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Suppression of FVIII-Specific Memory B Cells by Chimeric BAR Receptor-Engineered Natural Regulatory T Cells.

ABSTRACT: Anti-drug antibody formation poses tremendous obstacles for optimal treatment of hemophilia A (HA). In this study, we sought to utilize chimeric receptor-modified natural regulatory T cells (Tregs) to target FVIII-specific memory B cells, which are responsible for persistent anti-FVIII neutralizing antibodies (inhibitors) in HA patients. Thus, CD4+CD25 hi CD304+ natural Tregs were FACS sorted from naïve C57BL/6 mice and retrovirally transduced to express a chimeric B-cell antibody receptor (BAR) containing the immunodominant A2 domain of FVIII. Plasmablast-depleted (CD138 neg ) splenocytes from FVIII immunized FVIII-knockout HA mice served as the source for FVIII-specific memory B cells, which were specifically stimulated in vitro with FVIII and enumerated in a B-cell ELISPOT assays. Adding A2-BAR Tregs (1 per 150 splenocytes), but not conventional T cells, to the CD138- splenocytes significantly suppressed the formation of anti-FVIII antibody secreting cells (ASC), compared to the non-relevant OVA-BAR Tregs control group. The observation that A2-BAR Tregs can suppress the response to FVIII suggests that bystander suppression can occur in the local milieu in this system. Transwell experiments confirmed that the suppression was contact-dependent. Moreover, even in the presence of antibodies to FVIII (so-called inhibitors), similarly prepared CD4+CD25 hi CD127 low A2-BAR human natural Tregs completely suppressed polyclonal anti-FVIII ASC formation. In conclusion, we demonstrated in vitro that FVIII domain-expressing BAR Tregs could efficiently target and suppress FVIII-specific memory B cells.

SUBMITTER: Pohl AP

PROVIDER: S-EPMC7186411 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Suppression of FVIII-Specific Memory B Cells by Chimeric BAR Receptor-Engineered Natural Regulatory T Cells.

Pohl Alessandra De Paula AP Venkatesha Shivaprasad H SH Zhang Ai-Hong AH Scott David W DW

Frontiers in immunology 20200421

Anti-drug antibody formation poses tremendous obstacles for optimal treatment of hemophilia A (HA). In this study, we sought to utilize chimeric receptor-modified natural regulatory T cells (Tregs) to target FVIII-specific memory B cells, which are responsible for persistent anti-FVIII neutralizing antibodies (inhibitors) in HA patients. Thus, CD4+CD25 hi CD304+ natural Tregs were FACS sorted from naïve C57BL/6 mice and retrovirally transduced to express a ...[more]

PMID: 32373126

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Project description: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.

Dataset Information

Suppression of FVIII-Specific Memory B Cells by Chimeric BAR Receptor-Engineered Natural Regulatory T Cells.

Publications

Suppression of FVIII-Specific Memory B Cells by Chimeric BAR Receptor-Engineered Natural Regulatory T Cells.

Similar Datasets

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