Lori R. Covey

Professor

Department of Cell Biology and Neuroscience Division of Life Sciences Nelson Biological Labs - B319 604 Allison Road Busch Campus Piscataway, NJ 08854-8082 Phone: (732)445-4777 Fax: (732)445-587 0 covey@biology.rutgers.edu

Research Interests

Early Signals Regulating the Induction of Switch Recombination at the Immunoglobulin Heavy Chain Locus

Post-transcriptional Regulation of CD40 ligand (CD154) mRNA Expression during T cell Activation

Research Summary

Resting IgM+ B cells differentiate into antibody-producing cells of different immunoglobulin isotypes in response to a complex array of T cellinteractions. Activation signals are mediated by specific T cell cytokines as well as by physical or cognate interactions between ligands, and their respective receptors, located on the surface of T and B cells. Our laboratory is focused on understanding how interactions at the surface of theB cell influence its differentiation into antibody-producing cells. Our assay for differentiation is transcriptional activation (I region transcription) and switch recombination of the heavy chain alleles of both transformed B cell clones and primary peripheral B cells. We have been successful at studying a number of early events using as a model system an IgM+ B cell line (Ramos) that is activated and undergoes switch recombination to a limited extent in response to CD40 ligand (CD154) plus IL-4. Upon activation, these cells become transcriptionally active at the heavy chain locus in a defined, temporal pattern that initiates with the production of Ig1 transcripts.

In analyzing the results from multiple experiments it became quite clear that the transcriptional response of the four Ig subclasses was significantly distinct. This was observed with both Ramos B cells and IgM+ primary B cells prior to and after activation with IL-4 and/or CD154. These transcriptional responses strongly correlated with both the levels of mature transcripts and protein observed in both Ramos and peripheral B cells. Therefore, to establish whether these differences were a reflection of intrinsic differences between the individual promoters we cloned the four Ig promoters from the Ramos B cell line and tested them by transient transfection and luciferase expression. Using fragments that were >95% homologous we found a wide variation in the basal response of the promoters and the response to different stimuli. One significant difference was between the expression of the Ig1 and Ig3 promoters in unstimulated Ramos cells. Recently we have identified a 36 bp sequence that appears to be responsible for the differences in basal promoter activity between Ig1 and Ig3. We are currently identifying the specific transcription factors that bind the 36bp region within the Ig1 promoter region.

A separate ongoing project in the laboratory is to analyze lymphocyte function in a young girl (GP) with non-X-linked hyper-IgM syndrome. This project focuses on two distinct areas; 1) identification and characterization of the first naturally occurring polymorphism in CD154 (G219R), and 2) analysis of B cell function upon CD154 signaling. We identified a Gly-->Arg polymorphism in aa 219 in one copy of CD154 from GP and found that it bound CD40 with reduced avidity compared to wild type CD154. However, we did not detect qualitative differences in downstream signaling pathways that led to CD23 and CD80 upregulation, Ig transcription, or switch recombination. The most significant difference we observed was the reduced ability of soluble CD40 (sCD40) to block the interaction between CD40L/G219R as assayed by the upregulation of CD80 on B cells. These results suggest that naturally occurring polymorphisms in the CD154 molecule may affect the ability of CD40-mediated functions to be blocked by soluble CD40 or anti-CD154 mAb in the therapeutic treatment of disease and graft rejection.

In analyzing CD40-mediated functions in GP's B cells we have identified a subset of functions that are affected by the immune defect. Most dramatically is a complete shutdown of transcriptional activation of the Ig genes after T cell stimulation. In addition, we found diminished CD23, and CD154 responses to CD40L and IL-4 (CD23) or T cell activation (CD154). We are trying to understand the basis for immune dysfunction and determine whether it resides with a factor that is integral to multiple signal transduction pathways in the B cell.

The second active area of research in my laboratory is identifying post-transcriptional mechanisms that regulate CD40 ligand (CD40L or CD154) expression. The necessity for strict regulation of CD154 expression may be potentially dictated by a need to limit the interaction of activated CD4+ T cells with non-selected B cells and other CD40-expressing cells during an immune response. We found that CD154 mRNA is regulated post-transcriptionally throughout a time course of a-CD3 or a-CD-3 + a-CD28 activation. Early after activation we found that the message was very unstable and displayed a half-life very similar to the t1/2 of tnf-a mRNA. However, after approximately 12 hours of stimulation we began to observe that the CD154 message, and not the tnf-a message, was becoming increasingly stable. To complement these findings we have recently carried out nuclear run-on assays to demonstrate that the CD40L message is becoming stable at a time when the transcription of the gene has been significantly down-regulated. Because we know that surface CD40L expression becomes down-regulated by receptor-mediated endocytosis, integration of transcriptional and post-transcriptional mechanisms insures a continuous, but low level of CD40L expression on activated T cells late after activation.

Recently, we have identified a T cell activation-dependent complex (termed Complex I) that binds specifically to a region of the CD154 3'UTR and mediates message stability. We have demonstrated that this complex is composed of at least two proteins that have poly(CU) binding activity. The presence of these proteins in stimulated T cell extracts directly corresponds to a time when the CD154 mRNA becomes stabilized. We are interested in studying the expression of Complex I in T cells under different conditions of stimulation. We feel the regulated decay pathway of CD154 mRNA decay may be a novel, non-ARE pathway for regulating mRNA stability.

Recent Publications

Singh, K., J. Laughlin, P. A. Kosinski and L. R. Covey.  2004.  Nucleolin is a second component of the CD154 mRNA stability complex that regulates mRNA turnover in activated T cells.  J. Immunol. 173: 976-985.

Kosinski, P. A., J. Laughlin, K. Singh and L. R. Covey. 2003.  A complex containing polypyrimidine tract-binding protein is involved in regulating the stability of CD40 ligand (CD154) mRNA.  J. Immunol. 170:979-988.

Bhushan, A. and L. R. Covey. 2001.  CD40:CD40L interactions in X-linked and non-X-linked hyper-IgM syndromes. Immunolgic Res. 24:311-324.

Bhushan, A. and L. R. Covey. 2001.  CREB/ATF proteins enhance the basal and CD154- and IL-4-induced transcriptional activity of the human Ig1 promoter. Eur. J. Immunol. 31:653-664.

Kosinski, P, B. Barnhart, B., Z. Wang, G. S. Ford, , M. Kiledjian, and L. R. Covey. 2000. Identification of a complex that binds to the CD154 3'UTR: implications for a role in message stability during T cell activation. J. Immunol. 165:4478-4486.

Bhushan, A., B. Barnhart, S. Shone, C. Song, and L. R. Covey. 2000. A transcriptional defect underlies B lymphocyte dysfunction in a patient diagnosed with non-X-linked hyper-IgM syndrome. J. Immunol. 164:2871-2880.

Barnhart, B., G. S. Ford, A. Bhushan, C. Song, and L. R. Covey. 2000. A polymorphic CD40 ligand (CD154) molecule mediates CD40-dependent signaling but interferes with the ability of soluble CD40 to functionally block CD154:CD40 interactions. Immunology J. 99:54-61.

Ford, G. S., B. Barnhart, S. Shone, and L. R. Covey. 1999. Regulation of CD40 ligand (CD154) mRNA stability during T cell activation. J. Immunol. 162:4037-4044.

Ford, G. S., C. H. Yin, B. Barnhart, K. Sztam, and L. R. Covey. 1998. CD40 ligand exerts differential effects on the expression of Ig transcripts in subclones of an IgM+ human B cell lymphoma line. J. Immunol. 160:595-605.