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. 2017 Aug 23;14(1):42.
doi: 10.1186/s12977-017-0364-3.

Granulocytic Myeloid-Derived Suppressor Cells Suppress Virus-Specific CD8 + T Cell Responses During Acute Friend Retrovirus Infection

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Free PMC article

Granulocytic Myeloid-Derived Suppressor Cells Suppress Virus-Specific CD8 + T Cell Responses During Acute Friend Retrovirus Infection

Malgorzata Drabczyk-Pluta et al. Retrovirology. .
Free PMC article

Abstract

Background: Myeloid-derived suppressor cells (MDSCs) can suppress T cell responses in several different diseases. Previously these suppressive cells were observed to expand in HIV patients and in a mouse retrovirus model, yet their suppressive effect on virus-specific CD8+ T cells in vitro and in vivo has not been characterized thus far.

Results: We used the Friend retrovirus (FV) model to demonstrate that MDSCs expand and become activated during the late phase of acute FV infection. Only the subpopulation of granulocytic MDSCs (gMDSCs) but not monocytic MDSC suppressed virus-specific CD8+ T cell proliferation and function in vitro. gMDSCs expressed arginase 1, high levels of the inhibitory ligand PD-L1 and the ATP dephosphorylating enzyme CD39 on the cell surface upon infection. All three molecules were involved in the suppressive effect of the gMDSCs in vitro. MDSC depletion experiments in FV-infected mice revealed that they restrict virus-specific CD8+ T cell responses and thus affect the immune control of chronic retroviruses in vivo.

Conclusions: Our study demonstrates that MDSCs become activated and expand during the acute phase of retrovirus infection. Their suppressive activity on virus-specific CD8+ T cells may contribute to T cell dysfunction and the development of chronic infection.

Keywords: Arginase; CD8+ T cells; Friend virus; MDSC; PD-L1.

Figures

Fig. 1
Fig. 1
MDSCs expand during acute FV infection. B6 mice were infected i.v. with 20,000 SFFU of FV or left un-infected, and MDSC numbers were measured using flow cytometry. The total spleen cells were analyzed at various time points post infection. a The numbers of Ly6Ghigh Ly6Clow and b Ly6G Ly6Chigh per 1 × 106 live splenocytes and the frequencies of c CD80 expression on the surface of Ly6Ghigh Ly6Clow and Ly6G Ly6Chigh cells are displayed. At least 7 mice per group from five independent experiments were analyzed. Bars represent means with SD. For statistical analysis a Dunn’s test with the Benjamini–Hochberg correction for multiple testing (b) and an unpaired t test (c) were performed (*<0.05; **<0.005)
Fig. 2
Fig. 2
Granulocytic myeloid-derived suppressor cells inhibited CD8+ T cell proliferation. CD8+ T cells isolated from DbGagL TCR transgene mice were incubated with dendritic cells loaded with MHC class I-restricted FV-specific CD8+ T cell epitope peptide and co-incubated with either gMDSCs or mMDSCs (a). Representative histograms and percentages of CD8+ T cells after co-incubation with or without either gMDSC or mMDSCs (in relation 1 CD8+ to 10 MDSCs) from FV-infected mice are shown (b). CD8+ T cell proliferation was measured after co-incubation with different effector ratios of gMDSCs to CD8+ target cells (c). Frequencies of GzmB expressing CD43+CD8+ cells after incubation of CD8+ cells with gMDSCs or mMDSCs from FV-infected mice are shown (d). CD8+ T cells incubated with peptide loaded DC serve as a positive control, CD8+ T cells incubated with non-loaded DC serve as a negative control. At least three independent experiments were analyzed. Bars represent the mean with SD. For statistical analysis, an ANOVA multiple comparison test was carried out with the group of naïve mice as a reference (*<0.05; ***<0.0005). For statistical analysis a Dunn’s test with the Benjamini–Hochberg correction for multiple testing was performed (*<0.05; **<0.005)
Fig. 3
Fig. 3
Mechanisms of MDSC mediated suppression. B6 mice were infected i.v. with 20,000 SFFU of FV or left un-infected and the expression of Arg1 and NOS2 in the MDSCs was measured using flow cytometry for spleen cells at day 14 post infection. Frequencies of NOS2, Arg1, PD-L1, and CD39 expressing Ly6Ghigh Ly6Clow cells in the spleen are displayed (a). At least five mice per group from three independent experiments were analyzed. Bars represent the mean with SD. For statistical analysis an unpaired t test was performed (*<0.05; ***<0.0005). b CD8+ T cells isolated from DbGagL TCR transgenic mice were incubated with dendritic cells loaded with MHC class I-restricted FV-specific CD8+ T cell epitope peptide and co-incubated with gMDSCs in the ratio of 1 CD8+ to 10 gMDSCs with or without the addition of L-NMMA or nor-NOHA. Representative histograms and percentages of proliferating CD8+ T cell proliferation after co-incubation with or without gMDSCs in the presence or absence of L-NMMA/nor-NOHA are shown. At least three independent experiments were analyzed. Bars represent the mean with SD. c CD8+ T cells isolated from DbGagL TCR transgenic mice were incubated with dendritic cells loaded with MHC class I-restricted FV-specific CD8+ T cell epitope peptide and co-incubated with gMDSCs isolated from CD39 KO mice or from PD-L1 KO mice. Representative histograms and percentages of proliferating CD8+ T cells after co-incubation with or without gMDSCs isolated from B6, CD39 KO and PD-L1 KO FV infected mice are shown. At least three independent experiments were analyzed. The bars represent the mean with SD. For statistical analysis a Dunn’s test with the Benjamini–Hochberg correction for multiple testing was performed (*<0.05; **<0.005)
Fig. 4
Fig. 4
Depletion of MDSCs. B6 mice were infected i.v. with 20,000 SFFU of FV, and/or treated with 5FU or αLy6G. CD8+ numbers were measured using flow cytometry and viral loads were estimated for spleen cells at 14 days post infection. Experimental design of the MDSC depletion by administration of 5FU or αLy6G (a). b Representative dot plots of MDSC during FV infection after administration of 5FU, αLy6G or untreated. c The numbers of CD43+ CD8+ T cells, the numbers of FV-DbgagL class I tetramers positives CD8+CD43+, and the numbers of proliferating Ki67+CD43+CD8+ T cells per 1 × 106 live cells in the spleen are displayed. d The numbers of CD43+ CD8+ T cells expressing GzmB per 1 × 106 live cells in the spleen are displayed. e The numbers of viral loads (infectious centers per 1 × 106 cells) in the spleen are displayed. At least five mice per group from three independent experiments were analyzed. Bars represent the mean with SD. For statistical analysis a Dunn’s test with the Benjamini–Hochberg correction for multiple testing was performed (*<0.05; **<0.005)

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