Intercalated Cell Physiology Laboratory

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.

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.

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 T_REG 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.

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.

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.

Please see the NCBI website for recent publications.

Professor of Medicine and Physiology
Division of Renal Medicine
Woodruff Memorial Building (WMB), Room 338
1639 Pierce Drive, NE
Atlanta, GA  30322
Email Susan
Phone:  (404) 727-2525

Focus on Research: The role of renal intercalated cells in electrolyte balance and blood pressure regulation

Susan Wall, MD, is a physician-scientist who has made many major scientific contributions. Most notably, her laboratory was the first to report a role of intercalated cells, a minority cell type in kidney, in blood pressure regulation. She and her colleagues have described the mechanism of intercalated cell transporter regulation by aldosterone and angiotensin II and have characterized the downstream signaling cascades by which this regulation occurs. Since her initial observation that intercalated cells regulate blood pressure, a number of other groups throughout the world have become interested in this aspect of intercalated cell physiology. To explore these questions, the Wall Laboratory has used many techniques, such as renal tubules perfused in vitro, immunoblots, PCR and balance studies. In addition, her collaborators provide a wealth of expertise in other techniques, such as immunogold cytochemistry with morphometric analysis, immunofluorescence and immunohistochemistry.

Wall has been the recipient of a number of research awards, such as the 2014 Carl W. Gottschalk Distinguished Lecturer of the American Physiological Society (APS) and the 2011 Barry Brenner Lectureship of the ASN. Her laboratory has had uninterrupted NIH funding since 1993. She was a member of the CMBK NIH study section from 2003 to 2007.

Wall has had an important leadership role not only in running her laboratory, but also in serving as scientific director of the Emory Renal Division as well as professional societies, such as the American Society of Nephrology, the American Physiological Society, and the American Society for Clinical Investigation (ASCI). She was chair of the 2017 American Society of Nephrology Kidney Week meeting in New Orleans, as well as chair (2002-2005) and treasurer (1999-2002) of the Renal Section of the American Physiological Society. Moreover, in her capacity as Emory Representative to the ASCI (2002-present), she has been instrumental in facilitating the election of a number of physician scientists at Emory to that society. Wall has also served in an editorial capacity as associate editor for the American Journal of Physiology: Renal (2001 to 2013) and then as a member of the American Physiological Society Publications Committee (2013-2017). Finally, she is an active clinical nephrologist who attends on the renal consult services at Emory.