Separation and isolation of specific cell populations from solid immune organs are essential steps in immunological research. While antibody-labeling based cell sorting techniques, such as FACS, offer the possibility of isolating rare cells from a large background population, the cost and lengthy preparation time associated with these approaches remain burdensome for many researchers. Here, we report a rapid partitioning method that allows the separation of immune cells based on their size and density using a novel, spiral designed, microfluidic device (MFD) (Hou et al., 2016) Representing less than 0.5% of the total thymic cells, thymic epithelial cells (TECs) are critical for the generation of diverse T cell populations, essential for acquired immunity. Due to their rarity, separating TECs from the large pool of thymocytes is challenging. Using a MFD, we achieved substantial enrichment of TECs (up to 20X), high cell recovery (>2.5X of the traditional density gradient method) and excellent cell viability (>90%) within 7 minutes/thymus. High-purity TEC populations were obtained through further depletion of CD45+ thymic stromal cells using MACS. Thymus organoids reconstructed with TECs can support the development of both CD4+ and CD8+ T cells in vitro, proving the function of the isolated TECs. In addition, MFD could also be utilized to separate immune cell subtypes from spleen and lymph nodes. Notably, significant enrichment of CDllc+ MHCII+ dendritic cells (~15X) was achieved without RBC lysis and antibody labeling. In conclusion, our data suggest that the MFD partitioning method is efficient and cost-effective for rapid size-based separation of immune cells from primary and secondary immune organs for downstream applications.

Thymus involution, associated with aging or pathological insults, results in diminished output of mature T-cells. Restoring the function of a failing thymus is crucial to maintain effective T cell-mediated acquired immune response against invading pathogens. However, thymus regeneration and revitalization proved to be challenging, largely due to the difficulties of reproducing the unique 3D microenvironment of the thymic stroma that is critical for the survival and function of thymic epithelial cells (TECs). We developed a novel hydrogel system to promote the formation of TEC aggregates, based on the self-assembling property of the amphiphilic EAK16-II oligopeptides and its histidinylated analogue EAKIIH6. TECs were enriched from isolated thymic cells with density-gradient, sorted with fluorescence-activated cell sorting (FACS), and labeled with anti-epithelial cell adhesion molecule (EpCAM) antibodies that were anchored, together with anti-His IgGs, on the protein A/G adaptor complexes. Formation of cell aggregates was promoted by incubating TECs with EAKIIH6 and EAK16-II oligopeptides, and then by increasing the ionic concentration of the medium to initiate gelation. TEC aggregates embedded in EAK hydrogel can effectively promote the development of functional T cells in vivo when transplanted into the athymic nude mice.

A functional thymus is crucial to produce mature, self-tolerant T cells. Crosstalk between the residing thymic stromal cells, especially the predominant population of thymic epithelial cells (TECs), and the developing thymocytes is essential for thymopoiesis. The survival and proliferation of TECs depend on a unique 3 dimensional (3-D) configuration of the thymus microenvironment. Disorganization of TECs results in a loss of gene expressions essential for TEC growth and survival, as well as inability to produce functional T cells. Recapitulating the property of thymus has been proven to be challenging. In our study, we have incorporated EAK16-II, a low molecular weight peptide that self-assembles into beta-sheet fibrils, and its histidinylated analogue, to induce 3-D aggregation of TECs. When the adaptor complex comprised of anti-EpCAM IgG, anti-His and recombinant protein A/G molecules were mixed with TECs in the hydrogel, TECs were captured into small clusters. TECs cultured in this condition maintained their molecular properties and were viable up to 2 weeks. Furthermore, when the TEC clusters in the hydrogel were transplanted into athymic nude mice, T cell development was observed, and these newly generated T cells proliferated upon stimulation with allogenic cells. These results demonstrate that self-assembling EAK16-II/EAKIIH6 system with addition of tri-component adaptor complex may be a useful tool to organize TECs in a 3-D-like structure to support T cell development, suggesting a possibility to generate injectable mini thymus-like units to restore adaptive immune system.

One of the major obstacles in solid organ transplantation is to establish immune tolerance of allografts. While immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited, impermanent, and associated with severe side effects. As a primary immune organ essential to adaptive immunity, the thymus continuously generates a diverse population of naïve T-cells, which can effectively react to invading pathogens, but remains unresponsive to self-antigens. Many factors (e.g. infection, irradiation, drug treatments, or aging) can irreversibly compromise thymic function, resulting in immune deficiency, cancer, autoimmunity and other immunodysregulatory diseases. While numerous efforts have been made to modulate/rejuvenate thymic function, manipulating the thymus, either in vitro or in vivo, proves to be difficult. The major challenge is to reproduce its unique extracellular matrix microenvironment that is critical for the survival and function of thymic epithelial cells (TECs). TECs are the predominant population of thymic stromal cells that are essential for T-cell lineage determination and maturation. TECs cultured in the traditional 2-D culture rapidly lose their molecular properties and fail to grow, which prohibits them from undergoing genetic modification. We have recently developed a thymus decellularization technique, which allows us to reconstruct a functional thymus organoid de novo with isolated TECs. The 3-D thymic scaffolds can support the survival of TECs in vitro, and maintain their unique molecular properties. When transplanted into athymic nude mice, the bioengineered thymus organoids could effectively promote the homing of lymphocyte progenitors and support the development of a diverse, self-tolerant T-cell repertoire. The thymus-reconstructed nude mice could promptly reject skin allografts, and were able to mount antigen-specific humoral responses against ovalbumin upon immunization. Notably, tolerance to allogeneic skin grafts could be achieved by transplanting thymus organoids constructed with either TECs co-expressing both syngeneic and allogenic MHCs, or mixtures of donor and recipient TECs. Our results demonstrate the technical feasibility of inducing donor-specific allogeneic tolerance with bioengineered thymus organoids and highlight the clinical implications of this thymus reconstruction technique in solid organ transplantation and regenerative medicine.

Abstract Extended use of immunosuppression is a major setback in organ transplantation. Hence, establishment of donor specific immune unresponsiveness, without the need of immunosuppression, is critical and remains a major challenge. Immunological tolerance is known to occur through two main mechanisms: central (thymus) and peripheral. Our lab has recently reported that bioengineered thymus organoids, made of de-cellularized thymus scaffolds populated with thymic epithelial cells (TECs), can successfully support T-cell generation in athymic nude mice. Here, we aim to test the hypothesis that transplantation of bioengineered thymus, incorporated with donor TECs, can induce central tolerance to donor antigens. Thymus scaffold were reconstructed with either TECs harvested from F1 offspring of B6 (H-2b) and NOD (H-2b/g7) or TECs from both B6 and CBA/J (H-2k) mixed in 1:1 ratio. These reconstructed thymus organoids are transplanted to the B6. Nude recipient, that are subsequently challenged with syngeneic, allogeneic and third party skin graft. Results from recipients with transplanted bioengineered thymus consisting F1 TECs demonstrated successful engraftment of skin transplants from both syngeneic (B6) and allogeneic (B6.H-2b/g7) whereas, clear rejection of the third party (CBA/J). Similarly, we observed prolonged survival of allograft skin (CBA/J) than third party (Balb/C) in recipient mice with reconstructed thymus organoids containing TECS from both donor (B6) and recipient (Balb/c). Furthermore, immune unresponsiveness to allograft was shown in MLR assay. Overall, our findings suggest reconstructed thymus-containing donor TECs could potentially have clinical application to induce-donor specific immune tolerance.

238. Cell Therapeutic Approach Using Dental Pulp Stromal Cells for Duchenne Muscular Dystrophy Yuko N. Kasahara1,2, Mutsuki Kuraoka2, Hiromi H. Kinoh1,2, Chiaki Masuda1, Kiwamu Imagawa3, Katsuhiko Tachibana3, Shin’ichi Takeda2, Takashi Okada1,2 1Biochemistry and Molecular Biology, Nippon Medical School, Tokyo, Japan, 2Molecular Therapy, National Institute of Neuroscience, NCNP, Tokyo, Japan, 3JCR Pharmaceuticals Co., Ltd., Hyogo, Japan

One of the major obstacles in organ transplantation is to establish immune tolerance of allografts. Although immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited, transient, and associated with severe side effects. Induction of thymic central tolerance to allografts remains challenging, largely because of the difficulty of maintaining donor thymic epithelial cells in vitro to allow successful bioengineering. Here, the authors show that three-dimensional scaffolds generated from decellularized mouse thymus can support thymic epithelial cell survival in culture and maintain their unique molecular properties. When transplanted into athymic nude mice, the bioengineered thymus organoids effectively promoted homing of lymphocyte progenitors and supported thymopoiesis. Nude mice transplanted with thymus organoids promptly rejected skin allografts and were able to mount antigen-specific humoral responses against ovalbumin on immunization. Notably, tolerance to skin allografts was achieved by transplanting thymus organoids constructed with either thymic epithelial cells coexpressing both syngeneic and allogenic major histocompatibility complexes, or mixtures of donor and recipient thymic epithelial cells. Our results demonstrate the technical feasibility of restoring thymic function with bioengineered thymus organoids and highlight the clinical implications of this thymus reconstruction technique in organ transplantation and regenerative medicine.

For reasons not fully understood, patients with an organ-specific autoimmune disease have increased risks of developing autoimmune responses against other organs/tissues. We identified ICA69, a known β-cell autoantigen in Type 1 diabetes, as a potential common target in multi-organ autoimmunity. NOD mice immunized with ICA69 polypeptides exhibited exacerbated inflammation not only in the islets, but also in the salivary glands. To further investigate ICA69 autoimmunity, two genetically modified mouse lines were generated to modulate thymic ICA69 expression: the heterozygous ICA69(del/wt) line and the thymic medullary epithelial cell-specific deletion Aire-ΔICA69 line. Suboptimal central negative selection of ICA69-reactive T-cells was observed in both lines. Aire-ΔICA69 mice spontaneously developed coincident autoimmune responses to the pancreas, the salivary glands, the thyroid, and the stomach. Our findings establish a direct link between compromised thymic ICA69 expression and autoimmunity against multiple ICA69-expressing organs, and identify a potential novel mechanism for the development of multi-organ autoimmune diseases.

Abstract The essential role of tissue specific antigen (TSA) expression in medullary thymic epithelial cells (mTECs) in mediating central tolerance has been well-established. We have previously shown that mice with mTEC-specific insulin deletion develop autoimmune diabetes within 3 weeks postnatal. To further understand the mechanisms of mTEC-mediated self-tolerance, we generated the Aire-YFP mice by crossing R26R-YFP reporters with Aire-Cre transgenic mice. Three subsets of mTECs were isolated from these mice: the YFPLOWCD80LOWClass IILOW pre-Aire cells; the YFPHICD80HIClass IIHI Aire+ cells; and the YFPHICD80INTClass IIINT post-Aire cells. Diverse TSA transcripts were detected in Aire+ mTECs, but not, or more restricted in pre- and post-Aire subsets, respectively. To investigate the temporal differentiation of mTECs, we generated the Aire-Cre:R26R-DTR (Aire-DTR) mice, which marked both Aire+ and post-Aire mTECs with diphtheria toxin (DT) receptors. Consecutive administration of DT resulted in ablation of both subsets, whereas pre-Aire mTECs remained intact. Upon DT withdrawal, transcripts of Aire and TSAs remained absent at day 3, but became detectable at day 5. Interestingly, no autoimmunity was observed in Aire-DTR mice treated weekly with a single dose of DT, suggesting that the newly generated Aire+ mTECs could reestablish self-tolerance in a timely manner. Our data provide new insights into the temporal regeneration of mTECs and its importance in maintaining central tolerance.

Anti-insulin autoimmunity is one of the primary forces in initiating and progressing β-cell destruction in type 1 diabetes. While insulin expression in thymic medullary epithelial cells has been shown to be essential for establishing β-cell central tolerance, the function of insulin expression in antigen-presenting cells (APCs) of hematopoietic lineage remains elusive. With a Cre-lox reporter approach, we labeled Aire-expressing cells with enhanced yellow fluorescent proteins, and found that insulin expression in the spleen was restricted predominantly to a population of Aire(+)CD11c(int)B220(+) dendritic cells (DCs). Targeted insulin deletion in APCs failed to induce anti-islet autoimmunity in B6 mice. In contrast, elevated levels of T cell infiltration into islets were observed in B6(g7) congenic mice when insulin was specifically deleted in their CD11c-expressing DCs (B6(g7)·CD11c-ΔIns mice). Thus, insulin expression in BM-derived, Aire(+) tolerogenic DCs may play an essential role to prevent the activation and expansion of insulin-reactive T cells in the periphery.

Intracellular staining is a widely used flow cytometry (FCM)-based technique to detect the expression of cytoslio nucleic antigens. However, intracellular staining of cells expressing cytosolic fluorescent protein (FP) markers was proven to be problematic as significant loss of the FP-signal was routinely observed. Using splenocytes harvested from mice constitutively expressing the enhanced yellow fluorescent proteins (YFP) as a model, we modified the widely used intracellular staining protocol and successfully achieved simultaneous detection of both the nuclear proteins and YFP in T-regulatory cells. The improved protocol can be used to perform antibody-based intracellular characterization of FP-labeled target cells, while maintaining their fluorescent reporter signals for easy tracing and identification.

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance towards pancreatic beta-cells. To further explore the function of this ectopic insulin expression, we knocked out the mouse Ins2 gene specifically in the Aire-expressing medullary thymic epithelial cells (mTECs), without affecting its expression in the beta-cells. When further crossed to the Ins1 knockout background, both male and female pups (designated as ID-TEC mice for insulin-deleted mTEC) developed diabetes spontaneously around 3 weeks after birth. beta-cell-specific autoimmune destruction was observed, as well as islet-specific T cell infiltration. The presence of insulin-specific effector T cells was shown using ELISPOT assays and adoptive T cell transfer experiments. Results from thymus transplantation experiments proved further that depletion of Ins2 expression in mTECs was sufficient to break central tolerance and induce anti-insulin autoimmunity. Our observations may explain the rare cases of type 1 diabetes onset in very young children carrying diabetes-resistant HLA class II alleles. ID-TEC mice could serve as a new model for studying this pathology.

OBJECTIVE—To describe the ability of nonhuman primate endocrine pancreata to reestablish endogenous insulin production after chemical β-cell destruction. RESEARCH DESIGN AND METHODS—Eleven monkeys (Macaca fascicularis) were rendered diabetic with streptozotocin. Eight diabetic monkeys received intraportal porcine islet transplantation. RESULTS—Two monkeys transplanted after 75 days of type 1 diabetes showed recovery of endogenous C-peptide production a few weeks after transplantation, concomitant with graft failure. Histological analysis of the pancreas of these monkeys showed insulin-positive cells, single or in small aggregates, scattered in the pancreas and adjacent to ducts. Interestingly, numerous CK19+ cells costained with proinsulin and PDX-1 antibodies. Furthermore, the peculiar double phenotype glucagon-positive/GLUT2+ was observed. In these monkeys as well as in all others, the original islets showed no insulin staining. CONCLUSIONS—Our data provide evidence that, in nonhuman primates, the pancreas can reestablish endogenous insulin production after chemical β-cell destruction. This seems to be a nongeneralizable event with only 2 out of 11 monkeys recovering β-cell function. In these two monkeys, younger age and islet graft behavior might have played a role in triggering endogenous β-cell recovery.

Inspired by the articles presented in this issue of The Review of Diabetic Studies, we considered it useful to summa- rize the latest achievements and current challenges we face in the search for a cure of type 1 diabetes.In this editorial article, we took into account how the research landscape has changed in only a few years.While modern lifestyles impose new concerns, now we have a better knowledge of the various aspects of the disease that can be used to treat our young patients with more appropriate approaches, thereby eliminating old and obsolete prejudices.

Successful transplantation of tissue during solid organ and bone marrow transplantation relies on accurate determination of the human leukocyte antigen (HLA) phenotype of the potential donor(s) and recipient. Matching donor with recipient for a kidney transplant generally means finding a six-antigen match by looking at each of two alleles at HLA-A, -B, and -DR loci. For bone marrow transplantation the HLA-C and -DQ alleles are also considered. Molecular techniques, including sequencing, are capable of precisely defining HLA alleles. Because of the large number of possible allelic combinations there are numerous ambiguities associated with heterozygous genotypes even when sequence-based typing protocols are used. Sequencing-by-synthesis methodology employed by Pyrosequencing represents an improvement when applied to HLA genotyping that allows resolution of many ambiguous allelic pairs. Out-of-phase sequencing of HLA alleles by Pyrosequencing can resolve cis/trans ambiguities that would otherwise require the sequencing of isolated cloned DNAs. Single-nucleotide polymorphism typing of HLA for the presence of specific variants is also beneficial for monitoring HLA-encoded genetic risk to autoimmune diseases, such as celiac disease, rheumatoid arthritis, and type 1 diabetes mellitus.

Design of locus-specific primers for use during genetic analysis requires combining information from multiple sources and can be a time-consuming process when validating large numbers of assays. Data warehousing of genomic DNA sequences and genetic variations when coupled with software applications for optimizing the generation of locus-specific primers can increase the efficiency of assay development. Selection of oligonucleotide primers for PCR and Pyrosequencing (SOP3) software allows user-directed queries of warehoused data collected from the human and mouse genome sequencing projects. The software automates collection of DNA sequence flanking single-nucleotide polymorphisms (SNPs) as well as the incorporation of locus-associated functional information, such as whether the SNP occurs in an exon, intron, or untranslated region. SOP3 software accepts three types of user-directed input consisting of gene locus symbols, SNP reference sequence numbers, or chromosomal physical location. For human polymorphisms, SOP3 incorporates haplotype, ethnicity, and SNP validation attributes. The output is a list of oligonucleotide primers recommended for Pyrosequencing-based typing of genetic variations. SOP3 is available at the Division of Immunogenetics computational server found at http://imgen.ccbb.pitt.edu.