Allele-dependent similarity between viral and self-peptide presentation by HLA-B27 subtypes

Molecular mimicry is discussed as a possible mechanism that may contribute to the development of autoimmune diseases. It could also be involved in the differential association of the human major histocompatibility subtypes HLA-B(*)2705 and HLA-B(*)2709 with ankylosing spondylitis. These two subtypes differ only in residue 116 of the heavy chain (Asp in B(*)2705 and His in B(*)2709), but the reason for the differential disease association is not understood. Using x-ray crystallography, we show here that the viral peptide pLMP2 (RRRWRRLTV, derived from latent membrane protein 2 (residues 236-244) of Epstein-Barr virus) is presented by the B(*)2705 and B(*)2709 molecules in two drastically deviating conformations. Extensive structural similarity between pLMP2 and the self-peptide pVIPR (RRKWRRWHL, derived from vasoactive intestinal peptide type 1 receptor (residues 400-408)) is observed only when the peptides are presented by B(*)2705 because of a salt bridge between Arg(5) of both peptides and the subtype-specific heavy chain residue Asp(116). Combined with functional studies using pLMP2/pVIPR-cross-reactive cytotoxic T cell lines and clones, together with target cells presenting these peptides or a modified peptide analogue, our results reveal that a pathogen-derived peptide can exhibit major histocompatibility complex class I subtype-dependent, drastically distinct binding modes. Furthermore, the results demonstrate that molecular mimicry between pLMP2 and pVIPR in the HLA-B27 context is an allele-dependent property.     

Citrullination-dependent differential presentation of a self-peptide by HLA-B27 subtypes

Inflammatory processes are accompanied by the posttranslational modification of certain arginine residues within proteins to yield citrulline, although it is largely unknown how this modification influences antigen presentation. We employed crystallographic and functional studies to investigate whether the exchange of arginine to citrulline affects the display of a peptide by two human major histocompatibility antigen class I subtypes, HLA-B(*)2705 and HLA-B(*)2709. Both differ only in residue 116 within the peptide binding groove despite their differential association with ankylosing spondylitis, an inflammatory rheumatic disorder. The crystal structures described here show that a modified self-peptide, pVIPR-U5 (RRKWURWHL; U = citrulline), is presented by the two HLA-B27 molecules in distinct conformations. These binding modes differ not only drastically from each other but also from the conformations exhibited by the non-citrullinated peptide in a given subtype. The differential reactivity of HLA-B27-restricted cytotoxic T cells with modified or unmodified pVIPR supports the structural findings and shows that the presentation of citrullinated peptides has the potential to influence immune responses.     

Cutting edge: HLA-B27 acquires many N-terminal dibasic peptides: coupling cytosolic peptide stability to antigen presentation

Ag presentation by MHC class I is a highly inefficient process because cytosolic peptidases destroy most peptides after proteasomal generation. Various mechanisms shape the MHC class I peptidome. We define a new one: intracellular peptide stability. Peptides with two N-terminal basic amino acids are more stable than other peptides. Such peptides should be overrepresented in the peptidome of MHC class I-associated peptides. HLA-B27 binding peptides use anchor residue R at P2 and, although most amino acids are allowed, particular amino acids are overrepresented at P1, including R and K. We show that such N-terminal dibasic peptides are indeed more efficiently presented by HLA-B27. This suggests that HLA-B27 can present peptides from Ags present in fewer copies than required for successful peptide generation for other MHC class I molecules.     

Antigen presentation by neonatal murine spleen cells

The ability of murine neonatal spleen cells to present soluble antigen to T-helper cells and to produce growth factors in response to subsequent cellular interactions was studied. The T-helper-cell line (D10-G4.1) (D10), which is specific for the soluble antigen conalbumin presented on H-2-matched (H-2k) antigen-presenting cells, was used as cooperating and indicator cells in these cellular interactions. The D10 cells are TH2 T-helper cells which secrete the autocrine growth factor IL-4 and can also respond to exogenous IL-2 (T. R. Mosmann and R. L. Coffmann, Immunol. Today 8, 223, 1987). D10 cells require exogenous IL-1 for their proliferation and secrete, in addition to IL-4, IL-1 inducer factor and GM-CSF. The ability of neonatal spleen cells to present antigen and to stimulate D10 cells to produce IL-4 and proliferate is low. During antigen presentation there is an augmentation of IL-1 and IL-2 production by the antigen-presenting spleen cell population. However, neonatal spleen cells do not respond to the same levels as do adult spleen cells. The addition of exogenous IL-1 cannot repair the antigen presentation by neonatal cells. Experiments in which the antigen processing and presentation steps were separated from those requiring growth factor induction and secretion demonstrate that neonatal spleen cells are impaired in their ability to perform adequate antigen processing and presentation. The neonatal spleen cells are as competent as adult cells to cooperate with T-helper cells and secrete growth factors, provided antigen processing and presentation is performed by fully competent adult spleen cells. Experiments in which neonatal and adult antigen-presenting spleen cell populations were mixed, and others in which plastic adherent and nonadherent cells were separated, could not detect any suppressor mechanisms responsible for the low antigen presentation of neonatal cells. Thus, neonatal spleen cells are impaired in the initial stages of antigen processing and presentation. This impairment which leads to low levels of growth factor production is the major determinant in the ineffectual stimulation of T-helper cells by neonatal spleen cells.     

Alloreactive cytotoxic T-lymphocyte-defined HLA-B7 subtypes differ in peptide antigen presentation

We investigated T-cell-defined HLA-B7 subtypes using cDNA sequencing, analysis of bound peptides, and reactivity with a panel of alloreactive cytotoxic T-lymphocyte (CTL) clones. Three subtypes (HLA-B*0702, HLA-B*0703, and HLA-B*0705) differ in nucleotide and predicted amino acid sequence. CTL reactivity and pooled peptide sequencing show that these three HLA-B7 subtypes bind distinct but overlapping sets of peptides. In particular B*0702 expresses D pocket residue Asp 114 and binds peptides with P3 Arg, whereas B*0705 expresses D pocket residue Asn 114 and binds peptides with P3 Ala, Leu, and Met. Consistent with different peptide-binding specificities, three alloreactive CTL differentiate between cells expressing B*0702, B*0703, and B*0705 by detecting specific peptide/HLA-B7 complexes. In contrast, three other T-cell-defined HLA-B7 subtypes are identical to HLA-B*0702. The B*0702-expressing cell lines are differentiated by two of ten CTL clones. One CTL clone differentiates B*0702-expressing cells by their ability to present peptide antigen. Thus differences in peptide presentation can explain differential CTL recognition of cell lines expressing structurally identical and variant HLA-B7.     

Tumor antigen presentation by murine epidermal cells

The ability of epidermal Langerhans cells to present Ag for CD4-dependent immunity is well documented, and it has been hypothesized that Langerhans cells participate in the generation of immunity against incipient epidermal neoplasms by presentation of tumor-associated Ag in situ. This study examined the ability of murine epidermal cells (EC) to present tumor-associated Ag for the induction of in vivo antitumor immunity. Murine epidermal cells were deleted of Thy-1-bearing cells, cultured in 50 U/ml granulocyte-macrophage-CSF for 14 to 18 h, and pulsed with tumor fragments (TF) derived from S1509a-fibrosarcoma cells. These TF-pulsed EC were injected s.c. into syngeneic recipients at weekly intervals for a total of three immunizations and challenged with viable S1509a tumor cells 1 wk after the last immunization. Control animals received TF-pulsed allogeneic EC or EC treated identically but not pulsed with TF. EC that were pulsed with tumor cell fragments were able to induce protective immunity to tumor growth in vivo and to immunize for a significant delayed-type hypersensitivity response to injected tumor cells. The induction of antitumor immunity with TF-pulsed EC was genetically restricted, and culture of EC in granulocyte-macrophage-CSF was required for development of significant immunity. Furthermore, deletion of I-A+ cells by antibody and complement-mediated lysis eliminated the generation of immunity. Thus, I-A+ epidermal cells are capable of presenting S1509a tumor Ag for the generation of protective antitumor immunity in vivo.     

The Cys-67 residue of HLA-B27 influences cell surface stability, peptide specificity, and T-cell antigen presentation.

Cys-67 of HLA-B27 is located in the B pocket, which determines peptide-binding specificity. We analyzed effects of the Cys-67 --> Ser mutation on cell surface expression, peptide specificity, and T-cell recognition of HLA-B*2705. Surface expression was assessed with antibodies recognizing either native or unfolded HLA proteins. Whereas native B*2705 molecules predominated over unfolded ones, this ratio was reversed in the mutant, suggesting lower stability. Comparison of B*2705- and Cys-67 --> Ser-bound peptides revealed that the mutant failed to bind approximately 15% of the B*2705 ligands, while binding as many novel ones. Two peptides with Gln-2 found in both B*2705 and Cys-67 --> Ser are the first demonstration of natural B*2705 ligands lacking Arg-2. Other effects of the mutation on peptide specificity were: 1) average molecular mass of natural ligands higher than for B*2705, 2) bias against small residues at peptide position (P) 1, and 3) increased P2 permissiveness. The results suggest that the Cys-67 --> Ser mutation weakens B pocket interactions, leading to decreased stability of the mutant-peptide complexes. This may be partially compensated by interactions involving bulky P1 residues. The effect of the mutation on allorecognition was consistent with that on peptide specificity. Our results may aid understanding of the pathogenetic role of HLA-B27 in spondyloarthropathy.     

Imatinib mesylate and nilotinib affect MHC-class I presentation by modulating the proteasomal processing of antigenic peptides

Imatinib (IM) has been described to modulate the function of dendritic cells and T lymphocytes and to affect the expression of antigen in CML cells. In our study, we investigated the effect of the tyrosine kinase inhibitors IM and nilotinib (NI) on antigen presentation and processing by analyzing the proteasomal activity in CML cell lines and patient samples. We used a biotinylated active site-directed probe, which covalently binds to the proteasomally active beta-subunits in an activity-dependent fashion. Additionally, we analyzed the cleavage and processing of HLA-A3/11- and HLA-B8-binding peptides derived from BCR-ABL by IM- or NI-treated isolated 20S immunoproteasomes using mass spectrometry. We found that IM treatment leads to a reduction in MHC-class I expression which is in line with the inhibition of proteasomal activity. This process is independent of BCR-ABL or apoptosis induction. In vitro digestion experiments using purified proteasomes showed that generation of epitope-precursor peptides was significantly altered in the presence of NI and IM. Treatment of the immunoproteasome with these compounds resulted in an almost complete reduction in the generation of long precursor peptides for the HLA-A3/A11 and ?B8 epitopes while processing of the short peptide sequences increased. Treatment of isolated 20S proteasomes with serine-/threonine- and tyrosine-specific phosphatases induced a significant downregulation of the proteasomal activity further indicating that phosphorylation of the proteasome regulates its function and antigen processing. Our results demonstrate that IM and NI can affect the immunogenicity of malignant cells by modulating proteasomal degradation and the repertoire of processed T cell epitopes.     

HLA-B27 antigenicity: antibodies against the chemically synthesized 63-84 peptide from HLA-B27.1 display alloantigenic specificity and discriminate among HLA-B27 subtypes

A chemically synthesized peptide with an amino acid sequence identical to that of the segment spanning residue 63-84 of the major HLA-B27.1 subtype antigen has been obtained. Specific antibodies were raised in rabbits against this peptide, coupled to keyhole limpet hemocyanin carrier. These antibodies lysed lymphoblastoid cell lines expressing HLA-B27.1 in a complement-mediated cytotoxicity assay. They lysed neither B27-negative target cells, nor B27-positive cells expressing other B27 subtype antigens. Complement-mediated lysis of B27.1-positive targets was inhibited by free peptide and by peptide coupled to an unrelated carrier. In addition, the lytic action of the rabbit antiserum was blocked by a monoclonal antibody with no complement-activating capacity that under the conditions of the assay, was specific for HLA-B27. These results indicate that rabbit antibodies against the 63-84 peptide recognize the native HLA-B27.1 antigen; this antiserum is allospecific in character; and it discriminates among B27 subtypes. Thus the data provide direct evidence on the contribution of the hypervariable region spanning residues 63-84 to the alloantigenic specificity of HLA-B27.     

HLA-B27 subtypes differentially associated with disease exhibit conformational differences in solution

Human leukocyte antigen (HLA) class I molecules consist of a heavy chain, β₂-microglobulin, and a peptide that are noncovalently bound. Certain HLA-B27 subtypes are associated with ankylosing spondylitis (such as HLA-B*2705), whereas others (such as HLA-B*2709) are not. Both differ in only one residue (Asp116 and His116, respectively) in the F pocket that accommodates the peptide C-terminus. An isotope-edited IR spectroscopy study of these HLA-B27 subtypes complexed with the self-peptide RRKWRRWHL was carried out, revealing that the heavy chain is more flexible in the HLA-B*2705 than in the HLA-B*2709 subtype. In agreement with these experimental data, molecular dynamics simulations showed an increased flexibility of the HLA-B*2705 binding groove in comparison with that of the HLA-B*2709 subtype. This difference correlates with an opening of the HLA-B*2705 binding groove, accompanied by a partial detachment of the C-terminal peptide anchor. These combined results demonstrate how the deeply embedded polymorphic heavy-chain residue 116 influences the flexibility of the peptide binding groove in a subtype-dependent manner, a feature that could also influence the recognition of the HLA-B27 complexes by effector cells.     

Processing by accessory cells for presentation to murine T cells of apamin, a disulfide-bonded 18 amino acid peptide

Apamin, an 18 amino acid peptide with two disulfide bonds, elicits specific T cell proliferative responses in H-2d and H-2b mouse strains. We evaluated the processing requirement of this compact peptide by accessory cells for presentation to apamin-reactive T hybridoma cells (THC) by analyzing the IL-2 responses of 16 THC from apamin-primed BALB/c or C57BL/6 mice, to various forms of either native or chemically synthesized apamin analogs. These included: unfolded peptides (whose four sulfhydryl groups were blocked by acetamidomethyl residues), N-and/or C-truncated peptides, and an analog with a single amino acid substitution at position 10. Assessment of the Ag-specific THC responses in the presence of either live or formaldehyde-prefixed APC indicated the following: 1) all THC stringently required Ag processing; 2) in 8 of 16 cases, the simple unfolding of apamin was sufficient to eliminate the need for Ag processing, or even induced increased THC IL-2 responses (other cells required further antigenic alterations in addition to unfolding, or rare processing steps dependent on the integrity of the two disulfide bonds); and 3) for most THC, the Leu10 and the N terminus arm of apamin were shown to be critical for expression of the epitopes involved in T cell recognition. These data indicate that apamin, a natural peptide having an appropriate size for T cell triggering, acquires its antigenic conformation after a processing by APC which primarily involves an alteration of a disulfide bond-dependent peptide folding.     

Role of binding pockets for amino-terminal peptide residues in HLA-B27 allorecognition.

The peptide binding site of HLA-B27 and other class I Ag consists of a series of pockets that bind peptide side chains. Two of these pockets interact with the amino-terminal peptide residue (pocket A) and with the highly conserved second residue (pocket B). In this study, the role of pockets A and B in HLA-B27-specific T cell allorecognition has been analyzed. Four HLA-B27 mutants with single or double changes in pocket B (24T----A, 45E----M, 67C----V, and 24,67T,C----A,V) and three mutants with single changes in pocket A (163E----T, 167W----S, and 171Y----H) were constructed by site-directed mutagenesis and expressed in HMy2.C1R cells after DNA-mediated gene transfer. These transfectants were used as target cells in cytotoxicity assays with a series of HLA-B27-specific CTL. All the mutations analyzed affected allorecognition by a significant proportion of the CTL tested, but no single change abrogated recognition by all CTL. The global effects of each mutation on allorecognition were comparable to one another, except for the effect of the change at position 67, which was smaller. The behavior of individual CTL with the mutants was very diverse, ranging from CTL that did not recognize most of the mutants to CTL recognizing all of them. Thus, some alloreactive CTL can withstand drastic alterations in pockets A and B. Two CTL showed heteroclytic effects towards the V67 and M45 mutants. CTL behavior with the H171 mutant was closely parallel to that with the B*2703 subtype, having a single Y----H change at position 59. This parallelism correlates with the similar role of Tyr59 and Tyr171 in establishing hydrogen bonds with the amino termini of HLA-B27-bound peptides. The results demonstrate that altering the structure of pockets that interact with the amino-terminal first and second residues of HLA-B27-bound peptides significantly affects recognition by alloreactive CTL, and they strongly suggest widespread peptide involvement in HLA-B27 allorecognition.     

Epitope mapping of HLA-B27 and HLA-B7 antigens by using intradomain recombinants

To study the HLA-B7 and HLA-B27 antigenic determinants, hybrid genes between these two alleles were constructed by in vivo recombination in Escherichia coli. After transfection of these genes into P815 (high transfection efficiency recipient) murine cells, the bindings of Bw6, HLA-B7, and HLA-B27 allele-specific mAb were studied, as well as that of human anti-HLA-B7 and anti-HLA-B27 monospecific alloantisera. Most of the HLA-B7 antigenic determinants were assigned to the first external domain of the molecule. Four different epitopic areas could be defined: the Bw6 epitope was associated with residues 82 and 83; the BB7.1 epitope to amino acids 63, 67, and 70; the MB40.2 and MB40.3 epitope to amino acid sequence 177-180, and human alloantisera identified as an epitope associated with residue 9. HLA-B27 antigenicity studied by TM-1 mAb was found to involve residues 77 and 80 in the alpha-1 domain. Results obtained with human monospecific alloantisera allowed the definition of an additional allospecific site associated with the NH2 terminal part on the alpha-1 domain of HLA-B27. Epitope mapping fits with data obtained by sequence comparisons and is discussed with reference to the crystallographic three-dimensional structure of the HLA-A2 molecule.     

Antigen presentation by human antigen-presenting cells to antigen-specific xenogeneic murine T cells

Successful antigen presentation by xenogeneic human antigen-presenting cells (APC) to stimulate the proliferation of antigen-specific, keyhole limpet hemocyanin (KLH)-specific, ovalbumin (OVA)-specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)-specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen-specific murine T cells and xenogeneic human APC was given by experiments using antigen-specific murine T cell clones. The OVA-specific B10.S(9R) T cell line (9-0-A1) and PPD-specific B10.A(4R) T cell line (4-P-1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I-A compatible strains, while the PPD-specific human T cell line (Y-P-5) was stimulated by autologous human APC but not by murine APC. Anti-HLA-DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC-antigen-specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen-specific murine T cells through HLA-DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H-2 complex, while murine APC failed to present antigen to stimulate human antigen-specific T cells.     

Refined peptide HLA-B*3501 binding motif reveals differences in 9-mer to 11-mer peptide binding

 HLA-B^*3501 is associated with subacute thyroiditis and fast progression of AIDS. An important prerequisite to investigate the T-cell recognition of HLA-B^*3501-restricted antigens is the characterization of peptide-HLA-B^*3501 interactions. In this study, peptide-HLA-B^*3501 interactions were determined in quantitative peptide binding assays. The results were statistically analyzed to evaluate the influence of both anchor and nonanchor positions and the predictability of peptide binding. The binding data demonstrated that all anchor residues at position 2 and the C-terminus found in 9-mers functioned equally as anchors in 10-mers and 11-mers. These minimum requirements of peptide binding were refined by assessing positive and negative effects of nonanchor residues. Aliphatic hydrophobic residues at positions 3, 5, and 8 of 10-mers and position 3 of 11-mers significantly enhanced HLA-B^*3501 binding. Similar effects rendered aromatic, bulky residues, acidic or polar residues of 11-mers at position 1 as well as at positions 4, 8, and 10, respectively. Negative effects were observed for residues carrying positively charged side-chains at position 7 of 11-mers. The refined HLA-B^*3501 peptide binding motifs enhanced the identification of potential T-cell epitopes. The disparity between positive effects at the middle and C-terminal part (positions 5 – 8 and 10) of 11-mers and shorter peptides supports the extrusion of 11-mer residues at positions 5, 6, and 7, away from the HLA-B^*3501 binding cleft. Received: 29 May 1996 / Revised: 5 August 1996     

Species-specific differences in the toxicity of rhodamine 123 towards cultured mammalian cells

The toxicity of cationic fluorescent dye, rhodamine 123, towards a number of independently established cell lines from three different species, namely human, mouse, and Chinese hamster, has been examined. All of the cell lines from any one species that were examined were found to exhibit similar sensitivities towards rhodamine 123 and no appreciable differences were observed between the normal and transformed cell types. However, in comparison to the cells of human origin, mouse and Chinese hamster cell lines exhibited about 10-fold and 70-fold higher resistance, respectively, and these differences appeared to be species related. In contrast to rhodamine 123, no differences in relative toxicities for these cell lines were observed for the structurally related neutral dye, rhodamine B. Fluorescence studies with rhodamine 123 show that in comparison to mouse and Chinese hamster cells, the more sensitive human cells show much higher uptake/binding of the drug, and a good correlation was seen in these studies between the extent of dye uptake/binding and the relative sensitivities of cell lines to rhodamine 123. These results provide evidence that the observed species-related differences in cellular toxicities are due to differences in the cellular uptake/binding of the dye.     

TRIM27 negatively regulates NOD2 by ubiquitination and proteasomal degradation

NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohn's disease. We found that TRIM27 expression is increased in Crohn's disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus.We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.     

Structural aspects of chaperone-mediated peptide loading in the MHC-I antigen presentation pathway

Recognition of foreign and dysregulated antigens by the cellular innate and adaptive immune systems is in large part dependent on the cell surface display of peptide/MHC (pMHC) complexes. The formation of such complexes requires the generation of antigenic peptides, proper folding of MHC molecules, loading of peptides onto MHC molecules, glycosylation, and transport to the plasma membrane. This complex series of biosynthetic, biochemical, and cell biological reactions is known as “antigen processing and presentation”. Here, we summarize recent work, focused on the structural and functional characterization of the key MHC-I-dedicated chaperones, tapasin, and TAPBPR. The mechanisms reflect the ability of conformationally flexible molecules to adapt to their ligands, and are comparable to similar processes that are exploited in peptide antigen loading in the MHC-II pathway.