Heterogeneity of response to immune checkpoint blockade in hypermutated experimental gliomas

Intrinsic malignant brain tumors, such as glioblastomas are frequently resistant to immune checkpoint blockade (ICB) with few hypermutated glioblastomas showing response. Modeling patient-individual resistance is challenging due to the lack of predictive biomarkers and limited accessibility of tissue for serial biopsies. Here, we investigate resistance mechanisms to anti-PD-1 and anti-CTLA-4 therapy in syngeneic hypermutated experimental gliomas and show a clear dichotomy and acquired immune heterogeneity in ICB-responder and non-responder tumors. We made use of this dichotomy to establish a radiomic signature predicting tumor regression after pseudoprogression induced by ICB therapy based on serial magnetic resonance imaging. We provide evidence that macrophage-driven ICB resistance is established by CD4 T cell suppression and Treg expansion in the tumor microenvironment via the PD-L1/PD-1/CD80 axis. These findings uncover an unexpected heterogeneity of response to ICB in strictly syngeneic tumors and provide a rationale for targeting PD-L1-expressing tumor-associated macrophages to overcome resistance to ICB.


Statistics
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Michael Platten
Jan 12, 2020 Flow cytometry data was acquired using the BD FACSDIVA software (version 9) or Attune NxT Software (version 2.5). Microscopy data was collected using the ZEISS ZEN software. MRI data was analysed with Osrix, ImageJ and Amira imaging software.

October 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences
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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. Gene expression data that support the findings of this study has been deposited in the Gene Expression Omnibus repository and will be made available prior to publication (GSE129877). All additional data sets generated or analysed during this study are included in this published article (and its supplementary information files).
Sample size was calculated with the help of a biostatistician using R version 3.4.0. Assumptions for power analysis were as follows: alpha error: 5%; beta error: 20%. Values for standard deviation and differences between experimental groups were based on previous experiments (whenever a similar data type was available). In all other cases a pilot group size was used.
Two NR RNA samples were excluded from Nanostring analysis due to failed QC. For CIBER sort analysis of pre-PD-1 inhibitor GBM tissue of R and NR patients, 6 samples were excluded as these were obtained more than 6.5 months before the start of PD-1 therapy. In case animals had to be sacrificed prior to the pre-defined endpoint (due to weight loss or other termination criteria), they were excluded from any downstream analysis.
Key experiments (ICB response, macrophage phenotyping by flow cytometry, PD-L1 inhibition,) were all performed at least twice and data from one representative experiment are shown in this manuscript. All other experiments were performed once with biological replicates or technical replicates (as specified in figure legend).
Mice were matched into the groups according to age, sex and tumor size (measured by MRI or calliper) at the time of treatment start.
Intracranial tumor experiments were performed in a blinded manner (MRI, flow cytometric analyses). Note that full information on the approval of the study protocol must also be provided in the manuscript.

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Recruitment
Ethics oversight Note that full information on the approval of the study protocol must also be provided in the manuscript. no commonly misidentified lines from the ICLAC register were used in this study C57BL/6J wild-type (WT) mice were purchased from Charles River or Janvier Labs. Sex-and age-matched mice were used for further experiments. If not stated otherwise, female mice were used for the experiments. All mice were 7-12 weeks of age at use. Mice were kept under SPF conditions at the animal facility of the DKFZ Heidelberg.
the study did not involve wild animals the study did not involve samples collected from the field Animal experiments were performed according to the rules of the German Animal Welfare Act and were licensed by the regional authority Karlsruhe. This study did not involve any human material.
RNA sequencing data of a previously published patient cohort was analyzed (Zhao J et al., Nat. Med., 2019) RNA sequencing data of a previously published patient cohort was analyzed (Zhao J et al., Nat. Med., 2019) RNA sequencing data of a previously published patient cohort was analyzed (Zhao J et al., Nat. Med., 2019) nature research | reporting summary

October 2018
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A numerical value for number of cells or percentage (with statistics) is provided. Lymphocytes were defined by size and granularity in FSC-A vs. SSC-A plots. Subsequently, duplets were excluded in FSC-A vs FSC-H plots and dead cells were excluded by means of fixable viability dye positivity (except for apoptosis analysis). T cells were gated according to the lineage marker CD45 and CD3, then T cell types were gated in CD4 vs CD8 plots. Infiltrating Gl261 CD11b myeloid cells were gated as CD45highCD11b+, microglia as CD45lowCD11b+ of living single cells in CNS samples. Gating strategies of multi-parameter Gl261 TIL analysis is shown in Extended Data Figure 3. Boundaries between positive and negative cells were defined by use of fluorescence minus one (FMO) controls. Expression of cytokines, chemokine receptors and pro-and anti-inflammatory mediators was determined by calculating the median or mean fluorescence intensity.

Methodology
Block design. All animals within one experiment were subjected to MRI on the same day.
Animals were subjected to MRI 3 times during one experiment at inervals between 6 and 7 days. I.e. MRI was performed on days 13, 19, and 26 post surgery if not otherwise stated in the online methods and figure legends.