In or out of control: Modulating regulatory T cell homeostasis and function with immune checkpoint pathways.

Regulatory T cells (Tregs) are the master regulators of immunity and they have been implicated in different disease states such as infection, autoimmunity and cancer. Since their discovery, many studies have focused on understanding Treg development, differentiation, and function. While there are many players in the generation and function of truly suppressive Tregs, the role of checkpoint pathways in these processes have been studied extensively. In this paper, we systematically review the role of different checkpoint pathways in Treg homeostasis and function. We describe how co-stimulatory and co-inhibitory pathways modulate Treg homeostasis and function and highlight data from mouse and human studies. Multiple checkpoint pathways are being targeted in cancer and autoimmunity; therefore, we share insights from the clinic and discuss the effect of experimental and approved therapeutics on Treg biology.

B7-H7 (HHLA2) inhibits T-cell activation and proliferation in the presence of TCR and CD28 signaling.

Modulation of T-cell responses has played a key role in treating cancers and autoimmune diseases. Therefore, understanding how different receptors on T cells impact functional outcomes is crucial. The influence of B7-H7 (HHLA2) and CD28H (TMIGD2) on T-cell activation remains controversial. Here we examined global transcriptomic changes in human T cells induced by B7-H7. Stimulation through TCR with OKT3 and B7-H7 resulted in modest fold changes in the expression of select genes; however, these fold changes were significantly lower than those induced by OKT3 and B7-1 stimulation. The transcriptional changes induced by OKT3 and B7-H7 were insufficient to provide functional stimulation as measured by evaluating T-cell proliferation and cytokine production. Interestingly, B7-H7 was coinhibitory when simultaneously combined with TCR and CD28 stimulation. This inhibitory activity was comparable to that observed with PD-L1. Finally, in physiological assays using T cells and APCs, blockade of B7-H7 enhanced T-cell activation and proliferation, demonstrating that this ligand acts as a break signal. Our work defines that the transcriptomic changes induced by B7-H7 are insufficient to support full costimulation with TCR signaling and, instead, B7-H7 inhibits T-cell activation and proliferation in the presence of TCR and CD28 signaling.

Evaluation of ultra-low input RNA sequencing for the study of human T cell transcriptome.

Deeper understanding of T cell biology is crucial for the development of new therapeutics. Human naïve T cells have low RNA content and their numbers can be limiting; therefore we set out to determine the parameters for robust ultra-low input RNA sequencing. We performed transcriptome profiling at different cell inputs and compared three protocols: Switching Mechanism at 5' End of RNA Template technology (SMART) with two different library preparation methods (Nextera and Clontech), and AmpliSeq technology. As the cell input decreased the number of detected coding genes decreased with SMART, while stayed constant with AmpliSeq. However, SMART enables detection of non-coding genes, which is not feasible for AmpliSeq. The detection is dependent on gene abundance, but not transcript length. The consistency between technical replicates and cell inputs was comparable across methods above 1 K but highly variable at 100 cell input. Sensitivity of detection for differentially expressed genes decreased dramatically with decreased cell inputs in all protocols, support that additional approaches, such as pathway enrichment, are important for data interpretation at ultra-low input. Finally, T cell activation signature was detected at 1 K cell input and above in all protocols, with AmpliSeq showing better detection at 100 cells.

Targeting the CD40-CD40L pathway in autoimmune diseases: Humoral immunity and beyond.

CD40 is a TNF receptor superfamily member expressed on both immune and non-immune cells. Interactions between B cell-expressed CD40 and its binding partner, CD40L, predominantly expressed on activated CD4+ T cells, play a critical role in promoting germinal center formation and the production of class-switched antibodies. Non-hematopoietic cells expressing CD40 can also engage CD40L and trigger a pro-inflammatory response. This article will highlight what is known about the biology of the CD40-CD40L axis in humans and describe the potential contribution of CD40 signaling on both hematopoietic and non-hematopoietic cells to autoimmune disease pathogenesis. Additionally, novel therapeutic approaches to target this pathway, currently being evaluated in clinical trials, are discussed.

Endotoxin reduction in protein solutions using octyl ß-D-1-thioglucopyranoside wash on chromatography media.

Endotoxins are complex molecules and one of the most challenging impurities requiring separation in biopharmaceutical protein purification processes. Usually these contaminants are cleared during the downstream process, but if endotoxin interacts with the target protein it becomes difficult to remove. In the present study we identified a detergent, octyl-ß-D-1-thioglucopyranoside (OTG), that disrupted endotoxin-protein interactions. The integration of this detergent into washes on several chromatography media was demonstrated to provide a separation tool for decreasing endotoxin from target proteins. This study also examined the mechanism of OTG endotoxin-protein disruption through phase modification incubation and chromatographic studies. The non-ionic OTG wash was shown to break both hydrophobic and electrostatic interactions between the endotoxin and protein. This mechanism contrasts with the breaking of hydrophobic interactions by washing with known endotoxin decreasing Triton X-100 detergent. The difference in mechanisms likely results in the ability of OTG to decrease endotoxin to levels less than those resulting from a detergent wash such as Triton X-100. Finally, we show the impact of the OTG endotoxin removal tool on the biopharmaceutical industry. While maintaining monomer purity and activity levels, endotoxin removal from a fusion protein allowed for decreased background levels in a T cell functional assay. The lowered baseline of T cell responses allowed for more effective detection of molecular interaction with the cells. The detergent wash can be used to both decrease the overall level of endotoxin in a purified protein solution and to enable more effective screening of lead candidate molecules.

Evaluation of Cell Proliferation and Apoptosis in Immunotoxicity Testing.

Immunotoxicity testing is important in determining the toxic effects of various chemicals on the immune system. The immune system is a direct target of numerous toxicants, and the adverse effects include serious health complications such as susceptibility to infections, cancer, allergic reactions, and autoimmune diseases. One way to investigate the harmful effects of different chemicals is to study apoptosis and/or proliferation in immune cells. Apoptosis is defined as programmed cell death, and in general, this process helps in development and maintenance of tolerance and homeostasis. However, in the case of an insult by a toxicant, enhanced apoptosis of immune cells may cause immunosuppression resulting in the development of cancer and the inability to fight infections. Apoptosis is characterized by cell shrinkage, nuclear condensation, changes in cell membrane and mitochondria, DNA fragmentation, and protein degradation by caspases. Various methods are employed to investigate apoptosis, including direct measurement of apoptotic cells with flow cytometry and in situ labeling, as well as RNA, DNA, and protein assays that are indicative of apoptotic molecules. In addition to apoptosis, quantification of cell proliferation can provide important additional information about the effect of a toxicant upon various immune cell populations. In some cases, a toxicant may act as a mitogen pushing the immune cell into the different stages of the cell cycle. There are four stages of the active cell cycle: G1, S, G2, and M, with cell division occurring in M stage. Proliferation can be quantified by numerous methods, including staining with ki-67 or CFSE, BrdU labeling, MTT assay, and/or ATP quantification.

CD28 family of receptors inter-connect in the regulation of T-cells.

T-cell activation is mediated by a combination of signals from the antigen receptor (TCR) and co-receptors such as CD28, cytotoxic T-lymphocyte antigen-4 (CTLA-4), programmed cell death antigen 1 (PD-1), CD28H and others. Each is a member of the CD28 receptor gene family. CD28 sends positive signals that promote T-cell responses, while CTLA-4 and PD-1 limit responses. It is the balance between these positive and negative signals that determines the amplitude and level of T-cell responses. The regulatory role of other family members is also becoming the focus of increasing interest. The function of certain CD28 family members such as CTLA-4 and PD-1 is dependent the expression of CD28. Together, these findings have important implications in generation of immune responses and the application of anti-receptor blocking reagents in immunotherapy.

Helios Controls a Limited Subset of Regulatory T Cell Functions.

A subpopulation (60-70%) of Foxp3(+) regulatory T cells (Tregs) in both mouse and man expresses the transcription factor Helios, but its role in Treg function is still unknown. We generated Treg-specific Helios-deficient mice to examine the function of Helios in Tregs. We show that the selective deletion of Helios in Tregs leads to slow, progressive systemic immune activation, hypergammaglobulinemia, and enhanced germinal center formation in the absence of organ-specific autoimmunity. Helios-deficient Treg suppressor function was normal in vitro, as well as in an in vivo inflammatory bowel disease model. However, Helios-deficient Tregs failed to control the expansion of pathogenic T cells derived from scurfy mice, failed to mediate T follicular regulatory cell function, and failed to control both T follicular helper cell and Th1 effector cell responses. In competitive settings, Helios-deficient Tregs, particularly effector Tregs, were at a disadvantage, indicating that Helios regulates effector Treg fitness. Thus, we demonstrate that Helios controls certain aspects of Treg-suppressive function, differentiation, and survival.

Eos Is Redundant for Regulatory T Cell Function but Plays an Important Role in IL-2 and Th17 Production by CD4+ Conventional T Cells.

Eos belongs to the Ikaros family of transcription factors. It was reported to be a regulatory T cell (Treg) signature gene, to play a critical role in Treg suppressor functions, and to maintain Treg stability. We used mice with a global deficiency in Eos to re-examine the role of Eos expression in both Tregs and conventional T cells (Tconvs). Tregs from Eos-deficient (Eos(-/-)) mice developed normally, displayed a normal Treg phenotype, and exhibited normal suppressor function in vitro. Eos(-/-) Tregs were as effective as Tregs from wild-type (WT) mice in suppressing inflammation in a model of inflammatory bowel disease. Bone marrow (BM) from Eos(-/-) mice was as effective as that from WT mice in controlling T cell activation when used to reconstitute immunodeficient mice in the presence of scurfy fetal liver cells. Surprisingly, Eos was expressed in activated Tconvs and was required for IL-2 production, CD25 expression, and proliferation in vitro by CD4(+) Tconvs. Eos(-/-) mice developed more severe experimental autoimmune encephalomyelitis than WT mice, displayed increased numbers of effector T cells in the periphery and CNS, and amplified IL-17 production. In conclusion, our studies are not consistent with a role for Eos in Treg development and function but demonstrate that Eos plays an important role in the activation and differentiation of Tconvs.

IFN-α/ß receptor signaling promotes regulatory T cell development and function under stress conditions.

Type I IFNs are a family of cytokines with antiviral and immunomodulatory properties. Although the antiviral effects of IFNs are well characterized, their immunomodulatory properties are less clear. To specifically address the effects of type I IFNs on T regulatory cells (Tregs), we studied mixed bone marrow chimeras between wild-type and IFN-α/ß receptor (IFNAR) knockout (KO) mice, and heterozygous female mice expressing a Treg-specific deletion of the IFNAR. In these two models, IFNAR signaling promotes the development of the Treg lineage in the thymus and their survival in the periphery. IFNAR KO Tregs had a higher expression of the proapoptotic gene Bim and higher frequency of active caspase-positive cells. IFNAR KO Tregs from chimeric mice displayed a more naive phenotype, accompanied by lower levels of CD25 and phosphorylated STAT5. Therefore, in Tregs, IFNAR signaling may directly or indirectly affect phosphorylation of STAT5. In mixed chimeras with Scurfy fetal liver, Tregs derived from IFNAR KO bone marrow were unable to control T effector cell activation and tissue inflammation. Under stress conditions or in a competitive environment, IFNAR signaling may be required to maintain Treg homeostasis and function.

Engineered antigen-specific human regulatory T cells: immunosuppression of FVIII-specific T- and B-cell responses.

Expansion of human regulatory T cells (Tregs) for clinical applications offers great promise for the treatment of undesirable immune responses in autoimmunity, transplantation, allergy, and antidrug antibody responses, including inhibitor responses in hemophilia A patients. However, polyclonal Tregs are nonspecific and therefore could potentially cause global immunosuppression. To avoid this undesirable outcome, the generation of antigen-specific Tregs would be advantageous. Herein, we report the production and properties of engineered antigen-specific Tregs, created by transduction of a recombinant T-cell receptor obtained from a hemophilia A subject's T-cell clone, into expanded human FoxP3(+) Tregs. Such engineered factor VIII (FVIII)-specific Tregs efficiently suppressed the proliferation and cytokine production of FVIII-specific T-effector cells. Moreover, studies with an HLA-transgenic, FVIII-deficient mouse model demonstrated that antibody production from FVIII-primed spleen cells in vitro were profoundly inhibited in the presence of these FVIII-specific Tregs, suggesting potential utility to treat anti-FVIII inhibitory antibody formation in hemophilia A patients.

Staphylococcal enterotoxin B-induced microRNA-155 targets SOCS1 to promote acute inflammatory lung injury.

Staphylococcal enterotoxin B (SEB) causes food poisoning in humans. It is considered a biological weapon, and inhalation can trigger lung injury and sometimes respiratory failure. Being a superantigen, SEB initiates an exaggerated inflammatory response. While the role of microRNAs (miRNAs) in immune cell activation is getting increasing recognition, their role in the regulation of inflammatory disease induced by SEB has not been studied. In this investigation, we demonstrate that exposure to SEB by inhalation results in acute inflammatory lung injury accompanied by an altered miRNA expression profile in lung-infiltrating cells. Among the miRNAs that were significantly elevated, miR-155 was the most overexpressed. Interestingly, miR-155(-/-) mice were protected from SEB-mediated inflammation and lung injury. Further studies revealed a functional link between SEB-induced miR-155 and proinflammatory cytokine gamma interferon (IFN-γ). Through the use of bioinformatics tools, suppressor of cytokine signaling 1 (SOCS1), a negative regulator of IFN-γ, was identified as a potential target of miR-155. While miR-155(-/-) mice displayed increased expression of Socs1, the overexpression of miR-155 led to its suppression, thereby enhancing IFN-γ levels. Additionally, the inhibition of miR-155 resulted in restored Socs1expression. Together, our data demonstrate an important role for miR-155 in promoting SEB-mediated inflammation in the lungs through Socs1 suppression and suggest that miR-155 may be an important target in preventing SEB-mediated inflammation and tissue injury.

Multiple anti-inflammatory pathways triggered by resveratrol lead to amelioration of staphylococcal enterotoxin B-induced lung injury.

BACKGROUND AND PURPOSE: Inhalation of the superantigen,staphylococcal enterotoxin B (SEB), leads to the activation of the host T and invariant natural killer (iNK) T cells, thereby resulting in acute lung inflammation and respiratory failure but the underlying mechanism(s) of disease remain elusive, with limited treatment options. In this study, we investigated the therapeutic effectiveness of resveratrol, a plant polyphenol, during SEB-induced lung inflammation. EXPERIMENTAL APPROACH: C57BL/6 mice were exposed to SEB (50 µg·per mouse), administered intranasally, and were treated with resveratrol (100 mg·kg(-1)) before or after SEB exposure. Lung injury was studied by measuring vascular permeability, histopathological examination, nature of infiltrating cells, inflammatory cytokine induction in the bronchoalveolar fluid (BALF), apoptosis in SEB-activated T cells and regulation of SIRT1 and NF-κB signalling pathways. KEY RESULTS: Pretreatment and post-treatment with resveratrol significantly reduced SEB-induced pulmonary vascular permeability, and inflammation. Resveratrol significantly reduced lung infiltrating cells and attenuated the cytokine storm in SEB-exposed mice, which correlated with increased caspase-8-dependent apoptosis in SEB-activated T cells. Resveratrol treatment also markedly up-regulated Cd11b+ and Gr1+ myeloid-derived suppressor cells (MDSCs) that inhibited SEB-mediated T cell activation in vitro. In addition, resveratrol treatment was accompanied by up-regulation of SIRT1 and down-regulation of NF-κB in the inflammatory cells of the lungs. CONCLUSIONS AND IMPLICATIONS: The current study demonstrates that resveratrol may constitute a novel therapeutic modality to prevent and treat SEB-induced lung inflammation inasmuch because it acts through several pathways to reduce pulmonary inflammation.

Acute lung injury induced by Staphylococcal enterotoxin B: disruption of terminal vessels as a mechanism of induction of vascular leak.

The current hypothesis of alveolar capillary membrane dysfunction fails to completely explain the severe and persistent leak of protein-rich fluid into the pulmonary interstitium, seen in the exudative phase of acute lung injury (ALI). The presence of intact red blood cells in the pulmonary interstitium may suggest mechanical failure of pulmonary arterioles and venules. These studies involved the pathological and ultrastructural evaluation of the pulmonary vasculature in Staphylococcal enterotoxin B (SEB)-induced ALI. Administration of SEB resulted in a significant increase in the protein concentration of bronchoalveolar lavage fluid and vascular leak in SEB-exposed mice compared to vehicle-treated mice. In vivo imaging of mice demonstrated the pulmonary edema and leakage in the lungs of SEB-administered mice. The histopathological studies showed intense clustering of inflammatory cells around the alveolar capillaries with subtle changes in architecture. Electron microscopy studies further confirmed the diffuse damage and disruption in the muscularis layer of the terminal vessels. Cell death in the endothelial cells of the terminal vessels was confirmed with TUNEL staining. In this study, we demonstrated that in addition to failure of the alveolar capillary membrane, disruption of the pulmonary arterioles and venules may explain the persistent and severe interstitial and alveolar edema.

CD1d-independent activation of invariant natural killer T cells by staphylococcal enterotoxin B through major histocompatibility complex class II/T cell receptor interaction results in acute lung injury.

There are two important mechanisms of activation of invariant natural killer T cells (iNKT cells) by microbes: direct activation of the invariant T-cell receptor (TCR) by microbial glycolipids presented by CD1d and indirect activation, mediated by the responses of antigen-presenting cells to microbes. In this study, we provide evidence for a novel CD1d-independent direct activation of iNKT cells involving a microbial protein superantigen presented in the context of major histocompatibility complex class II (MHC-II), which plays a critical role in pathogenesis, thereby redefining the role of iNKT cells. Intranasal exposure to staphylococcal enterotoxin B (SEB) in C57BL/6 wild-type mice caused acute lung injury (ALI) characterized by vascular leak, cytokine storm, and infiltration of mononuclear cells in the lungs. In contrast, the vascular leak and inflammation were decreased by ~50% in NKT cell-deficient Jα18(-/-) and CD1d(-/-) mice following SEB exposure, which was reversed following adoptive transfer of iNKT cells into CD1d(-/-) mice. In vitro, SEB could directly stimulate iNKT cells in a CD1d-independent manner via MHC-II/TCR interaction, specifically involving Vß8. These studies not only demonstrate that iNKT cells can be activated directly by a bacterial protein superantigen independent of CD1d but also indicate that in addition to the conventional T cells, iNKT cells play a critical role in SEB-mediated ALI.

Do cannabinoids have a therapeutic role in transplantation?

Cannabinoids have emerged as powerful drug candidates for the treatment of inflammatory and autoimmune diseases due to their immunosuppressive properties. Significant clinical and experimental data on the use of cannabinoids as anti-inflammatory agents exist in many autoimmune disease settings, but virtually no studies have been undertaken on their potential role in transplant rejection. Here we suggest a theoretical role for the use of cannabinoids in preventing allograft rejection. The psychotropic properties of CB1 agonists limit their clinical use, but CB2 agonists may offer a new avenue to selectively target immune cells involved in allograft rejection. Moreover, development of mixed CB1/CB2 agonists that cannot cross the blood-brain barrier may help prevent their undesired psychotropic properties. In addition, manipulation of endocannabinoids in vivo by activating their biosynthesis and inhibiting cellular uptake and metabolism may offer another pathway to regulate immune response during allograft rejection.

Evaluation of apoptosis in immunotoxicity testing.

Immunotoxicity testing is important in determining the toxic effects of chemical substances, medicinal products, airborne pollutants, cosmetics, medical devices, and food additives. The immune system of the host is a direct target of these toxicants, and the adverse effects include serious health complications such as susceptibility to infections, cancer, allergic reactions, and autoimmune diseases. One way to investigate the harmful effects of different chemicals is to study apoptosis in immune cell populations. Apoptosis is defined as the programmed cell death, and in general, this process helps in development and maintains homeostasis. However, in the case of an insult by a toxicant, apoptosis of the immune cells can lead to immunosuppression resulting in the development of cancer and the inability to fight infections. Apoptosis is characterized by cell shrinkage, nuclear condensation, changes in cell membrane and mitochondria, DNA fragmentation into 200 base oligomers, and protein degradation by caspases. Various methods are employed in order to investigate apoptosis. These methods include direct measurement of apoptotic cells with flow cytometry and in situ labeling, as well as RNA, DNA, and protein assays that are indicative of apoptotic molecules.

Cannabinoid-induced apoptosis in immune cells as a pathway to immunosuppression.

Cannabinoids are a group of compounds present in Cannabis plant (Cannabis sativa L.). They mediate their physiological and behavioral effects by activating specific cannabinoid receptors. With the recent discovery of the cannabinoid receptors (CB1 and CB2) and the endocannabinoid system, research in this field has expanded exponentially. Cannabinoids have been shown to act as potent immunosuppressive and anti-inflammatory agents and have been shown to mediate beneficial effects in a wide range of immune-mediated diseases such as multiple sclerosis, diabetes, septic shock, rheumatoid arthritis, and allergic asthma. Cannabinoid receptor 1 (CB1) is mainly expressed on the cells of the central nervous system as well as in the periphery. In contrast, cannabinoid receptor 2 (CB2) is predominantly expressed on immune cells. The precise mechanisms through which cannabinoids mediate immunosuppression is only now beginning to be understood and can be broadly categorized into four pathways: apoptosis, inhibition of proliferation, suppression of cytokine and chemokine production and induction of T regulatory cells (T regs). Studies from our laboratory have focused on mechanisms of apoptosis induction by natural and synthetic cannabinoids through activation of CB2 receptors. In this review, we will focus on apoptotic mechanisms of immunosuppression mediated by cannabinoids on different immune cell populations and discuss how activation of CB2 provides a novel therapeutic modality against inflammatory and autoimmune diseases as well as malignancies of the immune system, without exerting the untoward psychotropic effects.

Cannabinoids as novel anti-inflammatory drugs.

Cannabinoids are a group of compounds that mediate their effects through cannabinoid receptors. The discovery of Δ9-tetrahydrocannabinol (THC) as the major psychoactive principle in marijuana, as well as the identification of cannabinoid receptors and their endogenous ligands, has led to a significant growth in research aimed at understanding the physiological functions of cannabinoids. Cannabinoid receptors include CB1, which is predominantly expressed in the brain, and CB2, which is primarily found on the cells of the immune system. The fact that both CB1 and CB2 receptors have been found on immune cells suggests that cannabinoids play an important role in the regulation of the immune system. Recent studies demonstrated that administration of THC into mice triggered marked apoptosis in T cells and dendritic cells, resulting in immunosuppression. In addition, several studies showed that cannabinoids downregulate cytokine and chemokine production and, in some models, upregulate T-regulatory cells (Tregs) as a mechanism to suppress inflammatory responses. The endocannabinoid system is also involved in immunoregulation. For example, administration of endocannabinoids or use of inhibitors of enzymes that break down the endocannabinoids, led to immunosuppression and recovery from immune-mediated injury to organs such as the liver. Manipulation of endocannabinoids and/or use of exogenous cannabinoids in vivo can constitute a potent treatment modality against inflammatory disorders. This review will focus on the potential use of cannabinoids as a new class of anti-inflammatory agents against a number of inflammatory and autoimmune diseases that are primarily triggered by activated T cells or other cellular immune components.