B cell depletion with anti-CD20 promotes neuroprotection in a BAFF-dependent manner in mice and humans.

Anti-CD20 therapy to deplete B cells is highly efficacious in preventing new white matter lesions in patients with relapsing-remitting multiple sclerosis (RRMS), but its protective capacity against gray matter injury and axonal damage is unclear. In a passive experimental autoimmune encephalomyelitis (EAE) model whereby TH17 cells promote brain leptomeningeal immune cell aggregates, we found that anti-CD20 treatment effectively spared myelin content and prevented myeloid cell activation, oxidative damage, and mitochondrial stress in the subpial gray matter. Anti-CD20 treatment increased B cell survival factor (BAFF) in the serum, cerebrospinal fluid, and leptomeninges of mice with EAE. Although anti-CD20 prevented gray matter demyelination, axonal loss, and neuronal atrophy, co-treatment with anti-BAFF abrogated these benefits. Consistent with the murine studies, we observed that elevated BAFF concentrations after anti-CD20 treatment in patients with RRMS were associated with better clinical outcomes. Moreover, BAFF promoted survival of human neurons in vitro. Together, our data demonstrate that BAFF exerts beneficial functions in MS and EAE in the context of anti-CD20 treatment.

Immunogenicity of COVID-19 vaccines and their effect on HIV reservoir in older people with HIV.

Older individuals and people with HIV (PWH) were prioritized for COVID-19 vaccination, yet comprehensive studies of the immunogenicity of these vaccines and their effects on HIV reservoirs are not available. Our study on 68 PWH and 23 HIV-negative participants aged 55 and older post-three vaccine doses showed equally strong anti-spike IgG responses in serum and saliva through week 48 from baseline, while PWH salivary IgA responses were low. PWH had diminished live-virus neutralization responses after two vaccine doses, which were 'rescued' post-booster. Spike-specific T cell immunity was enhanced in PWH with normal CD4+ T cell count, suggesting Th1 imprinting. The frequency of detectable HIV viremia increased post-vaccination, but vaccines did not affect the size of the HIV reservoir in most PWH, except those with low-level viremia. Thus, older PWH require three doses of COVID-19 vaccine for maximum protection, while individuals with unsuppressed viremia should be monitored for adverse reactions from HIV reservoirs.

Immunogenicity of COVID-19 vaccines and their effect on the HIV reservoir in older people with HIV.

Older individuals and people with HIV (PWH) were prioritized for COVID-19 vaccination, yet comprehensive studies of the immunogenicity of these vaccines and their effects on HIV reservoirs are not available. We followed 68 PWH aged 55 and older and 23 age-matched HIV-negative individuals for 48 weeks from the first vaccine dose, after the total of three doses. All PWH were on antiretroviral therapy (cART) and had different immune status, including immune responders (IR), immune non-responders (INR), and PWH with low-level viremia (LLV). We measured total and neutralizing Ab responses to SARS-CoV-2 spike and RBD in sera, total anti-spike Abs in saliva, frequency of anti-RBD/NTD B cells, changes in frequency of anti-spike, HIV gag/nef-specific T cells, and HIV reservoirs in peripheral CD4 + T cells. The resulting datasets were used to create a mathematical model for within-host immunization. Various regimens of BNT162b2, mRNA-1273, and ChAdOx1 vaccines elicited equally strong anti-spike IgG responses in PWH and HIV - participants in serum and saliva at all timepoints. These responses had similar kinetics in both cohorts and peaked at 4 weeks post-booster (third dose), while half-lives of plasma IgG also dramatically increased post-booster in both groups. Salivary spike IgA responses were low, especially in INRs. PWH had diminished live virus neutralizing titers after two vaccine doses which were 'rescued' after a booster. Anti-spike T cell immunity was enhanced in IRs even in comparison to HIV - participants, suggesting Th1 imprinting from HIV, while in INRs it was the lowest. Increased frequency of viral 'blips' in PWH were seen post-vaccination, but vaccines did not affect the size of the intact HIV reservoir in CD4 + T cells in most PWH, except in LLVs. Thus, older PWH require three doses of COVID-19 vaccine to maximize neutralizing responses against SARS-CoV-2, although vaccines may increase HIV reservoirs in PWH with persistent viremia.

Age-dependent gray matter demyelination is associated with leptomeningeal neutrophil accumulation.

People living with multiple sclerosis (MS) experience episodic CNS white matter lesions instigated by autoreactive T cells. With age, patients with MS show evidence of gray matter demyelination and experience devastating nonremitting symptomology. What drives progression is unclear and studying this has been hampered by the lack of suitable animal models. Here, we show that passive experimental autoimmune encephalomyelitis (EAE) induced by an adoptive transfer of young Th17 cells induced a nonremitting clinical phenotype that was associated with persistent leptomeningeal inflammation and cortical pathology in old, but not young, SJL/J mice. Although the quantity and quality of T cells did not differ in the brains of old versus young EAE mice, an increase in neutrophils and a decrease in B cells were observed in the brains of old mice. Neutrophils were also found in the leptomeninges of a subset of progressive MS patient brains that showed evidence of leptomeningeal inflammation and subpial cortical demyelination. Taken together, our data show that while Th17 cells initiate CNS inflammation, subsequent clinical symptoms and gray matter pathology are dictated by age and associated with other immune cells, such as neutrophils.

Neonatal LTßR signaling is required for the accumulation of eosinophils in the inflamed adult mesenteric lymph node.

Although eosinophils are important contributors to mucosal immune responses, mechanisms that regulate their accumulation in mucosal-associated lymphoid tissues remain ill-defined. Combining bone marrow chimeras and pharmacological inhibition approaches, here we find that lymphotoxin-beta receptor (LTßR) signaling during the neonatal period is required for the accumulation of eosinophils in the mesenteric lymph nodes (MLN) during an enteric viral infection in adult male and female mice. We demonstrate that MLN stromal cells express genes that are important for eosinophil migration and survival, such as Ccl-11 (eotaxin-1), Ccl7, Ccl9, and Cxcl2, and that expression of most of these genes is downregulated as a consequence of neonatal LTßR blockade. We also find that neonatal LTßR signaling is required for the generation of a rotavirus-specific IgA antibody response in the adult MLN, but eosinophils are dispensable for this response. Collectively, our studies reveal a role for neonatal LTßR signaling in regulating eosinophil numbers in the adult MLN.

Immunoglobulin A nephropathy is characterized by anticommensal humoral immune responses.

IgA nephropathy (IgAN) is a leading cause of kidney failure, yet little is known about the immunopathogenesis of this disease. IgAN is characterized by deposition of IgA in the kidney glomeruli, but the source and stimulus for IgA production are not known. Clinical and experimental data suggest a role for aberrant immune responses to mucosal microbiota in IgAN, and in some countries with high disease prevalence, tonsillectomy is regarded as standard-of-care therapy. To evaluate the relationship between microbiota and mucosal immune responses, we characterized the tonsil microbiota in patients with IgAN versus nonrelated household-matched control group participants and identified increased carriage of the genus Neisseria and elevated Neisseria-targeted serum IgA in IgAN patients. We reverse-translated these findings in experimental IgAN driven by BAFF overexpression in BAFF-transgenic mice rendered susceptible to Neisseria infection by introduction of a humanized CEACAM-1 transgene (B × hC-Tg). Colonization of B × hC-Tg mice with Neisseria yielded augmented levels of systemic Neisseria-specific IgA. Using a custom ELISPOT assay, we discovered anti-Neisseria-specific IgA-secreting cells within the kidneys of these mice. These findings suggest a role for cytokine-driven aberrant mucosal immune responses to oropharyngeal pathobionts, such as Neisseria, in the immunopathogenesis of IgAN. Furthermore, in the presence of excess BAFF, pathobiont-specific IgA can be produced in situ within the kidney.

Persistence of T Cell and Antibody Responses to SARS-CoV-2 Up to 9 Months after Symptom Onset.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces T cell, B cell, and Ab responses that are detected for several months in recovered individuals. Whether this response resembles a typical respiratory viral infection is a matter of debate. In this study, we followed T cell and Ab responses in 24 mainly nonhospitalized human subjects who had recovered from PCR-confirmed SARS-CoV-2 infection at two time points (median of 45 and 145 d after symptom onset). Ab responses were detected in 95% of subjects, with a strong correlation between plasma and salivary anti-spike (anti-S) and anti-receptor binding domain IgG, as well as a correlation between circulating T follicular helper cells and the SARS-CoV-2-specific IgG response. T cell responses to SARS-CoV-2 peptides were determined using intracellular cytokine staining, activation markers, proliferation, and cytokine secretion. All study subjects had a T cell response to at least one SARS-CoV-2 Ag based on at least one T cell assay. CD4+ responses were largely of the Th1 phenotype, but with a lower ratio of IFN-γ- to IL-2-producing cells and a lower frequency of CD8+:CD4+ T cells than in influenza A virus (IAV)-specific memory responses within the same subjects. Analysis of secreted molecules also revealed a lower ratio of IFN-γ to IL-2 and an altered cytotoxic profile for SARS-CoV-2 S- and nucleocapsid-specific responses compared with IAV-specific responses. These data suggest that the memory T cell phenotype after a single infection with SARS-CoV-2 persists over time, with an altered cytokine and cytotoxicity profile compared with long-term memory to whole IAV within the same subjects.

CCR6 Expression on B Cells Is Not Required for Clinical or Pathological Presentation of MOG Protein-Induced Experimental Autoimmune Encephalomyelitis despite an Altered Germinal Center Response.

B cells have been implicated in the pathogenesis of multiple sclerosis, but the mechanisms that guide B cell activation in the periphery and subsequent migration to the CNS remain incompletely understood. We previously showed that systemic inflammation induces an accumulation of B cells in the spleen in a CCR6/CCL20-dependent manner. In this study, we evaluated the role of CCR6/CCL20 in the context of myelin oligodendrocyte glycoprotein (MOG) protein-induced (B cell-dependent) experimental autoimmune encephalomyelitis (EAE). We found that CCR6 is upregulated on murine B cells that migrate into the CNS during neuroinflammation. In addition, human B cells that migrate across CNS endothelium in vitro were found to be CCR6+, and we detected CCL20 production by activated CNS-derived human endothelial cells as well as a systemic increase in CCL20 protein during EAE. Although mice that lack CCR6 expression specifically on B cells exhibited an altered germinal center reaction in response to MOG protein immunization, CCR6-deficient B cells did not exhibit any competitive disadvantage in their migration to the CNS during EAE, and the clinical and pathological presentation of EAE induced by MOG protein was unaffected. These data, to our knowledge, provide new information on the role of B cell-intrinsic CCR6 expression in a B cell-dependent model of neuroinflammation.

Siponimod therapy implicates Th17 cells in a preclinical model of subpial cortical injury.

Subpial demyelination is a specific hallmark of multiple sclerosis and a correlate of disease progression. Although the mechanism(s) that mediate pathogenesis in the subpial compartment remain unclear, it has been speculated that inflammation in the overlying meninges may be associated with subpial injury. Here we show that adoptive transfer of proteolipid protein-primed Th17 cells into SJL/J recipient mice induces subpial demyelination associated with microglial/macrophage activation, disruption of the glial limitans, and evidence of an oxidative stress response. This pathology was topologically associated with foci of immune cells in the meninges and occurred in the absence of measurable anti-myelin oligodendrocyte glycoprotein IgM or IgG antibodies. To test the role of brain-infiltrating leukocytes on subpial injury, we modulated sphingosine 1-phosphate (S1P) receptor1,5 activity with BAF312 (siponimod) treatment. Administration of BAF312, even after adoptively transferred T cells had entered the brain, significantly ameliorated clinical experimental autoimmune encephalomyelitis and diminished subpial pathology, concomitant with a selective reduction in the capacity of transferred T cells to make Th17 cytokines. We conclude that sustained subpial cortical injury is associated with the capacity for brain-resident T cells to produce Th17 cytokines, and this pathological process occurs in an S1P receptor1,5-dependent manner.

Early-life programming of mesenteric lymph node stromal cell identity by the lymphotoxin pathway regulates adult mucosal immunity.

Redundant mechanisms support immunoglobulin A (IgA) responses to intestinal antigens. These include multiple priming sites [mesenteric lymph nodes (MLNs), Peyer's patches, and isolated lymphoid follicles] and various cytokines that promote class switch to IgA, even in the absence of T cells. Despite these backup mechanisms, vaccination against enteric pathogens such as rotavirus has limited success in some populations. Genetic and environmental signals experienced during early life are known to influence mucosal immunity, yet the mechanisms for how these exposures operate remain unclear. Here, we used rotavirus infection to follow antigen-specific IgA responses through time and in different gut compartments. Using genetic and pharmacological approaches, we tested the role of the lymphotoxin (LT) pathway-known to support IgA responses-at different developmental stages. We found that LT-ß receptor (LTßR) signaling in early life programs intestinal IgA responses in adulthood by affecting antibody class switch recombination to IgA and subsequent generation of IgA antibody-secreting cells within an intact MLN. In addition, early-life LTßR signaling dictates the phenotype and function of MLN stromal cells to support IgA responses in the adult. Collectively, our studies uncover new mechanistic insights into how early-life LTßR signaling affects mucosal immune responses during adulthood.

Recirculating Intestinal IgA-Producing Cells Regulate Neuroinflammation via IL-10.

Plasma cells (PC) are found in the CNS of multiple sclerosis (MS) patients, yet their source and role in MS remains unclear. We find that some PC in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) originate in the gut and produce immunoglobulin A (IgA). Moreover, we show that IgA+ PC are dramatically reduced in the gut during EAE, and likewise, a reduction in IgA-bound fecal bacteria is seen in MS patients during disease relapse. Removal of plasmablast (PB) plus PC resulted in exacerbated EAE that was normalized by the introduction of gut-derived IgA+ PC. Furthermore, mice with an over-abundance of IgA+ PB and/or PC were specifically resistant to the effector stage of EAE, and expression of interleukin (IL)-10 by PB plus PC was necessary and sufficient to confer resistance. Our data show that IgA+ PB and/or PC mobilized from the gut play an unexpected role in suppressing neuroinflammation.

Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda-/- and Ung-/- Mice.

B cell-depleting therapies have been shown to ameliorate symptoms in multiple sclerosis (MS) patients; however, the mechanism of action remains unclear. Following priming with Ag, B cells undergo secondary diversification of their BCR, including BCR class-switch recombination (CSR) and somatic hypermutation (SHM), with both processes requiring the enzyme activation-induced (cytidine) deaminase. We previously reported that activation-induced (cytidine) deaminase is required for full clinical manifestation of disease in an animal model of MS (experimental autoimmune encephalomyelitis; EAE) provoked by immunization with the extracellular domain of recombinant human myelin oligodendrocyte glycoprotein (hMOG). In this study, we investigated the role of CSR versus SHM in the pathogenesis of EAE. We found that passive transfer of class-switched anti-MOG IgG1 Abs into hMOG-primed Aicda-/- mice is sufficient to fully rescue EAE disease. In addition, we found that the nature of the Ag is an important determinant of EAE severity in Aicda-/- mice because the lack of a diversified BCR does not affect the induction of EAE when immunized with the extracellular domain of rat MOG. To discriminate the effect of either CSR or SHM, we induced EAE in uracil DNA glycosylase-deficient mice (Ung-/-) that exhibit a defect primarily in CSR. We observed that Ung-/- mice exhibit milder clinical disease compared with control mice, concomitant with a reduced amount of anti-MOG IgG1 class-switched Abs that preserved normal affinity. Collectively, these results indicate that CSR plays an important role in governing the incidence and severity of EAE induced with hMOG but not rat MOG.

Integration of Th17- and Lymphotoxin-Derived Signals Initiates Meningeal-Resident Stromal Cell Remodeling to Propagate Neuroinflammation.

Tertiary lymphoid tissues (TLTs) have been observed in the meninges of multiple sclerosis (MS) patients, but the stromal cells and molecular signals that support TLTs remain unclear. Here, we show that T helper 17 (Th17) cells induced robust TLTs within the brain meninges that were associated with local demyelination during experimental autoimmune encephalitis (EAE). Th17-cell-induced TLTs were underpinned by a network of stromal cells producing extracellular matrix proteins and chemokines, enabling leukocytes to reside within, rather than simply transit through, the meninges. Within the CNS, interactions between lymphotoxin αß (LTαß) on Th17 cells and LTßR on meningeal radio-resistant cells were necessary for the propagation of de novo interleukin-17 responses, and activated T cells from MS patients expressed elevated levels of LTßR ligands. Therefore, input from both Th17 cells and the lymphotoxin pathway induce the formation of an immune-competent stromal cell niche in the meninges.

A Lymphotoxin/Type I IFN Axis Programs CD8+ T Cells To Infiltrate a Self-Tissue and Propagate Immunopathology.

Type I IFNs (IFN-I) are cytokines that can mediate both immune suppression and activation. Dendritic cells (DC) are significant producers of IFN-I, and depending on the context (nature of Ag, duration of exposure to Ag), DC-derived IFN-I can have varying effects on CD8(+) T cell responses. In this study, we report that in the context of a CD8(+) T cell response to a self-Ag, DC-intrinsic expression of IFN regulatory factor 3 is required to induce optimal proliferation and migration of autoreactive CD8(+) T cells, ultimately determining their ability to infiltrate a target tissue (pancreas), and the development of glucose intolerance in rat insulin promoter-glycoprotein (RIP-GP) mice. Moreover, we show that signals through the lymphotoxin-ß receptor (LTßR) in DC are also required for the proliferation of autoreactive CD8(+) T cells, the upregulation of VLA4/LFA1 on activated CD8(+) T cells, and their subsequent infiltration into the pancreas both in vitro and in vivo. Importantly, the defects in autoreactive CD8(+) T cell proliferation, accumulation of CD8(+) T cells in the pancreas, and consequent glucose intolerance observed in the context of priming by LTßR(-/-) DC could be rescued by exogenous addition of IFN-I. Collectively, our data demonstrate that the LTßR/IFN-I axis is essential for programming of CD8(+) T cells to mediate immunopathology in a self-tissue. A further understanding of the IFN-I/LTßR axis will provide valuable therapeutic insights for treatment of CD8(+) T cell-mediated autoimmune diseases.

A TNF-α-CCL20-CCR6 axis regulates Nod1-induced B cell responses.

Innate immune responses provoke the accumulation of leukocytes at sites of inflammation. In addition to monocytes and granulocytes, B cells also participate in antimicrobial innate immune responses; however, the mechanisms for accumulation of B cells to sites of inflammation are not well understood. To study B cell accumulation following systemic inflammation, we used a model synthetic ligand that stimulates a specific pattern recognition molecule, nucleotide-binding oligomerization domain-containing protein 1 (Nod1). Upon exposure to Nod1 agonists, both B cells and neutrophils rapidly accumulate within the spleen, and dendritic cells migrate into the periarterial lymphoid sheath. Nod1 stimulation led to a marked increase in several chemokines within the spleen, including CXCL13, CCL2, and CCL20. Whereas the lymphotoxin pathway was critical for the induction of the B cell chemoattractant CXCL13 in response to Nod1 agonists, B cell accumulation within the spleen following Nod1-induced systemic inflammation was independent of the lymphotoxin pathway. In contrast, a CCR6/CCL20 chemokine loop instructed rapid increase of B cells in the spleen in response to systemic administration of Nod1 agonists in a TNF-α-dependent manner. Moreover, CCR6 was required to regulate Nod1-mediated B cell responses. These results reveal a novel mechanism of B cells during inflammation and shed light on how B cells participate in innate immune responses to microbial stimulation.

BAI3, CDX2 and VIL1: a panel of three antibodies to distinguish small cell from large cell neuroendocrine lung carcinomas.

AIMS: Discriminating small-cell lung carcinoma (SCLC) from large-cell neuroendocrine carcinoma (LCNEC) rests on morphological criteria, and reproducibility has been shown to be poor. We aimed to identify immunohistochemical markers to assist this diagnosis. METHODS AND RESULTS: Gene expression profiling on laser captured frozen tumour samples from eight SCLC and eight LCNEC tumours identified a total of 888 differentially expressed genes (DEGs), 23 of which were validated by qRT-PCR. Antibodies to four selected gene products were then evaluated as immunohistochemical markers on a cohort of 173 formalin-fixed paraffin-embedded (FFPE) SCLC/LCNEC tumour samples, including 26 indeterminate tumours without a consensus diagnosis. Three markers, CDX2, VIL1 and BAI3, gave significantly different results in the two tumour types (P < 0.0001): CDX2 and VIL1 in combination (either marker positive) showed sensitivity and specificity of 81% for LCNEC while BAI3 showed 89% sensitivity and 75% specificity for SCLC. Of the 26 indeterminate tumours 15 (58%) showed an immunophenotype suggesting either SCLC or LCNEC, eight (31%) showed staining of both tumour types, and three (11%) were negative for all markers. CONCLUSION: A panel of three markers, BAI3, CDX2 and VIL1, is a useful adjunct in the diagnosis of these tumour types.

AID-expressing germinal center B cells cluster normally within lymph node follicles in the absence of FDC-M1+ CD35+ follicular dendritic cells but dissipate prematurely.

Upon activation with T-dependent Ag, B cells enter germinal centers (GC) and upregulate activation-induced deaminase (AID). AID(+) GC B cells then undergo class-switch recombination and somatic hypermutation. Follicular dendritic cells (FDC) are stromal cells that underpin GC and require constitutive signaling through the lymphotoxin (LT) ß receptor to be maintained in a fully mature, differentiated state. Although it was shown that FDC can be dispensable for the generation of affinity-matured Ab, in the absence of FDC it is unclear where AID expression occurs. In a mouse model that lacks mature FDC, as well as other LT-sensitive cells, we show that clusters of AID(+)PNA(+)GL7(+) Ag-specific GC B cells form within the B cell follicles of draining lymph nodes, suggesting that FDC are not strictly required for GC formation. However, later in the primary response, FDC-less GC dissipated prematurely, correlating with impaired affinity maturation. We examined whether GC dissipation was due to a lack of FDC or other LTß receptor-dependent accessory cells and found that, in response to nonreplicating protein Ag, FDC proved to be more critical for long-term GC maintenance. Our study provides a spatial-temporal analysis of Ag-specific B cell activation and AID expression in the context of a peripheral lymph node that lacks FDC-M1(+) CD35(+) FDC and other LT-sensitive cell types, and reveals that FDC are not strictly required for the induction of AID within an organized GC-like environment.

Substrain differences reveal novel disease-modifying gene candidates that alter the clinical course of a rodent model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis that is executed in animals by immunization with myelin Ag in adjuvant. The SJL/J autoimmune-prone strain of mouse has been used to model relapsing-remitting multiple sclerosis. However, significant variations in peak scores, timing of onset, and incidence are observed among laboratories, with the postacute (relapse) phase of the disease exhibiting significant inconsistency. We characterized two substrains of SJL/J mice that exhibit profoundly different EAE disease parameters. Induction of EAE in the first SJL/J substrain resulted in many cases of chronic EAE that was dominated by an aggressive B cell response to the immunizing Ag and to endogenous CNS Ags. In contrast, the other SJL/J substrain exhibited a relapsing-remitting form of EAE concomitant with an elevated number of cytokine-producing CD4(+) T cells in the CNS. Exploiting these interstrain differences, we performed a genome-wide copy number analysis on the two disparate SJL/J substrains and discovered numerous gene-dosage differences. In particular, one inflammation-associated gene, Naip1, was present at a higher copy number in the SJL/J substrain that exhibited relapsing-remitting EAE. These results demonstrate that substrain differences, perhaps at the level of genomic copy number, can account for variability in the postacute phase of EAE and may drive chronic versus relapsing disease.