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Viability and functionality of Cryopreserved Peripheral Blood Mononuclear Cells in 1

pediatric dengue 2

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Federico Perdomo-Celisa, Doris M. Salgadoa, b, Diana M. Castañedaa and Carlos F. 4

Narváeza# 5

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Programa de Medicina, Facultad de Salud, Universidad Surcolombiana, Neiva, 7

Colombiaa; Departamento de Pediatría, Hospital Universitario de Neiva, Colombiab. 8

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Running title: Cryopreservation efficiency in pediatric dengue 10

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# Address correspondence to Carlos F. Narváez, cfnarvaez@usco.edu.co 12

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CVI Accepted Manuscript Posted Online 9 March 2016Clin. Vaccine Immunol. doi:10.1128/CVI.00038-16Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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Abstract 19

Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used in 20

studies of dengue. In this disease, elevated frequency of apoptotic PBMCs has 21

been described, and molecules, such as soluble tumor necrosis factor (TNF)-22

related apoptosis-inducing ligand (sTRAIL), are involved. This effect of dengue 23

could affect the efficiency of PBMCs cryopreservation. Here, we evaluate viability 24

(trypan blue dye exclusion and amine-reactive dye staining) and functionality 25

(frequency of interferon [IFN]-γ producing T cells after polyclonal stimulation) of 26

fresh and cryopreserved PBMCs from children with dengue (in acute and 27

convalescence phase), children with other febrile illnesses, and healthy children as 28

controls. Plasma sTRAIL levels were also evaluated. The frequency of non-viable 29

PBMCs detected by both viability assays was positively correlated (r = 0.74, P < 30

0.0001). Cryopreservation particularly affected the PBMCs of children with dengue, 31

who had a higher frequency of non-viable cells than that of healthy and children 32

with other febrile illnesses (P ≤ 0.02) and PBMCs viability levels were restored in 33

the convalescent phase. In the acute phase, an increased frequency of CD3+CD8+ 34

amine+ cells was found before cryopreservation (P = 0.01). Except for B cells in 35

acute phase, cryopreservation usually did not affect the relative frequency of viable 36

PBMCs subpopulations. Dengue infection reduced the frequency of IFN-γ 37

producing CD3+ cells after stimulation, compared with healthy controls and 38

convalescence (P ≤ 0.003) and plasma sTRAIL correlated with this decreased 39

frequency in dengue (rho = −0.56, P = 0.01). Natural dengue infection in children 40

can affect the viability and functionality of cryopreserved PBMCs. 41

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Keywords: Dengue, TRAIL, viability, functionality, cryopreservation. 42

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Introduction 44

Cryopreservation is the maintenance of cells and biological tissues at low 45

temperatures and is based on the use of various media or solutions that form 46

hydrogen bonds with water molecules, preventing cellular damage. The low 47

temperatures allow the cell to enter a quiescent state in which cellular functions are 48

suspended, without affecting their intrinsic characteristics (1). Peripheral blood 49

mononuclear cells (PBMCs) are frequently cryopreserved for use in transplants or 50

immunological studies (2, 3). However, the cryopreservation process may affect 51

viability, phenotype, and cellular functionality due to factors such as inadequate 52

temperatures, the freezing protocol used, the expertise of the personnel, and 53

freezing time (4, 5). The disease of the individual from which the PBMCs come 54

also affects cryopreservation. For example, PBMCs from subjects infected with 55

human immunodeficiency virus (HIV) presented reduced viability after 56

cryopreservation, possibly due to the increased numbers of apoptotic cells 57

circulating during the course of the disease (6). Similar findings have been found in 58

the acute phases of diseases such as visceral leishmaniasis (7). Particularly for 59

HIV, great efforts have been undertaken to optimize the evaluation and 60

comparability of immune tests in cryopreserved PBMCs. Thus, studies evaluating 61

the efficiency of cryopreservation of PBMCs from patients with particular diseases 62

are greatly needed (8). 63

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Dengue is another infectious disease in which an elevated frequency of PBMCs 64

undergoing cellular death has been described (9). Dengue disease is caused by 65

the dengue virus (DV), transmitted by mosquitoes of the genus Aedes, and 66

constitutes a serious public health problem in tropical areas (10). A high frequency 67

of apoptotic PBMCs, particularly CD8+ T lymphocytes (TLs), circulate during the 68

acute phase (11). In some cases, the magnitude of cellular death has been 69

associated with clinically severe forms of disease (12). Although apoptosis 70

induction in PBMCs in the context of dengue infection is a mechanism to control 71

viral replication (13), the elevated frequencies of cells in different stages of cellular 72

death could affect the efficiency with which these cells are cryopreserved. Knowing 73

the efficiency of cryopreservation of PBMCs from children naturally infected with 74

dengue is critical to certain studies, such as the search for cellular correlates of 75

vaccine-induced protection. Here, we evaluate the viability and functionality of 76

cryopreserved PBMCs from children naturally infected with DV (both acute and 77

convalescent), and these PBMCs were compared with those from healthy children 78

or children who presented febrile pediatric infections other than dengue. The 79

soluble form of the TNF-related apoptosis-inducing ligand (sTRAIL) in plasma was 80

also determined to evaluate possible mechanisms associated with cellular 81

dysfunction of cryopreserved PBMCs. 82

Materials and methods 83

Ethics statement 84

This study was approved by the Ethics Committee at the Universidad 85

Surcolombiana (approval code: NCS-047) and the Hospital Universitario de Neiva 86

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(approval code: HUN-031). Written parents informed consent and informed assent 87

(for children older 6 years) were obtained from each of the children included. All 88

experiments followed the principles expressed in the Declaration of Helsinki. 89

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Patients and samples 91

This study was carried out in the Laboratorio de Infección e Inmunidad at 92

Universidad Surcolombiana and the Hospital Universitario de Neiva, Colombia. 93

Patients and healthy children were enrolled from February 2012 to January 2014. 94

Three groups of children, between 2 months and 14 years of age, were included in 95

this study: healthy (n = 14), other febrile ilnesses (OFI, n = 15), and infection with 96

DV (n = 20). Of the latter two groups, a blood sample in the acute phase (3–7 days 97

from the onset of symptoms) was taken. Additionally, for the children with dengue, 98

a second sample was taken 15 to 27 days from the onset of symptoms 99

(convalescent dengue [CD]). 100

Two to four milliliters of venous blood was collected in tubes containing 101

ethylenediaminetetraacetic acid (EDTA, BD Vacutainer®; Ref: 367861). Within the 102

first 4 hours after phlebotomy, the tubes were centrifuged at 300 x g, and the 103

plasma was collected and stored at −70ºC until the time of analysis. The cellular 104

fraction was used for the isolation of PBMCs, as described below. 105

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Diagnosis of DV infection 107

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For the diagnosis, classification, and clinical monitoring of dengue patients, the 108

revised guide of the World Health Organization (WHO) 2009 (14) was followed, 109

which classify the disease into dengue without warning signs (DNS), dengue with 110

warning signs (DWS), and severe dengue (SD). The diagnosis of infection was 111

confirmed by the presence of the viral non-structural protein 1 (NS1) and/or DV-112

specific immunoglobulin (Ig) M in plasma (assessed before and after five days from 113

the onset of symptoms, respectively). Children with OFI had diagnoses of 114

bronchiolitis, common cold, croup, or viral pharyngitis, in addition to negative tests 115

for the dengue types mentioned above. 116

The commercial enzyme-linked immunosorbent assay (ELISA) kits Dengue IgM 117

Capture (Ref: E-DEN01M), Dengue IgG Capture (Ref: E-DEN02G), and Dengue 118

Early (Ref: E-DEN02P) were used for the detection of DV-specific plasma IgM and 119

IgG and the viral protein NS1, respectively (all from Panbio®, Alere, Australia), 120

following the manufacturers’ instructions. For the type of infection (primary or 121

secondary), the relationship of DV-specific IgM/IgG in the plasma was determined, 122

taking a ratio ≤ 2 as a secondary infection, as previously reported (15). 123

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PBMCs isolation and cryopreservation 125

The isolation, freezing, and thawing of PBMCs were performed as has been 126

previously reported (16). Of note, this protocol has been frequently used (17-19). 127

After isolation, the PBMCs were washed twice with RPMI-1640 supplemented with 128

10% fetal bovine serum (FBS), penicillin 100 U/ml, streptomycin 100 μg/ml, and L-129

glutamine 2 mM (complete medium) (all obtained from Gibco®, Carlsbad, CA). For 130

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cryopreservation, the PBMCs were washed twice with complete medium, 131

resuspended, and counted by trypan blue staining as explained below. 132

Subsequently, the PBMCs were slowly resuspended in FBS containing 10% 133

dimethyl sulfoxide ([DMSO, ATCC®, Manassas, VA; Cat: 4-X], freezing medium) 134

pre-cooled to 4°C, at a cellular density of 5x106 cells/ml and were deposited into 135

polypropylene cryovials (Nunc™, Thermo Scientific™, Waltham, MA; Cat: 375418) 136

to be rapidly brought to −70°C in polystyrene containers which ensured a slow drop 137

in temperature. After 24 hours, the cryovials were transferred to a liquid nitrogen 138

tank (Thermo Scientific™, Waltham, MA; Cat: CY509106), where they remained 139

until analysis. The cryopreservation time ranged from 4 to 120 weeks. For their 140

thawing, the PBMCs were removed from the liquid nitrogen and incubated in a 141

preheated serological bath at 37ºC. Once thawed, the cells were rapidly 142

transferred to 15-ml polystyrene tubes (Falcon-BD™, San Jose, CA; Cat: 352099) 143

containing 10ml of cold complete medium to remove excess DMSO. Finally, the 144

cells were washed with complete medium and counted. To calculate the % 145

recovery of PBMCs, the number of cells obtained was compared before and after 146

cryopreservation using the following formula: (# PBMCs after thawing / # 147

cryopreserved PBMCs) x 100. 148

In a fraction of experiments, PBMCs from healthy volunteers had cellular death 149

induced by being frozen in excess DMSO (40% in FBS) and were used to 150

standardize the evaluation methods of cell viability and as positive controls in the 151

assays. 152

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Determination of PBMCs viability before and after cryopreservation 154

This study was designed to evaluate total viability and not a particular type of 155

cellular death. Cellular viability was evaluated using two methods: automated 156

counting using trypan blue dye exclusion and staining of cellular amines by flow 157

cytometry (FC). The trypan blue dye exclusion staining was performed following a 158

widely used protocol (16). A 1:1 (v / v) mixture of PBMCs suspension and 0.4% 159

trypan blue (Merck, Darmstadt, Germany; Cat: 111732) was incubated for 2 160

minutes at room temperature. Ten microliters of the mixture was deposited on 75 x 161

25 x 1.8 mm polymethyl methacrylate plates (Counting slides, Bio-Rad®, Hercules, 162

CA; Cat: 145-0011), and the plates were read in a TC20™ automated cell counter 163

(Bio-Rad®, Hercules, CA, Cat: 145-0102). The analysis was performed using 164

TC20™ data analyzer software (Bio-Rad®, Hercules, CA), adjusting the cell size 165

gate between 7 and 20μm. To corroborate the results, in all experiments, one 166

reading was also performed by conventional light microscopy. For this reading, 167

10μl of the same mixture was deposited in a Neubauer chamber and counted using 168

a Nikon Eclipse E100 optical microscope (Nikon®, Melville, NY). Counting was 169

performed by two trained observers, and the result was reported as the mean 170

value obtained by both. At least 40 cells were counted in each field (16). 171

To determine cellular viability by flow cytometry, a LIVE/DEAD® Fixable Dead Cell 172

Stain commercial kit (Invitrogen™, Waltham, MA; Cat: L34955) was used, following 173

the recommendations of the manufacturer. For this assay, 1x106 cells were 174

washed and resuspended in 1 ml of sterile 1X DPBS (Dulbecco's phosphate-175

buffered saline; Gibco®, Carlsbad, CA; Ref: 14190-144), stained with 1 μl of 176

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fluorescent reagent, and incubated for 30 minutes at 4°C while protected from light. 177

After washing with sterile DPBS and centrifugation at 196 x g, 10μl of Tritest™ 178

(anti-human CD3, clone SK7, anti-CD4, clone SK3, and anti-CD8, clone SK1, 179

labeled with peridinin chlorophyll protein complex [PerCP], fluorescein 180

isothiocyanate [FITC], and phycoerythrin [PE], respectively; BD™, San Jose, CA; 181

Cat: 340298) and 2μl of anti-CD19 PeCy7 (clone HIB19, BD™, San Jose, CA; Cat: 182

560728) were added, and the solution was incubated for 30 minutes at 4ºC while 183

protected from light. Finally, the cells were washed with 3 ml of FACS buffer (0.5% 184

bovine serum albumin [BSA] [Sigma-Aldrich®, St. Louis, MO; Cat: A7906], 0.02% 185

sodium azide [Merck, Darmstadt, Germany; Cat: 106688] in 1X PBS, filtered) and 186

fixed with 1% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). The 187

PBMCs were acquired on a FACS Canto II cytometer using FACS Diva v6.1.3 188

software (BD™, San Jose, CA) within one hour of completion of the staining. 189

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Evaluation of the functionality of cryopreserved PBMCs before and after 191

cryopreservation 192

The functionality of the PBMCs was evaluated for the capacity of CD3+CD4+ and 193

CD3+CD8+ cells to produce interferon-γ (IFN-γ) after being treated with polyclonal 194

stimuli. For this assay, 1x106 PBMCs/ml resuspended in complete medium were 195

stimulated with 50ng/ml of phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich®, 196

St. Louis, MO; Cat: P8139) and 500ng/ml of ionomycin (Sigma-Aldrich®, St. Louis, 197

MO; Cat: I0634) and incubated for 10 hours at 37ºC in 5% CO2, the last 5 hours in 198

the presence of 10μg/ml of Brefeldin A (Sigma-Aldrich®, St. Louis, MO; Cat: 199

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B7651). Then, the cells were washed and centrifuged at 196 x g, and 10μL of 200

Tritest™ were added (BD™, San Jose, CA; Cat: 340298). After 30 minutes of 201

incubation at 4ºC while protected from light, the cells were washed and 202

permeabilized with 300μl of Cytofix/Cytoperm™ (BD™, San Jose, CA; Cat: 203

554722) for 20 minutes at 4ºC. Subsequently, intracellular staining was performed 204

with anti-human IFN-γ labeled with allophycocyanin (APC) (clone 25723.11, BD™, 205

San Jose, CA; Cat: 341117), incubating for 30 minutes at 4ºC. Finally, the cells 206

were washed twice with 1X Perm/Wash™ solution (BD™, San Jose, CA; Cat: 207

554723) and acquired within one hour of completion of the staining. 208

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Detection of sTRAIL in plasma 210

The plasma sTRAIL concentration was evaluated by ELISA (Quantikine® Human 211

TRAIL/TNFSF10, R & D Systems®, Cat: DTRL00) following all of the 212

manufacturer's recommendations. The reported sensitivity of the test is 2.8 pg/ml. 213

The correlation coefficient of the standard curve was >99%, and the duplicate 214

variability was less than 10%. The mean optical density (OD 450nm) of the 215

negative controls was 0.055. Calculating the concentration of sTRAIL was obtained 216

by interpolation of the OD of the samples to a standard curve using 4-parameter 217

logistic regression with GraphPad Prism® software version 6.0. 218

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Statistical analysis 220

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The data are presented as medians and ranges. GraphPad Prism® 6.0 (GraphPad 221

Software, La Jolla, CA) software was used for the statistical analyses. The Mann-222

Whitney test was used to analyze two independent groups, and the Wilcoxon test 223

was used for paired data. To analyze more than two independent groups, the 224

Kruskal-Wallis test was used. If the Kruskal-Wallis P value was < 0.05, Dunn’s 225

multiple comparison test was used, according to each case. The degrees of 226

correlation between variables were determined with the Pearson and Spearman 227

tests. Fisher's test was used for frequency analysis. In all cases, a P value < 0.05 228

was taken as significant. 229

Results 230

Patients included 231

This study included 14 healthy children, 15 OFI, and 20 children with dengue (18 of 232

them matched to acute dengue [AD] and convalescent dengue [CD] phases). 233

Children with dengue were classified clinically as DWS (n = 12) and SD (n = 8) 234

(Table 1). As is known, bronchiolitis, common cold, croup, and viral pharyngitis are 235

common in children < 12 months old (20), explaining the lower median age of 236

children with OFI than that healthy and children with dengue (P < 0.0001, Dunn’s 237

post-hoc test). The children with OFI and dengue were included between the third 238

and seventh days of fever. Children with dengue had lower leukocyte and platelet 239

counts compared with those of OFI children (Table 1). The medians (ranges) of 240

aspartate aminotransferase (AST) in children with DWS and SD were 39 U/L (34–241

88) and 138 U/L (36–172), respectively (P = 0.01, Mann-Whitney test, data not 242

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shown). Alanine aminotransferase (ALT) was also significantly higher in children 243

with SD than in children with DWS (P = 0.01, Mann-Whitney test, data not shown). 244

In summary, the data presented in Table 1 support the adequate clinical 245

classification of the groups included. Of note, the medians (ranges) of the time 246

(weeks) of PBMCs cryopreservation were 25 (15–98), 49 (24–72), 95.5 (8–112), 247

and 27 (4–96) for the healthy children, OFI, AD, and CD, respectively, with no 248

significant differences between them (P = 0.1, Kruskal-Wallis test, data not shown). 249

250

The cellular viability assays that were used evaluated the same cellular 251

population 252

Automated cellular counting methods using dye exclusion with trypan blue and 253

staining of cellular amines were used to assess PBMCs viability. Both methods 254

detect increased permeability of the cell membrane as a viability marker and not a 255

particular type of cellular death (21). Figure 1A shows the frequency of non-viable 256

PBMCs detected by the two methods in the patients and in the positive viability 257

controls, where death was induced. Comparable frequencies were detected using 258

both methods (r = 0.74, P < 0.0001, Pearson test), which demonstrates their 259

capacity to identify the same cellular population. PBMCs were thawed and counted 260

to determine the % recovery with respect to the number of cells originally frozen. 261

As shown in Figure 1B, the median (range) recoveries were 83% (62–100), 84% 262

(70–99), 83.5% (70–100), and 87% (38–100) for the healthy children, OFI, AD, and 263

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CD, respectively, with no differences between the groups (P = 0.7, Kruskal-Wallis 264

test). 265

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Pediatric DV infection affects the viability of cryopreserved PBMCs 267

To establish whether natural DV infection in children affects the viability of 268

cryopreserved PBMCs, the frequencies of non-viable PBMCs before and after 269

freezing were evaluated. As previously reported (22), increased frequencies of 270

non-viable cells were found in PBMCs post-cryopreservation in all of the groups 271

analyzed (P ≤ 0.0001, Wilcoxon test, Fig. 2A), confirming the effect of the process 272

on cellular viability. The median (range) of non-viable PBMCs from healthy children 273

was low (4.6% [1–19]) after cryopreservation and comparable to that reported 274

previously (5, 23), demonstrating the efficiency of the freezing protocol used here 275

(Fig. 2A). After cryopreservation, there was a higher frequency of trypan+ PBMCs 276

in children with acute DV infection compared with healthy children (P = 0.0002, 277

Dunn’s post-hoc test, Fig. 2A). In convalescence, the values of trypan+ cells were 278

lower than in the acute phase (P = 0.0002, Dunn's post-hoc test, Fig. 2A) and 279

similar to those found in healthy children (P > 0.05, Dunn's post-hoc test, Fig. 2A). 280

Higher levels of trypan+ cells were also found in children with dengue than in those 281

with OFI (P = 0.02, Dunn’s post-hoc test, Fig. 2A), suggesting that this effect could 282

be virus-specific. Comparable results were obtained by amine staining, which was 283

performed simultaneously (data not shown). Cryopreservation particularly affected 284

PBMCs from children with dengue, as the relationship of the frequency of dead 285

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cryopreserved cells/dead fresh cells was at least 2 fold higher than that found in 286

the other groups (P < 0.0001, Dunn’s post-hoc test, Fig. 2A). Analysis of the 287

viability of cryopreserved PBMCs between children with DWS and children with SD 288

by amine-reactive dye and trypan blue staining showed no difference (P > 0.05, 289

Dunn’s post-hoc test, Fig. 2B and data not shown). In summary, in children 290

naturally infected with DV, there was a greater frequency of non-viable PBMCs 291

after cryopreservation, indicating a greater lability to this process. The frequency 292

was not associated with clinical severity. 293

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Phenotype of non-viable PBMCs from children with dengue 295

For identifying the type of cell that dies during dengue infection, the differences in 296

the frequencies of specific subpopulations of amine+ PBMCs between the AD 297

phase and the CD phase (AD/CD ratio) were evaluated by FC. Amine+ CD3+CD4+, 298

CD3+CD8+ (CD4+ and CD8+ TLs, respectively), CD19+ (B lymphocytes), and 299

CD3−CD19− (non-T non-B cells) cells were analyzed according to the strategy 300

shown in Figure 3A. Consistent with the previous results (Fig. 2), all of the PBMCs 301

populations had ratios greater than 1, indicating that death was higher in the acute 302

phase of infection (Figs. 3B and 3C). Before cryopreservation, the AD/CD ratio of 303

amine+ CD3+CD8+ cells was higher than that of amine+ CD3+CD4+ cells (P = 0.01, 304

Dunn’s post-hoc test, Fig. 3B). However, after the process, no differences in any of 305

the subpopulations evaluated were observed (P = 0.3, Kruskal-Wallis test, Fig. 3C). 306

These results suggest that in fresh PBMCs, the CD8+ TLs are particularly 307

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susceptible to death during the acute phase of infection. After cryopreservation, 308

this susceptibility is similar in all PBMCs subpopulations, fact explained for the 309

increase in the frequency of dead cells in the convalescence, due to the 310

cryopreservation process. 311

312

Cryopreservation usually conserved the relative frequencies of PBMCs 313

subpopulations 314

Subsequently, we evaluated the effects of the infection and cryopreservation on 315

the relative frequencies of living CD3+CD4+, CD3+CD8+, CD19+, and CD3−CD19− 316

cells. The relative frequencies of all of the analyzed populations were comparable 317

between the acute and convalescent phases, regardless of whether the cells were 318

fresh or cryopreserved (P ≥ 0.09, Mann-Whitney test, Fig. 4). Furthermore, the 319

relative frequencies of subpopulations of PBMCs were usually not affected by the 320

cryopreservation (Fig. 4), and only a lower frequency of viable CD19+ cells was 321

found after cryopreservation compared with before cryopreservation in AD (P = 322

0.02, Mann-Whitney test, Fig. 4C), indicating that this subpopulation could be more 323

labile to the cryopreservation. Therefore, cryopreservation generally maintains the 324

relative frequency of the different PBMCs subpopulations in children infected with 325

dengue. 326

327

Natural infection with DV affects the functionality of PBMCs 328

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The capacity of the cryopreserved PBMCs from children with dengue to produce 329

IFN-γ in response to stimulation with PMA-ionomycin was further evaluated. 330

Consistent with previous reports (24, 25), high CD4 down-regulation was found, so 331

that after stimulation, the CD4+ TLs were analyzed as CD3+CD8− cells. In healthy 332

controls, the medians (ranges) of IFN-γ producing CD3+CD8+ and CD3+CD8− TLs 333

were 5.7% (1.2–12.2) and 5.9% (2.7–13), respectively (Fig. 5), frequencies 334

consistent with previous reports (26). After cryopreservation, PBMCs from children 335

with dengue had lower frequencies of IFN-γ producing CD3+CD8+ (Fig. 5A) and 336

CD3+CD8− T cells after stimulation (Fig. 5B), compared with healthy children (P = 337

0.003 and P = 0.0001, respectively; Dunn’s post-hoc test). In convalescence, the 338

frequencies of IFN-γ producing CD4+ and CD8+ TLs were restored to levels 339

comparable with those of the healthy, indicating that the IFN-γ down-regulation 340

was virus-induced. Similar results were obtained before cryopreservation (data not 341

shown). This effect is not dependent on the used stimulus, as similar results were 342

observed when fresh PBMCs were treated with Staphylococcus aureus enterotoxin 343

B, a known super-antigen (n = 5, data not shown). Short protocols using PMA-344

ionomycin particularly stimulates memory T lymphocytes (26), which are low in 345

infants (under one year of age) (27), which would explain the low frequency of IFN-346

γ producing TLs also found in children with OFI (Figs. 5A and 5B). Of note, there 347

was no association between the frequencies of IFN-γ producing TLs after 348

polyclonal stimulation, both CD3+CD8+ and CD3+CD8−, with the clinical severity of 349

DV infection (DWS vs. SD, P ≥ 0.4, Mann-Whitney test, data not shown). In 350

summary, in acute phase natural DV infection decreased the frequency of IFN-γ 351

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producing TLs after in vitro stimulation, thus showing an effect on their 352

functionality. 353

Plasma sTRAIL correlates negatively with the low frequency of IFN-γ 354

producing T cells 355

To explore mechanisms explaining the high frequency of non-viable cryopreserved 356

PBMCs and the low functionality induced by natural DV infection in children, levels 357

of sTRAIL, a molecule associated with cellular dysfunction, were evaluated by 358

ELISA in children with dengue and OFI (in the sample of the same day that the 359

PBMCs were cryopreserved). The medians (ranges) in pg/ml for plasma sTRAIL 360

were 117 (14–253), 194 (87–314), and 113 (66–130) in children with OFI, DWS, 361

and SD, respectively (Fig. 6A). Of note, children with SD had lower levels of 362

sTRAIL than those of children with DWS (P = 0.01, Dunn’s post-hoc test, Fig. 6A). 363

No correlation between the frequency of dead cells and the respective plasma 364

sTRAIL levels in children with dengue or OFI was found (rho ≤ 0.2, P ≥ 0.4, 365

Spearman test, data not shown). However, plasma sTRAIL correlated negatively 366

and moderately with the low frequency of IFN-γ producing CD3+ TLs (CD4+ and 367

CD8+) in children with dengue (rho = −0.56, P = 0.01, Spearman test, Fig. 6B), but 368

not with OFI (rho = 0.4, P = 0.3, Spearman test, data not shown). These results 369

suggest that soluble factors such as sTRAIL may be partially involved in the 370

decreased functionality of TLs observed during acute DV infection. 371

372

Discussion 373

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In this study, the viability and functionality, before and after cryopreservation, of 374

PBMCs from children with dengue were evaluated. i. Cryopreserved PBMCs from 375

children with dengue had a higher frequency of non-viable cells than those from 376

healthy children or children with OFI. ii. Before cryopreservation of PBMCs from 377

children with dengue, the population with the highest frequency of dead cells was 378

that of CD8+ TLs. iii. Cryopreservation usually maintained the relative frequency of 379

PBMCs subpopulations from children with dengue. iv. Dengue virus infection in the 380

acute phase reduced the frequency of IFN-γ producing TLs after polyclonal 381

stimulation, and this inhibition was associated with increased plasma sTRAIL 382

levels. 383

The two methods that evaluated cellular viability had a strong positive correlation 384

(Fig. 1A), and the % of recoveries were similar between the study groups (Fig. 1B), 385

suggesting that the majority of the cells, regardless of their viability, were analyzed, 386

without significant cell loss during the freezing process. Consistent with what was 387

previously reported (22, 28), cryopreservation affected PBMCs viability in all of the 388

groups studied (Fig. 2A). Due to dehydration, mechanical and chemical stress, 389

intracellular crystallization, and thermal shock, the cryopreserved cells had reduced 390

viability after the process (1); in cryopreserved PBMCs frequencies of non-viable 391

cells of 5 to 10%, such as obtained here, are generally accepted in samples of 392

healthy individuals (28). 393

PBMCs from children with dengue had a higher frequency of death compared with 394

those of healthy children; PBMCs viability was restored in convalescence (Fig. 2A). 395

The frequency of non-viable cells in children with dengue found here is consistent 396

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with previous reports that used propidium iodide and annexin V (29). The 397

mechanisms by which DV induces cell death are not entirely clear and are 398

dependent on the cellular type analyzed. These mechanisms include i. 399

accumulation of viral proteins (30); ii. induction of the expression of CD137, a 400

death receptor (31); and iii. induction of cellular death directly by viral proteins (32). 401

In the dengue group, frequencies of dead cells were comparables in children with 402

or without antigenemia (positive detection of plasma NS1) (data not shown), 403

suggesting that other mechanisms in addition to the cell viral infection are 404

responsibles for the higher death levels found in dengue PBMCs. Activation-405

induced apoptosis (activation-induced cell death, AICD) would be a critical 406

mechanism for PBMCs death, as high expression levels of members of the TNF 407

receptor superfamily classically associated with cell death, such as CD95 (FAS) 408

and sTRAIL in PBMCs and the plasma of patients with the infection have been 409

shown (33-36). This mechanism modulates immune cell activation against the 410

virus, ensuring homeostasis (37). PBMCs death in acute DV infection has been 411

linked to disease severity (11, 12). In our study, this association was not found 412

(Fig. 2B), although it should be noted that patients with dengue without warning 413

signs were not included, which may behave differently than the more severe 414

hospitalized forms analyzed here. 415

Few studies have analyzed the PBMCs subpopulations particularly affected by 416

death in DV infection. Preceding cryopreservation, CD8+ TLs were those that died 417

in the acute phase of the infection in particular (Fig. 3B). In fresh PBMCs from 418

individuals with dengue, apoptotic antigen-specific CD8+ TLs have been detected 419

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(9), and this effect has been associated with AICD (11). Following the 420

cryopreservation, all of the subpopulations were affected similarly in the acute and 421

convalescent phases (Fig. 3C), possibly due to the effects of the cryopreservation 422

previously described. 423

In general, cryopreservation maintained the relative frequency of the principal 424

viable subpopulations of PBMCs in children with dengue (Fig. 4). However, a 425

decrease in the relative frequency of amine− CD19+ cells was found after 426

cryopreservation in the acute phase of infection (Fig. 4C). As a rapid and strong 427

response of antibody-secreting cells has been reported in the acute phase of 428

infection (38) and this type of cells are susceptible to cellular death (39), 429

cryopreservation could affect this population. Furthermore, studies analyzing the 430

effects of cryopreservation on the expression of differentiation markers and the 431

frequency of antigen-specific effector B cells from children with dengue are 432

necessary. 433

Multiple studies have evaluated the cellular functionality following PBMCs 434

cryopreservation. In healthy individuals, the performance of functional tests that 435

assess T and B memory cells by methods such as ELISPOT, after polyclonal 436

stimulation, is usually adecuate (40), but there is still controversy as to whether 437

these findings are similar in PBMCs of the sick (41). As has been previously noted, 438

the cryopreservation did not affect the functionality of cells from healthy children 439

(Fig. 5). However, children with dengue had a low frequency of IFN-γ producing 440

TLs after polyclonal stimulation, a number that was restored in the convalescent 441

phase (Fig. 5), suggesting an inhibitory effect of the virus on their functionality. DV 442

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can inhibit IFN-α production (42) but possibly not IFN-γ production, although other 443

Flaviviruses, such as West Nile virus, do inhibit the production of both interferon 444

types (43). However, the continuous activation of PBMCs during dengue infection 445

could force the cells to a state of exhaustion (44, 45). In addition, an inhibitory 446

effect of interleukin (IL)-10, a cytokine expressed during the infection, on the 447

secretion of other cytokines should also be considered (46). 448

High levels of sTRAIL were found in children with acute DV infection, and these 449

levels were lower in the severe cases (Fig. 6A). Previously, sTRAIL has been 450

attributed to an antiviral mechanism in dengue, given by the induction of apoptosis 451

or the production of type I IFN (47), reducing the cellular viral burden (35) and thus 452

explaining the higher levels of sTRAIL found in children who did not develop 453

severe forms of the infection (Fig. 6A) (36, 48). There was no correlation between 454

plasma sTRAIL and the frequency of dead cells (data not shown); however, a 455

negative correlation with the low frequency of IFN-γ producing TLs was detected 456

(Fig. 6B). Thus, sTRAIL could be implicated in mechanisms of functional T-cell 457

inhibition in dengue. Consistently, TRAIL can suppress signaling pathway 458

activation and TLs proliferation (49). Under T cell expansion, TRAIL may 459

participate in cell death signaling, process modulated by the celular FADD-like-460

Interleukin-1β-converting enzyme –inhibitory protein (c-FLIP) in this activated 461

subpopulation (50). Additionally, a negative correlation has been shown between 462

sTRAIL and the absolute count of TLs in dengue, which also supports their 463

possible relationship in vivo (48). In summary, after cryopreservation, the PBMCs 464

from children with acute DV infection had lower viability and functionality compared 465

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with PBMCs from healthy children, but these abnormalities were corrected during 466

convalescence. sTRAIL is partially implicated. Although future studies that include 467

antigen-specific T and B cells are more than necessary, this effect of dengue 468

infection should be taken into account when employing cryopreserved PBMCs. 469

470

Funding information 471

This work was funded by Colciencias, grant 112451929049 and the Vicerrectoría 472

de Investigación de la Universidad Surcolombiana, grant GI2015SAL04 (both to 473

Carlos F. Narváez). The funders had no role in study design, data collection and 474

interpretation, or the decision to submit the work for publication. 475

476

Acknowledgements 477

To all of the patients who participated in the study, to the Department of Pediatrics 478

of the Hospital Universitario de Neiva, and to Jairo A. Rodríguez and Rocío Vega 479

for their administrative support and the inclusion of patients. 480

481

References 482

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40. Truck J, Mitchell R, Thompson AJ, Morales-Aza B, Clutterbuck EA, 628 Kelly DF, Finn A, Pollard AJ. 2014. Effect of cryopreservation of peripheral 629 blood mononuclear cells (PBMCs) on the variability of an antigen-specific 630 memory B cell ELISpot. Hum Vaccin Immunother 10:2490-2496. 631

41. Kreher CR, Dittrich MT, Guerkov R, Boehm BO, Tary-Lehmann M. 2003. 632 CD4+ and CD8+ cells in cryopreserved human PBMC maintain full 633 functionality in cytokine ELISPOT assays. J Immunol Methods 278:79-93. 634

42. Green AM, Beatty PR, Hadjilaou A, Harris E. 2014. Innate immunity to 635 dengue virus infection and subversion of antiviral responses. J Mol Biol 636 426:1148-1160. 637

43. Morrison J, Aguirre S, Fernandez-Sesma A. 2012. Innate immunity 638 evasion by Dengue virus. Viruses 4:397-413. 639

44. Kahan SM, Wherry EJ, Zajac AJ. 2015. T cell exhaustion during persistent 640 viral infections. Virology 479-480:180-193. 641

45. Chunhakan S, Butthep P, Yoksan S, Tangnararatchakit K, Chuansumrit 642 A. 2015. Vascular leakage in dengue hemorrhagic Fever is associated with 643 dengue infected monocytes, monocyte activation/exhaustion, and cytokines 644 production. Int J Vasc Med 2015:917143. 645

46. Malavige GN, Jeewandara C, Alles KM, Salimi M, Gomes L, 646 Kamaladasa A, Jayaratne SD, Ogg GS. 2013. Suppression of virus 647 specific immune responses by IL-10 in acute dengue infection. PLoS Negl 648 Trop Dis 7:e2409. 649

47. Gandini M, Gras C, Azeredo EL, Pinto LM, Smith N, Despres P, da 650 Cunha RV, de Souza LJ, Kubelka CF, Herbeuval JP. 2013. Dengue virus 651 activates membrane TRAIL relocalization and IFN-alpha production by 652 human plasmacytoid dendritic cells in vitro and in vivo. PLoS Negl Trop Dis 653 7:e2257. 654

48. Limonta D, Torrentes-Carvalho A, Marinho CF, de Azeredo EL, de 655 Souza LJ, Motta-Castro AR, da Cunha RV, Kubelka CF, Nogueira RM, 656 de-Oliveira-Pinto LM. 2014. Apoptotic mediators in patients with severe 657 and non-severe dengue from Brazil. J Med Virol 86:1437-1447. 658

49. Lehnert C, Weiswange M, Jeremias I, Bayer C, Grunert M, Debatin KM, 659 Strauss G. 2014. TRAIL-receptor costimulation inhibits proximal TCR 660 signaling and suppresses human T cell activation and proliferation. J 661 Immunol 193:4021-4031. 662

50. Morales JC, Ruiz-Magana MJ, Ruiz-Ruiz C. 2007. Regulation of the 663 resistance to TRAIL-induced apoptosis in human primary T lymphocytes: 664 role of NF-kappaB inhibition. Mol Immunol 44:2587-2597. 665

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Figures Legends 669

Figure 1. The methods for evaluating cellular viability were comparable. A. 670

Correlation between the % of non-viable PBMCs determined by trypan blue and 671

amine staining. All children included in the study are shown. Pearson’s correlation, 672

P value and slope of the curve are shown. B. Percentage of cell recovery. The 673

median and respective ranges are shown. OFI: Children with other febrile illnesses; 674

AD: Acute dengue; CD: Convalescent dengue; ns: Not statistically significant by 675

the Kruskal-Wallis test. 676

Figure 2. Natural infection with DV affects the viability of cryopreserved 677

PBMCs. A. Frequencies of pre- and post-cryopreservation trypan+ PBMCs in the 678

groups analyzed. The delta (Δ) of the frequencies of post-/pre-cryopreservation 679

trypan+ cells is shown at the top of each group (for statistical purposes, the % 680

equal to zero were carried to one). B. Frequencies of post-cryopreservation amine+ 681

cells evaluated in the two clinical groups of children with dengue and healthy 682

controls. The median of each group and the P value of Dunn's post-hoc test are 683

shown. *P < 0.0001, Dunn’s post-hoc test AD vs. Healthy, OFI, and CD. OFI: Other 684

febrile illnesses; AD: Acute dengue; CD: Convalescent dengue; DWS: Dengue with 685

warning signs; SD: Severe dengue. 686

Figure 3. Dengue particularly affects the viability of fresh CD3+CD8+ cells. A. 687

Gating strategy for analyzing the viability of cryopreserved PBMCs subpopulations 688

by FC, in a child with dengue in both the acute phase and in the convalescent 689

phase. Relative frequencies of amine+ CD3+CD4+, CD3+CD8+, CD3−CD19+, and 690

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CD3−CD19− cells (non-T non-B cells, bottom panel) are shown. B. - C. Ratios of 691

the frequencies of amine+ CD3+CD4+, CD3+CD8+, CD3−CD19+, and CD3−CD19− 692

cells in the acute and the convalescent phase of infection by DV (AD/CD) before (n 693

= 5) (B) and after (n = 16) (C) cryopreservation. The medians and ranges are 694

shown. AD: Acute dengue; CD: Convalescent dengue; ns: Not statistically 695

significant. The P value of Dunn's post-hoc test is shown. 696

Figure 4. Cryopreservation generally maintains the relative frequency of the 697

major PBMCs subpopulations in children with dengue. Frequencies of amine− 698

CD3+CD4+ (A), CD3+CD8+ (B), CD3−CD19+ (C), and CD3−CD19− (D) cells pre- (n = 699

5) and post-cryopreservation (n = 16) in the acute and convalescent phases of DV 700

infection. The medians and their respective ranges are shown. AD: Acute dengue; 701

CD: Convalescent dengue; ns: Not statistically significant. The P value of the 702

Mann-Whitney test is shown in each case. 703

Figure 5. Natural infection with DV affects the functionality of cryopreserved 704

PBMCs. Frequencies of cryopreserved IFN-γ producing CD3+CD8+ cells (A) and 705

CD3+CD8− cells (B) after in vitro stimulation with PMA-ionomycin were analyzed by 706

FC. The horizontal lines indicate the median for each group. OFI: Children with 707

other febrile illnesses; AD: Acute dengue; CD: Convalescent dengue; ns: Not 708

statistically significant. The P value from Dunn's post-hoc test is shown in each 709

case. 710

Figure 6. sTRAIL is negatively associated with the frequency of IFN-γ 711

producing CD3+ cells after in vitro stimulation. A. Plasma sTRAIL levels in 712

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children with other febrile illnesses (OFI) and with acute DV infection (dengue 713

warning sign [DWS) and severe dengue [SD]) were evaluated by ELISA. The 714

dashed line indicates the detection limit of the assay. The solid horizontal lines 715

indicate the median. The P value of Dunn's post-hoc test is shown. B. Correlation 716

between plasma sTRAIL levels of children with dengue and the frequency of 717

cryopreserved IFN-γ producing CD3+ cells after treatment with PMA-ionomycin. 718

The P value and Spearman rank correlation test (rho) are displayed. 719

720

Table 1. Epidemiological and paraclinical characteristics of the children 721

included 722

Healthy (n=14)

OFI (n=15)

Dengue (n=20)

Age in months, median (range)a 60 (13–144) 2* 48 (7–88) Gender, male, n (%)b 8 (57.1) 7 (46.6) 14 (70) Illness day, median (range)c – 3 (3–6) 5 (3–7) Primary infection, n (%) – – 11 (55) Secondary infection, n (%) – – 9 (45) DWS, n (%) – – 12 (60) SD, n (%) – – 8 (40) Hematocrit, %, median (range)c ND 32.2 (28.9–39.4) 34.1 (11.8–47) Leukocyte, x 103/μl, median (range)c ND 13.8 (9–17.9) 4 (1.1–9.3)** Platelets, x 103/μl, median (range)c ND 360 (48–565) 58 (16–249)** ND: Not determined. 723

aKruskal-Wallis test, Dunn’s post-hoc test. bFisher test. cMann-Whitney test. 724

*P < 0.0001 versus healthy and Dengue. 725

**P ≤ 0.0007 versus OFI (other febrile illnesses). 726

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40

60

80

100

% o

f am

ine

- C

D19

+ c

ells

P=0.02

AD CD

ns

Pre Post Pre Post

0

20

40

60

80

100

% o

f am

ine-

CD

3+C

D8

+ c

ells

AD CD

ns ns

Pre Post Pre Post

0

20

40

60

80

100

% o

f am

ine-

CD

3- C

D19

- cells

ns ns

AD CD

A. B.

C. D.

on May 23, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

Healthy OFI AD CD0

2

4

6

8

10

12

14

16

18

20

% o

f IF

Nγ-

pro

ducin

g C

D3

+C

D8

+ c

ells

P=0.03P=0.003

Healthy OFI AD CD0

2

4

6

8

10

12

14

16

18

20

% o

f IF

Nγ-

pro

ducin

g C

D3

+C

D8

- cells

P=0.001P=0.0001

P=0.001 P=0.0001

A. B.

on May 23, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from

A. B.

OFI DWS SD0

50

100

150

200

250

300

350

Pla

sm

a s

TR

AIL

(p

g/m

L)

P=0.01

0 100 200 300 4000

2

4

6

8

10

12

Plasma sTRAIL (pg/mL)

% o

f IF

Nγ-p

rod

ucin

g C

D3

+ c

ells

rho= -0.56

P=0.01

on May 23, 2018 by guest

http://cvi.asm.org/

Dow

nloaded from