Highlights (maximum 85 characters, including spaces) 1. Absence of macrophages results in zigzag shaped pupils. 2. Macrophages do not prune pupillary membrane vessels during postnatal first week. 3. Macrophages engulf pigmented debris in pupillary edges. 4. Irises protrude between blood vessels in the absence of macrophages.

We describe a small lipid nanoparticle (SLNP)‐based nanovaccine platform and a new combination treatment regimen. Tumor antigen‐displaying, CpG adjuvant‐embedded SLNPs (OVAPEP‐SLNP@CpG) were prepared from biocompatible phospholipids and a cationic cholesterol derivative. The resulting nanovaccine showed highly potent antitumor efficacy in both prophylactic and therapeutic E.G7 tumor models. However, this vaccine induced T cell exhaustion by elevating PD‐L1 expression, leading to tumor recurrence. Thus, the nanovaccine was combined with simultaneous anti‐PD‐1 antibody treatment, but the therapeutic efficacy of this regimen was comparable to that of the nanovaccine alone. Finally, mice that showed a good therapeutic response after the first cycle of immunization with the nanovaccine underwent a second cycle together with anti‐PD‐1 therapy, resulting in suppression of tumor relapse. This suggests that the antitumor efficacy of combinations of nanovaccines with immune checkpoint blockade therapy is dependent on treatment sequence and the timing of each modality.

Pseudomonas fluorescens is an efficient platform for recombinant protein production. P. fluorescens has an ABC transporter secreting endogenous thermostable lipase (TliA) and protease, which can be exploited to transport recombinant proteins across the cell membrane. In this study, the expression vector pDART was constructed by inserting tliDEF, genes encoding the ABC transporter, along with the construct of the lipase ABC transporter recognition domain (LARD), into pDSK519, a widely used shuttle vector. When the gene for the target protein was inserted into the vector, the C-terminally fused LARD allowed it to be secreted through the ABC transporter into the extracellular medium. After secretion of the fused target protein, the LARD containing a hydrophobic C terminus enabled its purification through hydrophobic interaction chromatography (HIC) using a methyl-Sepharose column. Alkaline phosphatase (AP) and green fluorescent protein (GFP) were used to validate the expression, export, and purification of target proteins by the pDART system. Both proteins were secreted into the extracellular medium in P. fluorescens. In particular, AP was secreted in several Pseudomonas species with its enzymatic activity in extracellular media. Furthermore, purification of the target protein using HIC yielded some degree of AP and GFP purification, where AP was purified to almost a single product. The pDART system will provide greater convenience for the secretory production and purification of recombinant proteins in Gram-negative bacteria, such as Pseudomonas species.T he mass production of recombinant proteins continues to be an important issue in various industries (1, 2). In conventional methods, recombinant proteins are synthesized in prokaryotic cells, such as Escherichia coli, and are purified from the cell extract by biochemical means after cell lysis. A protein-manufacturing system that can simultaneously express and secrete recombinant proteins is far more efficient than conventional methods due to the reduced requirement for expensive extraction and purification procedures (3-6). Several features of Pseudomonas fluorescens, a Gram-negative psychrotrophic bacterium, make it one of the most useful organisms for recombinant protein production (7). P. fluorescens, living on the surfaces of most plants, is generally accepted as safe, since it has been consumed by humans for a long time (8). The safety of the ␣-amylase enzyme produced using P. fluorescens has been verified by pharmacological and toxicological studies in mice and rats (9). In addition to its biological safety, P. fluorescens is capable of withstanding various fermentation conditions in high-cell-density cultures and therefore can produce large quantities of recombinant proteins (10, 11). Several secretion systems exist natively in P. fluorescens, ranging from a type I secretion system (T1SS) to a type VI secretion system (T6SS) (12, 13). In particular, P. fluorescens has a type I secretion system that transports thermostable lipase, TliA, through the ATP-bin...

We describe a small lipid nanoparticle (SLNP)‐based nanovaccine platform and a new combination treatment regimen. Tumor antigen‐displaying, CpG adjuvant‐embedded SLNPs (OVAPEP‐SLNP@CpG) were prepared from biocompatible phospholipids and a cationic cholesterol derivative. The resulting nanovaccine showed highly potent antitumor efficacy in both prophylactic and therapeutic E.G7 tumor models. However, this vaccine induced T cell exhaustion by elevating PD‐L1 expression, leading to tumor recurrence. Thus, the nanovaccine was combined with simultaneous anti‐PD‐1 antibody treatment, but the therapeutic efficacy of this regimen was comparable to that of the nanovaccine alone. Finally, mice that showed a good therapeutic response after the first cycle of immunization with the nanovaccine underwent a second cycle together with anti‐PD‐1 therapy, resulting in suppression of tumor relapse. This suggests that the antitumor efficacy of combinations of nanovaccines with immune checkpoint blockade therapy is dependent on treatment sequence and the timing of each modality.

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