Alternative splicing of interleukin-7

Abstract: Alternative splicing of pre-mRNA is the primary source of creating proteome diversity in humans; it is therefore highly relevant to disease and therapy. Alternative splicing has been described for many cytokines and these splice variants appear to function as either agonists or as antagonists of the wildtype cytokine. Interleukin-7 (IL-7) plays a non-redundant role in early stages of normal B and T-cell development; IL-7-receptor signaling is required for the maintenance of T-cell homeostasis and memory T-cell formation. In the present study, we investigated IL-7 splice variant distribution in different human tissues and organs obtained from healthy individuals and patients suffering from cancer or chronic infections. Recombinant expression of an Interleukin-7 splice variant showed, as a paradigm, that IL-7 splice variants are biologically active defined by i) STAT5 phosphorylation, ii) prevention of apoptosis and iii) cellular differentiation. In paper I, we have investigated the relative distribution of Interleukin-7 receptor (IL-7R) molecules on different T-cell subsets in peripheral blood by using high content flow cytometry. Only IL-7R positive cells would be able to transmit an IL-7 or IL-7 isoform mediated signal. We analyzed peripheral blood mononuclear cells (PBMCs) from patients with MS in order to identify which immune cells express the IL-7R since Multiple Sclerosis (MS) is the first disease, in which differential IL-7R expression has been postulated due to variations in the IL-7R gene, defined by SNPs. The study was performed using 12 color flow cytometry including an anti-IL-7R monoclonal antibody to objectively enumerate IL-7R molecules on the single cell level. We were not able to identify any IL-7R positive immune cell subsets which showed statistically different composition in patients with MS as compared to healthy controls, but we were able to identify 19/189 immune cell subsets which were either increased or decreased in a significant fraction in patients with MS versus healthy controls. In contrast, we identified significant differences in IL-7R density, measured on a single cell level. 2/59 variables namely TCRalpha/beta+CD4+CD25-CD107a+ and TCRalpha/beta+CD4+CD25intermediate T-cells showed increased numbers of IL-7R molecules in patients with neurological disease. We proposed that the TCRalpha/beta+CD4+CD25-CD107a+ T-cell subset represents a differentiated, cytotoxic CD4+ T-cell population, which is associated with chronic antigen exposure in patients with MS. In paper II, we asked whether each IL-7R positive cell is able to transmit an IL-7 signal. Surprisingly, PBMCs obtained from patients with breast cancer showed IL-7 mediated signaling defects defined by STAT5 phosphorylation, despite the expression of IL-7Ralpha on the cell surface. This was associated with reduced level of IL-7Ralpha on CD8alpha/alpha+ and CD4+ T-cells. Cells with IL- 7R signaling defects were also impaired in cytokine production after PMA/Ionomycin stimulation. These data suggested that a detailed analysis of T-cell function, determined by IL-7 mediated signaling, may improve the design of biological therapy in patients with cancer and help to escort immunological strategies to improve anti-tumor T-cell reactivity. In paper III, we described a map of IL-7 variant expression in different human organs and tissues. We demonstrated aberrant expression of IL-7 splice variants in different tissues obtained from patients with cervical cancer lesions and Mycobacterium tuberculosis positive granuloma lesions obtained from individuals with latent TB. These data supported the observation that IL-7 is differently spliced in transformed cells or in inflammatory processes associated with M. tuberculosis infection. The functional impact of IL-7 isoforms on immune cells was demonstrated by gauging anti-apoptotic activity, bcl2 protein levels and STAT5 phosphorylation in peripheral T-cells and thymocytes, induced by an IL-7 variant lacking exon5. This suggested that alternatively splice variants of IL-7 are indeed biologically active. In paper IV, we demonstrated differentially spliced IL-7 in distinct anatomic areas of the brain. IL-7 and IL-7 splice variant proteins were able to shift differentiation of neural progenitor cells towards the glia cell lineage. Transcriptome analysis of IL-7 stimulated neural progenitor cells showed 58 differentially regulated genes; some of these genes were also involved in neural differentiation. Thus, IL-7 may play a significant role in neural development by participating in human brain architecture through glia cell formation. In conclusion, the work described in this thesis provides new insights into the biology of differentially spliced IL-7 in health and disease.