The Ikaros Family of Transcription Factors Regulates the Expression of the Transcriptional Repressor Bcl-6 in CD4+ T Cells
Authors: Cooley Ian D , Read Kaitlin A , Powell Michael D , Baker Chandra E , Sreekumar Bharath , Martin Emily , Vaughn Jennifer E , Oestreich Kenneth J 
Published: October 2019

Abstract
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.

Disclosures
No relevant conflicts of interest to declare.