Immune responses against tumors controlled by the actin cytoskeleton : lessons from primary immunodeficiencies

Abstract

The actin cytoskeleton and its regulators are vital for cellular processes such as cell motility and immune synapse formation. In this study, the aim was to dissect the role of two of those regulators, WASp and MKL1, in immune cells and their role in tumorigenesis. Additionally, we pharmacologically target an enzyme involved in fatty acid metabolism in monocytes and assess the impact on the actin cytoskeleton.

Deleterious mutations in WASp cause the Wiskott – Aldrich syndrome (WAS) and activating mutations cause X-linked neutropenia (XLN). Both immunodeficiencies are accompanied by a higher risk of developing malignancy. In paper I we investigated NK cell function when WASp is absent. We found that WASp-deficient NK cells were hyporesponsive to stimulation and failed to form immune synapses with tumor cells in vitro and to reject MHC Class I-deficient splenocytes in vivo. However, we observed no defect in lymphoma development or rejection in vivo. IL-2 is a cytokine known to stimulate NK cell cytotoxicity. When incubating NK cells with IL-2, we could rescue the functional defects of degranulation and cytokine secretion, as well as actin polarization to the synapse in vitro. Additionally, when injecting the IL-2 treated NK cells into WASp-deficient that have received MHC Class I-deficient splenocytes, we could also rescue the in vivo defect. Interestingly, we found that all the lymphomas we used in this study were capable of producing large amounts of IL-2 in vivo, which could be the reason that WASp-deficient NK cells were capable of handling the tumors similarly to wildtype. Therefore, we conclude that the malignancies observed in WAS are likely due to immune surveillance defects and can be circumvented by immunotherapy.

In paper II, we dissect the role of activating WASp mutations in NK cell and T cell functionality in the context of tumor development. We found that NK cells and T cells from mice with activating mutations WASpL272P and WASpI296T were capable of responding to receptor stimulation as well as form immune synapses in vitro. WASpL272P mice could reject lymphomas to the same extent as wildtype and had higher rejection rates of MHC Class Ideficient splenocytes in vivo. Additionally, NK cells from WASpL272P and WASpI296T mice had lower amounts of KLRG1, an inhibitory receptor. NK cells from XLN patients with the L270P mutation were hyporesponsive to stimulation with tumor cells but not to PMA/Ionomycin and had decreased KLRG1 expression. Interestingly, the patients’ T cells had increased amounts of KLRG1, high Granzyme B content and could respond to PMA/Ionomycin stimulation. There was also the appearance of a CD4+CD8low population of T cells in the patients. Therefore, the malignancies observed in XLN are more likely due to increased cell intrinsic transformational capacity than defects in immunosurveillance. While the cytoplasmic role of WASp is extensively studied, both in general and in the context of this thesis, the nuclear role of WASp is less known. In paper V, we sought to determine that role. We show that WASp is indeed present in the nucleus and mediates active transcription. Additionally, we observed that WASp drives transcriptional events associated with T cell development and does this through its regulation of T cell-specific transcription factor TCF1. We also found that WASp interacted with TCF1 but had no direct binding to it; the proteins could instead form a macromolecular complex.

Paper III, focuses on the actin regulator MKL1 and attempts to dissect its role in the development of Hodgkin’s lymphoma (HL). We investigated triplets with a deletion in the first intron of MKL1, two of which developed and were treated for HL. We found that the deletion induced increased expression of MKL1. In EBV-transformed B cells we generated from the triplets, we saw that the cells from the unaffected triplet (termed HL0) showed decreased aggregation, increased spreading and protrusion formation on slides, increased proliferation, and increased genomic instability. These phenotypic traits have all been connected to a more invasive cellular state. These findings, along with the increased expression of MKL1 in various lymphomas that we looked at, provide evidence for the link between MKL1 and lymphoma development.

The actin cytoskeleton can be hijacked by cancer cells to produce highly motile and invasive cells. In paper IV we used and inhibitor for the enzyme 15-lipoxygenase-1 (15-LOX-1) that mediates metabolism of fatty acids. We showed that the enzyme is necessary for the formation of podosomes in dendritic cells (DCs), which could contribute to invasion and metastasis. The DCs were also impaired in their migratory and endocytic capacity when treated with the inhibitor. T cell responses when using the inhibitor on DCs were only marginally affected.