The immune suppressive microenvironment affects efficacy of radio‐immunotherapy in brain metastasis
2021; Springer Nature; Volume: 13; Issue: 5 Linguagem: Inglês
10.15252/emmm.202013412
ISSN1757-4684
AutoresKatja Niesel, Michael Schulz, Julian Anthes, Tijna Alekseeva, Jadranka Macas, Anna Salamero‐Boix, Aylin Möckl, T. Oberwahrenbrock, Marco Lolies, Stefan Stein, Karl H. Plate, Yvonne Reiss, Franz Rödel, Lisa Sevenich,
Tópico(s)Cancer Immunotherapy and Biomarkers
ResumoArticle23 March 2021Open Access Source DataTransparent process The immune suppressive microenvironment affects efficacy of radio-immunotherapy in brain metastasis Katja Niesel Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Michael Schulz Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Biological Sciences, Faculty 15, Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Julian Anthes Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Tijna Alekseeva Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Jadranka Macas Institute of Neurology (Edinger Institute), University Hospital, Goethe University, Frankfurt am Main, Germany Frankfurt Cancer Institute (FCI), Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Anna Salamero-Boix Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Biological Sciences, Faculty 15, Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Aylin Möckl Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Timm Oberwahrenbrock Fraunhofer Institute for Translational Medicine and Pharmacology (ITMP), Frankfurt am Main, Germany Fraunhofer Cluster of Excellence Immune Mediated Diseases (CIMD), Frankfurt am Main, Germany Search for more papers by this author Marco Lolies Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Stefan Stein Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Karl H Plate Institute of Neurology (Edinger Institute), University Hospital, Goethe University, Frankfurt am Main, Germany Frankfurt Cancer Institute (FCI), Goethe University Frankfurt, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Germany and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Yvonne Reiss Institute of Neurology (Edinger Institute), University Hospital, Goethe University, Frankfurt am Main, Germany Frankfurt Cancer Institute (FCI), Goethe University Frankfurt, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Germany and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Franz Rödel Frankfurt Cancer Institute (FCI), Goethe University Frankfurt, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Germany and German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Radiotherapy and Oncology, Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Lisa Sevenich Corresponding Author [email protected] orcid.org/0000-0002-6543-6273 Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Frankfurt Cancer Institute (FCI), Goethe University Frankfurt, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Germany and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Katja Niesel Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Michael Schulz Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Biological Sciences, Faculty 15, Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Julian Anthes Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Tijna Alekseeva Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Jadranka Macas Institute of Neurology (Edinger Institute), University Hospital, Goethe University, Frankfurt am Main, Germany Frankfurt Cancer Institute (FCI), Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Anna Salamero-Boix Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Biological Sciences, Faculty 15, Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Aylin Möckl Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Timm Oberwahrenbrock Fraunhofer Institute for Translational Medicine and Pharmacology (ITMP), Frankfurt am Main, Germany Fraunhofer Cluster of Excellence Immune Mediated Diseases (CIMD), Frankfurt am Main, Germany Search for more papers by this author Marco Lolies Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Stefan Stein Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Search for more papers by this author Karl H Plate Institute of Neurology (Edinger Institute), University Hospital, Goethe University, Frankfurt am Main, Germany Frankfurt Cancer Institute (FCI), Goethe University Frankfurt, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Germany and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Yvonne Reiss Institute of Neurology (Edinger Institute), University Hospital, Goethe University, Frankfurt am Main, Germany Frankfurt Cancer Institute (FCI), Goethe University Frankfurt, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Germany and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Franz Rödel Frankfurt Cancer Institute (FCI), Goethe University Frankfurt, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Germany and German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Radiotherapy and Oncology, Goethe University Frankfurt, Frankfurt am Main, Germany Search for more papers by this author Lisa Sevenich Corresponding Author [email protected] orcid.org/0000-0002-6543-6273 Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany Frankfurt Cancer Institute (FCI), Goethe University Frankfurt, Frankfurt am Main, Germany German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Germany and German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Author Information Katja Niesel1, Michael Schulz1,2, Julian Anthes1, Tijna Alekseeva1, Jadranka Macas3,4, Anna Salamero-Boix1,2, Aylin Möckl1, Timm Oberwahrenbrock5,6, Marco Lolies1, Stefan Stein1, Karl H Plate3,4,7, Yvonne Reiss3,4,7, Franz Rödel4,7,8 and Lisa Sevenich *,1,4,7 1Institute for Tumor Biology and Experimental Therapy, Georg-Speyer-Haus, Frankfurt am Main, Germany 2Biological Sciences, Faculty 15, Goethe University Frankfurt, Frankfurt am Main, Germany 3Institute of Neurology (Edinger Institute), University Hospital, Goethe University, Frankfurt am Main, Germany 4Frankfurt Cancer Institute (FCI), Goethe University Frankfurt, Frankfurt am Main, Germany 5Fraunhofer Institute for Translational Medicine and Pharmacology (ITMP), Frankfurt am Main, Germany 6Fraunhofer Cluster of Excellence Immune Mediated Diseases (CIMD), Frankfurt am Main, Germany 7German Cancer Consortium (DKTK), Partner Site Frankfurt/Mainz, Germany and German Cancer Research Center (DKFZ), Heidelberg, Germany 8Department of Radiotherapy and Oncology, Goethe University Frankfurt, Frankfurt am Main, Germany *Corresponding author. Tel: +49 69 63395560; E-mail: [email protected] EMBO Mol Med (2021)13:e13412https://doi.org/10.15252/emmm.202013412 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The tumor microenvironment in brain metastases is characterized by high myeloid cell content associated with immune suppressive and cancer-permissive functions. Moreover, brain metastases induce the recruitment of lymphocytes. Despite their presence, T-cell-directed therapies fail to elicit effective anti-tumor immune responses. Here, we seek to evaluate the applicability of radio-immunotherapy to modulate tumor immunity and overcome inhibitory effects that diminish anti-cancer activity. Radiotherapy-induced immune modulation resulted in an increase in cytotoxic T-cell numbers and prevented the induction of lymphocyte-mediated immune suppression. Radio-immunotherapy led to significantly improved tumor control with prolonged median survival in experimental breast-to-brain metastasis. However, long-term efficacy was not observed. Recurrent brain metastases showed accumulation of blood-borne PD-L1+ myeloid cells after radio-immunotherapy indicating the establishment of an immune suppressive environment to counteract re-activated T-cell responses. This finding was further supported by transcriptional analyses indicating a crucial role for monocyte-derived macrophages in mediating immune suppression and regulating T-cell function. Therefore, selective targeting of immune suppressive functions of myeloid cells is expected to be critical for improved therapeutic efficacy of radio-immunotherapy in brain metastases. Synopsis This preclinical study demonstrates the potential of radiotherapy to sensitize breast cancer brain metastasis to checkpoint inhibition. Myeloid cells contribute to immune suppression affecting long-term survival and therefore represent a target for improved radio-immunotherapy. Radiotherapy increases CD8+ infiltration into breast cancer brain metastases. Reactivating T cells with immune checkpoint inhibition via anti-PD-1 in combination with radiotherapy improves survival and reduces tumor growth. Infiltration of blood borne PD-L1+ myeloid cells is increased after radio-immunotherapy and likely suppresses re-activated anti-tumor T cell immunity. The paper explained Problem Response rates to checkpoint inhibitors for poorly immunogenic cancers are low. Additionally, cancer cells metastasizing to the brain are protected from the adaptive immune system by the immune suppressive tumor microenvironment (TME). Therefore, many brain metastasis (BrM) patients cannot benefit from the advent of immunotherapies and are restricted to standard of care therapies with limited success rates. Results We found that cell types essential for successful immunotherapy via PD-1 blockade are present in murine breast cancer-derived BrM, including T cells and dendritic cells. T cells in the TME of a breast cancer BrM mouse model clonally expanded, indicating prior T-cell activation. A high proportion of T cells expressed PD-1, whereas tumor and myeloid cells recruited to the lesions expressed PD-L1. Classical fractionated whole brain radiotherapy (WBRT) increased the proportion of cytotoxic CD8+ T cells and prolonged survival transiently. Combination therapy of WBRT and PD-1 blockade increased T-cell infiltration and prevented the induction of compensatory inhibitory responses in lymphocytes induced by anti-PD-1 monotherapy. This led to reduced tumor progression and prolonged survival compared with WBRT alone. Analysis of combination-treated BrM revealed increased infiltration with PD-L1-positive myeloid cells recruited from the periphery. Impact We demonstrate that radiotherapy can sensitize low immunogenic BrM to checkpoint blockade. We showed that myeloid cells recruited from the periphery play a crucial role in regulating T-cell activation and are implicated in generating an immune suppressive environment, underlying the importance of investigating the role of these cells in acquired resistance to checkpoint blockade in more details. Introduction Immunotherapies that aim to reactivate immune responses against tumor cells have been implemented as standard therapies for different cancer entities (Pardoll, 2012). A major focus has been directed to strategies that block immune checkpoint molecules (Havel et al, 2019). Response rates to immune checkpoint blockade (ICB) depend on the mutational load of individual cancer types and the immune contexture of the respective tumor microenvironment (McGranahan et al, 2016; Mandal et al, 2019; Zhao et al, 2019). Preclinical and clinical studies revealed that hypermutated cancers with high infiltration of lymphoid effector cells show better response rates than immune excluded tumors with low mutational burden (Nishino et al, 2017; Zappasodi et al, 2018). Moreover, tumors with high content of immune suppressive cell types such as macrophages and neutrophils show low response rates to ICB (Nakamura & Smyth, 2020). Brain tumors are considered as immunologically cold tumors given the immune privileged status of the central nervous system (CNS) and the highly immune suppressive environment (Antonios et al, 2017; Quail & Joyce, 2017; Chongsathidkiet et al, 2018; Priego et al, 2018; Tomaszewski et al, 2019; Friebel et al, 2020; Pombo Antunes et al, 2020; Schulz et al, 2020). T-cell recruitment to brain metastases (BrM) is dependent on the primary tumor entity. Melanoma BrM show high T-cell content, whereas low-to-moderate T-cell recruitment is observed in breast cancer BrM (Harter et al, 2015; Berghoff et al, 2016; Friebel et al, 2020; Klemm et al, 2020). Clinical data revealed moderate response rates of ICB applied as monotherapy with melanoma and NSCLC patients showing highest response rates among BrM patients (Brahmer et al, 2015; Goldberg et al, 2016; Herbst et al, 2016; Reck et al, 2016; Adams et al, 2019). Despite the presence of innate and adaptive immune cell types in brain tumors, there is evidence that anti-tumor T-cell responses are inhibited by the highly immune suppressive brain tumor microenvironment (TME) even in the context of ICB (Aslan et al, 2020). It was reported that the efficacy of ICB in a melanoma mouse model depends on the presence of extracranial tumors and increased CD8+ T-cell trafficking into BrM (Taggart et al, 2018). The necessity of CD8+ T-cell priming and trafficking to CNS lesions to mount anti-tumor immune responses in synergy with ICB was further illustrated by a vascular endothelial growth factor-C (VEGF-C)-mediated modulation of the meningeal lymphatic system (Song et al, 2020). This indicates that immune modulation in BrM has the potential to overcome resistance to ICB. Given recent reports on synergistic anti-tumor effects of radio-immunotherapy (Koller et al, 2017) and the accumulating evidence that radiation can be used as an immune modulatory agent (Rodriguez-Ruiz et al, 2018; Sevenich, 2019; Schulz et al, 2020), we sought to evaluate the efficacy of radio-immunotherapy in the syngeneic breast-to-brain metastasis model 99LN-BrM (Bowman et al, 2016; Chae et al, 2019) with a particular focus on immune modulation induced by whole brain radiotherapy (WBRT) alone and in combination with ICB. Here, we show that WBRT sensitizes BrM to ICB by increasing the relative abundance of CD8+ T cells and by preventing the establishment of lymphoid cell-mediated immune suppressive effects. However, recurrent tumor lesions show accumulation of PD-L1+ monocyte-derived macrophages after radio-immunotherapy suggesting enhanced myeloid-mediated immune suppression to counteract T-cell reactivation in BrM. Results Cellular composition of the immune compartment in brain metastases We first characterized the cellular composition of brain metastatic lesions in the syngeneic breast-to-brain metastasis model 99LN-BrM (Appendix Fig S1). Flow cytometric analysis of macrodissected 99LN-BrM revealed that 50% of the cells in 99LN-BrM tumors are CD45-EpCAM+ tumor cells, while the remaining 50% are constituted by different tumor-infiltrating non-cancerous cell types. CD45+ leukocytes represented approximately 20% of the cells of the BrM-associated TME (Fig 1A, Appendix Fig S2). Within the immune compartment, myeloid cells (CD45+CD11b+ cells) represented the most abundant population (Fig 1B). Further classification of BrM-associated myeloid cells (Appendix Fig S2) revealed that brain-resident microglia (MG; CD45+CD11b+Ly6ClowLy6G−CD49d−) are the most prominent population, followed by blood-borne myeloid cells, including monocyte-derived macrophages (MDM; CD45+CD11b+Ly6ClowLy6G−CD49d+), inflammatory monocytes (CD45+CD11b+Ly6ChighLy6G−), and granulocytes (CD45+CD11b+Ly6CmedLy6G+) (Fig 1C). CD11c+MHCII+ cells represented another abundant population. Within this population, conventional dendritic cells 1 (cDC1: CD11c+MHCII+CD11b−CD24+) and 2 (cDC2: CD11c+MHCII+CD11b+) constituted 10 and 50%, respectively. For lymphocytes, we focussed our analysis on CD3+ T cells and B220+ B-cell populations that represented on average 8 and 12.5% of all CD45+ cells (Fig 1C). CD4+ and CD8+ T cells constituted on average 30 and 33% of the CD3+ T-cell population revealing a relatively high amount of double-negative (DN) CD3+ T cells in 99LN-BrM. This population was further stratified into γδ T cells (CD45+CD3+CD4−CD8-γδTCR+), NKT cells (CD45+CD3+CD4−CD8−DX5+), and a remaining DN CD3+ T-cell population (other DN) (Fig 1C). In contrast to previous findings (Chongsathidkiet et al, 2018), we did not observe significant differences in CD3+ T-cell numbers in peripheral blood isolated from BrM-bearing mice compared with tumor-free animals (Appendix Fig S3A). However, further stratification of T cells revealed decreased CD8+ T-cell numbers, whereas CD4+ T-cell numbers did not change (Appendix Fig S3B and C). Figure 1. Immune composition of the TME in breast-to-brain metastases A. Stacked column depicts proportions of cell types in the TME of 99LN-BrM analyzed by flow cytometry (n = 4). B. Stacked column depicts the relative amount of myeloid and lymphoid cells in 99LN-BrM analyzed by flow cytometry (n = 5). C. Stacked column depicts the relative amount of myeloid and lymphoid subpopulations in 99LN-BrM based on three flow cytometry panels: myeloid cells (n = 5), dendritic cells (n = 7), and lymphoid cells (n = 6). D. Principal component analysis of tumor-associated myeloid cells vs. blood monocytes and microglia from tumor-free mice (n = 3 per condition). E. Principal component analysis of tumor-infiltrating lymphocytes (TIL) vs blood lymphocytes from tumor-free mice (n = 3 per condition). F–I. Functional gene annotation of altered cellular pathways in (F) TAM-MG, (G) TAM-MDM, (H) TIL-CD4, and (I) TIL-CD8 compared with control cell types from tumor-free animals. Cutoffs: basemean > 20 and adjusted P-value (Padj) < 0.05. Adjusted P-values were obtained by Wald test and corrected for multiple testing using the Benjamini and Hochberg method. All DEGs based on Padj were subjected to analysis. Source data are available online for this figure. Source Data for Figure 1 [emmm202013412-sup-0005-SDataFig1.xlsx] Download figure Download PowerPoint Transcription programs in tumor-associated macrophages and lymphocytes in BrM We next performed RNA sequencing of different FACS purified myeloid and lymphoid populations isolated from brain metastatic lesions, normal brain, or peripheral blood (Dataset EV1). The purity of the sorted populations was validated by the expression of cell type-restricted markers (Fig EV1A and B). We observed pronounced transcriptional changes in tumor-associated cell populations compared with their normal cellular counterparts (Figs 1D and E, and EV1A–C and E, Dataset EV1). The majority of differentially expressed genes (DEG) was restricted to specific cell types. However, we also observed a considerable overlap of common DEG within the analyzed myeloid and lymphoid populations indicating the induction of core transcriptional programs in tumor-associated myeloid and lymphoid populations (Fig EV1D). Consistent with previous findings (Bowman et al, 2016; Klemm et al, 2020; Schulz et al, 2020), we observed loss of expression of microglial markers such as P2ry12, Tmem119, and Cx3cr1 in TAM-MG and increased expression of microglial markers in TAM-MDM compared with their normal cellular counterparts (Fig EV1A). The expression of T-cell markers was stable in TILs compared with blood lymphocytes. However, TIL-B cells acquired expression of Cd3, Cd4, and Cd8 and showed reduced expression of the B-cell markers Cd19 and Cd20 (Fig EV1B). Pathway analyses revealed that transcriptional programs in tumor-associated MG were associated with inflammation, cell motility, and proliferation. In contrast, house-keeping functions associated with tissue homeostasis were downregulated in TAM-MG compared with normal MG (Fig 1F). Likewise, TAM-MDM compared with blood monocytes showed induction of inflammatory responses, increased cell motility, and proliferation as well as association to wound repair processes. Moreover, changes in metabolism and signal transductions were observed in TAM-MDM (Fig 1G). Transcriptional programs in CD4+ T cells indicate the induction of immune responses, increased cell motility and apoptosis (Fig 1H). CD8+ TILs showed enrichment of pathways associated with proliferation, programmed cell death, and metabolism (Fig 1I). Both, CD4+ and CD8+ T cells, showed loss of pathways involved in blood coagulation and platelet formation (Fig 1H and I). Click here to expand this figure. Figure EV1. Transcription programs in tumor-associated myeloid and lymphoid populations Expression of MG and monocyte/MDM restricted genes in tumor-associated myeloid cells and control cells from tumor-free animals (n = 3; note: individual samples showed no expression for specific genes and are indicated on the x-axis). Values are depicted as normalized counts. Expression of T- and B-cell restricted genes in tumor-infiltrating lymphocytes and blood lymphocytes from tumor-free animals (n = 3; note: individual samples showed no expression for specific genes and are indicated on the x-axis). Values are depicted as normalized counts. Amount of significant DEG (cutoff: base mean (BM) > 20, Padj < 0.05) in different cell types based on RNAseq data of tumor-associated vs. control cell types (n = 3). Euler plots depict shared and unique DEGs (cutoff: BM > 20, Padj. < 0.05) in each cell type based on RNAseq data from tumor-associated vs. control cell types (n = 3). Unsupervised clustering of the top 100 DEG in control MG vs. TAM-MG, BL-Mono vs. TAM-MDM. BL-CD4 vs. TIL-CD4 and BL-CD8 vs. TIL-CD8 (cutoff: BM > 20, Padj. < 0.05) (n = 3). Selected genes are annotated. Data information: Adjusted P-values (Padj.) in (C–E) were obtained by Wald test and corrected for multiple testing using the Benjamini and Hochberg method. Source data are available online for this figure. Download figure Download PowerPoint BrM onset and progression are not dependent on T cells We employed a T-cell depletion strategy using CD4 and CD8 neutralizing antibodies to analyze the functional role of T cells in controlling BrM onset and progression. Treatment was commenced on day 7 after tumor cell inoculation with doses on three consecutive days followed by weekly injections of the neutralizing antibodies (Fig 2A). Flow cytometry confirmed a significant reduction of T-cell numbers in peripheral blood (Fig 2B and Appendix Fig S4). Moreover, histological assessment revealed depletion of CD3+ T cells in BrM with no effects on Iba1+ macrophage numbers (Fig 2C and D). However, T-cell depletion did not affect BrM onset or progression (Fig 2E–G). This finding suggests that TILs are ineffective in controlling disease progression. To address the activation status of tumor-infiltrating T cells, we queried the RNA sequencing data for the expression of a panel of effector and exhaustion markers (Wherry & Kurachi, 2015; Winkler & Bengsch, 2019) and observed prominent acquisition of an exhausted T-cell phenotype in both CD4+ and CD8+ T cells in BrM (Fig 2H). Figure 2. The effect of T-cell depletion on tumor onset and progression in 99LN-BrM Experimental design of T-cell depletion by αCD4 and αCD8a antibody treatment. Representative flow cytometry blots of blood samples from mice of both groups 3 weeks after treatment start showing successful depletion of CD45+CD3+ T cells in the αCD4 + αCD8 treatment group. Representative IHC images of CD3+ T cells and Iba1+ macrophages/MG in the isotype and αCD4 + αCD8 group. Scale bar; 100 µm. Quantification of Iba1+ and CD3+ cells in IHC sections of BrM from the isotype (n = 9) and αCD4 + αCD8 group (n = 7). Representative MRI pictures of BrM in early, medium, and late stage of both groups. Kaplan–Meier curves depict BrM-free survival of mice in the isotype and αCD4 + αCD8 group (Isotype n = 19, αCD4 + αCD8 n = 19). Tumor growth curves depict the increase in absolute BrM volume for each mouse in both groups over time (isotype n = 19, αCD4 + αCD8 n = 19). Heatmap displays the relative expression level of T-cell effector and exhaustion marker in TIL-CD4 and TIL-CD8 compared with blood lymphocytes. Values display the average per group based on vst values (BL-CD4 or BL-CD8 with n = 3 for each group) vs. BrM-associated TIL-CD4 or TIL-CD8 (n = 3 for each group). Data information: Numerical data in (D) are represented as scatter dot plot with line at mean ± SD. P-values were obtained by unpaired t-test in (D) or based on adjusted P-value (Padj) obtained by Wald test and corrected for multiple testing using the Benjamini and Hochberg method in (H) with *P < 0.05, **P < 0.01, and ***P < 0.001. Exact P-values can be found in Appendix Table S3 for (D) and the Data Source file associated with Fig 2 for (H). Source data are available online for this figure. Source Data for Figure 2 [emmm202013412-sup-0006-SDataFig2.zip] Download figure Download PowerPoint Blood-borne myeloid cells play a critical role in regulating T-cell activity We next sought to evaluate which cells within the BrM TME are involved in modulating T-cell activity. Quantitative real-time PCR (qRT-PCR) indicated high expression levels of PD-L1 in tumor cell lines, whereas PD-1 expression was low (Fig EV2A). Flow cytometry demonstrated that up to 40% of 99LN-BrM cells, 25% of TS1-BrM cells, and 90% of B16-F10 cells express PD-L1 in vitro (Fig EV2B). In vivo, PD-1 expression was found in lymphocytes, whereas PD-L1 expression was mostly associated with tumor cells (Fig EV2C). Flow cytometry confirmed that 40% of T cells expressed PD-1 with lower PD-1 expression on tumor cells and myeloid cells (Fig 3A). PD-L1 expression was highest on tumor cells followed by myeloid cells and T cells (Fig 3B). PD-L1 expression was almost absent on brain-resident myeloid cells in tumor-free animals but was strongly induced in tumor-associated myeloid cells (Fig 3C). Interestingly, we observed low PD-L1 expression on TAM-MG. In contrast, granulocytes, monocytes, and MDMs showed significantly higher PD-L1 expression (Fig 3D). We next queried the expression of genes involved in antigen presentation as well as co-regulatory factors with stimulatory and/or inhibitory functions in TAMs. This analysis revealed that the majority of genes associated with antigen presentation and regulation of T-cell activity show higher expression in TAM-MDM compared with TAM-MG (Fig 3E). Only the expression of the co-regulatory factor Cd112 and Vista was significantly higher in TAM-MG compared with TAM-MDM. In T cells, Ctla4 and Pd-1 were the most prominently expressed co-inhibitory receptors (Fig 3F). The critical role for TAM-MDM in regulating T-cell activity was further supported by functional annotation of enriched pathways in TAM-MG vs. TAM-MDM. Top 10 pathways in TAM-MG were associated with cell migration, angiogenesis, and wound repair mechanism, whereas pathways enriched in TAM-MDM were associated with immune modulation in particular regulation of lymphocyte activity (Fig 3G). We next performed multiplex immunofluorescence staining to evaluate the spatial distribution of TILs in BrM relative to tumor cells and TAMs using marker combinations that allow to discriminate TAM populations based on the expression of the macrophage marker Iba1 and the microglial marker Tmem119. Iba1+Tmem119+ MG were the most abundant population in the adjacent brain parenchyma and in the peri-tumor area. Infiltration of Iba1+Tmem119+ cells into BrM lesions was observed. However, Iba1+ cells with low or no Tmem119 expression were most prominent within tumor lesions, most likely representing BrM infiltrating TAM-MDM. We observed close proximity of most T cells to tumor cells and TAMs (Fig 3H and I). Interestingly, CD4+ and CD8+ T cells were localized closer to Iba1+Tmem119− TAMs compared with Iba1+Tmem119+ TAMs (Fig 3H and I) suggesting close proximity of T cells to TAMs associated with modulating T-cell functions in BrM. Click here to expand this figure. Figure EV2. Expression of PD-1 and PD-L1 by breast cance
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