Development of a reporter gene method to measure the bioactivity of anti-CD38 × CD3 bispecific antibody

Background A T cell-redirecting bispecific antibody consisting of a tumor-binding unit and a T cell-binding unit is a large group of antibody-based biologics against death-causing cancer diseases. The anti-CD38 × anti-CD3 bispecific antibody (Y150) is potential for treating multiple myeloma (MM). When developing a cell-based reporter gene bioassay to assess the activities of Y150, it was found that the expression of CD38 on the human T lymphocyte cells (Jurkat) caused the nonspecific activation which interfered with the specific T cells activation of mediated by the Y150 and CD38(+) tumor cells. Methods Here we first knocked-out the CD38 expression on Jurkat T cell line by CRISPR-Cas9 technology, then developed a stable monoclonal CD38(−) Jurkat T cell line with an NFAT-RE driving luciferase expressing system. Further based on the CD38(−) Jurkat cell, we developed a reporter gene method to assess the bioactivity of the anti-CD38 × anti-CD3 bsAb. Results Knocking out CD38 expression abolished the nonspecific self-activation of the Jurkat cells. The selected stable monoclonal CD38(−) Jurkat T cell line assured the robustness of the report genes assay for the anti-CD38 × anti-CD3 bsAb. The relative potencies of the Y150 measured by the developed reporter gene assay were correlated with those by the flow-cytometry-based cell cytotoxicity assay and by the ELISA-based binding assay. Conclusions The developed reporter gene assay was Mechanism of Action (MOA)-reflective for the bioactivity of anti-CD38 × anti-CD3 antibody, and suitable for the quality control for the bsAb product.

Identification of TSPAN4 as Novel Histamine H4 Receptor Interactor

The histamine H4 receptor (H4R) is a G protein-coupled receptor that is predominantly expressed on immune cells and considered to be an important drug target for various inflammatory disorders. Like most GPCRs, the H4R activates G proteins and recruits -arrestins upon phosphorylation by GPCR kinases to induce cellular signaling in response to agonist stimulation. However, in the last decade, novel GPCR-interacting proteins have been identified that may regulate GPCR functioning. In this study, a split-ubiquitin membrane yeast two-hybrid assay was used to identify H4R interactors in a Jurkat T cell line cDNA library. Forty-three novel H4R interactors were identified, of which 17 have also been previously observed in MYTH screens to interact with other GPCR subtypes. The interaction of H4R with the tetraspanin TSPAN4 was confirmed in transfected cells using bioluminescence resonance energy transfer, bimolecular fluorescence complementation, and co-immunoprecipitation. Histamine stimulation reduced the interaction between H4R and TSPAN4, but TSPAN4 did not affect H4R-mediated G protein signaling. Nonetheless, the identification of novel GPCR interactors by MYTH is a starting point to further investigate the regulation of GPCR signaling.

Zn-Doped CaP-Based Coatings on Ti–6Al–4V and Ti–6Al–7Nb Alloys Prepared by Magnetron Sputtering: Controllable Biodegradation, Bacteriostatic, and Osteogenic Activities

New TiNb-based alloys, such as Ti–6Al–7Nb, are currently being studied around the world as an alternative to other Ti alloys, e.g., instead of Ti–6Al–4V. We conducted a pilot study where thin (approximately 1.2 micron) CaP coatings containing low doses of Zn2+ (0.4–0.8 wt.%) were prepared by the radio frequency magnetron sputtering (RFMS) of Zn-hydroxyapatite (HA) target on Ti–6Al–4V and Ti–6Al–7Nb substrates and investigated their physicochemical properties, in vitro solubility, cytotoxicity, and antibacterial and osteogenic activities. The thickness of the obtained coatings was approximately 1.2–1.3 microns. Zn substitution did not result in roughness or structural or surface changes in the amorphous CaP coatings. The distributions of Ca, P, and Zn were homogeneous across the film thickness as shown by the EDX mapping of these elements. Zn doping of CaP coatings on both types of Ti-based alloys statistically influenced the results of the scratch-test. However, obtained values are satisfactory to use Zn-CaP coatings on biomedical implants. Increased Zn2+ release vs. tapered output of Ca and phosphate ions occurred during 5 weeks of an in vitro immersion test in 0.9% NaCl solution. Ti–6Al–7Nb alloy, unlike Ti–6Al–4V, promoted more linear biodegradation of CaP coatings in vitro. As a result, CaP-based surfaces on Ti–6Al–7Nb, compared with on Ti–6Al–4V alloy, augmented the total areas of Alizarin red staining in a 21-day culture of human adipose-derived mesenchymal stem cells in a statistically significant manner. Moreover, Zn–CaP coatings statistically reduced leukemic Jurkat T cell survival within 48 h of in vitro culture. Along with the higher solubility of the Zn–CaP surface, a greater reduction (4- to 5.5-fold) in Staphylococcus aureus growth was observed in vitro when 7-day extracts of the coatings were added into the microbial culture. Hence, Zn–CaP-coated Ti–6Al–7Nb alloy with controllable biodegradation as prepared by RFMS is a prospective material suitable for bone applications in cases where there is a risk of bacterial contamination with severe consequences, for example, in leukemic patients. Further research is needed to closely investigate the mechanical features and pathways of their solubility and antimicrobial, antitumor, and osteogenic activities.

CD3G or CD3D Knockdown in Mature, but Not Immature, T Lymphocytes Similarly Cripples the Human TCRαβ Complex

The human αβ T-cell receptor (TCR) is composed of a variable heterodimer (TCRαβ) and three invariant dimers (CD3γε, CD3δε, and ζζ/CD2472). The role of each invariant chain in the stepwise interactions among TCR chains along the assembly is still not fully understood. Despite the high sequence homology between CD3γ and CD3δ, the clinical consequences of the corresponding immunodeficiencies (ID) in humans are very different (mild and severe, respectively), and mouse models do not recapitulate findings in human ID. To try to understand such disparities, we stably knocked down (KD) CD3D or CD3G expression in the human Jurkat T-cell line and analyzed comparatively their impact on TCRαβ assembly, transport, and surface expression. The results indicated that TCR ensembles were less stable and CD3ε levels were lower when CD3γ, rather than CD3δ, was scarce. However, both defective TCR ensembles were strongly retained in the ER, lacked ζζ/CD2472, and barely reached the T-cell surface (<11% of normal controls) in any of the CD3 KD cells. This is in sharp contrast to human CD3γ ID, whose mature T cells express higher levels of surface TCR (>30% vs. normal controls). CD3 KD of human T-cell progenitors followed by mouse fetal thymus organ cultures showed high plasticity in emerging immature polyclonal T lymphocytes that allowed for the expression of significant TCR levels which may then signal for survival in CD3γ, but not in CD3δ deficiency, and explain the immunological and clinical disparities of such ID cases.

Increased Expression of Cytoskeleton Coordinator Protein MACF1 at the Immune Synapse during Jurkat T Cell Activation

Introduction: Activation of the T cell Receptor (TCR) by antigen on the surface of an Antigen Presenting Cell (APC) leads to a dramatic change in T cell morphology and formation of a specialized signaling interface with the APC known as the Immune Synapse (IS). The IS allows for controlled cytokine secretion and is an important regulator of T cell activation. IS formation has been found to be dysfunctional in many immune disorders including T cell acute lymphoblastic leukemia (ALL).The actin and microtubule (MT) cytoskeletons have individually been implicated in regulating both IS formation and TCR signaling, but the crosstalk between actin and MTs in T cells has not been well explored. We hypothesized that MACF1 (Microtubule Actin Crosslinking Factor 1), a giant cytoskeleton crosslinker from the spectraplakin family, is present at the IS following Jurkat T cell activation and coordinates actin and MTs. To investigate this, we used TIRFM (Total Internal Reflection Fluorescence Microscopy) and confocal microscopy to image MACF1 organization at the IS. Methods: Jurkat E6-1 cells were cultured in RPMI 1640 supplemented with 10% FBS and 1% penicillin/streptomycin. Glass coverslips were coated with Poly-L-Lysine 0.01% and then with 10 µg/mL anti-CD3 for 2 hours at 37 °C to create an activating surface. Cells were dropped carefully and allowed to spread on these coverslips at 37 °C for the indicated time. In the inhibition experiments,5 µM SB415286 or 5 µM PP2 was added to cells at 8 minutes following the initiation of activation and allowed to incubate for an additional 8 minutes. Cells were fixed with 3.7% paraformaldehyde following activation and then permeabilized with 0.1% Triton X-100. Cells were stained for anti-MACF1, anti-beta tubulin clone KMX-1, respective secondary antibodies, actin-stain 488 phalloidin, and DAPI (4',6-diamidino-2-phenylindole). Fluorescence of labeled cells was imaged using TIRFM with a Nikon Ti microscope and Andor Zyla CMOS camera with a 100x magnification objective (1.49 NA). TIRFM images were analyzed for Corrected Total Cell Fluorescence, which is the total fluorescence of the cell corrected for the background. Mann-Whitney U tests were used for all statistical comparisons. Cells were imaged on Leica SP5X laser confocal microscope with 63x objective for localization of MACF1 with actin and tubulin. Confocal images were deconvolved using the deconvolution lab2 plugin in FIJI (FIJI Is Just ImageJ). Results: Corrected Total Cell Fluorescence (CTCF) of MACF1 was higher at 4, 8 and 16 minutes following activation compared to the PLL control (Figures 1A and 1B). There was no significant difference in CTCF across the different activation times. To test whether MACF1 is dis-inhibited in Jurkat T cells during activation, we used SB415286 to inhibit Glycogen Synthase Kinase 3-Beta (GSK3B), a protein that inhibits MACF1 function, on PLL-only coverslips. We found MACF1 CTCF to be higher following SB415286 treatment compared to the control, indicating that MACF1 levels increase with GSK3B inhibition (Figure 1C). Thus, SB415286 inhibition of GSK3B mimics T cell activation where Akt upregulation downstream of TCR signaling inhibits GSK3B. To further test whether CD3 signaling activates MACF1, we stimulated Jurkat cells on anti-CD3 coated glass and then removed the activation signal with the src-kinase inhibitor PP2, which inhibits lymphocyte-specific protein tyrosine kinase (Lck) - an important regulator of early T cell signaling. MACF1 CTCF was lower after PP2 inhibition compared with the control, suggesting that continued CD3 stimulation is necessary for MACF1 accumulation at the IS (Figure 1D). We then used confocal microscopy to image the spatial distribution of MACF1 and assess whether it localizes with actin and MTs at the IS. We found that MACF1 puncta are present at the F-actin rich lamellipodial region of the IS and appear to localize with MT filaments, suggesting that MACF1 coordinates the two cytoskeleton components at the IS (Figure 2). Conclusion: Using high resolution microscopy, we show that MACF1 is upregulated at the IS during Jurkat T cell activation downstream of CD3 signaling through a GSK3B dependent pathway. At the IS, we found the novel localization of MACF1 with actin and MTs. This upregulation and expression of MACF1 during activation warrants more studies of the role MACF1 plays in T cell activation, cytoskeletal dynamics and IS function. Disclosures No relevant conflicts of interest to declare.

FLOW CYTOMETRY MULTIPLEXED METHOD FOR THE DETECTION OF NEUTRALIZING HUMAN ANTIBODIES TO THE NATIVE SARS-CoV-2 SPIKE PROTEIN

A correct identification of seropositive individuals for the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T cell line that stably expresses the full-length native spike S protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a ratio of the mean fluorescence intensities obtained by double- staining with the sera and a monoclonal antibody specific for EGFR. We show that the method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. It can also be employed to detect neutralizing activity in the sera of individuals. Finally, the method can be used in a multiplexed format to simultaneously measure all anti-S human immunoglobulin isotypes in blood and mucosal fluids including total saliva.