Recent thymic emigrants are preferentially incorporated only into the depleted T-cell pool

Recent thymic emigrants (RTEs) are the youngest subset of peripheral T cells, and they differ functionally and phenotypically from the rest of the naïve T-cell pool. RTEs are present in the peripheral T-cell pool throughout life but are the most common subset of T cells in neonates and adults recovering from lymphoablation. Using a murine model to study the homeostasis of RTEs, we show that under lymphoreplete conditions, RTEs are at a competitive disadvantage to already established mature naïve (MN) T cells. This disadvantage may be caused by a defect in survival, because RTEs may transduce homeostatic signals inefficiently, and their ability to survive is enhanced with increased expression of IL-7 receptor or B-cell lymphoma 2 (Bcl-2). Conversely, under lymphopenic conditions, enhanced proliferation by RTEs allows them to out-compete their MN T-cell counterparts. These results suggest that in times of need, such as in neonates or lymphopenic adults, RTEs perform well to fill the gaps in the peripheral T-cell pool, but when the periphery already is full, many RTEs are not incorporated into the pool of recirculating lymphocytes.Recent thymic emigrants (RTEs) are the youngest subset of naïve T cells, those that recently have entered the lymphoid periphery after development in the thymus. RTEs maintain T-cell diversity in the periphery (1), a particularly important contribution in the very young and in adults recovering from lymphopenia.The original paradigm held that the thymus minted fully functional T cells. However, it is becoming clear that RTEs in both humans and mice undergo postthymic maturation in the lymphoid periphery (2–6) before joining the mature naïve (MN) T-cell pool. RTEs and MN T cells differ in surface phenotype, and, as compared with MN T cells, stimulated RTEs generally proliferate less and secrete less cytokine. Studying RTEs has been facilitated by the characterization of a transgenic (Tg) model system (7) that allows unambiguous identification and live-cell purification of this subset. In mice Tg for GFP under control of the recombination activating gene 2 promoter (RAG2p-GFP Tg mice), RTEs are GFP⁺ peripheral T cells (2). The GFP label is brightest in the youngest RTEs (8) and decays over time until it can no longer be detected on T cells that have been in the lymphoid periphery for more than 3 wk (2).Throughout life, the peripheral T-cell pool is maintained at a relatively constant size despite continuous turnover and thymic export of about 1 × 10⁶ cells per day (9, 10). The T-cell pool is maintained at a size large enough to protect adequately against pathogens, with estimated 1 × 10⁶ antigen specificities (11), but small enough to avoid taxing the host''s resources. Homeostasis is maintained by competition for two limiting factors, IL-7 and MHC loaded with self-peptides (12, 13).Lymphopenia occurs in humans in a variety of clinical and disease settings, including after stem cell transplantation, chemotherapy, and HIV infection. Under lymphopenic conditions, the remaining peripheral T cells proliferate, replenishing the peripheral space and gradually acquiring an effector/memory phenotype. Murine models have demonstrated that lymphopenia can induce slower turnover driven by IL-7 and MHC (12, 13) and faster division driven by commensal gut antigens or IL-2/IL-15 (reviewed in ref. 12).Because RTEs provide the only source of new T-cell diversity for the naïve peripheral T-cell pool, their contribution is clearly desirable. It has been suggested that RTEs populate a distinct niche and are preferentially accepted into the naïve T-cell pool (14, 15). However, RTEs have slightly reduced IL-7 receptor (IL-7R) expression levels (2), suggesting they may not compete as efficiently as MN T cells for this survival factor. Using the RAG2p-GFP Tg RTE reporter system, we assessed the comparative homeostasis of RTEs and MN T cells and found that when the lymphoid periphery is full, RTEs are not accepted preferentially into the periphery and persist less well than MN T cells. However, under lymphopenic conditions, when RTEs would be particularly important for replenishing T-cell receptor (TCR) diversity, RTEs are able to fill the void effectively.     

Complementary costimulation of human T-cell subpopulations by cluster of differentiation 28 (CD28) and CD81

Cluster of differentiation 81 (CD81) is a widely expressed tetraspanin molecule that physically associates with CD4 and CD8 on the surface of human T cells. Coengagement of CD81 and CD3 results in the activation and proliferation of T cells. CD81 also costimulated mouse T cells that lack CD28, suggesting either a redundant or a different mechanism of action. Here we show that CD81 and CD28 have a preference for different subsets of T cells: Primary human naïve T cells are better costimulated by CD81, whereas the memory T-cell subsets and Tregs are better costimulated by CD28. The more efficient activation of naïve T cells by CD81 was due to prolonged signal transduction compared with that by CD28. We found that IL-6 played a role in the activation of the naïve T-cell subset by CD81. Combined costimulation through both CD28 and CD81 resulted in an additive effect on T-cell activation. Thus, these two costimulatory molecules complement each other both in the strength of signal transduction and in T-cell subset inclusions. Costimulation via CD81 might be useful for expansion of T cells for adoptive immunotherapy to allow the inclusion of naïve T cells with their broad repertoire.Activation of naïve T cells requires two independent signals. The first is generated by interaction of the TCR with its cognate antigen presented by the major histocompatibility complex. The second, costimulatory, signal is delivered through accessory molecules on the T-cell surface and is antigen independent (1). Once activated, naïve cells proliferate and generate effector cells that can migrate into inflamed tissues (2–4). After the initial response the majority of effector cluster of differentiation 4 (CD4) T cells die via apoptosis, leaving a small population of memory cells that can confer protection and give, upon a secondary challenge, a more rapid and vigorous response (5, 6). Memory cells respond optimally to lower doses of antigen, are less dependent on accessory cell costimulation, and display effector functions with faster kinetics compared with naïve T cells (7–10).CD28 is the major and best-studied T-cell costimulatory molecule, known to provide a powerful second signal for T-cell activation (11, 12). However, additional members of the CD28 family, such as ICOS and several members of the TNF family, such as CD27 and CD137 (4-1BB) are well known T-cell costimulatory molecules (13, 14). Less investigated is the costimulatory effect of the tetraspanin family of molecules, including CD9, CD53, CD63, CD81 and CD82 (15–24). Interestingly, costimulation either by CD9 or by CD81 occurs in CD28-deficient T cells, suggesting either a different or a redundant activation pathway (18, 19).Tetraspanins function as lateral organizers of their associated membrane proteins. Members of this family tend to associate both with each other, and with cell-type-specific partner proteins. For example, CD81 associates in B cells with CD19 (25) whereas in T cells it associates with CD4 and CD8 (26–28). Tetraspanins and their partners assemble in the membrane in tetraspanin-enriched microdomains (TEMs), which facilitate key cellular functions. Tetraspanins play a role in membrane fusion, cell migration, and in signal transduction (29–32). The role of CD81 as a costimulatory molecule is of special interest because it has been shown to localize at the site of interaction between B cells and T cells in a central supermolecular cluster (33).Here, we compared the costimulatory potential of CD81 to that of CD28. In doing so, we examined both the earliest steps of signal transduction, as well as the later events of T-cell activation. Also, we measured events both at the level of single cells and at the level of cell subpopulations. We found that the earliest steps of CD81-mediated signal transduction differ from those induced by CD28. Specifically, the costimulatory signal mediated through CD81 is more prolonged than that through CD28. In addition, we found that CD81 better costimulates the naïve T-cell subset than does CD28. Combined costimulation through both CD81 and CD28 results in a T-cell response that better includes the naïve T-cell subset.Adoptive T-cell transfer, the isolation and expansion of antigen-specific cells by costimulation with CD28, followed by reinfusion is a promising approach in the induction of anti-tumor immune response (34, 35). Interestingly, the adoptive transfer of naïve T cells was shown to mediate a superior antitumor immunity (36). Our study provides insight regarding the preferential activation of naive T cells and may be useful in manipulating diseases of the immune system, such as autoimmunity and cancer.     

Effects of lymphocyte profile on development of EBV-induced lymphoma subtypes in humanized mice

Epstein-Barr virus (EBV) infection causes both Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). The present study reveals that EBV-induced HL and NHL are intriguingly associated with a repopulated immune cell profile in humanized mice. Newborn immunodeficient NSG mice were engrafted with human cord blood CD34⁺ hematopoietic stem cells (HSCs) for a 8- or 15-wk reconstitution period (denoted ^8whN and ^15whN, respectively), resulting in human B-cell and T-cell predominance in peripheral blood cells, respectively. Further, novel humanized mice were established via engraftment of hCD34⁺ HSCs together with nonautologous fetal liver-derived mesenchymal stem cells (MSCs) or MSCs expressing an active notch ligand DLK1, resulting in mice skewed with human B or T cells, respectively. After EBV infection, whereas NHL developed more frequently in B-cell–predominant humanized mice, HL was seen in T-cell–predominant mice (P = 0.0013). Whereas human splenocytes from NHL-bearing mice were positive for EBV-associated NHL markers (hBCL2⁺, hCD20⁺, hKi67⁺, hCD20⁺/EBNA1⁺, and EBER⁺) but negative for HL markers (LMP1⁻, EBNA2⁻, and hCD30⁻), most HL-like tumors were characterized by the presence of malignant Hodgkin’s Reed–Sternberg (HRS)-like cells, lacunar RS (hCD30⁺, hCD15⁺, IgJ⁻, EBER⁺/hCD30⁺, EBNA1⁺/hCD30⁺, LMP⁺/EBNA2⁻, hCD68⁺, hBCL2⁻, hCD20^-/weak, Phospho STAT6⁺), and mummified RS cells. This study reveals that immune cell composition plays an important role in the development of EBV-induced B-cell lymphoma.Epstein Barr virus (EBV) infects human B lymphocytes and epithelial cells in >90% of the human population (1, 2). EBV infection is widely associated with the development of diverse human disorders that include Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphomas (NHL), including diffused large B-cell lymphoma (DLBCL), follicular B-cell lymphoma (FBCL), endemic Burkitt’s lymphoma (BL), and hemophagocytic lymphohistiocytosis (HLH) (3).HL is a malignant lymphoid neoplasm most prevalent in adolescents and young adults (4–6). Hodgkin/Reed–Sternberg (HRS) cells are the sole malignant cells of HL. HRS cells are characterized by CD30⁺/CD15⁺/BCL6⁻/CD20^+/− markers and appear large and multinucleated owing to multiple nuclear divisions without cytokinesis. Although HRS cells are malignant in the body, surrounding inflammatory cells greatly outnumber them. These reactive nonmalignant inflammatory cells, including lymphocytes, histiocytes, eosinophils, fibroblasts, neutrophils, and plasma cells, compose the vast majority of the tumor mass. The presence of HRS cells in the context of this inflammatory cellular background is a critical hallmark of the HL diagnosis (4). Approximately 50% of HL cases are EBV-associated (EBVaHL) (7–11). EBV-positive HRS cells express EBV latent membrane protein (LMP) 1 (LMP1), LMP2A, LMP2B, and EBV nuclear antigen (EBNA) 1 (EBNA1), but lack EBNA2 (latency II marker) (12). LMP1 is consistently expressed in all EBV-associated cases of classical HL (13, 14). LMP1 mimics activated CD40 receptors, induces NF-κB, and allows cells to become malignant while escaping apoptosis (15).The etiologic role of EBV in numerous disorders has been studied in humanized mouse models in diverse experimental conditions. Humanized mouse models recapitulate key characteristics of EBV infection-associated disease pathogenesis (16–24). Different settings have given rise to quite distinct phenotypes, including B-cell type NHL (DLBCL, FBCL, and unspecified B-cell lymphomas), natural killer/T cell lymphoma (NKTCL), nonmalignant lymphoproliferative disorder (LPD), extremely rare HL, HLH, and arthritis (16–24). Despite considerable efforts (16–24), EBVaHL has not been properly produced in the humanized mouse setting model, owing to inappropriate animal models and a lack of in-depth analyses. After an initial report of infected humanized mice, HRS-like cells appeared to be extremely rare in the spleens of infected humanized mice; however, the findings were inconclusive (18). Here we report direct evidence of EBVaHL or HL-like neoplasms in multiple humanized mice in which T cells were predominant over B cells. Our study demonstrates that EBV-infected humanized mice display additional EBV-associated pathogenesis, including DLBCL and hemophagocytic lymphohistiocytosis (16, 17).     

IL-33/ST2 axis promotes mast cell survival via BCLXL

Mast cells (MC) are potent innate immune cells that accumulate in chronically inflamed tissues. MC express the IL-33 receptor IL-1 receptor-related protein ST2 at high level, and this IL-1 family cytokine both activates MC directly and primes them to respond to other proinflammatory signals. Whether IL-33 and ST2 play a role in MC survival remains to be defined. In skin-derived human MC, we found that IL-33 attenuated MC apoptosis without altering proliferation, an effect mediated principally through the antiapoptotic molecule B-cell lymphoma-X large (BCLXL). Murine MC demonstrated a similar mechanism, dependent entirely on ST2. In line with these observations, St2^−/− mice exhibited reduced numbers of tissue MC in inflamed arthritic joints, in helminth-infected intestine, and in normal peritoneum. To confirm an MC-intrinsic role for ST2 in vivo, we performed peritoneal transfer of WT and St2^−/− MC. In St2^−/− hosts treated with IL-33 and in WT hosts subjected to thioglycollate peritonitis, WT MC displayed a clear survival advantage over coengrafted St2^−/− MC. IL-33 blockade specifically attenuated this survival advantage, confirming IL-33 as the relevant ST2 ligand mediating MC survival in vivo. Together, these data reveal a cell-intrinsic role for the IL-33/ST2 axis in the regulation of apoptosis in MC, identifying thereby a previously unappreciated pathway supporting expansion of the MC population with inflammation.Mast cells (MC) are tissue-resident effector cells that contribute both to innate and adaptive immunity (1). MC help defend against bacterial and helminthic infection (2, 3) and play a role in tissue remodeling (4, 5). MC can also contribute to a variety of allergic and nonallergic inflammatory diseases, such as anaphylaxis, atopic dermatitis, asthma, inflammatory arthritis, psoriasis, and multiple sclerosis (6, 7). Under many of these conditions, the number of MC in affected tissues increases by tenfold or more, amplifying the contribution of MC-derived mediators to ongoing inflammation (6–8). Regulation of the MC population is poorly understood, and therapeutic intervention to limit MC accumulation potentially could attenuate injury associated with inflammatory diseases (9, 10).The survival of mature MC in tissues depends on signals from neighboring cells (11, 12). The single most important mediator in this process is stem cell factor (SCF), a master regulator of MC proliferation, differentiation, survival, and activation. Mice with genetic mutations affecting SCF or its receptor Kit have few or no MC in healthy or inflamed tissues, whereas activating mutations of Kit give rise to pathologic mastocytosis (13–15). IL-3 is another well-recognized MC growth factor (16). SCF/Kit and IL-3 interface with several antiapoptotic pathways, up-regulating the antiapoptotic protein B-cell lymphoma-2 (BCL-2) and downregulating the proapoptotic protein Bim (14, 17–19). Aside from SCF/Kit and IL-3, other cytokines have been reported to support MC survival, but their effects appear to be restricted to specific MC subtypes. IL-4 promotes survival of intestinal MC via both BCL-2 and B-cell lymphoma-X large (BCLXL) but may suppress survival in human cord blood MC as well as murine bone marrow-derived MC (BMMC) (20–23). IL-10 can either promote or impair survival depending on the type of MC studied (21, 22).Recently, substantial attention has focused on the role of IL-33 in MC biology. A member of the IL-1 family, IL-33 is expressed primarily by stromal cells, such as epithelial cells, endothelial cells, and fibroblasts, as well as by certain hematopoietic cells, including MC themselves (24–26). IL-33 is an “alarmin” released upon cell necrosis and also can be secreted by live cells after mechanical stretching and via other incompletely defined mechanisms (27, 28). Its receptor ST2 (also called IL-1 receptor-related protein 1) is highly expressed by MC. Acting via ST2, IL-33 participates in MC activation in murine models of anaphylaxis and arthritis, serving both as a direct MC activator and as a factor that primes MC for enhanced cytokine release upon subsequent stimulation by IgE and IgG (29–32). IL-33 also prominently modulates the MC phenotype, promoting accumulation of MC protease 6 (the murine ortholog of human tryptase β) in vitro and in vivo (33).In cardiomyocytes and hepatocytes, IL-33 protects against apoptosis (34, 35), raising the possibility of an analogous effect in MC. Improved in vitro survival has been observed in human umbilical cord blood-derived MC exposed to IL-33 and in cultured murine MC under certain conditions, but this effect is incompletely characterized, and its relevance in vivo remains unknown (36, 37). In the present study, we show that IL-33 promotes the survival of both human and murine MC, mediated principally by up-regulation of the antiapoptotic factor BCLXL. In vivo, IL-33 and ST2 play a nonredundant role in promoting MC survival in peritoneum and potentially other sites. These data reveal a role for the IL-33/ST2 axis in the survival of tissue MC, particularly in the context of inflammation, raising the possibility that this axis could be targeted to limit the contribution of MC to chronic inflammatory diseases.     

Osteopontin expression by CD103? dendritic cells drives intestinal inflammation

Intestinal CD103⁻ dendritic cells (DCs) are pathogenic for colitis. Unveiling molecular mechanisms that render these cells proinflammatory is important for the design of specific immunotherapies. In this report, we demonstrated that mesenteric lymph node CD103⁻ DCs express, among other proinflammatory cytokines, high levels of osteopontin (Opn) during experimental colitis. Opn expression by CD103⁻ DCs was crucial for their immune profile and pathogenicity, including induction of T helper (Th) 1 and Th17 cell responses. Adoptive transfer of Opn-deficient CD103⁻ DCs resulted in attenuated colitis in comparison to transfer of WT CD103⁻ DCs, whereas transgenic CD103⁻ DCs that overexpress Opn were highly pathogenic in vivo. Neutralization of secreted Opn expressed exclusively by CD103⁻ DCs restrained disease severity. Also, Opn deficiency resulted in milder disease, whereas systemic neutralization of secreted Opn was therapeutic. We determined a specific domain of the Opn protein responsible for its CD103⁻ DC-mediated proinflammatory effect. We demonstrated that disrupting the interaction of this Opn domain with integrin α9, overexpressed on colitic CD103⁻ DCs, suppressed the inflammatory potential of these cells in vitro and in vivo. These results add unique insight into the biology of CD103⁻ DCs and their function during inflammatory bowel disease.Inflammatory bowel diseases (IBDs), including Crohn disease (CD) and ulcerative colitis (UC), are caused by excessive inflammatory responses to commensal microflora and other antigens present in the intestinal lumen (1). Intestinal dendritic cells (DCs) contribute to these inflammatory responses during human IBD, as well as in murine colitis models (2). DCs that reside in draining mesenteric lymph nodes (MLNs) are also crucial mediators of colitis induction (3) and may be grouped based on their surface CD103 (integrin αE) expression as CD11c^highCD103⁺ (CD103⁺ DCs) and CD11c^highCD103⁻ (CD103⁻ DCs) (4–6). CD103⁺ DCs are considered important mediators of gut homeostasis in steady state (4, 5, 7–9), and their tolerogenic properties are conserved between mice and humans (5). However, their role during intestinal inflammation is not well defined. Instead, CD103⁻ DC function has been described mostly during chronic experimental colitis (10–12). These cells secrete IL-23, IL-6, and IL-12 (10–12), contributing to the development of T helper (Th) 17 and Th1 cells, and are highly inflammatory during CD4⁺ T-cell transfer colitis (12) and during 2,4,6 trinitrobenzene sulfonic acid (TNBS)-induced chronic colitis (11). MLN CD103⁻ DCs cultured in the presence of LPS, a Toll-like receptor (TLR) 4 agonist, or R848, a TLR7 agonist, express higher levels of TNF-α and IL-6 (7, 12). In fact, these cells secrete IL-23 and IL-12 even in the absence of TLR stimulation (10). Both MLN CD103⁻ and CD103⁺ DC subsets are present in acute colitis (11, 13); however, their function, as well as their cytokine profile, during this phase of disease, reflecting colitis initiation, remains unknown.Recent studies suggest a proinflammatory role for the cytokine osteopontin (Opn) in TNBS- and dextran sulfate sodium (DSS)-induced colitis (14, 15), which are the models for CD and UC, respectively. Opn is expressed by DCs and other immune cell types, such as lymphocytes, during autoimmune responses (16–22), and its expression by DCs during autoimmunity contributes to disease severity (17–19, 21, 23). In addition, Opn expression is highly up-regulated in intestinal immune and nonimmune cells and in the plasma of patients with CD and UC (24–29), as well as in the colon and plasma of mice with experimental colitis (14, 15, 27, 30). Increased plasma Opn levels are related to the severity of CD inflammation (29), and certain Opn gene (Spp1) haplotypes are modifiers of CD susceptibility (31), indicating that Opn could be used as an IBD biomarker (27). In general, Opn affects DC biology during several inflammatory conditions (17–21, 32–37) and could be a potential therapeutic target in IBD.In this study, we initially asked whether Opn was expressed by MLN CD103⁻ and CD103⁺ DCs during colitis. We found that CD103⁻ DCs express excessive levels of Opn in addition to other proinflammatory cytokines. Conversely, CD103⁺ DCs express profoundly lower levels of Opn and are noninflammatory. Using adoptive transfer of purified specific DC subsets, we determined that MLN CD103⁻ DCs are critical mediators of acute intestinal inflammation and that their Opn expression is essential for their proinflammatory properties in both acute and chronic colitis. Furthermore, Opn-deficient and Opn-neutralized mice developed significantly milder disease. In addition, we constructed transgenic (Tg) mice overexpressing Opn only in DCs. These mice developed exaggerated colitis, and adoptive transfer of their CD103⁻ DCs into recipient mice dramatically exacerbated disease. Because Opn protein contains several domains interacting with various receptors, we defined a specific Opn domain significant for inducing proinflammatory properties in CD103⁻ DCs. Blockade of the interaction of this Opn domain [containing functional Ser-Leu-Ala-Tyr-Gly-Leu-Arg (SLAYGLR) sequence] with integrin α9 expressed on CD103⁻ DCs abrogated their proinflammatory profile and colitogenic effects in vivo.     

Design of a superior cytokine antagonist for topical ophthalmic use

IL-1 is a key inflammatory and immune mediator in many diseases, including dry-eye disease, and its inhibition is clinically efficacious in rheumatoid arthritis and cryopyrin-associated periodic syndromes. To treat ocular surface disease with a topical biotherapeutic, the uniqueness of the site necessitates consideration of the agent’s size, target location, binding kinetics, and thermal stability. Here we chimerized two IL-1 receptor ligands, IL-1β and IL-1Ra, to create an optimized receptor antagonist, EBI-005, for topical ocular administration. EBI-005 binds its target, IL-1R1, 85-fold more tightly than IL-1Ra, and this increase translates to an ∼100-fold increase in potency in vivo. EBI-005 preserves the affinity bias of IL-1Ra for IL-1R1 over the decoy receptor (IL-1R2), and, surprisingly, is also more thermally stable than either parental molecule. This rationally designed antagonist represents a unique approach to therapeutic design that can potentially be exploited for other β-trefoil family proteins in the IL-1 and FGF families.The IL-1 cytokines (IL-1α and IL-1β) are master mediators of inflammatory responses (1). IL-1β also regulates immune function through its role in T helper 17 (Th17) cell differentiation and maintenance (2, 3). IL-1 action has been implicated in numerous human diseases, including rheumatoid arthritis, Muckle–Wells syndrome, gout, type 2 diabetes, and stroke (4). Several natural mechanisms directly oppose the actions of IL-1, including a soluble and cell surface decoy receptor (IL-1R2), a natural antagonist (IL-1Ra), and a soluble signaling receptor (IL-1R1) (5). Therapeutics that block IL-1 based on these mechanisms have been developed (6–8).Recently, a nonoptimized formulation of anakinra (methionyl-IL-1Ra; Kineret) was shown to provide clinical benefit in dry-eye disease (DED) (9). Moderate to severe DED is a chronic inflammatory condition of the corneal surface that results in pain, discomfort, and epitheliopathy (as measured by fluorescein staining). Inability to maintain a proper tear film over the cornea (owing to a variety of etiologies) results in desiccating stress, which drives an inflammatory cascade (10, 11). IL-1 plays a central role in the initiation and maintenance of this cascade, as well as in the pain mediated by the corneal neural plexus. IL-1α and IL-1β protein are elevated in the lacrimal gland, tears, and the ocular surface in all forms of dry-eye disease (12), and their mRNA is increased in both humans and in rodent disease models (13, 14). Genetic ablation of IL-1R1, the primary receptor for IL-1α and IL-1β, can block the development of corneal staining in a Sjögren syndrome corneal epitheliopathy model (15), and topically administered anakinra can improve surface epithliopathy in a mouse dry-eye model (14). IL-1β is essential for Th17 cell differentiation and maintenance, and Th17 cells are likely the main effector cells that induce epithelial damage (2, 3). Genetic and pharmacologic studies have shown that IL-1β mediates, and IL-1Ra blocks, normal, inflammatory, and neuropathic pain sensations (16–19).The development of a topical biotherapeutic agent for DED presents a protein engineering challenge, requiring optimization for the biology and topical route of administration. Given the rapid turnover of tear volume (10% min⁻¹) (20), blocking the tissue-associated receptor would be preferred over blocking tear-associated ligands, and the agent’s target affinity should be maximized to minimize clearance and the frequency of dosing. Considering the corneal epithelial barrier, smaller molecules would be preferable, and ideally the agent would have sufficient thermal stability to allow a room temperature formulation, to optimize patient compliance. We considered two designs for small, receptor-targeted agents: improved versions of IL-1Ra (∼17 kDa) and small-format (e.g., single-chain variable fragment) anti–IL-1R1 antibodies. Both designs can be expressed in Escherichia coli, but given the straightforward isolation of anakinra as a soluble protein after E. coli expression, and the fact that the existing clinical data are from the use of anakinra, we focused on improved receptor antagonists. Here we describe the creation of a unique type of chimeric molecule that meets the foregoing criteria.     

Immunomodulatory effect of a decellularized skeletal muscle scaffold in a discordant xenotransplantation model

Decellularized (acellular) scaffolds, composed of natural extracellular matrix, form the basis of an emerging generation of tissue-engineered organ and tissue replacements capable of transforming healthcare. Prime requirements for allogeneic, or xenogeneic, decellularized scaffolds are biocompatibility and absence of rejection. The humoral immune response to decellularized scaffolds has been well documented, but there is a lack of data on the cell-mediated immune response toward them in vitro and in vivo. Skeletal muscle scaffolds were decellularized, characterized in vitro, and xenotransplanted. The cellular immune response toward scaffolds was evaluated by immunohistochemistry and quantified stereologically. T-cell proliferation and cytokines, as assessed by flow cytometry using carboxy-fluorescein diacetate succinimidyl ester dye and cytometric bead array, formed an in vitro surrogate marker and correlate of the in vivo host immune response toward the scaffold. Decellularized scaffolds were free of major histocompatibility complex class I and II antigens and were found to exert anti-inflammatory and immunosuppressive effects, as evidenced by delayed biodegradation time in vivo; reduced sensitized T-cell proliferative activity in vitro; reduced IL-2, IFN-γ, and raised IL-10 levels in cell-culture supernatants; polarization of the macrophage response in vivo toward an M2 phenotype; and improved survival of donor-derived xenogeneic cells at 2 and 4 wk in vivo. Decellularized scaffolds polarize host responses away from a classical TH1-proinflammatory profile and appear to down-regulate T-cell xeno responses and TH1 effector function by inducing a state of peripheral T-cell hyporesponsiveness. These results have substantial implications for the future clinical application of tissue-engineered therapies.Although replacement of airways (1–6) or urogenital tissue (7) using stem cell-based techniques has been achieved, engineering of functional contractile muscle tissue has only been partially explored (8–9). While debates have been generated around which myogenic progenitors (satellite cells, muscle stem cells, or myoblasts) should be used, fewer studies have focused on exploring which matrix could offer a better platform for regeneration (10, 11).Autologous tissue-engineered solutions have the major advantage of not requiring immunosuppression, but clinical applications are limited to static organs and tissues, such as skin, or those that can function through passive movement alone, such as trachea, heart valves, blood vessels, and bladder (4, 7, 12). The field continues to expand and tissue bioengineering has provided, or is close to delivering, functional human organ replacements elsewhere (6, 7, 13–17). The ability to produce innervated and revascularized muscles would hugely extend the possible applications of regenerative medicine (18–26).Decellularized skeletal muscle has been characterized by several groups, but its effect on cell-mediated immunity has not been studied (27–30). Here, we provide evidence that decellularized muscle scaffolds promote anti-inflammatory and immunosuppressive responses both in vitro and in vivo, down-regulate T-cell xeno responses and TH1 effector cytokines in vitro, and polarize the macrophage response in vivo toward an M2 phenotype (i.e., promote alternative pathway activation of macrophages).     

UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling

At our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca²⁺ response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain.The surface epithelium (epidermis) of skin provides barrier protection against dehydration and the potentially harmful external environment (1). Accordingly, skin is the site of first interaction between ambient environment and immunologically competent organismal structures, and also the site for sentient responses (2). Sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia (TG) are endowed with sensory transduction capacity for heat, cold, mechanical cues, itch, and pain, and their axons directly interface with skin epithelium (2–4).Against a background of suggestive findings (2, 5–7), we wondered whether the epidermis as a “forefront” of sensory signaling may function in sensitizing pain transduction in response to naturally occurring irritating cues. To elucidate mechanisms, we used a mouse sunburn model and induced a state of lowered sensory thresholds associated with tissue injury caused by UV radiation (8–10). UV-sunburn-evoked lowering of sensory thresholds shares major hallmarks of pathological pain, a valuable feature of this model. Skin tissue injury caused by UVB has been elucidated to be mediated by cytokines and chemokines, known from immunological responses, such as IL-1β and IL-6, which are also known to cause and facilitate pain (11–19). Another more recent study identified a proinflammatory chemokine, CXCL5, as proalgesic in response to UVB overexposure of rat and human skin (20). An exciting new arena pertaining to molecular mechanisms of the skin’s response to noxious UV was recently opened by an elegant study that reported the role of UVB-mediated damage to noncoding RNA molecules in the skin (21). Unraveling a molecular mechanism, the Toll-like receptor 3 gene was found critical in signaling the proinflammatory actions of the UVB-damaged noncoding RNA molecules. However, this study focused on molecular mechanisms of acute inflammation in the skin.We intended to identify pain mechanisms that mediate the pain associated with UVB-mediated tissue injury. Pain in response to external environmental cues has been understood better because of scientific progress in the field of transient receptor potential (TRP) ion channels that have been found responsive to such cues, and which were found expressed in DRG and TG peripheral sensory neurons, which are the cells believed to be the primary transducers. Indeed, TRPV1, one of the founding members of the TRPV channel subfamily, has been identified as relevant for pain, including pathological pain, response to thermal cues, and most recently for itch (22–31). However, TRPA1 (transient receptor potential ion channel, ankyrin subfamily, family member #1) and TRPM8 seem to be involved in transduction of pain-inducing stimuli as well (32–36).Also a family member of the TRPV subfamily, TRPV4 is a multimodally activated, nonselective cation channel that is involved in physiological pain evoked by osmotic and mechanical, but not thermal, cues (37–40). For pathological pain, it is relevant for inflammation- and nerve-damage-induced pain sensitization (41–43). Of note, Trpv4^−/− mice exhibit impaired skin-barrier function (44, 45). That said, TRPV4 is expressed in a number of different cell types, including robust expression in epidermal keratinocytes and also is detectable in skin-innervating sensory neurons. This “dual-location expression” of TRPV4 leaves the cellular mechanisms involved in the channel’s function and the functional contribution of environment-exposed keratinocytes vs. skin-innervating sensory neurons unclear.Against this background of dual-location TRPV4 expression and the role of TRPV4 in inflammatory and neuropathic pain, we now address whether epidermally derived TRPV4 is pathophysiologically relevant in sunburn pain and tissue damage. Using Trpv4 gene-targeted mice, selectively inducing targeting in postnatal keratinocytes, and topically applying selective TRPV4 inhibitors, we demonstrate that epidermal TRPV4 plays a prominent, hitherto unrecognized role in UVB-evoked skin tissue damage and pain of sunburn.     

Central domain of IL-33 is cleaved by mast cell proteases for potent activation of group-2 innate lymphoid cells

Interleukin-33 (IL-33) is an alarmin cytokine from the IL-1 family. IL-33 activates many immune cell types expressing the interleukin 1 receptor-like 1 (IL1RL1) receptor ST2, including group-2 innate lymphoid cells (ILC2s, natural helper cells, nuocytes), the major producers of IL-5 and IL-13 during type-2 innate immune responses and allergic airway inflammation. IL-33 is likely to play a critical role in asthma because the IL33 and ST2/IL1RL1 genes have been reproducibly identified as major susceptibility loci in large-scale genome-wide association studies. A better understanding of the mechanisms regulating IL-33 activity is thus urgently needed. Here, we investigated the role of mast cells, critical effector cells in allergic disorders, known to interact with ILC2s in vivo. We found that serine proteases secreted by activated mast cells (chymase and tryptase) generate mature forms of IL-33 with potent activity on ILC2s. The major forms produced by mast cell proteases, IL-33_95–270, IL-33_107–270, and IL-33_109–270, were 30-fold more potent than full-length human IL-33_1–270 for activation of ILC2s ex vivo. They induced a strong expansion of ILC2s and eosinophils in vivo, associated with elevated concentrations of IL-5 and IL-13. Murine IL-33 is also cleaved by mast cell tryptase, and a tryptase inhibitor reduced IL-33–dependent allergic airway inflammation in vivo. Our study identifies the central cleavage/activation domain of IL-33 (amino acids 66–111) as an important functional domain of the protein and suggests that interference with IL-33 cleavage and activation by mast cell and other inflammatory proteases could be useful to reduce IL-33–mediated responses in allergic asthma and other inflammatory diseases.Interleukin-33 (IL-33), previously known as nuclear factor from high endothelial venules or NF-HEV (1, 2), is an IL-1 family cytokine (3) that signals through the interleukin 1 receptor-like 1 (IL1RL1) receptor ST2 (4, 5) and induces expression of cytokines and chemokines in various immune cell types, including mast cells, basophils, eosinophils, Th2 lymphocytes, invariant natural killer T, and natural killer cells (3, 4, 6–8). Studies in IL-33–deficient mice indicate that IL-33 plays important roles in type-2 innate immunity and innate-type allergic airway inflammation (9–13). Indeed, IL-33 is a key activator of the recently described group-2 innate lymphoid cells (ILC2s, natural helper cells, nuocytes) (14–17). These cells control eosinophil homeostasis in blood and adipose tissue (18, 19) and produce extremely high amounts of the type-2 cytokines IL-5 and IL-13 in response to IL-33 (14–16). ILC2s also play important roles in allergic airway inflammation (20–24), atopic skin disease (25–28), helminth infection in the intestine (11, 12, 14–16), and influenza virus infection in the lungs (29, 30).Based on animal model studies and analyses of diseased tissues from patients, IL-33 has been proposed as a candidate therapeutic target for several important diseases, including asthma and other allergic diseases, rheumatoid arthritis, inflammatory bowel diseases, and cardiovascular diseases (4, 6). IL-33 is likely to play a critical role in asthma because the IL33 and IL1RL1/ST2 genes have been reproducibly identified as major susceptibility loci in several independent large-scale genome-wide association studies of human asthma (31, 32).Despite these important advances into the roles of IL-33, very little is known yet about the mechanisms regulating its activity. Full-length human IL-33 is a 270 amino acid protein localized in the nucleus of endothelial and epithelial cells in blood vessels and epithelial barrier tissues (1, 2, 33, 34), which associates with chromatin (2) and histones H2A-H2B, through a short chromatin-binding motif located in its N-terminal part (amino acids 40–58) (35). IL-33 can be released in the extracellular space upon cellular damage or necrotic cell death (36, 37), and it was thus proposed to function as an alarmin (alarm signal or endogenous danger signal), which alerts the immune system to tissue injury following trauma or infection (33, 36, 37).Proteases have been shown to regulate IL-33 activity. Full-length IL-33_1–270 is biologically active but processing by caspases after residue Asp₁₇₈ in the IL-1–like cytokine domain results in its inactivation (36, 37). In contrast, inflammatory proteases from neutrophils, cathepsin G, and elastase, process full-length IL-33 into mature forms that contain an intact IL-1–like cytokine domain and that have an increased biological activity compared with full-length IL-33_1–270 (38). Although neutrophils have been implicated in virus-induced exacerbations of asthma, they are unlikely to be involved in the processing of IL-33 during allergic inflammation. We therefore investigated the possibility that other cell types may be involved in this process. Mast cells, which are widely recognized for their roles as effector cells in allergic disorders, were good candidates because they interact directly with ILC2s in vivo (26) and they are strategically positioned close to vessel walls and epithelial surfaces exposed to the environment (39), the major sites of IL-33 expression (33, 34). We now demonstrate that activated human mast cells and purified mast cell proteases, tryptase and chymase, generate mature forms of IL-33 (IL-33_95–270, IL-33_107–270, and IL-33_109–270), which are ∼30-fold more potent than full-length IL-33_1–270 for activation of ILC2s ex vivo. These IL-33 mature forms are also potent inducers of ILC2s, eosinophils, and type-2 cytokines in vivo. Our study suggests that release of the C-terminal IL-1–like cytokine domain, through proteolytic maturation within the central cleavage/activation domain (amino acids 66–111), is important for full biological activity of IL-33.     

Initial viral load determines the magnitude of the human CD8 T cell response to yellow fever vaccination

CD8 T cells are a potent tool for eliminating intracellular pathogens and tumor cells. Thus, eliciting robust CD8 T-cell immunity is the basis for many vaccines under development. However, the relationship between antigen load and the magnitude of the CD8 T-cell response is not well-described in a human immune response. Here we address this issue by quantifying viral load and the CD8 T-cell response in a cohort of 80 individuals immunized with the live attenuated yellow fever vaccine (YFV-17D) by sampling peripheral blood at days 0, 1, 2, 3, 5, 7, 9, 11, 14, 30, and 90. When the virus load was below a threshold (peak virus load < 225 genomes per mL, or integrated virus load < 400 genome days per mL), the magnitude of the CD8 T-cell response correlated strongly with the virus load (R² ∼ 0.63). As the virus load increased above this threshold, the magnitude of the CD8 T-cell responses saturated. Recent advances in CD8 T-cell–based vaccines have focused on replication-incompetent or single-cycle vectors. However, these approaches deliver relatively limited amounts of antigen after immunization. Our results highlight the requirement that T-cell–based vaccines should deliver sufficient antigen during the initial period of the immune response to elicit a large number of CD8 T cells that may be needed for protection.CD8 T cells provide a powerful mechanism for elimination of intracellular pathogens and tumor cells. Accordingly, a major thrust of current vaccine research focuses on stimulating robust T-cell immunity for defense against infections such as HIV, malaria, tuberculosis, Ebola virus, herpes viruses, and hepatitis C virus (HCV) (1–8). Inducing effective CD8 T-cell immunity is also an important goal for cancer vaccines (9, 10). However, how antigen load affects the CD8 T-cell response has not been quantified in a detailed manner during a human immune response. In this study we address this question using the human live attenuated yellow fever vaccine (YFV-17D) vaccine.The dynamics of CD8 T-cell responses to intracellular infection have been extensively studied in model systems. Infection typically stimulates a rapid burst of proliferation in antigen-specific CD8 T cells with division occurring as quickly as once in 4–6 h (11). This expansion results in a large population of effector CD8 T cells that aid in clearance of infected cells. Although most (90–95%) of the effector CD8 T cells die, a small fraction differentiate to form long-term memory CD8 T cells (12). Detailed quantitative measurements of the dynamics of virus and the CD8 T-cell response to the YFV-17D vaccine allow us to characterize these basic features of the CD8 T-cell responses in humans. Additionally, tracking the dynamics of both virus and CD8 T cells over time in a large cohort allows us to explore the relationship between amount of antigen and the magnitude of expansion and answer the following questions: Is there a threshold amount of virus required to generate a response? Does the magnitude of the response increase proportionally, or does it saturate with viral load? Although a number of studies have considered the complex relationship between numbers of specific CD8 T cells and virus loads during the chronic phase of HIV and HCV infections (3, 13–23), very few studies (24–27) have investigated these questions in the context of the generation of immune response following acute infections and vaccination.We addressed these questions by measuring the dynamics of both virus and virus-specific CD8 T cells following immunization with the YFV-17D vaccine. The YFV-17D vaccine comprises a highly efficacious, live attenuated virus that causes an acute infection and stimulates a robust immune response conferring lifelong protection against the yellow fever virus (YFV) (28, 29). Because yellow fever is not endemic to the United States, immunization with YFV-17D induces a primary immune response (30, 31). Previous work with YFV-17D has identified CD8 T cells specific for some of the YFV epitopes and defined the stages of expansion, contraction, and memory maintenance (32–38). We now know that YFV stimulates a polyfunctional, broadly targeting, and long-lasting CD8 T-cell response. Of particular note, we have previously demonstrated that the magnitude of the total effector CD8 T-cell response against YFV can be measured using the Ki-67⁺ Bcl-2^lo HLA-DR⁺ CD38⁺ phenotype of activated T cells seen early after vaccination (38). In the current study, we followed a large cohort of 80 individuals with intensive sampling at days 0, 1, 2, 3, 5, 7, 9, 11, 14, 30, and 90 postvaccination to quantify viral load in plasma (39). Additionally, we quantified the magnitude of the YFV-specific effector CD8 T-cell response at days 0, 3, 7, 14, 30, and 90 postvaccination using the Ki-67⁺Bcl-2^lo phenotype. We find that different individuals have different virus loads following infection and generate CD8 T-cell responses of different sizes. This allows us to determine the relationship between virus load and magnitude of the CD8 T-cell response.The majority of vaccines that are currently under development use replication-incompetent or single-cycle vectors such as Modified Vaccinia Ankara, adenovirus, and DNA. Although these approaches are inherently safe, they may express and deliver relatively limited amounts of antigen. Our results emphasize the requirement that T-cell–based vaccines deliver sufficient antigen to elicit a large CD8 T-cell response that may be needed for protection.     

Hemolysis-induced lethality involves inflammasome activation by heme

The increase of extracellular heme is a hallmark of hemolysis or extensive cell damage. Heme has prooxidant, cytotoxic, and inflammatory effects, playing a central role in the pathogenesis of malaria, sepsis, and sickle cell disease. However, the mechanisms by which heme is sensed by innate immune cells contributing to these diseases are not fully characterized. We found that heme, but not porphyrins without iron, activated LPS-primed macrophages promoting the processing of IL-1β dependent on nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). The activation of NLRP3 by heme required spleen tyrosine kinase, NADPH oxidase-2, mitochondrial reactive oxygen species, and K⁺ efflux, whereas it was independent of heme internalization, lysosomal damage, ATP release, the purinergic receptor P2X7, and cell death. Importantly, our results indicated the participation of macrophages, NLRP3 inflammasome components, and IL-1R in the lethality caused by sterile hemolysis. Thus, understanding the molecular pathways affected by heme in innate immune cells might prove useful to identify new therapeutic targets for diseases that have heme release.Hemolysis, hemorrhage, and rhabdomyolysis cause the release of large amounts of hemoproteins to the extracellular space, which, once oxidized, release the heme moiety, a potentially harmful molecule due to its prooxidant, cytotoxic, and inflammatory effects (1, 2). Scavenging proteins such as haptoglobin and hemopexin bind hemoglobin and heme, respectively, promoting their clearance from the circulation and delivery to cells involved with heme catabolism. Heme oxygenase cleaves heme and generates equimolar amounts of biliverdin, carbon monoxide (CO) and iron (2). Studies using mice deficient for haptoglobin (Hp), hemopexin (Hx), and heme oxygenase 1 (HO-1) demonstrate the importance of these proteins in controlling the deleterious effects of heme. Both Hp^−/− and Hx^−/− mice have increased renal damage after acute hemolysis induced by phenyhydrazine (Phz) compared with wild-type mice (3, 4). Mice lacking both proteins present splenomegaly and liver inflammation composed of several foci with leukocyte infiltration after intravascular hemolysis (5). Hx protect mice against heme-induced endothelial damage improving liver and cardiovascular function (6–8). Lack of heme oxygenase 1 (Hmox1^−/−) causes iron overload, increased cell death, and tissue inflammation under basal conditions and upon inflammatory stimuli (9–15). This salutary effect of HO-1 has been attributed to its effect of reducing heme amounts as well as generating the cytoprotective molecules, biliverdin and CO.Heme induces neutrophil migration in vivo and in vitro (16, 17), inhibits neutrophil apoptosis (18), triggers cytokine and lipid mediator production by macrophages (19, 20), and increases the expression of adhesion molecules and tissue factor on endothelial cells (21–23). Heme cooperates with TNF, causing hepatocyte apoptosis in a mechanism dependent on reactive oxygen species (ROS) generation (12). Whereas heme-induced TNF production depends on functional toll-like receptor 4 (TLR4), ROS generation in response to heme is TLR4 independent (19). We recently observed that heme triggers receptor-interacting protein (RIP)1/3-dependent macrophage-programmed necrosis through the induction of TNF and ROS (15). The highly unstable nature of iron is considered critical for the ability of heme to generate ROS and to cause inflammation. ROS generated by heme has been mainly attributed to the Fenton reaction. However, recent studies suggest that heme can generate ROS through multiple sources, including NADPH oxidase and mitochondria (22, 24–27).Heme causes inflammation in sterile and infectious conditions, contributing to the pathogenesis of hemolytic diseases, subarachnoid hemorrhage, malaria, and sepsis (11, 13, 24, 28), but the mechanisms by which heme operates in different conditions are not completely understood. Blocking the prooxidant effects of heme protects cells from death and prevents tissue damage and lethality in models of malaria and sepsis (12, 13, 15). Importantly, two recent studies demonstrated the pathogenic role of heme-induced TLR4 activation in a mouse model of sickle cell disease (29, 30). These results highlight the great potential of understanding the molecular mechanisms of heme-induced inflammation and cell death as a way to identify new therapeutic targets.Hemolysis and heme synergize with microbial molecules for the induction of inflammatory cytokine production and inflammation in a mechanism dependent on ROS and Syk (24). Processing of pro–IL-1β is dependent on caspase-1 activity, requiring assembly of the inflammasome, a cytosolic multiprotein complex composed of a NOD-like receptor, the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC), and caspase-1 (31–33). The most extensively studied inflammasome is the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3). NLRP3 and pro–IL-1β expression are increased in innate immune cells primed with NF-κB inducers such as TLR agonists and TNF (34, 35). NLRP3 inflammasome is activated by several structurally nonrelated stimuli, such as endogenous and microbial molecules, pore-forming toxins, and particulate matter (34, 35). The activation of NLRP3 involves K⁺ efflux, increase of ROS and Syk phosphorylation. Importantly, critical roles of NLRP3 have been demonstrated in a vast number of diseases (34, 36). We hypothesize that heme causes the activation of the inflammasome and secretion of IL-1β. Here we found that heme triggered the processing and secretion of IL-1β dependently on NLRP3 inflammasome in vitro and in vivo. The activation of NLRP3 by heme was dependent on Syk, ROS, and K⁺ efflux, but independent of lysosomal leakage, ATP release, or cell death. Finally, our results indicated the critical role of macrophages, the NLRP3 inflammasome, and IL-1R to the lethality caused by sterile hemolysis.