Virus-transformed Lymphoblastoid Cell Lines Research Articles

DNA repair capacity measured by high throughput alkaline comet assays in EBV-transformed cell lines and peripheral blood cells from cancer patients and healthy volunteers

We collected peripheral blood (PB) from 556 patients with various types of cancer who had undergone radiotherapy and from 81 healthy volunteers. We exposed whole PB and Epstein-Barr virus-transformed lymphoblastoid cell lines (EBLs) derived from the PB mononucleocytes to X-irradiation (5 Gy). Using the alkaline comet assay, we measured the immediate DNA damage and, at 15 min, the % residual damage. In PB, the immediate damage was similar in patients and healthy volunteers while the % residual damage (mean ± S.D.) was significantly higher in patients with breast (54.3 ± A23.9), cervical (54.7 ± A23.9), head/neck (56.8 ± A24.4), lung (60.1 ± 23.5), or esophageal cancers (59.5 ± A33.7) than in healthy donors (42.9 ± 19.6) ( P < 0.05). We did not observe such differences in the EBV-transformed cell lines. Thus, radiation sensitivity of fresh PB cells measured by the alkaline comet assay was related to cancer status.

Allele-specific transcript quantification detects haplotypic variation in the levels of the SDF-1 transcripts

It has been suggested that SDF1-G801A, a single nucleotide polymorphism (SNP) in the 3' untranslated region (UTR) of the SDF1 gene, is associated with susceptibility to diseases such as AIDS and type-I diabetes. However, experimental studies examining the effect of SDF1-G801A on SDF-1 expression have not supported its functional importance. In this study, to examine whether other polymorphisms have a cis-acting effect on SDF1 expression, we carried out haplotype analyses of the SDF1 gene and the allele-specific transcript quantification utilizing Epstein-Barr virus-transformed lymphoblastoid cell lines with heterozygous genotype for SDF1-G801A. Haplotype-based analyses on the proportion of the allele-specific transcripts revealed the presence of haplotypes associated with a decreased amount of the transcripts. In addition, we observed haplotypic variation in response to dibutyl cyclic AMP and tetradecanoyl phorbol acetate that enhances the levels of SDF-1 transcripts probably through activation of transcription factors. Showing evidence that polymorphisms other than the SDF1-G801A have a cis-acting effect on expression of SDF-1 transcripts, the results of this study contribute to the interpretation of previous disease-association studies and to the selection of SNP markers for future studies. As shown in this study, allele-specific transcript quantification coupled with haplotype analyses can be an effective tool for detecting cis-acting polymorphisms in expressional regulation.

Activation of epitope-specific memory cytotoxic T lymphocyte responses by synthetic peptides.

Cytotoxic T lymphocytes (CTL) recognize antigens as short peptides selected for presentation by their ability to bind to MHC class I molecules. Polyclonal Epstein-Barr virus (EBV)-specific memory CTL responses, reactivated from blood lymphocytes of HLA-A11-positive individuals by stimulation with the autologous EBV-transformed lymphoblastoid cell line (LCL), are often dominated by reactivites directed to the peptide epitope IVTDFSVIK (IVT), corresponding to amino acids 416-424 of EBV nuclear antigen-4 (EBNA4). We now report the selective activation of IVT-specific CTL by stimulation of lymphocytes with the corresponding synthetic peptide. A more than 10-fold increase in frequency of CTL clones with this specificity (from 8% to 96%) was obtained when the peptide was presented by HLA-A11-transfected T2 cells (T2/A11). Titration of synthetic peptide in cytotoxic assay demonstrated that clones activated under these conditions are as efficient as clones activated by conventional LCL stimulations. Induction of memory CTL responses required low surface density of MHC: peptide complexes, since reactivation was achieved by stimulation with T2/A11 cells pulsed with concentrations of peptide that are suboptimal for induction of target cell lysis. This protocol of activation revealed the presence of IVT-specific CTL precursors in a donor that failed to mount an IVT-specific response upon stimulation with the autologous B95.8 virus-transformed LCL. The results suggest that stimulation with synthetic peptide epitopes can be efficiently used for induction of memory CTL responses, and may be particularly helpful for the selective expansion of subdominant CTL specificities.

In vitro induction of tumor-specific HLA class I-restricted CD8+ cytotoxic T lymphocytes from patients with locally advanced breast cancer by tumor antigen-pulsed autologous dendritic cells

Background. Early dissemination of treatment-resistant tumor cells remains the major cause of metastatic recurrence and death in breast cancer patients. Dendritic cells (DCs) are the most powerful antigen-presenting cells, and recently DC-based vaccination has shown great promise for the treatment of human malignancies by immunological intervention. Materials and methods. CD8+ T lymphocytes derived from peripheral blood mononuclear cells stimulated in vitro with autologous breast tumor antigen-pulsed DCs were tested for their ability to induce a HLA class I restricted cytotoxic T lymphocyte (CTL) response against autologous tumor cells. To correlate cytotoxic activity by CTL with T cell phenotype, two-color flow cytometric analysis of surface markers and intracellular cytokine expression was performed. Results. DC pulsed with breast tumor extracts consistently elicited a tumor-specific HLA class I restricted CTL response in vitro in three consecutive patients harboring locally advanced breast cancer. CTL expressed strong cytolytic activity against autologous tumor cells but did not lyse autologous Epstein Barr virus-transformed lymphoblastoid cell lines and showed variable cytotoxicity against the natural killer-sensitive cell line K-562. In all patients, two color flow cytometric analysis of surface markers and intracellular cytokine expression demonstrated that tumor-specific CTL exhibited an heterogeneous CD8+/CD56+ expression and a striking Th1 cytokine bias (IFNγ high/IL-4 low). Conclusions. Tumor lysate-pulsed DCs can consistently stimulate specific CD8+ CTLs able to kill autologous tumor cells in patients with locally advanced breast cancer in vitro. Tumor antigen-pulsed DC-based vaccinations may be appropriate for the treatment of residual and/or chemotherapy-resistant breast cancer refractory to standard salvage treatment modalities.

Induction of tumor-specific cytotoxicity in tumor infiltrating lymphocytes by HPV16 and HPV18 E7-pulsed autologous dendritic cells in patients with cancer of the uterine cervix

Objective To evaluate the potential of autologous dendritic cells (DC) pulsed with HPV16 and HPV18 E7 oncoprotein in restoring tumor-specific cytotoxicity in populations of tumor infiltrating lymphocytes (TIL) for adoptive immunotherapy of cervical cancer patients. Methods Full-length E7-pulsed DC-stimulated CD8 + T cells derived from peripheral blood (PBL) and from tumor tissues (TIL) were tested and compared for their ability to induce a HLA class-I-restricted cytotoxic T lymphocyte (CTL) response against autologous tumor cells. In addition, in order to correlate cytotoxic activity by CTL with a particular lymphoid subset, analysis of surface antigens and intracellular CD3 ζ chain and two-color flow cytometric analysis of intracellular cytokine expression (IFN-γ vs IL-4) at the single cell level were performed. Results DC stimulation induced powerful cytotoxicity against autologous tumor target cells by TIL-derived CD8 + T cells from all three cervical cancer patients, while autologous Epstein–Barr virus-transformed lymphoblastoid cell lines were not lysed. Killing of autologous tumor cells was higher by CD8 + T cells from TIL compared to PBL ( P > 0.01) and was more strongly inhibited by anti-HLA class I MAb ( P > 0.05). Phenotypically, all CTL populations were CD3 +/CD8 +, with higher levels of CD56 expression by TIL-derived CTL. Finally, although a marked Type 1 cytokine bias (i.e., IFN-γ high/IL-4 low) was observable in both PBL- and TIL-derived DC-stimulated CD8 + T cell populations, TIL-derived CD8 + T cells showed a higher percentage of IFN-γ-positive cells compared to PBL. Conclusions Full-length E7-pulsed DC can consistently restore strong CD8 + CTL responses against autologous HPV16- and HPV18-infected cervical cancer cells. DC-stimulated TIL may represent a superior source of tumor-specific CTL compared to PBL for adoptive T cell immunotherapy of patients harboring metastatic or recurrent cervical cancer refractory to standard treatment modalities.

Variation in radiation-induced apoptosis in ataxia telangiectasia lymphoblastoid cell lines

Purpose : To investigate and compare the ability of Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) from healthy individuals (normals) and ataxia telangiectasia (A-T) patients to undergo apoptosis after exposure to ionizing radiation. Materials and methods : Four normal and eight A-T LCL were exposed to doses of up to 20 Gy ionizing radiation. Apoptosis induction was studied 24 h after irradiation using three different methods: measurement of caspase-3 activity, PARP-1 cleavage and estimation of the sub-G 1 cell fraction. Results : Of the eight A-T LCL tested, all harbouring truncating ATM mutations, five had a higher level of spontaneous apoptosis than the normal LCL as assessed by the sub-G 1 cell fraction. Four of the eight A-T LCLs showed a similar level of radiation-induced apoptosis after exposure to 5 Gy as the normal LCL. The other four A-T LCL showed a greater radiation-induced apoptotic response, as assessed by at least one of the three techniques. Conclusions : LCL from A-T patients can undergo ionizing radiation-induced apoptosis in spite of a defect in ATM-p53-dependent signalling pathways. However, the apoptotic response is characterized by a large degree of variability between the A-T cell lines, the causes of which remain to be established.

DNA Damage and Repair in Lymphoblastoid Cell Lines from Normal Donors and Fragile X Syndrome Patients

Background Because lymphocytes from fragile X patients have been reported as hypersensitive to bleomycin-induced chromatid breaks and because the number of trinucleotide repeats in families with fragile X syndrome has a propensity to expand, we have investigated the possibility that fragile X cells may be hypersensitive to DNA damage and have a lower capacity for DNA repair. Methods Lymphocytes from normal and fragile X syndrome donors were immortalized by Epstein-Barr virus transformation. Characteristics of fragile X syndrome including the folate-sensitive fragile site on chromosome Xq27.3, length of CGG repeat expansion, and FMRP expression in Epstein-Barr virus-transformed lymphoblastoid cell lines were analyzed by standard cytogenetic methods, Southern blot, and Western blot, respectively. Analysis of DNA damage and repair induced by hydrogen peroxide, bleomycin, ethyl methanesulfonate, 4-nitroquinoline-N-oxide, etoposide, and mitomycin C was carried out by single-cell gel electrophoresis assay (known as comet assay). Results Lymphoblastoid cell lines from fragile X donors had a folate-sensitive fragile site on chromosome Xq27.3, no or low FMRP expression, and expansion of the CGG repeat. Results of comet assay showed that fragile X cells were not more sensitive to mutagen-induced DNA strand breaks and did not have lower DNA repair capacity in comparison with normal cells. Furthermore, one fragile X cell line showed hyposensitivity to DNA strand breaks induced by hydrogen peroxide, bleomycin, and ethyl methansulfonate. Conclusions The results of this study do not support the notion that CGG trinucleotide expansion in fragile X syndrome is caused by permanent deficiency in DNA repair.

The Siblings With Ischemic Stroke Study (SWISS) protocol.

BackgroundFamily history and twins studies suggest an inherited component to ischemic stroke risk. Candidate gene association studies have been performed but have limited capacity to identify novel risk factor genes. The Siblings With Ischemic Stroke Study (SWISS) aims to conduct a genome-wide scan in sibling pairs concordant or discordant for ischemic stroke to identify novel genetic risk factors through linkage analysis.MethodsScreening at multiple clinical centers identifies patients (probands) with radiographically confirmed ischemic stroke and a family history of at least 1 living full sibling with stroke. After giving informed consent, without violating privacy among other family members, the proband invites siblings concordant and discordant for stroke to participate. Siblings then contact the study coordinating center. The diagnosis of ischemic stroke in potentially concordant siblings is confirmed by systematic centralized review of medical records. The stroke-free status of potentially discordant siblings is confirmed by validated structured telephone interview. Blood samples for DNA analysis are taken from concordant sibling pairs and, if applicable, from 1 discordant sibling. Epstein-Barr virus-transformed lymphoblastoid cell lines are created, and a scan of the human genome is planned.DiscussionConducting adequately powered genomics studies of stroke in humans is challenging because of the heterogeneity of the stroke phenotype and the difficulty of obtaining DNA samples from clinically well-characterized members of a cohort of stroke pedigrees. The multicentered design of this study is intended to efficiently assemble a cohort of ischemic stroke pedigrees without invoking community consent or using cold-calling of pedigree members.

Responses to ultraviolet-B in cell lines from hereditary melanoma kindreds.

Ultraviolet-B (UV-B) triggers a cascade of events involving cell cycle control genes leading ultimately to DNA repair or apoptosis. The hypothesis examined here is that the genetic abnormality predisposing to melanoma affects the ability of the cell to respond appropriately to UV-B, so favouring mutagenesis. Epstein-Barr virus-transformed lymphoblastoid cell lines from hereditary melanoma kindreds were irradiated with UV-B, and changes in p53, p21 and Bcl-2 expression and cell cycle phase distribution were analysed. Twenty-two cell lines were tested: 12 carriers of melanoma susceptibility and 10 non-carriers (unaffected first degree relatives). At 24 h after irradiation with 50 J/m2, 15 of the 22 cell lines showed a rise in G2/M. After 400 J/m2, all the cell lines showed a reduction or loss of G2/M and 17 of the 22 showed an S phase delay. More carriers than noncarriers of melanoma susceptibility showed significant S phase delay after 50 J/m2 (seven out of 12 carriers versus two out of 10 non-carriers). Six of the 10 pairs (carrier versus non-carrier) tested showed discordant cell cycle responses; however the nature of the difference was not universal. Bcl-2 reduction was seen 4 h post-irradiation in all the carriers and non-carriers. The p53 and p21 responses, although showing some individual variations, were not related to carrier status. These results show individual variations in response to UV-B irradiation among cell lines from the members of hereditary melanoma kindreds, but no consistent differences between carriers and non-carriers of melanoma susceptibility.

Human T cell responses to HPV 16 E2 generated with monocyte-derived dendritic cells.

Persistent infection with human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical cancer. The E2 protein is required early in viral infection and therefore may serve as a useful immune target for a vaccine aimed at prevention or therapy of premalignant lesions. Dendritic cells (DC) prepared from monocytes and pulsed with bacterially produced HPV 16 E2 C-terminus protein were used to stimulate autologous T cells over several rounds of stimulation. T cells were tested for gamma-interferon release by ELISPOT and for cytotoxic activity by (51)chromium release assays. To generate E2-expressing target cells for cytotoxicity assays, we constructed a recombinant vaccinia virus encoding HPV 16 E2, which was used to infect autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL). The results show that DC pulsed with E2 C-terminus protein induce gamma-interferon-releasing T cells as demonstrated by ELISPOT. Furthermore, we demonstrate E2-specific lysis of vaccinia-E2 infected autologous LCL by CD8+ cytotoxic T lymphocytes (CTL). E2-specific CTL did not lyse untreated autologous LCL or LCL infected with wild-type vaccinia and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. In addition, T cells stimulated with DC in the absence of E2 failed to demonstrate lysis of vaccinia-E2-labeled targets. Phenotypically, CTL populations were CD3+/CD8+. These results will facilitate the study of naturally occurring T-cell responses to HPV E2 in patients with cervical intraepithelial neoplasia and the development of immunotherapeutic strategies designed to treat this and other HPV-associated diseases.

Dendritic cells efficiently cross-prime HLA class I-restricted cytolytic T lymphocytes when pulsed with both apoptotic and necrotic cells but not with soluble cell-derived lysates.

Dendritic cells (DC) have been shown to efficiently present antigen to CD8(+) cytolytic T cells (CTL) when pulsed with apoptotic cells as a source of cell-derived antigen. Such cross-priming could not be detected by the use of necrotic cells, while conflicting results have been reported for cell-derived soluble lysates. In this study, we reinvestigated this issue by using autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL) as a source of antigen to pulse monocyte-derived DC. Both autologous and HLA-mismatched allogeneic LCL have been used either in the form of apoptotic or of necrotic cells or of soluble cell lysates. At day 7, DC were co-cultured with the autologous CD8(+) lymphocyte fraction for an additional 9 days, in the presence of exogenous IL-2 (added after 48 h). At the end of the culture period, CD8(+) CTL efficiently lysed autologous LCL only when they had been co-cultured with DC pulsed with necrotic or apoptotic cells. That an efficient cross-priming of autologous CD8(+) cells could be induced by DC pulsed with apoptotic or necrotic LCL but not with cell lysates was further demonstrated in assays of IFN-gamma production in response to short-term re-stimulation of CD8(+) cells with LCL. In addition, LCL-specific CD8(+) cells could specifically lyse autologous DC that had been pulsed with LCL-derived antigens, further suggesting that DC presented exogenous antigens on HLA class I molecules.

Gene-modified spontaneous Epstein-Barr virus-transformed lymphoblastoid cell lines as autologous cancer vaccines: mutated p21 ras oncogene as a model.

The transfer of genes coding for tumor antigens (Ags) into Ag-presenting cells is a promising strategy to develop cancer vaccines for patients not limited by major histocompatibility complex restriction. To test whether autologous lymphoblastoid cell lines (LCL) can be used as an alternative to dendritic cells for Ag presentation, we established LCL by spontaneous growth in cyclosporin-containing medium in vitro (SP-LCL). SP-LCL, which have an advantage over B95-8-transfected LCL in that they carry no risk of introducing a new infectious agent when used for vaccination, served as a permanent source for transfection with an episomal Epstein-Barr virus-based expression vector encoding the Ki-ras p21 oncogene carrying a point mutation at codon 12 (muRas) as a model tumor Ag. The ability of muRas-transfected SP-LCL (muRas-LCL) to induce immunoreactivity was determined in vitro. After electroporation with an optimized protocol, muRas-LCL expressed mutated ras peptides for a considerable period of time (at least 8 weeks) on the cell surface. The transfection procedure did not affect the expression of the costimulatory molecules B7.1, intercellular adhesion molecule-1, and leukocyte function associated Ag-3 by SP-LCL. muRas-LCL were able to induce muRas-specific cytotoxic T lymphocytes from three of three healthy donors and one of one patient with pancreatic carcinoma. Our results suggest that the gene transfer of muRas sequences to SP-LCL leads to an endogenous processing of this Ag in the major histocompatibility complex class I pathway and to functional presentation of antigenic peptides to CD8+ T lymphocytes. Autologous SP-LCL can serve as an unlimited renewable source for autologous cellular vaccines and appear to be good candidates as presenters for a wide range of human tumor Ags.

Induction of human papillomavirus-specific CD4(+) and CD8(+) lymphocytes by E7-pulsed autologous dendritic cells in patients with human papillomavirus type 16- and 18-positive cervical cancer.

Human papillomavirus (HPV) type 16 (HPV 16) and HPV type 18 (HPV 18) are implicated in the induction and progression of the majority of cervical cancers. Since the E6 and E7 oncoproteins of these viruses are expressed in these lesions, such proteins might be potential tumor-specific targets for immunotherapy. In this report, we demonstrate that recombinant, full-length E7-pulsed autologous dendritic cells (DC) can elicit a specific CD8(+) cytotoxic T-lymphocyte (CTL) response against autologous tumor target cells in three patients with HPV 16- or HPV 18-positive cervical cancer. E7-specific CTL populations expressed strong cytolytic activity against autologous tumor cells, did not lyse autologous concanavalin A-treated lymphoblasts or autologous Epstein-Barr virus-transformed lymphoblastoid cell lines (LCL), and showed low levels of cytotoxicity against natural killer cell-sensitive K562 cells. Cytotoxicity against autologous tumor cells could be significantly blocked by anti-HLA class I (W6/32) and anti-CD11a/LFA-1 antibodies. Phenotypically, all CTL populations were CD3(+)/CD8(+), with variable levels of CD56 expression. CTL induced by E7-pulsed DC were also highly cytotoxic against an allogeneic HLA-A2(+) HPV 16-positive matched cell line (CaSki). In addition, we show that specific lymphoproliferative responses by autologous CD4(+) T cells can also be induced by E7-pulsed autologus DC. E7-specific CD4(+) T cells proliferated in response to E7-pulsed LCL but not unpulsed LCL, and this response could be blocked by anti-HLA class II antibody. Finally, with two-color flow cytometric analysis of intracellular cytokine expression at the single-cell level, a marked Th1-like bias (as determined by the frequency of gamma interferon- and interleukin 4-expressing cells) was observable for both CD8(+) and CD4(+) E7-specific lymphocyte populations. Taken together, these data demonstrate that full-length E7-pulsed DC can induce both E7-specific CD4(+) T-cell proliferative responses and strong CD8(+) CTL responses capable of lysing autologous naturally HPV-infected cancer cells in patients with cervical cancer. These results may have important implications for the treatment of cervical cancer patients with active or adoptive immunotherapy.

Epstein-Barr virus-transformed lymphoblastoid cell lines of ataxia telangiectasia patients are defective in X-ray-induced apoptosis

Purpose: To investigate and compare the propensity of EpsteinBarr virus (EBV)-transformed lymphoblastoid cell lines (LCL) obtained from unaffected healthy individuals and ataxia telangectasia (A-T) patients to undergo apoptosis after X-ray exposure. Material and methods: The LCL were exposed to 1-4Gy X-rays at a dose-rate of 1.36Gy/min. At various post-irradiation times (0, 24, 48 and 72h) the induction of apoptosis was analysed by: (1) monitoring the formation of high molecular weight (HMW) DNA fragments by field inversion pulse gel electrophoresis (FIGE); and (2) morphological characterization of apoptotic cells after fluorescence staining. In parallel, cell-cycle distribution, monitored by DNA flow cytometry, was investigated in these cells. Results: The LCL obtained from the A-T homozygotes were resistant to undergoing radiation-induced apoptosis during the observation time used. On the contrary, LCL from unaffected healthy controls displayed significant radiation-induced chromatin fragmentation seen at 48h and 72h after irradiation. In these cells, radiation-induced G1-arrest (24h post-irradiation) preceded chromatin cleavage. In A-T LCL, the defective G1-arrest was not followed by apoptosis. Conclusions: In spite of a defective cell-cycle control, EBVtransformed LCL of A-T patients compared with unaffected healthy controls do not undergo X-ray-induced apoptosis, at least during their first post-irradiation cell cycle.

Definition of unique and shared T-cell defined tumor antigens in human renal cell carcinoma.

From peripheral blood mononuclear cells of a patient with renal cell carcinoma (RCC), we isolated several T-cell clones, which efficiently lyse the autologous RCC cell line (LE-8915-RCC), but not the autologous Epstein Barr virus-transformed lymphoblastoid cell line. Most of the cytotoxic T lymphocyte (CTL) clones recognize HLA-A1-positive allogeneic RCC cell lines, indicating that HLA-A1 is the restricting element for these T cells. One CTL clone exclusively recognizes the autologous tumor cells. The HLA-A1-restricted CTL clones can be divided further into two subsets of T-cell clones, one blocked by an HLA-A1-specific monoclonal antibody, the other not. The reactivity of HLA-A1-restricted T-cell clone 6/135 was studied in greater detail. This T-cell clone also recognizes a number of melanoma cell lines, indicating that expression of the antigen seen by this CTL clone is not restricted to RCC. Strikingly, the antigen is not exclusively expressed by tumor cell lines, because primary cultures of proximal tubulus epithelium cells, adult mesangial cells, and normal breast epithelium cells are also lysed. These results corroborate the notion that renal carcinoma cells are immunogenic by virtue of a broadly distributed antigenic structure that may serve as a target for cytotoxic T cells and may be a potential candidate for tumor vaccine development.

Nijmegen breakage syndrome cells fail to induce the p53-mediated DNA damage response following exposure to ionizing radiation.

The functionality of the p53-mediated pathway, activated in response to DNA damage, has been assessed in primary fibroblast cell cultures and Epstein-Barr virus-transformed lymphoblastoid cell lines derived from Nijmegen breakage syndrome (NBS) patients. This autosomal recessive disease is characterized by microcephaly, growth and mental retardation, chromosomal instability, radiosensitivity, and high cancer incidence. The recent mapping of the NBS gene to chromosome 8q21 demonstrates that NBS is genetically distinct from ataxia telangiectasia (AT). Changes in p53 protein levels were significantly reduced and delayed in all the NBS fibroblast cell cultures and lymphoblastoid cell lines examined compared to normal cultures over a 4-h period postirradiation (5 Gy). The transcriptional activation of p21(WAF1/CIP1) mRNA was also lower in 12 NBS fibroblast cultures examined. In agreement with an abrogated p53 function, NBS cells exposed to ionizing radiation show an abnormal cell cycle arrest at G1-S and a prolonged accumulation of cells in the G2 phase. In contrast, exposure to the alkylating agent methyl methanesulfonate results in similar increases of p53 and p21(WAF1/CIP1) mRNA in both cell types. The ATM gene transcript was found to be expressed at similar levels in NBS and normal cells, whereas it was strongly reduced in the AT homozygote cells examined. These results suggest that the ATM gene product cannot substitute for that of the NBS gene in the signaling of cellular damage produced by ionizing radiation and that both are involved in the activation of p53. The suboptimal p53-mediated response could contribute to the high cancer risk and radiosensitivity seen in NBS patients.

Human histocompatibility leukocyte antigen (HLA)-G molecules inhibit NKAT3 expressing natural killer cells : Munz, C., Holmes, N., King, A., Loke, Y.W., Colonna, M., Schild, H., Rammensee, H.-G. Department of Immunology, Institute for Cell Biology, Auf der Morgenstelle 15, 72076 Tubingen, Germany [Journal of Experimental Medicine 185/3, 385–391 (1997)

The class I region of the human leukocyte antigen (HLA) complex includes genes encoding the classical transplantation antigens (HLA-A, -B, -C), at least three nonclassical class I genes (HLA-E, -F, and -G), and many class I pseudogenes (including HLA-7.5p). We have used probes from DNA within or flanking the HLA -A, -F, -G, and −7.5p genes to construct a physical linkage map that places the HLA-F, -G, and −7.5p loci in order with respect to HLA-A. The map was constructed using clamped homogeneous electric field pulsed-field gel electrophoresis. DNA was isolated from LCL 721 (A1:B8, A2:B5), a human Epstein-Barr virus-transformed lymphoblastoid cell line (LCL), and from two γ-irradiation-induced mutants of LCL 721 lacking complementary class I haplotypes. The physical linkage data place HLA-G closest to HLA-A and place HLA-7.5p between HLA-G and HLA-F. The map constructed supports a maximum distance of 490 kilobases between HLA-A and HLA-F. Human Immunology 31, 180–185 (1991).

Isolation of a T-Cell Clone Showing HLA-DRB1*0405-Restricted Cytotoxicity for Hematopoietic Cells in a Patient With Aplastic Anemia

AbstractThe existence of T cells capable of inhibiting in vitro hematopoiesis has been shown in aplastic anemia (AA), although whether such inhibition is mediated by a specific immune reaction involving an HLA allele remained unknown. We isolated a CD4+ Vβ21+ T-cell clone that was most dominant among Vβ21+ T cells in the bone marrow (BM) of an AA patient whose HLA-DRB1 alleles included 1501 and 0405. The T-cell clone named NT4.2 lysed an autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) and phytohemagglutinin-stimulated lymphocytes (PHA-blasts) as well as allogeneic LCLs sharing HLA-DRB1*0405. Cytotoxicity against LCL cells and PHA-blasts by NT4.2 was blocked by anti–HLA-DR monoclonal antibody (MoAb) or anti-CD3 MoAb. NT4.2 also lysed autologous BM mononuclear cells enriched with CD34+ cells that had been cultured for one week in the presence of colony-stimulating factors as well as allogeneic CD34+ cells of a normal individual carrying HLA-DRB1*0405, cultured in the same way. Moreover, NT4.2 strongly inhibited colony formation by hematopoietic progenitor cells derived from cultured CD34+ cells sharing HLA-DRB1*0405. These results indicate that the AA patient has T cells capable of killing hematopoietic cells in an HLA-DRB1*0405-restricted manner and that such cytotoxic T cells may contribute to the pathogenesis of AA.

Isolation of a T-Cell Clone Showing HLA-DRB1*0405-Restricted Cytotoxicity for Hematopoietic Cells in a Patient With Aplastic Anemia

The existence of T cells capable of inhibiting in vitro hematopoiesis has been shown in aplastic anemia (AA), although whether such inhibition is mediated by a specific immune reaction involving an HLA allele remained unknown. We isolated a CD4+ Vbeta21+ T-cell clone that was most dominant among Vbeta21+ T cells in the bone marrow (BM) of an AA patient whose HLA-DRB1 alleles included 1501 and 0405. The T-cell clone named NT4.2 lysed an autologous Epstein-Barr virus-transformed lymphoblastoid cell line (LCL) and phytohemagglutinin-stimulated lymphocytes (PHA-blasts) as well as allogeneic LCLs sharing HLA-DRB1*0405. Cytotoxicity against LCL cells and PHA-blasts by NT4.2 was blocked by anti-HLA-DR monoclonal antibody (MoAb) or anti-CD3 MoAb. NT4.2 also lysed autologous BM mononuclear cells enriched with CD34+ cells that had been cultured for one week in the presence of colony-stimulating factors as well as allogeneic CD34+ cells of a normal individual carrying HLA-DRB1*0405, cultured in the same way. Moreover, NT4.2 strongly inhibited colony formation by hematopoietic progenitor cells derived from cultured CD34+ cells sharing HLA-DRB1*0405. These results indicate that the AA patient has T cells capable of killing hematopoietic cells in an HLA-DRB1*0405-restricted manner and that such cytotoxic T cells may contribute to the pathogenesis of AA.

Langerhans cell histiocytosis-clinicopathological reappraisal and human leucocyte antigen association

We have examined the clinicopathological correlates of 74 patients with histologically confirmed Langerhans cell histiocytosis. Factors that influenced disease outcome included, three or more organ/systems being involved, a disease onset before the age of 2 years, the involvement of certain vital organs/systems such as liver/spleen, bone marrow and lungs, and male gender. The total number of involved organs/systems was the single most important determinant of disease outcome. Mortality rate in patients with three or more organs/systems involved, was 26%, as compared with 0% in the group with one or two organs/systems involved (chi 2 = 11.2, P = 0.008). There were no familial cases in our series, but we looked for a possible immunogenetic association by tissue typing 46 Caucasian sufferers and comparing the results with 117 controls. We used normal peripheral blood lymphocytes in 39 cases, Epstein-Barr virus-transformed lymphoblastoid cell lines in 12 cases, and both peripheral blood and Epstein-Barr virus-transformed lymphocytes in five cases. The HLA-B7 antigen was significantly increased Langerhans cell histiocytosis patients (19 of 46 = 41.3%) compared with 19 of 117 (16.2%) in the control group chi 2 = 11.2, relative risk = 3.6, P value after correction = 0.013). Attempt to stratify the disease into single-system or multisystem disease did not result in any significant association.