Differential rates of replacement of human dermal dendritic cells and macrophages during hematopoietic stem cell transplantation

Animal models of hematopoietic stem cell transplantation have been used to analyze the turnover of bone marrow–derived cells and to demonstrate the critical role of recipient antigen-presenting cells (APC) in graft versus host disease (GVHD). In humans, the phenotype and lineage relationships of myeloid-derived tissue APC remain incompletely understood. It has also been proposed that the risk of acute GVHD, which extends over many months, is related to the protracted survival of certain recipient APC. Human dermis contains three principal subsets of CD45⁺HLA-DR⁺ cells: CD1a⁺CD14⁻ DC, CD1a⁻CD14⁺ DC, and CD1a⁻CD14⁺FXIIIa⁺ macrophages. In vitro, each subset has characteristic properties. After transplantation, both CD1a⁺ and CD14⁺ DC are rapidly depleted and replaced by donor cells, but recipient macrophages can be found in GVHD lesions and may persist for many months. Macrophages isolated from normal dermis secrete proinflammatory cytokines. Although they stimulate little proliferation of naive or memory CD4⁺ T cells, macrophages induce cytokine expression in memory CD4⁺ T cells and activation and proliferation of CD8⁺ T cells. These observations suggest that dermal macrophages and DC are from distinct lineages and that persistent recipient macrophages, although unlikely to initiate alloreactivity, may contribute to GVHD by sustaining the responses of previously activated T cells.     

Myeloid cell–targeted STAT3 inhibition sensitizes head and neck cancers to radiotherapy and T cell–mediated immunity

The tumor microenvironment affects the outcome of radiotherapy against head and neck squamous cell carcinoma (HNSCC). We recently found that tolerogenic myeloid cells accumulate in the circulation of HNSCC patients undergoing radiotherapy. Here, we analyzed tumor-containing lymph node biopsies collected from these patients. After 2 weeks of radiotherapy, we found an increase in tumor-associated macrophages (TAMs) with activated STAT3, while CD8⁺ T cells were reduced as detected using multiplex IHC. Gene expression profiling indicated upregulation of M2 macrophage–related genes (CD163, CD206), immunosuppressive mediators (ARG1, LIF, TGFB1), and Th2 cytokines (IL4, IL5) in irradiated tumors. We next validated STAT3 as a potential target in human HNSCC-associated TAMs, using UM-SCC1 xenotransplants in humanized mice. Local injections of myeloid cell–targeted STAT3 antisense oligonucleotide (CpG-STAT3ASO) activated human DCs/macrophages and promoted CD8⁺ T cell recruitment, thereby arresting UM-SCC1 tumor growth. Furthermore, CpG-STAT3ASO synergized with tumor irradiation against syngeneic HPV⁺ mEERL and HPV^– MOC2 HNSCC tumors in mice, triggering tumor regression and/or extending animal survival. The antitumor immune responses were CD8⁺ and CD4⁺ T cell dependent and associated with the activation of antigen-presenting cells (DCs/M1 macrophages) and increased CD8⁺ to regulatory T cell ratio. Our observations suggest that targeted inhibition of STAT3 in tumor-associated myeloid cells augments the efficacy of radiotherapy against HNSCC.     

Uptake of Leishmania major Amastigotes Results in Activation and Interleukin 12 Release from Murine Skin–derived Dendritic Cells: Implications for the Initiation of Anti-Leishmania Immunity

Epidermal Langerhans cells (LC) are immature dendritic cells (DC) located in close proximity to the site of inoculation of infectious Leishmania major metacyclic promastigotes by sand flies. Using LC-like DC expanded from C57BL/6 fetal skin, we characterized interactions involving several developmental stages of Leishmania and DC. We confirmed that L. major amastigotes, but not promastigotes, efficiently entered LC-like DC. Parasite internalization was associated with activation manifested by upregulation of major histocompatibility complex (MHC) class I and II surface antigens, increased expression of costimulatory molecules (CD40, CD54, CD80, and CD86), and interleukin (IL)-12 p40 release within 18 h. L. major–induced IL-12 p70 release by DC required interferon γ and prolonged (72 h) incubation. In contrast, infection of inflammatory macrophages (Mφ) with amastigotes or promastigotes did not lead to significant changes in surface antigen expression or cytokine production. These results suggest that skin Mφ and DC are infected sequentially in cutaneous leishmaniasis and that they play distinct roles in the inflammatory and immune response initiated by L. major. Mφ capture organisms near the site of inoculation early in the course of infection after establishment of cellular immunity, and kill amastigotes but probably do not actively participate in T cell priming. In contrast, skin DC are induced to express increased amounts of MHC antigens and costimulatory molecules and to release cytokines (including IL-12 p70) by exposure to L. major amastigotes that ultimately accumulate in lesional tissue, and thus very likely initiate protective T helper cell type 1 immunity.     

Inherited human c-Rel deficiency disrupts myeloid and lymphoid immunity to multiple infectious agents

We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8⁺ T cells, memory CD4⁺ T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4⁺ T cells. In naive CD4⁺ T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient’s naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.     

Treg-mediated suppression of atherosclerosis requires MYD88 signaling in DCs

TLR activation on CD11c⁺ DCs triggers DC maturation, which is critical for T cell activation. Given the expansion of CD11c⁺ DCs during the progression of atherosclerosis and the key role of T cell activation in atherogenesis, we sought to understand the role of TLR signaling in CD11c⁺ DCs in atherosclerosis. To this end, we used a mouse model in which a key TLR adaptor involved in DC maturation, MYD88, is deleted in CD11c⁺ DCs. We transplanted bone marrow containing Myd88-deficient CD11c⁺ DCs into Western diet–fed LDL receptor knockout mice and found that the transplanted mice had decreased activation of effector T cells in the periphery as well as decreased infiltration of both effector T cells and Tregs in atherosclerotic lesions. Surprisingly, the net effect was an increase in atherosclerotic lesion size due to an increase in the content of myeloid-derived inflammatory cells. The mechanism involves increased lesional monocyte recruitment associated with loss of Treg-mediated suppression of MCP-1. Thus, the dominant effect of MYD88 signaling in CD11c⁺ DCs in the setting of atherosclerosis is to promote the development of atheroprotective Tregs. In the absence of MYD88 signaling in CD11c⁺ DCs, the loss of this protective Treg response trumps the loss of proatherogenic T effector cell activation.     

Vα24-invariant NKT cells mediate antitumor activity via killing of tumor-associated macrophages

Tumor infiltration with Vα24-invariant NKT cells (NKTs) associates with favorable outcome in neuroblastoma and other cancers. Although NKTs can be directly cytotoxic against CD1d⁺ cells, the majority of human tumors are CD1d^–. Therefore, the role of NKTs in cancer remains largely unknown. Here, we demonstrate that CD68⁺ tumor-associated monocytes/macrophages (TAMs) represented the majority of CD1d-expressing cells in primary human neuroblastomas. TAMs stimulated neuroblastoma growth in human cell lines and their xenografts in NOD/SCID mice via IL-6 production. Indeed, TAMs produced IL-6 in primary tumors and in the BM of patients with metastatic neuroblastoma. Gene expression analysis using TaqMan low-density arrays of 129 primary human neuroblastomas without MYCN amplification revealed that high-level expression of TAM-specific genes (CD14, CD16, IL6, IL6R, and TGFB1) was associated with poor 5-year event-free survival. While NKTs were not cytotoxic against neuroblastoma cells, they effectively killed monocytes pulsed with tumor cell lysate. The killing of monocytes was CD1d restricted because it was inhibited by a CD1d-specific mAb. Cotransfer of human monocytes and NKTs to tumor-bearing NOD/SCID mice decreased monocyte number at the tumor site and suppressed tumor growth compared with mice transferred with monocytes alone. Thus, killing of TAMs reveals what we believe to be a novel mechanism of NKT antitumor activity that relates to the disease outcome.     

Combined implantation of CD34+ and CD14+ cells increases neovascularization through amplified paracrine signalling

Cell therapy strategies that use adult peripheral blood‐derived CD34⁺ progenitor cells are hampered by low cell numbers and the infrequent cellular incorporation into the neovasculature. Hence, the use of CD34⁺ cells to treat ischaemic diseases is under debate. Interaction between CD34⁺ cells and CD14⁺ cells results in superior endothelial differentiation of CD14⁺ cells in vitro, indicating that cell therapy approaches utilizing both CD34⁺ and CD14⁺ cells may be advantageous in therapeutic neovascularization. Here, human CD34⁺ and CD14⁺ cells were isolated from adult peripheral blood and implanted subcutaneously into nude mice, using matrigel as the carrier. Combined implantation of human CD34⁺ and CD14⁺ cells resulted in superior neovascularization, compared to either cell type alone, albeit incorporation of human cells into the murine vasculature was not observed. Human CD34⁺ and CD14⁺ cells produced and secreted a pentad of pro‐angiogenic mediators, such as HGF, MCP‐1 and IL‐8, bFGF and VEGFa in monoculture. The production and secretion of pro‐angiogenic mediators by CD14⁺ cells was highly amplified upon incubation with conditioned medium from CD34⁺ cells. In vivo, neovascularization of matrigel implants did not rely on the endothelial differentiation and incorporation of CD34⁺ or CD14⁺ cells, but depended on the paracrine effects of IL‐8, MCP‐1, HGF, bFGF and VEGFa secreted by implanted cells. Administration of this growth factor/cytokine pentad using matrigel as a carrier results in cell recruitment and microvessel formation equal to progenitor cell‐induced neovascularization. These data provide new insights on neovascularization by cell therapy and may contribute to new strategies for the treatment of ischaemic diseases. Copyright © 2011 John Wiley & Sons, Ltd.     

Metabolic programming and PDHK1 control CD4+ T cell subsets and inflammation

Activation of CD4⁺ T cells results in rapid proliferation and differentiation into effector and regulatory subsets. CD4⁺ effector T cell (Teff) (Th1 and Th17) and Treg subsets are metabolically distinct, yet the specific metabolic differences that modify T cell populations are uncertain. Here, we evaluated CD4⁺ T cell populations in murine models and determined that inflammatory Teffs maintain high expression of glycolytic genes and rely on high glycolytic rates, while Tregs are oxidative and require mitochondrial electron transport to proliferate, differentiate, and survive. Metabolic profiling revealed that pyruvate dehydrogenase (PDH) is a key bifurcation point between T cell glycolytic and oxidative metabolism. PDH function is inhibited by PDH kinases (PDHKs). PDHK1 was expressed in Th17 cells, but not Th1 cells, and at low levels in Tregs, and inhibition or knockdown of PDHK1 selectively suppressed Th17 cells and increased Tregs. This alteration in the CD4⁺ T cell populations was mediated in part through ROS, as N-acetyl cysteine (NAC) treatment restored Th17 cell generation. Moreover, inhibition of PDHK1 modulated immunity and protected animals against experimental autoimmune encephalomyelitis, decreasing Th17 cells and increasing Tregs. Together, these data show that CD4⁺ subsets utilize and require distinct metabolic programs that can be targeted to control specific T cell populations in autoimmune and inflammatory diseases.     

Anti-TNF immunotherapy reduces CD8+ T cell–mediated antimicrobial activity against Mycobacterium tuberculosis in humans

The incidence of tuberculosis is increased during treatment of autoimmune diseases with anti-TNF antibodies. This is a significant clinical complication, but also provides a unique model to study immune mechanisms in human tuberculosis. Given the key role for cell-mediated immunity in host defense against Mycobacterium tuberculosis, we hypothesized that anti-TNF treatment impairs T cell–directed antimicrobial activity. Anti-TNF therapy reduced the expression in lymphocytes of perforin and granulysin, 2 components of the T cell–mediated antimicrobial response to intracellular pathogens. Specifically, M. tuberculosis–reactive CD8⁺CCR7^–CD45RA⁺ effector memory T cells (T_EMRA cells) expressed the highest levels of granulysin, lysed M. tuberculosis, and infected macrophages and mediated an antimicrobial activity against intracellular M. tuberculosis. Furthermore, T_EMRA cells expressed cell surface TNF and bound the anti-TNF therapeutic infliximab in vitro, making them susceptible to complement-mediated lysis. Immune therapy with anti-TNF was associated with reduced numbers of CD8⁺ T_EMRA cells and decreased antimicrobial activity against M. tuberculosis, which could be rescued by the addition of CD8⁺ T_EMRA cells. These results suggest that anti-TNF therapy triggers a reduction of CD8⁺ T_EMRA cells with antimicrobial activity against M. tuberculosis, providing insight into the mechanism whereby key effector T cell subsets contribute to host defense against tuberculosis.     

Granulocyte colony‐stimulating factor produces a decrease in IFNγ and increase in IL‐4 when administrated to healthy donors

Hematopoietic stem cells transplantation (HSCT) is the leading curative therapy for a variety of hematological and hereditary diseases; however, graft versus host disease (GVHD), an immunologic phenomenon that is favored by Th1 cytokines and cytotoxic cells from donors, is present frequently and is one of the most important causes of transplant related mortality. Peripheral blood HSCT is the preferred source of stem cells in almost 100% of the cases of autologous HSCT and in 70% of allogeneic transplants. The best mobilizing agent to get the stem cells out from the bone marrow is the Granulocyte‐Colony Stimulating Factor (G‐CSF). In this work, our main objective was to study a possible correlation between the graft cell dose and the patient's clinical outcome. We evaluated the immunologic changes produced by G‐CSF in the lymphocyte and cytokine profiles in allogeneic HSC donors. HSC from twelve donors were mobilized with G‐CSF at 16 μg/kg/day, for 5 days. Basal Peripheral Blood (BPB), Mobilized Peripheral Blood (MPB), and aphaeresis mononuclear cells (G‐MNC) samples were taken from all donors. Using flow cytometry, we quantified CD19⁺, CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, NK, NKT, DC1, and DC2 cells. Cytokines were determined by ELISA in culture supernatants. CD19⁺ (p = 0.001), DC1 (p < 0.002) and DC2 (p < 0.001) cells were increased in MPB with respect to BPB. An increase in Th2 cytokines such as (IL‐4) and a decrease in Th1 cytokines (IFNγ, IL‐2) were also found in MPB samples. In conclusion, Th1 and Th2 cytokines are relevant in predicting the clinical outcome after allogeneic peripheral blood HSCT. J. Clin. Apheresis 25:181–187, 2010. © 2010 Wiley‐Liss, Inc.     

The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α+ dendritic cells

Human BDCA3⁺ dendritic cells (DCs) were suggested to be homologous to mouse CD8α⁺ DCs. We demonstrate that human BDCA3⁺ DCs are more efficient than their BDCA1⁺ counterparts or plasmacytoid DCs (pDCs) in cross-presenting antigen and activating CD8⁺ T cells, which is similar to mouse CD8α⁺ DCs as compared with CD11b⁺ DCs or pDCs, although with more moderate differences between human DC subsets. Yet, no specific marker was known to be shared between homologous DC subsets across species. We found that XC chemokine receptor 1 (XCR1) is specifically expressed and active in mouse CD8α⁺, human BDCA3⁺, and sheep CD26⁺ DCs and is conserved across species. The mRNA encoding the XCR1 ligand chemokine (C motif) ligand 1 (XCL1) is selectively expressed in natural killer (NK) and CD8⁺ T lymphocytes at steady-state and is enhanced upon activation. Moreover, the Xcl1 mRNA is selectively expressed at high levels in central memory compared with naive CD8⁺ T lymphocytes. Finally, XCR1^−/− mice have decreased early CD8⁺ T cell responses to Listeria monocytogenes infection, which is associated with higher bacterial loads early in infection. Therefore, XCR1 constitutes the first conserved specific marker for cell subsets homologous to mouse CD8α⁺ DCs in higher vertebrates and promotes their ability to activate early CD8⁺ T cell defenses against an intracellular pathogenic bacteria.DCs are central to immune defenses in mammals. In mice, three subsets of DCs are resident of lymphoid organs (Crozat et al., 2010). Plasmacytoid DCs (pDCs) are professional producers of IFN-α and -β, contributing to immune defenses against viruses (Baranek et al., 2009). CD11b⁺ DCs preferentially prime CD4⁺ T cells and promote humoral immunity (Carter et al., 2006; Dudziak et al., 2007). CD8α⁺ DCs are endowed with a unique efficiency in priming CD8⁺ T cells and in cross-presenting exogenous antigens (Carter et al., 2006; Dudziak et al., 2007). CD8α⁺ DCs are required for the natural induction of strong CD8⁺ T cell responses against tumors (Hildner et al., 2008; Sancho et al., 2008) or West Nile virus (Hildner et al., 2008). Specific delivery of vaccine antigens to CD8α⁺ DCs is especially efficient for vaccination against intracellular pathogens or tumors (Bonifaz et al., 2004; Nchinda et al., 2008). Therefore, identification of human DC subsets functionally homologous to mouse CD8α⁺ DCs (CD8α⁺-type DCs) should be a major step forward for the design of innovative vaccination or immunotherapeutic strategies against cancer or infections (Crozat et al., 2010). So far, no conserved marker has been identified to specifically and unambiguously define CD8α⁺-type DCs in several mammalian species. Although human BDCA3⁺ and mouse CD8α⁺ DCs express the C-type lectin CLEC9A, which is known to be involved in cross-presentation in mice, this marker is also found on some human CD14⁺ monocytes and on mouse pDCs (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008). It is of note that no orthologue of CLEC9A has been identified yet in non mammalian vertebrate species.We have recently performed comparative genomics studies of human, mouse, and sheep DC subsets to help identify potential homologies between these cell types across mammalian species (Robbins et al., 2008; unpublished data). We found that human blood BDCA3⁺ DCs share a specific gene signature with mouse CD8α⁺ DCs and proposed that they could be human professional cross-presenting DCs (Robbins et al., 2008; Crozat et al., 2010). In this paper, we demonstrate that human BDCA3⁺ DCs are more potent than their BDCA1⁺ counterparts or than pDCs for CD8⁺ T cell activation through antigen cross-presentation. We established elsewhere that sheep lymph CD26⁺ DCs (Epardaud et al., 2004) are also equivalents to mouse CD8α⁺ DCs based on gene expression and functions such as superior efficacy for presentation of soluble antigen to CD8⁺ T cells (unpublished data). In this paper, we identify the XC chemokine receptor 1 (XCR1) as the first universal marker specifically expressed by the CD8α⁺-type DCs from three different mammalian species: ovine CD26⁺ DCs, mouse CD8α⁺ DCs, and human BDCA3⁺ DCs. We show that the Xcr1 gene is present and well conserved in all higher vertebrates from reptiles to human. The ligand of XCR1, chemokine (C motif) ligand 1 (XCL1), is specifically expressed by activated NK and CD8⁺ T cells in mouse and human. We show that Xcl1 mRNA is stored selectively in memory CD8⁺ T cells, allowing them to rapidly produce high levels of this chemokine upon stimulation. Finally, we show that XCR1^−/− mice have decreased CD8⁺ T cell responses to Listeria monocytogenes (Lm) associated with higher bacterial loads early after infection. Overall, our study strongly suggests an important and conserved role in mammals for XCR1 in the cross talk between NK or CD8⁺ T cells and CD8α⁺-type DCs, identifying this molecule as a novel tool to survey and target the DCs endowed with the best cross-presentation capacity across species.     

Galectin-9 prolongs the survival of septic mice by expanding tim-3-expressing natural killer T cells and PDCA-1+ CD11c+ macrophages

Introduction

Galectin-9 ameliorates various inflammatory conditions including autoimmune diseases by regulating T cell and macrophage/dendritic cell (DC) functions. However, the effect of galectin-9 on polymicrobial sepsis has not been assessed.

Methods

We induced polymicrobial sepsis by cecal ligation and puncture (CLP) in mice. The survival rate was compared between galectin-9- and PBS-treated CLP mice. An ELISA was used to compare the levels of various cytokines in the plasma and culture supernatants. Fluorescence-activated cell sorting analysis was further performed to compare the frequencies of subpopulations of spleen cells.

Results

Galectin-9 exhibited a protective effect in polymicrobial sepsis as demonstrated in galetin-9 transgenic mice and therapeutic galectin-9 administration. In contrast, such effect was not observed in nude mice, indicating the involvement of T cells in galectin-9-mediated survival prolongation. Galectin-9 decreased TNFα, IL-6, IL-10 and, high mobility group box 1 (HMGB1) and increased IL-15 and IL-17 plasma and spleen levels. Galectin-9 increased the frequencies of natural killer T (NKT) cells and PDCA-1⁺ CD11c⁺ macrophages (pDC-like macrophages) but did not change the frequency of CD4 or CD8 T cells, γδT cells or conventional DC. As expected, galectin-9 decreased the frequency of Tim-3⁺ CD4 T cells, most likely Th1 and Th17 cells. Intriguingly, many spleen NK1.1⁺ NKT cells and pDC-like macrophages expressed Tim-3. Galectin-9 increased the frequency of Tim-3-expressing NK1.1⁺ NKT cells and pDC-like macrophages. Galectin-9 further increased IL-17⁺ NK1.1⁺ NKT cells.

Conclusion

These data suggest that galectin-9 exerts therapeutic effects on polymicrobial sepsis, possibly by expanding NKT cells and pDC-like macrophages and by modulating the production of early and late proinflammatory cytokines.     

Altered innate immune profile in blood of systemic mastocytosis patients

BackgroundMast cells (MC) from systemic mastocytosis (SM) patients release MC mediators that lead to an altered microenvironment with potential consequences on innate immune cells, such as monocytes and dendritic cells (DC). Here we investigated the distribution and functional behaviour of different populations of blood monocytes and DC among distinct diagnostic subtypes of SM.MethodsOverall, we studied 115 SM patients ‐ 45 bone marrow mastocytosis (BMM), 61 indolent SM (ISM), 9 aggressive SM (ASM)‐ and 32 healthy donors (HD). Spontaneous and in vitro‐stimulated cytokine production by blood monocytes, and their plasma levels, together with the distribution of different subsets of blood monocytes and DCs, were investigated.ResultsSM patients showed increased plasma levels and spontaneous production by blood monocytes of IL1β, IL6, IL8, TNFα and IL10, associated with an exhausted ability of LPS + IFNγ‐stimulated blood monocytes to produce IL1β and TGFβ. SM (particularly ISM) patients also showed decreased counts of total monocytes, at the expense of intermediate monocytes and non‐classical monocytes. Interestingly, while ISM and ASM patients had decreased numbers of plasmacytoid DC and myeloid DC (and their major subsets) in blood, an expansion of AXL⁺ DC was specifically encountered in BMM cases.ConclusionThese results demonstrate an altered distribution of blood monocytes and DC subsets in SM associated with constitutive activation of functionally impaired blood monocytes and increased plasma levels of a wide variety of inflammatory cytokines, reflecting broad activation of the innate immune response in mastocytosis.     

Immunomodulatory effect of human bone marrow‐derived mesenchymal stromal/stem cells on peripheral blood T cells from rheumatoid arthritis patients

Rheumatoid arthritis (RA) is a Th1/Th17‐mediated autoimmune disease whose current treatment, consisting in the blockage of inflammatory cytokines by disease‐modifying antirheumatic drugs, is not effective for all patients. The therapeutic potential of mesenchymal stromal/stem cells' (MSCs) immunomodulatory properties is being explored in RA. Here, we investigate the effect of human bone marrow (BM)‐MSCs on the expression of cytokines involved in RA physiopathology by the distinct functional compartments of CD4⁺ and CD8⁺ T cells from RA patients. Peripheral blood mononuclear cells from healthy individuals (n = 6) and RA patients (n = 12) were stimulated with phorbol myristate acetate plus ionomycin and cultured in the presence/absence of BM‐MSCs. The expression of (interleukin) IL‐2, tumor necrosis factor alpha (TNF‐α), and interferon‐gamma (IFN‐γ) was evaluated in naive, central memory, effector memory, and effector CD4⁺ and CD8⁺ T cells, whereas IL‐6, IL‐9, and IL‐17 expression was measured in total CD4⁺ and CD8⁺ T cells. mRNA expression of IL‐4, IL‐10, transforming growth factor beta (TGF‐β), cytotoxic T‐lymphocyte‐associated antigen 4, and/or forkhead box P3 was quantified in fluorescence‐activated cell sorting‐purified CD4⁺ T cells, CD8⁺ T cells, and CD4⁺ Treg. BM‐MSCs inhibited the production of TNF‐α, IL‐17, IL‐6, IL‐2, IFN‐γ, and IL‐9 by T cells from RA patients, mainly by reducing the percentage of cells producing cytokines. This inhibitory effect was transversal to all T cell subsets analyzed. At mRNA level, BM‐MSCs increased expression of IL‐10 and TGF‐β by CD4⁺ and CD8⁺ T cells. BM‐MSCs displayed a striking inhibitory action over T cells from RA patients, reducing the expression of cytokines involved in RA physiopathology. Remarkably, BM‐MSC‐derived immunomodulation affected either naive, effector, and memory T cells.     

Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141⁺ DC subset. CD141⁺ DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-β, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c⁺ DC subset. Polyinosine-polycytidylic acid (poly I:C)–activated CD141⁺ DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8⁺ cytotoxic T lymphocytes than poly I:C–activated CD1c⁺ DCs. Importantly, CD141⁺ DCs, but not CD1c⁺ DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141⁺ DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8α⁺ DC subset. The data demonstrate a role for CD141⁺ DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.The essential role of DCs in the induction and regulation of immune responses to pathogens, self-antigens (Ags), and cancers is now well established. All DCs excel at processing and presenting Ag and priming naive T cell responses, but the complexity of DC subsets and their individual specialized functions is just becoming apparent (MacDonald et al., 2002; Villadangos and Schnorrer, 2007; Naik, 2008). Promising DC-based therapeutic vaccines have been described to treat malignancies and infections (Vulink et al., 2008), but the majority of these use in vitro–generated monocyte-derived DC (MoDC), and the physiological standing of this DC subtype is currently unclear. Understanding the emerging complexities of human DC subset biology is therefore essential to develop new vaccines and therapeutics targeting DC.The characterization and function of human DC subsets has been confounded by their rarity, the lack of distinctive markers, and limited access to human tissues. Human blood DCs comprise ∼1% of circulating PBMCs and have been classically defined as Ag-presenting leukocytes that lack other leukocyte lineage markers (CD3, 14, 15, 19, 20, and 56) and express high levels of MHC class II (HLA-DR) molecules (Hart, 1997). These can be broadly categorized into two groups: plasmacytoid CD11c⁻CD123⁺ DC and conventional or myeloid CD11c⁺CD123⁻ DC. We have described three further phenotypically distinct subsets of CD11c⁺ DC, defined by their expression of CD16, CD1c (BDCA-1), and CD141 (BDCA-3; MacDonald et al., 2002). Gene expression profiling and hierarchical clustering data has indicated that plasmacytoid DC and CD16⁺ DC arise from separate precursor cells, whereas the CD1c⁺ DC and CD141⁺ DC subsets appear to have a common origin and represent two different stages of a similar subset (Lindstedt et al., 2005). However, CD1c⁺ and CD141⁺ DCs each have unique gene expression profiles distinct from monocytes and MoDC, and this predicts that they have different functions (Dzionek et al., 2000; MacDonald et al., 2002; Lindstedt et al., 2005).The concept of distinct DC subtypes with unique capabilities to influence immunological outcomes is exemplified by the mouse CD8α⁻ and CD8α⁺ conventional DC subsets that reside in the lymph nodes and spleen (Villadangos and Schnorrer, 2007; Naik, 2008). The CD8α⁻ DC subset appears to be most effective at inducing Th2 responses (Maldonado-López et al., 1999; Pulendran et al., 1999) and processing and presenting Ag to CD4⁺ T cells via the MHC class II pathway (Pooley et al., 2001; Dudziak et al., 2007; Villadangos and Schnorrer, 2007). In contrast, the CD8α⁺ DC subset has a unique ability to take up dead or dying cells and to process and present exogenous Ag on MHC class I molecules to CD8⁺ T cells (i.e., cross-presentation; den Haan et al., 2000; Iyoda et al., 2002; Schnorrer et al., 2006). There is now substantial evidence that the CD8α⁺ DC subset plays a crucial role in the induction of protective CD8⁺ CTL responses that are essential for the eradication of cancers, viruses, and other pathogenic infections (Dudziak et al., 2007; Hildner et al., 2008; López-Bravo and Ardavín, 2008; Naik, 2008). The identification of the human DC subset with similar functional capacity would be a significant advance and would enable translation of mouse DC biology into clinical practice.Correlation of the human and mouse DC subsets has been hampered by differences in their defining markers (human DCs do not express CD8α). Interestingly, computational genome-wide expression profiling clustered human CD141⁺ DC and CD1c⁺ DC with the mouse CD8α⁺ and CD8α⁻ conventional DC subsets, respectively (Robbins et al., 2008). Human CD141⁺ DC and mouse CD8α⁺ DC share a number of phenotypic similarities, including expression of Toll-like receptor (TLR) 3 (Edwards et al., 2003; Lindstedt et al., 2005), the novel surface molecule Necl2 (nectin-like protein 2; Galibert et al., 2005), and the C-type lectin CLEC9A (Caminschi et al., 2008; Huysamen et al., 2008; Sancho et al., 2008). Thus, whether the human CD141⁺ DC subset is the human functional equivalent of the mouse CD8α⁺ DC subset has now become a major question for immunologists.CD141⁺ DCs constitute only ∼0.03% of human PBMCs and, although present in other human tissues, their low proportions and difficulties with aseptic human tissue access mean that they have never been isolated in sufficient quantity to study their function until now. We report the first detailed functional analysis of human CD141⁺ DCs in response to TLR3 stimuli and define their role in the induction of Th1 responses and cross-presentation.     

Immunophenotypic characterization of human monocyte subsets: possible implications for cardiovascular disease pathophysiology

Summary. Objectives: Monocytes include several subsets with different and sometimes divergent roles in immunity, atherogenesis and reparative processes. Objectives: We aimed to perform detailed immunophenotypic and functional characterization of human monocyte subsets. Patients/methods: Analysis of surface markers of blood and bone marrow monocyte subsets and functional characterization of blood monocyte subsets in healthy volunteers was performed using flow cytometry. Results: In the present study, we show the presence of three subsets which could be unequivocally distinguished by surface expression of CD14, CD16 and CCR2 as CD14⁺CD16^?CCR2⁺(Mon1), CD14⁺CD16⁺CCR2⁺(Mon2) and CD14^lowCD16⁺CCR2^?(Mon3) subsets. In comparison with the classic Mon1, the Mon2 subset had the highest expression of Tie2, CXCR4, CD163, CD115, receptors to inter‐cellular adhesion molecule‐1 (ICAM‐1), vascular endothelial growth factor (VEGF), and the highest surface levels of apolipoprotein B and ferritin. In contrast, Mon3 had maximal expression of VCAM‐1 receptors and CD204. The Mon2 and Mon3 subsets had significantly lower activity of the NFκB pathway than Mon1. Mon1 and Mon2 had similar phagocytic activity, which was significantly higher compared with Mon3. All three subsets were present in bone marrow, although the relative proportion of Mon2 in bone marrow was about 2.5‐fold higher compared with that seen in blood. Significant differences in cytokine production in response to endotoxin stimulation were observed between the three monocyte subsets. Conclusion: Given their immunophenotypic similarity, the newly characterized Mon2 population may represent the previously reported pluripotent progenitor/pro‐angiogenic monocytes.