Whole blood assessment of antigen specific cellular immune response by real time quantitative PCR: a versatile monitoring and discovery tool

Abstract

Background: Monitoring of cellular immune responses is indispensable in a number of clinical research areas, including microbiology, virology, oncology and autoimmunity. Purification and culture of peripheral blood mononuclear cells and rapid access to specialized equipment are usually required. We developed a whole blood (WB) technique monitoring antigen specific cellular immune response in vaccinated or naturally sensitized individuals.

Methods: WB (300 microl) was incubated at 37 degrees C with specific antigens, in the form of peptides or commercial vaccines for 5-16 hours. Following RNAlater addition to stabilize RNA, the mixture could be stored over one week at room temperature or at 4 degrees C. Total RNA was then extracted, reverse transcribed and amplified in quantitative real-time PCR (qRT-PCR) assays with primers and probes specific for cytokine and/or chemokine genes.

Results: Spiking experiments demonstrated that this technique could detect antigen specific cytokine gene expression from 50 cytotoxic T lymphocytes (CTL) diluted in 300 microl WB. Furthermore, the high sensitivity of this method could be confirmed ex-vivo by the successful detection of CD8+ T cell responses against HCMV, EBV and influenza virus derived HLA-A0201 restricted epitopes, which was significantly correlated with specific multimer staining. Importantly, a highly significant (p = 0.000009) correlation between hepatitis B surface antigen (HBsAg) stimulated IL-2 gene expression, as detectable in WB, and specific antibody titers was observed in donors vaccinated against hepatitis B virus (HBV) between six months and twenty years before the tests. To identify additional markers of potential clinical relevance, expression of chemokine genes was also evaluated. Indeed, HBsAg stimulated expression of MIP-1beta (CCL4) gene was highly significantly (p = 0.0006) correlated with specific antibody titers. Moreover, a longitudinal study on response to influenza vaccine demonstrated a significant increase of antigen specific IFN-gamma gene expression two weeks after immunization, declining thereafter, whereas increased IL-2 gene expression was still detectable four months after vaccination.

Conclusion: This method, easily amenable to automation, might qualify as technology of choice for high throughput screening of immune responses to large panels of antigens from cohorts of donors. Although analysis of cytokine gene expression requires adequate laboratory infrastructure, initial antigen stimulation and storage of test probes can be performed with minimal equipment and time requirements. This might prove important in "field" studies with difficult access to laboratory facilities.

Figure 1

Monitoring of CTL spiking by WB technology. CD8+ T cells from an HLA-A0201 restricted gp100_280–288 specific CTL clone were added to 300 μl WB from an unrelated donor in the presence of the specific or a control (Melan-A/MART-1_27–35) peptide at a 10 μg/ml concentration. Following 5 hour incubation at 37°C, RNAlater was added to the samples and total cellular RNA was extracted, reverse transcribed and amplified in the presence of primers and probes specific for IL-2, IFN-γ. The expression of the indicated genes from triplicate samples was analyzed by using, as reference, the expression of β-actin house keeping gene (y axes). Standard deviations, never exceeding 5% of the reported values were omitted. A threshold of 2-fold increase in specific gene expression over control values was considered as cut-off for the definition of positive responses. Numbers of CTL spiked into WB were reported on x axes. (triangles = IFN-γ gene; squares = IL-2 gene; filled symbols = specific peptide stimulation; empty symbols = control peptide stimulation).

Figure 2

Cytokine gene expression induced by HCMV, EBV and influenza virus derived HLA class I restricted antigenic peptides in WB of healthy donors. WB from two HLA-A0201+ healthy donors (panels A and B), seropositive for HCMV and EBV (300 μl) was incubated for 5 hours in the presence of HCMV pp65_495–503 (triangles), EBV LMP-2_426–434 (diamonds), BMLF-1_259–267 (squares) and IM_58–66 virus (crosses) derived peptides at 10 μg/ml final concentration. Melanocyte derived GP100_280–288 peptide (circles) was used as negative control. RNAlater was then added and total cellular RNA was purified, reverse transcribed and amplified in the presence of primers and probes specific for IFN-γ (full symbols) or IL-2 (empty symbols). Specific gene expression was analyzed by using, as reference, the expression of β-actin house keeping gene (y axes). A threshold of 2-fold increase in specific gene expression over control values was used as cut-off (dashed lines for IFN-γ and dotted lines for IL-2 gene expression, respectively). WB specimens of the same size (300 μl) from the same donors were simultaneously stained with the corresponding multimers and the number of antigen specific T cells (x axes) was evaluated and correlated with antigen driven gene expression data.

Figure 3

Correlation between expression of genes encoding cytokines and chemokines and anti HBsAg serum titers in vaccinated healthy donors. WB from donors naïve or vaccinated with HBsAg was incubated o/n in the presence of a 2 μg/ml concentration of HBsAg. Following addition of RNAlater, total cellular RNA was extracted, reverse transcribed and amplified by qRT-PCR in the presence of primers and probes specific for the indicated genes and β-actin house keeping gene (panels A-F). Cytokine and chemokine gene expression was evaluated by using, as reference, the expression of β-actin gene, as detailed in "materials and methods". Titers of anti HBsAg antibodies were measured by ELISA. Data regarding correlations between expression of MIP-1β (CCL4) and IFN-γ, IL-2 and TNF-α genes are shown in panels G-I. Linear regressions and 95% mean prediction intervals are reported in each panel.

Figure 4

WB monitoring of cellular immune response to vaccination against influenza virus. Eight healthy donors were vaccinated against influenza virus. WB specimens were obtained before vaccination (day 0) and 14–112 days afterwards. WB samples (300 μl) were incubated o/n in the presence of a 0.6 μg/ml concentration of influenza hemagglutinin. Values related to the expression of IFN-γ (panel A) or IL-2 genes (panel B) were calculated by using, as reference, the expression of β-actin house keeping gene (y axes).