Gene expression profiling in peripheral blood mononuclear cells of patients with common variable immunodeficiency: modulation of adaptive immune response following intravenous immunoglobulin therapy

Abstract

Background: Regular intravenous immunoglobulin treatment is used to replace antibody deficiency in primary immunodeficiency diseases; however the therapeutic effect seems to be related not only to antibody replacement but also to an active role in the modulation of the immune response. Common variable immunodeficiency is the most frequent primary immunodeficiency seen in clinical practice.

Methods: We have studied the effect of intravenous immunoglobulin replacement in patients with common variable immunodeficiency by evaluating the gene-expression profiles from Affimetrix HG-U133A. Some of the gene array results were validated by real time RT-PCR and by the measurement of circulating cytokines and chemokines by ELISA. Moreover we performed FACS analysis of blood mononuclear cells from the patients enrolled in the study.

Results: A series of genes involved in innate and acquired immune responses were markedly up- or down-modulated before therapy. Such genes included CD14, CD36, LEPR, IRF-5, RGS-1, CD38, TNFRSF25, IL-4, CXCR4, CCR3, IL-8. Most of these modulated genes showed an expression similar to that of normal controls after immunoglobulin replacement. Real time RT-PCR of selected genes and serum levels of IL-4, CXCR4 before and after therapy changed accordingly to gene array results. Interestingly, serum levels of IL-8 remained unchanged, as the corresponding gene, before and after treatment. FACS analysis showed a marked decrease of CD8+T cells and an increase of CD4+T cells following treatment. Moreover we observed a marked increase of CD23⁻CD27⁻IgM⁻IgG⁻ B cells (centrocytes).

Conclusions: Our results are in accordance with previous reports and provide further support to the hypothesis that the benefits of intravenous immunoglobulin therapy are not only related to antibody replacement but also to its ability to modulate the immune response in common variable immunodeficiency.

Figure 1. Genes modulated by IVIg infusion.

A. Graphical representation of the expression of the 23 genes returned to the levels of normal controls after IVIg infusion. B. FC expression, before and after therapy, of the genes represented in panel A with the corresponding colours.

Figure 2. Real time RT-PCR of some modulated genes confirms the results of gene array analysis.

Genes selected for validation in PBMC of CVID patients before IVIg treatment. IL8, RGS1 and CCR3 transcripts were increased, whereas TNFRSF17 transcript was decreased in CVID patients when compared to healthy donors. Relative expression levels were calculated for each sample after normalization against the housekeeping genes 18s rRNA, beta-actin and GAPDH. Experiments have been conducted in triplicates. Housekeeping genes: blue bar  = 18s rRNA; magenta bar: beta-actin; yellow bar: GAPDH.

Figure 3. Flow cytometric analysis of T and B cell populations in CVID patients before and after IVIg infusion.

Data are representative of all the subjects studied. A and B panels show the percentages of CD4+ and CD8+T cell populations before and after IVIg infusion, respectively. Percentages of naïve B cells, centrocytes and switched memory B cells before and after IVIg infusion are displayed in C vs D, E vs F and G vs H panels, respectively.

Figure 4. Serum levels of selected soluble mediators in CVID patients before and after IVIg infusion.

The histogram represents the mean of the results obtained in 30 patients. Results are expressed in pg/ml. p values calculated using the Student's t-test for paired samples.