PRIMARY FACULTY

Brian C. Schaefer, Ph.D.
Associate Professor
Microbiology & Immunology
 
4301 Jones Bridge Road
Bethesda MD 20814
Office: 301-295-3402
Fax: 301-295-1996
bschaefer@usuhs.mil

MOLECULAR MECHANISMS OF LYMPHOCYTE ACTIVATION

The research in my lab is focused on investigating signaling events that regulate antigen-dependent lymphocyte activation, with particular emphasis on T cell receptor activation of NF-B. Our experimental approach relies on combining imaging technologies with biochemical and cell biological techniques to understand how signals are transmitted within the cell. Over the next several years, we will focus on three major projects, outlined below.

1. Defining the kinetics and subcellular localization of protein-protein interactions between signaling intermediates in T cell receptor (TCR) activation of NF-B transcription factor proceeds via a complex cytoplasmic cascade, involving multiple phosphorylations and poly-ubiquitination events. These enzymatic modifications cause the reorganization of molecular complexes that are required intermediates in the activation of the IB kinase (IKK) complex, the enzyme complex is ultimately responsible for activating NF-B. We are using advanced confocal microscopy techniques (fluorescence resonance energy transfer [FRET], photo-activatable and photo-convertible fluorescent proteins) to define the molecular mechanisms and kinetics of cytoplasmic signal transmission from the TCR to the IKK complex.

2. Defining the molecular mechanisms whereby MALT1 and Bcl10 contribute to the etiology of MALT lymphomas. Activation of NF-B is required to stimulate B cell division in response to B cell receptor (BCR) ligation. Bcl10 and MALT1 are essential partners in this signaling pathway in normal B cells, and that perturbation of the normal signaling function of Bcl10 or MALT1 contributes to the etiology or maintenance of certain B lymphocyte non-Hodgkin lymphoma (B-NHL) malignancies, particularly marginal zone B cell lymphomas of the mucosa-associated lymphatic tissue (MALT lymphomas). We are currently defining subdomains of each protein that are required for NF-B activation and for protein-protein associations with other signaling partners (Rossman et al., 2006; Langel et al., 2008). Additionally, we are working with the cIAP2/MALT1 fusion proteins characteristic of many MALT lymphomas, investigating how this molecular fusion differs in its enzymatic activities from the wild-type MALT1 protein.


 
   

Figure 2. Three-dimensional fluorescent image of a mouse CD4+ T cell stained with antibodies against CD4 (blue), PKC (green) and Bcl10 (red). Note that PKC becomes enriched at the T cell/APC contact site, and that Bcl10 becomes clustered in discrete focal regions in the cytoplasm. The middle panel shows a bright field (DIC) image, allowing both the T cell and the stimulatory antigen-presenting cell (APC) to be visualized. Clicking on the link in the top panel will open the corresponding QuickTime movie, showing a 360º rotation of this cell.

 

Figure 3. Three-dimensional fluorescent image of a T cell/APC conjugate.A CD4+ T cell clone expressing PKC -CFP (blue) and Bcl10-YFP (green) is shown interacting with a stimulatory APC. The characteristic redistributions of PKC and Bcl10 are again seen, as in Figs 1-2. Antibody staining of tubulin (red) demonstrates re-orientation of the T cell microtubule organizing center (MTOC) towards the APC, in response to antigen stimulation. The middle panel shows a bright field (DIC) image. Clicking on the link in the top panel opens the corresponding QuickTime movie, showing a 360º rotation of this 3-D image.

3. Investigation of the role of specific NF-B signaling intermediates in T cell functional responses. Previously published data have reported that T cells lacking PKC, Bcl10, or Malt1 are unable to activate NF-B and enter the cell cycle. While we have confirmed that CD4+ T cells from PKC, Bcl10 and Malt1 knock-out mice show severe impairment of proliferation in response to TCR stimulation, we were surprised to observe that knock-out CD8+ T cells proliferate to a similar extent as wild-type cells in response to strong TCR signals. Further analyses of knock-out T cells from these animals yielded several findings at contrast with previous reports, most importantly that NF-B activation is only partially impaired in CD8+ T cells. Additionally, we showed that there is a graded degree of severity in T cell activation phenotypes, with PKC>Bcl10>>MALT1. Overall, our data suggest the existence of a significant TCR-regulated NF-B activation pathway in CD8+ T cells that is independent of PKC, Bcl10 and Malt1 (Kingeter et al., 2008). We are continuing these studies, attempting to identify signal transducers that are required for this residual/alternative NF-B activation in CD8+ T cells. We are also performing experiments to assess the functional capability of CD8+ T cells that develop in the absence of PKC, Bcl10, and Malt1, to assess whether or not these cells have a measurable defect in their ability to differentiate to CTL effectors. Together, these studies are contributing important information regarding the role of individual NF-B signal transducers and the TCR-to-NF-B signaling pathway in T cell biology.

Selected Publications

Resources