Cutting Edge: Polycomb Repressive Complex 1 Subunit Cbx4 Positively Regulates Effector Responses in CD8 T Cells.

CTL differentiation is controlled by the crosstalk of various transcription factors and epigenetic modulators. Uncovering this process is fundamental to improving immunotherapy and designing novel therapeutic approaches. In this study, we show that polycomb repressive complex 1 subunit chromobox (Cbx)4 favors effector CTL differentiation in a murine model. Cbx4 deficiency in CTLs induced a transcriptional signature of memory cells and increased the memory CTL population during acute viral infection. It has previously been shown that besides binding to H3K27me3 through its chromodomain, Cbx4 functions as a small ubiquitin-like modifier (SUMO) E3 ligase in a SUMO-interacting motifs (SIM)-dependent way. Overexpression of Cbx4 mutants in distinct domains showed that this protein regulates CTL differentiation primarily in an SIM-dependent way and partially through its chromodomain. Our data suggest a novel role of a polycomb group protein Cbx4 controlling CTL differentiation and indicated SUMOylation as a key molecular mechanism connected to chromatin modification in this process.

Differentiation of Memory CD8 T Cells Unravel Gene Expression Pattern Common to Effector and Memory Precursors.

Long-term immunological protection relies on the differentiation and maintenance of memory lymphocytes. Since the knowledge of memory generation has been centered on in vivo models of infection, there are obstacles to deep molecular analysis of differentiating subsets. Here we defined a novel in vitro CD8 T cell activation and culture regimen using low TCR engagement and cytokines to generate differentiated cells consistent with central memory-like cells, as shown by surface phenotype, gene expression profile and lack of cytotoxic function after challenge. Our results showed an effector signature expressed by in vitro memory precursors and their plasticity under specific conditions. Moreover, memory CD8 T cells conferred long-term protection against bacterial infection and slowed in vivo tumor growth more efficiently than effector cells. This model may allow further understanding of CD8 T cell memory molecular differentiation subsets and be suited for generating cells to be used for immunotherapy.

Leptin activation of mTOR pathway in intestinal epithelial cell triggers lipid droplet formation, cytokine production and increased cell proliferation.

Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-ß production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-ß production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma.

Cyclosporin A inhibits colon cancer cell growth independently of the calcineurin pathway.

Chronic inflammation is a risk factor for the development of colon cancer, providing genotoxic insults, growth and pro-angiogenic factors that can promote tumorigenesis and tumor growth. Immunomodulatory agents can interfere with the inflammation that feeds cancer, but their impact on the transformed cell is poorly understood. The calcium/calcineurin signaling pathway, through activation of NFAT, is essential for effective immune responses, and its inhibitors cyclosporin A (CsA) and FK506 are used in the clinics to suppress immunity. Moreover, the kinases GSK3ß and mTOR, modulated by PI-3K/Akt, can inhibit NFAT activity, suggesting a cross-talk between the calcium and growth factor signaling pathways. Both NFAT and mTOR activity have been associated with tumorigenesis. We therefore investigated the impact of calcineurin and PI-3K/mTOR inhibition in growth of human colon carcinoma cells. We show that despite the efficient inhibition of NFAT1 activity, FK506 promotes tumor growth, whereas CsA inhibits it due to a delay in cell cycle progression and induction of necroptosis. We found NFκB activation and mTORC1 activity not to be altered by CsA or FK506. Similarly, changes to mitochondrial homeostasis were equivalent upon treatment with these drugs. We further show that, in our model, NFAT1 activation is not modulated by PI3K/mTOR. We conclude that CsA slows cell cycle progression and induces necroptosis of human carcinoma cell lines in a TGFß-, NFAT-, NFκB- and PI3K/mTOR-independent fashion. Nevertheless, our data suggest that CsA, in addition to its anti-inflammatory capacity, may target transformed colon and esophagus carcinoma cells without affecting non-transformed cells, promoting beneficial tumoristatic effects.

TCR-dependent transformation of mature memory phenotype T cells in mice.

A fundamental goal in cancer research is the identification of the cell types and signaling pathways capable of initiating and sustaining tumor growth, as this has the potential to reveal therapeutic targets. Stem and progenitor cells have been implicated in the genesis of select lymphoid malignancies. However, the identity of the cells in which mature lymphoid neoplasms are initiated remains unclear. Here, we investigate the origin of peripheral T cell lymphomas using mice in which Snf5, a chromatin remodelling-complex subunit with tumor suppressor activity, could be conditionally inactivated in developing T cells. In this model of mature peripheral T cell lymphomas, the cell of origin was a mature CD44hiCD122loCD8⁺ T cell that resembled a subset of memory cells that has capacity for self-renewal and robust expansion, features shared with stem cells. Further analysis showed that Snf5 loss led to activation of a Myc-driven signaling network and stem cell transcriptional program. Finally, lymphomagenesis and lymphoma proliferation depended upon TCR signaling, establishing what we believe to be a new paradigm for lymphoid malignancy growth. These findings suggest that the self-renewal and robust proliferative capacities of memory T cells are associated with vulnerability to oncogenic transformation. Our findings further suggest that agents that impinge upon TCR signaling may represent an effective therapeutic modality for this class of lethal human cancers.

NFAT1 transcription factor is central in the regulation of tissue microenvironment for tumor metastasis.

Members of the nuclear factor of activated T cell (NFAT) family of transcription factors were originally described in T lymphocytes but later shown to be expressed in several immune and non-immune cell types. NFAT proteins can modulate cellular transformation intrinsically, and NFAT-deficient (NFAT1-/-) mice are indeed more susceptible to transformation than wild-type counterparts. However, the contribution of an NFAT1-/- microenvironment to tumor progression has not been studied. We have addressed this question by inoculating NFAT1-/- mice with B16F10 melanoma cells intravenously, an established model of tumor homing and growth. Surprisingly, NFAT1-/- animals sustained less tumor growth in the lungs after melanoma inoculation than wild-type counterparts. Even though melanoma cells equally colonize NFAT1-/- and wild-type lungs, tumors do not progress in the absence of NFAT1 expression. A massive mononuclear perivascular infiltrate and reduced expression of TGF-ß in the absence of NFAT1 suggested a role for tumor-infiltrating immune cells and the cytokine milieu. However, these processes are independent of an IL-4-induced regulatory tumor microenvironment, since lack of this cytokine does not alter the phenotype in NFAT1-/- animals. Bone marrow chimera experiments meant to differentiate the contributions of stromal and infiltrating cells to tumor progression demonstrated that NFAT1-induced susceptibility to pulmonary tumor growth depends on NFAT1-expressing parenchyma rather than on bone marrow-derived cells. These results suggest an important role for NFAT1 in radio-resistant tumor-associated parenchyma, which is independent of the anti-tumor immune response and Th1 versus Th2 cytokine milieu established by the cancer cells, but able to promote site-specific tumor growth.

Identification of dendritic antigen-presenting cells in the zebrafish.

In mammals, dendritic cells (DCs) form the key link between the innate and adaptive immune systems. DCs act as immune sentries in various tissues and, upon encountering pathogen, engulf and traffic foreign antigen to secondary lymphoid tissues, stimulating antigen-specific T lymphocytes. Although DCs are of fundamental importance in orchestrating the mammalian immune response, their presence and function in nonmammalian vertebrates is largely unknown. Because teleosts possess one of the earliest recognizable adaptive immune systems, we sought to identify antigen-presenting cells (APCs) in the zebrafish to better understand the potential origins of DCs and their evolutionary relationship to lymphocytes. Here we present the identification and characterization of a zebrafish APC subset strongly resembling mammalian DCs. Rare DCs are present in various adult tissues, and can be enriched by their affinity for the lectin peanut agglutinin (PNA). We show that PNA(hi) myeloid cells possess the classical morphological features of mammalian DCs as revealed by histochemical and ultrastructural analyses, phagocytose-labeled bacterial preparations in vivo, and exhibit expression of genes associated with DC function and antigen presentation, including il12, MHC class II invariant chain iclp1, and csf1r. Importantly, we show that PNA(hi) cells can activate T lymphocytes in an antigen-dependent manner. Together, these studies suggest that the cellular constituents responsible for antigen presentation are remarkably conserved from teleosts to mammals, and indicate that the zebrafish may serve as a unique model to study the origin of APC subsets and their evolutionary role as the link between the innate and adaptive immune systems.

Constitutive JAK3 activation induces lymphoproliferative syndromes in murine bone marrow transplantation models.

The tyrosine kinase JAK3 plays a well-established role during normal lymphocyte development and is constitutively phosphorylated in several lymphoid malignancies. However, its contribution to lymphomagenesis remains elusive. In this study, we used the newly identified activating JAK3A572V mutation to elucidate the effect of constitutive JAK3 signaling on murine lymphopoiesis. In a bone marrow transplantation model, JAK3A572V induces an aggressive, fatal, and transplantable lymphoproliferative disorder characterized by the expansion of CD8(+)TCRalphabeta(+)CD44(+)CD122(+)Ly-6C(+) T cells that closely resemble an effector/memory T-cell subtype. Compared with wild-type counterparts, these cells show increased proliferative capacities in response to polyclonal stimulation, enhanced survival rates with elevated expression of Bcl-2, and increased production of interferon-gamma (IFNgamma) and tumor necrosis factor-alpha (TNFalpha), correlating with enhanced cytotoxic abilities against allogeneic target cells. Of interest, the JAK3A572V disease is epidermotropic and produces intraepidermal microabscesses. Taken together, these clinical features are reminiscent of those observed in an uncommon but aggressive subset of CD8(+) human cutaneous T-cell lymphomas (CTCLs). However, we also observed a CD4(+) CTCL-like phenotype when cells are transplanted in an MHC-I-deficient background. These data demonstrate that constitutive JAK3 activation disrupts T-cell homeostasis and induces lymphoproliferative diseases in mice.

Regulation of CD8+ regulatory T cells: Interruption of the NKG2A-Qa-1 interaction allows robust suppressive activity and resolution of autoimmune disease.

Regulation of autoreactive CD4 T cells is essential to maintain self-tolerance and prevent autoimmune disease. Although CD8 T regulatory (Treg) cells that recognize self-peptides restricted by Qa-1 (HLA-E in humans) inhibit autoreactive CD4 cells and attenuate experimental autoimmune encephalomyelitis (EAE), the mechanism of this interaction is unclear. We generated Qa-1 mutant knock-in mice that impair Qa-1 binding to the T cell receptor (TCR) and CD94/NKG2A receptors. Analysis of these mice showed that TCR-dependent recognition of Qa-1-peptide complexes on target CD4 cells is essential for suppression by CD8 Treg cells. Further analysis revealed that genetic disruption of the Qa-1-CD94/NKG2A interaction unleashes robust CD8 Treg cell activity that completely abolishes development of EAE.

Dual roles for NFAT transcription factor genes as oncogenes and tumor suppressors.

Nuclear factor of activated T cells (NFAT) was first described as an activation and differentiation transcription factor in lymphocytes. Several in vitro studies suggest that NFAT family members are redundant proteins. However, analysis of mice deficient for NFAT proteins suggested different roles for the NFAT family of transcription factors in the regulation of cell proliferation and apoptosis. NFAT may also regulate several cell cycle and survival factors influencing tumor growth and survival. Here, we demonstrate that two constitutively active forms of NFAT proteins (CA-NFAT1 and CA-NFAT2 short isoform) induce distinct phenotypes in NIH 3T3 cells. Whereas CA-NFAT1 expression induces cell cycle arrest and apoptosis in NIH 3T3 fibroblasts, CA-NFAT2 short isoform leads to increased proliferation capacity and induction of cell transformation. Furthermore, NFAT1-deficient mice showed an increased propensity for chemical carcinogen-induced tumor formation, and CA-NFAT1 expression subverted the transformation of NIH 3T3 cells induced by the H-rasV12 oncogene. The differential roles for NFAT1 are at least partially due to the protein C-terminal domain. These results suggest that the NFAT1 gene acts as a tumor suppressor gene and the NFAT2 short isoform acts gene as an oncogene, supporting different roles for the two transcription factors in tumor development.

T-bet plays a key role in NK-mediated control of melanoma metastatic disease.

Antitumor responses depend on type 1 immunity, which is severely impaired in mice deficient for the T-box expressed in T cells (T-bet) transcription factor. Both T-bet-deficient (T-bet(-/-)) NK and CTL show defective function, which can be overcome by strong stimuli due to the expression of eomesodermin, another member of the T-box family. The effective response from T-bet(-/-) mice to viral infection and tumor initiation corroborates with these findings. However, T-bet(-/-) animals fail to control cancer metastasis and are, therefore, highly susceptible to tumor spread. The mechanism of T-bet-dependent resistance to metastatic disease is not known. In this study, we show that T-bet plays a role in inhibiting cancer metastasis by regulating the longevity and function of NK cells. Our data demonstrate that the absence of a proper innate immune response driven by NK cells in T-bet(-/-) mice precludes the initiation of a potent adaptive response to tumors. Adoptive transfer of wild-type activated NK cells protects T-bet(-/-) animals after melanoma challenge showing that reconstitution of the NK compartment in these mice is sufficient to mediate a significant reduction in tumor burden. Transfer of T-bet(-/-) A-NK cells fails to do so, due to their reduced in vivo survival, inefficient lysis of cancer cells, and poor IFN-gamma production. Taken together, these results show for the first time an irreplaceable role for T-bet in the NK-mediated cross-talk between innate and adaptive immune responses to metastatic disease.

Regulation of activated CD4+ T cells by NK cells via the Qa-1-NKG2A inhibitory pathway.

The ability of natural-killer cells to regulate adaptive immunity is not well understood. Here we define an interaction between the class Ib major histocompatibility complex (MHC) molecule Qa-1-Qdm on activated T cells responsible for adaptive immunity and CD94-NKG2A inhibitory receptors expressed by natural-killer cells by using Qa-1-deficient and Qa-1 knockin mice containing a point mutation that selectively abolishes Qa-1-Qdm binding to CD94-NKG2A receptors. The Qa-1-NKG2A interaction protected activated CD4+ T cells from lysis by a subset of NKG2A+ NK cells and was essential for T cell expansion and development of immunologic memory. Antibody-dependent blockade of this Qa-1-NKG2A interaction resulted in potent NK-dependent elimination of activated autoreactive T cells and amelioration of experimental autoimmune encephalomyelitis. These findings extend the functional reach of the NK system to include regulation of adaptive T cell responses and suggest a new clinical strategy for elimination of antigen-activated T cells in the context of autoimmune disease and transplantation.

The immunoregulatory effects of Qa-1.

The immune system is not only well equipped to control infections but also tightly controlled to prevent autoimmune disease. Despite the negative selection of T-cell clones in the thymus, mature T cells capable of recognizing self-antigens are present in every individual. Several types of specialized regulatory cells maintain homeostasis and prevent expansion of autoreactive T cells. In this issue of Immunological Reviews, the role of CD4+ regulatory T cells is extensively discussed. Suppression of T-cell responses by CD8+ T cells has received less attention. Here, we review research on Qa-1-restricted CD8+ regulatory T cells. We focus on the role of this class Ib major histocompatibility complex (MHC) molecule in both CD8+ regulatory T-cell activity and protection of activated T cells.

Osteopontin expression is essential for interferon-alpha production by plasmacytoid dendritic cells.

The observation that the T-bet transcription factor allows tissue-specific upregulation of intracellular osteopontin (Opn-i) in plasmacytoid dendritic cells (pDCs) suggests that Opn might contribute to the expression of interferon-alpha (IFN-alpha) in those cells. Here we show that Opn deficiency substantially reduced Toll-like receptor 9 (TLR9)-dependent IFN-alpha responses but spared expression of transcription factor NF-kappaB-dependent proinflammatory cytokines. Shortly after TLR9 engagement, colocalization of Opn-i and the adaptor molecule MyD88 was associated with induction of transcription factor IRF7-dependent IFN-alpha gene expression, whereas deficient expression of Opn-i was associated with defective nuclear translocation of IRF7 in pDCs. The importance of the Opn-IFN-alpha pathway was emphasized by its essential involvement in cross-presentation in vitro and in anti-herpes simplex virus 1 IFN-alpha response in vivo. The finding that Opn-i selectively coupled TLR9 signaling to expression of IFN-alpha but not to that of other proinflammatory cytokines provides new molecular insight into the biology of pDCs.

T-bet-dependent expression of osteopontin contributes to T cell polarization.

The osteopontin (Opn) glycoprotein has been implicated in diverse physiological processes, including vascularization, bone formation, and inflammatory responses. Studies of its role in immune responses has suggested that Opn can set the early stage of type-1 immune (cell-mediated) responses through differential regulation of IL-12 and IL-10 cytokine gene expression in macrophages. Although Opn has been suggested to play a role in the development of type-1 immunity, little is known about control of Opn gene expression. Here, we report that Opn gene expression in activated T cells, but not macrophages, is regulated by T-bet, a transcription factor that controls CD4+ T helper (Th1) cell lineage commitment. We also find that T-bet-dependent expression of Opn in T cells is essential for efficient skewing of CD4+ T and CD8+ T cells toward the Th1 and type 1 CD8+ T cells (Tc1) pathway, respectively. Taken together, these findings begin to delineate the genetic basis of Opn expression in T cells and further clarify the role of Opn in Th and Tc1 development.

NFATC2 transcription factor regulates cell cycle progression during lymphocyte activation: evidence of its involvement in the control of cyclin gene expression.

Upon antigen stimulation, lymphocytes enter in cell cycle and proliferate, and most of the activated T cells die by apoptosis. Many of the proteins that regulate lymphocyte activation are Under the control of transcription factors belonging to the NFAT family. As previously demonstrated, NFATC2-/- mice consistently showed a marked increase in lymphocyte proliferation. Here, we evaluate the role of NFATC2 in regulating lymphocyte proliferation and its involvement in the control of cell cycle progression during lymphocyte activation. NFATC2-/- lymphocytes, including CD4+ T cells and B cells, hyperproliferated upon stimulation when compared with NFATC2+/+ cells. Analysis of cell death demonstrated that NFATC2-/- lymphocytes displayed an increased rate of apoptosis after antigen stimulation in addition to the hyperproliferation. Cell cycle analysis after antigen stimulation showed that NFATC2-/- cultures contained more cycling cells when compared with NFATC2+/+ cultures, which is related to a shortening in time of cell division upon activation. Furthermore, hyperproliferation of NFATC2-/- lymphocytes is correlated to an overexpression of cyclins A2, B1, E, and F. Taken together, our results suggest that the NFATC2 transcription factor plays an important role in the control of cell cycle during lymphocyte activation and may act as an inhibitor of cell proliferation in normal cells.