Yongqiang Feng, Ph.D. - US grants

ABSTRACT The adaptive immune system provides remarkable defense against fast-evolving pathogens. In the meantime, tolerance mechanisms coevolved to prevent deleterious immune responses, among which regulatory T (Treg) cells play a dominant, active role. The adaptive immune system is established during postnatal development through continuous differentiation and egress of T and B cells into the peripheral lymphoid and non-lymphoid organs. It appears that the cells of the adaptive immune system that emerge at the early and late stages of life are programmed differently in their immunological function. Which factors are present at particular stages of life to program the function of immune cells differently is an unsolved mystery. How T and B cells developed at different stages of life respond to specific immunological challenges also remains to be fully explored. Revealing these features of the adaptive immune system will offer us better ways to manipulate immune cells for the treatment of immunological diseases. Genetic tracing tools are needed to address these important questions. However, despite significant progress in the field, tracing and investigating T and B cells emerging at given stages of life remain a technical challenge. To solve this issue, we propose a robust, versatile method to trace T and B cells during their early differentiation in experimental mice. In our preliminary experiments, we have successfully engineered this mouse strain. Specifically, we took advantage of transient Rag1 expression during early T and B cell differentiation and used it to drive the expression of three proteins to trace T and B cells with tamoxifen, to report Rag1 expression with fluorescent protein GFP, and to deplete Rag1-expressing cells with diphtheria toxin when necessary. We have validated all these genetic elements and their functions in the mice. In our proposed research, we will improve our methods to meet various experimental requirements. Furthermore, we will use Treg cells as a proof of concept of our technology in revealing the differential immunosuppressive activity and gene regulatory networks of Treg cells developed at the early and later stages of life in mouse models of autoimmune diseases. In summary, our study generates a robust, versatile genetic toolbox in experimental mice to report, trace, and acutely deplete developing T or B cells when necessary at given stages of life, offering a powerful approach to address many significant questions about adaptive immunity. Our research also produces insights into different Treg functions specified during postnatal development and reveals novel factors and mechanisms controlling Treg-mediated immune tolerance.

Identify novel modulators of regulatory T cell function from the dynamic cis-proteomes Regulatory T (Treg) cells are induced in the thymus or periphery to suppress effector T cells that not only cause autoimmune diseases but also kill tumors. Therefore, Treg cells serve as a therapeutic target to treat a variety of immunological diseases and cancer. Despite intensive investigations, methods to achieve this goal remain to be fully developed. Here, we aim identify novel factors to reprogram Treg immune suppressive function. To this end, we systematically examined the epigenetic and transcriptional mechanisms governing Treg function through Treg master regulator Foxp3. Treg cells are also regulated by environmental cues, including T cell antigen receptor (TCR) agonists and Interleukin-2 (IL-2), that act through transcriptional mechanisms to modulate Treg fitness, homing, and suppressive function. We hypothesize that opposing nuclear programs coordinate to interpret environmental cues, thus balancing Treg suppressive function. Identification and characterization of these programs will produce mechanistic insights into Treg function, leading to novel methods to modulate Treg suppressive activity. To test our hypothesis, we propose to develop a proteomics method to profile signal-induced dynamic protein components at the cis-regulatory elements governing Treg suppressive function. In our preliminary experiments, we adapted a proximity biotinylation?based proteomics method to identify the proteins at Foxp3-associated cis-regulatory elements in the steady state and after cells receiving TCR or IL-2 stimulation. These experiments uncovered unprecedented details of proteins potentially involved in the regulation of Foxp3-target gene expression at the resting state or upon TCR and IL-2 stimulation. As a proof of concept, these results demonstrate the feasibility of proximity biotinylation, when coupled with comparative proteomics, in unbiasedly profiling the nuclear proteins involved in signal-dependent transcriptional regulation. On the basis of these preliminary results, we propose to further define the temporal dynamics of the cis- proteomes in Treg cells after cells receiving TCR and IL-2 stimulation at the early, middle, and later stages of signal transduction. Our in-depth comparative analysis will reveal the proteins recruited to and depleted at Foxp3 targets in response to TCR or IL-2 stimulation. On the other side, we will integrate genetic perturbations and functional assays to identify novel regulators of Treg cell function from signal-dependent dynamic cis-proteomes. We will also determine the mechanisms by which these novel factors control Treg cell function by modulating their response to TCR or IL-2 signaling. Overall, we develop a comparative proteomics method to profile cis-proteomes in situ without engineering new cells or organisms. We will determine the temporal dynamics of the Treg cis-proteomes upon TCR and IL- 2 stimulation and use genome editing approach to uncover their roles in Treg immune suppressive function. These factors may serve as new targets to improve Treg-based treatment of autoimmune diseases.

Robust immune tolerance conferred by Foxp3 transcriptional regulation Regulatory T (Treg) cells actively suppress self-destructive T cells that cause a variety of autoimmune diseases. Despite significant progress, Treg-based treatment is significantly constrained by limited methods to improve Treg suppressive capacity. We aim to dissect the mechanistic processes of Treg induction and lineage maintenance to uncover the factors and mechanisms conferring robust immune suppressive function. Treg cells are induced by the convergence of environmental cues delivered to differentiating T cells with considerable variations, resulting in stochastic Treg development. Besides, the DNA sequences of the genetic elements regulating Treg lineage identity also vary among individuals. Given the enormously diverse T cell antigen receptors (TCRs) and target specificities, Treg cells are induced with many uncertainties. Once committed, Treg fate is maintained for extended suppressive function by cell-intrinsic and -extrinsic factors that constantly fluctuate. All these uncertainties raise a question about how robust immune tolerance is conferred by Treg cells. Study of Treg master regulator Foxp3 offers a unique approach to address this question, because Foxp3 expression centers Treg fate determination and function. We hypothesize that an adequate buffering capacity conferred by efficient Treg development and lineage stability opposes the genetic variations and immune perturbations. To test this hypothesis, we examined the Foxp3 enhancers that dictate Treg induction and lineage stability. Individual Foxp3 enhancers were known to play stage-specific roles in Foxp3 induction or maintenance. However, mice bearing their individual mutations develop mild if any immune dysregulation despite significant defects in Treg development or lineage stability. To solve this mystery, we examined the epigenetic mechanisms mediating Foxp3 expression and hypothesize that Foxp3 enhancers coordinate to enable efficient Treg induction or stable lineage identity for adequate Treg buffering capacity. We generated new mouse strains to test our hypotheses and found that deletion of two interacting Foxp3 enhancers caused fatal autoimmune diseases accompanied with severe defects of Treg induction or lineage stability. This result together with other studies delineates a full spectrum of Treg buffering capacity acquired through coordinating Foxp3 enhancers. In the proposed study, we will fully uncover the immunological consequences of mice with severely reduced Treg induction or lineage stability. We will develop new algorithms and use single cell RNA sequencing to assess Treg repertoire diversity and lineage stability to infer Treg buffering capacity. We will also use our newly developed mouse genetic tools to determine the role of continuous thymic Treg induction in maintaining the Treg buffering capacity. Overall, our study will uncover a full spectrum of Treg suppressive capacity conferred by Foxp3 transcriptional regulation. It will improve our basic understanding of Treg-related autoimmune diseases.