Henk E Viëtor

Lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells and is caused by methylation of the IFN-γ inducible promoter (PIV) of CIITA

Lack of MHC-mediated antigen presenting functions of fetal trophoblast cells is an important mechanism to evade maternal immune recognition. In this study we demonstrated that the deficiency in MHC expression and antigen presentation in the trophoblast cell lines JEG-3 and JAR is caused by lack of class II transactivator (CIITA) expression due to hypermethylation of its interferon-gamma (IFN-gamma)-responsive promoter (PIV). Circumvention of this lack of CIITA expression by introduction of exogenous CIITA induced cell surface expression of HLA-DR, -DP, and -DQ, leading to an acquired capacity to present antigen to antigen-specific T cells. Transfection of CIITA in JEG-3 cells also upregulated functional HLA-B and HLA-C expression. Noteworthy, this lack of IFN-gamma-mediated induction of CIITA was also found to exist in normal trophoblast cells expanded from chorionic villus biopsies. Together, these observations demonstrate that lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells.

Transcriptional control of MHC genes in fetal trophoblast cells

Tight control of MHC expression is essential for the outcome of a successful pregnancy. The lack of MHC class II and class I mediated antigen presentation by fetal trophoblast cells is an important mechanism to evade maternal immune recognition. Interestingly, the deficient expression of MHC class II molecules (HLA-DR, -DQ and -DP) and of the classical MHC class I molecules HLA-A and HLA-B is also noted after IFN-gamma treatment in trophoblast-derived cell lines. Our studies show that in trophoblast cell lines the IFN-gamma induced transactivation of HLA-A and HLA-B promoters is repressed. Furthermore, it was found that trophoblast cells lacked IFN-gamma mediated induction of the class II transactivator (CIITA). This lack of CIITA expression in trophoblast cells is due to CIITA promoter hypermethylation. In addition to lack of CIITA expression, trophoblast cells also displayed a repressed expression of RFX5. Together, these observations reveal a silencing of multiple activation pathways that are critical to the transcriptional control of MHC class II and class I antigen presentation functions by trophoblast cells.

Modulation of the T-cell compartment by blood transfusion : effect on cytotoxic and helper T lymphocyte precursor frequencies and T cell receptor V-beta usage

Recent data suggest that the favorable effect of pretransplant blood transfusion (BT) on transplant outcome depends on the HLA match. HLA-DR or haplotype shared transfusions lead to transplantation tolerance, and HLA-mismatched BT leads to immunization. The immunological mechanism involved is still unknown. To investigate the effect of HLA compatibility between blood donor and recipient on the T cell compartment, we determined the frequency of cytotoxic and helper T cell precursors specific for blood donor cells (n=20) and the T cell receptor Vbeta (TCRBV) repertoire of the CD4- and CD8-positive peripheral blood mononuclear cells before, at 2 weeks after, and at more than 10 weeks after BT (n=10). Patients had received one transfusion of a nonstored (<24 hr after withdrawal) erythrocyte concentrate without buffy coat containing on average 6x10(8) leukocytes. Eight patients shared an HLA-B and -DR antigen, nine patients shared one HLA-DR antigen, and three patients shared no HLA class II antigens with the blood donor. All patients showed a significant increase in both cytotoxic and helper T cell precursor frequencies against the blood donor 2 weeks after BT. In most patients, the frequencies reached pretransfusion levels again long after BT. In 5 of 10 patients, an expansion of one or more TCRBV families was observed in either the CD4 or CD8 compartment. This study demonstrates that BT, irrespective of the degree of HLA matching, induces activation of the T cell compartment. The degree of sharing of HLA antigens was not correlated with quantitative changes in cytotoxic T lymphocyte precursor or helper T lymphocyte precursor frequencies, or changes induced in the TCRBV repertoire. Cytotoxic and helper T lymphocyte precursor frequencies and TCRBV repertoire determined after BT do not give an indication for a state of tolerance prior to transplantation.

Antenatal care in pregnancies at risk of alloimmune thrombocytopenia: report of 19 cases in 16 families.

OBJECTIVE To assess accuracy of a management program in patients at risk for alloimmune thrombocytopenia (NAITP) and to describe perinatal outcomes. STUDY DESIGN Nineteen fetuses at risk of thrombocytopenia were identified using obstetric history, HLA type of the mother and fetal phenotyping in cases where paternal heterozygozity for the offending antigen was present. Cordocentesis was timed according to obstetric history and performed with safety precautions to prevent haemorrhage. High dose intravenous gamma globulin (IVIG) was administered to the mother in cases with a fetal platelet count < 100 x 10(9)/l. RESULTS The platelet antagonisms were distributed as follows: HPA-1a in 15 patients, HPA-5a in two, HPA-3a in one, with one further woman who had antibodies against a private antigen. All multigravidas (N = 18) had previously given birth to an infant with NAITP and two of those infants had experienced severe bleeding. Two fetuses were negative for the offending antigen. The median and mean platelet count at first cordocentesis was 26 and 75 x 10(9)/l respectively (range 3-276). A total of 46 cordocentesis were carried out, of which 37 were followed by platelet transfusions. Bleeding complications were not observed. IVIG was administered to eight mothers and two fetuses responded. Nine infants were delivered by caesarean section (CS) and 10 vaginally at a mean gestational age of 37 weeks (range 34-41). The median and mean platelet count at birth was 141.5 and 140 x 10(9)/l, respectively (range 36-314). Ultrasound examination, both ante- and postnatally, revealed no intracranial haemorrhages. There was one procedure related neonatal death and one infant suffered from convulsions in the neonatal period due to a sinus thrombosis, possibly related to the platelet transfusions. CONCLUSIONS When obstetric history is taken into account cordocentesis in NAITP can be postponed. Safety recommendations described in this study allow cordocentesis without bleeding complications. However, our study does not support routine cordocentesis in patients with a history of NAITP. Both the risks of cordocentesis, and the lack of prospective data on the magnitude of the risk of intrauterine or peripartal bleeding, should be considered.

Immunological tolerance in an HLA non-identical chimeric twin.

Blood group chimeric twins offer a unique opportunity to study immunological tolerance in humans. Although this condition is not as rare as previously considered, detailed immunological studies of blood group chimeras are lacking. We describe here a case of secondary chimerism in a dizygotic twin of opposite gender. The karyotypes of the cultured fibroblast confirmed the sex of each twin, all cells in the boy were 46, XY and all cells in the girl were 46, XX. Molecular HLA typing on fibroblasts revealed HLA-DR, DQ and DP disparities between the two siblings. Mixed lymphocyte culture (MLC) revealed a mutual absence of alloreactivity.

Alterations in cord blood leukocyte subsets of patients with severe hemolytic disease after intrauterine transfusion therapy

OBJECTIVES The aim of this study was to compare, at delivery, the cord blood mononuclear cells of infants with severe hemolytic disease who received intrauterine transfusion (IUT) therapy with the cord blood mononuclear cells of healthy nonimmunized control neonates. STUDY DESIGN The expression of leukocyte markers on CBMNC of 14 IUT-treated and 18 control neonates was analyzed by means of a panel of well-defined monoclonal antibodies and flow cytometry. RESULTS Patients with severe hemolytic disease requiring IUT treatment displayed significant altered expression of some leukocyte markers when compared with control subjects. The circulating CD34+ progenitor cells were significantly increased in comparison with cord blood of nonimmunized neonates. IUT-treated patients also showed a statistically significant decrease in natural killer (NK) cell associated markers (CD16, CD57, and CD69), which correlated with a lower expression of CD56. In these patients an increased expression of CD3/CD45RO and CD3/CD5 was also noted. Although these latter alterations were statistically significant in a single-parameter analysis, the significance disappeared after multi-parameter analysis because of a loss of statistical power. CONCLUSIONS Compared with nonimmunized healthy newborn infants, patients who underwent IUT also exhibited a down-regulation of NK cells and NK cell associated markers, as well as increased numbers of CD34+ progenitor cells.

Intrauterine transfusions influence fetal leukocyte counts and subsets

The objective of this study was to determine the effect of intravascular intrauterine transfusion (IUT) on fetal leukocyte counts and subsets. For this purpose, pre‐ and post‐transfusion blood samples of 81 fetuses, receiving a total of 253 IUTs, were compared. Immediately after the IUT procedure an average decrease in fetal leukocyte count of 4 per cent was observed. When corrected for the dilutional effect of IUT, the average increase in leukocyte count was 41 per cent (n=180), indicating that IUT resulted in a relative leukocytosis. This was in contrast to the statistically significant average decrease in platelet count of 62 per cent (P<0·0001) immediately after IUT, suggesting that the relative increase in leukocyte count was lineage‐specific. Differential leukocyte counts revealed that the changes in fetal leukocyte count, in terms of percentage, after IUT were the result of an increase in monocytes and basophils and a decrease in lymphocytes. Flow cytometric analysis demonstrated that the decrease in lymphocytes was evenly distributed among the different subpopulations and not the result of a specific down‐regulation of one or more lymphocyte subsets. We observed only a modest relation between the duration of the transfusion and the degree of relative leukocytosis, suggesting that the onset of the leukocytosis probably occurred within minutes after the start of the transfusion. The observed effects appeared transient since the pre‐transfusion leukocyte count between each consecutive IUT did not reveal significant alterations during the course of IUT treatment.

Immunomodulation induced by intrauterine transfusions

Intrauterine transfusion (IUT) therapy offers a unique model to study the immunological consequences of fetal exposure to donor alloantigens. IUT can result in immediate and short effects. Directly after IUT a relative leukocytosis was observed, which was evenly distributed among the different leukocyte subsets. After the course of IUT treatment a memory response against donor antigens was generated. This was also reflected by an increase in CD3/CD45RO+ T-cells and modulation of T cell receptor Vbeta (TCRBV) repertoire. However, a long term clinical follow-up study on IUT patients who received this treatment in the 1960s revealed no evidence of serious side effects. Furthermore, persistence of donor leukocytes and in vitro immunomodulation could not be observed.