CD antigens 2001.

This paper reviews the Seventh Human Leucocyte Differentiation Antigen (HLDA7) workshop. Due to the limitations of "blind" antibody screening, which had been evident at the previous meeting in 1996, participants at HLDA7 adopted a more selective approach to the choice of antibodies by identifying new CD specificities. This resulted in the addition of more than 80 new CD specificities. Plans for the eighth and subsequent workshops are also previewed.

CD antigens 2001.

The most recent Human Leucocyte Differentiation Antigen Workshop ("HLDA7") took place in 2000 in Harrogate, UK and the proceedings are about to be published (Leucocyte Typing VII). New Sections were introduced in this Workship (Dendritic cells, Stem/progenitor cells, Erythroid cells and Carbohydrate Structures) and monoclonal antibodies were selected for which at least some molecular data were already available (to avoid "blind" screening of reagents against known specificities). A total of more than 80 new CD specificities were established (previously the average was less than 30 new CD specificities per Workshop) and these are listed in this article. There is already evidence for the existence of many new leucocyte surface molecules for study at the next HLDA Workshop (in Adelaide in 2004), and we have listed in this article a number of such potential CD candidates (identified following the production of monoclonal antibodies or via gene cloning). There are also today an increasing number of lineage- and/or stage-restricted leucocyte-associated molecules localised within the cell cytoplasm (or nucleus): they will certainly prove of intense in the future for many laboratories studying human haematopoietic cells (regardless of whether a new "intracellular CD" categorisation scheme is devised for such molecules).

Measles virus infection causes transient depletion of activated T cells from peripheral circulation.

BACKGROUND: Natural measles virus infection as well as vaccination with attenuated measles virus induce temporary immunosuppression, which is responsible for part of the morbidity and mortality associated with measles. The underlying molecular mechanisms are not known. Recently, in vitro studies have revealed a marked increase of LFA-1 expression of lymphocytes in the presence of infectious measles virus. OBJECTIVES: In order to further investigate immune dysfunction in measles we analyzed the expression of leukocyte function-associated antigen 1 (LFA-1) on ex vivo derived circulating human T cells. STUDY DESIGN: Expression of LFA-1 was measured by flow cytometry using monoclonal antibodies directed against CD11a and CD18. LFA-1 expression was followed in the course of infection in four adult seronegative vaccinees and in four patients with natural measles. RESULTS: There was a remarkable loss of LFA-1-bright cells during natural measles and after measles vaccination. The number of LFA-1-bright cells reached a minimum on day 7-14 after vaccination, and at the onset of rash during natural measles infection, and approached normal levels within 3-5 weeks. CONCLUSION: It is suggested that measles virus infection interferes with lymphocyte trafficking and reallocation. Disruption of recirculation and random homing of lymphocytes might contribute to the immunosuppression, which is characteristic for measles virus infection.

Active specific immunotherapy of renal cell carcinoma: cellular and humoral immune responses.

We investigated the effect of vaccinating renal cell carcinoma (RCC) patients with irradiated autologous or allogeneic tumor cells and Newcastle disease virus (NDV) as adjuvant on cellular and humoral antitumor immunity. By Western blot analysis, we found that vaccination induced antibody formation in 33 of 34 patients against NDV proteins but not against tumor cell related antigens. NDV proteins detected had molecular weights of 53 kDa, 55-56 kDa, and 66 kDa. ADCC by patients' isolated PBMC and patients' sera against autologous or allogeneic tumor cells was not enhanced after vaccine treatment in a nonradioactive cytotoxicity assay. Target cells infected with NDV were lysed more effectively (p < 0.05) in ADCC after vaccination than noninfected targets. Natural cellular cytotoxicity of patients' isolated PBMC was not altered during vaccine treatment. Specific lysis rates against autologous and allogeneic RCC cells not exceeded 10% (effector:target ratio 50:1). Specific lysis of K-562 cells was > 20%; a slight decrease in lysis during vaccination was not significant. Numbers of lymphocyte subsets from patients' peripheral blood analyzed by FACS revealed significant expression of CD20+ (p < 0.02) and CD39+ (p < 0.03) cell numbers by vaccine therapy. Cytokine detection in patients' sera by ELISA showed significant increases (p < 0.05) for IFN-gamma and TNF-alpha but not for IFN-alpha four h post vaccination. Thus, immunomodulation with autologous or allogeneic RCC tumor cell vaccines is mainly due to cytokine induction, whereas tumor specific humoral or cellular responses are not detectable in patients' peripheral blood.

A novel DNA-binding regulatory factor is mutated in primary MHC class II deficiency (bare lymphocyte syndrome).

Regulation of MHC class II gene expression is an essential aspect of the control of the immune response. Primary MHC class II deficiency is a genetically heterogeneous disease of gene regulation that offers the unique opportunity of a genetic approach for the identification of the functionally relevant regulatory genes and factors. Most patients exhibit a characteristic defect in the binding of a nuclear complex, RFX, to the X box motif of MHC class II promoters. Genetic complementation of a B-lymphocyte cell line from such a patient with a cDNA expression library has allowed us to isolate RFX5, the regulatory gene responsible for the MHC class II deficiency. This gene encodes a novel DNA-binding protein that is indeed a subunit of the RFX complex. Mutations in the RFX5 gene have been characterized in two patients. Transfection of the patient's cells with the RFX5 cDNA repairs the binding defect and fully restores expression of all the endogenous MHC class II genes in vivo.

Coexpression of stem cell factor and its receptor c-Kit in human malignant glioma cell lines.

Stem cell factor (SCF), a hematopoietic growth factor, is the ligand of the tyrosine kinase receptor encoded by the c-kit proto-oncogene. Beside the important role of this receptor-ligand complex in hematopoiesis, gametogenesis and melanogenesis, SCF and its receptor have been shown to be expressed in the brain. We have studied the expression of SCF and c-kit in 20 human malignant glioma cell lines at the mRNA as well as at the protein level. In addition, recombinant human (rh) SCF was tested in [3H]thymidine uptake assays for a mitogenic effect on these cells. SCF and c-Kit proteins were detected in the cytoplasm of glioma cells by alkaline phosphatase-monoclonal anti-alkaline phosphatase immunostaining and Western blot analysis. However, neither SCF nor c-Kit were seen on the cell surface by flow cytometry. Furthermore, none of the proliferation assays showed a mitogenic effect for exogenously added rhSCF. Blocking studies using an anti-SCF antibody failed to demonstrate modulating effects on the growth of selected cell lines. These results suggest that SCF and c-Kit may mediate non-proliferative signals or may employ intracellular mechanisms for autocrine growth regulation of glioma cells.

Characterization of human TUR leukemia cells: continued cell cycle progression in the presence of phorbol ester is associated with resistance to apoptosis.

Human TUR leukemia cells were generated as a subclone of U937 monoblastoid leukemia cells. There was no obvious difference in the ultrastructure of both cell lines. Like in U937 cells, the expression of monocyte-specific surface markers such as CD14 was negligible in TUR cells. U937 cells and other human myeloid leukemia cell lines (HL-60, THP-1) can be induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to differentiate along the monocytic pathway. In contrast, exposure to TPA had no effect on the induction of the differentiation program in TUR cells. Thus, the presence of leukocyte integrins including CD11 and CD18, which are significantly induced during TPA-induced differentiation of HL-60, U937 and THP-1 cells, remained nearly unchanged at low levels in both TUR and TPA-treated TUR cells. Furthermore, while expression of major histocompatibility complex (MHC) class II antigens on U937 and TPA-treated U937 cells is barely detectable, there was a significantly constitutive expression of MHC class II, particularly human lymphocyte antigen (HLA-DR) on the surface of TUR and TPA-treated TUR cells. Exposure of human myeloid leukemia cells to TPA is also associated with growth arrest resulting either in a retrodifferentiation process or in programmed cell death. In contrast, TUR cells continued to proliferate in the presence of TPA although the proliferative capacity was continuously reduced by increasing concentrations of TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Cystic fibrosis-type mutational analysis in the ATP-binding cassette transporter signature of human P-glycoprotein MDR1.

Members of the ATP-binding cassette transporter superfamily such as the P-glycoproteins (MDR) and the cystic fibrosis transmembrane conductance regulator (CFTR) share conserved sequence motifs in their nucleotide binding fold that are the major targets for CFTR mutations in patients with cystic fibrosis. Cystic fibrosis-type mutations were introduced at analogous positions into the human MDR1 gene. Heterologous expression of wild-type or mutated MDR1 revealed similar mRNA transcript levels in Chinese hamster ovary K1 recipients, but the subsequent processing was defective for all mutations that give rise to severe cystic fibrosis in the case of CFTR. Functional multidrug transporter MDR1, however, was obtained when amino acid substitutions were introduced into a less conserved position of the ATP-binding cassette transporter signature (codon 536 in MDR1). The profile of cross-resistance and chemosensitization was modulated in these codon 536 variants, which suggests that this region is involved in the drug transport function of P-glycoprotein.

Production of interleukin-1 beta and interleukin-6 in hepatoblastoma.

Thrombocytosis and fever are frequent symptoms in children with hepatoblastoma. Interleukin-6 (IL-6) has been shown to mediate thrombocytosis and an acute-phase reaction including fever. We therefore investigated samples from 14 untreated patients with hepatoblastoma for this cytokine and in addition for interleukin-1 alpha (IL-1 alpha), interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha), all of which are known to induce IL-6 production. High serum levels of IL-6 were only found in 3/14 patients; the other cytokines were not detectable. In contrast, 12/14 tumors produced substantial amounts of IL-6 in primary cell culture, while IL-1 beta was found in 3/14 supernatants; IL-1 alpha and TNF-alpha were always negative. Immunoenzymatic staining of fresh tumors revealed that IL-6 is not produced by the tumor cells, but rather by surrounding fibroblasts and endothelial cells. In tumor cells only IL-1 beta, but neither IL-1 alpha, TNF-alpha nor IL-6, could be detected. In co-culture experiments with fibroblasts and endothelial cells, addition of hepatoblastoma cells enhanced IL-6 production. Including an IL-1 receptor antagonist abolished this effect incompletely. Our results suggest that tumor cells in hepatoblastoma induce IL-6 production in surrounding fibroblasts and endothelial cells by virtue of their endogenous secretion of IL-1 beta and supposedly some other, as yet unidentified, mediator.

Expansion of peripheral blood natural killer cells correlates with clinical outcome in cancer patients receiving recombinant subcutaneous interleukin-2 and interferon-alpha-2.

Natural killer (NK) cells are believed to contribute to the clinical efficacy of cancer immunotherapy using recombinant interleukin-2 (rIL-2) in humans. In previous trials of high-dose i.v. rIL-2, however, no correlation has been established between circulating NK cells and treatment response. Between January 1989 and October 1990, we treated a total of 47 outpatients with advanced tumors using low-dose s.c. rIL-2 and interferon-alpha-2 (rIFN-alpha). Therapy consisted of a 2-day rIL-2 pulse at 18 million IU/m2/day, followed by 6 weeks of rIL-2 (3.6 x 10(6)-4.8 x 10(6) IU/m2/day x 5 days/week) and rIFN-alpha (5 x 10(6)-6 x 10(6) U/m2 x 3/week). Before and after therapy, we phenotypically evaluated circulating lymphocytes and correlated them with clinical response. During 6-week therapy, peripheral blood lymphocytes bearing the CD56 (NK-cell-associated) surface antigen were increased significantly (p < or = 0.005) in treatment responders [complete response (CR) and partial response (PR), n = 10; 3.8-fold] and stable disease (SD) patients (n = 20; 2.1-fold), while patients with progressive disease (PD, n = 17) exhibited no significant expansion of circulating NK cells (p > 0.1). After one 6-week treatment cycle, CR/PR patients had significantly more peripheral NK cells, when compared with patients in SD (1.6-fold) and PD (1.9-fold) (p < 0.04). The overall number of circulating lymphocytes was also increased upon therapy (1.6-fold; p < or = 0.001), but remained independent of response (p > 0.4). These data demonstrate that s.c. rIL-2 and s.c. rIFN-alpha produce a significant increase in peripheral blood NK cells; this expansion correlates significantly with treatment response in advanced tumor patients receiving long-term combination immunotherapy at outpatient doses.

Low-dose interleukin-2 in combination with interferon-alpha effectively modulates biological response in vivo.

Phenotypic characterization of peripheral blood lymphocytes was performed in patients with advanced metastatic cancer receiving low-dose recombinant interleukin-2 (rIL-2) and recombinant interferon-alpha (rIFN-alpha) as subcutaneous home therapy. A total of 31 patients with progressive metastatic renal cell carcinoma, malignant melanoma, colorectal cancer, B-cell lymphoma, and Hodgkin's disease, were evaluated. Patients were treated with a combination of low-dose subcutaneous rIL-2 and rIFN-alpha, consisting of a 2-day rIL-2 pulse at 9.0 million IU/m2 twice daily, followed by 6 weeks of combined low-dose rIL-2 at 1.8 million IU/m2 twice daily, 5 days per week, and rIFN-alpha at 5.0 million U/m2 3 times per week. This treatment regimen resulted in an overall significant (p < 0.002) increase in peripheral blood lymphocyte subsets expressing CD3, CD8, CD16, CD25, and CD56. Expansion of peripheral blood natural killer (NK) cells was correlated to treatment response. Thus, treatment-related increase in CD56-positive lymphocytes was 1.8-fold higher in complete or partial responders when compared to progressive disease patients (p = 0.0). Increase in NK cells upon low-dose rIL-2 and rIFN-alpha was associated with a significant expansion (p = 0.0) of peripheral blood eosinophils (r = 0.71). Patient pretreatment using rIL-2, rIL-2 and rIFN-alpha, or chemotherapy abrogated the treatment-induced induction of NK cells and IL-2 receptor- (CD25) positive T lymphocytes, respectively. Peripheral blood NK cells were significantly decreased (p < 0.05) in patients developing neutralizing antibodies specific to rIL-2.

Clinical and preclinical evaluation of recombinant PEG-IL-2 in human.

High dose interleukin-2 alone or in combination with lymphokine activated killer (LAK) cells has demonstrated antitumor activity in a variety of malignant diseases. The currently formulated recombinant human interleukin-2 (IL-2) has limited solubility and short circulatory half life resulting in limited bioavailability. To improve the bioavailability of IL-2 the protein was covalently bound to activated Polyethylenglycol (PEG). We designed a phase I/II trial to evaluate the bioactivity of PEG-IL-2 in man, given as intravenous (iv) bolus injection every two weeks, and to determine safety, efficacy, and the maximum tolerated dose (MTD) in patients with advanced malignancies. Assessment of cytokine levels, phenotypic analyses and differential blood counts were performed to investigate the effects of PEG-IL-2 in-vivo. To compare in-vitro PEG-IL-2 activity to activities of IL-2 we evaluated proliferation, cytotolytic activity, morphology, and phenotype of cytokine activated lymphocytes. Among seven patients treated with PEG-IL-2, there was no objective remission, three patients exhibited stabilisation of disease. Four patients presented with further disease progression. Treatment-related toxicity was mild to moderate (mainly WHO grades I and II) in patients receiving dose levels up to 10 x 10(6) IU/m2 (maximum tolerated single dose in the outpatient setting). No toxic deaths occurred. In comparison to IL-2, the pharmacokinetic profile of PEG-IL-2 exhibited increased plasma levels and a decreased clearance (alpha and beta half-life estimates of 4 and 14 hours, respectively). The analysis of a variety of immunologic parameters demonstrated that PEG-IL-2 has significant biologic activity both in vitro, and in man.

Pretreatment natural killer antigen density correlates to clinical response in tumor patients receiving long-term subcutaneous recombinant interleukin-2 and recombinant interferon-alpha.

We evaluated density of the natural killer (NK) cell-associated CD56 antigen on circulating NK cells of 47 patients with advanced renal cell carcinoma. Patients received a combination of low-dose subcutaneous recombinant interleukin-2 (rIL-2) and recombinant interferon-alpha (rIFN-alpha) as home therapy. Antigen density of CD56 before therapy was 2.2-fold higher (P < 0.005) in patients who subsequently achieved a complete or partial remission when compared with patients who presented with progressive disease on therapy. After a 6-week treatment cycle, NK cells of treatment responders expressed significantly (2.1-fold; P < 0.005) more CD56 antigens than NK cells in nonresponding patients. These results suggested a potential role of both pre- and posttreatment NK antigen density levels as a biologic correlate to treatment response.

Expanded macrophage precursor populations in BXSB mice: possible reason for the increasing monocytosis in male mice.

The BXSB mouse spontaneously develops an autoimmune disease that resembles human systemic lupus erythematosus (SLE). During their lifetime, male BXSB mice show an increasing monocytosis in the peripheral blood as opposed to their female littermates. This monocytosis is unique among autoimmune-prone mice. To test the hypothesis that alterations at the stem cell level may be responsible for this monocytosis, myeloid bone marrow precursor cells were examined in both male and female BXSB mice from 4 to 40 weeks of age. The number of M-CSF responding stem cells (CFU-M) and the number of GM-CSF responding stem cells (CFU-GM) were higher than in all other inbred mouse strains tested. In addition, male BXSB mice developed a progressive increase of CFU-M and CFU-GM in the bone marrow during their lifetime, which paralleled the peripheral blood monocytosis. The monocytosis in male BXSB mice is the result of a further expansion of the strain-specific high number of macrophage precursors by intrinsic factors, which may be attributed to the influence of the Yaa factor. The sex-specific expanded mononuclear phagocyte system may promote the autoimmune process and may be one reason for the dramatic course of murine SLE in male BXSB mice.