A Simple-to-Perform ifn-γ mRNA Gene Expression Assay on Whole Blood Accurately Appraises Varicella Zoster Virus-Specific Cell-Mediated Immunity After Allogeneic Hematopoietic Stem Cell Transplantation

Abstract

Herpes zoster, which is due to the reactivation of Varicella zoster virus (VZV), is a leading cause of morbidity after allogeneic hematopoietic stem cell transplantation (allo-HSCT). While cell-mediated immunity (CMI) is critical to inhibiting VZV reactivation, CMI is not routinely assessed due to a lack of reliable tests. In this study, we aimed to evaluate VZV-specific CMI among allo-HSCT recipients (n = 60) and healthy individuals (HI, n = 17) through a panel of three immune functional assays after ex vivo stimulation by VZV antigen: quantification of (i) IFN-γ release in the supernatants, (ii) T-cell proliferation after a 7-day stimulation of peripheral blood mononuclear cells (PBMC), and (iii) measurement of the ifn-γ mRNA gene expression level after 24 h of stimulation of a whole-blood sample. VZV responsiveness was defined according to IFN-γ release from VZV-stimulated PBMC. Upon VZV stimulation, we found that allo-HSCT recipients at a median time of 6 [5-8] months post-transplant had lower IFN-γ release (median [IQR], 0.34 [0.12-8.56] vs. 409.5 [143.9-910.2] pg/ml, P <.0001) and fewer proliferating T cells (0.05 [0.01-0.57] % vs. 8.74 [3.12-15.05] %, P <.0001) than HI. A subset of allo-HSCT recipients (VZV-responders, n = 15/57, 26%) distinguished themselves from VZV-non-responders (n = 42/57, 74%; missing data, n = 3) by higher IFN-γ release (80.45 [54.3-312.8] vs. 0.22 [0.12-0.42] pg/ml, P <.0001) and T-cell proliferation (2.22 [1.18-7.56] % vs. 0.002 [0.001-0.11] %, P <.0001), suggesting recovery of VZV-specific CMI. Interestingly, VZV responders had a significant fold increase in ifn-γ gene expression, whereas ifn-γ mRNA was not detected in whole blood of VZV-non-responders (P <.0001). This study is the first to suggest that measurement of ifn-γ gene expression in 24-h-stimulated whole blood could be an accurate test of VZV-specific CMI. The routine use of this immune functional assay to guide antiviral prophylaxis at an individual level remains to be evaluated.

Keywords: antiviral prophylaxis; cell-mediated immunity; hematopoietic stem cell transplantation; immune functional assays; interferon gamma; varicella zoster virus.

Figure 1

VZV-specific cell-mediated immunity according to immunoassays on peripheral blood mononuclear cells. Peripheral blood mononuclear cells isolated (1 × 10⁵ cells) from healthy individuals (HI, n = 17) and allogeneic hematopoietic stem cell transplant recipients (allo-HSCT, n = 57) were stimulated by VZV antigen (inactivated supernatants from VZV-infected MRC-5 cells, 20 µg/ml) or supernatants from VZV-uninfected MRC-5 cells (control condition) and incubated for 7 days in duplicate. (A) IFN-γ release (pg/ml) in the supernatants was quantified using the ELLA nanofluidic system. Bars represent median values, black dotted line represents the lower limit of quantification (LLOQ = 0.17 pg/ml), and red dotted line represents the arbitrarily defined threshold of positivity of the assay, i.e., 4.83 pg/ml, corresponding to twice the maximum value observed in the control condition. All values below LLOQ have been set at 0.12 pg/ml corresponding to LLOQ/√2. (B) In respective wells, T-cell proliferation is expressed as the percentage of EdU⁺ proliferating cells (among CD3⁺ cells) measured by flow cytometry after EdU⁺ incorporation and completion of the Click-it^®. Groups were compared using Kruskal–Wallis test; ****P <.0001, NS: not significant. Red triangles represent recipients defined as VZV-responders, i.e., having recovered a VZV-specific cellular-mediated immunity according to the IFN-γ release assay. Clear triangles represent recipients who developed herpes zoster-related manifestations. (C) Correlation analysis between T-cell proliferation and IFN-γ release results obtained from the same well was performed using Spearman rank-correlation test; the P-values and the correlation coefficient are indicated. Data were missing for three VZV-stimulated allo-HSCT recipients.

Figure 2

Ex vivo whole blood VZV-stimulation and correlations between VZV-specific immune functional assays. Whole blood (100 µl) from healthy individuals (HI, n = 17) and allogeneic hematopoietic stem cell transplant recipients (allo-HSCT, n = 57) was stimulated 24 h by VZV antigen (inactivated supernatants from VZV-infected MRC-5 cells, 20 µg/ml) or supernatants from VZV-uninfected MRC-5 cells (control condition). (A) The relative quantification of ifn-γ gene expression levels is presented as fold change. Red triangles represent recipients defined as VZV responders, according to IFN-γ release in supernatants from VZV-stimulated peripheral blood mononuclear cells (see Figure 1 ). Clear triangles represent recipients who developed herpes zoster-related manifestations. Bars represent median and P-values were obtained using the Kruskal–Wallis test; ** P <.01, ****P <.0001. (B) Correlation between ifn-γ mRNA gene expression (fold change) and IFN-γ release in supernatants from VZV-stimulated PBMC quantified using the ELLA nanofluidic system, using Spearman rank-correlation test. (C) Correlation between ifn-γ mRNA gene expression (fold change) and percentage of EdU⁺ cells (among CD3⁺) measured by flow cytometry, using Spearman rank-correlation test. Data were missing for three VZV-stimulated recipients.