Au-Yeung Lab

Antigen Presenting Cell (APC)

T cells promote protective immune responses against pathogens such as viruses or bacteria, but can also react to self-antigens and promote autoimmune diseases. We study the T cell antigen receptor (TCR) signals that are required for activation of T cell immune responses. We know many of the signaling molecules involved, but not as much about the “amount” of signaling required for T cell activation. We use novel mouse model systems that take advantage of chemical-genetic approaches and fluorescent reporter genes to manipulate and monitor TCR signaling in primary T cells. Our goal is to better understand the TCR signaling requirements for T cell activation, so that we can develop new strategies for promoting better protective T cell responses, and for blocking detrimental T cell responses that cause disease.

Research

Diagram of general T cells+antigen

T cells are important immune cells that coordinate responses against pathogens, but can also react to one’s own tissues, causing autoimmunity. Activation of T cells requires signals triggered by the T cell antigen receptor (TCR). When the TCR recognizes a peptide presented by Major Histocompatibility Complex (MHC) proteins on Antigen Presenting Cells (APCs), a complex series of biochemical signals are induced within the T cell, resulting in gene transcription and T cell activation. It is important to understand how these signals drive T cell activation, so we can design better ways of promoting T cell immune responses to infections, and also find better strategies for preventing T cell activation in settings of autoimmunity.

Although these biochemical signals are detectable within seconds to minutes of TCR stimulation, it can take hours or days of TCR stimulation to observe some hallmarks of T cell activation. We are interested in figuring out what “amount” of TCR signaling is required for various types of T cell functions.

We want to know what the minimal TCR signaling requirements are for T cell activation. The lab uses techniques such as flow cytometry to analyze T cell activation markers and effector function, and analyze signal transduction of TCR signaling pathways using phospho-flow cytometry and western blotting. Our model systems are knockout and transgenic mice that enable us to tune the strength of TCR signals and to read-out how much TCR signaling T cells have experienced. Read more about our techniques and tools in the sections below.
Figure of TCR signals
Figure of AS system

Zap70 is a tyrosine kinase required for the major downstream signals triggered by the TCR. Studying the role of Zap70 in T cell biology has previously been difficult, because T cell development is blocked in Zap70 deficient mice, resulting in very few or no mature T cells. Human patients that lack ZAP-70 expression have a form of Severe Combined Immunodeficiency (SCID), in which there are few mature T cells that fail to signal properly through their TCRs.

Using a strategy developed by our collaborator (Kevan Shokat, UCSF), we generated a Zap70 mutant that retains kinase activity, but can be inhibited by a small molecule inhibitor. This drug (3-MB-PP1) is an analog of the kinase inhibitor PP1, is too bulky to fit in the Zap70 kinase domain, but is able to bind and inhibit the mutant Zap70 kinase. We call this mutant Zap70 analog-sensitive or Zap70(AS). We now have mice that express this Zap70(AS) mutant and continue to study the requirements for Zap70 activity in various stages of T cell maturation, activation, and effector function.

References (with hyperlinks):

  1. Au-Yeung BB, Levin, SE, Zhang C, Hsu LH, Cheng DA, Killeen N, Shokat KM, and Weiss A. A genetically selective inhibitor reveals a Zap70 catalytic-independent function in TREG cells. Nat Immunol 11:1085-92. 2010.
  2. Jenkins MR, Stinchcombe JC, Au-Yeung BB, Asano Y, Ritter AT, Weiss A and Griffiths GM. Distinct structural and catalytic roles for Zap70 in formation of the immunological synapse in CTL. eLife 2014;3:e01310. 2014.
  3. Au-Yeung BB, Melichar HJ, Ross JO, Cheng DA, Zikherman J, Shokat KM, Robey EA, and Weiss A.  Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development. Nat Immunol 15:687–694. 2014.

To visualize the relative “amount” of TCR signaling that is experienced by individual T cells during an immune response, we have recently taken advantage of a novel reporter transgene, which consists of the gene encoding GFP driven by the promoter and enhancer elements of the Nr4a (Nur77) gene.

Diagram of Nur77-GFP

Expression of the Nur77-GFP transgene is in relative proportion to the strength of TCR signaling. Weak or a short duration of TCR signaling will weakly upregulate GFP expression, while a strong and long-lasting TCR stimulus will drive robust GFP expression. We combined the Zap70(AS) and Nur77-GFP systems, and can modulate TCR signal strength and read out these differences in TCR signal intensity in the same T cells.

References:

  1. Zikherman J, Parameswaran R, and Weiss A. Endogenous antigen tunes the responsiveness of naive B cells but not T cells. Nature 489(7414):160-4. 2012
  2. Zikherman J, and Au-Yeung BB. The role of T cell receptor signaling thresholds in guiding T cell fate decisions. Curr Opin Immunol 33:43-8. 2015.

TCR signal strength Nur77-GFP reporter expression

Using these approaches, we have shown that there is a minimum amount of TCR signaling required for positive selection of immature T cells in the thymus, and for proliferative responses of mature T cells. It appears that no matter the TCR signal strength experienced by T cells, they accumulate a certain minimum amount of TCR signaling to reach a threshold.


When the strength of TCR signaling is decreased, this affects how many cells are able to reach the TCR signal threshold, but not the threshold itself. We continue to study the ramifications of this threshold in T cell biology.

References:

  1. Au-Yeung BB, Melichar HJ, Ross JO, Cheng DA, Zikherman J, Shokat KM, Robey EA, and Weiss A.  Quantitative and temporal requirements revealed for Zap70 catalytic activity during T cell development. Nat Immunol 15:687–694. 2014.
  2. Au-Yeung BB, Zikherman J, Mueller L, Cheng DA, Chen Y, Ashouri J, Shokat KM, and Weiss A.  A sharp TCR signal threshold for proliferation. Proc Natl Acad Sci USA 111(35):E3679-88. 2014.
  3. Au-Yeung BB, Smith GA, Mueller JL, Heyn CS, Garret Jaszczak R, Weiss A, and Zikherman J. IL-2 modulates the TCR signaling threshold for CD8 but not CD4 T cell proliferation on a single cell level. J Immunol 198:2445-2456. 2017.
  4. Zikherman J, and Au-Yeung BB. The role of T cell receptor signaling thresholds in guiding T cell fate decisions. Curr Opin Immunol 33:43-8. 2015.

Publications

Contact Us

Byron Au-Yeung

Principal Investigator: Byron Au-Yeung

Email: byron.au-yeung@emory.edu

Mailing address:

615 Michael St.

Whitehead Biomedical Research Building

Room 255

Atlanta, GA 30322