Estimating immunoregulatory gene networks in human herpesvirus type 6-infected T cells

The immune response to viral infection involves complex network of dynamic gene and protein interactions. We present here the dynamic gene network of the host immune response during human herpesvirus type 6 (HHV-6) infection in an adult T-cell leukemia cell line. Using a pathway-focused oligonucleotide DNA microarray, we found a possible association between chemokine genes regulating Th1/Th2 balance and genes regulating T-cell proliferation during HHV-6B infection. Gene network analysis using an integrated comprehensive workbench, VoyaGene, revealed that a gene encoding a TEC-family kinase, ITK, might be a putative modulator in the host immune response against HHV-6B infection. We conclude that Th2-dominated inflammatory reaction in host cells may play an important role in HHV-6B-infected T cells, thereby suggesting the possibility that ITK might be a therapeutic target in diseases related to dysregulation of Th1/Th2 balance. This study describes a novel approach to find genes related with the complex host-virus interaction using microarray data employing the Bayesian statistical framework.     

Identification of novel Th2-associated genes in T memory responses to allergens

Atopic diseases are associated with hyperexpression of Th2 cytokines by allergen-specific T memory cells. However, clinical trials with recently developed Th2 inhibitors in atopics have proven disappointing, suggesting underlying complexities in atopy pathogenesis which are not satisfactorily explained via the classical Th1/Th2 paradigm. One likely possibility is that additional Th2-associated genes which are central to disease pathogenesis remain unidentified. The aim of the present study was to identify such novel Th2-associated genes in recall responses to the inhalant allergen house dust mite. In contrast to earlier human microarray studies in atopy which focused on mitogen-activated T cell lines and clones, we concentrated on PBMC-derived primary T cells stimulated under more physiological conditions of low dose allergen exposure. We screened initially for allergen-induced gene activation by microarray, and validated novel genes in independent panels of subjects by quantitative RT-PCR. Kinetic analysis of allergen responses in PBMC revealed an early wave of novel atopy-associated genes involved in signaling which were coexpressed with IL-4 and IL-4R, followed by a later wave of genes encoding the classical Th2 effector cytokines. We further demonstrate that these novel activation-associated Th2 genes up-regulate in response to another atopy-associated physiological stimulus bacterial superantigen, but remain quiescent in nonphysiological responses in primary T cells or cell lines driven by potent mitogens, which may account for their failure to be detected in earlier microarray studies.     

A common factor regulates both Th1- and Th2-specific cytokine gene expression.

Murine T helper cell clones are classified into two distinct subsets, T helper 1 (Th1) and T helper 2 (Th2), on the basis of cytokine secretion patterns. Th1 clones produce interleukin-2 (IL-2), tumor necrosis factor-beta (TNF-beta) and interferon-gamma (IFN-gamma), while Th2 clones produce IL-4, IL-5, IL-6 and IL-10. These subsets differentially promote delayed-type hypersensitivity or antibody responses, respectively. The nuclear factor NF-AT is induced in Th1 clones stimulated through the T cell receptor-CD3 complex, and is required for IL-2 gene induction. The NF-AT complex consists of two components: NF-ATp, which pre-exists in the cytosol and whose appearance in the nucleus is induced by an increase of intracellular calcium, and a nuclear AP-1 component whose induction is dependent upon activation of protein kinase C (PKC). Here we report that the induction of the Th2-specific IL-4 gene in an activated Th2 clone involves an NF-AT complex that consists only of NF-ATp, and not the AP-1 component. On the basis of binding experiments we show that this 'AP-1-less' NF-AT complex is specific for the IL-4 promoter and does not reflect the inability of activated Th2 cells to induce the AP-1 component. We propose that NF-ATp is a common regulatory factor for both Th1 and Th2 cytokine genes, and that the involvement of PKC-dependent factors, such as AP-1, may help determine Th1-/Th2-specific patterns of gene expression.     

Low expression of the IL-23/Th17 pathway in atopic dermatitis compared to psoriasis

The classical Th1/Th2 paradigm previously defining atopic dermatitis (AD) and psoriasis has recently been challenged with the discovery of Th17 T cells that synthesize IL-17 and IL-22. Although it is becoming evident that many Th1 diseases including psoriasis have a strong IL-17 signal, the importance of Th17 T cells in AD is still unclear. We examined and compared skin biopsies from AD and psoriasis patients by gene microarray, RT-PCR, immunohistochemistry, and immunofluorescence. We found a reduced genomic expression of IL-23, IL-17, and IFN-gamma in AD compared with psoriasis. To define the effects of IL-17 and IL-22 on keratinocytes, we performed gene array studies with cytokine-treated keratinocytes. We found lipocalin 2 and numerous other innate defense genes to be selectively induced in keratinocytes by IL-17. IFN-gamma had no effect on antimicrobial gene-expression in keratinocytes. In AD skin lesions, protein and mRNA expression of lipocalin 2 and other innate defense genes (hBD2, elafin, LL37) were reduced compared with psoriasis. Although AD has been framed by the Th1/Th2 paradigm as a Th2 polar disease, we present evidence that the IL-23/Th17 axis is largely absent, perhaps accounting for recurrent skin infections in this disease.     

CD44 Reciprocally regulates the differentiation of encephalitogenic Th1/Th17 and Th2/regulatory T cells through epigenetic modulation involving DNA methylation of cytokine gene promoters, thereby controlling the development of experimental autoimmune encephalomyelitis

CD44 is expressed by a variety of cells, including glial and T cells. Furthermore, in the demyelinating lesions of multiple sclerosis, CD44 expression is chronically elevated. In this study, we demonstrate that targeted deletion of CD44 attenuated myelin oligodendrocyte glycoprotein peptide-induced experimental autoimmune encephalitomyelitis (EAE) through novel regulatory mechanisms affecting Th differentiation. Specifically, by developing chimeras and using adoptive transfer experiments, we noted that CD44 deficiency on CD4(+) T cells, but not other cells, conferred protection against EAE induction. CD44 expression played a crucial role in Th differentiation, inasmuch as deletion of CD44 inhibited Th1/Th17 differentiation while simultaneously enhancing Th2/regulatory T cell differentiation. In contrast, expression of CD44 promoted Th1/Th17 differentiation. When osteopontin and hyaluronic acid, the two major ligands of CD44, were tested for their role in Th differentiation, osteopontin, but not hyaluronic acid, promoted Th1/Th17 differentiation. Furthermore, activation of CD44(+) encephalitogenic T cells with myelin oligodendrocyte glycoprotein peptide led to demethylation at the ifnγ/il17a promoter region while displaying hypermethylation at the il4/foxp3 gene promoter. Interestingly, similar activation of CD44-deficient encephalitogenic T cells led to increased hypermethylation of ifnγ/il17a gene and marked demethylation of il4/foxp3 gene promoter. Together, these data suggested that signaling through CD44, in encephalitogenic T cells, plays a crucial role in the differentiation of Th cells through epigenetic regulation, specifically DNA methylation of Th1/Th17 and Th2 cytokine genes. The current study also suggests that molecular targeting of CD44 receptor to promote a switch from Th1/Th17 to Th2/regulatory T cell differentiation may provide a novel treatment modality against EAE.     

Proteomic profiling of surface proteins on Th1 and Th2 cells

We utilized mass spectrometry to profile cell surface protein differential expression on primary human T helper (Th1 and Th2) cells with the stable isotope labeling by amino acids in cell culture (SILAC) approach. Proteomic and microarray analyses were done concurrently and results were compared for 38 different genes. Although microarray studies displayed wide variability between donors for mRNA expression, these two approaches were shown to be corroborative for most gene products with the exception of a small subset of uncorrelated protein and message levels. The greatest differing Th1 to Th2 ratios were observed for BST2 (bone marrow stromal protein 2) and TRIM (T cell receptor interacting molecule). Both showed greater Th1 expression by proteomic methods, even though mRNA levels were approximately equal for both. To validate this method, we compared protein expression levels of a recently cloned molecule, B and T cell lymphocyte attenuator (BTLA), on Th1 and Th2 cell populations and showed greater protein expression on Th1 cells, which agrees with a previous analysis of higher BTLA mRNA expression in Th1 cells.(1).     

Mathematical modeling of allergy and specific immunotherapy: Th1-Th2-Treg interactions

Regulatory T cells (Treg) have recently been identified as playing a central role in the immune response to allergens and during allergen-specific immunotherapy. We have extended our previous mathematical model describing the nonlinear dynamics of Th1-Th2 regulation by including Treg cells and their major cytokines. We hypothesize that immunotherapy mainly acts on the T cell level and that the decisive process can be regarded as a dynamical phenomenon. The model consists of nonlinear differential equations which describe the proliferation and mutual suppression of different T cell subsets. The old version of the model was based upon the Th1-Th2 paradigm and is successful in describing the “Th1-Th2 switch” which was considered to be the decisive event during specific immunotherapy. In recent years, however, the Th1-Th2 paradigm has been questioned and therefore, we have investigated a modified model in order to account for the influence of a regulatory T cell type. We examined the extended model by means of numerical simulations and analytical methods. As the modified model is more complex, we had to develop new methods to portray its characteristics. The concept of stable manifolds of fixed points of a stroboscobic map turned out to be especially important. We found that when including regulatory T cells, our model can describe the events in allergen-specific immunotherapy more accurately. Our results suggest that the decisive effect of immunotherapy, the increased proliferation of Treg and suppression of Th2 cells, crucially depends on the administration of high dose injections in short intervals right before the maintenance phase sets in. Empirical protocols could therefore be improved by optimizing this step of therapy.     

Blimp-1 attenuates Th1 differentiation by repression of ifng, tbx21, and bcl6 gene expression

T cell-specific deletion of Blimp-1 causes abnormal T cell homeostasis and function, leading to spontaneous, fatal colitis in mice. Herein we explore the role of Blimp-1 in Th1/Th2 differentiation. Blimp-1 mRNA and protein are more highly expressed in Th2 cells compared with Th1 cells, and Blimp-1 attenuates IFN-gamma production in CD4 cells activated under nonpolarizing conditions. Although Blimp-1-deficient T cells differentiate normally to Th2 cytokines in vitro, Blimp-1 is required in vivo for normal Th2 humoral responses to NP-KLH (4-hydroxy-3-nitrophenylacetyl/keyhole lymphocyte hemocyanin) immunization. Lack of Blimp-1 in CD4 T cells causes increased IFN-gamma, T-bet, and Bcl-6 mRNA. By chromatin immunoprecipitation we show that Blimp-1 binds directly to a distal regulatory region in the ifng gene and at multiple sites in tbx21 and bcl6 genes. Our data provide evidence that Blimp-1 functions in Th2 cells to reinforce Th2 differentiation by repressing critical Th1 genes.     

A protein kinase A and Wnt-dependent network regulating an intermediate stage in epithelial tubulogenesis during kidney development

Genetic interactions regulating intermediate stages of tubulogenesis in the developing kidney have been difficult to define. A systems biology strategy using microarray was combined with in vitro/ex vivo and genetic approaches to identify pathways regulating specific stages of tubulogenesis. Analysis of the progression of the metanephric mesenchyme (MM) through four stages of tubule induction and differentiation (i.e., epithelialization, tubular organization and elongation and early differentiation) revealed signaling pathways potentially involved at each stage and suggested key roles for a number of signaling molecules. A screen of the signaling pathways on in vitro/ex vivo nephron formation implicated a unique regulatory role for protein kinase A (PKA), through PKA-2, in a specific post-epithelialization morphogenetic step (conversion of the renal vesicle to the S-shaped body). Microarray analysis not only confirmed this stage-specificity, but also highlighted the upregulation of Wnt genes. Addition of PKA agonists to LIF-induced nephrons (previously shown to be a Wnt/beta-catenin dependent pathway) disrupted normal tubulogenesis in a manner similar to PKA-agonist treated MM/spinal-cord assays, suggesting that PKA regulates a Wnt-dependent tubulogenesis step. PKA induction of canonical Wnt signaling during tubulogenesis was confirmed genetically using MM from Batgal-reporter mice. Addition of a Wnt synthesis inhibitor to activated PKA cultures rescued tubulogenesis. By re-analysis of existing microarray data from the FGF8, Lim1 and Wnt4 knockouts, which arrest in early tubulogenesis, a network of genes involving PKA, Wnt, Lhx1, FGF8, and hyaluronic acid signaling regulating the transition of nascent epithelial cells to tubular epithelium was derived, helping to reconcile in vivo and in vitro/ex vivo data.     

Effector CD4+ T cell expression signatures and immune-mediated disease associated genes

Genome-wide association studies (GWAS) in immune-mediated diseases have identified over 150 associated genomic loci. Many of these loci play a role in T cell responses, and regulation of T cell differentiation plays a critical role in immune-mediated diseases; however, the relationship between implicated disease loci and T cell differentiation is incompletely understood. To further address this relationship, we examined differential gene expression in na?ve human CD4+ T cells, as well as in in vitro differentiated Th1, memory Th17-negative and Th17-enriched CD4+ T cells subsets using microarray and RNASeq. We observed a marked enrichment for increased expression in memory CD4+ compared to na?ve CD4+ T cells of genes contained among immune-mediated disease loci. Within memory T cells, expression of disease-associated genes was typically increased in Th17-enriched compared to Th17-negative cells. Utilizing RNASeq and promoter methylation studies, we identified a differential regulation pattern for genes solely expressed in Th17 cells (IL17A and CCL20) compared to genes expressed in both Th17 and Th1 cells (IL23R and IL12RB2), where high levels of promoter methylation are correlated to near zero RNASeq levels for IL17A and CCL20. These findings have implications for human Th17 celI plasticity and for the regulation of Th17-Th1 expression signatures. Importantly, utilizing RNASeq we found an abundant isoform of IL23R terminating before the transmembrane domain that was enriched in Th17 cells. In addition to molecular resolution, we find that RNASeq provides significantly improved power to define differential gene expression and identify alternative gene variants relative to microarray analysis. The comprehensive integration of differential gene expression between cell subsets with disease-association signals, and functional pathways provides insight into disease pathogenesis.     

Prostaglandin E2 inhibits production of Th1 lymphokines but not of Th2 lymphokines.

PGE2 is known to inhibit IL-2 and IFN-gamma production from Th cells and is widely viewed as a general immunosuppressant. However, PGE2 was found not to inhibit IL-4 production from Th2 clones, and IL-5 production from these clones was slightly enhanced. The same results were obtained with short term T cell lines, which indicates that the lack of inhibition of IL-4 and IL-5 production by PGE2 is a general phenomenon. PGE2 functions by increasing cAMP levels through activation of adenylate cyclase. Despite its failure to inhibit lymphokine release, PGE2 was capable of increasing cAMP levels in Th2 cells, and forskolin, a direct activator of adenylate cyclase, also did not inhibit IL-4 or IL-5 production. These data indicate that the failure of PGE2 to inhibit IL-4 and IL-5 production was not due to an inability of PGE2 to induce an increase in intracellular cAMP, and suggested instead that the expression of IL-4 and IL-5 in Th2 cells is insensitive to elevated cAMP levels. When Th0 clones were examined, PGE2 was again found to differentially affect IL-2 and IL-4 production in three of five clones tested. In two additional Th0 clones, both IL-2 and IL-4 production were inhibited. These data suggest that lymphokine production may be regulated on two different levels. First, Th1- and Th2-associated lymphokines may be differentially sensitive to intracellular signals such as cAMP. Second, T cell subsets may exist, including subsets of Th0 cells, with different signaling pathways. In addition, our data suggest that PGE2 may play an important role in regulating the development of a response dominated by Th1- or Th2-associated lymphokines.     

The Metalloprotease ADAM12 Regulates the Effector Function of Human Th17 Cells

A key modulator of immune homeostasis, TGFβ has an important role in the differentiation of regulatory T cells (Tregs) and IL-17-secreting T cells (Th17). How TGFβ regulates these functionally opposing T cell subsets is not well understood. We determined that an ADAM family metalloprotease called ADAM12 is specifically and highly expressed in both Tregs and CCR6+ Th17 cells. ADAM12 is induced in vitro upon differentiation of naïve T cells to Th17 cells or IL-17-secreting Tregs. Remarkably, silencing ADAM12 expression in CCR6+ memory T cells enhances the production of Th17 cytokines, similar to suppressing TGFβ signaling. Further, ADAM12 knockdown in naïve human T cells polarized towards Th17/Treg cells, or ectopically expressing RORC, greatly enhances IL-17-secreting cell differentiation, more potently then inhibiting TGFβ signals. Together, our findings reveal a novel regulatory role for ADAM12 in Th17 cell differentiation or function and may have implications in regulating their aberrant responses during immune pathologies.     

Decision-making critical amino acids: role in designing peptide vaccines for eliciting Th1 and Th2 immune response

CD4 T cells play a cardinal role in orchestrating immune system. Differentiation of CD4 T cells to Th1 and Th2 effector subsets depends on multiple factors such as relative intensity of interactions between T cell receptor with peptide-major histocompatibility complex, cytokine milieu, antigen dose, and costimulatory molecules. Literature supports the critical role of peptide’s binding affinity to Human Leukocyte Antigens (HLAs) and in the differentiation of naïve CD4 T cells to Th1 and Th2 subsets. However, there exists no definite report addressing very precisely the correlation between physicochemical properties (hydrophobicity, hydrophilicity), pattern, position of amino acids in peptide and their role in skewing immune response towards Th1 and Th2 cells. This may play a significant role in designing peptide vaccines. Hence in the present study, we have evaluated the relationship between amino acid pattern and their influence in differentiation of Th1 and Th2 cells. We have used a data set of 320 peptides, whose role has been already established experimentally in the generation of either Th1 or Th2 immune response. Further, characterization was done based on binding affinity, promiscuity, amino acid pattern and binding conformation of peptides. We have observed that distinct amino acids in peptides elicit either Th1 or Th2 immunity. Consequently, this study signifies that alteration in the sequence and type of selected amino acids in the HLA class II binding peptides can modulate the differentiation of Th1 and Th2 cells. Therefore, this study may have an important implication in providing a platform for designing peptide-based vaccine candidates that can trigger desired Th1 or Th2 response.