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- 2020 (5) (entfernen)
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- natural killer cells (2)
- Apoptosis (1)
- CD19 (1)
- Cardiology (1)
- HIV-1 (1)
- Lactobacillus (1)
- Long non-coding RNAs (1)
- VHH (1)
- acute lymphoblastic leukemia (1)
- adoptive cancer immunotherapy (1)
Institut
- Georg-Speyer-Haus (5) (entfernen)
In the absence of an active prophylactic vaccine against HIV-1, passively administered, broadly neutralizing antibodies (bnAbs) identified in some chronically infected persons were shown to prevent HIV-1 infection in animal models. However, passive administration of bnAbs may not be suited to prevent sexual HIV-1 transmission in high-risk cohorts, as a continuous high level of active bnAbs may be difficult to achieve at the primary site of sexual transmission, the human vagina with its acidic pH. Therefore, we used Lactobacillus, a natural commensal in the healthy vaginal microbiome, to express bn nanobodies (VHH) against HIV-1 that we reported previously. After demonstrating that recombinant VHHA6 expressed in E. coli was able to protect humanized mice from mucosal infection by HIV-1Bal, we expressed VHHA6 in a soluble or in a cell-wall-anchored form in Lactobacillus rhamnosus DSM14870. This strain is already clinically applied for treatment of bacterial vaginosis. Both forms of VHHA6 neutralized a set of primary epidemiologically relevant HIV-1 strains in vitro. Furthermore, VHHA6 was still active at an acidic pH. Thus, lactobacilli expressing bn VHH potentially represent an attractive vector for the passive immunization of women in cohorts at high risk of HIV-1 transmission.
Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting. Hence, unlike autologous CAR-T cells, therapeutic CD19-CAR-NK cells can be generated as an off-the-shelf product from healthy donors. Nevertheless, genetic engineering of peripheral blood (PB) derived NK cells remains challenging and optimized protocols are needed. In our study, we aimed to optimize the generation of CD19-CAR-NK cells by retroviral transduction to improve the high antileukemic capacity of NK cells. We compared two different retroviral vector platforms, the lentiviral and alpharetroviral, both in combination with two different transduction enhancers (Retronectin and Vectofusin-1). We further explored different NK cell isolation techniques (NK cell enrichment and CD3/CD19 depletion) to identify the most efficacious methods for genetic engineering of NK cells. Our results demonstrated that transduction of NK cells with RD114-TR pseudotyped retroviral vectors, in combination with Vectofusin-1 was the most efficient method to generate CD19-CAR-NK cells. Retronectin was potent in enhancing lentiviral/VSV-G gene delivery to NK cells but not alpharetroviral/RD114-TR. Furthermore, the Vectofusin-based transduction of NK cells with CD19-CARs delivered by alpharetroviral/RD114-TR and lentiviral/RD114-TR vectors outperformed lentiviral/VSV-G vectors. The final generated CD19-CAR-NK cells displayed superior cytotoxic activity against CD19-expressing target cells when compared to non-transduced NK cells achieving up to 90% specific killing activity. In summary, our findings present the use of RD114-TR pseudotyped retroviral particles in combination with Vectofusin-1 as a successful strategy to genetically modify PB-derived NK cells to achieve highly cytotoxic CD19-CAR-NK cells at high yield.
Obtaining sufficient numbers of functional natural killer (NK) cells is crucial for the success of NK-cell-based adoptive immunotherapies. While expansion from peripheral blood (PB) is the current method of choice, ex vivo generation of NK cells from hematopoietic stem and progenitor cells (HSCs) may constitute an attractive alternative. Thereby, HSCs mobilized into peripheral blood (PB-CD34+) represent a valuable starting material, but the rather poor and donor-dependent differentiation of isolated PB-CD34+ cells into NK cells observed in earlier studies still represents a major hurdle. Here, we report a refined approach based on ex vivo culture of PB-CD34+ cells with optimized cytokine cocktails that reliably generates functionally mature NK cells, as assessed by analyzing NK-cell-associated surface markers and cytotoxicity. To further enhance NK cell expansion, we generated K562 feeder cells co-expressing 4-1BB ligand and membrane-anchored IL-15 and IL-21. Co-culture of PB-derived NK cells and NK cells that were ex-vivo-differentiated from HSCs with these feeder cells dramatically improved NK cell expansion, and fully compensated for donor-to-donor variability observed during only cytokine-based propagation. Our findings suggest mobilized PB-CD34+ cells expanded and differentiated according to this two-step protocol as a promising source for the generation of allogeneic NK cells for adoptive cancer immunotherapy.
Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.
Despite the great success of antiretroviral therapy, both in the treatment and prevention of HIV-1 infection, a vaccine is still urgently needed to end the epidemic. According to UNAIDS, in 2018, about 35% of HIV-1 infected persons did not receive antiretroviral therapy (ART), resulting in 1.7 million new infections in that year...