Follicular B helper T cells express CXC chemokine receptor 5, localize to B cell follicles, and support immunoglobulin production.

Chemokines and their receptors have been identified as major regulators controlling the functional organization of secondary lymphoid organs. Here we show that expression of CXC chemokine receptor 5 (CXCR5), a chemokine receptor required for B cell homing to B cell follicles, defines a novel subpopulation of B helper T cells localizing to follicles. In peripheral blood these cells coexpress CD45RO and the T cell homing CC chemokine receptor 7 (CCR7). In secondary lymphoid organs, CD4(+)CXCR5(+) cells lose expression of CCR7, which allows them to localize to B cell follicles and germinal centers where they express high levels of CD40 ligand (CD40L), a costimulatory molecule required for B cell activation and inducible costimulator (ICOS), a recently identified costimulatory molecule of the CD28 family. Thus, when compared with CD4(+)CD45RO(+)CXCR5(-) cells, CD4(+)CD45RO(+)CXCR5(+) tonsillar T cells efficiently support the production of immunoglobulin (Ig)A and IgG. In contrast, analysis of the memory response revealed that long-lasting memory cells are found within the CD4(+)CD45RO(+)CXCR5(-) population, suggesting that CXCR5(+)CD4 cells represent recently activated effector cells. Based on the characteristic localization within secondary lymphoid organs, we suggest to term these cells "follicular B helper T cells" (T(FH)).

Inhibition of IL-6-dependent growth of myeloma cells by an acidic peptide repressing the gp130-mediated activation of Src family kinases.

An acidic domain (AD) of gp130 was previously found to interact with the Src family kinase (SFK) Hck. Here, the influence of myristoylated peptides derived from this AD was assessed in the mouse myeloma cell line, 7TD1. The IL-6-dependent growth of 7TD1 cells was reduced by approximately 75%, if 100 microM of myristoylated 18mer peptide (18AD) was included in the growth medium, but was unaffected by a control peptide with scrambled sequence (18sc). A similar differential inhibition by peptides 18AD and 18sc was observed for the erythropoietin-dependent growth of BaF-EH cells expressing chimeric erythropoietin receptor-gp130 and human Hck and for the human myeloma cell line INA-6. While the peptide 18AD concentration inhibiting 50% was approximately 30 microM in 7TD1 and BaF-EH cells, peptide 18AD did not significantly inhibit growth of IL-6-independent MM1.S myeloma and OKT1 hybridoma cells or of BaF-EH cells supplied with IL-3. Treatment with 100 microM peptide 18AD caused the same degree or 60% of apoptosis induction as IL-6 deprivation in 7TD1 or INA-6 cells, respectively. Co-immunoprecipitation experiments revealed that peptide 18AD interfered with the association of Hck and gp130 in 7TD1 lysates in a concentration-dependent manner. IL-6-treatment of INA-6 cells induced the kinase activities of Fyn, Lyn and Hck, but not Src, and the IL-6-induced SFK activities were inhibited by peptide 18AD. Expression in 7TD1 cells of a kinase-inactive Hck mutant (K269R) elicited a dominant-negative effect on cell number increases providing further evidence that SFKs are required for gp130 signalling in myeloma cells.

Dual function of membrane-bound heat shock protein 70 (Hsp70), Bag-4, and Hsp40: protection against radiation-induced effects and target structure for natural killer cells.

CX+/CX- and Colo+/Colo- tumor sublines with stable heat shock protein 70 (Hsp70) high and low membrane expression were generated by fluorescence activated cell sorting of the parental human colon (CX2) and pancreas (Colo357) carcinoma cell lines, using an Hsp70-specific antibody. Two-parameter flow cytometry revealed that Hsp70 colocalizes with Bag-4, also termed silencer of death domain, not only in the cytosol but also on the plasma membrane. After nonlethal gamma-irradiation, the percentage of membrane-positive cells and the protein density of Hsp70 and Bag-4 were found to be strongly upregulated in carcinoma sublines with initially low expression levels (CX-, Colo-). Membrane expression of Hsp70 was also elevated in Bag-4 overexpressing HeLa cervix carcinoma cells when compared to neo-transfected cells. In response to gamma-irradiation, neo-transfected HeLa cells behaved like Hsp70/Bag-4 low-expressing CX- and Colo-, and Bag-4-transfected HeLa cells like Hsp70/Bag-4 high-expressing carcinoma sublines CX+ and Colo+. Immunoprecipitation studies further confirmed colocalization of Hsp70 and Bag-4 but also point to an association of Hsp70 and Hsp40 on the plasma membrane of CX+ and Colo+ cells; on CX- and Colo- tumor sublines, Hsp40 was detectable in the absence of Hsp70 and Bag-4. Other co-chaperones including Hsp60 and Hsp90 were neither found on the cell surface of CX+/CX-, Colo+/Colo- nor on HeLa neo-/HeLa Bag-4-transfected tumor cells. Functionally, Hsp70/Bag-4 and Hsp70/Hsp40 membrane-positive tumor cells appeared to be better protected against radiation-induced effects, including G2/M arrest and growth inhibition, on the one hand. On the other hand, membrane-bound Hsp70, but neither Bag-4 nor Hsp40, served as a recognition site for the cytolytic attack mediated by natural killer cells.

Drug testing in established cell lines by flow cytometric vitality measurements versus clonogenic assay.

The clonogenic assay is widely considered to be the most valid test for predicting tumor cell sensitivity to cytostatic drugs. In this study it was compared with early growth curves of human leukemic cell lines (HL-60, K562, Reh) after treatment with different types of cytostatic drugs (Adriamycin,cis-diamminedichloroplatinum(II):,1-beta-D-arabinofuranosyl- cytosine, and 5-fluorouracil) for 1 and 24 h. Following drug treatment two parallel cultures were started: a soft agar culture for the clonogenic assay; and a liquid suspension culture for vital cell counting by measuring esterase activity with fluorescein diacetate at different time points. The latter was recorded using flow cytometry during the following 3 days in 12-h intervals. For each drug concentration a survival factor was calculated from the growth curve between 24 and 72 h. This survival factor takes into account both the y intercept of the extrapolated growth curve and the slope of the growth curve. The dose-response curves resulting from either the survival factors or the clonogenic assay were always nearly identical. The results demonstrate that in established cell lines flow cytometric determination of vital cell increase rates provides a convenient alternative to the clonogenic assay for drug testing.

Abrogation of p53-induced cell cycle arrest by c-Myc: evidence for an inhibitor of p21WAF1/CIP1/SDI1.

The tumor-suppressor p53 inhibits cell cycle progression by direct transactivation of the p21WAF1/CIP1/SDI1 gene, which encodes a universal inhibitor of cyclin dependent kinases (cdk). The proto-oncogene product c-Myc induces cell cycle progression and, in the absence of survival factors, apoptosis. However, a direct link between the cell cycle machinery and c-Myc has not yet been established. We show that c-Myc has not yet been established. We show that c-Myc abrogates a p53-induced G1-arrest without elevating the expression of cdks or cyclins involved in the G1/S-transition. Instead, the results suggest that c-Myc interferes with the inhibitory action of p21 on cdk/cyclin-complexes by inducing a heat-labile inhibitor of p21. The inactivation of p21 and related cdk-inhibitors may explain several of the oncogenic actions of c-Myc, including the induction of proliferation, immortalisation and the inhibition of differentiation. Modulation of cdk activity by the induction of an inhibitor of cdk-inhibitors represents a novel mechanism of cell cycle regulation in mammalian cells.

HLA-class II antigens on hemopoietic and stromal cells in human micro long-term bone marrow cultures.

Using a complement-dependent cytotoxicity assay (CDC), we analyzed the presence of HLA-class II antigens on both stromal and hemopoietic cells in a miniaturized human long-term bone marrow culture system. 4-Hydroperoxycyclophosphamide (4-HC)-resistant hemopoietic stem cells capable of restoring in vitro hemopoiesis on irradiated stromal cell layers were HLA-DR, -DP, and -DQ negative. In addition, these cells failed to bind the monoclonal antibody (mAb) Tü 39, previously proposed as a candidate for the recognition of a novel class II antigen, "-DY." On the other hand, the formation of confluent stromal cell layers was inhibited by HLA-DR- or -DP-specific mAbs, but not by the HLA-DQ-specific mAb Tü 22. This suggests the presence of HLA-DR- and/or HLA-DP-positive, but HLA-DQ-negative stromal precursor cells.

Synergistic effects of heat and ET-18-OCH3 on membrane expression of hsp70 and lysis of leukemic K562 cells.

We previously reported that cell surface expression of hsp70, the major stress inducible member of the 70-kDa heat shock protein family, is inducible by nonlethal heat as well as by treatment with the membrane-interactive compound alkyl-lysophospholipid 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) selectively on human tumor cell lines. Plasma membrane expression of hsp70 increases selectively the sensitivity of tumor cells to lysis and, therefore, might play an important role in the antitumor immune response. Here, we demonstrate that a combined treatment consisting of sublethal heat (41.8 degrees C) and a noncytotoxic concentration of ET-18-OCH3 (25 micrograms/mL) results in a synergistic increase in the amount of cell membrane-bound hsp70 on leukemic K562 cells and on freshly isolated bone marrow of a chronic myelogeneous leukemia (CML) patient, but not on peripheral blood lymphocytes or CD34+ hematopoietic progenitor cells of healthy human individuals. Under these conditions the repopulating capacity of progenitor cells was not influenced. The increased hsp70 membrane expression on leukemic K562 cells results in a significantly increased sensitivity to lysis mediated by natural killer cells. In contrast to leukemic cells, the lysis of peripheral blood lymphocytes and CD34+ progenitor cells that lack expression of hsp70 on their plasma membrane was not negatively influenced by this treatment. A nonspecific disruption of the plasma membrane could be excluded, because treatment with a nontoxic concentration of the detergent Tween20 did not have an influence on hsp70 cell surface expression or on the sensitivity to lysis. Our findings might have further clinical implications with respect to purging of bone marrow from patients suffering from leukemia at sublethal conditions to induce a tumor-selective immune response.

Characterization of hemopoietic cell populations from human cord blood expressing c-kit.

Human cord blood or bone marrow cells expressing the CD34 surface antigen include a population of pluripotent progenitors. We identified and isolated a subpopulation of cells coexpressing CD34 and c-kit, a transmembrane receptor with tyrosine kinase activity. Novel monoclonal antibodies (16A6, 14A3, 3D6) directed against the extracellular domain of c-kit were used for immunofluorescence labeling and sorting of low-density mononuclear cells (MNCs) from umbilical cord blood and bone marrow. The frequency of c-kit-labeled MNCs from cord blood (mean 5.0% +/- 2.1%, n = 16) was similar to that from adult bone marrow (mean 3.7% +/- 1.3%, n = 4). On average, 1.4% of CD34-positive cells were recorded in cord blood and 2.1% in bone marrow MNCs. Roughly 60% of CD34-positive cells coexpressed c-kit. The ability of CD34+/c-kit+ cells to form multilineage colonies (CFU-GEMM) was assayed after sorting with an antibody that did not show any significant effect on c-kit ligand (RL) or granulocyte/macrophage colony-stimulating factor (GM-CSF)-induced colony formation. For CD34+/c-kit+ cells, we found a 20- to 50-fold enrichment as against total MNCs, and a 2-fold enrichment if compared with the CD34+/c-kit-population. To study expression of c-kit in lymphocytic precursors, monoclonal anti-CD7 or anti-CD10 antibodies were used simultaneously. In contrast to CD34-expressing cells, however, no consistent double-labeled subpopulation of lymphocytic cells was detected. Furthermore, coexpression of CD38 (73% +/- 14%, n = 4) or CD33 (29% +/- 12%, n = 5) on a majority of c-kit-positive cells showed their lineage commitment to erythropoiesis and granulocytopoiesis.

Cell membrane damage by ultrasound at different cell concentrations.

Cell membrane damage by ultrasound was studied in human nucleated cells in vitro at various concentrations. Suspensions of human blood cells, cells of a human leukemic cell line (Reh) and mixtures of nucleated cells with erythrocytes were exposed to continuous ultrasound of 782 kHz at a SPTA intensity of 15 W/cm2. The surviving nucleated cells without membrane damage were counted on the basis of exclusion of ethidium bromide using a flow cytometer. At high cell concentrations as present in whole blood, we observed no cell death, whereas below 5 X 10(7) cells/ml most of the granulocytes, stimulated lymphocytes and Reh cells were damaged. The concentration threshold below which cells were damaged seemed not to depend on the size of the cells, rather on the concentration of particles in the suspension.

Effect of 5-fluoro-2'-deoxyuridine (FdUrd) on 5-bromo-2'-deoxyuridine (BrdUrd) incorporation into DNA measured with a monoclonal BrdUrd antibody and by the BrdUrd/Hoechst quenching effect.

The purpose of this study was to improve the application of bromodeoxyuridine (BrdUrd) for the flow cytometric analysis of cell kinetics. In order to obtain a quantitative measure of the DNA synthesis rate (or the number of divided cells), BrdUrd should replace thymidine (dThd) completely in the newly synthesized DNA strands. The de novo synthesis of dThd monophosphate competing with BrdUrd incorporation was stopped by fluorodeoxyuridine (FdUrd). Cells of a human leukemic cell line (REH) were exposed to BrdUrd for either 20 min, 8 h, or 24 h. Bromodeoxyuridine incorporation was determined by a monoclonal antibody as well as by the BrdUrd/Hoechst (H) technique. Counterstaining of the DNA was performed with propidium iodide or ethidium bromide. DNA fluorescence was measured in both techniques with a two-parameter flow cytometer, the histograms being analyzed by computer. It was found that FdUrd is required in the BrdUrd/H technique for replacement of dThd at low BrdUrd concentrations and long incubation times. With short incubation periods, as used for detection by the monoclonal anti-BrdUrd antibody, FdUrd increases the incorporated BrdUrd amount when BrdUrd concentrations of 10 microM or less are applied.

[The mirror as a fixation aid for the fundus camera (author's transl)]. / Spiegel als Fixierhilfe für die Funduskamera.

A supplementary mirror is described for the fundus camera easily permitting correct adjusting of the central and temporal regions of the ocular fundus.

Simultaneous enrichment of HL-60 cells in G1 and separation from differentiating cells by centrifugal elutriation for studies on differentiation induction.

Cultures of the promyelocytic leukemia cell line HL-60 usually contain considerable numbers of spontaneously differentiating cells and are asynchronous in terms of cell-cycle phases. Counterflow centrifugal elutriation studies have been conducted to obtain a homogeneous cell population with regard to cell-cycle phases and stage of differentiation. Despite their small volume and probably because of their high buoyant density, differentiated cells are elutriated predominantly at higher flow rates. Accordingly, G1 cells elutriated at low flow rates are substantially free from spontaneously differentiating cells. By optimizing the technique, a population with approx. 90% G1 cells and less than 1% spontaneously differentiating cells was obtained. The yield in the fractions chosen was 5.1% of all cells recovered from elutriation. In culture, a cell population of this purity maintains a synchronous cell cycle for more than 2 days. This allows an exact determination of the time after induction when the first signs of differentiation occur. The presence of 1 microM retinoic acid (RA) causes the first significant increase of NBT-positive cells between the 24th and 27th h of culture.

Pteridines are produced during interleukin 2-induced T-cell proliferation and modulate transmission of this signal.

Pteridine levels of interleukin 2 (IL-2) receptor+ T-cell populations have been determined by HPLC after iodine oxidation; neopterin was monitored in the culture supernatants by radio-immunoassay. Upon addition of IL-2, cellular levels of biopterin and 6-hydroxymethylpterin rise transiently from 0.02 to 0.9 pmol/10(6) cells, cellular levels of neopterin from 1.5 to 4.1 pmol/10(6) cells. They peak at 8 and 13 h, respectively, after exposure to IL-2. Neopterin is not accumulated in the culture supernatant. DNA synthesis in T cells begins 10-12 h after adding the lymphokine and the portion of cells that undergo S-phase transition gradually increases during the subsequent 10 h. Entry into DNA synthesis phase is markedly accelerated if IL-2 is supplied together with tetrahydrobiopterin (0.8-1.6 X 10(-6) M) and the kinetics of entry into the S-phase transition during the period of 6-20 h become linear. This indicates that tetrahydrobiopterin modulation of IL-2 activity (Ziegler, I. et al. Naturwiss 72 (1985) 330) is an early event occurring during IL-2 signal transmission.

Vitality measurement using spectrum shift in Hoechst 33342 stained cells.

A bivariate flow cytometric technique has been developed in which Hoechst 33342 stained vital cells are discriminated from early damaged cells by differences in their fluorescence emission spectra. A discrete population of cells with intact cell membranes passing from vital to dead could be identified by using propidium iodide exclusion in combination with a concentration-independent spectrum shift of Hoechst fluorescence to longer wavelengths. The appearance after injury of two subsets of cells with intact membranes representing vital and early damaged cells, respectively, indicates two types of binding of Hoechst 33342 to these cells, presumably resulting from differences in chromatin structure. The method was tested in murine and human hemopoietic cell lines by cytotoxic treatment with cytosine arabinoside and interleukin 3 deprivation in factor-dependent lines. The power of the method was demonstrated by a cell cycle analysis of the early damaged cells after both cytotoxic treatment and growth factor deprivation.

Lactate production and amino acid incorporation in interleukin 3-dependent, factor-deprived hemopoietic murine cell lines.

Interleukin 3 (IL-3) stimulates the growth of various types of hemopoietic progenitors. In vitro, survival of a series of murine cell lines derived from either neoplastic or nonneoplastic hemopoietic tissue shows a strict IL-3 dependence. In order to test the implication of energy metabolism in this dependence as claimed in several studies, intracellular ATP levels as well as accumulative lactate release were measured in the murine hemopoietic lines FDC-P1, 32Dcl.23, DA-1, DA-3, NFS-60, and NFS-78. ATP levels showed little or no changes within 4-6 h of IL-3 starvation. In the absence of IL-3 the accumulative lactate release ranged from 1.4 to 2.6 mM, and in its presence values between 1.5 and 3.4 mM were recorded within 7 h. Only 32Dcl.23 showed an almost complete suppression of lactate release upon IL-3 withdrawal. The cell cycle times of these cell lines determined by flow cytometry ranged between 9 (DA-3) and 24 h (NFS-78). In the presence of IL-3 there was a significant inverse relationship between cell cycle times and lactate production. It is concluded that neither ATP generation nor the metabolic pathway of lactate production, although the latter correlated with proliferative activity in the studied cell lines, is controlled by IL-3. Furthermore, no control by IL-3 of essential amino acid incorporation into proteins was detected in cell lines 32Dcl.23 and NFS-60.

Measurement of efflux from G1-phase in a growth factor dependent cell line.

In order to test a mathematical model of G1/S-phase transition, the proliferative response of the murine myeloid interleukin 3 (IL-3) dependent cell line NFS-78 to graded reduction of IL-3 levels was measured. Exponentially growing cells were exposed to bromodeoxyuridine (BUdR), which replaces thymidine (TdR) in the DNA double strands during DNA synthesis. After incubation periods ranging from 3 to 36 h the cells were fixed and stained with a fluorescence dye mixture of Hoechst 33258 and ethidium bromide (EB) and subsequently analyzed in a two-parametrical flow cytometer. The BUdR-quenched TdR-specific Hoechst 33258 fluorescence of each cell provides information on the cell cycle location at the start of incubation and on whether or not a cell has divided. The DNA-specific EB fluorescence provides information on the actual cell cycle location at the end of the incubation period. From the 2-dimensional fluorescence distributions the efflux from G1-phase was calculated. Upon IL-3 reduction the cells showed accumulation in the G1-phase along with a reduction in the progression rate through the other phases of the cell cycle. By staining with the vital dye Hoechst 33342 as well as with propidium iodide (PI) it was further possible to show that cell death after IL-3 withdrawal occurred in all phases of the cell cycle.

Combination of BUdR-quenched Hoechst fluorescence with DNA-specific ethidium bromide fluorescence for cell cycle analysis with a two-parameter flow cytometer.

Stimulated or asynchronous L-cells were grown in a BUdR-medium, harvested and stained with a combination of 33258 Hoechst and ethidium bromide for analysis in a FACS II cell sorter. The u.v. laser line served as a light source for exciting the Hoechst fluorescence, the ethidium bromide fluorescence being excited mainly by energy transfer from the Hoechst dye. The quenched Hoechst fluorescence was analysed between 410 nm and 480 nm, the DNA specific EB fluorescence at beyond 630 nm. Thus, not only the actual location of each cell in the cycle could be determined, but also its initial location at time 0 of the experiment, together with its moment of division (BUdR-quenched Hoechst fluorescence). This method could become a powerful tool in many investigations dealing with cell cycle perturbations in culture.

Cell cycle analysis by combining the 5-bromodeoxyuridine/33258 Hoechst technique with DNA-specific ethidium bromide staining.

Cells were grown in 5-bromodeoxyuridine(BrdUrd)-containing medium, harvested and stained with a combination of 33258 Hoechst and ethidium bromide for analysis in a two-parametrical flow cytometer. Both fluorescences were excited by UV laser light, and ethidium bromide was additionally excited by energy transfer from the Hoechst dye. The separation of the two fluorescences proved to be excellent, the projected histograms showing a quality comparable to the quality obtained by single dye staining. The ethidium bromide fluorescence gives information as to where the cell is located within the cycle of DNA replication, while the BrdUrd-quenched Hoechst fluorescence gives information as to where in the cycle the cell was located at the beginning of BrdUrd incorporation. In this way information is obtained concerning the distance a cell traveled through the cycle during the BrdUrd incubation time. This method may become a powerful tool in many investigations dealing with cell cycle perturbations in culture.

Effect of the pulse length of ultrasound on cell membrane damage in vitro.

Suspended cells of a human lymphoblastic cell line were exposed to pulsed ultrasound of 775 kHz. The pulse lengths were varied between 16 and 1000 microseconds. The mark/space ratio was always kept at 1:1. Two ultrasound intensity levels were used: 3.6 and 6.4 W/cm2 spatial peak and temporal peak. After an exposure time of 5 min, cell membrane damage was measured cytometrically by a dye exclusion test. No membrane damage was observable at 16 microseconds, whereas, at pulse lengths of 1000 microseconds, about one-third of the cells were damaged.

Permeabilization of the plasma membrane of L1210 mouse leukemia cells using lithotripter shock waves.

Permeabilization of L1210 cells by lithotripter shock waves in vitro was monitored by evaluating the accumulation of fluorescein-labeled dextrans with a relative molecular mass ranging from 3,900-2,000,000. Incubation with labeled dextran alone caused a dose- and time-dependent increase in cellular fluorescence as determined by flow cytometry, with a vesicular distribution pattern in the cells consistent with endocytotic uptake. Shock wave exposure prior to incubation with labeled dextran revealed similar fluorescence intensities compared to incubation with labeled dextran alone. When cells were exposed to shock waves in the presence of labeled dextran, mean cellular fluorescence was further increased, indicating additional internalization of the probe. Confocal laser scanning microscopy confirmed intracellular fluorescence of labeled dextran with a diffuse distribution pattern. Fluorescence-activated cell sorting with subsequent determination of proliferation revealed that permeabilized cells were viable and able to proliferate. Permeabilization of the membrane of L1210 cells by shock waves in vitro allowed loading of dextrans with a relative molecular mass up to 2,000,000. Permeabilization of tumor cells by shock waves provides a useful tool for introducing molecules into cells which might be of interest for drug targeting in tumor therapy in vivo.