Adaptive Immune Response to Model Antigens Is Impaired in Murine Leukocyte-Adhesion Deficiency-1 Revealing Elevated Activation Thresholds In Vivo

Absence of β2 integrins (CD11/CD18) leads to leukocyte-adhesion deficiency-1 (LAD1), a rare primary immunodeficiency syndrome. Although extensive in vitro work has established an essential function of β2 integrins in adhesive and signaling properties for cells of the innate and adaptive immune system, their respective participation in an altered adaptive immunity in LAD1 patients are complex and only partly understood in vivo. Therefore, we investigated adaptive immune responses towards different T-dependent antigens in a murine LAD1 model of β2 integrin-deficiency (CD18−/−). CD18−/− mice generated only weak IgG responses after immunization with tetanus toxoid (TT). In contrast, robust hapten- and protein-specific immune responses were observed after immunization with highly haptenated antigens such as (4-hydroxy-3-nitrophenyl)21 acetyl chicken γ globulin (NP21-CG), even though regularly structured germinal centers with specificity for the defined antigens/haptens in CD18−/− mice remained absent. However, a decrease in the hapten/protein ratio lowered the efficacy of immune responses in CD18−/− mice, whereas a mere reduction of the antigen dose was less crucial. Importantly, haptenation of TT with NP (NP-TT) efficiently restored a robust IgG response also to TT. Our findings may stimulate further studies on a modification of vaccination strategies using highly haptenated antigens in individuals suffering from LAD1

Homing and homeostasis of T lymphocytes in CD18 deficient mice

Expressed on leukocytes, beta2 integrins (CD11/CD18) are involved in leukocyte function and trafficking. Using a CD18-deficient (CD18-/-) mouse model, the role of beta2 integrins for lymphocyte differentiation and recirculation was herein investigated. CD18-/- mice revealed a defect in distribution of naïve lymphocytes, which were reduced in numbers in axillary and inguinal lymph nodes (LN). In contrast, cervical LN (cLN) were signi¬ficantly enlarged and harbored elevated numbers of unconventional T cell receptor (TCR) alpha/beta or TCR gamma/delta double negative (DN) T cells. As demonstrated by adoptive transfer experiments, a selective homing of CD18-/- lymphocyte to pLN of wild type recipients was not the reason for this increase in cellularity. But unconventional DN T cells were found to be increased in cLNs due to a massive local expansion demonstrated by in vivo incorporation of thymidine analogue BrdU. In accordance, CD18-/- DN T cells showed an activated phenotype and recirculated through non-lymphoid organs resembling anti¬gen-experienced T and NKT cells. CD18-/- TCR alpha/beta DN T cells readily pro¬liferated upon IL-2, IL-7 and IL-15, known to regulate homeostasis or antigen-induced expansion of NKT and TCR gamma/delta T cells. In accordance with the assumption that CD18-/- TCR alpha/beta DN T cells are expanding effector cells, they also lacked suppressive function on proliferating WT T cell in vitro. Interestingly, CD18-/- TCR alpha/beta T cells revealed general characteristics of NKT cells but lacking re¬activity for CD1d/alpha-galactosylceramide showed that they were not type I NKT cells. Nevertheless, the possi¬bility that CD18-/- TCR alpha/beta DN T cells are a different subset of NKT cells cannot be fully excluded, currently. However, lack of IL-4 and IFN-gamma cytokine secretion after stimulation showed that CD18-/- TCR alpha/beta DN T cells may have different functional properties to NKT and TCR gamma/delta T cells in innate im¬munity. Collectively, DN T cells accumulated either due to antigen-dependent and/or homeostatic expansion in the periphery, likely driven by a cytokine imbalance in CD18-/- mice. The presented data indicates that CD18-/- TCR alpha/beta DN T cells, like NKT and TCR gamma/delta T cells, may be a unique subset of unconventional T cells at the interface to innate immunity accumulating compensatory to the impaired function of adaptive immunity in CD18-/- mice

Generation of human induced pluripotent stem cells from adipose-derived stromal/stem cells isolated from a 75-year-old patient

Human adipose-derived stromal/stem cells (hASCs) have been considered a valuable cell source for generating induced pluripotent stem cells (iPSCs). Adipose tissue is easy to obtain. Moreover, the isolated cells rapidly proliferate to reach the required number of cells. Some authors have already shown that iPSCs can be successfully obtained using adult hASCs. Nevertheless, little is known about the generation of iPSCs using hASCs isolated from the tissues of patients over the age of 70. In this study, we examined the generation of iPSCs from hASCs isolated from a 75-year-old man. We transduced hASCs with human transcription factors OCT4, SOX2, c-MYC and KLF4 and observed the formation of human embryonic stem cell (hESC)-like colonies. The efficiency of the reprogramming process was 0.08% at day 18 post-infection. Reprogrammed cells expressed pluripotent state-specific transcription factors OCT4, SOX2, NANOG and KLF4, and were able to differentiate into three germ layers in vitro

Cell Behavioral Changes after the Application of Magneto-Mechanical Activation to Normal and Cancer Cells

In vitro cell exposure to nanoparticles, depending on the applied concentration, can help in the development of theranostic tools to better detect and treat human diseases. Recent studies have attempted to understand and exploit the impact of magnetic field-actuated internalized magnetic nanoparticles (MNPs) on the behavior of cancer cells. In this work, the viability rate of MNP’s-manipulated cancerous (MCF-7, MDA-MB-231) and non-cancerous (MCF-10A) cells was investigated in three different types of low-frequency magnetic fields: static, pulsed, and rotating field mode. In the non-cancerous cell line, the cell viability decreased mostly in cells with internalized MNPs and those treated with the pulsed field mode. In both cancer cell lines, the pulsed field mode was again the optimum magnetic field, which together with internalized MNPs caused a large decrease in cells’ viability (50–55% and 70% in MCF-7 and MDA-MB-231, respectively) while the static and rotating field modes maintained the viability at high levels. Finally, F-actin staining was used to observe the changes in the cytoskeleton and DAPI staining was performed to reveal the apoptotic alterations in cells’ nuclei before and after magneto-mechanical activation. Subsequently, reduced cell viability led to a loss of actin stress fibers and apoptotic nuclear changes in cancer cells subjected to MNPs triggered by a pulsed magnetic field

TGF-β–dependent suppressive function of Tregs requires wild-type levels of CD18 in a mouse model of psoriasis

Dysfunctional Tregs have been identified in individuals with psoriasis. However, their role in the pathogenesis of the disease remains unclear. Here we explored the effect of diminished CD18 (β2 integrin) expression on the function of CD4+CD25+CD127– Tregs using the Cd18 hypomorphic (Cd18hypo) PL/J mouse model of psoriasis that closely resembles the human disease. We found that reduced CD18 expression impaired cell-cell contact between Tregs and DCs. This led to dysfunctional Tregs, which both failed to suppress the pathogenic T cells and promoted the onset and severity of the disease. This failure was TGF-β–dependent, as Tregs derived from Cd18hypo PL/J mice had diminished TGF-β1 expression. Adoptive transfer of Tregs expressing wild-type levels of CD18 into affected Cd18hypo PL/J mice resulted in a substantial improvement of the psoriasiform skin disease, which did not occur upon coinjection of the cells with TGF-β–specific neutralizing antibody. Our data indicate a primary dysfunction of Cd18hypo Tregs, allowing subsequent hyperproliferation of pathogenic T cells in the Cd18hypo PL/J mouse model of psoriasis. This study may provide a step forward in our understanding of the unique role of CD18 expression levels in avoiding autoimmunity

Wound-healing defect of CD18(−/−) mice due to a decrease in TGF-β(1) and myofibroblast differentiation

We studied the mechanisms underlying the severely impaired wound healing associated with human leukocyte-adhesion deficiency syndrome-1 (LAD1) using a murine disease model. In CD18(−/−) mice, healing of full-thickness wounds was severely delayed during granulation-tissue contraction, a phase where myofibroblasts play a major role. Interestingly, expression levels of myofibroblast markers α-smooth muscle actin and ED-A fibronectin were substantially reduced in wounds of CD18(−/−) mice, suggesting an impaired myofibroblast differentiation. TGF-β signalling was clearly involved since TGF-β(1) and TGF-β receptor type-II protein levels were decreased, while TGF-β(1) injections into wound margins fully re-established wound closure. Since, in CD18(−/−) mice, defective migration leads to a severe reduction of neutrophils in wounds, infiltrating macrophages might not phagocytose apoptotic CD18(−/−) neutrophils. Macrophages would thus be lacking their main stimulus to secrete TGF-β(1). Indeed, in neutrophil–macrophage cocultures, lack of CD18 on either cell type leads to dramatically reduced TGF-β(1) release by macrophages due to defective adhesion to, and subsequent impaired phagocytic clearance of, neutrophils. Our data demonstrates that the paracrine secretion of growth factors is essential for cellular differentiation in wound healing