Soluble molecules are key in maintaining the immunomodulatory activity of murine mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) possess both immuno-privileged and immuno-inhibitory properties that contribute to their therapeutic effects. Ex vivo expansion is required to obtain sufficient cells for therapy, but might also alter their immunological properties. To date there has been no systematic study of MSC immunobiology during extended culture. Here, we demonstrate that both immuno-privilege and immunosuppressive properties of MSCs change with increasing passage. We demonstrate that although MSCs exhibit powerful immunosuppressive effects through secretion of transforming growth factor-ß (TGF-ß) and induction of interleukin-10, these effects are diminished by a concomitant increase in MSC immunogenicity. Interferon-γ treatment for 3 days induced extendedly cultured MSCs to express significantly higher levels of major histocompatibility complex class I. In vivo, this results in cells that induce significant delayed-type hypersensitivity reactions in allogeneic recipients. Importantly, these effects are alleviated by isolation of the transplanted MSCs using a semi-permeable barrier. Under these conditions, even MSCs cultured through as many as 14 passages still exhibit immuno-inhibitory effects in vivo. Furthermore, the levels of anti-inflammatory molecule TGF-ß secreted by MSCs were maintained in the extended culture. These data shed light on the variable results of allogeneic MSCs in transplantation and suggest alternative strategies for prolonging the effect of allogeneic MSCs in cell-based therapy.

Gene expression profiling reveals the defining features of the classical, intermediate, and nonclassical human monocyte subsets.

New official nomenclature subdivides human monocytes into 3 subsets: the classical (CD14(++)CD16(-)), intermediate (CD14(++)CD16(+)), and nonclassical (CD14(+)CD16(++)) monocytes. This introduces new challenges, as monocyte heterogeneity is mostly understood based on 2 subsets, the CD16(-) and CD16(+) monocytes. Here, we comprehensively defined the 3 circulating human monocyte subsets using microarray, flow cytometry, and cytokine production analysis. We find that intermediate monocytes expressed a large majority (87%) of genes and surface proteins at levels between classical and nonclassical monocytes. This establishes their intermediary nature at the molecular level. We unveil the close relationship between the intermediate and nonclassic monocytes, along with features that separate them. Intermediate monocytes expressed highest levels of major histocompatibility complex class II, GFRα2 and CLEC10A, whereas nonclassic monocytes were distinguished by cytoskeleton rearrangement genes, inflammatory cytokine production, and CD294 and Siglec10 surface expression. In addition, we identify new features for classic monocytes, including AP-1 transcription factor genes, CLEC4D and IL-13Rα1 surface expression. We also find circumstantial evidence supporting the developmental relationship between the 3 subsets, including gradual changes in maturation genes and surface markers. By comprehensively defining the 3 monocyte subsets during healthy conditions, we facilitate target identification and detailed analyses of aberrations that may occur to monocyte subsets during diseases.

Lung CD103+ dendritic cells efficiently transport influenza virus to the lymph node and load viral antigen onto MHC class I for presentation to CD8 T cells.

The uptake, transport, and presentation of Ags by lung dendritic cells (DCs) are central to the initiation of CD8 T cell responses against respiratory viruses. Although several studies have demonstrated a critical role of CD11b(low/neg)CD103(+) DCs for the initiation of cytotoxic T cell responses against the influenza virus, the underlying mechanisms for its potent ability to prime CD8 T cells remain poorly understood. Using a novel approach of fluorescent lipophilic dye-labeled influenza virus, we demonstrate that CD11b(low/neg)CD103(+) DCs are the dominant lung DC population transporting influenza virus to the posterior mediastinal lymph node as early as 20 h postinfection. By contrast, CD11b(high)CD103(neg) DCs, although more efficient for taking up the virus within the lung, migrate poorly to the lymph node and remain in the lung to produce proinflammatory cytokines instead. CD11b(low/neg)CD103(+) DCs efficiently load viral peptide onto MHC class I complexes and therefore uniquely possess the capacity to potently induce proliferation of naive CD8 T cells. In addition, the peptide transporters TAP1 and TAP2 are constitutively expressed at higher levels in CD11b(low/neg)CD103(+) DCs, providing, to our knowledge, the first evidence of a distinct regulation of the Ag-processing pathway in these cells. Collectively, these results show that CD11b(low/neg)CD103(+) DCs are functionally specialized for the transport of Ag from the lung to the lymph node and also for efficient processing and presentation of viral Ags to CD8 T cells.

Antigen-specific effector CD8 T cells regulate allergic responses via IFN-γ and dendritic cell function.

BACKGROUND: Previous studies have shown that CD8 T cells can both prevent and cause allergic responses. However, the underlying mechanisms remain to be elucidated. OBJECTIVE: We aim to investigate the potential of CD8 T cells with different IFN-γ expressions to modulate the elicitation of allergic inflammation following ovalbumin (OVA) challenge and investigate the underlying mechanisms. METHODS: To study the role of IFN-γ in the effect of CD8 T cells, effector CD8 T cells from CD8 OVA transgenic (OT-I) mice and IFN-γ(-/-)OT-I mice were transferred to OVA-sensitized mice the day before 3 challenges with OVA. The effect on lung dendritic cells (DCs) exerted by CD8 T cells was studied with ex vivo culture of sorted DCs from treatment mice with CD4 T cells. RESULTS: Effector OT-I, but not IFN-γ(-/-)OT-I CD8 T cells, attenuated eosinophilia and mucus secretion in the lungs of sensitized mice in an antigen-specific manner. Effector IFN-γ(-/-)OT-I CD8 T cells displayed a Tc2-/Tc17-biased phenotype with weaker cytotoxicity and were able to both induce and exacerbate eosinophilia as well as neutrophilia. OT-I CD8 T cells increased the ability of lung CD11b(+)CD103(-) DCs to both prime the differentiation of naive OVA-specific CD4 T cells toward a T(H)1 phenotype and enhance IFN-γ production by antigen-experienced lung CD4 T cells. CONCLUSION: Effector CD8 T cells attenuate pulmonary inflammation and alter the ability of DCs within the allergic lung to polarize T cells to a T(H)1 phenotype during a T(H)2 response. In the absence of IFN-γ, CD8 T cells assume a Tc2-/Tc17-biased phenotype and potentiate inflammation.

Human CD8⁺ T cells drive Th1 responses through the differentiation of TNF/iNOS-producing dendritic cells.

TNF/iNOS-producing dendritic cells (Tip-DCs) have been shown to arise during inflammation and are important mediators of immune defense. However, it is still relatively unclear which cell types contribute to their differentiation. Here we show that CD8(+) T cells, through the interaction with DCs, can induce the rapid development of human monocytes into Tip-DCs that express high levels of TNF-α and iNOS. Tip-DCs exhibited T-cell priming ability, expressed high levels of MHC class II, upregulated co-stimulatory molecules CD40, CD80, CD86, toll-like receptors TLR2, TLR3, TLR4, chemokine receptors CCR1 and CX3CR1 and expressed the classical mature DC marker, CD83. Differentiation of monocytes into Tip-DCs was partially dependent on IFN-γ as blocking the IFN-γ receptor on monocytes resulted in a significant decrease in CD40 and CD83 expression and in TNF-α production. Importantly, these Tip-DCs were capable of further driving Th1 responses by priming naive CD4(+) T cells for proliferation and IFN-γ production and this was partially dependent on Tip-DC production of TNF-α and NO. Our study hence identifies a role for CD8(+) T cells in orchestrating Th1-mediating signals through the differentiation of monocytes into Th1-inducing Tip-DCs.

CD44high memory CD8 T cells synergize with CpG DNA to activate dendritic cell IL-12p70 production.

Protective memory CD8 T cell responses are generally associated with the rapid and efficient acquisition of CTL function. However, the ability of memory CD8 T cells to modulate immune responses through interactions with dendritic cells (DCs) during the early states of secondary Ag exposure is poorly understood. In this study, we show that murine Ag-specific CD44(high) CD8 T cells, representing CD8 T cells of the memory phenotype, potently activate DCs to produce high levels of IL-12p70 in conjunction with stimulation of DCs with the TLR 9 ligand, unmethylated CpG DNA. IL-12p70 production was produced predominantly by CD8alpha(+) DCs and plasmacytoid DCs, and mediated by CD8 T cell-derived cytokines IFN-gamma, GM-CSF, TNF-alpha, and surface CD40L. We also find that CD44(high) memory phenotype CD8 T cells were better DC IL-12p70 stimulators than CD44(low) naive phenotype CD8 T cells, and this was attributed to higher levels of IFN-gamma and GM-CSF produced by CD44(high) memory phenotype CD8 T cells during their Ag specific interaction with DCs. Our study identifies CpG DNA as the most effective TLR ligand that cooperates with CD8 T cells for DC IL-12p70 production, and suggests that effectiveness of memory CD8 T cells could be attributed to their ability to rapidly and effectively induce protective Th1 immunity during early stages of pathogen reinfection.

CD16 is indispensable for antibody-dependent cellular cytotoxicity by human monocytes.

Antibody-dependent cellular cytotoxicity (ADCC) is exerted by immune cells expressing surface Fcγ receptors (FcγRs) against cells coated with antibody, such as virus-infected or transformed cells. CD16, the FcγRIIIA, is essential for ADCC by NK cells, and is also expressed by a subset of human blood monocytes. We found that human CD16- expressing monocytes have a broad spectrum of ADCC capacities and can kill cancer cell lines, primary leukemic cells and hepatitis B virus-infected cells in the presence of specific antibodies. Engagement of CD16 on monocytes by antibody bound to target cells activated ß2-integrins and induced TNFα secretion. In turn, this induced TNFR expression on the target cells, making them susceptible to TNFα-mediated cell death. Treatment with TLR agonists, DAMPs or cytokines, such as IFNγ, further enhanced ADCC. Monocytes lacking CD16 did not exert ADCC but acquired this property after CD16 expression was induced by either cytokine stimulation or transient transfection. Notably, CD16+ monocytes from patients with leukemia also exerted potent ADCC. Hence, CD16+ monocytes are important effectors of ADCC, suggesting further developments of this property in the context of cellular therapies for cancer and infectious diseases.

The three human monocyte subsets: implications for health and disease.

Human blood monocytes are heterogeneous and conventionally subdivided into two subsets based on CD16 expression. Recently, the official nomenclature subdivides monocytes into three subsets, the additional subset arising from the segregation of the CD16+ monocytes into two based on relative expression of CD14. Recent whole genome analysis reveal that specialized functions and phenotypes can be attributed to these newly defined monocyte subsets. In this review, we discuss these recent results, and also the description and utility of this new segregation in several disease conditions. We also discuss alternative markers for segregating the monocyte subsets, for example using Tie-2 and slan, which do not necessarily follow the official method of segregating monocyte subsets based on relative CD14 and CD16 expressions.

Susceptibility and response of human blood monocyte subsets to primary dengue virus infection.

Human blood monocytes play a central role in dengue infections and form the majority of virus infected cells in the blood. Human blood monocytes are heterogeneous and divided into CD16(-) and CD16(+) subsets. Monocyte subsets play distinct roles during disease, but it is not currently known if monocyte subsets differentially contribute to dengue protection and pathogenesis. Here, we compared the susceptibility and response of the human CD16(-) and CD16(+) blood monocyte subsets to primary dengue virus in vitro. We found that both monocyte subsets were equally susceptible to dengue virus (DENV2 NGC), and capable of supporting the initial production of new infective virus particles. Both monocyte subsets produced anti-viral factors, including IFN-α, CXCL10 and TRAIL. However, CD16(+) monocytes were the major producers of inflammatory cytokines and chemokines in response to dengue virus, including IL-1ß, TNF-α, IL-6, CCL2, 3 and 4. The susceptibility of both monocyte subsets to infection was increased after IL-4 treatment, but this increase was more profound for the CD16(+) monocyte subset, particularly at early time points after virus exposure. These findings reveal the differential role that monocyte subsets might play during dengue disease.

CD40L-expressing CD8 T cells prime CD8alpha(+) DC for IL-12p70 production.

CD8alpha(+) DC are implicated as the principle DC subset for cross-presentation and cross-priming of cytotoxic CD8 T cell responses. In this study, we demonstrate another unique facet of the CD8alpha(+) DC and CD8 T cell relationship, by showing that CD8 T cells reciprocally activate CD8alpha(+) DC, but not CD8alpha(-) DC, for IL-12p70 production, the key Th1-promoting cytokine. This effect was observed during an antigen-specific interaction between DC and activated CD8 T cells, along with secondary TLR stimulation of DC by LPS. Activated CD8 T cells use a combination of IFN-gamma and CD40L, which is rapidly up-regulated post-stimulation, to prime DC for IL-12p70 production during an antigen-specific response. Our results suggest that the interaction between CD8alpha(+) DC and antigen-primed CD8 T cells may form an important component of Th1-mediated immunity through the induction of IL-12p70.

An essential role for IL-18 in CD8 T cell-mediated suppression of IgE responses.

The ability of CD8 T cells to suppress IgE responses is well established. Previously, we demonstrated that CD8 T cells inhibit IgE responses via the induction of IL-12, which promotes Th1 and suppresses Th2 responses. In this study, we show that IL-18 also plays an essential role in IgE suppression. In vitro, IL-18 synergized with IL-12 to promote Th1/T cytotoxic 1 and inhibit Th2/T cytotoxic 2 differentiation. OVA-specific TCR transgenic (OT-I) CD8 cells induced both IL-12 and IL-18 when cultured with OVA(257-264) peptide-pulsed dendritic cells. In vivo, IL-18(-/-) mice exhibited higher IgE and IgG1 levels compared with wild-type mice after immunization with OVA/alum. Furthermore, adoptive transfer of CD8 T cells from OVA-primed mice suppressed IgE responses in OVA/alum-immunized mice, but not in IL-18(-/-) mice. IgE suppression in IL-18(-/-) mice was restored if CD8 T cells were coadoptively transferred with IL-18-competent wild-type bone marrow dendritic cell progenitors, demonstrating an essential role of IL-18 in CD8 T cell-mediated suppression of IgE responses. The data suggest that CD8 T cells induce IL-18 production during a cognate interaction with APCs that synergizes with IL-12 to promote immune deviation away from the allergic phenotype. Our data identify IL-18 induction as a potentially useful target in immunotherapy of allergic disease.