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.

Lack of CIITA expression is central to the absence of antigen presentation functions of trophoblast cells and is caused by methylation of the IFN-gamma 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.

Sweat testing in hypomelanosis of Ito: divergent results reflecting genetic heterogeneity.

Sweat testing using a modified version of the method described by Minor was performed in 5 children affected with hypomelanosis of Ito. In 4 cases the areas of hypopigmentation were arranged in patterns following the lines of Blaschko, whereas in one case a phylloid pattern was observed. In 4 cases the hypomelanotic areas were shown to be anhidrotic. In one case of hypomelanosis following Blaschko's lines, sweat testing was normal and did not reveal any functional difference between the normally pigmented and the hypopigmented areas. These results provide a clinical illustration of the fact that hypomelanosis of Ito is not an entity but constitutes a cutaneous manifestation of various states of mosaicism.

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.

Survival of donor cells 25 years after intrauterine transfusion.

Persistence of donor leukocytes in the circulation of recipients of intrauterine transfusion (IUT) has been observed up to 5 years after birth. The aim of this study was to determine whether transfusions with nonirradiated, nonleukocyte-depleted donor blood during the fetal period resulted in long-term immunomodulation of the recipient. Twenty-four surviving IUT recipients between 1966 and 1976 were tested for autoimmune disease and autoantibodies at follow-up. Ten had sex-mismatched donors and were therefore informative for chimerism studies using fluorescence in situ hybridization (FISH). Seven female recipients could be tested for chimerism using a Y- chromosome-specific polymerase chain reaction (PCR) because they received at least 1 IUT from a male donor. Nine recipients could be studied for cytotoxic T-lymphocyte precursor (CTLp) and helper T-lymphocyte precursor (HTLp) frequencies because the original donors were available for testing. All surviving IUT recipients were in good health at the time of the examination, and routine laboratory testing revealed no abnormalities. None of the IUT recipients were chimeric as determined by FISH analysis, but Y-chromosome-specific sequences were detected by PCR in 6 of the 7 women. However, the CTLp and HTLp frequencies of the IUT recipients against the donors were comparable to those of the controls. The current study provides evidence that IUT can result in the persistence of donor cells in the recipient for a period longer than 20 years but that it is not associated with immunotolerance or with signs of chronic antigenic stimulation. (Blood. 2000;95:2709-2714)

Systemic sclerosis: new insights in autoimmunity.

The strong female predilection of systemic sclerosis, especially in women after their childbearing years, and the clinical and histopathological similarities with chronic graft-versus-host disease make systemic sclerosis an interesting subject of debate. Recent studies concerning the pathogenesis of this disease demonstrated the persistence of fetal cells in the maternal circulation in a majority of female patients. How or whether microchimerism is involved in the pathogenesis of systemic sclerosis remains to be elucidated. The present paper reviews the recent findings on the subject.

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 1960's revealed no evidence of serious side effects. Furthermore, persistence of donor leukocytes and in vitro immunomodulation could not be observed.

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 results 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.

Intrauterine transfusions affect fetal T-cell immunity.

Intrauterine transfusion (IUT) therapy is the treatment of choice in severe hemolytic disease of the fetus. This treatment automatically implies the introduction of alloantigens in the fetal circulation, which might potentially influence the unprimed fetal immune system. The present study provides evidence that the fetal immune system is indeed prone to modulations of the T-cell receptor BV (TCRBV) repertoire as a result of IUT treatment. Most notably, IUT therapy affects the composition of the CD4+ repertoire, whereas this effect may be obscured in the CD8+ subset. The CD8+ subset was found to be influenced by alterations of the TCRBV repertoire both in IUT patients and controls, suggesting that modulations in this subset could be the result of developmental influences. A more detailed analysis on the composition of the individual TCRBV families was performed by evaluating the distribution of the complementarity determining region 3 (CDR3) size lengths of [32P]-radiolabeled TCRBV transcripts. Using this technique, referred to as spectratyping, only marginal changes were observed in the CD4+ and CD8+ subset during the course of treatment and gestational development of both IUT-treated patients and controls. Therefore, the alterations in the overall TCRBV repertoire were of a quantitative rather than a qualitative nature. To evaluate whether the observed alterations in TCRBV usage-frequencies were a reflection of an allo-reactive response, a primed lymphocyte test (PLT) was performed in 3 IUT-treated patients. We observed that IUT, performed as early as 23 weeks of gestation, may induce the establishment of memory T cells against the IUT donor. However, there was no association between the observed changes in TCRBV repertoire and the magnitude of the secondary allo-reactive response.

Skewing of TCR V genes to CD4+ and CD8+ subsets of T lymphocytes is not determined by amino acid composition of CDR3.

TCR V genes show differing expression patterns, termed skewing, in CD4+ and CD8+ subsets of T lymphocytes. To determine which elements of the TCR V regions contribute to these observed TCR V gene skewing patterns, we have performed an in-depth analysis, taking advantage of RT-PCR and DNA sequencing, which was focused on the multi-member TCRBV6 gene family. These studies allowed us to evaluate the contributions of the various elements, that constitute the TCR beta chain variable region, to the observed TCR V gene skewing patterns. The results of these analyses revealed that within the TCRBV6 family individual members exhibited differing skewing patterns, i.e. TCRB6S7 was significantly skewed towards the CD4+ T cell subset, whereas TCRBV6S5 was significantly skewed towards the CD8+ subset. Scrutiny of the usage of TCRBV6 family members in combination with TCRBJ gene usage and amino acid composition of CDR3 did not reveal obvious structural characteristics which would explain the differing skewing patterns between TCRBV6S7 and TCRBV6S5. Further examination of these TCR V regions showed that the CDR1 and 2 regions within these TCRBV elements were composed of different amino acids. These observations suggests that these components contribute to the observed TCR V gene skewing patterns.

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.

Modulation of the T cell compartment by blood transfusion. Effect on cytotoxic and helper T lymphocyte precursor frequencies and T cell receptor Vbeta 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.

Induction of additional red cell alloantibodies after intrauterine transfusions.

BACKGROUND: The aim of this study was to determine the frequency and origin of additional alloantibodies directed against red cells (RBCs) after intrauterine transfusion (IUT). STUDY DESIGN AND METHODS: Between March 1987 and December 1992, fetuses with severe hemolytic disease (n = 91) received a total of 280 ultrasound-guided IUTs of RBCs from unrelated donors. The specificity of alloantibodies to RBCs in maternal serum was determined both before and after each IUT. If additional alloantibodies directed against RBCs were detected, their origin was determined by phenotyping the fetal, donor, and paternal RBCs for each particular antigen. The study included a control group of 69 pregnant women who underwent either amniocentesis or fetal blood sampling. RESULTS: Production of additional alloantibodies directed against RBC antigens was detected in 24 women (26%). The source of the immunizing antigen, either donor or fetus, was identified in 14 patients. The additional alloantibodies were directed against fetal antigens in 11 women and against donor antigens in 3. One additional alloantibody directed against donor antigen clearly reduced the survival of donor RBCs. The fetus and the donor shared the immunizing antigen in four cases, and in one case, the antibody occurred naturally. In five cases, the source of the immunizing antigen was not determined. In the control group, additional antibodies were detected in two patients. CONCLUSION: IUT therapy is associated with a high incidence of additional alloantibodies. In the majority of patients, the use of maternal RBCs for IUT would not have prevented additional formation of alloantibody to RBCs.

Hapten-specific T cell responses to 4-hydroxy-3-nitrophenyl acetyl. XIV. Carrier requirement for suppressor cell induction.

The carrier requirements for the induction of helper and suppressor T (Ts) cells were compared. Although H-2-linked Ir genes control the development of helper T cells and hapten-specific B cells, they do not influence Ts3 generation. That is, GL phi nonresponder C57BL/6 mice can generate NP-specific Ts3 cells after priming with NP-GL phi. The Ts3 cells generated under these conditions are functionally and phenotypically identical to the NP-specific Ts3 cells previously characterized. Furthermore, these Ts3 populations can be specifically depleted with a monoclonal anti-idiotope antibody prepared against monoclonal anti-NP antibodies. By using related polymers, carrier effects on Ts3 induction were noted. NP-D-GL and NP-Ficoll failed to induce Ts3 cells, whereas NP-L-GL induced this suppressor subset. The data demonstrate that Ts3 induction is independent of the carrier requirements involved in helper T cell induction and is not dependent upon B cell priming. The implications of these results with regard to the mechanisms of Ts3 induction are discussed.