Separation of antigen-specific lymphocytes. I. Enrichment of antigen-binding cells.

Normal mouse spleen cells were fractionated in dishes coated with thin layers of DNP-gelatin or NIP-gelatin, which were insoluble at 4 degrees C. Highly viable cells were recovered from the dishes by melting the gel at 37 degrees C. NIP3- gelatin layers bound approximately 0.1% and DNP4-gelatin layers 0.5% of normal spleen cells. Increasing numbers of low affinity cells were bound with increasing DNP density of the adsorbent. The binding to insoluble DNP-gelatin was hapten-specific since it was inhibited by DNP-lysine, soluble DNP-gelatin or DNP-BSA but not by soluble gelatin or bovine serum albumin (BSA). It was also inhibited by a polyvalent rabbit antimouse Ig. DNP-gelatin was detected on the surface of cells recovered from DNP-gelatin-coated dishes by 125-I-labeled anti-DNP Ig. The cell surface bound DNP-gelatin could be removed by treatment with collagenase. Collagenase treatment did not detectably affect cell viability or surface receptors. More than 90% of DNP-gelatin binding cells were labeled with a polyvalent 125-I-labeled antimouse Ig before or after collagenase treatment under conditions known to label B lymphocytes. Furthermore, the specific antigen-binding capacity of the purified cell populations could be demonstrated after treatment with collagenase. Purified DNP4-gelatin binding cells contained more than 100 times as many DNP-RFC than unfractionated cells. The enrichment of NIP-RFC in the cell population recovered from NIP3 gelatin-coated dishes was more than 200-fold.

Antigen-induced aggregation and modulation of receptors on hapten-specific B lymphocytes.

Mouse spleen cells were subjected to a fractionation procedure designed to enrich for 4-hydroxy-3-iodo-5-nitro-phenylacetyl (NIP)- or DNP-specific B lymphocytes, which depended on adherence of specific cells to a layer of hapten-gelatin at 4 degrees C, recovery of bound cells by melting, and digestion of adherent antigen by collagenase. A population of cells resulted which contained 90% typical B cells and 37% of cells capable of binding a fluorescent, haptenated polymeric protein. Fractionated cells were reacted in vitro with fluorescent conjugates of the specific haptens with polymerized flagellin [NIP-polymerized flagellin (POL)-tetramethylrhodamine isothiocyanate conjugate or DNP-POL-fluorescein isothiocyanate conjugate] under a variety of conditions, with the aim of investigating the behavior of Ig receptors on B lymphocytes after exposure to antigen; Experiments were performed with immunogenic and tolerogenic concentrations of antigen. Furthermore, four experimental designs were used, namely: (a) brief labeling with fluorescent antigen followed by culture without antigen (pulse design); (b) culture in the continuous presence of fluorescent antigen (continuous-labeling design); (c) culture in the continuous presence of nonlabeled antigen followed by labeling of unoccupied receptors by fluorescent antigen (receptor status design); and (d) culture with nonlabeled antigen for 2 h followed by incubation without further antigen for 20 h and labeling with fluorescent antigen (modulation design). Further insight into receptor occupancy and distribution was gained by the use of fluorescent antihapten and antiglobulin reagents. It was found that both immunogenic and tolerogenic antigen concentrations caused rapid patching and capping of the receptors to which they attached, followed by endocytosis and probably some shedding of Ig receptors. However, a proportion of cells continued to bear some cell surface antigen for 24 h. The immunogenic antigen concentration failed to completely remove the receptor coat from the cell surface. At all stages of immunogenesis, plentiful unoccupied receptors could be demonstrated. The tolerogenic concentration nearly saturated available receptors, and in its continuous presence, only few unoccupied or antigen-occupied surface receptors could be detected after 24 h of culture. Experiments of the modulation design showed that brief incubation with the tolerogenic concentration appeared to suppress receptor resynthesis, as few new receptors could be demonstrated after 20 h of further culture without antigen. Experiments were performed to determine whether fractionated cells prepared from spleens of 8-day-old mice showed an unusual tendency for modulation, even with immunogenic antigen concentrations. They were found to behave essentially like adult fractionated cells. The results are discussed in the framework of current theories of B-lymphocyte activation and tolerization.

Antigen-induced co-capping of IgM and IgD-like receptors on murine B cells.

Double fluorescence studies indicated that most mature lymphocytes of 11-52-wk-old mice possess both IgM and IgD-like surface immunoglobulins, while spleen cells from neonatal mice possess surface IgM only. These molecules cap independently with class-specific antisera, but co-cap when capping is induced by antigen. It is proposed that the two heavy chains on individual lymphocytes possess similar or identical antigen-combining sites.

Role of IgD in the immune response and tolerance. I. Anti-delta pretreatment facilitates tolerance induction in adult B cells in vitro.

Adult spleen cells from C57BL.Ige mice, which generally are resistant to in vitro tolerance induction in the B-cell compartment, became hyporesponsive (tolerant) when cultured with antigen in the presence of an anti-allotype serum. Both antigen and anti-delta had to be present for this effect, which was hapten-specific and did not occur in C57BL/L mice, which lack the Ig5-1 allotype of the delta-chain detected in this system. Preculture with anti-mu serum plus antigen, in contrast, did not cause tolerance induction in adult spleen B cells of either strain. These results suggest that the surface IgD may act as a failsafe receptor to prevent tolerance induction in adult B cells. Tolerance studies with spleen cells from mice with markedly reduced numbers of IgD+ve cells, because of regimen of repeated injections of anti-delta serum beginning at birth (delta-suppressed mice), confirmed the importance of membrane IgD in preventing tolerance, because such delta-suppressed mice were hypersusceptible to tolerance by antigen alone. Inasmuch as immature B cells lack IgD on their surface, these studies suggest that acquisition of IgD is an important maturational step in the ability of murine B cells to discriminate tolerogenic and immunogenic signals.

In vitro model for natural tolerance to self-antigens. Inhibition of the development of surface-immunoglobulin-negative lymphocytes into T-dependent responsive B cells by antigen.

Neonatal and adult splenic cell suspensions were labeled with fluorescein isothiocynate-anti-Ig and fractionated into surface-immunoglobulin- (s-Ig) positive and s-Ig-negative subpopulations by the fluorescence-activated cell sorter. The subpopulations were then tested by splenic focus assay for both frequency and tolerance susceptibility of clonable 2,4,-dinitrophenol (DNP) precursors. It was shown that both adult, and neonatal, s-Ig-negative subsets contained clonable DNP-specific B-cell precursors. However, because these precursors result in fewer clones secreting IgG, they appeared to be less mature than the s-Ig-positive precursors. In the absence of helper T cells, it was found that exposure of s-Ig-negative lymphocytes to tolerogen during the process in which they were acquiring surface receptors resulted in nearly total abrogation of potential DNP clones. This finding provides compelling evidence for clonal abortion.

Clonal analysis of B cells induced to secrete IgG by T cell-derived lymphokine(s).

To gain insight into how T cell-derived lymphokines induce the secretion of IgG in activated B cells, we performed a limiting dilution analysis, using murine splenic B cells incubated with lipopolysaccharide (LPS) and a T cell-derived B cell differentiating factor for IgG (BCDF gamma)-containing supernatant (SN). The results of this analysis indicate that such a SN induces a marked increase in the precursor frequency of IgG1-secreting cells and a modest increase in clone size. The precursors lack surface IgG and are committed to the differentiation pathway for IgG1 secretion after LPS activation, but before the addition of BCDF gamma-containing SN. The majority of IgG1-secreting clones arise independently from precursors of cells that secrete IgG3. Taken together, these results indicate that BCDF gamma directs differentiation of activated B cells to IgG1 secretion.

Growth of B-lymphocyte colonies in vitro.

In semisolid agar cultures containing mercaptoethanol, cells from the spleen, lymph nodes, marrow, peritoneal cavity, thoracic duct, and blood of normal mice generated clusters and colonies of up to 3,000 cells. Colony numbers and growth were markedly enhanced by the addition of sheep red cells. The frequency of colony-forming cells in the spleen or lymph nodes was 0.5-2.0%, and cluster forming cells were approximately five times more numerous. The mononuclear cells comprising these colonies had the electronmicroscopic morphology of immature lymphoid and plasma cells. The majority of the cells possessed Fc receptors, 61-69% reacted with anti-mu-serum and 4-11% with anti-gamma2-serum. Analysis of single cells from individual colonies indicated a higher frequency of the cells with membrane immunoglobulin and a clonal pattern of anti-mu or anti-gamma-reactivity. The clonal nature of colonies was supported by an analysis of NIP-binding cells in colonies grown from CBA spleen cells enriched for NIP-binding cells. Mass-harvested colony cells synthesized immunoglobulin in short-term liquid cultures. It is concluded that the colonies are clones of functionally active B-lymphoid cells.

Serological, biochemical, and functional identity of B cell-stimulatory factor 1 and B cell differentiation factor for IgG1.

By three criteria, we have demonstrated that B cell stimulatory factor (BSF-1) and B cell differentiation factor (BCDF-gamma) are the same lymphokine. Highly purified preparations of high performance liquid chromatography-purified or affinity-purified BSF-1 had BCDF-gamma activity but not BCDF-mu activity. A monoclonal anti-BSF-1 antibody coupled to Sepharose depleted both BSF-1 and BCDF-gamma activity but not BCDF-mu activity from two different T cell supernatants. Soluble monoclonal anti-BSF-1 blocked the BSF-1 and BCDF-gamma but not the BCDF-mu responses. These results suggest that BSF-1 acts on both resting and activated B cells to induce different effects.

Chronic neutropenia. A new canine model induced by human granulocyte colony-stimulating factor.

Normal dogs were treated with recombinant human granulocyte colony-stimulating factor (rhG-CSF) at 10 micrograms/kg/day for 30 d, which caused an initial neutrophilia, followed by a prolonged period of chronic neutropenia. A control dog treated with recombinant canine G-CSF (rcG-CSF) showed persistent neutrophilia over 3 mo. Serum from dogs during neutropenia contained an antibody to rhG-CSF, which neutralized the stimulatory effects of both rhG-CSF and rcG-CSF on dog marrow neutrophilic progenitor cell growth and on NFS-60 cell proliferation. 4 mo after discontinuation of rhG-CSF, the dogs' neutrophil counts returned to the normal range. Rechallenge with the rhG-CSF re-induced severe neutropenia in 1 wk. Neutropenia was transferred by plasma infusion from a neutropenic dog to a previously normal dog. These data suggest that human rhG-CSF immunizes normal dogs and thereby induces neutralization of endogenous canine G-CSF and neutropenia. This model system should allow more precise definition of the in vivo role of G-CSF.

Effects of bombesin antagonists on the growth of small cell lung cancer cells in vitro.

Small cell lung cancer (SCLC) produces several neuroendocrine peptides, including gastrin-releasing peptide (GRP), the mammalian equivalent of bombesin. There is some evidence to support the suggestion that GRP is an autocrine regulator of SCLC growth. Therefore, we have tested the effect of bombesin and two antagonists of bombesin on SCLC cell growth in a serum-free liquid tissue culture system. The antagonists used were analogues of substance P: spantide and (D-Arg1, D-Pro2, D-Trp7,9, Leu11) substance P. The cell lines used in this study all produced GRP-related peptides and one line had demonstrable GRP receptors. Exogenous bombesin did not cause any stimulation of growth in the liquid culture assay. The bombesin antagonists inhibited SCLC cell growth, but apparently not via the bombesin receptor. The bombesin used was biologically active because it stimulated the proliferation of Swiss 3T3 fibroblasts. The antagonists caused inhibition of this bombesin-induced proliferation, which was reversed by addition of excess bombesin. In addition, the antagonists and substance P alone stimulated proliferation of 3T3 cells, indicating that they may interact with another growth factor receptor on 3T3 cells. We conclude that growth of SCLC cells is not dependent on bombesin under all in vitro culture conditions because bombesin failed to stimulate growth in liquid cultures and the growth inhibition caused by bombesin antagonists was probably not mediated by the bombesin receptor.

Treatment of chemotherapy-induced neutropenia by subcutaneously administered granulocyte colony-stimulating factor with optimization of dose and duration of therapy.

In patients who have not received extensive prior chemotherapy or radiotherapy, it has been previously demonstrated that granulocyte colony-stimulating factor (G-CSF) abrogated the leukopenia following administration of melphalan (25 mg/m2). This study examined the necessity of a prechemotherapy period of G-CSF administration and the effect of varying the timing and duration of postchemotherapy G-CSF. Initially, patients received 0.3, 1.0, 3.0, and 10 micrograms/kg/d subcutaneously on days 1 to 5 and days 10 to 18. Melphalan was given on day 9. In the next portion of the study melphalan was administered on day 1 and G-CSF, 10 micrograms/kg/d, was administered by subcutaneous infusion on five schedules: (1) days 2 to 13; (2) days 8 to 13; (3) days 2 to 18; (4) days 8 to 18; (5) days -9 to -2 and 2 to 13. G-CSF produced a rapid and sustained elevation in neutrophil levels within 24 hours even when started 8 days after melphalan. This treatment was sufficient to abrogate the neutropenia in patients who had received no prior chemotherapy. It was not necessary to continue G-CSF for more than 7 days. G-CSF did not consistently alter the course of the thrombocytopenia that followed this dose of melphalan. G-CSF was well tolerated, although mild bone pain occurred and was reduced with acetaminophen. One of 22 patients developed cellulitis at an infusion site. We conclude that after melphalan chemotherapy, G-CSF may need to be given for only a short period to prevent chemotherapy-induced neutropenia, and that G-CSF induces a rapid rise in neutrophil levels even when started 8 days after melphalan administration.

Cloned stromal cell lines derived from human Whitlock/Witte-type long-term bone marrow cultures.

We report a human bone marrow culture technique that initially parallels the murine Whitlock/Witte culture system. As in the murine system, B cells predominate over other cell types, and all differentiation stages from pre-B to plasma cell are observed. Although these human long-term cultures pass through stages resembling phases I to III of murine Whitlock/Witte cultures, no outgrowth of nonadherent cells was seen after cultures had reached the "crisis" phase unless Epstein-Barr virus (EBV)-transformants appeared. The stromal cells persisted well beyond crisis, but they could not be maintained and passaged as cell lines, limiting their use in molecular analysis. Transfection of these stromal cells with plasmid DNA containing the simian virus 40 (SV40) early region yielded 124 cloned cell lines. Analysis of these lines showed that all expressed SV40 large T antigen, but they retained most phenotypic markers found on non-transformed stromal cells. When adherent and T-cell-depleted bone marrow cells were cultured on either nontransformed stromal layers or transformed cell lines they proliferated actively and soon yielded predominantly lymphoid nonadherent populations. Moreover, prolonged survival of acute lymphoblastic leukemia cells of pre-B phenotype was regularly achieved on both normal and transformed adherent cell layers. Although the liquid culture system favored lymphocytes, transformed stroma supported colony formation by both human and murine hemopoietic progenitors when marrow was added in agar medium. This was not explained by colony-stimulating factor (CSF) production, because striking heterogeneity in the levels of granulocyte CSF (G-CSF) and granulocyte-macrophage CSF (GM-CSF) secretion by the lines was noted. Some lines that did not produce detectable CSF demonstrated good support of fresh bone marrow growth and acute lymphoblastic leukemia (ALL) cell survival. The heterogeneity of these cell lines and their capacity to support hemopoiesis suggest that they will be useful in studying the molecular basis of in vitro lymphohemopoiesis in man.

Expression of granulocyte-colony stimulating factor and its receptor is regulated during the development of the human placenta.

The development of the placenta is dependent upon the regulated proliferation, invasion and differentiation of trophoblast. Expression of cytokines at the feto-maternal interface suggests that these molecules may participate in placentation. The expression of granulocyte-colony stimulating factor (G-CSF) and G-CSF receptor (G-CSFR) during the development of the human placenta was studied by immunohistochemistry using an anti-G-CSF monoclonal antibody (mAb) and two novel anti-G-CSFR mAbs. G-CSF was present in the stroma of fetal chorionic villi and maternal decidual tissues throughout pregnancy. G-CSFR was detected at high levels in fetal first and third, but not second trimester placental tissues. Staining for G-CSFR was undetectable in maternal decidual tissue from all gestational stages. In first trimester tissues, staining for placental G-CSFR was strongest in differentiated syncytiotrophoblast and invasive, extravillous cytotrophoblast, and weak staining was evident in undifferentiated cytotrophoblast. Immunohistochemical data suggesting temporal regulation of G-CSFR were corroborated by Western blotting and amplification by reverse transcription and PCR of G-CSFR mRNA. These data suggested that expression of G-CSFR in the human placenta is regulated both temporally and spatially, and that placental G-CSF is involved in paracrine regulation, and indicate a role for G-CSF and G-CSFR in trophoblast growth or function during placentation.

Monoclonal antibodies that recognize human granulocyte-macrophage colony-stimulating factor and neutralize its bioactivity in vitro.

We have produced monoclonal antibodies to bacterially synthesized, human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and have studied in detail the characteristics of three strongly neutralizing antibodies. The antibodies reacted with GM-CSF at high dilution (EC50 = 0.1-1.7 nM) in an indirect ELISA but did not react with murine GM-CSF or other cytokines. They also recognized glycosylated hGM-CSF produced by human lymphocytes. The antibodies were able to immunoprecipitate rhGM-CSF, but only reacted weakly with rhGM-CSF on Western blots, indicating that they recognized a conformation-dependent epitope. Cross-blocking studies showed that the three antibodies recognized overlapping epitopes. The antibodies inhibited binding of 125I-labeled rhGM-CSF to HL-60 cells at nanomolar concentrations and neutralized GM-CSF activity in two different bioassays. These antibodies thus provide a useful tool for analyzing the specificity of bioassays and for further studies of the production and function of GM-CSF in vitro and in vivo.

A simple method allowing DIC imaging in conjunction with confocal microscopy.

Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique (Barone-Nugent et al., 2002). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.

Anti-carbohydrate activity of T cell-reactive chicken anti-mouse immunoglobulin antibodies.

The specificity of chicken anti-mouse immunoglobulin F(ab')2 antibodies, which bind readily to T cells and have been claimed to detect immunoglobulin on T cells, was analyzed by indirect immunofluorescence. It ws found that only a minor proportion of the total antibody bound to T cells whereas the major fraction bound only to B cells. The T cell-reactive antibody cross-reacted with bovine IgG and therefore could be purified on this basis by affinity chromatography. T cell staining was inhibitable with nonimmunoglobulin glycoproteins as well as immunoglobulin, in contrast to staining with the B cell-specific antibody fraction, which was only inhibited by immunoglobulin. This suggested that the T cell staining was caused by anti-carbohydrate antibody. Inhibition of T cell staining was abolished by periodate oxidation of the inhibitors but not by proteolytic digestion. In addition, the antigenic determinant on the cell surface was not destroyed by autoclaving. Finally, antibody was absorbed on a mucin-Sepharose column and anti-T cell activity was found in the bound but not the unbound fraction. These results strongly suggest that the only anti-T cell activity in the chicken antibody is anti-carbohydrate antibody.

Distribution of surface IgM and IgD on single cells from lymphocyte subpopulations.

The surface immunoglobulin heavy chains on individual spleen cells fractionated by velocity sedimentation were studied using fluorescent antisera. In adult mice, cells bearing both mu and delta chains were found in all fractions. While there was an increase in the proportion of cells bearing mu only in the medium to large cell fractions, the majority of cells bearing mu only were small lymphocytes. Results obtained using 3-week-old mice were basically similar, but showed both a marked decrease in small mu + delta + cells and a marked increase in small mu + delta - cells when compared with adult animals.

Specific antibody-forming B-lymphocyte colonies. I. Distribution and nature of SRBC antibody-forming B-lymphocyte colonies in mouse lyphomyeloid organs.

Wehn normal mouse spleen or lymph node cells are cultured for 7 days in agar-medium containing 2-mercaptoethanol and sheep red blood cells (SRBC), approximately one B lymphocyte colony (BLC) develops per 50-100 cells seeded. Incubation of cultures for 3 h with guinea-pig complement at day 7, demonstrated that 0.05-0.25% of all BLC form specific antibody against SRBC (SRBC-AF-BLC). The SRBC specific colonies appear centrally in lytic plaques of 2-5 mm in diameter and cells recovered from individual SRBC-AF-BLC were shown to produce antibodies of the IgM class against SRBC. Cells forming SRBC-AF-BLC are absent in the new-born and infantile spleen but appear in adult mice with a frequency of 5, 10-25 and 25-70 per 10(6) bone marrow, spleen and lymph node cells respectively. specific immunization in vivo or in vitro does not greatly affect the number of SRBC-AF-BLC-forming cells. Cytolysis of spleen cells with anti-Ig serum plus complement prior to culture reduced the number of total BLC and that of specific SRBC-AF-BLC by 93% and 94% respectively. The peak sedimentation velocity of both SRBC-AF-BLC-forming cells and total BLC-forming cells was 3.5 mm/h. Spleen cells enriched 200-300 times for cells that bind specifically to the hapten NIP were not enriched for cells forming colonies with specific antibody production against NIP. The data indicate that the cells that give rise to specific antibody-forming colonies belong to a mature virgin B cell group of small Ig-positive B lymphocytes.

Endogenous haemopoietic growth factors in neutropenia and infection.

Haemopoietic growth factors (HGFs) are being administered to patients with neutropenic fever; however, little is known about the endogenous HGF response in these patients. Specific assays were used to study four HGFs, granulocyte (G-) CSF, granulocyte-macrophage (GM-) CSF, macrophage (M-) CSF and interleukin (IL-) 6 levels in the blood of patients with neutropenic fever (46 episodes). For comparison, levels were also measured in three control populations: normals (20), afebrile neutropenic (14), and bacteraemic but not neutropenic patients (20). In febrile patients, levels of G-CSF (median, range) (0.46, < 0.10-142 ng/ml). IL-6 (0.054, 0.005-24.3 ng/ml) and M-CSF (18.5, 9.9-79.1 ng/ml) were elevated compared with afebrile subjects (< 0.10, < 0.10-1.62 ng/ml). (0.008, 0.002-0.024 ng/ml) and (6.45, < 5.0-31.3 ng/ml) respectively. GM-CSF was not elevated (< 0.02, < 0.02-8.0 ng/ml) compared with afebrile subjects (0.021, < 0.02-0.20 ng/ml). Variables significantly associated (P < 0.05) with elevated cytokine levels were determined by multiple regression analyses. Factors associated with G-CSF elevation were fever, neutropenia, pathogen type and raised bilirubin and creatinine. In contrast, neutropenia was not associated with IL-6 elevation although there was an association between IL-6 elevation and fever, Gram-negative and fungal infections and raised creatinine and bilirubin. M-CSF elevation was associated with fever, renal impairment and known pathogen. Elevated G-CSF and IL-6 levels normalized rapidly (hours-days) with the resolution of infection, whereas M-CSF concentrations remained elevated for up to 10 d. Cytokine levels remained elevated in septic neutropenic patients who did not recover. In summary, G-CSF, IL-6 and M-CSF levels were significantly elevated in sepsis. In contrast, GM-CSF levels were not elevated. These studies should assist the development of therapeutic strategies using HGFs in the treatment of sepsis.