Intrathecal activation of CD8 + memory T cells in IgG4‐related disease of the brain parenchyma
2021; Springer Nature; Volume: 13; Issue: 8 Linguagem: Inglês
10.15252/emmm.202113953
ISSN1757-4684
AutoresMirco Friedrich, Niklas Kehl, Niko Engelke, Josephine Kraus, Katharina A.M. Lindner, Philipp Münch, Iris Mildenberger, Christoph Groden, Achim Gass, Nima Etminan, Marc Fatar, Andreas von Deimling, David Reuß, Michael Platten, Lukas Bunse,
Tópico(s)T-cell and B-cell Immunology
ResumoReport13 July 2021Open Access Source DataTransparent process Intrathecal activation of CD8+ memory T cells in IgG4-related disease of the brain parenchyma Mirco Friedrich DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Hematology, Oncology and Rheumatology, University Hospital Heidelberg, Heidelberg, Germany Search for more papers by this author Niklas Kehl DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Niko Engelke Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Josephine Kraus Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Katharina Lindner DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Faculty of Biosciences, Heidelberg University, Heidelberg, Germany Search for more papers by this author Philipp Münch DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Iris Mildenberger DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Christoph Groden Department of Neuroradiology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Achim Gass Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Nima Etminan Department of Neurosurgery, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Marc Fatar Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Andreas von Deimling Department of Neuropathology, Heidelberg University Hospital, Heidelberg, Germany DKTK CCU Neuropathology, DKFZ, Heidelberg, Germany Search for more papers by this author David Reuss Department of Neuropathology, Heidelberg University Hospital, Heidelberg, Germany DKTK CCU Neuropathology, DKFZ, Heidelberg, Germany Search for more papers by this author Michael Platten orcid.org/0000-0002-4746-887X DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Helmholtz Institute of Translational Oncology (HI-TRON), Mainz, Germany Immune Monitoring Unit, National Center for Tumor Diseases (NCT), Heidelberg, Germany Search for more papers by this author Lukas Bunse Corresponding Author [email protected] orcid.org/0000-0002-4490-7574 DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Mirco Friedrich DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Hematology, Oncology and Rheumatology, University Hospital Heidelberg, Heidelberg, Germany Search for more papers by this author Niklas Kehl DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Niko Engelke Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Josephine Kraus Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Katharina Lindner DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Faculty of Biosciences, Heidelberg University, Heidelberg, Germany Search for more papers by this author Philipp Münch DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Search for more papers by this author Iris Mildenberger DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Christoph Groden Department of Neuroradiology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Achim Gass Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Nima Etminan Department of Neurosurgery, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Marc Fatar Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Andreas von Deimling Department of Neuropathology, Heidelberg University Hospital, Heidelberg, Germany DKTK CCU Neuropathology, DKFZ, Heidelberg, Germany Search for more papers by this author David Reuss Department of Neuropathology, Heidelberg University Hospital, Heidelberg, Germany DKTK CCU Neuropathology, DKFZ, Heidelberg, Germany Search for more papers by this author Michael Platten orcid.org/0000-0002-4746-887X DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Helmholtz Institute of Translational Oncology (HI-TRON), Mainz, Germany Immune Monitoring Unit, National Center for Tumor Diseases (NCT), Heidelberg, Germany Search for more papers by this author Lukas Bunse Corresponding Author [email protected] orcid.org/0000-0002-4490-7574 DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany Search for more papers by this author Author Information Mirco Friedrich1,2, Niklas Kehl1,3, Niko Engelke3, Josephine Kraus3, Katharina Lindner1,4, Philipp Münch1, Iris Mildenberger1,3, Christoph Groden5, Achim Gass3, Nima Etminan6, Marc Fatar3, Andreas Deimling7,8, David Reuss7,8, Michael Platten1,3,9,10 and Lukas Bunse *,1,3 1DKTK Clinical Cooperation Unit (CCU) Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany 2Department of Hematology, Oncology and Rheumatology, University Hospital Heidelberg, Heidelberg, Germany 3Department of Neurology, MCTN, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 4Faculty of Biosciences, Heidelberg University, Heidelberg, Germany 5Department of Neuroradiology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 6Department of Neurosurgery, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany 7Department of Neuropathology, Heidelberg University Hospital, Heidelberg, Germany 8DKTK CCU Neuropathology, DKFZ, Heidelberg, Germany 9Helmholtz Institute of Translational Oncology (HI-TRON), Mainz, Germany 10Immune Monitoring Unit, National Center for Tumor Diseases (NCT), Heidelberg, Germany *Corresponding author. Tel: +49 6221 3858; E-mail: [email protected] EMBO Mol Med (2021)13:e13953https://doi.org/10.15252/emmm.202113953 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract IgG4-related disease (IgG4-RD) is a fibroinflammatory disorder signified by aberrant infiltration of IgG4-restricted plasma cells into a variety of organs. Clinical presentation is heterogeneous, and pathophysiological mechanisms of IgG4-RD remain elusive. There are very few cases of IgG4-RD with isolated central nervous system manifestation. By leveraging single-cell sequencing of the cerebrospinal fluid (CSF) of a patient with an inflammatory intracranial pseudotumor, we provide novel insights into the immunopathophysiology of IgG4-RD. Our data illustrate an IgG4-RD-associated polyclonal T-cell response in the CSF and an oligoclonal T-cell response in the parenchymal lesions, the latter being the result of a multifaceted cell–cell interaction between immune cell subsets and pathogenic B cells. We demonstrate that CD8+ T effector memory cells might drive and sustain autoimmunity via macrophage migration inhibitory factor (MIF)-CD74 signaling to immature B cells and CC-chemokine ligand 5 (CCL5)-mediated recruitment of cytotoxic CD4+ T cells. These findings highlight the central role of T cells in sustaining IgG4-RD and open novel avenues for targeted therapies. SYNOPSIS This translational study demonstrates the potential of single-cell profiling technologies to support clinicians in the diagnosis of rare autoimmune disorders while shedding new light into potential molecular pathomechanisms of intercellular communication in IgG4-related disease. T-cell - T-cell crosstalk facilitates recruitment of helper and cytotoxic CD4+ T-cells to manifestation sites of IgG4-related disease. T-cell-derived CXCL13 might regulate pathogenic B cell migration into the CNS and promote intrathecal accumulation of B-cells. Recurrent IgG4-RD in three intracranial manifestation sites is driven by a clonal T-cell response. The paper explained Problem IgG4-related disease (IgG4-RD) is an autoimmune disorder signified by infiltration of pathological plasma cells into a variety of organs. Clinical symptoms are diverse, and the underlying mechanisms that lead to IgG4-RD remain elusive. There are very few cases of IgG4-RD with isolated central nervous system manifestation, and treatment is unspecific and often not very successful. Results This paper aims at shedding new light into potential molecular mechanisms of cell-to-cell communication in IgG4-related disease. The authors propose the idea that the abnormal immune response in IgG4-RD is driven by single T-cell clonotypes. Impact This paper demonstrates the potential of single-cell profiling technologies to support clinicians in the diagnosis of rare autoimmune disorders. Future studies might incorporate results and hypotheses of this paper to develop new causal treatments against IgG4-RD. Introduction Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is the pathological consequence of aberrant infiltration of IgG4-restricted plasma cells into a variety of organs, most commonly the pancreas and lymph nodes (Stone et al, 2018; Perugino & Stone, 2020). These lesions are mostly accompanied by the excessive production of IgG4, resulting in elevated IgG4 serum levels and an increased IgG4/IgG ratio (Della-Torre et al, 2013). Parenchymal lesions of IgG4-RD in the central nervous system (CNS) are very rare, and the majority of these have additional systemic manifestations (Kuroda et al, 2019; Temmoku et al, 2020). The pathophysiological mechanisms of IgG4-related autoimmunity remain elusive, with many immune cell subsets described to be involved in disease progression (Baptista et al, 2017; Perugino & Stone, 2020). Results Here, we report on a 55-year-old male patient who was admitted to our hospital with progressive blurry vision and a monocular visual acuity reduction to 1% on the right side. At the time of admission, he was not taking any immunomodulatory medication. MRI scans showed a contrast-enhancing lesion within the right orbital cavity and optic channel (Fig EV1, 05/2019). According to the patient's medical history, in previous years, the patient had had progressive vertigo and right-sided hypoacusis followed by stereotactic radiosurgery for a right temporal/petrosal contrast-enhancing lesion (Fig EV1A, 09/2014). Following radiotherapy, the patient had developed structural epilepsy, a progressive sixth nerve palsy, and headache. A follow-up MRI scan had shown a progressive infiltration of the contrast-enhancing lesion extending into the right temporal lobe (Fig EV1A, 02/2015). Glucocorticoid therapy had been initiated followed by a partial resection of the lesion and adjacent temporal lobe. The histology of the resected lesion demonstrated no signs of malignancy, but unspecific inflammation. At the time of admission, all standard and extended diagnostics remained inconclusive and there were no extracranial disease manifestations found. However, we observed cerebrospinal fluid (CSF) pleocytosis accompanied by an isolated intrathecal immunoglobulin (Ig) production. In parallel to an intravenous glucocorticoid re-challenge (Fig EV1-EV5), we therefore performed exploratory CSF single-cell sequencing (CSF scSeq) and compared it to datasets of publicly available control and multiple sclerosis (MS) patients (Fig 1A–C) (Schafflick et al, 2020). We found a pleiotropic landscape of T cells, including naïve and effector CD8+ and CD4+ T cells (Fig 1A). Furthermore, the inflammatory pseudotumor (IPT) CSF exhibited a significantly increased abundance of naïve and non-switched memory B cells as compared to control and MS patient-derived CSF (Fig 1B and C). Pseudotime analysis of the patient's B-cell population revealed a trajectory from these naïve, cycling B cells to IgG4-restriced B cells (Fig 1D) (Trapnell et al, 2014). Click here to expand this figure. Figure EV1. Disease course of recurrent CNS-restricted inflammatory pseudotumor A. Top: Serial axial MR imaging (T1-weighted, contrast-enhanced) representative of the patient's disease course with indicated study dates. White arrowheads indicate disease manifestations as contrast enhancements. Bottom: Hematoxylin and eosin (H&E) stainings of representative resection tissues at indicated study dates. B. Sagittal MR imaging representative of the patient's disease course with indicated study dates. White arrows indicate optic nerve lesion pre and post steroid treatment. C, D. Longitudinal development of inflammation markers and immunoglobulin levels in serum and cerebrospinal fluid (CSF). Gray box indicates duration of methylprednisolone treatment C. left, serum C-reactive protein (CRP) [mg/dl] and leukocyte count [10E9/l]; right: serum levels of IgG, IgA, and IgM [g/l]. D. left, CSF total protein [mg/l] and total cell count [n/µl]; right, CSF levels of IgG, IgA, and IgM [mg/l]. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Histopathological features of IgG4-RD in recurrent CNS-restricted inflammatory pseudotumor A, B. Exemplary H&E staining (A) and IgG4 immunohistochemistry staining (B) of 2015 temporal lobe parenchyma, highlighting obliterative phlebitis and lymphocytic infiltration. C, D. Exemplary H&E staining (C) and Elastica van Gieson (EvG) staining (D) of 2019 optic nerve tissue, highlighting storiform fibrosis. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Histopathological consensus criteria of IgG4-RD A, B. Immunohistochemistry DAB staining of IgG4 (A) and total IgG (B) on resection tissues (study dates indicated). As suggested by the consensus statement on the pathology of IgG4-RD (Deshpande et al, 2012), three 40x fields with the highest number of IgG4+ and IgG+ cells each were selected, counted, and averaged within these fields. Cell counts as indicated. C. Quantification of IgG4+ and IgG+ cells from panels A-B. IgG4/IgG ratio shown on the right. Individual values, mean ± SEM. Source data are available online for this figure. Download figure Download PowerPoint Click here to expand this figure. Figure EV4. Automated IgG4 quantification and immunoglobulin ELISA in recurrent CNS-restricted inflammatory pseudotumor A–C. Immunohistochemistry DAB staining of IgG4 and total IgG on resection tissues (study dates indicated). Bottom, automatic quantification results as per default DAB parameters of QuPath 0.2.3 software. D. Immunoglobulin ELISA of IPT CSF, control CSF, and MS patient-derived CSF and respective sera. IgG4 and total IgG concentrations were measured [mg/dl]. IgG4/IgG ratio for CSF and serum shown on the right. Individual values, mean ± SD shown. n = 4 experimental repeats with technical replicates. Significance was assessed by two-way ANOVA analysis with Tukey's post hoc testing (*P < 0.05, **P < 0.01, ***P < 0.001). Download figure Download PowerPoint Click here to expand this figure. Figure EV5. Intraparenchymal IgG4-RD and peripheral T helper-like cells in IgG4-RD cerebrospinal fluid A. Immunofluorescence staining of 2015 resection tissue of GFAP (Alexa Fluor Plus 488, red) and IgG4 (Alexa Fluor Plus 546, green). DAPI (blue) was used for nuclear staining. 10× (left) and 20× (right) field shown. B. Stacked bar chart depicting mean relative expression levels of T helper cell-associated cytokines in cell subsets as identified by Seurat v4 reference mapping in IPT CSF as shown in Fig 1A. C. Dot plot of mean relative PDCD1 (x-axis) and CXCR5 (y-axis) transcript expression in CD4 T-cell subset in inflammatory pseudotumor CSF. Dotted lines indicate PDCD1+CXCR5− peripheral T helper (Tph)-like cells. TEM, T effector memory; TCM, T central memory. D. Heatmap depicting average relative expression of relevant transcripts in CD4 T-cell subset in inflammatory pseudotumor. PDCD1+ = mean relative PDCD1 expression > 0.5; CXCR5+ = mean relative CXCR5 expression > 0.5. Source data are available online for this figure. Download figure Download PowerPoint Figure 1. Comparative cerebrospinal fluid single-cell profiling of an inflammatory pseudotumor A. Uniform Manifold Approximation and Projection (UMAP) of sequenced single cells from inflammatory pseudotumor (IPT) CSF (n = 1 sample, n = 4,324 single cells, left), control CSF (n = 6 samples, n = 15,467 single cells, middle), and multiple sclerosis (MS) patient-derived CSF (n = 6, n = 18,412 cells, right). Indicated immune cell subsets as identified by Seurat v4 reference (ref) mapping. B. Stacked bar chart of relative B-cell abundances as identified by Seurat v4 reference mapping in inflammatory pseudotumor (IPT) CSF, control CSF, and MS patient-derived CSF. C. Circle plots representing the relative abundance of B-cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF, control CSF, and MS patient-derived CSF. D. Pseudotime analysis of intrathecal B-cell subsets in inflammatory pseudotumor CSF with the naïve B-cell cluster, identified by canonical markers, as root node. Percentage of cycling naïve B cells and IgG4 B cells depicted in pie charts. E. Retrospective immunohistochemistry DAB staining of IgG4 and IgG on archival temporal lobe resection tissue. As suggested by the consensus statement on the pathology of IgG4-RD (Deshpande et al, 2012), three 40x fields with the highest number of IgG4+ and IgG+ cells were selected, counted, and averaged within these fields. Cell counts as indicated. F. C-X-C motif chemokine 13 (CXCL13) concentrations measured by ELISA from inflammatory pseudotumor (IPT) CSF, control CSF, and MS patient-derived CSF. Individual values, mean ± SEM; n = 4 experiment repeats with technical replicates. G. Stacked bar chart depicting mean relative expression levels of T helper cell-associated cytokines in T-cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF as in (A). TEM, T effector memory; TCM, T central memory; Treg, regulatory T cell. H. Violin plot depicting relative expression levels of IL4R (left) and IL10RA (right) in B-cell subsets as identified by Seurat v4 reference mapping in inflammatory pseudotumor CSF. Data information: (B, C) Cell subsets as indicated by the legend on the right. Download figure Download PowerPoint As these findings were highly suggestive of IgG4-RD, we aimed to retrospectively validate the diagnosis in accordance with the consensus statement on the pathology of IgG4-RD (Deshpande et al, 2012): Strikingly, all resected tissues showed characteristic histological features such as dense lymphoplasmacytic infiltrate, obliterative phlebitis, storiform fibrosis, and IgG4 positivity (Fig EV2). Moreover, the IgG4/IgG ratio as determined by histology was greater than 0.4, thereby fulfilling the diagnostic criteria of IgG4-RD (Figs 1E and EV3 and EV4). Lastly, we found co-localization of astrocytes and IgG4+ cells, suggestive of a hitherto undescribed primary intraparenchymal manifestation (Fig EV5A). Based on the highly suggestive CSF scSeq results and in line with the clinical course with repeated sensitivity to glucocorticoids, the increased IgG4/IgG ratio as well as characteristic pathological features, the diagnosis of primary intracerebral IgG4-RD was confirmed. Based on the high abundance of naïve B cells on an IgG4 trajectory, suggestive for a strong recruitment of B cells into the CSF, we aimed to investigate the underlying mechanism. The chemokines C-X-C motif chemokine 13 (CXCL13) and CC motif chemokine ligand 21 (CCL21) are particularly known to regulate B-cell migration into the CNS and to promote intrathecal accumulation of B cells (Kowarik et al, 2012). Specifically, CXCL13 was suggested to play a role in the formation of ectopic lymphoid tissues within the CNS (Aloisi et al, 2008). Interestingly, CXCL13 CSF levels were remarkably higher in IPT CSF compared with control or MS patient-derived CSF (Fig 1F). CD4+ central memory and CD8+ effector memory T cells were the immune cell subset with the highest median expression of CXCL13 (Figs 1G and EV5B). Importantly, CD4+ memory T-cell subsets identified by Seurat v4 reference mapping expressed a T helper 2 cell (Th2)-associated cytokine profile including IL-4, IL-10, and IL-21. Previous studies suggest that Th2 cells drive the class switch toward IgG4 via IL-4 signaling (Baptista et al, 2017; Akiyama et al, 2018). Consistently, naïve B cells found in our IPT CSF dataset showed increased expression of IL4R. As IL-10 is suggested to preferentially promote class switch toward IgG4 over IgE (Jeannin et al, 1998), we found expression of IL10RA on all CSF-localized B-cell subsets in conjunction with a pronounced IL-10 expression in CD4+ memory T cells (Fig 1G and H). Our data suggested that the disease is driven by B cells in the CSF that are recruited to intraparenchymal lesions to become clonally expanding plasma cells secreting IgG4 that becomes detectable in the brain parenchyma and CSF. Several studies have assessed the pathological T-cell response in IgG4-RD, with a focus on Th2 cells (Zen et al, 2007; Tanaka et al, 2012; Müller et al, 2013; Heeringa et al, 2018) and, more recently, CD4+ cytotoxic T lymphocytes (CD4+ CTL) (Mattoo et al, 2016; Maehara et al, 2017) and PD-1hiCXCR5− peripheral T helper (Tph)-like cells (Rao et al, 2017; Kamekura et al, 2018). Interestingly, we found an increased abundance of CD4+ CTL in IPT CSF compared with control and MS patient-derived CSF (Fig 2A), while 10% of CD4+ T cells in IPT CSF were Tph-like cells (Fig EV5C and D). However, CSF T-cell repertoire was polyclonal (Fig 2B), arguing against recruitment of antigen-specific T cells. Most CD4+ T cells demonstrated expression of TCF7 consistent with a dysfunctional state, while most CD8+ T cells exhibited a phenotype reflective of cytotoxic activity, expressing granzyme and granulysin (Fig 2C) (Li et al, 2019). We therefore aimed to further characterize the functional interactions between pathogenic B cells and highly abundant T-cell clusters. Strikingly, we found the strongest predicted cell–cell interactions to be between pathogenic naïve, intermediate and memory B cells and CD8+ T effector memory cells (CD8 TEM) as well as a direct T-cell–T-cell crosstalk between CD8 TEM and CD4+ memory cells, while plasmablasts did not show potent intercellular interactions (Fig 2D). Analysis of co-expressed interaction partner molecules revealed that—in addition to canonical mediators of B-cell maturation, such as CD40—CD40LG and ALOX5—ALOX5AP (Nagashima et al, 2011)—the most highly co-expressed molecules were CD74 on B cells and its ligand macrophage migration inhibitory factor (MIF) on CD8+ and CD4+ T cells, which has recently been described as B-cell chemokine that might be responsible for the migration of pathogenic B cells to IgG4-RD manifestation sites as well as their aberrant proliferation (Shi et al, 2006; Klasen et al, 2014, 2018; Della-Torre et al, 2020) (Fig 2E–G). Lastly, we found signals of T-cell–T-cell crosstalk via CC-chemokine ligand 5 (CCL5) on CD8 TEM binding to C-C chemokine receptor type 4/5 (CCR4/5)-expressing T cells that might facilitate recruitment of helper and cytotoxic CD4+ T cells that have been shown to mediate inflammation in IgG4-RD (Fig 2E–G) (Mattoo et al, 2016; Mattoo et al, 2017; Perugino et al, 2021). Figure 2. CNS manifestation of IgG4-RD with distinct cytotoxic T-cell–B-cell interactions A. Relative abundances of cell types identified by Seurat v4 reference mapping as in Fig 1A in IPT CSF compared with control or MS patient-derived CSF. Boxplot depicting 25th–75th percentiles with median shown as central band and whiskers extending from minimum to maximum values. FC, fractional difference. B. TCR repertoire of IPT CSF as analyzed by single-cell VDJ sequencing. Top, all sequenced and paired TCR clonotypes shown, clonotypes ordered counterclockwise according to abundance. Bottom, clonotype frequency stratified by T-cell subset. C. Feature plot of UMAP of single cells from pseudotumor CSF shown in Fig 1A, depicting cell-wise representations of indicated transcripts. Relative expression shown. D. Heatmap of cell–cell interaction analysis depicting top predicted interactions based on receptor-ligand co-expression and reference-based cell subsets. Interaction z-score shown. E. Dot plot representation of top 30 receptor–ligand interactions based on molecule co-expression and reference-based cell subsets. Mean relative expression of both interaction partners (dot color) and interaction P-value (dot size) shown. P-values are derived from one-sided permutation tests and refer to the enrichment of the interacting ligand–receptor pair in each of the interacting pairs of cell types. F. Relative expression of CD40, CD74, and CCL5 on indicated combined meta-clusters classified by reference-based cell identification as shown in Fig 1A. G. Circos plot representation of highlighted cell–cell interactions in IPT CSF between indicated cell subsets. Colored receptor–ligand interaction pairs from (E-F). Download figure Download PowerPoint We hypothesized that IgG4-plasma cell maturation mediated by dysfunctional T helper cells is followed by T-cell–T-cell crosstalk and a clonal cytotoxic T-cell response as effector arm of IgG4-related autoimmunity. We here propose a novel pivotal role for CD8 TEM in driving and sustaining IgG4-RD via distinct cell–cell interactions and chemoattraction of pathogenic B cells and cytotoxic CD4+ T cells. However, we observed a polyclonal TCR repertoire in the CSF in both T-cell compartments (Fig 2B). We therefore extracted FFPE-derived DNA from all available IgG4-RD lesions (2015 temporal lobe parenchyma, 2018 cavernous sinus, and 2019 optic nerve) and performed targeted TCR beta immune repertoire sequencing (TCRB-Seq). Interestingly, we found an oligoclonal TCR repertoire in all parenchymal lesions compared with the polyclonal repertoire in the CSF (Fig 3A). Repertoire distribution analysis indicated that hyperexpanded clones dominated especially the early and latest lesions (Fig 3B). When adjusting clonotype diversity for differences in library sizes across samples by rarefaction analysis, T-cell clonality consistently increased over time (2015 < 2018 < 2019; Fig 3C). TCR sequence overlap between lesions revealed greatest overlap between 2015 and 2018, followed by 2015 and 2019 as measured by Morisita's overlap index (Fig 3D). A total of 17 clonotypes were shared between all sites and time points of disease manifestation (Fig 3E), resulting in a number of predominant TCRB amino acid motifs over the course of the disease (Fig 3F). Strikingly, longitudinal tracking of the most abundant clonotypes revealed dominance of two clones at first resection (2015: CSARVDYNEQFF: 49.27%, CASSQEYSPYEQYF: 38.33%). While productive frequency of clone CSARVDYNEQFF decreased over time (2018: 9.12%, 2019: 0.07%), clone CASSQEYSPYEQYF hyperexpanded, resulting in an almost completely monoclonal disease at re-recurrence in 2019 (83.47% productive frequency; Fig 3G and H). Taken together, these finding
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