Fabrice Renner

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 ≤1.8 mg/dl and 32 recipients with a serum creatinine of ≥2.0 mg/dl more than 100 days 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 (P = 0.017). 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 (P < 0.001), low CD3+CD8+CD28−Foxp3+ (P = 0.002), CD3+CD4+DR+ (P = 0.002), CD3+CD8+DR+ T (P = 0.005) and CD19+ B PBL (P = 0.005), and low lineage−HLA‐DR+CD11c+CD123− DC1 (P = 0.006). 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.

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.

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, n = 44) up to 1 year 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 (4 months, 1 year; P < 0.0005) which was associated with a decreased CD28 expression (1 year, P = 0.02) and CD4 cell interleukin 2 (IL‐2) response (4 months, P < 0.0005). 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.

The Risk of Polyomavirus-Associated Graft Nephropathy Is Increased by a Combined Suppression of CD8 and CD4 Cell-Dependent Immune Effects

Polyomavirus-associated graft nephropathy (PAN) has emerged as a significant risk factor for kidney graft loss. We analyzed intracellular cytokine responses for possible protective versus permissive immunologic effects on BK-virus replication. One hundred five renal transplant patients included in a prospective single-center study were randomized to receive cyclosporine mycophenolate mofetil (MMF) (CM: n = 31), tacrolimus (Tac)/MMF (TM: n = 32) or Tac/MMF with conversion to everolimus (TErl; n = 32). Ten patients were not randomized (NR) due to contraindications to MMF. The immunosuppressive therapy was monitored pre- and posttransplantation at 4, 12, and 24 months using triple fluorescence flow cytometry for intracellular interleukin (Il)-2 Il-4 and interferon (IFN)-γ production in phorbol myristate acetate- and lipopolysaccharide- stimulated lymphocyte cultures. BK viremia screening was performed by reverse-transcriptase polymerase chain reaction testing on days 0, 14, 30, 60, 90, 120, 180, 270, 360, and 720. Seven of 105 (6.7%) patients developed biopsy-proven PAN (CM: n = 1, TM: n = 3, TErl: n = 2, NR: n = 1), among whom 4 lost their grafts (TM: n = 1, TErl: n = 2, NR: n = 1). Twenty-one of 105 (20.0%) patients had documented BK viremia. BK viremia which preceded PAN in all cases, was significantly associated with TM immunosuppression: 4/31 (12.9%) CM: 11/32 (34.4%) TM; 5/32 (15.6%) TErl, and 1/10 (10.0%) NR patients (P = .034). BK-viremic patients showed significantly diminished CD8(+) T-cell Il-2 production at 120 days (P = .011) and 1 year posttransplantation (P = .014) compared with non-BK-viremic patients. Patients with PAN displayed significantly lower CD4(+) T-cell Il-4 responses at 1 and 2 years after transplantation (1 year: P = .007; 2 years: P = .001) with diminished IFN-γ responses at 1 year after transplantation (P = .011). Our analysis showed the incidence of BK viremia to be increased among patients with defective cytotoxic CD8(+) T-cell -dependent immune reactivity. Recipients who progressed from BK viremia to overt PAN showed an additional immunologic defect in CD4(+) T-cell function. Patients on a Tac- plus MMF-based immunosuppression were at higher risk to develop BK viremia.

Increased pretransplantation plasma kynurenine levels do not protect from but predict acute kidney allograft rejection.

Indoleamine 2,3-dioxygenase (IDO), an enzyme expressed in many cell types, catalyses degradation of tryptophan (Trp) to kynurenine (Kyn) and may exert immunosuppressive functions, mediated mainly by kynurenines. Therefore, increased Kyn concentrations would be expected to protect allografts from rejection. We conducted this study to examine whether Kyn has predictive value for kidney graft outcome. End-stage renal disease patients (n = 210) demonstrated an increased Kyn/Trp ratio compared with healthy controls (n = 30). Both Kyn and Trp levels were significantly higher in patients who subsequently developed acute rejection than in patients who did not (p < 0.001 and p < 0.001, respectively). Furthermore, pretransplantation Kyn and Trp plasma concentrations were significantly different in patients who went on to develop acute rejection (high values) or acute tubular necrosis (low values) (p = 0.007 and p = 0.021, respectively). After transplantation Kyn levels decreased. Approximately 3 days before biopsy-confirmed rejection, Kyn was significantly increased in patients with rejection compared with those without rejection (p < 0.001). Contrary to expectation, high Kyn plasma levels before transplantation were not predictive of low rejection risk. Although informative in overall terms, at the present stage, Kyn levels do not allow the concise risk differentiation of individual patients.

Impact of maintenance immunosuppressive regimens – balance between graft protective suppression of immune functions and a near physiological immune response

The Symphony study showed superior 1‐year kidney graft outcome in patients on immunosuppression with tacrolimus/mycophenolate mofetil (Tacr/MMF). To analyze whether differences in clinical outcome between maintenance regimens may be explained by their impact on clinically relevant immune parameters, we assessed CD4 helper activity, immunoglobulin‐secreting cell (ISC) formation, neopterin, sCD30, and intracellular cytokine production in a prospective study in 77 renal transplant recipients treated with cyclosporine A/azathioprine (CsA/Aza), CsA/MMF, Tacr/Aza or Tacr/MMF at 2 years post‐transplant. Tacr‐ compared with CsA‐based immunosuppression was independently associated with increased IL‐2 (P < 0.0001, CD4 cells; P = 0.014, CD8 cells) and CD4 cell IL‐4 responses (P = 0.046; stepwise logistic regression) resulting in physiological responses in Tacr/Aza patients as compared with 25 healthy controls. MMF versus Aza treatment was proven to be an independent variable associated with suppression of CD4 cell IL‐10 responses (P = 0.008), B‐cell IL‐6R expression (P < 0.0001) and ISC formation [P = 0.020, staphylococcus cowan strain I (SAC I); P = 0.021, pokeweed mitogen (PWM)]. Our data suggest that Tacr/MMF had the most effective impact on graft protective Th2 responses (enhanced CD4 cell IL‐4 by Tacr, decreased CD4 cell IL‐10 responses by MMF) and suppression of B‐cell functions (MMF), whereas Tacr/Aza was associated with physiological IL‐2 and IL‐4 and stronger humoral responses which may reduce the risk of infectious disease complications.

One hundred six live kidney donors in a single German transplantation center: renal, physical, and psychological follow-up.

OBJECTIVE The objective of this study was to analyze the psychological and physical status as well as renal outcomes of 106 live kidney donors between 1993 and 2003. METHODS We performed general and nephrological examinations, including measurements of creatinine clearance (ClCr), proteinuria, and 24-hour blood pressure monitoring. We evaluated the psychological and general health situation using the standardized SF-36 questionnaire. RESULTS We evaluated 69/106 (65%) live kidney donors at 5.3 ± 0.4 years after donation. The reason for the 37 drop-outs were unknown current address (n = 21), refusal of study participation (n = 14), and death due to accident and suicide (n = 2). In the 69 donors renal function was well preserved: serum creatinine 1.3 ± 0.0 mg/dL; ClCr 81 ± 2 mL/min; postdonation to predonation ClCr ratio 0.73 ± 0.02; and proteinuria 104 ± 11 mg/d. None of the donors experienced renal failure, although 36/69 (52%) patients have developed de novo hypertension. Compared with normotensive donors, the hypertensive subgroup was significantly older at the time of donation (50.7 ± 1.4 vs 46.4 ± 1.6 years; P = .010) and had a longer interval since donation (6.4 ± 0.2 vs 3.9 ± 0.1 years; P = .001). SF-36 questionnaire results in live kidney donors showed higher scores regarding physical (54.3 ± 0.8 vs 49.3 ± 0.1; P = .048) and psychological health (53.8 ± 0.6 vs 50.7 ± 0.1; P = .043) compared with the average German population. CONCLUSION Our cohort of live kidney donors showed good renal outcomes and superior SF-36 scores in both physical and psychological health compared with the German population. The risk of de novo hypertension increased with age and time after donation. Blood pressure screening should be regularly performed especially in the long term after donation.

Association of pretransplant soluble glycoprotein 130 (sgp130) plasma levels and posttransplant acute tubular necrosis in renal transplant recipients.

Objective. Previously, we reported that high pretransplant sIL-6R plasma levels are associated with posttransplant acute tubular necrosis (ATN). In this study, we examined associations of pretransplant plasma levels of sgp130 with ATN. Patients and Methods. Pretransplant serum creatinine (Cr), plasma neopterin, and sgp130 levels were studied in 105 first renal transplant recipients who received grafts from deceased donors. Although 57 patients had immediate and sustained graft function, ATN was diagnosed in 30 patients within the first 11.3±7.8 posttransplant days and acute rejection in 18 patients during the first 27.1±27.6 days. Results. Pretransplant serum Cr and plasma neopterin were similar in the three patient groups. Pretransplant sgp130 plasma levels, however, were significantly lower in patients with ATN than in patients with immediate graft function (P=0.004) or acute rejection (P=0.009). Multivariable logistic regression analysis for ATN showed an odds ratio of 4.3 of patients with pretransplant sgp130 less than or equal to 250 pg/mL with posttransplant ATN (P=0.006). Conclusion. Patients at risk of ATN showed immediately before transplantation low anti-inflammatory sgp130, suggesting a contribution of the IL-6 cytokine family to the development of ATN. It might be useful to measure IL-6 family plasma levels pretransplant to identify patients who are at an increased risk of developing ATN.