Nicholaus Zavazava

Inhibition of anti-HLA-B7 alloreactive CTL by affinity-purified soluble HLA.

The objective of this study was to elucidate the interaction of naturally occurring soluble MHC class I molecules with alloreactive CTL and to discuss its possible relevance to graft acceptance. An anti-HLA-B7 specific CTL-line, BV.B7, was generated in vitro. On phenotyping the cells after 6 weeks, 80% were found to be CD8+, 14% CD4+ and 6% CD8+CD4+. CD4+ CTL were depleted using immunomagnetic beads precoated with an anti-CD4 antibody. Of the recovered CTL greater than 96% were CD8+. A total of 12 HLA-B7 target cell lines and PHA blasts tested were specifically lysed in a 51Cr-release assay. Soluble HLA class I molecules were isolated on affinity chromatography columns using the anti-HLA-B7 ME 1 and the anti-heavy chain W6/32 monoclonal antibodies. Antigen purity was confirmed by analysis on SDS-PAGE gels. CTL were preincubated with 0.1-1.8 micrograms/ml soluble HLA for 30 min at 37 degrees C and subsequently tested for cytotoxicity in the 51Cr-release assay; 1.1 micrograms/ml HLA-B7 molecules reduced CTL cytotoxicity by 50% whereas non-B7 HLA had no effect. Further, CTL cytotoxicity was reduced by preincubation with anti-CD8, anti-TcR, and anti-CD3 antibodies. We anticipate a possible down-regulatory role of soluble HLA on CTL in allogeneic transplantation.

Soluble HLA class I molecules: biological significance and clinical implications

Soluble class I human leukocyte antigens (sHLAs) have been detected in serum, sweat, lymphatic fluid, urine and cerebrospinal fluid. Their biological function has, however, remained a puzzle. The physiological concentration of sHLA varies more than tenfold depending on the phenotype of the individual, and is significantly upregulated in various diseases and during inflammation. This suggests that sHLAs might serve as a marker of pathological changes. Recent experiments have shown that, in vitro, sHLAs can modulate T-cell reactivity and induce cell-activated apoptosis, implicating sHLAs in the induction and maintenance of peripheral tolerance. Therefore, sHLAs have the therapeutic potential to induce tolerance to transplants.

MHC-Related Odors in Humans

Up to now, MHC-related odors have been only described for rodents Yamazaki et al., 1991; Roser et al., 1991). Nevertheless, it has been speculated that a similar phenomenon may also occur in humans (Beauchamp et al., 1985; Boyse et al., 1987). Individual specific body odors do indeed play a role in human self-perception (Porter and Moore, 1981; Lord and Kasprzak, 1989) and recognition of offspring (Porter et al., 1983; Kaitz et al., 1987), but there is no information available on the biological basis of these odors.

Different in vivo tolerogenicity of MHC class I peptides.

The efficacy of MHC class I‐derived peptides to induce tolerance was tested in a cardiac transplantation model. Two 25‐mer peptides from the polymorphic region of the DA class I molecule (RT1.Aa) were synthesized by F‐moc chemistry and injected intrathymically or intraperitoneally into LEW (RT1.1) responder animals. Intrathymic treatment of the recipient animals with peptide 1 (residues 56–80) accompanied by intraperitoneal treatment with peptide 4 (residues 96–120) led to indefinite survival of allogeneic DA cardiac allografts (n = 7; >100 days). The tolerogenicity of both peptides differed according to the site of inoculation, as donor‐specific tolerance was only observed after administration of peptide 1 into the thymus and injection of peptide 2 into the abdominal cavity of LEW recipients, but not vice versa. Donor‐specific tolerance was confirmed in vivo by grafting of full‐thickness skin and in vitro by appropriate proliferation and cytotoxicity assays using donor and third‐party rats. Donor‐specific tolerance was associated with up‐regulation of interleukin‐4, transforming growth factor β, and interleukin‐10 gene expression within cardiac allografts, thus suggesting intrathymic clonal deletion and external suppression with expansion of T‐helper 2‐type lymphocytes as the underlying mechanisms of tolerance induction. J. Leukoc. Biol. 65: 16‐27; 1999.

In vitro cultivation and immunogenicity of human cardiac valve endothelium.

Abstract Endothelial cells were derived from aortic and mitral valves (n = 17) by collagenase digestion and subsequently cultivated in RPMI medium supplemented with 20% fetal calf serum. The cells were stained in an alkaline phosphatase‐anti‐alkaline phosphatase stain for the expression of MHC Class I and Class II antigens, ICAM‐1, ELAM‐1, F VIII, and H/Y. The endothelium showed a strong expression of Class I, H/Y, and ICAM‐1 molecules, and weak expression of MHC Class II molecules. In contrast to vascular endothelium that is known to express F VIII constitutively, cardiac valve endothelium was found to be negative. F VIII and ELAM‐1 were only expressed after stimulation with recombinant interferon‐gamma. To analyze the immunogenicity of valve endothelium, cells were used as stimulator cells in a mixed cell culture reaction using lymphocytes as responder cells. Endothelial cells had a 2 to 3 times higher stimulatory effect than peripheral blood lymphocytes. These data allow speculation on whether the observed degeneration of homografts can be reduced if HLA matching is performed prior to valve implantation.

Cross-matches on donor cadaver retinal pigment epithelial cells in corneal risk patients

Abstract• BackgroundAllografts can be rejected either through the antibody-mediated or cellular pathways. The objective of this study was to look at the extent of antibody formation in patients awaiting re-keratoplasty using cross-matches on cadaver retinal pigment epithelial (RPE) cells.• MethodsCadaverRPE cells were derived by trypsin digestion from donor eyes (n=1200). After 3 days of cell cultivation, the cells were adherent and began to lose their pigment. By day 7 most cells were clear and grew as a polygonal monolayer. MHC class I expression by RPE cells was studied by the W6/32 (anti-HLA-A, B, C) monoclonal antibody (MoAb) and that of class II (HLA-DR) by the 136 MoAb. Normal RPE cells express few class I and no detectable class II antigens. For the induction of MHC expression, cells were subsequently stimulated with 250 U/ml of recombinant gamma interferon for 5 days. Cells were used for tissue typing and also for cross-matches with recipient serum. Cross-matches were subsequently performed and measured by flow cytometry.• ResultsBoth class I and class II antigens were strongly enhanced, as could be shown by immunohistochemical staining. Some 20% of those patients awaiting rekeratoplasty (n=60) were positive for anti-HLA antibodies. In one case anti-DR3 antibodies were detected in a recipient who had had several rejection episodes after keratoplasty.• ConclusionsRPE cells are not only useful for cadaver post-mortem HLA typing but also for donor-specific cross-matches. The degree of antibody formation after keratoplasty in rejecting patients was, however, low. This may imply that anti-HLA antibodies are not the major cause of corneal graft loss after keratoplasty.

DNA-based HLA class II postmortem typing: evaluation of different techniques for prospective corneal allografting.

Abstract• Background: The objective of this study was to establish DNA-based HLA-DR postmortem tissue typing techniques in order to improve the quality and quanity of fully HLA-typed corneas for prospective allografting. •Methods: Four hundred and thirty-seven cornea donors were investigated. DNA was derived from cultivated retinal pigment epithelial cells by spin column purification or a salting out technique, and from scleral tissue by a very simple boiling method. Donors were typed by hybridization of polymerase chain reaction (PCR) products with sequence-specific oligodesoxynucleotide probes (PCR-SSOP) or by PCR with sequence-specific primers (PCR-SSP). Twenty-two of the donors were pretyped by serology. • Results: We observed high concordance (96%) between the results of DNA-based postmortem typing and the serological lymphocytotoxicity test. Furthermore, the distribution of the HLA-DR specificities that were detected correlated well with the distribution in a control population. • Conclusions: The results of this study demonstrate that both PCR-SSP and PCR-SSOP allow prospective allocation of HLA class II-matched corneas with high accuracy.

Immunfunktionelle Charakterisierung der adaptiven und non-adaptiven Immunantwort nach Anwendung des WOFIE-Konzeptes am Beispiel der vollallogenen Herztransplantation in der Ratte

Hintergrund: In Anlehnung an das inzwischen klinisch etablierte WOFIE-Konzept sollten am Beispiel der heterotopen Herztransplantation die an der an der Induktion von Langzeitorganakzeptanz beteiligten spezifischen und unspezifischen Effektorzellennaher charakterisiert werden.

Die Bedeutung der Allopeptid-induzierten Expression von Fas-Ligand (FasL) im Parenchym allogener Herztransplantate als Ursache der spenderspezifischen Toleranzinduktion

Die allogene Transplantatabstosung bedient sich zweier unterschiedlicher Wege der Antigenerkennung, zum einen der direkten Prasentation allogener Haupthistokompatibilitatsantigene (MHC) auf Transplantatzielzellen an T-Lymphozyten und zum anderen der indirekten Prasentation immunogener Spenderantigene auf empfangereigenen antigenprasentierenden Zellen (APCs) an Effektorzellen der Empfangerimmunantwort [1, 2].

Pathomorphologische und immunfunktionelle Charakterisierung Transplant-gegen-Wirt-induzierter Schädigungen nicht-lymphatischer Zielorgane nach solider Organtransplantation im Rattenmodell

Die willkurlich limitierte Augmentation der Transplantat-gegen-Wirt Reaktion (GvHR) durch simultane Knochenmark (KM)- und Organtransplantation desselben Spenders wird derzeit in experimentellen und klinischen Studien erprobt [1]. Das Konzept der iatrogenen Verstarkung der GvHR basiert auf der Annahme einer, durch zusatzliche Gabe immunkompetenter Zellen des Spenders, verbesserten Interaktion der spender- und empfangerspezifischen Immunantwort [2]. Die temporar erhohte Prasenz spenderspezifischer Antigene korreliert mit der Suppression der empfangerspezifischen Immunantwort gegen Transplantatantigene und ermoglicht, so die konzeptuelle Uberlegung, die Langzeitakzeptanz transplantierter Organe [3]. Das Risiko der durch die zusatzliche KMTx vermittelten Induktion einer akuten GvH-Erkrankung scheint, nach den bisher vorliegenden klinischen Ergebnissen gering zu sein.