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Progesterone (P4) and glucocorticoid (GC) play crucial roles in the immunoregulation of a mother to accept and maintain a semi-allogenic fetus. P4 concentration increases during pregnancy and becomes much higher in the placenta than in the other peripheral tissues, wherein the concentration of cortisol (COR), the most abundant GC and a strong immunosuppressor, remains uniform throughout the rest of the body. Here, we evaluated the effect of a high-P4 environment on pregnant immunity by comparing it with COR. Naïve T cell proportion increased transiently in peripheral blood of pregnant women just after delivery and decreased after one month. T cells stimulated with superantigen toxic-shock-syndrome-1 (TSST-1) in the presence of P4 stayed in the naïve state and did not increase, irrespective of the presence of COR, and reactive T cells could not survive. Treatment of T cells with P4 without T cell receptor (TCR) stimulation transiently suppressed T cell activation and proliferation, whereas the levels remain unaltered if P4 was not given before stimulation. Comparison of the engraftment and response against specific antigens using hu-PBL-NOG-hIL-4-Tg mice showed that P4-pretreated lymphocytes preserved CD62L expression and engrafted effectively in the spleen. Moreover, they produced antigen-specific antibodies, whereas COR-pretreated lymphocytes did not. These results suggest that a high-P4 environment suppresses T cell activation and induces T cell migration into lymphoid tissues, where they maintain the ability to produce anti-pathogen antibodies, whereas COR does not preserve T cell function. The mechanism may be pivotal in maintaining non-fetus-specific T cell function in pregnancy.

Immune-humanized mouse models are indispensable tools for evaluating human immune responses and testing immune-based therapies; however, the induction of robust human antigen-specific immunoglobulin G (IgG) production remains limited due to species-specific incompatibilities. To investigate how human interleukin-4 (IL-4) promotes antibody responses, we employed NOG-hIL-4 transgenic mice (NOG-hIL-4-Tg), which constitutively express human IL-4, and transplanted them with human peripheral blood mononuclear cells (PBMCs). Flow cytometry, histological analysis, and next-generation sequencing were used to assess human lymphocyte maintenance, receptor repertoire diversity, class switching, and antigen-specific responses following immunization with peptide antigens. Sustained human B cell maintenance correlated with defined supraphysiological ranges of plasma hIL-4 concentrations. T and B cell receptor repertoire analyses demonstrated stable clonal diversity for one month after engraftment, followed by contraction by three months. Immunoglobulin repertoire profiling revealed IL-4–dependent class switching to IgG, with early predominance of IgG3 that declined over time and a gradual increase in IgG1, though subclass distribution varied among donors. Immunization induced antigen-reactive IgG, although many clones displayed low-affinity or cross-reactive binding, consistent with limited affinity maturation. Histological examination revealed tertiary lymphoid structure–like accumulations of human B and T cells within the spleen, without fully developed germinal centers. These findings demonstrate that human IL-4 expression in NOG-hIL-4-Tg mice supports human B cell survival, class switching, and partial IgG maturation, providing a relevant platform for studying human humoral immunity and evaluating antibody-based immunotherapies.

Immune checkpoint inhibitors highlight the importance of anticancer immunity. However, their clinical utility and safety are limited by the low response rates and adverse effects. We focused on progesterone (P4), a hormone produced by the placenta during pregnancy, because it has multiple biological activities related to anticancer and immune regulation effects. P4 has a reversible immune regulatory function distinct from that of the stress hormone cortisol, which may drive irreversible immune suppression that promotes T cell exhaustion and apoptosis in patients with cancer. Because the anticancer effect of P4 is induced at higher than physiological concentrations, we aimed to develop a new anticancer drug by encapsulating P4 in liposomes. In this study, we prepared liposome-encapsulated anti-programmed death ligand 1 (PD-L1) antibody-conjugated P4 (Lipo-anti-PD-L1-P4) and evaluated the effects on the growth of MDA-MB-231 cells, a PD-L1-expressing triple-negative breast cancer cell line, in vitro and in NOG-hIL-4-Tg mice transplanted with human peripheral blood mononuclear cells (humanized mice). Lipo-anti-PD-L1-P4 at physiological concentrations reduced T cell exhaustion and proliferation of MDA-MB-231 in vitro . Humanized mice bearing MDA-MB-231 cells expressing PD-L1 showed suppressed tumor growth and peripheral tissue inflammation. The proportion of B cells and CD4+ T cells decreased, whereas the proportion of CD8+ T cells increased in Lipo-anti-PD-L1-P4-administrated mice spleens and tumor-infiltrated lymphocytes. Our results suggested that Lipo-anti-PD-L1-P4 establishes a systemic anticancer immune environment with minimal toxicity. Thus, the use of P4 as an anticancer drug may represent a new strategy for cancer treatment.

Coccomyxa subellipsoidea KJ (C-KJ) is a green alga with unique immunoregulatory characteristics. Here, we investigated the mechanism underlying the modification of T cell function by C-KJ components. The water-soluble extract of C-KJ was fractionated into protein (P) and sugar (S) fractions acidic (AS), basic (BS), and neutral (NS). These fractions were used for the treatment of peripheral blood mononuclear cells stimulated with toxic shock syndrome toxin-1. Transcriptome analysis revealed that both P and AS enhanced the expression of the genes encoding metallothionein (MT) family proteins, inflammatory factors, and T helper (Th) 17 cytokine and suppressed that of those encoding Th2 cytokines in stimulated T cells. The kinetics of MT1 and MT2A gene expression showed a transient increase in MT1 and maintenance of MT2A mRNA after T cell stimulation in the presence of AS. The kinetics of Th17-related cytokine secretion in the early period were comparable to those of MT2A mRNA. Furthermore, our findings revealed that static, a STAT-3 inhibitor, significantly suppressed MT2A gene expression. These findings suggest that the expression of MTs is involved in the immune regulatory function of C-KJ components, which is partially regulated by Th17 responses, and may help develop innovative immunoregulatory drugs or functional foods.

Coccomyxa subellipsoidea KJ (C-KJ) is a green alga with unique immunoregulatory characteristics. Here, we investigated the mechanism underlying the modification of T cell function by C-KJ components. The water-soluble extract of C-KJ was fractionated into protein (P) and sugar (S) fractions acidic (AS), basic (BS), and neutral (NS). These fractions were used for the treatment of peripheral blood mononuclear cells stimulated with toxic shock syndrome toxin-1. Transcriptome analysis revealed that both P and AS enhanced the expression of the genes encoding metallothionein (MT) family proteins, inflammatory factors, and T helper (Th) 17 cytokine and suppressed that of those encoding Th2 cytokines in stimulated T cells. The kinetics of MT1 and MT2A gene expression showed a transient increase in MT1 and maintenance of MT2A mRNA after T cell stimulation in the presence of AS. The kinetics of Th17-related cytokine secretion in the early period were comparable to those of MT2A mRNA. Furthermore, our findings revealed that static, a STAT-3 inhibitor, significantly suppressed MT2A gene expression. These findings suggest that the expression of MTs is involved in the immune regulatory function of C-KJ components, which is partially regulated by Th17 responses, and may help develop innovative immunoregulatory drugs or functional foods.

Immune-humanized mice provide valuable experimental models for evaluating immune-based therapies, yet the induction of human antigen-specific IgG production remains limited due to species-specific incompatibilities. Our previous work demonstrated that NOG-hIL-4-Tg mice, which express human interleukin-4 (IL-4), support human T and B cell maintenance and enable antigen-specific IgG production following transplantation of human peripheral blood mononuclear cells (PBMCs). In this study, we investigated how IL-4 enhances antibody responses in this model. Flow cytometry and histological analysis revealed that human B cell maintenance was associated with specific plasma hIL-4 concentration ranges at supraphysiological levels. T and B cell receptor repertoire analysis using next-generation sequencing showed that clonal diversity remained largely conserved for one month and decreased to three months post-engraftment. Immunoglobulin repertoire profiling confirmed IgG class switching in an IL-4-concentration-dependent manner. Among the IgG subclasses, IgG3 increased during the first and second months and then decreased thereafter. In contrast, IgG1 tended to increase over time; however, the proportion of IgG subclass varied among individual donors. Following immunization with two distinct peptide antigens, the mice produced enhanced levels of antigen-reactive IgG. However, many B cell clones also exhibited weak responses to unrelated third-party antigens, likely reflecting insufficient affinity maturation. Histological evaluation showed tertiary lymphoid structure (TLS)-like accumulations of B and T cells in the spleen, although fully developed germinal centers were absent. Taken together, these findings demonstrate that NOG-hIL-4-Tg mice maintain human B cells within a regulated IL-4 environment, promote early formation of splenic tertiary lymphoid structures, and support IgG production characterized by clonal expansion, class switching, and somatic hypermutation. These results confirm that human IL-4 expression supports human antibody responses in the PBL-NOG-hIL-4-Tg mouse system. (278)

In most cases of sporadic colorectal cancers, tumorigenesis is a multistep process, involving genomic alterations in parallel with morphologic changes. In addition, accumulating evidence suggests that the human gut microbiome is linked to the development of colorectal cancer. Here we performed fecal metagenomic and metabolomic studies on samples from a large cohort of 616 participants who underwent colonoscopy to assess taxonomic and functional characteristics of gut microbiota and metabolites. Microbiome and metabolome shifts were apparent in cases of multiple polypoid adenomas and intramucosal carcinomas, in addition to more advanced lesions. We found two distinct patterns of microbiome elevations. First, the relative abundance of Fusobacterium nucleatum spp. was significantly ( P  < 0.005) elevated continuously from intramucosal carcinoma to more advanced stages. Second, Atopobium parvulum and Actinomyces odontolyticus , which co-occurred in intramucosal carcinomas, were significantly ( P  < 0.005) increased only in multiple polypoid adenomas and/or intramucosal carcinomas. Metabolome analyses showed that branched-chain amino acids and phenylalanine were significantly ( P  < 0.005) increased in intramucosal carcinomas and bile acids, including deoxycholate, were significantly ( P  < 0.005) elevated in multiple polypoid adenomas and/or intramucosal carcinomas. We identified metagenomic and metabolomic markers to discriminate cases of intramucosal carcinoma from the healthy controls. Our large-cohort multi-omics data indicate that shifts in the microbiome and metabolome occur from the very early stages of the development of colorectal cancer, which is of possible etiological and diagnostic importance. Colorectal cancer stages are associated with distinct microbial and metabolomic profiles that could shed light on cancer progression.

Epithelial–mesenchymal transition (EMT)—a fundamental process in embryogenesis and wound healing—promotes tumor metastasis and resistance to chemotherapy. While studies have identified signaling components and transcriptional factors responsible in the TGF-β-dependent EMT, whether and how intracellular metabolism is integrated with EMT remains to be fully elucidated. Here, we showed that TGF-β induces reprogramming of intracellular amino acid metabolism, which is necessary to promote EMT in non-small cell lung cancer cells. Combined metabolome and transcriptome analysis identified prolyl 4-hydroxylase α3 (P4HA3), an enzyme implicated in cancer metabolism, to be upregulated during TGF-β stimulation. Further, knockdown of P4HA3 diminished TGF-β-dependent changes in amino acids, EMT, and tumor metastasis. Conversely, manipulation of extracellular amino acids induced EMT-like responses without TGF-β stimulation. These results suggest a previously unappreciated requirement for the reprogramming of amino acid metabolism via P4HA3 for TGF-β-dependent EMT and implicate a P4HA3 inhibitor as a potential therapeutic agent for cancer.Through metabolome and transcriptome analyses, Nakasuka et al find that TGF-β-induced epithelial–mesenchymal transition (EMT) in non-small cell lung cancer cells is associated with reprogramming of amino acid metabolism. They also identify P4HA3 as a key enzyme involved in these changes altogether providing insights into potential mechanisms of metastasis.

Vascular endothelial growth factor A (VEGF-A) plays pivotal roles in regulating tumor angiogenesis as well as physiological vascular function. The major VEGF-A isoforms, VEGF-A121 and VEGF-A165, in serum, plasma, and platelets have not been exactly evaluated due to the lack of the appropriate assay system. Antibodies against human VEGF-A121 and VEGF-A165 (hVEGF-A121 and hVEGF-A165) were successfully produced and Enzyme-Linked ImmunoSorbent Assay (ELISA) for hVEGF-A121 and hVEGF-A165 were separately created by these monoclonal antibodies. The measurement of recombinant hVEGF-A121 and hVEGF-A165 by the created ELISA showed no cross-reaction between hVEGF-A121 and hVEGF-A165 in conditioned media from HEK293 cells transfected with either hVEGF-A121 or hVEGF-A165 expression vector. The levels of VEGF-A121 and VEGF-A165 in serum, plasma, and platelets from 59 healthy volunteers proved that VEGF-A121 level was higher than VEGF-A165 in both plasma and serum in all the cases. VEGF-A121 or VEGF-A165 in serum represented higher level than that in plasma. In contrast, the level of VEGF-A165 was higher than VEGF-A121 in platelets. The newly developed ELISAs for hVEGF-A121 and hVEGF-A165 revealed different ratios of VEGF isoforms in serum, plasma, and platelets. Measuring these isoforms in combination provides useful information as biomarkers for diseases involving VEGF-A121 and VEGF-A165.