Congenic hematopoietic stem cell transplantation promotes survival of heart allografts in murine models of acute and chronic rejection.

The ability to induce tolerance would be a major advance in the field of solid organ transplantation. Here, we investigated whether autologous (congenic) hematopoietic stem cell transplantation (HSCT) could promote tolerance to heart allografts in mice. In an acute rejection model, fully MHC-mismatched BALB/c hearts were heterotopically transplanted into C57BL/6 (CD45.2) mice. One week later, recipient mice were lethally irradiated and reconstituted with congenic B6 CD45.1 Lin-Sca1+ckit+ cells. Recipient mice received a 14-day course of rapamycin both to prevent rejection and to expand regulatory T cells (Tregs). Heart allografts in both untreated and rapamycin-treated recipients that did not undergo HSCT were rejected within 33 days (median survival time = 8 days for untreated recipients, median survival time = 32 days for rapamycin-treated recipients), whereas allografts in HSCT-treated recipients had a median survival time of 55 days (P < 0.001 vs. both untreated and rapamycin-treated recipients). Enhanced allograft survival following HSCT was associated with increased intragraft Foxp3+ Tregs, reduced intragraft B cells, and reduced serum donor-specific antibodies. In a chronic rejection model, Bm12 hearts were transplanted into C57BL/6 (CD45.2) mice, and congenic HSCT was performed two weeks following heart transplantation. HSCT led to enhanced survival of allografts (median survival time = 70 days vs. median survival time = 28 days in untreated recipients, P < 0.01). Increased allograft survival post-HSCT was associated with prevention of autoantibody development and absence of vasculopathy. These data support the concept that autologous HSCT can promote immune tolerance in the setting of allotransplantation. Further studies to optimize HSCT protocols should be performed before this procedure is adopted clinically.

Treatment with Angiotensin-(1-7) Prevents Development of Oral Papilloma Induced in K-ras Transgenic Mice.

Oral Squamous Cell Carcinoma (OSCC) is the most common malignant cancer affecting the oral cavity. It is characterized by high morbidity and very few therapeutic options. Angiotensin (Ang)-(1-7) is a biologically active heptapeptide, generated predominantly from AngII (Ang-(1-8)) by the enzymatic activity of angiotensin-converting enzyme 2 (ACE 2). Previous studies have shown that Ang-(1-7) counterbalances AngII pro-tumorigenic actions in different pathophysiological settings, exhibiting antiproliferative and anti-angiogenic properties in cancer cells. However, the prevailing effects of Ang-(1-7) in the oral epithelium have not been established in vivo. Here, we used an inducible oral-specific mouse model, where the expression of a tamoxifen-inducible Cre recombinase (CreERtam), which is under the control of the cytokeratin 14 promoter (K14-CreERtam), induces the expression of the K-ras oncogenic variant KrasG12D (LSLK-rasG12D). These mice develop highly proliferative squamous papilloma in the oral cavity and hyperplasia exclusively in oral mucosa within one month after tamoxifen treatment. Ang-(1-7) treated mice showed a reduced papilloma development accompanied by a significant reduction in cell proliferation and a decrease in pS6 positivity, the most downstream target of the PI3K/Akt/mTOR signaling route in oral papilloma. These results suggest that Ang-(1-7) may be a novel therapeutic target for OSCC.

Invariant NKT Cell Activation Is Potentiated by Homotypic trans-Ly108 Interactions.

Invariant NKT (iNKT) cells are innate lymphocytes that respond to glycolipids presented by the MHC class Ib molecule CD1d and are rapidly activated to produce large quantities of cytokines and chemokines. iNKT cell development uniquely depends on interactions between double-positive thymocytes that provide key homotypic interactions between signaling lymphocyte activation molecule (SLAM) family members. However, the role of SLAM receptors in the differentiation of iNKT cell effector subsets and activation has not been explored. In this article, we show that C57BL/6 mice containing the New Zealand Black Slam locus have profound alterations in Ly108, CD150, and Ly9 expression that is associated with iNKT cell hyporesponsiveness. This loss of function was only apparent when dendritic cells and iNKT cells had a loss of SLAM receptor expression. Using small interfering RNA knockdowns and peptide-blocking strategies, we demonstrated that trans-Ly108 interactions between dendritic cells and iNKT cells are critical for robust activation. LY108 costimulation similarly increased human iNKT cell activation. Thus, in addition to its established role in iNKT cell ontogeny, Ly108 regulates iNKT cell function in mice and humans.

Oxidized Low-Density Lipoprotein Loading of Macrophages Downregulates TLR-Induced Proinflammatory Responses in a Gene-Specific and Temporal Manner through Transcriptional Control.

Hypercholesterolemia is a key risk factor for atherosclerosis and leads to the uptake of native and oxidized low-density lipoprotein (oxLDL) by macrophages (Mϕs) and foam cell formation. Inflammatory processes accompany Mϕ foam cell formation in the artery wall, yet the relationship between Mϕ lipid loading and their response to inflammatory stimuli remains elusive. We investigated proinflammatory gene expression in thioglycollate-elicited peritoneal Mϕs, bone marrow-derived Mϕs and dendritic cells, and RAW264.7 cells. Loading with oxLDL did not induce peritoneal Mϕ apoptosis or modulate basal-level expression of proinflammatory genes. Upon stimulation of TLR4, the rapid induction of IFN-ß was inhibited in cells loaded with oxLDL, whereas the induction of other proinflammatory genes by TLR4 (LPS), TLR3 (polyriboinosinic-polyribocytidylic acid), TLR2 (Pam3CSK4), and TLR9 (CpG) remained comparable within the first 2 h. Subsequently, the expression of a subset of proinflammatory genes (e.g., IL-1ß, IL-6, CCL5) was reduced in oxLDL-loaded cells at the level of transcription. This phenomenon was partially dependent on NF erythroid 2-related factor 2 (NRF2) but not on nuclear liver X receptors α and ß (LXRα,ß), peroxisome proliferator-activated receptor-γ (PPARγ), and activating transcription factor 3 (ATF3). LPS-induced NF-κB reporter activity and intracellular signaling by NF-κB and MAPK pathways were comparable in oxLDL-loaded Mϕs, yet the binding of p65/RelA (the prototypic NF-κB family member) was reduced at IL-6 and CCL5 promoters. This study revealed that oxLDL loading of Mϕs negatively regulates transcription at late stages of TLR-induced proinflammatory gene expression and implicates epigenetic mechanisms such as histone deacetylase activity.

Inhibition of the Fibrinogen-Like Protein 2:FcγRIIB/RIII immunosuppressive pathway enhances antiviral T-cell and B-cell responses leading to clearance of lymphocytic choriomeningitis virus clone 13.

Persistent viruses evade immune detection by interfering with virus-specific innate and adaptive antiviral immune responses. Fibrinogen-like protein-2 (FGL2) is a potent effector molecule of CD4+  CD25+  FoxP3+ regulatory T cells and exerts its immunosuppressive activity following ligation to its cognate receptor, FcγRIIB/RIII. The role of FGL2 in the pathogenesis of chronic viral infection caused by lymphocytic choriomeningitis virus clone-13 (LCMV cl-13) was assessed in this study. Chronically infected fgl2+/+ mice had increased plasma levels of FGL2, with reduced expression of the maturation markers, CD80, CD86 and MHC-II on macrophages and dendritic cells and impaired production of neutralizing antibody. In contrast, fgl2-/- mice or fgl2+/+ mice that had been pre-treated with antibodies to FGL2 and FcγRIIB/RIII and then infected with LCMV cl-13 developed a robust CD4+ and CD8+ antiviral T-cell response, produced high titred neutralizing antibody to LCMV and cleared LCMV. Treatment of mice with established chronic infection with antibodies to FGL2 and FcγRIIB/RIII was shown to rescue the number and functionality of virus-specific CD4+ and CD8+ T cells with reduced total and virus-specific T-cell expression of programmed cell death protein 1 leading to viral clearance. These results demonstrate an important role for FGL2 in viral immune evasion and provide a rationale to target FGL2 to treat patients with chronic viral infection.

Elevated Levels of Interferon-α Act Directly on B Cells to Breach Multiple Tolerance Mechanisms Promoting Autoantibody Production.

OBJECTIVE: Elevated levels of serum interferon-α (IFNα) and the disruption of B cell tolerance are central to systemic lupus erythematosus (SLE) immunopathogenesis; however, the relationship between these 2 processes remains unclear. The purpose of this study was to investigate the impact of elevated IFNα levels on B cell tolerance mechanisms in vivo and determine whether any changes observed were due to the direct effect of IFNα on B cells. METHODS: Two classical mouse models of B cell tolerance were used in conjunction with an adenoviral vector encoding IFNα to mimic the sustained elevations of IFNα seen in SLE. The role of B cell IFNα signaling, T cells, and Myd88 signaling was determined using B cell-specific IFNα receptor-knockout, CD4+ T cell-depleted, or Myd88-knockout mice, respectively. Flow cytometry, enzyme-linked immunosorbent assay, real-time quantitative polymerase chain reaction, and cell cultures were used to study the effects of elevated IFNα on the immunologic phenotype. RESULTS: Elevation of serum IFNα disrupts multiple B cell tolerance mechanisms and leads to autoantibody production. This disruption was dependent upon B cell expression of IFNα receptor. Many of the IFNα-mediated alterations also required the presence of CD4+ T cells as well as Myd88, suggesting that IFNα acts directly on B cells to modify their response to Myd88 signaling and their ability to interact with T cells. CONCLUSION: The results provide evidence that elevated IFNα levels act directly on B cells to facilitate autoantibody production and further highlight the importance of IFN signaling as a potential therapeutic target in SLE.

Immersive Virtual Reality and Cadaveric Bone are Equally Effective in Skeletal Anatomy Education: A Randomized Crossover Noninferiority Trial.

OBJECTIVE: Immersive virtual reality (IVR) technology is transforming medical education. Our aim was to compare the effectiveness of IVR with cadaveric bone models in teaching skeletal anatomy. DESIGN: A randomized crossover noninferiority trial was conducted. SETTING: Anatomy laboratory of a large medical school. PARTICIPANTS: Incoming first-year medical students. Participants were randomized to IVR or cadaveric groups studying upper limb skeletal anatomy, and then were crossed over to use the opposite tool, to study lower limb skeletal anatomy. Participants in both groups completed a pre-and postintervention knowledge test. The primary endpoint of the study was change in performance from the pre-to postintervention knowledge test. Surveys were completed to assess participant's impressions on IVR as an educational tool. RESULTS: Fifty first-year medical students met inclusion criteria and were randomized. Among all students, the average score on the preintervention knowledge test was 14.6% (standard deviation (SD) = 18.2%) and 25.0% (SD = 17%) for upper and lower limbs, respectively. Percentage increase in scores between pre-and postintervention knowledge test, was 15.0% in the upper limb IVR group, and 16.7% for upper limb cadaveric bones (p = 0.286). For the lower limb, score increase was 22.6% in the IVR and 22.5% in the cadaveric bone group (p = 0.936). 79% of participants found that IVR was most valuable for teaching 3-dimensional orientation, anatomical relationships, and key landmarks. Majority of participants were favorable towards combination use of traditional methods and IVR technology for learning skeletal anatomy (LSM>3). CONCLUSIONS: In this randomized controlled trial, there was no significant difference in knowledge after using IVR or cadaveric bones for skeletal anatomy education. These findings have further implications for medical schools that face challenges in acquiring human cadavers and cadaveric parts.

Quantitative analysis of KLF4 and SOX2 expression in oral carcinomas reveals independent association with oral tongue subsite location and histological grade.

BACKGROUND: Stemness factors associated with tumorigenesis in different types of cancers have not been specifically studied in oral tongue SCC (OTSSC). Here, we aimed to quantify expression levels and distribution of KLF4 and SOX2, two relevant stemness factors, in oral SCC including OTSCC samples from different subsites. METHODS AND RESULTS: We determined KLF4 and SOX2 expression levels by immunostaining 35 biopsies of OSCC. Stained wholeslide images were digitized and subjected to automatic cell detection and unbiased quantification using Qupath software. We found statistically significant reduction in KLF4 positive cells density (p= 0.024), and fraction (p= 0.022) in OTSCC from tongue borders compared with other tongue subsites. Instead, quantitative SOX2 analysis did not show differences in expression levels between OTSCC from the borders versus OTSCC developed in others subsites. Notably SOX2 expression was revealed increased in moderately and poorly differentiated OSCC compared with well differentiated ones (positive cells density p= 0.025, fraction p= 0.006). No significant correlation between KLF4 and SOX2 expression was observed, neither in OSCC nor in OTSCC. CONCLUSIONS: KLF4 and SOX2 exhibit opposite expression profiles regarding subsite localization and differentiation level in OSCC. Our study prompts future OTSCC prospective studies looking for clinical prognosis to incorporate detailed subsite information in the analysis.

Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins.

Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic genome engineering applications, but extension to primary human B cells remains limited. Here we report a method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In summary, we demonstrate a powerful and scalable research tool for functional genetic studies of human B cell biology that may have further applications in engineered B cell therapeutics.

The presence of anti-nuclear antibodies alone is associated with changes in B cell activation and T follicular helper cells similar to those in systemic autoimmune rheumatic disease.

BACKGROUND: Diagnosis of systemic autoimmune rheumatic diseases (SARD) relies on the presence of hallmark anti-nuclear antibodies (ANA), many of which can be detected years before clinical manifestations. However, ANAs are also seen in healthy individuals, most of whom will not develop SARD. Here, we examined a unique cohort of asymptomatic ANA+ individuals to determine whether they share any of the cellular immunologic features seen in SARD. METHODS: Healthy ANA- controls and ANA+ (ANA ≥1:160 by immunofluorescence) participants with no SARD criteria, with at least one criterion (undifferentiated connective tissue disease (UCTD)), or meeting SARD classification criteria were recruited. Peripheral blood cellular immunological changes were assessed by flow cytometry and transcript levels of BAFF, interferon (IFN)-induced and plasma cell-expressed genes were quantified by NanoString. RESULTS: A number of the immunologic abnormalities seen in SARD, including changes in peripheral B (switched memory) and T (iNKT, T regulatory, activated memory T follicular helper) subsets and B cell activation, were also seen in asymptomatic ANA+ subjects and those with UCTD. The extent of these immunologic changes correlated with ANA titer or the number of different specific ANAs produced. Principal component analysis of the cellular data indicated that a significant proportion of asymptomatic ANA+ subjects and subjects with UCTD clustered  with patients with early SARD, rather than ANA- healthy controls. CONCLUSIONS: ANA production is associated with altered T and B cell activation even in asymptomatic individuals. Some of the currently accepted cellular features of SARD may be associated with ANA production rather than the immunologic events that cause symptoms in SARD.