CD83 Antibody Inhibits Human B Cell Responses to Antigen as well as Dendritic Cell-Mediated CD4 T Cell Responses.

Anti-CD83 Ab capable of Ab-dependent cellular cytotoxicity can deplete activated CD83+ human dendritic cells, thereby inhibiting CD4 T cell-mediated acute graft-versus-host disease. As CD83 is also expressed on the surface of activated B lymphocytes, we hypothesized that anti-CD83 would also inhibit B cell responses to stimulation. We found that anti-CD83 inhibited total IgM and IgG production in vitro by allostimulated human PBMC. Also, Ag-specific Ab responses to immunization of SCID mice xenografted with human PBMC were inhibited by anti-CD83 treatment. This inhibition occurred without depletion of all human B cells because anti-CD83 lysed activated CD83+ B cells by Ab-dependent cellular cytotoxicity and spared resting (CD83-) B cells. In cultured human PBMC, anti-CD83 inhibited tetanus toxoid-stimulated B cell proliferation and concomitant dendritic cell-mediated CD4 T cell proliferation and expression of IFN-γ and IL-17A, with minimal losses of B cells (<20%). In contrast, the anti-CD20 mAb rituximab depleted >80% of B cells but had no effect on CD4 T cell proliferation and cytokine expression. By virtue of the ability of anti-CD83 to selectively deplete activated, but not resting, B cells and dendritic cells, with the latter reducing CD4 T cell responses, anti-CD83 may be clinically useful in autoimmunity and transplantation. Advantages might include inhibited expansion of autoantigen- or alloantigen-specific B cells and CD4 T cells, thus preventing further production of pathogenic Abs and inflammatory cytokines while preserving protective memory and regulatory cells.

CMRF-56(+) blood dendritic cells loaded with mRNA induce effective antigen-specific cytotoxic T-lymphocyte responses.

There are numerous transcriptional, proteomic and functional differences between monocyte-derived dendritic cells (Mo-DC) and primary blood dendritic cells (BDC). The CMRF-56 monoclonal antibody (mAb) recognizes a cell surface marker, which is upregulated on BDC following overnight culture. Given its unique ability to select a heterogeneous population of BDC, we engineered a human chimeric (h)CMRF-56 IgG4 mAb to isolate primary BDC for potential therapeutic vaccination. The ability to select multiple primary BDC subsets from patients and load them with in vitro transcribed (IVT) mRNA encoding tumor antigen might circumvent the issues limiting the efficacy of Mo-DC. After optimizing and validating the purification of hCMRF-56(+) BDC, we showed that transfection of hCMRF-56(+) BDC with mRNA resulted in efficient mRNA translation and antigen presentation by myeloid BDC subsets, while preserving superior DC functions compared to Mo-DC. Immune selected and transfected hCMRF-56(+) BDC migrated very efficiently in vitro and as effectively as cytokine matured Mo-DC in vivo. Compared to Mo-DC, hCMRF-56(+) BDC transfected with influenza matrix protein M1 displayed superior MHC peptide presentation and generated potent antigen specific CD8(+) T-cell recall responses, while Wilms tumor 1 (WT1) transfected CMRF-56(+) BDC generated effective primary autologous cytotoxic T-cell responses. The ability of the combined DC subsets within hCMRF-56(+) BDC to present mRNA delivered tumor antigens merits phase I evaluation as a reproducible generic platform for the next generation of active DC immune therapies.

Reactivated CD4+Tm cells of T1D patients and siblings display an exaggerated effector phenotype with heightened sensitivity to activation-induced cell death.

Dysfunction in effector memory has been proposed to contribute to autoimmunity in type 1 diabetes (T1D). Using a unique cohort of age- and sex-matched T1D patients, nonaffected siblings, and unrelated control children, we undertook a detailed analysis of proliferation, activation, effector responses, and apoptosis in reactivated CD4(+)Tm cells during T-cell receptor stimulation. Across cohorts, there was no difference in the proliferation of reactivated CD4(+)Tm cells. In T1D patients and siblings, CD4(+)Tm cells easily acquired the activated CD25(+) phenotype and effectively transitioned from a central (CD62L(+)Tcm) to an effector memory (CD62L(-)Tem) phenotype with an elevated cytokine "signature" comprising interferon (IFN)-γ and interleukin-10 in T1D patients and IFN-γ in siblings. This amplified Tem phenotype also exhibited an exaggerated immune shutdown with heightened sensitivity to activation-induced cell death and Fas-independent apoptosis. Apoptosis resulted in the elimination of one-half of the effector memory in T1D patients and siblings compared with one-third of the effector memory in control subjects. These data suggest genetic/environment-driven immune alteration in T1D patients and siblings that manifests in an exaggerated CD4(+)Tem response and shutdown by apoptosis. Further immunological studies are required to understand how this exaggerated CD4(+)Tem response fits within the pathomechanisms of T1D and how the effector memory can be modulated for disease treatment and/or prevention.

Immunosuppressive properties of mesenchymal stromal cell cultures derived from the limbus of human and rabbit corneas.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) cultivated from the corneal limbus (L-MSCs) provide a potential source of cells for corneal repair. In the present study, we investigated the immunosuppressive properties of human L-MSCs and putative rabbit L-MSCs to develop an allogeneic therapy and animal model of L-MSC transplantation. METHODS: MSC-like cultures were established from the limbal stroma of human and rabbit (New Zealand white) corneas using either serum-supplemented medium or a commercial serum-free MSC medium (MesenCult-XF Culture Kit; Stem Cell Technologies, Melbourne, Australia). L-MSC phenotype was examined by flow cytometry. The immunosuppressive properties of L-MSC cultures were assessed using mixed leukocyte reactions. L-MSC cultures were also tested for their ability to support colony formation by primary limbal epithelial (LE) cells. RESULTS: Human L-MSC cultures were typically CD34⁻, CD45⁻ and HLA-DR⁻ and CD73⁺, CD90⁺, CD105⁺ and HLA-ABC⁺. High levels (>80%) of CD146 expression were observed for L-MSC cultures grown in serum-supplemented medium but not cultures grown in MesenCult-XF (approximately 1%). Rabbit L-MSCs were approximately 95% positive for major histocompatibility complex class I and expressed lower levels of major histocompatibility complex class II (approximately 10%), CD45 (approximately 20%), CD105 (approximately 60%) and CD90 (<10%). Human L-MSCs and rabbit L-MSCs suppressed human T-cell proliferation by up to 75%. Conversely, L-MSCs from either species stimulated a 2-fold to 3-fold increase in LE cell colony formation. CONCLUSIONS: L-MSCs display immunosuppressive qualities in addition to their established non-immunogenic profile and stimulate LE cell growth in vitro across species boundaries. These results support the potential use of allogeneic L-MSCs in the treatment of corneal disorders and suggest that the rabbit would provide a useful pre-clinical model.

Improved Protein-A separation of V(H)3 Fab from Fc after papain digestion of antibodies.

Antibody-binding fragments (Fab) are generated from whole antibodies by treatment with papain and can be separated from the Fc component using Protein-A affinity chromatography. Commercial kits are available, which facilitate the production and purification of Fab fragments; however, the manufacturer fails to report that this method is inefficient for antibodies with V(H)3 domains as a result of the intrinsic variable region affinity for Protein-A. A commercially available, modified Protein-A resin (MabSelect SuRe) has been engineered for greater stability. Here, we report that an additional consequence of the modified resin is the ability to purify V(H)3 family Fab fragments, which cannot be separated effectively from other components of the papain digest by traditional Protein-A resin. This improvement of a commonly used procedure is of significance, as increasingly, therapeutic antibodies are being derived from human origin, where V(H)3 is the most abundantly used variable region family.

A method for rapid, ligation-independent reformatting of recombinant monoclonal antibodies.

Recombinant monoclonal antibodies currently dominate the protein biologics marketplace. The path from target antigen discovery and screening, to a recombinant therapeutic antibody can be time-consuming and laborious. We describe a set of expression vectors, termed mAbXpress, that enable rapid and sequence-independent insertion of antibody variable regions into human constant region backbones. This method takes advantage of the In Fusion cloning system from Clontech, which allows ligation-free, high-efficiency insertion of the variable region cassette without the addition of extraneous amino acids. These modular vectors simplify the antibody reformatting process during the preliminary evaluation of therapeutic or diagnostic candidates. The resulting constructs can be used directly for transient or amplifiable, stable expression in mammalian cells. The effectiveness of this method was demonstrated by the creation of a functional, fully human anti-human CD83 monoclonal antibody.

Antibody to the dendritic cell surface activation antigen CD83 prevents acute graft-versus-host disease.

Allogeneic (allo) hematopoietic stem cell transplantation is an effective therapy for hematological malignancies but it is limited by acute graft-versus-host disease (GVHD). Dendritic cells (DC) play a major role in the allo T cell stimulation causing GVHD. Current immunosuppressive measures to control GVHD target T cells but compromise posttransplant immunity in the patient, particularly to cytomegalovirus (CMV) and residual malignant cells. We showed that treatment of allo mixed lymphocyte cultures with activated human DC-depleting CD83 antibody suppressed alloproliferation but preserved T cell numbers, including those specific for CMV. We also tested CD83 antibody in the human T cell-dependent peripheral blood mononuclear cell transplanted SCID (hu-SCID) mouse model of GVHD. We showed that this model requires human DC and that CD83 antibody treatment prevented GVHD but, unlike conventional immunosuppressants, did not prevent engraftment of human T cells, including cytotoxic T lymphocytes (CTL) responsive to viruses and malignant cells. Immunization of CD83 antibody-treated hu-SCID mice with irradiated human leukemic cell lines induced allo antileukemic CTL effectors in vivo that lysed (51)Cr-labeled leukemic target cells in vitro without further stimulation. Antibodies that target activated DC are a promising new therapeutic approach to the control of GVHD.

Reduced intensity conditioning for allogeneic hematopoietic stem-cell transplant determines the kinetics of acute graft-versus-host disease.

BACKGROUND: Preparative myeloablative conditioning regimens for allogeneic hematopoietic stem-cell transplantation (HSCT) may control malignancy and facilitate engraftment but also contribute to transplant related mortality, cytokine release, and acute graft-versus-host disease (GVHD). Reduced intensity conditioning (RIC) regimens have decreased transplant related mortality but the incidence of acute GVHD, while delayed, remains unchanged. There are currently no in vivo allogeneic models of RIC HSCT, limiting studies into the mechanism behind RIC-associated GVHD. METHODS: We developed two RIC HSCT models that result in delayed onset GVHD (major histocompatibility complex mismatched (UBI-GFP/BL6 [H-2]-->BALB/c [H-2]) and major histocompatibility complex matched, minor histocompatibility mismatched (UBI-GFP/BL6 [H-2]-->BALB.B [H-2])) enabling the effect of RIC on chimerism, dendritic cell (DC) chimerism, and GVHD to be investigated. RESULTS: In contrast with myeloablative conditioning, we observed that RIC-associated delayed-onset GVHD is characterized by low production of tumor necrosis factor-alpha, maintenance of host DC, phenotypic DC activation, increased T-regulatory cell numbers, and a delayed emergence of activated donor DC. Furthermore, changes to the peritransplant milieu in the recipient after RIC lead to the altered activation of DC and the induction of T-regulatory responses. Reduced intensity conditioning recipients suffer less early damage to GVHD target organs. However, as donor cells engraft, activated donor DC and rising levels of tumor necrosis factor-alpha are associated with a later onset of severe GVHD. CONCLUSIONS: Delineating the mechanisms underlying delayed onset GVHD in RIC HSCT recipients is vital to improve the prediction of disease onset and allow more targeted interventions for acute GVHD.

Activated circulating dendritic cells after hematopoietic stem cell transplantation predict acute graft-versus-host disease.

BACKGROUND: Dendritic cells (DC) are central to the development of acute graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (alloHSCT). We hypothesized that DC activation status determines the severity of GVHD and that activated DC may be detected in the circulation prior to clinical presentation of GVHD. METHODS: Following transplant, blood samples were obtained twice weekly from alloHSCT patients. Myeloid (CD11c+) and plasmacytoid (CD123hi) DC were enumerated by flow cytometry, and activated myeloid DC were identified using the CMRF-44 monoclonal antibody. RESULTS: Of 40 alloHSCT patients, 26 developed acute GVHD. Severity of GVHD was associated with low total blood DC counts (P=0.007) and with low myeloid and plasmacytoid DC numbers (P=0.015 and 0.003). The CMRF-44 antigen was expressed on blood CD11c+ DC in all cases prior to GVHD onset, whereas of the 14 patients without GVHD, seven had no CMRF-44+ CD11c DC. Patients with CMRF-44+ CD11c+ DC in more than 20% of samples were more likely to subsequently develop acute GVHD (P=0.001, odds ratio=37.1), while patients who developed grade 2-4 GVHD had prior higher percentages of CMRF-44+ CD11c+ DC compared to grade 0-1 GVHD patients (P=0.001). CMRF-44 expression on >7.9% CD11c+ DC predicted for subsequent development of GVHD with a sensitivity of 87.5% and specificity of 79.2%. CONCLUSIONS: Activation status, as assessed by CMRF-44 antigen expression, of blood CD11c+ DC is highly associated with acute GVHD and these cells may be targets for therapeutic intervention.

Expression of human DEC-205 (CD205) multilectin receptor on leukocytes.

DEC-205 (CD205) belongs to the macrophage mannose receptor family of C-type lectin endocytic receptors and behaves as an antigen uptake/processing receptor for dendritic cells (DC). To investigate DEC-205 tissue distribution in human leukocytes, we generated a series of anti-human DEC-205 monoclonal antibodies (MMRI-5, 6 and 7), which recognized epitopes within the C-type lectin-like domains 1 and 2, and the MMRI-7 immunoprecipitated a single approximately 200 kDa band, identified as DEC-205 by mass spectrometry. MMRI-7 and another DEC-205 mAb (MG38), which recognized the epitope within the DEC-205 cysteine-rich and fibronectin type II domain, were used to examine DEC-205 expression by human leukocytes. Unlike mouse DEC-205, which is reported to have predominant expression on DC, human DEC-205 was detected by flow cytometry at relatively high levels on myeloid blood DC and monocytes, at moderate levels on B lymphocytes and at low levels on NK cells, plasmacytoid blood DC and T lymphocytes. MMRI-7 F(ab')2 also labeled monocytes, B lymphocytes and NK cells similarly excluding reactivity due to non-specific binding of the mAb to FcgammaR. Tonsil mononuclear cells showed a similar distribution of DEC-205 staining on the leukocytes. DEC-205-specific semiquantitative immunoprecipitation/western blot and quantitative reverse transcriptase-PCR analysis established that these leukocyte populations expressed DEC-205 protein and the cognate mRNA. Thus, human DEC-205 is expressed on more leukocyte populations than that were previously assumed based on mouse DEC-205 tissue localization studies. The broader DEC-205 tissue expression in man is relevant to clinical DC targeting strategies and DEC-205 functional studies.

Eighth Leucocyte Differentiation Antigen Workshop DC section summary.

Dendritic cells (DC) are specialist antigen presenting cells that play a role in the initiation of innate and adaptive immune response. At the seventh Human Leucocyte Differentiation Antigen workshop, these intriguing cell populations were included as a separate lineage of leucocytes. This paper reports the studies performed in the eighth Human Leucocyte Differentiation Antigen workshop as part of the DC section. Many investigators currently focus on DC that are derived from a number of different leucocyte populations, including those that are differentiated in vitro and cells that are purified ex vivo. The DC section assessed the surface expression of different leucocyte surface molecules on a range of different DC populations. The results summarise the expression of each molecule on dendritic cell populations and differences between different DC preparations. Eleven new CDs were allocated on the basis of monoclonal antibodies and molecular information that identify known cell surface molecules expressed by dendritic cells. This paper gives a brief review of the work that was performed during the HLDA8 and a summary of the CDs represented by submitted mAb.

In vitro depletion of tissue-derived dendritic cells by CMRF-44 antibody and alemtuzumab: implications for the control of Graft-versus-host disease.

Graft-versus-host disease (GvHD), a life-threatening complication of bone marrow transplantation, is initiated by donor T cells reacting to recipient dendritic cells (DC). GvHD can be controlled by attenuating donor T cells, but few strategies exist to target DC, particularly resident tissue DC, despite recent evidence of their importance. In this report, CMRF-44, a mouse monoclonal IgM reactive to human DC, is tested against human Langerhans cells (LC) in vitro. CMRF-44 antigen is expressed at low level on fresh LC but is up-regulated 40-60-fold during migration. CMRF-44 and complement kill more than 97% of migratory LC in vitro and inhibit allostimulation by LC up to 95%. In comparison, alemtuzumab, which binds CD52, reacts weakly with primary LC and fails to induce significant lysis with complement (less than 5%). These results highlight the potential of new therapeutic antibodies active against tissue DC to control graft-versus-host reactions.

Human T lymphoblasts and activated dendritic cells in the allogeneic mixed leukocyte reaction are susceptible to NK cell-mediated anti-CD83-dependent cytotoxicity.

CD83 is a marker of dendritic cell (DC) differentiation/activation and its expression in the mouse thymus contributes to CD4(+) T lymphocyte development. Its extrathymic role remains unclear despite the functional effects observed with CD83 fusion proteins or CD83 antibody and recent reports of potential ligands. We investigated the previously observed and presumed functional blockade of the allogeneic mixed leukocyte reaction (MLR) with rabbit polyclonal anti-CD83 (RA83). RA83 inhibition of T lymphocyte proliferation stimulated with allogeneic immature monocyte-derived DC (iMoDC) was confirmed. However, we found it was due to antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells in the responder T cell preparation. The likely targets of the ADCC were MoDC that had up-regulated CD83 during the MLR. Using a (51)Cr-release assay, we confirmed that CD83(+) MoDC, but not CD83(-) MoDC, are lysed by NK cells in the presence of RA83. However, prior fixation of the stimulator MoDC in the allogeneic MLR did not abrogate RA83 inhibition, indicating that cells from the responder T lymphocyte preparation, involved in the MLR proliferative response, also expressed CD83. We found, after 3-4 days of culture with allogeneic MoDC, a subset of CD3(+) cells had up-regulated CD83 and CD25. These were blasting T cells and, when isolated from the MLR, were found to be lysed by autologous NK cells in the presence of RA83. Thus, CD83 is expressed by responding T cells as well as by stimulating cells in the MLR and both are susceptible to anti-CD83-mediated ADCC.

CMRF-44 antibody-mediated depletion of activated human dendridic cells: a potential means for improving allograft survival.

BACKGROUND: Activated dendritic cells (DC) initiate immune responses by presenting antigen, including alloantigen from tissue grafts, to T lymphocytes. The potential to deplete or inactivate differentiated-activated DC during allogeneic transplantation represents a new approach to immunosuppression. METHODS: The authors investigated the potential of the monoclonal antibody CMRF-44, which has specificity for a DC-associated differentiation-activation antigen, to induce complement-mediated lysis of activated human DC. Peripheral blood mononuclear cells (PBMC), or purified DC preparations, were cultured overnight to activate endogenous DC, resulting in the expression of CMRF-44 antigen and CD83. These were then treated with CMRF-44 and complement. Depletion of activated DC was monitored by flow cytometry. RESULTS: Eighty-nine percent of activated (CD83+) DC in cultured PBMC were depleted by treatment with CMRF-44 and autologous serum (AS) (complement source; mean percentage of CD83+-CD14--CD19- cells=0.06%; cf 0.50% for heat-inactivated AS controls, P<0.0005, n=7). Ninety-five percent of cultured purified myeloid DC were depleted by this treatment, compared with only 43% of similarly treated lymphoid DC. Overnight culture also increases CMRF-44 antigen on a proportion of B cells and mononuclears, but only 24% of these cells were depleted. This treatment considerably reduced the ability of PBMC to stimulate allogeneic CD4+ CD45RA+ T lymphocytes. Similarly, the T-cell proliferative responses to recall and naive antigens were significantly reduced. CONCLUSIONS: CMRF-44 may be a suitable candidate for a new selective immunosuppressive strategy, targeting differentiated-activated but not resting DC. It may have applications in preventing GVHD in allogeneic bone marrow transplantation and facilitate immunoacceptance of solid organ allografts.

Single step enrichment of blood dendritic cells by positive immunoselection.

Dendritic cells (DC) for cancer immunotherapy protocols are generated most commonly by in vitro differentiation of monocytes with exogenous cytokines (Mo-DC). However, Mo-DC differ in their molecular phenotype and function from blood DC (BDC). Clinical isolation of BDC has been limited to the use of density gradients, which result in low yields of variable purity. We have developed a DC enrichment platform, which uses the CMRF-44 (IgM) or CMRF-56 (IgG) monoclonal antibodies (mAb) to select BDC that express these antigens after a short overnight incubation. After culture of peripheral blood mononuclear cells (PBMC) in autologous/AB serum, biotinylated CMRF-44 was used to select DC in a single step immuno-magnetic bead procedure; this produced populations containing up to 99% CMRF-44(+) cells, including up to 67% CMRF-44(+) CD14(-) CD19(-) DC, from an initial starting population of approximately 0.5%. We observed consistent differences in the purities obtained from individual donors with a mean of 54% CMRF-44(+) cells (range 19-99%). Similar results were obtained using biotinylated CMRF-56 mAb, an antibody identifying a comparable population in cultured PBMC. We recovered an average of 54% and 66% of the available BDC in separations performed with the CMRF-44 and CMRF-56 mAb, respectively. The reproducibility of the procedure and the ability to perform it in a closed sterile system makes it suitable for clinical use. Larger scale preparations starting from apheresis derived PBMC will produce sufficient BDC for immunotherapy protocols. The purified BDC elicited strong allogeneic mixed leukocyte reactions and HLA classes II- and I-restricted antigen-specific primary immune responses.

Characterization of human blood dendritic cell subsets.

Dendritic cells (DCs) are key antigen-presenting cells for stimulating immune responses and they are now being investigated in clinical settings. Although defined as lineage-negative (Lin(-)) HLA-DR(+) cells, significant heterogeneity in these preparations is apparent, particularly in regard to the inclusion or exclusion of CD14(+), CD16(+), and CD2(+) cells. This study used flow cytometry and a panel of monoclonal antibodies (mAbs), including reagents from the 7th Leukocyte Differentiation Antigen Workshop, to define the cellular composition of 2 standardized peripheral blood mononuclear cell (PBMCs)-derived Lin(-) HLA-DR(+) preparations. Lin(-) cells were prepared from PBMCs by depletion with CD3, CD14, CD19, CD11b, and either CD16 or CD56 mAbs. Analysis of the CD16-replete preparations divided the Lin(-) HLA-DR(+) population into 5 nonoverlapping subsets (mean +/- 1 SD): CD123 (mean = 18.3% +/- 9.7%), CD1b/c (18.6% +/- 7.6%), CD16 (49.6% +/- 8.5%), BDCA-3 (2.7% +/- 1.4%), and CD34 (5.0% +/- 2.4%). The 5 subsets had distinct phenotypes when compared with each other, monocytes, and monocyte-derived DCs (MoDCs). The CD85 family, C-type lectins, costimulatory molecules, and differentiation/activation molecules were also expressed differentially on the 5 Lin(-) HLA-DR(+) subsets, monocytes, and MoDCs. The poor viability of CD123(+) DCs in vitro was confirmed, but the CD16(+) CD11c(+) DC subset also survived poorly. Finally, the individual subsets used as stimulators in allogeneic mixed leukocyte reactions were ranked by their allostimulatory capacity as CD1b/c > CD16 > BDCA-3 > CD123 > CD34. These data provide an opportunity to standardize the DC populations used for future molecular, functional and possibly even therapeutic studies.

Spontaneous generation and survival of blood dendritic cells in mononuclear cell culture without exogenous cytokines.

Studies on purified blood dendritic cells (DCs) are hampered by poor viability in tissue culture. We, therefore, attempted to study some of the interactions/relationships between DCs and other blood cells by culturing unseparated peripheral blood mononuclear cell (PBMC) preparations in vitro. Flow cytometric techniques were used to undertake a phenotypic and functional analysis of DCs within the cultured PBMC population. We discovered that both the CD11c(+) and CD11c(-) CD123(hi) DC subsets maintained their viability throughout the 3-day culture period, without the addition of exogenous cytokines. This viability was accompanied by progressive up-regulation of the surface costimulatory (CD40, CD80, CD86) and activation (CMRF-44, CMRF-56, CD83) molecules. The survival and apparent production of DCs in PBMC culture (without exogenous cytokines) and that of sorted DCs (with cytokines) were evaluated and compared by using TruCOUNT analysis. Absolute DC counts increased (for CD123(hi) and CD11c(+) subsets) after overnight culture of PBMCs. Single-cell lineage depletion experiments demonstrated the rapid and spontaneous emergence of "new" in vitro generated DCs from CD14(+)/CD16(+) PBMC radioresistant precursors, additional to the preexisting ex vivo DC population. Unlike monocyte-derived DCs, blood DCs increased dextran uptake with culture and activation. Finally, DCs obtained after culture of PBMCs for 3 days were as effective as freshly isolated DCs in stimulating an allogeneic mixed leukocyte reaction.