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CD8+ T cell immunity to SARS-CoV-2 has been implicated in COVID-19 severity and virus control. Here, we identified nonsynonymous mutations in MHC-I-restricted CD8+ T cell epitopes after deep sequencing of 747 SARS-CoV-2 virus isolates. Mutant peptides exhibited diminished or abrogated MHC-I binding in a cell-free in vitro assay. Reduced MHC-I binding of mutant peptides was associated with decreased proliferation, IFN-γ production and cytotoxic activity of CD8+ T cells isolated from HLA-matched COVID-19 patients. Single cell RNA sequencing of ex vivo expanded, tetramer-sorted CD8+ T cells from COVID-19 patients further revealed qualitative differences in the transcriptional response to mutant peptides. Our findings highlight the capacity of SARS-CoV-2 to subvert CD8+ T cell surveillance through point mutations in MHC-I-restricted viral epitopes.

Hematopoietic stem and progenitor cells (HSPCs) derived from human induced pluripotent stem cells (hiPSCs) hold great promise for disease modeling, drug screens, and eventually cell therapy approaches. During in vitro differentiation of hiPSCs into hematoendothelial progenitors, the emergence of CD34-positive cells indicates a critical step of lineage specification. To facilitate the monitoring of hematopoietic differentiation of hiPSCs, we established fluorescent reporter cells for the stem and progenitor cell marker CD34. An IRES-GFP (internal ribosome entry site green fluorescent protein) construct was introduced by CRISPR/Cas9 into the 3' untranslated region of one endogenous CD34 allele. Single-cell clones were generated after excision of the floxed puromycin resistance cassette by Cre recombination and correct insertion was confirmed by genotyping polymerase chain reaction and Southern blot. To validate their functionality, the reporter hiPSCs were in vitro differentiated toward CD34+ cells using the STEMdiff Hematopoietic Kit combined with short-term inhibition of GSK3 (glycogen synthase kinase 3). All cells expressing nuclear GFP were positive for cell surface CD34, thus allowing the direct monitoring of the differentiation of hiPSCs into CD34+ cells either by flow cytometry or confocal microscopy. After fluorescence-activated cell sorting, cells displaying high GFP expression exhibited increased colony-forming potential in the MethoCult colony-forming unit assays as compared with CD34+ cells obtained by magnetic-activated cell sorting. In summary, we have generated functional CD34 GFP reporter hiPSCs, which not only permit label-free separation of HSPCs, but also tracing of the emergence and fate of CD34+ progenitors at the single-cell level.

SummaryFollowing vascular injury or activation, endothelial cells (ECs) participate in the modulation of haemostasis and fibrinolysis. Viscoelastic tests (VETs) are a potent bedside monitoring tool that reports haemostatic parameters in real time. However, VETs neglect the influence of the surrounding endothelium. Our aim was therefore to establish an assay that incorporates ECs in a whole blood VET and to assess the impact of ECs on coagulation parameters. Outgrowth endothelial cells (OECs) and human umbilical vein endothelial cells (HUVECs) were seeded onto microbeads to create transferable EC-microcarriers. Microbeads were then added to citrated whole blood in the measurement cup of a thromboelastometry device (ROTEM). After the addition of CaCl2 (star-TEM®) to the blood sample (NATEM assay), standard ROTEM parameters were analysed. Scanning electron microscopy (SEM) was carried out to visualise the interactions of the beads, whole blood components and the ROTEM pin after clotting. SEM showed that the added microbeads were effectively incorporated into the final blood clot. In the presence of activated ECs, the clotting time (CT) of the blood was shortened fourfold compared to that in uncoated control beads. A significant reduction in CT was also observed in the presence of unstimulated ECs. Interestingly, CT was also reduced by the addition of purified EC culture supernatant. CT shortening was prevented by incubating the supernatant with an inhibiting antibody against tissue factor (TF). Our findings demonstrate that ECs can be incorporated into a ROTEM assay via coated microbeads, and whole blood clotting initiation is accelerated by non-activated and activated ECs.

Vascularization of engineered tissues is one of the current challenges in tissue engineering. Several strategies aim to generate a prevascularized scaffold which can be implanted at sites of injury or trauma. Endothelial cells derived from peripheral blood (outgrowth endothelial cells, OECs) display promising features for vascular tissue engineering, including their autologous nature, capacity for proliferation and ability to form mature vessels. In this study we investigated the ability of OECs to form vascular structures in co-culture with adipose-derived stem cells (ASCs) in a fibrin matrix. Using microcarrier beads coated with OECs, we showed ingrowth of endothelial cells in the fibrin scaffold. Furthermore, co-cultures with ASCs induced vessel formation, as evidenced by immunostaining for CD31. The degradation of fibrin is at least in part mediated by expression of matrix metalloproteinase-14. Moreover, we showed OEC/ASC-induced vessel-like structure formation even in the absence of microcarrier beads, where increasing amounts of ASCs resulted in a denser tubular network. Our data add new insights into co-culture-induced vessel formation of outgrowth endothelial cells within a fibrin matrix in an autologous system. Copyright © 2012 John Wiley & Sons, Ltd.

// Anna-Maria Husa 1, 2 , Željana Magic 1 , Malin Larsson 3 , Tommy Fornander 4 , Gizeh Perez-Tenorio 1 1 Department of Clinical and Experimental Medicine, Division of Oncology, Linkoping University, Linkoping, Sweden 2 Current address: CCRI, Children’s Cancer Research Institute, St. Anna Kinderkrebsforschung e.V., Vienna, Austria 3 Bioinformatics Infrastructure for Life Sciences (BILS) and Department of Physics, Chemistry and Biology, Linkoping University, Linkoping, Sweden 4 Department of Oncology, Karolinska University Hospital and Karolinska Institute, Stockholm, Sweden Correspondence to: Gizeh Perez-Tenorio, e-mail: gizeh.perez-tenorio@liu.se Keywords: EPHB2, EPH family, TaqMan array, gene expression, protein expression Received: July 03, 2015 Accepted: January 23, 2016 Published: February 8, 2016 ABSTRACT The EPH and ephrins function as both receptor and ligands and the output on their complex signaling is currently investigated in cancer. Previous work shows that some EPH family members have clinical value in breast cancer, suggesting that this family could be a source of novel clinical targets. Here we quantified the mRNA expression levels of EPH receptors and their ligands, ephrins, in 65 node positive breast cancer samples by RT-PCR with TaqMan ® Micro Fluidics Cards Microarray. Upon hierarchical clustering of the mRNA expression levels, we identified a subgroup of patients with high expression, and poor clinical outcome. EPHA2, EPHA4, EFNB1, EFNB2, EPHB2 and EPHB6 were significantly correlated with the cluster groups and particularly EPHB2 was an independent prognostic factor in multivariate analysis and in four public databases. The EPHB2 protein expression was also analyzed by immunohistochemistry in paraffin embedded material (cohort 2). EPHB2 was detected in the membrane and cytoplasmic cell compartments and there was an inverse correlation between membranous and cytoplasmic EPHB2. Membranous EPHB2 predicted longer breast cancer survival in both univariate and multivariate analysis while cytoplasmic EPHB2 indicated shorter breast cancer survival in univariate analysis. Concluding: the EPH/EFN cluster analysis revealed that high EPH/EFN mRNA expression is an independent prognostic factor for poor survival. Especially EPHB2 predicted poor breast cancer survival in several materials and EPHB2 protein expression has also prognostic value depending on cell localization.

Vascularization of tissue-engineered constructs is essential to provide sufficient nutrient supply and hemostasis after implantation into target sites. Co-cultures of adipose-derived stem cells (ASC) with outgrowth endothelial cells (OEC) in fibrin gels were shown to provide an effective possibility to induce vasculogenesis in vitro. However, the mechanisms of the interaction between these two cell types remain unclear so far. The aim of this study was to evaluate differences of direct and indirect stimulation of ASC-induced vasculogenesis, the influence of ASC on network stabilization and molecular mechanisms involved in vascular structure formation. Endothelial cells (EC) were embedded in fibrin gels either containing non-coated or ASC-coated microcarrier beads as well as ASC alone. Moreover, EC-seeded constructs incubated with ASC-conditioned medium were used in addition to constructs with ASC seeded on top. Vascular network formation was visualized by green fluorescent protein expressing cells or immunostaining for CD31 and quantified. RT-qPCR of cells derived from co-cultures in fibrin was performed to evaluate changes in the expression of EC marker genes during the first week of culture. Moreover, angiogenesis-related protein levels were measured by performing angiogenesis proteome profiler arrays. The results demonstrate that proximity of endothelial cells and ASC is required for network formation and ASC stabilize EC networks by developing pericyte characteristics. We further showed that ASC induce controlled vessel growth by secreting pro-angiogenic and regulatory proteins. This study reveals angiogenic protein profiles involved in EC/ASC interactions in fibrin matrices and confirms the usability of OEC/ASC co-cultures for autologous vascular tissue engineering.

Background aims Human endothelial progenitor cells (EPC) play an important role in regenerative medicine and contribute to neovascularization on vessel injury. They are usually enriched from peripheral blood, cord blood and bone marrow. In human fat tissue, EPC are rare and their isolation remains a challenge. Methods Fat tissue was prepared by collagenase digestion, and the expression of specific marker proteins was evaluated by flow cytometry in the stromal vascular fraction (SVF). For enrichment, magnetic cell sorting was performed with the use of CD133 microbeads and EPC were cultured until colonies appeared. A second purification was performed with CD34; additional isolation steps were performed with the use of a combination of CD34 and CD31 microbeads. Enriched cells were investigated by flow cytometry for the expression of endothelial specific markers, by Matrigel assay and by the uptake of acetylated low-density lipoprotein. Results The expression pattern confirmed the heterogeneous nature of the SVF, with rare numbers of CD133+ detectable. EPC gained from the SVF by magnetic enrichment showed cobblestone morphology of outgrowth endothelial cells and expressed the specific markers CD31, CD144, vascular endothelial growth factor (VEGF)R2, CD146, CD73 and CD105. Functional integrity was confirmed by uptake of acetylated low-density lipoprotein and the formation of tube-like structures on Matrigel. Conclusions Rare EPC can be enriched from human fat tissue by magnetic cell sorting with the use of a combination of microbeads directed against CD133, an early EPC marker, CD34, a stem cell marker, and CD31, a typical marker for endothelial cells. In culture, they differentiate into EC and hence could have the potential to contribute to neovascularization in regenerative medicine.

Vascularization of engineered tissues is one of the current challenges in tissue engineering. Several strategies aim to generate a prevascularized scaffold which can be implanted at sites of injury or trauma. Endothelial cells derived from peripheral blood (outgrowth endothelial cells, OECs) display promising features for vascular tissue engineering, including their autologous nature, capacity for proliferation and ability to form mature vessels. In this study we investigated the ability of OECs to form vascular structures in co-culture with adipose-derived stem cells (ASCs) in a fibrin matrix. Using microcarrier beads coated with OECs, we showed ingrowth of endothelial cells in the fibrin scaffold. Furthermore, co-cultures with ASCs induced vessel formation, as evidenced by immunostaining for CD31. The degradation of fibrin is at least in part mediated by expression of matrix metalloproteinase-14. Moreover, we showed OEC/ASC-induced vessel-like structure formation even in the absence of microcarrier beads, where increasing amounts of ASCs resulted in a denser tubular network. Our data add new insights into co-culture-induced vessel formation of outgrowth endothelial cells within a fibrin matrix in an autologous system.

The Eph receptors belong to a large family of receptors tyrosine kinase. The Ephs and their ligands ephrins are aberrantly expressed in cancer. However, due their complex signaling and promiscuous interactions, a wide screening of the entire family will be important to determine their clinical value. Purpose: The Eph receptor tyrosine kinase family has been implicated in breast cancer but their role in tumor progression remains elusive. Therefore we aimed to profile the Eph receptors and their ligands ephrins in breast cancer samples to define them as potential clinical markers or therapeutic targets. Experimental design: Gene expression of Eph/ephrins was quantified by RT-PCR using a TaqMan® Micro Fluidics Cards Microarray custom designed for 21 Eph/ephrin family members: EphA1-A8, EphB1-B4 and EphB6, ephrinA1-A5, ephrinB1-B3 and 2 endogenous controls: GAPDH and HPRT1 in 70 breast cancer tumors. Results: We found that high expression of EphBs, and particularly EphB2, was associated to worse outcome among postmenopausal patients with lymph node infiltration. Additionally high EphB2 expression correlated with HER2 positive status. Conclusions: Our results suggest that the EphB receptor family may have prognostic value for postmenopausal breast cancer patients with lymph nodal infiltration. We will continue this study in a larger patient material in order to better evaluate the therapeutic impact of this family. Citation Format: Gizeh Perez-Tenorio, Anna-Maria Husa, Olle Stål. Clinical potential of the Eph/ephrin profile in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3819. doi:10.1158/1538-7445.AM2014-3819