Transfusable neutrophil progenitors as cellular therapy for the prevention of invasive fungal infections

Abstract

The use of mature neutrophil (granulocyte) transfusions for the treatment of neutropenic patients with invasive fungal infections (IFIs) has been the focus of multiple clinical trials. Despite these efforts, the transfusion of mature neutrophils has resulted in limited clinical benefit, likely owing to problems of insufficient numbers and the very short lifespan of these donor cells. In this report, we employed a system of conditionally immortalized murine neutrophil progenitors that are capable of continuous expansion, allowing for the generation of unlimited numbers of homogenous granulocyte-macrophage progenitors (GMPs). These GMPs were assayed in vivo to demonstrate their effect on survival in 2 models of IFI: candidemia and pulmonary aspergillosis. Mature neutrophils derived from GMPs executed all cardinal functions of neutrophils. Transfused GMPs homed to the bone marrow and spleen, where they completed normal differentiation to mature neutrophils. These neutrophils were capable of homing and extravasation in response to inflammatory stimuli using a sterile peritoneal challenge model. Furthermore, conditionally immortalized GMP transfusions significantly improved survival in models of candidemia and pulmonary aspergillosis. These data confirm the therapeutic benefit of prophylactic GMP transfusions in the setting of neutropenia and encourage development of progenitor cellular therapies for the management of fungal disease in high-risk patients.

Keywords: Aspergillus; Candida; differentiation; fungi; innate immunity; neutropenia.

Figure 1.

Granulocyte monocyte progenitors (GMPs) transduced with the ERHoxb8 system mature and respond to C. albicans in vitro. (A) Schematic illustrating the ERHoxb8 cassette insertion into murine mononuclear cells which results in the conditional immortalization of GMPs in the presence of estrogen and stem cell factor. With the removal of estrogen from the media, GMPs mature into functional neutrophils in vitro. (B) Representative Wright Giemsa images of GMPs and polymorphonuclear cells (PMNs) demonstrating morphological differences between cell states. Scale bar = 10 μm. (C) PMN maturation as demonstrated by CD11b and GR-1 staining by flow cytometry. (D) UBC-GFP expressing neutrophils were co-incubated with C. albicans expressing dTomato for 1 hour. Neutrophils were then labelled with DAPI and images were taken with a Zeiss 780 confocal microscope. Scale bar 20 μm.

Figure 2.

GMPs undergo cell division in vitro and in vivo to mature into functional PMNs. (A) Schematic demonstrating the loss of fluorescence in GMPs labelled with the cell tracing dye CFSE. (B) GMPs and PMNs were stained with CFSE for 20 minutes before washing. Mean fluorescence intensity was measured via flow cytometry. The relationship between cell division and time was calculated for GMPs in media containing estrogen, R² = 0.9998. (C) Wild type C57BL/6 mice were irradiated and injected with 20 million UBC-GPF GMPs labelled with a far-red fluorescent CFSE dye. ERHoxb8 progenitors undergo 5 cell divisions as demonstrated by CFSE labelling from cells harvested from the bone marrow and spleen. Mean fluorescence intensity measured by flow cytometry. (D) Wild type C57BL/6 mice were irradiated as described and injected with 20 million UBC-GFP or 20 million primary PMNs harvested from CD45.1^STEM mice. Each day following transfusion, cells from the bone marrow (BM), spleen, and peripheral blood (PB) were harvested from each group. Flow cytometry was used to identify CD45.1^STEM and UBC-GFP PMNS. N = 4 mice with UBC-GFP each day, 3 mice with CD45.1^STEM on day 1 and 2, 2 mice on day 3. Mean and SEM shown, analyzed by one-way ANOVA with Kruskal-Wallis post-test. (E) To examine UBC-GFP PMN recruitment, thioglycolate was injected in the intraperitoneal cavity as a method of inflammation one day prior to PMN harvest from the peritoneal fluid (PF). Fluorescence was measured by flow cytometry. N = 5 mice on day 2 and day 6, 4 mice on day 4. Mean and SD shown, analyzed by one way ANOVA with Turkey’s post test **p = 0.0025, ***p = 0.0006

Figure 3.

GMPs differentiate into functional neutrophils in vivo and extend host survival during C. albicans infection. (A) Schematic illustrating murine challenge. Briefly, wild type C56BL/6 mice were irradiated (XRT) as described and injected with 20 million UBC-GFP GMPs retro-orbitally with a single loading dose or with the initial loading dose and an additional daily dose thereafter. Mice were challenged with 800 C. albicans intravenously four days after the loading dose of GMPs. (B) Survival curve of mice infected with C. albicans. N=4-8 mice per group. Images created with BioRender.com

Figure 4.

Transfused neutrophils respond to C. albicans in the kidneys but do not eliminate fungi. C57BL/6 mice were irradiated and injected with GMPs as described previously. Mice were then infected with 500 C. albicans intravenously and kidneys were harvested on day 2 and day 3 post infection. (A) Histology sections of infected kidneys with and without GMP transfusion. (B) Percent scoring of inflammation and (C) cortical fungal burden from kidney histology sections. Pooled kidney sections from 3 experiments, 12 mice per group. Unpaired t-test, p = 0.0011 in inflammation, p = 0.0058 in cortical fungal burden. (D) Colony-forming units (CFUs) of C. albicans from kidneys of mice with and without GMP supplementation. Represents data from one experiment, 6 mice per group. Mann-Whitney test, n.s. between plus and minus GMPs at day 2 and day 3.

Figure 5.

GMPs extend host survival in an inhalation model of Aspergillus fumigatus infection. (A) UBC-GFPs allowed to mature into functional neutrophils were challenged with A. fumigatus for 1 hour in vitro. Images demonstrate neutrophils interacting with A. fumigatus filaments. Scale bar = 20 μm. (B) Schematic demonstrating wild type C57BL/6 murine challenge with A. fumigatus conidia. Briefly, mice were irradiated (XRT) and transfused with GMPs with one loading dose or with an initial loading dose plus an additional daily dose for four days. Mice were then anesthetized and given 200 A. fumigatus conidia via inhalation and mice were followed for survival. (C) Survival curve of mice infected with A. fumigatus. n=5-6 mice per group. Images created with BioRender.com