T cell factor-1 controls the lifetime of CD4+ CD8+ thymocytes in vivo and distal T cell receptor α-chain rearrangement required for NKT cell development.

Natural killer T (NKT) cells are a component of innate and adaptive immune systems implicated in immune, autoimmune responses and in the control of obesity and cancer. NKT cells develop from common CD4+ CD8+ double positive (DP) thymocyte precursors after the rearrangement and expression of T cell receptor (TCR) Vα14-Jα18 gene. Temporal regulation and late appearance of Vα14-Jα18 rearrangement in immature DP thymocytes has been demonstrated. However, the precise control of lifetime of DP thymocytes in vivo that enables distal rearrangements remains incompletely defined. Here we demonstrate that T cell factor (TCF)-1, encoded by the Tcf7 gene, is critical for the extended lifetime of DP thymocytes. TCF-1-deficient DP thymocytes fail to undergo TCR Vα14-Jα18 rearrangement and produce significantly fewer NKT cells. Ectopic expression of Bcl-xL permits Vα14-Jα18 rearrangement and rescues NKT cell development. We report that TCF-1 regulates expression of RORγt, which regulates DP thymocyte survival by controlling expression of Bcl-xL. We posit that TCF-1 along with its cofactors controls the lifetime of DP thymocytes in vivo

Ectopic expression of Bcl-x_L in developing TCF-1-deficient thymocytes rescues Vα14-Jα18 rearrangements and NKT cells.

<p>(<b>A</b>) Flow cytometry of thymocytes from WT, TCF-1-KO, Bcl-x_L-Tg and TCF-1-KO Bcl-x_L-Tg mice showing CD4⁻CD8⁻ (double-negative, DN), CD4⁺CD8⁺ (double positive, DP), CD4 single-positive (SP) and CD8SP thymocytes. <b>Top</b>, dot plots are representative of at least 4 different experiments. <b>Bottom</b>, graphs with DP cell percentages and cell numbers from 4 mice per group are shown (mean and s.e.m.). Numbers over dot plots refer to total thymocyte cell numbers. (<b>B</b>) Flow cytometry of thymocytes showing percent of gated CD1d-tetramer+ TCRβ+ NKT cells from WT, Bcl-x_L-Tg and TCF-1-KO Bcl-x_L-Tg mice. <b>Top</b>, dot plots are representative of at least 4 different experiments. <b>Bottom</b>, graphs with NKT cell percentages and cell numbers from 4 mice per group are shown (mean and s.e.m.). (<b>C</b>) Relative expression of Vα14-Jα18 rearrangements from WT and TCF-1-KO DP cells (n = 3). (<b>D</b>) Semiquantitative PCR of cDNA (1∶1, 1∶2, 1∶4 dilutions) from DP cells of WT, TCF-1-KO, Bcl-x_L-Tg and TCF-1-KO Bcl-x_L-Tg mice showing Vα14-Jα18 and control Vα14-Cα rearrangements (n = 3). *<i>P</i><.05; ***, <i>P</i><.001.</p

TCF-1 is expressed in NKT cells in a developmentally relevant manner.

<p>(<b>A</b>) Flow cytometry of thymocytes (<b>top</b>) and hepatic lymphocytes (<b>bottom</b>) from wild-type (WT) C57BL/6 mice showing TCF-1 intracellular expression versus the control antibody (<b>right</b>) in NKT cells (gated in <b>left</b> as CD1d-tetramer+ TCRβ+ cells, numbers indicate percentage of NKT cells). (<b>B</b>) Flow cytometry of CD1d-tetramer positive thymocytes (<b>top</b>) and hepatic lymphocytes (<b>bottom</b>) from WT mice stained with anti-CD44 and anti-NK1.1 to assess NKT developmental stages 1–3, as depicted in <b>left</b>. <b>Right</b>, TCF-1 intracellular expression among stages. Data are from a representative experiment out of four WT mice analyzed. (<b>C</b>) Flow cytometry of CD1d-tetramer positive thymocytes (<b>top</b>) and hepatic lymphocytes (<b>bottom</b>) from WT mice showing TCF1 intracellular expression versus the control antibody (<b>right</b>) in NKT1 and NKT2 NKT cells (gated in <b>left</b>, numbers indicate percentage of NKT cells). Data are from a representative experiment out of four WT mice analyzed.</p

TCF-1-deficient mice have reduced proportion and number of NKT cells.

<p>Flow cytometry of thymocytes showing percent of gated CD1d-tetramer+ TCRβ+ NKT cells from wild-type (WT) and TCF-1 knock-out (KO) mice (<b>A</b>) and from WT, Vα14-Tg and TCF-1-KO Vα14-Tg mice (<b>B</b>). Representative dot plots and graphs with cell percentages and cell numbers from 4–6 mice per group are shown (mean and s.e.m.). Numbers over dot plots refer to total thymocyte cell numbers; *<i>P</i><.05; **, <i>P</i><.01; ***, <i>P</i><.001.</p

Transcription factors and target genes of pre-TCR signaling

Almost 30 years ago pioneering work by the laboratories of Harald von Boehmer and Susumo Tonegawa provided the first indications that developing thymocytes could assemble a functional TCRβ chain-containing receptor complex, the pre-TCR, before TCRα expression. The discovery and study of the pre-TCR complex revealed paradigms of signaling pathways in control of cell survival and proliferation, and culminated in the recognition of the multifunctional nature of this receptor. As a receptor integrated in a dynamic developmental process, the pre-TCR must be viewed not only in the light of the biological outcomes it promotes, but also in context with those molecular processes that drive its expression in thymocytes. This review article focuses on transcription factors and target genes activated by the pre-TCR to drive its different outcomes.Work in CL-R and JA laboratory has been supported by the Ramón y Cajal and I3 Researchers Programs (CL-R), research grants from the Spanish Government (SAF2009-08066, SAF2012-36535 to CL-R; and BFU2008-01070, SAF2011-24268 to JA), Fundació la Marató TV3 (080730, 122530 to CL-R and JA), the Marie Curie International Reintegration Program of the European Union (MCIRG516308 to CL-R), the Spanish Ministry of Health (ISCIII-RETIC RD06/0009-FEDER), and Generalitat de Catalunya (2009SGR601, 2014SGR1153). CL-R is a recipient of the ICREA Acadèmia Award (Generalitat de Catalunya)

Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5

Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) β activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.C. López-Rodríguez was supported by the Ramón y Cajal and I3 Researchers Programmes and grants from the Spanish Government (SAF2006-04913 and SAF2009-08066) and European Union (MCIRG516308). J. Aramburu was supported by grants from the Spanish Government (BFU2008-01070 and SAF2011-24268), and Distinció de la Generalitat de Catalunya per a la Promoció de la Recerca Universitària. Research in the laboratories of C. López-Rodríguez and J. Aramburu is also supported by Fundació la Marató TV3 (030230/31, 080730), Spanish Ministry of Health (ISCIII-RETIC RD06/0009-FEDER), and Generalitat de Catalunya (2009 SGR 601). M. Buxadé was supported by a Beatriu de Pinós postdoctoral contract from Generalitat de Catalunya. S. Iborra was supported by a Sara Borrell postdoctoral contract from the Ministry of Health of Spain. J. Minguillón and R. Berga-Bolaños were supported by predoctoral fellowships from the Spanish Government

Gene expression induced by Toll-like receptors in macrophages requires the transcription factor NFAT5

Toll-like receptors (TLRs) engage networks of transcriptional regulators to induce genes essential for antimicrobial immunity. We report that NFAT5, previously characterized as an osmostress responsive factor, regulates the expression of multiple TLR-induced genes in macrophages independently of osmotic stress. NFAT5 was essential for the induction of the key antimicrobial gene Nos2 (inducible nitric oxide synthase [iNOS]) in response to low and high doses of TLR agonists but is required for Tnf and Il6 mainly under mild stimulatory conditions, indicating that NFAT5 could regulate specific gene patterns depending on pathogen burden intensity. NFAT5 exhibited two modes of association with target genes, as it was constitutively bound to Tnf and other genes regardless of TLR stimulation, whereas its recruitment to Nos2 or Il6 required TLR activation. Further analysis revealed that TLR-induced recruitment of NFAT5 to Nos2 was dependent on inhibitor of κB kinase (IKK) β activity and de novo protein synthesis, and was sensitive to histone deacetylases. In vivo, NFAT5 was necessary for effective immunity against Leishmania major, a parasite whose clearance requires TLRs and iNOS expression in macrophages. These findings identify NFAT5 as a novel regulator of mammalian anti-pathogen responses.C. López-Rodríguez was supported by the Ramón y Cajal and I3 Researchers Programmes and grants from the Spanish Government (SAF2006-04913 and SAF2009-08066) and European Union (MCIRG516308). J. Aramburu was supported by grants from the Spanish Government (BFU2008-01070 and SAF2011-24268), and Distinció de la Generalitat de Catalunya per a la Promoció de la Recerca Universitària. Research in the laboratories of C. López-Rodríguez and J. Aramburu is also supported by Fundació la Marató TV3 (030230/31, 080730), Spanish Ministry of Health (ISCIII-RETIC RD06/0009-FEDER), and Generalitat de Catalunya (2009 SGR 601). M. Buxadé was supported by a Beatriu de Pinós postdoctoral contract from Generalitat de Catalunya. S. Iborra was supported by a Sara Borrell postdoctoral contract from the Ministry of Health of Spain. J. Minguillón and R. Berga-Bolaños were supported by predoctoral fellowships from the Spanish Government