As the targets of chimeric antigen receptor (CAR)–T cells expand to a variety of cancers, autoimmune diseases, viral infections, and fibrosis, there is an increasing demand for identifying new antigens and designing new CARs that can be effectively activated. However, the rational selection of antigens and the design of CARs are limited by a lack of knowledge regarding the molecular mechanism by which CARs are activated by antigens. Here, we present data supporting a “size exclusion” model explaining how antigen signals are transmitted across the plasma membrane to activate the intracellular domains of CARs. In this model, antigen engagement with CAR results in a narrow intermembrane space that physically excludes CD45, a bulky phosphatase, out of the CAR zone, thus favoring CAR phosphorylation by kinases, which further triggers downstream pathways leading to T cell activation. Aligned with this model, increasing the size of CAR extracellular domains diminished CAR-T activation both in vitro and in a mouse lymphoma model; membrane-proximal epitopes activated CAR-Ts better than membrane-distal epitopes. Moreover, increasing the size of CD45 by antibody conjugation enhanced the activation of CARs that recognize membrane-distal epitopes. Consistently, CAR-Ts expressing CD45RABC, the larger isoform, were activated to a higher level than those expressing a smaller isoform CD45RO. Together, our work revealed that CAR-T activation depends on the size difference between the CAR-antigen pair and CD45; the size of CAR, antigen, and CD45 can thus be targets for tuning CAR-T activation.

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A previous study found that an AAAG-rich Oligodeoxynucleotide (ODN), designated as MS19, could lessen the acute lung inflammatory injury (ALII) in mice infected by influenza viruses. Bioinformatics analysis found that MS19 is consensus with the binding site of interferon regulatory factor 5 (IRF5) in the regulatory elements of pro-inflammatory genes. This study established a septic peritonitis model in Institute of Cancer Research (ICR) mice infected with Escherichia coli (E. coli), and found that MS19 prolonged the survival of the mice and down-regulated the expression of inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor α (TNF-α). In cultured RAW264.7 cells, MS19 significantly reduced the expression of iNOS, IRF5, IL-6, and TNF-α and inhibited the nuclear translocation of IRF5. This data may provide a new insight for understanding how MS19 reduces the excessive inflammatory responses in sepsis.

Mixed infection of porcine circovirus type 2 (PCV2) and foot-and-mouth disease virus (FMDV) is devastating to swine populations. To develop an effective vaccine that can protect the pigs from the infection of PCV2 and FMDV, we used the neutralizing B cell epitope region (aa 135-160) of FMDV to replace the regions aa 123-151 and aa 169-194 of the PCV2b Cap protein to generate a recombinant protein designated as Capfb. The Capfb protein was expressed in Escherichia coli system and the purified Capfb protein assembled into virus-like particles (VLPs) through dialysis. The ability of the Capfb protein to induce effective immune response against FMDV and PCV2b was tested in mice and guinea pigs. The results showed that the Capfb-VLPs could elicit anti-PCV2b and anti-FMDV antibody response in mice and guinea pigs without inducing antibodies against decoy epitope. Moreover, the Capfb-VLPs could enhance the percentage and activation of B cells in lymph nodes when the mice were stimulated with inactivated FMDV or PCV2b. These data suggested that the Capfb-VLPs could be an efficacious candidate antigen for developing a novel PCV2b-FMDV bivalent vaccine.

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The success of using immune checkpoint inhibitors to treat cancers implies that inhibiting an immunosuppressive cytokine, such as TGF-2, could be a strategy to develop novel adjuvants for microbial vaccines. To develop nucleic acid based TGF-2 inhibitors, we designed three antisense oligonucleotides, designated as TIO1, TIO2, and TIO3, targeting the conserve regions identical in human and mouse TGF-2 mRNA 3 ′-untranslated region. In cultured immune cells, TIO3 and TIO1 significantly reduced the TGF-2 mRNA expression and protein production. In mice, the TIO3 and TIO1, when formulated in various microbial vaccines, significantly enhanced the antibody response to the vaccines, and the TIO3-adjuvanted influenza virus vaccine induced effective protection against the influenza virus challenge. In the immunized mice, TIO3 formulated in microbial vaccines dramatically reduced surface-bound TGF-2 expression on CD4 + T cells and CD19 + B cells in the lymph node (LN) cells and spleen cells; up-regulated the expression of CD40, CD80, CD86, and MHC II molecules on CD19 + B cells and CD11c + dendritic cells; and promoted IFNproduction in CD4 + T cells and CD8 + T cells in the LN cells. Overall, TIO3 or TIO1 could be used as a novel type of adjuvant for facilitating the microbial vaccines to elicit more vigorous and persistent antibody response by interfering with TGF-2 expression.

Summary The excessive activation of interferon regulatory factor 7 (IRF7) promotes the development of acute lung injury (ALI) caused by influenza A virus (IAV). However, the deficiency of IRF7 increases the susceptibility to deadly IAV infection in both humans and mice. To test whether the attenuation rather than the abolishment of IRF7 activity in local infectious sites could alleviate IAV‐induced ALI, we established IAV‐infected mouse model and trachea/lung‐tissue culture systems, and designed two IRF7‐interfering oligodeoxynucleotides, IRF7‐rODN M1 and IRF7‐rODN A1, based on the mouse and human consensus sequences of IRF7‐binding sites of Ifna/IFNA genes, respectively. In the model mice, we found a close relationship between the IAV‐induced ALI and the level/activity of IRF7 in local infectious sites, and also found that the reduced IRF7 level or activity in the lungs of mice treated with IRF7‐rODN M1 led to decreased mRNA levels of Ifna genes, reduced neutrophil infiltration in the lungs and prolonged survival of mice. Furthermore, we found that the effects of IRF7‐rODN M1 on alleviating IAV‐induced ALI could be correlated to the reduced translocation of IRF7, caused by the IRF7‐rODN M1, from cytosol to nucleus in IAV‐infected cells. These data suggest that the proper attenuation of IRF7 activity in local infectious sites could be a novel approach for treating IAV‐induced ALI.