Effects of methionine deficiency on B7H3-DAP12-CAR-T cells in the treatment of lung squamous cell carcinoma.

Lung squamous cell carcinoma (LUSC) is a subtype of lung cancer for which precision therapy is lacking. Chimeric antigen receptor T-cells (CAR-T) have the potential to eliminate cancer cells by targeting specific antigens. However, the tumor microenvironment (TME), characterized by abnormal metabolism could inhibit CAR-T function. Therefore, the aim of this study was to improve CAR-T efficacy in solid TME by investigating the effects of amino acid metabolism. We found that B7H3 was highly expressed in LUSC and developed DAP12-CAR-T targeting B7H3 based on our previous findings. When co-cultured with B7H3-overexpressing LUSC cells, B7H3-DAP12-CAR-T showed significant cell killing effects and released cytokines including IFN-γ and IL-2. However, LUSC cells consumed methionine (Met) in a competitive manner to induce a Met deficiency. CAR-T showed suppressed cell killing capacity, reduced cytokine release and less central memory T phenotype in medium with lower Met, while the exhaustion markers were up-regulated. Furthermore, the gene NKG7, responsible for T cell cytotoxicity, was downregulated in CAR-T cells at low Met concentration due to a decrease in m5C modification. NKG7 overexpression could partially restore the cytotoxicity of CAR-T in low Met. In addition, the anti-tumor efficacy of CAR-T was significantly enhanced when co-cultured with SLC7A5 knockdown LUSC cells at low Met concentration. In conclusion, B7H3 is a prospective target for LUSC, and B7H3-DAP12-CAR-T cells are promising for LUSC treatment. Maintaining Met levels in CAR-T may help overcome TME suppression and improve its clinical application potential.

Enhancement of humoral and cellular immunity in mice against Japanese encephalitis virus using a DNA prime-protein boost vaccine strategy.

A synthetic multi-epitope gene containing critical epitopes of the Japanese encephalitis virus (JEV) envelope gene was cloned into both prokaryotic and eukaryotic expression vectors. The recombinant plasmid and purified recombinant protein (heterologously expressed in Escherichia coli) were used as immunogens in a mouse model. The results indicate that both the recombinant protein and the DNA vaccine induce humoral and cellular immune responses. Neutralising antibody titres in mice in the pcDNA-TEP plus rEP group increased considerably relative to mice immunised using either pcDNA-TEP or rEP alone (P<0.05). Furthermore, the highest levels of interleukin (IL)-2, interferon-gamma and IL-4 were induced following priming with the DNA vaccine and boosting with the recombinant protein. Together these findings demonstrate that a DNA-recombinant protein prime-boost vaccination strategy can produce high levels of antibody and trigger significant T cell responses in mice, highlighting the potential value of such an approach in the prevention of JEV infection.

Immune responses of recombinant adenoviruses expressing immunodominant epitopes against Japanese encephalitis virus.

Japanese encephalitis virus (JEV), which belongs to the family Flaviviridae, causes infection of the central nervous system in humans and equines and stillbirths in swine. In the present report, we constructed and characterized the immune responses conferred by recombinant adenoviruses expressing JEV E epitopes (six amino acid residues 60-68, 327-333, 337-345, 373-399, 397-403 and 436-445 in E, designated TEP). Seven groups (n = 10) of female BALB/c mice received intramuscular (IM) or oral immunization with the recombinant adenoviruses twice at 2-week intervals. Intramuscular immunization of mice with rAd-TEP generated greater titers of anti-JEV antibodies and JEV neutralizing activity than in animals with oral injection. It statistically significant differences were found in anti-JEV antibody titers and JEV neutralizing activity induced by IM immunization with rAd-TEP at a dose of 1 x 10(8.0)TCID50 when compared with the doses tested (3 x 10(7.0) and 1 x 10(7.0)TCID50) IM inoculation of rAd-TEP. Splenocytes from mice immunized intramuscularly with rAd-TEP secreted the largest amounts of interferon-gamma and interleukin-2 and moderate amounts of interleukin-4 in the presence of JEV. It demonstrates that IM immunization with rAd-TEP induced the highest level of cell-mediated immune responses and the higher level of JEV-specific humoral immune responses than oral immunization. Then we further evaluated the protective efficacy of the recombinants in swine. All swine were protected from viral challenge with IM rAd-TEP at 1 x 10(10.0)TCID50, even though the neutralizing antibody titers were lower than those in the group inoculated with inactivated vaccine. Our findings indicate that rAd-TEP might be an attractive candidate vaccines for preventing JEV infection.