Journal: Hepatology Communications

Article Title: Activation of Liver X Receptor α Sensitizes Mice to T‐Cell Mediated Hepatitis

doi: 10.1002/hep4.1584

Figure Lengend Snippet: Production of IFN‐γ is required for the sensitization of LXRα‐KI mice to ConA‐induced hepatitis. (A,B) Female WT and LXRα‐KI mice received a tail‐vein injection of 200 μg anti‐IFN‐γ antibody or the control isotype IgG 1 hour before the ConA (10 mg/kg) injection. The mice were sacrificed 24 hours after the ConA injection. (A) Liver histology with the quantification of necrotic area shown on the right. (B) Serum levels of ALT and AST (n = 4‐5 per group). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01.

Article Snippet: For in vivo neutralization of IFN‐γ, mice were injected intravenously with 200 μg InVivoMAb anti‐mouse IFN‐γ or the isotype control IgG (see Supporting Table ) from Bio X Cell (West Lebanon, NH) 1 hour before the ConA injections.

Techniques: Injection, Control

Journal: Frontiers in immunology

Article Title: Analysis of the mechanisms regulating soluble PD-1 production and function in human NK cells.

doi: 10.3389/fimmu.2023.1229341

Figure Lengend Snippet: FIGURE 1 Characterization of the isolated recombinant sPD-1 protein. (A) Gel-based proteomic analysis of purified sPD-1. Left panel: Coomassie staining of the isolated recombinant sPD-1 revealed a band of approximately 20 kDa corresponding to sPD-1 molecular weight. BSA sample was used as a control. Right panel: western blot analysis allowed detection of the recombinant sPD-1 protein with both the anti-Streptavidin-HRP and anti-PD-1 antibodies. (B) SKNAS and IMR-32 cell lines were analysed for PD-L1 and PD-L2 expression. Only PD-L1 could be detected on the surface of both cell lines. (C) Analysis of recombinant sPD-1 binding to PD-L1 ligand. Flow cytometry analysis demonstrated that recombinant sPD-1 was able to bind PD-L1 expressed by SKNAS and IMR-32 cell lines. (D) SKNAS were incubated with PBS, IgG1 Isotype or Atezolizumab and then analysed for PD- L1 expression. Abrogation of PD-L1 signal occurred only when tumor cells were treated with Atezoliumab (left panel). Binding of sPD-1 to tumor expressed PD-L1 was abrogated when NB cells were previously incubated with Atezolizumab. (E) Incubation of PD-L1+ SKNAS and IMR-32 cell lines with recombinant sPD-1 resulted in a decreased PD-L1 signal. MFI from ctrl (not stained) samples was subtracted from their corresponding stained samples. Values are mean ± SEM. Statistical significance has been determined by Paired T test, p< 0.01 **; p<0.05 *.

Article Snippet: Binding of Nivolumab (A2002, Selleckchem) to PD-1 was performed incubating stimulated NK cells with IgG4 control Isotype (DDXCH04P-100, Novus Biological) or Nivolumab for 30 min at 4°C and then with anti-PD1 for 30 min at 4°C.

Techniques: Isolation, Recombinant, Staining, Molecular Weight, Control, Western Blot, Expressing, Binding Assay, Flow Cytometry, Incubation

Journal: Frontiers in immunology

Article Title: Analysis of the mechanisms regulating soluble PD-1 production and function in human NK cells.

doi: 10.3389/fimmu.2023.1229341

Figure Lengend Snippet: FIGURE 2 Recombinant sPD-1 can modulate NK cell effector function. (A) Control and stimulated NK cells were analysed for membrane PD-1 and PD-L1 expression. Membrane-bound PD-1 was detected only in stimulated NK cells compared to control cells. Conversely, while Ctrl NK cells expressed PD-L1, it was barely detectable on stimulated NK cells. One representative experiment has been shown. Quantifications of four different experiments have been reported. Values are mean ± SEM. Statistical significance has been determined by Paired T test, p< 0.01 **; p< 0.001 ***. (B) NB cell lines were incubated with stimulated NK cells, expressing PD-1, with or without recombinant sPD-1. An increase in cytotoxicity toward both SKNAS (n=7) and IMR-32 (n=8) cell lines at different Effector (E) Target (T) ratios was observed only when recombinant sPD-1 was present in the co-culture. Data have been compared using paired T test, p< 0.05 *; p< 0.01 **, p< 0.001 ***. (C) Similarly, in the presence of recombinant sPD-1 an increase in degranulation (left and middle panels) and accumulation of IFN-g (right panel) of NK cells were observed. Values are mean ± SEM. Statistical significance has been determined by Paired T test, p< 0.01 **, p< 0.001 ***. (D) Comparison of sPD-1 and Nivolumab treatments on cytotoxicity of stimulated NK cells. Incubation with Nivolumab abrogated PD-1 signal compared to Ctrl and IgG4 samples (left panel). Increase of cytotoxicity toward SKNAS cell line at different E:T ratio was observed in both the analysed conditions (treated or not with Nivolumab) only in the presence of sPD-1. Values are mean ± SEM. Data have been compared using paired T test, p< 0.05 *; p< 0.01 **, p< 0.001 ***.

Article Snippet: Binding of Nivolumab (A2002, Selleckchem) to PD-1 was performed incubating stimulated NK cells with IgG4 control Isotype (DDXCH04P-100, Novus Biological) or Nivolumab for 30 min at 4°C and then with anti-PD1 for 30 min at 4°C.

Techniques: Recombinant, Control, Membrane, Expressing, Incubation, Co-Culture Assay, Comparison

Journal: International Journal of Biological Sciences

Article Title: The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

doi: 10.7150/ijbs.67203

Figure Lengend Snippet: A schematic diagram of the process of verifying hypotheses to identify a new immune system built up by FSCs in early pregnancy. (A) The developing progress of the fetal immune system. Igs-secreting B cell progenitors and pre pro-B cells were detected only in the fetal liver around 9 weeks of gestation, and mature B cells expressing B cell receptors were found in various fetal tissues after 18 weeks of gestation. In addition, maternal IgG can be transferred to the fetus through the umbilical cord after 15 weeks of gestation. However, it is not known whether the fetus produces Igs before the innate and adaptive immune system are formed. Adapted from with permission from AAAS. (B) To investigate the mechanism of self-protection by FSCs before establishing the innate and adaptive immune system by immune cells in early pregnancy, we established hypotheses and prepared a flow chart to verify them. (1) We confirmed whether Igs, particularly NAbs, including IgG3, which act on the innate immune system, were secreted from FSCs under a typical 2D culture condition. (2) We established an ex-vivo culture condition that simulates in vivo environment of FSCs to induce the secretion of NAbs from them. We confirmed the secretions of Igs and NAbs from FSCs Ex-vivo (NAbs-FSCs) cultured in this culture condition and compared them with FSCs Control cultured in a typical 2D culture condition. (3) We confirmed NAbs, proteins related to the promotion of Ig secretion, and complement proteins in NAbs-FSC-EVs secreted from NAbs-FSCs cultured in a newly established ex-vivo culture condition to induce the secretion of NAbs and compared them with FSCs Control -EVs. In addition, we confirmed the difference in induction aspects according to the culture conditions by comparing the content of HLA-G proteins inducing immune tolerance and various anti-viral proteins induced by IFN in NAbs-FSC-EVs with FSCs Control -EVs. Moreover, we compared and analyzed the induction aspects of NAbs such as IgG3 and IgM in NAbs-FSC-EVs and FSCs Control -EVs through Exoview®. This verification process confirmed that FSCs cultivated in a newly established ex-vivo culture condition could secrete NAbs, complement proteins, and various anti-viral proteins, which could construct the new immune system to protect themselves from external infectious agents at the cellular level.

Article Snippet: Labelling Abs that consist of anti-syntenin Alexa-555, anti-human IgG3 Alexa Fluor 488 (Novus Biologicals), and anti-human IgM Alexa Fluor 647 Abs (BioLegend), were used.

Techniques: Expressing, Ex Vivo, In Vivo, Cell Culture, Control, Construct

Journal: International Journal of Biological Sciences

Article Title: The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

doi: 10.7150/ijbs.67203

Figure Lengend Snippet: Analysis of NAbs expression and secretion characteristics of FSCs cultured in the novel ex-vivo culture conditions for inducing the secretion of Nabs. (A) A schematic diagram to verify NAbs expression and secretion potential in FSCs (FSCs Ex-vivo ) cultured under new ex-vivo culture conditions. (B) Flow cytometry results comparing the expression patterns of stem cell-specific markers in FSCs cultured in FSCs Control and FSCs Ex-vivo . The results showed that the expression aspects of MSC markers (CD73, CD90, and CD105) and ESC marker (SSEA4) were very similar in FSCs Control and FSCs Ex-vivo . (C) Flow cytometry results comparing the cell membrane and intracellular expression patterns of Igs and NAbs in FSCs Control and FSCs Ex-vivo . The expression of membrane-bound IgM is rather higher in FSCs Control than FSCs Ex-vivo , but the expressions of intracellular IgM and IgG3 (NAbs) are much higher in FSCs Ex-vivo than FSCs Control . The expression pattern of IgG is similar in FSCs Control and FSCs Ex-vivo . (D) Results of protein antibody microarray analysis comparing the relative expression levels of Igs in the culture supernatants of FSCs Control and FSCs Ex-vivo . Compared to FSCs Control , FSCs Ex-vivo secreted higher levels of Igs. Error bars represent standard deviation. *, P <0.05; **, P<0.01; ***, P<0.001; Student's t-test.

Article Snippet: Labelling Abs that consist of anti-syntenin Alexa-555, anti-human IgG3 Alexa Fluor 488 (Novus Biologicals), and anti-human IgM Alexa Fluor 647 Abs (BioLegend), were used.

Techniques: Expressing, Cell Culture, Ex Vivo, Flow Cytometry, Control, Marker, Membrane, Microarray, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

doi: 10.7150/ijbs.67203

Figure Lengend Snippet: Comparative Analysis of IgG3 and IgM in culture medium and EVs from various cells

Article Snippet: Labelling Abs that consist of anti-syntenin Alexa-555, anti-human IgG3 Alexa Fluor 488 (Novus Biologicals), and anti-human IgM Alexa Fluor 647 Abs (BioLegend), were used.

Techniques: Concentration Assay, Ex Vivo, Co-Culture Assay

Journal: International Journal of Biological Sciences

Article Title: The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

doi: 10.7150/ijbs.67203

Figure Lengend Snippet: Characterization of NAb-secreting FSCs-derived EVs (NAbs-FSC-EVs). (A) A schematic diagram verifying whether NAbs are contained in NAbs-FSC-EVs secreted from NAbs-FSCs cultured under new ex-vivo culture conditions. (B) The results of NTA on NAbs-FSC-EVs and FSC Control -EVs secreted from FSCs Control cultured on a typical 2D plate. The concentrations, average sizes, and distribution of FSC Control -EVs and NAbs-FSC-EVs are almost similar. There is no significant difference in EV secretion characteristics according to the culture conditions. (C) SEM (left) and TEM (right) images of FSC Control -EVs and NAbs-FSC-EVs, respectively. There are no significant differences in shapes and size distributions in both EVs according to the culture conditions. (D) The results of flow cytometry analysis to compare the expression patterns of exosome markers (CD9, CD63, and CD81) in FSC Control -EVs and NAbs-FSC-EVs. The expression aspects that CD63 and CD81 are expressed at higher levels than CD9 are similar in both EVs. (E) The results of co-expression analysis of exosome markers (CD9, CD63, CD81, and syntenin) in FSC Control -EVs and NAbs-FSC-EVs using ExoView®. They show expression patterns very similar to the flow cytometry results in Figure D, and these common expression patterns appear to be a unique characteristic of FSCs-derived EVs. (F) The results of flow cytometry analysis comparing the presence of IgG and NAbs (IgG3 and IgM) in FSC Control -EVs and NAbs-FSC-EVs. (G) The results of exosomal cargo analysis to compare the content of NAbs in FSC Control -EVs and NAbs-FSC-EVs using ExoView®. Similar to the flow cytometry results in Figure F, they show that NAbs-FSC-EVs contain higher levels of NAbs than FSC Control -EVs. (H) The results of analyzing the patterns of NAbs content in both EVs captured by each exosomal marker using Exoview®. (I) The results of protein antibody microarray analysis comparing relative expression levels of B cell-specific proteins related to the promotion of Ig transcription induced in FSC Control -EVs and NAbs-FSC-EVs. They show that B cell-specific proteins related to the promotion of Ig transcription are induced in NAbs-FSC-EVs at higher levels than FSC Control -EVs. (J) The results of analyzing the concentrations of IgG3 and IgM contained in both EVs obtained from the subculture of FSCs Control and NAbs-FSCs using each ELISA kit. IgG3 and IgM were not detected in all FSC Control -EVs, but the contents of IgG3 and IgM in NAbs-FSC-EVs gradually increased as the subculture progressed. Error bars represent standard deviation. *, P <0.05; **, P<0.01; ***, P<0.001; Student's t-test; N.D., Not Detected.

Article Snippet: Labelling Abs that consist of anti-syntenin Alexa-555, anti-human IgG3 Alexa Fluor 488 (Novus Biologicals), and anti-human IgM Alexa Fluor 647 Abs (BioLegend), were used.

Techniques: Derivative Assay, Cell Culture, Ex Vivo, Control, Flow Cytometry, Expressing, Marker, Microarray, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Biological Sciences

Article Title: The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

doi: 10.7150/ijbs.67203

Figure Lengend Snippet: Overview of HLA-G-based immune tolerance environment and NAbs-based self-defense mechanism induced by hormones secreted during pregnancy. During pregnancy, the fetus must protect itself from various foreign pathogens and the maternal immune system. Protection from the maternal immune system is achieved by an immune tolerance environment induced by various HLA-G isoforms secreted mainly by EVTs, which continuously infiltrate the maternal decidua. The transcription, alternative splicing, and secretion of soluble HLA-G isoforms, which can induce extensive immune tolerance, are induced by the pregnancy-related hormones hCG and PR , and the transcription and expression of membrane-bound HLA-G, which induces local immune tolerance, is also upregulated by IFN stimulation (the left panel). On the other hand, we suggest that before the immune system establishment, the fetal self-defense mechanism against foreign pathogens can be induced by various factors, including IL-10, Pax-5, NF-κB, and Oct-1/2, that promote the transcription of Igs, especially NAbs, such as IgG3 and IgM, which have the most outstanding anti-viral effector functions, through the pathway triggered by ER, another pregnancy-related hormone secreted from TBCs (the right panel). Therefore, our results demonstrate that hormones secreted during pregnancy may induce the different immune systems that fully protect the fetus from maternal immune system and foreign antigens.

Article Snippet: Labelling Abs that consist of anti-syntenin Alexa-555, anti-human IgG3 Alexa Fluor 488 (Novus Biologicals), and anti-human IgM Alexa Fluor 647 Abs (BioLegend), were used.

Techniques: Alternative Splicing, Expressing, Membrane

Journal: International Journal of Biological Sciences

Article Title: The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

doi: 10.7150/ijbs.67203

Figure Lengend Snippet: Suggested Primal Immune System as a Self Defense Mechanism of Fetal Stem Cells before the establishment of innate and adaptive immune systems. Since virus invasion and infection into host cells are affected by the host's innate and adaptive immune responses, it was recognized that the fetus was very susceptible to viral infection before forming innate and adaptive immune systems. However, our results for FSCs in early pregnancy suggest that the fetus possesses very complicated and sophisticated self-defense mechanisms (primal immune system) at the cellular level exist in addition to the protective function of the placental TBC, which was considered as the only barrier to protect the fetus from external infections so far. (A) Extracellular defense mechanism by secretion of NAbs and complement proteins. Our results show that FSCs in early pregnancy can produce and secrete various NAbs, including IgP (fetal IgG3) polyreactive to unexperienced antigens, along with complement proteins to eliminate external pathogens immediately by CDC and ADCC, before umbilical cord generation, the main delivery route of maternal IgG, and before the complete development of B cells. In particular, IgP secreted from early pregnancy FSCs was proved to have the primal Ig repertoire that can instantly recognize various antigenic epitopes pathogens. Furthermore, IgP has the most outstanding Fc effector function to induce ADCC by binding with the highest affinity to Fcγ receptor of NK cells. These functions of IgP may contribute to much effective removal of the infectious agents. Also, we demonstrated that itPG-FSCs could secrete various NAbs such as IgP, IgM, and IgA as they contained factors, including Igll1, Pax5, and Cstf, that promote the secretion of soluble Igs. This finding may indicate that the primal immune system can establish the protective mechanism against various infection routes such as the upper respiratory tract in addition to the placenta and maternal blood. (B) The defense mechanism in the cellular and endosomal membranes to inhibit virus entry and replication by IFN-inducing proteins. Over the past decade, several IFN-inducible proteins, including interferon-inducible transmembrane family (IFITMs), ArfGAP with dual pleckstrin homology (PH) domains 2 (ADAP2), gamma-interferon-inducible lysosome/endosome-localized thiolreductase (GILT), and LY6E, have been known to regulate the infectious entry of various viruses . Still, few studies have been performed on the fetus during pregnancy. However, we proved through new ex-vivo culture conditions containing HSC/UCB-MSC CO -EVs that the expressions of IFN-inducible proteins such as IFITM3, LY6E, and HLA-G were increased in fetal stem cells by increased IFN stimulation. Our results that itPG-FSC-EVs contained a higher level of LY6E and IFITM3 than FSC-EVs suggest that FSCs also have a sophisticated self-protection mechanism that suppresses the viral infection by LY6E mainly expressed in the plasma membrane at the beginning of virus entry and by IFITM3 expressed in the endosomal membrane after cell entry. In addition, membrane-bound HLA-G, whose expression is increased by IFN stimulation, may inhibit the relative expression of receptors that can be used as entry pathways of viruses and protect infected host cells by inducing delayed immune response through trogocytosis by contact with immune cells . (C) Intercellular defense mechanism to inhibit virus transmission by activating anti-viral autophagy. Transient Receptor Potential Mucolipin Subfamily (TRPMLs) are proteins constituting endosome cation channels and perform various physiological functions. TRPML1 is receiving attention as a target molecule that inhibits the fusion of the SARS-CoV-2 envelopes and endosomes , and a novel role of TRPML2, only expressed in the recycled endosome, in the innate immune response was recently revealed , but there is still little study on fetal stem cells during pregnancy. From our results, the expression of TRPML2 was increased in itPG-FSC EVs. We intend to suggest a new mechanism that the viral antigen information degraded by IFITMs upregulated by interferon stimulation acts as a pattern recognition receptor (PRR), thereby increasing TRPML2 expression , . In addition, upregulated TRPML2 may contribute to the intracellular protective mechanism that can prevent the replication of infected inhibits virus by binding to the endosomes containing the virus entering the cell, thereby inhibiting the entry of the virus into the cell nucleus and by inducing the degradation through anti-viral autophagy (lysosomal degradation).

Article Snippet: Labelling Abs that consist of anti-syntenin Alexa-555, anti-human IgG3 Alexa Fluor 488 (Novus Biologicals), and anti-human IgM Alexa Fluor 647 Abs (BioLegend), were used.

Techniques: Virus, Infection, Binding Assay, Ex Vivo, Clinical Proteomics, Membrane, Expressing, Transmission Assay

Journal: International Journal of Biological Sciences

Article Title: The Different Immune Responses by Age Are due to the Ability of the Fetal Immune System to Secrete Primal Immunoglobulins Responding to Unexperienced Antigens

doi: 10.7150/ijbs.67203

Figure Lengend Snippet: Suggested Immune System for the whole life. To date, the antibody-based humoral immune system against foreign pathogen infection is divided into three categories according to the infection stage . The first is the immediate innate immune response by NAbs already present in the body before antigen exposure, and it acts as the first line of defense against infectious agents. The second is an extrafollicular "innate-like" antibody response induced within the early few days after antigen exposure, which serves to eliminate the infection temporarily until the specialized antibody response matures. Lastly, it is an acquired antibody response that appears delayed 1-2 weeks after infection by highly assembled antibodies through somatic cell hypermutation and class switch recombination processes. However, we found that the primal immune system by IgP existed before the fetal immune system establishment during pregnancy, and the adaptabilities of innate and adaptive immune systems against foreign antigens depend on the primal repertoire that was experienced and formed during this period. (A) Primal Immunity. According to the study for healthy fetuses in early pregnancy , circulating B lymphocytes in fetal blood at 12 weeks of gestation have a diverse BCR repertoire, and immunoglobulin heave chain variable region gene (IGHV)-containing clones analyzed at 26 weeks of gestation are similar to those of healthy infants. It was confirmed that it exists in proportion, but this can be called the primary repertoire. On the contrary, the repertoire of NAbs, including IgG3 secreted from FSCs obtained around 10 weeks of pregnancy, which we confirmed through this study, has not experienced any antigens except for themselves, and in other words, can respond to any antigens. Therefore, it can be a primal repertoire, and it will be gradually diversified to the primary repertoires with antigen-specific diversities due to modifying factors such as Abs transmitted from the mother, food and environmental factors, vaccinations, infections, or diseases . (B) Innate Immunity. The primary repertoire formed from the Primal repertoire in early pregnancy plays a critical role in manifesting symptoms after infection in the later innate and adaptive immune systems. Younger children who have a relatively similar repertoire to the Primal repertoire can react immediately to new antigens and eliminate them before the adaptive immune system acts. However, since birth, the number of naïve B-1 cells that secrete natural antibodies acting on the innate immune system, the ability to secrete antibodies, and the adaptability of the repertoire to new antigens gradually decrease with age. The ability to cope with new viruses, such as SARS-CoV-2, also decreases with age . For this reason, many children and younger generations have asymptomatic infections of COVID-19, and even young children are well adapted to the mutant virus , while the older, the more symptomatic and severe the proportion of patients increases. (C) Adaptive Immunity. The evasion mechanisms to the initial innate immunity of all viruses, including SARS-CoV-2, and the resulting delayed priming of T cell responses and adaptive immunity are receiving attention as the main factors that increase the severity and fatality of COVID-19 with age . In other words, as the naïve T cell pool gradually decreases with age, it is difficult to generate a T cell response capable of recognizing a new antigen. This assumption can be supported by the findings that systemic excessive immune pathology, including cytokine storms, is caused by an explosive innate immune response activated to replace the delayed adaptive immune system . In particular, the delayed adaptive immune response can cause taste and olfactory abnormalities in asymptomatic and mild patients accounting for more than half of SARS-CoV-2 infections , so the most effective prevention and treatment for various infections including SARS-CoV-2 It can be said that the strategy lies in an effective innate immune system capable of triggering a normal adaptive immune response. From this point of view, it is expected that our research results, which first proposed the concept of the primal immune system before the formation of the innate immune system, will contribute to finding a fundamental solution to protect humanity against unknown infectious agents in the future. Another important finding in our results is that IgA is also contained in itPG-FSC-EVs secreted from FSCs in early pregnancy. It can be expected to provide an expanded variety of applicability to various infection pathways, such as the upper respiratory tract in the existing IgG-centered Ab therapeutics.

Article Snippet: Labelling Abs that consist of anti-syntenin Alexa-555, anti-human IgG3 Alexa Fluor 488 (Novus Biologicals), and anti-human IgM Alexa Fluor 647 Abs (BioLegend), were used.

Techniques: Infection, Clone Assay, Mutagenesis, Virus

Journal: bioRxiv

Article Title: Lupus IgA1 autoantibodies synergize with IgG to enhance plasmacytoid dendritic cell responses to RNA-containing immune complexes

doi: 10.1101/2023.09.07.556743

Figure Lengend Snippet: Surface staining and flow cytometry analysis performed on PBMCs from healthy control (HC) donors. (A) Representative histograms of FcαR ( red ) and FcγRIIa ( blue ) expression on pDCs compared to mIgG1 isotype control ( grey ), with detection by the same secondary antibody. (B) Paired analysis between monocyte and pDC FcαR (left) and FcγRIIa (right) surface staining (n=12 HC donors). (C) Binding of IgA and IgG to pDCs was assessed using biotinylated human heat-aggregated IgA or heat-aggregated IgG and fluorescently labeled streptavidin for visualization by flow cytometry, Streptavidin (SA) alone ( grey ), heat-aggregated IgA ( red ) and heat-aggregated IgG ( blue ). (D) Direct ex vivo binding of IgA and IgG to pDCs was assessed by staining with anti-IgA ( red ) or anti-IgG antibody ( blue ) directly ex vivo. Control ( grey ) has no anti-IgA or anti-IgG detection antibody. ( A-D ) Staining was performed on thawed PBMCs and pDCs gated as shown in Supplementary Fig. 2B. (B) Ratio paired t-test (**** p<0.0001).

Article Snippet: To generate aggregated IgA or IgG, biotinylated IgG (Novus NBP1-96855) or IgA (Novus NBP1-97181) was heated at a concentration of 2 mg/mL for 35-45 min at 65 C. After live dead staining, heat aggregated antibody was diluted in staining buffer to a final concentration of 0.5 mg/mL, incubated with PBMCs for 30 min at 37C.

Techniques: Staining, Flow Cytometry, Control, Expressing, Binding Assay, Labeling, Ex Vivo

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: T cells isolated from healthy human donors were activated with anti-CD3/28 beads and IL-2 was added on Day 3. A PD-1, PD-L1, and PD-L2 expression were measured via flow cytometry over 15 days for N = 8 in CD4+ and N = 5 in CD8 + T cells. B Day 4 purified CD8 + T cells were restimulated with autologous irradiated PBMC and anti-CD3 (1 μg/mL) ± anti-CD28 (1 μg/mL) in the presence of control antibody, anti-PD-1, or anti-PD-L1 at 10 μg/mL for 20 hr. Death was assessed via flow cytometry by propidium iodide staining. Single dot correlates with one human donor. Connecting line compares change in untreated and treated samples within the same donor. N = 3. C Diagram illustrating the proxy APC (pAPC) design created with a 1:1:1 ratio of anti-CD3, anti-CD28, and IgG1κ or recombinant human Fc-chimera PD-L1. D Freshly isolated CD4+ or CD8+ human T cells were activated at a 1:1 cell:bead ratio with IgG or PD-L1 beads and monitored via flow cytometry for CD69 expression after 24 hr ( N = 3 CD4 + , N = 5 CD8 + ) and CD25 expression after 72 hr ( N = 3 CD4 + , N = 6 CD8 + ). E Blastogenesis patterns of unactivated, IgG, or PD-L1 activated cells on Day 3 are demonstrated by dot plots assessing forward scatter (FSC) (x-axis) and side scatter (SSC) (y-axis). The progressive changes in FSC monitored via flow cytometry over 3 days are summarized for CD4+ and CD8 + T cells. N = 3 CD4 + , N = 5 CD8 + .

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Isolation, Expressing, Flow Cytometry, Purification, Irradiation, Control, Staining, Recombinant

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A CD8+ or CD4 + T cells were restimulated on Day 4 or Day 11 with p328-IgG of p328-L1 beads at a 1:1 bead:cell ratio and monitored for cell loss via flow cytometry 24 hr later using propidium iodide. N = 8 CD4+ early, N = 11 CD4+ late, N = 9 CD8+ early, N = 18 CD8+ late. B The difference in % cell loss between p328-IgG and p328-L1 beads in ( A ) is plotted for each donor, cell type, and cell stage evaluated. (same N as ( A ). C RICD assays set-up in ( A ) were examined on the flow cytometer using propidium iodide to calculate the percentage of live cells remaining after 24 hr of restimulation. N = 7 CD4+ early, N = 9 CD4+ late, N = 12 CD8+ early, N = 8 CD8+ late. D PD-L1 dose response was determined using the standard RICD setup amd a new bead set that gradually decreased PD-L1 concentration on the bead from 33% to 0%. Day 4 CD4+ and CD8 + T cells were utilized in this experiment. N = 12 CD4 + , N = 10 CD8 + , statistical analysis was calculated with two-way ANOVA. E Diagram representing RICD setup and creation of secondary bead set that was ligated 100% to IgG1κ (control secondary bead) or 67% IgG1κ and 33% recombinant PD-L1, matching the PD-L1 protein concentration on the p328-L1 primary bead. F Day 4 CD8+ and CD4 + T cells were restimulated with a 2:1 bead:cell ratio comprising a 1:1 ratio of the primary bead and 1:1 ratio of the secondary bead. RICD was monitored as in ( A ) N = 12.

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Flow Cytometry, Concentration Assay, Control, Recombinant, Protein Concentration

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A Representative histogram of data displaying demarcation of AnnexinV hi cells -/+ bead restimulation. B Day 4 and Day 11 CD4+ and CD8 + T cells were left unstimulated or were restimulated with p328-IgG or p328-L1 beads for 6 hr before being stained with AnnexinV and propidium iodide and read on the flow cytometer. N = 6. C Representative flow cytometric histogram of PI cell cycle phases based on DNA content. D Day 4 and Day 11 CD4+ and CD8 + T cells were left unstimulated or were restimulated with p328-IgG or p328-L1 beads for 18 hr before being permeabilized and stained with propidium iodide to determine % of cells in the subG1 phase. N = 8 CD4+ early, N = 8 CD4+ late, N = 10 CD8+ early, N = 9 CD8+ late. E Analysis using gated cells from ( D ) was performed to quantify changes in the G1 phase. F Cell phase characterization from ( D ) was analyzed to determine the PD-L1 induced difference in subG1 and G1 phases.

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Staining, Flow Cytometry

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A CD8+ or CD4 + T cells were restimulated on Day 4 with soluble anti-CD3 (OKT3) at 100 ng/mL or anti-CD3/CD28 coated beads at a 1:1 bead:cell ratio. Cell loss/apoptosis was quantified 18–24 hr later after propidium iodide staining and flow cytometry. N = 5 CD4+ early, N = 6 CD4+ late, N = 5 CD8+ early, N = 6 CD8+ late. B Day 4 and Day 11 CD4+ and CD8 + T cells from N = 5 individual donors were stained for CD28 and PD-1 expression and compared via flow cytometry. C Diagram illustrating restimulation beads conjugated with or without anti-CD28, with p328-IgG used to maintain total protein concentration. D CD8+ or CD4 + T cells were restimulated on Day 4 with beads illustrated in ( C ) at a 1:1 bead:cell ratio, monitored for cell loss 24 hr later using propidium iodide using flow cytometry. E The difference in cell loss induced by restimulation with beads -/+ anti-CD28 (leaving anti-CD3 + /- PD-L1-Fc constant) was plotted for every donor ( N = 10).

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Staining, Flow Cytometry, Expressing, Protein Concentration

Journal: Cell Death & Disease

Article Title: PD-1 protects expanding human T cells from premature restimulation-induced cell death by modulating TCR and CD28 signaling

doi: 10.1038/s41419-026-08530-6

Figure Lengend Snippet: A Day 4 CD4+ and CD8 + T cells ( N = 4 donors each) were restimulated with p328-IgG or p328-L1 beads at a 1:1 bead:cell ratio for 5 hr prior to lysate collection. Control cells were left unstimulated or treated with 1 μM STX. After cell lysis and protein standardization, donor lysates were pooled for each stimulation condition and analyzed on the apoptosis protein array. Representative spots for differentially expressed proteins are shown. Protein signal intensity from membrane imaging was analyzed and normalized using QuickSpots software. T cells were restimulated as in ( A ), lysed in RIPA buffer and separated by SDS-PAGE to quantify FASL ( B ) and survivin ( C ). Data are representative of experiments for 3–4 separate donors. ( D ) Day 4 CD4+ and CD8 + T cells were treated with 10 μg/mL anti-FASL blocking antibody for 1 hr prior to restimulation with the quad bead set. Cells were restimulated for 24 hr and stained with propidium iodide; % viability was assessed by flow cytometry. N = 7 CD4 + , N = 5 CD8 + .

Article Snippet: Isotype control antibodies were paired with the corresponding blocking antibody at 10 μg/mL: IgG1κ (BioXCell, #BP0297) or IgG4 (BioXCell, #CP148).

Techniques: Control, Lysis, Protein Array, Membrane, Imaging, Software, SDS Page, Blocking Assay, Staining, Flow Cytometry

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Humoral immunity remains largely intact in irAE patients, while seronegative RA patients have increased atypical B cell frequency and autoantibodies. (A) Expression of IgM and IgD on CD27 - CD24 - CD38 - B cells. Right, percentage of IgD + CD27 - CD24 - CD38 - naïve B cells and IgM - IgD - CD27 - CD24 - CD38 - double-negative B cells. (B) Expression of CD27 and CD38 on CD19 + B cells. Right, percentages of CD27 hi CD38 hi antibody-secreting cells (ASCs) and CD27 + CD38 - memory B cells. (C) Expression of CD138 on CD27 hi CD38 hi ASCs. Right, percentage of CD138 + ASCs and CD138 - ASCs. (D) Expression of CD11c and CD21 on CD19 + B cells. Right, percentage of CD11c + CD21 - CD19 + B cells. A-D, HC (n = 63), irAE (n = 33), RAC (n = 46) ICI, (n = 20). (E-G) GSEA was performed on the B cells between irAE and ICI. (E) Significantly enriched pathways in B cells from irAE and ICI. GSEA plots of IFN-α and IFN-γ response (F), and oxidative phosphorylation (G). (H) The volcano plot of the citrullinated or non-citrullinated relative IgG or IgM-isotype autoantigen levels comparing RA vs HC, irAE vs ICI, irAE vs HC, or irAE vs RA. The autoantigens were labeled when p < 0.01. (I) Immunoglobulin isotype levels in the plasma were measured by multiplex assay, HC (n = 22), irAE (n = 34), RAC (n = 46), ICI (n = 26). (J) CXCL13 levels in the plasma were measured by ELISA, HC (n = 20), irAE (n = 34) RAC (n = 47), ICI (n = 23). (K) BAFF levels in the plasma were measured by multiplex assay, HC (n = 21), irAE (n = 34), RAC (n = 47), ICI (n = 18). Data in graphs represent mean ± SEM, Significance was tested by one-way ANOVA (A-D, I-K), logistic regression (H).

Article Snippet: 100 μg/mL pembrolizumab (Keytruda) or Isotype control IgG4 (BioXcell, Cat# CP147) was added into the culture media 2 days after stimulation, and B cells were further cultured for another 5 days.

Techniques: Expressing, Phospho-proteomics, Immunopeptidomics, Labeling, Clinical Proteomics, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Pembrolizumab does not substantially affect B cell activation and antibody production ex vivo (A-C) Human naïve B cells were isolated from PBMCs and cultured under indicated conditions for 7 days; Pembrolizumab or isotype control IgG4 was added on Day 2, n = 8. (A) Expression of CD38 and CD27 on B cells. Right, percentages of CD27 + CD38 - , CD27 + CD38 + B cells. (B) Expression of CD138 on CD27 + CD38 + B cells. Right, percentage of CD138 + CD27 + CD38 + B cells. (C) Different immunoglobulin isotypes were measured in the culture supernatant of (A-B) by multiplex assay. (D-F) B cells from HuPD-1 mice were isolated and cultured with LPS, IL4, BAFF, or ODN2006, anti-IgM, IL-21, IL-4 or R848, anti-IgM, anti-CD40, IL-21, IFN-γ for 3 days, pembrolizumab or isotype control was added on Day 1. (D) Expression of IgG2c on activated B cells. Right, percentage of IgG2c + B cells, n = 5. (E) Expression of IgG1 on activated B cells. Right, percentage of IgG1 + B cells, n = 5. (F) Different immunoglobulin isotypes in the supernatant of were measured by multiplex assay, n = 5. Data in graphs represent mean ± SEM, Significance was tested by Two-way ANOVA.

Article Snippet: 100 μg/mL pembrolizumab (Keytruda) or Isotype control IgG4 (BioXcell, Cat# CP147) was added into the culture media 2 days after stimulation, and B cells were further cultured for another 5 days.

Techniques: Activation Assay, Ex Vivo, Isolation, Cell Culture, Control, Expressing, Multiplex Assay

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: PD-1 inhibition does not have substantial impact on B cells ex vivo (A) Human naïve B cells were isolated from healthy donor PBMCs and stimulated with different conditions; Pembrolizumab (Keytruda) or isotype control IgG4 was added into the culture media on day 2, mean fluorescence intensity (MFI) of CD86 on B cells was measured, n = 8. (B-F) B cells were isolated from HuPD-1 mice, labeled with CTV, and cultured under 3 different conditions: LPS, rmIL-4, BAFF or Anti-IgM, CpG ODN, rmIL-21, rmIL-4, 100 rhIL-2 or R848, anti-CD40, anti-IgM, rmIL-21, rmIFN-γ for 3 days. Pembrolizumab or isotype control IgG4 was added on day 1, n = 5. (B) Summary of human PD-1 MFI on B cells from different groups. (C) Expression of CD138 on activated B cells. Right, percentage of CD138 + B cells. (D) Summaries of activation marker CD69 and CD86 MFIs on B cells from different groups. (E) Summaries of CD71 and CD98 MFIs on B cells from different groups. (F) Representative flow cytometry plot of CTV dilution on B cells. Right, a summary of the proliferation index of B cells from different groups. Data in graphs represent mean ± SEM, Significance was tested by Two-way ANOVA.

Article Snippet: 100 μg/mL pembrolizumab (Keytruda) or Isotype control IgG4 (BioXcell, Cat# CP147) was added into the culture media 2 days after stimulation, and B cells were further cultured for another 5 days.

Techniques: Inhibition, Ex Vivo, Isolation, Control, Fluorescence, Labeling, Cell Culture, Expressing, Activation Assay, Marker, Flow Cytometry

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Inflammatory cytokines enriched in irAE patients promote both CD4 + and CD8 + T cell functions (A) Summaries of the percentage of CD45RA - CD8 + T cells at day 1 or day 5 of the culture, n = 6. (B) Summary of the percentage of Ki67 + in CD45RA - CD8 + T cells, n = 4. (C) Expression of CD69 on cultured CD8 + T cells at day 1. Right, CD69 mean fluorescence intensity (MFI) relative to the control in each experiment, n = 5. (D) Summary of the CD25 MFI relative to the control in each experiment, n = 5. (E) Summary of the percentage of Granzyme B + CD8 + T cells, n = 5. (F) Summary of the percentage of CD45RA - CD4 + T cells at day 5 of the culture, n = 4. (G) Summary of the percentage of Ki67 + CD45RA - CD4 + T cells, n = 4. (H-L) CD4 + T cells were isolated from healthy donor PBMCs, stimulated with 10 μg/mL plate-coated anti-human CD3 and anti-human CD28 in the presence of vehicle control (control), 100 ng/mL IFN-α, 100 ng/mL IL-6, 100 ng/mL IL-12, or the combination of them for 5 days, followed by restimulating with PMA/Ionomycin/Monensin for 5 h. (H) Expression of TNF-α in CD45RA - CD4 + T cells. Right, percentage of TNF-α + cells in CD45RA - CD4 + T cells, n =4. (I) Expression of IL-2 and IL-4 in CD45RA - CD4 + T cells. Right, percentage of IL-4 + and IL-2 + cells in CD45RA - CD4 + T cells, n = 3. (J-L) Summaries of the percentages of TNF-α + (J), IL-4 + (K), or IL-2 + (L) cells in CD38 + CXCR5 - or CD38 - CXCR5 + CD4 + T cells at day 5, n = 4. (M-Q) PBMCs from irAE patients were stimulated with 10 μg/mL plate-coated anti-human CD3 and anti-human CD28 in presence of IgG1 isotype control or a combination of 50 μg/mL anti-human IL-6R, 50 μg/mL anti-human IL-12p40 and 50 μg/mL anti-human IFNAR1 for 3 days, n = 9. (M) Expression of granzyme B and perforin in CD8 + T cells. Right, percentage of granzyme-B + perforin + CD8 + T cells. (N) MFI of MTG in CD8 + T cells. (O, P) MFI of GluCy5 (O), TMRM and MTDR (P), and MTG (Q) in CD4 + T cells. Data in graphs represent mean ± SEM, Significance was tested by One-way ANOVA (A-L), and paired Student’s T-test (N-Q).

Article Snippet: 100 μg/mL pembrolizumab (Keytruda) or Isotype control IgG4 (BioXcell, Cat# CP147) was added into the culture media 2 days after stimulation, and B cells were further cultured for another 5 days.

Techniques: Expressing, Cell Culture, Fluorescence, Control, Isolation

Journal: medRxiv

Article Title: Inflammatory arthritis immune related adverse events represent a unique autoimmune disease entity primarily driven by T cells, but likely not autoantibodies

doi: 10.1101/2025.06.06.25328991

Figure Lengend Snippet: Inflammatory cytokines enriched in irAE patients promote both CD4 + and CD8 + T cell functions (A-G) CD8 + T cells were isolated from healthy donor PBMCs and cultured in the plate coated with 10 μg/mL anti-human CD3 and anti-human CD28 for indicated days in the presence of vehicle control (control), 100 ng/mL IFN-α, 100 ng/mL IL-6, 100 ng/mL IL-12, or the combination of IFN-α, IL-6 and IL-12. (A) Summaries of the percentage of CD38 + CD127 - CD8 + T cells at day 1 and day 5, n = 5. (B) CD69 expression was measured on day 5, and the mean fluorescence intensity (MFI) of CD69 was summarized on the right, n = 4. (C) CD25 expression was measured on day 5, and the MFI of CD25 was summarized on the right, n = 4. (D) CD8 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, expression of granzyme B and IFN-γ in CD8 + T cells were examined by flow cytometry. Right, percentage of granzyme B + IFN-γ + CD8 + T cells, n = 5. (E-G) CD8 + T cells were cultured for 5 days. MFIs (all relative to those in control condition) of MTDR (E), MTG (F), and GluCy5 (G) were summarized, n = 5. (H-L) CD4 + T cells were isolated from the PBMCs of HC, cultured in the plate coated with 10 μg/mL anti-human CD3 and anti-human CD28 for indicated days in the presence of 100 ng/mL IFN-α, 100 ng/mL IL-6, 100 ng/mL IL-12, control or the combination of them. (H) CD4 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, and IL-21 level was examined in the CD45RA - CD4 + T cells, n =3. (I) Activated CD4 + T cells were restimulated with 10 μg/mL plate-coated anti-human CD3 and anti-human CD28 with monensin for 6 h, and expression of CXCL13 was measured in CD45RA - CD4 + T cells, n = 4. (J) CD38 + CXCR5 - or CD38 - CXCR5 + CD4 + T cells were examined at day 5, and the percentages were summarized, n = 4. (K) Percentage of CXCL13 + cells in CD38 + CXCR5 - or CD38 - CXCR5 + CD4 + T cells at day 5, n = 4. (L) Percentage of IL-21 + cells in CD38 + CXCR5 - or CD38 - CXCR5 + CD4 + T cells at day 5, n = 3. (M-S) PBMCs from the irAE patients were cultured in the plate coated with 10 μg/mL anti-CD3 and anti-CD28 in the presence of IgG1 isotype control or 50 μg/mL anti-human IL-6R, 50 μg/mL anti-human IL-12p40 and 50 μg/mL anti-human IFNAR1 for 3 days, n = 9. (M) Expression of CD38 and CD127 on CD8 + T cells. Right, percentage of CD38 + CD127 - CD8 + T cells. (N, O) CD8 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, (N) Expression of Granzyme B and IFN-γ. Right, percentage of granzyme B + IFN-γ + CD8 + T cells, (O) Expression of perforin and IFN-γ. Right, percentage of perforin + IFN-γ + CD8 + T cells. (P, Q) MFIs of GluCy5 (P), TMRM, and MTDR (Q) in CD8 + T cells were presented. (R, S) CD4 + T cells activated for 5 days were restimulated with PMA/Ionomycin/Monensin for 5 h, (R) Expression of IL-21 and IL-2. Right, percentage of IL-21 + IL-2 + CD4 + T cells. (S) Expression of CD38 and CXCR5 on CD4 + T cells. Right, percentage of CD38 + CXCR5 - CD4 + T cells. Data in graphs represent mean ± SEM, Significance was tested by One-way ANOVA (A-L), and paired Student’s t-test (M-S).

Article Snippet: 100 μg/mL pembrolizumab (Keytruda) or Isotype control IgG4 (BioXcell, Cat# CP147) was added into the culture media 2 days after stimulation, and B cells were further cultured for another 5 days.

Techniques: Isolation, Cell Culture, Control, Expressing, Fluorescence, Flow Cytometry

Journal: Cell reports

Article Title: Inhibitory affinity modulation of FcγRIIA ligand binding by glycosphingolipids by inside-out signaling

doi: 10.1016/j.celrep.2021.109142

Figure Lengend Snippet: (A) DMF-differentiated HL60 cells expressing FcγRIIA (HL60–2A) treated with β-glucan exhibited decreased affinity for hIgG-coated RBCs in the presence of LacCer C24 compared with LacCer C16 or vehicle. Non-specific binding as measured with RBCs without hIgG was negligible. (B) Lyn knockdown HL60–2A cells (Lyn-1) showed increased affinity for hIgG-coated RBCs compared to scRNA control in the presence of β-glucan but not in dextran. (C) SHP-1 knockdown HL60–2A cells showed similar results as Lyn-1 knockdown cells, with the addition of β-glucan leading to increased 2D affinity of knockdown cells for IgG. Conversely, there was a significant drop in affinity upon SHP-1 knockdown in the presence of dextran control. All measurements in (B) and (C) were in the presence of LacCer C24. Non-specific binding controls without hIgG are shown. A c K a values represent ligand-receptor density normalized adhesion frequencies. Data are average ± SD, and individual values are plotted. *p < 0.05, **p < 0.01, ***p < 0.001 using an unpaired Student’s t test.

Article Snippet: SA-coated RBCs were then incubated with 20 μg/mL biotinylated human IgG1 (Novus Biologicals, NBP1–96855) for 1 hr at 15°C while shaking.

Techniques: Expressing, Binding Assay, Knockdown, Control

Journal: Cell reports

Article Title: Inhibitory affinity modulation of FcγRIIA ligand binding by glycosphingolipids by inside-out signaling

doi: 10.1016/j.celrep.2021.109142

Figure Lengend Snippet: (A) DMF-differentiated HL60 cells expressing FcγRIIA (HL60–2A) were treated with vehicle or C24, dextran or β-glucan, and isotype or functional blocking FcγRIIA antibody (IV.3) as indicated. Cells were seeded on immobilized BSA-anti-BSA under static conditions and stained with phalloidin. The area of spread cells and representative images are shown. (B) DMF-differentiated HL60, HL60–2A with scRNA, Lyn shRNA-1 (Lyn-1), or Lyn shRNA-2 (Lyn-2) were evaluated as in (A). (C–E) Human neutrophils (C), HL60–2A cells (D) pretreated with C24 and β-glucan, or HL60–2A cells with indicated shRNAs (E) were treated with IV.3 followed with luminol and anti-mouse F(ab’) to induce cross-linking (XL), and ROS generation (relative light units [RLUs]) was monitored over time. Representative plots (left) and the peak level of ROS (right) for each condition that was normalized to the average of the F(ab’) untreated control are shown. (F) HL60 cells expressing wild-type FcγRIIA (WT) or FcγRIIA ΔITAM mutant were treated with LacCer C24 and β-glucan, dextran, IV.3, or isotype control as indicated and incubated with IgG-opsonized fluorescein isothiocyanate (FITC)-latex beads. Phagocytic index (percentage of FITC-positive cells over total cells) analyzed by FACS is shown. Bar graphs represent fold change compared to vehicle + dextran for (A), native for (B), and WT + dextran for (F). Dotted line represents the value of untreated HL60 for (A), (B), and (F) and no F(ab’) control for (C), (D), and (E). Data are average ± SEM, and individual values are plotted. *p < 0.05 using the Kruskal-Wallis test.

Article Snippet: SA-coated RBCs were then incubated with 20 μg/mL biotinylated human IgG1 (Novus Biologicals, NBP1–96855) for 1 hr at 15°C while shaking.

Techniques: Expressing, Functional Assay, Blocking Assay, Staining, shRNA, Control, Mutagenesis, Incubation

Journal: Cell reports

Article Title: Inhibitory affinity modulation of FcγRIIA ligand binding by glycosphingolipids by inside-out signaling

doi: 10.1016/j.celrep.2021.109142

Figure Lengend Snippet:

Article Snippet: SA-coated RBCs were then incubated with 20 μg/mL biotinylated human IgG1 (Novus Biologicals, NBP1–96855) for 1 hr at 15°C while shaking.

Techniques: Purification, Control, Recombinant, Phagocytosis Assay, Immunoprecipitation, Knock-Out, Software

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: ICAM-1 IgG autoantibody and sICAM-1 levels across healthy individuals and those with severe COVID-19, high BMI, and autoimmune conditions. (A) Dot plot graph of AUC on the log axis of the optical density at 450 nm measurement by ELISA for determining the levels of IgG antibodies targeting ICAM-1 within plasma from healthy individuals (n = 40), individuals with severe COVID-19 (n = 40), individuals with a high BMI over 30 kg/m 2 (n = 50), and individuals with select autoimmune conditions, SLE or RA (n = 39). (B) Dot plot graph of concentration (pg/mL) on the log axis measured by ELISA for determining the levels of sICAM-1 within plasma from healthy individuals (n = 40), individuals with severe COVID-19 (n = 40), individuals with a high BMI over 30, (n = 50), and individuals with autoimmune conditions SLE or RA (n = 39). Statistical significance was determined using the Mann-Whitney test. P value results are shown.

Article Snippet: Standards were prepared as followed prepared in duplicate: IgG (1-001-A, Lot#WAB0822071; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgA (NBP1-97039-1 Lot# 35592; Novus Biologicals) was diluted to 312.5 ng/mL followed by subsequent 1:2 dilutions until 4.88 ng/mL, IgM (DDXCH05P Lot# DDxCH04-010; Novus Biologicals) was diluted to 1,250 ng/mL followed by subsequent 1:2 dilutions until 19.531 μg/mL, IgG1 (DDXCH01P lot# DDXCH01-048; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgG2 (DDXCH02P Lot#DDxCH02P-100; Novus Biologicals) was diluted to 2500 ng/mL followed by subsequent 1:1.25 dilutions until 524.3 ng/mL, IgG3 (DDXCH03P Lot#DDXCH03-014; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, IgG4 (DDXCH04P Lot#DDXCH04-036; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, and standards were diluted in 0.1 M sodium bicarbonate.

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Concentration Assay, MANN-WHITNEY

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: Correlation of anti-ICAM-1 IgG autoantibody levels with age. (A) Dot plot graph of the AUC of the optical density at 450 nm on the log y-axis of IgG antibodies targeting ICAM-1 within plasma compared with age (x-axis) from healthy pediatric individuals ranging in age from 6 mo to 15 yr old (n = 37). (B) Dot plot graph of the AUC of the optical density at 450 nm on the log y-axis of IgG antibodies targeting ICAM-1 within plasma compared with age (x-axis) from healthy adult individuals ranging in age from 22 to 75 yr old (n = 40). Statistical significance was determined using the Pearson correlation test. P value results are shown.

Article Snippet: Standards were prepared as followed prepared in duplicate: IgG (1-001-A, Lot#WAB0822071; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgA (NBP1-97039-1 Lot# 35592; Novus Biologicals) was diluted to 312.5 ng/mL followed by subsequent 1:2 dilutions until 4.88 ng/mL, IgM (DDXCH05P Lot# DDxCH04-010; Novus Biologicals) was diluted to 1,250 ng/mL followed by subsequent 1:2 dilutions until 19.531 μg/mL, IgG1 (DDXCH01P lot# DDXCH01-048; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgG2 (DDXCH02P Lot#DDxCH02P-100; Novus Biologicals) was diluted to 2500 ng/mL followed by subsequent 1:1.25 dilutions until 524.3 ng/mL, IgG3 (DDXCH03P Lot#DDXCH03-014; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, IgG4 (DDXCH04P Lot#DDXCH04-036; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, and standards were diluted in 0.1 M sodium bicarbonate.

Techniques: Clinical Proteomics

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: High-resolution IgG epitope mapping of ICAM-1 autoantibodies using ICAM-1 peptide microarrays. (A) Heatmap of z scores for ICAM-1 epitope binding for individual samples (n = 20). ICAM-1 domains 1 through 5 are annotated above the heatmap. (B) Line graphs of median group z scores of ICAM-1 autoantibody binding to individual ICAM-1 peptides in the peptide array. Epitopes of recurrent high binding were qualified as median z scores of ≥0.95 to represent 1 SD ±0.05 above the median, points in red. (C) Table of recurrent high binding peptides, with associated z score and sequence. (D) Table of high binding clusters with associated peptide IDs, z score range, and sequence. Overlapping sequence motifs are bolded, and the common leucine threonine epitope is underlined.

Article Snippet: Standards were prepared as followed prepared in duplicate: IgG (1-001-A, Lot#WAB0822071; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgA (NBP1-97039-1 Lot# 35592; Novus Biologicals) was diluted to 312.5 ng/mL followed by subsequent 1:2 dilutions until 4.88 ng/mL, IgM (DDXCH05P Lot# DDxCH04-010; Novus Biologicals) was diluted to 1,250 ng/mL followed by subsequent 1:2 dilutions until 19.531 μg/mL, IgG1 (DDXCH01P lot# DDXCH01-048; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgG2 (DDXCH02P Lot#DDxCH02P-100; Novus Biologicals) was diluted to 2500 ng/mL followed by subsequent 1:1.25 dilutions until 524.3 ng/mL, IgG3 (DDXCH03P Lot#DDXCH03-014; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, IgG4 (DDXCH04P Lot#DDXCH04-036; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, and standards were diluted in 0.1 M sodium bicarbonate.

Techniques: Binding Assay, Peptide Microarray, Sequencing

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: Immunoglobulin isotype and subclass determination of anti-ICAM-1 antibodies. (A) Dot plot graph of concentration (µg/mL) on the log y-axis obtained by ELISA for determining the levels of immunoglobulin isotype class within high-autoantibody-presenting plasma, defined as the top 16 positive samples by qualitative ELISA regardless of disease group (n = 16). (B) Dot plot graph of concentration (µg/mL) obtained by ELISA for determining the levels of IgG subclass within high-autoantibody-presenting plasma, defined as the top 16 positive samples by qualitative ELISA regardless of disease group (n = 16). Statistical significance was determined using the Mann-Whitney test. P value results are shown.

Article Snippet: Standards were prepared as followed prepared in duplicate: IgG (1-001-A, Lot#WAB0822071; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgA (NBP1-97039-1 Lot# 35592; Novus Biologicals) was diluted to 312.5 ng/mL followed by subsequent 1:2 dilutions until 4.88 ng/mL, IgM (DDXCH05P Lot# DDxCH04-010; Novus Biologicals) was diluted to 1,250 ng/mL followed by subsequent 1:2 dilutions until 19.531 μg/mL, IgG1 (DDXCH01P lot# DDXCH01-048; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgG2 (DDXCH02P Lot#DDxCH02P-100; Novus Biologicals) was diluted to 2500 ng/mL followed by subsequent 1:1.25 dilutions until 524.3 ng/mL, IgG3 (DDXCH03P Lot#DDXCH03-014; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, IgG4 (DDXCH04P Lot#DDXCH04-036; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, and standards were diluted in 0.1 M sodium bicarbonate.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, MANN-WHITNEY

Journal: ImmunoHorizons

Article Title: ICAM-1 autoantibodies detected in healthy individuals and cross-react with functional epitopes

doi: 10.1093/immhor/vlaf025

Figure Lengend Snippet: Anti-ICAM-1 IgG autoantibodies interact with protein glycans. (A) SDS-PAGE separation of ICAM-1 under nonreducing conditions. Lane 1 shows ICAM-1 protein, lane 2 shows ICAM-1 in reaction buffer without PNGase F, and lane 3 shows ICAM-1 with PNGase F. Molecular masses are displayed on the left margin in kDa. (B) Dot plot graph of the AUC of the optical density at 450 nm on the log y-axis obtained by ELISA for determining the levels of ICAM-1 IgG autoantibodies targeting ICAM-1 with glycans (control) compared with enzymatically deglycosylated ICAM-1 (PNGASE) within plasma with high levels of detected ICAM-1 autoantibodies, defined as the top 8 positive samples by qualitative ELISA regardless of disease group (n = 8). Statistical significance was determined using the Wilcoxon matched pairs signed rank test. P value results are shown.

Article Snippet: Standards were prepared as followed prepared in duplicate: IgG (1-001-A, Lot#WAB0822071; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgA (NBP1-97039-1 Lot# 35592; Novus Biologicals) was diluted to 312.5 ng/mL followed by subsequent 1:2 dilutions until 4.88 ng/mL, IgM (DDXCH05P Lot# DDxCH04-010; Novus Biologicals) was diluted to 1,250 ng/mL followed by subsequent 1:2 dilutions until 19.531 μg/mL, IgG1 (DDXCH01P lot# DDXCH01-048; Novus Biologicals) was diluted to 500 ng/mL followed by subsequent 1:2 dilutions until 7.813 ng/mL, IgG2 (DDXCH02P Lot#DDxCH02P-100; Novus Biologicals) was diluted to 2500 ng/mL followed by subsequent 1:1.25 dilutions until 524.3 ng/mL, IgG3 (DDXCH03P Lot#DDXCH03-014; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, IgG4 (DDXCH04P Lot#DDXCH04-036; Novus Biologicals) was diluted to 2,500 ng/mL followed by subsequent 1:2 dilutions until 39.063 ng/mL, and standards were diluted in 0.1 M sodium bicarbonate.

Techniques: SDS Page, Enzyme-linked Immunosorbent Assay, Control, Clinical Proteomics