HLA-A9 antibodies and epitopes

Fifty pregnancy alloantisera directed towards HLA-A23 and/or A24 antigens were investigated serologically in titration studies against the three sequenced HLA-A9 specificities, A23 (A*2301), A24 (A*2402) and A2403 (A*2403). The reaction patterns of the antisera fell into five categories which allowed the three HLA-A9 specificities to be easily differentiated. Based on the various titre cytotoxicity scores of the antisera five possible antibody specificities were defined: anti-A23; -A24; -A23/24; -A24/2403 and anti-A23/24/2403. One antiserum crossreacted with HLA-A1 and A24. Inspection of the amino acid sequences of 136 HLA-A, B and C molecules allowed the prediction of five unique epitopes corresponding to these antibody specificities, a possible epitope unique to A2403 and confirmation of a likely epitope shared by A1 and A24. These, together with the previously suggested epitopes HLA-A9/ A2/A28 and A1/A23/A24 together with the presence of Bw4 on the three HLA-A9 antigens suggests that the HLA-A9 family of antigens is characterized by a minimum of nine serologically definable epitopes.     

Specific lysis of murine cells expressing HLA molecules by allospecific human and murine H-2-restricted anti-HLA T killer lymphocytes

The lysis by human and murine anti-HLA cytolytic T lymphocytes (CTL) of murine cells expressing class I HLA molecule after gene transfection has been studied using two different murine cells: LMTK- and P815-HTR-TK-. Weak but significant HLA-A11-specific lysis was found occasionally with human CTL on the HLA-A11+ L cells. On the contrary, P815-A11 or P815-A2 cells were lysed strongly and specifically by HLA-A11 or HLA-A2-specific human CTL. The T8+T4- phenotype of the effector cells was confirmed and the reaction was inhibited by anti-HLA class I monoclonal antibodies. Despite their higher sensitivity to human CTL, the P815-HLA+ cells did not express higher levels of HLA antigens than L cells, and the presence or the absence of human beta 2 microglobulin was irrelevant. Anti-human LFA-1 antibodies abrogated the lysis of P815-A11+ cells showing that the LFA-1 receptor which is apparently lacking on the L cell surface was on the contrary expressed on P815 cells. On the other hand, murine anti-HLA CTL have been prepared by immunizing mice against syngeneic HLA-A11+ L cells. They lysed very efficiently and specifically these cells, but appeared completely devoid of activity against human HLA-A11 target cells. This barrier was apparently due to the H-2 restriction of these H-2k anti-HLA murine CTL, as shown by their inability to lyse allogeneic H-2d cells expressing HLA-A11, and by the blocking of their activity by anti H-2k antibodies. By contrast, xenogeneic anti-HLA CTL obtained by immunizing murine lymphocytes against human cells lysed both human and murine HLA+ cells but they reacted with a monomorphic epitope of the HLA molecule in a nonrestricted way. These results show that human cells lyse very efficiently P815 murine cells expressing HLA class I antigens; the higher sensitivity of P815 cells compared to L cells is probably due to the presence of a LFA-1 receptor on these cells; a class I molecule of human origin can be seen as an H-2-restricted minor histocompatibility antigen in another species.     

RG1, a new murine monoclonal antibody recognizing a "supertypic" determinant on HLA-A molecules

Abstract: Monomorphic and polymorphic anti-HLA monoclonal antibodies (mAb) are valuable reagents for assessment of the structural and functional importance of different class I determinants. We have generated a new mAb, RG1, reacting with an epitope variably expressed on normal and leukemic hematopoietic cells of different lineages. Immunoprecipitation of the RG1 antigen disclosed a bimolecular complex characteristic of class I proteins. The RG1 epitope was expressed on an HLA-A2 transfected cell line but not on cells transfected with HLA-E, -F or -G molecules. MAb reactivity with reference B-lymphoblastoid cell lines and HLA typing of RG1 reactive and unreactive cells demonstrated that the epitope was expressed in conjunction with defined HLA-A molecules. Cells expressing HLA-A2, -A24(9) and -A68(28) proteins were brightly stained with RG1 whereas mAb binding to HLA-A1, -A11 and a split of A3 molecules was significantly lower. In contrast, the RG1 epitope was apparently not expressed on HLA-A23(9), -A25(10), -A26(10), -A29(19), -A30(19), -A31(19), -A32(19), -A33(19) and some HLA-A3 molecules. Based on class I α sequence data, these results suggest that the RG1 epitope is localized to a region of the α2 helix accessible to the T cell receptor for antigen on cytotoxic T lymphocytes. Lys in position 144 and His in position 151 are apparently critical for RG1 binding.     

Transfection of HLA genes using genomic DNA

Mouse L cells expressing HLA genes are potentially useful for producing and analyzing monoclonal antibodies (mAb) to HLA molecules. This paper describes the preparation of transfectants using uncloned human DNA and three methods to isolate the HLA-expressing transfectants. Transfectant libraries were made by contransfecting mouse thymidine kinase (tk)-deficient L cells with a calcium phosphate precipitate containing genomic DNA and tk plasmid DNA. Transfectants expressing HLA genes were isolated using these methods: immunomagnetism, replicate-plating combined with cellular enzyme-linked immunoassay, and sorting using a fluorescence-activated cell sorter. Two HLA-A2 transfectants were isolated using immunomagnetism, two HLA-A24 transfectants by replicate plating, and one HLA-Bw60 transfectant by FACS. However, no transfectants were isolated that stably expressed class II genes. The class I transfectants have been useful in characterizing several locally prepared mAbs which bind to monomorphic determinants on class I HLA molecules. Two of the transfectant lines, one expressing HLA-A2 (8001) and the other HLA-A24 (8008), have been included in the collection of lines distributed for use in the Eleventh International Histocompatibility Workshop.     

Comprehensive analysis of the frequency of recognition of melanoma-associated antigen (MAA) by CD8 melanoma infiltrating lymphocytes (TIL): implications for immunotherapy.

Fifty-nine tumor-infiltrating lymphocyte (TIL) cultures established from melanoma-invaded lymph nodes were screened for recognition of 28 melanoma-associated antigens (MAA) in association with31 HLA molecules. Twenty-three (39%) TIL lines reacted to at least one melanoma antigen. Melanosomal proteins were recognized by 19 TIL populations and the most prominent responses against these proteins were directed against Melan-A/MART-1 (mainly in association with HLA-A*0201) and gp100 (in association with diverse HLA contexts). Ten TIL populations reacted against 10 tumor-specific antigens, in association with 8 different HLA molecules. HLA-A*0201 and B*3501-restricted responses were the most frequent with, respectively, 17 and 7 responses directed against 5 distinct antigens. Unexpectedly, the recognition by TIL of different MAA was frequently restricted by a single HLA in individual tumors, and there was no evidence for the existence of dominant MAA epitopes between tumors,except for Melan-A/MART-1 antigen. This analysis also led to the detection of 21 new HLA-peptide complexes recognized by melanoma TIL. This study, which is to our knowledge the most comprehensive analysis of TIL specificity to tumor antigens, has several implications for the design of immunotherapeutic strategies based on immunization against selected tumor epitopes.     

Differential immunogenicity of paternal HLA Class I antigens in pregnant women

More insight into the differential immunogenicity of human leukocyte antigen (HLA) mismatches will be beneficial for donor selection in clinical transplantation. In this study the immunogenicity of HLA antigens was analyzed by examining the antibody profiles in women who have been pregnant. In total 888 women, who had pregnancy induced HLA alloantibodies, were included in this study, while 413 women who had not been immunized by their pregnancy, served as controls. First it was analyzed whether women expressing particular HLA antigens are more likely to produce HLA alloantibodies. Next we determined whether certain HLA mismatches of their children are more immunogenic than other ones. Finally we studied whether the immunogenicity of specific HLA mismatches is dependent on the HLA phenotype of the women. Women expressing HLA-A3, HLA-A32, and HLA-B21 are more likely to produce alloantibodies whereas women expressing HLA-B13 and HLA-B17 have a significantly lower incidence of alloantibodies compared with women expressing other HLA antigens. Children with HLA-A2 or HLA-B5 mismatches induced alloantibodies significantly more often whereas children with HLA-A30, -A31 or -A33 and HLA-A28 induced alloantibodies significantly less often than children with other HLA class I mismatches. Finally we could demonstrate that the immunogenicity of a particular HLA mismatch is dependent on the HLA phenotype of the women. Information on the differential immunogenicity of HLA mismatches may be of benefit for the determination of acceptable and taboo mismatches in the case of donor selection for (highly sensitized) patients.     

Anti-HLA-A2 and -A28 monoclonal antibody: production and study of the cross-reaction

An anti-HLA-A2 and -A28 monoclonal antibody, XV.17, has been prepared by immunizing a Balb/c mouse with PBL. This XV.17 monoclonal antibody is a cytotoxic IgM. Its reactivity was tested by lymphocytotoxicity test and indirect immunofluorescence technique, in parallel with an alloantiserum ORA having the same anti-HLA-A2, -A28 reactivity pattern, against different panels. Family studies were undertaken. Absorptions-elutions and cytofluorometry experiments were performed to study the cross-reaction. The XV. 17 monoclonal antibody is cytotoxic against all the HLA-A2 and -A28 tested cells, and is absorbed by HLA-Aw23 and -Aw24 cell susepnsions.     

Evaluation of individual specificities of class I HLA on platelets by a newly developed monoclonal antibody

In order to quantify each specific HLA-A or-B antigen on platelets, a monoclonal antibody against HLA heavy chains was developed and designated as 2F2 monoclonal antibody. This monoclonal antibody reacted on Western blot with platelet HLA from each 10 individuals with different HLA phenotypes and precipitated all ³⁵S-methionine-labeled HLA-A and -B antigens from three different Epstein-Barr Virus-transformed lymphoblastoid cell lines. The results indicate that the 2F2 monoclonal antibody recognizes an epitope shared by different HLA-A and -B antigens. The quantitative variation of specific HLA antigens on platelets was then studied in nine different donors by isoelectric-focusing gel electrophoresis and immunoblot using the 2F2 monoclonal antibody. The results of our studies showed that the shared HLA antigens such as A2, B35, and B62, varied three- to fivefold among different individuals and individual HLA-A or -B antigen was not equally expressed on a person's platelets. The relative quantities of specific HLA-A and -B antigens on lymphocytes were also noted to be the same as those on platelets. The finding suggests that differential expression of HLA specificities may not be restricted to platelets but is a more general phenomenon including other nucleated cells.     

Enhancement of immunogenicity of Jeg3 cells by ectopic expression of HLA-A*0201 and CD80

PROBLEM: The choriocarcinoma cell line Jeg3 suppresses immunity in vitro by secretion of soluble factors like leukemia inhibitory factor suppressing leukocyte activation. The cells lack expression of classical human leukocyte antigen (HLA)-A and -B alleles but express some HLA-C, and non-classical HLA-G and -E. Upon binding to killing inhibitory receptor on natural killer (NK) cells, HLA-G prevents activation of cytolytic activity. We investigated whether Jeg3 cells are capable of immune stimulation after complementation with classical HLA and T cell costimulatory signal CD80. METHOD OF STUDY: Jeg3 cells were transduced to express HLA-A*0201 and/or CD80. Parental Jeg3 or transfectants Jeg3-A2, Jeg3-CD80 or Jeg3-CD80-A2 were used to stimulate allogeneic resting and activated peripheral blood lymphocytes (PBL). The different cell lines were loaded with a HLA-A2-restricted Epstein-Barr virus (EBV) recall antigen peptide epitope and antigen presenting ability was examined. T cell lines specific for Jeg3 and transfectants were generated from HLA-A2 matched and nonmatched donors and compared for expansion, phenotypes and cytolytic activity. RESULTS: While all Jeg3 cell lines induced only marginal proliferation of resting T cells, phytohemagglutinin (PHA)-activated T cells were stimulated by CD80 or CD80-A2 expressing Jeg3. Only the transfectant Jeg3-CD80-A2 was capable of specific T cell stimulation by EBV recall antigen presentation. T cell lines of HLA-A2 non-matched donors stimulated with the Jeg3 transfectants showed significant expansion only when HLA-A2 and the costimulus CD80 were present. T cells from HLA-A2 positive donors did not expand significantly or differentially. No NK cells grew under any condition. In Jeg3-CD80-A2 stimulated T cells lines CD8+ cells expanded preferentially. These T cells exerted cytolytic activity toward all Jeg3 cell lines. CONCLUSION: Our data suggest that, in spite of immunosuppressive mechanisms, proliferative and cytolytic T cell responses are induced by Jeg3 cells when classical HLA- and/or costimulatory signals are present on the cells.     

An HLA class I specific monoclonal antibody that fails to bind to all HLA-A antigens

A monoclonal antibody, MHM.5, specific for HLA class I antigens, bound to lymphocytes of all donors tested and was thought to bind to a monomorphic determinant. When the antibody was used to precipitate 35S methionine labeled HLA class I molecules from lymphoid cells, which were then isoelectric focused, it was found that the HLA-A1,A2 and A3 antigens were not precipitated. Similarly, MHM.5, which is IgG1, failed to block complement mediated lysis by alloantisera specific for HLA-A1, 2 and 3, and most other HLA-A antigens. HLA-Aw24, A25, and A32, and all other HLA-B and C typing reactions tested were blocked. Thus the antibody binds to an epitope that is lacking on most A antigens, but present on Aw24, A25, A32 and all B and C locus antigens. Comparison of the published amino acid sequences of HLA-A2, A3, Aw24, A28, Cw3, B7, and B40 suggests some possible sites for this epitope.     

Two cytotoxic human-human hybridoma antibodies to HLA: TrAH10 (anti A3.1) and TrAG2 (anti B7,Bw42)

Two cytotoxic human-human hybridoma IgM antibodies to HLA were generated by EBV transformation of PBMC from multiparous women and fusion of EBV transformed cells with the human fusion partners KR4 or KR12. Both mAbs required the sensitive immunomagnetic cytotoxicity method to display killing of freshly prepared PBMC. One mAb (TrAH10) was specific for HLA-A3. Strikingly, TrAH10 reacted much more strongly with lymphoblastoid cell lines of HLA-A3.1 than of the rare variant HLA-A3.2, previously detected by cytotoxic T cells. Thus, in the microcytotoxicity test, the titer of concentrated TrAH10 was approximately 2000 times higher for A3.1 as compared to A3.2, and a clear difference was also observed in radioimmunoassay. Since the two HLA-A3 variants differ by only two amino acids at positions 152 and 156 of the alpha 2-domain's alpha-helix, the epitopes defined by the mAb TrAH10 and HLA-A3.1 specific cytotoxic T cells must be closely related. The observations with TrAH10 suggest that the HLA polymorphism detected by human mAbs may turn out to be as extensive as the T-cell defined HLA polymorphism. The other mAb (TrAG2) bound B7 and Bw42 with equal strength, and in addition bound weakly to some cells that were Bw22 or B39. Magnetic polymerbeads coated with affinity purified human mAbs TrAH10 or TrAG2 formed rosettes with EBV transformed cells carrying relevant HLA antigens; however, rosette formation with freshly isolated PBMC was very weak and unsuitable as a typing assay.     

Identification of HLA-A24-restricted CTL epitope encoded by the matrix protein pp65 of human cytomegalovirus

The activation of a specific cellular immune response against human cytomegalovirus (CMV) is an important key factor to solving CMV infection after bone marrow transplantation (BMT). In the present study, our purpose was to identify the HLA-A24-restricted cytotoxic T cell (CTL) epitope from the CMV immunogenic matrix protein pp65. We selected five CMV pp65 peptides with HLA-A24 binding motif from the HLA peptide binding predictions web site. Peptide binding assay was performed using biotinylated HLA-A24-restricted MAGE-1 peptide as a reference peptide and transporter associated with antigen processing (TAP)-deficient T2-A24 cells expressing high level of HLA-A24 protein as target cells. After co-incubation of biotinylated MAGE-1 and titrated amounts of competitor peptides with T2-A24 cells, the binding of each peptide was analyzed on a flow cytometer. Peptide binding assay showed that three out of five peptides derived from CMV pp65 bound to T2-A24 cells with various affinity levels. CTL induction assay using peptide-pulsed DC derived from eight HLA-A24(+) donors revealed that the peptide (QYDPVAALF) with the highest affinity was able to elicit potent CTLs which killed peptide-pulsed TISI cells. These CTLs were found to show the killing activity against human fibroblast cells transduced with both HLA-A*2402 and CMV pp65 cDNAs, and CMV-infected HLA-A24(+) fibroblast cells. These results suggested that the peptide (QYDPVAALF) is one of HLA-A24-restricted CTL epitope derived from CMV pp65 protein and may be of therapeutic value in peptide-based vaccines against CMV infection in BMT patients.     

Differences in the antigens recognized by cytolytic T cells on two successive metastases of a melanoma patient are consistent with immune selection

We have studied the patterns of antigens recognized by autologous cytolytic T lymphocytes (CTL) on two melanoma cell lines derived from metastases that were removed from patient LB33 at several years distance. Cell line LB33-MEL. A was obtained after surgery in 1988. A large number of CTL clones directed against LB33-MEL. A was obtained with blood lymphocytes collected from the patient in 1990. In vitro selection of melanoma cells that were resistant to these CTL clones indicated that at least five different antigens were recognized on LB33-MEL. A by autologous CTL. Four of these antigens were found to be presented by HLA-A28, B13, B44 and Cw6, respectively. The patient remained disease-free until 1993 when a metastasis was detected and was used to obtain cell line LB33-MEL. B. This cell line proved resistant to lysis by all the CTL clones directed against the LB33-MEL. A cells and showed no expression of HLA class I molecules except for HLA-A24. Using LB33-MEL. B cells to stimulate blood lymphocytes collected from the patient in 1994 we derived CTL clones that lysed these cells. All these CTL clones recognized a new antigen presented by HLA-A24. These results suggest that in patient LB33 the melanoma cells may have lost the expression of several HLA molecules under the selective pressure of an anti-tumor CTL response.     

Expression of an unusual Bw4 epitope by a subtype of HLA-B8 [B*0802]

Abstract: The primary structure of a variant HLA-B8 antigen has been determined by cDNA cloning and sequencing. The variant, B*0802 differs, from the common B*0801 subtype at positions 77–83 of the α₁ helix that determine the Bw4 and Bw6 public epitopes. Whereas B*080l has the common Bw6 motif, B*0802 has the Bw4 motif found in B*13 and B*44 allotypes. Serological analysis of B cell lines expressing B*0802 and of a B*0802 transfectant made with the HLA-A, B negative cell line 721.221 shows that B*0802 reacts with Bw4-specific antibodies, but at a level much lower than expected for Bw4 positive HLA-B allotypes.     

HLA class I association with progression to end-stage renal disease in patients from Zulia,Venezuela

The aim of this study was to determine HLA associations with progression to end-stage renal disease (ESRD) in the mixed Zulian population in Venezuela, regardless of other factors. A retrospective study to determine HLA Class I association was performed on 188 patients with ESRD due to different types of glomerulonephritis, and 202 healthy controls. Patients and control groups were serologically typed by Terasaki microlymphocytotoxicity technique using commercial Class I plates including 26 HLA-A and 48 HLA-B specificities. The antigens positively associated to the ESRD were: HLA-B38, B51, B53 and B62. Negatively associated antigens were: HLA-A9, B12, B17, B40 and B48. The haplotypes positively associated were: HLA-A2-B51, A2-B53, A23-B38 and A68-B38. The negatively associated haplotypes were: HLA-A2-B12, A2-B48, A9-B35 and A28-B40. The high Odds ratio observed and its statistical corroboration reflect the strength of the described association between HLA antigens and ESRD. Further molecular studies should clarify types and subtypes of the HLA class I alleles involved in the progression to ESRD.     

HLA antigens: rabbit antisera reacting with all A series or all B series specificities

Papain-solubilized HLA antigens giving only two bands of 34 000 mol.wt. and 11 000 mol.wt. by sodium dodecyl sulfate (SDS) gel electrophoresis and isolated from the cultured human lymphoblastoid cell line RPMI 4265, have been used to prepare antisera in rabbits. Antisera were raised against soluble products of the A (or 1st) series of HLA, bearing the determinant HLA-A2 (A2 substance), or against a mixture of products of the B (or 2nd) series of HLA, bearing the determinants HLA-B7 and HLA-B 12 (B7-12 substances). Rabbit antisera to A2 substance reacted primarily with A2 substance on Ouchterlony analysis, showing an apparent spur of cross-reactivity with B7-12 substances. Rabbit antisera to B7-12 substances reacted primarily with B7-12 substances, giving a spur of cross-reactivity with the A2 material. Neither antiserum precipitated β-₂microglobulin. Both types of sera reacted with membrane molecules of 43 000 mol.wt. and 11 000 mol.wt. by immune precipitation and gel electrophoresis in SDS of detergent-solubilized radiolabeled membranes from cultured cell lines, as predicted for sera directed towards HLA antigens. F(ab′)₂ fragments of the antibodies blocked the complement-mediated cytotoxicity of all HLA alloantisera tested for human peripheral blood lymphocytes. After absorption with B7-12 substances, F(ab′)₂ fragments of antisera to A2 substance only blocked the cytotoxicity of HLA alloantisera to A series specificities. After absorption with A2 substance, F(ab′)₂ fragments of rabbit antisera to B7-12 substances only blocked the cytotoxicity of HLA alloantisera to B series specificities. The results prove the existence of shared antigenic determinants between all members of the same series. These findings support the genetic evidence that A series HLA antigens are allelic products of a single locus, while B series HLA antigens are allelic products of a separate locus, by establishing some invariance of structure, presumably amino acid sequence, between members of the same series. The apparent cross-reactivity between A2 substance and B7-12 substances, and the ability of the unabsorbed F(ab′)₂, preparations to block the cytotoxicity of all HLA alloantisera, suggests that some determinants are common to both HLA loci. This may be considered to support the hypothesis that the two loci arose by gene duplication.     

Quantification of antibody (IgY) titers in hen eggs following immunization and their use in detecting cell surface molecules on nitrocellulose membranes

HLA-A*0201 alpha chain and beta2m were expressed from a prokaryotic system, and after refolding and purification, the alpha chain and beta2m were used to immunize eight laying hens. The titer of egg yolk antibody against alpha chain increased from 10(2) to 10(5.3) The titer of egg yolk antibody against beta2m increased from 10(1) to 10(4.7). The extent of titer increase is similar between the two antigens. An average of 135 mg purified polyclonal antibody (IgY) can be easily obtained from one egg yolk. The use of egg collection rather than serum collection is compatible with modern animal protection regulations. An average of 28 eggs were obtained from a laying hen every month, with a total amount of 3780 mg immunoglobulin extracted from one immunized hen every month, which would be equivalent to 630 mL of serum or 1260 mL of blood per month. Chickens are an optimal host for the production of polyclonal antibodies with high titer and high yield. Purified IgY was labeled with horseradish peroxidase and reacted with PBMC on nitrocellulose membranes indicating that the antibody can bind to the native conformation of class I HLA molecule on PBMC.     

The HLA-DR phenotype of the responder is predictive of humoral response against HLA class I antigens

Recent studies suggest that the immunogenicity of an human leukocyte antigen (HLA) incompatibility should be considered in the context of the HLA phenotype of the recipient. The HLA-DR phenotype of the responder is thought to be predictive for the strength of the alloimmune response. In order to analyze the humoral response against HLA class I antigens in the context of the HLA-DR phenotype of the responder, we selected all HLA-DR homozygous Dutch patients that were present on the Eurotransplant waiting list between 1967 and 2000 (n=1,317 patients). By logistic regression it was determined whether antibody production against a specific HLA class I antigen is associated with a particular HLA-DR antigen in the patient. Furthermore, it was analyzed whether a patient, expressing a particular HLA-DR antigen, preferentially produces antibodies against particular HLA class I antigens. The results demonstrate that patients, homozygous for a certain HLA-DR antigen, cannot be considered high or low responders when analyzing the antibody response in terms of panel reactive antibody (PRA) value. However, a correlation can be found between the HLA-DR phenotype of the patient and the specific antibody response against HLA class I antigens. For example, antibodies against HLA-A10, -A11, -A19, and -B35 are produced more frequently by HLA-DR6 positive individuals, whereas antibodies against HLA-A3, -B5, -B7, -B8, and -B12 are produced more frequently by HLA-DR4 positive individuals. These data confirm that the HLA-DR phenotype of the responder plays a determinative role in the immunogenicity of mismatched HLA antigens. The results indicate that selection of HLA class I mismatches of the donor in the context of the HLA-DR phenotype of the responder might reduce the incidence of humoral graft rejection and minimize the sensitization grade of retransplant candidates.     

Specific engagement of the CD94/NKG2-A killer inhibitory receptor by the HLA-E class Ib molecule induces SHP-1 phosphatase recruitment to tyrosine-phosphorylated NKG2-A: evidence for receptor function in heterologous transfectants

It has been recently demonstrated that the CD94/NKG2-A killer inhibitory receptor (KIR) specifically recognizes the HLA-E class Ib molecule. Moreover, the apparent CD94-mediated specific recognition of different HLA class Ia allotypes, transfected into the HLA-defective cell line 721.221, indeed depends on their selective ability to concomitantly stabilize the surface expression of endogenous HLA-E molecules, which confer protection against CD94/NKG2-A⁺ effector cells. In the present study, we show that a selective engagement of the CD94/NKG2-A inhibitory receptor with a specific monoclonal antibody (mAb) (Z199) was sufficient to induce tyrosine phosphorylation of the NKG2-A subunit and SHP-1 recruitment. These early biochemical events, commonly related to negative signaling pathways, were also detected upon the specific interaction of NK cells with an HLA-E⁺ 721.221 transfectant (.221-AEH), and were prevented by pre-incubation of .221-AEH with an anti-HLA class I mAb. Furthermore, mAb cross-linking of the CD94/NKG2-A receptor, segregated from other NK-associated molecules by transfection into a rat basophilic leukemia cell line (RBL-2H3), promoted tyrosine phosphorylation of NKG2-A and co-precipitation of SHP-1, together with an inhibition of secretory events triggered via FcϵRI. Remarkably, interaction of CD94/NKG2-A⁺ RBL cells with the HLA-E⁺ .221-AEH transfectant specifically induced a detectable association of SHP-1 with NKG2-A, constituting a more formal evidence for the receptor-HLA class I interaction.     

Establishment of shared antigen reactive cytotoxic T lymphocyte using co-stimulatory molecule introduced autologous cancer cells

Cytotoxic T lymphocytes (CTLs) play an essential role in immunological responses for tumor rejection. In the past decade, many tumor-associated antigens (TAAs) have been identified predominantly in melanomas. Several clinical trials based on such antigenic peptides with or without adjuvants brought about partially favorable results, suggesting that identification of more immunogenic TAAs is needed. We show here the successful establishment of human leukocyte antigen (HLA)-A24-restricted CTL (TcLHK2 line1) from a pleural effusion of lung cancer patient, using B7.1 (CD80) transduced autologous lung cancer cells as an antigen-presenting cell (APC). TcLHK2 line1 recognized autologous lung adenocarcinoma cell line LHK2 in an HLA-A24-restricted fashion. Moreover, this CTL line also recognized allogeneic HLA-A24-positive lung adenocarcinoma cell line, gastric carcinoma cell line and melanoma cell line. These data raise the possibility that co-stimulatory molecule B7.1 (CD80) plays important role to overcome the immunological tolerance. Furthermore, TcLHK2 line1 is a useful tool for the identification of widely expressed shared antigens restricted by HLA-A24. Further analysis of this CTL and autologous cancer cell line will bring about novel TAAs.