Planned immunization of human volunteers with HLA-DR incompatible lymphocytes

Aiming at the production of anti HLA-DR test sera, eight healthy human volunteers were immunized by repeated intradermal injections of lymphocytes which were selected to be incompatible for one HLA-DR antigen, and matched as well as possible for HLA-A,-B,-C antigens. One out of 3 recipients immunized exclusively against HLA-DR produced lyrnphocytotoxic HLA-DR antibodies. The remaining 5 recipients were immunized against 1 or more HLA-A,-B,-C antigens in addition to one HLA-DR antigen. After 3 immunizations, 3 of these reacted with strong HLA-A or -B antibody production; however, only one showed a parallel anti HLA-DR antibody response detectable by complement dependent lymphocytotoxicity.
Testing of the recipient sera in the antibody dependent cell-mediated cytotoxicity (ADCC) assay revealed that 6 of the 8 recipients did react early to the immunizations with HLA specific antibody production. However, in spite of repeated booster injections it was not possible to obtain more than the above-mentioned 2 sera with HLA-DR antibodies strong enough to react in the lymphocytotoxicity microtechique.     

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.     

HLA-D and -DR antigens on human amniotic fluid cells. II. Heterogeneous expression of HLA-DR and other cell surface markers

To evaluate further the feasibility of HLA typing for prenatal diagnosis, we tested human amniotic fluid cells (AFC), known to express HLA-A, -B, and -C antigens, for the presence of HLA-DR antigens using type-specific antisera in the microcytotoxicity assay and a monoclonal antibody directed against the common HLA-DR structure (cDR) in indirect immunofluorescence. Prenatal typing of HLA-DR on AFC in the microcytotoxicity test was possible in only one out of eight families studied. The detected DR2 antigen was confirmed by postnatal typings of cord blood lymphocytes. Thereafter, 23 different AFC cultures were tested with monoclonal antibodies in indirect immunofluorescence. Only six cultures were partially positive (23-35% fluorescent cells) with the monoclonal cDR antibody while all AFC cultures demonstrated strong positive fluorescence (68-100%) with a monoclonal antibody against the common HLA-A, -B, and -C structure (cHLA). These data suggest that only a small subpopulation of AFC expresses class II (HLA-DR) antigens in contrast to the nearly ubiquitous expression of class I (HLA-A, -B, and -C) antigens. Furthermore, the heterogeneous expression of cell surface antigens within the various AFC cultures was substantiated with monoclonal antibodies directed toward cell surface antigens of the OKT, OKM, and Lyt series that have been found to be characteristic for subpopulations of lymphoid and hemapoetic cells. Thus, at present, HLA-DR typing is not reliable for prenatal diagnosis.     

Allotyping for HLA class I using plasma as antigen source

Immunoadsorption of soluble HLA class I antigens onto immunobeads, one-dimensional iso-electric focusing of these proteins and subsequent immunoblotting allows a biochemical identification of HLA class I allotypes. The distinct protein bands can be clearly attributed to particular HLA antigens and are comparable to those observed after detergent solubilization of membrane-bound HLA antigens. Segregation analysis showed that the biochemically detected pattern of soluble class I gene products followed Mendelian inheritance. However, antigens such as HLA-A1, -A2, -B8, and -B51 were not always clearly detectable, a phenomenon attributable to either different plasma concentrations of these HLA antigens or variable affinity of the monoclonal antibody used to capture class I antigens. These results show that in principle allotyping of HLA class I using plasma as the antigen source is feasible, but with the limitation that some antigens may not be easily detected in some individuals.     

Evaluation of humoral immune response to donor HLA after implantation of cellularized versus decellularized human heart valve allografts

We have evaluated the development of antibodies in response to donor allograft valve implant in patients who received cellularized and decellularized allografts and determined possible immunogenic epitopes considered responsible for antibodies reactivity. Serum samples from all recipients who received cellularized allografts or decellularized allografts were collected before valve replacement and at 5, 10, 30 and 90 days post-operatively and frozen until required. Tests were performed using the Luminex-based single human leukocyte antigen (HLA)-A, -B, -C and HLA-DR, -DQ antigen microsphere assay. To determine possible immunogenic epitopes, we used the HLAMatchmaker (HLAMM) software if applicable. Decellularized grafts elicited lower levels of anti-HLA class I and II antibody formation after implantation than cellularized allografts. All patients from cellularized group presented donor-specific antibodies class I and II within 3 months of observation period. In HLAMM analysis, the cellularized group had significantly higher numbers of immunogenic epitopes than decellularized group for both class I and II (p: 0.002 - cl I / p: 0.009 - cl II / p: 0.004 - cl I and II). Our findings demonstrate that the anti-HLA antibodies detected in the cellularized group were against donor HLA possible immunogenic epitopes and that in the decellularized group the anti-HLA antibodies were not against donor HLA possible immunogenic epitopes. These findings lead us to suggest that choosing sodium dodecyl sulfate decellularization process is the best alternative to decrease the immunogenicity of allograft valve transplant.     

Major histocompatibility complex status in breast carcinogenesis and relationship to apoptosis

Major histocompatibility complex (MHC) molecules are of central importance in regulating the immune response against tumors. In this study we used immunohistochemistry to study human leukocyte antigen (HLA) class I and II antigen expression in normal breast tissues and benign, preneoplastic, primary, and metastatic breast lesions using antibodies against beta-2-microglobulin (beta2-m), heavy-chain, and HLA-DR antigens. Whereas all normal tissues and benign lesions were positive for beta2-m and HLA-A, -B, and -C antigens, total loss of HLA class I antigens was found in 37% (11 of 30) of in situ carcinomas, in 43% (56 of 131) of the primary tumors, and in 70% (31 of 45) of the lymph node metastases. HLA-DR was also underexpressed in breast cancer cells; thus 20% (6 of 30) of in situ carcinomas, 15% of invasive carcinomas (20 of 131), and only 1 metastatic case were positive for this antigen. Both HLA class I and II antigen expression were more frequently down-regulated in metastatic lesions than in primary breast lesions (P <0.05), and a tendency toward a simultaneous defective expression of HLA class I and II antigens was observed in primary carcinomas (P = 0.07). However, no correlation was found between the expression of any of the aforementioned molecules and pathological parameters or survival. Interestingly, HLA class I expression was expressed more frequently in tissues with high apoptotic activity and was significantly associated with the expression of the proapoptotic bax gene (P = 0.02), and was inversely associated with expression of the antiapoptotic bcl-2 gene (P = 0.03). We conclude that alterations in HLA class I and II antigen expression are early events in breast carcinogenesis and play significant roles in metastatic progression. In addition, their expression is correlated with apoptosis-regulating proteins, which may influence the cytotoxicity of T cells against HLA class I-specific tumor antigens.     

HC restricted dual specific inhibition of mixed leukocyte culture reactions by human HLA antibody molecules

A human alloantiserum was found which selectively inhibits responding cells in mixed leukocyte culture reactions. Inhibition was achieved by pre-incubation of responder cells in the antiserum followed by washing. The serum showed dual specificity as an inhibiting agent. First, inhibition was restricted to HLA-B7 or -B40 positive stimulator cells, specificities against which the antiserum also had cytotoxic activity. Second, inhibition was almost exclusively associated with the presence of the phenotype HLA-A1, -B8 on the responder cells The HLA associated specificity for responder cells was unexpected since no alloantibody activity directed to responder alloantigens could be detected by conventional serological-methods. The antiserum donor had not been immunized with HLA-A1, -B8 antigens nor with known crossreactive antigens. Furthermore, the serum donor did not carry HLA-A1, -B8 antigens herself. The inhibiting substance in the antiserum had physicochemical properties of IgG and was specifically reactive with HLA-B7 positive platelets. Pepsin digest preparations were not inhibitory. Fc receptor positive responder cells were required for inhibition. Responder cells, preincubated with the antiserum, suppressed the response of cells not incubated with the antiserum. Three possible explanations of these results are discussed: specific binding of the Fc part of the antibody with Fc receptors of responder cells, specific activation of suppressor cells and cross-reactivity.     

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.     

A new murine lymphocytotoxic monoclonal antibody recognizing HLA-A2, -A28 and -A9

Monoclonal antibodies recognizing polymorphic as well as monomorphic epitopes on HLA antigens are important tools for understanding the immuno-biology of HLA molecules. We immunized BALB/c mice with a HLA-A2 transfectant and screened for hybridomas which reacted with a HLA-A2 trans-fectant but not with a HLA-B75 transfectant. After subcloning by limiting dilution four times, a hybridoma secreting a monoclonal antibody (mAb) (IgG 2a, kappa) designated 1–145 was established. 1–145 reacted with Epstein-Barr virus transformed B lymphoblastoid cell lines (B cell lines) which expressed HLA-A2, -A28, -A23 and -A24. The titer of 1–145 in culture supernatant against HLA-A2 and -A28 antigens was similar and the titer against HLA-A23 was lower. 1–145 reacted with cells expressing HLA-A24 but the titer against HLA-A24 antigens was even lower than that againt HLA-A23 antigens. The HLA-A24 antigens on the peripheral blood lymphocytes were not detected by 1–145 possibly due to the lower expression compared to the B cell lines. These differences of the titers were reflected to microlymphocytotoxic-ity assay in which 1–145 culture supernatant lysed all PBLs expressing HLA-A2.-A28 and -A23 but did not lyse PBLs expressing HLA-A24. Published deduced amino acid sequence data of HLA class I molecules indicate that Lys in position 127 may be critical for 1–145 binding.     

A limited number of HLA epitopes are recognized from HLA class I-specific antibodies detected in the serum of sensitized patients

The goal of this study was to evaluate the epitope specificity of HLA class I-specific antibodies detected in the serum of sensitized patients awaiting retransplantation. The study group consisted of 22 sensitized from previous graft patients, who produced stable IgG HLA class I-specific antibodies. A total of 60 serum samples were screened and analyzed by two techniques in parallel: the antihuman globulin augmented CDC (AHG-CDC) technique and an ELISA technique. All recipients and donors were typed for class I HLA antigens by a standard lymphocytotoxicity technique. The epitope identification was based on class I HLA antigens sequencing, where the multiple immunogenic epitopes are differentially shared among various HLA antigens. The unique epitope configuration on one HLA antigen represents the private epitope of the specific HLA antigen while epitopes shared by more than one HLA antigen represent public determinants. In some HLA antigens (HLA-A1), more than one private epitope has been defined, while in others (HLA-B35, -B51), the private epitopes are not yet known. In a total of 36 antibody reactivity patterns, the majority of the definable IgG HLA class I-specific antibodies corresponded to the A-locus (75%), and only 25% had specificities against the B-locus antigens, although the number of incompatibilities concerning both loci were almost identical (29 for the HLA antigens of the A-locus and 26 for those of B-locus). All patients produced HLA class I-specific antibodies with specificities against the private epitopes of the immunogenic mismatched HLA antigen(s). In 6/21 cases (28.6%), HLA class I alloreactivity spreading to nongraft HLA antigens was detected and 9 public (shared) immunogenic alloepitopes were recognized. In conclusion, appling the epitope analysis of HLA class I-specific antibodies produced by sensitized from previous graft patients, we were able to define the immunogenic alloepitopes. We consider that the immunogenic alloepitopes, during transplantation course, are mainly private epitopes of mismatched HLA antigens and, in certain cases, shared epitopes between the donor alloantigens and other HLA antigens. This knowledge may offer the potential of transplanting sensitized patients through improved donor selection.     

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.     

Identification of acceptable HLA mismatches in immunized patients using single-antigen-expressing cell lines

Sera of highly sensitized patients (HSP) contain complex human leukocyte antigen (HLA) antibodies, minimizing the chance to identify crossmatch-negative donors. Expression of 3-6 HLA class I antigens on lymphocytes hampers identification of acceptable mismatches (AMs) by conventional screening (C-SCR). The single-antigen-expressing cell line (SAL) concept circumvents this problem. As a proof of principle, 26 sera of sensitized patients were tested by flow cytometry for immunoglobulin G antibodies against 16 HLA-A and -B SALs. Results were compared with C-SCR. Mostly, SAL reactions confirmed presence/absence of HLA antibodies. While C-SCR sometimes failed to provide unambiguous antibody specificity, we defined 24 new HLA antibody specificities with SALs and proposed 33 new AM by non-reactivity with SALs. Thus, the SAL concept is useful for confirmation/identification of AM and will enhance transplantation of HSP.     

Genes in the HLA class I region may contribute to the HLA class II-associated genetic susceptibility to multiple sclerosis

In order to analyze whether loci in the human leukocyte antigen (HLA) class I region may contribute to the HLA class II-associated genetic susceptibility to multiple sclerosis (MS), we examined selected microsatellite markers in 177 Nordic sib-pair families, 222 British sib-pair families, 323 sporadic Norwegian MS patients and 386 Norwegian controls. All samples were, in addition, genotyped for the HLA-DR DQ haplotype, and the Norwegian case-control samples were also typed for HLA-A and -B loci. In the Norwegian sporadic MS patients association was seen with HLA-A, HLA-B, and with the D6S265 marker, located 100 kb centromeric to HLA-A. Associations with HLA-A and D6S265 loci were also suggested when restricting the analysis to HLA-DR15 haplotypes. In the sib-pair data a similar trend was seen with marker D6S265. Higher genotypic relative risk (GRR) was found for individuals who carry both HLA-DR15 and -A3 (GRR = 15), compared to those who carry only HLA-DR15 (GRR = 7), only HLA-A3 (GRR = 3) or none of these alleles (GRR = 1). The highest risk was conferred by a combination of HLA-DR15 and -A3 (odds ratio (OR) = 5.2). These results suggest that HLA-A or a gene in linkage disequilibrium with it may contribute to the HLA class II-associated genetic susceptibility to MS.     

Development of class I-specific helper-inducer T-cell clone

A unique alloreactive human helper-inducer T-cell clone which reacts with the class I HLA antigen was characterized. The clone was derived from a mixed lymphocyte culture between cells from a HLA-A2-; B35,w51; Bw4,Bw6; Cw4,-; DR1,4; DQw1,-; DRw53 responder and a HLA-A2,3; B7,14; Bw6; Cw8,-; DR7,-; DQw2,w3; DRw52 stimulator. When screened against a panel of stimulator cells, this clone was found to have a specificity towards a stimulator HLA-B14 and consisted entirely of Leu-4, Leu-3, and 4B4 positive T cells, which is a T helper-inducer phenotype. Using monoclonal antibodies directed towards various monomorphic class I and II HLA epitopes in an inhibition experiment, it was found that both monoclonal antibodies w6/32 and 4E (recognizing class I monomorphic and HLA-B epitopes, respectively) inhibited proliferation. However, monoclonal antibodies BBm.1 and L227 (directed against beta 2-microglobulin and Ia-like molecule, respectively) had no inhibitory effect. Functional evaluation of this clone demonstrated helper activity on the proliferation of MLC response. The helper-induction effect on MLC cultures remained intact following irradiation of the clone for either 500 or 2000 rad suggesting that helper function of the clone was radiation resistant. The helper activity of this clone was MHC nonrestricted since it enhanced proliferation of the original responder and stimulator MLC as well as third and fourth party individuals. By CTLL cell line proliferation assay, this clone was found to release IL-2. When assayed (day 1 to 7) for class II HLA products expression following stimulation, HLA-DRw53 was consistently expressed by the clone. However, HLA-DR1, DQw1 were found to be expressed simultaneously on day 2 through day 7 and HLA-DR4, DQw3 on day 2 and 3. These data demonstrate, for the first time, the generation of a class I specific helper-inducer T-cell clone in MLC response.     

Permissible and immunogenic HLA-A mismatches: cytotoxic T-cell precursor frequencies reflect graft survival data.

Analysis of the in vivo immunogenicity of single HLA mismatches, in the context of a patient's own human leukocyte antigen (HLA) phenotype, has been used to define permissible and immunogenic HLA mismatches. Kidney graft survival in the case of permissible mismatches was similar to that of completely HLA matched combinations, whereas immunogenic mismatches lead to a significantly poorer graft survival. The present study tested whether such permissible and immunogenic HLA mismatches are reflected in the in vitro cytotoxic T-lymphocyte (CTL) allorepertoire. Limiting dilution experiments were performed to analyze the number of precursor CTL directed against individual HLA class I antigens. In general, the frequency of CTLp directed against permissible HLA-A antigens (n = 70, mean frequency 27 CTLp per million peripheral blood lymphocytes [PBL]) was found to be significantly lower compared with the CTLp directed against immunogenic HLA-A antigens (n = 73, mean frequency 59 CTLp per million PBL). The difference was found both in healthy individuals and a population of renal transplant candidates. These results were confirmed by a retrospective analysis of CTLp frequencies performed between partly mismatched unrelated bone marrow donors and their potential recipients. In conclusion, on the population level the permissible and immunogenic HLA-A mismatches are indeed reflected in the CTL allorepertoire. However, due to the big overlap of the CTLp frequencies in these populations, the permissible or immunogenic nature of a mismatch for a particular patient should be determined on an individual basis.     

HLA-A33 and -B44 and susceptibility to postherpetic neuralgia (PHN)

HLA class I and class II alleles of 32 Japanese patients with postherpetic neuralgia (PHN) and 136 healthy controls were analyzed by serological (class I) and DNA (class II) typing for any significance in the susceptibility to varicella-zoster virus (VZV). We recognized positive associations of the development of PHN with the HLA class I antigens HLA-A33 and -B44, and the HLA-A33-B44 haplotype. This haplotype is tightly linked to DRB1*1302 in a Japanese healthy population. However, no significant association between PHN and HLA class II alleles was observed with no linkage of the HLA haplotype HLA-A33-B44 to HLA-DRB1*1302 in the patients with PHN. These findings suggest that HLA class I gene may genetically control the immune response against VZV in the pathogenesis of PHN.     

Relationship Between HLA Antigens and Infectious Agents in Contributing Towards the Development of Graves' Disease

Graves' disease (GD) is an autoimmune thyroid disorder which is associated with the human leucocyte antigens HLA-DR3 and DQA1^* O501 in Caucasians. We have explored the possibility that some patients with certain HLA specificities develop anti-HLA antibodies which are correlated with environmental factors that may contribute to the development of GD. We studied 40 GD patients and 157 healthy individuals (controls). Serology was used to type HLA-A, -B, -Cw, and -DR antigens. The frequencies of these antigens in relation to lymphocytotoxic anti-HLA-A-B-Cw-DR antibodies and two environmental factors (Yersinia enterocolitica and Coxsackie B virus) were determined. The frequencies of HLA-B15, -B21 and DR3 antigens were increased, whereas HLA-DR5 antigen was decreased in GD patients. A significant association between HLA-DR3 antigen and lymphocytotoxic antibodies was observed, i. e., IgGs from GD patients were cytotoxic to HLA-DR3+ normal B cells. Following absorption with Yersinia enterocolitica or Coxsackie-B-virus, only Coxsackie-B virus completely inhibited the lymphocytotoxic reactions against HLA-DR3⁺ B cells. Besides confirming the association of HLA-DR3 with GD, this study also suggests the role of Coxsackie-reative HLA-DR3 antibodies as contributing factors to the pathogenesis of the disease.     

Comparison of the humoral and cytotoxic T-lymphocyte response to individual HLA class I alloantigens in highly immunized patients

In order to investigate the correlation between activation of cytotoxic T-lymphocyte precursor (CTLp) and the formation of antibodies to alloantigens, we studied 21 highly sensitized patients waiting for a kidney transplantation. Both antibody reactivity and CTLp frequencies of these patients were determined against 88 individual HLA class I alloantigens. A high or low CTLp frequency against a certain HLA-A or -B alloantigen was not correlated with the presence or absence of the antibodies to that antigen. Mismatched antigens, towards which the patient had not formed antibodies, can induce either a higher or a lower frequency of CTLp compared to mismatches towards which the patients had formed antibodies. The possible implications of this lack of correlation between the T- and B-cell allorepertoires with regard to donor selection for (highly immunized) patients is discussed.