T cells are key mediators of graft tolerance/rejection, development of autoimmunity, and the anticancer response. Consequently, differentially modifying the T cell response is a major therapeutic target. Most immunomodulatory approaches have focused on cytotoxic agents, cytokine modulation, monoclonal antibodies, mitogen activation, adoptive cell therapies (including CAR-T cells). However, these approaches do not persistently reorient the systemic immune response thus necessitating continual therapy. Previous murine studies from our laboratory demonstrated that the adoptive transfer of polymer-grafted (PEGylated) allogeneic leukocytes resulted in the induction of a persistent and systemic tolerogenic state. Further analyses demonstrated that miRNA isolated from the secretome of polymer-modified or control allogeneic responses effectively induced either a tolerogenic (TA1 miRNA) or proinflammatory (IA1 miRNA) response both in vitro and in vivo that was both systemic and persistent. In a murine Type 1 diabetes autoimmune model, the tolerogenic TA1 therapeutic effectively attenuated the disease process via the systemic upregulation of regulatory T cells while simultaneously downregulating T effector cells. In contrast, the proinflammatory IA1 therapeutic enhanced the anticancer efficacy of naïve PBMC by increasing inflammatory T cells and decreasing regulatory T cells. The successful development of this secretome miRNA approach may prove useful treating both autoimmune diseases and cancer.

This study introduces a flexible format for tolerogenic vaccination that incorporates IFN-β and neuroantigen (NAg) in the Alum adjuvant. Tolerogenic vaccination required all three components, IFN-β, NAg, and Alum, for inhibition of experimental autoimmune encephalomyelitis (EAE) and induction of tolerance. Vaccination with IFN-β + NAg in Alum ameliorated NAg-specific sensitization and inhibited EAE in C57BL/6 mice in pretreatment and therapeutic regimens. Tolerance induction was specific for the tolerogenic vaccine Ag PLP178-191 or myelin oligodendrocyte glycoprotein (MOG)35–55 in proteolipid protein– and MOG-induced models of EAE, respectively, and was abrogated by pretreatment with a depleting anti-CD25 mAb. IFN-β/Alum–based vaccination exhibited hallmarks of infectious tolerance, because IFN-β + OVA in Alum–specific vaccination inhibited EAE elicited by OVA + MOG in CFA but not EAE elicited by MOG in CFA. IFN-β + NAg in Alum vaccination elicited elevated numbers and percentages of FOXP3+ T cells in blood and secondary lymphoid organs in 2D2 MOG-specific transgenic mice, and repeated boosters facilitated generation of activated CD44high CD25+ regulatory T cell (Treg) populations. IFN-β and MOG35–55 elicited suppressive FOXP3+ Tregs in vitro in the absence of Alum via a mechanism that was neutralized by anti–TGF-β and that resulted in the induction of an effector CD69+ CTLA-4+ IFNAR+ FOXP3+ Treg subset. In vitro IFN-β + MOG–induced Tregs inhibited EAE when transferred into actively challenged recipients. Unlike IFN-β + NAg in Alum vaccines, vaccination with TGF-β + MOG35-55 in Alum did not increase Treg percentages in vivo. Overall, this study indicates that IFN-β + NAg in Alum vaccination elicits NAg-specific, suppressive CD25+ Tregs that inhibit CNS autoimmune disease. Thus, IFN-β has the activity spectrum that drives selective responses of suppressive FOXP3+ Tregs.

Autoimmune destruction of the pancreatic islets in Type 1 diabetes is mediated by both increased proinflammatory (Teff) and decreased regulatory (Treg) T lymphocytes resulting in a significant decrease in the Treg: Teff ratio. The non-obese diabetic (NOD) mouse is an excellent in vivo model for testing potential therapeutics for attenuating the decrease in the Treg: Teff ratio and inhibiting disease pathogenesis. Here we show for the first time that a bioreactor manufactured therapeutic consisting of a complex of miRNA species (denoted as TA1) can effectively reset the NOD immune system from a proinflammatory to a tolerogenic state thus preventing or delaying autoimmune diabetes. Treatment of NOD mice with TA1 resulted in a systemic broad-spectrum upregulation of tolerogenic T cell subsets with a parallel downregulation of Teff subsets yielding a dramatic increase in the Treg: Teff ratio. Moreover, the murine-derived TA1 was highly effective in the inhibition of allorecognition of HLA-disparate human PBMC. TA1 demonstrated dose-responsiveness and exhibited equivalent or better inhibition of allorecognition driven proliferation than etanercept (a soluble TNF receptor). These findings demonstrate that miRNA-based therapeutics can effectively attenuate or arrest autoimmune disease processes and may be of significant utility in a broad range of autoimmune diseases including Type 1 diabetes.

BackgroundGlycophorin C (GPC) is necessary in the maintenance of red blood cell structure. Severe autoimmune hemolytic anemia and hemolytic disease of the fetus and newborn (HDFN) have been associated with Gerbich (Ge) blood group system antigens expressed on GPC. Previous in vitro studies with cord blood progenitor cells have shown that anti-Ge suppresses erythropoiesis.Study Design and MethodsHere, we evaluated the K562 erythroleukemic cell line to study the cellular effects of a murine anti-GPC. Cell proliferation was evaluated after treatment with anti-GPC. Flow cytometry was used to evaluate exofacial phosphatidylserine (PS) expression and cell viability (propidium iodide binding). Cell morphology was evaluated under light microscopy with cytospin preparations stained with May-Grunwald Giemsa.ResultsAnti-GPC dramatically inhibited K562 proliferation and increased PS expression, consistent with cytoplasmic blebbing, suggesting evidence of apoptosis. Z-VAD-FMK, an inhibitor of classical apoptosis, was unable to reverse the suppressive effect of anti-GPC. However, hemin was able to attenuate growth suppression.ConclusionTogether, the data suggest that anti-GPC suppresses erythroid proliferation through the induction of nonclassical apoptosis.

Organ and tissue transplantations (including blood transfusions) are a critical care component for many life-threatening diseases. In transfusion and transplantation medicine, the concept of “self” is of crucial importance. Immunological “self” in transfusion medicine is primarily mediated by the ABO/RhD blood group antigens, though several hundred blood groups exist that can cause problems especially in the chronically transfused patient. For most other tissues, “self” is imparted by the major histocompatibility complex (MHC) proteins which provide a means for identifying, targeting and eliminating foreign (allorecognition) or diseased cells while preserving normal tissue. If the differences between donor and recipient occur only at minor MHC molecules (or non-ABO blood group antigens) or the transplanted antigens are only weakly antigenic or immunogenic, successful engraftment may result. In contrast, if significant differences in the exceedingly polymorphic MHC loci are present, or other highly antigenic (e.g., ABO) or immunogenic antigens are present, allorecognition of the donor tissue occurs leading to rejection. Importantly, while rejection may be manifested as either Host versus Graft or Graft versus Host Disease (HVGD and GVHD, respectively), both are mediated by T lymphocyte (T cell) activation, differentiation and proliferation consequent to allorecognition. [Cote et al., 2001] In this chapter, we will demonstrate how we have utilized flow cytometry to measure the induction of tolerance and/or anergy by polymer-mediated immunocamouflage using in vitro and in vivo models of allorecognition.

Red blood cell (RBC) transfusions are an important clinical intervention. However, RBC express hundreds of non-ABO antigens making alloimmunization a significant risk. RhD expression is the most immunologically important non-ABO antigen. Availability of RhD(-) blood, often problematic in North America and Europe, is a significant issue in Asia and Africa where RhD(-) blood is uncommon (<0.5% of supply). The immunocamouflage of RhD is readily accomplished by the covalent grafting of methoxypoly(ethylene glycol) [mPEG] to the RBC membrane. To determine if RhD immunocamouflage would inhibit its immunologic recognition, an in vitro RhD-sensitized antigen presentation assay using PBMC and dendritic cells (DC) from RhD-sensitized women was used. The immunological effects of polymer grafting to an immunodominant RhD peptide, purified RhD protein and intact RhD(+) RBC were examined via T cell proliferation and cytokine release assays. At Day 11, PEGylation significantly attenuated T cell proliferation arising from RhD peptide (~80 → 5%), protein (36 → 0.2%) and intact RBC (33 → 1.4%). Cytokine secretion was similarly blunted following PEGylation of the purified protein or intact RBC. These data support the immunomodulatory effects of PEGylation and the potential utility of this technology in transfusion medicine - especially in situations where RhD(-) blood is rare or in short supply.

Grafting of methoxypoly(ethylene glycol) (mPEG) to cells and biomaterials is a promising non-pharmacological immunomodulation technology. However, due to the labile nature of cells, surface-plasma interactions are poorly understood; hence, a latex bead model was studied. PEGylation of beads resulted in a density and molecular weight dependent decrease in total adsorbed protein with a net reduction from (159.9±6.4) ng cm(-2) on bare latex to (18.4±0.8) and (52.3±5.3) ng cm(-2) on PEGylated beads (1 mmol L(-1) of 2 or 20 kD SCmPEG, respectively). SDS-PAGE and iTRAQ-MS analysis revealed differential compositions of the adsorbed protein layer on the PEGylated latex with a significant reduction in the compositional abundance of proteins involved in immune system activation. Thus, the biological efficacy of immunocamouflaged cells and materials is mediated by both biophysical obfuscation of antigens and reduced surface-macromolecule interactions.

The induction of anergy or tolerance to prevent allorecognition is of clinical interest. To this end, the effects of methoxypoly(ethylene glycol) [mPEG] grafting to allogeneic lymphocytes on proliferation and phenotype (Th17 and Treg) was examined in vitro and in vivo. Control studies demonstrated that PEGylation did not affect cells viability or proliferation (mitogen) potential. Conditioned media (1° MLR) collected at 72 h from resting PBMC demonstrated no immunomodulatory effects whereas the control MLR demonstrated significant (p < 0.001) pro-proliferative potential and significantly increased in IL-2, TNF-α, and INF-γ. However, 1° media from either resting mPEG-PBMC or the PEGylated MLR resulted in a significant inhibitory effect (p < 0.001) in the 2° MLR and no increase in cytokines. PEGylation of donor murine splenocytes resulted in significant in vivo immunosuppressive effects in H2-disparate mice. While unmodified allogeneic splenocytes resulted in a significant in vivo decrease in Treg and increased Th17 lymphocytes, PEGylated allogeneic splenocytes yielded significantly increased Tregs and baseline levels of Th17 lymphocytes. This effect was persistent to at least 30 days post challenge and was not reversed by unmodified allogeneic cells. These studies demonstrate that PEGylation of allogeneic lymphocytes induced an immunoquiescent state both in vitro and in vivo.

降低血型抗原的异体免疫水平对于慢性输血病人有很重要的临床意义.利用甲氧基聚乙二醇修饰红细胞的策略,可以免疫修饰血型抗原以降低红细胞的异体免疫水平.本研究显示,抗体识别非ABO的水平随甲氧基聚乙二醇的分子大小和浓度的升高而显著下降,分子量大的多聚体的免疫保护作用比分子量小的显著.同时,动物实验表明,甲氧基聚乙二醇修饰的异种红细胞(羊红细胞)以及同种不同品系(C57B1/6)或自体(Balb/c)红细胞的体内免疫原性均明显降低.与对照相比,小鼠接受甲氧基聚乙二醇修饰的绵羊血,发现抗绵羊红细胞的抗体产生降低了90％.与对照相比,两次输入甲氧基聚乙二醇修饰的绵羊血的小鼠,抗绵羊血抗体产生降低了80％.尤为重要的是,甲氧基聚乙二醇修饰的自体血细胞没有诱导新抗原的产生,也没有IgG或IgM应答.以上结果表明,聚合物对红细胞的免疫修饰可以安全有效地降低异体免疫的水平,具有潜在的输血医学临床应用价值.

Developing a practical means of reducing alloimmunization in chronically transfused patients would be of significant clinical benefit. Immunocamouflaging red blood cells (RBCs) by membrane grafting of methoxypoly(ethylene glycol) (mPEG) may reduce the risk of allo-immunization. The results of this study showed that antibody recognition of non-ABO antigens was significantly reduced in an mPEG-dose- and polymer size-dependent manner, with higher molecular weight mPEGs providing better immunoprotection. Furthermore, in vivo immunogenicity was significantly reduced in mice serially transfused with mPEG-modified xenogeneic (sheep; sRBCs), allogeneic (C57Bl/6), or syngeneic (Balb/c) RBCs. Following a primary transfusion of sRBCs, mice receiving mPEG-sRBCs showed a >90% reduction in anti-sRBC IgG antibody levels. After two transfusions, mice receiving mPEG-sRBCs showed reductions of >80% in anti-sRBC IgG levels. Importantly, mPEG-modified autologous cells did not induce neoantigens or an immune (IgG or IgM) response. These data suggest that the global immunocamouflage of RBCs by polymer grafting may provide a safe and cost-effective means of reducing the risk of alloimmunization.

BACKGROUND: Anti-glycophorin C (G PC), blood group antibodies of which cause hemolytic disease of the fetus and newborn (HDFN), is a potent inhibitor of erythroid progenitor cell growth. The cellular mechanism for growth inhibition has not been characterized.STUDY DESIGN AND METHODS: K562 cells were incubated in the presence of either anti-GPO, an immunoglobulin G isotype control, an inhibitor of actin polymerization called cytochalasin D with anti-GPO, or cytochalasin D alone. The JC-1 cationic dye was used to detect mitochondrial depolarization and the activity of the mitogen-activated protein kinases was assessed by Western blotting.RESULTS: Anti-GPC inhibits the activity of extracellular regulated kinase (ERK)1/2 within 10 minutes but does not alter the activity of p38 or c-Jun N-terminal kinase. After 24 hours there was a significant loss of mitochondrial membrane potential compared to isotype control treated cells. Both the ERK1/2 inhibition and the loss of mitochondrial potential were prevented by pretreatment with cytochalasin D.CONCLUSION: A cell surface antibody can cause anemia by altering the signaling pathways in erythroid cells by promoting depolarization of mitochondria via cytoskeletal rearrangement. The observation that neonates with anti-GPO HDFN are unresponsive to erythropoietin can be explained by the antibody inhibiting a protein kinase through which this hematopoietic growth factor achieves its effects.

Hematopoiesis is controlled by the interplay between transcription factors and environmental signals. One of the primary determinants of the T-lineage choice is Delta-like (DL)-Notch signaling, which promotes T-cell development and inhibits B-cell development. We have found that the transcription factor HEBAlt is up-regulated in early hematopoietic precursors in response to DL-Notch signaling and that it can promote early T-cell development. Here, we identified a population of lineage-negative Sca-1⁻c-kit(+) (LK) cells in the mouse fetal liver that rapidly gave rise to myeloid cells and B cells but exhibited very little T-cell potential. However, forced expression of HEBAlt in these precursors restored their ability to develop into T cells. We also showed that Ikaros and Notch1 are up-regulated in response to HEBAlt over-expression and that activated Notch1 enhances the ability of LK cells to enter the T-cell lineage. Furthermore, the myeloid transcription factor C/EBPα is down-regulated in response to HEBAlt. We therefore propose that HEBAlt plays a role in the network that enforces the T-lineage fate and limits myeloid fate during hematopoiesis.

Hematopoietic development is controlled by combinatorial interactions between E-protein transcription factors and other lineage regulators that operate in the context of gene-regulatory networks. The E-proteins HEB and E2A are critical for T cell and B cell development, but the mechanisms by which their activities are directed to different genes in each lineage are unclear. We found that a short form of HEB, HEBAlt, acts downstream of Delta-like (DL)-Notch signaling to promote T cell development. In this paper, we show that forced expression of HEBAlt in mouse hematopoietic progenitors inhibited B cell development, but it allowed them to adopt a myeloid fate. HEBAlt interfered with the activity of E2A homodimers and with the expression of the transcription factor Pax5, both of which are critical for B cell development. However, when combined with DL-Notch signaling, HEBAlt enhanced the generation of T cell progenitors at the expense of myeloid cells. The longer form of HEB, HEBCan, also inhibited E47 activity and Pax5 expression, but it did not collaborate with DL-Notch signaling to suppress myeloid potential. Therefore, HEBAlt can suppress B cell or myeloid potential in a context-specific manner, which suggests a role for this factor in maintaining T lineage priming prior to commitment.