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Nonclassical MHC class Ib-restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum.

Nagarajan NA, Gonzalez F, Shastri N - Nat. Immunol. (2012)

Bottom Line: We found that inhibition of ERAAP rapidly induced presentation of the peptide FYAEATPML (FL9) by the MHC class Ib molecule Qa-1(b).MHC class Ib-restricted cytolytic effector cells specifically eliminated ERAAP-deficient cells in vitro and in vivo.Thus, nonclassical Qa-1(b)-peptide complexes direct cytotoxic T cells to targets with defective antigen processing in the endoplasmic reticulum.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, California, USA.

ABSTRACT
The aminopeptidase ERAAP is essential for trimming peptides presented by major histocompatibility complex (MHC) class I molecules. Inhibition of ERAAP by cytomegalovirus results in evasion of the immune response by this virus, and polymorphisms in ERAAP are associated with autoimmune disorders. How normal ERAAP function is monitored is unknown. We found that inhibition of ERAAP rapidly induced presentation of the peptide FYAEATPML (FL9) by the MHC class Ib molecule Qa-1(b). Antigen-experienced T cells specific for the Qa-1(b)-FL9 complex were frequent in naive mice. Wild-type mice immunized with ERAAP-deficient cells mounted a potent CD8(+) T cell response specific for Qa-1(b)-FL9. MHC class Ib-restricted cytolytic effector cells specifically eliminated ERAAP-deficient cells in vitro and in vivo. Thus, nonclassical Qa-1(b)-peptide complexes direct cytotoxic T cells to targets with defective antigen processing in the endoplasmic reticulum.

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Wild-type CD8 T cells respond to pMHC Ia and pMHC Ib complexes expressed by ERAAP-deficient cells(a-b) Spleen cells from C57BL/6J (WT) mice, immunized with ERAAP-/- splenocytes, were restimulated in vitro to generate cytotoxic T lymphocyte (CTL) lines. IFN-γ production by the CTL lines was measured after incubation with the indicated splenic APCs. (a) Representative flow cytometry plots show IFN-γ produced by WT anti-ERAAP-/- CTLs. Numbers indicate % of IFN-γ+ cells among all the CD8+ cells in the sample. (b) The percentage of CD8+IFN-γ+ cells in cultures of individual immunized mice. Each symbol represents one mouse. One representative of three independent experiments is shown. (c-e) The lacZ response of BEko8Z hybridoma cells to the indicated LPS-stimulated spleen cells used as APCs. In (d) LPS-stimulated spleen cells were treated with the aminopeptidase inhibitor, leucinethiol and the BEko8Z hybridoma response measured. Data shown is representative of three independent experiments.
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Figure 1: Wild-type CD8 T cells respond to pMHC Ia and pMHC Ib complexes expressed by ERAAP-deficient cells(a-b) Spleen cells from C57BL/6J (WT) mice, immunized with ERAAP-/- splenocytes, were restimulated in vitro to generate cytotoxic T lymphocyte (CTL) lines. IFN-γ production by the CTL lines was measured after incubation with the indicated splenic APCs. (a) Representative flow cytometry plots show IFN-γ produced by WT anti-ERAAP-/- CTLs. Numbers indicate % of IFN-γ+ cells among all the CD8+ cells in the sample. (b) The percentage of CD8+IFN-γ+ cells in cultures of individual immunized mice. Each symbol represents one mouse. One representative of three independent experiments is shown. (c-e) The lacZ response of BEko8Z hybridoma cells to the indicated LPS-stimulated spleen cells used as APCs. In (d) LPS-stimulated spleen cells were treated with the aminopeptidase inhibitor, leucinethiol and the BEko8Z hybridoma response measured. Data shown is representative of three independent experiments.

Mentions: The immune system efficiently detects differences between self- and non-self. Therefore, to identify immunologically significant changes caused by ERAAP deficiency, we immunized wild-type (C57BL/6J, H-2b) mice with MHC-matched spleen cells from ERAAP-deficient mice (14; referred to as ERAAP-KO in the text). We generated wild-type anti-ERAAP-KO CD8+ T cell lines by restimulating host spleen cells with ERAAP-KO antigen-presenting cells (APCs) in vitro. Similar to our earlier report17, CD8+ T cell lines generated from immunized mice responded strongly by producing interferon-γ (IFN-γ) and tumor necrosis factor (TNF) when cultured with ERAAP-deficient APCs compared to the background responses to self-APCs (Fig. 1a,b, Supplementary Fig. 1). Comparable fractions (30% versus 25% on average) of the same CD8+ T cells responded to ERAAP-deficient cells that also lacked any classical H2-Kb or H2-Db MHC Ia molecules (18; ERAAP-MHC Ia-TKO, Fig. 1a,b). MHC I molecules were nevertheless required for the IFN-γ response because APCs lacking ERAAP as well as β2-microglobulin (β2m; β2m-ERAAP-DKO), an essential structural subunit all MHC I molecules19,20, failed to stimulate the same wild-type anti-ERAAP-KO CD8+ T cells (Fig 1b). Likewise, APCs deficient in ERAAP as well as the TAP transporter (9; TAP-ERAAP-DKO), also failed to stimulate the same CD8+ T cells (Fig. 1b), demonstrating that their responses required peptide(s) transported into the ER. We therefore conclude that loss of ERAAP function results in the presentation of immunologically distinct peptides by MHC Ia and MHC Ib molecules to wild-type CD8+ T cells.


Nonclassical MHC class Ib-restricted cytotoxic T cells monitor antigen processing in the endoplasmic reticulum.

Nagarajan NA, Gonzalez F, Shastri N - Nat. Immunol. (2012)

Wild-type CD8 T cells respond to pMHC Ia and pMHC Ib complexes expressed by ERAAP-deficient cells(a-b) Spleen cells from C57BL/6J (WT) mice, immunized with ERAAP-/- splenocytes, were restimulated in vitro to generate cytotoxic T lymphocyte (CTL) lines. IFN-γ production by the CTL lines was measured after incubation with the indicated splenic APCs. (a) Representative flow cytometry plots show IFN-γ produced by WT anti-ERAAP-/- CTLs. Numbers indicate % of IFN-γ+ cells among all the CD8+ cells in the sample. (b) The percentage of CD8+IFN-γ+ cells in cultures of individual immunized mice. Each symbol represents one mouse. One representative of three independent experiments is shown. (c-e) The lacZ response of BEko8Z hybridoma cells to the indicated LPS-stimulated spleen cells used as APCs. In (d) LPS-stimulated spleen cells were treated with the aminopeptidase inhibitor, leucinethiol and the BEko8Z hybridoma response measured. Data shown is representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 1: Wild-type CD8 T cells respond to pMHC Ia and pMHC Ib complexes expressed by ERAAP-deficient cells(a-b) Spleen cells from C57BL/6J (WT) mice, immunized with ERAAP-/- splenocytes, were restimulated in vitro to generate cytotoxic T lymphocyte (CTL) lines. IFN-γ production by the CTL lines was measured after incubation with the indicated splenic APCs. (a) Representative flow cytometry plots show IFN-γ produced by WT anti-ERAAP-/- CTLs. Numbers indicate % of IFN-γ+ cells among all the CD8+ cells in the sample. (b) The percentage of CD8+IFN-γ+ cells in cultures of individual immunized mice. Each symbol represents one mouse. One representative of three independent experiments is shown. (c-e) The lacZ response of BEko8Z hybridoma cells to the indicated LPS-stimulated spleen cells used as APCs. In (d) LPS-stimulated spleen cells were treated with the aminopeptidase inhibitor, leucinethiol and the BEko8Z hybridoma response measured. Data shown is representative of three independent experiments.
Mentions: The immune system efficiently detects differences between self- and non-self. Therefore, to identify immunologically significant changes caused by ERAAP deficiency, we immunized wild-type (C57BL/6J, H-2b) mice with MHC-matched spleen cells from ERAAP-deficient mice (14; referred to as ERAAP-KO in the text). We generated wild-type anti-ERAAP-KO CD8+ T cell lines by restimulating host spleen cells with ERAAP-KO antigen-presenting cells (APCs) in vitro. Similar to our earlier report17, CD8+ T cell lines generated from immunized mice responded strongly by producing interferon-γ (IFN-γ) and tumor necrosis factor (TNF) when cultured with ERAAP-deficient APCs compared to the background responses to self-APCs (Fig. 1a,b, Supplementary Fig. 1). Comparable fractions (30% versus 25% on average) of the same CD8+ T cells responded to ERAAP-deficient cells that also lacked any classical H2-Kb or H2-Db MHC Ia molecules (18; ERAAP-MHC Ia-TKO, Fig. 1a,b). MHC I molecules were nevertheless required for the IFN-γ response because APCs lacking ERAAP as well as β2-microglobulin (β2m; β2m-ERAAP-DKO), an essential structural subunit all MHC I molecules19,20, failed to stimulate the same wild-type anti-ERAAP-KO CD8+ T cells (Fig 1b). Likewise, APCs deficient in ERAAP as well as the TAP transporter (9; TAP-ERAAP-DKO), also failed to stimulate the same CD8+ T cells (Fig. 1b), demonstrating that their responses required peptide(s) transported into the ER. We therefore conclude that loss of ERAAP function results in the presentation of immunologically distinct peptides by MHC Ia and MHC Ib molecules to wild-type CD8+ T cells.

Bottom Line: We found that inhibition of ERAAP rapidly induced presentation of the peptide FYAEATPML (FL9) by the MHC class Ib molecule Qa-1(b).MHC class Ib-restricted cytolytic effector cells specifically eliminated ERAAP-deficient cells in vitro and in vivo.Thus, nonclassical Qa-1(b)-peptide complexes direct cytotoxic T cells to targets with defective antigen processing in the endoplasmic reticulum.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cell Biology, Division of Immunology and Pathogenesis, University of California, Berkeley, California, USA.

ABSTRACT
The aminopeptidase ERAAP is essential for trimming peptides presented by major histocompatibility complex (MHC) class I molecules. Inhibition of ERAAP by cytomegalovirus results in evasion of the immune response by this virus, and polymorphisms in ERAAP are associated with autoimmune disorders. How normal ERAAP function is monitored is unknown. We found that inhibition of ERAAP rapidly induced presentation of the peptide FYAEATPML (FL9) by the MHC class Ib molecule Qa-1(b). Antigen-experienced T cells specific for the Qa-1(b)-FL9 complex were frequent in naive mice. Wild-type mice immunized with ERAAP-deficient cells mounted a potent CD8(+) T cell response specific for Qa-1(b)-FL9. MHC class Ib-restricted cytolytic effector cells specifically eliminated ERAAP-deficient cells in vitro and in vivo. Thus, nonclassical Qa-1(b)-peptide complexes direct cytotoxic T cells to targets with defective antigen processing in the endoplasmic reticulum.

Show MeSH
Related in: MedlinePlus