T Cells in the Brain: Using Immunosequencing to Understand Their Role in Neurological Disorders

An image of T cells to depict their role in neurological disease.
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    The brain is considered an immune-privileged region due to the exclusion of the peripheral immune system by the blood–brain barrier. (1,2) Normal immune functions are performed by microglia—the tissue-resident macrophages of the brain—and adaptive immune cells, such as T cells and B cells, are normally restricted to the meninges surrounding the brain.

    Despite this segregation between brain-resident and adaptive immune cells, there is a growing emphasis on understanding the role of the adaptive immune system in normal brain function. (1) In addition, any breakdown of the blood–brain barrier can allow adaptive immune cells into the brain, resulting in neuroinflammation and autoimmunity.

    T cells and B cells are critical effectors of the adaptive immune system that recognize antigens through their respective T-cell or B-cell receptors (TCRs and BCRs). Their normal function is to detect and mount an immune response to neutralize pathogens; however, they can sometimes incorrectly recognize and react to self-antigens, resulting in the immune system attacking the self (autoimmunity). It has become more apparent that the presence of these cells in the brain is a feature of several neurological diseases. (3)

    Understanding if and how T and B cells in the brain relate to neurological diseases is key to more fully understanding the pathology and potential diagnosis and treatment of these diseases and disorders. The immunoSEQ® Assay is a powerful, yet flexible and cost-effective, end-to-end large-scale immunosequencing technology that has been used by researchers studying Parkinson’s disease, Alzheimer’s disease, major depressive disorder (MDD), brain tumors, and more to help understand the potential role of both T and B cells in these disorders and diseases.

    What is immunosequencing?

    Before we dive into how immunosequencing advances neurology research, it’s helpful to understand what immunosequencing is and how the immunoSEQ Assay works.

    In brief, immunosequencing is a next-generation sequencing-based technology allowing characterization of TCRs and BCRs at high throughput. With up to 1015 possible unique TCRs and BCRs in a single repertoire, this allows unprecedented insight into the massive complexity of the adaptive immune system. (4) The estimated theoretical diversity of the TCR repertoire is greater than 1015 distinct αβ receptors or clonotypes, which is many more sequence combinations than the number of T cells in an individual. (4)

    The immunoSEQ Assay is capable of capturing the enormous depth and breadth of the adaptive immune system and eliminates the problem of amplification bias commonly associated with multiplex PCR. The immunoSEQ Assay sequences the highly variable complementarity determining region 3 (CDR3) of TCRs and BCRs. Because this region is so variable, it acts as a unique barcode for each cell, allowing you to identify and track specific T- and B-cell populations over time and between samples (Figure 1).

    Figure 1. Recombination of the V, (D), and J genes of TCRs and BCRs creates a unique barcode for each cell, allowing the immunoSEQ Assay to profile the adaptive immune system.

    For more details on how this technology works, view our webinar on Exploring Repertoire Development and Dynamics. You can find more information on various techniques to profile the immune repertoire in our previous article: Immunosequencing Methods: Which is Right for You?

    Studying the adaptive immune system in the brain with the immunoSEQ Assay

    Here, we explore some examples of how the immunoSEQ Assay has been used to better understand the role of T and B cells in the brain in neurodegenerative disorders, mood disorders, and cancer.

    Neurodegenerative disorders

    The adaptive immune system has been shown to be important in several neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis. (5)

    Neuroinflammation and autoimmunity mediated by T and B cells may contribute to the irreversible neuronal loss for which these diseases are known. (6) There are several ways in which lymphocytes can contribute to neurotoxicity:

    1. Activation of cytotoxic CD8+ T cells by the presentation of disease-related self-antigens by neurons, resulting in autoimmunity;
    2. Activation of CD4+ T cells via the presentation of self-antigens by antigen-presenting cells, including dendritic cells, macrophages, and B cells, resulting in neuroinflammation;
    3. Activation of B cells by CD4+ T cells, leading to an autoantibody response and neuronal death (Figure 2). (6–8)

    Figure 2. Potential roles of adaptive immune cells in neuron damage and death. Adapted from Tan et al. (6) (CC BY 4.0)

    The immunoSEQ Assay allows you to identify and track disease-specific T- or B-cell clones to determine if they play a role in disease, (9) compare TCR or BCR repertoires between disease and non-disease states to identify disease-specific T- or B-cell markers, (10) and track T- and B-cell populations longitudinally to understand how diseases progress over time. (11,12)

    Here (Table 1), we’ve highlighted how the immunoSEQ Assay has been implemented to help further understand the role of T and B cells in neurodegenerative disorders.

    Table 1: T-cell metrics assessed via large-scale immunosequencing in neurodegenerative diseases

    T-cell metricSample typeDetails of T-cell metric analyzedReference
    Receptor diversityCultured PBMCs and ex vivo CD4+ T cellsAssessed the diversity of ⍺-synuclein-specific TCRs and compared with diversity of pathogen-related TCRsSinghania et al. (9)
    Identify and track disease-specific clonesCultured PBMCs and ex vivo CD4+ T cellsIdentified and characterized ⍺-synuclein-specific T cellsSinghania et al. (9)
    Identify and track disease-specific clonesCSF and blood mononuclear cellsAnalyzed persistence of B-lineage cells producing oligoclonal IgG found in the CSF over 18 monthsTomescu-Baciu et al. (11)
    Repertoire properties and overlap between samplesBrain lesions and sorted peripheral blood cellsAssessed the overlap in proliferating T cells vs. non-proliferating T cells in brain lesionsJelcic et al. (13)
    Repertoire properties and overlap between samplesPBMCsAssessed whether there was expansion of JCV-specific T or B cells at the time of PML identificationBertoli et al. (14)
    Repertoire properties and overlap between samplesSorted CD4+ and CD8+ cellsT-cell repertoire dynamics assessed throughout pregnancyRamien et al. (12)
    CSF, cerebrospinal fluid; IgG, immunoglobulin G; JCV, JC virus; PBMC, peripheral blood mononuclear cell; PML, progressive multifocal leukoencephalopathy.

    Here, we look at one of these studies in more detail.

    Analyzing the role of anti-⍺-synuclein T cells in Parkinson’s disease

    Immunosequencing can identify potential disease-related T-cell signatures by identifying and tracking specific T-cell clones, including clones associated with a specific disease-related antigen. Such a disease signature may be used in clinical research trials to determine if the T cells correlate with important disease characteristics, such as disease progression or treatment response.

    For example, anti-⍺-synuclein (⍺-syn) T cells have been identified in patients with Parkinson’s disease. (6) A recent study used the immunoSEQ TCRβ Assay to characterize ⍺-syn-specific T cells in patients with Parkinson’s disease for the first time. (9) Among six patients, the results showed a surprisingly diverse TCR repertoire; no shared TCRs were identified between the patients (Figure 3). The authors suggest that the lack of shared clones is likely because of human leukocyte antigen (HLA) differences, as participants were not matched for HLA and displayed unique HLA types.

    This study provides the first characterization of ⍺-syn-peptide-specific TCR clonotypes in individuals with Parkinson’s disease. Future studies using HLA-matched subjects could identify shared clones that can be used to track changes during disease progression.

    Figure 3. Overlap heatmap of ⍺-syn-peptide-specific TCRs between patients with Parkinson’s disease, with darker blue representing a higher number of overlapping clones, and white representing no overlap. Adapted from Singhania et al. (9)

    Mood disorders

    Dysfunctions in the adaptive immune system have been implicated in several psychiatric and mood disorders, including major depressive disorder (MDD), attention deficit hyperactivity disorder (ADHD), and autism spectrum disorder (ASD). (13–17) For example, immunosuppressive regulatory T cells have been found to be dysfunctional in subjects with MDD, (16) and some reports have shown a reduced percentage of CD4+ T cells in people with ASD. (17)

    Determining the role of T cells in major depressive disorder

    To determine the role of T cells in MDD, the TCR repertoires of five anti-depressant-free MDD subjects and five depression-free controls were analyzed using the immunoSEQ Assay. (18) The results revealed a less diverse CD4+ T-cell repertoire in subjects with MDD. In particular, T cells from subjects with MDD showed higher use of some TCR Vβ families (Figure 4A). The frequencies of different Vβ5.1-expressing CD4+ T-cell clones in each subject were determined. In this study, results from the immunoSEQ Assay were consistent with those of flow-cytometry-based analysis of a larger, 40-person cohort. An example from one subject is shown in Figure 4B. Of the top five Vβ5.1-expressing CD4+ T-cell clones, no clones were shared between subjects. (18)

    Figure 4. (A) Sequencing of the TCRβ repertoire showed how Vβ usage within CD4+ T cells differs between patients with MDD and matched controls. (B) Example of the frequencies of the top five Vβ5.1-expressing CD4+ T-cell clones in one patient with MDD. CTR, control. Adapted from Patas et al. (18) (CC BY 4.0)

    Cancer

    Immunosequencing has been instrumental in characterizing the immune infiltrate in patients with brain metastases, a deadly progressive outcome of many solid tumors. If treatment is attempted, it usually involves surgery and/or radiation, both of which are fraught with complications. Immunotherapy, which has played a major role in improving survival for patients with many cancer types, has also shown activity in patients with brain metastases. Understanding how tumor-infiltrating T and B cells contribute to tumor growth and survival could pave the way for improved therapies.

    Monitoring the T-cell repertoire in glioblastoma patients undergoing immunotherapy as a predictor of clinical outcome

    Immunosequencing can also be used to quantitatively assess the overlap of TCR sequences between tissues and longitudinally to study disease progression and response to treatment.

    Hsu et al. (19) used the immunoSEQ Assay to monitor the T-cell repertoire in brain tumors of 15 glioblastoma subjects. They compared the tumor T-cell repertoire with that in the peripheral blood before and after treatment with autologous tumor lysate-pulsed dendritic cell (DC) immunotherapy, while also following tumor progression. The results showed that the level of unique TCR β-chain sequence overlap found between the peripheral blood and the tumor prior to treatment correlated with survival outcomes (Figure 5).

    Figure 5. Monitoring the T-cell repertoire of tumors and peripheral blood in glioblastoma subjects treated with autologous tumor lysate-pulsed DC vaccination. (A) Overlap of TCR between the initial tumor and PBMC at pre-treatment (pre-T), post-treatment (post-T), and relapse shows a correlation with survival. (B) Assessment of the TCR overlap between the recurrent tumor and PBMC at pre-treatment (pre-T), post-treatment (post-T), and relapse also shows a correlation with survival. Adapted from Hsu et al. (19)

    The study highlights the potential to leverage immunosequencing to identify markers of clinical outcomes in cancer patients undergoing immunotherapy, which can be useful for selecting patients for clinical studies.

    Immune-related adverse events in brain cancer

    Another important aspect of immunotherapy in cancer is the emergence of immune-related adverse events (irAEs), some of which can be severe or even fatal. One rare yet often deadly adverse event is encephalitis, i.e. inflammation of the brain characterized by the infiltration of T cells.

    The immunoSEQ Assay was used to understand the role of T cells in a subject with metastatic melanoma who experienced fatal encephalitis following anti-programmed cell death protein 1 (PD-1) treatment. An analysis of inflamed and non-inflamed brain tissue revealed that inflamed tissue had an extremely high T-cell fraction. Approximately 70% of the tissue was made up of infiltrating T cells.

    Interestingly, one TCRβ (CASSFPSGSYEQYF) comprised almost one-fifth (19.6%) of all infiltrating T cells in the inflamed tissue and displayed high sequence homology with an Epstein–Barr virus (EBV)-associated clone (Figure 6, sequence in orange). These results suggest that checkpoint blockade treatment triggered the EBV-associated clone to expand in brain tissue, leading to fatal encephalitis. These results shed light on the mechanism of irAEs and may ultimately identify immune-related markers of risk for irAEs (Figure 6). (20)

    Figure 6. TCR tracking over time and across tissues of EBV-specific clonotypes and non-EBV-specific clonotypes. Adapted from Johnson et al. (20)

    Potential role of immunosequencing in identifying biomarkers and treatment targets for neurological disorders

    It is generally agreed that early detection and treatment holds the most promise to change the disease course for neurological disorders. T-cell signatures have the potential to act as markers of disease and allow early identification. For example, in one subject with Parkinson’s disease, ⍺-synuclein-specific T cells were identified more than a decade before diagnosis. (21) T cells reactive against ⍺-synuclein have been identified previously in subjects with Parkinson’s disease, and it is thought that this could be a marker for the disease, which is characterized in part by the presence of intraneuronal aggregates, known as Lewy bodies, composed of ⍺-synuclein. (22)

    More research is needed to determine if a T-cell signature can act as a biomarker of early Parkinson’s disease. However, T-cell signatures have been successful in distinguishing between individuals exposed and unexposed to specific pathogens, including cytomegalovirus (23) and SARS-CoV-2. (24)

    Understanding the impact of COVID-19 on the brain

    While COVID-19 is predominantly a respiratory disease, exposure to SARS-CoV-2 has been shown to have neurological symptoms, including anosmia, encephalopathy, and ischemic stroke. (25,26) In addition, 18% of COVID-19 patients developed mental health issues including depression, anxiety, or dementia within 3 months of diagnosis. (27) Studies into SARS-CoV-2 show it affects pathways relevant in neurodegenerative diseases such as Parkinson’s disease, leading to speculation that infection may play a role in the development of neurodegenerative diseases. (28)

    In order to study the effects of SARS-CoV-2 infection on the brain, including the development of neurodegenerative diseases, it is critical for researchers to determine past exposure to SARS-CoV-2. In fact, with the immunoSEQ® T-MAP™ COVID offering, researchers can determine prior exposure to SARS-CoV-2, using the same underlying technology that powers the first and only T-cell-based clinical test for COVID-19 to have received emergency use authorization.

    Studying the adaptive immune system in the brain with immunosequencing

    Using immunosequencing to characterize the T- and B-cell repertoires has allowed greater insight into the role of the adaptive immune system across various brain diseases and disorders. Such studies can not only unravel the role of the adaptive immune system in disease pathology but also accelerate research in immune therapies and biomarkers of disease, progression, and treatment response.

    The immunoSEQ Assay is ideal for researchers who are looking to explore the immune system in neurological disorders, as collecting and analyzing immunoSEQ Data requires no special equipment or expertise in immunology. We can walk you through every step of the process, from experimental design to getting you publication-ready data.

    The immunoSEQ Analyzer offers a user-friendly suite of analysis tools that can perform complex calculations and comparisons between samples and generate publication-ready figures. In addition, our open-access immuneACCESS® database contains immunoSEQ Data from >13,000 human and >1000 mouse samples, allowing you to compare your immunoSEQ Data with that of others. The database also includes several healthy control datasets, allowing you to compare disease states with healthy controls.

    We’re here to help you accelerate your research; if you’d like more information, you can visit our immunoSEQ Assay products page. If you have questions about how to get started, please get in touch with our product team.

    For Research Use Only. Not for use in diagnostic procedures.

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