Leo P. de Waal

Alloimmunization against the platelet-specific Zwa antigen, resulting in neonatal alloimmune thrombocytopenia or posttransfusion purpura, is associated with the supertypic DRw52 antigen including DR3 and DRw6

The strong association between HLA-DR3 and Zwa alloimmunization in Zwa-negative mothers, resulting in the production of anti-Zwa antibodies and ultimately in neonatal alloimmune thrombocytopenic purpura (NAITP) in the newborn, could be confirmed. In addition, we have shown an equally strong association between HLA-DR3 and Zwa alloimmunization in Zwa-negative recipients of blood transfusions, resulting in posttransfusion purpura (PTP). However, the strongest association both in mothers of NAITP patients and in PTP patients was observed with the supertypic DRw52 antigen. In fact, all individuals carried this antigen. Normally, DRw52 includes DR3, DR5, DRw6, and DRw8 almost completely. However, in our material, DRw52 included only DR3 and DRw6 with one exception. Another interesting observation is that all PTP patients were women with previous pregnancies without any clinical signs of a bleeding disorder in their children. This indicates that alloimmunization against Zwa during pregnancy is DR3- and/or DRw52-positive women does not always lead to the development of NAITP, but these women may be at risk for the development of PTP after blood transfusion.

Long-term detection of microchimaerism in peripheral blood after pretransplantation blood transfusion

Renal allograft survival is prolonged after pretransplantation blood transfusion. The aim of this study was to test retrospectively the development and persistence of microchimaerism after pretransplantation blood transfusion and to assess whether the type of blood transfusion (partially matched [= sharing of at least one HLA‐B and one HLA‐DR antigen between blood donor and recipient] versus mismatched) influences the (continued) presence of donor‐type cells. A sensitive nested PCR technique based on HLA‐DRB1 allele‐specific amplification using sequence‐specific primers (detection level: one donor cell among 105 recipient cells) for detection of donor cells was implemented in our laboratory. We studied 21 patients for microchimaerism in the peripheral blood compartment, following blood transfusion. Our preliminary data show that microchimaerism was detectable up to 8 weeks after blood transfusion. In all patients receiving a partially matched blood transfusion, donor‐type cells were detected in the first week after transfusion, in 7/8 patients 2–4 weeks after transfusion, and in some patients up to 8 weeks after transfusion. After mismatched transfusion a tendency to shorter duration of microchimaerism was observed.

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.

HLA-A29 subtypes and birdshot chorioretinopathy

J Central Laboratory ofthe Netherlands Red Cross Blood Transfusion Service and Laboratory for Experimental and Clinical Immunology, University of Amsterdam, Amsterdam, The Netherlands 2 Department of Ophthalmology, Erasmus University, Rotterdam, The Netherlands 3 Department of Ophthalmo-Immunology of the Netherlands Ophthalmic Research Institute, Amsterdam, The Netherlands 4 Department of Haematology, University of Gent, Gent, Belgium 5 Department of Ophthalmology, University of Gent, Gent, Belgium

Conventional alloantisera can recognize the same HLA-B27 polymorphism as detected by cytotoxic T lymphocytes.

Subtypes of B27 have been identified by cytotoxic T lymphocytes, biochemistry, molecular biology, and murine monoclonal antibodies. In the present study we describe seven B27 subtype-specific pregnancy sera. The reaction pattern of these B27 subtype-specific sera closely parallels the recognition pattern of B27 subtype-specific cytotoxic T lymphocytes. Because the complete amino acid sequence of the studied B27 subtypes (B27W, B27K, B27C, B27D) is known, it can be determined which amino acid substitutions are responsible for recognition by subtype-specific sera and by cytotoxic T lymphocytes, respectively. It is proposed that B27 subtype-specific sera and B27 subtype-specific cytotoxic T lymphocytes recognize the same epitopes or that a single amino acid change can induce multiple antigenic determinants, which are recognized differentially by antibodies and T cells.

An alternative conditioning regimen for induction of specific skin graft tolerance across full major histocompatibility complex barriers.

Previously, we developed a well-tolerated single-day protocol for induction of stable multilineage chimerism and permanent donor-specific tolerance across major histocompatibility complex (MHC) barriers, with preservation of the hosts normal immune responses. In our murine model, recipient mice were treated with a single dose of anti-CD3, anti-CD4, low dose total body irradiation (TBI; 3-6 Gy) and allogeneic bone marrow cells. An alternative cytoreductive strategy that is well-recognized in bone marrow transplantation, but has not been evaluated extensively in organ allograft recipients, involves the use of a combined chemotherapeutic drug treatment. The present data show that conditioning with low dose TBI, in a MHC-disparate donor-recipient combination, can be successfully substituted by a combined single low-dose dimethyl myleran (DMM)/cyclophosphamide (CY) therapy, resulting in both stable, mixed chimerism and specific skin graft tolerance.

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.

Specific Tolerance Induction and Organ Transplantation

Induction of tolerance to histocompatibility antigens of an organ donor would eliminate the need for long-term administration of nonspecific immunosuppressive drugs associated with an increased risk of infection and malignancies. Recently, we established a murine model in which recipient mice were treated with a single dose of anti-CD3, anti-CD4, low dose of total body irradiation (TBI) and allogeneic bone marrow cells. Our results clearly demonstrate that stable multilineage mixed chimerism, immunocompetence and permanent donor-specific skin graft tolerance across full major histocompatibility (MHC) barriers can be successfully achieved in this way. The observations that the preparative regimen and skin transplantation can be performed on the same day, and that a significant reduction in irradiation dose is sufficient in haploidentical donor-recipient combinations (MHC-sharing effect), bring our protocol closer to clinical use.

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.

Donor T Cells Are Responsible for the Blood Transfusion Effect Across an H-2 Class I Disparity

Donor-specific blood transfusion has a favorable effect on the survival of renal transplants. However, the mechanism underlying this effect is not known. We have adopted a mouse model to study the influence of donor-specific spleen cell transfusion on T-cell reactivity measured in vivo by skin graft rejection and DTH-reaction, and in vitro by the determination of cytotoxic T-lymphocyte precursor (CTLp) frequencies. The results indicated that the induction of donor-specific suppression in C57B1/6 (B6) recipient mice by spleen cell transfusion depends on the H2 mismatches between transfusion donor and recipient (manuscript in preparation). Donor-specific spleen cell transfusion across a class I H2 mutant difference (bm 1, bm 1l) resulted in a significant prolongation of skin allograft survival and in a severe depression of the alloantigen-specific CTLp frequency. The same result was observed across a class II H2 mutant difference (bm 12). However, donor-recipient mismatching for class I and class II H2 antigens completely abolished the immunosuppressive effects. Furthermore, we observed that prolongation of skin allograft survival always correlated with a marked decrease in CTLp frequency whereas no correlation was found with DTH suppression.