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Massive thymic deletion results in systemic autoimmunity through elimination of CD4+ CD25+ T regulatory cells.

Shih FF, Mandik-Nayak L, Wipke BT, Allen PM - J. Exp. Med. (2004)

Bottom Line: Extensive thymic deletion resulted in lymphopenia and elimination of CD4+ CD25+ regulatory T cells (Tregs), but spared some CD4+ T cells expressing endogenous TCR, which oligoclonally expanded in the periphery.Thus, our studies demonstrated that central tolerance can paradoxically result in systemic autoimmunity through differential susceptibility of Tregs and autoreactive T cells to thymic deletion.Therefore, too little or too much negative selection to a self-antigen can result in systemic autoimmunity and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Incomplete deletion of KRN T cells that recognize the ubiquitously expressed self-antigen glucose-6-phosphate-isomerase (GPI) initiates an anti-GPI autoimmune cascade in K/BxN mice resulting in a humorally mediated arthritis. Transgenic (Tg) expression of a KRN T cell receptor (TCR) agonist under the major histocompatibility complex class II promoter resulted in thymic deletion with loss of anti-GPI T and B cell responses and attenuated arthritis course. However, double Tg mice succumbed to systemic autoimmunity with multiorgan inflammation and autoantibody production. Extensive thymic deletion resulted in lymphopenia and elimination of CD4+ CD25+ regulatory T cells (Tregs), but spared some CD4+ T cells expressing endogenous TCR, which oligoclonally expanded in the periphery. Disease was transferred by these T cells and prevented by cotransfer of CD4+ CD25+ Tregs. Moreover, we extended our findings to another TCR system (anti-hen egg lysozyme [HEL] TCR/HEL mice) where similarly extensive thymic deletion also resulted in disease. Thus, our studies demonstrated that central tolerance can paradoxically result in systemic autoimmunity through differential susceptibility of Tregs and autoreactive T cells to thymic deletion. Therefore, too little or too much negative selection to a self-antigen can result in systemic autoimmunity and disease.

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CD4+ CD25+ T cells from KRNk/g7 mice are Tregs and are thymically derived. (A) Sorted CD4+ CD25− (open and solid bars) and CD4+ CD25+ (dotted and striped bars) T cells from pooled LNs and spleens from three KRNk/k (open and dotted bars) and three KRNk/g7 (solid and striped bars) mice were analyzed for HPRT, CD25, and FoxP3 expression by quantitative PCR and normalized to housekeeping gene HPRT. (B) Sorted CD4+ CD25+ and CD4+ CD25− T cells were purified from pooled LNs and spleens of seven KRNk/g7 mice. CD4+ CD25+ T cells (solid bars), CD4+ CD25− (open bars), and mixed CD4+ CD25+/CD4+ CD25− (1:1; striped bars) were cultured at 5 × 104/well with graded doses of GPI(281–293) peptide and 2 × 105 irradiated T-depleted H-2k/g7 splenocytes in 96-well round-bottom plates for 72 h with 0.2 μCi [3H]thymidine in the last 18 h. Each point represents the mean of duplicate wells with error bars indicating SD. (C) Thymocytes from 4-wk-old KRNk/k and KRNk/g7 mice were analyzed by flow cytometry with CD4-APC, CD8-FITC, and CD25-PE. Live cells were identified by propidium iodide exclusion. Histograms of CD25 expression of CD4+ SP thymocytes are presented with the percentage of CD25hi cells indicated on the marker. Data are representative of three mice. (D) Sorted CD4+ CD25− (open bars) and CD4+ CD25+ (solid bars) were purified from five KRNk/g7 mice and analyzed for HPRT, CD25, and FoxP3 expression by quantitative PCR and normalized to HPRT.
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fig5: CD4+ CD25+ T cells from KRNk/g7 mice are Tregs and are thymically derived. (A) Sorted CD4+ CD25− (open and solid bars) and CD4+ CD25+ (dotted and striped bars) T cells from pooled LNs and spleens from three KRNk/k (open and dotted bars) and three KRNk/g7 (solid and striped bars) mice were analyzed for HPRT, CD25, and FoxP3 expression by quantitative PCR and normalized to housekeeping gene HPRT. (B) Sorted CD4+ CD25+ and CD4+ CD25− T cells were purified from pooled LNs and spleens of seven KRNk/g7 mice. CD4+ CD25+ T cells (solid bars), CD4+ CD25− (open bars), and mixed CD4+ CD25+/CD4+ CD25− (1:1; striped bars) were cultured at 5 × 104/well with graded doses of GPI(281–293) peptide and 2 × 105 irradiated T-depleted H-2k/g7 splenocytes in 96-well round-bottom plates for 72 h with 0.2 μCi [3H]thymidine in the last 18 h. Each point represents the mean of duplicate wells with error bars indicating SD. (C) Thymocytes from 4-wk-old KRNk/k and KRNk/g7 mice were analyzed by flow cytometry with CD4-APC, CD8-FITC, and CD25-PE. Live cells were identified by propidium iodide exclusion. Histograms of CD25 expression of CD4+ SP thymocytes are presented with the percentage of CD25hi cells indicated on the marker. Data are representative of three mice. (D) Sorted CD4+ CD25− (open bars) and CD4+ CD25+ (solid bars) were purified from five KRNk/g7 mice and analyzed for HPRT, CD25, and FoxP3 expression by quantitative PCR and normalized to HPRT.

Mentions: Despite the activated phenotype of the CD4+ T cells derived from KRN/G7mTg7k/g7 mice, only 6% of these CD4+ T cells were CD25hi, whereas 20% of CD4+ T cells from KRNk/g7 mice were CD25hi (Fig. 4 C). As KRN T cells were activated by endogenous GPI in KRNk/g7 mice, the enhanced CD4+ CD25+ T cells may reflect peripherally activated T cells or CD4+ CD25+ Tregs. To distinguish between these two possibilities, we assessed the expression of the forkhead transcription factor, FoxP3, by quantitative PCR. FoxP3 had been identified as critical in the development of CD4+ CD25+ Tregs (10, 11). CD4+ CD25− and CD4+ CD25+ T cells were sorted from pooled LN cells and splenocytes from three to five KRNk/k and KRNk/g7 mice. Expression of CD25 and FoxP3 were analyzed by quantitative PCR and normalized to the housekeeping gene HPRT. FoxP3 was expressed in low abundance but correlated with CD25 expression in both KRNk/k and KRNk/g7 mice (Fig. 5 A). Moreover, to show that these are indeed Tregs, we assayed the ability of CD4+ CD25+ T cells from KRNk/g7 mice to suppress the proliferation of their CD4+ CD25− counterparts. CD4+ CD25+ T cells proliferated poorly in response to GPI compared to the response elicited in CD4+ CD25− T cells. The addition of CD4+ CD25+ Tregs at a 1:1 ratio reduced proliferation by 70–85% (Fig. 5 B). Hence, CD4+ CD25+ T cells from KRNk/g7 mice expressed FoxP3 and functioned as Tregs.


Massive thymic deletion results in systemic autoimmunity through elimination of CD4+ CD25+ T regulatory cells.

Shih FF, Mandik-Nayak L, Wipke BT, Allen PM - J. Exp. Med. (2004)

CD4+ CD25+ T cells from KRNk/g7 mice are Tregs and are thymically derived. (A) Sorted CD4+ CD25− (open and solid bars) and CD4+ CD25+ (dotted and striped bars) T cells from pooled LNs and spleens from three KRNk/k (open and dotted bars) and three KRNk/g7 (solid and striped bars) mice were analyzed for HPRT, CD25, and FoxP3 expression by quantitative PCR and normalized to housekeeping gene HPRT. (B) Sorted CD4+ CD25+ and CD4+ CD25− T cells were purified from pooled LNs and spleens of seven KRNk/g7 mice. CD4+ CD25+ T cells (solid bars), CD4+ CD25− (open bars), and mixed CD4+ CD25+/CD4+ CD25− (1:1; striped bars) were cultured at 5 × 104/well with graded doses of GPI(281–293) peptide and 2 × 105 irradiated T-depleted H-2k/g7 splenocytes in 96-well round-bottom plates for 72 h with 0.2 μCi [3H]thymidine in the last 18 h. Each point represents the mean of duplicate wells with error bars indicating SD. (C) Thymocytes from 4-wk-old KRNk/k and KRNk/g7 mice were analyzed by flow cytometry with CD4-APC, CD8-FITC, and CD25-PE. Live cells were identified by propidium iodide exclusion. Histograms of CD25 expression of CD4+ SP thymocytes are presented with the percentage of CD25hi cells indicated on the marker. Data are representative of three mice. (D) Sorted CD4+ CD25− (open bars) and CD4+ CD25+ (solid bars) were purified from five KRNk/g7 mice and analyzed for HPRT, CD25, and FoxP3 expression by quantitative PCR and normalized to HPRT.
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fig5: CD4+ CD25+ T cells from KRNk/g7 mice are Tregs and are thymically derived. (A) Sorted CD4+ CD25− (open and solid bars) and CD4+ CD25+ (dotted and striped bars) T cells from pooled LNs and spleens from three KRNk/k (open and dotted bars) and three KRNk/g7 (solid and striped bars) mice were analyzed for HPRT, CD25, and FoxP3 expression by quantitative PCR and normalized to housekeeping gene HPRT. (B) Sorted CD4+ CD25+ and CD4+ CD25− T cells were purified from pooled LNs and spleens of seven KRNk/g7 mice. CD4+ CD25+ T cells (solid bars), CD4+ CD25− (open bars), and mixed CD4+ CD25+/CD4+ CD25− (1:1; striped bars) were cultured at 5 × 104/well with graded doses of GPI(281–293) peptide and 2 × 105 irradiated T-depleted H-2k/g7 splenocytes in 96-well round-bottom plates for 72 h with 0.2 μCi [3H]thymidine in the last 18 h. Each point represents the mean of duplicate wells with error bars indicating SD. (C) Thymocytes from 4-wk-old KRNk/k and KRNk/g7 mice were analyzed by flow cytometry with CD4-APC, CD8-FITC, and CD25-PE. Live cells were identified by propidium iodide exclusion. Histograms of CD25 expression of CD4+ SP thymocytes are presented with the percentage of CD25hi cells indicated on the marker. Data are representative of three mice. (D) Sorted CD4+ CD25− (open bars) and CD4+ CD25+ (solid bars) were purified from five KRNk/g7 mice and analyzed for HPRT, CD25, and FoxP3 expression by quantitative PCR and normalized to HPRT.
Mentions: Despite the activated phenotype of the CD4+ T cells derived from KRN/G7mTg7k/g7 mice, only 6% of these CD4+ T cells were CD25hi, whereas 20% of CD4+ T cells from KRNk/g7 mice were CD25hi (Fig. 4 C). As KRN T cells were activated by endogenous GPI in KRNk/g7 mice, the enhanced CD4+ CD25+ T cells may reflect peripherally activated T cells or CD4+ CD25+ Tregs. To distinguish between these two possibilities, we assessed the expression of the forkhead transcription factor, FoxP3, by quantitative PCR. FoxP3 had been identified as critical in the development of CD4+ CD25+ Tregs (10, 11). CD4+ CD25− and CD4+ CD25+ T cells were sorted from pooled LN cells and splenocytes from three to five KRNk/k and KRNk/g7 mice. Expression of CD25 and FoxP3 were analyzed by quantitative PCR and normalized to the housekeeping gene HPRT. FoxP3 was expressed in low abundance but correlated with CD25 expression in both KRNk/k and KRNk/g7 mice (Fig. 5 A). Moreover, to show that these are indeed Tregs, we assayed the ability of CD4+ CD25+ T cells from KRNk/g7 mice to suppress the proliferation of their CD4+ CD25− counterparts. CD4+ CD25+ T cells proliferated poorly in response to GPI compared to the response elicited in CD4+ CD25− T cells. The addition of CD4+ CD25+ Tregs at a 1:1 ratio reduced proliferation by 70–85% (Fig. 5 B). Hence, CD4+ CD25+ T cells from KRNk/g7 mice expressed FoxP3 and functioned as Tregs.

Bottom Line: Extensive thymic deletion resulted in lymphopenia and elimination of CD4+ CD25+ regulatory T cells (Tregs), but spared some CD4+ T cells expressing endogenous TCR, which oligoclonally expanded in the periphery.Thus, our studies demonstrated that central tolerance can paradoxically result in systemic autoimmunity through differential susceptibility of Tregs and autoreactive T cells to thymic deletion.Therefore, too little or too much negative selection to a self-antigen can result in systemic autoimmunity and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA.

ABSTRACT
Incomplete deletion of KRN T cells that recognize the ubiquitously expressed self-antigen glucose-6-phosphate-isomerase (GPI) initiates an anti-GPI autoimmune cascade in K/BxN mice resulting in a humorally mediated arthritis. Transgenic (Tg) expression of a KRN T cell receptor (TCR) agonist under the major histocompatibility complex class II promoter resulted in thymic deletion with loss of anti-GPI T and B cell responses and attenuated arthritis course. However, double Tg mice succumbed to systemic autoimmunity with multiorgan inflammation and autoantibody production. Extensive thymic deletion resulted in lymphopenia and elimination of CD4+ CD25+ regulatory T cells (Tregs), but spared some CD4+ T cells expressing endogenous TCR, which oligoclonally expanded in the periphery. Disease was transferred by these T cells and prevented by cotransfer of CD4+ CD25+ Tregs. Moreover, we extended our findings to another TCR system (anti-hen egg lysozyme [HEL] TCR/HEL mice) where similarly extensive thymic deletion also resulted in disease. Thus, our studies demonstrated that central tolerance can paradoxically result in systemic autoimmunity through differential susceptibility of Tregs and autoreactive T cells to thymic deletion. Therefore, too little or too much negative selection to a self-antigen can result in systemic autoimmunity and disease.

Show MeSH
Related in: MedlinePlus