bluuebunny/crossref_metadata_2025 huggingface dataset:
Pairwise interactions govern the effects of complex immune adjuvant combinations
Authors: Pandey Surya , Gruenbaum Adam , Kanashova Tamara , Mertins Philipp , Cluzel Philippe , Chevrier Nicolas
Published: January 2023

The immune system decides upon actions in response to complex combinations of microbial inputs. Dozens of innate immune sensing pathways can collectively respond to combinations of microbial and immunomodulatory agents such as adjuvants. We currently lack a clear understanding for the combinatorial logic that governs how the interplay between higher-order combinations of microbial or adjuvant signals shape immune responses. Here, using in vitro coculture experiments and statistical analyses, we uncover an intrinsic property of pathogen-sensing pathways whereby the effects of higher-order combinations of stimuli on immune responses can be explained by single and pairwise effects. Surprisingly, this property of combinatorial innate sensing is mechanistically rooted in the molecular organization underlying pathogen-sensing pathways. Indeed, we find that the outputs of mouse and human innate immune dendritic cells upon triplet stimulations are dictated by single and pairwise effects at the levels of transcription, chromatin and protein secretion. Furthermore, we exploit this simplifying property of combinatorial pathogen sensing to develop and characterize cell-based immunotherapies with potent anti-tumor properties in mouse models using combinations of adjuvants. We conclude that the processing of complex mixtures of microbial or adjuvant inputs by immune cells is governed by pairwise effects, which suggests a path towards the rationale selection of adjuvant combinations to alter immunity.
Promoting anti-tumor immunity via bispecific T cell engaging cytokine (biteokine) therapy
Authors: Do Priscilla , Perdue Lacey A. , Chyong Andy , Henry Curtis J. , Porter Christopher C. , Dreaden Erik C.
Published: January 2023

Bispecific T cell engager (BiTE) therapy bypasses the need for natural recognition of tumor cells by cytotoxic T cells, inducing potent anti-tumor immunity. Currently, the FDA approved BiTE, Blinatumomab (Blin), provides an alternative to standard chemotherapy in patients with relapsed/refractory or MRD+ Acute Lymphoblastic Leukemia (ALL) and is in clinical trials for other B cell malignancies. Blin targets malignant B cells using the antigen binding domain of an αCD19 antibody linked to the antigen binding domain of a T cell-activating αCD3 antibody. Despite progress with Blin clinically, complete response is 39–69%, and a significant proportion of patients are refractory or relapse. To improve this outcome, we have engineered a nanoparticle-based assembly and screening approach to capitalize on cytokine-enhancement of BiTE activity. We found that IL-12 significantly improves Blin-induced B-ALL cell lysis in vitro and, using nanoparticles capable of displaying ~142 antibodies/particle, we generated 28 unique formulations with varying densities and ratios of antibodies to target CD19, activate CD3, and localize IL-12 in a non-neutralizing fashion. We screened these particles in parallel using a co-culture assay for target cell lysis and T cell expansion by flow cytometry. Through regression modeling of these data, we can select-for and optimize architectures that maximize target cell lysis and T cell proliferation. Our preliminary results indicate that optimally lytic architectures favor high αCD3 to αCD19 ratios and are improved linearly with increasing IL-12. This work seeks to innovate and enhance bispecific T cell engager therapy with a modular platform that can be used broadly in the treatment of cancer and autoimmunity.
Tri-functional T-cell engagers target immune checkpoint inhibitors PD-1 and TIGIT to enhance CD8 effector T-cell functions in chronically SHIV-infected rhesus macaques
Authors: Promsote Wanwisa , Ambrozak David , Almasri Cassandra , Liu Catherine , DeMouth Megan , Hataye Jason , Pegu Amarendra , Mascola John R. , Koup Richard
Published: January 2023

Checkpoint inhibitors, such as PD-1 and TIGIT, are crucial dysregulators of CD8 T cell function during chronic HIV/SIV infections. Importantly, these checkpoint inhibitors are highly expressed on the surface of CD4 T cells that harbor latent HIV. We have previously demonstrated that an anti-HIV/anti-CD3 bispecific T-cell engager (BiTE) can be used to redirect follicular CD8 (fCD8) T cells to kill HIV infected cells in vitro. Here, we hypothesize that adding an extra specificity to target PD-1 or TIGIT to the BiTE will further enhance the functional activities of CD8 T cells and simultaneously lower the threshold for reactivation of HIV latently infected cells. By using HIV-infected cell lines and primary CD4 T cells isolated from chronically SHIV-infected lymph nodes (LN) as target cells in in vitro and ex vivo killing assays, we found that trifunctional anti-HIV/anti-CD3-anti-TIGIT or anti-HIV/anti-CD3-anti-PD1 increased (1) T-cell specific activation, (2) antigen-specific CD107a degranulation, levels of granzyme B, cytokine/chemokine release by CD8 T cells, and (3) the killing capability of functionally compromised CD8 T cells when compared to the BiTE. When administered at 100 mcg/kg subcutaneously to chronically SHIV-infected animals, we demonstrated that anti-HIV/anti-CD3-anti-TIGIT could safely be used to augment activation and expansion of CD8 effector T cells that further resulted in a better elimination of the SHIV-infected cells as shown by the reduction in cell-associated SHIV gag DNA and RNA. Together, our results indicate that trifunctional T cell engagers targeting checkpoint inhibitors may serve as novel immunotherapeutic strategies to eliminate infected cells in HIV infected individuals.
Reduced T cell priming to vaccination in microbially-experienced (‘dirty’) mice
Authors: Sjaastad Frances V , Huggins Matthew A , Swanson Whitney , Kucaba Tamara A , Hamilton Sara E. , Griffith Thomas S
Published: January 2023

Successful vaccination strategies can provide lifelong immunity against infectious diseases and cancer. Vaccines are widely considered one of the greatest advancements in public health; however, recent failures in vaccine development indicate our inadequate understanding of the immune response to vaccination. There has been increased attention recently regarding the limited translation of preclinical findings generated using specific pathogen free (SPF) laboratory mice to humans. One potential reason for the difference between laboratory mice and humans lies in maturation status of the immune system at the time of challenge. We have been using a ‘dirty’ mouse model, where SPF laboratory mice are cohoused with pet store mice to permit pathogen transfer and immune system maturation, to investigate the naive T cell response after peptide vaccination with Trivax (polyI:C and anti-CD40 mAb) or Complete Freund’s Adjuvant (CFA). Despite having no differences in the number of antigen-specific naïve CD4 and CD8 T cell precursors, the expansion of these CD4 and CD8 T cell populations in cohoused (CoH) B6 mice was significantly reduced (compared to SPF B6 mice) following vaccination with either Trivax or CFA. Furthermore, memory recall responses to cognate antigen – as measured by proliferative capacity or cytokine production – were reduced in the CoH mice. Evaluation of the dendritic cell compartment revealed reduced expression of costimulatory molecules (e.g., CD80/CD86) on DC from CoH mice shortly after vaccination. One consequence of the muted T cell priming in CoH mice was reduced antitumor immunity after vaccination. Collectively, our data show the potential limitation of exclusive use of SPF mice when testing vaccine efficacy.
Effect of CRISPR/Cas9-mediated PD-1-disrupted primary human third-generation CAR-T cells targeting EGFRvIII on human glioblastoma cell growth
Authors: Nakazawa Tsutomu , Natsume Atsushi , Nishimura Fumihiko , Matsuda Ryosuke , Nakamura Mitsutoshi , Nakagawa Ichiro , Motoyama Yasushi , Park Young Soo , Tsujimura Takahiro , Wakabayashi Toshihiko , Nakase Hiroyuki
Published: January 2023

Glioblastoma (GBM) is the most common and malignant brain tumor. As GBM is resistant to standard treatment, immunotherapy might be a promising alternative. Chimeric antigen receptor (CAR) is an artificially modified fusion protein that can be engineered to direct the specificity and function of T cells against tumor antigens. The antitumor effects of EGFRvIII-targeting CAR-T (EvCAR-T) cells are limited in GBM, on account of the interaction between programmed cell death protein 1 (PD-1) on activated EvCAR-T cells and its ligands on target tumor cells. Antibodies against PD-1 have shown limitations, so permanent deletion of the PD-1 gene in CAR-T cells might be a promising solution. Therefore, in the present study, PD-1-disrupted EvCAR-T cells were established for the first time with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing tool. We determined the disruption efficiency of the human PD-1 gene in EvCAR-T cells and evaluated the antitumor effect of PD-1-disrupted EvCAR-T cells against human GBM cells. The results showed that our designed-sgRNA/Cas9 expression vectors precisely disrupted the target region of PD-1 and inhibited the expression of PD-1 in EvCAR-T cells. Further, the PD-1-disrupted EvCAR-T cells also had a growth inhibitory effect on EGFRvIII-expressing GBM cells. Thus, this study provides a simple, easy, and clinical applicable strategy for inducing PD-1-disrupted EvCAR-T cells, which could potentially be useful for improving the efficacy of EvCAR-T cell-based adoptive immunotherapy for GBM, and even other cancers.
Macrophages genetically engineered to secrete IL-12 induce tumor killing and immune activation in human tumor slice cultures
Authors: Kreuser Shannon A , Brempelis Katherine J , Ladabie Kevin P , Cowan Courtney M , Chinn Harrison K , Davis Amira , Pillarisetty Venu G , Crane Courtney A
Published: January 2023

Cell-based immunotherapies for cancer, such as chimeric antigen receptor (CAR) T-cells, are effective against certain hematologic malignancies, but less reliably overcome the immunosuppressive tumor microenvironment (TME) of solid tumors to effectively cause cancer remission. Preclinical studies testing interleukin-12 (IL-12), an immune stimulating cytokine that activates T-cells and NK cells, as an anti-tumor agent have been promising. However, clinical trials have demonstrated that IL-12 is toxic if delivered systemically. To achieve local, low-dose delivery of IL-12, we have made genetically engineered macrophages (GEMs) to produce IL-12. In vitro, IL-12 GEMs stably produce IL-12 for at least one month. To evaluate their ability to stimulate an immune response and induce tumor cell death in an intact immune environment, we co-cultured them with tumor slices from both human colorectal cancer liver metastases and pancreatic ductal adenocarcinoma. In both tumor subtypes, the addition of IL-12 GEMs increased tumor cell death and generated an inflammatory response within the tumor slice, as evidenced by an increase in interferon-gamma and C-X-C Motif Chemokine Ligand 9 and 10, when compared to control GEMs. We demonstrate our GEM platform as a promising way to locally and sustainably deliver IL-12 to solid tumors to overcome the immunosuppressive TME.
Interferon gamma signaling regulates the generation of influenza-specific memory B cells
Authors: Risley Chris A , Stone Sara L , Scharer Christopher D. , Boss Jeremy M , Lund Frances E
Published: January 2023

Memory B cells(Bmem), which can rapidly differentiate into antibody secreting cells (ASC) upon secondary challenge, are key mediators of humoral immunity. However, the signals that govern the formation and function of these cells remain poorly understood. To date, most Bmem studies have been performed with alum-adjuvanted proteins that induce type 2 immune responses. This differs significantly from the type I and II interferon (IFN) response generated after viral infection. Using a murine model of influenza, we showed that Bmem cell intrinsic expression of the IFNγ-inducible transcription factor (TF) T-bet was required for Bmem secondary ASC responses following viral challenge. Here, we demonstrate that B cell-specific deletion of the IFNγR and the IFNg-induced TF STAT1 also affects the flu-specific Bmem compartment and greatly diminishes Bmem recall responses. We demonstrate that T-bet is expressed by a subset of all flu-specific Bmem, including those expressing IgG1 and IgM receptors. Therefore, we hypothesized that T-bet expression could be used to subset the Bmem cells into those that did or did not receive prior IFNg and STAT1 programming signals. Sorting Bmem from flu-infected mice, we show that Bmem cells with the highest levels of T-bet differentiate quickly and robustly following ex vivo stimulation. By contrast, those Bmem expressing lower levels of T-bet differentiate with delayed kinetics. Taken together, these data support the notion that B cell intrinsic IFNγR signaling, mediated by STAT1, drives formation of a distinct population of T-bet expressing effector memory B cells that are programmed to rapidly differentiate following re-exposure to antigen.
Human Lymph: Formation Transport and Immunological role
Authors: Santambrogio Laura , Clement Cristina C , Stern Lawrence J.
Published: January 2023

The pre-nodal afferent lymph is the fluid which directly derives from the extracellular milieu from every parenchymal organ and, as it continues to circulate between the cells, it collects products deriving from the organ metabolism/catabolism. A comprehensive qualitative and quantitative investigation of the self-antigenic repertoire transported by the human lymph and its role in immunological recognition is still missing and is the focus of this investigation.

Using advanced proteomics and biochemical approaches; we sequenced the proteome/degradome/peptidome of the human lymph and compared it with the MHC II peptidome displayed by HLA-DR1+ dendritic cells.

Analysis of more than 3000 sequences identified self-peptides derived from both intracellular and extracellular proteins revealing the variety of catabolic products transported by human lymph. Quantitative analysis established that at least some of these peptides are present in the circulating lymph in nanomolar concentration. Processing enzymes were identified by reference to a database of experimentally determined cleavage sites. We found that self-antigens processed by tissue-specific proteases, including matrix metalloproteases, caspases, and granzymes, and carried by lymph contribute to the MHC II self-peptidome presented by conventional dendritic cells in vivo. Our work brings new attention to the role of “immunological self-recognition” as a dynamic interaction between dendritic cells and the lymph-transported metabolic/catabolic products from parenchymal organ derived from tissue growth, remodeling, and physiological apoptosis.
Oral feeding as well as intravenous administration of biodegradable nanoparticles containing antigen and dexamethasone induces antigen-specific immune tolerance
Authors: Kim Sang-Hyun , Park Ha-Eun , Kim Kyungjae , Lee Chong-Kil
Published: January 2023

Polymeric nanoparticles containing both antigens and drugs have remarkable advantages because they can specifically deliver the antigens and the drugs to phagocytes such as dendritic cells and macrophages, thereby reducing the potential systemic side effects of the drugs. In the present study, we fabricated polymeric nanoparticles (NP) containing both ovalbumin (OVA) and dexamethasone (Dex) (NP[OVA+Dex]) using biodegradable and biocompatible polymer poly(lactic-co-glycolic acid) (PLGA), after which their immunomodulatory effects were examined. Immature dendritic cells treated with NP[OVA+Dex] were profoundly suppressed in both MHC class II- and class I-restricted OVA peptide presentation. In addition, mice injected with NP[OVA+Dex] were profoundly suppressed in generating OVA-specific cytotoxic T cells and also in producing OVA-specific IgG with concomitant increase of regulatory T cells. Furthermore, feeding of mice with NP[OVA+Dex] induced OVA-specific immune tolerance, as demonstrated by significant decrease in generating OVA-specific immune responses upon subsequent intravenous challenge with the nanoparticles containing OVA only. The present study demonstrates that oral feeding as well as intravenous injection of PLGA nanoparticles encapsulated with both antigen and Dex is a useful means for inducing antigen-specific immune tolerance.
Podocyte-targeting autoimmunity promotes diabetic nephropathy progression
Authors: Li Limin , Li Shan , Liu Ying , Zhang Mingchao , Zen Ke
Published: January 2023

Diabetic nephropathy (DN) is the leading cause of kidney failure worldwide. High levels of advanced glycation end products (AGEs) derived from chronic hyperglycemia occur in diabetes. As AGEs are highly antigenic, it promotes us to hypothesize that AGEs may exert an autoimmune response in DN development. First, we demonstrated that podocytes have the ability to present antigens under inflammatory condition. Second, we confirmed the presence of AGEs-recognizing autoantibodies in the serum of DN patients, not nondiabetic individuals or diabetic patients without nephropathy. Third, we showed that the autoantobodies in sera of DN patients specifically identify kidney biopsies of DN patients, not nondiabetic individuals or diabetic patients without nephropathy. In particular, the autoantobodies recognize signal specificity in podocytes from DN patients. Finally, employing the kidney tissue sections from DN patients as antigen to screen serum samples from diabetic or DN patients, we found that significant DN patients (48%) but not diabetic patients without nephropathy (<5%) exhibited podocyte labeling with autoantibodies. These results suggest that podocyte-targeting autoimmunity may plays an important role in promoting immune-mediated DN progression.
Effects of FcμR expression on B cells activation
Authors: Blandino Rebecca A , Lou Zheng , Cook Sarah , Sciammas Roger , Baumgarth Nicole
Published: January 2023

FcμR is present on the cell surface of B cells as well as other antigen presenting cells where they bind the secreted form of IgM (sIgM). Our previous studies have shown that follicular B cells internalize sIgM continuously in vivo and that lack of sIgM or the FcμR on B cells results in diminished IgG responses following influenza virus infection. We aim to determine the mechanisms underlying the need for sIgM in the development of IgG responses. For this we generated HELB-FcmR−/− and HELB-FcmR+/+ mice. In vitro stimulation of purified B cells from these mice with HEL-OVA conjugate in the presence of OVA-specific OTII CD4 T cells showed an antigen-dose-dependent stimulation of both types of B cells. However, FcmR−/− B cells proliferated less at intermediate and low doses of antigen compared to controls, while they proliferated similar as wild type B cells following high-dose antigen stimulation. Intravenous injection of CD45.1-expressing C57BL/6 mice with B cells from either FcmR-deficient or -sufficient CD45.2+ HELB mice together with sheep red blood cells conjugated to HEL showed no significant differences in HEL-specific serum IgM or IgG, or frequencies of HEL-specific B cells between mice receiving either type of B cell, as assessed by ELISA and FACS, respectively. Ongoing studies are aimed at determining whether antigen-dose affects the need for FcmR expression in vivo. For that we have created mixed bone marrow (BM) irradiation chimeras with 2% BM from CD45.2 HELB or HELB-FcmR−/− mice, and 98% CD45.1 wild type BM. To-date our studies suggest that FcmR-IgM interaction enhances B cell proliferation when antigen is limiting. Future studies are aimed at testing this conclusion and explore how sIgM-FcmR interaction enhanced B cell responses.
The E3 Ubiquitin Ligase Itch Restricts the Magnitude of Antigen-Driven B Cell responses
Authors: Moser Emily K , Roof Jennifer , Dybas Joseph , Spruce Lynn , Seeholzer Stephen , Cancro Michael P , Oliver Paula M
Published: January 2023

More than 23 million Americans suffer from autoimmune disease, driven in large part by germinal center (GC)-derived autoantibodies. In GC B cells, somatic hypermutation of immunoglobulin (Ig) genes can render autoreactivity, but cells bearing autoreactive Ig receptors are eliminated due to strict selection of GC B cells by mechanisms that are still poorly defined. The E3 ubiquitin ligase Itch prevents the emergence of autoimmune disease and autoantibodies in humans and mice, and patients lacking Itch develop debilitating, multi-faceted, potentially fatal, autoimmune disease; yet how Itch regulates GC B cell fate or function has not previously been explored. By studying spontaneous and immunization-induced GC B cell responses in Itch deficient mice, we now show that Itch directly limits B cell activity to shape antibody responses. While Itch-deficient mice displayed normal numbers of pre-immune B cell populations, they showed elevated numbers of antigen-experienced B cells. Mixed bone marrow chimeras revealed that Itch acts within B cells to limit follicular and GC B cell numbers. Proteomic profiling of acutely activated B cells uncovered that Itch limited abundance of a subset of proteins involved in cell cycle and mTORC1 activity. We next found that B cells lacking Itch showed increased proliferation, glycolytic capacity, and mTORC1 activation in vitro and in vivo. Moreover, stimulation of these cells by in vivo immunization resulted in elevated numbers of GC B cells, plasma cells, and serum IgG. These results support a novel role for Itch in limiting B cell metabolism and proliferation to suppress GC B cell responses, supporting the idea that overly-exuberant GC B cell responses may contribute to autoimmunity in Itch deficiency.
Genetic contribution to loss of B cell anergy during development of autoimmunity
Authors: Smith Mia J , Rihanek Marynette , Stensland Zachary , Sarapura Virginia , Gottlieb Peter , Cambier John C
Published: January 2023

Background

Development of autoimmunity requires the activation of autoantigen-reactive T and B cells that are normally silenced by immune tolerance mechanisms. Many potentially dangerous autoreactive B cells are silenced by anergy, wherein cells persist in the periphery in an unresponsive state.

Goals of Study

Determine whether perturbation of self-reactive anergic B cells occurs during development of autoimmunity, and whether this is associated with autoimmunity risk alleles that may act by compromising B cell anergy.

Results

Using a novel enrichment method, we assessed the status of self-reactive B cells in peripheral blood of type 1 diabetic (T1D) and autoimmune thyroid disease (AITD) patients, as well as healthy controls. We observed a >90% reduction in anergic autoreactive B cells in new-onset autoimmune patients, as well as in pre-diabetic subjects and some first degree relatives (FDRs). In new-onset T1D and mice, absence of anergic insulin-reactive B cells from peripheral blood corresponded with their activation and migration to the pancreas and pancreatic lymph node. To assess any genetic contribution to subversion of B cell tolerance, we genotyped FDRs of T1D patients with normal or low levels of anergic B cells. We found that the high risk HLA:DR4-DQ8 haplotype and certain non-HLA risk alleles (PTPN2, PTPN22, IKZF3, and INS) relevant to B and T-cell development and signaling are associated with loss of anergy.

Conclusions

Findings reveal a breach in B cell anergy occurs early in development in multiple autoimmune disorders in humans, and demonstrate an association between loss of anergy and autoimmunity risk alleles.
Ikaros regulates gene expression programs required for proper BCR-activation in mature B cells
Authors: Rodriguez Princess D , Howell William , Boyd Joseph , Frietze Seth , Schjerven Hilde
Published: January 2023

The zinc-finger (ZnF) transcription factor Ikaros (encoded by Ikzf1) plays an essential role in regulating transcriptional programs required for B cell development, as Ikzf1null/null mice do not develop B cells. Ikaros contains two ZnF domains which function in DNA-binding and protein-protein interactions, respectively. Recently, mutations within the central DNA-binding ZnF domain of the IKZF1 gene has been linked to autoimmunity. Likewise, our murine model containing a germ-line deletion of the fourth ZnF (Ikzf1DF4/DF4) displays a B cell hyper-reactive (HR) phenotype, with B cells activated by anti-IgM without the requirement for a second co-stimulatory signal. Although Ikaros has been well-studied in early developing B cells, how loss-of-function mutations in Ikzf1 impacts downstream effector functions of mature B cells, remains largely unknown. Here, we investigated the role of Ikaros in regulating an epigenetic program required for co-stimulation-restricted activation of B cells. We utilized transcriptomic and epigenomic approaches (RNA-seq and ATAC-seq) to define transcriptional programs which dynamically change upon ex vivo stimulation and investigated their Ikaros-dependency. Interestingly, we found that the largest subset of genes deregulated between wt and Ikzf1DF4/DF4 B cells, occur prior to stimulation. In addition, CUT&RUN performed in wt B cells shows Ikaros binding to a large subset of genes expressed upon stimulation with anti-IgM and CD40L. Together, these results demonstrate that Ikaros regulates the transcriptional landscape of BCR-activated mature B cells. In the future, we will be integrating single cell RNA-seq analysis to investigate B cell heterogeneity in wt and Ikzf1DF4/DF4 B cells.
Autoantibody Prevalence in Cerebrospinal Fluid in Autoimmune Encephalitis and Related Disorders: The Need for a More Targeted Screening Approach
Authors: Klonoski Josh , Haven Thomas R , Peterson Lisa K
Published: January 2023

Detection of a neural autoantibody can confirm diagnosis and guide patient management and treatment. Recent recommendations suggest concurrent serum and cerebrospinal fluid (CSF) testing. However, most studies on prevalence of these autoantibodies have focused on serum testing. Herein we performed a retrospective review of CSF specimens submitted to Mayo Clinic Laboratories for CSF panel (Encephalopathy, Dementia, Paraneoplastic or Epilepsy) testing. Autoimmune encephalitis-associated and other autoimmune neurologic disorder-associated autoantibody prevalence was evaluated in conjunction with ordering patterns for CSF testing only or paired with serum testing to evaluate the clinical relevance of autoantibody testing in CSF.

An overall positivity rate of 4.63% (112/2420) was observed, with 95.54% (107/112) CSF specimens positive for a single autoantibody. The CSF positivity rates were highest for antibodies associated with autoimmune encephalitis (GAD65>NMDA>GABA-B>LGI1>AMPA>VGKC) except CASPR2 which was not detected, while antibodies associated with paraneoplastic syndrome were less prevalent (ANNA-1/Hu>PCA-1/Yo>CRMP-5/CV2>AGNA-1/SOX-1>PCA-2) or not detected (amphiphysin, ANNA-2/Ri, ANNA-3 and PCA-Tr). Most CSF specimens (62.56%; n=1514) did not have a paired serum submitted for testing. Of 906 CSF/serum pairs, only 49 were CSF autoantibody positive. Rarely were antibodies identified in CSF with negative serum results while antibodies were more frequently identified in serum with negative CSF results. These data indicate that test utilization for neural autoantibodies in CSF could be substantially improved by basing ordering practices on clinical manifestations and autoantibody prevalence.
The tetraspanin CD53 impacts splenic B cell migration and function
Authors: Greenberg Zev Joshua , Schuettpelz Laura Graves
Published: January 2023

Tetraspanins are a family of transmembrane proteins important for membrane organization and regulation of cellular migration, adhesion, and activation (Hemler, M.E., Nat. Rev. Mol. Cell Biol., 2005). The tetraspanin CD53 has been implicated in B cell development and function, and human deficiency of CD53 results in recurrent infections and reduced serum immunoglobulin (Mollinedo, F. et al., Clin. Diagn. Lab. Immunol., 1997). We have previously reported a novel function for CD53 in supporting early B cell development by maintaining IL-7 signaling (Greenberg, Z.J., et al., J. Immunol., (in press)). However, characterization of CD53 in peripheral B cell subsets has not been described. We found that Cd53−/− mice have significantly fewer splenic transitional 1 (T1, 48% reduced, p<0.01) and follicular B (Fo, 30% reduced, p<0.05) cells than wild type (WT) littermate controls. Congruent with human data, Cd53−/− mice have reduced resting serum IgG and IgM (40% reduced, p<0.01) compared to WT. CD53 has been shown to interact with integrin α4 and we found in vitro chemotaxis of both cultured primary B cells and B220-enriched splenocytes towards CXCL12 was significantly impaired in the absence of CD53 (45% reduced, p<0.005). The lymph nodes of naïve Cd53−/− mice were smaller (35% reduced, p<0.05) with significantly fewer B cells (65% reduced, p<0.0001) than WT, furthermore suggesting that CD53 may be important for B cell localization. Interestingly, surface expression of CXCR4 and VLA-4 was significantly upregulated on Cd53−/− T1, T2, and marginal zone B cells (15% increased, p<0.05). In summary, we hypothesize that CD53 maintains naïve chemotactic signaling in splenic B cell populations and is necessary for proper B cell localization.
Type III interferon networks in systemic lupus erythematosus
Authors: Barnas Jennifer L , Albrecht Jennifer , Meednu Nida , Nandedkar-Kulkarni Neha D , O’Connell Mary , Looney R John , Anolik Jennifer H
Published: January 2023

Background

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of nucleic acid autoantibodies and immune complex deposition resulting in organ damage. A type I interferon (IFN) gene signature historically attributed to IFN-α is found in cells from SLE patients and associated with disease activity. Type III IFN (IFN-λ) is relatively understudied, but is expressed in SLE renal tubule epithelial cells (RTEC) and found in patient serum. The objective of this study was to determine if IFN-λ1 allows cross-talk between human RTEC and B cells, which may be relevant for lupus nephritis pathogenesis.

Methods

SLE peripheral blood B cell phenotyping was completed by flow cytometry (n=26). RNA was isolated for RT-PCR from primary human RTEC and B cells stimulated with Toll-like receptor (TLR) agonists. CD19+ cells were stimulated with or without IFN-λ1. IFN-λ1 protein was measured by ELISA in supernatant and serum.

Results

SLE IFN-λ1 serum levels positively correlate (p=0.006) with peripheral blood IgD- CD27- CD24- CD21- B cells, a subset which expresses CD11c and T-bet and was expanded in SLE. Healthy B cells treated with IFN-λ1 express IFIT1, ISG15 and IRF7 mRNA. IFN-λ1 promoted plasma cell differentiation in B cells activated with TLR7 agonists. IFN-λ1 mRNA and protein secretion increase in RTEC with TLR3 stimulation.

Conclusions

RTEC, which constitute the bulk of the kidney, produce IFN-λ1 upon TLR3 stimulation. B lymphocytes express an IFN signature in response to IFN-λ1. IFN-λ1 promotes plasma cells in TLR7-activated B cells. These findings suggest the potential for type III IFN networks between RTEC and B cells in the renal microenvironment which may contribute to SLE and lupus nephritis pathogenesis.
Identification of T cell epitopes in sarcoidosis
Authors: Greaves Sarah A , Falta Michael , Santos Radleigh , Pinilla Clemencia , Grunewald Johan , Fontenot Andrew
Published: January 2023

Sarcoidosis is an inflammatory granulomatous disease that primarily affects the lung. It has a worldwide distribution and affects individuals of all ages. Despite the prevalence of the disease, the cause of sarcoidosis remains unknown. Evidence suggests that CD4+ T cells in the bronchoalveolar lavage (BAL) of sarcoidosis patients are instrumental in disease progression. Löfgren’s syndrome (LS) is an acute form of sarcoidosis with a specific set of inflammatory symptoms. In patients with LS, disease susceptibility has been associated with expression of HLA-DR3 and an expansion of BAL CD4+ T cells expressing T cell receptor (TCR) α-chain variable region (TRAV) 12-1 and TCR β-chain variable region (TRBV) 2. These oligoclonal T cell populations accumulate within the lung and disappear with disease resolution. We hypothesize that in these HLA-DR3+ LS patients, TRAV12-1/TRBV2 CD4+ T cell clones accumulate and expand in the lung in response to unknown etiologic antigens. We have identified a set of expanded and related TRAV12-1/TRBV2 αβTCRs derived from the BAL CD4+ T cells of DR3+ LS patients. We screened T cell hybridomas expressing these disease-relevant TCRs with decapeptide positional scanning libraries and identified stimulatory mimotopes for this set of TCRs. We identified several naturally occurring peptides derived from relevant fungal and bacterial species that stimulate the LS TCRs. Validation of these peptides as pathogenic T cell epitopes in LS is currently underway. Peptides that stimulate LS-associated TCRs have not been discovered until now, making this a major novel achievement in the field. Identification of an etiologic antigen in LS will narrow our search of the stimuli driving sarcoidosis in the US.
Platelet extracellular vesicles drive inflammasome-IL1β-dependent lung injury in Sickle Cell Disease
Authors: Sundd Prithu , Vats Ravi , Brzoska Tomasz , Bennewitz Margaret , Jimenez Maritza , Pradhan-Sundd Tirthadipa , Gladwin Mark
Published: January 2023

Intraerythrocytic polymerization of hemoglobin S promotes hemolysis and vaso-occlusive events in the microvasculature of sickle cell disease (SCD) patients. Although platelet-neutrophil aggregates-dependent vaso-occlusion is known to occur in the lung and contribute to lung injury, the etiological mechanisms that trigger acute chest syndrome are largely unknown. Here, we use intravital imaging of the lung in transgenic humanized SCD mice and in vitro imaging of SCD patient blood flowing through a microfluidic system to identify the innate-immune mechanism that promotes platelet-neutrophil aggregate-dependent lung vaso-occlusion and injury in SCD. Platelet-inflammasome activation led to generation of IL-1β and caspase-1 carrying platelet extracellular vesicles (EVs) that bind to neutrophils and promote platelet-neutrophil aggregation in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. The inflammasome activation, platelet EV generation and platelet-neutrophil aggregation were enhanced by the presence of lipopolysaccharide at a nanogram dose in SCD but not control human blood. Inhibition of the inflammasome effector caspase-1 or IL-1β pathway attenuated platelet EV generation, prevented platelet-neutrophil aggregation, and restored microvascular blood flow in lung arterioles of SCD mice in vivo and SCD human blood in microfluidics in vitro. These results are the first to identify that platelet-inflammasome dependent shedding of IL-1β and caspase-1 carrying platelet EVs promote lung vaso-occlusion in SCD. The current findings also highlight the therapeutic potential of targeting the platelet-inflammasome dependent innate immune pathway to prevent acute chest syndrome in SCD.
Autoinflammatory Eye Disease Due to NLRP3 Inflammasome-Associated Mutations and Excessive IL-1β Responses may be Driven by Ocular Exposure to Commensals
Authors: Siak Jay , St. Leger Anthony J , Mattapallil Mary , Magone M. Teresa , Zein Wadih , Alehashemi Sara , Raychaudhuri Kumarkrishna , Meng Guangxun , Fuss Ivan , Strober Warren , Goldbach-Mansky Raphaela , Caspi Rachel R
Published: January 2023

In previous studies we have shown that the healthy murine ocular surface is colonized by a commensal, Corynebacterium mastitidis, that elicits IL-17 from γδ T cells and promotes local host defense. Based on that, we hypothesized that ocular inflammation in patients with NLRP3-associated mutations, including those with Cryopyrin Associated Periodic Syndrome (CAPS), may be triggered by an overactive response to their own eye surface commensals. We found that, unlike wild type mice, mice with a CAPS mutant NLRP3 gene and increased IL-1β and IL-17 responses, develop conjunctival neutrophilia and ocular inflammation following ocular exposure to C. mast. Single cell transcriptomics confirmed that conjunctival γδ T cells were the major source of IL-17 and identified increased amounts of ccr2 and s100a8 mRNA, accounting for the neutrophil infiltration. In parallel studies of patients with inflammasome-associated mutations (NLRP3 or CARD8 mutations) we found that patient PBMCs cultured with C. mast produced increased amounts of IL-17A compared to healthy controls (C. mast. status of subjects: unknown). Patient immune cells (conjunctival and PBMC) showed upregulation of RORC, BATF and STAT3 as well as interferon-related genes (IRF1, STAT1). However, unlike in mice, IL-17-related genes appeared to be more upregulated in αβ than in γδ T cells, suggesting that in patients this response may be mediated by other cells, and/or that other commensals than in mice may be the stimulus. Thus, commensals can elicit an exaggerated immune response in humans and mice with NLRP3-related autoinflammatory disease. If indeed the conjunctivitis in CAPS patients reflects a response to their own commensals, adjunct antimicrobial therapy may be considered.
Altered CD4+ T cell Transcriptional and Epigenetic Programming Define Changes in Sarcoidosis Disease Phenotypes
Authors: Tu Ting-Hui Clair , Knapp Jennifer , Harmacek Laura , Magallon Roman , Vestal Brian , Leach Sonia , Danhorn Thomas , Li Lily , Gillespie May , MacPhail Kristyn , Riley Christina , Barkes Briana , Elliott Jill , Arger Nicholas , Sommer Anne , Powers Linda , Werner Brenda , Fingerlin Tasha , Maier Lisa , Koth Laura , Hamzeh Nabeel , O’Connor Brian
Published: January 2023

Sarcoidosis is an inflammatory disease, which is characterized by granulomas in the lungs, extrapulmonary inflammation, and can cause death by lung fibrosis. It is known that CD4+ T cell lung population increases in Sarcoidosis. However, the immunopathology remains unclear and no biomarker has been identified. Here we use RNA-seq to examine gene expression in CD4+ T cells of differential Sarcoidosis phenotypes and healthy donors. Our data showed that BATF, a Tfh inducer/key factor in Th17 differentiation was up-regulated while BCL6, a Th17 repressor is down-regulated in RNA-seq. IRF4 and BCL6 were identified as potential transcriptional upstream regulators using IPA. IL-4 and fibrosis related genes were differentially expressed with altered disease phenotype, indicating that type 2 immune response may participate in disease phenotype progression for development of fibrosis. In addition, we used ChIP-seq and ATAC-seq to examine genome-wide epigenetic histone signatures and global chromatin accessibility associated with Sarcoidosis phenotypes. TF binding-site analysis of ATAC-seq peaks revealed an enrichment of BATF binding motifs, highlighting the utility of integrating epigenetic and transcriptional signatures to suggest molecular drivers of Sarcoidosis. Our findings identified several potential immune related biomarkers and molecular mechanisms that may drive disease progression.
Detection of T cells Reactive to Hybrid Insulin Peptides in Subjects at Risk for Type 1 Diabetes
Authors: Baker Rocky Lee , Delong Thomas , Rewers Marian J , Gottlieb Peter , Haskins Kathryn
Published: January 2023

Our lab recently established that Hybrid Insulin Peptides (HIPs) are antigenic ligands for diabetogenic T cell clones in the NOD mouse model of autoimmune diabetes. Importantly, we have found that the phenotype of HIP-reactive T cells present in the peripheral blood of NOD mice is indicative of ongoing autoimmunity in the pancreas. In the human setting, we demonstrated that T cells reactive to HIPs are present in the residual islets of type 1 diabetic (T1D) organ donors and more recently, in PBMC from T1D patients. Using an IFN-g ELISPOT assay to test T cell reactivity in the peripheral blood of T1D patients or control subjects to a panel of 16 HIPs, we demonstrated that 70% of the responding patients showed reactivity to multiple HIPs. To determine whether the presence of HIP-reactive T cells might serve as a biomarker of disease activity in humans, we are following longitudinally a cohort of subjects positive for multiple islet autoantibodies (Ab+), individuals that are at high risk for developing T1D. Using IFN-g ELISPOT and CFSE dilution assays, our data indicate that HIP-reactive T cells with an inflammatory phenotype can be detected before disease onset. In one Ab+ individual that showed HIP reactivity, we used single-cell RNA sequencing to study the phenotype and TCR usage of HIP-responding T cells. We observed that T cells with a regulatory phenotype (FoxP3+, CTLA-4+, LAG3+) can be observed when a proliferative (but not inflammatory) response is detected. In this ongoing study, we are reporting for the first time that HIP-reactive T cells can be observed in the peripheral blood of subjects at risk for T1D, suggesting that the balance of inflammatory vs. regulatory signals of HIP-reactive T cells could be potential indicators of disease activity.
Myasthenia gravis: From single cell signatures to cancer diagnosis
Authors: Ingelfinger Florian , Kramer Michael , Utz Sebastian , Mundt Sarah , Krishnarajah Sinduya , Galli Edoardo , Lutz Mirjam , Jurado Nicole Puertas , Widmer Corinne , Reichen Ina Camille , Piccoli Luca , Sallusto Federica , Schneiter Didier , Opitz Isabelle , Jung Hans Heinrich , Lanzavecchia Antonio , Schreiner Bettina , Becher Burkhard
Published: January 2023

Myasthenia gravis is a rare but archetypic autoimmune disease that is characterized by the autoantibody-mediated disruption of the neuromuscular junction leading to a skeletal muscle weakness. Immunomodulatory treatment options for Myasthenia gravis patients are largely unspecific, include suppression of the entire immune compartment and are often accompanied by severe side effects.

In order to identify novel biomarkers for more targeted and effective therapeutic approaches, we combined high-dimensional mass and flow cytometry with supervised and unsupervised machine-learning algorithms. Analysis of the peripheral immune compartment of Myasthenia gravis patients and healthy controls revealed a cellular immune signature consisting of inflammatory memory T helper cells with a defined cytokine profile. The abundance of the identified leukocytes in the blood strongly correlated with the patients clinical disease activity, far better than auto-Ab titers. Moreover, we were able to locate T cells with the defined signature enriched in the inflamed thymus of Myasthenia gravis patients – the key organ for the induction and maintenance of the autoimmune disease. Lastly, using an unbiased pattern recognition approach, we identified lymphomas in a subset of Myasthenia gravis patients, further highlighting the potential of the applied analysis tools.
CpG methylation in the TGFB1 and IL-6 gene promoters serve as surrogate biomarkers for diagnosis of IBD in children
Authors: Ahmad Ali , Samarani Suzanne , Dupont Claire-Lucas , Marcel Valerie , Mack David , Seidman Ernest , Deslandres Colette , Jantchou Prevost , Amre Devendra
Published: January 2023

Crohn’s disease (CD) and ulcerative colitis (UC) are recurrent, chronic forms of inflammatory bowel diseases (IBD), the diagnosis of which can entail long delays. The delays can lead to disease presentation with complications and high morbidity. Therefore, better diagnostic markers for IBD are required. We investigated whether methylation marks in the TGFB1 and IL-6 gene promoters could be of diagnostic utility in IBD. To this end, we conducted a case-control study using peripheral blood DNA samples from 67 CD and 37 UC pediatric patients, and from 43 age-matched healthy controls. The patients were recruited from three pediatric gastroenterology clinics across Canada. The DNA samples were processed to identify methylation sites (CpG) across the promoter regions of the TGFB1 and IL-6 genes using the Sequenom technology. After implementing quality control measures, and initial non-parametric univariate (Man-Whitney U test) analyses, multivariate logistic regression analyses was carried out to identify models with the best fit (Akaike Information Criteria) and the greatest discriminatory capabilities (Area Under the Receiver Operating Curve). Logistic regression analysis showed that a model comprising 14 TGFB1 CpG sites had high discriminatory ability to separate CD from controls (AUC=0.94). Similarly, a logistic model comprising 9 CpG sites in the TGFB1 gene had near perfect ability to discriminate between UC and controls (AUC=0.99). A logistic model comprising three CpG sites in the TGFB1 gene had moderate ability (AUC=0.81) to discriminate between CD and UC. In conclusion, CpG methylation in the TGFB1 gene promoter has high discriminative power for identifying CD and UC, and could serve as an important diagnostic markers.
Single cell profiling illustrates down-regulation of GM42031 in macrophages and microglia as a potential mechanism of neuroinflammation in transgenic GFAP-gp120 mice
Authors: Wilson Kiesha Maria , Miranda Kathryn , Kaul Marcus , Nagarkatti Prakash S. , Nagarkatti Mitzi
Published: January 2023

Over thirty percent of individuals infected with human immunodeficiency virus type 1 (HIV) worldwide suffer from HIV-associated neurocognitive disorder (HAND). GFAP-gp120 transgenic (Tg) mice serve as an excellent model system for HAND because they manifest the same neuropathological attributes observed in the brains of AIDS patients. In this study, we sought to identify novel genes associated with the GFAP-gp120 neurodegenerative mouse model. Von Frey filament and hot plate tests on 8 month old GFAP-gp120 mice resulted in a significant increase in mechanical and thermal nociception in Tg versus wild-type mice. Neuropathology was confirmed via immunohistochemistry of MAP2, Synaptophysin, Iba-1, and GFAP. The MAP2 and Synaptophysin allowed us to visualize dendritic density and synaptic loss. Infiltration of macrophages was observed with anti-Iba-1 and astrocytosis with anti-GFAP Abs. Additionally, single cell RNA sequencing (scRNASeq) of encephalitogenic mononuclear cells illustrated 10 distinct clusters of cells. These clusters included: astrocytes, neurons, oligodendrocytes, endothelial cells, microglia, and macrophages. Differential expression analysis showed decreased expression of GM42031 in Tg macrophages and microglia. This locus is currently unclassified and thought to be either a long non coding RNA (lncRNA) or a gene for a NYNRIN-like protein. Until now, this particular RNA has not been associated with the GFAP-gp120 disease model. However, there has been some association of GM42031 with macrophage regulation in Parkinson’s disease and colitis models. Together our study provides novel insights into the immunopathology of HAND that may form the basis for development of effective therapeutics.