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CD8 + memory T cells (T M ) are crucial for long-term protection from infections and cancer. Multiple cell types and cytokines are involved in the regulation of CD8 + T cell responses and subsequent T M formation. Besides their direct antiviral effects, type I interferons (IFN-I) modulate CD8 + T cell immunity via their action on several immune cell subsets. However, it is largely unclear how nonimmune cells are involved in this multicellular network modulating CD8 + T M formation. Fibroblastic reticular cells (FRCs) form the 3D scaffold of secondary lymphoid organs, express the IFN-I receptor (IFNAR), and modulate adaptive immune responses. However, it is unclear whether and how early IFNAR signals in lymph node (LN) FRCs affect CD8 + T M differentiation. Using peptide vaccination and viral infection, we studied CD8 + T M differentiation in mice with an FRC-specific IFNAR deletion (FRC ΔIFNAR ). We show here that the differentiation of CD8 + TCR-transgenic T cells into central memory cells (T CM ) is enhanced in peptide-vaccinated FRC ΔIFNAR mice. Conversely, vesicular stomatitis virus infection of FRC ΔIFNAR mice is associated with impaired T CM formation and the accumulation of vesicular stomatitis virus specific double-positive CD127 hi KLRG-1 hi effector memory T cells. In summary, we provide evidence for a context-dependent contribution of FRC-specific IFNAR signaling to CD8 + T M differentiation., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Cerebral infections are restrained by a complex interplay of tissue-resident and recruited peripheral immune cells. Whether innate lymphoid cells (ILCs) are involved in the orchestration of the neuroinflammatory dynamics is not fully understood. Here, we demonstrate that ILCs accumulate in the cerebral parenchyma, the choroid plexus, and the meninges in the onset of cerebral Toxoplasma gondii infection. Antibody-mediated depletion of conventional natural killer (cNK) cells and ILC1s in the early stage of infection results in diminished cytokine and chemokine expression and increased cerebral parasite burden. Using cNK- and ILC1-deficient murine models, we demonstrate that exclusively the lack of ILC1s affects cerebral immune responses. In summary, our results provide evidence that ILC1s are an early source of IFN-γ and TNF in response to cerebral T. gondii infection, thereby inducing host defense factors and initiating the development of a neuroinflammatory response., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers., (© 2021 Wiley-VCH GmbH.)

Targeted antigen delivery to cross-presenting dendritic cells (DC) in vivo efficiently induces T effector cell responses and displays a valuable approach in vaccine design. Antigen is delivered to DC via antibodies specific for endocytosis receptors such as DEC-205 that induce uptake, processing, and MHC class I- and II-presentation. Efficient and reliable conjugation of the desired antigen to a suitable antibody is a critical step in DC targeting and among other factors depends on the format of the antigen. Chemical conjugation of full-length protein to purified antibodies is one possible strategy. In the past, we have successfully established cross-linking of the model antigen ovalbumin (OVA) and a DEC-205-specific IgG2a antibody (αDEC-205) for in vivo DC targeting studies in mice. The first step of the protocol is the purification of the antibody from the supernatant of the NLDC (non-lymphoid dendritic cells)-145 hybridoma by affinity chromatography. The purified antibody is activated for chemical conjugation by sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate) while at the same time the sulfhydryl-groups of the OVA protein are exposed through incubation with TCEP-HCl (tris (2-carboxyethyl) phosphine hydrochloride). Excess TCEP-HCl and sulfo-SMCC are removed and the antigen is mixed with the activated antibody for overnight coupling. The resulting αDEC-205/OVA conjugate is concentrated and freed from unbound OVA. Successful conjugation of OVA to αDEC-205 is verified by western blot analysis and enzyme-linked immunosorbent assay (ELISA). We have successfully used chemically crosslinked αDEC-205/OVA to induce cytotoxic T cell responses in the liver and to compare different adjuvants for their potential in inducing humoral and cellular immunity following in vivo targeting of DEC-205 + DC. Beyond that, such chemically coupled antibody/antigen conjugates offer valuable tools for the efficient induction of vaccine responses to tumor antigens and have been proven to be superior to classical immunization approaches regarding the prevention and therapy of various types of tumors.

The survival of peripheral T cells is dependent on their access to peripheral LNs (pLNs) and stimulation by IL-7. In pLNs fibroblastic reticular cells (FRCs) and lymphatic endothelial cells (LECs) produce IL-7 suggesting their contribution to the IL-7-dependent survival of T cells. However, IL-7 production is detectable in multiple organs and is not restricted to pLNs. This raises the question whether pLN-derived IL-7 is required for the maintenance of peripheral T cell homeostasis. Here, we show that numbers of naive T cells (T N ) remain unaffected in pLNs and spleen of mice lacking Il7 gene activity in pLN FRCs, LECs, or both. In contrast, frequencies of central memory T cells (T CM ) are reduced in FRC-specific IL-7 KO mice. Thus, steady state IL-7 production by pLN FRCs is critical for the maintenance of T CM , but not T N , indicating that both T cell subsets colonize different ecological niches in vivo., (© 2020 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

NKp46 + innate lymphoid cells (ILC) modulate tissue homeostasis and anti-microbial immune responses. ILC development and function are regulated by cytokines such as Interleukin (IL)-7 and IL-15. However, the ILC-intrinsic pathways translating cytokine signals into developmental programs are largely unknown. Here we show that the anti-apoptotic molecule cellular FLICE-like inhibitory protein (c-FLIP) is crucial for the generation of IL-7/IL-15-dependent NKp46 + ILC1, including conventional natural killer (cNK) cells, and ILC3. Cytokine-induced phosphorylation of signal transducer and activator of transcription 5 (STAT5) precedes up-regulation of c-FLIP, which protects developing NKp46 + ILC from TNF-induced apoptosis. NKp46 + ILC-specific inactivation of c-FLIP leads to the loss of all IL-7/IL-15-dependent NKp46 + ILC, thereby inducing early-onset chronic colitis and subsequently microbial dysbiosis; meanwhile, the depletion of cNK, but not NKp46 + ILC1/3, aggravates experimental colitis. In summary, our data demonstrate a non-redundant function of c-FLIP for the generation of NKp46 + ILC, which protect T/B lymphocyte-sufficient mice from intestinal inflammation.

Vaccination is the most efficient strategy to protect from infectious diseases and the induction of a protective immune response not only depends on the nature of the antigen, but is also influenced by the vaccination strategy and the co-administration of adjuvants. Therefore, the precise monitoring of adjuvant candidates and their immune modulatory properties is a crucial step in vaccine development. Here, one central aspect is the induction of appropriate humoral and cellular effector mechanisms. In our study we performed a direct comparison of two promising candidates in adjuvant development, the STING activator bis-(3,5)-cyclic dimeric adenosine monophosphate (c-di-AMP) and the Toll-like receptor ligand formulation poly(I:C)/CpG. These were evaluated in C57BL/6 mice using the model antigen ovalbumin (OVA) in subcutaneous vaccination with soluble protein as well as in a dendritic cell (DC) targeting approach (αDEC-OVA). Strikingly, c-di-AMP as compared to poly(I:C)/CpG resulted in significantly higher antigen-specific IgG antibody levels when used in immunization with soluble OVA as well as in antigen targeting to DC. In vaccination with soluble OVA, c-di-AMP induced a significantly stronger CTL, Th1 and IFNγ-producing CD8 + memory T cell response than poly(I:C)/CpG. The response was CTL and Th1 cell dominated, a profile shared by both adjuvants. In the context of targeting OVA to DC, c-di-AMP induced significantly increased Th1 and Th2 cell responses as compared to poly(I:C)/CpG. Interestingly, the Th1 response dominated the overall T cell response only when c-di-AMP was used, indicating a distinct modulatory property of c-di-AMP when the DC targeting immunization approach was exploited. Taken together, we describe superior properties of c-di-AMP as compared to poly(I:C)/CpG in subcutaneous vaccination with soluble antigen as well as antigen targeting to DC. This indicates exceptionally effective adjuvant properties for c-di-AMP and provides compelling evidence of its potential for further adjuvant development, especially also when using DC targeting approaches., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Epigenetic mechanisms may play a major role in the biological embedding of early-life stress (ELS). One proposed mechanism is that glucocorticoid (GC) release following ELS exposure induces long-lasting alterations in DNA methylation (DNAm) of important regulatory genes of the stress response. Here, we investigate the dynamics of GC-dependent methylation changes in key regulatory regions of the FKBP5 locus in which ELS-associated DNAm changes have been reported., Results: We repeatedly measured DNAm in human peripheral blood samples from 2 independent cohorts exposed to the GC agonist dexamethasone (DEX) using a targeted bisulfite sequencing approach, complemented by data from Illumina 450K arrays. We detected differentially methylated CpGs in enhancers co-localizing with GC receptor binding sites after acute DEX treatment (1 h, 3 h, 6 h), which returned to baseline levels within 23 h. These changes withstood correction for immune cell count differences. While we observed main effects of sex, age, body mass index, smoking, and depression symptoms on FKBP5 methylation levels, only the functional FKBP5 SNP (rs1360780) moderated the dynamic changes following DEX. This genotype effect was observed in both cohorts and included sites previously shown to be associated with ELS., Conclusion: Our study highlights that DNAm levels within regulatory regions of the FKBP5 locus show dynamic changes following a GC challenge and suggest that factors influencing the dynamics of this regulation may contribute to the previously reported alterations in DNAm associated with current and past ELS exposure.

In lymphopenic mice, T cells become activated and undergo lymphopenia-induced proliferation (LIP). However, not all T cells are equally sensitive to lymphopenia. Several lymphopenia-insensitive T cell clones were described and their non-responsiveness was mainly attributed to clone-specific properties. Here, we provide evidence for an additional, host-dependent mechanism restraining LIP of lymphopenia-insensitive CD4 + T cells. We show that such cells undergo LIP in lymphopenic mice lacking IFN-γ receptor (IFN-γR) expression, a process, which is promoted by the autocrine action of T cell-derived IFN-γ. Additionally, LIP of lymphopenia-insensitive CD4 + T cells requires an intact microflora and is accompanied by the massive accumulation of IL-6 and dendritic cells (DCs). Consistent with these results, IL-6 neutralization and the DC-specific restoration of IFN-γR expression are both sufficient to restrict LIP. Hence, the insensitivity of CD4 + T cells to lymphopenia relies on cell-intrinsic properties and a complex interplay between the commensal microflora, IL-6, IFN-γR + DCs, and T cell-derived IFN-γ.

This paper describes the methods and experiment protocols for estimation of the human ankle impedance during turning and straight line walking. The ankle impedance of two human subjects during the stance phase of walking in both dorsiflexion plantarflexion (DP) and inversion eversion (IE) were estimated. The impedance was estimated about 8 axes of rotations of the human ankle combining different amounts of DP and IE rotations, and differentiating among positive and negative rotations at 5 instants of the stance length (SL). Specifically, the impedance was estimated at 10%, 30%, 50%, 70% and 90% of the SL. The ankle impedance showed great variability across time, and across the axes of rotation, with consistent larger stiffness and damping in DP than IE. When comparing straight walking and turning, the main differences were in damping at 50%, 70%, and 90% of the SL with an increase in damping at all axes of rotation during turning.

Introduction: The finite element method (FEM) is an engineering resource applied to calculate the stress and deformation of complex structures, and has been widely used in orthodontic research. With the advantage of being a non-invasive and accurate method that provides quantitative and detailed data on the physiological reactions possible to occur in tissues, applying the FEM can anticipate the visualization of these tissue responses through the observation of areas of stress created from applied orthodontic mechanics., Objective: This article aims at reviewing and discussing the stages of the finite element method application and its applicability in Orthodontics., Results: FEM is able to evaluate the stress distribution at the interface between periodontal ligament and alveolar bone, and the shifting trend in various types of tooth movement when using different types of orthodontic devices. Therefore, it is necessary to know specific software for this purpose., Conclusions: FEM is an important experimental method to answer questions about tooth movement, overcoming the disadvantages of other experimental methods.

Introduction: Nicotine is harmful to angiogenesis, osteogenesis and synthesis of collagen., Objective: The aim of this study was to investigate the effect of nicotine on bone remodeling during orthodontic movement in rats., Methods: Eighty male Wistar rats were randomly divided into three groups: Group C (control), group CM (with orthodontic movement) and group NM (nicotine with orthodontic movement) groups. The animals comprising groups C and CM received 0.9% saline solution while group NM received nicotine solution (2 mg/kg). A nickel-titanium closed-coil spring was used to induce tooth movement. The animals were euthanized and tissue specimens were histologically processed. Blood vessels, Howship's lacunae and osteoclast-like cells present in the tension and compression areas of periodontal ligaments were quantified. The extent of bone formation was evaluated under polarized light, to determine the percentage of immature/mature collagen., Results: It was observed lower blood vessel densities in the NM group in comparison to the CM group, three (p < 0.001) and seven (p < 0.05) days after force application. Osteoclast-like cells and Howship's lacunae in the NM group presented lower levels of expression, in comparison to the CM group, with significant differences on day 7 (p < 0.05 for both variables) and day 14 (p < 0.05 for osteoclast-like cells and p < 0.01 for Howship's lacunae). The percentage of immature collagen was increased in the NM group in comparison to the CM group, with a statistically significant difference on day 3 (p < 0.05), day 7 (p < 0.001), day 14 (p < 0.001) and day 21 (p < 0.001)., Conclusions: Nicotine affects bone remodeling during orthodontic movement, reducing angiogenesis, osteoclast-like cells and Howship's lacunae, thereby delaying the collagen maturation process in new bone matrix.

Chronic lymphocytic leukemia (CLL) is predominantly a disease of accumulation rather than rapid proliferation. To date, no cell lines exist, as CLL cells undergo rapid apoptosis when cultured in vitro, suggesting that a favorable in vivo microenvironment is required. To identify survival signals we cultured primary CLL peripheral blood mononuclear cells (PBMCs) at high density, which has previously been shown to dramatically improve survival. Using antibody arrays we measured the level of 42 cytokines in culture supernatants and showed that inerleukin-6 (IL-6), IL-8, CXCL2 and CCL2 were highly up-regulated in culture. This is the first report to describe a role for CCL2 and CXCL2 in CLL cell survival. Importantly, CXCL2, IL-6 and IL-8 were significantly up-regulated in primary patient plasma. The addition of either CXCL2 or CCL2 enhanced CLL cell survival, while antibodies blocking these chemokines reduced survival. Co-culture of CLL cells and PBMC accessory cells separated by transwells provided a similar degree of survival protection compared to normal culture, whereas CLL cells cultured alone died rapidly. Interestingly, CCL2 and CXCL2 appeared to be produced by CLL cells but only when co-cultured with accessory cells. Thus, we speculate that accessory cells release soluble factors that promote the production of these pro-survival chemokines from CLL cells and physical interactions are not required. Our data support the concept that the CLL microenvironment is critical, and suggests that soluble factors are more important than physical interactions.

Bone formation is essential to orthodontic tooth movement and bone is formed by collagen. To analyze the collagen maturation process on bone matrix neoformed under nonsteroidal and steroidal treatment during orthodontic tooth movement by polarized microscopy, male Wistar rats (n = 90) were randomly divided into three groups (n = 30): C (control), NSAID (potassium diclofenac) and SAID (disodic phosphate dexamethasone). The animals of the C group received 0.9% saline solution; NSAID group received 5 mg/kg potassium diclofenac (CATAFLAM®); and SAID group received 2 mg/kg phosphate dissodic dexamethasone (DEXANIL®). Animals were sacrificed 3, 7 or 14 days after the placement of orthodontic appliances and the upper first molars were processed histologically and stained with picrosirius. Bone formation was evaluated under polarized light microscopy and 4.5 Image Pro-Plus® software calculated the percentage of immature/mature collagen present in the groups. On the third days after force application, SAID and NSAID groups showed greater proportion of immature collagen than C group. On the seventh and fourteenth days, there was a lower proportion of mature collagen only in the SAID group (P < 0.001). These data demonstrate that dexamethasone delays the collagen maturation process in established bone matrix., (Copyright © 2010 Wiley-Liss, Inc.)