bluuebunny/crossref_metadata_2025 huggingface dataset:
Importance of microRNAs and gut microbiota in the characterization of a phenotypic drift in lupus-prone MRL/lpr mice.
Authors: Puig Xavier Cabana , Bond Jacob M , Wang Zhuang , Dai Rujuan , Lu Ran , Lin Amy , Oakes Vanessa , Rizzo Amy , Swarwout Brianna , Abdelhamid Leila , Mao Jiangdi , Prakash Meeta , Sangmeister Constanza , Cheung Nathaniel , Cowan Catharine R , Reilly Christopher M , Sun Sha , Ahmed S Ansar , Luo Xin M
Published: January 2023

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease. The cause of SLE is not only genetic; in fact, environmental factors may play a more important role in disease development. The MRL/lpr mouse model lupus-like symptoms due to multiple SLE susceptible loci in the MRL background, and offers an accelerated model compared to the MRL parent strain due to the Faslpr mutation. Recently, our laboratory witnessed a loss of disease phenotype in our in-house colony of MRL/lpr mice. We thus compared mice newly obtained from The Jackson Laboratory (JAX; Stock Number 000485) to our in-house mice. The single-nucleotide polymorphism (SNP) analysis showed no genetic drift, suggesting that environmental factors could be triggering a phenotypic drift. Surprisingly, the newly purchased JAX mice also had attenuation of glomerulonephritis. Even though JAX mice manifested similar attenuation of lupus nephritis, our in-house colony showed differences in organ weights. Furthermore, males showed a significantly higher level of anti-double stranded DNA auto-IgG consistent with germinal center maturation. In addition, in-house males had significantly higher levels of microRNA-21 and microRNA-183 explaining spleen size difference. Moreover, the composition of gut microbiota was different between in-house and new JAX mice at early age, with many groups of bacteria differing at later time points, which might explain some of the phenotypic differences. These results suggest that microRNAs and gut microbiota might be responsible for the phenotypic differences of MRL/lpr mice in JAX and our colonies as they were genetically identical. On the other hand, the attenuation of nephritis in both groups requires further investigation.

Supported by R01AR073240
Dissecting the effect of a novel hypomorphic IL2RB mutation on immune dysregulation.
Authors: Bailey Olivia , Garcia-Perez Josselyn E , Baxter Ryan M , Cabrera-Martinez Berenice , Lui Victor G , Fernandez Isabel Z , Foster Mary J , Gessner Rachel , Gapin Laurent , Pietras Eric M , Kedl Ross , Hsieh Elena
Published: January 2023

Human inborn errors of immunity (IEIs) are a class of genetic disorders caused by monogenic germline mutations that impair the function of the encoded protein. We recently described a novel IEI due to an IL2RB homozygous mutation (p.Pro222_Gln225Del) in two siblings who suffered from multi-organ autoimmunity and CMV susceptibility. This mutation resulted in decreased cell surface protein expression and increased serum IL-2/15 levels, with concomitant increased baseline STAT5 phosphorylation (pSTAT5) but poor pSTAT5 response to IL-2 and IL-15 stimulation. Counterintuitively, this IL-2Rβ hypomorphic mutation led to an expansion of memory CD8+ T and immature CD56bright NK cells, which express the highest levels of IL-2Rβ and require IL-2/15 signaling for survival and differentiation. Yet, their maturation and function appeared impaired, suggesting that both intrinsic (hypomorphic receptor) and extrinsic (serum cytokine milieu) mechanisms were at play. To further interrogate these receptor instrinsic/extrinsic mechanisms we developed a mouse model that harbors the homolog mutation (Il2rbMut/Mut). When mutant bone marrow cells reconstituted a WT host, serum IL-2/15 levels and IL-2Rβ protein surface expression partially normalized, though IL-2/15-induced signaling remained hypofunctional. Additionally, the dysregulated mutant IL-2Rβ-driven CD8+ T cell immunophenotype normalized but the NK phenotype did not. These data highlight the asymmetrical effect that a partial IL-2Rβ defect has on CD8 T and NK cell IL-2/15-dependent signaling and downstream cellular processes. These results imply that strategies to modify serum IL-2/15 levels may serve as a therapeutic approach to treating IL-2/15 signaling defects.

Supported by grants from NIH (T32 AI074491, K23 AR070897)
IgA deficiency destabilizes immunological homeostasis towards intestinal microbiota and increases the risk of systemic immune dysregulation
Authors: Silverman Michael A , Conrey Peyton E , Denu Lidiya , O’Boyle Kaitlin C , Tanes Ceylan , Green Jamal , Bittinger Kyle , Allman David , Lubin Jean-Bernard , Duranova Tereza , Oldridge Derek , Henrickson Sarah
Published: January 2023

Mammals produce large quantities of mucosal and systemic antibodies that maintain the intestinal barrier, shape the intestinal microbiome and promote lifelong mutualism with commensal microbes. Here, we developed an integrated host-commensal approach combining microbial flow cytometry and 16S rRNA gene sequencing to define the population of microbes that induce mucosal and systemic antibodies with CyTOF analysis in pediatric selective Immunoglobulin A (IgA) deficient and household control siblings to determine the impacts of IgA deficiency on host cellular immune phenotype. In healthy controls, mucosal secretory IgA and IgM antibodies coat an overlapping subset of microbes, predominantly Firmicutes and Proteobacteria. Serum IgG antibodies target a similar consortium of fecal microbes, revealing connections between mucosal and systemic antibody networks. Furthermore, we find broad systemic immune dysregulation in a subset of children and mice lacking IgA, including enhanced IgG targeting of fecal microbiota, elevated levels of inflammatory and allergic cytokines and alterations in T cell activation state. Thus, IgA tunes systemic interactions between the host and commensal microbiota. Understanding how IgA affects baseline immune tone has implications for predicting and preventing autoimmune, inflammatory and allergic diseases, as well as providing improved prognostic guidance to patients with IgA deficiency.

Supported by CHOP Microbiome Pilot Grant K08AI135091 Burroughs Wellcome Fund, the American Academy of Allergy, Asthma, and Immunology, the Immune Deficiency Foundation, the Primary Immune Deficiency Treatment Consortium and Chan Zuckerberg Initiative
Profiling the myeloid compartment of PBMC in active tuberculosis reveals substantial changes in CD14+ cells and upregulation of CD16 in pro-inflammatory dendritic cells
Authors: Burel Julie G , Hillman Hannah , Khan Nabeela , Singhania Akul , Dubelko Paige , Casals Ferran Soldevilla , Tippalagama Rashmi , deSilva Aruna D , Scriba Thomas J , Taplitz Randy , Seumois Gregory , Vijayanand Pandurangan , Hedrick Catherine C , Sette Alessandro , Peters Bjoern
Published: January 2023

Tuberculosis (TB) is the second leading infectious killer after COVID-19 worldwide. Gene expression analysis of whole blood and peripheral blood mononuclear cells (PBMC) from TB infected versus uninfected donors suggests that myeloid cells contribute to the immune signature of TB infection. In this study, we carried out the largest cellular and molecular profiling of the circulating myeloid compartment in the context of Mycobacterium tuberculosis (Mtb) infection to date. Using flow cytometry and RNA sequencing, we isolated and interrogated the transcriptomic profile of myeloid cell subsets isolated from PBMC in a cohort of active TB (ATB) patients with paired sampling at diagnosis and mid-treatment, as well as from Mtb sensitized (IGRA+) and unsensitized (IGRA−) healthy individuals. We identified an increased frequency of CD14+ CD16− and CD14+ CD16+ myeloid cells in ATB at diagnosis with upregulated expression of interferon signaling genes that significantly overlapped with previously reported blood TB signatures. In CD14+ CD16+ cells, there was an additional increased expression of MHC-II related genes, which could be traced down to a subset of pro-inflammatory dendritic cells (DCs), namely CD14+ CD163+ DC3. This cell population significantly upregulated CD16 in ATB at diagnosis, thus displaying a CD14+ CD16+ phenotype, similarly to intermediate monocytes. This result also suggests CD16 might play an important role in inflammatory DC function in ATB. Thus, our study demonstrates quantitative and qualitative changes in CD14+ myeloid cells that are contributing to blood TB signatures. Additionally, it reveals phenotypic overlaps between subsets of monocytes and DCs in human blood that may hold disease relevance.

Supported by a grant from NIH (U19 AI118626)
PD-1 inhibition on pulmonary ILC2s promotes TNF-α production and restricts progression of metastatic melanoma tumor growth
Authors: Howard Emily Diane , Hurrell Benjamin P , Helou Doumet Georges , Quach Christine , Painter Jacob D , Shafiei-Jahani Pedram , Fung Marshall , Sharpe Arlene H , Akbari Omid
Published: January 2023

Pulmonary metastatic melanoma is an aggressive form of cancer that is highly resistant to treatment and research efforts targeting this cancer are severely lacking. While pulmonary Group 2 Innate lymphoid cells (ILC2s) represent one of the major tissue-resident innate lymphoid cell populations at steady state and are key drivers of cytokine secretion in their occupational niche, their role in pulmonary cancer progression remains unclear. As the programmed cell death protein-1 (PD-1) plays a major role in cancer immunotherapy and immunoregulatory properties, here we investigated the specific effect of PD-1 inhibition on ILC2s during a pulmonary murine model of melanoma cancer metastasis. We demonstrate that murine models of B16 tumor growth upregulate PD-1 expression on pulmonary ILC2s and that this expression severely promotes tumor growth. Conversely, utilizing targeted murine adoptive transfers we demonstrate that PD-1 inhibition on ILC2s specifically and significantly inhibits tumor growth. Furthermore, the results revealed that PD-1 inhibition upregulated both murine and human ILC2 TNF-α production and secretion, a cytotoxic cytokine that we demonstrate has the ability to directly induce tumor cell death and apoptosis independent of adaptive immunity. Together, the results of these studies may lead to a better understanding of ILC2s and their role in metastatic melanoma, as well as providing a foundation for further elucidation of their anti-tumor mechanisms in cancer progression.

Supported by grants from NIH (R01 ES025786, R01 ES021801, R01 HL144790, R01 HL151493, R01 AI145813 and R01 HL151769)
Innate immune mechanisms underlying sex differences in COVID-19
Authors: Agrawal Anshu , Agrawal Sudhanshu , Salazar Jon , Nguyen James , Rahmatpanah Farah
Published: January 2023

COVID-19 exhibits a sex-bias with males showing signs of more severe disease and hospitalizations compared to females. The mechanisms are not clear but differential immune responses, particularly the initial innate immune response, between sexes may be playing a role. The early innate immune responses to SARS-CoV2 have not been studied because of the gap in timing between the patient getting infected, showing symptoms, and getting the treatment. The primary objective of the present study was to compare the response of DCs and monocytes from males and females to SARS-CoV2 24h after infection. To investigated this, PBMCs from healthy young individuals were stimulated in vitro with the virus. Our results indicate that DCs and monocytes from females displayed increased activation compared to males. In addition, females secreted significantly higher levels of IFN-α and IL-29 early while the secretion of IFN-α was delayed and prolonged in males. Further investigations revealed that the secretion of CXCL-10, a chemokine associated with lung complications, was higher in males than females. The PBMCs from females also displayed increased induction of CTLs. Bulk RNA-seq analysis indicates that IFN related pathways may be upregulated in females at baseline. Altogether, our results suggest that decreased activation of pDCs, mDCs and monocytes, the delayed and prolonged IFN-α secretion along with increased CXCL-10 secretion may be responsible for the severity of COVID-19 in males.

The study is supported by grants R00RG2352 and R01RG3735 to AA from University of California office of the President (UCOP).
Serine Arginine Splicing Factor 7 (SRSF7) is a critical regulator of innate immune activation in macrophages
Authors: Scott Haley M , Wagner Allison R , West Kelsi , Watson Robert , Patrick Kristin L
Published: January 2023

Macrophages are immune cells that receive signals from pathogens and respond by reprogramming gene expression to activate an innate immune response. We currently have a poor understanding of how this response is fine-tuned at a post-transcriptional level. To address this, we turned to recent proteomics studies that identified mRNA processing as a pathway enriched for differentially phosphorylated proteins in macrophages infected with the important human pathogen Mycobacterium tuberculosis (Mtb). We became interested in one of these mRNA processing proteins, a splicing regulatory factor called SRSF7 (formerly known as 9G8), that was differentially phosphorylated at three serine residues during early Mtb infection. Transcriptomics analysis of SRSF7 knockdown RAW 264.7 macrophage-like cells revealed that SRSF7 is required for proper expression of a group of innate immune transcripts including the antiviral genes Mx1 and Ifit3 and the cytosolic DNA sensor Zbp1. Consistent with this phenotype, SRSF7 KD macrophages are permissive to Vesicular Stomatitis Virus (VSV) hyper replication. On the other hand, overexpression of SRSF7 in RAW 264.7 cells leads to elevated ISG abundance in resting cells and enhanced ability to restrict VSV replication. Curiously, it appears that certain ISGs are more reliant on SRSF7 than others, suggesting that SRSF7 acts at the level of the RNA itself. We have started to implicate phosphorylation of SRSF7 in these phenotypes. Together, these studies provide some of the best evidence to date that pathogen sensing cascades can functionalize RNA binding proteins during macrophage activation and highlight a previously unappreciated role for SRSF7 in controlling macrophage antiviral responses.

Supported by grants from 1F31GM143893-01 NIH Predoctoral Fellowship and R35GM133720 from NIH/NIGMS.
Human KIR+ CD8+ T cells target pathogenic T cells in Celiac disease and are active in autoimmune diseases and COVID-19
Authors: Li Jing , Zaslavsky Maxim , Su Yapeng , Guo Jing , Sikora Michael , van Unen Vincent , Christophersen Asbjørn , Chiou Shin-Heng , Chen Liang , Li Jiefu , Ji Xuhuai , Wilhelmy Julie , McSween Alana , Palanski Brad , Mallajosyula Venkata , Bracey Nathan , Dhondalay Gopal , Bhamidipati Kartik , Pai Joy , Kipp Lucas , Dunn Jeffrey , Hauser Stephen , Oksenberg Jorge , Satpathy Ansuman , Robinson William H , Steinmetz Lars , Khosla Chaitan , Utz Paul , Sollid Ludvig , Chien Yueh-Hsiu , Heath James , Fernandez-Becker Nielsen , Nadeau Kari , Saligrama Naresha , Davis Mark
Published: January 2023

Previous reports show a small subset of CD8+ T cells expressing Ly49 proteins in mice can suppress autoimmunity in a model of demyelinating disease. Here we find a markedly increased frequency of CD8+ T cells expressing inhibitory Killer cell Immunoglobulin like Receptors (KIR), the human equivalent of the Ly49 family, in the blood and inflamed tissues of various human autoimmune diseases. Increased KIR+ CD8+ T cells in the gut also correlate with disease activity in Celiac disease (CeD) patients. Moreover, KIR+ CD8+ T cells can efficiently eliminate pathogenic gliadin-specific CD4+ T cells from CeD patients’ leukocytes in vitro. Together with gene expression data, this shows that these cells are the likely equivalent of the mouse Ly49+ CD8+ T cells. Furthermore, we observe elevated levels of KIR+ CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 and influenza-infected patients, and this correlates with disease severity and vasculitis in COVID-19. Single-cell RNA and parallelized TCR sequencing reveals that expanded KIR+ CD8+ T cells from these different diseases and healthy subjects display shared phenotypes and similar T cell receptor sequences. Selective ablation of the murine counterpart in virus-infected mice leads to exacerbated autoimmunity developed after infection. These results characterize a regulatory CD8+ T cell subset in humans which we hypothesize functions to control pathogenic cells in autoimmune and infectious diseases, with important implications for the cellular dynamics and possible therapeutic approaches to suppress unwanted autoimmunity.

Supported by National Institutes of Health U19-AI057229 Howard Hughes Medical Institute Stanford Diabetes Research Center (P30DK116074)
Acid β-glucosidase insufficiency activates the C-X-C Motif Chemokine Ligand 9/CXCR3 axis leading to T cell mediated inflammation in Gaucher disease
Authors: Pandey Manoj K , Magnusen Albert Frank , Trivedi Vyoma Snehal , Hatton Shelby Loraine , Rani Reena , Nyamajenjere Tsitsi Carol , Magnusen Daniel Nii Aryee , McKay Mary Ashley , Woods Christopher , DiPasquale Betsy Ann , Köhl Jörg , Grabowski Gregory Alex
Published: January 2023

Gaucher disease (GD) is caused by GBA1 mutations that lead to decreased activity of lysosomal acid β-glucosidase and abnormal tissue accumulation of its substrate, glucosylceramide (GC). Monocyte lineage cells, e.g., macrophages (Mφs) and dendritic cells (DCs) are prominent disease effectors due to their massive accumulation of GC resulting in “Gaucher cells.” Interaction of Gaucher cells and T lymphocytes trigger massive secretion of IFNγ, TNFα, IL6, IL12p40, IL12p70, IL23, and IL17A/F, that leads to the tissue destruction in GD. The exact mechanisms that trigger excess tissue T cells recruitment in GD are still unclear. CXCR3+ T cells are enriched at inflammatory sites in patients with rheumatoid arthritis and multiple sclerosis. Using D409V/null (Gba19V/−) GD mouse model, this study uncovered the role of CXCR3 receptor and its C-X-C Motif Chemokine Ligand 9 (CXCL9) in induction of excess tissue recruitment of T cells in GD. Intracellular FACS staining of Mφs and DCs from Gba19V/− mice showed elevated amounts of CXCL9. CD4+ T and the CD8+ T cells purified from Gba19V/− mice showed increased amounts of CXCR3. Ex vivo and in vivo chemotaxis experiments, evaluated CXCL9’s recruitment of Gba19V/− T cells. Blockade of T-cell CXCL9 receptor using antibodies to CXCR3 caused marked reduction in their CXCL9-mediated chemotaxis in Gba19V/− mice. These data suggest that Gba1 defects and the resultant deficiency of acid β-glucosidase activate CXCL9-CXCR3 axis for enhanced tissue Tells recruitment of T cells in GD. Collectively, our results indicate a critical role for CXCR3 mediated T cell transmigration to sites of inflammation. Molecular targeting of CXCL9/CXCR3 axis may provide needed anti-inflammatory therapies in human patients with GD.

Supported by Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229, USA
Aiolos restrains the acquisition of cytotoxic features by CD4+ T cells
Authors: Jones Devin M , Read Kaitlin A , Pokhrel Srijana , Hales Emily , Eisele Caprice , Warren Robert T , Collins Patrick , Freud Aharon , Oestreich Kenneth J
Published: January 2023

Classically, CD4+ T cells have been defined as “helper” type cells, providing aid to other immune cell populations via the secretion of cytokines and direct cell-cell interactions. More recently, a subset of CD4+ T cells with cytotoxic capabilities, termed CD4+ cytotoxic T lymphocytes (CD4-CTLs), have been observed in both mice and humans, performing protective functions in the settings of infection and cancer while conversely contributing to the pathogenesis of autoimmunity. Despite their well-documented importance in several disease contexts, the complete mechanisms that underlie their differentiation and function remain unknown. Here, we identify the Ikaros family member, Aiolos, as a novel regulator of CD4+ CTL differentiation and function. We find that Aiolos deficiency results in increased expression of key CD4+ CTL transcription factors and effector molecules both in vitro and in an in vivo murine model of influenza infection. Mechanistically, we find that Aiolos deficiency results in increased IL-2/STAT5 signaling, supporting a repressive role for Aiolos in CD4+ CTL differentiation via negative regulation of the IL-2/STAT5 pathway. Collectively, this work identifies Aiolos as a novel negative regulator of CD4+ cytotoxic gene programming, and thus may represent a therapeutic target for the treatment of autoimmune diseases and enhanced anti-tumor immunity.

Sponsored by a grant from NIAID (NIH-RO1 AI134972) and funds through The Ohio State University College of Medicine
The role of ERK2 in regulating germinal center B cell fate decisions
Authors: Negron Austin Joseph , Abdul-Baki Nawal Samer , Perez Rebecca , Perez Christina , Bartsch Brenden L , Forsthuber Thomas G
Published: January 2023

The regulation of B cell activation, differentiation, and survival is a crucial control point for the maintenance of B cell tolerance in the germinal center (GC) and involves the precise coordination of several complex intracellular signaling pathways. Dysregulation of these pathways can lead to the generation and persistence of autoreactive memory B cells and antibody-secreting cells (ASCs), resulting in autoimmune disease. The mitogen-activated protein kinase (MAPK) pathway is central to signals required for GC B cell selection and differentiation, such as those received through the B cell receptor (BCR) and C-X-C chemokine receptor type 4 (CXCR4), respectively. Extracellular signal-regulated kinases 1 and 2 (ERK1/2), important effectors of this pathway, play a major role in B cell activation and differentiation. Though ERK2 specifically regulates the pro-apoptotic protein, Bmf, its role in regulating GC B cell fate is unknown. Our results show that peripheral Erk2-deficient B cells express DEC205, which is associated with follicular T helper cell interactions and CD80, which is involved in co-stimulation and immunoglobulin class switching. Thus, we hypothesize that ERK2 regulates several aspects of GC B cell selection. Our results elucidate key molecular mechanisms underlying the role of ERK2 in B cell differentiation, potentially offering new approaches to treating B cell-driven autoimmune diseases.

Supported by grant R21AI44731 from the NIH/NIAID (TGF), and partially funded by the UTSA president’s distinguished researcher fellowship and NIH/NIGMS RISE grant GM60655 (A.N.).
BCL6-dependent TCF-1+ progenitor cells maintain effector and helper CD4 T cell responses to persistent antigen
Authors: Xia Yu , Sandor Katalin , Pai Joy A , Daniel Bence , Raju Saravanan , Wu Renee , Hsiung Sunnie , Qi Yanyan , Yangdon Tenzin , Okamoto Mariko , Schreiber Robert D , Murphy Kenneth M , Satpathy Ansuman , Egawa Takeshi
Published: January 2023

Shortly after priming, the fate of activated CD4 T cells is segregated into BCL6+ follicular helper T (Tfh) and BCL6− effector (Teff) cells. However, it remains unknown how these subsets are sustained in the presence of chronic antigen stimulation. Using a combination of single cell- and population-based approaches, we show that in chronic viral infection, activated CD4 T cells differentiate into BCL6-dependent TCF-1+ progenitor cells with superior capacity to expand and give rise to both Teff and Tfh. They share properties with progenitor-exhausted CD8 T cells and are required for the continued generation of Teff cells as antigen persists. In response to tumors, an analogous CD4 T cell population develops in draining lymph nodes. Our study reveals the heterogeneity and plasticity of CD4 T cells upon encountering persistent antigen and highlights their population dynamics through a stable bipotent intermediate state.
Effect of Tritrichomonas foetus, Tf-31 (a cattle isolate) and Tff6 (a feline isolate) on TLR signaling pathway in cervical epithelial cells.
Authors: Kucknoor Ashwini S , Lavergne Randall
Published: January 2023

Bovine trichomonosis is caused by Tritrichomonas foetus, which is widely prevalent in 33 US states, and is a reportable disease due to potential huge economic losses to cattle industry. T. foetus also causes chronic large-bowel diarrhoea in cats, complicated not only by its chronicity but for its difficulty to treat, with few registered or effective products available. T. foetus persistently colonize the lower genital tract of bulls but usually do not cause either major clinical signs or inflammation while vaginal inflammation is a common disease of the dairy cows’ reproductive tract. Mechanisms of immune response to this pathogen are poorly studied. This study for the first time, reports the effect of T. foetus; Tf-31-cattle isolate and Tff6-cat isolate, on activation of toll-like receptors (TLRs) and inflammatory response in vaginal epithelial cells. The expression levels of serial effectors related to the TLRs-MyD88/NF-κB signaling pathway (TLR2, TLR4, MyD88, NF-κB, IL-1β, IL-6, TNF-α, and IL-10) were measured with real-time polymerase chain reaction (RT-PCR), ELISA, and Western blot analyses. Of the 6 cell-membrane TLRs tested, TLR2 and TLR4 were activated by cells, as noted by increased mRNA expression levels of TLR2 and TLR4 in both Tf31 and Tff6 treated cervical epithelial cells relative to control cells. ELISA assays showed up-regulation on IL-10, both in Tf-31 and Tff6 treated epithelial cells. Treatment with inhibitors of MyD88 signaling pathway abolished the upregulation of downstream cytokines. These data indicate that T. foetus isolates activate TLR-MyD88 signaling pathway to induce innate immune response. However, further detailed in vitro and in vivo studies are required to confirm these findings.

Supported by Biology department
Breakdown of the blood brain barrier in Experimental Cerebral Malaria is caused by CD8+ T cells with low affinity TCRs
Authors: Cornwall Douglas , Jacobs Jesica , Kolawole Elizabeth M , Evavold Brian D , Lamb Tracey J
Published: January 2023

Malaria is a worldwide health concern killing over 400,000 people each year, many of them children under 5 years old. One severe outcome of Plasmodium infection is cerebral malaria caused by a dysregulation of the immune response in the central nervous system. Using an established mouse model of cerebral malaria called experimental cerebral malaria(ECM) using Plasmodium berghei ANKA (PbA) infections of C57BL/6J mice have shown that breakdown of the blood brain barrier is mediated by IFN-g producing CD8 T cells. Here, using novel techniques to determine the affinity of the MHC:peptide:TCR interaction we have discovered that although there are high affinity cells in the population trafficking to the CNS, surprisingly a large majority of these PbA-reactive pathogenic CD8 T cells have low affinity TCRs, and a large percentage of them cannot be detected by tetramer. Importantly we show that these low affinity cells are also producing IFN-g suggesting they are pathogenic. Through adoptive transfer experiments we show that high and low affinity T cells remain stable in their affinity profile. Furthermore we show here that low affinity cells are needed for breakdown of the blood brain barrier – CD8 T cells bearing high affinity TCRs for the GAP50 peptide of Plasmodium have an impaired ability to cause ECM compared to low affinity CD8 T cells. This work demonstrates that low affinity cells are the major component of the immunopathology causing ECM when infected with PbA and contrasts with current dogma which suggests that high affinity cells are the main cells responding in these types if infections.

Supported by grants from NIH (R01 AI167422)
Epithelial HNF4A shapes the intraepithelial lymphocyte compartment via direct regulation of immune signaling molecules
Authors: Lei Xuqiu , Carneiro Natalia Ketelut , Galia Liraz , Wilson Ruth , Vierbuchen Tim , Chen Yongzhi , Ward Doyle , Fitzgerald Katherine A
Published: January 2023

Hepatocyte Nuclear Factor 4A (HNF4A) is a highly conserved orphan nuclear receptor that has been associated with ulcerative colitis. Mice lacking Hnf4a specifically in the intestinal epithelial cells (IECs) are more susceptible to DSS-induced colitis and develop spontaneous colonic inflammation with aging. However, the mechanisms how HNF4A maintains intestinal homeostasis and protects against colitis remain unclear. Here we demonstrate that epithelial HNF4A shapes the intraepithelial lymphocyte (IEL) compartment by selectively promoting the development of natural IEL subsets that are TCRgd+ or TCRab+ CD8aa+. Mechanistically, HNF4A serves as a transcription factor that directly regulates epithelial expression of immune signaling molecules involved in the IEC-IEL crosstalk. Those molecules include the butyrophilin-like (Btnl) molecules Btnl1 and Btnl6. Further, while HNF4A is stably expressed along the intestinal tract, these molecules are more abundant in the small intestine, which correlates with the distribution of natural IELs. We propose HNF4A paralog, HNF4G, which is restricted to the small intestine, cooperates with HNF4A in driving expression of signaling molecules and accounts for the geographic specificity. Moreover, HNF4A-BTNL regulatory axis is conserved in Caco2 cells of human colorectal origin. Collectively, our findings underscore the importance of HNF4A as a conserved transcription factor controlling IEC-IEL crosstalk and suggest that HNF4A maintains intestinal homeostasis through regulation of the IEL compartment.
Spatially mapping T cell receptors and transcriptomes
Authors: Liu Sophia , Iorgulescu Julian Bryan , Li Shuqiang , Barrera-Lopez Irving A , Borji Mehdi , Shanmugam Vignesh , Morriss Julia W , Garcia Zoe N , Murray Evan , Braun David A , Livak Kenneth J , Wu Catherine J , Chen Fei
Published: January 2023

T cells mediate antigen-specific immune responses to disease through the specificity and diversity of their T-cell receptors (TCRs). Although determining the spatial distributions of T cell clonotypes in tissues is essential to understanding T cell maturation and behavior, spatial sequencing methods remain unable to profile the TCR repertoire. We have developed Slide-TCR-seq, a 10-μm-resolution method to sequence whole transcriptomes and TCRs within the spatial context of intact tissues. We first confirmed the ability of Slide-TCR-seq to map the characteristic architecture of T cells and their receptors in mouse spleen. In human reactive lymph node and tonsil, we identified spatially distinct TCR repertoires both between germinal center (GC) and non-GC regions as well as between GCs. We then spatially profiled T cell clonotypes and their infiltration in renal cell carcinoma specimens before and following immune checkpoint blockade, which identified uniquely distributed T cell clonotypes and transcriptional states, highlighting the infiltration and expression heterogeneity both between clonotypes and within a T cell clonotype. Our method is anticipated to facilitate dissection of the immune microenvironment, yielding insights into the complex spatial relationships between T cell clonotypes, neighboring cell types, and gene expression that drive T cell development and response to disease.

This work was supported in part by the NIH U19AI082630, the Dana-Farber/Harvard Cancer Center Kidney Cancer SPORE (NCI P50CA101942), and the NCI IMAT grant.
A rapid and fully automated in vitro micronucleus assay using imaging flow cytometry and convolutional neural network analysis
Authors: Mendoza Maria Gracia Garcia , Sutton Alex , Kong Raymond , Rodrigues Matthew , Pugsley Haley
Published: January 2023

Micronuclei (MN) originate from whole chromosomes or chromosome fragments that lag behind during cell division and fail to be incorporated into one of the two main nuclei. As a result, scoring MN using the well-established in vitro micronucleus assay evaluates the ability of chemicals or other agents to induce DNA damage. This technique is typically performed by manual microscopy, which can be time-consuming and prone to variability. Additionally, automated methods lack cytoplasmic visualization when using slide-scanning microscopy, and conventional flow cytometry doesn’t provide visual confirmation of MN.

The ImageStream®X Mk II (ISX) imaging flow cytometer combines the high-resolution imagery of microscopy with conventional flow cytometry’s speed and statistical robustness in a single system. Previously, we developed a rapid and automated MN assay based on high-throughput image capture and feature-based image analysis using IDEAS® Software. However, the feature-based analysis was not readily applicable to multiple cell lines and chemicals, so we developed a deep learning method based on convolutional neural networks to score imaging flow cytometry data in both the cytokinesis-blocked and unblocked versions of the MN assay using Amnis® AI Software.

Our current study validates our previously established assay and analyses using three different chemicals (Mitomycin C, Cyclophosphamide, and Eugenol) and three different cell lines (TK6, L5178Y, CHO-K1). Here, we demonstrate how using Amnis AI to score imagery acquired on the ISX provides a rapid and fully automated in vitro MN assay with improved accuracy, reproducibility, and time-to-results in toxicity and biodosimetry applications across multiple cell lines.
Derivation of a parsimonious Tuberculosis gene signature using the digital NanoString nCounter platform.
Authors: Kaipilyawar Vaishnavi , Zhao Yue , Wang Xutao , Joseph Noyal M , Babu Senbagavalli Prakash , Hochberg Natasha S , Sarkar Sonali , Horsburgh Charles , Ellner Jerrold J , Johnson William Evan , Salgame Padmini
Published: January 2023

Rationale

Currently, there are several host immune response-based biomarkers to triage individuals suspected to have Tuberculosis (TB), to predict TB treatment failure and to predict risk of progression from latent TB infection (LTBI) to TB disease. However, validation of these biomarkers in diverse populations, with well-defined clinical endpoints, has been scarce. Moreover, a robust platform to validate TB biomarkers is essential to the development of a universal diagnostic or prognostic clinical test.

Objectives

NanoString nCounter technology is an amplification-free, highly sensitive digital detection platform that directly measures gene expression of up to 800 targets. Here, we investigated whether NanoString technology could serve as a platform to extensively validate existing TB biomarkers, in a new cohort from South India.

Methods

A NanoString Codeset that probes 107 genes (NS-TB107), comprising 12 published TB gene signatures and 6 house-keeping genes was developed to quantify RNA from whole blood samples of 80 pulmonary TB patients and 102 individuals with LTBI. The TBSignatureProfiler software was used to score samples for each signature. An ensemble of machine-learning algorithms were used to derive a parsimonious gene signature.

Results

Gene signatures present in NS-TB107 had statistically significant power to distinguish TB from LTBI in our cohort. A six-gene set (NANO6) derived from this cumulative data, when tested on 10 publicly-available transcriptomic datasets, was highly diagnostic for active TB.

Conclusions

The NanoString nCounter system provides an efficient platform to validate a panel of existing TB biomarkers and to derive a highly-diagnostic parsimonious candidate TB biomarker.

This project has been funded by Award No. USB-31150-XX-13 of the US Civilian Research & Development Foundation (CRDF Global) and by the National Science Foundation under Cooperative Agreement No. OISE-9531011 with Federal funds from the Government of India’s (GOI) Department of Biotechnology (DBT), the Indian Council of Medical Research (ICMR), the United States National Institutes of Health (NIH), National Institute of Allergy and Infectious Diseases (NIAID), Office of AIDS Research (OAR), and distributed in part by CRDF Global.
StarBright Dyes:-Build bigger better panels with superior dyes excitable by the violet and ultraviolet lasers.
Authors: Blundell Michael , Sanderson Sharon , Huang Leon , Cichewicz Magdalena , Li Lei
Published: January 2023

StarBright UltraViolet and StarBright Violet Dyes are the brightest ultraviolet and violet laser excitable fluorescent dyes specifically developed for flow cytometry. Their narrow excitation and emission mean that despite the increased brightness there is no loss of resolution due to excessive spillover and spreading. StarBright Dyes can be pre-mixed, fixed and have no requirement for a special buffer when multiplexing, providing researchers with the flexibility to build bigger, better panels.

Here we demonstrate this flexibility by comparing freshly made and premixed antibody cocktails from Bio-Rad Antibodies to immunophenotype human peripheral blood. Cells were stained using common staining buffers with acquisition on the ZE5 Cell Analyzer to show how bigger multiplex panels can be built with ease.
Expanding the size of multicolor panels with ease with new StarBright Blue and StarBright Yellow Dyes.
Authors: Blundell Michael , Sanderson Sharon , Huang Leon , Cichewicz Magdalena , Li Lei
Published: January 2023

Bio-Rad is committed to improving multicolor flow cytometry and is excited to announce a novel series of StarBright Dyes, exceptionally bright fluorescent nanoparticles developed for use with flow cytometry antibodies. These bright dyes have the additional benefits of narrow excitation and emission, high stability, minimal lot-to-lot variation, no requirement for a special buffer and can be fixed making them ideal for inclusion in multicolor immunophenotyping panels containing multiple antibodies conjugated to StarBright Dyes and traditional fluorescent dyes.

Here we show how new blue (488 nm) and yellow/green (561 nm) laser excitable StarBright Dyes can be incorporated into multicolor panels in both conventional and full spectrum flow cytometry to give unrivalled resolution, flexibility and choice. Available by the end of 2022 these new dyes will be available conjugated to our highly cited, validated antibodies.
DEFINING THE ROLE FOR THE GUT MICROBIOME IN THE CLINICAL EFFICACY OF SULFASALAZINE THERAPY FOR IBD ASSOCIATED SPONDYLOARTHRITIS
Authors: Lima Svetlana Ferreira , Rupert Amanda , Viladomiu Monica , Marderstein Andrew , Pires Silvia , Putzel Gregory , Woo Viola , Funez-dePagnier Gabriela , Jin Wen-Bing , Guo Chun-Jun , Scherl Ellen , Longman Randy S
Published: January 2023

Joint inflammation (spondyloarthritis, SpA), is the most common extra-intestinal manifestation of inflammatory bowel disease (IBD), but the specific role for therapies targeting SpA is not well defined. Sulfasalazine (SAS) is a prodrug composed of two chemical moieties, 5-aminosalicylate and the anti-folate antibiotic sulfapyridine, with efficacy in peripheral arthritis. Our study aims to evaluate the role for the gut microbiome in clinical response of SpA to SAS and to define microbial mechanisms targeted by SAS.

We longitudinally follow IBD patients with SpA who have a medical indication for SAS therapy. Clinical data and fecal samples from 22 patients were collected before initiation of SAS and at week 12 after initiation of SAS. The fecal microbiome of SAS-responders was distinct from that observed in non-responders and 6 pre-treatment microbial markers (including the short chain fatty acid (SCFA) producer Faecalibacterium prausnitzii) predicted SAS-response (AUC=0.9). Fecal metabolome of SAS responders had lower thymine and higher deoxyuridine compared to non-responders consistent with evidence of a folate trap in response to SAS treatment. SAS therapy in SPF mouse-model of chemically-induced colitis alleviated colitis in GPR 109a- and 43-dependent fashion consistent with a synergistic role for SCFA. In vitro and in vivo models revealed SAS direct regulation of F. prausnitzii transcription and metabolic function and its impact on host immune response.

Collectively, these findings highlight the potential role for microbial diagnostics to improve SAS efficacy, and drug modulation of microbial markers to potentiate therapy for IBD patients with SpA.

Supported by New York Crohn’s Foundation
Bispecific CD8 Treg modulators regulate a novel regulatory CD8 T cell network and eliminate pathogenic CD4 T cells in live cell co-culture system
Authors: Gardell Jennifer L , Crane Courtney , Bowser Justin , Fasnacht Rachael , Julien Susan , Maurer Meghan E , Templeton Megan , Therriault Jon , Yang Su Jung , Swiderek Kristine M
Published: January 2023

INTRODUCTION

Others have described a subset of CD8 T cells (CD8 Treg) with immunosuppressive characteristics in inflammatory disease settings. CD8 Treg activation through canonical T cell receptors results in their oligoclonal expansion and perforin dependent elimination of pathogenic CD4 T cells. We have described the CD8 Treg network in Celiac patients and its potential to eliminate pathogenic CD4 T cells. Here we describe bispecific CD8 Treg modulators that activate CD8 Tregs, resulting in pathogenic CD4 T cell death.

METHODS

Novel bispecific CD8 Treg modulators were tested for target specificity by Octet and cell binding. The functional impact of CD8 Treg modulators was evaluated using flow and Luminex assays, and in a live cell co-culture system using Celiac patient derived CD8 Tregs and gliadin activated CD4 T cell targets. CD8 Treg modulators with specific molecular formats were then ranked for functional efficacy.

RESULTS

We tested a panel of bispecific CD8 Treg modulators with monovalent binding affinities in the low nanomolar range that selectively bind the CD8 Treg subset. Within 48 hours of CD8 Treg modulator addition to T cell co-cultures, CD8 Tregs had a rapid increase of cytolytic markers and degranulation in response to CD4 T cell targets. We observed a concomitant increase of activated CD4 T cell death and decrease of pro-inflammatory cytokines.

CONCLUSIONS

Our results indicate that a targeted approach to engage and activate CD8 Treg reduces pathogenic CD4 T cells. As this newly described CD8 Treg cell network is observed in many autoimmune diseases, we postulate that bispecific CD8 T cell modulators may represent a novel, selective and broadly applicable therapeutic approach for the treatment of human autoimmune diseases.
Therapeutic regulation of autoreactive B cells using bispecific molecules
Authors: Griffith Amber Simone , Apley Kyle , Downes Grant , Johnson Stephanie , Ross Patrick , DeKosky Brandon , Farrell Mark , Berkland Cory , Kendall Peggy L
Published: January 2023

Type 1 diabetes (T1D) occurs when autoreactive T cells destroy insulin-producing beta cells. Anti-insulin B cells promote this disease, acting as essential antigen-presenting cells. Rituximab, which globally depletes B cells, provided temporary preservation of insulin production in early onset T1D, but increases susceptibility to infection. Antigen-specific immune therapy (ASIT) may be a viable option for treating T1D by targeting anti-insulin B cells for removal from the B cell repertoire. In this study we investigate the efficacy of soluble antigen arrays containing insulin (SAgAins) developed by the Berkland lab. These constructs consist of a polyethylene glycol (PEG) backbone with four linker arms for bioconjugation. We conducted in vitro experiments to investigate the effects of constructs containing insulin and a trisaccharide ligand (CD22L) for inhibitory B cell receptor CD22 on transgenic anti-insulin B cells. We found that incubating insulin-PEG with anti-insulin B cells stimulates proliferation and CD86 upregulation. However, when CD22L is added (CD22L-PEG-insulin) the expression of CD86 is dampened. This suggests that this novel construct harnesses the regulatory power of CD22 to dampen activation of B cells in an autoantigen-specific manner.
Impact of Swine Leukocyte Antigen on renal endothelial cells in pig-to-non-human primate xenotransplantation in vitro
Authors: Wang Zheng-Yu , Reyes Luz , Estrada Jose , Tector Mathew , Tector Alfred
Published: January 2023

BACKBROUND

The development of a pig lacking the GGTA1, B4GNT2 and CMAH genes (TKO) that produce the three known glycan xenoantigens has provided the decreased background human antibody binding to TKO cells. It can be easily detected for antibody binding to sufficiently low levels of any additional xenoantigens expressed on the pig cells, including swine leukocyte antigen (SLA). The object of this study is to evaluate the role of SLA class I and II in the pig-to-non-human primate (NHP) xenotransplantation in vitro at an organ-specific cell level using renal endothelial cells (REC).

METHODS

Characteristics of genetically-edited REC and Interferon gamma (IFN-γ)-activated REC were analyzed using flow cytometry. Rhesus IgG/IgM binding to REC with or without SLA class I, or SLA class II expression were examined using flow cytometric crossmatch and complement-dependent cytotoxicity assay (CDC).

RESULTS

SLA I-deficient REC with GGTA1/B4GNT2/CMAH TKO background had significantly lower Levels of antibody binding (p=0.02 and p=0.0001, respectively for IgG and IgM) using 25% rhesus sera (n=14). SLA class II expression on SLA I-deficient GGTA1/B4GNT2 REC was induced following 3-day treatment of IFN-γ. These cells with SLA class II expression had significantly increased IgM binding by crossmatch (p=0.002), and had significantly high levels of cell lysis by CDC assay (p<0.0001) using 25% rhesus sera (n=11).

CONCLUSIONS

Gene editing of the SLA class I and II on pig organs might protect against chronic antibody-mediated rejection via reducing antibody binding to REC in pig-to-NHP renal xenotransplantation. Porcine REC cell lines will be a useful reagent for the further study of xenoimmunology.

Supported by Partial 5R01AI126322
Targeting temporal metabolic needs of T follicular helper cells to treat cGVHD utilizing a mitochondrial pyruvate carrier inhibitor
Authors: Mohamed Fathima A , Rhee Stephanie Y , Ly Joanna , Aguilar Ethan G , Sage Peter T , Schumacher Tanner , Thangavelu Govindarajan , Zaiken Michael C , Liu Juan , Mereddy Venkatram , Locasale Jason W , Blazar Bruce R
Published: January 2023

The curative potential of allogeneic hematopoietic stem cell transplantation is limited by development of chronic graft versus host disease (cGVHD) and metabolic regulation of immune cells is a promising therapeutic target. During cGVHD, interactions between T follicular helper (TFH) and germinal center B (GCB) cells result in production of alloreactive class-switched antibodies that attack host tissues, causing multi-organ damage and tissue fibrosis. We show the metabolic dependencies of TFH change throughout cGVHD progression and targeting mitochondrial pyruvate transport is an effective strategy to mitigate disease. Pyruvate, the end product of glycolysis, is transported by the mitochondrial pyruvate carrier (MPC) for use in the tricarboxylic acid cycle and oxidative phosphorylation (OXPHOS). TFH require glycolysis in order to induce a GC reaction, both in an immunization model and in an in vivo systemic cGVHD model with bronchiolitis obliterans (BO). Furthermore, Seahorse data from TFH extracted early in disease development in the BO model demonstrate increased dependency on glycolysis. But Seahorse data also indicates TFH undergo a shift away from glycolysis and toward OXPHOS as cGVHD continues. Based on the central role of MPC in tying pyruvate to multiple metabolic pathways, we examined an inhibitor of MPC (7ACC1) in reducing TFH activity. Administration of 7ACC1 resulted in inhibition of GCB production of class switched antibody in an in vitro assay. These findings were additionally supported in vivo, whereby daily treatment after establishment of cGVHD resulted in a decrease in disease severity. These data provide evidence that inhibiting mitochondrial pyruvate transport is a viable therapeutic strategy for cGVHD.

This work was supported by grants from National Institutes of Health, National Institute of Allergy, and Infectious Diseases R37 AI34495, T32 AI007313; National Heart, Lung, and Blood Institute R01 HL56067, R01 HL11879, R01 HL115114; National Cancer Institute P01 CA065493