Type 2 immunity is a stereotyped host response to allergens and parasitic helminths that is sustained in large part by the cytokines IL-4 and IL-13. Recent advances have called attention to the contributions by innate cells in initiating adaptive immunity, including a novel lineage-negative population of cells that secretes IL-13 and IL-5 in response to the epithelial cytokines IL-25 and IL-33. Here, we use IL-4 and IL-13 reporter mice to track lineage-negative innate cells that arise during type 2 immunity or in response to IL-25 and IL-33 in vivo. Unexpectedly, lineage-negative IL-25 (and IL-33) responsive cells are widely distributed in tissues of the mouse and are particularly prevalent in mesenteric lymph nodes, spleen, and liver. These cells expand robustly in response to exogenous IL-25 or IL-33 and after infection with the helminth Nippostrongylus brasiliensis, and they are the major innate IL-13-expressing cells under these conditions. Activation of these cells using IL-25 is sufficient for worm clearance, even in the absence of adaptive immunity. Widely dispersed innate type 2 helper cells, which we designate Ih2 cells, play an integral role in type 2 immune responses.T ype 2 immune responses are important for the control of infections at mucosal barriers and the development of allergic inflammation. These responses are characterized by eosinophilia, elevated IgE, goblet cell metaplasia with enhanced mucus production, and smooth muscle hyperreactivity, all of which rely critically on production of the canonical type 2-associated cytokines IL-4, IL-5, and IL-13 (1, 2). Although adaptive Th2 cells and follicular T cells are important sources of these cytokines (3), various innate cells, including eosinophils, basophils, and mast cells, have also been implicated as producers of these cytokines in various model systems (1, 2, 4, 5). More recently, the cytokines IL-25 and IL-33, members of the IL-17 and IL-1 cytokine families, respectively, were found to induce type 2 cytokine production when administered to mice, implicating these cytokines in the initiation of type 2 immune responses (6, 7). IL-25 and IL-33 are expressed by epithelial cells, macrophages, and possibly other cell types (8), and they are expressed at elevated levels during infection with parasitic helminths (9, 10) or after challenge with allergens (9, 11). Administration of exogenous IL-25 or IL-33 to mice leads to markedly enhanced levels of IL-4, IL-5, and IL-13 and many of the tissue features of a type 2 immune response (6, 7). Conversely, deficiency in IL-25 leads to diminished IL-4, IL-5, and IL-13 production and variable delays in worm clearance in different helminth models (12, 13). Similarly, mice unable to respond to IL-33 because of deficiency in the T1-ST2 subunit of the IL-33 receptor display diminished Th2-associated cytokines and decreased granuloma formation after injection of Schistosoma mansoni eggs (14).Some of the original descriptions of these cytokines as well as more recent reports have noted the capacity of exogenous ...

Signaling by Toll-like receptors (TLRs) on intestinal epithelial cells (IECs) is critical for intestinal homeostasis. To visualize epithelial expression of individual TLRs in vivo, we generated five strains of reporter mice. These mice revealed that TLR expression varied dramatically along the length of the intestine. Indeed, small intestine (SI) IECs expressed low levels of multiple TLRs that were highly expressed by colonic IECs. TLR5 expression was restricted to Paneth cells in the SI epithelium. Intestinal organoid experiments revealed that TLR signaling in Paneth cells or colonic IECs induced a core set of host defense genes, but this set did not include antimicrobial peptides, which instead were induced indirectly by inflammatory cytokines. This comprehensive blueprint of TLR expression and function in IECs reveals unexpected diversity in the responsiveness of IECs to microbial stimuli, and together with the associated reporter strains, provides a resource for further study of innate immunity.

Interleukin (IL)-17A plays an important role in host defense against a variety of pathogens and may also contribute to the pathogenesis of autoimmune diseases. However, precise identification and quantification of the cells that produce this cytokine in vivo have not been performed. We generated novel IL-17A reporter mice to investigate expression of IL-17A during Klebsiella pneumoniae infection and during experimental autoimmune encephalomyelitis, conditions previously demonstrated to potently induce IL-17A production. In both settings, the majority of IL-17A was produced by non-CD4+ T cells, particularly γδ T cells, but also invariant NKT cells and other CD4−CD3ε+ cells. As measured in dual-reporter mice, IFN-γ-producing Th1 cells greatly outnumbered IL-17A-producing Th17 cells throughout both challenges. Production of IL-17A by cells from unchallenged mice or by non-T cells under any condition was not evident. Administration of IL-1β and/or IL-23 elicited rapid production of IL-17A by γδ T cells, invariant NKT cells and other CD4−CD3ε+ cells in vivo, demonstrating that these cells are poised for rapid cytokine production and likely comprise the major sources of this cytokine during acute immunologic challenges.

Autoinflammatory disease and hyperinflammatory syndromes represent a growing number of diseases associated with inappropriately controlled inflammation in multiple organs. Systemic inflammation commonly results from dysregulated activation of innate immune cells, and therapeutic targeting of the Interleukin-1 beta (IL-1β)–pathway has been used to ameliorate some of these diseases. Some hyperinflammatory syndromes, however, such as hemophagocytic lymphohistiocytosis and the newly classified proteasome disability syndromes, are refractory to such treatments, suggesting that other factors or environmental stressors may be contributing. In comparing two cytokine reporter mouse strains, we identify interferon gamma (IFN-γ) as a mediator of systemic autoinflammatory disease. Chronically elevated levels of IFN-γ resulted in progressive multi-organ inflammation and two copies of the mutant allele resulted in increased mortality accompanied by myeloproliferative disease. Disease was alleviated by genetic deletion of T-bet. These studies raise the possibility that therapeutics targeting the IFN-γ pathway might be effective in hyperinflammatory conditions refractory to IL-1β-targeted therapies.

Infant HIV infection and maternal coinfection with HIV and PM negatively influence antibody responses to TT, but not those to malarial antigens, in infants. Antimalarial antibodies rarely showed protective associations with morbidity in infants and were more often a marker for malaria exposure and risk of infection.

A synthetic multistage, multi-epitope Plasmodium falciparum malaria antigen (FALVAC-1A) was designed and evaluated in silico, and then the gene was constructed and expressed in Escherichia coli. The FALVAC-1A protein was purified by inclusion body isolation, followed by affinity and ion exchange chromatography. Although FALVAC-1A was a synthetic antigen, it folded to a specific, but as yet incompletely defined, molecular conformation that was stable and comparable from lot to lot. When formulated with four different adjuvants, FALVAC-1A was highly immunogenic in rabbits, inducing not only ELISA reactivity to the cognate antigen and most of its component epitopes, but also in vitro activity against P. falciparum parasites as demonstrated by inhibition of sporozoite invasion, antibody dependent cellular inhibition and the immunofluorescence assay.

FALVAC-1A is a second-generation multitarget, multiepitope synthetic candidate vaccine against Plasmodium falciparum, incorporating elements designed to yield a stable and immunogenic molecule. Characteristics of the immunogenicity of FALVAC-1A were evaluated in congenic (H-2 b , H-2 k , and H-2 d ) and outbred strains of mice. The influences of four adjuvants (aluminum phosphate, QS-21, Montanide ISA-720, and copolymer CRL-1005) on different aspects of the immune response were also assessed. FALVAC-1A generated strong antibody responses in all mouse strains. The highest mean enzyme-linked immunosorbent assay (ELISA) antibody concentrations against FALVAC-1A were observed in the outbred ICR mice, followed by B10.BR, B10.D2, and C57BL/6 mice, though this order varied for the different adjuvants, with no statistical differences between mouse strains. In all mouse strains, the highest anti-FALVAC-1A antibody titers in ELISAs were induced by FALVAC-1A in copolymer and ISA-720 formulations, followed by QS-21 and AlPO4. These antibodies were of all four subclasses, though immunoglobulin G1 (IgG1) predominated, with the exception of FALVAC-1A with the QS-21 adjuvant, which induced predominantly IgG2c responses. Both sporozoites and blood stages of P. falciparum were recognized by anti-FALVAC-1A sera in the immunofluorescence assay. In addition to antibody, cellular immune responses were detected; these responses were studied by examining spleen cells producing gamma interferon and interleukin-4 in enzyme-linked immunospot assays. In summary, FALVAC-1A was found to be highly immunogenic and elicited functionally relevant antibodies that can recognize sporozoites and blood-stage parasites in diverse genetic backgrounds.

Background: Acute myeloid leukemia (AML) chimeric antigen receptor (CAR) T cell therapies are at early stages of testing in human clinical trials. We previously described the design of a CD33-specific dimerizing agent regulated immunoreceptor complex (DARIC33) that, in the presence of rapamycin (RAPA), switches from an "OFF" to "ON" state that activates T cells in response to tumor antigen. Here, we describe RAPA controlled activation and anti-AML activity of DARIC33 using human AML xenograft NSG mouse models and GMP-compliant T cell manufacturing methodologies. We find that low nanomolar whole blood concentrations of RAPA, below levels used for immunosuppression, are needed for DARIC33 to become active in vivo and exhibit potent CD33-specific anti-AML activity. Methods: Clinical scale, DARIC33 and control cell products were manufactured by Seattle Children's Therapeutics (SC-DARIC33) by stimulating equal numbers of CD4+ and CD8+ T cells with anti-CD3/CD28 microbeads in closed gas-permeable culture vessels followed by lentiviral vector transduction and expansion in serum-free media supplemented with IL7, IL15 and IL21. The activation and anti-AML activity of SC-DARIC33 assayed in vitro by measuring cytokine production or lysis of chromium labeled target cells in the presence or absence of RAPA. NSG mice inoculated with either 1x10 6 luciferase expressing MV4-11 (CD33+) or 0.5x10 6 Raji cells expressing a huCD33 transgene (Raji.CD33, CD19+/CD33+) were treated with SC-DARIC33 and RAPA. Concentrations of RAPA in mouse blood were quantified by LC-MS/MS (Charles River). Results: Donor matched SC-DARIC33 and control CD19 CAR T cell products exhibited similar surface markers of engraftment fitness (CD62L+CD45RA+) and capacity for anti-tumor (CD27+CCR7+) effector function. Following coculture of SC-DARIC33 and Raji.CD33 cells without RAPA, concentrations of IL-2, TNF-α or IFN-γ were not increased in comparison to Raji.CD33 cells cocultured with mock T cell products. However, when exposed to 1 nM RAPA and Raji.CD33 targets, SC-DARIC33 produced cytokines in quantities similar to (Fig A). To determine the concentration of RAPA required to activate SC-DARIC33 in patients, DARIC33 T cells, CD33+ AML targets, and graded concentrations of RAPA were added to allogeneic human whole blood samples and plasma was recovered after 24 hours of incubation. RAPA addition increased IFN-g release with an apparent EC50 = 2.6nM (Fig B). Anti-AML SC-DARIC33 activity and whole blood [RAPA WB] in tumor bearing mice were determined and compared to pediatric RAPA pharmacokinetic models to select appropriate clinical RAPA dose schedules. NSG mice inoculated with Raji.CD33 tumors treated with either 1x10 7 CD19 CAR T cells or 3x10 7 SC-DARIC33 T cells, followed by RAPA 0.1 mg/kg IP QOD, exhibited stringent control of tumor growth, demonstrating a 3:1 cell dose equivalency (Fig C). AML progression was also inhibited when NSG mice were inoculated with MV411 AML cells and subsequently treated with 10 7 SC-DARIC33 followed by 0.01 mg/kg RAPA QD (Fig D). Blood samples from tumor bearing mice obtained on day 15, 2 hours post RAPA administration, showed [RAPA WB] = 2.3 ± 1.3 ng/mL, overlapping with the in vitroand indicating very low concentrations of RAPA effectively modulate the "OFF-to-ON" state transition. Population PK models simulating various RAPA doses and schedules in pediatric patients found oral daily dosing of 0.5 mg/m 2 RAPA will achieve [RAPA WB] = 1-3 ng/mL in most patients (Fig E). Conclusion: Evaluation of GMP cell products and RAPA PK demonstrate that very low doses of RAPA are sufficient to regulate SC-DARIC33. To establish safety of SC-DARIC33 in humans, an upcoming phase 1 trial clinical trial evaluating SC-DARIC33 in pediatric AML patients will test escalating cell doses followed by low-dose RAPA administration from post T cell infusion days 2-21 using a Bayesian optimal interval (BOIN) design. Peripheral blood samples will be monitored for CD33+ myeloid cell recovery after cessation of RAPA dosing. These data will establish safety and support the feasibility of SC-DARIC33 CAR T cells to be reversibly modulated in an "OFF-ON-OFF" fashion by intermittent low-dose RAPA administration. Figure 1 Figure 1. Disclosures Price: bluebird, bio: Current Employment. Zhang: bluebird, bio: Current Employment. Sundaram: bluebird, bio: Current Employment. Lewis: bluebird, bio: Current Employment. Bilic: C4 Therapeutics: Current Employment. Xia: bluebird, bio: Current Employment. Krostag: bluebird, bio: Ended employment in the past 24 months. So: bluebird, bio: Current Employment. Martin: bluebird, bio: Current Employment. Leung: bluebird, bio: Ended employment in the past 24 months. Astrakhan: bluebird, bio: Current Employment. Pogson: bluebird, bio: Current Employment. Jarjour: bluebird, bio: Current Employment. Jensen: bluebird, bio: Research Funding.