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Maintenance immunosuppressive therapy with everolimus preserves humoral immune responses
original article
http://www.kidney-international.org & 2010 International Society of Nephrology
Maintenance immunosuppressive therapy with everolimus preserves humoral immune responses Geertrude H. Struijk1, Robert C. Minnee1,2, Sven D. Koch1, Aeilko H. Zwinderman3, Karlijn A.M.I. van Donselaar-van der Pant1, Mirza M. Idu2, Ineke J.M. ten Berge1 and Frederike J. Bemelman1 1
Renal Transplant Unit, Department of Nephrology, Division of Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 2Department of Surgery, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands and 3Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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. Kidney International (2010) 78, 934–940; doi:10.1038/ki.2010.269; published online 11 August 2010 KEYWORDS: everolimus; immunosuppression; mycophenolate; pneumococcus; renal transplantation; vaccination.
Correspondence: Geertrude H. Struijk, Renal Transplant Unit, Department of Nephrology, Division of Internal Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Room F4-263, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected] Received 22 January 2010; revised 4 June 2010; accepted 9 June 2010; published online 11 August 2010 934
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.1–3 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 The use of live vaccines is contraindicated. Despite these guidelines, there continues to be a gap between guidelines and clinical practice.5 A main reason for non-vaccination is the absence of a doctors’ recommendation.6 This may be owing to concerns that vaccination might trigger allograft rejection.7,8 However, this concern is unfounded.9,10 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.11–13 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 Recently, Candon et al.10 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)-g T-cell responses were conserved. Willcocks et al.15 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 rejection3,16 and has a different side effect profile compared with CsA and MPA.17,18 The effect of everolimus on human responses to vaccination in vivo has not been studied until now. Kidney International (2010) 78, 934–940
original article
GH Struijk et al.: Immune responses under everolimus
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 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.
immunocyanin-specific IgG antibody levels, was significantly reduced in all patient groups as compared with healthy control individuals (Po0.0005). There was no difference in response between the patient groups. Secondary humoral response after vaccination with TT
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/MPAtreated 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 (Po0.0005; Figure 1b), in patients receiving P/CsA (Po0.005), and in patients treated with P/everolimus (Po0.0005). 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 (Po0.05, Po000.5, respectively). Patients treated with P/MPA also showed a significantly decreased response as compared with patients receiving P/everolimus (Po0.005). In patients receiving P/everolimus, the humoral response against TT was not reduced compared with healthy control individuals.
RESULTS Study population
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. Primary humoral response after vaccination with immunocyanin
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 (Po0.0005; Figure 1a) and in patients receiving P/everolimus (Po0.05). 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
Secondary humoral response after vaccination with PPS
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/MPAtreated 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 (Po0.0005; Figure 1c), in patients receiving P/CsA (Po0.0005), and in patients treated with P/everolimus (Po0.0005). In patients treated with P/MPA,
Table 1 | Characteristics of patients and healthy control individuals
Number of patients Age (years, median range) Male (n, %) First/second transplant Acute rejection (%) End-stage renal disease Unknown Adult polycystic kidney disease Renal vascular disease Pyelonephritis Vasculitis Glomerular Other Renal function GFRa (ml/min)
P/CsA
P/MPA
P/everolimus
HC
P-value
12 58 (34–72) 10 (83) 11/1 0
12 60 (30–70) 9 (75) 11/1 0
12 50 (27–68) 8 (67) 12/0 0
13 55 (42–63) 8 (62) — —
NS NS NS NS
2 2 1 2 1 3 1
1 3 5 1 1 — 1
1 1 4 1 — 5 —
49 (27–93)
59 (26–100)
59 (30–85)
NA
NS
Abbreviations: 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.
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GH Struijk et al.: Immune responses under everolimus
Day 14
2 Day 14
Day 0
3 2
–2 –3
–2 –3 Day 0
Log IgG anti-PPS (U/l)
***
3
0 –1
Day 14
4
2 1 0 Day 0
Log IgG anti-TT (kU/l)
0 –1
2 NS
1
Day 14
3
NS
2 1 0 Day 0
P/CsA
0 –1 –2 –3
Day 14
4
***
1
Day 0
Log IgG anti-PPS (U/l)
**
1
Day 14
Day 0
2
2
Day 0
Log IgG anti-PPS (U/l)
3
*
4
Log IgG anti-immunocyanin (U/l)
Day 0
NS
5
Log IgG anti-TT (kU/l)
2
4
TT-specific cellular responses in vitro, as assessed by enzymelinked 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). 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
Day 14
4
***
3 2 1 0 Day 0
P/MPA
5
Day 14
***
4 3 2 Day 0
2
Day 14
***
1 0 –1 –2 –3
Day 14
Day 0
Log IgG anti-PPS (U/l)
3
5
Cellular responses after in vitro stimulation with TT
Log IgG anti-immunocyanin (U/l)
NS
4
Log IgG anti-immunocyanin (U/l)
5
Log IgG anti-TT (kU/l)
Log IgG anti-TT (kU/l)
Log IgG anti-immunocyanin (U/l)
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 PPSspecific IgG antibody levels, was significantly reduced in patients receiving P/CsA as compared with healthy control individuals (Po0.05). Patients treated with P/MPA showed a significantly decreased response as compared with healthy control individuals (Po0.0005) and with patients receiving P/CsA (Po0.05) or P/everolimus (Po0.005). In patients receiving P/everolimus, the humoral response against PPS was not reduced compared with healthy control individuals.
Day 14
4
***
3 2 1 0 Day 0
P/everolimus
Day 14
HC
Figure 1 | Humoral responses against immunocyanin, TT, and PPS. Humoral responses against (a) immunocyanin, (b) tetanus toxoid (TT), and (c) pneumococcal polysaccharide (PPS). Mean antigen-specific immunoglobulin (Ig) G antibody levels before and 14 days after vaccination were analyzed by enzyme-linked immunosorbent assay. Statistical comparison of the antigen-specific IgG levels before and 14 days after vaccination: *Po0.05, **Po0.005, ***Po0.0005. The first column shows the prednisolone (P)/cyclosporine-treated patients; the second column shows patients treated with P/MPA; the third column shows patients receiving P/everolimus; the last column shows the healthy control individuals. NS, not significant.
Table 2 | Number of IL-2-, IFN-c-, and IL-4-producing cells after in vitro stimulation with tetanus toxoid IL-2
P/CsA P/MPA P/everolimus HC
IFN-c
IL-4
Day 0
Day 14
Day 0
Day 14
Day 0
Day 14
0.8±1.5 3.4±3.2 2.8±4.6 2.8±3.0
4.7±5.8** 5.4±6.1 6.7±7.5* 14.0±7.0***
5.2±10.8 1.4±2.9 10.6±20.6 11.1±12.4
10.0±10.6* 5.2±5.1 6.4±8.6 38.1±30.1**
0.2±0.4 0.1±0.4 2.0±3.1 0.3±0.5
6.3±11.7* 5.0±5.8* 5.6±7.2* 19.8±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: *Po0.05, **Po0.005, ***Po0.0005. Results were corrected for background by deducting the responses from the culture with medium only.
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from healthy control individuals (Po0.0005) as well as from patients treated with P/CsA (Po0.005) or P/everolimus (Po0.05) 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 postand 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-g-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-g 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-g-producing cells increased significantly in the healthy control individuals (Po0.005) and in patients treated with P/CsA (Po0.05). There was no significant increase in the mean number of IFN-g-producing cells in patients receiving P/MPA or P/everolimus. The IFN-g 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 (Po0.0005) and in all patient groups: P/CsA (Po0.05); P/MPA (Po0.05); and P/everolimus (Po0.05). 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-g-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 ¼ 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 ¼ 0.50, data not shown).
B-cell phenotype
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 (Po0.0005), 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 naı¨ve (IgD þ CD27) B cells, non-switched memory (IgD þ CD27 þ ) B cells, and switched memory (IgDCD27 þ ) B cells within the total B-cell population between either of the patient groups and healthy control individuals (data not shown). A fair degree of correlation was found between the absolute number of B cells and IgG levels against TT after vaccination both in healthy control individuals and in P/everolimus-treated patients (r ¼ 0.38 and 0.31, respectively; data not shown). No significant correlation was found between absolute B cells or B-cell subset counts and IgG levels against PPS. DISCUSSION
Here, we show that immunosuppressive drug therapy consisting of P and everolimus allows for a significant rise in the antigen-specific IgG antibody level in the primary humoral immune response, and leaves secondary T-celldependent and T-cell-independent immune responses intact. However, treatment with P and MPA completely prevents the generation of antibodies not only in primary but also in secondary humoral immune responses. Treatment with P and CsA inhibits the primary humoral immune response, but permits significant rises in the antigen-specific IgG antibody level in secondary immune responses. Cellular immune responses against TT in vitro are significantly inhibited by all these drug combinations, compared with healthy control individuals. Patients treated with P/MPA show severely reduced circulating B-cell numbers, which are normally distributed between the main B-cell subsets. Previously, we found no effect of CsA in combination with P on primary T-cell-dependent humoral responses,14 which may be explained by the lower drug exposure as compared with the strict areas under the curve (AUC)targeted drug dosing in the present study.19 The P/CsA
Table 3 | Number of total lymphocytes and B-lymphocyte subsets
P/CsA P/MPA P/everolimus HC
Lymphocytes
B cells
Naive B cells
Non-switched memory B cells
Switched memory B cells
1.8±1.0 1.6±0.5 1.8±0.7 2.0±0.4
0.07±0.06 0.02±0.02*** 0.07±0.05 0.14±0.08
0.040±0.038 0.013±0.015** 0.041±0.027 0.071±0.046
0.012±0.012 0.002±0.001*** 0.013±0.011 0.031±0.039
0.018±0.017 0.004±0.003*** 0.017±0.013 0.026±0.013
Abbreviations: CsA, cyclosporine; HC, healthy control individuals; MPA, mycophenolate sodium; P, prednisolone. Statistical comparison of the mean (±s.d.) absolute number ( 109/l): *Po0.05, **Po0.005, ***Po0.0005 versus HC. Kidney International (2010) 78, 934–940
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regimen significantly inhibited antigen-specific cellular responses against TT in vitro, which may be explained by the property of CsA to inhibit transcription of several cytokines, in particular IL-2 and IFN-g. Indeed, IFN-g production was reduced in patients treated with calcineurin inhibitors compared with healthy control individuals in one study,20 which was not confirmed in two other studies,10,21 probably owing to differences in drug exposure. The drug combination P/MPA completely inhibited the capacity to mount both primary and secondary humoral immune responses, which is in agreement with previous studies.14,22 MPA may have suppressed the humoral immune responses to some extent via its effect on T cells, as cellular responses against TT in vitro were severely inhibited during treatment with P/MPA. However, the T-cell-independent humoral response against PPS was also severely reduced, also suggesting at least a direct effect on B cells. The reduced number of circulating memory B cell may have contributed to this, as has been shown in patients after splenectomy.23 In vitro, MPA has been shown to inhibit B-cell proliferation and IgG production, and to induce apoptosis in B cells.24 In P/everolimus-treated patients, the primary and secondary humoral immune responses showed a significant rise in the antigen-specific IgG antibody level after vaccination. This is in line with recent findings of Araki et al.25 showing that administration of everolimus to non-human primates even enhanced memory T-cell responses to vaccination with modified vaccinia virus. The renal transplant recipients receiving P/everolimus are still protected against rejection of their allograft may be because to the fact that everolimus and other mammalian target of rapamycin-inhibitors induce regulatory T cells, which specifically inhibit allo-specific responses.26–28 Our findings are in agreement with those of Willcocks et al.,15 who demonstrated similar humoral responses after vaccination with PPS and influenza vaccines in renal transplant recipients treated with a sirolimus-based as compared with a calcineurin inhibitor-based immunosuppressive regimen. Here, we show that everolimus even permits a primary immune response. However, the drug did lower the antigen-specific cellular responses in vitro. In contrast to previous reports,10,15 we studied a homogenous cohort of renal transplant recipients, who received everolimus with only a minimal dose of P (10 mg) and a strict AUC-targeted drug dosing.19 In the P/everolimus-treated patients, there was a significant increase in the IL-2 secretion, measured by ELISPOT, after vaccination. We found a correlation between the number of IL-2-producing cells and the IgG levels against TT, suggesting that mainly these cells determine the TT-specific antibody response, which is in agreement with data by Litjens et al.29 regarding vaccination with the T-celldependent Hepatitis B surface antigen. As has also been demonstrated in two other studies,10,21 we did not find a correlation between IFN-g production in vitro and humoral responses against TT in healthy control individuals or in any 938
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of the patient groups. In healthy control individuals, we found a correlation between the number of TT-specific IL-4-producing cells and IgG levels against TT 14 days after vaccination, which supports the importance of T-cell help for B-cell differentiation and IgG production by plasma cells. We did not find a difference in the percentage of naı¨ve B cells, non-switched memory B cells, and switched memory B cells between either of the patient groups and healthy control individuals. Only in P/everolimus-treated patients a significant relationship between the number of switched memory B cells and TT-specific IgG antibody levels was found, which is in agreement with data found in HIVinfected patients.30 The number of individuals tested in each group may have been too small to detect more significant relationships. Several studies indicate that maintenance of immunological memory is impaired in immunosuppressed transplant recipients. Renal transplant recipients vaccinated against hepatitis A experienced a much more rapid decline of antibodies as compared with healthy control individuals.31 Renal transplant recipients with preexisting immunity, who were treated with maintenance therapy consisting of CsA, azathioprine, and/or P, and who received a booster vaccination at least 6 months after renal transplantation, all developed protective tetanus antibody levels. However, 1 year later, although still in the protective range, titers were significantly lower compared with healthy control individuals.32 These observations suggest that protective immunity that is induced after vaccination wanes more rapidly. Although this issue was not addressed in the present study, the finding of only minimal numbers of IFN-g-producing cells before vaccination in all patient groups points towards that direction. In conclusion, double immunosuppressive drug therapy consisting of P and everolimus allows for a significant rise in the antigen-specific IgG antibody level in the primary humoral immune response and leaves secondary T-celldependent and T-cell-independent immune responses intact. The drug combination P/CsA decreases the capacity to mount a primary immune response, but allows for significant rises in the antigen-specific IgG antibody level in secondary immune responses. However, both primary and secondary humoral responses are severely disturbed in renal transplant recipients receiving the drug combination P/MPA, implying that vaccination of these patients will not result in a protective immune response. MATERIALS AND METHODS Study population The current study is a sub-study nested in an open randomized multicenter trial in renal transplant recipients, to asses the long-term effects of three different immunosuppressive agents, containing CsA, MPA, or everolimus. Renal transplant recipients between 18 and 70 years, receiving a first or second renal transplantation were eligible. The following patients were excluded: recipients of a simultaneous pancreas kidney transplant or a double kidney transplant, a third or fourth transplant, or a transplant from an Kidney International (2010) 78, 934–940
GH Struijk et al.: Immune responses under everolimus
human leukocyte antigen-identical sib; those having 450% (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 mg h/ml and for everolimus 150 mg 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. Vaccinations 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 mg 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 mg 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 antigenspecific antibody production and -cellular reactivity. Serology IgG antibody levels against PPS, TT, and immunocyanin were measured using enzyme-linked immunosorbent assay as described previously.13,14,33,34 Antigen-specific antibody titers are represented as the ratio between post- and pre-vaccination antibody levels. Kidney International (2010) 78, 934–940
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B-cell phenotyping 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 41C 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 CD3CD19 þ CD20 þ 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. ELISPOT assay 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 371C and 5% CO2 for 4 h in the absence or presence of TT (15 Lf/ ml RIVM, Bilthoven, The Netherlands) or phytohaemagglutinin (0.1 mg/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-g-specific antibodies as described in the manufacturer’s protocol (Mabtech, Nacka Strand, Sweden). Preincubated 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-g) or 48 h (IL-4) in a cell incubator at 371C 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. Statistical analysis 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, withingroup 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 939
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variables within a group were assessed using Spearman’s rank correlation test. A P-value of o0.05 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. DISCLOSURE
GH Struijk et al.: Immune responses under everolimus
15.
16.
17.
All the authors declared no competing interests. 18.
ACKNOWLEDGMENTS
We thank Frederiek de Wilde for excellent technical assistance as well as Marianne van de Pol, Dr Rene´ 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.
19.
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21.
22.
REFERENCES 1. Boggild AK, Sano M, Humar A et al. Travel patterns and risk behavior in solid organ transplant recipients. J Travel Med 2004; 11: 37–43. 2. Fishman JA, Rubin RH. Infection in organ-transplant recipients. N Engl J Med 1998; 338: 1741–1751. 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: 1532–1540. 4. Guidelines for vaccination of solid organ transplant candidates and recipients. Am J Transplant 2004; 4(Suppl 10): 160–163. 5. Harris K, Baggs J, Davis RL et al. Influenza vaccination coverage among adult solid organ transplant recipients at three health maintenance organizations, 1995–2005. Vaccine 2009; 27: 2335–2341. 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: 1047. 7. Blumberg EA, Brozena SC, Stutman P et al. Immunogenicity of pneumococcal vaccine in heart transplant recipients. Clin Infect Dis 2001; 32: 307–310. 8. Vilchez RA, McCurry K, Dauber J et al. Influenza virus infection in adult solid organ transplant recipients. Am J Transplant 2002; 2: 287–291. 9. Cainelli F, Vento S. Infections and solid organ transplant rejection: a cause-and-effect relationship? Lancet Infect Dis 2002; 2: 539–549. 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: 2346–2354. 11. ten Berge RJ, Schellekens PT, 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: 20–32. 12. ten Berge RJ, Schellekens PT, 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: 33–46. 13. van der Heyden AA, Bloemena E, Out TA et al. The influence of immunosuppressive treatment on immune responsiveness in vivo in kidney transplant recipients. Transplantation 1989; 48: 44–47. 14. Rentenaar RJ, van Diepen FN, Meijer RT et al. Immune responsiveness in renal transplant recipients: mycophenolic acid severely depresses humoral immunity in vivo. Kidney Int 2002; 62: 319–328.
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Willcocks LC, Chaudhry AN, Smith JC et al. The effect of sirolimus therapy on vaccine responses in transplant recipients. Am J Transplant 2007; 7: 2006–2011. Webster AC, Lee VW, Chapman JR 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: 1234–1248. Andres V, Castro C, Campistol JM. Potential role of proliferation signal inhibitors on atherosclerosis in renal transplant patients. Nephrol Dial Transplant 2006; 21(Suppl 3): iii14–iii17. Meier-Kriesche HU, Steffen BJ, Hochberg AM et al. Mycophenolate mofetil versus azathioprine therapy is associated with a significant protection against long-term renal allograft function deterioration. Transplantation 2003; 75: 1341–1346. Bemelman FJ, de Maar EF, Press RR et al. Minimization of maintenance immunosuppression early after renal transplantation: an interim analysis. Transplantation 2009; 88: 421–428. Lindemann M, Witzke O, Lutkes P et al. ELISpot assay as a sensitive tool to detect cellular immunity following influenza vaccination in kidney transplant recipients. Clin Immunol 2006; 120: 342–348. Ballet C, Roussey-Kesler G, Aubin JT et al. Humoral and cellular responses to influenza vaccination in human recipients naturally tolerant to a kidney allograft. Am J Transplant 2006; 6: 2796–2801. Smith KG, Isbel NM, Catton MG et al. Suppression of the humoral immune response by mycophenolate mofetil. Nephrol Dial Transplant 1998; 13: 160–164. Wasserstrom H, Bussel J, Lim LC et al. Memory B cells and pneumococcal antibody after splenectomy. J Immunol 2008; 181: 3684–3689. Heidt S, Roelen DL, Eijsink C et al. Effects of immunosuppressive drugs on purified human B cells: evidence supporting the use of MMF and rapamycin. Transplantation 2008; 86: 1292–1300. Araki K, Turner AP, Shaffer VO et al. mTOR regulates memory CD8 T-cell differentiation. Nature 2009; 460: 108–112. Coenen JJ, Koenen HJ, van RE et al. Rapamycin, and not cyclosporin A, preserves the highly suppressive CD27+ subset of human CD4+CD25+ regulatory T cells. Blood 2006; 107: 1018–1023. Nikolaeva N, Bemelman FJ, Yong SL et al. Rapamycin does not induce anergy but inhibits expansion and differentiation of alloreactive human T cells. Transplantation 2006; 81: 445–454. Thomson AW, Turnquist HR, Raimondi G. Immunoregulatory functions of mTOR inhibition. Nat Rev Immunol 2009; 9: 324–337. Litjens NH, Huisman M, Hijdra D et al. IL-2 producing memory CD4+ T lymphocytes are closely associated with the generation of IgG-secreting plasma cells. J Immunol 2008; 181: 3665–3673. Hart M, Steel A, Clark SA et al. Loss of discrete memory B cell subsets is associated with impaired immunization responses in HIV-1 infection and may be a risk factor for invasive pneumococcal disease. J Immunol 2007; 178: 8212–8220. Gunther M, Stark K, Neuhaus R et al. Rapid decline of antibodies after hepatitis A immunization in liver and renal transplant recipients. Transplantation 2001; 71: 477–479. Huzly D, Neifer S, Reinke P et al. Routine immunizations in adult renal transplant recipients. Transplantation 1997; 63: 839–845. Korver K, Zeijlemaker WP, Schellekens PT 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: 241–251. Rodrigo MJ, Miravitlles M, Cruz MJ 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: 168–172.
Kidney International (2010) 78, 934–940
http://www.kidney-international.org & 2010 International Society of Nephrology
Maintenance immunosuppressive therapy with everolimus preserves humoral immune responses Geertrude H. Struijk1, Robert C. Minnee1,2, Sven D. Koch1, Aeilko H. Zwinderman3, Karlijn A.M.I. van Donselaar-van der Pant1, Mirza M. Idu2, Ineke J.M. ten Berge1 and Frederike J. Bemelman1 1
Renal Transplant Unit, Department of Nephrology, Division of Internal Medicine, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands; 2Department of Surgery, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands and 3Department of Clinical Epidemiology, Biostatistics and Bioinformatics, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands
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. Kidney International (2010) 78, 934–940; doi:10.1038/ki.2010.269; published online 11 August 2010 KEYWORDS: everolimus; immunosuppression; mycophenolate; pneumococcus; renal transplantation; vaccination.
Correspondence: Geertrude H. Struijk, Renal Transplant Unit, Department of Nephrology, Division of Internal Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Room F4-263, 1105 AZ Amsterdam, The Netherlands. E-mail: [email protected] Received 22 January 2010; revised 4 June 2010; accepted 9 June 2010; published online 11 August 2010 934
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.1–3 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 The use of live vaccines is contraindicated. Despite these guidelines, there continues to be a gap between guidelines and clinical practice.5 A main reason for non-vaccination is the absence of a doctors’ recommendation.6 This may be owing to concerns that vaccination might trigger allograft rejection.7,8 However, this concern is unfounded.9,10 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.11–13 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 Recently, Candon et al.10 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)-g T-cell responses were conserved. Willcocks et al.15 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 rejection3,16 and has a different side effect profile compared with CsA and MPA.17,18 The effect of everolimus on human responses to vaccination in vivo has not been studied until now. Kidney International (2010) 78, 934–940
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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 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.
immunocyanin-specific IgG antibody levels, was significantly reduced in all patient groups as compared with healthy control individuals (Po0.0005). There was no difference in response between the patient groups. Secondary humoral response after vaccination with TT
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/MPAtreated 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 (Po0.0005; Figure 1b), in patients receiving P/CsA (Po0.005), and in patients treated with P/everolimus (Po0.0005). 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 (Po0.05, Po000.5, respectively). Patients treated with P/MPA also showed a significantly decreased response as compared with patients receiving P/everolimus (Po0.005). In patients receiving P/everolimus, the humoral response against TT was not reduced compared with healthy control individuals.
RESULTS Study population
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. Primary humoral response after vaccination with immunocyanin
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 (Po0.0005; Figure 1a) and in patients receiving P/everolimus (Po0.05). 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
Secondary humoral response after vaccination with PPS
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/MPAtreated 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 (Po0.0005; Figure 1c), in patients receiving P/CsA (Po0.0005), and in patients treated with P/everolimus (Po0.0005). In patients treated with P/MPA,
Table 1 | Characteristics of patients and healthy control individuals
Number of patients Age (years, median range) Male (n, %) First/second transplant Acute rejection (%) End-stage renal disease Unknown Adult polycystic kidney disease Renal vascular disease Pyelonephritis Vasculitis Glomerular Other Renal function GFRa (ml/min)
P/CsA
P/MPA
P/everolimus
HC
P-value
12 58 (34–72) 10 (83) 11/1 0
12 60 (30–70) 9 (75) 11/1 0
12 50 (27–68) 8 (67) 12/0 0
13 55 (42–63) 8 (62) — —
NS NS NS NS
2 2 1 2 1 3 1
1 3 5 1 1 — 1
1 1 4 1 — 5 —
49 (27–93)
59 (26–100)
59 (30–85)
NA
NS
Abbreviations: 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.
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Day 14
2 Day 14
Day 0
3 2
–2 –3
–2 –3 Day 0
Log IgG anti-PPS (U/l)
***
3
0 –1
Day 14
4
2 1 0 Day 0
Log IgG anti-TT (kU/l)
0 –1
2 NS
1
Day 14
3
NS
2 1 0 Day 0
P/CsA
0 –1 –2 –3
Day 14
4
***
1
Day 0
Log IgG anti-PPS (U/l)
**
1
Day 14
Day 0
2
2
Day 0
Log IgG anti-PPS (U/l)
3
*
4
Log IgG anti-immunocyanin (U/l)
Day 0
NS
5
Log IgG anti-TT (kU/l)
2
4
TT-specific cellular responses in vitro, as assessed by enzymelinked 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). 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
Day 14
4
***
3 2 1 0 Day 0
P/MPA
5
Day 14
***
4 3 2 Day 0
2
Day 14
***
1 0 –1 –2 –3
Day 14
Day 0
Log IgG anti-PPS (U/l)
3
5
Cellular responses after in vitro stimulation with TT
Log IgG anti-immunocyanin (U/l)
NS
4
Log IgG anti-immunocyanin (U/l)
5
Log IgG anti-TT (kU/l)
Log IgG anti-TT (kU/l)
Log IgG anti-immunocyanin (U/l)
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 PPSspecific IgG antibody levels, was significantly reduced in patients receiving P/CsA as compared with healthy control individuals (Po0.05). Patients treated with P/MPA showed a significantly decreased response as compared with healthy control individuals (Po0.0005) and with patients receiving P/CsA (Po0.05) or P/everolimus (Po0.005). In patients receiving P/everolimus, the humoral response against PPS was not reduced compared with healthy control individuals.
Day 14
4
***
3 2 1 0 Day 0
P/everolimus
Day 14
HC
Figure 1 | Humoral responses against immunocyanin, TT, and PPS. Humoral responses against (a) immunocyanin, (b) tetanus toxoid (TT), and (c) pneumococcal polysaccharide (PPS). Mean antigen-specific immunoglobulin (Ig) G antibody levels before and 14 days after vaccination were analyzed by enzyme-linked immunosorbent assay. Statistical comparison of the antigen-specific IgG levels before and 14 days after vaccination: *Po0.05, **Po0.005, ***Po0.0005. The first column shows the prednisolone (P)/cyclosporine-treated patients; the second column shows patients treated with P/MPA; the third column shows patients receiving P/everolimus; the last column shows the healthy control individuals. NS, not significant.
Table 2 | Number of IL-2-, IFN-c-, and IL-4-producing cells after in vitro stimulation with tetanus toxoid IL-2
P/CsA P/MPA P/everolimus HC
IFN-c
IL-4
Day 0
Day 14
Day 0
Day 14
Day 0
Day 14
0.8±1.5 3.4±3.2 2.8±4.6 2.8±3.0
4.7±5.8** 5.4±6.1 6.7±7.5* 14.0±7.0***
5.2±10.8 1.4±2.9 10.6±20.6 11.1±12.4
10.0±10.6* 5.2±5.1 6.4±8.6 38.1±30.1**
0.2±0.4 0.1±0.4 2.0±3.1 0.3±0.5
6.3±11.7* 5.0±5.8* 5.6±7.2* 19.8±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: *Po0.05, **Po0.005, ***Po0.0005. Results were corrected for background by deducting the responses from the culture with medium only.
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from healthy control individuals (Po0.0005) as well as from patients treated with P/CsA (Po0.005) or P/everolimus (Po0.05) 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 postand 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-g-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-g 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-g-producing cells increased significantly in the healthy control individuals (Po0.005) and in patients treated with P/CsA (Po0.05). There was no significant increase in the mean number of IFN-g-producing cells in patients receiving P/MPA or P/everolimus. The IFN-g 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 (Po0.0005) and in all patient groups: P/CsA (Po0.05); P/MPA (Po0.05); and P/everolimus (Po0.05). 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-g-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 ¼ 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 ¼ 0.50, data not shown).
B-cell phenotype
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 (Po0.0005), 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 naı¨ve (IgD þ CD27) B cells, non-switched memory (IgD þ CD27 þ ) B cells, and switched memory (IgDCD27 þ ) B cells within the total B-cell population between either of the patient groups and healthy control individuals (data not shown). A fair degree of correlation was found between the absolute number of B cells and IgG levels against TT after vaccination both in healthy control individuals and in P/everolimus-treated patients (r ¼ 0.38 and 0.31, respectively; data not shown). No significant correlation was found between absolute B cells or B-cell subset counts and IgG levels against PPS. DISCUSSION
Here, we show that immunosuppressive drug therapy consisting of P and everolimus allows for a significant rise in the antigen-specific IgG antibody level in the primary humoral immune response, and leaves secondary T-celldependent and T-cell-independent immune responses intact. However, treatment with P and MPA completely prevents the generation of antibodies not only in primary but also in secondary humoral immune responses. Treatment with P and CsA inhibits the primary humoral immune response, but permits significant rises in the antigen-specific IgG antibody level in secondary immune responses. Cellular immune responses against TT in vitro are significantly inhibited by all these drug combinations, compared with healthy control individuals. Patients treated with P/MPA show severely reduced circulating B-cell numbers, which are normally distributed between the main B-cell subsets. Previously, we found no effect of CsA in combination with P on primary T-cell-dependent humoral responses,14 which may be explained by the lower drug exposure as compared with the strict areas under the curve (AUC)targeted drug dosing in the present study.19 The P/CsA
Table 3 | Number of total lymphocytes and B-lymphocyte subsets
P/CsA P/MPA P/everolimus HC
Lymphocytes
B cells
Naive B cells
Non-switched memory B cells
Switched memory B cells
1.8±1.0 1.6±0.5 1.8±0.7 2.0±0.4
0.07±0.06 0.02±0.02*** 0.07±0.05 0.14±0.08
0.040±0.038 0.013±0.015** 0.041±0.027 0.071±0.046
0.012±0.012 0.002±0.001*** 0.013±0.011 0.031±0.039
0.018±0.017 0.004±0.003*** 0.017±0.013 0.026±0.013
Abbreviations: CsA, cyclosporine; HC, healthy control individuals; MPA, mycophenolate sodium; P, prednisolone. Statistical comparison of the mean (±s.d.) absolute number ( 109/l): *Po0.05, **Po0.005, ***Po0.0005 versus HC. Kidney International (2010) 78, 934–940
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regimen significantly inhibited antigen-specific cellular responses against TT in vitro, which may be explained by the property of CsA to inhibit transcription of several cytokines, in particular IL-2 and IFN-g. Indeed, IFN-g production was reduced in patients treated with calcineurin inhibitors compared with healthy control individuals in one study,20 which was not confirmed in two other studies,10,21 probably owing to differences in drug exposure. The drug combination P/MPA completely inhibited the capacity to mount both primary and secondary humoral immune responses, which is in agreement with previous studies.14,22 MPA may have suppressed the humoral immune responses to some extent via its effect on T cells, as cellular responses against TT in vitro were severely inhibited during treatment with P/MPA. However, the T-cell-independent humoral response against PPS was also severely reduced, also suggesting at least a direct effect on B cells. The reduced number of circulating memory B cell may have contributed to this, as has been shown in patients after splenectomy.23 In vitro, MPA has been shown to inhibit B-cell proliferation and IgG production, and to induce apoptosis in B cells.24 In P/everolimus-treated patients, the primary and secondary humoral immune responses showed a significant rise in the antigen-specific IgG antibody level after vaccination. This is in line with recent findings of Araki et al.25 showing that administration of everolimus to non-human primates even enhanced memory T-cell responses to vaccination with modified vaccinia virus. The renal transplant recipients receiving P/everolimus are still protected against rejection of their allograft may be because to the fact that everolimus and other mammalian target of rapamycin-inhibitors induce regulatory T cells, which specifically inhibit allo-specific responses.26–28 Our findings are in agreement with those of Willcocks et al.,15 who demonstrated similar humoral responses after vaccination with PPS and influenza vaccines in renal transplant recipients treated with a sirolimus-based as compared with a calcineurin inhibitor-based immunosuppressive regimen. Here, we show that everolimus even permits a primary immune response. However, the drug did lower the antigen-specific cellular responses in vitro. In contrast to previous reports,10,15 we studied a homogenous cohort of renal transplant recipients, who received everolimus with only a minimal dose of P (10 mg) and a strict AUC-targeted drug dosing.19 In the P/everolimus-treated patients, there was a significant increase in the IL-2 secretion, measured by ELISPOT, after vaccination. We found a correlation between the number of IL-2-producing cells and the IgG levels against TT, suggesting that mainly these cells determine the TT-specific antibody response, which is in agreement with data by Litjens et al.29 regarding vaccination with the T-celldependent Hepatitis B surface antigen. As has also been demonstrated in two other studies,10,21 we did not find a correlation between IFN-g production in vitro and humoral responses against TT in healthy control individuals or in any 938
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of the patient groups. In healthy control individuals, we found a correlation between the number of TT-specific IL-4-producing cells and IgG levels against TT 14 days after vaccination, which supports the importance of T-cell help for B-cell differentiation and IgG production by plasma cells. We did not find a difference in the percentage of naı¨ve B cells, non-switched memory B cells, and switched memory B cells between either of the patient groups and healthy control individuals. Only in P/everolimus-treated patients a significant relationship between the number of switched memory B cells and TT-specific IgG antibody levels was found, which is in agreement with data found in HIVinfected patients.30 The number of individuals tested in each group may have been too small to detect more significant relationships. Several studies indicate that maintenance of immunological memory is impaired in immunosuppressed transplant recipients. Renal transplant recipients vaccinated against hepatitis A experienced a much more rapid decline of antibodies as compared with healthy control individuals.31 Renal transplant recipients with preexisting immunity, who were treated with maintenance therapy consisting of CsA, azathioprine, and/or P, and who received a booster vaccination at least 6 months after renal transplantation, all developed protective tetanus antibody levels. However, 1 year later, although still in the protective range, titers were significantly lower compared with healthy control individuals.32 These observations suggest that protective immunity that is induced after vaccination wanes more rapidly. Although this issue was not addressed in the present study, the finding of only minimal numbers of IFN-g-producing cells before vaccination in all patient groups points towards that direction. In conclusion, double immunosuppressive drug therapy consisting of P and everolimus allows for a significant rise in the antigen-specific IgG antibody level in the primary humoral immune response and leaves secondary T-celldependent and T-cell-independent immune responses intact. The drug combination P/CsA decreases the capacity to mount a primary immune response, but allows for significant rises in the antigen-specific IgG antibody level in secondary immune responses. However, both primary and secondary humoral responses are severely disturbed in renal transplant recipients receiving the drug combination P/MPA, implying that vaccination of these patients will not result in a protective immune response. MATERIALS AND METHODS Study population The current study is a sub-study nested in an open randomized multicenter trial in renal transplant recipients, to asses the long-term effects of three different immunosuppressive agents, containing CsA, MPA, or everolimus. Renal transplant recipients between 18 and 70 years, receiving a first or second renal transplantation were eligible. The following patients were excluded: recipients of a simultaneous pancreas kidney transplant or a double kidney transplant, a third or fourth transplant, or a transplant from an Kidney International (2010) 78, 934–940
GH Struijk et al.: Immune responses under everolimus
human leukocyte antigen-identical sib; those having 450% (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 mg h/ml and for everolimus 150 mg 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. Vaccinations 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 mg 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 mg 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 antigenspecific antibody production and -cellular reactivity. Serology IgG antibody levels against PPS, TT, and immunocyanin were measured using enzyme-linked immunosorbent assay as described previously.13,14,33,34 Antigen-specific antibody titers are represented as the ratio between post- and pre-vaccination antibody levels. Kidney International (2010) 78, 934–940
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B-cell phenotyping 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 41C 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 CD3CD19 þ CD20 þ 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. ELISPOT assay 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 371C and 5% CO2 for 4 h in the absence or presence of TT (15 Lf/ ml RIVM, Bilthoven, The Netherlands) or phytohaemagglutinin (0.1 mg/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-g-specific antibodies as described in the manufacturer’s protocol (Mabtech, Nacka Strand, Sweden). Preincubated 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-g) or 48 h (IL-4) in a cell incubator at 371C 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. Statistical analysis 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, withingroup 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 939
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variables within a group were assessed using Spearman’s rank correlation test. A P-value of o0.05 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. DISCLOSURE
GH Struijk et al.: Immune responses under everolimus
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All the authors declared no competing interests. 18.
ACKNOWLEDGMENTS
We thank Frederiek de Wilde for excellent technical assistance as well as Marianne van de Pol, Dr Rene´ 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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Kidney International (2010) 78, 934–940