The monoclonal antibody CR11-351 discriminates HLA-A2 variants identified by T cells

Serologic and immunochemical assays have shown that the monoclonal antibody (MoAb) CR11-351 recognizes a determinant expressed by HLA-A2 and A28 alloantigens. The MoAb CR11-351 blocks the cytotoxicity of some, but not all, anti-HLA-A2 and anti-HLA-A28 alloantisera tested. These findings suggest that each allospecificity consists of several determinants, only some of which are spatially close to the determinant defined by the MoAb CR11-351. The binding of the MoAb CR11-351 to HLA-A2 lymphoid cells is not effected by their precoating with the HLA-A, B-specific MoAb CR10-214, Q6/64, and 6/31 but is enhanced by at least 20% by the MoAb CR10-131, CR10-402 and by the beta 2-m-specific MoAb NAMB-1. The MoAb CR11-351 did not react with one of four HLA-A2 variants which are indistinguishable with conventional anti-HLA-A2 sera, but are not recognized by "normal" HLA-A2-restricted cytotoxic T cells and possess structurally distinct HLA-A2 heavy chains. Therefore the MoAb CR11-351 provides the first evidence of a serologically detectable difference between the four HLA-A2 variants and "normal" HLA-A2 antigens.     

Clonal T lymphocyte recognition of the fine structure of the HLA-A2 molecule

A human alloimmune cytotoxic T lymphocyte (CTL) clone (4E4) was generated against the HLA-A2 molecule. Lysis of 51Cr-labeled HLA-A2 target cells was blocked by monoclonal antibodies (mAb), including mAb PA2.1 (anti-HLA-A2), mAb BB7.2 (anti-HLA-A2), mAb 4B (anti-HLA-A2-plus-A28), mAb MA2.1 (anti-HLA-A2-plus-B17), and mAb W6/32 (anti-HLA-A,B,C), which are directed against different serologic epitopes on the HLA-A2 molecule. However, HLA-A2 mutant lines lacking the serologic epitope recognized by mAb BB7.2 (anti-HLA-A2) were efficiently lysed by CTL 4E4. Thus, although mAb may block cytolysis, the HLA-A2 epitope recognized the 4E4 CTL clone is distinct from the HLA-A2-specific epitope recognized by serologic reagents. Moreover, analysis of HLA-A2 population variants revealed that only the predominant HLA-A2.1 subtype molecule was recognized by CTL 4E4. No cross-reactivity on other, biochemically related HLA-A2 population subtypes was observed, including HLA-A2.2 cells (Hill, CVE, ZYL, M7), HLA-A2.3 cells (TENJ, DK1), or HLA-A2.4 cells (CLA, KNE). This CTL clone appears to recognize a single epitope and, like monoclonal antibody counterparts, can be used to discriminate among immunogenic cellular and serologic epitopes on closely related HLA-A2 molecules. On the basis of the known sequence changes in mutant and subtype HLA-A2 molecules, it appears that the sequence spanning residues 147 to 157 may be critical for cellular recognition of this Class I MHC molecule.     

Syngeneic anti-idiotypic antisera to murine monoclonal antibodies to monomorphic and polymorphic determinants of HLA class I antigens

BALB/c mice were immunized with syngeneic anti-HLA class I monoclonal antibodies. The latter included the anti-HLA-A2, A28 monoclonal antibody (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to HLA-B antigens and the MoAb CR10-215 and CR11-115 to the same (or spatially close) monomorphic determinant. Anti-idiotypic antibodies could be detected in bleedings obtained 3 days after the first booster, increased in titer in bleedings obtained after the second booster, and persisted at high levels in subsequent bleedings. The four anti-HLA class I MoAb did not differ in their ability to elicit syngeneic anti-idiotypic antibodies. Cross-blocking studies with a panel of anti-HLA class I, anti-HLA class II, and anti-human melanoma-associated antigen (MAA) MoAb showed that the anti-MoAb CR10-215 and anti-MoAb CR11-115 antisera contain only antibodies to private idiotopes, whereas the anti-HLA MoAb CR11-351 and anti-MoAb Q6/64 antisera also contain antibodies to public idiotopes. The latter are expressed by the anti-HLA class I MoAb CR11-351, Q1/28, Q6/64, and 6/31, and by the anti-HLA class II MoAb Q5/6, Q5/13, 127, and 441. Public idiotopes were not detected on the nine anti-MAA MoAb tested. Public idiotopes do not interfere with the binding of anti-HLA MoAb with the corresponding antigenic determinants. On the other hand private idiotopes are located within the antigen-combining site, because anti-idiotypic antisera specifically inhibit the binding of the corresponding immunizing anti-HLA class I MoAb to cultured human lymphoid cells in a dose-dependent manner. Analysis by isoelectric focusing of the anti-HLA class I MoAb antisera showed that the spectrotype of the anti-MoAb CR11-351 antiserum comprises four components that focus in the pH 6.9 to 6.2 range, the spectrotype of anti-MoAb Q6/64 antiserum comprises three components that focus in the pH 6.5 to 6.1 range, the spectrotype of the anti-MoAb CR10-215 antiserum comprises three components that focus in the pH 6.4 to 6.1 range, and the spectrotype of the anti-MoAb CR11-115 antiserum comprises three components that focus in the pH 6.6 to 6.4 range.     

Serological expression after sequential double transfection with purified HLA-11 gene of mouse fibroblasts carrying human beta-2 microglobulin

A genomic cosmid library constructed from DNA from a genotyped individual (JF = HLA-A11, Cw–, B38/A26, Cw7, B51) was screened for clones containing class I histocompatibility genes. Among these clones, one was found to carry a 4.8 kb Hind III fragment which is highly correlated with HLA-A11. This clone was used to transfect LMTK⁺ cultured mouse fibroblast transformants expressing human beta-2 microglobulin. The human beta-2 microglobulin heavy chain-associated determinant was positively detected by the M18 monoclonal antibody. HLA-A11 expression on these doubly transformed cells was specifically demonstrated by complement-dependent cytotoxicity with HLA-A11 + A3-specific but not with HLA-A3-specific monoclonal antibodies. Absorption studies with human alloantisera confirmed the presence on these cells of HLA-A11 determinants and of cross-reacting determinants which absorbed anti-HLA-A1 and –A3 alloantisera. The JF5-J27 transfected cell expressed both heavy and light chains of human class I histocompatibility genes.Abbreviations used in this paper ₂m beta-2 microglobulin - CTL cytolytic T lymphocytes - FCS fetal calf serum - HAT hypoxanthine-azaguanine-thymidine - kb kilobase pair - MHC major histocompatibility complex - MoAb monoclonal antibodies - PBL peripheral blood lymphocytes - PEG polyethylene glycol - r correlation coefficient This study is dedicated to the memory of Jean-Jacques Metzger.     

Lack of a role of monocytes in the inhibition by monoclonal antibodies to monomorphic and polymorphic determinants of HLA class I antigens of PHA-P-induced peripheral blood mononuclear cell proliferation

This study aimed at characterizing the mechanism(s) underlying the regulatory role of distinct determinants of HLA Class I antigens in PHA-P-induced T cell proliferation and the involvement of monocytes in this phenomenon. The anti-HLA-A2,A28 monoclonal antibodies (MoAb) CR11-351, the MoAb Q6/64 to a determinant restricted to the gene products of the I antigens HLA-B locus, and the MoAb CR10-215 and W6/32 to distinct monomorphic determinants of HLA Class I antigens were found to inhibit PHA-P-induced peripheral blood mononuclear cell (PBMC) proliferation in a dose-dependent fashion. The inhibition is specific and reflects neither inhibition of PHA-P binding to cells nor a toxic effect of the anti-HLA Class I MoAb. The latter differed in the concentration required to induce inhibition, in the influence of the concentration of PHA-P used as mitogen, in the differential effect on the donors used as a source of PBMC, and/or in the requirement of the Fc portion to induce inhibition. At variance with the information in the literature, the inhibitory effect of anti-HLA Class I MoAb on PHA-P-induced PBMC proliferation neither reflected their interaction with accessory cells nor was mediated by suppressor factors released by monocytes stimulated with PHA-P in the presence of anti-HLA Class I MoAb. Therefore, the regulatory role of HLA Class I antigens in T cell proliferation is not likely to be mediated by monocytes and/or factors released from them, but may reflect an involvement of these molecules in T cell activation pathways.     

Role of HLA class I and class II antigens in activation and differentiation of B cells

The monoclonal antibodies (MoAb) CR10-214, CR11-115, and Q1/28 to distinct monomorphic determinants of HLA class I antigens, the MoAb CL413 and PTF29.12 recognizing monomorphic determinants of HLA-DR antigens, the anti-HLA-DQw1 MoAb KS11, the anti-HLA-DPw1 MoAb B7/21, and the anti-HLA-DR,DP MoAb CR11-462 were tested for their ability to modulate human B-lymphocyte proliferation and maturation to IgM-forming cells. Purified tonsillar B cells were stimulated with Staphylococcus aureus bacteria of the Cowan first strain (SAC) or anti-human mu-chain xenoantibodies, as well as in growth factor- or T-cell-dependent activation cultures. The B-cell proliferative responses induced by SAC or by mitogenic concentrations of anti-mu-chain xenoantibodies were inhibited by some of the anti-HLA class I and anti-HLA class II monoclonal antibodies tested. The same antibodies were effective inhibitors of the proliferation of B cells stimulated with interferon-gamma (IFN-gamma) or interleukin-2 (IL-2) and with submitogenic concentrations of anti-mu-chain xenoantibodies. The proliferation induced by IL-2 of SAC-preactivated B cells was inhibited by some of the anti-HLA class II monoclonal antibodies, but not by the anti-HLA class I monoclonal antibodies tested. This inhibition appeared to reflect at least in part a direct effect on later events of the B-cell activation cascade, since some anti-HLA class II monoclonal antibodies still exerted considerable inhibitory activity when added together with IL-2 to SAC-preactivated B cells after the third day of culture. Anti HLA-DR, DQ, and DP monoclonal antibodies consistently inhibited the IgM production induced in B cells by T cells alone, T cells plus pokeweed mitogen (PWM), SAC plus IL-2, or IL-2 alone. In contrast, two of the three anti-HLA class I monoclonal antibodies tested inhibited the IgM production in cultures stimulated with SAC plus IL-2 and one the IgM production induced by IL-2 alone, but none of them had inhibitory effects on T-cell dependent IgM production. The results reported herein indicate that HLA class II molecules directly participate in different phases of the B-cell activation cascade. In addition, our data also suggest that HLA class I molecules can be involved in the events leading to B-cell proliferation and differentiation into immunoglobulin-secreting cells.     

Syngeneic anti-idiotypic antisera to murine anti-HLA class II monoclonal antibodies

BALB/c mice have been immunized with six anti-HLA Class II monoclonal antibodies (MoAb); the latter included MoAb CR11-462, Q5/6, and Q5/13 to monomorphic determinants, the anti-HLA-DR1,4,w6,w8,w9 MoAb AC1.59, the anti-HLA-DRw9 MoAb AB7ae9, and the anti-HLA-DQw3 MoAb AC6G. The six monoclonal antibodies markedly differ in their ability to induce anti-idiotypic antibodies, because the latter were not detected in the sera from the mice immunized with the MoAb AB7ae9 and with the MoAb AC6G. The MoAb AC1.59 and CR11-462 elicited antibodies to private idiotypes, and the MoAb Q5/6 and Q5/13 elicited antibodies to private and public idiotypes. The titer of the latter in the anti-MoAb Q5/6 antiserum appears to be markedly lower than that of the former ones; no marked difference was detected in the titer of the two types of antibodies in the anti-MoAb Q5/13 antiserum. Blocking experiments with a panel of monoclonal antibodies showed that the MoAb Q5/13 shares idiotypes with the anti-HLA Class I MoAb Q5/6, 127, and 441 and with the anti-HLA Class I MoAb CR11-351, Q1/28, Q6/64, and 6/31, but does not share idiotypes with any of the nine anti-human melanoma-associated antigen MoAb tested. The spectrotypes of the anti-MoAb CR11-462 and anti-MoAb Q5/13 antisera comprise two major components in the pH 6.2 to 6.7 range, that of the anti-MoAb Q5/6 antiserum comprises two major components in the pH 6.5 to 6.8 range, and that of the anti-MoAb AC1.59 antiserum comprises a number of components in the pH 5.6 to 7.2 range.     

Antibody-induced association between discrete regions of HLA class I and II antigens

The anti-HLA-DR + DP monoclonal antibody (MoAb) CR11-462 was unexpectedly found to cross-inhibit the binding to B lymphoid cells of the anti-HLA Class I MoAb CR10-215 and CR11-115. The latter two antibodies recognized the same or spatially close antigenic determinant. The cross-blocking of anti-HLA Class I MoAb CR10-215 and CR11-115 by MoAb CR11-462 reflects neither its contamination by anti-HLA Class I antibodies nor its cross-reactivity with HLA Class I antigens. On the other hand, the cross-blocking appears to reflect redistribution of HLA Class II antigens by the MoAb CR11-462, since the MoAb CR10-215 and CR11-115 are not susceptible to blocking when lymphoid cells are treated with 0.025% glutaraldehyde or are coated with Fab' fragments of the MoAb CR11-462. Furthermore, immunoprecipitates from B lymphoid cells preincubated with the MoAb CR11-462 before solubilization contain HLA Class I antigens. Therefore, these results have shown for the first time an antibody-induced association between discrete regions of HLA Class I and Class II antigens on the membrane of B lymphoid cells.     

Differential regulatory role of monomorphic and polymorphic determinants of histocompatibility leukocyte antigen class I antigens in monoclonal antibody OKT3-induced T cell proliferation

Monoclonal antibodies (mAb) to monomorphic and polymorphic determinants on the heavy chain of histocompatibility leukocyte antigen (HLA) class I antigens inhibit mAb OKT3-induced T cell proliferation, whereas the anti-beta 2-microglobulin mAb NAMB-1 does not affect it. The inhibitory effect of anti-HLA class I mAb is specific, is not an Fc-mediated phenomenon, does not require accessory cells, and does not involve early stages of T cell activation. Distinct determinants of HLA class I antigens regulate T cell proliferation by different mechanisms, because the anti-HLA-A2, A28 mAb CR11-351, and the mAb W6/32 to a framework determinant of HLA class I antigens block interleukin 2 (IL-2) secretion and IL-2 receptor expression, whereas the mAb CR10-215 to a monomorphic determinant blocks only IL-2 receptor expression. The mAb CR10-215 and W6/32 induced a 50% of maximal inhibition of T cell proliferation, when added after 27 and 12 hr, respectively, of incubation of peripheral blood mononuclear cells with mAb OKT3. On the other hand, the mAb CR11-351 inhibited T cell proliferation even when added after 38 hr of incubation of peripheral blood mononuclear cells with mAb OKT3 and was the only one to inhibit proliferation of cycling T lymphocytes. It is suggested that HLA class I antigens regulate T cell proliferation by interacting with cell-surface molecules involved in T cell activation. The differential inhibitory activity of the anti-HLA class I monoclonal antibodies tested may reflect the different ability of the corresponding determinants to interact with activation molecules.     

HLA-DR antigens and mitogenic factors released by PWM-stimulated T lymphocytes share the framework determinant recognized by the monoclonal antibody Q5/13

Human T lymphocytes release factors which enhance the mitogenic response of B lymphocytes to PWM. These mitogenic factors share with HLA-DR antigens the framework determinant recognized by the monoclonal antibody Q5/13 (MoAb Q5/13) since adsorption of T-cell medium with an excess of insolubilized MoAb Q5/13 significantly reduces the enhancing activity of the T-cell supernatant on the proliferative response of B cells to PWM. On the other hand, incubation of the T-cell supernatant with an excess of insolubilized anti-HLA-A,B MoAb Q1/28 did not effect the activity of the T-cell supernatant in the proliferative assay.     

Monoclonal antibodies to HLA-A,B, and Ia-like antigens inhibit colony formation by human myeloid progenitor cells

Hybridomas derived from the fusion of murine myeloma cells with splenocytes from mice immunized with human cultured lymphoid cells secreted monoclonal antibodies to human cell surface antigens. Serologic and immunochemical assays showed that 4 monoclonal antibodies (Ab Q2/47, Q2/61, Q2/70, Q2/80) recognize framework determinants of Ia-like antigens and 1 monoclonal antibody (Ab Q1/28) reacts with determinants expressed on the heavy chain of HLA-A,B antigens. Both anti-HLA-A,B and anti-Ia-like antigen monoclonal antibodies caused complement-dependent inhibition of granulocyte-macrophage colony formation by human bone marrow grown in soft agar. Mixing experiments excluded the possibility of an indirect effect on progenitor cells by lysis of auxiliary cells. These results indicate that human myeloid progenitor cells express HLA-A,B and Ia-like antigens.     

Ontogeny of human Ia antigens

Indirect immunofluorescence (IIP) staining of tissues from human fetuses (ages ranging from 8 to 32 weeks of intrauterine life) with monoclonal antibodies (MoAb) to monomorphic determinants of Ia antigens and HLA-A,B,C antigens has shown that both types of antigens are already detectable in tissues of 8-week-old fetuses. Ia antigens and HLA-A,B,C antigens reach their almost-complete tissue distribution after 32 and 24 weeks of intrauterine life, respectively. The structure of Ia antigens synthesized by fetal thymus cells is similar to that of B-lymphoid cell-derived Ia antigens. Ia antigen-bearing thymic fetal cells can stimulate allogeneic lymphocytes in mixed lymphocyte reactions (MLRs). These reactions are blocked by monoclonal antibodies to monomorphic determinants of human Ia antigens and of HLA-A,B, antigens.     

Regulatory role of a monomorphic determinant of HLA Class I antigens in T cell proliferation

The role of HLA Class I antigens in T cell proliferation was investigated by using the anti-HLA Class I monoclonal antibodies (MoAb) CR10-215, CR10-325, and CR11-115. MoAb CR10-215 and CR11-115 recognize the same (or spatially close) monomorphic determinant, which is distinct and spatially distant from that reacting with MoAb CR10-325. Addition of MoAb CR10-215 and CR11-115 to cultures of peripheral blood mononuclear cells stimulated with MoAb OKT3, MoAb Pan T2, PHA, or PPD inhibited cell proliferation. The blocking is specific in that the anti-HLA Class I MoAb CR10-325 and the Pan T MoAb Pan T1 had no effect on the proliferation. The inhibitory activity of MoAb CR10-215 and CR11-115 does not reflect i) toxic effects, ii) induction of suppressor cells and factors, iii) blocking of the binding of mitogens to lymphocytes, iv) inhibition of the production of interleukin 1 (IL 1) and interleukin 2 (IL 2), or v) function of IL 2 receptor. Anti-HLA Class I MoAb were able to inhibit the proliferation of purified, Tac-, T cells. The inhibited cells did not express Tac antigen, as assayed by direct immunofluorescence, with MoAb anti-Tac, but released a normal amount of IL 2 in culture medium. These results indicate that monomorphic determinants of the HLA Class I complex are involved in the regulation of T cell proliferation. The effect appears to occur at the level of IL 2 receptor expression.     

Comparison of one-dimensional IEF patterns for serologically detectable HLA-A and B allotypes

A new mouse monoclonal antibody (MoAb) 4E, which detects an epitope shared by HLA-B locus antigens, together with the MoAb W6/32, detecting a common HLA, B, C, determinant, and the MoAb 4B, detecting HLA-A2 and A28, were used to isolate HLA-A and -B antigens in sequential immunoprecipitation. The HLA antigens obtained from metabolically labeled cell extracts of B-lymphoblastoid cell lines or from phytohemagglutinin (PHA) activated peripheral blood lymphocytes were compared by one-dimensional isoelectric focusing (1D-IEF). The IEF banding patterns obtained with native HLA antigens segregated in a family with HLA. Neuraminidase treatment of isolated antigens reduced the number of bands to one or two, simplifying the analysis of characteristic patterns. Thus, we have cataloged IEF banding patterns for the majority of the serologically recognized HLA-A and -B allotypes obtained from 57 unrelated American Caucasians. While no HLA-A locus or HLA-B locus specific banding patterns were observed, the HLA-A antigens had, in general, slightly higher pl values than the HLA-B antigens. HLA-C antigens could not be detected in this assay system. The polymorphism detected by IEF banding patterns was as extensive as the serologically detected polymorphism identified by classical HLA serology. Moreover, variants for some HLA allotypes could be detected by this method. In addition to previously recognized A2 variants, new variants were identified for HLA-A1, A26, and Bw44. Each A1 and Bw44 variant was associated with particular haplotypes. The HLA-A2 antigens occurred on 43 HLA haplotypes in the unrelated Caucasian population. Only one of each A2 variants was identified in this population.     

Cross-reactivity between human and murine lymphocyte antigens

The anti-H-2 alloantiserum D-32 [(BlO.A(2R) × C3H.SW) anti-C3H] is cytolytic to human lymphocytes. Fab₂ blocking assays, indirect immunoprecipitation and sequential immunoprecipitation experiments showed that the anti-H-2 alloantiserum D-32 recognizes antigenic determinants which are expressed on the heavy chain of subpopulations of HLA-A, B antigens. These determinants are different from those defining the serological polymorphism of the HLA-A, B, C system, are the same as or spatially close to those recognized by the anti-HLA-A, B monoclonal antibody Q6/64 and are expressed on rabbit, rat or guinea pig lymphocytes.     

Molecular characterization of HLA-A,B homologues in owl monkeys and other nonhuman primates

Mouse anti-HLA-A, B monoclonal antibodies have been used to study the homologues of HLA-A, B antigens in other primate species. Immunoprecipitates of primate histocompatibility antigens from extracts of radioactively labeled lymphocytes were analyzed by SDS-polyacrylamide electrophoresis. Primate histocompatibility antigens appear to have similar molecular structure to human HLA antigens. Owl monkeys, which react polymorphically with some monomorphic anti-HLA antibodies, showed biochemical differences which correlated with the serological polymorphism. An antibody (W6/32) which only reacts with the HLA/β ₂-microglobulin complex in humans and not with the free HLA heavy chain has the reverse specificity in some owl monkeys.     

Enhancing effect of anti-HLA class I monoclonal antibodies on T cell proliferation induced via CD2 molecule

The mAb 131 to a determinant preferentially expressed on the gene products of the HLA-A locus, the mAb Q6/64 and 4E to determinants preferentially expressed on the gene products of the HLA-B locus, the anti-HLA-A2,A28 mAb CR11-351, HO-2, HO-3, HO-4, and KS1, and the anti-HLA-B7 cross-reacting group mAb KS4 enhanced proliferation of T cells in most, if not all, the PBMC preparations stimulated with the anti-CD2 mAb 9-1 + 9.6. The mAb CR10-215, W6/32, and 6/31 to distinct monomorphic determinants of HLA class I antigens enhanced CD2-induced T cell proliferation in at most 30% of the PBMC preparations tested. The anti human beta 2-microglobulin (beta 2-mu) mAb NAMB-1 displayed no detectable effect on the proliferation of T cells stimulated with the mAb 9-1 + 9.6. The enhancing effect of anti-HLA class I mAb is specific, is dose dependent, is not abrogated by the addition of exogenous IL-1 and IL-2 to the cultures, and reflects the interaction of anti-HLA class I mAb with T cells. Enhancement of CD2 mediated proliferation of T cells is not a unique property of anti-HLA class I mAb, since the anti-HLA class II mAb Q5/6 and Q5/13 also had a similar effect. Analysis of the kinetics of the enhancing effect of anti-HLA class I mAb suggests that they modulate an early event of T cell activation and may affect the interaction of T cells with mAb 9-1. Phenotyping of T lymphocytes activated by mAb 9-1 + 9.6 in the presence of anti-HLA class I mAb suggests that the enhancing effect of anti-HLA class I mAb may reflect the recruitment of a higher percentage of T cells. The present study has shown for the first time that certain, but not all, the determinants of the HLA class I molecular complex are involved in the proliferation of T cells stimulated with the anti-CD2 mAb 9-1 + 9.6. Furthermore, the inhibitory effect of mAb CR11-351, KS1, Q6/64, and W6/32 on the proliferation of T cells stimulated with mAb OKT3 or with mAb BMA 031 indicates that the same determinants of HLA class I antigens play a differential regulatory role in T cell proliferation induced via the CD2 and CD3 pathway.     

Characterization of a T-cell clone recognizing idiotypes as tumor-associated antigens

Although heterogeneous T cells recognizing idiotypic determinants have been demonstrated to occur spontaneously in vitro or to be expanded by immunization with antigen or idiotype, their in vitro propagation and cloning was not successful. These previous studies have relied extensively on soluble immunoglobulin to induce proliferation of idiotype-specific T cells. This report describes a unique approach to obtain a stable T-cell clone specific for a monoclonal beta 2-6 fructosan binding myeloma ABPC48 (BALB/c origin), bearing well-defined A48 regulatory idiotopes. Following repeated immunizations with ABPC48 myeloma protein of C.B/R3 mice (H-2d, VHb, CHa), which differ only in the VH locus from BALB/c mice (H-2d, VHa CHa), several stable T-cell clones were obtained after stimulation in vitro with ABPC48 myeloma cells. The proliferation of a T-cell clone A48.B2 was observed with irradiated myeloma cells or hybridomas producing antibodies bearing A48 idiotype encoded by genes deriving from the VH 441-4 family. Proliferation of the clone did not occur with soluble ABPC48 myeloma protein or with Sepharose 4B-bound ABPC48 myeloma protein. Both anti-A48Id and anti-Iad monoclonal antibodies can specifically inhibit the proliferation of this clone when stimulated with ABPC48 myeloma cells. These results demonstrate recognition of idiotypes on B-cell tumours by T cells and implicate the role of class II major histocompatibility complex determinants in this cellular interaction.     

Identification of a cyclin B1-derived CTL epitope eliciting spontaneous responses in both cancer patients and healthy donors

With the aim to identify cyclin B1-derived peptides with high affinity for HLA-A2, we used three in silico prediction algorithms to screen the protein sequence for possible HLA-A2 binders. One peptide scored highest in all three algorithms, and the high HLA-A2-binding affinity of this peptide was verified in an HLA stabilization assay. By stimulation with peptide-loaded dendritic cells a CTL clone was established, which was able to kill two breast cancer cell lines in an HLA-A2-dependent and peptide-specific manner, demonstrating presentation of the peptide on the surface of cancer cells. Furthermore, blood from cancer patients and healthy donors was screened for spontaneous T-cell reactivity against the peptide in IFN-γ ELISPOT assays. Patients with breast cancer, malignant melanoma, or renal cell carcinoma hosted powerful and high-frequency T-cell responses against the peptide. In addition, when blood from healthy donors was tested, similar responses were observed. Ultimately, serum from cancer patients and healthy donors was analyzed for anti-cyclin B1 antibodies. Humoral responses against cyclin B1 were frequently detected in both cancer patients and healthy donors. In conclusion, a high-affinity cyclin B1-derived HLA-A2-restricted CTL epitope was identified, which was presented on the cell surface of cancer cells, and elicited spontaneous T-cell responses in cancer patients and healthy donors.     

In vitro-isolated human cytotoxic T-lymphocyte clones detect variations in serologically defined HLA antigens

T cells of two donors, JR (HLA-A23, 29; B7,7; G; DRw5) and HG (HLA-A2, 23; B40, w44; Cw4), were stimulated with cells from an HLA homozygous lymphoblastoid cell line JY (HLA-A2, 2; B7,7, C-, DRw4, 6) and cloned by limiting dilution after the third stimulation. Two cytotoxic T-cell (CTL) clones, JR-2-16 (from donor JR) and HG-31 (from donor HG), were used for detailed studies. The results of a panel study using lymphocytes from HLA-typed individuals and a study with two HLA recombinant families indicate that the antigens recognized by the CTL clones JR-2-16 and HG-31 were highly associated with HLA-A2 and HLA-B7, respectively. Blocking studies with a monoclonal antibody recognizing a framework determinant on HLA-A, -B and-C antigens and a monoclonal antibody reacting with HLA-A2 support the notion that JR-2-16 and HG-31 interact with the HLA-A2 and the HLA-B7 antigens per se. However, these clones did not recognize the HLA-A2 and HLA-B7 of all donors typed for these antigens, suggesting that the HLA-A2 and HLA-B7 antigens of these particular donors are variants of the serologically defined HLA antigens. These results indicate that in vitro-derived human CTL clones detect variations in the serologically defined allospecificities and can be used as reagents to elucidate the polymorphism of HLA antigens further.Abbreviations used in this paper: CTL cytotoxic - T lymphocytes - BSA bovine serum albumin - PHA phytohemagglutinin - Con A concanavalin A.