Immune stimulation mediated by autoantigen binding sites within small nuclear RNAs involves Toll-like receptors 7 and 8

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies to certain cellular macromolecules, such as the small nuclear ribonucleoprotein particles (snRNPs), which had been considered to be passive targets of the autoimmune response. SLE is also characterized by the increased expression of type I interferon (IFN), which appears to be associated with the development and severity of disease. Here, we show that specific, highly conserved RNA sequences within snRNPs can stimulate Toll-like receptors (TLRs) 7 and 8 as well as activate innate immune cells, such as plasmacytoid dendritic cells (pDCs), which respond by secreting high levels of type I IFN. SLE patient sera containing autoantibodies to snRNPs form immune complexes that are taken up through the Fc receptor γRII and efficiently stimulate pDCs to secrete type I IFNs. These results demonstrate that a prototype autoantigen, the snRNP, can directly stimulate innate immunity and suggest that autoantibodies against snRNP may initiate SLE by stimulating TLR7/8

Functionality and Cell Senescence of CD4/ CD8-Selected CD20 CAR T Cells Manufactured Using the Automated CliniMACS Prodigy® Platform

Clinical studies using autologous CAR T cells have achieved spectacular remissions in refractory CD19+ B cell leukaemia, however some of the patient treatments with CAR T cells failed. Beside the heterogeneity of leukaemia, the distribution and senescence of the autologous cells from heavily pretreated patients might be further reasons for this. We performed six consecutive large-scale manufacturing processes for CD20 CAR T cells from healthy donor leukapheresis using the automated CliniMACS Prodigy® platform. Starting with a CD4/CD8-positive selection, a high purity of a median of 97% T cells with a median 65-fold cell expansion was achieved. Interestingly, the transduction rate was significantly higher for CD4+ compared to CD8+ T cells and reached in a median of 23%. CD20 CAR T cells showed a good specific IFN-γ secretion after cocultivation with CD20+ target cells which correlated with good cytotoxic activity. Most importantly, 3 out of 5 CAR T cell products showed an increase in telomere length during the manufacturing process, while telomere length remained consistent in one and decreased in another process. In conclusion, this shows for the first time that beside heterogeneity among healthy donors, CAR T cell products also differ regarding cell senescence, even for cells manufactured in a standardised automated process

RNA-based, transient modulation of gene expression in human haematopoietic stem and progenitor cells

Modulation of gene expression is a useful tool to study the biology of haematopoietic stem and progenitor cells (HSPCs) and might also be instrumental to expand these cells for therapeutic approaches. Most of the studies so far have employed stable gene modification by viral vectors that are burdensome when translating protocols into clinical settings. Our study aimed at exploring new ways to transiently modify HSPC gene expression using non-integrating, RNA-based molecules. First, we tested different methods to deliver these molecules into HSPCs. The delivery of siRNAs with chemical transfection methods such as lipofection or cationic polymers did not lead to target knockdown, although we observed more than 90% fluorescent cells using a fluorochrome-coupled siRNA. Confocal microscopic analysis revealed that despite extensive washing, siRNA stuck to or in the cell surface, thereby mimicking a transfection event. In contrast, electroporation resulted in efficient, siRNA-mediated protein knockdown. For transient overexpression of proteins, we used optimised mRNA molecules with modified 5'- and 3'-UTRs. Electroporation of mRNA encoding GFP resulted in fast, efficient and persistent protein expression for at least seven days. Our data provide a broad-ranging comparison of transfection methods for hard-to-transfect cells and offer new opportunities for DNA-free, non-integrating gene modulation in HSPCs

INH-ODNs are not toxic for human PBMCs.

<p>(A) PBMCs (4x10⁵ cells/well) were cultured with INH-ODNs alone (10 μM) or with a combination of CpG-DNA 2006 (100 nM) and titrated amounts of INH-ODNs (0.01, 0.1, 1, 10 μM). Medium in the absence of cells (medium) and cells cultured in the absence of CpG-DNA 2006 or INH-ODNs (mock) served as negative controls. Bovine LDH was used as positive control (pos. contr.). The extracellular LDH-content was determined after 48h of culture. Error bars represent SD of three individual cultures. The experiment was repeated with imiquimod (5 μg/ml) using cells from a different donor (data not shown). INH-ODNs were again not toxic. (B) PBMCs (4x10⁵ cells/well) were not stimulated (mock) or stimulated with CpG-ODN 2006 (100 nM) or a combination of CpG-ODN 2006 (100 nM) and titrated amounts of INH-ODNs (0.01, 0.1, 1, 10 μM) as indicated. The intracellular ATP-content was determined after 48h of culture. Error bars represent SD of three individual cultures. The experiment was repeated twice with cells from another donor with similar results.</p

IL-6 release by CpG-ODN-activated human B-cells is significantly more prevented by G-modified INH-ODNs.

<p>(A) B-cells (5–10x10⁴ cells/well) were stimulated with CpG-ODN 2006 (100 nM) in the absence (CpG) or presence of titrated amounts of INH-ODNs (0.1, 1, 10 μM) for 24h. IL-6 was determined in the culture supernatant by ELISA. To determine whether INH-ODNs influence IL-6 release per se, the highest dose (10 μM) of each INH-ODN was also evaluated without TLR-mediated stimulation. Data represent mean and SD of three independent experiments, each experiment was performed with cells from a different donor (each bar represents n = 4–9 cultures). (B) IL-6 content of the culture supernatants of the experiment described in (A) was additionally determined after 5 days of culture. (C) depicts the intracellular ATP-content of the cells described in (B). Data represent mean and SD of three independent experiments, each experiment was performed with cells from a different donor (each bar represents n = 4–9 cultures). ^#p<0.05, ANOVA compared to CpG-ODN 2006; *p<0.05, ANOVA compared to INH-ODN 2088 for G-modified INH-ODNs 21595, 20844 and 24888 or compared to INH-ODN 21158 for G-modified INH-ODN 24987 and 24991; ^§p<0.05, ANOVA compared to mock in the absence of a TLR-stimulus.</p