KP1 (CD 68) staining of malignant melanomas

The monoclonal antibody KP1, which recognizes the CD 68 antigen on macrophages and myeloid precursors, was tested on 28 malignant (primary and metastatic) melanomas, 28 naevi, and 17 skin biopsies showing either normal (10) or hyperplastic melanocytes (7). Sixteen of 20 primary melanomas and six of eight metastatic melanomas showed variable numbers of KP1 positive tumour cells. All but five benign melanocytic proliferations (two Spitz naevi and three intradermal naevi), as well as normal and hyperplastic melanocytes were negative. These results indicate that difficulties may occur with the use of KP1 in the differential diagnosis between melanomas and neoplasms derived from histiocytes-macrophages, and that the expression of CD 68 antigen might be related to tumour progression in melanocytic cells.     

Use of monoclonal antibody KP1 for identifying normal and neoplastic human mast cells.

The monoclonal antibody KP1 (CD68) was used to stain normal and neoplastic monocytes and macrophages in routinely processed, paraffin wax embedded tissue: mast cells also exhibited strong, consistent cytoplasmic immunoreactivity. Light microscopic findings were corroborated by electron microscopical and immunocytochemical findings. The predominant sites of immunoreactivity were the specific intracytoplasmic granules of the mast cells. All mast cell subtypes--that is, normal and reactive mast cells, such as those in lymph nodes exhibiting chronic non-specific lymphadenitis, and malignant or neoplastic mast cells in various types of mastocytosis--reacted with this antibody. This finding is of diagnostic importance, because mast cell proliferation could be mistaken for histiocyte proliferation. It also supports the hypothesis that mast cells derive from the bone marrow.     

Angiomyolipoma of the kidney. Immunoreactivity with HMB-45. Light- and electron-microscopic findings

Immunoreactivity with HMB-45 has recently been described in renal angiomyolipoma, a tumour of smooth muscle cells. HMB-45 is a monoclonal antibody that reacts specifically with melanosomes. In order to determine whether the tumour cells contain melanosomes and synthesize melanin, seven tumours were studied by light microscopy and immunohistochemically with the antibodies HMB-45, KP1 (CD68), PG-M1 (CD68), Ki-M1P, anti-lysozyme, anti-smooth-muscle actin, anti-vimentin, anti-S100 protein and KL1 (anti-keratin). Two tumours were also studied by electronmicroscopy and one by immuno-electronmicroscopy. Histochemical investigation for dopa oxidase was performed on cryostat sections. The tumours contained varying numbers of HMB-45-positive muscle cells. Reactivity was noted in lysosomal granules and rough endoplasmic reticulum. Typical premelanosomes were found in the tumour cells by electronmicroscopy. Groups of tumour cells stained for dopa oxidase. The tumour cells were not reactive for lysozyme, but reacted with KP1, PG-M1 and Ki-M1P. Immuno-electronmicrosopy showed that reactivity for KP1 was located within lysosomal granules. The findings show that the tumour cells of renal angiomyolipoma contain premelanosomes and that they are able to synthesize melanin, because they contain dopa oxidase. Immunoreactivity with KP1, PG-M1 and Ki-M1P can be attributed, in the absence of staining for lysozyme, to the large number of lysosomal granules. The tumour cells were not found to be related to macrophages or myeloid cells.     

PG-M1: A New Monoclonal Antibody Directed against a Fixative-Resistant Epitope on the Macrophage-Restricted Form of the CD68 Molecule

A new anti-macrophage monoclonal antibody (PG-M1) was produced by immunizing BALB/c mice with fresh spleen cells from a patient with Gaucher's disease. PG-M1 reacts strongly with a fixative-resistant epitope of an intracytoplasmic molecule, selectively expressed by virtually all macrophages of the human body. Although attempts to immunoprecipitate the molecule recognized by PG-M1 have failed so far, the reactivity of the antibody with COS-1 and WOP cells transfected with a human complementary DNA clone encoding for the CD68 antigen suggests that PG-M1 is a new member of the CD68 cluster. However, unlike other CD68 antibodies (KP1, EBM11, etc.), which react with both macrophages and myeloid cells, PG-M1 detects a fixative-resistant epitope on the macrophage-restricted form of the CD68 antigen. In 957 routinely fixed, paraffin-embedded samples, PG-M1 showed a more restricted reactivity with elements of the monocyte/macrophage lineage than the previously described monoclonal antibodies MAC-387 (anti-calgranulins), KP1 (CD68) and Ki-M1P. Among hematological malignancies, PG-M1 only labels acute leukemias of M4 and M5 type and rare examples of malignant histiocytosis/true histiocytic sarcoma. In contrast, acute leukemias of the M1, M2, M3, M6, M7, and L1-L3 types, non-Hodgkin's lymphomas, and Hodgkin and Reed-Sternberg cells of Hodgkin's disease are consistently PG-M1-negative. In the daily diagnostic practice, PG-M1 seems to be particularly valuable for the diagnosis of myelomonocytic or monocytic leukemia and neoplasms of true histiocytic origin in routine paraffin sections.     

CD68 reactivity of non-macrophage derived tumours in cytological specimens.

AIMS--To investigate the presence of the macrophage associated antigen CD68 in non-haematopoietic tumours. METHODS--Cytological specimens from non-macrophage derived tumours were stained using the alkaline phosphatase anti-alkaline phosphatase immunocytochemical method (APAAP) and three monoclonal anti-CD68 antibodies, Y1/82A, EBM11, and KP1. RESULTS--Reactivity of malignant cells with one or more of the antibodies was seen in 11 out of 40 adenocarcinomas and in one of seven poorly differentiated carcinomas; other neoplasms, including 10 cases of squamous carcinoma, three of malignant melanoma, and four of oat cell carcinoma were negative. Monoclonal antibody KP1 gave the strongest staining and reacted with the highest proportion of neoplastic cells. CONCLUSIONS--CD68 is expressed in a proportion of epithelial tumours although the labelling is usually less intense than in macrophages. Anti-CD68 antibodies should therefore be used as part of a panel in the diagnosis of poorly differentiated neoplasms in cytological material.     

Diagnosis of myelomonocytic and macrophage neoplasms in routinely processed tissue biopsies with monoclonal antibody KP1.

A new monoclonal antibody, KP1, against the CD68 antigen, which labels macrophages and other members of the mononuclear phagocyte lineage in routinely processed tissue sections, has been used to stain a range of lymphoid, histiocytic, and myelomonocytic proliferations. All 20 neoplasms of myeloid, myelomonocytic, and presumed macrophage derivation reacted with antibody KP1. None of the 22 cases of T cell neoplasia had positive reactions. Although 14 of 41 B lineage lymphomas and leukaemias were stained by antibody KP1, staining was usually confined to small dots of reactivity, in contrast to the strong and extensive cytoplasmic staining seen in the neoplasms of myeloid and macrophage/monocyte origin. Furthermore, positive B cell neoplasms were almost all small cell proliferations, which are unlikely to be confused with myelomonocytic malignancies. It was concluded that antibody KP1 is a valuable addition to a panel of monoclonal antibodies for phenotyping lymphomas, particularly in routinely fixed tissues. It should assist the pathologist in the recognition of extramedullary presentation of leukaemia, aid in the diagnosis of suspected cases of true histiocytic neoplasia, and allow for quantitation of macrophages infiltrating lymphomas and other solid tumors.     

Production of anti-CD14 monoclonal antibodies using CD14 expressing COS cells as immunizing antigen

CD14 is a leukocyte surface molecule expressed on monocytes but not on lymphocytes. Recently, CD14 molecule was demonstrated to function as a receptor for endotoxin. CD14 specific monoclonal antibody (MAb), therefore, can be used to identify monocytes and study the host defense mechanism to bacterial endotoxin. To produce MAb against CD14 protein, in this study cDNA encoding CD14 protein and COS cell expression systems were used to prepare CD14 expressing COS cells. The CD14 transfectants were then used as antigen for mouse immunization. The spleen cells of the immunized mouse were then fused with myeloma cells by conventional hybridoma technique. By using this strategy, 5 hybridroma clones secreting antibody specific for CD14 molecule were generated within one fusion. The generated CD14 MAbs were strongly positive with monocytes, weakly positive with neutrophils but negative with lymphocytes. In addition, the generated CD14 MAb blocked the binding of lipopolysaccharide (LPS) to the CD14 molecules. These CD14 MAbs could be used to enumerate peripheral blood monocytes as well as using referent CD14 MAb. We, therefore, introduce an alternative method for preparation of antigen for production of monoclonal antibody. This type of antigen is a very effective antigen for the production of monoclonal antibodies against cell surface molecules.     

Primary nasal mucosal melanoma in Brazil: clinicopathologic and immunohistochemical study of 12 patients

Primary nasal melanoma is a rare tumor of unknown etiopathogenesis that occurs in adult and elderly patients usually diagnosed at advanced stages. The aim of this study was to analyze the clinicopathologic and immunohistochemical characteristics of 12 cases of primary nasal melanomas in Brazil. Twelve cases of primary nasal melanoma were analyzed histologically and by immunohistochemistry using the antibodies S-100 protein, HMB-45, Melan-A, CD63 (NKI/C3), CD68/KP1, fatty acid synthase (FASN), and Ki-67. The mean age of the patients was 60 years, and 7 of 12 patients were men. Microscopically, 10 cases presented level III of invasion; 4 were amelanotic; and in 7, cells were epithelioid. S-100 protein and FASN were positive in all cases, whereas 9, 8, 7, and 6 cases were positive for HMB-45, Melan-A, CD63 (NKI/C3), and CD68/KP1, respectively. Ki-67 labeling index ranged from 11.45% to 28.5% of positive cells. S-100 protein is more frequently expressed in nasal melanomas than in HMB-45, Melan-A, CD63 (NKI/C3), and CD68/KP1. FASN seems to be involved in the pathogenesis of nasal melanomas, and also, it can be helpful to confirm the diagnosis.     

CD163: a specific marker of macrophages in paraffin-embedded tissue samples

Paraffin-section immunohistochemical analysis was performed using a monoclonal antibody against CD163 to evaluate the antibody's usefulness in identifying cells of monocyte/macrophage lineage in normal and neoplastic conditions. Normal human tissue samples and samples from 211 hematopoietic disorders and 115 nonhematopoietic neoplasms were examined. The distribution of KP1 and PG-M1, monoclonal antibodies to the macrophage-associated CD68 antigen, also were evaluated for comparison. CD163 immunoreactivity was observed in resident macrophages of all normal tissue samples except splenic white pulp macrophages and germinal center tingible body macrophages. Among hematopoietic disorders and nonhematopoietic neoplasms, CD163 expression was restricted largely to cases of chronic myelomonocytic leukemia, histiocytic sarcoma, sinus histiocytosis with massive lymphadenopathy, and littoral cell angioma. Acute myeloid leukemias (AMLs) with monocytic differentiation were CD163- with the exception of 1 case of acute monoblastic leukemia. Most myeloid sarcomas also were CD163-. Compared with the CD68 antibodies, CD163 demonstrated greater specificity as a marker of disorders of monocyte/macrophage origin. However, immunohistochemical evaluation of CD163 expression does not seem to be a sensitive means of determining monocytic differentiation in AMLs in paraffin sections or establishing a diagnosis of myeloid sarcoma.     

Comparison of different methodologies and cryostat versus paraffin sections for chromogenic immunohistochemistry

Immunohistochemistry (IHC) specifically localizes proteins in cells and tissues, but methodologies vary widely. Therefore, we performed a methodological IHC optimization and validation study. First, we compared advantages and disadvantages of cryostat sections versus paraffin sections. Second, we compared and optimized antigen retrieval in paraffin sections using citrate buffer and Tris/EDTA buffer. Third, aminoethyl carbazole (AEC) and 3,3'-diaminobenzidine (DAB) were tested as horseradish peroxidase (HRP) substrates to obtain a water-insoluble coloured end product to visualize antigens. Fourth, secondary antibodies conjugated with either mono-HRP or poly-HRP were compared. The study was performed using serial sections of human tonsil. IHC was performed with primary antibodies against endothelial cell marker CD31, smooth muscle actin (SMA), chemokine stromal-derived factor-1α (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4), macrophage marker CD68 and proliferation marker Ki67. DAB rather than AEC, and cryostat sections rather than paraffin sections gave optimum staining at highest primary antibody dilutions, whereas tissue morphology in paraffin sections was superior. Loss of antigenicity in paraffin sections by formaldehyde fixation, heat and/or masking of epitopes was counteracted by antigen retrieval but not for all antigens. Two out of six antigens (CD31 and CD68) could not be retrieved irrespective time and type of retrieval. Tris-EDTA was superior to citrate buffer for antigen retrieval. The use of mono-HRP or poly-HRP depended on the affinity of the primary antibody for its antigen. We conclude that IHC methodology optimization and validation are crucial steps for each antibody and each research question.     

A new monoclonal antibody (3A5) that recognises a fixative resistant epitope on tissue macrophages and monocytes.

AIMS--To develop a monoclonal antibody specific for human macrophages in routinely processed material. METHODS--The monoclonal antibody was derived from a mouse popliteal lymph node after subcutaneous immunisation in the footpad with fragments of human spleen depleted of lymphocytes and erythrocytes. RESULTS--3A5 is a monoclonal antibody reactive with macrophages, monocytes, and histiocytes in routinely processed (formalin fixed, paraffin wax embedded) human tissue specimens. Unlike the well known panmacrophage marker KP1 (CD68), neither dendritic cells (interdigitating cells, Langerhans' cells, and microglia) nor myeloid, lymphoid, or epithelial cells stained with 3A5. CONCLUSION--As the staining pattern of 3A5 is restricted, compared with other macrophage markers and the recognised epitope survives common fixation and embedding procedures, 3A5 is a valuable marker for histiocytes and macrophages in routine diagnostic applications.     

Epithelial membrane antigen (EMA) or MUC1 expression in monocytes and monoblasts

AIMS: Epithelial membrane antigen (EMA) or MUC1 belongs to a heterogeneous group of heavily glycosylated proteins and is expressed in most normal and epithelial neoplastic cells. EMA is also expressed in plasma cells, anaplastic large cell lymphoma (Ki-1 antigen), malignant histiocytosis and erythroleukaemia. In 1996, Cheong et al. (Hematology 1996; 1: 223) demonstrated the positive expression of EMA in monoblasts. Since there were very few useful markers for differentiating subtypes of acute myeloid leukaemia with a monocytic component from the those without, a study was conducted to evaluate the prevalence of EMA expression and its relationship with known markers for monocytic-macrophage lineage (CD11c, CD14 and intracellular CD68) in monocytes and monoblasts. METHODS: EMA detection was performed by flow cytometry in monocytes and monoblasts. EMA expression was compared with other known markers of monocytic-macrophage lineage (CD11c, CD14 and intracellular CD68). Samples of purified monocytes were obtained from 20 healthy volunteers. Twenty-two cases of monocytic AML (M4 and M5) were studied and controls were selected from 20 cases of acute lymphoblastic leukaemia (ALL) and 18 cases of non-monocytic AML (M0, M1, M2, M3, and M7). RESULTS: EMA was shown to be expressed strongly on the surface of all purified monocytes. EMA expression was observed on blast cells in 18/22 (81.8%) cases of AML M4 and M5, but not in that of non-monocytic AML or ALL. In this study EMA monoclonal antibody has demonstrated a strong association (P<0.001) with all the other known markers of monocytic-macrophage lineage in acute leukaemia subtypes. EMA had also shown 100% specificity and 81.8% sensitivity in the diagnosis of AML M4 and M5. CONCLUSIONS: The monoclonal antibody EMA (clone E29) is a useful marker in the classification of acute myeloid leukaemia and can be used as a supplementary analysis for the diagnosis of acute leukemia with monocytic involvement.     

Distribution of the CD68 macrophage/myeloid associated antigen

The distribution of the pan-macrophage CD68 antigen, recognized by six different monoclonal antibodies, was examined in human blood, tissue, and cell lines using APAAP staining and Western blotting. All antibodies stained monocytes and macrophages, but labelling of neutrophils, basophils, and lymphocytes was seen with some of the reagents. In addition, the CD68 antibodies demonstrated a variety of staining patterns on some non-haemopoietic cells. The subtle differences between the reactions of the different antibodies suggested that the CD68 antigen may be heterogeneous, possibly due to differences in glycosylation. While CD68 antibodies are very useful markers of the macrophage/myeloid series, the presence of small amounts of the antigen on some lymphoid and non-haemopoietic cells means that care should be taken when using them for the diagnosis of tumours of unknown origin.     

Immunophenotypic profile of myeloid cells in granulocytic sarcoma by immunohistochemistry. Correlation with blast differentiation in bone marrow

The present study was designed to evaluate the lineage differentiation (particularly monocytic differentiation) of immature myeloid cells in granulocytic sarcoma (GS) by immunohistochemistry and correlate the results with lineage differentiation of blasts in the bone marrow and to determine the degree of maturation of the infiltrating myeloid cells in GS by immunohistochemistry using CD34 and HLA-DR. Immunohistochemical stains were performed on paraffin-embedded tissue from 17 GS lesions with lineage-associated markers: myeloperoxidase, CD68 (KP1), CD68 (PG-Ml), glycophorin A, factor VIII, and CD56; and with markers for blasts and immature myeloid cells: CD34 and HLA-DR. Our results show that positive staining with PG-M1, but not KP1, suggests monocytic differentiation of myeloid cells in GS and correlates with the monocytic differentiation of blasts in the bone marrow. Expression of CD56 is frequent in GS, especially when the marrow blasts have monocytic differentiation, and should not be interpreted as a primary natural-killer cell process. The immature myeloid cells in GS are frequently HLA-DR positive. However, CD34 positivity of the immature myeloid cells is relatively uncommon, except in cases with underlying myelodysplastic syndrome or chronic myelogenous leukemia.     

Monoclonal antibody AMH152 reacts with human monocytes in culture and with inflammatory macrophages.

Monoclonal antibodies (mAb) raised against human peritoneal macrophages were selected for their non-reactivity with freshly sampled blood cells. One of these mAb, AMH152, initially non-reactive, bound to monocytes after 18 h of culture, a property which was not shared by an unrelated antibody of the same isotype (IgG1). The induction of the expression of the antigen detected by AMH152 on monocytes in culture was not influenced by the addition of serum or by the substrate used, plastic that favoured adhesion or teflon bags. Overnight incubation at 4 degrees C in adhesion conditions did not enable antigen expression. A 1-h treatment with phorbol myristate acetate or formyl-methionyl-leucyl-phenylalanine did not increase AMH152 binding. Culturing monocytes with cycloheximide tended to inhibit antigen expression. These observations suggested that antigen expression represents an active phenomenon, requiring protein synthesis. The antigen recognized by mAb AMH152 could be visualized on sections of formalin-fixed and paraffin-embedded tissues. Macrophages of healthy lymphoid organs and tissues that expressed CD68 antigen failed to bind AMH152. In contrast, chronic inflammatory lesions, like those of sarcoidosis, tuberculosis and cat scratch disease, contained epithelioid and multinucleated giant cells that reacted with AMH152. In serous exudates of cancer metastases, 10-40% of macrophages were also stained. The antigenic material was essentially present at the cell periphery. Thus, mAb AMH152 recognized a surface antigen, detectable on paraffin-embedded tissue sections, and which accompanied differentiation of monocytes into inflammatory cells. The expression of this antigen on monocytes in culture suggests that these cells underwent an activation process, even when maintained for some hours in teflon bags and in a serum-free medium.     

Chronic myelomonocytic leukemia: The role of bone marrow biopsy immunohistology.

The World Health Organization criteria for diagnosing chronic myelomonocytic leukemia (CMML) are largely based on findings observed in the peripheral blood and bone marrow aspirate. A specific diagnostic role for the bone marrow biopsy has not been adequately explored. We examined whether bone marrow biopsy supplemented by immunohistochemistry may be helpful in distinguishing CMML from cases of chronic myelogenous leukemia and atypical chronic myeloid leukemia (aCML). We immunostained 25 cases of CMML with paraffin reactive antibodies which included CD68 (KP1), CD68R (PG-M1), and CD163, and compared the results with those observed in six cases of chronic myelogenous leukemia and in three cases of atypical CML. In addition, we examined whether CD34 immunohistochemistry could be useful in separating cases of CMML with less than 10% blasts (type-1) from cases of CMML with blasts accounting for 10-19% (type-2), and cases of CMML in acute transformation to acute myeloid leukemia (blasts > or = 20%). The presence of nodules of plasmacytoid monocytes was investigated by CD123 staining. CD42b was used to highlight abnormal megakaryocytes. Our results demonstrate significant differences between the groups. CD34 analysis allowed separating CMML type-1 from type-2 and the former from CMML in acute transformation. CD123-positive plasmacytoid monocyte nodules were found only in CMML and not in the other two disease groups. Overlap between CMML and the other two groups were observed with CD68 immunostaining. CD68R was more restricted to bone marrow macrophages and monocytes than CD68, but the differences between CMML and chronic myelogenous leukemia or atypical CML were still not significant. Although CD42b immunostaining facilitated the detection of dwarf megakaryocytes often present in CMML, the distinction between those and the small forms seen in chronic myelogenous leukemia was still problematic.     

Activation of peripheral blood monocytes and macrophages in Kawasaki disease: Ultrastructural and immunocytochemical investigation

Monocytes/macrophages are considered to play an important role in the pathogenesis of Kawasaki disease (KD). However, the morphological and Immunocytochemical features of the cells in acute KD have not been investigated. The ultrastructural and Immunocytochemical characteristics of peripheral blood CD14+ monocytes/macrophages sorted by a magnetic cell sorter (MACS) during the course of KD were, therefore, studied to evaluate their role in the disease pathogenesis. Electron microscopy showed that CD14+ monocytes/macrophages from patients with acute KD had nuclei with complex shapes, apparent nucleoli and abundant intracytoplasmlc granules, some of which were positive for acid phosphatase. The quantity of intracy-toplasmic granules was correlated with disease severity, in terms of the duration of fever, maximum level of C-reactive protein and the presence of coronary artery lesions (CAL), suggesting that the monocytes/macrophages were activated and showed increased phagocytosis. Immunocytochemical staining of smears made from cell suspensions of sorted CD14+ monocytes/macrophages was carried out using a monoclonal antibody against tumor necrosis factor (TNF)-α. The cytoplasm of monocytes/macrophages from patients with acute KD was strongly positive in comparison to that of cells from control subjects, suggesting that intracytoplasmic granules secrete TNF-α.     

Maturation of macrophages from peripheral blood monocytes in Kawasaki disease: immunocytochemical and immunoelectron microscopic study

Kawasaki disease (KD) is regarded as a cytokine-associated disorder. Despite intensive investigation into the etiology of KD, this remains unclear, although monocytes and macrophages are thought to play an important role. We examined peripheral blood monocytes using a monoclonal antibody, PM-2K, which recognizes mature macrophages but not monocytes. This study was conducted in 12 patients with KD, three patients with sepsis and 12 control subjects. Approximately 8% of whole peripheral blood monocytes from patients with acute KD were observed to be PM-2K positive. Approximately 15-20% of peripheral blood CD14+ monocytes from these patients were positive for PM-2K antibody (as determined by immunoelectron microscopy). PM-2K-positive monocytes had significantly fewer numbers of intracytoplasmic peroxidase-positive granules than monocytes from control subjects. In contrast, PM-2K-negative monocytes from patients with acute KD had a significantly greater number of peroxidase-positive granules in the cytoplasm than in those from controls. Monocytes from patients with sepsis displayed PM-2K immunocytochemical staining, similar to that in monocytes from patients with KD. These results suggest that during the acute stage of KD, monocytes partly differentiate into macrophages in the peripheral circulation.     

Antigen-Specific Regulation of CD1 Expression in Humans

During the last decade the CD1 family of cell surface glycoproteins has been implicated in the presentation of nonpeptide antigens in man. Recent findings by our group indicate that CD1 molecules also can be involved in the presentation of certain bacterial proteins. However, CD1a, b, and c (group 1 CD1 molecules) are not present at significant levels on circulating monocytes unless their expression is induced by cytokines such as granulocyte–macrophage colony-stimulating factor (GM-CSF). In this study we investigated the cell surface expression of CD1 molecules following the antigenic stimulation in vivo via immunization of healthy volunteers with tetanus toxoid vaccine and in vitro cell cultures using the same antigen. Both the in vivo and in vitro studies demonstrated clear up-regulation of the surface expression of CD1a, b, and c on monocytes as a result of antigenic stimulation with tetanus toxoid, supporting the idea that CD1 molecules participate in the presentation of this protein antigen in man. In vitro, antigen-triggered expression of these molecules was mediated by GM-CSF, since neutralization of this cytokine with specific antibody totally abrogated CD1a, b, and c expression. In contrast to the group 1 CD1 molecules, CD1d was found to be constitutively expressed on the majority of circulating monocytes and B lymphocytes prior to immunization. There was no effect of antigenic stimulation with tetanus toxoid on the cell surface expression of CD1d, suggesting major differences in regulation of the expression and function of the different CD1 molecules in humans. Altogether our results point to antigen-driven up-regulation of CD1a, b, and c expression on human monocytes that is mediated by GM-CSF and no effect on CD1d expression.