Treatment of hereditary angioedema with nanofiltered C1-esterase inhibitor concentrate (Cetor®): multi-center phase II and III studies to assess pharmacokinetics, clinical efficacy and safety.

From 1997, plasma-derived C1-inhibitor concentrate (Cetor®) has been available to HAE and AAE patients. Recently, a virus reducing 15 nm nanofiltration step has been introduced in the production process. A randomized, double-blind controlled cross-over study was performed to compare the pharmacokinetics (PK) of nanofiltered (C1-INH-NF) with conventional C1-inhibitor (C1-INH). Efficacy and safety were investigated in an open-label, on-demand and a prophylactic study. No differences in pharmacokinetic parameters between C1-INH and C1-INH-NF were found (13 non-symptomatic HAE patients). Both C1-inhibitor products equally increased plasma C4 levels. In the on-demand study, 14 acute angioedema attacks in 8 patients were analyzed. In the prophylactic study, 1 AAE and 5 HAE patients experienced in total 31 attacks during 748 observation days. In total 180,000 units of C1-INH-NF were administered. No product-related adverse events occurred, and no anti-C1-antibodies were induced. Nanofiltration in the production process of C1-inhibitor did not affect the pharmacokinetics, efficacy, and safety.

Donor-specific immunological non-responsiveness after liver transplantation.

Since the rate of immunological losses of liver allograft after the immediate posttransplant period is much lower than in other organs, we studied the immune responses against donor HLA antigens in 18 patients with a good long-term outcome to determine whether the development of a state of immunological non-responsiveness to donor antigens might account for this favorable outcome. The reactivity against donor spleen cells was measured before and 2 years after transplantation. The reactivity in mixed lymphocyte culture (MLC) and the frequencies of cytotoxic T cell precursors (CTLp) were determined. Responses against third-party spleen cells were determined concurrently to exclude a generalized reduction of immunocompetence due to chronic immunosuppressive treatment. Before orthotopic liver transplantation, the majority of patients had normal T cell responses against donor antigens that were comparable to those against third-party antigens. Two years after transplantation, donor-specific MLC non-reactivity had developed in 10 of the 18 (56%) patients. In addition, 15 of 18 (83%) patients had developed donor-specific cytotoxic T cell (CTL) non-responsiveness; 2 had reduced numbers of CTLp against both donor and third-party cells, while the remaining patient had maintained reactivity against donor antigens. In conclusion, donor-specific non-responsiveness is present in the majority of patients 2 years after successful liver transplantation, but occurs predominantly at the CTL level.

A substantial level of mixed chimerism is required for the induction of permanent transplantation tolerance.

The induction of donor-specific transplantation tolerance is a major goal in organ transplantation, in order to eliminate the requirement for lifelong immunosuppressive therapy. Previously, we have developed a murine bone marrow transplantation model in which recipient mice were treated with a single dose of anti-CD3 and low dose whole body irradiation (WBI). Transfusion of donor bone marrow cells across a full H-2 disparity resulted in induction of high levels of stable mixed chimerism, specific T cell non-responsiveness and indefinite skin allograft survival. The present study has focused on manipulation of the level of chimerism in this model by varying the number of infused bone marrow cells, varying the dose of WBI and addition of syngeneic bone marrow cells. Our results indicate that a substantial level of chimerism is needed for induction of transplantation tolerance. In addition, in the semi-allogeneic donor-recipient combination an even lower dose of WBI was sufficient of engraftment of bone marrow cells and subsequent tolerance induction. We suggest that sharing of MHC antigens between donor and recipient might play an important role in facilitating the development of chimerism and tolerance in organ transplantation.

Comparison of T cell responses in patients with a long-term surviving renal allograft versus a long-term surviving liver allograft. It's a different world.

The aim of the present study was to analyze whether acquired transplantation tolerance had developed in patients with a long-term surviving renal or liver allograft. Analysis of antidonor cytotoxic T cell precursor frequencies was performed in 31 renal allograft recipients and 9 liver allograft recipients with good graft function 2 years after transplantation. The results demonstrated that, before transplantation, normal antidonor T cell responses were generated in both groups of patients. Two years after transplantation, donor-specific CTL nonresponsiveness had developed in a minority of the renal transplant recipients. In contrast, 8 out of 9 liver transplant recipients showed donor-specific mixed lymphocyte culture and CTL nonresponsiveness. These findings indicate that development of donor-specific T cell nonresponsiveness is not a common event after kidney transplantation, whereas liver transplantation seems to induce, at least in vitro, a state of donor-specific T cell nonresponsiveness.

Missing T-cell receptor V beta families following blood transfusion. The role of HLA in development of immunization and tolerance.

Previously, we showed that donor-specific CTL nonresponsiveness occurs in transfused recipients sharing one HLA haplotype (or at least one HLA-B and one HLA-DR antigen) with the blood donor. The aim of the present study was to disclose the distinct effects of BT on the T-cell receptor repertoire and to analyze which factors determine the tolerizing versus immunizing properties of BT. We show here that recipients of HLA-sharing BT develop not only donor-specific CTL nonresponsiveness posttransfusion, but also a significant decrease in the usage of one to three V beta families as shown by PCR. In contrast, recipients of non-HLA-sharing BT remained donor-specific CTL responders and did not decrease the usage of V beta families. In addition, these patients generated high-affinity CTL for donor antigens which could not be blocked by anti-CD8 mAb. Our results show that major alterations occur in the CTL and TCR V beta repertoire following BT. We hypothesize that the fate of transfused allogeneic lymphocytes in the host is based on the degree of sharing of HLA antigens with the host. This relationship determines the ultimate outcome of BT: immunization versus tolerization.

Anti-CD3 treatment facilitates engraftment of full H-2-disparate donor bone marrow cells and subsequent skin allograft tolerance.

The aim of the present study was to induce engraftment of full H-2-disparate donor bone marrow cells and the development of subsequent transplantation tolerance. To this end, recipient H-2b mice were treated with anti-CD3 and on the same day received 6 Gy whole body irradiation as well as donor bone marrow cells (H-2d). Anti-CD3 treatment was chosen because it results in suppression of T cell function and in the release of CSF associated with enhancement of donor bone marrow engraftment. Stable, long-term chimerism measured in peripheral blood and mesenteric lymph nodes was obtained using this preparative regimen. In contrast, the use of anti-CD3 F(ab')2 fragments failed to induce donor bone marrow cell engraftment, suggesting indeed an important role of anti-CD3-mediated growth factor production in marrow engraftment. To overcome the side effects of anti-CD3 treatment (cytokine release syndrome), anti-CD4 was given 1 day before the treatment protocol. Omission of anti-CD3 resulted in failure of donor bone marrow engraftment, indicating the essential role of anti-CD3 treatment in marrow engraftment. Skin transplantation performed 2 and 6 months after this well-tolerated conditioning regimen showed indefinite survival of first and second grafts, respectively. In addition, specific CTL nonresponsiveness developed, demonstrating the presence of classical transplantation tolerance across a full H-2 barrier.

High-affinity cytotoxic T lymphocytes after non-HLA-sharing blood transfusion--the other side of the coin.

Previously, we have shown that pretransplantation blood transfusion modulates the T cell repertoire to a great extent. Patients receiving a BT from a donor sharing one HLA haplotype with the patient (HLA-sharing BT) develop CTL nonresponsiveness against cells of the BT donor and show a selective decrease in the usage of T cell receptor V beta families. The present study has focused on the analysis of the T cell repertoire in patients receiving an HLA mismatched (non-HLA-sharing) BT. CTL precursor frequencies were measured against single class I-mismatched antigens in split-well analysis. In addition, blocking studies of CTL-target cell interaction were performed with anti-CD8 monoclonal antibodies. The results demonstrate that non-HLA-sharing BT immunizes and induces the generation of CD8 independent, high-affinity CTL against immunogenic class I-mismatched antigens. Such HLA class I antigens might become nonacceptable mismatches in subsequent organ transplantation.

cis-acting regulatory elements abrogate allele-specific HLA class I gene expression in healthy individuals.

Pedigree analysis of a Dutch family revealed the presence of an HLA-A "blanc" allele segregating with the HLA-B8, -Cw7, -DR3, -DR52, -DQ2 haplotype. Precipitation studies, using selected mAb and sera directed against conserved epitopes of HLA class I products, failed to detect the expression of a corresponding HLA-A locus product. cDNA nucleotide sequence analysis of the HLA-A "blanc" specificity showed that the obtained sequence was identical to the authentic HLA-A1 gene revealing no mutations, deletions or recombinations that could influence translation or transport of a putative translation product to the cell surface. Mitogen stimulation, EBV transformation, or treatment with rIFN-gamma and rTNF-alpha did not induce HLA-A1 expression. Furthermore, cell-mediated lympholysis analysis revealed that individuals carrying the nonexpressed HLA-A1 gene could mount a strong anti-HLA-A1 T cell response, indicating that HLA-A1 was not expressed during T cell ontogeny. Therefore, this study describes for the first time the allele-specific down-regulation of the expression of a classical HLA class I gene segregating in healthy individuals.

Pretransplantation blood transfusion revisited.

BACKGROUND: Blood transfusion before organ transplantation has a beneficial effect on allograft survival; the mechanism of this effect has remained a mystery. In murine models, the presence of common histocompatibility antigens in the blood donor and the recipient favors the induction of allograft tolerance. METHODS: To investigate the effect of HLA compatibility between blood donor and recipient on the induction of allograft tolerance, we determined the relative frequency of cytotoxic T-lymphocyte precursors specific for donor cells before and at several times after blood transfusion in 23 patients awaiting a first renal transplant. We correlated the results with the presence of shared HLA antigens. RESULTS: T-cell nonresponsiveness against donor cells developed after blood transfusion in 10 of the 23 patients. Tolerance developed only if the blood donor and the recipient had one HLA haplotype or at least one HLA-B and one HLA-DR antigen in common (as was observed in 9 of these 10 patients). Tolerance developed relatively late after blood transfusion (one to two months) and was long-lasting. No decline in the T-cell response against donor alloantigens was observed in any of the 13 patients who received transfusions without having HLA-antigen compatibility with the donor. CONCLUSIONS: Blood transfusion in which there is a common HLA haplotype or shared HLA-B and HLA-DR antigens induces tolerance to donor antigens. This finding may lead to the development of new strategies with which to induce tolerance for transplantation after blood transfusion. Perhaps transplant donors will be selected not only by HLA-antigen matching, but also on the basis of acceptable HLA-antigen mismatches associated with T-cell non-responsiveness induced by selected blood transfusion.

Frequency analysis of HLA-specific cytotoxic T lymphocyte precursors in humans.

Frequencies of HLA class 1-specific cytotoxic T lymphocyte precursors (CTLp) from 33 responders were determined in 115 responder/stimulator combinations. In each combination there was a single HLA-A or HLA-B antigen mismatch. A wide range of CTLp frequency (CTLpf) values was found for most A and B locus antigens. Some A locus antigens appeared less immunogenic than other A locus antigens. The effect of additional C locus differences was negligible. The relationship between responder and stimulator HLA antigens is of minor importance because HLA-specific CTLpf against crossreactive (CREG) and subtype antigens were not significantly lower than CTLpf against non-CREG antigens. The CTLpf did not correlate with Bw4 or Bw6 mismatches. The existence of a broad range of values for HLA class I-specific CTLpf is of general interest. We have arbitrarily subdivided the CTLpf values into high, medium, low, and very low. In about 20% of the combinations the HLA-specific CTLpf were low or not even detectable in our assay. In contrast, HLA-specific CTLpf in combinations with multiple HLA antigen differences were regularly high. Our results confirm the high values of allospecific CTLpf in general but simultaneously point to unexpected variations. Frequency analysis of HLA-specific CTLp may be considered a new parameter in clinical transplantation for the selection of appropriate donor/recipient pairs.

Different requirements for the regulation of transplantation tolerance induction for allogeneic versus xenogeneic major histocompatibility complex antigens.

One way to solve the problem of human donor organ shortage is the use of animal organs. Therefore, it is important to study the T-cell response against xenogeneic major histocompatibility complex (MHC) antigens. In the present study, we have used HLA-B27 transgenic mice in a xenogeneic transplantation model. The results indicate that both transgenic skin transplantation and intravenous (IV) injection of transgenic spleen cells can reverse specific T-cell low responsiveness against the transgenic HLA-B27 antigen into high responsiveness in vivo and in vitro. In contrast, IV injection of spleen cells across an allogeneic H-2 class I disparity results in transplantation tolerance. Thus, despite T-cell low responsiveness against the transgenic HLA-B27 antigen, IV injection of transgenic HLA-B27 disparate lymphocytes does not tolerize, but rather immunizes for the xeno-MHC antigen.

Induction of transplantation tolerance by intravenous injection of allogeneic lymphocytes across an H-2 class II mismatch. Different mechanisms operate in tolerization across an H-2 class I vs. H-2 class II disparity.

Previously, we have shown that the intravenous (i.v.) injection of allogeneic lymphocytes across an H-2 class I-mutant disparity leads to specific skin allograft tolerance caused by irradiation-sensitive donor T cells, which function as veto cells. In the present study, we show that the i.v. injection of H-2 class II-incompatible spleen cells also results in specific skin allograft tolerance. However, tolerance induction depends on the presence of irradiation-resistant non-T cells in the donor cell inoculum. Thus, different mechanisms operate in tolerance induction across an H-2 class I vs. H-2 class II mismatch. I.v. injection of allogeneic spleen cells across an H-2 class I plus class II disparity does not result in skin allograft tolerance. Finally, our data show that transfusion-induced suppression of the delayed-type hypersensitivity response against alloantigens does not correlate with skin allograft tolerance induced by i.v. injected allogeneic lymphocytes. In conclusion, the type of H-2 mismatch between transfusion donor and recipient not only determines the occurrence of allograft tolerance but also the mechanism leading to tolerization.

Allograft tolerance induction in adult mice associated with functional deletion of specific CTL precursors.

Rejection of H-2 class I bm 1 mutant skin allografts by B6 recipient mice is mediated by a population of CD8+ anti-bm1 cytotoxic T-lymphocytes that produces and consumes its own T helper factor in response to bm1 skin allografts (dual function CTL). Previously we have demonstrated that transfusion of allogeneic lymphocytes across an H-2 class I disparity induces specific long-lasting skin allograft survival. We now show that intravenous injection of allogeneic spleen cells across the bm1 mutant disparity results in a temporary decrease of donor-reactive CTLp in the spleen of recipient mice, lasting for approximately five weeks. The sponge matrix allograft model was used to show that allograft tolerance is caused by a specific functional clonal deletion of CTLp within the allograft. We propose that dual function CTL are vetoed by donor T cells, resulting in skin allograft tolerance.

Mechanism of skin allograft enhancement across an H-2 class I mutant difference. Evidence for involvement of veto cells.

Intravenous injection of spleen cells across mutant class I H-2 incompatibility results in a drastic donor-specific prolongation of skin allograft survival and a marked decrease in the donor-specific cytotoxic T lymphocyte precursor (CTLp) frequency. This immunosuppressive effect depends on the presence of radiosensitive T cells in the donor cell inoculum. It was excluded that a graft-vs.-host reaction was responsible for the observed effects. In mixing experiments, spleen cells from animals transfused with allogeneic lymphocytes could not suppress a normal CTL response against the alloantigen, despite an excess of putative recipient-derived spleen suppressor cells. The data are compatible with the idea that donor T cells function as veto cells which inactivate recipient CTLp directed against the alloantigen expressed by the veto cell.