ELISpot assay as a sensitive tool to detect cellular immunity following influenza vaccination in kidney transplant recipients

Clinical Immunology (2006) 120, 342 — 348

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ELISpot assay as a sensitive tool to detect cellular immunity following influenza vaccination in kidney transplant recipients ¨tkes b, Monika Lindemann a,1, Oliver Witzke b,1, Peter Lu Melanie Fiedler c, Ernst Kreuzfelder a, Thomas Philipp b, Michael Roggendorf c, Hans Grosse-Wilde a,* a

Institute of Immunology, University Hospital, Virchowstrasse 171, 45122 Essen, Germany Department of Nephrology, University Hospital, 45122 Essen, Germany c Institute of Virology, University Hospital, 45122 Essen, Germany b

Received 26 January 2006; accepted with revision 9 March 2006 Available online 21 April 2006

KEYWORDS Cellular immunity ELISpot Lymphocyte transformation test Influenza vaccination Kidney transplantation

Abstract Enhanced cellular immunity following influenza vaccination has been undetectable in kidney transplant recipients so far. Protection from influenza is dependent on cellular and humoral immunity. Aim of the study was to investigate immune responses before and after vaccination with influenza A and B antigens in 65 kidney transplant recipients. A significant increase in proliferative responses was only observed towards influenza B (P b 0.0001) by lymphocyte transformation test. The enzyme-linked immunospot (ELISpot) assay was more sensitive and detected significant, 3- to 5-fold increases (P b 0.0001) in interferon-g secretion using influenza A and B antigens. Furthermore, influenza antibody titers increased significantly (P b 0.0001). At month 1 post-vaccination 85% of patients displayed specific cellular, and 95% or 92% humoral immunity against influenza A and B, respectively. Thus, applying the sensitive ELISpot assay, influenza-specific cellular immunity could be detected for the first time in kidney transplant recipients after vaccination. D 2006 Elsevier Inc. All rights reserved.

Introduction

* Corresponding author. Fax: +49 201 723 5906. E-mail address: [email protected] (H. Grosse-Wilde). 1 Both authors contributed equally to this work.

The recent occurrence of avian influenza A (subtype H5N1) infections in man drew attention to influenza infection and prevention in general. Infections with human influenza viruses continue to cause a substantial burden of disease. Worldwide, every year about 5% of adults develop symp-

1521-6616/$ — see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2006.03.002

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tomatic influenza infection and more than half a million people are estimated to die from influenza-associated complications such as pneumonia or cardiopulmonary disease [1,2]. The incidence of complications is severely increased in risk groups such as patients with end-stage renal disease, e.g., the pulmonary infectious mortality rate was approximately twofold higher in kidney transplant recipients and 14- to 16-fold higher in dialysis patients as compared to healthy controls [3]. Furthermore, influenza infection can precipitate kidney transplant rejection [4]. A cost-effective way to protect against influenza-associated complications is vaccination [5]. Therefore, annual immunizations against human influenza A and B viruses are recommended for patients at risk such as transplant recipients [6—8]. Apart from antibodies which neutralize viral glycoproteins, cellular immunity is mandatory to eliminate infected cells and thereby eradicate the virus [9]. Thus, humoral and cellular immunity are necessary for protection against the disease. CD4+ T cells provide help both to B cells for antibody production and to CD8+ cytotoxic T cells for their proliferation. However, especially cellular immunity is diminished in solid organ recipients as compared to controls. There were some case reports on this patient cohort suffering from influenza infection despite vaccination [10], and it was speculated that the complex interaction of both humoral and cellular immunity may have been defective in these patients due to the chronic immunosuppressive drug therapy. In the clinical routine, kidney transplant recipients are not monitored for the success of influenza vaccination. However, several studies showed an increase in specific antibody titers in these patients after vaccination [11—16]. On the contrary, enhanced cellular immunity after influenza vaccination was not demonstrable so far applying lymphocyte transformation test (LTT) [17,18]. Furthermore, the enzyme-linked immunospot (ELISpot) assay that determines cellular immunity on a single cell level has not been used to measure influenza immunity in this patient cohort. To assess cellular immune responses in patients after kidney transplantation, LTT and ELISpot assays measuring influenza-specific lymphocyte proliferation and interferon (IFN)-g secretion were established. Cellular and humoral influenza immunity prior to and post-vaccination were compared in kidney transplant recipients. It was the aim of this study (I) to define if vaccination can induce cellular immunity towards influenza viruses in these patients, (II) to correlate LTT, ELISpot, and antibody results, and (III) to investigate the influence of different immunosuppressive drugs, cellular or clinical parameters and human leukocyte antigens (HLA) on influenza immunity.

antigens [split A/Beijing/262/95 (H1N1) X-127 (A/H1N1), A/ Sydney/5/97 (H3N2) IVR-108 (A/H3N2), and B/Yamanshi/ 166/98 (B), InflusplitR SSW, lot 9916A9, SmithKline Beechem Pharma, Munich, Germany]. Inclusion criteria were: absence of acute infection, no rejection episode within the last 3 months, changes in serum creatinine level within 3 months b15%, interval to kidney transplantation at least 4 months, no pregnancy. After informed consent, each subject received one dose of the vaccine (0.5 ml) by intradeltoideal administration, containing 15 Ag hemagglutinin antigen of each split. Assays to detect influenza-immunity and analyses of leukocyte subpopulations were performed immediately prior to immunization, 1 month and 1 year after immunization, respectively. 37 patients were treated with cyclosporine A (100—150 ng/ml whole blood trough level, tested by FPIA Abbott TDxTM (monoclonal) assay, Abbott Laboratories Limited, Wiesbaden, Germany) and 22 with tacrolimus (4— 10 ng/ml whole blood trough level, tested by Abbott IMxTM Tacrolimus assay, Abbott Laboratories Limited). Six patients did not receive calcineurin inhibitors (non-calcineurininhibitor group) but azathioprine (n = 5) or mycophenolate mofetil (MMF, n = 1). Additionally, all but four patients received prednisone prior to and at month 1 post-vaccination (median dose 0.08, range 0—0.17 mg/kg/day). A group of healthy controls (n = 5) was also vaccinated against influenza and assessed for specific immunity.

Materials and methods

Lymphocyte transformation test Cellular in vitro responses towards the three influenzaderived proteins which were applied for vaccinations (0.06, 0.13, 0.25, 0.5, 1, and 2 mg/ml per split antigen, monobulks provided by SmithKline Beechem Pharma) and towards four mitogens [phytohemagglutinin, concanavalin A, pokeweed mitogen, and anti-CD3 monoclonal antibody (OKT3)] [19] were quantified by LTT. In brief, peripheral blood mononuclear cells (PBMC) from 20 ml heparinized blood were collected after Ficoll-PaqueTM density gradient (Amersham Pharmacia Biotech, Uppsala, Sweden) centrifugation and 50,000 fresh cells were incubated with influenza antigens and mitogens in 200 ml of cell culture medium (RPMI 1640, GIBCO, Life Technologies, Paisley, UK, with 10% of inactivated pooled human serum, Institute of Transfusion Medicine, University Hospital, Essen, Germany) per well of microtiter plates (378C, 5% CO2). Antigen and mitogen cultures were grown in sixtuplicates for 5 days and quadruplicates for 3 days, respectively. For the last 16 h, the cultures were labeled with 1 mCi H3 thymidine per well (TRA.120, specific activity 5 Ci/mmol, Amersham, Buckinghamshire, UK). The cultures were then harvested (Micro96 Harvester, Skatron Instruments, Transby, Norway) onto filter pads (Wallac, Turku, Finland), and the incorporated radioactivity was quantified by liquid scintillation counting (1205 Betaplate, Wallac).

Probands ELISpot assay Standard influenza immunization as recommended by the German advisory board for vaccination (STIKO) [7] was performed in 65 kidney-transplanted, clinically stable patients (26 female, 39 male, median age 52, range 27—74 years) using a cocktail of three influenza-derived protein

The secretion of IFN-g was detected by ELISpot, using first and biotinylated second antibodies at concentrations of 10 and 2 mg/ml, respectively (clone 1-D1K and biotinylated clone 7-B6-1, Mabtech, Nacka, Sweden) as described [20].

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MultiScreen-HA plates were coated with 60 mL of the first antibody for 2 h at 378C in 5% CO2, washed three times, and blocked with cell culture medium. Then, duplicates of 200,000 fresh PBMC per 200 mL culture were stimulated by the three influenza-derived proteins (0.5 mg/ml per split). After 48 h incubation at 378C the ELISpot plates were washed six times. Captured cytokines were detected by incubation for 2 h with 60 mL of the second antibody. Following six washings, 100 mL of avidin—biotin—peroxidase complex (ABC Vectastain-Elite kit, Vector Laboratories, Burlingame, CA, U.S.A.) were added at a dilution of 1:100. ELISpot plates were incubated at room temperature for 1 h and washed six times. After adding the substrate 3-amino-9-ethyl-carbazole (Sigma, St. Louis, MO, U.S.A.) red spots appeared within 4 min. The color reaction was then stopped. Numbers of spots were analyzed by the Bioreader 2000 (Biosys, Karben, Germany). Cellular immunity was assumed if influenzaspecific spots were at least 3-fold higher than autologous controls (stimulation index z3) [21]. For comparison, ELISpot assays were performed twice using frozen cells in parallel (n = 3 vaccinated, healthy controls). ELISpot results utilizing either fresh or frozen PBMC showed that numbers of influenza-specific (stimulated-autologous) spots did not differ significantly. However, the evaluation of results in frozen cells was hampered by an increase in autologous IFN-g production (5 vs. 0.5 spots in fresh PBMC, median values). Furthermore, the inter-assay variability between the two tests using frozen cells was 11% (median value).

Determination of antibody titers Titration of IgG antibodies against influenza A and B was performed by immune fluorescence method [22] according to the manufacturer’s instructions (Fluorimmun-Influenza A and B, Labor Koch und Merk, Ochsenhausen, Germany). Here, the same WHO-recommended influenza strains were used as for vaccinations and cellular assays. Antibody titers were expressed as the reciprocal of the serum dilution. Titers of N320 were considered positive and protective.

Analysis of leukocyte subpopulations Leukocyte subpopulations were measured by flow cytometry (Calibur, Becton Dickinson, Heidelberg, Germany) as described [19].

HLA typing HLA-class I (A, B, C) typings were performed by serology (standard microcytotoxicity assays), HLA-class II (DR, DQ) typings either by serology or by molecular genetics as published [23].

values each. Reactions pre- and post-vaccination were compared by Wilcoxon matched pairs test, those in patients and healthy controls by Mann—Whitney U test. The correlation analyses of LTT, ELISpot, and antibody results and of cellular or clinical parameters (prednisone dose, creatinine level, interval to transplantation, patient age) with influenza immunity were performed by Spearman test. The influence of immunosuppressive therapy, concomitant disease, and HLA antigens was also analyzed by Mann—Whitney U test. For comparisons of the multiple HLA antigens, Bonferroni correction was applied according to Svejgaard et al. [24]. A value of P b 0.05 was considered as statistically significant.

Results Immune response after vaccination Cellular in vitro responses towards influenza antigens were increased in 65 kidney-transplanted patients at month 1 vs. pre-vaccination, and returned to pre-vaccination levels 1 year after vaccination. In detail, LTT reactions to influenza B antigen and ELISpot responses to all three influenza split antigens and their mixture (influenza A and B) were approximately 3- to 5-fold increased (P b 0.0001 each) at month 1 (Fig. 1A, B, Fig. 2). The median frequency of influenza reactive cells as determined by ELISpot — using a mixture of influenza A and B split antigens — was 5/200,000 (0.0025%) prior to vaccination, 25/200,000 (0.013%) at month 1, and 5/200,000 (0.0025%) 1 year after vaccination. On the contrary, LTT reactions towards influenza A/H1N1 and A/H3N2 antigens and mitogens (Fig. 1A, D) did not differ significantly pre- and at month 1 post-vaccination. Humoral immunity against influenza A and B also displayed a significant increase (P b 0.0001) by three and two titer levels (median values), respectively (Fig. 1C). Considering individual courses, LTT responses towards influenza B antigen increased in 63% of vaccinees, and ELISpot responses towards influenza B antigen in 75% and towards the mixture of influenza A and B split antigens in 82%, respectively (month 1 after vs. pre-immunization, Table 1). As determined by ELISpot, cellular immunity towards the mixture of split antigens was present in 85% at month 1 post-vaccination. At that time, 95% and 92% of patients displayed antibody titers of N320 against influenza A and B, respectively. In healthy controls, LTT responses towards the mixture of influenza split antigens were approximately 3-fold higher than in transplant patients (12,434 vs. 3856 cpm increment at month 1, data represent median values). Furthermore, ELISpot reactions towards this mixture were significantly increased (P b 0.01 each) as compared to patients (45 vs. 5 and 81 vs. 25 spots per 200,000 PBMC at day 0 and month 1, respectively). Finally, antibody titers at month 1 postvaccination were comparable in controls and patients.

Statistical analysis 3

For analysis of LTT data, the second highest H thymidine uptake of antigen or mitogen cultures was chosen as published [19,20]. Counts per minute (cpm) increment values were generated as stimulated minus autologous proliferation. Spots increment were calculated considering median

Correlation analyses of LTT, ELISpot, and antibody results Cellular in vitro immune responses, i.e., LTT and ELISpot using influenza split antigens, and LTT mitogen were mutually correlated (P b 0.05), both at day 0 and month

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Figure 1 Cellular and humoral reaction towards influenza antigens (A—C) and mitogens (D) in 65 kidney-transplanted patients. Data represent median and interquartile range, either pre- (white bars) or 1 month post-vaccination (black bars). LTT, lymphocyte transformation test; INF, influenza; IFN, interferon; PHA, phytohemagglutinin; ConA, concanavalin A; PWM, pokeweed mitogen; OKT3, anti-CD3 monoclonal antibody; anot tested: n = 1. Antibody titers are given as reciprocal of serum dilution. *P b 0.0001 (Wilcoxon test).

1. Here, the most significant findings were observed for the correlation of either LTT or ELISpot reactions towards the different split antigens, respectively (median r value: 0.62, P b 0.0001). On the contrary, we could not detect a significant correlation of cellular and humoral influenza immunity. Immune response and immunosuppressive therapy

Figure 2 Interferon-g ELISpot pre- and at month 1 postinfluenza vaccination in a kidney transplant patient. (A) Negative (autologous) control, (B) stimulation by influenza A/ H1N1, (C) influenza A/H3N2, (D) influenza B, (E) mixture of all three influenza split antigens, (F) phytohemagglutinin (positive control).

Vaccination-induced changes in cellular immune responses as measured by ELISpot were significantly (P b 0.05) more pronounced in cyclosporine A as compared to tacrolimus or non-calcineurin-inhibitor treated patients (delta spots increment at month 1 vs. baseline: influenza B: 11 vs. 3, mixture of influenza A and B split antigens: 24 vs. 14, data present median values, Fig. 3A). Moreover, increases in antibody titers were significantly (P b 0.05) reduced in the non-calcineurin-inhibitor as compared to the cyclosporine A and tacrolimus group (delta influenza A antibody titer 960 vs. 2240, Fig. 3B). LTT antigen and mitogen responses, however, did not differ significantly between the cyclosporine A and tacrolimus groups while both were relatively low in the non-calcineurin-inhibitor group. The differences between the three treatment groups cannot be attributed to the number of CD4+ T cells (absolute or relative); values were comparable in all groups. Furthermore, prednisone treatment appears as a factor significantly influencing LTT but not ELISpot or antibody responses. LTT reactions towards influenza B antigen were negatively correlated with prednisone dose both at baseline and at month 1 (r = 0.31, P = 0.01, and r = 0.37, P =

346 Table 1

M. Lindemann et al. Number of patients with an increase in cellular and humoral influenza immunity at month 1 vs. pre-immunization

Immunosuppression

Cyclosporine A (n = 37) Tacrolimus (n = 22) NCI (n = 6) Sum (n = 65)

Cellular immunity

Humoral immunity

LTT

ELISpot

INF B

INF B

INF A + B

INFA

INF B

26 13 2 41

32 13 4 49

32 16 5 53

35a 21 5 61a

32a 18 3 53a

LTT, lymphocyte transformation test; INF, influenza; NCI, non-calcineurin-inhibitor. a Not tested: n = 1.

0.003, respectively, Fig. 4). Interestingly, spontaneous lymphocyte proliferation was also significantly correlated with prednisone dose at both points in time. Correlation of immune response and cellular, clinical, and genetic parameters In addition, we could detect a significant positive correlation of absolute CD4+ T cell counts and LTT and ELISpot reactions (e.g., LTT and ELISpot towards a mixture of all split

antigens: r = 0.39 and r = 0.59 at day 0, P b 0.005 each; r = 0.31 and r = 0.29 at month 1, P b 0.05 each, respectively). Correlation of cellular in vitro function with absolute CD8+ T cell counts, however, was less pronounced. Moreover, patient age and influenza immunity showed a trend towards a negative correlation. Kidney transplant function as assessed by serum creatinine level (median 1.3, range 0.6—5.4 mg/dl), interval to transplantation (median 6 years, range 4 months—19 years), and the presence of hypertension or diabetes (n = 47 or n = 12, respectively) could, however, not be defined as factors influencing influenza immunity. Finally, it was observed that individuals with vs. without the HLA-DR5 antigen displayed lower increases in influenzaspecific ELISpot responses (delta spots at month 1 minus pre-vaccination for influenza A/H3N2: P = 0.005, P after Bonferroni correction = 0.05). On the contrary, the HLA antigen did not influence LTT responses or antibody titers.

Discussion Our data indicate that a specific cellular and humoral immune response to influenza vaccination is detectable in the majority of kidney-transplanted patients despite receiving intense immunosuppression. The IFN-g-specific ELISpot appears more sensitive than the LTT in determining vaccination induced changes of cellular influenza-immunity. In this study, the definition of positive cellular immunity in the ELISpot assay was chosen in accordance with current literature, setting a stimulation index of 3 as a threshold

Figure 3 Vaccination-induced changes in ELISpot reactions (A) and antibody titers (B) in 65 kidney-transplanted patients stratified according to the immunosuppressive drug regimen. Data represent median and interquartile range (delta month 1 post- minus pre-vaccination). 5Cyclosporine A (n = 37), tacrolimus (n = 22), non-calcineurin-inhibitor treatment (n = 6). INF, influenza; IFN, interferon; anot tested: n = 1; *P b 0.05 for cyclosporine A vs. tacrolimus + non-calcineurin-inhibitor group, yP b 0.05 for cyclosporine A + tacrolimus vs. noncalcineurin-inhibitor group (Mann—Whitney U test).

Figure 4 Spearman correlation analysis of prednisone dose and LTT reaction towards influenza B antigen at month 1 postvaccination. The continuous line represents the regression line, the fractioned ones the 95% confidence interval.

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[21]. But, actually there is no consensus on a definition of protective cellular immunity against influenza viruses, especially not in immunocompromised patients. Assuming that cellular immunity would be positive and protective in 85% (55/65) of vaccinees, the remaining ones could be at risk of influenza infections. However, due to the limited size of this study, an estimation of the clinical significance of positive cellular in vitro results cannot be obtained. It is assessed that at least 300 influenza-vaccinated transplant recipients have to be analyzed to yield a definite answer to the question which magnitude of cellular immunity is needed for protection against the disease. As compared to healthy controls, the strength of cellular reactions was significantly decreased in kidney transplanted patients. Another recent study [25] also applied the ELISpot for measuring cell-mediated influenza immunity in immunocompromised patients. Here, six allogeneic stem cell recipients were vaccinated against influenza 3 months posttransplantation and ELISpot assays were performed prior to and 1 month post-vaccination. In contrast to our data, no increase of IFN-g responses could be shown in this cohort. This difference is most likely attributable to the fact that stem cell recipients at that stage post-transplantation generally experience a more profound immunosuppression than clinically stable kidney transplant recipients. Cellular in vitro reactions were positively correlated, especially LTT and IFN-g ELISpot reactions towards the different influenza split antigens. This could be explained most likely by cross-reactivity of the antigens used. Presumably, cellular reactions are directed partly against components common to all influenza A and B viruses. This phenomenon has already been described concerning proliferative responses towards different serotypes of influenza A [17]. Furthermore, influenza-specific cellular reactions were positively correlated with LTT mitogen results indicating that those transplanted patients with a better preserved unspecific immune function also respond better to specific stimuli. Cellular and humoral immunity towards influenza, however, did not correlate; this is not unexpected as these two components of the specific immune system are most likely differentially regulated, e.g., on a genetic basis as also observed for immune responses against the hepatitis B virus [23]. This finding is confirmed by a paper of Briggs et al. [18] who were unable to find a correlation between LTT results and antibody titers following influenza vaccination. Extending these data, we could now show that influenza-specific IFN-g secretion was not correlated with antibody titers. Similar to our data on the virus splits A/H1N1 and A/H3N2, Rytel et al. [17] and Briggs et al. [18] reported that LTT did not increase post-influenza vaccination despite a significant increase in antibody titers in kidney allograft recipients. In the present study, however, LTT responses towards the influenza B split antigen were significantly increased at month 1 vs. pre-vaccination. But overall, changes in LTT responses post-vaccination were less profound than those in influenza-specific ELISpot responses and antibody titers. In our study, cellular immune reactions depended on the type of immunosuppression used. Tacrolimus seems to act more suppressive on cellular antiviral in vitro reactions than cyclosporine A as defined by the ELISpot data. These two drugs belong to a class of immunosuppressive agents called

calcineurin inhibitors which are described to act specifically on T cells [26]. It is known from clinical and experimental observations [27—29] that tacrolimus is a more potent immunosuppressive agent which is reflected by our in vitro results on T cell function. The weak vaccination-induced ELISpot and antibody responses in recipients treated only with azathioprine or MMF may reflect the selection of a special group of transplant patients with generally less reactive T and B cells; responses to mitogens were also relatively low in this treatment group. The negative correlation of antigen-specific T cell proliferation with prednisone dose is consistent with published data describing a corticosteroid-induced inhibition of IL-2 production [26,30]. In contrast to previous studies [31,32] where HLA-class I or II restricted influenza peptides were used, we stimulated PBMC by protein antigens. Thus, we did not selectively measure either CD8+ or CD4+ cell responses. The IFN-g producing cells stimulated by protein antigens should predominantly — but not exclusively — belong to the CD4+ cell subset which is strengthened by the significant correlation of CD4+ cell counts with ELISpot reactions at baseline and at month 1 post-vaccination. This assumption is confirmed by intracellular cytokine staining experiments where CD8+ and CD8 (CD4+) PBMC fractions were stimulated by an influenza protein antigen [33]. It could be shown that the CD4+ subset contributed to approximately 80% of IFN-g production. Previous associations of influenza antibody titers with the HLA-B16 [34], -B35 [35,36], -DR7, or DQ6 [37] antigen could not be confirmed in our patient cohort. However, we describe here, for the first time, a decreased influenza-specific IFN-g secretion in carriers of the HLA-DR5 antigen. Presumably, this HLA-class II antigen binds less effectively to influenza peptides and thereby leads to a decreased immune response. However, the significance of this finding has to be determined in a larger patient cohort. In conclusion, our study using the ELISpot assay shows that influenza vaccination can induce influenza-specific cellular immunity in the majority of kidney transplant recipients. Thus, ELISpot may be a good tool to sensitively monitor the success of vaccinations in immunocompromised patients and to determine factors influencing cellular immunity in this group.

Acknowledgments We thank Dr. M. Haase, Sa ¨chsisches Serumwerk Dresden, Germany, for kindly providing the influenza vaccine and monobulks of the split antigens. In addition, we are grateful to U. Bu ¨ttner, M. Huben, B. Nyadu, and S. Wortmann for their excellent technical assistance. This work was partly supported by the IFORES program, project 107-506-0, of the Medical Faculty, University Hospital of Essen.

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