Maintenance immunosuppressive therapy with everolimus preserves humoral immune responses
2010; Elsevier BV; Volume: 78; Issue: 9 Linguagem: Inglês
10.1038/ki.2010.269
ISSN1523-1755
AutoresGeertrude H. Struijk, Robert C. Minnee, Sven D. Koch, Aeilko H. Zwinderman, K.A.M.I. van Donselaar–van der Pant, Mirza M. Idu, Ineke J. M. ten Berge, Fréderike J. Bemelman,
Tópico(s)Transplantation: Methods and Outcomes
ResumoWhile the guidelines for vaccination in renal transplant recipients recommend the use of pneumococcal polysaccharide (PPS) and tetanus toxoid (TT), their efficacy in immunocompromised renal transplant recipients is not known. Here we tested the effect of everolimus on immune responses after vaccination by measuring the capacity of 36 stable renal transplant recipients to mount cellular and humoral responses after vaccination. Twelve patients in each treatment arm received immunosuppressive therapy consisting of prednisolone (P) plus cyclosporine (CsA), mycophenolate sodium (MPA), or everolimus. Patients were vaccinated with the T-cell-dependent antigens immunocyanin and TT, and the T-cell-independent PPS. Treatment with CsA partially inhibited and MPA completely abolished the capacity to mount a primary humoral response, whereas everolimus left this largely intact. Recall responses were inhibited by MPA only. All drug combinations inhibited cellular responses against TT. In patients treated with MPA, B-cell numbers were severely reduced. Thus, combined with P, treatment with MPA completely disturbed primary and secondary humoral responses. Everolimus or CsA allowed the boosting of T-cell-dependent and -independent secondary humoral responses. Treatment with everolimus allowed a primary response. While the guidelines for vaccination in renal transplant recipients recommend the use of pneumococcal polysaccharide (PPS) and tetanus toxoid (TT), their efficacy in immunocompromised renal transplant recipients is not known. Here we tested the effect of everolimus on immune responses after vaccination by measuring the capacity of 36 stable renal transplant recipients to mount cellular and humoral responses after vaccination. Twelve patients in each treatment arm received immunosuppressive therapy consisting of prednisolone (P) plus cyclosporine (CsA), mycophenolate sodium (MPA), or everolimus. Patients were vaccinated with the T-cell-dependent antigens immunocyanin and TT, and the T-cell-independent PPS. Treatment with CsA partially inhibited and MPA completely abolished the capacity to mount a primary humoral response, whereas everolimus left this largely intact. Recall responses were inhibited by MPA only. All drug combinations inhibited cellular responses against TT. In patients treated with MPA, B-cell numbers were severely reduced. Thus, combined with P, treatment with MPA completely disturbed primary and secondary humoral responses. Everolimus or CsA allowed the boosting of T-cell-dependent and -independent secondary humoral responses. Treatment with everolimus allowed a primary response. Current immunosuppressive drug regimens after renal transplantation have improved allograft survival at the expense of more profound immunosuppression, as apparent from an increased risk of infectious complications.1.Boggild A.K. Sano M. Humar A. et al.Travel patterns and risk behavior in solid organ transplant recipients.J Travel Med. 2004; 11: 37-43Crossref PubMed Scopus (70) Google Scholar, 2.Fishman J.A. Rubin R.H. Infection in organ-transplant recipients.N Engl J Med. 1998; 338: 1741-1751Crossref PubMed Scopus (1287) Google Scholar, 3.Vitko S. Margreiter R. Weimar W. et al.Everolimus (Certican) 12-month safety and efficacy versus mycophenolate mofetil in de novo renal transplant recipients.Transplantation. 2004; 78: 1532-1540Crossref PubMed Scopus (143) Google Scholar Guidelines for immunizations in renal transplant patients have been established by the American Society of Transplantation, recommending immunization with pneumococcal polysaccharide (PPS), tetanus toxoid (TT), and influenza.4.Guidelines for vaccination of solid organ transplant candidates and recipients.Am J Transplant. 2004; 4: 160-163Google Scholar The use of live vaccines is contraindicated. Despite these guidelines, there continues to be a gap between guidelines and clinical practice.5.Harris K. Baggs J. Davis R.L. et al.Influenza vaccination coverage among adult solid organ transplant recipients at three health maintenance organizations, 1995–2005.Vaccine. 2009; 27: 2335-2341Crossref PubMed Scopus (28) Google Scholar A main reason for non-vaccination is the absence of a doctors' recommendation.6.Lanternier F. Henegar C. Mouthon L. et al.Low influenza-vaccination rate among adults receiving immunosuppressive therapy for systemic inflammatory disease.Ann Rheum Dis. 2008; 67: 1047Crossref PubMed Scopus (47) Google Scholar This may be owing to concerns that vaccination might trigger allograft rejection.7.Blumberg E.A. Brozena S.C. Stutman P. et al.Immunogenicity of pneumococcal vaccine in heart transplant recipients.Clin Infect Dis. 2001; 32: 307-310Crossref PubMed Scopus (56) Google Scholar, 8.Vilchez R.A. McCurry K. Dauber J. et al.Influenza virus infection in adult solid organ transplant recipients.Am J Transplant. 2002; 2: 287-291Crossref PubMed Scopus (189) Google Scholar However, this concern is unfounded.9.Cainelli F. Vento S. Infections and solid organ transplant rejection: a cause-and-effect relationship?.Lancet Infect Dis. 2002; 2: 539-549Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 10.Candon S. Thervet E. Lebon P. et al.Humoral and cellular immune responses after influenza vaccination in kidney transplant recipients.Am J Transplant. 2009; 9: 2346-2354Crossref PubMed Scopus (86) Google Scholar Another reason might be doubts about efficacy in immunosuppressed patients. Different immunosuppressive regimens vary in their effects on cellular and humoral responses after vaccination. Most studies on humoral responses after vaccination have been performed in the prednisolone (P), azathioprine, and cyclosporine (CsA) era.11.ten Berge R.J. Schellekens P.T. Surachno S. et al.The influence of therapy with azathioprine and prednisone on the immune system of kidney transplant recipients.Clin Immunol Immunopathol. 1981; 21: 20-32Crossref PubMed Scopus (29) Google Scholar, 12.ten Berge R.J. Schellekens P.T. Surachno S. et al.A longitudinal study on the effects of azathioprine and high doses of prednisone on the immune system of kidney-transplant recipients.Clin Immunol Immunopathol. 1982; 24: 33-46Crossref PubMed Scopus (17) Google Scholar, 13.van der Heyden A.A. Bloemena E. Out T.A. et al.The influence of immunosuppressive treatment on immune responsiveness in vivo in kidney transplant recipients.Transplantation. 1989; 48: 44-47Crossref PubMed Scopus (8) Google Scholar Previously, we demonstrated that stable renal transplant recipients treated with double immunosuppressive therapy consisting of P and CsA mount normal antigen-specific antibody responses after vaccination with keyhole limpet hemocyanin and TT. In contrast, patients treated with mycophenolic acid (MPA), as part of a triple immunosuppressive maintenance regimen showed severely reduced antibody responses after vaccination.14.Rentenaar R.J. van Diepen F.N. Meijer R.T. et al.Immune responsiveness in renal transplant recipients: mycophenolic acid severely depresses humoral immunity in vivo.Kidney Int. 2002; 62: 319-328Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Recently, Candon et al.10.Candon S. Thervet E. Lebon P. et al.Humoral and cellular immune responses after influenza vaccination in kidney transplant recipients.Am J Transplant. 2009; 9: 2346-2354Crossref PubMed Scopus (86) Google Scholar showed diminished antibody responses to influenza vaccination in renal transplant recipients treated with steroids, MPA, or azathioprine, in combination with tacrolimus, CsA, or sirolimus, whereas interferon (IFN)-γ T-cell responses were conserved. Willcocks et al.15.Willcocks L.C. Chaudhry A.N. Smith J.C. et al.The effect of sirolimus therapy on vaccine responses in transplant recipients.Am J Transplant. 2007; 7: 2006-2011Crossref PubMed Scopus (36) Google Scholar demonstrated similar efficacy of vaccination with PPS and influenza in renal transplant recipients treated with a calcineurin inhibitor- or sirolimus-containing immunosuppressive regimen. Everolimus, like sirolimus, is a mammalian target of rapamycin inhibitor, which is effective in the prevention of renal allograft rejection3.Vitko S. Margreiter R. Weimar W. et al.Everolimus (Certican) 12-month safety and efficacy versus mycophenolate mofetil in de novo renal transplant recipients.Transplantation. 2004; 78: 1532-1540Crossref PubMed Scopus (143) Google Scholar, 16.Webster A.C. Lee V.W. Chapman J.R. et al.Target of rapamycin inhibitors (sirolimus and everolimus) for primary immunosuppression of kidney transplant recipients: a systematic review and meta-analysis of randomized trials.Transplantation. 2006; 81: 1234-1248Crossref PubMed Scopus (281) Google Scholar and has a different side effect profile compared with CsA and MPA.17.Andres V. Castro C. Campistol J.M. Potential role of proliferation signal inhibitors on atherosclerosis in renal transplant patients.Nephrol Dial Transplant. 2006; 21: iii14-iii17PubMed Google Scholar, 18.Meier-Kriesche H.U. Steffen B.J. Hochberg A.M. et al.Mycophenolate mofetil versus azathioprine therapy is associated with a significant protection against long-term renal allograft function deterioration.Transplantation. 2003; 75: 1341-1346Crossref PubMed Scopus (130) Google Scholar The effect of everolimus on human responses to vaccination in vivo has not been studied until now. We recently performed a randomized prospective trial in renal transplant recipients starting on induction therapy with an interleukin (IL)-2 receptor antagonist, followed by a triple immunosuppressive regimen. Immunosuppression was tapered after 6 months to double therapy with P combined with CsA, mycophenolate sodium (MPA), or everolimus.19.Bemelman F.J. de Maar E.F. Press R.R. et al.Minimization of maintenance immunosuppression early after renal transplantation: an interim analysis.Transplantation. 2009; 88: 421-428Crossref PubMed Scopus (57) Google Scholar In this study, we tightly monitored exposure to CsA, MPA, and everolimus. In a subgroup of these patients, we assessed the capacity to mount cellular and humoral immune responses after vaccination. In addition, phenotyping of peripheral B cells was performed to analyze the effects of the different immunosuppressive regimens on B-cell numbers and differentiation in vivo. A total of 36 renal transplant recipients, 12 in each treatment arm, and 13 healthy control individuals were included in this study. Baseline clinical characteristics are summarized in Table 1. Patient groups and healthy control individuals were comparable for age and sex. There was no difference in renal function between the patient groups and no acute rejection episodes had occurred.Table 1Characteristics of patients and healthy control individualsP/CsAP/MPAP/everolimusHCP-valueNumber of patients12121213Age (years, median range)58 (34–72)60 (30–70)50 (27–68)55 (42–63)NSMale (n, %)10 (83)9 (75)8 (67)8 (62)NSFirst/second transplant11/111/112/0—NSAcute rejection (%)000—NSEnd-stage renal disease Unknown211 Adult polycystic kidney disease231 Renal vascular disease154 Pyelonephritis211 Vasculitis11— Glomerular3—5 Other11—Renal function GFRaGFR estimated by the modification of diet in renal disease. (ml/min)49 (27–93)59 (26–100)59 (30–85)NANSAbbreviations: CsA, cyclosporine; GFR, glomerular filtration rate; HC, healthy control individuals; MPA, mycophenolate sodium; NA, not available; NS, not significant; P, prednisolone.a GFR estimated by the modification of diet in renal disease. Open table in a new tab Abbreviations: CsA, cyclosporine; GFR, glomerular filtration rate; HC, healthy control individuals; MPA, mycophenolate sodium; NA, not available; NS, not significant; P, prednisolone. Mean (±s.d.) immunocyanin-specific immunoglobulin (Ig) G antibody levels before vaccination were 737±456 in healthy control individuals; 897±513 in P/CsA-treated patients; 542±332 in P/MPA-treated patients; and 392±201 in P/everolimus-treated patients. Following vaccination, mean antigen-specific IgG antibody levels rose significantly in healthy control individuals (P<0.0005; Figure 1a) and in patients receiving P/everolimus (P<0.05). In patients treated with P/CsA or P/MPA, there was no significant increase in antigen-specific IgG antibody levels. The primary humoral response against immunocyanin, measured as the difference between post- and pre-vaccination immunocyanin-specific IgG antibody levels, was significantly reduced in all patient groups as compared with healthy control individuals (P<0.0005). There was no difference in response between the patient groups. Mean (±s.d.) TT-specific IgG antibody levels before vaccination were 2.35±1.86 in healthy control individuals; 1.31±1.56 in P/CsA-treated patients; 1.24±1.46 in P/MPA-treated patients; and 0.87±1.13 in P/everolimus-treated patients. Following vaccination, mean antigen-specific IgG antibody levels rose significantly in healthy control individuals (P<0.0005; Figure 1b), in patients receiving P/CsA (P<0.005), and in patients treated with P/everolimus (P<0.0005). In patients treated with P/MPA, there was no significant increase in antigen-specific IgG antibody levels. The secondary humoral response against TT, measured as the difference between post- and pre-vaccination TT-specific IgG antibody levels, was significantly reduced in patients treated with P/CsA and P/MPA as compared with healthy control individuals (P<0.05, P<000.5, respectively). Patients treated with P/MPA also showed a significantly decreased response as compared with patients receiving P/everolimus (P<0.005). In patients receiving P/everolimus, the humoral response against TT was not reduced compared with healthy control individuals. Mean (±s.d.) PPS-specific IgG antibody levels before vaccination were 138.4±162.5 in healthy control individuals; 50.3±45.9 in P/CsA-treated patients; 46.2±35.2 in P/MPA-treated patients; and 102.4±82.4 in P/everolimus-treated patients. Following vaccination, mean antigen-specific IgG antibody levels rose significantly in healthy control individuals (P<0.0005; Figure 1c), in patients receiving P/CsA (P<0.0005), and in patients treated with P/everolimus (P<0.0005). In patients treated with P/MPA, there was no significant increase in antigen-specific IgG antibody levels. The secondary humoral response against PPS, measured as the difference between post- and pre-vaccination PPS-specific IgG antibody levels, was significantly reduced in patients receiving P/CsA as compared with healthy control individuals (P<0.05). Patients treated with P/MPA showed a significantly decreased response as compared with healthy control individuals (P<0.0005) and with patients receiving P/CsA (P<0.05) or P/everolimus (P<0.005). In patients receiving P/everolimus, the humoral response against PPS was not reduced compared with healthy control individuals. TT-specific cellular responses in vitro, as assessed by enzyme-linked immunospot (ELISPOT) assay, are shown in Table 2. Non-specific stimulation with phytohaemagglutinin showed no difference in the number of responsive peripheral blood mononuclear cells (PBMCs) at both time points for all cytokines either in the healthy control individuals or in any patient group (data not shown).Table 2Number of IL-2-, IFN-γ-, and IL-4-producing cells after in vitro stimulation with tetanus toxoidIL-2IFN-γIL-4Day 0Day 14Day 0Day 14Day 0Day 14P/CsA0.8±1.54.7±5.8**5.2±10.810.0±10.6*0.2±0.46.3±11.7*P/MPA3.4±3.25.4±6.11.4±2.95.2±5.10.1±0.45.0±5.8*P/everolimus2.8±4.66.7±7.5*10.6±20.66.4±8.62.0±3.15.6±7.2*HC2.8±3.014.0±7.0***11.1±12.438.1±30.1**0.3±0.519.8±11.4***Abbreviations: CsA, cyclosporine; HC, healthy control individuals; IFN, interferon; IL, interleukin; MPA, mycophenolate sodium; P, prednisolone.Statistical comparison of the mean (±s.d.) number of cytokine-secreting cells before and 14 days after vaccination: *P<0.05, **P<0.005, ***P<0.0005. Results were corrected for background by deducting the responses from the culture with medium only. Open table in a new tab Abbreviations: CsA, cyclosporine; HC, healthy control individuals; IFN, interferon; IL, interleukin; MPA, mycophenolate sodium; P, prednisolone. Statistical comparison of the mean (±s.d.) number of cytokine-secreting cells before and 14 days after vaccination: *P<0.05, **P<0.005, ***P<0.0005. Results were corrected for background by deducting the responses from the culture with medium only. The mean (±s.d.) numbers of IL-2-producing cells before vaccination were: healthy control individuals 2.8±3.0, P/CsA 0.8±1.5, P/MPA 3.4±3.2, and P/everolimus 2.8±4.6. After vaccination, the number of IL-2-producing cells in PBMCs from healthy control individuals (P<0.0005) as well as from patients treated with P/CsA (P<0.005) or P/everolimus (P<0.05) showed a significant increase. In contrast, patients treated with P/MPA did not show a significant increase in IL-2-producing cells after vaccination. In all patient groups, the IL-2 response, represented as the difference between post- and pre-vaccination numbers of cytokine-producing cells per 105 PBMCs, was significantly less as compared with healthy control individuals. Before vaccination, in most healthy control individuals, IFN-γ-producing cells were detectable upon stimulation with TT in vitro (11.1±12.4). In contrast, in most patients, no or only minimal numbers of cells producing IFN-γ were detectable before vaccination: P/CsA, 5.2±10.8; P/MPA, 1.4±2.9; and P/everolimus, 10.6±20.6. At 14 days after vaccination, the mean number of IFN-γ-producing cells increased significantly in the healthy control individuals (P<0.005) and in patients treated with P/CsA (P<0.05). There was no significant increase in the mean number of IFN-γ-producing cells in patients receiving P/MPA or P/everolimus. The IFN-γ response, measured as the difference between post- and pre-vaccination numbers of cytokine-producing cells per 105 PBMCs, was significantly reduced in all patient groups as compared with healthy control individuals. In healthy control individuals and in all patient groups, no or only minimal numbers of cells producing IL-4 were detectable before vaccination: healthy control individuals, 0.3±0.5; P/CsA, 0.2±0.4; P/MPA, 0.1±0.4; and P/everolimus, 2.0±3.1. At 14 days after vaccination, there was a significant increase in the number of IL-4-producing cells in healthy control individuals (P<0.0005) and in all patient groups: P/CsA (P<0.05); P/MPA (P<0.05); and P/everolimus (P<0.05). As compared with healthy control individuals, the IL-4 response, as measured by the difference in cytokine-producing cells post- and pre-vaccination, was significantly inhibited in all patient groups. In contrast to the IFN-γ-producing cell numbers, the number of TT-specific IL-2-producing cells at 14 days after vaccination showed a fair correlation with the levels of IgG antibodies against TT after vaccination in healthy control individuals as well as in P/everolimus-treated patients (r=0.38 and 0.42, respectively; data not shown). For IL-4-producing cells, a correlation was found in the healthy control individuals only (r=0.50, data not shown). Mean (±s.d.) lymphocyte counts, total B cells, and B-cell subsets are listed in Table 3. There was no significant difference between total lymphocyte counts in either of the patient groups compared with the healthy control individuals. The absolute B-cell number was significantly lower in patients treated with P/MPA compared with healthy control individuals (P<0.0005), whereas patients treated with P/CsA or P/everolimus had slightly less B-cell numbers, though not-significant, in peripheral blood as compared with healthy control individuals. There was no difference in the percentage naïve (IgD+CD27-) B cells, non-switched memory (IgD+CD27+) B cells, and switched memory (IgD-CD27+) B cells within the total B-cell population between either of the patient groups and healthy control individuals (data not shown).Table 3Number of total lymphocytes and B-lymphocyte subsetsLymphocytesB cellsNaive B cellsNon-switched memory B cellsSwitched memory B cellsP/CsA1.8±1.00.07±0.060.040±0.0380.012±0.0120.018±0.017P/MPA1.6±0.50.02±0.02***0.013±0.015**0.002±0.001***0.004±0.003***P/everolimus1.8±0.70.07±0.050.041±0.0270.013±0.0110.017±0.013HC2.0±0.40.14±0.080.071±0.0460.031±0.0390.026±0.013Abbreviations: CsA, cyclosporine; HC, healthy control individuals; MPA, mycophenolate sodium; P, prednisolone.Statistical comparison of the mean (±s.d.) absolute number ( × 109/l): *P<0.05, **P<0.005, ***P<0.0005 versus HC. Open table in a new tab Abbreviations: CsA, cyclosporine; HC, healthy control individuals; MPA, mycophenolate sodium; P, prednisolone. Statistical comparison of the mean (±s.d.) absolute number ( × 109/l): *P<0.05, **P<0.005, ***P 50% (current or historic) panel reactive antibodies; and patients who were pregnant or unwilling to use adequate contraception during the study. Patients were block-randomized per participating hospital by a computer-generated randomization table. For the sub-study, only patients who were included in one of the participating centers (Amsterdam), who were not vaccinated against TT and PPS in the previous 5 years, and who were in their second year after transplantation with a stable glomerular filtration rate of at least 25 ml for the last 6 months were eligible. Subsequent, eligible patients visiting the outpatient renal transplantation clinic were asked to participate until the maximum number of participants per group was reached. A total of 36 patients were enrolled in the sub-study, 12 in each arm. They were on double immunosuppressive maintenance therapy consisting of P with CsA, MPA, or everolimus from 6 months after transplantation. The patients all received P 10 mg per day orally. Drug exposure to CsA, MPA, and everolimus were monitored by calculating AUC. Target values of AUCs for CsA were 3250 ng h/ml. The AUC targets for MPA were 70–85 μg h/ml and for everolimus 150 μg h/ml. In all, 13 healthy individuals, matched for age and sex, were included in the sub-study to serve as healthy control individuals. Exclusion criteria were: use of immunosuppressive drugs and vaccination against TT and PPS in the previous 5 years. The healthy individuals were recruited via an advertisement. The sub-study was approved by the local medical ethics committee and separate written informed consent was obtained. Concomitant administration of three vaccines was performed at the outpatient renal transplantation clinic by an independent physician. Vaccination with immunocyanin (Immucothel-, Biosyn Arzneimittel GmbH, Fellbach, Germany), a stable modification of the blood pigment hemocyanin from a sea snail (Megathura crenulata), was performed to study the primary immune response to a protein antigen. The vaccine was supplied as a lyophilized powder, containing 1000 μg immunocyanin, which was dissolved in NaCl before administration. TT (containing at least 80 IE TT per ml) was obtained from Aventis Pasteur MSD (Brussels, Belgium). This protein antigen elicits a secondary T-cell-dependent immune response. Polyvalent Pneumococcal vaccine (Pneumovax-, Merck Sharp and Dohme, Haarlem, The Netherlands), is the 23-valent polysaccharide, sterile, liquid vaccine containing 25 μg of each of the following capsular polysaccharides: types 1–5, 6B, 7F, 8, 9V, 10A, 11A, 12F, 18C, 19A, 19F, 20, 22, 23F, and 33F. By this vaccination the secondary immune response to PPS was tested, which is mainly T-cell independent. One milligram of immunocyanin was administered subcutaneously. A total of 1 ml of Pneumovax- and 0.5 ml of TT were administered in the deltoid muscle of separate arms. Before as well as 14 days after vaccination, blood was drawn to analyze antigen-specific antibody production and -cellular reactivity. IgG antibody levels against PPS, TT, and immunocyanin were measured using enzyme-linked immunosorbent assay as described previously.13.van der Heyden A.A. Bloemena E. Out T.A. et al.The influence of immunosuppressive treatment on immune responsiveness in vivo in kidney transplant recipients.Transplantation. 1989; 48: 44-47Crossref PubMed Scopus (8) Google Scholar, 14.Rentenaar R.J. van Diepen F.N. Meijer R.T. et al.Immune responsiveness in renal transplant recipients: mycophenolic acid severely depresses humoral immunity in vivo.Kidney Int. 2002; 62: 319-328Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 33.Korver K. Zeijlemaker W.P. Schellekens P.T. et al.Measurement of primary in vivo IgM- and IgG-antibody response to KLH in humans: implications of pre-immune IgM binding in antigen-specific ELISA.J Immunol Methods. 1984; 74: 241-251Crossref PubMed Scopus (39) Google Scholar, 34.Rodrigo M.J. Miravitlles M. Cruz M.J. et al.Characterization of specific immunoglobulin G (IgG) and its subclasses (IgG1 and IgG2) against the 23-valent pneumococcal vaccine in a healthy adult population: proposal for response criteria.Clin Diagn Lab Immunol. 1997; 4: 168-172PubMed Google Scholar Antigen-specific antibody titers are represented as the ratio between post- and pre-vaccination antibody levels. Circulating B-cell subsets were determined at day 0. PBMCs were isolated from peripheral blood by standard Ficoll (Fresenius Kabi Norge AS, Oslo, Norway) density-gradient centrifugation and cryopreserved until analysis. Then, they were thawed and resuspended in phosphate-buffered saline containing 0.5% (w/v) bovine serum albumin and 0.01% (w/v) NaN3 (PBA). For surface staining, 300.000 PBMCs were incubated with fluorescent-labeled conjugated monoclonal antibodies (concentrations according to manufacturer's instructions) for 30 min at 4°C protected from light. AntiCD27-PE, IgD-FITC, CD19-PerCP-Cy5.5, CD20-AP,C and CD3-Pe-Cy7 were all purchased from BD Biosciences, San Jose, CA, USA. Next, cells were washed in PBA and subsequently counted. Analysis was performed using a FACS CANTO flowcytometer and BD FACSDiva (both BD Biosciences) and FlowJo software (Treestar, Ashland, OR, USA). A peripheral white blood cell count and differentiation was performed in all participants. Absolute B-cell counts were calculated by the percentage of CD3-CD19+CD20+ cells multiplied by the absolute lymphocyte number. The absolute B-cell subset counts were calculated as the percentage of B-cell subset obtained from flowcytometry multiplied by the absolute B-cell count. Cytokine production by PBMCs after antigen-specific stimulation with TT was analyzed by ELISPOT assay. PBMCs were isolated from peripheral blood by standard Ficoll density-gradient centrifugation and cryopreserved until analysis. PBMCs from patients and healthy control individuals were thawed, washed, and diluted to a concentration of 2 × 106 cells/ml in RPMI 1640 with L-glutamin (Gibco BRL, Life Technologies, Paisley, UK) supplied with 10% heat-inactivated fetal calf serum (Gibco). Cells were pre-incubated at 37°C and 5% CO2 for 4 h in the absence or presence of TT (15 Lf/ml RIVM, Bilthoven, The Netherlands) or phytohaemagglutinin (0.1 μg/ml) in round bottom tubes (Micronic, McMurray, PA, USA). PVDF-based membrane plates (Millipore, Billerica, MA, USA) were coated with IL-4-, IL-2-, or IFN-γ-specific antibodies as described in the manufacturer's protocol (Mabtech, Nacka Strand, Sweden). Pre-incubated cells were diluted to a concentration of 1 × 105 cells/ml and transferred to the coated plates (in triplicates) and left for 24 (IL-2 and IFN-γ) or 48 h (IL-4) in a cell incubator at 37°C and 5% CO2. After the incubation period, the plates were washed and cytokine production by PBMCs was detected by incubating the plates with biotinylated secondary monoclonal antibodies, streptavidin-ALP (Mabtech) and BCIP/NBT substrate (Sigma-Aldrich, St. Louis, MO, USA). Spots were counted using ELISPOT analysis software (A.EL.VIS GmbH, Hannover, Germany) and expressed as positive cells per 105 stimulated PBMCs. The ELISPOT response in phytohaemagglutinin-stimulated cultures served as positive control and PBMCs cultured with medium only as negative control. ELISPOT responses of the TT cultures were corrected for background by subtraction of the responses from the medium only culture. After logtransformation, between-group differences of continuous variables were analyzed using an analysis of variance test or with a weighted least squares analysis. After logtransformation, within-group differences of continuous variables were analyzed using a paired t-test. The Bonferroni correction was used to correct for multiple comparisons. Univariate correlations between different variables within a group were assessed using Spearman's rank correlation test. A P-value of <0.05 was considered statistically significant. For statistical analyses, GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) and SPSS 16.0 (SPSS, Chicago, IL, USA) software programs were used. We thank Frederiek de Wilde for excellent technical assistance as well as Marianne van de Pol, Dr René Lutter, and Dr Theo Out for analytical support, and helpful comments and discussions. This study was funded by RISET, by a grant from the RISET consortium (Sixth Framework Programme of the European Commission; http://www.risetfp6.org). Disclaimer: We declare that all authors have contributed to, seen, and approved the final version of this paper and that the paper is not submitted or accepted elsewhere.
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