T-cell repertoire of normal, rejected, and pathological corneas: phenotype and function.

The specific immune mechanisms of corneal graft rejection are not completely understood. Recently, the technique of growing T-cell lines from rejected allografts using recombinant IL-2 has enabled the cells involved in allograft rejection to be recognized. In the present study, this method was applied for the extraction and propagation of T lymphocytes from rejected, normal, and diseased corneas. T-cell lines could successfully be grown from rejected and normal corneas, but not from corneas with keratoconus or pseudophakic bullous keratopathy. The phenotypic repertoire of the grown cells was studied by FACS scan analysis. Rejected corneas were invaded by a mixture of activated CD4+ and CD8+ T-cell lines, with one population being predominant. In normal corneas only activated CD8+ cells with cytotoxic function were cultured. No cells were obtained from diseased corneas. The in vitro function of cell lines was assessed by primed lymphocyte testing. The present study shows that the technique of propagating invading T-cell lines from organ grafts can be applied to human corneas, offering a new approach to understanding the immunological mechanisms occurring inside this immune tissue.

Homozygous human TAP peptide transporter mutation in HLA class I deficiency.

Human lymphocyte antigen (HLA) class I proteins of the major histocompatibility complex are largely dependent for expression on small peptides supplied to them by transporter associated with antigen processing (TAP) protein. An inherited human deficiency in the TAP transporter was identified in two siblings suffering from recurrent respiratory bacterial infections. The expression on the cell surface of class I proteins was very low, whereas that of CD1a was normal, and the cytotoxicity of natural killer cells was affected. In addition, CD8+ alpha beta T cells were present in low but significant numbers and were cytotoxic in the most severely affected sibling, who also showed an increase in CD4+CD8+ T cells and gamma delta T cells.

[T-lymphocyte population in the normal human cornea]. / Population T-lymphocytaire de la cornée humaine normale.

T-lymphocytes of the normal human cornea (10 donors) were studied from corneal fragments cultured in IL-2 medium. Their phenotypic repertoire was performed by FACS analysis using the following monoclonal antibodies: CD3, CD19, CD4, CD29, CD18, CD16, CD56, CD25, TCR alpha/beta, TCR alpha/delta. Functional analysis was done by proliferation assays (PLT). T cell growth was more often obtained from peripheral (n = 7/10) than central corneal parts (n = 3/10). Most of the cultured cells carried the CD8 marker, were activated (HLA-DR+) and had a cytotoxic function (CD 18+). All their T cell-receptors were alpha/beta type. No NK cells could be detected. No specific proliferation was observed when tested on a panel of HLA typed presenting cells. This study demonstrated activated T-lymphocytes in the normal human cornea. These activated, cytotoxic CD8+ lymphocytes could participate in the particular immunological characteristics of the eye.

Susceptibility to relapsing polychondritis is associated with HLA-DR4.

OBJECTIVE: To determine the frequency of HLA class II antigens in Caucasian central European patients with relapsing polychondritis (RP). METHODS: HLA class I, DR, and DQ specificities were identified in 41 patients with RP, and the frequencies were compared with those in 204 healthy, unrelated control subjects. HLA typing was performed using the standard complement-dependent microcytotoxicity assay. HLA-DR genotyping of 12 DR4-positive RP patients and 57 controls was performed by allele-specific oligonucleotide probing after amplification of genomic DNA by polymerase chain reaction. RESULTS: A significant increase in DR4 antigen frequency was found in the patients (56.1%) as compared with that in healthy controls (25.5%) (Pcorr < 0.001). Genotyping of DR4-positive patients and controls revealed no predominance of any DR4 subtype. CONCLUSION: There are important clinical similarities and overlaps between RP and rheumatoid arthritis (RA). In RA, the association with DR4 has been well established. Our findings show that although there is a DR4 association with RP, the situation is sufficiently distinct from that of RA to imply considerable differences in pathogenesis of the two conditions.

A new DR14 allele (DRB1*1411) containing a short DR11 sequence and its haplotypic association.

In the present work, we describe a new DR14 allele. Sequencing of its second DRB1 exon showed it to be DRB1*1404 from codon numbers 9 to 56 and 61 to 86, and DRB1*11 from codons 57 to 60 inclusive.

Establishment and characterization of B-like lymphoblastoid cell lines by long-term culture of primary explants from human myasthenic thymus.

Using a simple method of long-term culture, it was possible to obtain B-like lymphoblastoid cell lines (LyCLs) from myasthenic thymuses. Successful cultures were carried out from 14 out of 15 hyperplastic thymuses and in 1 out of 3 myasthenic thymoma, whereas none of the 8 control thymuses, nor the 2 Myasthenia gravis-associated normally involuted thymuses, nor the Myasthenia gravis-associated lymphoma gave rise to LyCL. All the LyCLs secreted immunoglobulins (Ig), either IgG or IgM. None of these Ig reacted with acetylcholine receptor or with other antigens known to be often involved in autoimmune diseases. EBV antigens were found in all the LyCLs as well as in the corresponding donors at the time of thymectomy. HLA characterization of some LyCLs and the corresponding donors showed that class II MHC antigens were expressed normally or with mild differences. However, 86% of the LyCL tested did not express class I MHC antigens.

DP epitope mapping by using T-cell clones.

To determine whether a correlation exists between the genomic HLA class II DP DNA polymorphism and cell surface expression and to detect the DP epitopes responsible for alloreactivity, anti-DP T-cell clones were generated against new PLT blank RFLP DPa and DPb-defined specificities. The clones were tested on the 10th IHWS B-LCLs and on local panel cells. Oligotyping of the tested cells made it possible to (a) correlate the DPa specificity with the DPB1*0402 specificity and (b) split DPb into DPB1*1001 and DPB1*1401. By comparing DNA sequences of the second exon to panel reactivity, the epitopes responsible for DPB1*1001 and 1401 were defined and attributed to beta-chain residues contributing to peptide selection inside the HLA groove. However, DNA sequences could not explain anti-DPa allospecificity, indicating that another structure not yet definable may be involved.

Complexity of the HLA-DP region: RFLP analysis versus PLT typing and oligotyping.

A total of 84 individuals were DP typed in parallel with the restriction fragment length polymorphism (RFLP) analysis and the primed lymphocyte test (PLT). Whereas 80% of the cases gave concordant results, the other 20% showed discrepancies for one of the two alleles carried by the typed individuals. Oligotyping, PCR-RFLP and sequencing confirmed the results found by PLT. The 20% discordant results obtained with RFLP led us to conclude that RFLP typing cannot replace PLT typing. From a more general point of view, the RFLP analysis revealed the DP region to be more complex than expected since for each given PLT DP defined specificity, more than one RFLP DP haplotype could be determined. These were possibly induced by crossing-overs or gene conversion events.

Analysis of HLA specificity of human monoclonal antibodies by cytofluorimetry and cell ELISA.

In this study we report on the characterization of cytotoxic human monoclonal antibodies (HmAb) detecting polymorphic HLA class II specificities using cytofluorimetric analysis in combination with micro cell ELISA. In both techniques, five anti-HLA HmAb were tested against HLA-transfected murine L cells as target cells and the bound antibody was detected, either by cytofluorimetry or by cell ELISA reader, after addition of fluoresceinated or peroxidase-conjugated anti-human IgG+IgM antibodies, respectively. The results demonstrate that HmAb directed against HLA-DR, -DQ and -DP molecules can be efficiently discriminated by cytofluorimetry and cell ELISA, which appear to be highly sensitive and perfectly comparable to the standard cytotoxicity assay.