Donor-specific Antibody Production Research Articles

Immune Function Testing and Donor-Specific Antibody Production among Heart Transplant Patients

De novo donor-specific antibody (DSA) production has been correlated to the development of antibody-mediated rejection after heart transplantation. It is unclear whether the Cylex ImmuKnow (IK) assay - a rapid, quantitative assessment of T-cell-mediated immune function designed to monitor immunosuppression response - can be a useful tool in overall immunosuppression and identification of patients at risk for de novo DSA production. We hypothesize that, among heart transplant recipients, increased immune function activity as measured by the IK assay would correlate with de novo DSA production. We conducted a single center cohort study including all consecutive single organ heart transplant recipients from 2014 to 2018. The IK assay categorized subjects into low, moderate, or high immune response levels at 6, 12, and 24-month intervals. De novo DSA production based on human leukocyte antigen antibody testing was correlated to IK immune response levels at these time points. 122 single organ heart transplant recipients were included. High immune response levels were noted in 10%, 9%, and 12% of subjects at 6, 12, and 24-months, respectively. Compared to baseline, de novo DSA production was noted in 21%, 23%, and 25% of subjects at the respective time intervals. 6-month IK category did not correlate with DSA production at 6 (r = -0.03, p=0.9), 12 (r = -0.03, p=0.9), or 24 months (r = 0.15, p=0.3). Similar findings were noted at 12 and 24 months. Being in a high IK category at any interval was not associated with de novo DSA production at any time point (p>0.05). Univariate analysis of demographic factors, induction agents used, and co-morbidities (including EMV or CBV infection status) did not reveal significant associations with de novo DSA production. High T-cell-mediated immune response levels, a possible indicator of under-suppression, as measured by the IK assay were not associated with de novo DSA production after heart transplantation.

Pseudomonas Aeruginosa Infection of Lung Transplants Breaks Established Tolerance through B Cell-Intrinsic Innate Immune Signaling That Promotes Intragraft Plasma Cell Generation and Donor Specific Antibody Accumulation

Antibody mediated rejection (AMR) has become an increasingly recognized form of lung transplant rejection. Our group recently identified Pseudomonas aeruginosa infection as a risk factor for clinical AMR but whether infection is sufficient for donor specific antibody (DSA) production and AMR remains unknown. Here we ask in a mouse model of orthotopic lung allograft transplantation whether airway infection with P. aeruginosa promotes DSA generation and AMR. Balb/c lungs were transplanted into the following recipients: B6 WT (wildtype), uMT KO (No B cells), AID/uS KO (Have B cells but are unable to secrete Ig), CD19 Cre , CD19 Cre x TLR4fl/fl and CD19 Cre x MyD88fl/fl. Lung recipients were immunosuppressed with costimulatory blockade on post-operative day (POD) 0 (CD40L Ab) and POD 2 (CTLA4Ig) to induce allograft acceptance. On POD 21 recipients received intratracheal saline or 2 x 105 pfu/g body weight of live P. aeruginosa (clinical isolate M57-15) and then treated with ciprofloxacin from POD 22 to 25. On POD 42 peripheral blood was assessed for DSA and graft tissue evaluated for C4d deposition or quantitated for plasma cells (PC) by flow cytometric analysis (FACS). P. Aeruginosa infection stimulated high amounts of DSA generation and marked C4d deposition. In contrast, C4d deposition was nearly absent in uMT and AID/uS KO recipients. Infected recipients with TLR4 or MyD88 targeted-deficiency in B cells had sharply lower DSA when compared to WT controls. P. aeruginosa infection drove a 4-fold expansion of allograft resident PCs that was largely dependent on maintaining B cell TLR4-MyD88 signaling. In contrast, the spleen and bone marrow had only modest changes in PC abundance despite infection. Surprisingly, re-transplantation of P. aeruginosa-infected lung allografts into secondary uMT KO recipients revealed that lung allograft-resident PCs are capable of producing large amounts of DSA. Collectively, these data show that P. aeruginosa infection abrogates established lung transplant tolerance through engaging innate immune signaling pathways within the B cell compartment. Additionally, we make the novel observation that the lung is a suitable environment for PC development with implications for clinical AMR.

Is Anti-Thymocyte Globulin for Sensitized Patients Undergoing Heart Transplantation Effective?

Anti-thymocyte globulin (ATG) has been used as an induction agent in heart transplant (HTx). Early studies suggest that ATG can decrease de novo donor specific antibody (DSA) production after HTx. It has not been established as to the efficacy of ATG in various levels of sensitized pts undergoing HTx. Between 2010-18, we assessed 370 sensitized pts who were divided into panel reactive antibody (PRA) groups including a control group without sensitization but who received ATG: Group A: 0% (control); Group B: 1-25%; Group C: 26-50%; and Group D: >50%. Pts received ATG immediately post-transplant x5 days. Blood was drawn for antibody detection at baseline, 1, 3, 6, and 12 months after HTx. Groups were assessed for freedom from 1-year survival, rejection (any treated rejection (ATR), acute cellular rejection (ACR), and antibody mediated rejection (AMR)), freedom from non-fatal major adverse cardiac events (NF-MACE: myocardial infarction, percutaneous coronary intervention/angioplasty, new congestive heart failure, pacemaker/implantable cardioverter-defibrillator placement, and stroke), and freedom from cardiac allograft vasculopathy (CAV). Pts in Groups C and D had decreased freedom from DSA compared to the Control. Furthermore, there was lower freedom from AMR in pts in Group D compared to Control. There were no differences in outcome between each Group and the Control in 1-year survival, freedom from CAV, freedom from NF-MACE, freedom from any treated rejection, and freedom from ACR (see Table). ATG induction may be beneficial in sensitized patients with PRA under 50% but did not appear to decrease the development of AMR and DSA in those pts with PRA >50% compared to control. Larger studies and longer follow-up will need to be performed to assess ATG's impact on antibodies and the development of chronic rejection (CAV).

Single fixed low-dose rituximab as induction therapy suppresses de novo donor-specific anti-HLA antibody production in ABO compatible living kidney transplant recipients.

This study was conducted to evaluate de novo donor-specific anti-human leukocyte antigen (HLA) antibody (dnDSA) production leading to antibody-mediated rejection (ABMR) after rituximab induction in non-sensitized ABO-compatible living kidney transplantation (ABO-CLKTx). During 2008–2015, 318 ABO-CLKTx were performed at the Department of Surgery III at Tokyo Women’s Medical University Hospital. To reduce confounding factors, we adopted a propensity score analysis, which was applied with adjustment for age, gender, duration of pretransplant dialysis, HLA mismatch count, preformed DSA, non-insulin-dependent diabetes mellitus, immunosuppressive treatment, and estimated glomerular filtration rate (eGFR) on postoperative day 7. Using a propensity score matching model (1:1, 115 pairs), we analyzed the long-term outcomes of 230 ABO-CLKTx recipients retrospectively. Recipients were classified into a rituximab-treated (RTX-KTx, N = 115) group and a control group not treated with rituximab (C-KTx, N = 115). During five years, adverse events, survival rates for grafts and patients, and incidence of biopsy-proven acute rejection (BPAR) and dnDSA production for the two groups were monitored and compared. All recipients in the RTX-KTx group received rituximab induction on preoperative day 4 at a single fixed low dose of 100 mg; the CD19+ B cells were eliminated completely before surgery. Of those recipients, 13 (11.3%) developed BPAR; 1 (0.8%) experienced graft loss. By contrast, of C-KTx group recipients, 25 (21.7%) developed BPAR; 3 (2.6%) experienced graft loss. The RTX-KTx group exhibited a significantly lower incidence of BPAR (P = .041) and dnDSA production (13.9% in the RTX-KTx group vs. 26.9% in the C-RTx group, P = .005). Furthermore, lower incidence of CMV infection was detected in the RTX-KTx group than in the C-KTx group (13.9% in the RTX-KTx group vs. 27.0% in the C-KTx group, P = .014). No significant difference was found between groups for several other factors: renal function (P = .384), graft and patient survival (P = .458 and P = .119, respectively), and the respective incidences of BK virus infection (P = .722) and leukopenia (P = .207). During five-year follow-up, single fixed low-dose rituximab therapy is sufficient for ensuring safety, reducing rejection, and suppressing dnDSA production for immunological low-risk non-sensitized ABO-CLKTx.

Update on C1 Esterase Inhibitor in Human Solid Organ Transplantation.

Complement plays important roles in both ischemia-reperfusion injury (IRI) and antibody-mediated rejection (AMR) of solid organ allografts. One approach to possibly improve outcomes after transplantation is the use of C1 inhibitor (C1-INH), which blocks the first step in both the classical and lectin pathways of complement activation and also inhibits the contact, coagulation, and kinin systems. C1-INH can also directly block leukocyte-endothelial cell adhesion. C1-INH contrasts with eculizumab and other distal inhibitors, which do not affect C4b or C3b deposition or noncomplement pathways. Authors of reports on trials in kidney transplant recipients have suggested that C1-INH treatment may reduce IRI and delayed graft function, based on decreased requirements for dialysis in the first month after transplantation. This effect was particularly marked with grafts with Kidney Disease Profile Index ≥ 85. Other clinical studies and models suggest that C1-INH may decrease sensitization and donor-specific antibody production and might improve outcomes in AMR, including in patients who are refractory to other modalities. However, the studies have been small and often only single-center. This article reviews clinical data and ongoing trials with C1-INH in transplant recipients, compares the results with those of other complement inhibitors, and summarizes potentially productive directions for future research.

To Evaluate Anti-HLA Antibodies Sensitization in Pre- and Post-renal Transplant Patient’s Serum: A Retrospective Case Series

Introduction: In India, patients of renal failure are dependent on live related or unrelated donor. Because of poor financial condition patients do not go for DSA (Donor Specific Antibody) detection using Luminex. In absence of screening of de-novo production of DSA and don’t get proper management. As a result of which patient undergo acute rejection.
 Case Series: Here we are presenting 5 acute rejection cases comparing there DSA in pre-transplant and post-transplant sera using solid-phase assays.5 renal transplanted patients undergone acute and hyperacute rejection (Banff's classification) were considered for presented case series. Collected serum (pre and post-transplant on day of rejection) from each patient was subjected to detection of anti-HLA antibody using Luminex-PRA.
 Conclusion: Presence of donor-specific anti-HLA antibody with their titer was detected in pre and post-transplant serum. It is found that the strength of DSA is directly proportional to graft life.

The PI3Kδ selective inhibitor AS2541019 suppresses donor-specific antibody production in rat cardiac and non-human primate renal allotransplant models

Long-term graft survival after organ transplantation is difficult to achieve because of the development of chronic rejection. One cause of chronic rejection arises from antibody-mediated rejection (AMR), which is dependent on the production of donor-specific antibodies (DSA). Current immunosuppression in organ transplantation is effective in preventing acute T cell-mediated rejection, but the risk of DSA production and graft loss due to AMR remains unchanged. Phosphatidylinositol-3-kinase p110δ (PI3Kδ), a member of the family of PI3K lipid kinases, is a key mediator of B cell activation, proliferation and antibody production. AS2541019 is a novel PI3Kδ selective inhibitor that prevents antibody production by inhibiting B cell immunity. The purpose of this study was to evaluate the inhibitory effect of AS2541019 on DSA production in preclinical rodent and non-human primate allotransplant models. Concomitant administration of AS2541019 with tacrolimus and mycophenolate mofetil (MMF) inhibited de novo DSA production in an ACI-to-Lewis rat cardiac allotransplant model. To predict the efficacy of AS2541019 in clinical practice, we evaluated its effects in cynomolgus monkeys. AS2541019 inhibited B cell proliferation and major histocompatibility complex (MHC) class II expression on B cells in cynomolgus monkeys. Oral administration of AS2541019 inhibited MHC class II expression on peripheral B cells and anti-tetanus toxoid antibody production. In cynomolgus monkey renal allotransplant model, concomitant administration of AS2541019 with tacrolimus and MMF significantly inhibited de novo DSA production. Together, our findings indicate that the PI3Kδ selective inhibitor AS2541019 is a potential candidate for preventing AMR development by inhibiting DSA production.

Everolimus-based Immunosuppression Possibly Suppresses Mean Fluorescence Intensity Values of De Novo Donor-specific Antibodies After Primary Kidney Transplantation

PurposeWe evaluated de novo donor-specific antibody (DSA) production of everolimus (EVR)-based immunosuppression for primary kidney transplant recipients involved in the A1202 study at our institute. MethodsFrom March 2008 to August 2009, 24 recipients were prospectively randomized into 2 groups. The EVR group received reduced cyclosporin A and EVR. The standard protocol (STD) group received standard cyclosporin A and mycophenolate mofetil. Both groups received basiliximab and steroids. De novo DSA was identified using LABScreen single antigen beads (One Lambda, Canoga Park, Calif., United States). Mean fluorescence intensity (MFI) values > 1000 were considered positive. P < .05 was considered significant. ResultsGraft survival was 100% in the EVR group and 90.9% in the STD group. All patients remained on the primary protocol in the EVR group, but 3 patients in the STD group (27.3%) were converted to tacrolimus due to DSA and non-adherence. Estimated glomerular filtration rate was similar in both groups. No EVR group recipients and 9.1% of STD group recipients were treated for T-cell-mediated rejection. No recipients of the EVR group exhibited peritubular capillaritis, while 9.1% in STD group developed chronic active antibody-mediated rejection. LABScreen revealed an accumulative class II DSA production rate of 15.4% in the EVR group and 18.3% in the STD group at 10 years. When the MFI cut-off level was set to 6000, anti-HLA antibody and de novo DSA-free survival was significantly better in the EVR group. ConclusionsEVR-based immunosuppression provided equivalent or even better clinical outcomes. EVR suppressed de novo DSA production at 10 years follow-up; however, further follow-up is inevitable.

Allosensitization after transplant failure: the role of graft nephrectomy and immunosuppression - a retrospective study.

There are conflicting data about the role of transplant nephrectomy and immunosuppression withdrawal on the development of allosensitization and the impact on re-transplantation. We divided 109 first graft recipients into two groups according to whether they underwent nephrectomy (NX+, n=61) or their graft was left insitu (NX-, n=48). Sera were assessed for HLA-A/B/Cw/DR/DQ antibodies at the time of NX/transplant failure and after 3, 6, 12, 24months. The NX+ group showed a higher rate of donor specific antibody (DSA) and non-DSA human leukocyte antigen (HLA) antibody production at all the time points. Multivariable analysis showed that nephrectomy was a strong, independent risk factor for the development of DSAs after 12 and 24months (P=0.005 and 0.008). In the NX- group, low tacrolimus levels correlated with DSA formation (AUC 0.817, P=0.002; best cut-off level 2.9ng/ml). Analysis with a standardized pool of UK donors showed a more difficult grade of HLA matchability following nephrectomy compared with the NX- group. Nephrectomy is followed by the long-term production of DSA and non-DSA HLA antibodies and negatively impacts on the chances of finding a HLA-compatible kidney. Tacrolimus levels ≥3ng/ml are protective against the development of allosensitization and could facilitate re-transplantation in the NX- group.

Use of Bortezomib in the Treatment of Antibody Mediated Rejection (AMR) in Pediatric Heart Recipients

Purpose No single agent has been found to adequately treat AMR after heart transplant (HTx). Bortezomib, which can reduce donor-specific antibody (DSA) production by elimination of plasma cells, has been used as adjunctive AMR treatment in adults, but its use in pediatric HTx recipients has not been well described. Methods Single center retrospective review of 27 consecutive patients (<21yo) diagnosed with AMR and treated with bortezomib from 1/2011-10/2017. Clinical and immunogenic data were reviewed. Results Median age at HTx was 36 months (IQR 9-108) and time from HTx to AMR was 308 days (35-2231). Majority of cases (81.5%) had an underlying diagnosis of CHD. Diagnosis was by biopsy in 23 patients (11 pAMR1, 11 pAMR2, and 1 pAMR3), while 4 were based on clinical evidence. Echocardiogram showed systolic dysfunction in 8 (29.6%). All 27 patients had circulating DSAs at diagnosis. Each received at least 1 course of bortezomib (1.3 mg/m2 x4 doses), while 10 had 2 courses, and 2 had 3 courses. Other intensive treatments for AMR were also administered. DSA production decreased in patients receiving bortezomib, more noticeably for HLA-I than HLA-II, with partial rebound noted after treatment.(Figure). Follow-up biopsy (median 68 days [41-122] after diagnosis) showed resolution of AMR in 21 (78%). At median follow-up of 603 days (79-1400), graft survival was 63%; 2 required retransplant (1 directly attributed to AMR) and 8 died (4 directly attributed to AMR). Adverse events leading to discontinuation or adjustment of dosing included headache (2), thrombocytopenia (2), elevated transaminases (3), neuropathy (2), and flash pulmonary edema leading to ARDS/death (1). Conclusion Bortezomib can reduce DSA production and may be a viable adjunctive therapeutic option for AMR in pediatric HTx recipients. However, risk of adverse effects requires close monitoring, with early intervention to adjust or discontinue dosing. Further study is needed to determine the precise role of bortezomib in pediatric AMR. No single agent has been found to adequately treat AMR after heart transplant (HTx). Bortezomib, which can reduce donor-specific antibody (DSA) production by elimination of plasma cells, has been used as adjunctive AMR treatment in adults, but its use in pediatric HTx recipients has not been well described. Single center retrospective review of 27 consecutive patients (<21yo) diagnosed with AMR and treated with bortezomib from 1/2011-10/2017. Clinical and immunogenic data were reviewed. Median age at HTx was 36 months (IQR 9-108) and time from HTx to AMR was 308 days (35-2231). Majority of cases (81.5%) had an underlying diagnosis of CHD. Diagnosis was by biopsy in 23 patients (11 pAMR1, 11 pAMR2, and 1 pAMR3), while 4 were based on clinical evidence. Echocardiogram showed systolic dysfunction in 8 (29.6%). All 27 patients had circulating DSAs at diagnosis. Each received at least 1 course of bortezomib (1.3 mg/m2 x4 doses), while 10 had 2 courses, and 2 had 3 courses. Other intensive treatments for AMR were also administered. DSA production decreased in patients receiving bortezomib, more noticeably for HLA-I than HLA-II, with partial rebound noted after treatment.(Figure). Follow-up biopsy (median 68 days [41-122] after diagnosis) showed resolution of AMR in 21 (78%). At median follow-up of 603 days (79-1400), graft survival was 63%; 2 required retransplant (1 directly attributed to AMR) and 8 died (4 directly attributed to AMR). Adverse events leading to discontinuation or adjustment of dosing included headache (2), thrombocytopenia (2), elevated transaminases (3), neuropathy (2), and flash pulmonary edema leading to ARDS/death (1). Bortezomib can reduce DSA production and may be a viable adjunctive therapeutic option for AMR in pediatric HTx recipients. However, risk of adverse effects requires close monitoring, with early intervention to adjust or discontinue dosing. Further study is needed to determine the precise role of bortezomib in pediatric AMR.

Dynamic BCMA Expression by Alloreactive B Cells Coupled with Donor Specific Antibody Production during De Novo Alloantibody Responses

Purpose BCMA, a member of the tumor necrosis factor receptor superfamily (TNFRSF17), is selectively expressed during plasma cell differentiation while being nearly absent on naive and memory B cells. Therefore, BCMA appears as a highly selective antigen for mature B-cell targeting. The current study investigated BCMA expression by peripheral B cells and plasma cells during the development of de novo alloantibody responses. Methods A mouse model of allo-sensitization induced by skin allograft was used to study BCMA expression by B cell subsets and plasma cells. Blood samples were collected weekly for measurement of anti-HLA.A2 IgM and IgG titers. PBMCs and splenic lymphocytes were analyzed in FACS for identification of B cell subsets expressing surface BCMA. Splenic B-cells were isolated using EasySep B-cell isolation kit. mRNA expression of TNFRSF17 was studied in quantitative PCR. Results qPCR demonstrated a significant increase in TNFRSF17 mRNA expression by splenic B-cells isolated from HLA.A2 sensitized mice (p=0.004 vs. naive control). Multi-perimeter FACS analysis of splenic lymphocytic cells showed that cell surface BCMA (CD269) was expressed by the majority of CD38+CD138+ plasma cells (60-80%), and by members of CD19+CD23+sIgD+ mature B-cells. CD269 was scarcely expression by early transitional B-cells (CD93+/CD19+/CD23-). Following skin grafting BCMA was increasingly expressed on mature B cells and plasma cells at Days 14 (p=0.03 vs. naive control mice), 21(p=0.004) and 28(p=0.0001) PTx. Increase in BCMA expression by B cells was associated with high DSA IgG titers in the blood, indicating plasmablast differentiation. Conclusion Our data demonstrate that BCMA are increasingly expressed by B/plasma cells during the development of de novo alloantibody responses. Thus, targeting BCMA may serve as a new treatment strategy for desensitization in HLA highly sensitized transplant patients.

Bronchus-associated lymphoid tissue-resident Foxp3+ T lymphocytes prevent antibody-mediated lung rejection.

Antibody-mediated rejection (AMR) is a principal cause of acute and chronic failure of lung allografts. However, mechanisms mediating this oftentimes fatal complication are poorly understood. Here, we show that Foxp3+ T cells formed aggregates in rejection-free human lung grafts and accumulated within induced bronchus-associated lymphoid tissue (BALT) of tolerant mouse lungs. Using a retransplantation model, we show that selective depletion of graft-resident Foxp3+ T lymphocytes resulted in the generation of donor-specific antibodies (DSA) and AMR, which was associated with complement deposition and destruction of airway epithelium. AMR was dependent on graft infiltration by B and T cells. Depletion of graft-resident Foxp3+ T lymphocytes resulted in prolonged interactions between B and CD4+ T cells within transplanted lungs, which was dependent on CXCR5-CXCL13. Blockade of CXCL13 as well as inhibition of the CD40 ligand and the ICOS ligand suppressed DSA production and prevented AMR. Thus, we have shown that regulatory Foxp3+ T cells residing within BALT of tolerant pulmonary allografts function to suppress B cell activation, a finding that challenges the prevailing view that regulation of humoral responses occurs peripherally. As pulmonary AMR is largely refractory to current immunosuppression, our findings provide a platform for developing therapies that target local immune responses.

Donor-specific anti-HLA antibody production following pediatric ABO-incompatible heart transplantation.

ABO-i heart transplantation can be performed in infants with end-stage heart failure to increase organ availability. The development of newly detected DSAs is associated with decreased cardiac graft survival, and the effect of ABO-i transplantation on DSA production is unknown. We examined DSA production and rejection frequency in infant recipients of ABO-i and ABO-c heart transplants via a retrospective cohort study of infant heart transplant recipients transplanted at a single pediatric center between January 2004 and November 2014. Patients were included if they were less than 1year of age at transplant and had a minimum of 6months follow-up. DSA positivity was examined under two categories, either the lowest level detectable (MFI>500) or a level presumed to have clinical relevance in our immunogenetics laboratory (MFI > 5000). Of 52 patients, 36 received ABO-c transplants and 16 received ABO-i transplants. Compared to ABO-c recipients, the ABO-i group showed a consistent but statistically non-significant finding of less frequent ndDSA positivity (69.4% ABO-c vs 43.8% ABO-i with MFI >500, P=0.122; 41.7% ABO-c vs 25% ABO-i with MFI >5000, P=0.353). Additionally, ABO-i patients were less likely to have any form of rejection (12.5% vs 47.2%, P=0.027) or acute cellular rejection (6.3% vs 38.9%, P=0.021). Our data suggest that infants receiving ABO-i heart transplants may be less likely to develop ndDSAs or have rejection compared to same age ABO-c recipients. Larger multicenter studies are needed to confirm results from this single center study.

Benefits of a loading dose of tacrolimus on graft survival of kidney transplants in nonhuman primates

We examined the benefit of a loading dose of tacrolimus on the production of donor-specific antibodies (DSA), occurrence of acute rejection (AR) episodes, graft survival, and histological evidence of antibody-mediated rejection (ABMR) after kidney transplantation in nonhuman primates. Eight cynomolgus monkeys were assigned to two groups (n = 4 each): a maintenance dose-group, orally administered 1 mg/kg/day tacrolimus from the day of transplantation; and a loading-dose group, orally administered 2 mg/kg/day tacrolimus for 21 days after transplantation followed by 1 mg/kg/day. The monkeys were observed for up to 178 days after transplantation. Plasma creatinine was monitored over time. Recipients with increased plasma creatinine levels of >2 mg/dL received anti-acute rejection therapy. In the maintenance dose-group, DSA production, frequent AR episodes, and histological evidence of ABMR were observed in all recipients. Three of four recipients did not survive until the end of the observation period. In the loading-dose group, two recipients showed DSA production, frequent AR episodes and histological evidence of ABMR, while the remaining two had no DSA, AR episodes, or ABMR. Our findings indicate that a loading dose of tacrolimus may prevent DSA production, occurrence of AR events and ABMR, and prolong graft survival following kidney transplantation in monkeys.

T Follicular Regulatory Cells and Antibody Responses in Transplantation.

De novo donor-specific antibody (DSA) formation is a major problem in transplantation, and associated with long-term graft decline and loss as well as sensitization, limiting future transplant options. Forming high-affinity, long-lived antibody responses involves a process called the germinal center (GC) reaction, and requires interaction between several cell types, including GC B cells, T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. T follicular regulatory cells are an essential component of the GC reaction, limiting its size and reducing nonspecific or self-reactive responses.An imbalance between helper function and regulatory function can lead to excessive antibody production. High proportions of Tfh cells have been associated with DSA formation in transplantation; therefore, Tfr cells are likely to play an important role in limiting DSA production. Understanding the signals that govern Tfr cell development and the balance between helper and regulatory function within the GC is key to understanding how these cells might be manipulated to reduce the risk of DSA development.This review discusses the development and function of Tfr cells and their relevance to transplantation. In particular how current and future immunosuppressive strategies might allow us to skew the ratio between Tfr and Tfh cells to increase or decrease the risk of de novo DSA formation.

Effective suppression of donor specific antibody production by Cathepsin S inhibitors in a mouse transplantation model

Donor-specific antibodies (DSA) are a major risk factor for antibody-mediated rejection (ABMR) in solid organ transplantation, and ABMR remains a medical challenge. Therefore, effective anti-ABMR therapies are needed to improve overall graft survival. Cathepsin S (Cat S) is an essential protease for antigen peptide loading onto lysosomal/endosomal major histocompatibility complex (MHC) class II molecules to promote antigen presentation. Cat S deficiency produces immuno-deficient phenotypes including a suppressed humoral immune response, and Cat S inhibition reportedly prevents autoimmunity. However, little is known about the effects of Cat S inhibitors on organ transplantation, especially ABMR. Here, we report the pharmacological profile of novel Cat S inhibitors, AS2761325 and AS2863995, and explore their preventive potential on DSA production and acute rejection in a mouse cardiac transplantation model. Cat S inhibitors potently inhibited upregulation of antigen peptide loading MHC class II expression on the surface of splenic B cells and suppressed ovalbumin-induced T cell-dependent antibody production in mice. In a mouse cardiac transplantation model, oral administration of AS2761325 monotherapy inhibited DSA production without affecting graft survival. When combined with a suboptimal dose of tacrolimus, AS2761325 significantly prolonged graft survival. The more potent Cat S inhibitor AS2863995 also prolonged graft survival and almost completely suppressed DSA production. These results suggest that Cat S inhibitors may be promising ABMR prophylaxis drug candidates. Combination therapy comprising a Cat S inhibitor and calcineurin inhibitors may be a more effective immunosuppressive maintenance therapy for controlling both cell-mediated and antibody-mediated rejection.

Immunosuppression Related to De Novo Donor-Specific HLA Antibody Production after Renal Transplantation

Aim One of the major factors influencing graft loss is antibody-mediated rejection caused by the production of donor-specific HLA antibodies (DSA). The optimization of multidrug combination therapy using immunosuppressants is required in order to prevent DSA production. We herein evaluated the relationships among clinical variants including immunosuppressant levels and the prediction of DSA production after renal transplantation (Tx). Methods Living renal transplant patients (n=160) at our institute who received standard immunosuppressant protocols with basiliximab, tacrolimus (Tac), mycophenolate mofetil (MMF), and steroids between July 2004 and September 2013 were enrolled. The production of serum anti-HLA antibodies at one-year post-Tx was analyzed by solid-phase bead-based assays using Luminex. The HLA specificity of DSA was evaluated based on the data of donor HLA typing. In the present study, preformed DSA and de novo DSA were defined as those detected pre-Tx and at one-year post-Tx, and those detected at one-year post-Tx only, respectively. Risk factors for DSA production were statistically evaluated using clinical variables including Tac trough levels and the incidence of virus infections. Results DSA production at one-year post-Tx was observed in 15 patients including 9 with preformed DSA and 7 with de novo DSA. Preformed and de novo DSA were detected in one patient. De novo DSA production was more likely to occur in patients with low serum Tac levels (cut-off value: 3.2 ng/mL), in those who ceased MMF medication, and in those who acquired CMV infection within one-year post-Tx (p=0.008, 0.044, and 0.050, respectively). Conclusions The appropriate control of Tac trough levels, continuation of MMF intake, and prevention of CMV infection were found to be important for avoiding de novo DSA production at one-year post-Tx in the present study.

Role of Graft-derived Graft-versus-Host T cells in Facilitating Multilineage Blood Chimerism after Human Intestinal Transplantation

Long-term multilineage chimerism often appears in blood following human intestinal transplantation (ITx), especially in multivisceral transplant (MVTx) recipients. Patients with donor T cell macrochimerism (>4%) in blood, frequently without graft-versus-host disease (GVHD), displayed less graft rejection and donor-specific antibody (DSA) production and slower graft T cell replacement by the recipient. To address the hypothesis that the balance of graft-versus-host (GvH) and host-versus-graft (HvG) alloreactivity might underly these relationships, we analyzed the phenotype, repertoire and alloreactivity of donor- and recipient-derived T cells in the intestinal allograft, peripheral blood and bone marrow (BM) of ITx recipients. We identified GvH and HvG clones on the basis of high throughput TCRβ CDR3 sequencing from pre-Tx donor and recipient mixed lymphocyte reactions as we have described1. Enrichment of GvH compared to HvG clones in early intestinal biopsies and absence of Class I DSA were associated with donor T cell macrochimerism. Local expansion of GvH clones in the graft was likely driven by uniformly rapid replacement of recipient antigen presenting cells (APCs) within allografts early post-Tx. Cumulative frequency of GvH clones in blood early post-Tx was positively correlated with the peak level of donor T cell chimerism (Fig.1).Unlike donor ileal lymphocytes, which display a tissue-resident memory phenotype, long-term circulating donor T cells were markedly enriched for the recent thymic emigrant phenotype compared with recipient cells, and GvH clones were undetectable among these late circulating naïve donor-derived T cells. Enrichment of donor circulating naïve B cells and T cells and recurrent myeloid chimerism late post-Tx are consistent with our observation that donor-derived hematopoietic stem and progenitor cells are present in intestinal allografts and are detectable in the circulation of patients with macrochimerism. Multilineage chimerism might reflect a lymphohematopoietic GvH response (LGVHR) migrating from recipient circulation to the BM, making “space” for engraftment of progenitor cells from the graft. Indeed, 0.0356% of CD45+ cells in the BM of an MVTx patient on day 105 post-Tx were donor-derived, among which 72.9% were T cells and 1.37% were CD34+ progenitors. TCR sequencing of sorted donor cells in recipient BM identified donor-mappable clones, among CD4s of which 60% by frequency were GvH-reactive. GvH CD4 cells did not show marked enrichment in donor-mappable PBMCs on the same day. (Fig.2)Together, these data suggest that donor GvH-reactive T cells expanding within the graft in response to recipient APCs entering the graft migrate into the recipient circulation and BM, playing a key role in promoting and maintaining mixed chimerism. LGVHR without GVHD in ITx recipients suggests a novel approach to achieving intestinal allograft tolerance. 1. Zuber et al., 2016 Science Immunology. The study was funded by the National Institute of Allergy and Infectious Diseases grant P01 AI106697. Research reported in this study was performed in the CCTI Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056.

Increased CD40L+PD-1+ follicular helper T cells (Tfh) as a biomarker for predicting calcineurin inhibitor sensitivity against Tfh-mediated B-cell activation/antibody production after kidney transplantation.

It is unclear to what extent the development of follicular helper T cells (Tfh) and de novo donor-specific human leukocyte antigen antibody (DSA) production could be influenced by immunosuppressive agents, particularly calcineurin inhibitor (CNI; cyclosporine or tacrolimus), after kidney transplantation. Here, the effects of immunosuppressive agents on Tfh-mediated B-cell activation and antibody production were investigated. In vitro circulating Tfh (cTfh; memory CD4+CXCR5+)/B-cell (CD19+) co-culture assays revealed that CNI considerably inhibited cTfh-mediated B-cell activation and IgG antibody secretion through the suppression of IL-21 and IL-2. Both IL-21 and CD40L up-regulated IL-2 receptors (CD25) on B cells, and anti-CD25 antibody induced apoptosis of activated B cells, resulting in the inhibition of IgG production. The frequency of cTfh-expressed CD40L and PD-1 was elevated in patients with de novo DSA 1 year after transplantation. The degree of inhibition by CNI was dependent on Staphylococcal enterotoxin B-induced CD40L+PD-1+ cTfh up-regulation level. Our data demonstrate that CD40L+PD-1+cTfh could be a marker to implicate individual difference in CNI sensitivity for Tfh-mediated B-cell activation in kidney transplantation.

Triptolide inhibits donor-specific antibody production and attenuates mixed antibody-mediated renal allograft injury.

Donor-specific antibodies (DSAs) are major mediators of renal allograft injury, and strategies to inhibit DSAs are important in promoting long-term graft survival. Triptolide exhibits a wide spectrum of antiinflammatory and immunosuppressive activities, and in autoimmune diseases it inhibits autoantibody levels. In this study, we investigated the suppressive role of triptolide in the generation of DSAs in transplant recipients. We found that triptolide treatment of skin allograft recipients in mice significantly suppressed the development of circulating anti-donor-specific IgG and effectively alleviated DSA-mediated renal allograft injury, which led to prolonged allograft survival. In vitro studies revealed that triptolide inhibited the differentiation of B cells into CD138+ CD27++ plasma cells; reduced the levels of IgA, IgG, and IgM secreted by plasma cells; and repressed somatic hypermutation and class switch recombination of B cells. Moreover, triptolide-treated recipients showed reduced numbers of B cells, plasma cells, and memory B cells in spleens and decreased numbers of T, B, natural killer (NK) cells, and macrophages infiltrating grafts. These findings highlight the importance of triptolide in suppressing DSAs and establish triptolide as a novel therapeutic agent for antibody-mediated allograft rejection.