Jacob Rachmilewitz

Parental imprinting of the human H19 gene

It has only recently become clear that genetic imprinting plays an important role in human embryogenesis and in processes leading to the development of pediatric cancers and other human diseases. Using a unique human tissue, the androgenetic complete hydatidiform mole, we established that the maternally inherited allele of the imprinted H19 gene is expressed. Our results also show that the paternal allele of the human IGF‐II gene, a gene suspected to be parentally imprinted in humans, is expressed.

TLR3 signaling in a hepatoma cell line is skewed towards apoptosis

Toll‐like receptors (TLRs) recognize pathogen‐associated molecular patterns (PAMPS) leading to the activation of the innate immune response and subsequently to the shaping of the adaptive immune response. Of the known human TLRs, TLR3, 7, 8, and 9 were shown to recognize nucleic acid ligands. TLR3 signaling is induced by double‐stranded (ds)RNA, a molecular signature of viruses, and is mediated by the TRIF (TIR domain‐containing adaptor‐inducing IFNβ) adaptor molecule. Thus, TLR3 plays an important role in the host response to viral infections. The liver is constantly exposed to a large variety of foreign substances, including pathogens such as HBV (hepatitis B virus) and HCV (hepatitis C virus), which frequently establish persistent liver infections. In this work, we investigated the expression and signaling pathway of TLR3 in different hepatoma cell lines. We show that hepatocyte lineage cells express relatively low levels of TLR3 mRNA. TLR3 signaling in HEK293 cells (human embryonic kidney cells) activated NF‐κB and IRF3 (interferon regulatory factor 3) and induced IFNβ (interferon β) promoter expression, which are known to lead to pro‐inflammatory cytokine secretion. In Huh7 cells, there was only a short‐term IRF3 activation, and a very low level of IFNβ expression. In HepG2 cells on the other hand, while no induction of pro‐inflammatory factors was observed, signaling by TLR3 was skewed towards the induction of apoptosis. These results indicate preferential induction of the apoptotic pathway over the cytokine induction pathway by TLR3 signaling in hepatocellular carcinoma cells with potential implications for therapeutic strategies. J. Cell. Biochem. 100: 1301–1312, 2007.

Differential Regulation of Th1/Th2 Cytokine Responses by Placental Protein 14

The potency of TCR signaling during primary CD4+ T cell activation influences initial cytokine expression patterns and subsequent polarization toward either Th1 or Th2 subsets. In this study, we demonstrate that the T cell inhibitor placental protein 14 (PP14; glycodelin) preferentially inhibits Th1 cytokine responses and chemokine expression when present during ex vivo priming of CD4+ T cells. PP14 synergizes with exogenously added IL-4 in skewing T cell responses. Significantly, PP14 impairs the down-regulation of GATA-3 transcriptional regulator expression that normally accompanies T cell activation, which is a prerequisite for Th1 development. Taken together, these data document for the first time the ability of PP14 to skew Th responses.

Placental protein 14 regulates selective B cell responses

Placental protein 14 (PP14) is a glycoprotein of the lipocalin family that acts as a negative regulator in T cell receptor-mediated activation. In this study, we investigated PP14s potential role in regulating B cell activation. While PP14-inhibited B cell proliferation, IgM secretion and the surface expression of MHC class II, the expression of other surface molecules, such as CD69 and CD86, were unaffected. These observed effects were independent of the anti-IgM concentration used for stimulation, regardless of the presence of either T cells or IL-4, and persisted when B cells were stimulated by stimuli, which circumvent early events during B cell Ag receptor (BCR) activation, namely, protein kinase C activators in combination with Ca(2+) ionophore. Interestingly, we demonstrated that PP14s inhibitory characteristics are reminiscence of that achieved by independent ligation of CD19 using anti-CD19 mAb. Together with our previously reported effects on T cells, these findings identify PP14 as a soluble regulatory factor capable of interacting with both T and B cells in a carbohydrate-dependent manner and as a result it can affect both cellular and humoral immune responses.

Toll‐like receptor 3 signaling attenuates liver regeneration

The current model for liver regeneration suggests that cell damage triggers Toll‐like receptor (TLR) signaling via MyD88, leading to the induction of nuclear factor κB (NF‐κB) and secretion of inflammatory cytokines that in turn prime liver regeneration. TLR3 is unique among TLRs in that it signals through TRIF (TIR domain‐containing adaptor‐inducing interferon‐β), not through MyD88, and may lead to activation of either the inflammatory or apoptotic pathway. The inflammatory pathway leads to NF‐κB activation, whereas the apoptotic pathway, believed to be mediated by Rip3, leads to caspase‐8 activation. In this study, we explored the role of TLR3 in liver regeneration by comparing the response to 70% partial hepatectomy of TLR3wt and TLR3−/− mice. We found that following partial hepatectomy, TLR3−/− mice demonstrated earlier hepatocyte proliferation. Furthermore, within the first hours, we observed a dramatic TLR3‐dependent NF‐κB activation and an increase in Rip3 levels in hepatocytes, accompanied by caspase‐8 activation but without an apoptotic outcome. Conclusion: TLR3 plays an inhibitory role in the priming of liver regeneration, thus reinforcing the role of the innate immune system in balancing tissue regeneration. (HEPATOLOGY 2009.)

Human Embryonic Stem Cells Suppress T Cell Responses via Arginase I-Dependent Mechanism

Human embryonic stem cells (hESCs) can proliferate extensively in culture and give rise to progeny of the three germ layers. Several reports suggested that mouse and hESCs may attenuate immune responses. In this study, we focused on the mechanism by which hESCs inhibit T cell responses. Using coculture experiments, we demonstrate that hESCs inhibit cytokine secretion and T cell proliferation in response to potent T cell activators. Furthermore, we show that hESCs downmodulate the TCR-associated CD3-ζ chain. These effects are maintained when hESCs are replaced by their conditioned media and can be restored by the addition of l-arginine to hESC-conditioned media or by treatment of hESCs with a specific arginase inhibitor. Moreover, we show arginase-I expression and activity in hESCs. We further demonstrate that mouse ESCs (mESCs) similarly inhibit T cell activation via arginase I, suggesting an evolutionary conserved mechanism of T cell suppression by ESCs. In addition, we demonstrate that arginase I expression is not limited to ESCs in culture, but can also be detected in the inner cell mass and the trophectoderm of preimplantation mouse embryos and hESC-derived trophectoderm cells. Finally, T cells infiltrating ESC-derived teratomas have significantly lower levels of CD3-ζ chain. Collectively, the data indicate a role for ESC-arginase I activity in the attenuation of T cell activation.

CTLA-4 · FasL Induces Alloantigen-Specific Hyporesponsiveness

The APC:T cell interface can be effectively targeted with immunotherapeutic proteins. We previously described a unique trans signal converter protein, CTLA-4 · Fas ligand (FasL), that has the inherent capacities to tether the T cell inhibitor FasL (CD95 ligand) to the surfaces of B7 (CD80 and CD86)-positive APC (via CTLA-4:B7 interaction), and in so doing, to simultaneously interfere with B7-to-CD28 T cell activation signals. Given the continuing need for agents capable of inducing allograft tolerance without generalized immunosuppression, we have explored in depth the functional activity of CTLA-4 · FasL in human allogeneic MLR. CTLA-4 · FasL inhibits 1° MLR and induces specific hyporesponsiveness in 2° MLR, with both effects only partially reversible with exogenous IL-2. Moreover, the presence of exogenous IL-2 during the 1° MLR does not affect the induction of hyporesponsiveness upon restimulation. Furthermore, CTLA-4 · FasL enables partial activation of allostimulated T cells, reduces the fraction of actively dividing cells, and increases the percentage of dead cells among dividing T cells. Taken together, these findings suggest that CTLA-4 · FasL-mediated inhibition of secondary alloantigenic responses involves both anergy induction and clonal deletion. Thus, CTLA-4 · FasL, a paradigmatic trans signal converter protein, manifests unique functional properties and emerges as a potentially useful immunotherapeutic for modulating alloresponsiveness.

CTLA-4 · FasL Induces Early Apoptosis of Activated T Cells by Interfering with Anti-Apoptotic Signals

The fusion protein CTLA-4 · FasL, a paradigmatic “trans signal converter protein”, can attach to APC surfaces and in effect convert B7-activating costimulator signals into inhibitory Fas receptor-generated signals. The present study investigates CTLA-4 · FasL’s mechanism of action. A combination of p27kip and proliferating cell nuclear Ag Western blot and propidium iodide flow cytometric analysis showed no CTLA-4 · FasL effect on cell cycle entry and progression, pointing away from the kind of classical anergy associated with CTLA-4 · Ig. Significantly, CTLA-4 · FasL elicited apoptosis (as detected by annexin-V/propidium iodide costaining) as early as 24 h after T cell activation, suggesting that some coordinate signaling might be capacitating the Fas receptor. Significantly, CTLA-4 · FasL, but not CTLA-4 · Ig, anti-Fas mAb, or the two in combination, abrogated the usual increase in expression of the anti-apototic protein, cFLIP. Furthermore, activation of caspases 8 and 3 were not affected by CTLA-4 · FasL. These findings suggest a model for CTLA-4 · FasL action wherein there is coordinate triggering of a death receptor and suppression of a proapoptotic protein.

Normal and Atypical Butyrylcholinesterases in Placental Development, Function, and Malfunction

Abstract1. In utero exposure to poisons and drugs (e.g., anticholinesterases, cocaine) is frequently associated with spontaneous abortion and placental malfunction. The major protein interacting with these compounds is butyrylcholinesterase (BuChE), which attenuates the effects of such xenobiotics by their hydrolysis or sequestration. Therefore, we studied BuChE expression during placental development.2. RT-PCR revealed both BuChEmRNA and acetylcholinesterase (AChE) mRNA throughout gestation. However, cytochemical staining detected primarily BuChE activity in first-trimester placenta but AChE activity in term placenta.3. As the atypical variant of BuChE has a narrower specificity for substrates and inhibitors than the normal enzyme, we investigated its interactions with α-solanine and cocaine, and sought a correlation between the occurrence of this variant and placental malfunction.4. Atypical BuChE of serum or recombinant origin presented >10-fold weaker affinities than normal BuChE for cocaine and α-solanine. However, BuChE in the serum of a heterozygote and a homozygous normal were similar in their drug affinities. Therefore, heterozygous serum or placenta can protect the fetus from drug or poison exposure, unlike homozygous atypical serum or placenta.5. Genotype analyses revealed that heterozygous carriers of atypical BuChE were threefold less frequent among 49 patients with placental malfunction than among 76 controls or the entire Israeli population. These observations exclude heterozygote carriers of atypical BuChE from being at high risk for placental malfunction under exposure to anticholinesterases.

Inhibition of Effector Function but Not T Cell Activation and Increase in FoxP3 Expression in T Cells Differentiated in the Presence of PP14

Background T-helper polarization of naïve T cells is determined by a complex mechanism that involves many factors, eventually leading to activation of Th1, Th2, or Th17 responses or alternatively the generation of regulatory T cells. Placental Protein 14 (PP14) is a 28 kDa glycoprotein highly secreted in early pregnancy that is able to desensitize T cell receptor (TCR) signaling and modulate T cell activation. Methodology/Principal Findings Prolonged antigen-specific stimulation of T cells in the presence of PP14 resulted in an impaired secretion of IFN-γ, IL-5 and IL-17 upon restimulation, although the cells proliferated and expressed activation markers. Furthermore, the generation of regulatory CD4+CD25highFoxp3+ T cells was induced in the presence of PP14, in both antigen-specific as well as polyclonal stimulation. In accordance with previous reports, we found that the induction of FoxP3 expression by PP14 is accompanied by down regulation of the PI3K-mTOR signaling pathway. Conclusions/Significance These data suggest that PP14 arrests T cells in a unique activated state that is not accompanied with the acquisition of effector function, together with promoting the generation of regulatory T cells. Taken together, our results may elucidate the role of PP14 in supporting immune tolerance in pregnancy by reducing T cell effector functions along with augmenting Treg differentiation.