Involvement of metabotropic glutamate receptors in regulation of immune response in the Pacific oyster Crassostrea gigas.

Metabotropic glutamate receptors (mGluRs) play a pivotal role in the neuroendocrine-immune regulation. In this study, eight mGluRs were identified in the Pacific Oyster Crassostrea gigas, which were classified into three subfamilies based on genetic similarity. All CgmGluRs harbor variable numbers of PBP1 domains at the N-terminus. The sequence and structural features of CgmGluRs are highly similar to mGluRs in other species. A uniformly upregulated expression of CgmGluRs was observed during D-shaped larval stage compared to early D-shaped larval stage. The transcripts of CgmGluRs were detectable in various tissues of oyster. Different CgmGluR exhibited diverse expression patterns response against different PAMP stimulations, among which CgmGluR5 was significantly downregulated under these stimulations, reflecting its sensitivity and broad-spectrum responsiveness to microbes. Following LPS stimulation, the mRNA expression of CgmGluR5 and CgCALM1 in haemocytes was suppressed within 6 h and returned to normal levels by 12 h. Inhibition of CgmGluR5 activity resulted in a significant reduction in CgCALM1 expression after 12 h. Further KEGG enrichment analysis suggested that CgmGluR5 might modulate calcium ion homeostasis and metabolic pathways by regulating CgCALM1. This research delivers the systematic analysis of mGluR in the Pacific Oyster, offering insights into evolutionary characteristics and immunoregulatory function of mGluR in mollusks.

Unveiling the functional diversity of ionotropic glutamate receptors in the Pacific oyster (Crassostrea gigas) by systematic studies.

Ionotropic glutamate receptors (iGluRs), pivotal in mediating excitatory neurosignals within the central nervous system, are instrumental in environmental stress responses. In this investigation, 12 iGluRs identified in the Pacific oyster are herein designated as CgiGluRs, and further categorized into three distinct subfamilies based on their transmembrane domains. Cross-species evolutionary analysis unveiled a high degree of conservation in the sequence and structural attributes of these CgiGluRs. These receptors are ubiquitously distributed across various tissues, with pronounced expression in the oyster's mantle, labial palps, and gills, underlining their integral role in the oyster's environmental sensing mechanisms. Post the D-shaped larval stage, a marked upward trend in CgiGluRs expression was observed, denoting their critical involvement in oyster development beyond this phase. Exposure to five metals-cadmium (Cd), copper (Cu), zinc (Zn), mercury (Hg), and lead (Pb)-elicited a significant upregulation of CgGRIA4 expression, indicating a robust response to metal stress. A KEGG enrichment analysis on 142 genes, exhibiting parallel expression trends with CgGRIA4 under metal stress, suggests that CgGRIA4 could augment excitatory signal transmission by activating glutamatergic and dopaminergic synapses, thereby contributing to the metal stress response in the oyster. This inquiry not only bolsters our comprehension of the iGluRs gene family in metal stress response but also paves the way for future exploration of its cardinal role in cellular signaling and environmental adaptability.

Research Progress in Boron-Modified Phenolic Resin and Its Composites.

As one of the most successful modified phenolic resins, boron-modified phenolic resin (BPF) has excellent heat resistance and ablative resistance, good mechanical and wear resistance, and flame retardancy. BPF and its composites can be widely used in areas such as aerospace, weapons and equipment, automobile brakes, and fire retardants. In this review, the current state of development of BPF and its composites is presented and discussed. After introducing various methods to synthesize BPF, functionalization of BPF is briefly summarized. Particular emphasis is placed on general methods used to fabricate BPF-based composites and the heat resistance, ablative resistance, mechanical property, wear resistance, flame retardancy, and water resistance of BPF-based composites. Finally, the challenges of this research area are summarized and its future outlook is prospected.

Phylogeography of the desert scorpion illuminates a route out of Central Asia.

A comprehensive understanding of phylogeography requires the integration of knowledge across different organisms, ecosystems, and geographic regions. However, a critical knowledge gap exists in the arid biota of the vast Asian drylands. To narrow this gap, here we test an "out-of-Central Asia" hypothesis for the desert scorpion Mesobuthus mongolicus by combining Bayesian phylogeographic reconstruction and ecological niche modeling. Phylogenetic analyses of one mitochondrial and three nuclear loci and molecular dating revealed that M. mongolicus represents a coherent lineage that diverged from its most closely related lineage in Central Asia about 1.36 Ma and underwent radiation ever since. Bayesian phylogeographic reconstruction indicated that the ancestral population dispersed from Central Asia gradually eastward to the Gobi region via the Junggar Basin, suggesting that the Junggar Basin has severed as a corridor for Quaternary faunal exchange between Central Asia and East Asia. Two major dispersal events occurred probably during interglacial periods (around 0.8 and 0.4 Ma, respectively) when climatic conditions were analogous to present-day status, under which the scorpion achieved its maximum distributional range. M. mongolicus underwent demographic expansion during the Last Glacial Maximum, although the predicted distributional areas were smaller than those at present and during the Last Interglacial. Development of desert ecosystems in northwest China incurred by intensified aridification might have opened up empty habitats that sustained population expansion. Our results extend the spatiotemporal dimensions of trans-Eurasia faunal exchange and suggest that species' adaptation is an important determinant of their phylogeographic and demographic responses to climate changes.

Evolution and phylogenetic diversity of the aquaporin gene family in arachnids.

Water flux across cells predominantly occurs through the pore formed by the aquaporin channels. Since water balance is one of the most important challenges to terrestrial animals, aquaporin evolution and diversity is known to play roles in animal terrestrialisation. Arachnids (Arthropoda: Chelicerata: Arachnida) are the second most diverse group and represent the pioneer land colonists in animals; however, there remains no thorough investigation on aquaporin evolution and diversity in this evolutionarily important lineage. Here we reported a phylogenetic study of aquaporin evolution and diversity using genomic data from 116 arachnid species covering almost all (15/16) extant orders. A previously unrecognised subfamily related to aquaporin-4 (i.e. Aqp4-like subfamily) via phylogenetic analysis was identified, suggesting certain underestimate of the arachnid aquaporin diversity in earlier studies probably due to limited taxonomic sampling. Further analysis indicates that this subfamily emerged deep within the life tree of arthropods. Gene tree of another Aqp4-like subfamily (PripL) shows an unexpected basal split between acariform mites (Acariformes) and other arachnids. A closer inspection demonstrated that the PripL evolved quickly and has been under differential selection pressure in acariform mites. Evidence is provided that the evolutionarily ancient Glp subfamily (i.e. aquaglyceroporin) is significantly expanded in terrestrial arachnids compared with their marine relatives. Finally, in spite of the phylogenetic diversity, there exists conservation of some exons in size, functional domain, and intron-insertion phase: an 81-bp and a 218-bp exon, respectively, in apq4-like and glp genes across Eumetazoa lineages including arachnids and human beings. Both exons encode the carboxyl-terminal NPA motif, implying the coding and splicing pressure during hundreds of million years of animal evolution. Hypotheses were tested to explore the possible link between these findings and arachnid terrestrialisation.

Long non-coding RNAs are involved in immune resistance to Aeromonas hydrophila in black carp (Mylopharyngodon piceus).

A growing number of studies identified long non-coding RNAs (lncRNAs) to be closely associated with immune function through the regulation of immune cell differentiation and immune cell effector function. Here we tested whether lncRNAs are involved in immune function in black carp (Mylopharyngodon piceus) through the exposure to Aeromonas hydrophila and analysis of the spleen gene expression response using RNA-seq. A total of 9036 lncRNAs were identified with high confidence. Differential expression analysis identified a total of 3558 DElncRNAs (Differential expression lncRNA) involved in A. hydrophila infection and 4526 target genes corresponding to DElncRNAs. After screening 4526 target genes in the InnateDB database, a total of 150 immunity genes were identified. After GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis of the obtained immunity genes, the Toll-like receptor (TLR) signaling pathway, TLR2, TLR3, TLR5, and TLR8 were identified as particularly significant in A. hydrophyla-resistant black carp. At the same time, the Ras signaling pathway was particularly enriched in the spleen of susceptible black carp. Analysis of PPI (protein-protein interaction) networks of the obtained immune genes identified SRC (SRC Proto-Oncogene), MYD88 (Myeloid differentiation primary response 88), MAPK3 (Mitogen-Activated Protein Kinase 3), MYC (MYC Proto-Oncogene) as main hub genes regulated by lncRNA and possibly mediating a mechanism of susceptibility to bacteria. These results establish a functional role of lncRNAs and a mechanistic base for the immune response in black carp resistant to A. hydrophila.

Interleukin 1 receptor type I (IL-1RI) is involved in the innate immune response of olive flounder (Paralichthys olivaceus) to resist pathogens.

The pleiotropic cytokine IL -1 is involved in important immune responses such as thymocyte proliferation and B cell growth and differentiation. Activation of the IL -1 pathway requires its functional receptor IL -1RI, making IL -1RI the critical point of the IL -1 pathway. In-depth study of IL -1RI will help to understand the immune mechanism involved in IL -1. In this study, we identified the cDNA of the IL -1RI gene of olive flounder (PoIL-1RI). The total length of the PoIL-1RI cDNA is 2490 bp, the open reading frame is 1689 bp long and encodes a protein of 562 amino acids. The protein has three Ig domains and a typical TIR domain, as in other mammals and fish. We found that PoIL-1RI is widely expressed in the tissues studied and shows a significant immune response after stimulation with bacteria and pathogen-associated molecular patterns (PAMPs) both in vitro and in vivo. After PoIL-1RI was overexpressed in olive flounder embryonic cell line (FEC), pro-inflammatory cytokines (IL -1ß, IL -6, IL -8, TNF-α) and interferon (IFN-α, IFN-γ) were significantly upregulated. And we found that after overexpressing PoIL-1RI in FEC, the antibacterial ability of FEC was significantly stronger than that of the control group, and we found that overexpression of PoIL-1RI gene significantly increased the activity of NF-κB signaling pathway. These results suggest that PoIL-1RI plays an important role in innate immune response.

Diel rhythm of urotensin I mRNA expression and its involvement in the locomotor activity and appetite regulation in olive flounder Paralichthys olivaceus.

Urotensin I (UI), a member of the corticotropin-releasing hormone family of peptides, regulates a diverse array of physiological functions, including appetite regulation, defensive behavior and stress response. In this study, firstly, the tissue-specific distribution of UI mRNA in olive flounder (Paralichthys olivaceus) was characterized and we found that UI mRNA was highly expressed in caudal neurosecretory system (CNSS) tissue. Secondly, alignment analysis found that a conserved cAMP response binding (CREB) site and a TATA element were located in the proximal promoter of UI gene. In addition, treatment of forskolin activatated cAMP-CREB pathway and induced the up-regulation of UI mRNA in cultured CNSS, suggesting the role of CREB in regulating the UI mRNA expression. Furthermore, plasma UI concentration and UI mRNA in CNSS showed obvious daily rhythm, with higher values in the daytime while lower values in the nighttime. Thirdly, using bold personality (BP) and shy personality (SP) flounder as an animal model, we found that flounder exhibited significantly higher locomotor activity in the nighttime than in the daytime (P < 0.001), and BP flounder showed significantly higher locomotor activity (P < 0.001) compared with SP flounder both in the daytime and nighttime. Analysis of feeding behavior revealed that BP flounder showed a shorter latency to feed and more attacks to prey. Furthermore, the qPCR and immunohistochemistry results showed that BP flounder expressed significantly lower level of UI mRNA and protein in CNSS tissue. Collectively, our study suggested that the UI plays an important role in locomotor activity and appetite regulation, which provides a basis for understanding the mechanism of defensive behavior and animal personality in flounder.

Comparative transcriptome and methylome analysis of the hindbrain in olive flounder (Paralichthys olivaceus) considering individual behavior-type and oxygen metabolism.

In previous studies we employed multiple behavior assays, including propensity to feed, simulated trawl capture and escape response, to prove the presence of bold and shy personality (BP,SP) in olive flounder (Paralichthys olivaceus). However, the molecular mechanism of the different personality has not been elucidated. In this study, firstly, we found that the SP flounder had lower red blood cell count (RBC) and haemoglobin concentration (HBG) than BP flounder. Secondly, the transcriptomic profiles of the hindbrain in flounder with distinct personality were compared. A total of 144 differently expressed genes (DEGs) were identified, including 70 up-regulated and 74 down-regulated genes in SP flounder compared with BP flounder. Genes involved in hypoxia stress were detected in SP flounder, accompanied with down-regulation of ribosomal RNA synthesis. In addition, genes related with calcium signaling pathway, including endothelin, b-Fos, c-Fos and c-Jun were up-regulated in SP flounder. Furthermore, personality-related genes including UI, CCK, c-Fos showed significantly higher level in SP flounder than in BP flounder. GO enrichment analysis indicated that the GO categories "the tight junction pathway" and "lipid transport or localization pathway" were enriched in SP flounder, suggesting that the central nervous system homeostasis would be compromised. Thirdly, using a simple and scalable DNA methylation profiling method (MethylRAD), which allows for methylation analysis for DEGs in RNA-seq, we found that only part of gene expression was negatively associated with promoter methylation. Altogether, our study will not only lay a foundation for further studies on animal personality but also facilitate the selective breeding of olive flounder in aquaculture.

Myeloid differentiation factor 88 (Myd88) is involved in the innate immunity of black carp (Mylopharyngodon piceus) defense against pathogen infection.

Myeloid differentiation factor 88 (MyD88) is an important transduction protein in the Toll-like receptor signaling pathway. In this study, we identified the cDNA of the MpMyD88 gene in black carp. We found that MpMyD88 was widely distributed in the tissues tested and showed significant immune responses both in vitro and in vivo after stimulation with bacterial and pathogen-associated molecular patterns. After MpMyD88 overexpression/silencing, proinflame-matory cytokines (TNF-α, IFN-α, IL-6, and IL-8) also showed significant up-regulation/down-regulation. Moreover, we found that the antibacterial ability of cells over-expressing MpMyD88 was significantly stronger than that of control cells, while that of silenced MpMyD88 was significantly lower than that in control cells. Besides, we found that the overexpression of MpMyD88 significantly increased the activity of NF-κB. These results indicate that MpMyD88 plays an important role in the innate immune response.

Cloning, functional analysis, and microRNA-induced negative regulation of growth arrest and DNA damage-inducible 45 γ (Gadd45g) in grass carp (Ctenopharyngodon idella).

Growth arrest and DNA damage-inducible 45 gamma (Gadd45g) is a member of Gadd45 gene family of immunological proteins in mammals. Herein, we identified and characterised Gadd45g from grass carp. The cDNA spans over 1189 bp, with an open reading frame of 480 bp encoding a 159 amino acid protein. CiGadd45g mRNAs were expressed in all tissues investigated, with abundant expression in liver, kidney, heart, brain, blood and skin. Following infection with Aeromonas hydrophila, CiGadd45g expression was upregulated in these immune-related tissues (gill, liver, spleen, intestine, kidney and head kidney). Immune-related cytokines (p38 and JNK) and proinflammatory cytokines (IL-8, IFN-1 and TNF-α) were activated by CiGadd45g. CiGadd45g and downstream genes were regulated by microRNA miR-429b. These results indicate that CiGadd45g plays an important immune role in the response to A. hydrophila infection in grass carp.

Transcriptome analysis and histopathology of black carp (Mylopharyngodon piceus) spleen infected by Aeromonas hydrophila.

Aeromonas hydrophila causes serious economic losses to the black carp (Mylopharyngodon piceus) industry. In this study, we analyzed the spleen of disease-resistant and susceptible black carp by RNA-seq. Overall, a total of 5243 terms were enriched in the gene ontology (GO) analysis, and 323 related pathways were found in the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. A total of 1935 differentially expressed genes were found and were primarily involved in cell adhesion, pathogen recognition, cellular immunity, cytokines, complement systems, and iron transport. Sixteen of the differently expressed genes involved in the immune response and the accuracy of the transcriptome data were further validated by quantitative real-time PCR (qRT-PCR). We observed Tissue sections of the spleen infected with A. hydrophila and the control group and found that the spleen of the infected group had necrosis.

Growth arrest and DNA damage inducible 45-beta activates pro-inflammatory cytokines and phagocytosis in the grass carp (Ctenopharyngodon idella) after Aeromonas hydrophila infection.

Growth arrest and DNA damage inducible 45-beta (Gadd45B) is essential for mitogen-activated protein kinases (MAPK) activities, and involved in regulating growth, apoptosis, and DNA demethylation. In the present study, the cDNA of gcGadd45Ba and gcGadd45Bb in grass carp was identified. And the expression levels show that they were widely distributed in the tested tissues and showed significant immune responses both in vitro and in vivo after challenge with bacteria and pathogen-associated molecular patterns (PAMPs). Overexpression of Gadd45B significantly induced the expression of pro-inflammatory cytokines (IL-1ß, IL-8, and TNF-α) and enhanced the phagocytosis activation of grass carp blood cells. These results indicate that Gadd45B plays an important role in innate immune responses.

Enhanced suppression of polyclonal CD8+25+ regulatory T cells via exosomal arming of antigen-specific peptide/MHC complexes.

Compared with CD4+25+ regulatory T cells (Tregs), the mechanisms for natural, polyclonal CD8+25+ Treg immune suppression have been significantly less studied. We previously showed that polyclonal T cells can acquire antigen-specific targeting activity through arming with exosomal peptide-MHC (pMHC). In this study, we assessed the suppressive effect of CD8+25+ Tregs or CD8+25+ Tregs armed with ovalbumin (OVA)-specific exosomes on other immune cells and OVA-specific dendritic cell (DCOVA)-stimulated antitumor immunity. We demonstrate that CD8+25+ Tregs inhibit T cell proliferation in vitro in a cell contact-dependent fashion but independent of the expression of immunosuppressive IL-10, TGF-ß, and CTLA-4. CD8+25+ Tregs anergize naïve T cells upon stimulation by up-regulating T cell anergy-associated Egr2 and down-regulating IL-2 production. Tregs also anergize DCs by preventing DC maturation through the down-regulation of Iab, CD80, CD86, and inflammatory cytokines, leading to defects in T cell stimulation. Moreover, CD8+25+ Tregs inhibit CTLs through inducing CTL death via perforin-mediated apoptosis and through reducing effector CTL cytotoxic activity via down-regulating CTL perforin-production and degranulation. In addition, we show that CD8+25+ Tregs suppress DCOVA-stimulated CTL responses in priming and effector phases and inhibit immunity against OVA-expressing CCLOVA lung cancer. Remarkably, polyclonal CD8+25+ Tregs armed with OVA-specific exosomal pMHC class-II (pMHC-II), or pMHC class-I (pMHC-I) complexes exert their enhanced inhibition of CTL responses in the priming and the effector phases, respectively. Taken together, our investigation reveals that assigning antigen specificity to nonspecific polyclonal CD8+25+ Tregs for enhanced immune suppression can be achieved through exosomal pMHC arming. This principle may have a great effect on Treg-mediated immunotherapy of autoimmune diseases.

Natural CD8⁺25⁺ regulatory T cell-secreted exosomes capable of suppressing cytotoxic T lymphocyte-mediated immunity against B16 melanoma.

Natural CD4(+)25(+) and CD8(+)25(+) regulatory T (Tr) cells have been shown to inhibit autoimmune diseases. Immune cells secrete exosomes (EXOs), which are crucial for immune regulation. However, immunomodulatory effect of natural Tr cell-secreted EXOs is unknown. In this study, we purified natural CD8(+)25(+) Tr cells from C57BL/6 mouse naive CD8(+) T cells, and in vitro amplified them with CD3/CD28 beads. EXOs (EXO(Tr)) were purified from Tr cell's culture supernatants by differential ultracentrifugation and analyzed by electron microscopy, Western blot and flow cytometry. Our data showed that EXO(Tr) had a "saucer" or round shape with 50-100 nm in diameter, contained EXO-associated markers LAMP-1 and CD9, and expressed natural Tr cell markers CD25 and GITR. To assess immunomodulatory effect, we i.v. immunized C57BL/6 mice with ovalbumin (OVA)-pulsed DCs (DC(OVA)) plus Tr cells or EXO(Tr), and then assessed OVA-specific CD8(+) T cell responses using PE-H-2K(b)/OVA tetramer and FITC-anti-CD8 antibody staining by flow cytometry and antitumor immunity in immunized mice with challenge of OVA-expressing BL6-10OVA melanoma cells. We demonstrated that DC(OVA)-stimulated CD8(+) T cell responses and protective antitumor immunity significantly dropped from 2.52% to 1.08% and 1.81% (p<0.05), and from 8/8 to 2/8 and 5/8 mice DC(OVA) (p<0.05) in immunized mice with co-injection of Tr cells and EXO(Tr), respectively. Our results indicate that natural CD8(+)25(+) Tr cell-released EXOs, alike CD8(+)25(+) Tr cells, can inhibit CD8(+) T cell responses and antitumor immunity. Therefore, EXOs derived from natural CD4(+)25(+) and CD8(+)25(+) Tr cells may become an alternative for immunotherapy of autoimmune diseases.

Optimal TLR9 signal converts tolerogenic CD4-8- DCs into immunogenic ones capable of stimulating antitumor immunity via activating CD4+ Th1/Th17 and NK cell responses.

Abstract TLR ligands have been reported to promote DC maturation and enhance CD8+ CTL responses. We have demonstrated previously that CD4-8- DCs secreting TGF-beta stimulate CD4+ Tr1 cell responses. Here, we have assessed whether TLR4 and TLR9 signaling through LPS and CpG stimulation can convert CD4-8- DC-induced tolerance. We demonstrate that immature OVA-pulsed CD4-8- DCs cultured in medium with LPS (2 microg/ml) and CpG (5 microg/ml) for 8 h became mature DCs (DCOVA) with no TGF-beta secretion. CpG-treated, CD4-8- DCOVA-secreting IL-6/IL-15 induced IFN-gamma/IL-17-secreting/T-bet- and ROR-gammat-expressing CD4+ Th1/Th17, whereas LPS-treated CD4-8- DCOVA stimulated IFN-gamma-secreting/T-bet-expressing CD4+ Th1 responses. The former also significantly stimulated more efficient OVA-specific CD8+ T cell responses and antitumor immunity against OVA-expressing BL6-10OVA tumor cells than the latter (P<0.05). CpG-treated, CD4-8- DCOVA-stimulated CD4+ Th1/Th17 cell responses and antitumor immunity were found to be reduced by using neutralizing anti-IL-6, IL-15, and NK1.1 antibodies in wild-type C57BL/6 mice, IL-15R-/- mice for immunization, or CD4-8- (IL-6-/-) DCOVA for immunization in C57BL/6 mice. Interestingly, in vitro-generated CD4+ Th17 cells significantly enhanced LPS-treated, CD4-8- DCOVA-induced in vivo antitumor immunity via increasing CD8+ CTL responses (P<0.05), although they did not show any direct killing activity against tumor cells in vitro. In addition, prolonged 48 h CpG-treated CD4-8- DCOVA dramatically diminished its cytokine secretion, stimulatory effect, and antitumor immunity. Taken together, our data demonstrate an effect of conversion of tolerogenic DCs into immunogenic ones capable of stimulating antitumor immunity via activating CD4+ Th1/Th17 and NK cell responses by optimal CpG signaling, which may advance current understanding of the importance of TLR9 signaling in a DC-based cancer vaccine.

CD40 ligation converts TGF-beta-secreting tolerogenic CD4-8- dendritic cells into IL-12-secreting immunogenic ones.

CD40L, the ligand for CD40 on dendritic cells (DCs), plays an important role in maturation and activation of DCs leading to induction of immune responses. Our previous studies showed that the mouse splenic CD4(-)8(-) DCs are tolerogenic and capable of stimulating suppressive type 1 CD4(+) regulatory T (Tr1) cell responses via TGF-beta secretion. In this study, we investigated whether CD40 ligation is able to convert tolerogenic CD4(-)8(-) DCs into immunogenic ones by in vitro treatment of DCs with anti-CD40 antibody. Our data showed that in vitro CD40 ligation with anti-CD40 antibody converted TGF-beta-secreting tolerogenic CD4(-)8(-) DCs into IL-12-secreting immunogenic ones capable of stimulating type 1 CD4(+) helper T (Th1) and CD8(+) cytotoxic T lymphocyte (CTL) responses leading to induction of antitumor immunity. In addition, in vivo CD40 ligation by intratumoral injection of adenoviral vector AdVCD40L expressing CD40 ligand also induced tumor growth inhibition and regression of established P815 tumors with infiltration of tolerogenic CD4(-)8(-) DCs. Therefore, our data provide new information for and may thus have useful impacts in CD40 ligation-based immunotherapy of cancer.

Active CD4+ helper T cells directly stimulate CD8+ cytotoxic T lymphocyte responses in wild-type and MHC II gene knockout C57BL/6 mice and transgenic RIP-mOVA mice expressing islet beta-cell ovalbumin antigen leading to diabetes.

CD4+ helper T (Th) cells play crucial role in priming, expansion and survival of CD8+ cytotoxic T lymphocytes (CTLs). However, how CD4+ Th cell's help is delivered to CD8+ T cells in vivo is still unclear. We previously demonstrated that CD4+ Th cells can acquire ovalbumin (OVA) peptide/major histocompatibility complex (pMHC I) and costimulatory CD80 by OVA-pulsed DC (DC(OVA)) stimulation, and then stimulate OVA-specific CD8+ CTL responses in C57BL/6 mice. In this study, we further investigated CD4+ Th cell's effect on stimulation of CD8 CTL responses in major histocompatibility complex (MHC II) gene knockout (KO) mice and transgenic rat insulin promoter (RIP)-mOVA mice with moderate expression of self OVA by using CD4+ Th cells or Th cells with various gene deficiency. We demonstrated that the in vitro DC(OVA)-activated CD4+ Th cells (3 x 10(6) cells/mouse) can directly stimulate OVA-specific CD8+ T-cell responses in wild-type C57BL/6 mice and MHC II gene KO mice lacking CD4+ T cells. A large amount of CD4+ Th cells (12 x 10(6) cells/mouse) can even overcome OVA-specific immune tolerance in transgenic RIP-mOVA mice, leading to CD8+ CTL-mediated mouse pancreatic islet destruction and diabetes. The stimulatory effect of CD4+ Th cells is mediated by its IL-2 secretion and CD40L and CD80 costimulations, and is specifically delivered to OVA-specific CD8+ T cells in vivo via its acquired pMHC I complexes. Therefore, the above elucidated principles for CD4+ Th cells will have substantial implications in autoimmunity and antitumor immunity, and regulatory T-cell-dependent immune suppression.

Acquired pMHC I complexes greatly enhance CD4(+) Th cell's stimulatory effect on CD8(+) T cell-mediated diabetes in transgenic RIP-mOVA mice.

CD4(+) helper T (Th) cells play pivotal roles in induction of CD8(+) CTL immunity. However, the mechanism of CD4(+) T cell help delivery to CD8(+) T cells in vivo is still elusive. In this study, we used ovalbumin (OVA)-pulsed dendritic cells (DC(OVA)) to activate OT-II mouse CD4(+) T cells, and then studied the help effect of these CD4(+) T cells on CD8(+) cytotoxic T lymphocyte (CTL) responses. We also examined CTL mediated islet beta cell destruction which led to diabetes in wild-type C57BL/6 mice and transgenic rat insulin promoter (RIP)-mOVA mice expressing beta cell antigen OVA with self OVA-specific tolerance, respectively. In adoptive transfer experiments, we demonstrated that help, in the form of peptide/major histocompatibility complex (pMHC) I acquired from DC(OVA) by DC(OVA) activation, was required for induction of OVA-specific CTL responses in C57BL/6 mice. However, in combination with TCR transgenic OT-I mouse CD8(+) T cells, the tolerogenic dosage of CD4(+) Th cells with acquired pMHC I, but not CD4(+) K(b-/-) Th cells without acquired pMHC I were able to cause diabetes in 8/10 (80%) RIP-mOVA mice. This study thus expands the current knowledge in T cell-mediated autoimmunity and provides insight into the nature of CD4(+) T cell-mediated help in CD8(+) CTL induction.

Type 1 CD8+ T cells are superior to type 2 CD8+ T cells in tumor immunotherapy due to their efficient cytotoxicity, prolonged survival and type 1 immune modulation.

CD8+ cytotoxic T (Tc) cells play a crucial role in host immune responses to cancer, and in this context, adoptive CD8+ Tc cell therapy has been studied in numerous animal tumor models. Its antitumor efficacy is, to a large extent, determined by the ability of Tc cells to survive and infiltrate tumors. In clinical trials, such in vitro-activated T cells often die within hours to days, and this greatly limits their therapeutic efficacy. CD8+ Tc cells fall into two subpopulations based upon their differential cytokine secretion. In this study, we in vitro generated that ovalbumin (OVA)-pulsed dendritic cell (DCOVA)-activated CD8+ type 1 Tc (Tc1) cells secreting IFN-gamma, and CD8+ type 2 Tc (Tc2) cells secreting IL-4, IL-5 and IL-10, which were derived from OVA-specific T cell receptor (TCR) transgenic OT I mice. We then systemically investigated the in vitro and in vivo effector function and survival of Tc1 and Tc2 cells, and then assessed their survival kinetics after adoptively transferred into C57BL/6 mice, respectively. We demonstrated that, when compared to CD8+ Tc2, Tc1 cells were significantly more effective in perforin-mediated cytotoxicity to tumor cells, had a significantly higher capacity for in vivo survival after the adoptive T cell transfer, and had a significantly stronger therapeutic effect on eradication of well-established tumors expressing OVA in animal models. In addition, CD8+ Tc1 and Tc2 cells skewed the phenotype of CD4+ T cells toward Th1 and Th2 type, respectively. Therefore, the information regarding the differential effector function, survival and immune modulation of CD8+ Tc1 and Tc2 cells may provide useful information when preparing in vitro DC-activated CD8+ T cells for adoptive T cell therapy of cancer.