Postergroup No. 1/5
Solid organ transplantation
Form of presentation:
HLA incompatible renal transplantation across Bw4/Bw6 alleles in two patients
M. Mishra (Lucknow, UP, GB)
HLA incompatible (HLAi) renal transplantation may be an option for highly sensitised patients with reportedly a superior survival compared to long term maintenance dialysis. Risk stratification for potential recipients in the United Kingdom is often performed as per British Society for Histocompatibility and Immunogenetics (BSHI) guidelines, which involves comprehensive evaluation by a combination of Complement dependent cytotoxicity crossmatch (CDCXM), Flowcytometry crossmatch (FCXM) and Luminex single antigen bead (SAB) assay, and correlation with sensitization history. Transplanting successfully across a broad specificity such as HLA- Bw4 or Bw6 may prove more difficult, because non–DSA reacting with Bw4 or Bw6 epitopes could have an additive effect and greater overall reactivity. The maintenance immunosuppression protocol comprised of standard triple drug regimen which includes prednisolone, Mycophenolate Mufti and Tacrolimus. In this paper the workup leading to successful outcome of two HLAi transplants is presented.
Evaluation of soluble Human Leukocyte Antigen-G levels in kidney transplantation patients
V. Grimaldi (Napoli, IT)
The expression of human leukocyte antigen (HLA)-G, a non-classical HLA class I molecule and its soluble forms (sHLA-G) are found to improve graft acceptance. However, there are previous data on the correlation of sHLA-G and graft rejection, as well as data on the correlation with viral infections (HCV) in kidney transplanted patients. We evaluated the expression of sHLA-G levels on both patients on a waiting list for kidney transplantation and on patients returned to the list after the first transplantation by comparing them with blood donors as a control group. In addition, we investigated the correlation between sHLA-G levels and HCV infections. Sera of 67 patients on the waiting list for kidney transplantation, n=43 with anti-HCV and n=24 without anti-HCV were analyzed. Among all these patients n=39 were on thr waiting list for the first transplantation while n=28 were patients were returned to list due to graft loss. The control group included n=23 blood donors with anti-HCV (n=13) and without anti-HCV (n=10). Serum samples were tested for anti-HCV by using chemiluminescent immunoassays (CMIA) (ARCHITECT, Abbott Diagnostics, Wiesbaden, Germany) followed by confirmatory testing for HCV (Immunoblot assays, INNO-LIA, Innogenetics, Ghent, Belgium). The serum levels of sHLA-G were analyzed by Enzyme-Linked Immunosorbent Assay (ELISA, BioVendor, Czech Republic). Preliminary data revealed that sHLA-G levels obtained from patient sera were higher compared to controls. In addition, we found that serum sHLA-G levels were significantly higher in patients returned to the waiting list with respect to patients at first transplantation (p=0.004). No significant correlation was observed for HCV infection among groups.Interestingly, we found high levels of sHLA-G in transplanted patients compared to patients at first transplantation. Previous data seem to be consistent with the hypothetic protective function of HLA-G in the post transplantation period. Therefore, our findings might be the result of ischemia and re-perfusion injury suggesting a possible use of sHLA-G levels as a marker for measuring the state of kidney allograft acceptance.
The contribution of MICA-129 Val/Met polymorphism on graft rejection and CMV infection in simultaneous pancreas and kidney transplant patients – one-year follow-up
R. Michita (Essen, DE)
Simultaneous transplantation of pancreas and kidney (SPK) is an established therapy for diabetes type-I patients suffering from chronic renal failure. Emerging evidence suggest that other antigens besides HLA mismatches may play a role in immune cell activation, graft rejection and survival. The polymorphic MHC class I chain-related sequence A (MICA) gene encodes a membrane-bound protein that binds to the NKG2D receptor activating NK and T cells whereas soluble forms of MICA (sMICA) impair the effector functions of these cells. Importantly, a single nucleotide polymorphism (SNP) rs1051792 at position 454A/G of exon 3 causes a valine (val) to methionine (met) exchange at codon 129, which influences MICA expression patterns and binding affinity to NKG2D. The clinical relevance of this dimorphism in solid transplantation is still not clear. Therefore we genotyped this MICA SNP in 50 SPK patients and donors and measured sMICA levels once during one-year follow-up in 18 patients. We evaluate the effect of val/met mismatch situation with regard to CMV infection and graft rejection in the one-year follow up. Distribution of allele (p=0.261) and genotype (p=0.411) frequencies of 129 val/met were similar between donors and recipients. We observed that the val-mismatched patients (n=7) had a shorter CMV infection-free-survival than the remaining patients [p=0.004; hazard ratio (HR) 7.36; 95% CI 1.47-36.9]. Similarly, a shorter kidney rejection-free survival (p=0.012; HR 3.64, CI 1.24-10.6) were observed in val mismatched patients. Interestingly, the one-year follow-up sMICA levels could hardly be detected in val mismatched patients (N=4, range: 0-45 pg/ml), whereas other patients revealed substantial amount of sMICA (N=14, range: 269-1331 pg/ml; p<0.001). Our study gives for the first time evidence that the functionally relevant mismatch situation of MICA at position 129 has an impact on kidney allograft recognition and CMV infection in the first year post SPK transplantation.
Immune status assay-an approach to evaluate immunosuppression in kidney transplanted patients
M. Alheim (Stockholm, SE)
A major challenge in transplantation medicine today is to develop an assay to detect patients with high risk of transplant rejection or infection due to inadequate level of immunosuppression. To measure drug concentrations of immune-modulating agents in vivo may not be sufficient to achieve an effective immune suppressive treatment as metabolism of these drugs can vary between patients. The aim here is to develop a flow cytometry based assay for immune monitoring of patients after transplantation. In initial experiments, heparinized whole blood cells from ten patients with various immunodeficiencies and ten healthy controls were collected and treated with polyclonal immune-stimulating agents such as pokeweed mitogen (PWM), Concanavalin A (ConA) and Staphylococcal Entertoxin A and B (SEA/SEB) or medium alone for 1-3 days. The frequencies of CD3+/CD4+ T cells expressing the activation markers CD69, CD134, CD25, CD71, and HLA-DR were analyzed by flow cytometry (Immune Status Assay; ISA). The results were compared with the previously described 7-days reference FASCIA-assay. The stimulation index (SI) for each activation marker was calculated and compared with data obtained with FASCIA. Patients were divided into two groups, “negative” (n=5) or “normal to high” (n=5) according to FASCIA assay status. Preliminary results show significant differences between patients in these two groups for all studied activation markers using Con A as a stimulation agent for 2 or 3 days; CD134 (d2. p=0.0457, d3. p=0.0156), CD25 (p=0.0133, p=0.0064), CD69 (p=0.0054, p=0.0209), CD71 (p=<0.0001, p=0.0071), HLA-DR (p=0.0027, p=0.0236). The data from ISA indicates that 2 days of immune stimulation with Con A is sufficient for discrimination between responsive and non-immune responsive patients. In vitro activation for 1 day or with PWM or SEA/SEB 2-3 days did not equally well discriminate between patients with low and normal immune activation status. A larger cohort of kidney patients, selected for living donor transplantation, will now be tested with ISA before and after transplantation. Taken together, we here demonstrate a novel assay (ISA) which shows excellent correlation with current reference assay evaluating immune stimulation. This assay requires little hands-on time and results are available within 2-3 days.
CX3CL1 is significantly upregulated in biopsies from acutely rejecting kidney transplant recipients.
M. Xavier (Porto, PT)
The chemokine CX3CL1 can act as both chemoattractant and adhesion molecule and is expressed on the apical surface of tubular epithelium in human renal transplant biopsy specimens procured during acute rejection (Rej) but also by endothelial and mesangial cells. We studied CX3CL1 on transplant aspiration biopsies (AB) and its evolution post-Rej. First cadaver kidney transplant recipients were studied and divided into three groups: I, stable cases, where AB was done between 7 and 10 days post-transplantation, and which proved Rej-free for at least six months (n=42), group II, Rej cases with AB done the first day of diagnosis (confirmed by a classical tru-cut biopsy) done concomitantly, and group III post successful treatment of Rej (n=5) all coming from group II, done one week post treatment completion. The AB samples were cytocentrifuged and the CX3CL1 immunostaining was done following APAAP methodology. Neither differed significantly by comparing patient demographics or immunosuppressive treatments. The results are expressed as absolute positive cells (abs), the ratio of positive cells for kidney cells (+/R) and ratio for mononuclear immune cells (+/LM). For group I, abs 10.1 ±14.4, +/R 0.028 ±0.046 and +/LM 0.038 ±0.087; group II, abs 80.7 ±76.6, +/R 0.22 ±0.23, +/LM 0.39 ±0.23; group III, abs 17.6 ±18.5, +/R 0.024 ±0.019, +/LM 0.16 ±0.29. Group I was lower than II, abs (p=0.0001), +/R (p=0.0001), +/LM (p=0.0001). Group II was higher than III, abs (p=0.057), +/R (p=0.006), and +/LM (p=0.12). No differences were observed by comparing I versus III, respectively for abs, +/R and +/LM, p=0.32, p=0.42 and p=0.43. The negative predictive value for acute rejection recurring to abs < 20 was 97.3% and positive predictive value was 53.8%. We confirm the significant association of CX3CL1 with acute rejection in human kidney transplants and we show for the first time its rapid normalization following successful treatment.
HLA-G expression variability as indicator for renal transplant outcome
U. Kanga (New Delhi, IN)
HLA-G is a non-classical HLA molecule known to play a crucial role in allogeneic situations like pregnancy and allo-transplantation. Higher HLA-G expression is related with better transplant outcome. The study aimed to investigate the role of HLA-G in North Indian patients undergoing renal transplantation. A total of 39 recipient-donor pairs were enrolled in the study and results were compared with 50 healthy controls. Of the enrolled recipient-donor pairs, 11 recipients experienced graft rejection episodes. The HLA-G exon8-14bp indel polymorphism was evaluated by PCR-SSP and sequencing. Serum soluble HLA-G levels (pre-transplant, day 15, 30, 60, and 90) were measured using ELISA. The distribution of HLA-G exon8-14bp genotypes among the healthy controls was, ins/del-46%, del/del-24% and ins/ins-30%. Among the recipients, the genotype distribution was evaluated on segregation into rejection and non-rejection groups. A higher percentage of recipients in the non-rejection group carried the ins/ins genotype as compared to the rejection group (32.1% vs 20% respectively). Distribution of the del/del genotype was similar in both groups. The median sHLA-G level was 31.7 U/ml in healthy controls. sHLA-G levels were higher in the individuals with ins/ins genotype in comparison to del/del or ins/del genotype. The sHLA-G levels were significantly higher in transplant recipients when compared with healthy controls. Among recipient cohort that experienced a rejection episode, sHLA-G levels were lower (median-105.4 U/ml) as compared to recipients maintaining well-functioning grafts (WFG) (median-260.4 U/ml). The sHLA-G levels were better maintained throughout the post-transplant period in the WFG group. In the rejection group, the sHLA-G levels improved only after anti-rejection therapy, however it remained lower than WFG. It is evident that HLA-G 3’UTR genotyping and serial monitoring of sHLA-G levels in serum/plasma may be useful tests for predicting any adverse event like rejection.
Impact of sensitization events on HLA alloimmunization in kidney transplantation candidates
N. Katalinic (Rijeka, HR)
Alloantibodies against human leukocyte antigens (HLA) may develop after exposure to blood transfusions, pregnancy and/or previous transplantation. HLA antibodies represent an important immunological barrier to successful organ transplantation. The aim of our study was to assess the frequency of exposure to different sensitizing events (SEs) and evaluate their effect on HLA alloimmunization in kidney transplantation candidates. We performed retrospective analysis of the HLA antibody screening results in 163 patients on kidney transplant waiting list in Clinical Hospital Center Rijeka tested from March 2012 until the end of December 2015. The screening of HLA antibodies has been performed periodically every three months, four times per year by CDC and Luminex assays. Information on earlier and recent SEs were obtained from transplantation candidates, their nephrologists and medical records. A total of 163 patients, 114 (69.94%) were exposed to one or more SEs. The most common SE were blood transfusions. At least one red blood cell unit was received by 92 patients (56.44%). Pregnancies were reported in 57 females (34.97%) and previously transplants were recorded in 24 recipients (14.72%). In 105 patients (64.42%), regardless of a history of SEs, HLA antibodies were not detected by CDC nor by Luminex techniques. HLA alloimmunization occured in 48 patients (29.45%) exposed to SEs and in ten patients (6.13%) without history of SEs. In 21 patients (36.21%) they were detected by CDC and Luminex (CDC+LUM+) while in additional 37 candidates (63.79%), including all sensitized patients without known SEs, HLA antibodies were detected by Luminex only (CDC-LUM+). Exposure of patients on the waiting list to SEs represents a risk factor to developing HLA antibodies. Blood transfusions are the most frequent and the most susceptible to our influence. They should be minimized or avoided in transplantation candidates whenever is possible.
Acute antibody mediate rejection due to pre-rransplant HLA-DP donor specific antibodies
I. Kovacevic Vojtusek (Zagreb, HR)
Here we report two patients who developed biopsy proven acute antibody mediated rejection due to pre-transplant HLA-DP donor specific antibodies. The first patient, a 40 year old woman, sensitized following two pregnancies, received transfusions vPRA 90%. The second patient, a 36 year old woman, sensitized following pregnancy; vPRA 6%. HLA antibody screens of serum samples was performed by Luminex using Lifecodes LSA Class I and Class II SAB kits (Immucor, USA). HLA class I and class II molecular typing including retrospective high resolution HLA-DPB1 typing, was performed by a PCR-SSP genotyping method (Olerup SSP®, Sweden). After a standard Eurotransplant allocation procedure both patients received a deceased donor kidney transplant immunologically selected according to the patients' profile and negative T+B crossmatch by complement-dependent cytotoxicity (CDC), without and with DTT, at the time of transplant. Both patients were negative in Luminex screening for donor mismatched alleles at HLA-A, -B, -C, -DRB1 and -DQB1 loci. Both received standard induction therapy, consisting of interleukin-2 receptor antagonist basiliximab and steroids, continued with triple maintainance therapy with tacrolimus, mycophenolate mofetil and steroids. Both had delayed graft function and after the biopsy results were suspected for ABMR. As both patients were transplanted without HLA-A, B, C, DRB1, and DQB1 DSAs, in order to determine the cause of acute ABMR, we expanded the HLA typing of patients and their donors to DPB1 locus and re-analyzed the pre-transplant sera and sera samples drawn at the time of the ABMR diagnosis for the presence of HLA-DP DSAs. The analysis revealed that both patients had HLA-DP DSAs with an avarege MFI value of 5000. Furthermore, retrospective analysis demonstrated that donor-specific DP antibodies were present in all tested pre-transplant samples. Patients were treated with steroids 0.5 gram per day for three days and plasmapheresis. Both improved their kidney function in relatively short period of time. In both cases, there were no other donor-specific HLA alloantibodies, suggesting that the HLA-DP specific antibodies may be directly pathogenic. HLA-DP matching might become relevant for renal transplantation in patients with pre-transplant HLA-DP antibodies.
Clinical significance of complement-binding donor-specific antibodies after kidney transplantation
M. Rodova (Prague, CZ)
Recent studies have reported a worse clinical outcome after kidney transplantation in patients having complement-activating donor-specific antibodies (DSA) in comparison with non-complement-binding DSA. Besides the C1q technique, a newly available method detects the C3d-binding capacity of antibodies. The purpose of this study was to compare the two approaches for detection of complement-activating antibodies (C1q vs. C3d) and to correlate these results with findings in graft biopsies which were performed due to deterioration of graft function. 14 kidney recipients, who received kidney grafts between 2012 and 2016 and had de novo DSA detected by solid-phase assays after transplantation, were retested by the Luminex Single Antigen IgG and C3d assays. Patient age was 46.7 ± 7.1 years, 11 (79%) recipients were retransplanted and seven (50%) were sensitized pre-transplant. The median time of serum testing was 13 days post-transplant. The concordance rate between immunofluorescent detection of diffuse C4d deposits in peritubular capillaries and the positivity in the C3d and C1q tests was 92.8% and 57% respectively. The agreement between the two vendors (OneLambda and Immucor) in detection of DSA on the Single Antigen level was 85.7% for HlA class I and 100% for class II. We conclude that the C3d binding assay reliably identifies de novo DSA with complement-activating capacity in kidney transplant recipients with elevated risk of graft loss.
Optimization of donor
N. Svetlicky (Ramat Gan, IL)
The pre-transplant complement-dependent cytotoxicity (CDC) cross-match (Xm) assay is a gold standard method for organ rejection prediction. There are three possible tissue sources for lymphocyte separation: lymph nodes, spleen and peripheral blood. A long established procedure for cell purification is using nylon wool column T and B cells separation. This method is convenient and cost effective, but has been found to be impractical when the lymphocyte source is donor's peripheral blood. In most of cases, results of CDC with nylon wool separated lymphocytes from peripheral blood are un-interpretable for cadaver sources due to high background non-specific cell mortality. However, the logistics of obtaining lymph nodes is more complicated and hence final result is usually much later than that of peripheral blood. The aim of this study was to determine the most effective way to obtain T and B lymphocytes from donor's blood. Toward this end, we compared four methods of separation with regard to yield, purity and convenience: RosetteSep gradient negative selection (StemCell), MACS MicroBeads magnetic beads positive separation (Miltenyi Biotec), MagniSort magnetic beads negative selection (eBioscience) and MACSprep HLA magnetic beads negative separation (Miltenyi Biotec). We found a significant decrease in concentration of lymphocytes in blood of deceased organ donors compared to living donors (2.7 x10^6/mL vs. 0.57 x10^6/mL). Each separation method had its advantages and disadvantages. Overall in our hands, we found the MACSprep HLA kit advantageous especially in terms of yield, purity and convenience. In addition, we compared CDC Xm results with cells that were separated from blood versus cells that were derived from lymph node of the same donor. We found no difference in T cell Xm results; however the B cell CDC sensitivity was much higher with peripheral blood (50% positive vs. 15%). These results requisite additional consideration in the analysis of cross-matches.
Impact of C1q binding to HLA donor specific antibodies on the mortality and the development of chronic rejection (CLAD) in lung transplantation
C. PICARD (MARSEILLE, FR)
We investigated the impact of C1q binding to de novo HLA Donor Specific Antibodies (DSA) on the mortality and the occurrence of chronic rejection (CLAD) in 192 Lung transplantation (LTx) recipients from the Marseille Lung Transplant Center between December 2006 and December 2013 and with at least 30-day survival. Complement-binding antibodies were detected retrospectively for patients with de novo DSA at M1 and M3 after LTx, using the C1q Luminex assays. CD16 engagement were assessed within PBMC effector cells by flowcytometry analysis of the decrease of MFI CD16 at the surface of CD3-CD56 NK cell subsets exposed to allogeneic target cells in presence of DSA.
During the study period, 45 LTx recipients developed CLAD (including 8 patients (17%) with RAS and 39 (83 %) with bronchiolitis obliterans) and median overall survival was 35 (+/- 30) months. DSA were detected in 31% of LTx at M1 and 18% at M3. The cumulative DSA MFI (cMFI) level of de novo DSA at M1 were not associated to persistence of DSA at M3 (p=0.40). ROC curve analysis showed that DSA MFI threshold >17,000 was associated with death with 50% sensitivity and 85% specificity. Only DSA at M3 were associated with lower survival (p=0.02) and CLAD occurrence (p=0.01). C1q bound 52% and 33% of DSA at M1 and M3, respectively. Among ten C1q+DSA at M3, eight C1q+ DSA were persistent and two were de novo DSA. The cMFI value of DSA at M1> 10,500 were correlated with C1q binding with 89% sensitivity and 69% specificity. C1q DSA at M1 and M3 were not correlated with clinical occurrence. Interesting, the engagement of CD16 by LTx DSA were very low whatever the MFI intensity, the time after LTx and the clinical occurrence.
Finally, these data show that biochemical and biophysical characteristics of LTx DSA are different to these of DSA in other organ transplantation. They should be confirmed by another larger LTx cohort.