Biology of inerleukin-7 variant isoforms

Abstract

The interleukin-7 (IL-7) gene produces nine alternatively spliced mRNA variants. Until now, the clinical and biological significance of alternatively spliced IL-7 products has not been well defined. In this thesis, we investigated IL-7 gene alternative splicing in association with chronic inflammation and infection. This thesis also investigated the biology of alternatively spliced IL- 7 variants in T-cell subsets and CD14+ monocytes. We identified for the first time the alternatively spliced IL-7δ5 variant protein among IL-7 protein variants in healthy and diseased human tissue.

In paper I, we investigated the extent and impact of IL-7 pre-mRNA alternative splicing in Peripheral Blood Mononuclear Cells (PBMCs) from 16 patients with multiple sclerosis and 15 healthy individuals by transcript specific amplification of interleukin-7 and the interleukin-7 receptor gene. The relative abundance of the gene transcripts was correlated to health and disease (MS) status and also with the IL-7R SNP rs_6897932, which has been shown to affect alternative splicing of the IL-7R gene. Flow cytometric analysis of PBMC samples was carried out to enumerate abundance of immune cell subsets in healthy and diseased individuals along with the enumeration of IL-7R molecules on single cells to identify correlation with specific IL- 7 alternative splicing patterns. This was the first study to i) investigate patterns of IL-7 and IL- 7R in healthy human PBMCs, ii) show that the IL-7 alternatively spliced variant lacking exon 4 (IL-7δ4), and not the canonical IL-7, constitutes the major transcript of IL-7 in human PBMCs. This was also the first report to identify a novel IL-7R spliced variant lacking exon 5, 6 and 7. We observed changes in relative distribution of IL-7 and IL-7R spliced variants in healthy individuals and patients with MS.

In paper II, we investigated IL-7 and IL-7R alternative splicing in a rhesus macaque model. The study aimed to identify the extent of IL-7 and IL-7R pre mRNA alternative splicing and to gauge its impact in chronic inflammation i.e in M. tuberculosis (MTB) infection in a vaccination setting defined by clinical endpoints (improved survival after challenge with virulent MTB). This enabled to study the IL-7 protein directly in lung tissue. Fifteen rhesus macaques were divided in BCG vaccine groups and controls, followed by a challenge with a virulent MTB strain. PBMC samples before the vaccination, as well as before and after the virulent MTB challenge, along with tissues (lung with and without lesions, pulmonary hilus lymph nodes and spleen) from macaques which succumbed to MTB infection were analysed for IL-7 and IL-7R pre-mRNA alternative splicing. Measurement of IL-7, IL-17 and TGF-β at the site of infection (lung) was performed by immunohistochemistry and correlated with survival. The results showed that the spliced variants of IL-7 are similar in humans and rhesus macaque PBMCs; yet the relative distribution of IL-7 transcripts is different i.e. IL-7c and not the IL-7δ4 constituted the most frequent transcript in macaque PBMCs. The IL-7R gene exhibited extensive alternative splicing associated with soluble IL-7R in lung and lymph nodes. The study could correlate increased survival of rhesus macaques upon MTB challenge to increased local production of IL-7c and IL-17a as well as decreased TGF-β in lung lesions of non-human primates exhibiting increased survival.

In paper III, we elaborated on the data produced in the first 2 reports and investigated the effect of IL-7δ5 variant on T-cells. A mono-specific (affinity-purified) polyclonal antibody recognising exclusively IL-7δ5 (and no other IL-7 isoforms) was developed using a junction peptide forming the unique junction of the IL-7δ5 protein. The binding affinity and off-rate of IL-7 and IL-7δ5 (using a soluble IL-7 receptor, sIL-7R) was examined by surface plasmon resonance on a biacore platform in order to study mechanistic properties of IL-7δ5 and IL-7R interaction. Sorted CD4+ and CD8+ T-cell subsets were stimulated with IL-7δ5 or IL-7c (along with unstimulated controls) and changes in the transcriptome were studied using a microarray gene expression platform. The effect of IL-7δ5 and IL-7c on T-cell subsets was investigated based on the phosphorylation of signalling transducers and BCL-2 induction using flow cytometry. This report identified IL-7δ5 production, for the first time, in situ using a monospecific anti IL-7δ5 antibody in granuloma and lymph node sections from patients with infections (M. tuberculosis and HIV). IL-7 and IL-7δ5 protein expression analysis indicated a different distribution in situ, both proteins induced similar gene expression pattern in CD4+ and CD8+ T-cell subsets. IL-7δ5 showed slightly higher affinity to IL-7R, based on the SPR assay and induced BCL-2 in both CD4+ and CD8+ subsets earlier (3 days versus 10 days as compared to IL-7c), suggesting a superior agonistic activity based on BCL-2 induction.

In paper IV, we investigated the effects of the IL-7c versus the IL-7δ5 spliced variant in regard to CD14 monocyte functions. Sorted CD14+ cells from healthy donors were stimulated with IL-7c or IL-7δ5 (along with unstimulated controls in triplicates) and changes in gene expression levels after 72 hours were measured by an RNA microarray platform; cell culture supernatants were examined for differential production of immune mediator cytokines and chemokines by a luminex assay. IL-7δ5 induced a pro-inflammatory profile in CD14+ monocytes, associated with upregulation of oxidised low density lipoprotein receptor 1 (OLR1) gene, confirmed by flow cytometry. In order to investigate IL-7δ5 protein expression, we elaborated on findings in paper III and performed immunohistochemistry to identify IL-7δ5 protein production in association with healthy and corresponding transformed tissues. We were able to show that IL- 7δ5 can be identified in both, human healthy and diseased tissue; IL-7δ5 can be produced by epithelial cells as well as by CD68+ tissue residing.