Ashish Banerjee

c-Rel is required for the development of thymic Foxp3+ CD4 regulatory T cells

During thymopoiesis, a unique program of gene expression promotes the development of CD4 regulatory T (T reg) cells. Although Foxp3 maintains a pattern of gene expression necessary for T reg cell function, other transcription factors are emerging as important determinants of T reg cell development. We show that the NF-κB transcription factor c-Rel is highly expressed in thymic T reg cells and that in c-rel−/− mice, thymic T reg cell numbers are markedly reduced as a result of a T cell–intrinsic defect that is manifest during thymocyte development. Although c-Rel is not essential for TGF-β conversion of peripheral CD4+CD25− T cells into CD4+Foxp3+ cells, it is required for optimal homeostatic expansion of peripheral T reg cells. Despite a lower number of peripheral T reg cells in c-rel−/− mice, the residual peripheral c-rel−/− T reg cells express normal levels of Foxp3, display a pattern of cell surface markers and gene expression similar to those of wild-type T reg cells, and effectively suppress effector T cell function in culture and in vivo. Collectively, our results indicate that c-Rel is important for both the thymic development and peripheral homeostatic proliferation of T reg cells.

Coordinating TLR-activated signaling pathways in cells of the immune system

Toll‐like receptor (TLR) signaling leads to the activation of mitogen‐activated protein kinase and nuclear factor‐κB signaling pathways. While the upstream signaling events initiated at the level of adaptors and the activation of the downstream signaling pathways have received a lot of attention, our understanding of how these signaling pathways are coordinated to regulate gene expression is poorly understood. This review gives a selective overview on our current understanding of signaling downstream of TLRs, with an emphasis on how the upstream kinases like the mitogen‐activated protein kinase kinase kinases (TAK1 and Tpl2) and inhibitor of κ‐B kinase (IKK) coordinate the signaling events that steer the course of an immune response.

Regulating B-cell activation and survival in response to TLR signals

Following encounters with microbes, cellular activation programs that involve the control of proliferation and survival are initiated in follicular B cells either via the B‐cell receptor in a specific antigen‐defined manner, or through Toll‐like receptors (TLRs) that recognize specific microbial products. This review summarizes and discusses recent findings that shed light on how the nuclear factor κB pathway controls and coordinates B‐cell division and survival following TLR4 engagement.

NF-κB subunit specificity in hemopoiesis

Summary:  Although the diverse functions served by the nuclear factor‐κB (NF‐κB) pathway in virtually all cell types are typically employed to deal with stress responses, NF‐κB transcription factors also play key roles in the development of hemopoietic cells. This review focuses on how NF‐κB transcription factors control various aspects of thymic T‐cell and myeloid cell differentiation that include its roles in hemopoietic precursors, conventional αβ T cells, CD4+ regulatory T cells, natural killer T cells, γδ T cells, macrophages, and dendritic cells.

c-Rel Controls Multiple Discrete Steps in the Thymic Development of Foxp3+ CD4 Regulatory T Cells

The development of natural Foxp3+ CD4 regulatory T cells (nTregs) proceeds via two steps that involve the initial antigen dependent generation of CD25+GITRhiFoxp3−CD4+ nTreg precursors followed by the cytokine induction of Foxp3. Using mutant mouse models that lack c-Rel, the critical NF-κB transcription factor required for nTreg differentiation, we establish that c-Rel regulates both of these developmental steps. c-Rel controls the generation of nTreg precursors via a haplo-insufficient mechanism, indicating that this step is highly sensitive to c-Rel levels. However, maintenance of c-Rel in an inactive state in nTreg precursors demonstrates that it is not required for a constitutive function in these cells. While the subsequent IL-2 induction of Foxp3 in nTreg precursors requires c-Rel, this developmental transition does not coincide with the nuclear expression of c-Rel. Collectively, our results support a model of nTreg differentiation in which c-Rel generates a permissive state for foxp3 transcription during the development of nTreg precursors that influences the subsequent IL-2 dependent induction of Foxp3 without a need for c-Rel reactivation.

Modulating T regulatory cells in cancer: how close are we?

Regulatory T cells (Tregs) are a specialized subset of CD4 T cells that have an indispensable role in maintaining immune homeostasis and tolerance. Although studies in mice and humans have clearly highlighted that the absence of these cells results in severe autoimmunity and inflammation, increased Treg numbers and/or function is not always beneficial. This is best exemplified in certain cancers where increased Tregs promote cancer progression by interfering with immune surveillance. Conversely, in other types of cancers that have an inflammatory component, Tregs can inhibit cancer progression by dampening inflammation. In this review article, we provide a historical perspective of the discovery of Tregs, followed by a summary of the existing literature on the role of Tregs in malignancy.

Illumina WG-6 BeadChip strips should be normalized separately

BackgroundIllumina Sentrix-6 Whole-Genome Expression BeadChips are relatively new microarray platforms which have been used in many microarray studies in the past few years. These Chips have a unique design in which each Chip contains six microarrays and each microarray consists of two separate physical strips, posing special challenges for precise between-array normalization of expression values.ResultsNone of the normalization strategies proposed so far for this microarray platform allow for the possibility of systematic variation between the two strips comprising each array. That this variation can be substantial is illustrated by a data example. We demonstrate that normalizing at the strip-level rather than at the array-level can effectively remove this between-strip variation, improve the precision of gene expression measurements and discover more differentially expressed genes. The gain is substantial, yielding a 20% increase in statistical information and doubling the number of genes detected at a 5% false discovery rate. Functional analysis reveals that the extra genes found tend to have interesting biological meanings, dramatically strengthening the biological conclusions from the experiment. Strip-level normalization still outperforms array-level normalization when non-expressed probes are filtered out.ConclusionPlots are proposed which demonstrate how the need for strip-level normalization relates to inconsistent intensity range variation between the strips. Strip-level normalization is recommended for the preprocessing of Illumina Sentrix-6 BeadChips whenever the intensity range is seen to be inconsistent between the strips. R code is provided to implement the recommended plots and normalization algorithms.

The acetyltransferase HAT1 moderates the NF-κB response by regulating the transcription factor PLZF

To date, the activities of protein kinases have formed the core of our understanding of cell signal transduction. Comprehension of the extent of protein acetylation has raised expectations that this alternate post-transcriptional modification will be shown to rival phosphorylation in its importance in mediating cellular responses. However, limited instances have been identified. Here we show that signalling from Toll-like or TNF-α receptors triggers the calcium/calmodulin-dependent protein kinase (CaMK2) to activate histone acetyltransferase-1 (HAT1), which then acetylates the transcriptional regulator PLZF. Acetylation of PLZF promotes the assembly of a repressor complex incorporating HDAC3 and the NF-κB p50 subunit that limits the NF-κB response. Accordingly, diminishing the activity of CaMK2, the expression levels of PLZF or HAT1, or mutating key residues that are covalently modified in PLZF and HAT1, curtails control of the production of inflammatory cytokines. These results identify a central role for acetylation in controlling the inflammatory NF-κB transcriptional programme.

Tumor progression locus 2 (Tpl2) deficiency does not protect against obesity-induced metabolic disease.

Obesity is associated with a state of chronic low grade inflammation that plays an important role in the development of insulin resistance. Tumor progression locus 2 (Tpl2) is a serine/threonine mitogen activated protein kinase kinase kinase (MAP3K) involved in regulating responses to specific inflammatory stimuli. Here we have used mice lacking Tpl2 to examine its role in obesity-associated insulin resistance. Wild type (wt) and tpl2−/− mice accumulated comparable amounts of fat and lean mass when fed either a standard chow diet or two different high fat (HF) diets containing either 42% or 59% of energy content derived from fat. No differences in glucose tolerance were observed between wt and tpl2−/− mice on any of these diets. Insulin tolerance was similar on both standard chow and 42% HF diets, but was slightly impaired in tpl2−/− mice fed the 59% HFD. While gene expression markers of macrophage recruitment and inflammation were increased in the white adipose tissue of HF fed mice compared with standard chow fed mice, no differences were observed between wt and tpl2−/− mice. Finally, a HF diet did not increase Tpl2 expression nor did it activate Extracellular Signal-Regulated Kinase 1/2 (ERK1/2), the MAPK downstream of Tpl2. These findings argue that Tpl2 does not play a non-redundant role in obesity-associated metabolic dysfunction.

Distinct roles in NKT cell maturation and function for the different transcription factors in the classical NF-κB pathway.

The nuclear factor (NF)‐κB signalling pathway is known to be critical for natural killer T (NKT) cell differentiation; however, the role of individual NF‐κB transcription factors and the precise developmental stages that they control remain unclear. We have investigated the influence of the classical NF‐κB transcription factors NF‐κB1, c‐Rel and RelA on NKT cell development and function, using gene‐deleted mice. Individually, none of these factors were essential for the requirement of NF‐κB signalling in early NKT cell development before NK1.1 expression, in contrast to earlier reports in which the classical NF‐κB pathway was globally disrupted. Instead, we found that each factor played a non‐redundant role in later stages of NKT cell maturation and function. Although NF‐κB1 deficiency resulted in a moderate reduction in mature NK1.1+ NKT cells, this was found to be more subtle than previously reported. RelA deficiency had a more profound effect on the NK1.1+ stage of NKT cell development, whereas c‐Rel‐deficient mice had normal NKT cell numbers. All three factors (NF‐κB1, RelA and c‐Rel) were necessary for normal NKT cell cytokine production. Notably, IL‐17, which is produced by a specific subset of NKT cells (NKT‐17 cells), defined as NK1.1−CD4−, was not impaired by a lack of these individual NF‐κB transcription factors, nor was this subset depleted, suggesting that NKT‐17 cells are regulated independently of the NF‐κB pathway. Thus, individual NF‐κB family members have a largely redundant role in early NKT cell development, but each of them has an important and distinct role in NKT cell maturation and/or function.