bluuebunny/crossref_metadata_2025 huggingface dataset:
A single-tube, 44-marker CyTOF assay to assess antigen-specific immunity in whole blood human samples with data analysis solution
Authors: Mujib Shariq , Li Stephen , Zabinyakov Nick , Loh Christina
Published: February 2023

Monitoring the immune response in the setting of infectious disease and cancer is critical to assess disease status and targets of immune therapy. CyTOF® mass cytometry enables multiplex cellular phenotyping with more than 50 markers, making it ideal for comprehensive immune profiling. CyTOF technology utilizes antibodies tagged with unique monoisotopic metals, resulting in distinct signals that provide a high-resolution multiparametric landscape of a single cell. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated, dried-down, 30-marker antibody cocktail for immune profiling of human whole blood and PBMC by CyTOF. Paired with Maxpar Pathsetter™ software, stained samples are automatically resolved into 37 immune populations including major lineages and their subsets. In this study, we expanded the 30-marker assay to a 44-marker panel including exhaustion markers such as PD-1 and CTLA-4, co-stimulation markers 4-1BB and ICOS, and intracellular cytoplasmic markers IFN-γ, TNF-α, IL-2, perforin and granzyme B to assess cellular function in PMA/ionomycin-stimulated whole blood cultures. We modified the existing Maxpar Pathsetter model to automate the analysis of the expanded panel and report on additional functional parameters such as T cell exhaustion and cytokine production. Next, we applied this panel to whole blood stimulated with CMV peptides to investigate antigen-specific immune responses in a viral infection model in concert with in-depth phenotypic assessment. Collectively, we demonstrate the flexibility of the Maxpar Direct Immune Profiling Assay to incorporate additional surface and intracellular markers to study antigen-specific immunity in the context of whole blood immune profiling.

For RUO.
Practical Cell Counting Method Selection to Increase the Quality of Cell Counting Results
Authors: Chan Leo , Qiu Jean
Published: February 2023

The importance of cell counting has increased significantly in the last decade due to the major advances in the fields of cell and gene therapy, biologics production, and regenerative medicine. This has necessitated the development of a standardized approach to cell counting assays. In the recent years, the U.S. Food and Drug Administration (FDA), in collaboration with the National Institute of Standards and Technology (NIST) and the International Organization for Standardization (ISO), has launched an effort to standardize cell counting methods to improve the confidence in cell counting measurements.

There is a wide range of biological sample types, various formulations, and bioprocessing steps for cell and gene therapy products. Furthermore, there are no ground truth reference materials for live cells makes determining the accuracy of cell counting difficult defined in the ICH Q2 (R1). Therefore, in order to increase the confidence of cell counting results, it is critical to follow the two recently published ISO cell counting standards to ensure cell counting methods are tailored to the specific sample and purpose.

In this work, we will provide insights and guidance for the ISO standards for method selection. The important aspects of the ISO cell counting standards have been distilled to six key factors for selecting the cell counting methods. Attention to these details will allow researchers to more easily adhere to the ISO recommendations to ensure high quality cell counting measurements, and in doing so, sites can meticulously document their rationale, process, and data results.
Development of a dried pre-formulated antibody panel (7 colors/17antibodies) for the identification of human innate lymphoid cells by flow cytometry
Authors: Lacaille Vashti , Miloud Tewfik
Published: February 2023

Innate lymphoid cells (ILC) play an important role in innate immunity, lymphoid organogenesis and tissue remodeling. They form a heterogeneous population that can be categorized into 3 groups (i.e. ILC1, ILC2 and ILC3) based on their phenotype and/or distinct patterns of cytokine production. The identification and enumeration of different blood ILC subsets is important to understand immune regulation in disease models such as allergy, cancer and inflammation. Flow Cytometry is the method of choice for analysis of ILCs in human blood. However, as ILC frequencies may range below 0.1% of white blood cells, considerably elevated sample volumes and/or cell-enrichment is required. Furthermore, due to the lack of specific markers for definition of ILCs, at least 15 antibodies are required for mere identification of ILCs, including a lineage-exclusion channel to exclude most of the leukocytes. Based on current knowledge in human ILC biology, we have composed a respective set of markers to design a dry unitized 17 antibodies/7-color panel (CD294/Lineage/CD117/Nkp46/CD127/CD161/CD45) for flow cytometry. The panel in dry DURAClone format allows for the reliable identification of all ILCs subsets in whole blood or peripheral blood mononuclear cell fractions (PBMCs) in concordance with current scientific opinion. Due to the elimination of the pipetting of 17 antibodies, the method can substantially reduce variability and error related to sample preparation. Furthermore, the streamlined workflow can facilitate inter-laboratory transfer of the assays in multi-site studies.
Targeting SLP76:ITK interaction separates GVHD from GVL in allo-HSCT
Authors: Karimi Mobin A , Mammadli Mahinbanu Mahinbanu , Huang Weishan , Harris Rebecca , Weeks Samuel , May Adriana , Gentile Teresa , Henty Ridilla Jessica L. , August Avery , Karimi Mobin
Published: February 2023

Allogeneic hematopoietic cell transplantation (allo-HCT) is a curative therapy for relapsed hematological malignancies due to graft-versus-leukemia (GVL) activity mediated by alloreactive donor T cells. However, graft-versus-host disease (GVHD) is also primarily mediated by the same donor T cells. Here we assessed the effect of attenuating TCR-mediated SLP76:ITK interaction in T cell-mediated GVL vs. GVHD effects after allo-HSCT. Neither CD8+ T cells nor CD4+ donor T cells from mice expressing a tyrosine to phenylalanine mutation at position 145 (Y145F) of the adapter protein SLP-76 caused GVHD, T cells cells preserved GVL effects after allogeneic transplantation. SLP76Y145FKI CD8+ and CD4+ donor T cells also produce less inflammatory cytokines and show decreased migration to GVHD target organs such as the liver and small intestine, while maintaining GVL efficacy against primary B-cell acute lymphoblastic leukemia (B-ALL). We also report the development of a novel peptide that can specifically inhibit SLP76 and ITK interactions, which results in decreased phosphorylation of PLCγ1 and ERK, and decreased cytokine production in human T cells. This peptide inhibited donor T cell-mediated GVHD while maintaining GVL effects. Altogether, our data suggest that inhibiting SLP76:ITK interaction could be a therapeutic strategy to reduce GVHD while retaining the beneficial GVL effects after allo-HSCT treatment.
TCF-1 acts as a positive regulator in alloactivated T cells, and TCF-1 deficiency reduces GVHD yet maintains GVT responses
Authors: Harris Rebecca , Karimi Mobin A
Published: February 2023

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for hematological malignancies. Mature donor T cells in this graft help kill residual malignant cells (graft-versus-tumor or GVT effect). However, donor T cells also damage healthy host tissue, causing graft-versus-host disease (GVHD) in about 30–70% of allo-HSCT patients. These alloactivated T cells proliferate, migrate to target organs (liver, skin, and small intestine), and produce cytokines during GVHD. Our work focuses on modulating T cell signaling to separate GVHD from GVT, as GVHD is the main clinical barrier to widespread use of allo-HSCT.

T Cell Factor-1 (TCF-1) is critical for T cell development and activation, but its role in alloactivated and mature T cells is unclear. Using a mouse model of MHC-mismatched allo-HSCT leading to GVHD, we examined the role of TCF-1 in this context. Loss of TCF-1 specifically in T cells led to reduced severity and persistence of GVHD, and reduced host tissue damage from both CD4 and CD8 T cells, yet maintained GVT effects. Cytokine production, proliferation, and survival of donor T cells, as well as donor T cell phenotype, were all affected by loss of TCF-1. Therefore, TCF-1 controls mature T cell phenotype and function during allo-HSCT, and is indispensable for GVHD damage. TCF-1 is dispensable for GVT effects, because loss of this factor does not disrupt antitumor immunity. Thus, loss of TCF-1 separates detrimental GVHD damage from beneficial GVT effects.

This work will help to improve outcomes for allo-HSCT patients, who have poor survival due to GVHD and increased risk of deadly infections. This research also enhances our basic understanding of how alloactivated mature T cells are regulated during allo-HSCT.
IL-33 is a critical early activation and differentiation signal for alloreactive CD4+ T cells that enables their infiltration into GVHD target tissues
Authors: Dwyer Gaelen K , Mathews Lisa , Lucas Anna , Blazar Bruce R , Shlomchik Warren , Turnquist Heth Roderick
Published: February 2023

Graft vs host disease (GVHD) is a lethal complication after allogeneic stem cell transplantation (alloSCT), where donor T cells destroy host tissues. To create space for donor cells and prevent their rejection, alloSCT protocols use recipient conditioning that augments the stromal-derived cytokine interleukin (IL)-33 in the lymphoid organs (LO) and tissues. While we have established that IL-33 is a clinically-relevant signal in GVHD, mechanisms by which it contributes to alloimmunity are not well defined. Herein, we used IL-33 receptor, ST2, deficient B6 mice as T cell donors to elucidated IL-33-mediated mechanisms in the early activation and differentiation of alloreactive T cells. In our model, we transferred equal ratios of B6 Cd4-Cre x St2fl/fl (ST2ko) and Cd4-Cre (ST2wt) donor T cells into irradiated B6 and BALB/c recipients. Donor T cells in the LO and tissues were characterized at day 1–3, 5, and 7 post alloSCT. BALB/c recipients exhibited blunted early activation and proliferation of alloresponsive ST2ko CD4+ T cells compared to ST2wt CD4+ T cells in LO of the same recipient. ST2ko T cells in allogeneic recipients also failed to differentiation into Tbethi and CXCR3hi Th1 cells able to infiltrate into target tissues and cause GVHD. RNAseq analysis confirmed the need for IL-33-signaling for alloreactive Th1 generation as ST2KO CD4+ cells exhibited reduced IFNγ, Tbet, CXCR3, and instead expressed higher levels of Th2 and regulatory molecules (Foxp3, IL10, Gata3). IL-33 signaling was not required for the non-alloantigen driven lymphopenic expansion by CD4+ T cells in syngeneic recipients. These data establish a previously unappreciated and targetable role for IL-33 in effective alloAg-reactive donor Th1 generation after alloSCT.
Invariant NKT cells promote expansion of novel CXCR3+CCR4+CD8+ T cells in the liver following allogeneic hepatocyte transplant
Authors: Ringwald Bryce Alan , Zimmerer Jason M , Chaudhari Sachi , Han Jing , Peterson Chelsea M , Warren Robert T , Hart Madison , Abdel-Rasoul Mahmoud , Bumgardner Ginny L
Published: February 2023

Hepatocyte transplant (HcTx) is a treatment for metabolic diseases and a bridge to liver transplant but is limited by immunogenicity. Our prior work identified the critical role of CD4-dependent and CD4-independent CD8+ T cells in HcTx rejection. Herein, we evaluated the influence of invariant natural killer T cells (iNKTs), abundant in the liver, upon CD8+ T cell responses in the presence and absence of CD4+ T cells. To investigate this, C57BL/6 (wild-type; WT) or iNKT deficient Jα18 KO (H-2b) received FVB/N (H-2q) HcTx. Cohorts were depleted of CD4+ T cells and/or reconstituted with iNKTs. Recipients were evaluated for cytotoxicity, CD8+ T cell phenotype, and HcTx rejection. By comparing CD4-sufficient WT and Jα18 KO recipients, we found that the presence of iNKTs significantly enhanced in vivo CD8-mediated cytotoxicity (83±3% vs 23±3%; p<0.001) but did not affect survival (10 days vs 14 days; p=ns). However, in CD4-deficient recipients, iNKTs were critical, since without CD4+ T cells and iNKTs, CD8-mediated in vivo cytotoxicity was completely abrogated (31±3% vs 2±1%; p<0.001) and HcTx survival was significantly prolonged (14 days vs 56 days; p<0.001). iNKTs enhanced the quantity of a novel CD8+ T cell subset, CD44+IFN-γ+CXCR3+CCR4+CD8+ T cells, in the liver (2-fold) in all HcTx recipients (p<0.03 for all comparisons). CXCR3+CCR4+CD8+ T cells, when compared to CXCR3+CCR4−CD8+ T cells, express higher IFN-γ (32±3% vs 9±1%; p<0.001) and TNF-α (37±3% vs 6±1%; p<0.001). They also exhibit greater in vivo cytotoxicity (13±1% vs 5±1%; p<0.001) and mediate rapid HcTx rejection in RAG1 KO recipients (5 days vs 16 days; p=0.02). We show that iNKTs enhance the expansion of highly cytotoxic CXCR3+CCR4+CD8+ T cells that mediate rapid rejection of HcTx.
Xenogeneic Sertoli cells express complement inhibitory proteins necessary for their survival of hyperacute rejection
Authors: Washburn Rachel , Kaur Gurvinder , Dufour Jannette
Published: February 2023

Transplantation is used to treat organ failure associated with various diseases, however there is a severe shortage of organs acceptable for transplant. Utilization of pig organs offers an endless supply of transplantable tissue, but xenografts are rapidly destroyed by hyperacute rejection (HAR). The primary humoral mediator of HAR is the activation of complement (C″) by preformed-antibody binding to xenoantigens, causing a proteolytic cascade that results in cell lysis by membrane attack complex (MAC) insertion. Sertoli cells (SCs) are immunoregulatory testicular cells that survive as xenografts for an extended period without the use of immunosuppressive drugs. The goal of this study was to examine the mechanisms for SC survival of C″. Neonatal porcine SCs (NPSCs) transplanted into rats survived as xenografts while porcine islets (NPIs) did not. Immunohistochemistry of the surviving grafts show deposited C3, but not MAC, suggesting that NPSCs inhibit C″ after activation. An in vitro human serum (HS)-C″ cytotoxicity assay confirmed that NPSCs survive activated human C″. Analysis of NPSC expression of complement inhibitory proteins (CIP) revealed higher levels of membrane cofactor protein (MCP) and decay accelerating factor (DAF) as compared to NPIs, which are killed by C″. Knocking down expression of MCP and DAF using shRNA confirmed their importance in NPSC survival of C″. Less than 10% of these cells survived exposure to the HS-C″ cytotoxicity assay, indicating MCP and DAF are important in NPSC xenograft survival in vitro.
Selective expansion of regulatory T cells by kidney-tethered IL2 mutein
Authors: Larkin Bridget M , Visweswaraiah Jyothsna , Li Bilian , Pradhan Komal , Rowe Michael , Rajani Gaurav , Nagy-Domonkos Celesztina , Higginson-Scott Nathan , Viney Joanne L , Otipoby Kevin L , Shaheen Hussam H , Mascanfroni Ivan
Published: February 2023

Regulatory T cells (Treg) play a critical role in maintaining graft tolerance following organ transplantation, and increased numbers of Tregs in solid-tissue grafts such as the kidney are associated with improved graft survival and function. Therapeutic approaches that increase Tregs offer a promising alternative to current standard-of-care treatment consisting of broad-acting immunosuppressants and their attendant side effects. While efforts to expand Tregs ex vivo for infusion into patients have shown promise, they present manufacturing and administration challenges. Low dose (LD) interleukin 2 (IL-2) drives Treg proliferation and function via the heterotrimeric IL-2 receptor expressed on Tregs and has been shown to expand Tregs in vivo, including in the context of transplantation. However, IL-2 can also activate other immune cells including conventional T cells and Natural Killer (NK) cells, which express IL-2Rb/CD122 and IL-2g/CD132, and this could accelerate rejection. We have enhanced IL-2 selectivity for Treg by introducing mutations that increase affinity for CD25 and decrease affinity for CD122/CD132, resulting in Treg specific expansion. Here, we demonstrate that this IL-2 Mutein (IL-2M) can be “tethered” to the kidney via a bifunctional antibody targeting a tubule-specific protein. This kidney-tethered IL-2M maintains Treg selectivity and localizes to the kidney with extended tissue PK. Moreover, in a CD34-NSG mouse model, animals dosed with kidney-tethered IL-2M exhibited prolonged kidney Treg expansion compared to a systemic, non-tethered IL-2M. This approach has the potential to substantially improve kidney graft acceptance while reducing adverse effects and improving patient quality of life.
IL-12 abrogates mechanisms of immune suppression by B-ALL
Authors: Hunter Rae , Henry Curtis J. , Porter Christopher
Published: February 2023

Despite major advance in therapeutic options for treating patients with acute lymphoblastic leukemia (ALL), including the use of immunotherapies, 15–20% of patients still succumb to disease. Given the high mortality rates, particularly in the context of immune-based therapies, there is still a knowledge gap regarding how leukemia cells evade immune surveillance.

In published studies, we have shown that interleukin-12 (IL-12) secreted by B-leukemia cells stimulate T-cell mediated immunity. We found that recombinant IL-12 treated WT and Rag1−/− mice, which lack B- and T-cells but still have innate immune cells, had prolonged survival as compared to untreated mice. These results demonstrate that IL-12 increases the anti-leukemia immunosurveillance capacity of innate and adaptive immune cells.

Mechanistically, we observed when T-cells were stimulated in ALL supernatant in vitro there is a reduction in the surface expression of the T-cell activation marker, CD44, and marker of degranulation, CD107b. Macrophages co-cultured with leukemia cells were also unable to significantly upregulate the activation markers, CD80 and CD86, compared to macrophages in media alone.

We then explored combination therapeutics in vitro to override ALL immune suppression. Considering the potency of IL-12 alone and Blinatumomab, a bispecific T-cell engager (BiTE) antibody used to treat patients with relapsed and refractory disease, we determined how combining these therapies impacted B-ALL cytotoxicity when cultured with T-cells. Impressively, IL-12 augmented the efficacy of Blinatumomab resulting in more T-cell mediated killing of leukemia cells. Our results demonstrate the therapeutic potential of IL-12 as a treatment for ALL.
Robust Anti-SARS-CoV-2 Humoral and Cellular Immunity through cGAMP-loaded Microparticle Adjuvanted Subunit Vaccination
Authors: Sandor Adam , Varma Devika M , Batty Cole J , Baxter Victoria K , Sakar Sanjay , Thompson Michael A , Gao Hao , Liang Kaixin , Chou Wei-Chun , Bachelder Eric M , Heise Mark T. , Ainslie Kristy M , Ting Jenny P
Published: February 2023

In less than one year, the COVID-19 pandemic has infected over 95 million people with greater than two million deaths globally. Vaccines against the SARS-CoV-2 virus offer the best chance at curbing the severity and spread of COVID-19. While initial COVID-19 vaccine candidates have demonstrated great promise and efficacy, it is unknown how broad and durable those responses will be. Furthermore, eliciting strong antiviral immune responses will prove critical against future emerging strains. Previously, we demonstrated that cyclic-AMP-GMP (cGAMP) encapsulated within acetalated dextran microparticles (MPs) adjuvants influenza subunit antigens to strongly protect against influenza viral challenge when used as an intramuscular vaccination in a mouse and ferret model. Based on these studies, we hypothesized that vaccination with the stabilized spike protein of SARS-CoV-2 (S2P) and cGAMP MPs would lead to protective anti-SARS-CoV-2 immune responses. We demonstrate that S2P vaccination with cGAMP MPs lead to increased total anti-SARS-CoV-2 IgG with a strong Th1 skewed IgG2c antibody response compared to other clinically relevant adjuvants. Vaccination using cGAMP MPs also increased the frequency of activated Th1 antigen-specific T cell responses compared to controls and other vaccine adjuvants, as determined by IL-2 and INFg ELISPOTs. Finally, vaccination using cGAMP MPs lead to comparable SARS-CoV-2 neutralizing antibody production and protection against mouse adapted SARS-CoV-2 infection. These studies demonstrate that vaccination with S2P and cGAMP MPs lead to a protective humoral response and improved antigen-specific T cell responses compared to other clinically relevant adjuvants.
Development and Characterization of Potential Neutralizaing Antibodies to SARS-CoV-2 from Phage Display Human Antibody Library System
Authors: Park In Ho , Lee Sang Pil , Yoon Sunha , Shin Ji-Young , Oh Jooyeon , Lee Hoojung , Jeon Eenkyung , Park Bum-Chan , Shin Jeon-Soo
Published: February 2023

Since the outbreak of Wuhan, China, in 2019, SARS-CoV-2 infection has been increasing globally, and as of January 2021, it has exceeded about 85 million people, with about 2 million deaths. Despite Covid-19 vaccines urgently approved for creating population (herd) immunity, the first need for the infected patients is a treatment. In this perspective, neutralizing antibody is the first concern because it is significant for symptom relief and rapid recovery of patients without therapeutic chemicals.

Phage display human antibody library technology is a potential tool screening neutralizing antibodies specific to the receptor-binding domain, RBD of the spike protein of SARS-CoV-2 virus entry to host cells.

Fifteen antibodies specific to RBD were selected through Ymax®-ABL and Patient-Immune Library and characterized through ELISA, immune fluorescence assay, epitope mapping. Some of the selected antibodies were improved in the spike-neutralizing property through in vitro affinity maturation. Here, the COVID-19 therapeutic values of two promising candidates, both of which recognize different epitope sites on RBD, were functionally proved by using both in vitro cell and in vivo mouse models.
Costimulatory blockade modifies germinal center B cell function in NZB/W F1 mice
Authors: Raparia Chirag , Zeumer-Spataro Leilani , Quach Tam D , Choi Seung-Chul , Morel Laurence , Davidson Anne
Published: February 2023

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with significant morbidity and mortality - demanding further examination of disease reducing treatments. Previously, we showed that a 2-week course of combined CTLA4Ig and anti-CD40L costimulatory blockade prevented loss of tolerance to dsDNA in NZB/W mice while maintaining immune function against exogenous antigen, explained by an altered selection of autoreactive B cells in germinal centers (GC). Here, we examined both B and T cells to clarify if restoration of tolerance is due to a change in GC B cell profile or a decrease in T cell help. 3 months after treatment, large germinal centers persisted in the treated group, but due to smaller spleen size total GC B cells and TFH Cells were decreased compared with controls. The metabolic profile of splenic B and T cells showed a decreased spare respiratory capacity in both subsets and a decrease in B cell glycolysis among the treated group. RNA-seq analysis of GC dark zone and light zone B cells from treated mice showed decreased expression of markers of oxidative phosphorylation and cell proliferation and increased expression of signaling molecules downstream of BCR, including PLCγ, compared with untreated controls. GC B cell stimulation assays showed a delay in phosphorylation of PLCγ in response to anti-IgG in treated mice. These data show a long-lasting effect of short-term costimulatory blockade on both B and T cells with metabolic remodeling of GC B cells and an alteration of BCR signaling. Since both a profound decrease in BCR signaling and an increase in B cell glycolysis have been reported to enhance loss of tolerance in lupus prone mice, our findings help to explain the restoration of tolerance observed after treatment.
Correlation of autoantigen-specific Treg frequency with development of spontaneous organ-specific autoimmunity in a mouse model of uveitis
Authors: Yin Mianmian , Hiam Kamir , Proekt Irina , Chan Jackie , Hu Yongmei , Lowell Clifford A , Gould Douglas B , Anderson Mark S , DeFranco Anthony L
Published: February 2023

About 50% of AireGW/+Lyn−/− mice developed autoimmune uveitis, and this was accompanied by the expansion of interphotoreceptor retinoid-binding protein (IRBP) specific CD4+T cells. Moreover, the response to IRBP was necessary for disease initiation, as deletion of IRBP prevented uveitis. Mass cytometry (CyTOF) analysis of CD4+T cells in the retina of the mice with uveitis identified activated T cell subsets characterized as Ly6Chi effectors, PD-1hi effectors, and Treg. To study cellular mechanisms that promoted development of uveitis, we did single-cell RNA sequencing (scRNA-seq) of eye-draining lymph node (LN) CD4+T cells that recognize the P2 epitope of IRBP (amino acid 271–290). Uniform manifold approximation and projection (UMAP) analysis showed that these CD4+T cells included distinct subsets that were Ly6c1hi , Pdcd1hi and Treg, with the Treg population being overrepresented in the mice without disease. Pseudotime analysis indicated that the Ly6c1hi T cell subset represented an earlier stage in activation, whereas the Pdcd1hi subset represented a later stage. Similar analysis of P2–specific CD4+T cells from the retina of mice with uveitis identified 6 subsets of P2-specific T cells, and was largely consistent with the CyTOF analysis of total CD4+ T cells from the retina. Pseudotime analysis indicated that toward the earlier time were cells characterized by being proliferative (Mki67hi), followed by a Lag3hi subset and at other end of the pseudotime axis by a Lag3hi / Pdcd1hi subset. Our results are consistent with the hypothesis that the fraction of Treg within the IRBP-specific CD4+ T cells in the draining LN determines whether these genetically susceptible mice develop eye-specific autoimmunity or are protected.
Contribution of a Hybrid Insulin Peptide-reactive T cell to Diabetes progression in the NOD mouse.
Authors: Lin Yong , Kong Yuelin , Igyarto Botond Z , Zurawski Gerard , Zurawski Sandra , Bettini Maria , Bettini Matthew
Published: February 2023

Type 1 Diabetes (T1D) is a T cell-mediated autoimmune disease characterized by the immune destruction of pancreatic β cells. The search for natural equivalents to the mimetopes that can stimulate diabetogenic T cell clones from the Non-Obese Diabetic (NOD) mouse model of T1D led to the realization that hybrid peptides (HP) formed by the joining of β cell granule protein-derived peptide fragments are highly stimulatory to some clones. These hybrid peptides were later identified in vivo and proposed as a new class of post-translationally modified autoantigens mediating this disease. Recent data suggest high concentrations of these proteins in β cell lysosomes during granule turnover can lead to the hybridization of peptide fragments which may then be presented on MHC molecules. In the thymus, medullary thymic epithelial cells (mTECs) mediate T cell tolerance through promiscuous gene expression, but may not mediate HP-reactive T cell tolerance. We hypothesize that HP-reactive T cells preferentially escape the thymus and contribute significantly to T1D progression. We attempted to enhance T cell central tolerance to 2.5HIP, a hybrid insulin peptide formed from Proinsulin and Chromogranin A, the only two antigens known to be critical for T1D progression in the NOD mice, to better understand its impact on T1D incidence. Using an antibody-peptide conjugation system, we targeted 2.5HIP to a subset of thymic resident DCs to enhance presentation of 2.5HIP in early neonatal thymus. In neonatal BDC2.5 TCR-transgenic mice we found that a single dose of antibody-2.5HIP enhanced antigen-specific thymocyte apoptosis and regulatory T cell development. Importantly, a single dose in wild-type neonates resulted in a significant delay of diabetes onset.
Anti-TRIM21 antibody seropositivity is related to B-cell abnormalities in systemic lupus erythematosus
Authors: Kunishita Yosuke , Yoshimi Ryusuke , Komiya Takaaki , Sakurai Natsuki , Sugiyama Yumiko , Takase-Minegishi Kaoru , Kirino Yohei , Nagaoka Shouhei , Nakajima Hideaki
Published: February 2023

Background/Purpose

TRIM21 is one of the autoantigens which react with anti-SS-A antibody (Ab). Previous studies have shown that TRIM21 dysfunction promotes aberrant B-cell differentiation in systemic lupus erythematosus (SLE). Here we examined the relationship between anti-TRIM21 Ab and clinical and immunological characteristics in SLE patients.

Methods

Twenty-seven patients with SLE before immunosuppressive therapies and four healthy controls (HC) were enrolled in the study. Serum anti-TRIM21 Ab levels were measured using enzyme-linked immunosorbent assays. The serum levels of cytokines and immunoglobulins were measured using cytometer beads arrays. The protein expression levels of TRIM21 in peripheral blood mononuclear cells (PBMCs) were evaluated by western blotting.

Results

Nineteen and 8 patients showed seronegativity and seropositivity for anti-TRIM21 Ab, respectively. There were no significant differences in the background parameters, including SLE activity and laboratory data. The serum levels of interferon (IFN)-α2 were significantly higher in patients with anti-TRIM21 Ab as compared with those without anti-TRIM21 Ab (p = 0.03). The levels of IgG1, IgG2, and IgA were significantly higher in patients with anti-TRIM21 Ab as compared with those without anti-TRIM21 Ab (p = 0.005, 0.04 and 0.01, respectively). The PBMCs of patients with anti-TRIM21 Ab showed a significantly higher expression of TRIM21 protein as compared with those of patients without anti-TRIM21 Ab.

Conclusion

Anti-TRIM21 Ab seropositivity is related to B-cell abnormalities and type I IFN overproduction in SLE patients, which may be due to the inhibitory effect of anti-TRIM21 Ab on TRIM21 functions.
Pertussis toxin inhibits encephalitogenic T cell infiltration and promotes a B cell driven disease during TH17-EAE
Authors: Maria Zahra , Turner Emma , Axtell Robert C.
Published: February 2023

Pertussis toxin (PTX) is required as a co-adjuvant to induce disease in active Experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. The objective of this study was to investigate the role of PTX during adoptive transfer TH17-EAE and observe alterations in the disease phenotype. TH17-EAE was induced by transferring myelin-specific TH17 cells, harvested from MOG35-55 immunized wild type (WT) donor mice, into WT or B cell deficient (μMT) recipient mice. The recipient mice also received PTX or PBS on days 0 and 2 and monitored for paralysis. In WT mice, PTX significantly reduced disease severity and delayed onset of paralysis. PTX reduced accumulation of pathogenic T helper cells and myeloid cells in the CNS, which we found to be associated with PTX inhibition of chemokine receptor dependent trafficking. Strikingly, our data shows that PTX promotes the accumulation of B cells in the CNS and elevates the capacity of B cells to present antigen to T cells in the periphery, suggesting an altered disease toward a B cell-dependent pathology. We found that without PTX treatment, the severity of disease was equivalent between WT and μMT mice. In contrast, with PTX treatment, the μMT mice had significantly less disease compared to the WT mice. In conclusion, our data suggests that during TH17-EAE, PTX restricts the trafficking of pathogenic T helper cells and myeloid cells into the CNS and thereby delays the disease onset. However, not only PTX does not alter trafficking of B cells into the CNS it also stimulates a more inflammatory B cell phenotype. This study provides novel insights to interactions between TH17 and B cells in mice and has defined a new model to study the pathologic functions of B cells during multiple sclerosis.
Gene Edited Human CD4+ T Cells Reveal that Loss of PTPN22 Function Enhances Mildly Autoreactive T Cell Responses
Authors: Anderson Warren R , Barahmand-pour-Whitman Farbia , Linsley Peter S , Cerosaletti Karen , Rawlings David
Published: February 2023

A genetic variant in the gene PTPN22 (R620W, rs2476601) is strongly associated with increased risk for multiple autoimmune diseases. PTPN22 is a non-receptor tyrosine phosphatase that negatively regulates proximal T cell receptor (TCR) signaling. PTPN22 deficiency increases TCR signaling in mouse and human T cells. Mouse models of the variant similarly exhibit increased TCR signaling, while primary T cells from human carriers of the variant exhibit decreased signaling. Here, we utilize gene editing in primary human T cells to address these discordant findings and explore how the risk variant impacts TCR signaling. Using Crispr/Cas9 nucleases and DNA repair templates, we established a robust platform for precise homology-directed-repair based gene editing of PTPN22, allowing the generation of PTPN22 risk edited, control edited (silent modification), and knock-out T cells from the same donor. In contrast to control edited cells, PTPN22 risk edited T cells exhibit increased activation in response to TCR engagement with higher expression of activation markers including CD40L and CD25, mimicking PTPN22 KO cells. Further, using lentiviral delivery of transgenic TCRs (derived from autoreactive T cells of T1D patients), we demonstrate that loss of PTPN22 leads to enhanced TCR signaling in weakly autoreactive, but not strongly autoreactive T cells, causing increased phosphorylation of S6 kinase, enhanced proliferation, and greater Th1 skewing. Taken together, these findings suggest that direct effects of the PTPN22 rs2476601 variant represents a loss of function in T cells, increasing TCR signaling in mildly autoreactive T cells, potentially expanding the pool of autoreactive T cells and skewing them toward an inflammatory phenotype.
Oxytocin is required for remote learning. Students need a bonding-hormone to improve their learning.
Authors: Fazal Nadeem , Howley Jamie , Brehm Danielle
Published: February 2023

Humans being a social animal need physical presence of their own kind to learn and educate themselves. COVID-19 has increased the need and desire for oxytocin more than any time in history of education. The purpose of this research project was to look at how pandemic-induced remote learning has reduced students’ social interaction, and impacted their education. With the ability to turn off audio and camera, struck in unproductive environments, students feel not only disengaged, isolated but overly anti-social. For our research, we recruited 25 undergraduate biomedical students at the University of Plymouth, England to survey their experiences with online learning, engagement with the technology and how it has affected their ability to learn in the void of physical presence. Our preliminary data suggests that (84%) of these students agreed that online/remote learning has impacted learning and made them crave for bonding to their classmates. However, for a small percentage (16%) said it did not make any difference. This initially highlights the issue that online learning is of the students seem to have no effect. These cohort of the students also seem to identify themselves as “introverts” and they also seem to have enjoyed being less sociable. We conclude that a statistically significant number of students proved to need a social environment conducive to learning and current pandemic limited that aspect. A social and a societal element of a happy civil environment is needed for not only healthy mind and body but also for a healthy and fruitful education world. A well-known bonding hormone “Oxytocin” is a prerequisite for human learning environment, and we don’t see the virtual world to take that over in any time soon.
CARTOONING AS AN EFFECTIVE TOOL FOR TEACHING EXPERIMENTAL DESIGN
Authors: Bernard Aimee Pugh , Thompson Scott B
Published: February 2023

Medical education and scientific thinking often involve understanding complex experimental methods. We utilized cartooning as an active learning method for understanding complex experimental methods from a diverse array of scientific articles in an undergraduate scientific thinking course. Here we describe our implementation of cartooning and how it can be used as tool to enhance clarity, instruction, and analysis.

A cartoon is a drawing or series of drawings used to illustrate an action or subject. In our implementation as a teaching tool, students created cartoons of experimental methods from a variety of assigned readings using a digital platform to visualize their understanding of the experimental procedures. Cartoons created on the same experimental method by different individuals are rarely identical, thus exposing the entire class to a diversity of understanding. When students produce cartoons demonstrating differences in experimental methods, the flexible nature of cartooning enables editing or the addition of details to improve the accuracy of the illustrated procedure. Additionally, over the course of the semester the level of detail and clarity of the cartoons evolves thus serving as qualitative assessment of student understanding of experimental procedures throughout the course.

Cartooning enables students to visualize their understanding of complex scientific methods and to think critically about the experimental design process. Visualization of how students represent experimental methods reinforces the deeply thoughtful and creative nature of experimental design as well as facilitates the assessment of student growth and evolving comprehension of the scientific method.
AAI Curriculum Ad Hoc Committee Presentation: The Development of AAI Recommendations for an Undergraduate Course in Immunology
Authors: Amiel Eyal , Swanson-Mungerson Michelle Anne , Bottaro Andrea , Carr William H , Varga Steven M , Bose Nandita , Jameson Julie M , Porter Edith
Published: February 2023

The subject of immunology is a conceptually intricate and rapidly expanding field of study that is vitally important to biomedical science undergraduate education. Identifying the “essential” components of an undergraduate immunology (UGI) course can be daunting due to the varying post-graduate pathways students take. To address the need for a guiding framework for teaching UGI, the American Association of Immunologists (AAI) Education Committee commissioned the AAI Curriculum Ad Hoc Committee to develop a cohesive document identifying the core curricular components deemed essential for UGI courses, and opportunities to meet the needs of diverse student populations. In this presentation, we will share the process by which this effort was accomplished, with the hope of illuminating both the successes and challenges of our work to the education community, and inspiring similar efforts in other areas. We will cover the essential and reiterative steps of this undertaking by detailing: the collaboration with various stakeholders, including undergraduate, medical school, and industry partners; the evaluation of potential curricular components, including outreach surveys and small working groups from the AAI community; the refinement of the recommendations, including an organized virtual workshop with open participation from stakeholders; the development of a statement on inclusive and antiracist teaching; the determination of a dissemination plan; and the identification of future needs and goals for the process. We believe that sharing this process will be insightful for educators in the field of immunology, and more broadly to any educator with an interest in the process of inclusive and comprehensive curriculum development.
The decision to pursue a degree in immunology may be influenced by the timing at which students are introduced to the field
Authors: Anderson Ashlyn E , Redden David , Buzzelli Nicholas , Giordano-Mooga Samantha , Loyd Christine , Justement Louis B , Bruns Heather A.
Published: February 2023

The decision to pursue an academic plan of study is influenced by several factors. Educational and personal experiences through childhood, adolescence, and young adulthood can have a powerful impact on the academic preferences of students. Immunology, a popular field of study at the graduate level, is largely ignored at the undergraduate level. Identifying the type and timing of experiences that influence the choice of current undergraduate and graduate students to pursue immunology is critical for developing and refining immunology curricula. To identify potential influencing factors, students were surveyed about their exposure to the immune system or immunology-related topics. There were 283 total respondents (45% immunology majors, 55% other science majors) of which 39% were graduate students and 61% were undergraduate students. There were no significant differences in the demographics of immunology majors compared to non-majors. Although there is no difference between immunology majors and non-majors in the number of respondents having an educational or personal experience involving immunology, an educational experience was more influential than a personal experience for both graduate (85%) and undergraduate (66%) immunology majors. However, more undergraduates (33.7%) pursue immunology based upon a personal experience compared to graduate students (14.6%). Importantly, students who chose to pursue an immunology degree were introduced to immunology at a later academic stage compared to students who are not pursuing an immunology degree. These data suggest that timing may be more influential than the type of exposure in influencing the choice to pursue immunology.
Using practice tests as a vaccine to desensitize test anxiety in vulnerable students during pandemic induced lock-down.
Authors: Burtley Aundriana , Emeakoroha Uzoma , Nguyen Minh-Thu , Simmons Jazmyne Durrah , Jhatu Prabjyot , Salamanca Sonia , Fazal Nadeem
Published: February 2023

Field of education psychology seeks bridging gap between research and theory on teaching and classroom practice. Students’ struggle when content matter gets increasingly intense especially when subjects need plenty of rote memory. Covid-19 took a heavy toll on students who were already prone to test anxiety. The current research hypothesized that utilization of practice tests for first-year pharmacy microbiology and immunology course will enhance students learning, understanding and comprehension of complicated material. Since pandemic anxiety added to pre-existing anxiety among pharamacy students we used this measure to mitigate. A cohort of sixty students taking a 4-credit course of Microbiology/Immunology were recruited in this study. Students’ voluntarily declared their fears of superadded anxiety levels because of pandemic. A pool of exam ‘questions collected over the last five years were accurately screened and 300 questions were selected based on the criteria of lectures topics, difficulty level and meeting the specific objectives for every lecture that was presented to students and covered in the exams. Two groups were analyzed based upon their use of practice exams offered and their willingness to utilize them. The data based upon their performance was collected at the end of final examination to assess the benefit of study guide to students. Likert-type Ordinary Scale data analysis was used to determine if hypothesis was supported. The analysis of primary endpoint indicated that majority of students (69.87%) who used the practice exams performed better. In the feedback they reportedly strongly agreed that it helped them to make their study more focused and improved their pre-existing anxiety levels during pandemic.
The global digital divide: Younger and older teachers at odds in this COVID19-induced remote teaching experiment.
Authors: Fazal Nadeem , Brehm Danielle , Alakbarova Gulgaz , Childs Porscha J , Finn Emily , Delt Eva , Beamon Jessica , Howley Jamie
Published: February 2023

COVID-19 was anticipated to target aging population and surely it did in world of health, but did collateral damage in education. Lack of preparedness and digital divide became more obvious among old-aged teachers as compared to younger faculty. The later found themselves more equipped with skill sets and more motivated to embrace the realities of the virtual world. We studied a cohort of teachers across the spectrum of age or years of service compared to younger or newly hired faculty. We solicited teachers in both cohorts from Asia, Europe and Americas and surveyed their responses to a given set of questions. We also recruited a group of students to independently and anonymously verify if they noticed any major effect in their learning process. Our global data showed a clear digital divide among the aged and younger faculty. Both groups found themselves unprepared and lacking necessary skill set to deal with this unprecedented, sudden and mandatory online education being forced upon them. However, younger faculty reported to have adapted to the onslaught of technology in an abrupt and timely manner as compared to their senior colleagues. Mostly, both group of teachers considered the learning experience at best as “on job training module”. High anxiety levels with fear of lack of performance, proper delivery methods also played a role in this panic-stricken educational world. Both group of teachers also reported lack of historical perspective and pilot studies for the use of technological gadgets as one of the artifacts in this experiment. The students being at the recipient end also reported in their surveys that the haste and rush of delivery and assessment methods negatively influenced the teaching methods and reciprocally their learning process.
Using an Indigenous framework to contextualise Immunology education within the Health Sciences setting
Authors: Sika-Paotonu Dianne
Published: February 2023

Indigenous Māori and Pacific children and young people remain disproportionately affected by Acute Rheumatic Fever (ARF) and Rheumatic Heart Disease (RHD), with rates in Aotearoa/New Zealand and the Pacific Region of significant concern. ARF is the body’s autoimmune response to an untreated Group A Streptococcal (GAS) throat or skin infection. If multiple or severe episodes of ARF occur and are left untreated, this can lead to permanent cardiac damage known as RHD. RHD remains a major cause of morbidity and mortality globally. In the absence of a GAS vaccine, the best recommended treatment and management approach consists of painful monthly intramuscular injections of Benzathine Penicillin G (BPG) administered to the upper thigh area, and given for at least 10 years or more. A new Penicillin for ARF/RHD prevention is urgently needed. The purpose of this work was to employ the use of an Indigenous Pacific framework within the undergraduate Health Sciences education setting to support learning and teaching efforts appropriate to vulnerable communities affected by ARF and RHD. The Talanoa approach was utilised within the classroom setting to support discussions relating to ARF and RHD and vulnerable communities that was appropriate and respectful to the Indigenous cultural setting. Well-received by students, this approach was not only applicable to the cultural setting for those communities affected by ARF/RHD in Aotearoa/New Zealand, but also helped with contextualisation, supporting courteous class discussions with relevant in-class learning opportunities and experiences. This work highlighted the importance of Indigenous frameworks for supporting Immunology education within the Health Sciences education setting.