The transcriptional repressor Bcl-6 is linked to the development of both CD4+ T follicular helper (TFH) and central memory T (TCM) cells. Here, we demonstrate that in response to decreased IL-2 signalling, T helper 1 (TH1) cells upregulate Bcl-6 and co-initiate TFH- and TCM-like gene programs, including expression of the cytokine receptors IL-6Rα and IL-7R. Exposure of this potentially bi-potent cell population to IL-6 favours the TFH gene program, whereas IL-7 signalling represses TFH-associated genes including Bcl6 and Cxcr5, but not the TCM-related genes Klf2 and Sell. Mechanistically, IL-7-dependent activation of STAT5 contributes to Bcl-6 repression. Importantly, antigen-specific IL-6Rα+IL-7R+ CD4+ T cells emerge from the effector population at late time points post influenza infection. These data support a novel role for IL-7 in the repression of the TFH gene program and evoke a divergent regulatory mechanism by which post-effector TH1 cells may contribute to long-term cell-mediated and humoral immunity.

Bcl-6 (B cell lymphoma-6) is a transcriptional repressor required for the differentiation of T follicular helper (TFH) cell populations. Currently, the molecular mechanisms underlying the transcriptional regulation of Bcl-6 expression are unclear. Here, we have identified the Ikaros zinc finger (IkZF) transcription factors Aiolos and Ikaros as novel regulators of Bcl-6. We found that increased expression of Bcl-6 in CD4+ T helper cell populations correlated with enhanced enrichment of Aiolos and Ikaros at the Bcl6 promoter. Furthermore, overexpression of Aiolos or Ikaros, but not the related family member Eos, was sufficient to induce Bcl6 promoter activity. Intriguingly, STAT3, a known Bcl-6 transcriptional regulator, physically interacted with Aiolos to form a transcription factor complex capable of inducing the expression of Bcl6 and the TFH-associated cytokine receptor Il6ra. Importantly, in vivo studies revealed that the expression of Aiolos was elevated in antigen-specific TFH cells compared to that observed in non-TFH effector T helper cells generated in response to influenza infection. Collectively, these data describe a novel regulatory mechanism wherein STAT3 and the IkZF transcription factors Aiolos and Ikaros cooperate to regulate Bcl-6 expression.

Scope The long-term effect of exposure to relevant dietary levels of genistein (GEN) on estrogen receptor-positive (ER+) human breast cancer (MCF-7) progression after GEN withdrawal in athymic mice xenograft model was studied. Materials and methods Feeding studies were conducted to determine the estrogenic effect of diets on MCF-7 tumor growth: 1) implantation (19 weeks) and withdrawal (6 weeks) of 17β-estradiol (E2); 2) dietary GEN 500 and 750 ppm during treatment/withdrawal for 23/10 and 15/9 weeks, respectively; and, 3) dietary soy protein isolate (SPI) containing GEN 180 ppm for 31/9 weeks of treatment/withdrawal. MCF-7 tumors grew fast in the presence of E2 implantation and abruptly regressed completely after E2 withdrawal. At different rates, dietary GEN alone (500 and 750 ppm) and GEN (180 ppm)-containing SPI stimulated MCF-7 tumor growth. After removal of the stimulus diet, tumors induced by 750 ppm GEN, but not 500 ppm GEN or SPI, regressed completely. The protein expression of epidermal growth factor receptor 2 (HER2) was higher in the GEN- and SPI-induced non-regressing (GINR) tumors compared to MCF-7 and E2 controls. Conclusion Long-term consumption of low GEN doses (≤500 ppm) promotes MCF-7 tumor growth and results in GINR tumors with more aggressive and advanced growth phenotypes.

Non-digestible carbohydrates (NDC(4)) have been used as a low-calorie sweetener and prebiotics that stimulate the growth of certain intestinal bacteria that support healthy colon conditions. In this study, we examined the dietary effect of commercially available NDCs on estrogen receptor positive (ER+) human breast cancer. We conducted a feeding study of fructooligosaccharides (FOSs), Fibersol 2 (F2; digestion resistant maltodextrin), Hi-Maize (HM; high amylose cornstarch), and Frutafit (FF; a range of powdered inulins) (5% in diet, w/w) to evaluate their effects on the growth of ER(+) human breast cancer (MCF-7) tumors in the presence of 17β-estradiol (E(2)) using an athymic xenograft model. F2, HM, and FOSs supplementation significantly reduced E(2)-stimulated MCF-7 tumor growth by inhibiting cellular proliferation (Ki-67) and increasing apoptosis (M30) in tumors. F2, HM, and FOSs treatments also lowered serum E(2) level and reduced uterine weight compared to the control diet. NDCs treatments downregulated relative mRNA expression of the E(2)-responsive gene markers pS2, bcl2, bcl-xL, and cyclin D1 in MCF-7 tumors. In conclusion, the NDC intake may have a protective effect against ER(+) tumors by inhibiting cellular proliferation and increasing apoptosis.

CD4+ T cell subtype specialization depends upon unique lineage-defining transcription factors that direct T helper cell development by both activating cell-specific gene expression programs and repressing alternative T helper cell fates. One such example is the transcriptional repressor Bcl-6, required for the development of T follicular helper (TFH) cell populations. Here, we identify the Ikaros zinc finger (IkZF) transcription factors Ikaros and Aiolos as novel regulators of Bcl-6 expression. We find that increased expression of Bcl-6 in T helper cells is associated with enhanced enrichment of Aiolos and Ikaros at the Bcl6 promoter. Additionally, we demonstrate that signal transducer and activator of transcription 3 (STAT3) physically interacts with Aiolos, suggesting a cooperative role for these factors in the activation of Bcl-6 expression. Mechanistically, the association of Ikaros, Aiolos, and STAT3 with the Bcl6 promoter was accompanied by chromatin remodeling events consistent with gene activation including increased histone acetylation. Importantly, expression of Aiolos and Ikaros was elevated in in vivo generated TFH cell populations compared to other IkZF family members. Collectively, these data describe a novel regulatory mechanism wherein STAT3 and the IkZF transcription factors Ikaros and Aiolos cooperate to regulate Bcl-6 expression. Elucidating the complex network of signals and factors that regulate Bcl-6 expression may enhance the potential to design more efficacious vaccines and develop novel immunotherapeutic approaches due to the wide-ranging importance of this transcriptional regulator.

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.

During the course of an immune response, multiple effector T helper cell types emerge to combat the initial infection, with subsequent emergence of long-term CD4+ T memory cells to ensure that a second exposure to the same pathogen will not cause disease. CD4+ T helper cell populations that are critical for this process include T helper 1 (TH1) and T follicular Helper (TFH) effector cells and long-lasting central memory T (TCM) cells. Previous work has demonstrated a level of flexibility between effector TH1 and TFH-like cell populations, with the respective gene programs regulated by alterations in interleukin 2 (IL-2) signaling. Here, we demonstrate that in response to decreased IL-2 signaling, effector TH1 cells are capable of initiating the expression of both TFH and TCM-like gene programs, including cell surface expression of IL-6Ra and IL-7R. Importantly, exposure of these cells to either IL-6 or IL-7 augments or inhibits the TFH gene program, respectively, suggesting a potential mechanism whereby cytokine availability may influence TFH or TCM cell function. Consistent with this model, we have identified a novel population of IL-6Ra+IL-7R+ cells that emerge at late time points post-infection in an in vivo mouse model of influenza infection. Further research into the temporal and spatial expression of IL-2, IL-6, and IL-7 in the modulation of these cells represents an exciting avenue of research with wide-ranging therapeutic implications.

Members of the Ikaros Zinc Finger Transcription Factor (IkZF) family are known regulators of hematopoiesis, including the development of CD4+ T helper cell populations. Our recent work established that the IkZF family member Aiolos cooperates with STAT3 to positively regulate Bcl-6 expression in T follicular helper (Tfh) cells. Given the conservation between members of the STAT and IkZF families, and their roles in T helper cell development, we hypothesized that additional IkZF/STAT interactions may regulate the differentiation of other T helper cell subsets. Here, we find that IL-2 signaling induces expression of the IkZF factor Eos in Th1 cells. Knockdown of Eos results in a significant reduction in the expression of signature Th1 genes including Prdm1, Il2ra, and Ifng. Mechanistically, we find that Eos and STAT5 physically interact and form a transcriptional complex that is capable of inducing Prdm1 expression. In support of these data, we also find that Eos and STAT5 are co-enriched at the regulatory regions of Th1 genes including Prdm1 and Il2ra. Taken together, these findings are suggestive of a novel, cooperative role for the IkZF family member Eos and STAT5 in regulating the Th1 differentiation program.