Differential binding to HLA-C of p50-activating and p58-inhibitory natural killer cell receptors

Natural killer (NK) cell cytotoxicity is regulated in large part by the expression of NK cell receptors able to bind class I major histocompatibility complex glycoproteins. The receptors associated with recognition of HLA-C allospecificities are the two-domain Ig-like molecules, p50 and p58 proteins, with highly homologous extracellular domains but differing in that they have either an activating or inhibitory function, respectively, depending on the transmembrane domain and cytoplasmic tails that they possess. We have compared the binding to HLA-Cw7 of an inhibitory p58 molecule, NKAT2, the highly homologous activating p50 molecule, clone 49, and a second activating p50 molecule, clone 39, which has homologies to both NKAT1 and NKAT2. NKAT2 binds to HLA-Cw7 with very rapid association and dissociation rates. However, the p50 receptors bind only very weakly, if at all, to HLA-C. The molecular basis of this difference is analyzed, and the functional significance of these observations is discussed.

Effect of genetic background on the contribution of New Zealand Black loci to autoimmune lupus nephritis

Autoimmune diseases such as systemic lupus erythematosus are complex genetic traits with contributions from major histocompatibility complex (MHC) genes and multiple unknown non-MHC genes. Studies of animal models of lupus have provided important insight into the immunopathogenesis of disease, and genetic analyses of these models overcome certain obstacles encountered when studying human patients. Genome-wide scans of different genetic crosses have been used to map several disease-linked loci in New Zealand hybrid mice. Although some consensus exists among studies mapping the New Zealand Black (NZB) and New Zealand White (NZW) loci that contribute to lupus-like disease, considerable variability is also apparent. A variable in these studies is the genetic background of the non-autoimmune strain, which could influence genetic contributions from the affected strain. A direct examination of this question was undertaken in the present study by mapping NZB nephritis-linked loci in backcrosses involving different non-autoimmune backgrounds. In a backcross with MHC-congenic C57BL/6J mice, H2z appeared to be the strongest genetic determinant of severe lupus nephritis, whereas in a backcross with congenic BALB/cJ mice, H2z showed no influence on disease expression. NZB loci on chromosomes 1, 4, 11, and 14 appeared to segregate with disease in the BALB/cJ cross, but only the influence of the chromosome 1 locus spanned both crosses and showed linkage with disease when all mice were considered. Thus, the results indicate that contributions from disease-susceptibility loci, including MHC, may vary markedly depending on the non-autoimmune strain used in a backcross analysis. These studies provide insight into variables that affect genetic heterogeneity and add an important dimension of complexity for linkage analyses of human autoimmune disease.

Complementary mutations in an antigenic peptide allow for crossreactivity of autoreactive T-cell clones

T cells recognize antigen by formation of a trimolecular complex in which the T-cell receptor (TCR) recognizes a specific peptide antigen within the groove of a major histocompatibility complex (MHC) molecule. It has generally been assumed that T-cell recognition of two distinct MHC–antigen complexes is due to similarities in the three-dimensional structure of the complexes. Here we report results of experiments examining the crossreactivity of TCRs recognizing the myelin basic protein peptide MBPp85–99 and several of its analogs in the context of MHC. We demonstrate that single conservative amino acid substitutions of the antigenic peptide at the predominant TCR contact residues at positions 91 and 93 totally abrogate reactivity of specific T-cell clones. Yet, when a conservative substitution is made at position 91 concomitant with a substitution at position 93, the T-cell clones regain reactivity equivalent with that of the original stimulating peptide. Thus, the exact nature of the amino acid side chains engaging one TCR functional pocket may change the apparent selectivity of the other predominant TCR functional pocket, thus suggesting a remarkable degree of receptor plasticity. This ability of the TCR–MHC–peptide complex to undergo conformational changes provides a conceptual framework for reconciling the apparent paradox of the extreme selectivity of the TCR and its remarkable crossreactivity with different MHC–peptide complexes.

Visualizing the dynamics of T cell activation: Intracellular adhesion molecule 1 migrates rapidly to the T cell/B cell interface and acts to sustain calcium levels

T cell recognition typically involves both the engagement of a specific T cell receptor with a peptide/major histocompatibility complex (MHC) and a number of accessory interactions. One of the most important interactions is between the integrin lymphocyte function-associated antigen 1 (LFA-1) on the T cell and intracellular adhesion molecule 1 (ICAM-1) on an antigen-presenting cell. By using fluorescence video microscopy and an ICAM-1 fused to a green fluorescent protein, we find that the elevation of intracellular calcium in the T cell that is characteristic of activation is followed almost immediately by the rapid accumulation of ICAM-1 on a B cell at a tight interface between the two cells. This increased density of ICAM-1 correlates with the sustained elevation of intracellular calcium in the T cell, known to be critical for activation. The use of peptide/MHC complexes and ICAM-1 on a supported lipid bilayer to stimulate T cells also indicates a major role for ICAM-1/LFA-1 in T cell activation but, surprisingly, not for adhesion, as even in the absence of ICAM-1 the morphological changes and adhesive characteristics of an activated T cell are seen in this system. We suggest that T cell antigen receptor-mediated recognition of a very small number of MHC/peptide complexes could trigger LFA-1/ICAM-1 clustering and avidity regulation, thus amplifying and stabilizing the production of second messengers.

Identification of rat susceptibility loci for adjuvant-oil-induced arthritis

One intradermal injection of incomplete Freund’s adjuvant-oil induces a T cell-mediated inflammatory joint disease in DA rats. Susceptibility genes for oil-induced arthritis (OIA) are located both within and outside the major histocompatibility complex (MHC, Oia1). We have searched for disease-linked non-MHC loci in an F2 intercross between DA rats and MHC-identical but arthritis-resistant LEW.1AV1 rats. A genome-wide scan with microsatellite markers revealed two major chromosome regions that control disease incidence and severity: Oia2 on chromosome 4 (P = 4 × 10−13) and Oia3 on chromosome 10 (P = 1 × 10−6). All animals homozygous for DA alleles at both loci developed severe arthritis, whereas all those homozygous for LEW.1AV1 alleles were resistant. These results have general implications for situations where nonspecific activation of the immune system (e.g., incomplete Freund’s adjuvant-oil) causes inflammation and disease, either alone or in conjunction with specific antigens. They may also provide clues to the etiology of inflammatory diseases in humans, including rheumatoid arthritis.

The vav exchange factor is an essential regulator in actin-dependent receptor translocation to the lymphocyte–antigen-presenting cell interface

During the interaction of a T cell with an antigen-presenting cell (APC), several receptor ligand pairs, including the T cell receptor (TCR)/major histocompatibility complex (MHC), accumulate at the T cell/APC interface in defined geometrical patterns. This accumulation depends on a movement of the T cell cortical actin cytoskeleton toward the interface. Here we study the involvement of the guanine nucleotide exchange factor vav in this process. We crossed 129 vav−/− mice with B10/BR 5C.C7 TCR transgenic mice and used peptide-loaded APCs to stimulate T cells from the offspring. We found that the accumulation of TCR/MHC at the T cell/APC interface and the T cell actin cytoskeleton rearrangement were clearly defective in these vav+/− mice. A comparable defect in superantigen-mediated T cell activation of T cells from non-TCR transgenic 129 mice was also observed, although in this case it was more apparent in vav−/− mice. These data indicate that vav is an essential regulator of cytoskeletal rearrangements during T cell activation.

αβ T cell receptor interactions with syngeneic and allogeneic ligands: Affinity measurements and crystallization

Cellular immunity is mediated by the interaction of an αβ T cell receptor (TCR) with a peptide presented within the context of a major histocompatibility complex (MHC) molecule. Alloreactive T cells have αβ TCRs that can recognize both self- and foreign peptide–MHC (pMHC) complexes, implying that the TCR has significant complementarity with different pMHC. To characterize the molecular basis for alloreactive TCR recognition of pMHC, we have produced a soluble, recombinant form of an alloreactive αβ T cell receptor in Drosophila melanogaster cells. This recombinant TCR, 2C, is expressed as a correctly paired αβ heterodimer, with the chains covalently connected via a disulfide bond in the C-terminal region. The native conformation of the 2C TCR was probed by surface plasmon resonance (SPR) analysis by using conformation-specific monoclonal antibodies, as well as syngeneic and allogeneic pMHC ligands. The 2C interaction with H-2Kb-dEV8, H-2Kbm3-dEV8, H-2Kb-SIYR, and H-2Ld-p2Ca spans a range of affinities from Kd = 10−4 to 10−6M for the syngeneic (H-2Kb) and allogeneic (H-2Kbm3, H-2Ld) ligands. In general, the syngeneic ligands bind with weaker affinities than the allogeneic ligands, consistent with current threshold models of thymic selection and T cell activation. Crystallization of the 2C TCR required proteolytic trimming of the C-terminal residues of the α and β chains. X-ray quality crystals of complexes of 2C with H-2Kb-dEV8, H-2Kbm3-dEV8 and H-2Kb-SIYR have been grown.

The role of zinc in the binding of killer cell Ig-like receptors to class I MHC proteins

The binding of killer cell Ig-like Receptors (KIR) to their Class I MHC ligands was shown previously to be characterized by extremely rapid association and dissociation rate constants. During experiments to investigate the biochemistry of receptor–ligand binding in more detail, the kinetic parameters of the interaction were observed to alter dramatically in the presence of Zn2+ but not other divalent cations. The basis of this phenomenon is Zn2+-induced multimerization of the KIR molecules as demonstrated by BIAcore, analytical ultracentrifugation, and chemical cross-linking experiments. Zn2+-dependent multimerization of KIR may be critical for formation of the clusters of KIR and HLA-C molecules, the “natural killer (NK) cell immune synapse,” observed at the site of contact between the NK cell and target cell.

Evolution by the birth-and-death process in multigene families of the vertebrate immune system

Concerted evolution is often invoked to explain the diversity and evolution of the multigene families of major histocompatibility complex (MHC) genes and immunoglobulin (Ig) genes. However, this hypothesis has been controversial because the member genes of these families from the same species are not necessarily more closely related to one another than to the genes from different species. To resolve this controversy, we conducted phylogenetic analyses of several multigene families of the MHC and Ig systems. The results show that the evolutionary pattern of these families is quite different from that of concerted evolution but is in agreement with the birth-and-death model of evolution in which new genes are created by repeated gene duplication and some duplicate genes are maintained in the genome for a long time but others are deleted or become nonfunctional by deleterious mutations. We found little evidence that interlocus gene conversion plays an important role in the evolution of MHC and Ig multigene families.

Induction of alloantigen-specific tolerance by B cells from CD40-deficient mice.

Interaction between CD40 on B cells and CD40 ligand molecules on T cells is pivotal for the generation of a thymus-dependent antibody response. Here we show that B cells deficient in CD40 expression are unable to elicit the proliferation of allogeneic T cells in vitro. More importantly, mice immunized with CD40-/- B cells become tolerant to allogeneic major histocompatibility complex (MHC) antigens as measured by a mixed lymphocyte reaction and cytotoxic T-cell assay. The failure of CD40-/- B cells to serve as antigen presenting cells in vitro was corrected by the addition of anti-CD28 mAb. Moreover, lipopolysaccharide stimulation, which upregulates B7 expression, reversed the inability of CD40-/- B cells to stimulate an alloresponse in vitro and abrogated the capacity of these B cells to induce tolerance in vivo. These results suggest that CD40 engagement by CD40 ligand expressed on antigen-activated T cells is critical for the upregulation of B7 molecules on antigen-presenting B cells that subsequently deliver the costimulatory signals necessary for T-cell proliferation and differentiation. Our experiments suggest a novel strategy for the induction of antigen-specific tolerance in vivo.

The crystal structure of human glycosylation-inhibiting factor is a trimeric barrel with three 6-stranded beta-sheets.

Glycosylation-inhibiting factor (GIF) is a cytokine that is involved in the regulation of IgE synthesis. The crystal structure of recombinant human GIF was determined by the multiple isomorphous replacement method. The structure was refined to an R factor of 0.168 at 1.9 angstrom resolution. The overall structure is seen to consist of three interconnected subunits forming a barrel with three 6-stranded beta-sheets on the inside and six alpha-helices on the outside. There is a 5-angstrom-diameter "hole" through the middle of the barrel. The barrel structure of GIF in part resembles other "trefoil" cytokines such as interleukin 1 and fibroblast growth factor. Each subunit has a new class of alpha + beta sandwich structure consisting of two beta-alpha-beta motifs. These beta-alpha-beta motifs are related by a pseudo-twofold axis and resemble both interleukin 8 and the peptide binding domain of major histocompatibility complex protein, although the topology of the polypeptide chain is quite different.

HLA-G expression during preimplantation human embryo development.

HLA-G is a nonclassical class I major histocompatibility complex molecule with a restricted pattern of expression that includes the placental extravillus cytotrophoblast cells in direct contact with maternal tissues. Circumstantial evidence suggests that HLA-G may play a role in protection of the semiallogeneic human fetus. We examined whether HLA-G is expressed during the critical period of preimplantation human development and whether expression of this molecule could be correlated with the cleavage rate of embryos. Using reverse transcription PCR on surplus human embryos and unfertilized oocytes from patients undergoing in vitro fertilization we detected HLA-G heavy chain mRNA in 40% of 148 of blastocysts tested. The presence of HLA-G mRNA was also detected in unfertilized oocytes and in early embryos, but not in control cumulus oophorus cells. beta 2-Microglobulin mRNA was also found in those embryos expressing HLA-G. In concordance with our mRNA data, a similar proportion of embryos stained positive for HLA-G utilizing a specific monoclonal antibody. Interestingly, expression of HLA-G mRNA was associated with an increased cleavage rate, as compared to embryos lacking HLA-G transcript. Thus, HLA-G could be a functional homologue of the mouse Qa-2 antigen, which has been implicated in differences in the rate of preimplantation embryo development. To our knowledge, the presence of HLA-G mRNA and protein in human preimplantation embryos and oocytes has not been reported previously. The correlation of HLA-G mRNA expression with cleavage rate suggests that this molecule may play an important role in human pre-embryo development.

Intracellular survival of apicomplexan parasites and host cell modification

The intracellular stages of apicomplexan parasites are known to extensively modify their host cells to ensure their own survival. Recently, considerable progress has been made in understanding the molecular details of these parasite-dependent effects for Plasmodium-, Toxoplasma- and Theileria-infected cells. We have begun to understand how Plasmodium liver stage parasites protect their host hepatocytes from apoptosis during parasite development and how they induce an ordered cell death at the end of the liver stage. Toxoplasma parasites are also known to regulate host cell survival pathways and it has been convincingly demonstrated that they block host cell major histocompatibility complex (MHC)-dependent antigen presentation of parasite epitopes to avoid cell-mediated immune responses. Theileria parasites are the masters of host cell modulation because their presence immortalises the infected cell. It is now accepted that multiple pathways are activated to induce Theileria-dependent host cell transformation. Although it is now known that similar host cell pathways are affected by the different parasites, the outcome for the infected cell varies considerably. Improved imaging techniques and new methods to control expression of parasite and host cell proteins will help us to analyse the molecular details of parasite-dependent host cell modifications.

Novel multiple sclerosis susceptibility loci implicated in epigenetic regulation.

We conducted a genome-wide association study (GWAS) on multiple sclerosis (MS) susceptibility in German cohorts with 4888 cases and 10,395 controls. In addition to associations within the major histocompatibility complex (MHC) region, 15 non-MHC loci reached genome-wide significance. Four of these loci are novel MS susceptibility loci. They map to the genes L3MBTL3, MAZ, ERG, and SHMT1. The lead variant at SHMT1 was replicated in an independent Sardinian cohort. Products of the genes L3MBTL3, MAZ, and ERG play important roles in immune cell regulation. SHMT1 encodes a serine hydroxymethyltransferase catalyzing the transfer of a carbon unit to the folate cycle. This reaction is required for regulation of methylation homeostasis, which is important for establishment and maintenance of epigenetic signatures. Our GWAS approach in a defined population with limited genetic substructure detected associations not found in larger, more heterogeneous cohorts, thus providing new clues regarding MS pathogenesis.