Direct demonstration of binding of aggregated mouse IgG1 to the 40-kDa Fc receptor for IgG (Fc gamma RII) in both high and low responders.

Several directly fluorochrome-conjugated murine monoclonal antibodies (mAb) of the IgG1 subclass and directed against B or T cell antigens were found to bind to monocytes via the 40-kDa Fc receptor for IgG (Fc gamma RII). As expected from the established polymorphism of Fc gamma RII, strong staining was observed in about 75% of individuals. In the remaining 25% staining was clearly weaker, but could be definitely demonstrated with a mAb against the B cell-specific CD19 differentiation antigen. Specificity of binding to Fc gamma RII was confirmed by the ability to block the binding of the CD19 mAb by pre-incubation with aggregated IgG1 or with mAb against Fc gamma RII. The extent of T cell proliferation induced with a CD3 mAb of the IgG1 isotype (a-Leu 4), which is dependent on the interaction of monocyte Fc gamma RII with the Fc portion of the CD3 mAb, exactly correlated with the amount of binding to Fc gamma RII in all individuals. Proliferation was dose-dependent for both high and low responders; cells of low responders did not proliferate at concentrations below 16 ng/ml of mAb, whereas there was a small but unequivocal proliferation at higher concentrations. These results confirm that monocytes from previously characterized "non-responders" are able to bind aggregated murine IgG1 via Fc gamma RII. They also demonstrate that directly labeled mAb can cause extensive nonspecific staining which may not be excluded by the use of control antibodies of the same isotype.

Flow cytometric detection of cytoplasmic antigens in acute leukemias: implications for lineage assignment.

This study aimed at optimizing the conditions for flow cytometric detection of myeloperoxidase (MPO), cytoplasmic CD3 (cCD3), and cytoplasmic CD22 (cCD22), which seem to be more reliable lineage-associated markers in acute leukemia than surface antigens. Fixation methods employing saponin as detergent resulted in accurate detection of MPO and cCD3, whereas cCD22 was detectable only after buffered-formaldehyde-acetone fixation. MPO was detected in 16/17 AML, but only in 1/6 ALL, the MPO positive ALL being also CD13 positive. MPO was detectable in 3/4 AML with T-lymphoid features; a case of myeloid antigen-positive T-ALL, however, was MPO-negative. cCD3 was expressed only in T-ALL, and five cases of lymphoid antigen-positive AML were cCD3-negative. We suggest that these flow cytometric assays are useful for the lineage assignment of poorly differentiated leukemias and contribute to the identification of truly biphenotypic acute leukemias.

Identification of binding site for heparin and other polysulfated glycosaminoglycans on human thrombocytes.

Heparin and other polysulfated glycosaminoglycans (GAGPS) can evoke an immune response when complexed to the surface of platelets in vivo. We have previously reported on the detection and characteristics of such antibodies leading to thrombocytopenia. Here we report data concerning the biochemical characterization of the heparin-/GAGPS-binding site on human thrombocytes. By means of heparin affinity chromatography, GAGPS chromatography and subsequent 'Western blotting' of specifically eluted proteins we were able to detect two proteins of apparent molecular mass of 207 and 170 kilodaltons, which could be reduced to 57 and 142 kilodaltons, respectively. The 170-kilodalton glycoprotein was stained by the carbohydrate specific PAS-reagent and by surface labeling with 3H. The second molecule binding to heparin (GAGPS)-Sepharose was detected by platelet surface labeling with 125I, protein-specific staining Coomassie brilliant blue and PAS staining. Binding studies using enzyme-labeling techniques with five monoclonal antibodies (MCA) against platelet surface proteins GPI, GPIIb/IIIa complex, GPIIIa and CRI (C3b receptor) revealed specific interaction of the 170-kilodalton heparin-binding glycoprotein with an MCA to GPI, suggesting identity or immunological cross-reactivity of these two moieties. No specific reactions were observed between the 207-kilodalton glycoprotein and any of the MCA tested. For further evaluation of the antigenicity of separated platelet proteins, an ELISA in microtiter plates was developed, and a cellular sandwich ELISA for serological detection of antibodies against whole native thrombocytes or GAGPS-derivatized proteins was set up.(ABSTRACT TRUNCATED AT 250 WORDS)

Simultaneous flow cytometric analysis of surface markers and nuclear Ki-67 antigen in leukemia and lymphoma.

The monoclonal antibody Ki-67 identifies an antigen present during the late G1, S, G2, and M phases of the cell cycle, whereas resting cells do not express this antigen. Immunostaining with Ki-67 provides a simple method with which to determine the growth fraction of a malignant cell population without requiring a laborious procedure or use of radioactive materials. Thus far, detection of Ki-67-positive cells by flow cytometry was limited because of nuclear location of the antigen. In this study, periodate-lysine-paraformaldehyde (PLP) fixation of cells in suspension, labeling with Ki-67, and the subsequent flow cytometric analysis of the tumor growth fraction is described. Fixation with PLP at -10 degrees C for 15 min rendered the plasma membrane permeable without destroying cell surface antigens. Thus double immunofluorescence studies using both a surface marker and Ki-67 could be performed. This offers the additional advantage of being able to define the phenotype of proliferating cells. This method was applied to determine the growth fraction in peripheral blood and bone marrow samples of patients with leukemia and non-Hodgkin's lymphoma. The results of Ki-67 studies in 91 patients are shown. A wide variability of individual Ki-67 values was observed within each entity. Use of this flow cytometric procedure substantially facilitates the quantification of proliferating cells in pathological blood and bone marrow samples.

Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease.

The aim of this study was to investigate to which extent acute leukemias could be monitored for residual disease by using atypical antigen combinations as leukemia-related markers. Atypical antigenic features were determined by double color flow cytometry and included coexpression of lymphoid and myeloid related antigens, unphysiological coexpression of immature and mature antigens, and lack of an antigen that is normally expressed during maturation. Atypical immunophenotypes were detected in 35 of 68 patients with AML (51.5%) and 15 of 24 patients with ALL (62.5%). When 12 patients with leukemia-associated markers were again analyzed at relapse, the relevant antigen combinations were retained in 11 of them. The sensitivity of this two color flow cytometric assay as determined in dilution experiments was 1 in 10(3) to 10(4) cells. Follow-up studies of bone marrow samples revealed that, after induction chemotherapy cells with leukemia-associated markers were detectable in several patients at a frequency of 0.5 to 4%, but only patients in whom the cells with atypical antigens never disappeared suffered from relapse. In contrast, patients who became negative for the atypical cells remained in complete remission (median remission duration after the first negative bone marrow assessment by flow cytometry 52 weeks, range 20-102). We conclude that atypical antigen combinations, which are present in a meaningful number of acute leukemias, are a valuable means of monitoring acute leukemia patients during follow-up. This flow cytometric approach can complement other strategies to get a more accurate definition of remission in acute leukemia.

The biological and clinical significance of the KI-67 growth fraction in multiple myeloma.

We tested the significance of the Ki-67 plasma cell growth fraction in 49 bone marrow samples from 42 patients with multiple myeloma (MM). As a new approach to study myeloma cell proliferation, strong positivity of the CD38 antigen as plasma cell related feature was simultaneously evaluated with nuclear Ki-67 expression in a flow cytometric double immunofluorescence assay. Mean Ki-67 values were significantly higher in MM at relapse (22.4 per cent +/- 10.4) as compared with MM at diagnosis (11.9 per cent +/- 8.4, p less than 0.005) and plateau-phase (10.0 per cent +/- 5.5, p less than 0.001), respectively. Serial observations in six patients confirmed this change in cell kinetic behaviour during the course of the disease. Elevated Ki-67 values correlated significantly with stage III (versus stage I, p less than 0.05), beta-2-microglobulin serum levels greater than 6 (p less than 0.001), plasmablastic morphology (p less than 0.001), and diploid myeloma cell DNA-content (p less than 0.005). No correlation was found between Ki-67 and immunoglobulin isotypes as well as immunophenotypic features (expression of CD10, CD33, and CD56) of myeloma cells. Clinically, six of seven patients with Ki-67 greater than 14 per cent at diagnosis had an unfavourable course (primary resistant disease or early relapse), and three of four patients with elevated Ki-67 values at plateau-phase relapsed within 3 months. Our results demonstrate the usefulness of Ki-67 in determining proliferative activity in MM and emphasize its value in the evaluation of the risk profile of MM patients.

An immunohistologic study of the distribution and status of activation of head and neck tumor infiltrating leukocytes.

We examined tumor infiltrating leukocytes (TIL) in frozen sections of 28 biopsies from squamous cell carcinomas of the head and neck (SCCHN). In so doing, we used monoclonal antibodies (MoAb) directed against various leukocyte antigens. As defined by HLe-1+ cells, leukocyte infiltration was present in all biopsies. The amount of HLe-1+ cells was more often greater in stage III than in stage IV lesions. Most of the TIL were identified as CD5+ T-lymphocytes. In contrast, CD19+ B-cells were sparse in most biopsies. CD14+ monocytes/macrophages were found in only a few specimens. The relative proportion of CD4+ T-helper cells was higher than or at least equal to CD8+ suppressor/cytotoxic cells in all samples tested. Interleukin-2 (IL-2) receptor+ lymphocytes were evident in 13 of 22 biopsies stained for CD25 reactivity, and were more often observed in stage III than in stage IV tumors. All biopsies from recurrent tumors had no detectable IL-2 receptor+ cells. Our findings provide evidence for a positive correlation between a greater amount of TIL in earlier stages of SCCHN. The presence of IL-2+ lymphocytes suggests that SCCHN may be capable of activating resting lymphocytes for further IL-2-induced proliferation.

Diagnostic and prognostic value of immunohistological bone marrow examination: results in 212 patients with lymphoproliferative disorders.

Cryostat sections of 246 consecutive bone marrow biopsies from 212 patients with lymphoproliferative disease were investigated using a panel of monoclonal antibodies (MOAb's) and an immunoperoxidase technique. Bone marrow involvement was demonstrated by immunohistological examination in 121/160 patients (76%) with non-Hodgkin lymphomas (NHL) and 16/23 patients (70%) with plasma cell malignancies; the definite immunological diagnosis could be performed in 77% and 88%, respectively. Reactivity with the MoAb Ki-67 correlated with clinical parameters: in all cases exhibiting more than 5% positive cells an unfavourable course was seen, independent of the histological subtype. Another MoAb of potential prognostic relevance is KiM 4 b, which reacts with follicular dendritic cells (FDC). Besides the presence of FDC in germinal center tumors (CB/CC and CC-NHL) we found FDC in a minority of cases with B-CLL (5/44) and IC lymphoma (4/18). In the latter group 3/4 patients showed a favourable clinical course (vs 2/14 without FDC). The MoAb Tü 1 could discriminate between the lymphoplasmocytoid (11/12 positive) and the lymphoplasmocytic (0/6 positive) subtype of IC lymphoma and has proven of diagnostic importance. Expression of IL-2 receptors, detected by MoAb anti-Tac (CD 25), was demonstrated on leukemic cells from patients with hairy cell leukemia (100%), B-CLL (82%), IC (61%), CC (50%) and CB/CC lymphoma (50%). A considerable number of reactive T-lymphocytes (5%-60% of tissue cells) were identified among the neoplastic B cells with a predominance of CD4+ cells in most cases with NHL, whereas the CD4+/CD8+ ratio was significantly lower in myelomas and non-infiltrated bone marrows. The potential meaning of these findings is discussed. The immunohistological bone marrow analysis represents an important additional method in the diagnostic procedures of lymphoproliferative diseases involving the bone.

Murine epidermal Langerhans cells express significant amounts of class I major histocompatibility complex antigens.

Epidermal Langerhans cells (LC) are leukocytes that express major histocompatibility complex (MHC) class II antigens and function as antigen-presenting and accessory cells. Caughman et al. [Caughman, S. W., Sharrow, S. O., Shimada, S., Stephany, D., Mizuochi, T., Rosenberg, A. S., Katz, S. I. & Singer, A. (1986) Proc. Natl. Acad. Sci. USA 83, 7438-7442] reported that LC are deficient in surface expression of MHC class I antigens, implying a specialization of these cells to class II-restricted antigen presentation. To readdress this obviously important issue, we have studied murine epidermal sheets prepared from B6 X BALB/c----B6 bone marrow chimeras 5 months after irradiation and bone marrow reconstitution. This enabled us to distinguish class I of LC from that of surrounding keratinocytes. When sheets were analyzed by immunofluorescence microscopy with monoclonal antibodies specific for donor class I antigens, donor-derived LC but not LC of recipient origin were stained. Appropriate controls for antibody isotype and MHC haplotype were negative. LC in epidermal cell suspensions, prepared from normal BALB/c and BALB/cBy mice (MHC haplotype d), were analyzed by flow cytometry as well as immunofluorescence microscopy. LC were stained by monoclonal antibodies to class I antigens of haplotype d, but not by isotype-matched control antibodies to class I antigens of haplotype k. We also found that LC were virtually depleted from epidermal cell suspensions by treatment with monoclonal antibodies to class I antigens of haplotype d and complement but not by treatment with control monoclonal antibodies and complement. Our data, therefore, show that LC express MHC class I molecules on their surface.

Phenotypic and functional lymphocyte recovery after CD34+-enriched versus non-T cell-depleted autologous peripheral blood stem cell transplantation.

To determine the effect of CD34+ selection on immune recovery after high-dose chemo/radiotherapy in the setting of autologous stem cell transplantation (ASCT), we analyzed quantitative and qualitative lymphocyte reconstitution for up to 1 year post-transplantation in 27 consecutive adult patients receiving either CD34+-enriched or unmanipulated autologous stem cell (SC) grafts. Pretransplant immunological parameters were identical for both treatment groups. Total lymphocyte counts as well as CD3+ T cells provided a similar course of recovery in both cohorts, returning to baseline values within the first 3 months. There were no significant differences in the reconstitution kinetics of CD4+, CD8+, CD45RA+, and CD45RO+ T cells. CD4+ and CD45RA+ T cells between the two groups were significantly decreased within the first 6 months, returning to pretransplant baseline values by 1 year. Although within the first 3 months the majority of CD3+ cells were activated as demonstrated by expression of HLA-DR, we observed a significant loss of CD25+ T cells in both groups within the first 6 months. B cell numbers returned to baseline values within 3 months but in vivo B cell function measured by serum immunoglobulin M (IgM) and IgA levels did not recover as early as 6 months post-transplantation. T cell function measured by proliferation in response to the lectins phytohemagglutinin (PHA) and Concanavalin A (ConA) and to alloantigens in the mixed lymphocyte reaction (MLR) was significantly impaired, but tended to return to pretransplant baseline values by 1 year. Although preliminary, our results provide strong evidence that T cell depletion (TCD) by CD34+ enrichment using the CellPro device does not result in delayed phenotypic immune reconstitution after autologous peripheral blood stem cell transplantation (PB-SCT). Even in the absence of a high thymic T cell regenerative capacity in adults, T cell numbers and subset distributions were restored within the time frame studied. T and B cell function, however, remained significantly impaired for a prolonged period of time (>6 months after SCT) with a more profound defect in patients autografted with CD34+-enriched SC.

A comparative study of the in vitro immunomodulatory activity of human intact immunoglobulin (7S IVIG), F(ab')2 fragments (5S IVIG) and Fc fragments. Evidence for post-transcriptional IL-2 modulation.

During the past few decades intravenous immunoglobulin (IVIG) has been used successfully in the treatment of various immunoregulatory disorders. Treatment results have been attributed to immunomodulation mainly via Fc receptors or by anti-idiotypic antibodies to disease-causing autoantibodies. From the present study it is clearly evident that 7S IVIG (intact immunoglobulin) as well as 5S IVIG [F(ab')2 fragments] and Fc fragments have a potent immunomodulatory capacity. We demonstrate that mainly 7S IVIG inhibits alloantigen-induced T-cell proliferation and generation of cytotoxic T lymphocytes. Reduced interleukin-2 (IL-2) protein levels in culture supernatants of IVIG-supplemented mixed lymphocyte reactions (MLR) but unchanged IL-2 mRNA levels strongly argue in favour of a post-transcriptional interference of IVIG with cytokines and/or cytokine production. Interferon-gamma (IFN-gamma), soluble IL-2 receptor (sIL-2R) and monokines such as IL-1 beta, IL-6, IFN-alpha and tumour necrosis factor (TNF-alpha) were not affected by IVIG supplementation to MLR. Fc fragments were superior to F(ab')2-containing IVIG (5S and 7S IVIG) in inhibiting lectin stimulation of peripheral blood mononuclear cells (PBMC), whereas natural killer (NK) cytotoxicity was primarily inhibited by Fc-bearing IVIG (7S IVIG and Fc fragments), suggesting multiple mechanisms of IVIG immunomodulatory activity.