Learning from primary immunodeficiencies to treat cancer

Abstract

The immune system is a fine-tuned network of cells, tissues, organs and biological processes working together in order to protect our body. Immunodeficiency disorders occur when the body’s immune system is reduced or absent and can be characterized as primary, if the cause of disease is genetic; or secondary, if the cause of disease are extrinsic factors. Primary immunodeficiencies (PID) are in general rare, severe, and in many cases lethal. During my PhD, I aimed to investigate the role of dendritic cells (DCs) and natural killer (NK) cells in a primary immunodeficiency named Wiskott-Aldrich Syndrome (WAS). Moreover, I aimed to translate the findings from WAS to cancer treatment.

In paper I, we investigated how WAS protein (WASp) affects migration in DCs. In vitro assays, using microchannels and micropillars, showed that WASp-deficient DCs had increased migration speed while DCs expressing an overactive mutation in WASp, identified in X-linked neutropenia (XLN) patients, had similar average speed but increased speed fluctuations, reduced displacement, and atypical rounded morphology. Surprisingly, in vivo studies using an ear inflammation model, WT, WASp-deficient and XLN DCs migrated to the draining lymph nodes at the same extent, but deeper analysis by microscopy showed that WASp-deficient and XLN DCs localize differently in the draining lymph nodes, compared to WT DCs. Microscopy analysis revealed that XLN DCs had reduced cell area but formed larger podosome structures when compared to WT DCs. These results suggest that WASp activity regulates DC migration and that DCs have a remarkable adaptation for migration under inflammatory conditions in vivo.

In paper II, we examined how WASp affects DC cross-presentation to T cells. Using two different skin pathology models, challenge with Der p 2 or Leishmania major (L. major), we show that WASp-deficient mice had an accumulation of dendritic cells in the skin and increased expansion of interferon (IFN)γ-producing CD8+ T cells in the draining lymph node and spleen. Specific deletion of WASp in dendritic cells led to expansion of CD8+ T cells at the expense of CD4+ T cells. WASp-deficient dendritic cells induced increased cross-presentation to CD8+ T cells by activating Rac2 that maintains a near neutral pH of phagosomes. Our data reveal an intricate balance between activation of WASp and Rac2 signaling pathways in dendritic cells.

In paper III, we used the findings from paper II to develop a DC-based vaccine for cancer therapy. Here, we used the small molecule CK666 to inhibit signaling via WASp and Actin related protein 2/3 (Arp2/3) to enhance DC mediated killing of tumor cells. Treating DCs with CK666 in vitro during antigen uptake and processing of ovalbumin (OVA), murine and human DCs showed decreased phagosomal acidification that induced increased proliferation of OVA-specific OT-I CD8 T cells in vitro and in vivo. Furthermore, using the B16-mOVA melanoma tumor model, we show that mice injected with CK666-treated DCs and OVA-specific OT-I CD8 T cells showed higher rejection of tumor when compared to mice receiving non-treated DCs. This resulted in prolonged survival of tumor-bearing mice receiving CK666-treated DCs. Mice survival is extended when CK666-treated DCs are combined with checkpoint inhibitor anti-PD1. Our data suggests that the small molecule inhibitor CK666 is a good candidate to enhance DC cross-presentation for cancer therapy.

In paper IV, we studied the anti-tumor responses of natural killer (NK) cells and T cells in XLN. Here, we examined NK and T cells from two XLN patients harboring the WASp L270P mutation. XLN patient NK and T cells had increased Granzyme B content and elevated degranulation and IFNγ production when compared to healthy control cells. Murine XLN T cells had normal degranulation and cytokine response whereas XLN NK cells showed an enhanced response. When compared to WT mice, XLN mice showed reduced growth of B16 melanoma and increased capacity to reject MHC class I-deficient cells. Together, our data suggests that cytotoxic cells with constitutively active WASp have an increased capacity to respond to and kill tumor cells.

In summary, the work presented in this thesis show that both DCs and NK cells are very important in starting an immune response and defects in their cytoskeleton dramatically affects their function. Still, cytoskeleton rearrangement and its outcome depend greatly on the context. Both WAS-deficiency and WASp-overactive patients suffer from negative effects however, we can learn from them and focus on the positive consequences in order to help cancer patients.