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CVI05513-11 REVISED VERSION (2) 1

Performance of the QuantiFERON®-cytomegalovirus (CMV) Assay for Detecting 2

and Estimating the Magnitude and Functionality of the CMV-Specific IFN-γ CD8+ 3

T-Cell Response in Allogeneic Stem Cell Transplant Recipients 4

María Ángeles Clari,1† Beatriz Muñoz-Cobo,1† Carlos Solano,2,3 Isabel Benet,2,3 Elisa 5

Costa,1 María José Remigia,2 Dayana Bravo,1 Paula Amat,2 and David Navarro1,4* 6

7

1Microbiology Service Hospital Clínico Universitario, Valencia, Spain; 2Hematology 8

and Medical Oncology Service, Hospital Clínico Universitario, Valencia, Spain; 9

3Department of Medicine, School of Medicine, University of Valencia, Valencia, Spain; 10

4Department of Microbiology, School of Medicine, University of Valencia, Valencia, 11

Spain. 12

*Correspondence: David Navarro, Microbiology Service, Hospital Clínico 13

Universitario, and Department of Microbiology, School of Medicine, Av. Blasco Ibáñez 14

17, 46010 Valencia, Spain. Phone: 34(96)3864657; Fax: 34(96)3864173; E-mail: 15

david.navarro@uv. 16

†These authors contributed equally to the present work 17

RUNNING TITLE: QuantiFERON®-CMV assay for evaluating CMV-specific CD8+ 18

T-cell immunity 19

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.Clin. Vaccine Immunol. doi:10.1128/CVI.05633-11 CVI Accepts, published online ahead of print on 29 February 2012

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ABSTRACT 24

The performance of the QuantiFERON®-cytomegalovirus (CMV) assay was compared 25

to that of a flow-cytometry intracellular cytokine staining method (ICS) for detecting 26

CMV-specific IFN-γ-producing CD8+ T-cell responses in allogeneic stem cell 27

transplant recipients (allo-SCT) and for estimating their magnitude and functionality. A 28

total of 90 whole blood specimens from 23 allo-SCT recipients was analyzed by both 29

methods. Overall, the percentage of specimens that yielded concordant results by both 30

methods was 68.8% (κ=0.691; 95% CI 0.548-0.835), and the sensitivity of the 31

QuantiFERON®-CMV assay for detecting positive IFN-γ T-cell responses (>0.2 32

UI/mL), taking the ICS method as the reference, was 76.3%. The magnitude of IFN-γ 33

CD8+ T-cell responses to CMV-specific peptides measured with the QuantiFERON®-34

CMV assay correlated significantly (σ=0.695; P=<0.001) with that of the total IFN-γ 35

CD8+ T cells, functional dual (IFN-γ/TNF-α, σ=0.652; P=<0.001, and IFN-γ/CD107a, 36

σ=0.690; P=<0.001) and trifunctional (IFN-γ/TNF-α/CD107a; σ=0.679; P=>0.001) 37

CMV-specific CD8+ T-cell responses as quantitated by ICS. In summary, the data 38

indicated that the QuantiFERON®-CMV assay is less sensitive than the ICS method for 39

detecting CMV-specific IFN-γ CD8+ T-cell responses in the allo-SCT setting; 40

Nevertheless, it allowed the estimation of the total and polyfunctional CMV-specific 41

IFN-γ CD8+ T-cell responses in specimens testing positive by both methods. 42

43

KEY WORDS: Cytomegalovirus, QuantiFERON®-CMV assay, Intracellular cytokine 44

staining, CMV-specific IFN-γ-producing CD8+ T-cell response, allogeneic stem cell 45

transplantation. 46

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INTRODUCTION 48

The assessment of the magnitude and the functionality of T-cell immunity against 49

cytomegalovirus (CMV) is emerging as a clinically useful tool for the therapeutic 50

management of CMV infection in the allogeneic stem cell transplantation setting (allo-51

SCT) (7,13). Monitoring CMV-specific CD8+ or CD4+ T-cell responses may allow for 52

the optimization of pre-emptive antiviral therapy regimens on an individual basis and 53

the identification of patients who may benefit from prophylactic antiviral or adoptive T-54

cell transfer therapeutic strategies (1,7,13). In recent years, several methods have been 55

developed for the ex vivo quantitation and functional characterization of T-cell 56

responses, among which flow cytometry for surface immunophenotyping and 57

intracellular cytokine staining (ICS) is currently considered the “gold standard” (13). 58

The QuantiFERON®-CMV assay (Cellestis Ltd., Melbourne, Australia) is a 59

commercially available test that allows the inference of the size of the CMV-specific T-60

cell response by quantitating the level of IFN-γ, mostly produced by CMV-specific 61

CD8+ T cells, upon the stimulation of whole blood with a number of immunogenic 62

peptides mapped within IE-1, IE-2, pp65, pp50, and gB and restricted by several 63

widespread HLA-I haplotypes (17). The performance of the QuantiFERON®-CMV 64

assay has been mostly assessed in the solid organ transplant (SOT) setting (see 4 for 65

review). Recently published data, although preliminary, lend support to the suitability of 66

the QuantiFERON®-CMV assay for monitoring the CMV-specific IFN-γ-producing 67

CD8+ T-cell response in allo-SCT recipients (2). Nevertheless, it is largely unknown 68

how this method compares to ICS assays for such purpose. In previous studies we 69

reported on the development and clinical utility of an ICS assay for the quantitation of 70

CMV-specific IFN-γ-producing CD8+ and CD4+ T cells (11,12,14-16). The assay was 71

found to reliably predict protection from the development of CMV DNAemia in allo-72


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SCT recipients, and it has recently been used in the setting of a novel strategy for the 73

guidance of pre-emptive antiviral therapy based on combined virological and 74

immunological monitoring (12). In the current study, we compared the performance of 75

the QuantiFERON®-CMV assay with that of our ICS method for detecting and 76

quantitating CMV-specific IFN-γ-producing CD8+ T-cell responses in allo-SCT 77

recipients. 78

MATERIAL AND METHODS 79

Patients and specimens. A total of 90 whole blood samples obtained from 23 non-80

consecutive patients (median age, 53 years; range, 20 to 71 years old; 16 males and 7 81

females) who underwent peripheral blood (n=20) or umbilical cord blood (n=3) allo-82

SCT at the Hospital Clínico Universitario of Valencia between February 2010 and June 83

2011 were analyzed in this study. The underlying diseases of the patients were myeloid 84

acute leukemia (n=10), non-Hodgkin’s lymphoma (n=7), chronic lymphocytic leukemia 85

(n=2), multiple myeloma (n=2), or myelodysplastic syndrome (n=1). The types of 86

transplant were related/matched (n=10), unrelated/matched (n=9), 87

unrelated/mismatched (n=3), and related/mismatched (n=1). The conditioning regimen 88

was non-myeloablative (n=17) or myeloablative (n=6), and the paired CMV serostatus 89

of the donors (D) and recipients R) was: D+/R+ (n=9), D-/R+ (n=9), D+/R- (n=2) and 90

D-/R- (n=3). The study was approved by the Ethics Committees. All patients gave their 91

informed consent to participate in the study. 92

CMV serology. The CMV serological testing of the donors and recipients was 93

performed using the DiaSorin LIAISON® CMV IgG assay (DiaSorin, Saluggia, Italy), 94

following the recommendations of the manufacturer. 95


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CMV plasma DNAemia quantitation. The CMV DNA load in the plasma was 96

quantitated by real-time PCR (Abbott CMV PCR Kit, produced by Qiagen GmbH, 97

Hilde, Germany for Abbott diagnostics, DesPlaines, Illinois, USA). The PCR reactions 98

were performed using the m2000RT system (Abbott Molecular, Illinois, USA), as 99

previously described (3). The DNA extractions were performed using 500 µL of plasma 100

on the m24 SP instrument (Abbott Diagnostics, Illinois, USA). 101

Enumeration of CMV-specific IFN-γ-producing CD8+ T-cells. Quantitation of the 102

total number of CMV-specific IFN-γ-producing CD8+ T cells, and selectively 103

bifunctional (IFN-γ/TNF-α, IFN-γ/CD107a) and trifunctional (IFN-γ/TNF-α/CD107a) 104

CMV-specific IFN-γ-producing CD8+ T cells was performed by ICS. Whole blood (0.5 105

mL) was simultaneously stimulated with two sets of 15-mer overlapping peptides 106

encompassing the sequence of the pp65 and IE-1 CMV proteins (1 μg/mL/peptide), 107

obtained from JPT peptide Technologies GmbH (Berlin, Germany), in the presence of 1 108

μg/mL of co-stimulatory mAbs to CD28 and CD49d, an anti-CD107a mAb coupled 109

with APC, brefeldin (5 µg/mL), and monensin (1 µM) for 6 h at 37 ºC. The cells were 110

washed in PBS-2% FCS, lysed in BD FACS lysis solution, stained with surface markers 111

(anti-CD8-PerCP-Cy5-5 and anti-CD3-APC-Cy7), permeabilized (BD FACS 112

Permeabilizing solution 2), washed, and finally stained for intracellular cytokines (anti-113

IFN-γ-FITC and anti-TNF-α-PE). All antibodies and solutions were purchased from 114

Becton Dickinson (San Jose, CA, USA). The cells were stored at 4 ºC in PBS-1% 115

formaldehyde, acquired within 4 h in a BD FCSCantoII flow cytometer (BD 116

Biosciences Immunocytometry Systems, San Jose, CA) and analyzed with the software 117

program Infinicyt (Cytognos, Salamanca, Spain). The negative controls (absence of 118

peptide stimulation) were processed in parallel for all experiments. The total number of 119

each CD8+ T-cell subpopulation was calculated by multiplying the corresponding 120


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percentage of CMV-specific T cells (after background subtraction) by the absolute 121

number of CD8+ T cells. Specific responses were considered as those that were >0.1% 122

for each population. The CMV-specific IFN-γ-producing CD8+ T cells were enumerated 123

in parallel using the BD Fastimmune kit (BD-Beckton Dickinson and Company-124

Biosciences, San Jose, CA, USA) as previously described (11,12,14-16), but employing 125

the BD FCSCantoII flow cytometer and the software program Infinicyt for the analyses. 126

The levels of IFN-γ-producing CD8+ T cells (total number) measured by the two 127

abovementioned procedures were comparable (data not shown). 128

QuantiFERON®-CMV assay. The QuantiFERON®-CMV assay (Cellestis Ltd. 129

Melboure Australia) was performed according to the manufacturer’s instructions. 130

Briefly, 1 mL aliquots of heparinized whole blood were collected in three 131

QuantiFERON® CMV collection tubes, one containing a number of CMV 132

immunogenic peptides (CMV antigen), another with no peptides (nil control), and the 133

other one containing a polyclonal stimulating antigen (phytohemagglutinin) (mitogen 134

control). The tubes were shaken vigorously for 5 s and then incubated for 18-24 h at 37 135

ºC. The supernatants were then harvested and the levels of IFN-γ were measured by 136

ELISA. A standard curve was generated in each run. The results of the assay were 137

interpreted according to the criteria established by the manufacturers: (i) <0.2 IU/mL 138

(CMV minus Nil) and ≥0.5 IU/mL (mitogen minus nil)-non-reactive-; (ii) ≥0.2 IU/mL 139

(CMV minus nil) and any value of mitogen minus nil-reactive-; (iii) <0.2 IU/mL (CMV 140

minus nil) and <0.5 IU/mL (mitogen minus nil)-indeterminate. According to the 141

manufacturer, indeterminate results are not interpretable. 142

Statistical analysis. The data were analyzed with the aid of the statistical package 143

SPSS version 17.0 (SPSS, North Chicago, IL). Differences between the medians were 144

compared using the Mann-Whitney U test. The Spearman’s rank test was used to 145


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analyze correlations between continuous variables. Two-sided exact P values are 146

reported. A P value < 0.05 was considered statistically significant. 147

RESULTS 148

A total of 90 blood samples from 23 allo-SCT recipients were analyzed by ICS and by 149

the QuantiFERON®-CMV assay. The data are summarized in Table 1. Fifty-five 150

samples (61.1%) from 17 patients had detectable CMV-specific IFN-γ-producing CD8+ 151

T-cell responses by ICS; of these, 42 (76.3%) from 13 patients were reactive (>0.2 152

UI/mL), and 13 (from 4 patients) yielded negative (n=10) or indeterminate (n=3) 153

results in the QuantiFERON®-CMV assay. As shown in Figure 1, specimens testing 154

positive in the QuantiFERON®-CMV assay displayed significantly higher (P=0.001) 155

numbers of CMV-specific IFN-γ-producing CD8+ T cells, as measured by ICS, than 156

those that tested negative or indeterminate. As shown in Table 1, 35 specimens from six 157

patients lacked detectable CMV-specific IFN-γ-producing CD8+ T-cell responses, as 158

determined by ICS. Of these, only one sample yielded a positive result in the 159

QuantiFERON®-CMV assay (2.5 IU/mL). All patients (and/or their donors) with 160

negative results in the QuantiFERON®-CMV assays throughout the study period 161

displayed at least one HLA-I specificity able to present one of the immunogenic 162

peptides included in the QuantiFERON®-CMV assay pool (data not shown). A large 163

number of specimens gave indeterminate results in the QuantiFERON®-CMV assay 164

(n=17; 18.8%); most of these samples (n=14) had no detectable CMV-specific IFN-γ-165

producing CD8+ T-cell responses, as measured by ICS. Overall, the percentage of 166

specimens that yielded concordant results by both methods was 68.8% (κ=0.691; 95% 167

CI 0.548-0.835), and the sensitivity of the QuantiFERON®-CMV assay for detecting 168

positive IFN-γ T-cell responses, as determined by ICS, was 76.3%. Three specimens 169

that tested positive in the ICS assay displayed IFN-γ levels within the range of >0.1-170


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<0.2 IU/mL in the QuantiFERON®-CMV assay. Thus, decreasing the cut-off level for a 171

positive result to >0.1 IU/mL resulted in a slight increase in the sensitivity of the 172

QuantiFERON®-CMV assay (81.8%). Of note was the fact that 15 out of the 42 173

specimens that tested positive in the QuantiFERON®-CMV assay gave optical density 174

values above the upper limit of quantification of the assay, and thus had to be further 175

diluted in order to determine precisely the levels of IFN-γ. 176

We next assessed to what extent the QuantiFERON®-CMV assay allowed the size 177

and functional diversity estimation of CMV-specific IFN-γ-producing CD8+ T-cell 178

responses as determined by ICS. For this, we performed a correlation analysis of the 179

levels of IFN-γ measured in the QuantiFERON®-CMV assay with the number of 180

different functional populations of IFN-γ-producing CD8+ T cells quantitated in the ICS 181

assay for 42 specimens that showed detectable responses by both methods. It is of note 182

that 20 out of these 42 specimens were obtained during episodes of non-treated (n=11 183

from 3 patients) or ganciclovir-treated (n=9 from 2 patients) CMV plasma DNAemia. In 184

addition, the remaining 22 specimens were obtained from eight patients who had a prior 185

episode of CMV DNAemia. As shown in Figure 2, the magnitude of IFN-γ responses to 186

the CMV-specific peptides measured with the QuantiFERON®-CMV assay significantly 187

correlated (σ=0.695; P=<0.0001) with that quantitated by ICS for the total IFN-γ-188

producing CD8+ T cells. Furthermore, the IFN-γ levels determined by the 189

QuantiFERON®-CMV assay also significantly correlated with the number of functional 190

dual (IFN-γ/TNF-α, σ=0.652; P=<0.001, and IFN-γ/CD107a, σ=0.690; P=<0.0001) and 191

trifunctional (IFN-γ/TNF-α/CD107a; σ=0.679; P=<0.0001) CMV-specific CD8+ T cells. 192

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DISCUSSION 195

The QuantiFERON®-CMV assay is the only commercially available method for 196

measuring CMV-specific IFN-γ-producing CD8+ T-cell responses. This assay has 197

previously been evaluated in SOT and allo-SCT recipients (2,4,5,8,10,17,18). 198

Preliminary data supported the clinical utility of this method for assessing the risk of 199

late-onset CMV end-organ disease in SOT recipients (5) and for predicting the 200

occurrence of CMV DNAemia in allo-SCT patients (2). Nevertheless, information on 201

how the QuantiFERON®-CMV assay correlates with ICS methods for estimating the 202

magnitude and assessing the functionality of CMV-specific CD8+ T-cell responses in 203

transplant recipients is still scarce. In the current study, we compared the performance 204

of the QuantiFERON®-CMV assay with that of an ICS method developed by our group 205

that has been proven to be clinically useful in the management of active CMV infection 206

in the allo-SCT setting (11,12,14-16). Overall, we found both methods to yield 207

concordant qualitative results in around 70% of specimens. This finding was not 208

entirely unexpected as 18 out of the 21 peptides included in the CMV tube of the 209

QuantiFERON®-CMV assay map within pp65 (n=15) and IE-1 (n=3). When the results 210

in QuantiFERON®-CMV assay were interpreted as indicated by the manufacturers 211

(positive responses if IFN-γ levels >0.2 UI/mL), the sensitivity of the assay, considering 212

the ICS method as the reference assay, was 76.37%. The sensitivity of the assay slightly 213

improved (81.8%) when the cut-off threshold for positive IFN-γ responses was lowered 214

to 0.1 UI/mL, as previously suggested (5). All patients (and/or their donors) who tested 215

negative in the QuantiFERON®-CMV assay throughout the study period displayed at 216

least one HLA-I variant included among the specificities covered by the test. The 217

specificity of the QuantiFERON®-CMV assay approached 100%. In fact, there was only 218

one specimen that gave a positive result in the QuantiFERON®-CMV assay (2.5 IU/mL) 219


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but which tested negative using the ICS method; this could have been a false positive 220

result, since a follow-up specimen of the same patient obtained 3 days later tested 221

negative in both assays. The QuantiFERON®-CMV assay gave a large number of 222

indeterminate results. This observation is in keeping with previously published data (2). 223

Most of the specimens that yielded indeterminate results (14 out of 17), and those that 224

tested negative, had undetectable CMV-specific IFN-γ-producing CD8+ T-cell 225

responses as measured by ICS. 226

The levels of IFN-γ measured in the QuantiFERON®-CMV assay were found to 227

correlate significantly with the total number of CMV-specific IFN-γ-producing CD8+ T 228

cells quantitated in the ICS assay. This observation is in accordance with the data 229

reported in a recently published study in which the QuantiFERON®-CMV assay was 230

compared to an ICS method using a library of CMV peptides mapped within pp65 as 231

the stimulating antigen (2). 232

Recent data seem to favor the idea that polyfunctional rather than monofunctional 233

CMV-specific CD8+ T cells, in particular those with the ability to produce cytokines 234

with antiviral properties such as IFN-γ and TNF-α and to simultaneously display 235

cytotoxic activity (cells expressing CD107a), are crucial in the protection from and 236

resolution of episodes of active CMV infection in the allo-SCT setting (6,10,19). The 237

QuantiFERON®-CMV assay cannot distinguish between different functional CD8+ T-238

cell populations producing IFN-γ in response to CMV replication. In this context, we 239

were interested to assess to what extent the QuantiFERON®-CMV assay could allow 240

the estimation of levels of CMV-specific polyfunctional IFN-γ-producing CD8+ T cells. 241

We found that IFN-γ levels determined by the QuantiFERON®-CMV assay 242

significantly correlated with the number of CMV-specific functional dual (IFN-γ/TNF-243

α, and IFN-γ/CD107a) and trifunctional (IFN-γ/TNF-α/CD107a) CD8+ T cells. It should 244


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be stressed that the above correlations were observed in a very precise virological 245

setting, i.e., in patients with an ongoing episode of active CMV infection or in patients 246

who had recently resolved an episode of CMV DNAemia. In this context, we observed 247

that the expansion and contraction of monofunctional and polyfunctional CMV-specific 248

IFN-γ-producing CD8+ T-cell responses elicited by CMV replication followed different 249

kinetic patterns and also showed wide variations on an individual basis (9). It is thus 250

likely that the ability of the QuantiFERON®-CMV assay to accurately estimate the size 251

of CMV-specific polyfunctional IFN-γ-producing CD8+ T-cell responses may 252

ultimately depend upon the past and current status of CMV replication. The current 253

study has two main limitations; First, both assays are not entirely comparable, as they 254

are of distinct nature and, most importantly, employ different stimulating antigens (see 255

Table 2); Second, the ICS method taken as the reference standard for measuring CMV-256

specific CD8+ T-cell responses in the current study lacked extensive inter-laboratory 257

validation. Despite these limitations, we are convinced that studies comparing the 258

performance of QuantiFERON®-CMV assay with that of ICS methods with proven 259

reliability to assess and quantitate CMV-specific IFN-γ-producing CD8+ T-cell 260

responses conferring protection against CMV infection are of major clinical interest. 261

Table 2 summarizes the characteristics, advantages and limitations of the 262

QuantiFERON®-CMV assay and the ICS method developed by our group. In summary, 263

the data presented herein indicated that the QuantiFERON®-CMV assay, although less 264

sensitive than our ICS method, allowed the estimation of total and polyfunctional CMV-265

specific IFN-γ-producing CD8+ T-cell-responses in specimens testing positive by both 266

methods. We wonder whether the use of overlapping pp65 and IE-1 peptide pools in the 267

QuantiFERON®-CMV assay instead of a mix of immunogenic CMV peptides would 268

increase the sensitivity of the method. Further studies should be undertaken to confirm 269


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our observations, and most importantly to assess the clinical utility of the 270

QuantiFERON®-CMV assay in this setting; In this context, it seems essential to 271

establish threshold levels of IFN-γ associated with protection from or the resolution of 272

episodes of active CMV infection and CMV end-organ disease. 273

ACKNOWLEDGMENTS 274

We thank Matilde Pastor and Amanda Mataix for their technical assistance. This 275

research study was supported by a grant (09/1117) from FIS (Fondo de Investigaciones 276

Sanitarias, Ministerio de Sanidad y Consumo, Spain). The QuantiFERON®-CMV assay 277

reagents were kindly provided by Alere (Barcelona, Spain). 278

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Marrow Transplant. Jan 17. [Epub ahead of print]. 342

15. Tormo, N., C. Solano, I. Benet, M.A. Clari, J. Nieto, R. de la Cámara, J. 343

López, N. López-Aldeguer, J.C. Hernández-Boluda, M.J. Remigia, A. 344

Garcia-Noblejas, C. Gimeno, and D. Navarro. 2010. Lack of prompt 345

expansion of cytomegalovirus pp65 and IE-1-specific IFNgamma CD8+ and 346

CD4+ T cells is associated with rising levels of pp65 antigenemia and DNAemia 347

during pre-emptive therapy in allogeneic hematopoietic stem cell transplant 348

recipients. Bone Marrow Transplant. 45:543-549. 349

16. Tormo, N., C. Solano, I. Benet, J. Nieto, R. de la Cámara, A. Garcia-350

Noblejas, M.A. Clari, M. Chilet, J. López, J.C. Hernández-Boluda, M.J. 351

Remigia, and D. Navarro. 2010. Kinetics of cytomegalovirus (CMV) pp65 and 352

IE-1-specific IFNgamma CD8+ and CD4+ T cells during episodes of viral 353

DNAemia in allogeneic stem cell transplant recipients: potential implications for 354

the management of active CMV infection. J. Med. Virol. 82:1208-1215. 355

17. Walker, S., C. Fazou, T. Crough, R. Holdsworth. P. Kiely, M. Veale, S. Bell, 356

A. Gailbraith, K. McNeil, S. Jones, and R. Khanna. 2007. Ex vivo monitoring 357

of human cytomegalovirus-specific CD8+ T-cell responses using 358

QuantiFERON-CMV. Transpl. Infect. Dis. 9:165-170. 359

18. Westall, G.P., N.A. Mifsud, and T. Kotsimbos. 2008. Linking CMV serostatus 360

to episodes of CMV reactivation following lung transplantation by measuring 361

CMV-specific CD8+ T-cell immunity. Am. J. Transplant. 8:1749-1754. 362

19. Zhou, W., J. Longmate, S.F. Lacey, J.M. Palmer, G.Gallez-Hawkins, 363

L.Thao, R. Spielberger, R. Nakamura, S.J. Forman, J.A. Zaia, and D.J. 364

Diamond. 2009. Impact of donor CMV status on viral infection and 365


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reconstitution of multifunction CMV-specific T cells in CMV-positive transplant 366

recipients. Blood. 113:6465-6476. 367

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FIGURE LEGENDS 389

390

FIGURE 1. The number of cytomegalovirus pp65/IE-1-specific IFN-γ-producing 391

CD8+ T cells (total) enumerated by flow cytometry for intracellular cytokine staining 392

(ICS) in the specimens that tested positive, negative or intermediate in the 393

QuantiFERON®-CMV assay. The data are given as log10 values, each dot represents a 394

single measurement and the bars represent median values. 395

FIGURE 2. Correlation between the number (log10) of pp65/IE-1-specific IFN-γ-396

producing CD8+ T cells enumerated by flow cytometry for intracellular cytokine 397

staining (ICS) and IFN-γ levels (log10) measured in the QuantiFERON®-CMV assay. 398

The data of 42 whole blood specimens (from 13 allogeneic stem cell transplant 399

recipients) testing tested positive in both methods are depicted. (A) Total number of 400

IFN-γ-producing CD8+ T cells; (B) dual IFN-γ/TNF-α CD8+ T cells; (C) dual IFN-401

γ/CD107a CD8+ T cells; (D) trifunctional (IFN-γ/TNF-α/CD107a) CD8+ T cells. 402

403

404

. 405

406

407

408

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412

413

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415

416

417

418

419

420

421

422

423

424

425

426

427

428

429

430

431

432

433

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435

436


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FIGURE 1.

g 10

cells

/μl)

1

2

CD

8+ T ce

lls (l

og

0

MV

-spe

cific

IFN

-γ

-1

QuatiFERON® CMV assay result

Indeterminate Non-reactive Reactive

CM

-2


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A B

FIGURE 2.

T ce

lls (l

og10

cel

ls/μ

l)

1,5

2,0

2,5

3,0

D8+

T ce

lls (l

og10

cel

ls/ μ

l) 1

2

CM

V-s

peci

fic IF

N-γ

CD

8+ T

0,0

0,5

1,0

1,5-s

peci

fic IF

N- γ

/TN

F-α

CD

-1

0

IFN-γ level (log10 UI/ml)

-2 -1 0 1 2 3

C


-3 -2 -1 0 1 2 3

CM

V-



C D

T ce

lls (l

og10

cel

ls/μ

l)

1

2

T ce

lls (l

og10

cel

ls/μ

l)

1

2

ecifi

c IF

N-γ

/CD

107

CD

8+ T

-1

0

-spe

cific

trifu

nctio

nal C

D8+

-1

0

-3 -2 -1 0 1 2 3

CM

V-s

p

-2



-3 -2 -1 0 1 2 3

CM

V-




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TABLE 1. Detection of cytomegalovirus-specific IFN-γ-producing CD8+ T cells by

intracelullar cytokine staining (ICS) and the QuantiFERON®-CMV assay in whole

blood specimens from allogeneic stem cell transplant recipients

______________________________________________________________________

Result of the ICS method Result of the QuantiFERON®-CMV

(no. of specimens) (no. of specimens)

______________________________________

Positive Negative Indeterminate

______________________________________________________________________

Positive (55) 42 10 3

Negative (35) 1 20 14

______________________________________________________________________


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TABLE 2. Characteristics, advantages and limitations of the intracelular cytokine

staining method (ICS) used in the current study and the QuantiFERON®-

cytomegalovirus (CMV) Assay for evaluation of CMV-specific T-cell immunity

Method ICS (Intracellular cytokine

staining)

QuantiFERON®-cytomegalovirus

(CMV) Assay

Specimen/

volume

Whole blood/1 mL Whole blood/3 mL

Results

available within

8-10 h. 24-48 h.

CMV antigen Two peptide pools consisting

15 mer overlapping peptides

spanning the entire sequences

of pp65 and IE-1

21 peptides mapped within IE-1, IE-

2, pp65, pp50, and gB, and restricted

by several widespread HLA-I

variants (HLA A1,A2,A3,A24,

HLAB7, B8, B27,B35,B44,B52)

Knowledge of

HLA genotype

Not necessary Not necessary

Allows

differentiation

between CD8+

and CD4+ T

cells

Yes No (mostly detects CD8+ T cells)

Allows Yes Yes (IFN-γ only)


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functional

analysis of T

cells

Allows

phenotypic

analysis of T

cells

Yes No

Major

advantages

High sensitivity/ Precise

enumeration of CMV-specific

T cells/ Proven clinical utility

Highly standardized/Can be

performed in any center/ Simple to

perform

Major

limitations

Lack of standardization/Need

access to a flow cytometer

Suboptimal sensitivity (76% taking

ICS as the reference procedure and a

threshold of 0.2 UI/mL, as

recommended by the manufacturers)/

High percentage (~20%) of

indeterminate specimens/ Need to

dilute ~30% of positive samples for

precise quantitation/Lack of studies

demonstrating clinical utility in the

Allo-SCT setting


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