VH2+ Antigen-Experienced B Cells in the Cerebrospinal Fluid Are Expanded and Enriched in Pediatric Anti-NMDA Receptor Encephalitis.

Pediatric and adult autoimmune encephalitis (AE) are often associated with Abs to the NR1 subunit of the N-methyl-d-aspartate (NMDA) receptor (NMDAR). Very little is known regarding the cerebrospinal fluid humoral immune profile and Ab genetics associated with pediatric anti-NMDAR-AE. Using a combination of cellular, molecular, and immunogenetics tools, we collected cerebrospinal fluid from pediatric subjects and generated 1) flow cytometry data to calculate the frequency of B cell subtypes in the cerebrospinal fluid of pediatric subjects with anti-NMDAR-AE and controls, 2) a panel of recombinant human Abs from a pediatric case of anti-NMDAR-AE that was refractory to treatment, and 3) a detailed analysis of the Ab genes that bound the NR1 subunit of the NMDAR. Ag-experienced B cells including memory cells, plasmablasts, and Ab-secreting cells were expanded in the pediatric anti-NMDAR-AE cohort, but not in the controls. These Ag-experienced B cells in the cerebrospinal fluid of a pediatric case of NMDAR-AE that was refractory to treatment had expanded use of variable H chain family 2 (VH2) genes with high somatic hypermutation that all bound to the NR1 subunit of the NMDAR. A CDR3 motif was identified in this refractory case that likely drove early stage activation and expansion of naive B cells to Ab-secreting cells, facilitating autoimmunity associated with pediatric anti-NMDAR-AE through the production of Abs that bind NR1. These features of humoral immune responses in the cerebrospinal fluid of pediatric anti-NMDAR-AE patients may be relevant for clinical diagnosis and treatment.

Neuron-binding antibody responses are associated with Black ethnicity in multiple sclerosis during natalizumab treatment.

Multiple sclerosis is an inflammatory degenerative condition of the central nervous system that may result in debilitating disability. Several studies over the past twenty years suggest that multiple sclerosis manifests with a rapid, more disabling disease course among individuals identifying with Black or Latin American ethnicity relative to those of White ethnicity. However, very little is known about immunologic underpinnings that may contribute to this ethnicity-associated discordant clinical severity. Given the importance of B cells to multiple sclerosis pathophysiology, and prior work showing increased antibody levels in the cerebrospinal fluid of Black-identifying, compared to White-identifying multiple sclerosis patients, we conducted a cohort study to determine B cell subset dynamics according to both self-reported ethnicity and genetic ancestry over time. Further, we determined relationships between ethnicity, ancestry, and neuron-binding IgG levels. We found significant associations between Black ethnicity and elevated frequencies of class-switched B cell subsets, including memory B cells; double negative two B cells; and antibody-secreting cells. The frequencies of these subsets positively correlated with West African genetic ancestry. We also observed significant associations between Black ethnicity and increased IgG binding to neurons. Our data suggests significantly heightened T cell-dependent B cell responses exhibiting increased titres of neuron-binding antibodies among individuals with multiple sclerosis identifying with the Black African diaspora. Factors driving this immunobiology may promote the greater demyelination, central nervous system atrophy and disability more often experienced by Black-, and Latin American-identifying individuals with multiple sclerosis.

Neuronal binding by antibodies can be influenced by low pH stress during the isolation procedure.

Low pH stress and its influence on antibody binding is a common consideration among chemists, but is only recently emerging as a consideration in Immunological studies. Antibody characterizations in Multiple Sclerosis (MS), an autoimmune disease of the Central Nervous System (CNS) has revealed that antibodies in the cerebrospinal fluid (CSF) of patients with Multiple Sclerosis bind to myelin-related and non-myelin antigen targets. Many laboratories have used molecular biology techniques to generate recombinant human antibodies (rhAbs) expressed by individual B cells from healthy donors and patients with systemic autoimmune disease to identify antigen targets. This approach has been adapted within the Neuroimmunology research community to investigate antigen targets of individual B cells in the CSF of MS patients. Our laboratory determines which antibodies to clone based on their immunogenetics and this method enriches for cloning of rhAbs that bind to neurons. However, newer technologies to assist in purification of these rhAbs from culture supernatants use an acidic elution buffer which may enhance low pH stress on the antibody structure. Our laboratory routinely uses a basic elution buffer to purify rhAbs from culture supernatants to avoid low pH stress to the antibody structure. Our goal was to investigate whether acidic elution of our rhAbs using Next Generation Chromatography would impact the rhAbs' ability to bind neurons. The limited data presented here for two neuron-binding rhAbs tested indicated that acidic elution buffers used during rhAb purification impacted the ability of rhAbs with low CDR3 charge to maintain binding to neuronal targets. Reproducibility in a larger panel of rhAbs and factors underlying these observations remain untested.

Sex-based differences in effector cells of the adaptive immune system during Alzheimer's disease and related dementias.

Neurological conditions such as Alzheimer's disease (AD) and related dementias (ADRD) present with many challenges due to the heterogeneity of the related disease(s), making it difficult to develop effective treatments. Additionally, the progression of ADRD-related pathologies presents differently between men and women. With two-thirds of the population affected with ADRD being women, ADRD has presented itself with a bias toward the female population. However, studies of ADRD generally do not incorporate sex-based differences in investigating the development and progression of the disease, which is detrimental to understanding and treating dementia. Additionally, recent implications for the adaptive immune system in the development of ADRD bring in new factors to be considered as part of the disease, including sex-based differences in immune response(s) during ADRD development. Here, we review the sex-based differences of pathological hallmarks of ADRD presentation and progression, sex-based differences in the adaptive immune system and how it changes with ADRD, and the importance of precision medicine in the development of a more targeted and personalized treatment for this devastating and prevalent neurodegenerative condition.

Demographics and baseline disease characteristics of Black and Hispanic patients with multiple sclerosis in the open-label, single-arm, multicenter, phase IV CHIMES trial.

BACKGROUND: Black/African American patients with multiple sclerosis (BpwMS) and Hispanic/Latino patients with multiple sclerosis (HpwMS), who historically have been underrepresented in multiple sclerosis (MS) clinical trials, exhibit greater disease severity and more rapid disease progression than White patients with MS (WpwMS). The lack of diversity and inclusion in clinical trials, which may be due to barriers at the system, patient and study levels, impacts the ability to effectively assess risks, benefits and treatment responses in a generalized patient population. METHODS: CHIMES (Characterization of Ocrelizumab in Minorities With Multiple Sclerosis), an open-label, single-arm, multicenter, phase IV study of self-identified BpwMS and HpwMS aged 18-65 years with relapsing MS and an Expanded Disability Status Score (EDSS) of ≤5.5, was developed in collaboration with patients with MS, national advocacy groups and clinical researchers. Patients were enrolled at study centers across the US, including Puerto Rico, and 1 site in Kenya. RESULTS: A total of 182 patients enrolled in CHIMES: 113 (62.1%) were BpwMS, and 69 (37.9%) were HpwMS; the mean (SD) baseline EDSS score was 2.4 (1.4), and 62.6% of patients were treatment naive. Using the pooled non-BpwMS/HpwMS group in the OPERA ocrelizumab trials as a reference population, patients enrolled in CHIMES were younger, had a higher mean body mass and had a greater T2 lesion volume but similar T2 lesion number on MRI. CONCLUSION: BpwMS and HpwMS have been consistently underrepresented in clinical trials, limiting the understanding of disease biology and response to treatment in this population. Data from the CHIMES study revealed differences in demographics and some baseline disease characteristics and disease burden between BpwMS and HpwMS vs WpwMS. These differences could have an impact when assessing clinical outcomes in BpwMS and HpwMS. GOV IDENTIFIER: NCT04377555.

Understanding humoral immunity and multiple sclerosis severity in Black, and Latinx patients.

People identified with Black/African American or Hispanic/Latinx ethnicity are more likely to exhibit a more severe multiple sclerosis disease course relative to those who identify as White. While social determinants of health account for some of this discordant severity, investigation into contributing immunobiology remains sparse. The limited immunologic data stands in stark contrast to the volume of clinical studies describing ethnicity-associated discordant presentation, and to advancement made in our understanding of MS immunopathogenesis over the past several decades. In this perspective, we posit that humoral immune responses offer a promising avenue to better understand underpinnings of discordant MS severity among Black/African American, and Hispanic/Latinx-identifying patients.

Autoantigen microarrays reveal myelin basic protein autoantibodies in morphea.

BACKGROUND: Morphea is an autoimmune, sclerosing skin disorder. Despite the recent emphasis on immune dysregulation in morphea, the role of autoantibodies in morphea pathogenesis or utility as biomarkers are poorly defined. METHODS: Autoantigen microarray was used to profile autoantibodies from the serum of participants from the Morphea in Adults and Children (MAC) cohort. Clinical and demographic features of morphea patients with myelin basic protein (MBP) autoantibodies were compared to those without. MBP immunohistochemistry staining was subsequently performed in morphea skin to assess for perineural inflammation in areas of staining. Immunofluorescence staining on mouse brain tissue was also performed using patient sera and mouse anti-myelin basic protein antibody to confirm the presence of MBP antibodies in patient sera. RESULTS: Myelin basic protein autoantibodies were found in greater frequency in morphea (n = 50, 71.4%) compared to systemic sclerosis (n = 2, 6.7%) and healthy controls (n = 7, 20%). Patients with MBP antibodies reported pain at higher frequencies. Morphea skin biopsies, highlighted by immunohistochemistry, demonstrated increased perineural inflammation in areas of MBP expression. Immunofluorescence staining revealed an increased fluorescence signal in myelinated areas of mouse brain tissue (i.e. axons) when incubated with sera from MBP antibody-positive morphea patients compared to sera from MBP antibody-negative morphea patients. Epitope mapping revealed target epitopes for MBP autoantibodies in morphea are distinct from those reported in MS, and included fragments 11-30, 41-60, 51-70, and 91-110. CONCLUSIONS: A molecular classification of morphea based on distinct autoantibody biosignatures may be used to differentially classify morphea. We have identified anti-MBP as a potential antibody associated with morphea due to its increased expression in morphea compared to healthy controls and systemic sclerosis patients.

CSF-Derived CD4+ T-Cell Diversity Is Reduced in Patients With Alzheimer Clinical Syndrome.

BACKGROUND AND OBJECTIVES: Patients with Alzheimer dementia display evidence of amyloid-related neurodegeneration. Our focus was to determine whether such patients also display evidence of a disease-targeting adaptive immune response mediated by CD4+ T cells. To test this hypothesis, we evaluated the CSF immune profiles of patients with Alzheimer clinical syndrome (ACS), who display clinically defined dementia. METHODS: Innate and adaptive immune profiles of patients with ACS were measured using multicolor flow cytometry. CSF-derived CD4+ and CD8+ T-cell receptor repertoire genetics were measured using next-generation sequencing. Brain-specific autoantibody signatures of CSF-derived antibody pools were measured using array technology or ELISA. CSF from similar-age healthy controls (HCs) was used as a comparator cohort. RESULTS: Innate cells were expanded in the CSF of patients with ACS in comparison to HCs, and innate cell expansion increased with age in the patients with ACS, but not HCs. Despite innate cell expansion in the CSF, the frequency of total CD4+ T cells reduced with age in the patients with ACS. T-cell receptor repertoire genetics indicated that T-cell clonal expansion is enhanced, and diversity is reduced in the patients with ACS compared with similar-age HCs. DISCUSSION: Examination of CSF indicates that CD4+ T cell-mediated adaptive immune responses are altered in patients with ACS. Understanding the underlying mechanisms affecting adaptive immunity will help move us toward the goal of slowing cognitive decline.

Neutrophil Extracellular Trap Formation Potential Correlates with Lung Disease Severity in COVID-19 Patients.

Severe lung inflammation is common in life-threatening coronavirus disease 2019 (COVID-19). This study tested the hypothesis that polymorphonuclear (PMN, neutrophil) phenotype early in the course of disease progression would predict peak lung disease severity in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is increasingly evident that PMN activation contributes to tissue injury resulting from extracellular reactive oxygen species generation, granule exocytosis with release of proteases, neutrophil extracellular trap (NET) formation, and release of cytokines. The current study focuses on PMN activation in response to SARS-CoV-2 infection, specifically, the association between NETs and lung disease. This is a prospective cohort study at an academic medical center with patients enrolled within 4 days of admission at 3 tertiary hospitals: Clements University Hospital, Parkland Memorial Hospital, and Children's Health in Dallas, TX. Patients were categorized as having minimal or moderate to severe lung disease based on peak respiratory support. Healthy donor controls matched for age, sex, race, and ethnicity were also enrolled. Neutrophils from COVID-19 patients displayed greater IL-8 expression, elastase release, and NET formation as compared with neutrophils from healthy donors. Importantly, neutrophils from COVID-19 patients had enhanced NET formation in the absence of any additional stimulus, not seen in PMN from healthy donors. Moreover, PMA-elicited NET formation by circulating PMN correlated with severity of lung disease. We speculate that neutrophil immuno-phenotyping can be used to predict lung disease severity in COVID-19 patients.

Outcome and Immune Correlates of a Phase II Trial of High-Dose Interleukin-2 and Stereotactic Ablative Radiotherapy for Metastatic Renal Cell Carcinoma.

PURPOSE: This phase II clinical trial evaluated whether the addition of stereotactic ablative radiotherapy (SAbR), which may promote tumor antigen presentation, improves the overall response rate (ORR) to high-dose IL2 (HD IL2) in metastatic renal cell carcinoma (mRCC). PATIENTS AND METHODS: Patients with pathologic evidence of clear cell renal cell carcinoma (RCC) and radiographic evidence of metastasis were enrolled in this single-arm trial and were treated with SAbR, followed by HD IL2. ORR was assessed based on nonirradiated metastases. Secondary endpoints included overall survival (OS), progression-free survival (PFS), toxicity, and treatment-related tumor-specific immune response. Correlative studies involved whole-exome and transcriptome sequencing, T-cell receptor sequencing, cytokine analysis, and mass cytometry on patient samples. RESULTS: Thirty ethnically diverse mRCC patients were enrolled. A median of two metastases were treated with SAbR. Among 25 patients evaluable by RECIST v1.1, ORR was 16% with 8% complete responses. Median OS was 37 months. Treatment-related adverse events (AE) included 22 grade ≥3 events that were not dissimilar from HD IL2 alone. There were no grade 5 AEs. A correlation was observed between SAbR to lung metastases and improved PFS (P = 0.0165). Clinical benefit correlated with frameshift mutational load, mast cell tumor infiltration, decreased circulating tumor-associated T-cell clones, and T-cell clonal expansion. Higher regulatory/CD8+ T-cell ratios at baseline in the tumor and periphery correlated with no clinical benefit. CONCLUSIONS: Adding SAbR did not improve the response rate to HD IL2 in patients with mRCC in this study. Tissue analyses suggest a possible correlation between frameshift mutation load as well as tumor immune infiltrates and clinical outcomes.

The Role of B Cells in Primary Progressive Multiple Sclerosis.

The success of ocrelizumab in reducing confirmed disability accumulation in primary progressive multiple sclerosis (PPMS) via CD20-targeted depletion implicates B cells as causal agents in the pathogenesis of PPMS. This review explores the possible mechanisms by which B cells contribute to disease progression in PPMS, specifically exploring cytokine production, antigen presentation, and antibody synthesis. B cells may contribute to disease progression in PPMS through cytokine production, specifically GM-CSF and IL-6, which can drive naïve T-cell differentiation into pro-inflammatory Th1/Th17 cells. B cell production of the cytokine LT-α may induce follicular dendritic cell production of CXCL13 and lead indirectly to T and B cell infiltration into the CNS. In contrast, production of IL-10 by B cells likely induces an anti-inflammatory effect that may play a role in reducing neuroinflammation in PPMS. Therefore, reduced production of IL-10 may contribute to disease worsening. B cells are also capable of potent antigen presentation and may induce pro-inflammatory T-cell differentiation via cognate interactions. B cells may also contribute to disease activity via antibody synthesis, although it's unlikely the benefit of ocrelizumab in PPMS occurs via antibody decrement. Finally, various B cell subsets likely promulgate pro- or anti-inflammatory effects in MS.

Molecular Level Characterization of Circulating Aquaporin-4 Antibodies in Neuromyelitis Optica Spectrum Disorder.

OBJECTIVE: To determine whether distinct aquaporin-4 (AQP4)-IgG lineages play a role in neuromyelitis optica spectrum disorder (NMOSD) pathogenesis, we profiled the AQP4-IgG polyclonal serum repertoire and identified, quantified, and functionally characterized distinct AQP4-IgG lineages circulating in 2 patients with NMOSD. METHODS: We combined high-throughput sequencing and quantitative immunoproteomics to simultaneously determine the constituents of both the B-cell receptor (BCR) and the serologic (IgG) anti-AQP4 antibody repertoires in the peripheral blood of patients with NMOSD. The monoclonal antibodies identified by this platform were recombinantly expressed and functionally characterized in vitro. RESULTS: Multiple antibody lineages comprise serum AQP4-IgG repertoires. Their distribution, however, can be strikingly different in polarization (polyclonal vs pauciclonal). Among the 4 serum AQP4-IgG monoclonal antibodies we identified in 2 patients, 3 induced complement-dependent cytotoxicity in a model mammalian cell line (p < 0.01). CONCLUSIONS: The composition and polarization of AQP4-IgG antibody repertoires may play an important role in NMOSD pathogenesis and clinical presentation. Here, we present a means of coupling both cellular (BCR) and serologic (IgG) antibody repertoire analysis, which has not previously been performed in NMOSD. Our analysis could be applied in the future to clinical management of patients with NMOSD to monitor disease activity over time as well as applied to other autoimmune diseases to facilitate a deeper understanding of disease pathogenesis relative to autoantibody clones.

T Cell Receptor Repertoires Acquired via Routine Pap Testing May Help Refine Cervical Cancer and Precancer Risk Estimates.

Cervical cancer is the fourth most common cancer and fourth leading cause of cancer death among women worldwide. In low Human Development Index settings, it ranks second. Screening and surveillance involve the cytology-based Papanicolaou (Pap) test and testing for high-risk human papillomavirus (hrHPV). The Pap test has low sensitivity to detect precursor lesions, while a single hrHPV test cannot distinguish a persistent infection from one that the immune system will naturally clear. Furthermore, among women who are hrHPV-positive and progress to high-grade cervical lesions, testing cannot identify the ~20% who would progress to cancer if not treated. Thus, reliable detection and treatment of cancers and precancers requires routine screening followed by frequent surveillance among those with past abnormal or positive results. The consequence is overtreatment, with its associated risks and complications, in screened populations and an increased risk of cancer in under-screened populations. Methods to improve cervical cancer risk assessment, particularly assays to predict regression of precursor lesions or clearance of hrHPV infection, would benefit both populations. Here we show that women who have lower risk results on follow-up testing relative to index testing have evidence of enhanced T cell clonal expansion in the index cervical cytology sample compared to women who persist with higher risk results from index to follow-up. We further show that a machine learning classifier based on the index sample T cells predicts this transition to lower risk with 95% accuracy (19/20) by leave-one-out cross-validation. Using T cell receptor deep sequencing and machine learning, we identified a biophysicochemical motif in the complementarity-determining region 3 of T cell receptor ß chains whose presence predicts this transition. While these results must still be tested on an independent cohort in a prospective study, they suggest that this approach could improve cervical cancer screening by helping distinguish women likely to spontaneously regress from those at elevated risk of progression to cancer. The advancement of such a strategy could reduce surveillance frequency and overtreatment in screened populations and improve the delivery of screening to under-screened populations.

Clinical Features, Treatment Strategies, and Outcomes in Hospitalized Children With Immune-Mediated Encephalopathies.

BACKGROUND: Autoimmune encephalitis (AE) and acute disseminated encephalomyelitis (ADEM) are immune-mediated brain conditions that can cause substantial neurological sequalae. Data describing the clinical characteristics, treatments, and neurological outcomes for these conditions are needed. METHODS: This is a single-center retrospective review of children diagnosed with AE or ADEM over a nine-year period with discharge outcomes measured by the Modified Rankin Score. RESULTS: Seventy-five patients (23 with ADEM and 52 with AE) were identified. Patients with ADEM had a higher percentage of abnormal magnetic resonance imaging findings (100% vs 60.8%; P < 0.001) and a shorter time from symptom onset to diagnosis (6 vs 14 days; P = 0.024). Oligoclonal bands and serum and cerebrospinal fluid inflammatory indices were higher in patients with AE. Nearly all patients received corticosteroids followed by plasmapheresis or intravenous immunoglobulin, and treatment strategies did not differ significantly between groups. Second-line immune therapies were commonly used in patients with AE. Finally, patients with AE had trends toward longer hospital lengths of stay (21 vs 13 days) and a higher percentage of neurological disability at hospital discharge (59.6% vs 34.8%). CONCLUSIONS: Although patients with ADEM and AE may have similar presenting symptoms, we found significant differences in the frequency of imaging findings, symptom duration, and laboratory and cerebrospinal fluid profiles, which can assist in distinguishing between the diagnoses. Patients in both groups were treated with a combination of immunomodulating therapies, and neurological disability was common at hospital discharge.

Reelin depletion protects against autoimmune encephalomyelitis by decreasing vascular adhesion of leukocytes.

Neuroinflammation as a result of immune cell recruitment into the central nervous system (CNS) is a key pathogenic mechanism of multiple sclerosis (MS). However, current anti-inflammatory interventions depleting immune cells or directly targeting their trafficking into the CNS can have serious side effects, highlighting a need for better immunomodulatory strategies. We detected increased Reelin concentrations in the serum of patients with MS, resulting in increased endothelial permeability to leukocytes through increased nuclear factor κB-mediated expression of vascular adhesion molecules. We thus investigated the prophylactic and therapeutic potential of Reelin immunodepletion in experimental autoimmune encephalomyelitis (EAE) and further validated the results in Reelin knockout mice. Removal of plasma Reelin by either approach protected against neuroinflammation and largely abolished the neurological consequences by reducing endothelial permeability and immune cell accumulation in the CNS. Our findings suggest Reelin depletion as a therapeutic approach with an inherent good safety margin for the treatment of MS and other diseases where leukocyte extravasation is a major driver of pathogenicity.

Biophysicochemical motifs in T cell receptor sequences as a potential biomarker for high-grade serous ovarian carcinoma.

We previously showed, in a pilot study with publicly available data, that T cell receptor (TCR) repertoires from tumor infiltrating lymphocytes (TILs) could be distinguished from adjacent healthy tissue repertoires by the presence of TCRs bearing specific, biophysicochemical motifs in their antigen binding regions. We hypothesized that such motifs might allow development of a novel approach to cancer detection. The motifs were cancer specific and achieved high classification accuracy: we found distinct motifs for breast versus colorectal cancer-associated repertoires, and the colorectal cancer motif achieved 93% accuracy, while the breast cancer motif achieved 94% accuracy. In the current study, we sought to determine whether such motifs exist for ovarian cancer, a cancer type for which detection methods are urgently needed. We made two significant advances over the prior work. First, the prior study used patient-matched TILs and healthy repertoires, collecting healthy tissue adjacent to the tumors. The current study collected TILs from patients with high-grade serous ovarian carcinoma (HGSOC) and healthy ovary repertoires from cancer-free women undergoing hysterectomy/salpingo-oophorectomy for benign disease. Thus, the classification task is distinguishing women with cancer from women without cancer. Second, in the prior study, classification accuracy was measured by patient-hold-out cross-validation on the training data. In the current study, classification accuracy was additionally assessed on an independent cohort not used during model development to establish the generalizability of the motif to unseen data. Classification accuracy was 95% by patient-hold-out cross-validation on the training set and 80% when the model was applied to the blinded test set. The results on the blinded test set demonstrate a biophysicochemical TCR motif found overwhelmingly in women with HGSOC but rarely in women with healthy ovaries, strengthening the proposal that cancer detection approaches might benefit from incorporation of TCR motif-based biomarkers. Furthermore, these results call for studies on large cohorts to establish higher classification accuracies, as well as for studies in other cancer types.

B cells migrate into remote brain areas and support neurogenesis and functional recovery after focal stroke in mice.

Lymphocytes infiltrate the stroke core and penumbra and often exacerbate cellular injury. B cells, however, are lymphocytes that do not contribute to acute pathology but can support recovery. B cell adoptive transfer to mice reduced infarct volumes 3 and 7 d after transient middle cerebral artery occlusion (tMCAo), independent of changing immune populations in recipient mice. Testing a direct neurotrophic effect, B cells cocultured with mixed cortical cells protected neurons and maintained dendritic arborization after oxygen-glucose deprivation. Whole-brain volumetric serial two-photon tomography (STPT) and a custom-developed image analysis pipeline visualized and quantified poststroke B cell diapedesis throughout the brain, including remote areas supporting functional recovery. Stroke induced significant bilateral B cell diapedesis into remote brain regions regulating motor and cognitive functions and neurogenesis (e.g., dentate gyrus, hypothalamus, olfactory areas, cerebellum) in the whole-brain datasets. To confirm a mechanistic role for B cells in functional recovery, rituximab was given to human CD20+ (hCD20+) transgenic mice to continuously deplete hCD20+-expressing B cells following tMCAo. These mice experienced delayed motor recovery, impaired spatial memory, and increased anxiety through 8 wk poststroke compared to wild type (WT) littermates also receiving rituximab. B cell depletion reduced stroke-induced hippocampal neurogenesis and cell survival. Thus, B cell diapedesis occurred in areas remote to the infarct that mediated motor and cognitive recovery. Understanding the role of B cells in neuronal health and disease-based plasticity is critical for developing effective immune-based therapies for protection against diseases that involve recruitment of peripheral immune cells into the injured brain.

T and B cell subsets differentially correlate with amyloid deposition and neurocognitive function in patients with amnestic mild cognitive impairment after one year of physical activity.

Individuals with amnestic mild cognitive impairment (aMCI) experience cognitive declines in learning and memory greater than expected for normal aging, and are at a high risk of dementia. We previously reported that sedentary aMCI patients exhibited neuroinflammation that correlated with brain amyloid beta (Aß) burden, as determined by 18F-florbetapir positron emission tomography (PET). These aMCI patients enrolled in a one-year randomized control trial (AETMCI, NCT01146717) to test the beneficial effects of 12 months of moderate-to-high intensity aerobic exercise training (AET) or stretching/toning (ST) control intervention on neurocognitive function. A subset of aMCI participants had PET imaging, cognitive testing, and immunophenotyping of cerebrospinal fluid (CSF) and peripheral blood after AET or ST interventions. As adaptive immune responses were similar between AET and ST groups, we combined AET/ST into a general 'physical activity' (PA) group and compared Aß burden, cognitive function, and adaptive immune cell subsets to sedentary lifestyle before intervention. We found that PAinduced immunomodulation of CD4+ and CD8+ T cells in CSF correlated with changes in Aß burden in brain regions associated with executive function. Furthermore, after PA, cognitive scores on tests of memory, processing speed, attention, verbal fluency, and executive function were associated with increased percent representation of circulating naïve B + T cells. We review the literature on aMCI-related cognition and immune changes as they relate to exercise, and highlight how our preliminary data suggest a complex interplay between the adaptive immune system, physical activity, cognition, and Aß burden in aMCI.

Variations of the lung microbiome and immune response in mechanically ventilated surgical patients.

Mechanically ventilated surgical patients have a variety of bacterial flora that are often undetectable by traditional culture methods. The source of infection in many of these patients remains unclear. To address this clinical problem, the microbiome profile and host inflammatory response in bronchoalveolar lavage samples from the surgical intensive care unit were examined relative to clinical pathology diagnoses. The hypothesis was tested that clinical diagnosis of respiratory tract flora were similar to culture positive lavage samples in both microbiome and inflammatory profile. Bronchoalveolar lavage samples were collected in the surgical intensive care unit as standard of care for intubated individuals with a clinical pulmonary infection score of >6 or who were expected to be intubated for >48 hours. Cytokine analysis was conducted with the Bioplex Pro Human Th17 cytokine panel. The microbiome of the samples was sequenced for the 16S rRNA region using the Ion Torrent. Microbiome diversity analysis showed the culture-positive samples had the lowest levels of diversity and culture negative with the highest based upon the Shannon-Wiener index (culture positive: 0.77 ± 0.36, respiratory tract flora: 2.06 ± 0.73, culture negative: 3.97 ± 0.65). Culture-negative samples were not dominated by a single bacterial genera. Lavages classified as respiratory tract flora were more similar to the culture-positive in the microbiome profile. A comparison of cytokine expression between groups showed increased levels of cytokines (IFN-g, IL-17F, IL-1B, IL-31, TNF-a) in culture-positive and respiratory tract flora groups. Culture-positive samples exhibited a more robust immune response and reduced diversity of bacterial genera. Lower cytokine levels in culture-negative samples, despite a greater number of bacterial species, suggest a resident nonpathogenic bacterial community may be indicative of a normal pulmonary environment. Respiratory tract flora samples were most similar to the culture-positive samples and may warrant classification as culture-positive when considering clinical treatment.

VDJServer: A Cloud-Based Analysis Portal and Data Commons for Immune Repertoire Sequences and Rearrangements.

Background: Recent technological advances in immune repertoire sequencing have created tremendous potential for advancing our understanding of adaptive immune response dynamics in various states of health and disease. Immune repertoire sequencing produces large, highly complex data sets, however, which require specialized methods and software tools for their effective analysis and interpretation. Results: VDJServer is a cloud-based analysis portal for immune repertoire sequence data that provide access to a suite of tools for a complete analysis workflow, including modules for preprocessing and quality control of sequence reads, V(D)J gene segment assignment, repertoire characterization, and repertoire comparison. VDJServer also provides sophisticated visualizations for exploratory analysis. It is accessible through a standard web browser via a graphical user interface designed for use by immunologists, clinicians, and bioinformatics researchers. VDJServer provides a data commons for public sharing of repertoire sequencing data, as well as private sharing of data between users. We describe the main functionality and architecture of VDJServer and demonstrate its capabilities with use cases from cancer immunology and autoimmunity. Conclusion: VDJServer provides a complete analysis suite for human and mouse T-cell and B-cell receptor repertoire sequencing data. The combination of its user-friendly interface and high-performance computing allows large immune repertoire sequencing projects to be analyzed with no programming or software installation required. VDJServer is a web-accessible cloud platform that provides access through a graphical user interface to a data management infrastructure, a collection of analysis tools covering all steps in an analysis, and an infrastructure for sharing data along with workflows, results, and computational provenance. VDJServer is a free, publicly available, and open-source licensed resource.