Complement: a unique innate immune sensor for danger signals.

T-helper cells that produce interleukin-17 (T(H)17 cells) are a recently identified CD4(+) T-cell subset with characterized pathological roles in autoimmune diseases. The nuclear receptors retinoic-acid-receptor-related orphan receptors α and γt (RORα and RORγt, respectively) have indispensible roles in the development of this cell type. Here we present SR1001, a high-affinity synthetic ligand-the first in a new class of compound-that is specific to both RORα and RORγt and which inhibits T(H)17 cell differentiation and function. SR1001 binds specifically to the ligand-binding domains of RORα and RORγt, inducing a conformational change within the ligand-binding domain that encompasses the repositioning of helix 12 and leads to diminished affinity for co-activators and increased affinity for co-repressors, resulting in suppression of the receptors' transcriptional activity. SR1001 inhibited the development of murine T(H)17 cells, as demonstrated by inhibition of interleukin-17A gene expression and protein production. Furthermore, SR1001 inhibited the expression of cytokines when added to differentiated murine or human T(H)17 cells. Finally, SR1001 effectively suppressed the clinical severity of autoimmune disease in mice. Our data demonstrate the feasibility of targeting the orphan receptors RORα and RORγt to inhibit specifically T(H)17 cell differentiation and function, and indicate that this novel class of compound has potential utility in the treatment of autoimmune diseases.

Suppression of TH17 Differentiation and Autoimmunity by a Synthetic ROR Ligand

Dietary modulation of immune function by beta-glucans.

TCRαβ thymocytes differentiate into either CD8αβ(+) cytotoxic T lymphocytes or CD4(+) helper T cells. This functional dichotomy is controlled by key transcription factors, including the helper T cell master regulator ThPOK, which suppresses the cytolytic program in major histocompatibility complex (MHC) class II-restricted CD4(+) thymocytes. ThPOK continues to repress genes of the CD8 lineage in mature CD4(+) T cells, even as they differentiate into effector helper T cell subsets. Here we found that the helper T cell fate was not fixed and that mature, antigen-stimulated CD4(+) T cells terminated expression of the gene encoding ThPOK and reactivated genes of the CD8 lineage. This unexpected plasticity resulted in the post-thymic termination of the helper T cell program and the functional differentiation of distinct MHC class II-restricted CD4(+) cytotoxic T lymphocytes.

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Transcriptional reprogramming of mature CD4+ helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes

The differential expression of IL-4 and IL-13 and its impact on type-2 immunity.

We examined the immunological actions of Sophy β-glucan (Ikewaki N., et al. United States Patent 6956120 and Japan Patent 2004-329077), a type of β-1,3–1,6 glucan produced by the black yeast Aureobasidium pullulans (A. pullulans) strain AFO-202, currently available commercially as a health food supplement, using different human in vitro experimental systems. Sophy β-glucan significantly (P<0.01) stimulated the 3H-thymidine incorporation rates (marker of DNA synthesis) in human peripheral blood mononuclear cells (PBMCs) obtained from normal adult donors, in vitro. Enzyme-linked immunoassays (EIAs) revealed that Sophy β-glucan stimulated the production of interleukin-8 (IL-8) or soluble Fas (sFas), but not that of IL-1β, IL-2, IL-6, IL-12 (p70+40), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) or soluble Fas ligand (sFasL), in either cultured PBMCs or cells of the human monocyte-like cell line, U937. The induction by Sophy β-glucan of DNA synthesis in PBMCs was completely blocked by the addition of monoclonal antibodies (mAbs) to CD11a, CD54, human leukocyte antigen-class II (HLA-class II), Toll-like receptor-2 (TLR-2), and Toll-like receptor-4 (TLR-4). In these blocking experiments using the mAbs, the main differences in the results between PBMCs and U937 cells were that the mAbs against TLR-2 and TLR-4 did not block the Sophy β-glucan -induced production of IL-8 in the U937 cells. Furthermore, a mAb to the β-glucan receptor, Dectin-1, significantly (P<0.05) blocked the Sophy β-glucan-induced DNA synthesis in the PBMCs, and Sophy β-glucan -induced production of IL-8 in the U937 cells. The Sophy β-glucan-induced production of IL-8 in the U937 cells was significantly (P<0.01) blocked by the conventional protein kinase C (PKC) inhibitor Go6976, the novel PKC inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89, and the protein tyrosine kinase (PTK) inhibitor herbimycin A. Among these, the blocking effect of the novel PKC (PKC delta isoenzyme) inhibitor Rottlerin was the most pronounced. Studies employing reverse transcriptase-polymerase chain reaction (RT-PCR) showed that Sophy β-glucan stimulated the expression of IL-8 mRNA in the U937 cells, and that this induction was inhibited by Rottlerin. Sophy β-glucan also blocked the stimulator cell induction of DNA synthesis and IFN-γ production in the responder cells in a one-way mixed lymphocyte reaction (MLR) using allogenic PBMCs. Interestingly, immunoglobulin G (IgG), but not IgM to Sophy β-glucan was detected in the sera derived from normal adult donors and from the umbilical cord blood of neonates. Taken together, these findings strongly suggest that the Sophy β-glucan may have unique immune regulatory or enhancing properties that could be exploited by the health food, medical and pharmaceutical industries.

Immunological Actions of Sophy β-Glucan (β-1,3-1,6 Glucan), Currently Available Commercially as a Health Food Supplement

Conventional vaccines to combat COVID-19 through different approaches are at various stages of development. The complexity of COVID-19 such as the potential mutations of the virus leading to antige...

https://www.tandfonline.com/doi/pdf/10.1080/21645515.2021.1880210

β-glucans: wide-spectrum immune-balancing food-supplement-based enteric (β-WIFE) vaccine adjuvant approach to COVID-19.

The transcription factor GATA-3 has been shown to play an important role for the in vitro induction of Th2 cells. To clarify how the in vivo immune response is governed under GATA-3 function, we generated double-transgenic mice by crossing GATA-3 transgenic mice with ovalbumin (OVA)specific TCR transgenic mice. After immunization with OVA, the double-transgenic mice showed increased expression of GATA-3 in antigen-reactive fresh CD4 + T cells, and higher production of IL-5 and IL-13 in cultured spleen cells in the presence of cognate antigen without any polarizing conditions for Th2 cells. Moreover, the immunized double-transgenic mice showed a higher increase of in vivo secretion of IL-5 and IL-13 in bronchoalveolar lavage fluid after OVA aerosol challenging. The serum levels of OVA-specific IgG1, IgE and IgA antibodies were much higher in the immunized double-transgenic mice than TCR transgenic mice. These findings provide direct evidence that antigen-stimulated CD4 + T cells in the immunized mice have already been committed into Th2 cells producing IL-5 and IL-13 selectively through enhanced GATA-3 expression in vivo, thereby inducing higher production of antigen-specific antibody for three isotypes other than IgM.

Evidence of GATA-3-dependent Th2 commitment during the in vivo immune response.

Growth factor-dependent gut intraepithelial lymphocyte (IEL) cell lines were established from a long-term in vitro culture of BALB/c IEL with syngeneic irradiated spleen cells in the presence of concanavalin A-stimulated spleen supernatant fluids. The cell lines were preferentially consisted of very limited thymoindependent subsets of IEL; i.e., Thy-1+CD5-TCR alpha beta+CD4+CD8 alpha+beta- (double-positive; DP) IEL and Thy-1+CD5-TCR alpha beta+CD4-CD8 alpha+beta- (CD8 single-positive; CD8 SP) IEL. The CD8 SP IEL cell line had cytotoxic activities and was triggered to proliferate by T-cell receptor (TCR)-directed stimuli. The DP IEL cell line expressed high levels of the CD3-TCR alpha beta, exhibited cytotoxic activity in redirected lysis assays, and had perforin in the cytoplasm, indicating the functional maturity of this cell line. However, the DP IEL cell line did not proliferate in response to TCR alpha beta-directed stimuli, which indicated that TCR alpha beta-mediated signalling was able to initiate cytotoxic function but not to induce proliferation of the DP IEL cell line. Although both cell lines were shown to have functional competence, they expressed J11d antigen which marks immaturity in thymocyte differentiation pathways. These results indicate that the established thymoindependent DP and CD8 SP IEL cell lines have unique properties distinct from DP thymocytes and CD8 SP peripheral T cells. Together with a recent report on freshly isolated DP IEL (10), the unique properties of the DP IEL cell line seems to support the notion that DP IEL may undergo a unique maturation process in the gut microenvironment.

Unique Properties of a Cytotoxic CD4+CD8+ Intraepithelial T-Cell Line Established from the Mouse Intestinal Epithelium

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell-lines. Whereas recombinant tumor necrosis factor-alpha (rTNF-alpha) slightly up-regulated CD93 expression on the U937 cells, recombinant interleukin-1beta (rIL-1beta), recombinant interleukin-2 (rIL-2), recombinant interferon-gamma (rIFN-gamma) and lipopolysaccharide (LPS) had no effect. Taken together, these findings indicate that the regulation of CD93 expression on these cells involves the PKC isoenzymes.

Nobunao Ikewaki

Papers

Immunological Actions of Sophy β-Glucan (β-1,3-1,6 Glucan), Currently Available Commercially as a Health Food Supplement

β-glucans: wide-spectrum immune-balancing food-supplement-based enteric (β-WIFE) vaccine adjuvant approach to COVID-19.

Evidence of GATA-3-dependent Th2 commitment during the in vivo immune response.

Unique Properties of a Cytotoxic CD4+CD8+ Intraepithelial T-Cell Line Established from the Mouse Intestinal Epithelium

Regulation of CD93 cell surface expression by protein kinase C isoenzymes