CD8alpha+ dendritic cells originate from the CD8alpha- dendritic cell subset by a maturation process involving CD8alpha, DEC-205, and CD24 up-regulation.

CD8alpha+ and CD8alpha- dendritic cells (DCs) have been considered as independent DC subpopulations both ontogenetically and functionally during recent years. However, it has been demonstrated that both DC subsets can be generated from a single precursor population, supporting the concept that they do not represent separate DC lineages. By using highly purified splenic CD8alpha- DCs, which were injected intravenously and traced by means of an Ly5.1/Ly5.2 transfer system, this study shows that CD8alpha- DCs acquired the phenotypic characteristics of CD8alpha+ DCs, by a differentiation process involving CD8alpha, DEC-205, and CD24 up-regulation, paralleled by the down-regulation of CD11b, F4/80, and CD4. These data demonstrate that CD8alpha+ DCs derive from CD8alpha- DCs, and strongly support that CD8alpha- and CD8alpha+ DCs represent different maturation or differentiation stages of the same DC population. Therefore, CD8alpha+ DCs would represent the last stage of DC differentiation, playing an essential role in the induction of T-cell responses, due to their antigen-presenting potential, cross-priming ability, and capacity to secrete large amounts of key cytokines such as interferon gamma and interleukin-12.     

Differentiation of myeloid dendritic cells into CD8alpha-positive dendritic cells in vivo

Bone marrow-derived dendritic cells (DC) represent a family of antigen-presenting cells (APC) with varying phenotypes. For example, in mice, CD8alpha(+) and CD8alpha(-) DC are thought to represent cells of lymphoid and myeloid origin, respectively. Langerhans cells (LC) of the epidermis are typical myeloid DC; they do not express CD8alpha, but they do express high levels of myeloid antigens such as CD11b and FcgammaR. By contrast, thymic DC, which derive from a lymphoid-related progenitor, express CD8alpha but only low levels of myeloid antigens. CD8alpha(+) DC are also found in the spleen and lymph nodes (LN), but the origin of these cells has not been determined. By activating and labeling CD8alpha(-) epidermal LC in vivo, it was found that these cells expressed CD8alpha on migration to the draining LN. Similarly, CD8alpha(-) LC generated in vitro from a CD8 wild-type mouse and injected into the skin of a CD8alphaKO mouse expressed CD8alpha when they reached the draining LN. The results also show that CD8alpha(+) LC are potent APC. After migration from skin, they localized in the T-cell areas of LN, secreted high levels of interleukin-12, interferon-gamma, and chemokine-attracting T cells, and they induced antigen-specific T-cell activation. These results demonstrate that myeloid DC in the periphery can express CD8alpha when they migrate to the draining LN. CD8alpha expression on these DC appears to reflect a state of activation, mobilization, or both, rather than lineage. (Blood. 2000;96:1865-1872)     

Development of thymic and splenic dendritic cell populations from different hemopoietic precursors.

The antigen-presenting dendritic cells (DCs) found in mouse lymphoid tissues are heterogeneous. Several types of DCs have been identified on the basis of the expression of different surface molecules, including CD4, CD8alpha, and DEC-205. Previous studies by the authors showed that the mouse intrathymic lymphoid-restricted precursors (lin(-)c-kit(+)Thy-1(low)CD4(low)) can produce DCs in the thymus and spleen upon intravenous transfer, suggesting a lymphoid origin of these DCs. In the current study, the potential for DC production by the newly identified bone marrow (BM) common lymphoid precursors (CLPs), common myeloid precursors (CMPs), and committed granulocyte and macrophage precursors was examined. It was found that both the lymphoid and the myeloid precursors had the potential to produce DCs. All the different DC populations identified in mouse thymus and spleen could be produced by all these precursor populations. However, CLPs produced predominantly the CD4(-)CD8alpha(+) DCs, whereas CMPs produced similar numbers of CD4(-)CD8alpha(+) and CD4(+)CD8alpha(-) DCs, although at different peak times. On a per cell basis, the CLPs were more potent than the CMPs at DC production, but this may have been compensated for by an excess of CMPs over CLPs in BM. Overall, this study shows that the expression of CD8alpha does not delineate the hemopoietic precursor origin of DCs, and the nature of the early precursors may bias but does not dictate the phenotype of the DC product.     

Identification of CD8alpha+CD11c- lineage phenotype-negative cells in the spleen as committed precursor of CD8alpha+ dendritic cells

CD8alpha+ dendritic cells (DCs) represent a functionally distinct DC subset in vivo, which plays a critical role in initiating various cellular immune responses. However, the committed precursor of CD8alpha+ DCs remains to be identified. We reported here that murine splenic CD8alpha+CD11c- lineage phenotype (Lin)- cells could differentiate into CD8alpha+ DCs in vivo after intravenous transplantation. Immunohistochemistry staining showed that donor-derived DCs mainly located in T-cell areas of the spleen. Functionally, these CD8alpha+CD11c-Lin- cell-derived DCs were capable of stimulating allogenic T-cell response, as well as secreting bioactive interleukin 12 p70 and interferon gamma. Freshly isolated CD8alpha+CD11c-Lin- cells expressed CC chemokine receptor (CCR)2, CCR5, and CCR7 messenger RNA, whereas CD8alpha+ DCs derived from CD8alpha+CD11c-Lin- cells further obtained the expression of CCR6 and macrophage-derived chemokine. Flow cytometry analysis showed that CD8alpha+CD11c-Lin- cells were identified in bone marrow and lymph nodes. Moreover, transplanted splenic CD8alpha+CD11c-Lin- cells could also home to thymus and lymph nodes and were capable of developing into CD8alpha+ DCs in these locations. However, CD8alpha+CD11c-Li- cells failed to differentiate into CD8alpha- DCs, T cells, natural killer cells, or other myeloid lineage cells in irradiated chimeras. Taken together, all these findings suggest that CD8alpha+CD11c-Lin- cells are a committed precursor of CD8alpha+ DCs.     

Mouse CD11c(+) B220(+) Gr1(+) plasmacytoid dendritic cells develop independently of the T-cell lineage

The developmental origin of dendritic cells (DCs) is controversial. In the mouse CD8alpha(+) and CD8alpha(-) DC subsets are often considered to be of lymphoid and myeloid origin respectively, although evidence on this point is conflicting. Very recently a novel CD11c(+) B220(+) DC subset has been identified that appears to be the murine counterpart to interferon alpha (IFNalpha)-producing human plasmacytoid DCs (PDCs). We show here that CD11c(+) B220(+) mouse PDCs, like human PDCs, are present in the thymus and express T lineage markers such as CD8alpha and CD4. However, the intrathymic development of PDCs can be completely dissociated from immature T lineage cells in mixed chimeras established with bone marrow cells from mice deficient for either Notch-1 or T-cell factor 1, two independent mutations that severely block early T-cell development. Our data indicate that thymic PDCs do not arise from a bipotential T/DC precursor.     

CD8alpha+ mouse spleen dendritic cells do not originate from the CD8alpha- dendritic cell subset

Although previous studies had indicated that the CD8alpha- and CD8alpha+ subtypes of murine dendritic cells (DCs) differ in immediate origin, a recent study found that intravenous transfer of CD8alpha- DCs led to CD8alpha+ DCs in the spleen several days later, suggesting a direct precursor-product relationship. We have repeated these experiments with a balance sheet approach. We find that though a few CD8alpha+ DCs can be generated in such experiments, this is a rare event and could be the result of a contaminant precursor. Most of the immediate precursors of CD8alpha+ DCs are cells that lack the phenotype of a recognizable DC. CD8alpha- DCs and CD8alpha+ DCs are not precursor-product related, though these sublineages may be connected further upstream.     

Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression

Mouse spleen contains CD4+, CD8alpha+, and CD4-/CD8alpha- dendritic cells (DCs) in a 2:1:1 ratio. An analysis of 70 surface and cytoplasmic antigens revealed several differences in antigen expression between the 3 subsets. Notably, the Birbeck granule-associated Langerin antigen, as well as CD103 (the mouse homologue of the rat DC marker OX62), were specifically expressed by the CD8alpha+ DC subset. All DC types were apparent in the T-cell areas as well as in the splenic marginal zones and showed similar migratory capacity in collagen lattices. The 3 DC subtypes stimulated allogeneic CD4+ T cells comparably. However, CD8alpha+ DCs were very weak stimulators of resting or activated allogeneic CD8+ T cells, even at high stimulator-to-responder ratios, although this defect could be overcome under optimal DC/T cell ratios and peptide concentrations using CD8+ F5 T-cell receptor (TCR)-transgenic T cells. CD8alpha- or CD8alpha+ DCs presented alloantigens with the same efficiency for lysis by cytotoxic T lymphocytes (CTLs), and their turnover rate of class I-peptide complexes was similar, thus neither an inability to present, nor rapid loss of antigenic complexes from CD8alpha DCs was responsible for the low allostimulatory capacity of CD8alpha+ DCs in vitro. Surprisingly, both CD8alpha+ DCs and CD4-/CD8- DCs efficiently primed minor histocompatibility (H-Y male antigen) cytotoxicity following intravenous injection, whereas CD4+ DCs were weak inducers of CTLs. Thus, the inability of CD8alpha+ DCs to stimulate CD8+ T cells is limited to certain in vitro assays that must lack certain enhancing signals present during in vivo interaction between CD8alpha+ DCs and CD8+ T cells.     

Dendritic cell potentials of early lymphoid and myeloid progenitors

It has been proposed that there are at least 2 classes of dendritic cells (DCs), CD8alpha(+) DCs derived from the lymphoid lineage and CD8alpha(-) DCs derived from the myeloid lineage. Here, the abilities of lymphoid- and myeloid-restricted progenitors to generate DCs are compared, and their overall contributions to the DC compartment are evaluated. It has previously been shown that primitive myeloid-committed progenitors (common myeloid progenitors [CMPs]) are efficient precursors of both CD8alpha(+) and CD8alpha(-) DCs in vivo. Here it is shown that the earliest lymphoid-committed progenitors (common lymphoid progenitors [CLPs]) and CMPs and their progeny granulocyte-macrophage progenitors (GMPs) can give rise to functional DCs in vitro and in vivo. CLPs are more efficient in generating DCs than their T-lineage descendants, the early thymocyte progenitors and pro-T cells, and CMPs are more efficient DC precursors than the descendant GMPs, whereas pro-B cells and megakaryocyte-erythrocyte progenitors are incapable of generating DCs. Thus, DC developmental potential is preserved during T- but not B-lymphoid differentiation from CLP and during granulocyte-macrophage but not megakaryocyte-erythrocyte development from CMP. In vivo reconstitution experiments show that CLPs and CMPs can reconstitute CD8alpha(+) and CD8alpha(-) DCs with similar efficiency on a per cell basis. However, CMPs are 10-fold more numerous than CLPs, suggesting that at steady state, CLPs provide only a minority of splenic DCs and approximately half the DCs in thymus, whereas most DCs, including CD8alpha(+) and CD8alpha(-) subtypes, are of myeloid origin. (Blood. 2001;97:3333-3341)     

Effect of aging on bone marrow-derived murine CD11c+CD4-CD8alpha- dendritic cell function

Dendritic cells (DCs) are actively used as cellular adjuvant in cancer immunotherapy. However, although DC immunotherapies primarily target the elderly population, little is known about the effect of aging on DC functions. Here, we compared the T-cell stimulation, cytokine production, and tumor surveillance functions of bone marrow-derived CD11c(+)CD4(-)CD8alpha(-) DCs of old and young C57BL/6 mice. Old immature bone marrow-derived CD4(-)CD8alpha(-) DCs (imDCs) were 4 times less effective than were young DCs in stimulating syngeneic CD4(+) T-cell proliferation. Old imDCs also have decreased DC-specific/intracellular adhesion molecule type 3-grabbing, nonintegrin (DC-SIGN) expression compared to young DCs. Interestingly, mice treated with the ovalbumin peptide-pulsed young DCs exhibited significantly greater tumor regression than with ovalbumin peptide-pulsed old DCs. Old terminally differentiated bone marrow-derived DCs (tDC) also have increased interleukin-10, but decreased interleukin-6 and tumor necrosis factor-alpha production. Taken together, these results have important implications in the clinical application of DC-based tumor immunotherapy in elderly persons.     

CD8alpha-11b+ dendritic cells but not CD8alpha+ dendritic cells mediate cross-tolerance toward intestinal antigens

Cross-presentation is a critical process by which antigen is displayed to CD8 T cells to induce tolerance. It is believed that CD8alpha+ dendritic cells (DCs) are responsible for cross-presentation, suggesting that the CD8alpha+ DC population is capable of inducing both cross-priming and cross-tolerance to antigen. We found that cross-tolerance against intestinal soluble antigen was abrogated in C57BL/6 mice lacking mesenteric lymph nodes (MLNs) and Peyer patches (PPs), whereas mice lacking PPs alone were capable of developing CD8 T-cell tolerance. CD8alpha-CD11b+ DCs but not CD8alpha+ DCs in the MLNs present intestinal antigens to relevant CD8 T cells, while CD8alpha+ DCs but not CD8alpha-CD11b+ DCs in the spleen exclusively cross-present intravenous soluble antigen. Thus, CD8alpha-CD11b+ DCs in the MLNs play a critical role for induction of cross-tolerance to dietary proteins.     

Impaired generation of bone marrow CD34-derived dendritic cells with low peripheral blood subsets in patients with myelodysplastic syndrome

Myelodysplastic syndrome (MDS) is a stem cell disorder characterized by ineffective haematopoiesis and blood cytopenias. The present study investigated the potential of bone marrow CD34(+) progenitors in MDS patients to proliferate and differentiate into dendritic cells (DCs) in a cytokine-supplemented liquid culture system and analysed the status of blood DC subsets in these patients. CD34(+) progenitors had low potential to generate DCs in vitro, as the number of DCs obtained from one CD34(+) cell was significantly lower compared with controls (median value 0.2 vs. 4, P = 0.003). In patients, the survival and proliferation of CD34(+) cells in culture was not correlated to the degree of apoptosis. Phenotypically and functionally CD34(+)-derived DCs were similar in MDS patients and normal subjects. The percentage of both circulating DC subsets in patients was extremely diminished compared with controls (myeloid DC: 0.10 +/- 0.10% vs. 0.35 +/- 0.13%, P < 0.001; plasmacytoid DC: 0.11 +/- 0.10% vs. 0.37 +/- 0.14%, P < 0.001). In cases with the 5q deletion both CD34-derived DCs and blood DCs harboured the cytogenetic abnormality. Our results indicate that, in MDS, the production of DCs is affected by the neoplastic process resulting in ineffective 'dendritopoiesis' with low blood DC precursor numbers. This quantitative DC defect probably contributes to the poor immune response against infectious agents and to the escape of the malignant clone from immune recognition with disease progression towards acute leukaemia.     

Vasoactive intestinal peptide generates human tolerogenic dendritic cells that induce CD4 and CD8 regulatory T cells

Induction of antigen-specific tolerance is critical for autoimmunity prevention and immune tolerance maintenance. In addition to their classical role as sentinels of the immune response, dendritic cells (DCs) play important roles in maintaining peripheral tolerance through the induction/activation of regulatory T (T(reg)) cells. The possibility of generating tolerogenic DCs opens new therapeutic perspectives in autoimmune/inflammatory diseases. Characterizing endogenous factors that contribute to the development of tolerogenic DCs is highly relevant. We here report that the immunosuppressive neuropeptide vasoactive intestinal peptide (VIP) induces the generation of human tolerogenic DCs with the capacity to generate CD4 and CD8 T(reg) cells from their respective naive subsets. The presence of VIP during the early stages of DC differentiation from blood monocytes generates a population of IL-10-producing DCs unable to fully mature after the effects of inflammatory stimuli. CD4 T(reg) cells generated with VIP-differentiated DCs resemble the previously described Tr1 cells in terms of phenotype and cytokine profile. CD8 T(reg) cells generated with tolerogenic VIP DCs have increased numbers of IL-10-producing CD8(+)CD28(-)-CTLA4(+) T cells. CD4 and CD8 T(reg) cells primarily suppress antigen-specific T(H)1-mediated responses. Therefore, the possibility of generating or expanding ex vivo tolerogenic DC(VIPs) opens new therapeutic perspectives for treating autoimmune diseases and graft-versus-host disease after allogeneic transplantation in humans.     

Myeloid blood CD11c(+) dendritic cells and monocyte-derived dendritic cells differ in their ability to stimulate T lymphocytes

Dendritic cells (DCs) initiate and direct immune responses. Recent studies have defined different DC populations, therefore we undertook this study comparing 2 types of myeloid DCs: blood CD11c(+) DCs and in vitro monocyte-derived DCs (Mo-DCs), which are both candidates as cellular adjuvants for cancer immunotherapy. Blood CD11c(+) DCs were prepared by cell sorting from peripheral blood mononuclear cells cultured overnight in RPMI 1640 medium supplemented with autologous or pooled AB serum. Mo-DCs were prepared in the same medium using granulocyte macrophage-colony-stimulating factor (GM-CSF)/interleukin 4 (IL-4) and differentiated/activated with lipopolysaccharide or monocyte-conditioned medium (ActMo-DCs). Morphologically, differences between the DC preparations were noted both at a light and and electron microscopic level. Blood CD11c(+) DCs expressed similar levels of HLA-DR, CD40, CD86, and CD83 as Mo-DCs. CD209 was present on Mo-DCs but not on blood CD11c(+) DCs. Blood CD11c(+) DCs generated a lower proliferative mixed leukocyte response (MLR) than Mo-DCs. Blood CD11c(+) DCs loaded with 0.1 microg/mL tetanus toxoid (TT)-generated greater T lymphocyte proliferative responses than did Mo-DCs or ActMo-DCs, but when loaded with higher TT concentrations no difference in T lymphocyte proliferative response was observed. Keyhole limpet hemocyanin (KLH)-loaded blood CD11c(+) DCs generated greater T lymphocyte proliferative responses than Mo-DCs or ActMo-DCs. Allogeneic MLR- or KLH-specific responses induced by blood CD11c(+) DCs generated more Th1 effectors than the responses induced by Mo-DCs or ActMo-DCs. These data establish several differences in the properties of blood CD11c(+) DCs, Mo-DCs, and ActMo-DCs, which suggest that blood DCs merit further consideration as DC preparations for clinical programs are evolved.