Lack of cytomegalovirus (CMV)-specific cell-mediated immune response using QuantiFERON-CMV assay in CMV-seropositive healthy volunteers: fact not artifact

The QuantiFERON-CMV (QF) assay measures cell-mediated immunity against cytomegalovirus (CMV-CMI), which is particularly useful in individuals susceptible to CMV infection such as transplant patients. A positive QF result identifies patients that are better protected against CMV infection. However, the significance of a negative QF result in CMV-seropositive individuals needs to be clarified. CMV-CMI was analyzed in healthy subjects using the QF assay, and, in parallel, the Flow-cytometric Assay of Specific Cell-mediated Immune response in Activated whole blood (FASCIA). FASCIA assay measures T-cell proliferation using CMV lysate as stimulus whereas QF assay use a mix of peptides. A total of 93 healthy volunteers were enrolled, and 13/71 CMV-seropositive individuals (18.3%) showed humoral/cellular discordance using QF assay (CMV+ QF−). Interestingly, with FASCIA assay CD4+ and CD8+ T-cell proliferations were lower in CMV+ QF− than in CMV+ QF+ individuals. Furthermore, CMV+ QF− volunteers had a lower level of anti-CMV IgG than CMV+ QF+ subjects. Discordant CMV+ QF− volunteers can be defined as low responder individuals since they show lower CMV-specific humoral and cellular immune responses in comparison to CMV+ QF+ individuals. Immune discordance shows the high heterogeneity of immunity to CMV in healthy subjects.

. Demographic characteristics of the study population. SD, standard deviation; CMV−, CMV− seronegative, CMV+; CMV−seropositive.  www.nature.com/scientificreports www.nature.com/scientificreports/ found that it had a good discriminatory power (AUC: 0.87, 95% CI 0.78-0.95; p < 0.001). The value with the highest sensibility (0.69) and specificity (0.92) was 3.2 IU/mL of anti-CMV IgG. The positive and negative predictive values for this cut-off were 0.97 and 0.40, respectively. The low negative predictive value indicated that only 40% of individuals with an anti-CMV IgG level lower than 3.2 IU/mL were CMV+ QF−. Therefore, a low specific IgG level was not a reliable indicator for identifying discordant individuals.
Combination of markers: IgG CMV level and proliferative response. We then analyzed the combination of IgG CMV level (IU/mL) and proliferation capacity within CMV+ QF+ and CMV+ QF− individuals. As it is shown in Fig. 4, all the CMV+ QF− subjects had a low IgG CMV level and low lymphocyte proliferation, whereas CMV+ QF+ individuals exhibited a high heterogeneity in both parameters. Specifically, discordant CMV+ QF− individuals had an anti-CMV IgG level lower than 4 IU/mL, CD4+ lower than 400 cells/µL and CD8+ lower than 20 cells/µL.
Other parameters. We also analyzed whether CMV+ QF+ and CMV+ QF− showed differences in other parameters, such as sex, age or HLA alleles. Discordant CMV+ QF− subjects showed a tendency to be older than CMV+ QF+ individuals (47.9 vs. 42.7 years old; p = 0.087). With respect to sex, the male and females were not distributed differently between the two groups, with the frequency of females being 31.0% (18/58) in CMV+ QF+ and 38.5% (5/13) in CMV+ QF− subjects (Chi-square test p = 0.744). HLA allele frequencies were not significantly different between CMV+ QF+ and CMV+ QF− individuals (data not shown).
Given that IFNG quantified by QF assay is released by CMV-specific CD8+ T cells, we analyzed this subpopulation in the discordant CMV− QF+ group. For this purpose, we used dextramer technology, which quantifies the number of CMV-specific CD8+ T cells. Since the 5 discordant CMV− QF+ individuals had the HLA-A*02 allele, we used the HLA-A*02 dextramer. As the reference group we selected 4 CMV+ QF+ HLA-A*02 subjects with a similar level of IFNG to CMV− QF+ individuals. In addition, one CMV+ QF+ subject with the HLA-A*01 allele and the lowest level of IFNG was also included in the reference group Strikingly, none of the discordant CMV− QF+ volunteers had a detectable CMV-specific CD8+ T-cell subpopulation, whereas all CMV+ QF+ individuals, even the one with an IFNG level of 0.6 IU/mL, showed this subset (Fig. 6).

Discussion
This study evaluates humoral/cellular CMV immunity discordance in CMV-seropositive healthy volunteers using, in parallel, two techniques that measure CMV-CMI in different ways (IFNG release vs. lymphocyte proliferation). The objective was to evaluate whether a negative QF result in CMV-seropositive individuals (CMV+ QF−) is an artifact of the QF assay, as suggested by some authors 15,17 , or it identifies individuals with immunological characteristics different from non-discordant individuals (CMV+ QF+). We found that, compared to the CMV+ QF+ individuals, the CMV+ QF− subjects are low-responder individuals since they have a reduced CMV-specific cellular response, showing a lower CD4+ and CD8+ T-cell proliferation as well as a lower anti-CMV IgG level. This indicates that a negative QF result in CMV-seropositive individuals is not associated with technical artifacts of the QF assay, but represents a well differentiated group.
When using the QF assay to measure CMV-specific T-cell response, we found that around 19% of healthy volunteers have humoral/cellular discordance (either CMV+ QF− or CMV− QF+). This is in line with findings previously reported by other authors in healthy subjects using other techniques to determine cellular immune response 10,18,19 . Discordance between serology and T-cell response has also been described in transplant patients, where it has shown to be related to the risk of CMV reactivation after transplantation 8,9,12,20 .
The difference in T-cell proliferation between CMV+ QF− and CMV+ QF+ individuals was more pronounced in CD4+ cells than in CD8+ T cells. In fact, unexpectedly, some CMV+ QF+ individuals had no CD8+ T cell proliferation with CMV lysate, which might be due to the fact that CMV lysate induces a weaker major histocompatibility class I-mediated response 19 . The discordant CMV+ QF− individuals show reduced specific immune response that is not only restricted to cellular immune response, but also to humoral immunity. Therefore, we can say that the discordance in CMV-seropositive individuals identifies a group of low responder subjects. One possible explanation for the dysfunctional response in CMV+ QF− individuals might be related to the relevant role of CD4+ T cells in the development of an appropriate antiviral immune response. It is well known that CD4+ T cells are necessary to help mount an effective specific memory CD8+ T cell response and are crucial to promote isotype switching and the production of high-affinity immunoglobulins IgG by B cells 21,22 . In this line, it has been recently published that CMV-specific CD4+ T cells and anti-CMV IgG are directly correlated 12 . Similar correlation have been reported in healthy individuals after vaccination against Hepatitis B virus 23 . Therefore, we can speculate that the impaired proliferation of CD4+ T cells with CMV lysate in CMV+ QF− subjects might influence isotype switching, thus producing a lower level of anti-CMV IgG and a lower number of CMV-specific CD8+ T cells.
In addition, discordant CMV+ QF− individuals show not only reduced specific immunity to CMV, but also against superantigen SEA + SEB. Both toxins bind in a non-specific manner to MHC class II on antigen presentation cells as well as to the T-cell antigen receptor of CD4+ T cells, producing a massive proliferation of T cells www.nature.com/scientificreports www.nature.com/scientificreports/ and release of proinflammatory cytokines 24,25 . Therefore, the significantly lower proliferation of CD4+ T cells with superantigen that we observed in CMV+ QF− individuals would support the role of CD4+ T cells in the discordance in CMV-seropositive individuals.
However, we also found some discordant individuals in the CMV-seronegative group since five individuals had a positive QF result, in spite of lacking IgG CMV. We cannot rule out the possibility of false negative anti-CMV IgG test results, as reported by Sester et al. 18 , since the serological analyses in CMV-individuals were not repeated. However, strikingly, none of them had CD4+ or CD8+ T-cell proliferation with CMV lysate or detectable CMV-specific CD8+ T-cell subpopulation with dextramer. Therefore, where does the IFNG quantified by the QF assay come from? According to Sester et al. 18 , one explanation might be related to an acute CMV infection in these CMV− QF+ subjects. However, the level of anti-CMV IgM was negative. Another option could be that cells other than CD8+ T cells (i.e., CMV-specific Th1 CD4+ T cells) can produce IFNG in the QF assay, since it has been reported that the peptide length distinction is not absolute and CD4+ T cells can be stimulated by antigen presented on HLA class I molecules 26 . Other alternative explanations could be related to the cross-reactivity with T cells specific for other herpes viruses or unrelated viruses 27,28 or even that the 24 h stimulation of the QF assay led to the in vitro priming of CD8+ T cells 19 .
Our study has some limitations. First, in spite of our results, we cannot rule out the possibility that the negative QF result in the CMV+ QF− individuals is due to the fact that these individuals recognize other CMV antigens not represented by the peptides included in QuantiFERON-CMV. However, this is unlikely since, according to the manufacturer of the QF assay, the HLA-restricted CMV peptides of the test cover more than 98% of the population. Second, CMV lysate stimulation may provide more reliable information regarding CD4+ cells than CD8+ T cells and might not be the most suitable antigen to compare the CD8+ T-cell response of the QF assay. However, the relevant point is the significantly lower CD4+ T-cell proliferation we found in CMV+ QF− than in CMV+ QF+ individuals. Third, CMV− QF+ individuals might represent some false negative IgG test results due to the low sensitivity of some commercial IgG tests. However, in spite of this limitation, determination of serological status is the gold standard in clinical routine to classify solid organ and stem cell transplant patients according to the risk of CMV infection 3,4 . www.nature.com/scientificreports www.nature.com/scientificreports/ In summary, healthy CMV+ QF− volunteers show a lower CMV-specific immunity in comparison to CMV+ QF+ individuals, either in humoral or cellular specific immunity. This discordance shows the high heterogeneity of immune response to CMV in healthy subjects. This natural heterogeneity might have clinical consequences if immunocompromised transplant patients and hematopoietic stem cell donors present this immune discordance, for whom these immunological techniques provide useful information. In this regard, interventional studies and clinical trials have demonstrated the utility of the QF assay in transplant patients, where it helps to personalize therapy against CMV after transplantation 29,30 . In addition, this study also demonstrates that a negative QF result in CMV-seropositive individuals is not an artifact but a reality.

Materials and Methods
Donor characteristics and study design. This cross-sectional study was carried out with healthy volunteers from the Blood Transfusion Center and Tissue and Cells Establishment of Cordoba. The participants were recruited from October 2017 to June 2019. A single blood sample was taken from each donor and CMVserostatus and CMV-CMI response (using the QF and FASCIA assays) was determined. Informed consent was obtained from each of the subjects and the Ethics Committee of the Reina Sofia Hospital approved the study. This study was conducted in accordance with the Declaration of Helsinki.

Determination of anti-CMV IgG and IgM antibodies.
Anti-CMV IgG antibodies were determined by two commercially available methods: a semiquantitative assay (expressed as arbitrary units; AU/mL) and a quantitative assay (expressed as international units; IU/mL). In the semiquantitative assay, antibodies were analyzed by chemoluminescence at the Microbiology Unit of the Reina Sofia Hospital (Diasorin SA, Madrid, Spain). The samples with an anti-CMV IgG concentration lower than 12.0 AU/mL were considered CMV-seronegative. In the quantitative method (DIAsource Immunoassays SA, Louvain-la-Neuve, Belgium), a calibration curve and the World Health Organization international standard was used. This analysis was carried out in our lab at the IMIBIC research center.
In discordant volunteers in the CMV-seronegative group, a semiquantitative determination of anti-CMV IgM was also performed. QuantiFERON-CMV assay. The QuantiFERON-CMV (QF) assay was performed according to the manufacturer's instructions (Cellestis, a QIAGEN company, Melbourne, Australia). In brief, 1 mL of heparinized whole blood was collected in 3 QF blood collection tubes. The tubes contained either (i) a mix of 22 CMV peptides; (ii) a negative control (no antigens); or (iii) a positive mitogen control (containing phytohemagglutinin) 31 . All individuals included in the study had HLA class I alleles capable of binding CMV peptides. After collection, the tubes were shaken vigorously and incubated for 16-24 h at 37 °C. Supernatants were subsequently harvested and analyzed for IFNG (IU/mL) by standard ELISA. A result for the CMV antigen was "Positive" when the CMV antigen response minus the negative control response was higher than 0.2 IU/mL of IFNG. According to the manufacturer's instructions, a result was "Indeterminate" when the IFNG level in the CMV antigen tube minus the negative control was less than 0.2 IU/mL and the IFNG level in the mitogen tube (once the negative control was subtracted) was less than 0.5 IU/mL. FASCIA assay. The FASCIA assay was performed as previously reported 32 . In the assay, whole blood was diluted at 1:9 in RPMI supplemented with L-glutamine, penicillin and streptomycin. The blood/medium mixture was stimulated with CMV viral lysate (5 μg/mL) (Microbix Biosystems Inc, Canada), Pokeweed mitogen (PWM) (5 μg/mL) (Sigma-Aldrich, USA), Staphylococcus aureus enterotoxins A and B (SEA + SEB) (0.1 μg/mL of each) (Sigma-Aldrich, USA) or RPMI (unstimulated control) in sterile polypropylene Falcon 12 ×75 mm tubes to a final volume of 500 μL. Pokeweed mitogen (PWM) is a lectin from Phytolacca americana that promotes polyclonal differentiation of B cells. The staphylococcal enterotoxins A and B are considered superantigens that stimulate large populations of T cells. The tubes were incubated for 7 days at 37 °C, 5% CO 2 and 95% humidity. After this time, the supernatant was collected and stored at −80 °C. The cells were subsequently stained with anti-CD3 AF700 (BD Biosciences, USA) and anti-CD8 APC (Miltenyi Biotec, Germany). In addition, the medium tube and PWM tube were stained with anti-CD19 Viogreen (Miltenyi Biotec, Germany). All the tubes were incubated for 10 minutes in the dark at room temperature. The erythrocytes were then lysed with Test lysis buffer (Beckman Coulter, USA) and the samples were centrifuged at 1800 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in 450 μL of phosphate buffered saline with 1% of bovine serum albumin (PBSA). Blast numbers were acquired for 80 seconds in an LSR Fortessa flow cytometer (Beckman Coulter, CA, USA).
The absolute number of proliferating cells was calculated using a Trucount tube (BD Biosciences, CA, USA), as previously reported 32 . The values of the unstimulated samples (with RPMI) were subtracted from the values of the stimulated samples.
Statistical analysis. The statistical analysis was performed using IBM SPSS Statistics 24.0 software (SPSS Inc., Chicago IL, USA). Categorical variables were compared using the Chi-square or Fisher tests. Comparison of quantitative variables was assessed using the Mann-Whitney U test. Values were considered statistically significant when the p-value was <0.05. Graphic presentation was performed with GraphPad Prism 7 (GraphPad Sofware Inc).

Data availability
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.