Reciprocal relationship of T regulatory cells and monocytic myeloid-derived suppressor cells in LP-BM5 murine retrovirus-induced immunodeficiency

Abstract

Immunomodulatory cellular subsets, including myeloid-derived suppressor cells (MDSCs) and T regulatory cells (Tregs), contribute to the immunosuppressive tumour microenvironment and are targets of immunotherapy, but their role in retroviral-associated immunosuppression is less well understood. Due to known crosstalk between Tregs and MDSCs in the tumour microenvironment, and also their hypothesized involvement during human immunodeficiency virus/simian immunodeficiency virus infection, studying the interplay between these immune cells during LP-BM5 retrovirus-induced murine AIDS is of interest. IL-10-producing FoxP3+ Tregs expanded after LP-BM5 infection. Following in vivo adoptive transfer of natural Treg (nTreg)-depleted CD4+T-cells, and subsequent LP-BM5 retroviral infection, enriched monocytic MDSCs (M-MDSCs) from these nTreg-depleted mice displayed altered phenotypic subsets. In addition, M-MDSCs from LP-BM5-infected nTreg-depleted mice exhibited increased suppression of T-cell, but not B-cell, responses, compared with M-MDSCs derived from non-depleted LP-BM5-infected controls. Additionally, LP-BM5-induced M-MDSCs modulated the production of IL-10 by FoxP3+ Tregs in vitro. These collective data highlight in vitro and for the first time, to the best of our knowledge, in vivo reciprocal modulation between retroviral-induced M-MDSCs and Tregs, and may provide insight into the immunotherapeutic targeting of such regulatory cells during retroviral infection.

Fig. 1.

Induction of IL-10-producing FoxP3⁺ Tregs following LP-BM5 infection. FoxP3–GFP mice (a, b) or 10BiT mice (c–e) were infected with LP-BM5 and splenocytes were stained from uninfected (D0) and 14 days p.i. (D14) mice. All flow cytometry plots (a–c) are representative with accompanying histogram means ± sd (a–e) of three to four mice from a single experiment. All data are representative of at least three independent experiments (a–e). (a) Flow cytometry plots and frequency of GFP⁺(FoxP3⁺) cells of CD4⁺ cells. (b) GITR, CD39 and ICOS expression in GFP⁺(FoxP3⁺)CD4⁺ Tregs from D14 mice (shaded), D0 mice (dashed line) or fluorescence minus one (FMO) control (solid line). The accompanying histogram indicates the mean fluorescence intensities (MFI) of GFP⁺(FoxP3⁺)CD4⁺ Tregs. (c) Flow cytometry dot plots and histogram frequencies of Thy1.1⁺(IL-10⁺) of FoxP3⁺CD4⁺ cells. (d) Absolute numbers per spleen of CD4⁺FoxP3⁺Thy1.1⁺cells. (e) Thy1.1 (IL-10) expression by CD4⁺FoxP3⁺Thy1.1⁺ cells. *P < 0.05; **P < 0.01; ns, not significant.

Fig. 2.

AT of nTreg-depleted CD4⁺T-cells during LP-BM5 infection enhances the myeloid compartment. (a) AT protocol. Splenocytes were isolated from naı¨ve FoxP3–GFP mice, positively selected for CD4⁺T-cells, and remained unsorted or were sorted to deplete GFP⁺(FoxP3⁺)CD4⁺ Tregs. Purified CD4 T-cells (1 × 10⁷), non-depleted (CD4⁺ AT) or nTreg-depleted (nTreg-depl. CD4⁺ AT), underwent AT into naı¨ve TCRα^{− / −} mice. Recipient (Recip.) mice were infected, in parallel with B6 mice, 48 h after transfer with LP-BM5 retrovirus or left as uninfected controls. All mice were assessed at 5 weeks p.i. (b–d) All flow cytometry dot plots are representative, and the graphs are cumulative, with points indicating individual mice from all the experiments. Solid black horizontal lines indicate means. Flow cytometry plots and summary of CD11b⁺GR-1⁺ cells (b), CD11b⁺Ly6G⁺cells (c), CD11b⁺Ly6C⁺cells (d): uninfected B6 (▪) and TCRα − / −  (•) mice; infected B6 (▪), CD4⁺ AT (○), or nTreg-depleted CD4⁺ AT (shaded circle) mice. *P < 0.05; **P < 0.01; ns, not significant.

Fig. 3.

Depletion of nTregs during LP-BM5 infection alters the M-MDSC phenotype. M-MDSCs were enriched by Ly6G bead depletion, followed by CD11b bead enrichment, from individual mice at 5 weeks p.i. All flow cytometry dot plots (a, b) are representative and the accompanying graphs (a–c) sum up a total of six independent experiments, with points indicating individual mice. Solid black horizontal lines indicate means. (a) Flow cytometry dot plot and graphic summary of CD11b and Ly6C expression of M-MDSCs. (b) Ly6C expression and MFI of M-MDSCs: infected B6 (dotted line), CD4⁺ AT (dashed line), and nTreg-depleted CD4⁺ AT (shaded) mice, and isotype control (solid line). (c) Frequency of Ly6C^± /Lo, Ly6C^+/Mid and Ly6C^+/Hi M-MDSC subpopulations. The means ± sd of individual mice from six aggregated experiments are given. **P < 0.01; #P < 0.05 in comparison with B6; ‡P < 0.05 in comparison with CD4⁺ AT; ns, not significant.

Fig. 4.

Suppression of T-cell, but not B-cell, responses is enhanced by M-MDSCs from nTreg-depleted mice. M-MDSCs were co-cultured with naı¨ve responder cells in a suppression assay and stimulated with lipopolysaccharide (LPS) (B-cell) (a–d) or anti-CD3/CD28 (T-cell) (e–h), as described previously (Green et al., 2013). (a–d) Responder : suppressor cell (R : S) ratio of 1 : 0.33; (e–h) R : S of 1 : 0.166. All histograms represent the means ± sd of triplicate samples and are representative of at least three independent experiments. The percent suppression against B-cell (a) or T-cell (e) proliferative responses by M-MDSCs, as measured by incorporation of [³H]thymidine, is shown. The percent suppression (b, f) and effect on suppression (blockade) (c, g) by addition of the iNOS inhibitor l-NIL on B-cell (b, c) or T-cell (f, g) responsiveness is depicted. nd, No detectable suppression. Suppression assay-derived supernatants, from co-cultures of responder cells with B-cell (d) or T-cell (h) stimuli, per above, with or without M-MDSCs, were collected to measure the amount of NO production using Griess reagent for nitrite. nd, not detectable NO. *P < 0.05; **P < 0.01; ns, not significant.

Fig. 5.

LP-BM5-induced M-MDSCs modulate Treg function. Responder cells from naı¨ve FoxP3–GFP mice (a) or 10BiT mice (b, c) were stimulated with anti-CD3/CD28 and cultured alone (control) or co-cultured with M-MDSCs (+ MDSC) for 3 days. Flow cytometry contour plots are representative and bar graphs are the means ± sd of triplicate samples of a representative experiment. Results are representative of at least three independent experiments. (a) Flow cytometry contour plots and frequency of GFP⁺(FoxP3⁺) cells of all CD4⁺ cells, in cultures with no treatment (No Rx) or with the iNOS inhibitor l-NIL. (b) Flow cytometry contour plot and frequency of IL-10 production (Thy1.1) of FoxP3⁺CD4⁺ cells, in untreated (No Rx) and l-NIL-treated cultures. (c) MFI of Thy1.1 of FoxP3⁺CD4⁺Thy1.1⁺ cells in untreated (No Rx) and l-NIL-treated cultures. * P < 0.05; * * P < 0.01; NS, not significant.