Giovanni Battista Ferrara

Evidence that celiac disease is primarily associated with a DC locus allelic specificity.

Sixty patients with celiac disease were typed by radioimmunoassay for the specificities HLA-DR3, HLA-DR7, and for an allelic specificity, DC3, of the HLA-associated DC locus. We found that celiac disease is primarily associated with the DC determinant. The previously described associations with DR3, DR7, B8, B13, and A1 can be explained by decreasing degrees of linkage disequilibrium with DC3.

CTLA‐4 is constitutively expressed on tumor cells and can trigger apoptosis upon ligand interaction

CTLA‐4 (CD152) is a cell surface receptor that behaves as a negative regulator of the proliferation and the effector function of T cells. We have previously shown that CTLA‐4 is also expressed on neoplastic lymphoid and myeloid cells, and it can be targeted to induce apoptosis. In our study, we have extended our analysis and have discovered that surface expression of CTLA‐4 is detectable by flow cytometry on 30 of 34 (88%) cell lines derived from a variety of human malignant solid tumors including carcinoma, melanoma, neuroblastoma, rhabdomyosarcoma and osteosarcoma (but not in primary osteoblast‐like cultures). However, by reverse transcriptase‐PCR, CTLA‐4 expression was detected in all cell lines. We have also found, by immunohistochemistry, cytoplasmic and surface expression of CTLA‐4 in the tumor cells of all 6 osteosarcoma specimens examined and in the tumour cells of all 5 cases (but only weakly or no positivity at all in neighbouring nontumor cells) of ductal breast carcinomas. Treatment of cells from CTLA‐4‐expressing tumor lines with recombinant forms of the CTLA‐4‐ligands CD80 and CD86 induced apoptosis associated with sequential activation of caspase‐8 and caspase‐3. The level of apoptosis was reduced by soluble CTLA‐4 and by anti‐CTLA‐4 scFvs antibodies. The novel finding that CTLA‐4 molecule is expressed and functional on human tumor cells opens up the possibility of antitumor therapeutic intervention based on targeting this molecule.

HLA-DP typing by DNA amplification and hybridization with specific oligonucleotides.

HLA-DP genotyping was performed using dot-blot analysis with synthetic oligonucleotide probes. Fourteen probes were designed based on the known sequence variations in the polymorphic segments of the DP beta second exon. Each probe was tested against genomic DNA amplified by the polymerase chain reaction, using DP beta-specific primers. A total of 45 HLA homozygous B-cell lines, selected from the Tenth International Histocompatability Workshop and pretyped for the known DP omega specificities, were analyzed. Different hybridization patterns were found for each DP omega specificity. The oligonucleotide hybridization performed on DP omega-negative B-cell lines gave a pattern distinct from those of known DP omega specificities, indicating the presence of novel DP allelic sequences. The use of sequence-specific oligonucleotides combined with DNA amplification allows a simple and reliable genotyping of DP antigens.

The distribution of DR5, MT2, and MB3 specificities on human Ia subsets.

Human Ia molecules were isolated from cells of LG-38, an HLA-homozygous lymphoid cell line of DR5 specificity. Three Ia subsets could be distinguished and separated by using specific alloantisera and a monoclonal antibody with polymorphic reactivity. These subsets carried the specificities DR5 and MT2, MT2 alone and MB3 alone. The structure of the three molecular species was analyzed by microfingerprinting. The subset carrying only MT2 was similar, in both the component α and β chains, to the major subset carrying DR5 and MT2, whereas the subset carrying MB3 was distinct in both chains from the other two subsets. These data are compatible with our previous findings obtained for the products of two Ia loci closely linked to the DR locus and provisionally called DC and BR; they also support the conclusion that the subset carrying only MT2 is an allelic product of the BR locus, whereas the MB3 subset is an allelic product of the DC locus. MT2 appears to be a shared specificity of DR and BR loci products.

Molecular identification of human Ia antigens coded for by a gene locus closely linked toHLA-DR locus

Human Ia(-like) specificities controlled by gene loci other thanHLA-DR were searched for at the molecular level in cells of human B-cell-type cell lines which carrytwo established DR specificities. Chevalier cells of DRw3 and 7 and U698M cells of DRw2 and 4 were used. Their Ia molecules were partially purified, radioiodinated and analyzed for Ia specificities by the direct binding and sequential binding assays with a selected panel of human Ia alloantisera. It was possible in both the cell lines to define a third subset of Ia molecules carrying a new specificity in addition to two Ia subsets carrying the established DR specificities. The new specificity was detected by putative anti-DRw4 and anti-DRw7 antisera and was closely associated with DRw4 and DRw7 at population level. It was thus designated provisionally as BR4X7. These results suggest that the BR4X7 specificity is coded for by a separateIa locus closely linked toHLA-DR locus. The determinant(s) responsible for BR4X7 was located on the small subunit of Ia molecules.

Immunotoxins Containing Recombinant Anti-CTLA-4 Single-Chain Fragment Variable Antibodies and Saporin: In Vitro Results and In Vivo Effects in an Acute Rejection Model

Immunotoxins containing recombinant human-derived single-chain fragment variable (scFv) reagents (83 and 40) against CTLA-4 (CD152) linked to saporin, a ribosome-inactivating protein, were prepared and tested on CD3/CD28-activated T lymphocytes, MLRs, CTLA-4-positive cell lines, and hemopoietic precursors. Immunotoxins induced apoptosis in activated T lymphocytes and were able to specifically inhibit MLR between T lymphocytes and dendritic cells. The 83-saporin immunotoxin also inhibited the T cell activation in an MLR between T lymphocytes and an EBV-positive lymphoblastoid B cell line. Toxicity tests on hemopoietic precursors showed little or no effects in inhibiting colonies’ growth. As the 83 scFv Ab was reactive also with activated mouse T lymphocytes, 83-saporin was tested in a model of tumor rejection consisting of C57BL/6 mice bearing a murine H.end endothelioma cell line, derived from DBA/2 mice. The lymphoid infiltration due to the presence of the tumor was reduced to a high extent, demonstrating that the immunotoxin was actually available and active in vivo. Thus, taking the results altogether, this study might represent a new breakthrough for immunotherapy, showing the possibility of targeting CTLA-4 to kill activated T cells, using conjugates containing scFv Abs and type 1 ribosome-inactivating protein.

A safe blood transfusion procedure for immunization against major histocompatibility complex determinants in man.

SUMMARY A standardized procedure is proposed for deliberate immunizations against human major histocompatibility complex determinants. The data presented demonstrate its effectiveness and, by using a number of necessary precautions, this procedure has proven to be very safe. The following points are especially important: (1) exclusive utilization of regular blood donors as immunizers; (2) use of whole blood as an immunizing agent; and (3) use of small immunizing stimuli rather than large transfusions. This procedure can be recommended for the production of monospecific anti-HLA antisera and it may be useful if and when a deliberate transfusion policy for prospective kidney recipients is adopted. Because of the increasing complexity of the HLA system, planned immunizations become more and more necessary if extensive selections and absorptions of sera from multiparous women are to be avoided. Also, there is evidence indicating the beneficial effect of blood transfusion on the outcome of kidney transplants (1–5). This may create in the future the requirement for deliberate transfusion also in these cases. A standardized immunization procedure in volunteer recipients has been undertaken by our group during the past 9 years. Based on the considerable amount of data collected, we can confidently recommend this procedure as both safe and effective in eliciting anti-HLA antibodies.

Reassessment of HLA association with celiac disease in special reference to the DP association

Patients with the late-onset form of celiac disease have been studied for HLA association by conventional serology (DR and DQ typing) and by oligonucleotide probing with gene amplification (DP typing). Patients and controls were sampled in the Bologna area of northern Italy. Almost all patients were positive for DQw2 (94%), being DR3 positive (72%) and/or DR7 positive (65%). The proportion of DR3/7 heterozygotes in the patients was significantly increased over that expected from the Hardy-Weinberg equilibrium. No positive association with DR5 and no significant increase of DR5/7 heterozygotes were observed. Among the DP alleles reported to exhibit an association with celiac disease in other populations, only DPB3 showed a moderate increase of a borderline significance, not attributable to a linkage disequilibrium with DQw2.

Analysis of HLA-G expression in breast cancer tissues.

Among the different mechanisms by which cancer can elude the immune system, alterations in the expression of human leukocyte antigen (HLA) class I molecules on tumor cells may play a crucial role by impairing the HLA molecules interaction with T and natural killer (NK) cells specific receptors. More recently, aberrant expression of HLA-G has been described in different tumor tissues in addition to HLA class I downregulation. The HLA-G molecule is a nonclassical HLA class I antigen selectively expressed by trophoblast and thymic epithelial cells. Several studies reported that the HLA-G function might represent an additional mechanism of tumor immune escape, mainly inhibiting NK and cytotoxic T-cell activity. Here we report the analysis of HLA-G expression both at RNA level by reverse transcriptase-polymerase chain reaction and at protein level by Western blot and immunohistochemistry in 25 breast cancer patient tissues. The aim of this study was to elucidate the HLA-G gene expression pattern in breast tumor tissues and correlate it with HLA class I alterations. Our results demonstrated that HLA-G molecules expression was never found even in a group of patients revealing HLA class I total loss, and that HLA-G is not expressed in breast cancer tissue with a low-tumor grade (G1-G2) and minimal stromal contamination.

A new approach to HLA-DPB1 typing combining DNA heteroduplex analysis with allele-specific amplification and enzyme restriction

Allelic polymorphism of HLA-class II antigens plays a key role in the regulation of the immune response and in transplantation immunity. The allelic diversity of these antigens can now be analyzed at the DNA level after amplification by polymerase chain reaction. In this study we apply a simple technique based on the electrophoretic analysis of DNA heteroduplexes to the typing of HLA-DPB1 alleles. In order to increase its resolution, a group-specific amplification was used which subdivides the 19 HLA-DPB1 alleles in two non-overlapping families. A separate analysis was then performed within each group of alleles. This approach allowed an unequivocal one-step typing of the alleles belonging to group 1 which comprises few alleles of high frequency. Some group 2 alleles require, as a further step, the test with a restriction enzyme. The combination of more than one technique represents, in our opinion, the easiest way to solve the micropolymorphism of class II alleles. We conclude that this method, which is very simple, quick, and accurate and does not require probes, may become the method of choice for HLA-DPB1 typing.