Cell Surface Expression Of Costimulatory Molecules Research Articles

Cigarette Smoke Exposure Impairs Dendritic Cell Maturation and T Cell Proliferation in Thoracic Lymph Nodes of Mice

Robbins CS, Franco F, Mouded M, Cernadas M, Shapiro SD. J Immunol. 2008;180(10):6623–6628 PURPOSE OF THE STUDY. Airborne antigens are processed and presented by respiratory tract dendritic cells (DCs). The purpose of this study was to determine the consequences of cigarette-smoke exposure on DC function in mice. METHODS. Mice were exposed to cigarette smoke 5 days per week for 1 month. There was also a control group of mice unexposed to smoke. Mice then underwent intratracheal injection of ovalbumin-fluorescein isothiocyanate (FITC) or ovalbumin and were killed at varying time points thereafter depending on the aspect of DC function being studied (migration, maturation, or T-cell activation). Lung cells were isolated, and the numbers of DCs were determined in exposed and unexposed mice by flow cytometry. Migration of lung DCs to thoracic lymph nodes (TLNs) was assessed by measuring CD11c+FITC+ cells. Maturation of TLN DCs after ovalbumin exposure was then determining by assessing expression of cell-surface costimulatory molecules (CD80, CD86, MHCII, CD40). T-cell proliferation was assessed by measuring T-cell–derived cytokine production by TLN cells in vitro. The staining profile was assessed to measure T-cell proliferation in vivo. RESULTS. The number of lung DCs was decreased among mice exposed to cigarette smoke. Exposed mice did not exhibit decreased migration of lung DCs to TLNs, as evidenced by similar numbers of TLN CD11c+FITC+ cells as in unexposed mice. Although no differences were observed in migration, smoking suppressed DC maturation in the lymph nodes as demonstrated by decreased cell-surface expression of MHC class II and the costimulatory molecules CD80 and CD86. T-cell proliferation was impaired in smoke-exposed mice, evidenced by reduced interleukin 2 production from CD4 T cells as compared with controls. T-cell proliferation was also decreased in vivo: smoke-exposed mice had a lower percentage of T cells undergoing ≥6 cycles or cell division compared with controls. CONCLUSIONS. These findings suggest that cigarette smoke is associated with defects in DC maturation and suppressed thoracic lymph node CD4+ T-cell proliferation in mice. REVIEWER COMMENTS. This study suggests that cigarette-smoke exposure in mice leads to impaired DC maturation and thereby diminished T-cell clonal expansion in the TLNs. The findings imply that, although the T-cell effector program remains intact, fewer cells might be available to perform those duties. The authors asserted that the observed effects on DC function might serve as a potential mechanism for cigarette smoke causing impaired tumor surveillance. In addition, it is suggested that impaired CD4+ T-cell function might predispose patients with obstructive lung disease to infections and exacerbations. Additional studies will help to elucidate the specific mechanisms by which cigarette-smoke exposure exerts its harmful immunologic effects.

Diesel-Enriched Particulate Matter Functionally Activates Human Dendritic Cells

Epidemiologic studies have associated exposure to airborne particulate matter (PM) with exacerbations of asthma. It is unknown how different sources of PM affect innate immunity. We sought to determine how car- and diesel exhaust-derived PM affects dendritic cell (DC) activation. DC development was modeled using CD34+ hematopoietic progenitors. Airborne PM was collected from exhaust plenums of Fort McHenry Tunnel providing car-enriched particles (CEP) and diesel-enriched particles (DEP). DC were stimulated for 48 hours with CEP, DEP, CD40-ligand, or lipopolysaccharide. DC activation was assessed by flow cytometry, enzyme-linked immunosorbent assay, and standard culture techniques. DEP increased uptake of fluorescein isothiocyanate-dextran (a model antigen) by DC. Diesel particles enhanced cell-surface expression of co-stimulatory molecules (e.g., CD40 [P < 0.01] and MHC class II [P < 0.01]). By contrast, CEP poorly affected antigen uptake and expression of cell surface molecules, and did not greatly affect cytokine secretion by DC. However, DEP increased production of TNF, IL-6, and IFN-gamma (P < 0.01), IL-12 (P < 0.05), and vascular endothelial growth factor (P < 0.001). In co-stimulation assays of PM-exposed DC and alloreactive CD4+ T cells, both CEP and DEP directed a Th2-like pattern of cytokine production (e.g., enhanced IL-13 and IL-18 and suppressed IFN-gamma production). CD4+ T cells were not functionally activated on exposure to either DEP or CEP. Car- and diesel-enriched particles exert a differential effect on DC activation. Our data support the hypothesis that DEP (and to a lesser extent CEP) regulate important functional aspects of human DC, supporting an adjuvant role for this material.

Sequential delivery of maturation stimuli increases human dendritic cell IL-12 production and enhances tumor antigen-specific immunogenicity

Background Despite the increasing use of dendritic cells (DCs) in clinical trials, questions regarding the optimal means of DC preparation, in particular how to achieve optimal maturation, remain unanswered. We hypothesized that delivering two separate sequential maturation signals to DC in vitro, mimicking the process of DC maturation that occurs in vivo, would enhance the ability of DCs to generate antigen-specific effector T cells in an experimental in vitro antimelanoma model. Materials and methods Human monocyte-derived DCs were transfected with mRNA encoding melanoma-associated antigen Mart-1 (MART) or influenza M1 matrix protein (M1). After mRNA transfection, DCs were left untreated or exposed to different maturation stimuli either added simultaneously or delivered sequentially 18 h after first stimulation. Phenotypic DC cell-surface marker changes and IL-12 secretion were analyzed. Specific antigen presentation by DCs was measured by IFN-γ release Elispot assay using a CD8 + MART peptide-specific T cell clone. RNA-transfected and treated DCs were cultured with autologous naive T cells and the induction of antigen-specific effector T cells were assessed by IFN-γ release Elispot assay. Results DCs transfected and matured had increased cell-surface expression of CD40 and costimulatory molecules CD80, and CD86. DCs matured and further treated by soluble CD40 ligand (sCD40L) had a 10- and 2-fold increase in MART antigen presentation compared to untreated (immature) DCs and DCs treated only with a first maturation signal, respectively (Elispot P = 0.02). Delivery of sequential maturation stimuli resulted in maximal DC IL-12 secretion compared to simultaneous stimuli. Last, generation of antigen-specific effector T cells more than doubled with the sequential addition of sCD40L to mature DC stimulators (Elispot P = 0.009). Conclusions Maturation of DCs following mRNA transfection increases expression of cell-surface costimulatory molecules. Delivery of a second sequential maturation stimulus enhances antigen presentation, increases IL-12 secretion, and augments immunogenicity as evidenced by generation of tumor antigen-specific effector T cells. This strategy should be considered in the future development of RNA-based DC vaccine strategies for the treatment of cancer.

Prolongation of cardiac allograft survival using dendritic cells treated with NF-kB decoy oligodeoxyribonucleotides.

Dendritic cells (DC) classically promote immune responses but can be manipulated to induce antigen-specific hyporesponsiveness in vitro. The expression of costimulatory molecules (CD40, CD86, CD80) at the DC cell surface correlates with their capacity to induce or suppress immune responses. Expression of these molecules is associated with NF-kB-dependent transcription of their genes. DC tolerogenicity has been associated with impaired NF-kB-dependent transcription of costimulatory genes as well as NF-kB translocation to the nucleus. In this report, we demonstrate that double-stranded oligodeoxyribonucleotides containing binding sites for NF-kB (NF-kB ODN) are efficiently incorporated by bone marrow-derived DC and specifically inhibit NF-kB-dependent transcription of a reporter gene. Moreover, exposure of DC to the oligonucleotide decoys inhibited lipopolysaccharide (LPS)-induced nitric oxide production, a marker of DC maturation. Treatment of bone marrow-derived DC progenitors with NF-kB ODN selectively suppressed the cell-surface expression of costimulatory molecules without interfering with MHC class I or class II expression. Furthermore, NF-kB ODN DC induced allogeneic donor-specific hyporesponsiveness in mixed leukocyte cultures, and this was associated with inhibition of Th1-type cytokine production. Finally, infusion of NF-kB ODN-modified bone marrow-derived DC into allogeneic recipients prior to heart transplantation resulted in significant prolongation of allograft survival in the absence of immunosuppression. Specific interference with NF-kB and other transcriptional pathways involved in immune stimulation in DC using ODN decoy approaches could be one means to promote tolerance induction in organ transplantation.

Measurement of lymphoproliferation at the single-cell level by flow cytometry.

The recognition phase of the cell-mediated immune response is studied in vitro by monitoring responses of CD4+ T cells, cultured in the presence of specific antigen. Conventional cultures include mononuclear cells separated from peripheral blood by a complex and time-consuming procedure. Several investigators (, , , , , , ) have suggested the use of whole-blood cultures which is easier to perform and involve less manipulations, thus reducing the risk of contamination, preactivation, or selective depletion or enrichment of cell subsets. The cell-mediated immune response involves a broad spectrum of measurable mechanisms. Multiparameter flow cytometry analysis permits the simultaneous detection of cell surface and intracellular molecules, as well as light-scatter characteristics of individual cells. In this chapter, a method is described for the measurement of proliferative responses in whole-blood cultures, using flow cytometric detection of lymphoblast development. The method is simple and reproducible, and can be combined with immunostaining for the detection of cytokine production, cell-surface expression of costimulatory molecules, and DNA synthesis.