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Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity

Abstract

Tumor-associated macrophages are composed of distinct populations arising from monocytes or tissue macrophages, with a poorly understood link to disease pathogenesis. Here, we demonstrate that mouse monocyte migration was supported by glutaminyl-peptide cyclotransferase-like (QPCTL), an intracellular enzyme that mediates N-terminal modification of several substrates, including the monocyte chemoattractants CCL2 and CCL7, protecting them from proteolytic inactivation. Knockout of Qpctl disrupted monocyte homeostasis, attenuated tumor growth and reshaped myeloid cell infiltration, with loss of monocyte-derived populations with immunosuppressive and pro-angiogenic profiles. Antibody targeting of the receptor CSF1R, which more broadly eliminates tumor-associated macrophages, reversed tumor growth inhibition in Qpctl−/− mice and prevented lymphocyte infiltration. Modulation of QPCTL synergized with anti-PD-L1 to expand CD8+ T cells and limit tumor growth. QPCTL inhibition constitutes an effective approach for myeloid cell-targeted cancer immunotherapy.

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Fig. 1: QPCTL protects MCPs from DPP4-mediated inactivation in vivo.
Fig. 2: QPCTL function is essential for monocyte homeostasis.
Fig. 3: QPCTL sustains monocyte infiltration and tumor growth.
Fig. 4: CSF1R targeting reverses tumor inhibition in Qpctl−/− mice.
Fig. 5: QPCTL modulation reshapes the tumor myeloid infiltrate.
Fig. 6: Qpctl loss in chemokine-producing cells is sufficient to inhibit tumor growth.
Fig. 7: Qpctl loss improves CD8+ T cell responses upon PD-L1 blockade.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request. Mouse reference genome GRCm38 and GENCODE is a publicly available database used for single-cell RNA-seq analysis. Material requests should be made to the corresponding authors. Source data are provided with this paper.

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Acknowledgements

The authors acknowledge the following people for technical and intellectual support: Y. Lu, J. Wu, J. Payandeh, I. Lehoux and protein structure/purification teams; B. Haley, J. Lill, W. Sandoval and P. Liu; S. Warming, U. Segal, V. Arumugam, R. Asuncion, T. Scholl, M. Dempsey, K. Veliz, M. Lamoureux, W. Ortiz, M. Reich and animal care-takers and veterinarians; Y. Chestnut, M. Singh and A. Gutierrez; pathology, necropsy and antibody engineering/production teams; A. Cordrey and the cell culture team; and W. Fu for careful reading of the manuscript. This work was funded by Genentech. Illustrations were generated by the authors or adapted from Servier medical art (France) or BioRender.com.

Author information

Authors and Affiliations

Authors

Contributions

R.B.d.S. and M.L.A. were responsible for conceptualization. R.B.d.S., R.M.L., S.W., J.O., V.J., Y.W., W.P., C. Everett, J.N., D.A., J.Z., Z.M., J.M.S., M.M. and S.R. were responsible for investigation and methodology development. R.B.d.S., R.M.L., X.P.-J., S.W., J.O., C. Everett, J.N. and J.Z. were responsible for data analysis. C.L., Y.-C.H., J.T.K., I.H., T.H.P. and M.R.-G. were responsible for resources. R.B.d.S., J.M.S., M.M., C. Eidenschenk, I.M. and M.L.A. were responsible for supervision. R.B.d.S. was responsible for writing the original draft. R.B.d.S., C. Eidenschenk., S.R., I.M. and M.L.A. were responsible for review and editing of the manuscript, with edits from all authors.

Corresponding authors

Correspondence to Rosa Barreira da Silva, Ira Mellman or Matthew L. Albert.

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All authors are current or former employees of Genentech and may be shareholders of Roche.

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Nature Immunology thanks Alexandre Boissonnas and Thorsten Mempel for their contribution to the peer review of this work. Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Monocyte migration is not modulated by DPP4.

a, Quantification of MCPs in plasma of naive WT mice (n = 5). b, NH2-terminal amino acid sequence of MCPs highlighting the presence of a glutamine (Q) in the first position and a proline (P) in the second position. c, Monocyte quantification in blood (n = 5), spleen (n = 4 (+/+) or 5 (–/–)) and bone marrow (n = 4 (+/+) or 5 (–/–)) of naive Dpp4+/+ and Dpp4−/− littermates. NS, P > 0.05. d, Monocyte quantification in tumors extracted from WT mice treated with Ctrl chow or DPP4i (Hepa1-6, n = 11 (Ctrl) or 9 (DPP4i); CT26 (n = 11); B16F10 (n = 10); LL/2 (n = 10)). NS, P > 0.05. e, Human (h)CCL7 was incubated in the absence or presence of hDPP4. Molecular weight profiles of resulting species are shown in Daltons (Da). f, Schematic representation of antibodies that detect MCP forms: anti-pE recognizes NH2-terminal cyclization and anti-ΔQP is specific for the truncated chemokine. Anti-total antibody recognizes the chemokine independently of its N-terminal modification. g, Cross-reactivity profile of anti-mouse (m)CCL7 capture antibodies against mCCL7 forms. AF456 recognizes mCCL7 independently of its N-terminal modification (total); 1G2 binds preferentially to pE-CCL7 and 2F3 binds preferentially to ΔQP-CCL7. Detection was done with biotinylated anti-mCCL7 AF456. AEB, average enzyme per bead. h, Cross-reactivity profile of mCCL2 capture antibodies against mCCL7 and mCCL2 forms. Detection was done with biotinylated anti-mCCL2 MAB479. i, Lower limit of detection (LLOD) of antibodies used to analyze mCCL2 and mCCL7 PTMs. Bars are medians and symbols represent individual mice. Data shown is representative (a,c,g,h) or pooled from 2 experiments (d). All experiments were repeated independently (≥ twice). Statistical analysis was done using unpaired, nonparametric Mann-Whitney U-test.

Source data

Extended Data Fig. 2 Regulation of MCP function by QPCTL and DPP4.

a, Glutaminyl cyclase activity in serum from the indicated mouse genotypes (n = 18 (WT), 6 (Qpctl–/–, Qpct–/–Qpctl–/–) or 12 (Qpct–/–). NS, P > 0.05, *P < 0.0001. b, Quantification of CCL2 PTMs in plasma from Qpctl–/– and Qpctl+/+ littermates bearing Qpctl–/– and Qpctl+/+ LL/2 tumors, respectively. (n = 9 (Qpctl–/– Total) or 10 (all other groups)). *P = 0.004, **P < 0.0001. c, Quantification of total CCL7 in serum (n = 7 (Qpctl+/+) or 11 (Qpctl–/–)) and plasma (n = 5) from Qpctl+/+ and Qpctl–/– littermates. *P = 0.008, **P < 0.0001. d, Quantification of mCCL7 PTMs in plasma of naive Qpctl–/– and Dpp4–/–Qpctl–/– littermates (n = 9). *P = 0.0002, **P < 0.0001. e, Migration of THP-1 cells to hCCL7 forms placed on the right side of a u-migration chamber. Cellular trajectories moving towards to (blue) or away from (green) the chemokine are shown. f, Akt phosphorylation (pAkt) in THP-1 cells after incubation with media (lane 1); Q-CCL7 (lanes 2 to 4) or ∆QP-CCL7 (lanes 9 and 10). Antagonist activity of ∆QP-CCL7 was evaluated by measuring pAkt following incubation with 10nM of Q-CCL7 and the indicated doses of ∆QP-CCL7 (lanes 5 to 8) or following pre-incubation with ∆QP-CCL7 (lane 11, red asterisk). Bars are medians and symbols represent individual mice. Data shown are representative experiments. All experiments were performed independently (≥ twice) besides f, which was performed once. Statistical analysis was done using unpaired, nonparametric Mann-Whitney U-test.

Source data

Extended Data Fig. 3 Model of chemokine-mediated peritoneal inflammation.

a,b,c, WT mice were intraperitoneally injected with PBS () or mCCL7 forms. a, Gating strategy for identification of peritoneal leukocytes. b, Frequency of monocytes (n = 9 (, Q and pE) or 7 (∆QP)). NS, P > 0.05, *P = 0.0008, **P = 0.0005. c, Number of neutrophils, eosinophils, B cells, T cells and NK cells (n = 9 (, Q and pE) or 7 (∆QP)). d, Mouse Q-CCL7 was intraperitoneally injected in Dpp4+/+ or Dpp4–/– littermates. Number of peritoneal monocytes is depicted (n = 5 ( Dpp4+/+, Q-CCL7 Dpp4+/+, Q-CCL7 Dpp4–/–) or 4 ( Dpp4–/–)). *P = 0.03, **P = 0.008. Bars are medians and, symbols represent individual mice. Data shown is representative (d) or pooled from 2 experiments (b and c). All experiments were repeated (≥ twice). Statistical analysis was done using unpaired, nonparametric Mann-Whitney U-test.

Source data

Extended Data Fig. 4 Identification of leukocytes in mouse tissues.

a,b,c, Gating strategy for identification of leukocytes in mouse a, blood, b, spleen and c, bone marrow.

Extended Data Fig. 5 Qpctl loss in both hematopoietic or stromal compartments compromises monocytes but not lymphocytes or tissue macrophages.

a, Identification of tissue macrophages in liver, lung and brain sections from naive Qpctl+/+ and Qpctl–/– littermates. Scale is 100µM. Representative images of 5 mice per genotype (experiment done once). b, Leukocyte quantification in blood from Qpctl+/+ and Qpctl–/– littermates (n = 16 (Qpctl+/+) or 19 (Qpctl–/–)). NS, P > 0.05. c, Quantification of splenic patrolling monocytes (CD11b+CD115+Ly6CCX3CR1+) in Qpctl+/+ and Qpctl–/– littermates (n = 10 (Qpctl+/+) or 9 (Qpctl–/–)). *P = 0.03. d,e, Chimeras were generated by reconstituting lethally irradiated WT or Qpctl–/– mice with WT or Qpctl–/– hematopoietic progenitors. d, Monocyte quantification in blood (n = 17 (WT→WT), 19 (Qpctl–/–Qpctl–/–) or 20 (WT→Qpctl–/–, Qpctl–/–→WT) and spleen (n = 19 (WT→WT, Qpctl–/–Qpctl–/–) or 20 (WT→Qpctl–/–, Qpctl–/–→WT). *P < 0.0001. e, Quantification of CCL7 PTMs in plasma (Total CCL7, n = 18 (WT→WT, Qpctl–/–Qpctl–/–) or 19 (WT→Qpctl–/–, Qpctl–/–→WT); pE-CCL7, n = 19 (Qpctl–/–Qpctl–/–, WT→Qpctl–/–) or 20 (WT→WT, Qpctl–/–→WT); ∆QP-CCL7, n = 19 (Qpctl–/–Qpctl–/–, Qpctl–/–→WT) or 20 (WT→WT, WT→Qpctl–/–). NS, P > 0.05, *P = 0.0025, **P = 0.001, ***P = 0.0008, ****P < 0.0001. f, WT (CD45.1) and Qpctl–/– (CD45.2) monocytes were co-transferred into WT (CD45.1/CD45.2) hosts. Quantification of transferred monocytes into the peritoneal cavity following pE-CCL7 injection, normalized to the ratio of WT and Qpctl–/– monocytes transferred (n = 9). g, Quantification of splenic monocytes in Qpctl+/+ and Qpctl–/– littermates treated with Ctrl chow or DPP4i (n = 10). *P = 0.0002. h, Quantification of monocytes in blood and spleen of WT, Dpp4–/–, Qpctl–/– and Dpp4–/–Qpctl–/– littermates (n = 10 (Dpp4–/–) or 9 (WT, Qpctl–/– and Dpp4–/–Qpctl–/–)). *P = 0.0056, **P < 0.0001. Bars are medians and symbols represent individual mice. Data shown is representative (f), or pooled from 2-3 experiments (b,c,d,e,g,h). All experiments were repeated (≥ twice) besides a. Statistical analysis was done using unpaired, nonparametric Mann-Whitney U-test.

Source data

Extended Data Fig. 6 Characterization of EO771 and LL/2 mouse tumor models.

a, Quantification of Ccl2, Ccl7 and Qpctl RNA expression in LL/2 and EO771 cells. (n = 2 (LL/2) or 1 (EO771) sequenced bulk samples, bars represent means). b, WT mice were inoculated with EO771 (n = 6) or with LL/2 (n = 8) cells. Frequency of tumor-associated leukocytes was quantified at day 14. Bars are medians and symbols individual mice. c, Gating strategy for the identification of leukocytes in tumors. d,e, Bulk CRISPR/Cas9-edited Qpctl–/– LL/2 and EO771 cells were generated. d, Quantification of pE-CCL7 in the cell supernatants, plotted as percentage of total CCL7, measured in the same samples. e, In vitro growth curves of tumor cell lines (n = 2 (EO771) or 1 (LL/2) technical replicates). - parental cell line; ntc - non-targeted control. Lines are means. Data shown are representative experiments. All experiments were repeated independently (≥ twice), except a, which was done once.

Source data

Extended Data Fig. 7 Loss or inhibition of QPCTL impairs monocyte migration.

a, Qpctl+/+ and Qpctl–/– littermates were inoculated with Qpctl+/+ or Qpctl–/– EO771 cells, respectively. Quantification of tumor-associated leukocytes (n = 6). *P = 0.009. b, Gating strategy to identify tumor-infiltrating WT CD45.1+ monocytes transferred into LL/2 tumor-bearing mice. c,d, WT mice received Ctrl vehicle or QPCTLi for 4 days before LL/2 or EO771 inoculation (preventative) or at day 7 after LL/2 inoculation (therapeutic). Quantification of c, splenic monocytes (n = 8 (LL/2 preventative), 8 (EO771 ctrl), 9 (EO771 QPCTLi), 10 (LL/2 Ctrl therapeutic) or 6 (LL/2 QPCTLi therapeutic)), *P = 0.02, **P = 0.004, ***P = 0.0002, and d, tumor-infiltrating macrophages (n = 8 (LL/2 preventative), 8 (EO771 Ctrl), 9 (EO771 QPCTLi), 10 (LL/2 Ctrl therapeutic) or 6 (LL/2 QPCTLi therapeutic)). NS, P > 0.05, *P = 0.01. e,f, WT mice treated with Ctrl Isotype or anti-CSF1R blocking antibody were inoculated with e, EO771 (n = 4), NS, P > 0.05, *P = 0.03 or f, LL/2 cells (n = 7 (Isotype) or 8 (anti-CSF1R)). NS, P > 0.05, *P = 0.0003. Quantification of leukocytes in tumors excised e, 14 days or f, 19 days after tumor cell inoculation. Bars are medians and symbols represent individual mice. Data shown are representative experiments. All experiments were repeated independently (≥ twice). Statistical analysis was done using unpaired, nonparametric Mann-Whitney U-test.

Source data

Extended Data Fig. 8 Single cell RNA-seq analysis of LL/2 immune infiltrates.

a,b,c,d, Qpctl–/– and Qpctl+/+ littermates were inoculated with Qpctl–/– or Qpctl+/+ LL/2 tumors, respectively. On day 14 after tumor inoculation, tumors were excised and processed for single-cell RNA-seq. a, Number of cells recovered per sample (n = 3). b, UMAP plots of tumor immune infiltrates, from merged samples (n = 6, three samples per genotype). c, Violin plots representing quality control measurements. d, Heatmap showing normalized expression of top 10 expressed genes in each cluster. Bars are medians and symbols represent individual mice. Mono/Mac/DCs: Monocyte/Macrophage/Dendritic Cells.

Extended Data Fig. 9 Loss of Qpctl remodels the tumor myeloid compartments.

a,b,c,d,e,f,g,h,i,j, Qpctl–/– and Qpctl+/+ littermates were inoculated with Qpctl–/– or Qpctl+/+ LL/2 tumors, respectively. On day 14 after tumor inoculation, tumors were excised and processed for single-cell RNA-seq. a, Heatmap showing normalized expression of top 10 expressed genes in each Monocyte/Macrophage/DC cluster. b, Dot plots of selected markers in monocyte populations. Dot size indicates proportion of cells in each cluster expressing a gene, color shading indicates the relative level of gene expression. c, Violin plot representing Ly6c2 expression across Monocyte/Macrophage/DC clusters. d, Violin plot representing the expression of a blood Ly6C+ monocyte signature across Monocyte/Macrophage/DC clusters. e, Dot plots of selected markers in dendritic cell populations. Dot size indicates proportion of cells in each cluster expressing a gene, color shading indicates the relative levels of gene expression. f, Violin plots representing the expression of indicated genes across macrophage clusters. g, Similarity score between the Mac_Ki67 cluster and the other Monocyte/Macrophage/DC clusters. h,i, Violin plots representing the expression of indicated gene signatures across macrophage clusters. j, Violin plot representing Csf1r expression across macrophage clusters.

Extended Data Fig. 10 Loss of Ccl2 and Ccl7 mimics loss of Qpctl in LL/2 tumors.

a,b, CRISPR/Cas9-edited Ccl2–/–Ccl7–/– LL/2 cells were generated. a, Quantification of MCPs in the cell supernatants (n = 1). b, WT mice were inoculated with WT or Ccl2–/–Ccl7–/– LL/2 cells. CCL7 quantification in plasma of naive and tumor-bearing mice (n = 5 (naive and day 14) and 10 (day 19)). *P = 0.008, **P < 0.0001. c,d, WT mice were inoculated with WT, Qpctl–/– or Ccl2–/–Ccl7–/– LL/2 cells. c, Tumor growth measurements (n = 10). Gray lines represent individual mice and overlay of fitting spline cubic curves for each group is shown. *P < 0.0001. d, Tumor weight at day 14 (n = 5). *P = 0.03, **P = 0.008. e,f,g, WT mice were inoculated as described in (c). e, Identification of Ly6C+ monocytic cells (red circle) and F4/80hi macrophages (blue circle) among tumor leukocytes (gated on live, singlets, CD45+CD11b+Ly6G). f, Quantification of Ly6C+ monocytic cells and macrophages (n = 5). g, Frequency of F4/80+MHCII+ macrophages (n = 5). *P = 0.02, **P = 0.008. h, Qpctl+/+ and Qpctl–/– EO771 bearing mice were treated with Ctrl Isotype or anti-CD8 depletion antibody. Identification of CD8+ T cells among CD3+ T cells in blood, 2 days after antibody injection. i, Qpctl+/+ and Qpctl–/– littermates were inoculated with Qpctl+/+ or Qpctl–/– EO771 tumor cells, respectively, and treated with Ctrl Isotype or anti-PD-L1 blocking antibody. Frequency of tumor-associated GzmB+CD8+ T cells (n = 12 (Qpctl+/+) or 14 (Qpctl–/–)). NS, P > 0.05. Bars are medians and symbols represent individual mice. Data shown is representative (a,b,c,d,f,g,h) or pooled from 2 experiments (i). All experiments were repeated independently (≥ twice). Statistical analysis was done using unpaired, nonparametric Mann-Whitney U-test (b,d,g,i) or Two-way ANOVA with Tukey correction (c).

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Barreira da Silva, R., Leitao, R.M., Pechuan-Jorge, X. et al. Loss of the intracellular enzyme QPCTL limits chemokine function and reshapes myeloid infiltration to augment tumor immunity. Nat Immunol 23, 568–580 (2022). https://doi.org/10.1038/s41590-022-01153-x

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