Fernando Cruz-Guilloty

Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells.

Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (Prf1) transcription, and repressed re-expression of memory CTL markers Bcl6 and IL-7Ralpha. Binding of Eomes and STAT5 to Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Ralpha and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Ralpha repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Ralpha-deficient effector CD8(+) T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1(+) and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.

Biochemical and functional characterization of Orai proteins.

Stimulation of immune cells triggers Ca2+ entry through store-operated Ca2+ release-activated Ca2+ channels, promoting nuclear translocation of the transcription factor NFAT. Through genome-wide RNA interference screens in Drosophila, we and others identified olf186-F (Drosophila Orai, dOrai) and dStim as critical components of store-operated Ca2+ entry and showed that dOrai and its human homologue Orai1 are pore subunits of the Ca2+ release-activated Ca2+ channel. Here we report that Orai1 is predominantly responsible for store-operated Ca2+ influx in human embryonic kidney 293 cells and human T cells and fibroblasts, although its paralogue Orai3 can partly compensate in the absence of functional Orai1. All three mammalian Orai are widely expressed at the mRNA level, and all three are incorporated into the plasma membrane. In human embryonic kidney 293 cells, Orai1 is glycosylated at an asparagine residue in the predicted second extracellular loop, but mutation of the residue does not compromise function. STIM1 and Orai1 colocalize after store depletion, but Orai1 does not associate detectably with STIM1 in glycerol gradient centrifugation or coimmunoprecipitation experiments. Glutamine substitutions in two conserved glutamate residues, located within predicted transmembrane helices of Drosophila Orai and human Orai1, greatly diminish store-operated Ca2+ influx, and primary T cells ectopically expressing mutant E106Q and E190Q Orai1 proteins show reduced proliferation and cytokine secretion. Together, these data establish Orai1 as a predominant mediator of store-operated calcium entry, proliferation, and cytokine production in T cells.

Runx3 and T-box proteins cooperate to establish the transcriptional program of effector CTLs

Activation of naive CD8+ T cells with antigen induces their differentiation into effector cytolytic T lymphocytes (CTLs). CTLs lyse infected or aberrant target cells by exocytosis of lytic granules containing the pore-forming protein perforin and a family of proteases termed granzymes. We show that effector CTL differentiation occurs in two sequential phases in vitro, characterized by early induction of T-bet and late induction of Eomesodermin (Eomes), T-box transcription factors that regulate the early and late phases of interferon (IFN) γ expression, respectively. In addition, we demonstrate a critical role for the transcription factor Runx3 in CTL differentiation. Runx3 regulates Eomes expression as well as expression of three cardinal markers of the effector CTL program: IFN-γ, perforin, and granzyme B. Our data point to the existence of an elaborate transcriptional network in which Runx3 initially induces and then cooperates with T-box transcription factors to regulate gene transcription in differentiating CTLs.

The NFAT Family: Structure, Regulation, and Biological Functions

Publisher Summary This chapter sheds light on the structure, regulation, and biological functions of the NFAT family, which is a family of transcription factors. The primordial NFAT family member NFAT5/TonEBP is expressed ubiquitously in mammalian cells and regulates the response to hypertonic stress. NFAT5 is also likely to be involved in regulating diverse other biological programs. The DNA binding domains of all NFAT and NFкB/Rel family members have two domains, an N-terminal specificity domain involved in making base specific DNA contacts, and a C-terminal domain involved in dimer formation. Together these domains constitute the Rel homology region (RHR) common to all members of the extended NFAT/NFкB/Rel family. NFAT proteins can also function as dimeric transcription factors at quasi-palindromic sites that resemble NFкB binding sites. The calcium regulated NFAT proteins are activated by ligand binding to a variety of cell surface receptors. The common feature of the receptors is their ability to activate phosphatidylinositol specific phospholipase C (PLC), thereby inducing calcium influx across the plasma membrane. NFAT dependent gene transcription is exquisitely sensitive to changes in intracellular calcium concentration. Even in the continuous presence of stimulus, levels may oscillate depending on specific parameters of receptor occupancy and desensitization. Interaction with calcineurin is central to the calcium responsiveness of NFAT1-4. The major calcineurin docking site on NFAT is located at the N-terminus of the regulatory domain and has the consensus sequence PxIxIT. Substitution of the PxIxIT sequence of NFAT1 with a higher affinity version obtained by peptide selection increases the basal sensitivity of NFAT.

The NFAT Family

Publisher Summary This chapter sheds light on the structure, regulation, and biological functions of the NFAT family, which is a family of transcription factors. The primordial NFAT family member NFAT5/TonEBP is expressed ubiquitously in mammalian cells and regulates the response to hypertonic stress. NFAT5 is also likely to be involved in regulating diverse other biological programs. The DNA binding domains of all NFAT and NFкB/Rel family members have two domains, an N-terminal specificity domain involved in making base specific DNA contacts, and a C-terminal domain involved in dimer formation. Together these domains constitute the Rel homology region (RHR) common to all members of the extended NFAT/NFкB/Rel family. NFAT proteins can also function as dimeric transcription factors at quasi-palindromic sites that resemble NFкB binding sites. The calcium regulated NFAT proteins are activated by ligand binding to a variety of cell surface receptors. The common feature of the receptors is their ability to activate phosphatidylinositol specific phospholipase C (PLC), thereby inducing calcium influx across the plasma membrane. NFAT dependent gene transcription is exquisitely sensitive to changes in intracellular calcium concentration. Even in the continuous presence of stimulus, levels may oscillate depending on specific parameters of receptor occupancy and desensitization. Interaction with calcineurin is central to the calcium responsiveness of NFAT1-4. The major calcineurin docking site on NFAT is located at the N-terminus of the regulatory domain and has the consensus sequence PxIxIT. Substitution of the PxIxIT sequence of NFAT1 with a higher affinity version obtained by peptide selection increases the basal sensitivity of NFAT.

Chapter 254 – The NFAT Family: Structure, Regulation, and Biological Functions

Publisher Summary This chapter sheds light on the structure, regulation, and biological functions of the NFAT family, which is a family of transcription factors. The primordial NFAT family member NFAT5/TonEBP is expressed ubiquitously in mammalian cells and regulates the response to hypertonic stress. NFAT5 is also likely to be involved in regulating diverse other biological programs. The DNA binding domains of all NFAT and NFкB/Rel family members have two domains, an N-terminal specificity domain involved in making base specific DNA contacts, and a C-terminal domain involved in dimer formation. Together these domains constitute the Rel homology region (RHR) common to all members of the extended NFAT/NFкB/Rel family. NFAT proteins can also function as dimeric transcription factors at quasi-palindromic sites that resemble NFкB binding sites. The calcium regulated NFAT proteins are activated by ligand binding to a variety of cell surface receptors. The common feature of the receptors is their ability to activate phosphatidylinositol specific phospholipase C (PLC), thereby inducing calcium influx across the plasma membrane. NFAT dependent gene transcription is exquisitely sensitive to changes in intracellular calcium concentration. Even in the continuous presence of stimulus, levels may oscillate depending on specific parameters of receptor occupancy and desensitization. Interaction with calcineurin is central to the calcium responsiveness of NFAT1-4. The major calcineurin docking site on NFAT is located at the N-terminus of the regulatory domain and has the consensus sequence PxIxIT. Substitution of the PxIxIT sequence of NFAT1 with a higher affinity version obtained by peptide selection increases the basal sensitivity of NFAT.