Interaction of mature CD3+CD4+ T cells with dendritic cells triggers the development of tertiary lymphoid structures in the thyroid

Ectopic expression of CC chemokine ligand 21 (CCL21) in the thyroid leads to development of lymphoid structures that resemble those observed in Hashimoto thyroiditis. Deletion of the inhibitor of differentiation 2 (Id2) gene, essential for generation of CD3-CD4+ lymphoid tissue-inducer (LTi) cells and development of secondary lymphoid organs, did not affect formation of tertiary lymphoid structures. Rather, mature CD3+CD4+ T cells were critical for the development of tertiary lymphoid structures. The initial stages of this process involved interaction of CD3+CD4+ T cells with DCs, the appearance of peripheral-node addressin-positive (PNAd+) vessels, and production of chemokines that recruit lymphocytes and DCs. These findings indicate that the formation of tertiary lymphoid structures does not require Id2-dependent conventional LTis but depends on a program initiated by mature CD3+CD4+ T cells.     

Differentiation and expansion of lentivirus vector-marked dendritic cells derived from human CD34(+) cells

The in vitro genetic manipulation of dendritic cells (DCs) for the expression of foreign proteins or peptides will assist in the development of immunotherapeutic approaches to treat cancer, immunological disorders, and/or infectious diseases. Reports have shown the expansion and differentiation of CD34(+) progenitor cells into mature DCs. In this article we describe the differentiation and expansion of lentivirus vector-marked DCs from umbilical cord blood, bone marrow, and cytokine-mobilized peripheral blood CD34(+) cells in the presence of GM-CSF, TNF-alpha, SCF, Flt-3, and IL-4. Lentivirus-marked DCs expressed high levels of enhanced green fluorescent protein and the characteristic DC surface markers CD1a, CD83, HLA-DR, and CD80. Transduced DCs activated allogeneic CD3(+) T cells as efficiently as control (nontransduced) DCs in mixed lymphocyte reactions. These results demonstrate the potential utility of lentivirus-transduced DCs in future immunotherapy protocols.     

Short-term liquid storage of CD14+ monocytes, CD11c+, and CD123+ precursor dendritic cells produced by leukocytapheresis

BACKGROUND: This prospective study compared white blood cell (WBC) storage in polyvinylchloride (PVC) bags and in polyolefin (POF) bags. After leukapheresis, CD14+ monocytes, CD11c+, and CD123+ precusor dendritic cells (DCs) were analyzed under platelet (PLT) storage conditions. STUDY DESIGN AND METHODS: Twenty-five leukapheresis procedures were performed on blood cell separators (AS.TEC204 [PVC; Fresenius HemoCare GmbH] and the COBE Spectra [POF, Gambro BCT]). Blood cell counts, glucose, lactic acid, pO(2), pCO(2), and pH were measured in mononuclear cell (MNC) harvests on Days 0, 1, 3, and 5. WBCs were analyzed by flow cytometry. RESULTS: The WBC yields of the AS.TEC204 harvests were 25 percent higher (13.4 x 10(9) +/- 2.7 x 10(9) WBCs) compared to the COBE Spectra (9.9 x 10(9) +/- 2.5 x 10(9) WBCs). During 5-day storage, WBC counts (PVC bags) decreased significantly (24%). Storage in POF bags showed more consistent results (WBC loss, 6%). Loss of CD14+ monocytes and CD11c+ precursor DCs did not differ significantly in leukapheresis products. CD123+ precursor DCs stored in PVC bags dropped by more than 90 percent (POF bags, 24%). Lactic acid concentrations exceeded 20 mmol per L after 24 hours in PVC bags and after 72 hours in POF bags. The mean pH value on Day 5 was 6.2 (PVC) and 6.3 (POF). On Day 1, the product glucose concentration decreased by 76 percent after storage in PVC bags and by 16 percent in POF bags. CONCLUSIONS: Storage of MNCs within 72 hours in the original harvest container assures stable WBC content and is easy to perform. POF bags should be preferred in the case of extended WBC storage. Patient studies should clarify changes in efficiency of hematopoietic reconstitution that might occur over time during MNC storage.     

High level of retrovirus-mediated gene transfer into dendritic cells derived from cord blood and mobilized peripheral blood CD34+ cells

Dendritic cells (DCs), the most potent antigen-presenting cells, can be generated from CD34+ hematopoietic stem cells and used for generating therapeutic immune responses. To develop immunotherapy protocols based on genetically modified DCs, we have investigated the conditions for high-level transduction of a large amount of CD34+-derived DCs. Thus, we have used an efficient and clinically applicable protocol for the retroviral transduction of cord blood (CB) or mobilized peripheral blood (MPB) CD34+ cells based on infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral vectors carrying the nls-LacZ reporter gene. Infected cells have been subsequently cultured under conditions allowing their dendritic differentiation. The results show that using a growth factor combination including granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha plus interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DCs derived from CB or MPB CD34+ cells can be transduced. Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Transduced DCs retain normal immunophenotype and potent T cell stimulatory capacity. Finally, by using a semisolid methylcellulose assay for dendritic progenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduced. Such a highly efficient retrovirus-mediated gene transfer into CD34+-derived DCs makes it possible to envision the use of this methodology in clinical trials.     

A simple two-step culture system for the large-scale generation of mature and functional dendritic cells from umbilical cord blood CD34+ cells

BACKGROUND: In vitro generated dendritic cells (DCs) are widely used as adjuvants in cancer immunotherapy. The major sources for DC generation are monocytes and CD34+ cells. CD34+-derived DCs are less frequently used in clinical applications because it requires complex generation methods. Here a simple method for the large-scale generation of mature functional DCs from umbilical cord blood–derived CD34+ cells is described.
STUDY DESIGN AND METHODS: CD34+ cells were first expanded with a combination of early acting growth factors in a medium containing autologous plasma. In the second step the DC precursors were further either enriched by plastic adherence or sorted on a cell sorter and differentiated as DCs. DCs generated by both methods were compared for their morphology, phenotype, and different functional variables.
RESULTS: This culture system provided a large-scale expansion of CD34+ cells giving a mean fold increase of 615. The majority of the expanded cells were interstitial DC precursors, that is, CD14+-positive cells. In vitro generated immature DCs could be matured into functional DCs by appropriate maturation stimuli. DCs generated by the plastic adherence method had a better cytokine profile and strong mixed leukocyte reaction compared to those generated by cell sorting.
CONCLUSION: A two-step culture system provides a large-scale expansion of CD34+ cells with a preferential lineage commitment toward CD14+ cells. Enrichment of these precursors with a simple plastic adherence technique results in generation of large numbers of mature, functional DCs. This method of in vitro DC generation will have applications in cancer immunotherapy.     

Efficacy of dendritic cell generation for clinical use: recovery and purity of monocytes and mature dendritic cells after immunomagnetic sorting or adherence selection of CD14+ starting populations

Immunotherapy with monocyte-derived dendritic cells (Mo-DCs) is applied to an increasing number of patients requiring large-scale production of clinical-grade dendritic cells with standardized Mo-DC generation protocols. In many countries, e.g., in Germany, Mo-DCs are legally considered medicinal products, which must be produced under Good Manufacturing Practice (GMP) conditions by an institution holding an official production license. Plastic adherence, immunomagnetic selection of CD14(+) monocytes and depletion of CD2(+) and CD19(+) cells are used to enrich monocytes for Mo-DC culture. The latter two have received approval of the European Union (CE). However, enrichment by plastic adherence is well-established and commonly used for clinical and research Mo-DC applications. The various plastic materials, nevertheless, have not been officially approved for monocyte selection for clinical use. In the present study therefore, we compared three methods for enrichment of CD14(+) monocytes with regard to efficiency of enrichment, yield of monocyte-derived functional mature dendritic cells, cost effectiveness, and handling. We demonstrate that CD14 selection and CD2 and CD19 depletion yield similar results regarding purity of mature DEs MoDCs (97-99% vs. 64-97%) and their immunostimulatory capacity. However, cell preparations cultured after CD14 selection possessed 91% to 97% CD14(+) cells, whereas CD2-/and DC19-depleted preparations contained only 8% to 57% CD14(+) cells. Thus, positive selection requires smaller culture volumes to generate equal numbers of Mo-DCs. Both methods gave better results than plastic adherence. In conclusion, of the techniques examined, CD14 selection of monocytes gave the best results regarding reproducibility, yield, and purity of the resulting monocytes and mature Mo-DCs.     

Circulating precursors of human CD1c+ and CD141+ dendritic cells

Two subsets of conventional dendritic cells (cDCs) with distinct cell surface markers and functions exist in mouse and human. The two subsets of cDCs are specialized antigen-presenting cells that initiate T cell immunity and tolerance. In the mouse, a migratory cDC precursor (pre-CDC) originates from defined progenitors in the bone marrow (BM). Small numbers of short-lived pre-CDCs travel through the blood and replace cDCs in the peripheral organs, maintaining homeostasis of the highly dynamic cDC pool. However, the identity and distribution of the immediate precursor to human cDCs has not been defined. Using a tissue culture system that supports the development of human DCs, we identify a migratory precursor (hpre-CDC) that exists in human cord blood, BM, blood, and peripheral lymphoid organs. hpre-CDCs differ from premonocytes that are restricted to the BM. In contrast to earlier progenitors with greater developmental potential, the hpre-CDC is restricted to producing CD1c⁺ and CD141⁺ Clec9a⁺ cDCs. Studies in human volunteers demonstrate that hpre-CDCs are a dynamic population that increases in response to levels of circulating Flt3L.Conventional DCs (cDCs) induce immunity or tolerance by capturing, processing, and presenting antigen to T lymphocytes (Banchereau and Steinman, 1998). In the mouse, cDCs are short-lived cells, whose homeostasis in lymphoid and nonlymphoid tissues is critically dependent on continual replenishment from circulating pre-CDC (Liu et al., 2007; Liu and Nussenzweig, 2010). Murine pre-CDCs are BM-derived cells that are present in very small numbers in the blood but increase in response to Flt3L injection (Liu et al., 2007, 2009). pre-CDCs have a very short dwell time in the blood, 65% of these cells leave the circulation within 1 min after leaving the BM (Liu et al., 2007, 2009). Upon leaving the circulation, pre-CDCs seed tissues where they differentiate to cDCs, which divide further under the control of Flt3L (Liu et al., 2007, 2009). Thus, in addition to the BM and blood, mouse pre-CDCs are also found in peripheral lymphoid organs and nonlymphoid tissues (Naik et al., 2006; Bogunovic et al., 2009; Ginhoux et al., 2009; Liu et al., 2009; Varol et al., 2009).Mouse cDCs can be divided into two major subsets, CD11b⁺ DCs and CD8⁺/CD103⁺ DCs that differ in their microanatomic localization, cell surface antigen expression, antigen-processing activity, and ability to contribute to immune responses to specific pathogens (Merad et al., 2013; Murphy, 2013). Despite these important differences, both CD11b⁺ and CD8⁺/CD103⁺ cDC subsets of mouse DCs are derived from the same immediate precursor (pre-CDC) that expresses CD135 (Flt3), the receptor for Flt3L, a cytokine that is critical to DC development in vivo (McKenna et al., 2000; Waskow et al., 2008).Similar to the mouse, humans have two major subsets of cDCs. CD141 (BDCA3)⁺Clec9a⁺ DCs (CD141⁺ cDC herein) appear to be the human counterpart of mouse CD8⁺/CD103⁺ DCs, expressing XCR1, Clec9a, IRF8, and TLR3 and producing IL-12 (Robbins et al., 2008; Bachem et al., 2010; Crozat et al., 2010; Jongbloed et al., 2010; Poulin et al., 2010; Haniffa et al., 2012). CD1c (BDCA1)⁺ cDCs appear to be more closely related to mouse CD11b⁺ DCs, expressing IRF4, inducing Th17 differentiation upon A. fumigatus challenge, and imprinting intraepithelial homing of T cells (Robbins et al., 2008; Crozat et al., 2010; Schlitzer et al., 2013; Yu et al., 2013). In the mouse, the superior ability of CD8⁺/CD103⁺ DCs to cross-present exogenous antigens to CD8⁺ T cells is attributed to both differential antigen uptake (Kamphorst et al., 2010) and to increased expression of proteins and enzymes that facilitate MHC class I presentation (Dudziak et al., 2007). Human CD141⁺ cDCs are more efficient than CD1c⁺ cDCs in cross-presentation (Bachem et al., 2010; Crozat et al., 2010; Jongbloed et al., 2010; Poulin et al., 2010), but this difference appears to result from differences in antigen uptake and cytokine activation rather than a specialized cell-intrinsic program (Segura et al., 2012; Cohn et al., 2013; Nizzoli et al., 2013).Both CD1c⁺ cDCs and CD141⁺ cDCs are present in human blood and peripheral tissues. Each subset in the blood resembles its tissue counterpart in gene expression but appears less differentiated (Haniffa et al., 2012; Segura et al., 2012; Schlitzer et al., 2013). These observations are consistent with the idea that less differentiated human cDCs travel through the blood to replenish the cDC pool in the peripheral tissues (Collin et al., 2011; Segura et al., 2012; Haniffa et al., 2013). Others have postulated the existence of a less differentiated circulating DC progenitor based on absence of CD11c, expression of CD123, and response to Flt3L (O’Doherty et al., 1994; Pulendran et al., 2000), but the progenitor potential of these putative precursors that produced large amounts of IFN-α was never tested directly and they appear to correspond at least in part to plasmacytoid DCs (Grouard et al., 1997; Siegal et al., 1999). Thus, whether there is an immediate circulating precursor restricted to human immature and mature CD1c⁺ and CD141⁺ cDCs is not known.Here, we report the existence of a migratory pre-CDC in humans (hpre-CDC) that develops from committed DC progenitors (hCDPs) in the BM (Lee et al., 2015) and is the immediate precursor of both CD1c⁺ and CD141⁺ cDCs, but not pDCs or monocytes. hpre-CDCs are present in BM, cord, and peripheral blood, as well as peripheral lymphoid tissues. In studies of human volunteers, Flt3L injection induces expansion of hpre-CDCs in the circulation. Thus, human cDC precursors constitute a dynamic circulating population whose homeostasis is regulated by Flt3L, a cytokine that is responsive to inflammatory and infectious agents.     

Efficient transduction of mature CD83+ dendritic cells using recombinant adenovirus suppressed T cell stimulatory capacity

We have developed a culture method for the foreign serum-free generation of highly immunostimulatory, CD83+ human dendritic cells (DC). In this study, we evaluated the feasibility and consequences of endogenously expressing antigens in mature DC using adenoviral vectors. Transduction of DC with Ad-EGFP demonstrated endogenous fluorescence in 50-85% of CD83+ DC. Ad-transduced DC stimulated the proliferation of allogeneic CD8+ and CD4+ T cells at low DC: T cell ratios. However, at high DC: T cell ratios the stimulatory capacity of Ad-transduced DC was suppressed. This immunosuppressive effect was confirmed by demonstrating that the stimulatory function of untreated DC could be suppressed in a dose-dependent manner by addition of Ad-transduced DC. Furthermore, transwell experiments suggested that direct cell contact was required. Taken together, our results demonstrate the feasibility of efficiently expressing antigens in CD83+ DC using adenoviruses. However, immunosuppressive effects must be considered and carefully studied before Ad-transduced DC are employed for clinical trials. Gene Therapy (2000) 7, 249-254.     

小干扰RNA抑制成熟树突细胞表面抗原CD80/CD86的表达

背景：成熟树突细胞可通过其表面抗原CD80/CD86来启动Th细胞活化,促使Th细胞向Th1细胞分化减少、向Th2细胞方向分化增多。〈br〉 目的：探讨小干扰RNA抑制哮喘小鼠成熟树突细胞表面抗原CD80/CD86表达后对Th1及Th2型细胞因子干扰素γ、白细胞介素4分泌的影响。方法：建立哮喘小鼠模型,分离哮喘小鼠骨髓成熟树突细胞,流式细胞术检测表面标记物 CD11c、CD80、CD86的阳性表达率;设计合成CD80/CD86相关小干扰RNA转染哮喘小鼠成熟树突细胞,荧光定量PCR、流式细胞术检测干扰前后成熟树突细胞中CD80/CD86 mRNA和相应蛋白的表达,将干扰组、未干扰组、转染试剂对照组的成熟树突细胞分别与小鼠T淋巴细胞共培养,ELISA检测上清液中Th1及Th2型细胞因子干扰素γ、白细胞介素4分泌水平。结果与结论：①正常组成熟树突细胞的CD80/CD86的表达与哮喘组相比均明显降低（均P＜0.05）。②小干扰RNA转染哮喘小鼠成熟树突细胞后,干扰组CD80/CD86 mRNA及其蛋白表达水平较其他组均明显降低（均P＜0.05）。③小干扰RNA转染后干扰组共培养体系中干扰素γ水平明显高于其他组（均P＜0.05）,白细胞介素4水平则明显低于其他组（均P ＜0.05）。提示哮喘小鼠成熟树突细胞高表达CD80/CD86,小干扰RNA特异性抑制哮喘小鼠成熟树突细胞中CD80/CD86的表达,能增加干扰素γ并减少白细胞介素4的分泌,从而纠正Th1/Th2失衡。     

CD4+ CD25+ regulatory T cells control T helper cell type 1 responses to foreign antigens induced by mature dendritic cells in vivo

Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II-restricted interferon gamma-producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.     

In vivo depletion of CD4+CD25+ regulatory T cells enhances the antigen-specific primary and memory CTL response elicited by mature mRNA-electroporated dendritic cells.

We previously described mRNA electroporation as an efficient gene delivery method to introduce tumor-antigens (Ag) into murine immature dendritic cells (DC). Here, we further optimize the protocol and evaluate the capacity of mRNA-electroporated DC as a vaccine for immunotherapy. First, the early DC maturation kinetics and the effect of different lipopolysaccharide incubation periods on the phenotypic maturation profile of DC are determined. Next, we show that either immature or mature DC are equally well electroporated and express and present the transgene at a comparable level after electroporation. We point out that the mRNA electroporation results in a negative effect on the interleukin (IL)-12p70, IL-6, and tumor necrosis factor-alpha secretion after maturation. Nevertheless, mRNA-electroporated DC induce an effective cytotoxic T lymphocyte (CTL) response in vivo. Mature electroporated DC are significantly more potent in eliciting an Ag-specific CD8+ CTL response compared to their immature electroporated counterparts. In addition, a significant improvement in CTL response is obtained both in the primary and in the memory effector phases when CD4+CD25+ regulatory T cells (Treg) are depleted in vivo prior to immunization. These findings are further substantiated in tumor protection experiments and hold convincing evidence for the merit of Treg cell depletion prior to immunization with mRNA-electroporated DC.     

Generation of mature human myelomonocytic cells through expansion and differentiation of pluripotent stem cell–derived lin–CD34+CD43+CD45+ progenitors

Basic research into human mature myelomonocytic cell function, myeloid lineage diversification and leukemic transformation, and assessment of myelotoxicity in preclinical drug development requires a constant supply of donor blood or bone marrow samples and laborious purification of mature myeloid cells or progenitors, which are present in very small quantities. To overcome these limitations, we have developed a protocol for efficient generation of neutrophils, eosinophils, macrophages, osteoclasts, DCs, and Langerhans cells from human embryonic stem cells (hESCs). As a first step, we generated lin^–CD34⁺CD43⁺CD45⁺ hematopoietic cells highly enriched in myeloid progenitors through coculture of hESCs with OP9 feeder cells. After expansion in the presence of GM-CSF, these cells were directly differentiated with specific cytokine combinations toward mature cells of particular types. Morphologic, phenotypic, molecular, and functional analyses revealed that hESC-derived myelomonocytic cells were comparable to their corresponding somatic counterparts. In addition, we demonstrated that a similar protocol could be used to generate myelomonocytic cells from induced pluripotent stem cells (iPSCs). This technology offers an opportunity to generate large numbers of patient-specific myelomonocytic cells for in vitro studies of human disease mechanisms as well as for drug screening.     

Effects of pentoxifylline on differentiation, maturation, and function of human CD14+ monocyte-derived dendritic cells

Pentoxifylline (PTX) is a nonspecific phosphodiesterase inhibitor which has potent immunoregulatory and antiinflammatory effects. Although its immunomodulation property has been recognized, it is not clear whether PTX could affect dendritic cells (DCs), the most efficient antigen-presenting cells. The purpose of this study was to determine whether PTX could suppress DC differentiation, maturation, and its associated functions. Immature DCs (iDCs) were generated from human peripheral blood mononuclear cell CD14+ monocytes cultured with granulocyte macrophage colony stimulating factor and interleukin-4 for 5 days. PTX concentration-dependently suppressed the expression of iDC differentiation markers including CD54, CD80, CD86, and human leukocyte antigen-DR. In addition, PTX also inhibited DC maturation marker CD83 expression after stimulating DCs with lipopolysaccharide. Furthermore, PTX inhibited the antigen-uptake ability of DCs when tested by fluorescein isothiocyanate-dextran endocytosis assay. PTX significantly reduced the production of TNF-alpha and IFN-gamma in mature DCs (mDCs). Consequently, PTX-treated mDCs showed a reduced activity of mDC-induced T-cell allostimulation and proliferation by mixed-lymphocyte reaction (MLR) assay. Therefore, PTX significantly inhibits CD14+ monocyte-derived DC differentiation, maturation, antigen-uptake ability of iDCs, and antigen-presentation ability of mDCs possibly due to the suppression of TNF-alpha and IFN-gamma production. These results suggested that inhibitory effects of PTX on DCs may contribute its antiinflammatory and immunoregulatory functions.     

Making dendritic cells from the inside out: lentiviral vector-mediated gene delivery of granulocyte-macrophage colony-stimulating factor and interleukin 4 into CD14+ monocytes generates dendritic cells in vitro

We have evaluated a one-hit lentiviral transduction approach to genetically modifying monocytes in order to promote autocrine and paracrine production of factors required for their differentiation into immature dendritic cells (DCs). High-titer third-generation self-inactivating lentiviral vectors expressing granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) efficiently achieved simultaneous and persistent codelivery of the transgenes into purified human CD14+ monocytes. Coexpression of GM-CSF and IL-4 in CD14+ cells was sufficient to induce their differentiation into a DC-like phenotype, as evidenced by their morphology, immature immunophenotypic profile (CD14-, CD1a+, CD80+, CD86+, MHC-I+, MHC-II+), and their ability to further develop into a mature phenotype (CD83+) on further treatment with soluble CD40 ligand. Mixed lymphocyte reactions showed that the T cell-stimulating activity of lentivirus-modified DCs was superior to that of DCs grown by conventional methods. Lentivirus-modified DCs displayed efficient antigen-specific, MHC class I-restricted stimulation of autologous CD8+ T cells, as shown by IFN-gamma production and CTL assays. DCs coexpressing GM-CSF and IL-4 could be kept metabolically active and viable in culture for 14 days in the absence of exogenously added growth factors, unlike conventionally produced DCs. Coexpression of FLT3 ligand did not improve the viability, expansion, or immunologic performance of lentivirus-modified DCs. This article demonstrates the proof-of-concept to genetically convert monocytes to DC-type antigen-presenting cells with lentiviral vectors.     

Direct expansion of functional CD25+ CD4+ regulatory T cells by antigen-processing dendritic cells

An important pathway for immune tolerance is provided by thymic-derived CD25+ CD4+ T cells that suppress other CD25- autoimmune disease-inducing T cells. The antigen-presenting cell (APC) requirements for the control of CD25+ CD4+ suppressor T cells remain to be identified, hampering their study in experimental and clinical situations. CD25+ CD4+ T cells are classically anergic, unable to proliferate in response to mitogenic antibodies to the T cell receptor complex. We now find that CD25+ CD4+ T cells can proliferate in the absence of added cytokines in culture and in vivo when stimulated by antigen-loaded dendritic cells (DCs), especially mature DCs. With high doses of DCs in culture, CD25+ CD4+ and CD25- CD4+ populations initially proliferate to a comparable extent. With current methods, one third of the antigen-reactive T cell receptor transgenic T cells enter into cycle for an average of three divisions in 3 d. The expansion of CD25+ CD4+ T cells stops by day 5, in the absence or presence of exogenous interleukin (IL)-2, whereas CD25- CD4+ T cells continue to grow. CD25+ CD4+ T cell growth requires DC-T cell contact and is partially dependent upon the production of small amounts of IL-2 by the T cells and B7 costimulation by the DCs. After antigen-specific expansion, the CD25+ CD4+ T cells retain their known surface features and actively suppress CD25- CD4+ T cell proliferation to splenic APCs. DCs also can expand CD25+ CD4+ T cells in the absence of specific antigen but in the presence of exogenous IL-2. In vivo, both steady state and mature antigen-processing DCs induce proliferation of adoptively transferred CD25+ CD4+ T cells. The capacity to expand CD25+ CD4+ T cells provides DCs with an additional mechanism to regulate autoimmunity and other immune responses.     

不同树突状细胞对CD4~+CD25~+Foxp3~+调节性T细胞体外扩增的影响

目的利用不同种类树突状细胞(dendritic cells,DC)体外扩增获得表型和功能稳定的CD4+CD25+Foxp3+调节性T细胞(Treg)。方法免疫磁珠法(MACS)分离Balb/c小鼠CD4+CD25+调节性T细胞,利用与调节性T细胞同基因或异基因成熟DC、未成熟DC和调节性DC刺激其扩增,流式细胞术测定其纯度和表型。以CD4+CD25-T细胞作为反应细胞,验证扩增前后Treg细胞的免疫抑制功能。结果MACS分离的CD4+CD25+调节性T细胞纯度达到(95.38±1.82)%,同基因和异基因DC都能有效刺激Treg细胞体外扩增,其中同基因成熟DC扩增效果最为明显。而且同基因成熟DC扩增后CD4+CD25+调节性T细胞纯度达到(94.16±1.88)%,而且高表达Foxp3分子。当CD4+CD25+调节性T细胞与效应T细胞比例为1∶1时,能够有效的抑制效应T细胞的增殖,而且,同基因成熟DC扩增的CD4+CD25+调节性T细胞的抑制效果比新分离的Treg效果更好。结论同基因成熟DC能够体外扩增表型和功能稳定的Treg细胞。     

Freeze-thawing procedures have no influence on the phenotypic and functional development of dendritic cells generated from peripheral blood CD14+ monocytes

Little is known about the potential influence of cryopreservation on the biologic activities of dendritic cells (DCs). In this study, we examined the effects of freeze-thawing on the phenotypic and functional development of human DCs obtained from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood CD14+ cells. CD14+ cells were cultured, immediately or after freeze-thawing, with granulocyte-macrophage CSF and interleukin-4 for 9 days, and then with added tumor necrosis factor-alpha for another 3 days. For both fresh and freeze-thawed monocytes, immature DCs harvested on day 6 and mature DCs harvested on day 9 of culture were examined under the same conditions. Cells were compared with regard to their 1) capacities for antigen endocytosis and chemotactic migration (immature DCs), and 2) allogeneic mixed lymphocyte reaction and antigen-specific cytotoxic T lymphocyte responses (mature DCs). Freeze-thawing did not affect the viability or subsequent maturation of DCs at any stage of development. Furthermore, essentially no difference was observed in phenotype or function between cells generated from fresh or cryopreserved/thawed cells. Although this study design was limited with the use of fetal bovine serum, the observation still suggests that freeze-thawing does not affect viability, phenotype, subsequent maturation, or functions of DCs at any stage of maturation.     

Infusion of unpulsed dendritic cells derived from granulocyte/macrophage colony-stimulating factor-mobilized peripheral blood CD34+ cells and monocytes in patients with advanced carcinoma

Dendritic cells are potent antigen-presenting cells that are reduced in number and function in cancer patients. The infusion of dendritic cells pulsed with tumor-associated antigens has demonstrated antitumor immunologic activity. The effects of dendritic cells derived from granulocyte/macrophage colony-stimulating factor (GM-CSF)-mobilized peripheral blood CD34(+) cell and monocyte precursors when administered without antigen pulsing was examined. Patients with metastatic pancreatic and colorectal cancer received GM-CSF for 5 days. Blood was collected by a 250-ml phlebotomy. Dendritic cells were derived from CD34(+) cells with culture in GM-CSF, tumor necrosis factor-alpha, and serum-free media or from monocytes with culture in GM-CSF, interleukin-4, and autologous serum. From 2.0 to 9.4 x 10(6) dendritic cells were generated from CD34(+) cells and from 71 x 10(6) to 210 x 10(6) dendritic cells were generated from monocytes. Dendritic cells generated from CD34(+) cells expressed more CD1a than dendritic cells generated from monocytes; the ability to stimulate mixed lymphocyte reactions in vitro was not significantly different. Six patients received a single intravenous infusion of up to 5 x 10(6) autologous CD34(+) cell derived, and 6 patients, up to 50 x 10(6) monocyte-derived dendritic cells. The infusion was well tolerated. Increases in skin test reactivity and peripheral blood proliferative responses to the recall antigen, candida, were observed after the infusion of dendritic cells of both derivations. Changes in skin test reactivity and peripheral blood proliferative responses to tumor-associated peptides, including Ras and Muc1, were not. Significant numbers of functionally competent dendritic cells can be generated from patients with advanced carcinoma after GM-CSF mobilization. The infusion of these dendritic cells has nonspecific immunomodulatory activity that may have clinical significance.