Transcriptomic analysis reveals optimal cytokine combinations for SARS-CoV-2-specific T cell therapy products.

Adoptive T cell immunotherapy has been used to restore immunity against multiple viral targets in immunocompromised patients after bone-marrow transplantation and has been proposed as a strategy for preventing coronavirus 2019 (COVID-19) in this population. Ideally, expanded severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-virus-specific T cells (CSTs) should demonstrate marked cell expansion, T cell specificity, and CD8+ T cell skewing prior to adoptive transfer. However, current methodologies using IL-4 + IL-7 result in suboptimal specificity, especially in CD8+ cells. Using a microexpansion platform, we screened various cytokine cocktails (IL-4 + IL-7, IL-15, IL-15 + IL-4, IL-15 + IL-6, and IL-15 + IL-7) for the most favorable culture conditions. IL-15 + IL-7 optimally balanced T cell expansion, polyfunctionality, and CD8+ T cell skewing of a final therapeutic T cell product. Additionally, the transcriptomes of CD4+ and CD8+ T cells cultured with IL-15 + IL-7 displayed the strongest induction of antiviral type I interferon (IFN) response genes. Subsequently, microexpansion results were successfully translated to a Good Manufacturing Practice (GMP)-applicable format where IL-15 + IL-7 outperformed IL-4 + IL-7 in specificity and expansion, especially in the desirable CD8+ T cell compartment. These results demonstrate the functional implications of IL-15-, IL-4-, and IL-7-containing cocktails for therapeutic T cell expansion, which could have broad implication for cellular therapy, and pioneer the use of RNA sequencing (RNA-seq) to guide viral-specific T cell (VST) product manufacturing.

The Interactions Between Autoinflammation and Type 2 Immunity: From Mechanistic Studies to Epidemiologic Associations.

Autoinflammatory diseases are a group of clinical syndromes characterized by constitutive overactivation of innate immune pathways. This results in increased production of or responses to monocyte- and neutrophil-derived cytokines such as interleukin-1ß (IL-1ß), Tumor Necrosis Factor-α (TNF-α), and Type 1 interferon (IFN). By contrast, clinical allergy is caused by dysregulated type 2 immunity, which is characterized by expansion of T helper 2 (Th2) cells and eosinophils, as well as overproduction of the associated cytokines IL-4, IL-5, IL-9, and IL-13. Traditionally, type 2 immune cells and autoinflammatory effectors were thought to counter-regulate each other. However, an expanding body of evidence suggests that, in some contexts, autoinflammatory pathways and cytokines may potentiate type 2 immune responses. Conversely, type 2 immune cells and cytokines can regulate autoinflammatory responses in complex and context-dependent manners. Here, we introduce the concepts of autoinflammation and type 2 immunity. We proceed to review the mechanisms by which autoinflammatory and type 2 immune responses can modulate each other. Finally, we discuss the epidemiology of type 2 immunity and clinical allergy in several monogenic and complex autoinflammatory diseases. In the future, these interactions between type 2 immunity and autoinflammation may help to expand the spectrum of autoinflammation and to guide the management of patients with various autoinflammatory and allergic diseases.

Systematic evaluation of nine monogenic autoinflammatory diseases reveals common and disease-specific correlations with allergy-associated features.

BACKGROUND: Monogenic autoinflammatory diseases (AID) are caused by mutations in innate immune genes. The effects of these mutations on allergic inflammation are unknown. OBJECTIVES: We investigated allergic, immunological and clinical phenotypes in FMF (familial Mediterranean fever), CAPS (cryopyrin-associated periodic syndrome), TRAPS (tumour necrosis factor receptor-associated periodic syndrome), HIDS (hyper-IgD syndrome), PAPA (pyogenic arthritis, pyoderma gangrenosum and acne), DADA2 (deficiency of adenosine deaminase 2), HA20 (haploinsufficiency of A20), CANDLE (chronic atypical neutrophilic dermatosis, lipodystrophy, elevated temperature) and SAVI (STING-associated vasculopathy of infancy). METHODS: In this cross-sectional study, clinical data were assessed in 425 patients with AID using questionnaires and chart reviews. Comparator data were obtained from public databases. Peripheral blood mononuclear cells obtained from 55 patients were stimulated and CD4+ cytokine production assessed. RESULTS: Clinical laboratory features of Type 2 immunity were elevated in CAPS but reduced in most AID, particularly DADA2. Physician-diagnosed allergic diseases were prevalent in multiple AID, including CAPS and DADA2. T helper 2 (Th2) cells were expanded in CAPS, TRAPS and HIDS; Th9 cells were expanded in HA20. CONCLUSIONS: CAPS is characterised by an enhanced Type 2 signature, whereas FMF and CANDLE are associated with reduced Type 2 responses. DADA2 is associated with reduced Type 2 responses but a high rate of physician-diagnosed allergy. Therefore, NLRP3-driven autoinflammation may promote Type 2 immunity, whereas AID like DADA2 may manifest clinical phenotypes that masquerade as allergic disorders. Further investigations are needed to determine the contribution of autoinflammation to allergic clinical and immunological phenotypes, to improve the treatment of patients with AID.

Transduction of Salivary Gland Acinar Cells with a Novel AAV Vector 44.9.

The loss of salivary gland function caused by radiation therapy of the head and neck or autoimmune disease such as Sjögren's syndrome is a serious condition that affects a patient's quality of life. Due to the combined exocrine and endocrine functions of the salivary gland, gene transfer to the salivary glands holds the potential for developing therapies for disorders of the salivary gland and the expression of therapeutic proteins via the exocrine pathway to the mouth, upper gastrointestinal tract, or endocrine pathway, systemically, into the blood. Recent clinical success with viral vector-mediated gene transfer for the treatment of irradiation-induced damage to the salivary glands has highlighted the need for the development of novel vectors with acinar cell tropism able to result in stable long-term transduction. Previous studies with adeno-associated virus (AAV) focused on the submandibular gland and reported mostly ductal cell transduction. In this study, we have screened AAV vectors for acinar cell tropism in the parotid gland utilizing membrane-tomato floxed membrane-GFP transgenic mice to screen CRE recombinase encoding AAV vectors of different clades to rapidly identify capsid isolates able to transduce salivary gland acinar cells. We determined that AAVRh10 and a novel isolate found as a contaminant of a laboratory stock of simian adenovirus SV15, AAV44.9, are both able to transduce parotid and sublingual acinar cells. Persistence and localization of transduction of these AAVs were tested using vectors encoding firefly luciferase, which was detected 6 months after vector administration. Most luciferase expression was localized to the salivary gland compared to that of distal organs. Transduction resulted in robust secretion of recombinant protein in both blood and saliva. Transduction was species specific, with AAVRh10 having stronger transduction activity in rats compared with AAV44.9 or AAV2 but weaker in human primary salivary gland cells. This work demonstrates efficient transduction of parotid acinar cells by AAV that resulted in secretion of recombinant protein in both serum and saliva.

Ca2+ Influx Channel Inhibitor SARAF Protects Mice From Acute Pancreatitis.

BACKGROUND & AIMS: Pancreatitis is characterized by increased influx of Ca2+ into acinar cells, by unknown mechanisms. Inhibitors of Ca2+ influx channels could be effective in treating acute pancreatitis, but these have deleterious side effects that can result in death. We investigated the expression patterns and functions of acinar cell Ca2+ channels and factors that regulate them during development of acute pancreatitis, along with changes in the channel inactivator store-operated calcium entry-associated regulatory factor (SARAF). We investigated whether SARAF is a target for treatment of acute pancreatitis and its status in human with pancreatitis. METHODS: We generated mice that expressed SARAF tagged with hemagglutinin, using CRISPR/Cas9 gene editing, and isolated acinar cells. We also performed studies with Saraf-/- mice, Sarafzf/zf mice, mice without disruption of Saraf (control mice), and mice that overexpress fluorescently labeled SARAF in acinar cells. We analyzed interactions between stromal interaction molecule 1 (STIM1) and SARAF in HEK cells stimulated with carbachol using fluorescence resonance energy transfer microscopy and immunoprecipitation. Mice were given injections of caerulein or L-arginine to induce pancreatitis. Pancreatic tissues and blood samples were collected and levels of serum amylase, trypsin, tissue damage, inflammatory mediators, and inflammatory cells were measured. We performed quantitative polymerase chain reaction analyses of pancreatic tissues from 6 organ donors without pancreatic disease (controls) and 8 patients with alcohol-associated pancreatitis. RESULTS: Pancreatic levels of Ca2+ influx channels or STIM1 did not differ significantly between acinar cells from mice with vs. without pancreatitis. By contrast, pancreatic levels of Saraf messenger RNA and SARAF protein initially markedly increased but then decreased during cell stimulation or injection of mice with caerulein, resulting in excessive Ca2+ influx. STIM1 interacted stably with SARAF following stimulation of HEK or mouse acinar cells with physiologic levels of carbachol, but only transiently following stimulation with pathologic levels of carbachol, leading to excessive Ca2+ influx. We observed reduced levels of SARAF messenger RNA in pancreatic tissues from patients with pancreatitis, compared with controls. SARAF knockout mice developed more severe pancreatitis than control mice after administration of caerulein or L-arginine, and pancreatic acinar cells from these mice had significant increases in Ca2+ influx. Conversely, overexpression of SARAF in acini reduced Ca2+ influx, eliminated inflammation, and reduced severity of acute pancreatitis. CONCLUSIONS: In mice with pancreatitis, SARAF initially increases but is then degraded, resulting in excessive, pathological Ca2+ influx by acinar cells. SARAF knockout mice develop more severe pancreatitis than control mice, whereas mice that express SARAF from a transgene in acinar cells develop less-severe pancreatitis. SARAF therefore appears to prevent pancreatic damage during development of acute pancreatitis. Strategies to stabilize or restore SARAF to acinar cells might be developed for treatment of pancreatitis.

Homer2 and Homer3 modulate RANKL-induced NFATc1 signaling in osteoclastogenesis and bone metabolism.

The receptor activator of nuclear factor-kappa B ligand (RANKL) induces osteoclastogenesis by induction of Ca2+ oscillation, calcineurin activation and translocation into the nucleus of nuclear factor of activated T cells type c1 (NFATc1). Homer proteins are scaffold proteins. They regulate Ca2+ signaling by modulating the activity of multiple Ca2+ signaling proteins. Homers 2 and 3, but not Homer1, also independently affect the interaction between NFATc1 and calcineurin. However, to date, whether and how the Homers are involved in osteoclastogenesis remains unknown. In the present study, we investigated Homer2 and Homer3 roles in Ca2+ signaling and NFATc1 function during osteoclast differentiation. Deletion of Homer2/Homer3 (Homer2/3) markedly decreased the bone density of the tibia, resulting in bone erosion. RANKL-induced osteoclast differentiation is greatly facilitated in Homer2/3 DKO bone marrow-derived monocytes/macrophages (BMMs) due to increased NFATc1 expression and nuclear translocation. However, these findings did not alter RANKL-induced Ca2+ oscillations. Of note, RANKL treatment inhibited Homer proteins interaction with NFATc1, but it was restored by cyclosporine A treatment to inhibit calcineurin. Finally, RANKL treatment of Homer2/3 DKO BMMs significantly increased the formation of multinucleated cells. These findings suggest a novel potent mode of bone homeostasis regulation through osteoclasts differentiation. Specifically, we found that Homer2 and Homer3 regulate NFATc1 function through its interaction with calcineurin to regulate RANKL-induced osteoclastogenesis and bone metabolism.

STIM1 holds a STING in its (N-terminal) tail.

The Ca2+ sensor STIM1 is essential for adaptive immune responses, yet patients with hypomorphic STIM1 mutations develop both immunodeficiency and autoimmunity, implying that STIM1 also restrains immune responses. This study by Srikanth et al demonstrates that STIM1 tethers STING, a major inducer of the interferon (IFN) response, to the endoplasmic reticulum (ER) to prevent constitutive STING activation.

Modulation of Cl- signaling and ion transport by recruitment of kinases and phosphatases mediated by the regulatory protein IRBIT.

IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl--sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl- (Cl- in). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl- in Mutational analysis suggested that the phosphorylation status of Ser232, Ser233, and Ser235 was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser232, Ser233, or Ser235 produced NBCe1-B in the conformations pSer233/pSer235, pSer232/pSer235, or pSer232/pSer233, respectively. The activity of the pSer233/pSer235 form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl- in The properties of the pSer232/pSer235 form were similar to those of wild-type NBCe1-B, whereas the pSer232/pSer233 form was partially active, further activated by IRBIT, but retained inhibition by Cl- in Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser65, which affected Cl- in sensing by the 32GXXXP36 motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser12, which affected Cl- in sensing by the 194GXXXP198 motif. Ser232, Ser233, and Ser235 are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO3 - secretion.

TRPM3/TRPV4 regulates Ca2+-mediated RANKL/NFATc1 expression in osteoblasts

Mechanical stress plays an important role in the regulation of bone turnover. However, the mechanism underlying hypo-osmotic stress-induced cellular response in osteoblasts remains poorly understood. In this study, we investigated the effect of hypotonic stress on the expression of bone remodeling factors, including the receptor activator of nuclear factor-kappa B ligand (RANKL) and the nuclear factor of activated T cells type c1 (NFATc1) in primary mouse osteoblasts and MC3T3-E1 cells. Hypo-osmotic stress induced significant increases in RANKL mRNA expression and intracellular Ca2+ concentration ([Ca2+]i) from the extracellular space. Hypo-osmotic stress-induced effects on [Ca2+]i and RANKL and NFATc1 protein expression were decreased by antagonists of transient receptor potential melastatin 3 (TRPM3) and vanilloid 4 (TRPV4). Agonists of TRPM3 and TRPV4 activated [Ca2+]i and RANKL and NFATc1 protein expression. Furthermore, genetic suppression of Trpm3 and Trpv4 reduced hypo-osmotic stress-induced effects in mouse osteoblasts. These results suggest that hypo-osmotic stress induces increases in [Ca2+]i through TRPM3 and TRPV4 to regulate RANKL and NFATc1 expression in mouse osteoblastic cells and that mechanical stress-activated TRP channels may play a critical role in bone remodeling.

The TRPCs, Orais and STIMs in ER/PM Junctions.

The Ca(2+) second messenger is initiated at ER/PM junctions and propagates into the cell interior to convey the receptor information. The signal is maintained by Ca(2+) influx across the plasma membrane through the Orai and TRPC channels. These Ca(2+) influx channels form complexes at ER/PM junctions with the ER Ca(2+) sensor STIM1, which activates the channels. The function of STIM1 is modulated by other STIM isoforms like STIM1L, STIM2 and STIM2.1/STIM2ß and by SARAF, which mediates the Ca(2+)-dependent inhibition of Orai channels. The ER/PM junctions are formed at membrane contact sites by tethering proteins that generate several types of ER/PM junctions, such as PI(4,5)P2-poor and PI(4,5)P2-rich domains. This chapter discusses several properties of the TRPC channels, the Orai channels and the STIMs, their key interacting proteins and how interaction of the STIMs with the channels gates their activity. The chapter closes by highlighting open questions and potential future directions in this field.

Airborne allergens induce protease activated receptor-2-mediated production of inflammatory cytokines in human gingival epithelium.

OBJECTIVE: In reaching the airways inhaled allergens pass through and contact with the oral mucosa. Although they are often responsible for initiating asthmatic attacks, it is unknown whether airborne allergens can also trigger chronic inflammation of gingival epithelial cells leading to chronic periodontitis. In this study, we investigated the inflammatory responses of human gingival epithelial cells (HGECs) to airborne allergens, particularly German cockroach extract (GCE) with a focus on calcium signaling. DESIGN: HGECs isolated from healthy donors were stimulated with GCE. Intracellular Ca(2+) concentration ([Ca(2+)]i) was measured with Fura-2-acetoxymethyl ester (Fura-2/AM) staining. Expression of inflammatory cytokines interleukin (IL)-8, IL-1ß, IL-6, and NOD-like receptor family, pyridine domain-containing (NLRP) 3 was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: GCE promoted increase in the [Ca(2+)]i in a dose-dependent manner. Depletion of endoplasmic reticulum (ER) Ca(2+) by the ER Ca(2+) ATPase inhibitor thapsigargin (Tg) but not the depletion of extracellular Ca(2+) abolished the GCE-induced increase in [Ca(2+)]i. Treatment of phospholipase C (PLC) inhibitor (U73122) or 1,4,5-trisinositolphosphate (IP3) receptor inhibitor (2-APB) also prevented GCE-induced increase in [Ca(2+)]i. Protease activated receptor (PAR)-2 activation mainly mediated the GCE-induced increase in [Ca(2+)]i and enhanced the expression of IL-8, NLRP3, IL-1ß, and IL-6 in HGECs. CONCLUSIONS: GCE activates PAR-2, which can induce PLC/IP3-dependent Ca(2+) signaling pathway, ultimately triggering inflammation via the production of pro-inflammatory cytokines such as IL-1ß, IL-6, IL-8, and NLRP 3 in HGECs.

Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis.

The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca(2+) signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP(3)) and IP(3)-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP(3) and evaluated IP(3)-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca(2+) signaling proteins such as IP(3) receptors (IP(3)Rs), plasma membrane Ca(2+) ATPase, and sarco/endoplasmic reticulum Ca(2+) ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP(3) was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP(3) levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP(3)Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP(3)Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP(3) levels and the IP(3)-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.

RANKL-mediated reactive oxygen species pathway that induces long lasting Ca2+ oscillations essential for osteoclastogenesis.

RANKL (receptor activator of NF-kappaB ligand) induces osteoclastogenesis by activating multiple signaling pathways in osteoclast precursor cells, chief among which is induction of long lasting oscillations in the intracellular concentration of Ca(2+) ([Ca(2+)](i)). The [Ca(2+)](i) oscillations activate calcineurin, which activates the transcription factor NFATc1. The pathway by which RANKL induces [Ca(2+)](i) oscillations and osteoclastogenesis is poorly understood. Here we report the discovery of a novel pathway induced by RANKL to cause a long lasting increase in reactive oxygen species (ROS) and [Ca(2+)](i) oscillations that is essential for differentiation of bone marrow-derived monocytes into osteoclasts. The pathway includes RANKL-mediated stimulation of Rac1 to generate ROS, which stimulate phospholipase Cgamma1 to evoke [Ca(2+)](i) oscillations by stimulating Ca(2+) release from the inositol 1,4,5-trisphosphate pool and STIM1-regulated Ca(2+) influx. Induction and activation of the pathway is observed only after 24-h stimulation with RANKL and lasts for at least 3 days. The physiological role of the pathway is demonstrated in mice with deletion of the Peroxiredoxin II gene and results in a mark increase is ROS and, consequently, a decrease in bone density. Moreover, bone marrow-derived monocytes in PrxII(-/-) primary culture show increased ROS and spontaneous [Ca(2+)](i) oscillations. These findings identify the primary RANKL-stimulated pathway to trigger the late stages of osteoclastogenesis and regulate bone resorption.

Alteration of RANKL-induced osteoclastogenesis in primary cultured osteoclasts from SERCA2+/- mice.

RANKL is essential for the terminal differentiation of monocytes/macrophages into osteoclasts. RANKL induces long-lasting oscillations in the intracellular concentration of Ca(2+) ([Ca(2+)](i)) only after 24 h of stimulation. These Ca(2+) oscillations play a switch-on role in NFATc1 expression and osteoclast differentiation. Which Ca(2+) transporting pathway is induced by RANKL to evoke the Ca(2+) oscillations and its specific role in RANKL-mediated osteoclast differentiation is not known. This study examined the effect of a partial loss of sarco/endoplasmic reticulum Ca(2+) ATPase type 2 (SERCA2) on osteoclast differentiation in SERCA2 heterozygote mice (SERCA2(+/-)). The BMD in the tibias of SERCA2(+/-) mice increased >1.5-fold compared with wildtype mice (WT). RANKL-induced [Ca(2+)](i) oscillations were generated 48 h after RANKL treatment in the WT mice but not in the SERCA2(+/-) bone marrow-derived macrophages (BMMs). Forty-eight hours after RANKL treatment, there was a lower level of NFATc1 protein expression and markedly reduced translocation of NFATc1 into the nucleus during osteoclastogenesis of the SERCA2(+/-) BMMs. In addition, RANKL treatment of SERCA2(+/-) BMMs incompletely induced formation of multinucleated cells, leading to reduced bone resorption activity. These results suggest that RANKL-mediated induction of SERCA2 plays a critical role in the RANKL-induced [Ca(2+)](i) oscillations that are essential for osteoclastogenesis.