Françoise Vuillier

Benchmarking a luciferase complementation assay for detecting protein complexes

1Department of Genetics, Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri, USA. 2British Columbia Cancer Agency, Canada’s Michael Smith Genome Sciences Centre, Vancouver, British Columbia, Canada. 3Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, California, USA. 4Center for Biomolecular Science and Engineering, University of California Santa Cruz, Santa Cruz, California, USA. 5Brain Tumor Research Center, Department of Neurosurgery, Helen Diller Family Comprehensive Cancer Center, University of California San Francisco, Santa Cruz, California, USA. 6Howard Hughes Medical Institute, Santa Cruz, California, USA. 7Department of Psychiatry, Washington University School of Medicine, St. Louis, Missouri, USA. e-mail: twang@genetics.wustl.edu or jcostello@cc.ucsf.edu

Evaluation of residual disease in B-cell chronic lymphocytic leukemia patients in clinical and bone-marrow remission using CD5-CD19 markers and PCR study of gene rearrangements.

We evaluated minimal residual disease (MRD) in 23 CD5 + B-chronic lymphocytic leukemia (CLL) patients who achieved clinico-hematological remission confirmed by bone-marrow biopsy. MRD was evaluated by dual marker analysis flow-cytometry using CD5 and CD19 markers, and by the study of Ig heavy chain gene rearrangements using the fast polymerase chain reaction (PCR). According to our laboratory conditions patients were considered to be in complete phenotypic remission when total CD19+ cells were < 25% and the ratio of CD5 + CD19 + /CD19 + cells was < 25%. According to these strict criteria only 9 of the 23 patients were in complete phenotypic remission. In order to evaluate the sensitivity of the above method, PCR analysis of the configuration of the Ig heavy chain gene region was performed in 12 of these patients. Five of 7 patients in complete phenotypic remission retained a detectable monoclonal rearrangement of the Ig heavy chain gene. For the remaining 5 patients in partial phenotypic remission, only one failed to show a monoclonal band and this is probably explained by the presence of an unusual gene rearrangement. In conclusion, this study suggests that PCR is more sensitive than dual marker flow-cytometry for evaluation of residual disease and that it is indeed possible to achieve complete remission at the molecular level, in B-CLL. Nevertheless, we suggest a word of caution as this was a retrospective study, and samples were not assessed before treatment. Thus the possibility that apparent molecular remission might correspond to unusual gene rearrangements cannot be completely excluded in these cases.

Natural killer (NK) cell activity during HIV infection: a decrease in NK activity is observed at the clonal level and is not restored after in vitro long-term culture of NK cells.

NK cell activity is impaired in HIV‐infected patients. The mechanisms behind the altered NK functions are not clear, and conflicting data concerning NK and antibody‐dependent cellular cytotoxicity (ADCC) activity have been reported. In order to investigate whether this impairment is also observed at the clonal level and whether it is related to a defect at the target cell binding and/or the post‐binding level, we evaluated highly purified NK cell lines and cloned NK cells obtained from 22 HIV‐infected patients at different stages of disease and compared them with normal controls for their ability to: (i) kill K‐562 and U‐937 cell lines using a 51Cr release assay; (ii) bind and kill K‐562 and U‐937 cells at the single cell binding level; (iii) release NK cytotoxic factor (NKCF), tumour necrosis factor‐alpha (TNF‐α) and interferon‐gamma (IFN‐γ); (iv) kill anti‐IgM preincubated Daudi cell line (ADCC activity). This study with cloned NK cells or NK cell lines from HIV‐infected individuals showed: (i) a decrease in their lytic capability against target cell lines; (ii) a low ability to form conjugates with K‐562 and U‐937 cell lines with respect to controls; (iii) a decreased ability to kill bound target cells; (iv) low levels of released NKCF, TNF‐α and IFN‐γ after incubation with U‐937 cells. Taken together, these findings suggest that the impaired NK cell function during HIV infection is also observed at the clonal level and is related to defects both at the target and post‐binding levels. However, the precise mechanisms remain to be determined. The inability to restore normal NK activity after long‐term culture in the presence of high levels of recombinant IL‐2 is in agreement with the hypothesis of a ‘general anergic process’ during HIV infection.

Functional monocyte‐derived dendritic cells can be generated in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL) remains an incurable disease. Although modern available treatments are able to induce disease regression, relapse almost inexorably occurs. Therefore, novel therapeutic strategies aimed at reducing the disease relapse rate are very much needed. Among these, the induction of tumour‐associated antigen‐specific cytotoxic T lymphocytes (CTL), through either DNA vaccines or injection of idiotype pulsed dendritic cells (DCs), has been actively investigated with encouraging preliminary results in B‐cell malignancies. As the CLL B lymphocyte characteristically expresses low amounts of surface immunoglobulin (Ig) and T cells from these patients have been reported to display impaired functional activity, there are concerns related to the possibility of generating specific cytotoxic antitumoral T cells in this disease. In addition, no information is presently available regarding the functional ability of CLL‐derived DCs. In the present work, freshly purified monocytes from CLL patients and normal donors were induced to differentiate in granulocyte–macrophage colony‐stimulating factor (GM‐CSF) and interleukin (IL)‐4 serum‐free medium and compared for their morphological, phenotypic and functional characteristics. Our results demonstrate that: (1) functional DCs can be generated from CLL patients with similar phenotype and function to those observed from normal donors; (2) in contrast to normal control subjects, monocyte‐derived DCs from CLL patients spontaneously secrete endogenous IL‐10; and (3) interferon (IFN)‐γ in combination with CD40L plays a major role in priming DCs from CLL patients for IL‐12 and IL‐15 production. Overall, these results indicate that it is possible to derive functionally competent DCs from circulating monocytes in CLL patients.

EVER Proteins, Key Elements of the Natural Anti-Human Papillomavirus Barrier, Are Regulated upon T-Cell Activation

Human papillomaviruses (HPV) cause a variety of mucosal and skin lesions ranging from benign proliferations to invasive carcinomas. The clinical manifestations of infection are determined by host-related factors that define the natural anti-HPV barrier. Key elements of this barrier are the EVER1 and EVER2 proteins, as deficiency in either one of the EVER proteins leads to Epidermodysplasia Verruciformis (EV), a genodermatosis associated with HPV-induced skin carcinoma. Although EVERs have been shown to regulate zinc homeostasis in keratinocytes, their expression and function in other cell types that may participate to the anti-HPV barrier remain to be investigated. In this work, we demonstrate that EVER genes are expressed in different tissues, and most notably in lymphocytes. Interestingly, in contrast to the skin, where EVER2 transcripts are hardly detectable, EVER genes are both abundantly expressed in murine and human T cells. Activation of CD4+ and CD8+ T cells via the TCR triggers a rapid and profound decrease in EVER expression, accompanied by an accumulation of free Zn2+ ions. Thus, EVER proteins may be involved in the regulation of cellular zinc homeostasis in lymphocytes. Consistent with this hypothesis, we show that the concentration of Zn2+ ions is elevated in lymphoblastoid cells or primary T cells from EVER2-deficient patients. Interestingly, we also show that Zn2+ excess blocks T-cell activation and proliferation. Therefore, EVER proteins appear as key components of the activation-dependent regulation of Zn2+ concentration in T cells. However, the impact of EVER-deficiency in T cells on EV pathogenesis remains to be elucidated.

Do CLL B cells correspond to naive or memory B-lymphocytes? Evidence for an active Ig switch unrelated to phenotype expression and Ig mutational pattern in B-CLL cells

Recent work suggests that chronic lymphocytic leukemia (B-CLL) expressing unmutated immunoglobulin V genes could correspond to the proliferation of naive B cells whereas those expressing mutated genes, may correspond to the proliferation of post-germinal center B cells. Current data from gene profiling expression have failed to demonstrate a clear-cut distinction between these two forms of B-CLL disease. In the present study, we have investigated the complete VH nucleotide sequence and the presence of RNA transcripts from different CH domains in 25 B-CLL patients. Our results demonstrate that: (1) expression of IgD is not related to the mutational frequency and activation of the isotype switch pathway; (2) isotype switch, leading to simultaneous expression at the transcriptional and protein level of IgM, IgD, IgG and IgA, occurs in a small percentage of patients, and (3) different mechanisms such as VDJ duplication and trans-splicing or RNA splicing of long nuclear transcript, could be involved in isotype switch. Our results highlight the difficulty in assigning a normal counterpart to B-CLL cells and raise the possibility that a different B cell development pathway, independent from classical germinal centers, might exist in B-CLL.

Impaired proliferative capacity and abnormal cytokine profile of naive and memory CD4 T cells from HIV-seropositive patients.

Purified naive and memory CD4 T cells from healthy donors, HIV+ asymptomatic carriers and AIDS patients were examined for their proliferative activity and their pattern of cytokine secretion (IL‐4, IL‐6, interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α)) upon stimulation with phytohaemagglutinin (PHA), phorbol myristate acetate (PMA) and cross‐linked anti‐CD3 MoAb, in the presence of recombinant IL‐2 (rIL‐2). We found a decrease in the proliferative capacity of naive CD4 T cells following stimulation with PHA and PMA, and a sharp decline in this response upon cross‐linked anti‐CD3 stimulation in both subsets, although it predominated in the naive subpopulation. In AIDS patients, less pronounced impairment of thymidine uptake by the naive subset was found upon PHA and cross‐linked anti‐CD3 MoAb stimulation. In addition, an altered secretion pattern of the different cytokines was observed, consisting of abnormal secretion of IL‐6 by both naive and memory cells, an abnormal pattern of IFN‐γ secretion and frequent loss of detectable lL‐4 production by HIV patients. These abnormalities were even more pronounced in AIDS patients than in the asymptomatic carriers. Overall, our results extend previous reports indicating functional impairment of memory CD4 subsets in HIV+ subjects by showing that this impairment involves naive CD4 T cells.

Enhancement of HIV-specific cytotoxic T lymphocyte responses by zidovudine (AZT) treatment

Zidovudine or 3′‐azido‐2′‐3′‐dideoxy‐thymidine (AZT) is an antiviral drug widely used to treat HIV‐infected patients. Because cytotoxic T lymphocytes (CTL) are thought to contribute actively to resistance against HIV‐induced disease, we studied sequentially 10 HIV‐infected individuals under zidovudine treatment for a period of 6–12 months. For a given patient all lymphocyte suspensions corresponding to the complete zidovudine therapy period were tested on the same day and on the same target cells. Patients were selected for expression of HLA‐A2 and/or HLA‐A3 class I transplantation antigen. HLA‐restricted cytotoxicity specific for env, gag and nef HIV proteins was quantified for each patient at 6 week intervals. The data clearly indicated that zidovudine has a beneficial effect on the CTL response during the first 6–12 weeks of treatment, inducing cytotoxicity levels up to 100‐fold stronger than base line. This effect was usually short lived. However, patients who maintained strong levels of cytotoxicity had better clinical and survival outlook than patients who had lost all detectable cytotoxic lymphocytes. It is proposed that AZT, among other effects, delays the onset of disease in HIV‐infected patients by contributing to the stimulation of the HIV specific CTL response.

Differential requirements for HIV‐1 replication in naive and memory CD4 T cells from asymptomatic HIV‐1 seropositive carriers and AIDS patients

One of the major routes for modulating HIV‐1 expression by infected T cells is through the control of transcription initiation from the HIV‐1 long terminal repeat (LTR), which is regulated either by ils own viral gene products or by several cellular DNA‐binding proteins induced during T cell activation. Previous work reported preferential HIV‐1 infection and replication of memory CD4 T eells from infected individuals, which was explained either by a higher viral burden of this subset or by differences between naive and memory cells in the activation of the general transcription machinery involved in HIV‐1 replication. In this work, we have studied HIV‐1 replication by highly purified naive and memory CD4 T eells from asymptomatic seropositive carriers (ASC) and AIDS patients following different activation signals. Our results demonstrate that viral replication in memory cells from ASC was observed after mitogenic (phytohaemagglutinin (PHA) and/or phorbol myristate acetate (PMA)) recombinant tumour necrosis factor‐alpha (rTNF‐α) and CD3‐mediated activation. In contrast, in naive subsets, early viral replication was almost exclusively observed upon CD3‐mediated activation. AIDS patients are characterized by similar levels of viral replication in both subsets after PHA and soluble or immobilized anti‐CD3 MoAb activation. However, naive subsets from AIDS patienis still displayed differential requirements since they failed to replicate HIV‐1 after treatmenl with PMA and rTNF‐a. Taken together, these results provide evidence thai HIV‐1 replication in CD4+ T cells from infected individuals is a function of the differentiation stage of the eells, the disease stage of the patient and the aetivation signal employed.

CD4+ lymphocytes from HIV-infected patients display impaired CD45-associated tyrosine phosphatase activity which is enhanced by anti-oxidants.

It has been proposed that signal transduction defects may, at least partially, account for the functional impairment of CD4+ lymphocytes during HIV‐1 infection. Recently, we have demonstrated that unresponsive CD4+ lymphocytes from these patients had reduced protein tyrosine phosphorylation after CD3 engagement, and that this defect was associated with constitutively altered levels of p56lck and p59fynkinases. Since CD45 is essential for T cell receptor (TCR) and CD2‐mediated activation of protein tyrosine kinases, we study here CD45‐associated tyrosine phosphatase activity in resting and activated CD4 T cells from HIV‐infected patients. We found a significant decrease in the basal and post‐activation phosphatase activity of CD45 which correlated well with impairment of proliferative responses. In addition, decreased levels of cellular thiols observed in resting CD4+ lymphocytes from these patients suggested a disturbed redox status. Although expression levels of CD45 were decreased in most patients, a significant recovery of phosphatase activity and proliferative responses was observed in most patients by preincubating cells with N‐acetyl‐L‐cysteine and β2‐mercaptoethanol. In some patients, anti‐oxidant treatment failed to significantly enhance phosphatase activity and proliferative responses. The low responses of purified CD4+ lymphocytes from these patients were associated with a high ratio of apoptotic cell death which did not appear to be influenced by anti‐oxidant treatment.