Different impact of pretransplant anti-HLA antibodies detected by Luminex in highly sensitized renal transplanted patients.

It is well know that anti-HLA antibodies are an important obstacle in kidney transplantation. Our aim was to study the clinical impact of pretransplant donor specific anti-HLA antibodies (HLA-DSA), in highly sensitized (HS) patients. We analyzed retrospectively the day-of-transplant sera by Luminex Single Antigen Assay (LSA) in HS patients, and the results were correlated with episodes of humoral and cellular rejection as well as with graft and patient survival. All HS subjects received the same induction therapy and rejection episodes were biopsy proven. Thirteen patients (56.5%) preformed HLA-DSA, and we observed higher incidence of acute rejection in aforementioned patients than in the pre-transplant negatives DSA recipients (77% versus 30%, P = 0.03). The one-year graft survival was significantly reduced in positive pre-transplant HLA-DSA patients (60% versus 100%, P = 0.01 Breslow). The positive predicted value of HLA-DSA in relation to rejection reached 100% if patients lost their previous graft in the first year after transplant. Among anti-HLA antibodies present in patients before transplant, HLA-DSA were significantly associated with high risk of acute humoral and cellular rejection and reduced graft survival in posttransplant outcome. The negative impact of these antibodies was even higher when patients suffered an early loss of the previous transplant.

Identification of a new HLA-A*11:78N allele by polymerase chain reaction sequence-based typing.

Nucleotide sequence of HLA-A*11:78N allele was different from that of HLA-A*11:01:01 by two nucleotides deletion at positions 286 and 287, resulting in reading frameshift and has premature stop codon at position 73 in exon 2.

Description and molecular modeling of two novel HLA alleles: HLA-A*0343 and A*0345.

We report the identification of two novel human leucocyte antigen (HLA) in two Caucasian individuals. HLA-A*0343 differs from A*03010101 by four changes at nucleotides 411-414 (CCGG-->TGAA) and by a point mutation at position 418 (G-->C). These differences lead to two amino acid substitutions at codon 114, where arginine has changed into negatively charged glutamic acid, and at codon 116, where aspartic acid has changed into positively charged histidine. Molecular modeling showed that these changes have a profound influence on the overall charge of the F pocket of the groove, resulting in potentially important changes in the peptide repertoire. HLA-A*0345 was found in a hematological female patient candidate to bone marrow transplantation. This new variant differs from HLA-A*03010101 at position 845 (C-->A) encoding an amino acid change of threonine to asparagine at codon 258 located in the alpha3 domain. Molecular modeling does not suggest a substantial role of this substitutions on the interaction with beta2-microglobulin or CD8.

Detection, isolation, and characterization of alpha-fetoprotein-specific T cell populations and clones using MHC class I multimer magnetic sorting.

alpha-fetoprotein (AFP) is a fetal protein specifically reexpressed in 50% of hepatocellular carcinomas. This protein could serve as a tumor-associated antigen for immunotherapy purpose. The aim of our work was to analyze the presence of AFP-specific T cell populations in peripheral blood mononuclear cells (PBMCs) from cirrhotic patients with or without hepatocellular carcinoma. Using peptide-major histocompatibility complex class I multimers, AFP-specific populations corresponding to 3 previously described human leukocyte antigen (HLA)-A*0201 major histocompatibility complex class I epitopes (AFP137, AFP158, and AFP325) were sorted magnetically from CD8 positive cells without prior stimulation with the target antigen. T cell populations specific for 1 peptide (AFP158) were frequent, whereas populations corresponding to peptide AFP137 were rare and absent for peptide AFP325. We also isolated and fully characterized T cell clones specific for AFP137 and AFP158 peptides. We show that these clones can be used to monitor dendritic cell loading with peptides and could be useful for future immunotherapy protocols.

[Construction and primary application of two HBV-HLA-A*2402-peptide tetramers].

AIM: To construct two soluble HLA-A*2402-peptide tetramers and detect the HBV-specific cytotoxic T lymphocytes (CTLs) with the constructed HLA-A*2402-peptide tetramers. METHODS: After proteins HLA-A*2402-BSP and beta2m were made an effective prokanyotical expression, the purified proteins were refolded into HLA-A*2402-beta2m-peptide complexes in the presence of two antigenic peptides (Hepatitis B virus Pol756-764 or Core117-125) with dilution method. Then the complexes were biotinylated by BirA enzyme and purified by gel-filtration chromatography. The tetramers were generated by mixing the complex with PE-Streptavidin in the molar ratio of 5:1. FCM can and cell quest software were used to analyze the stained PBMCs. RESULTS: Two biotinylated HBV-HLA-A*2402-peptide complexes were identified by Western blot and they were shown to have natural conformation by Dot-ELISA and ELISA. CONCLUSION: The constructed HLA-A*2402-peptide tetramers can detect the HBV-specific CTLs in the patients with self-limited acute HBV infection.

A robust method for production of MHC tetramers with small molecule fluorophores.

Tetramers of major histocompatibility complex molecules (MHC) are now well-established reagents for the detection of antigen-specific T cells by flow cytometry. MHC tetramers are prepared by mixing enzymatically biotinylated MHC molecules with commercial preparations of streptavidin, usually conjugated to a fluorescent phycobiliprotein such as phycoerythrin (PE) or allophycocyanin (APC). While data obtained with MHC tetramers prepared with small molecule fluorophores has been reported, considerable lot-to-lot variation among conventional streptavidin conjugates to small molecules prevents routine preparation of such reagents. We now report robust preparation of MHC tetramers with small molecule fluorophores, using a recombinant mutant of streptavidin incorporating a carboxy-terminal cysteine in each of the four identical subunits that is conjugated to maleimide derivatives of any of several small molecule fluorophores. These reagents significantly expand the versatility of the MHC tetramer methodology.

In situ detection of antigen-specific tumor-infiltrating lymphocytes using newly designed tetramers.

We described a new process for the design of HLA tetramers using soluble MHC class I molecules purified from Epstein Barr Virus-transformed B cells. This method does not rely on genetic engineering and presents a significant advantage in view of the polymorphism of MHC class I molecules because tetramers can be produced with any HLA molecule. Here, we showed that our HLA-A*0201 tetramers provided experimental results similar to those obtained with tetramers made with recombinant MHC molecules. Moreover, they can be used to efficiently identify peptide-specific T cells from ex vivo PBMCs as well as from lymphocytes infiltrating human tumor. This innovative and simple method could be widely adopted, specially in diagnostic procedures for monitoring peptide-based immunotherapy.

High frequency of altered HLA class I phenotypes in laryngeal carcinomas.

The exact frequency of HLA class I losses in human tumors is unknown. We have previously shown that primary breast and colorectal carcinomas frequently lose HLA class I molecules (88% and 73%, respectively). Now we report that this phenomenon is also a frequent event in laryngeal carcinomas. Of a total of 76 laryngeal carcinomas analyzed, 66% of the tumors showed an alteration in HLA class I phenotype. These altered HLA phenotypes were classified as total HLA loss (10.52%) (phenotype I); HLA-A locus-specific loss (13.15%) (phenotype IIIa); HLA-B locus-specific loss (10.52%) (phenotype IIIb); HLA allelic loss (27.63%) (phenotype IV); and HLA-A and B locus loss (3.9%). Comparison of histopathological parameters with HLA expression showed that poorly differentiated tumors had the lowest levels of HLA class I expression (p < 0.05).

High frequency of cytomegalovirus-specific cytotoxic T-effector cells in HLA-A*0201-positive subjects during multiple viral coinfections.

How the cellular immune response copes with diverse antigenic competition is poorly understood. Responses of virus-specific cytotoxic T lymphocytes (CTL) were examined longitudinally in an individual coinfected with human immunodeficiency virus type 1 (HIV-1), Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CTL responses to all 3 viruses were quantified by limiting dilution analysis and staining with HLA-A*0201 tetrameric complexes folded with HIV-1, EBV, and CMV peptides. A predominance of CMV-pp65-specific CTL was found, with a much lower frequency of CTL to HIV-1 Gag and Pol and to EBV-BMLF1 and LMP2. The high frequency of CMV-specific CTL, compared with HIV-1- and EBV-specific CTL, was confirmed in an additional 16 HLA-A*0201-positive virus-coinfected subjects. Therefore, the human immune system can mount CTL responses to multiple viral antigens simultaneously, albeit with different strengths.

Monomorphic HLA class I-(non-A, non-B) expression on Ewing's tumor cell lines, modulation by TNF-alpha and IFN-gamma.

In this study, the expression of polymorphic and non-polymorphic MHC antigens in Ewing's tumor (ET) cells was examined by surface staining, Western blots and transcriptional analysis. Cell lines derived from Ewing's tumors largely lack polymorphic HLA class Ia antigens of both the HLA-A and the HLA-B loci but binding of monomorphic HLA antibodies indicates significant expression of HLA-C locus antigens and/or HLA class Ib molecules. HLA Ib molecules encoded by the HLA-E, -F or -G loci with a molecular mass of less than 44 kDa were not detected in lysates of either constitutive or TNF-alpha plus IFN-gamma treated ET cells. Two representative ET cell lines with either detectable HLA-A, -B antigens (A673) or absolutely non-detectable HLA-A, -B antigens (SK-ES-1) were further subjected to transcriptional analysis. A673 mRNA hybridized with HLA-A, -B, -C and HLA-E-specific probes in Northern blots. By contrast, mRNA specific for HLA-A, -B, -C was negative in SK-ES-1 but TNF-alpha plus IFN-gamma reconstituted HLA-A, -B, -C transcription in this cell line. HLA-E was transcribed in A673 but not in SK-ES-1. Combining mRNA and surface expression of HLA class Ia molecules results in a highly variable pattern of defective HLA class I expression in this type of neuroectodermal tumor. The involvement of the ET-specific fusion transcript EWS/Fli-1 in modulating the HLA-A and -B locus antigens is likely to occur by the upregulation of c-myc in these tumors. The exceptionally constant expression of HLA-C or some other non-A, non-B antigens (reactive with defined monoclonal antibodies) implies important consequences on tumor-cell resistance against specific CTL and NK activity in vivo.

A comparison of flow cytometry and immunohistochemistry in human colorectal cancers.

In human colorectal cancer it has been reported that some tumours lack the HLA-ABC antigens. This has been interpreted as reflecting tumour escape from the immune system. Earlier data have been obtained by immunohistochemistry. In this study, we compared the expression of HLA-ABC, HLA-DR, CD80 (B7-1) and CD54 (ICAM-1) in 20 tumours using both a conventional immunohistochemistry two-layer technique and multiparameter flow cytometry, gating on an epithelial cell marker. Colorectal cancer tissue used in flow cytometry was dissociated with collagenase, deoxyribonuclease and hyaluronidase. The intensity of expression of HLA-ABC, HLA-DR and CD80 was unaffected by the enzymes, but CD54 was decreased by 30%. The reproducibility of flow cytometry was good. Microscopy of sections revealed that about 5% of each tumour sample consisted of normal epithelium, but even after correction for this, flow cytometry was superior to immunohistochemistry in 33 out of 80 cases, and showed that tumours described as HLA-ABC negative by immunohistochemistry were in fact weakly positive for HLA-ABC. We conclude that flow cytometry and immunohistochemistry are complementary, and that flow cytometry is superior to immunohistochemistry for detecting antigens/epitopes present in low amounts.

Identification and characterization of multiple HLA-A24-restricted HIV-1 CTL epitopes: strong epitopes are derived from V regions of HIV-1.

HIV-1-specific CTL has a crucial role in the elimination of the virus. However, a restricted number of common HLA class I alleles has been studied for their presentation of HIV-1 CTL epitopes. We have attempted to identify HIV-1 CTL epitopes presented by HLA-A*2402 using reverse immunogenetics. Fifty-three HLA-A*2402-binding HIV-1 peptides were used to induce specific CTL in PBL of four HIV-1-infected individuals carrying HLA-A24. Twelve peptides were strongly suggested to be HLA-A*2402-restricted HIV-1 CTL epitopes because these peptides induced the specific CTL that killed both the target cells pulsed with the specific peptides and those infected with the vaccinia HIV-1 recombinant virus in at least one HIV-1-infected individual. Of these epitopes, 11 were confirmed by the generation of the specific CTL clones. Six were the Env epitopes while three, one, and one were derived from Gag, Pol, and Nef proteins, respectively. Analysis of 12 HIV-1-infected individuals using these peptides showed that 5 derived from the Env protein and one from the Nef protein were strong epitopes. These strong epitopes were derived from the diverse region of HIV-1 while weak epitopes were conserved in the HIV-1 clade B strain. Analysis of CTL recognition of mutations in these strong epitopes suggested that the mutations in the Env epitopes may critically influence CTL recognition in vivo.

HLA antigen expression in enteropathy associated T cell lymphoma.

AIMS: To investigate the occurrence of abnormal patterns of HLA-ABC and HLA-DR expression in enteropathy associated T cell lymphoma and to relate such abnormalities to the Epstein Barr virus (EBV) status of the tumours. METHODS: Eleven enteropathy associated T cell lymphomas were immunostained with HC10 (HLA-ABC heavy chain) and TAL 1B5 (HLA-DR alpha chain) monoclonal antibodies and polyclonal anti-beta 2 microglobulin (beta 2m, the HLA-ABC light chain) antibodies. In situ hybridisation for EBV using EBER probes was performed on all cases. RESULTS: Tumour cells of two of 11 patients were EBER positive. One of these showed partial, and the other, complete loss of beta 2m. HLA-DR expression was undetectable in both patients. Of the remaining nine EBER negative tumours, two were HLA-ABC heavy chain negative or showed only occasional positive cells and five of nine showed partial or complete loss of the HLA-ABC light chain, beta 2m. Seven of the nine cases were either negative for HLA-DR or showed weak expression in a proportion of tumour cells. CONCLUSIONS: These data show that low or absent HLA-ABC and HLA-DR antigen expression occurs commonly in enteropathy associated T cell lymphoma. These abnormal patterns of HLA expression may be associated with escape from immune attack which, in a minority of patients, could be directed against EBV antigens.

Reduction of cellular rejection and increase in longer-term survival after heart transplantation after HLA-DR matching.

HLA matching in cardiac transplants is perceived as being logistically difficult. We studied 1135 consecutive primary cardiac allografts between 1980 and 1994 to assess the effect of HLA mismatching on long-term graft survival and cellular rejection episodes within 3 months of transplantation. We found a significant association between HLA-DR mismatching and the number of episodes of rejection (no mismatch 0.80 [SE 0.13], one mismatch 1.22 [0.06], two mismatches 1.42 [0.06], p < 0.05). We found a similar correlation between the total number of biopsy specimens showing evidence of cellular rejection and HLA-DR mismatch. The time between operation and the first rejection episode shortened with increasing HLA-DR mismatch (no mismatch 85.5 [37.3] days, one mismatch 43.1 [8.1], two mismatches 24.1 [2.9], p < 0.05). Furthermore, the proportion of patients with no evidence of rejection correlated with HLA-DR incompatibility. A significant association between improved graft survival and HLA-DR mismatching was found over 1, 5, and 10 years after transplantation (no mismatch 1 year 92%, 5 years 83%, 10 years 76%, one mismatch 1 year 81%, 5 years 73%, 10 years 59%, and two mismatches 78% 1 year, 5 years 70%, and 10 years 52%, p = 0.02). Increased efforts to prospectively HLA match patients has resulted in 25% of patients transplanted between January and May 1995 (n = 13/52) receiving grafts matched for HLA-DR. HLA matching reduces the frequency and severity of acute cardiac allograft rejection and improves graft survival for up to 10 years. Our preliminary results suggest that it is possible to use HLA matching prospectively for our selection of recipients.

Effect of anchor residue modifications on the stability of HLA-A11/peptide complexes.

MHC class I antigens bind peptides derived from endogenous proteins and present them to cytotoxic T lymphocytes. This binding is selective and shows high allele specificity. Peptides binding to HLA-A11 contain a hydrophobic or a small polar amino acid at position 2 and a lysine at the carboxy terminus. Peptide analogues, derived from previously identified high affinity peptides and carrying amino acid substitutions in position 2, were used to determine the requirements for formation of stable HLA-A11/peptide complexes. By kinetic analysis we were able to discriminate among apparent and true binders. Only analogues carrying in position 2 the amino acids valine, threonine and isoleucine formed stable complexes with HLA-A11 with a half life > or = 72 hours.

[Immunoproliferative small intestinal disease (IPSID): an unusual form of gastrointestinal lymphoma]. / Immunoproliferative Dünndarmerkrankung (IPSID): eine seltene Form gastrointestinaler Lymphome.

Worsening of long-lasting diarrhea, abdominal discomfort and weight loss were main symptoms in a 27-year-old Moroccan woman who had lived in Germany for 18 years. Pseudomonas, salmonella and lamblia cysts were found in stools. Histological examination of the gastrointestinal tract showed immunoproliferative small intestinal disease (IPSID), characterized by atrophy of the villi and lymphoplasmocytic infiltrates. alpha 1-heavy chains were found immunohistologically in the biopsy specimen, but not in serum, urine or jejunal juice. HLA-typing gave evidence of A9. Antibiotic treatment was successful for almost one year. Clinical, histological and immunological diagnosis of IPSID in an African woman living for nearly 20 years in Europe shows that, besides environmental factors, genetic disposition is an essential factor in the development of IPSID.

An HLA-A11-specific motif in nonamer peptides derived from viral and cellular proteins.

T lymphocytes recognize their antigenic targets as peptides associated with major histocompatibility complex molecules. The HLA-A11 allele, a preferred restriction element for Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocyte responses, presents an immunodominant epitope derived from the EBV nuclear antigen 4. Subpicomolar concentrations of a synthetic nonamer peptide, IVTDFSVIK, corresponding to amino acids 416-424 of the EBV nuclear antigen 4 sequence, can sensitize phytohemagglutinin-stimulated blasts to lysis by EBV-specific HLA-A11-restricted cytotoxic T-lymphocytes. We show that micromolar concentrations of this peptide induce assembly and surface expression of HLA-A11 in an A11-transfected subline of the peptide transporter mutant cell line T2. Using the IVTDFSVIK peptide and a series of synthetic nonamer peptides, differing from the original sequence by single amino acid substitutions, we have defined a motif for HLA-A11-binding peptides. This predicts the presence of a hydrophobic amino acid in position 2, amino acids with small side chains in positions 3 and 6, and a lysine in position 9. Using this motif, we have identified a peptide in the carboxyl-terminal end of wild-type p53, ELNEALELK, which is able to induce HLA-A11 assembly as efficiently as the IVTDFSVIK viral peptide.