CD47 restricts antiviral function of alveolar macrophages during influenza virus infection

CD47 is an ubiquitously expressed surface molecule with significant impact on immune responses. However, its role for antiviral immunity is not fully understood. Here, we revealed that the expression of CD47 on immune cells seemed to disturb the antiviral immune response as CD47-deficient mice (CD47−/−) showed an augmented clearance of influenza A virus (IAV). Specifically, we have shown that enhanced viral clearance is mediated by alveolar macrophages (aMФ). Although aMФ displayed upregulation of CD47 expression during IAV infection in wildtype mice, depletion of aMФ in CD47−/− mice during IAV infection reversed the augmented viral clearance. We have also demonstrated that CD47 restricts hemoglobin (HB) expression in aMФ after IAV and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, with HB showing antiviral properties by enhancing the IFN-β response. Our study showed a negative role for CD47 during antiviral immune responses in the lung by confining HB expression in aMФ

Influenza virus infection enhances tumour-specific CD8+ T-cell immunity, facilitating tumour control.

Influenza A virus (IAV) can cause severe respiratory infection leading to significant global morbidity and mortality through seasonal epidemics. Likewise, the constantly increasing number of cancer diseases is a growing problem. Nevertheless, the understanding of the mutual interactions of the immune responses between cancer and infection is still very vague. Therefore, it is important to understand the immunological cross talk between cancer and IAV infection. In several preclinical mouse models of cancer, including melanoma and colorectal cancer, we observed that IAV infection in the lung significantly decreased the tumour burden. Concomitantly, tumour-specific CD8+ T-cells are strongly activated upon infection, both in the tumour tissue and in the lung. CD8+ T-cell depletion during infection reverses the reduced tumour growth. Interestingly, IAV infection orchestrated the migration of tumour-specific CD8+ T-cells from the tumour into the infected lung. Blocking the migration of CD8+ T-cells prevented the anti-tumoural effect. Thus, our findings show that viral respiratory infection has significant impact on the anti-tumour CD8+ T-cell response, which will significantly improve our understanding of the immunological cross talk between cancer and infection

IAV infection suppresses tumour growth.

(a-d) 1 x 105 B16F1 (B16) tumour cells were transplanted subcutaneously (s.c.) into the right flank per mouse. 4 days after tumour transplantation, mice were infected intranasally with 150 PFU/mL Influenza A/PR/8/34 (IAV). (a) Viral load was measured by M1 expression relative to RPS9 expression in the lungs of infected mice with or without B16 tumour 8 days post infection (dpi). n = 9 for B16+IAV, n = 6 for IAV (b) Viral load as measured from homogenized lung supernatants by counting plaque forming units (PFU) from plaque assay 8 dpi. (c) Body weight change after IAV infection. Data from 2 experiments with 4–6 mice per group per experiment. (d-g) 1 x 105 B16 (d and g), CT26 (e) or 5 x 105 Lewis Lung Carcinoma (LLC) (f) tumour cells were transplanted s.c. and mice were infected as described in a) with (d-f) IAV or (g) inactivated IAV. The tumour volume was measured everyday once palpable upon infection. d&e: Data from 4 experiments with 3–4 mice per group per experiment, f: n = 4, g: Data from 2 experiments with 4 mice per group per experiment. (h) Viral load as measured by M1 expression relative to RPS9 expression in the tumour or lung of infected mice 12 days post tumour transplantation. (i) B16 tumours were transplanted s.c. with 1 x 105 tumour cells and mice were infected intravenously with Friend Virus (FV) 4 days post tumour transplantation. The tumour volume was scored everyday once palpable. n = 4. Error bars represent SEM. Statistical tests on tumour growth development were performed as Two-way-ANOVA, in Sidak’s multiple comparisons test when two groups were compared or Tukey’s multiple comparisons test when more than two groups were compared. Viral titres were compared with non-parametric t tests. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001, ns = not significant.</p

Infection at a later timepoint still enables tumour growth restriction but previous infection reverses the effect.

(a) B16F1 (B16) tumour cells were injected into mice. Influenza A/PR/8/34 (IAV) infection followed either 4 days (“early infection”) or 10 days (“late infection”) post tumour transplantation. (b) Tumour growth is shown. Data from 2 experiments with 3–4 mice per group per experiment. (c) Frequencies of Granzyme B (GzmB) expressing CD8+ T-cells in the tumour 18 (late) or 12 (early) days post tumour cell injection. (d) Frequencies of GzmB expressing CD8+ T-cells in the lung 18 (late infection) or 12 (early infection) days post tumour cell injection. (e) Mice were infected 3 days before (d-3) or 4 days post tumour transplantation. Data from 2 experiments with 4 mice per group per experiment. Error bars represent SEM. Statistical tests between two groups were performed as Student’s t-tests. Statistical tests on tumour growth development were performed as Two-way-ANOVA, followed by Tukey’s multiple comparisons test. * = p<0.05, ** = p<0.01.</p

FV infection induced splenomegaly.

Spleen weights were taken 12 days after tumour cell transplantation and 8 days after infection, respectively. 4 mice per group Error bars represent SEM. Significance was tested in Tukey’s multiple comparisons test. * = p (TIF)</p

Tumour-specific CD8⁺ T-cells are recruited to the IAV-infected lung.

(a) Cytokine concentrations in the tumour and lung were analysed by Luminex analysis 12 days post tumour cell injection in mice infected with IAV. (b) CT26 tumours were transplanted into BALB/c mice, which were then infected with Influenza A/PR/8/34 (IAV). CT26 tumour derived CD8+ T-cells from Thy1.1 mice were adoptively transferred into infected and non-infected mice. (c) Two days after adoptive transfer, the numbers of Thy1.1+ CD8+ T-cells were analysed in the lung, spleen and tumour. (d) B16 tumour-bearing mice were infected with IAV (150 PFU/mL) as described. Mice were treated with FTY720 every second day upon infection to avoid lymphocyte egress from lymphoid organs. Data from 2 experiments with 4 mice per group per experiment. Error bars represent SEM. Statistical tests between two groups were performed as Student’s t-tests. Statistical tests on tumour growth development were performed as Two-way-ANOVA, in Tukey’s multiple comparisons test. * = p<0.05, *** = p<0.001, **** = p<0.0001. ns = not significant.</p

Individual tumour size developments.

Tumour growth from each individual mouse based on experiments shown in Fig 1. (TIF)</p

CXCL9 and CXCL11 are not enhanced in the tumour after IAV infection.

B16 tumours were transplanted and mice were infected as described in Fig 1. RNA was extracted from tumours and lungs 12 days post tumour transplantation, corresponding with 8 days post infection. mRNA levels of Cxcl9 (a) or Cxcl11 (b) were measured relative to Rps9 levels. n.d. = not detectable. Error bars represent SEM. (TIF)</p

IAV infection suppresses tumour growth dose dependently.

1 x 105 B16F1 (B16) tumour cells were transplanted subcutaneously (s.c.) into the right flank per mouse. 4 days after tumour transplantation, mice were infected intranasally with Influenza A/PR/8/34 (IAV). The tumour volume was measured everyday once palpable upon infection. Data from 2 experiments with 3–4 mice per group per experiment. Error bars represent SEM. Statistical tests on tumour growth development were performed as Two-way-ANOVA followed by Tukey’s multiple comparisons test. **** = p (TIF)</p

IAV infection impedes differentiation of T-cell exhaustion.

B16F1 (B16) tumour-bearing mice were infected with Influenza A/PR/8/34 (IAV) (150 PFU/mL) as described. 8 days post infection mice were sacrificed for flow cytometry analysis. (a) Frequencies of PD-1 expressing gp100-specific CD8+ T-cells in the tumour. (b) Representative dot plots of CD8+ T-cells expressing PD-1 and TIM-3. (c) Frequencies of PD-1high TIM-3+ or PD-1int TIM-3- of gp100-specific CD8+ T-cells in the tumour. (d) Frequencies of Granzyme B (GzmB) expressing cells of the populations described in c. (e) Frequencies of Thymocyte selection-associated high mobility group box protein (TOX) expressing gp100-specific CD8+ T-cells in the tumour. Data from 2 experiments are shown. Error bars represent SEM. Statistical tests between two groups were performed as Student’s t-tests. * = p<0.05, ** = p<0.01, *** = p<0.001.</p