Effective Multi-lineage Engraftment in a Mouse Model of Fanconi Anemia Using Non-genotoxic Antibody-Based Conditioning.

Conditioning chemotherapy is used to deplete hematopoietic stem cells in the recipient's marrow, facilitating donor cell engraftment. Although effective, a major issue with chemotherapy is the systemic genotoxicity that increases the risk for secondary malignancies. Antibody conjugates targeting hematopoietic cells are an emerging non-genotoxic method of opening the marrow niche and promoting engraftment of transplanted cells while maintaining intact marrow cellularity. Specifically, this platform would be useful in diseases associated with DNA damage or cancer predisposition, such as dyskeratosis congenita, Schwachman-Diamond syndrome, and Fanconi anemia (FA). Our approach utilizes antibody-drug conjugates (ADC) as an alternative conditioning regimen in an FA mouse model of autologous transplantation. Antibodies targeting either CD45 or CD117 were conjugated to saporin (SAP), a ribosomal toxin. FANCA knockout mice were conditioned with either CD45-SAP or CD117-SAP prior to receiving whole marrow from a heterozygous healthy donor. Bone marrow and peripheral blood analysis revealed equivalent levels of donor engraftment, with minimal toxicity in ADC-treated groups as compared with cyclophosphamide-treated controls. Our findings suggest ADCs may be an effective conditioning strategy in stem cell transplantation not only for diseases where traditional chemotherapy is not tolerated, but also more broadly for the field of blood and marrow transplantation.

Guide Swap enables genome-scale pooled CRISPR-Cas9 screening in human primary cells.

CRISPR-Cas9 screening allows genome-wide interrogation of gene function. Currently, to achieve the high and uniform Cas9 expression desirable for screening, one needs to engineer stable and clonal Cas9-expressing cells-an approach that is not applicable in human primary cells. Guide Swap permits genome-scale pooled CRISPR-Cas9 screening in human primary cells by exploiting the unexpected finding that editing by lentivirally delivered, targeted guide RNAs (gRNAs) occurs efficiently when Cas9 is introduced in complex with nontargeting gRNA. We validated Guide Swap in depletion and enrichment screens in CD4+ T cells. Next, we implemented Guide Swap in a model of ex vivo hematopoiesis, and identified known and previously unknown regulators of CD34+ hematopoietic stem and progenitor cell (HSPC) expansion. We anticipate that this platform will be broadly applicable to other challenging cell types, and thus will enable discovery in previously inaccessible but biologically relevant human primary cell systems.

Incorporation of Phosphonate into Benzonaphthyridine Toll-like Receptor 7 Agonists for Adsorption to Aluminum Hydroxide.

Small molecule Toll-like receptor 7 (TLR7) agonists have been used as vaccine adjuvants by enhancing innate immune activation to afford better adaptive response. Localized TLR7 agonists without systemic exposure can afford good adjuvanticity, suggesting peripheral innate activation (non-antigen-specific) is not required for immune priming. To enhance colocalization of antigen and adjuvant, benzonaphthyridine (BZN) TLR7 agonists are chemically modified with phosphonates to allow adsorption onto aluminum hydroxide (alum), a formulation commonly used in vaccines for antigen stabilization and injection site deposition. The adsorption process is facilitated by enhancing aqueous solubility of BZN analogs to avoid physical mixture of two insoluble particulates. These BZN-phosphonates are highly adsorbed onto alum, which significantly reduced systemic exposure and increased local retention post injection. This report demonstrates a novel approach in vaccine adjuvant design using phosphonate modification to afford adsorption of small molecule immune potentiator (SMIP) onto alum, thereby enhancing co-delivery with antigen.

Phase I/II Trial of StemRegenin-1 Expanded Umbilical Cord Blood Hematopoietic Stem Cells Supports Testing as a Stand-Alone Graft.

Clinical application of umbilical cord blood (UCB) as a source of hematopoietic stem cells for transplantation is limited by low CD34+ cell dose, increased risk of graft failure, and slow hematopoietic recovery. While the cell dose limitation is partially mitigated by using two UCB units, larger-dosed single units would be preferable. We have evaluated the feasibility and safety of StemRegenin-1 (SR-1), an aryl hydrocarbon receptor antagonist that expands CD34+ cells, by placing one of the two units in expansion culture. SR-1 produced a 330-fold increase in CD34+ cells and led to engraftment in 17/17 patients at a median of 15 days for neutrophils and 49 days for platelets, significantly faster than in patients treated with unmanipulated UCB. Taken together, the marked expansion, absence of graft failure, and enhanced hematopoietic recovery support testing of SR-1 expansion as a stand-alone graft and suggest it may ameliorate a limitation of UCB transplant.

Inhibition of the Inositol Kinase Itpkb Augments Calcium Signaling in Lymphocytes and Reveals a Novel Strategy to Treat Autoimmune Disease.

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease.

A new TLR2 agonist promotes cross-presentation by mouse and human antigen presenting cells.

Cross-presentation is the process by which professional APCs load peptides from an extracellularly derived protein onto class I MHC molecules to trigger a CD8(+) T cell response. The ability to enhance this process is therefore relevant for the development of antitumor and antiviral vaccines. We investigated a new TLR2-based adjuvant, Small Molecule Immune Potentiator (SMIP) 2.1, for its ability to stimulate cross-presentation. Using OVA as model antigen, we demonstrated that a SMIP2.1-adjuvanted vaccine formulation induced a greater CD8(+) T cell response, in terms of proliferation, cytokine production and cytolytic activity, than a non-adjuvanted vaccine. Moreover, using an OVA-expressing tumor model, we showed that the CTLs induced by the SMIP2.1 formulated vaccine inhibits tumor growth in vivo. Using a BCR transgenic mouse model we found that B cells could cross-present the OVA antigen when stimulated with SMIP2.1. We also used a flow cytometry assay to detect activation of human CD8(+) T cells isolated from human PBMCs of cytomegalovirus-seropositive donors. Stimulation with SMIP2.1 increased the capacity of human APCs, pulsed in vitro with the pp65 CMV protein, to activate CMV-specific CD8(+) T cells. Therefore, vaccination with an exogenous antigen formulated with SMIP2.1 is a successful strategy for the induction of a cytotoxic T cell response along with antibody production.

Using Poly I:C as an adjuvant does not induce or exacerbate models of systemic lupus erythematosus.

Subunit vaccines are typically poorly immunogenic when administered alone, and require adjuvants for robust responses. Triggering TLRs to boost antigen-specific adaptive immunity is an attractive approach to increase the potency and quality of vaccines. However, recent reports suggest that alterations in TLR expression are associated with the pathogenesis of inflammatory and autoimmune diseases. To compare genetic studies with adjuvant studies, we examined whether stimulation through a TLR agonist induces or increases the autoimmune phenotype of healthy or autoimmune mice. C57BL/6, MRL/lpr, and Fcγr2b-deficient mice were dosed i.p. with Poly I:C every other day for 3 weeks, and monitored for signs of autoimmunity over 3 months. A separate group of mice was vaccinated three times i.m. with rPA anthrax antigen with or without Poly I:C with 2 weeks between doses. Immunized groups exhibited robust responses to vaccine and C57BL/6 and MRL/lpr mice showed a statistically significant increase in anti-rPA IgG responses in the presence of Poly I:C. Interestingly, Fcγr2b-/- mice showed increases with the base rPA vaccine, which was not significantly increased when Poly I:C was used as an adjuvant. In the chronically dosed groups, we also observed subtle alterations in levels of total antibody and some autoantibodies. However, there were no statistically significant differences in autoimmune syndrome, as measured by proteinurea, kidney pathology, weight loss, and mortality, with Poly I:C administration in chronic or vaccination mode. Taken together, these results suggest that administration of TLR3 agonists in chronic or vaccination mode does not induce or exacerbate models of systemic lupus erythematosus.

Rational design of small molecules as vaccine adjuvants.

Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.

Mst1 directs Myosin IIa partitioning of low and higher affinity integrins during T cell migration.

Chemokines promote T cell migration by transmitting signals that induce T cell polarization and integrin activation and adhesion. Mst1 kinase is a key signal mediator required for both of these processes; however, its molecular mechanism remains unclear. Here, we present a mouse model in which Mst1 function is disrupted by a hypomorphic mutation. Microscopic analysis of Mst1-deficient CD4 T cells revealed a necessary role for Mst1 in controlling the localization and activity of Myosin IIa, a molecular motor that moves along actin filaments. Using affinity specific LFA-1 antibodies, we identified a requirement for Myosin IIa-dependent contraction in the precise spatial distribution of low and higher affinity LFA-1 on the membrane of migrating T cells. Mst1 deficiency or Myosin inhibition resulted in multipolar cells, difficulties in uropod detachment and mis-localization of low affinity LFA-1. Thus, Mst1 regulates Myosin IIa dynamics to organize high and low affinity LFA-1 to the anterior and posterior membrane during T cell migration.

Topical SMIP-7.7, a toll-like receptor 7 agonist, protects against genital herpes simplex virus type-2 disease in the guinea pig model of genital herpes.

BACKGROUND: Development of more effective therapies for genital herpes simplex virus type-2 (HSV-2) infections remains a priority. The toll-like receptors (TLR) are attractive targets for the immunomodulation of primary and recurrent genital herpes infection. The guinea pig model of genital HSV-2 disease was therefore used to evaluate the efficacy of a new TLR-7 agonist, SMIP-7.7. METHODS: The effects of SMIP-7.7 at concentrations between 0.90% and 0.09% were compared to the vehicle control or Aldara(®) (3M Health Care Limited, Northridge, CA, USA) as treatment for genital HSV-2 infections. Following intravaginal inoculation of Hartley guinea pigs with 10(6) pfu HSV-2 (MS strain), animals were treated intravaginally beginning at 36 h post-infection. Animals were evaluated for acute disease, acute virus replication, recurrent disease and shedding, as well as infection of the dorsal root ganglia. RESULTS: Treatment with SMIP-7.7 significantly decreased mean total lesion scores during primary infection (all doses, P<0.01 compared with vehicle control, and similar to Aldara(®)). Vaginal virus titres were reduced in treated animals compared with vehicle control (P<0.001 for each treatment versus vehicle control on day 4). Treatment with SMIP-7.7 also significantly decreased the number of recurrent lesion days, the number of days with recurrent virus shedding and the infection of the dorsal root ganglia compared to the vehicle control, and was similar to Aldara(®). As opposed to Aldara(®), SMIP-7.7 did not induce fever or weight loss during treatment. CONCLUSIONS: SMIP-7.7 improves the outcome of primary and recurrent HSV-2 disease comparable to Aldara(®) but without some of the side effects associated with Aldara(®).

Eupalinilide E inhibits erythropoiesis and promotes the expansion of hematopoietic progenitor cells.

Hematopoietic stem cells (HSCs) are the progenitor cells that give rise to all blood cells. The ability to control HSC differentiation has the potential to improve the success of bone marrow transplants and the production of functional blood cells ex vivo. Here we performed an unbiased screen using primary human CD34(+) hematopoietic stem and progenitor cells (HSPCs) to identify natural products that selectively control their differentiation. We identified a plant-derived natural product, eupalinilide E, that promotes the ex vivo expansion of HSPCs and hinders the in vitro development of erythrocytes. This activity was additive with aryl hydrocarbon receptor (AhR) antagonists, which are also known to expand HSCs and currently in clinical development. These findings reveal a new activity for eupalinilide E, and suggest that it may be a useful tool to probe the mechanisms of hematopoiesis and improve the ex vivo production of progenitors for therapeutic purposes.

The intramembrane protease Sppl2a is required for B cell and DC development and survival via cleavage of the invariant chain.

B cell development requires tight regulation to allow for the generation of a diverse repertoire while preventing the development of autoreactive cells. We report, using N-ethyl-N-nitrosourea (ENU)-induced mutagenesis, the identification of a mutant mouse (chompB) with a block in early B cell development. The blockade occurs after the transitional 1 (T1) stage and leads to a decrease in mature B cell subsets and deficits in T cell-dependent antibody responses. Additionally, chompB mice have decreases in myeloid dendritic cells (DCs). The mutation was mapped to the intramembrane protease signal peptide peptidase-like 2a (Sppl2a), a gene not previously implicated in immune cell development. Proteomic analysis identified the invariant chain (CD74) as a key substrate of Sppl2a and suggests that regulated intramembrane proteolysis of CD74 by Sppl2a contributes to B cell and DC survival. Moreover, these data suggest that modulation of Sppl2a may be a useful therapeutic strategy for treatment of B cell dependent autoimmune disorders.

An image-based screen identifies a small molecule regulator of megakaryopoiesis.

Molecules that control the lineage commitment of hematopoietic stem cells (HSCs) may allow the expansion of enriched progenitor populations for both research and therapeutic uses. In an effort to better understand and control the differentiation of HSCs to megakaryocytes, we carried out an image-based screen of a library of 50,000 heterocycles using primary human CD34(+) cells. A class of naphthyridinone derivatives was identified that induces the differentiation of common myeloid progenitors (CMP) to megakaryocytes. Kinase profiling and subsequent functional assays revealed that these compounds act through inhibition of platelet-derived growth factor receptor (PDGFR) signaling in CMPs. Such molecules may ultimately have clinical utility in the treatment of thrombocytopenia.

Euphohelioscopin A is a PKC activator capable of inducing macrophage differentiation.

To identify small molecules that selectively control hematopoietic stem cell differentiation, we performed an unbiased screen using primary human CD34(+) cells. We identified a plant-derived natural product, euphohelioscopin A, capable of selectively differentiating CD34(+) cells down the granulocyte/monocytic lineage. Euphohelioscopin A also inhibits proliferation and induces differentiation of the myeloid leukemia cell lines THP-1 and HL-60. Mechanistic studies revealed that euphohelioscopin A is an activator of protein kinase C (PKC), and that the promonocytic effects of this natural product are mediated by PKC activation. In addition to shedding insights into normal hematopoiesis, this work may ultimately facilitate the application of stem cell therapies to a host of myeloid dysfunctions.

Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling.

Clinical hematopoietic transplantation outcomes are strongly correlated with the numbers of cells infused. Anticipated novel therapeutic implementations of hematopoietic stem cells (HSCs) and their derivatives further increase interest in strategies to expand HSCs ex vivo. A fundamental limitation in all HSC-driven culture systems is the rapid generation of differentiating cells and their secreted inhibitory feedback signals. Herein we describe an integrated computational and experimental strategy that enables a tunable reduction in the global levels and impact of paracrine signaling factors in an automated closed-system process by employing a controlled fed-batch media dilution approach. Application of this system to human cord blood cells yielded a rapid (12-day) 11-fold increase of HSCs with self-renewing, multilineage repopulating ability. These results highlight the marked improvements that control of feedback signaling can offer primary stem cell culture and demonstrate a clinically relevant rapid and relatively low culture volume strategy for ex vivo HSC expansion.

Identification of a high-affinity ligand that exhibits complete aryl hydrocarbon receptor antagonism.

The biological functions of the aryl hydrocarbon receptor (AHR) can be delineated into dioxin response element (DRE)-dependent or -independent activities. Ligands exhibiting either full or partial agonist activity, e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin and α-naphthoflavone, have been demonstrated to potentiate both DRE-dependent and -independent AHR function. In contrast, the recently identified selective AHR modulators (SAhRMs), e.g., 1-allyl-3-(3,4-dimethoxyphenyl)-7-(trifluoromethyl)-1H-indazole (SGA360), bias AHR toward DRE-independent functionality while displaying antagonism with regard to ligand-induced DRE-dependent transcription. Recent studies have expanded the physiological role of AHR to include modulation of hematopoietic progenitor expansion and immunoregulation. It remains to be established whether such physiological roles are mediated through DRE-dependent or -independent pathways. Here, we present evidence for a third class of AHR ligand, "pure" or complete antagonists with the capacity to suppress both DRE-dependent and -independent AHR functions, which may facilitate dissection of physiological AHR function with regard to DRE or non-DRE-mediated signaling. Competitive ligand binding assays together with in silico modeling identify N-(2-(1H-indol-3-yl)ethyl)-9-isopropyl-2-(5-methylpyridin-3-yl)-9H-purin-6-amine (GNF351) as a high-affinity AHR ligand. DRE-dependent reporter assays, in conjunction with quantitative polymerase chain reaction analysis of AHR targets, reveal GNF351 as a potent AHR antagonist that demonstrates efficacy in the nanomolar range. Furthermore, unlike many currently used AHR antagonists, e.g., α-naphthoflavone, GNF351 is devoid of partial agonist potential. It is noteworthy that in a model of AHR-mediated DRE-independent function, i.e., suppression of cytokine-induced acute-phase gene expression, GNF351 has the capacity to antagonize agonist and SAhRM-mediated suppression of SAA1. Such data indicate that GNF351 is a pure antagonist with the capacity to inhibit both DRE-dependent and -independent activity.

Tonic BCR signaling represses receptor editing via Raf- and calcium-dependent signaling pathways.

Light chain receptor editing is an important mechanism that prevents B cell self-reactivity. We have previously shown that tonic signaling through the BCR represses RAG expression at the immature B cell stage, and that initiation of light chain rearrangements occurs in the absence of these tonic signals in an in vitro model of B cell development. To further test our hypothesis we studied the effect of itpkb deficiency (itpkb(-/-) mice) or Raf hyper-activation (Raf-CAAX transgenic mice), two mutations that enhance BCR signaling, on receptor editing in an in vivo model. This model relies on transferring bone marrow from wild-type or mutant mice into mice expressing an anti-kappa light chain transgene. The anti-kappa transgene induces receptor editing of all kappa light chain expressing B cells, leading to a high frequency of lambda light chain expressing B cells. Anti-κ transgenic recipients of bone marrow from itpkb(-/-) or Raf-CAAX mice showed lower levels of editing to λ light chain than did non-transgenic control recipients. These results provide evidence in an in vivo model that enhanced BCR signaling at the immature B cell stage of development suppresses light chain receptor editing.

Aryl hydrocarbon receptor antagonists promote the expansion of human hematopoietic stem cells.

Although practiced clinically for more than 40 years, the use of hematopoietic stem cell (HSC) transplants remains limited by the ability to expand these cells ex vivo. An unbiased screen with primary human HSCs identified a purine derivative, StemRegenin 1 (SR1), that promotes the ex vivo expansion of CD34+ cells. Culture of HSCs with SR1 led to a 50-fold increase in cells expressing CD34 and a 17-fold increase in cells that retain the ability to engraft immunodeficient mice. Mechanistic studies show that SR1 acts by antagonizing the aryl hydrocarbon receptor (AHR). The identification of SR1 and AHR modulation as a means to induce ex vivo HSC expansion should facilitate the clinical use of HSC therapy.

Regulation of immune cell development through soluble inositol-1,3,4,5-tetrakisphosphate.

The membrane lipid phosphatidylinositol-3,4,5-trisphosphate (PtdInsP(3)) regulates membrane receptor signalling in many cells, including immunoreceptor signalling. Here, we review recent data that have indicated essential roles for the soluble PtdInsP(3) analogue inositol-1,3,4,5-tetrakisphosphate (InsP(4)) in T cell, B cell and neutrophil development and function. Decreased InsP(4) production in leukocytes causes immunodeficiency in mice and might contribute to inflammatory vasculitis in Kawasaki disease in humans. InsP(4)-producing kinases could therefore provide attractive drug targets for inflammatory and infectious diseases.