Targeting myeloid-derived suppressor cells promotes antiparasitic T-cell immunity and enhances the efficacy of PD-1 blockade

Zhang, Chuanshan; Wang, Hui; Aji, Tuerganaili; Li, Zhide; Li, Yinshi; Ainiwaer, Abidan; Rousu, Zibigu; Li, Jing; Wang, Maolin; Deng, Bingqing; duolikun, Adilai; Kang, Xuejiao; Zheng, Xuran; Yu, Qian; Shao, Yingmei; Zhang, Wenbao; Vuitton, Dominique A.; Tian, Zhigang; Sun, Haoyu; Wen, Hao

doi:10.1038/s41467-024-50754-7

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Published: 27 July 2024

Targeting myeloid-derived suppressor cells promotes antiparasitic T-cell immunity and enhances the efficacy of PD-1 blockade

Chuanshan Zhang ORCID: orcid.org/0000-0002-3241-2224^1,2,3^na1,
Hui Wang ORCID: orcid.org/0000-0002-9308-6186^1,3,4^na1,
Tuerganaili Aji^3,5^na1,
Zhide Li¹^na1,
Yinshi Li^1,2,
Abidan Ainiwaer^1,2,
Zibigu Rousu^1,2,
Jing Li²,
Maolin Wang^1,5,
Bingqing Deng^1,2,
Adilai duolikun^1,2,
Xuejiao Kang^1,2,
Xuran Zheng^1,2,
Qian Yu^1,2,
Yingmei Shao⁵,
Wenbao Zhang^1,4,
Dominique A. Vuitton⁶,
Zhigang Tian ORCID: orcid.org/0000-0002-5512-6378⁷,
Haoyu Sun ORCID: orcid.org/0000-0002-7506-8806^7,8,9 &
…
Hao Wen ORCID: orcid.org/0000-0001-7144-220X¹

Nature Communications volume 15, Article number: 6345 (2024) Cite this article

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Abstract

Immune exhaustion corresponds to a loss of effector function of T cells that associates with cancer or chronic infection. Here, our objective was to decipher the mechanisms involved in the immune suppression of myeloid-derived suppressor cells (MDSCs) and to explore the potential to target these cells for immunotherapy to enhance checkpoint blockade efficacy in a chronic parasite infection. We demonstrated that programmed cell-death-1 (PD-1) expression was significantly upregulated and associated with T-cell dysfunction in advanced alveolar echinococcosis (AE) patients and in Echinococcus multilocularis-infected mice. PD-1 blockade ex vivo failed to reverse AE patients’ peripheral blood T-cell dysfunction. PD-1/PD-L1 blockade or PD-1 deficiency had no significant effects on metacestode in mouse model. This was due to the inhibitory capacities of immunosuppressive granulocytic MDSCs (G-MDSCs), especially in the liver surrounding the parasite pseudotumor. MDSCs suppressed T-cell function in vitro in an indoleamine 2, 3 dioxygenase 1 (IDO1)-dependent manner. Although depleting MDSCs alone restored T-cell effector functions and led to some limitation of disease progression in E. multilocularis-infected mice, combination with PD-1 blockade was better to induce antiparasitic efficacy. Our findings provide preclinical evidence in support of targeting MDSC or combining such an approach with checkpoint blockade in patients with advanced AE.

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Introduction

Immune exhaustion corresponds to a loss of effector function of T cells that is associated with cancer or chronic infection¹. Exhausted T cells exhibit high and sustained expression of inhibitory molecules such as cytotoxic T-lymphocyte-associated protein 4 (CTLA4), programmed cell death 1 (PD-1), T-cell immunoreceptor with Ig and ITIM domains (TIGIT) and lymphocyte activation gene-3 (LAG3)². Interference with checkpoint inhibitors has revolutionised the field of cancer therapy^3,4,5. However, resistance to checkpoint blockade may limit the efficacy of these treatments, and the mechanisms involved in this resistance are not fully understood⁶. Parasite infections may be used as models to further decipher the mechanisms of resistance and thus find complementary treatments that can restore sensitivity to checkpoint blockade. Parasites such as Schistosoma mansoni (S. mansoni), Leishmania infantum, and Taenia crassiceps (T. crassiceps) have been shown to suppress effective immune responses by upregulating PD-1/PD-1 ligands (PD-L1) on host macrophages or myeloid-derived suppressor cells (MDSCs), which, in turn, inhibit activated T cells⁷. Recent reports have shown that Echinococcus multilocularis (E. multilocularis) infection is associated with the expression of “checkpoint” receptors which limit the activity of parasite-specific lymphocytes^8,9,10. Upregulation of PD-1 is associated with immunosuppression in the livers of infected mice, and in vivo PD-1 blockade enhances host immune responses to control E. multilocularis growth at the early and middle stages of infection^11,12; however, the efficacy of PD-1 or PDL1 blockade at late stages of infection remains to be demonstrated. Our recent data also showed that TIGIT and LAG3 function as checkpoints to mediate T-cell exhaustion in the process of E. multilocularis immune escape at the late stage of infection^9,10. In addition, we found that TIGIT⁺ T cells exhibited increased PD-1 expression in patients with alveolar echinococcosis (AE)⁹. AE is a zoonotic parasitic disease caused by the larvae of E. multilocularis that seriously endangers human health¹³. AE is characterised by a slow but progressive tumour-like growth of metacestodes (larval stage) mainly in the liver¹⁴. The role of immunosuppression in the control of E. multilocularis development has been demonstrated in experimental models and in individuals with impaired immunity, such as AIDS patients or patients who underwent transplantation or were treated with immunosuppressive drugs or biological agents¹⁴. The variable clinical outcomes of AE patients depend on the immunological status of host cells, and we and others found that the specific immunological profile associated with T-cell functional impairment plays an important role in the established tolerance state in chronic AE^8,15,16. Currently, AE treatment relies only on surgery and albendazole antiparasitic treatment. In addition to allowing us to study the mechanisms of checkpoint blockade, using the ‘E. multilocularis model’ may provide a therapeutic paradigm for treating AE patients.

Multiple suppressive mechanisms, including the accumulation of MDSCs, have been implicated in chronic inflammation and also play a major role in the negative regulation of immune responses during infection and cancer^6,17. Mounting evidence has shown that MDSCs, which are the major immunosuppressive cells residing within the hepatic immune microenvironment, are characterised by their ability to contribute to T-cell exhaustion through multiple pathways in chronic inflammation, such as the secretion of arginase 1 (Arg-1), nitric oxide synthase 2 (Nos2 or iNOS) and indoleamine 2, 3-dioxygenase (IDO) or the expression of immunoregulatory molecules^{18,19,20,21,22}. An increase in the MDSC count is related to PD-1 or PD-L1 inhibitor resistance during cancer, infection, and autoimmunity^23,24,25. Furthermore, MDSCs expand during helminth infection and may contribute to the immunosuppressive and chronic characteristics of infections caused by S. japonicum, T. crassiceps or Heligmosomoides polygyrus bakeri²⁶. Interfering with the function of MDSC alone or in combination with checkpoint blockade may represent a potentially effective therapeutic approach for eliminating adult worms and inducing immunity to reinfection²⁶. Previous studies have indicated that MDSCs play a key role in downregulating the immune response of T cells in a mouse model of Echinococcus granulosus (E. granulosus) infection^27,28. However, the involvement of MDSCs in T-cell dysfunction and their role in resistance to checkpoint blockade has never been studied in E. multilocularis infection.

In this study, we analysed the PD-1 expression profile and functional roles of PD-1 in intrahepatic and peripheral T cells in both AE patients and E. multilocularis-infected mice, and then defined the key roles of MDSCs and the resistance mechanisms to PD-1 blockade in anti-echinococcal immunity. We demonstrated that PD-1 blockade failed to reverse the functional exhaustion of T cells in AE patients ex vivo and failed to inhibit liver metacestode growth, due to the compensatory accumulation of immunosuppressive granulocytic MDSCs (G-MDSCs) in the inflamed liver microenvironment in patients with AE and model mice. Depletion of MDSCs alone enhances the T-cell response. In combination with PD-1 blockade, it also significantly reduces metacestode growth. In addition, we found the effects of IDO1 expression in MDSCs on the immunosuppression of T cells in vitro. Our study suggests that such combination therapy or even depleting MDSCs alone represents a promising therapeutic approach for patients with late-stage AE.

Results

High expression of PD-1 and functional exhaustion of T cells in AE patients

We performed a 10 × 5’ single-cell RNA sequencing (scRNA-seq) analysis of CD45⁺ cells from four donors with AE using paired liver tissues and peripheral blood (blood) from a public database described previously (accession no. HRA000553; https://figshare.com/s/f2a4578be27c2cb767f9). We observed 8 major cell subtypes, namely, CD4⁺ T cells, CD8⁺ T cells, B cells, NK cells, NKT cells, monocytes, MDSCs_like and macrophage, each of which were identified by their signature genes (Fig. 1a–d). It was apparent that there were higher proportions of T and NK cells than of other cell subsets in the liver and of monocyte cells in the blood. The results showed that the inhibitory molecule PD-1 was mainly expressed in liver-derived T lymphocytes and exhausted CD8⁺ T-cell subsets, exhibiting the highest expression in both the liver tissue close to the parasitic lesions (close liver tissue [CLT]) and distant to the lesions (distant liver tissue [DLT]) (Fig. 1e, related statistics). Among the T-cell subclusters, CD4_c12_PDCD1 and CD8_c5_PDCD1, corresponding to exhausted CD4⁺ T cells and exhausted CD8⁺ T cells, respectively, were enriched in the CLT and DLT (Fig. 1f–i, related statistics).

**Fig. 1: Single-cell RNA sequencing analysis of PD-1 expression and distribution in T-cell subsets from the PB, DLT and CLT in AE patients.**

We then confirmed the gene expression of PD-1 and its ligands PD-L1 and PD-L2 at the mRNA level by quantitative real-time polymerase chain reaction (qRT-PCR) in liver samples from AE patients. Our results showed that the PD-1, PD-L1 and PD-L2 genes were upregulated in the CLT of AE patients (Supplementary Fig. S1). We also evaluated the expression of PD-1 in T cells through flow cytometry. The percentage, mean fluorescence intensity (MFI) and absolute count of PD-1⁺CD4⁺ T cells and PD-1⁺CD8⁺ T cells were significantly higher in the CLT than in the DLT according to paired analysis (Fig. 2a, b). PD-1 was colocalized with CD4⁺ T cells and CD8⁺ T cells in the CLT, as determined by multiple-colour fluorescence staining of snap-frozen tissue sections (Fig. 2c).

**Fig. 2: PD-1 expression and dysfunction of liver T cells in AE patients.**

To understand the influence of PD-1 expression on T-cell functionality, we examined the cytokine profiles of the T cells in the CLTs of AE patients. We observed that liver-infiltrating PD-1⁺CD4⁺ T cells and PD-1⁺CD8⁺ T cells produced less interferon-gamma (IFN-γ) and tumour necrosis factor-α (TNF-α) than PD-1^-CD4⁺ T cells and PD-1^-CD8⁺ T cells, respectively (Fig. 2d–e). Moreover, we found that peripheral blood CD4⁺ T cells and CD8⁺ T cells from AE patients also expressed higher levels of PD-1 than those from healthy donors (HDs) (Fig. 3a, b). In addition, in the blood of AE patients, PD-1⁺CD4⁺ T cells and PD-1⁺CD8⁺ T cells expressed lower levels of cytokines such as granzyme B, IFN-γ, and TNF-α than PD-1^-CD4⁺ T cells and PD-1^-CD8⁺ T cells respectively (Fig. 3c, d). These results suggested that liver-infiltrating and blood T cells from AE patients expressed higher PD-1 levels, which was associated with functional impairment.

**Fig. 3: PD-1 expression and dysfunction of peripheral blood T cells in AE patients.**

High PD-1 expression correlates with the functional exhaustion of T cells in murine E. multilocularis infection

In experimental mice, infiltrating lymphocytes surrounding the E. multilocularis inoculum could only be observed at week 2 post-infection, and few protoscoleces (PSCs) from the hepatic portal vein inoculum were still present. At weeks 12 and 24 post-infection, the parasitic pseudotumor mass included many vesicular structures embedded within thick fibrous tissue, and the periphrastic area was composed of inflammatory cells and fibrous tissue (Fig. 4a).

**Fig. 4: High PD-1 expression and dysfunction of liver T cells in a mouse model of *E. multilocularis* infection.**

We investigated PD-1 expression on T cells in the livers of infected mice. The percentage of PD-1⁺CD4⁺ T cells was significantly increased at weeks 2, 12 and 24, and there was a higher percentage of PD-1⁺CD4⁺ T cells after 24 weeks; moreover, PD-1 expression on CD8⁺ T cells was significantly elevated at 24 weeks (Fig. 4b, c). In addition, the PD-1 MFI of T cells (both CD4⁺ and CD8⁺) was higher in infected mice than in uninfected mice at week 24 (Fig. 4d). Furthermore, we analysed the activation and proliferation of CD4⁺ T cells and CD8⁺ T cells via the expression of CD44, CD69 and Ki67, and found that PD-1⁺ T cells (both CD4⁺ and CD8⁺) were more activated and proliferative than their PD-1^- counterparts after 24 weeks of infection (Fig. 4e–g). We also found that a lower percentage of liver-infiltrating PD-1⁺ T cells (both CD4⁺ and CD8⁺) expressed IFN-γ and TNF-α than PD-1- T cells (Fig. 4h, i).

Consistent with our findings in liver T cells, the percentage of splenic PD-1⁺ T cells (both CD4⁺ and CD8⁺) increased at 24 weeks after E. multilocularis infection (Supplementary Fig. S2a, b). In addition, the PD-1 MFI of CD4⁺ T cells was higher in infected mice than in control mice at week 24 (Supplementary Fig. S2c). Splenic PD-1⁺ T cells (both CD4⁺ and CD8⁺) were more activated and proliferative than their PD-1^- counterparts in mice after 24 weeks of infection (as indicated by the expression of CD69, CD44 and Ki67; Supplementary Fig. S2d–f). Furthermore, splenic PD-1⁺ T cells (both CD4⁺ and CD8⁺) showed significantly impaired cytokine secretion. However, the percentage of splenic PD-1⁺ T cells (both CD4⁺ and CD8⁺) that expressed granzyme B and CD107α was higher than that of PD-1^- T cells (Supplementary Fig. S2g, h). Collectively, these results indicated that high PD-1 expression was associated with functional exhaustion of liver and splenic T cells (both CD4⁺ and CD8⁺) in late-stage E. multilocularis-infected mice.

PD-1 blockade or deficiency has no direct effect on disease progression or T-cell function in murine E. multilocularis infection

To further evidence the role of PD-1 on T cells (both CD4⁺ and CD8⁺) during E. multilocularis infection, we treated E. multilocularis-infected mice with anti-PD-1 mAb or anti-PD-L1 mAb. Anti-PD-1 or anti-PD-L1 blockade alone failed to inhibit the growth of metacestodes (Supplementary Figs. S3a–c, S4a–c), although the percentages of single-positive TNF-α and double-positive TNF-α and IL-2 CD4⁺ T cells and the percentage of double-positive IFN-γ and TNF-α CD8⁺ T cells were increased in the livers of anti-PD-1-treated mice (Supplementary Figs. S3d, S5). The percentages of single-positive IL-2 and double-positive TNF-α and IL-2 CD4⁺ T cells and the percentages of single- or double-positive IFN-γ and IL-2, double-positive IFN-γ and TNF-α CD8⁺ T cells, and of CD8⁺ T cells expressing granzyme B, IFN-γ, TNF-α and IL-2, were increased in the spleens of anti-PD-L1-treated mice (Supplementary Fig. S4d). However, the percentages of double-positive TNF-α and IL-2 splenic T cells (both CD4⁺ and CD8⁺) and the percentage of single-positive TNF-α splenic CD8⁺ T cells were significantly lower in anti-PD-1-treated mice than in WT mice (Supplementary Fig. S3e). In addition, PD-1 blockade did not affect the expression of CD69 on T cells or the phenotype of Tem cells (both CD4⁺ and CD8⁺) (Supplementary Fig. S3f–i).

Consistent with the results observed in PD-1-blockade mice, we found that PD-1 deficiency did not inhibit liver metacestode growth in infected PD-1^-/- mice at 2, 12 and 24 weeks. Whole liver weight and lesion weight were not significantly different between PD-1^-/- mice and wild-type (WT) mice (Fig. 5a-c). PD-1^-/- mice exhibited a significantly higher percentage of CD69⁺CD4⁺ T cells in the liver than WT mice at 24 weeks post-infection. In contrast, PD-1 deficiency did not affect the expression of CD69 on CD8⁺ T cells or the phenotype of effector memory CD44⁺CD62L^- T cells (Tem, both CD4⁺ and CD8⁺) (Fig. 5d, e), which play an essential role in protecting against parasitic infections through its own effector functions such as cytotoxicity and cytokine secretion^29,30,31. The percentage of single-positive IFN-γ CD4⁺ T cells was increased in the livers of PD-1-deficient mice (Fig. 5f). However, the percentages of single- or double-positive TNF-α and IL-2 CD4⁺ T cells and the percentages of double-positive IFN-γ and IL-2 and triple-positive IFN-γ, TNF-α and IL-2 CD8⁺ T cells were significantly lower in the livers of PD-1-deficient mice than in those of WT mice (Fig. 5f). Compared with those in WT mice, splenic T cells (both CD4⁺ and CD8⁺) in PD-1^-/- mice showed no significant activation, effector memory phenotype or degranulation (Supplementary Fig. S6a, b). The percentage of double-positive TNF-α and IL-2 CD4⁺ T cells was significantly lower in the spleens of PD-1-deficient mice than in those of WT mice (Supplementary Fig. S6c).

**Fig. 5: PD-1 deficiency does not influence disease progression or T-cell dysfunction in a mouse model of *E. multilocularis* infection.**

Importantly, to determine whether in vitro blockade of PD-1 on T cells could reactivate their functional responsiveness, peripheral blood mononuclear cells (PBMCs) were isolated from AE patients and restimulated with anti-CD3e/CD28 in the presence of anti-PD-1 mAb. No significant increase in IFN-γ or TNF-α levels was observed in CD4⁺ T cells or CD8⁺ T cells upon the addition of anti-PD-1 mAb, indicating that PD-1 blockade could not restore cytokine secretion by exhausted CD4⁺ T cells or CD8⁺ T cells from AE patients ex vivo (Supplementary Fig. S7). Taken together, these data indicated that PD-1 blockade or deficiency could not reverse the exhaustion of T cells in AE patients or mouse models to enhance anti-echinococcal immunity.

MDSCs accumulation and T-cell suppression in AE patients and murine E. multilocularis infection

To determine whether myeloid cell recruitment contributes to immune suppression by inhibiting the T-cell response, we analysed mRNA expression data in paired liver tissue (CLT versus DLT) from 6 AE patients studied in our previous report (accession no. GSE124362). Gene set enrichment analysis (GSEA) revealed a marked upregulation of genes associated with myeloid leukocyte migration in the CLT compared to the DLT (Supplementary Fig. S8a). These upregulated genes were associated with myeloid cell chemotaxis, such as CCL1, CCL2, and CXCR2; myeloid cell activation and expansion, such as VEGFA, S100A8, S100A9, and S100A12; and myeloid leukocyte differentiation, such as CSF3R (Supplementary Fig. S8b). Furthermore, Gene Ontology (GO) analysis revealed that the upregulated genes, including CD33, S100A8, S100A9, IL-10, IDO1, and NOS2 in the CLT from AE patients were enriched in myeloid leukocyte migration, myeloid leukocyte activation, myeloid leukocyte-mediated immunity, which are related to the characteristics, recruitment and immunosuppressive function of MDSCs (Supplementary Fig. S8c–e). Our results also showed that the phenotype and functional markers of G-MDSCs (e.g., CD33, CD11b, CD15, IDO1, S100A8, S110A9, and S100A12) were upregulated at the mRNA level in the whole liver cell mixture from CLTs (Supplementary Fig. S1). Moreover, flow cytometry revealed that the absolute numbers of MDSCs and G-MDSCs were significantly increased in the CLT of AE patients, while the absolute number of monocytic MDSCs (M-MDSCs) was not (Supplementary Fig. 6a–c), in agreement with results from scRNA-seq analysis (Fig. 1a–d).

**Fig. 6: MDSC accumulation in the livers of chronic AE patients, uninfected mice, *E. multilocularis*-infected wild type and PD-1-deficient mice.**

We next evaluated the accumulation of local and peripheral MDSCs in WT mice at 24 weeks post-infection. An enrichment of MDSCs (e.g., M-MDSCs) was observed in the liver of WT mice after infection. In addition, splenic MDSCs (both G-MDSCs and M-MDSCs) were more abundant in infected WT mice than in uninfected mice at week 24 (Fig. 6d–f). Importantly, we observed a statistically significant increase in both the percentage and absolute number of MDSCs and G-MDSCs in the liver and spleen of PD-1^-/- mice compared to those of WT mice at 24 weeks post-infection. However, there was no significant difference in monocytic MDSCs (M-MDSCs) between PD-1^-/- mice and WT mice (Fig. 6d–f and Supplementary Fig. S9a, b). These results suggested that abundant MDSCs, including G-MDSCs, limited the adaptive anti-echinococcal effector function of T cells and that depleting MDSCs alone or in conjunction with anti-PD-1 could be a potential therapeutic strategy to promote PD-1 blockade efficacy while boosting anti-echinococcal immune response.

MDSCs depletion enhances activation and reduces exhaustion of T cell and potentiates the efficacy of PD-1 blockade in murine E. multilocularis infection

We next investigated the role MDSCs and the effect of depleting MDSCs on T cell function and disease progression in chronic E. multilocularis model. The combination of anti-PD-1 and anti-Gr-1 mAb significantly decreased liver metacestode growth, whereas anti-PD-1 monotherapy did not affect growth (Fig. 7a–c). In addition, anti-Gr-1 treatment alone significantly depleted hepatic G-MDSCs, M-MDSCs and splenic G-MDSCs, and partially limited parasite progression in the liver; however, the observed decrease in liver parasite weight was not significant (Supplementary Fig. S10 and Fig. 7a–c). The combined treatment significantly increased the percentages of CD69⁺CD8⁺ T cells, liver Tem cells (both CD4⁺ and CD8⁺) and splenic CD8⁺ Tem cells in mice treated with anti-PD-1 or non-related IgG alone. There was also an increase in liver Tem cells (both CD4⁺ and CD8⁺), IFN-γ CD4⁺ T cells and IL-2 CD8⁺ T cells percentages after anti-Gr-1 treatment alone (Fig. 7d–f and Supplementary Fig. S11a, b). However, significantly greater levels of IFN-γ and IL-2 were produced by liver T cells (both CD4⁺ and CD8⁺) following combined treatment than following treatment with anti-PD-1, anti-Gr-1 or non-related IgG alone. In addition, splenic T cells (both CD4⁺ and CD8⁺) produced high levels of IFN-γ following the combined treatment (Fig. 7f and Supplementary Fig. S11c). Importantly, anti-Gr-1 treatment elicited a strong synergistic effect with anti-PD-1 to markedly boost the level of IFN-γ in liver T cells (both CD4⁺ and CD8⁺) (Fig. 7f). Collectively, these results indicated that depleting MDSCs partially restores effector immunity against E. multilocularis larvae. In addition, it strongly synergises with PD-1 blockade to significantly reverse the exhaustion of T cells, at least partially via the restoration of their IFN-γ and IL-2 production, including locally, in the infected liver.

**Fig. 7: PD-1 blockade in combination with MDSC depletion enhanced the function of T cells and delayed disease progression in mice after persistent *E. multilocularis* infection.**

We then targeted MDSCs with anti-Gr-1 mAb starting at 13 weeks in PD-1^-/- mice after infection. Whole liver weight and lesion weight were significantly lower in PD-1^-/- mice treated with anti-Gr-1 mAb than in PD-1^-/- mice (Fig. 8a–c). In addition, the large difference in lesion weight (and total liver weight) within groups is closely related to the growth characteristics of the parasite itself (Fig. 8c). Anti-Gr-1 treatment significantly increased the percentage of liver CD69⁺CD4⁺ T cells, splenic CD69⁺CD8⁺ T cells and Tem cells (both CD4⁺ and CD8⁺) in PD-1^-/- mice although the absolute number of liver T cells (both CD4⁺ and CD8⁺) showed no increase (Fig. 8d–f and Supplementary Fig. S12a, b). Furthermore, significantly increased percentages of single- or double-positive IFN-γ and IL-2 CD4⁺ T cells were present among the liver T cells from PD-1^-/- mice treated with anti-Gr-1 mAb; significantly increased percentages of double-positive IFN-γ and granzyme B or IL-2, triple-positive IFN-γ, IL-2 and granzyme B or TNF-α CD8⁺ T cells, and of CD8⁺ T cells expressing granzyme B, IFN-γ, TNF-α and IL-2, were present in the liver from PD-1^-/- mice treated with anti-Gr-1 mAb (Fig. 8g). In addition, high percentages of single-positive IL-2, double-positive IL-2 and IFN-γ or TNF-α, and triple-positive IFN-γ, TNF-α and IL-2 CD4⁺ T cells were present in the spleen following anti-Gr-1 treatment; high percentages of single- or double-positive IFN-γ and IL-2, double-positive IFN-γ and TNF-α, and triple-positive IFN-γ, IL-2 and granzyme B or TNF-α CD8⁺ T cells were also present in the spleens of the infected mice following anti-Gr-1 treatment (Supplementary Fig. S12c). These data also showed that abundant MDSCs, including G-MDSCs, limited the adaptive anti-echinococcal effector function of T cells and that depleting MDSCs inhibited metacestode growth in PD-1-deficient mice after infection.

**Fig. 8: MDSC depletion enhanced the function of T cells and delayed disease progression in PD-1-deficient mice after persistent *E. multilocularis* infection.**

G-MDSCs suppress the T-cell response mainly via IDO1 in AE patients and murine E. multilocularis infection

Based on the data presented thus far, we found that the mRNA levels of IDO1 and NOS2 as critical mediators of the suppressive phenomenon, increased in the CLT from AE patients (Supplementary Fig. S8). Immunohistochemistry was subsequently used to determine the protein expression levels of CD33, IDO1 and INOS in liver tissues from AE patients. There were greater numbers of CD33⁺ cells, IDO1⁺ cells and INOS⁺ cells in the inflammatory infiltrate of the periphrastic area in the CLT samples than in the DLT samples (Fig. 9a–d), and the percentages of hepatic CD33⁺ cells and IDO1⁺ cells were significantly positively associated with fluorodeoxyglucose (FDG) uptake via positron emission tomography (PET)-CT, a marker of metabolic activity of the lesions in the patient liver; however, the percentage of INOS⁺ cells was not associated with this marker (Fig. 9e, f). Furthermore, IDO1 was colocalized with CD33⁺ cells in the CLT (Fig. 9g). We also found that the absolute number of IDO1⁺ MDSCs was significantly greater in the livers of PD-1^-/- mice after 24 weeks of infection (Fig. 9h). These data suggested that MDSCs might impair the function of T cells by producing IDO1.

**Fig. 9: Liver-infiltrating G-MDSCs upregulate IDO1, which inhibits T-cell function.**

To further verify that IDO1 was indeed important for suppression, we measured T-cell function in G-MDSCs obtained from the spleens of E. multilocularis-infected mice in the presence of an IDO1inhibitor in vitro. There was a significant increase in IFN-γ and TNF-α levels after the addition of 1-methyl-Trp (1-MT), confirming that G-MDSCs exert their suppressive effects through the expression of IDO1 (Fig. 9i).

Discussion

The T-cell population is one of the main contributors to resistance against tumours, intracellular viruses and parasitic infection^32,33. The functional deficiency of T cells is associated with pathogen progression and contributes to disease pathogenesis^9,16,34 In the present study, a late-stage E. multilocularis infection model is used to better understand the mechanisms involved in resistance to checkpoint blockade and how this resistance might be overcome by combined specific biotherapy. The results demonstrated that reversing checkpoint inhibition is not sufficient to restore an efficient immune response by the host and that MDSCs are also key actors, at least partially by an IDO1-dependent mechanism, and should be targeted simultaneously to alleviate the deleterious effect of T-cell exhaustion on the progression of AE lesions. Moreover, targeting MDSCs alone showed a certain level of therapeutic potential. These findings are consistent with those obtained through other models of T-cell exhaustion and checkpoint inhibition, such as malignant tumours or other types of infection^20,23,25. In addition, they provide experimental evidence for the ability of MDSCs-based therapy and especially of therapies which combine checkpoint blockadeand MDSC-targeting to treat patients with a life-threatening parasitic disease.

PD-1 is a central regulator of T-cell exhaustion, a state of T-cell dysfunction that may occur during many chronic infections. With regard to echinococcosis, PD-1 was found to be highly expressed in the hepatic T cells of AE patients and mice infected with E. multilocularis^9,35. Significantly higher levels of sPD-L1 were observed in patients with cystic echinococcosis (CE) than in healthy controls³⁶, which suggests that sPD-L1 levels can be a predictive biomarker of recurrence after surgery³⁷. In this study, we demonstrated that upregulation of PD-1 expression leads to T-cell exhaustion in the liver, blood and spleen as E. multilocularis infection progresses to the chronic stage. However, our study revealed that PD-1-deficient mice or anti-PD-1/anti-PD-L1 treatment failed to control established metacestode at such later stages (12–24 weeks). In addition, PD-1 blockade did not restore cytokine secretion by exhausting T cells from the blood of AE patients ex vivo. However, several studies have shown that anti-PD-L1 or combined with albendazole treatment potently inhibited E. multilocularis growth in an intraperitoneal (secondary) or oral (primary) infection mouse model at early (0–4 weeks) or later (6–14 weeks) stages. This effect was associated with increased effector T-cell function and decreased regulatory T-cell (Treg)/helper T-cell (Th2) responses^11,12. Recent studies on cancer, viral and parasitic infections have also reported better activity for anti-PD-1 given early in the disease course^25,38,39. These and our present data suggest that the timing of blockade relative to infection (early versus later) is likely a key factor in determining whether PD-1 blockade will be beneficial versus detrimental in any given infection or cancer setting⁴⁰. For example, in a tumour model, early anti-PD-1 treatment elicited robust antitumour immunity by enhancing T-cell tumour infiltration, whereas treatment of late-stage tumours in the same model was ineffective and associated with infiltration of G-MDSCs that correlated with a lack of response to anti-PD-1 therapy²⁵. PD-1 blockade may exert different effects depending on the degree of T-cell exhaustion. Our results showed that liver CD4⁺ T cells and CD8⁺ T cells harvested from infected mice at the late stage (24 weeks) exhibited a higher level of PD-1 expression than those harvested from mice at the early and middle stages of infection (2 and 12 weeks). Several studies have also demonstrated that the effector function of T cells expressing intermediate levels of PD-1 can be restored by PD-1 blockade, while the function of T cells expressing high levels of PD-1 cannot be restored^3,7,41. A smaller tumour burden usually indicates a better therapeutic response to immune checkpoint inhibitor treatment and longer overall or progression-free survival^4,5,42,43,44. Hence, the results of our parasitic model suggest that high metacestode burden and PD-1 expression level at later stages might be associated with an immunosuppressive microenvironment around liver lesions, which determines the low efficacy of anti-PD-1/anti-PD-L1 treatment.

Our findings of resistance to checkpoint blockade may also be related to differences in the sites of lesions (i.e., liver versus peritoneum). Interestingly, data from previous reports and this study revealed that blocking the PD-1 pathway restores the effector function of intraperitoneal T cells infiltrating peritoneal lesions but not that of local T cells harvested from the liver after natural infection^11,12. Importantly, we found that local T cells taken from the liver exhibited higher PD-1 expression than those taken from the periphery (i.e., PBMCs or spleen) in AE patients as well as in a mouse model at the late stage of infection, suggesting that the cells present within the chronic inflammatory hepatic microenvironment may be more ‘exhausted’ than those circulating in the periphery and/or subjected to more ‘exhausting’ influences from other cell types, such as G-MDSCs^7,41. These data indicate that differences in local immune characteristics and the organ microenvironment are important factors to consider when predicting responsiveness to PD-1 blockade therapy, suggesting that organ-specific strategies are important for treating organ-specific diseases. These results support the need to better define populations of T cells at late stages of chronic infection or in tumours when considering the impact that PD-1 blockade could have on the course of infection or cancer^45,46. The particular ‘tolerogenic’ functions of cell populations that reside in the liver have long been emphasised⁴⁷. Supporting the particularities of the hepatic immunological microenvironment, we found that at the parasitic site of lesions in the liver, a subset of highly PD-1-expressing CD4⁺ T cells and CD8⁺ T cells displayed a tissue-resident memory (Trm) cell phenotype characterised by the expression of typical surface markers (i.e., CD44 and CD69) in mice. These cells stably reside in tissues and play a critical role in local immunosurveillance or in restraining immunopathology^48,49. PD-1 expression was found to be elevated on Trm cells localised at the target sites of different chronic inflammatory diseases or tumours, displaying more signs of exhaustion than recirculating memory T cells^50,51. Furthermore, other key factors (e.g., MDSCs) in the inflammatory hepatic microenvironment can promote T-cell exhaustion locally and contribute to anti-PD-1/anti-PD-L1 resistance in tumours as well as infectious diseases^52,53,54. Our enrichment analysis also highlighted the role of myeloid leukocyte migration, differentiation and activation, which are related to the characteristics, recruitment and immunosuppressive function of myeloid cells (i.e., MDSCs) in the livers of AE patients.

One of the major characteristics of MDSCs is their ability to inhibit the immune response of T cells^55,56,57. Here, we found that targeting MDSCs with anti-Gr-1 alone induced T-cell immune activation and moderately delayed the growth of metacestode; furthermore, targeting PD-1 and MDSCs together was superior to either single-agent treatment, and effectively improved the efficacy of PD-1 deficiency or PD-1 blockade therapy in our experimental model. As a result, the combined treatment increased the proportion of liver-infiltrating T cells producing IFN-γ (but not TNF-α and IL-2) significantly more than anti-Gr-1 alone (Fig. 7f), providing a major source of IFN-γ that confers host protection during chronic infection. Increased production of IFN-γ is a marker for the transition from a T exhaustive phenotype to a T effector cell phenotype⁵⁸. In addition, a robust immune response by IFN-γ-producing T cells is necessary to control parasite replication and/or growth in a number of parasitic infections, and loss of this cytokine also leads to a loss of these protective mechanisms⁵⁹. Our and others’ studies have demonstrated that IFN-γ production is critical for preventing disease progression in chronic E. multilocularis model and AE patients^60,61,62,63. In addition, our data suggest that G-MDSCs could play a major role in the resistance to checkpoint blockade we observed and, more generally, in the immunosuppression of T cells and in the chronic characteristics of E. multilocularis infection. Similarly, a study in late Mesocestoides vogae infection demonstrated that the accumulation of G-MDSCs had greater suppressive effects than M-MDSCs⁶⁴. There were a few reports of MDSCs which expand and suppress immune responses in Schistosoma, Leishmania donovani, and Toxoplasma gondii (T. gondii) infections^{65,66,67,68,69,70}, and a significant increase in MDSCs and their role in inhibiting the differentiation and activity of T helper 2 cells in a NO-dependent manner has been shown during persistent infection with E. granulosus^27,28. The presence of MDSCs may be one of the main contributors to the immunosuppressive liver microenvironment; MDSCs were shown to correlate with decreased efficacy of immune checkpoint therapy (e.g., anti-PD-1/anti-PD-L1) in colorectal cancer⁵³.

Previous work also demonstrated that MDSCs exert their antagonistic effect on T cells by depleting amino acids via arginase 1 (ARG1) and 2 (ARG2) and IDO1, or by upregulating INOS and inhibitory cytokines (e.g., IL-10)^21,56,71,72. In our study, the expression levels of IDO1 and INOS, rather than ARG1/2, were significantly increased in the livers of AE patients, and IDO1 expression was colocalized with CD33⁺ cells in the livers of AE patients. IDO⁺ MDSCs were also increased in the livers of PD-1-deficient infected mice. The expression level of IDO1, rather than that of INOS or IL-10, in the periphrastic area of the liver in AE patients was positively correlated with lesion metabolic activity according to PET-CT. Some studies have shown that the lack of INOS expression by peritoneal and splenic macrophages rather than MDSCs, confers a certain degree of resistance against persistent chronic infection with E. multilocularis^73,74; moreover, no IL-10 was detected when polymorphonuclear granulocytes (PMN, the phenotype of which overlaps with G-MDSCs⁷⁵) from AE patients were cultured with metacestode vesicles^76,77,78. In addition, elevated IDO1 expression promotes immunosuppression and AE progression by inhibiting the activation, clonal expansion and differentiation of T cells⁷⁹. Similarly, E. granulosus antigens (e.g., recombinant Eg10, EgB, mMDH) can upregulate IDO1 expression to induce host immune tolerance and promote chronic infection^80,81. We also showed that inhibition of IDO1 by 1-MT significantly attenuated MDSC-mediated suppression of T-cell function and enhanced IFN-γ and TNF-α secretion by T cells in vitro. This finding strongly suggested that the immunosuppressive activity of MDSCs in E. multilocularis infection is IDO1-dependent and leads to anti-PD-1 resistance. Future experiments will evaluate the therapeutic significance of combining IDO1-specific inhibitors with immune checkpoint blockade to prevent disease progression during persistent parasitic infection. High expression of IDO in dendritic cells reportedly increases Leishmania spp. infection and facilitates pathogen survival as well^82,83. The inhibition of increased IDO activity was shown to be involved in antiparasitic mechanisms during T. gondii infection⁸⁴. Overexpression of IDO1 also facilitates T-cell anergy in noninfectious disease models^85,86.

In summary, our study demonstrated that the resistance to anti-PD-1 checkpoint interventions observed in chronic E. multilocularis infection is mainly attributed to the accumulation of G-MDSCs around liver lesions and their substantial inhibitory effects on T-cell immune function, in part via an alternative IDO1-dependent mechanism. The removal of G-MDSC-mediated immunosuppression by anti-Gr-1 antibody administration can effectively restore T-cell function and potentiate PD-1 blockade therapy. These findings provide additional support for the mechanisms of T-cell exhaustion through checkpoint inhibition associated with G-MDSC IDO1-dependent immune suppression, as suggested in other models, both in cancer and in other types of chronic infection. In addition, they provide preclinical evidence in support of targeting MDSC or combining such an approach with the check-point blockade in patients with advanced AE, a severe disease with only limited treatment options.

Methods

Ethics statement

This study protocol was approved by the ethics committee of the First Affiliated Hospital of Xinjiang Medical University (N° S20130418-3), and all patients provided informed written consent in accordance with the Declaration of Helsinki (1975) of the World Medical Association. All the data of the human subjects are summarised in Supplementary Data 2.

Human donors

Liver specimens were obtained from 64 patients with AE who underwent liver resection. Patients with immune suppression-associated conditions, as described by Chauchet et al. ¹⁶, were excluded. AE diagnosis was confirmed in all patients by liver biopsies. One specimen was taken close to the parasitic lesion, including the metacestode (CLT, ‘close liver tissue’, ~ 0.5 cm from lesion), and/or one was taken from the macroscopically normal liver distant from the lesion (DLT, ‘distant liver tissue’, at least 2 cm distant from lesion), as described in our previous publication⁹. Peripheral blood samples (PB) were obtained from 33 AE patients and from 34 healthy age- and sex-matched controls. The number of liver and blood samples, types of measurements, and purpose of comparisons are given in Supplementary Data 1.

Liver pathology and immunohistochemistry

Liver tissue samples were fixed in 4% paraformaldehyde (PFA)/PBS for 48 hr. Dehydrated, paraffin-embedded liver tissues were cut into 5- μm sections, which were then processed for pathological evaluation or immunohistochemistry. The sections were subjected to staining and microscopically examined, and photographs were taken using a digital image-capture system (Olympus, Tokyo, Japan).

For pathological analysis, sections were stained with haematoxylin and eosin (H&E) to evaluate infiltrating cells and liver immune microenvironment formation according to guidelines for clinical samples.

For immunohistochemistry analysis, the sections were deparaffinized and then subjected to heat-mediated antigen-retrieval in Tris-EDTA buffer (Solarbio, Beijing, China). The sections were blocked for 1 hr at room temperature in PBS with 10% goat serum (blocking buffer) and then incubated at 4 °C overnight with primary antibodies in blocking buffer (anti-human CD33, 1:100, Abcam; anti-human IDO1, 1:500, Abcam; anti-human INOS, 1:75, Abcam, Cambridge, UK). The next day, the sections were washed three times for 5 min and incubated for 2 hr at room temperature with a secondary antibody (goat anti-rabbit F(ab’)2-HRP, Abcam, Cambridge, UK). Staining was developed using a 3, 3’ diaminobenzidine (DAB) substrate kit (Abcam, Cambridge, UK) according to the manufacturer’s instructions. A total of 3-5 fields/sections/samples were assessed at × 400 magnification using cellSens Dimension software (Olympus, Japan) for computerised quantification, and the results are expressed as the intensity of positive staining per field.

Immunofluorescence

For immunofluorescence analysis, the frozen sections of liver tissue were stained with primary antibodies (anti-human CD4, 1:250, Abcam; anti-human CD8, 1:250, Abcam; anti-PD-1, 1:200, Abcam; anti-human CD33, 1:200, Abcam; anti-human IDO1, 1:500, Abcam, Cambridge, UK) at 4 °C in blocking buffer overnight and subsequently stained with secondary antibodies (Alexa Fluor® 488-labelled anti-rat IgG, Cell Signalling Technology; Alexa Fluor® 488-labelled anti-mouse IgG, Cell Signalling Technology; Alexa Fluor® 555-labelled anti-mouse IgG, Cell Signalling Technology, Boston, United States) for 2 hr at room temperature. 4’, 6-Diamidino-2-phenylindole (DAPI) was used for nuclear staining. Images were acquired by a confocal laser scanning fluorescence microscope (Leica, Oskar-Barnack-Straße). Positive cells were quantified by ImagePro Plus software (Media Cybernetics, Rockville, United States) and are expressed as the mean number of positive cells in five fields at 200 × magnification.

Isolation of mononuclear cells from human liver tissues

Close- and distant-liver tissue-infiltrating cells were obtained from paired fresh liver tissue samples of AE patients as previously described⁸⁷. For isolation of liver mononuclear cells, liver tissue was cut into small pieces and digested in RPMI 1640 supplemented with 0.05% collagenase IV (Sigma-Aldrich), 0.02% DNase I (Roche, Basel), and 20% FBS (HyClone) at 37 °C for 1 h. Dissociated cells were filtered through a 150 µm mesh and separated by Ficoll (Solarbio, Beijing, China) centrifugation, and the mononuclear cells were washed and resuspended in RPMI 1640 supplemented with 10% FBS.

Isolation of mononuclear cells from human peripheral blood

Peripheral blood mononuclear cells (PBMCs) were purified from blood samples by Ficoll (Solarbio, Beijing, China) density gradient centrifugation as previously described⁸⁸. In brief, 6–8 mL blood prediluted 1:1 in PBS was carefully added to 3 ml of Ficoll liquid in a 15 mL tube and then was centrifuged at 400 × g for 30 min at 25–27 °C. PBMCs were washed and resuspended in RPMI 1640 supplemented with 10% FBS.

Flow cytometry

A single-cell suspension of leukocytes from human samples was resuspended in RPMI 1640 containing 10% FBS, 1000 mg/mL penicillin and 1000 µ/mL streptomycin (HyClone, Beijing, China). For cell surface staining, 0.5 × 10⁶ cells per tube were stained for surface markers using antibodies for 30 min at 4 °C in the dark. For intracellular staining, 1 × 10⁶ cells per tube were cultured in the presence of Cell Stimulation Cocktail (eBioscience, San Diego, United States), according to the manufacturer’s instructions and incubated at 37 °C for 4 h. The cells were first stained for surface markers using antibodies for 30 min at 4 °C in the dark. The cells were then suspended in a permeabilization buffer containing antibodies according to the manufacturer’s instructions (BioLegend, San Diego, United States). The antibodies used herein are listed in Supplementary Data 3. An LSRFortessa flow cytometer (BD Immunocytometry Systems, San Diego, United States) was used to acquire the data, which were analysed with the FlowJo software 7.6.1 (Treestar, San Carlos, United States).

A single-cell suspension of mononuclear cells from mouse samples was incubated in PBS (0.2% BSA and 0.1% sodium azide) in the presence of neutralising monoclonal antibodies against CD16:CD32 (Fc Block; Biolegend, San Diego, United States) for 20 min at 4 °C prior to staining. For cell surface staining, 0.5 × 10⁶ cells per tube were stained for surface markers using specific antibodies in PBS buffer for 30 min at 4 °C in the dark. The following phenotypes were used to determine the MDSC populations: CD45⁺CD11b⁺Gr-1⁺ for MDSCs; CD45⁺CD11b⁺Gr-1⁺Ly6C^low for G-MDSCs; and CD45⁺CD11b⁺Gr-1⁺Ly6C⁺ for M-MDSCs^89,90. The protocol for intracellular staining was the same as that described above. The antibodies used herein are listed in Supplementary Data 3. The samples were then analysed using an LSRFortessa flow cytometer and the FlowJo software 7.6.1 (Treestar, San Carlos, United States).

Ex vivo T-cell function antibody blockade

PBMCs were isolated from AE patients and restimulated with anti-human CD3 (2.5 μg/ml, BD Biosciences) and anti-human CD28 (1.25 μg/ml, BD Biosciences) at 37 °C in the presence of anti-human PD-1 mAb (5 µg/ml, R&D Systems) or isotype control (R&D Systems). After 20 h of incubation and 6 h of brefeldin A (1000 dilution, BD Biosciences), the cells were harvested to determine the intracellular cytokine production (IFN-γ and TNF-α) of CD4⁺ T cells and CD8⁺ T cells using flow cytometry.

Mice

Wild-type (WT) female C57BL/6 (B6) and BALB/c mice were purchased from Beijing Vital River Experimental Animal Technology Co., Ltd. PD-1^-/- mice, which were originally generated by Dr. Tasuku Honjo (Kyoto University, Kyoto, Japan), were kindly provided by Dr. Yuzhang Wu (Army Medical University, Chongqing, China). All mice were maintained under specific pathogen-free conditions according to the guidelines for experimental animals established by the Animal Care and Use Committee and the Ethical Committee of the First Affiliated Hospital of Xinjiang Medical University (N° 20140411-05).

Mouse model of E. multilocularis infection and in vivo blockade

Mice were inoculated via the hepatic portal vein with live protoscoleces (PSCs) in saline as previously described⁸, whereas control mice were injected with isotonic saline.

At necropsy, the whole weight of the liver was measured using an electronic scale; metacestode tissue was carefully removed from the liver lobes by using microsurgery forceps and scalpel blades. The metacestode tissue, which was free of host tissue, was immediately weighed on an electronic scale. The formation of all parasitic foci was observed on haematoxylin and eosin (H&E)-stained sections and the number of infectious foci with metacestode structures (the confluent lesions) was counted in all liver lobes as previously described⁸.

For in vivo blockade, mice bearing metacestodes of 1.5–3 mm² after 12 weeks were randomised into treatment groups and treated with anti-PD-1 mAb (200 μg, clone RMP1-14, BioXcell, West Lebanon, USA), anti-PD-L1 mAb (200 μg, clone 10 F.2H11, BioXcell), or isotype-control antibody (mouse IgG1, clone 2A3 or LTF-2, BioXcell) by intraperitoneal (i.p.) injection two or three times per week for 12 weeks. For depletion of MDSCs, mice were injected with 200 μg of anti-Ly6G/Ly6C (Gr-1) mAb (clone RB6-8C5, BioXcell) or isotype control antibody (clone LTF-2, BioXcell) i.p. two times per week for 12 weeks. For treatment experiments with a combination of anti-PD-1 mAb and anti-Ly6G/Ly6C (Gr-1) mAb, mice were intraperitoneally injected. According to the above dose and time points, control mice received both control isotypes.

Isolation of mononuclear cells from mouse liver and spleen

A single-cell suspension of mononuclear cells was isolated from the liver and spleen as described previously. Livers were homogenised and mononuclear cells were separated from hepatocytes by centrifugation with 40% Percoll (GE Healthcare, Pittsburgh, United States). The spleens were crushed between two glass slides, suspended in PBS containing 0.2% BSA, and passed through a 74 μm nylon mesh. Red blood cells were depleted using red blood lysis buffer (BioLegend, San Diego, United States). The cells were then manually counted on a haemocytometer.

In vitro coculture of T cells and G-MDSCs

Splenic CD4⁺ T cells or CD8⁺ T cells were enriched from PD-1^-/- mice by positive magnetic selection (Miltenyi, Germany). Splenic G-MDSCs were enriched from E. multilocularis-infected mice by positive magnetic selection (Miltenyi, Germany). Purified CD4⁺ or CD8⁺ T cells (5 × 10⁵/ml) were cocultured with G-MDSCs (10⁵/ml) for 48 h in the presence of anti-mouse CD3 (2.5 μg/ml, eBiosciences) and anti-mouse CD28 (1.25 μg/ml, eBiosciences). The IDO1 inhibitor 1-MT (5 μg/ml, Sigma-Aldrich®, United States) or medium alone was added to the culture. Coculture supernatants were collected for IFN-γ and TNF-α analysis by enzyme-linked immunosorbent assays (ELISA).

Enzyme-linked immunosorbent assays (ELISAs)

The culture supernatants were used for the measurement of IFN-γ and TNF-α. The concentrations of cytokines were measured using ELISA kits according to the manufacturer’s instructions (Thermo Fisher Scientific).

Quantitative reverse transcription-PCR (qRT-PCR) assays

Total RNA was extracted from liver tissue using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. M-MLV Transcriptase was used for reverse transcription according to the manufacturer’s instructions (Thermo Fisher Scientific, Waltham, MA). qRT-PCR was performed in a thermocycler (iQ5 Bio-Rad, Hercules, CA) with the SYBR Green PCR premix (TaKaRa, Dalian) according to the manufacturer’s protocol. The PCR primers used herein are listed in Supplementary Data 3. The results were normalised with respect to the value obtained for the housekeeping gene GAPDH. The △Ct was calculated by subtracting the Ct value of the housekeeping gene (GAPDH) from that of the target gene △Ct. The △△Ct was calculated by subtracting the △Ct of the experimental group target gene from the △Ct of the control group target gene. Relative mRNA expression was calculated by the 2^−△△Ct method.

Data analysis

Single-cell sequences for 4 AE patients were retrieved from a public database (accession no. HRA000553) reported in our previous study⁹¹. Hepatic mRNA expression profiles for 6 AE patients were retrieved from the GEO database (GSE124362) reported in our previous study⁹. Gene set enrichment analysis (GSEA) was performed using the Broad Institute tool GSEA v.2.2.1. In addition, hierarchical clustering of genes and arrays was performed using the R software heatmap package.

Statistical analysis

Student’s t test was used to compare two groups, and one-way analysis of variance (ANOVA) was used to compare three or more groups. Pearson’s correlation was used to calculate the strength of a relationship. All the data are presented as the mean ± standard deviation (SD). p < 0.05 was considered to indicate statistical significance.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The RNA sequencing data in this study are available in the figshare database under accession code HRA000553 (https://figshare.com/s/f2a4578be27c2cb767f9). The mRNA expression data in this study are available in the Gene Expression Omnibus database under accession code GSE124362 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE124362). The key reagents or resources used in this study are listed in Supplementary Data 3. Source data are provided with this paper.

Code availability

The data analysis pipeline in our study is described on the Seurat official website and clusterProfiler official website. The code can be found in Supplementary Data 4.

Change history

27 August 2024
A Correction to this paper has been published: https://doi.org/10.1038/s41467-024-51832-6

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Acknowledgements

We thank all members of our laboratory for their support and helpful discussions, the Yuzhang Wu laboratory at Chongqing for providing PD-1^-/- mice (originally generated by Dr. Tasuku Honjo, Japan), and the Lu Shi from the CRE Life Institute, Beijing. This research was supported by the National Key Research and Development Programme of China (2021YFC2300800 to CSZ, 2021YFC2300801 to CSZ, 2021YFC2300802 to CSZ), Natural Science Foundation of Xinjiang Uygur Autonomous Region (2022D01D60 to CSZ), National Natural Science Foundation of China (82160396 to CSZ, 81830066 to WBZ, 81760368 to CSZ, 82160397 to WH, 82060370 to JL), Xinjiang Uygur Autonomous Region “Tianshan Talent” Training Programme (2022TSYCCX0106 to CSZ), and Cultivation projects of the National Science Fund for Distinguished Young Scholars (xyd2021J003 to CSZ). Mice images were drawn by hand using Procreate software.

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These authors contributed equally: Chuanshan Zhang, Hui Wang, Tuerganaili Aji, Zhide Li.

Authors and Affiliations

State Key Laboratory of Pathogenesis, Prevention and Treatment of High Incidence Diseases in Central Asia, Xinjiang Medical University; Clinical Medicine Research Institute, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang, P. R. China
Chuanshan Zhang, Hui Wang, Zhide Li, Yinshi Li, Abidan Ainiwaer, Zibigu Rousu, Maolin Wang, Bingqing Deng, Adilai duolikun, Xuejiao Kang, Xuran Zheng, Qian Yu, Wenbao Zhang & Hao Wen
Basic Medical College, Xinjiang Medical University, Urumqi, Xinjiang, P. R. China
Chuanshan Zhang, Yinshi Li, Abidan Ainiwaer, Zibigu Rousu, Jing Li, Bingqing Deng, Adilai duolikun, Xuejiao Kang, Xuran Zheng & Qian Yu
Key Laboratory of High Incidence Disease Research in Xingjiang, Ministry of Education, Xinjiang Medical University, Urumqi, Xinjiang, P. R. China
Chuanshan Zhang, Hui Wang & Tuerganaili Aji
Xinjiang Key Laboratory of Echinococcosis, Clinical Medicine Research Institute, The First Affiliated Hospital of Xinjiang Medical University, and WHO Collaborating Centre on Prevention and Case Management of Echinococcosis, Urumqi, Xinjiang, P. R. China
Hui Wang & Wenbao Zhang
Department of Hepatic Hydatid and Hepatobiliary Surgery, Digestive and Vascular Surgery Centre, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang, P. R. China
Tuerganaili Aji, Maolin Wang & Yingmei Shao
WHO-Collaborating Centre for the Prevention and Treatment of Human Echinococcosis, Department of Parasitology, University Bourgogne Franche-Comté (EA 3181) and University Hospital, Besançon, France
Dominique A. Vuitton
Hefei National Research Center for Physical Sciences at Microscale, the CAS Key Laboratory of Innate Immunity and Chronic Disease, School of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, Anhui, P. R. China
Zhigang Tian & Haoyu Sun
Department of Immunology, School of Basic Medical Sciences, Fudan University, Shanghai, China
Haoyu Sun
Institute of Immunology, University of Science and Technology of China, Hefei, Anhui, P. R. China
Haoyu Sun

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Chuanshan Zhang
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Hui Wang
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Tuerganaili Aji
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Zhide Li
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Yinshi Li
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Jing Li
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Contributions

Conceived and designed the study: C.S.Z., H.W., J.L. H. Y. S and Z.G.T.; performed the experiments: C.S.Z., H.W., Z.D.L., J.L., M.L.W., Y.S.L., B.Q.D., A.D.L., X.J.K., X.R.Z., Z.B.G.R., A.B.D.A. nd Q.Y.; analysed the data: C.S.Z., H.W. and Y.S.L.; contributed to technical or materials related issues: T.E.G., Y.M.S. and W.B.Z.; draughted and revised the manuscript: C.S.Z., H. Y. S., D. A. V., H.W. and Z.G.T.; supervised the study: C.S.Z., Z.G.T. and H.W.; All authors reviewed and approved the manuscript.

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Correspondence to Chuanshan Zhang, Haoyu Sun or Hao Wen.

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Zhang, C., Wang, H., Aji, T. et al. Targeting myeloid-derived suppressor cells promotes antiparasitic T-cell immunity and enhances the efficacy of PD-1 blockade. Nat Commun 15, 6345 (2024). https://doi.org/10.1038/s41467-024-50754-7

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Received: 02 August 2023
Accepted: 18 July 2024
Published: 27 July 2024
DOI: https://doi.org/10.1038/s41467-024-50754-7

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