Correction: Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

[This corrects the article DOI: 10.1371/journal.ppat.1008560.].

Downregulation of Swine Leukocyte Antigen Expression Decreases the Strength of Xenogeneic Immune Responses towards Renal Proximal Tubular Epithelial Cells.

Xenotransplantation reemerged as a promising alternative to conventional transplantation enlarging the available organ pool. However, success of xenotransplantation depends on the design and selection of specific genetic modifications and on the development of robust assays allowing for a precise assessment of tissue-specific immune responses. Nevertheless, cell-based assays are often compromised by low proliferative capacity of primary cells. Proximal tubular epithelial cells (PTECs) play a crucial role in kidney function. Here, we generated immortalized PTECs (imPTECs) by overexpression of simian virus 40 T large antigen. ImPTECs not only showed typical morphology and phenotype, but, in contrast to primary PTECs, they maintained steady cell cycling rates and functionality. Furthermore, swine leukocyte antigen (SLA) class I and class II transcript levels were reduced by up to 85% after transduction with lentiviral vectors encoding for short hairpin RNAs targeting ß2-microglobulin and the class II transactivator. This contributed to reducing xenogeneic T-cell cytotoxicity (p < 0.01) and decreasing secretion of pro-inflammatory cytokines such as IL-6 and IFN-γ. This study showed the feasibility of generating highly proliferative PTECs and the development of tissue-specific immunomonitoring assays. Silencing SLA expression on PTECs was demonstrated to be an effective strategy to prevent xenogeneic cellular immune responses and may strongly support graft survival after xenotransplantation.

Repertoire characterization and validation of gB-specific human IgGs directly cloned from humanized mice vaccinated with dendritic cells and protected against HCMV.

Human cytomegalovirus (HCMV) causes serious complications to immune compromised hosts. Dendritic cells (iDCgB) expressing granulocyte-macrophage colony-stimulating factor, interferon-alpha and HCMV-gB were developed to promote de novo antiviral adaptive responses. Mice reconstituted with a human immune system (HIS) were immunized with iDCgB and challenged with HCMV, resulting into 93% protection. Immunization stimulated the expansion of functional effector memory CD8+ and CD4+ T cells recognizing gB. Machine learning analyses confirmed bone marrow T/CD4+, liver B/IgA+ and spleen B/IgG+ cells as predictive biomarkers of immunization (≈87% accuracy). CD8+ and CD4+ T cell responses against gB were validated. Splenic gB-binding IgM-/IgG+ B cells were sorted and analyzed at a single cell level. iDCgB immunizations elicited human-like IgG responses with a broad usage of various IgG heavy chain V gene segments harboring variable levels of somatic hypermutation. From this search, two gB-binding human monoclonal IgGs were generated that neutralized HCMV infection in vitro. Passive immunization with these antibodies provided proof-of-concept evidence of protection against HCMV infection. This HIS/HCMV in vivo model system supported the validation of novel active and passive immune therapies for future clinical translation.

Immunogenetics of xenotransplantation.

Xenotransplantation may become the highly desired solution to close the gap between the availability of donated organs and number of patients on the waiting list. In recent years, enormous progress has been made in the development of genetically engineered donor pigs. The introduced genetic modifications showed to be efficient in prolonging xenograft survival. In this review, we focus on the type of immune responses that may target xeno-organs after transplantation and promising immunogenetic modifications that show a beneficial effect in ameliorating or eliminating harmful xenogeneic immune responses. Increasing histocompatibility of xenografts by eliminating genetic discrepancies between species will pave their way into clinical application.

Generating low immunogenic pig pancreatic islet cell clusters for xenotransplantation.

Xenotransplantation of pancreatic islets offers a promising alternative to overcome the shortage of allogeneic donors. Despite significant advances, either immune rejection or oxygen supply in immune protected encapsulated islets remains major bottlenecks for clinical application. To decrease xenogeneic immune responses, we generated tissue engineered swine leucocyte antigen (SLA)-silenced islet cell clusters (ICC). Single-cell suspensions from pancreatic islets were generated by enzymatic digestion of porcine ICCs. Cells were silenced for SLA class I and class II by lentiviral vectors encoding for short hairpin RNAs targeting beta2-microglobulin or class II transactivator, respectively. SLA-silenced ICCs-derived cells were then used to form new ICCs in stirred bioreactors in the presence of collagen VI. SLA class I silencing was designed to reach a level of up to 89% and class II by up to 81% on ICCs-derived cells. Xenogeneic T cell immune responses, NK cell and antibody-mediated cellular-dependent immune responses were significantly decreased in SLA-silenced cells. In stirred bioreactors, tissue engineered islets showed the typical 3D structure and insulin production. These data show the feasibility to generate low immunogenic porcine ICCs after single-cell engineering and post-transduction islet reassembling that might serve as an alternative to allogeneic pancreatic islet cell transplantation.

Triple (GGTA1, CMAH, B2M) modified pigs expressing an SLA class Ilow phenotype-Effects on immune status and susceptibility to human immune responses.

Porcine xenografts lacking swine leukocyte antigen (SLA) class I are thought to be protected from human T cell responses. We have previously shown that SLA class I deficiency can be achieved in pigs by CRISPR/Cas9-mediated deletion of ß2 -microglobulin (B2M). Here, we characterized another line of genetically modified pigs in which targeting of the B2M locus did not result in complete absence of B2M and SLA class I but rather in significantly reduced expression levels of both molecules. Residual SLA class I was functionally inert, because no proper differentiation of the CD8+ T cell subset was observed in B2Mlow pigs. Cells from B2Mlow pigs were less capable in triggering proliferation of human peripheral blood mononuclear cells in vitro, which was mainly due to the nonresponsiveness of CD8+ T cells. Nevertheless, cytotoxic effector cells developing from unaffected cell populations (eg, CD4+ T cells, natural killer cells) lysed targets from both SLA class I+ wildtype and SLA class Ilow pigs with similar efficiency. These data indicate that the absence of SLA class I is an effective approach to prevent the activation of human CD8+ T cells during the induction phase of an anti-xenograft response. However, cytotoxic activity of cells during the effector phase cannot be controlled by this approach.

Towards Reduction or Substitution of Cytotoxic DMSO in Biobanking of Functional Bioengineered Megakaryocytes.

Donor platelet transfusion is currently the only efficient treatment of life-threatening thrombocytopenia, but it is highly challenged by immunological, quality, and contamination issues, as well as short shelf life of the donor material. Ex vivo produced megakaryocytes and platelets represent a promising alternative strategy to the conventional platelet transfusion. However, practical implementation of such strategy demands availability of reliable biobanking techniques, which would permit eliminating continuous cell culture maintenance, ensure time for quality testing, enable stock management and logistics, as well as availability in a ready-to-use manner. At the same time, protocols applying DMSO-based cryopreservation media were associated with increased risks of adverse long-term side effects after patient use. Here, we show the possibility to develop cryopreservation techniques for iPSC-derived megakaryocytes under defined xeno-free conditions with significant reduction or complete elimination of DMSO. Comprehensive phenotypic and functional in vitro characterization of megakaryocytes has been performed before and after cryopreservation. Megakaryocytes cryopreserved DMSO-free, or using low DMSO concentrations, showed the capability to produce platelets in vivo after transfusion in a mouse model. These findings propose biobanking approaches essential for development of megakaryocyte-based replacement and regenerative therapies.

Repeated Freezing Procedures Preserve Structural and Functional Properties of Amniotic Membrane for Application in Ophthalmology.

For decades, the unique regenerative properties of the human amniotic membrane (hAM) have been successfully utilized in ophthalmology. As a directly applied biomaterial, the hAM should be available in a ready to use manner in clinical settings. However, an extended period of time is obligatory for performing quality and safety tests. Hence, the low temperature storage of the hAM is a virtually inevitable step in the chain from donor retrieval to patient application. At the same time, the impact of subzero temperatures carries an increased risk of irreversible alterations of the structure and composition of biological objects. In the present study, we performed a comprehensive analysis of the hAM as a medicinal product; this is intended for a novel strategy of application in ophthalmology requiring a GMP production protocol including double freezing-thawing cycles. We compared clinically relevant parameters, such as levels of growth factors and extracellular matrix proteins content, morphology, ultrastructure and mechanical properties, before and after one and two freezing cycles. It was found that epidermal growth factor (EGF), transforming growth factor beta 1 (TGF-ß1), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), hyaluronic acid, and laminin could be detected in all studied conditions without significant differences. Additionally, histological and ultrastructure analysis, as well as transparency and mechanical tests, demonstrated that properties of the hAM required to support therapeutic efficacy in ophthalmology are not impaired by dual freezing.

Silencing of HLA class I on primary human hepatocytes as a novel strategy for reduction in alloreactivity.

In contrast to the whole liver, primary hepatocytes are highly immunogenic. Thus, alternative strategies of immunomodulation after hepatocyte transplantation are of special interest. Silencing of HLA class I expression is expected to reduce the strength of allogeneic immune responses and to improve graft survival. In this study, primary human hepatocytes (PHH) were isolated using a two-step-collagenase perfusion-technique and co-cultured with allogeneic lymphocytes in terms of a mixed lymphocyte hepatocyte culture. Expression of HLA class I on PHH was silenced using lentiviral vectors encoding for ß2-microglobulin-specific short hairpin RNA (shß2m) or non-specific shRNA (shNS) as control. The delivery of shß2m into PHH caused a decrease by up to 96% in ß2m transcript levels and a down-regulation of HLA class I cell surface expression on PHH by up to 57%. Proliferative T cell alloresponses against HLA-silenced PHH were significantly lower than those observed form fully HLA-expressing PHH. In addition, significantly lower secretion of pro-inflammatory cytokines was observed. Levels of albumin, urea and aspartate-aminotransferase did not differ in supernatants of cultured PHH. In conclusion, silencing HLA class I expression on PHH might represent a promising approach for immunomodulation in the transplant setting without compromising metabolic function of silenced hepatocytes.

Characterization of induced pluripotent stem cell-derived megakaryocyte lysates for potential regenerative applications.

Recently, platelet-derived growth factors present in lysates became an extreme interest in the field of regenerative medicine such as in wound healing and as substitutes to foetal bovine serum in xeno-free cell culture systems. However, the generation of such platelet lysates completely depends on the availability of platelet donors. In this study, the possibility to use in vitro-generated megakaryocytes derived from induced pluripotent stem cells (iPSCs) as a cell source for typical platelet growth factors was investigated. Therefore, the presence and levels of those factors were characterized in in vitro-produced megakaryocytes. In comparison with platelets, in vitro-generated megakaryocytes showed a multifold increased content in transcript and protein levels of typical platelet growth factors including platelet-derived growth factors (PDGFs), transforming growth factor (TGF)-1ß, vascular endothelial cell factor (VEGF)-A, epidermal growth factor (EGF), insulin-like growth factor (IGF)-1 and tissue factor (TF). Hence, iPSC-derived megakaryocytes may serve as an efficient cell source for a donor-independent generation of growth factor-rich lysates with a broad application potential in innovative cell culture systems and regenerative therapies.

Hepatocyte-induced CD4+ T cell alloresponse is associated with major histocompatibility complex class II up-regulation on hepatocytes and suppressible by regulatory T cells.

Hepatocyte transplantation is a promising therapeutic approach for various liver diseases. Despite the liver's tolerogenic potential, early immune-mediated loss of transplanted cells is observed, and longterm acceptance has not been achieved yet. Patients deemed tolerant after liver transplantation presented an increased frequency of regulatory T cells (Tregs), which therefore also might enable reduction of posttransplant cell loss and enhance longterm allograft acceptance. We hence characterized hepatocyte-induced immune reactions and evaluated the immunomodulatory potential of Tregs applying mixed lymphocyte cultures and mixed lymphocyte hepatocyte cultures. These were set up using peripheral blood mononuclear cells and primary human hepatocytes, respectively. Polyclonally expanded CD4+ CD25high CD127low Tregs were added to cocultures in single-/trans-well setups with/without supplementation of anti-interferon γ (IFNγ) antibodies. Hepatocyte-induced alloresponses were then analyzed by multicolor flow cytometry. Measurements indicated that T cell response upon stimulation was associated with IFNγ-induced major histocompatibility complex (MHC) class II up-regulation on hepatocytes and mediated by CD4+ T cells. An indirect route of antigen presentation could be ruled out by use of fragmented hepatocytes and culture supernatants of hepatocytes. Allospecific proliferation was accompanied by inflammatory cytokine secretion. CD8+ T cells showed early up-regulation of CD69 despite lack of cell proliferation in the course of coculture. Supplementation of Tregs effectively abrogated hepatocyte-induced alloresponses and was primarily cell contact dependent. In conclusion, human hepatocytes induce a CD4+ T cell alloresponse in vitro, which is associated with MHC class II up-regulation on hepatocytes and is susceptible to suppression by Tregs. Liver Transplantation 24 407-419 2018 AASLD.

Human Effector Memory T Helper Cells Engage with Mouse Macrophages and Cause Graft-versus-Host-Like Pathology in Skin of Humanized Mice Used in a Nonclinical Immunization Study.

Humanized mice engrafted with human hematopoietic stem cells and developing functional human T-cell adaptive responses are in critical demand to test human-specific therapeutics. We previously showed that humanized mice immunized with long-lived induced-dendritic cells loaded with the pp65 viral antigen (iDCpp65) exhibited a faster development and maturation of T cells. Herein, we evaluated these effects in a long-term (36 weeks) nonclinical model using two stem cell donors to assess efficacy and safety. Relative to baseline, iDCpp65 immunization boosted the output of effector memory CD4+ T cells in peripheral blood and lymph nodes. No weight loss, human malignancies, or systemic graft-versus-host (GVH) disease were observed. However, for one reconstitution cohort, some mice immunized with iDCpp65 showed GVH-like signs on the skin. Histopathology analyses of the inflamed skin revealed intrafollicular and perifollicular human CD4+ cells near F4/80+ mouse macrophages around hair follicles. In spleen, CD4+ cells formed large clusters surrounded by mouse macrophages. In plasma, high levels of human T helper 2-type inflammatory cytokines were detectable, which activated in vitro the STAT5 pathway of murine macrophages. Despite this inflammatory pattern, human CD8+ T cells from mice with GVH reacted against the pp65 antigen in vitro. These results uncover a dynamic cross-species interaction between human memory T cells and mouse macrophages in the skin and lymphatic tissues of humanized mice.

Towards the Manufacture of Megakaryocytes and Platelets for Clinical Application.

Platelet transfusions are used in standard clinical practice to prevent hemorrhage in patients suffering from thrombocytopenia or platelet dysfunctions. Recently, a constant rise on the demand of platelets for transfusion has been registered. This may be associated with several factors including demographic changes, population aging as well as incidence and prevalence of hematological diseases. In addition, platelet-regenerative properties have been started to be exploited in different areas such as tissue remodeling and anti-cancer therapies. These new applications are also expected to increase the future demand on platelets. Thus, in vitro generated platelets may constitute a highly desirable alternative to meet the rising demand on platelets. Several factors have been considered in the road trip of producing in vitro megakaryocytes and platelets for clinical application. From selection of the cell source, differentiation protocols and culture conditions to the design of optimal bioreactors, several strategies have been proposed to maximize production yields while preserving functionality. This review summarizes new advances in megakaryocyte and platelet differentiation and their production upscaling.

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.

Discovery of immunodominant T-cell epitopes reveals penton protein as a second immunodominant target in human adenovirus infection.

BACKGROUND: Human adenovirus (HAdV) infections remain a significant cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Efficient antiviral T-cell responses are necessary to clear infection, which is hampered by delayed immune reconstitution and medical immunosuppression after HSCT. Protective immunity may be conferred by adoptive transfer of HAdV-specific T cells. For identification of patients at risk and monitoring of treatment responses diligent assessment of anti-HAdV cellular immune responses is crucial. The HAdV-derived protein hexon has been recognized as a major immunodominant target across HAdV species. We aimed at identifying further targets of protective anti-HAdV immune response and characterizing immunogenic epitopes. METHODS: Nineteen candidate nonamers from hexon and penton proteins were identified by epitope binding prediction. Peptides were synthesized and tested for in vivo immunogenicity by screening peripheral blood mononuclear cells from healthy volunteers (n = 64) and HAdV-infected stem cell recipients (n = 26) for memory T cells recognizing the candidate epitopes in the context of most common HLA alleles. RESULTS: Functional CD8+ T cells recognizing seven epitopes were identified, among them four penton-derived and two hexon-derived peptides. The HLA-A*01-restricted penton-derived peptide STDVASLNY (A01PentonSTDV) and HLA-A*02-restricted hexon-derived peptide TLLYVLFEV (A02HexonTLLY) were recognized by more than half of the persons carrying the respective HLA-type. CONCLUSIONS: Thus, the HAdV-derived penton protein is a novel major target of the anti-HAdV immune response. Identification of new immunodominant epitopes will facilitate and broaden immune assessment strategies to identify patients suitable for T-cell transfer. Knowledge of additional target structures may increase T-cell recovery in manufacturing processes.

Dendritic cell-mediated immune humanization of mice: implications for allogeneic and xenogeneic stem cell transplantation.

De novo regeneration of immunity is a major problem after allogeneic hematopoietic stem cell transplantation (HCT). HCT modeling in severely compromised immune-deficient animals transplanted with human stem cells is currently limited because of incomplete maturation of lymphocytes and scarce adaptive responses. Dendritic cells (DC) are pivotal for the organization of lymph nodes and activation of naive T and B cells. Human DC function after HCT could be augmented with adoptively transferred donor-derived DC. In this study, we demonstrate that adoptive transfer of long-lived human DC coexpressing high levels of human IFN-α, human GM-CSF, and a clinically relevant Ag (CMV pp65 protein) promoted human lymphatic remodeling in immune-deficient NOD.Rag1(-/-).IL-2rγ(-/-) mice transplanted with human CD34(+) cells. After immunization, draining lymph nodes became replenished with terminally differentiated human follicular Th cells, plasma B cells, and memory helper and cytotoxic T cells. Human Igs against pp65 were detectable in plasma, demonstrating IgG class-switch recombination. Human T cells recovered from mice showed functional reactivity against pp65. Adoptive immunotherapy with engineered DC provides a novel strategy for de novo immune reconstitution after human HCT and a practical and effective tool for studying human lymphatic regeneration in vivo in immune deficient xenograft hosts.

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.

miR-155 is associated with the leukemogenic potential of the class IV granulocyte colony-stimulating factor receptor in CD34⁺ progenitor cells.

Granulocyte colony-stimulating factor (G-CSF) is a major regulator of granulopoiesis on engagement with the G-CSF receptor (G-CSFR). The truncated, alternatively spliced, class IV G-CSFR (G-CSFRIV) has been associated with defective differentiation and relapse risk in pediatric acute myeloid leukemia (AML) patients. However, the detailed biological properties of G-CSFRIV in human CD34(+) hematopoietic stem and progenitor cells (HSPCs) and the potential leukemogenic mechanism of this receptor remain poorly understood. In the present study, we observed that G-CSFRIV-overexpressing (G-CSFRIV(+)) HSPCs demonstrated an enhanced proliferative and survival capacity on G-CSF stimulation. Cell cycle analyses showed a higher frequency of G-CSFRIV(+) cells in the S and G2/M phase. Also, apoptosis rates were significantly lower in G-CSFRIV(+) HSPCs. These findings were shown to be associated with a sustained Stat5 activation and elevated miR-155 expression. In addition, G-CSF showed to further induce G-CSFRIV and miR-155 expression of peripheral blood mononuclear cells isolated from AML patients. A Stat5 pharmacological inhibitor or ribonucleic acid (RNA) interference-mediated silencing of the expression of miR-155 abrogated the aberrant proliferative capacity of the G-CSFRIV(+) HSPCs. Hence, the dysregulation of Stat5/miR-155 pathway in the G-CSFRIV(+) HSPCs supports their leukemogenic potential. Specific miRNA silencing or the inhibition of Stat5-associated pathways might contribute to preventing the risk of leukemogenesis in G-CSFRIV(+) HSPCs. This study may promote the development of a personalized effective antileukemia therapy, in particular for the patients exhibiting higher expression levels of G-CSFRIV, and further highlights the necessity of pre-screening the patients for G-CSFR isoforms expression patterns before G-CSF administration.

Generation of lentivirus-induced dendritic cells under GMP-compliant conditions for adaptive immune reconstitution against cytomegalovirus after stem cell transplantation.

BACKGROUND: Reactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs ("SmyleDCpp65") that accelerated antigen-specific T cell development. METHODS: Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65) that were cryopreserved and thawed. RESULTS: Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated. CONCLUSION: These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.

Semaphorin 3A alters endothelial cell immunogenicity by regulating Class II transactivator activity circuits.

BACKGROUND: Endothelial cells (ECs) play a pivotal role in the allogeneic immune response upon transplantation. Semaphorin 3A (Sema3A) was implicated in the modulation of EC growth, but its effects on immunogenicity were not previously investigated. STUDY DESIGN AND METHODS: ECs were transduced with a lentiviral vector encoding for the green fluorescence protein (GFP) sequence under the control of a Class II transactivator (CIITA)-dependent promoter. Upon stimulation of nonmodified ECs with recombinant Sema3A protein, mRNA and protein levels of CIITA, HLA-DR, and Sema3A receptors were evaluated. An enzyme-linked immunosorbent assay was developed to quantify Sema3A levels in the sera of kidney-transplanted patients. RESULTS: Sema3A stimulation of lentiviral vector encoding for the GFP sequence ECs caused a significant up regulation of the transgene expression, indicating an increase in CIITA levels. Stimulation of nonmodified ECs with Sema3A resulted in an up regulation of CIITA expression, which was associated with enhanced HLA-DR levels and an increase in alloreactive CD4+ T-cell proliferation. Sema3A receptor expression was enhanced by CIITA, establishing a positive feedback loop. Higher levels of Sema3A were observed in sera of patients presenting with organ rejection. CONCLUSION: This study links Sema3A signaling in ECs with increased CIITA levels and higher HLA-DR expression, resulting in CD4+ T-cell activation, which might have important implications for tissue and organ transplantation.