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Journal of Clinical Virology 61 (2014) 65–73

Contents lists available at ScienceDirect

Journal of Clinical Virology

journa l homepage: www.e lsev ier .com/ locate / j cv

ormalizing ELISPOT responses to T-cell counts: A novel approach foruantification of HCMV-specific CD4+ and CD8+ T-cell responses inidney transplant recipients

andra A. Calarotaa, Antonella Chiesaa, Lucia Scaramuzzib, Kodjo M.G. Adzasehouna,iuditta Comolli a,c, Filippo Mangioneb, Pasquale Espositob, Fausto Baldanti a,∗

Molecular Virology Unit, Microbiology and Virology Department, Fondazione IRCCS Policlinico San Matteo, Via Taramelli 5, 27100 Pavia, ItalyNephrology, Dialysis and Transplantation Unit, Fondazione IRCCS Policlinico San Matteo, Viale Golgi 19, 27100 Pavia, ItalyExperimental Research Laboratories, Biotechnology Area, Fondazione IRCCS Policlinico San Matteo, Viale Golgi 19, 27100 Pavia, Italy

r t i c l e i n f o

rticle history:eceived 20 March 2014eceived in revised form 23 May 2014ccepted 29 May 2014

eywords:uman cytomegalovirusLISPOT-cell countsnterferon-�idney transplantation

a b s t r a c t

Background: Human cytomegalovirus (HCMV) is the most common opportunistic virus infection in solidorgan transplant recipients. The analysis of HCMV-specific T-cell immunity after organ transplant is ofrelevant clinical interest.Objectives: To analyze HCMV-specific CD4+ and CD8+ T-cell responses in healthy subjects and kidneytransplant recipients (KTR).Study design: HCMV-specific T-cell responses were evaluated by interferon-� (IFN-�) enzyme-linkedimmunospot (ELISPOT) using overlapping 15-mer peptide pools of immediate early (IE)-1, IE-2, phos-phoprotein 65 (pp65) (for stimulation of both CD4+ and CD8+ T-cell responses) and a pool of 34 shortpeptides (8–12 amino acids in length, for stimulation of CD8+ T-cell responses). ELISPOT results werenormalized to T-cell subset counts and their correlations with a reported dendritic cell (DC)-based assay,which simultaneously quantifies HCMV-specific CD4+ and CD8+ T-cell responses, were analyzed.Results: HCMV-seropositive KTR showed higher ELISPOT responses compared to HCMV-seropositivehealthy subjects. IE-1 and pp65 ELISPOT responses were mediated mainly by CD8+ T-cells and, to alesser extent, CD4+ T cells; IE-2 peptides appear to stimulate CD56+ cells (natural killer cells). In HCMV-seropositive healthy subjects, ELISPOT results (expressed either as net spots/million cells or normalized

to the corresponding T-cell count) significantly correlated with the DC assay. However, in HMCV-seropositive KTR, only normalized ELISPOT responses to overlapping 15-mer peptide pools significantlycorrelated with DC-assay responses.Conclusions: The normalized ELISPOT represents a novel and simple approach for quantifying and moni-toring HCMV-specific CD4+ and CD8+ T-cell responses in KTR.

© 2014 Elsevier B.V. All rights reserved.

. Background

In immunocompetent subjects, human cytomegalovirusHCMV) establishes life-long latent infection and the T cell-

ediated immune response is crucial for controlling virus

Abbreviations: HCMV, human cytomegalovirus; KTR, kidney transplant recip-ents; IFN-�, interferon-�; ELISPOT, enzyme-linked immunospot; IE, immediatearly; pp65, phosphoprotein 65; DC, dendritic cells; SOT, solid organ transplant;CS, intracellular cytokine staining; aa, amino acids; PBMC, peripheral blood mono-uclear cells; IQR, interquartile range; NK, natural killer.∗ Corresponding author. Tel.: +39 0382 502283; fax: +39 0382 502599.

E-mail addresses: f.baldanti@smatteo.pv.it, fausto.baldanti@unipv.itF. Baldanti).

ttp://dx.doi.org/10.1016/j.jcv.2014.05.017386-6532/© 2014 Elsevier B.V. All rights reserved.

reactivations [1]. Despite the use of antiviral therapy, HCMVremains a significant cause of morbidity and mortality in solidorgan transplant (SOT) recipients [2,3]. Clearly, HCMV-specificT-cell immunity plays a crucial role in defining the clinical outcome[4].

Several methods have been developed to evaluate HCMV-specific T-cell responses, such as HLA-peptide tetramer stain-ing, intracellular cytokine staining (ICS), the enzyme-linkedimmunospot (ELISPOT) assay, and an HCMV-specific enzyme-linked immunosorbent-based interferon gamma (IFN-�) release

assay (Quantiferon-CMV assay) [5]. The ICS and ELISPOT assaysare not limited by HLA-restriction; CD4+ T-cell determination canbe obtained using as the stimulus infected cell lysate or longsynthetic peptides (13–22 amino acids [aa]), while CD8+ T-cell

66 S.A. Calarota et al. / Journal of Clinical Virology 61 (2014) 65–73

Table 1Characteristics of kidney transplant recipients.

Pt no. Age(years)a

Sex HLA HCMV IgGat Tx (D/R)

HCMVIgGa

Dayspost-Txa

Inductiontherapy

Immunosuppressivetherapya

HCMV DNA(copies/mlblood)a

Peak HCMVDNA (copies/mlblood) post-Tx(days post-Tx)

1 50 M A3,A11,B7,B65 D+/R+ + 30 Anti-CD25 mAb Tac/MMF/steroid 4700 4700 (30)2 38 F A3,A26,B7,B35 D+/R+ + 54 Anti-CD25 mAb Tac/MMF/steroid <50 150 (19)3 62 M A2,A24,B35,B50 D+/R+ + 71 Anti-CD25 mAb Tac/MMF/steroid 25,400 25,400 (71)4 58 M A30,A33,B8,B65 D+/R+ + 96 Anti-CD25 mAb CsA/MMF/steroid 550 10,300 (59)5 75 M A3,A24,B7,B35 D+/R+ + 98 Anti-CD25 mAb Tac/MMF/steroid 100b 23,400 (77)6 70 F A1,A33,B44,B57 D+/R+ + 110 ATG Tac/MMF/steroid 5600b 39,200 (64)7 59 M A11,A29,B44,B51 D+/R+ + 115 Anti-CD25 mAb Tac/MMF/steroid 4300 8500 (46)8 66 M A3,A33,B47,B65 D+/R+ + 119 Anti-CD25 mAb Tac/MMF/steroid 150 3000 (50)9 44 M A1,A24,B40,B44 D+/R+ + 136 ATG Tac/MMF/Evr <50 <50

10 65 M A1,A26,B38,B52 D+/R+ + 144 Anti-CD25 mAb Tac/MMF/steroid <50 200 (53)11 51 M A2,A3,B35,B62 D+/R+ + 251 Anti-CD25 mAb Tac/steroid <50b 3,400,000 (127)12 26 F A3,B7,B18 D+/R+ + 292 Anti-CD25 mAb Tac/MMF/steroid <50 20,750 (55)13 50 F A2,B46,B58 D+/R+ + 321 Anti-CD25 mAb Tac/MMF/steroid <50 <5014 57 M A2,A3,B18,B57 D+/R+ + 365 ATG Tac/MMF/Evr <50 <5015 60 M A1,B8,B37 D+/R+ + 374 Anti-CD25 mAb Tac/MMF/steroid 550 1200 (170)16 73 M A1,A29,B8,B51 D+/R+ + 394 ATG Tac/MMF/steroid 200 1100 (56)17 71 M A2,B14,B51 D+/R+ + 404 ATG Tac/MMF/Evr <50 3500 (109)18 41 F A11,A68,B35,B39 D+/R+ + 426 Anti-CD25 mAb Tac/MMF/steroid <50 4500 (60)19 77 M A1,A30,B13,B35 D+/R- + 376 ATG Tac/MMF <50 166,300 (33)20 55 F A1,A29,B8,B13 D+/R- + 470 Anti-CD25 mAb CsA/MMF/steroid <50 478,600 (99)21 41 M A3,B35,B45 D-/R- − 20 ATG Tac/MMF/steroid <50 <5022 46 M A2,A24,B7,B35 D-/R- − 57 Anti-CD25 mAb Tac/MMF/steroid <50 <5023 26 F A2,A24,B18,B35 D-/R- − 173 ATG Tac/MMF/Evr <50 <50

M, male; F, female; HLA, human leukocyte antigen; HCMV, human cytomegalovirus; Tx, transplantation; D/R, donor/recipient; mAb, monoclonal antibody; ATG, anti-t ine; Ev

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hymocyte globulin; Tac, tacrolimus; MMF, mycophenolate mofetil; CsA, cyclospora At the time of immunologic testing.b Patients were receiving valgancyclovir.

etermination is obtained using short synthetic peptides (8–10a). A method aimed at stimulating both CD4+ and CD8+ T cellsith HCMV-infected autologous dendritic cells (DC) (referred to

s the DC assay) has been developed and used to monitor SOTecipients [6–8]. However, its major limitations are the technicalomplexity (including the potential difficulty in generating autol-gous monocyte-derived immature DC from severely leukopenicatients) and its long turnaround time.

The IFN-� ELISPOT has been used for the detection of HCMV-pecific T-cell responses [9–11]. However, the ELISPOT does notasily distinguish between CD4+ and CD8+ T-cell responses unlesseparate antigens to detect CD4+ and CD8+ responses or lympho-yte subset depletion are used. Overlapping 15-mer peptide poolspanning entire target proteins represent a good compromise fortimulating both CD8+ and CD4+ T-cell responses in a number ofpplications [12–14].

. Objectives

We analyzed HCMV-specific CD4+ and CD8+ T-cell responses inealthy subjects and kidney transplant recipients (KTR) by IFN-�LISPOT using HCMV overlapping 15-mer peptide pools, as well ashort peptides, and results normalized to T-cell subset counts. Theerformance of the normalized ELISPOT was compared with the DCssay.

. Study design

.1. Healthy subjects and immunocompromised patients

Twenty-three healthy subjects (median age 39 [range 25–67]ears, 8 males/15 females) volunteered to give blood samples. Sub-

ects (17 HCMV seropositive and six HCMV seronegative) wereecruited among laboratory personnel and blood bank donors.lood samples from 23 KTR (transplanted between October 2011nd September 2013) were analyzed. The demographic, clinical and

r, everolimus.

virological characteristics of the KTR are presented in Table 1. Noneof the KTR developed HCMV disease.

3.2. Peripheral blood samples

Blood was collected in EDTA (BD Vacutainer, Plymouth, UK) forviral genome quantification and determination of T-cell subsets.Blood was also collected in heparin (BD Vacutainer) for isolationof peripheral blood mononuclear cells (PBMC) by standard den-sity gradient centrifugation. Ten million PBMC were resuspendedin culture medium [RPMI 1640 medium supplemented with 2 mMl-glutamine, 100 U/ml penicillin, 100 �g/ml streptomycin and 10%heat-inactivated fetal calf serum (Euroclone, Milan, Italy)] and usedfor the DC assay, while the remaining PBMC were cryopreserved forthe ELISPOT assay.

3.3. HCMV DNA

HCMV DNA levels were quantified in fresh whole blood by real-time PCR (lower limit of detection 50 HCMV DNA copies/ml blood)as described [15].

3.4. T-cell subsets

Fresh whole blood was stained with anti-CD3-PC5, anti-CD45-FITC, anti-CD4-RD1 and anti-CD8-ECD monoclonal antibodies(Beckman Coulter, Milan, Italy). After lysis of red blood cells, CD3+,CD3+CD4+ and CD3+CD8+ T-cell counts were determined by flowcytometry (Navios, Beckman Coulter) using Flow-Count Fluoro-spheres (Beckman Coulter).

3.5. Synthetic peptides

Peptide pools, 15 aa in length with an 11 aa overlap, span-ning full-length IE-1 (120 peptides), IE-2 (143 peptides) and pp65(138 peptides) HCMV proteins (JPT Peptide Technologies, Berlin,

f Clinical Virology 61 (2014) 65–73 67

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Fig. 1. HCMV-specific IFN-� ELISPOT responses. PBMC from HCMV-seropositivehealthy subjects (healthy HCMV+, n = 17), HCMV-seropositive kidney transplantrecipients (KTR HCMV+, n = 20) and nine HCMV-seronegative controls (controlsHCMV-, n = 6 healthy subjects and n = 3 KTR) were evaluated by ELISPOT in responseto peptide pools (15 amino acids in length with an 11 amino acid overlap) repre-senting full length IE-1 (A), IE-2 (B) and pp65 (C) HCMV proteins. ELISPOT responsesto IE-1, IE-2 and pp65 were summed to calculate the response to 15-mer peptide

S.A. Calarota et al. / Journal o

ermany) were used at a final concentration of 0.25 �g/ml for eachndividual peptide in the corresponding pool. An HCMV peptideool containing 34 peptides (JPT Peptide Technologies), 8–12 aa in

ength, each corresponding to a defined HLA class I-restricted T-cellpitope from HCMV [16], denoted as the HCMV peptide pool (class), was used at a final concentration of 1 �g/ml for each individualeptide.

.6. ELISPOT assay

Human IFN-� ELISPOT kits (Diaclone, Cedex, France) andultiScreen-IP 96-well plates (Merck Millipore, Darmstadt,ermany) were used as described [17]. PBMC (1 × 105 cells/well)ere stimulated (in duplicate) with the corresponding HCMVeptide pool, or phytohaemagglutinin (5 �g/ml; positive con-rol; Sigma–Aldrich, St. Louis, MO, USA), or culture medium onlynegative control). Spots were counted using an automated AIDLISPOT reader system (AutoImmun Diagnostika GmbH, Strass-erg, Germany). The mean number of spots from duplicate wellsas adjusted to 1 × 106 PBMC (the number of PBMC used in co-

ulture with DC in the DC assay; see Section 3.8). Results wereresented as net spots/million PBMC calculated by subtracting theumber of spots in wells with culture medium only from the num-er of spots in wells from each HCMV peptide pool (referred tos the net ELISPOT response). ELISPOT results were also normal-zed to T-cell counts as follows: (net number of spots/1 × 106) × theorresponding T-cell count (referred to as the normalized ELISPOTesponse).

.7. Depletion of CD8+, CD4+ or CD56+ cells

ELISPOT was also performed following CD8+, CD4+ or CD56+

ell depletion from PBMC by using CD8, CD4 or CD56 MicroBeadsMiltenyi Biotec, Bergisch Gladbach, Germany) according to manu-acturer’s instructions. Magnetic separations were performed using

S columns (Miltenyi Biotec). The depleted fractions contained1% of target cells, as determined by flow cytometry.

.8. DC assay

HCMV-specific CD4+ and CD8+ T cells were measured usingutologous, monocyte-derived HCMV-infected immature DC asescribed [6–8]. Briefly, immature DC were infected with anndotheliotropic and leukotropic HCMV strain VR1814 (at a mul-iplicity of infection of 10) or mock-infected. HCMV-infected or

ock-infected DC (5 × 104) were then co-cultured with autolo-ous PBMC (1 × 106) in the presence of brefeldin A (Sigma–Aldrich).BMC were tested for ICS by flow cytometry. The percentagef HCMV-specific CD4+ and CD8+ T cells producing IFN-� wasalculated by subtracting the value of PBMC incubated with mock-nfected DC from the value of PBMC incubated with HCMV-infectedC. The number of HCMV-specific T cells was determined by mul-

iplying the percentages of HCMV-specific T cells positive for IFN-�y the corresponding T-cell count.

.9. Data analysis

Analyses were performed using GraphPad Prism 5 (GraphPad

oftware, CA, USA). Differences between medians were determinedy using the Mann–Whitney U test for unpaired data and thepearman’s test was used for correlation analysis. All tests werewo-tailed. A p value <0.05 was considered statistically significant.

pools (D). PBMC were also evaluated by ELISPOT in response to an HCMV peptidepool (class I; 8–12 amino acids in length, representing defined HCMV-derived HLAclass I-restricted T-cell epitopes) (E). Differences between medians were determinedusing the Mann–Whitney U test.

6 f Clinical Virology 61 (2014) 65–73

4

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Fig. 2. HCMV-specific IFN-� ELISPOT responses after in vitro lymphocyte subsetdepletion. Peripheral blood mononuclear cells (PBMC), CD8-depleted PBMC, CD4-depleted PBMC and CD56-depleted PBMC from five HCMV-seropositive healthysubjects were analyzed by ELISPOT in response to peptide pools (15 amino acids

8 S.A. Calarota et al. / Journal o

. Results

.1. T-cell counts and HCMV DNAemia

The median CD3+, CD4+ and CD8+ T-cell counts in healthy sub-ects was 1597 (interquartile range [IQR]: 1238–2403), 921 (IQR:38–1397) and 556 (IQR: 387–891) cells/�l, respectively, while

n KTR it was 876 (IQR: 615–1299, p < 0.001), 477 (IQR: 260–647,< 0.001) and 393 (IQR: 262–619, p = 0.018), cells/�l, respectively.CMV DNA was not detected in any of the 23 healthy subjects,hile it was detected in nine HCMV-seropositive KTR (Table 1).

.2. HCMV-specific ELISPOT responses

The median responses to IE-1 (p = 0.027), IE-2 (p = 0.493),p65 (p = 0.113), the three overlapping 15-mer peptide poolsp = 0.013) and the HCMV peptide pool (class I) (p = 0.067)ere higher in HCMV-seropositive KTR compared to HCMV-

eropositive healthy subjects (Fig. 1A–E). The overall medianLISPOT response detected in nine HCMV-seronegative con-rols (6 healthy subjects and 3 KTR) was 5 (IQR: 0–20) netpots/million PBMC. Both HCMV-seropositive healthy subjects andTR showed significantly higher levels of ELISPOT responses com-ared to HCMV-seronegative controls (p ≤ 0.002, Fig. 1A–E). Theedian and IQR responses are presented in Supplemental Table

.

.3. ELISPOT responses after in vitro lymphocyte subset depletion

CD8+ or CD4+ T-cell depletion decreased the median IE-1esponse by 55% and 38%, respectively, while a 26% decrease wasbserved after CD56+ (natural killer [NK]) cell depletion (Fig. 2A).owever, the median IE-2 response decreased after CD8+ or CD4+

-cell depletion by 27% and 54%, respectively, while a maximalecrease (75%) was observed after depletion of CD56+ cells (Fig. 2B).D8+ or CD4+ T-cell depletion decreased the median pp65 responsey 64% and 40%, respectively, while a 4% decrease was observedfter depletion of CD56+ cells (Fig. 2C). CD8+ T-cell depletioneduced by 85% the median HCMV peptide pool (class I) response,hile depletion of CD4+ or CD56+ cells did not affect the response

Fig. 2D).

.4. HCMV-specific DC-assay responses

The median CD4+ T-cell response tended to be higher in HCMV-eropositive healthy subjects compared to HMCV-seropositive KTRp = 0.057, Fig. 3A). The median CD8+ T-cell response detected inCMV-seropositive healthy subjects was 13.3 T cells/�l, while inCMV-seropositive KTR it was 19.57 T cells/�l (p = 0.796, Fig. 3B).he median CD4+ and CD8+ T-cell responses detected in nineCMV-seronegative controls were 0.12 (IQR: 0–0.31) and 0.04

IQR: 0.03–0.13) T cells/�l, respectively. Compared to HCMV-eronegative controls, both HCMV-seropositive healthy subjectsnd KTR showed significantly higher levels of CD4+ and CD8+ T-cellesponses (p < 0.001, Fig. 3A and B). The median and IQR responsesre presented in Supplemental Table 1. Fig. 3C–F presents repre-entative FACS plots.

.5. ELISPOT responses to 15-mer peptides vs. DC-assay responses

In HCMV-seropositive healthy subjects, the combined IE-1, IE-, and pp65 net ELISPOT responses significantly correlated with:

D4+ (p = 0.028) as well as CD8+ (p < 0.001) DC-assay responsesFig. 4A), the sum of CD4+ plus CD8+ DC-assay responses (p < 0.001,ig. 4B), and the CD3+ DC-assay response (p = 0.003, Fig. 4C). Inddition, the combined IE-1, IE-2, and pp65 normalized ELISPOT

in length with an 11 amino acid overlap) representing full length IE-1 (A), IE-2 (B)and pp65 (C) HCMV proteins and an HCMV peptide pool (class I; 8–12 amino acids inlength, representing defined HCMV-derived HLA class I-restricted T-cell epitopes)(D).

S.A. Calarota et al. / Journal of Clinical Virology 61 (2014) 65–73 69

Fig. 3. HCMV-specific DC-assay responses. PBMC from HCMV-seropositive healthy subjects (healthy HCMV+, n = 17), HCMV-seropositive kidney transplant recipients (KTRHCMV+, n = 20) and nine HCMV-seronegative controls (controls HCMV-, n = 6 healthy subjects and n = 3 KTR) were evaluated by the DC assay. Results are expressed as absolutecounts of CD4+ (A) and CD8+ (B) T cells specific for HCMV-infected DC. Differences between medians were determined using the Mann–Whitney U test. Representative FACSplots of CD4+ and CD8+ IFN-�+ T cells after stimulation with mock-infected DC or HCMV-infected DC are shown in a healthy HCMV+ (C), a KTR HCMV+ (D), a healthy HCMV−(E) and a KTR HCMV− (F). Cells were first gated on CD3+ T cells and then the percentage of IFN-�-producing CD4+ T cells or IFN-�-producing CD8+ T cells was obtained.

70 S.A. Calarota et al. / Journal of Clinical Virology 61 (2014) 65–73

Fig. 4. Comparison of IFN-� ELISPOT responses to 15-mer peptides with DC-assay responses. Comparisons were made between ELISPOT responses to 15-mer peptide pools(IE-1, IE-2 and pp65 HCMV proteins) and DC-assay T-cell responses in 17 HCMV-seropositive healthy subjects (healthy HCMV+) (A–F) and in 20 HCMV-seropositive kidneyt ots/m( or CDT ach g

rCroH

ransplant recipients (KTR HCMV+) (G–L). ELISPOT responses are expressed as net spE and K) or CD3+ (F and L) T-cell count. DC-assay responses are expressed as CD4+

-cell responses. Correlation coefficient and p value (Spearman’s test) are given in e

esponses significantly correlated with: CD4+ (p = 0.006, Fig. 4D),

D8+ (p = 0.004, Fig. 4E), and CD3+ (p < 0.001, Fig. 4F) DC-assayesponses, respectively. In contrast, no significant correlations werebserved between net ELISPOT responses and the DC assay inCMV-seropositive KTR (Fig. 4G–I and Supplemental Table 2).

illion PBMC (A–C and G–I) or normalized to the corresponding CD4+ (D and J), CD8+

8+ T-cell responses, the summed CD4+ and CD8+ T-cell responses as well as CD3+

raph.

However, the combined IE-1, IE-2, and pp65 normalized ELISPOT

responses correlated with: CD4+ (p = 0.013, Fig. 4J), CD8+ (p = 0.065,Fig. 4K), and CD3+ (p = 0.048, Fig. 4L) DC-assay responses, respec-tively. The coefficients and p values are presented in SupplementalTable 3.

S.A. Calarota et al. / Journal of Clinical Virology 61 (2014) 65–73 71

Fig. 5. Comparison of IFN-� ELISPOT response to HCMV peptide pool (class I) and CD8+ DC-assay response. The ELISPOT response to an HCMV peptide pool (class I; 8–12a cell eph transs relatio

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mino acids in length, representing defined HCMV-derived HLA class I-restricted T-ealthy subjects (healthy HCMV+) (A and B) and in 20 HCMV-seropositive kidneypots/million PBMC (A and C) or normalized to the CD8+ T-cell count (B and D). Cor

.6. ELISPOT response to HCMV peptide pool (class I) vs. CD8+

C-assay response

In HCMV-seropositive healthy individuals, the HCMV peptideool (class I) ELISPOT response, expressed as either net or nor-alized response, significantly correlated with the CD8+ DC-assay

esponse (p = 0.007 and p = 0.001, respectively, Fig. 5A and B). Sim-larly, in HCMV-seropositive KTR, the CD8+ DC-assay responseorrelated with the HCMV peptide pool (class I) ELISPOT responsexpressed as either net (p = 0.026, Fig. 5C) or normalized (p = 0.008,ig. 5D) response.

. Discussion

In HCMV-seropositive healthy subjects ELISPOT responseseither net or normalized) to 15-mer peptide pools (mainly to IE-and pp65) and HCMV peptide pool (class I) correlated with theC-assay response. In HCMV-seropositive KTR, HCMV peptide pool

class I) ELISPOT data (either net or normalized response) correlatedith the CD8+ DC-assay response but only normalized ELISPOT

esponses to 15-mer peptide pools (mainly to pp65 and IE-1) cor-elated with the DC-assay response. Our findings in KTR furtherupport the importance of post-transplant T-lymphocyte subsetonitoring [18].Although pp65 and IE-1 proteins have been recognized as the

ajor targets of the T-cell response to HCMV [19–23], other HCMVroteins are recognized by T cells [24,25]. In this study, we alsoocused on IE-2, because together with IE-1 is an important indi-ator of viral reactivation [26]. Early studies have shown that

ealthy subjects and SOT recipients respond to IE-2 protein, as mea-ured with the T-cell proliferation assay [27,28]. We found that theedian ELISPOT response to pp65 was the highest followed by IE-1

nd the lowest was IE-2, that ELISPOT responses to pp65 and IE-1

itopes) was compared with the CD8+ DC-assay response in 17 HCMV-seropositiveplant recipients (KTR HCMV+) (C and D). ELISPOT responses are expressed as netn coefficient and p value (Spearman’s test) are given in each graph.

were mediated mainly by CD8+ T cells and, to a lesser extent, CD4+ Tcells. However, IE-2 peptides appear to stimulate IFN-� secretion byNK cells more efficiently than IE-1 and pp65 peptides and this find-ing may partially explain why we did not observe any correlationbetween the IE-2 ELISPOT response and DC assay. For the evaluationof HCMV CD8+ T-cell responses, we used a pool of 34 peptides rep-resenting a broad range of HCMV epitopes with a wide range of HLAclass I specificities [16,25]. In our KTR cohort, all patients displayedat least one HLA class I variant included among the specificitiescovered by the 34 peptides.

HCMV T-cell responses measured with the DC assay have beencompared with short-term (24 h) assays using ICS in SOT recipientsand showed overall a good correlation [29,30]. In the present study,we compared the ELISPOT with the DC assay. The assays are not100% equivalent, as they employ different stimulating and detec-tion procedures. Moreover, the ELISPOT quantifies IFN-� secretedby the cell, while the DC assay measures IFN-� blocked within thecell; ELISPOT results include all IFN-�-producing cells within thePBMC sample, while in the DC assay, IFN-� produced only by CD4+

and CD8+ T cells is quantified. However, our results indicate thatthere is a good correlation between the normalized ELISPOT andthe DC assay in KTR. The DC assay requires a high volume of blood(needed for the generation of autologous DC) compared to ELISPOTand longer turnaround time (7 days vs. 2 days). In addition, com-pared to the DC assay, the ELISPOT is relatively easy to perform andbetter suited to analyses with frozen samples. Thus, the high sensi-tivity and specificity of the normalized ELISPOT assay, the 10-foldlower volume of blood required as well as the faster turnaroundtime are added benefits in the clinical setting.

HCMV-seropositive healthy individuals devote about 10% oftheir circulating memory T-cell compartment to HCMV antigens[24] and these cells are mostly effector memory T cells [31]. Inour cohort of HCMV-seropositive KTR, ELISPOT responses were

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2 S.A. Calarota et al. / Journal o

igher than in HCMV-seropositive healthy subjects. It is possi-le that immunosuppressive treatment leads to repetitive antigenxposure in KTR that, despite suppression, can drive expansionf HCMV-specific T cells resulting in a high frequency of HCMV-pecific T-cell responses.

Studies have emphasized the role of CD8+ and CD4+ T cells inontrolling HCMV infection after transplantation. While HCMV-pecific CD8+ T cells are important during the early period followingOT, HCMV-specific CD4+ T cells seem to play a major role in long-erm control of viral replication [5,7,8,32–36]. Our study showedhat the normalized ELISPOT can be efficiently used to detect bothD4+ and CD8+ T-cell responses. Thus, it can be used to assesshe CD4/CD8 T-cell response balance after transplantation. Theesults presented here have some limitations since they are cross-ectional, and the small number of KTR studied, as well as, the timerom transplantation to blood sample collection was different forach patient. Consequently, a robust relationship of the detectedCMV T-cell responses with HCMV DNAemia cannot be demon-

trated.In conclusion, the normalized ELISPOT represents a novel and

imple approach for quantification and monitoring of HCMV CD4+

nd CD8+ T-cell responses in KTR. An ongoing longitudinal studyill determine whether this approach may help to identify patients

t risk for viral complications after transplantation.

uthor contributions

Sandra A. Calarota performed laboratory testing, data analysisnd writing of the paper. Antonella Chiesa performed laboratoryesting and data collection. Kodjo M.G. Adzasehoun and Giudittaomolli performed laboratory testing. Lucia Scaramuzzi, Filippoangione and Pasquale Esposito performed patient recruitment,

ample and clinical data collection and writing of the paper. Faustoaldanti participated in the study design, supervised experimentalctivity, contributed to the writing of the paper and raised funds.ll authors have approved the final manuscript.

unding

This work was supported by the Ministero della Salute, Fon-azione IRCCS Policlinico San Matteo Ricerca Corrente grant 80207.

ompeting interests

None declared.

thical approval

The study was approved by the Fondazione IRCCS Policlinico Sanatteo Ethical Committee (protocol number P-20110032770) andritten informed consent was obtained from all participants.

cknowledgements

We thank Daniela Sartori for careful preparation of theanuscript and Laurene Kelly for revision of the English.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at http://dx.doi.org/10.1016/j.jcv.2014.05.017.

eferences

[1] Crough T, Khanna R. Immunobiology of human cytomegalovirus: from benchto bedside. Clin Microbiol Rev 2009;22(1):76–98.

[

al Virology 61 (2014) 65–73

[2] Fishman JA. Infection in solid-organ transplant recipients. N Engl J Med2007;357(25):2601–14.

[3] Humar A, Snydman D. AST Infectious Diseases Community of PracticeCytomegalovirus in solid organ transplant recipients. Am J Transplant2009;9(Suppl. 4):S78–86.

[4] Kotton CN. CMV: prevention, diagnosis and therapy. Am J Transplant2013;13(Suppl. 3):24–40.

[5] Egli A, Humar A, Kumar D. State-of-the-art monitoring of cytomegalovirus-specific cell-mediated immunity after organ transplant: a primer for theclinician. Clin Infect Dis 2012;55(12):1678–89.

[6] Lozza L, Lilleri D, Percivalle E, Fornara C, Comolli G, Revello MG, et al. Simul-taneous quantification of human cytomegalovirus (HCMV)-specific CD4+ andCD8+ T cells by a novel method using monocyte-derived HCMV-infected imma-ture dendritic cells. Eur J Immunol 2005;35(6):1795–804.

[7] Gerna G, Lilleri D, Fornara C, Comolli G, Lozza L, Campana C, et al. Monitoringof human cytomegalovirus-specific CD4 and CD8 T-cell immunity in patientsreceiving solid organ transplantation. Am J Transplant 2006;6(10):2356–64.

[8] Gerna G, Lilleri D, Chiesa A, Zelini P, Furione M, Comolli G, et al. Virologic andimmunologic monitoring of cytomegalovirus to guide preemptive therapy insolid-organ transplantation. Am J Transplant 2011;11(11):2463–71.

[9] Godard B, Gazagne A, Gey A, Baptiste M, Vingert B, Pegaz-Fiornet B, et al.Optimization of an elispot assay to detect cytomegalovirus-specific CD8+ Tlymphocytes. Hum Immunol 2004;65(11):1307–18.

10] Nickel P, Bold G, Presber F, Biti D, Babel N, Kreutzer S, et al. High levels ofCMV-IE-1-specific memory T cells are associated with less alloimmunity andimproved renal allograft function. Transpl Immunol 2009;20(4):238–42.

11] Abate D, Saldan A, Fiscon M, Cofano S, Paciolla A, Furian L, et al. Evalua-tion of cytomegalovirus (CMV)-specific T cell immune reconstitution revealedthat baseline antiviral immunity, prophylaxis, or preemptive therapy but notantithymocyte globulin treatment contribute to CMV-specific T cell reconsti-tution in kidney transplant recipients. J Infect Dis 2010;202(4):585–94.

12] Kiecker F, Streitz M, Ay B, Cherepnev G, Volk HD, Volkmer-Engert R, et al. Anal-ysis of antigen-specific T-cell responses with synthetic peptides – what kind ofpeptide for which purpose? Hum Immunol 2004;65(5):523–36.

13] Draenert R, Altfeld M, Brander C, Basgoz N, Corcoran C, Wurcel AG, et al. Com-parison of overlapping peptide sets for detection of antiviral CD8 and CD4 Tcell responses. J Immunol Methods 2003;275(1-2):19–29.

14] Maecker HT, Dunn HS, Suni MA, Khatamzas E, Pitcher CJ, Bunde T, et al. Useof overlapping peptide mixtures as antigens for cytokine flow cytometry. JImmunol Methods 2001;255(1–2):27–40.

15] Gerna G, Baldanti F, Torsellini M, Minoli L, Viganò M, Oggionnis T, et al. Eval-uation of cytomegalovirus DNAaemia versus pp65-antigenaemia cutoff forguiding preemptive therapy in transplant recipients: a randomized study.Antivir Ther 2007;12(1):63–72.

16] Walker S, Fazou C, Crough T, Holdsworth R, Kiely P, Veale M, et al. Ex vivomonitoring of human cytomegalovirus-specific CD8+ T-cell responses usingQuantiFERON-CMV. Transpl Infect Dis 2007;9(2):165–70.

17] Calarota SA, Chiesa A, Zelini P, Comolli G, Minoli L, Baldanti F. Detection ofEpstein–Barr virus-specific memory CD4+ T cells using a peptide-based cul-tured enzyme-linked immunospot assay. Immunology 2013;139(4):533–44.

18] Calarota SA, Zelini P, De Silvestri A, Chiesa A, Comolli G, Sarchi E, et al. Kineticsof T-lymphocyte subsets and posttransplant opportunistic infections in heartand kidney transplant recipients. Transplantation 2012;93(1):112–9.

19] Wills MR, Carmichael AJ, Mynard K, Jin X, Weekes MP, Plachter B, et al. Thehuman cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is domi-nated by structural protein pp65: frequency, specificity, and T-cell receptorusage of pp65-specific CTL. J Virol 1996;70(11):7569–79.

20] Kern F, Surel IP, Faulhaber N, Frömmel C, Schneider-Mergener J, Schöne-mann C, et al. Target structures of the CD8(+)-T-cell response to humancytomegalovirus: the 72-kilodalton major immediate-early protein revisited. JVirol 1999;73(10):8179–84.

21] McLaughlin-Taylor E, Pande H, Forman SJ, Tanamachi B, Li CR, Zaia JA, et al.Identification of the major late human cytomegalovirus matrix protein pp65as a target antigen for CD8+ virus-specific cytotoxic T lymphocytes. J Med Virol1994;43(1):103–10.

22] Kern F, Faulhaber N, Frömmel C, Khatamzas E, Prösch S, Schönemann C,et al. Analysis of CD8 T cell reactivity to cytomegalovirus using protein-spanning pools of overlapping pentadecapeptides. Eur J Immunol 2000;30(6):1676–82.

23] Slezak SL, Bettinotti M, Selleri S, Adams S, Marincola FM, Stroncek DF. CMV pp65and IE-1 T cell epitopes recognized by healthy subjects. J Transl Med 2007;5:17.

24] Sylwester AW, Mitchell BL, Edgar JB, Taormina C, Pelte C, Ruchti F, et al. Broadlytargeted human cytomegalovirus-specific CD4+ and CD8+ T cells dominate thememory compartments of exposed subjects. J Exp Med 2005;202(5):673–85.

25] Elkington R, Walker S, Crough T, Menzies M, Tellam J, Bharadwaj M, et al. Ex vivoprofiling of CD8+-T-cell responses to human cytomegalovirus reveals broad andmultispecific reactivities in healthy virus carriers. J Virol 2003;77(9):5226–40.

26] Sinclair J, Sissons P. Latency and reactivation of human cytomegalovirus. J GenVirol 2006;87(Pt 7):1763–79.

27] van Zanten J, Harmsen MC, van der Meer P, van der Bij W, van Son WJ, van derGiessen M, et al. Proliferative T cell responses to four human cytomegalovirus-

specific proteins in healthy subjects and solid organ transplant recipients. JInfect Dis 1995;172(3):879–82.

28] He H, Rinaldo Jr CR, Morel PA. T cell proliferative responses to five humancytomegalovirus proteins in healthy seropositive individuals: implications forvaccine development. J Gen Virol 1995;76(Pt 7):1603–10.

f Clinic

[

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[

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[

S.A. Calarota et al. / Journal o

29] Lilleri D, Zelini P, Fornara C, Comolli G, Gerna G. Inconsistent responses ofcytomegalovirus-specific T cells to pp65 and IE-1 versus infected dendritic cellsin organ transplant recipients. Am J Transplant 2007;7(8):1997–2005.

30] Zelini P, Lilleri D, Comolli G, Rognoni V, Chiesa A, Fornara C, et al. Humancytomegalovirus-specific CD4(+) and CD8(+) T-cell response determination:comparison of short-term (24 h) assays vs long-term (7-day) infected dendriticcell assay in the immunocompetent and the immunocompromised host. ClinImmunol 2010;136(2):269–81.

31] Torti N, Oxenius A. T cell memory in the context of persistent herpes viralinfections. Viruses 2012;4(7):1116–43.

32] Mattes FM, Vargas A, Kopycinski J, Hainsworth EG, Sweny P, Nebbia G, et al.Functional impairment of cytomegalovirus specific CD8 T cells predicts high-level replication after renal transplantation. Am J Transplant 2008;8(5):990–9.

[

al Virology 61 (2014) 65–73 73

33] Bunde T, Kirchner A, Hoffmeister B, Habedank D, Hetzer R, Cherepnev G,et al. Protection from cytomegalovirus after transplantation is correlated withimmediate early 1-specific CD8 T cells. J Exp Med 2005;201(7):1031–6.

34] Sester M, Sester U, Gärtner B, Heine G, Girndt M, Mueller-Lantzsch N, et al.Levels of virus-specific CD4 T cells correlate with cytomegalovirus controland predict virus-induced disease after renal transplantation. Transplantation2001;71(9):1287–94.

35] Egli A, Binet I, Binggeli S, Jäger C, Dumoulin A, Schaub S, et al. Cytomegalovirus-

specific T-cell responses and viral replication in kidney transplant recipients. JTransl Med 2008;6:29.

36] Sund F, Lidehäll AK, Claesson K, Foss A, Tötterman TH, Korsgren O, et al. CMV-specific T-cell immunity, viral load, and clinical outcome in seropositive renaltransplant recipients: a pilot study. Clin Transplant 2010;24(3):401–9.