Defects in Long-Term APC Repopulation Ability of Adult Human Bone Marrow Hematopoietic Stem Cells (HSCs) Compared with Fetal Liver HSCs.

Immunodeficient mice reconstituted with immune systems from patients, or personalized immune (PI) mice, are powerful tools for understanding human disease. Compared with immunodeficient mice transplanted with human fetal thymus tissue and fetal liver-derived CD34+ cells administered i.v. (Hu/Hu mice), PI mice, which are transplanted with human fetal thymus and adult bone marrow (aBM) CD34+ cells, demonstrate reduced levels of human reconstitution. We characterized APC and APC progenitor repopulation in human immune system mice and detected significant reductions in blood, bone marrow (BM), and splenic APC populations in PI compared with Hu/Hu mice. APC progenitors and hematopoietic stem cells (HSCs) were less abundant in aBM CD34+ cells compared with fetal liver-derived CD34+ cell preparations, and this reduction in APC progenitors was reflected in the BM of PI compared with Hu/Hu mice 14-20 wk posttransplant. The number of HSCs increased in PI mice compared with the originally infused BM cells and maintained functional repopulation potential, because BM from some PI mice 28 wk posttransplant generated human myeloid and lymphoid cells in secondary recipients. Moreover, long-term PI mouse BM contained functional T cell progenitors, evidenced by thymopoiesis in thymic organ cultures. Injection of aBM cells directly into the BM cavity, transgenic expression of hematopoietic cytokines, and coinfusion of human BM-derived mesenchymal stem cells synergized to enhance long-term B cell and monocyte levels in PI mice. These improvements allow a sustained time frame of 18-22 wk where APCs and T cells are present and greater flexibility for modeling immune disease pathogenesis and immunotherapies in PI mice.

Human stem cell-derived thymic epithelial cells enhance human T-cell development in a xenogeneic thymus.

BACKGROUND: Generation of thymic tissue from pluripotent stem cells would provide therapies for acquired and congenital thymic insufficiency states. OBJECTIVES: This study aimed to generate human thymic epithelial progenitors from human embryonic stem cells (hES-TEPs) and to assess their thymopoietic function in vivo. METHODS: This study differentiated hES-TEPs by mimicking developmental queues with FGF8, retinoic acid, SHH, Noggin, and BMP4. Their function was assessed in reaggregate cellular grafts under the kidney capsule and in hybrid thymi by incorporating them into swine thymus (SwTHY) grafts implanted under the kidney capsules of immunodeficient mice that received human hematopoietic stem and progenitor cells (hHSPCs) intravenously. RESULTS: Cultured hES-TEPs expressed FOXN1 and formed colonies expressing EPCAM and both cortical and medullary thymic epithelial cell markers. In thymectomized immunodeficient mice receiving hHSPCs, hES-TEPs mixed with human thymic mesenchymal cells supported human T-cell development. Hypothesizing that support from non-epithelial thymic cells might allow long-term function of hES-TEPs, the investigators injected them into SwTHY tissue, which supports human thymopoiesis in NOD severe combined immunodeficiency IL2Rγnull mice receiving hHSPCs. hES-TEPs integrated into SwTHY grafts, enhanced human thymopoiesis, and increased peripheral CD4+ naive T-cell reconstitution. CONCLUSIONS: This study has developed and demonstrated in vivo thymopoietic function of hES-TEPs generated with a novel differentiation protocol. The SwTHY hybrid thymus model demonstrates beneficial effects on human thymocyte development of hES-TEPs maturing in the context of a supportive thymic structure.

Mixed xenogeneic porcine chimerism tolerizes human anti-pig natural antibody-producing cells in a humanized mouse model.

BACKGROUND: A major obstacle to the success of organ transplantation from pigs to humans, necessitated by the shortage of human organs, is robust humoral immune rejection by pig-reactive human antibodies. Mixed xenogeneic hematopoietic chimerism induces xenoreactive B cell tolerance in rodents, but whether mixed pig/human chimerism could induce tolerance of human B cells to pig xenoantigens is unknown. METHODS: We investigated this question using a humanized mouse model in which durable mixed (pig-human) xenogeneic chimerism can be established. RESULTS: Human natural anti-pig cytotoxic antibodies, predominantly IgM, are detectable in non-chimeric humanized mouse serum, and pig-reactive antibodies were reduced in mixed chimeric versus non-chimeric humanized mice. This difference required persistent mixed chimerism and was not due to the adsorption of antibodies on pig cells in vivo. Furthermore, human B cells from spleens of mixed chimeric mice produced lower levels of anti-pig antibodies when stimulated in vitro compared with those from non-chimeric mice. CONCLUSIONS: Our findings demonstrate that mixed chimerism reduces human natural antibodies to pig xenoantigens, providing the first in vivo evidence of human B cell tolerance induction by mixed xenogeneic chimerism and supporting further evaluation of this approach for inducing human B cell tolerance to xenografts.

Ig-like transcript 4 as a cellular receptor for soluble complement fragment C4d.

Complement regulation leads to the generation of complement split products (CSPs) such as complement component (C)4d, a marker for disease activity in autoimmune syndromes or antibody-mediated allograft rejection. However, the physiologic role of C4d has been unknown. By screening murine thymoma BW5147 cells expressing a cDNA library generated from human monocyte-derived dendritic cells with recombinant human C4d, we identified Ig-like transcript (ILT)4 and ILT5v2 as cellular receptors for C4d. Both receptors, expressed on monocytes, macrophages, and dendritic cells, also interacted with the CSPs C3d, C4b, C3b, and iC3b. However, C4d did not bind to classic complement receptors (CRs). Interaction between cell surface-resident ILT4 and soluble monomeric C4d resulted in endocytosis of C4d. Surprisingly, binding of soluble ILT4 to C4d covalently immobilized to a cellular surface following classic complement activation could not be detected. Remarkably, C4d immobilized to a solid phaseviaits intrinsic thioester conferred a dose-dependent inhibition of TNF-α and IL-6 secretion in monocytes activatedviaFc-cross-linking of up to 50% as compared to baseline. Similarly, C4d conferred an attenuation of intracellular Ca(2+)flux in monocytes activatedviaFc-cross-linking. In conclusion, ILT4 represents a scavenger-type endocytotic CR for soluble monomeric C4d, whereas attenuation of monocyte activation by physiologically oriented C4d on a surface appears to be dependent on a yet to be identified C4d receptor.-Hofer, J., Forster, F., Isenman, D. E., Wahrmann, M., Leitner, J., Hölzl, M. A., Kovarik, J. K., Stockinger, H., Böhmig, G. A., Steinberger, P., Zlabinger, G. J. Ig-like transcript 4 as a cellular receptor for soluble complement fragment C4d.

Eicosanoid modulation by the short-chain fatty acid n-butyrate in human monocytes.

n-Butyrate deriving from bacterial fermentation in the mammalian intestine is a key determinant in gastrointestinal homeostasis. We examined the effects of this short-chain fatty acid and Toll-like receptor 2 (TLR) and TLR4 engagement on inflammatory/immunity-associated genes, cyclo-oxygenases (COXs), prostaglandins (PGs) and leukotrienes (LTs) in human monocytes. Before RNA isolation, freshly isolated human monocytes were co-incubated for different time-points with 1 mm n-butyrate alone or in combination with bacterial stimuli. Based on a knowledge-driven approach, a signature of 180 immunity/inflammation-associated genes was picked and real-time PCR analysis was performed. Pathway analysis was carried out using a web-based database analysing program. Based on these gene expression studies the findings were evaluated at the protein/mediator level by Western blot analysis, FACS and ELISA. Following co-incubation with n-butyrate and lipopolysaccharide, key enzymes of the eicosanoid pathway, like PTGS2 (COX-2), TXS, ALOX5, LTA4H and LTC4S, were significantly up-regulated compared with stimulation with lipopolysaccharide alone. Furthermore, release of the lipid mediators PGE(2), 15d-PGJ(2), LTB(4) and thromboxane B(2) was increased by n-butyrate. Regarding signalling, n-butyrate had no additional effect on mitogen-activated protein kinase and interfered differently with early and late phases of nuclear factor-κB signalling. Our results suggest that among many other mediators of eicosanoid signalling n-butyrate massively induces PGE(2) production by increasing the expression of PTGS2 (COX-2) in monocytes following TLR4 and TLR2 activation and induces secretion of LTB(4) and thromboxane B(2). This underscores the role of n-butyrate as a crucial mediator of gut-specific immunity.

The zymogen granule protein 2 (GP2) binds to scavenger receptor expressed on endothelial cells I (SREC-I).

The pancreatic zymogen granule membrane protein (GP2) is expressed by pancreatic acinar cells and M cells of the ileum. GP2 is the closest related homologue of the urine resident Tamm-Horsfall protein (THP). Recently, it was shown that THP is a ligand of various scavenger receptors (SRs). Therefore, we were interested, if GP2 has similar properties. cDNA of different SRs was stably transfected into a murine thymoma cell line. GP2 was recombinantly expressed, purified and biotinylated. Binding or uptake of GP2 by transfected cells or monocyte-derived dendritic cells (moDCs) was analyzed by flow-cytometry. GP2 is a binding partner of the scavenger receptor expressed on endothelial cells I (SREC-I) but not of SR-AI and SR-BI. The dissociation constant (K(d)) of GP2 binding to SREC-I is 41.3nM. SREC transfected cells are able to internalize GP2. moDCs express SREC-I and also bind and internalize GP2. Inhibition of SREC-I on moDCs with anti-SREC-I antibodies does not result in a decreased GP2 binding. Interaction of GP2 with SREC-I and uptake might have profound effects in antigen clearance and mediation of the immune response. In addition to SREC-I other presently unknown receptors for GP2 on DCs might be involved in this process.

Impaired anti-inflammatory efficacy of n-butyrate in patients with IBD.

BACKGROUND: The intestinal mucosa of patients with inflammatory bowel diseases (IBD) characteristically shows a high degree of inflammation when compared to healthy subjects. This appears to be attributable to an imbalance in local reactivity of inflammatory cells. In the present study, we tested the hypothesis that immune cells from patients with IBD are less sensitive to anti-inflammatory agents in the gut as exemplified by the short-chain fatty acid (SCFA) n-butyrate. MATERIAL AND METHODS: Peripheral blood mononuclear cells (PBMC) of patients with IBD (22 Crohn`s Disease, CD; 9 Ulcerative Colitis, UC) and 20 healthy individuals were stimulated through TLR-4 and TLR-2 engagement, respectively, and the anti-inflammatory activity of n-butyrate (0·06-1 mM) on cytokine production (IL-1ß, IL-10, IL-12/23p40, TNF-α) was assessed. Inhibition curves were generated, and effective doses (ED20-ED80) were determined. RESULTS: Hyperresponsiveness to TLR-2 activation reflected by increased IL-12/23p40 and TNF-α production was observed in patients with IBD. To inhibit the release of IL-12/23p40 from PBMC after activation via TLR2-agonists, higher concentrations of n-butyrate were required in patients with IBD , when compared to healthy subjects. With regard to TLR-4 activation, PBMC from patients with IBD and controls were equally responsive to the immunoregulatory effects of n-butyrate. Further analysis revealed that the impaired sensitivity of PBMC to the anti-inflammatory action of n-butyrate was independent from hyperreactivity of immunocompetent cells. CONCLUSIONS: Impaired sensitivity to the inhibitory action of n-butyrate in IBD may constitute a determinant in the pathogenesis of these inflammatory diseases.

Host antimicrobial proteins as endogenous immunomodulators.

Host defense mechanisms are multilayered and involve physical as well as chemical barriers, antimicrobial factors as well as a broad set of immunocompetent cells. The mode of action of antimicrobial factors is variable, ranging from opsonisation and agglutination to direct killing of pathogens. In the last years it has become increasingly clear that some of these factors act as endogenous ligands that bind to distinct host receptors, as for example pathogen recognition receptors (PRRs), thereby influencing distinct immunological processes like chemotaxis, modulation of phagocytosis, dendritic cell maturation or the production of cytokines. By that way, these factors are implicated to protect the host by preventing and clearing of microbial infections.

Fasting metabolism modulates the interleukin-12/interleukin-10 cytokine axis.

A crucial role of cell metabolism in immune cell differentiation and function has been recently established. Growing evidence indicates that metabolic processes impact both, innate and adaptive immunity. Since a down-stream integrator of metabolic alterations, mammalian target of rapamycin (mTOR), is responsible for controlling the balance between pro-inflammatory interleukin (IL)-12 and anti-inflammatory IL-10, we investigated the effect of upstream interference using metabolic modulators on the production of pro- and anti-inflammatory cytokines. Cytokine release and protein expression in human and murine myeloid cells was assessed after toll-like receptor (TLR)-activation and glucose-deprivation or co-treatment with 5'-adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators. Additionally, the impact of metabolic interference was analysed in an in-vivo mouse model. Glucose-deprivation by 2-deoxy-D-glucose (2-DG) increased the production of IL-12p40 and IL-23p19 in monocytes, but dose-dependently inhibited the release of anti-inflammatory IL-10. Similar effects have been observed using pharmacological AMPK activation. Consistently, an inhibition of the tuberous sclerosis complex-mTOR pathway was observed. In line with our in vitro observations, glycolysis inhibition with 2-DG showed significantly reduced bacterial burden in a Th2-prone Listeria monocytogenes mouse infection model. In conclusion, we showed that fasting metabolism modulates the IL-12/IL-10 cytokine balance, establishing novel targets for metabolism-based immune-modulation.

HSC extrinsic sex-related and intrinsic autoimmune disease-related human B-cell variation is recapitulated in humanized mice.

B cells play a major role in antigen presentation and antibody production in the development of autoimmune diseases, and some of these diseases disproportionally occur in females. Moreover, immune responses tend to be stronger in female vs male humans and mice. Because it is challenging to distinguish intrinsic from extrinsic influences on human immune responses, we used a personalized immune (PI) humanized mouse model, in which immune systems were generated de novo from adult human hematopoietic stem cells (HSCs) in immunodeficient mice. We assessed the effect of recipient sex and of donor autoimmune diseases (type 1 diabetes [T1D] and rheumatoid arthritis [RA]) on human B-cell development in PI mice. We observed that human B-cell levels were increased in female recipients regardless of the source of human HSCs or the strain of immunodeficient recipient mice. Moreover, mice injected with T1D- or RA-derived HSCs displayed B-cell abnormalities compared with healthy control HSC-derived mice, including altered B-cell levels, increased proportions of mature B cells and reduced CD19 expression. Our study revealed an HSC-extrinsic effect of recipient sex on human B-cell reconstitution. Moreover, the PI humanized mouse model revealed HSC-intrinsic defects in central B-cell tolerance that recapitulated those in patients with autoimmune diseases. These results demonstrate the utility of humanized mouse models as a tool to better understand human immune cell development and regulation.

CTLA4-Ig preserves thymus-derived T regulatory cells.

BACKGROUND: CTLA-4 immunoglobulin fusion proteins (CTLA4-Ig) suppress immune reactions by blocking the T-cell costimulatory CD28-CD80-86 pathway and are used in clinical trials for diseases featuring exaggerated T-cell reactivity including autoimmune diseases and allograft rejection. However, because CTLA4-Ig has been suspected to interfere with T regulatory (Treg) cell homeostasis and function, recently, substantial concerns on CTLA4-Ig's potentially antitolerogenic effects have been raised. METHODS: We tested immunoregulatory CTLA4-Ig explicitly for its effect on Treg cell numbers, frequencies and function in an in vitro murine major histocompatibility complex mismatched setting using C57BL/6 bone marrow-derived dendritic cells as stimulators of allogeneic Balb/c Foxp3 T cells, which allowed for tracing Treg cells in a straightforward fashion. RESULTS: The presence of CTLA4-Ig in mixed leukocyte reactions-while dampening the global proliferative response of allostimulated Balb/c T cells-resulted in a relative increase of the frequency of thymus-derived CD4CD25Foxp3 Treg cells with intact suppressive activity. This relative increase was caused by a selective inhibitory effect of CTLA4-Ig on proliferating conventional T cells, whereas the proliferative capacity of Treg cells in cell cultures remained unaffected. Additionally, in the presence of CTLA4-Ig, the frequency of apoptosis was decreased in these cells. CONCLUSION: Our findings unequivocally demonstrate that CTLA4-Ig does not negatively affect Treg cell frequencies and function in vitro.

Quantification of α-galactosidase activity in intact leukocytes.

BACKGROUND: Mutations of the α-galactosidase (α-Gal) A gene in Fabry disease lead to a severe disturbance in glycosphingolipid catabolism. The atypical clinical picture of Fabry disease hampers diagnosis, resulting in a delayed start of therapy. Current tests utilize leukocyte lysates to evaluate the activity of α-Gal A. It has never been investigated whether cell homogenisation is necessary. METHODS: Isolated leukocyte subsets were incubated with the α-Gal substrate methylumbelliferyl-α-D galactopyranosid (MU-Gal) and substrate conversion was measured by fluorimetry. Specificity of the reaction was evaluated using the α-Gal inhibitor deoxygalactonojirimycin (DGJ). The novel procedure was compared to the standard method. A reference population and Fabry patients were tested. RESULTS: Substrate conversion in intact leukocytes was a function of substrate concentration, cell number and time and could be inhibited by DGJ. Monocytes showed the highest enzyme activity among leukocyte populations. The novel procedure highly correlated with the standard method. Both Fabry hemizygotes and heterozygotes showed reduced substrate conversion. CONCLUSION: We here present a novel sensitive, fast and simple procedure for the evaluation of α-Gal activity suitable to identify enzyme deficiencies in Fabry patients. Furthermore, we show for the first time that leukocyte subtypes have different α-Gal activities.