Immunology
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Ellis Reinherz

Department of Medicine, Harvard Medical School
Dana-Farber Cancer Institute
44 Binney Street
HIM 419
Boston, MA 02115
Tel: 617-632-3412
Fax: 617-632-3351
email:ellis_reinherz@dfci.harvard.edu
16 Postdoctoral Fellows

 Ellis Reinherz

The Laboratory of Immunobiology continues to focus on T lymphocyte recognition of antigen as well as the process of T cell activation and differentiation. As in previous years, particular emphasis has been placed on studying the structure-function relationship of the T cell receptor (TCR) for antigen in major histocompatibility complex (MHC) products (CD3-Ti), CD2 and CD4.

The TCR consists of multiple transmembrane polypeptide chains on the surface of T lymphocytes. The disulfide-linked a-b heterodimer (Ti) is the clonally unique component that possesses a recognition site for antigen in the context of the MHC. Sequence analysis of a and b subunits strongly argues that their ectodomains form a recognition unit reminiscent of an immunoglobulin Fab. This notion has been confirmed in crystallographic studies of TCR subunit fragments. On the other hand, the invariant CD3 components (g, d, e, z and h) possess lengthy cytoplasmic tails containing ITAMs (immune cell tyrosine based activation motifs) and are involved in signal transduction. To date, most of the attributes of TCR recognition have been studied largely indirectly because of the intimate membrane association of this complex.

To understand the process by which T cells recognize pathogens in explicit molecular terms, recent efforts have begun to focus on the structural nature of the T cell receptor (TCR). However, many efforts to express soluble TCR a-b heterodimers in both prokaryotic and eukaryotic systems have been hampered by inefficient pairing of a and b subunits in the absence of their respective transmembrane regions and associated CD3 components. We have recently developed a methodology to overcome this obstacle by adding 30 amino acid-long segments to the carboxy termini of a and b extracellular domains via a thrombin cleavable flexible linker. These peptide segments (Base-p1 for a and Acid-p1 for b) were previously shown to selectively associate to form a stable heterodimer coiled coil termed the leucine zipper. Homodimeric structures are not favored due to the electrostatic repulsion among amino acid side chains. Furthermore, the yield of these engineered proteins was 5-10 fold greater than that of the TCR expressed in the absence of the synthetic leucine zipper. Through the use of a panel of mAbs directed at native a and b epitopes within constant and variable regions, it was further shown that the fusion heterodimer was native. We have now developed a way to make soluble a-b TCRs in a form which permits them to crystallize. This accomplishment should make it possible to discern the structural basis of TCR function including antigen recognition in the near term.

CD4 is the T cell co-receptor specific for class II MHC proteins. It appears to contact with nonpolymorphic regions of class II molecules, either directly in an adhesion interaction or in a ternary complex with the TCR. CD4 is linked intracellularly with p56lck, a SRC-like tyrosine kinase. Association of CD4 with class II MHC-TCR complex brings this kinase into the TCR signaling pathway. The Laboratory continues to investigate the structural basis of CD4 class II interaction as well as CD4-gp120 interaction using site-directed mutagenesis in conjunction with a high resolution CD4 structure.

The CD2 molecule on T lymphocytes is a transmembrane surface glycoprotein which facilitates cell-cell contact. The amino terminal domain of CD2 (domain 1) mediates its adhesion function by binding to LFA-3 (CD58), another cell surface glycoprotein widely expressed on various cell types including hematopoietic and epithelial cells. Both CD2 and CD58 are members of the Ig gene superfamily. The importance of CD2 function in the normal human immune response has been well documented 1) for the process of cognate recognition involving helper T cells and antigen presenting cells; 2) for the cytolytic effector function of natural killer (NK) cells and cytotoxic T lymphocytes (CTL); and 3) as a basis of the thymocyte-thymic epithelial cell interaction. Moreover, in T lymphocytes perturbation of the extracellular segment of CD2 with specific mAbs leads to activation via a TCR-dependent mechanism. Also, engagement of CD2 by its ligand CD58 together with mAbs specific for the membrane proximal second domain of CD2 (domain 2) can activate T cells indicating that coordinate binding of ligands to both domains of CD2 generates an intracellular activation signal. We continue to investigate the various physiologic ligands of CD2 as well as the structural basis for the CD2-CD58 interaction. A recent discovery in collaboration with the Ritz laboratory shows that CD2 upregulates IL-12 responses explaining why CD2 has a pivotal role in facilitating TH1 type directed inflammation.

 

Papers & Publications:

  1. Ghendler Y, Teng M-K, Liu J-H, Witte T, Liu J, Kim KS, Kern P, Chang H-C, Wang J-H, Reinherz EL. Differential thymic selection outcomes stimulated by focal structural alteration in peptide/MHC ligands. Proc Natl Acad Sci USA 1998; 95:10061-10066.
  2. Li J, Nishizawa K, An W, Hussey RE, Lialios FE, Salgia R, Sunder-Plassmann R, Reinherz EL. A cdc15-like adaptor protein (CD2BP1) interacts with the CD2 cytoplasmic domain and regulates CD2-triggered adhesion. EMBO J. 1998; 17:7320-7336.
  3. Wang JH, Smolyar A, Tan K, Liu JH, Kim M, Sun ZJ, Wagner G, Reinherz EL. Structure of a heterophilic adhesion complex between human CD2 and CD58 (LFA-3) counter-receptors. Cell 1999; 97:791-803.
  4. Reinherz EL, Tan K, Tang L, Kern P, Liu J-H, Xiong Y, Hussey RE, Smolyar A, Hare B, Zhang R, Joachimiak A, Chang H-C, Wagner G, Wang J-H. The structure of a T cell receptor in complex with peptide and MHC class II. Science 1999; 286:1913-1921.
  5. Sun Z-YJ, Kim KS, Wagner G, Reinherz EL. Mechanisms contributing to T cell receptor signaling and assembly revealed by the solution structure of an ectodomain fragment of the CD3eg heterodimer. Cell 2001; 105:913-923.
  6. Wang J-H, Meijers R, Xiong Y, Liu J-H, Sakihama T, Zhang R, Joachimiak A, Reinherz EL. Crystal structure of the human CD4 N -terminal two domain fragment complexed to a class II MHC molecule. Proc Natl Acad Sci USA. 2001; 98:10799-10804.
  7. Kim MK, Sun Z-Y J, Byron O, Campbell G, Wagner G, Wang J-H, Reinherz EL. Molecular dissection of the CD2-CD58 counterreceptor interface identifies CD2 Tyr86 and CD58 Lys34 residues as the functional 'hot spot'. J Mol Biol., 2001; 312:711-720.
  8. Sasada T, Ghendler Y, Neveu JM, LaneWS, Reinherz EL. A naturally processed mitochondrial self-peptide in complex with thymic MHC functions as a selecting ligand for a viral-specific T cell receptor. J Exp Med. 2001; 194:883-892.
  9. Zhang C W-H, Chishti Y, Hussey RE, Reinherz EL. Expression, purification and characterization of recombinant HIV gp140: the gp41 ectodomain of HIV or SIV is sufficient to maintain the retroviral envelope glycoprotein as a trimer. J Biol Chem. 2001; 276:39577-39585.
  10. Kim M, Chen B, Hussey RE, Chishti Y, Monte fiori D, Hoxie JA, Byron O, Ca mpbell G, Harrison SC, Reinherz EL. The stoichiometry of trimeric SIV glycoprotein interaction with CD4 differs from that of anti-envelope antibody Fab fragments. J Biol Chem. 2001; 276:42667-42676.
  11. Moody AM, Chui D, Reche PA, Priatel JJ, Marth JD, Reinherz EL. Developmentally regulated glycosylation of the CD8ab coreceptor stalk modulates ligand binding. Cell 2001; 107:501-512.
  12. Tibaldi EV, Salgia R, Reinherz EL. CD2 molecules distribute to the uropod during T cell scanning: Implications for cellular activation and immune surveillance. Proc Natl Acad Sci USA., 2002; 99:7582-7587.