Rolf Weimer

Post‐Transplant sCD30 and Neopterin as Predictors of Chronic Allograft Nephropathy: Impact of Different Immunosuppressive Regimens

Immunological monitoring for chronic allograft nephropathy (CAN) is of great potential interest. We assessed serum soluble CD30 (sCD30) together with in vitro Th2‐type responses (IL‐4, IL‐10, CD4 helper activity) and neopterin in a prospective study of 84 renal transplant recipients with 2‐year follow‐up. Patients were randomized to CsA/Aza, CsA/MMF and Tacr/Aza, respectively, to analyze the effect of immunosuppression on posttransplant sCD30 and neopterin. ATG induction and acute rejections did not alter sCD30 levels whereas CMV disease was associated with transient upregulation of sCD30 (p = 0.003 at 4 months) and sustained upregulation of neopterin (corrected for graft function (Neo/CR) p = 0.005 at 2 years). Tacr versus CsA treatment proved to be an independent variable associated with downregulation of 1‐year sCD30, which was positively related to Neo/CR (p = 0.007 and 0.01, respectively; logistic regression). Importantly, increased 1‐year sCD30 and Neo/CR were associated with decreased glomerular filtration rate at 2 years (p = 0.02 and p < 0.0005, respectively) and evidence of CAN (p < 0.0005). High 1‐year sCD30 could not be attributed to enhanced Th2‐type responses and was not associated with HLA antibody formation. Our data suggest that elevated sCD30 and neopterin predict graft deterioration by CAN. Tacr effectively downregulates these responses and might be of advantage in patients with elevated sCD30 or neopterin.

Observational support for an immunoregulatory role of CD3+CD4+CD25+IFN‐γ+ blood lymphocytes in kidney transplant recipients with good long‐term graft outcome

There is evidence that interferon‐gamma (IFN‐γ)‐dependent interactions of dendritic cell (DC), T regulatory (Treg), and T suppressor (Ts) subpopulations contribute to allograft acceptance. We measured DC subsets, CD3+CD4+CD25+ (Treg phenotype) and CD3+CD8+CD28− (Ts phenotype) peripheral blood lymphocytes (PBL) expressing Foxp3, Th1 or Th2 cytokines, peripheral T‐ and B‐cell counts, and plasma cytokines in 33 kidney transplant recipients with a serum creatinine of ≤1.8u2003mg/dl and 32 recipients with a serum creatinine of ≥2.0u2003mg/dl more than 100u2003days post‐transplant. Cell subsets were measured in whole blood using four‐color flow cytometry. Patients with increased creatinine had less frequently detectable CD3+CD4+CD25+IFN‐γ+ PBL than patients with good graft function (Pu2003=u20030.017). In patients with good graft function, CD3+CD4+CD25+IFN‐γ+ PBL were associated with high Foxp3+, IL‐2+, IL‐12+, IL‐4+, and IL‐10+ CD3+CD4+CD25+ T PBL (Pu2003<u20030.001), low CD3+CD8+CD28−Foxp3+ (Pu2003=u20030.002), CD3+CD4+DR+ (Pu2003=u20030.002), CD3+CD8+DR+ T (Pu2003=u20030.005) and CD19+ B PBL (Pu2003=u20030.005), and low lineage−HLA‐DR+CD11c+CD123− DC1 (Pu2003=u20030.006). Patients with impaired graft function did not show these associations. Additional flow cytometric analysis confirmed strong co‐expression of IFN‐γ and Foxp3 by CD4+CD25+ PBL particularly in patients with good graft function. Our data support an immunoregulatory role of CD3+CD4+CD25+Foxp3+IFN‐γ+ cells in a subgroup of transplant recipients with good graft acceptance.

Pretransplant CD4 helper function and interleukin 10 response predict risk of acute kidney graft rejection.

In a prospective study of 80 patients, we investigated the association of acute kidney graft rejection with pretransplant T helper/suppressor activity, B-cell responses, and in vitro cytokine secretion. Patients CD4+ or CD8+ T cells were cocultured with control B cells and pokeweed mitogen for 6 days. SAC I was used for T cell- and monocyte-independent B-cell stimulation and pokeweed mitogen was used for T cell-dependent B-cell stimulation. B-cell differentiation was assessed in a reverse hemolytic plaque assay. Cytokine responses of T cells (interleukin [IL]-2, IL-10, gamma-interferon) and B cells/monocytes (IL-6, IL-8, tumor necrosis factor-alpha, granulocyte-macrophage colony-stimulating factor) were determined in culture supernatants using ELISA. Subsets of CD4+ T cells, CD8+ T cells, and B cells were assessed by flow cytometry. None of 12 patients with pretransplant CD4 helper defects (CD4 helper activity < 10%) had acute rejection episodes, in contrast to 32 of 68 (47%) patients with normal pretransplant CD4 helper function (P = 0.001). Patients with pretransplant CD4 helper defects also had better 1-year graft function than patients without CD4 helper defects (serum creatinine 1.2 +/- 0.1 mg/dl and 1.7 +/- 0.1 mg/dl, respectively, P < 0.05). Pretransplant IL-10 responses were significantly associated with the occurrence of acute rejection episodes (P = 0.001) and impaired 1-year graft function (P < 0.001). All 14 patients with low pretransplant IL-10 responses (< 100 pg/ml) had 1-year serum creatinine values of < 1.5 mg/dl. Pretransplant B-cell defects and B cell/monocyte-derived cytokine secretion were not related to the incidence of graft rejection or infectious complications. Pretransplant CD8 suppressor-effector (CD11b+), cell counts were significantly associated with the occurrence of infections (P < 0.05). These results show that pretransplant CD4 helper defects and low IL-10 responses predict a low risk of graft rejection, whereas Th1 (IL-2, gamma-interferon) and B-cell/monocyte responses are not of predictive value.

Pre-transplant Th1 and post-transplant Th2 cytokine patterns are associated with early acute rejection in renal transplant recipients

Abstract:u2002 In this retrospective study, we tried to define pre‐ and post‐transplant immunological parameters that identify patients at risk for early acute rejection. Lymphocyte subpopulations and plasma levels of cytokines and neopterin were determined pre‐ and post‐transplant in 32 renal transplant recipients with biopsy‐proven early acute graft rejection. Recipients without early acute rejection served as controls. High pre‐transplant interferon‐γ (IFN‐γ) plasma levels (pu200a=u200a0.006), consistently high levels of neopterin early post‐transplant (pu200a=u200a0.008), a post‐transplant switch from a Th1 to a Th2 cytokine pattern with decreasing IFN‐γ (pu2003=u20030.02), low CD8+ lymphocyte counts (pu200a=u200a0.006) and consistently high CD19+ B lymphocyte counts were associated with acute rejection. Our data suggest that patients with a pre‐transplant Th1 and an early post‐transplant Th2 cytokine pattern are pre‐disposed for early acute rejection.

Posttransplant sCD30 as a Predictor of Kidney Graft Outcome

Background. Reliable markers for assessing the biological effect of immunosuppressive drugs and identification of transplant recipients at risk of developing rejection are not available. Methods. In a prospective multicenter study, we investigated whether posttransplant measurement of the T-cell activation marker soluble CD30 (sCD30) can be used for estimating the risk of graft loss in kidney transplant recipients. Pre- and posttransplant sera of 2322 adult deceased-donor kidney recipients were tested for serum sCD30 content using a commercial enzyme-linked immunosorbent assay. Results. sCD30 decreased posttransplant and reached a nadir on day 30. Patients with a high sCD30 of more than or equal to 40 U/mL on day 30 showed a subsequent graft survival rate after 3 years of 78.3±4.1%, significantly lower than the 90.3±1.0% rate in recipients with a low sCD30 on day 30 of less than 40 U/mL (log-rank P<0.001; Cox hazard ratio 2.02, P<0.001). Although an association was found between pre- and posttransplant sCD30 levels, patients with high sCD30 on posttransplant day 30 demonstrated significantly lower 3-year graft survival irrespective of the pretransplant level. Conclusions. Our data suggest that posttransplant measurement of sCD30 on day 30 is a predictor of subsequent graft loss in kidney transplant recipients and that sCD30 may potentially serve as an indicator for adjustment of immunosuppressive medication.

Dendritic cell deficiency in the blood of kidney transplant patients on long-term immunosuppression: results of a prospective matched-cohort study.

Evidence from in vitro studies suggests that immunosuppressive drugs interfere with key functions of dendritic cells (DCs), but the in vivo relevance of these findings is elusive. We prospectively analyzed the major DC precursor subsets in the blood of kidney transplant recipients on long‐term immunosuppression (≥1 year). A total of 87 patients were compared to 87 age‐ and sex‐matched controls. Total DC numbers and the precursor subsets, myeloid type 1 DCs, myeloid type 2 DCs (mDC1, mDC2) and plasmacytoid DCs (pDCs) were identified by four color flow cytometry. Long‐term immunosuppression was associated with significant reduction of all major DC subsets in comparison to healthy controls (mDC1 p < 0.001; mDC2 p < 0.0001; two‐tailed Mann‐Whitney U‐test) with the strongest negative impact on pDCs (p < 0.00001). In contrast, total leukocyte numbers were not significantly affected. Analysis of the relative impact of different agents revealed a significant impact of prednisolone on pDCs (p = 0.009) and mDCs2 (p = 0.006). The functional relevance of pDC deficiency was confirmed independently by Interferon‐alpha analysis after Toll‐like receptor 7 (p ≤ 0.001) and 9 (p < 0.05) stimulation. These results indicate for the first time a profound negative impact of long‐term immunosuppression on major DC subsets in kidney transplant recipients. DC deficiency may have important implications with respect to viral infections and tumor development.

Association of Kidney Graft Loss With De Novo Produced Donor-Specific and Non-Donor-Specific HLA Antibodies Detected by Single Antigen Testing

Background The association of donor-specific HLA antibodies (DSA) with kidney graft failure has been addressed previously; however, the majority of studies were based on small numbers of patients with graft failure. Methods We investigated 83 patients with failed kidney transplants for a possible association of de novo development and persistence or loss of pre-existing DSA with graft failure. Single Antigen Bead assay-detected DSA and non-DSA antibodies were compared between patients with graft loss and matched controls with functioning grafts. Results The incidence of weak de novo DSA or non-DSA at a mean fluorescence intensity of 500 or higher was higher in the graft loss than in the nonrejector group (76% vs 40%, P < 0.001). Because of the low number of patients developing de novo DSA, the DSA results did not reach statistical significance (only 22% of patients with graft loss developed de novo DSA). However, at all cutoffs, there was a significantly higher rate of graft loss in patients with de novo non-DSA. The incidence of strong pretransplant DSA that persist after transplantation was higher in the graft loss group (10% vs 1%, P = 0.034). When C1q-binding ability in sera of rejectors and nonrejectors with posttransplant de novo or persistent DSA was compared, none of the nonrejectors demonstrated C1q positivity, whereas 43% of patients with graft loss showed C1q-positive antibodies, although not necessarily donor-specific (P < 0.001). Conclusions Our data show that the posttransplant presence of persisting or de novo HLA antibodies, especially if C1q binding, is associated with graft loss, even if the antibodies are not specific for mismatched donor HLA.

Mycophenolate mofetil-based immunosuppression and cytokine genotypes: effects on monokine secretion and antigen presentation in long-term renal transplant recipients.

Background. It has been suggested that increased monocyte responses might play a role in chronic allograft rejection. Methods. We investigated in vitro monokine responses in 112 patients with long-term stable kidney graft function (ST patients; n=80, non-mycophenolate mofetil [MMF]; n=32, MMF) and 25 patients with chronic renal transplant rejection (CR patients; non-MMF). Interleukin 10 and tumor necrosis factor (TNF)-&agr; promoter gene polymorphisms were tested by polymerase chain reaction and sequence-specific primers; antigen-presenting capacity (AC) of monocytes was tested by incubation with staphylococcal superantigens (SEA, SEE, SED). Results. Although non-MMF–based immunosuppression in ST patients did not result in compromised AC or lipopolysaccharide (LPS)-stimulated monokine responses compared with healthy controls, we found MMF therapy to be associated with significantly reduced TNF-R1 expression on monocytes (P <0.001), suppressed AC (P <0.02, SED), and suppressed LPS-stimulated IL-1&bgr;, IL-10, and TNF-&agr; secretion (P <0.01). Coinciding with a significantly higher steroid dosage in CR patients, IL-6 receptor and TNF-R1 expression on monocytes were down-regulated (P ≤0.02) and AC was suppressed in CR compared with ST (non-MMF) patients (P <0.01, SED; P <0.05, SEE). However, LPS-stimulated monokine secretion was not decreased or even enhanced (IL-6, granulocyte-macrophage colony-stimulating factor [GM-CSF]; P <0.05). Enhanced in vitro IL-10 responses (>500 pg/mL) were found predominantly in non-MMF–treated patients with the IL-10 genotype GCC (GCC: 23/62 [37%], non-GCC: 2/27 [7%], P <0.005; GCC and non-MMF: 22/47 [47%], GCC and MMF: 1/15 [7%], P <0.005]. Conclusion. Steroids and azathioprine did not sufficiently suppress monokine responses, whereas MMF treatment might inhibit chronic graft rejection because of suppression of TNF-R1 expression and vigorous inhibition of monokine secretion. MMF treatment may especially be indicated in patients with the IL-10 “high-producer” genotype GCC.

ATG induction therapy: long‐term effects on Th1 but not on Th2 responses

Antithymocyte globulin (ATG) induction therapy is associated with an increased long‐term risk of infection‐ and cancer‐related death. To analyze long‐term effects of ATG induction on lymphocyte function, we prospectively assessed CD4 helper function, B‐cell/monocyte and cytokine responses in 84 renal transplant recipients (ATG, nu2003=u200344) up to 1u2003year post‐transplant. A PWM‐driven allogeneic coculture system was used to assess helper function of CD4+ T cells and T‐cell‐dependent B‐cell responses. SAC I was used for T‐cell‐independent stimulation of B‐cell cultures. In vitro cytokine secretion and serum soluble CD30 (sCD30) were determined by enzyme‐linked immunosorbent assay (ELISA). ATG induced a persistent decrease of peripheral blood lymphocyte counts compared with non‐ATG treatment because of a predominant decrease of CD4+ T cells (4u2003months, 1u2003year; Pu2003<u20030.0005) which was associated with a decreased CD28 expression (1u2003year, Pu2003=u20030.02) and CD4 cell interleukin 2 (IL‐2) response (4u2003months, Pu2003<u20030.0005). However, Th2 responses (CD4 help, CD4 cell IL‐4 and IL‐10 responses, sCD30), which proved to be predictive of graft outcome, were not affected, and neither was the secretion of the lymphoma growth factors IL‐6 and IL‐10 by B cells and monocytes. Our data show that ATG induction therapy in immunological high‐risk patients induces a profound long‐term decrease in cell counts and Th1 but not Th2 responses of CD4+ T cells which may explain long‐term effects on infection and post‐transplant lymphoproliferative disease (PTLD) incidence because of inadequate T‐cell control.

Evaluation of T-cell receptor repertoires in patients with long-term renal allograft survival

The mechanisms underlying long‐term acceptance of kidney allografts in humans under minimal or no maintenance immunosuppression are poorly understood. We analyzed the T‐cell receptor (TCR) repertoires in circulating T cells of patients with long‐term (≥9 years) renal allograft survival with (LTS‐IS) and without immunosuppression (LTS‐NoIS). T cells of LTS patients exhibited strongly altered TCR Vß usage, including an increased frequency of oligoclonality and a decreased frequency of polyclonality. All 3 LTS‐NoIS and 12 of 16 LTS‐IS patients demonstrated oligoclonality in at least three or more TCR Vß families, and the frequency of oligoclonality in these patients was significantly higher as compared to patients with well‐functioning grafts at 3 years (p < 0.005 both), an uncomplicated course during the first year (p < 0.0001, both), acute rejection (p < 0.0001, both), chronic allograft nephropathy at 7 (p < 0.0001, both) or 13 years (p < 0.0001, both), dialysis patients (p < 0.0001, both) or healthy controls (p < 0.0001, both). In contrast to LTS patients, all other studied patient groups exhibited a polyclonal TCR repertoire. Our data indicate that TCR alteration is a common feature of long‐term allograft outcome, which might be explained by clonal deletion, exhaustion of alloreactive T cells or predominant expression of particular T‐cell subpopulations, such as regulatory T cells.