A NOVel ELISPOT assay to quantify HLA-specific B cells in HLA-immunized individuals.

Quantification of the humoral alloimmune response is generally achieved by measuring serum HLA antibodies, which provides no information about the cells involved in the humoral immune response. Therefore, we have developed an HLA-specific B-cell ELISPOT assay allowing for quantification of B cells producing HLA antibodies. We used recombinant HLA monomers as target in the ELISPOT assay. Validation was performed with human B-cell hybridomas producing HLA antibodies. Subsequently, we quantified B cells producing HLA antibodies in HLA-immunized individuals, non-HLA-immunized individuals and transplant patients with serum HLA antibodies. B-cell hybridomas exclusively formed spots against HLA molecules of corresponding specificity with the sensitivity similar to that found in total IgG ELISPOT assays. HLA-immunized healthy individuals showed up to 182 HLA-specific B cells per million total B cells while nonimmunized individuals had none. Patients who were immunized by an HLA-A2-mismatched graft had up to 143 HLA-A2-specific B cells per million total B cells. In conclusion, we have developed and validated a highly specific and sensitive HLA-specific B-cell ELISPOT assay, which needs further validation in a larger series of transplant patients. This technique constitutes a new tool for quantifying humoral immune responses.

A multiplex bead array analysis to monitor donor-specific cytokine responses after withdrawal of immunosuppression in HLA-identical living related kidney transplant patients.

Immune reactivity after HLA-identical living related (LR) kidney transplantation can be caused by minor histocompatibility antigen and non-HLA antigen mismatches between donor and recipient. In our center, HLA-identical LR kidney transplant recipients receive azathioprine (AZA) or mycophenolate mofetil (MMF) in combination with corticosteroids for 1 year after transplantation. Thereafter, AZA or MMF was withdrawn, and the patients were treated with steroid monotherapy as maintenance therapy. We questioned whether withdrawal of AZA or MMF affected the donor-specific lymphocyte proliferation and cytokine production. Donor and third-party T-cell reactivities were determined by mixed lymphocyte reactions and by cytokine production using multiplex bead array technique. The donor and third-party proliferative capacities were not affected after withdrawal of AZA or MMF. Thirteen of 17 cytokines were detected by the multiplex bead array technique. No differences were observed after third-party induced cytokine production after withdrawal of AZA or MMF. However, production of donor-specific interferon-gamma and macrophage inflammatory protein-1beta increased after discontinuation of AZA or MMF, but no clinically relevant acute rejection was observed. In conclusion, after HLA-identical LR kidney transplantation, donor-specific cytokine responses can be found when AZA or MMF therapy is discontinued. The clinical relevance of this phenomenon is still not evident.

Immune monitoring after kidney transplantation.

During the last decennium, research in the field of organ transplantation changed from studying mechanisms of acute rejection shortly after transplantation to understanding why patients benefit from their grafts only for a limited time, because of irreversible chronic rejection and serious side effects of immunosuppression. To avoid side effects, tapering or even withdrawal of immunosuppression in transplant recipients is warranted, provided this is not accompanied with graft loss. Noninvasive cell mediated immune tests could be helpful to identify transplant recipients in whom the immunosuppressive load can be safely reduced, and to identify patients at risk for chronic rejection. In the present report, we describe cellular assays to determine donor-specific responses in peripheral blood mononuclear cells from transplant recipients taken before transplantation and during tapering or withdrawal of immunosuppression.

Donor-reactive cytokine production after HLA-identical living related kidney transplantation: a protein-array analysis.

In the present pilot study, we investigated which proteins are produced after donor stimulation of peripheral blood mononuclear cells from recipients of HLA-identical living related kidney transplant. We used a protein-array analysis to determine cytokines, chemokines, and growth factors in supernatant from donor-stimulated mixed lymphocyte reaction cultures. Autologous cultures were considered to be negative controls. In 38 out of 42 proteins (90%), the donor response was higher compared with the autologous response. Therefore, we concluded that even after HLA-identical living related kidney transplantation we could measure a donor-reactive response, which we assumed was directed toward minor histocompatibility or non-HLA antigens.

The direct and indirect allogeneic presentation pathway during acute rejection after human cardiac transplantation.

Alloreactive T cells may be activated via a direct or an indirect antigen presentation pathway. We questioned whether the frequency of interferon (IFN)-gamma producing cells determined by enzyme-linked immunospot (ELISPOT) assay is an effective tool to monitor the direct and/or indirect presentation pathway. Secondly, we wondered whether early and late acute rejection (AR) are associated with both pathways. Before (n = 15), during (n = 18) and after (n = 16) a period of AR, peripheral blood mononuclear cell (PBMC) samples were tested from 13 heart transplant recipients. The direct presentation pathway was always present. The number of IFN-gamma producing cells reactive to this pathway increased significantly (P = 0.04) during AR and the number decreased (P = 0.005) after AR therapy. In contrast, the indirect allogeneic presentation pathway was present in only eight of 18 AR samples. When the indirect presentation pathway was detectable, it increased significantly during AR. Five of eight of these AR occurred more than 6 months after transplantation. The ELISPOT assay, enumerating alloreactive IFN-gamma producing cells, is a valuable tool to determine the reactivity via both the direct and the indirect presentation pathway. The direct presentation pathway always plays a role in AR, while the indirect pathway contributes especially to late AR.

Granzyme B ELISPOT assay determines the cytotoxic T lymphocyte precursor frequency after HLA-identical living-related kidney transplantation.

A major goal in organ transplantation is to define the optimal immunosuppressive dose. Recently, we demonstrated that the frequency of cytotoxic T lymphocytes (CTLpf) identifies patients in whom the immunosuppressive load can be safely reduced. However, in peripheral blood mononuclear cells (PBMC) from HLA-identical living-related kidney transplant patients, no donor-specific CTLpf can be measured. The determination of the functional activity of cytotoxic T lymphocytes (CTLs) could be an alternative method for the CTLpf. Granzyme B (GrB) is present in the granules of CTLs and is involved in the direct lethal hit of donor target cells. Therefore, we wondered whether the GrB ELISPOT assay is an alternative method to determine the activity of CTLs after HLA-identical living-related kidney transplantation. We measured the number of GrB producing cells (pc) against donor PBMC and third-party PBMC in PBMC from HLA-identical patients who were reduced in their immunosuppression from 100% to 50% azathioprine with 5 to 10 mg/day prednisone. We found low numbers of GrB pc before reduction of immunosuppression, as only 20% of the patients' PBMC responded to donor cells, whereas 57% of the patients' PBMC responded to donor cells after reduction of immunosuppression. After third-party stimulation, the number of GrB pc increased after tapering the immunosuppressive load (P = .03). Our results demonstrate that the GrB ELISPOT assay might be used as an alternative for the CTLpf after HLA-identical living-related kidney transplantation.

Reduction of immunosuppressive load in renal transplant recipients with a low donor-specific cytotoxic T-lymphocyte precursor frequency is safe.

BACKGROUND: Tapering of immunosuppressive medication is indicated to prevent long-term side effects. Recently, we have shown that renal transplant recipients can safely be converted from calcineurin inhibitors to MMF or AZA when their donor-specific cytotoxic T-lymphocyte precursor frequencies (CTLpf) are below 10/10(6) PBMC. We wondered whether a low CTLpf also had predictive value when immunosuppressive medication was reduced in patients only on MMF or AZA and steroid medication. METHODS: Renal transplant recipients with stable renal function at least 2 years after transplantation and with low (<10/10(6) PBMC) CTLpf were included. Their MMF or AZA dose was reduced to 75% and to 50% of the original dose at 4 months and 8 months after inclusion. Endpoint of the study was 12 months after inclusion or developing acute rejection. RESULTS: Forty-five patients have reached the 1-year follow up endpoint. Their median time after transplantation was 4.2 years (range 2.0-15.5 years). Acute rejection was seen in one patient only (who had discontinued all his medication). CONCLUSION: In patients with low CTLpf long after kidney transplantation, a 50% reduction of immunosuppression is safe and further decreasing their immunosuppressive load is the obvious next step.

Down-regulated donor-specific T-cell reactivity during successful tapering of immunosuppression after kidney transplantation.

Stable cadaveric renal transplant patients were routinely converted from cyclosporin A (CsA) to either azathioprine (AZA) or mycophenolate mofetil (MMF) 1 year after transplantation to reduce the side effects of long-term immunosuppressive therapy. Thereafter, the AZA and MMF dose was gradually tapered to 50% at 2 years after transplantation. We questioned whether a reduction of immunosuppressive treatment results in a rise of donor-specific T-cell reactivity. Before transplantation (no immunosuppression), 1 year (high dose immunosuppression) and 2 years (low dose immunosuppression) after transplantation, the T-cell reactivity of peripheral blood mononuclear cells (PBMC) against donor and third-party spleen cells was tested in mixed lymphocyte cultures (MLC) and against tetanus toxoid (TET) to test the general immune response. We also measured the frequency of donor and third-party reactive helper (HTLpf) and cytotoxic (CTLpf) T-lymphocyte precursors in a limiting dilution assay. Donor-specific responses, calculated by relative responses (RR = donor/third-party reactivity), were determined. Comparing responses after transplantation during high dose immunosuppression with responses before transplantation (no immmunosuppression), the donor-specific MLC-RR (P = 0.04), HTLp-RR (P = 0.04) and CTLp-RR (P = 0.09) decreased, while the TET-reactivity did not change. Comparing the responses during low dose with high dose immunosuppression, no donor- specific differences were found in the MLC-RR, HTLp-RR and CTLp-RR, although TET-reactivity increased considerably (P = 0.0005). We observed a reduction in donor-specific T-cell reactivity in stable patients after renal transplantation during in vivo high dose immunosuppression. Tapering of the immunosuppressive load had no rebound effect on the donor-specific reactivity, while it allowed recovery of the response to nominal antigens.

Selective attention to a facial feature with and without facial context: an ERP-study.

The present experiment addressed the question whether selectively attending to a facial feature (mouth shape) would benefit from the presence of a correct facial context. Subjects attended selectively to one of two possible mouth shapes belonging to photographs of a face with a happy or sad expression, respectively. These mouths were presented randomly either in isolation, embedded in the original photos, or in an exchanged facial context. The ERP effect of attending mouth shape was a lateral posterior negativity, anterior positivity with an onset latency of 160-200 ms; this effect was completely unaffected by the type of facial context. When the mouth shape and the facial context conflicted, this resulted in a medial parieto-occipital positivity with an onset latency of 180 ms, independent of the relevance of the mouth shape. Finally, there was a late (onset at approx. 400 ms) expression (happy vs. sad) effect, which was strongly lateralized to the right posterior hemisphere and was most prominent for attended stimuli in the correct facial context. For the isolated mouth stimuli, a similarly distributed expression effect was observed at an earlier latency range (180-240 ms). These data suggest the existence of separate, independent and neuroanatomically segregated processors engaged in the selective processing of facial features and the detection of contextual congruence and emotional expression of face stimuli. The data do not support that early selective attention processes benefit from top-down constraints provided by the correct facial context.

Pretransplant donor-specific helper T cell reactivity as a tool for tailoring the individual need for immunosuppression.

BACKGROUND: A reliable immunological assay for quantification of donor-specific alloreactivity to identify patients at risk for future allograft rejection would be a helpful tool in organ transplantation. Therefore, we questioned whether the T cell reactivity in patients measured before transplantation was predictive for the occurrence of acute rejection during the first year after kidney transplantation. METHODS: The pretransplant T cell reactivity of peripheral blood mononuclear cells to donor and third-party antigens was tested in mixed lymphocyte cultures, and to tetanus toxoid. In addition, we measured the frequency of donor and third-party reactive helper T lymphocyte precursor and cytotoxic T lymphocyte precursors using limiting dilution analysis. RESULTS: Patients who experienced acute rejection had significantly higher donor-specific mixed lymphocyte cultures responses (n=38; median stimulation index): 113 vs. 15, P=0.005) and helper T lymphocyte precursor frequency (n=37; median 194/106 vs. 62/106, P=0.009) measured before transplantation compared to patients without acute rejection. All patients with a low mixed lymphocyte culture response (stimulation index

Mycophenolic acid trough levels after kidney transplantation in a cyclosporine-free protocol.

Twenty-seven stable kidney transplant recipients treated with cyclosporine and prednisone were converted to mycophenolate mofetil (MMF) and prednisone 1 year after transplantation. After conversion the patients were treated with a standard daily dose of 1 g MMF b.i.d. and 10 mg prednisone for 4 months. Thereafter, two MMF dose reductions were performed with a 4-month interval. Mycophenolic acid (MPA) trough levels were measured at regular intervals. A relation was found between MPA trough levels and MMF dose. The median MPA trough level for patients treated with 1 g MMF b.i.d. was 4.3 microg/ml (0.95-15.5) and 3.0 microg/ml (0.73-7.8) for patients treated with 750 mg b.i.d. (P = 0.0002). The MPA trough levels further decreased from 3.0 to 2.3 microg/ml (0.6-6.63) in patients treated with 500 mg MMF b.i.d. (P = 0.01). Dose reduction of MMF from 1 g to 750 mg b.i.d. could be performed without acute rejections. A further dose reduction to 500 mg b.i.d. elicited 3 rejections. Patients experiencing an acute rejection had a median MPA trough level of 2.3 microg/ml (1.26-3.38) compared to 3.8 microg/ml (1.48-6.52) in patients without an acute rejection (P = 0.25). We conclude that there is a significant relation between MPA trough levels and MMF dose. MPA trough levels were not predictive of rejection in the present study.

Donor-specific T-cell reactivity identifies kidney transplant patients in whom immunosuppressive therapy can be safely reduced.

BACKGROUND: To reduce the side effects of long-term immunosuppressive therapy, stable renal transplant patients were routinely converted from cyclosporine to either azathioprine or mycophenolate mofetil. Thereafter, the azathioprine and mycophenolate mofetil dose was reduced to 75% at 4 months and to 50% at 8 months after conversion. We questioned whether the T-cell reactivity before conversion was able to predict which patients could be safely converted and tapered in their immunosuppressive load, while remaining free from acute rejection. METHODS: Before conversion, the T-cell reactivity of peripheral blood mononuclear cells against donor and third-party spleen cells were tested in mixed lymphocyte cultures. We measured the frequency of donor and third-party reactive helper T-lymphocyte (HTLpf) and cytotoxic T-lymphocyte (CTLpf) precursors and their avidity for HLA class I antigens using limiting dilution analysis. Peripheral blood mononuclear cells were also stimulated with tetanus toxoid to test the general immune response. RESULTS: The tetanus toxoid response, reactivity to donor and third-party cells as measured in mixed lymphocyte cultures and HTLpf, and the avidity of cytotoxic T-lymphocyte precursors were not predictive for the development of acute rejection. However, significant differences were found in donor-specific CTLpf before conversion, between patients with and without acute rejection after conversion in immunosuppression. The donor-specific CTLpf was significantly lower in patients without compared to those with acute rejection (P=0.01). Additionally, when no CTLpf was detectable before conversion, acute rejection did not occur after conversion. Acute rejection was only diagnosed in patients with detectable CTLpf before conversion. CONCLUSION: The number of donor-specific cytotoxic T-lymphocytes identifies patients in whom the immunosuppressive load can be safely reduced.

Randomized study on the conversion of treatment with cyclosporine to azathioprine or mycophenolate mofetil followed by dose reduction.

BACKGROUND: The introduction of cyclosporine (CsA) in kidney transplantation has improved early graft survival. However, its long-term use is associated with impairment of renal function and increased cardiovascular risk factors. To avoid CsA-related long-term adverse effects, patients were converted to either azathioprine (AZA) or mycophenolate mofetil (MMF) 1 year after transplantation. METHODS: Between September 1995 and January 1997, 64 stable renal transplant recipients on CsA and prednisone treatment were included in a prospective, randomized study. Patients were randomized for conversion of CsA to 2 mg/kg AZA (n=30) or 1 g of MMF twice daily (n=34). All patients remained on low-dose steroids. To decrease the total immunosuppressive load, a dose reduction in MMF and AZA was performed at 4 and again at 8 months after conversion. Mycophenolic acid trough levels were measured at regular intervals. RESULTS: After conversion, a decrease in serum creatinine was found for both groups: for MMF, 132 to 109 micromol/L (P=0.016); and for AZA, 123 to 112 micromol/L (P<0.0001). After conversion, more acute rejections occurred in the AZA group (11/30) compared to the MMF group (4/34) (P=0.04). Dose reduction of MMF to 500 mg twice daily and of AZA to 1.0 mg/kg elicited three rejections in both groups. The incidence of side effects and infections were similar. CONCLUSION: Discontinuation of CsA spared renal function. In patients converted to MMF significantly less rejections occurred compared to patients converted to AZA. Furthermore, dose reduction of both AZA and MMF is possible in the majority (72%) of the patients.

A longitudinal study of TGF-beta1 protein levels in renal allograft recipients converted from CsA to MMF or AZA.

Cyclosporine (CsA) is thought to enhance transforming growth factor (TGF)-beta1 production in vitro and in vivo and this may have a negative effect on long-term graft survival. Therefore, we studied TGF-beta1, plasma levels in 30 patients before kidney transplantation, after transplantation during CsA treatment and after conversion from CsA to azathioprine (AZA) or mycophenolate mofetil (MMF). We questioned whether TGF-beta1 plasma levels would decrease after the discontinuation of CsA and whether the TGF-beta1 plasma levels did correlate with CsA trough levels and kidney function, measured by serum creatinine levels. TGF-beta1 plasma levels measured 1 yr after transplantation were lower compared to levels measured before transplantation, however not significantly (p = 0.08). After conversion from CsA to MMF or AZA, a slight increase was observed in some patients, but in the total group TGF-beta1 levels remained unaffected. No correlation was found between the TGF-beta1 levels and CsA trough levels nor with creatinine levels. In conclusion, we did not observe higher TGF-beta1 plasma levels in plasma levels of patients receiving CsA treatment compared to blood from the same patients while on AZA or MMF.

Effect of mycophenolate mofetil on erythropoiesis in stable renal transplant patients is correlated with mycophenolic acid trough levels.

BACKGROUND: Both mycophenolate mofetil (MMF) and azathioprine (AZA) are immunosuppressive drugs that inhibit purine synthesis. In theory, MMF selectively inhibits lymphocyte proliferation, while AZA has well-known effects on red blood cells and thrombocytes as well. In renal transplant recipients we replaced CsA therapy by MMF in an attempt to reduce the immunosuppressive load 1 year after kidney transplantation. During this study we observed the effect of MMF on haematological parameters such as haemoglobin (Hb), leukocytes, and thrombocytes. METHODS: One year after kidney transplantation 26 stable patients were converted from cyclosporin A (CsA) to MMF (2 g/day). Thereafter, these patients were tapered twice in their MMF dose from 2 g to 1.5 g (4 months after conversion) and from 1.5 to 1 g (8 months after conversion) per day. The Hb levels, leukocyte and thrombocyte counts, and mycophenolic acid (MPA) trough levels were routinely measured. RESULTS: After conversion from CsA to MMF not only creatinine levels and the number of leukocytes, but also the haemoglobin (Hb) level significantly decreased in 21/26 patients (P=0.0004). In eight patients the Hb level dropped more than 1 mmol/l (=1.61 g/dl). Only in two of eight patients was an explanation for blood loss found. The effect on Hb level did not ameliorate after the first MMF dose reduction to 1.5 g/day. After tapering the MMF dose to 1 g/day, the Hb approached the pre-conversion level. Not only the MMF dose but also the mycophenolic acid (MPA) trough level correlated with the Hb level. CONCLUSIONS: After conversion from CsA to MMF 1 year after kidney transplantation, a decrease in Hb level and leukocyte count was observed. The MPA trough level correlated also with the Hb level. The effect on the Hb level was reversible after dose reduction. This finding suggests that MMF exerts a negative effect on erythropoietic cells.

Mycophenolic acid plasma concentrations in kidney allograft recipients with or without cyclosporin: a cross-sectional study.

BACKGROUND: Combining cyclosporin (CsA) and prednisone with mycophenolate mofetil (MMF) results in a significant reduction in the rate of biopsy-proven acute rejection after kidney transplantation. This is achieved with a standard daily MMF dosage of 2 or 3 g. Whether monitoring of the pharmacologically active metabolite mycophenolic acid (MPA) will lead to improved safety and efficacy is unclear. METHODS: We monitored MPA trough levels in 18 kidney transplant recipients treated with CsA, prednisone, and MMF (63 samples) and in 11 patients (31 samples) treated with prednisone and MMF only, in a cross-sectional study. All patients were at least 3 months after transplantation with stable graft function. All patients were treated with 2 g MMF for at least 3 months and 10 mg prednisone. RESULTS: The MPA trough levels in the CsA-treated patients were significantly lower (P<0.0001; Mann-Whitney) than those in patients on MMF and prednisone only (mean MPA levels 1.98+/-0.12 vs 4.38+/-0.40 mg/l respectively). CONCLUSIONS: Although all patients were treated with an identical MMF dose, a significant difference was found in the MPA trough levels between CsA- vs non-CsA-treated patients. This suggests that CsA influences the MPA trough level. The level at which CsA affects the MPA trough levels is unclear.