Identifizierung von Tumor-assoziierten Antigenen in IDH-mutierten Gliomen

Gliomas are tumors of the central nervous system which are classified by the World Health Organization (WHO) from grade I to grade IV, according to the degree of malignancy as defined by histopathological and molecular criteria. Mutations in the isocitrate dehydrogenase (IDHmut) genes are a common cha- racteristic for lower grade gliomas (LGGs, WHO◦II/◦III) and have been shown to be a prognostic marker for a favorable clinical outcome. The metabolic and biologic consequences by IDHmut-induced epigentic alterations have changed our perceptions of gliomas and demonstrated the demand to consider IDHmut LGGs separately. Despite a favourable prognosis, IDHmut LGGs remain deadly since there is still a lack of effective therapies. For that reason, immunotherapeutic approaches are gaining increasing attention. Due to the limited number of known T cell targets in IDHmut gliomas, we aimed to elucidate the reper- toire of spontaneous T cell responses in IDHmut LGGs by performing an unbiased proteomic approach. We systematically analyzed the proteome of IDHmut LGG samples (n = 4) by fractionating tumor tissue lysates and testing resulting fractions by IFN-γ enzyme linked immunospot (ELISpot) assay for recogni- tion by the patients T cell repertoire. Immunogenic tumor protein fractions were subsequently analyzed by quantitative mass spectrometry resulting in 2897 identified proteins. Based on a thorough filter pro- cess 79 proteins have been selected as potential target antigens and were validated in IFN-γ ELISpots by means of synthetic in silico predicted 50-mer peptides. 26 of these were recognized by autologous T cells and were tested in further IDHmut LGG patients as well as in healthy donors. Tumor-specific T cell responses in up to 50 % of IDHmut LGG patients were observed for CRKII, CFL1, CNTN1, NME2, and TKT. Beside the immunogenicity of the antigens, we further characterized their role in oncogenesis, the antigen-specificity as well as the expression levels in the tumor and on glioma stem-like cells (GSCs). By using immunohistochemistry and gene expression analysis we found that four out of five of the most immunogenic tumor-associated antigens (CRKII, CFL1, CNTN1 and NME2) were expressed in astro- cytic and oligodendroglial tumors as well as in IDHmut GSCs, while being almost absent in normal brain tissues. Finally, we identified HLA-A*02-restricted reactive epitopes for CRKII (ALALEVGEL), NME2 (MVWEGLNVV), and TKT (FLAEAELLNL) which are recognized by up to 1.5 % of antigen-specific pe- ripheral cytotoxic T cells in IDHmut LGG patients. By analyzing the repertoire of T cell target antigens in IDHmut LGG patients, we identified four novel immunogenic antigens and even confirmed their expression on IDHmut GSCs, highlighting their potential as T cell targets for the development of new immunotherapeutic approaches

When Immune Cells Turn Bad—Tumor-Associated Microglia/Macrophages in Glioma

As a substantial part of the brain tumor microenvironment (TME), glioma-associated microglia/macrophages (GAMs) have an emerging role in tumor progression and in controlling anti-tumor immune responses. We review challenges and improvements of cell models and highlight the contribution of this highly plastic cell population to an immunosuppressive TME, besides their well-known functional role regarding glioma cell invasion and angiogenesis. Finally, we summarize first therapeutic interventions to target GAMs and their effect on the immunobiology of gliomas, focusing on their interaction with T cells

Tumor specific regulatory T cells in the bone marrow of breast cancer patients selectively upregulate the emigration receptor S1P1

Regulatory T cells (Treg) hamper anti-tumor T-cell responses resulting in reduced survival and failure of cancer immunotherapy. Among lymphoid organs, the bone marrow (BM) is a major site of Treg residence and recirculation. However, the process governing the emigration of Treg from BM into the circulation remains elusive. We here show that breast cancer patients harbour reduced Treg frequencies in the BM as compared to healthy individuals or the blood. This was particularly the case for tumor antigen-specific Treg which were quantified by MHCII tumor peptide loaded tetramers. We further demonstrate that decreased Treg distribution in the BM correlated with increased Treg redistribution to tumor tissue, suggesting that TCR triggering induces a translocation of Treg from the BM into tumor tissue. Sphingosine-1-phosphate receptor 1 (S1P1)-which is known to mediate exit of immune cells from lymphoid organs was selectively expressed by tumor antigen-specific BM Treg. S1P1 expression could be induced in Treg by BM-resident antigen-presenting cells (BMAPCs) in conjunction with TCR stimulation, but not by TCR stimulation or BMAPCs alone and triggered the migration of Treg but not conventional T cells (Tcon) to its ligand Sphingosine-1-phosphate (S1P). Interestingly, we detected marked S1P gradients between PB and BM in breast cancer patients but not in healthy individuals. Taken together, our data suggest a role for S1P1 in mediating the selective mobilization of tumor specific Treg from the BM of breast cancer patients and their translocation into tumor tissue

Identification of CRKII, CFL1, CNTN1, NME2, and TKT as Novel and Frequent T-Cell Targets in Human IDH-Mutant Glioma

Purpose:Successful immunotherapies for IDH(mut )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 IDH(mut )gliomas (n = 4) were performed. Fractions were tested by IFN gamma ELISpot assay for recognition through patients' T cells. Proteins of immunogenic fractions were identified by mass spectrometry and validated byin silico-predicted synthetic long peptides in patients of origin, additional IDH(mut)glioma patients (n = 16), and healthy donors (n = 13). mRNA and protein expression of immunogenic antigens was analyzed in tumor tissues and IDH(mut)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 IDH(mut)glioma patients. Most immunogenic tumor-associated antigens (TAA) were expressed in IDH(mut)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 IDH(mut)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 IDH(mut )tumors and GSCs. (C)2018 AACR

Tumor-Specific Regulatory T Cells from the Bone Marrow Orchestrate Antitumor Immunity in Breast Cancer

Endogenous antitumor effector T-cell responses and immune-suppressive regulatory T cells (Treg) critically influence the prognosis of patients with cancer, yet many of the mechanisms of how this occurs remain unresolved. On the basis of an analysis of the function, antigen specificity, and distribution of tumor antigen-reactive T cells and Tregs in patients with breast cancer and transgenic mouse tumor models, we showed that tumor-specific Tregs were selectively activated in the bone marrow (BM) and egressed into the peripheral blood. The BM was constantly depleted of tumor-specific Tregs and was instead a site of increased induction and activity of tumor-reactive effector/memory T cells. Treg egress from the BM was associated with activation-induced expression of peripheral homing receptors such as CCR2. Because breast cancer tissues express the CCR2 ligand CCL2, the activation and egress of tumor antigen-specific Tregs in the BM resulted in the accumulation of Tregs in breast tumor tissue. Such immune compartmentalization and redistribution of T-cell sub-populations between the BM and peripheral tissues were achieved by vaccination with adenoviral vector-encoded TRP-2 tumor antigen in a RET transgenic mouse model of spontaneous malignant melanoma. Thus, the BM simultaneously represented a source of tumor-infiltrating Tregs and a site for the induction of endogenous tumor-specific effector T-cell responses, suggesting that both antitumor immunity and local immune suppression are orchestrated in the BM

Identification of KIF11 as a Novel Target in Meningioma

Kinesins play an important role in many physiological functions including intracellular vesicle transport and mitosis. The emerging role of kinesins in different cancers led us to investigate the expression and functional role of kinesins in meningioma. Therefore, we re-analyzed our previous microarray dataset of benign, atypical, and anaplastic meningiomas (n = 62) and got evidence for differential expression of five kinesins (KIFC1, KIF4A, KIF11, KIF14 and KIF20A). Further validation in an extended study sample (n = 208) revealed a significant upregulation of these genes in WHO°I to °III meningiomas (WHO°I n = 61, WHO°II n = 88, and WHO°III n = 59), which was most pronounced in clinically more aggressive tumors of the same WHO grade. Immunohistochemical staining confirmed a WHO grade-associated upregulated protein expression in meningioma tissues. Furthermore, high mRNA expression levels of KIFC1, KIF11, KIF14 and KIF20A were associated with shorter progression-free survival. On a functional level, knockdown of kinesins in Ben-Men-1 cells and in the newly established anaplastic meningioma cell line NCH93 resulted in a significantly inhibited tumor cell proliferation upon siRNA-mediated downregulation of KIF11 in both cell lines by up to 95% and 71%, respectively. Taken together, in this study we were able to identify the prognostic and functional role of several kinesin family members of which KIF11 exhibits the most promising properties as a novel prognostic marker and therapeutic target, which may offer new treatment options for aggressive meningiomas

Identification of KIF11 as a novel target in meningioma

Kinesins play an important role in many physiological functions including intracellular vesicle transport and mitosis. The emerging role of kinesins in different cancers led us to investigate the expression and functional role of kinesins in meningioma. Therefore, we re-analyzed our previous microarray dataset of benign, atypical, and anaplastic meningiomas (n = 62) and got evidence for differential expression of five kinesins (KIFC1, KIF4A, KIF11, KIF14 and KIF20A). Further validation in an extended study sample (n = 208) revealed a significant upregulation of these genes in WHO°I to °III meningiomas (WHO°I n = 61, WHO°II n = 88, and WHO°III n = 59), which was most pronounced in clinically more aggressive tumors of the same WHO grade. Immunohistochemical staining confirmed a WHO grade-associated upregulated protein expression in meningioma tissues. Furthermore, high mRNA expression levels of KIFC1, KIF11, KIF14 and KIF20A were associated with shorter progression-free survival. On a functional level, knockdown of kinesins in Ben-Men-1 cells and in the newly established anaplastic meningioma cell line NCH93 resulted in a significantly inhibited tumor cell proliferation upon siRNA-mediated downregulation of KIF11 in both cell lines by up to 95% and 71%, respectively. Taken together, in this study we were able to identify the prognostic and functional role of several kinesin family members of which KIF11 exhibits the most promising properties as a novel prognostic marker and therapeutic target, which may offer new treatment options for aggressive meningiomas