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.

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.

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.

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.

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.

Human anti-HLA-DQw2 monoclonal antibody secreted by an Epstein-Barr-virus--transformed lymphoblastoid cell line: assessment of the monoclonality, allospecificity, and target.

IgM molecules were purified by the use of anti-IgM antibody-coupled Sepharose from the culture supernatant of an Epstein-Barr-virus-transformed lymphoblastoid cell line, MP1, that secretes alloantibodies possessing HLA-DQw2 specificity as defined by the cytotoxicity assay. The obtained IgM preparation was labeled with radioactive iodine-125I and fractionated by gel filtration. It contained pentameric IgM and smaller oligomeric IgMs. When tested by the direct cellular binding assay against a panel of HLA-typed cell lines, they all showed the DR3 and DR7 association pattern characteristic of DQw2. A weak but significant binding was detected for DR1, DR6, and DR9. On isoelectrofocusing, MP1 pentameric IgM gave a restricted banding pattern comparable to monoclonal IgM obtained from a patient with Waldenström's syndrome. Moreover, the pattern was identical to that of IgM purified from the culture supernatant of a defined hybrid clone, 162, that was generated by fusing MP1 cells with heteromyeloma D33 cells. The target class II molecules showed the dimeric structure that conforms to DQw2 molecules.

A high proportion of sera of heroin addicts possesses anti-HLA class I and class II reactivity.

The anti-HLA reactivity of sera from 210 heroin addicts was tested by the direct binding with 125I-labeled preparations of HLA class I and class II molecules purified from human B-cell lines of various HLA haplotypes. A high proportion (81.7%) of the sera tested possessed anti-HLA class I and II reactivity. The reactivity did not show any allospecificity and was apparently mediated by antibodies. The control included 100 healthy blood donors, 25 male homosexuals positive for anti-HIV (human immunodeficiency virus) antibodies, and 25 patients positive for HBsAg (hepatitis B surface antigen). Of these controls, only one of the healthy blood donors was positive for anti-HLA reactivity (P much less than 0.001). Among heroin addicts, the reactivity was independent of the presence of either HBsAg or anti-HIV antibodies in the serum.

Immunochemistry of the HLA class II molecules isolated from a mouse cell transfected with DQ alpha and beta genes from a DR4 haplotype.

Cells from a mouse B lymphoma were transfected by DQ alpha and DQ beta genes derived from a DR4 haplotype. Quantitatively, the resulting expression of human class II molecules was similar to that of human B lymphoblastoid cell lines. Qualitatively, the transformant class II molecules differed from "normal" class II molecules in their carbohydrate moiety. As for their antigenic specificity, they were shown to carry two determinants previously identified on DQ molecules controlled by DR4 haplotypes, i.e., DQw3 and DCHON. The transformant molecules did not carry a third DR4-associated specificity, DC5 (equivalent to TA10), and must possess a structure allelic to DC5. However, no corresponding alloantigenic specificity was detected by a screening of relevant alloantisera.

Serological and functional analysis of the epitope clusters on Leu3/T4 antigen.

Monoclonal antibodies (MoAbs) were generated against cells or cell membrane glycoproteins of a human T-cell line, HPB-ALL. Five, designated MCN 1, 3, 12, 19, and 29, were found to be specific to helper/inducer T-cells; they gave a positive membrane staining to approximately 48% of peripheral blood lymphocytes and 84% of thymocytes and these proportions did not change upon costaining with Leu 3a, a known anti-helper/inducer T-cell MoAb. Furthermore, their reaction pattern with a panel of human lymphoid cell lines was identical to that of Leu 3a. A reciprocal binding blocking test showed that the epitopes reactive with the MCN MoAbs are divided into three separate clusters. The MCN 3- and Leu 3a-reactive epitopes formed a cluster and they appeared to be the same epitope. This cluster was well separated from that represented by the MCN 1-reactive epitope. The MCN 12-, 19-, and 29-reactive epitopes could be assigned to a third cluster. MCN 12 and 19 were probably toward the same epitope. A sequential binding test indicated that the three epitope clusters reside on the molecule carrying Leu 3a-defined epitope, i.e., the Leu3/T4 antigen. On the functional analysis, MCN 3 gave a profound inhibitory effect on T-cell proliferative response to MHC class II antigens, whereas other MCN MoAbs did not show any modifying effect on the T-cell function.