I-Ag7 ß56/57 polymorphisms regulate non-cognate negative selection to CD4+ T cell orchestrators of type 1 diabetes.

Genetic susceptibility to type 1 diabetes is associated with homozygous expression of major histocompatibility complex class II alleles that carry specific beta chain polymorphisms. Why heterozygous expression of these major histocompatibility complex class II alleles does not confer a similar predisposition is unresolved. Using a nonobese diabetic mouse model, here we show that heterozygous expression of the type 1 diabetes-protective allele I-Ag7 ß56P/57D induces negative selection to the I-Ag7-restricted T cell repertoire, including beta-islet-specific CD4+ T cells. Surprisingly, negative selection occurs despite I-Ag7 ß56P/57D having a reduced ability to present beta-islet antigens to CD4+ T cells. Peripheral manifestations of non-cognate negative selection include a near complete loss of beta-islet-specific CXCR6+ CD4+ T cells, an inability to cross-prime islet-specific glucose-6-phosphatase catalytic subunit-related protein and insulin-specific CD8+ T cells and disease arrest at the insulitis stage. These data reveal that negative selection on non-cognate self-antigens in the thymus can promote T cell tolerance and protection from autoimmunity.

Transcriptional and chromatin profiling of human blood innate lymphoid cell subsets sheds light on HIV-1 pathogenesis.

Innate lymphoid cells (ILCs) are a diverse population of cells that include NK cells and contribute to tissue homeostasis and repair, inflammation, and provide protection from infection. The interplay between human blood ILCs, as well as their responses to HIV-1 infection, remains poorly understood. This study used transcriptional and chromatin profiling to explore these questions. Transcriptional profiling and flow cytometry analysis support that there are four main ILC subsets found in human blood. Unlike in mice, human NK cells expressed the tissue repair protein amphiregulin (AREG). AREG production was induced by TCF7/WNT, IL-2, and IL-15, and inhibited by TGFB1, a cytokine increased in people living with HIV-1. In HIV-1 infection, the percentage of AREG+ NK cells correlated positively with the numbers of ILCs and CD4+ T cells but negatively with the concentration of inflammatory cytokine IL-6. NK-cell knockout of the TGFB1-stimulated WNT antagonist RUNX3 increased AREG production. Antiviral gene expression was increased in all ILC subsets from HIV-1 viremic people, and anti-inflammatory gene MYDGF was increased in an NK-cell subset from HIV-1-infected people whose viral load was undetectable in the absence of antiretroviral therapy. The percentage of defective NK cells in people living with HIV-1 correlated inversely with ILC percentage and CD4+ T-cell counts. CD4+ T cells and their production of IL-2 prevented the loss of NK-cell function by activating mTOR. These studies clarify how ILC subsets are interrelated and provide insight into how HIV-1 infection disrupts NK cells, including an uncharacterized homeostatic function in NK cells.

Simultaneous evaluation of treatment efficacy and toxicity for bispecific T-cell engager therapeutics in a humanized mouse model.

Immuno-oncology (IO)-based therapies such as checkpoint inhibitors, bi-specific antibodies, and CAR-T-cell therapies have shown significant success in the treatment of several cancer indications. However, these therapies can result in the development of severe adverse events, including cytokine release syndrome (CRS). Currently, there is a paucity of in vivo models that can evaluate dose-response relationships for both tumor control and CRS-related safety issues. We tested an in vivo PBMC humanized mouse model to assess both treatment efficacy against specific tumors and the concurrent cytokine release profiles for individual human donors after treatment with a CD19xCD3 bispecific T-cell engager (BiTE). Using this model, we evaluated tumor burden, T-cell activation, and cytokine release in response to bispecific T-cell-engaging antibody in humanized mice generated with different PBMC donors. The results show that PBMC engrafted NOD-scid Il2rgnull mice lacking expression of mouse MHC class I and II (NSG-MHC-DKO mice) and implanted with a tumor xenograft predict both efficacy for tumor control by CD19xCD3 BiTE and stimulated cytokine release. Moreover, our findings indicate that this PBMC-engrafted model captures variability among donors for tumor control and cytokine release following treatment. Tumor control and cytokine release were reproducible for the same PBMC donor in separate experiments. The PBMC humanized mouse model described here is a sensitive and reproducible platform that identifies specific patient/cancer/therapy combinations for treatment efficacy and development of complications.

Prostaglandin E2 stimulates cAMP signaling and resensitizes human leukemia cells to glucocorticoid-induced cell death.

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.

Enhanced development of functional human NK cells in NOD-scid-IL2rgnull mice expressing human IL15.

Human innate immunity plays a critical role in tumor surveillance and in immunoregulation within the tumor microenvironment. Natural killer (NK) cells are innate lymphoid cells that have opposing roles in the tumor microenvironment, including NK cell subsets that mediate tumor cell cytotoxicity and subsets with regulatory function that contribute to the tumor immune suppressive environment. The balance between effector and regulatory NK cell subsets has been studied extensively in murine models of cancer, but there is a paucity of models to study human NK cell function in tumorigenesis. Humanized mice are a powerful alternative to syngeneic mouse tumor models for the study of human immuno-oncology and have proven effective tools to test immunotherapies targeting T cells. However, human NK cell development and survival in humanized NOD-scid-IL2rgnull (NSG) mice are severely limited. To enhance NK cell development, we have developed NSG mice that constitutively expresses human Interleukin 15 (IL15), NSG-Tg(Hu-IL15). Following hematopoietic stem cell engraftment of NSG-Tg(Hu-IL15) mice, significantly higher levels of functional human CD56+ NK cells are detectable in blood and spleen, as compared to NSG mice. Hematopoietic stem cell (HSC)-engrafted NSG-Tg(Hu-IL15) mice also supported the development of human CD3+ T cells, CD20+ B cells, and CD33+ myeloid cells. Moreover, the growth kinetics of a patient-derived xenograft (PDX) melanoma were significantly delayed in HSC-engrafted NSG-Tg(Hu-IL15) mice as compared to HSC-engrafted NSG mice demonstrating that human NK cells have a key role in limiting the tumor growth. Together, these data demonstrate that HSC-engrafted NSG-Tg(Hu-IL15) mice support enhanced development of functional human NK cells, which limit the growth of PDX tumors.

An RNAi therapeutic targeting hepatic DGAT2 in a genetically obese mouse model of nonalcoholic steatohepatitis.

Nonalcoholic steatohepatitis (NASH) is a severe liver disorder characterized by triglyceride accumulation, severe inflammation, and fibrosis. With the recent increase in prevalence, NASH is now the leading cause of liver transplant, with no approved therapeutics available. Although the exact molecular mechanism of NASH progression is not well understood, a widely held hypothesis is that fat accumulation is the primary driver of the disease. Therefore, diacylglycerol O-acyltransferase 2 (DGAT2), a key enzyme in triglyceride synthesis, has been explored as a NASH target. RNAi-based therapeutics is revolutionizing the treatment of liver diseases, with recent chemical advances supporting long-term gene silencing with single subcutaneous administration. Here, we identified a hyper-functional, fully chemically stabilized GalNAc-conjugated small interfering RNA (siRNA) targeting DGAT2 (Dgat2-1473) that, upon injection, elicits up to 3 months of DGAT2 silencing (>80%-90%, p < 0.0001) in wild-type and NSG-PiZ "humanized" mice. Using an obesity-driven mouse model of NASH (ob/ob-GAN), Dgat2-1473 administration prevents and reverses triglyceride accumulation (>85%, p < 0.0001) without increased accumulation of diglycerides, resulting in significant improvement of the fatty liver phenotype. However, surprisingly, the reduction in liver fat did not translate into a similar impact on inflammation and fibrosis. Thus, while Dgat2-1473 is a practical, long-lasting silencing agent for potential therapeutic attenuation of liver steatosis, combinatorial targeting of a second pathway may be necessary for therapeutic efficacy against NASH.

Prosurvival kinase PIM2 is a therapeutic target for eradication of chronic myeloid leukemia stem cells.

A major obstacle to curing chronic myeloid leukemia (CML) is the intrinsic resistance of CML stem cells (CMLSCs) to the drug imatinib mesylate (IM). Prosurvival genes that are preferentially expressed in CMLSCs compared with normal hematopoietic stem cells (HSCs) represent potential therapeutic targets for selectively eradicating CMLSCs. However, the discovery of such preferentially expressed genes has been hampered by the inability to completely separate CMLSCs from HSCs, which display a very similar set of surface markers. To overcome this challenge, and to minimize confounding effects of individual differences in gene expression profiles, we performed single-cell RNA-seq on CMLSCs and HSCs that were isolated from the same patient and distinguished based on the presence or absence of BCR-ABL. Among genes preferentially expressed in CMLSCs is PIM2, which encodes a prosurvival serine-threonine kinase that phosphorylates and inhibits the proapoptotic protein BAD. We show that IM resistance of CMLSCs is due, at least in part, to maintenance of BAD phosphorylation by PIM2. We find that in CMLSCs, PIM2 expression is promoted by both a BCR-ABL-dependent (IM-sensitive) STAT5-mediated pathway and a BCR-ABL-independent (IM-resistant) STAT4-mediated pathway. Combined treatment with IM and a PIM inhibitor synergistically increases apoptosis of CMLSCs, suppresses colony formation, and significantly prolongs survival in a mouse CML model, with a negligible effect on HSCs. Our results reveal a therapeutically targetable mechanism of IM resistance in CMLSCs. The experimental approach that we describe can be generally applied to other malignancies that harbor oncogenic fusion proteins or other characteristic genetic markers.

Noninvasive Determination of CMV Serostatus From Dried Buccal Swab Samples: Assay Development, Validation, and Application to 1.2 Million Samples.

BACKGROUND: Buccal swab sampling constitutes an attractive noninvasive alternative to blood drawings for antibody serostatus assays. Here we describe a method to determine the cytomegalovirus immunoglobulin G (CMV IgG) serostatus from dried buccal swab samples. METHODS: Upon solubilization, CMV IgG is determined by an ELISA assay specifically adapted to cope with low IgG concentrations. The derived CMV titer is normalized against the total protein concentration to adjust for incorrectly or less efficiently sampled buccal swabs. Assay parameters were optimized on a set of 713 samples. RESULTS: Validation with 1784 samples revealed distinct results for > 80% of samples with 98.6% specificity and 99.1% sensitivity. Based on the analysis of 1.2 million samples we derived age- and sex-stratified CMV prevalence statistics for Germany, Poland, United Kingdom, and Chile. To confirm accuracy of the assay in routine operation, the CMV status of 6518 donors was reassessed by independent laboratories based on conventional blood samples revealing 96.9% specificity and 97.4% sensitivity. CONCLUSIONS: The assay accurately delivers the CMV IgG serostatus from dried buccal swab samples for > 80% of the participants. Thereby it provides a noninvasive alternative to plasma-based CMV monitoring for nondiagnostic purposes such as hematopoietic stem cell transplantation donor screening or population studies.

A rapid, sensitive, and reproducible in vivo PBMC humanized murine model for determining therapeutic-related cytokine release syndrome.

Immunotherapy is a powerful treatment strategy being applied to cancer, autoimmune diseases, allergies, and transplantation. Although therapeutic monoclonal antibodies (mAbs) have demonstrated significant clinical efficacy, there is also the potential for severe adverse events, including cytokine release syndrome (CRS). CRS is characterized by the rapid production of inflammatory cytokines following delivery of therapy, with symptoms ranging from mild fever to life-threating pathology and multi-organ failure. Overall there is a paucity of models to reliably and accurately predict the induction of CRS by immune therapeutics. Here, we describe the development of a humanized mouse model based on the NOD-scid IL2rgnull (NSG) mouse to study CRS in vivo. PBMC-engrafted NSG, NSG-MHC-DKO, and NSG-SGM3 mice were used to study cytokine release in response to treatment with mAb immunotherapies. Our data show that therapeutic-stimulated cytokine release in these PBMC-based NSG models captures the variation in cytokine release between individual donors, is drug dependent, occurs in the absence of acute xeno-GVHD, highlighting the specificity of the assay, and shows a robust response following treatment with a TGN1412 analog, a CD28 superagonist. Overall our results demonstrate that PBMC-engrafted NSG models are rapid, sensitive, and reproducible platforms to screen novel therapeutics for CRS.

Human Anti-HIV-1 gp120 Monoclonal Antibodies with Neutralizing Activity Cloned from Humanized Mice Infected with HIV-1.

Broadly neutralizing, anti-HIV-1 gp120 mAbs have been isolated from infected individuals, and there is considerable interest in developing these reagents for Ab-based immunoprophylaxis and treatment. As a means to identify potentially new anti-HIV Abs, we exploited humanized NOD-scid IL2rγnull mice systemically infected with HIV-1 to generate a wide variety of Ag-specific human mAbs. The Abs were encoded by a diverse range of variable gene families and Ig classes, including IgA, and several showed significant levels of somatic mutation. Moreover, the isolated Abs not only bound target Ags with similar affinity as broadly neutralizing Abs, they also demonstrated neutralizing ability against multiple HIV-1 clades. The use of humanized mice will allow us to use our knowledge of HIV-1 gp120 structure and function, and the immune response targeting this protein, to generate native human prophylactic Abs to reduce the infection and spread of HIV-1.

Cutting Edge: Early Attrition of Memory T Cells during Inflammation and Costimulation Blockade Is Regulated Concurrently by Proapoptotic Proteins Fas and Bim.

Apoptosis of CD8 T cells is an essential mechanism that maintains immune system homeostasis, prevents autoimmunity, and reduces immunopathology. CD8 T cell death also occurs early during the response to both inflammation and costimulation blockade (CoB). In this article, we studied the effects of a combined deficiency of Fas (extrinsic pathway) and Bim (intrinsic pathway) on early T cell attrition in response to lymphocytic choriomeningitis virus infection and during CoB during transplantation. Loss of Fas and Bim function in Bcl2l11-/-Faslpr/lpr mice inhibited apoptosis of T cells and prevented the early T cell attrition resulting from lymphocytic choriomeningitis virus infection. Bcl2l11-/-Faslpr/lpr mice were also resistant to prolonged allograft survival induced by CoB targeting the CD40-CD154 pathway. These results demonstrate that both extrinsic and intrinsic apoptosis pathways function concurrently to regulate T cell homeostasis during the early stages of immune responses and allograft survival during CoB.

Kaposi Sarcoma-Associated Herpesvirus Glycoprotein H Is Indispensable for Infection of Epithelial, Endothelial, and Fibroblast Cell Types.

Kaposi sarcoma-associated herpesvirus (KSHV) is an emerging pathogen and is the causative infectious agent of Kaposi sarcoma and two malignancies of B cell origin. To date, there is no licensed KSHV vaccine. Development of an effective vaccine against KSHV continues to be limited by a poor understanding of how the virus initiates acute primary infection in vivo in diverse human cell types. The role of glycoprotein H (gH) in herpesvirus entry mechanisms remains largely unresolved. To characterize the requirement for KSHV gH in the viral life cycle and in determination of cell tropism, we generated and characterized a mutant KSHV in which expression of gH was abrogated. Using a bacterial artificial chromosome containing a complete recombinant KSHV genome and recombinant DNA technology, we inserted stop codons into the gH coding region. We used electron microscopy to reveal that the gH-null mutant virus assembled and exited from cells normally, compared to wild-type virus. Using purified virions, we assessed infectivity of the gH-null mutant in diverse mammalian cell types in vitro Unlike wild-type virus or a gH-containing revertant, the gH-null mutant was unable to infect any of the epithelial, endothelial, or fibroblast cell types tested. However, its ability to infect B cells was equivocal and remains to be investigated in vivo due to generally poor infectivity in vitro Together, these results suggest that gH is critical for KSHV infection of highly permissive cell types, including epithelial, endothelial, and fibroblast cells.IMPORTANCE All homologues of herpesvirus gH studied to date have been implicated in playing an essential role in viral infection of diverse permissive cell types. However, the role of gH in the mechanism of KSHV infection remains largely unresolved. In this study, we generated a gH-null mutant KSHV and provided evidence that deficiency of gH expression did not affect viral particle assembly or egress. Using the gH-null mutant, we showed that gH was indispensable for KSHV infection of epithelial, endothelial, and fibroblast cells in vitro This suggests that gH is an important target for the development of a KSHV prophylactic vaccine to prevent initial viral infection.

Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) support preclinical studies of human pathogens, allograft rejection, and human T-cell function. However, a major limitation of PBMC engraftment is development of acute xenogeneic graft- versus-host disease (GVHD) due to human T-cell recognition of murine major histocompatibility complex (MHC). To address this, we created 2 NOD- scid IL-2 receptor subunit γ ( IL2rg) null (NSG) strains that lack murine MHC class I and II [NSG-ß-2-microglobulin ( B2M) null ( IA IE)null and NSG -( Kb Db) null ( IAnull)]. We observed rapid human IgG clearance in NSG- B2Mnull ( IA IE) null mice whereas clearance in NSG -( Kb Db) null ( IAnull) mice and NSG mice was comparable. Injection of human PBMCs into both strains enabled long-term engraftment of human CD4+ and CD8+ T cells without acute GVHD. Engrafted human T-cell function was documented by rejection of human islet allografts. Administration of human IL-2 to NSG -( Kb Db) null ( IAnull) mice via adeno-associated virus vector increased human CD45+ cell engraftment, including an increase in human regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data document that NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD and enable evaluation of human antibody therapeutics targeting human T cells.-Brehm, M. A., Kenney, L. L., Wiles, M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

Humanized mouse models of immunological diseases and precision medicine.

With the increase in knowledge resulting from the sequencing of the human genome, the genetic basis for the underlying differences in individuals, their diseases, and how they respond to therapies is starting to be understood. This has formed the foundation for the era of precision medicine in many human diseases that is beginning to be implemented in the clinic, particularly in cancer. However, preclinical testing of therapeutic approaches based on individual biology will need to be validated in animal models prior to translation into patients. Although animal models, particularly murine models, have provided significant information on the basic biology underlying immune responses in various diseases and the response to therapy, murine and human immune systems differ markedly. These fundamental differences may be the underlying reason why many of the positive therapeutic responses observed in mice have not translated directly into the clinic. There is a critical need for preclinical animal models in which human immune responses can be investigated. For this, many investigators are using humanized mice, i.e., immunodeficient mice engrafted with functional human cells, tissues, and immune systems. We will briefly review the history of humanized mice, the remaining limitations, approaches to overcome them and how humanized mouse models are being used as a preclinical bridge in precision medicine for evaluation of human therapies prior to their implementation in the clinic.

AK002, a Humanized Sialic Acid-Binding Immunoglobulin-Like Lectin-8 Antibody that Induces Antibody-Dependent Cell-Mediated Cytotoxicity against Human Eosinophils and Inhibits Mast Cell-Mediated Anaphylaxis in Mice.

INTRODUCTION: Pathologic accumulation and activation of mast cells and eosinophils are implicated in allergic and inflammatory diseases. Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is an inhibitory receptor selectively expressed on mast cells, eosinophils and, at a lower extent, basophils. When engaged with an antibody, Siglec-8 can induce apoptosis of activated eosinophils and inhibit mast cell activation. AK002 is a humanized, non-fucosylated IgG1 anti-Siglec-8 antibody undergoing clinical investigation for treatment of allergic, inflammatory, and proliferative diseases. Here we examine the human tissue selectivity of AK002 and evaluate the in vitro, ex vivo, and in vivo activity of AK002 on eosinophils and mast cells. METHODS: The affinity of AK002 for Siglec-8 and CD16 was determined by biolayer interferometry. Ex vivo activity of AK002 on human eosinophils from blood and dissociated human tissue was tested in apoptosis and antibody-dependent cell-mediated cytotoxicity (ADCC) assays. The in vivo activity of a murine precursor of AK002 (mAK002) was tested in a passive systemic anaphylaxis (PSA) humanized mouse model. RESULTS: AK002 bound selectively to mast cells, eosinophils and, at a lower level, to basophils in human blood and tissue and not to other cell types examined. AK002 induced apoptosis of interleukin-5-activated blood eosinophils and demonstrated potent ADCC activity against blood eosinophils in the presence of natural killer cells. AK002 also significantly reduced eosinophils in dissociated human lung tissue. Furthermore, mAK002 prevented PSA in humanized mice through mast cell inhibition. CONCLUSION: AK002 selectively evokes potent apoptotic and ADCC activity against eosinophils and prevents systemic anaphylaxis through mast cell inhibition.

Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy.

Establishment of an in vivo small animal model of human tumor and human immune system interaction would enable preclinical investigations into the mechanisms underlying cancer immunotherapy. To this end, nonobese diabetic (NOD).Cg- PrkdcscidIL2rgtm1Wjl/Sz (null; NSG) mice were transplanted with human (h)CD34+ hematopoietic progenitor and stem cells, which leads to the development of human hematopoietic and immune systems [humanized NSG (HuNSG)]. HuNSG mice received human leukocyte antigen partially matched tumor implants from patient-derived xenografts [PDX; non-small cell lung cancer (NSCLC), sarcoma, bladder cancer, and triple-negative breast cancer (TNBC)] or from a TNBC cell line-derived xenograft (CDX). Tumor growth curves were similar in HuNSG compared with nonhuman immune-engrafted NSG mice. Treatment with pembrolizumab, which targets programmed cell death protein 1, produced significant growth inhibition in both CDX and PDX tumors in HuNSG but not in NSG mice. Finally, inhibition of tumor growth was dependent on hCD8+ T cells, as demonstrated by antibody-mediated depletion. Thus, tumor-bearing HuNSG mice may represent an important, new model for preclinical immunotherapy research.-Wang, M., Yao, L.-C., Cheng, M., Cai, D., Martinek, J., Pan, C.-X., Shi, W., Ma, A.-H., De Vere White, R. W., Airhart, S., Liu, E. T., Banchereau, J., Brehm, M. A., Greiner, D. L., Shultz, L. D., Palucka, K., Keck, J. G. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy.

Survival Advantage of Both Human Hepatocyte Xenografts and Genome-Edited Hepatocytes for Treatment of α-1 Antitrypsin Deficiency.

Hepatocytes represent an important target for gene therapy and editing of single-gene disorders. In α-1 antitrypsin (AAT) deficiency, one missense mutation results in impaired secretion of AAT. In most patients, lung damage occurs due to a lack of AAT-mediated protection of lung elastin from neutrophil elastase. In some patients, accumulation of misfolded PiZ mutant AAT protein triggers hepatocyte injury, leading to inflammation and cirrhosis. We hypothesized that correcting the Z mutant defect in hepatocytes would confer a selective advantage for repopulation of hepatocytes within an intact liver. A human PiZ allele was crossed onto an immune-deficient (NSG) strain to create a recipient strain (NSG-PiZ) for human hepatocyte xenotransplantation. Results indicate that NSG-PiZ recipients support heightened engraftment of normal human primary hepatocytes as compared with NSG recipients. This model can therefore be used to test hepatocyte cell therapies for AATD, but more broadly it serves as a simple, highly reproducible liver xenograft model. Finally, a promoterless adeno-associated virus (AAV) vector, expressing a wild-type AAT and a synthetic miRNA to silence the endogenous allele, was integrated into the albumin locus. This gene-editing approach leads to a selective advantage of edited hepatocytes, by silencing the mutant protein and augmenting normal AAT production, and improvement of the liver pathology.

The Presence and Preferential Activation of Regulatory T Cells Diminish Adoptive Transfer of Autoimmune Diabetes by Polyclonal Nonobese Diabetic (NOD) T Cell Effectors into NSG versus NOD-scid Mice.

NOD-scid.Il2rg(null) (NSG) mice are currently being used as recipients to screen for pathogenic autoreactive T cells in type 1 diabetes (T1D) patients. We questioned whether the restriction of IL-2R γ-chain (Il-2rγ)-dependent cytokine signaling only to donor cells in NSG recipients differently influenced the activities of transferred diabetogenic T cells when they were introduced as a monoclonal/oligoclonal population versus being part of a polyclonal repertoire. Unexpectedly, a significantly decreased T1D transfer by splenocytes from prediabetic NOD donors was observed in Il-2rγ(null)-NSG versus Il-2rγ-intact standard NOD-scid recipients. In contrast, NOD-derived monoclonal/oligoclonal TCR transgenic ß cell-autoreactive T cells in either the CD8 (AI4, NY8.3) or CD4 (BDC2.5) compartments transferred disease significantly more rapidly to NSG than to NOD-scid recipients. The reduced diabetes transfer efficiency by polyclonal T cells in NSG recipients was associated with enhanced activation of regulatory T cells (Tregs) mediated by NSG myeloid APC. This enhanced suppressor activity was associated with higher levels of Treg GITR expression in the presence of NSG than NOD-scid APC. These collective results indicate NSG recipients might be efficiently employed to test the activity of T1D patient-derived ß cell-autoreactive T cell clones and lines, but, when screening for pathogenic effectors within polyclonal populations, Tregs should be removed from the transfer inoculum to avoid false-negative results.

Growth factors for angiogenesis in peripheral arterial disease.

BACKGROUND: Peripheral artery disease (PAD) is associated with a high clinical and socioeconomic burden. Treatments to alleviate the symptoms of PAD and decrease the risks of amputation and death are a high societal priority. A number of growth factors have shown a potential to stimulate angiogenesis. Growth factors delivered directly (as recombinant proteins), or indirectly (e.g. by viral vectors or DNA plasmids encoding these factors), have emerged as a promising strategy to treat patients with PAD. OBJECTIVES: To assess the effects of growth factors that promote angiogenesis for treating people with PAD of the lower extremities. SEARCH METHODS: The Cochrane Vascular Information Specialist searched the Specialised Register (June 2016) and CENTRAL (2016, Issue 5). We searched trial registries for details of ongoing or unpublished studies. We also checked the reference lists of relevant publications and, if necessary, tried to contact the trialists for details of the studies. SELECTION CRITERIA: We included randomised controlled trials comparing growth factors (delivered directly or indirectly) with no intervention, placebo or any other intervention not based on the growth factor's action in patients with PAD of the lower extremities. The primary outcomes were limb amputation, death and adverse events. The secondary outcomes comprised walking ability, haemodynamic measures, ulceration and rest pain. DATA COLLECTION AND ANALYSIS: Two review authors independently selected trials and assessed the risk of bias. We used outcomes of the studies at low risk of bias for the main analysis and of all studies in the sensitivity analyses. We calculated odds ratios (OR) for dichotomous outcomes and mean differences for continuous outcomes with 95% confidence intervals (CI). We evaluated statistical heterogeneity using the I2 statistic and Cochrane's Q test. We conducted meta-analysis for the overall effect and for each growth factor as a subgroup analysis using OR in a fixed-effect model. We evaluated the robustness of the results in a sensitivity analysis using risk ratio (RR) and/or a random-effects model. We also assessed the quality of the evidence for each outcome. MAIN RESULTS: We included 20 trials in the review and used 14 studies (on approximately 1400 participants) with published results in the analyses. Six published studies compared fibroblast growth factors (FGF), four studies hepatocyte growth factors (HGF) and another four studies vascular endothelial growth factors (VEGF), versus placebo or no therapy. Six of these studies exclusively or mainly investigated participants with intermittent claudication and eight studies exclusively participants with critical limb ischaemia. Follow-up generally ranged from three months to one year. Two small studies provided some data at 2 years and one of them also at 10 years.The direction and size of effects for growth factors on major limb amputations (OR 0.99, 95% CI 0.71 to 1.38; 10 studies, N = 1075) and death (OR 0.99, 95% CI 0.69 to 1.41; 12 studies, N = 1371) at up to two years are uncertain. The quality of the evidence is low due to risk of bias and imprecision (at one year, moderate-quality evidence due to imprecision). However, growth factors may decrease the rate of any limb amputations (OR 0.56, 95% CI 0.31 to 0.99; 6 studies, N = 415). The quality of the evidence is low due to risk of bias and selective reporting.The direction and size of effects for growth factors on serious adverse events (OR 1.09, 95% CI 0.79 to 1.50; 13 studies, N = 1411) and on any adverse events (OR 1.10, 95% CI 0.73 to 1.64; 4 studies, N = 709) at up to two years are also uncertain. The quality of the evidence is low due to risk of bias and imprecision (for serious adverse events at one year, moderate-quality evidence due to imprecision).Growth factors may improve haemodynamic measures (low-quality evidence), ulceration (very low-quality evidence) and rest pain (very low-quality evidence) up to one year, but they have little or no effect on walking ability (low-quality evidence). We did not identify any relevant differences in effects between growth factors (FGF, HGF and VEGF). AUTHORS' CONCLUSIONS: The results of this review do not support the use of therapy with the growth factors FGF, HGF or VEGF in people with PAD of the lower extremities to prevent death or major limb amputation or to improve walking ability. However, the use of these growth factors may improve haemodynamic measures and decrease the rate of any limb amputations (probably due to preventing minor amputations) with an uncertain effect on adverse events; an improvement of ulceration and rest pain is very uncertain. New trials at low risk of bias are needed to generate evidence with more certainty.