Plasmodium infection inhibits the expansion and activation of MDSCs and Tregs in the tumor microenvironment in a murine Lewis lung cancer model

Abstract

Background: A major challenge in the development of effective cancer immunotherapy is the ability of tumors and their microenvironment to suppress immune cells through immunosuppressive cells such as myeloid -derived suppressor cells and regulatory T cells. We previously demonstrated that Plasmodium infection promotes innate and adaptive immunity against cancer in a murine Lewis lung cancer model but its effects on immunosuppressive cells in the tumor microenvironment are unknown.

Methods: Whole Tumors and tumor-derived sorted cells from tumor-bearing mice treated with or without plasmodium infected red blood cells were harvested 17 days post tumor implantation and analyzed using QPCR, western blotting, flow cytometry, and functional assays. Differences between groups were analyzed for statistical significance using Student's t-test.

Results: Here we found that Plasmodium infection significantly reduced the proportions of MDSCs and Tregs in the lung tumor tissues of the treated mice by downregulating their recruiting molecules and blocking cellular activation pathways. Importantly, CD8+ T cells isolated from the tumors of Plasmodium-treated mice exhibited significantly higher levels of granzyme B and perforin and remarkably lower levels of PD-1.

Conclusion: We reveal for the first time, the effects of Plasmodium infection on the expansion and activation of MDSCs and Tregs with a consequent elevation of CD8+T cell-mediated cytotoxicity within the tumor microenvironment and hold great promise for the development of effective immunotherapeutic strategies.

Keywords: Lung cancer; MDSC; PD-1; Recruiting molecules; Tregs.

Conflict of interest statement

Ethics approval

The animal experiment facilities were approved by the Guangdong Provincial Department of Science and Technology, and complied with the guidelines of the Animal Care Committee, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. All efforts were made to minimize animal suffering.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Downregulation of the population of MDSCs in the tumor microenvironment by Plasmodium. a Gating protocol for the identification of MDSC subsets. b Gated polymorphonuclear and monocytic MDSCs in the tumor tissues of uninfected tumor-bearing mice (LR, control). c Gated polymorphonuclear and monocytic MDSCs in the tumor tissues of Py-infected tumor-bearing mice (treated, LP). d Comparison of the total MDSC population in the tumor tissues of both the treated and the control groups. e The relative proportions of polymorphonuclear and monocytic MDSC subsets in the tumor tissues of tumor-bearing mice treated with infected and uninfected RBCs. f Tumors harvested from tumor-bearing mice treated or untreated with Plasmodium 18 days after tumor inoculation. A total of 50,000 live events was recorded and analyzed per sample. The average number of MDSCs gated out were 2000 cells and 5000 cells for (LP) and (LR) respectively

Fig. 2

Effects of Plasmodium on the population of Tregs in the tumor microenvironment. a Gating protocol for CD4+ cells. b Gated CD25+FOXP3+ Treg population in untreated tumor tissues. c Gated CD25+FOXP3+ Treg population in treated tumor tissues. d The populations of Tregs in the tumors were compared. Plasmodium infection significantly downregulated the populations of Tregs in the tumor tissues of tumor-bearing mice compared to the control mice. ***P < 0.001

Fig. 3

Expression levels of key MDSC- and Treg-recruiting cytokines and chemokines in the tumor tissues assessed by qRT-PCR and western blotting. The relative mRNA expression levels of GM-CSF (a), G-CSF (b), M-CSF (c), IL-1β (d), VEGF (e), IL-6 (f), IFN-g (g) IL-14 (h), IL-13 (i), CCL-17 (i) TGF-β (j), CCL-17 (k), and CCL-22 (l) in the tumor tissues of Py-infected tumor-bearing mice compared with those of the control mice. Western blot results of CCR4 protein expression in the tumor tissues (m). β-Actin was used as a loading control. Plasmodium infection significantly decreased the relative mRNA levels of key MDSC and Treg cytokines and chemokines in the tumor tissues of Py-infected tumor-bearing mice compared to the control mice

Fig. 4

Effects of Plasmodium infection on MDSC signal transduction and downstream protein expression. The expression levels of pSTAT1, pSTAT3, pSTAT5, pSTAT6, NF-κB, Survivin, and S100A9 were assessed by qRT-PCR and western blot analysis. a The protein expression levels of pSTAT1, pSTAT3, pSTAT5, pSTAT6, NF-KB, Survivin and S100A9 in MDSCs from the infected and control mice, as determined by western blotting. b, c qRT-PCR analysis of Survivin and S100A9 expression of the sorted MDSCs. d The protein expression of pSTAT3 in the two MDSC subsets was assessed. (e) Purity of the sorted MDSCs. β-Actin was used as a loading control for both the qRT-PCR and western blot analysis. *P < 0.05. MM, monocytic MDSC; PM, polymorphonuclear MDSC

Fig. 5

Effects of Plasmodium infection on the expression of immunosuppressive molecules by sorted MDSCs. The MDSC expression of IL-10, arginase 1, NOS2, and ROS was assessed by qRT-PCR and functional assays. Relative mRNA expression levels of IL-10 (a), NOS2 (b), and arginase 1 (c) of MDSCs isolated from the tumor tissues of Py-treated and untreated tumor-bearing mice. The levels of ROS (d) and arginase activity (e) were detected using a DCFDA Cellular ROS Detection Assay Kit (Abcam; cat. # ab113851) and Arginase Activity Assay Kit (Abcam; cat. # ab180877), respectively. **P < 0.01, ****P < 0.0001

Fig. 6

Effects of Plasmodium infection on CD8+ T cells in the tumor microenvironment. a Flow cytometry gating protocol for CD8+ T cell sorting. b Comparison of the mRNA expression of PD-1 on sorted CD8+ T cells. c mRNA expression of granzyme B (d) and perforin by the sorted CD8+ T cells. e Protein expression of PD-1 on sorted CD8+ T cells determined by western blotting. **P < 0.01

Fig. 7

Plasmodium infection inhibits tumor cytokines and chemokines secretions through exosomes-like vesicles. To determine whether or not cell-to-cell contact was required for the inhibition of tumor cytokines, we co-cultured LLC cells with either infected RBCs (Li), uninfected RBCs (LRB), exosomes isolated from uninfected mice (LN), exosomes isolated from Py-infected mice (LP), exosomes isolated from tumor-bearing mice infected with Py (LLP), or plasma from Py-infected mice after exosomes have been isolated (LPL). Cells were cultured for either 24 or 48 h and subsequently harvested for RNA extraction and QPCR analysis. Our QPCR results suggest that infected RBCs does not communicate with tumor cells through cell-to-cell contact. Exosome-like vesicles significantly inhibited tumor release of GMCSF(a), IL-10(b), IL-6(c), CCL-17(d), and CCL-22(e) at the mRNA levels