Histological studies on the development of porcine tonsils after birth

Tonsils form the topographically first immune barrier of an organism against the invasion of pathogens. We used histology to study the development of tonsils of pigs after birth. At birth, the tonsils consist of diffuse lymphoid tissue without any lymphoid follicle aggregations. At the age of 7 days, lymphoid follicles appeared in the soft palate tonsil. The lymphoid layer of the nasopharyngeal tonsil, soft palate tonsil, and lingual tonsil became thicker, and lymphoid follicles in the lamina propria were clearly visible at the age of 21 days. Secondary lymphoid follicles were present in the nasopharyngeal tonsil at the age of 50 days, and in the soft palate tonsil at the age of 120 days. Dendritic cells (DCs), CD3⁺ T cells and IgA⁺ B cells in the soft palate tonsil, nasopharyngeal tonsil and lingual tonsil increased continuously, especially during the first 21 days. The results suggested that tonsils have an important role in local immune defense against invading antigens after birth and will be beneficial for understanding the mechanisms of immunity in these animals after nasal and oral vaccination.     

Overexpression of IL-10 in C2D Macrophages Promotes a Macrophage Phenotypic Switch in Adipose Tissue Environments

Adipose tissue macrophages are a heterogeneous collection of classically activated (M1) and alternatively activated (M2) macrophages. Interleukin 10 (IL-10) is an anti-inflammatory cytokine, secreted by a variety of cell types including M2 macrophages. We generated a macrophage cell line stably overexpressing IL-10 (C2D-IL10) and analyzed the C2D-IL10 cells for several macrophage markers after exposure to adipocytes compared to C2D cells transfected with an empty vector (C2D-vector). C2D-IL10 macrophage cells expressed more CD206 when co-cultured with adipocytes than C2D-vector cells; while the co-cultured cell mixture also expressed higher levels of Il4, Il10, Il1β and Tnf. Since regular C2D cells traffic to adipose tissue after adoptive transfer, we explored the impact of constitutive IL-10 expression on C2D-IL10 macrophages in adipose tissue in vivo. Adipose tissue-isolated C2D-IL10 cells increased the percentage of CD206⁺, CD301⁺, CD11c⁻CD206⁺ (M2) and CD11c⁺CD206⁺ (M1b) on their cell surface, compared to isolated C2D-vector cells. These data suggest that the expression of IL-10 remains stable, alters the C2D-IL10 macrophage cell surface phenotype and may play a role in regulating macrophage interactions with the adipose tissue.     

Direct antibacterial activity of CD8+/CD4+ T-cells in ginbuna crucian carp,Carassius auratus langsdorfii

Cytotoxic T cells (CTLs) constitute an important component of the specific effector mechanism in killing against microbial-infected or transformed cells. In addition to these activities, recent studies in mammals have suggested that CTLs can exhibit direct antimicrobial activity. Therefore, the present investigation was conducted to find out the microbicidal activity of CD8α⁺ T cells of ginbuna crucian carp, Carassius auratus langsdorfii. The CD8α⁺ T cells from immunised ginbuna exhibited the antibacterial activity against both facultative intracellular bacteria and extracellular bacteria. The maximum reduction of viable count of pathogens was recorded with effector (sensitized) cells and target (bacteria) ratio of 10:1 co-incubated for a period of 1–2 h at 26 °C when effector cells were derived from ginbuna 7 days after one booster dose at 15th day of primary sensitization/immunisation. Sensitized CD8α⁺ T cells are found to kill 92.1 and 98.9% of Lactococcus garvieae and Edwardsiella tarda, respectively. No significant difference in the bacterial killing activity could be recorded against facultative intracellular bacteria and extracellular bacteria. The specificity study indicated the non-specific killing of bacteria. CD8α⁺ T cells from E. tarda immunised ginbuna exhibited 40% of non-specific killing activity against L. garvieae and those from L. garvieae immunised ginbuna showed 42.7% of non-specific killing activity against E. tarda. Furthermore, CD4⁺ T cells also killed 88% and 95.7% of L. garvieae and E. tarda, respectively. In addition to T cell subsets, surface IgM⁺ cells also killed both types of pathogens. Therefore, the present study demonstrated the direct antibacterial activity of CD8α⁺, CD4⁺ T-cells and surface IgM⁺ cells in fish.     

Identification of MS4A3 as a reliable marker for early myeloid differentiation in human hematopoiesis

Information of myeloid lineage-related antigen on hematopoietic stem/progenitor cells (HSPCs) is important to clarify the mechanisms regulating hematopoiesis, as well as for the diagnosis and treatment of myeloid malignancies. We previously reported that special AT-rich sequence binding protein 1 (SATB1), a global chromatin organizer, promotes lymphoid differentiation from HSPCs. To search a novel cell surface molecule discriminating early myeloid and lymphoid differentiation, we performed microarray analyses comparing SATB1-overexpressed HSPCs with mock-transduced HSPCs. The results drew our attention to membrane-spanning 4-domains, subfamily A, member 3 (Ms4a3) as the most downregulated molecule in HSPCs with forced overexpression of SATB1. Ms4a3 expression was undetectable in hematopoietic stem cells, but showed a concomitant increase with progressive myeloid differentiation, whereas not only lymphoid but also megakaryocytic-erythrocytic progenitors were entirely devoid of Ms4a3 expression. Further analysis revealed that a subset of CD34⁺CD38⁺CD33⁺ progenitor population in human adult bone marrow expressed MS4A3, and those MS4A3⁺ progenitors only produced granulocyte/macrophage colonies, losing erythroid colony- and mixed colony-forming capacity. These results suggest that cell surface expression of MS4A3 is useful to distinguish granulocyte/macrophage lineage-committed progenitors from other lineage-related ones in early human hematopoiesis. In conclusion, MS4A3 is useful to monitor early stage of myeloid differentiation in human hematopoiesis.     

NKp46+ Innate Lymphoid Cells Dampen Vaginal CD8 T Cell Responses following Local Immunization with a Cholera Toxin-Based Vaccine

Innate and adaptive immune cells work in concert to generate efficient protection at mucosal surface. Vaginal mucosa is an epithelial tissue that contains innate and adaptive immune effector cells. Our previous studies demonstrated that vaginal administration of Cholera toxin -based vaccines generate antigen-specific CD8 T cells through the stimulation of local dendritic cells (DC). Innate lymphoid cells (ILC) are a group of lymphocytes localized in epithelial tissues that have important immune functions against pathogens and in tissue homeostasis. Their contribution to vaccine-induced mucosal T cell responses is an important issue for the design of protective vaccines. We report here that the vaginal mucosa contains a heterogeneous population of NKp46⁺ ILC that includes conventional NK cells and ILC1-like cells. We show that vaginal NKp46⁺ ILC dampen vaccine-induced CD8 T cell responses generated after local immunization. Indeed, in vivo depletion of NKp46⁺ ILC with anti-NK1.1 antibody or NKG2D blockade increases the magnitude of vaginal OVA-specific CD8 T cells. Furthermore, such treatments also increase the number of DC in the vagina. NKG2D ligands being expressed by vaginal DC but not by CD8 T cells, these results support that NKp46⁺ ILC limit mucosal CD8 T cell responses indirectly through the NKG2D-dependent elimination of vaginal DC. Our data reveal an unappreciated role of NKp46⁺ ILC in the regulation of mucosal CD8 T cell responses.     

Efficient,Long Term Production of Monocyte-Derived Macrophages from Human Pluripotent Stem Cells under Partly-Defined and Fully-Defined Conditions

Human macrophages are specialised hosts for HIV-1, dengue virus, Leishmania and Mycobacterium tuberculosis. Yet macrophage research is hampered by lack of appropriate cell models for modelling infection by these human pathogens, because available myeloid cell lines are, by definition, not terminally differentiated like tissue macrophages. We describe here a method for deriving monocytes and macrophages from human Pluripotent Stem Cells which improves on previously published protocols in that it uses entirely defined, feeder- and serum-free culture conditions and produces very consistent, pure, high yields across both human Embryonic Stem Cell (hESC) and multiple human induced Pluripotent Stem Cell (hiPSC) lines over time periods of up to one year. Cumulatively, up to ∼3×10⁷ monocytes can be harvested per 6-well plate. The monocytes produced are most closely similar to the major blood monocyte (CD14⁺, CD16^low, CD163⁺). Differentiation with M-CSF produces macrophages that are highly phagocytic, HIV-1-infectable, and upon activation produce a pro-inflammatory cytokine profile similar to blood monocyte-derived macrophages. Macrophages are notoriously hard to genetically manipulate, as they recognise foreign nucleic acids; the lentivector system described here overcomes this, as pluripotent stem cells can be relatively simply genetically manipulated for efficient transgene expression in the differentiated cells, surmounting issues of transgene silencing. Overall, the method we describe here is an efficient, effective, scalable system for the reproducible production and genetic modification of human macrophages, facilitating the interrogation of human macrophage biology.     

Helminth Infection and Commensal Microbiota Drive Early IL-10 Production in the Skin by CD4+ T Cells That Are Functionally Suppressive

The skin provides an important first line of defence and immunological barrier to invasive pathogens, but immune responses must also be regulated to maintain barrier function and ensure tolerance of skin surface commensal organisms. In schistosomiasis-endemic regions, populations can experience repeated percutaneous exposure to schistosome larvae, however little is known about how repeated exposure to pathogens affects immune regulation in the skin. Here, using a murine model of repeated infection with Schistosoma mansoni larvae, we show that the skin infection site becomes rich in regulatory IL-10, whilst in its absence, inflammation, neutrophil recruitment, and local lymphocyte proliferation is increased. Whilst CD4⁺ T cells are the primary cellular source of regulatory IL-10, they expressed none of the markers conventionally associated with T regulatory (T_reg) cells (i.e. FoxP3, Helios, Nrp1, CD223, or CD49b). Nevertheless, these IL-10⁺ CD4⁺ T cells in the skin from repeatedly infected mice are functionally suppressive as they reduced proliferation of responsive CD4⁺ T cells from the skin draining lymph node. Moreover, the skin of infected Rag^-/- mice had impaired IL-10 production and increased neutrophil recruitment. Finally, we show that the mechanism behind IL-10 production by CD4⁺ T cells in the skin is due to a combination of an initial (day 1) response specific to skin commensal bacteria, and then over the following days schistosome-specific CD4⁺ T cell responses, which together contribute towards limiting inflammation and tissue damage following schistosome infection. We propose CD4⁺ T cells in the skin that do not express markers of conventional T regulatory cell populations have a significant role in immune regulation after repeated pathogen exposure and speculate that these cells may also help to maintain skin barrier function in the context of repeated percutaneous insult by other skin pathogens.     

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.     

Characteristics and clinical significance of CD163+/CD206+M2 mono-macrophage in the bladder cancer microenvironment

The tumor microenvironment may recruit monocytes, with a protumoral macrophage phenotype (M2) that plays an important role in solid tumor progression and metastasis. Therefore, it is necessary to understand the characteristics of these cells for cancer prevention and treatment. Bladder cancer tissue samples and paracarcinoma tissues samples were collected, and the expression of CD163⁺ cells in tumor tissues was observed. Then, we observed the expression of infiltrating CD45⁺CD14⁺CD163⁺ cell subset and analyzed the molecular expressions related to immunity and angiogenesis. C57/BL6 mice were inoculated subcutaneously, and dynamic changes of CD11b⁺F4/80⁺CD206⁺ mononuclear macrophages expression for tumor-bearing mice were detected. The results showed that the proportion of CD45⁺CD14⁺CD163⁺ mono-macrophage subset infiltrated by tumor tissue was significantly higher than that in paracarcinoma tissues. In bladder cancer tissue, the expression rate of CD40 in CD45⁺CD14⁺CD163^- mono-macrophage subset was significantly lower than that in CD45⁺CD14⁺CD163⁺ mono-macrophage subset. Similar results were found in the paracarcinoma tissues. We found that, as the proportion of CD11b⁺F4/80⁺CD206⁺ mono-macrophages increased gradually, the difference was statistically significant. CD163⁺/CD206⁺ mono-macrophages in bladder cancer microenvironment are abnormally elevated, and these cells are closely related to tumor progression. CD40 may be an important molecule that exerts biological function in this subset.     

Age-related changes in the distribution and frequency of myeloid and T cell populations in the small intestine of calves

Mucosal dendritic cells (DCs) play a key role in discriminating between dietary antigens, commensal microflora and pathogens but little is known regarding age-related changes in mucosal DC populations. We analyzed lymphoid and myeloid populations within the epithelium and lamina propria (LP) of the ileum and jejunum of weaned calves (6 months old) and compared their frequency and distribution with newborn calves (3–5 weeks old). CD4, CD8 and γδ TcR T cells and CD11c^HiMHC Class II⁺ myeloid cell frequency were significantly different when comparing ileum and jejunum of weaned calves. In particular, the number of CD8 and γδ TcR T cells, and CD11c^HiCD14⁺ macrophages was significantly greater in the ileum but CD11c⁺ and CD11b⁺ myeloid cell distribution was similar throughout the mucosal epithelium of the small intestine. Furthermore, significant age-related changes were apparent when comparing the frequency and abundance of mucosal leukocyte subpopulations in newborn and weaned calves. Total mucosal leukocytes (CD45⁺) increased significantly with age in both ileum and jejunum and much of this increase was attributed to mucosal T cells (CD3⁺). In particular, CD4 T cells and NK cells increased significantly in the jejunum and CD8, and γδ TcR T cells increased significantly with age throughout the small intestine. In contrast, CD11c^HiMHC Class II⁺ myeloid cells remained numerically unchanged with age but DCs (CD13⁺, CD26⁺, CD205⁺) were enriched and macrophages (CD14⁺, CD172a⁺) were depleted in older animals. Therefore, regional differences between ileal and jejunal mucosal leukocytes changed with age and there was also a marked age-dependent change in the composition of mucosal myeloid cells. These observations have significant implications for host responses to both pathogens and commensal microflora.     

Immune reconstitution in lymphoid tissue following potent antiretroviral therapy

During untreated HIV-1 infection, a chronic state of immune activation and inflammation develops at the lymphoid tissue sites of viral replication. The early effect of potent combination drug therapy is a reduction in peripheral viral burden and a reduction in the production of inflammatory and type 1 cytokines. Further along in treatment there are trends toward normalization in the frequencies of CD8⁸ T-cells, CD4⁺ CD45RA⁺ cells, as well as CD4⁺ CD45R0⁺ cells. Finally, the CD1a⁺ dendritic cell network is re-established and germinal centers are reformed. Although this restoration of the lymphoid dynamic form is coupled to a reconstitution of peripheral blood T-cell function in vitro and by skin testing, sterilizing immunity to HIV-1 does not develop. Furthermore there is no heightened development of cytotoxic CD8⁺ T-cell function at the site of HIV-1 latency. This is evidenced by a massive recrudescence of HIV-1 viral replication within lymphoid tissue when therapy is stopped. The development of supplemental therapies, which reconstitute anti-HIV-1 immunity, will be required. Specific defects in anti-HIV-1 activity which occur in lymphoid tissue during infection include a downregulation of perform expression by cytotoxic T-cells, the down regulation of the TCR signal transducing chain CD3ζ, and inadequate CD4⁺ T-cell help within the tissue compartment of immune regeneration.     

CD8+ T cells Are Preferentially Activated during Primary Low Dose Leishmania major Infection but Are Completely Dispensable during Secondary Anti-Leishmania Immunity

We previously showed that CD8⁺ T cells are required for optimal primary immunity to low dose Leishmania major infection. However, it is not known whether immunity induced by low dose infection is durable and whether CD8⁺ T cells contribute to secondary immunity following recovery from low dose infection. Here, we compared primary and secondary immunity to low and high dose L. major infections and assessed the influence of infectious dose on the quality and magnitude of secondary anti-Leishmania immunity. In addition, we investigated the contribution of CD8⁺ T cells in secondary anti-Leishmania immunity following recovery from low and high dose infections. We found that the early immune response to low and high dose infections were strikingly different: while low dose infection preferentially induced proliferation and effector cytokine production by CD8⁺ T cells, high dose infection predominantly induced proliferation and cytokine production by CD4⁺ T cells. This differential activation of CD4⁺ and CD8⁺ T cells by high and low dose infections respectively, was imprinted during in vitro and in vivo recall responses in healed mice. Both low and high dose-infected mice displayed strong infection-induced immunity and were protected against secondary L. major challenge. While depletion of CD4⁺ cells in mice that healed low and high dose infections abolished resistance to secondary challenge, depletion of CD8⁺ cells had no effect. Collectively, our results show that although CD8⁺ T cells are preferentially activated and may contribute to optimal primary anti-Leishmania immunity following low dose infection, they are completely dispensable during secondary immunity.     

TIM-3 shuttled by MV3 cells-secreted exosomes inhibits CD4+ T cell immune function and induces macrophage M2 polarization to promote the growth and metastasis of melanoma cells

This study is sought to determine the physiological mechanisms by which exosomes-encapsulated TIM-3 derived from melanoma cells might mediate CD4⁺ T cell immune function and macrophage M2 polarization in melanoma. Initially, exosomes were isolated from the human skin-derived melanoma cell line MV3for analysis of TIM-3 expression pattern. Next, the exosomes sourced from MV3 cells manipulated with sh-TIM-3 were co-incubated with CD4⁺ T cells to detect CD4⁺ T cell proliferation and MV3 cell migration and invasion, to observe the macrophage M2 polarization, and to determine levels of several EMT-related factors. Finally, melanoma nude mouse models were established to study the in vivo modulatory effects of TIM-3 from MV3 cells-derived exosomes. MV3 cells-derived exosomes inhibited CD4⁺ T cell immune function and promoted macrophage M2 polarization in melanoma. Our results revealed the abundance of TIM-3 in MV3 cells-derived exosomes. Of importance, silencing of TIM-3 shuttled by MV3 cells-derived exosomes improved CD4⁺ T cell immune function and inhibited macrophage M2 polarization to attenuate the growth and metastasis of melanoma cells. Collectively, MV3 cells-derived exosomes-loaded TIM-3 suppressed CD4⁺ T cell immune function and induced macrophage M2 polarization to improve occurrence and development of melanoma, therefore providing us with a potential therapeutic target for effectively combating melanoma.     

T-Cell Populations and Cytokine Expression Are Impaired in Thymus and Spleen of Protein Malnourished BALB/c Mice Infected with Leishmania infantum

Visceral leishmaniasis (VL) is a parasitic infectious disease that causes significant morbidity and mortality in the tropical and subtropical regions of the world. Although infections with visceralizing Leishmania may be asymptomatic, factors such as undernutrition increase the likelihood of progressing to clinical disease. Protein malnutrition, the most deleterious cause of malnutrition in developing countries, has been considered as a primary risk factor for the development of clinical VL. However, data regarding the immunological basis of this association are scarce. With the aim to analyze the effects of protein malnutrition on Leishmania infantum infection, we used BALB/c mice subjected to control or low protein isocaloric diets. Each animal group was divided into two subgroups and one was infected with L. infantum resulting in four study groups: animals fed 14% protein diet (CP), animals fed 4% protein diet (LP), animals fed 14% protein diet and infected (CPi), and animals fed 4% protein diet and infected (LPi).The susceptibility to L. infantum infection and immune responses were assessed in terms of body and lymphoid organ weight, parasite load, lymphocyte subpopulations, and cytokine expression. LPi mice had a significant reduction of body and lymphoid organ weight and exhibited a severe decrease of lymphoid follicles in the spleen. Moreover, LPi animals showed a significant decrease in CD4⁺CD8⁺ T cells in the thymus, whereas there was an increase of CD4⁺ and CD8⁺ T cells percentages in the spleen. Notably, the cytokine mRNA levels in the thymus and spleen of protein malnourished-infected animals were altered compared to the CP mice. Protein malnutrition results in a drastic dysregulation of T cells and cytokine expression in the thymus and spleen of L. infantum-infected BALB/c mice, which may lead to defective regulation of the thymocyte population and an impaired splenic immune response, accelerating the events of a normal course of infection.     

A multiparameter approach to monitor disease activity in collagen-induced arthritis

Introduction

Recent accumulating evidence indicates a crucial involvement of macrophage lineage in the pathogenesis of systemic sclerosis (SSc). To analyze the assembly of the monocyte/macrophage population, we evaluated the expression of CD163 and CD204 and various activated macrophage markers, in the inflammatory cells of the skin and in the peripheral blood mononuclear cells (PBMCs) derived from patients with SSc.

Methods

Skin biopsy specimens from 6 healthy controls and 10 SSc patients (7 limited cutaneous SSc and 3 diffuse cutaneous SSc) were analyzed by immunohistochemistry using monoclonal antibody against CD68 (pan-macrophage marker), CD163 and CD204. Surface and/or intracellular protein expression of CD14 (marker for monocyte lineage), CD163 and CD204 was analysed by flow cytometry in PBMCs from 16 healthy controls and 41 SSc patients (26 limited cutaneous SSc and 15 diffuse cutaneous SSc). Statistical analysis was carried out using Mann-Whitney U test for comparison of means.

Results

In the skin from SSc patients, the number of CD163⁺ cells or CD204⁺ cells between the collagen fibers was significantly larger than that in healthy controls. Flow cytometry showed that the population of CD14⁺ cells was significantly greater in PBMCs from SSc patients than that in healthy controls. Further analysis of CD14⁺ cells in SSc patients revealed higher expression of CD163 and the presence of two unique peaks in the CD204 histogram. Additionally, we found that the CD163⁺ cells belong to CD14^brightCD204⁺ population.

Conclusions

This is the first report indicating CD163⁺ or CD204⁺ activated macrophages may be one of the potential fibrogenic regulators in the SSc skin. Furthermore, this study suggests a portion of PBMCs in SSc patients abnormally differentiates into CD14^brightCD163⁺CD204⁺ subset. The subset specific to SSc may play an important role in the pathogenesis of this disease, as the source of CD163⁺ or CD204⁺ macrophages in the skin.