B cell aplasia caused by “on-target off-tumor” toxicity is one of the clinical side effects during CD19-targeted chimeric antigen receptor (CAR) T (CD19-CAR-T) cells treatment for B cell malignancies. Persistent B cell aplasia was observed in all patients with sustained remission, which increased the patients’ risk of infection. Some patients even died due to infection. To overcome this challenge, the concept of incorporating an inhibitory CAR (iCAR) into CAR-T cells was introduced to constrain the T cells response once an “on-target off-tumor” event occurred. In this study, we engineered a novel KIR/PD-1-based inhibitory CAR (iKP CAR) by fusing the extracellular domain of killer cell immunoglobulin-like receptors (KIR) 2DL2 (KIR2DL2) and the intracellular domain of PD-1. We also confirmed that iKP CAR could inhibit the CD19 CAR activation signal via the PD-1 domain and CD19-CAR-T cells bearing an iKP CAR (iKP-19-CAR-T) exerted robust cytotoxicity in vitro and antitumor activity in the xenograft model of CD19+HLA-C1− Burkitt’s lymphoma parallel to CD19-CAR-T cells, whilst sparing CD19+HLA-C1+ healthy human B cells both in vitro and in the xenograft model. Meanwhile, iKP-19-CAR-T cells exhibited more naïve, less exhausted phenotypes and preserved a higher proportion of central memory T cells (TCM). Our data demonstrates that the KIR/PD-1-based inhibitory CAR can be a promising strategy for preventing B cell aplasia induced by CD19-CAR-T cell therapy.

Neural cell adhesion molecule (NCAM) is involved in cell multi-directional differentiation, but its role in osteoblast differentiation is still poorly understood. In the present study, we investigated whether and how NCAM regulates osteoblastic differentiation. We found that NCAM silencing inhibited osteoblast differentiation in pre-osteoblastic MC3T3-E1 cells. The function of NCAM was further confirmed in NCAM-deficient mesenchymal stem cells (MSCs), which also had a phenotype with reduced osteoblastic potential. Moreover, NCAM silencing induced decrease of Wnt/β-catenin and Akt activation. The Wnt inhibitor blocked osteoblast differentiation, and the Wnt activator recovered osteoblast differentiation in NCAM-silenced MC3T3-E1 cells. We lastly demonstrated that osteoblast differentiation of MC3T3-E1 cells was inhibited by the PI3K-Akt inhibitor. In conclusion, these results demonstrate that NCAM silencing inhibited osteoblastic differentiation through inactivation of Wnt/β-catenin and PI3K-Akt signaling pathways.

Pancreatic carcinoma (PC) is one of the most common malignancies. Chimeric antigen receptor (CAR)-modified T cells has achieved remarkable efficacy in the treatment of hematological malignancies. However, lack of tumor-specific targets and the existence of inhibitory factors limit the function of CAR T cells when treating solid tumors. 4.1R has been reported to suppress the anti-tumor activity of T cell responses. In this study, we investigated the anti-tumor activity of 4.1R deletion in natural killer group 2D (NKG2D)-CAR T cells against PC. The CAR T cells were obtained by transfecting T cells with lentiviral vector carrying NKG2D-CAR, NC-NKG2D-CAR, or KD2-NKG2D-CAR. In vitro, NKG2D-CAR T cells showed higher cytotoxicity than Mock T cells. However, compared to NKG2D-CAR T cells, furtherly higher cytotoxicity against PC cells in a dose-dependent manner was found in KD2-NKG2D-CAR T cells. In addition, the proliferation rate and cytotoxic activity of KD2-NKG2D-CAR T cells were significantly higher than those of NKG2D-CAR T cells. Besides, the inhibitory receptors PD-1 and TIM-3 were expressed in lower level on KD2-NKG2D-CAR T cells. In vivo, KD2-NKG2D-CAR T cells suppressed tumor growth more effectively in a xenograft model compared to NKG2D-CAR T cells. Mechanistically, 4.1R regulated CAR T cell function via activating ERK signaling pathway. Therefore, the study provides a new idea to enhance the anti-tumor efficiency of CAR T therapy.

Mesenchymal stem cell (MSC) therapy is a promising prospect for the treatment of Alzheimer's disease (AD); however, the underlying mechanisms by which MSCs mediate positive effects are still unclear. We speculated that MSCs mediate microglial autophagy and enhance the clearance of Aβ. To test this hypothesis, we cultured BV2 microglial cells with umbilical cord mesenchymal stem cells conditioned medium (ucMSCs-CM) in the presence or absence of Aβ25-35 oligomers. We investigated BV2 cell proliferation, cell death, and Aβ25-35 phagocytosis as well as protein expression levels of LC3, Beclin-1, p62, insulin-degrading enzyme (IDE), and neprilysin (Nep) with western blotting. The results showed that ucMSCs-CM inhibited the proliferation and decreased cell death of BV2 cells induced by Aβ25-35. ucMSCs-CM also promoted the phagocytosis of Aβ25-35 by BV2 cells and changed the expression of autophagy-related proteins LC3, Beclin-1, and p62. Treatment also upregulated the expression of Aβ-degrading enzymes IDE and Nep. Furthermore, the culture medium in BV2 cells with Aβ25-35 and ucMSCs-CM prevented neuronal cell SH-SY5Y from cell death compared to control medium without ucMSCs-CM. Altogether, these data suggested that ucMSCs-CM protect microglial and neuronal cells from Aβ25-35-induced cell death and promote Aβ phagocytosis by modulating autophagy and enhancing the expression of Aβ-degrading enzymes in microglia.

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