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CD1-reactive natural killer T cells are required for development of systemic tolerance through an immune-privileged site.

Sonoda KH, Exley M, Snapper S, Balk SP, Stein-Streilein J - J. Exp. Med. (1999)

Bottom Line: Therefore, this model for immune-privileged site-mediated tolerance provided us with an excellent format for studying the role of NKT cells in the development of tolerance.Significantly, CD1-reactive NKT cells were not required for intravenously induced systemic tolerance, thereby establishing that different mechanisms mediate development of tolerance to antigens inoculated by these routes.A critical role for NKT cells in the development of systemic tolerance associated with an immune-privileged site suggests a mechanism involving NKT cells in self-tolerance and their defects in autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.

ABSTRACT
Systemic tolerance can be elicited by introducing antigen into an immune-privileged site, such as the eye, or directly into the blood. Both routes of immunization result in a selective deficiency of systemic delayed type hypersensitivity. Although the experimental animal model of anterior chamber-associated immune deviation (ACAID) occurs in most mouse strains, ACAID cannot be induced in several mutant mouse strains that are coincidentally deficient in natural killer T (NKT) cells. Therefore, this model for immune-privileged site-mediated tolerance provided us with an excellent format for studying the role of NKT cells in the development of tolerance. The following data show that CD1-reactive NKT cells are required for the development of systemic tolerance induced via the eye as follows: (a) CD1 knockout mice were unable to develop ACAID unless they were reconstituted with NKT cells together with CD1(+) antigen-presenting cells; (b) specific antibody depletion of NKT cells in vivo abrogated the development of ACAID; and (c) anti-CD1 monoclonal antibody treatment of wild-type mice prevented ACAID development. Significantly, CD1-reactive NKT cells were not required for intravenously induced systemic tolerance, thereby establishing that different mechanisms mediate development of tolerance to antigens inoculated by these routes. A critical role for NKT cells in the development of systemic tolerance associated with an immune-privileged site suggests a mechanism involving NKT cells in self-tolerance and their defects in autoimmunity.

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Analysis of NKT and NK cells as efferent regulatory DTH cells in the LAT assay. (A) Flow cytometry confirmation of NKT and NK cell depletion in vitro. Column-enriched splenic T cells were harvested from B6 mice 7 d after OVA (ac) inoculation. All NK1.1+ cells were removed from the T cell–enriched populations with a magnetic field after treatment with a mixture of FITC–anti-NK1.1, biotin–anti-Ly49C mAbs, and anti–pan-NK cell conjugated Microbeads, and anti-FITC and streptavidin MicroBeads. CyChrome 5–conjugated anti–TCR β chain and FITC-conjugated anti-NK1.1 cells are shown in the dot plots for cells before and after treatment. The percentage of labeled cells is shown next to the square and rectangle for NKT and NK cells, respectively. (B) The effect of NKT and NK cell depletion on efferent regulation of DTH. Ear swelling was measured 24 h after cell transfer of the various cell mixtures (indicated below the abscissa for each bar) into the ear pinnae of naive syngeneic mice (five per group), and is shown on the ordinate. Significant differences (P ≤ 0.05) are indicated by an asterisk.
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Figure 4: Analysis of NKT and NK cells as efferent regulatory DTH cells in the LAT assay. (A) Flow cytometry confirmation of NKT and NK cell depletion in vitro. Column-enriched splenic T cells were harvested from B6 mice 7 d after OVA (ac) inoculation. All NK1.1+ cells were removed from the T cell–enriched populations with a magnetic field after treatment with a mixture of FITC–anti-NK1.1, biotin–anti-Ly49C mAbs, and anti–pan-NK cell conjugated Microbeads, and anti-FITC and streptavidin MicroBeads. CyChrome 5–conjugated anti–TCR β chain and FITC-conjugated anti-NK1.1 cells are shown in the dot plots for cells before and after treatment. The percentage of labeled cells is shown next to the square and rectangle for NKT and NK cells, respectively. (B) The effect of NKT and NK cell depletion on efferent regulation of DTH. Ear swelling was measured 24 h after cell transfer of the various cell mixtures (indicated below the abscissa for each bar) into the ear pinnae of naive syngeneic mice (five per group), and is shown on the ordinate. Significant differences (P ≤ 0.05) are indicated by an asterisk.

Mentions: To confirm that NKT cells do not have a direct role in efferent regulation of DTH in another way, we depleted cells expressing NK1.1 antigens from the regulator cell population before testing in a LAT assay using specific Ab treatment and magnetic beads selection (Fig. 4 A). Treated cells were then assessed by flow cytometry before cotransferring with primed T cells and OVA-pulsed PECs to the ear pinnae. The results of the LAT assay showed that the NK and NKT cell–depleted populations retained their DTH-regulatory capability. Thus, the efferent-regulatory cell is a conventional NK1.1− T cell. It is known that activated NKT cells can downregulate their NK1.1 molecules in vitro 35. Therefore, we were aware that the in vivo–activated NKT cell may not express NK1.1, and the Ab depletion of NK1.1+ cells treatment may not work. However, as also shown in Fig. 1, after OVA was inoculated in the ac NK1.1+ T cells were clearly present in the spleen. Therefore, together with our observations that CD1− T cells (from CD1+ NKT cell–reconstituted CD1 KO mice) can function as regulators of DTH (Fig. 3 C), these data confirm that NKT cells are not direct efferent regulators of DTH in ACAID.


CD1-reactive natural killer T cells are required for development of systemic tolerance through an immune-privileged site.

Sonoda KH, Exley M, Snapper S, Balk SP, Stein-Streilein J - J. Exp. Med. (1999)

Analysis of NKT and NK cells as efferent regulatory DTH cells in the LAT assay. (A) Flow cytometry confirmation of NKT and NK cell depletion in vitro. Column-enriched splenic T cells were harvested from B6 mice 7 d after OVA (ac) inoculation. All NK1.1+ cells were removed from the T cell–enriched populations with a magnetic field after treatment with a mixture of FITC–anti-NK1.1, biotin–anti-Ly49C mAbs, and anti–pan-NK cell conjugated Microbeads, and anti-FITC and streptavidin MicroBeads. CyChrome 5–conjugated anti–TCR β chain and FITC-conjugated anti-NK1.1 cells are shown in the dot plots for cells before and after treatment. The percentage of labeled cells is shown next to the square and rectangle for NKT and NK cells, respectively. (B) The effect of NKT and NK cell depletion on efferent regulation of DTH. Ear swelling was measured 24 h after cell transfer of the various cell mixtures (indicated below the abscissa for each bar) into the ear pinnae of naive syngeneic mice (five per group), and is shown on the ordinate. Significant differences (P ≤ 0.05) are indicated by an asterisk.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195676&req=5

Figure 4: Analysis of NKT and NK cells as efferent regulatory DTH cells in the LAT assay. (A) Flow cytometry confirmation of NKT and NK cell depletion in vitro. Column-enriched splenic T cells were harvested from B6 mice 7 d after OVA (ac) inoculation. All NK1.1+ cells were removed from the T cell–enriched populations with a magnetic field after treatment with a mixture of FITC–anti-NK1.1, biotin–anti-Ly49C mAbs, and anti–pan-NK cell conjugated Microbeads, and anti-FITC and streptavidin MicroBeads. CyChrome 5–conjugated anti–TCR β chain and FITC-conjugated anti-NK1.1 cells are shown in the dot plots for cells before and after treatment. The percentage of labeled cells is shown next to the square and rectangle for NKT and NK cells, respectively. (B) The effect of NKT and NK cell depletion on efferent regulation of DTH. Ear swelling was measured 24 h after cell transfer of the various cell mixtures (indicated below the abscissa for each bar) into the ear pinnae of naive syngeneic mice (five per group), and is shown on the ordinate. Significant differences (P ≤ 0.05) are indicated by an asterisk.
Mentions: To confirm that NKT cells do not have a direct role in efferent regulation of DTH in another way, we depleted cells expressing NK1.1 antigens from the regulator cell population before testing in a LAT assay using specific Ab treatment and magnetic beads selection (Fig. 4 A). Treated cells were then assessed by flow cytometry before cotransferring with primed T cells and OVA-pulsed PECs to the ear pinnae. The results of the LAT assay showed that the NK and NKT cell–depleted populations retained their DTH-regulatory capability. Thus, the efferent-regulatory cell is a conventional NK1.1− T cell. It is known that activated NKT cells can downregulate their NK1.1 molecules in vitro 35. Therefore, we were aware that the in vivo–activated NKT cell may not express NK1.1, and the Ab depletion of NK1.1+ cells treatment may not work. However, as also shown in Fig. 1, after OVA was inoculated in the ac NK1.1+ T cells were clearly present in the spleen. Therefore, together with our observations that CD1− T cells (from CD1+ NKT cell–reconstituted CD1 KO mice) can function as regulators of DTH (Fig. 3 C), these data confirm that NKT cells are not direct efferent regulators of DTH in ACAID.

Bottom Line: Therefore, this model for immune-privileged site-mediated tolerance provided us with an excellent format for studying the role of NKT cells in the development of tolerance.Significantly, CD1-reactive NKT cells were not required for intravenously induced systemic tolerance, thereby establishing that different mechanisms mediate development of tolerance to antigens inoculated by these routes.A critical role for NKT cells in the development of systemic tolerance associated with an immune-privileged site suggests a mechanism involving NKT cells in self-tolerance and their defects in autoimmunity.

View Article: PubMed Central - PubMed

Affiliation: Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114, USA.

ABSTRACT
Systemic tolerance can be elicited by introducing antigen into an immune-privileged site, such as the eye, or directly into the blood. Both routes of immunization result in a selective deficiency of systemic delayed type hypersensitivity. Although the experimental animal model of anterior chamber-associated immune deviation (ACAID) occurs in most mouse strains, ACAID cannot be induced in several mutant mouse strains that are coincidentally deficient in natural killer T (NKT) cells. Therefore, this model for immune-privileged site-mediated tolerance provided us with an excellent format for studying the role of NKT cells in the development of tolerance. The following data show that CD1-reactive NKT cells are required for the development of systemic tolerance induced via the eye as follows: (a) CD1 knockout mice were unable to develop ACAID unless they were reconstituted with NKT cells together with CD1(+) antigen-presenting cells; (b) specific antibody depletion of NKT cells in vivo abrogated the development of ACAID; and (c) anti-CD1 monoclonal antibody treatment of wild-type mice prevented ACAID development. Significantly, CD1-reactive NKT cells were not required for intravenously induced systemic tolerance, thereby establishing that different mechanisms mediate development of tolerance to antigens inoculated by these routes. A critical role for NKT cells in the development of systemic tolerance associated with an immune-privileged site suggests a mechanism involving NKT cells in self-tolerance and their defects in autoimmunity.

Show MeSH
Related in: MedlinePlus