Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens ^1,2 as well as in the control of tolerogenic responses ³ in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) ⁴, plasmacytoid DC (pDC) ⁵ and the IFN-producing killer DC (IKDC) ^6,7. The cDC subset is the most prominent DC subset in the lung ⁸. The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (β2) that is also expressed on monocytes, macrophages, neutrophils and some B cells ⁹. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c⁺ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4/80, an splenic macrophage marker, the CD11c^hiMHC-II^lo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor ¹¹. In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung. Download video file.^{(55M, mov)}     

Impaired function and delayed regeneration of dendritic cells in COVID-19

Disease manifestations in COVID-19 range from mild to severe illness associated with a dysregulated innate immune response. Alterations in function and regeneration of dendritic cells (DCs) and monocytes may contribute to immunopathology and influence adaptive immune responses in COVID-19 patients. We analyzed circulating DC and monocyte subsets in 65 hospitalized COVID-19 patients with mild/moderate or severe disease from acute illness to recovery and in healthy controls. Persisting reduction of all DC subpopulations was accompanied by an expansion of proliferating Lineage⁻HLADR⁺ cells lacking DC markers. Increased frequency of CD163⁺ CD14⁺ cells within the recently discovered DC3 subpopulation in patients with more severe disease was associated with systemic inflammation, activated T follicular helper cells, and antibody-secreting cells. Persistent downregulation of CD86 and upregulation of programmed death-ligand 1 (PD-L1) in conventional DCs (cDC2 and DC3) and classical monocytes associated with a reduced capacity to stimulate naïve CD4⁺ T cells correlated with disease severity. Long-lasting depletion and functional impairment of DCs and monocytes may have consequences for susceptibility to secondary infections and therapy of COVID-19 patients.     

Differential expansion, activation and effector functions of conventional and plasmacytoid dendritic cells in mouse tissues transiently infected with Listeria monocytogenes

Dendritic cells (DC) are crucial in generating immunity to infection. Here we characterize changes in DC in terms of number, activation and effector functions, focusing on conventional DC (cDC) and plasmacytoid DC (pDC), in Listeria-infected mice. Kinetic studies showed a subset- and tissue-specific expansion of cDC and upregulation of CD80 and CD86 on splenic and mesenteric lymph node (MLN) cDC after intragastric infection. Expansion of pDC was more prolonged than cDC, and pDC upregulated CD86 and MHC-II, but not CD80, in both the spleen and MLN. cDC were an important source of IL-12 but not TNF-alpha during infection, while pDC made neither of these cytokines. Instead other CD11c(int) cells produced these cytokines. Using five-colour flow cytometry and double intracellular cytokine staining, we detected phenotypically similar CD11c(int)CD11b(+)Gr1(+) cells with distinct capacities to produce TNF-alpha/IL-12 or TNF-alpha/iNOS (inducible nitric oxide synthase) in Listeria-infected tissues. IL-12p70 was also produced by sorted CD11c(hi) and CD11c(int)CD11b(+)Gr1(+) cells. Furthermore, production of TNF-alpha, iNOS and IL-12 was differentially dependent on cellular localization of the bacteria. Cytosol-restricted bacteria induced TNF-alpha and iNOS-producing cells, albeit at lower frequency than wild-type bacteria. In contrast, IL-12 was induced only with wild-type bacteria. These data provide new insight into the relative abundance and function of distinct CD11c-expressing populations during the early stage of Listeria infection.     

Expression of high-affinity IgE receptor on human peripheral blood dendritic cells in children

Background

In a mouse model of viral induced atopic disease, expression of FcεRI on dendritic cells is critical. While adult human conventional (cDC) and plasmacytoid (pDC) dendritic cells have been shown to express FcεRI, it is not known if this receptor is expressed in childhood and how its expression is governed by IgE.

Methods

Following informed consent of subjects (n = 27, aged 12–188 months), peripheral blood was stained for surface expression of CD19, ILT7, CD1c, IgE, FcεRI and analyzed by flow cytometry (cDC: CD19⁻ ILT7⁻ CD1c⁺; pDC: CD19⁻ ILT7⁺ CD1c⁻). Total and specific serum IgE levels to food and inhalant allergens were determined by ImmunoCAP, and the relationship between FcεRI expression on dendritic cells and sensitization, free IgE, cell bound IgE, and age was determined.

Results

Independent of sensitization status, FcεRI expression was noted on cDC and pDC as early as 12 months of age. Serum IgE level correlated with expression of FcεRI on cDC, but not pDC. Based on the concentration of IgE, a complex relationship was found between surface bound IgE and expression of FcεRI on cDC. pDC exhibited a linear relationship of FcεRI expression and bound IgE that was consistent through all IgE concentrations.

Conclusions

In children, FcεRI expression on cDC and pDC is modulated differently by serum and cell bound IgE. IgE governance of FcεRI expression on cDC depends upon a complex relationship. Further studies are needed to determine the functional roles of FcεRI on cDC and pDC.     

Plasmacytoid dendritic cells are inefficient in activation of human regulatory T cells

Background

Dendritic cells (DC) play a key role in initiation and regulation of immune responses. Plasmacytoid DC (pDC), a small subset of DC, characterized as type-I interferon producing cells, are critically involved in anti-viral immune responses, but also mediate tolerance by induction of regulatory T cells (Treg). In this study, we compared the capacity of human pDC and conventional DC (cDC) to modulate T cell activity in presence of Foxp3⁺ Treg.

Principal Findings

In coculture of T effector cells (Teff) and Treg, activated cDC overcome Treg anergy, abrogate their suppressive function and induce Teff proliferation. In contrast, pDC do not break Treg anergy but induce Teff proliferation even in coculture with Treg. Lack of Treg-mediated suppression is independent of proinflammatory cytokines like IFN-α, IL-1, IL-6 and TNF-α. Phenotyping of pDC-stimulated Treg reveals a reduced expression of Treg activation markers GARP and CTLA-4. Additional stimulation by anti-CD3 antibodies enhances surface expression of GARP and CTLA-4 on Treg and consequently reconstitutes their suppressive function, while increased costimulation with anti-CD28 antibodies is ineffective.

Conclusions/Significance

Our data show that activated pDC induce Teff proliferation, but are insufficient for functional Treg activation and, therefore, allow expansion of Teff also in presence of Treg.     

Respiratory Syncytial Virus-Induced Activation and Migration of Respiratory Dendritic Cells and Subsequent Antigen Presentation in the Lung-Draining Lymph Node

In the respiratory tract, different dendritic cell (DC) populations guard a tight balance between tolerance and immunity to infectious or harmless materials to which the airways are continuously exposed. For infectious and noninfectious antigens administered via different routes, different subsets of DC might contribute during the induction of T-cell tolerance and immunity. We studied the impact of primary respiratory syncytial virus (RSV) infection on respiratory DC composition in C57BL/6 mice. We also tracked the migration of respiratory DC to the lymph nodes and studied antigen presentation by lung-derived and lymph node-resident DC to CD4⁺ and CD8⁺ T cells. We observed a massive influx of mainly CD103⁻ CD11b^high CD11c⁺ conventional DC (cDC) and plasmacytoid DC during the first 7 days of RSV infection, while CD103⁺ CD11b^low CD11c⁺ cDC disappeared from the lung. The two major subsets of lung tissue DC, CD103⁺ CD11b^low CD11c⁺ and CD103⁻ CD11b^high CD11c⁺ cDC, both transported RSV RNA to the lung-draining lymph node. Furthermore, these lung-derived cDC subsets as well as resident LN DC, which did not contain viral RNA, displayed viral antigen by major histocompatibility complex class I and class II to CD8⁺ and CD4⁺ T cells. Taken together, our data indicate that during RSV infections, at least three DC subsets might be involved during the activation of lymph node-homing naïve and memory CD4⁺ and CD8⁺ T cells.Respiratory syncytial virus (RSV) constitutes a major health burden for infants, elderly people, and immunocompromised individuals (16, 19). The virus infects most children in their first year of life and is the main cause of severe lower respiratory tract infections in infants (19). Despite many decades of research, the immune response to RSV is still not completely understood. Infection with RSV leads to poor development of immunity, and recurrent infections are common (23). In mice, it was found that RSV induces virus-specific CD8⁺ T-cell responses in the lung that are functionally impaired (10). It has been suggested that a functional inactivation of CD8⁺ T cells by RSV could be a reason for the short-lived immune response. Furthermore, we and others have previously shown that human monocyte-derived dendritic cells (DC) can be infected with RSV, which results in a strong inhibition of their ability to support proliferative responses and induction of effector function in naïve T cells (11, 12). An early vaccine trial with formalin-inactivated RSV in alum administered intramuscularly elicited a memory immune response that caused a strong aberrant secondary immune response in vaccinees upon natural exposure with live virus. This resulted in a high rate of morbidity in the vaccinated children (31). These observations underscore the necessity to understand the components of the immune response that are protective during RSV infections and the need to understand the mechanism by which protective immunity can be elicited for the development of an effective and safe vaccine.DC play an important role in the initiation of both the innate and adaptive immune responses to pathogens including RSV (3). They are a heterogeneous population of cells represented by two main subsets, the myeloid or “conventional” CD11c⁺ DC (cDC) and the CD11c^low/mPDCA-1⁺ plasmacytoid DC (pDC) (47, 52). cDC can be further divided based on the expression of surface markers and anatomic location. cDC in the tissue and cDC in lymph nodes (LN) appear to be different subsets arising from different pools of progenitor cells and with specialized functions (13, 17, 30, 33, 46). In the mouse lung, two major cDC populations are derived from blood monocytes. CD11c⁺ major histocompatibility complex class II (MHC-II)-positive (MHC-II⁺) CD103⁻ CD11b^high cDC (CD11b^hi cDC) are localized in the parenchyma. These cells are the main producers of chemokines and are important for the recruitment of leukocytes (4). A second cDC population, CD11c⁺ MHC-II⁺ CD103⁺ CD11b^low cDC (CD103⁺ cDC), is located directly underneath the airway epithelium. These CD103⁺ cDC express the integrin α_Eβ₇; therefore, they are found mainly at the basal lamina of the bronchial epithelia and arterioles, which express E-cadherin, the ligand for α_Eβ₇. Furthermore, CD103⁺ cDC express the tight-junction proteins ZO-2 and claudin-7, which enables them to sample the airways with their extensions (45). In the lung-draining LN, in addition to pDC, at least two steady-state populations of cDC are present, which are characterized by the expression or absence of CD8α. In contrast to the lung tissue DC, these cells enter the LN from the blood, and they are directly derived from a bone marrow precursor (38, 39, 41). In addition, minor fractions of tissue-derived cDC also access draining LN in the steady state (28). Several studies have addressed the roles of different DC subsets that are present in the tissue and LN draining the infection site. In spleen and skin-draining LN, the role of CD8α⁺ cDC seems to be important for the initiation of anti-ovalbumin and antiviral CD8⁺ T-cell responses (6, 26, 35). In mice exposed to innocuous (ovalbumin) or infectious (influenza virus) antigen, functional specialization was described for CD103⁺ and CD11b^hi lung cDC subsets. CD11b^hi cDC presented intranasally administered ovalbumin or influenza virus antigen mainly to naïve CD4⁺ T cells, while CD103⁺ cDC were important for the induction of CD8⁺ T-cell responses (14, 32).The ability of DC to present or cross-present antigens depends on the type of antigenic materials and the uptake mechanism used by antigen-presenting cells. Hence, different pathogens and innocuous antigens might be differently presented by different DC subsets. We studied the kinetics of lung DC migration and repopulation during primary RSV infection in C57BL/6 mice. We found that upon RSV infection, CD103⁺ cDC disappeared from the lung, while there was a net increase in numbers of CD11b^hi cDC, pDC, and macrophages. Within the first 48 h after virus exposure, both CD103⁺ and CD11b^hi cDC rapidly migrated to the lung-draining mediastinal LN (MLN), while this accumulation was absent in the non-lung-draining axillary LN. The migrating cDC showed the highest level of expression of the costimulatory molecules CD40, CD80, and CD86, which are necessary for T-cell stimulation, compared to the MLN-resident cDC. Furthermore, the migrating cDC transported viral RNA to the MLN and were capable of stimulating RSV-specific CD4⁺ and CD8⁺ T-cell responses. Resident cDC in the LN were uniformly negative for viral RNA. However, resident cDC in the LN did present viral antigen to CD8⁺ and CD4⁺ T cells via MHC-I and MHC-II, respectively.     

Modulation of respiratory dendritic cells during Klebsiella pneumonia infection

Background

Klebsiella pneumoniae is a leading cause of severe hospital-acquired respiratory tract infections and death but little is known regarding the modulation of respiratory dendritic cell (DC) subsets. Plasmacytoid DC (pDC) are specialized type 1 interferon producing cells and considered to be classical mediators of antiviral immunity.

Method

By using multiparameter flow cytometry analysis we have analysed the modulation of respiratory DC subsets after intratracheal Klebsiella pneumonia infection.

Results

Data indicate that pDCs and MoDC were markedly elevated in the post acute pneumonia phase when compared to mock-infected controls. Analysis of draining mediastinal lymph nodes revealed a rapid increase of activated CD103⁺ DC, CD11b⁺ DC and MoDC within 48 h post infection. Lung pDC identification during bacterial pneumonia was confirmed by extended phenotyping for 120G8, mPDCA-1 and Siglec-H expression and by demonstration of high Interferon-alpha producing capacity after cell sorting. Cytokine expression analysis of ex vivo-sorted respiratory DC subpopulations from infected animals revealed elevated Interferon-alpha in pDC, elevated IFN-gamma, IL-4 and IL-13 in CD103⁺ DC and IL-19 and IL-12p35 in CD11b⁺ DC subsets in comparison to CD11c⁺ MHC-class II^low cells indicating distinct functional roles. Antigen-specific naive CD4⁺ T cell stimulatory capacity of purified respiratory DC subsets was analysed in a model system with purified ovalbumin T cell receptor transgenic naive CD4⁺ responder T cells and respiratory DC subsets, pulsed with ovalbumin and matured with Klebsiella pneumoniae lysate. CD103⁺ DC and CD11b⁺ DC subsets represented the most potent naive CD4⁺ T helper cell activators.

Conclusion

These results provide novel insight into the activation of respiratory DC subsets during Klebsiella pneumonia infection. The detection of increased respiratory pDC numbers in bacterial pneumonia may indicate possible novel pDC functions with respect to lung repair and regeneration.     

PDL1 Expression on Plasma and Dendritic Cells in Myeloma Bone Marrow Suggests Benefit of Targeted anti PD1-PDL1 Therapy

In this study we set out to investigate whether anti PDL1 or PD–1 treatment targeting the immune system could be used against multiple myeloma. DCs are important in regulating T cell responses against tumors. We therefore determined PDL1 and PDL2 expression on DC populations in bone marrow of patients with plasma cell disorders using multicolour Flow Cytometry. We specifically looked at CD141⁺ and CD141^- myeloid and CD303⁺ plasmacytoid DC. The majority of plasma cells (PC) and DC subpopulations expressed PDL1, but the proportion of positive PDL1+ cells varied among patients. A correlation between the proportion of PDL1⁺ PC and CD141⁺ mDC was found, suggesting both cell types could down-regulate the anti-tumor T cell response.     

Plasmacytoid DC from aged mice down-regulate CD8 T cell responses by inhibiting cDC maturation after Encephalitozoon cuniculi infection

Age associated impairment of immune function results in inefficient vaccination, tumor surveillance and increased severity of infections. Several alterations in adaptive immunity have been observed and recent studies report age related declines in innate immune responses to opportunistic pathogens including Encephalitozoon cuniculi. We previously demonstrated that conventional dendritic cells (cDC) from 9-month-old animals exhibit sub-optimal response to E. cuniculi infection, suggesting that age associated immune senescence begins earlier than expected. We focused this study on how age affects plasmacytoid DC (pDC) function. More specifically how aged pDC affect cDC function as we observed that the latter are the predominant activators of CD8 T cells during this infection. Our present study demonstrates that pDC from middle-aged mice (12 months) suppress young (8 week old) cDC driven CD8 T cell priming against E. cuniculi infection. The suppressive effect of pDC from older mice decreased maturation of young cDC via cell contact. Aged mouse pDC exhibited higher expression of PD-L1 and blockade of their interaction with cDC via this molecule restored cDC maturation and T cell priming. Furthermore, the PD-L1 dependent suppression of cDC T cell priming was restricted to effector function of antigen-specific CD8 T cells not their expansion. To the best of our knowledge, the data presented here is the first report highlighting a cell contact dependent, PD-L1 regulated, age associated defect in a DC subpopulation that results in a sub-optimal immune response against E. cuniculi infection. These results have broad implications for design of immunotherapeutic approaches to enhance immunity for aging populations.     

Human CD141+ dendritic cells generated from adult peripheral blood monocytes

Human CD141⁺ dendritic cells (DCs), specialized for cross-presentation, have been extensively studied in the development of DC-based therapy against cancer. A series of attempts was made to generate CD141⁺ DCs from cord blood CD34⁺ hematopoietic progenitors to overcome the practical limitation of in vivo rareness. In the present study, we identified a culture system that generates high CD141⁺ DCs. After culture of CD14⁺ monocytes in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 for 8 days, CD141 was detected on cells that adhered to the bottom of the culture plate. The attached cells exhibited typical features of immature monocyte-derived DCs (moDCs), except for higher CD86 expression, more dendrites and higher granularity compared with those that did not attach. With 3 additional days of culture, increased CD141 expression on the cells was retained along with adhesion ability and partial expression of CLEC9A, a c-type lectin receptor. Furthermore, the cells exhibited effective uptake of dead cells. Interestingly, the attached moDCs differently responded to polyinosinic:polycytidylic acid (poly I:C) stimulation as well as a mixed lymphocyte reaction. Collectively, our findings show that human CD141⁺ DCs can be sufficiently generated from peripheral blood CD14⁺ monocytes, potentiating further investigation into generation of higher yields of cross-priming human DCs in vitro.     

Adenovirus efficiently transduces plasmacytoid dendritic cells resulting in TLR9-dependent maturation and IFN-alpha production

BACKGROUND: Recombinant replication-deficient adenoviral vectors (recAd) are attractive candidates for DNA vaccination approaches because they are able to activate the innate and adaptive immune systems. Here we explore the ability of recAd to transduce and activate subsets of dendritic cells, namely plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC). METHODS: DC were derived from bone marrow precursors in vitro with the help of FLT3-ligand. Sorted populations of pDC and cDC were infected with recAd at various multiplicities of infection. Transduction efficiency, phenotypic maturation and production of IFN-alpha as well as IL-6 were assessed. Additionally, activation of DC and induction of cytotoxic T lymphocytes (CTL) were determined in vivo. The role of Toll-like receptor (TLR) 9 in recAd recognition was investigated as it has previously been shown that DNA viruses are recognized via this receptor. RESULTS: RecAd can efficiently transduce pDC as well as cDC in vitro. Both DC subsets mature and produce IFN-alpha upon interaction with recAd. In the absence of TLR9, activation and cytokine production was only detected in cDC but not in pDC. Importantly, induction of CD8+ CTL following in vivo injection of recAd was similar in TRL9-deficient mice when compared with wildtype controls. CONCLUSIONS: RecAd can efficiently transduce and activate both pDC and cDC. pDC required TLR9 to detect the presence of recAd whereas cDC also recognized recAd independently of TLR9. These unique immunostimulatory properties support the future development of recombinant Ad as a vector for DNA vaccine approaches.     

Distinct responses of splenic dendritic cell subsets to infection with Listeria monocytogenes: maturation phenotype, level of infection, and T cell priming capacity ex vivo

To determine the relative contributions of DC subsets in the development of protective immunity to Listeria monocytogenes we examined the relationship between maturation, bacterial burden, and T cell priming capacity of four well characterized subsets of splenic DC following infection with Lm. CD8α⁺, CD4⁺, and CD8α⁻CD4⁻ DC and the B220⁺ plasmacytoid DC (pDC) were compared for abundance and costimulatory molecule expression at 24, 48, and 72 h post i.v. infection. We further determined the bacterial burden associated with each DC subset and their relative capacities to prime CD8⁺ T cells at 24 hpi. The CD8α⁺ DC displayed the highest level of maturation, association with live bacteria, and T cell activation potential. Second, the CD4⁺ DC were also mature, yet were associated with fewer bacteria, and stimulated T cell proliferation, but not IFN-γ production. The CD8α⁻CD4⁻ DC showed a modest maturation response and were associated with a high number of bacteria, but failed to induce T cell proliferation ex vivo. pDC displayed a strong maturation response, but were not associated with detectable bacteria and also failed to stimulate T cell activation. Finally, we measured the cytokine responses in these subsets and determined that IL-12 was produced predominantly by the CD8⁺ DC, correlating with the ability of this subset DC to induce IFN-γ production in T cells. We conclude that Listeria-specific CD8⁺ T cell activation in the spleen is most effectively achieved by infection-induced maturation of the CD8α⁺ DC subset.     

Effect of Foot-and-Mouth Disease Virus Infection on the Frequency,Phenotype and Function of Circulating Dendritic Cells in Cattle

Foot-and-mouth disease virus (FMDV) is a highly contagious virus that causes one of the most devastating diseases in cloven-hoofed animals. Disease symptoms develop within 2 to 3 days of exposure and include fever and vesicular lesions on the tongue and hooves. Dendritic cells (DC) play an essential role in protective immune responses against pathogens. Therefore, investigating their role during FMDV infection would lead to a better understanding of host-pathogen interactions. In this study, following infection of cattle with FMDV, we investigated the frequency and function of conventional (cDC) and plasmacytoid DC (pDC) in blood by using multi-color flow cytometry. We show that the frequency of cDC and pDC increased following FMDV infection and peaked 3 to 4 days post-infection. During peak viremia, the cattle became lymphopenic, the expression of MHC class II molecules on cDC and pDC was dramatically down-regulated, the processing of exogenous antigen by cDC and pDC was impaired, and there was an increase in IL-10 production by DC and monocytes. Notably, after clearance of FMDV from the blood, MHC class II expression returned to pre-infection levels. Altogether, our study demonstrates that in cattle, FMDV inhibits the function of DC, thereby retarding the initiation of adaptive immune responses, potentially enhancing virus shedding during the acute phase of infection.     

A Higher Frequency of CD14+CD169+ Monocytes/Macrophages in Patients with Colorectal Cancer

Objective

Monocytes and macrophages can infiltrate into tumor microenvironment and regulate the progression of tumors. This study aimed at determining the frequency of different subsets of circulating monocytes and tumor infiltrating macrophages (TIMs) in patients with colorectal cancer (CRC).

Methods

The frequency of different subsets of circulating monocytes was characterized in 46 CRC patients and 22 healthy controls (HC) by flow cytometry. The frequency of different subsets of macrophages was analyzed in TIMs from 30 tumor tissues and in lamina propria mononuclear cells (LPMCs) from 12 non-tumor tissues. The concentrations of plasma cytokines and carcinoembryonic antigen (CEA) were determined. The potential association of these measures with the values of clinical parameters was analyzed.

Results

In comparison with that in the HC, the percentages of circulating CD14⁺CD169⁺, CD14⁺CD169⁺CD163⁺ and CD14⁺CD169⁺CD206⁺ monocytes and TIMs CD14⁺CD169⁺ as well as IL-10⁺CD14⁺CD169⁺, but not IL-12⁺ CD14⁺CD169⁺ macrophages were significantly increased, accompanied by higher levels of plasma IL-10 in the CRC patients. The percentages of CD14⁺CD169⁺ circulating monocytes and TIM macrophages were associated with the stage of disease and correlated positively with the levels of plasma IL-10 and CEA in CRC patients.

Conclusion

Our data suggest that an increase in the frequency of CD14⁺CD169⁺ cells may be associated with the development and progression of CRC and is concomitant rise of both, pro-tumor (M2-like, IL-10 producing) and anti-tumor (M1-like, IL-12 producing) monocytes and infiltrating macrophages. The frequency of CD14⁺CD169⁺ circulating monocytes and infiltrating macrophages may serve as a biomarker for evaluating the pathogenic degrees of CRC.     

IL-10 Conditioning of Human Skin Affects the Distribution of Migratory Dendritic Cell Subsets and Functional T Cell Differentiation

In cancer patients pervasive systemic suppression of Dendritic Cell (DC) differentiation and maturation can hinder vaccination efficacy. In this study we have extensively characterized migratory DC subsets from human skin and studied how their migration and T cell-stimulatory abilities were affected by conditioning of the dermal microenvironment through cancer-related suppressive cytokines. To assess effects in the context of a complex tissue structure, we made use of a near-physiological skin explant model. By 4-color flow cytometry, we identified migrated Langerhans Cells (LC) and five dermis-derived DC populations in differential states of maturation. From a panel of known tumor-associated suppressive cytokines, IL-10 showed a unique ability to induce predominant migration of an immature CD14⁺CD141⁺DC-SIGN⁺ DC subset with low levels of co-stimulatory molecules, up-regulated expression of the co-inhibitory molecule PD-L1 and the M2-associated macrophage marker CD163. A similarly immature subset composition was observed for DC migrating from explants taken from skin overlying breast tumors. Whereas predominant migration of mature CD1a⁺ subsets was associated with release of IL-12p70, efficient Th cell expansion with a Th1 profile, and expansion of functional MART-1-specific CD8⁺ T cells, migration of immature CD14⁺ DDC was accompanied by increased release of IL-10, poor expansion of CD4⁺ and CD8⁺ T cells, and skewing of Th responses to favor coordinated FoxP3 and IL-10 expression and regulatory T cell differentiation and outgrowth. Thus, high levels of IL-10 impact the composition of skin-emigrated DC subsets and appear to favor migration of M2-like immature DC with functional qualities conducive to T cell tolerance.     

Comparative analysis of circulating dendritic cell subsets in patients with atopic diseases and sarcoidosis

Background

Dendritic cells (DCs) are professional antigen-presenting cells that play a crucial role in the initiation and modulation of immune responses. Human circulating blood DCs are divided into two major subsets: myeloid DCs (mDCs); and plasmacytoid DCs (pDCs). Furthermore, mDCs are subdivided into two subsets: Th1-promoting mDCs (mDC1s); and Th2-promoting mDCs (mDC2s). Although CD1a, CD1c, and CD141 are generally used for classifying mDC subsets, their adequacy as a specific marker remains unclear. We performed this study to compare circulating mDC, pDC, mDC1, and mDC2 subsets between Th1- and Th2-mediated diseases using CD1a and CD141, and to analyze the adequacy of CD1a and CD141 as a marker for mDC1s and mDC2s, respectively.

Methods

Thirty patients with sarcoidosis, 23 patients with atopic diseases, such as atopic bronchial asthma, and 23 healthy subjects as controls were enrolled in this study. Peripheral blood DC subsets were analyzed with flow cytometry according to expressions of CD11c, CD123, CD1a, and CD141. For functional analysis, we measured interleukin (IL) 12p40 levels produced by the sorted mDC subsets.

Results

The sarcoidosis group showed decreased total DC (P < 0.05) and mDC counts (P < 0.05) compared to controls. The atopy group showed decreased CD1a⁺mDC count (P < 0.05), and increased CD1a^-mDC count (P < 0.05) compared to controls. CD141⁺mDC count in the atopy group was higher than controls (P < 0.05). Sorted CD1a⁺mDCs produced higher levels of IL-12p40 than CD1a^-mDCs (P = 0.025) and CD141⁺mDCs (P = 0.018).

Conclusions

We conclude that decreased count of CD1a⁺mDC and increased count of CD141⁺mDC may reflect the Th2-skewed immunity in atopic diseases. The results of IL-12 levels produced by the sorted mDC subsets suggested the adequacy of CD1a and CD141 as a marker for mDC1 and mDC2, respectively, in vivo.