Immunological Barrier Research Articles

Lymphatic Endothelial Cells Are Essential Components of the Subcapsular Sinus Macrophage Niche

In lymph nodes, subcapsular sinus macrophages (SSMs) form an immunological barrier that monitors lymph drained from peripheral tissues. Upon infection, SSMs activate B and natural killer T(NKT) cells while secreting inflammatory mediators. Here, we investigated the mechanisms regulating development and homeostasis of SSMs. Embryonic SSMs originated from yolk sac hematopoiesis and were replaced by a postnatal wave of bone marrow (BM)-derived monocytes that proliferated to establish the adult SSM network. The SSM network self-maintained by proliferation with minimal BM contribution. Upon pathogen-induced transient deletion, BM-derived cells contributed to restoring the SSM network. Lymphatic endothelial cells (LECs) were the main source of CSF-1 within the lymph node and conditional deletion of Csf1 in adult LECs decreased the network of SSMs and medullary sinus macrophages (MSMs). Thus, SSMs have a dual hematopoietic origin, and LECs are essential to the niche supporting these macrophages.

Montmorillonite improved the intestinal mucosal barrier functions of laying hens in late production.

This study was conducted to investigate the effects of dietary supplementation with montmorillonite (MMT) on performance, intestinal endotoxin concentration, gut mucosal oxidation status, intestinal morphology and permeability, and immunological barrier function of laying hens during late production. Four hundred and eighty 75-week-old laying hens (Lohmann Brown) were randomly assigned to five treatments with eight replicates per treatment and 12 hens in each replicate. The hens were fed the basal diet supplemented with 0 (control), 0.3, 0.6, 0.9, or 1.2g MMT/kg for 70days. Compared with the control, supplemented with 0.9g MMT/kg increased egg mass significantly (p<0.05) during weeks 1-5 of the experiment. Supplemented with 0.6 and 0.9g MMT/kg also increased the endotoxin concentration in the ileal digesta (p<0.05), but decreased the MDA concentration in the ileum significantly (p<0.05). The T-AOC in the jejunum of the group fed 0.3g MMT/kg was significantly increased (p<0.05). Compared with the control, the villus height:crypt depth of ileum from the groups fed 0.6, 0.9, and 1.2g MMT/kg increased significantly (p<0.05). The sIgA concentration of jejunum in the groups fed 0.6 and 0.9g MMT/kg was higher (p<0.05) than the control. The MMT supplementation linearly increased (p<0.05) the mRNA expression of claudin-1 and claudin-5 in the jejunum. Dietary MMT supplementation down-regulated the mRNA expression of NF-κB P65 and TNF-α in the jejunum in a linear and quadratic manner (p<0.05). The IL-1β mRNA expression of jejunum in the group fed 0.6g MMT/kg was lower (p<0.05) than the control. In conclusion, dietary supplementation with MMT may improve the gut barrier functions and suggests that 0.9g/kg of MMT in diets may be the optimal supplemental level for laying hens in late production.

660 Recapitulating T cell infiltration in psoriasis for patient-specific drug testing models

The skin is an immune organ that serves as a physical and immunological barrier against bacteria, fungi, viruses, haptens, and protein antigens. Inflammatory skin diseases can be induced by immune reactions to these stimuli. Therefore, understanding skin immune responses is essential to understand the pathogenesis of inflammatory skin diseases and to discover new targets for treatment. Drug testing studies for inflammatory skin diseases are currently restricted to mouse models, 2D cell culture systems, or 3D skin constructs stimulated with cytokines. In this study, we developed an inflammatory skin disease model by incorporating disease-relevant immune cells into 3D skin constructs. To validate our model, we examined the effect of the epidermis on T cell migration and activation in 3D skin constructs and the capability of infiltrating T cells to reproduce the psoriatic phenotype. To determine the effect of the epidermis on T cell migration, we mechanically removed the epidermis from some of the constructs and monitored the migration of CD4+ T cells. As a result, the constructs with the epidermis exhibited a significantly higher number of CD4+ T cells with deeper penetration into the constructs. In addition, the presence of the epidermis resulted in higher expression of CD69 on T cells, suggesting the activation of T cells. With the ability to recapitulate T cell infiltration, we next developed a psoriasis model by incorporating polarized Th1/Th17 cells or patient derived CCR6+CLA+ skin homing T cells into the constructs. The skin constructs infiltrated by these cells showed higher IFN-γ and IL-17A secretion and a psoriatic epidermal phenotype. Taken together, we developed T-cell incorporated 3D skin constructs as a novel in vitro model to investigate epidermal-T cell interactions, as well as the biology of inflammatory skin diseases.

Filaggrin and atopic march.

There is an increasing number of experimental, genetic and clinical evidence of atopic dermatitis expression as a pre-condition for later development of other atopic diseases such as asthma, food allergy and allergic rhinitis. Atopic dermatitis is a heterogeneous, recurrent childhood disease, also present in the adult age. It is increasingly attributed to systemic features and is characterized by immunological and skin barrier integrity and function dysregulation. To maintain the protective function of the skin barrier, in particular the maintenance of pH, hydration and antimicrobial functions, the filaggrin, among others, plays a significant role. Filaggrin is a multifunctional, histidine-rich, insoluble protein. The lack of filaggrin is associated with various cutaneous (e.g. ichthyosis vulgaris, allergic contact dermatitis) and non-cutaneous (e.g. diabetes, inflammatory conditions of the gastrointestinal tract) diseases and may be a result of genetic, immunological factors combined with environmental factors. In this review we summarised (emphasized) recent findings in understanding the role of filaggrin in atopic dermatitis and other diseases, participants in the atopic march.

Tumor cell oxidative metabolism as a barrier to PD-1 blockade immunotherapy in melanoma

The tumor microenvironment presents physical, immunologic, and metabolic barriers to durable immunotherapy responses. We have recently described roles for both T cell metabolic insufficiency as well as tumor hypoxia as inhibitory mechanisms that prevent T cell activity in murine tumors, but whether intratumoral T cell activity or response to immunotherapy varies between patients as a function of distinct metabolic profiles in tumor cells remains unclear. Here, we show that metabolic derangement can vary widely in both degree and type in patient-derived cell lines and in ex vivo analysis of patient samples, such that some cells demonstrate solely deregulated oxidative or glycolytic metabolism. Further, deregulated oxidative, but not glycolytic, metabolism was associated with increased generation of hypoxia upon implantation into immunodeficient animals. Generation of murine single-cell melanoma cell lines that lacked either oxidative or glycolytic metabolism showed that elevated tumor oxygen consumption was associated with increased T cell exhaustion and decreased immune activity. Moreover, melanoma lines lacking oxidative metabolism were solely responsive to anti-PD-1 therapy among those tested. Prospective analysis of patient sample immunotherapy revealed that oxidative, but not glycolytic, metabolism was associated with progression on PD-1 blockade. Our data highlight a role for oxygen as a crucial metabolite required for the tumor-infiltrating T cells to differentiate appropriately upon PD-1 blockade, and suggest that tumor oxidative metabolism may be a target to improve immunotherapeutic response.

Intestinal Barrier Function-Non-alcoholic Fatty Liver Disease Interactions and Possible Role of Gut Microbiota.

Non-alcoholic fatty liver disease (NAFLD) is a metabolic stress liver injury that is closely related to obesity, insulin resistance, type 2 diabetes, atherosclerosis, and metabolic syndrome. The pathological features are diffuse hepatic vesicular steatosis, including non-alcoholic steatohepatitis, liver fibrosis, and even liver cancer. A variety of pathological outcomes cause serious harm to human health. At present, an increasing number of researchers are investigating the pathogenesis of NAFLD from the perspective of changes in the function of the intestinal barrier. The physical, chemical, immunological, and microbiological barriers in the intestinal tract constitute the complete intestinal barrier, which plays an important defensive role against the invasion of harmful substances from the intestines. Protecting the function of the intestinal barrier is a new way to treat NAFLD and its related diseases. In this perspective, we summarized the current knowledge of the role of the intestinal barrier in NAFLD.

Structural assessments in decellularized extracellular matrix of porcine semilunar heart valves: Evaluation of cell niches.

Tissue-engineered heart valves aim to reproduce the biological properties of natural valves with anatomically correct structure and physiological performance. The closest alternative to creating an ideal heart valve substitute is to use decellularized porcine heart valves, due to their anatomy and availability. However, the immunological barrier and the structural maintenance limit the long-term physiological performance of decellularized porcine heart valves. This study investigated the extracellular matrix (ECM) structure of aortic and pulmonary porcine valves decellularized by a low concentration sodium dodecyl sulfate (SDS)-based method in order to determine the ECM scaffold (ECMS) conditions related to remodeling potential. To assess the structures of the leaflets and conduits of the heart valves, ECM components and their organization were evaluated by histology, biochemical analysis (BC), scanning electron microscopy, multiphoton microscopy, tensile test, immunofluorescence labeling (IF), and Raman microspectroscopy used to draw a profile of the cell niches. Histology and multiphoton imaging of decellularized aortic and pulmonary leaflets and conduits revealed a collagen and elastin histoarchitecture with rearrangement, loosening fibers, and glycosaminoglycan depletion confirmed by biochemistry quantification. The potential cytotoxicity of SDS residues was eliminated after 10 wash cycles. The mechanical properties of the structure of the valve indicated a functional resistance of decellularized ECM. The IF demonstrated the presence of basement membrane, suggesting a potential structure for host cell attachment. The RM analysis showed evidence of molecular interactions, suggesting conservation of the chemical composition, particularly among the protein molecular structures. The structural analyses performed in the semilunar porcine heart valves demonstrate that decellularized ECMS has structural properties that support physiological performance and potential host tissue integration. In fact, decellularized leaflet scaffolds were prone to cell interaction after human adipose-derived stromal cell seeding and culturing. Further analysis of biocompatibility, particularly the ECM-cell interaction, can elucidate the remodeling process, in preserved decellularized heart valve scaffold.

Oral administration of Lactobacillus rhamnosus GG to newborn piglets augments gut barrier function in pre-weaning piglets.

To understand the effects of Lactobacillus rhamnosus GG (ATCC 53103) on intestinal barrier function in pre-weaning piglets under normal conditions, twenty-four newborn littermate piglets were randomly divided into two groups. Piglets in the control group were orally administered with 2 mL 0.1 g/mL sterilized skim milk while the treatment group was administered the same volume of sterilized skim milk with the addition of viable L. rhamnosus at the 1st, 3rd, and 5th days after birth. The feeding trial was conducted for 25 d. Results showed that piglets in the L. rhamnosus group exhibited increased weaning weight and average daily weight gain, whereas diarrhea incidence was decreased. The bacterial abundance and composition of cecal contents, especially Firmicutes, Bacteroidetes, and Fusobacteria, were altered by probiotic treatment. In addition, L. rhamnosus increased the jejunal permeability and promoted the immunologic barrier through regulating antimicrobial peptides, cytokines, and chemokines via Toll-like receptors. Our findings indicate that oral administration of L. rhamnosus GG to newborn piglets is beneficial for intestinal health of pre-weaning piglets by improving the biological, physical, and immunologic barriers of intestinal mucosa.

British Society for Gene and Cell Therapy Autumn Conference Friday 23 November 2018 Regent's University Conference Centre, London, UK www.bsgct.org

British Society for Gene and Cell Therapy Autumn Conference Friday 23 November 2018 Regent's University Conference Centre, London, UK www.bsgct.org

High-resolution quantitative proteomics applied to the study of the specific protein signature in the sputum and saliva of active tuberculosis patients and their infected and uninfected contacts

Our goal was to establish panels of protein biomarkers that are characteristic of patients with microbiologically confirmed pulmonary tuberculosis (TB) and their contacts, including latent TB-infected (LTBI) and uninfected patients. Since the first pathogen-host contact occurs in the oral and nasal passages the saliva and sputum were chosen as the biological fluids to be studied. Quantitative shotgun proteomics was performed using a LTQ-Orbitrap-Elite platform. For active TB patients, both fluids exhibited a specific accumulation of proteins that were related to complement activation, inflammation and modulation of immune response. In the saliva of TB patients, a decrease of in proteins related to glucose and lipid metabolism was detected. In contrast, the sputum of uninfected contacts presented a specific proteomic signature that was composed of proteins involved in the perception of bitter taste, defense against pathogens and innate immune response, suggesting that those are key events during the initial entry of the pathogen in the host. SignificanceThis is the first study to compare the saliva and sputum from active TB patients and their contacts. Our findings strongly suggest that TB patients show not only an activation of processes that are related to complement activation and modulation of inflammation but also an imbalance in carbohydrate and lipid metabolism. In addition, those individuals who do not get infected after direct exposure to the pathogen display a typical proteomic signature in the sputum, which is a reflection of the secretion from the nasal and oral mucosa, the first immunological barriers that M. tuberculosis encounters in the host. Thus, this result indicates the importance of the processes related to the innate immune response in fighting the initial events of the infection.

Novel insights into the glia limitans of the olfactory nervous system.

Olfactory ensheathing cells (OECs) are often described as being present in both the peripheral and the central nervous systems (PNS and CNS). Furthermore, the olfactory nervous system glia limitans (the glial layer defining the PNS-CNS border) is considered unique as it consists of intermingling OECs and astrocytes. In contrast, the glia limitans of the rest of the nervous system consists solely of astrocytes which create a distinct barrier to Schwann cells (peripheral glia). The ability of OECs to interact with astrocytes is one reason why OECs are believed to be superior to Schwann cells for transplantation therapies to treat CNS injuries. We have used transgenic reporter mice in which glial cells express DsRed fluorescent protein to study the cellular constituents of the glia limitans. We found that the glia limitans layer of the olfactory nervous system is morphologically similar to elsewhere in the nervous system, with a similar low degree of intermingling between peripheral glia and astrocytes. We found that the astrocytic layer of the olfactory bulb is a distinct barrier to bacterial infection, suggesting that this layer constitutes the PNS-CNS immunological barrier. We also found that OECs interact with astrocytes in a similar fashion as Schwann cells in vitro. When cultured in three dimensions, however, there were subtle differences between OECs and Schwann cells in their interactions with astrocytes. We therefore suggest that glial fibrillary acidic protein-reactive astrocyte layer of the olfactory bulb constitutes the glia limitans of the olfactory nervous system and that OECs are primarily "PNS glia."

Why Potential Living Kidney Donors Do Not Proceed for Donation: A Single-Center Experience

BackgroundLiving donor kidney transplantation is the best type of renal replacement therapy. However, large numbers of potential living kidney donors (LKDs) are declined because of various reasons. The aim of this study is to define and quantify the reasons for declining potential LKDs. MethodsAll potential LKDs evaluated at our center between September 2008 and December 2016 were reviewed. Data were collected from the electronic database. ResultsA total of 2090 potential LKDs were evaluated, with an average age of 32 years (range, 18–67 years) and men constituting 72.6%. A total of 675 (32.3%) were accepted for donor nephrectomy. Living kidney donation did not proceed in 830 (39.7%): 661 (79.6%) because of donor-related reasons and 169 (20.4%) because of recipient-related reasons. Donor-related reasons included medical contraindications (61.7%), immunological barriers (23.1%), surgical contraindications (7.9%), and psychosocial reasons (7.3%). A total of 585 (28.0%) potential LKDs voluntarily withdrew themselves at variable time points during the evaluation process, even after being accepted for donation. Male and young (18–35 years) potential LKDs were more likely to withdraw compared with female and older (>35 years) potential LKDs (34.3% vs 11.4%, P < .005 and 29.6% vs 24.5%, P = .02, respectively). ConclusionsDespite the large number of potential LKDs, medically complex donors are increasing, and a significant proportion decided to withdraw at some point during the evaluation process. The latter highlights the need to increase public awareness about living donation, to perform more careful initial screening and targeted educational programs, and to provide continuous support for potential LKDs.

Immune responses at the maternal-fetal interface.

Pregnancy poses an immunological challenge because a genetically distinct (nonself) fetus must be supported within the pregnant female for the required gestational period. Placentation, or the establishment of the fetally derived placenta, is a common strategy used by eutherian mammals to protect the fetus and promote its growth. However, the substantial morphological differences of the placental architecture among species suggest that the process of placentation results from convergent evolution. Although there are considerable similarities in placental function across placental mammals, there are important differences that arise owing to species-specific immunological (and other biological) constraints. This Review focuses on the immunological similarities and differences that occur at the maternal-fetal interface in the context of human and mouse pregnancies. We discuss how the decidua and placenta of these different species form key immunological barriers that sustain maternal tolerance yet generate innate immune responses that prevent microbial infections.

Immunological barriers in ABO-incompatible kidney transplantation: How to overcome

Background: Various desensitization strategies are used to overcome immunologic barriers such as anti-human leukocyte antigen-donor-specific antibody (HLA-DSA) and anti-ABO blood group incompatibility. Materials and Methods: Three index cases of ABO-incompatible (ABO-i) kidney transplantation from living-related donors are discussed. Each recipient was evaluated by complement-dependent cytotoxicity crossmatch, flow cytometric crossmatch, lysate-based crossmatch, and single-antigen bead Luminex assay and anti-A, B isoagglutinin titer. The desensitization protocol included 500 mg of rituximab, followed by 2–10 sessions of plasmapheresis with basiliximab/antithymocyte globulin induction. Results: Case 1: A patient received the third transplant as ABO-i transplant, with no anti-HLA-DSA. Anti-ABO antibody titers were immunoglobulin M (IgM)/IgG 1:128/1:256. He had successful desensitization to anti-AB titer of 11000) resulting in graft loss. Conclusions: Careful evaluation of immunological barriers before ABO-i transplantation should be done, especially when both anti-HLA and ABO sensitization are present.

Staphylococcus aureus: an underestimated factor in the pathogenesis of atopic dermatitis?

Atopic dermatitis is a common, recurrent pruritic dermatosis with a complex pathogenesis. It has been associated with disordered patterns of immunological response and impaired epithelial barrier integrity. These features predispose the patients to robust colonization of skin lesions by Staphylococcus aureus. Virulence factors of S. aureus (e.g. superantigens, α- and δ-toxin, protein A) have been shown to exacerbate and perpetuate the course of atopic dermatitis. Novel therapeutic options with potential for restoring natural microbiome composition are being elaborated and may enter clinical practice in the future.

Isolation, Culture, and Characterization of Primary Mouse Epidermal Keratinocytes.

Epidermis, the outermost layer of the skin, plays a critical role as both a physical and immunological barrier protecting the internal tissues from external environmental insults, such as pathogenic bacteria, fungi, viruses, UV irradiation, and water loss. Epidermal keratinocytes (KC), the predominant cell type in the skin epidermis, are in the front line of skin defense. Here we describe methods to isolate and culture primary epidermal KC from neonatal and adult mouse skin and describe in vitro assays to study and characterize KC proliferation and differentiation and pro-inflammatory responses to viral products and UVB irradiation. These methods will be useful for researchers in the field of epidermal biology to set up in vitro assays to study the barrier and pro-inflammatory function of epidermal keratinocytes.

Microneedle-Mediated Vaccine Delivery to the Oral Mucosa.

The oral mucosa is a minimally invasive and immunologically rich site that is underutilized for vaccination due to physiological and immunological barriers. To develop effective oral mucosal vaccines, key questions regarding vaccine residence time, uptake, adjuvant formulation, dose, and delivery location must be answered. However, currently available dosage forms are insufficient to address all these questions. An ideal oral mucosal vaccine delivery system would improve both residence time and epithelial permeation while enabling efficient delivery of physicochemically diverse vaccine formulations. Microneedles have demonstrated these capabilities for dermal vaccine delivery. Additionally, microneedles enable precise control over delivery properties like depth, uniformity, and dosing, making them an ideal tool to study oral mucosal vaccination. Select studies have demonstrated the feasibility of microneedle-mediated oral mucosal vaccination, but they have only begun to explore the broad functionality of microneedles. This review describes the physiological and immunological challenges related to oral mucosal vaccine delivery and provides specific examples of how microneedles can be used to address these challenges. It summarizes and compares the few existing oral mucosal microneedle vaccine studies and offers a perspective for the future of the field.

Immunosuppression overcomes insulin- and vector-specific immune responses that limit efficacy of AAV2/8-mediated insulin gene therapy in NOD mice

We report the restoration of euglycaemia in chemically induced diabetic C57BL/6 mice and spontaneously diabetic Non Obese Diabetic (NOD) mice by intravenous systemic administration of a single-stranded adeno-associated virus (ssAAV2/8) codon optimised (co) vector encoding furin cleavable human proinsulin under a liver-specific promoter. There were no immunological barriers to efficacy of insulin gene therapy in chemically induced C57BL/6 mice, which enjoyed long-lasting correction of hyperglycaemia after therapy, up to 250 days. Euglycaemia was also restored in spontaneously diabetic NOD mice, although these mice required a 7–10-fold higher dose of vector to achieve similar efficacy as the C57BL/6 mice and the immunodeficient NODscid mice. We detected CD8+ T cell reactivity to insulin and mild inflammatory infiltration in the livers of gene therapy recipient NOD mice, neither of which were observed in the treated C57BL/6 mice. Efficacy of the gene therapy in NOD mice was partially improved by targeting the immune system with anti-CD4 antibody treatment, while transfer of NOD mouse AAV2/8-reactive serum to recipients prevented successful restoration of euglycaemia in AAV2/8-HLP-hINSco-treated NODscid mice. Our data indicate that both immune cells and antibodies form a barrier to successful restoration of euglycaemia in autoimmune diabetic recipient mice with insulin gene therapy, but that this barrier can be overcome by increasing the dose of vector and by suppressing immune responses.

Advances in liver xenotransplantation.

This review highlights advances in liver xenotransplantation, focusing on immunologic barriers and mechanisms underlying graft failure and recipient demise, and discussion of recent in-vivo results. Pig to primate models of liver xenotransplantation have been plagued by thrombocytopenia, anemia, and coagulopathy. It is now known that platelet sequestration is mediated by liver sinusoidal endothelial cells and Kupffer cells in part by asialoglycoprotein receptor 1-driven mechanisms. Xenoantigens, specifically N-glycolylneuraminic acid, play a role in graft injury as well as red blood cell consumption. Finally incompatibilities between coagulation cascade molecules contribute to lethal coagulopathy, but can be counteracted with genetic modifications and coagulation factor supplementation. Survival has markedly increased with this strategy. An increased understanding of the cellular mechanisms responsible for failure of in-vivo pig to primate liver xenotransplant models has led to improved outcomes, and this recent success supports initial clinical application.

Conditioning for Hematopoietic Stem Cell Transplantation Using Antibody-Drug Conjugate Targeting CD45 Permits Engraftment across Immunologic Barriers

INTRODUCTION: Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapy for benign and malignant hematologic diseases. It also holds great promise as treatment for infectious and autoimmune disease and for autologous gene therapy. However, due to the toxicity of conditioning regimens for HSCT, it is generally reserved for the most life-threatening diagnoses, such as acute leukemia. Targeted approaches to conditioning may improve the safety of HSCT and extend its applicability to other diseases. Recently, conditioning regimens were described consisting of treatment with anti-CD45.2 or anti-cKit antibodies conjugated to the ribosome-inhibiting protein saporin (CD45-SAP and cKit-SAP, respectively) (Palchaudhuri et al (2016), Nat Biotechnol, Czechowicz et al (2016), ASH Abstract). These were reported to deplete murine hematopoietic stem cells (HSCs) and permit engraftment of syngeneic HSCs with minimal toxicity. However, the ability of CD45-SAP and cKit-SAP to permit HSCT across immunologic barriers without chemotherapy or radiation, or to target malignant cells expressing these receptors, are potential applications that were not explored.METHODS: We compared CD45-SAP and cKit-SAP conditioning in syngeneic (B6 to B6) and allogeneic (CB6F1 to B6) murine transplantation models. Donor chimerism in peripheral blood (PB) and complete blood counts (CBCs) were assessed monthly during these experiments. Successful HSC engraftment was confirmed by serial transplantation experiments. Human CD45-SAP and cKit-SAP conjugates were also tested for their ability to target MOLM13 AML cells using XTT viability assays, and in targeting cord-blood derived HSCs using colony forming cell assays.RESULTS: We confirmed that CD45-SAP dosed at 75 µg per mouse could effectively deplete B6 mice of HSCs and condition them for syngeneic HSCT as reported (Palchaudhuri et al (2016), Nat Biotechnol), with normal-range CBCs and stable donor PB chimerism of approximately 80% at 6 months post-transplant. Donor-derived HSC in CD45-SAP treated mice successfully engrafted upon transplantation to secondary B6 recipients. cKit-SAP dosed at 10 µg per mouse was sufficient for HSC depletion, but robust syngeneic engraftment required dosing at 50 µg per mouse. Notably, CD45-SAP treatment combined with CD4+and CD8+ T cell depletion permitted haploidentical transplantation from CB6F1 donors to B6 recipients, with normal-range CBCs and stable donor PB chimerism of approximately 70% at 3 months post-transplant. Finally, human CD45-SAP conjugates could successfully target MOLM13 leukemia cells and cord-blood derived HSCs in vitro.CONCLUSIONS: Transplantation in mice across immunological barriers is achievable using CD45-SAP conditioning, with high-level donor chimerism without chemotherapy or radiation. Furthermore, human CD45-SAP conjugates can successfully target human leukemia cell lines and primary HSCs in vitro. Further studies are underway in three areas. First, by combining T and NK cell depletion with CD45-SAP conditioning, we are attempting full-mismatch HSCT in mice. Second, using humanized mouse models, we will assess the ability of CD45-SAP to target human leukemia cells and HSCs in vivo. Finally, we are exploring using HSCT with CD45-SAP conditioning as a treatment in mouse models of autoimmune disease. DisclosuresRettig:Amphivena Therapeutics: Research Funding; Novimmune: Research Funding.