Changes in cytokine production and composition of peripheral blood leukocytes during pregnancy are not associated with a difference in the proliferative immune response to the fetus.

We analyzed peripheral blood from women at term pregnancy for leukocyte composition, in vitro proliferative responses and cytokine production after nonspecific and fetus-specific stimulation. Maternal peripheral blood mononuclear cells (PBMCs) were collected and stimulated with umbilical cord blood (UCB) of the mother's own child, third-party UCB, nonspecific stimulus phytohemagglutinin, and anti-CD3 antibody, with PBMCs of nonpregnant women (cPBMC) as controls. Nine combinations of patient, child, third party child, and controls were selected on basis of sharing one human leukocyte antigen (HLA)-DR antigen. The response of mPBMC upon specific stimulation with fetal antigens was similar to that of cPBMC. No differences were found when comparing the mother's response upon stimulation to her own child with stimulation to that with a control child. Nonspecific stimulation with phytohemagglutinin and anti-CD3 antibody did not reveal a difference in proliferation rate between mPBMC and cPBMC. However, mPBMC contained a higher percentage of CD14(+) cells (p = 0.001) and activated T cells (CD25(dim), p < 0.0001), but a lower percentage CD16(-)CD56(bright) natural killer (NK) cells (p = 0.001) and CD16(+)CD56(+) NK cells (p = 0.003). mPBMC produced more interleukin (IL)-6, IL-10, and IL-17 compared with cPBMC (p < 0.05). We found differences in lymphocyte composition and cytokine production between mPBMC and cPBMC. These differences did not result in quantitative changes in proliferative responses during pregnancy compared with responses in nonpregnant controls.

Discontinuation of calcineurin inhibitors treatment allows the development of FOXP3+ regulatory T-cells in patients after kidney transplantation.

This study investigated specific gene expression profiles in patients with donor-specific cytotoxic-hyporesponsiveness, reflected by cytotoxic T-lymphocyte precursor frequency (CTLpf). The effect of calcineurin inhibitor (CNI) withdrawal was studied on markers for cytotoxicity (perforin, granzyme B), apoptosis (Fas,FasL), Th1 and Th2 cytokines (IL-2, IL-10), Th1 and Th2 transcription factors (T-bet, GATA 3), Th17 transcription factor and cytokine (RORγt, IL-17), and for immune regulation/activation (CD25, FOXP3). Peripheral blood samples from renal allograft recipients (n = 18) more than two yr after transplantation with stable renal function were analyzed before and four months after CNI withdrawal. Additionally, systolic and diastolic blood pressure, cholesterol, serum creatinine and proteinuria were evaluated, and no significant differences were measured before and after CNI withdrawal. However, CNIs' discontinuation influenced peripheral gene expression profiles. After CNI withdrawal, the mRNA expression of Granzyme B, Perforin, Fas, FasL, T-bet, GATA3 and CD25 were significantly lower than during CNI treatment. After CNI discontinuation, donor-specific CTLpf decreased, while FOXP3 expression discriminated between detectable and non-detectable donor-specific cytolysis reactivity; FOXP3 transcript values were highest in absence of donor-specific cytotoxicity (p < 0.01). Our study shows CNI withdrawal in stable kidney transplant recipients two yr after transplantation is safe. Moreover, discontinuation of CNIs' treatment allows FOXP3+ regulatory T-cells development, resulting in a significant decrease of anti-donor immune reactivity.

Human decidual tissue contains differentiated CD8+ effector-memory T cells with unique properties.

During pregnancy, maternal lymphocytes at the fetal-maternal interface play a key role in the immune acceptance of the allogeneic fetus. Recently, CD4(+)CD25(bright) regulatory T cells have been shown to be concentrated in decidual tissue, where they are able to suppress fetus-specific and nonspecific immune responses. Decidual CD8(+) T cells are the main candidates to recognize and respond to fetal HLA-C at the fetal-maternal interface, but data on the characteristics of these cells are limited. In this study we examined the decidual and peripheral CD8(+) T cell pool for CD45RA, CCR7, CD28, and CD27 expression, using nine-color flow cytometry. Our data demonstrate that decidual CD8(+) T cells mainly consist of differentiated CD45RA(-)CCR7(-) effector-memory (EM) cells, whereas unprimed CD45RA(+)CCR7(+) naive cells are almost absent. Compared with peripheral blood EM CD8(+) T cells, the decidual EM CD8(+) T cells display a significantly reduced expression of perforin and granzyme B, which was confirmed by immunohistochemistry of decidual tissue sections. Interestingly, quantitative PCR analysis demonstrates an increased perforin and granzyme B mRNA content in decidual EM CD8(+) T cells in comparison with peripheral blood EM CD8(+) T cells. The presence of high levels of perforin and granzyme B mRNA in decidual EM T cells suggests that decidual CD8(+) T cells pursue alternative means of EM cell differentiation that may include a blockade of perforin and granzyme B mRNA translation into functional perforin and granzyme B proteins. Regulation of decidual CD8(+) T cell differentiation may play a crucial role in maternal immune tolerance to the allogeneic fetus.

Preeclampsia is associated with increased cytotoxic T-cell capacity to paternal antigens.

OBJECTIVE: During an uncomplicated pregnancy the conceptus is a semiallogeneic entity in which rejection is prevented by suppression of the maternal immune system. We hypothesized that this suppression is disturbed in patients with preeclampsia and that a maternal immune response to fetal (foreign/paternal) antigens in the fetal-maternal interface may be responsible for local inflammation, with subsequent endothelial dysfunction and systemic disease. STUDY DESIGN: Blood samples were obtained from 14 women with preeclampsia (cases), 14 gestational-age and parity-matched women with uncomplicated pregnancies (controls), and their partners. We determined the partner-specific cytotoxic T-lymphocyte precursor frequency (CTLpf) and the CTLpf directed to unrelated partners with uncomplicated pregnancies. We measured the CTLpf in peripheral blood mononuclear cells (PBMCs) from cases and controls using limited-dilution assays. In addition, proliferation was tested in a mixed-lymphocyte culture (MLR). RESULTS: The partner-specific CTLpf was significantly higher in cases compared with controls (median, 183 [15-338] vs 67 [9-232] per million PBMCs, P = .02). In contrast, in women with uncomplicated pregnancies, the partner-specific CTLpf was down-regulated compared with the CTLpf directed to an unrelated partner who fathered uncomplicated pregnancies (P = .02). No difference was found in partner-specific MLR response between cases and controls. CONCLUSION: These results suggest that women with preeclampsia have a higher cytotoxic T-cell response to paternal antigens compared with pregnant controls. This insufficiently suppressed immune response may eventually lead to the development of preeclampsia.

Fetal-maternal HLA-C mismatch is associated with decidual T cell activation and induction of functional T regulatory cells.

Human leukocyte antigen-C (HLA-C) is the only polymorphic classical histocompatibility antigen expressed by fetal trophoblasts at the fetal-maternal interface. Interactions between HLA-C and decidual natural killer (NK) cells may facilitate trophoblast invasion into maternal tissue. Thus far no evidence has been provided that decidual T cells specifically recognize and respond to fetal alloantigens at the fetal-maternal interface. In this study, we show that pregnancies containing a HLA-C mismatched child induce an increased percentage of CD4(+)CD25(dim) activated T cells in decidual tissue. In addition, HLA-C mismatched pregnancies exhibit a decidual lymphocyte response to fetal cells and contain functional CD4(+)CD25(bright) regulatory T cells in decidual tissue, whereas HLA-C matched pregnancies do not. This suggests that decidual T cells specifically recognize fetal HLA-C at the fetal-maternal interface but are prevented from inducing a destructive immune response in uncomplicated pregnancies.

Deficient TNF-alpha and IFN-gamma production correlates with nondetectable donor-specific cytotoxicity after clinical kidney transplantation.

BACKGROUND: We previously reported that no cytotoxic T-lymphocyte precursor frequencies (CTLpf) were found in 60% of patients on azathioprine or mycophenolate mofetil+Pred long after kidney transplantation. We questioned whether the absence of donor-specific CTLpf was associated with low levels of stimulatory Th1 (tumor necrosis factor [TNF]-alpha, interferon [IFN]-gamma) or high levels of regulatory Th2 (interleukin [IL]-4, IL-6, IL-10) cytokines. METHODS: In this study, peripheral blood monocyte cells (PBMC) were stimulated with irradiated donor cells. After 7 days, cytokine production was determined by cytokine bead array, and CTLpf by limiting dilution assay. RESULTS: Patients with detectable CTLpf (> or =10/10(6) PBMC) had significantly higher levels of TNF-alpha (P=0.04) and IFN-gamma (P=0.02) than patients with nondetectable CTLpf (<10/10(6) PBMC). Donor-reactive IL-4, IL-6, and IL-10 production was comparable in both patient groups. Additionally, CTLpf was positively correlated with TNF-alpha (rs=0.54, P=0.0003) and IFN-gamma (rs=0.64, P<0.0001) production. CONCLUSION: The absence of donor-specific CTLp after transplantation correlates with low levels of stimulatory cytokines, not with elevated levels of regulatory cytokines.

Expression of NK cell receptors on decidual T cells in human pregnancy.

Specific receptors enable NK cells to discriminate between cells with normal expression of MHC class I and cells that have low or absent expression of MHC class I molecules. In addition to NK cells, these receptors can be expressed on T cell subsets, mainly on CD8+ T cells but also on gammadeltaTCR+ T cells and CD4+ T cells. Although the function of NK cell receptor expression on T cells is not completely understood, various studies have shown that they are involved in down regulation of T cell receptor (TCR)-mediated activation and influence effector functions, like cytotoxicity and cytokine production. The aim of this study was to analyze expression of NK cell receptors on peripheral blood and decidual T cells during human pregnancy using flow cytometry. We demonstrate that a proportion of decidual T cells express HLA-C specific killer immunoglobulin-like receptors (KIRs). Furthermore, a small proportion of decidual T cells express the HLA-E specific CD94-NKG2A inhibitory and CD94-NKG2C activating receptors. Decidual KIR+ and CD94-NKG2+ T cells mainly display a CD3+CD4-CD8- phenotype. However, decidual tissue also contains higher percentages of KIR and CD94-NKG2 expressing CD4+ and CD8+ T cells compared to peripheral blood. So far, the functional capacities of decidual T cells expressing the NK cell receptors are unknown but NK cell receptor expression on decidual T cells may provide an alternative means by which decidual T cells distinguish self (maternal) cells from allogeneic fetal cells, and act to modulate the decidual immune response.

Differential immunomodulatory effects of fetal versus maternal multipotent stromal cells.

Protective mechanisms are likely to be present at the fetomaternal interface because fetus-specific alloreactive T cells present in the decidua do not harm the fetus. We tested the immunosuppressive capacity of maternal and fetal multipotent stromal cells (MSC). Single cell suspensions were made from second-trimester amnion, amniotic fluid, and decidua. Culture-expanded cells were identified as MSC based on phenotype and multilineage potential. Coculture of MSC in a primary mixed lymphocyte culture of unrelated responder-stimulator combinations resulted in a dose-dependent inhibition of proliferation. Fetal MSC demonstrated a significantly higher inhibition compared with maternal MSC. This stronger inhibition by fetal MSC was even more prominent in a secondary mixed lymphocyte reaction (MLR) with primed alloreactive T cells. Analysis of cytokine production revealed that fetal MSC produced significantly more interleukin (IL)-10 and vascular endothelial growth factor than maternal MSC. Cell-cell contact is needed for part of the inhibitory effects of MSC. In addition, soluble factors play a role because blocking experiments with anti-IL-10 revealed that the inhibition of the MLR response by fetal MSC is mainly mediated by IL-10. For maternal MSC, other soluble factors seem to be involved. Fetal MSC derived from the fetomaternal interface have a stronger inhibitory effect on naive and antigen-experienced T cells compared with maternal MSC, which is probably related to their higher IL-10 production.

Tapering immunosuppressive therapy significantly improves in vivo cutaneous delayed type hypersensitivity responses.

Immunosuppressive therapy affects cell-mediated immunity and thereby increases the frequency of infections and malignancies in transplanted patients. We questioned whether reducing the immunosuppressive dose in stable kidney transplant patients has an in vivo effect on cutaneous delayed type hypersensitivity responses (DTH) reflecting cell-mediated immunity. We measured DTH responses to recall antigens (Tetanus, Diphteria, Streptococcus, Tuberculin, Candida, Trychophyton, Proteus, glycerin control) on the volar surface of the forearm in patients before and after successful reduction (50%) of the dose of mycophenolate mofetil (MMF) or azathioprine (AZA). In addition, we tested healthy individuals who were age- and sex-matched to the patient group. Results of the skin reaction test were calculated as the sum in millimeters (mm) of all positive reactions (score), and as the number of positive antigens. Patients treated with a high dose of MMF or AZA had a significantly lower test score compared to healthy controls (p=0.01). Also the number of positive antigens was reduced in patients compared to healthy controls (p=0.02). After reduction of the MMF or AZA dose, the test score and the number of positive antigens increased significantly (p=0.02, p=0.01, respectively) to comparable scores of healthy controls. Additionally, the mycophenolic acid (MPA) trough level was negatively correlated with the test score (p=0.006) and number of positive antigens (p=0.004). In conclusion, successful tapering of the MMF or AZA dose in kidney transplant patients more than 2 years after transplantation favorably affects the in vivo DTH response, reflecting an improvement of the general immunity, facilitating the defense against infection and malignancies.

Evidence for a selective migration of fetus-specific CD4+CD25bright regulatory T cells from the peripheral blood to the decidua in human pregnancy.

During pregnancy, the maternal immune system has to tolerate the persistence of fetal alloantigens. Many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. Murine studies suggest that CD4(+)CD25(+) T cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. Previous studies by our group demonstrate that a significantly higher percentage of activated T cells and CD4(+)CD25(bright) T cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy. In this study, we examined the phenotypic and functional properties of CD4(+)CD25(bright) T cells derived from maternal peripheral blood and decidual tissue. Depletion of CD4(+)CD25(bright) T cells from maternal peripheral blood demonstrates regulation to third party umbilical cord blood cells comparable to nonpregnant controls, whereas the suppressive capacity to umbilical cord blood cells of her own child is absent. Furthermore, maternal peripheral blood shows a reduced percentage of CD4(+)CD25(bright)FOXP3(+) and CD4(+)CD25(bright)HLA-DR(+) cells compared with peripheral blood of nonpregnant controls. In contrast, decidual lymphocyte isolates contain high percentages of CD4(+)CD25(bright) T cells with a regulatory phenotype that is able to down-regulate fetus-specific and fetus-nonspecific immune responses. These data suggest a preferential recruitment of fetus-specific regulatory T cells from maternal peripheral blood to the fetal-maternal interface, where they may contribute to the local regulation of fetus-specific responses.

After discontinuation of calcineurin inhibitors, tapering of mycophenolate mofetil further impairs donor-directed cytotoxicity.

BACKGROUND: Recently, we described a significant decrease in donor-specific cytotoxic T-lymphocyte precursor frequency (CTLpf) after discontinuation of calcineurin inhibitors (CNI), while the proliferative capacity in mixed lymphocyte culture (MLC), and the number of interferon-gamma (IFN-gamma) producing cells (pc) in Elispot remained unchanged. METHODS: We tested T-cell reactivity in CNI free patients with stable renal graft function, on mycophenolate mofetil (MMF) or azathioprine (AZA) plus prednisone, who were tapered to 50% of their MMF or AZA dose. RESULTS: Furthermore, tapering of the MMF or AZA dose resulted in a decrease of donor-reactive CTLpf in all patients with detectable CTLpf. Detectable numbers decreased from a median of 32 to 8 CTLp/10(6) peripheral blood mononuclear cell (PBMC). No effect on third-party reactive CTLpf was found, while the T-cell reactivity to donor and third-party cells as tested in MLC and in IFN-gamma Elispot was not affected either by tapering of immunosuppression. Third-party reactivity was significantly higher than donor-specific reactivity in all tests. A control group showed no changes in any of the in vitro assays. CONCLUSION: Both withdrawal of CNI and tapering of MMF or AZA dose decreases the donor-specific CTLpf. Our data suggest that reduction of immunosuppression results in a specific decrease of donor-directed cytotoxic capacity of immunocompetent cells, while their proliferation and cytokine production capacity remained unchanged. Immunosuppression hinders development of cytotoxic non-responsiveness.

Calcineurin inhibitor withdrawal in stable kidney transplant patients decreases the donor-specific cytotoxic T lymphocyte precursor frequency.

BACKGROUND: In a prospective study, calcineurin inhibitors (CNI) were withdrawn in patients two years after kidney transplantation. We questioned whether stopping CNI had an effect on the donor-specific reactivity, as CNI might hinder immune responses leading to graft acceptance. METHODS: We measured the donor-specific cytotoxic T lymphocyte (CTL) precursor frequency (CTLpf) in 54 patients before and after withdrawal of CNI. In addition, the T-cell reactivity of PBMC to donor and third-party antigens was tested in MLR, and in IFNgamma-Elispot. Reactivity to tetanus toxoid (TET) was studied as well. RESULTS: Donor-specific CTLpf significantly decreased after CNI withdrawal (P=0.0001). In contrast, no difference was observed in third-party reactive CTLpf, donor and third-party reactive MLR and IFNgamma-Elispot. Proliferative responses and the number of IFNgamma-producing cells to TET also decreased after CNI withdrawal. The decrease in CTLpf correlated with the time between the two blood samples (before and after stopping CNI, P=0.05). This decrease was caused by stopping CNI, because there was no correlation between CTLpf and the duration of the CNI treatment after transplantation. Moreover, the percentage of regulatory T cells in the peripheral blood increased after CNI withdrawal. CONCLUSIONS: We report here that after withdrawal of CNI the donor-specific CTLpf decreases. We hypothesize that CNI suppress regulatory mechanisms that have the potential to down-regulate donor-specific CTL responses and reactivity to TET.

Differential effect of calcineurin inhibitors, anti-CD25 antibodies and rapamycin on the induction of FOXP3 in human T cells.

BACKGROUND: The transcription factor FOXP3 has been identified as the molecule associated with the regulatory function of CD25+ T cells. METHODS: To understand the biology of FOXP3+ T cells in allogeneic reactions, we measured FOXP3 mRNA expression levels in allostimulated CD25 cells and CD25 cells and in peripheral blood mononuclear cells (PBMC). The effect of immunosuppressive drugs on FOXP3 expression was studied in mixed lymphocyte reactions (MLR) in the presence and absence of calcineurin inhibitors (CNI), alphaCD25 mAb, and Rapamycin (Rapa), and analyzed in biopsies from cardiac allograft recipients during acute rejection by quantitative (Q)-PCR. RESULTS: FOXP3 mRNA expression was restricted to the CD25 population that inhibited the proliferation of allostimulated CD25 cells. In the MLR FOXP3 was readily induced after allostimulation. Kinetic examination of the MLR showed a 10-20-fold higher FOXP3 mRNA expression level after 5 days of culture. The CNI Cyclosporin and Tacrolimus, and alphaCD25 mAb inhibited in vitro induced FOXP3 gene transcription (range 70%-90%), whereas Rapa did not inhibit the induction. After clinical heart transplantation the highest FOXP3 mRNA expression levels were measured in biopsies during acute rejection (P=0.03). CONCLUSIONS: The high FOXP3 mRNA levels during allogeneic responses in vivo and in vitro suggests that regulatory activities of CD25 T cells or the generation of these cells is an intrinsic part of activation. CNI and alphaCD25 mAb in contrast to Rapa, did interfere with this immunosuppressive counter-mechanism and as a result might have an inhibitory effect to tolerance induction after transplantation.

The granzyme B and interferon-gamma enzyme-linked immunospot assay as alternatives for cytotoxic T-lymphocyte precursor frequency after renal transplantation.

BACKGROUND: The interferon (IFN)-gamma enzyme-linked immunospot (ELISPOT) assay has gained increased popularity as a surrogate marker of cytotoxic T-lymphocyte (CTL) activity. However, the functional activity of CTL might be a more relevant surrogate marker of CTL. Therefore, the authors wondered whether the granzyme B (GrB) ELISPOT assay is a better marker for determining the number of CTL than the IFN-gamma ELISPOT assay. METHOD.: Peripheral blood mononuclear cells (PBMC) from 19 kidney transplant patients were stimulated with donor cells or third-party cells. The authors determined the CTL precursor frequency (CTLpf) and simultaneously measured the number of IFN-gamma- and GrB-producing cells (pc) by ELISPOT assay. RESULTS: In all three different assays, the reactivity to donor cells was significant lower than the reactivity to third-party cells: CTLpf, median: 9 versus 60/10(6) PBMC (P=0.0002); number of IFN-gamma pc: 10 versus 90/10(6) PBMC (P=0.0001); number of GrB pc: 60 versus 205/10(6) PBMC (P=0.05). When the authors compared the CTLpf after third-party stimulation with the corresponding ELISPOT results, they found a positive correlation between the CTLpf and the number of IFN-gamma pc (r(s)=0.47, P=0.05). No correlation was found between the CTLpf and the number of GrB pc (r(s)=0.23, P=0.36). However, when they compared the donor-specific CTLpf with the corresponding ELISPOT results, no correlation with the ELISPOT for IFN-gamma (r(s)=0.10, P=0.69) or GrB (r(s)=-0.24, P=0.34) was found. CONCLUSIONS: The authors feel that the CTLpf, as a measure of the actual endpoint of cytolytic activity and independent of the pathway of killing, remains the "gold standard" for determining donor-specific cytolytic activity after clinical organ transplantation.

Tapering immunosuppression in recipients of living donor kidney transplants.

We have previously suggested that the in vitro donor-specific cytotoxic T-lymphocyte precursor (CTLp) assay can guide us to identify patients in which the immunosuppressive load can be tapered. In a clinical trial we had observed that a low (<10/10(6) PBMC) frequency of these CTLp was predictive for an uneventful rejection-free clinical course in patients that were converted from calcineurin inhibitors to mycophenolate mofetil or azathiopine. In the present prospective study in 81 stable kidney transplant recipients, already converted from calcineurin inhibitors, we measured CTLp frequencies and reduced the immunosuppressive load on a routine basis when CTLp were <10/10(6) PBMC. Donor-specific cytotoxicity could not be measured in 50/81 patients, while their reactivity against third-party lymphocytes was not impaired. These 50 patients were tapered in their immunosuppression. Only in one patient, who had stopped all his medication, was a rejection episode diagnosed. We conclude that in patients with a low donor-specific CTLp frequency it is safe to reduce the immunosuppression.

Formation of donor-specific human leukocyte antigen antibodies after kidney transplantation: correlation with acute rejection and tapering of immunosuppression.

BACKGROUND: Before kidney transplantation, a serological crossmatch is routinely performed between donor and recipient to prevent hyperacute rejection by donor-specific anti-human leukocyte antigen (HLA) antibodies. After transplantation, the presence of these antibodies is not routinely monitored. We wanted to know whether donor-specific anti-HLA antibodies are detectable during acute rejection (AR), before or after reduction of immunosuppression in kidney transplant recipients who were converted from cyclosporine A (CsA) to the less nephrotoxic azathioprine (AZA) or mycophenolate mofetil (MMF) at 1 year after transplantation. METHODS: Plasma samples were collected before transplantation, at several time points after transplantation, and during AR. Antibodies were measured in 29 patients: 5 patients with AR during the first year after transplantation (before conversion), 14 patients with AR after conversion or dose-reduction of AZA or MMF, and a control group of 10 patients without AR during a follow-up of 2 years (1 year before and 1 year after conversion of immunosuppression). Antibodies were measured by complement-dependent cytotoxicity assay, enzyme-linked immunosorbent assay (ELISA), and flow-cytometry in a crossmatch with donor spleen cells. RESULTS: Donor-specific antibodies were not detectable after transplantation in the control group without AR, nor in patients with AR shortly after transplantation during CsA therapy. After conversion from CsA to AZA or MMF, antibodies appeared only in one patient after graft failure followed by transplantectomy and in patients during AR on AZA but not on MMF therapy. CONCLUSION: In this patient group, we could not detect donor-specific antibodies during CsA treatment, not even at the time of AR using three different techniques. Donor-specific antibodies were primarily present during AR in patients converted from CsA to AZA and were not found in the sera from patients converted to MMF.

Transfusion-associated graft vs. host disease after donor-specific leukocyte transfusion before kidney transplantation.

Transfusion-associated graft vs. host disease (TA-GVHD) is a well-known but rare complication that follows infusion of histo-incompatible lymphoid cells, often seen in individuals with impaired cellular immunity. However, we present here a case report of fatal TA-GVHD in a 'presumed' immunocompetent patient after transfusion of a freshly isolated buffycoat from a relative as part of our protocol to prepare the patient for living-related kidney transplantation. To confirm the diagnosis of TA-GVHD, a polymerase chain reaction was used to detect donor cells in various affected tissues. Furthermore, the immune reactivity of the patient against donor and vice versa was tested on samples taken before transfusion using limiting dilution assays. Our patient received a transfusion with blood from a donor who was homozygous at the human leukocyte antigen (HLA) class I loci. Despite incompatibility for HLA class II, infused donor T lymphocytes were not rejected and became engrafted. The patient did not have cytotoxic T lymphocytes to reject the donor cells. DNA polymorphism studies on several organ biopsies confirmed the presence of infiltrating cells of donor origin. This report illustrates the possibility, in the general patient population, of developing TA-GVHD from whole blood transfusion. In the case of pre-transplant blood transfusion, the patient and donor have to be HLA-typed and special care should be taken in the situation of donor homozygosity for HLA class I, even in the presence of HLA class II incompatibility. Protocols in which donor-specific blood or bone marrow transfusions are given in an attempt to modulate the immune system should exclude these combinations.