Agonistic CD40 therapy induces tertiary lymphoid structures but impairs responses to checkpoint blockade in glioma

Gliomas are brain tumors characterized by an immunosuppressive microenvironment. Immunostimulatory agonistic CD40 antibodies (αCD40) are in clinical development for solid tumors, but are yet to be evaluated for glioma. Here, we demonstrate that systemic delivery of αCD40 in preclinical glioma models induces the formation of tertiary lymphoid structures (TLS) in proximity of meningeal tissue. In treatment-naïve glioma patients, the presence of TLS correlates with increased T cell infiltration. However, systemic delivery of αCD40 induces hypofunctional T cells and impairs the response to immune checkpoint inhibitors in pre-clinical glioma models. This is associated with a systemic induction of suppressive CD11b+ B cells post-αCD40 treatment, which accumulate in the tumor microenvironment. Our work unveils the pleiotropic effects of αCD40 therapy in glioma and reveals that immunotherapies can modulate TLS formation in the brain, opening up for future opportunities to regulate the immune response.


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Life sciences study design
All studies must disclose on these points even when the disclosure is negative. No sample size calculation was performed to predetermine sample size in either in vivo/ex vivo or in vitro experiments. Sample size for in vivo/ex vivo experiments was determined based on previous experience with biological variation in the in vivo models used (PMID: 26363010, 26645582) and accounting for the 3R principle. The same applies to in vitro experiments, which were conducted with murine cells freshly isolated from naive mice. We believe that the chosen sample size is sufficient since our results are reproducible and consistent with respect to statistical differences between the treatment groups.
For in vivo experiments, mice were excluded from survival analysis if they died or were sacrificed for reasons other than brain tumor growth e.g. a few mice got an adverse reaction in direct association with the fourth administration of agonistic CD40 mAb. For flow cytometry analysis, samples were excluded if too few events were recorded for reliable analysis.
In vivo studies and in vitro assays were repeated multiple times (at least two independent experiments) and by different scientists to ensure reproducibility. The manuscript only includes the data which was reproducible.
For in vivo/ex vivo studies, allocation of mice to each treatment group was random. Mice of similar ages were randomly divided in treatment groups at the start of each experiment. Since the mice were of similar age and background, further randomization was not necessary. For in vitro assays testing the effect of agonistic CD40 mAb or CD11b neutralizing mAb, randomization was not necessary, since cells derived from the same mice were divided in two groups and treated with either the aforementioned antibodies or with the respective isotype controls.
The investigators were not blinded during group allocation, treatments and data collection, because there were rare side effects related to repeated administration of agonistic CD40 mAbs that needed to be carefully monitored. However, the investigators were blinded during data analysis.
All antibodies used in this study for immunofluorescence staining or flow cytometry are listed in Supplementary table 3,  Supplementary table 4 and Supplementary table 5. Information regarding fluorochromes, clone, dilution, catalog number and company are also reported in the above mentioned Supplementary tables.
Relevant publication or validations assays for all antibodies used for immunofluorescence staining or flow cytometry can be found on the RRID portal (https://scicrunch.org/resources) or on the manufacturer's website. For easy access to this information, the research resource identifiers (RRIDs) and catalogue numbers of all antibodies used for are provided in Supplementary The cell lines used in this study were not authenticated.
The cell lines routinely tested negative for mycoplasma contamination.
No misidentified cell lines were used in this study.
This study did not involve wild animals.
This study did not involve the use of field-collected samples All animal experiments were approved by the Uppsala County regional ethics committee (permits C1/14, C26/15, N164/15 and 5.8.18-19429/2019), and were performed according to the guidelines for animal experimentation and welfare of Uppsala University.
The histological and molecular tumor characteristics of the cohorts are shown in Supplementary Table 1. No additional patients were recruited to the study. Glioma samples from treatment-naïve patients containing meningeal tissue were selected from biobanks and anonymized. This part of the study was authorized by the regional Ethics Committee of Uppsala, Sweden (DNR 2010/291).
No additional participants were recruited to the study. Glioma samples from treatment-naïve patients containing meningeal tissues were selected from biobanked material. This selection was considered necessary since our observations from mouse models indicated that tertiary lymphoid structures were mainly present in meningeal regions. Results from the histological analysis were not associated with any clinical data or survival data, and correlated only to the presence of tertiary lymphoid structures and the amount of T cells infiltrating the tumor. It is possible that tertiary lymphoid structures were present in meningeal regions in other parts of the brain that have not been included in our examination. Nevertheless, our results give a clear indication that the prevalence of TLS, and the likelihood of identifying these structures in a given tissue, is associated with the number of enhanced T cells in the tumor.
The study was approved by the institutional review board at the Uppsala University Hospital, Uppsala, Sweden (DNR2010/05), the regional Ethics Committee of Uppsala, Sweden (DNR2015/089, DNR 2010/291) and the Ethics Committee of Western Sweden (EPN/DNR: 559-12). A statement that all participation was based on informed consent has been added to the Methods section.