Elevated memory T-cell conversion in a preclinical mouse model of hemophilia A.

One of the major challenges in the choice of the best therapeutic approach for the treatment of patients affected by hemophilia A (HA) is the definition of criteria predicting the formation of factor VIII (FVIII) neutralizing antibodies, called inhibitors. Both genetic and environmental elements influencing the immune response toward FVIII have been identified but still not all the factors causing the pathological rejection of FVIII have been identified. Since there is a connection between coagulation and inflammation, here we assessed the role played by the FVIII deficiency in shaping the humoral and cellular response toward an antigen other than FVIII itself. To this aim, we challenged both HA and wild-type (WT) mice with either FVIII or ovalbumin (OVA) and followed antigen-specific antibody level, immune cell population frequency and phenotype up to 9 weeks after the last antigen booster. The activation threshold was evaluated in vitro by stimulating the murine T cells with a decreasing dose of α-CD3. The humoral response to FVIII was similar between the two groups while both the in vivo and in vitro experiments highlighted an antigen-independent sensitivity of HA compared with WT T cells causing an increase in memory T-cell conversion and proliferation capability.

Dissection of pleiotropic effects of variants in and adjacent to F8 exon 19 and rescue of mRNA splicing and protein function.

The pathogenic significance of nucleotide variants commonly relies on nucleotide position within the gene, with exonic changes generally attributed to quantitative or qualitative alteration of protein biosynthesis, secretion, activity, or clearance. However, these changes may exert pleiotropic effects on both protein biology and mRNA splicing due to the overlapping of the amino acid and splicing codes, thus shaping the disease phenotypes. Here, we focused on hemophilia A, in which the definition of F8 variants' causative role and association to bleeding phenotypes is crucial for proper classification, genetic counseling, and management of affected individuals. We extensively characterized a large panel of hemophilia A-causing variants (n = 30) within F8 exon 19 by combining and comparing in silico and recombinant expression analyses. We identified exonic variants with pleiotropic effects and dissected the altered protein features of all missense changes. Importantly, results from multiple prediction algorithms provided qualitative results, while recombinant assays allowed us to correctly infer the likely phenotype severity for 90% of variants. Molecular characterization of pathogenic variants was also instrumental for the development of tailored correction approaches to rescue splicing affecting variants or missense changes impairing protein folding. A single engineered U1snRNA rescued mRNA splicing of nine different variants and the use of a chaperone-like drug resulted in improved factor VIII protein secretion for four missense variants. Overall, dissection of the molecular mechanisms of a large panel of HA variants allowed precise classification of HA-affected individuals and favored the development of personalized therapeutic approaches.

Hemostasis and endothelial functionality: the double face of coagulation factors.

Hemostasis is a sophisticated sequence of events aimed at repairing vessel injury. This process occurs in combination with angiogenesis, which leads to new blood vessel formation, helping in wound repair and facilitating tissue healing. The fine mechanisms that regulate hemostasis and angiogenesis are well described, but for a long time, coagulation factors (CF) have been considered merely players in the coagulation cascade. However, evidence from several experiments highlights the crucial functions of these CF in regulating endothelial functionality, especially in the angiogenic process. Some of these CF (e.g., thrombin and tissue factor) have been widely investigated and have been described as triggering intracellular signaling related to endothelial cell (EC) functionality. For others (e.g., factor VIII and thrombomodulin), potential receptors and molecular mechanisms have not been fully elucidated but some data show their potential to induce EC response. This review focuses on the emerging roles of selected CF in regulating EC functions, highlighting in particular their ability to activate signaling pathways involved in angiogenesis, migration, proliferation and endothelial barrier stability.

Factor VIII promotes angiogenesis and vessel stability regulating extracellular matrix proteins.

Not available.

Therapeutic potential of fetal liver cell transplantation in hemophilia A mice.

Hemophilia A (HA) cell therapy approaches in pediatric individuals require suitable factor (F)VIII-producing cells for stable engraftment. Liver sinusoidal endothelial cells (LSEC) and hematopoietic stem cells (HSC) have been demonstrated to be suitable for the treatment of adult HA mice. However, after transplantation in busulfan (BU)-conditioned newborn mice, adult LSEC/HSC cannot efficiently engraft, while murine fetal liver (FL) hemato/vascular cells from embryonic day 11-13 of gestation (E11-E13), strongly engraft the hematopoietic and endothelial compartments while also secreting FVIII. Our aim was to investigate the engraftment of FL cells in newborn HA mice to obtain a suitable "proof of concept" for the development of a new HA treatment in neonates. Hence, we transplanted FL E11 or E13 cells and adult bone marrow (BM) cells into newborn HA mice with or without BU preconditioning. Engraftment levels and FVIII activity were assessed starting from 6 weeks after transplantation. FL E11-E13+ BU transplanted newborns reached up to 95% engraftment with stable FVIII activity levels observed for 16 months. FL E13 cells showed engraftment ability even in the absence of BU preconditioning, while FL E11 cells did not. BM BU transplanted newborn HA mice showed high levels of engraftment; nevertheless, in contrast to FL cells, BM cells cannot engraft HA newborns in BU non-conditioning regimen. Finally, none of the transplanted mice developed anti-FVIII antibodies. Overall, this study sheds some light on the therapeutic potential of healthy FL cells in the cure of HA neonatal/pediatric patients.

Gut Microbiota Association with Diverticular Disease Pathogenesis and Progression: A Systematic Review.

INTRODUCTION: Growing evidence supports the role of the intestinal microbiome in the development of different intestinal and extraintestinal diseases. Diverticular disease (DD) is one of the most common disorders in western countries. In the last years, different articles have suggested a possible role of the intestinal microbiome in DD pathogenesis and in the development of acute diverticulitis (AD). This systematic review aimed to clarify the current knowledge on the role of the intestinal microbiome in colonic diverticulitis in different stages according to the 2009 PRISMA guidelines. MATERIALS AND METHODS: Two independent reviewers searched the literature in a systematic manner through online databases, including Medline, Scopus, Embase, Cochrane Oral Health Group Specialized Register, ProQuest Dissertations and Theses Database, and Google Scholar. Patients with any stage of disease were included. The Newcastle-Ottawa scale for case-control and cohort studies was used for the quality assessment of the selected articles. RESULTS: Overall, nine studies were included in the review. Only one article was focused on patients with AD, while all other articles only considered patients with DD without acute inflammation signs. Enterobacteriaceae seems to be the microbiota most associated with the disease, followed by Bifidobacteria. CONCLUSIONS: All the included studies showed great heterogeneity in population characteristics and sampling methods. Therefore, given the high prevalence of colonic diverticulitis in the general population, further studies are needed to clarify the role of the intestinal microbiome, paving the way to new target therapies with important social implications.

Cardiac Differentiation Promotes Focal Adhesions Assembly through Vinculin Recruitment.

Cells of the cardiovascular system are physiologically exposed to a variety of mechanical forces fundamental for both cardiac development and functions. In this context, forces generated by actomyosin networks and those transmitted through focal adhesion (FA) complexes represent the key regulators of cellular behaviors in terms of cytoskeleton dynamism, cell adhesion, migration, differentiation, and tissue organization. In this study, we investigated the involvement of FAs on cardiomyocyte differentiation. In particular, vinculin and focal adhesion kinase (FAK) family, which are known to be involved in cardiac differentiation, were studied. Results revealed that differentiation conditions induce an upregulation of both FAK-Tyr397 and vinculin, resulting also in the translocation to the cell membrane. Moreover, the role of mechanical stress in contractile phenotype expression was investigated by applying a uniaxial mechanical stretching (5% substrate deformation, 1 Hz frequency). Morphological evaluation revealed that the cell shape showed a spindle shape and reoriented following the stretching direction. Substrate deformation resulted also in modification of the length and the number of vinculin-positive FAs. We can, therefore, suggest that mechanotransductive pathways, activated through FAs, are highly involved in cardiomyocyte differentiation, thus confirming their role during cytoskeleton rearrangement and cardiac myofilament maturation.

Deciphering the Ets-1/2-mediated transcriptional regulation of F8 gene identifies a minimal F8 promoter for hemophilia A gene therapy.

A major challenge in the development of a gene therapy for hemophilia A (HA) is the selection of cell type- or tissue-specific promoters to ensure factor VIII (FVIII) expression without eliciting an immune response. As liver sinusoidal endothelial cells (LSECs) are the major FVIII source, understanding the transcriptional F8 regulation in these cells would help optimize the minimal F8 promoter (pF8) to efficiently drive FVIII expression. In silico analyses predicted several binding sites (BS) for the E26 transformation-specific (Ets) transcription factors Ets-1 and Ets-2 in the pF8. Reporter assays demonstrated a significant up-regulation of pF8 activity by Ets-1 or Ets-1/Est-2 combination, while Ets2 alone was ineffective. Moreover, Ets-1/Ets-2-DNA binding domain mutants (DBD) abolished promoter activation only when the Ets-1 DBD was removed, suggesting that pF8 up-regulation may occur through Ets-1/Ets-2 interaction with Ets-1 bound to DNA. pF8 carrying Ets-BS deletions unveiled two Ets-BS essential for pF8 activity and response to Ets overexpression. Lentivirus-mediated delivery of GFP or FVIII cassettes driven by the shortened promoters led to GFP expression mainly in endothelial cells in the liver and to long-term FVIII activity without inhibitor formation in HA mice. These data strongly support the potential application of these promoters in HA gene therapy.

Bio-Engineering of Pre-Vascularized Islet Organoids for the Treatment of Type 1 Diabetes.

Lack of rapid revascularization and inflammatory attacks at the site of transplantation contribute to impaired islet engraftment and suboptimal metabolic control after clinical islet transplantation. In order to overcome these limitations and enhance engraftment and revascularization, we have generated and transplanted pre-vascularized insulin-secreting organoids composed of rat islet cells, human amniotic epithelial cells (hAECs), and human umbilical vein endothelial cells (HUVECs). Our study demonstrates that pre-vascularized islet organoids exhibit enhanced in vitro function compared to native islets, and, most importantly, better engraftment and improved vascularization in vivo in a murine model. This is mainly due to cross-talk between hAECs, HUVECs and islet cells, mediated by the upregulation of genes promoting angiogenesis (vegf-a) and ß cell function (glp-1r, pdx1). The possibility of adding a selected source of endothelial cells for the neo-vascularization of insulin-scereting grafts may also allow implementation of ß cell replacement therapies in more favourable transplantation sites than the liver.

Thyroid hormone inhibits hepatocellular carcinoma progression via induction of differentiation and metabolic reprogramming.

BACKGROUND & AIMS: Only limited therapeutic options are currently available for hepatocellular carcinoma (HCC), making the development of effective alternatives essential. Based on the recent finding that systemic or local hypothyroidism is associated with HCC development in humans and rodents, we investigated whether the thyroid hormone triiodothyronine (T3) could inhibit the progression of HCCs. METHODS: Different rat and mouse models of hepatocarcinogenesis were investigated. The effect of T3 on tumorigenesis and metabolism/differentiation was evaluated by transcriptomic analysis, quantitative reverse transcription PCR, immunohistochemistry, and enzymatic assay. RESULTS: A short treatment with T3 caused a shift in the global expression profile of the most aggressive preneoplastic nodules towards that of normal liver. This genomic reprogramming preceded the disappearance of nodules and involved reprogramming of metabolic genes, as well as pro-differentiating transcription factors, including Kruppel-like factor 9, a target of the thyroid hormone receptor ß (TRß). Treatment of HCC-bearing rats with T3 strongly reduced the number and burden of HCCs. Reactivation of a local T3/TRß axis, a switch from Warburg to oxidative metabolism and loss of markers of poorly differentiated hepatocytes accompanied the reduced burden of HCC. This effect persisted 1 month after T3 withdrawal, suggesting a long-lasting effect of the hormone. The antitumorigenic effect of T3 was further supported by its inhibitory activity on cell growth and the tumorigenic ability of human HCC cell lines. CONCLUSIONS: Collectively, these findings suggest that reactivation of the T3/TRß axis induces differentiation of neoplastic cells towards a more benign phenotype and that T3 or its analogs, particularly agonists of TRß, could be useful tools in HCC therapy. LAY SUMMARY: Hepatocellular carcinoma (HCC) represents an important challenge for global health. Recent findings showed that systemic or local hypothyroidism is associated with HCC development. In rat models, we showed that administration of the thyroid hormone T3 impaired HCC progression, even when given at late stages. This is relevant from a translational point of view as HCC is often diagnosed at an advanced stage when it is no longer amenable to curative treatments. Thyroid hormones and/or thyromimetics could be useful for the treatment of patients with HCC.

Biological and clinical implications of BIRC3 mutations in chronic lymphocytic leukemia.

BIRC3 is a recurrently mutated gene in chronic lymphocytic leukemia (CLL) but the functional implications of BIRC3 mutations are largely unexplored. Furthermore, little is known about the prognostic impact of BIRC3 mutations in CLL cohorts homogeneously treated with first-line fludarabine, cyclophosphamide, and rituximab (FCR). By immunoblotting analysis, we showed that the non-canonical nuclear factor-κB pathway is active in BIRC3-mutated cell lines and in primary CLL samples, as documented by the stabilization of MAP3K14 and by the nuclear localization of p52. In addition, BIRC3-mutated primary CLL cells are less sensitive to flu-darabine. In order to confirm in patients that BIRC3 mutations confer resistance to fludarabine-based chemoimmunotherapy, a retrospective multicenter cohort of 287 untreated patients receiving first-line FCR was analyzed by targeted next-generation sequencing of 24 recurrently mutated genes in CLL. By univariate analysis adjusted for multiple comparisons BIRC3 mutations identify a poor prognostic subgroup of patients in whom FCR treatment fails (median progression-free survival: 2.2 years, P<0.001) similar to cases harboring TP53 mutations (median progression-free survival: 2.6 years, P<0.0001). BIRC3 mutations maintained an independent association with an increased risk of progression with a hazard ratio of 2.8 (95% confidence interval 1.4-5.6, P=0.004) in multivariate analysis adjusted for TP53 mutation, 17p deletion and IGHV mutation status. If validated, BIRC3 mutations may be used as a new molecular predictor to select high-risk patients for novel frontline therapeutic approaches.

Multiparameter flow cytometric detection and quantification of senescent cells in vitro.

It has been over half a century since cellular senescence was first noted and characterized, and yet no consensus senescent marker has been reliably established. This challenge is compounded by the complexity and heterogenic phenotypes of senescent cells. This necessitates the use of multiple biomarkers to confidently characterise senescent cells. Despite cytochemical staining of senescence associated-beta-galactosidase being a single marker approach, as well as being time and labour-intensive, it remains the most popular detection method. We have developed an alternative flow cytometry-based method that simultaneously quantifies multiple senescence markers at a single-cell resolution. In this study, we applied this assay to the quantification of both replicative and induced senescent primary cells. Using this assay, we were able to quantify the activity level of SA ß-galactosidase, the expression level of p16INK4a and γH2AX in these cell populations. Our results show this flow cytometric approach to be sensitive, robust, and consistent in discriminating senescent cells in different cell senescence models. A strong positive correlation between these commonly- used senescence markers was demonstrated. The method described in this paper can easily be scaled up to accommodate high-throughput screening of senescent cells in applications such as therapeutic cell preparation, and in therapy-induced senescence following cancer treatment.

iPSC-Derived Liver Organoids: A Journey from Drug Screening, to Disease Modeling, Arriving to Regenerative Medicine.

Liver transplantation is the most common treatment for patients suffering from liver failure that is caused by congenital diseases, infectious agents, and environmental factors. Despite a high rate of patient survival following transplantation, organ availability remains the key limiting factor. As such, research has focused on the transplantation of different cell types that are capable of repopulating and restoring liver function. The best cellular mix capable of engrafting and proliferating over the long-term, as well as the optimal immunosuppression regimens, remain to be clearly well-defined. Hence, alternative strategies in the field of regenerative medicine have been explored. Since the discovery of induced pluripotent stem cells (iPSC) that have the potential of differentiating into a broad spectrum of cell types, many studies have reported the achievement of iPSCs differentiation into liver cells, such as hepatocytes, cholangiocytes, endothelial cells, and Kupffer cells. In parallel, an increasing interest in the study of self-assemble or matrix-guided three-dimensional (3D) organoids have paved the way for functional bioartificial livers. In this review, we will focus on the recent breakthroughs in the development of iPSCs-based liver organoids and the major drawbacks and challenges that need to be overcome for the development of future applications.

Decreasing TfR1 expression reverses anemia and hepcidin suppression in ß-thalassemic mice.

Iron availability for erythropoiesis and its dysregulation in ß-thalassemia are incompletely understood. We previously demonstrated that exogenous apotransferrin leads to more effective erythropoiesis, decreasing erythroferrone (ERFE) and derepressing hepcidin in ß-thalassemic mice. Transferrin-bound iron binding to transferrin receptor 1 (TfR1) is essential for cellular iron delivery during erythropoiesis. We hypothesize that apotransferrin's effect is mediated via decreased TfR1 expression and evaluate TfR1 expression in ß-thalassemic mice in vivo and in vitro with and without added apotransferrin. Our findings demonstrate that ß-thalassemic erythroid precursors overexpress TfR1, an effect that can be reversed by the administration of exogenous apotransferrin. In vitro experiments demonstrate that apotransferrin inhibits TfR1 expression independent of erythropoietin- and iron-related signaling, decreases TfR1 partitioning to reticulocytes during enucleation, and enhances enucleation of defective ß-thalassemic erythroid precursors. These findings strongly suggest that overexpressed TfR1 may play a regulatory role contributing to iron overload and anemia in ß-thalassemic mice. To evaluate further, we crossed TfR1+/- mice, themselves exhibiting iron-restricted erythropoiesis with increased hepcidin, with ß-thalassemic mice. Resultant double-heterozygote mice demonstrate long-term improvement in ineffective erythropoiesis, hepcidin derepression, and increased erythroid enucleation in relation to ß-thalassemic mice. Our data demonstrate for the first time that TfR1+/- haploinsufficiency reverses iron overload specifically in ß-thalassemic erythroid precursors. Taken together, decreasing TfR1 expression during ß-thalassemic erythropoiesis, either directly via induced haploinsufficiency or via exogenous apotransferrin, decreases ineffective erythropoiesis and provides an endogenous mechanism to upregulate hepcidin, leading to sustained iron-restricted erythropoiesis and preventing systemic iron overload in ß-thalassemic mice.

A functional assay for the clinical annotation of genetic variants of uncertain significance in Diamond-Blackfan anemia.

Diamond-Blackfan anemia (DBA) is a rare genetic hypoplasia of erythroid progenitors characterized by mild to severe anemia and associated with congenital malformations. Clinical manifestations in DBA patients are quite variable and genetic testing has become a critical factor in establishing a diagnosis of DBA. The majority of DBA cases are due to heterozygous loss-of-function mutations in ribosomal protein (RP) genes. Causative mutations are fairly straightforward to identify in the case of large deletions and frameshift and nonsense mutations found early in a protein coding sequence, but diagnosis becomes more challenging in the case of missense mutations and small in-frame indels. Our group recently characterized the phenotype of lymphoblastoid cell lines established from DBA patients with pathogenic lesions in RPS19 and observed that defective pre-rRNA processing, a hallmark of the disease, was rescued by lentiviral vectors expressing wild-type RPS19. Here, we use this complementation assay to determine whether RPS19 variants of unknown significance are capable of rescuing pre-rRNA processing defects in these lymphoblastoid cells as a means of assessing the effects of these sequence changes on the function of the RPS19 protein. This approach will be useful in differentiating pathogenic mutations from benign polymorphisms in identifying causative genes in DBA patients.

A humanized mouse model of liver fibrosis following expansion of transplanted hepatic stellate cells.

Hepatic stellate cells (HSCs) are major contributors to liver fibrosis, as hepatic injuries may cause their transdifferentiation into myofibroblast-like cells capable of producing excessive extracellular matrix proteins. Also, HSCs can modulate engraftment of transplanted hepatocytes and contribute to liver regeneration. Therefore, understanding the biology of human HSCs (hHSCs) is important, but effective methods have not been available to address their fate in vivo. To investigate whether HSCs could engraft and repopulate the liver, we transplanted GFP-transduced immortalized hHSCs into immunodeficient NOD/SCID mice. Biodistribution analysis with radiolabeled hHSCs showed that after intrasplenic injection, the majority of transplanted cells rapidly translocated to the liver. GFP-immunohistochemistry demonstrated that transplanted hHSCs engrafted alongside hepatic sinusoids. Prior permeabilization of the sinusoidal endothelial layer with monocrotaline enhanced engraftment of hHSCs. Transplanted hHSCs remained engrafted without relevant proliferation in the healthy liver. However, after CCl4 or bile duct ligation-induced liver damage, transplanted hHSCs expanded and contributed to extracellular matrix production, formation of bridging cell-septae and cirrhosis-like hepatic pseudolobules. CCl4-induced injury recruited hHSCs mainly to zone 3, whereas after bile duct ligation, hHSCs were mainly in zone 1 of the liver lobule. Transplanted hHSCs neither transdifferentiated into other cell types nor formed tumors in these settings. In conclusion, a humanized mouse model was generated by transplanting hHSCs, which proliferated during hepatic injury and inflammation, and contributed to liver fibrosis. The ability to repopulate the liver with transplanted hHSCs will be particularly significant for mechanistic studies of cell-cell interactions and fibrogenesis within the liver.

In Atp7b-/- Mice Modeling Wilson's Disease Liver Repopulation With Bone Marrow-Derived Myofibroblasts or Inflammatory Cells and Not Hepatocytes Is Deleterious.

In Wilson's disease, Atp7b mutations impair copper excretion with liver or brain damage. Healthy transplanted hepatocytes repopulate the liver, excrete copper, and reverse hepatic damage in animal models of Wilson's disease. In Fah-/- mice with tyrosinemia and α-1 antitrypsin mutant mice, liver disease is resolved by expansions of healthy hepatocytes derived from transplanted healthy bone marrow stem cells. This potential of stem cells has not been defined for Wilson's disease. In diseased Atp7b-/- mice, we reconstituted bone marrow with donor cells expressing green fluorescent protein reporter from healthy transgenic mice. Mature hepatocytes originating from donor bone marrow were identified by immunostaining for green fluorescence protein and bile canalicular marker, dipeptidylpeptidase-4. Mesenchymal and inflammatory cell markers were used for other cells from donor bone marrow cells. Gene expression, liver tests, and tissues were analyzed for outcomes in Atp7b-/- mice. After bone marrow transplantation in Atp7b-/- mice, donor-derived hepatocytes containing bile canaliculi appeared within weeks. Despite this maturity, donor-derived hepatocytes neither divided nor expanded. The liver of Atp7b-/- mice was not repopulated by donor-derived hepatocytes: Atp7b mRNA remained undetectable; liver tests, copper content, and fibrosis actually worsened. Restriction of proliferation in hepatocytes accompanied oxidative DNA damage. By contrast, donor-derived mesenchymal and inflammatory cells extensively proliferated. These contributed to fibrogenesis through greater expression of inflammatory cytokines. In Wilson's disease, donor bone marrow-derived cells underwent different fates: hepatocytes failed to proliferate; inflammatory cells proliferated to worsen disease outcomes. This will help guide stem cell therapies for conditions with proinflammatory or profibrogenic microenvironments.

A Novel Platform for Immune Tolerance Induction in Hemophilia A Mice.

Hemophilia A (HA) is an X-linked bleeding disease caused by factor VIII (FVIII) deficiency. We previously demonstrated that FVIII is produced specifically in liver sinusoid endothelial cells (LSECs) and to some degree in myeloid cells, and thus, in the present work, we seek to restrict the expression of FVIII transgene to these cells using cell-specific promoters. With this approach, we aim to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. To increase the target specificity of FVIII expression, we included miRNA target sequences (miRTs) (i.e., miRT-142.3p, miRT-126, and miRT-122) to silence expression in hematopoietic cells, endothelial cells, and hepatocytes, respectively. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory T cell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA.

The Dendritic Cell Major Histocompatibility Complex II (MHC II) Peptidome Derives from a Variety of Processing Pathways and Includes Peptides with a Broad Spectrum of HLA-DM Sensitivity.

The repertoire of peptides displayed in vivo by MHC II molecules derives from a wide spectrum of proteins produced by different cell types. Although intracellular endosomal processing in dendritic cells and B cells has been characterized for a few antigens, the overall range of processing pathways responsible for generating the MHC II peptidome are currently unclear. To determine the contribution of non-endosomal processing pathways, we eluted and sequenced over 3000 HLA-DR1-bound peptides presented in vivo by dendritic cells. The processing enzymes were identified by reference to a database of experimentally determined cleavage sites and experimentally validated for four epitopes derived from complement 3, collagen II, thymosin ß4, and gelsolin. We determined that self-antigens processed by tissue-specific proteases, including complement, matrix metalloproteases, caspases, and granzymes, and carried by lymph, contribute significantly to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Additionally, the presented peptides exhibited a wide spectrum of binding affinity and HLA-DM susceptibility. The results indicate that the HLA-DR1-restricted self-peptidome presented under physiological conditions derives from a variety of processing pathways. Non-endosomal processing enzymes add to the number of epitopes cleaved by cathepsins, altogether generating a wider peptide repertoire. Taken together with HLA-DM-dependent and-independent loading pathways, this ensures that a broad self-peptidome is presented by dendritic cells. This work brings attention to the role of "self-recognition" as a dynamic interaction between dendritic cells and the metabolic/catabolic activities ongoing in every parenchymal organ as part of tissue growth, remodeling, and physiological apoptosis.

Kupffer Cell Transplantation in Mice for Elucidating Monocyte/Macrophage Biology and for Potential in Cell or Gene Therapy.

Kupffer cells (KC) play major roles in immunity and tissue injury or repair. Because recapitulation of KC biology and function within liver will allow superior insights into their functional repertoire, we studied the efficacy of the cell transplantation approach for this purpose. Mouse KC were isolated from donor livers, characterized, and transplanted into syngeneic recipients. To promote cell engraftment through impairments in native KC, recipients were preconditioned with gadolinium chloride. The targeting, fate, and functionality of transplanted cells were evaluated. The findings indicated that transplanted KC engrafted and survived in recipient livers throughout the study period of 3 months. Transplanted KC expressed macrophage functions, including phagocytosis and cytokine expression, with or without genetic modifications using lentiviral vectors. This permitted studies of whether transplanted KC could affect outcomes in the context of acetaminophen hepatotoxicity or hepatic ischemia-reperfusion injury. Transplanted KC exerted beneficial effects in these injury settings. The benefits resulted from cytoprotective factors including vascular endothelial growth factor. In conclusion, transplanted adult KC were successfully targeted and engrafted in the liver with retention of innate immune and tissue repair functions over the long term. This will provide excellent opportunities to address critical aspects in the biogenesis, fate, and function of KC within their native liver microenvironment and to develop the cell and gene therapy potential of KC transplantation.