Higher Mean Fluorescence Intensity Research Articles

#1016 Kidney transplantation in highly sensitized patients with preformed donor-specific antibodies through personalized delisting strategies

Abstract Background and Aims This study explores a personalized delisting strategy enabling kidney transplantation in highly sensitized patients with preformed donor-specific anti-HLA antibodies (DSA) trying to minimize the risk of antibody-mediated rejection (ABMR). Method Retrospective single-centre analysis of 50 kidney transplant recipients with preformed DSA (preDSA) after employing a delisting strategy to enhance their access to transplantation, and without received a pre-transplant or immediate post-transplant desensitization protocol. The delisting approach focused on allowing less deleterious antibodies according to their mean fluorescence intensity (MFI), anti-HLA class and C1q study results. The strategy consisted on eliminating as prohibited HLA antigens those recognized by antibodies with the lowest MFI in the following order: 1st- MFI < 5000; 2nd- MFI < 10000; 3rd-Any MFI. Additionally, delisting prioritized a single locus in the following order: 1st- one of the HLA class I loci (A, B, or Cw); 2nd- the rest of the class I loci; 3rd- HLA-DP, followed by DR. C1q studies were performed before delisting and transplantation was contraindicated in the presence of DSA detected in the C1q assay. Two comparative cohorts included 50 sensitized recipients without pre-transplant DSA (SwoDSA) and 50 non-sensitized recipients (NS). Results The delisting strategy allowed transplantation with preformed DSA and demonstrated comparable rejection rates to SwoDSA and NS groups (16%, 14% and 8%, respectively; log-rank = 0.28). However, the occurrence of acute ABMR was 12% in the preDSA group, significantly higher than in SwoDSA (1%) and NS (0%) (log-rank = 0.0093). The immunological risk assumed in delisting correlated with ABMR incidence (patients with ABMR had significantly higher sum MFI [mean ± SD: 9301 ± 6340 vs 4492 ± 5641; p = 0.049]), emphasizing the importance of tailored strategies. DSA persistence after transplantation correlated with higher MFI and increased ABMR risk. Of note, 16% of preDSA patients developed de novo DSA versus only the 4% of SwoDSA and 0% of NS patients (p = 0.004). After a mean follow-up of 3.1 ± 2.1 years, the 1-, 3- and 5-year death-censored allograft survival rates were 100%, 90% and 78%, respectively, in the preDSA group; 98%, 84% and 84% in the SwoDSA group and 100% in NS group (log-rank = 0.02). Conclusion The present study demonstrates the feasibility and acceptable outcomes of kidney transplantation in highly sensitized patients with preformed DSA through a personalized delisting strategy without using desensitization protocols.

The efficacy and safety of protein A immunoadsorption combined with rituximab treatment for highly sensitized patients undergoing haplo-hematopoietic stem cell transplantation

Objective: To investigate the efficacy and safety of protein A immunoadsorption (PAIA) combined with rituximab (RTX) in highly sensitized patients who underwent haplo-hematopoietic stem cell transplantation (haplo-HSCT) . Methods: The clinical data of 56 highly sensitized patients treated with PAIA and RTX before haplo-HSCT at the First Affiliated Hospital of Soochow University and Soochow Hopes Hematonosis Hospital between March 2021 and June 2023 were retrospectively analyzed. The number of human leukocyte antigen (HLA) antibody types and the mean fluorescence intensity (MFI), humoral immunity, adverse reactions during adsorption, and survival within 100 days before and after adsorption were measured. Results: After receiving the PAIA treatment, the median MFI of patients containing only HLA Ⅰ antibodies decreased from 7 859 (3 209-12 444) to 3 719 (0-8 275) (P<0.001), and the median MFI of HLA Ⅰ+Ⅱ antibodies decreased from 5 476 (1 977-12 382) to 3 714 (0-11 074) (P=0.035). The median MFI of patients with positive anti-donor-specific antibodies decreased from 8 779 (2 697-18 659) to 4 524 (0-15 989) (P<0.001). The number of HLA-A, B, C, DR, and DQ antibodies in all patients decreased after the PAIA treatment, and the differences were statistically significant (A, B, C, DR: P<0.001, DQ: P<0.01). The humoral immune monitoring before and after the PAIA treatment showed a significant decrease in the number of IgG and complement C3 (P<0.001 and P=0.002, respectively). Forty-four patients underwent HLA antibody monitoring after transplantation, and the overall MFI and number of antibody types decreased. However, five patients developed new antibodies with low MFI, and nine patients continued to have high MFI. The overall survival, disease-free survival, non-recurrent mortality, and cumulative recurrence rates at 100 days post-transplantation were 83.8%, 80.2%, 16.1%, and 4.5%, respectively. Conclusions: The combination of PAIA and RTX has a certain therapeutic effect and good safety in the desensitization treatment of highly sensitive patients before haplo-HSCT.

Impact of donor-specific antibody with low mean fluorescence intensity on allograft outcomes in kidney transplant

BackgroundImmune-mediated rejection is the most common cause of allograft failure in kidney transplant (KT) patients. Exposure to alloantigen, including human leukocyte antigen (HLA), results in the production of donor-specific antibodies (DSA). There are limited data about low levels of mean fluorescence intensity (MFI) DSA, especially post-transplantation. This study evaluated allograft outcomes in KT patients with low MFI DSA. MethodsFrom January 2007 to December 2021, KT patients who were tested for post-transplant DSA at Ramathibodi Hospital, Bangkok, Thailand, with the DSA MFI ≤ 1000 were evaluated. These KT patients were categorized into two groups: very low DSA (VLL; MFI = 1–500) and low DSA (LL; MFI = 501–1000). All KT patients were evaluated for the primary outcomes, such as the incidence of acute rejection, serum creatinine levels at one and five years after transplantation as well as allograft and patient survivals. ResultsAmong 36 KT patients 25 were included as those with VLL and 11 as those with LL. The LL group had significantly higher T-cell mediated allograft rejection (TCMR) than the VLL group (45% vs. 12%, P = 0.04). In addition, 10 patients, 5 in the VLL group and 5 in the LL group developed antibody-mediated allograft rejection (ABMR). Both TCMR and ABMR were confirmed by biopsy results. There was a trend toward higher MFI in KT patients with ABMR than without ABMR (P = 0.22). At 5 post-transplant years, serum creatinine, allograft and patient survivals were comparable between these two groups. Furthermore, the univariate and multivariate analyzes revealed that the LL group was a high risk for rejection. ConclusionLow MFI DSA values after transplantation may be associated with a higher incidence of rejection, but this finding did not show differences in allograft and patient survival in this study's analysis.

Pre-transplant donor specific antibodies in ABO incompatible kidney transplantation - data from the Swiss transplant cohort study.

Living donor (LD) kidney transplantation in the setting of ABO blood group incompatibility (ABOi) has been previously reported to be associated with increased risk for antibody-mediated rejection (ABMR). It is however unclear if the presence of pre-transplant donor specific antibodies (DSA) works as an additive risk factor in the setting of ABOi and if DSA positive ABOi transplants have a significantly worse long-term outcome as compared with ABO compatible (ABOc) DSA positive transplants. We investigated the effect of pre-transplant DSA in the ABOi and ABOc setting on the risk of antibody-mediated rejection (ABMR) and graft loss in a cohort of 952 LD kidney transplants. We found a higher incidence of ABMR in ABOi transplants as compared to ABOc transplants but this did not significantly affect graft survival or overall survival which was similar in both groups. The presence of pre-transplant DSA was associated with a significantly increased risk of ABMR and graft loss both in the ABOi and ABOc setting. We could not detect an additional risk of DSA in the ABOi setting and outcomes were comparable between DSA positive ABOi and ABOc recipients. Furthermore, a combination of DSA directed at both Class I and Class II, as well as DSA with a high mean fluorescence intensity (MFI) showed the strongest relation to ABMR development and graft loss. The presence of pre-transplant DSA was associated with a significantly worse long-term outcome in both ABOi and ABOc LD kidney transplants and our results suggests that the risk associated with pre-transplant DSA is perhaps not augmented in the ABOi setting. Our study is the first to investigate the long-term effects of DSA in the ABOi setting and argues that pre-transplant DSA risk could potentially be evaluated similarly regardless of ABO compatibility status.

Targeting Distinct Epitopes of FCRL5 Using CAR-T Generated with Sequences Derived from Newly Identified FCRL5 Humanized Antibodies Resulted in Potent Anti-Myeloma Activity in Vitro and In Vivo

Despite improvement in treatment options for multiple myeloma (MM) patients, including targeted immunotherapies, MM remains incurable. Given the potential for a single CAR-T treatment to generate long-term remission and the optimal expression profile of FCLR5 (expressed on most MM with limited expression on normal cells), we generated new FCLR5 antibodies and obtained single chain variable fragment (scFv) sequences to develop FCRL5 CAR-Ts. The extracellular domain of FCRL5 has 9 Ig domain subunits (D1-9). We immunized ATX mice (Alloy Therapeutics), genetically engineered to produce humanized antibodies, with human membrane proximal D9 of FCRL5 since cell surface membrane proximal epitopes enhance membrane synapse interactions. Engaging D1-8 could also be effective for targeting FCRL5 by CAR-T, so we immunized mice with full-length FCRL5 and screened for clones binding to D1-8. Because we found FCRL5 expression on primary MM cells was universal but modest and variable, we prioritized screening the 40 antibodies we identified (23 bound to D9 and 17 bound to D1-8) for binding to MOLP-2 cells, known to endogenously express low levels of FCRL5 using flow cytometry. The thirteen best binders (highest mean fluorescence intensity; MFI) were next tested for binding to the MM.1S cell line modified to overexpress FCRL5. All 13 antibodies bound with high efficiency. We generated CAR-T (4-1BB CD3z) for the top seven clones (four D9; three D1-8 binders) and assessed cytolytic activity on MOLP-2 target cells modified to express luciferase (MOLP2-CG) and OPM2 cells modified to express luciferase and high levels of full length human FCRL5 (OPM2-CG-FCRL5). Effector (E) CAR-T cells were incubated with target (T) cells at a range of E:T ratios for 48 hours and bioluminescence (BLI) was used to determine cytolytic activity. Most clones showed high killing activity. We tested clones 977 (D1-8), 942, 947 and 980 (D9 binders) for in vivo studies due to highest performance in vitro. NSG mice were engrafted with 1x10 6 OPM2-CG-FCRL5 cells i.v. into tail veins and treated 14 days later i.v. with 2x10 6 FCRL5 CAR-T or BCMA CAR-T, the current lead CAR-T target in MM as an independent positive control. We found significant extension of survival compared to controls (Kaplan Meier, 942: P&amp;lt;.001, 947 P&amp;lt;.003, 977 P&amp;lt;.0002, 980 P&amp;lt;.008 BCMA P&amp;lt;.001) and reduced tumor burden (longitudinal BLI) in all CAR-T treated mice compared to untreated and mice treated with non-transduced T cell controls in this aggressive tumor model ( Figure 1). Co-targeting BCMA and FCRL5 may prevent antigen escape since it targets cells expressing either or both FCRL5 and BCMA. We generated a bi-targeted CAR targeting both BCMA and FCRL5 (clone D1-8 977). To confirm each scFv was active, we ran in vitro killing assays using BCMA knockout (KO)-OPM2-CG FCRL5 (BCMA-FCRL5+), OPM2-CG (BCMA+ FCLR5-) targets and found killing of each cell type by our bi-targeted CAR-T, confirming both scFvs were active. BCMA-FCRL5 CAR-T killed OPM2-CG FCRL5 and MOLP-2-CG cells (both BCMA+FCRL5+) but not BCMA KO OPM2 (BCMA-FCLR5-) cells. The best performing BCMA CAR-T in the clinic targets two BCMA epitopes- our scFv's targeting different domains of FCLR5 opens the opportunity for bi-targeting the FCLR5 protein itself. We generated an FCRL5-FCRL5 bi-targeted CAR-T using clone 977 (D1-8) and 980 (D9) and demonstrate activity of this CAR-T on OPM2-CG-FCRL5 cells; ongoing studies will determine if each scFv is active and whether in vivo activity is superior to single epitope targeting in vivo. Finally, blocking studies of the D9 antibodies showed potential for multiple epitopes within that domain which may facilitate co-targeting D9. We tested four FCRL5 CAR-T (4-1BB CD3z) predicted to target distinct D9 epitopes and observed high cytolytic activity in killing assays using OPM2-CG-FCRL5 target cells. We tested each in vivo by injecting NSG mice in with OPM2-CG and 18 days later treated with 2x10 6 FCRL5 CAR-T. As above, we observed significant extension of survival and reduced tumor burden compared to controls. Future studies will assess feasibility of D9 co-targeting. Together, our data demonstrate efficacy of FCRL5 CAR-T using novel sequences targeting FCLR5 both within and outside of D9 and support development of an FCRL5 CAR-T clinical candidate for use in relapsed/refractory MM patients.

Post-transplant donor-specific anti-HLA antibodies with a higher mean fluorescence intensity are associated with graft fibrosis in pediatric living donor liver transplantation.

The roles of post-transplant anti-HLA donor specific antibody (DSA) in pediatric liver transplantation (LT), including therapeutic strategies, remain controversial. This study aimed to identify the risks of post-transplant DSA for graft fibrosis progression in pediatric living donor LT (LDLT). We retrospectively evaluated 88 LDLT pediatric cases between December 1995 and November 2019. DSAs were assessed with single antigen bead test. Graft fibrosis was histopathologically scored with METAVIR and the centrilobular sinusoidal fibrosis system. Post-transplant DSAs were detected in 37 (52.9%) cases at 10.8 (1.3-26.9) years post-LDLT. The histopathological examination of 32 pediatric cases with post-transplant DSA revealed that 7 (21.9%) with a high DSA-MFI (≥9,378) showed graft fibrosis progression (≥F2). No graft fibrosis was observed in the subjects with a low DSA-MFI. The risk factors for developing graft fibrosis in pediatric cases with post-transplant DSA were an older graft age (>46.5 years old), lower platelet count (<10.7 × 104/ml) and higher Fib4 index (>0.7807, recipient age; >1.8952, donor age). Limited efficacy of additional immunosuppressants was observed in DSA positive pediatric cases. In conclusion, pediatric cases with a high DSA-MFI and risk factors should undergo a histological examination. The appropriate treatment for post-transplant DSA in pediatric LT needs to be determined.

The Prognostic Value of Eryptosis Parameters in the Cerebrospinal Fluid for Cerebral Vasospasm and Delayed Cerebral Ischemia Formation

Delayed cerebral ischemia (DCI) and cerebral vasospasm (VS.) contribute to poor outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH). The pathophysiology of DCI is not fully understood, and this has hindered the adoption of a uniform definition. Reliable diagnostic tests and effective evidence-based treatment are lacking. This study explored the possibility of using eryptosis parameters in the cerebrospinal fluid (CSF) as a marker for early detection of VS and DCI. Twenty-one SAH patients were recruited and treated at Kharkiv Regional Hospital. The occurrences of DCI and VS were also recorded. Flow cytometry was used to assess eryptosis indices in the CSF by analyzing phosphatidylserine externalization in erythrocytes using annexin V staining and evaluating reactive oxygen species generation using 2,7-dichlorodihydrofluorescein (DCF) diacetate staining. The percentage of annexin-positive red blood cells (RBCs) in the VS group was significantly higher than that in the non-VS group (P=0.0017). Furthermore, higher values of this index were significantly associated with DCI formation (P<0.0001). Patients with VS had higher mean fluorescence intensity values of DCF in RBCs compared to patients without VS (P=0.0258). Patients with DCI also had higher mean fluorescence intensity values of DCF in RBCs (P=0.0282). A higher percentage of annexin-positive RBCs following 3days of aSAH was correlated with poor 3-month neurological outcomes (r=0.7). Our findings indicate a strong correlation between eryptosis level and DCI in a sizable series of patients with aSAH. Correlations between eryptosis indicators in the CSF and clinical and radiological manifestations suggest that eryptosis parameters are promising diagnostic biomarkers for DCI.

Donation type and the effect of pre-transplant donor specific antibodies - Data from the Swiss Transplant Cohort Study.

The type of donation may affect how susceptible a donor kidney is to injury from pre-existing alloimmunity. Many centers are, therefore, reluctant to perform donor specific antibody (DSA) positive transplantations in the setting of donation after circulatory death (DCD). There are, however, no large studies comparing the impact of pre-transplant DSA stratified on donation type in a cohort with a complete virtual cross-match and long-term follow-up of transplant outcome. We investigated the effect of pre-transplant DSA on the risk of rejection, graft loss, and the rate of eGFR decline in 1282 donation after brain death (DBD) transplants and compared it to 130 (DCD) and 803 living donor (LD) transplants. There was a significant worse outcome associated with pre-transplant DSA in all of the studied donation types. DSA directed against Class II HLA antigens as well as a high cumulative mean fluorescent intensity (MFI) of the detected DSA showed the strongest association with worse transplant outcome. We could not detect a significant additive negative effect of DSA in DCD transplantations in our cohort. Conversely, DSA positive DCD transplants appeared to have a slightly better outcome, possibly in part due to the lower mean fluorescent intensity (MFI) of the pre-transplant DSA. Indeed when DCD transplants were compared to DBD transplants with similar MFI (<6.5k), graft survival was not significantly different. Our results suggest that the negative impact of pre-transplant DSA on graft outcome could be similar between all donation types. This suggests that immunological risk assessment could be performed in a similar way regardless of the type of donor kidney transplantation.

Human leukocyte antigen antibodies and leukocyte antigen/killer-cell immunoglobulin-like receptor genes are important in transplant immunology in the liver

Many mechanisms have been proposed to explain the hypothetical state of hepatic tolerance, which is described by eventual imbalances or deregulation in the balance of cytokines, mediators, effectors, and regulatory cells in the complex milieu of the liver. In this section, we will comment on the importance of donor-specific anti-human leukocyte antigen (HLA) antibodies (DSA) as well as the compatibility and pairings of HLA and killer-cell immunoglobulin-like receptor (KIR) genotypes in the evolution of liver transplantation. Thus, HLA compatibility, viral infections, and HLA-C/KIR combinations have all been linked to liver transplant rejection and survival. There have been reports of increased risk of acute and chronic rejection with ductopenia, faster graft fibrosis, biliary problems, poorer survival, and even de novo autoimmune hepatitis when DSAs are present in the recipient. Higher mean fluorescence intensity (MFI) values of the DSAs and smaller graft size were associated with poorer patient outcomes, implying that high-risk patients with preformed DSAs should be considered for selecting the graft placed and desensitization methods, according to the investigators. Similarly, in a combined kidney-liver transplant, a pretransplant with a visible expression of several DSAs revealed that these antibodies were resistant to treatment. The renal graft was lost owing to antibody-mediated rejection (AMR). The HLA antigens expressed by the transplanted liver graft influenced antibody elimination. Pathologists are increasingly diagnosing AMR in liver transplants, and desensitization therapy has even been employed in situations of AMR, particularly in patients with DSAs in kidney-hepatic transplants and high-class II MFI due to Luminex. In conclusion, after revealing the negative impacts of DSAs with high MFI, pretransplant virtual crossmatch techniques may be appropriate to improve evolution; however, they may extend cold ischemia periods by requiring the donor to be typed.

Assessment of donor specific IgG Anti-HLA subclasses before and after kidney transplantation in Upper Egypt: A Single Center Study

Donor specific antibodies (DSAs) are known as the leading cause of antibody mediated rejection (AMR), graft loss in kidney transplant (KT) recipients. DSAs characteristics, as immunoglobulin (Ig) classes, subclasses, and strength, are important to assess the immunological risk, early prediction of AMR, and therefor proper management. This longitudinal, case control study included 32 KT recipients at Assiut University Urology Hospital and 10 age and sex matched normal subjects as the control group. Total IgG, its subclasses and anti-human leukocyte antigen (anti-HLA) panel reactive antibody (PRA) were detected pre-transplantation (pre-TX), at 6-12- and 24-36-months post-TX. Rejection occurred in 4 recipients, 3 of them had high total IgG, IgG1 and/or IgG3. IgG2 and IgG4 were normal in all recipients. There were preformed anti-DSAs antibodies in 3/32 recipients (9.4%). Of these, two recipients became negative with no rejection occurred. The third recipient had high post-TX mean fluorescence intensity (MFI) and AMR occurred. The pre-TX PRA was negative in 29/32 recipients (90.6%). The PRA was negative in 8/29 recipients (27.6%) and the remaining 21/29 recipients (72.4%) developed de novo DSAs post-TX (MFI < 3000->10000). Rejection occurred with both low and high MFI. In 11 recipients, anti-HLA class I and II were not different between pre-TX, 3-6- and 24-36 months post-TX with no rejection occurred. The frequency and median levels of total IgG, IgG1 and IgG3 were increased in all recipients 24-36 months post-TX when compared with their levels pre-TX and 6-12 months post-TX in the 11 recipients and with the control group. The graft survival time significantly decreased in recipients with positive post-TX class I PRA. In conclusion, preformed DSAs may persist post-TX or turn negative. De novo DSAs developed post-TX even in non-sensitized recipients. Serum total IgG, IgG1 and IgG3 frequency increase 2-3 years post-TX.

Low Incidence of Acute Antibody-Mediated Rejection after HLA Desensitization in Living Donor Kidney Transplant Recipients.

Desensitization allows the performance of human leukocyte antigen (HLA)-incompatible transplants. However, the incidence of acute rejection (AR) is high. This study aims to analyze the incidence of AR after transplantation with HLA-incompatible living donors in patients who underwent desensitization. Patients were immunosuppressed with tacrolimus, mycophenolic acid derivatives, and steroids after being desensitized with rituximab, plasma exchange, and/or immunoadsorption with specific cytomegalovirus immunoglobulins. A negative complement-dependent cytotoxicity or flow cytometry crossmatch and a donor-specific antibody titer < 1000 mean fluorescence intensity (MFI) were used to determine desensitization efficacy. A total of 36 patients underwent desensitization, and 27 (75%) were transplanted. After a follow-up of 58 ± 58 months (Min−Max: 0.13−169.5), five episodes of AR occurred: two antibody-mediated and three T-cell-mediated. No differences were found in baseline calculated panel-reactive antibodies (cPRA), class I or II MFI, number of antibodies, or Relative Intensity Scale (RIS) between AR and non-AR patients. Patients with antibody-mediated AR had higher cPRA (NS), MFI class I (p = 0.07) and class II (p = 0.006), and RIS (p = 0.01). The two patients with antibody-mediated AR and one patient with T-cell-mediated AR lost their grafts. In conclusion, the incidence of acute antibody-mediated rejection after desensitization was 7.4%, which occurred early post-transplantation in patients with high MFI and was associated with early graft loss.

Successful desensitization of high level donor-specific anti-HLA antibody in patients with hematological diseases receiving haploidentical allografts.

Donor-specific anti-human leukocyte antigen (HLA) antibody (DSA) is associated with a higher incidence of graft failure and mortality in HLA-mismatched allograft settings. However, the optimal protocol of desensitization for patients with positive DSA remains uncertain. We investigated the effectiveness of a desensitization protocol, including rituximab, high-dose intravenous immunoglobulin (IVIG), and a single session of plasma exchange (PE), for haploidentical allograft recipients with a high mean fluorescence intensity (MFI) level of DSA (≥ 5,000). Eleven patients with hematological disease who had positive DSA (median, 11,676, range 5387-20,435) were desensitized by the protocol. All of the patients achieved hematopoietic recovery. The median times for neutrophil and platelet engraftment were 13 (range, 11-26) days and 19 (range, 11-90) days, respectively. Grade II-IV acute graft-versus-host disease (GVHD) was seen in one patient and was controlled completely. Chronic cutaneous GVHD was seen in eight patients. Nine patients are alive with good performance so far. One patient suffered extramedullary relapse, and one patient died of transplantation-associated thrombotic microangiopathy. The 1-year probability of overall survival was 81.8%. These results suggest that successful desensitization could be obtained by a combination of rituximab, high-dose IVIG, and PE for haploidentical allograft recipients with high MFI levels of DSA.

Abstract 3436: Novel multifunctional tetravalent CD38 NKp46 FLEX NK࣪ engagers actively target and kill multiple myeloma cells

Abstract Given that CD38 is a clinically validated target for NK cell mediated cytotoxicity in multiple myeloma, we sought to leverage our FLEX NKTrademark platform to create a NK engager antibody targeting CD38. FLEX NKTrademark is a proprietary platform for production of tetravalent multifunctional NK engager antibodies with a novel FLEX linker to allow for simultaneous binding of both the targeted cancer cells and NK cells. NK engagement and activation is mediated through a binder directed against the natural cytotoxicity receptor NKp46. With the FLEX NKTrademark scaffold, we created two novel tetravalent NK cell engagers targeting CD38, one with a wild type (WT) Fc and one with a mutant null Fc. Both CD38 NKp46 engagers showed dose dependent binding to CD38 expressing multiple myeloma cell lines MM1S and KMS11 and no binding to a CD38 knock out MM.1S cell line. Both engagers also bound multiple myeloma cell lines with ~ 2-fold higher mean fluorescence intensity than anti-CD38 monoclonal antibody (mAb)or daratumumab alone. Epitope mapping studies for our anti-CD38 mAb using alanine scanning mutagenesis showed that amino acid S274 on CD38, critical for binding to daratumumab, is not important for our anti-CD38 antibody binding to CD38, suggesting a distinct epitope detected by our antibody. Interestingly, the NKp46 monoclonal antibody alone without a functional Fc induced peripheral blood NK cell cytolysis of the multiple myeloma cells, consistent with a prior report that NKp46 plays a key role in NK-cell mediated killing of myeloma cells. Both CD38 NKp46 engagers showed further enhanced dose dependent NK cell redirected cytolysis and degranulation against multiple myeloma cells compared to anti-NKp46 monoclonal antibody. The CD38 NKp46 engagers demonstrated higher cytotoxicity than the anti-CD38 binder mAb or daratumumab alone. The CD38 NKp46 engager with the WT Fc is more potent in induction of TNF-α and IFN-ɣ production compared to daratumumab and the engager with the mutant Fc. No IL-1β or IL-6 was induced by the engagers or daratumumab. Daratumumab treatment resulted in NK cell fratricide, while no fratricide was observed with the CD38-NKp46 NK engager with the mutant Fc. In peripheral blood mononuclear cell hemato-toxicity studies depletion of monocytes and NK cells were observed with daratumumab but no depletion was observed with the CD38 NKp46 engager with the mutant Fc. These results suggest that the CD38 NKp46 engagers have a favorable NK cell engager profile for targeting CD38 expressing multiple myeloma that’s distinct from daratumumab. Citation Format: Liang Lin, Hao-Ming Chang, Cecilia Nakid, Stanley Frankel, Dennis Wu, Jean Kadouche, Daniel Teper, Ofer Mandelboim, Jean-Christophe Bories, Antonio Arulanandam, Wei Li. Novel multifunctional tetravalent CD38 NKp46 FLEX NK࣪ engagers actively target and kill multiple myeloma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3436.

I Got It from My Mamma: Sensitization by Transplacental Transfer of Maternal HLA Antibodies Managed with Manual Exchange Transfusion

<h3>Introduction</h3> During pregnancy antibodies are passed from mother to fetus with many known immunologic benefits. There is no data about the implications of this on pediatric transplant immunology. We report a case of transplacental transfer of maternal Human Leukocyte Antigen (HLA) antibodies affecting cardiac transplant candidacy in an infant with cardiomyopathy. <h3>Case Report</h3> A 3-week-old Amish boy with a homozygous MYBPC3 mutation causing severe cardiomyopathy admitted for refractory heart failure symptoms was referred for heart transplant evaluation. Labs were notable for high levels of high Mean Fluorescence Intensity (MFI) HLA antibodies (Panel of Reactive Antibodies (PRA) Class I: 74%, Class II: 80%) with no known sensitizing events. Maternal history was remarkable for multiple miscarriages raising the question that his HLA antibodies could be a result of maternal HLA sensitization with transplacental antibody transfer versus active antibody production by the child. Subsequent antibody testing of his mother's blood and breast milk revealed nearly identical HLA antibody profiles to that of the patient. Given the patient's prohibitively small size for antibody removal by plasmapheresis, we elected to perform double volume whole blood exchange transfusions with serial monitoring of his HLA antibodies. Considering his severe ventricular dysfunction, exchange transfusions were performed manually via the push pull method over 6 hours. He ultimately underwent 6 exchange transfusions over the course of 7 weeks. Serial antibody testing revealed a progressive decline in all his HLA antibody levels to <3,000 MFI with only mild rebound after each transfusion consistent with tissue redistribution rather than active antibody production. He was successfully transplanted with a negative PRA (Class I: 0%, Class II: 0%) without post-transplant complications. <h3>Summary</h3> We believe this is the first report demonstrating transplacental acquisition of maternal HLA antibodies in an infant successfully removed via serial whole blood exchange transfusions enabling successful cardiac transplantation.

High Keratin-7 Expression in Benign Peri-Tumoral Prostatic Glands Is Predictive of Bone Metastasis Onset and Prostate Cancer-Specific Mortality.

Simple SummaryKeratin-7 overexpression has been associated with poor prognosis in several cancers. To determine its prognostic relevance in prostate cancer, cohorts of patients presenting with localized or advanced tumors were investigated by either immunohistochemistry or immunofluorescence. Keratin-7 immunostaining was absent in localized tumors and rare in advanced tumors. By contrast, it was abundantly detected in basal cells within benign glands surrounding localized tumors. Importantly, high keratin-7 expression in benign peri-tumoral glands was correlated with shorter bone metastasis-free survival and increased risk of cancer-specific mortality. This study establishes keratin-7 expression in localized prostate cancer specimens as a promising biomarker of disease progression.Background: New predictive biomarkers are needed to accurately predict metastasis-free survival (MFS) and cancer-specific survival (CSS) in localized prostate cancer (PC). Keratin-7 (KRT7) overexpression has been associated with poor prognosis in several cancers and is described as a novel prostate progenitor marker in the mouse prostate. Methods: KRT7 expression was evaluated in prostatic cell lines and in human tissue by immunohistochemistry (IHC, on advanced PC, n = 91) and immunofluorescence (IF, on localized PC, n = 285). The KRT7 mean fluorescence intensity (MFI) was quantified in different compartments by digital analysis and correlated to clinical endpoints in the localized PC cohort. Results: KRT7 is expressed in prostatic cell lines and found in the basal and supra-basal compartment from healthy prostatic glands and benign peri-tumoral glands from localized PC. The KRT7 staining is lost in luminal cells from localized tumors and found as an aberrant sporadic staining (2.2%) in advanced PC. In the localized PC cohort, high KRT7 MFI above the 80th percentile in the basal compartment was significantly and independently correlated with MFS and CSS, and with hypertrophic basal cell phenotype. Conclusion: High KRT7 expression in benign glands is an independent biomarker of MFS and CSS, and its expression is lost in tumoral cells. These results require further validation on larger cohorts.

Donor-derived Cell-free DNA Complements De Novo Class II DSA in Detecting Late Alloimmune Injury Post Kidney Transplantation.

Background.We sought to evaluate the association between de novo donor-specific antibodies (dnDSAs) class and their mean fluorescence intensity (MFI) with donor-derived cell-free DNA (dd-cfDNA), aiming to further clarify the biomarker utility of these noninvasive tests in relation to renal allograft function and histology.Methods.The study included kidney transplant recipients (n = 171) who underwent surveillance testing with DSA and dd-cfDNA as part of their clinical care between September 2017 and December 2019 at our center.Results.We identified dnDSA in 43 patients (25%) at a median of 4.63 y (IQR, 1.5–7) posttransplant. The presence of DSA with MFI >2500 was associated with a median dd-cfDNA of 0.96% (IQR, 0.26–2.95) significantly higher than in patients with DSA MFI <2500 (0.28%; IQR, 0.19–0.39) or without detectable DSA (0.22%; IQR, 0.17–0.37; P < 0.001). Class II dnDSAs were the most prevalent dnDSA (88.3%), the majority with MFI >2500 (82.9%). Patients with DQ-dnDSAs (47.4%) had higher MFI and dd-cfDNA levels than other class II dnDSAs. By comparison, all patients that developed only class I DSAs had MFI <2500 and a low dd-cfDNA. In addition, the serum creatinine was 1.55 ± 0.48 mg/dL in those dnDSA-negative, 1.15 ± 0.37 mg/dL in those with dnDSA MFI <2500, and 1.53 ± 0.66 mg/dL in those with dnDSA MFI >2500 (P = 0.05). After multivariate adjustment, an elevated dd-cfDNA was independently associated with the presence of dnDSA with MFI ≥2500. We identified that both dd-cfDNA and dnDSAs were strongly associated with antibody-mediated rejection, whereas for individual Banff histological lesions, DSA MFIs ≥2500 had the strongest association with C4d staining score and dd-cfDNA >1% with microvascular inflammation.Conclusions.Our study identifies class II dnDSA as being strongly associated with late alloimmune injury post kidney transplant independent of allograft dysfunction and shows that dd-cfDNA may complement the clinical significance of dnDSAs.

Clinical Significance of C3d Assay in Kidney Transplant Recipients With Donor-Specific Anti–Human Leukocyte Antigen Antibodies

Antibody-mediated rejection (AMR) is a major cause of allograft loss in kidney transplant. Although donor-specific anti-human leukocyte antigen antibody (DSA) is a key cause of AMR, not all patients with DSA are diagnosed as having AMR and show poor allograft outcomes. This study aimed to evaluate clinical significance of C3d-binding activity in patients with DSA identified by single-antigen bead (SAB) assay. A total of 168 recipients screened for DSA from 2015 to 2018 were enrolled. Among them, 52 patients had DSA confirmed by SAB assay. Sera were tested using the C3d assay on Luminex platform. AMR was defined by kidney allograft biopsy results using Banff 2015 criteria. Of 52 patients, C3d-binding DSAs were detected in 22 patients (42.3%). Indication allograft biopsy was performed in 35 patients, with 31 (88.6%) diagnosed as having AMR. Patients with C3d-binding DSA had more class II SAB-DSA (73.3% vs 100%, P=.015) and showed significantly higher mean (SD) fluorescence intensity of class II SAB-DSA than the C3d-binding DSA(-) group (9606.7 [6096.6] vs 1921.0 [1483.8], P < .001). There was a positive correlation in the highest mean fluorescence intensity between class II SAB-DSA and class II C3d-binding DSA (r=0.70, P < .001). Patients with C3d-binding DSA showed worse death-censored graft survival than those with non-C3d-binding DSA (P=.023). This study showed that presence of C3d-binding DSA was significantly associated with allograft loss in SAB-DSA-positive patients. Further trials are warranted.

Crossmatch, Donor-specific Antibody Testing, and Immunosuppression in Simultaneous Liver and Kidney Transplantation: A Review.

Since the introduction of simultaneous liver-kidney transplantation (SLKT) in the 1960s, the potential for immunological protection from the liver allograft to a simultaneously transplanted kidney has been recognized. Due to expanded indications and changes in allocation policies, there has been increased utilization of SLKT. Despite growing experience, a lack of consensus exists regarding the extent of the immunological privilege of the liver the role for donor-specific HLA antibody (DSA) and crossmatch testing, and appropriateness of modern immunosuppression protocols in SLKT recipients. This review provides a detailed analysis of SLKT outcomes in the context of these factors, suggesting that although the liver can reduce the incidence of antibody-mediated rejection, attention should be given to liver allograft function, previous failed transplants, and other risk factors in pretransplant risk assessment. Current methods of DSA and crossmatch testing in SLKT are also discussed, and the role of specific DSA (high mean fluorescence intensity antibody, C1q+ binding) and their potential importance in posttransplant risk assessment are examined. Finally, trends in SLKT immunosuppression are discussed, including the use of nondepleting agents for induction and de-escalating use of steroids for maintenance immunosuppression. Ongoing research, including multicenter or randomized trials, will be necessary to optimize immune-related outcomes in SLKT recipients.

Prevalence and Risk Factors of Antibodies to Class I and II Human Leukocyte Antigens in Haploidentical Allograft Candidates: A Prospective Study on 3805 Subjects

The aim of this study is to investigate the prevalence and risk factors of anti-human leukocyte antigen (HLA) antibodies in haploidentical candidates.This study was completed at Peking University People's Hospital, Beijing China. We performed a prospective analysis of patients with hematological diseases concerning the prevalence and risk factors of anti-HLA antibodies. Patients were enrolled between July 2015 - December 2019. Serum was collected for PRAs test within 1 month before haploidentical transplantation. The risk factors, such as age, sex, total transfusion, red blood cell (RBC) transfusion, platelet (PLT) transfusion, pregnancy, disease duration and diagnosis were collected. Univariate and multivariate logistic regression analyses were performed to evaluate the risk factors of anti-HLA antibodies.Six hundred and eighty (17.9%) patients were positive for panel reactive antibodies (PRA)-class I, 360 (9.5%) for class II, 768 (20.2%) class I or II, and 272 (7.1%) positive for class I and II both. Multivariate analysis indicated that female was related to higher risk of having PRAs for class I (P = 0.011), class I or II (P = 0.009), anti-HLA-A (P = 0.015), anti-HLA-DP (P = 0.048) and also for having higher mean fluorescence intensity (MFI) (2000 or more) of PRAs in class I (P = 0.020) and class I or II (P = 0.005). Compared to patients with myelodysplastic syndrome (MDS), patients with acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), aplastic anemia (AA) had a lower incidence for PRAs in class I, class II, class I or II, class I and II, anti-HLA-A. anti-HLA-B, anti-HLA-C, anti-HLA-DQ, anti-HLA-DR, anti-HLA-DP (Table 1). Prior pregnancy was a risk factor for PRAs (P < 0.001), and no previous pregnancy group having lower MFI of PRAs in class I (P = 0.001) and class I or II (P = 0.004). PLT transfusion (more than 4 times) rleted with a higher prevalence of PRAs (P < 0.001), and also had a higher MFI of PRAs in class II (P < 0.001), class I and II (P < 0.001). Patients with RBC transfusion (more than 3 times) had a higher prevalence of PRAs in class I (P = 0.001), class II (P = 0.029), class I or II (P < 0.001), anti-HLA-A (P = 0.001), anti-HLA-B (P < 0.001), anti-HLA-C (P = 0.007), anti-HLA-DQ (P < 0.001) and anti-HLA-DR (P = 0.011). In addition, diseases duration (8 months or more) was also associated with higher MFI of PRAs in class I (P = 0.023) and class I or II (P = 0.004).Subgroup analysis showed that 11.7% of pediatric patients were positive for PRAs in class I; 19.2% of adults, 17.9% of elder patients; 12.4% of males; 26.1% of females; 21.0% of patients with AML; 10.5% of patients with acute lymphoblastic leukemia (ALL); 18.9% of patients with AA; 30.3% of patients with MDS; 16.6% of patients with other hematological diseases. The positive rate of class II PRAs in children was 4.3%; 11.1% for adults; 9.5% for elder patients; 5.5% for males; 15.4% for females; 11.4% for patients with AML; 5.2% for patients with ALL; 10.3% for patients with AA; 17.2% for patients with MDS; 6.6% of patients with other hematological diseases. Multivariate analysis showed that, in children, PLT transfusion and diagnosis were the two main risk factors of PRAs in class I and class II (P < 0.001, P = 0.017). In adults, diagnosis (P = 0.003), transfusion (P < 0.001) and pregnancy (P < 0.001) were the three main factors associated with PRAs in class I and transfusion (P < 0.001) and pregnancy (P < 0.001) were the two main factors associated with PRAs in class II. In males, PLT transfusion (P < 0.001) and diagnosis (P < 0.001) were the two main factors associated with PRAs in class I and class II. In ALL subgroup, gender (P = 0.026, P = 0.048), pregnancy (P < 0.001) and transfusion (P < 0.001) were the three main factors associated with PRAs in class I and II. In AA subgroup, gender (P = 0.004) and PLT transfusion (P < 0.001) were risk factors for class I PRAs, pregnancy (P = 0.008) and PLT transfusion (P = 0.003) were risk factors for class II PRAs. In elder patients, females, AML, MDS and other diseases subgroup, transfusion and pregnancy were the two main factors associated with PRAs in class I and class II.Our results indicated that female sex, diagnosis, pregnancy, transfusion, disease duration were independent risk factors of anti-HLA antibodies in haploidentical allograft candidates, which provided evidence for best haploidentical donor selection. The risk factors of anti-HLA antibodies were different among total patients and those of cases in different subgroups. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Food additive E407a stimulates eryptosis in adose-dependent manner.

Concerns about the biosafety of the common food additive E407a have been raised. It has been demonstrated to induce intestinal inflammation, accompanied by activation of apoptosis, upon oral exposure. Thus, it is of interest to investigate how E407a affects eryptosis, asuicidal cell death mode of red blood cells. To evaluate the effects of semi-refined carrageenan (E407a) on eryptosis. Flow cytometry was employed to assess eryptosis in blood exposed to various concentrations of E407a (0 g/L, 1 g/L, 5 g/L, and 10 g/L) during incubation for 24 h by analyzing phosphatidylserine externalization in erythrocytes using annexinV staining and via evaluating reactive oxygen species (ROS) generation using 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA). In addition, the eryptosis indices mentioned above were determined in rats orally administered E407a at adose of 140 mg/kg weight for 2weeks. Confocal scanning laser microscopy was performed to visualize cell membrane scrambling. Oral intake of E407a for 2weeks by rats was not associated with membrane scrambling in erythrocytes. However, ROS overproduction was observed. Meanwhile, incubation of blood with various concentrations of semi-refined carrageenan resulted in adose-dependent promotion of eryptosis, evidenced by the enhanced percentage of annexinV-positive erythrocytes and higher mean fluorescence intensity (MFI) values of annexinV-FITC in all erythrocytes. The highest concentration of E407a promotes astatistically significant increase in ROS generation in erythrocytes, suggesting the role of ROS-mediated induction of eryptosis in this case. Incubation of blood with the food additive E407a leads to the activation of eryptosis in adose-dependent manner. ROS-mediated mechanisms are partially responsible for E407a-induced eryptosis.