Ikaros Family Of Transcription Factors Research Articles

The Ikaros Family of Transcription Factors Regulates the Expression of the Transcriptional Repressor Bcl-6 in CD4+ T Cells

Background:CD4+ T helper cells play critical roles in the regulation of pathogen-specific immune responses and immune tolerance. The formation of each individual T helper cell subset is dictated by the expression of a unique gene program. These gene programs are regulated by both the cytokine environment and cell-intrinsic, cytokine-responsive "lineage-defining" transcription factors, which imprint the conserved gene expression programs characteristic of a given T helper cell lineage. The transcriptional repressor Bcl-6 is one such factor, and has been identified as the lineage-defining transcription factor for the T follicular helper (TFH) cell subset. TFH cells participate in the generation of humoral immunity by providing help to B cells, which are responsible for the production of pathogen-neutralizing antibodies. Interestingly, Bcl-6 expression has also been implicated in the formation of CD4+ central memory T (TCM) cells, which play a critical role in long-term cell-mediated immunity. Recently, our laboratory has demonstrated that Bcl-6 expression can be induced in effector T helper 1 (TH1) cells in response to decreased interleukin 2 (IL-2) signaling. Consequently, TH1 cells are capable of upregulating Bcl-6-dependent TFH- and TCM-like gene programs, suggesting that these cells may be able to contribute to aspects of long-term humoral and cell-mediated immunity. Despite these insights, the upstream factor(s) that directly control the expression of Bcl-6 remain largely unknown. Preliminary RNAseq analysis indicated that the expression of members of the Ikaros family of zinc-finger transcription factors, which have been shown to play important roles in regulating gene expression during hematopoiesis, correlated with that of Bcl-6 in TH1 and TFH/TCM-like cells. As such, we hypothesized that Ikaros-family proteins may contribute to the regulation of Bcl-6 expression.Methods:Naïve CD4+ T cells isolated from the spleens and lymph nodes of 5-8 week old C57BL/6 mice were stimulated with α-CD3 and α-CD28 in TH1 polarizing conditions. Following the generation of TH1 cells, these cells were split into either high IL-2 conditions to maintain the TH1 phenotype or into low IL-2 conditions to induce the TFH/TCM-like phenotype. Total cellular RNA, total cellular protein, and chromatin samples were isolated for analysis.Results:In this study, we demonstrate that the Ikaros family members, Ikaros and Aiolos, are preferentially expressed in TFH/TCM-like cells when compared to TH1 cells. siRNA knockdown demonstrates that the expression of Bcl-6 correlates with that of Ikaros and/or Aiolos. To define the molecular mechanisms that lead to the aforementioned findings, we used chromatin immunoprecipitation (ChIP) assays to show that Ikaros and Aiolos directly bind to the Bcl-6 promoter region. Interestingly, co-immunoprecipitation experiments reveal that Ikaros and Aiolos physically interact, suggesting that they may act cooperatively to promote Bcl-6 expression. Finally, Ikaros and Aiolos siRNA experiments show that reduced expression of these transcription factors correlates with a reduction in the expression of a number of canonical TFH and TCM genes.Conclusion:Collectively, these results demonstrate that the Ikaros family members Ikaros and Aiolos are IL-2-sensitive transcription factors that positively regulate Bcl-6 expression and that of key TFH and TCM genes. These data support the possibility that Ikaros and Aiolos may be critical factors in the induction of the TFH and TCM cell types and thus, potentially, in the regulation of long-term humoral and cell-mediated immunity. DisclosuresNo relevant conflicts of interest to declare.

Variations in mRNA and protein levels of Ikaros family members in pediatric T cell acute lymphoblastic leukemia.

Pediatric T cell acute lymphoblastic leukemia (T-ALL) is a highly heterogeneous disease in which the cells share phenotypic characteristics with normal human thymocytes. The Ikaros family of transcription factors includes five members that are required for normal T cell development and are implicated in leukemogenesis. The goal of this work was to correlate the pattern of expression of Ikaros family members with the phenotype of the T-ALL cells. We obtained twenty-four samples from pediatric T-ALL patients and used multi-parameter flow cytometry to characterize each sample, comparing the phenotype of the leukemic cells with normal human thymocytes. Then, we defined the expression levels of each Ikaros family member to determine whether the mRNA levels or splicing or protein levels were similar to the normal patterns seen during human T cell development. Multi-parameter analysis of the phenotype of T-ALL cells revealed that each patient's cells were unique and could not be readily correlated with stages of T cell development. Similarly, the pattern of Ikaros expression varied among patients. In most patients, Ikaros mRNA was the dominant family member expressed, but some patients' cells contained mostly Helios, Aiolos, or Eos mRNA. Despite that most patients had elevated mRNA levels of Ikaros family members and unique patterns of mRNA splicing, most patients had significantly reduced protein levels of Ikaros and Aiolos. Our analysis of the cell phenotype and Ikaros expression levels in T-ALL cells revealed the extent of heterogeneity among patients. While it is rarely possible to trace leukemic cells to their developmental origin, we found distinct patterns of Ikaros family mRNA levels in groups of patients. Further, mRNA and protein levels of Ikaros and Aiolos did not correlate, indicating that mRNA and protein levels are regulated via distinct mechanisms.

Expression patterns of Ikaros family members during positive selection and lineage commitment of human thymocytes.

The Ikaros family of transcription factors is essential for normal T-cell development, but their expression pattern in human thymocytes remains poorly defined. Our goal is to determine how protein levels of Ikaros, Helios and Aiolos change as human thymocytes progress through the positive selection and lineage commitment stages. To accomplish this goal, we used multi-parameter flow cytometry to define the populations in which positive selection and lineage commitment are most likely to occur. After human thymocytes express CD3 and receive positive selection signals, the cells down-regulate expression of CD4 to become transitional single-positive (TSP) CD8+ thymocytes. At this stage, there was a transient increase in the Ikaros, Helios and Aiolos protein levels. After the TSP CD8+ developmental stage, some thymocytes re-express CD4 and become CD3hi double-positive thymocytes before down-regulating CD8 to become mature single-positive CD4+ thymocytes. Except for regulatory T cells, Helios protein levels declined and Aiolos protein levels transiently increased during CD4+ T-cell maturation. For thymocytes progressing toward the CD8+ T-cell lineage, TSP CD8+ thymocytes increase their expression of CD3 and maintain high levels of Aiolos protein as the cells complete their maturation. In summary, we defined the TSP CD8+ developmental stage in human T-cell development and propose that this stage is where CD4/CD8 lineage commitment occurs. Ikaros, Helios and Aiolos each undergo a transient increase in protein levels at the TSP stage before diverging in their expression patterns at later stages.

Functional Characterization of the role of Eos (lkzf4) in CD4+ and CD8+ T cells

Abstract Eos is a member of the Ikaros family of transcription factors and is preferentially expressed in T regulatory cells (Treg), and activated T conventional (Tconv) cells. The role of Eos in Treg function is controversial. One study using siRNA knock down of Eos demonstrated that Eos was critical for Treg suppressor function. In contrast, Treg from mice with a global deficiency of Eos had normal Treg phenotype and function in vitro and in vivo, but had a deficiency in IL-2 production with an enhanced Th17 response. To further dissect the function of Eos in CD4+ and CD8+Tconv cells, we evaluated the immune responses of Eosfl/flX CD4-Cre mice. IL-2 production in vitro by both CD4+ and CD8+ T cells was diminished upon polyclonal stimulation of cells from Eosfl/flX CD4-Cre mice. EAE was much more severe in the Eosfl/fl X CD4-Cre mice as compared to WT mice. The percentages of CD4+, Foxp3−, IL17+, IFNg+ T cells were significantly higher in spleen, draining lymph nodes, and spinal cord from Eosfl/fl X CD4-cre mice when compared to WT mice. However, the percentages of Treg cells were significantly higher in the WT mice. We also determined if Eos plays any role in the activation of CD8+ cells during LCMV Armstrong infection. On day 9 following infection, no differences between Eos−/− and WT mice were observed in the expansion, expression of activation markers, and cytokine production by tetramer+ LCMV-peptide-specific T cells. Virus was completely cleared on day 7 and anti-viral CTL activity both in vitro and in vivo was similar in the Eos−/− mice and WT mice. Eos appears to play a major role in regulating IL-2 production in vitro in both CD4+ and CD8+ Tconv, but only modulates CD4+, but not CD8+, T effector function in vivo. This Research was supported by the DIR, NIAID, NIH.

IKZF2, a Novel Target of MSI2 RNA-Binding Protein Plays an Oncogenic Role in Myeloid Leukemia

Deregulated epigenetic program is found in many cancers, and genetic aberrations of histone methyltransferases contribute to transformation in myeloid leukemias. Post-transcriptional regulation in leukemia has recently been highlighted as a novel way for maintaining the leukemia stem cell (LSC) program. We have recently demonstrated that Msi2 is required for LSC function in a murine MLL-AF9 leukemia model. We determined that MSI2 maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting and retaining efficient translation of critical MLL regulated transcription factors including Hoxa9, Myc and Ikzf2. Despite extensive studies implicating Myc and Hoxa9 in leukemia, the role for Ikzf2 in myeloid leukemia is not known.Ikzf2 is a member of the Ikaros transcription factor family and regulates lymphocyte development by controlling regulatory T-cell function. Ikzf2 is highly expressed in Hematopoietic Stem Cells (HSC) and to investigate if Ikzf2 is involved in HSC function, we utilized mice that have a specific deletion of Ikzf2 in the hematopoietic system through the Vav-cre system. We found similar frequencies for different populations in the stem, progenitor and mature cells in the bone marrow of Ikzf2f/f and Ikzf2D/D mice. Colony assays of isolated Lin- Sca1+ c-Kit+ (LSK) cells from Ikzf2f/f and Ikzf2D/D mice resulted in a comparable number of myeloid progenitor colonies. Furthermore, noncompetitive transplant of Ikzf2f/f and Ikzf2D/D bone marrow cells showed similar chimerism after 34 months indicating that I kzf2D/D mice have normal HSC function and hematopoiesis.To interrogate the role of Ikzf2 in acute myeloid leukemia we utilized the MLL-AF9 retroviral transduction model. Intracellular flow cytometry showed that IKZF2 is highly expressed in the LSC population compared to the non-LSCs. We then transduced Ikzf2f/f and Ikzf2D/D LSK cells with MLL-AF9 and found that Ikzf2 deletion results in a ten-fold reduction in colony formation compared to Ikzf2f/f cells. Transplantation of transduced cells results in delayed leukemia progression with reduced disease burden. Secondary transplantation of the initiation experiment exhibited a significant delay in leukemogenesis in the Ikzf2D/D compared to the Ikzf2f/f mice (median survival of 32 and 19.5 days, respectively). The role for Ikzf2 in maintenance was assessed with an inducible puro-creER system, which resulted in 80% decrease in viable cell number within 24hrs of 4-hydroxytamoxifen (4-OHT) treatment. Flow cytometric analysis showed that the Ikzf2-deficient cells had increased apoptosis and differentiation, shown by AnnexinV/7-AAD and Mac1 expression respectively. Furthermore, inducible deletion of Ikzf2 using puro-creER system in vivo revealed that Ikzf2 deletion leads to a delay in leukemia after tamoxifen administration in mice. These results indicate that Ikzf2 is required for both leukemia initiation and maintenance.To determine a role for IKZF2 in human leukemia cells, we performed Ikzf2 knockdown experiments with shRNAs in Kasumi-1, KG1, KCL22 and MOLM13 cells. Ikzf2 depletion resulted in decreased cell growth and increased apoptosis compared to cells infected with scramble shRNA.To determine the mechanism for how IKZF2 controls leukemia cell survival and self-renewal, we performed gene expression profiling of the Ikzf2-deficient Vav-cre LSCs and demonstrated enrichment in signatures for self-renewal loss, increased differentiation, loss of Myc-regulated genes and loss for targets of Hoxa9 and Meis1. Further analysis overlapping our MSI2 HITS-CLIP data and our differentially regulated genes revealed a strong enrichment suggesting that the MSI2 bound targets are transcriptionally regulated by IKZF2. Lastly, ATAC-sequencing of Ikzf2f/f and Ikzf2D/D LSCs revealed alterations in chromatin accessibility that correlated closely with differentially expressed genes. Utilizing the ATAC-seq data we predicted that HOXA9 and MYC sites were significantly altered. We validated that MYC RNA and protein levels were reduced in both murine and human AML cell lines.In contrast to its known tumor suppressor role in hypodiploid B-ALL and T-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell initiation and maintenance. Thus, we provide evidence that Ikzf2 can regulate c-MYC expression helping in maintaining the stem cell self-renewal program in LSCs. DisclosuresNo relevant conflicts of interest to declare.

Transcription Factor Repertoire of Homeostatic Eosinophilopoiesis.

The production of mature eosinophils (Eos) is a tightly orchestrated process with the aim to sustain normal Eos levels in tissues while also maintaining low numbers of these complex and sensitive cells in the blood. To identify regulators of homeostatic eosinophilopoiesis in mice, we took a global approach to identify genome-wide transcriptome and epigenome changes that occur during homeostasis at critical developmental stages, including Eos-lineage commitment and lineage maturation. Our analyses revealed a markedly greater number of transcriptome alterations associated with Eos maturation (1199 genes) than with Eos-lineage commitment (490 genes), highlighting the greater transcriptional investment necessary for differentiation. Eos-lineage-committed progenitors (EoPs) were noted to express high levels of granule proteins and contain granules with an ultrastructure distinct from that of mature resting Eos. Our analyses also delineated a 976-gene Eos-lineage transcriptome that included a repertoire of 56 transcription factors, many of which have never previously been associated with Eos. EoPs and Eos, but not granulocyte-monocyte progenitors or neutrophils, expressed Helios and Aiolos, members of the Ikaros family of transcription factors, which regulate gene expression via modulation of chromatin structure and DNA accessibility. Epigenetic studies revealed a distinct distribution of active chromatin marks between genes induced with lineage commitment and genes induced with cell maturation during Eos development. In addition, Aiolos and Helios binding sites were significantly enriched in genes expressed by EoPs and Eos with active chromatin, highlighting a potential novel role for Helios and Aiolos in regulating gene expression during Eos development.

Eos Is Redundant for Regulatory T Cell Function but Plays an Important Role in IL-2 and Th17 Production by CD4+ Conventional T Cells.

Eos belongs to the Ikaros family of transcription factors. It was reported to be a regulatory T cell (Treg) signature gene, to play a critical role in Treg suppressor functions, and to maintain Treg stability. We used mice with a global deficiency in Eos to re-examine the role of Eos expression in both Tregs and conventional T cells (Tconvs). Tregs from Eos-deficient (Eos(-/-)) mice developed normally, displayed a normal Treg phenotype, and exhibited normal suppressor function in vitro. Eos(-/-) Tregs were as effective as Tregs from wild-type (WT) mice in suppressing inflammation in a model of inflammatory bowel disease. Bone marrow (BM) from Eos(-/-) mice was as effective as that from WT mice in controlling T cell activation when used to reconstitute immunodeficient mice in the presence of scurfy fetal liver cells. Surprisingly, Eos was expressed in activated Tconvs and was required for IL-2 production, CD25 expression, and proliferation in vitro by CD4(+) Tconvs. Eos(-/-) mice developed more severe experimental autoimmune encephalomyelitis than WT mice, displayed increased numbers of effector T cells in the periphery and CNS, and amplified IL-17 production. In conclusion, our studies are not consistent with a role for Eos in Treg development and function but demonstrate that Eos plays an important role in the activation and differentiation of Tconvs.

Generation of Helios reporter mice and an evaluation of the suppressive capacity of Helios+ regulatory T cells in vitro

Helios is a member of the Ikaros transcription factor family and has been reported to be a marker of thymus-derived regulatory T cells (Treg). Helios is an intracellular protein, however, and hence cannot be used as a marker to separate living Tregs. To solve this problem, we generated Helios reporter mice in which Helios+ cells selectively express Venus, a variant of green fluorescent protein. Most of the Tregs in the thymus expressed Helios, whereas its expression was varied in peripheral lymphoid organs. The Helios+ Treg-population was superior in ability to suppress both antigen-specific and TCR-stimulated T cell responses. We also showed that Helios+ Tregs inhibited the cytokine production by T cells more efficiently than Helios- Tregs. We conclude that Helios reporter mouse strain is a useful tool to study function of Helios and that Helios+ Tregs represent the highly suppressive population.

Msi2 Maintains the MLL Leukemia Stem Cell Regulatory Program

Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. LSCs are characterized by their gain of a self-renewal program that is normally associated with hematopoietic stem cells (HSCs). Previously we have shown that the RNA binding protein, Msi2 contributes to both HSC and myeloid leukemia function. Elevated MSI2 expression predicts a poor prognosis in a variety of leukemias and shRNA-mediated depletion in human AML cell lines reduces proliferation, increases differentiation and induces apoptosis. Despite these in vitroand correlative studies, MSI2’s molecular mechanism is not known and its role in LSC function has not been assessed.To elucidate MSI2’s role in LSC function, we utilized the MLL-AF9 leukemia mouse model. Initially we found MSI2 was elevated in the LSC enriched compartment (c-KitHigh cells) compared to non-LSCs (c-KitLow cells) based on flow cytometric intracellular staining. Therefore, to establish a model to study Msi2 and its contribution to myeloid LSCs, we have utilized the Msi2 conditional knockout mice that we previously crossed (Msi2f/f) into an Mx1-Cre background to generate the Msi2Δ/Δallele (injection of polyinositol-polycytosine; pIpC). In order to test if Msi2 is critical for MLL-AF9 mediated initiation, we transduced control Msi2f/f and Msi2Δ/ΔLin- Sca1+ c-Kit+cells (LSKs) with MLL-AF9 expressing retroviruses co-expressing GFP.Msi2 deleted LSKs or granulocyte-monocyte progenitors (GMPs) transduced with MLL-AF9 demonstrated delayed leukemogenesis with dramatically reduced diseased burden. Msi2 deficient leukemias were found to have a 4-fold reduced phenotypic LSC population and were more differentiated based on cellular morphology. Msi2 deficient leukemias failed to transplant into secondary recipients demonstrating that Msi2 is required for maintaining LSCs. Deletion of Msi2after leukemia engraftment led to a delay in leukemogenesis indicating that Msi2 is also important for leukemia maintenance.Gene expression profiling of the Msi2 ablated LSCs resulted in a loss of the HSC/LSC program and an increase in differentiation gene sets. The gene signature from the Msi2 deleted murine LSCs (121 genes) was overlapped and subjected to unsupervised clustering with the gene expression profiles from 336 AML patients (ECOG1900 dataset). This analysis resulted in distinct clusters that had differential MSI2 expression and the MSI2“high” cluster predicted a worse clinical outcome when compared to the other clusters.Overlapping of the differential transcriptional analysis of the Msi2 deleted murine LSCs with our global MSI2 direct mRNA targets (HITS-CLIP) led us to identify that MSI2 binds to transcripts that are associated with the downstream MLL self-renewal program, including Myc and Ikzf2. Ikzf2 is a member of the Ikaros transcription factor family and is known to regulate lymphocyte development by controlling regulatory T-cell function and chromatin remodeling. Ikzf2 shRNA mediated depletion resulted in reduced colony formation, decreased proliferation and increased apoptosis. The MLL associated targets were also reduced, which included Bcl-2 and Hoxa9. In contrast to its tumor suppressor role in hypodiploid B-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell maintenance. Thus, we provide evidence that MSI2 maintains the oncogenic LSC epigenetic program and the rationale for clinically targeting MSI2 in myeloid leukemia. DisclosuresNo relevant conflicts of interest to declare.

Helios defines T cells being driven to tolerance in the periphery and thymus.

The expression of the Ikaros transcription factor family member, Helios, has been shown to be associated with T-cell tolerance in both the thymus and the periphery. To better understand the importance of Helios in tolerance pathways, we have examined the expression of Helios in TCR-transgenic T cells specific for the gastric H(+) /K(+) ATPase, the autoantigen target in autoimmune gastritis. Analysis of H(+) /K(+) ATPase-specific T cells in mice with different patterns of H(+) /K(+) ATPase expression revealed that, in addition to the expression of Helios in CD4(+) Foxp3(+) regulatory T (Treg) cells, Helios is expressed by a large proportion of CD4(+) Foxp3(-) T cells in both the thymus and the paragastric lymph node (PgLN), which drains the stomach. In the thymus, Helios was expressed by H(+) /K(+) ATPase-specific thymocytes that were undergoing negative selection. In the periphery, Helios was expressed in H(+) /K(+) ATPase-specific CD4(+) T cells following H(+) /K(+) ATPase presentation and was more highly expressed when T-cell activation occurred in the absence of inflammation. Analysis of purified H(+) /K(+) ATPase-specific CD4(+) Foxp3(-) Helios(+) T cells demonstrated that they were functionally anergic. These results demonstrate that Helios is expressed by thymic and peripheral T cells that are being driven to tolerance in response to a genuine autoantigen.

Ikaros and aiolos undergo different post-transcriptional regulation in mice and humans (HEM4P.249)

Abstract The Ikaros family of transcription factors is important for T cell development and leukemogenesis. In mice, homozygous expression of an Ikaros isoform that is unable to bind DNA leads to a complete block in T cell development, while heterozygous expression of the same Ikaros isoform results in leukemogenesis. To better understand the roles of Ikaros family members in human and murine T cell development, we used quantitative RT-PCR and Western blot analysis to determine the mRNA and protein levels of Ikaros family members in FACS-purified murine and human thymocytes. As thymocytes mature from the double negative (DN) to double positive (DP) stages, Ikaros mRNA levels increase 3.0±1.8 fold (p<0.01, n=6) in mice and 4.4±1.7 fold (p<0.001, n=5) in humans. Similarly, Aiolos mRNA levels increase 27±2.3 fold (p<0.001, n=6) in mice and 18±2.8 fold (p<0.001, n=5) in humans. However, during the same DN to DP transition, protein levels only increase significantly in mice for both Ikaros (2.5±0.79 fold, p<0.001, n=6) and Aiolos (11±5.4 fold, p<0.01, n=5). These results suggest that Ikaros and Aiolos undergo different post-transcriptional control in murine and human thymocytes. Further studies are needed to understand the method of this post-transcriptional control, and how it regulates human T cell development and leukemogenesis.

Simultaneous presentation of JAK2 V617F mutation-related essential thrombocythemia and B-cell chronic lymphocytic leukemia.

Simultaneous presentation of JAK2 V617F mutation-related essential thrombocythemia and B-cell chronic lymphocytic leukemia.

CD8+ Treg - From Mouse To Man

T cell-mediated regulation of the immune response to self and foreign antigens is essential to maintain immune homeostasis and prevent autoimmune tissue destruction. The majority of studies addressing this issue, both in mice and humans, have focused on the contribution of CD4+CD25+FOXP3+ regulatory T cells. The contribution of regulatory CD8+ T cells has not been appreciated until recently. Recent studies have identified a small subset of IL-15 dependent CD8+ regulatory T cells (Treg) that is essential for maintenance of self- tolerance and prevention of autoimmune disease in mice. Expression of a triad of cell surface markers – CD44+CD122+Ly49+ – has been used to distinguish and purify CD8+Treg (Kim et al., Nature 2010; Kim et al., PNAS 2011).Here we have defined the human homologue of CD8+ Treg. The Ly49 receptor, identified as a stable surface marker on CD8+ Treg, is a member of a multigenic/multiallelic receptor family recognizing classical MHC class I molecules. The functional homologue of murine Ly49 is the killer cell immunoglobulin-like receptor (KIR). Our analysis has revealed that, while expression of KIR subtype combinations appears to be stochastic and co-expression of these KIR receptors is random, the inhibitory KIR2DL2/3 and KIR3DL1 subtypes are dominantly expressed by human CD3+CD8+ T cells. Similar to murine CD8+ Treg, CD8+ T cells that express KIR2DL2/3 or KIR3DL1 also express CD44 and CD122. Moreover, consistent with murine CD8+ Treg, incubation with IL-15 results in activation and proliferation of KIR+CD8+ T cells that maintain a stable surface phenotype. Gene array analyses in mice has indicated that Helios, a highly conserved zinc finger transcription factor and member of the Ikaros family of transcription factors, is expressed by CD44+CD122+Ly49+ CD8+ Treg. Helios is involved in T cell development and expressed by ∼70% of FoxP3+CD4+ Treg but not by mature B cells, dendritic cells or myeloid cells. Our analyses identified a Helios+ subset in the CD8+ Treg population, which (compared to the Helios- subset) embodies many of the functional characteristics of CD8+ Treg in mice. Co-expression of Helios is also apparent in some KIR+CD8+ T cells in human samples. In mice, we have shown that CD8+ Treg target CD4 TFH cells and thus maintain self-tolerance. In vitro suppression assays revealed that KIR+CD8+ cells but not KIR–CD8+ cells confer inhibitory activity on CD4+CXCR5+ TFHtarget cells in humans.Taken together, our findings implicate KIR+CD8+ cells as the human homologue of murine CD8+ Treg, including expression of transcription factor Helios, responsiveness to IL-15 and suppression of CD4+ TFH cells. Understanding the genetic and biological features of this CD8+ T cell subset in humans opens the possibility of exploiting their regulatory activity for the development of immunotherapy in the context of autoimmune disease and cancer. Disclosures:No relevant conflicts of interest to declare.

Lenalidomide Promotes CRBN-Mediated Ubiquitination and Degradation of IKZF1 and IKZF3

Lenalidomide is a highly effective drug for the treatment of multiple myeloma and has activity in additional B cell lymphomas. Lenalidomide has been shown to bind the CRBN-DDB1 E3 ubiquitin ligase, but it is unknown how lenalidomide alters the activity of this enzyme complex, and how this leads to therapeutic efficacy. We used a combination of quantitative proteomic approaches to demonstrate that lenalidomide acts by a novel mechanism of action for a therapeutic agent: in multiple myeloma cells, lenalidomide increases the binding of two substrates, IKZF1 (Ikaros) and IKZF3 (Aiolos), to the CRBN substrate adaptor; increases the ubiquitination of these substrates; and causes the targeted degradation of these transcription factors that are essential for the differentiation and survival of plasma cells including multiple myeloma cells.To identify targets of the CRBN-DDB1 ubiquitin ligase that are altered by lenalidomide, we applied SILAC (stable isotope labeling by amino acids in cell culture)-based quantitative mass spectrometry studies to globally assess changes in ubiquitination and proteome levels in the multiple myeloma cell line MM1S. Two members of the Ikaros transcription factor family, IKZF1 and IKZF3, were differentially ubiquitinated and decreased after lenalidomide treatment. Subsequent validation experiments in various cell lines demonstrated that lenalidomide, thalidomide, and pomalidomide cause a decrease of endogenous and ectopically expressed IKZF1 and IKZF3 protein levels but not mRNA levels. Furthermore, we confirmed that IKZF1 and IKZF3 bind CRBN in the presence of lenalidomide, supporting CRBN's role as a substrate adaptor. Consistent with this, shRNA mediated knockdown or overexpression of a CRBN mutant (CRBNYWAA) that does not bind lenalidomide abrogated lenalidomide's effect on IKZF1 and IKZF3. Moreover, CRBN promoted IKZF3 ubiquitination in vitro in the presence of lenalidomide, demonstrating that it is an enzymatic substrate.Using deletion mutants of IKZF3 we identified a 58-amino-acid degron in the N-terminal zinc finger domain that is sufficient for lenalidomide-induced degradation. Based on sequence alignment of that region between lenalidomide responding Ikaros proteins IKZF1 and IKZF3 vs. non-responding IKZF2, IKZF4 and IKZF5 we substituted a single amino acid (IKZF3Q147H) that prevented binding of IKZF3 to CRBN and conferred resistance to lenalidomide induced degradation.IKZF1 and IKZF3 are essential transcription factors for terminal B cell differentiation. We evaluated the biological effects of IKZF1 and IKZF3 loss using shRNAs in a variety of cell lines. IKZF1 and IKZF3 specific shRNAs inhibited the growth of multiple myeloma cell lines while lenalidomide insensitive cell lines derived from other hematopoietic neoplasms were unaffected. Similarly, a dominant negative IKZF3 mutant resulted in growth inhibition of MM1S cells. In contrast, expression of IKZF3Q147Hconferred lenalidomide resistance to MM1S cells.Lenalidomide induces IL-2 expression and release in T cells. We found that lenalidomide induced a dose-dependent decrease of IKZF1 and IKZF3 protein levels in primary human T cells. Previous studies have shown that IKZF3 is a transcriptional repressor of IL-2. To further evaluate the effect of IKZF3 loss, we transduced primary human T cells with shRNAs targeting either IKZF3 or control. IL2 RNA levels increased 3.3 fold after lenalidomide treatment in T cells expressing control shRNAs. In contrast, the baseline IL2 RNA level in T cells transduced with IKZF3 specific shRNAs was 3.7 fold higher compared to controls and this effect could not be further stimulated by lenalidomide.In conclusion, selective targeting of two lymphoid transcription factors, IKZF1 and IKZF3, explains lenalidomide's selective growth inhibition in multiple myeloma and likely other B cell lymphomas as well as its immunomodulatory effects in T cells. Furthermore, selective ubiquitination and degradation of specific targets provides a novel mechanism of therapeutic activity for proteins that are not otherwise amenable to small-molecule inhibition. Disclosures:Ebert:Celgene: Membership on an entity's Board of Directors or advisory committees.

The Ikaros Transcription Factor Regulates Responsiveness to IL-12 and Expression of IL-2 Receptor Alpha in Mature, Activated CD8 T Cells

The Ikaros family of transcription factors is critical for normal T cell development while limiting malignant transformation. Mature CD8 T cells express multiple Ikaros family members, yet little is known about their function in this context. To test the functions of this gene family, we used retroviral transduction to express a naturally occurring, dominant negative (DN) isoform of Ikaros in activated CD8 T cells. Notably, expression of DN Ikaros profoundly enhanced the competitive advantage of activated CD8 T cells cultured in IL-12, such that by 6 days of culture, DN Ikaros-transduced cells were 100-fold more abundant than control cells. Expression of a DN isoform of Helios, a related Ikaros-family transcription factor, conferred a similar advantage to transduced cells in IL-12. While DN Ikaros-transduced cells had higher expression of the IL-2 receptor alpha chain, DN Ikaros-transduced cells achieved their competitive advantage through an IL-2 independent mechanism. Finally, the competitive advantage of DN Ikaros-transduced cells was manifested in vivo, following adoptive transfer of transduced cells. These data identify the Ikaros family of transcription factors as regulators of cytokine responsiveness in activated CD8 T cells, and suggest a role for this family in influencing effector and memory CD8 T cell differentiation.

Helios Induces Epigenetic Silencing of Il2 Gene Expression in Regulatory T Cells

Regulatory T cells (Tregs) play a critical role in maintaining immune tolerance and preventing autoimmune disease. Tregs express the transcription factor Foxp3, which acts as a master regulator of their differentiation and controls their capacity to suppress T cell responses. Tregs have an intrinsically anergic phenotype and do not produce IL-2 or proliferate upon stimulation ex vivo. Recent studies identified that Helios, a member of the Ikaros family of transcription factors, is expressed in Tregs. However, its specific function is not fully understood. In this study, we show that Helios regulates IL-2 production in Tregs by suppressing Il2 gene transcription. Loss of Helios in Tregs breaks their anergic phenotype and results in derepression of the Il2 locus, allowing Tregs to display increased baseline proliferation and to produce IL-2 following stimulation. Conversely, forced expression of Helios in CD4(+)Foxp3(-) T cells results in a loss of their normal ability to produce IL-2. Helios acts by binding to the Il2 promoter and inducing epigenetic modifications that include histone deacetylation. We also show that loss of Helios in Tregs results in decreased Foxp3 binding to the Il2 promoter, indicating that Helios promotes binding of Foxp3 to the Il2 promoter. Interestingly, the loss of Helios in Tregs also causes a decrease in suppressive capacity. Our results identify Helios as a key regulator of Il2 expression in Tregs, contributing to the maintenance of the anergic phenotype.

FOXP3 + T cell subset characterized by Helios, FOXP3 and CD45RO in patients with melanoma

Human CD4+FOXP3+T cells in peripheral blood mononuclear cells (PBMCs) are separated into three subsets according to the function and cell-surface phenotype as CD45RO+FOXP3low nonregulatory T cells, CD45RO+FOXP3high effector Tregs, and CD45RO-FOXP3low naive Tregs. We previously reported that all the FOXP3+ T cell subsets were increased in PBMCs from patients with melanoma compared with age-matched healthy controls. It remains elusive how these cells increase and where they originate. Recently, Helios, an Ikaros family of transcription factors, is recognized as a new marker for FOXP3+ T cells. Helios divides FOXP3+ T cells into two subpopulations, FOXP3+Helios+T cells and FOXP3+Helios-T cells. All of the thymic Tregs express Helios. In the periphery, 70% of Tregs express Helios while the remaining 30% do not. Previous reports indicate that FOXP3+Helios+Tregs have more suppressive characteristics compared to FOXP3+Helios-Tregs. On the other hand, FOXP3+Helios-Tregs have the capacity to produce effector cytokines. To investigate where the origin of increased subsets of FOXP3+T cells in patients with melanoma, we evaluated the expression of Helios in these subsets of freshly stained PBMCs by flow cytometry and cytokine production in patients with melanoma compared with healthy controls. We also anlalyzed Helios expression in tumor infiltrating lymphocytes. Our data suggested that antitumor immunity were activated in patients with melanoma. The interpretaion of these results will be discussed. JSID AbstractsJournal of Dermatological ScienceVol. 69Issue 2Preview Full-Text PDF

Aiolos promotes TH17 differentiation by directly silencing Il2 expression

CD4(+) interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) are instrumental in the immune response to pathogens. However, an overactive T(H)17 response results in tissue inflammation and autoimmunity, and therefore it is important to identify the molecular mechanisms that control the development of T(H)17 cells. IL-2 suppresses such development, but how IL-2 production is actively suppressed during T(H)7 differentiation is not understood. Here we report that under T(H)17-polarizing conditions, the transcription factors STAT3 and AhR upregulated the expression of Aiolos, a member of the Ikaros family of transcription factors. Using Aiolos-deficient mice, we demonstrated that Aiolos silenced the Il2 locus, promoting T(H)17 differentiation in vitro and in vivo. Thus, we have identified a module in the transcriptional program of T(H)17 cells that actively limits IL-2 production and promotes their differentiation.

α-MSH-Stimulated Tolerogenic Dendritic Cells Induce Functional Regulatory T Cells and Ameliorate Ongoing Skin Inflammation

The neuropeptide α-melanocyte-stimulating hormone (α-MSH) is a well-known mediator of skin pigmentation. More recently, it has been shown that α-MSH also exerts a strong anti-inflammatory and immunosuppressive activity. To elucidate the mechanisms underlying α-MSH-induced immunosuppression, we investigated whether α-MSH affects dendritic cell/T cell communication, as especially this interaction has an important role in the regulation of immune responses. Here, we show that α-MSH, by binding to MC-1R, induced tolerogenic dendritic cells, which were capable of expanding CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in vitro, as well as in vivo. Notably, those α-MSH-induced Tregs were functional as they efficiently inhibited cutaneous contact allergy and ongoing psoriasis-like skin inflammation in mice. Furthermore, α-MSH induced tolerogenic dendritic cells capable of generating functional Tregs in human blood. Interestingly, human Tregs expanded via α-MSH-stimulated dendritic cells suppressed the proliferation and cytokine secretion of pathogenic T-helper-17 (Th17) cells from individuals with psoriasis. Taken together, these data indicate that α-MSH induced immunosuppressive Tregs in vitro and in vivo, which inhibited disease progression in a mouse model of psoriasis-like skin inflammation and suppressed the activation and proliferation of effector T cells from subjects with psoriasis.

Helios+ FoxP3+ naturally occurring regulatory T cells are peripherally expanded in active systemic lupus erythematosus

BackgroundNaturally occurring FoxP3+ regulatory T cells (Tregs) are pivotal in induction and maintenance of tolerance to self and foreign antigens. Even though intensively studied, their role in systemic lupus erythematosus...