bluuebunny/crossref_metadata_2025_split huggingface dataset:
Tim-3 expression in Treg controls viral persistence and effector T cell response during chronic LCMV infection.
Authors: Nieves-Rosado Hector M , Banerjee Hridesh , Kane Lawrence P 
Published: August 2023

Abstract
Tim-3, a surface protein, is upregulated on Treg during chronic viral infections. During HCV infection in humans there is an increase in frequency of Tim-3+ Treg, which have increased proliferation, survival, and expression of immunosuppressive cytokines like IL-10, compared to Tim-3− Treg. Similarly, it was recently reported that there is an increase in Tim-3+ Treg in people with HIV regardless of viral suppression. However, the role of regulatory T cells (Treg) during chronic viral infection has not been fully defined. We hypothesize that Tim-3 promotes an effector phenotype on Treg during chronic viral infection, which limits the virus-specific T cell response and impairs viral clearance. Using an inducible Treg-specific Tim-3 loss-of-function (Tim-3 Treg KO) model, we found a significant decrease in morbidity during LCMV chronic infection. Tim-3 Treg KO mice mounted a higher virus-specific T cell response and had lower viral burden in liver, and kidney at 30 days post-infection. Interestingly, we found that there was a decrease in the frequency of PD-1+Tim-3+ and PD-1+Tox+ LCMV-specific CD8 T cells. Lack of Tim-3 expression on Treg limited their ability to upregulate IL-10, TGFb, CD39, PD-1, CD25, CTLA-4 and CD44. Our findings demonstrate that modulation of a single surface protein in Treg can lead to a reduction in viral burden and enhance LCMV-specific T cell response. These studies may also help to identify Tim-3 directed therapies for the management of persistent infections and other chronic illnesses.
Supported by grants from NIH (T32GM008208, R01 AI138504)
Role of germinal center response in the antibody responses against SARS-CoV-2 spike protein
Authors: Khalil Jumana A T , Miyauchi Kosuke , Yamamoto Yuichiro , Noguchi Kohji , Takayama Kazuo , Kubo Masato 
Published: August 2023

Abstract
Spike-based vaccination strategies, including mRNA, effectively induce antibody responses against SARS-CoV-2. However, the breadth of antibody responses raised by Wuhan spike covering newly emerging variants remains unclear. Here, we investigate how the loss of function of GC responses and IL-4 signal influence the antibody and memory B cell responses against spike proteins of SARS-CoV-2 variants. In mouse model, immunization of C57BL/6J and C3H mice with trimeric recombinant spike protein of Wuhan-CoV-2 efficiently induced the production of antibodies capable of recognizing the receptor binding domain of delta, and omicron (BA1, and BA5) variants. The third boosting with Wuhan spike protein was also effective in inducing the antibodies to recognize omicron variants, but the boosting with BA5 was only effective for BA5, not Wuhan. We tested the neutralizing activity of these antibodies by the in vitro binding assay using psuedo-virus and human bronchial organoids infection system. These antibodies effectively blocked the infection of Wuhan and delta variants, but the protection was reduced for omicron variants. We further evaluated the contribution of the GC response and IL-4 using Bcl6 deficient mice (Bcl6f/f CD4-cre, Bcl6f/f CD4ER-cre) or B cell-specific deficient mice of the IL-4 receptor(R) (Il4rΔB mice). The results indicated that Bcl6 deficient mice and the mice lacking IL-4R signaling in B cells markedly reduced initial antibody responses and diminished protectivity of the antibodies against the variant virus. In conclusion, our results indicated that the GC response and IL-4 signal provides a quantitative checkpoint optimizing the breadth of anti-SARS-CoV2 protective antibody response.
Inulin enriched diet reinforces cancer immunosurveillance via γδ T cell activation
Authors: Boucher Emilie , Plazy Caroline , Lavie-Richard Mathias , Suau Antonia , Mangin Irène , Cornet Muriel , Aldebert Delphine , Toussaint Bertrand , Hannani Dalil 
Published: August 2023

Abstract
Gut microbiota is now recognized as a key player impacting the host’s anti-cancer immunosurveillance and ability to respond to immunotherapy. Its optimal modulation for preventive and therapeutic purposes is very appealing. Since diet is one of the most powerful modulators of the microbiota, nutritional interventions could be leveraged to improve host immunity. The present study aims to analyze the anticancer properties of inulin, a fiber known to promote bacteria described as immunostimulatory. The effects of inulin complementation were tested in mice fed an inulin-enriched diet for 2 weeks before tumor implantation. In the three tumor models used (melanoma, fibrosarcoma, and colorectal cancer), inulin consumption inhibited tumor growth by 80% compared to controls. Tumor-infiltrated immunity analysis showed that inulin triggers an enhanced Th1-polarized CD4+ CD8+ and γδ T cell-mediated antitumor response. In the gut, inulin consumption favored Bifidobacterium relative abundance, and strengthened gut immunity, by increasing the proportion of colonic IFNγ-producing γδ intra-epithelial T lymphocytes as well as the expression of genes involved in inflammation. In addition, we showed that the inulin-mediated anti-tumor effect is gut microbiota- and γδ T cell-dependent, as both antibiotic treatment and in vivo blocking of γδ TcR abrogate inulin efficacy. Overall, our data identified γδ T cells as a critical immune subset, mandatory for inulin-mediated antitumor immunity in vivo. This supports and rationalizes the use of a prebiotic approache as well as the development of immunotherapies targeting γδ T cells in cancer prevention and treatment.
This work is supported by GEFLUC Dauphiné-Savoie, Ligue contre le Cancer Comité Isère, Ligue contre le Cancer Comité Savoie, Université Grenoble Alpes IDEX Initiatives de Recherche Stratégiques. Emilie Boucher is supported by a grant salary from the French Ministry of Higher Education, Research and Innovation.
Ex vivo activation and fold-expansion of human regulatory T cells negatively correlates with preservation of beta cell function in adolescents with type 1 diabetes
Authors: Bender Christine , Hu Alex , Wiedeman Alice , De Castro Grace , Sietsema William K , Gitelman Stephen , Griffin Kurt , Long Alice 
Published: August 2023

Abstract
Type 1 diabetes (T1D) is an autoimmune disease marked by immune destruction of insulin-producing β-cells of the pancreas. In an effort to halt disease progression, autologous polyclonal expanded regulatory T cells (expTregs) were adoptively transferred into adolescents with recent-onset T1D (Sanford/Lisata Therapeutics Trex Phase 2 clinical trial, NCT02691247). Overall, expTreg therapy (1–24×106 cells/kg) did not preserve residual β-cell function, as measured by change in insulin C-peptide AUC-mean over 2 years compared to placebo (p=0.39) regardless of dose (p=0.27). Consistent with a more activated phenotype, expTregs expressed more CD25, CD38, HLA-DR, and CXCR3 than endogenous Tregs by flow cytometry, and bulk-RNAseq revealed 2377 genes (related to metabolism, cell cycle, and immune response) up-regulated in expTregs compared to endogenous Tregs. In subjects from the high-dose group (&gt;10×106 cells/kg), there was a transient increase of total Tregs one week after infusion (p=0.003), which had an activated memory phenotype, consistent with infused expTreg accounting for this difference. However, the increase in the activation of Tregs post-treatment was associated with poorer outcome (p=0.03), and the extent of expansion of the expTregs ex vivo (range 24.3–643-fold) negatively correlated with C-peptide change (p=0.01) in treated subjects regardless of Treg dose. Yet, neither purity nor suppressive function correlated with outcome, suggesting differences in endogenous Treg qualities leading to increased expansion may influence outcome. These results suggest that expTreg quantity alone does not influence outcome; instead, both endogenous and expTreg quality may be important factors modulating T1D progression.
High-throughput Western Blot for in vitro and in vivo assessment of human Ig specific cleaving enzymes
Authors: Le Nam , Pellerin Alex , Sanmarco Liliana , Green Tobias , Borah Minasri , Mande Purvi , Newton Andita , Hoyt Emily , Anderson Jordan , Bengeri Soumya , Higginson-Scott Nathan , Otipoby Kevin , Mascanfroni Ivan 
Published: August 2023

Abstract
Antibodies play a vital role in the body’s immune defense mechanism. However, pathogenic Ig antibodies are thought to contribute to several autoimmune conditions and transplant rejection. Proteases derived from human pathogens can specifically cleave IgG into F(ab’)2 and Fc fragments, thus allowing for rapid clearance of IgG. This unique trait suggests a novel opportunity to use these molecules to treat autoantibody mediated diseases.
Using our IMPACT platform leveraging machine learning, we have engineered Ig proteases that specifically cleave IgG, and treatment with these proteases causes cleavage below the hinge region, which can result in the generation of the following degradation products: 1) Fab’2 and Fc fragments lacking effector function; or 2) single chain cleaved fragments with reduced effector functions. To detect these species, we developed a method using JESS, an automated high throughput western blot instrument. Using this system, we can detect intact, single cleaved and fully cleaved IgG in a time and dose dependent manner in both in vitro and in vivo assays.
In conclusion, we successfully optimized a high throughput assay format that is highly specific and sensitive in evaluating the cleavage activity of different Ig proteases and could be a powerful tool to assess efficacy of IgG cleaving enzymes in the clinic.
Selective targeting of pathogenic auto-reactive VH4–34 B cells with a rationally developed anti-VH4–34 antibody offers a new therapeutic approach for autoimmune disorders
Authors: Paliwal Shalini , Thakkar Dipti , Chin Wen Jie , Lee Warren , Neo Jacklyn J Y , Tay Lavina , Hanson Brendon , Paszkiewicz Konrad , Ingram Piers , Boyd-Kirkup Jerome 
Published: August 2023

Abstract
B cells expressing B cell receptors (BCRs) specific for self-antigens are normally eliminated, or made anergic, through negative selection during B cell development. VH4–34 is an intrinsically autoreactive antibody (and BCR) heavy chain variable region that includes a germline encoded hydrophobic patch in framework region 1 (FR1). Autoantigens recognized by VH4–34 antibodies include N-acetyl lactosamine chains displayed on RBCs. In healthy individuals, &lt;1% of B cells express VH4–34 encoded BCRs but during the loss of self-tolerance, characteristic of pathogenic disorders, the hydrophobic region of VH4–34 reacts to self-antigen, erroneously driving BCR activation and expansion.
Expansion of VH4–34+ B cells and increased serum concentrations of VH4–34 antibodies has been implicated in systemic lupus erythematosus (50%) and virtually all cases of cold agglutinin disease. To date there have been no therapies against VH4–34 due to challenges of specifically targeting VH4–34 while avoiding other VH regions.
We applied Hummingbird Bioscience’s Rational Antibody Discovery approach to develop anti-VH4–34 antibodies that bind to a computationally predicted and lineage conserved epitope in the hydrophobic FR1 region. The lead antibody, HMBD-011, demonstrates selective and high affinity binding to all tested VH4–34 antibodies, with no off-target binding. The binding epitope of HMBD-011 was confirmed by mutagenesis to be within the FR1 region of VH4–34. Further, HMBD-011 shows selective binding to cells that express VH4–34 BCRs. In vivo, HMBD-011 treatment resulted in rapid elimination of VH4–34+ antibodies and cells.
In conclusion, HMBD-011 represents a promising precision therapy for VH4–34 driven autoimmune disorders.
STAT1 gain of function mutation impairs immune response to viral infections
Authors: Philips Rachael Laura , Liao Yi-Chu , Morrison Tasha , Chaitankar Vijender , Yao Chen , Oikonomou Vasileios , Milner Joshua , Lionakis Michail S , O’Shea John J 
Published: August 2023

Abstract
The coordinated effort of the immune system in host defense is dependent upon the proper release and interpretation of different cytokine signals. Several examples of monogenic diseases targeting the JAK-STAT pathway reveal there is still a critical need to understand basic principles of cytokine signaling and cytokine output. For example, patients with STAT1 gain-of-function (GOF) mutations exhibit a type 1/IFN-gamma bias that antagonizes a type 3/IL-17 immune response important for controlling fungal infection, yet patients also, paradoxically, exhibit chronic and sometimes lethal viral infections. Using a novel conditional knock in STAT1-GOF mouse model, we demonstrate that STAT1-GOF mice respond poorly to viral infections that are easily controlled by WT mice, such as MCMV and LCMV-Armstrong. High-dimensional flow cytometry and scRNA-seq reveal an impaired NK and CD8 T cell effector response in STAT1-GOF mice that is not simply due to an exhaustion phenotype. Instead, STAT1-GOF mice respond poorly to viral infections due to an unexpected defect in IFN-gamma production early during the innate immune response, leading to a cytokine storm, immunopathology, and an impaired adaptive immune response. scATAC/RNA-seq uncovers altered usage of different STAT complexes in STAT1-GOF mice, leading to a misinterpretation of early innate-stimulating cytokines. These results provide insight into why patients with interferonopathies or elevated interferons respond poorly to viral infections and highlight the importance of properly tuning STAT1 during an immune response.
R.L.P. was supported by a Postdoctoral Research Associate (PRAT) fellowship from the National Institute of General Medical Sciences (NIGMS), award number 1Fi2GM137942-01
Single cell transcriptomes from HCMV+ donors reveal a unique NKG2CHigh subset that represents true memory NK cells
Authors: Khalil Mohamed , Malarkannan Subramaniam , Terhune Scott 
Published: August 2023

Abstract
Infection with human cytomegalovirus (HCMV) can result in severe morbidity and mortality to the developing fetus and immunocompromised individuals. Natural killer (NK) cells are cytotoxic lymphocytes that are required to manage and control HCMV infections. The NKG2C receptor expressed by NK cells recognizes and responds to infected cells expressing HLA-E loaded with HCMV UL40-derived peptide. These responding NKG2C+ NK cells have been referred to as ‘adaptive NK’ because they possess features of adaptive immunity and are found at greater percentages in HCMV seropositive individuals. The molecular mechanisms by which adaptive NK cells are generated and their transcriptomic signatures remain unknown. To investigate this, we obtained healthy adult spleens from four HCMV+ and four HCMV− donors and their NK cells were sorted and captured for single-cell RNA-sequencing (scRNA-seq). Donor median age was 59 [IQR 48.5–56.5], 50% (n=4) were female, 50% (n=2) of females were HCMV seropositive and 50% (n=2) of females were HCMV seronegative. Using scRNA-seq, we observed elevated expression and cell numbers of NKG2C in HCMV+ donors when compared to HCMV-donors. We identified a distinct population of NKG2C+ adaptive NK cells displaying elevated levels of CD52, B3GAT1, FCGR3A, CD3E, and IL7R. In addition, our developmental trajectory analysis of adaptive NKG2C+ NK cells revealed a unique branchpoint of true memory NK cells expressing elevated levels of NKG2C and IL7R. Here, we demonstrate that HCMV infection can induce the formation of adaptive NKG2C+ NK cells that display a unique transcriptional and developmental profile. These findings have the potential to influence the future application of adaptive NK cells in cellular immunotherapies.
This work is supported by the National Center for Advancing Translational Sciences, National Institutes of Health, through grant number TL1TR001437
Oral tablet vaccination against SARS-CoV-2 spike boosts cross-reactive serum and mucosal neutralizing antibodies following mRNA vaccination: a phase 2 clinical trial
Authors: Johnson Susan , Jegede Clara B , Gutierrez Samanta , Greco Susan , Cummings James , Gottlieb Keith A , Tucker Sean N 
Published: August 2023

Abstract
Current SARS-CoV-2 intramuscular vaccines do not induce the breadth of immunity needed to halt the development of new variants. New strategies that elicit immunity at the site of infection could induce cross-reactive mucosal responses that decrease transmission.
We developed a room-temperature stable oral tablet vaccine composed of recombinant adenovirus type 5 expressing the spike gene of SARS-CoV-2. In an open label phase 2 clinical trial (NCT05067933), 66 subjects, either naïve or previously mRNA vaccinated, received 2 doses of a low or a high dose 28 days apart. The vaccine was well tolerated with a benign safety profile.
Among previously vaccinated subjects, oral tablet vaccination was able to boost serum IgG and IgA at 29 and 57 days post-vaccination. The serum neutralizing antibodies increased 1.6-fold, from 481 AU/ml to 778 AU/ml at Day 57. The subjects with lower starting titers had greater increases after oral boosting. Most subjects in the vaccinated cohorts had an increase in SARS-CoV-2 IgA in the nose or saliva. This mucosal IgA was not only cross-reactive against SARS-CoV-2 variants but also SARS-CoV-1 and MERS. These samples demonstrated surrogate neutralizing activity against both Wuhan and omicron BA4/5, despite the vaccine expressing Wuhan antigens, highlighting the ability of mucosal vaccination to elicit cross-reactive responses.
Overall, this vaccine was safe and well tolerated. It induced cross-reactive immunity in both the serum and more importantly, in the mucosa, the site of infection. This offers an ideal platform for boosting immune responses that could decrease transmission rates and extend protection to emerging variants. It also offers unique advantages in distribution to curb future pandemics.
An in vivo overexpression system to study the functions of complement activated products in a mouse model of lupus
Authors: Wang Fang , Wu Yee ling 
Published: August 2023

Abstract
Complement cleavage products generated during activation serve a wide range of functions from immune defense against microbes to homeostasis. However, the functions of complement activated products such as C4a, albeit present at high levels in lupus, remain unknown. Studying the functions of complement activated products is challenging. Studies using purified complement proteins can be confounded by minute contaminants with potent effects. Transgenic animals often provide more unequivocal insights. However, the generation of transgenic animals is highly involved and time-consuming, especially when backcrossing to genetically susceptible backgrounds is required for the development of diseases. Here, as an illustration for studying the functions of a complement activated product in vivo, we employed hydrodynamic injections to deliver expression plasmids encoding C4a to mouse liver. This technique allows hepatocytes to synthesize and directly secrete C4a into the circulation without disrupting intact C4. Using this system, we were able to detect exogenous C4a protein &gt;2 months post-injection in the lupus prone MRL/lpr mice. Preliminary phenotyping on MRL/lpr mice overexpressing C4a revealed two immunologically relevant features: 1) the frequency of T cells was reduced and 2) the levels of autoantibodies against double-strand DNA were elevated comparing with vector controls. These data suggest that C4a can potentially modulate lupus manifestations. While detailed analyses are ongoing to determine the functions of C4a in lupus, our study illustrates that in vivo overexpression using hydrodynamic injections represents a relevant approach for studying the functions of complement activated products in disease models.
supported by the Lupus Research Alliance
Spatial immunoprofiling of tissue sections with ChipCytometry&[trade]
Authors: Sindelar Tim , Stanslowsky Nancy , Northcutt Adam 
Published: August 2023

Abstract
Highly multiplexed spatial biomarker analysis has demonstrated the potential to advance our current understanding of the immune system and its role in cancer, from tumor initiation to metastatic progression. Previously, a trade-off between plex and spatial context meant that our understanding of immune cell involvement in cancer was limited to providing deep information on either cell phenotypes or their spatial context, but not both. ChipCytometry is a novel, highly multiplexed technology that preserves both plex and spatial context to deeply profile immune cell diversity at single-cell resolution. ChipCytometry uses commercially available antibodies and combines iterative immuno-fluorescent staining with high-dynamic range imaging to profile dozens of protein biomarkers in a single tissue specimen. Cellular phenotypes are identified with via flow cytometry-like hierarchical gating from standard multichannel OME-TIFF images, compatible with a variety of computational tools being developed for multiplexed analysis and visualization. Here, we use ChipCytometry to identify and quantify key immune cell subtypes in FFPE tissues. The results show precise expression levels for each biomarker in the assay in each individual cell in the sample, while maintaining spatial positioning of each cell. Spatial analysis reveals quantifiable heterogeneity of immune cell infiltration within the tumor samples, demonstrating the utility of the ChipCytometry platform for the in-depth immune profiling in FFPE samples.
High-Throughput Antibody Discovery Using Barcode Enabled Antigen Mapping (BEAM
Authors: Shahi Payam , Adams Bruce , Reyes Daniel , Maheshwari Shamoni , Mousavi Nima , Krishnan Sreenath , Ramen Nandhini , Tsai FuNien , Kumar Poornasree , Finnegan Peter , Fernandes Sunjay , Bali Gunmeet Kaur , Terry Jessica 
Published: August 2023

Abstract
Multi-analyte single cell technologies have potential to greatly accelerate antibody discovery by identifying antigen-specific B cells. Barcode Enabled Antigen Mapping (BEAM) provides a high-throughput, multimodal analysis of antigen-bound B cells (BEAM-Ab) using 10x Genomics Chromium Single Cell Immune Profiling v2 Solution and novel computational approaches to discover B-cell receptor (BCR) sequences for further functional characterization.
To demonstrate the antigen sensitivity of BEAM-Ab, we spiked in 5% Hen Egg Lysozyme (HEL) and 5% gp120 transgenic B cells in 90% wild type non-transgenic splenocytes. The cells were screened using a panel of barcoded antigens and then CD19+PE+ (antigen+) B cells were isolated using flow cytometry. Single cell gene expression, BCR, and antigen barcode analysis indicated clonotype specificity to the respective antigens in the sorted cells. Furthermore, we demonstrated BEAM-Ab specificity by analyzing a convalescent COVID patient sample against an antigen panel containing five different COVID antigens and a negative control. Analysis of this COVID sample suggests the clonal expansion of B cells recognizing the ectodomain of the wild type SARS-CoV2 spike protein, receptor binding domain of spike protein, and the ectodomain of the D614G mutant spike protein. We did not observe any clonal expansion to Omicron-specific antigen or the non-specific negative control in our dataset.
Our data demonstrates that BEAM-Ab is an effective antibody discovery tool. BEAM-Ab unlocks the ability to rapidly screen large numbers of samples with an accurate single cell resolution and antigen specificity, with the entire workflow generating the candidate sequences just one week after sample processing.