Abstract

Fungal specific CD154+ T-cells have been described as a biomarker in invasive aspergillosis. The influence of sample storage on the detection of these cells was assessed. Six-hour delay prior to PBMC isolation is associated with an 18% decrease of cell viability and alterations of the cellular composition of the sample. This results in 87% reduction of CD154+A. fumigatus specific cells due to reduced assay sensitivity and increased background values in unstimulated samples. If prompt cell measurement is not feasible, isolated PBMCs can be frozen (at −20°C and −80°C) and processed later with comparable assay reliability (mean value fresh vs. thawing: 0.126, 0.133; Pearson-Coefficient: 0.962).

Introduction

Invasive aspergillosis (IA) is a common opportunistic fungal infection in immunocompromised patients. Early and reliable diagnosis is pivotal in order to improve therapeutic outcomes.1,2 It has become clear that T-cell immunity significantly contributes to the defence against mould infections.3 Recently, it was shown that the quantification of fungus-specific CD154+ T cells may provide a novel diagnostic assay for the rapid identification of invasive pulmonary mould infections.4 CD154 is a T-cell activation marker transiently expressed on CD4+ cells upon stimulation of their T-cell receptor. Different studies showed that ex vivo stimulation of mononuclear cells followed by flow cytometric quantification of CD154-positive cells is a sensitive and convenient approach for the assessment of antigen-specific T-cell response.5,6,7 Since treatment trials with Aspergillus-specific T-cells are being developed, monitoring of the frequency of circulating Aspergillus-specific T-cells in patients at risk, especially after T-cell transfer will receive special attention.8 In invasive mould infections, the sensitivity and specificity of this assay for the occurrence of invasive fungal infections (IFI) was reported to exceed the results of currently available blood tests including galactomannan or polymerase chain reaction.4 For integrating this approach in the diagnostic routine, however, further assessment is required to determine the reliability of the assay depending on the preanalytical sample handling. Sample shipment to specialized centers may cause preanalytic delays and may thus impact the reliability of the results provided by the assay. In this study, we therefore examined the impact of different preanalytical storage periods and conditions on the outcome of the quantification of CD154+A. fumigatus-specific T cells.

Materials and methods

After obtaining written informed consent (approved by the Ethics Committee of the University of Wuerzburg), 18 ml of EDTA whole blood were collected from healthy volunteers. Peripheral blood mononuclear cells (PBMCs) were purified from 26 healthy donors (9 male, 17 female, mean age 23.7 years) by Ficoll gradient centrifugation. The assay was performed as previously described.4,6 In brief, 1 × 106 cells (100 μl) were plated in each well of a 96-well plate and stimulated overnight with 5 μg of an A. fumigatus mycelial lysate (Miltenyi Biotec) or 2 μl PepTivator EBV Consensus (Miltenyi Biotec) and 1 μl CD28 costimulatory antibody. Detection of CD154-positive and IFNγ-positive T cells was performed using the Inside Stain Kit (Miltenyi Biotec). Cells were measured on a FACS Calibur flow cytometer. The frequency of A. fumigatus-specific T-helper cells was calculated by subtracting the percentage of CD154+/CD4+ cells in a well cultured with the CD28 costimulatory antibody only from the mean percentage of CD154+/CD4+ cells in wells cultured with the costimulatory antibody and the mycelial lysate.

Donors whose frequency of A. fumigatus-specific T cells exceeded the mean value of the cohort were enrolled in a time course experiment. Blood was drawn every two hours and stored at room temperature (RT) or 4°C for up to 6 h prior to conducting the assay. Additionally, 1 × 107 PBMCs purified from the immediately processed blood were frozen in RPMI 1640 + 20% autologous serum + 8% DMSO. After freezing at −20°C for 24–48 h or −80°C for 6–8 days, stimulation and quantification of A. fumigatus-specific cells were performed as described above.

Results

An average frequency of 0.12% (range: 0%–0.43%) A. fumigatus-specific CD154+/CD4+ T cells was found in immediately purified PBMC samples obtained from healthy donors. Using technical duplicates for each donor, the mean intra-assay difference for the frequency of A. fumigatus-specific T cells was 0.01% with a maximum of 0.07%. This is indicative for a reliable assay setup allowing a highly reproducible quantification of A. fumigatus-specific cells.

Six donors with an A. fumigatus-specific T-cell frequency of ≥0.12% were enrolled in the second part of the study. Their mean A. fumigatus-specific T-cell frequency in freshly obtained and processed samples was 0.26%. Storing the blood samples at either RT or 4°C for 2 h prior to the isolation of the PBMCs, this frequency declined to 0.15% (P = .13) and 0.16% (P = .04), respectively. After a 6-h preanalytic storage period at RT or 4°C, an even lower mean frequency of A. fumigatus-specific T-cells was found (0.10%, P = .05 and 0.10%, P = .03, respectively; Fig. 1A).

Figure 1.

Prompt sample processing is crucial for reliable quantification of A.fumigatus- specific T-cells. A) Frequencies of CD154+/CD4+A. fumigatus-specific T-cells in 6 healthy subjects using immediately processed whole blood samples (0 h) or samples stored at RT or 4°C for 2 or 6 hours. Horizontal bars indicate mean values. B) Unspecific background frequencies of CD154+/CD4+ T-cells in samples cultured with CD28 costimulatory antibody only. C) Difference of the percentage of CD154+/CD4+ in samples stimulated with A. fumigatus mycelial lysate plus CD28 costimulatory antibody and samples cultured with the costimulatory antibody only. D)–E) Frequencies of CD154+/CD4+ (D) and IFN-gamma**+/CD3+ (E) EBV-specific T-cells in 6 additional donors using freshly isolated PBMCs and blood samples stored at RT for 6 hours. Significance testing was performed using the paired two-sided Student's t-test. P > .1 ns, P < .1 ▪, P < .05 *.

Figure 1.

Prompt sample processing is crucial for reliable quantification of A.fumigatus- specific T-cells. A) Frequencies of CD154+/CD4+A. fumigatus-specific T-cells in 6 healthy subjects using immediately processed whole blood samples (0 h) or samples stored at RT or 4°C for 2 or 6 hours. Horizontal bars indicate mean values. B) Unspecific background frequencies of CD154+/CD4+ T-cells in samples cultured with CD28 costimulatory antibody only. C) Difference of the percentage of CD154+/CD4+ in samples stimulated with A. fumigatus mycelial lysate plus CD28 costimulatory antibody and samples cultured with the costimulatory antibody only. D)–E) Frequencies of CD154+/CD4+ (D) and IFN-gamma**+/CD3+ (E) EBV-specific T-cells in 6 additional donors using freshly isolated PBMCs and blood samples stored at RT for 6 hours. Significance testing was performed using the paired two-sided Student's t-test. P > .1 ns, P < .1 ▪, P < .05 *.

Unspecific background signals needed to be considered. Therefore, the frequency of CD154+/CD4+ cells in samples cultured with the CD28 costimulatory antibody, without the lysate, was also determined. In immediately processed samples the average background frequency was 0.03%. After 2-h sample storage at RT or 4°C, this frequency increased to 0.08% and 0.05%. During the following 4 hours, a further rise of the background frequency was observed (0.10% and 0.08%; Fig. 1B). The poor detection of A. fumigatus-specific T cells after preanalytic sample storage in combination with this increased unspecific background frequency of CD154+/CD4+ cells resulted in a markedly declined overall sensitivity of the assay after 2 hours and a completely reduced sensitivity after 6 hours (Fig. 1C). Similarly, the detection rates of CD154+/CD4+ cells (Fig. 1D) and IFNγ+/CD3+ cells (Fig. 1E) in EBV-stimulated PBMCs declined after 6-h sample storage at RT, suggesting an assay impairment by preanalytic delay, which is not antigen specific. Accordingly, cell viability and frequencies of CD14+ antigen presenting cells were reduced in samples stored for 6 h at either RT or 4°C (Fig. 2B–C).

Figure 2.

Freezing of immediately purified PBMCs is recommended if immediate sample stimulation and measurement is not feasible A) Mean frequencies of CD154+/CD4+A. fumigatus-specific T-cells in healthy subjects using immediately processed whole blood samples (0 h), samples stored at RT or 4°C for 6 hours, or PBMCs purified immediately after the blood draw, but frozen for 24 to 48 hours prior to conducting the assay (Fr/Th). Error bars indicate the standard deviation (n = 6). B) Mean percentage of vital cells determined by trypan blue exclusion and FSC/SSC values in flow cytometry (live gate). C) Mean frequencies of CD14+ antigen presenting cells. D) Frequencies of CD154+/CD4+A. fumigatus-specific T-cells in healthy subjects (n = 10) using freshly isolated PBMCs and PBMCs isolated promptly and frozen at −20°C for 24 to 48 hours (n = 10) or at −80°C for 6 to 8 days (n = 6). Dotted lines indicate the highest intra-assay deviations observed in technical replicates using freshly isolated cells (Fig. 1A). P > .1 ns, P < .1 ▪, P < .05 *.

Figure 2.

Freezing of immediately purified PBMCs is recommended if immediate sample stimulation and measurement is not feasible A) Mean frequencies of CD154+/CD4+A. fumigatus-specific T-cells in healthy subjects using immediately processed whole blood samples (0 h), samples stored at RT or 4°C for 6 hours, or PBMCs purified immediately after the blood draw, but frozen for 24 to 48 hours prior to conducting the assay (Fr/Th). Error bars indicate the standard deviation (n = 6). B) Mean percentage of vital cells determined by trypan blue exclusion and FSC/SSC values in flow cytometry (live gate). C) Mean frequencies of CD14+ antigen presenting cells. D) Frequencies of CD154+/CD4+A. fumigatus-specific T-cells in healthy subjects (n = 10) using freshly isolated PBMCs and PBMCs isolated promptly and frozen at −20°C for 24 to 48 hours (n = 10) or at −80°C for 6 to 8 days (n = 6). Dotted lines indicate the highest intra-assay deviations observed in technical replicates using freshly isolated cells (Fig. 1A). P > .1 ns, P < .1 ▪, P < .05 *.

PBMCs were isolated from the same donors and frozen promptly after the blood draw; however, these were stimulated and measured later. Subtracting the unspecific background, a mean frequency of 0.21% A. fumigatus-specific T cells was detected. These results were basically identical to the ones found in immediately processed cells from the same donor (Fig 2A). Similarly, cell viability and frequency of CD14+ cells remained largely unchanged compared to freshly isolated cells (Fig. 2B–C).

To further test the reliability of the quantification of A. fumigatus-specific T cells after freezing of PBMCs, the assay was comparatively performed using frozen and thawed PBMCs from 16 healthy donors (Fig. 2D). Ten samples were stored at −20°C for 24–48 h, and 6 were frozen at −80°C for 6–8 days. In all cases, the difference of the frequencies of A. fumigatus-specific T cells in both samples was lower than the highest intra-assay deviation observed in technical replicates using freshly isolated cells (0.07%).

Discussion

In summary, A. fumigatus-specific T cells can be quantified in a highly reliable manner using freshly purified PBMC samples. The mean values and the range of A. fumigatus-specific T-cell frequencies were comparable to the observations in a previous study.4 Our data, however, are indicative for a highly reduced sensitivity of the assay if the blood samples are not processed appropriately. As functional T-cell assays rely on the interaction of antigen presenting cells and T-helper cells, it is quite understandable that a dysbalance of the cell subsets, reduced viability, or functionality may impair the results. Several studies evaluating the performance of T-cell assays in infectious diseases highlighted that blood collection, sample storage, and blood shipment conditions are crucial for reliable test performances.9,10,11 Particularly, the time between blood collection and sample processing appears to be the most critical parameter greatly affecting cell recovery and function.9,10,11,12 This observation is explained by increasing granulocyte contamination of the PBMC isolates due to granulocyte aggregation, reduced T-cell responsiveness, or biochemical alterations caused by an increasing fragility of erythrocytes and granulocytes.10,12,13,14 Given the relatively lengthy assay procedure for the quantification of A. fumigatus specific T cells and the circumstances of sample shipment, our observations highlight significant logistical challenges for using this assay in the diagnostic routine. Our results demonstrate that freezing of immediately isolated PBMCs at the patient's local center with subsequent storage and shipment is easily feasible and may contribute to overcome multiple challenges. This optimization will require widely harmonized protocols for sample collection, PBMC isolation, and shipment. Hence, further standardization of functional T-cell assays in infectious diseases is strongly recommended.

Funding

This work was supported by the Interdisciplinary Centre for Clinical Research (IZKF) Wuerzburg (grant number Z-3/56 to SW), the Bavarian Immune Therapy Network (BayImmuNet, to AJU) and the TR SFB124 (HE).

Declaration of interest

AJU has received support for travel to meetings from Astellas and Basilea. He is a consultant and on the speakers’ bureaus of Astellas, Gilead, MSD, and Pfizer. He has also received support for travel and accommodation from Astellas, Boehringer Ingelheim, Gilead, MSD, and Pfizer for activities unrelated to this study. His institution has received grants from Astellas, Gilead, MSD, and Pfizer. None were related to this work.

The other authors have nothing to disclose.

Meetings where the information has previously been presented

Parts of this study have been presented at the Interscience Conference of Antimicrobial Agents and Chemotherapy (San Diego, 2015).

References

1.
Karthaus
M
,
Buchheidt
D
,
Invasive aspergillosis: new insights into disease, diagnostic and treatment
,
Curr Pharm Des.
 ,
2013
,
19
,
3569
3594
2.
Cakir
FB
,
Cakir
E
,
Berrak
SG
et al
,
Invasive respiratory aspergillosis is a treatable disease with early diagnosis and aggressive therapy
,
Pediatr Hematol Oncol.
 ,
2010
,
27
,
422
434
3.
Romani
L
,
Immunity to fungal infections
,
Nat Rev Immunol.
 ,
2011
,
11
,
275
288
4.
Bacher
P
,
Steinbach
A
,
Kniemeyer
O
et al
,
Fungus-specific CD4(+) T cells for rapid identification of invasive pulmonary mold infection
,
Am J Respir Crit Care Med.
 ,
2015
,
191
,
348
352
5.
Stuehler
C
,
Nowakowska
J
,
Bernardini
C
et al
,
Multispecific Aspergillus T cells selected by CD137 or CD154 induce protective immune responses against the most relevant mold infections
,
J Infect Dis.
 ,
2015
,
211
,
1251
1261
6.
Bacher
P
,
Kniemeyer
O
,
Teutschbein
J
et al
,
Identification of immunogenic antigens from Aspergillus fumigatus by direct multiparameter characterization of specific conventional and regulatory CD4+ T cells
,
J Immunol.
 ,
2014
,
193
,
3332
3343
7.
Bacher
P
,
Scheffold
A
,
Flow-cytometric analysis of rare antigen-specific T cells
,
Cytometry A.
 ,
2013
,
83
,
692
701
8.
Bacher
P
,
Jochheim-Richter
A
,
Mockel-Tenbrink
N
et al
,
Clinical-scale isolation of the total Aspergillus fumigatus-reactive T-helper cell repertoire for adoptive transfer
,
Cytotherapy.
 ,
2015
,
17
,
1396
1405
9.
Bull
M
,
Lee
D
,
Stucky
J
et al
,
Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials
,
J Immunol Methods.
 ,
2007
,
322
,
57
69
10.
Jeurink
PV
,
Vissers
YM
,
Rappard
B
et al
,
T cell responses in fresh and cryopreserved peripheral blood mononuclear cells: kinetics of cell viability, cellular subsets, proliferation, and cytokine production
,
Cryobiology.
 ,
2008
,
57
,
91
103
11.
McKenna
KC
,
Beatty
KM
,
Vicetti Miguel
R
et al
,
Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function
,
J Immunol Methods.
 ,
2009
,
341
,
68
75
12.
Afonso
G
,
Scotto
M
,
Renand
A
et al
,
Critical parameters in blood processing for T-cell assays: validation on ELISpot and tetramer platforms
,
J Immunol Methods.
 ,
2010
,
359
,
28
36
13.
Schmielau
J
,
Finn
OJ
,
Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients
,
Cancer Res.
 ,
2001
,
61
,
4756
4760
14.
Mallone
R
,
Mannering
SI
,
Brooks-Worrell
BM
et al
,
Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society
,
Clin Exp Immunol.
 ,
2011
,
163
,
33
49