Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.

During immunoglobulin class switch recombination (CSR), activation induced cytidine deaminase (AID) induces DNA double strand breaks into transcribed, repetitive DNA elements called switch sequences. The mechanism that promotes the binding of AID specifically to switch regions remains to be elucidated. We have used a proteomic screen that employs in vivo biotinylation of AID and have identified the splicing regulator polypyrimidine tract binding protein-2 (PTBP2) as an AID interactor. Short hairpin RNA-mediated knock-down of PTBP2 in B cells led to a striking reduction in binding of AID to transcribed switch regions that resulted in marked impairment of CSR. PTBP2 is thus an effector of CSR that promotes binding of AID to switch region DNA.

The ability of activation-induced cytidine deaminase (AID) to efficiently mediate class-switch recombination (CSR) is dependent on its phosphorylation at Ser38; however, the trigger that induces AID phosphorylation and the mechanism by which phosphorylated AID drives CSR have not been elucidated. Here we found that phosphorylation of AID at Ser38 was induced by DNA breaks. Conversely, in the absence of AID phosphorylation, DNA breaks were not efficiently generated at switch (S) regions in the immunoglobulin heavy-chain locus (Igh), consistent with a failure of AID to interact with the endonuclease APE1. Additionally, deficiency in the DNA-damage sensor ATM impaired the phosphorylation of AID at Ser38 and the interaction of AID with APE1. Our results identify a positive feedback loop for the amplification of DNA breaks at S regions through the phosphorylation- and ATM-dependent interaction of AID with APE1.

Background: Rheumatoid factors (RFs) are autoantibodies associated with rheumatoid arthritis. They can be detected in normal individuals, although transiently. This dichotomy has led to questions about the origins and types of RFs. Recently it has been shown that B cells that produce RFs only do so when activated by two signals, one from engagement of the B-cell receptor and the other from recognition of a pathogen-associated molecular pattern through a Toll-like receptor (TLR). These autoantibodies thus link the innate and acquired immune responses. Objective: Through a review of the literature, an examination of the current knowledge of RF induction is presented. The focus is on a discussion of a beneficial or detrimental role for RFs in normal individuals and in those with chronic disease. Results: What makes RF ‘good’ in some cases and ‘bad’ in others may reflect the type of RF produced. Low-affinity polyreactive IgM RFs are probably beneficial as they aid in the clearance of immune complexes that are more efficiently cleared, and the RF B cell can act as an antigen-presenting cell and stimulate host defense. However, large amounts of high-affinity RFs found in patients with chronic disease may be harmful by participation in a vicious cycle of autoantibody production by stimulation of self lymphocytes, and/or deposition in blood vessels thus causing vasculitis. Conclusions: Whether RFs are beneficial or detrimental depends on the context in which they are expressed, the type and amount of RF produced, whether the response is perpetuated by TLR ligation and whether other cells are stimulated either directly or indirectly by RF-positive B cells.

Vitamin D receptor (VDR) is present in multiple blood cells, and the hormonal form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is essential for the proper functioning of the immune system. The role of retinoic acid receptor α (RARα) in hematopoiesis is very important, as the fusion of RARα gene with PML gene initiates acute promyelocytic leukemia where differentiation of the myeloid lineage is blocked, followed by an uncontrolled proliferation of leukemic blasts. RARα takes part in regulation of VDR transcription, and unliganded RARα acts as a transcriptional repressor to VDR gene in acute myeloid leukemia (AML) cells. This is why we decided to examine the effects of the combination of 1,25D and all-trans-retinoic acid (ATRA) on VDR gene expression in normal human and murine blood cells at various steps of their development. We tested the expression of VDR and regulation of this gene in response to 1,25D or ATRA, as well as transcriptional activities of nuclear receptors VDR and RARs in human and murine blood cells. We discovered that regulation of VDR expression in humans is different from in mice. In human blood cells at early stages of their differentiation ATRA, but not 1,25D, upregulates the expression of VDR. In contrast, in murine blood cells 1,25D, but not ATRA, upregulates the expression of VDR. VDR and RAR receptors are present and transcriptionally active in blood cells of both species, especially at early steps of blood development.

Current regenerative strategies used for cartilage repair rely on biomaterial functionality as a scaffold for cells that may have potential in chondrogenic differentiation. The purpose of the research was to investigate the biocompatibility of enzymatically treated alginate/chitosan hydrosol sponges and their suitability to support chondrogenic differentiation of human adipose derived multipotent stromal cells (hASCs). The alginate/chitosan and enzyme/alginate/chitosan sponges were formed from hydrosols with various proportions and were used as a biomaterial in this study. Sponges were tested for porosity and wettability. The porosity of each sponge was higher than 80%. An equal dose of alginate and chitosan in the composition of sponges improved their swelling ability. It was found that equal concentrations of alginate and chitosan in hydrosols sponges assure high biocompatibility properties that may be further improved by enzymatic treatment. Importantly, the high biocompatibility of these biomaterials turned out to be crucial in the context of hydrosols' pro-chondrogenic function. After exposure to the chondrogenic conditions, the hASCs in N/A/C and L/A/C sponges formed well developed nodules and revealed increased expression of collagen type II, aggrecan and decreased expression of collagen type I. Moreover, in these cultures, the reactive oxygen species level was lowered while superoxide dismutase activity increased. Based on the obtained results, we conclude that N/A/C and L/A/C sponges may have prospective application as hASCs carriers for cartilage repair.

Background: Ovarian cancer (OC) is one of the most lethal cancers in women. The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25D3, calcitriol) has anticancer activity in several cancers, including ovarian cancer, but the required pharmacological doses may cause hypercalcemia. We hypothesized that newly developed, low calcemic, vitamin D analogs (an1,25Ds) may be used as anticancer agents instead of calcitriol in ovarian cancer cells. Methods: We used two patient-derived high-grade serous ovarian cancer (HGSOC) cell lines with low (13781) and high (14433) mRNA expression levels of the gene encoding 1,25-dihydroxyvitamin D3 24-hydroxylase CYP24A1, one of the main target genes of calcitriol. We tested the effect of calcitriol and four structurally related series of an1,25Ds (PRI-1906, PRI-1907, PRI-5201, PRI-5202) on cell number, viability, the expression of CYP24A1, and the vitamin D receptor (VDR). Results: CYP24A1 mRNA expression increased in a concentration-dependent manner after treatment with all compounds. In both cell lines, after 4 h, PRI-5202 was the most potent analog (in 13781 cells: EC50 = 2.98 ± 1.10 nmol/L, in 14433 cells: EC50 = 0.92 ± 0.20 nmol/L), while PRI-1907 was the least active one (in 13781 cells: EC50 = n/d, in 14433 cells: EC50 = n/d). This difference among the analogs disappeared after 5 days of treatment. The 13781 cells were more sensitive to the an1,25Ds compared with 14433 cells. The an1,25Ds increased nuclear VDR levels and reduced cell viability, but only in the 13781 cell line. Conclusions: The an1,25Ds had different potencies in the HGSOC cell lines and their efficacy in increasing CYP24A1 expression was cell line- and chemical structure-dependent. Therefore, choosing sensitive cancer cell lines and further optimization of the analogs’ structure might lead to new treatment options against ovarian cancer.

SummaryAlcoholic liver disease and hepatitis C are associated with the production of autoantibodies such as rheumatoid factors (RF), which bind to IgG and can aid in host defence, but are also associated with pathological conditions such as rheumatoid arthritis. Because little is known about the role of RF in liver disease, we characterized the RF production that either occurred spontaneously in response to alcohol consumption or was induced by injection of an Escherichia coli glycolipoprotein in C57Bl/6 mice. Whereas severe liver damage was induced by carbon tetrachloride (CCl4), minimal damage was caused by chronic alcohol consumption. Liver damage was monitored by measurements of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Circulating RF was induced in response to chronic alcohol consumption; the latter probably involved Toll-like receptor ligation. In contrast, CCl4-induced damage was not associated with RF induction. However, concurrent treatment with an E. coli glycolipoprotein macromolecule that induced RF, protected against CCL4-induced liver damage as measured by a highly significant decrease (P = 0·008) at 4 weeks in AST and ALT. RF induced by E. coli glycolipoprotein correlated with 'protection' from liver damage, indicating that the RF autoimmune response does not necessarily exacerbate liver disease.