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• W2058058730 abstract "The contribution of memory B cells in alloreactive humoral responses remains poorly understood. Here we tested the presence of circulating alloreactive memory B cells in 69 patients with end-stage renal disease under renal replacement therapy, using an in vitro memory B cell–stimulation assay combined with identification of IgG human leukocyte antigen (HLA) antibodies in culture supernatant. HLA antibody–producing memory B cells were evidenced only in patients carrying serum HLA antibodies following multiple classical HLA-immunizing events. In patients with a previous renal allograft, alloreactive memory B cells could be detected ranging from 6 to 32 years (mean 13.2 years) after transplantation. HLA antibodies produced by memory B cells were also detected in the corresponding sera and showed a restricted reactivity, targeting only a few epitopes shared by several HLA antigens. In contrast, serum HLA antibodies, not associated with the detection of specific memory B cells, showed a broader pattern of specificities. Thus, expansion and survival of alloreactive memory B cells is alloantigen driven, and their frequency is related to the ‘strength’ of HLA immunization. The contribution of memory B cells in alloreactive humoral responses remains poorly understood. Here we tested the presence of circulating alloreactive memory B cells in 69 patients with end-stage renal disease under renal replacement therapy, using an in vitro memory B cell–stimulation assay combined with identification of IgG human leukocyte antigen (HLA) antibodies in culture supernatant. HLA antibody–producing memory B cells were evidenced only in patients carrying serum HLA antibodies following multiple classical HLA-immunizing events. In patients with a previous renal allograft, alloreactive memory B cells could be detected ranging from 6 to 32 years (mean 13.2 years) after transplantation. HLA antibodies produced by memory B cells were also detected in the corresponding sera and showed a restricted reactivity, targeting only a few epitopes shared by several HLA antigens. In contrast, serum HLA antibodies, not associated with the detection of specific memory B cells, showed a broader pattern of specificities. Thus, expansion and survival of alloreactive memory B cells is alloantigen driven, and their frequency is related to the ‘strength’ of HLA immunization. Antibody-mediated rejection owing to donor-specific human leukocyte antigen (HLA) alloantibodies (DSA) represents the primary cause of late kidney allograft loss.1Einecke G. Sis B. Reeve J. et al.Antibody-mediated microcirculation injury is the major cause of late kidney transplant failure.Am J Transplant. 2009; 9: 2520-2531Crossref PubMed Scopus (536) Google Scholar, 2Gaston R.S. Cecka J.M. Kasiske B.L. et al.Evidence for antibody-mediated injury as a major determinant of late kidney allograft failure.Transplantation. 2010; 90: 68-74Crossref PubMed Scopus (389) Google Scholar, 3Lefaucheur C. Loupy A. Hill G.S. et al.Preexisting donor-specific HLA antibodies predict outcome in kidney transplantation.J Am Soc Nephrol. 2010; 21: 1398-1406Crossref PubMed Scopus (625) Google Scholar, 4Mohan S. Palanisamy A. Tsapepas D. et al.Donor-specific antibodies adversely affect kidney allograft outcomes.J Am Soc Nephrol. 2012; 23: 2061-2071Crossref PubMed Scopus (201) Google Scholar Several therapies aiming at depleting B cells or antibodies are currently used to prevent/control antibody–mediated rejection and improve graft survival in patients carrying DSA. Although such therapies appear to give better access to transplantation and a survival advantage as compared with patients still awaiting a transplant,5Marfo K. Lu A. Ling M. et al.Desensitization protocols and their outcome.Clin J Am Soc Nephrol. 2011; 6: 922-936Crossref PubMed Scopus (190) Google Scholar, 6Montgomery R.A. Lonze B.E. King K.E. et al.Desensitization in HLA-incompatible kidney recipients and survival.N Engl J Med. 2011; 365: 318-326Crossref PubMed Scopus (502) Google Scholar their effectiveness has not been formally proven. As preventing the development of DSA appears necessary to reduce transplant rejection, there is a real need to better characterize humoral alloreactive responses in patients awaiting a kidney transplant. During immune responses toward protein antigens, both memory B cells and plasma cells are generated. Continuous synthesis of antibodies by long-lived plasma cells can provide durable humoral protection through the production of high-affinity antibodies that may persist for decades (serological memory).7McHeyzer-Williams L.J. McHeyzer-Williams M.G. Antigen-specific memory B cell development.Annu Rev Immunol. 2005; 23: 487-513Crossref PubMed Scopus (563) Google Scholar, 8Elgueta R. de Vries V.C. Noelle R.J. The immortality of humoral immunity.Immunol Rev. 2010; 236: 139-150Crossref PubMed Scopus (79) Google Scholar, 9Good-Jacobson K.L. Tarlinton D.M. Multiple routes to B-cell memory.Int Immunol. 2012; 24: 403-408Crossref PubMed Scopus (41) Google Scholar Upon reencounter with the antigen, memory B cells can rapidly differentiate into short-lived and long-lived plasma cells, thus enhancing the titers of specific antibodies. The mechanisms sustaining memory B cell and plasma cell survival for such remarkably long periods of time are still poorly defined. A critical question, particularly under conditions in which the antigen is cleared, is whether the pool of long-lived plasma cells is replenished through plasma cell differentiation from memory B cells. The lack of correlation observed between the frequency of peripheral-specific memory B cells and antibody levels10Amanna I.J. Carlson N.E. Slifka M.K. Duration of humoral immunity to common viral and vaccine antigens.N Engl J Med. 2007; 357: 1903-1915Crossref PubMed Scopus (827) Google Scholar, 11Leyendeckers H. Odendahl M. Lohndorf A. et al.Correlation analysis between frequencies of circulating antigen-specific IgG-bearing memory B cells and serum titers of antigen-specific IgG.Eur J Immunol. 1999; 29: 1406-1417Crossref PubMed Scopus (110) Google Scholar argues against such a replenishment. Nevertheless, CD20 monoclonal antibody (rituximab) treatment, which depletes all mature CD20+ B cells, including memory B cells but not plasma cells, does have an impact on the titer of specific antibodies in various clinical settings such as rheumatoid arthritis,12Cambridge G. Leandro M.J. Edwards J.C. et al.Serologic changes following B lymphocyte depletion therapy for rheumatoid arthritis.Arthritis Rheum. 2003; 48: 2146-2154Crossref PubMed Scopus (404) Google Scholar systemic lupus erythematosus,13Vallerskog T. Gunnarsson I. Widhe M. et al.Treatment with rituximab affects both the cellular and the humoral arm of the immune system in patients with SLE.Clin Immunol. 2007; 122: 62-74Crossref PubMed Scopus (197) Google Scholar antineutrophil cytoplasmic antibody–associated vasculitis,14Smith R.M. Jones R.B. Guerry M.J. et al.Rituximab for remission maintenance in relapsing antineutrophil cytoplasmic antibody-associated vasculitis.Arthritis Rheum. 2012; 64: 3760-3769Crossref PubMed Scopus (199) Google Scholar or lymphoma.15van der Kolk L.E. Baars J.W. Prins M.H. et al.Rituximab treatment results in impaired secondary humoral immune responsiveness.Blood. 2002; 100: 2257-2259PubMed Google Scholar In the transplantation setting, the relative contribution of memory B cells and plasma cells in the production of HLA antibodies (HLA-Abs) is also still ill-defined, although alloreactive memory B cells,16Mulder A. Eijsink C. Kardol M.J. et al.Identification, isolation, and culture of HLA-A2-specific B lymphocytes using MHC class I tetramers.J Immunol. 2003; 171: 6599-6603Crossref PubMed Scopus (46) Google Scholar, 17Han M. Rogers J.A. Lavingia B. et al.Peripheral blood B cells producing donor-specific HLA antibodies in vitro.Hum Immunol. 2009; 70: 29-34Crossref PubMed Scopus (49) Google Scholar, 18Zachary A.A. Kopchaliiska D. Montgomery R.A. et al.HLA-specific B cells: I. A method for their detection, quantification, and isolation using HLA tetramers.Transplantation. 2007; 83: 982-988Crossref PubMed Scopus (60) Google Scholar, 19Heidt S. Roelen D.L. de Vaal Y.J. et al.A NOVel ELISPOT assay to quantify HLA-specific B cells in HLA-immunized individuals.Am J Transplant. 2012; 12: 1469-1478Crossref PubMed Scopus (58) Google Scholar as well as bone marrow–derived plasma cells,20Perry D.K. Pollinger H.S. Burns J.M. et al.Two novel assays of alloantibody-secreting cells demonstrating resistance to desensitization with IVIG and rATG.Am J Transplant. 2008; 8: 133-143Crossref PubMed Scopus (71) Google Scholar have been detected in HLA-immunized patients. HLA-specific B cells have been identified and quantified using the ELISPOT technique.19Heidt S. Roelen D.L. de Vaal Y.J. et al.A NOVel ELISPOT assay to quantify HLA-specific B cells in HLA-immunized individuals.Am J Transplant. 2012; 12: 1469-1478Crossref PubMed Scopus (58) Google Scholar, 20Perry D.K. Pollinger H.S. Burns J.M. et al.Two novel assays of alloantibody-secreting cells demonstrating resistance to desensitization with IVIG and rATG.Am J Transplant. 2008; 8: 133-143Crossref PubMed Scopus (71) Google Scholar, 21Lynch R.J. Silva I.A. Chen B.J. et al.Cryptic B cell response to renal transplantation.Am J Transplant. 2013; 13: 1713-1723Crossref PubMed Scopus (60) Google Scholar ELISPOT assays require significant quantities of antigen in the form of recombinant HLA monomers of single specificity.19Heidt S. Roelen D.L. de Vaal Y.J. et al.A NOVel ELISPOT assay to quantify HLA-specific B cells in HLA-immunized individuals.Am J Transplant. 2012; 12: 1469-1478Crossref PubMed Scopus (58) Google Scholar Recently, an ELISPOT assay based on the use of donor-derived fibroblasts as allogenic targets was reported.21Lynch R.J. Silva I.A. Chen B.J. et al.Cryptic B cell response to renal transplantation.Am J Transplant. 2013; 13: 1713-1723Crossref PubMed Scopus (60) Google Scholar Owing to technical complexities, such assays cannot be easily used on a routine basis. To better characterize the nature of the alloreactive humoral response in end-stage renal disease (ESRD) patients carrying serum HLA-Abs and awaiting a kidney transplant, we studied whether circulating alloreactive memory B cells could be detected in such patients. We used an assay based on the polyclonal stimulation of peripheral memory B cells, similar to that used in the conventional ELISPOT technique, combined with multiplex bead array identification of HLA-reactive antibodies produced by alloreactive memory B cells. We used this assay in patients with or without a history of HLA-sensitizing events. In addition, the specificity of identified HLA-Abs produced by these stimulated B cells was evaluated at the epitope level in comparison with that of HLA-Abs present in the serum. We analyzed alloreactive memory B cells using a modified assay of B-cell stimulation previously described.22Crotty S. Aubert R.D. Glidewell J. et al.Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system.J Immunol Methods. 2004; 286: 111-122Crossref PubMed Scopus (355) Google Scholar We used cells from ESRD patients under renal replacement therapy and awaiting a kidney transplant. In these patients, plasmablasts accounted for a very low percentage of circulating B cells (IgD-CD38hi: 0.5±0.9% of total CD19+ cells). To differentiate memory B cells into plasmablasts, we cultured total peripheral blood mononuclear cells (PBMCs) with a polyclonal stimulation cocktail. Unstimulated control cultures in the presence of only interleukin (IL)-2 were also performed. After 6 days of culture, the percentage of plasmablasts increased to 36.3±24.4% (Figure 1a). Plasmablasts were not detected in control cultures. In preliminary experiments, in vitro cultures lasting from 6 to 12 days were tested. The maximum concentration of immunoglobulin (Ig)G in culture supernatants was observed after 10 days of culture; this condition was selected for further experiments. The presence of peripheral alloreactive memory B cells in the PBMCs was assessed by detecting HLA-Abs in culture supernatants using a HLA multiplex bead array. The specificity and the mean fluorescence intensity (MFI) rank of HLA-Abs measured in the supernatants (SN-HLA-Abs) was compared with those of HLA-Abs detected in the serum (s-HLA-Abs) collected on the same day as circulating PBMCs (Figure 1b). We screened three populations of patients (Figure 2), namely patients with no HLA-Abs (group 1), patients with HLA-Abs but without a history of immunizing events (group 2), and patients with HLA-Abs and a history of pregnancy, transfusion, and/or previous transplantation (group 3). The three groups did not differ with respect to the underlying causes of ESRD or previous immunosuppressive therapy to treat primary nephropathy (Table 1). However, patients in group 3 were more frequently women than those in group 1 and group 2. They expressed more frequently class II HLA-Abs and showed a higher percentage of positive single-antigen flow beads—i.e., a greater diversity of HLA-Ab specificities as compared with patients in group 2.Table 1Baseline characteristics of the three groups of patientsCharacteristics of patientsGroup 1, no s-HLA-Ab (n =18)Group 2, s-HLA-Ab+/no IE (n=12)Group 3, s-HLA-Ab+/IE+ (n=39)PAge of recipient, years (mean±s.d.)48.0±12.241.9±13.146.0±13.50.46Sex, n (% female)4 (22.2)1 (8.3)24 (61.5)0.0003Cause of ESRD Diabetes2 (11.1)2 (16.7)2 (5.1)0.32 Glomerulonephritis7 (38.9)3 (25.0)11 (28.2)0.64 Hypertensive nephropathy2 (11.1)0 (0)1 (2.6)0.57 Interstitial nephritis3 (16.7)3 (25.0)7 (18.0)0.82 Polycystic disease2 (11.1)1 (7.7)4 (10.3)0.96 Unknown2 (11.1)2 (16.7)11 (28.2)0.31 Other0 (0)1 (7.7)3 (7.7)0.47Previous immunosuppression for nephropathy1 (5.6)0 (0)5 (12.8)0.33Previous transplant, n (%)4 (22.2)—20 (51.3)0.039Transfusion, n (%)9 (50.0)—31 (81.6)0.015Pregnancy, n (%)3/4 (75.0)—22/24 (91.7)0.32No immunizing event, n (%)9 (50.0)12 (100)—s-HLA -Ab class I, n (%)—7 (58.3)30 (83.3)0.07s-HLA -Ab class II, n (%)—5 (41.7)28 (77.8)0.019s-HLA -Ab class I, MFI max—3658±29727413±63100.10s-HLA -Ab class II, MFI max—2410±24828497±78020.17Positive class I beads, %—2.2±2.515.3±18.8*0.021Positive class II beads, %—2.1±4.417.8±21.2*0.015Abbreviations: ESRD, end-stage renal disease; IE, immunizing event; s-HLA-Ab, serum HLA antibodies. P values in boldface are statistically significant. Open table in a new tab Abbreviations: ESRD, end-stage renal disease; IE, immunizing event; s-HLA-Ab, serum HLA antibodies. P values in boldface are statistically significant. SN-HLA-Abs were detected in 18 out of 39 patients of group 3 (Figure 2). No SN-HLA-Abs were detected in patients from group 1 and group 2. Patients with or without SN-HLA-Abs had similar percentages of CD19+ peripheral B cells and similar distribution of B-cell subsets, including memory B cells (Supplementary Figure S1 online). After 6 days of stimulation, the percentage of plasmablasts did not differ (mean±s.d.: 21.0±19.1 vs. 25.6±21.7, P=0.5 in SN-HLA-Ab-positive and -negative patients, respectively). Download .pdf (1.23 MB) Help with pdf files Supplementary Information In group 3, the proportions of patients with one, two, or three different types of immunizing events differed between SN-HLA-Ab-positive and -negative subgroups: 22.2, 38.9, and 38.9% vs. 42.9, 52.4, and 4.7%, respectively (P=0.028; Figure 3). Accordingly, the mean number of immunizing events was higher—2.2±0.8 vs. 1.6±0.6, P=0.018—in patients who exhibited alloreactive memory B cells as compared with those who did not. Moreover, patients with SN-HLA-Abs had a higher maximal HLA-Ab MFI in the serum, as well as a greater percentage of positive single-antigen flow beads (Table 2). No relation between the detection of SN-HLA-Abs and other clinical parameters such as previous immunotherapy (for nephropathy or previous transplantation) or primary cause of ESRD could be evidenced (Table 2). Of note, the frequency of transplant removal was higher in SN-HLA-Ab-positive (75%) than -negative (37.5%) patients, but this difference was not statistically significant (Table 2).Table 2Characteristics of group 3 patients with supernatant HLA-AbsSN-HLA-Ab+ n=18SN-HLA-Ab- n=21PAge years, mean±s.d.48.3±11.344.0±15.10.33Sex female, n (%)14 (77.8)10 (47.6)0.05Cause of ESRD Diabetes1 (5.6)1 (4.8)1.00 Glomerulonephritis6 (33.3)5 (23.8)0.72 Hypertensive nephropathy0 (0)1 (4.8)1.00 Interstitial nephritis2 (11.1)5 (23.8)0.42 Polycystic nephropathy2 (11.1)2 (9.5)1.00 Unknown5 (27.8)6 (28.6)1.00 Other2 (11.1)1 (4.8)0.59Previous immunosuppression for nephropathy4 (22.2)aSteroids plus cyclophosphamide or azathioprine for lupus nephritis (n=3) or Henoch Schönlein purpura (n=1).1 (4.7)bSteroids only for focal and segmental glomerulosclerosis.0.16Previous transplant, n (%)12 (66.7)8 (38.1)0.075 None, n (%)6 (33.3)13 (61.9) One, n (%)8 (44.4)6 (28.6)0.65 Two or more, n (%)4 (22.3)2 (9.5) Transplant removal, n (%)8/12 (75.0)3/8 (37.5)0.36Pregnancy, n (%)13/14 (92.9)9/10 (90)0.80Transfusion, n (%)14 (77.8)17 (85)0.56s-HLA -Ab class I, MFI max11273±50854147±54350.003s-HLA -Ab class II, MFI max14077±69073383±43260.0002s-HLA -Ab class I or II, MFI max16495±36024810±5590<0.0001Positive class I beads, %27.4±18.57.9±14.90.002Positive class II beads, %33.0±22.47.0±12.0<0.0001Abbreviations: ESRD, end-stage renal disease; MFI, mean fluorescence intensity; s-HLA-Ab, serum HLA antibodies; SN-HLA-Ab, supernatant HLA antibodies. P values in boldface are statistically significant.a Steroids plus cyclophosphamide or azathioprine for lupus nephritis (n=3) or Henoch Schönlein purpura (n=1).b Steroids only for focal and segmental glomerulosclerosis. Open table in a new tab Abbreviations: ESRD, end-stage renal disease; MFI, mean fluorescence intensity; s-HLA-Ab, serum HLA antibodies; SN-HLA-Ab, supernatant HLA antibodies. P values in boldface are statistically significant. In addition, among the nine patients with a transplant in situ, five still received immunosuppression (consisting only of prednisone, 5–10 mg per day in all cases): three SN-HLA-Ab-positive patients (75%) and two SN-HLA-Ab-negative patients (40%). The only HLA-immunizing event that could be precisely dated was a previous transplantation. Twelve patients with SN-HLA-Abs had received one or more previous transplants. In nine of them, we found, in supernatants, antibodies specific for alloantigens from a previous transplant (Table 3 and Supplementary Figure S2 online). In all cases, donor-specific SN-HLA-Abs were also present in the corresponding serum (Supplementary Figure S2 online). In the three remaining patients (#M, N, and Q, Table 3), the presence of alloantibodies reactive to a previous graft could not be excluded owing to the lack of HLA-DQ and -DP typing of the donors. The delay between the previous transplant and the day of testing ranged from 6 to 32 years (mean 13.2±8.4 years), highlighting a very long survival of these alloantibody-producing B cells detected in our assay. Of note, such SN-HLA-Abs specific for antigens of a previous graft were detected in five cases after graft removal (#A, C, I, K, and O; Supplementary Table S1 online).Table 3Analysis of supernatant HLA-Abs at the antigen and epitope levels in group 3PatientImmunizing eventsaR: red cell transfusion, P: pregnancy, T: previous transplant; in brackets: lack of HLA-DQ or -DP typing of a previous allograft donor. (Donor typing)Number of previous transplantHLA reactivity of SN-HLA-AbsbAs assessed by multiplex bead array. (*MM of a previous transplant)SN-HLA-Abs also detected in serumEplets targeted by SN-HLA-AbscAccording to HLAMatchmaker algorithm; for each eplet, the numbers indicate the exposed polymorphic residues and the letters represent polymorphic residues in the 3.0 Å patch of the HLA molecule. (*shared by a MM of a previous transplant)HLA antigens expressing the identified eplet(s)Eplets targeted both by SN-HLA-Abs and s-HLA-AbsART1 A23-24*-25-32-38-49-51-52-53- 57-58-59-63 B13-27-37-44-47-77all82LR*all82LR*BRT1 A23* B7-8-13-27-37-42-48-55-59-60-61-72-81 DQ5 DP2-23all except DQ5125SQonly DQ5—CRPT (DP unknown)2 B62-64-65-71 Cw6-18 DQ5* DP1-2-3-5-6-9-10-13-14-15-17-18-23-28Cw6-Cw18 DQ5DQ eplets 57LD*/185I*DP eplets 84VG-56ED-76V-35LVDQ5* all DP except DP2-DP23—DRPT1 B13-18-27-37-38-39-47*-65-67B13-18-27-37-38-47*-6776ED*-158TB27-37-47* B38-39-6776ED*FT (DQ-DP unknown)2 A1*-3-11-23-24-25-29-30-31-32*-33-34-36-43-66-74-80 B76 Cw7 DR8-11-12-13-14-17-18 DQ2allA and B eplets 62QE*-167DG*-113YR*DR eplets 12 STS-14GEYDP eplets 45GES/56LPAall class I except A25-34-43-66-74, Cw7 all DR all DQ45GESIRPT1 B57*-58all63GEN*all63GEN*JRPT1 A1*-36all44KM*all44KM*KRT3 B8-18-35-37-38-39-48-51*-52-53-59-62*-71-72-75-76-77-78all66QIF*B8-35-51*-53-59-7866QIF*ORT1 DR53*DR53*48YQ6*DR53*48YQ6*MRPT (DQ unknown)1 DQ7-8-9all55PPPall—NRPT (DQ unknown)1 DQ4-7-8-9all52PL/140T2all52PL/140T2QRPT (DQ-DP unknown)2 DQ2-4-7-8-9 DP1-3-5-6-9-10-11-13-14-17-19allDQ eplets 84QL2DP eplets 84DEAV—84QL284DEAVERP0 B8-35-51-53-59-78all66QIFall66QIFGP0 A80 B73all56EA8056EHP0 A1-36all44KMall44KMLRP0 A25 DQ7A25DQ eplets 45EVDQ7—PRP0 DR4-7-12-13-15-53 DQ5-6-7-8-9all except DR7-15-53DR eplets 96YL4-25YRL-67IK-48YQ6-31QFHY-26QF3/67IR/67GDTDQ eplets 55PPP/66EV/67VVT/70RT/71VRT/74ELall except DQ5-655PPP/66EV/67VVT/70RT/71VRT/74ELRP0DR1DR1———Abbreviations: MM, mismatch; SN-HLA-Ab, supernatant HLA antibodies.a R: red cell transfusion, P: pregnancy, T: previous transplant; in brackets: lack of HLA-DQ or -DP typing of a previous allograft donor.b As assessed by multiplex bead array.c According to HLAMatchmaker algorithm; for each eplet, the numbers indicate the exposed polymorphic residues and the letters represent polymorphic residues in the 3.0 Å patch of the HLA molecule. Open table in a new tab Abbreviations: MM, mismatch; SN-HLA-Ab, supernatant HLA antibodies. We compared in group 3 the target HLA specificities and the MFI of SN-HLA-Abs with those of s-HLA-Abs of the same patients. In supernatants, we observed antibodies to class I alloantigens only in eight patients, to class II alloantigens only in six patients, and to both class I and II alloantigens in four patients (Figure 2). We found fewer antigenic specificities for class I and II alloantibodies in supernatants when compared with the corresponding serum: 9±9 class I or class II specificities in supernatants versus 34±21 in serum (P<0.0001; Supplementary Table S1 online, Figures 4 and 5). All SN-HLA-Ab-positive patients except one had both class I and class II alloantibodies in the serum. Most SN-HLA-Abs were also present in the serum collected on the same day (86% on average; Supplementary Figure S3 online). Alloantibodies targeting one or two mismatched antigens from a previous donor (underlined bold antigens in Supplementary Table S1 and Figures S2, S4, and S5 online) were found concomitantly in the supernatants and in the serum of the nine previously transplanted patients with known HLA typing of the donors. In four cases, these antibodies were detected in the serum together with one to six additional donor-specific specificities present in serum only.Figure 5Comparison between supernatant and serum alloantibodies in patient E. Specificity of alloantibodies in a 45-year-old woman on the day of her first transplant. SN-HLA-Abs target the 66QIF epitope. Most HLA class I antibodies (n=45) detected in the serum are not found in SN and target, in addition to 6QIF, five other epitopes: 66RN, 44RT, 82LR, 138MI and 90D. For purpose of clarity, the MFI of specificities found in SN were multiplied by 10. SN, supernatant; SN-HLA-Abs, HLA antibodies measured in the supernatants.View Large Image Figure ViewerDownload (PPT) When assessing the strength of different HLA specificities by means of their respective MFI, we did not find the same hierarchy in the supernatants and in the serum from the same patients (Supplementary Table S1 online). In other words, specificities with the highest reactivity were distinct in the supernatants and the serums. For example (Supplementary Figure S4 online), in patient F who had received two previous transplants, the HLA-A1 antibody targeting a mismatched antigen of the second donor was the highest ranked antibody in the culture supernatant but not in the serum. The anti-A32 directed against the first donor showed one of the weakest MFI in the supernatant and a medium MFI in the serum. To evaluate the repertoire of SN-HLA-Abs detected in group 3 patients, we took advantage of the HLAMatchmaker algorithm, which allows identifying HLA functional epitopes or eplets targeted by these antibodies according to their HLA reactivity pattern23Duquesnoy R.J. Antibody-reactive epitope determination with HLAMatchmaker and its clinical applications.Tissue Antigens. 2011; 77: 525-534Crossref PubMed Scopus (66) Google Scholar (see Materials and Methods). Such eplets are constituted of two to five highly energetic amino acid residues, including at least a polymorphic one, at the surface of the HLA molecule in an antibody-accessible location.24Duquesnoy R.J. A structurally based approach to determine HLA compatibility at the humoral immune level.Hum Immunol. 2006; 67: 847-862Crossref PubMed Scopus (205) Google Scholar They represent key elements of structural HLA epitopes that can induce the formation of specific antibodies. In 14 patients (78%), the pattern of supernatants’ alloantibody reactivities was restricted to only one or two shared eplets (Table 3 and Supplementary Table S1 online: #A, B, D, E, G, H, I, J, K, L, M, N, O, and Q; Figure 4), accounting in 9 cases (#A, E, H, I, J, K, M, N, and Q) for all HLA antigenic specificities detected (Figure 4, i.e., patient A in Supplementary Figure S5 online). In 13 patients (72%), at least one eplet targeted by SN-HLA-Abs was also targeted by s-HLA-Abs (Supplementary Figure S6 online). In contrast, in seven patients, some eplets were targeted exclusively by SN-HLA-Abs, corresponding in six cases to HLA-antigenic specificities identified only in the supernatants (Supplementary Figure S6 and Table S1 online and Table 3: #B, C, D, L, M, and P). The number of eplets targeted by s-HLA-Abs was significantly higher, ranging from 1 to >10 (median (range): 7 (1–15) eplets in sera vs. 1 (0–7) eplets in supernatants, P<0.0001) (Supplementary Table S1 online and Figures 4 and 5). Interestingly, in patient F, different eplets of the HLA class I mismatched antigens from previous donors were targeted both by supernatant and serum alloantibodies (Supplementary Table S1 online). In 8 of the 12 re-transplanted recipients, SN-HLA-Abs were directed against mismatched eplets between donor and recipient (patients #A, C, D, F, I, J, K, and O; eplets marked with * in Table 3 and Supplementary Table S1 and Figure S7 online). These donor-specific eplets were also targeted by s-HLA-Abs in six of these eight patients (75%). In this study, we describe, in a group of patients awaiting a kidney transplant, the detection of SN-HLA-Abs in an in vitro culture assay reflecting the presence of peripheral alloreactive memory B cells. This in vitro memory B-cell assay was adapted from the procedure of Crotty et al.22Crotty S. Aubert R.D. Glidewell J. et al.Tracking human antigen-specific memory B cells: a sensitive and generalized ELISPOT system.J Immunol Methods. 2004; 286: 111-122Crossref PubMed Scopus (355) Google Scholar developed to track antigen-specific IgG-producing memory B cells by ELISPOT.25Mamani-Matsuda M. Cosma A. Weller S. et al.The human spleen is a major reservoir for long-lived vaccinia virus-specific memory B cells.Blood. 2008; 111: 4653-4659Crossref PubMed Scopus (120) Google Scholar It is based on the use of polyclonal mitogens in combination with CpG, a TRL9 ligand, which triggers preferential proliferation and differentiation of memory B cells into plasma cells in the absence of B cell–receptor stimulation.26Bernasconi N.L. Traggiai E. Lanzavecchia A. Maintenance of serological memory by polyclonal activation of human memory B cells.Science. 2002; 298: 2199-2202Crossref PubMed Scopus (1069) Google Scholar We chose as a readout the measurement of IgG SN-HLA-Abs using a highly sensitive multiplex bead array technology. Such a readout, reflecting the production of HLA-Abs by activated memory B cells, has been successfully used by others in HLA-sensitized patients,17Han M. Rogers J.A. Lavingia B. et al.Peripheral blood B cells producing donor-specific HLA antibodies in vitro.Hum Immunol. 2009; 70: 29-34Crossref PubMed Scopus (49) Google Scholar, 18Zachary A.A. Kopchaliiska D. Montgomery R.A. et al.HLA-specific B cells: I. A method for their detection, quantification, and isolation using HLA tetramers.Transplantation. 2007; 83: 982-988Crossref PubMed Scopus (60) Google Scholar, 19Heidt S. Roelen D.L. de Vaal Y.J. et al.A NOVel ELISPOT assay to quantify HLA-specific B cells in HLA-immunized individuals.Am J Transplant. 2012; 12: 1469-1478Crossref PubMed Scopus (58) Google Scholar, 20Perry D.K. Pollinger H.S. Burns J.M. et al.Two novel assays of alloantibody-secreting cells demonstrating resistance to desensitization with IVIG and rATG.Am J Transplant. 2008; 8: 133-143Crossref PubMed Scopus (71) Google Scholar allowing the characterization of the specificity of HLA-Ab-producing B cells on the basis of the reactivity of SN-HLA-Abs with HLA ant" @default.
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• W2058058730 title "Restricted specificity of peripheral alloreactive memory B cells in HLA-sensitized patients awaiting a kidney transplant" @default.
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