Summary: Our understanding of the role of T cells in human disease is undergoing revision as a result of the discovery of T-helper 17 (Th17) cells, a unique CD4+ T-cell subset characterized by production of interleukin-17 (IL-17) IL-17 is a highly inflammatory cytokine with robust effects on stromal cells in many tissues Recent data in humans and mice suggest that Th17 cells play an important role in the pathogenesis of a diverse group of immune-mediated diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, and asthma Initial reports also propose a role for Th17 cells in tumorigenesis and transplant rejection Important differences, as well as many similarities, are emerging when the biology of Th17 cells in the mouse is compared with corresponding phenomena in humans As our understanding of human Th17 biology grows, the mechanisms underlying many diseases are becoming more apparent, resulting in a new appreciation for both previously known and more recently discovered cytokines, chemokines, and feedback mechanisms Given the strong association between excessive Th17 activity and human disease, new therapeutic approaches targeting Th17 cells are highly promising, but the potential safety of such treatments may be limited by the role of these cells in normal host defenses against infection

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Th17 cells in human disease

Ca2+ entry into cells of the peripheral immune system occurs through highly Ca2+-selective channels known as CRAC (calcium release-activated calcium) channels. CRAC channels are a very well-characterized example of store-operated Ca2+ channels, so designated because they open when the endoplasmic reticulum (ER) Ca2+ store becomes depleted. Physiologically, Ca2+ is released from the ER lumen into the cytoplasm when activated receptors couple to phospholipase C and trigger production of the second messenger inositol 1,4,5-trisphosphate (IP3). IP3 binds to IP3 receptors in the ER membrane and activates Ca2+ release. The proteins STIM and ORAI were discovered through limited and genome-wide RNAi screens, respectively, performed in Drosophila cells and focused on identifying modulators of store-operated Ca2+ entry. STIM1 and STIM2 sense the depletion of ER Ca2+ stores, whereas ORAI1 is a pore subunit of the CRAC channel. In this review, we discuss selected aspects of Ca2+ signaling in cells of the immune syste...

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Molecular Basis of Calcium Signaling in Lymphocytes: STIM and ORAI

The human genome contains 40 voltage-gated potassium channels (KV) which are involved in diverse physiological processes ranging from repolarization of neuronal or cardiac action potentials, over regulating calcium signaling and cell volume, to driving cellular proliferation and migration. KV channels offer tremendous opportunities for the development of new drugs for cancer, autoimmune diseases and metabolic, neurological and cardiovascular disorders. This review first discusses pharmacological strategies for targeting KV channels with venom peptides, antibodies and small molecules and then highlights recent progress in the preclinical and clinical development of drugs targeting KV1.x, KV7.x (KCNQ), KV10.1 (EAG1) and KV11.1 (hERG) channels.

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Voltage-gated potassium channels as therapeutic targets

1.1 Progress and challenges in the new age of engineering immunity
The immune system plays a critical role in our health. No other component of human physiology plays a decisive role in as diverse an array of maladies, from deadly diseases with which we are all familiar to equally terrible esoteric conditions: HIV, malaria, pneumococcal and influenza infections; cancer; atherosclerosis; autoimmune diseases such as lupus, diabetes, and multiple sclerosis. The importance of understanding the function of the immune system and learning how to modulate immunity to protect against or treat disease thus cannot be overstated. Fortunately, we are entering an exciting era where the science of immunology is defining pathways for the rational manipulation of the immune system at the cellular and molecular level, and this understanding is leading to dramatic advances in the clinic that are transforming the future of medicine.1,2 These initial advances are being made primarily through biologic drugs– recombinant proteins (especially antibodies) or patient-derived cell therapies– but exciting data from preclinical studies suggest that a marriage of approaches based in biotechnology with the materials science and chemistry of nanomaterials, especially nanoparticles, could enable more effective and safer immune engineering strategies. This review will examine these nanoparticle-based strategies to immune modulation in detail, and discuss the promise and outstanding challenges facing the field of immune engineering from a chemical biology/materials engineering perspective.



1.1.1 Key cellular actors in the immune system 
A brief summary of the cellular players in the immune response is worthwhile to preface the many immunomodulatory approaches described in this review. The immune system can be viewed at a high level as a collection of mobile cells that include members that traffic throughout the body in search of invading pathogens as well as cells that reside as sentinels at portals of entry (i.e. the airways, skin, gastrointestinal tract, etc.).3 These cells belong to one of two major arms, the innate immune system and adaptive immune system. Innate immune cells such as neutrophils and macrophages are poised to rapidly respond to pathogen invasion, expressing receptors that recognize conserved molecular motifs characteristic of bacteria, viruses, and fungi, to quickly phagocytose (internalize) microbes and secrete reactive oxygen species or cytokines that provide an immediate response to invading pathogens. The adaptive immune system is comprised of T-cells and B-cells, including CD4+ helper T-cells that secrete cytokines to direct the functions of innate cells, killer cells, and B-cells; and CD8+ killer T-cells that recognize and destroy infected or transformed cells. B-cells play a canonical role in vaccine responses by producing antibodies that bind to and neutralize the ability of microbes to invade host cells and/or promote their phagocytosis. The adaptive immune system is so named because of the clonal nature of T and B lymphocytes– each T-cell and B-cell expresses a unique T-cell receptor or B-cell receptor, respectively, which is generated in part by a process of stochastic DNA recombination, enabling the pool of lymphocytes the potential to recognize any microbial antigen they may encounter.4 When a T- or B-cell binds an antigen (essentially, any biological molecule from a microbe that is recognized by a T-cell receptor (TCR) or B-cell receptor (BCR)), this triggers massive proliferation of the antigen-specific cell, generating a pool of effectors within ~7 days following exposure. These effector T-cells and B-cells play an important role in backing up innate immune defenses to clear the invading pathogen. Following pathogen clearance, the majority of these cells (~90%) undergo programmed cell death, leaving a small pool of differentiated memory cells that can remain for the lifetime of the individual, to provide rapid recall protection if the same microbe is ever encountered again.5 A final key group of immune cells are the antigen presenting cells (APCs), and particularly a critical APC known as the dendritic cell, which is responsible for activating naive T-cells (and in some cases B-cells).6,7 Dendritic cells (DCs) are innate-like cells that reside in all peripheral tissues, and which act as sentinels, collecting antigens from the surrounding fluid and staying on constant alert for “danger signals”- molecular motifs signifying tissue damage or pathogen invasion. DCs and other immune cells express a host of receptors that specifically recognize danger signals to trigger their activation; the most studied among these receptors are the Toll-like receptors.8 If activated by danger signals, DCs migrate from their home tissue through the lymphatic vessels to local draining lymph nodes, where they physically present antigen to T-cells and B-cells. For T-cell activation, this is through the loading of short (8–15 amino acids) peptide fragments of antigens into the cleft of major histocompatibility complex (MHC) molecules displayed on the DC surface. These peptides are surveyed by the TCRs of T-cells, and on finding a cognate peptide, T-cells become activated by the DC to proliferate and carry out their effector functions.

The vastly complex set of cellular interactions summarized above (greatly oversimplified) is the network of interest to those interested in immune engineering, and in this review we aim to summarize the myriad ways in which materials scientists, chemical engineers, bioengineers, chemists, and physicists (often in collaboration with immunologists) are using nanomaterials as powerful tools to probe or manipulate immune responses for therapeutic ends. To set the stage for the rest of the review, we will briefly discuss two of the areas where synthetic nanoparticles have the prospect to play a significant role in the ongoing revolution of immunology in medicine– vaccines and active immunotherapy.

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Synthetic Nanoparticles for Vaccines and Immunotherapy

For more than 25 years, it has been widely appreciated that Ca2+ influx is essential to trigger T-lymphocyte activation. Patch clamp analysis, molecular identification, and functional studies using blockers and genetic manipulation have shown that a unique contingent of ion channels orchestrates the initiation, intensity, and duration of the Ca2+ signal. Five distinct types of ion channels--Kv1.3, KCa3.1, Orai1+ stromal interacting molecule 1 (STIM1) [Ca2+-release activating Ca2+ (CRAC) channel], TRPM7, and Cl(swell)--comprise a network that performs functions vital for ongoing cellular homeostasis and for T-cell activation, offering potential targets for immunomodulation. Most recently, the roles of STIM1 and Orai1 have been revealed in triggering and forming the CRAC channel following T-cell receptor engagement. Kv1.3, KCa3.1, STIM1, and Orai1 have been found to cluster at the immunological synapse following contact with an antigen-presenting cell; we discuss how channels at the synapse might function to modulate local signaling. Immuno-imaging approaches are beginning to shed light on ion channel function in vivo. Importantly, the expression pattern of Ca2+ and K+ channels and hence the functional network can adapt depending upon the state of differentiation and activation, and this allows for different stages of an immune response to be targeted specifically.

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The functional network of ion channels in T lymphocytes

Autoreactive memory T lymphocytes are implicated in the pathogenesis of autoimmune diseases. Here we demonstrate that disease-associated autoreactive T cells from patients with type-1 diabetes mellitus or rheumatoid arthritis (RA) are mainly CD4+ CCR7- CD45RA- effector memory T cells (T(EM) cells) with elevated Kv1.3 potassium channel expression. In contrast, T cells with other antigen specificities from these patients, or autoreactive T cells from healthy individuals and disease controls, express low levels of Kv1.3 and are predominantly naive or central-memory (T(CM)) cells. In T(EM) cells, Kv1.3 traffics to the immunological synapse during antigen presentation where it colocalizes with Kvbeta2, SAP97, ZIP, p56(lck), and CD4. Although Kv1.3 inhibitors [ShK(L5)-amide (SL5) and PAP1] do not prevent immunological synapse formation, they suppress Ca2+-signaling, cytokine production, and proliferation of autoantigen-specific T(EM) cells at pharmacologically relevant concentrations while sparing other classes of T cells. Kv1.3 inhibitors ameliorate pristane-induced arthritis in rats and reduce the incidence of experimental autoimmune diabetes in diabetes-prone (DP-BB/W) rats. Repeated dosing with Kv1.3 inhibitors in rats has not revealed systemic toxicity. Further development of Kv1.3 blockers for autoimmune disease therapy is warranted.

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Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases

Clinical treatment of neuropathic pain can be achieved with a number of different drugs, some of which interact with all members of the voltage-gated sodium channel (NaV1) family. However, block of central nervous system and cardiac NaV1 channels can cause dose-limiting side effects, preventing many patients from achieving adequate pain relief. Expression of the tetrodotoxinresistant NaV1.8 subtype is restricted to small-diameter sensory neurons, and several lines of evidence indicate a role for NaV1.8 in pain processing. Given these features, NaV1.8 subtypeselective blockers are predicted to be efficacious in the treatment of neuropathic pain and to be associated with fewer adverse effects than currently available therapies. To facilitate the identification of NaV1.8-specific inhibitors, we stably expressed the human NaV1.8 channel together with the auxiliary human β1 subunit (NaV β1) in human embryonic kidney 293 cells. Heterologously expressed human NaV1.8/NaV β1 channels display biophysical properties...

A high-capacity membrane potential FRET-based assay for NaV1.8 channels.

The role of ion channels in cell physiology is regulated by processes occurring after protein biosynthesis, which are critical for both channel function and targeting of channels to appropriate cell compartments. Here we apply biochemical and electrophysiological methods to investigate the role of the high-conductance, calcium-activated potassium (Maxi-K) channel C-terminal domain in channel tetramerization, association with the beta1 subunit, trafficking of the channel complex to the cell surface, and channel function. No evidence for channel tetramerization, cell surface expression, or function was observed with Maxi-K(1)(-)(323), a construct truncated three residues after the S(6) transmembrane domain. However, Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) are able to form tetramers and to associate with the beta1 subunit. Maxi-K(1)(-)(343)-beta1 and Maxi-K(1)(-)(441)-beta1 complexes are efficiently targeted to the cell surface and cannot be pharmacologically distinguished from full-length channels in binding experiments but do not form functional channels. Maxi-K(1)(-)(651) forms tetramers and associates with beta1; however, the complex is not present at the cell surface, but is retained intracellularly. Maxi-K(1)(-)(651) surface expression and channel function can be fully rescued after coexpression with its C-terminal complement, Maxi-K(652)(-)(1113). However coexpression of Maxi-K(1)(-)(343) and Maxi-K(1)(-)(441) with their respective C-terminal complements did not rescue channel function. Together, these data demonstrate that the domain(s) in the Maxi-K channel necessary for formation of tetramers, coassembly with the beta1 subunit, and cell surface expression resides within the S(0)-S(6) linker domain of the protein, and that structural constraints within the gating ring in the C-terminal region can regulate trafficking and function of constructs truncated in this region.

Hans Guenther Knaus

Papers

Kv1.3 channels are a therapeutic target for T cell-mediated autoimmune diseases

A high-capacity membrane potential FRET-based assay for NaV1.8 channels.

Role of the C-terminus of the high-conductance calcium-activated potassium channel in channel structure and function.