Immunoglobulin G (IgG) antibodies against donor human leukocyte antigens (HLA) are monitored in the pre-and post-transplant period due to their established role in predicting rejection and renal allograft survival. However, the role of immunoglobulin M (IgM) anti-HLA donor-specific antibodies (DSA) is not fully understood, especially in highly-sensitized patients undergoing direct transplantation. We designed this study to determine whether IgM DSA predicts rejection episodes and/or graft failure. Samples from 92 patients who had undergone HLA-antibody incompatible transplants were tested at 5 time points: days -8 (pre-plasmapheresis), 0, 7, 14, and 30 using Luminex microbead assay with ethylenediaminetetraacetic acid containing wash buffer (LABScreen®, One Lambda, Canoga Park, CA). IgM was defined positive if the mean fluorescence values were greater than 2000. Presence of pre- and post-transplant IgM was correlated with early antibody mediated rejection episodes (within 30 days post-transplantation) and graft failure. Statistical analyses were performed using SPSS IBM software. Graft survival estimates were death-censored. The presence of pre-transplant IgM DSA did not predict rejection (p=0.83) or graft failure (p=0.424). The post-transplant IgM DSA levels peaked at day 14 (similar to IgG DSA levels). Presence of IgM DSA post-transplant (de novo and resynthesis) was not associated with rejection (p=0.83). However, post-transplant IgM was associated with graft failure (p=0.037). This study shows additional testing of post-transplant IgM DSA over and above IgG is important as post-transplant IgM DSA is associated with graft failure.

The aim of this study was to determine whether the influenza vaccine induces the development of anti-human leukocyte antigen (HLA) and anti-major histocompatibility complex class I - chain A (MICA) antibodies. We determined the presence or de novo development of anti-HLA and anti-MICA antibodies in 3 groups of patients vaccinated against influenza: A) 42 healthy adults; B) 40 end-stage kidney disease patients; C) 25 kidney transplant recipients; and, D) 22 healthy adults who refused vaccination. Serum samples per subject were obtained: prior to vaccination, one week after vaccination, and on a monthly basis for 6 months. They were analyzed by LABScreen® Single Antigen, Luminex. The proportions of de novo antibodies (anti-HLA and anti-MICA) in the 4 groups were 2.4%, 17.5%, 20%, and 0%, respectively. Some patients developed the antibodies later, unrelated to the vaccine. We found preformed antibodies (anti-HLA and anti-MICA) in 67%, 78%, 88%, and 27% of cases in groups A, B, C, and D, respectively. The presence of preformed antibodies was the only predictive factor for the development of de novo antibodies. In conclusion, the development of de novo anti-HLA and anti-MICA antibodies after an external stimulus other than HLA antigens is possible. However, not all cases can be attributed to the vaccine.

Single antigen bead (SAB) and complement-binding assays are commonly used to monitor immunization in transplant patients. Like all new diagnostic assays, some considerations have to be appreciated to avoid a biased utilization. By truly decreasing antibody concentration, SAB monitoring in sensitized patients experiencing apheresis offers a good opportunity to explore analytical interference. We explored analytical artifacts by analyzing the role of prozone and saturation effects through a concrete example of a single patient who experienced immunoadsorption. We then assessed, on a larger cohort, the link between an accurate assessment of mean fluorescence intensity (MFI) and the C1q and C3d binding assays. Finally, we compared MFI with the two main available SAB assays. After immunoadsorption, the MFI of some antibodies unexpectedly rose. We showed that this increase was due, in part, to both a prozone effect and a saturation of the beads. Dithiothreitol treatment appeared to be an efficient way to avoid a prozone effect. The analysis of dilution profile was an interesting tool to detect a saturation effect. The comparison of the two main SABs revealed a systematic difference of 3000 MFI. MFI was a strong predictor of C1q and C3d positivity. Complement-positive antibodies had a higher MFI (p<0.01). Despite the great contribution of SAB assays in anti-HLA antibody assessment, the knowledge of analytical interference is necessary to avoid any misleading conclusions. With regard to the interference between MFI and complement-binding assays, their place in risk stratification has to be clarified.

The highly-sensitized kidney transplant candidate with no available living donors remains at a major disadvantage with decreased access and worse outcomes post-transplant. We have previously reported our initial data on both pre-transplant and post-transplant desensitization. We observed only a modest decline in unacceptable antigens with pretransplant intravenous immunoglobin (IVIG) and rituximab. Due to these observations, we have focused on a peri-operative post-transplant desensitization protocol in our program. Beginning in 2006, we implemented a simple point-based algorithm [variables included: panel reactive antibody (PRA) status; flow cytometric crossmatch (FCXM); and delayed graft function] to identify kidney transplant recipients who would undergo peri-operative plasmapheresis/IVIG to abrogate preformed antibody-mediated allograft rejection (AMR). Our previous results suggested acceptable 5-year outcomes. Here, in an expanded population (N=66), we report an overall death-censored graft survival of 73% at a mean follow-up of 8.5 years post-transplant. Our patients were largely African American (85%) and regrafts (39%), with a median PRA of 88%, and a mean T- and B-FCXM of 97 mean channel shifts (MCS) and 117 MCS, respectively. Although acute AMR rates were acceptable (12%), 22% of patients developed chronic AMR. A pre-transplant T-cell FCXM of > 200 MCS (p=0.02) or presence of donor specific antibodies (DSA) at most recent follow-up (p=0.02) were associated with graft loss. Current studies with revised protocols utilizing additional DSA information, surveillance biopsies, and proteasome inhibition are ongoing.

Acute rejection in human leukocyte antigen (HLA)-matched kidney transplant recipients is uncommon and the mechanisms involved are not well understood. Data from 6-antigen HLA-matched recipients transplanted between 1994 and 2014 were analyzed to identify the incidence, risk factors, and outcomes associated with biopsy-proven acute rejection (BPAR). A total of 278 HLA-matched recipients were identified, of which, 155 (55.8%) received a graft from a sibling donor. Ten patients (3.6%) experienced BPAR over a median follow-up of 10 years (0.41 cases per 100 person-years). Median time to rejection was 36.5 months (standard deviation ±56.4). Recipients who experienced rejection did not differ from those who did not in terms of age, gender, ethnicity, induction agent, panel reactive antibody, or sensitizing events. Acute cellular, antibody-mediated, and mixed rejection occurred in 5, 3, and 2 patients, respectively. The most common biopsy classification was Banff IA (n=4). Four out of 10 patients had documented nonadherence to maintenance immunosuppression. Thirty percent of HLA-matched recipients who rejected had graft loss and 10% died, compared to 30.8% graft loss and 28.4% deaths in non-rejectors (p=0.57 and 0.20, respectively). In conclusion, acute cellular and antibody-mediated rejection are infrequent in HLA-matched kidney transplant recipients. Nonadherence appears to be relatively common among those experiencing rejection. Acute rejection was not associated with higher graft loss or death. The pathogenesis of acute rejection in HLA-matched recipients remains to be determined.

Donor-specific anti-human leukocyte antigen antibodies (DSA) are associated with antibody-mediated rejection (AMR) in kidney transplantation, but the spectrum of graft injury seen in patients with DSA ranges from no damage to florid rejection. Since immunoglobulin G (IgG) antibodies with cytotoxic potential can be distinguished by their binding complement fraction C1q, the level of C1q-binding IgG (C1q+) DSA may be useful for stratifying risk or diagnosing AMR. We therefore investigated the value of IgG and C1q+ DSA in predicting pathologic features of AMR on kidney biopsies. We tested the associations between DSA at different cut-off levels and pathologic features of AMR on biopsy in a cohort of consecutive, highly-sensitized patients transplanted after December 2014 who had 1-, 3-, and 6-month protocol kidney biopsies and monitoring for IgG and C1q+ DSA. Eight patients with cPRA >90% and negative flow crossmatch underwent kidney transplant and completed six months of follow-up contributing 23 pairs of biopsy/ serum samples for analysis. C1q+ DSA was significantly associated with C4d finding on biopsy at mean fluorescence intensity (MFI) cut-offs of >100 (p=0.046) and >300 (p=0.008) and showed superior positive and negative predictive value in comparison to conventional IgG DSA. C1q+ DSA also showed significant association and good predictive value for any AMR feature on biopsy (p=0.003, for >100 MFI; p=0.005 for >300 MFI), while IgG DSA showed no association. In a small cohort of high cPRA transplant recipients, C1q+ DSA outperformed IgG DSA as an indicator of AMR biopsy findings. Including C1q+ DSA testing in post-transplant DSA monitoring of highly-sensitized patients may aid the timely diagnosis of AMR.

Recent literature suggests that a positive crossmatch adversely impacts outcomes in simultaneous liver-kidney transplant (SLKT). The aim of this study was to evaluate outcomes of SLKT with a positive flow crossmatch (+FCXM) at our center. We retrospectively analyzed all of the SLKTs between January 1, 2000, and September 30, 2010. A total of 2793 kidney transplants and 892 liver transplants were performed in this time period, of which, 31 were SLKT (3%). Seven of the 31 (22%) SLKTs had a +FCXM. There were 3 major adverse events: 1 patient mortality at 9 months with liver failure; 1 allograft nephrectomy for primary nonfunction secondary to hyper-acute rejection; and, 1 recurrent liver allograft rejection with eventual graft loss and death at 26 months post-transplant. The median follow-up time was 34 months. The 3-year overall SLKT patient survival in the negative FCXM (-FCXM) patients was 85% compared with 71% in the +FCXM group. The rates of acute liver and kidney rejection were 6% and 10%, respectively, in the -FCXM group compared to 14% and 28%, respectively, in the +FCXM group. A very strongly +FCXM with a mean channel shift above 4 times the positive cut-off and the presence of multiple strong donor-specific antibodies (DSA) with mean fluorescence intensity (MFI) above 10,000 were associated with poorer outcome. In conclusion, in patients with very strongly +FCXM with high MFI DSA, proceeding with the transplantation leads to poor outcomes.

Development of acute antibody-mediated rejection (AMR) is associated with graft loss and can occur both early (<3 months) and late (>3 months) post-transplant. Treatment and prognosis differ in early and late AMR. Herein, we present a single-center experience using high-dose intravenous immunoglobulin (IVIg) (2g/kg) for the treatment of late AMR. All kidney recipients with negative T- and B-cell flow crossmatch at transplant and biopsy-proven late AMR were included (2009-2013, n=126). All patients were treated with IVIg at 2g/kg over divided doses and high-dose intravenous methylprednisolone. Variables collected included demographics, Banff 2007 renal allograft biopsy scoring criteria, and laboratory values. Multivariable Cox proportional hazard regression was used to identify factors predictive of graft loss. Median age was 46 years, with 60% male and 47.6% African American. Median time from transplant to rejection was 3.8 years. Baseline serum creatinine was 1.6 mg/dl and median serum creatinine at diagnosis was 2.6 mg/dl. Fifty-eight patients (46%) eventually lost their grafts at a median of 12 months (interquartile range: 4-21) from diagnosis. Serum creatinine >5.3 mg/dl at time of diagnosis was associated with a 94% probability of graft loss, and after controlling for multiple recipient and donor factors, only serum creatinine and urine protein creatinine ratio at diagnosis were predictive of graft loss. Late AMR has a poor prognosis, with 46% graft loss at a median follow-up of 12 months. Serum creatinine was a better predictor of subsequent graft failure than histological characteristics in late AMR. These findings help inform treatment plans as well as prognosis.

BK polyomavirus infection and de novo donor-human leukocyte antigen (HLA) specific antibodies (dnDSA) are two well-known and distinct complications occurring after kidney transplantation. Recent literature suggests an association between the two events. This study aims to examine the relationship between BK viremia (BKV) and dnDSA and to identify potential risk factors for dnDSA following BKV in kidney transplant recipients. A retrospective review of 1019 recipients from Houston Methodist Hospital was conducted. All patients underwent routine screening for BKV and dnDSA. Median follow-up was 44 months. BKV was detected in 186 (18%) patients at a median of 107 (82-205) days post-transplant. dnDSA occurred in 283 (28%) patients at a median of 272 (62-575) days post-transplant. Of the 69 dnDSA-positive/BKV-positive patients, dnDSA detection occurred after BKV onset in 46 patients. Thus, 46 (28%) previously DSA-negative patients later became dnDSA-positive following BKV, not significantly different from the rate seen in BKV-negative patients (26%; p=0.5). Median time to DSA detection following BKV onset was 232 days (interquartile range, 119-460) post-BKV detection. Multivariate analysis revealed a greater number of HLA mismatches and viral clearance as risk factors for development of dnDSA following BKV, whereas delayed graft function was associated with a lower risk of dnDSA. In conclusion, despite being considered a result of over-immunosuppression, BKV can still be followed by dnDSA in a substantial proportion of patients. Monitoring for dnDSA in patients being managed for BKV may be warranted.

Antibody-mediated rejection (AMR) remains a problem without a reliable treatment in the care of kidney transplant patients. We proposed and tested a program of screening for donor specific antibodies (DSA) to initiate treatment of patients before AMR was detected and to prevent its occurrence. Starting in April 2012, we stratified patients into high-, medium-, and low-risk groups for the development of DSA and instituted a program of screening for and treatment of these antibodies. We used a historic control group of patients transplanted at our center as a comparator and looked at rates of DSA testing and development as well as rates of development of AMR, cell-mediated rejection, and graft loss. 614 patients were transplanted under the protocol compared with 266 patients in the control group. Length of follow-up was similar in both groups. The group undergoing DSA screening had lower rates of DSA development (17.6% versus 24.8%, p=0.016) and that DSA was found at a significantly earlier time post-transplant (147 versus 248 days, p=0.02). Incidence of AMR was dramatically lower in the screened group (1.3% versus 8.6%, p<0.0001) with no grafts lost due to AMR. AMR was found to occur at an average of 181 days post-transplant. Rates of acute cellular rejection did not decrease in a manner similar to AMR rates. In conclusion, a program of universal risk-stratified DSA testing in kidney transplant patients can dramatically reduce rates of AMR and virtually eliminate graft loss due to AMR.

The relationship between circulating pre-transplant immunoglobulin G (IgG) antibodies to donor human leukocyte antigen (HLA) -C locus determined antigens alone and acute rejection, kidney allograft function, and graft survival is not fully defined. Also, the impact of circulating pre-transplant IgG antibodies to donor HLA-C locus antigens alone on these outcomes has not been compared with the impact of circulating pre-transplant IgG antibodies to donor HLA-A or -B locus antigens. We conducted a retrospective review of records of 1252 kidney allograft recipients transplanted at our center between January 2010 and January 2016 to identify patients with circulating pre-transplant IgG antibodies directed at kidney donor HLA-A, -B, or -C locus determined antigens. Antibodies were detected and reported using the LABScreen Single Antigen Bead assay with microbeads coated with single HLA class I antigens. Pre-transplant and post-transplant data were collected and the graft outcomes of 16 kidney graft recipients with antibodies to HLA-C locus antigens were compared to the outcomes in 56 recipients with antibodies to HLA-A or -B locus determined antigens. The one-year acute rejection rate was 6% in those with donor-specific antibodies (DSA) to HLA-C locus antigens and 20% in those with DSA to HLA-A or -B locus antigens. The graft survival rate was 100% in those with DSA to HLA-C locus antigens and 95% in those with DSA to HLA-A or -B locus antigens. None of the numerical differences were statistically significant (p>0.05). The presence of circulating pre-transplant IgG antibodies directed at kidney donor HLA-C locus antigens alone may not be associated with an increased risk of acute rejection or a decreased graft survival rate. Our observations support the concept that circulating pre-transplant IgG antibodies directed at kidney donor HLA-C locus antigens alone do not negatively impact kidney allograft outcomes and that the mean fluorescence intensities of the antibodies directed at HLA-C locus alone should not be used to list unacceptable HLA-C locus antigens for kidney allocation. A study with a larger cohort is needed to investigate our hypothesis.

We report three interesting cases concerning antibody-mediated rejection (AMR), associated or not with anti-donor-specific antibodies, and detection of implicated molecular epitopes. The first report presents a case of intra-allele sensitization. The second case presents an interesting case concerning Luminex mean fluorescence intensity (MFI) levels considered to be low risk antibodies (<1000), but producing AMR. The third case occurred after a second kidney transplantation mediated by antibodies directed against HLA-C antigens (MFI<1000) in the previous transplantation (which was considered to be an indicator of low-risk of AMR).

A significant barrier to long-term transplant success is the development of de novo donor-specific human leukocyte antigen (HLA) antibodies. The best approach to minimize the risk of developing such antibodies is an HLA identical transplant, but the likelihood of finding such an organ is very low. The alternative is to identify "permissible mismatches" - HLA antigen mismatches that are less likely to induce an immune response. In the past few years, it has become clear that matching at the "epitope level" is the likely solution; however, we are still struggling with how to define HLA epitopes. Herein, we provide a short review of the development of the epitope concept within the HLA field, with the hope that it sheds light on present knowledge. We follow with our personal opinion on where the future is leading us.

Organ transplantation overcomes conservative therapy to improve patient longevity. Despite the improvement of donor-recipient compatibility tests and intensified immunosuppressive agents, long-term graft survival remains poor because of acute and chronic injury driven by immunologic and non-immunologic factors. The significant immunological barrier for graft longevity is antibody-mediated rejection. Antibodies reactive to donor-specific human leukocyte antigens (HLA) have been shown to have adverse effects on the transplanted organ. However, there is minimal or controversial data supporting a pathogenic effect of antibodies against non-HLAs. This review discusses non-HLA antibodies and relevant antigen targets that have been uncovered by molecular medicine and correspond with acute rejection and chronic allograft injury. Updated proteomic evaluation may improve our knowledge of the immune response by enhancing immunologic epitope determination outside the scope of HLA.

Antibody-mediated rejection in solid organ transplantation is associated with significant organ dysfunction and allograft loss. Donor-specific antibodies against human leukocyte antigens (HLAs) have been a major focus for research, clinical testing, and therapies. Recently, non-HLA autoantibodies to various endothelial antigens including angiotensin II type 1 receptor, endothelin-1 type A receptor, Major Histocompatibility Complex Class 1-Related Chain A, perlecan, and collagen V are emerging as both potential mediators of allograft dysfunction and targets for intervention. Incorporation of non-HLA antibodies into clinical practice is currently not standardized due to a lack of consensus regarding the pathogenic effects on the allograft. Treatment strategies for non-HLA antibodies are evolving and remain an area that warrants further investigation.