Lars Rogge

Transcript imaging of the development of human T helper cells using oligonucleotide arrays.

Many pathological processes, including those causing allergies and autoimmune diseases, are associated with the presence of specialized subsets of T helper cells at the site of inflammation. Understanding the genetic program that controls the functional properties of T helper type 1 (Th1) versus T helper type 2 (Th2) cells may provide insight into the pathophysiology of inflammatory diseases. We compared the gene-expression profiles of human Th1 and Th2 cells using high-density oligonucleotide arrays with the capacity to display transcript levels of 6,000 human genes. Here we analyse the data sets derived from five independent experiments using statistical algorithms. This approach resulted in the identification of 215 differentially expressed genes, encoding proteins involved in transcriptional regulation, apoptosis, proteolysis, and cell adhesion and migration. A subset of these genes was further upregulated by exposure of differentiated Th1 cells to interleukin-12 (IL-12), as confirmed by kinetic PCR analysis, indicating that IL-12 modulates the effector functions of Th1 cells in the absence of antigenic stimulation. Functional assays and in vivo expression of selected genes have validated the biological relevance of our study. Our results provide new insight into the transcriptional program controlling the functional diversity of subsets of T helper cells.

Integrin LFA-1 interacts with the transcriptional co-activator JAB1 to modulate AP-1 activity

Integrin adhesion receptors transduce signals that control complex cell functions which require the regulation of gene expression, such as proliferation, differentiation and survival. Their intracellular domain has no catalytic function, indicating that interaction with other transducing molecules is crucial for integrin-mediated signalling. Here we have identified a protein that interacts with the cytoplasmic domain of the β2 subunit of the αL/β2 integrin LFA-1. This protein is JAB1 (Jun activation domain-binding protein 1), a coactivator of the c-Jun transcription factor. We found that JAB1 is present both in the nucleus and in the cytoplasm of cells and that a fraction of JAB1 colocalizes with LFA-1 at the cell membrane. LFA-1 engagement is followed by an increase of the nuclear pool of JAB1, paralleled by enhanced binding of c-Jun-containing AP-1 complexes to their DNA consensus site and increased transactivation of an AP-1-dependent promoter. We suggest that signalling through the LFA-1 integrin may affect c-Jun-driven transcription by regulating JAB1 nuclear localization. This represents a new pathway for integrin-dependent modulation of gene expression.

Regulation of the IL-12/IL-12R axis: a critical step in T-helper cell differentiation and effector function

Summary: Interleukin (IL)‐12 is required for the development of T‐helper (Th)1 cells, which have been shown to be important for protective cell‐mediated immune responses against a variety of intracellular pathogens. Recent studies have clarified the sources and the regulation ofIL‐12 production leading to Th1 development against microbes. Expression of IL‐12R is necessary for maintaining IL‐12 responsiveness and controlling Thl lineage commitment. Advances in this area have included a broader understanding of the factors involved in the regulation of the IL‐12Rβ2 signaling component. Expression of this receptor subunit in humans is critically influenced by IL‐12 and type I interferons. IL‐12 signaling results in STAT4 activation and interferon (IFN)‐γ production. Recent evidence suggests that IL‐12 also modulates a number of genes involved in leukocyte trafficking. Thus, IL‐12 is not only an important proinflammatory cytokine, which induces production of IFN‐γ and subsequent activation of phago‐cytic cells but also plays a major role in regulating the migration and proper positioning of effector cells.

In vivo expansion of naive and activated CD4+CD25+FOXP3+ regulatory T cell populations in interleukin-2-treated HIV patients.

HIV-1 infection is characterized by a progressive decline in CD4+ T cells leading to a state of profound immunodeficiency. IL-2 therapy has been shown to improve CD4+ counts beyond that observed with antiretroviral therapy. Recent phase III trials revealed that despite a sustained increase in CD4+ counts, IL-2-treated patients did not experience a better clinical outcome [Abrams D, et al. (2009) N Engl J Med 361(16):1548–1559]. To explain these disappointing results, we have studied phenotypic, functional, and molecular characteristics of CD4+ T cell populations in IL-2-treated patients. We found that the principal effect of long-term IL-2 therapy was the expansion of two distinct CD4+CD25+ T cell populations (CD4+CD25loCD127loFOXP3+ and CD4+CD25hiCD127loFOXP3hi) that shared phenotypic markers of Treg but could be distinguished by the levels of CD25 and FOXP3 expression. IL-2-expanded CD4+CD25+ T cells suppressed proliferation of effector cells in vitro and had gene expression profiles similar to those of natural regulatory CD4+CD25hiFOXP3+ T cells (Treg) from healthy donors, an immunosuppressive T cell subset critically important for the maintenance of self-tolerance. We propose that the sustained increase of the peripheral Treg pool in IL-2-treated HIV patients may account for the unexpected clinical observation that patients with the greatest expansion of CD4+ T cells had a higher relative risk of clinical progression to AIDS.

Type I interferons and the Th1/Th2 paradigm.

During the past year significant advances have been made in our understanding of the factors contributing to the differentiation of CD4+ T helper cell subsets. These have been driven, in part, by the realization that cytokines from the innate immune response, such as interleukin-12 (IL-12) and interferons (IFNs), play a critical role in T cell subset differentiation. This review covers some of the most recent data concerning the divergent role that IFNs have in the differentiation of human versus mouse T helper cell subsets. In this review we discuss the molecular basis for the specie-specific effect of type I IFN on the selective induction of Th1 type immune responses. Furthermore, since IFN-beta is used in the treatment of multiple sclerosis (MS) we discuss the potential effects of such treatment and the value of the Th1/Th2 paradigm in MS.

The COP9 Signalosome Regulates Skp2 Levels and Proliferation of Human Cells

The COP9 signalosome (CSN) is a conserved, multisubunit complex first identified as a developmental regulator in plants. Gene inactivation of single CSN subunits results in early embryonic lethality in mice, indicating that the CSN is essential for mammalian development. The pleiotropic function of the CSN may be related to its ability to remove the ubiquitin-like peptide Nedd8 from cullin-RING ubiquitin ligases, such as the SCF complex, and therefore regulate their activity. However, the mechanism of CSN regulatory action on cullins has been debated, since, paradoxically, the CSN has an inhibitory role in vitro, while genetic evidence supports a positive regulatory role in vivo. We have targeted expression of CSN subunits 4 and 5 in human cells by lentivirus-mediated small hairpin RNA delivery. Down-regulation of either subunit resulted in disruption of the CSN complex and in Cullin1 hyperneddylation. Functional consequences of CSN down-regulation were decreased protein levels of Skp2, the substrate recognition subunit of SCFSkp2, and stabilization of a Skp2 target, the cyclin-dependent kinase inhibitor p27Kip1. CSN down-regulation caused an impairment in cell proliferation, which could be partially reversed by suppression of p27Kip1. Moreover, restoring Skp2 levels in CSN-deficient cells recovered cell cycle progression, indicating that loss of Skp2 in these cells plays an important role in their proliferation defect. Our data indicate that the CSN is necessary to ensure the assembly of a functional SCFSkp2 complex and therefore contributes to cell cycle regulation of human cells.

Regulation of the IL-12 receptor β2 subunit by soluble antigen and IL-12in vivo

Continuous administration of soluble protein antigen to BALB/c mice inhibits the development of Th1 and induces selective differentiation of Th2 cells. Here we show that interleukin (IL)‐12, administered together with soluble protein through a mini‐osmotic pump implanted s.c., not only prevents the inhibition of Th1 cell development, but stimulates higher interferon (IFN)‐γ production than in mice receiving IL‐12 alone. In parallel to co‐stimulation of Th1 cell development, co‐administration of IL‐12 blocks the Th2 response induced by soluble protein. IL‐12 administered in adjuvant with antigen or intraperitoneally 2 days after the immunization does not break the inhibition of Th1 but can still decrease the Th2 response induced by pretreatment with soluble protein antigen. In contrast to IL‐12, co‐administration of IL‐2 or IFN‐γ does not affect the diversion to Th2 induced by soluble antigen. Thus IL‐12, but not IL‐2 nor IFN‐γ, converts in vivo the inhibitory signal for Th1 cell development delivered by soluble antigen into an immunogenic one, while blocking a positive signal for Th2 cell differ entiation. A molecular basis for the co‐stimulation of Th1 priming and the prevention of Th2 differentiation by IL‐12 in vivo is provided by the observation that transcripts encoding the IL‐12 receptor β2 chain, which is required for IL‐12 signaling and Th1 cell development, are selectively inhibited by soluble antigen but are enhanced by IL‐12 co‐administration.

Gene expression profiling in immune cells using microarray.

The recent development of DNA microarray, which offers the opportunity to study the expression of thousands of individual genes simultaneously in different biological systems, has provided new insights into the immune system. Examples discussed in this review include molecular descriptions of the differentiation program of T helper (Th) cells into Th1 and Th2 pathways and the genetic program underlying maturation of dendritic cells. It is anticipated that this new information can be used to understand gene function in both physiological and pathological conditions of the immune system.

Integration of Distinct Intracellular Signaling Pathways at Distal Regulatory Elements Directs T-bet Expression in Human CD4+ T Cells

T-bet is a key regulator controlling Th1 cell development. This factor is not expressed in naive CD4+ T cells, and the mechanisms controlling expression of T-bet are incompletely understood. In this study, we defined regulatory elements at the human T-bet locus and determined how signals originating at the TCR and at cytokine receptors are integrated to induce chromatin modifications and expression of this gene during human Th1 cell differentiation. We found that T cell activation induced two strong DNase I-hypersensitive sites (HS) and rapid histone acetylation at these elements in CD4+ T cells. Histone acetylation and T-bet expression were strongly inhibited by cyclosporine A, and we detected binding of NF-AT to a HS in vivo. IL-12 and IFN-γ signaling alone were not sufficient to induce T-bet expression in naive CD4+ T cells, but enhanced T-bet expression in TCR/CD28-stimulated cells. We detected a third HS 12 kb upstream of the mRNA start site only in developing Th1 cells, which was bound by IL-12-induced STAT4. Our data suggest that T-bet locus remodeling and gene expression are initiated by TCR-induced NF-AT recruitment and amplified by IL-12-mediated STAT4 binding to distinct distal regulatory elements during human Th1 cell differentiation.

IL-12 receptor regulation in IL-12-deficient BALB / c and C57BL / 6 mice

Immunization with protein antigen in complete Freunds adjuvant (CFA) induces Th1 cells in BALB / c and C57BL / 6 (B6) mice. Pretreatment with the same protein in soluble form induces Th2 cells in BALB / c but not in B6 mice and inhibits Th1 cell development in both. We have previously shown that inhibition of Th1 in BALB / c mice correlates with the down‐regulation of transcripts encoding the IL‐12 receptor β2 (IL‐12Rβ2) chain, which is required for IL‐12 signaling and Th1 cell development. We now demonstrate that IL‐12‐deficient BALB / c mice, when primed with antigen in CFA, mount a Th2 instead of the Th1 response which develops in wild‐type mice. Conversely, IL‐12‐deficient B6 mice fail to develop Th2 cells. Thus, a default Th2 development is induced by antigen priming in IL‐12‐deficient BALB / c but not B6 mice. IL‐12Rβ2 transcripts are still expressed in antigen‐restimulated CD4+ T cells from IL‐12‐deficient BALB / c and B6 mice and they are similarly reduced by pretreatment with soluble antigen, suggesting that intrinsic strain differences in IL‐12R regulation do not account for the differential polarization to the Th2 pathway. IL‐4 is not required for down‐regulation of IL‐12Rβ2 transcripts and inhibition of Th1 development in mice pretreated with soluble protein, as shown by their reduction in IL‐4‐deficient BALB / c mice.