Effects of replacing fishmeal with soybean meal on the immune and antioxidant capacity, and intestinal metabolic functions of red swamp crayfish Procambarus clarkii.

Excess utilization of plant protein sources in animal feed has been found to adversely affect the antioxidant properties and immunity of animals. While the role of gut microbes in plant protein-induced inflammation has been identified in various models, the specific mechanisms regulating gut microbes in crustaceans remain unclear. Accordingly, this study was designed to investigate the effects of replacing fishmeal with soybean meal (SM) on the hepatopancreas antioxidant and immune capacities, and gut microbial functions of crayfish, as well as the potential microbial regulatory mechanisms. 750 crayfish (4.00 g) were randomly divided into five groups: SS0, SS25, SS50, SS75, and SS100, and fed diets with different levels of soybean meal substituted for fishmeal for six weeks. High SM supplementation proved detrimental to maintaining hepatopancreas health, as indicated by an increase in hemolymph MDA content, GPT, and GOT activities, the observed rupture of hepatopancreas cell basement membranes, along with the decreased number of hepatopancreatic F cells. Moreover, crayfish subjected to high SM diets experienced obvious inflammation in hepatopancreas, together with up-regulated mRNA expression levels of nfkb, alf, and tlr (p<0.05), whereas the lzm mRNA expression level exhibited the highest value in the SS25 group. Furthermore, hepatopancreas antioxidant properties highly attenuated by the level of dietary SM substitution levels, as evidenced by the observed increase in MDA content (p<0.05), decrease in GSH content (p<0.05), and inhabitation of SOD, CAT, GPx, and GST activities (p<0.05), along with down-regulated hepatopancreas cat, gpx, gst, and mmnsod mRNA expression levels via inhibiting nrf2/keap1 pathway. Functional genes contributing to metabolism identified that high SM diets feeding significantly activated lipopolysaccharide biosynthesis, revealing gut dysfunction acted as the cause of inflammation. The global microbial co-occurrence network further indicated that the microbes contributing more to serum indicators and immunity were in module eigengene 17 (ME17). A structural equation model revealed that the genes related to alf directly drove the serum enzyme activities through microbes in ME17, with OTU399 and OTU533 identified as major biomarkers and classified into Proteobacteria that secrete endotoxins. To conclude, SM could replace 25 % of fishmeal in crayfish diets without negatively affecting immunity, and antioxidant capacity. Excessive SM levels contributed to gut dysfunction and weakened the innate immune system of crayfish.

PD-L1 on dendritic cells attenuates T cell activation and regulates response to immune checkpoint blockade.

Immune checkpoint blockade therapies have shown clinical promise in a variety of cancers, but how tumor-infiltrating T cells are activated remains unclear. In this study, we explore the functions of PD-L1 on dendritic cells (DCs), which highly express PD-L1. We observe that PD-L1 on DC plays a critical role in limiting T cell responses. Type 1 conventional DCs are essential for PD-L1 blockade and they upregulate PD-L1 upon antigen uptake. Upregulation of PD-L1 on DC is mediated by type II interferon. While DCs are the major antigen presenting cells for cross-presenting tumor antigens to T cells, subsequent PD-L1 upregulation protects them from killing by cytotoxic T lymphocytes, yet dampens the antitumor responses. Blocking PD-L1 in established tumors promotes re-activation of tumor-infiltrating T cells for tumor control. Our study identifies a critical and dynamic role of PD-L1 on DC, which needs to be harnessed for better invigoration of antitumor immune responses.

Targeting IFNα to tumor by anti-PD-L1 creates feedforward antitumor responses to overcome checkpoint blockade resistance.

Many patients remain unresponsive to intensive PD-1/PD-L1 blockade therapy despite the presence of tumor-infiltrating lymphocytes. We propose that impaired innate sensing might limit the complete activation of tumor-specific T cells after PD-1/PD-L1 blockade. Local delivery of type I interferons (IFNs) restores antigen presentation, but upregulates PD-L1, dampening subsequent T-cell activation. Therefore, we armed anti-PD-L1 antibody with IFNα (IFNα-anti-PD-L1) to create feedforward responses. Here, we find that a synergistic effect is achieved to overcome both type I IFN and checkpoint blockade therapy resistance with the least side effects in advanced tumors. Intriguingly, PD-L1 expressed in either tumor cells or tumor-associated host cells is sufficient for fusion protein targeting. IFNα-anti-PD-L1 activates IFNAR signaling in host cells, but not in tumor cells to initiate T-cell reactivation. Our data suggest that a next-generation PD-L1 antibody armed with IFNα improves tumor targeting and antigen presentation, while countering innate or T-cell-driven PD-L1 upregulation within tumor.

Targeting Tertiary Lymphoid Structures for Tumor Immunotherapy.

Tumor microenvironments (TME) are usually immunosuppressive and prevent lymphocyte priming. Recent clinical trials have shown that cancer immunotherapy such as immune checkpoint inhibitors can induce unprecedented durable responses in patients with a variety of cancers. Tertiary lymphoid structures (TLS) can form inside or adjacent to tumor tissues due to persistent inflammation. The formation of TLS facilitates lymphocyte trafficking and infiltration into tumor tissues. It can also support effective antigen presentation and lymphocyte activation. Thus, TLS have become an intriguing target to manipulate antitumor immunity. Several therapeutics targeting TLS have been developed and shown promising antitumor effects in various mouse models. In this chapter, we describe the general approach to establish transplantable mouse tumor models for the study of immunotherapy. We introduce the strategies for therapy through systemic or local treatment targeting TLS. We also present approaches to evaluate the antitumor immune responses provoked by the therapies.

PD-L1 on host cells is essential for PD-L1 blockade-mediated tumor regression.

Programmed death-ligand 1 (PD-L1) expression on tumor cells is essential for T cell impairment, and PD-L1 blockade therapy has shown unprecedented durable responses in several clinical studies. Although higher expression of PD-L1 on tumor cells is associated with a better immune response after Ab blockade, some PD-L1-negative patients also respond to this therapy. In the current study, we explored whether PD-L1 on tumor or host cells was essential for anti-PD-L1-mediated therapy in 2 different murine tumor models. Using real-time imaging in whole tumor tissues, we found that anti-PD-L1 Ab accumulates in tumor tissues, regardless of the status of PD-L1 expression on tumor cells. We further observed that, while PD-L1 on tumor cells was largely dispensable for the response to checkpoint blockade, PD-L1 in host myeloid cells was essential for this response. Additionally, PD-L1 signaling in defined antigen-presenting cells (APCs) negatively regulated and inhibited T cell activation. PD-L1 blockade inside tumors was not sufficient to mediate regression, as limiting T cell trafficking reduced the efficacy of the blockade. Together, these findings demonstrate that PD-L1 expressed in APCs, rather than on tumor cells, plays an essential role in checkpoint blockade therapy, providing an insight into the mechanisms of this therapy.