Masako Kohyama

Analysis of cytokine producing activity of Intestinal intraepithelial T cells from TCR β-chain and δ-chain mutant mice

Intestinal intraepithelial T cells (IELs) expressing either 78 TCR or αβ TCR have been proposed to play an important role in the regulation of intestinal epithelia by producing cytokines that directly influence the adjoining intestinal epithelial cell (IEC) functions. To illuminate this issue, we utilized TCR mutant mice to obtain γδ IELs, αβ IELs and mixed γδ and αβ IELs from corresponding αβ T‐cell‐deficient (β−/‐), γδ T‐cell‐deficient (δ−/‐) and wild‐type (WT) littermate mice. The production of IFN‐γ by these IELs as well as the mRNA for IFN‐γ, TGF‐α, TGF‐β1, TNF‐α and TNF‐β in these IELs, in conjunction with the effect of produced cytokines on the expression of class II MHC molecules by the in vitro cell line IEC‐6, were investigated. IFN‐γ and TGF‐α specific mRNA were detectable in all freshly isolated γδ, αβ and WT IELs. In addition to the IFN‐γ and TGF‐α mRNA, αβ and WT IELs that had been activated in culture plates coated with anti‐CD3 mAb contained mRNA for TGF‐β1 and TNF‐β proteins. In the cultured γδ IELs, however, the signals for IFN‐γ and TGF‐α transcripts were weak, and mRNA for the latter two cytokines was almost undetectable. Supernatants from in vitro culturing of αβ and WT IELs but not γδ IELs induced class II MHC gene expression in IEC‐6, whereas, in the presence of anti‐IFN‐γ mAb, the same culture supernatants failed to do so. In fact, the concentration of IFN‐γ in supernatants from αβ and WT IEL cultures was ten‐ to twentyfold higher than that in the supernatant from the γδ IEL culture. Finally, TGF‐α specific mRNA was not detectable in the γδ and αβ IELs even after in vitro activation. These results indicate that αβ IELs are superior to γδ IELs in the ability to produce IFN‐γ, TGF‐β1, TGF‐α and TNF‐β through TCR crosslinking primary in vitro stimulation.

IL-4-mediated development of TGF-β1-producing cells from naïve CD4+ T cells through a STAT6-independent mechanism

Transforming growth factor‐β1 (TGF‐β1) is an inhibitory cytokine increasingly recognized as a key factor for immuno‐regulation. The function of IL‐4 in the regulation of TGF‐β1 production from T cells has been reported previously; however, the precise molecular mechanism still remains to be elucidated. For a better understanding of the mechanism involved in regulation, we have investigated a relationship between the STAT6‐dependent pathway and TGF‐β1 production from naïve T cells. TCR crosslinking initiates TGF‐β1 production in CD4+ T cells, and IL‐4‐mediated signaling enhances the TGF‐β1 production from naïve CD4+ T cells. The IL‐4‐mediated up‐regulation of TGF‐β1 production from naïve CD4+ T cells is elicited in STAT6‐deficient (STAT6 KO) mice, but not in IL‐4 receptor‐deficient (IL‐4R KO) mice. These results clearly demonstrate that a STAT6‐independent pathway is working in IL‐4‐mediated enhancement of TGF‐β1 production from naïve CD4+ T cells. Moreover, the addition of IL‐4 showed no additive effect on TGF‐β1 promoter‐mediated transcription stimulated by TCR. Therefore, we hypothesize that the IL‐4‐mediated signaling does not work directly on the transcription of the TGF‐β1 gene, but rather regulates the expansion of TGF‐β1‐secreting T cells.

Selective induction of CD8+ T cell functions by single substituted analogs of an antigenic peptide: distinct signals for IL-10 production

The CD8+ T cell clone 5F1 produces interleukin 10 (IL‐10) and interferon γ (IFN‐γ) in response to stimulation with a peptide corresponding to region 142–149 of bovine αs1‐casein (p142–149). Ninety analog peptides derived from p142–149 with single amino acid substitutions of putative T cell receptor contact residues were prepared to examine whether production of IL‐10 and IFN‐γ by 5F1 can be altered by stimulation with these peptides. We found that some peptides triggered only IL‐10 production whereas others induced production of IFN‐γ alone or both of these cytokines. Peptides inducing IFN‐γ production triggered both cytotoxicity and a proliferative response, whereas peptides inducing production of IL‐10 but not IFN‐γ triggered neither of these responses. Our results clearly demonstrate that the signaling pathway required for IL‐10 production in CD8+ T cells differs from that required for IFN‐γ production. The distinct cellular signals for IL‐10 production appear to be independent of those for cytotoxicity and the proliferative response of CD8+ T cells.

Oral administration of antigen does not influence the proliferation and IFN-γ production of responsive CD8+ T cells but enables to establish T cell clones with different lymphokine production profile.

Feeding of a whole casein diet, which abolished the αs1-casein-specific proliferation and IFN-γ productivity of CD4+ T cells, did not affect the proliferative response of CD8+ T cells with regard to the antigen dose response, cell dose response, kinetics of the proliferation and epitope specificity, as well as IFN-γ production. To assess the characteristics of the CD8+ T cells, we established αs1-casein-specific CD8+ T cell clones from both casein-fed and control mice. The established clones produced different amount of IFN-γ and IL-10, and one clone derived from the casein-fed mice produced a remarkable amount of IL-10. The clones from casein-fed mice produced considerable amounts of TGF-β, while those from control mice produced only small amounts. The possible role of CD8+ T cells in oral tolerance is discussed.

Manipulation of Cytokine Production from CD8+ T Cells by Means of Amino Acid-Substituted Analogs of a Peptide Antigen

Manipulation ofCD8+T-cellresponses specific for an exogenous antigen by epitope variants would be advantageous to develop a novel means of antigen-specific immune regulation. We previously established a CD8+ T cell clone named 5F1 which is specific for peptide 142–149 (p142–149) of as1-casein, a major milk allergen, and these cells produce interleukin 10 (IL- 10) and interferon γ(IFN-γ) upon antigenic stimulation. Some of the analog peptides derived from p142–149 with single amino-acid substitutions triggered only IL- 10 production whereas others induced production of IFN-γ alone or both of these cytokines. These results demonstrate that the signaling pathway involved in induction of IL-10 production in CD8+ T cells differs from that for IFN-production. Our findings illustrate that cytokine production from CD8+ T cells can be manipulated by using single amino-acid substituted analogs of an antigenic peptide.

Suppressive Lymphokines Produced by CD8+ T Cells

Immunological suppression is partially mediated by suppressive lymphokines such as IL-10 and γ-IFN. Both lymphokines were produced by all the CD8+ T cell clones used in this study. Recombinant IL-10 inhibited the proliferation of CD4+ type 1 helper T cell clones and CD8+ T cell clones, but not that of CD4+ type 2 T cell clones. These results suggest the important role of CD8+ T cells in negatively regulating immune response. Anti-IL-10 antibodies enhanced the proliferative response of the CD8+ T cell clones, indicating that CD8+ T cells suppressed their own proliferation by secreting IL-10. Our study suggests the important role of IL-10 in immune regulation by CD8+ T cells and in their activity.