Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs).

Mesenchymal stromal cells (MSCs) have a multilineage differentiation potential and provide immunosuppressive and antimicrobial functions. Murine as well as human MSCs restrict the proliferation of T cells. However, species-specific differences in the underlying molecular mechanisms have been described. Here, we analyzed the antiparasitic effector mechanisms active in murine MSCs. Murine MSCs, in contrast to human MSCs, could not restrict the growth of a highly virulent strain of Toxoplasma gondii (BK) after stimulation with IFN-γ. However, the growth of a type II strain of T. gondii (ME49) was strongly inhibited by IFN-γ-activated murine MSCs. Immunity-related GTPases (IRGs) as well as guanylate-binding proteins (GBPs) contributed to this antiparasitic effect. Further analysis showed that IFN-γ-activated mMSCs also inhibit the growth of Neospora caninum, a parasite belonging to the apicomplexan group as well. Detailed studies with murine IFN-γ-activated MSC indicated an involvement in IRGs like Irga6, Irgb6 and Irgd in the inhibition of N. caninum. Additional data showed that, furthermore, GBPs like mGBP1 and mGBP2 could have played a role in the anti-N. caninum effect of murine MSCs. These data underline that MSCs, in addition to their regenerative and immunosuppressive activity, function as antiparasitic effector cells as well. However, IRGs are not present in the human genome, indicating a species-specific difference in anti-T. gondii and anti-N. caninum effect between human and murine MSCs.

Heat-shock protein 70-dependent dendritic cell activation by 5-aminolevulinic acid-mediated photodynamic treatment of human glioblastoma spheroids in vitro.

BACKGROUND: T-cell responses contribute to the anti-tumoural effect of photodynamic therapy (PDT). For such responses to occur, dendritic cells (DCs) have to migrate to the tumour, take up tumour antigens and respond to danger signals with maturation, before they engage in T-cell activation. Here, we have studied the effect of 5-aminolevulinic acid (ALA)-mediated PDT on DCs in vitro in a human spheroid model of glioblastoma (GB). METHODS: Spheroids of the GB cell lines U87 and U251 were treated with ALA/PDT, and effects on attraction, uptake of tumour antigens and maturation of DCs were studied. To block heat-shock protein-70 (HSP-70) on the spheroids, neutralising antibodies were used. RESULTS: 5-Aminolevulinic acid /PDT-treated GB spheroids attracted DCs that acquired tumour antigens from the spheroids effectively. Moreover, co-culture with ALA/PDT-treated spheroids induced DC maturation as indicated by the upregulation of CD83 and co-stimulatory molecules as well as increased T-cell stimulatory activity of the DCs. Heat-shock protein-70 was upregulated on the spheroids after ALA/PDT treatment. Uptake of tumour antigens and DC maturation induced by the ALA/PDT-treated spheroids were inhibited when HSP-70 was blocked. CONCLUSION: ALA/PDT treatment of glioma spheroids promotes the three initial steps of the afferent phase of adaptive immunity, which is at least partially mediated by HSP-70.

Dendritic cell generation and CD4+ CD25high FOXP3+ regulatory t cells in human head and neck carcinoma during radio-chemotherapy.

BACKGROUND: Regulatory T cells (Treg) and dendritic cells (DC) play an important role in tumor immunity and immune escape. However, their interplay and the effects of anti-cancer therapy on the human immune system are largely unknown. METHODS: For DC generation, CD14⊃+ monocytes were enriched by immunomagnetic selection from peripheral blood of advanced head and neck squamous cell carcinoma (HNSCC) patients and differentiated into immature DC using GM-SCF and IL-4. DC maturation was induced by addition of TNFα. The frequency of CD4⊃+CD25⊃highFOXP3⊃+ Treg in HNSCC patients was analyzed before and after radio-chemotherapy (RCT) by four-color flow cytometry. RESULTS: In HNSCC patients, the frequency of Treg (0.33 ± 0.06%) was significantly (p = 0.001) increased compared to healthy controls (0.11 ± 0.02%), whereas RCT had variable effects on the Treg frequency inducing its increase in some patients and decrease in others. After six days in culture, monocytes of all patients had differentiated into immature DC. However, DC maturation indicated by CD83 up-regulation (70.7 ± 5.5%) was successful only in a subgroup of patients and correlated well with lower frequencies of peripheral blood Treg in those patients. CONCLUSION: The frequency of regulatory T cells is elevated in HNSCC patients and may be modulated by RCT. Monocyte-derived DC in HNSCC patients show a maturation deficiency ex vivo. Those preliminary data may have an impact on multimodality clinical trials integrating cellular immune modulation in patients with advanced HNSCC.

Phenotypic and functional comparison of monocytes from cord blood and granulocyte colony-stimulating factor-mobilized apheresis products.

OBJECTIVE: It is well established that T cells are effector cells in graft-vs-host disease (GVHD), yet the contribution of graft monocytes is less well characterized. Therefore, monocytes in cord blood (CB) and granulocyte colony-stimulating factor-mobilized apheresis products (G-AP), two stem cell grafts associated with reduction of acute and chronic GVHD and relative reduction of acute GVHD, respectively, were compared phenotypically and functionally. MATERIALS AND METHODS: The frequencies, phenotypes, and pinocytosis activities of monocytes from CB and G-AP were determined by flow cytometry and their allostimulatory potential in a primary mixed leukocyte reaction. RESULTS: G-AP contained significantly more monocytes than CB (24.9% +/- 7.1% vs 8.8% +/- 1.5% CD14+ and 62.4 +/- 27.5 x 10(6) vs 0.9 +/- 0.2 x 10(6) CD14+ cells/mL). Monocytes from both sources revealed similar phenotypes. They expressed CD4, CD11a, CD11b, CD11c, CD18, CD32, CD33, CD45R0, CD48, CD50, CD54, CD58, CD64, CD86, CD102, CD116, CD123, and HLA-DR; showed no expression of CD1a and CD83; and weak expression of CD16, CD45RA, and CD80. The levels of CD80 and CD86 expression were comparable; however, in contrast to G-AP monocytes, CB monocytes lacked CD40. There was no difference in pinocytosis activity and allostimulatory capacity of CB and G-AP monocytes. CONCLUSIONS: Monocytes in CB and G-AP are phenotypically and functionally comparable. The only difference observed is the lack of CD40 on CB monocytes.

Identification of an alternatively spliced transcript of human interleukin-4 lacking the sequence encoded by exon 2.

The expression of interleukin-4 (IL-4) mRNA of human peripheral blood mononuclear cells stimulated with phytohemagglutinin (PHA) for 15 hours was analyzed by reverse transcription and subsequent polymerase chain reaction (RT/PCR). These analyses revealed an additional smaller fragment that hybridizes with an IL-4 cDNA probe and an oligonucleotide that is specific for a fragment lacking the sequence encoded by exon 2. Sequencing of this fragment demonstrates that it is generated from an alternatively spliced transcript of the IL-4 gene with the sequence encoded by exon 2 being skipped. Skipping of exon 2 does not result in a frame shift but would delete part of the mature protein (48 bp coding for amino acid residues 22 to 37), including Cys24 but not a region directly involved in receptor binding. Differential splicing of other exons or exon combinations has not been observed. The data suggest that the alternatively spliced transcript is not generated by a splicing or PCR error and is not detectable solely because of the high sensitivity of RT/PCR, but in contrast, argue for a physiological role of the transcript and its potentially encoded protein.

Necrotic cell-derived high mobility group box 1 attracts antigen-presenting cells but inhibits hepatocyte growth factor-mediated tropism of mesenchymal stem cells for apoptotic cell death.

Tissue damage due to apoptotic or necrotic cell death typically initiates distinct cellular responses, leading either directly to tissue repair and regeneration or to immunological processes first, to clear the site, for example, of potentially damage-inducing agents. Mesenchymal stem cells (MSC) as well as immature dendritic cells (iDC) and monocytes migrate to injured tissues. MSC have regenerative capacity, whereas monocytes and iDC have a critical role in inflammation and induction of immune responses, including autoimmunity after tissue damage. Here, we investigated the influence of apoptotic and necrotic cell death on recruitment of MSC, monocytes and iDC, and identified hepatocyte growth factor (HGF) and the alarmin high mobility group box 1 (HMGB1) as key factors differentially regulating these migratory responses. MSC, but not monocytes or iDC, were attracted by apoptotic cardiomyocytic and neuronal cells, whereas necrosis induced migration of monocytes and iDC, but not of MSC. Only apoptotic cell death resulted in HGF production and HGF-mediated migration of MSC towards the apoptotic targets. In contrast, HMGB1 was predominantly released by the necrotic cells and mediated recruitment of monocytes and iDC via the receptor of advanced glycation end products. Moreover, necrotic cardiomyocytic and neuronal cells caused an HMGB1/toll-like receptor-4-dependent inhibition of MSC migration towards apoptosis or HGF, while recruitment of monocytes and iDC by necrosis or HMGB1 was not affected by apoptotic cells or HGF. Thus, the type of cell death differentially regulates recruitment of either MSC or monocytes and iDC through HGF and HMGB1, respectively, with a dominant, HMGB1-mediated role of necrosis in determining tropism after tissue injury.

Dermal dendritic cells associated with T lymphocytes in normal human skin display an activated phenotype.

The CMRF-44 and CD83 (HB15) antigens are associated with functional maturation and activation of blood dendritic cells (DC). We describe the expression of these antigens on freshly isolated epidermal Langerhans cells and dermal DC as well as the distribution of CD83+/ CMRF-44++-activated DC within sections of normal human skin. Fresh Langerhans cells were prepared by standard techniques and large numbers of enriched (25%-55%), viable dermal DC were obtained using an improved collagenase treatment protocol with density gradient enrichment. Freshly isolated Langerhans cells and dermal DC had similar costimulator and activation antigen expression, and both stimulated moderate levels of allogeneic T lymphocyte proliferation as determined in the 7 d mixed leukocyte reaction. In situ labeling of DC within skin sections revealed a population of CD83 and CMRF-44 positive dermal cells of which most (approximately 75%) were in intimate contact with CD3+ T lymphocytes, especially in the adnexal regions. In contrast, only 25%-30% of the more numerous CD1a++ dermal DC population were directly apposed to T lymphocytes. The CMRF-44++ dermal DC population stimulated an allogeneic mixed leukocyte reaction, confirming their identity as DC. These data, plus comparative data obtained for migratory dermal DC, suggest that only a small proportion of dermal DC have been triggered to a more advanced state of differentiation or activation. The striking association of the activated dermal DC population with T lymphocytes suggests that communication between these two cell types in situ may occur early in the immune response to cutaneous antigen.

Human dendritic cells express functional interleukin-7.

Interleukin-7 (IL-7) supports the proliferation of mature T lymphocytes, however, the cellular source of IL-7 for T lymphocyte activation has not been well established. We therefore investigated whether human peripheral blood dendritic cells (DC) produce IL-7 as a contribution towards T lymphocyte activation. Human CMRF-44+/CD14-/CD19- low density DC, purified after overnight tissue culture, contained IL-7 transcripts, detected by direct cell reverse transcription-polymerase chain reaction. Intracytoplasmic staining confirmed IL-7 protein in at least a subpopulation of cultured low density DC. In contrast, resting/immature DC, isolated directly by immunodepletion of lineage marker positive cells, contained no IL-7 mRNA. Thus, the expression of IL-7 by DC follows the pattern described previously for CD80, CD86 and CD40. However, tissue culture of purified resting/immature DC, in contrast to CD80, CD86 and CD40, failed to induce IL-7 transcripts. The functional importance of DC IL-7 expression was demonstrated in an allogeneic mixed leukocyte reaction (MLR). Neutralising mAb to IL-7 significantly inhibited T lymphocyte proliferation when low DC numbers were used, but at higher stimulator numbers, anti-IL-7 mAb failed to inhibit an allogeneic MLR. This suggests, that when DC are in excess, other co-stimulatory pathways can compensate for the lack of IL-7. Addition of IL-7 to a MLR caused a significant increase in the proliferative response stimulated by monocytes and B lymphocytes but not by DC. These data support the concept of an initial phase of antigen uptake by DC followed by the optimisation of DC co-stimulatory potential. The co-stimulatory repertoire expressed, including IL-7, may be regulated by exogenous stimuli, thereby ensuring DC flexibility in mounting a response appropriate to the environmental changes.

Functional competence of dendritic cells in human umbilical cord blood.

Dendritic cells (DC) are the main stimulators of primary T cell responses. Very little is known about DC in cord blood (CB), and whether they are involved in the low incidence and severity of GVHD following CB transplantation. Here, CBDC were identified as a HLA-DR+/lineage marker (lin; CD3, CD11b, CD14, CD16, CD19, CD34, CD56 and glycophorin A antigens) negative population, representing 0.3 +/- 0.1% (mean +/- s.d.; n = 15) of CB mononuclear cells. CBDC expressed the CD4, CD11a, CD18, CD45RA, CD50 and CD54 antigens but revealed no expression of the CD1a, CD11c, CD40, CD45R0, CD58, CD83, CD86 and CD102 antigens. Immunomagnetically enriched CBDC showed potent allostimulatory activity for CB T cells. Thus, CBDC are functionally competent and resemble in their immature/resting state CD11c- DC in peripheral blood.

An eight-fold ex vivo expansion of long-term culture-initiating cells from umbilical cord blood in stirred suspension cultures.

Simultaneous ex vivo expansion of different progenitor cell types may be beneficial for cord blood (CB) transplantation, to overcome a potential limitation due to restricted cell numbers. Therefore, 1.5 x 10(6) CD34+ cells isolated from fresh or thawed CB samples were inoculated in a large-scale stirred suspension bioreactor and cultured in the presence of Flt3-L, SCF and IL-3. At days 0, 7, 10, 14, 21 and 28, the spinner cultures were analyzed for viable cells, colony-forming cells (CFC), including erythroid burst-forming unit (BFU-E), granulocyte-macrophage colony-forming unit (CFU-GM) and granulocyte-erythrocyte-megakaryocyte-monocyte colony-forming unit (CFU-GEMM) as well as long-term culture-initiating cells (LTC-IC). Expansion of thawed CD34+ cells resulted in a substantial amplification of total cells (maximal at day 28: 154 +/- 132-fold), CFC (maximal at day 14: 45 +/- 36-fold), CFU-GM (maximal at day 14: 88 +/- 85-fold), CFU-GEMM (maximal at day 7: 4 +/- 2-fold) and of LTC-IC (maximal at day 10: 8 +/- 3-fold). There was no significant difference between fresh and thawed CD34+ cells. These results demonstrate that simultaneously committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be substantially amplified from CD34+-enriched CB samples in large-scale stirred suspension cultures within 7-14 days without exhausting the proliferative potential and, thus, it may be possible to improve CB transplantation by ex vivo generated cells.

The effect of different thawing methods, growth factor combinations and media on the ex vivo expansion of umbilical cord blood primitive and committed progenitors.

Assuming a threshold of 2 x 10(7) nucleated cells (NC)/kg body weight required for transplantation and 10 +/- 5 x 10(8) NC per cord blood (CB) unit (n = 1828, July 1997), 100%, 65% and 25% of the CB units stored in the CB Bank Düsseldorf contain sufficient NC to engraft patients of 10 kg, 35 kg and 50-70 kg, respectively. Thus, there is a potential limitation for the use of CB in adults which, however, may be overcome by ex vivo expansion of cells important in the different phases of engraftment. Therefore, four combinations of SCF, Flt3-L, IL-3, erythropoietin and GM-CSF as well as three media were evaluated for their capacity to amplify hematopoietic progenitors. A prerequisite for expansion was the significantly higher recovery of CD34+ cells, colony-forming cells (CFC) and long-term culture-initiating cells (LTC-IC) by thawing cryopreserved CB units with an isotonic albumin/dextran solution. When CD34+ CB cells were cultured with the four cytokine combinations in H5100 medium, all combinations promoted an expansion of total cells (43 to 356-fold) and CFC (49 to 462-fold) within 7 days, however, early progenitors as defined by mixed-colony formation (CFU-GEMM) were substantially amplified only with SCF, Flt3-L plus IL-3 (94.3 +/- 62.4-fold). H5100 medium or a serum-free medium supplemented with SCF, Flt3-L plus IL-3 were superior to 20% FCS/RPMI-1640 medium in the expansion of all progenitor cell types and were similarly effective in supporting the amplification of total cells, CFC, CFU-GM, BFU-E/CFU-E and LTC-IC (maximum at day 7: 6.7 +/- 3.4-fold and 5.5 +/- 0.5-fold, respectively). However, the serum-free medium promoted a significantly higher expansion of CFU-GEMM (176.9 +/- 81.7-fold) than H5100 medium (83.5 +/- 26.2-fold) at day 7 and only under serum-free conditions, CFU-GEMM were maintained over 14 days in tissue culture. These results demonstrate that committed progenitors as well as the more immature CFU-GEMM and LTC-IC can be substantially amplified at the same time without exhausting the proliferative potential.

The influence of hormones on CD44 expression in endometrial and breast carcinomas.

The expression of distinct variant isoforms of the cell surface glycoprotein CD44 (CD44v) has been found to be associated with metastatic potential of rodent adenocarcinoma cells and with an altered prognosis in several types of human cancer. In hormone-dependent gynecological cancers, different CD44v expression patterns have been observed. The influence of ovarian steroid hormones and their antagonists on CD44v expression is still unclear, since there are only retrospective correlation studies so far. Therefore, we examined the CD44 mRNA expression in a standardized stimulation experiment in a number of breast and endometrial carcinoma cell lines varying in estrogen receptor (ER) status. Higher CD44 overall expression was observed in ER positive endometrial and breast carcinoma cell lines when compared to corresponding ER negative cell lines. The number and composition of alternatively spliced isoforms showed no clear correlation to the ER expression status. Three CD44v isoforms were detected in all cell lines expressing CD44v, two of which have not been reported previously in normal endometrial cells. These isoforms may have specific functions in this type of carcinoma. In the second part of the study, the influence of (anti-) hormones on CD44 expression in endometrial carcinoma cell lines was examined. CD44 overall expression showed an increase when the cells were grown in medium containing fetal calf serum (FCS) as compared to cells maintained in medium-free of FCS. CD44 expression was transiently increased by estradiol (1 h). The CD44 splice pattern of endometrial cancer cell lines RL95-2 and Hec-1-A, after treatment with (anti-) hormones showed constant and high expression rates for distinct CD44v-isoforms such as CD44E (CD44v8-v10). Only certain weakly expressed isoforms changed their expression level during the experimental period, but no direct correlation to hormone treatment was observed. In conclusion, estradiol or FCS increase CD44 overall expression, but there seems to be no direct influence of ovarian steroid hormones on the CD44v splice machinery in endometrial carcinoma cell lines.

Identification of cord blood dendritic cells as an immature CD11c- population.

Dendritic cells (DC) are the main stimulators of primary T-cell responses and, thus, probably play a role in the immune reactions after stem cell transplantation. Very little is known about DC in cord blood (CB) and about their potential involvement in the low incidence and severity of acute graft-versus-host disease after CB transplantation. Here, CBDC were identified as a HLA-DR+ cell population, lacking the CD3, CD11b, CD14, CD16, CD19, CD34, CD56, and glycophorin A lineage markers (lin). This lin-/HLA-DR+ population represented 0.3% +/- 0.1% (mean +/- SD; range, 0.1% to 0. 6%; n = 15) of CB mononuclear cells, and CB contained 5.4 +/- 3.2 x 10(3) CBDC/mL (1.8 to 13.0 x 10(3); n = 15). CBDC expressed CD4, CD11a, CD18, CD45RA, CD50, CD54, and CD123, but showed no expression of CD1a, CD11c, CD33, CD40, CD45R0, CD80, CD83, and CD86 and only limited expression of CD58, CD102, and CD116. Despite this immature phenotype, immunomagnetically lin--enriched CBDC were potent stimulators of allogeneic CB T cells. As few as 266 +/- 107 (193 to 530; n = 10) lin-/HLA-DR+ CBDC stimulated a significant response. However, CBDC failed to take up protein or peptide antigens. Thus, in CB there is a prevalence of a DC subpopulation, resembling the CD11c- DC identified in tonsils, the so-called plasmacytoid T cells, which may exert a function distinct from the CD11c+ DC subpopulation.

Human dendritic cells activate T lymphocytes via a CD40: CD40 ligand-dependent pathway.

The CD40:CD40 ligand (CD40L) interaction provides T lymphocyte-mediated help for B lymphocyte and monocyte function but has also been shown to serve as a co-stimulus for T lymphocyte activation. In this report, we studied the regulation of CD40 expression and its functional relevance for the human dendritic cell (DC) stimulation of T lymphocytes. Only a small subpopulation of directly isolated blood DC expressed CD40. However, CD40 was rapidly up-regulated by culture, and its expression was further enhanced by interleukin (IL)-1 alpha, IL-1 beta, IL-3, tumor necrosis factor-alpha and granulocyte/macrophage-colony-stimulating factor. Expression of CD40L on DC was not detected. The proliferation of T lymphocytes in an allogeneic mixed leukocyte reaction, stimulated by blood DC or epidermal Langerhans cells, was significantly reduced in the presence of the CD40 immunoglobulin (CD40Ig) fusion protein or CD40L monoclonal antibodies. Cross-linking of CD40 on directly isolated DC with mouse CD40L trimer (mCD40LT) markedly augmented CD80 and CD86 up-regulation. Nevertheless, the same cross-linking mCD40LT inhibited DC stimulated T lymphocyte proliferation. When CD40Ig was added simultaneously with CTLA-4Ig, only minimal and variable additional inhibition of DC-stimulated allogeneic T lymphocyte proliferation and IL-2 secretion was observed, compared to each fusion protein alone. These results suggest that both CD80/CD86-dependent and -independent components of DC-T lymphocyte CD40:CD40L co-stimulation exist and further emphasize that the majority of blood DC have to differentiate or be activated to express co-stimulatory molecules.

One-step molecular HLA-DR prescreening employing a set of 14 sequence specific oligonucleotides in a non-radioactive tetramethylammonium chloride hybridization protocol.

A novel non-radioactive protocol for molecular generic HLA-DR typing is introduced, employing sequence specific oligonucleotides (SSOs) enzymatically 3'-labelled with biotin-14-dATP via terminal deoxynucleotidyltransferase in a tetramethylammonium chloride hybridization procedure. The detection reaction is carried out, using streptavidin conjugated horseradish peroxidase which is bound to the SSOs, in combination with a light emitting detection system. Fourteen SSOs and one control SSO are employed for generic HLA-DR typing in a one-step protocol. In order to demonstrate the suitability of this procedure, 5 homozygous typing cell lines and samples of 11 pretyped individuals which include most serologically defined HLA-DR specificities (DR1, 2, 3, 4, 11, 12, 6, 7, 8, 9, 10, 52, and DR53) are analysed with the panel of 14 SSOs. The typing results show that this protocol, which avoids the use of radioisotopes, combines high specificity and easy handling. It also allows typing of poorly amplified samples because even after longer exposition times no false positive signals were observed and is particularly suitable for routine molecular HLA-DR typing on the generic level. In addition it can easily be adapted to DP and DQ typing or DR subtyping.

Identification of a novel dendritic cell surface antigen defined by carbohydrate specific CD24 antibody cross-reactivity.

Dendritic cells (DC) are characterized as leucocytes that lack mature lineage specific markers and stimulate naive T-lymphocyte proliferation in vitro and in vivo. The mouse heat stable antigen (HSA) participates in T lymphocyte co-stimulation and is expressed by DC isolated from thymus, skin and spleen. The human HSA homologue, CD24, is predominantly expressed by B lymphocytes and granulocytes, but its expression on DC has not been studied in detail. CD24 clearly participates in B-lymphocyte signalling but co-stimulatory activity for T lymphocytes has not yet been described. We have examined the expression of CD24 on human peripheral blood DC populations isolated directly or following in vitro culture. The CD24 antigen was absent from blood DC however, cross-reactive sialylated carbohydrate epitopes were detected on DC with some CD24 monoclonal antibodies (mAb). These CD24 mAb define a protein surface antigen, which is expressed by an immature or resting subpopulation of peripheral blood DC and is down-regulated following activation differentiation in vitro.

cDNA cloning of human DEC-205, a putative antigen-uptake receptor on dendritic cells.

Dendritic cells (DC) are specialist antigen presenting cells which capture antigens in the periphery, migrate centrally, and present the processed antigens in the context of major histocompatibility complex and appropriate co-stimulatory molecules to T lymphocytes for the initiation of an immune response. DEC-205 has been identified as a putative antigen-uptake receptor, which is expressed abundantly on mouse DC. The recently cloned mouse DEC-205 cDNA predicts a molecular structure which has a marked similarity to the macrophage mannose receptor. Using reverse transcriptase-polymerase chain reaction (RT-PCR) and cDNA library screening, we obtained the full coding region of human DEC-205 cDNA from the Hodgkin's disease-derived L428 cell line. The predicted protein structure is a type I transmembrane protein of 1722 amino acids consisting of a signal peptide, cysteine-rich domain, fibronectin type II domain, ten carbohydrate recognition-like domains, transmembrane domain, and a cytoplasmic tail. Human DEC-205 is 77% identical to the mouse protein with completely conserved cysteines. The DEC-205 gene (LY75) was mapped to chromosome band 2q24 by somatic cell hybrid panel analysis and fluorescent in situ hybridization. Northern blot analysis detected 7.8 and 9.5 kilobase DEC-205 transcripts in myeloid, B lymphoid, and Hodgkin's disease-derived cell lines. RT-PCR analysis indicated that immature blood DC contain a barely detectable amount of DEC-205 transcripts but these were markedly increased upon differentiation/activation.