Calcineurin Controls Drug Tolerance, Hyphal Growth, and Virulence in Candida dubliniensis▿†

Candida dubliniensis is an emerging pathogenic yeast species closely related to Candida albicans and frequently found colonizing or infecting the oral cavities of HIV/AIDS patients. Drug resistance during C. dubliniensis infection is common and constitutes a significant therapeutic challenge. The calcineurin inhibitor FK506 exhibits synergistic fungicidal activity with azoles or echinocandins in the fungal pathogens C. albicans, Cryptococcus neoformans, and Aspergillus fumigatus. In this study, we show that calcineurin is required for cell wall integrity and wild-type tolerance of C. dubliniensis to azoles and echinocandins; hence, these drugs are candidates for combination therapy with calcineurin inhibitors. In contrast to C. albicans, in which the roles of calcineurin and Crz1 in hyphal growth are unclear, here we show that calcineurin and Crz1 play a clearly demonstrable role in hyphal growth in response to nutrient limitation in C. dubliniensis. We further demonstrate that thigmotropism is controlled by Crz1, but not calcineurin, in C. dubliniensis. Similar to C. albicans, C. dubliniensis calcineurin enhances survival in serum. C. dubliniensis calcineurin and crz1/crz1 mutants exhibit attenuated virulence in a murine systemic infection model, likely attributable to defects in cell wall integrity, hyphal growth, and serum survival. Furthermore, we show that C. dubliniensis calcineurin mutants are unable to establish murine ocular infection or form biofilms in a rat denture model. That calcineurin is required for drug tolerance and virulence makes fungus-specific calcineurin inhibitors attractive candidates for combination therapy with azoles or echinocandins against emerging C. dubliniensis infections

Magnetic targeting of adoptively transferred tumour-specific nanoparticle-loaded CD8+ T cells does not improve their tumour infiltration in a mouse model of cancer but promotes the retention of these cells in tumour-draining lymph nodes

[Background] Adoptive T cell-transfer (ATC) therapy is a highly promising cancer-treatment approach. However, in vivo-administered T cells tend to disperse, with only a small proportion reaching the tumour. To remedy this, magnetic targeting of T cells has been recently explored. Magnetic nanoparticles (MNPs) functionalised with antibodies were attached to effector T cells and magnetically recruited to tumour sites under MRI guidance. In this study, we investigated whether 3-aminopropyl-triethoxysilane (APS)-coated MNPs directly attached to CD8+ T cell membranes could also magnetically target and accumulate tumour-specific CD8+ T cells in solid tumours using an external magnetic field (EMF). As it has been shown that T cells associated with APS-coated MNPs are retained in lymph nodes (LNs), and tumour-draining LNs are the most common sites of solid-tumour metastases, we further evaluated whether magnetic targeting of APS-MNP-loaded CD8+ T cells could cause them to accumulate in tumour-draining LNs.[Results] First, we show that antigen-specific CD8+ T cells preserve their antitumor activity in vitro when associated with APS-MNPs. Next, we demonstrate that the application of a magnetic field enhanced the retention of APS-MNP-loaded OT-I CD8+ T cells under flow conditions in vitro. Using a syngeneic mouse model, we found similar numbers of APS-MNP-loaded OT-I CD8+ T cells and OT-I CD8+ T cells infiltrating the tumour 14 days after cell transfer. However, when a magnet was placed near the tumour during the transfer of tumour-specific APS-MNP-loaded CD8+ T cells to improve tumour infiltration, a reduced percentage of tumour-specific T cells was found infiltrating the tumour 14 days after cell transfer, which was reflected in a smaller reduction in tumour size compared to tumour-specific CD8+ T cells transferred with or without MNPs in the absence of a magnetic field. Nonetheless, magnet placement near the tumour site during cell transfer induced infiltration of activated tumour-specific CD8+ T cells in tumour-draining LNs, which remained 14 days after cell transfer.[Conclusions] The use of an EMF to improve targeting of tumour-specific T cells modified with APS-MNPs reduced the percentage of these cells infiltrating the tumour, but promoted the retention and the persistence of these cells in the tumour-draining LNs.This work was supported in part by Grants from the Spanish Ministry of Economy, Industry and Competitiveness (SAF-2014-54057-R and SAF-2017-82223-R to DFB). L Sanz-Ortega and Y Portilla receive predoctoral FPU Grants (FPU13/05037 and FPU15/06170 respectively) from the Spanish Ministry of Economy.We acknowledge support of the publication fee by the CSIC Open Access Support Initiative through its Unit of Information Resources for Research (URICI)Peer reviewe