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Control of amino-acid transport by antigen receptors coordinates the metabolic reprogramming essential for T cell differentiation.

Sinclair LV, Rolf J, Emslie E, Shi YB, Taylor PM, Cantrell DA - Nat. Immunol. (2013)

Bottom Line: T cells responded to antigen by upregulating expression of many amino-acid transporters, but a single System L ('leucine-preferring system') transporter, Slc7a5, mediated uptake of LNAAs in activated T cells.The metabolic catastrophe caused by loss of Slc7a5 reflected the requirement for sustained uptake of the LNAA leucine for activation of the serine-threonine kinase complex mTORC1 and for expression of the transcription factor c-Myc.Control of expression of the System L transporter by pathogens is thus a critical metabolic checkpoint for T cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Signalling and Immunology, University of Dundee, Dundee, UK.

ABSTRACT
T lymphocytes must regulate nutrient uptake to meet the metabolic demands of an immune response. Here we show that the intracellular supply of large neutral amino acids (LNAAs) in T cells was regulated by pathogens and the T cell antigen receptor (TCR). T cells responded to antigen by upregulating expression of many amino-acid transporters, but a single System L ('leucine-preferring system') transporter, Slc7a5, mediated uptake of LNAAs in activated T cells. Slc7a5- T cells were unable to metabolically reprogram in response to antigen and did not undergo clonal expansion or effector differentiation. The metabolic catastrophe caused by loss of Slc7a5 reflected the requirement for sustained uptake of the LNAA leucine for activation of the serine-threonine kinase complex mTORC1 and for expression of the transcription factor c-Myc. Control of expression of the System L transporter by pathogens is thus a critical metabolic checkpoint for T cells.

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Slc7a5fl/flCD4-Cre mice(a) PCR analysis of genomic DNA isolated from the indicate thymocytes showing Slc7a5 floxed (FL), wild-type (WT) and deleted (DEL) PCR products. (b) Thymocyte (left) and thymocyte subset (middle) numbers of Slc7a5fl/fl and Slc7a5fl/flCD4-Cre mice, the right panel shows flow cytometric analysis of NKT cells from the indicated mice (c) CD98 in thymic subsets. (d,e) Cellular analysis of spleen and lymph node from Slc7a5fl/fl and Slc7a5fl/flCD4-Cre mice. (d) Flow cytometry analysis of CD98, CD62L and CD44 levels in lymph node T cells. (e) Total numbers of spleen (left) and brachial lymph node cells (center left). Flow cytometry analysis of CD4 and CD8 expression by lymph node T cells (center right) or Foxp3 and CD25 expression by splenic T cells (right). (f) immunoblot analysis with Slc7a5 antibodies of naïve and 20 h TCR stimulated Slc7a5fl/fl and Slc7a5fl/flCD4-Cre CD8+ T cells. (g)3H-phenylalanine uptake by Slc7a5fl/fl and Slc7a5fl/flCD4-Cre CD8+ TCR stimulated (20 h) T cells, p=0.0003. Uptake performed in the presence or absence of cold competitor 10 mM Leu to quench. (b-e). Representative data from 8-12 week mice, 3 mice per group; (f) representative of 3 experiments. SMC1 is loading control. (g) 3 mice per group.
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Figure 4: Slc7a5fl/flCD4-Cre mice(a) PCR analysis of genomic DNA isolated from the indicate thymocytes showing Slc7a5 floxed (FL), wild-type (WT) and deleted (DEL) PCR products. (b) Thymocyte (left) and thymocyte subset (middle) numbers of Slc7a5fl/fl and Slc7a5fl/flCD4-Cre mice, the right panel shows flow cytometric analysis of NKT cells from the indicated mice (c) CD98 in thymic subsets. (d,e) Cellular analysis of spleen and lymph node from Slc7a5fl/fl and Slc7a5fl/flCD4-Cre mice. (d) Flow cytometry analysis of CD98, CD62L and CD44 levels in lymph node T cells. (e) Total numbers of spleen (left) and brachial lymph node cells (center left). Flow cytometry analysis of CD4 and CD8 expression by lymph node T cells (center right) or Foxp3 and CD25 expression by splenic T cells (right). (f) immunoblot analysis with Slc7a5 antibodies of naïve and 20 h TCR stimulated Slc7a5fl/fl and Slc7a5fl/flCD4-Cre CD8+ T cells. (g)3H-phenylalanine uptake by Slc7a5fl/fl and Slc7a5fl/flCD4-Cre CD8+ TCR stimulated (20 h) T cells, p=0.0003. Uptake performed in the presence or absence of cold competitor 10 mM Leu to quench. (b-e). Representative data from 8-12 week mice, 3 mice per group; (f) representative of 3 experiments. SMC1 is loading control. (g) 3 mice per group.

Mentions: Systemic deletion of Slc7a5 causes embryonic lethality (unpublished data). However, mice with a single functional Slc7a5 allele deleted are born at normal Mendelian frequency and have normal peripheral lymphocyte subpopulations (data not shown). T cells haplo-insufficient for Slc7a5 underwent normal blastogenesis and proliferation in response to TCR triggering and IL-2. Slc7a5+/− T cells showed the expected reduction in Slc7a5 mRNA and protein expression and also had a 50% reduction in System L transporter activity while maintaining normal glutamine uptake (Supplementary Fig. 1). To further probe the importance of Slc7a5 in T cells we generated Slc7a5fl/flCD4-Cre mice to delete the Slc7a5 gene in CD4+CD8+ double-positive (DP) thymocytes and all subsequent T cell populations. The deletion of Slc7a5 alleles in Slc7a5fl/flCD4-Cre T cells was confirmed by genomic PCR analysis (Fig. 4a). Slc7a5fl/flCD4-Cre mice had normal numbers and frequency of conventional αβ T cells and NKT cells in the thymus (Fig. 4b). Slc7a5 forms a heterodimer with CD98 and one question we wished to address was the impact of Slc7a5 deletion on CD98 expression. Slc7a5fl/flCD4-Cre DP and single-positive (SP) thymocytes expressed approximately 50% less CD98 as compared to wild-type cells (Fig. 4c), whereas CD98 levels were reduced 2 fold in Slc7a5 peripheral T cells compared to control cells (Fig. 4d). Slc7a5fl/flCD4-Cre mice also had normal numbers and frequency of naïve CD4+ and CD8+ T cell subsets in the spleen and lymph node and a normal frequency of FoxP3 regulatory T cells (Fig. 4e). We also used a Vav-Cre transgene to delete Slc7a5 in hematopoietic progenitors in the bone marrow30. These Slc7a5fl/flVav-Cre mice had normal thymocyte numbers and a normal distribution of CD4 and CD8 double-negative, DP and SP subsets. They also had normal numbers and frequency of peripheral T lymphocyte subpopulations, B lymphocytes and NK cells (Supplementary Fig. 2).


Control of amino-acid transport by antigen receptors coordinates the metabolic reprogramming essential for T cell differentiation.

Sinclair LV, Rolf J, Emslie E, Shi YB, Taylor PM, Cantrell DA - Nat. Immunol. (2013)

Slc7a5fl/flCD4-Cre mice(a) PCR analysis of genomic DNA isolated from the indicate thymocytes showing Slc7a5 floxed (FL), wild-type (WT) and deleted (DEL) PCR products. (b) Thymocyte (left) and thymocyte subset (middle) numbers of Slc7a5fl/fl and Slc7a5fl/flCD4-Cre mice, the right panel shows flow cytometric analysis of NKT cells from the indicated mice (c) CD98 in thymic subsets. (d,e) Cellular analysis of spleen and lymph node from Slc7a5fl/fl and Slc7a5fl/flCD4-Cre mice. (d) Flow cytometry analysis of CD98, CD62L and CD44 levels in lymph node T cells. (e) Total numbers of spleen (left) and brachial lymph node cells (center left). Flow cytometry analysis of CD4 and CD8 expression by lymph node T cells (center right) or Foxp3 and CD25 expression by splenic T cells (right). (f) immunoblot analysis with Slc7a5 antibodies of naïve and 20 h TCR stimulated Slc7a5fl/fl and Slc7a5fl/flCD4-Cre CD8+ T cells. (g)3H-phenylalanine uptake by Slc7a5fl/fl and Slc7a5fl/flCD4-Cre CD8+ TCR stimulated (20 h) T cells, p=0.0003. Uptake performed in the presence or absence of cold competitor 10 mM Leu to quench. (b-e). Representative data from 8-12 week mice, 3 mice per group; (f) representative of 3 experiments. SMC1 is loading control. (g) 3 mice per group.
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Figure 4: Slc7a5fl/flCD4-Cre mice(a) PCR analysis of genomic DNA isolated from the indicate thymocytes showing Slc7a5 floxed (FL), wild-type (WT) and deleted (DEL) PCR products. (b) Thymocyte (left) and thymocyte subset (middle) numbers of Slc7a5fl/fl and Slc7a5fl/flCD4-Cre mice, the right panel shows flow cytometric analysis of NKT cells from the indicated mice (c) CD98 in thymic subsets. (d,e) Cellular analysis of spleen and lymph node from Slc7a5fl/fl and Slc7a5fl/flCD4-Cre mice. (d) Flow cytometry analysis of CD98, CD62L and CD44 levels in lymph node T cells. (e) Total numbers of spleen (left) and brachial lymph node cells (center left). Flow cytometry analysis of CD4 and CD8 expression by lymph node T cells (center right) or Foxp3 and CD25 expression by splenic T cells (right). (f) immunoblot analysis with Slc7a5 antibodies of naïve and 20 h TCR stimulated Slc7a5fl/fl and Slc7a5fl/flCD4-Cre CD8+ T cells. (g)3H-phenylalanine uptake by Slc7a5fl/fl and Slc7a5fl/flCD4-Cre CD8+ TCR stimulated (20 h) T cells, p=0.0003. Uptake performed in the presence or absence of cold competitor 10 mM Leu to quench. (b-e). Representative data from 8-12 week mice, 3 mice per group; (f) representative of 3 experiments. SMC1 is loading control. (g) 3 mice per group.
Mentions: Systemic deletion of Slc7a5 causes embryonic lethality (unpublished data). However, mice with a single functional Slc7a5 allele deleted are born at normal Mendelian frequency and have normal peripheral lymphocyte subpopulations (data not shown). T cells haplo-insufficient for Slc7a5 underwent normal blastogenesis and proliferation in response to TCR triggering and IL-2. Slc7a5+/− T cells showed the expected reduction in Slc7a5 mRNA and protein expression and also had a 50% reduction in System L transporter activity while maintaining normal glutamine uptake (Supplementary Fig. 1). To further probe the importance of Slc7a5 in T cells we generated Slc7a5fl/flCD4-Cre mice to delete the Slc7a5 gene in CD4+CD8+ double-positive (DP) thymocytes and all subsequent T cell populations. The deletion of Slc7a5 alleles in Slc7a5fl/flCD4-Cre T cells was confirmed by genomic PCR analysis (Fig. 4a). Slc7a5fl/flCD4-Cre mice had normal numbers and frequency of conventional αβ T cells and NKT cells in the thymus (Fig. 4b). Slc7a5 forms a heterodimer with CD98 and one question we wished to address was the impact of Slc7a5 deletion on CD98 expression. Slc7a5fl/flCD4-Cre DP and single-positive (SP) thymocytes expressed approximately 50% less CD98 as compared to wild-type cells (Fig. 4c), whereas CD98 levels were reduced 2 fold in Slc7a5 peripheral T cells compared to control cells (Fig. 4d). Slc7a5fl/flCD4-Cre mice also had normal numbers and frequency of naïve CD4+ and CD8+ T cell subsets in the spleen and lymph node and a normal frequency of FoxP3 regulatory T cells (Fig. 4e). We also used a Vav-Cre transgene to delete Slc7a5 in hematopoietic progenitors in the bone marrow30. These Slc7a5fl/flVav-Cre mice had normal thymocyte numbers and a normal distribution of CD4 and CD8 double-negative, DP and SP subsets. They also had normal numbers and frequency of peripheral T lymphocyte subpopulations, B lymphocytes and NK cells (Supplementary Fig. 2).

Bottom Line: T cells responded to antigen by upregulating expression of many amino-acid transporters, but a single System L ('leucine-preferring system') transporter, Slc7a5, mediated uptake of LNAAs in activated T cells.The metabolic catastrophe caused by loss of Slc7a5 reflected the requirement for sustained uptake of the LNAA leucine for activation of the serine-threonine kinase complex mTORC1 and for expression of the transcription factor c-Myc.Control of expression of the System L transporter by pathogens is thus a critical metabolic checkpoint for T cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Signalling and Immunology, University of Dundee, Dundee, UK.

ABSTRACT
T lymphocytes must regulate nutrient uptake to meet the metabolic demands of an immune response. Here we show that the intracellular supply of large neutral amino acids (LNAAs) in T cells was regulated by pathogens and the T cell antigen receptor (TCR). T cells responded to antigen by upregulating expression of many amino-acid transporters, but a single System L ('leucine-preferring system') transporter, Slc7a5, mediated uptake of LNAAs in activated T cells. Slc7a5- T cells were unable to metabolically reprogram in response to antigen and did not undergo clonal expansion or effector differentiation. The metabolic catastrophe caused by loss of Slc7a5 reflected the requirement for sustained uptake of the LNAA leucine for activation of the serine-threonine kinase complex mTORC1 and for expression of the transcription factor c-Myc. Control of expression of the System L transporter by pathogens is thus a critical metabolic checkpoint for T cells.

Show MeSH
Related in: MedlinePlus