immortalized but non-tumorigenic lymphoblastoid cell lines (LCL). The cell lines maintain the genome in a latent form with only limited parts of the genome expressed and none or very little virus is produced (Menezes 1976). Some of the latently transcribed EBV genes code for different components of the EBV nuclear antigens (EBNA) expressed in the cell nucleus of all EBV-carrying cells (Reedman 1973).

Epstein-Barr virus-carrying B cells in the blood during acute infectious mononucleosis give rise to lymphoblastoid lines in vitro by release of transforming virus and by proliferation

In accordance with earlier studies, we detected higher numbers of Epstein-Barr virus (EBV)-carrying lymphocytes (B-EBV) in the blood of acute infectious mononucleosis (IM) patients and higher amounts of transforming EBV particles in the saliva compared to healthy seropositive individuals. B cells grew in cultures seeded with the low and high density IM lymphocytes. The majority of B cells which grew acquired the infection in vitro (2-step outgrowth), because addition of virus neutralizing antibodies considerably reduced the emergence of lymphoblastoid cell lines (LCLs). Only the minority of the explanted B-EBV cells proliferated. The antiviral drug phosphonoformate (PFA) did not influence the frequency of 2-step LCLs in the IM cultures. This may indicate that a large proportion of EBV carrying B cells have already entered the viral productive cycle in vivo and passed the PFA-sensitive stage at the time of explantation. Earlier experiments with blood of healthy seropositive individuals showed an inhibitory effect of PFA on the generation of LCLs. One healthy individual who entered this study as a control, probably had a reactivated EBV infection as judged by the anti-EA activity in his serum and the high level of virus in his saliva. He had antibodies against EBV nuclear antigens (EBNA), and therefore he did not have a primary infection at the time of the test. Judged by the number of wells with B cell growth, the frequency of virus-carrying B cells in his blood was low. It seems that anti-EBV immunity can control the number of infected B cells in the blood, but does not influence the virus load in the epithelial cells.

Induction of K562 lysis in autologous LGL and dense T cell cocultures

Human large granular lymphocytes (LGL) were cultured with autologous dense T lymphocytes for 0-8 days. The mixed LGL dense T cell cultures had enhanced lytic activity against K562 on day 4, as compared to LGL cultured alone. Dense T cells cultured alone had no K562 lytic activity. The mixed LGL dense T cell cultures were reseparated on day 4 into low dense SRBC- and SRBC+ and into high dense subsets. Both low dense subsets did lyse K562 whereas the high dense subset did not lyse K562. Since the LGL population was the low dense SRBC- cells, the results suggested that the day 4 low dense SRBC+ cytotoxic population in the mixed LGL dense T cell culture originated from noncytotoxic dense T cells. Dense T cells were also separated into OKT8- and OKT8+ subsets and cultured together with LGL for 0-8 days. The LGL dense OKT8- T cell coculture had enhanced K562 lytic activity while the LGL dense OKT8+ T cell coculture had no enhanced lytic activity, as compared to LGL cultured alone. These results indicated that LGL interacted with the dense OKT8- T cells to produce the enhanced K562 lysis.

Surface Marker Characterization of EBV-Carrying Cells in Blood of Healthy Seropositive Individuals

We have determined the phenotype of EBV-carrying cells in human blood by establishing spontaneous LCLs from populations selected according to three B cell markers, B1, B2, and 35.1C5. LCLs grew from the B1-positive, B2-positive, B2-negative and 35.1C5-positive population. Thus, EBV-carrying cells in blood of seropositive individuals are restricted to the B cell lineage. These B cells may or may not express the B2 epitope, and they may be derived from the lymph node mantle zone or the splenic marginal zone. These in vivo EBV-carrying cells could enter the viral productive cycle and transform coresident B cells during the initial 3 days in vitro. The characteristic of the LCLs with regard to these 3 B cell markers did not correspond to the original B cell population from which they were derived.