Biomedical subjects

Lars Kjer-Nielsen

Publications and source records attributed to Lars Kjer-Nielsen.

27 records · Page 2Linked to original sources

PubMed2004

Crystal structure of the human T cell receptor CD3 epsilon gamma heterodimer complexed to the therapeutic mAb OKT3.

The CD3 epsilon gamma heterodimer is essential for expression and function of the T cell receptor. The crystal structure of the human CD3 epsilon gamma heterodimer is described to 2.1-A resolution complexed with OKT3, a therapeutic mAb that not only activates and tolerizes mature T cells but also induces regulatory T cells. The mode of CD3 epsilon gamma dimerization provides a general structural basis for CD3 assembly and maps candidate T cell antigen receptor docking sites, including a duplicated linear region rich in acidic residues that is unique to human CD3 epsilon. OKT3 binds to an atypically small area of CD3 epsilon and has a low affinity for the isolated CD3 epsilon gamma heterodimer. The structure of the OKT3/CD3 epsilon gamma complex has implications for T cell signaling and therapeutic design.

Amino Acid Sequence↗

PubMed2004

Functional and structural characteristics of NY-ESO-1-related HLA A2-restricted epitopes and the design of a novel immunogenic analogue.

NY-ESO-1, a commonly expressed tumor antigen of the cancer-testis family, is expressed by a wide range of tumors but not found in normal adult somatic tissue, making it an ideal cancer vaccine candidate. Peptides derived from NY-ESO-1 have shown preclinical and clinical trial promise; however, biochemical features of these peptides have complicated their formulation and led to heterogeneous immune responses. We have taken a rational approach to engineer an HLA A2-restricted NY-ESO-1-derived T cell epitope with improved formulation and immunogenicity to the wild type peptide. To accomplish this, we have solved the x-ray crystallographic structures of HLA A2 complexed to NY-ESO (157-165) and two analogues of this peptide in which the C-terminal cysteine residue has been substituted to alanine or serine. Substitution of cysteine by serine maintained peptide conformation yet reduced complex stability, resulting in poor cytotoxic T lymphocyte recognition. Conversely, substitution with alanine maintained complex stability and cytotoxic T lymphocyte recognition. Based on the structures of the three HLA A2 complexes, we incorporated 2-aminoisobutyric acid, an isostereomer of cysteine, into the epitope. This analogue is impervious to oxidative damage, cysteinylation, and dimerization of the peptide epitope upon formulation that is characteristic of the wild type peptide. Therefore, this approach has yielded a potential therapeutic molecule that satiates the hydrophobic F pocket of HLA A2 and exhibited superior immunogenicity relative to the wild type peptide.

Adult↗

PubMed2003

A naturally selected dimorphism within the HLA-B44 supertype alters class I structure, peptide repertoire, and T cell recognition.

HLA-B*4402 and B*4403 are naturally occurring MHC class I alleles that are both found at a high frequency in all human populations, and yet they only differ by one residue on the alpha2 helix (B*4402 Asp156-->B*4403 Leu156). CTLs discriminate between HLA-B*4402 and B*4403, and these allotypes stimulate strong mutual allogeneic responses reflecting their known barrier to hemopoeitic stem cell transplantation. Although HLA-B*4402 and B*4403 share >95% of their peptide repertoire, B*4403 presents more unique peptides than B*4402, consistent with the stronger T cell alloreactivity observed toward B*4403 compared with B*4402. Crystal structures of B*4402 and B*4403 show how the polymorphism at position 156 is completely buried and yet alters both the peptide and the heavy chain conformation, relaxing ligand selection by B*4403 compared with B*4402. Thus, the polymorphism between HLA-B*4402 and B*4403 modifies both peptide repertoire and T cell recognition, and is reflected in the paradoxically powerful alloreactivity that occurs across this "minimal" mismatch. The findings suggest that these closely related class I genes are maintained in diverse human populations through their differential impact on the selection of peptide ligands and the T cell repertoire.

Alleles↗

PubMed2003

A structural basis for the selection of dominant alphabeta T cell receptors in antiviral immunity.

We have examined the basis for immunodominant or "public" TCR usage in an antiviral CTL response. Residues encoded by each of the highly selected genetic elements of an immunodominant clonotype recognizing Epstein-Barr virus were critical to the antigen specificity of the receptor. Upon recognizing antigen, the immunodominant TCR undergoes extensive conformational changes in the complementarity determining regions (CDRs), including the disruption of the canonical structures of the germline-encoded CDR1alpha and CDR2alpha loops to produce an enhanced fit with the HLA-peptide complex. TCR ligation induces conformational changes in the TCRalpha constant domain thought to form part of the docking site for CD3epsilon. These findings indicate that TCR immunodominance is associated with structural properties conferring receptor specificity and suggest a novel structural link between TCR ligation and intracellular signaling.

Amino Acid Sequence↗

PubMed2002

The production, purification and crystallization of a soluble heterodimeric form of a highly selected T-cell receptor in its unliganded and liganded state.

T-cell antigen receptors (TcRs) are heterodimeric cell-surface receptors that play a pivotal role in the cellular immune response. The TcR interacts specifically with a peptide-laden major histocompatability complex (pMHC). A human TcR has been characterized that interacts with an immunodominant epitope, FLRGRAYGL, from the Epstein-Barr virus, a ubiquitous human pathogen, in complex with HLA-B8. Despite the vast TcR repertoire, this TcR is found in up to 10% of the total T-cell population in seropositive HLA-B8+ individuals. In this report, this highly selected TcR is characterized by expressing in Escherichia coli, refolding, purifying and crystallizing the receptor. In addition, the HLA-B8-FLRGRAYGL complex has been expressed in E. coli, refolded and shown to be functionally active. Using native gel electrophoresis, the refolded TcR is shown to be capable of binding specifically to the refolded HLA-B8-FLRGRAYGL and this TcR has been crystallized in complex with the pMHC. The crystals of the unliganded and liganded TcR diffract to 1.5 and 2.5 A, respectively.

Amino Acid Sequence↗

PubMed2002

GRIP domain-mediated targeting of two new coiled-coil proteins, GCC88 and GCC185, to subcompartments of the trans-Golgi network.

The GRIP domain is a targeting sequence found in a family of coiled-coil peripheral Golgi proteins. Previously we demonstrated that the GRIP domain of p230/golgin245 is specifically recruited to tubulovesicular structures of the trans-Golgi network (TGN). Here we have characterized two novel Golgi proteins with functional GRIP domains, designated GCC88 and GCC185. GCC88 cDNA encodes a protein of 88 kDa, and GCC185 cDNA encodes a protein of 185 kDa. Both molecules are brefeldin A-sensitive peripheral membrane proteins and are predicted to have extensive coiled-coil regions with the GRIP domain at the C terminus. By immunofluorescence and immunoelectron microscopy GCC88 and GCC185, and the GRIP protein golgin97, are all localized to the TGN of HeLa cells. Overexpression of full-length GCC88 leads to the formation of large electron dense structures that extend from the trans-Golgi. These de novo structures contain GCC88 and co-stain for the TGN markers syntaxin 6 and TGN38 but not for alpha2,6-sialyltransferase, beta-COP, or cis-Golgi GM130. The formation of these abnormal structures requires the N-terminal domain of GCC88. TGN38, which recycles between the TGN and plasma membrane, was transported into and out of the GCC88 decorated structures. These data introduce two new GRIP domain proteins and implicate a role for GCC88 in the organization of a specific TGN subcompartment involved with membrane transport.

Amino Acid Sequence↗

PubMed2002

The structure of HLA-B8 complexed to an immunodominant viral determinant: peptide-induced conformational changes and a mode of MHC class I dimerization.

EBV is a ubiquitous human pathogen that chronically infects up to 90% of the population. Persistent viral infection is characterized by latency and periods of viral replication that are kept in check by a strong antiviral CTL response. Despite the size of the EBV genome, CTL immunity focuses on only a few viral determinants but expands a large primary and memory response toward these epitopes. In unrelated HLA-B8(+) individuals, the response to the immunodominant latent Ag FLRGRAYGL from Epstein Barr nuclear Ag 3A is largely comprised of CTL clones with identical conserved alphabeta TCR structures. To better understand the structural correlates of Ag immunodominance and TCR selection bias, we have solved the crystal structure of the HLA-B8-FLRGRAYGL peptide complex to a resolution of 1.9 A. The structure confirms the importance of P3-Arg, P5-Arg, and P9-Leu as dominant anchor residues involved in peptide binding to HLA-B8. A bulged conformation of the bound peptide provides a structural basis for the critical role of the P7-Tyr residue in T cell recognition. The peptide also induces backbone and side-chain conformational changes in HLA-B8 that are transmitted along the peptide-binding groove in a domino effect. The HLA-B8-FLRGRAYGL complex crystallizes as a dimer in the asymmetric unit and is oriented such that both peptide ligands are projected in the same plane suggesting a higher order arrangement of MHC-peptide complexes that could be involved in formation of the class I Ag-loading complex or in T cell activation.

Antigens, Viral↗

PubMed2002

Identification of a dominant self-ligand bound to three HLA B44 alleles and the preliminary crystallographic analysis of recombinant forms of each complex.

A naturally processed and presented ligand that is shared by human leukocyte antigen (HLA) B*4402, B*4403 and B*4405 molecules has been identified in peptides isolated from immunoaffinity purified HLA B44 complexes. This peptide derived from HLA DPalpha residues 46-54, an endogenous product of HLA DP expressed in the cell line Hmy2.C1R, is a prominent peptide in the mass spectra of species isolated as bound peptides from each allele when the three HLA B44 subtypes were introduced as transfected gene products. Recombinant truncated forms of HLA B*4405(1-276), HLA B*4403(1-276), HLA B*4402(1-276) and beta(2)-microglobulin have been prepared as inclusion bodies in Escherichia coli and refolded in the presence of the DPalpha(46-54) peptide and purified by a combination of size exclusion and anion exchange chromatography. This material was determined to be correctly folded based on detection of a conformational epitope recognized by the W6/32 monoclonal antibody. Large, plate-like crystals of the three complexes were produced using polyethylene glycol as the precipitant. All the crystals belong to the space group P2(1)2(1)2(1) with unit cell dimensions of approximately a=51, b=82, c=110 A. The crystals of three B44/DPalpha complexes diffracted to a resolution of 1.9 A or better. For the first time, using this natural, high abundance ligand of the HLA B44 molecules we have successfully expressed and refolded the three HLA B44 molecules and produced crystals amenable to structural studies.

Alleles↗

PubMed2002

The 1.5 A crystal structure of a highly selected antiviral T cell receptor provides evidence for a structural basis of immunodominance.

Despite a potential repertoire of >10(15) alphabeta T cell receptors (TcR), the HLA B8-restricted cytolytic T cell response to a latent antigen of Epstein-Barr virus (EBV) is strikingly limited in the TcR sequences that are selected. Even in unrelated individuals this response is dominated by a single highly restricted TcR clonotype that selects identical combinations of hypervariable Valpha, Vbeta, D, J, and N region genes. We have determined the 1.5 A crystal structure of this "public" TcR, revealing that five of the six hypervariable loops adopt novel conformations providing a unique combining site that contains a deep pocket predicted to overlay the HLA B8-peptide complex. The findings suggest a structural basis for the immunodominance of this clonotype in the immune response to EBV.

Amino Acid Sequence↗