Common variable immunodeficiency revisited: normal generation of naturally occurring dendritic cells that respond to Toll-like receptors 7 and 9.

Patients with common variable immunodeficiency (CVID) have reduced numbers and frequencies of dendritic cells (DCs) in blood, and there is also evidence for defective activation through Toll-like receptors (TLRs). Collectively, these observations may point to a primary defect in the generation of functional DCs. Here, we measured frequencies of plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) in peripheral blood of 26 CVID patients and 16 healthy controls. The results show that the patients have reduced absolute counts of both subsets. However, the decreased numbers in peripheral blood were not reflected in reduced frequencies of CD34(+) pDC progenitors in the bone marrow. Moreover, studies at the single cell level showed that DCs from CVID patients and healthy controls produced similar amounts of interferon-α or interleukin-12 and expressed similar levels of activation markers in response to human cytomegalovirus and ligands for TLR-7 and TLR-9. The study represents the most thorough functional characterization to date, and the first to assess bone marrow progenitor output, of naturally occurring DCs in CVID. In conclusion, it seems unlikely that CVID is secondary to insufficient production of naturally occurring DCs or a defect in their signalling through TLR-7 or TLR-9.

Targeting B cell leukemia with highly specific allogeneic T cells with a public recognition motif.

The possibility that allogeneic T cells may be targeted to leukemia has important therapeutic implications. As most tumor antigens represent self-proteins, high-avidity tumor-specific T cells are largely deleted from the repertoire of the patient. In contrast, T cells from major histocompatibility complex (MHC)-mismatched donors provide naïve repertoires wherein such cells have not been systematically eliminated. Yet, evidence for peptide degeneracy or poly-specificity warrants caution in the use of foreign human leukocyte antigen (HLA) or peptide complexes as therapeutic targets. Here, we cocultured HLA-A(*)0201-negative T cells with autologous dendritic cells engineered to present HLA-A(*)0201 complexed with a peptide from the B cell antigen CD20 (CD20p). HLA-A(*)0201/CD20p pentamer-reactive CD8(+) T cells were readily obtained from all donors. The polyclonal cells showed exquisite peptide and MHC specificity, and efficiently killed HLA-A(*)0201-positive B cells, including primary chronic lymphocytic leukemia cells. The T cell receptor (TCR) sequences displayed a novel type of conservation, with extensive homology in the TCR ß chain complementarity-determining region 3 and in J, but not V, region. This is surprising, as the donors were HLA disparate and their TCR repertoires are expected to show little overlap. The results demonstrate the first public recognition motif for an allogeneic HLA/peptide complex. The allo-restricted T cells or TCRs could provide graft-versus-leukemia in the absence of graft-versus-host disease.

Dendritic cells engineered to express defined allo-HLA peptide complexes induce antigen-specific cytotoxic T cells efficiently killing tumour cells.

Most tumour-associated antigens (TAA) are non-mutated self-antigens. The peripheral T cell repertoire is devoid of high-avidity TAA-specific cytotoxic T lymphocytes (CTL) due to self-tolerance. As tolerance is major histocompatibility complex-restricted, T cells may be immunized against TAA presented by a non-self human leucocyte antigen (HLA) molecule and transferred to cancer patients expressing that HLA molecule. Obtaining allo-restricted CTL of high-avidity and low cross-reactivity has, however, proven difficult. Here, we show that dendritic cells transfected with mRNA encoding HLA-A*0201, efficiently present externally loaded peptides from the antigen, Melan-A/MART-1 to T cells from HLA-A*0201-negative donors. CD8(+) T cells binding HLA-A*0201/MART-1 pentamers were detected already after 12 days of co-culture in 11/11 donors. The majority of cells from pentamer(+) cell lines were CTL and efficiently killed HLA-A*0201(+) melanoma cells, whilst sparing HLA-A*0201(+) B-cells. Allo-restricted CTL specific for peptides from the leukaemia-associated antigens CD33 and CD19 were obtained with comparable efficiency. Collectively, the results show that dendritic cells engineered to express defined allo-HLA peptide complexes are highly efficient in generating CTL specifically reacting with tumour-associated antigens.

Assay for monitoring in vitro selective depletion strategies in allogeneic stem cell transplantation.

BACKGROUND: GvHD is a serious and potentially life-threatening side-effect of allogeneic BMT, caused by alloreactive cells attacking normal host cells. A number of different approaches have been attempted to remove allo-activated cells from the graft prior to transplantation. When developing such assays, there is a need to control for unwanted removal of cells, as well as depletion efficiency related to activation kinetics. METHODS: The specific activation induced by the superantigens SEB and TSST-1 of T cells with defined Vbeta chains was utilized to follow activation of bystander cells and the kinetics of specific cellular activation by flow cytometry. RESULTS: The activation marker CD69 was up-regulated on bystander T cells, and was only transiently highly expressed on the specific T cells, making this marker unreliable for removal of alloreactive cells. In contrast, CD25 was found only on specifically activated T cells and was stably expressed over several days. However, it was not detected on all specific cells until day 6. Likewise, proliferation occurred only in T cells expressing the expected Vbeta chains, with all activated cells having undergone at least one cell cycle by day 4. DISCUSSION: In conclusion, our assay demonstrates that only temporary bystander activation occurs when polyclonally activating T cells by SEB or TSST-1, and that CD25, but not CD69, can be used for removal of specifically activated cells. Furthermore, this assay is useful for monitoring methods aiming at specific removal of cycling cells.

[Dendritic cells--strong candidates for immunotherapy]. / Dendrittiske celler--sterke kandidater for immunterapi.

Why are immune responses primarily directed towards infectious agents, and how can the immune system be manipulated to attack for instance malignant cells? The role of the dendritic cells in the immune system may provide the answers. We present a review of a field in which results from basic science are rapidly applied in clinical trials. We searched the Medline database using the terms dendritic cells combined with ontogeny, subpopulations, vaccine or review. Results from our own experimental work are also described. The cited studies show that dendritic cells take up material from their surroundings and migrate to lymphoid tissue where the material is presented to T-cells. Dendritic cells have the ability to selectively direct immune responses towards potentially harmful agents such as bacteria and viruses. Clinical trials show that vaccines based on the use of dendritic cells induce tumor-specific immunity and clinical remission. Experiments conducted by the authors and others indicate the existence of subpopulations of dendritic cells with specialized functions. Dendritic cells play a central role in the initiation of immune responses and may be used to manipulate the immune system. Their use in the treatment of diseases such as cancer is highly promising.

Primitive human hematopoietic progenitor cells express receptors for granulocyte-macrophage colony-stimulating factor.

Most cytokines act only synergistically in assays of primitive progenitor cell proliferation, and effects have usually been observed first after prolonged cell culture. Studies reporting that primitive progenitors lack receptors for a number of cytokines, including granulocyte-macrophage colony stimulating factor (GM-CSF), could indicate that several "synergistic" cytokines primarily affect cells that have differentiated in vitro. Here, however, we show that freshly isolated primitive progenitor cells (CD34hi CD38-) express receptors for GM-CSF at levels 20%-30% of granulo-monocytic progenitors. Although GM-CSF had minimal effects on the survival or proliferation of primitive progenitors when added alone, the cytokine enhanced stem cell factor (SCF) induced cell cycle entry in the first generation. The effect was not observed when cells were incubated sequentially with SCF and GM-CSF. The results suggest that the synergistic effects of GM-CSF are mediated directly on primitive progenitor cells and that the cytokine may be useful to enhance cell cycle entry of hematopoietic stem cells.

Insulin-like Growth Factor-1 (IGF-1) is a Costimulator of the Expansion of Lineage Committed Cells Derived from Peripheral Blood Mobilized CD34(+) Cells in Multiple Myeloma Patients.

The effect of insulin-like growth factor-1 (IGF-1) on highly enriched human apheresis CD34(+) progenitor cells was investigated in vitro. The progenitor cells were mobilized by treatment with cyclophosphamide + granulocyte-colony stimulating factor (G-CSF) in patients with multiple myeloma. CD34(+) cells were cultured for 7 days in serumfree medium containing stem cell factor (SCF), granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin-3 (IL-3), and this is referred to as cytokine-dependent proliferation. After 7 days of cytokine-dependent proliferation the total number of viable cells increased 1.6-8.2 times, and subsets of cells expressing the granulocyte marker CD15, the myelomonocytic marker CD64 and the erythrocyte phenotype CD71(high)/CD64(-) were detected among the in vitro cultured cells. Addition of G-CSF together with SCF + IL-3 + GM-CSF increased the number of CD15(+) and CD64(+) cells, but without altering the number of erythroid cells. IGF-1 caused a dose-dependent increase in the number of CD15(+), CD64(+) and CD71(high)/CD64(-) cells, and this increase was detected when cells were cultured in both SCF + IL-3 + GM-CSF alone and G-CSF + SCF + IL-3 + GM-CSF. A minor subset of CD34(+) cells could still be detected among in vitro cultured cells and the number of CD34(+) cells was not altered by adding G-CSF and/or IGF-1. Morphologically recognizable mature granulocytes or erythroid cells could not be detected for any of the combinations investigated. We conclude that IGF-1 can enhance the in vitro proliferation of committed progenitor cells derived from apheresis CD34(+) cells.

Insulin-like growth factor-1 (IGF-1) has a costimulatory effect on proliferation of committed progenitors derived from human umbilical cord CD34+ cells.

The effects of insulin-like growth factor-1 (IGF-1) on highly enriched human umbilical cord CD34+ cells were investigated in vitro. CD34+ cells were cultured in serum-free medium containing stem cell factor (SCF), GM-CSF, and interleukin-3 (IL-3). Culture of CD34+ cells for one week in the presence of these cytokines resulted in a dose-dependent increase in total cell number. Addition of G-CSF together with SCF+IL-3+GM-CSF increased the proliferation of myelopoietic cells as determined by the number of cells expressing the myelomonocytic marker CD64 and the granulocytic marker CD15 without significantly altering the number of CD34+ cells in the cultures. In the presence of G-CSF, IGF-1 induced a dose-dependent increase in the total cell number and a moderate but significant increase in the percentages of CD15+, CD64+ cells with sustained CD34+ cell proliferation. We conclude that IGF-1 can enhance the in vitro proliferation of committed progenitor cells derived from umbilical cord CD34+ cells.

Dendritic cell ontogeny: a human dendritic cell lineage of myeloid origin.

Dendritic cells (DC) have been thought to represent a family of closely related cells with similar functions and developmental pathways. The best-characterized precursors are the epidermal Langerhans cells, which migrate to lymphoid organs and become activated DC in response to inflammatory stimuli. Here, we demonstrate that a large subset of DC in the T cell-dependent areas of human lymphoid organs are nonactivated cells and belong to a separate lineage that can be identified by high levels of the interleukin 3 receptor alpha chain (IL-3Ralphahi). The CD34+IL-3Ralphahi DC progenitors are of myeloid origin and are distinct from those that give rise to Langerhans cells in vitro. The IL-3Ralphahi DC furthermore appear to migrate to lymphoid organs independently of inflammatory stimuli or foreign antigens. Thus, DC are heterogeneous with regard to function and ontogeny.

AC133, a novel marker for human hematopoietic stem and progenitor cells.

AC133 is one of a new panel of murine hybridoma lines producing monoclonal IgG antibodies (mAbs) to a novel stem cell glycoprotein antigen with a molecular weight of 120 kD. AC133 antigen is selectively expressed on CD34(bright) hematopoietic stem and progenitor cells (progenitors) derived from human fetal liver and bone marrow, and blood. It is not detectable on other blood cells, cultured human umbilical vein endothelial cells (HUVECs), fibroblast cell lines, or the myeloid leukemia cell line KG1a by standard flow cytometric procedures. All of the noncommitted CD34(+) cell population, as well as the majority of CD34(+) cells committed to the granulocytic/monocytic pathway, are stained with AC133 antibody. In vitro clonogenicity assays have demonstrated that the CD34(+)AC133(+) double-positive population from adult bone marrow contains the majority of the CFU-GM, a proportion of the CFU-Mix, and a minor population of BFU-E. The CD34(dim) and AC133(-) population has been shown to contain the remaining progenitor cells. AC133-selected cells engraft successfully in a fetal sheep transplantation model, and human cells harvested from chimeric fetal sheep bone marrow have been shown to successfully engraft secondary recipients, providing evidence for the long-term repopulating potential of AC133(+) cells. A cDNA coding for AC133 antigen has been isolated, which codes for a polypeptide consisting of 865 amino acids (aa) with a predicted size of 97 kD. This antigen is modeled as a 5-transmembrane molecule, a structure that is novel among known cell surface structures. AC133 antibody provides an alternative to CD34 for the selection and characterization of cells necessary for both short- and long-term engraftment, in transplant situations, for studies of ex vivo expansion strategies, and for gene therapy.

Sustained, retransplantable, multilineage engraftment of highly purified adult human bone marrow stem cells in vivo.

Data from many laboratory and clinical investigations indicate that CD34+ cells comprise approximately 1% of human bone marrow (BM) mononuclear cells, including the progenitor cells of all the lymphohematopoietic lineages and lymphohematopoietic stem cells (stem cells). Because stem cells are an important but rare cell type in the CD34+ cell population, investigators have subdivided the CD34+ cell population to further enrich stem cells. The CD34+/CD38- cell subset comprises less than 10% of human CD34+ adult BM cells (equivalent to < 0.1% of marrow mononuclear cells), lacks lineage (lin) antigens, contains cells with in vitro replating capacity, and is predicted to be highly enriched for stem cells. The present investigation tested whether the CD34+/CD38- subset of adult human marrow generates human hematopoiesis after transfer to preimmune fetal sheep. CD34+/ CD38- cells purified from marrow using immunomagnetic microspheres or fluorescence-activated cell sorting generated easily detectable, long-term, multilineage human hematopoiesis in the human-fetal sheep in vivo model. In contrast, transfer of CD34+/CD38+ cells to preimmune fetal sheep generated only short-term human hematopoiesis, possibly suggesting that the CD34+/CD38+ cell population contains relatively early multipotent hematopoletic progenitor cells, but not stem cells. This work extends the prior in vitro evidence that the earliest cells in fetal and adult human marrow lack CD38 expression. In summary, the CD34+/ CD38- cell population has a high capacity for long-term multilineage hematopoietic engraftment, suggesting the presence of stem cells in this minor adult human marrow cell subset.

Granulocytic and monocytic differentiation of CD34hi cells is associated with distinct changes in the expression of the PU.1-regulated molecules, CD64 and macrophage colony-stimulating factor receptor.

The present study investigated the possibility that macrophage colony-stimulating factor (M-CSF) responsiveness of hematopoietic progenitor cells is regulated at the level of receptor expression and that M-CSF receptor (M-CSFR) may be used as an early marker of monocyte lineage commitment. Immunofluorescence measurements with an anti-M-CSFR antibody showed that 44% +/- 5% of CD34hi cells expressed the receptor. The M-CSFR was present on progenitor cells that were positive for the granulo-monocytic marker CD64, but not on primitive, erythroid, or lymphoid progenitors. The CD34hiCD64+ population could be divided into subsets of M-CSFRhi and M-CSFRlo cells. In addition, a subset of CD34hiCD64-M-CSFRhi cells was found. CD34+ cells that were positive for M-CSFR, CD64, or both gave rise exclusively to granulo-monocytic cells, and 65% of the granulomonocytic colony-forming cells in the CD34+ population were recovered from these cells. Approximately 70% of the colony-forming cells (CFCs) derived from CD34hiM-CSFRhi cells were macrophage colony-forming units (CFU-M), whereas 91% of the CFCs in the CD34hiCD64+M-CSFRlo population were granulocyte colony-forming units (CFU-G). The M-CSFRhi cells with the highest frequency of colony-forming and bipotent cells and largest average colony size were found in the CD64- subset, indicating that M-CSFR appears earlier than CD64 during monocyte development. After 60 hours in culture, a subset of the CD34hiM-CSFRhi cells had downmodulated M-CSFR (29% to 38%). This population gave rise almost exclusively to granulocytes, whereas the cells that remained M-CSFRhi gave rise exclusively to monocytes. In all experiments, the M-CSFRhi population responded to M-CSF, whereas minimal responses were observed among M-CSFRlo cells. These results suggest that M-CSF target specificity among human hematopoietic progenitor cells is determined by lineage-specific regulation of the M-CSFR and show that M-CSFR is a useful marker to discriminate between monocytic and granulocytic progenitor cells.

Expression and function of receptors for stem cell factor and erythropoietin during lineage commitment of human hematopoietic progenitor cells.

The aim of the present study was to determine whether stem cell factor (SCF) and erythropoietin (EPO) act differently on defined subsets of progenitor cells, and if potential differences correlate with the receptor density on each subset. To investigate this possibility directly, we optimized conditions for the identification and purification of homogeneous progenitor cell subpopulations from human bone marrow. Populations containing 40% and 44% colony forming cells (CFCs) with 99% and 95% purity for the granulomonocytic and erythroid lineage, respectively, were sorted on the basis of differential expression of CD34, CD64, and CD71. In addition, a population containing 67% CFCs, of which 29-43% were CFU-MIX, was sorted from CD34hi CD38loCD50+ cells. Purified progenitor cell subsets were compared directly for responsiveness to SCF and EPO using a short-term proliferation assay. Expression of the receptors for SCF and EPO were then examined on each subset using a flow cytometer modified for high-sensitivity fluorescence measurements. The results show that EPO induces extensive proliferation of erythroid progenitor cells, but has no effect on the proliferation or survival of primitive or granulomonocytic progenitors, even when used in combination with other cytokines. The majority of erythroid progenitor cells furthermore stained positively with anti-EPO receptor (EPO-R) monoclonal antibodies, whereas other progenitor cells were negative. SCF alone induced extensive proliferation of erythroid progenitor cells, and had a stronger synergistic effect on primitive than on granulo-monocytic progenitors. In spite of these differences in SCF activity, there were no significant differences in SCF-R expression between the progenitor subsets. These results suggest that the selective action of EPO on erythropoiesis is determined by lineage-restricted receptor expression, whereas there are additional cell-type specific factors that influence progenitor cell responses to SCF.

CD64/Fc gamma RI is a granulo-monocytic lineage marker on CD34+ hematopoietic progenitor cells.

The aim of this study was to identify markers specific for granulo-monocytic commitment of progenitor cells. Large panels of antibodies were screened for selective staining of subsets of CD34+ cells from fetal and adult bone marrow. Flow cytometric analysis showed that CD64/fc gamma RI was undetectable on noncommitted progenitor cells (CD34++, CD38-/lo, HLA-DR+) and expressed on a subset of lineage-committed progenitors (CD34+, CD38+) with higher mean orthogonal light scatter than the remaining CD34+ cells. The CD34+, CD64+ cells were CD19- and the majority were CD45RA+, CD71lo, suggesting that CD64 recognized granulomonocytic progenitor cells. Specificity of CD64 for the granulo-monocytic lineage was shown by demonstrating that colonies arising from CD34+, CD64+ cells consisted of 98% +/- 2% colony-forming unit-granulocyte-macrophage (CFU-GM) in semisolid medium containing stem cell factor (SCF), interleukin-3 (IL-3), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (EPO). In contrast, 63% +/- 15% of the colonies from the CD34+, CD64- cells were burst-forming unit-erythroid/colony-forming unit-erythroid (BFU-E/CFU-E). Furthermore, four-color immunofluourescence and cell sorting was used to analyze the progeny of cells cultured in liquid medium containing identical cytokines as used in the semisolid medium. This analysis showed that CD34+, CD64+ cells gave rise to 83% +/- 10% granulo-monocytic cells whereas progeny of the CD34+, CD64- cells contained 81% +/- 11% erythroid cells. Neutrophils as well as basophils and monocytes/macrophages were present in the cultures from CD34+, CD64+ cells, showing that this population contains progenitors of most types of granulo-monocytic cells. Two widely used myeloid markers, CD13 and CD33, were not myeloid-specific, because both were clearly positive on noncommitted progenitor cells. Of 40 antigens tested, CD15 was the only other marker fulfilling the criteria of a myeloid-specific marker. However, at concentrations of CD15 that did not induce aggregation, CD15+ cells constituted less than 50% of the CD34+, CD64+ cells. Furthermore, the CD34+, CD15- cells showed more than 50% higher CD34 mean fluorescence intensity than the CD64+, CD15+ cells, indicating that CD64 appears earlier than CD15 during differentiation. Thus, among a large number of antigens screened, CD64 was the most useful for the identification and purification of granulo-monocytic progenitor cells.

The "common stem cell" hypothesis reevaluated: human fetal bone marrow contains separate populations of hematopoietic and stromal progenitors.

There is a long-standing controversy as to whether a single bone marrow (BM)-derived cell can differentiate along both hematopoietic and stromal lineages. Both primitive hematopoietic and stromal progenitor cells in human BM express the CD34 antigen but lack expression of other surface markers, such as CD38. In this study we examined the CD34+, CD38- fraction of human fetal BM by multiparameter fluorescence-activated cell sorting (FACS) analysis and single-cell sorting. CD34+, C38- cells could be divided into HLA-DR+ and HLA-DR- fractions. After single-cell sorting, 59% of the HLA-DR+ cells formed hematopoietic colonies. In contrast, the CD34+, CD38-, HLA-DR- cells were much more heterogeneous with respect to their light scatter properties, expression of other hematopoietic markers (CD10, CD36, CD43, CD49b, CD49d, CD49e, CD50, CD62E, CD90w, CD105, and CD106), and growth properties. Single CD34+, CD38-, HLA-DR- cells sorted into individual culture wells formed either hematopoietic or stromal colonies. The presence or absence of CD50 (ICAM-3) expression distinguished hematopoietic from stromal progenitors within the CD34+, CD38-, HLA-DR- population. The CD50+ fraction had light scatter characteristics and growth properties of hematopoietic progenitor cells. In contrast, the CD50- fraction lacked hematopoietic progenitor activity but contained clonogenic stromal progenitors at a mean frequency of 5%. We tested the hypothesis that cultures derived from single cells with the CD34+, CD38-, HLA-DR- phenotype could differentiate along both a hematopoietic and stromal lineage. The cultures contained a variety of mesenchymal cell types and mononuclear cells that had the morphologic appearance of histiocytes. Immunophenotyping of cells from these cultures indicated a stromal rather than a hematopoietic origin. In addition, the growth of the histiocytic cells was independent of the presence or the absence of hematopoietic growth factors. Based on sorting more than 30,000 single cells with the CD34+, CD38-, HLA-DR- phenotype into individual culture wells, and an analysis of 864 stromal cultures initiated by single CD34+ BM cells, this study does not support the hypothesis of a single common progenitor for both hematopoietic and stromal lineages within human fetal BM.

Expression of cell surface markers during differentiation of CD34+, CD38-/lo fetal and adult bone marrow cells.

Even though there has been considerable progress in the phenotypic characterization of CD34+ bone marrow cells, there is still limited knowledge about the cell phenotypes corresponding to functional terms such as colony-forming cells, burst-forming cells, long-term culture-initiating cells, and high-proliferative potential cells. In this study, we performed a detailed analysis of phenotypic characteristics of subsets of CD34+ cells. We compared cells from adult and fetal bone marrow to investigate whether reported functional differences are reflected in the cellular phenotypes. CD34+, CD38-/lo, HLA-DR+ cells, which have been shown to contain the most immature hematopoietic progenitor cells, stained as a homogeneous population with most monoclonal antibodies (mAbs). The antigens sLex, CD32, and CD7 were, however, heterogeneously expressed in the CD38-/lo population. Phenotypic differences in the CD34+, CD38-/lo population was found when comparing adult and fetal bone marrow cells. Adult bone marrow CD34+, CD38-/lo cells stained more brightly with CD4, Thy-1, and CD49b and more dimly with CD32 than the same population in fetal bone marrow. Certain antigens that have previously been regarded as lineage-specific were found on the CD34+, CD38-/lo, HLA-DR+ cells in both fetal and adult bone marrow. These included CD52, CD13, and CD33. The markers that were found to be most useful in discriminating between subsets of lineage-committed cells within the CD34+, CD38+ population included the B cell marker CD19 and the granulomonocytic marker CD64. The phenotypic analysis presented here should provide a basis for establishing a better link between functional and phenotypic characteristics of hematopoietic progenitor cells.

Activation of human monocytes and granulocytes by monoclonal antibodies to glycosylphosphatidylinositol-anchored antigens.

The present study investigated possible receptor-like characteristics of glycosylphosphatidylinositol (GPI)-linked antigens on human monocytes and granulocytes by measuring cytoplasmic calcium fluxes and the oxidative burst in cells following cross-linking of GPI-linked antigens. Cross-linking of cell-bound anti-CD14, -CDw52 and -CD55 induced cytoplasmic calcium fluxes and oxidative bursts in unprimed human monocytes similar to those observed following Fc gamma R cross-linking. In granulocytes primed with 200 mM N-formyl-Met-Leu-Phe (FMLP), cross-linking of cell-bound anti-CD16, -CD24, -CD59 and -CD67 led to calcium fluxes and activation of the oxidative burst. The oxidative bursts mediated by GPI-linked antigens were stronger than those induced by 200 nM FMLP, even though FMLP induced a larger increase in cytoplasmic calcium concentration. The responses were likely to be independent of Fc gamma R interactions as F(ab')2 fragments of IgG or IgM antibodies were used in the experiments. Activating effects of monoclonal antibody to GPI-linked antigens were not observed in cells from patients with paroxysmal nocturnal hemoglobinuria, which are deficient in GPI-linked antigens. In addition, treatment with GPI-specific phospholipase C led to inhibition of cell activation through GPI-linked antigens but not through transmembrane receptors. Cross-linking of a number of non-GPI-linked antigens (CD11a, CD18, CD31, CD35, CD43, and CD45) neither induced calcium fluxes, nor activated the oxidative burst. The results indicate that most, if not all, GPI-linked surface glycoproteins on myeloid cells are capable of mediating cell activation and suggest that the GPI anchor is a structure facilitating signal transduction.

CD53, a protein with four membrane-spanning domains, mediates signal transduction in human monocytes and B cells.

CD53 is a member of a novel family of molecules with four presumably membrane-spanning domains. The structure and functional characteristics of these molecules indicate that they may play an important role in transmembrane communication. We therefore investigated whether CD53 is involved in activation of human leukocytes. Cross-linking of cell-bound F(ab')2 fragments of two different anti-CD53 mAb with F(ab')2 anti-mouse Ig led to cytoplasmic calcium fluxes in B cells, monocytes, and granulocytes and activation of the monocyte oxidative burst. These responses were specific for CD53, as cross-linking of CD11a, CD18, CD35, CD43, CD44, CD45, or CDw50 did not induce leukocyte activation. Low concentrations of staurosporine (10 to 20 nM) completely inhibited PMA-mediated activation, but had no effect on CD53-mediated calcium fluxes and inhibited only partially CD53-mediated oxidative burst. This suggests that CD53-mediated signaling is largely independent of protein kinase C. CD53-mediated calcium fluxes were inhibited by high concentrations of staurosporine (300 to 500 nM) but not by ADP-ribosylating toxins, suggesting dependence on tyrosine kinases rather than GTP-binding proteins. The results indicate that CD53, like several other leukocyte Ag with four membrane-spanning regions, has the ability to mediate cell activation, and support the view that these molecules are involved in transmembrane communication.