Research InterestsAdaptive Immunity, Developmental Immunology, T-cells
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There are two main lines of inquiry that we focus on in our laboratory. Our research aims to 1) define the molecular mechanisms that drive hematopoietic stem cells to develop into T cells or dendritic cells in the thymus, and 2) to understand how certain subsets of T cells are programmed to function in the specialized environments of the female reproductive tract . We approach this question through the lens of transcriptional regulation, with a focus on the E protein transcription factors HEBAlt and HEBCan.
T cell commitment can be defined as the point at which a developing cell loses the ability adopt any other fate. This process occurs in a specialized organ called the thymus. The earliest precursors to enter the thymus are not yet committed to the T cell lineage but also have the potential to become dendritic cells or natural killer cells. Upregulation of the T-lineage genes needed for commitment to the T-cell lineage and for exclusion of other lineage choices is induced in part by the interaction of the Notch1 receptor with the Delta-like ligands expressed on thymic stromal cells. One of the transcriptional regulators induced by Notch signaling is HEBAlt. We have shown that HEBAlt promotes T cell development in two different ways: 1) by inhibiting dendritic cell development, and 2) by upregulating genes required for pre-TCR signaling, which is critical for committed T cell precursors to survive and continue to differentiate. We have also found that HEB factors play an important role in the development of gamma-delta T cells, which are found in large numbers in barrier tissues such as the skin, intestine, lung, and vagina. We have found that whereas HEBAlt is completely silenced in alpha-beta T cells it remains on at low levels in gamma-delta T cells, and that perturbation of HEB factors can influence the specific types of gamma-delta T cells that develop.
There are currently three main projects ongoing in the laboratory:
- Regulation of T cell development by HEBAlt and HEBCan. We are comparing the lineage choice and developmental trajectory of thymocytes mice that are wildtype for HEB (express endogenous HEBAlt and HEBCan), are deficient in HEB (express neither HEBAlt nor HEBCan), or express HEBAlt only (no HEBCan, transgenic for HEBAlt). Since the germline allele for HEB-deficiency is embryonic lethal, we are using two in vitro model systems to study the development of T cells, dendritic cells, and natural killer cells from precursors isolated from these animals. The first approach is fetal thymic organ culture, in which E14.5 fetal thymic lobes are dissected and cultured for progressive time periods, followed by analysis of developmental stages and lineages by flow cytometry and quantitative realtime PCR for gene expression. The second approach is to use OP9-DL co-cultures, which create the environment needed for T cell development from fetal liver hematopoietic stem cells by providing strong Delta-like signals. We are currently breeding mice that conditionally lack endogenous HEB factors specifically in T cells, with or without transgenic HEBAlt, to study their roles in T cell development in adult mice. Recently we have performed an RNAseq screen from T cells precursors derived by OP9-DL1 co-cultures, which provide a mechanistic basis for our observations that HEB factors are critical for Notch signaling, pre-TCR signaling, growth, and survival.
- Origins and development of thymic dendritic cells. One of the roads not taken in the journey towards the T cell lineage is toward becoming a thymic dendritic cell. Thymic dendritic cells reside within the thymus and play an important role in mediating negative selection of potentially autoreactive T cells. However, the developmental pathway that produces these cells is unknown. There are three types of thymic dendritic cells: CD8α+ cDCs (classical dendritic cells), CD8α- cDCs, and pDCs (plasmacytoid dendritic cells). There is great debate over whether these cells develop in the bone marrow or spleen and migrate into the thymus, or whether they develop within the thymus. We have identified a possible precursors of thymic pDCs in the thymus, the DN1d cells. Thymic pDCs and DN1d cells which share high expression of the transcriptional regulators Spi-B, HEBCan, and Notch1, whereas DN1e and cDCs express high levels of the E protein antagonist Id2. By intrathymic and intravenous injection studies, we have shown that DN1d and DN1e cells can both develop into DCs in the thymus in vivo. We have also shown that low levels of DL Notch ligands similar to those expressed in the thymic medulla, where thymic DCs reside, is permissive for DC development in vitro. Current experiments are focused on determining the types of DCs that can be generated in vivo from these subsets, as well as the influence of specific transcription factors and signaling molecules on their development within the thymic environment.
- Generation and function of vaginal and uterine gamma-delta T cells. Our studies of T cell development in FTOC suggest that HEB factors are important in directing a second wave of fetal gamma-delta T cell development, corresponding to the subset that populates the vagina, uterus, and lung. It is known that in the lung, these cells produce IL-17 and are protective against certain infections. However, their roles in the vaginal and uterine tissues are unclear. In a new project, we have embarked on a characterization of the functions of gamma-delta T cells in the complex environments of the vagina and uterus. The epithelial compartment of the vagina and uterus are both extensively remodeled during the estrus cycle in response to the sex hormones estradiol and progesterone. It has been previously shown that sex hormones affect the macrophages and neutrophils in the vagina and uterus, but their influence on T cells is unknown. We therefore treat the mice with estradiol or progesterone, and analyze the numbers, percentages, localization, and activation status of the local gamma-delta cells by flow cytometry, immunofluorescence, and quantitative realtime PCR. Another layer of complexity is present in the vaginal environment, in that it is populated by commensal bacteria which are collectively referred to as the microbiome. These bacteria are essential to maintain a healthy vaginal environment that is conducive to reproduction and also function to compete out pathogenic bacteria. Therefore, we are treating mice with antibiotics to monitor the response of the gamma-delta T cells to perturbation of the microbiome. Finally, we are collaborating with the laboratory of Dr. Scott Gray Owen to assess the response of vaginal and uterine gamma-delta T cells to colonization of the reproductive tract by specific pathogenic bacteria. Once the roles of these gamma-delta T cells have been established, we will analyze the ability of gamma-delta T cells to carry out their proper functions in the presence or absence of HEBAlt and/or HEBCan in conditional knockout and transgenic mice.
Publications and Awards
- Braunstein, M., Anderson, M.K. (2012) HEB in the Spotlight: Transcriptional Regulation of T-Cell Specification, Commitment, and Developmental Plasticity. Clinical and Developmental Immunology. DOI: 10.1155/2012/678705.
- Moore, A.J., Sarmiento, J., Mohtashami, M., Braunstein, M., Zuniga-Pflucker, J.C., and Anderson, M.K. (2012) Transcriptional priming for intrathymic precursors for dendritic cell development. Development. 139:373-384.
- Braunstein, M. and Anderson, M.K. (2011) HEB-deficient T-cell precursors lose T-cell potential and adopt an alternative pathway of differentiation. Molecular and Cellular Biology, 31(5):971-82.
- Braunstein, M., Rajkumar, P., Claus, C.L., Vaccarelli, G., Moore, A.J., Wang, D., and Anderson, M.K. (2010) HEBAlt enhances the T-cell potential of fetal myeloid-biased precursors. International Immunology. 22(12):963-972.
- Wang, D., Claus, C.L., Rajkumar, P., Braunstein, M., Moore, A.J., Sigvardsson, M., and Anderson, M.K. (2010) Context-dependent regulation of hematopoietic lineage choice by HEBAlt. Journal of Immunology. 185(7):4109-17.
- Braunstein, M., and Anderson, M.K. (2010) Developmental progression of fetal HEB-/- precursors to the pre-T cell stage is restored by HEBAlt. European Journal of Immunology. 40(11):3173-3182.
- Wang, D., Claus, C.L., Vaccarelli, G., Schmitt, T., Zuniga-Pflucker, J.C., Rothenberg, E.V. and Anderson, M.K. (2006) The bHLH transcription factor HEBAlt is expressed in early T cell precursors and enhances progression through T cell development. Journal of Immunology, 177:109-119.