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 (T_FH) cell subset. T_FH 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 (T_CM) 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 (T_H1) cells in response to decreased interleukin 2 (IL-2) signaling. Consequently, T_H1 cells are capable of upregulating Bcl-6-dependent T_FH- and T_CM-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 T_H1 and T_FH/T_CM-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 T_H1 polarizing conditions. Following the generation of T_H1 cells, these cells were split into either high IL-2 conditions to maintain the T_H1 phenotype or into low IL-2 conditions to induce the T_FH/T_CM-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 T_FH/T_CM-like cells when compared to T_H1 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 T_FH and T_CM 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 T_FH and T_CM genes. These data support the possibility that Ikaros and Aiolos may be critical factors in the induction of the T_FH and T_CM cell types and thus, potentially, in the regulation of long-term humoral and cell-mediated immunity.