Elena M. Gallo

Lymphocyte calcium signaling from membrane to nucleus.

Ca2+ signals control a variety of lymphocyte responses, ranging from short-term cytoskeletal modifications to long-term changes in gene expression. The identification of molecules and channels that modulate Ca2+ entry into T and B lymphocytes has both provided details of the molecular events leading to immune responses and raised controversy. Here we review studies of the pathways that allow Ca2+ entry, the function of Ca2+ in the regulation of cell polarity and motility and the principles by which Ca2+-dependent transcription regulates lymphocyte function.

The gene encoding early growth response 2, a target of the transcription factor NFAT, is required for the development and maturation of natural killer T cells

The influence of signals transmitted by the phosphatase calcineurin and the transcription factor NFAT on the development and function of natural killer T (NKT) cells is unclear. In this report, we demonstrate that the transcription factor early growth response 2 (Egr2), a target gene of NFAT, was specifically required for the ontogeny of NKT cells but not that of conventional CD4+ or CD8+ T cells. NKT cells developed normally in the absence of Egr1 or Egr3, which suggests that Egr2 is a specific regulator of NKT cell differentiation. We found that Egr2 was important in the selection, survival and maturation of NKT cells. Our findings emphasize the importance of the calcineurin-NFAT-Egr2 pathway in the development of the NKT lymphocyte lineage.

Murine CD4+CD25+ Regulatory T Cells Fail to Undergo Chromatin Remodeling Across the Proximal Promoter Region of the IL-2 Gene

CD4+CD25+ regulatory T cells (Treg) acquire unique immunosuppressive properties while maintaining an anergy phenotype when activated in vitro under conditions that induce IL-2 production and proliferation in conventional CD4+ T cells. We investigated the mechanism underlying one component of this naturally anergic phenotype, the inability of the Treg cells to produce IL-2 following activation. Analysis of freshly isolated murine CD4+CD25+ Treg and conventional CD4+CD25− T cells following PMA/ionomycin stimulation demonstrated no differences in inducible AP-1 formation, an important transcriptional complex in regulating IL-2 gene expression. Although p38 MAPK and ERK1/2 protein kinases were phosphorylated with similar kinetics, we observed diminished activation of JNK in the CD4+CD25+ Treg cells. However, lentiviral-mediated reconstitution of the JNK pathway using a constitutively active construct did not overcome the block in IL-2 synthesis. Using a PCR-based chromatin accessibility assay we found that the minimal IL-2 promoter region of CD4+CD25+ Treg cells, unlike conventional CD4 T cells, did not undergo chromatin remodeling following stimulation, suggesting that the inability of CD4+CD25+ Treg cells to secrete IL-2 following activation is controlled at the chromatin level.

OX40 Ligand-Transduced Tumor Cell Vaccine Synergizes with GM-CSF and Requires CD40-Apc Signaling to Boost the Host T Cell Antitumor Response

Efficient T cell priming by GM-CSF and CD40 ligand double-transduced C26 murine colon carcinoma is not sufficient to cure metastases in a therapeutic setting. To determine whether a cellular vaccine that interacts directly with both APC and T cells in vivo might be superior, we generated C26 carcinoma cells transduced with the T cell costimulatory molecule OX40 ligand (OX40L) either alone (C26/OX40L) or together with GM-CSF (C26/GM/OX40L), which is known to activate APC. Mice injected with C26/OX40L cells displayed only a delay in tumor growth, while the C26/GM/OX40L tumor regressed in 85% of mice. Tumor rejection required granulocytes, CD4+, CD8+ T cells, and APC-mediated CD40-CD40 ligand cosignaling, but not IFN-γ or IL-12 as shown using subset-depleted and knockout (KO) mice. CD40KO mice primed with C26/GM/OX40L cells failed to mount a CTL response, and T cells infiltrating the C26/GM/OX40L tumor were OX40 negative, suggesting an impairment in APC-T cell cross-talk in CD40KO mice. Indeed, CD4+ T cell-depleted mice failed to mount any CTL activity against the C26 tumor, while treatment with agonistic mAb to CD40, which acts on APC, bypassed the requirement for CD4+ T cells and restored CTL activation. C26/GM/OX40L cells cured 83% of mice bearing lung metastases, whereas C26/OX40L or C26/GM vaccination cured only 28 and 16% of mice, respectively. These results indicate the synergistic activity of OX40L and GM-CSF in a therapeutic setting.

Thymocyte negative selection is mediated by protein kinase C- and Ca2+-dependent transcriptional induction of Bim of cell death.

The processes of positive and negative selection in the thymus both determine the population of T cells that will enter the peripheral immune system and eliminate self-reactive T cells by apoptosis. Substantial evidence indicates that TCR signal intensity mediates this cell fate choice: low-intensity signals lead to survival and differentiation, whereas high-intensity signals generated by self-Ag lead to cell death. The molecular mechanism by which these graded signals are converted to discrete outcomes is not understood. Positive selection requires the Ca2+-dependent phosphatase calcineurin, whereas negative selection requires the proapoptotic Bcl-2 family member Bcl-2-interacting mediator of cell death (Bim). In this study, we investigated the regulation of Bim expression and the role of Ca2+ in mediating negative selection. Our results show that transcription is necessary for both negative selection and Bim induction. Surprisingly, we also found that Ca2+ is necessary for Bim induction. Induction of bim transcription appears to involve protein kinase C, but not calcineurin, JNK, p38 MAPK, or MEK. These results localize the decision point in positive vs negative selection to a step downstream of Ca2+ signaling and suggest that negative selection signals induce Ca2+-dependent bim transcription through PKC.

Calcineurin sets the bandwidth for discrimination of signals during thymocyte development

At critical times in development, cells are able to convert graded signals into discrete developmental outcomes; however, the mechanisms involved are poorly understood. During thymocyte development, cell fate is determined by signals originating from the αβ T-cell receptor. Low-affinity/avidity interactions between the T-cell receptor and peptide–MHC complexes direct differentiation to the single-positive stage (positive selection), whereas high-affinity/avidity interactions induce death by apoptosis (negative selection). Here we show that mice deficient in both calcineurin and nuclear factor of activated T cells (NFAT)c2/c3 lack a population of preselection thymocytes with enhanced ability to activate the mitogen-activated protein kinase (Raf–MEK–ERK) pathway, and fail to undergo positive selection. This defect can be partially rescued with constitutively active Raf, indicating that calcineurin controls MAPK signalling. Analysis of mice deficient in both Bim (which is required for negative selection) and calcineurin revealed that calcineurin-induced ERK (extracellular signal-regulated kinase) sensitization is required for differentiation in response to ‘weak’ positive selecting signals but not in response to ‘strong’ negative selecting signals (which normally induce apoptosis). These results indicate that early calcineurin/NFAT signalling produces a developmental period of ERK hypersensitivity, allowing very weak signals to induce positive selection. This mechanism might be generally useful in the discrimination of graded signals that induce different cell fates.

Selective role of calcineurin in haematopoiesis and lymphopoiesis

The calcineurin/NFAT (nuclear factor of activated T‐cells) signalling pathway is essential for many aspects of vertebrate development and is the target of the widely used immunosuppressive drugs FK506 and cyclosporine A. The basis for the therapeutic specificity of these drugs has remained unclear, as calcineurin is expressed ubiquitously. By inactivating calcineurin during haematopoietic development, we found that although this signalling pathway has an important, non‐redundant role in the regulation of lymphocyte developmental checkpoints, it is not essential for the development of blood myeloid lineages. These studies have shown that the specificity of calcineurin inhibitors arises from the selective use of calcineurin at distinct developmental stages. The requirement for calcineurin/NFAT in the development of the adaptive but not of the innate immune system is consistent with the idea that the evolutionary appearance of this pathway was involved in the emergence of vertebrates.