PAT Project 4

Uniformed Services University

Development of transgenic mouse models for studying in vivo polysaccharide- and protein-specific Ig responses to Streptococcus p


Fluorescence microscopy for both static and live imaging of the spatiotemporal and functional behavior of distinct cell types in secondary lymphoid organs (e.g. lymph node and spleen) following murine in vivo immunization represents a powerful tool for dissecting the mechanism of a humoral immune response to intact pathogens and vaccines. A major limitation to this approach is the relatively few numbers of B and T cells expressing specificity for any given antigen, and thus the difficulty in finding them in tissue sections. This problem has been largely overcome through the use of B or T cells from mice in which a rearranged B cell receptor (BCR) or T cell receptor (TCR) transgene has been introduced. These mice provide a relatively large source of antigen-specific B or T cells that can be transferred into recipient mice prior to immunization, and then identified either through prior live cell labeling, antibody recognition of allotype differences between donor (transgenic) and recipient mice, fluorescence-labeled intact antigen (B cells) or MHC-peptide tetramers (T cells), or fluorescence-labeled anti-idiotype monoclonal antibodies (B or T cells).

Experimental Plan

We have been investigating the cellular parameters that underlie the distinct humoral (protein- and polysaccharide-specific) immune responses to intact Streptococcus pneumoniae, capsular type 14 (Pn14) versus a soluble conjugate vaccine of protein and polysaccharide (PPS14) derived from Pn14, or purified PPS14 alone (see Project#2). To better understand the mechanistic parameters underlying these Ig responses we are producing transgenic mice that will express large numbers of 1) B cells specific for PPS14, 2) B cells specific for pneumococcal surface protein A [PspA], and 3) T cells specific for MHC-II-PspA peptide. In addition we have engineered intact Pn14 to express a PspA-cOVA peptide fusion protein in its cell wall in order to utilize T cells from cOVA peptide-specific TCR transgenic mice available in our mouse colony. We have also obtained anti-idiotype mAbs that recognize the PPS14-specific and PspA-specific BCR, respectively, being used to create our transgenic mice, in order to eventually identify the transgenic B cells. The rearranged genomic DNA for the PPS14- and PspA-specific variable heavy and light chain Ig genes have already been cloned and placed in suitable vectors to begin production of transgenic mice.

These transgenic models will allow us to probe more deeply into the mechanisms underlying protein- and polysaccharide-specific humoral immune responses to both whole bacteria and soluble vaccines in an intact murine host and investigate approaches to improve vaccine immunogenicity.

References (Review Articles)

  • 1. Mebius, R. E., and G. Kraal. 2005. Structure and function of the spleen. Nat Rev Immunol 5:606-616.
  • 2. Germain, R. N., M. J. Miller, M. L. Dustin, and M. C. Nussenzweig. 2006. Dynamic imaging of the immune system: progress, pitfalls and promise. Nat Rev Immunol 6:497-507.
  • 3. Allen, C. D., T. Okada, and J. G. Cyster. 2007. Germinal-center organization and cellular dynamics. Immunity 27:190-202.
  • 4. Kraal, G., and R. Mebius. 2006. New insights into the cell biology of the marginal zone of the spleen. Int Rev Cytol 250:175-215.
  • 5. Cahalan, M. D. 2008. Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs. Annu. Rev. Immunol. 26:585-626.
  • 6. Harnett, M. M. 2007. Laser scanning cytometry: understanding the immune system in situ. Nat Rev Immunol 7:897-904.
  • 7. Harwood, N. E., and F. D. Batista. 2008. New insights into the early molecular events underlying B cell activation. Immunity 28:609-619.
  • 8. Alvarez, D., E. H. Vollmann, and U. H. von Andrian. 2008. Mechanisms and consequences of dendritic cell migration. Immunity 29:325-342.
  • 9. Lopez-Bravo, M., and C. Ardavin. 2008. In vivo induction of immune responses to pathogens by conventional dendritic cells. Immunity 29:343-351.
  • 10. Roozendaal, R., R. E. Mebius, and G. Kraal. 2008. The conduit system of the lymph node. Int Immunol 20:1483-1487.
  • 11. Bousso, P. 2008. T-cell activation by dendritic cells in the lymph node: lessons from the movies. Nat Rev Immunol.
  • 12. Batista, F. D., and N. E. Harwood. 2009. The who, how and where of antigen presentation to B cells. Nat Rev Immunol 9:15-27.
  • 13. Sarris, M., and A. G. Betz. 2009. Shine a light: imaging the immune system. Eur J Immunol 39:1188-1202.

Pathology Contact Info

Uniformed Services University of the Health Sciences
Department of Pathology
4301 Jones Bridge Road
Bethesda, Maryland 20814-4799
Room: B3154
Phone: (301) 295-3450
fax: (301) 295-1640