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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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Effect of NKT cell reconstitution on ability of CD1 KO mice to develop ACAID. (A) Flow cytometry confirmation of NK and NKT cell depletion in vitro. Spleen cells from F1 mice were treated with FITC-conjugated anti-NK1.1 mAb, biotin-conjugated Ly49C mAbs, and MicroBead-conjugated anti–mouse pan-NK cell Ab before treatment with anti-FITC MicroBeads and streptavidin MicroBeads, and exposure to a magnetic field to remove NK and NKT cells. The negatively selected cells, and the similarly treated whole spleen cell population not exposed to the magnetic field, were stained with CyChrome 5–conjugated anti–TCR β chain mAb and analyzed by flow cytometry. Fluorescence for CyChrome 5–TCR β chain and FITC-NK1.1 are shown on the ordinate and abscissa, respectively. The percentage of cells within the NKT cell (square) and the NK cell (rectangle) quadrants are listed in the blocks before (whole splenocytes, type 1) and after (NK and NKT depleted, type 2) depletion. (B) Flow cytometry confirmation of CD1+ cell depletion. 7 d after reconstitution with whole spleen cells (type 1) or NK and NKT cell–depleted spleen cells (type 2, Fig. 3 A), five mice from each reconstituted CD1 KO mouse group were inoculated (ac) with OVA. Column-enriched splenic T cells were harvested 1 wk after ac inoculation, and CD1+ cells were removed. Cells were stained with biotin-conjugated anti-CD1 (1B1), then treated with streptavidin MicroBeads and applied to MiniMACS columns to deplete CD1+ cells. Flow cytometry–generated graphs show the relative number of cells (ordinate) versus the increasing fluorescence channels (abscissa) that identify PE-streptavidin-biotin–conjugated anti-CD1 mAb–labeled cells. “KO & WT control” shows the fluorescence pattern of splenic T cells from unreconstituted CD1 KO (open) or WT (shaded) mice; “reconstituted KO” shows the pattern of CD1+ cells after reconstitution; “cells for LAT” shows the pattern of CD1+ cells after negative selection. The percentage of CD1+ cells is indicated in each of the lower blocks. (C) LAT assay for CD1− T cell regulator function. CD1− T cells from reconstituted CD1 KO mice (regulator) were cotransferred with effector and stimulator cells from F1 mice into the ear pinnae of naive F1 mice. Adoptively transferred regulator cells from reconstituted mice that did not receive ac inoculation were used as a control. Ear swelling measurements (24 h after ear challenge) are shown on the ordinate, and the identity of the cell mixture inoculated into the ear pinnae for each group (five per group) is indicated below the abscissa. Significant differences (P ≤ 0.05) are indicated by an asterisk.
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Figure 3: Effect of NKT cell reconstitution on ability of CD1 KO mice to develop ACAID. (A) Flow cytometry confirmation of NK and NKT cell depletion in vitro. Spleen cells from F1 mice were treated with FITC-conjugated anti-NK1.1 mAb, biotin-conjugated Ly49C mAbs, and MicroBead-conjugated anti–mouse pan-NK cell Ab before treatment with anti-FITC MicroBeads and streptavidin MicroBeads, and exposure to a magnetic field to remove NK and NKT cells. The negatively selected cells, and the similarly treated whole spleen cell population not exposed to the magnetic field, were stained with CyChrome 5–conjugated anti–TCR β chain mAb and analyzed by flow cytometry. Fluorescence for CyChrome 5–TCR β chain and FITC-NK1.1 are shown on the ordinate and abscissa, respectively. The percentage of cells within the NKT cell (square) and the NK cell (rectangle) quadrants are listed in the blocks before (whole splenocytes, type 1) and after (NK and NKT depleted, type 2) depletion. (B) Flow cytometry confirmation of CD1+ cell depletion. 7 d after reconstitution with whole spleen cells (type 1) or NK and NKT cell–depleted spleen cells (type 2, Fig. 3 A), five mice from each reconstituted CD1 KO mouse group were inoculated (ac) with OVA. Column-enriched splenic T cells were harvested 1 wk after ac inoculation, and CD1+ cells were removed. Cells were stained with biotin-conjugated anti-CD1 (1B1), then treated with streptavidin MicroBeads and applied to MiniMACS columns to deplete CD1+ cells. Flow cytometry–generated graphs show the relative number of cells (ordinate) versus the increasing fluorescence channels (abscissa) that identify PE-streptavidin-biotin–conjugated anti-CD1 mAb–labeled cells. “KO & WT control” shows the fluorescence pattern of splenic T cells from unreconstituted CD1 KO (open) or WT (shaded) mice; “reconstituted KO” shows the pattern of CD1+ cells after reconstitution; “cells for LAT” shows the pattern of CD1+ cells after negative selection. The percentage of CD1+ cells is indicated in each of the lower blocks. (C) LAT assay for CD1− T cell regulator function. CD1− T cells from reconstituted CD1 KO mice (regulator) were cotransferred with effector and stimulator cells from F1 mice into the ear pinnae of naive F1 mice. Adoptively transferred regulator cells from reconstituted mice that did not receive ac inoculation were used as a control. Ear swelling measurements (24 h after ear challenge) are shown on the ordinate, and the identity of the cell mixture inoculated into the ear pinnae for each group (five per group) is indicated below the abscissa. Significant differences (P ≤ 0.05) are indicated by an asterisk.

Mentions: To confirm whether the defect in the CD1 KO mice that led to the failure of ACAID was actually the NKT cell deficiency, we reconstituted CD1 KO mice with whole spleen cells from WT mice (F1) (containing both NKT cells and CD1+ APCs), or spleen cells immunomagnetically depleted of NK1.1+ lymphocytes (that still contained CD1+ APCs). The successful depletion of NKT and NK cells was confirmed by flow cytometry analysis (Fig. 3 A). 7 d after reconstitution, the mice were inoculated (ac) with OVA, and after 8 d spleen cells were harvested for T cell enrichment. A typical profile of enriched splenic T cells from reconstituted CD1 KO mice (Fig. 3 B, bottom left panel) shows that ∼4.9% of T cells were donor derived (CD1+). (Total of CD1+ cells in the non-T splenic cells was 25%; data not shown.) To analyze the regulatory potential of the host CD1− T cells, the spleen cells were further negatively selected against CD1 expression to yield populations that were CD1− (Fig. 3 B, bottom right panel). These cells were then assessed as regulator cells in the LAT assay.


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)

Effect of NKT cell reconstitution on ability of CD1 KO mice to develop ACAID. (A) Flow cytometry confirmation of NK and NKT cell depletion in vitro. Spleen cells from F1 mice were treated with FITC-conjugated anti-NK1.1 mAb, biotin-conjugated Ly49C mAbs, and MicroBead-conjugated anti–mouse pan-NK cell Ab before treatment with anti-FITC MicroBeads and streptavidin MicroBeads, and exposure to a magnetic field to remove NK and NKT cells. The negatively selected cells, and the similarly treated whole spleen cell population not exposed to the magnetic field, were stained with CyChrome 5–conjugated anti–TCR β chain mAb and analyzed by flow cytometry. Fluorescence for CyChrome 5–TCR β chain and FITC-NK1.1 are shown on the ordinate and abscissa, respectively. The percentage of cells within the NKT cell (square) and the NK cell (rectangle) quadrants are listed in the blocks before (whole splenocytes, type 1) and after (NK and NKT depleted, type 2) depletion. (B) Flow cytometry confirmation of CD1+ cell depletion. 7 d after reconstitution with whole spleen cells (type 1) or NK and NKT cell–depleted spleen cells (type 2, Fig. 3 A), five mice from each reconstituted CD1 KO mouse group were inoculated (ac) with OVA. Column-enriched splenic T cells were harvested 1 wk after ac inoculation, and CD1+ cells were removed. Cells were stained with biotin-conjugated anti-CD1 (1B1), then treated with streptavidin MicroBeads and applied to MiniMACS columns to deplete CD1+ cells. Flow cytometry–generated graphs show the relative number of cells (ordinate) versus the increasing fluorescence channels (abscissa) that identify PE-streptavidin-biotin–conjugated anti-CD1 mAb–labeled cells. “KO & WT control” shows the fluorescence pattern of splenic T cells from unreconstituted CD1 KO (open) or WT (shaded) mice; “reconstituted KO” shows the pattern of CD1+ cells after reconstitution; “cells for LAT” shows the pattern of CD1+ cells after negative selection. The percentage of CD1+ cells is indicated in each of the lower blocks. (C) LAT assay for CD1− T cell regulator function. CD1− T cells from reconstituted CD1 KO mice (regulator) were cotransferred with effector and stimulator cells from F1 mice into the ear pinnae of naive F1 mice. Adoptively transferred regulator cells from reconstituted mice that did not receive ac inoculation were used as a control. Ear swelling measurements (24 h after ear challenge) are shown on the ordinate, and the identity of the cell mixture inoculated into the ear pinnae for each group (five per group) is indicated below the abscissa. Significant differences (P ≤ 0.05) are indicated by an asterisk.
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Related In: Results  -  Collection

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Figure 3: Effect of NKT cell reconstitution on ability of CD1 KO mice to develop ACAID. (A) Flow cytometry confirmation of NK and NKT cell depletion in vitro. Spleen cells from F1 mice were treated with FITC-conjugated anti-NK1.1 mAb, biotin-conjugated Ly49C mAbs, and MicroBead-conjugated anti–mouse pan-NK cell Ab before treatment with anti-FITC MicroBeads and streptavidin MicroBeads, and exposure to a magnetic field to remove NK and NKT cells. The negatively selected cells, and the similarly treated whole spleen cell population not exposed to the magnetic field, were stained with CyChrome 5–conjugated anti–TCR β chain mAb and analyzed by flow cytometry. Fluorescence for CyChrome 5–TCR β chain and FITC-NK1.1 are shown on the ordinate and abscissa, respectively. The percentage of cells within the NKT cell (square) and the NK cell (rectangle) quadrants are listed in the blocks before (whole splenocytes, type 1) and after (NK and NKT depleted, type 2) depletion. (B) Flow cytometry confirmation of CD1+ cell depletion. 7 d after reconstitution with whole spleen cells (type 1) or NK and NKT cell–depleted spleen cells (type 2, Fig. 3 A), five mice from each reconstituted CD1 KO mouse group were inoculated (ac) with OVA. Column-enriched splenic T cells were harvested 1 wk after ac inoculation, and CD1+ cells were removed. Cells were stained with biotin-conjugated anti-CD1 (1B1), then treated with streptavidin MicroBeads and applied to MiniMACS columns to deplete CD1+ cells. Flow cytometry–generated graphs show the relative number of cells (ordinate) versus the increasing fluorescence channels (abscissa) that identify PE-streptavidin-biotin–conjugated anti-CD1 mAb–labeled cells. “KO & WT control” shows the fluorescence pattern of splenic T cells from unreconstituted CD1 KO (open) or WT (shaded) mice; “reconstituted KO” shows the pattern of CD1+ cells after reconstitution; “cells for LAT” shows the pattern of CD1+ cells after negative selection. The percentage of CD1+ cells is indicated in each of the lower blocks. (C) LAT assay for CD1− T cell regulator function. CD1− T cells from reconstituted CD1 KO mice (regulator) were cotransferred with effector and stimulator cells from F1 mice into the ear pinnae of naive F1 mice. Adoptively transferred regulator cells from reconstituted mice that did not receive ac inoculation were used as a control. Ear swelling measurements (24 h after ear challenge) are shown on the ordinate, and the identity of the cell mixture inoculated into the ear pinnae for each group (five per group) is indicated below the abscissa. Significant differences (P ≤ 0.05) are indicated by an asterisk.
Mentions: To confirm whether the defect in the CD1 KO mice that led to the failure of ACAID was actually the NKT cell deficiency, we reconstituted CD1 KO mice with whole spleen cells from WT mice (F1) (containing both NKT cells and CD1+ APCs), or spleen cells immunomagnetically depleted of NK1.1+ lymphocytes (that still contained CD1+ APCs). The successful depletion of NKT and NK cells was confirmed by flow cytometry analysis (Fig. 3 A). 7 d after reconstitution, the mice were inoculated (ac) with OVA, and after 8 d spleen cells were harvested for T cell enrichment. A typical profile of enriched splenic T cells from reconstituted CD1 KO mice (Fig. 3 B, bottom left panel) shows that ∼4.9% of T cells were donor derived (CD1+). (Total of CD1+ cells in the non-T splenic cells was 25%; data not shown.) To analyze the regulatory potential of the host CD1− T cells, the spleen cells were further negatively selected against CD1 expression to yield populations that were CD1− (Fig. 3 B, bottom right panel). These cells were then assessed as regulator cells in the LAT assay.

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