Embryonic Stem Cell-Derived Factors Inhibit T Effector Activation and Induce T Regulatory Cells by Suppressing PKC-θ Activation

Embryonic stem cells (ESCs) possess immune privileged properties and have the capacity to modulate immune activation. However, the mechanisms by which ESCs inhibit immune activation remain mostly unknown. We have previously shown that ESC-derived factors block dendritic cell maturation, thereby indirectly affecting T cell activation. Here, we show that ESC-derived factors also directly affect T cell activation. We provide the first demonstration that ESC-derived factors significantly down-regulated the expressions of IL-2 and IFN-γ, while markedly up-regulating the expression of IL-10, TGF-β, and Treg transcription factor Foxp3 in CD4+ CD25+ T cells. Furthermore, ESC-derived factors robustly suppressed T cell proliferation in response to the protein kinase C-θ (PKC-θ) activator phorbol 12-myristate 13-acetate (PMA). Western blot analysis indicated that ESC-derived factors prevented PKC-θ phosphorylation without influencing total PKC-θ levels. Moreover, IκB-α degradation was abrogated, confirming absence of PKC-θ activity. The impact of ESC-derived factors on PKC-θ activation appeared to be specific since other upstream T cell signaling components were not affected. In conclusion, ESCs appear to directly impact T cell activation and polarization by negatively regulating the PKC-θ pathway

Repurposing the oncolytic virus VSV∆51M as a COVID-19 vaccine

The coronavirus disease 2019 (COVID-19) pandemic imposes an urgent and continued need for the development of safe and cost-effective vaccines to induce preventive responses for limiting major outbreaks around the world. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we repurposed the VSV∆51M oncolytic virus platform to express the spike receptor-binding domain (RBD) antigen. In this study, we report the development and characterization of the VSV∆51M-RBD vaccine. Our findings demonstrate successful expression of the RBD gene by the VSV∆51M-RBD virus, inducing anti-RBD responses without attenuating the virus. Moreover, the VSV∆51M-RBD vaccine exhibited safety, immunogenicity, and the potential to serve as a safe and effective alternative or complementary platform to current COVID-19 vaccines

ESC-derived factors inhibit PMA mediated PKC-θ activation in T cells.

<p><i>a)</i> C57BL/6 splenocytes were stained with CellTrace Violet Cell Proliferation dye and activated with 50 ng/ml of PMA for 24 hours in the presence of 0.23 mg/ml of ESC- derived factors (without pre-treatment). Cells were harvested, washed with PBS and examined for proliferation by flow cytometry. <i>b)</i> C57BL/6 splenocytes were pre-treated over night with 0.23 mg/ml of ESC-derived factors and stimulated with 50 ng/ml of PMA for the indicated periods of time. Subsequently, the cells were harvested and lysed. Lysates were examined by western blotting for PKC-θ phosphorylation (Thre 538), and total PKC-θ. <i>c)</i> C57BL/6 CD3+ T cells were pre-treated overnight with 0.23 mg/ml ESC-derived factors and stimulated with 50 ng/ml of PMA for the indicated periods of time. Subsequently, the cells were harvested and lysed. Lysates were examined by western blotting for IκB-α degradation. Results are representative of 4 separate experiments.</p

ESC-derived factors modulate T helper responses during an allogeneic immune response.

<p>A one way mixed lymphocyte reaction was performed, C57BL/6 splenocytes were used as responders and CD1 splenocytes as stimulators in the presence of ESC-derived factors or vehicle control. Cells were harvested at the indicated time points and total RNA was isolated. Subsequently, cDNA was synthesized and used to carry out QPCR to examine the expression of cytokines and master regulator transcription factors of T helper cells. <i>a)</i> IL-2, <i>b)</i> IFN-γ, <i>c)</i> TGF-β, <i>d)</i> T-bet, <i>e)</i> Foxp3. Responders alone were used as baseline. Results are representative of 3 separate experiments. Data points represent mean ± SD. * indicates p value≤0.05. White bars represent results obtained from vehicle treated MLRs and black bars indicate results obtained from ESC-derived factor treated MLRs.</p

ESC-derived factors in combination with cyclosporin A enhance inhibition of allogeneic immune activation.

<p>A one way mixed lymphocyte reaction was performed, C57BL/6 splenocytes were used as responders and CD1 splenocytes as stimulators in the presence of vehicle control, ESC-derived factors and CsA. Moreover, ESC-derived factors were used in combination with CsA to determine whether they can complement one another in preventing allo-immune activation.</p

ESC-derived factors skew T cell helper responses towards T regulatory cells.

<p><i>a)</i> C57BL/6 splenocytes were pre-treated over night with ESC-derived factors and stimulated with PMA and Ionomycin or anti-CD3/anti-CD28 for 6 hours. Protein transport inhibitor cocktail was added to the cells 1 hour following stimulation. Cells were harvested and stained for surface CD8. After washing, the cells were fixed, permeabilized and stained for intracellular IFN-γ. <i>b)</i> C57BL/6 splenocytes were treated with ESC-derived factors and stimulated with anti-CD3 and anti-CD28 for 3 days. The cells were harvested and stained for CD3, CD4 and CD25. Subsequently, cells were fixed, permeabilized and stained for Foxp3. Gates were set on CD3+ followed by CD4+ cells. Results are representative of at least 3 separate experiments.</p

ESC-conditioned media and cellular factors from ESC-extracts inhibit T cell proliferation in response to anti-CD3/anti-CD28 stimulation.

<p><i>a)</i> C57BL/6 splenocytes were labeled with CFSE and activated with anti-CD3/anti-CD28 in RPMI media (unstimulated cells presented in panel 1 and stimulated cells in panel 2). Cells were also activated in 50% RPMI along with 50% or 100% fresh unconditioned media (panels 3 and 4), 50% RPMI along with 50% or 100% mouse embryonic fibroblast-conditioned media (MEF-CM, panels 5 and 6), and 50% RPMI along with 50% or 100% mouse ESC-conditioned medium (ESC-CM, panels 7 and 8). After 48 hours the cells were analyzed by flow cytometry for proliferation. <i>b)</i> ESCs were grown in feeder free cultures, harvested and lysed by sonication. Cell membrane, mitochondria and nucleus were removed by centrifuging the sonicate at 15000 g for 15 minutes. Proliferation of B6 splenocytes stimulated with anti-CD3/anti-CD28 in RPMI media was assessed using extraction buffer alone (vehicle, panel 1), lysates from C2C12 cells (Control-Factors) or increasing concentration of ESC-derived factors (ESC-Factors). Results are representative of 4 separate experiments.</p

ESC-derived factors specifically inhibit PKC-θ activation without affecting upstream signaling molecules.

<p>C57BL/6 splenocytes were pre-treated with ESC-derived factors overnight and stimulated with anti-CD3/anti-CD28 for the indicated periods of time. Subsequently, the cells were harvested and lysed. Lysates were examined by western blotting for PLC-γ, AKT and PKC-θ phosphorylation. Results are representative of 3 separate experiments.</p

Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion.

The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed ("functional"), or unlicensed ("hypofunctional"). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m-/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab')2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans

Mouse Nkrp1-Clr gene cluster sequence and expression analyses reveal conservation of tissue-specific MHC-independent immunosurveillance.

The Nkrp1 (Klrb1)-Clr (Clec2) genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b), Nkrp1c (Klrb1c), and Clr-c (Clec2f) genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d) and Clr-g (Clec2i) showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells), as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner