Generation of HLA-Universal iPSC-Derived Megakaryocytes and Platelets for Survival Under Refractoriness Conditions.

Platelet (PLT) transfusion is indispensable to maintain homeostasis in thrombocytopenic patients. However, PLT transfusion refractoriness is a common life-threatening condition observed in multitransfused patients. The most frequent immune cause for PLT transfusion refractoriness is the presence of alloantibodies specific for human leukocyte antigen (HLA) class I epitopes. Here, we have silenced the expression of HLA class I to generate a stable HLA-universal induced pluripotent stem cell (iPSC) line that can be used as a renewable cell source for the generation of low immunogenic cell products. The expression of HLA class I was silenced by up to 82% and remained stable during iPSC cultivation. In this study, we have focused on the generation of megakaryocytes (MK) and PLTs from a HLA-universal iPSC source under feeder- and xeno-free conditions. On d 19, differentiation rates of MKs and PLTs with means of 58% and 76% were observed, respectively. HLA-universal iPSC-derived MKs showed polyploidy with DNA contents higher than 4n and formed proPLTs. Importantly, differentiated MKs remained silenced for HLA class I expression. HLA-universal MKs produced functional PLTs. Notably, iPSC-derived HLA-universal MKs were capable to escape antibody-mediated complement- and cellular-dependent cytotoxicity. Furthermore, HLA-universal MKs were able to produce PLTs after in vivo transfusion in a mouse model indicating that they might be used as an alternative to PLT transfusion. Thus, in vitro produced low immunogenic MKs and PLTs may become an alternative to PLT donation in PLT-based therapies and an important component in the management of severe alloimmunized patients.

miR-145 Is a Promising Therapeutic Target to Prevent Cornea Scarring.

Corneal scarring is an expected outcome of corneal injury or infection and is one of the major causes for visual loss. The formation of light-scattering myofibroblasts is thought to be the underlying cause of corneal haze formation. Recently, microRNA (miRNA) gene therapies have been proposed as novel approach for complex processes such as fibrosis and scarring. In this study, we focused on the role of miR-145 in corneal myofibroblast differentiation and function. Analysis of human corneal scar tissue and transforming growth factor (TGF)-ß1-induced corneal myofibroblasts showed a 13- and 4-fold increase of miR-145, respectively, compared with healthy cornea and nonstimulated fibroblasts (p<0.01). Furthermore, myofibroblasts showed an increase in α-smooth muscle actin (α-SMA) expression and a decreased expression of Kruppel-like factor 4 (KLF4). These results indicated that TGF-ß1 increases miR-145 expression, which indirectly induces α-SMA expression via downregulation of KLF4, a known negative regulator of α-SMA. Consistently, miR-145 silencing in corneal myofibroblasts using a specific antimiR resulted in increased KLF4 and strongly decreased α-SMA expression. In addition, miR-145 inhibition also significantly decreased myofibroblast contractility, migratory capacity, and TGF-ß1 secretion, which are all thought to contribute to corneal scarring. Hence, miR-145 plays an important role in TGF-ß1-stimulated corneal myofibroblast differentiation and activation, which can be reversed by miR-145 silencing. Therefore, we suggest miR-145 as a promising therapeutic target for miRNA-based gene therapy to prevent or treat visual loss caused by corneal fibrosis.

miR-145 Contributes to Hypertrophic Scarring of the Skin by Inducing Myofibroblast Activity.

Hyperthrophic scarring of the skin is caused by excessive activity of skin myofibroblasts after wound healing and often leads to functional and/or aesthetic disturbance with significant impairment of patient quality of life. MicroRNA (miRNA) gene therapies have recently been proposed for complex processes such as fibrosis and scarring. In this study, we focused on the role of miR-145 in skin scarring and its influence in myofibroblast function. Our data showed not only a threefold increase of miR-145 levels in skin hypertrophic scar tissue but also in transforming growth factor ß1 (TGF-ß1)-induced skin myofibroblasts compared with healthy skin or nontreated fibroblasts (p < 0.001). Consistent with the upregulation of miR-145 induced by TGF-ß1 stimulation of fibroblasts, the expression of Kruppel-like factor 4 (KLF4) was decreased by 50% and α-smooth muscle actin (α-SMA) protein expression showed a threefold increase. Both could be reversed by miR-145 inhibition (p < 0.05). Restoration of KLF4 levels equally abrogated TGF-ß1-induced α-SMA expression. These data demonstrate that TGF-ß1 induces miR-145 expression in fibroblasts, which in turn inhibits KLF4, a known inhibitor of α-SMA, hence upregulating α-SMA expression. Furthermore, treatment of myofibroblasts with a miR-145 inhibitor strongly decreased their α-1 type I collagen expression, TGF-ß1 secretion, contractile force generation and migration. These data demonstrate that upregulation of miR-145 plays an important role in the differentiation and function of skin myofibroblasts. Additionally, inhibition of miR-145 significantly reduces skin myofibroblast activity. Taken together, these results suggest that miR-145 is a promising therapeutic target to prevent or reduce hypertrophic scarring of the skin.

Prevention of rejection of allogeneic endothelial cells in a biohybrid lung by silencing HLA-class I expression.

Variability in Human Leukocyte Antigens (HLA) remains a hurdle to the application of allogeneic cellular products. Due to insufficient autologous endothelial cell harvesting for the biohybrid lung, allogeneic human cord blood derived endothelial cells (HCBEC) were used for the endothelialization of poly-4-methyl-1-pentene (PMP) gas exchange membranes. Therefore, HLA class I expression was silenced stably in HCBECs to prevent rejection. The capacity of HLA class I-silenced HCBEC to abrogate allogeneic immune responses, their functional properties and suitability for endothelialization of PMP membranes were investigated. Delivery of ß2-microglobulin (ß2m)-specific shRNAs reduced ß2m mRNA levels by up to 90% and caused a knockdown of HLA class I expression by up to 85%. HLA-silenced HCBEC abrogated T-cell responses and escaped antibody-mediated complement-dependent cytotoxicity. The EC phenotype and cytokine secretion profiles between HLA-expressing or -silenced HCBEC remained unaltered. EC specific activation (e.g. ICAM) and thrombogenic markers (e.g. thrombomodulin) remained unaffected by HLA-silencing, but their expression was upregulated by TNFα-stimulation. Furthermore, HLA-silenced HCBECs showed high proliferation rates and built an EC monolayer onto PMP membranes. This study represents a new therapeutic concept in the field of cell and organ transplantation and may bring the bioartificial lung as an alternative to lung transplantation closer to reality.

Secreted semaphorin 5A activates immune effector cells and is a biomarker for rheumatoid arthritis.

OBJECTIVE: To investigate the role of the multifunctional protein semaphorin 5A (Sema5A) in modulating cellular immune responses and as a biomarker in rheumatoid arthritis (RA). METHODS: A soluble form of recombinant Sema5A was used to assess its effect on the functions of primary T cells and natural killer (NK) cells isolated from the peripheral blood of healthy donors. Cell proliferation and expression of transcription factors were determined by flow cytometry. Cytokine secretion was analyzed using Luminex technology. Serum samples obtained from 145 patients with RA and control serum samples obtained from healthy individuals or patients with non-RA rheumatic diseases were analyzed for the presence of secreted Sema5A, using enzyme-linked immunosorbent assays and immunoblotting. RESULTS: Soluble Sema5A strongly increased T cell and NK cell proliferation and induced the secretion of proinflammatory Th1/Th17 cytokines. Accordingly, Sema5A stimulation caused significant up-regulation of T-bet and retinoic acid receptor-related orphan nuclear receptor γt levels in T cells. In addition, significantly elevated levels of secreted Sema5A were detected in the serum of patients with RA compared with control serum. Sema5A levels were highest in patients with RA who were positive for the RA biomarker anti-cyclic citrullinated peptide (P < 0.001 versus patients with systemic lupus erythematosus and patients with Sjögren's syndrome) and correlated with the levels of rheumatoid factor. CONCLUSION: Soluble Sema5A is a potent activator of T cells and NK cells in vitro, and high serum levels of Sema5A are associated with RA. Taken together, the results indicate that Sema5A contributes to the pathogenesis of RA through antigen-independent T cell and NK cell activation. Hence, Sema5A is a promising complementary biomarker for the diagnosis of RA.

HLA-universal platelet transfusions prevent platelet refractoriness in a mouse model.

Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA class I antigens constitutes a significant clinical problem. Thus, it would be desirable to have PLT units lacking HLA antigens on the cell surface. Previously, we showed that the generation of functional HLA class I-silenced (HLA-universal) PLTs from CD34(+) cells, using a short hairpin RNA (shRNA) to target ß2-microglobulin (ß2m) transcripts, is feasible. Here, we assessed the capacity of HLA-silenced PLTs to escape HLA antibody-mediated cytotoxicity in vitro and in vivo. Generation of megakaryocytes (MKs) and PLTs was performed by thrombopoietin-mediated differentiation of HLA-silenced CD34(+) cells within 10 days. Lymphocytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) reporter assays using anti-HLA antibodies and a mouse model for PLT refractoriness were used to assess the immune-evasion capability of HLA-universal MKs and PLTs. To mimic PLT refractoriness in vivo, NOD/SCID/IL-2Rγc(-/-) mice were injected with specific anti-HLA antibodies followed by the infusion of 1 × 10(6) HLA-universal MKs. In vivo PLT generation was evaluated by flow cytometry using anti-CD42a and CD61 antibodies. Cells expressing a nonspecific shRNA were used as control. Lymphocytotoxicity and ADCC reporter assays showed that HLA silencing protects MKs against HLA antibody-mediated complement-dependent and cell-mediated cytotoxicity. In lymphocytotoxicity assays, 80-90% of HLA-expressing MKs but only 3% of HLA-silenced MKs were lysed. In the circulation of mice, HLA-expressing and HLA-silenced MKs showed PLT production in the absence of anti-HLA antibodies, with human PLT frequencies of up to 0.5% within the PLT population. However, in presence of anti-HLA antibodies HLA-expressing MKs were rapidly cleared from the circulation of mice, whereas HLA-silenced MKs escaped HLA antibody-mediated cytotoxicity and produced PLTs that were detectable up to 11 days. Our data show that HLA-silenced PLTs are efficiently protected against HLA antibody-mediated cytotoxicity and prevent PLT refractoriness in vivo. Provision of HLA-silenced PLTs may become an important component in the management of patients refractory to PLT transfusion.

Semaphorin 7A protein variants differentially regulate T-cell activity.

BACKGROUND: Semaphorin 7A (Sema7A) carries the John-Milton-Hagen human blood group antigen on red blood cells and shows molecular diversity. It is known that Sema7A has immunomodulatory functions, but its regulatory effects on T-cell activation are not completely understood. In this study, the functional role of the R461C Sema7A polymorphism on T-cell responses was investigated. STUDY DESIGN AND METHODS: Soluble recombinant wild-type Sema7A (Sema7A_wt) and its R461C variant (Sema7A_R461C) were produced in human embryonic kidney cells. Specific assays were performed to determine the effects of Sema7A_wt and Sema7A_R461C on T-cell activation in terms of proliferation, phenotypic alterations, granzyme B transcript levels, and secretion of proinflammatory cytokines. RESULTS: Sema7A_wt did not affect T-cell activity, but Sema7A_R461C led to marked antigen-independent activation of T cells. In the presence of antigen stimulation, Sema7A_R461C had a major costimulatory effect on T-cell response. Upon Sema7A_R461C stimulation, CD4+ T cells strongly proliferated and exhibited a cytotoxic phenotype with significant up regulation of granzyme B transcripts (up to 220-fold), even in the absence of antigen stimulation. Antibody blocking studies indicated that Sema7A_R461C-mediated T-cell activation is largely ß1 integrin dependent. CONCLUSION: These data demonstrate that Sema7A_R461C, unlike wild-type Sema7A, causes differential regulation of T-cell responses. Since Sema7A has important immunomodulatory functions in inflammatory responses, it might play a key role in autoimmune diseases and other major disorders. Further studies are needed to elucidate the regulatory role of Sema7A and its variants.