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Title: Stochastic Cytokine Expression Induces Mixed T Helper Cell States}
Article:
Abstract:
During eukaryotic development, the induction of a lineage-specific transcription factor typically drives differentiation of multipotent progenitor cells, while repressing that of alternative lineages. This process is often mediated by some extracellular signaling molecules, such as cytokines that can bind to cell surface receptors, leading to activation and/or repression of transcription factors. We explored the early differentiation of naive CD4 T helper( Th) cells into Th1 versus Th2 states by counting single transcripts and quantifying immunofluorescence in individual cells. Contrary to mutually exclusive expression of antagonistic transcription factors, we observed their ubiquitous co-expression in individual cells at high levels that are distinct from basal-level co-expression during lineage priming. We observed that cytokines are expressed only in a small subpopulation of cells, independent from the expression of transcription factors in these single cells. This cell-to-cell variation in the cytokine expression during the early phase of T helper cell differentiation is significantly larger than in the fully differentiated state. Upon inhibition of cytokine signaling, we observed the classic mutual exclusion of antagonistic transcription factors, thus revealing a weak intracellular network otherwise overruled by the strong signals that emanate from extracellular cytokines. These results suggest that during the early differentiation process CD4 T cells acquire a mixed Th1/Th2 state, instructed by extracellular cytokines. The interplay between extracellular and intracellular signaling components unveiled in Th1/Th2 differentiation may be a common strategy for mammalian cells to buffer against noisy cytokine expression.
Introduction:
A multipotent progenitor cell can differentiate into a particular lineage by turning on the expression of a lineage-specific transcription factor, which coordinates the expression of a defined set of target genes. Numerous examples of such toggle-switch-like cell fate decisions have been observed in the differentiation of hematopoietic cells[1]. For example, common myeloid progenitor cells differentiate into granulocyte-monocyte progenitor versus megakaryocyte-erythrocyte progenitor cells based on expression of PU. 1 versus Gata1[2]; naive CD4 T cells differentiate into Th1 versus Th2 driven by the expression of Tbet or Gata3[3]–[6]. Antagonistic transcription factors are therefore believed to be expressed exclusively in the pertinent cell types, or co-expressed at basal levels in hematopoietic progenitors prior to commitment to “prime” the cells for rapid deployment of transcription factors to execute a particular lineage program[7]. For instance, common myeloid progenitors can co-express low levels of PU1 and GATA1 during lineage priming[8]–[12], though their expression is mutually exclusive in the fully committed state[7]. In addition to transcription factors that reside within the cell, the signaling network governing cell differentiation often comprises extracellular components, such as cytokines that can bind to cell surface receptors leading to activation and/or repression of transcription factors. In many previous studies, where the goal has been attaining a relatively homogenous population of differentiated cells, high concentrations of cytokines were added to the culture media to bias the cellular decision process toward one particular cell fate[2],[3],[6],[13]. In this work, we studied gene regulation during the early stage of cell differentiation to delineate the interplay between extracellular cytokines and intracellular transcription factors in single cells, using CD4 T helper cells as a model system. Contrary to previous studies where cellular fate was biased artificially[2],[3],[6],[13], we sought to avoid this bias by exploring the spontaneous differentiation of naive CD4 T cells in the absence of exogenously added cytokines. Tbet, encoded by Tbx21, is the master transcription factor of Th1 differentiation associated with production of the hallmark cytokine IFNγ[3], whereas Gata3 is the master transcription factor of Th2 differentiation associated with IL4 production[5]. In terminally differentiated individual CD4 T cells, the expression of Tbx21 and Gata3 is mutually exclusive[14],[15]. This is attributed to positive feedback loops and cross-inhibitory interactions in the regulatory network( Figure 1A). This network consists of two types of interactions: those that depend on cytokine signaling and those that are cytokine-independent and involve only intracellular players including transcription factors. Specifically, Tbet activates Ifng[16], and extracellular IFNγ can induce Tbx21 via receptor signaling[17]. Tbet also induces itself independently of signaling via cytokine receptors[18]. Similarly, Gata3 activates Il4[19],[20] and extracellular IL4 can induce Gata3[21]. Furthermore, Gata3 can be auto-induced independently of signaling via cytokine receptors[19],[22]. Finally, Tbet silences Il4[16], Gata3 silences Ifng[23],[24], and Tbet blocks the function of Gata3 through direct protein–protein interactions[25], leading to cross-inhibitory interactions. To quantify the number of Tbx21 and Gata3 transcripts in activated CD4 T cells, we isolated total CD4+ cells from C57BL/6 mice. CD4 cells were then activated by culturing them in wells coated with anti-CD3 and anti-CD28 antibodies, in the absence of polarizing cytokines or neutralizing antibodies against cytokines, such that CD4 T cells would choose their cell fates without being biased. We performed single-molecule fluorescent in situ hybridization( smFISH)[26] combined with immunofluorescence to quantify transcripts and protein levels in individual cells( Figures S1 and S2).
Discussion:
The large variability of cytokine expression between individual cells is very striking. Previous studies on unicellular organisms such as genetically identical populations of bacteria and yeast have observed lower variations in gene expression[35]–[38]. We were thus intrigued by the different mechanism of gene regulation in mammalian cells, where complex chromatin modeling that is not available to unicellular organisms may play an important role. Using a two-state model of transcription, where a gene needs to transition from an inactive to an active state before transcription can occur, the steady state of transcript density can be approximated to a Gamma distribution under the limiting case of short but infrequent bursts of mRNA synthesis[39]. We therefore fitted cells expressing cytokine transcripts to Gamma distributions. From the fitting, we can deduce that the average transcriptional burst size of Ifng is 159 and that of Il4 is 176( Figure 6A–B). Compared to the transcription factors, which have average burst size of 18 transcripts for Tbx21 and 36 transcripts for Gata3( Figure 6C–D), the transcriptional bursts are much larger for cytokine genes, which are comparable to the very bursty 7×-tetO construct in the work of Raj et al.[39]. As cytokine molecules are produced and secreted to the cell culture media, a uniform cytokine milieu is established because diffusion of cytokine molecules is not rate-limiting( Figure S13), leading to up-regulation of transcription factors ubiquitously. It is interesting to note that although the production of cytokine molecules is highly heterogeneous amongst cells, the expression of transcription factors as a read-out is less variable because of averaging effect from mixing cytokine in the extracellular environment. The interplay between extracellular cytokines and intracellular transcription factors may be a common strategy for mammalian cells to buffer transcriptional noise that is otherwise intrinsic to the cells. While cytokine expression appears to be decoupled from transcription factors in individual cells, we wondered how cytokine expression is regulated at the population level—for instance, how a cell population turns on IFNγ but not IL4 when cultured under Th1-favoring conditions with supplement of antibody against IL4. We quantified the expression of Ifng in cells treated with anti-IFNγ and the expression of Il4 in cells treated with anti-IL4. We found that the number of cytokine-expressing cells and hence the mean of cytokine transcripts decreased when neutralizing antibody is added to the cell culture( Figure S23). Therefore, when cytokines are sequestered, not only the respective transcription factor gets down-regulated, but the expression of the cytokine itself is also down-regulated. This observation suggests that although the expression of a cytokine is not positively correlated with the expression of its respective transcription factor in individual cells, the expression of cytokine in the entire cell population is still in concert with the expression level of the transcription factor. We postulate that transcription factor may be largely responsible for de-condensing the cytokine locus during early activation of CD4 T cells. While switching an inactive gene to the active state is a stochastic process in individual cells, the average of gene activation events is still deterministically controlled by the amount of transcription factors. As cell differentiation progresses, the activation of cytokine genes eventually becomes more ubiquitous and depends on the local concentration of active transcription factors, leading to higher positive correlation between a cytokine gene and the respective transcription factor in fully differentiated cells[27],[33],[40],[41]. In the light of our work, it will be interesting to delineate the underlying molecular mechanisms governing cytokine gene expression. In addition, given sufficient technological advances, it will be interesting to perform time-lapse experiments to track stochastic cytokine expression in individual cells over a time-course to visualize how these rare cytokine-producing cells arise and evolve over time. It will also be helpful to study the single-cell transcriptome of these cells to quantify how different these cells are from other cells. Insights from such experiments will shed light on the interplay between extracellular cytokines and the intracellular transcription factor on the fate specification of single cells. We note that mixed Th1–Th2 phenotypes were also observed concurrently by two other groups, using different experimental approaches[42],[43].
During the development of a multicellular organism , the progenitor cells , which have the potential to become any of several different cell lineages with specialized functions , commit and differentiate into one particular lineage . This differentiation of progenitors is driven by the induction of lineage-specific transcription factors , molecules that regulate gene expression . This process is often mediated by extracellular signaling molecules , including a class of molecules called cytokines that can bind to cell surface receptors , activating and/or repressing transcription factors . Here we explored the early differentiation of naive T helper ( Th ) cells , an important class of T lymphocytes that help effector immune cells to defend the body against various pathogens . We measured both mRNA and protein levels of cytokines and transcription factors in individual cells . In particular , mRNA levels were measured with single-molecule resolution . Contrary to the expression of only one set of lineage-specific transcription factors , we observed ubiquitous high-level co-expression of antagonistic transcription factors in individual cells . We found that cytokines are expressed only in a small subpopulation of cells , independent from the expression of transcription factors in individual cells . When cytokine signaling is inhibited , each cell expressed only one of the antagonistic transcription factors at high levels . This reveals a weak intracellular network that is otherwise overruled by the strong signals that emanate from extracellular cytokines . These results suggest that during the early differentiation process T helper cells acquire a mixed Th1/Th2 state , instructed by extracellular cytokines . The interplay between extracellular and intracellular signaling components unveiled in Th1/Th2 differentiation may be a common strategy for mammalian cells to buffer against noisy cytokine expression .