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T. Shahin et al.
Effects of IL6ST mutations on T-cell differentiation and function
As cells from PP498L and PN404Y exhibited particularly aber- rant IL-6 and IL-27 signaling, we hypothesized an impact on T-cell differentiation. Despite a history of recurrent infections, both patients had rather reduced CD4+ memo- ry T cells and lower CCR6 expression in CD4+ memory T cells and total CD4+ T cells (particularly in PP498L; P<0.05) (Figure 5A and B and Online Supplementary Figure S3A and B).5 However, CXCR3 expression was reduced only in CD4+ T cells of PP498L, while CD4+ memory T cells from PN404Y showed increased expression of CCR4 and CRTh2. To investigate changes in Th-cell phenotypes, we ana- lyzed chemokine receptor-expression linked to Th-cell homing and function.21,22 t-Distributed Stochastic Neighbor Embedding (TSNE) revealed that predominantly CD4+CCR6+, but less so CD4+CCR6– Th-cells, were reduced in both patients (Figure 5C). Moreover, although both patients showed normal numbers of total CD8+ memory T cells (Figure 5D), CCR6 expression within CD8+ memory T cells was reduced (particularly in PP498L; P<0.05) (Figure 5E and F and Online Supplementary Figure S3C and D). Furthermore, both patients showed reduced frequencies of RORγt+ (particularly in PP498L; P<0.05) but not TBET+CD8+ T cells (Online Supplementary Figure S4A). The CD8+RORγt+ memory T cells co-expressed CCR6 and intermediate level of TBET (Online Supplementary Figure S4B and C). These results demonstrate that functional IL- 6 signaling in human T cells is required for normal devel- opment of both CCR6-expressing CD4+ and CD8+ T cells. In addition, PP498L showed reduced CD8+ CXCR3+ T-cell frequencies (Figure 5E).
Both patients had normal frequencies of peripheral reg- ulatory T cells (Online Supplementary Figure S5). However, in peripheral blood CD4+ memory cells, and more evi- dently in total CD4+ T cells, we noted reduced Th17- enriched23 CCR6+ CCR4+ CXCR3– frequencies and lower Th1-enriched CCR6–CCR4–CXCR3+ frequencies only in PP498L (Figure 6A). On the other hand, Th2-enriched CCR6– CCR4+ CXCR3– frequencies were significantly increased in PN404Y. To confirm chemokine receptor-based enrichments, and to evaluate also the composition of T-cell phenotypes inside chemokine receptor-enriched compartments, we analyzed subset specific transcription factor expression at the protein level (Online Supplementary Figure S6). TBET, GATA3 and RORγt expression fell into normal ranges, with elevated expression of GATA3 inside Th2 and Th17-enriched subsets of PN404Y only (Online Supplementary Figure S7). Interestingly, at the functional level, PP498L showed reduced IL-17A-producing CD4+ mem- ory T-cell numbers. Furthermore, PP498L showed reduced IFNγ-producing and low IL-10-producing (P=0.0667) CD4+ memory T-cell frequencies while IL-4 production was in the normal range (Figure 6B-E). Besides these aberrations in CD4+ T cells, PP498L had significantly less IFNγ-producing CD8+ memory T cells (Figure 6F and G) indicating that, in vivo, the IL-27-specific signaling defects shown to be more severe in PP498L affect both CD4+ and CD8+ T-cell composi- tion and effector function.
Discussion
In this study, we show an intriguingly high degree of phenotypic similarity between the clinical manifestations
exhibited by 2 unrelated patients with cytokine selective IL6ST-loss of function mutations. Both patients present with elevated IgE, eosinophilia, recurrent infections (including invasive infections, severe lung pathology, and keratitis), and skeletal abnormalities (abnormal skull form/craniosynostosis and scoliosis). This is strikingly reminiscent of key features of HIES due to STAT3 variants or the recently studied ZNF341 deficiency,24,25 whereas they are less in common with other forms including DOCK8-deficiency26,27 and other deficiencies leading to high levels of serum IgE28 (Table 2). While STAT3 is a downstream transcription factor for several signaling pathways, including IL-10, IL-21 and IL-23, the mutations in IL6ST encoding GP130, constrict the defect to selected signaling pathways and suggest that defects in IL-6 and IL- 11 signaling dominate the HIES phenotype, whereas addi- tional defects in IL-27, LIF and OSM signaling might con- tribute to the individual immunopathology. The two mutations affecting GP130 (p.P498L and p.N404Y) are 95 amino acids apart, yet both are on the highly conserved membrane-proximal ectodomain of the protein, known to play a crucial role in signal transduction and downstream JAK activation.29-31 Both variants show stable GP130 sur- face expression, preserving a partially intact quaternary protein structure, allowing GP130 to bind its ligands, and maintaining downstream signaling of cytokines such as LIF.
The effect of individual GP130 variants may depend on various factors, including the genetic variant itself, the cytokine levels, as well as GP130 expression and recep- tor/co-receptor stochiometries in different cell types, explaining the cell type specific responses. We have addressed these factors by plasmid transfection combined with cytokine stimulation assays that cover a range of concentrations, as well as by comparing diverse types of primary immune cells. We found that the GP130P498L vari- ant has a significant impact on IL-6, IL-11, IL-27 and LIF signaling in the HEK293 transfection assay and impaired downstream signaling upon stimulation with IL-6 and IL- 27 in primary T cells, T lymphoblasts and EBV-LCLs, as well as IL-6 and IL-11 in fibroblasts. Yet, in fibroblasts, sig- naling upon OSM (and to some extent IL-27 and LIF) stim- ulation was reduced but not completely abrogated. STAT1 that was phosphorylated in response to IL-27 and OSM in healthy donor fibroblasts showed decreased phosphoryla- tion in PP498L fibroblasts. Furthermore, in response to IL-27, STAT1, STAT3 and STAT4 phosphorylation was compro- mised in T lymphoblasts of PP498L, demonstrating a general defect in signal transduction.
IL-6 is a key cytokine responsible for the activation and differentiation of both T and B cells, as well as pro-inflam- matory cues, including the acute-phase response.12 Therefore, the absence of an IL-6 response in PP498L explains his high susceptibility to pulmonary infections with no fevers. In contrast, aberrant IL-11 signaling underlies the observed bone manifestations in both patients with sup- porting evidence from IL11RA-deficient patients that pres- ent with craniosynostosis and delayed tooth eruption.14,15 IL-27 promotes differentiation of CD4+ T cells towards the Th1 phenotype, promotes IFNγ+IL-10+FOXP3– T-helper cell differentiation, and enhances CD8+ T-cell responses by increasing proliferation and effector functions such as IFNγ production and cytolytic activity.13,32-36 Defects in these immune functions were particularly seen in PP498L who had completely aberrant IL-27 signaling in primary T
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haematologica | 2019; 104(3)