Generation of thymic tissue from pluripotent stem cells would provide therapies for acquired and congenital thymic insufficiency states.
This study aimed to generate human thymic epithelial ...progenitors from human embryonic stem cells (hES-TEPs) and to assess their thymopoietic function in vivo.
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.
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.
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.
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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.
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.
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.
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.
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.
Our findings unequivocally demonstrate that CTLA4-Ig does not negatively affect Treg cell frequencies and function in vitro.
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.
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 phase via its intrinsic thioester conferred a dose‐dependent inhibition of TNF‐α and IL‐6 secretion in monocytes activated via Fc‐cross‐linking of up to 50% as compared to baseline. Similarly, C4d conferred an attenuation of intracellular Ca2+ flux in monocytes activated via Fc‐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. FASEB J. 30, 1492–1503 (2016). www.fasebj.org
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.
•Increased human B-cell reconstitution is seen in female compared to male mice in multiple humanized mouse models.•The PI mouse model recapitulates HSC-intrinsic autoimmune defects from T1D and RA bone marrow donors.
Abstract 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.
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 (Kd) 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.
Eur J Clin Invest 2011; 41 (3): 291–298
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‐1β, 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.
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