Immune dynamics in the CNS and its barriers during homeostasis and disease.

The central nervous system (CNS) has historically been viewed as an immunologically privileged site, but recent studies have uncovered a vast landscape of immune cells that reside primarily along its borders. While microglia are largely responsible for surveying the parenchyma, CNS barrier sites are inhabited by a plethora of different innate and adaptive immune cells that participate in everything from the defense against microbes to the maintenance of neural function. Static and dynamic imaging studies have revolutionized the field of neuroimmunology by providing detailed maps of CNS immune cells as well as information about how these cells move, organize, and interact during steady-state and inflammatory conditions. These studies have also redefined our understanding of neural-immune interactions at a cellular level and reshaped our conceptual view of immune privilege in this specialized compartment. This review will focus on insights gained using imaging techniques in the field of neuroimmunology, with an emphasis on anatomy and CNS immune dynamics during homeostasis, infectious diseases, injuries, and aging.

Immune-related encephalitis after immune checkpoint inhibitor therapy.

BACKGROUND: Immune checkpoint inhibitors (ICI) have revolutionized cancer treatment but can trigger immune-related encephalitis. We report one of the largest case series of patients with immune-related encephalitis and review of the literature. METHODS: Retrospective series of patients with immune-related encephalitis and literature review. RESULTS: Fourteen patients with cancer treated with ICI (50% combination therapy) developed immune-related encephalitis. Diagnostic testing revealed cerebral spinal fluid (CSF) lymphocytic pleocytosis (85%) and elevated protein (69%), abnormal brain magnetic resonance imaging(MRI) (33%) or brain FDG-PET (25%), electroencephalogram (EEG) abnormalities (30%), and autoantibodies (31%). Encephalitis treatment included: corticosteroids (86%), intravenous immunoglobulin (IVIg) (36%), plasmapheresis (7%), and rituximab (29%). There were no deaths and 12 patients had significant recovery, although long-term complications were observed. All patients discontinued ICI. Longitudinal follow-up demonstrated anti-cancer response to ICI at 3 months (85%) and 6 months post-ICI initiation (77%). A literature review identified 132 patients with immune-related encephalitis. Most were treated with PD-1 inhibitors (18% combination). Common abnormalities included elevated CSF protein (84%) or pleocytosis (77%), abnormal brain MRI (65%), or autoantibodies (47%). Nearly all were treated with corticosteroids, many required additional therapy with IVIg (26%) or rituximab (12%). Most patients had clinical improvement (81%) but a minority (10%) had a clinical relapse after completing corticosteroid taper. ICIs were resumed in 7 patients (5%), with relapse in 3. CONCLUSIONS AND RELEVANCE: Immune-related encephalitis is treatable and improves with corticosteroids in most cases but may require additional immunosuppression. Re-emergence of encephalitis is rare and does not typically result in adverse outcomes, and this should be considered in neurological immune-related adverse event management guidelines.

Macrophages redirect phagocytosis by non-professional phagocytes and influence inflammation.

Professional phagocytes (such as macrophages) and non-professional phagocytes (such as epithelial cells) clear billions of apoptotic cells and particles on a daily basis. Although professional and non-professional macrophages reside in proximity in most tissues, whether they communicate with each other during cell clearance, and how this might affect inflammation, is not known. Here we show that macrophages, through the release of a soluble growth factor and microvesicles, alter the type of particles engulfed by non-professional phagocytes and influence their inflammatory response. During phagocytosis of apoptotic cells or in response to inflammation-associated cytokines, macrophages released insulin-like growth factor 1 (IGF-1). The binding of IGF-1 to its receptor on non-professional phagocytes redirected their phagocytosis, such that uptake of larger apoptotic cells was reduced whereas engulfment of microvesicles was increased. IGF-1 did not alter engulfment by macrophages. Macrophages also released microvesicles, whose uptake by epithelial cells was enhanced by IGF-1 and led to decreased inflammatory responses by epithelial cells. Consistent with these observations, deletion of IGF-1 receptor in airway epithelial cells led to exacerbated lung inflammation after allergen exposure. These genetic and functional studies reveal that IGF-1- and microvesicle-dependent communication between macrophages and epithelial cells can critically influence the magnitude of tissue inflammation in vivo.

ShcA regulates late stages of T cell development and peripheral CD4+ T cell numbers.

T cell development in the thymus is a highly regulated process that critically depends upon productive signaling via the preTCR at the ß-selection stage, as well as via the TCR for selection from the CD4(+)CD8(+) double-positive stage to the CD4 or CD8 single-positive stage. ShcA is an adapter protein expressed in thymocytes, and it is required for productive signaling through the preTCR, with impaired signaling via ShcA leading to a developmental block at the ß-selection checkpoint. However, the role of ShcA in subsequent stages of T cell development has not been addressed. In this study, we generated transgenic mice (CD4-Cre/ShcFFF mice) that specifically express a phosphorylation-defective dominant-negative ShcA mutant (ShcFFF) in late T cell development. Thymocytes in CD4-Cre/ShcFFF mice progressed normally through the ß-selection checkpoint, but displayed a significant reduction in the numbers of single-positive CD4(+) and CD8(+) thymocytes. Furthermore, CD4-Cre/ShcFFF mice, when bred with transgenic TCR mouse strains, had impaired signaling through the transgenic TCRs. Consistent with defective progression to the single-positive stage, CD4-Cre/ShcFFF mice also had significant peripheral lymphopenia. Moreover, these CD4-Cre/ShcFFF mice develop attenuated disease in CD4(+) T cell-dependent experimental autoimmune encephalomyelitis, a mouse model of multiple sclerosis. Collectively, these data identify an important role for the adapter protein ShcA in later stages of thymic T cell development and in peripheral T cell-dependent events.

Utility of Protein Microarrays for Detection of Classified and Novel Antibodies in Autoimmune Neurologic Disease.

BACKGROUND AND OBJECTIVES: Neural antibodies are detected by tissue-based indirect immunofluorescence assay (IFA) in Mayo Clinic's Neuroimmunology Laboratory practice, but the process of characterizing and validating novel antibodies is lengthy. We report our assessment of human protein arrays. METHODS: Assessment of arrays (81% human proteome coverage) was undertaken using diverse known positive samples (17 serum and 14 CSF). Samples from patients with novel neural antibodies were reflexed from IFA to arrays. Confirmatory assays were cell-based (CBA) or line blot. Epitope mapping was undertaken using phage display immunoprecipitation sequencing (PhiPSeq). RESULTS: Control positive samples known to be reactive with linear epitopes of intracellular antigens (e.g., ANNA-1 [anti-Hu]) were readily identified by arrays in 20 of 21 samples. By contrast, 10 positive controls known to be enriched with antibodies against cell surface protein conformational epitopes (e.g., GluN1 subunit of NMDA-R) were indistinguishable from background signal. Three antibodies, previously characterized by other investigators (but unclassified in our laboratory), were unmasked in 4 patients using arrays (July-December 2022): Neurexin-3α, 1 patient; regulator of gene protein signaling (RGS)8, 1 patient; and seizure-related homolog like 2 (SEZ6L2), 2 patients. All were accompanied by previously reported phenotypes (encephalitis, 1; cerebellar ataxia, 3). Patient 1 had subacute onset of seizures and encephalopathy. Neurexin-3α ranked high in CSF (second ranked neural protein) but low in serum (660th overall). Neurexin-3α CBA was positive in both samples. Patient 2 presented with rapidly progressive cerebellar ataxia. RGS8 ranked the highest neural protein in available CSF sample by array (third overall). RGS8-specific line blot was positive. Patients 3 and 4 had rapidly progressive cerebellar ataxia. SEZ6L2 was the highest ranked neural antigen by arrays in all samples (CSF, 1, serum, 2; Patient 3, ranked 9th overall in CSF, 11th in serum; Patient 4, 6th overall in serum]). By PhIPSeq, diverse neurexin-3α epitopes (including cell surface) were detected in CSF from patient 1, but no SEZ6L2 peptides were detected for serum or CSF samples from Patient 3. DISCUSSION: Individualized autoimmune neurologic diagnoses may be accelerated using protein arrays. They are optimal for detection of intracellular antigen-reactive antibodies, though certain cell surface-directed antibodies (neurexin-3α and SEZ6L2) may also be detected.

Hematopoietic pannexin 1 function is critical for neuropathic pain.

Neuropathic pain symptoms respond poorly to available therapeutics, with most treated patients reporting unrelieved pain and significant impairment in daily life. Here, we show that Pannexin 1 (Panx1) in hematopoietic cells is required for pain-like responses following nerve injury in mice, and a potential therapeutic target. Panx1 knockout mice (Panx1-/-) were protected from hypersensitivity in two sciatic nerve injury models. Bone marrow transplantation studies show that expression of functional Panx1 in hematopoietic cells is necessary for mechanical hypersensitivity following nerve injury. Reconstitution of irradiated Panx1 knockout mice with hematopoietic Panx1-/- cells engineered to re-express Panx1 was sufficient to recover hypersensitivity after nerve injury; this rescue required expression of a Panx1 variant that can be activated by G protein-coupled receptors (GPCRs). Finally, chemically distinct Panx1 inhibitors blocked development of nerve injury-induced hypersensitivity and partially relieved this hypersensitivity after it was established. These studies indicate that Panx1 expressed in immune cells is critical for pain-like effects following nerve injury in mice, perhaps via a GPCR-mediated activation mechanism, and suggest that inhibition of Panx1 may be useful in treating neuropathic pain.

Unexpected phenotype of mice lacking Shcbp1, a protein induced during T cell proliferation.

T cell development and activation are highly regulated processes, and their proper execution is important for a competent immune system. Shc SH2-domain binding protein-1 (Shcbp1) is an evolutionarily conserved protein that binds to the adaptor protein ShcA. Studies in Drosophila and in cell lines have strongly linked Shcbp1 to cell proliferation, embryonic development, growth factor signaling, and tumorigenesis. Here we show that Shcbp1 expression is strikingly upregulated during the ß-selection checkpoint in thymocytes, and that its expression tightly correlates with proliferative stages of T cell development. To evaluate the role for Shcbp1 during thymic selection and T cell function in vivo, we generated mice with global and conditional deletion of Shcbp1. Surprisingly, the loss of Shcbp1 expression did not have an obvious effect during T cell development. However, in a mouse model of experimental autoimmune encephalomyelitis (EAE), which depends on CD4(+) T cell function and mimics multiple features of the human disease multiple sclerosis, Shcbp1 deficient mice had reduced disease severity and improved survival, and this effect was T cell intrinsic. These data suggest that despite the striking upregulation of Shcbp1 during T cell proliferation, loss of Shcbp1 does not directly affect T cell development, but regulates CD4(+) T cell effector function in vivo.