IKZF3/Aiolos Is Associated with but Not Sufficient for the Expression of IL-10 by CD4+ T Cells.

The expression of anti-inflammatory IL-10 by CD4+ T cells is indispensable for immune homeostasis, as it allows T cells to moderate their effector function. We previously showed that TNF-α blockade during T cell stimulation in CD4+ T cell/monocyte cocultures resulted in maintenance of IL-10-producing T cells and identified IKZF3 as a putative regulator of IL-10. In this study, we tested the hypothesis that IKZF3 is a transcriptional regulator of IL-10 using a human CD4+ T cell-only culture system. IL-10+ CD4+ T cells expressed the highest levels of IKZF3 both ex vivo and after activation compared with IL-10-CD4+ T cells. Pharmacological targeting of IKZF3 with the drug lenalidomide showed that IKZF3 is required for anti-CD3/CD28 mAb-mediated induction of IL-10 but is dispensable for ex vivo IL-10 expression. However, overexpression of IKZF3 was unable to upregulate IL-10 at the mRNA or protein level in CD4+ T cells and did not drive the transcription of the IL10 promoter or putative local enhancer constructs. Collectively, these data indicate that IKZF3 is associated with but not sufficient for IL-10 expression in CD4+ T cells.

Anti-TNF treatment negatively regulates human CD4+ T-cell activation and maturation in vitro, but does not confer an anergic or suppressive phenotype.

TNF-blockade has shown clear therapeutic value in rheumatoid arthritis and other immune-mediated inflammatory diseases, however its mechanism of action is not fully elucidated. We investigated the effects of TNF-blockade on CD4+ T cell activation, maturation, and proliferation, and assessed whether TNF-inhibitors confer regulatory potential to CD4+ T cells. CyTOF and flow cytometry analysis revealed that in vitro treatment of human CD4+ T cells with the anti-TNF monoclonal antibody adalimumab promoted IL-10 expression in CD4+ T cells, whilst decreasing cellular activation. In line with this, analysis of gene expression profiling datasets of anti-TNF-treated IL-17 or IFN-γ-producing CD4+ T cells revealed changes in multiple pathways associated with cell cycle and proliferation. Kinetics experiments showed that anti-TNF treatment led to delayed, rather than impaired T-cell activation and maturation. Whilst anti-TNF-treated CD4+ T cells displayed some hyporesponsiveness upon restimulation, they did not acquire enhanced capacity to suppress T-cell responses or modulate monocyte phenotype. These cells however displayed a reduced ability to induce IL-6 and IL-8 production by synovial fibroblasts. Together, these data indicate that anti-TNF treatment delays human CD4+ T-cell activation, maturation, and proliferation, and this reduced activation state may impair their ability to activate stromal cells.

The cholesterol biosynthesis pathway regulates IL-10 expression in human Th1 cells.

The mechanisms controlling CD4+ T cell switching from an effector to an anti-inflammatory (IL-10+) phenotype play an important role in the persistence of chronic inflammatory diseases. Here, we identify the cholesterol biosynthesis pathway as a key regulator of this process. Pathway analysis of cultured cytokine-producing human T cells reveals a significant association between IL-10 and cholesterol metabolism gene expression. Inhibition of the cholesterol biosynthesis pathway with atorvastatin or 25-hydroxycholesterol during switching from IFNγ+ to IL-10+ shows a specific block in immune resolution, defined as a significant decrease in IL-10 expression. Mechanistically, the master transcriptional regulator of IL10 in T cells, c-Maf, is significantly decreased by physiological levels of 25-hydroxycholesterol. Strikingly, progression to rheumatoid arthritis is associated with altered expression of cholesterol biosynthesis genes in synovial biopsies of predisposed individuals. Our data reveal a link between sterol metabolism and the regulation of the anti-inflammatory response in human CD4+ T cells.

TNF Blockade Maintains an IL-10+ Phenotype in Human Effector CD4+ and CD8+ T Cells.

CD4+ and CD8+ effector T cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine IL-10. However, the underlying cellular mechanisms that regulate expression of IL-10 in different T cell subpopulations are not yet fully elucidated. We recently showed that TNF inhibitors (TNFi) promote IL-10 expression in human CD4+ T cells, including IL-17+ CD4+ T cells. Here, we further characterized the regulation of IL-10 expression via blockade of TNF signaling or other cytokine/co-stimulatory pathways, in human T cell subpopulations. Addition of the TNFi drug adalimumab to anti-CD3-stimulated human CD4+ T cell/monocyte cocultures led to increased percentages of IL-10+ cells in pro-inflammatory IL-17+, IFNγ+, TNFα+, GM-CSF+, and IL-4+ CD4+ T cell subpopulations. Conversely, exogenous TNFα strongly decreased IL-10+ cell frequencies. TNF blockade also regulated IL-10 expression in CD4+ T cells upon antigenic stimulation. Using time course experiments in whole peripheral blood mononuclear cell (PBMC) cultures, we show that TNF blockade maintained, rather than increased, IL-10+ cell frequencies in both CD4+ and CD8+ T cells following in vitro stimulation in a dose- and time-dependent manner. Blockade of IL-17, IFNγ, IL-6R, or CD80/CD86-mediated co-stimulation did not significantly regulate IL-10 expression within CD4+ or CD8+ T cell subpopulations. We show that TNF blockade acts directly on effector CD4+ T cells, in the absence of monocytes or CD4+ CD25highCD127low regulatory T cells and independently of IL-27, resulting in higher IL-10+ frequencies after 3 days in culture. IL-10/IL-10R blockade reduced the frequency of IL-10-expressing cells both in the presence and absence of TNF blockade. Addition of recombinant IL-10 alone was insufficient to drive an increase in IL-10+ CD4+ T cell frequencies in 3-day CD4+ T cell/monocyte cocultures, but resulted in increased IL-10 expression at later time points in whole PBMC cultures. Together, these data provide additional insights into the regulation of IL-10 expression in human T cells by TNF blockade. The maintenance of an IL-10+ phenotype across a broad range of effector T cell subsets may represent an underappreciated mechanism of action underlying this widely used therapeutic strategy.

Candidate driver genes involved in genome maintenance and DNA repair in Sézary syndrome.

Sézary syndrome (SS) is a leukemic variant of cutaneous T-cell lymphoma (CTCL) and represents an ideal model for study of T-cell transformation. We describe whole-exome and single-nucleotide polymorphism array-based copy number analyses of CD4(+) tumor cells from untreated patients at diagnosis and targeted resequencing of 101 SS cases. A total of 824 somatic nonsynonymous gene variants were identified including indels, stop-gain/loss, splice variants, and recurrent gene variants indicative of considerable molecular heterogeneity. Driver genes identified using MutSigCV include POT1, which has not been previously reported in CTCL; and TP53 and DNMT3A, which were also identified consistent with previous reports. Mutations in PLCG1 were detected in 11% of tumors including novel variants not previously described in SS. This study is also the first to show BRCA2 defects in a significant proportion (14%) of SS tumors. Aberrations in PRKCQ were found to occur in 20% of tumors highlighting selection for activation of T-cell receptor/NF-κB signaling. A complex but consistent pattern of copy number variants (CNVs) was detected and many CNVs involved genes identified as putative drivers. Frequent defects involving the POT1 and ATM genes responsible for telomere maintenance were detected and may contribute to genomic instability in SS. Genomic aberrations identified were enriched for genes implicated in cell survival and fate, specifically PDGFR, ERK, JAK STAT, MAPK, and TCR/NF-κB signaling; epigenetic regulation (DNMT3A, ASLX3, TET1-3); and homologous recombination (RAD51C, BRCA2, POLD1). This study now provides the basis for a detailed functional analysis of malignant transformation of mature T cells and improved patient stratification and treatment.

The Interplay Between Monocytes/Macrophages and CD4(+) T Cell Subsets in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by inflammation of the synovial lining (synovitis). The inflammation in the RA joint is associated with and driven by immune cell infiltration, synovial hyperproliferation, and excessive production of proinflammatory mediators, such as tumor necrosis factor α (TNFα), interferon γ (IFNγ), interleukin (IL)-1ß, IL-6, and IL-17, eventually resulting in damage to the cartilage and underlying bone. The RA joint harbors a wide range of immune cell types, including monocytes, macrophages, and CD4(+) T cells (both proinflammatory and regulatory). The interplay between CD14(+) myeloid cells and CD4(+) T cells can significantly influence CD4(+) T cell function, and conversely, effector vs. regulatory CD4(+) T cell subsets can exert profound effects on monocyte/macrophage function. In this review, we will discuss how the interplay between CD4(+) T cells and monocytes/macrophages may contribute to the immunopathology of RA.

TNF-α blockade induces IL-10 expression in human CD4+ T cells.

IL-17+ CD4+ T (Th17) cells contribute to the pathogenesis of several human inflammatory diseases. Here we demonstrate that TNF inhibitor (TNFi) drugs induce the anti-inflammatory cytokine IL-10 in CD4+ T cells including IL-17+ CD4+ T cells. TNFi-mediated induction of IL-10 in IL-17+ CD4+ T cells is Treg-/Foxp3-independent, requires IL-10 and is overcome by IL-1ß. TNFi-exposed IL-17+ CD4+ T cells are molecularly and functionally distinct, with a unique gene signature characterized by expression of IL10 and IKZF3 (encoding Aiolos). We show that Aiolos binds conserved regions in the IL10 locus in IL-17+ CD4+ T cells. Furthermore, IKZF3 and IL10 expression levels correlate in primary CD4+ T cells and Aiolos overexpression is sufficient to drive IL10 in these cells. Our data demonstrate that TNF-α blockade induces IL-10 in CD4+ T cells including Th17 cells and suggest a role for the transcription factor Aiolos in the regulation of IL-10 in CD4+ T cells.