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The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space.

Salmonella enterica is a versatile vaccine carrier for heterologous antigens. One strategy for vaccine antigen delivery is the use of live attenuated S. enterica strains that translocate heterologous antigens into antigen-presenting cells by means of type III secretion systems (T3SS). The feasibility of this approach has been demonstrated in various experimental vaccination studies. The efficacy of recombinant live vaccines is critically influenced by the optimal level of attenuation and many other factors. For the rational design of approaches involving translocation by T3SS, additional parameters are the level of expression of the heterologous antigens and the selection of carrier proteins for the delivery of antigens to desirable subcellular compartments of the target cell. We deployed the Salmonella pathogenicity island 2 (SPI2)-encoded T3SS for antigen delivery. The SPI2-T3SS and effector proteins are encoded by members of the large SsrAB regulon, including promoters with highly variable strength of expression. We investigated the effect of various in vivo-activated promoters of the SsrAB regulon on the efficacy of recombinant Salmonella vaccines. We observed that the use of promoters with higher strength results in greater synthesis of recombinant antigens and greater stimulation of T-cell responses in cell culture assays for the stimulation of T cells by the model antigen ovalbumin. In contrast, in vaccination experiments, promoters with a low level of expression resulted in the induction of higher amounts of T cells reactive to the model antigen listeriolysin. These results demonstrate that high-level expression of heterologous antigens does not necessarily result in optimal stimulation of immune responses.

Immunization with particulate Ag effectively induces antitumor and antiviral T cell-mediated immunity. Immature dendritic cells (DCs) efficiently internalize, process, and present a variety of particulate Ags; however, previously published data suggest that both the uptake of soluble Ag through micropinocytosis, and phagocytosis of particulates are significantly curtailed in activated DC populations. In this study, we demonstrate that although macropinocytosis of soluble Ag is diminished following DC activation, subsets of DCs in activated DC populations retain the ability to actively phagocytose particulate Ags. Live cell imaging of activated DCs reveals that phagocytosis of particulates can result in cytoskeletal remodeling and perinuclear lysosome cluster disruption in a time-dependent manner. Interestingly, our results suggest that in activated DC populations, presentation of phagocytosed particulate Ags is dependent on the nature of the activation signal. These results provide direct evidence of functional heterogeneity in DC populations and contribute to the development of particle-based immunization strategies.

Liposomes have been widely used as drug delivery systems for many years. However, they are of limited use as delivery systems for subunit vaccines due to their low immunogenicity. Here we examine the effect of incorporating the adjuvant Quil A into liposomes on the type of particles produced, on the ability of the different particles to incorporate antigen and on the ability of the different particles to stimulate murine bone-marrow-derived dendritic cells (DC) and lymphocytes. The incorporation of increasing amounts of Quil A, from 20% to 70% of the total lipid into liposomes, reduces the size of the particles that form in aqueous dispersion and decreases antigen incorporation and uptake by DC. Interestingly, the particles with 20% Quil A were more toxic to cells in culture than the particles containing 70% Quil A, and the 20% particles were also more immunostimulatory., (Copyright 2004 Australasian Society for Immunology Inc.)

Dendritic cells expanded from mouse bone marrow (BMDC) with granulocyte/macrophage-colony-stimulating factor have potent T cell-stimulatory properties both in vitro and in vivo. This has been well documented for major histocompatibility complex (MHC) class II-restricted responses, and more recently using peptide-loaded and protein-pulsed DC for CD8 responses following adoptive transfer in mice. An unresolved question concerns the capacity of BMDC to present exogenous antigen on MHC class I molecules, an unconventional mode of MHC class I loading for which there is now considerable evidence, particularly in macrophages. Here, we show that BMDC exhibit high levels of macropinocytosis driven by constitutive membrane ruffling activity. Up to one-third of actively ruffling and macropinocytosing BMDC transferred pinocytosed horseradish peroxidase into the cytosol following a 15-min pulse, suggesting that they might be capable of presenting exogenous soluble antigen on MHC class I molecules. We show that BMDC presented exogenous ovalbumin to a T cell hybridoma more effectively, more rapidly, and at lower exogenous antigen concentrations than BM macrophages on a cell-for-cell basis. Presentation was TAP dependent, brefeldin A sensitive, and blocked by inhibitors of proteasomal processing, demonstrating use of the classical MHC class I pathway. Although effective presentation of exogenous antigen by BMDC occurred in the absence of agents which stimulate macropinocytosis, treatment with phorbol myristate acetate (PMA) enhanced both pinocytosis and MHC class I presentation by BMDC. Finally, PMA-stimulated BMDC exposed to exogenous ovalbumin in vitro were able to prime an antigen-specific cytotoxic T lymphocyte response following adoptive transfer in vivo.

In the present work, we explored the cytokine-dependent regulation of bone marrow-derived mast cell (BMMC) antigen-presenting cell (APC) function, and co-stimulation requirements, and analyzed the nature of antigens presented to T cells. We observed an up-regulation of the APC function of mast cells induced by granulocyte/macrophage-colony-stimulating factor (GM-CSF) and a complete abrogation by interferon (IFN)-gamma. Expression of co-stimulatory molecules CD80 and CD86 was suggested by the ability of mast cells to activate purified lymph node-derived T cells. Indeed, addition of the fusion protein mCTLA4-Ig strongly inhibited antigen presentation by mast cells to normal T cells and to the T cell hybridoma 3DO-54.8. The regulatory mechanisms of APC function by GM-CSF and IFN-gamma were investigated by measuring CD80 and CD86 transcripts in mast cells. GM-CSF-treated must cells showed a strong increase in the expression of both CD80 and CD86 transcripts, whereas in IFN-gamma-treated mast cells, this expression was completely abrogated. Thus, up- and down-regulation of CD80 and CD86 expression by GM-CSF and IFN-gamma is directly correlated to the APC function. In addition, we analyzed antigen presentation by mast cells of endogenous self-antigens. Mast cells failed to activate anti-I-A or anti-I-E-specific T cell hybridomas and alloreactive T cells in primary mixed lymphocyte reactions (MLR). Furthermore, mast cells did not present the mouse beta 2-microglobulin (m beta 2-m) peptide 25-40, constitutively expressed on B cells. However, mast cells, especially those treated with GM-CSF, activated an anti-m beta 2-m-specific T cell hybridoma in the presence of exogenous peptide. The minor lymphocyte-stimulating antigen-1 Mls-1a is a viral superantigen (vSAG) encoded by the the mouse mammary tumor provirus-7 (MMTV-7). Mast cells, despite a reasonable amount of major histocompatibility complex class II on the cell surface and the presence of MMTV transcripts predicted to encode the vSAG, cannot stimulate in vivo or in vitro V beta 6+ T cells specific for Mls-1a. In contrast, mast cells could present the exogenous bacterial SAG, staphylococcal enterotoxin B (SEB), to specific V beta 8+ T cells. The selective ability of mast cells to present exogenous antigens may have physiological relevance in that mast cells could participate in immune response regulatory mechanisms by discriminating self from nonself.

An immunomorphological and immunochemical study was performed to elucidate the pattern of carbohydrate antigens and their relationships to the cluster differentiation (CD) 68 epitopes on macrophages derived from human bone marrow and milk. Core and backbone antigens recognized by lectins from Bauhinia purpurea (BPA), Helix pomatia (HPA), Arachis hypogaea (PNA), Glycine max. (SBA), Griffonia simplicifolia (GSA-I-B4), Lycopersicon esculentum (LEA) and Erythrina cristagalli (ECA) were expressed by both macrophage populations. Additionally, they exhibited various peripheral type 1 and type 2 carbohydrate antigens. In bone marrow trephine biopsies, the number of macrophages stained by the CD68-specific monoclonal antibody PG-M1 exceeded significantly (range 30-40%) the subpopulation expressing SBA, GSA-I-B4, and ECA binding sites as well as the Lewisa antigen. This result is very interesting since, from in vitro studies GSA-I-B4 and SBA are known to react especially with activated macrophages. Western blotting experiments on milk macrophage lysates revealed that ECA, GSA-I-B4, BPA, PNA and MAA visualize a 110 kDa band isographic with the CD68 antigen detected by PG-M1, KP1 and Ki-M1P monoclonal antibodies. These antibodies recognize peptide epitopes as shown by enzyme-linked immunosorbent assays after biochemical modification of milk macrophage lysates. This result is in keeping with the assumption that the CD68 antigen consists of a highly glycosylated mucin-type glycoprotein comprising various differentiation-dependent epitopes.

Two proteins, a recombinant malaria protein (R32NS1) and conalbumin, were encapsulated in separate liposomes. The mechanisms of presentation of unencapsulated and liposome-encapsulated R32NS1 and conalbumin to antigen-specific T-cell clones were investigated in in vitro antigen presentation assays using murine bone marrow-derived macrophages (BMs) as antigen-presenting cells. A much lower concentration of liposomal antigen than of unencapsulated antigen was required for T-cell proliferation. Liposome-encapsulated conalbumin required intracellular processing by BMs for antigen-specific T-cell proliferation, as determined by inhibition with chloroquine, NH4Cl, leupeptin, brefeldin A, monensin, antimycin A, NaF, and cycloheximide and by treatment of BMs with glutaraldehyde. Liposome-encapsulated conalbumin therefore follows the classical intracellular antigen processing pathway described for protein antigens. Similarly, unencapsulated conalbumin also required intracellular processing for presentation to antigen-specific T cells. In contrast, both unencapsulated R32NS1 and liposome-encapsulated R32NS1 were presented to T cells by BMs without undergoing internalization and intracellular processing. These results suggest that the antigen itself is the major element that determines whether a requirement exists for intracellular processing of liposomal antigens by macrophages.

It has previously been shown that bone marrow-generated dendritic cells (DC) are potent stimulators in allogeneic mixed leukocyte reactions and are capable of activating naive CD4+ T cells in situ in an antigen-specific manner. In this study we have investigated whether bone marrow-generated DC are capable of inducing antigen-specific CD8+ T cell responses in vivo. Initial attempts to induce specific cytotoxic T lymphocyte (CTL) responses in mice injected with bone marrow-generated DC pulsed with ovalbumin (OVA) peptide were frustrated by the presence of high levels of nonspecific lytic activity, which obscured, though not completely, the presence of Ag-specific CTL. Using conditions that effectively differentiate between antigen-specific and nonspecific lytic activity, we have shown that bone marrow-generated DC pulsed with OVA peptide are potent inducers of OVA-specific CTL responses in vivo, compared with splenocytes or RMA-S cells pulsed with OVA peptide, or compared with immunization with free OVA peptide mixed with adjuvant. Antibody-mediated depletion experiments have shown that the cytotoxic effector cells consist primarily of CD8+ cells, and that induction of CTL in vivo is dependent on CD4+ as well as on CD8+ T cells. These results provide the basis for exploring the role of bone marrow-generated DC in major histocompatibility class I-restricted immune responses, and they provide the rationale for using bone marrow-generated DC in CTL-mediated immunotherapy of cancer and infectious diseases.

The mechanisms of endothelin-1-induced histamine release were examined and compared with those responsible for antigen-induced release by using passively sensitized mouse bone marrow-derived mast cells and various drugs that may influence histamine release. The following results were obtained: (1) Although islet-activating protein potently inhibited endothelin-1-induced histamine release, it did not affect the antigen-induced release. (2) Histamine release induced by endothelin-1 was relatively more sensitive to ethylenediaminetetraacetic acid than that induced by antigen, although extracellular Ca2+ is a requisite for both types of the release. (3) (8R*, 9S*, 11S*)-(-)-9- hydroxy-9-methoxycarbonyl-8-methyl-2,3,9,10-tetrahydro-8,11-epoxy-1H,8H, 11H-2, 7b,11a-triazadibenzo[a,g]cycloocta[c,d,e]trinden-1-one and (8R*, 9S*, 11S*)-(-)-9-hydroxy-9-methoxycarbonyl-8-methyl-14-n-propoxy-2,3,9,10-tet rahydro - 8,11-epoxy-1H,8H,11H-2,7b,11a-triazadibenzo[a,g]cycloocta-[c ,d,e] trinden-1-one, which possibly inhibit protein kinases, strongly inhibited antigen-induced histamine release, while these drugs alone did not inhibit endothelin-1-induced release. (4) Staurosporine, a non-selective protein kinase inhibitor, prevented the elevation of cytosolic Ca2+ concentrations induced by antigen, whereas that induced by endothelin-1 was not influenced; histamine release induced by either stimulus was greatly inhibited by the drug. These results indicate and/or suggest that some biological events induced by endothelin-1 leading to histamine release are different from those involved in the histamine release induced by antigen.

The morphology of osteoclasts, primary cells that resorb bone, is well documented; however, the precise details of their terminal differentiation remains obscure. To date, the only morphological criterion for identifying activated functional osteoclasts has been the presence of ruffled borders. We have developed a rat bone marrow culture system in which osteoclast-like cells formed. These cells fulfilled most of the criteria of osteoclasts, and when they were reseeded on calcified tissue, formed numerous resorption lacunae in vitro. To find an immunological marker for functional osteoclasts, we have used these cells in a functional state as antigens for the preparation of monoclonal antibodies (mAb) that reacted with rat osteoclasts; we obtained mAb Ch1 and Ch2. Interestingly, these mAbs reacted with the marginal portion of authentic osteoclasts, where they attached to the bone surface on frozen sections. The reactivity of Ch1 to rat osteoclasts was more restricted than that of Ch2: Ch1 reacted with few tartrate-resistant acid phosphatase (TRAP)-positive cells on a culture plate. These TRAP-positive cells (including mono- and multinucleated cells) were, however, converted to Ch1-positive cells when they were reseeded on calcified tissues. These findings suggested that the antigen recognized by the Ch1 antibody was induced by some factors of matrix proteins released from calcified tissues.

This paper describes a new role for mast cells as being able to present Ag to immune T cells. A mouse bone marrow-derived mast cell population obtained after 3 wk of culture in a conditioned medium has been shown to express a variety of membrane-associated Ag, including MHC class II and class I Ag, CD23, CD32, high affinity receptor for IgE, and CD4. Expression of MHC class II molecules was up-regulated upon stimulation with LPS but not with IFN-gamma and was down-regulated after exposure of mast cells to IL-3 treatment. We have demonstrated that mast cells were able to present native Ag as well as immunogenic peptides to MHC class II-restricted T cell hybridoma. The inhibition of Ag presentation after mast cells have been treated with ammonia suggests that Ag catabolism in intracytoplasmic compartment as a key step in Ag handling takes place in these cells. The MHC class II molecule is the restricting element for the presentation of OVA and the lambda repressor from bacteriophage lambda to a panel of specific T cell hybridomas, as demonstrated by the blocking effect of anti-MHC class II mAb on the Ag-presenting function. A characteristic feature of mast cells is the generation of a narrower immunogenic peptide repertoire as compared with A20 and LBB 3.4.16, a B lymphoma cell line, and a B cell hybridoma, respectively. This novel function of mast cells brings to a much closer connection inflammatory and immunologic processes and sheds new light on the biology of mast cells and particularly on the specific allergic responses.

Stem cell factor (SCF) is encoded at the Sl locus of the mouse and is the ligand for the c-kit receptor. Recombinant rat SCF164 (rrSCF164) induces proliferation and promotes maturation of mouse mast cells in vitro and in vivo and can also induce c-kit receptor-dependent mouse mast cell degranulation. We now report that in both quiescent and non-quiescent mouse bone marrow-derived cultured mast cells (BMCMC) rrSCF164 induces increased mRNA levels for the "early response genes" c-fos, c-jun and junB but has only slight effects on the expression of junD. Recombinant mouse interleukin-3 (IL-3) also promotes proliferation of both quiescent and non-quiescent BMCMC. However, IL-3 induces increased expression of c-fos and junB only in quiescent BMCMC. Cross-linking of Fc epsilon receptor type I (Fc epsilon RI) on BMCMC by IgE and specific antigen induces a pattern of early gene expression very similar to that induced by rrSCF164. However, BMCMC stimulated through the Fc epsilon RI did not proliferate and, in comparison to control BMCMC, exhibited significantly decreased proliferation in response to rrSCF164 or IL-3. These results indicate that stimulation of BMCMC proliferation by IL-3 or rrSCF164 induces distinct patterns of early response gene expression and suggest that the proliferative effects of these growth factors may be mediated through distinct signal transduction pathways. Our data also point to previously unappreciated similarities between the effects of signaling through the c-kit receptor or the Fc epsilon RI on mast cell expression of fos and jun genes.

Recently, we produced monoclonal antibodies reacting specifically with the reticular meshwork (RM) of lymphoid tissues, and demonstrated that, in the splenic white pulp of normal mouse, the antigenic heterogeneity of RM was associated with the segregation of the T and B lymphocytes. In the present study, we attempted to visualize further the interaction between splenic RM and T and B lymphocytes transferred into severe combined immunodeficient (SCID) mice. The splenic white pulp of naive SCID mice, containing a few T and B cells, showed little tendency for T-B segregation and antigenic diversity of RM. Transfer of spleen or bone marrow cells from normal mice resulted in complete recovery of lymphocyte populations, showing not only a clear segregation of T and B lymphocytes but also a remarkable antigenic diversity of RM. The same results were obtained following the transfer of spleen or bone marrow cells from the nude mouse. Next, we transferred purified T lymphocytes to one group of SCID mice and B cells to another. In mice given T cells, a few B cells were observed in the white pulp; T lymphocytes lodged not only in the inner periarterial lymphatic sheath (PALS) but also in the outer PALS and follicles. In the animals to which B cells were transferred, T cells were few and the homing of B cells occurred only into their proper compartments, such as the outer PALS, follicles and marginal zone, but not in the inner PALS. Thus, B cells can home into their proper compartments of the splenic white pulp independently of T lymphocytes.

Previously we characterized by immunohistology a group of rat anti-mouse thymic stromal mAbs (MTS 12, 32, 33, 35, and 37), which recognized novel plasma membrane determinants on both thymic stromal cells (TSCs) and thymocytes. The present study investigates in more detail this incidence of shared phenotype by an extensive flow cytometric analysis of MTS mAb reactivity on TSCs, thymocyte subsets, peripheral lymphocytes, and bone marrow cells. Examination of freshly isolated or cultured heterogeneous TSCs and TSC clones confirmed that the mAb identified plasma membrane molecules on distinct subsets of these cells. All but MTS 12 reacted with epithelial cells. Triple-labelling illustrated that MTS 32, 33, and 37 were also reactive with more than 90% of total thymocytes, but varied in their distribution on the four major CD4 and CD8 defined subsets. MTS 12, staining with thymic vascular endothelium by immunohistology, labelled more than 95% of each subset. MTS 35 reactivity in each subset correlated strongly with only the immature populations. Examination of peripheral lymphocytes by triple- and double-labelling unexpectedly showed that MTS 33, 35, and 37 did not recognize peripheral T cells but labelled all B cells. MTS 32 was negative for B cells, but positive for all CD8+ T cells, yet only a subset of CD4+ T cells. Further, MTS 33, 35, and 37 were present on a significant percentage of bone marrow cells. MTS 12 reacted with virtually all peripheral T and B cells, and about 50% bone marrow leukocytes. Collectively these results reveal the same novel epitopes on different thymic cell types and subsets thereof, highlighting specific similarities between cells of apparently diverse lineages. These findings may be of importance in the delineation of intercellular communications within the thymus and emphasize the integrated nature of the microenvironment in this organ.

Natural suppressor (NS) activity has been identified in several sites of active hematopoiesis. In this study we characterized NS activity in murine bone marrow (BM) using monoclonal antibodies (mAbs) to interleukin 3 (IL-3) receptor-associated antigen (IL-3RAA) and various cytokines that exert a strong influence on hematopoiesis or lymphocyte interaction. NS activity of BM cells of relatively low density was enhanced by IL-3 or granulocyte-macrophage colony-stimulating factor (GM-CSF). When the BM cells were separated into IL-3RAA+ cells and IL-3RAA- cells, the IL-3RAA+ cells demonstrated potent NS activity, whereas IL-3RAA- cells had either no or weak NS activity. The IL-3RAA+ cells showed non-T- and non-B-cell phenotype and had high affinity to wheat germ agglutinin (WGA), a marker for hematopoietic progenitors. In assays for hematopoietic activity, it appeared that the early differentiating progenitors (day 8 spleen colony-forming units [CFU-S], granulocyte-macrophage colony-forming units [CFU-GM]) were enriched in the IL-3RAA+ cell population, whereas more immature multipotent progenitors (day 12 CFU-S, granulocyte erythrocyte macrophage megakaryocyte colony-forming units [CFU-GEMM]) were contained in the IL-3RAA- cell population. Both suppressor cells and IL-3RAA+ cells spontaneously developed from the IL-3RAA- cell population. These findings suggest that NS cells in murine BM are early hematopoietic progenitors and are probably committed to the myeloid lineage. Hybridoma cells established between the IL-3RAA+ cells and BW5147 cells produced suppressor factor(s). This finding suggests that the NS cells produce soluble mediator(s) that may be responsible for their suppressive action.

Hematopoietic stem cells (HSCs) are defined in mice by three activities: they must rescue lethally irradiated mice (radioprotection), they must self-renew, and they must restore all blood cell lineages permanently. We initially demonstrated that HSCs were contained in a rare (approximately 0.05%) subset of bone marrow cells with the following surface marker profile: Thy-1.1lo Lin- Sca-1+. These cells were capable of long-term, multi-lineage reconstitution and radioprotection of lethally irradiated mice with an enrichment that mirrors their representation in bone marrow, namely, 1,000-2,000-fold. However, the experiments reported did not exclude the possibility that stem cell activity may also reside in populations that are Thy-1.1-, Sca-1-, or Lin+. In this article stem cell activity was determined by measuring: (a) radioprotection provided by sorted cells; (b) long-term, multi-lineage reconstitution of these surviving mice; and (c) long-term, multi-lineage reconstitution by donor cells when radioprotection is provided by coinjection of congenic host bone marrow cells. Here we demonstrate that HSC activity was detected in Thy-1.1+, Sca-1+, and Lin- fractions, but not Thy-1.1-, Sca-1-, or Lin+ bone marrow cells. We conclude that Thy-1.1lo Lin- Sca-1+ cells comprise the only adult C57BL/Ka-Thy-1.1 mouse bone marrow subset that contains pluripotent HSCs.

We have previously defined a subset of High Proliferative Potential Colony Forming Cells (HPP-CFC), derived from murine marrow purified for early progenitors expressing the Stem Cell Antigen (SCA+) and lacking terminal lineage markers (Lin-), which are responsive to multiple cytokines in combination. Stem Cell Factor (SCF), a multipotent growth factor which is the ligand for c-kit, synergizes with these multiple factor combinations to increase colony number and size. SCF is thus a potent mitogen with direct action on early hematopoietic progenitor cells. These data are consistent with a model of stromal control of hematopoiesis via elaboration of multiple cytokines in locally high concentration, with SCF playing a central role.

Human hematopoietic colony-forming cells (CFC) express the CD34 antigen (CD34+) as well as differentiation antigens such as CD33 and HLA-DR. CD34+ cells that do not express these latter differentiation antigens have been shown to contain few CFC in direct culture, but generate increasing numbers of CFC when cultured over a marrow stromal cell layer in the long-term culture system. In this study we determined if CD34+ cells with low or absent expression of CD33 and a novel antigen, 7B9 (CD34+CD33-7B9-), could be distinguished from CD34+ cells expressing these antigens (CD34+CD33+7B9+) based on their proliferative responses to interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) in a short-term liquid culture system. These two populations were separated by fluorescence-activated cell sorting, cultured with IL-3 (10 ng/mL), G-CSF (100 ng/mL), or IL-3 and G-CSF, and 3H-thymidine uptake was measured. CD34+CD33-7B9- cells proliferated in the presence of IL-3, but not G-CSF. However, a synergistic response to the combination of IL-3 and G-CSF was seen in most experiments. In contrast, CD34+CD33+7B9+ cells proliferated in the presence of either IL-3 or G-CSF but did not display an additive or synergistic response to the combination of IL-3 and G-CSF. In colony-forming assays performed before and after liquid culture, the CD34+CD33-7B9- cells in two experiments contained 0.3% and 2.2% of all sorted marrow CFC before liquid culture and generated 40-fold and ninefold increases in the number of granulocyte-macrophage colony-forming units (CFU-GM), respectively, after liquid culture with IL-3 and G-CSF. In contrast, the CD34+CD33+7B9+ cells contained 99.7% and 97.8% of all sorted marrow CFC before liquid culture and had no change or a threefold increase in the number of CFU-GM, respectively, after liquid culture with IL-3 and G-CSF. Single-cell liquid cultures containing IL-3 and G-CSF with cells that were either CD34+CD33-7B9- and depleted of mature lymphoid cells (CD34+lin-) or were CD34+lin+ showed that a higher proportion of wells containing a CD34+lin- cell gave rise to one or more CFC (8.7%) than did wells containing a CD34+lin+ cell (2.9%), with the responding cells in the former population giving rise to an average of 2.9 +/- 0.6 CFC and in the latter population, 2.0 +/- 1.0 CFC.(ABSTRACT TRUNCATED AT 400 WORDS)

Hemopoietic stem cell (CFUs) proliferation is controlled by regulatory activities (stimulator and inhibitor) produced by bone marrow macrophages. Previously it has been shown that antigen administration stimulates CFUs proliferation. The data obtained in this study show the possible mechanism of antigen-induced stimulation of CFUs proliferation. 3-4 days after antigen injection bone marrow cells of BDF1 mice cease to produce inhibitory activity in contrast to similar cells of control animals. Therefore, increased CFUs proliferation in immunized mice can be due to decreased production of inhibitory activity and resulting abundance of stimulating factors. In BAlB/c mice CFUs proliferation is not changed after antigen injection and their bone marrow cells continue to synthesize inhibitory substances. Differentiation of CFUs into committed blood precursor cells may depend on the proliferation level in CFUs population since activation of CFUs proliferation in immunized BDF1 mice is accompanied by a decreased number of CFU-GM and CFU-M but an increased number of BFU-E. It should be noted that intact BAlB/c mice show a high level of CFUs proliferation similar to that of immunized BDF1 mice.

Transplantation of immunocompetent cells present within allogeneic bone marrow has been associated with the elimination of residual host leukemia, both in animal tumor models and in patients receiving marrow transplants for leukemia. This observation has been called the "graft-versus-leukemia effect." We have attempted to study this phenomenon in vitro by characterizing the cytolytic response of T cells from normal donors after in vitro activation with allogeneic leukemic cells. As expected, most T cells that react against an allogeneic patient's leukemic cells recognize their foreign HLA antigens and lyse the patient's nonleukemic remission lymphoid cells. In addition, we have shown that a small fraction of the T cells recognize and lyse foreign leukemic targets without lysis of nonmalignant remission targets from the same leukemic patient. These T cells have been isolated and characterized as CD3+, CD4+ cells expressing the alpha/beta T cell receptor (TCR). Their lysis appears to reflect specific antigen recognition mediated via the CD3-TCR complex and interactions involving the CD4 receptor. Some of these "leukemic specific" T cell lines, which are restricted by HLA class II molecules, can also lyse occasional nonleukemic cells from certain unrelated donors. This recognition appears to involve crossreactive determinants shared by the leukemic cells and the unrelated allogeneic nonleukemic cells. These specific interactions may represent an in vitro model of the graft-versus-leukemia effect.

Initial and (or) subsequent exposures of animals to selected antigens result in impressive accumulations of eosinophil granulocytes at the site of challenge. When it has been studied, the exudation is accompanied by an increase in medullary eosinophilopoiesis. The medullary response is proportional to the amount of antigen used to induce the exudate as shown by responses to single challenge injections with graded doses of a soluble antigen preparation from schistosome eggs (SEA). Attempts were made to mimic the continuous antigen challenge of chronic schistosomiasis by use of osmotic minipumps and intermittent injections with SEA. These did not result in prolonged increases of marrow eosinophilopoiesis for reasons which remain unclear.

Various macrophage-containing preparations were tested for their ability to increase the antigen-specific proliferative response of murine T-lymphocytes. The preparations examined included: peritoneal exudate cells (PEC) from mice injected with mineral oil or thioglycolate; fresh bone-marrow cells; bone marrow cells grown in culture for up to 11 days; normal spleen cells, and spleen cells from mice injected with mineral oil. The best proliferative response was obtained when the lymphocytes were supplemented with 30% spleen cells from mice injected with mineral oil. When spleen cells from mineral oil injected mice are compared with those of spleen cells from normal mice, it is evident that mineral oil given i.p. activates the spleen macrophages. Although the number and percentage of macrophages in the spleen does not increase following mineral oil injection, the activities of some of their enzymes (acid phosphatase and beta-glucuronidase) increase while others do not change (Cathepsin D and lysozyme). Furthermore, the Fc-dependent phagocytic activity of spleen macrophages and their spreading on plastic culture dishes is increased after mineral oil treatment. We conclude that the activation of spleen macrophages caused by an i.p. injection of mineral oil also induces the changes in their antigen-presenting apparatus. Consequently, macrophages from spleens of mineral oil-injected mice are most suitable cell preparations for antigen presentation to T-lymphocytes.

An investigation of the immunological accessory cell function of in vitro bone marrow derived colony forming cells has been made. Data are presented to show that such cells can present immunogenic stimuli to lymphocytes and release lymphostimulatory products that in turn replace certain macrophage functions in the immune response. The appearance of these two activities is not correlated. Considerable heterogeneity exists in terms of the ability of different populations of cells to present distinct carbohydrate antigens in immunogenic form. In addition, that different macrophage cell populations can alter the affinity of antibody produced by an optimally stimulated primed spleen cell population is reported.

We describe an antigen(s) characterized by a heteroantiserum raised in rabbits against mature human granulocytes. This antigen was found on neutrophils, monocytes, platelets, acute and chronic myelocytic leukemia cells and on granulocyte-macrophage progenitor cells grown in agar. It was not found on lymphocytes, eosinophils, erythrocytes, or erythroid progenitor cells. On the basis of tissue distribution and absorption studies, the antigen (tentatively designated the "myelo-monocytic" antigen) is distinct from antigens previously identified on human neutrophils. Restriction of the "myelo-monocytic" antigen to normal and malignant cells of the myelo-monocytic series suggests that it may represent a normal differentiation antigen of the myelo-monocytic lineage.

Expression of a rabbit T-cell specific antigen can be induced on bone marrow lymphocytes following exposure to an RNA extract obtained from the thymuses of young rabbits. The presence of the antigen was demonstrated using goat anti-rabbit T-cell serum in a complement-dependent cytotoxicity assay. The T-cell antigen first appeared 3 h after addition of the thymus RNA to bone marrow cell cultures and the maximum number of cells expressing the T-cell antigen was observed within 24 h. RNA obtained from a source other than the thymus was found to be ineffective in inducing expression of the T-cell antigen. The induction of the antigen appears to be dependent on the presence of intact thymus RNA, as RNase treatment but not trypsin treatment, destroyed the ability of the RNA to induce the T-cell antigen.

Tumor-specific antigens capable of eliciting a response from autologous lymphocytes have been described in mouse plasmacytoma systems. This paper presents evidence of similar antigens in human myeloma. Myeloma plasma cells isolated from the bone marrow of 27 patients stimulated autologous and allogeneic peripheral blood lymphocytes (PBL) from myeloma patients in mixed leukocyte culture. Plasma cells isolated fromt the bone marrow of normal patients or patients with benign plasmacytosis failed to stimulate PBL in mixed leukocyte culture. Similar results were found in passive cytotoxicity assays with the use of chinken red blood cells (CRBC) coated with 3M KC1 plasma cell extracts from myeloma patients. PBL from myeloma patients caused 30 to 80 percent chromium-51 release from tanned chromium-labeled plasma cell extract-coated CRBC targets, whereas PBL from normal patients or patients with benign plasmacytosis caused only 10 to 25 percent chromium-51 release. This study indicates the presence of material resembling tumor-specific antigens on human myeloma plasma cells. Immune response to suce antigens is elicited in autologous and allogeneic myeloma patients.

Eosinophilia is usually regarded as a T cell-dependent hemopoietic response, although in most instances of immune responses by T cells there is no accompanying eosinophilia. BALB/c mice immunized with keyhole limpet hemocyanin (KLH) in complete Freund's adjuvant (CFA) were found not to develop eosinophilia, whereas pretreatment of such mice with 150 mg/kg cyclophosphamide (CY) induced marked eosinophilia in the blood (1000 to 3000/mm3) and bone marrow. CY as well as CY and KLH in CFA cause a leukocytosis and neutrophilia, but significant eosinophilia was seen only in mice receiving both CY and KLH-CFA. The eosinophilic response depended on the dose of CY and peaked 13 days after immunization, and was preceded by a bone marrow eosinophilia that peaked 8 days after immunization. No eosinophilia occurred in BALB/c nu/nu mice after CY and KLH-CFA but did occur in the nu/+ littermates. This model in mice of eosinophilia in response to CY followed by a nonparasite antigen should be useful in the analysis of T cell signals that might regulate eosinophilia.

A role for antigen in the generation of fully mature splenic type B cells has been shown. In adoptive transfer experiments, cells from bone marrow or fetal liver required a longer period to give an anti-sheep red blood cell plaque-forming cell (PFC) response than those from spleen. This delay was not overcome by allowing the cells a 7-day sojourn in the irradiated host before antigen challenge. A two-stage protocol was designed in which the in vivo generation of fully mature cells could be measured by their ability to give PFC in lipopolysaccharide-stimulated cultures in vitro. These experiments showed that a critical factor which influences the final differentiation of bone marrow or fetal liver cells into mature, splenic type B cells is exposure to antigen.

In order to determine the cell type responsible for the antigen-binding reaction in the bone marrow and spleen of mice, cells derived from pure in vitro derived colonies of neutrophils, eosinophils, macrophage-megakaryocytes and B lymphocytes were tested for their ability to bind fluorescent protein antigens. Only B lymphocytes bound antigen. An unexpectedly high percentage of bone marrow B lymphocytes (20%) bound a given antigen. This frequency was considerably higher than that found for spleen cells. As might be expected from such high binding frequencies, some cells bound two fluorchromated antigens when these are added together. As a direct test of the clonality of antigen binding to bone marrow B lymphocytes, whole colonies of B cells were tested for antigen binding of two non-cross-reacting protein antigens. The frequency of antigen-binding clones, including double antigen-binding clones, reflects exactly the frequencies observed for dispersed colony B cells and for in vivo derived Ig-bearing bone marrow B cells. The frequency of double antigen-binding colonies was equal to the product of the frequencies of the colonies binding each of the two antigens alone. No 'mixed' colonies containing single binding cells for each antigen were found. Thus, the ability to bind any two given antigens is a clonally distributed property of the bone marrow B lymphocyte population. Heterogenous receptors for multiple antigen binding on each cell are either randomly distributed among the B cell population, or homogenous antigen-binding receptors on each cell have a random chance of cross-reaction with the two antigens tested.

This paper describes the use of immunoenzymatic techniques (and in particular a recently developed immuno-alkaline phosphatase procedure) for labelling haematological samples with monoclonal antibodies. Since cells are smeared and fixed before staining it is possible to combine optimal preservation of cellular detail with visualization of positive labelling. Additional advantages over conventional immunofluorescent procedures for detecting cellular antigens include the fact that samples may be stored for long periods both before and after staining, and that double labelling may readily be performed (either by combining immunoenzymatic staining with T cell rosetting or by performing immunoperoxidase and immuno-alkaline phosphatase techniques sequentially). Furthermore, these methods may be applied to samples containing too few cells for conventional examination (e.g. samples of cerebrospinal fluid). A total of 16 different antigens (including HLA-DR, common ALL antigen and antigens associated with T cells, B cells, erythroid cells and megakaryocytes) were demonstrated by immuno-enzymatic staining on a range of normal and neoplastic haematological samples. It is concluded that this approach to cellular antigen labelling is of potential value not only in routine haematological diagnosis, but also for research in many immunological and haematological fields.

Mouse bone marrow mast cells sensitized with monoclonal IgE and activated with specific antigen released 2.8 +/- 0.5 ng of platelet-activating factor (1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) (PAF-acether)/ 10(6) cells. The PAF-acether was identified by its ability to aggregate fully aspirin-treated washed rabbit platelets in the presence of an adenosine diphosphate (ADP)-scavenger complex, by its co-chromatography with [3H]-labeled semi-synthetic PAF-acether and synthetic 1-0-octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, and by its inactivation by phospholipases A2, C, and D and not by lipase A1. The antigen-initiated release of PAF-acether, leukotriene C4 (LTC4), and leukotriene B4 (LTB4), and the secretion of the granule marker beta-hexosaminidase were not diminished by washing the cells before challenge, indicating that they were due to the interaction of antigen with the IgE fixed on the cell membrane and not to phagocytosis of immune complexes formed in the fluid phase. The parallel antigen-induced dose-response relationship, along with the superimposable time-course of the extracellular appearance, of beta-hexosaminidase, PAF-acether, and both leukotrienes indicated that the origin of these diverse mediators was from a common cell type with IgE-Fc receptors. Ethanol extraction of antigen-stimulated bone marrow-derived mast cells revealed the early transient appearance of a cell-associated platelet-aggregating activity, the action of which on platelets, like PAF-acether, was independent of ADP and arachidonic acid metabolism. The cell-associated activity contained a novel product that eluted at 13 min during high performance liquid chromatography (HPLC) (solvent hexane:n-propanol:water, 46:46:8), permitting resolution from PAF-acether and lyso-PAF-acether (1-O-alkyl-sn-glyceryl-3-phosphorylcholine), which eluted at 29 min and 30 min, respectively. The cell-associated material, which differs from lyso-PAF-acether, the putative precursor of PAF-acether, in being active in the bioassay on platelets may represent a newly recognized intermediate in the generation of PAF-acether. As the transiently present cell-associated intermediate has not been previously recognized, its detection may depend upon the relatively unique properties of the bone marrow-derived mast cell system in which IgE-dependent activation leading to product generation is complete within 5 min.(ABSTRACT TRUNCATED AT 400 WORDS)

The presence of neutrophil surface antigens on maturing bone marrow granulocytes was examined by indirect immunofluorescence and cellular immunoadsorption using heterologous antibody to mature neutrophil granulocytes. The results show that bone marrow neutrophils possess surface antigens that appear during cell maturation from myeloblasts. Fluorescent antibody capping and patching, indicators of antigen mobility, were more pronounced in mature than in immature cells. Neutrophil surface antigen development could also be demonstrated during granulocyte maturation in vitro.

As it is previously shown (Tsyrlova et al., 1986), the level of humoral immune response is not only determined by the reaction of peripheral lymphoid system on antigenic effect, but also is bound up with the observed stem blood cell (SBC) proliferation in bone marrow (Frindel et al., 1976, Kozlov et al., 1982). Dynamics of label accumulation in bone marrow was examined when injecting antigen--sheep red blood cells labeled by radioisotope 51Cr, 125I. The peak of label accumulation in bone marrow, accompanied by the increase in proliferative SBC, was observed on the 3rd day after antigen injection. Furthermore in the course of immunization 51Cr labeled macrophage assortment was changed in time in such a way that a greater number of macrophages was accumulated in bone marrow on the 3rd and 4th days in the immunized animals in comparison with the intact ones. The macrophages in bone marrow are likely to take part in antigen uptake and to mediate its effect on SBC proliferation.

Bone marrow-derived macrophages (BMM phi) were grown in a liquid culture system in the presence of L cell-conditioned medium as a source of colony-stimulating factor. After a 4-h pulse with antigen, cultured irradiated BMM phi were capable of presenting the antigen to primed T cells as assessed in a T cell proliferation assay. Proliferation was optimal when BMM phi were used between days 5 and 8 of bone marrow cell culture. T cells of Lyt1 and Lyt123 phenotype had to be present at the start of the culture period to yield an optimal response. Conventional antisera and monoclonal antibodies directed against the H-2 I region and the I-A subregion, respectively, proved inhibitory in this system. Cultured BMM phi from low-responder strains failed to present antigens under immune response gene control in a form that was immunogenic to T lymphocytes. Cultured BMM phi might thus serve as a source of antigen-presenting cells in the study of cell-cell interaction and immune response gene regulatory mechanisms.

The peculiarities of carefully studied immunoglobulin receptors of the specialized clones of B-lymphocytes are described. These latter are precursors of antibody-forming cells but play only a limited role in the recognition of foreign antigens which is realized by T-lymphocytes. The differentiation of T- and B-lymphocytes from the single precursor, a bone marrow stem hemopoietic cell, proceeds in the bone marrow itself, thymus and peripheral lymphoid organs in several discrete stages. At each of these stages the cells have definite properties and, in some cases, may be separated one from another. The modern data are given concerning the properties of cells at every stage of antigen-independent differentiation of T- and B-lymphocytes which proceeds during embryonic and postnatal development, its alternative pathways, factors of regulation, role of local microenvironment and possibilities of modelling in vitro.

A monoclonal antibody of IgM class that defines the antigen present on human peripheral blood granulocytes was produced and characterized. This monoclonal antibody (IF10) was made from a single fusion between P3-X63-Ag8-U1 (P3U1) myeloma cells and splenocytes from a BALB/c mouse immunized against human cultured monocytoid cell line THP-1 cells. IF10-defined antigen(s) was expressed on the cells of granulocyte lineage such as peripheral blood granulocytes and metamyelocytes, myelocytes, promyelocytes, and a part of myeloblasts in the normal bone marrow, whereas it was not detected on resting and activated T lymphocytes, B lymphocytes, adherent monocytes, and thymocytes. The IF10-defined antigen was also expressed on cultured monocytoid cell lines as well as myeloid and myeloid/erythroid (K-562) cell lines. T lymphoblastoid cell lines, particularly those representing the T cells at an early thymocyte level, and a part of null cell lines were also reactive to IF10. Therefore, IF10 may be a unique monoclonal antibody that defines an antigen(s) which is expressed on almost whole stages of granulocytes and early stages of macrophages and T-cell lineages, and possibly during very early stages of erythroid lineage.