Peripheral blood but not tissue dendritic cells express CD52 and are depleted by treatment with alemtuzumab

CAMPATH antibodies recognize CD52, a phosphatidylinositol-linked membrane protein expressed by mature lymphocytes and monocytes. Since some antigen-presenting dendritic cells (DCs) differentiate from a monocytic progenitor, we investigated the expression of CD52 on dendritic cell subsets. Four-color staining for lineage markers (CD3, 14, 16, 19, 20, 34, and 56), HLA-DR, CD52, and CD123 or CD11c demonstrated that myeloid peripheral blood (PB) DCs, defined as lineage(-)HLA-DR(+)CD11c(+), express CD52, while expression by CD123(+) lymphoid DCs was variable. Depletion of CD52(+) cells from normal PB strongly inhibited their stimulatory activity in an allogeneic mixed lymphocyte reaction and also reduced the primary autologous response to the potent neoantigen keyhole limpet hemocyanin. CD52 is thus expressed by a myeloid subset of PBDCs that is strongly allostimulatory and capable of initiating a primary immune response to soluble antigen. Administration of alemtuzumab, a humanized monoclonal antibody against CD52, to patients with lymphoproliferative disorders or as conditioning for hematopoietic stem cell transplantation resulted in a marked reduction in circulating lineage(-)HLA-DR(+) DCs (mean 31-fold reduction, P =.043). Analysis of monocyte-derived DCs in vitro revealed a reduction in CD52 expression during culture in granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4, with complete loss following activation-induced maturation with lipopolysaccharide. In contrast to the findings in PB, epidermal and small-intestine DCs did not express CD52, suggesting either that transit from blood to epidermis and gut is associated with loss of CD52 or that DCs in these tissues originate from another population of cells.     

Bone marrow and peripheral blood dendritic cells from patients with multiple myeloma are phenotypically and functionally normal despite the detection of Kaposi's sarcoma herpesvirus gene sequences

Multiple myeloma (MM) cells express idiotypic proteins and other tumor-associated antigens which make them ideal targets for novel immunotherapeutic approaches. However, recent reports show the presence of Kaposi's sarcoma herpesvirus (KSHV) gene sequences in bone marrow dendritic cells (BMDCs) in MM, raising concerns regarding their antigen-presenting cell (APC) function. In the present study, we sought to identify the ideal source of DCs from MM patients for use in vaccination approaches. We compared the relative frequency, phenotype, and function of BMDCs or peripheral blood dendritic cells (PBDCs) from MM patients versus normal donors. DCs were derived by culture of mononuclear cells in the presence of granulocyte-macrophage colony-stimulating factor and interleukin-4. The yield as well as the pattern and intensity of Ag (HLA-DR, CD40, CD54, CD80, and CD86) expression were equivalent on DCs from BM or PB of MM patients versus normal donors. Comparison of PBDCs versus BMDCs showed higher surface expression of HLA-DR (P =.01), CD86 (P =. 0003), and CD14 (P =.04) on PBDCs. APC function, assessed using an allogeneic mixed lymphocyte reaction (MLR), demonstrated equivalent T-cell proliferation triggered by MM versus normal DCs. Moreover, no differences in APC function were noted in BMDCs compared with PBDCs. Polymerase chain reaction (PCR) analysis of genomic DNA from both MM patient and normal donor DCs for the 233-bp KSHV gene sequence (KS330233) was negative, but nested PCR to yield a final product of 186 bp internal to KS330233 was positive in 16 of 18 (88.8%) MM BMDCs, 3 of 8 (37.5%) normal BMDCs, 1 of 5 (20%) MM PBDCs, and 2 of 6 (33.3%) normal donor PBDCs. Sequencing of 4 MM patient PCR products showed 96% to 98% homology to the published KSHV gene sequence, with patient specific mutations ruling out PCR artifacts or contamination. In addition, KHSV-specific viral cyclin D (open reading frame [ORF] 72) was amplified in 2 of 5 MM BMDCs, with sequencing of the ORF 72 amplicon revealing 91% and 92% homology to the KSHV viral cyclin D sequence. These sequences again demonstrated patient specific mutations, ruling out contamination. Therefore, our studies show that PB appears to be the preferred source of DCs for use in vaccination strategies due to the ready accessibility and phenotypic profile of PBDCs, as well as the comparable APC function and lower detection rate of KSHV gene sequences compared with BMDCs. Whether active KSHV infection is present and important in the pathophysiology of MM remains unclear; however, our study shows that MMDCs remain functional despite the detection of KSHV gene sequences.     

Natural cytotoxicity to autologous antigen-pulsed dendritic cells in multiple myeloma

To analyse autologous lymphocyte cytolytic activities of potential importance for cell-based immunotherapy in multiple myeloma (MM), in vitro differentiated dendritic cells (DCs) loaded with patient-specific monoclonal immunoglobulin (mIg) were used as autologous target cellsin cytotoxicity assays. Effector populations consisted of purified natural killer (NK) cells (CD56+, CD3-) and T cells (CD3+). The MM patients' NK cells cultured in the presence of interleukin 2 (IL-2) showed pronounced cytotoxic activity towards autologous mature DCs. Autologous MM DC targets displayed similar susceptibility to NK cell lysis, compared with allogeneic control DC targets, despite high surface expression of self major histocompatibility complex (MHC) antigens. However, some degree of classic MHC class I-mediated negative regulation was implicated in the NK-DC interactions, as indicated by class I blocking experiments. NK-mediated lysis was also discerned towards primary autologous MM cells. The results indicated that the major effector mechanism was mediated through the perforin-granzyme exocytosis pathway. In conclusion, NK cells from MM patients displayed significant and consistent cytotoxicity towards autologous mature DCs, suggesting that innate immunity could be implicated in MM and may influence the outcome of the administration of tumour antigen-pulsed DCs in treatment trials.     

Successful cross-presentation of allogeneic myeloma cells by autologous alpha-type 1-polarized dendritic cells as an effective tumor antigen in myeloma patients with matched monoclonal immunoglobulins

For wide application of a dendritic cell (DC) vaccination in myeloma patients, easily available tumor antigens should be developed. We investigated the feasibility of cellular immunotherapy using autologous alpha-type 1-polarized dendritic cells (αDC1s) loaded with apoptotic allogeneic myeloma cells, which could generate myeloma-specific cytotoxic T lymphocytes (CTLs) against autologous myeloma cells in myeloma patients. Monocyte-derived DCs were matured by adding the αDC1-polarizing cocktail (TNFα/IL-1β/IFN-α/IFN-γ/poly-I:C) and loaded with apoptotic allogeneic CD138(+) myeloma cells from other patients with matched monoclonal immunoglobulins as a tumor antigen. There were no differences in the phenotypic expression between αDC1s loaded with apoptotic autologous and allogeneic myeloma cells. Autologous αDC1s effectively took up apoptotic allogeneic myeloma cells from other patients with matched subtype. Myeloma-specific CTLs against autologous target cells were successfully induced by αDC1s loaded with allogeneic tumor antigen. The cross-presentation of apoptotic allogeneic myeloma cells to αDC1s could generate CTL responses between myeloma patients with individual matched monoclonal immunoglobulins. There was no difference in CTL responses between αDC1s loaded with autologous tumor antigen and allogeneic tumor antigen against targeting patient's myeloma cells. Our data indicate that autologous DCs loaded with allogeneic myeloma cells with matched immunoglobulin can generate potent myeloma-specific CTL responses against autologous myeloma cells and can be a highly feasible and effective method for cellular immunotherapy in myeloma patients.     

树突细胞负载骨髓瘤抗原介导的免疫反应

目的 研究负载肿瘤抗原的树突细胞能否诱导特异性细胞霉T淋巴细胞反应。方法 用多发性骨髓瘤患者骨髓CD34^ 细胞诱生树突细胞，并将骨髓瘤冻融物冲击致敏树突细胞。采用MTT法检测骨髓瘤抗原致敏及未致敏树突细胞诱导的自身T细胞对不同靶细胞(患者骨髓瘤细胞、K562细胞株)的杀伤率。结果 骨髓瘤冻融物致敏树突细胞诱导的自身T细胞对患者骨髓瘤细胞的杀伤远大于对K562细胞的杀伤。结论 患者骨髓瘤冻融物致敏的树突细胞能有效诱导自身T细胞发生特异性抗肿瘤免疫。     

Alpha-type 1-polarized dendritic cells loaded with apoptotic allogeneic myeloma cell line induce strong CTL responses against autologous myeloma cells

To induce a potent cytotoxic T lymphocyte (CTL) response, various tumor antigens should be loaded onto dendritic cells (DCs). In multiple myeloma (MM), it is difficult to obtain a sufficient number of autologous tumor cells as a source of tumor antigens in the clinical setting. We investigated the feasibility of immunotherapy in patients with MM, using myeloma-specific CTLs generated in vitro by alpha-type 1-polarized DCs (αDC1s) loaded with the ultraviolet B-irradiated allogeneic myeloma cell line, ARH77. αDC1s significantly increased the expression of several costimulatory molecules without differences in loading with tumor antigens. αDC1s showed a high production of interleukin-12 during maturation and after subsequent stimulation with CD40L but were not significantly affected by loading tumor antigens. Myeloma-specific CTLs against autologous myeloma cells from MM patients were induced by αDC1s pulsed with apoptotic ARH77 cells. Our data indicate that autologous DCs loaded with an allogeneic myeloma cell line can generate potent myeloma-specific CTL responses against autologous myeloma cells and might provide a practical method for cellular immunotherapy in patients with MM.     

Expansion of FOXP3high regulatory T cells by human dendritic cells (DCs) in vitro and after injection of cytokine-matured DCs in myeloma patients

CD4(+)CD25(+)FOXP3(+) regulatory T cells (Treg's) play an important role in the maintenance of immune tolerance. The mechanisms controlling the induction and maintenance of Treg's in humans need to be defined. We find that human myeloid dendritic cells (DCs) are superior to other antigen presenting cells for the maintenance of FOXP3(+) Treg's in culture. Coculture of DCs with autologous T cells leads to an increase in both the number of Treg's, as well as the expression of FOXP3 protein per cell both in healthy donors and myeloma patients. DC-mediated expansion of FOXP3(high) Treg's is enhanced by endogenous but not exogenous interleukin-2 (IL-2), and DC-T-cell contact, including the CD80/CD86 membrane costimulatory molecules. DCs also stimulate the formation of Treg's from CD25(-) T cells. The efficacy of induction of Treg's by DCs depends on the nature of the DC maturation stimulus, with inflammatory cytokine-treated DCs (Cyt-DCs) being the most effective Treg inducers. DC-induced Treg's from both healthy donors and patients with myeloma are functional and effectively suppress T-cell responses. A single injection of cytokine-matured DCs led to rapid enhancement of FOXP3(+) Treg's in vivo in 3 of 3 myeloma patients. These data reveal a role for DCs in increasing the number of functional FOXP3(high) Treg's in humans.     

Induction of HM1.24 peptide-specific cytotoxic T lymphocytes by using peripheral-blood stem-cell harvests in patients with multiple myeloma

HM1.24 antigen is preferentially overexpressed in multiple myeloma (MM) cells but not in normal cells. To explore the potential of HM1.24 as a target for cellular immunotherapy, we selected 4 HM1.24-derived peptides that possess binding motifs for HLA-A2 or HLA-A24 by using 2 computer-based algorithms. The ability of these peptides to generate cytotoxic T lymphocytes (CTLs) was examined in 20 healthy donors and 6 patients with MM by a reverse immunologic approach. Dendritic cells (DCs) were induced from peripheral-blood mononuclear cells of healthy donors or peripheral-blood stem-cell (PBSC) harvests from patients with MM, and autologous CD8(+) T cells were stimulated with HM1.24 peptide-pulsed DCs. Both interferon-gamma-producing and cytotoxic responses were observed after stimulation with either HM1.24-126 or HM1.24-165 peptides in HLA-A2 or HLA-A24 individuals. The peptide-specific recognition of these CTLs was further confirmed by tetramer assay and cold target inhibition assay. Importantly, HM1.24-specific CTLs were also induced from PBSC harvests from patients with MM and these CTLs were able to kill MM cells in an HLA-restricted manner. These results indicate the existence of functional DCs and HM1.24-specific CTL precursors within PBSC harvests and provide the basis for cellular immunotherapy in combination with autologous PBSC transplantation in MM.     

Functional comparison of DCs generated in vivo with Flt3 ligand or in vitro from blood monocytes: differential regulation of function by specific classes of physiologic stimuli

Dendritic cells (DCs) are a family of leukocytes that initiate T- and B-cell immunity against pathogens. Migration of antigen-loaded DCs from sites of infection into draining lymphoid tissues is fundamental to the priming of T-cell immune responses. In humans, the major peripheral blood DC (PBDC) types, CD1c+ DCs and interleukin 3 receptor-positive (IL-3R+) plasmacytoid DCs, are significantly expanded in vivo with the use of Flt3 ligand (FL). DC-like cells can also be generated from monocyte precursors (MoDCs). A detailed comparison of the functional potential of these types of DCs (in an autologous setting) has yet to be reported. Here, we compared the functional capacity of FL-expanded CD1c+ PBDCs with autologous MoDCs in response to 3 different classes of stimuli: (1) proinflammatory mediators, (2) soluble CD40 ligand trimer (CD40L), and (3) intact bacteria (Escherichia coli). Significant differences in functional capacities were found with respect to changes in phenotype, migratory capacity, cytokine secretion, and T-cell stimulation. MoDCs required specific stimuli for the expression of functions. They responded vigorously to CD40L or E coli, expressing cytokines known to regulate interferon-gamma (IFN-gamma) in T cells (IL-12p70, IL-18, and IL-23), but required prostaglandin E2 (PGE2) during stimulation to migrate to chemokines. In contrast, PBDCs matured in response to minimal stimulation, rapidly acquired migratory function in the absence of PGE2-containing stimuli, and were low cytokine producers. Interestingly, both types of DCs were equivalent with respect to stimulation of allogeneic T-cell proliferation and presentation of peptides to cytotoxic T lymphocyte (CTL) lines. These distinct differences are of particular importance when considering the choice of DC types for clinical applications.     

Tumor lysate-specific cytotoxic T lymphocytes in multiple myeloma: promising effector cells for immunotherapy

The idiotype protein, secreted by myeloma plasma cells, is a tumor-specific but weak antigen. Idiotype-based immunotherapy has been explored in myeloma patients with disappointing results. It is conceivable that myeloma cells contain a multitude of tumor antigens that can more effectively stimulate antitumor T cells. To explore the possibility of using whole myeloma cells as a source of tumor antigens for immunotherapy, the current study was undertaken to generate and examine the function of myeloma-specific cytotoxic T lymphocytes (CTLs) by using dendritic cells (DCs) pulsed with myeloma cell lysates as stimulating cells. After repeated stimulation, specific CTL lines, containing CD4(+) and CD8(+) T cells, were generated from myeloma patients. Our results show that these T cells not only recognized and lysed autologous myeloma protein-pulsed DCs, they also killed autologous primary myeloma cells. Occasionally, CTLs responded to autologous idiotype-pulsed DCs and to allogeneic primary myeloma cells. No cytolytic activity, however, was detected against autologous lymphocytes including B cells, suggesting that the T cells acted specifically against myeloma cells. Cytotoxicity against target cells was major histocompatibility complex class 1 and, to a lesser extent, class 2 restricted and was dependent mainly on the perforin-mediated pathway. CTLs secreted predominantly interferon-gamma and tumor necrosis factor-alpha on antigenic stimulation, indicating a type 1 T-cell subset. These findings represent the first demonstration that tumor cell lysate-primed CTLs kill only myeloma cells, not autologous lymphocytes. This provides a rationale for myeloma cell-based immunotherapy in multiple myeloma.     

Efficient lentiviral transduction of human cord blood CD34(+) cells followed by their expansion and differentiation into dendritic cells

OBJECTIVE: To support immune reconstitution after cord blood transplantation, immunotherapy using gene-modified dendritic cells (DCs), the most potent antigen-presenting cells, can be a powerful strategy for preventing infection and recurrence. To investigate the applicability of lentiviral vector-transduced DCs compared to retroviral vectors, we transduced umbilical cord blood (CB) CD34(+) cells, then expanded and differentiated them into DCs. MATERIALS AND METHODS: We transduced CB CD34(+) cells by vesicular stomatitis virus G-protein pseudotyped self-inactivating lentiviral vector or retroviral vectors carrying the enhanced green fluorescent protein gene. The cells were expanded in the stroma-dependent culture system and transferred to the culture condition for developing DCs. The efficiency of transduction and expression of the transgene in severe combined immunodeficiency (SCID) mice-repopulating cells (SRCs) and DCs were compared between lentiviral vector and retroviral vectors. Induced DCs were cocultured with allogeneic or autologous T cells to test the ability to present antigens. RESULTS: CB CD34(+) cells transduced by lentiviral vector and expanded ex vivo sustained stable transgene expression and multipotentiality by assessing SRCs assay and clonogenic assay of bone marrow cells from the transplanted mice. DCs derived from these cells expressed green fluorescent protein and surface markers CD1a, CD80, and HLA-DR and showed potent allo-stimulatory activity as well as nontransduced DCs did. On the other hand, we did not detect transgene expression in SRCs and DCs transduced by retroviral vectors. CONCLUSION: Gene-modified DCs derived from ex vivo expanded CB CD34(+) cells transduced by lentiviral vector will be useful in future immunotherapy protocols.     

Characterization of dendritic cell differentiation pathways from cord blood CD34(+)CD7(+)CD45RA(+) hematopoietic progenitor cells

To better characterize human dendritic cells (DCs) that originate from lymphoid progenitors, the authors examined the DC differentiation pathways from a novel CD7(+)CD45RA(+) progenitor population found among cord blood CD34(+) cells. Unlike CD7(-)CD45RA(+) and CD7(+)CD45RA(-) progenitors, this population displayed high natural killer (NK) cell differentiation capacity when cultured with stem cell factor (SCF), interleukin (IL)-2, IL-7, and IL-15, attesting to its lymphoid potential. In cultures with SCF, Flt3 ligand (FL), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF)-alpha (standard condition), CD7(+)CD45RA(+) progenitors expanded less (37- vs 155-fold) but yielded 2-fold higher CD1a(+) DC percentages than CD7(-)CD45RA(+) or CD7(+)CD45RA(-) progenitors. As reported for CD34(+)CD1a(-) thymocytes, cloning experiments demonstrated that CD7(+)CD45RA(+) cells comprised bipotent NK/DC progenitors. DCs differentiated from CD7(-)CD45RA(+) and CD7(+)CD45RA(+) progenitors differed as to E-cadherin CD123, CD116, and CD127 expression, but none of these was really discriminant. Only CD7(+)CD45RA(+) or thymic progenitors differentiated into Lag(+)S100(+) Langerhans cells in the absence of exogenous transforming growth factor (TGF)-beta 1. Analysis of the DC differentiation pathways showed that CD7(+)CD45RA(+) progenitors generated CD1a(+)CD14(-) precursors that were macrophage-colony stimulating factor (M-CSF) resistant and CD1a(-)CD14(+) precursors that readily differentiated into DCs under the standard condition. Accordingly, CD7(+)CD45RA(+) progenitor-derived mature DCs produced 2- to 4-fold more IL-6, IL-12, and TNF-alpha on CD40 ligation and elicited 3- to 6-fold higher allogeneic T-lymphocyte reactivity than CD7(-)CD45RA(+) progenitor-derived DCs. Altogether, these findings provide evidence that the DCs that differentiate from cord blood CD34(+)CD7(+)CD45RA(+) progenitors represent an original population for their developmental pathways and function. (Blood. 2000;96:3748-3756)     

Phagocytosis of co-developing neutrophil progenitors by dendritic cells in a culture of human CD34<Superscript>+</Superscript> cells with granulocyte colony-stimulating factor and tumor necrosis factor-α

Tumor necrosis factor-alpha (TNF-alpha) has been shown to induce the differentiation of CD34(+) cells toward dendritic cells (DCs). We have previously shown that DCs are co-generated from human CD34(+) cells during erythroid or megakaryocytic differentiation in the presence of TNF-alpha, and those DCs are able to stimulate autologous T cell proliferation. The aim of this study was to learn whether the co-stimulation of granulocyte colony-stimulating factor (G-CSF) and TNF-alpha would generate neutrophil progenitors and DCs together from human CD34(+) cells, and if this was the case, to clarify the phenotypic and functional characteristics of these DCs. When highly purified human CD34(+) cells were cultured for 7 days with G-CSF alone, the generated cells predominantly expressed a granulocyte marker, CD15, and then differentiated into neutrophils after 14 days of culture. The addition of TNF-alpha with G-CSF markedly decreased the number of CD15(+) cells without affecting the total number of cells during 7 days of culture. Almost one third of the generated cells were positive for CD11c and CD123. Furthermore, CD11c(+) cells were found to phagocytose CD15(+) cells and were able to induce allogeneic, but not autologous, T cell proliferation in the mixed lymphocyte reaction (MLR). On the other hand, the CD11c(+) cells generated by TNF-alpha and cytokines capable of inducing erythroid differentiation were able to stimulate autologous T cells. There was a difference in the expression of CD80, CD83 and CD86 among CD11c(+) cells induced by G-CSF plus TNF-alpha and those generated by interleukin-3, stem cell factor, and erythropoietin plus TNF-alpha. These results indicate that the co-stimulation of human CD34(+) cells with G-CSF and TNF-alpha induces the phagocytosis of co-developing neutrophil progenitors by DCs, and the stimulatory effects of these DCs on autologous T cells is different from that of DCs generated from CD34(+) cells during erythroid differentiation.     

Efficient Ex Vivo Generation of Human Dendritic Cells from Mobilized CD34p+ Peripheral Blood Progenitors

We tried to efficiently generate human dendritic cells (DCs) from CD34+ peripheral blood hematopoietic progenitor cells mobilized by high-dose chemotherapy and subsequent administration of granulocyte colony-stimulating factor, using a liquid suspension culture system. Among various combinations, the combination of c-kit ligand, flt-3 ligand, c-mpl ligand (TPO), and interleukin (IL)-4 most potently generated the number of CD1a+CD14- DCs in cultures containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor alpha (TNF-alpha). The delayed addition of IL-4 on day 6 of culture gave rise to an additional increase in the yield of CD1a+CD14-DCs that were characterized by the expression of HLA-ABC, HLA-DR, CD80, CD86, and CD83. The majority of the sorted CD1a-CD14+ cells derived from 6-day culture of CD34+ cells gave rise to CD1a+CD14- DCs and CD1a-CD14+ macrophages on day 12 of culture in the presence and absence of IL-4, respectively. These findings suggest that IL-4 promotes the differentiation of CD1a- CD14+ cells derived from mobilized CD34+ peripheral blood hematopoietic progenitors to CD1a+ CD14- DCs. The majority of these DCs expressed CD68 but not the Langerhans-associated granule antigen, a finding that suggests they emerge through the monocyte differentiation pathway. The addition of TPO and IL-4 to cultures did not affect the potential of DCs to stimulate the primary allogeneic T-cell response. These findings demonstrated that the combination of c-kit ligand plus flt-3 ligand plus TPO with GM-CSF plus TNF-alpha, followed by IL-4, is useful for ex vivo generation of human DCs from mobilized CD34+ peripheral blood progenitors.     

1,25-Dihydroxyvitamin D(3) inhibits dendritic cell differentiation and maturation in vitro

OBJECTIVE: Because of its potent immunosuppressive properties in vitro as well as in vivo, we studied the effect of 1,25-dihydroxyvitamin D(3) (calcitriol) on differentiation, maturation, and function of dendritic cells (DC). MATERIALS AND METHODS: Monocyte-derived DCs were generated with GM-CSF plus IL-4, and maturation was induced by a 2-day exposure to TNFalpha. DCs were derived from CD34(+) progenitors using SCF plus GM-CSF plus TNFalpha. For differentiation studies, cells were exposed to calcitriol at concentrations of 10(-)(9)- 10(-7) M at days 0, 6, and 8, respectively. The obtained cell populations were evaluated by morphology, phenotype, and function. RESULTS: When added at day 0, calcitriol blocked DC differentiation from monocytes and inhibited the generation of CD1a(+) cells from progenitor cells while increasing CD14(+) cells. Exposure of immature DCs to calcitriol at day 6 resulted in a loss of the DC-characteristic surface molecule CD1a, downregulation of the costimulatory molecules CD40 and CD80, and MHC class II expression, whereas the monocyte/macrophage marker CD14 was clearly reinduced. In addition, calcitriol hindered TNFalpha-induced DC maturation, which is usually accompanied with induction of CD83 expression and upregulation of costimulatory molecules. In contrast, the mature CD83(+) DCs remained CD1a(+)CD14(-) when exposed to calcitriol. The capacity of cytokine-treated cells to stimulate allogeneic and autologous T cells and to take up soluble antigen was inhibited by calcitriol. CONCLUSION: The potent suppression of DC differentiation, the reversal of DC phenotype, and function in immature DCs, as well as the inhibition of DC maturation by calcitriol, may explain some of its immunosuppressive properties.     

Adenovirus-mediated delivery of p53 results in substantial apoptosis to myeloma cells and is not cytotoxic to flow-sorted CD34(+) hematopoietic progenitor cells and normal lymphocytes

Multiple myeloma (MM) is an incurable disease; therefore, there is a need for new modalities of treatment for this disease. We designed a study to test the sensitivity of MM cell lines, freshly isolated myeloma cells, and CD34(+) hematopoietic progenitor cells to adenovirus-mediated delivery of wild-type p53 (Ad-p53). Replication-deficient Ad-p53, previously used in phase I-II clinical trial for treatment of patients with solid tumors, was used in this study. Myeloma cells from seven MM cell lines with mutated or w.t. p53 and varying expression of bcl-2 were used. Fresh myeloma cells (CD38(bright)CD45(-)) and fresh CD34(+) hematopoietic stem cells and CD34(-) cells were purified by flow sorting of apheresis collections of MM patients undergoing high-dose chemotherapy and stem cell rescue. The effect of Ad-p53 on colony-forming unit granulocyte-macrophage (CFU-GM) and burst-forming unit erythroid (BFU-E) colony formation in methylcellulose was tested on purified CD34(+) and CD34(-) cells to evaluate bone marrow toxicity.Myeloma cells from cell lines, or freshly isolated myeloma cells, were sensitive to Ad-p53 only if they had mutated p53 and had low expression of bcl-2. CD34(+) cells were resistant to Ad-p53-mediated apoptosis, and CFU-GM and BFU-E colony formation was not affected by treatment with Ad-p53.Ad-p53 is a potent inducer of apoptosis in MM cell lines and in freshly isolated myeloma cells expressing low levels of bcl-2. Ad-p53 is not overtly cytotoxic to normal hematopoietic stem cells or normal lymphocytes; therefore, it could be considered for a phase I clinical trial of MM patients with mutated p53.     

Immunologic mechanisms of extracorporeal photochemotherapy in chronic graft-versus-host disease

Extracorporeal photochemotherapy (ECP) has been shown to be an effective therapy for patients with acute and chronic graft-versus-host disease (GVHD) following allogeneic bone marrow transplantation, but its biologic mechanism is not understood. We reported that clinical response to ECP was associated not only with normalization of skewed CD4/CD8 ratios but also with an increase in CD3(-)/CD56(+) natural killer cells and a decrease in the number of CD80(+) and CD123(+) circulating dendritic cells (DCs). To further elucidate the effects of ECP on activated lymphocyte subpopulations and the interaction between effector lymphocytes and antigen-presenting DCs, we isolated and characterized DC populations from patients with chronic GVHD undergoing ECP therapy. Antigen-presenting activity of DCs was measured as proliferation of antigen-stimulated autologous and allogeneic T cells by mixed-lymphocyte reaction (MLR). In MLR assays the proliferation of T cells was decreased in all 10 patients by a mean of 84% (range, 75%-95%; P < or =.002) after a 2-day cycle of ECP and longitudinally over the 12-month course of therapy. Immunophenotypic analysis of DC populations revealed a preponderance of DC1 monocytic dendritic cells in all patients before the initiation of ECP. Nine of 10 patients demonstrated a shift from DC1 to DC2 and as a concordant shift from a predominantly Th1 (interleukin-2 [IL-2], interferon-gamma) to Th2 (IL-4, IL-10) cytokine profile after ECP, and 8 of 10 had a clinical response to ECP. Our results suggest that ECP alters alloreactivity by affecting allo-targeted effector T cells and antigen-presenting DCs.     

Ex vivo induction of multiple myeloma-specific cytotoxic T lymphocytes

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by immunosuppression. In this study, we identified factors in patients' bone marrow (BM) sera inhibiting autologous anti-MM immunity and developed an ex vivo strategy for inducing MM-specific cytotoxic T lymphocytes (CTLs). We found that sera from BM of MM patients inhibited induction of dendritic cells (DCs), evidenced by both phenotype and only weak stimulation of T-cell proliferation. Anti-vascular endothelial growth factor (anti-VEGF) and/or anti-interleukin 6 (anti-IL-6) antibodies neutralized this inhibitory effect, confirming that VEGF and IL-6, at least in part, mediate immunosuppression in MM patients. To induce MM-specific CTLs ex vivo, immature DCs were generated by culture of adherent mononuclear cells in medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4 for 5 days and then cocultured with apoptotic MM bodies in the presence of tumor necrosis factor alpha (TNF-alpha) for 3 days to induce their maturation. Autologous BM or peripheral blood mononuclear cells were stimulated weekly with these DCs, and cytotoxicity was examined against the MM cells used to pulse DCs. DCs cultured with apoptotic bodies stimulated significantly greater T-cell proliferation (stimulation index [SI] = 23.2 at a T-DC ratio of 360:1) than T cells stimulated by MM cells only (SI = 5.6), DCs only (SI = 9.3), or MM lysate-pulsed DCs (SI = 13.5). These CTLs from MM patients demonstrated specific cytotoxicity (24.7% at the effector-target [E/T] ratio of 40:1) against autologous primary MM cells. These studies therefore show that CTLs from MM patients can recognize and lyse autologous tumor cells and provide the framework for novel immunotherapy to improve patient outcome in MM.     

Allogeneic T cells induce rapid CD34+ cell differentiation into CD11c+CD86+ cells with direct and indirect antigen-presenting function

Dendritic cells (DCs) derive from CD34+ cells or monocytes and stimulate alloimmune responses in transplantation. We hypothesized that the interaction between CD34+ cells and allogeneic T cells would influence the function of hematopoietic stem cells (HSCs). Cord blood (CB) CD34+ cells proliferated briskly in response to allogeneic, but not autologous, T cells when mixed with irradiated T cells for 6 days in vitro. This proliferation was significantly inhibited by an anti-HLA class II monoclonal antibody (mAb), by an anti-TNFalpha mAb, or by CTLA4-Ig. Allogeneic T cells induced the differentiation of CD34+ progenitors into cells with the morphology of dendritic monocytic precursors and characterized by the expression of HLA-DR, CD86, CD40, CD14, and CD11c, due to an endogenous release of TNFalpha. Cotransplantation of CD34+ cells with allogeneic T cells into nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice resulted in a greater engraftment of myeloid CD1c+ dendritic cells compared with cotransplantation with autologous T cells. In vitro, CD34+ cell-derived antigen-presenting cells (APCs) were functionally capable of both direct and indirect presentation of alloantigens. Based on these findings, we hypothesize that in HSC transplantation the initial cross talk between allogeneic T cells and CD34+ cells may result in the increased generation of APCs that can present host alloantigens and possibly contribute to the development of graft-versus-host disease.     

Efficient presentation of tumor idiotype to autologous T cells by CD83(+) dendritic cells derived from highly purified circulating CD14(+) monocytes in multiple myeloma patients

To generate mature and fully functional CD83(+) dendritic cells derived from circulating CD14(+) cells highly purified from the leukapheresis products of multiple myeloma patients.CD14(+) monocytes were selected by high-gradient magnetic separation and differentiated to immature dendritic cells with granulocyte-macrophage colony-stimulating factor and interleukin-4 for 6-7 days and then induced to terminal maturation by the addition of tumor necrosis factor-alpha or stimulation with CD40 ligand. Dendritic cells were characterized by immunophenotyping, evaluation of soluble antigens uptake, cytokine secretion, capacity of stimulating allogeneic T cells, and ability of presenting nominal antigens, including tumor idiotype, to autologous T lymphocytes. Phenotypic analysis showed that 90% +/- 6% of cells recovered after granulocyte-macrophage colony-stimulating factor and interleukin-4 stimulation expressed all surface markers typical of immature dendritic cells and demonstrated a high capacity of uptaking soluble antigens as shown by the FITC-dextran assay. Subsequent exposure to maturation stimuli induced the downregulation of CD1a and upregulation of CD83, HLA-DR, costimulatory molecules and induced the secretion of large amounts of interleukin-12. Mature CD83(+) cells showed a diminished ability of antigen uptake whereas they proved to be potent stimulators of allogeneic T cells in a mixed lymphocyte reaction. Monocyte-derived dendritic cells, pulsed before the addition of maturation stimuli, were capable of presenting soluble proteins such as keyhole limpet hemocyanin and tetanus toxoid to autologous T cells for primary and secondary immune response, respectively. Conversely, pulsing of mature (CD83(+)) dendritic cells was less efficient for the induction of T-cell proliferation. More importantly, CD14(+) cells-derived dendritic cells stimulated autologous T-cell proliferation in response to a tumor antigen such as the patient-specific idiotype. Moreover, idiotype-pulsed dendritic cells induced the secretion of interleukin-2 and gamma-interferon by purified CD4(+) cells. T-cell activation was better achieved when Fab immunoglobulin fragments were used as compared with the whole protein. When dendritic cells derived from CD14(+) cells from healthy volunteers were analyzed, we did not find any difference with samples from myeloma patients as for cell yield, phenotypic profile, and functional characteristics. These studies demonstrate that mobilized purified CD14(+) cells represent the optimal source for the production of a homogeneous cell population of mature CD83(+) dendritic cells suitable for clinical trials in multiple myeloma.