Different roles of PD-L1 and FasL in immunomodulation mediated by human placenta-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) derived from either bone marrow (BMSCs) or placenta (PMSCs) have the capacity to suppress immune responses to mitogenic and allogeneic stimulations. Both cell contact and soluble factor dependent mechanisms have been proposed to explain this immunosuppression. This study explored the roles of some of cell surface molecules expressed on human PMSCs (hPMSCs) in hPMSC mediated immunomodulation. hPMSCs strongly suppressed mitogen and allogeneic peripheral mononuclear cells (PBMCs) induced T cell activation and proliferation. hPMSCs constituently expressed programmed death-ligand 1 (PD-L1) and Fas ligand (FasL) molecules. Neutralising antibodies to-PD-L1 and FasL significantly reduced the suppressive effect of hPMSCs on T cell proliferation. However, only anti-PD-L1 antibody partially restored early T cell activation suppressed by hPMSCs. Anti-FasL antibody but not anti-PD-L1 antibody reduced apoptosis of activated T cell indicating that FasL molecule plays a role in inducing apoptosis of activated T cells, although overall hPMSCs diminished T cell apoptosis. Different effects of PD-L1 and FasL molecules on T cell activation and activated T cell apoptosis suggest that these two molecules influence T cell response at different stages. hPMSCs significantly prevented activated T cells from going into S phase. Both antibodies to PD-L1 and FasL had significant effect on reversing the effect of hPMSCs on cell cycles. hPMSCs reduced INF-γ but increased IL-10 production by mitogen activated T cells. Both antibodies partially abolished the effect of hPMSCs on INF-γ and IL-10 production. These data demonstrated that PD-L1 and FasL molecules play significant roles in immunomodulation mediated by hPMSCs. This study provides a rational basis for modulation of negative costimulators on hPMSCs to increase their immunosuppressive properties in their therapeutic applications.     

Kinetics of phytohemaglutinin-induced IFN-γ and TNF-α expression in peripheral blood mononuclear cells from patients with chronic hepatitis B after liver transplantation

AIM: To study the association between host immunity and hepatitis B virus (HBV) recurrence after liver transplantation. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 40 patients with hepatitis B and underwent orthotopic liver transplantation (OLT) before and 2, 4, 8 wk after surgery. After being cultured in vitro for 72 h, the levels of INF-gamma and TNF-alpha in culture supernatants were detected with ELISA. At the same time, the quantities of HBV DNA in serum and PBMCs were measured by real time PCR. RESULTS: The levels of INF-gamma and TNF-alpha in PBMC culture supernatants decreased before and 2, 4 wk after surgery in turns (INF-gamma 155.52+/-72.32 ng/L vs 14.76+/-9.88 ng/L vs 13.22+/-10.35 ng/L, F = 6.946, P = 0.027 < 0.05; TNF-alpha 80.839+/-46.75 ng/L vs 18.59+/-17.29 ng/L vs 9.758+/-7.96 ng/L, F = 22.61, P = 0.0001 < 0.05). The levels of INF-gamma and TNF-alpha were higher in groups with phytohemagglutinin (PHA) than in those without PHA before surgery. However, the difference disappeared following OLT. Furthermore, INF-gamma and TNF-alpha could not be detected in most patients at wk 4 and none at wk 8 after OLT. The HBV detection rate and virus load in PBMC before and 2, 4 wk after surgery were fluctuated (HBV detected rate: 51.4%, 13.3%, 50% respectively; HBV DNA: 3.55+/-0.674 log10 copies/mL vs 3.00+/-0.329 log10 copies/mL vs 4.608+/-1.344 log10 copies/mL, F = 7.582, P = 0.002 < 0.05). HBV DNA in serum was 4.48+/-1.463 log10 copies/mL before surgery and <10(3) copies/mL after OLT except for one with 5.72 x 10(6) copies/mL 4 wk after OLT who was diagnosed as HBV recurrence. The levels of INF-gamma and TNF-alpha were lower in patients with a high HBV load than in those with a low HBV load (HBV DNA detected/undetected in PBMCs: IFN-gamma 138.08+/-72.44 ng/L vs 164.24+/-72.07 ng/L, t = 1.065, P = 0.297 > 0.05, TNF-alpha 80.75+/-47.30 ng/L vs 74.10+/-49.70 ng/L, t = 0.407, P = 0.686 > 0.05; HBV DNA positive/negative: IFN-gamma 136.77+/-70.04 ng/L vs 175.27+/-71.50 ng/L, t = 1.702, P = 0.097 > 0.05; TNF-alpha 75.37+/-43.02 ng/L vs 81.53+/-52.46 ng/L, t = 0.402, P = 0.690 > 0.05). CONCLUSION: The yielding of INF-gamma and TNF-alpha from PBMCs is inhibited significantly by immunosuppressive agents following OLT with HBV load increased, indicating that the impaired immunity of host is associated with HBV recurrence after OLT.     

Aging increases mitochondrial DNA damage and oxidative stress in liver of rhesus monkeys

While the mechanisms of cellular aging remain controversial, a leading hypothesis is that mitochondrial oxidative stress and mitochondrial dysfunction play a critical role in this process. Here, we provide data in aging rhesus macaques supporting the hypothesis that increased oxidative stress is a major characteristic of aging and may be responsible for the age-associated increase in mitochondrial dysfunction. We measured mitochondrial DNA (mtDNA) damage by quantitative PCR in liver and peripheral blood mononuclear cells of young, middle age, and old monkeys and show that older monkeys have increases in the number of mtDNA lesions. There was a direct correlation between the amount of mtDNA lesions and age, supporting the role of mtDNA damage in the process of aging. Liver from older monkeys showed significant increases in lipid peroxidation, protein carbonylations and reduced antioxidant enzyme activity. Similarly, peripheral blood mononuclear cells from the middle age group showed increased levels in carbonylated proteins, indicative of high levels of oxidative stress. Together, these results suggest that the aging process is associated with defective mitochondria, where increased production of reactive oxygen species results in extensive damage at the mtDNA and protein levels. This study provides valuable data based on the rhesus macaque model further validating age-related mitochondrial functional decline with increasing age and suggesting that mtDNA damage might be a good biomarker of aging.     

Human lymphocyte activation assay: An in vitro method for predictive immunotoxicity testing

Preclinical immunotoxicity assessments may be performed during pharmaceutical drug development in order to identify potential cause for concern prior to use in the clinic. The in vivo T-dependent antibody response (TDAR) is widely used in this regard, given its sensitivity to known immunosuppressive compounds, but may be impractical early in drug development where quantities of test article are limited. The goal of the current work is to develop an in vitro human cell-based assay that is sensitive to immunosuppression, uses relatively small quantities of test article, and is simple to perform with moderate to high throughput. Ideally, this assay would require the cooperation of multiple cellular compartments to produce a response, similar to the TDAR. Although the Mishell–Dutton assay (in vitro mouse splenic sheep red blood cell response) has been used for this purpose, it shows considerable inter-laboratory variability, and rodent cells are used which leads to potential difficulty in translation of findings to humans. We have developed an assay that measures an influenza antigen-specific response using frozen-stored human peripheral blood mononuclear cells, which we have termed the human lymphocyte activation (HuLA) assay. The HuLA assay is sensitive to cyclosporine, dexamethasone, rapamycin, mycophenolic acid, and methotrexate at concentrations within their respective therapeutic ranges. Although proliferation is the primary endpoint, we demonstrate that flow cytometry approaches may be used to characterize the proliferating lymphocyte subsets. Flu antigen-specific proliferation in the HuLA assay primarily involves both CD4⁺ and CD8⁺ T-lymphocytes and B-lymphocytes, although other lymphocyte subsets also proliferate. In addition, flu-specific antibody-secreting cells can be measured in this assay by ELISPOT, a response that is also sensitive to known immunosuppressive compounds. The HuLA assay represents a relatively straightforward assay with the capability of detecting immune suppression in human cells and can be applied to compound ranking and immunotoxicity assessment.     

COVID-19 adenovirus vector vaccine induces higher interferon and pro-inflammatory responses than mRNA vaccines in human PBMCs,macrophages and moDCs

BackgroundDuring the COVID-19 pandemic multiple vaccines were rapidly developed and widely used throughout the world. At present there is very little information on COVID-19 vaccine interactions with primary human immune cells such as peripheral blood mononuclear cells (PBMCs), monocyte-derived macrophages and dendritic cells (moDCs).MethodsHuman PBMCs, macrophages and moDCs were stimulated with different COVID-19 vaccines, and the expression of interferon (IFN-λ1, IFN-α1), pro-inflammatory (IL-1β, IL-6, IL-8, IL-18, CXCL-4, CXCL-10, TNF-α) and Th1-type cytokine mRNAs (IL-2, IFN-γ) were analyzed by qPCR. In addition, the expression of vaccine induced spike (S) protein and antiviral molecules were studied in primary immune cells and in A549 lung epithelial cells.ResultsAdenovirus vector (Ad-vector) vaccine AZD1222 induced high levels of IFN-λ1, IFN-α1, CXCL-10, IL-6, and TNF-α mRNAs in PBMCs at early time points of stimulation while the expression of IFN-γ and IL-2 mRNA took place at later times. AZD1222 also induced IFN-λ1, CXCL-10 and IL-6 mRNA expression in monocyte-derived macrophages and DCs in a dose-dependent fashion. AZD1222 also activated the phosphorylation of IRF3 and induced MxA expression. BNT162b2 and mRNA-1273 mRNA vaccines failed to induce or induced very weak cytokine gene expression in all cell models. None of the vaccines enhanced the expression of CXCL-4. AZD1222 and mRNA-1273 vaccines induced high expression of S protein in all studied cells.ConclusionsAd-vector vaccine induces higher IFN and pro-inflammatory responses than the mRNA vaccines in human immune cells. This data shows that AZD1222 readily activates IFN and pro-inflammatory cytokine gene expression in PBMCs, macrophages and DCs, but fails to further enhance CXCL-4 mRNA expression.     

Allelic expression patterns of KIR3DS1 and 3DL1 using the Z27 and DX9 antibodies

KIR3DL1 is one of the best-characterised inhibitory NK cell receptors. Unusually, one common allele at the 3DL1 locus encodes an activating receptor known as 3DS1. There is genetic evidence for a protective role of 3DS1 in certain viral diseases, but there has been uncertainty about expression of the 3DS1 protein. Using transfection, we show that surface expression of 3DS1 is reliant on the adaptor protein DNAX-activating protein 12 (DAP12). KIR3DS1 was recognised by the antibody Z27, a reagent that also detects KIR3DL1 but no other killer immunoglobulin-like receptor (KIR) molecule. Z27 stained 3DS1 on the surface of fresh circulating NK cells from 3DS1/3DS1 homozygotes. By double-staining with Z27 and DX9, an antibody specific for 3DL1, we obtained evidence that in 3DS1/3DL1 heterozygous donors significant numbers of NK cells express 3DS1 without co-expressing 3DL1 and that NK cells expressing both alleles are difficult to detect.     

Vaccination of sheep with Quil-A® adjuvant expands the antibody repertoire to the Fasciola MF6p/FhHDM-1 antigen and administered together impair the growth and antigen release of flukes

Fasciolosis continues to be a major cause of economic losses in the livestock industry and a growing threat to humans. The limited spectrum of effective anthelmintics and the appearance of resistances urge the need for developing an effective vaccine. Most studies have been focused on the use of TH₁-polarizing adjuvants and the use of recombinant Fasciola critical molecules and, despite the efforts, no reproducible protections have been achieved. The F. hepatica MF6p/FhHDM-1 protein is a heme-binding protein also reported to have immunomodulatory properties, constituting a promising target for vaccination and/or as target for the development of new flukicides. Thus, in this study, we investigated the effects of the TH₁-polarizing adjuvant Quil A® on sheep immune response to MF6p/FhHDM-1, and the vaccine potential of both native and synthetic forms of this protein against ovine fasciolosis. Subcutaneous injection of Quil A® alone, i.e., without co-injecting any antigen, expands the antibody repertoire to MF6p/FhHDM-1 triggered by a subsequent primoinfection with metacercariae. This effect was not observed with aluminum hydroxide, the most frequently adjuvant used in commercial vaccines. On the other hand, vaccination with synthetic MF6p/FhHDM-1 in Quil A® prompted a 2–4-week delay in the antibody response induced in sheep by a challenge experimental infection. Moreover, fluke populations stablished showed stunted growth and low antigen release probably due to reduced metabolic activity. These observations suggest that primary circulating antibodies induced by the immunization had harmful effects on fluke development. Such effects could not be demonstrated to be associated to TH₁ immune response linked events (production of IgG₂ isotype antibodies and IFN-γ).     

Targeting V-ATPase in primary human monocytes by archazolid potently represses the classical secretion of cytokines due to accumulation at the endoplasmic reticulum

The macrolide archazolid inhibits vacuolar-type H(+)-ATPase (V-ATPase), a proton-translocating enzyme involved in protein transport and pH regulation of cell organelles, and potently suppresses cancer cell growth at low nanomolar concentrations. In view of the growing link between inflammation and cancer, we investigated whether inhibition of V-ATPase by archazolid may affect primary human monocytes that can promote cancer by sustaining inflammation through the release of tumor-promoting cytokines. Human primary monocytes express V-ATPase, and archazolid (10–100 nM) increases the vesicular pH in these cells. Archazolid (10 nM) markedly reduced the release of pro-inflammatory (TNF-α, interleukin-6 and -8) but also of anti-inflammatory (interleukin-10) cytokines in monocytes stimulated with LPS, without affecting cell viability up to 1000 nM. Of interest, secretion of interleukin-1β was increased by archazolid. Comparable effects were obtained by the V-ATPase inhibitors bafilomycin and apicularen. The phosphorylation of p38 MAPK and ERK-1/2, Akt, SAPK/JNK or of the inhibitor of NFκB (IκBα) as well as mRNA expression of IL-8 were not altered by archazolid in LPS-stimulated monocytes. Instead, archazolid caused endoplasmic reticulum (ER) stress response visualized by increased BiP expression and accumulation of IL-8 (and TNF-α) at the ER, indicating a perturbation of protein secretion. In conclusion, by interference with V-ATPase, archazolid significantly affects the secretion of cytokines due to accumulation at the ER which might be of relevance when using these agents for cancer therapy.