Dynamics and epitope specificity of anti-human leukocyte antibodies following renal allograft nephrectomy.

BACKGROUND: A considerable proportion of patients awaiting kidney transplantation is immunized by previous transplantation(s). We investigated how allograft nephrectomy (Nx) and withdrawal of maintenance immunosuppression (WD-MIS) in patients with a failed renal allograft contribute to allosensitization. METHODS: HLA antibodies (HLAabs) were analyzed before and after Nx and/or WD-MIS using a single antigen bead assay. Patients were grouped as follows: (A) Nx and concomitant WD-MIS (n = 28), (B) Nx (n = 14) and (C) WD-MIS (n = 12). In a subgroup of patients, the epitope specificity of HLAabs was determined by adsorption and elution of sera with recombinant single HLA allele-expressing cell lines. RESULTS: Following Nx and/or WD-MIS, HLAabs were detectable in 100, 100 and 92% of patients in Groups A, B and C, respectively. In patients of all groups, de novo donor-specific HLAabs (DSAs) were found. After Nx, an increase in the breadth [percent panel reactive antibody (%PRA)] and mean fluorescence intensity of class I HLAabs was predominant. In contrast, an increase of class II HLAabs prevailed following WD-MIS. Experimental analysis of the epitope specificities revealed that 64% of the class I HLAabs classically denoted as non-DSA were donor epitope-specific HLAabs (DESA). CONCLUSIONS: Both Nx and WD-MIS contribute to alloimmunization with differing patterns concerning class I and II HLAabs. Nx preferentially increased class I HLAabs and most of the observed class I HLAabs were DESA. Considering that class I, but not class II, HLA molecules are constitutively expressed, our results support the hypothesis that the increase of HLAabs following Nx might have been caused by removal of the adsorbing donor tissue (sponge hypothesis).

Complement-fixing antibodies against denatured HLA and MICA antigens are associated with antibody mediated rejection.

BACKGROUND: We have found antibodies against denatured HLA class I antigens in the serum of allograft recipients which were not significantly associated with graft failure. It is unknown whether transplant recipients also have denatured HLA class II and MICA antibodies. The effects of denatured HLA class I, class II, and MICA antibodies on long-term graft outcome were further investigated based on their ability to fix complement c1q. MATERIALS AND METHODS: In this 4-year retrospective cohort study, post-transplant sera from 975 kidney transplant recipients were tested for antibodies against denatured HLA/MICA antigens and these antibodies were further classified based on their ability to fix c1q. RESULTS: Thirty percent of patients had antibodies against denatured HLA class I, II, or MICA antigens. Among them, 8.5% and 21.5% of all patients had c1q-fixing and non c1q-fixing antibodies respectively. There was no significant difference on graft survival between patients with or without antibodies against denatured HLA/MICA. However, when these antibodies were further classified according to their ability to fix c1q, patients with c1q-fixing antibodies had a significantly lower graft survival rate than patients without antibodies or patients with non c1q-fixing antibodies (p=0.008). In 169 patients who lost renal grafts, 44% of them had c1q-fixing antibodies against denatured HLA/MICA antigens, which was significantly higher than that in patients with functioning renal transplants (25%, p<0.0001). C1q-fixing antibodies were more significantly associated with graft failure caused by AMR (72.73%) or mixed AMR/CMR (61.9%) as compared to failure due to CMR (35.3%) or other causes (39.2%) (p=0.026). CONCLUSIONS: Transplant recipients had antibodies against denatured HLA class I, II, and MICA antigens. However, only c1q-fixing antibodies were associated with graft failure which was related to antibody mediated rejection.

Pretransplant virtual PRA and long-term outcomes of kidney transplant recipients.

Virtual panel-reactive antibodies (vPRA) have been implemented to gauge sensitization worldwide. It is unclear how it associates with long-term outcomes, and its correlation with peak (pPRA) or actual (aPRA) has not been studied. We retrospectively reviewed data from 18- to 65-year-old kidney-only transplant patients during 1.1.1996-31.7.2011 in our center. PRAs were calculated based on solid-phase techniques. Of the 726 qualified cases, regardless of the PRA type, sensitized patients (PRA > 5%) had more females and previous transplant. Highly sensitized (HS, PRA > 50%) had longer waiting time, lower transplant rate, less living donor, more delayed graft function, and acute rejection. The conformity between vPRA and pPRA in HS was 75%, 57% between pPRA and aPRA. Forty-three percent (61/142) patients whose pPRA was >5% had no detectable aPRA and maintained similar outcomes as sensitized patients. Multivariate analysis showed consistently lower death-censored graft survival in HS defined by vPRA [HR 2.086 (95% CI 1.078-4.037), P < 0.05] and pPRA [HR 2.139 (95% CI 1.024-4.487), P < 0.05]. Both vPRA and pPRA provided reliable way estimating sensitization and predicting long-term graft survival, while aPRA might underestimate true sensitization. vPRA might be the most objective parameter to gauge sensitization.

The genetic predisposition of natural killer cell to BK virus-associated nephropathy in renal transplant patients.

BK virus (BKV) infection represents a serious complication in renal transplant patients resulting in BKV-associated nephropathy and subsequent allograft loss. Natural killer cells are crucial in the antiviral immune response; however, an understanding of the role of natural killer cells in protection against BKV is limited. To elucidate whether killer-cell immunoglobulin-like receptors and their interaction between donor-/recipient-related ligands have a role in BKV infection, we performed genotyping analysis in 48 kidney transplant recipients with a history of severe BKV infection/BKV-associated nephropathy and 110 recipients with stable renal function and no BKV reactivation. Of interest, we found that telomeric gene content motif was significantly associated with severe course of BKV infection/BKV-associated nephropathy and detected significantly higher percentage of patients with BKV-associated nephropathy carrying low numbers of activating receptors compared with the control group. Detailed analysis of each single receptor revealed significantly lower frequencies of the activating receptor KIR3DS1 in patients with BKV infection/nephropathy as compared with the controls. Thus, our study supports protective effects of activating receptors in BKV infection and suggest natural killer-cell-related genetic predisposition to the development of BKV-associated nephropathy.

Luminex(®) and its applications for solid organ transplantation, hematopoietic stem cell transplantation, and transfusion.

SUMMARY: The detection of antibodies against the human leukocyte antigen (HLA) complex has become indispensable in every clinical practice. The development of solid-phase assays like the Luminex allows the standardized measurement of anti-HLA antibodies (HLAab) with high sensitivity, albeit the relevance for some clinical settings remains a matter of debate. In this review we aim to describe the principle of Luminex-based antibody detection, including two modifications that allow identifying solely complement-activating antibodies. We then describe three applications for Luminex: i) detection of HLAab preceding solid-organ transplantation and monitoring of donor-specific antibodies posttransplant as a risk factor for antibody-mediated rejection; ii) presence of HLAab in recipients as a risk for graft failure in hematopoietic stem cell transplantation, especially in haploidentical or mismatched transplantations; iii) role of HLAab in blood transfusion including refractory thrombocytopenia and selection of suitable platelet donors, transfusion-related lung injury after plasma transfusion, and immunization against HLA after red blood cell transfusion despite leukodepletion. Although the Luminex platform constitutes a potent technology for HLA antibody detection, some drawbacks require the well-educated analysis and interpretation of data in critical cases. In addition, Luminex has become an important tool to identify clinically relevant antibodies.

Immune response to an adjuvanted influenza A H1N1 vaccine (Pandemrix(®)) in renal transplant recipients.

BACKGROUND: In the course of the influenza A H1N1 pandemic, transplanted patients were recommended to receive vaccination. In the present study, we evaluated the immune response to an adjuvanted influenza A H1N1 vaccine (Pandemrix®) in renal allograft recipients. METHODS: Sixty patients and 22 healthy controls participated in a prospective observational study and received a single dose of Pandemrix®. H1N1 antibody titres as well as anti-HLA antibodies were determined before and after vaccination. In 19 patients, a booster vaccination was performed and the outcome of all vaccinated renal allograft recipients (n = 107) in our clinic was reviewed. RESULTS: Two out of sixty patients had an elevated influenza A H1N1 titre before vaccination. Of the remaining 58 patients, only 20/58 (34.5%) developed a protective immune response in contrast to 20/22 (91%) of the control group. After booster vaccination, a protective titre was present in 8/19 (42%) of patients. Of the 107 patients, 6 (5.6%) developed new donor-specific HLA antibodies after vaccination. CONCLUSIONS: These data suggest that Pandemrix® does not provide a protective immune response in the majority of kidney transplant recipients. Therefore, for new vaccines, efficacy as well as safety profiles should be evaluated in this subgroup of patients.

Comparison between bortezomib and rituximab in the treatment of antibody-mediated renal allograft rejection.

BACKGROUND: Antibody-mediated rejection (ABMR) following kidney transplantation is associated with poor allograft survival. Conventional treatment based on plasmapheresis (PPH) and the administration of intravenous immunoglobulins (IVIG) is not satisfactory. Two compounds, more specifically targeting B cells and plasma cells, may help to improve the prognosis: rituximab, a B-cell-depleting monoclonal antibody, and bortezomib, a proteasome inhibitor causing apoptosis of plasma cells. METHODS: Starting in February 2009, we treated 10 consecutive patients with ABMR according to current Banff criteria with one cycle of bortezomib [1.3 mg/m(2) intravenously (i.v.), Day 1, 4, 8, 11]. This group was compared to a historical control group of patients (n = 9) treated with a fixed single dose of rituximab (500 mg i.v.). All patients received PPH (6×) and IVIG (30 g). Patients with acute ABMR additionally received methylprednisolone (3 × 500 mg i.v.). RESULTS: Patient survival in both groups was 100%. At 18 months after treatment, graft survival was 6/10 in the bortezomib group as compared to 1/9 functioning grafts in the rituximab group (P = 0.071). Renal function was superior in patients treated with bortezomib as compared to rituximab-treated patients (serum creatinine at 9 months: 2.5 ± 0.6 versus 5.1 ± 2.1 mg/dL, P = 0.008). Proteinuria was not different between both groups (9 months: 1.3 ± 1.0 versus 1.6 ± 1.6 g/day, P = n.s.). CONCLUSIONS: Treatment of ABMR with bortezomib in addition to standard therapy was partially effective, whereas treatment with a fixed dose of rituximab in addition to standard therapy with PPH and IVIG did not result in sufficient long-term graft survival. In the future, new strategies including the combination of both substances and the application of higher doses must be discussed.

Prognostic value and cost-effectiveness of different screening strategies for HLA antibodies prior to kidney transplantation.

HLA antibody screening is conducted routinely prior to kidney transplantation, but the comparative prognostic value and cost-effectiveness of different methods are unclear. Pre-transplant sera of 141 patients transplanted between 1998 and 2000 were screened by ELISA and Luminex assays, and antibody specificities of reactive sera determined using bead array techniques. ELISA screening detected donor-specific antibodies (DSA) in 19 patients, who had a higher incidence of impaired graft function (60% vs. 20%, p = 0.04) and antibody-mediated rejection (AMR) within 90 d after transplantation (AMR, 35% vs. 5%, p = 0.02). Luminex screening detected eight additional patients with DSA, among those one with AMR. Six of eight patients with Luminex-only-DSA reported no prior immunizing events. Death-censored graft survival was shorter only in patients with DSA and AMR (median, 1.7 yr instead of between 9.5 and 11.0 yr for patients without DSA or patients with DSA but no AMR, p < 0.001). Material costs per detected clinically relevant DSA were about 57% higher for Luminex screening, but this increase could be avoided by modifying the cut-off recommended by the manufacturer. Conclusively, specification of antibodies only in sera reactive in screening tests was cost-effective to prevent shortened graft survival. Preformed DSA were only harmful if AMR was diagnosed within 90 d after transplantation.

High levels of CMV-IE-1-specific memory T cells are associated with less alloimmunity and improved renal allograft function.

BACKGROUND: Cytomegalovirus (CMV) infection has been associated with allograft rejection in solid organ transplantation. However, the immunologic mechanisms behind this observation have not been elucidated. One proposed mechanism is direct cross-reactivity of antiviral T-cells with allogeneic MHC/peptide complexes, a process termed heterologous immunity. Another model favours indirect stimulation of alloimmunity by CMV-induced proinflammatory cytokines and upregulation of MHC class II and adhesion molecules. Recently, we found that protection from CMV disease was correlated with high levels of CMV-immediate early-1 (IE-1) specific IFN-gamma-producing T-cell responses in heart and lung transplant recipients. The aim of this study was to define the relation of CMV-specific T-cell responses to acute rejection, donor-reactive memory T cells, and allograft function after kidney transplantation. METHODS: To address this issue, IFN-gamma-producing T-cell responses following ex-vivo stimulation with pools of overlapping peptides representing the CMV pp65 and IE-1 proteins, as well as donor-reactive IFN-gamma-producing T-cells were determined at multiple time points before (pre-Tx) and during the first 6 months posttransplant (post-Tx) in 36 kidney transplant recipients using an enzyme linked immunoabsorbent spot assay (ELISPOT). RESULTS: CMV-specific T cells were not exclusively detectable in CMV seropositive patients, as 3/12 seronegative patients had significant pre- and post-Tx pp65/IE-1-specific T-cell responses. In patients with detectable anti-CMV antibody or T-cell responses, no difference in CMV-specific T-cell frequencies was found between patients with versus without acute rejection. However, early (week 1, r=0.457, p=0.037) and average IE-1-specific T-cell responses (r=-0.415, p=0.032) during 6 months post-Tx showed a significant inverse correlation with average post-Tx donor-reactive T-cell responses. Furthermore, average post-Tx IE-1-specific T-cell responses correlated significantly with 6 and 12 months glomerular filtration rate (GFR). In contrast, pp65-specific T-cell responses did not correlate with donor-reactive T cells or graft function. Only 2/36 patients developed CMV disease, both showing very weak IE-1-specific T-cell responses during the whole monitoring period. CONCLUSION: No evidence for heterologous immunity could be found in patients with high levels of CMV-specific T cells. On the contrary, less alloreactivity and improved graft function were found in patients with strong IE-1-specific T-cell responses. These results emphasize the importance of immediate early antigens (IE) as targets for T-cell immunity to CMV. We hypothesize that IE-1-specific T cells might effectively suppress IE-1-induced indirect effects such as inflammation and upregulation of MHC class II and adhesion molecules.

Angiotensin II type 1-receptor activating antibodies in renal-allograft rejection.

BACKGROUND: Antibodies against HLA antigens cause refractory allograft rejection with vasculopathy in some, but not all, patients. METHODS: We studied 33 kidney-transplant recipients who had refractory vascular rejection. Thirteen had donor-specific anti-HLA antibodies, whereas 20 did not. Malignant hypertension was present in 16 of the patients without anti-HLA antibodies, 4 of whom had seizures. The remaining 17 patients had no malignant hypertension. We hypothesized that activating antibodies targeting the angiotensin II type 1 (AT1) receptor might be involved. RESULTS: Activating IgG antibodies targeting the AT1 receptor were detected in serum from all 16 patients with malignant hypertension and without anti-HLA antibodies, but in no other patients. These receptor-activating antibodies are subclass IgG1 and IgG3 antibodies that bind to two different epitopes on the second extracellular loop of the AT1 receptor. Tissue factor expression was increased in renal-biopsy specimens from patients with these antibodies. In vitro stimulation of vascular cells with an AT1-receptor-activating antibody induced phosphorylation of ERK 1/2 kinase and increased the DNA binding activity of the transcription factors activator protein 1 (AP-1) and nuclear factor-kappaB. The AT1 antagonist losartan blocked agonistic AT1-receptor antibody-mediated effects, and passive antibody transfer induced vasculopathy and hypertension in a rat kidney-transplantation model. CONCLUSIONS: A non-HLA, AT1-receptor-mediated pathway may contribute to refractory vascular rejection, and affected patients might benefit from removal of AT1-receptor antibodies or from pharmacologic blockade of AT1 receptors.

Dissection of the CMV specific T-cell response is required for optimized cardiac transplant monitoring.

Despite the success of antivirals in preventing clinically overt CMV disease in cardiac allograft recipients, sub-clinical active CMV infection remains a major concern because of its association with allograft rejection and vasculopathy. The measurement of CMV specific T-cell responses is a promising approach to assessing this situation. For simplicity, class-I MHC/peptide-multimers staining CD8 T-cells directly are often used but this ignores a much wider range of responses including the whole CD4 T-cell compartment. CD4 T-cells, however, were recently shown to be critical to reducing CMV load early after transplantation. To determine how extensive T-cell responses to CMV are, the responses to two dominant CMV proteins, IE-1 and pp65, were dissected in detail accounting for T-cell lineage, frequencies, epitope recognition and changes over time in more than 25 heart transplant recipients. Cross-sectional results from over 30 healthy CMV-carriers were analyzed for comparison. Responses were unexpectedly complex, with considerable inter-individual variation in terms of dominance, breadth, and recognized epitopes. Whereas the use of MHC/peptide-multimers for clinical CD8 T-cell response monitoring alone can be justified in some situations, short term T-cell activation combined with intracellular cytokine staining was clearly found to be of more general usefulness. The performance of IFN-gamma, TNF-alpha, or IL-2 as single read-outs in identifying activated T-cells was examined and confirmed that the frequently used IFN-gamma was best suited. These results should be used to inform the design of clinically applicable and diagnostically useful approaches to monitoring CMV specific responses in heart transplant recipients.

Architectural B-cell organization in skeletal muscle identifies subtypes of dermatomyositis.

OBJECTIVE: To study the B-cell content, organization, and existence of distinct B-cell subpopulations in relation to the expression of type 1 interferon signature related genes in dermatomyositis (DM). METHODS: Evaluation of skeletal muscle biopsies from patients with adult DM (aDM) and juvenile DM (jDM) by histology, immunohistochemistry, electron microscopy, and quantitative reverse-transcription PCR. RESULTS: We defined 3 aDM subgroups-classic (containing occasional B cells without clusters), B-cell-rich, and follicle-like aDM-further elucidating IM B-lymphocyte maturation and immunity. The quantity of B cells and formation of ectopic lymphoid structures in a subset of patients with aDM were associated with a specific profile of cytokines and chemokines involved in lymphoid neogenesis. Levels of type 1 interferon signature related gene expression paralleled B-cell content and architectural organization and link B-cell immunity to the interferon type I signature. CONCLUSION: These data corroborate the important role of B cells in DM, highlighting the direct link between humoral mechanisms as key players in B-cell immunity and the role of type I interferon-related immunity.

Evaluation of hepatocyte growth factor as a sensitive marker for early detection of acute renal allograft rejection.

BACKGROUND: It has been shown that hepatocyte growth factor (HGF), besides its well-established hepatotrophic effect in liver regeneration, is involved in the regeneration of the kidney after injury. In the present study we investigated whether HGF can serve as a marker for detection of acute rejection in the early posttransplantation period. METHODS: HGF levels were determined in pre- and posttransplant sera (up to day 21) of 26 recipients with biopsy-proven acute rejection, 30 recipients with acute tubular necrosis (ATN), and 32 recipients without posttransplant complications. RESULTS: Although no association was found between pretransplant HGF and death-censored functional graft survival, receiver operating characteristic (ROC) curves demonstrated that HGF measured during the entire posttransplant study period, and especially on days 3 to 5, was a good marker for differentiating recipients who subsequently developed acute rejection from recipients with an uncomplicated course (P<0.0001, specificity 87%, sensitivity 84%). HGF measured from day 3 until day 21 posttransplantation, and especially on days 7 to 9, was also a sensitive marker for differentiating recipients with ATN from recipients with an uncomplicated course (P<0.0001). If considered in combination with sCD30, the diagnostic value of HGF was further improved. While 73% of samples from patients with impending rejection were positive for both HGF and sCD30, 94% of samples from nonrejecting patients were double-negative and none of the samples from this group fell into the double-positive category (P<0.0001). CONCLUSIONS: Our data suggest that HGF measured during the early posttransplant period might be a useful parameter for early detection of acute renal allograft rejection.

KIR/HLA ligand incompatibility in kidney transplantation.

BACKGROUND: The polymorphic family of killer-cell immunoglobulin-like receptors (KIRs) consists of activating and inhibitory receptors expressed by natural killer (NK) cells and effector T cells that recognize human leukocyte antigen (HLA) class I ligands. It has been suggested that KIR/HLA incompatibility exerts beneficial effects in hematopoietic stem cell transplantation. METHODS: To elucidate whether certain receptor-ligand combinations between recipient KIR and donor HLA antigens lead to enhanced alloreactivity of NK cells associated with acute rejection (aRx) after kidney transplantation, we analyzed the entirety of matches/mismatches between KIR genes and known HLA ligands for aRx patients (n=105) compared to patients with stable renal function (n=119). RESULTS: Whereas HLA-C ligand incompatibility between donor and recipient has no influence on aRx, grafts derived from donors homozygous for HLA-C group 2 alleles seem to demonstrate a better outcome (P=0.052). Additionally, a higher number of inhibitory receptors in the recipient's genotype (P=0.042), a significant higher number of matches for the receptors KIR2DL2/DS2 (P=0.004), as well as a higher number of mismatches for KIR2DL3 (P=0.014) could be observed for patients with stable renal function. CONCLUSION: Our data illustrate that certain KIR/HLA class I ligand combinations between donor and recipient might influence graft short-term outcome after renal transplantation.

Treatment of Antibody-Mediated Renal Allograft Rejection: Improving Step by Step.

Throughout the past years we stepwise modified our immunosuppressive treatment regimen for patients with antibody-mediated rejection (ABMR). Here, we describe three consecutive groups treated with different regimens. From 2005 until 2008, we treated all patients with biopsy-proven ABMR with rituximab (500 mg), low-dose (30 g) intravenous immunoglobulins (IVIG), and plasmapheresis (PPH, 6x) (group RLP, n = 12). Between 2009 and June 2010, patients received bortezomib (1.3 mg/m2, 4x) together with low-dose IVIG and PPH (group BLP, n = 11). In July 2010, we increased the IVIG dose and treated all subsequent patients with bortezomib, high-dose IVIG (1.5 g/kg), and PPH (group BHP, n = 11). Graft survival at three years after treatment was 73% in group BHP as compared to 45% in group BLP and 25% in group RLP. At six months after treatment median serum creatinine was 2.1 mg/dL, 2.9 mg/dL, and 4.2 mg/dL in groups BHP, BLP, and RLP, respectively (p = 0.02). Following treatment, a significant decrease of donor-specific HLA antibody (DSA) mean fluorescence intensity from 8467 ± 6876 to 5221 ± 4711 (p = 0.01) was observed in group BHP, but not in the other groups. Our results indicate that graft survival, graft function, and DSA levels could be improved along with stepwise modifications to our treatment regimen, that is, the introduction of bortezomib and high-dose IVIG treatment.

Treatment of Acute Antibody-Mediated Renal Allograft Rejection With Cyclophosphamide.

BACKGROUND: Antibody-mediated rejection (AMR) is a major risk for renal allograft survival. Throughout decades, cyclophosphamide treatment has been proven to be effective in patients with antibody-associated autoimmune diseases. We investigated whether cyclophosphamide combined with plasmapheresis and intravenous immunoglobulins is an option for patients with AMR. METHODS: Between March 2013 and November 2015, we initiated treatment of 13 consecutive patients with biopsy-proven acute AMR with intravenous cyclophosphamide pulses (15 mg/kg adapted to age and renal function) at 3-week intervals, PPH (6×), and high-dose intravenous immunoglobulin (1.5 g/kg). Treatment was completed after 6 cyclophosphamide pulses or in case of return to baseline serum creatinine together with reduction of donor-specific HLA antibodies (DSA) below 500 mean fluorescence intensity. RESULTS: Eleven of 13 patients completed treatment. Median follow-up was 18 (12-44) months. At the end of follow-up, graft survival was 77% (10/13). The 3 graft losses were caused at least in part by nonadherence and premature termination of treatment. Serum creatinine increased from 1.7±0.4 mg/dL at 3 months before diagnosis to 3.7±2.4 mg/dL at diagnosis (P = 0.01), and decreased to 2.1 ± 0.7 mg/dL at 3 months after diagnosis (P = 0.01). In 7 (64%) of 11 patients, who completed treatment, DSA decreased, in 4 (36%) of 11 DSA were below 500 mean fluorescence intensity after treatment. Dose reductions had to be performed in 3 of 13 patients for leukopenia. We observed 14 hospitalizations in 9 of 13 patients. CONCLUSIONS: To our knowledge, this is the first systematic report on cyclophosphamide-based treatment of acute AMR based on modern diagnostics. Treatment was effective and relatively safe. Future studies will show, whether cyclophosphamide proves to be a valuable alternative for the treatment of AMR.

Clinical Outcome of Patients With De Novo C1q-Binding Donor-Specific HLA Antibodies After Renal Transplantation.

BACKGROUND: De novo donor specific anti-HLA antibodies (dnDSA) may cause graft loss in renal transplant recipients. The capability to bind the complement may help to stratify the risk for inferior outcomes associated with dnDSA. We developed a modified C1q-binding assay and hypothesized that C1q-binding dnDSA could differentiate between indolent and harmful dnDSA causing antibody-mediated rejection (AMR) and graft loss. METHODS: We retrospectively identified 59 renal transplant recipients who developed dnDSA and had serum available and complete follow-up. All patients were analyzed for C1q-binding dnDSA at the time of dnDSA detection, and 1-year later or at time of AMR. AMR-positive patients were also tested 6 to 12 months before the event if IgG dnDSA was present. RESULTS: Thirty-seven of 59 dnDSA patients developed AMR during 5.9 ± 3.1 years follow-up. AMR-positive patients had more dnDSA with a significant higher frequency of class I, a higher frequency and a higher mean fluorescence intensity value of C1q-dnDSA at all time-points. Death-censored AMR-free and allograft survivals were significantly lower in C1q-dnDSA patients. In multivariate analysis, C1q-dnDSA was an independent risk factor for AMR. CONCLUSIONS: C1q-binding dnDSA is associated with inferior outcomes, yet not in all patients. Nevertheless, C1q-dnDSA was shown to be an independent risk factor of AMR and graft loss and may be a useful tool to stratify the immunological risk for AMR.

Modified ELISPOT technique--highly significant inverse correlation of post-Tx donor-reactive IFNgamma-producing cell frequencies with 6 and 12 months graft function in kidney transplant recipients.

BACKGROUND: Upcoming trials for immunosuppression minimization and tolerance induction require the development of reliable in vitro assays for monitoring cellular alloimmunity in transplant patients. The IFN-gamma ELISPOT assay represents a promising tool for monitoring alloreactive memory/effector T cells. As T lymphopenia is a common finding during the early post-transplant (post-Tx) period, the IFN-gamma ELISPOT technique was here modified by using ELISPOT responder cells with enhanced percentage and standardized number of 200,000 T cells per well. METHODS: Peripheral blood mononuclear cells (PBMNC) of kidney transplant recipients were depleted of CD14+ and CD15+ cells to increase the percentage of T cells from average 47.8% to 71.5% before transplantation (pre-Tx) and from 39.7% to 74.9% post-Tx. The assay was tested in a population of 23 de novo renal transplant patients for clinical relevance. Before and at 2-3 times during the first 6 months post-Tx, IFN-gamma-producing donor-reactive as well as recall antigen-reactive cell frequencies (Candida, tuberculin, tetanus) were determined and correlated with outcome. RESULTS: Pre-Tx donor-reactive ELISPOT frequencies were enhanced in patients with acute rejection compared to non-rejectors. Moreover, mean post-Tx donor-reactive ELISPOT frequencies showed a highly significant inverse correlation with renal function at 6 and 12 months. In contrast, recall antigen-reactive ELISPOT frequencies did not correlate with outcome. CONCLUSION: Our results suggest that the modified donor-reactive ELISPOT approach might provide a useful surrogate marker for renal transplant outcome with possible utility especially in T-lymphopenic patients.