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Chimeric antigen receptor (CAR)-T cell therapy is an innovative treatment for CD19-expressing lymphomas. CAR-T cells are primarily manufactured via lentivirus transfection or transposon electroporation. While anti-tumor efficacy comparisons between the two methods have been conducted, there is a current dearth of studies investigating the phenotypes and transcriptome alterations induced in T cells by the two distinct manufacturing methods. Here, we established CAR-T signatures using fluorescent imaging, flow cytometry, and RNA-sequencing. A small fraction of CAR-T cells that were produced using the PiggyBac transposon (PB CAR-T cells) exhibited much higher expression of CAR than those produced using a lentivirus (Lenti CAR-T cells). PB and Lenti CAR-T cells contained more cytotoxic T cell subsets than control T cells, and Lenti CAR-T cells presented a more pronounced memory phenotype. RNA-sequencing further revealed vast disparities between the two CAR-T cell groups, with PB CAR-T cells exhibiting greater upregulation of cytokines, chemokines, and their receptors. Intriguingly, PB CAR-T cells singularly expressed IL-9 and fewer cytokine release syndrome-associated cytokines when activated by target cells. In addition, PB CAR-T cells exerted faster in vitro cytotoxicity against CD19-expressing K562 cells but similar in vivo anti-tumor efficacy with Lenti CAR-T. Taken together, these data provide insights into the phenotypic alterations induced by lentiviral transfection or transposon electroporation and will attract more attention to the clinical influence of different manufacturing procedures.

Discoidin domain receptor 1 (DDR1) has been demonstrated to be able to promote tumor invasion and metastasis and being closely related to tumor immune infiltration. However, DDR1 has rarely been studied in gastric cancer. Here, we primarily evaluated DDR1 expression in gastric cancer and its cell lines using multiple databases. Subsequently, the cancer prognosis was investigated in relation to DDR1 expression. After analysis, we discovered that DDR1 was highly expressed and significantly connected with poor prognosis in gastric cancer. To comprehensively understand the molecular mechanism of DDR1, we explored genes and proteins interacting with DDR1 in gastric cancer using databases. Additionally, we found that the expression level of DDR1 was inversely correlated with immune infiltration and significantly relative to various immune cell markers. Overall, DDR1 was implicated in invasion, metastasis, and immune infiltration of gastric cancer. Inhibition of DDR1 may have the potential to alleviate the strong invasiveness and metastasis of advanced gastric cancer. Meanwhile, immune exclusion by DDR1 may also provide a new strategy for improving the efficacy of immune checkpoints inhibitors (ICIs), such as programmed cell death protein 1 (PD-1) antibody.

Kunitz-type protease inhibitors (KPIs) are ubiquitously found in many organisms, and participate in various physiological processes. However, their function in insects remains to be elucidated. In the present study, we characterized and functionally analyzed silkworm KPI5. Sequence analysis showed that KPI5 contains 85 amino acids with six conserved cysteine residues, and the P1 site is a phenylalanine residue. Inhibitory activity and stability analyses indicated that recombinant KPI5 protein significantly inhibited the activity of chymotrypsin and was highly tolerant to temperature and pH. The spatio-temporal expression profile analysis showed that KPI5 was synthesized in the fat body and secreted into the hemolymph. In vivo induction analysis showed that the expression of KPI5 in the fat body was significantly upregulated by pathogen-associated molecular patterns (PAMPs). Binding assays suggested that KPI5 can bind to pathogens and PAMPs. In vitro pathogen growth inhibition assay and encapsulation analysis indicated that KPI5 can neither kill pathogenic bacteria directly nor promote the encapsulation of agarose beads by silkworm hemocytes. Recombinant protein injection test and CRISPR/Cas9-mediated knockdown showed that KPI5 promotes the expression of antimicrobial peptides (AMPs) in the fat body. Moreover, the survival rate of individuals in the KPI5 knockdown group was significantly lower than that of the control group after pathogen infection. Phenoloxidase (PO) activity assays showed that KPI5 significantly inhibited the hemolymph PO activity and melanization induced by PAMPs. These findings suggested that KPI5 plays a dual regulatory role in innate immunity by promoting the expression of antimicrobial peptides in the fat body and inhibiting hemolymph melanization. Our study furthers the understanding of the function of insect KPIs and provides new insights into the regulatory mechanism of insect immune homeostasis.

The SIRPαFc fusion protein can block the immunosuppressive CD47-SIRPα signal between macrophages and tumor cells as a decoy receptor and has demonstrated its immunotherapeutic efficacy in various tumors. However, its clinical application was limited because of the potential hematologic toxicity. The heptapeptide “TKKTLRT” is a collagen-binding domain (CBD) which can bind collagen specifically. Herein, we aim to improve the tumor targeting of SIRPαFc and therefore avoid its unnecessary exposure to normal cells through synthesizing a TKKTLRT–SIRPαFc conjugate. Experiments at molecular and cellular levels indicate that the TKKTLRT–SIRPαFc conjugate-derived collagen-binding affinity and the introduction of CBD did not impact the CD47-binding affinity as well as its phagocytosis-promoting effect on NSCLC cells. In vivo distribution experiments showed that CBD–SIRPαFc accumulated in tumor tissue more effectively compared to unmodified SIRPαFc, probably due to the exposed collagen in the tumor vascular endothelium and stroma resulting from the abnormal vessel structure. On an A549 NSCLC nude mouse xenograft model, CBD–SIRPαFc presented more stable and effective antitumor efficacy than SIRPαFc, along with significantly increased CD11b+F4/80+ macrophages especially MHC II+ M1 macrophages within tumors. All of these results revealed that CBD brought a tumor-targeting ability to the SIRPαFc fusion protein, which contributed to the enhanced antitumor immune response. Altogether, the CBD–SIRPαFc conjugate may have the potential to be an effective tumor immunotherapy with improved antitumor efficacy but less non-tumor-targeted side effect.

Serine protease inhibitors of Kazal-type (SPINKs) were widely identified in vertebrates and invertebrates, and played regulatory roles in digestion, coagulation, and fibrinolysis. In this study, we reported the important role of SPINK7 in regulating immune defense of silkworm, Bombyx mori. SPINK7 contains three Kazal domains and has 6 conserved cysteine residues in each domain. Quantitative real-time PCR analyses revealed that SPINK7 was exclusively expressed in hemocytes and was upregulated after infection with two fungi, Saccharomyces cerevisiae and Candida albicans. Enzyme activity inhibition test showed that SPINK7 significantly inhibited the activity of proteinase K from C. albicans. Additionally, SPINK7 inhibited the growth of three fungal spores, including S. cerevisiae, C. albicans, and Beauveria bassiana. The pathogen-associated molecular patterns (PAMP) binding assays suggested that SPINK7 could bind to β-D-glucan and agglutinate B. bassiana and C. albicans. In vitro assays were performed using SPINK7-coated agarose beads, and indicated that SPINK7 promoted encapsulation and melanization of agarose beads by B. mori hemocytes. Furthermore, co-localization studies using immunofluorescence revealed that SPINK7 induced hemocytes to aggregate and entrap the fungi spores of B. bassiana and C. albicans. Our study revealed that SPINK7 could recognize fungal PAMP and induce the aggregation, melanization, and encapsulation of hemocytes, and provided valuable clues for understanding the innate immunity and cellular immunity in insects.

The activation of TLR7 signaling in T cells accelerates antigen-specific responses. Such responses play an essential role in eliminating viral infections and can be anti-tumorigenic. However, the underlying mechanisms of how TLR7 can promote the optimal function of CD8+ T cells remain unclear. To investigate how TLR signaling directly contributes to CD8+ T cell functions, we examine the activation of cellular TLR7-related pathways and functional and metabolic alterations in TLR7-stimulated T cells during T cell receptor (TCR) signaling. In the present study, we investigated the activation of CD8+ T cells in response to direct stimulation by TLR7 ligands. TLR7 stimulation could promote the effector functions of purified CD8+ T cells in vitro. The TLR7-induced activation of CD8+ T cells occurs if CD8+ T cells were primed by αCD3 activation and increasingly expressed TLR7. MyD88 and AKT-mTOR signaling plays a critical role in TLR7-induced T cell activation. In addition to the upregulation of immune-related genes, metabolic alterations in CD8+ T cells, including the upregulation of glucose uptake and glycolysis, occurred by TLR7 stimulation. Glycolysis was found to be regulated by the AKT-mTOR pathway and a downstream transcription factor IRF4. Blocking glycolysis by either direct glucose deprivation or modulating the mTOR pathway and IRF4 expression was found to impair T cell activation and functions. Taken together, the activation of TLR7 signaling promotes the effector functions of CD8+ T cells by enhancing cellular glycolysis.

Toll-like receptors (TLRs) play a crucial role in activation of innate immunity, which is essential for inducing effective adaptive immune responses. Our previous studies have shown that toll-like receptor 2 (TLR2) is required to induce effective virus-specific T-cell responses against hepatitis B virus (HBV) in vivo. However, the contribution of TLR2 activation to adaptive immunity and HBV clearance remains to be clarified. In this study, we explored the hydrodynamic injection (HI) mouse models for HBV infection and examined how the TLR2 agonist Pam3CSK (P3C) influences HBV control and modulates HBV-specific T-cell response if applied in vivo. We found that TLR2 activation by P3C injection leads to the rapid but transient production of serum proinflammatory factors interleukin-6 and tumor necrosis factor-α and activation of CD8+ T cells in vivo. Then, the anti-HBV effect and HBV-specific T-cell immunity were investigated by TLR2 activation in the mouse models for persistent or acute HBV infections using HBV plasmids pAAV-HBV1.2 and pSM2, respectively. Both P3C application at early stage and pre-activation promoted HBV clearance, while only TLR2 pre-activation enhanced HBV-specific T-cell response in the liver. In the mouse model for acute HBV infection, P3C application had no significant effect on HBV clearance though P3C significantly enhanced the HBV-specific T-cell response. Collectively, TLR2 pre-activation enhances HBV-specific T-cell responses and accelerates HBV clearance in HI mouse models. Thus, the modulation of host immune status by TLR2 agonists may be explored for immunotherapeutic strategies to control HBV infection.