Rat Anti-Mouse Cd19 Heterohybridoma [2E4]

Anti-CD19 chimeric antigen receptor (CAR) T cells showed significant antileukemic activity in B-precursor acute lymphoblastic leukemia (ALL). Allogeneic, HLA-mismatched off-the-shelf third-party donors may offer ideal fitness of the effector cells, but carry the risk of graft-versus-host disease. Knockout (KO) of the endogenous T-cell receptor (TCR) in CD19-CAR-T cells may be a promising solution. Here, we induced a CRISPR/Cas9-mediated KO of the TCR chain in combination with a second-generation retroviral CAR transduction including a 4-1BB costimulatory domain in primary T cells. This tandem engineering led to a highly functional population of TCR-KO-CAR-T cells with strong activation (CD25, interferon gamma), proliferation, and specific killing upon CD19 target recognition. TCR-KO-CART cells had a balanced phenotype of central memory and effector memory T cells. KO of the endogenous TCR in T cells strongly ablated alloreactivity in comparison with TCR-expressing T cells. In a patient-derived xenograft model of childhood ALL TCR-KO-CAR-T cells clearly controlled CD19(+) leukemia burden and improved survival in vivo. However, coexpression of endogenous TCR plus CAR led to superior persistence of T cells and significantly prolonged leukemia control in vivo, confirmed by a second in vivo model using the leukemia cell line NALM6. These results point toward an essential role of the endogenous TCR for longevity of the response at the price of alloreactivity. In conclusion, anti-CD19 CAR T cells with a CRISPR/Cas9-mediated TCR-KO are promising candidates for nonmatched thirdparty adoptive T-cell transfer with high antileukemic functionality in the absence of alloreactivity, but long-term persistence in vivo is better in the presence of the endogenous TCR.

Regulatory B cells (Bregs) are unique subpopulations of B cells with immune-regulating or immune-suppressing properties and play a role in peripheral tolerance. Due to the current limitations of human Breg studies among periodontal diseases, in the present study, we tried to analyze the change in circulating Bregs, pro-inflammatory, and anti-inflammatory cytokines in patients with periodontitis. Peripheral blood from 55 patients with stage 2 periodontitis and 20 healthy controls was analyzed using flow cytometry to evaluate the frequency of CD19(+)CD24(+)CD38(+)Breg cells. ELISA was used to assess the serum levels of the pro-inflammatory cytokines, including interleukins (IL)-1 beta, IL-6, TNF-alpha, and anti-inflammatory cytokines including IL-10, IL-35, and TGF-beta. Increased proportions of Breg cells were observed in patients with stage 2 periodontitis compared to controls. Serum levels of cytokines were significantly higher in patients with periodontitis compared to controls. A significant positive correlation was observed between the frequencies of Breg cells and IL35 levels, IL10 levels, and TGF-beta. In conclusion, our results suggest that the increase in peripheral Breg cells and serum cytokine levels among periodontitis patients seems to be closely associated with disease progression, a possible link between periodontitis, and systemic inflammatory process.