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Supplementary Figure 1 from Tumor-Associated Galectin-3 Modulates the Function of Tumor-Reactive T Cells

Supplementary Figure 2 from Tumor-Associated Galectin-3 Modulates the Function of Tumor-Reactive T Cells

Supplementary Figure 4 from Tumor-Associated Galectin-3 Modulates the Function of Tumor-Reactive T Cells

Supplementary Figure 3 from Tumor-Associated Galectin-3 Modulates the Function of Tumor-Reactive T Cells

The efficacious detection of pathogens and prompt induction of innate immune signaling serve as a crucial component of immune defense against infectious pathogens. Over the past decade, DNA-sensing receptor cyclic GMP-AMP synthase (cGAS) and its downstream signaling adaptor stimulator of interferon genes (STING) have emerged as key mediators of type I interferon (IFN) and nuclear factor-κB (NF-κB) responses in health and infection diseases. Moreover, both cGAS-STING pathway and pathogens have developed delicate strategies to resist each other for their survival. The mechanistic and functional comprehension of the interplay between cGAS-STING pathway and pathogens is opening the way for the development and application of pharmacological agonists and antagonists in the treatment of infectious diseases. Here, we briefly review the current knowledge of DNA sensing through the cGAS-STING pathway, and emphatically highlight the potent undertaking of cGAS-STING signaling pathway in the host against infectious pathogenic organisms.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death in the world. Besides genetic causes, colonic inflammation is one of the major risk factors for CRC development, which is synergistically regulated by multiple components, including innate and adaptive immune cells, cytokine signaling, and microbiota. The complex interaction between CRC and the gut microbiome has emerged as an important area of current CRC research. Metagenomic profiling has identified a number of prominent CRC-associated bacteria that are enriched in CRC patients, linking the microbiota composition to colitis and cancer development. Some microbiota species have been reported to promote colitis and CRC development in preclinical models, while a few others are identified as immune modulators to induce potent protective immunity against colitis and CRC. Mechanistically, microbiota regulates the activation of different immune cell populations, inflammation, and CRC via crosstalk between innate and adaptive immune signaling pathways, including nuclear factor kappa B (NF-κB), type I interferon, and inflammasome. In this review, we provide an overview of the potential interactions between gut microbiota and host immunity and how their crosstalk could synergistically regulate inflammation and CRC, thus highlighting the potential roles and mechanisms of gut microbiota in the development of microbiota-based therapies to prevent or alleviate colitis and CRC.

Over the past decade, immunotherapy has emerged as one of the most promising approaches to cancer treatment. The use of immune checkpoint inhibitors has resulted in impressive and durable clinical responses in the treatment of various cancers. Additionally, immunotherapy utilizing chimeric antigen receptor (CAR)-engineered T cells has produced robust responses in blood cancers, and T cell receptor (TCR)-engineered T cells are showing promising results in the treatment of solid cancers. Despite these noteworthy advancements in cancer immunotherapy, numerous challenges remain. Some patient populations are unresponsive to immune checkpoint inhibitor therapy, and CAR T cell therapy has yet to show efficacy against solid cancers. In this review, we first discuss the significant role that T cells play in the body’s defense against cancer. We then delve into the mechanisms behind the current challenges facing immunotherapy, starting with T cell exhaustion due to immune checkpoint upregulation and changes in the transcriptional and epigenetic landscapes of dysfunctional T cells. We then discuss cancer-cell-intrinsic characteristics, including molecular alterations in cancer cells and the immunosuppressive nature of the tumor microenvironment (TME), which collectively facilitate tumor cell proliferation, survival, metastasis, and immune evasion. Finally, we examine recent advancements in cancer immunotherapy, with a specific emphasis on T-cell-based treatments.

Cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) play critical roles in the innate immunity against infectious diseases and are required to link pathogen DNA sensing to immune responses. However, the mechanisms by which cGAS-STING-induced cytokines suppress the adaptive immune response against malaria infections remain poorly understood. Here, cGAS-STING signaling is identified to play a detrimental role in regulating anti-malaria immunity. cGAS or STING deficiency in mice markedly prolongs mouse survival during lethal malaria Plasmodium yoelii nigeriensis N67C infections by reducing late interleukin (IL)-6 production. Mechanistically, cGAS/STING recruits myeloid differentiation factor 88 (MyD88) and specifically induces the p38-dependent signaling pathway for late IL-6 production, which, in turn, expands CD11b

Innate immunity is the first defense system against invading pathogens. Toll-like receptors (TLRs) are well-defined pattern recognition receptors responsible for pathogen recognition and induction of innate immune responses. Since their discovery, TLRs have revolutionized the field of immunology by filling the gap between the initial recognition of pathogens by innate immune cells and the activation of the adaptive immune response. TLRs critically link innate immunity to adaptive immunity by regulating the activation of antigen-presenting cells and key cytokines. Furthermore, recent studies also have shown that TLR signaling can directly regulate the T cell activation, growth, differentiation, development, and function under diverse physiological conditions. This review provides an overview of TLR signaling pathways and their regulators and discusses how TLR signaling, directly and indirectly, regulates cell-mediated immunity. In addition, we also discuss how TLR signaling is critically important in the host’s defense against infectious diseases, autoimmune diseases, and cancer.

Glioblastoma (GBM) stem cells are resistant to cancer therapy, and therefore responsible for tumor progression and recurrence after conventional therapy. However, the molecular mechanisms driving the maintenance of stemness and dedifferentiation are poorly understood. In this study, we identified plant homeodomain finger-containing protein 20 (PHF20) as a crucial epigenetic regulator for sustaining the stem cell-like phenotype of GBM. It is highly expressed in GBM and tightly associated with high levels of aggressiveness of tumors and potential poor prognosis in GBM patients. Knockout of PHF20 inhibits GBM cell proliferation, as well as its invasiveness and stem cell-like traits. Mechanistically, PHF20 interacts with WDR5 and binds to the promoter regions of WISP1 for its expression. Subsequently, WISP1 and BGN act in concert to regulate the degradation of β-Catenin. Our findings have identified PHF20 as a key driver of GBM malignant behaviors, and provided a potential target for developing prognosis and therapy.

Glioma is one of the most common malignant tumors of the central nervous system and is characterized by extensive infiltrative growth, neovascularization, and resistance to various combined therapies. In addition to heterogenous populations of tumor cells, the glioma stem cells (GSCs) and other nontumor cells present in the glioma microenvironment serve as critical regulators of tumor progression and recurrence. In this review, we discuss the role of several resident or peripheral factors with distinct tumor-promoting features and their dynamic interactions in the development of glioma. Localized antitumor factors could be silenced or even converted to suppressive phenotypes, due to stemness-related cell reprogramming and immunosuppressive mediators in glioma-derived microenvironment. Furthermore, we summarize the latest knowledge on GSCs and key microenvironment components, and discuss the emerging immunotherapeutic strategies to cure this disease.

The methyl farnesoate receptor (MfR) orchestrates aspects of reproduction and development such as male sex determination in branchiopod crustaceans. Phenotypic endpoints regulated by the receptor have been well-documented, but molecular interactions involved in receptor activation remain elusive. We hypothesized that the MfR subunits, methoprene-tolerant transcription factor (Met) and steroid receptor coactivator (SRC), would be expressed coincident with the timing of sex programming of developing oocytes by methyl farnesoate in daphnids. We also hypothesized that methyl farnesoate activates MfR assembly. Met mRNA was expressed rhythmically during the reproductive cycle, with peak mRNA accumulation just prior period of oocytes programming of sex. Further, we revealed evidence that Met proteins self-associate in the absence of methyl farnesoate, and that the presence of methyl farnesoate stimulates dissociation of Met multimers with subsequent association with SRC. Results demonstrated that the Met subunit is highly dynamic in controlling the action of methyl farnesoate through temporal variation in its expression and availability for receptor assembly.

Previous studies have shown that insulin receptor substrate (IRS)1 and IRS2 mediate proliferative and antiapoptotic signaling through the IL-4R in 32D cells; however their role in regulating normal B cell responses is not clear. To investigate the role of IRS2 in normal B cell function, we developed IRS2 transgenic (Tg) mice on the C57BL/6 background. Western blot analysis revealed a 2-fold elevation in IRS2 protein levels in Tg+ mice compared with littermate controls and a 3-fold increase in basal tyrosine phosphorylated IRS2 in the absence of IL-4 stimulation. IL-4-induced tyrosine phosphorylation of IRS2 was elevated in Tg+ B cells, whereas IL-4-induced phosphorylation of STAT6 was similar between Tg+ and Tg− B cells. Tg expression of IRS2 had little effect on IL-4-mediated proliferation and no effect on protection from apoptosis. However, production of IgE and IgG1 by Tg+ B cells using standard in vitro conditions was diminished 50–60%. Because Ig production in vitro is known to be highly cell concentration-dependent, we performed experiments at different cell concentrations. Interestingly, at very low B cell concentrations (1000–5000 B cells/well), IgE and IgG1 production by Tg+ B cells was greater than that of controls, whereas at higher cell concentrations (10,000–20,000 cells/well) Ig production by Tg+ B cells was less than controls. Furthermore, in vivo immunization with OVA-alum or goat anti-IgD resulted in elevated serum IgE levels in the Tg+ mice. These results indicate that overexpression of IRS2 alters the B cell intrinsic density-dependence of IgE and IgG1 production in vitro and enhances IgE responses in vivo.

A mutation in the human (hu) IL-4Rα, Q576R, has been linked with allergy in humans. Increased sensitivity of patients cells with this mutation to IL-4 suggest that a Q576R change enhances IL-4 signaling. To directly test this hypothesis, we analyzed the ability of huIL-4Rα cDNA bearing the Q576R and Y575F mutations to signal tyrosine phosphorylation, DNA-binding activity, proliferation, protection from apoptosis, and CD23 induction in response to huIL-4 in murine cells. Responses generated by the Q576R and Y575F mutants were similar to those of the wild-type receptor, using various concentrations of huIL-4 and times of stimulation. These results indicate that neither the Q576R nor the Y575F mutations have a significant direct effect on IL-4 signal transduction, and that hypersensitive induction of CD23 in cells derived from human allergy patients may be due to different and/or additional alterations in the IL-4 signaling pathway.

The interleukin (IL)-4 receptor alpha-chain (IL-4Ralpha) contains a sequence motif (488PLVIAGNPAYRSFSD) termed the insulin IL-4 receptor motif (I4R motif). Mutation of the central Tyr497 to Phe blocks the tyrosine phosphorylation of the insulin receptor substrate 1 (IRS1) and diminishes proliferation in response to IL-4. Recent data suggest that the I4R motif encodes binding sites for several protein tyrosine binding (PTB) domain-containing proteins such as IRS1 and Shc and potentially for the Src homology 2 domain of signal transducer and activator of transcription 6 (STAT6). To analyze the function of the I4R motif in regulating IL-4 signaling, we changed conserved residues upstream and downstream of the central Tyr to Ala in the human IL-4Ralpha. We analyzed the ability of these constructs to signal the tyrosine phosphorylation of IRS2 and STAT6, the induction of DNA binding activity, and CD23 induction in response to human IL-4 (huIL-4) in transfected M12.4.1 cells. Mutagenesis of residues downstream of Tyr497, such as Arg498 or Phe500, to Ala had no effect on any of these responses, suggesting that the I4R motif may not be important for functional Src homology 2 domain interactions. However, mutagenesis of Pro488 to Ala (P488A) greatly diminished the tyrosine phosphorylation of IRS2 and abolished tyrosine phosphorylation of STAT6, induction of DNA binding activity, and CD23 induction in response to huIL-4. By contrast, a P488G mutant signaled these responses to huIL-4. Mutagenesis of hydrophobic amino acids previously shown to contact the PTB domain of IRS1, Leu489 or Ile491, to Ala had only minimal effects on responses to huIL-4. However, changing both Leu498 and Ile491 to Ala greatly diminished the tyrosine phosphorylation of IRS2 and abolished STAT6 activation. Taken together, these results indicate the important role of the I4R motif in regulating IRS docking and suggest that I4R docking to a PTB domain-containing protein regulates activation of the STAT6 pathway.

Cytokines play an essential role in the regulation of lymphocyte survival and growth. We have analyzed the pathways activated by IL-2 that lead to protection from apoptosis and cell proliferation. IL-2 can act as a long-term growth factor in 32D cells expressing the wild-type human (hu)IL-2Rβ. By contrast, cells expressing a truncated form of the huIL-2Rβ, which is able to induce Bcl-2 and c-myc expression but not STAT5 activation, were not protected from apoptosis by IL-2; consequently, they could not be grown long term in the presence of IL-2. However, IL-2 promoted cell cycle progression in cells bearing the truncated huIL-2Rβ with percentages of viable cells in the G0/G1, S, and G2/M phases similar to cells expressing the wild-type huIL-2Rβ. Transplantation of a region from the erythropoietin receptor, which contains a docking site for STAT5 (Y343) to the truncated huIL-2Rβ, restored the ability of IL-2 to signal both activation of STAT5 and protection from apoptosis. By contrast, transplantation of a region from the huIL-4Rα containing STAT6 docking sites did not confer protection from apoptosis. These results indicate that the IL-2-induced cell cycle progression can be clearly distinguished from protection from apoptosis and that STAT5 participates in the regulation of apoptosis.

IL-4 binds to a cell surface receptor complex that consists of the IL-4 binding protein (IL-4R alpha) and the gamma chain of the IL-2 receptor complex (gamma c). The receptors for IL-4 and IL-2 have several features in common; both use the gamma c as a receptor component, and both activate the Janus kinases JAK-1 and JAK-3. In spite of these similarities, IL-4 evokes specific responses, including the tyrosine phosphorylation of 4PS/IRS-2 and the induction of CD23. To determine whether sequences within the cytoplasmic domain of the IL-4R alpha specify these IL-4-specific responses, we transplanted the insulin IL-4 receptor motif (I4R motif) of the huIL-4R alpha to the cytoplasmic domain of a truncated IL-2R beta. In addition, we transplanted a region that contains peptide sequences shown to block Stat6 binding to DNA. We analyzed the ability of cells expressing these IL-2R-IL-4R chimeric constructs to respond to IL-2. We found that IL-4 function could be transplanted to the IL-2 receptor by these regions and that proliferative and differentiative functions can be induced by different receptor sequences.

Hemoglobin levels are significantly elevated in the crustacean Daphnia magna by juvenoid hormones. The present study was undertaken to identify the specific globin (hb) genes that are induced by juvenoids and to identify putative juvenoid response elements (JREs) that may mediate this induction. Gene product of globin 2 (hb2), but not globin 1 and globin 3, was robustly elevated following juvenoid treatment of daphnids. A candidate JRE, located in the promoter of hb2, bound activated factor(s) in response to juvenoid treatment of daphnids. This hormone-induced protein:JRE interaction was robust when daphnids were reared at high oxygen tension but was inhibited when daphnids were reared under low pO2, implying that hypoxia might act to disrupt juvenoid-mediated endocrine signaling. The candidate JRE consists of a steroid/retinoid-response element-like core adjacent to a 5′ AT-rich extension and thus bears resemblance to response elements that bind monomeric nuclear receptors. The induction of hb2 mRNA levels by juvenoid treatment occurred rapidly (within 4 h of exposure) and was not attenuated by treatment of daphnids with cycloheximide. In contrast, cycloheximide treatment did block hormone-mediated elevations in hemoglobin protein levels. Thus, induction of hb2 by juvenoids was not dependent upon the synthesis of secondary transcription factors that bound the JRE but was likely due to activation of the gene directly by the juvenoid-receptor complex. Affinity pull-down experiments with nuclear proteins extracted from juvenoid-treated daphnids using the JRE as bait yielded a 52 kDa candidate for a monomeric nuclear receptor in D. magna that may mediate the regulatory activity of juvenoids.

U.S. Environmental Protection Agency is charged with developing a screening and testing paradigm for detecting endocrine toxicity of chemicals that are subject to regulation under the Food Quality Protection and the Safe Drinking Water Acts. In this study, we developed and evaluated a screening assay that could be employed to detect juvenoid-related endocrine-modulating activity in an invertebrate species. Juvenoid activity, anti-juvenoid activity, and juvenoid potentiator activity of chemicals was assessed using the water flea Daphnia magna. Male sex determination is under the regulatory control of juvenoid hormone, presumably methyl farnesoate, and this endpoint was used to detect juvenoid modulating activity of chemicals. Eighteen chemicals were evaluated for juvenoid agonist activity. Positive responses were detected with the juvenoid hormones methyl farnesoate and juvenile hormone III along with the insect growth regulating insecticides pyriproxyfen, fenoxycarb, and methoprene. Weak juvenoid activity also was detected with the cyclodiene insecticide dieldrin. Assays performed repetitively with compounds that gave either strong positive, weak positive, or negative response were 100% consistent indicating that the assay is not prone to false positive or negative responses. Five candidate chemicals were evaluated for anti-juvenoid activity and none registered positive. Four chemicals (all trans-retinoic acid, methoprene, kinoprene, bisphenol A) also were evaluated for their ability to potentiate the activity of methyl farnesoate. All registered positive. Results demonstrate that an in vivo assay with a crustacean species customarily employed in toxicity testing can be used to effectively screen chemicals for juvenoid-modulating activity.

Regulatory T cells play an important role in the maintenance of immunological self-tolerance by suppressing immune responses against autoimmune diseases and cancer. Little is known, however, about the nature of the physiological target antigens for CD4(+) regulatory T (Treg) cells. Here we report the identification of the LAGE1 protein as a ligand for tumor-specific CD4(+) Treg cell clones generated from the tumor-infiltrating lymphocytes (TILs) of cancer patients. Phenotypic and functional analyses demonstrated that they were antigen-specific CD4(+) Treg cells expressing CD25 and GITR molecules and possessing suppressive activity on the proliferative response of naive CD4(+) T cells to anti-CD3 antibody stimulation. Ligand-specific activation and cell-cell contact were required for TIL102 Treg cells to exert suppressive activity on CD4(+) effector cells. These findings suggest that the presence of tumor-specific CD4(+) Treg cells at tumor sites may have a profound effect on the inhibition of T cell responses against cancer.

IL-4 is an important B cell survival and growth factor. IL-4 induced the tyrosine phosphorylation of IRS2 in resting B lymphocytes and in LPS- or CD40L-activated blasts. Phosphorylated IRS2 coprecipitated with the p85 subunit of PI 3' kinase in both resting and activated cells. By contrast, association of phosphorylated IRS2 with GRB2 was not detected in resting B cells after IL-4 treatment although both proteins were expressed. However, IL-4 induced association of IRS2 with GRB2 in B cell blasts. The pattern of IL-4-induced recruitment of p85 and GRB2 to IRS2 observed in B cells derived from STAT6 null mice was identical to that observed for normal mice. While IL-4 alone does not induce activation of MEK, a MEK1 inhibitor suppressed the IL-4-induced proliferative response of LPS-activated B cell blasts. These results demonstrate that costimulation of splenic B cells alters IL-4-induced signal transduction independent of STAT6 leading to proliferation. Furthermore, proliferation induced by IL-4 in LPS-activated blasts is dependent upon the MAP kinase pathway.