PTEN loss in glioma cell lines leads to increased extracellular vesicles biogenesis and PD-L1 cargo in a PI3K-dependent manner.

Phosphatase and Tensin Homologue (PTEN) is one of the most frequently lost tumor suppressors in cancer and the predominant negative regulator of the PI3K/AKT signaling axis. A growing body of evidence has highlighted the loss of PTEN with immuno-modulatory functions including the upregulation of the programmed death ligand-1 (PD-L1), an altered tumor derived secretome that drives an immunosuppressive tumor immune microenvironment (TIME), and resistance to certain immunotherapies. Given their roles in immunosuppression and tumor growth, we examined whether the loss of PTEN would impact the biogenesis, cargo, and function of extracellular vesicles (EVs) in the context of the anti-tumor associated cytokine interferon-γ (IFN-γ). Through genetic and pharmacological approaches, we show that PD-L1 expression is regulated by JAK/STAT signaling, not PI3K signaling. Instead, we observe that PTEN loss positively upregulates cell surface levels of PD-L1 and enhances the biogenesis of EVs enriched with PD-L1 in a PI3K-dependent manner. We demonstrate that because of these changes, EVs derived from glioma cells lacking PTEN have a greater ability to suppress T cell receptor (TCR) signaling. Taken together, these findings provide important new insights into how the loss of PTEN can contribute to an immunosuppressive TIME, facilitate immune evasion, and highlight a novel role for PI3K signaling in the regulation of EV biogenesis and the cargo they contain.

Adaptive immune cells are necessary for SARS-CoV-2-induced pathology.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the ongoing global pandemic associated with morbidity and mortality in humans. Although disease severity correlates with immune dysregulation, the cellular mechanisms of inflammation and pathogenesis of COVID-19 remain relatively poorly understood. Here, we used mouse-adapted SARS-CoV-2 strain MA10 to investigate the role of adaptive immune cells in disease. We found that while infected wild-type mice lost ~10% weight by 3 to 4 days postinfection, rag-/- mice lacking B and T lymphocytes did not lose weight. Infected lungs at peak weight loss revealed lower pathology scores, fewer neutrophils, and lower interleukin-6 and tumor necrosis factor-α in rag-/- mice. Mice lacking αß T cells also had less severe weight loss, but adoptive transfer of T and B cells into rag-/- mice did not significantly change the response. Collectively, these findings suggest that while adaptive immune cells are important for clearing SARS-CoV-2 infection, this comes at the expense of increased inflammation and pathology.

Monoclonal antibodies binding to different epitopes of CD20 differentially sensitize DLBCL to different classes of chemotherapy.

Introduction: Rituximab (R), an anti-CD20 monoclonal antibody (mAb) and the world's first approved antibody for oncology patients, was combined with the CHOP chemotherapy regimen and markedly improved the prognosis of all B- cell-derived lymphomas, the most common hematological malignancy worldwide. However, there is a 35% disease recurrence with no advancement in the first-line treatment since R was combined with the archetypal CHOP chemotherapy regimen nearly 30 years ago. There is evidence that R synergizes with chemotherapy, but the pharmacological interactions between R and CHOP or between newer anti-CD20 mAbs and CHOP remain largely unexplored. Methods: We used in vitro models to score pharmacological interactions between R and CHOP across various lymphoma cell lines. We compared these pharmacological interactions to ofatumumab, a second-generation anti-CD20 mAb, and CHOP. Lastly, we used RNA-sequencing to characterize the transcriptional profiles induced by these two antibodies and potential molecular pathways that mediate their different effects. Results: We discovered vast heterogeneity in the pharmacological interactions between R and CHOP in a way not predicted by the current clinical classification. We then discovered that R and ofatumumab differentially synergize with the cytotoxic and cytostatic capabilities of CHOP in separate distinct subsets of B-cell lymphoma cell lines, thereby expanding favorable immunochemotherapy interactions across a greater range of cell lines beyond those induced by R-CHOP. Lastly, we discovered these two mAbs differentially modulate genes enriched in the JNK and p38 MAPK family, which regulates apoptosis and proliferation. Discussion: Our findings were completely unexpected because these mAbs were long considered to be biological and clinical equivalents but, in practice, may perform better than the other in a patient-specific manner. This finding may have immediate clinical significance because both immunochemotherapy combinations are already FDA-approved with no difference in toxicity across phase I, II, and III clinical trials. Therefore, this finding could inform a new precision medicine strategy to provide additional therapeutic benefit to patients with B-cell lymphoma using immunochemotherapy combinations that already meet the clinical standard of care.

Degrading the signal amplifier: ITK as a target for targeted protein degradation.

In this issue of Cell Chemical Biology, Jiang and colleagues show for the first time that the Tec kinase ITK can be targeted using PROTAC approaches. This new modality has implications for the treatment of T cell lymphomas, but also potentially for the treatment of T cell-mediated inflammatory diseases, that depend on ITK signaling.

Cell-autonomous requirement for ACE2 across organs in lethal mouse SARS-CoV-2 infection.

Angiotensin-converting enzyme 2 (ACE2) is the cell-surface receptor for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). While its central role in Coronavirus Disease 2019 (COVID-19) pathogenesis is indisputable, there remains significant debate regarding the role of this transmembrane carboxypeptidase in the disease course. These include the role of soluble versus membrane-bound ACE2, as well as ACE2-independent mechanisms that may contribute to viral spread. Testing these roles requires in vivo models. Here, we report humanized ACE2-floxed mice in which hACE2 is expressed from the mouse Ace2 locus in a manner that confers lethal disease and permits cell-specific, Cre-mediated loss of function, and LSL-hACE2 mice in which hACE2 is expressed from the Rosa26 locus enabling cell-specific, Cre-mediated gain of function. Following exposure to SARS-CoV-2, hACE2-floxed mice experienced lethal cachexia, pulmonary infiltrates, intravascular thrombosis and hypoxemia-hallmarks of severe COVID-19. Cre-mediated loss and gain of hACE2 demonstrate that neuronal infection confers lethal cachexia, hypoxemia, and respiratory failure in the absence of lung epithelial infection. In this series of genetic experiments, we demonstrate that ACE2 is absolutely and cell-autonomously required for SARS-CoV-2 infection in the olfactory epithelium, brain, and lung across diverse cell types. Therapies inhibiting or blocking ACE2 at these different sites are likely to be an effective strategy towards preventing severe COVID-19.

Interleukin-2-inducible T-cell kinase (Itk) signaling regulates potent noncanonical regulatory T cells.

Regulatory T cells (Tregs) play an important role in controlling autoimmunity and limiting tissue damage and inflammation. IL2-inducible T cell kinase (Itk) is part of the Tec family of tyrosine kinases and is a critical component of T cell receptor mediated signaling. Here, we showed that either genetic ablation of Itk signaling or inhibition of Itk signaling pathways resulted in increased frequency of "noncanonical" CD4+ CD25- FOXP3+ Tregs (ncTregs), as well as of "canonical" CD4+ CD25+ FOXP3+ Tregs (canTregs). Using in vivo models, we showed that ncTregs can avert the formation of acute graft-versus-host disease (GVHD), in part by reducing conventional T cell proliferation, proinflammatory cytokine production, and tissue damage. This reduction in GVHD occurred without disruption of graft-versus-leukaemia (GVL) effects. RNA sequencing revealed that a number of effector, cell adhesion, and migration molecules were upregulated in Itk-/- ncTregs. Furthermore, disrupting the SLP76: ITK interaction using a specific peptide inhibitor led to enhanced Treg development in both mouse and primary human cells. This peptide inhibitor also significantly reduced inflammatory cytokine production in primary GVHD patient samples and mouse T cells without causing cell death or apoptosis. We provide evidence that specifically targeting Itk signaling could be a therapeutic strategy to treat autoimmune disorders.

Genetic Analysis of iNKT Cell Development and Function.

Natural killer T (NKT) cells are among the immediate and early responding immune cells and are important players in autoimmune diseases and tumor immunity. This unique subset of T cells shares properties of natural killer cells and T cells. Proper identification and characterization of NKT cell subsets is essential to understand the function and involvement of these understudied immune cells in various diseases. This review aims to summarize the known methods for identifying and characterizing NKT cells. NKT cells are divided into Type I (or invariant) and Type II, with either limited or broad TCR repertoires, respectively, that generally respond to glycolipids presented on the nonclassical MHC, CD1d. Type I NKT cells or invariant NKT cells (iNKT) are the most well studied and can be further subdivided into NKT1, NKT2, or NKT17 populations, classified based on their functional capacity. Conversely, less is known about Type II NKT cells because they have a more diverse TCR repertoire which make them hard to identify. However, genetic analyses have shed light on the development and function of all NKT subsets, which aids in their characterization. Further exploration of the role of NKT cells in various diseases will reveal the intricacies and importance of their novel functions.

Human Wnt/ß-Catenin Regulates Alloimmune Signaling during Allogeneic Transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is one of the most widely applied forms of adoptive immunotherapy for the treatment of hematological malignancies. Detrimental graft-versus-host disease (GVHD), but also beneficial graft-versus-leukemia (GVL) effects occurring after allo-HSCT are largely mediated by alloantigen-reactive donor T cells in the graft. Separating GVHD from GVL effects is a formidable challenge, and a greater understanding of donor T cell biology is required to accomplish the uncoupling of GVHD from GVL. Here, we evaluated the role of ß-catenin in this process. Using a unique mouse model of transgenic overexpression of human ß-catenin (Cat-Tg) in an allo-HSCT model, we show here that T cells from Cat-Tg mice did not cause GVHD, and surprisingly, Cat-Tg T cells maintained the GVL effect. Donor T cells from Cat-Tg mice exhibited significantly lower inflammatory cytokine production and reduced donor T cell proliferation, while upregulating cytotoxic mediators that resulted in enhanced cytotoxicity. RNA sequencing revealed changes in the expression of 1169 genes for CD4, and 1006 genes for CD8+ T cells involved in essential aspects of immune response and GVHD pathophysiology. Altogether, our data suggest that ß-catenin is a druggable target for developing therapeutic strategies to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment.

Targeting SLP76:ITK interaction separates GVHD from GVL in allo-HSCT.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative therapy for hematological malignancies, due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells. However, graft-versus-host disease (GVHD) is also mediated by these cells. Here, we assessed the effect of attenuating TCR-mediated SLP76:ITK interaction in GVL vs. GVHD effects after allo-HSCT. CD8+ and CD4+ donor T cells from mice expressing a Y145F mutation in SLP-76 did not cause GVHD but preserved GVL effects against B-ALL cells. SLP76Y145FKI CD8+ and CD4+ donor T cells also showed less inflammatory cytokine production and migration to GVHD target organs. We developed a novel peptide to specifically inhibit SLP76:ITK interactions, resulting in decreased phosphorylation of PLCγ1 and ERK, decreased cytokine production in human T cells, and separation of GVHD from GVL effects. Altogether, our data suggest that inhibiting SLP76:ITK interaction could be a therapeutic strategy to separate GVHD from GVL effects after allo-HSCT treatment.

Effect of Perfluorooctanesulfonic acid (PFOS) on immune cell development and function in mice.

Perfluoroctanesulfonate (PFOS) belongs to a larger family of compounds known as Per- and polyfluoroalkyl substances (PFAS). The strength of the carbon-fluorine bond makes PFOS extremely resistant to environmental degradation. Due to its persistent nature, research has been directed to elucidating possible health effects of PFOS on humans and laboratory animals. Here we have explored the effects of PFOS exposure on immune development and function in mice. We exposed adult mice to 3 and 1.5 µg/kg/day of PFOS for 2 and 4 weeks, respectively, and examined the effects of PFOS exposure on populations of T cells, B cells, and granulocytes. These doses of PFOS resulted in serum levels of approximately 100 ng/mL with no weight loss during exposure. We find that PFOS does not affect T-cell development during this time. However, while PFOS exposure reduced immune cell populations in some organs, it also led to an increase in the numbers of cells in others, suggesting possible relocalization of cells. We also examined the effect of PFOS on the response to influenza virus infection. We find that exposure to PFOS at 1.5 µg/kg/day of PFOS for 4 weeks does not affect weight loss or survival, nor is viral clearance affected. Analysis of antibody and T cell specific antiviral responses indicate that at this concentration, PFOS does not suppress the immune cell development or antigen specific immune response.

Targeting Interleukin-2-Inducible T-Cell Kinase (ITK) Differentiates GVL and GVHD in Allo-HSCT.

Allogeneic hematopoietic stem cell transplantation is a potentially curative procedure for many malignant diseases. Donor T cells prevent disease recurrence via graft-versus-leukemia (GVL) effect. Donor T cells also contribute to graft-versus-host disease (GVHD), a debilitating and potentially fatal complication. Novel treatment strategies are needed which allow preservation of GVL effects without causing GVHD. Using murine models, we show that targeting IL-2-inducible T cell kinase (ITK) in donor T cells reduces GVHD while preserving GVL effects. Both CD8+ and CD4+ donor T cells from Itk-/- mice produce less inflammatory cytokines and show decrease migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). Itk-/- T cells exhibit reduced expression of IRF4 and decreased JAK/STAT signaling activity but upregulating expression of Eomesodermin (Eomes) and preserve cytotoxicity, necessary for GVL effect. Transcriptome analysis indicates that ITK signaling controls chemokine receptor expression during alloactivation, which in turn affects the ability of donor T cells to migrate to GVHD target organs. Our data suggest that inhibiting ITK could be a therapeutic strategy to reduce GVHD while preserving the beneficial GVL effects following allo-HSCT treatment.

Interleukin-2-Inducible T-Cell Kinase Deficiency Impairs Early Pulmonary Protection Against Mycobacterium tuberculosis Infection.

Interleukin-2 (IL-2) inducible T-cell kinase (ITK) is a non-receptor tyrosine kinase highly expressed in T-cell lineages and regulates multiple aspects of T-cell development and function, mainly through its function downstream of the T-cell receptor. Itk deficiency can lead to CD4 lymphopenia and Epstein-Bar virus (EBV)-associated lymphoproliferation and recurrent pulmonary infections in humans. However, the role of the ITK signaling pathway in pulmonary responses in active tuberculosis due to Mtb infection is not known. We show here that human lungs with active tuberculosis exhibit altered T-cell receptor/ITK signaling and that Itk deficiency impaired early protection against Mtb in mice, accompanied by defective development of IL-17A-producing γδ T cells in the lungs. These findings have important implications of human genetics associated with susceptibility to Mtb due to altered immune responses and molecular signals modulating host immunity that controls Mtb activity. Enhancing ITK signaling pathways may be an alternative strategy to target Mtb infection, especially in cases with highly virulent strains in which IL-17A plays an essential protective role.

Amino acid starvation sensing dampens IL-1ß production by activating riboclustering and autophagy.

Activation of the amino acid starvation response (AAR) increases lifespan and acute stress resistance as well as regulates inflammation. However, the underlying mechanisms remain unclear. Here, we show that activation of AAR pharmacologically by Halofuginone (HF) significantly inhibits production of the proinflammatory cytokine interleukin 1ß (IL-1ß) and provides protection from intestinal inflammation in mice. HF inhibits IL-1ß through general control nonderepressible 2 kinase (GCN2)-dependent activation of the cytoprotective integrated stress response (ISR) pathway, resulting in rerouting of IL-1ß mRNA from translationally active polysomes to inactive ribocluster complexes-such as stress granules (SGs)-via recruitment of RNA-binding proteins (RBPs) T cell-restricted intracellular antigen-1(TIA-1)/TIA-1-related (TIAR), which are further cleared through induction of autophagy. GCN2 ablation resulted in reduced autophagy and SG formation, which is inversely correlated with IL-1ß production. Furthermore, HF diminishes inflammasome activation through suppression of reactive oxygen species (ROS) production. Our study unveils a novel mechanism by which IL-1ß is regulated by AAR and further suggests that administration of HF might offer an effective therapeutic intervention against inflammatory diseases.

Self-Assembly Protein Nanogels for Safer Cancer Immunotherapy.

Soluble antigen-based cancer vaccines have poor retention in tissues along with suboptimal antigen processing by dendritic cells. Multiple booster doses are often needed, leading to dose-limiting systemic toxicity. A versatile, immunomodulatory, self-assembly protein nanogel vaccine is reported that induces robust immune cell response at lower antigen doses than soluble antigens, an important step towards biomaterials-based safer immunotherapy approaches.

IL-2-inducible T cell kinase tunes T regulatory cell development and is required for suppressive function.

IL-2-inducible T cell kinase (ITK) is a key signaling mediator downstream of TCR, mediating T cell positive selection, as well as innate T cell and CD4(+) Th2/Th17 differentiation. In this article, we show that ITK also negatively tunes IL-2-induced expansion of conventional Foxp3-expressing regulatory T cells (Tregs). In vivo, Treg abundance is inversely correlated with ITK expression, and inducible Treg development is inversely dependent on ITK kinase activity. While Treg development normally requires both hematopoietic and thymic MHC class 2 (MHC2) expression, the absence of ITK allows Treg development with MHC2 expression in either compartment, with preference for selection by thymic MHC2, suggesting a gatekeeper role for ITK in ensuring that only Tregs selected by both thymic and hematopoietic MHC2 survive selection. Although ITK suppresses Treg development and is not required for maintenance of neuropilin-1-positive natural Tregs in the periphery, it is indispensable for Treg functional suppression of naive CD4(+) T cell-induced colitis in Rag(-/-) recipients. ITK thus regulates the development and function of Tregs.

Dendritic cell-MHC class II and Itk regulate functional development of regulatory innate memory CD4+ T cells in bone marrow transplantation.

MHC class II (MHCII)-influenced CD4(+) T cell differentiation and function play critical roles in regulating the development of autoimmunity. The lack of hematopoietic MHCII causes autoimmune disease that leads to severe wasting in syngeneic recipients. Using murine models of bone marrow transplantation (BMT), we find that MHCII(-/-)→wild-type BMT developed disease, with defective development of innate memory phenotype (IMP, CD44(hi)/CD62L(lo)) CD4(+) T cells. Whereas conventional regulatory T cells are unable to suppress pathogenesis, IMP CD4(+) T cells, which include conventional regulatory T cells, can suppress pathogenesis in MHCII(-/-)→wild-type chimeras. The functional development of IMP CD4(+) T cells requires hematopoietic but not thymic MHCII. B cells and hematopoietic CD80/86 regulate the population size, whereas MHCII expression by dendritic cells is sufficient for IMP CD4(+) T cell functional development and prevention of pathogenesis. Furthermore, the absence of Tec kinase IL-2-inducible T cell kinase in MHCII(-/-) donors leads to preferential development of IMP CD4(+) T cells and partially prevents pathogenesis. We conclude that dendritic cells-MHCII and IL-2-inducible T cell kinase regulate the functional development of IMP CD4(+) T cells, which suppresses the development of autoimmune disorder in syngeneic BMTs.

Evaluation of the stability, bioavailability, and hypersensitivity of the omega-3 derived anti-leukemic prostaglandin: Δ(12)-prostaglandin J3.

Previous studies have demonstrated the ability of an eicosapentaenoic acid (EPA)-derived endogenous cyclopentenone prostaglandin (CyPG) metabolite, Δ(12)-PGJ3, to selectively target leukemic stem cells, but not the normal hematopoietic stems cells, in in vitro and in vivo models of chronic myelogenous leukemia (CML). Here we evaluated the stability, bioavailability, and hypersensitivity of Δ(12)-PGJ3. The stability of Δ(12)-PGJ3 was evaluated under simulated conditions using artificial gastric and intestinal juice. The bioavailability of Δ(12)-PGJ3 in systemic circulation was demonstrated upon intraperitoneal injection into mice by LC-MS/MS. Δ(12)-PGJ3 being a downstream metabolite of PGD3 was tested in vitro using primary mouse bone marrow-derived mast cells (BMMCs) and in vivo mouse models for airway hypersensitivity. ZK118182, a synthetic PG analog with potent PGD2 receptor (DP)-agonist activity and a drug candidate in current clinical trials, was used for toxicological comparison. Δ(12)-PGJ3 was relatively more stable in simulated gastric juice than in simulated intestinal juice that followed first-order kinetics of degradation. Intraperitoneal injection into mice revealed that Δ(12)-PGJ3 was bioavailable and well absorbed into systemic circulation with a Cmax of 263 µg/L at 12 h. Treatment of BMMCs with ZK118182 for 12 h resulted in increased production of histamine, while Δ(12)-PGJ3 did not induce degranulation in BMMCs nor increase histamine. In addition, in vivo testing for hypersensitivity in mice showed that ZK118182 induces higher airways hyperresponsiveness when compared Δ(12)-PGJ3 and/or PBS control. Based on the stability studies, our data indicates that intraperitoneal route of administration of Δ(12)-PGJ3 was favorable than oral administration to achieve effective pharmacological levels in the plasma against leukemia. Δ(12)-PGJ3 failed to increase histamine and IL-4 in BMMCs, which is in agreement with reduced airway hyperresponsiveness in mice. In summary, our studies suggest Δ(12)-PGJ3 to be a promising bioactive metabolite for further evaluation as a potential drug candidate for treating CML.

Cutting edge: innate memory CD8+ T cells are distinct from homeostatic expanded CD8+ T cells and rapidly respond to primary antigenic stimuli.

Innate memory phenotype (IMP) CD8(+) T cells are nonconventional αß T cells exhibiting features of innate immune cells and are significantly increased in the absence of ITK. Their developmental path and function are not clear. In this study, we show hematopoietic MHC class I (MHCI)-dependent generation of Ag-specific IMP CD8(+) T cells using bone marrow chimeras. Wild-type bone marrow gives rise to IMP CD8(+) T cells in MHCI(-/-) recipients, resembling those in Itk(-/-) mice, but distinct from those derived via homeostatic proliferation, and independent of recipient thymus. In contrast, MHCI(-/-) bone marrow does not lead to IMP CD8(+) T cells in wild-type recipients. OTI IMP CD8(+) T cells generated via this method exhibited enhanced early response to Ag without prior primary stimulation. Our findings suggest a method to generate Ag-specific "naive" CD8(+) IMP T cells, as well as demonstrate that they are not homeostatic proliferation cells and can respond promptly in an Ag-specific fashion.

A unique role for ITK in survival of invariant NKT cells associated with the p53-dependent pathway in mice.

Invariant NKT (iNKT) cells play important roles in the immune response. ITK and TXK/RLK are Tec family kinases that are expressed in iNKT cells; the expression level of ITK is ∼7-fold higher than that of TXK. Itk(-/-) mice have reduced iNKT cell frequency and numbers, with defects in development and cytokine secretion that are exacerbated in Itk/Txk double-knockout mice. In contrast, there is no iNKT cell defect in Txk(-/-) mice. To determine whether ITK and TXK play distinct roles in iNKT cell development and function, we examined mice that overexpress TXK in T cells at levels similar to Itk. Overexpression of TXK rescues the maturation and cytokine secretion of Itk(-/-) iNKT cells, as well as altered expression of transcription factors T-bet, eomesodermin, and PLZF. In contrast, the increased apoptosis observed in Itk(-/-) splenic iNKT cells is not affected by TXK overexpression, likely due to the lack of effect on the elevated expression of p53 regulated proapoptotic pathways Fas, Bax, and Bad in those cells. Supporting this idea, p53(-/-) and Bax(-/-) mice have increased splenic iNKT cells. Our results suggest that TXK plays an overlapping role with ITK in iNKT cell development and function but that ITK also has a unique function in the survival of iNKT cells, likely via a p53-dependent pathway.