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Representative images of immunohistochemical stainings of the isotype controls IgG and IgG1 in normal brain (NB) tissues, astrocytomas (WHO{degree sign}II: n = 10; WHO{degree sign}III: n = 10), and oligodendrogliomas (WHO{degree sign}II: n = 10; WHO{degree sign}III: n = 10). Scale bar: 50 µm.

TissueQuest gating strategy and Kaplan-Meier plots of significant clinical parameters.

(A) IFN-� ELISpot raw data of all HLA-A*02:01 patients analyzed (n = 7) showing the IFN-� spots / 1x105 T-cells for patient cells against TAAs vs. patient cells against the negative control (human immunodeficiency virus (HIV) gag/pol, 9 amino acids). Grey bars indicate a significantly increased immunogenicity compared to negative control (solid line). (B) Homogenous levels of stimulation while comparing the total immune response by mean of IFN-� spot numbers among astrocytomas (red) and oligodendrogliomas (blue) to a certain reactive epitope. (n.s., not significant, *, p < 0.05; **, p < 0.01)

IFN-� ELISpot raw data to validate antigen immunogenicity in patients of origin. Antigens with a significant increased immunogenicity (grey bars and asterisks) or 1.3-fold increased levels of immune responses (grey bars) were further validated in a bigger study sample of healthy individuals and lower-grade glioma patients. Solid line represents mean background of negative control (IgG1). (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

DNA methylation analysis in primary TCGA LUADs and comparative analysis of methylation of all CpG sites in the primary and metastatic tumors.

Further information on antibodies, differentially expressed genes, clinical characteristics, survival analysis, mutations, and methylation.

Purpose:To provide a better understanding of the interplay between the immune system and brain metastases to advance therapeutic options for this life-threatening disease.Experimental Design:Tumor-infiltrating lymphocytes (TIL) were quantified by semiautomated whole-slide analysis in brain metastases from 81 lung adenocarcinomas. Multi-color staining enabled phenotyping of TILs (CD3, CD8, and FOXP3) on a single-cell resolution. Molecular determinants of the extent of TILs in brain metastases were analyzed by transcriptomics in a subset of 63 patients. Findings in lung adenocarcinoma brain metastases were related to published multi-omic primary lung adenocarcinoma The Cancer Genome Atlas data (n = 230) and single-cell RNA-sequencing (scRNA-seq) data (n = 52,698).Results:TIL numbers within tumor islands was an independent prognostic marker in patients with lung adenocarcinoma brain metastases. Comparative transcriptomics revealed that expression of three surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) was closely associated with TIL numbers. Their expression was not only prognostic in brain metastasis but also in primary lung adenocarcinoma. Correlation with scRNA-seq data revealed that brain metastases with high expression of surfactant genes might originate from tumor cells resembling alveolar type 2 cells. Methylome-based estimation of immune cell fractions in primary lung adenocarcinoma confirmed a positive association between lymphocyte infiltration and surfactant expression. Tumors with a high surfactant expression displayed a transcriptomic profile of an inflammatory microenvironment.Conclusions:The expression of surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) defines an inflamed subtype of lung adenocarcinoma brain metastases characterized by high abundance of TILs in close vicinity to tumor cells, a prolonged survival, and a tumor microenvironment which might be more accessible to immunotherapeutic approaches.

Differentially expressed genes between the T cell infiltration groups and boxplots of gene expression.

Boxplots comparing the infiltration of TIL subsets for preoperative treatments (chemotherapy or other systemic therapy) as well as for the comparison of synchronous vs. metachronous brain metastases.

Correlation plots of T cell subtypes.

Details on the gene expression, single-cell sequencing, and copy number alteration data.

Purpose: Successful immunotherapies for IDHmut gliomas require better knowledge of T-cell target antigens. Here, we elucidated their antigen repertoire recognized by spontaneous T-cell responses using an unbiased proteomic approach.Experimental Design: Protein fractionations of tissue lysates from IDHmut gliomas (n = 4) were performed. Fractions were tested by IFNγ ELISpot assay for recognition through patients' T cells. Proteins of immunogenic fractions were identified by mass spectrometry and validated by in silico-predicted synthetic long peptides in patients of origin, additional IDHmut glioma patients (n = 16), and healthy donors (n = 13). mRNA and protein expression of immunogenic antigens was analyzed in tumor tissues and IDHmut glioma stem-like cells (GSC). HLA-A*02–restricted T-cell epitopes were functionally determined by short peptides and numbers of antigen-specific T cells by HLA-peptide tetramer analysis.Results: A total of 2,897 proteins were identified in immunogenic tumor fractions. Based on a thorough filter process, 79 proteins were selected as potential T-cell antigens. Twenty-six of these were recognized by the patients’ T cells, and five of them (CRKII, CFL1, CNTN1, NME2, and TKT) in up to 56% unrelated IDHmut glioma patients. Most immunogenic tumor-associated antigens (TAA) were expressed in IDHmut gliomas and GSCs, while being almost absent in normal brain tissues. Finally, we identified HLA-A*02–restricted epitopes for CRKII, NME2, and TKT that were recognized by up to 2.82% of antigen-specific peripheral cytotoxic T cells in IDHmut glioma patients.Conclusions: By analyzing the repertoire of T-cell target antigens in IDHmut glioma patients, we identified five novel immunogenic TAAs and confirmed their expression on IDHmut tumors and GSCs. Clin Cancer Res; 24(12); 2951–62. ©2018 AACR.

(A) Applied gating strategy (one representative patient, NCH645) for the expression analysis of tumor-associated antigens in IDHmut lower-grade glioma and secondary glioblastoma GSCs, analyzed by flow cytometry in regard to an adequate isotype control. (B) Fluorescence intensity measurements for the isotype control and the antigen expressing (positive) GSC population, showing counts in percentage [%] over the mean PE-intensity.(C) Gating strategy, including the markers pacific orange (PO), CD3, and CD8, used for quantification of antigen-specific peripheral cytotoxic CD3+CD8+ T-cells by flow cytometry and (D) antigen-loaded pMHC I-tetramers (PE-conjugated) and the respective positive (Influenza M1) as well as negative control HLA-A*02-tetramers. GSCs: glioma stem-like cells, HIV: human immunodeficiency virus, HLA: human leukocyte antigen, MHC: major histocompatibility complex, PE: Phycoerythrin, pos: positive, US: unstained.

Graphical representation of IFN-� ELISpots using peripheral blood mononuclear T cells, co-cultivated with dendritic cells, pulsed with autologous tumor lysate fractions of (A) the first (1st) PF2D dimension and corresponding (B) second (2nd) PF2D dimension of patients NCH1390, NCH612, and NCH519a. Solid line represents mean background of negative control (PBL). F: fraction, PBL: peripheral blood lymphocytes (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

Peptide information of potential T cell target antigens, tested by IFN-γ ELISpot

Example of HE and immunofluorescent stainings.

Clinical data of IDHmut LGG patients

Usage of RNA Seq TCGA data of IDHmut LGG tumors to compare mRNA expression levels with T-cell infiltration. Significant increased expression of NME2 in the TCGA IDHmut LGG dataset was shown to be positively correlated with the expression of CD3E (encoding CD3-ε as part of the CD3 T-cell receptor complex) (r = 0.2352). Stronger correlation in the histology of astrocytomas (r = 0.3693**) than in oligodendrogliomas (r = 0.1919**). (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

(A) Semi-quantitative evaluation on immunohistochemically stained sections of the frequency of tumor cells expressing the potential T-cell target antigens CRKII, CFL1, NME2, and CNTN1 in astrocytoma (WHO{degree sign}II: n = 10, WHO{degree sign}III: n = 10) and (B) oligodendroglioma (WHO{degree sign}II: n = 10, WHO{degree sign}III: n = 10) tumor tissues.

Representative multicolor stainings of CFL1, CRKII, NME2, and CNTN1 on acetone-fixed cryosections with markers for common cell types in IDHmut LGGs: anti-GFAP (tumor cells, #Z0334, DAKO), anti-CD68 (microglia/ macrophages, #M0718, DAKO), anti-CD31 (endothelial cells, #223609, BD Pharmingen). Detection was performed by using fluorochrom-conjugated secondary antibodies (anti-mouse AF647 (#A-21463, Invitrogen), anti-rabbit AF555 (#A-21428, Invitrogen), DAPI (#D1306, ThermoFisher), and the Zenon AF488 mouse IgG1 Labeling Kit (#Z25001, ThermoFisher)) according to the manufacturer`s protocol (scale bar: 50 µM, scale bar zoom: 10 µM).

(A) Significantly increased immune responses in IDHmut lower-grade glioma patients (P) vs. healthy donors (HD) seen for the potential T-cell target antigens CRKII, CFL1, CNTN1, NME2, and TKT. Homogenous levels of stimulation while comparing IFN-� spot numbers among astrocytomas (red) and oligodendrogliomas (blue) to a certain TAA as well as (B) the total immune response. (C) IFN-� ELISpot raw data showing the spot count / 1x105 T-cells upon stimulation with autologous dendritic cells loaded with antigens (CRKII, CFL1, CNTN1, NME2, or TKT, black bar) vs. negative control (IgG1, grey bar) for each patient (P) (n = 16). Highlighted in green: significant immune responses (p < 0.05) with an > 2-fold increased spot count compared to the negative control (IgG) (*, p < 0.05; **, p < 0.01; ***, p < 0.001)

Purpose: Successful immunotherapies for IDHmut gliomas require better knowledge of T-cell target antigens. Here, we elucidated their antigen repertoire recognized by spontaneous T-cell responses using an unbiased proteomic approach. Experimental Design: Protein fractionations of tissue lysates from IDHmut gliomas (n = 4) were performed. Fractions were tested by IFNγ ELISpot assay for recognition through patients' T cells. Proteins of immunogenic fractions were identified by mass spectrometry and validated by in silico-predicted synthetic long peptides in patients of origin, additional IDHmut glioma patients (n = 16), and healthy donors (n = 13). mRNA and protein expression of immunogenic antigens was analyzed in tumor tissues and IDHmut glioma stem-like cells (GSC). HLA-A*02–restricted T-cell epitopes were functionally determined by short peptides and numbers of antigen-specific T cells by HLA-peptide tetramer analysis. Results: A total of 2,897 proteins were identified in immunogenic tumor fractions. Based on a thorough filter process, 79 proteins were selected as potential T-cell antigens. Twenty-six of these were recognized by the patients’ T cells, and five of them (CRKII, CFL1, CNTN1, NME2, and TKT) in up to 56% unrelated IDHmut glioma patients. Most immunogenic tumor-associated antigens (TAA) were expressed in IDHmut gliomas and GSCs, while being almost absent in normal brain tissues. Finally, we identified HLA-A*02–restricted epitopes for CRKII, NME2, and TKT that were recognized by up to 2.82% of antigen-specific peripheral cytotoxic T cells in IDHmut glioma patients. Conclusions: By analyzing the repertoire of T-cell target antigens in IDHmut glioma patients, we identified five novel immunogenic TAAs and confirmed their expression on IDHmut tumors and GSCs. Clin Cancer Res; 24(12); 2951–62. ©2018 AACR.

Purpose: To provide a better understanding of the interplay between the immune system and brain metastases to advance therapeutic options for this life-threatening disease. Experimental Design: Tumor-infiltrating lymphocytes (TIL) were quantified by semiautomated whole-slide analysis in brain metastases from 81 lung adenocarcinomas. Multi-color staining enabled phenotyping of TILs (CD3, CD8, and FOXP3) on a single-cell resolution. Molecular determinants of the extent of TILs in brain metastases were analyzed by transcriptomics in a subset of 63 patients. Findings in lung adenocarcinoma brain metastases were related to published multi-omic primary lung adenocarcinoma The Cancer Genome Atlas data (n = 230) and single-cell RNA-sequencing (scRNA-seq) data (n = 52,698). Results: TIL numbers within tumor islands was an independent prognostic marker in patients with lung adenocarcinoma brain metastases. Comparative transcriptomics revealed that expression of three surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) was closely associated with TIL numbers. Their expression was not only prognostic in brain metastasis but also in primary lung adenocarcinoma. Correlation with scRNA-seq data revealed that brain metastases with high expression of surfactant genes might originate from tumor cells resembling alveolar type 2 cells. Methylome-based estimation of immune cell fractions in primary lung adenocarcinoma confirmed a positive association between lymphocyte infiltration and surfactant expression. Tumors with a high surfactant expression displayed a transcriptomic profile of an inflammatory microenvironment. Conclusions: The expression of surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) defines an inflamed subtype of lung adenocarcinoma brain metastases characterized by high abundance of TILs in close vicinity to tumor cells, a prolonged survival, and a tumor microenvironment which might be more accessible to immunotherapeutic approaches.