Neutrophils Which Migrate to Lymph Nodes Modulate CD4+ T Cell Response by a PD-L1 Dependent Mechanism

It is well known that neutrophils are rapidly recruited to a site of injury or infection and perform a critical role in pathogen clearance and inflammation. However, they are also able to interact with and regulate innate and adaptive immune cells and some stimuli induce the migration of neutrophils to lymph nodes (LNs). Previously, we demonstrated that the immune complex (IC) generated by injecting OVA into the footpad of OVA/CFA immunized mice induced the migration of OVA+ neutrophils to draining LNs (dLNs). Here we investigate the effects of these neutrophils which reach dLNs on CD4+ T cell response. Our findings here strongly support a dual role for neutrophils in dLNs regarding CD4+ T cell response modulation. On the one hand, the CD4+ T cell population expands after the influx of OVA+ neutrophils to dLNs. These CD4+ T cells enlarge their proliferative response, activation markers and IL-17 and IFN-γ cytokine production. On the other hand, these neutrophils also restrict CD4+ T cell expansion. The neutrophils in the dLNs upregulate PD-L1 molecules and are capable of suppressing CD4+ T cell proliferation. These results indicate that neutrophils migration to dLNs have an important role in the homeostasis of adaptive immunity. This report describes for the first time that the influx of neutrophils to dLNs dependent on IC presence improves CD4+ T cell response, at the same time controlling CD4+ T cell proliferation through a PD-L1 dependent mechanism

Class-B CpG-ODN Formulated With a Nanostructure Induces Type I Interferons-Dependent and CD4+ T Cell-Independent CD8+ T-Cell Response Against Unconjugated Protein Antigen

There is a need for new vaccine adjuvant strategies that offer both vigorous antibody and T-cell mediated protection to combat difficult intracellular pathogens and cancer. To this aim, we formulated class-B synthetic oligodeoxynucleotide containing unmethylated cytosine-guanine motifs (CpG-ODN) with a nanostructure (Coa-ASC16 or coagel) formed by self-assembly of 6-0-ascorbyl palmitate ester. Our previous results demonstrated that mice immunized with ovalbumin (OVA) and CpG-ODN formulated with Coa-ASC16 (OVA/CpG-ODN/Coa-ASC16) elicited strong antibodies (IgG1 and IgG2a) and Th1/Th17 cellular responses without toxic systemic effects. These responses were superior to those induced by a solution of OVA with CpG-ODN or OVA/CpG-ODN formulated with aluminum salts. In this study, we investigated the capacity of this adjuvant strategy (CpG-ODN/Coa-ASC16) to elicit CD8+ T-cell response and some of the underlying cellular and molecular mechanisms involved in adaptive response. We also analyzed whether this adjuvant strategy allows a switch from an immunization scheme of three-doses to one of single-dose. Our results demonstrated that vaccination with OVA/CpG-ODN/Coa-ASC16 elicited an antigen-specific long-lasting humoral response and importantly-high quality CD8+ T-cell immunity with a single-dose immunization. Moreover, Coa-ASC16 promoted co-uptake of OVA and CpG-ODN by dendritic cells. The CD8+ T-cell response induced by OVA/CpG-ODN/Coa-ASC16 was dependent of type I interferons and independent of CD4+ T-cells, and showed polyfunctionality and efficiency against an intracellular pathogen. Furthermore, the cellular and humoral responses elicited by the nanostructured formulation were IL-6-independent. This system provides a simple and inexpensive adjuvant strategy with great potential for future rationally designed vaccines

Development and validation of a high performance liquid chromatography method for oligodeoxynucleotides determination in a novel coagel-based formulation

The therapeutic benefit of phosphorothioate oligodeoxynucleotides (PS-ODN) containing immune stimulatory sequences has been demonstrated in animal models of cancer and infection. Several tools are available for the determination of these oligonucleotides in biological samples and pharmaceutical preparations, including UV spectroscopy, dye binding, isotopic tracing, capillary gel electrophoresis (CGE), hybridization-based enzyme-linked immunosorbent assay (ELISA), and chromatography techniques. However, due to inter-assay variability and accuracy problems associated with the afore mentioned methods, we have developed and validated an isocratic high performance liquid chromatographic (HPLC) for analytical determination of PS-ODN containing unmethylated CpG motifs (CpG-ODN). Validation under Food and Drug Administration (FDA) guidelines of the analytical parameters include: linearity (r2 0.9996), LOD (0.86 μg/ml) and LOQ (6.25 μg/ml), intra (0.19–3.37%) and inter-day precision (0.63–3.75%) expressed as relative standard deviation (RSD), and robustness parameters (less than 2.80%). Using this method, recoveries ranging from 89.9% to 99.9% were obtained. Thus, this method provides a simple, sensitive, precise and reproducible examination which can be readily adapted for the assessment of CpG-ODN in different pharmaceutical preparations

CpG-ODN + IFN-γ confer pro- and anti-inflammatory properties to peritoneal macrophages in aged mice

Aging is accompanied by a disturbance in the homeostasis of the immune system. However, research into the behavior of macrophages in aging has shown disagreements about the functional status of these cells in aged mice. In this work, we studied the influence of aging on macrophage functions by evaluating the pro- and anti-inflammatory parameters of peritoneal macrophages preserved in their natural microenvironment. Resident peritoneal macrophages from old mice, in the context of their natural milieu, were found to respond with a similar phenotype and functional pattern to macrophages from young mice. In addition, we evaluated the macrophage response to CpG-ODN, a well-known Th1 promoter. CpG-ODN + IFN-γ were able to activate not only nitric oxide to initiate the inflammatory response, but also IL-12 in resident and inflammatory peritoneal macrophages from aged mice in the context of their natural milieu, although some quantitative differences were found in IL-10 and IL-12 secretion. With this stimulus, NO secretion and arginase activation were maintained in peritoneal macrophages during aging. These results will help to elucidate potential immunization strategies with CpG-ODN in the elderly.Fil: Liscovsky, Miriam Veronica. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina; ArgentinaFil: Ranocchia, Romina Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Bioquímica Clínica E Inmunología; Argentina; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Alignani, Diego O.. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina; ArgentinaFil: Gorlino, Carolina Virginia. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina; ArgentinaFil: Moron, Victor Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Bioquímica Clínica E Inmunología; Argentina; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Maletto, Belkys A.. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina; ArgentinaFil: Pistoresi, Maria Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones En Bioquímica Clínica E Inmunología; Argentina; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentin

Aging Impairs the Ability of Conventional Dendritic Cells to Cross-Prime CD8⁺ T Cells upon Stimulation with a TLR7 Ligand

<div><p>The aging process is accompanied by altered immune system functioning and an increased risk of infection. Dendritic cells (DCs) are antigen-presenting cells that play a key role in both adaptive and innate immunity, but how aging affects DCs and their influence on immunity has not been thoroughly established. Here we examined the function of conventional DCs (cDCs) in old mice after TLR7 stimulation, focusing on their ability to cross-prime CD8⁺ T cells. Using polyU, a synthetic ssRNA analog, as TLR7 ligand and OVA as an antigen (Ag) model, we found that cDCs from old mice have a poor ability to stimulate a CD8⁺ T cell-mediated cytotoxic response. cDCs from old mice exhibit alterations in Ag-processing machinery and TLR7 activation. Remarkably, CD8α⁺ cDCs from old mice have an impaired ability to activate naïve CD8⁺ T cells and, moreover, a lower capacity to mature and to process exogenous Ag. Taken together, our results suggest that immunosenescence impacts cDC function, affecting the activation of naïve CD8⁺ T cells and the generation of effector cytotoxic T cells.</p></div

Aged splenic cDCs have impaired ability to cross-prime naïve CD8⁺ T cells <i>in vitro</i>.

<p>Total (A-D) or CD8α⁺ (E) cDCs purified from young and old mice were incubated with 1 mg/mL OVA mixed with 20 μg/mL polyU/DO for 90 minutes. Additional cDCs from young and old mice were incubated with RPMI or OVA as control. cDCs were then washed and cultured for 3 days with CFSE-labeled CD8β⁺ T cells isolated from the spleen of OT-I mice at different DC:T cell ratios. After culture, T cell proliferation and CD25 expression were analyzed by flow cytometry. (A) Representative histograms of T cell proliferation are shown from 1:1 ratio. (B, E) Percentages of proliferating T cells, (C) CD25 expression and (D) IFN-γ content in culture supernatants, determined by ELISA. Data show the mean ± SEM. *p < 0.05, **p < 0.01. Results are representative of 3 independent experiments (3–4 mice/age group/experiment).</p