Allele- and temperature-dependency of in vitro HLA class I assembly.

Allelic variations of in vitro HLA class I assembly have been investigated in both the absence and the presence of binding peptides by flow cytometry using human leukocyte antigen (HLA) class I alpha chains isolated by alkali treatment from cultured HLA homozygous B cells and polystyrene beads coated with anti-HLA class I alpha chain antibodies specific to the C-terminal segment (anti-HLA class I beads). The specificity of assembly was temperature dependent, while the stability of the assembled complex depended on the bound peptide. The efficiency of assembly was allele dependent and primarily ruled by the binding affinity of alpha chains with beta(2)m. Thus, an allele hierarchy could be defined for the binding of HLA-B alpha chain with beta(2)-microglobulin: B7, B18 > B35, B62 > B27, B51. Allele and temperature dependency was found in HLA class I reassembly on acid treated B cells. The HLA class I proteins, reassembled with specific single peptides, could be efficiently transferred to anti-HLA class I beads. These findings would be used to produce microspheres coupled at high surface density with oriented single-peptide loaded HLA class I molecules and also to improve the preparation efficiency of HLA class I tetramers by the use of site-specific biotinylation.

Spreading of HIV-specific CD8+ T-cell repertoire in long-term nonprogressors and its role in the control of viral load and disease activity.

Long-term non-progressors (LTNP) represent a minority of human immunodeficiency virus (HIV) infected individuals characterized by stable or even increasing CD4+ T-cell count and by stronger immune responses against HIV than progressors. In this study, HIV-specific effector CD8+ T cells, as detected by both a sensitive ex vivo enzyme-linked immunospot (ELISPOT) assay and specific major histocompatibility complex (MHC) peptide tetramers, were at a low frequency in the peripheral blood of LTNP, and recognized a lower number of HIV peptides than their memory resting cell counterparts. Both factors may account for the lack of complete HIV clearance by LTNP, who could control the viral spread, and displayed a higher magnitude of cytotoxic T lymphocyte (CTL) responses than progressors. By combining cell purification and ELISPOT assays this study demonstrates that both effector and memory resting cells were confined to a CD8+ population with memory CD45RO+ phenotype, with the former being CD28- and the latter CD28+. Longitudinal studies highlighted a relatively stable HIV-specific effector repertoire, viremia, and CD4+ T-cell counts, which were all correlated with maintenance of nonprogressor status. In conclusion, the analysis of HIV-specific cellular responses in these individuals may help define clear correlates of protective immunity in HIV infection.

Polystyrene beads coated with antibodies directed to HLA class I intracytoplasmic domain: the use in quantitative measurement of peptide-HLA class I binding by flow cytometry.

Protein-reactive, conformation-independent anti-peptide antibodies were raised in rabbits against a C-terminal sequence SDSAQGSDVSLA, common to most HLA-A and -B locus products. Antibodies were coupled to 4.5-microm polystyrene beads through the Fc portion by the use of protein A. The antibody-coupled beads showed a high capacity to bind HLA-A and -B proteins as well as their alpha chains by the intracytoplasmic domain, keeping the extracellular domains solvent exposed. The density of HLA class I proteins bound on the beads was approximately the same as that on cultured B cells. The antibody beads made it possible to quantitate peptide-HLA class I binding, i.e., in vitro HLA class I assembly by flow cytometry. The assembly rate determined by the provisionally called flow cytometric HLA class I assay was 15%-19% for the reassembly of dissociated HLA class I proteins with the released selfpeptides. With single synthetic peptides, the highest rate so far obtained was 6.5%. The assay specificity and reproducibility were satisfactory.

Two brc-abl junction peptides bind HLA-A3 molecules and allow specific induction of human cytotoxic T lymphocytes.

Intracellular processing of the products of the bcr-abl junction region in CML Philadelphia chromosomes would generate novel peptides which, if they are capable of binding to HLA-class I molecules, would be potential targets of a cytotoxic T cell response. The 18 nonamers corresponding to the b2-a2 and b3-a2 fusions and differing from the parental bcr and abl sequences for at least one amino acid have been synthesized and tested for binding with HLA class I alpha chain preparations from HLA-homozygous B lymphoblastoid cell lines. Two peptides derived from the b3-a2 junction bound to HLA-A3 and elicited detectable specific CTL responses in vitro. The binding affinity of one of the two peptides could be increased by appropriate substitutions of the anchor residues with those of the known HLA-A3 anchor motifs. More important, the modified peptide had increased capacity to prime a specific CTL response in vitro. The interaction with HLA-A3 of these two peptides and their substitution derivatives seems to be promising for target trials aimed at eliciting a specific CD8 T cell response against CML.

Presentation of peptides by cultured monocytes or activated T cells allows specific priming of human cytotoxic T lymphocytes in vitro.

The conditions favouring effective specific cytotoxic T lymphocyte (CTL) priming have been exploited to set up a simple and reproducible method to induce a primary CTL response in vitro. We report that cultured monocytes, as well as activated T cells, pulsed with exogenous HLA-A2 binding immunogenic peptides, can induce primary peptide-specific CTL responses in vitro in a Th-independent manner. Primary viral peptide-induced CTL were HLA-A2 restricted, and recognized both peptide-pulsed target cells and targets infected with recombinant vaccinia virus expressing viral endogenous antigens. In addition, both cultured monocytes and activated T cells primed peptide-specific CD8+ T cells depleted from the CD45RO+ memory cell fraction. The efficiency of CTL priming by monocytes was dependent upon the strong up-regulation of class I, adhesion and co-stimulatory molecules occurring spontaneously upon in vitro culture. The inability of unseparated peripheral blood mononuclear cells to mount a peptide-specific CTL response could be reverted by direct co-stimulation of responding CD8+ T cells by soluble B7.1 or a stimulatory anti-CD28 antibody, that allowed a specific response to take place. Although co-stimulation via the B7-CD28 interaction appeared sufficient to trigger CTL responses, it was not essential for CTL priming, since neither anti-B7.1 mAb nor soluble CTLA-4 inhibited induction of primary CTL response. This new method for induction of specific CD8+ T cell response in vitro may be exploited in adoptive immunotherapy in cancer or in HIV-infected patients.

Augmentation of the affinity of HLA class I-binding peptides lacking primary anchor residues by manipulation of the secondary anchor residues.

A direct binding assay has been used to investigate the effect of the secondary anchor residues on peptide binding to class I proteins of the major histocompatibility complex. Based on predictions from a previous chemometric approach, synthetic peptide analogues containing unnatural amino acids were synthesized and tested for B*2705 binding. Hydrophobic unnatural amino acids such as alpha-naphthyl- and cyclohexyl-alanine were found to be excellent substituents in the P3 secondary anchor position giving peptides with very high B*2705-binding affinity. The binding to B*2705 of peptides optimized for their secondary anchor residues, but lacking one of the P2 or P9 primary anchor residues was also investigated. Most such peptides did not bind, but one peptide, lacking the P2 Arg residue generally considered essential for binding to all B27 subtypes, was found to bind quite strongly. These findings demonstrate that peptide binding to class I proteins is due to a combination of all the anchor residues, which may be occupied also by unnatural amino acids-a necessary step towards the development of peptidic or non-peptidic antagonists for immunomodulation.

Differences in peptide-binding specificity of two ankylosing spondylitis-associated HLA-B27 subtypes.

Two HLA-B27 subtypes, B*2702 and B*2705, both associated with ankylosing spondylitis, were tested for binding affinity with a panel of polyalanine model nonapeptides carrying Arg at position 2 (P2) and a series of different amino acids at position 9 (P9). The alpha chains were isolated from BTB(B*2705), C1R/B*2702 (a B*2702 transfectant cell line) and from the NW (B*2702) cell line that has a peculiar peptide presentation behavior. Peptide binding was measured by the HLA alpha chain refolding assay. The results obtained show that: 1) Peptides with basic residues (Arg and Lys) and also aliphatic (Leu) and aromatic (Phe and Tyr) peptides at P9 have a similar high affinity in the binding to B*2705; 2) B*2702 binds well to P9 aliphatic and aromatic peptides but only very weakly to P9 basic peptides. Since both B*2702 and B*2705 are associated with AS the presumed arthritogenic peptide is hypothesized to have an aromatic or aliphatic residue at position 9. Peptides with basic residues in this position would be excluded as candidates because of their low binding affinity with B*2702.

The peptide-binding specificity of HLA-B27 subtype (B*2705) analyzed by the use of polyalanine model peptides.

Model peptides have been used to quantitate the effect on HLA-B*2705 binding of the spacing between primary anchor residues, the type of amino acid accepted in the P9 anchor position, and the type of amino acid accepted in the "secondary anchor positions" P3 and P7. Peptide binding was measured by the HLA class I alpha-chain-refolding assay. The results obtained show that (a) Among the model peptides differing in the spacing between anchor residues, the nonamer with Arg in P2 and Lys in P9 (R2, K9) has the maximum binding with B*2705 molecule. The decamer, with an extra Ala inserted between Arg and Lys (R2, K10), has much lower binding, and still lower binding is observed for the octamer, where an Ala is removed (R2, K8). (b) Besides the "classic" Lys and Arg, several other aminoacids such as Tyr, Leu, Ala, and Gln can be accepted in P9, but with significant differences in binding affinity. (c) Different amino acids in P3 have an influence on peptide binding. Trp and Phe have a favorable influence, whereas Lys and Val appear to hinder the binding. Some variations are seen also for different amino acids in P7.

Exploring myelin basic protein for HLA class I-binding sequences.

In view of the increasing evidence of the involvement of CD8+ T cells in the pathogenesis of multiple sclerosis (MS), we have scanned the sequence of the myelin basic protein (MBP), using 162 overlapping nonapeptides, for HLA-class I binding sites. Peptide binding was measured using the recently reported HLA class I alpha-chain-refolding assay, and the following HLA allelic products were analyzed: HLA-A2 (*0201, *0204), B27 (*2705), B35, B51 and B62. A considerable number of binding peptides were distinguished for each of the allelic products tested. In addition, three interesting points emerged. The first was the identification of several binding peptides which did not contain the known anchor motifs. The second was the evidence that several peptides showed a promiscuous binding profile, being able to bind to different HLA class I molecules that were either allelic or non allelic. The third was that in several cases two consecutive peptides could bind to the same HLA molecule.

The importance of secondary anchor residue motifs of HLA class I proteins: a chemometric approach.

In this paper we report a chemometric approach to Quantitative Structure-Activity Relationship (QSAR) analysis applied to a study of the binding of peptides to Major Histocompatibility Complex (MHC) class I proteins. Peptides which possess the known primary anchor residue motif for HLA-B27 binding do not necessarily bind to HLA-B27 proteins. Secondary anchor residues are also involved, but it is not yet clear which amino acids are required or in which positions. A classic approach to this problem would be to synthesize multiple peptides each varying by a single amino acid from a starting peptide, and test them for their binding properties. Not only is this approach inefficient, but it is essentially unable to provide information about possible mutual interactions of amino acid residues in different positions. Using a statistical design to select the most informative compounds to use in the QSAR study, it was possible to analyse the effects on HLA-B27 peptide binding of different amino acids in four positions by means of only nine peptides. The relative binding activity of these peptides could then be modeled mathematically to provide information about the relative contribution of each of the four positions and to suggest a new peptide with high binding affinity. Our results demonstrate the usefulness of the chemometric strategy for studying peptides of interest in molecular immunology.

HLA-A2-binding peptides cross-react not only within the A2 subgroup but also with other HLA-A-locus allelic products.

Seven A2-binding peptides were tested by the HLA class I alpha-chain refolding assay previously described for their direct binding to HLA class I alpha chains derived from a panel of 18 HLA-homozygous B-cell lines of various HLA specificities, including four A2 subtypes: A*0201, A*0204, A*0205, and A*0206. All but one test peptide possessed the major anchor residue motifs, L-V, L-L, or I-L, of A2(A*0201)/A2(A*0205)-binding peptides or the closely related motifs, I-V or V-V. This cell panel analysis confirmed the high A2 allele specificity of the test peptides, but also revealed the existence of a broad cross-binding within the A2 subgroup. Most peptides bound to the alpha chains of the A2 subtypes tested, although their binding patterns showed differences. Furthermore, the A2-binding peptides carrying the I-V or V-V motif were found to cross-react also outside of the A2 subtypes, probably with A24, A26, A28, and A29. Other A-locus allelic products, A1, A3, A11, A30, and A31, and the B-locus allelic products carried by the cells tested were essentially negative, although a few exceptions were seen.

The peptide binding specificity of HLA-B27 subtypes.

Five HLA-B27 subtypes, B*2701, B*2703, B*2704, B*2705, and B*2706, were tested for direct binding with twenty-six synthetic nonapeptides carrying the primary anchor residue motifs (combination of amino residues at positions 2 and 9) relevant to B*2705. The peptide sequences were derived from human HSP89 alpha, P53 and MBP. The alpha chains were immunospecifically isolated from LH (B*2701), CH (B*2703), WE1 (B*2704), BTB (B*2705), and LIE (B*2706) cells and their peptide binding was measured by the HLA class I alpha chain refolding assay. The data obtained indicated that the B27 subtypes tested can bind a common set of peptides carrying several different anchor residue motifs. The motifs, R-K and R-R, reported for B*2705 and a new motif H-R were accepted by B*2703, B*2704, and B*2706, but not by B*2701. However, other motifs, including known B*2702 and/or B*2705 motifs, R-H, R-L, R-A, and R-F, and a new motif found here, R-G, were apparently accepted by all B27 subtypes tested. The observed cross-peptide binding in the B27 subgroup is compatible with the so-called arthritogenic peptide hypothesis in the pathogenesis of ankylosing spondylitis.

Anchor residue motifs of HLA class-I-binding peptides analyzed by the direct binding of synthetic peptides to HLA class I alpha chains.

The binding characteristics of the primary anchor residue motifs reported for HLA-A2 (A*0201, A*0205) and HLA-B27 (B*2705) alleles were investigated by a direct binding assay of the pertinent synthetic peptides to HLA class I alpha chains derived from a panel of HLA homozygous B-cell lines of various HLA phenotypes, including four A2 subtypes. The assay is based on a serologic detection of the conformational change of HLA class I alpha chains induced by binding to specific peptides in the presence of beta 2m. It is applicable to test a large number of HLA allelic products and synthetic peptides. Assay data confirmed the high allele specificity of the anchor residue motifs tested, but also revealed the intra- and interlocus cross-reactivity of these motifs. In the case of A2 anchor motifs, not only a broad cross-reactivity within the A2 subgroup, but also cross-reactivities with A24, A26, A28, and A29 were observed. With B27 anchor motifs, an interlocus cross-reactivity with A3 and A31 was seen. Several peptides, even though they carried A2 or B27 major anchor residue motifs, failed to bind to the relevant alpha chains, suggesting that the presence of a primary anchor residue motif is necessary for HLA class-I-peptide binding but is not by itself sufficient to guarantee binding.

Unfolded HLA class I alpha chains and their use in an assay of HLA class-I-peptide binding.

Unfolded HLA class I alpha chains were isolated from B-cell lysates by alkaline denaturation and subsequent gel filtration and used for the detection of HLA class-I-peptide binding. Binding to specific peptides in the presence of excess beta 2-microglobulin induced the unfolded alpha chains to refold and acquire a conformation that is specific to folded alpha chains. This conformational change was measured by a specific RIA that involves inhibition of the binding of 125I-labeled HLA-A2 alpha/beta dimers and rabbit anti-HLA-B7 serum absorbed with beta 2-microglobulin. This assay procedure does not require labeling of either test peptides or test class I proteins and does not seem to have specificity degeneracy. It is applicable to the detection of peptide binding by all HLA class I allelic proteins. Evaluation of the assay conditions and HLA allelic specificity of the peptide binding defined by the use of synthetic peptides are described here, including the technical details, specificity, and reproducibility.

HLA class I binding of synthetic nonamer peptides carrying major anchor residue motifs of HLA-B27 (B*2705)-binding peptides.

Eight nonamer peptides that comply with the major anchor residue motifs (the combination of amino acid residues at positions 2 and 9), R - K and R - R, of HLA-B27 (B*2705)-binding peptides were synthesized and tested for their direct binding to HLA class I alpha chains by the HLA class I alpha chain refolding assay previously described. One was a known B27 (B*2705)-binding heat shock protein peptide, HSP89 alpha (201-209), and the other seven were derived from the sequence of wild-type P53, a human tumor suppressor protein. A total of 36 HLA class I allospecificities were tested. HSP89 alpha (201-209) and two P53 peptides, P53 (362-370) and P53 (378-386), all possessing the motif R - K, bound strongly to B27 (B*2705) alpha chains. A weak binding was seen for P53 (272-280) and P53 (334-342), both showing the motif R - R. Most of these B27-binding peptides were found to bind to A3 alpha chains as well. In addition, P53 (173-181) and P53 (334-342), both with the R - R motif, showed substantial binding with A31 alpha chains. All the peptides carrying the motif R - K also showed weak binding with A31 alpha chains. The remaining two peptides, P53 (201-209) and P53 (282-290), with the motif R - R, did not show significant binding with any of the alpha chains tested. This study demonstrates both the specificity of peptide binding to a given HLA allelic product and the occurrence of cross-peptide-binding between the allelic products of different HLA loci.

The specificity and efficiency of endogenous peptides in the induction of HLA class I alpha chain refolding.

The specificity and efficiency of endogenous peptides in the HLA class I binding have been investigated by the use of a simple procedure that is based on the serological detection of the folding of HLA class I alpha chains that is induced by the binding to specific peptides in the presence of beta 2 microglobulin. HLA class I proteins were solubilized with a nonionic detergent from cultured HLA homozygous B lymphoblastoid cells and dissociated by alkaline denaturation. The resulting unfolded alpha chains were isolated by gel filtration at a neutral pH. The unfolded alpha chains showed a high refolding capacity and specificity when tested in the presence of an excess beta 2 microglobulin against endogenous peptides extracted by alkaline or acid treatment from cultured B lymphoblastoid cells of various HLA class I phenotypes. Cells of identical or overlapping HLA phenotypes clearly showed shared peptides, whereas such peptide sharing was rarely, if at all, seen between cells of non-overlapping HLA phenotypes. The efficiency of endogenous peptides in the induction of refolding was high; at an estimated concentration of 0.2 microM or less, a strong refolding effect was observed.

Reassessment of HLA association with celiac disease in special reference to the DP association.

Patients with the late-onset form of celiac disease have been studied for HLA association by conventional serology (DR and DQ typing) and by oligonucleotide probing with gene amplification (DP typing). Patients and controls were sampled in the Bologna area of northern Italy. Almost all patients were positive for DQw2 (94%), being DR3 positive (72%) and/or DR7 positive (65%). The proportion of DR3/7 heterozygotes in the patients was significantly increased over that expected from the Hardy-Weinberg equilibrium. No positive association with DR5 and no significant increase of DR5/7 heterozygotes were observed. Among the DP alleles reported to exhibit an association with celiac disease in other populations, only DPB3 showed a moderate increase of a borderline significance, not attributable to a linkage disequilibrium with DQw2.

Arg74 in HLA-DRB1 and DRB3 controls a DR3-related epitope.

TR81 is a specificity closely related to or identical with DR3. In Caucasoids two amino acids, Tyr at position 26 and Arg at position 74 of HLA class II DR beta chains, have been found to be associated with the presence of TR81. Recently, a variant of DRB1*03 identified in American Blacks has been shown to possess Arg at position 74 but Phe at position 26. This codon combination is found to be present in four other cell lines where it still specifies the TR81 determinant. This suggests that the TR81 specificity is uniquely dependent on the presence of Arg at position 74.

Electron microscopic study of cultured cells from the murine hair tissues: cell growth and differentiation.

The cultured hair cells from 4-day-old C3H mice were studied by electron microscopy. The hair roots isolated from the skin by collagenase digestion were dispersed into a cell suspension by treatment with a mixture of trypsin and ethylenediaminetetraacetate. The cells were cultured in MCDB-153 (a medium containing seven growth factors) for 1, 3, 6 or 13 days. The number of cultured cells on day 3 was twice that on day 1, and stayed at the same level until day 13. By electron microscopy, some of the cells cultured for 1 day were seen to be undifferentiated and others already showed differentiation into various hair structures. Such differentiated cells disappeared on day 3 and most of the cells cultured for 3 days were undifferentiated. Cell cultured for 6 days were differentiated showing inner root sheath cell, hair cortical cell and medulla cell structures. The characteristics of these cultured cells corresponded well to those of in vivo cells of the hair tissues from the back skin of 7-day-old C3H mice. On day 13 degeneration occurred in the cultured cells. In none of these cultures were mesenchymal cells, such as fibroblasts, found. The present electron microscopic study reveals that immature cells obtained from mouse hair tissues proliferate in vitro and differentiate into several subpopulations corresponding to those of in vivo cell layers of hair tissues. The present culture technique may be useful for studies of hair cell growth and differentiation.