Sensitized Transplant Patients Research Articles

Bone marrow derived long-lived plasma cell phenotypes are heterogeneous and can change in culture

Bone marrow-derived long-lived plasma cells (LLPCs) are thought to be a major source of alloantibody in sensitized transplant patients. However, studies of LLPCs have been hampered not only by the fact that they are rare and difficult to isolate and culture but also due to the lack of consensus regarding a definitive cell-surface phenotype. The goal of the current study was to determine if LLPCs have a specific, stable cell-surface phenotype. PCs were isolated from high-volume (120cc) bone marrow aspirates that were enriched first by negative selection then positive selection using anti-CD38 antibody-coated beads and purified by cell sorting. A typical isolation resulted in >100,000 PCs that were sorted into 4 populations with variable numbers of PCs: CD19+/CD138+/CD38Hi (64.1% of the PCs), CD19-/CD138+/CD38Hi (20.9%), CD19+/CD138-/CD38Hi (10.7%), and CD19-/CD138-/CD38Hi (4.3%). The purity of each subset was 96–99%. Each subset contained PCs secreting IgG and IgA. Measles- and tetanus-specific PCs (i.e. putative IgG secreting, antigen-specific LLPCs). LLPCs were identified in both the CD19+/CD138+/CD38Hi and CD19-/CD138+/CD38Hi subsets and in the smaller CD138- subsets (when pooled). Thus, all CD38Hi subsets contained LLPCs. Cultured PCs maintained viability (>50%) and function and could be retrieved for analyses. During 7 days of culture, cell surface expression changed from baseline in many PCs. For example, approximately 20% of CD19 + CD138+/CD38Hi cells (the largest PC subset) became CD19-. CFSE assays showed no division and only a small percentage of LLPCs were Ki-67 positive suggesting that the cells did not divide in culture and that the antibody detected was not from plasmablasts. We conclude that human bone marrow LLPCs have a heterogeneous expression of CD19 and CD138, which can change during cell culture. The fact that LLPCs were found in all four subsets raises the possibility that a large percentage of PCs in the bone marrow may be LLPCs.

Methods to Detect MHC-Specific IgE in Mice and Men.

Humoral immunity is a major barrier limiting long-term outcome after organ transplantation. Especially, the production of antibodies directed against donor HLA/MHC antigens (i.e. donor-specific antibodies (DSA)) leading to antibody-mediated rejection (ABMR) is considered to be a major factor negatively affecting allograft survival. DSAs of the IgG isotype are routinely measured in transplant patients. However, not all patients diagnosed with IgG-DSA develop ABMR events. Therefore, research in better understanding the mechanisms of ABMR is of great importance. We recently demonstrated the production of MHC-specific IgE upon allograft rejection in mice and in transplant patients. IgE is classically connected with allergy and is known to be important for the humoral defense against helminths and worms. However, its role in autoimmune diseases and cancer has been reported recently as well. The concentration of IgE in blood is extremely low compared to other antibody isotypes. Therefore, detection of MHC-specific IgE from serum requires methods of high sensitivity. Since MHC-specific IgG—typically present at much higher serum levels—develops as well, high specificity is also required of IgE detection methods. In the murine model we developed an enzyme linked immunosorbent assay (ELISA) using MHC monomers for measurement of MHC-specific IgE, allowing us to distinguish between specificities of antibodies against different class I and class II antigens. For measurement of functional activity of MHC-specific IgE in vitro, a release assay using a rat basophil cell line (RBL-2H3) was established. For functional analysis of MHC-specific IgE in vivo, a cutaneous hypersensitivity reaction assay was adapted for this purpose using MHC monomers. Humanized RBL-2H3 cells transfected with cDNA coding for the human-high affinity IgE receptor were used for functionality measurement of donor-specific IgE in sensitized transplant patients. For detection of HLA-specific IgE, a bead assay was adapted, using beads expressing single HLA antigens. The aim of this publication is to demonstrate currently established methods for the detection and characterization of MHC-specific IgE in the murine and human setting.

Preoperative carfilzomib and lulizumab based desensitization prolongs graft survival in a sensitized non-human primate model

Sensitized patients are difficult to transplant due to pre-formed anti-donor immunity. We have previously reported successful desensitization using carfilzomib and belatacept in a non-human primate (NHP) model. Here we evaluated selective blockade of the co-stimulatory signal (CD28-B7) with Lulizumab, which preserves the co-inhibitory signal (CTLA4-B7). Five maximally MHC-mismatched pairs of NHPs were sensitized to each other with two sequential skin transplants. Individuals from each pair were randomized to either desensitization with once-weekly Carfilzomib (27mg/m2 IV) and Lulizumab (12.5mg/kg SC) over four weeks, or no desensitization (Control). NHPs then underwent life-sustaining kidney transplantation from their previous skin donor. Rhesus-specific anti-thymocyte globulin was used as induction therapy and immunosuppression maintained with tacrolimus, mycophenolate, and methylprednisolone. Desensitized subjects demonstrated a significant reduction in donor-specific antibody, follicular helper T cells (CD4+PD-1+ICOS+), and proliferating B cells (CD20+Ki67+) in the lymph nodes. Interestingly, regulatory T cell (CD4+CD25+CD127lo) frequency was maintained after desensitization in addition to increased frequency of naïve CD4 T cells (CCR7+CD45RA+) and naïve B cells (IgD+CD27-CD20+) in circulation. This was associated with significant prolongation in graft survival (MST= 5.8 ± 4.0 vs. 64.8 ± 36.3; p<0.05) and lower antibody-mediated rejection scores compared to control animals. However, all desensitized animals eventually developed AMR and graft failure. Desensitization with CFZ and Lulizumab improves allograft survival in allosensitized NHPs, by transient control of the germinal center and shifting of the immune system to a more naive phenotype. This regimen may translate into clinical practice to improve outcomes of highly sensitized transplant patients.

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.

Highly HLA Sensitized Kidney Transplant Patients in a Transplant Center

IntroductionApproximately 10% to 30% of patients on renal transplant waiting lists are sensitized, which gives them more time on the waiting list. Transplantation in this setting has a greater risk of rejection and decreased graft survival. New strategies of donor allocation through virtual crossmatching and optimization of immunosuppressive therapies in induction and maintenance have allowed the allocation of organs for this population, which in other circumstances would not be chosen for a kidney transplant. ObjectiveTo describe the experience of renal transplantation in highly sensitized patients with a panel reactive antibody of >80% in a transplant center, through virtual crossmatching, discarding unacceptable antigens, and without desensitization treatment. MethodsAn observational, descriptive, retrospective case series study was conducted on highly sensitized kidney transplant patients with a panel reactive antibody of ≥80% from 2010 to 2016. ResultsA total of 10 highly sensitized transplant patients were identified. Six patients were women, all of whom had a history of pregnancy; all patients had undergone blood transfusions, and 40% had undergone a first transplant. Average time spent on dialysis was 148.5 months, and on the waiting list, 45.8 months. Average follow-up was 42 months (range, 10–84 months). The estimated glomerular filtration rate by the Chronic Kidney Disease Epidemiology Collaboration method at year 1 was 75 mL/min/1.73 m2 body surface. Nine patients at 1 year posttransplantation had graft and patient survivals of 100%, as did 5 patients at >3 years posttransplantation. ConclusionsRenal transplantation based on virtual crossmatching is a good alternative for highly sensitized patients.

Intravenous Immunoglobulins in the Prevention of Rejection of a Second or Third Kidney Graft

BackgroundIntravenous immunoglobulin (IVIg) reduces acute rejection episodes in kidney transplantation, but adverse events (AEs) are common. The aim of this study was to assess whether human IVIg enhances immunosuppressive effects without increasing AEs in the prevention of acute kidney graft rejection. MethodsPatients receiving a second or third kidney graft were treated with standard immunosuppressant therapy with (n = 18) or without (n = 10) IVIg. The primary efficacy endpoint was biopsy-proven acute rejection (BPAR) rate at 3 months, and secondary endpoints included acute rejection rate at 12 months, intensity of rejection, and patient survival. ResultsPatients in the experimental arm received 3 infusions of IVIg. The BPAR rate decreased with IVIg versus standard immunosuppression alone over 12 months of follow-up. Experimental versus control rates of survival without BPAR were 94% versus 63% and 82% versus 63% at 3 and 12 months. The intensity of the acute rejection episodes (BANFF 97 grade) was similar between groups. One patient from each group died during the 12-month follow-up period. Treatment-emergent AEs were reported in 100% and 94.4% of the control and experimental arms. Most AEs were considered unrelated or unlikely to be related to treatment. ConclusionsThis study supports the efficacy and safety of IVIg in highly sensitized transplant patients for improving transplant rates and reducing graft rejection episodes.

Successful desensitization with proteasome inhibition and costimulation blockade in sensitized nonhuman primates.

The detrimental effects of donor-directed antibodies in sensitized transplant patients remain a difficult immunologic barrier to successful organ transplantation. Antibody removal is often followed by rebound. Proteasome inhibitors (PIs) deplete antibody-producing plasma cells (PCs) but have shown marginal benefit for desensitization. In an allosensitized nonhuman primate (NHP) model, we observed increased germinal center (GC) formation after PI monotherapy, suggesting a compensatory PC repopulation mediated via GC activation. Here we show that costimulation blockade (CoB) targets GC follicular helper T (Tfh) cells in allosensitized NHPs. Combined PI and CoB significantly reduces bone marrow PCs (CD19+CD20-CD38+), Tfh cells (CD4+ICOS+PD-1hi), and GC B cells (BCL-6+CD20+); controls the homeostatic GC response to PC depletion; and sustains alloantibody decline. Importantly, dual PC and CoB therapy prolongs rejection-free graft survival in major histocompatibility complex incompatible kidney transplantation without alloantibody rebound. Our study illustrates a translatable desensitization method and provides mechanistic insight into maintenance of alloantibody sensitization.

Successful Desensitization With Combination of Costimulation Blockade and Bortezomib Via Regulating Plasma Cells and Follicular Helper T Cells in Sensitized Rhesus Model.

[Background] Preformed donor specific antibody (DSA) affects a significant population of sensitized transplant patients, portending a poorer prognosis in graft survival. We investigated the effect of proteasome inhibitor and costimulation blockades (COB) on DSA responses and plasma cells in pre-sensitized rhesus macaques in order to develop an effective desensitization regimen. [Method] All recipients received a fully mismatched skin graft. Grafts were all rejected within 2 weeks without treatment. Animals received Bortezomib alone (N=3, 1.33mg/m2), Belatacept/2C10R4 (N=3, 20mg/kg), or combined Belatacept/2C10R4/Bortezomib (N=3) twice a week for 4 weeks upon stabilization of their DSA levels. T and B cell phenotypes and DSA were assessed with flow cytometry. Bone marrow plasma cells were evaluated by ELISPOT. Sensitized animals received kidney transplantation from the same donor as their skin graft with or without desensitization in the presence of induction/maintenance immunuosuppression (Basiliximab/Prograf/Cellcept). [Results] The combination of COB and bortezomib showed the most efficient reduction of BM plasma cells compared to Bortezomib alone and Belatacept/2C10R4 treatment (9.6±1.2 vs. 25.6±12.2 and 45.8±0.6%; p<0.05). Triple drug treatment also showed the greatest reduction of serum DSA. Isotype switched IgG+ B cells were reduced by combined treatment that could reflect Tfh cell activity. Finally, we confirmed that LN follicular helper T cells (Tfh; PD-1hiICOShiCD4+) were reduced with triple treatment.Figure: No Caption available.Renal allografts without desensitization showed accelerated rejection (N=5, MST=3.6±2.7). In contrast, desensitization with triple treatment has resulted in no signs of rejection on POD 22. [Conclusion] Desensitization with Bortezomib alone moderately reduces plasma cells but induces germinal center responses reflected by Tfh cell populations. However, targeting costimulation signals via Belatacept (CTLA4-Ig) and 2C10R4 (anti-CD40L) in conjunction with Bortezomib profoundly reduced Tfh cell populations, plasma cell number, and serum DSA.

Effect of Mycophenolic Acid and Bortezomib on Purified Human B Cells: An In Vitro Study of Long-Term Functionally Stable MICA-Sensitized Renal Recipients

We sought to develop a B-cell in vitro culture system and test B cells isolated from sensitized kidney recipients and healthy controls, and assess the effectiveness of proteasome inhibitors and mycophenolic acid on antibody secretion and cell apoptosis. CD19(+) B cells and CD19(+)CD27(+) memory B-cell subsets were detected from peripheral blood mononuclear cells obtained from 6 MICA-sensitized kidney recipients and 6 healthy controls. Peripheral blood B cells were isolated and cultured with CpG2006, PMA, MICA antigen, B-cell activating factor, CD40 ligand (CD40L), human recombinant IL-2 (rhuIL-2), rhuIL-10, rhuIL-4, and rhuIL-21. After culturing for 7 days, we tested several variables of B-cell activity including differentiation, apoptosis, and IgM production. We also assessed the effects of 2 immunosuppressive drugs (mycophenolic acid and bortezomib) on antibody secretion and cellular apoptosis. Kidney recipients had a lower ratio of CD19+ B cells in peripheral blood mononuclear cells than did healthy controls. However, the percentage of CD19(+)CD27(+) B cells was higher in kidney recipients than in healthy controls. In the cell stimulation culture system, the ratio of CD19(+) B cells, CD19(+)CD27(+) B cells, and CD19(+)CD138(+) B cells increased after culturing for 7 days compared with unstimulated controls. In addition, the percentage of apoptotic B cells decreased, and antibody production increased in sensitized transplant patients and healthy controls. Treatment with bortezomib or mycophenolic acid induced B-cell apoptosis and inhibited secretion of antibodies. This study describes establishment of a B-cell in vitro culture system, showing that B cells may be stimulated to secrete antibodies. The study also provides an assay for studying B cells in vitro. This study provides information suggesting that bortezomib and mycophenolic acid can inhibit B-cell antibody secretion.

HLA class II DQA and DQB epitopes: Recognition of the likely binding sites of HLA-DQ alloantibodies eluted from recombinant HLA-DQ single antigen cell lines

Donor-specific antibodies (DSA) in sera of sensitized transplant patients are often produced against the specific epitopes on mismatched HLA antigens. In this study, we selected sera from 30 kidney transplant patients with DSA and AMR to define DQ epitopes. Using adsorption and elution assays, we identified 18 antibody reaction patterns to define 6 new epitopes and to confirm 12 previously defined epitopes. In one patient case, one mismatched antigen produced 3 different antibodies and, in another, antibodies were produced against the alpha and beta chains of the same antigen. For some sera, a single epitope can explain reactions for 27 of the 29 DQ beads in the single antigen panel. Several studies highlighted the prevalence of anti-DQ antibodies. In 2011, Almeshari et al. observed DQ DSA in 34/46 (74%) of rejection episodes - 44 patients had DSA and 20 lost their graft due to AMR. Other studies have shown a high prevalence of anti-DQ antibodies and an association with adverse effects on the graft. We conclude that analysis of the epitopes of the DQ antibodies using Adsorption/Elution and testing on single antigen DQ beads helps to better understand the specificities and cross-reactions of DQ antibodies in transplant patients.

Pretransplant Antithymocyte Globulin Has Increased Efficacy in Controlling Donor-Reactive Memory T Cells in Mice

Antibody-mediated lymphocyte depletion is frequently used as induction therapy in sensitized transplant patients. Although T cells with an effector/memory phenotype remain detectable after lymphoablative therapies in human transplant recipients, the role of preexisting donor-reactive memory in reconstitution of the T cell repertoire and induction of alloimmune responses following lymphoablation is poorly understood. We show in a mouse cardiac transplantation model that antidonor immune responses following treatment with rabbit antimouse thymocyte globulin (mATG) were dominated by T cells derived from the preexisting memory compartment. Administration of mATG 1 week prior to transplantation (pre-TP) was more efficient in targeting preexisting donor-reactive memory T cells, inhibiting overall antidonor T cell responses, and prolonging heart allograft survival than the commonly used treatment at the time of transplantation (peri-TP). The failure of peri-TP mATG to control antidonor memory responses was due to faster recovery of preexisting memory T cells rather than their inefficient depletion. This rapid recovery did not depend on T cell specificity for donor alloantigens suggesting an important role for posttransplant inflammation in this process. Our findings provide insights into the components of the alloimmune response remaining after lymphoablation and may help guide the future use of ATG in sensitized transplant recipients.

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Aim Therapies exist for the bulk removal of antibodies in sensitized transplant patients, but there are no means to specifically deplete antibodies that recognize a specific HLA molecule. Current bulk antibody removal procedures may leave a patient compromised immunologically and more susceptible to opportunistic infections. The goal of this project was to create specific HLA-A∗02:01 removal matrices and demonstrate their ability to capture defined anti-HLA antibodies. Methods Soluble class I HLA-A∗02:01 molecules were produced in a native conformation in mammalian cells, purified by affinity chromatography, coupled to a Sepharose matrix, and loaded into a column enclosure. Various columns were created and tested with an automated chromatography system by initial injection of various HLA antibodies under different loading conditions. After an extensive column wash, the bound antibodies were released by a pH shift and quantitated. Loading and elution profiles were recorded. Results Initial tests show that HLA columns maintain their structural integrity and function. Multiple passes of the antibody W6/32, recognizing only intact HLA molecules, resulted in consistent and repeatable binding patterns. During the entire performance process, several parameters were identified, determining column saturation, capacity and efficiency values. Specificity was shown by removing a typical A∗02:01 recognition pattern from an A2 sera. In addition, treatment conditions were established to reuse the device. Conclusions Anti-HLA antibody removal devices are highly efficient in selectively depleting HLA antibodies. Capable of removing up to 1 mg per ml of matrix depending on the avidity of the antibody makes this device very efficient for research studies of HLA antibodies and provides promise for therapeutic applications. Furthermore, this device might also find an application in HLA diagnostics, allowing for the deconvolution of serologic samples and providing a new way to evaluate high PRA patients.

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Many sensitized transplant patients have HLA-specific antibodies due to previous graft failures, blood transfusions, and pregnancies. These humoral responses are typically polyclonal in nature, antibody composition is distinct to each individual, and it is difficult to precisely determine antibody specificity. Distinct antibodies cannot be readily isolated for individual characterization. In this study we isolate distinct anti-HLA antibodies to evaluate their phenotypic and functional traits. After characterizing sera from highly sensitized patients with a single antigen assay, sera were passed over HLA-specific separation devices made by coupling purified soluble HLA to a sepharose matrix. A scheme of multiple separation rounds involving loading and elution cycles with different HLA allele combinations was applied. This process resulted in mono-specific patterns of HLA reactivity measured by a Luminex-based bead assay. Our results show that complex patterns of sera reactivity can be reduced to more simplified outputs allowing better interpretation of individual antibody populations. Separation of IgG, IgA, and IgM further assisted in the interpretation of these responses. Furthermore, this technique was also of use in evaluating false positive/negative assay signals, strengthening otherwise weak antibody reactivity, and eliminating background interference. Antibody concentration, isotype, epitope specificity, cross-reactivity, and the ability to fix complement have all been implicated as factors that contribute to the pathogenicity of anti-HLA antibodies following organ transplantation. The deconvolution of antibody reactivity patterns can assist in the assessment of risk factors for immunologic rejection as well as provide new insights into anti-HLA antibody profiling and epitope structure analysis.

Pre-transplant administration of antithymocyte globulin increases its efficacy in inhibiting anti-donor memory T cell responses (126.9)

Abstract Antithymocyte globulin (ATG) is a lymphoablative agent used in transplant patients. In this study, we tested how murine analog of ATG (mATG)affects pre-existent donor-reactive memory T cells. mATG treatment of heart allograft recipients on d.-7 and -4 (pre-TP)led to better outcome than treatment on d.0 and d.4 (peri-TP). Pre-TP mATG resulted in lower frequencies of donor-specific IFNγ-secreting T cells than peri-TP mATG(72000 vs 12500spots/million CD44hi cells). Studies in recipients injected with tracer subsets of donor-reactive T cells showed that while both mATG regimens depleted naïve T cells equally well, pre-TP mATG was four times more efficient in depleting pre-existing memory T cells. IFNγ production by donor-specific memory T cell was diminished by 99% with pre-TP mATG and only by 70% with peri-TP mATG. Transfer of TCR transgenic Ld-reactive 2C memory CD8 cells into recipients of Ld+ or Ld- heart allografts showed that memory T cells resistance to peri-TP mATG depletion depended on the systemic inflammation after transplantation but not on their antigen specificity. In summary,pre-TP ATG is more efficient in targeting pre-existing alloreactive memory T cells and anti-donor T cell responses. Our findings may guide the future use of ATG in sensitized transplant patients.

T cells reconstituting the periphery in thymoglobulin treated kidney transplant recipients are preferentially donor-senescent when compared to basiliximab treated patients (141.25)

Abstract Induction with anti-thymocyte globulin (ATG) results in better 1 yr renal transplant outcomes than basiliximab in immunologically sensitized transplant patients. This observation contrasts with the potential homeostatic proliferation of surviving donor-reactive T cells in the post-ATG lymphopenic environment. Donor-specific reactivity was measured longitudinally in 8 (n=4 for basiliximab, n=4 for ATG) kidney transplant recipients who completed a 6 mo follow-up. Peripheral blood cells at pretransplant and monthly posttransplant were tested for donor-reactivity by IFN-γ ELISPOT assays. Patients induced with ATG or basiliximab had similar decreases in their absolute lymphocyte counts (and total T cells) at 1 wk post-tx. While these counts returned to near baseline by 3 mo in basiliximab treated patients, counts in ATG-treated patients were 61% of baseline at 6 mo. Mean post-tx donor-specific alloreactive T cell frequencies at 6 mo follow-up were similar to pretransplant levels for basiliximab patients but were &amp;lt;20% of pretransplant levels for ATG-treated patients. Furthermore, peripheral T cells in ATG-treated patients are not preferentially reconstituted with RO+ memory cells as might be expected in an environment promoting homeostatic proliferation. These data suggest that reconstitution of the periphery in ATG-treated patients is not preferentially donor-reactive.

ICOS-Dependent and -Independent Functions of Memory CD4 T Cells in Allograft Rejection

Donor-reactive memory T cells undermine the survival of transplanted organs through multiple pathways. We have previously reported that memory CD4 T cells resist treatment with anti-CD154 antibody and donor-specific transfusion (DST/MR1) and promote cardiac allograft rejection via generation of effector CD4 T cells and alloantibody. We hypothesized that the helper functions of memory CD4 T cells are independent of T-cell costimulation through CD154 but instead are regulated by alternative costimulatory pathways. This study investigated how blocking ICOS/B7RP-1 interactions affects functions of donor-reactive memory CD4 T cells. Treatment with blocking anti-ICOS mAb synergized with DST/MR1 and prolonged mouse cardiac allograft survival despite the presence of donor-reactive memory CD4 T cells. While blocking ICOS did not diminish the expansion of preexisting memory CD4 T cells or the induction of allospecific effector T cells, it did inhibit recruitment of the activated memory and effector T cells into the graft. In addition, anti-ICOS mAb treatment in combination with DST/MR1 prevented help provided by memory CD4 T cells for production of donor-specific IgG antibody. These results demonstrate the potential efficacy of ICOS blockade in sensitized transplant patients and provide the foundation for rational use of ICOS blockade in combination with other graft-prolonging strategies.

Donor‐specific memory CD4 T cells provide help for alloantibody production in the absence of CD40/CD154 interactions and germinal center formation

CD40 signaling is essential for immunoglobulin class switching in response to T‐dependent antigens. We investigated whether memory CD4 T cell help can be delivered in the absence of CD40/CD154 interactions.We used either polyclonal alloreactive memory CD4 T cells from C57BL/6 (B6, H‐2b) recipients of C3H (H‐2k) skin allografts or TCR transgenic Mar memory CD4 T cells reactive to male HYDby peptide. Wild type B6 or CD40−/− female mice injected with naïve or memory CD4 T cells received heterotopic heart transplants from male C3H donors, donor‐reactive serum alloAb titers were determined by flow cytometry.We observed that memory but not naïve CD4 T cells induced high titers of donor‐specific IgG Ab in wild type B6 recipients treated with anti‐CD154 mAb MR1. After adoptive transfer into CD40−/− mice, memory, but not naïve CD4 cells provided help to CD40−/− B cells to produce donor‐reactive IgG1, IgG2a, IgG2b and IgG3 as early as 1 week post transplant. Despite high titers of serum IgG, PNA and B220 staining revealed no germinal center formation in the spleens of CD40−/− recipients containing memory CD4 T cells. AlloAb production was not affected by the treatment of CD40−/− recipients with MR1 Ab or anti‐ICOS mAb, indicating that the memory CD4 T cell help to CD40−/− B cells was independent of CD154 engagement by an alternative ligand or ICOS/B7RP‐1 pathway.In summary, donor‐specific memory CD4 T cells can initiate alloAb response in CD40‐deficient heart allograft recipients. Thus, therapeutic interruption of CD40/CD154 signaling may fail to efficiently block de novo alloantibody production in T cell sensitized transplant patients.

Proposed Relationship Between Intravenous Immunoglobulin and Thrombosis in Renal Transplant Recipients

To report 2 cases of intravenous human immunoglobulin (IVIG)-associated thrombosis in kidney transplant patients. Both cases involved female patients presenting to Henry Ford Hospital in Detroit for renal transplantation. Patient 1 presented with systemic lupus erythematosus, positive for both anticardiolipin and anti-DNA antibody. Patient 2, postnephrectomy, was found to have moderate hyperhomocysteinemia, with a total plasma homocysteine level of 3.9 mg/L. Both patients were considered highly sensitized and at high risk for rejection due to the presence of either high panel reactive antibody or a positive B cell flow cytometry crossmatch, in addition to other risk factors. Therefore, IVIG was considered a viable treatment option to be included in induction therapy. IVIG was administered both peri- and postoperatively, and both patients experienced immediate graft function with good urine output. Within 24 hours following transplantation, elevations in serum creatinine and decreases in urine output were seen. Subsequently, kidney exploration was performed and palpable thrombi in renal arteries and veins were detected. Immediate nephrectomy was performed in both cases. Currently, evidence derived from case reports highlights numerous risk factors for IVIG-associated thrombosis, one of which appears to be a hypercoagulable state. It has also been reported that some IVIG products contain amounts of anticardiolipin antibodies; these antibodies may potentiate thrombosis in the presence of hypercoagulable states, such as hyperhomocysteinemia or antiphospholipid syndrome. In these 2 patients, the Naranjo probability scale indicated that there was a possible association between the thrombotic events and the use of IVIG. Prospective trials evaluating the safety of IVIG in highly sensitized transplant patients are scarce. Therefore, it is imperative that the benefits and risks be weighed when considering the use of IVIG in highly sensitized transplant patients.

HLA Class I Epitopes: Recognition of Binding Sites by mAbs or Eluted Alloantibody Confirmed With Single Recombinant Antigens

Development of beads coated with single recombinant HLA antigens has permitted the confirmation and further definition of HLA class I epitopes. In this study, monoclonal antibodies (mAbs) or alloantibodies eluted from recombinant cell lines were tested for reactivity with Luminex beads individually coated with 79 recombinant HLA class I single antigen (rHLA SA). Published amino acid sequences were used to map epitopes common to sets of antigens reactive with each antibody. While several epitopes have already been demonstrated, this study confirmed them by adsorption of allosera with transfectants or SA beads having a single HLA antigen and specific binding of the eluted antibody on SA beads. The allosera and mAbs used in this study recognized a total of at least 58 HLA class I epitopes, as demonstrated by their different adsorption/reactivity patterns. Of these, 25 epitopes were characterized by a single unique common amino acid, 30 shared 2 signature amino acids in close proximity, and 3 epitopes involved 3 specific amino acids in a non-linear sequence. Since these epitopes may be targets for antibody-mediated allograft rejection, epitope analysis should complement HLA and CREG assignment for defining complex antibodies and identifying suitable donors for highly sensitized transplant patients.

Lymphoid Sequestration of Alloreactive Memory CD4 T Cells Promotes Cardiac Allograft Survival

Memory T cells specific for donor Ags present a unique challenge in transplantation. In addition to expressing robust immune responses to a transplanted organ, memory T cells may be resistant to the effects of immunosuppressive therapies used to prolong graft survival. In this study, we explore the possibility of controlling deleterious donor-reactive memory CD4 T cells through lymphoid sequestration. We showed that sphingosine 1-phosphate receptor-1 agonist FTY720 induces relocation of circulating memory CD4 T cells into secondary lymphoid organs. Lymphoid sequestration of these donor-reactive memory CD4 T cells prolonged survival of murine heterotopic cardiac allografts and synergizes with conventional costimulatory blockade to further increase graft survival. Despite limited trafficking, memory CD4 T cells remain capable of providing help for the induction of anti-donor CD8 T cell and alloantibody responses. Elimination of antidonor humoral immunity resulted in indefinite allograft survival proving the pathogenicity of alloantibody under these conditions. Overall, this is the first demonstration that FTY720 influences memory CD4 T cell trafficking and attenuates their contribution to allograft rejection. The data have important implications for guiding FTY720 therapy and for designing combinatorial strategies aimed at prolonging allograft survival in sensitized transplant patients with donor-specific memory T cells.