Upregulation of CD47 Is a Host Checkpoint Response to Pathogen Recognition

Abstract

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

Keywords: CD47; host response; immune checkpoint; innate immunity; pathogen recognition receptors.

FIG 1

CD47 is broadly upregulated in immune cell types in response to several types of infection. (A and B) Comparison of CD47 median fluorescence intensities (MFI) on splenic hematopoietic cell subsets from naive mice and female (A.BY × C57BL/6)F₁ mice infected intravenously with 2 × 10⁴ SFFU Friend virus at 3 days postinfection (A) or female C57BL/6 mice infected intravenously with 2 × 10⁶ PFU LCMV-WE at 2 days postinfection (B). (C) Female C57BL/6 mice inoculated intraperitoneally with 10⁵ PFU La Crosse virus at 2 days postinfection. (D) CD47 expression levels analyzed from the publicly available gene expression data set from SARS-CoV-2 infection of A549 human lung tumor cells (GEO accession number GSE147507) (n = 10) comparing mock-infected (n = 13) with SARS-CoV-2 (USA-WA1/2020)-infected cells (n = 6). (E) Comparison of CD47 MFI on hematopoietic cells from Borrelia burgdorferi-GFP-infected human PBMCs 24 and 48 h after in vitro infection, compared to naive controls. GFP was used under infection conditions to identify cells with intracellular Borrelia infection (shaded). (F) Comparison of CD47 MFI on human CD19⁺ B cells 24 h after in vitro infection with Salmonella enterica serovar Typhi strain Ty2 (Ty2 WT) or Salmonella enterica serovar Typhi strain ΔfliC (Ty2 ΔfliC), which lacks flagella, compared to naive controls. Statistical analyses were done by Student’s t tests for panels A to D and by one-way analysis of variance (ANOVA) with a multiple-comparison posttest for panels E and F (ns [not significant], P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Error bars represent standard errors of the means (SEM).

FIG 2

Stimulation of pathogen-associated molecular patterns upregulates CD47 surface expression. (A) MFI of CD47 surface expression on human PBMC monocytes and dendritic cells after 48 h of stimulation with either 1 μg/ml MDP or 1 μg/ml CL264 or with no stimulation. The results are from one of three experiments with two different donors. All 3 experiments showed consistent effects. Statistics were done by a paired two-way t test with Bonferroni correction. (B) CD47 MFI on human total PBMCs from 4 separate donors stimulated with titrated concentrations of R848 from 0.1 μg/ml to 10 μg/ml, as indicated, for 48 h. Statistics were done by a paired two-way t test with Bonferroni correction. (C) Mice (n = 5/group) were injected intraperitoneally with 1 mg/kg R848 for 3 days, and on day 3, splenocytes were isolated and macrophages and DCs were analyzed for CD47 MFI. Statistics were done by an unpaired two-way t test. (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Error bars represent SEM.

FIG 3

CD47 is involved in innate licensing of adaptive immune responses in HCV patient clinical samples. (A) Comparison of CD47 expression from Affymetrix array profiles of liver biopsy specimens from two healthy controls and six patients with acute HCV infection (P = 0.03 by an unpaired two-way t test) (NCBI GEO accession number GSE38597). (B and C) Comparison of CD47 expression by CyTOF MFI on CD14⁺ monocytes (B) and HLA-DR⁺ DC subpopulations (C) isolated from HCV-infected sofosbuvir-treated patients before the initiation of treatment (Pre), midway through treatment (Mid), and after treatment (Post) compared to healthy controls. (D) Comparison of CD47 expression on SIRPα^lo versus SIRPα^hi DCs from healthy control and HCV patients. Statistics were done by one-way ANOVA with multiple-comparison posttests (ns, P > 0.05; *, P < 0.05; **, P < 0.01). Error bars represent SEM.

FIG 4

Plasma from HCV-infected patients is sufficient to increase CD47 expression on monocyte-derived DCs. (A) Quantitative comparison of CD47 CyTOF MFI of monocyte-derived DCs matured in culture medium containing plasma samples from the sofosbuvir HCV patient cohort before the initiation of treatment (Pre), midway through treatment (Mid), and after treatment (Post) compared to healthy controls. (B to D) Plasma collected from the sofosbuvir HCV patient cohort was analyzed by Luminex assays and normalized as described in Materials and Methods for TNF-α (B), CXCL10 (C), and IFN-α (D). (E) Comparison of CD47 MFI on HLA-DR⁺ DCs 72 h after stimulation in vitro with 10 μg/ml TNF-α, 100 μg/ml CXCL10, and 100 μg/ml IFN-α as single treatments or in combination. Statistics were done by one-way ANOVA with multiple-comparison posttests (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). Error bars represent SEM. Comparisons not labeled are not significantly different.

FIG 5

In vivo CD47 blockade in LCMV infection and CD47 genetic inactivation in M. tuberculosis infection. (A to C) Female C57BL/6 mice 8 to 12 weeks old were treated by intraperitoneal injection with either 100 μg anti-CD47 or an isotype control antibody at days −2, −1, 0, +1, and +2 relative to the day of intravenous infection (day 0 [D0]) with 2 × 10⁶ PFU LCMV-WE. (A) Mice were euthanized at 3 dpi, and LCMV PFU from spleen and liver were determined as described in Materials and Methods. (B) LCMV viremia levels were determined from blood samples by plaque-forming assays for control and anti-CD47-treated mice at 8 and 12 dpi. (C) CD8⁺ T cell counts in blood samples from control and anti-CD47-treated mice were analyzed by flow cytometry at 0, 8, 10, and 12 dpi. Statistics were done by unpaired two-way t tests (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001). Error bars represent SEM. (D) Human peripheral blood monocyte-derived macrophages from four different donors were infected in vitro with M. tuberculosis (Mtb) (pantothenate/lysine mutant strain) fluorescently labeled with pHrodo (to distinguish infected from uninfected cells) in triplicate and stained with anti-CD47 at 24 h postinfection. Flow cytometry was used to measure CD47 MFI in cells from uninfected cultures (naive) compared to both infected and uninfected cells from M. tuberculosis-infected cultures. Statistics were done by one-way ANOVA with multiple-comparison posttests (ns, P > 0.05; *, P < 0.05). (E) Both male and female C57BL/6 WT (n = 16) and C57BL/6.CD47^−/− (n = 23) mice were analyzed for survival (humane endpoints) following M. tuberculosis infection by inhalation. The difference between C57BL/6 WT and C57BL/6.CD47^−/− mice was statistically significant (****, P < 0.001 by a log rank Mantel-Cox test from pooled data from three independent experiments). (F) Analysis of M. tuberculosis CFU from lungs and spleens of endpoint animals. Statistical analyses were done by Student’s t tests, and two-sided P values are shown (*, P < 0.05; **, P < 0.01) with standard deviations.