CD4+ T cells orchestrate the adaptive immune response in vertebrates. While both experimental and modeling work has been conducted to understand the molecular genetic mechanisms involved in CD4+ T cell responses and fate attainment, the dynamic role of intrinsic (produced by CD4+ T lymphocytes) versus extrinsic (produced by other cells) components remains unclear, and the mechanistic and dynamic understanding of the plastic responses of these cells remains incomplete. In this work, we studied a regulatory network for the core transcription factors involved in CD4+ T cell-fate attainment. We first show that this core is not sufficient to recover common CD4+ T phenotypes. We thus postulate a minimal Boolean regulatory network model derived from a larger and more comprehensive network that is based on experimental data. The minimal network integrates transcriptional regulation, signaling pathways and the micro-environment. This network model recovers reported configurations of most of the characterized cell types (Th0, Th1, Th2, Th17, Tfh, Th9, iTreg, and Foxp3-independent T regulatory cells). This transcriptional-signaling regulatory network is robust and recovers mutant configurations that have been reported experimentally. Additionally, this model recovers many of the plasticity patterns documented for different T CD4+ cell types, as summarized in a cell-fate map. We tested the effects of various micro-environments and transient perturbations on such transitions among CD4+ T cell types. Interestingly, most cell-fate transitions were induced by transient activations, with the opposite behavior associated with transient inhibitions. Finally, we used a novel methodology was used to establish that T-bet, TGF-β and suppressors of cytokine signaling proteins are keys to recovering observed CD4+ T cell plastic responses. In conclusion, the observed CD4+ T cell-types and transition patterns emerge from the feedback between the intrinsic or intracellular regulatory core and the micro-environment. We discuss the broader use of this approach for other plastic systems and possible therapeutic interventions. Bird Shannyn birdgirl@arcticmail.com University of Nebraska-Lincoln 2016-10-28T14:33:21Z 2017-02-25T18:06:48Z

Name: Interferon gamma - environmental

Gene Name: IFNG

UNIPROT ID: P01579

Gene ID: 3458

 2016-12-22T20:57:41Z

Name: Interleukin-21 - environmental

Gene Name: IL21

UNIPROT ID: Q9HBE4

Gene ID: 59067

 2016-12-26T18:59:02Z

Name: Interleukin-4 - environmental

Gene Name: IL4

UNIPROT ID: P05112

Gene ID: 3565

 2016-12-26T19:04:35Z

Name: Transforming growth factor beta-1 - environmental

Gene Name: TGFB1

UNIPROT ID: P01137

Gene ID: 7040

 2016-12-27T13:13:49Z

Name: Interleukin-10 - environmental

Gene Name: IL10

UNIPROT ID: P22301

Gene ID: 3586

 2016-12-26T11:47:53Z

Name: Interleukin-2 - environmental

Gene Name: IL2

UNIPROT ID: P60568

Gene ID: 3558

 2016-12-26T19:02:51Z

Name: Nuclear receptor ROR-gamma

Gene Name: RORC

UNIPROT ID: P51449

Gene ID: 6097

 2016-10-28T15:11:08Z

Name: Interferon gamma

Gene Name: IFNG

UNIPROT ID: P01579

Gene ID: 3458

 2016-12-22T20:57:41Z

Name: B-cell lymphoma 6 protein

Gene Name: BCL6

UNIPROT ID: P41182

Gene ID: 604

 2016-10-28T15:15:00Z

Name: Trans-acting T-cell-specific transcription factor GATA-3

Gene Name: GATA3

UNIPROT ID: P23771

Gene ID: 2625

 2016-12-21T17:07:46Z

Name: T-box transcription factor TBX21

Gene Name: TBX21

UNIPROT ID: Q9UL17

Gene ID: 30009

 2016-10-28T15:15:00Z

Name: Interleukin-2

Gene Name: IL2

UNIPROT ID: P60568

Gene ID: 3558

 2017-02-25T17:59:08Z

Name: Interleukin-21

Gene Name: IL21

UNIPROT ID: Q9HBE4

Gene ID: 59067

 2016-12-26T18:59:02Z

Name: Interleukin-9

Gene Name: IL9

UNIPROT ID: P15248

Gene ID: 3578

 2016-10-28T15:15:00Z

Name: Interleukin-10

Gene Name: IL10

UNIPROT ID: P22301

Gene ID: 3586

 2016-12-26T11:47:53Z

Name: Interleukin-4

Gene Name: IL4

UNIPROT ID: P05112

Gene ID: 3565

 2016-12-26T19:04:35Z

Name: Transforming growth factor beta-1

Gene Name: TGFB1

UNIPROT ID: P01137

Gene ID: 7040

 2017-02-25T17:59:08Z

Name: Forkhead box protein P3

Gene Name: FOXP3

UNIPROT ID: Q9BZS1

Gene ID: 50943

 2017-02-25T17:59:08Z

IL21 activates RORGT in conjunction with TGFB. BCL6, FOXP3, GATA3, and TBET are inhibitors of RORGT which express dominance over IL21.

TBET sequesters runx1 and runx3 in order to prevent activation of RORGT.

IL21 indirectly activates RORGT.

TGFB activates RORGT gene expression.

FOXP3 binds to RORGT and inhibits binding of runx1. This prevents RORGT-runx1 action, including inhibiting binding to IL17.

GATA3 binds to rorc in order to inhibit RORGT.

BCL6 inhibits RORGT-induced luciferase activity.

 S_11 1 S_16 1 S_6 1 S_17 1 S_8 1 S_7 1

TBET, IFNG, and IFNGe activate IFNG. TGFB, BCL6, and GATA3 are inhibitors of IFNG which express dominance over IFNG and TBET. IL10, IL21, IL4, and IL9 are negative regulators of IFNG which express dominance over IFNG, IFNGe, and TBET.

T-bet transactivates the IFNγ gene.

IL21 inhibits IFNG by repressing eomesodermin expression.

IL9 indirectly inhibits IFNG.

TGFB inhibits priming of the IFNG gene through inhibition of Stat4.

IFNG indirectly activates itself.

IL10 indirectly inhibits IFNG through STAT3.

Gata3 binds to runx3. This prevents runx3-mediated activation of IFNG.

IFNGe indirectly activates IFNG.

Bcl6−/− mice cells observed an increase in IFNG production, indicating that BCL6 is a negative regulator of IFNG.

IL4 indirectly inhibits IFNG.

 S_8 1 S_11 1 S_12 1 S_6 1 S_7 1 S_16 1 S_13 1 S_14 1 S_1 1 S_11 1 S_12 1 S_13 1 S_14 1 S_4 1 S_11 1 S_12 1 S_6 1 S_7 1 S_16 1 S_13 1 S_14 1

IL21 activates BCL6. IL2, TBET, and TGFB are negative regulators of BCL6 that express dominance over IL21.

TBET recruits BCL6 to the Ifng locus.

IL21 induces BCL6 expression.

TGFB indirectly inhibits BCL6.

IL2 inhibits BCL6 expression in TH1 cells.

 S_11 1 S_8 1 S_9 1 S_16 1

GATA3 activates itself. IL2 is a positive regulator of GATA3 which acts in conjunction with IL4. BCL6, IFNG, IL21, TBET, and TGFB are all inhibitors of GATA3 which express dominance over GATA3 and IL4.

TBET in conjunction with runx3 inhibit gata3 binding to target genes.

TGFB indirectly inhibits GATA3.

CD4+ T cells treated with IL21 observe a decrease in GATA3 activity.

IFNG indirectly inhibits GATA3.

GATA3 establishes a positive feedback loop that sustains its own activation.

BCL6 is a postranscriptional inhibitor for GATA3.

IL2 indirectly activates GATA3 through IL4.

IL4 indirectly activates GATA3 through STAT6.

 S_7 1 S_8 1 S_6 1 S_4 1 S_16 1 S_11 1 S_14 1 S_9 1 S_8 1 S_6 1 S_4 1 S_16 1 S_11 1

TBET and IFNG activate TBET. BCL6, GATA3, IL21, IL4, and IL9 are inhibitors of TBET which express dominance over IFNG and TBET.

TBET works in cooperation with stat1 to maintain T cell levels and for T cell memory.

IL9 indirectly inhibits TBET.

IL21 inhibits TBET expression.

Naive T cells induced TBET expression after stimulation of IFNG.

GATA3 inhibits TBET via a protein-protein interaction.

BCL6 binds to the TBET promoter.

IL4 indirectly inhibits TBET.

 S_8 1 S_12 1 S_14 1 S_11 1 S_7 1 S_6 1 S_4 1 S_12 1 S_14 1 S_11 1 S_7 1 S_6 1

IL2 and IL2e are both positive regulators of IL2. IL21, IL10, and IFNG inhibit IL2 and express dominance over IL2 and IL2e. FOXP3 is a negative regulator of IL2 which also is dominant over IL2.

IL21 inhibits IL2 production.

IL2e indirectly activates IL2 expression .

IFNG inhibits IL2 by altering the histone modifications in the IL2 promoter to make it inaccessible.

IL10 inhibits T cell proliferation and IL-2 production.

FOXP3 binds to nfat and inhibits nfat from forming a complex with ap-1. FOXp3 also binds to runx1 to prevent runx1-mediated activation of IL2.

IL2 indirectly activates its own production.

 S_18 1 S_4 1 S_13 1 S_11 1 S_9 1 S_4 1 S_17 1 S_13 1 S_11 1

RORGT, BCL6, IL21, and IL21e are positive regulators of IL21. IL4, IL9, IL2, IL10, and IFNG are negative regulators of IL21 that express dominance over BCL6, RORGT, IL21, and IL21e.

IL21 activation of it's own expression is dependent on stat3 and rorgt.

IL9 indirectly inhibits IL21.

IFNG inhibits IL-21-mediated STAT3 phosphorylation.

IL21 is inhibited by IL10.

IL21e activation of IL21 expression is dependent on stat3 and rorgt. .

RORGT indirectly activates IL21.

BCL6 indirectly activates IL21.

IL2 and IL21 have opposing differentiation functions in CD8 T cell.

IL4 inhibits IL21-induced plasma cell differentiation.

 S_2 1 S_14 1 S_9 1 S_4 1 S_12 1 S_13 1 S_6 1 S_14 1 S_9 1 S_4 1 S_12 1 S_13 1 S_11 1 S_14 1 S_9 1 S_4 1 S_12 1 S_13 1 S_3 1 S_14 1 S_9 1 S_4 1 S_12 1 S_13 1

IL4 activates IL9 in the presence of TGFB, or in conjunction with either IL10 or IL2. IFNG and IL21 are inhibitors of IL9 which express dominance over IL4.

TGFB indirectly enhances IL9 production.

IL21 indirectly inhibits IL9 production.

IFNG has been found to significantly inhibit IL-9 production from Th9 T cells

IL10 stimulates the production of IL9 by CD4 T reg cells.

IL2 is crutial for the differentiation of CD4+ T cells.

IL4 enhances IL9 production through STAT6.

 S_14 1 S_16 1 S_13 1 S_9 1 S_4 1 S_11 1

IL10 activates itself in conjunction with either GATA3, IFNG, IL21, or TGFB. IL10e also activates IL10.

IL21 indirectly activates IL10 production.

TGFB indirectly activates IL10 production.

Th1 cells require STAT1 activation via IFNG.

Environmental IL10 activates STAT3, a positive regulator for IL10.

IL10 activates STAT3, a positive regulator for IL10.

GATA3 binds to the IL10 promoter.

 S_13 1 S_11 1 S_16 1 S_4 1 S_7 1 S_15 1

IL4e activates IL4. GATA3 is a positive regulator of IL4 which acts in conjunction with either IL2 or IL4. IFNG and IL21 are inhibitors of IL4 which express dominance over IL4e and GATA3. TBET is a negative regulator of IL4 which is dominant over GATA3.

TBET inhibits IL4 gene expression.

IL21 indirectly inhibits IL4.

IFNG inhibits IL-4-induced cytokine expression by CD8 T cells.

GATA3 binds to the IL4 enhancer region and requires stat5 to activate IL4.

IL2 indirectly activates IL4 through STAT5.

IL4e indirectly activates IL4 production.

IL4 indirectly activates its own production.

 S_5 1 S_4 1 S_11 1 S_7 1 S_9 1 S_14 1 S_8 1 S_4 1 S_11 1

FOXP3, TGFB, and TGFBe all activate TGFB. IL21 is a negative regulator of TGFB which expresses dominance over FOXP3 and TGFB.

TGFB indirectly activates its own expression.

IL21 indirectly inhibits TGFB.

FOXP3 is a predicted activator for TGFB expression

TGFBe indirectly activates TGFB production.

 S_10 1 S_17 1 S_11 1 S_16 1 S_11 1

IL2 activates FOXP3 in conjunction with either FOXP3 or TGFB. IL21 and RORGT inhibit FOXP3 and express dominance over IL2.

T3GFB upregulates FOXP3 via SMAD3.

IL21 impairs Treg proliferation by regulating different checkpoints of the FOXP3 Treg chain.

FOXP3 upreguates its own production by upregulating IL2 and TGFB signaling components.

RORGT interacts with the FOXP3 promoter.

IL2 upregulates FOXP3 through a STAT-dependent mechanism.

 S_9 1 S_16 1 S_17 1 S_3 1 S_11 1