NFAT Activation Research Articles

A novel functional IKBKE variant activating NFAT in a patient with polyarthritis and a remittent fever

BackgroundIKBKE is a negative regulator of T cell activation and one of the key activators of type I interferon (IFN) and NFκB signaling via non-classical pathways. The upstream single nucleotide polymorphism of IKBKE (rs2297550-G) is a genome-wide association study risk variant of systemic lupus erythematosus, and is associated with decreased IKBKE expression in T cells by expression quantitative trait locus analysis.Case presentationA 48-year-old female had a remittent fever, arthritis, and oral ulcers for 20 years. She had a poor response to corticosteroids or disease-modifying antirheumatic drugs, including the tumor necrosis factor-α antagonist, etanercept, and the anti-interleukin-6 receptor antibody, tocilizumab.MethodShe participated in the Initiative on Rare and Undiagnosed Disease (IRUD), and whole-exome sequencing (WES) was performed. Functional analyses were conducted by transfecting the identified variants into reporter cells to assess the activation of NFAT and NFκB signaling. Additionally, peripheral blood RNA- sequencing (RNA-seq) data were compared with those from healthy individuals to evaluate the gene expression profiles of immune cells.ResultWES identified a novel heterozygous c.1877G>A, p(Cys626Tyr) variant in IKBKE. Functional analysis indicated that this variant led to increased activity of NFAT (p = 0.015) and decreased activity of NFκB and type I IFN (p = 0.00068 and 0.00044, respectively). The patient had a remarkably low proportion of Naïve CD4 T cells. RNA-seq of peripheral blood immune cell subsets revealed significant differences in gene expression, especially in T cells.ConclusionA novel functional heterozygous variant in IKBKE is described in a patient with a remittent fever and arthritis. The data suggest that IKBKE is an important negative regulator of inflammation, particularly in T cells, and this IKBKE variant might be the underlying cause of a novel autoinflammatory pathology.

CAR-T cells based on a TCR mimic nanobody targeting HPV16 E6 exhibit antitumor activity against cervical cancer

The E6 and E7 oncoproteins of human papillomavirus (HPV) are considered promising targets for HPV-related cancers. In this study, we evaluated novel T cell receptor mimic (TCRm) nanobodies targeting E629-38 peptide complexed with human leukocyte antigen (HLA)-A*02:01 in the chimeric antigen receptor (CAR) format. We isolated two dromedary camel nanobodies, F5 and G9, through phage display screening. F5 bound more efficiently to the complex expressed on cells, including peptide pulsed T2, overexpressed 293E6, and cervical cancer lines CaSki and SS4050, compared to G9. CAR-T cells based on the F5 nanobody specifically killed target cells, including 293E6, CaSki, and SS4050 in vitro, through activation of NFAT and NF-κB signaling. Importantly, F5 CAR-T cells inhibited the growth of CaSki and SS4050 tumor xenografts in mice. These findings demonstrate that HPV-16+ cervical cancer can be targeted by F5 nanobody-based CAR-T cells, offering a valuable alternative strategy for treating HPV-16+ malignancies.

Type I IFN Induces TCR-dependent and -independent Antimicrobial Responses in γδ Intraepithelial Lymphocytes.

Intraepithelial lymphocytes (IELs) expressing the TCRγδ survey the intestinal epithelium to limit the invasion of microbial pathogens. The production of type I IFN is a central component of an antiviral immune response, yet how these proinflammatory cytokines contribute to γδ IEL effector function remains unclear. Based on the unique activation status of IELs and their ability to bridge innate and adaptive immunity, we investigated the extent to which type I IFN signaling modulates γδ IEL function. Using an ex vivo culture model, we find that type I IFN alone is unable to drive IFN-γ production, yet low-level TCR activation synergizes with type I IFN to induce IFN-γ production in murine γδ IELs. Further investigation into the underlying molecular mechanisms of costimulation revealed that TCRγδ-mediated activation of NFAT and JNK is required for type I IFN to promote IFN-γ expression in a STAT4-dependent manner. Whereas type I IFN rapidly upregulates antiviral gene expression independent of a basal TCRγδ signal, neither tonic TCR triggering nor the presence of a TCR agonist was sufficient to elicit type I IFN-induced IFN-γ production invivo. However, bypassing proximal TCR signaling events synergized with IFNAR/STAT4 activation to induce γδ IEL IFN-γ production. These findings indicate that γδ IELs contribute to host defense in response to type I IFN by mounting a rapid antimicrobial response independent of TCRγδ signaling, and may produce IFN-γ in a TCR-dependent manner under permissive conditions.

Therapeutic Inhibition of LincRNA-p21 Protects Against Cardiac Hypertrophy.

Cardiac hypertrophy is an adaptive response to pressure overload aimed at maintaining cardiac function. However, prolonged hypertrophy significantly increases the risk of maladaptive cardiac remodeling and heart failure. Recent studies have implicated long noncoding RNAs in cardiac hypertrophy and cardiomyopathy, but their significance and mechanism(s) of action are not well understood. We measured lincRNA-p21 RNA and H3K27ac levels in the hearts of dilated cardiomyopathy patients. We assessed the functional role of lincRNA-p21 in basal and surgical pressure-overload conditions using loss-of-function mice. Genome-wide transcriptome analysis revealed dysregulated genes and pathways. We labeled proteins in proximity to full-length lincRNA-p21 using a novel BioID2-based system. We immunoprecipitated lincRNA-p21-interacting proteins and performed cell fractionation, ChIP-seq (chromatin immunoprecipitation followed by sequencing), and co-immunoprecipitation to investigate molecular interactions and underlying mechanisms. We used GapmeR antisense oligonucleotides to evaluate the therapeutic potential of lincRNA-p21 inhibition in cardiac hypertrophy and associated heart failure. lincRNA-p21 was induced in mice and humans with cardiomyopathy. Global and cardiac-specific lincRNA-p21 knockout significantly suppressed pressure overload-induced ventricular wall thickening, stress marker elevation, and deterioration of cardiac function. Genome-wide transcriptome analysis and transcriptional network analysis revealed that lincRNA-p21 acts in trans to stimulate the NFAT/MEF2 (nuclear factor of activated T cells/myocyte enhancer factor-2) pathway. Mechanistically, lincRNA-p21 is bound to the scaffold protein KAP1 (KRAB-associated protein-1). lincRNA-p21 cardiac-specific knockout suppressed stress-induced nuclear accumulation of KAP1, and KAP1 knockdown attenuated cardiac hypertrophy and NFAT activation. KAP1 positively regulates pathological hypertrophy by physically interacting with NFATC4 to promote the overactive status of NFAT/MEF2 signaling. GapmeR antisense oligonucleotide depletion of lincRNA-p21 similarly inhibited cardiac hypertrophy and adverse remodeling, highlighting the therapeutic potential of inhibiting lincRNA-p21. These findings advance our understanding of the functional significance of stress-induced long noncoding RNA in cardiac hypertrophy and demonstrate the potential of lincRNA-p21 as a novel therapeutic target for cardiac hypertrophy and subsequent heart failure.

NFAT activation by FKBP52 promotes cancer cell proliferation by suppressing p53.

FK506-binding protein 52 (FKBP52) is a member of the FKBP family of proline isomerases. FKBP52 is up-regulated in various cancers and functions as a positive regulator of steroid hormone receptors. Depletion of FKBP52 is known to inhibit cell proliferation; however, the detailed mechanism remains poorly understood. In this study, we found that FKBP52 depletion decreased MDM2 transcription, leading to stabilization of p53, and suppressed cell proliferation. We identified NFATc1 and NFATc3 as transcription factors that regulate MDM2 We also found that FKBP52 associated with NFATc3 and facilitated its nuclear translocation. In addition, calcineurin, a well-known Ca2+ phosphatase essential for activation of NFAT, plays a role in MDM2 transcription. Supporting this notion, MDM2 expression was found to be regulated by intracellular Ca2+ Taken together, these findings reveal a new role of FKBP52 in promoting cell proliferation via the NFAT-MDM2-p53 axis, and indicate that inhibition of FKBP52 could be a new therapeutic tool to activate p53 and inhibit cell proliferation.

Metabolic regulator LKB1 controls adipose tissue ILC2 PD-1 expression and mitochondrial homeostasis to prevent insulin resistance

Adipose tissue group 2 innate lymphoid cells (ILC2s) help maintain metabolic homeostasis by sustaining type 2 immunity and promoting adipose beiging. Although impairment of the ILC2 compartment contributes to obesity-associated insulin resistance, the underlying mechanisms have not been elucidated. Here, we found that ILC2s in obese mice and humans exhibited impaired liver kinase B1 (LKB1) activation. Genetic ablation of LKB1 disrupted ILC2 mitochondrial metabolism and suppressed ILC2 responses, resulting in exacerbated insulin resistance. Mechanistically, LKB1 deficiency induced aberrant PD-1 expression through activation of NFAT, which in turn enhanced mitophagy by suppressing Bcl-xL expression. Blockade of PD-1 restored the normal functions of ILC2s and reversed obesity-induced insulin resistance in mice. Collectively, these data present the LKB1-PD-1 axis as a promising therapeutic target for the treatment of metabolic disease.

Akap5 links synaptic dysfunction to neuroinflammatory signaling in a mouse model of infantile neuronal ceroid lipofuscinosis.

Palmitoylation and depalmitoylation represent dichotomic processes by which a labile posttranslational lipid modification regulates protein trafficking and degradation. The depalmitoylating enzyme, palmitoyl-protein thioesterase 1 (PPT1), is associated with the devastating pediatric neurodegenerative condition, infantile neuronal ceroid lipofuscinosis (CLN1). CLN1 is characterized by the accumulation of autofluorescent lysosomal storage material (AFSM) in neurons and robust neuroinflammation. Converging lines of evidence suggest that in addition to cellular waste accumulation, the symptomology of CLN1 corresponds with disruption of synaptic processes. Indeed, loss of Ppt1 function in cortical neurons dysregulates the synaptic incorporation of the GluA1 AMPA receptor (AMPAR) subunit during a type of synaptic plasticity called synaptic scaling. However, the mechanisms causing this aberration are unknown. Here, we used the Ppt1-/- mouse model (both sexes) to further investigate how Ppt1 regulates synaptic plasticity and how its disruption affects downstream signaling pathways. To this end, we performed a palmitoyl-proteomic screen, which provoked the discovery that Akap5 is excessively palmitoylated at Ppt1-/- synapses. Extending our previous data, in vivo induction of synaptic scaling, which is regulated by Akap5, caused an excessive upregulation of GluA1 in Ppt1-/- mice. This synaptic change was associated with exacerbated disease pathology. Furthermore, the Akap5- and inflammation-associated transcriptional regulator, nuclear factor of activated T cells (NFAT), was sensitized in Ppt1-/- cortical neurons. Suppressing the upstream regulator of NFAT activation, calcineurin, with the FDA-approved therapeutic FK506 (Tacrolimus) modestly improved neuroinflammation in Ppt1-/- mice. These findings indicate that the absence of depalmitoylation stifles synaptic protein trafficking and contributes to neuroinflammation via an Akap5-associated mechanism.

Nsp1 facilitates SARS-CoV-2 replication through calcineurin-NFAT signaling.

Cyclosporine A (CsA), commonly used to inhibit immune responses, is also known to have anti-SARS-CoV-2 activity, but its mode of action remains elusive. Here, we provide a model to explain how CsA antagonizes SARS-CoV-2 through three critical proteins: DDX5, NFAT1, and Nsp1. DDX5 is a cellular facilitator of SARS-CoV-2 replication, and NFAT1 controls the production of DDX5. Nsp1 is a viral protein absent from the mature viral particle and capable of activating the function of NFAT1 and DDX5. CsA and similar agents suppress Nsp1, NFAT1, and DDX5 to exert their anti-SARS-CoV-2 activity either alone or in combination with Paxlovid.

Role of Signal-Transducing Adaptor Protein-1 for T Cell Activation and Pathogenesis of Autoimmune Demyelination and Airway Inflammation.

Signal-transducing adaptor protein (STAP)-1 is an adaptor protein that is widely expressed in T cells. In this article, we show that STAP-1 upregulates TCR-mediated T cell activation and T cell-mediated airway inflammation. Using STAP-1 knockout mice and STAP-1-overexpressing Jurkat cells, we found that STAP-1 enhanced TCR signaling, resulting in increased calcium mobilization, NFAT activity, and IL-2 production. Upon TCR engagement, STAP-1 binding to ITK promoted formation of ITK-LCK and ITK-phospholipase Cγ1 complexes to induce downstream signaling. Consistent with the results, STAP-1 deficiency reduced the severity of symptoms in experimental autoimmune encephalomyelitis. Single-cell RNA-sequencing analysis revealed that STAP-1 is essential for accumulation of T cells and Ifng and Il17 expression in spinal cords after experimental autoimmune encephalomyelitis induction. Th1 and Th17 development was also attenuated in STAP-1 knockout naive T cells. Taken together, STAP-1 enhances TCR signaling and plays a role in T cell-mediated immune disorders.

Novel chimeric antigen receptor-expressing T cells targeting the malignant mesothelioma-specific antigen sialylated HEG1.

The selection of highly specific target antigens is critical for the development of clinically efficient and safe chimeric antigen receptors (CARs). In search of diagnostic marker for malignant mesothelioma (MM), we have established SKM9-2 monoclonal antibody (mAb) which recognizes a MM-specific molecule, sialylated Protein HEG homolog 1 (HEG1), with high specificity and sensitivity. In this study, to develop a novel therapeutic approach against MM, we generated SKM9-2 mAb-derived CARs that included the CD28 (SKM-28z) or 4-1BB (SKM-BBz) costimulatory domain. SKM-28z CAR-T cells showed continuous growth and enhanced Tim-3, LAG-3, and PD-1 expression in vitro, which might be induced by tonic signaling caused by self-activation; however, these phenotypes were not observed in SKM-BBz CAR-T cells. In addition, SKM-BBz CAR-T cells exhibited slightly stronger in vitro killing activity against MM cell lines than SKM-28z CAR-T cells. More importantly, only SKM-BBz CAR-T cells, but not SKM-28z CAR-T cells, significantly inhibited tumor growth in vivo in a MM cell line xenograft mouse model. Gene expression profiling and reporter assays revealed differential signaling pathway activation; in particular, SKM-BBz CAR-T cells exhibited enhanced NF-kB signaling and reduced NFAT activation. In addition, SKM-BBz CAR-T cells showed upregulation of early memory markers, such as TCF7 and CCR7, as well as downregulation of pro-apoptotic proteins, such as BAK1 and BID, which may be associated with phenotypical and functional differences between SKM-BBz and SKM-28z CAR-T cells. In conclusion, we developed novel SKM9-2-derived CAR-T cells with the 4-1BB costimulatory domain, which could provide a promising therapeutic approach against refractory MM.

Establishment of a Reporter System to Monitor FcRγ-Dependent Activation of NFAT

Establishment of a Reporter System to Monitor FcRγ-Dependent Activation of NFAT

Induction of IL-2 by interleukin-12 p40 homodimer and IL-12, but not IL-23, in microglia and macrophages: Implications for multiple sclerosis

The level of IL-2 increases markedly in serum and central nervous system (CNS) of patients with multiple sclerosis (MS) and animals with experimental allergic encephalomyelitis (EAE). However, mechanisms by which IL-2 is induced under autoimmune demyelinating conditions are poorly understood. The present study underlines the importance of IL-12p40 homodimer (p402), the so-called biologically inactive molecule, in inducing the expression of IL-2 in mouse BV-2 microglial cells, primary mouse and human microglia, mouse peritoneal macrophages, RAW264.7 macrophages, and T cells. Interestingly, we found that p402 and IL-12p70 (IL-12), but not IL-23, dose-dependently induced the production of IL-2 and the expression of IL-2 mRNA in microglial cells. Similarly, p402 also induced the activation of IL-2 promoter in microglial cells and RAW264.7 cells. Among various stimuli tested, p402 was the most potent stimulus followed by IFN-γ, bacterial lipopolysaccharide, HIV-1 gp120, and IL-12 in inducing the activation of IL-2 promoter in microglial cells. Moreover, p402, but not IL-23, increased NFATc2 mRNA expression and the transcriptional activity of NFAT. Furthermore, induction of IL-2 mRNA expression by over-expression of p40, but not by p19, cDNA indicated that p40, but not p19, is responsible for the induction of IL-2 mRNA in microglia. Finally, by using primary microglia from IL to 12 receptor β1 deficient (IL-12Rβ1-/-) and IL-12 receptor β2 deficient (IL-12Rβ2-/-) mice, we demonstrate that p402 induces the expression of IL-2 via IL-12Rβ1, but not IL-12Rβ2. In experimental autoimmune encephalomyelitis, an animal model of MS, neutralization of p402 by mAb a3-1d led to decrease in clinical symptoms and reduction in IL-2 in T cells and microglia. These results delineate a new biological function of p402, which is missing in the so-called autoimmune cytokine IL-23, and raise the possibility of controlling increased IL-2 and the disease process of MS via neutralization of p402.

IMPA2 promotes basal-like breast cancer aggressiveness by a MYC-mediated positive feedback loop

Basal-like breast cancer (BLBC) is the most aggressive subtype with poor prognosis; however, the mechanisms underlying aggressiveness in BLBC remain poorly understood. In this study, we showed that in contrast to other subtypes, inositol monophosphatase 2 (IMPA2) was dramatically increased in BLBC. Mechanistically, IMPA2 expression was upregulated due to copy number amplification, hypomethylation of IMPA2 promoter and MYC-mediated transcriptional activation. IMPA2 promoted MI-PI cycle and IP3 production, and IP3 then elevated intracellular Ca2+ concentration, leading to efficient activation of NFAT1. In turn, NFAT1 up-regulated MYC expression, thereby fulfilling a positive feedback loop that enhanced aggressiveness of BLBC cells. Knockdown of IMPA2 expression caused the inhibition of tumorigenicity and metastasis of BLBC cells in vitro and in vivo. Clinically, high IMPA2 expression was strongly correlated with large tumor size, high grade, metastasis and poor survival, indicating poor prognosis in breast cancer patients. These findings suggest that IMPA2-mediated MI-PI cycle allows crosstalk between metabolic and oncogenic pathways to promote BLBC progression.

Abstract B119: First-in-class orally active pharmacological inhibitors of TRPV6 for the treatment of advanced prostate cancer

Abstract The highly calcium selective ion channel TRPV6, has pronouced overexpression in many prostate cancers, is a promotor of prostate cancer growth and has been proposed as a prognostic marker in prostate cancer. A novel series of first-in-class TRPV6 inhibitors has been developed by the Queensland Emory Drug Discovery Initiative (QEDDI), a drug discovery and development group aiming to collaborativey translate academic biomedical research into new medicines. The preclinical lead drug candidate QED-203, is orally active and potently inhibits (low nM) calcium influx mediated by TRPV6 as assessed by both Ca2+ probe and electrophysiology assays, and TRPV6-driven NFAT activation as assessed by a luciferase reporter assay. Using RNAseq and qPCR analysis TRPV6 pharmacological inhibition and/or siRNA-mediated silencing was shown to remodel the expression of genes related to both NFAT and WNT signalling, as well as ER stress and the cell cycle. In prostate cancer cell lines that had increased levels of TRPV6, TRPV6 inhibition promoted cell cycle arrest, decreased proliferation and promoted apoptosis. Proliferation of enzalutamide resistant prostate cancer cell lines was also inhibited by QED-203. QED-203 exhibited greater potency over enzalutamide, darolutamide and apalutamide [androgen receptor targeting agents (ARTA)] in prostate cancer cell lines with clinically relevant AR mutations or those with the ARV7 splice variant. QED-203 appeared well suited to once-a-day oral dosing with a high bioavailability and an advantageous pharmacokinetic profile. In preclinical models, QED-203 was well tolerated in rodents and had similar in vivo efficacy to enzalutamide in a castrated LNCaP mouse model of prostate cancer as assessed by tumour growth and PSA levels. Increases in urine calcium levels and gene expression changes consistent with TRPV6 inhibition in isolated xenograft tumours, confirmed QED-203 TRPV6 target engagement in vivo. These studies suggest that the orally active TRPV6 inhibitor QED-203 represents a first-in-class mechanism to treat prostate cancer patients whose tumours have become resistant to current ARTA therapies. Pharmacological inhibitors of TRPV6, could be particularly valuable during the advanced stages of prostate cancer, where there is a clear unmet clinical need. Citation Format: Gregory Monteith, Kimberley Beaumont, Rebecca Pouwer, Rebecca Farrow, Matthew McLachlan, Mei Yeh, Therese Johnson, Raphael Rahmani, Claire Levrier, Hasanthi Wijesekera, Malika Kumarasiri, Grant Stuchbury, Terrie-Anne Cock, Andrew Harvey, Brian Dymock. First-in-class orally active pharmacological inhibitors of TRPV6 for the treatment of advanced prostate cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B119.

Dual Functions of Mitochondrial Calcium Uniporter in T Cell Alloimmunity

Despite pharmacological prophylaxis using calcineurin inhibitors, graft-versus-host disease (GVHD) still occurs in patients undergoing allogeneic hematopoietic stem cell transplantation. However, the mechanism that underlies the breakthrough of alloreactive T cell responses following calcium signaling inhibition remains poorly defined. The mitochondrial calcium uniporter (MCU), which is located in the inner mitochondrial membrane, controls Ca2+ uptake into the mitochondria matrix. Past dogma suggested that lack of mitochondria calcium uptake would compromise T cell immune response due to suboptimal NFAT activation upon TCR ligation. However, the exact role of MCU in the regulation of alloreactive T cell responses has not been previously determined. Here, we demonstrate dual functions of MCU in alloreactive T cell response and GVHD maintenance, highlighting MCU as a potential novel target to manipulate T cell alloreactivity. Using T cell-specific Mcu conditional knockout C57/BL6 (B6) mice (Mcu-cKO), we found that compared to WT T cells, B6 Mcu-cKO T cells induced less severe GVHD in Balb/c mice, with prolonged survival and significantly lower number of donor effector cells in the spleen and liver early after transplantation. Surprisingly, these Mcu-cKO T cell recipients developed severe liver GVHD by 60 days after transplantation, as evidenced by extensive lymphocytic infiltration in the liver, while to a lesser extent in the skin and intestine in these GVHD mice. Flow cytometric analysis of donor T cells, which were harvested between 5 and 8 weeks after transplantation, showed that Mcu-cKO T cells retained the IFN-g- and GM-CSF-producing capacity, but acquired stronger capacity of producing the cytotoxic molecule granzyme B. To comprehensively delineate the role of MCU in allogeneic T cell response, we used MHC-identical but minor histocompatibility antigen (miHA)-mismatched B6-to-Balb/b GVHD model, in which alloreactive CD8+ T cells can be identified with H60 peptide/MHC class-I dimer staining. Similarly, loss of Mcu in T cells caused less severe GVHD, attributed to the decreased number of H60+CD8+ effector T cells that showed impaired proliferation capacity. RNA-seq analysis of liver-infiltrating H60+CD8+ T cells revealed that Mcu ablation resulted in decreased expression of cell proliferation genes (Pclaf, Cdc45, Top2a, Cdca8, etc), increased expression of genes (Cd7, Bcl2, CD96, Eomes, Ezh1, etc) associated with memory T cells. These results demonstrated that Mcu is required for the expansion of alloreactive T cells, but restricts their memory-associated transcriptional programs during GVHD maintenance. Further characterization showed these alloreactive Mcu-cKO T cells produced higher levels of tissue migration molecules (CCR9, CCR5, etc) and lower death molecule (Lpin1) than their WT counterparts. Using gain-of-function strategy of retroviral Mcu overexpression in T cells (McuOE), we found McuOE CD8+ T cells displayed enhanced effector differentiation represented by higher expression of effector molecules (Ifng), decreased expression of stemness and memory associated molecules (Tcf7, Id3), and reduced expression of migration molecules (CCR9, CXCR3, etc). The cultured McuOE CD8+ T cells contracted earlier than WT. These results were further validated in vivo using the B6-to-Balb/c GVHD model. These data demonstrate a negative regulatory role of MCU in the tissue migration and persistence of alloreactive T cells. Notably, human T cells from patients with severe GVHD produced lower levels of MCU protein compared to that of non-GVHD patients. Our findings identified for the first time the dual functions of MCU in the regulation of T cell alloimmunity. MCU-mediated mitochondria calcium uptake promotes early expansion of alloreactive T cells but restricts the expression of memory-associated gene programs and tissue migration molecules. Strategies of enhancing MCU functionality in alloreactive T cells could potentially reduce their memory potential and tissue infiltration capacity, thereby reducing GVHD.

Optogenetic Control of Oncogenic Signaling in B-Cell Malignancies

Background: B-cell receptor (BCR) signals are essential determinants of survival and proliferation throughout normal B-cell development. In B-cell malignancies, these signals are frequently generated by oncogenic mimics of the BCR signaling pathway. For instance, oncogenes in B-ALL, derived from B-cell precursors, typically mimic survival signals from a constitutively active pre-BCR, while tonic and chronic active BCR signaling were identified in mature B-cell lymphomas. Oncogenic BCR-signaling represents an important target of therapeutic intervention: for instance, small molecule inhibitors of SYK (e.g. entospletinib) and BTK (e.g. ibrutinib) have been developed to disrupt oncogenic BCR signaling in mature B-cell lymphomas. Besides SYK and BTK tyrosine kinases, oncogenic BCR-signaling leads to activation of PLCG2, which initiates the release of Ca 2+ from the ER into the cytoplasm. Thereby, calcium flux is decoded by NFATC1 or NF-kB based on fast (NFATC1) or slow (NF-kB) frequencies of Ca 2+-signals. Significance: To interrogate Ca 2+-signals as critical integration point of oncogenic BCR-signaling, we engineered cell lines and PDX with a GCaMP6s-biosensor, which allows tracing of calcium signaling in single cells over time ( Figure A). To cover B-cell malignancies from multiple stages of B-cell development, we engineered B-ALL (pro- and pre-B cells), mantle cell lymphoma (MCL, naïve B-cells), Burkitt's lymphoma (germinal center), DLBCL (post-GC), multiple myeloma (terminally differentiated plasma cells) and Hodgkin's disease (“crippled” BCR-deficient GC-B cells). Strikingly, B-cell malignancies exhibit autonomous Ca 2+-oscillations, which decreased in their frequency from 20 mHz (pro- and pre-B), 11 mHz (naïve), 4 mHz (germinal center) to 0 mHz in post-GC and terminally differentiated plasma cells based on the differentiation stage of their cell of origin ( Figure A). Results: Given the striking differences in oncogenic BCR-signaling as measured by autonomous Ca 2+ oscillations, we developed an optogenetic system to control the frequency and amplitude of Ca 2+ oscillations by blue light pulses ( Figure B). To model frequency-modulated Ca 2+ signaling in B-cells, we engineered murine pre-B cells carrying an optogenetic tool termed OptoCRAC, which enables reversible activation and inactivation of the plasma membrane Ca 2+ channel Orai1 by intermittent blue light irradiation. Time-lapse imaging of a fluorescent Ca 2+ reporter (R-CaMP1.07) confirmed rapid and reversible Ca 2+ influx in response to intermittent blue light pulses. To assess the phenotypic consequence of high-frequency Ca 2+ oscillations, we developed an LED-array-based optogenetic platform for cell culture plates. While low-frequency Ca 2+ oscillations (0.5 mHz) did not impact cell viability, high-frequency Ca 2+ oscillations (20 mHz) rapidly induced NFAT activation (5 min), and eventually cell death within 72 h ( Figure B). Mechanism and conclusions: To elucidate the mechanistic contribution of NFATC1 in B-cell death induced by fast Ca 2+ oscillations, we tested whether genetic deletion of Nfatc1 is sufficient to rescue B-cell death upon fast Ca 2+ oscillation induced by 20 mHz intermittent blue light pulses. As expected, while B-ALL cells retaining intact Nfatc1 rapidly decreased cell viability, the cells with Cre-mediated deletion persistently remained in cell culture upon 20 mHz Ca 2+ oscillation. In addition to genetic deletion of Nfatc1, we also found that inducible expression of mutant activators of NF-kB signaling were able to rescue B-cells from cell death induced by high-frequency oscillations: Concurrent expression of Card11 L232LI, MYD88 L265P and IKK2 S177E/S181E not only reduced the frequency of autonomous Ca 2+ oscillations but also enabled B-cell survival despite delivery of light-pulses and Ca 2+ oscillations at a fast 20 mHz frequency. We conclude that autonomous Ca 2+ oscillations represent a critical integration point of oncogenic BCR-signaling in B-cell malignancies and that optogenetic control of BCR-downstream Ca 2+ oscillations will reveal previously unrecognized vulnerabilities that can be exploited to increase efficacy of BCR-signaling inhibitors (e.g. ibrutinib, entospletinib, idelalisib) that are currently being used for the treatment of B-cell lymphomas.

Separation of B- and T-Cell-Specific Signaling Molecules Prevents Oncogenic Transformation in Lymphoid Malignancies

Background and significance: Despite the highly homologous structure and function of proteins controlling signal transduction from the BCR and TCR, B- and T-cells have evolved separate sets of linker and kinase molecules and mechanisms to ensure that their expression is strictly separated between B- and T-lymphoid lineages. While this is the case among normal B- and T-cells, we and others showed that the separation of B- and T-cell-specific signaling molecules is often blurred in lymphoid malignancies. For instance, B-ALL, CLL, and MCL often aberrantly express the T-cell kinase ZAP70, whereas PTCLs and γδTCR CTCLs often express SYK or the ITK-SYK fusion. Here we investigated the mechanisms driving the aberrant cross-lineage expression of BCR- and TCR-downstream signaling molecules and how the promiscuous expression of these molecules contributes to the malignant transformation of lymphoid malignancies. Results: We have previously shown that the B-cell-specific kinase SYK and the T-cell-specific kinase ZAP70 are segregated to prevent autoimmunity and malignant transformation of pre-germinal center B-cells. In pre-malignant B-cells, SYK drives negative selection through Ca 2+-signaling via BTK-BLNK phosphorylation and downstream activation of NFAT. The aberrant expression of ZAP70 diverts SYK signaling towards CD19-PI3K, weakening negative selection, and consequently permitting the expansion of pre-malignant cells. This represents a novel key mechanism of aberrantly expressed signaling molecules in the etiology and persistence of B-lymphoid malignancy. We previously showed that co-expression of the T-cell kinase ZAP70 with the B-cell kinase SYK dramatically accelerated the development of fatal disease in a mouse model of CLL, consistent with poor clinical outcomes of ZAP70 + CLL in patients. To elucidate the role of ZAP70 co-expression in the initiation of B-ALL, pre-B-cells were isolated from Zap70 -/- mice and transformed ex vivo with BCR-ABL1 and NRAS G12D to model B-ALL. These cells showed significantly impaired colony-forming capacity compared to transformed wild-type cells. To corroborate these results, we crossed BCR-ABL1-transgenic with Zap70 -/- mice. Next, patient-derived B-ALL and MCL xenografts (PDX) were engineered to carry a homozygous knock-in of dTAG at the C-terminus of ZAP70. Upon degradation of ZAP70-dTAG, consequences on cellular fitness and proliferation were assessed, as well as the downstream activation of Ca 2+-signaling and NFAT activation ( Figure 1). Additionally, a mouse strain carrying a bulky ATP-analog-sensitive variant of Zap70 (M413A) was crossed with the p190 strain to assess the effect of ZAP70 inhibition after the manifestation of B-cell leukemia. Since the expression of ZAP70 increases upon malignant transformation of pre-germinal center B-cells, we used Genomic Locus Proteomics (GLoPro) to identify transcription factors driving this transcriptional upregulation ( Figure 1). By comparing malignant B- and T-cell lines, which either express only SYK or ZAP70 or co-express them, we were able to isolate specific mediators of aberrant transcription of ZAP70, including β-catenin/WNT signaling. ChIP-qPCR, CRISPR/Cas9-mediated knockout, as well as dTAG, were used to validate the binding of transcription factors identified by GLoPro to the ZAP70 enhancers and their role in ZAP70 expression. Apart from ZAP70, we identified multiple aberrantly co-expressed T-cell signaling proteins by comparative proteomic analysis of malignant B- and T-cell lines. These included LCK, PLCγ1, and SLP-76, while the BTK-homolog ITK is completely absent in B-cell lines. In future work, we aim to elucidate the transcriptional regulation and consequences of the loss of these aberrantly co-expressed proteins. Further, we will perform a CRISPR/Cas9-based switch of BTK and ITK to explore the effects of forced co-expression of these segregated proteins. Conclusions: We conclude that malignant pre-germinal center B-cells increase the expression of ZAP70 through the formation of aberrant transcription machinery. Furthermore, our work elucidates the role of ZAP70 not only for the initiation but also for the maintenance of B-cell leukemia. Other T-cell signaling molecules are also aberrantly expressed in B-cell malignancies and further investigation will highlight their transcriptional regulation and role during and after malignant transformation.

STIM-mediated calcium influx regulates maintenance and selection of germinal center B cells.

Positive selection of high-affinity germinal center (GC) B cells is driven by antigen internalization through their B cell receptor (BCR) and presentation to follicular helper T cells. However, the requirements of BCR signaling in GC B cells remain poorly understood. Store-operated Ca2+ entry, mediated by stromal interacting molecule 1 (STIM1) and STIM2, is the main Ca2+ influx pathway triggered by BCR engagement. Here, we showed that STIM-deficient B cells have reduced B cell competitiveness compared with wild-type B cells during GC responses. B cell-specific deletion of STIM proteins decreased the number of high-affinity B cells in the late phase of GC formation. STIM deficiency did not affect GC B cell proliferation and antigen presentation but led to the enhancement of apoptosis due to the impaired upregulation of anti-apoptotic Bcl2a1. STIM-mediated activation of NFAT was required for the expression of Bcl2a1 after BCR stimulation. These findings suggest that STIM-mediated survival signals after antigen capture regulate the optimal selection and maintenance of GC B cells.

A Novel Allogeneic Rituximab-Conjugated Gamma Delta T Cell Therapy for the Treatment of Relapsed/Refractory B-Cell Lymphoma.

Chimeric antigen receptor T cell (CAR-T) therapy has been applied in the treatment of B-cell lymphoma; however, CAR-T manufacturing requires virus- or non-virus-based genetic modification, which causes high manufacturing costs and potential safety concerns. Antibody-cell conjugation (ACC) technology, which originated from bio-orthogonal click chemistry, provides an efficient approach for arming immune cells with cancer-targeting antibodies without genetic modification. Here, we applied ACC technology in Vγ9Vδ2 T (γδ2 T) cells to generate a novel off-the-shelf CD20-targeting cell therapy ACE1831 (rituximab-conjugated γδ2 T cells) against relapsed/refractory B-cell lymphoma. ACE1831 exhibited superior cytotoxicity against B-cell lymphoma cells and rituximab-resistant cells compared to γδ2 T cells without rituximab conjugation. The in vivo xenograft study demonstrated that ACE1831 treatment strongly suppressed the aggressive proliferation of B-cell lymphoma and prolonged the survival of tumor-bearing mice with no observed toxicity. Mass spectrometry analysis indicated that cell activation receptors including the TCR complex, integrins and cytokine receptors were conjugated with rituximab. Intriguingly, the antigen recognition of the ACC-linked antibody/receptor complex stimulated NFAT activation and contributed to ACE1831-mediated cytotoxicity against CD20-expressing cancer cells. This study elucidates the role of the ACC-linked antibody/receptor complex in cytotoxicity and supports the potential of ACE1831 as an off-the-shelf γδ2 cell therapy against relapsed/refractory B-cell lymphoma.

Glycogen synthase kinase 3 controls T-cell exhaustion by regulating NFAT activation.

Cellular immunity mediated by CD8+ T cells plays an indispensable role in bacterial and viral clearanceand cancers. However, persistent antigen stimulation of CD8+ T cells leads to an exhausted or dysfunctional cellular state characterized by the loss of effector function and high expression of inhibitory receptors during chronic viral infection and in tumors. Numerous studies have shown that glycogen synthase kinase 3 (GSK3) controls the function and development of immune cells, but whether GSK3 affects CD8+ T cells is not clearly elucidated. Here, we demonstrate that mice with deletion of Gsk3α and Gsk3β in activated CD8+ T cells (DKO) exhibited decreased CTL differentiation and effector function during acute and chronic viral infection. In addition, DKO mice failed to control tumor growth due to the upregulated expression of inhibitory receptors and augmented T-cell exhaustion in tumor-infiltrating CD8+ T cells. Strikingly, anti-PD-1 immunotherapy substantially restored tumor rejection in DKO mice. Mechanistically, GSK3 regulates T-cell exhaustion by suppressing TCR-induced nuclear import of NFAT, thereby in turn dampening NFAT-mediated exhaustion-related gene expression, including TOX/TOX2 and PD-1. Thus, we uncovered the molecular mechanisms underlying GSK3 regulation of CTL differentiation and T-cell exhaustion in anti-tumor immune responses.