Cranioencephalic functional lymphoid units in glioblastoma

Abstract

The present invention relates to the field of diagnosis and therapy of brain diseases such as brain tumors. More specifically, the present invention relates to a cranial bone-derived immune cell population comprising a) effector memory CD8+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow, b) naïve CD8+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA+, and CCR7+, c) effector memory CD4+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow, d) naïve CD4+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA+, and CCR7+, and e) regulatory T cells CD45+, CD3+, TCRgd-, CD56-, CD4+, CD25+, and CD127low / -, wherein compared to blood and non-cranial bone marrow the effector memory CD8+ T cells according to a) are increased whereas the naïve CD8+ T cells according to b) are reduced, wherein compared to blood and non-cranial bone marrow the effector memory CD4+ T cells according to c) are increased whereas the naïve CD4+ T cells according to d) are reduced, and wherein the regulatory T cells according to e) are reduced. The present invention also relates to effector memory CD8+ and CD4+ T cells as specified above as well to therapeutic and diagnostic applications of such immune cell population and effector memory T cells.

Description

[0001] Cranioencephalic functional lymphoid units in glioblastoma

[0002] The present invention relates to the field of diagnosis and therapy of brain diseases such as brain tumors. More specifically, the present invention relates to a cranial bone-derived immune cell population comprising a) effector memory CD8+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow, b) naive CD8+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA+, and CCR7+, c) effector memory CD4+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow, d) naive CD4+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA+, and CCR7+, and e) regulatory T cells CD45+, CD3+, TCRgd-, CD56-, CD4+, CD25+, and CD1271ow / -, wherein compared to blood and non-cranial bone marrow the effector memory CD8+ T cells according to a) are increased whereas the naive CD8+ T cells according to b) are reduced, wherein compared to blood and non-cranial bone marrow the effector memory CD4+ T cells according to c) are increased whereas the naive CD4+ T cells according to d) are reduced, and wherein the regulatory T cells according to e) are reduced. The present invention also relates to effector memory CD8+ and CD4+ T cells as specified above as well to therapeutic and diagnostic applications of such immune cell population and effector memory T cells.

[0003] While recent research connects brain function and immunosurveillance to guardian immune cells assembling on the outer borders of the brain, the overall view on how brain immunity works in health and disease is currently amended. Structures like the choroid plexus, the meninges, and the dural sinuses act as a neuro-immune interface hosting innate and adaptive immune cells (J. Rustenhoven et al. 2021; K. Mollgard et al. 2023; G. Castellani et al. 2023). Brain drainage is provided by the central nervous lymphatic system (A. Louveau et al. 2015) and vascular channels connect the overlying skull bone marrow with the underlying brain surface (F. Herisson et al. 2018). Consequently, cerebrospinal fluid can instruct cranial hematopoiesis in response to brain injury and inflammation, and therefore provides tailored populations of locally derived immune cells to the scene (JA Mazzitelli et al. 2022).

[0004] As the overall concept continues to evolve, there is much more to learn about the specific settings during malignant disease, for example, in the most malignant brain cancer, glioblastoma, that remains uniformly lethal with a median survival of less than two years. Despite the efforts dedicated to studying the microenvironment, genomic factors, and epigenetic impacts of primarily ineffective immunotherapies in the brain tumor tissue (JH Sampson et al. 2020), it remains elusive how what the adjacent structures of the neuro-immune interface are and how they function.

[0005] In particular, there remains a need for identifying the prevalence, characteristics and disease- related function of the immune cell repertoire in newly-diagnosed glioblastoma patients or other patients suffering from brain diseases.

[0006] The technical problem underlying the present invention may be seen as the provision of means and methods for complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the embodiments and described herein below.

[0007] The present invention relates to a cranial bone-derived immune cell population and subpopulations as well as cell types comprised therein which are involved in an immune response of a subject suffering from a brain disease referred to herein against said disease.

[0008] Thus, the present invention relates to a cranial bone-derived immune cell population comprising: a) effector memory CD8+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow; b) naive CD8+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA+, and CCR7+; c) effector memory CD4+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow; d) naive CD4+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA+, and CCR7+; and e) regulatory T cells CD45+, CD3+, TCRgd-, CD56-, CD4+, CD25+, and CD1271ow / -; wherein compared to blood and non-cranial bone marrow the effector memory CD8+ T cells according to a) are increased whereas the naive CD8+ T cells according to b) are reduced; wherein compared to blood and non-cranial bone marrow the effector memory CD4+ T cells according to c) are increased whereas the naive CD4+ T cells according to d) are reduced; and wherein the regulatory T cells according to e) are reduced.

[0009] It is to be understood that in the specification and in the embodiments, “a” or “an” can mean one or more of the items referred to in the following depending upon the context in which it is used. Thus, for example, reference to “an” item can mean that at least one item can be utilized.

[0010] As used in the following, the terms “have”, “comprise”, or “include” and its grammatical variations are meant to have a non-limiting meaning or a limiting meaning. Thus, having a limiting meaning these terms may refer to a situation in which, besides the feature introduced by these terms, no other features are present in an embodiment described, i.e., the terms have a limiting meaning in the sense of “consisting of’ or “essentially consisting of’. Having a nonlimiting meaning, the terms refer to a situation where besides the feature introduced by these terms, one or more other features are present in an embodiment described.

[0011] Further, as used in the following, the terms “preferably”, “more preferably”, “most preferably”, “particularly”, “more particularly”, “typically”, and “more typically” are used in conjunction with features in order to indicate that these features are preferred features, i.e., the terms shall indicate that alternative features may also be envisaged in accordance with the invention.

[0012] Further, it will be understood that the term “at least one” as used herein means that one or more of the items referred to following the term may be used in accordance with the invention. For example, if the term indicates that at least one item shall be used this may be understood as one item or more than one item, i.e., two, three, four, five or any other number. Depending on the item the term refers to the skilled person understands as to what upper limit the term may refer, if any.

[0013] The term "about" in the context of the present invention means ± 50%, ± 20%, ± 10%, ± 5%, ± 2 % or ± 1% from the indicated parameters or values. This also takes into account usual deviations caused by measurement techniques and the like.

[0014] The term “cranial bone” as used herein refers to all flat bones of the skull. Typically, these bones comprise a spongy, trabecular structure where marrow resides called diploe between their inner and outer layers of dense bone. The diploe contains many vessels including blood and lymphatic vessels. Cranial bone as referred to herein is, preferably, cranial bone being located in close proximity to a part of the brain suffering from a brain disease as referred to herein elsewhere. Preferably, said brain disease-proximal cranial bone is located adjacent to the circumference of the intracerebral brain disease area, preferably, within a distance of at most about 3 cm, at most about 2 cm, at most about 1 cm or at most about 0.5 cm. Preferably, said area in proximity to brain tissue affected by a brain disease can be identified by imaging, preferably, by PET-, CT-, MR-imaging or combinations thereof.

[0015] A “brain disease” in accordance with the present invention may be any disease affecting brain cells and tissue. Typically, the brain disease of the invention is a disease that involves inflammation or other reactions of the immune system. Preferably, the said disease is a brain tumor or a neuroinflammatory disease. More preferably, said brain tumor is (i) a brain tumor selected from the group consisting of adult-type diffuse gliomas, preferably astrocytoma, oligodendroglioma or glioblastoma; pediatric-type low-grade gliomas, preferably diffuse astrocytoma, angiocentric glioma, polymorphous low-grade neuroepithelial tumor of the young or diffuse low-grade glioma; pediatric-type diffuse high-grade gliomas, preferably diffuse midline glioma, diffuse hemispheric glioma, diffuse pediatric-type high-grade glioma or infanttype hemispheric glioma; circumscribed astrocytic gliomas, preferably pilocytic atrocytoma, high-grade astrocytoma with piloid features, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, chordoid glioma or astroblastoma; glioneuronal and neuronal tumors, preferably ganglioglioma, gangliocytoma, desmoplastic infantile ganglioglioma, desmoplastic infantile astrocytoma, dysembryoplastic neuroepithelial tumour, diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters, papillary glioneuronal tumour, rosette-forming glioneuronal tumor, myxoid glioneuronal tumour, diffuse leptomeningeal glioneuronal tumour, multinodular and vacuolating neuronal tumour, dysplastic cerebellar gangliocytoma, central neurocytoma, extraventricular neurocytoma or cerebellar liponeurocytoma; ependymal tumors, preferably ependymoma, myxopapillary ependymoma or subependymoma; choroid plexus tumors, preferably choroid plexus papilloma, atypical choroid plexus papilloma or choroid plexus carcinoma; embryonal tumors, preferably medulloblastoma, atypical teratoid / rhabdoid tumor, cribriform neuroepithelial tumor, embryonal tumor with multilayered rosettes, CNS neuroblastoma, CNS tumor with BCOR-internal tandem duplication or CNS embryonal tumor; pineal tumors, preferably pineocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, papillary tumor of the pineal region or desmoplastic myxoid tumor of the pineal region; meningioma, preferably meningioma grades 1 to 3; mesenchymal, non-meningothelial tumors involving the CNS, preferably solitary fibrous tumor, cavernous hemangioma, capillary hemangioma, arteriovenous malformation, hemangioblastoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, spindle cell rhabdomyosarcoma, intracranial mesenchymal tumor, CIC-rearranged sarcoma, primary intracranial sarcoma, Ewing sarcoma, Mesenchymal chondrosarcoma, chondrosarcoma, dedifferentiate chondrosarcoma, or chordoma; melanocytic tumors, preferably meningeal melanocytosis, meningeal melanomatosis, meningeal melanocytioma or meningeal melanoma; hematolymphoid tumors involving the CNS, preferably primary diffuse large B-cell lymphoma of the CNS, lymphomatoid granulomatosis, intravascular large B-cell lymphoma, MALT lymphoma of the dura, lymphoplasmacytic lymphoma, follicular lymphoma, anaplastic large cell lymphoma, T- cell lymphoma, NK / T-cell lymphoma, Erdheim-Chester disease, Rosai-Dorfman disease, Juvenile xanthogranuloma, Langerhans cell histiocytosis or histiocytic sarcoma; germ cell tumors, preferably mature teratoma, immature teratoma, teratoma with somatic-type malignancy, germinoma, embryonal carcinoma, yolk sac tumors, choriocarcinoma or mixed germ cell tumor; tumors of the sellar region, preferably adamantinomatous craniopharyngioma, papillary craniopharyngioma, pituicytoma, granular cell tumor of the sellar region, spindle cell oncocytoma, pituitary adenoma, pituitary neuroendocrine tumor and pituitary blastoma, or (ii) is a metastasized secondary brain tumor selected from the group consisting of lung cancer, breast cancer, melanoma, colorectal cancer, kidney cancer, thyroid cancer and uterine cancer.

[0016] More preferably, the neuroinflammatory disease is selected from the group consisting of: multiple sclerosis, vasculitis, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, stroke, migraine, epilepsy and traumatic brain injuries.

[0017] The aforementioned diseases and symptoms thereof are well known to the person skilled in the art and are described in standard textbooks of medicine. A neuroinflammatory disease according to the invention may also be a neurodegenerative disease such as Alzheimer's disease or Parkinson's disease and the like.

[0018] The term “immune cell population” as referred to herein relates to population of different cell types being cell types of the immune system which are derived, i.e., which may be, preferably, isolated from, a cranial bone sample. Such a cell population shall be typically cultivated ex vivo or shall be used as a starting material for obtaining other cells or cell populations (e.g., subpopulations), preferably, those specified elsewhere herein. Therefore, a “cranial bone- derived immune cell population” according to the present invention is, preferably, an isolated immune cell population. As specified elsewhere herein, said immune cell population has, preferably, been derived from a cranial bone sample. Preferably, said cranial bone sample is from an area in proximity to brain tissue affected by a brain disease. Yet, the immune cell population may also be merely derivable from such a cranial bone sample, i.e., may not have been isolated, but be still present in said cranial bone. It will be understood that the cells of the immune cell population or any subpopulation or cell types comprised therein referred to in accordance with the present invention are, preferably, viable cells.

[0019] The different cell types of said immune cell population comprise, among others, effector memory CD8+ T cells expressing biomarkers as specified above, naive CD8+ T cells expressing biomarkers as specified above, effector memory CD4+ T cells expressing biomarkers as specified above, naive CD4+ T cells expressing biomarkers as specified above, and regulatory T cells expressing biomarkers as referred to above. For the immune cell population of the invention, it is characteristic that the effector memory CD4+ and CD8+ T cells are increased while the naive CD4+ and CD8+ T cells and the regulatory T cells are reduced compared to a reference immune cell population comprising theses cell types from blood or non-cranial, distal bone marrow such as iliac crest. The immune cell population of the present invention may also contain further immune cells such as other T cells, e.g., stem celllike T cells, central memory T cells or tissue resident T cells, B cells, NK cells, myeloid cells, and the like. The immune cell population may also comprise other non-immune cells associated with immune cells or immune cell actions such as lymphatic and / or blood vessel endothelial cells.

[0020] As discussed before, the immune cell population of the present invention may be isolated from a subject suffering from a brain disease as described elsewhere herein. The term “subject” refers to animals, preferably mammals, and, more preferably, humans.

[0021] The term “effector memory CD8+ T cells” as used herein refers to CD8+ T cells, which are key providers of cytotoxic actions against pathogens or diseased cells. In the immune cell population of the present invention, theses T cells, preferably, exhibit cytotoxic activity against the brain tissue cells affected by the brain disease. Preferably, the effector memory CD8+ T cells are derived from the naive CD8+ T cells comprised in the immune cell population of the invention. The effector memory CD8+ T cells referred to herein are characterized by exhibiting the surface protein biomarkers CD45, CD3, and CD8 and non-exhibiting the surface protein biomarkers TCRgd, CD56, CD45RA, and CCR7. The effector memory CD8+ T cells of the invention are further characterized by increased prevalence compared to blood and non-cranial bone marrow effector memory CD8+ T cells, and, preferably, by an increased expression of a further biomarker S1PR1. This biomarker is specific for the effector memory CD8+ cells present in the immune cell population derived from cranial bone samples from areas proximal to a brain disease as referred to herein.

[0022] The term “naive CD8+ T cells” as used herein refers to non-effector memory CD8+ T cells present in the immune cell population. These cells are characterized by exhibiting the surface biomarkers CD45, CD3, CD8, CD45RA, and CCR7 while not exhibiting the surface biomarkers TCRgd- and CD56. These naive T cells are, preferably, capable of converting into the aforementioned effector memory CD8+ T cells.

[0023] The term “effector memory CD4+ T cells” as used herein refers to CD4+ T cells, which are responsible for supporting as T helper cells cytotoxic actions against pathogens or diseased cells. In the immune cell population of the present invention, theses T cells, preferably, exhibit their supportive activity against the brain tissue cells affected by the brain disease. Preferably, the effector memory CD4+ T cells are derived from the naive CD4+ T cells comprised in the immune cell population of the invention. The effector memory CD4+ T cells referred to herein are characterized by exhibiting the surface protein biomarkers CD45, CD3, and CD4 and nonexhibiting the surface protein biomarkers TCRgd, CD56, CD45RA, and CCR7. The effector memory CD4+ T cells of the invention are further characterized by increased prevalence compared to blood and non-cranial bone marrow effector memory CD4+ T cells, and, preferably, by an increased expression of a further biomarker S 1PR1. This biomarker is specific for the effector memory CD4+ cells present in the immune cell population derived from cranial bone samples from areas proximal to a brain disease as referred to herein

[0024] The term “naive CD4+ T cells” as used herein refers to non-effector memory CD4+ T cells present in the immune cell population. These cells are characterized by exhibiting the surface biomarkers CD45, CD3, CD4, CD45RA, and CCR7 while not exhibiting the surface biomarkers TCRgd- and CD56. These naive T cells are, preferably, capable of converting into the aforementioned effector memory CD4+ T cells.

[0025] The term “regulatory T cells” as used herein refers to CD4+ T cells, which are characterized are characterized by exhibiting the surface biomarkers CD45, CD3, CD4, and CD25, while not exhibiting the surface biomarkers TCRgd, CD56, and CD127. CD127 may sometimes be present at low levels on these cells. The term “biomarker” as used herein refers to a molecule present in a cell, which indicates certain properties of the cell, i.e., a biomarker may indicate that, e.g., an immune cell of the immune cell population of the invention is an effector memory CD8+ T cell or a naive CD8+ T cell and the like. It will be understood that and as described above for characterization of the different cell types referred to herein one or more biomarkers may be required. The presence or absence or the expression of the biomarkers may be determined by detecting either the protein on the cells surface or any intermediate product that occurs during transcription, translation or processing of the said protein. Thus, the biomarker according to the invention may be, preferably, the surface protein as present or absent on the cells or any pre- or pre-pro protein, any cleavage fragment occurring during maturation of the protein by processing, or any RNA transcript encoding the protein, such as hnRNA, mRNA and the like. Depending on the molecular nature of the biomarker to be detected, different detection techniques and agents may be envisaged. For example, a surface protein to be used as biomarker may be determined by an antibody while an mRNA to be used as a biomarker may be determined by a nucleic acid amplification technique such as RT-PCR or be next generation sequencing techniques. Further details on how the biomarkers referred to in accordance with the present invention may be determined are described elsewhere herein.

[0026] The term “CD45” as used herein, refers to the cluster of differentiation 45 encoded in humans by the PTPRC gene. CD45 is also known as protein tyrosine phosphatase, receptor type, C (PTPRC). It is a member of the protein tyrosine phosphatase family and is involved in a variety of cellular processes including cell growth, differentiation, mitotic cycle and oncogenic transformation. CD45 is also an essential regulator of T cell and B cell antigen receptor signaling. Eight isoforms produced by alternative splicing are known and several orthologues of CD45 have been reported in various animal species.

[0027] The CD45 protein referred to in accordance with the present invention is, preferably, human CD45 having an amino acid sequence as deposited under UniProt accession number P08575. It will be understood that the term “CD45” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD45 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human CD45 protein, preferably over the entire length of the said CD45 proteins, respectively. The degree of identity between two amino acid sequences in accordance with the present invention can be determined, in general, by algorithms well known in the art. Preferably, the degree of identity is to be determined by comparing two optimally aligned sequences over a comparison window, where the fragment of amino acid sequence in the comparison window may comprise additions or deletions (e.g., gaps or overhangs) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment. The percentage is calculated by determining the number of positions at which the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm disclosed by Smith, by the homology alignment algorithm of Needleman, by the search for similarity method of Pearson, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FAST, PASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI) or by visual inspection. Given that two sequences have been identified for comparison, GAP and BESTFIT are preferably employed to determine their optimal alignment and, thus, the degree of identity. Preferably, the default values of 5.00 for gap weight and 0.30 for gap weight length are used. Variants referred to above may be allelic variants or any other species-specific homologs, paralogs, or orthologues. Variants referred to above may be allelic variants or any other species-specific homologs, paralogs, or orthologues.

[0028] The term “CD3” as used herein, refers to the cluster of differentiation 3 encoded in humans by the CD3G gene. CD3 is also known as T cell surface glycoprotein CD3 delta chain which is part of the TCR-CD3 complex present on the T cell surface. It plays an essential role in adaptive immune response. Two isoforms produced by alternative splicing are known and several orthologues of CD3 have been reported in various animal species.

[0029] The CD3 protein referred to in accordance with the present invention is, preferably, human CD3 having an amino acid sequence as deposited under UniProt accession number P04234. It will be understood that the term “CD3” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD3 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human CD3 protein, preferably over the entire length of the said CD3 proteins, respectively.

[0030] The term “TCRgd” as used herein refers to gamma delta T-cell receptor. The TCR is a disulfi delinked membrane-anchored heterodimeric protein normally consisting of the highly variable alpha and beta chains expressed as part of a complex with the invariant CD3 chain molecules. The TCRgd is an alternative form of the TCR formed by variable gamma and delta chains.

[0031] The TCRgd protein referred to in accordance with the present invention is, preferably, human TCRgd having an amino acid sequence as deposited under NCBI accession number NG_001336.2 (gamma chain) and NG_001332.3 (delta chain). It will be understood that the term “TCRgd” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned TCRgd protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human TCRgd protein, preferably over the entire length of the said TCRgd proteins, respectively.

[0032] The term “CD56” as used herein refers to Neural cell adhesion molecule (NCAM). It is a homophilic binding glycoprotein expressed, inter alia, cells of the hematopoietic system. Here, the expression of CD56 is mostly associated with natural killer cells but also gamma delta (y5) T cells and activated CD8+ T cells.

[0033] The CD56 protein referred to in accordance with the present invention is, preferably, human CD56 having an amino acid sequence as deposited under UniProt accession number P13591. It will be understood that the term “CD56” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD56 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human CD56 protein, preferably over the entire length of the said CD56 proteins, respectively.

[0034] The term “CD8” as used herein, refers to the T cell surface glycoprotein CD8 beta chain encoded in humans by the CD8 gene. CD8 is an integral membrane glycoprotein that plays an essential role in the immune response and serves multiple functions in responses against both external and internal offenses. In T-cells, functions primarily as a co-receptor for MHC class I molecule :peptide complex. It interacts simultaneously with the T-cell receptor (TCR) and the MHC class I proteins presented by antigen presenting cells (APCs). Eight isoforms produced by alternative splicing are known and several orthologues of CD8 have been reported in various animal species.

[0035] The CD8 protein referred to in accordance with the present invention is, preferably, human CD8 having an amino acid sequence as deposited under UniProt accession number P10966. It will be understood that the term “CD8” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD8 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human CD8 protein, preferably over the entire length of the said CD8 proteins, respectively.

[0036] The term “CD4” as used herein, refers to the T cell surface glycoprotein 4 encoded in humans by the CD4 gene. CD4 is an integral membrane glycoprotein that plays an essential role in the immune response and serves multiple functions in responses against both external and internal offenses. In T-cells, functions primarily as a co-receptor for MHC class II molecule:peptide complex. It interacts simultaneously with the T-cell receptor (TCR) and the MHC class II proteins presented by antigen presenting cells (APCs). CD4 also participates in the development of T-helper cells in the thymus and triggers the differentiation of monocytes into functional mature macrophages. Several orthologues of CD4 have been reported in various animal species.

[0037] The CD4 protein referred to in accordance with the present invention is, preferably, human CD4 having an amino acid sequence as deposited under UniProt accession number P01730. It will be understood that the term “CD4” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD4 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human CD4 protein, preferably over the entire length of the said CD4 proteins, respectively.

[0038] The term “CD45RA” as used herein refers to Protein tyrosine phosphatase, receptor type, C also known as PTPRC. It is a type I transmembrane protein that is present in various iso forms on all differentiated hematopoietic cells (except erythrocytes and plasma cells). CD45RA has been shown to be an essential regulator of T- and B-cell antigen receptor signaling. It functions either directly via interaction with components of the antigen receptor complexes or by activating various src family kinases required for the antigen receptor signaling.

[0039] The CD45RA protein referred to in accordance with the present invention is, preferably, human CD45RA having an amino acid sequence as deposited under UniProt accession number PP08575. It will be understood that the term “CD45RA” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD45RA protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human CD45RA protein, preferably over the entire length of the said CD45RA proteins, respectively.

[0040] The term “CCR7” as used herein refers to C-C chemokine receptor 7. CCR7 is a transmembrane protein with 7 transmembrane domains, which is coupled with heterotrimeric G proteins, which transduce the signal downstream through various signaling cascades. The main function of the receptor is to guide immune cells to immune organs (lymph nodes, thymus, spleen) by detecting specific chemokines, which these tissues secrete.

[0041] The CCR7 protein referred to in accordance with the present invention is, preferably, human CCR7 having an amino acid sequence as deposited under UniProt accession number P32248. It will be understood that the term “CCR7” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CCR7 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human CCR7 protein, preferably over the entire length of the said CCR7 proteins, respectively.

[0042] The term “CD25” as used herein refers to the interleukin-2 receptor alpha chain, which is a protein involved in the assembly of the high-affinity Interleukin-2 receptor. It consists of alpha (IL2RA), beta (IL2RB) and the common gamma chain (IL2RG). The receptor interacts with interleukin-2, a cytokine involved, inter alia, in immune homeostasis.

[0043] The CD25 protein referred to in accordance with the present invention is, preferably, human CD25 having an amino acid sequence as deposited under UniProt accession number P01589. It will be understood that the term “CD25” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD25 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human CD25 protein, preferably over the entire length of the said CD25 proteins, respectively.

[0044] The term “CD 127” as used herein refers to the interleukin-7 receptor subunit alpha (IL7R alpha) also known as Cluster of Differentiation 127. IL7R alpha is a type I cytokine receptor and is a subunit of the functional interleukin-7 receptor and thymic stromal lymphopoietin (TSLP) receptors.

[0045] The CD 127 protein referred to in accordance with the present invention is, preferably, human CD127 having an amino acid sequence as deposited under UniProt accession number P16871. It will be understood that the term “CD127” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned CD 127 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human CD 127 protein, preferably over the entire length of the said CD127 proteins, respectively.

[0046] The term “S1PR1” as used herein refers to sphingosine 1-phosphate receptor 1 and belongs into a class of G protein-coupled receptors that are targets of the lipid signaling molecule Sphingosine- 1-phosphate. The sphingosine- 1-phosphate receptors are involved in regulation of biological processes such as cell proliferation, angiogenesis, migration, cytoskeleton organization, endothelial cell chemotaxis, immune cell trafficking and mitogenesis. Sphingosine- 1-phosphate receptors are also involved in immune-modulation and directly involved in suppression of innate immune responses from T cells. Moreover, S1PR1 has been suggested to be required for immune cells that want to leave lymphatic organs.

[0047] The S1PR1 protein referred to in accordance with the present invention is, preferably, human S1PR1 having an amino acid sequence as deposited under UniProt accession number P21453. It will be understood that the term “S1PR1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned S1PR1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human S1PR1 protein, preferably over the entire length of the said S1PR1 proteins, respectively.

[0048] The term “expressing” or “expression” as referred to herein means that the biomarker referred to is transcribed, translated into protein including pre-pro-protein variants, and matures into the surface biomarker protein exhibited on a cell of interest referred to herein. A “+” following a biomarker indicates that the biomarker is expressed by the cell, whereas a indicates that the biomarker is not expressed by the cell. As discussed elsewhere herein, expression can be determined at various levels of the process including the RNA level and the protein level. Preferably, it is envisaged to determine the biomarkers, however, as protein biomarkers exhibited on the cell surface of the cells of interest. Determining a biomarker as referred to herein relates to determine its presence, absence or abundance. Thus, determining comprises qualitative and quantitative determination of a biomarker, i.e., the term encompasses the determination of the presence or absence or the determination of the absolute or relative amount of said biomarker. The term further encompasses measuring the amount or concentration, preferably, semi-quantitatively or quantitatively.

[0049] The biomarker may be determined based on any of its specific physical or chemical properties that can be directly or indirectly measured. Direct measuring relates to measuring the amount or concentration of the biomarker based on a signal which is obtained from the biomarker molecule itself and the intensity of which directly correlates with the number of molecules of the biomarker present in the cell. Such a signal - sometimes referred to herein as intensity signal - may be obtained, e.g., by measuring an intensity value of a specific physical or chemical property of the biomarker molecule. Indirect measuring includes measuring of a signal obtained from a secondary component, i.e., a component not being the biomarker molecule itself. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained and / or modified by all standard mathematical operations.

[0050] In accordance with the present invention, determining the presence or absence or the amount of a biomarker can be achieved by all known means for determining such presence or absence or amount in a cell. Said means comprise immunological techniques. In immunological assay, e.g., a signal will be developed, which is indicative for the presence or absence of the protein. Moreover, the signal strength can, preferably, be correlated directly or indirectly (e.g., reverseproportional) to the amount of the biomarker present in a cell. Biomarkers on single cells may be determined, e.g., by using cell sorting techniques which count the cells exhibiting a desired biomarker or biomarker combination, such as fluorescence activated cell sorting (FACS), microfluidic cell sorting or magnetic cell sorting (MACS) techniques may be used. The immune cell population of the present invention may be, preferably, derived from the cranial bone sample by isolating CD45+ cells from the sample using a cell separation technique. More preferably, said cell separation technique is magnetic cell separation, preferably magnetic activated cell sorting (MACS). Specific cell types such as those referred to herein, i.e., effector memory CD8+ T cells, effector memory CD4+ T cells, naive CD8+ cells, naive CD4+ T cells or other regulatory T cells, can be isolated from the immune cell population by further cell sorting steps using biomarkers or biomarker combinations for said specific cell types. The specific cell types can also be isolated or enriched by using cultivation and / or selection techniques when cultivating the isolated immune cell population of the invention. Typically, the cultivation and / or enrichment of specific cells from the immune cell population of the invention may be performed, e.g., in activation media containing different supplements that vary depending on the type of cells to be expanded. Suitable expansion media are generally known and commercially available.

[0051] Preferably, for determining a biomarker as referred to herein, a binding molecule shall be used that specifically binds to the biomarker of the invention when present on or in the cell of interest. The binding molecule, typically, can be detected either by a detectable label present in the binding molecule or by a secondary binding molecule that specifically binds to the first binding molecule and comprises a detectable label. The binding molecule can be exposed to the biomarker in solution or while the binding molecule is immobilized on a solid support. A binding molecule refers in this context to any molecule that is capable of specifically binding to the biomarker to be detected. The binding molecule is selected based on the type of analysis to be conducted. Binding molecules include but are not limited to aptamers, antibodies, adnectins, ankyrins, antibody mimetics and other protein scaffolds, small molecules, nucleic acids, lectins, affybodies, nanobodies, avimers, and peptidomimetics.

[0052] An “antibody” in accordance with the present invention may encompass all types of antibodies, which specifically bind to the biomarker protein. Preferably, the antibody of the present invention is a monoclonal antibody, a polyclonal antibody, a single chain antibody, a chimeric antibody or any fragment or derivative of such antibodies being still capable of binding to the biomarker protein specifically.

[0053] The term “antigen binding fragment” refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term “antigen binding fragment” include a fragment antigen binding (Fab) fragment, a Fab’ fragment, a F(ab’)2 fragment, a heavy chain antibody, a single-domain antibody (sdAb), a single-chain fragment variable (scFv), a fragment variable (Fv), a VH domain, a VL domain, a single domain antibody, a nanobody, an IgNAR (immunoglobulin new antigen receptor), a di-scFv, a bispecific T-cell engager (BITEs), a dual affinity re-targeting (DART) molecule, a triple body, a diabody, a single-chain diabody, an alternative scaffold protein, and a fusion protein thereof.

[0054] An “adnectin” as used herein, refers to a synthetic binding protein, also known as monobody, based on the 10thfibronectin type III (10Fn3) domain. It is a member of the immunoglobulin superfamily and contains a “beta sandwich” protein fold that bears striking resemblance to an antibody domain. As such, adnectins represent a simple and robust alternative to antibodies for creating target-binding proteins. A major advantage of adnectins over conventional antibodies is that adnectins can readily be used as genetically encoded intracellular inhibitors, that is one can express an adnectin inhibitor in a cell of choice by simply transfecting the cell with an adnectin expression vector.

[0055] An “ankyrin” as used herein, refers to a family of proteins that comprise binding sites for a wide range of membrane proteins. Ankyrins contain four functional domains: (i) an N-terminal domain with 24 tandem ankyrin repeats that are responsible for the recognition of multiple membrane proteins, (ii) a central domain that binds to spectrin, (iii) a death domain that binds to proteins involved in apoptosis, and (iv) a C-terminal regulatory domain that is highly variable between different anykrin proteins. Ankyrins are encoded in humans by three genes, which in turn produce multiple proteins through alternative splicing.

[0056] “Antibody mimetics” as used herein, refer to compounds, which can specifically bind antigens, similar to an antibody, but are not structurally related to antibodies. Usually, antibody mimetics are artificial peptides or proteins with a molar mass of about 3 to 20 kDa, which comprise one, two or more exposed domains specifically binding to an antigen. Examples include inter alia the LACI-Dl (lipoprotein-associated coagulation inhibitor); affilins, e.g. human-y B crystalline or human ubiquitin; cystatin; Sac7D from Sulfolobus acidocaldctriiis lipocalin and anticalins derived from lipocalins; DARPins (designed ankyrin repeat domains); SH3 domain of Fyn; Kunits domain of protease inhibitors; monobodies, e.g. the 10thtype III domain of fibronectin; adnectins: knottins (cysteine knot miniproteins); atrimers; evibodies, e.g., CTLA4-based binders, affibodies, e.g. three-helix bundle from Z-domain of protein A from Staphylococcus aureus,' Trans-bodies, e.g. human transferrin; tetranectins, e.g. monomeric or trimeric human C-type lectin domain; microbodies, e.g. trypsin-inhibitor-II; affilins; armadillo repeat proteins. Nucleic acids and small molecules are sometimes considered antibody mimetics as well (aptamers), but not artificial antibodies, antibody fragments and fusion proteins composed from these. Common advantages over antibodies are better solubility, tissue penetration, stability towards heat and enzymes, and comparatively low production costs.

[0057] A ’’scaffold protein” as used herein refers to a specific protein whose main function is to mediate protein complexes. Scaffold proteins usually have multiple protein domains that mediate binding to other proteins. Examples of scaffold proteins include but are not limited to the protein inaD from rhabdomeres of Drosophila melanogaster or titin, a protein found in muscles.

[0058] The term “lectin” as used herein, refers to carbohydrate-binding proteins that are highly specific for sugar groups. They occur ubiquitously in nature and may bind to soluble carbohydrates or carbohydrate moieties that are part of a glycoprotein or glycolipid. Lectins typically agglutinate certain cells and / or precipitate glycoconjugates. As such, they find use in medicine, particularly for blood typing. Lectins are also used in neuroscience for anterograde labelling to trace the path of efferent axons.

[0059] An “affibody” as used herein are small, highly robust proteins with high affinity to target proteins. In contrast to antibodies, affibodies are composed of alpha helices and lack disulphide bridges. In particular, they are based on a three-helix bundle domain with 58 amino acids and have a molar mass of about 6 kDa. They can be expressed in soluble and proteolytically stable forms in various host cells on its own or via fusion with other protein partners. Affibodies can be used for protein purification, enzyme inhibition, research reagents for protein capture and detection, diagnostic imaging, and targeted therapy.

[0060] The term “nanobody” as used herein refers to tiny, recombinantly produced antigen binding fragments, typically consisting of a single monomeric variable antibody domain. Although nanobodies lack the light chains and heavy chain constant domain, the antigen-binding capacity remains similar to that of conventional antibodies. Typically, the complementarity-determining region 3 (CDR3) of nanobodies is similar or even longer than that of human variable domain of the heavy immunoglobulin chain (VH). They can form finger-like structures to recognize cavities or hidden epitopes that are not available to monoclonal antibodies, a feature that enhances the binding affinity and specificity of nanobodies.

[0061] An “avimer” (short of avidity multimer) as used herein refers to artificial proteins with multiple binding sites for specific binding to certain antigens. They are not structurally related to antibodies and thus, are classified as antibody mimetics. Typically, they consist of two or more peptide sequences of 30 to 35 amino acids, connected by linker peptides. The individual sequences are derived from A domains of various membrane receptors and have a rigid structure, stabilised by disulfide bonds and calcium. Each A domain can bind to a certain epitope of the target protein. The combination of domains binding to different epitopes of the same protein increases affinity to this protein, an effect known as avidity. Avimers are widely used in early detection in tissue imaging, treatment, and study on carcinogenesis.

[0062] The term “peptidomimetic” as used herein, refers to compound that mimics one or more structural aspects or biological activities of a naturally-occurring polypeptide, but which comprises one or more non-peptide or non-naturally occurring chemical structures or bonds. Peptidomimetics are frequently used to mimic the biological action of a peptide, thus they may be small protein-like chain designed to mimic one or more peptides. Peptidomimetics are often synthesized based on existing peptides of interest with one or more modifications to alter the molecule's structure or properties. Modifications can change the peptide molecule's stability, half-life, biological activity, absorption, or side-effects (e.g., toxicity, solubility, hydrophobicity, side-chain charge, or flexibility) of a peptide. Peptidomimetics can be useful as medicaments or drug-like compounds developed rationally, or based on modification of an existing peptide with known or putative biological activity.

[0063] A “detectable label” as referred to herein, which may be used in accordance with the invention include gold particles, latex beads, acridan ester, luminol, ruthenium, enzymatically active labels, radioactive labels, magnetic labels, e.g., magnetic beads, including paramagnetic and superparamagnetic labels, and fluorescent labels. Enzymatically active labels include, e.g., horseradish peroxidase, alkaline phosphatase, beta-Galactosidase, Luciferase, and derivatives thereof. Suitable substrates for detection include di-amino-benzidine (DAB), 3,3'-5,5'- tetramethylbenzidine, NBT-BCIP (4-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3- indolyl-phosphate. A suitable enzyme- substrate combination may result in a coloured reaction product, fluorescence or chemiluminescence, which can be measured according to methods known in the art (e.g., using a light-sensitive film or a suitable camera system). As for measuring the enzymatic reaction, the criteria given above apply analogously. Typical fluorescent labels include, e.g., fluorescent proteins (such as GFP and its derivatives), Cy3, Cy5, Texas Red, Fluorescein, the Alexa dyes, brilliant violet or brilliant ultraviolet. Also, the use of quantum dots as fluorescent labels is contemplated. Typical radioactive labels include 35S, 1251, 32P, 33P, and the like. A radioactive label can be detected by any method known and appropriate, e.g., a light-sensitive film or a phosphor imager. Suitable labels may also be or comprise tags, such as biotin, digoxygenin, His-, GST-, FLAG-, GFP-, MYC-tag, influenza A virus hemagglutinin (HA), maltose binding protein, and the like.

[0064] The term “blood” refers to peripheral blood from the same subject from which the cranial bone- derived immune cell population is obtained. The said blood may be used as a source for obtaining reference immune cell population for determining whether the cell types within the cranial bone-derived immune cell population according to the invention are increased or reduced.

[0065] The term “non-cranial bone marrow” refers to bone marrow from bones other than skull bones of the same subject from which the cranial bone-derived immune cell population is obtained. Preferably, said bone marrow is from distal bones, more preferably, from the hip bones, such as from iliac crest. The said non-cranial bone marrow may be used as a source to obtain a reference immune cell population for determining whether the cell types within the cranial bone-derived immune cell population according to the invention are increased or reduced.

[0066] The term “increased” as used herein refers to an increase in a parameter and, preferably, an increase of the number of cells relative to a reference or an increase in an amount or abundance relative to a reference. Said increase shall be, preferably, statistically significant. Whether an increase is statistically significant can be determined by statistical tests well known to those skilled in the art including, e.g., regression analysis, determination of confidence intervals, p- value determination, likelihood ratio test, Student's t-test, Mann-Whitney-U test etc. Details may be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983.

[0067] The term “reduced” as used herein refers to a decrease in a parameter and, preferably, a decrease of the number of cells relative to a reference or a decrease in an amount or abundance relative to a reference. Said decrease shall be, preferably, statistically significant. Whether a decrease is statistically significant can be determined by statistical tests well known to those skilled in the art.

[0068] Advantageously, in the studies underlying the present invention, it was found that a population of immune cells, which is found in brain disease-proximal areas of cranial bone is immunologically active vis-a-vis said brain disease. In particular, focal imaging and also in histological fine tissue analysis show a clear enrichment of CD45+ immune cells in the diploe of cranial bone in proximity to glioblastoma. Tissue or vital cells were obtained from glioblastoma patients from peripheral blood mononuclear cells (PBMC), tumor tissue, and bone tissue of the skull. As a further control, distal bone marrow from iliac crest biopsy was investigated. Subsequently, immune cells (CD45+) from these sample materials were enriched by MACS. Profiles of these cells collected by flow cytometry show T cells as the dominant fraction of immune cells in the skull bone. Furthermore, CD8+ T cells conventionally referred to as cytotoxic T cells or T killer cells were analyzed in more detail. The following observations were made:

[0069] (i) There was a significant enrichment of CD8 effector-memory T cells in the cranial bone of glioblastoma patients compared to the peripheral blood of glioblastoma patients. This was accompanied by the statistically significantly increased tumor reactivity of the cranial bone- derived CD8+ killer cells in the ELISpot assay. In the vital state (ViaDye red-), these cells were characterized by the following immune profile: CD45+CD3+ TCRgd-CD56-CD8+CD45RA- CCR7-. At the same time, there was a statistically significant reduction in naive CD8 T cells in glioblastoma cranial bone, which in the vital state (ViaDye red-) were characterized by the following immune biomarker profile: CD45+CD3+ TCRgd-CD56-CD8+CD45RA+CCR7+.

[0070] (ii) Important for the distinction from the more distant “distal” bone marrow was a statistically significant increase in the expression of S1PR1 on T cells from the cranial bone compared to peripheral blood or distal bone marrow. After MACS separation of CD45+ cells in the vital state (7AAD-), these cells were characterized by the following immune profile: CD3+S1PR1+. S1P1 receptors (such as S1RP1) are required when immune cells want to leave lymphoid organs. This finding was not present in the distal bone marrow of glioblastoma patients (see also, Chongsathidkiet et al., Nat Med 2018). This was accompanied by the finding of high tumor reactivity in CD8+ T cells from cranial bone, compared to CD8+ T cells from the blood or the distal bone marrow.

[0071] (iii) There was also a statistically significant enrichment of CD4 effector-memory T cells in the glioblastoma cranial bone compared to the peripheral blood of the glioblastoma patient. These cells are characterized in the vital state (ViaDye red-) were characterized by the following immune biomarker profile: CD45+CD3+ TCRgd-CD56-CD4+CD45RA-CCR7-. At the same time, there was a statistically significant reduction in naive CD4 T cells in the glioblastoma cranial bone, which in the vital state (ViaDye red-) was characterized by the following immune biomarker profile: CD45+CD3+TCRgd-CD56-CD4+CD45RA+CCR7+.

[0072] (iv) In addition, there was a statistically significant reduction in vital regulatory T cells (Tregs) in the glioblastoma cranial bone among the CD3+CD56- T cells, i.e., cells with the following immune biomarker profile: CD45+CD3+ TCRgd- CD56-CD4+CD25+CD1271ow / -.

[0073] The preferential distribution of these T cells substantiated the findings that the proximal cranial bone hosts functional adaptive immunological niches. In line with this notion, the presence of heterogeneous and well-defined subpopulations of T cells as described above indicates that the cranial bone may serve as a major site for homing and differentiation of effector T cell subsets.

[0074] It has been also shown that cranial bone-derived CD8+T cells are more resilient since they respond more durably to stimulation and expand more reliably ex vivo than CD8+T cells from peripheral blood (PBMCs) or from the tumor tissue. Gene expression analysis revealed that cranial bone-derived CD8+T cells are enriched for active effector types that are tumor-reactive, clonotypes with CD8+T cells are shared between cranial bone and the tumor. However, the tumor possesses fewer active effector phenotypes and more exhausted phenotypes. Using a CXCR4-PET ligand as a clinical surrogate marker for immune cell enrichment, it could be shown that (calvarial) enhancement in the proximal cranial bone indicates better patient survival.

[0075] Thanks to these findings, the diagnosis and treatment of various brain disease requiring immune responses will greatly benefit. Diseased areas of the brain may be identified by the presence of the cranial bone-derived immune cell population of the invention and / or the effector memory T cells comprised thereby. Moreover, the knowledge of the location of such an immune cell population, which at least contribute the body-own defense against the brain disease, is highly valuable for applying directed therapies. In particular, it may be detrimental for the amelioration or cure of a brain disease if such cells would be removed by, e.g., surgery or would be destroyed by irradiation therapies. Moreover, the said cells being immunologically active may be either stimulated for improving the immune response against the brain disease or they may be used in transplantation approaches for eliciting an immune response. Yet, the cells may also be genetically modified, e.g., for generating CAR T cells and the like and, thus, for developing new therapeutic approaches towards curing brain disease.

[0076] The definitions and explanations of the terms apply mutatis mutandis to all embodiments characterized herein below, too.

[0077] The present invention also relates to a cranial bone-derived effector memory CD8+ T cell expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA-, and CCR7-, wherein said T cells are further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow. Preferably, said cranial bone-derived effector memory CD8+ T cell has been derived from the immune cell population of the present invention as specified elsewhere herein. Preferably, said cranial bone-derived effector memory CD8+ T cell is an isolated T cell. More preferably, the said cell is an isolated cell, i.e., it is isolated from the subject from which it originates and, most preferably, isolated from the immune cell population of the invention.

[0078] The present invention also relates to a cranial bone-derived effector memory CD4+ T cell expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA-, and CCR7-, wherein said T cells are further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow. Preferably, said cranial bone-derived effector memory CD4+ T cell has been derived from the immune cell population of the present invention as specified elsewhere herein. Preferably, said cranial bone-derived effector memory CD4+ T cell is an isolated T cell. More preferably, the said cell is an isolated cell, i.e., it is isolated from the subject from which it originates and, most preferably, isolated from the immune cell population of the invention. The present invention also provides for a method of isolating the cranial bone-derived immune cell population and its subpopulations or cell types comprised therein.

[0079] In particular, the present invention also contemplates a method for providing a cranial bone- derived immune cell population of the present invention or any subpopulation thereof comprising the step of isolating CD45+ cells from a cranial bone sample from an area in proximity to brain tissue affected by a brain disease.

[0080] CD45+ cells shall be, preferably, isolated from the sample using a cell separation technique. More preferably, said cell separation technique is magnetic cell separation, preferably magnetic activated cell sorting (MACS). Subpopulations of cells within the immune cell population obtained or specific cell types such as those referred to herein, i.e., effector memory CD8+ T cells, effector memory CD4+ T cells, naive CD8+ cells, naive CD4+ T cells or other regulatory T cells, can be isolated from the immune cell population by further cell sorting steps using biomarkers or biomarker combinations for said specific cell types. Yet, the specific cell types can also be isolated or enriched by using cultivation and / or selection techniques when cultivating the isolated immune cell population of the invention. Typically, the cultivation and / or enrichment of specific cells from the immune cell population of the invention is performed in activation media containing different supplements that vary depending on the type of cells to be expanded. Suitable expansion media are generally known and commercially available.

[0081] Preferably, said subpopulation is (i) a cranial bone-derived effector memory CD8+ T cell or (ii) a cranial bone-derived effector memory CD4+ T cell of the invention.

[0082] A particular preferred method of isolating the cranial bone-derived immune cell population of the present invention is described in the accompanying Examples, below.

[0083] The present invention also relates to therapeutic as well as diagnostic uses of the immune cell population of the invention and any subpopulation thereof such as the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of the invention.

[0084] Accordingly, the present invention relates to a cranial bone-derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell of the invention for use in treating and / or preventing a brain disease in a subject suffering therefrom. The term “treating” as used herein refers to any improvement, cure or amelioration of the brain disease, i.e., a brain tumor or a neuroinflammatory disease as referred to herein. It will be understood that treatment may not occur in 100% of the subjects to which the cranial bone- derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell of the invention have been administered. The term, however, requires that the treatment occurs in a statistically significant portion of subjects (e.g., a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by a person skilled in the art using various well-known statistic evaluation tools known in the art.

[0085] The term “preventing” as used herein refers to significantly reducing the likelihood with which the brain disease, i.e., brain tumor or neuroinflammatory disease, develops in a subject within a defined window (prevention window). The preventive window, typically, starts from the administration of the cranial bone-derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell of the invention, e.g., by application of an antibody, onwards. Typically, the prevention window is within 3 to 6 months, 3 to 12 months, within 1 to 3 years or within 4 to 10 years. However, it will be understood that the preventive window may, dependent on the kind of medicament, also be several years up to the entire life time. The prevention window depends on the amount of cranial bone-derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell of the invention, which will be targeted by or which will be used as a treatment. It will be understood that prevention may not occur in 100% of the subjects to which the cells have been administered or in which the cells have been targeted. The term, however, requires that the prevention occurs in a statistically significant portion of subjects as discussed elsewhere herein.

[0086] The cranial bone-derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell of the invention may be pre-treated before administration. It will be understood that the cell population or cells according to the invention may be further modified for therapeutic approaches. For example, T cells may be further activated and / or differentiated by applying cytokines or they may be genetically modified to by T cell receptor engineering to improve target recognition and / or killing capabilities. Typically, the said cells may be used in autologous therapeutic approaches. Typically, autologous T cells are envisaged according to the invention. Said autologous T cells can be genetically modified by introducing a polynucleotide encoding the CAR protein via retroviral or lentiviral transfections. Moreover, the T cells may also be modified by other genetic approaches such as CRISPR / Cas technology. However, a heterologous treatment using the cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell of the invention, i.e., administration to a different subject from which they were obtained, is also envisaged in accordance with the present invention.

[0087] The cranial bone-derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell of the invention may be formulated as a cell-based medicament for autologous applications. Such a cell-based medicament is, preferably, for topical or systemic administration. It is to be understood that the formulation of a cell-based medicament takes place under GMP standardized conditions or the like in order to ensure quality, pharmaceutical security, and effectiveness of the medicament. A therapeutically effective dosage refers to an amount to be used that prevents, ameliorates or treats the symptoms accompanying a disease or condition referred to in this specification. Therapeutic efficacy and toxicity can be determined by standard pharmaceutical procedures. The dosage regimen will be, typically, determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment. The cell-based medicament referred to herein is administered at least once in order to treat or ameliorate or prevent a disease or condition recited in this specification. However, the said cell-based medicament may be administered more than one time.

[0088] Preferably, said cell population or cell is to be used with an additional therapeutic agent.

[0089] More preferably, the said additional therapeutic agent is selected from the group consisting of: Temozolomide, Carmustine (BCNU), Lomustine (CCNU), Bevacizumab, Avelumab, Nivolumab, Pembrolizumab, Ipilimumab, Tremelimumab, Retifanlimab, Cemiplimab, MK- 3475, Oleclumab, Atezolizumab, Avelumab, Dostarlizumab, Durvalumab, IL-2, IL-12, Interferon-alpha, NT-17, Rindopepimut, SurVaxM, DSP-7888, VXM01, IMA950, EO2401, APVAC1, APVAC2, ICT-107 DC, Audencel, DCVaxL, ATL-DC, AdV-tk, Ad-RTS-hIL-12, M032, rQNestin34.5v.2, G47delta, DNX-2401, CRAd-Survivin-pk7, PVSRIPO, Vocimagene amiretrorepvec (TOCA 511), Extended-release 5-fluorocytosine (Toca FC), Fludeoxyglucose F-18, Paclitaxel, Carboplatin, Hydroxyurea, Capecitabine, DM-CHOC-PEN, VAL-083, Topotecan, Aldoxorubicin, Cyclophosphamide, Etoposide, Crizotinib, Imatinib, Neratinib, Sunitinib, Ponatinib, Acalabrutinib, Axitinib, Cediranib, Olaparib, Cabozantinib, Pazopanib, Tivozanib, Beta-elemene, Afatinib, Dacomitinib, Epitinib, Erlotinib, Sirolimus, Temsirolimus, Gefitinib, Osimertinib, WSD0922-FU, Infigratinib, Dovitinib, Pazopanib, Lapatinib, AEE788, Regorafinib, Tesevatinib, Abemaciclib, GLR2007, Palbociclib, Ribociclib, Zotiraciclib, Buparlisib, Fimepinostat, Paxalisib, Perifosine, Metformin, Sapanisertib, Vistusertib, Veliparib, Pamiparib, Olaparib, AZD1390, Alisertib, Fingolimod, Crenolanib, Trametinib, Dabrafenib, Chlorpromazine, Chloroquine, Lisavanbulin, Cabazitaxel, Mebendazole, Ortataxel, Belinostat, Vorinostat, Ixazomib, Marizomib, Nabucasin, WP1066, AMG-232, Celecoxib, Ralimetinib, ONC201, ONC206, Atorvastatin, Dexanabinol, Dimethyl fzmarate, Disulfiram, Epacodastat, Capmatinib, Evofosfamide, Galunisertib, Indoximod, Ketoconazole, LB 100, Mipsagargin, Mibefradil, Pexidartinib, Plerixafor, Posoconazole, PT2385, Selinexor, Tipifarnisb, Ascorbate, Cilengitide, Chlorogenic acid, and Chlorotoxin.

[0090] Also more preferably, the additional therapeutic agent is selected from the group consisting of: anti-angiogenic therapy, chemotherapy, immunotherapy, surgery, radiotherapy, preferably, whole-brain irradiation, fractionated radiotherapy, radio surgery, proton therapy or a combination thereof, anti-angiogenic therapy, alternating electric field therapy, cytokine-based therapy, chimeric antigen receptor (CAR) T-cell therapy, vaccine-based therapy, viral vector therapy, oncolytic virus therapy and therapy with immunosuppressive agents.

[0091] The aforementioned therapeutic agents and therapies have been reported or indicated to be effective in treating different brain disease envisaged in accordance with the present invention. Based on the findings underlying this invention, therapeutic efficacy of the said therapies shall be further enhanced by using them together with the cranial bone-derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell of the invention for therapy of a brain disease in accordance with the present invention.

[0092] The present invention also relates to a cranial bone-derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell of the invention for use in aiding therapy involving physical treatment of a brain disease in a subject suffering from said brain disease.

[0093] Preferably, said physical treatment involves surgery or irradiation.

[0094] Surgery as referred to herein is, typically, brain surgery. Irradiation as referred to herein encompasses different radiation-based therapies such as whole-brain irradiation, fractionated radiotherapy, radio surgery, proton therapy and a combination thereof.

[0095] The term “aiding” as used herein relates to providing guidance for said physical treatments in order to improve the therapeutic results. The said improvement may be an increase in therapeutic efficacy of a physical treatment applied and / or a reduction of undesired side- effects. By identifying the immune cell population and the effector memory CD8+ and CD4+ T cells in brain disease proximal areas of the cranial bone, the present invention provides for an aid for physical therapy. In particular, as will be understood from the findings of the present invention, the immune cell population of the present invention and its subpopulations is involved in the auto-immunological response of the organism against the brain disease. Accordingly, knowing the position of the said immune cell population in the cranial bone shall be helpful not only for applying therapies which are based on isolating the cells of the immune cell population, such as autologous T cell therapies or stimulating these cells in vivo, but also for physical treatments. In particular, by the identification of the valuable immune cell population of the present invention and its subpopulations, the destruction and / or removal of cranial bone areas can be prevented, which harbor these valuable cells.

[0096] The present invention also relates to a cranial bone-derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell for use in assessing a brain disease in a subject.

[0097] The term “assessing” as used herein refers to diagnosing, including staging or differentially diagnosing, monitoring and / or predicting, including risk stratifying, a brain disease as referred to herein. Diagnosing as used herein refers to determining whether a subject suffers from the brain disease, or not. Staging refers to determining the grade or severity of the brain disease. Monitoring refers to repeatedly diagnosing the brain disease over a predefine period of time, e.g., to assess success of a therapy. Predicting the brain disease refers an assessment of the development of the brain disease within a predictive window in the future or to stratify the brain disease with respect to a certain (future) predefined end point or outcome, or not. This may include determining the likelihood with which the subject shall develop a certain end point or outcome. As will be understood by those skilled in the art, an assessment is usually not intended to be correct for 100% of the subjects to be investigated. The term, however, requires that the assessment is correct for a statistically significant portion of the subjects (e.g., a cohort in a cohort study). Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools.

[0098] For the purpose of assessing the brain disease in accordance with the present invention, the presence or absence or the abundance of the cranial bone-derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell may be determined in a sample of cranial bone from a subject suspected to suffer from a brain disease as mentioned herein. The sample is, preferably, from an area which is in proximity of the area of the brain affected by the brain disease. The presence, absence or the abundance of the cranial bone-derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell may be determined by detecting the presence, absence or abundance of one or more of the different cell types of the immune cell population based on the biomarkers referred to elsewhere herein. The biomarkers can be detected by the binding molecules referred to elsewhere herein. Besides for analyzing samples of cranial bone, the presence or absence or the abundance of the cranial bone-derived immune cell population, a cranial bone-derived effector memory CD8+ T cell, or a cranial bone-derived effector memory CD4+ T cell may be also determined in vivo.

[0099] It follows from the above that the present invention also relates to a binding molecule or a combination of binding molecules for use in assessing a brain disease wherein said binding molecule specifically binds to one or more of the biomarkers of an effector memory CD8+ T cell, an effector memory CD4+ T cell, a naive CD8+ T cell, a naive CD4+ T cell and / or a regular T cell as specified elsewhere herein.

[0100] Moreover, the present invention provides for a kit for assessing a brain disease, wherein said kit comprises a binding molecule or combination of binding molecules which specifically bind to one or more of the biomarkers of an effector memory CD8+ T cell, an effector memory CD4+ T cell, a naive CD8+ T cell, a naive CD4+ T cell and / or a regular T cell as specified elsewhere herein.

[0101] The term “kit” as used herein refers to collection of the aforementioned components, typically, provided in separately or within a single container. The container also typically comprises instructions for carrying out the uses and methods of the present invention. These instructions may be in the form of a manual or may be provided by a computer program code, which is capable of carrying out or supports the determination of the biomarker(s) referred to herein when implemented on a computer or a data processing device. The computer program code may be provided on a data storage medium or device such as an optical storage medium (e.g., a Compact Disc) or directly on a computer or data processing device or may be provided in a download format such as a link to an accessible server or cloud. Moreover, the kit may usually comprise standards for reference amounts of biomarkers for calibration purposes as described elsewhere herein in detail. The kit according to the present invention may also comprise further components which are necessary for carrying out the method of the invention such as solvents, buffers, washing solutions and / or reagents required for detection of the released second molecule. The present invention also relates to therapeutic as well as diagnostic methods of the immune cell population of the invention and any subpopulation thereof such as the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of the invention.

[0102] The present invention relates to a method for treating and / or preventing a brain disease comprising the step of administering to a subject suffering therefrom a therapeutically effective amount of the cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell, or the cranial bone-derived effector memory CD4+ T cell of the invention.

[0103] Preferably, the said the cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell, or the cranial bone-derived effector memory CD4+ T cell of the invention is administered together with a further therapeutic agent against the brain disease as specified elsewhere herein.

[0104] The present invention also relates to a method of aiding a treatment of a brain disease in a subject suffering from a brain disease, said method comprising: a) identifying in an image of the brain disease-proximal cranial bone of the subject bone areas comprising the cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell, or the cranial bone-derived effector memory CD4+ T cell of the invention; and b) aiding the said therapy by recommending to prevent those bone areas from physical treatment; or c) aiding the said therapy by recommending to specifically target those bone areas by pharmaceutical treatment.

[0105] Preferably, said physical treatment involves surgery or irradiation as described elsewhere herein. Also preferably, said pharmaceutical treatment involved administration of a therapeutic agent as specified elsewhere herein or is selected from the group consisting of an interferon, preferably, iFa-2b, iFaconl, iFa-n3, iFP-la, iFP-lb, iFy-lb, or pegIFa-2b, an interleukin, preferably, aldesleukin (proleukin) or oprelvekin (neumega), a colony-stimulating factor, preferably, filgrastin (neupogen), pegfilgrastim (neulasta), or sargramostim (leukine), glatiramer (Copaxone), elapegademase, plerixafor, trilaciclib, and PEG-ademase. Preferably, said bone areas in step a) are identified by allocating data indicating the presence, absence or abundance of the cranial bone-derived immune cell population, the cranial bone- derived effector memory CD8+ T cell, or the cranial bone-derived effector memory CD4+ T cell of the invention in bone areas proximal to the brain area affected by the brain disease to the image. It will be understood that this step is, preferably, assisted by automation, e.g., by using an imaging device and a data processing device such as a computer. The data processing device may be equipped with image evaluation and recognition algorithms including those using artificial intelligence. Typically, an image of the brain disease-proximal cranial bone of the subject displays graphically the morphological structure of the brain disease-proximal cranial bone. The image may be, e.g., an X-ray, CT-, MR-, or PET- image or a combination or an image fusion thereof. In order to identify the location of the cranial bone-derived immune cell population or any subpopulation or cell type comprised therein as referred to herein in the morphological structure displayed on the image, data indicating the presence, absence or abundance of said population, subpopulation or cells shall be allocated to the image such that they indicate their positioning the morphological structure. Such data may represent fluorescence, X-ray, CT-, MR-, or PET- image data or data representing a combination or an image fusion thereof indicating the presence, absence or abundance of the cells due to detectable labels for biomarkers present on or in said cells. Typically, radiotracers may be used for indicating individual cell types. Details on suitable biomarkers and detection agents therefor are to be found elsewhere herein. Such data may be allocated to the image data, i.e., the data may be integrated into the image such that the localization of the cells is indicated in the image.

[0106] The present invention contemplates a method for assessing a brain disease in a subject suspected to suffer or suffering therefrom comprising the steps of: a) identifying in an image of the brain disease-proximal cranial bone of the subject bone areas comprising the cranial bone-derived immune cell population, the cranial bone- derived effector memory CD8+ T cell, or the cranial bone-derived effector memory CD4+ T cell of the invention; and b) assessing the brain disease based on the identified cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell, or the cranial bone- derived effector memory CD4+ T cell of the invention.

[0107] Finally, the present invention also relates to a therapeutic agent for use in treating and / or preventing the brain disease in a subject, wherein said therapeutic agent is capable of stimulating an anti-disease immune response against the brain disease and wherein said therapeutic agent is to be administered into the areas within cranial bone comprising the cranial bone-derived immune cell population of the present invention.

[0108] Preferably, said therapeutic agent is a therapeutic agent as specified elsewhere herein. These therapeutic agents are, preferably, known therapeutic agents effective for treating the brain disease referred to herein. However, thanks to the identification of the immune cell population of the present invention and the subpopulations and cells referred to herein, it is possible to apply such therapeutic agents to areas within cranial bone hosting cells that are capable of eliciting an immune response against the brain disease.

[0109] The following are particular preferred embodiments of the present invention:

[0110] Embodiment 1 : A cranial bone-derived immune cell population comprising: a) effector memory CD8+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow; b) naive CD8+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA+, and CCR7+; c) effector memory CD4+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow; d) naive CD4+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA+, and CCR7+; and e) regulatory T cells CD45+, CD3+, TCRgd-, CD56-, CD4+, CD25+, and CD1271ow / -; wherein compared to blood and non-cranial bone marrow the effector memory CD8+ T cells according to a) are increased whereas the naive CD8+ T cells according to b) are reduced; wherein compared to blood and non-cranial bone marrow the effector memory CD4+ T cells according to c) are increased whereas the naive CD4+ T cells according to d) are reduced; and wherein the regulatory T cells according to e) are reduced.

[0111] Embodiment 2: The cranial bone-derived immune cell population of embodiment 1, wherein said immune cell population is an isolated cell population. Embodiment 3: The cranial bone-derived immune cell population of embodiment 1 or 2, wherein said immune cell population is derived from a cranial bone sample from an area in proximity to brain tissue affected by a brain disease.

[0112] Embodiment 4: The cranial bone-derived immune cell population of any one of embodiments 1 to 3, wherein said brain disease is a brain tumor or a neuroinflammatory disease.

[0113] Embodiment 5: The cranial bone-derived immune cell population of embodiment 4, wherein said brain tumor is

[0114] (i) a brain tumor selected from the group consisting of: adult-type diffuse gliomas, preferably astrocytoma, oligodendroglioma or glioblastoma; pediatric-type low-grade gliomas, preferably diffuse astrocytoma, angiocentric glioma, polymorphous low-grade neuroepithelial tumor of the young or diffuse low-grade glioma; pediatric-type diffuse high-grade gliomas, preferably diffuse midline glioma, diffuse hemispheric glioma, diffuse pediatric-type high-grade glioma or infant-type hemispheric glioma; circumscribed astrocytic gliomas, preferably pilocytic atrocytoma, high-grade astrocytoma with piloid features, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, chordoid glioma or astroblastoma; glioneuronal and neuronal tumors, preferably ganglioglioma, gangliocytoma, desmoplastic infantile ganglioglioma, desmoplastic infantile astrocytoma, dysembryoplastic neuroepithelial tumour, diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters, papillary glioneuronal tumour, rosette-forming glioneuronal tumor, myxoid glioneuronal tumour, diffuse leptomeningeal glioneuronal tumour, multinodular and vacuolating neuronal tumour, dysplastic cerebellar gangliocytoma, central neurocytoma, extraventricular neurocytoma or cerebellar liponeurocytoma; ependymal tumors, preferably ependymoma, myxopapillary ependymoma or subependymoma; choroid plexus tumors, preferably choroid plexus papilloma, atypical choroid plexus papilloma or choroid plexus carcinoma; embryonal tumors, preferably medulloblastoma, atypical teratoid / rhabdoid tumor, cribriform neuroepithelial tumor, embryonal tumor with multilayered rosettes, CNS neuroblastoma, CNS tumor with BCOR-internal tandem duplication or CNS embryonal tumor; pineal tumors, preferably pineocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, papillary tumor of the pineal region or desmoplastic myxoid tumor of the pineal region; meningioma, preferably meningioma grades 1 to 3; mesenchymal, non-meningothelial tumors involving the CNS, preferably solitary fibrous tumor, cavernous hemangioma, capillary hemangioma, arteriovenous malformation, hemangioblastoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, spindle cell rhabdomyosarcoma, intracranial mesenchymal tumor, CIC-rearranged sarcoma, primary intracranial sarcoma, Ewing sarcoma, Mesenchymal chondrosarcoma, chondrosarcoma, dedifferentiate chondrosarcoma, or chordoma; melanocytic tumors, preferably meningeal melanocytosis, meningeal melanomatosis, meningeal melanocytioma or meningeal melanoma; hematolymphoid tumors involving the CNS, preferably primary diffuse large B-cell lymphoma of the CNS, lymphomatoid granulomatosis, intravascular large B-cell lymphoma, MALT lymphoma of the dura, lymphoplasmacytic lymphoma, follicular lymphoma, anaplastic large cell lymphoma, T- cell lymphoma, NK / T-cell lymphoma, Erdheim-Chester disease, Rosai-Dorfman disease, Juvenile xanthogranuloma, Langerhans cell histiocytosis or histiocytic sarcoma; germ cell tumors, preferably mature teratoma, immature teratoma, teratoma with somatic-type malignancy, germinoma, embryonal carcinoma, yolk sac tumors, choriocarcinoma or mixed germ cell tumor; tumors of the sellar region, preferably adamantinomatous craniopharyngioma, papillary craniopharyngioma, pituicytoma, granular cell tumor of the sellar region, spindle cell oncocytoma, pituitary adenoma, pituitary neuroendocrine tumor and pituitary blastoma, or (ii) is a metastasized secondary brain tumor selected from the group consisting of lung cancer, breast cancer, melanoma, colorectal cancer, kidney cancer, thyroid cancer and uterine cancer.

[0115] Embodiment 6: The cranial bone-derived immune cell population of embodiment 4, wherein the neuroinflammatory disease is selected from the group consisting of multiple sclerosis, vasculitis, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, stroke, migraine, epilepsy and traumatic brain injuries.

[0116] Embodiment 7: The cranial bone-derived immune cell population of any one of embodiments 1 to 6, wherein said area in proximity to brain tissue affected by a brain disease has been identified by imaging, preferably, by PET-, CT-, MR-imaging, or and / or combinations thereof.

[0117] Embodiment 8: The cranial bone-derived immune cell population of any one of embodiments 1 to 7, wherein said cranial bone sample id from an area which is located adjacent to the circumference of the intracerebral brain disease area, preferably, within a distance of at most about 3 cm, at most about 2 cm, at most about 1 cm or at most about 0.5 cm.

[0118] Embodiment 9: The cranial bone-derived immune cell population of any one of embodiments 1 to 8, wherein said immune cell population has been derived from the cranial bone sample by isolating CD45+ cells from the sample using a cell separation technique.

[0119] Embodiment 10: The cranial bone-derived immune cell population of embodiment 9, wherein said cell separation technique is magnetic cell separation, preferably magnetic activated cell sorting (MACS).

[0120] Embodiment 11 : A cranial bone-derived effector memory CD8+ T cell expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow.

[0121] Embodiment 12: The cranial bone-derived effector memory CD8+ T cell of embodiment 11, wherein said cell has been derived from the immune cell population of any one of embodiments 1 to 10.

[0122] Embodiment 13 : A cranial bone-derived effector memory CD4+ T cell expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow.

[0123] Embodiment 14: The cranial bone-derived effector memory CD4+ T cell of embodiment 11, wherein said cell has been derived from the immune cell population of any one of embodiments 1 to 10.

[0124] Embodiment 15: A method for providing an immune cell population of any one of embodiments 1 to 10 or any subpopulation thereof comprising the step of isolating CD45+ cells from a cranial bone sample from an area in proximity to brain tissue affected by a brain disease.

[0125] Embodiment 16: The method of embodiment 15, wherein said subpopulation is (i) a cranial bone-derived effector memory CD8+ T cell of embodiment 11 or 12 or (ii) a cranial bone- derived effector memory CD4+ T cell of embodiment 13 or 14.

[0126] Embodiment 17: A cranial bone-derived immune cell population of any one of embodiments 1 to 10, a cranial bone-derived effector memory CD8+ T cell of embodiment 11 or 12, or a cranial bone-derived effector memory CD4+ T cell of embodiment 13 or 14 for use in treating and / or preventing a brain disease in a subject suffering therefrom.

[0127] Embodiment 18: The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 17, wherein said brain disease is a brain tumor or a neuroinflammatory disease.

[0128] Embodiment 19: The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 18, wherein said brain tumor is (i) a brain tumor selected from the group consisting of: adult-type diffuse gliomas, preferably astrocytoma, oligodendroglioma or glioblastoma; pediatric-type low-grade gliomas, preferably diffuse astrocytoma, angiocentric glioma, polymorphous low-grade neuroepithelial tumor of the young or diffuse low-grade glioma; pediatric-type diffuse high-grade gliomas, preferably diffuse midline glioma, diffuse hemispheric glioma, diffuse pediatric-type high-grade glioma or infant-type hemispheric glioma; circumscribed astrocytic gliomas, preferably pilocytic atrocytoma, high-grade astrocytoma with piloid features, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, chordoid glioma or astroblastoma; glioneuronal and neuronal tumors, preferably ganglioglioma, gangliocytoma, desmoplastic infantile ganglioglioma, desmoplastic infantile astrocytoma, dysembryoplastic neuroepithelial tumour, diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters, papillary glioneuronal tumour, rosette-forming glioneuronal tumor, myxoid glioneuronal tumour, diffuse leptomeningeal glioneuronal tumour, multinodular and vacuolating neuronal tumour, dysplastic cerebellar gangliocytoma, central neurocytoma, extraventricular neurocytoma or cerebellar liponeurocytoma; ependymal tumors, preferably ependymoma, myxopapillary ependymoma or subependymoma; choroid plexus tumors, preferably choroid plexus papilloma, atypical choroid plexus papilloma or choroid plexus carcinoma; embryonal tumors, preferably medulloblastoma, atypical teratoid / rhabdoid tumor, cribriform neuroepithelial tumor, embryonal tumor with multilayered rosettes, CNS neuroblastoma, CNS tumor with BCOR-internal tandem duplication or CNS embryonal tumor; pineal tumors, preferably pineocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, papillary tumor of the pineal region or desmoplastic myxoid tumor of the pineal region; meningioma, preferably meningioma grades 1 to 3; mesenchymal, non-meningothelial tumors involving the CNS, preferably solitary fibrous tumor, cavernous hemangioma, capillary hemangioma, arteriovenous malformation, hemangioblastoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, spindle cell rhabdomyosarcoma, intracranial mesenchymal tumor, CIC-rearranged sarcoma, primary intracranial sarcoma, Ewing sarcoma, Mesenchymal chondrosarcoma, chondrosarcoma, dedifferentiate chondrosarcoma, or chordoma; melanocytic tumors, preferably meningeal melanocytosis, meningeal melanomatosis, meningeal melanocytioma or meningeal melanoma; hematolymphoid tumors involving the CNS, preferably primary diffuse large B-cell lymphoma of the CNS, lymphomatoid granulomatosis, intravascular large B-cell lymphoma, MALT lymphoma of the dura, lymphoplasmacytic lymphoma, follicular lymphoma, anaplastic large cell lymphoma, T- cell lymphoma, NK / T-cell lymphoma, Erdheim-Chester disease, Rosai-Dorfman disease, Juvenile xanthogranuloma, Langerhans cell histiocytosis or histiocytic sarcoma; germ cell tumors, preferably mature teratoma, immature teratoma, teratoma with somatic-type malignancy, germinoma, embryonal carcinoma, yolk sac tumors, choriocarcinoma or mixed germ cell tumor; tumors of the sellar region, preferably adamantinomatous craniopharyngioma, papillary craniopharyngioma, pituicytoma, granular cell tumor of the sellar region, spindle cell oncocytoma, pituitary adenoma, pituitary neuroendocrine tumor and pituitary blastoma, or

[0129] (ii) is a metastasized secondary brain tumor selected from the group consisting of lung cancer, breast cancer, melanoma, colorectal cancer, kidney cancer, thyroid cancer and uterine cancer.

[0130] Embodiment 20: The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 18, wherein the neuroinflammatory disease is selected from the group consisting of multiple sclerosis, vasculitis, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, stroke, migraine, epilepsy and traumatic brain injuries.

[0131] Embodiment 21 : The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of any one of embodiments 17 to 20, wherein said cell population or cell is to be used with an additional therapeutic agent.

[0132] Embodiment 22: The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 21, wherein the additional therapeutic agent is selected from the group consisting of: Temozolomide, Carmustine (BCNU), Lomustine (CCNU), Bevacizumab, Avelumab, Nivolumab, Pembrolizumab, Ipilimumab, Tremelimumab, Retifanlimab, Cemiplimab, MK- 3475, Oleclumab, Atezolizumab, Avelumab, Dostarlizumab, Durvalumab, IL-2, IL-12, Interferon-alpha, NT-17, Rindopepimut, SurVaxM, DSP-7888, VXM01, IMA950, EO2401, APVAC1, APVAC2, ICT-107 DC, Audencel, DCVaxL, ATL-DC, AdV-tk, Ad-RTS-hIL-12, M032, rQNestin34.5v.2, G47delta, DNX-2401, CRAd-Survivin-pk7, PVSRIPO, Vocimagene amiretrorepvec (TOCA 511), Extended-release 5-fluorocytosine (Toca FC), Fludeoxyglucose F-18, Paclitaxel, Carboplatin, Hydroxyurea, Capecitabine, DM-CHOC-PEN, VAL-083, Topotecan, Aldoxorubicin, Cyclophosphamide, Etoposide, Crizotinib, Imatinib, Neratinib, Sunitinib, Ponatinib, Acalabrutinib, Axitinib, Cediranib, Olaparib, Cabozantinib, Pazopanib, Tivozanib, Beta-elemene, Afatinib, Dacomitinib, Epitinib, Erlotinib, Sirolimus, Temsirolimus, Gefitinib, Osimertinib, WSD0922-FU, Infigratinib, Dovitinib, Pazopanib, Lapatinib, AEE788, Regorafinib, Tesevatinib, Abemaciclib, GLR2007, Palbociclib, Ribociclib, Zotiraciclib, Buparlisib, Fimepinostat, Paxalisib, Perifosine, Metformin, Sapanisertib, Vistusertib, Veliparib, Pamiparib, Olaparib, AZD1390, Alisertib, Fingolimod, Crenolanib, Trametinib, Dabrafenib, Chlorpromazine, Chloroquine, Lisavanbulin, Cabazitaxel, Mebendazole, Ortataxel, Belinostat, Vorinostat, Ixazomib, Marizomib, Nabucasin, WP1066, AMG-232, Celecoxib, Ralimetinib, ONC201, ONC206, Atorvastatin, Dexanabinol, Dimethyl fzmarate, Disulfiram, Epacodastat, Capmatinib, Evofosfamide, Galunisertib, Indoximod, Ketoconazole, LB 100, Mipsagargin, Mibefradil, Pexidartinib, Plerixafor, Posoconazole, PT2385, Selinexor, Tipifarnisb, Ascorbate, Cilengitide, Chlorogenic acid, and Chlorotoxin.

[0133] Embodiment 23 : The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 21, wherein the additional therapeutic agent is selected from the group consisting of anti-angiogenic therapy, chemotherapy, immunotherapy, surgery, radiotherapy, preferably, whole-brain irradiation, fractionated radiotherapy, radio surgery, proton therapy or a combination thereof, anti-angiogenic therapy, alternating electric field therapy, cytokine-based therapy, chimeric antigen receptor (CAR) T-cell therapy, vaccine-based therapy, viral vector therapy, oncolytic virus therapy and therapy with immunosuppressive agents.

[0134] Embodiment 24: A cranial bone-derived immune cell population of any one of embodiments 1 to 10, a cranial bone-derived effector memory CD8+ T cell of embodiment 11 or 12, or a cranial bone-derived effector memory CD4+ T cell of embodiment 13 or 14 for use in aiding therapy involving physical treatment of a brain disease in a subject suffering from said brain disease.

[0135] Embodiment 25: The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 24, wherein said physical treatment involves surgery or irradiation.

[0136] Embodiment 26: The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 24 or 25, wherein said brain disease is a brain tumor or a neuroinflammatory disease

[0137] Embodiment 27: The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 26, wherein said brain tumor is

[0138] (i) a brain tumor selected from the group consisting of: adult-type diffuse gliomas, preferably astrocytoma, oligodendroglioma or glioblastoma; pediatric-type low-grade gliomas, preferably diffuse astrocytoma, angiocentric glioma, polymorphous low-grade neuroepithelial tumor of the young or diffuse low-grade glioma; pediatric-type diffuse high-grade gliomas, preferably diffuse midline glioma, diffuse hemispheric glioma, diffuse pediatric-type high-grade glioma or infant-type hemispheric glioma; circumscribed astrocytic gliomas, preferably pilocytic atrocytoma, high-grade astrocytoma with piloid features, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, chordoid glioma or astroblastoma; glioneuronal and neuronal tumors, preferably ganglioglioma, gangliocytoma, desmoplastic infantile ganglioglioma, desmoplastic infantile astrocytoma, dysembryoplastic neuroepithelial tumour, diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters, papillary glioneuronal tumour, rosette-forming glioneuronal tumor, myxoid glioneuronal tumour, diffuse leptomeningeal glioneuronal tumour, multinodular and vacuolating neuronal tumour, dysplastic cerebellar gangliocytoma, central neurocytoma, extraventricular neurocytoma or cerebellar liponeurocytoma; ependymal tumors, preferably ependymoma, myxopapillary ependymoma or subependymoma; choroid plexus tumors, preferably choroid plexus papilloma, atypical choroid plexus papilloma or choroid plexus carcinoma; embryonal tumors, preferably medulloblastoma, atypical teratoid / rhabdoid tumor, cribriform neuroepithelial tumor, embryonal tumor with multilayered rosettes, CNS neuroblastoma, CNS tumor with BCOR-internal tandem duplication or CNS embryonal tumor; pineal tumors, preferably pineocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, papillary tumor of the pineal region or desmoplastic myxoid tumor of the pineal region; meningioma, preferably meningioma grades 1 to 3; mesenchymal, non-meningothelial tumors involving the CNS, preferably solitary fibrous tumor, cavernous hemangioma, capillary hemangioma, arteriovenous malformation, hemangioblastoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, spindle cell rhabdomyosarcoma, intracranial mesenchymal tumor, CIC-rearranged sarcoma, primary intracranial sarcoma, Ewing sarcoma, Mesenchymal chondrosarcoma, chondrosarcoma, dedifferentiate chondrosarcoma, or chordoma; melanocytic tumors, preferably meningeal melanocytosis, meningeal melanomatosis, meningeal melanocytioma or meningeal melanoma; hematolymphoid tumors involving the CNS, preferably primary diffuse large B-cell lymphoma of the CNS, lymphomatoid granulomatosis, intravascular large B-cell lymphoma, MALT lymphoma of the dura, lymphoplasmacytic lymphoma, follicular lymphoma, anaplastic large cell lymphoma, T- cell lymphoma, NK / T-cell lymphoma, Erdheim-Chester disease, Rosai-Dorfman disease, Juvenile xanthogranuloma, Langerhans cell histiocytosis or histiocytic sarcoma; germ cell tumors, preferably mature teratoma, immature teratoma, teratoma with somatic-type malignancy, germinoma, embryonal carcinoma, yolk sac tumors, choriocarcinoma or mixed germ cell tumor; tumors of the sellar region, preferably adamantinomatous craniopharyngioma, papillary craniopharyngioma, pituicytoma, granular cell tumor of the sellar region, spindle cell oncocytoma, pituitary adenoma, pituitary neuroendocrine tumor and pituitary blastoma, or (ii) is a metastasized secondary brain tumor selected from the group consisting of lung cancer, breast cancer, melanoma, colorectal cancer, kidney cancer, thyroid cancer and uterine cancer.

[0139] Embodiment 28: The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 28, wherein the neuroinflammatory disease is selected from the group consisting of: multiple sclerosis, vasculitis, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, stroke, migraine, epilepsy and traumatic brain injuries.

[0140] Embodiment 29: A cranial bone-derived immune cell population of any one of embodiments 1 to 10, a cranial bone-derived effector memory CD8+ T cell of embodiment 11 or 12, or a cranial bone-derived effector memory CD4+ T cell of embodiment 13 or 14 for use in assessing a brain disease in a subject.

[0141] Embodiment 30: The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 29, wherein said assessing comprises diagnosing, monitoring and or predicting the said brain disease.

[0142] Embodiment 31 : The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 30, wherein said brain disease is a brain tumor or a neuroinflammatory disease.

[0143] Embodiment 32: The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 31, wherein said brain tumor is

[0144] (i) a brain tumor selected from the group consisting of: adult-type diffuse gliomas, preferably astrocytoma, oligodendroglioma or glioblastoma; pediatric-type low-grade gliomas, preferably diffuse astrocytoma, angiocentric glioma, polymorphous low-grade neuroepithelial tumor of the young or diffuse low-grade glioma; pediatric-type diffuse high-grade gliomas, preferably diffuse midline glioma, diffuse hemispheric glioma, diffuse pediatric-type high-grade glioma or infant-type hemispheric glioma; circumscribed astrocytic gliomas, preferably pilocytic atrocytoma, high-grade astrocytoma with piloid features, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, chordoid glioma or astroblastoma; glioneuronal and neuronal tumors, preferably ganglioglioma, gangliocytoma, desmoplastic infantile ganglioglioma, desmoplastic infantile astrocytoma, dysembryoplastic neuroepithelial tumour, diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters, papillary glioneuronal tumour, rosette-forming glioneuronal tumor, myxoid glioneuronal tumour, diffuse leptomeningeal glioneuronal tumour, multinodular and vacuolating neuronal tumour, dysplastic cerebellar gangliocytoma, central neurocytoma, extraventricular neurocytoma or cerebellar liponeurocytoma; ependymal tumors, preferably ependymoma, myxopapillary ependymoma or subependymoma; choroid plexus tumors, preferably choroid plexus papilloma, atypical choroid plexus papilloma or choroid plexus carcinoma; embryonal tumors, preferably medulloblastoma, atypical teratoid / rhabdoid tumor, cribriform neuroepithelial tumor, embryonal tumor with multilayered rosettes, CNS neuroblastoma, CNS tumor with BCOR-internal tandem duplication or CNS embryonal tumor; pineal tumors, preferably pineocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, papillary tumor of the pineal region or desmoplastic myxoid tumor of the pineal region; meningioma, preferably meningioma grades 1 to 3; mesenchymal, non-meningothelial tumors involving the CNS, preferably solitary fibrous tumor, cavernous hemangioma, capillary hemangioma, arteriovenous malformation, hemangioblastoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, spindle cell rhabdomyosarcoma, intracranial mesenchymal tumor, CIC-rearranged sarcoma, primary intracranial sarcoma, Ewing sarcoma, Mesenchymal chondrosarcoma, chondrosarcoma, dedifferentiate chondrosarcoma, or chordoma; melanocytic tumors, preferably meningeal melanocytosis, meningeal melanomatosis, meningeal melanocytioma or meningeal melanoma; hematolymphoid tumors involving the CNS, preferably primary diffuse large B-cell lymphoma of the CNS, lymphomatoid granulomatosis, intravascular large B-cell lymphoma, MALT lymphoma of the dura, lymphoplasmacytic lymphoma, follicular lymphoma, anaplastic large cell lymphoma, T- cell lymphoma, NK / T-cell lymphoma, Erdheim-Chester disease, Rosai-Dorfman disease, Juvenile xanthogranuloma, Langerhans cell histiocytosis or histiocytic sarcoma; germ cell tumors, preferably mature teratoma, immature teratoma, teratoma with somatic-type malignancy, germinoma, embryonal carcinoma, yolk sac tumors, choriocarcinoma or mixed germ cell tumor; tumors of the sellar region, preferably adamantinomatous craniopharyngioma, papillary craniopharyngioma, pituicytoma, granular cell tumor of the sellar region, spindle cell oncocytoma, pituitary adenoma, pituitary neuroendocrine tumor and pituitary blastoma, or (ii) is a metastasized secondary brain tumor selected from the group consisting of lung cancer, breast cancer, melanoma, colorectal cancer, kidney cancer, thyroid cancer and uterine cancer.

[0145] Embodiment 33: The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of embodiment 31, wherein the neuroinflammatory disease is selected from the group consisting of multiple sclerosis, vasculitis, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, stroke, migraine, epilepsy and traumatic brain injuries.

[0146] Embodiment 34: A method of aiding a treatment of a brain disease in a subject suffering from a brain disease, said method comprising: a) identifying in an image of the brain disease-proximal cranial bone of the subject bone areas comprising the cranial bone-derived immune cell population of any one of embodiments 1 to 10, the cranial bone-derived effector memory CD8+ T cell of embodiment 11 or 12, or the cranial bone-derived effector memory CD4+ T cell of embodiment 13 or 14; and b) aiding the said therapy by recommending to prevent those bone areas from physical treatment; or c) aiding the said therapy by recommending to specifically target those bone areas by pharmaceutical treatment.

[0147] Embodiment 35: The method of embodiment 34, wherein said physical treatment involves surgery or irradiation and wherein said pharmaceutical treatment involved administration of a therapeutic agent as specified in embodiment 22 or 23 or is selected from the group consisting of: an interferon, preferably, iFa-2b, iFaconl, iFa-n3, iFP-la, iF|3-lb, iFy-lb, or pegIFa-2b, an interleukin, preferably, aldesleukin (proleukin) or oprelvekin (neumega), a colony-stimulating factor, preferably, filgrastin (neupogen), pegfilgrastim (neulasta), or sargramostim (leukine), glatiramer (Copaxone), elapegademase, plerixafor, trilaciclib, and PEG-ademase.

[0148] Embodiment 36: The method of embodiment 34 or 35, wherein said bone areas in step a) are identified by allocating data indicating the presence, absence or abundance of the cranial bone- derived immune cell population of any one of embodiments 1 to 10, the cranial bone-derived effector memory CD8+ T cell of embodiment 11 or 12, or the cranial bone-derived effector memory CD4+ T cell of embodiment 13 or 14 in bone areas proximal to the brain area affected by the brain disease to the image.

[0149] Embodiment 37: A therapeutic agent for use in treating and / or preventing the brain disease in a subject, wherein said therapeutic agent is capable of stimulating an anti-disease immune response against the brain disease and wherein said therapeutic agent is to be administered into the areas with cranial bone comprising the cranial bone-derived immune cell population of any one of embodiments 1 to 10.

[0150] Embodiment 38: The therapeutic agent for use of embodiment 37, wherein said therapeutic agent is a therapeutic agent as specified in embodiment 22 or 23 or is selected from the group consisting of: an interferon, preferably, iFa-2b, iFaconl, iFa-n3, iFP-la, iFP-lb, iFy-lb, or pegIFa-2b, an interleukin, preferably, aldesleukin (proleukin) or oprelvekin (neumega), a colony-stimulating factor, preferably, filgrastin (neupogen), pegfilgrastim (neulasta), or sargramostim (leukine), glatiramer (Copaxone), elapegademase, plerixafor, trilaciclib, and PEG-ademase.

[0151] Embodiment 39: The therapeutic agent for use of embodiment 37 or 38, wherein said brain disease is a brain tumor or a neuroinflammatory disease

[0152] Embodiment 40: The therapeutic agent for use of embodiment 39, wherein said brain tumor is (i) a brain tumor selected from the group consisting of: adult-type diffuse gliomas, preferably astrocytoma, oligodendroglioma or glioblastoma; pediatric-type low-grade gliomas, preferably diffuse astrocytoma, angiocentric glioma, polymorphous low-grade neuroepithelial tumor of the young or diffuse low-grade glioma; pediatric-type diffuse high-grade gliomas, preferably diffuse midline glioma, diffuse hemispheric glioma, diffuse pediatric-type high-grade glioma or infant-type hemispheric glioma; circumscribed astrocytic gliomas, preferably pilocytic atrocytoma, high-grade astrocytoma with piloid features, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, chordoid glioma or astroblastoma; glioneuronal and neuronal tumors, preferably ganglioglioma, gangliocytoma, desmoplastic infantile ganglioglioma, desmoplastic infantile astrocytoma, dysembryoplastic neuroepithelial tumour, diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters, papillary glioneuronal tumour, rosette-forming glioneuronal tumor, myxoid glioneuronal tumour, diffuse leptomeningeal glioneuronal tumour, multinodular and vacuolating neuronal tumour, dysplastic cerebellar gangliocytoma, central neurocytoma, extraventricular neurocytoma or cerebellar liponeurocytoma; ependymal tumors, preferably ependymoma, myxopapillary ependymoma or subependymoma; choroid plexus tumors, preferably choroid plexus papilloma, atypical choroid plexus papilloma or choroid plexus carcinoma; embryonal tumors, preferably medulloblastoma, atypical teratoid / rhabdoid tumor, cribriform neuroepithelial tumor, embryonal tumor with multilayered rosettes, CNS neuroblastoma, CNS tumor with BCOR-internal tandem duplication or CNS embryonal tumor; pineal tumors, preferably pineocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, papillary tumor of the pineal region or desmoplastic myxoid tumor of the pineal region; meningioma, preferably meningioma grades 1 to 3; mesenchymal, non-meningothelial tumors involving the CNS, preferably solitary fibrous tumor, cavernous hemangioma, capillary hemangioma, arteriovenous malformation, hemangioblastoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, spindle cell rhabdomyosarcoma, intracranial mesenchymal tumor, CIC-rearranged sarcoma, primary intracranial sarcoma, Ewing sarcoma, Mesenchymal chondrosarcoma, chondrosarcoma, dedifferentiate chondrosarcoma, or chordoma; melanocytic tumors, preferably meningeal melanocytosis, meningeal melanomatosis, meningeal melanocytioma or meningeal melanoma; hematolymphoid tumors involving the CNS, preferably primary diffuse large B-cell lymphoma of the CNS, lymphomatoid granulomatosis, intravascular large B-cell lymphoma, MALT lymphoma of the dura, lymphoplasmacytic lymphoma, follicular lymphoma, anaplastic large cell lymphoma, T- cell lymphoma, NK / T-cell lymphoma, Erdheim-Chester disease, Rosai-Dorfman disease, Juvenile xanthogranuloma, Langerhans cell histiocytosis or histiocytic sarcoma; germ cell tumors, preferably mature teratoma, immature teratoma, teratoma with somatic-type malignancy, germinoma, embryonal carcinoma, yolk sac tumors, choriocarcinoma or mixed germ cell tumor; tumors of the sellar region, preferably adamantinomatous craniopharyngioma, papillary craniopharyngioma, pituicytoma, granular cell tumor of the sellar region, spindle cell oncocytoma, pituitary adenoma, pituitary neuroendocrine tumor and pituitary blastoma, or (ii) is a metastasized secondary brain tumor selected from the group consisting of lung cancer, breast cancer, melanoma, colorectal cancer, kidney cancer, thyroid cancer and uterine cancer.

[0153] Embodiment 41 : The therapeutic agent for use of embodiment 39, wherein the neuroinflammatory disease is selected from the group consisting of multiple sclerosis, vasculitis, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, stroke, migraine, epilepsy and traumatic brain injuries.

[0154] All references cited throughout this specification are herewith incorporated by reference in their entirety as well as with respect to the specifically mentioned disclosure content.

[0155] FIGURES

[0156] Fig 1 : Immune cell aggregates in the cranial bone of glioblastoma patients, a-g, Clinical presentation, a, PET-computed tomography (PET-CT) visualizing clinical CXCR4 radiolabeling (bright) with 68Ga-pentixafor in a coronal plane. Arrowhead depicts focally bridging contact between tracer-enhanced glioblastoma parenchyma (gb) and locally accumulated radiolabel in the cranial bone compartment. Additional findings include demarcation of the nasopharyngeal mucosa and parts ofWaldeyer’s pharyngeal ring, b, Clinical CXCR4 PET-CT in an axial plane. Secondary fusion with magnetic resonance imaging (MRI) data exposes anatomy of brain and surrounding structures. Arrowhead in the magnified inset depicts nodular enhancement in the cranial bone ipsilateral to the gb. Note the lower unconnected radiolabeling of the dural sinus, c, 3D-reconstruction of PET-CT data from (b). Arrowhead depicts focal CXCR4 radiolabeling in the skull in juxtaposition to the intracerebral gb. Note the unconnected aspects of the neuro-immune interface of the dural sinus and bystander radiolabeling of the skin and head and neck lymphatic system, d, Schematic representation of a craniotomy, e, Photograph of a representative fresh bone specimen used for the study (scale bar: 5 mm). The magnified inset represents a drawing of the inner configuration of its spongy structure. Note, in a,b,f, repetitive patterns of CXCR4 radiolabel exposing focally connecting brain and bone in individual disease settings of glioblastoma patients (GB PAT) 1- 6. g, By contrast, ocal enhancement of CXCR4 radiolabeling was not observed in the cranial bone of non-tumor control patients (NTC patients 1-4). Upper panel: axial planes. Lower panel: 3D-reconstruction of the respective PET-CT data, h-m, Microscopic anatomy. 3D-rendering of representative light-sheet microscopy data obtained from whole-mount preparations of fixed and optically cleared clinical cranial bone (CB) fragments of non-tumor control (h, NTC PAT5) and glioblastoma patients (j, GB PAT7). Arrowheads indicate CD34+ vessels. CD45+ immune cells are rare in the NTC and abundant in the cranial bone of GB (note asterisk in j). Parallel immunofluorescence analysis of tissue sections confirms low cellular aspects (DAPI indicates cellular nuclei) of fatty, aged marrow in the control cranial bone (i; NTC PAT5) that contrasts with the extended gathering of CD45+ immune cells surrounding CD34+ or CD146+ microvessels in the diploe of glioblastoma patients. Note abundant accumulation of CXCR4 in the dense immune cell aggregates of cranial bone of glioblastoma patients (k, GB PAT8). Scale bar sizes indicated. 1, Quantification of cranial bone cavities (n=12 of two GB patients; n=20 of three NTC patients) revealed increased cellular numbers (5,336 vs. 281 DAPI+ nuclei per cavity) and frequencies of CD45+ immune cells (25.97+11,73 vs. 4.52+4,52%) in GB vs. NTC patient-derived tissue, m, Cartoon illustrating data and derivation of vital cells from the tissue for further analysis in the study. Fig. 2: Composition and tumor reactivity of cranial bone immune cells, a, Single cell analysis encompasses a total of n=82,320 vital, CD45+ immune cells that were enriched by magnetic activated cell sorting (MACS) from surgical samples of cranial bone (CB: n=8, GB and n=5, NTC) and tumor tissue (n=6), as well as from peripheral blood mononuclear cells (PBMCs; n=5, GB and n=5, NTC). b, Uniform manifold approximation and projection (UMAP) of Harmony-integrated and harmonized, respective scRNA-seq data. Clusters of cellular identities as indicated, derived by SingleR- (Aran et al., Nat Immunol, 2019) and marker-based annotation of cell types. Inset visualizes expression of the CXCR4 gene, c-e, i, Flow cytometrybased 25-Color Immunoprofiling Assay (Full Spectrum Cytometer (Cytek Aurora), cFluor® Reagent Kit (18C)). d, Bubble plot summarizing distribution of common immune cells by patient CB sample, d, Dot plots illustrate the gating strategy for characterization of CD8+ T cell phenotypes, e, Graphs present frequency (%) of T cell phenotypes in paired samples. Two- tailed paired t-test: p values indicated; ns, not significant, f-h, Enzyme-linked Immunospot (ELISpot) assay, f, Experimental strategy and representative bright field microscopic appearance of interferone gamma (IFN-y) secreting CD8+ T cells in the assay, g, Graph presents ELISpot counts split by sample source of a representative patient (GB PATH). One-way ANOVA corrected for multiple comparisons: p values indicated; ns, not significant, g, Graph summarizes mean data from ELISpot assays of n=9 glioblastoma patients. Specific antitumor counts were determined by antibody-blocking of major histocompatibility (MHC)-independent IFN-y spots. Paired data and p values indicated. Two-tailed paired t-test: ns, not significant, i, Data shows distribution of effector memory CD8+ T cell phenotypes (top) and composition of T cells (bottom, median values of n=4 patients per sources) in samples from CB vs. iliac crest- derived distal bone marrow (dBM). Two-tailed paired t-test: ns, not significant, j, Graphs display cytometric frequencies of T cells exposing cell surface expression of sphingosine 1- phosphate receptor 1 (S1PR1), separated by sample source in indicated patients: p values indicated; ns, not significant. One-way ANOVA corrected for multiple comparisons, k, Illustration indicates active lymphocyte egress kinetics in the process of adaptive immunity in the CB. 1, ELISpot assay scoring IFN-y spots from various intra-individual CD8+ T cell sources in co-culture with paired autologous, primary glioblastoma cells. Note the specific abrogation of activity in the cranial bone-derived T cells by antibodies against MHC I / II, which contrasts to the unspecific activity of T cells derived from the PBMCs. *, ****, p<0.001; ns, not significant. One-way ANOVA corrected for multiple comparisons, n=4 technical replicates.

[0157] Fig. 3: Phenotyping of immune cells, a, Representative flow cytometry dot plots demonstrating gating strategy for detection of immune cell phenotypes by the 25-Color Immunoprofiling Assay (Full Spectrum Cytometer (Cytek Aurora), cFluor® Reagent Kit (18C)). Nomenclature of T cell subsets as indicated, b, Stack plots visualize distribution of CD4+ T cell phenotypes by patient and sample source (left). Graphs present frequency (%) of T cell phenotypes in paired samples. Two-tailed paired t-test: p values indicated; ns, not significant, c, Graph presents frequency (%) of regulatory T cell phenotypes in paired samples among CD3+CD56- T cells, i.e., cells with the following immune profile: CD45+CD3+ TCRgd- CD56- CD4+CD25+CD1271ow / -. Two-tailed paired t-test: p value indicated.

[0158] Fig. 4: a, Illustration of experimental approach used to monitor T cell aggregation. Expanded CD8+ T cells were restimulated (RESTIM) and allowed to rest in intermediary phases. Graphs display quantification of clusters forming at indicated time points, n = 3 technical replicates, patient 11. b, Resilience assay. Data represent successful rounds of restimulation. Experiment conducted in triplicates per patient and source.

[0159] Fig. 5: a, Top ten differentially expressed genes (ranked by log2(FC)) comparing tumor-shared expanding clonotypes (es) versus nonexpanding singlets (ne) in the CB. DEGs detected by FindMarkers() Seurat function, per default setting (two-sided Wilcoxon rank-sum test). Gene expression cutoff set to a minimum of 20% of cells, b, Frequency of predicted tumor reactivity in individual CD8+ T cells by predicTCR, visualized as bar plots per source aligning top 50 CD8+ clonotypes by frequency, (n) patients per source: PBMC (2 patients), dBM (3 patients), CB (6 patients), tumor (6 patients), c, Frequency of predicted tumor reactivity among tumor- shared CD8+ T cell clonotypes, by source, d, Stacked bar plots visualizing CD8+ T cell phenotypes among tumor-reactive T cell clonotypes shared between CB and tumor, split per source (CB vs Tumor). Difference in active T cell effector types (NK-like CD8, activated effCD8, effCD8, effCD8 (SLAMF6+), effCD8 (Cytokine+) and terminal effCD8 T cells) and exhausted effector types (exhausted effCD8) between the two sources highlighted.

[0160] Fig. 6: a, Illustration of sites assessed for PET-CT / MRI-specific Pentixafor labeling, b, Presurgical CXCR4 PET-CT data, secondary MRI fused, showing examples with (dark gray label, left) and without (light gray label, right) radiotracer enhancement in the CB at initial diagnosis. Insets magnify selected CB areas. Arrowheads point to radiotracer enhancement, c, Kaplan-Meier survival plot of patients with glioblastoma. Censored data and P value indicated, log-rank (Mantel-Cox) test.

[0161] EXAMPLES

[0162] The invention will be illustrated by the following Examples. The Examples shall not be construed as limiting the scope of the invention. Example 1: General methods and materials

[0163] Human biosampling

[0164] Cranial bone specimens, tumor tissue, and whole blood samples were collected from patients at the Department of Neurosurgery and Spine Surgery of the University Hospital Essen. Nontumor controls presented with non-neoplastic intracranial diseases, i.e., subdural hematoma, cavernous malformation or normal-pressure hydrocephalus. Tumor patients were newly diagnosed, chemo- / radiotherapy-naive and their tissue samples were classified as IDH-wildtype glioblastoma CNS WHO grade 4 by board-certified neuropathologists. At the time of surgery, no patient suffered from diseases associated with known immune reactivity, e.g., acute infection or chronic inflammation. Bone chips of the calvaria derived during craniotomy from unplanned, intra-surgically required extensions of the burr hole or during necessary additional temporo- basal decompression after craniotomy. Tumor tissue samples were obtained using neuronavigation (Brainlab, Germany) and originated from contrast-enhancing, 5-ALA fluorescent, non-necrotic tumor areas. Tumor tissue and paired bone specimens were preserved intra-surgically in sterile Dulbecco’s Modified Eagle Medium (DMEM) / F12 (Gibco, #11320033), supplemented with antibiotics / antimycotics (2%, Gibco, #15240062). Matched venous blood was collected during surgery or within 24 h thereafter. Bone marrow aspirates from iliac crest were derived under anesthesia, immediately before brain tumor surgery using standard clinical sampling kits and heparin-buffered transport tubes for immediate processing in the lab. Clinical specimens were registered at the Westdeutsche Biobank Essen (WBE, University Hospital Essen, University of Duisburg -Essen, Germany; approval #22-WBE-137). Postoperatively, all patients received a CT scan of the neurocranium within 24 h, and tumor patients an additional MRI scan within 72 h. Written informed consent was obtained by all participants of this study; all procedures were performed in accordance with the Declaration of Helsinki and were approved by the local ethics committee (approval #19-8706-BO, #22-10564- BO).

[0165] Clinical CXCR4 radiolabeling

[0166] PET-CT imaging data were obtained from six patients with suspected glioblastoma who underwent presurgical [68Ga]Ga-CXCR4 ([68Ga]Ga-Pentixafor) radiolabeling as part of clinical care. Glioblastoma was confirmed post-hoc by neuropathological diagnosis of surgically resected tumor tissue. Preparation of the radiotracer and PET-CT imaging was performed inhouse at the Department of Nuclear Medicine of the University Hospital Essen. [68Ga]Ga- Pentixafor was administered intravenously (i.v.) with an activity of 2.4+0.1 MBq / kg and glioblastoma patients were imaged 61.8+19.3 minutes thereafter (Fig. la-c). Non tumor control patients (Conn’s syndrome, n = 4) received i.v. [68Ga]Ga-Pentixafor, during clinical workup, with an activity of 2.06+0.24 MBq / kg and were imaged 80.3+11 min thereafter, respectively (Fig. lf,g). Imaging of all patients was performed on a Siemens Vision PET-CT scanner (Siemens Healthineers, Germany). Data interpretation and CT / MRI fusion was performed and verified by board-certified nuclear medicine personnel using the Syngo. Via software (Siemens Healthineers) or Brainlab cranial navigation software.

[0167] Tumor tissue processing

[0168] Clinical samples arrived at the lab within 30 min after resection in ice-cold transport media. Tumor tissues were mechanically minced and underwent previously reported standard procedures for the derivation of primary cell cultures or to create single cells. Briefly, single cell suspensions were prepared by homogenizing the tissue in Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco, #12440053) with 0.11 DMC U / mL neutral protease (NP, Nordmark Biochemicals, Germany, #S3030112) at 37 °C for approximately 30 minutes in a shakerincubator. Tissue dissociation was supported by intermittent resuspension. Cell suspension was filtered through a 35 pm cell strainer (Falcon, #352235) and was washed twice in phosphate- buffered saline (PBS, pH 7.4, Gibco, #14190169) supplemented with 0.04% bovine serum albumin (BSA, Miltenyi Biotec, Germany, #130-091-376) for further use.

[0169] Bone sample processing

[0170] Bone chips were transferred to a 15 mL canonical tube and repeatedly flushed with pre-warmed (37 °C) 0.11 DMC U / mL NP in IMDM for 10-15 minutes to facilitate release of cells from the diploe. In a final step, bone chips were flushed with PBS supplemented with 0.04% BSA and the combined suspension was filtered through a 35 pm cell strainer. The cell suspension was centrifuged (10 minutes, 300 x g) and washed once in PBS with 0.04% BSA, for further use. If the available bone sample size exceeded the required amount for cell isolation, a fraction of the tissue was immediately flash-frozen in liquid nitrogen and stored at -80 °C for subsequent histological studies.

[0171] Whole mount staining and optical clearing of human cranial bone fragments

[0172] Clinical bone chips (Fig. ld,e) were fixed in 4% PFA (in PBS, pH = 7.4) over night at 4-8°C. Samples were transferred to 5 mL tubes filled with blocking buffer (5% DMSO, 0.1% Tween20, 1% BSA, and 5 mM EDTA in PBS) and incubated for two days at room temperature. Immunofluorescence labeling was performed with PE / Dazzle594 anti-human CD45 (1 :200, Biolegend, #304052) and AlexaFluor647 anti-human CD34 (1 :200, Biolegend, #343508) antibodies in blocking buffer for five days at room temperature. Subsequently, antibody solution was removed and samples were washed twice with 5% DMSO and 0.1% Tween20 in PBS for one day at room temperature, respectively. Optical tissue clearing was performed as described previously (Gruneboom et al., Nat Metab 2019). Briefly, samples were dehydrated in increasing ethanol concentrations of 50%, 70% and 100% at room temperature for one day, respectively. For optical clearing, samples were transferred to ethyl cinnamate (eCi, Sigma Aldrich, #112372-100G) and incubated at room temperature until complete transparency was achieved.

[0173] Light-Sheet Fluorescence Microscopy (LSFM) of human cranial bone eCi-cleared bone fragments via LSFM (Fig. lh,j) where imaged using a LaVision BioTec Ultramicroscope Blaze (Miltenyi / LaVision BioTec, Germany) with a supercontinuum white light laser (460-800 nm), 7 individual combinable excitation and emission filters covering 450 nm to 865 nm, an AndorNeo sCMOS Camera with a pixel size of 6.5x6.5 pm2, and 1.1 x (NA 0.1), 4 x (NA 0.35), 12 x (NA 0.53) objectives with a magnification changer ranging from 0.66 x to 30 x. For image acquisition, cleared samples were immersed in eCi in a quartz cuvette and excited at 500 / 20 nm for visualizing tissue autofluorescence and detected using a 535 / 30 nm band-pass emission filter. CD45-PE-Dazzle594 was excited at 560 / 40 nm and detected via a 650 / 50 nm band-pass emission filter. For CD34-AlexaFluor647, a 630 / 60 nm excitation band-pass filter and a 680 / 30 nm detection band-pass filter were used. Because the excitation optics of the microscope provide a light-sheet thickness of 4-24 pm (adjustable NA), the Z-step size was set to 5 or 10 pm depending on the selected light-sheet NA. The optical zoom factor varied from 4 x to 12 x depending on the objectives combined with a digital zoom factor of 1 x. LSFM data processing was performed using the visualization tools of the software Imaris (Bitplane), version 9.7.1.

[0174] Tissue processing for immunofluorescence confocal imaging of cranial bone

[0175] Cranial bone samples from glioblastoma and non-tumor control patients (Fig. 1 i,k) were fixed with ice-cold 4% methanol-free formaldehyde (Thermo) overnight with rotation at 4 °C. Bone samples underwent a 14-day decalcification process in 10% EDTA (pH 8, Sigma) at RT with stirring, followed by dehydration (overnight) and paraffin embedding. Paraffin blocks were cut into 10 pm sections using a pfm slide 4004M sledge microtome. Sections were dried, deparaffinized, rehydrated and antigen-retrieved according to manufacturer’s instructions (Agilent Technologies Deutschland GmbH). Prior to antibody incubation, sections were blocked and permeabilized with TBS (0.1M Tris, 0.15 M NaCl, pH 7.5) containing 0.05% Tween-20, 20% DMSO (Sigma) and 10% donkey serum (Jackson ImmunoResearch) for 15 min at RT. For immunolabelling, all antibodies and DAPI were diluted in DAKO EnVision FLEX diluent (Agilent Technologies). Primary antibodies (rat anti-CD45 (Bio-Rad), goat anti- CD146 (R&D), rabbit anti-CXCR4 (ThermoFisher Scientific) were applied overnight in a 1 :25 dilution. Secondary antibodies (Donkey anti-rabbit 488, anti-goat 555 anti-rat 594, (all Biotium)) were incubated for 5-hours (1 :25 dilution), and DAPI (Thermo Fischer Scientific) was applied prior to mounting using the Vectashield Hard Set Antifade Mounting Medium (Vector Laboratories, Inc). Labelled sections were imaged on a Leica Stellaris 8 laser scanning confocal microscope equipped with 2x HyD-S, 2x HyD-X and one HyD-R detectors and 2 laser lines (405 and white-light laser) using a 20x multiple-immersion objective (NA 0.75, FWD 0.680 mm) at 400Hz, 8-bit with 1024x1024 resolution. Immunolabelled cells were quantified using Image J (Fig 11, m).

[0176] Blood and bone marrow sample processing

[0177] Patient venous blood and iliac crest bone marrow samples arrived at room temperature in 7.5 mL EDTA vacutainers (Sarstedt, Germany, #01.1605.001) or heparin containing tubes for immediate isolation of peripheral blood mononuclear cells (PBMCs) or bone marrow mononuclear cells (BMMCs). PBMCs and BMMCs were harvested by density gradient centrifugation using Histopaque®-1077 (Sigma-Aldrich, #10771-100ML) according to the manufacturer’s protocol. Cells were washed twice in PBS supplemented with 0.04% BSA for further use.

[0178] Selection and preservation Q CD45+cells

[0179] Single cell suspensions from tumor tissue, bone and blood were enriched for vital CD45+cells, either before or after cryopreservation, by the REAlease® CD45 (TIL) MicroBead Kit (Miltenyi Biotec, #130-121-563) according to the manufacturer’s instructions (Fig. 2a). Anti- CD45 antibodies were subsequently removed from the selected cells following the manufacturer’s protocol. Cells were immediately used for further analysis or cryopreserved at -150 °C in 50% resuspension media (40% FBS in IMDM) and 50% freezing media (30% DMSO + 40% FBS in IMDM), according to lOx Genomics protocol (#CG00039).

[0180] Single cell RNA sequencing and analysis

[0181] Cell suspensions with > 85% viable cells after sequential thawing, as assessed by trypan blue exclusion, were further processed for single cell RNA sequencing (scRNA-seq). The Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (Dual Index) (lOx Genomics, #CG0000315 Rev C) was used according to the manufacturer’s instructions. Following quality control (2100 Bioanalyzer, Agilent), paired-end sequencing of pooled libraries was conducted on one flow cell lane per patient, using a NovaSeq 6000 system (Illumina). Read alignment of raw reads was performed to the hg38 human reference genome and feature-barcode matrices were obtained by the application of Cell Ranger (v.7.0.1) with default parameters. Further analyses were performed in the R environment (v4.2.0). Digital expression matrices were analyzed using the Seurat package (v.4.3.0). Cells with low counts (nFeature_RNA < 200), high counts (nFeature_RNA > 6000), and cells with high percentages of mitochondrial genes (> 15%) were removed. SCTransform was applied for normalization and doublets were identified using DoubletFinder (v.2.0.3). For subsequent analysis, all Seurat objects of the patients were merged and SCTransform was applied to regress out the mitochondrial read percentage per cell. Integration of data was achieved using Harmony (v.0.1.1), followed by cell cycle scoring. Singlets were subsetted and SCTransform was reapplied, regressing out mitochondrial read as well as cell cycle scores. Data integration was performed again using Harmony, followed by the FindNeighbors (dims = 1 :20) and FindClusters functions lesolutetion = 1.1) in the Seurat package. Data visualization was carried out by uniform manifold approximation and projection (UMAP) via RunUMAP (dims = 1 :20). After the removal of doublets, we obtained a dataset with a total of n = 82,320 cells with an average of 2,841 genes, 10,142 UMIs and 3.7% mitochondrial expression detected per cell.

[0182] Cell type annotation (Fig. 2b) of the integrated dataset was performed by combining SingleR (v.1.10.0) and marker-based cell type identification using the Find AllMarkers function in Seurat. Based on the SingleR annotation, expression of canonical markers and subsequent literature research, distinct cell types were identified (Fig. 2b). Expression of canonical marker gene sets in the annotated cell types was further confirmed and visualized by applying gene set enrichment scores (AUCell score, v.1.18.1).

[0183] Cytometric immune cell phenotyping

[0184] Comprehensive immunoprofiles of CD45+ cells (Fig 2c-e,i,j; Fig. 3a-c) were obtained using the Cytek® 25-Color Immunoprofiling Assay, cFluor® Reagent Kit (18C) (Cytek Biosciences, CA, USA), with 18 cFluor reagents offered by Cytek, supplemented with 7 antibodies from BioLegend (San Diego, CA, USA) by manufacturer instructions. Cryoconserved single cell suspensions from cranial bone, tumor, blood and iliac-crest bone marrow were sequentially thawed and washed twice prior to antibody-labelling. Immunolabeling was performed according to the manufacturer’s protocol. All samples were measured on a Cytek Aurora flow cytometer configured in the 5L setup (16UV-16V-14B-10YG-8R) and profiled using the provided system software package (Cytek).

[0185] Ex vivo expansion Q T cells

[0186] CD45+-enriched cells from the tumor tissue, blood, cranial bone, and iliac crest bone marrow were used for short-term in vitro expansion of T cells. Expansion was performed in T cell activation media, consisting of RPMI 1640 (Gibco, #72400021), supplemented with human AB serum (10%, Sigma-Aldrich, #H5667), sodium pyruvate (1 mM, Gibco, #11360039), 0- mercaptoethanol (50 pM, Gibco, #21985023), antibiotic-antimycotic (1%), recombinant IL-2 (1000 U / mL, #200-02), IL-15 (10 ng / mL, #200-15) and IL-21 (10 ng / mL, #200-21, all Peprotech), similarly to57. T cells were allowed to expand for 14-21 days in 96-well plates (Coming, #3596) in the presence of human T-activator CD3 / CD28 / CD137 Dynabeads (Gibco, #11163D) in a 1:5-10 bead:cell ratio. Prior to subsequent analyses, CD8+T cells were enriched by magnetic separation (Miltenyi Biotec, #130-096-495) and immediately used or stored at - 150 °C until further use.

[0187] ELISpot assays

[0188] The Enzyme-Linked ImmunoSpot (ELISpot) assay was used to detect cellular release of Interferon-y (R&D Systems, Minneapolis, MN, USA, #SEL285 and #EL285) (Fig. 2f-h,l). Experiments were performed using a 2:1 effector Target ratio by incubating 15,000-20,000 effector CD8+T cells and 7,500-10,000 autologous tumor cells in 96-well plates. Autologous, short-term expanded glioblastoma cells (tumor cells) were used at passages 4-12 and stimulated with IFN-y (1 pg / mL, Peprotech #300-02) for 24 h prior to the co-incubation. T cells were allowed to rest in culture media with reduced cytokine concentrations (20 U / mL IL-2, 1 ng / mL IL-15, 1 ng / mL IL-21) for at least 3 days and in cytokine-free media over night before coincubation. T cells and autologous tumor cells were co-incubated for 48 h and the respective ELISpot assays were performed according to the manufacturer’s instructions. MHCI / II blockade was achieved by pre-incubating autologous tumor cells for 1 h prior to the assay with 10 pg / mL anti HLA-DR (clone L243) and 10 pg / mL anti HLA-A,B,C (W6 / 32) antibodies (Biolegend, #307648 and #311428). Wells without tumor cells served as control for the experiments (CD8+T cells only). Wells with tumor cells only and without T cells were also similarly to control for background signals. Positive controls consisted of T cells activated by T cell activator Dynabeads. Spots for each condition were determined from 3-4 wells using Imaged software analysis.

[0189] Example 2: Accumulation of immune cells in the proximal cranial bone

[0190] To assess potential presence of hematopoietic niches within the skull bone marrow, the radiolabeling of the C-X-C motif chemokine receptor 4 (CXCR4) as a surrogate marker (Sugiyama, Kohara and Nagasawa, Immunity 2006) was analyzed in glioblastoma patients receiving presurgical [68Ga]Ga-Pentixafor tracer (Lapa et al., Theranostics 2016) during positron emission tomography (PET) (Fig. la-c). Comparing with PET data derived from nontumor controls (Fig. 1g), a pronounced labeling in the cranial bone of the glioblastoma patients was noted, where ageing bone marrow was instead expected to exhibit stronger tissue attrition and a reduced regenerative potential of aged hematopoietic cells (Pinho and Frenette, Nature Rev Mol Cell Biol, 2019). The individual patterns of radiolabeling in the bone repeatedly extended to adjacent tracer accumulations within the tumor parenchyma (Lapa et al., Theranostics, 2016; Jacobs et al., Eur J Nucl Med Mol Imaging, 2022), or were found in ipsilateral association with bridging meningeal structures (Fig. la,b). Connections from the various intracerebral tumor locations to other aspects of the neuro-immune interface, i.e., the choroid plexus or to dural sinuses were not observed (Fig. 1c).

[0191] Intrigued by this finding, surplus fragments of fresh bone chips were assessed derived from clinical craniotomies under informed consent (Fig. Id). The surgical approach of a craniotomy adjacent to the intracerebral tumor mass is indicated (i) to derive tissue for routine diagnosis and (ii) for tumor resection according to the guideline-based standards of care (Tan et al., CA Cancer J Clin 2020; Wen et al., Neuro-Oncol 2020). Whole-mount three-dimensional lightsheet fluorescence microscopy (Gruneboom et al., Nat Metabol, 2019) and standard immunohistochemical analysis of tissue sections were used to expose the cellular content of the spongy diploe within the flat bones in which marrow characteristically resides (Fig. le). Nontumor control samples displayed ageing marrow with low cellularity and adiposity (Pinho and Frenette, Nature Rev Mol Cell Biol, 2019) (Fig. lh,i). The diploe of glioblastoma patients contrasted with aggregates of immune cells. Extended patches of CD45+ cell populations were observed in the surrounding of CXCR4 expression forming solid arrangements with CD34+ and CD146+ microvessels in the cancellous bone (Fig. lj-1), indicative for bone marrow stromal cell niches (Sugiyama, Kohara and Nagasawa, Immunity 2006). These data indicated a co- morbid process where immune cells accumulate locally, in the proximal cranial bone of glioblastoma patients.

[0192] Example 3: Effector T cell enrichment and local tumor-reactivity delineate focused immunopathology

[0193] Prior landscape analyses of the glioblastoma microenvironment have not considered immune cell niches in the cranial bone, e.g., (Friebel et al., Cell 2020; Klemm et al., Cell 2020; Lu et al., Nature Commun, 2021; Yeo et al., Nature Immunol, 2022). Therefore, CD45+ immune cells were extracted by magnetic activated cell sorting (MACS) (Fig. Im) of fresh samples from glioblastoma and five non-tumor control patients including surgical bone, glioblastoma tumor tissue, and peripheral blood mononuclear cells (PBMCs) to obtain their cell RNA sequencing (scRNA-seq) profiles (Fig. 2a, b). Additionally, the immune profiles of vital CD45+ cells were obtained by flow cytometry and decided to further specify the most abundant population of T cells in the samples (Fig 2c and Fig. 3). Naive T cell accumulation has previously been described in the bone marrow of newly diagnosed intracranial tumor subjects as a mode of T cell dysfunction (Chongsathidkiet et al., Nature Med 2018). Notably, this (and similar) studies did not investigate T cells from the regional / proximal cranial bone, but rather relied on distal bone marrow aspirates derived from the patient’s iliac crest, or from femurs and tibias of animal models of disease. Considering that bone marrow can be a priming site for T cell responses (Feuerer et al., Nature Medicine 2003) and speculating on a distinct spatial relationship of cranial T cells with the adjacent encephalic tumor mass, it was asked if the proximal cranial bone of glioblastoma patients contained tumor-reactive cells. Cranial bone cytometry-based immunoprofiling revealed, in comparison to matched PBMCs, decreasing fractions of naive and regulatory T cell lineages, while effector memory CD4+ and CD8+ T cell phenotypes increased (Fig. 2d,e; Fig. 3b, c). This suggested a functional response of cranial bone immune cells to the tumor growth. To validate the concept, freshly isolated CD8+ effector T cells were expanded for scoring of tumor reactive interferon-gamma (IFN-g) secretion by enzyme-linked immunospot (ELISpot) assay upon physical contact with autologous glioblastoma cells. A specific response of the T cells from cranial bone was observed that could be abrogated by antibodies against major histocompatibility complex (MHC) molecules, indicating adaptive recognition of tumor antigens. The extent of the response was significantly higher than that of the patient’s PBMC-derived T cells, even more pronounced than that of tumor-derived T cells in the assay (Fig. 2f,g). This indicated an increased presence of tumor-reactive CD8+ T cells in the proximal cranial bone of glioblastoma patients compared to matched intra-tumoral or peripheral blood CD8+ T cells (Fig. 2h). To further correlate these findings, quadruplicate biosamples were collected from glioblastoma patients involving the CB, PBMC, tumor tissue, and iliac crest-derived distal bone marrow (dBM). No differences were found in the fractions of effector memory cells between CB and dBM (Fig. 2i), but significantly more T cells from the cranial bone expressed sphingosine 1-phosphate receptor 1 (S1PR1) on the cell surface (Fig. 2j), suggesting active lymphocyte egress kinetics in the process of adaptive immunity in the CB (Fig. 2k) (Spiegel and Milstien, Nat Rev Immunol, 2011). ELISpot assays on tumor-reactive IFN-g secretion, furthermore, revealed strong and specific responses in CD8+ T cells of the CB that were not mirrored by paired CD8+ T cells of the dBM (Fig. 21).

[0194] The following immune cells were found in disease proximal cranial bone of glioblastoma patients: Effector memory CD8+ T cells expressing CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA-, and CCR7-, and compared to effector memory CD8+ T cells from blood or iliac crest increased expression of the biomarker S1PR1, naive CD8+ T cells expressing CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA+, and CCR7+, effector memory CD4+ T cells expressing CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA-, and CCR7-, and compared to effector memory CD8+ T cells from blood or iliac crest increased expression of the biomarker S1PR1, naive CD4+ T cells expressing CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA+, and CCR7+, and regulatory T cells CD45+, CD3+, TCRgd-, CD56-, CD4+, CD25+, and CD1271ow / -. Compared to blood and iliac crest, the effector memory CD8+ and CD4+ T cells were increased whereas the naive CD8+ and CD4 T cells were less abundant. The regulatory T cells were also reduced (Fig. 3). In summary, the local enrichment of tumor-reactive effector T cells in the proximal cranial bone of glioblastoma patients suggests vivid and focused immunopathology that is not mirrored by systemic components of the immune system.

[0195] Example 4: Monitoring of T cell aggregation

[0196] T cells were expanded from CD45+ enriched cells in T cell activation media (RPMI 1640 (Gibco, 72400021), human AB serum (10%; Sigma-Aldrich, H5667), sodium pyruvate (1 mM; Gibco, 11360039), P-mercaptoethanol (50 pM; Gibco, 21985023), antibiotic-antimycotic (1%), recombinant IL-2 (1000 U ml- 1, 200-02), IL- 15 (10 ng ml-l, 200-15) and IL-21 (10 ng ml-l, 200-21, all Peprotech). T cells were expanded for 14-21 days in 96-well plates (Coming, 3596) with human T-activator CD3 / CD28 / CD137 Dynabeads (Gibco, 11163D) in a 1 :5-10 bead-to-cell ratio.

[0197] T cell activation (a) was monitored by T cell clustering / aggregation during restimulation. Expanded T cells rested in reduced cytokine conditions for at least 72 h. Resilience assay (b) evaluated CD8+T cell fitness by counting successful restimulation cycles. Restimulation cycles involved seeding 10,000 T cells in 96-well plates in activation media and CD3 / CD28 / CD137 Dynabeads (1 :2, bead-to-cell ratio). Three 14-day-restimulation cycles, followed by 7 days of rest were performed in triplicates per patient and source. A restimulation cycle was successful if the mean cell count (across all three wells) exceeded the input of 10,000 cells per well.

[0198] It has been shown that cranial bone-derived CD8+T cells are more resilient since they respond more durably to stimulation and expand more reliably ex vivo than CD8+T cells from peripheral blood (PBMCs) or from the tumor tissue (Fig. 4).

[0199] Example 5: Mapping of tumor-reactive phenotypes scRNA-seq data were obtained using the Chromium Next GEM Single Cell 5' Reagent Kit v2 (lOx Genomics). For integrating scVDJ information, TCRA / TCRB nucleotide sequences were assigned to T cells using Cell Ranger’s filtered contig annotation data and combineExpression function of scRepertoire (v.1.11.0). Differentially expressed genes (DEGs) were detected from tumor-shared clones ( > 2 cells) versus nonexpanding singlets in the CB niche using Seurat’s FindMarkers() function with min.pct=0.2 (20% of cells). The top 10 DEGs (ranked by log2(FC)) were visualized using Seurat’s VlnPlot. Probability of reactivity was predicted by predicTCR model under xgboost (vl.7.4), averaged for each clonotype, and threshold was determined using Fisher-Jenks natural break optimization. Clones with reactivity scores above threshold were designated as reactive and vice versa. Reactivity was mapped onto the annotated scRNA-seq data and the relative composition of the shared tumor-reactive phenotypes visualized as stacked barplot.

[0200] Expanding T cells in the proximal cranial bone displays a CD8+T cell-typic gene expression profile. Cranial bone-derived CD8+T cells are enriched for active effector types that are tumor- reactive. They share clonotypes with CD8+T cells in the tumor, where they possess fewer active effector phenotypes and more exhausted phenotypes (Fig. 5).

[0201] Example 6: PET-CT imaging

[0202] PET-CT imaging data (University Hospital Wuerzburg) complemented data were derived from presurgical 68Ga-labeled CXCR4 (Pentixafor) radiolabeling of patients with glioblastoma as part of clinical care at the University Hospital Essen. Intravenous (i.v.) administration of Pentixafor in Wurzburg and Essen used activities of 1.94 ± 0.41 MBq kg1and 2.38 ± 0.39 MBq kg1, respectively, followed by imaging 72 ± 14 min and 65 ± 19 min thereafter (mean± s.d.). Integrated data (n = 19 histologically confirmed glioblastoma) underwent blinded consensus read by board-certified nuclear radiologists from both centers, using equal range settings. Cranial and calvarial enhancement was defined as focal uptake in the tumor-adjacent CB and absence of uptake in the contralateral reference point. Bridging tracer enhancement was classified as clearly distinguishable tracer transition between tumor and CB exceeding brain background uptake. Tracer uptake in the skin or in the venous sinuses was not assessed. Vision PET-CT scanner and CT-MRI fusion was conducted by board- certified nuclear medicine personnel using syngo.via (Siemens Healthineers) or Brainlab’s cranial navigation software (iPlanNet). Statistical analysis of survival data was executed in SPSS (v.29.0.2.0).

[0203] It has been shown that using Pentixafor® (a CXCR4-PET ligand) as a clinical surrogate marker for immune cell enrichment, (calvarial) enhancement in the proximal cranial bone indicates better patient survival (Fig. 6). CITED LITERATURE

[0204] J. Rustenhoven et al., “Functional characterization of the dural sinuses as a neuroimmune interface”. Cell 184, 1000-1016 el 027 (2021);

[0205] K. Mollgard et al., “A mesothelium divides the subarachnoid space into functional compartments”. Science 379, 84-88 (2023);

[0206] G. Castellani, T. Croese, J. M. Peralta Ramos, M. Schwartz, “Transforming the understanding of brain immunity”. Science 380, eabo7649 (2023);

[0207] A. Louveau et al., “Structural and functional features of central nervous system lymphatic vessels”. Nature 523, 337-341 (2015);

[0208] F. Herisson et al., “Direct vascular channels connect skull bone marrow and the brain surface enabling myeloid cell migration”. Nat Neurosci 21, 1209-1217 (2018);

[0209] J. A. Mazzitelli et al., “Cerebrospinal fluid regulates skull bone marrow niches via direct access through dural channels“. Nat Neurosci 25, 555-560 (2022);

[0210] J.H. Sampson et al., “Brain immunology and immunotherapy in brain tumours”. Nat Rev Cancer 20, 12-25 (2020);

[0211] P. Chongsathidkiet et al., “Sequestration of T cells in bone marrow in the setting of glioblastoma and other intracranial tumors”. Nat Med 24, 1459-1468 (2018);

[0212] D. Aran et al. “Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage”. Nat Immunol 20, 163-172 (2019);

[0213] T. Sugiyama et al. “Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches”. Immunity 25, 977-988 (2006);

[0214] C. Lapa, et al. “(68)Ga-Pentixafor-PET / CT for Imaging of Chemokine Receptor 4 Expression in Glioblastoma”. Theranostics 6, 428-434 (2016);

[0215] S. Pinho and P.S. Frenette, “Haematopoietic stem cell activity and interactions with the niche”. Nat Rev Mol Cell Biol 20, 303-320 (2019); S.M. Jacobs et al. “CXCR4 expression in glioblastoma tissue and the potential for PET imaging and treatment with [(68)Ga]Ga-Pentixafor / [(177)Lu]Lu-Pentixather”. Eur J Nucl Med Mol Imaging 49, 481-491 (2022);

[0216] A.C. Tan et al. “Management of glioblastoma: State of the art and future directions”. CA Cancer J Clin 70, 299-312 (2020);

[0217] P.Y. Wen et al. “Glioblastoma in adults: a Society for Neuro-Oncology (SNO) and European Society of Neuro-Oncology (EANO) consensus review on current management and future directions”. Neuro Oncol 22, 1073-1113 (2020);

[0218] A. Gruneboom et al. “A network of trans-cortical capillaries as mainstay for blood circulation in long bones”. Nat Metab 1, 236-250 (2019);

[0219] E. Friebel et al. “Single-Cell Mapping of Human Brain Cancer Reveals Tumor-Specific Instruction of Tissue-Invading Leukocytes”. Cell 181, 1626-1642 el620 (2020);

[0220] F. Klemm et al. “Interrogation of the Microenvironmental Landscape in Brain Tumors Reveals Disease-Specific Alterations of Immune Cells”. Cell 181, 1643-1660 el617 (2020);

[0221] I.N. Lu et al. “Tumor-associated hematopoietic stem and progenitor cells positively linked to glioblastoma progression”. Nat Commun 12, 3895 (2021);

[0222] A.T. Yeo et al. “Single-cell RNA sequencing reveals evolution of immune landscape during glioblastoma progression”. Nat Immunol 23, 971-984 (2022);

[0223] M. Feuerer, et al. “Bone marrow as a priming site for T-cell responses to blood-borne antigen”. Nat Med 9, 1151-1157 (2003);

[0224] S. Spiegel and S. Milstien, “The outs and the ins of sphingosine- 1 -phosphate in immunity”. Nat Rev Immunol 11, 403-415 (2011).

Claims

Claims1. A cranial bone-derived immune cell population comprising: a) effector memory CD8+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow; b) naive CD8+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA+, and CCR7+; c) effector memory CD4+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow; d) naive CD4+ T cells expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA+, and CCR7+; and e) regulatory T cells CD45+, CD3+, TCRgd-, CD56-, CD4+, CD25+, and CD1271ow / -; wherein compared to blood and non-cranial bone marrow the effector memory CD8+ T cells according to a) are increased whereas the naive CD8+ T cells according to b) are reduced; wherein compared to blood and non-cranial bone marrow the effector memory CD4+ T cells according to c) are increased whereas the naive CD4+ T cells according to d) are reduced; and wherein the regulatory T cells according to e) are reduced.

2. The cranial bone-derived immune cell population of claim 1, wherein said immune cell population is derived from a cranial bone sample from an area in proximity to brain tissue affected by a brain disease.

3. A cranial bone-derived effector memory CD8+ T cell expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD8+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow.

4. A cranial bone-derived effector memory CD4+ T cell expressing the following biomarkers CD45+, CD3+, TCRgd-, CD56-, CD4+, CD45RA-, and CCR7-, wherein said T cells are, preferably, further characterized by an increased expression of the biomarker S1PR1 compared to effector memory T cells having the aforementioned biomarker profile from blood or non-cranial bone marrow.

5. A method for providing an immune cell population of any one of claims 1 or 2 or any subpopulation thereof, a cranial bone-derived effector memory CD8+ T cell of claim 3, or a cranial bone-derived effector memory CD4+ T cell of claim 4 comprising the step of isolating CD45+ cells from a cranial bone sample from an area in proximity to brain tissue affected by a brain disease.

6. A cranial bone-derived immune cell population of claim 1 or 2, a cranial bone-derived effector memory CD8+ T cell of claim 3, or a cranial bone-derived effector memory CD4+ T cell of claim 4 for use in treating and / or preventing a brain disease in a subject suffering therefrom.

7. The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of claim 6, wherein said cell population or cell is to be used with an additional therapeutic agent.

8. A cranial bone-derived immune cell population of claim 1 or 2, a cranial bone-derived effector memory CD8+ T cell of claim 3, or a cranial bone-derived effector memory CD4+ T cell of claim 4 for use in aiding therapy involving physical treatment of a brain disease in a subject suffering from said brain disease.

9. A cranial bone-derived immune cell population of claim 1 or 2, a cranial bone-derived effector memory CD8+ T cell of claim 3, or a cranial bone-derived effector memory CD4+ T cell of claim 4 for use in assessing a brain disease in a subject.

10. The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of claim 9, wherein said assessing comprises diagnosing, monitoring and or predicting the said brain disease.

11. A method of aiding a treatment of a brain disease in a subject suffering from a brain disease, said method comprising:a) identifying in an image of the brain disease-proximal cranial bone of the subject bone areas comprising the cranial bone-derived immune cell population of claim 1 or 2, a cranial bone-derived effector memory CD8+ T cell of claim 3, or a cranial bone-derived effector memory CD4+ T cell of claim 4; and b) aiding the said therapy by recommending to prevent those bone areas from physical treatment; or c) aiding the said therapy by recommending to specifically target those bone areas by pharmaceutical treatment.

12. A therapeutic agent for use in treating and / or preventing the brain disease in a subject, wherein said therapeutic agent is capable of stimulating an anti-disease immune response against the brain disease and wherein said therapeutic agent is to be administered into the areas with cranial bone comprising the cranial bone-derived immune cell population of claim 1 or 2.

13. The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell of any one of claims 1 to 10, the method of claim 11 or the therapeutic agent for use of claim 12, wherein said brain disease is a brain tumor or a neuroinflammatory disease14. The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell, the method or the therapeutic agent for use of claim 13, wherein said brain tumor is(i) a brain tumor selected from the group consisting of adult-type diffuse gliomas, preferably astrocytoma, oligodendroglioma or glioblastoma; pediatric-type low-grade gliomas, preferably diffuse astrocytoma, angiocentric glioma, polymorphous low-grade neuroepithelial tumor of the young or diffuse low-grade glioma; pediatric-type diffuse high-grade gliomas, preferably diffuse midline glioma, diffuse hemispheric glioma, diffuse pediatric-type high-grade glioma or infant-type hemispheric glioma; circumscribed astrocytic gliomas, preferably pilocytic atrocytoma, high-grade astrocytoma with piloid features, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, chordoid glioma or astroblastoma; glioneuronal and neuronal tumors, preferably ganglioglioma, gangliocytoma, desmoplastic infantile ganglioglioma, desmoplastic infantile astrocytoma, dysembryoplastic neuroepithelial tumour, diffuse glioneuronal tumour with oligodendroglioma-like features and nuclear clusters, papillary glioneuronal tumour, rosette-forming glioneuronal tumor, myxoid glioneuronal tumour, diffuse leptomeningeal glioneuronal tumour, multinodular and vacuolating neuronal tumour, dysplastic cerebellar gangliocytoma, centralneurocytoma, extraventricular neurocytoma or cerebellar liponeurocytoma; ependymal tumors, preferably ependymoma, myxopapillary ependymoma or subependymoma; choroid plexus tumors, preferably choroid plexus papilloma, atypical choroid plexus papilloma or choroid plexus carcinoma; embryonal tumors, preferably medulloblastoma, atypical teratoid / rhabdoid tumor, cribriform neuroepithelial tumor, embryonal tumor with multilayered rosettes, CNS neuroblastoma, CNS tumor with BCOR-internal tandem duplication or CNS embryonal tumor; pineal tumors, preferably pineocytoma, pineal parenchymal tumor of intermediate differentiation, pineoblastoma, papillary tumor of the pineal region or desmoplastic myxoid tumor of the pineal region; meningioma, preferably meningioma grades 1 to 3; mesenchymal, non-meningothelial tumors involving the CNS, preferably solitary fibrous tumor, cavernous hemangioma, capillary hemangioma, arteriovenous malformation, hemangioblastoma, embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, rhabdomyosarcoma, spindle cell rhabdomyosarcoma, intracranial mesenchymal tumor, CIC-rearranged sarcoma, primary intracranial sarcoma, Ewing sarcoma, Mesenchymal chondrosarcoma, chondrosarcoma, dedifferentiate chondrosarcoma, or chordoma; melanocytic tumors, preferably meningeal melanocytosis, meningeal melanomatosis, meningeal melanocytioma or meningeal melanoma; hematolymphoid tumors involving the CNS, preferably primary diffuse large B-cell lymphoma of the CNS, lymphomatoid granulomatosis, intravascular large B-cell lymphoma, MALT lymphoma of the dura, lymphoplasmacytic lymphoma, follicular lymphoma, anaplastic large cell lymphoma, T-cell lymphoma, NK / T-cell lymphoma, Erdheim-Chester disease, Rosai-Dorfman disease, Juvenile xanthogranuloma, Langerhans cell histiocytosis or histiocytic sarcoma; germ cell tumors, preferably mature teratoma, immature teratoma, teratoma with somatic-type malignancy, germinoma, embryonal carcinoma, yolk sac tumors, choriocarcinoma or mixed germ cell tumor; tumors of the sellar region, preferably adamantinomatous craniopharyngioma, papillary craniopharyngioma, pituicytoma, granular cell tumor of the sellar region, spindle cell oncocytoma, pituitary adenoma, pituitary neuroendocrine tumor and pituitary blastoma, or(ii) is a metastasized secondary brain tumor selected from the group consisting of: lung cancer, breast cancer, melanoma, colorectal cancer, kidney cancer, thyroid cancer and uterine cancer.

15. The cranial bone-derived immune cell population, the cranial bone-derived effector memory CD8+ T cell or the cranial bone-derived effector memory CD4+ T cell, the method or the therapeutic agent for use of claim 13, wherein the neuroinflammatory disease is selected from the group consisting of multiple sclerosis, vasculitis,5 Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, stroke, migraine, epilepsy and traumatic brain injuries.

Images