Inhibition of the CCL2 receptor, CCR2, enhances tumor response to immune checkpoint therapy

Immunotherapies targeting the PD-1/PD-L1 axis are now a mainstay in the clinical management of multiple cancer types, however, many tumors still fail to respond. CCL2 is highly expressed in various cancer types and has been shown to be associated with poor prognosis. Inhibition or blockade of the CCL2/CCR2 signaling axis has thus been an area of interest for cancer therapy. Here we show across multiple murine tumor and metastasis models that CCR2 antagonism in combination with anti-PD-1 therapy leads to sensitization and enhanced tumor response over anti-PD-1 monotherapy. We show that enhanced treatment response correlates with enhanced CD8+ T cell recruitment and activation and a concomitant decrease in CD4+ regulatory T cell. These results provide strong preclinical rationale for further clinical exploration of combining CCR2 antagonism with PD-1/PD-L1-directed immunotherapies across multiple tumor types especially given the availability of small molecule CCR2 inhibitors and antibodies.


Ecological, evolutionary & environmental sciences study design
All studies must disclose on these points even when the disclosure is negative. Methods n/a Involved in the study ChIP-seq Flow cytometry

MRI-based neuroimaging
If participants were not allocated into experimental groups, state so OR describe how participants were allocated to groups, and if allocation was not random, describe how covariates were controlled.
Briefly describe the study. For quantitative data include treatment factors and interactions, design structure (e.g. factorial, nested, hierarchical), nature and number of experimental units and replicates.
Describe the research sample (e.g. a group of tagged Passer domesticus, all Stenocereus thurberi within Organ Pipe Cactus National Monument), and provide a rationale for the sample choice. When relevant, describe the organism taxa, source, sex, age range and any manipulations. State what population the sample is meant to represent when applicable. For studies involving existing datasets, describe the data and its source.
Note the sampling procedure. Describe the statistical methods that were used to predetermine sample size OR if no sample-size calculation was performed, describe how sample sizes were chosen and provide a rationale for why these sample sizes are sufficient.
Describe the data collection procedure, including who recorded the data and how.
Indicate the start and stop dates of data collection, noting the frequency and periodicity of sampling and providing a rationale for these choices. If there is a gap between collection periods, state the dates for each sample cohort. Specify the spatial scale from which the data are taken If no data were excluded from the analyses, state so OR if data were excluded, describe the exclusions and the rationale behind them, indicating whether exclusion criteria were pre-established.
Describe the measures taken to verify the reproducibility of experimental findings. For each experiment, note whether any attempts to repeat the experiment failed OR state that all attempts to repeat the experiment were successful.
Describe how samples/organisms/participants were allocated into groups. If allocation was not random, describe how covariates were controlled. If this is not relevant to your study, explain why.
Describe the extent of blinding used during data acquisition and analysis. If blinding was not possible, describe why OR explain why blinding was not relevant to your study.
Describe the study conditions for field work, providing relevant parameters (e.g. temperature, rainfall).
State the location of the sampling or experiment, providing relevant parameters (e.g. latitude and longitude, elevation, water depth).
Describe the efforts you have made to access habitats and to collect and import/export your samples in a responsible manner and in compliance with local, national and international laws, noting any permits that were obtained (give the name of the issuing authority, the date of issue, and any identifying information).
Describe any disturbance caused by the study and how it was minimized. Tick this box to confirm that the raw and calibrated dates are available in the paper or in Supplementary Information.

Ethics oversight
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Pre-validated antibodies were purchased from well recognized vendors and reported by other researchers. We based specificity on their provided description and data sheets. Anti-PD-1 and isotype control were validated by the manufacturer, Bristol-Myers Squibb. Titrations were performed by lab personnel to determine optimal dilution concentrations.

N/A
Provide provenance information for specimens and describe permits that were obtained for the work (including the name of the issuing authority, the date of issue, and any identifying information).
Indicate where the specimens have been deposited to permit free access by other researchers.
If new dates are provided, describe how they were obtained (e.g. collection, storage, sample pretreatment and measurement), where they were obtained (i.e. lab name), the calibration program and the protocol for quality assurance OR state that no new dates are provided.
Identify the organization(s) that approved or provided guidance on the study protocol, OR state that no ethical approval or guidance was required and explain why not.
Mus musculus, C67BL/6, female, ordered from Charles River. Mice were received at 6 weeks old and allowed to acclimate for at least one week in sterile micro isolator cages with constant temperature and humidity. Mice had free access to food and water.
Wild animals were not used in this study.
Field-collected samples were not used in this study.

nature research | reporting summary
April 2020

Ethics oversight
Note that full information on the approval of the study protocol must also be provided in the manuscript.

Human research participants
Policy information about studies involving human research participants Population characteristics

Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript.

Clinical data Policy information about clinical studies
All manuscripts should comply with the ICMJEguidelines for publication of clinical research and a completedCONSORT checklist must be included with all submissions.

Clinical trial registration
Study protocol

Data collection
Outcomes Dual use research of concern Policy information about dual use research of concern

Hazards
Could the accidental, deliberate or reckless misuse of agents or technologies generated in the work, or the application of information presented in the manuscript, pose a threat to:

Experiments of concern
Does the work involve any of these experiments of concern: No Yes Mice were housed in specific-pathogen-free conditions and cared for in accordance with US National Institutes of Health guidelines, and all procedures were approved by the University of Colorado Denver Animal Care and Use Committee and carried out according to approved protocols.
Describe the covariate-relevant population characteristics of the human research participants (e.g. age, gender, genotypic information, past and current diagnosis and treatment categories). If you filled out the behavioural & social sciences study design questions and have nothing to add here, write "See above." Describe how participants were recruited. Outline any potential self-selection bias or other biases that may be present and how these are likely to impact results.
Identify the organization(s) that approved the study protocol. ChIP-seq Data deposition Confirm that both raw and final processed data have been deposited in a public database such as GEO.
Confirm that you have deposited or provided access to graph files (e.g. BED files) for the called peaks.

Data access links
May remain private before publication.

Files in database submission
Genome browser session Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.
For "Initial submission" or "Revised version" documents, provide reviewer access links. For your "Final submission" document, provide a link to the deposited data.
Provide a list of all files available in the database submission.
Provide a link to an anonymized genome browser session for "Initial submission" and "Revised version" documents only, to enable peer review. Write "no longer applicable" for "Final submission" documents.
Describe the experimental replicates, specifying number, type and replicate agreement.
Describe the sequencing depth for each experiment, providing the total number of reads, uniquely mapped reads, length of reads and whether they were paired-or single-end.
Describe the antibodies used for the ChIP-seq experiments; as applicable, provide supplier name, catalog number, clone name, and lot number.
Specify the command line program and parameters used for read mapping and peak calling, including the ChIP, control and index files used.
Describe the methods used to ensure data quality in full detail, including how many peaks are at FDR 5% and above 5-fold enrichment.
Describe the software used to collect and analyze the ChIP-seq data. For custom code that has been deposited into a community repository, provide accession details.
Tumors were mechanically dissociated in Click's media in the absence of mercaptoethanol or L-glutamine (Irvine Scientific). Cells were digested for 1 hour at 37°C with 500 units/ml collagenase type II and IV and 20 "g/ml DNase (Worthington Biochemical). The digested tissue suspension was then filtered through a 100 "m strainer. Filtered cells were carefully layered into a centrifuge tube containing 5 ml Lympholyte-M (Cedarlane). The cells were centrifuged at 1500g for 20 min., then the interface lymphocyte layer was carefully removed. The cells were washed prior to staining.
All samples were run on the CyAn ADP flow cytometer, acquired using Summit software.
Analysis was performed using FlowJo software.
Cell samples were not subjected to cell sorting. Immune populations were determined through singlet, live-dead, and CD45+ cell gating.
Singlet, live cells were gated for CD45+ cells. CD8 T cells were gated on live, CD3+/CD8+ double-positive cells. The cells were then further classified based on the expression of PD-1 and Lag-3. Neutrophils were gated by the expression of Ly-6Ghi/ CD11b+. Macrophages were gated as F4/80+/CD11bhi population and MERTKhi/CD64hi population. Monocytes were confirmed with two population gates as F4/80lo/CD11b+ and MERTKlo/CD64+. Cells were further analyzed for expression of PD-1 and Lag-3. For CD4 T cell analysis, singlet, live, CD45+ were gated for the CD4+/CD8-population. Further classification of activated CD4 T cells as CD25+/FoxP3-and regulatory CD4 T cells as FoxP3+ was performed. Samples of the flow gating strategy are provided in the Supplementary.