Antigen density quantification of cell-surface immunotherapy targets by flow cytometry: Multi-antigen assay of neuroblastoma bone marrow metastasis.

The central role of target antigen density on chimeric antigen receptor T cell potency highlights the need for accurate measurement of antigen levels on clinical tumor samples. Here, we present a protocol for quantifying antigen density for six cell-surface antigens on neuroblastoma cells metastatic to bone marrow. We describe steps for patient sample acquisition, flow cytometry panel development, instrument setup, and compensation and detail procedures for running clinical samples and data analysis. For complete details on the use and execution of this protocol, please refer to Heitzeneder et al. (2022).1.

COVID-19 Adenoviral Vector Vaccination Elicits a Robust Memory B Cell Response with the Capacity to Recognize Omicron BA.2 and BA.5 Variants.

Following the COVID-19 pandemic, novel vaccines have successfully reduced severe disease and death. Despite eliciting lower antibody responses, adenoviral vector vaccines are nearly as effective as mRNA vaccines. Therefore, protection against severe disease may be mediated by immune memory cells. We here evaluated plasma antibody and memory B cells (Bmem) targeting the SARS-CoV-2 Spike receptor-binding domain (RBD) elicited by the adenoviral vector vaccine ChAdOx1 (AstraZeneca), their capacity to bind Omicron subvariants, and compared this to the response to mRNA BNT162b2 (Pfizer-BioNTech) vaccination. Whole blood was sampled from 31 healthy adults pre-vaccination and 4 weeks after dose one and dose two of ChAdOx1. Neutralizing antibodies (NAb) against SARS-CoV-2 were quantified at each time point. Recombinant RBDs of the Wuhan-Hu-1 (WH1), Delta, BA.2, and BA.5 variants were produced for ELISA-based quantification of plasma IgG and incorporated separately into fluorescent tetramers for flow cytometric identification of RBD-specific Bmem. NAb and RBD-specific IgG levels were over eight times lower following ChAdOx1 vaccination than BNT162b2. In ChAdOx1-vaccinated individuals, median plasma IgG recognition of BA.2 and BA.5 as a proportion of WH1-specific IgG was 26% and 17%, respectively. All donors generated resting RBD-specific Bmem, which were boosted after the second dose of ChAdOx1 and were similar in number to those produced by BNT162b2. The second dose of ChAdOx1 boosted Bmem that recognized VoC, and 37% and 39% of WH1-specific Bmem recognized BA.2 and BA.5, respectively. These data uncover mechanisms by which ChAdOx1 elicits immune memory to confer effective protection against severe COVID-19.

An optimized flow cytometry protocol for simultaneous detection of T cell activation induced markers and intracellular cytokines: Application to SARS-CoV-2 immune individuals.

Antigen (ag)-specific T cell analysis is an important step for investigation of cellular immunity in many settings, such as infectious diseases, cancer and vaccines. Multiparameter flow cytometry has advantages in studying both the rarity and heterogeneity of these cells. In the cellular immunologist's toolbox, the expression of activation-induced markers (AIM) following antigen exposure has made possible the study and sorting of ag-specific T cells without using human leukocyte antigen (HLA)-multimers. In parallel, assessing the cytokine profile of responding T cells would support a more comprehensive description of the ongoing immune response by providing information related to cell function, such as polarization and effector activity. Here, a method and flow cytometry panel were optimized to combine the detection of activated CD4+ and CD8+ T cells in a TCR-dependent manner with the evaluation of cytokine production by intracellular staining, without affecting the positivity of activation markers. In particular, the expression of CD134 (OX40) and CD69 have been tested in conjunction with intracellular (ic) CD137 (4-1BB) to detect SARS-CoV-2 Spike protein-specific activated T cells. In our setting, CD134 provided minimal contribution to detect the pool of AIM+ T cells, whereas a key role was described for ic-CD69 which was co-expressed with ic-CD137 in both CD4+ and CD8+ lymphocytes. Moreover, the analysis of TCR-triggered cytokine-producing T cells (IFNγ, TNFα and IL-2 were assessed) further confirmed the capacity of ic-CD69 to identify functionally responsive antigen-specific T cells which were often largely negative or weakly positive for CD134 expression. In parallel, the use of CD45RA, CCR7 and CXCR5 allowed us to describe the T cell matuarion curve and detect T follicular helper (Tfh) CD4+ cells, including the antigen specific activated subsets. In conclusion, we optimized a method and flow cytometry panel combining assessment of activation induced markers and intracellular cytokines that will be useful for measuring TCR stimulation-dependent activation of CD4+ and CD8+ T cells.

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants.

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.

GPC2-CAR T cells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity.

Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (∼5,000 molecules/cell, range 1-6 × 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors.

CAR T cells with dual targeting of CD19 and CD22 in adult patients with recurrent or refractory B cell malignancies: a phase 1 trial.

Despite impressive progress, more than 50% of patients treated with CD19-targeting chimeric antigen receptor T cells (CAR19) experience progressive disease. Ten of 16 patients with large B cell lymphoma (LBCL) with progressive disease after CAR19 treatment had absent or low CD19. Lower surface CD19 density pretreatment was associated with progressive disease. To prevent relapse with CD19- or CD19lo disease, we tested a bispecific CAR targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) in a phase I clinical trial ( NCT03233854 ) of adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) and LBCL. The primary end points were manufacturing feasibility and safety with a secondary efficacy end point. Primary end points were met; 97% of products met protocol-specified dose and no dose-limiting toxicities occurred during dose escalation. In B-ALL (n = 17), 100% of patients responded with 88% minimal residual disease-negative complete remission (CR); in LBCL (n = 21), 62% of patients responded with 29% CR. Relapses were CD19-/lo in 50% (5 out of 10) of patients with B-ALL and 29% (4 out of 14) of patients with LBCL but were not associated with CD22-/lo disease. CD19/22-CAR products demonstrated reduced cytokine production when stimulated with CD22 versus CD19. Our results further implicate antigen loss as a major cause of CAR T cell resistance, highlight the challenge of engineering multi-specific CAR T cells with equivalent potency across targets and identify cytokine production as an important quality indicator for CAR T cell potency.

Lymphocyte Subset Counts in COVID-19 Patients: A Meta-Analysis.

A reduced peripheral blood absolute lymphocyte count with an elevated neutrophil count has been a consistent observation in hospitalized coronavirus disease 2019 (COVID-19) patients. In this brief meta-analysis, the reduction of lymphocyte subset counts in COVID-19 patients was investigated across 20 peer-reviewed studies meeting criteria for reporting lymphocyte subset counts and COVID-19 disease severity. CD4+ T cell, CD8+ T cell, B cell, NK cell, and total lymphocyte cell counts all showed statistically significant reduction in patients with severe/critical COVID-19 disease compared to mild/moderate disease. T-cell subsets showed the largest standardized magnitude of change. In some studies, multivariate analysis has shown that CD4 and/or CD8 T-cells counts are independently predictive of patient outcomes. © 2020 International Society for Advancement of Cytometry.

A G{alpha}i-GIV molecular complex binds epidermal growth factor receptor and determines whether cells migrate or proliferate.

Cells respond to growth factors by either migrating or proliferating, but not both at the same time, a phenomenon termed migration-proliferation dichotomy. The underlying mechanism of this phenomenon has remained unknown. We demonstrate here that Galpha(i) protein and GIV, its nonreceptor guanine nucleotide exchange factor (GEF), program EGF receptor (EGFR) signaling and orchestrate this dichotomy. GIV directly interacts with EGFR, and when its GEF function is intact, a Galpha(i)-GIV-EGFR signaling complex assembles, EGFR autophosphorylation is enhanced, and the receptor's association with the plasma membrane (PM) is prolonged. Accordingly, PM-based motogenic signals (PI3-kinase-Akt and PLCgamma1) are amplified, and cell migration is triggered. In cells expressing a GEF-deficient mutant, the Galphai-GIV-EGFR signaling complex is not assembled, EGFR autophosphorylation is reduced, the receptor's association with endosomes is prolonged, mitogenic signals (ERK 1/2, Src, and STAT5) are amplified, and cell proliferation is triggered. In rapidly growing, poorly motile breast and colon cancer cells and in noninvasive colorectal carcinomas in situ in which EGFR signaling favors mitosis over motility, a GEF-deficient splice variant of GIV was identified. In slow growing, highly motile cancer cells and late invasive carcinomas, GIV is highly expressed and has an intact GEF motif. Thus, inclusion or exclusion of GIV's GEF motif, which activates Galphai, modulates EGFR signaling, generates migration-proliferation dichotomy, and most likely influences cancer progression.

Activation of Galphai3 triggers cell migration via regulation of GIV.

During migration, cells must couple direction sensing to signal transduction and actin remodeling. We previously identified GIV/Girdin as a Galphai3 binding partner. We demonstrate that in mammalian cells Galphai3 controls the functions of GIV during cell migration. We find that Galphai3 preferentially localizes to the leading edge and that cells lacking Galphai3 fail to polarize or migrate. A conformational change induced by association of GIV with Galphai3 promotes Akt-mediated phosphorylation of GIV, resulting in its redistribution to the plasma membrane. Activation of Galphai3 serves as a molecular switch that triggers dissociation of Gbetagamma and GIV from the Gi3-GIV complex, thereby promoting cell migration by enhancing Akt signaling and actin remodeling. Galphai3-GIV coupling is essential for cell migration during wound healing, macrophage chemotaxis, and tumor cell migration, indicating that the Galphai3-GIV switch serves to link direction sensing from different families of chemotactic receptors to formation of the leading edge during cell migration.

Computational modeling reveals how interplay between components of a GTPase-cycle module regulates signal transduction.

Heterotrimeric G protein signaling is regulated by signaling modules composed of heterotrimeric G proteins, active G protein-coupled receptors (Rs), which activate G proteins, and GTPase-activating proteins (GAPs), which deactivate G proteins. We term these modules GTPase-cycle modules. The local concentrations of these proteins are spatially regulated between plasma membrane microdomains and between the plasma membrane and cytosol, but no data or models are available that quantitatively explain the effect of such regulation on signaling. We present a computational model of the GTPase-cycle module that predicts that the interplay of local G protein, R, and GAP concentrations gives rise to 16 distinct signaling regimes and numerous intermediate signaling phenomena. The regimes suggest alternative modes of the GTPase-cycle module that occur based on defined local concentrations of the component proteins. In one mode, signaling occurs while G protein and receptor are unclustered and GAP eliminates signaling; in another, G protein and receptor are clustered and GAP can rapidly modulate signaling but does not eliminate it. Experimental data from multiple GTPase-cycle modules is interpreted in light of these predictions. The latter mode explains previously paradoxical data in which GAP does not alter maximal current amplitude of G protein-activated ion channels, but hastens signaling. The predictions indicate how variations in local concentrations of the component proteins create GTPase-cycle modules with distinctive phenotypes. They provide a quantitative framework for investigating how regulation of local concentrations of components of the GTPase-cycle module affects signaling.