Author: Si-Han Hai

Abstract:

When samples with highly fluorescent proteins, dyes, or probes are used, the signals they generate are of extremely high intensity, posing a risk of signal saturation. Such saturated signals lead to inaccurate compensation or unmixing, undermining the quality of data analysis. Even when in the absence of signal saturation, high fluorophore brightness can cause signal spreading errors, complicating the accurate identification of co-expressed signals. To tackle this issue, the Agilent NovoCyte Opteon spectral flow cytometer offers a solution with user-adjustable laser power. By appropriately reducing the laser power, saturation signals can be lowered to within the detection range and minimize the spreading error from high-brightness signals. This adjustment improves the accuracy of unmixing and enhances overall data reliability. Several applications were used to evaluate the performance of the NovoCyte Opteon at different laser power settings. A key characteristic of these applications is the bright signals associated with fluorescent proteins or various fluorescent probes, while some markers in multicolor immunophenotyping exhibit relatively weak expression. 

Author: Toby Astill, Connie Inlay

Abstract: Immune profiling has emerged as a critical tool to reveal predictive biomarkers, and standardized high-parameter assays are essential to enable reproducibility across time, cohorts and sites. Efficient and reproducible panel preparation is critical for high-dimensional single-cell analysis workflows such as mass cytometry (CyTOF™ technology). Traditional liquid antibody panels require careful handling and extended preparation times and are prone to variability and stability issues during storage and shipping. Dry lyophilized antibody pellets address these challenges by offering pre-aliquoted stable reagents that streamline workflows, reduce technical variation and improve operational efficiency. This approach minimizes hands-on time, ensures consistent antibody concentrations and enhances long-term stability, making it particularly valuable for complex panels and large-scale studies. 

In this study, lyophilized CyTOF antibody panels, which may contain up to 50 metal-tagged antibodies, were evaluated against equivalent liquid antibody controls. PBMC samples were stained with lyophilized panels as small as four antibodies up to 38 antibodies and acquired on a CyTOF XT instrument. Experimental data confirmed that lyophilization preserves antibody functionality, even in complex staining workflows. Lyophilized panels demonstrated significant time savings, reducing preparation time from approximately two hours to <1 minute, while lowering technical variation well below clinical flow cytometry standards at <2% (as measured by %CV of marker signal intensity). Compatibility testing for intracellular staining and multiplex barcoding showed performance equivalent to liquid controls. Long-term stability studies further verified that lyophilized reagents aged over two years at 4 °C retained full functional equivalence to freshly prepared controls. 

These findings establish lyophilized panels as a reliable, reproducible and efficient solution for high-parameter mass cytometry workflows, enabling standardization, time savings and complex immune profiling studies while improving data quality. 

Author: Toby Astill

Abstract: 

Transcriptomics and proteomics provide synergistic information about the cell types, states and functions present in a tissue sample. Integrating spatial proteomics with transcriptomics allows for improved identification of functional protein networks, and a deeper understanding of cellular organization. However, following up to several days of transcriptomics acquisition, many spatial proteomics methodologies that use sequential acquisition of data further degrades the quality of the samples and eventual data. Imaging Mass Cytometry (IMC ) technology is a high-plex spatial biology imaging technique that enables deep characterization of the diversity and complexity of tissue microenvironments. IMC supports detailed assessment of cell phenotype and function using 40-plus metal-tagged antibodies simultaneously on a single slide without artifacts associated with fluorescence-based spectral overlap, tissue autofluorescence and multiple acquisition cycles. Here, we showcase the application of IMC assays directly on slides following 10x Genomics Xenium acquisition. This novel approach enables simultaneous spatial  transcriptomics and proteomics within the same single cell, thus delivering more comprehensive cellular signatures at a single-cell level. We demonstrate the utility of this new approach in liver cancer tissues

Author: Preksha Gupta

Abstract: 

Droplet microfluidics has emerged as a critical component of several high-throughput single cell analysis techniques in biomedical research and diagnostics. While there has been significant progress in the individual assays being developed, multi-parametric optical sensing of droplets and their encapsulated contents remains challenging. The current common approach of microscopy based high-speed imaging of droplets is technically complex and requires expensive instrumentation limiting their large-scale adoption. Here we have adapted the principles of the widely established technique of flow cytometry to a novel optofluidic setup – the OptiDrop platform, with on-chip detection of scatter and multiple fluorescence signals from microfluidic droplets and their contents. Furthermore, the detected signals are analyzed real-time for triggering on-demand droplet sorting. The highly customizable on-chip optical fiber-based droplet signal detection and sorting system enables simplified, miniaturized, low-cost, multi-parametric sensing of optical signals with high sensitivity and single cell resolution within each droplet.

Author: Evan Davis

Abstract:

Unmixing Algorithm in CytoFLEX mosaic Spectral Detection Module

Our approach includes two algorithms: LSM and our Poisson hybrid. LSM is well-known in the industry. The Poisson hybrid is useful for larger panels with multiple fluorochromes, overlapping dyes, or varied sample types, even when antibodies are not fully titrated. It may reduce spreading errors in these scenarios.

Author: Evan Davis

Abstract:

Extracellular vesicles (EVs) are a heterogeneous family of membranous particles released by all cells, providing crucial insights into various physiological processes and diseases. To better understand EVs, researchers must characterize them.

However, analyzing these EVs is challenging due to their heterogeneity, size, refractive index, and complexity. For instance, EVs can range in size from 20 nm up to 5 μm for the larger apoptotic bodies. The CytoFLEX nano flow cytometer is for research use only (RUO). It has high sensitivity, capable of detecting nanoparticles via violet side scatter as small as 40 nm* and provides better sensitivity and resolution for the six fluorescent detection channels. Here, we present the immunophenotypic characterization using a 5-color panel to identify different types of EVs in platelet-poor plasma (PPP) samples.

Author: Rita Rozmarynowycz

Abstract:

Stable cell line development is a critical process for developing and manufacturing biologics, generating disease-relevant cellular models, and creating the next - generation of cell & gene therapies.

• The utility of new cell models (patient derived iPSCs), the development of new genome editing technologies (CRISPR) and the deployment of automation and AI for high - throughput screening are creating a greater demand for scaling cell line development capabilities and workflows.

• Bio - Techne has developed the Pala Cell Sorter and Single Cell Dispenser that incorporates microfluidics, flow cytometry, and liquid dispensing in a portable, easy to use, and gentle device for generating high rates of single cell derived clones.

• The Pala makes it simple to generate hundreds of clones needed for downstream screening for optimizing the development of stable cell lines.

Author: Nori Ueno

Abstract:

Manual antibody cocktail preparation for high-dimensional flow cytometry introduces variability and consumes valuable hands-on time, limiting reproducibility across experiments and users. In this study, we evaluated an automated workflow for antibody mastermix preparation using Curiox C-Free™ Pluto workstation, a programmable benchtop liquid handler, benchmarking its performance against traditional manual methods across 10- and 24-color panels. Whole blood samples from five to six healthy donors were stained using cocktails prepared either manually or by the automated system, and processed via both centrifuge-based and centrifuge-less sample preparation protocols.

Across all tested conditions, staining quality as measured by Stain Index (SI) was comparable or superior with automated prep. Key markers across lymphocyte and myeloid subsets showed tight clustering of fluorescence intensity, and inter-donor consistency was preserved. Importantly, coefficient of variation in replicate cocktail preps was consistently below 5%, and SI variation across donors remained within acceptable limits for immunophenotyping studies. The integration of fixed-volume scripting and real-time liquid handling validation contributed to the robustness of the process. In addition, the automated prep significantly reduced overall turnaround time—from over 24 minutes manually to approximately 16 minutes hands-off workflow —while eliminating user variability and pipetting fatigue.

The workflow also facilitated the preparation of FMOs and single-color controls with no additional pipetting steps, and enabled reproducible scaling of panels using pre-validated CSV templates. Combined with centrifuge-free processing, this approach supports fully automated immunostaining pipelines with minimal user intervention. Our findings demonstrate that automated antibody mastermix preparation is not only feasible but advantageous for high-throughput, high-dimensional cytometry workflows where consistency, traceability, and speed are critical.

Author: Jack Panopoulos

Abstract: Automated computational methods for analyzing flow cytometry data have been around for over a decade. The number and functionality of these tools continues to grow annually with many algorithms addressing a variety of individual points along the analysis workflow. A chief category among these tools are the myriad clustering algorithms used for identifying populations within a given data set. Clustering algorithms provide two powerful advantages over manual gating: speed and the ability to identify additional complex phenotypes from multi-dimensional data. Despite these advantages, wide variety, and free availability of these algorithms, their use as a standard tool for routine analysis of high dimensional flow cytometry data is limited and disparate. 

Widespread adoption of clustering algorithms for day-to-day use is hampered by the lack of granular knowledge about algorithmic settings, difficulties associated with reproducibility, and an inability to easily understand the phenotypes of the output populations. Fortunately, several new algorithms have addressed the first two problems with auto-adjustment of settings, and implementation of a non-random seed to ensure consistent reproducibility for the same sets of files. However, phenotypic naming, that is, the strict identification of what numbered populations actually are, remains a major obstacle for implementation of these tools for routine analysis. 

Recently, auto-phenotyping tools have been developed to help name clustering-derived populations using human readable symbols. In general, the user inputs a series of known phenotypic names and then a set of expression thresholds for the defining markers, to help the algorithm categorize the cluster names. Known populations are given the phenotype’s name, unknown populations or undefined populations whose marker expression is under or over the threshold values typically get assigned an integer and a “-“ or a “+”. Though these methods make a substantial and necessary attempt to solve the human-machine language barrier, they are subjective, the naming schematic must be reinitiated or adjusted with each new panel, and the phenotypic distinction between populations that lie outside threshold bounds is difficult to understand. Worse, these algorithms are abstracted from the clustering tools themselves, adding another layer set of inputs and preventing a more streamlined experience as occurs with manual gating. 

Herein we describe the ability to map existing manual gates and their associated population names to influence the naming of algorithmically-derived outputs. We have developed a software component that maps existing manual gating hierarchies onto the clustering results. The clustering-derived populations get filtered and hierarchical cutoffs determined by the human expert, maintaining phenotypic consistency across all analyses. Importantly, the structured naming schematic that results from this implementation can help with the SOULCAP initiative, where well-defined systematic naming of known (and unknown) populations is core component of their mission. Instead of “population 21” stagnating as the bane of our existence, the population’s identity becomes something familiar to the end user interpreting the data: “CD8+ T cells IFN+ cluster 21”.

Author: Mengsha Gong

Abstract:

Rabbit IgG Fc amino acid sequences are highly similar to human IgG1 Fc, especially at the Fcγ receptor (FcγR) binding sites, and Biolayer Interferometry (BLI) kinetic analysis of rabbit IgG and human IgG1 showed equivalent interactions with human FcγRs. Recombinant Rabbit IgG antibody completely devoid of binding to human Fcγ can be used to reduce false positives caused by human Fcγ receptors in bioassays. We analyzed the rabbit Fc–human FcγR interface by antibody modeling and identified some novel mutations that disrupt the interface of IgGs and FcγRs. Rabbit IgG Fc domains can be engineered to contain a limited number of mutations such that human FcγR interactions are completely abolished while human FcRn interactions and rabbit Fc stability are unaffected.  Here, we describe a mutated rabbit Fc region designed to significantly reduce potential non-specific antibody staining caused by IgG receptors in various applications, including flow cytometric analysis of human cells. 

Author: Alexander Elias Zambidis

Abstract:

Accurate spectral unmixing is a critical step for flow cytometry data analysis and requires a single stain control for every fluorescent parameter used in an experiment. Generating single stain controls with cells can often be difficult when labeling a target protein that is of low abundance or a cell type of low frequency. For this reason, compensation particles are often used instead of cells for making single stain controls due to their uniformity and brightness of a positive population. However, it has been observed that when specific fluorochromes are bound to compensation particles, they produce an emission spectrum that differs from when those same fluorochromes are bound to cells. These incongruencies between emission spectra result in unmixing errors within the resulting flow cytometry data. Due to the rapid increase in number of detectors on current spectral flow cytometers and of resolvable fluorescent parameters within a single panel, it is no longer advisable for a flow cytometer practitioner to manually correct for inaccuracies in a normalized spectral matrix in panels of high complexity. As a result, generating accurate single stain controls is a significant step to prevent unmixing error.

In the current study, we reasoned that a bispecific reagent that binds to an abundant cell surface protein and simultaneously captures a fluorochrome-conjugated antibody would overcome the limitations that are inherent to cell-based single stain controls. Using previously designed antibodies and nanobodies, we engineered a novel set of bispecific reagents by linking a human anti-CD45 and mouse anti-IgG variable region. We refer to this new bispecific tool as CaptureBody (CB) and highlight the benefits of its final nanobody-based design. We demonstrate that CaptureBodies stain cells with similar efficiency as primary staining with a commercially available human anti-CD45 antibody. Additionally, we show that CaptureBodies enhance the use of cells for generating single stain controls by creating a positive population that is abundant, uniform, and bright. 

Author: Kristine Valenteros

Abstract:

Cell Mimics as Reliable Controls for Consistent, Reproducible Flow Cytometry Assays in Cell Therapy

Swetha Pratyusha Gunturu, Subhanip Biswas,Kanwal Palla, Louisa D’Lima, Armando Martinez

Slingshot Biosciences, Inc.

Background:

Flow cytometry is essential for immune profiling, but lack of standardized controls limits reproducibility and assay reliability. Synthetic cell mimics expressing defined biomarkers provide a consistent, flexible alternative to biological controls, enabling integration into diverse assays, including lymphocyte subset, T-cell activation, exhaustion, CAR-T detection, and post-infusion biomarker analysis. Their stability, reproducibility, and multicolor panel compatibility improve assay calibration, gating validation, and immune monitoring.

Methods:

Two lots of lyophilized and cryopreserved PBMC controls were evaluated for lot-to-lot consistency against TruCytes™ Lymphocyte Subset Cell Mimic Controls using a Cytek® Aurora CS. To assess stability, one lot of TruCytes controls underwent an accelerated stability study, while two additional lots were monitored in an ongoing real-time study. TruCytes performance was also tested with commercially available assay kits to confirm compatibility. Accuracy was evaluated by spiking TruCytes™ Lymphocyte Subsets into cryopreserved PBMCs and comparing the measured frequencies with the expected target values.

Results:

TruCytes™ cell mimics demonstrated superior lot-to-lot reproducibility and stability compared with PBMC controls, with CVs of 0.1%–5.7% versus 1.6%–36.6%. Closed-vial stability extended up to 36 months, supporting long-term assay reliability. The cell mimics were also fully compatible with commercially available assay kits, demonstrating robust performance across different panel configurations. Accuracy testing confirmed minimal bias (≤2.0%) across evaluated subsets, indicating reliable recovery of expected population proportions under spike-in conditions.

Conclusion:

TruCytes™ Lymphocyte Subset Cell Mimic Controls offer a reliable, reproducible, and long-term solution for flow cytometry standardization. Their robust performance and compatibility with commercial antibody panels reduce reliance on variable PBMC controls, supporting consistent assay development, quality control, and scalable laboratory workflows.

Author: Yongping Zhong

Abstract: 

Monoclonal antibodies (mAbs) are critical reagents for flow cytometry, enabling quantitative single-cell detection of surface and intracellular targets. However, antibodies generated without considering native antigen conformation and cellular context may fail during flow validation. The OHSU Flow Cytometry and Monoclonal Antibody Shared Resource (FCMAbSR) provides an integrated platform linking antigen selection, immunization strategy, hybridoma production, and flow cytometry–based screening to accelerate development of novel antibodies optimized for flow applications. Antigen strategies include whole-cell immunization to preserve native epitopes, recombinant proteins for controlled specificity, and peptides for epitope-focused targeting. Subtractive immunization approaches are used to reduce background responses and enrich for antibodies recognizing unique or differentially expressed antigens. Hybridoma candidates are screened by immunohistochemistry (IHC), ELISA and multiparameter flow cytometry to rapidly prioritize clones with strong specificity and robust staining performance in complex samples. This workflow streamlines discovery and validation of functional antibodies for research and translational flow cytometry assays.

Author: Andrew Konecny

Abstract:

At home blood collection devices can facilitate study participation of patients who have difficulty attending routine clinic visits such as remote and rural cohorts. Numerous studies have established that self-administered (at home) blood collection devices (S-ABCD) are suitable to interrogate serum or lysed samples by proteomics and sequencing approaches. It is unclear whether live, single cell analyses such as flow cytometry are also feasible. Here we assess the if the Tasso+ device, a lancet-enabled S-ABCD, can be used for the analysis of immune cells with high parameter flow cytometry (n = 16). We found that time-matched blood collected via S-ABCD and venipuncture were highly congruent in regards to immune cell composition and phenotype. Shipment of samples via United States Postal Service resulted in some alterations within the myeloid compartment, but strikingly we found that T cells numbers, subsets, and phenotypes remained remarkably stable compared to venipuncture and non-shipped samples. The shipped S-ABCD samples had a median viability of 82% (IQR: 79% – 85%; n = 16) with a shipping transit range of 1 to 5 days and ambient temperature range of 50 – 77 F. These data highlight the feasibility of using S-ABCDs to remotely interrogate immune cell subsets and phenotypes. Additionally, our study also provides insight to guide study design around collection variables. 

Authors: Alex Heubeck, Vaishnavi Parthasarathy*, Veronica Hernandez, Tyanna Stuckey, Blessing Musgrove, Julian Reading

Abstract:

Cryopreserved peripheral blood mononuclear cells (PBMCs) are widely used for immune profiling in clinical trials; however, standard PBMC isolation and storage practices pose logistical challenges, particularly in resource-limited settings. These limitations hinder timely sample processing and longitudinal immune monitoring. To address these constraints, we implemented a whole blood fixation protocol that rapidly stabilizes surface protein epitopes, enabling high-dimensional spectral flow cytometry without immediate processing. Two 35-marker panels were designed and optimized to characterize major immune populations, including T cell subsets, B cells, NK cells, monocytes, dendritic cells, and granulocytes. Panel development focused on antibody clone compatibility with fixed antigens, minimizing non-specific binding, and ensuring reliable detection of chemokine receptors. Performance was evaluated by comparing fresh and fixed whole blood samples and assessing long-term stability in cryopreserved fixed blood.

Comparative analysis demonstrated robust performance of fixed samples, with coefficients of variation (CV) of 2% for neutrophils, 6.5% for myeloid cells, 8.7% for NK cells, 6% for T cells, and 9% for B cells. Long-term stability was confirmed by using cryopreserved fixed blood from a healthy donor over 18 months, maintaining consistent white blood cell frequencies (CV = 1%).

These findings support fixed whole blood as a scalable and practical approach for deep immunophenotyping and longitudinal immune monitoring in clinical trials, particularly in resource-limited environments.

Author: Morgan Boyd

Abstract: 

Abatacept is a CTLA-4 Ig fusion molecule that blocks CD28-mediated T cell activation. Trials of abatacept in individuals with type 1 diabetes (T1D) have yielded mixed clinical outcomes and inconsistent correlations between immune parameters and clinical response. Thus, the DREAM-T mechanistic study was performed in individuals recently diagnosed with T1D who received weekly subcutaneous abatacept therapy for 12 weeks with intensive immune profiling and a 6-month quantitative response (QR) measure integrating age and C-peptide to compare observed versus expected (with and without abatacept) disease progression. DREAM-T participants displayed a mean positive QR (p < 0.05), indicating 12 weeks of abatacept therapy significantly delayed T1D progression. Longitudinal PBMC samples were characterized by flow and mass cytometry and bulk RNA sequencing. While flow analyses were consistent with previous trial findings and showed reduced total T regulatory cell (Treg) ICOS+ expression as early as two weeks into treatment, we did not find transcriptional-level changes to Treg ICOS expression in our RNA sequencing data. To address these differences, we designed a CyTOF panel to differentially quantify surface and intracellular expression of co-stimulatory markers. We found a reduction in intracellular ICOS and CTLA-4 two weeks into treatment in CD4+ Treg and T conventional cells, maintained throughout therapy. Interestingly, while we see a decline in surface ICOS expression, we do not see significant changes to surface CTLA-4 expression. The observed immune modulations return to baseline values one-year post-treatment, suggesting a direct effect of abatacept on co-stimulation pathways that are not sustained following treatment. The intracellular- versus surface-level differential findings expand upon our current understanding of the mechanism of action of abatacept, which may predominantly affect CTLA-4 recycling over surface-level expression.

Author: Kai Herko

Abstract:

Approximately 90% of Type 1 Diabetes (T1D) patients have circulating T1D-associated autoantibodies, which are often present years before disease onset. Therefore, identifying and characterizing autoantibody responses can be key in predicting T1D development and progression. Autoantibodies against human Tetraspanin 7 (TSPAN7) have been detected in individuals with T1D and at-risk individuals but were significantly associated with IA-2 autoantibodies and had a lower prevalence. Furthermore, the precise protein target of these autoantibodies (TSPAN7, formerly called GLIMA38) was only recently identified. Consequently, the study of TSPAN7 as a beta cell autoantigen has been minimal. Autoreactive CD4 T cells contribute to the progression of T1D by infiltrating pancreatic tissue, where they secrete inflammatory cytokines, leading to reduced function and facilitate destruction of insulin-secreting beta cells.  Because T cell and autoantibody targets are known to overlap, we investigated TSPAN7 as a CD4+ T cell antigen in T1D.   

Prior studies suggest that TSPAN7 autoantibodies are associated with the HLA-DR4/DQ8 haplotype. Despite that association, T cell epitopes within TSPAN7 have not been identified. We designed and synthesized a set of 30 overlapping (20mer with 12mer overlap) peptides representing the full-length native TSPAN7 protein sequence plus 24 additional peptides with likely post-translational modifications (PTM). These were divided into 6 pools of 9 peptides each, and used to stimulate and expand TSPAN7-specific T cells from the peripheral blood of DQ8-positive T1D donors. Following 21 days of in vitro culture expansion, we performed sequential screens for Activation Induced Marker (AIM) positive responses, using CD154 as the primary activation marker. For wells with positive responses, CD154-positive T cells were sorted at 4 cells per well and expanded, yielding TSPAN7-specific CD4 T cell lines. The HLA restriction of each line was determined through T cell proliferation assays using immortalized B cell lines from an individual with bare lymphocyte syndrome transfected to express single HLA molecules. Through this approach, we identified five TSPAN7 epitopes, encompassing both native and PTM peptides. TSPAN7-specific CD4 T cell lines were restricted by DQ8, DRB1*03:01, and DRB1*04:01. The majority (71%) of acquired lines were DRB1*04:01 restricted, corresponding to the previously reported association between TSPAN7 autoantibodies and DQ8/DR4. 

Establishing the identity of the HLA/peptide pairs capable of eliciting CD4 T cell activation and proliferation and acquiring T Cell lines corresponding to those specificities provides important tools for dissecting TPAN7-specific responses in individuals with T1D.  Our ongoing work seeks to validate the disease relevance of TSPAN7-specific T cell responses and to assess their association with TSPAN7 autoantibodies. Antigen specific T1D therapies will likely rely on epitope identification and mapping of disease-specific autoantigens, such as TSPAN7. Given the presence of TSPAN7 autoantibodies in antibody positive individuals who have not yet developed T1D, understanding TSPAN7 recognition could provide insights about the loss of self-tolerance. Monitoring TSPAN7 recognition could improve the accuracy of clinical T1D diagnostics and contribute to development of antigen-specific T1D immunotherapies.

Author: Nicole Potchen

Abstract:

T cells play a key role in driving as well as resolving tissue inflammation. Whether the compositional and functional properties of the T cell compartment change as tissues progress from a mildly to severely inflamed state is still poorly understood. Inflammation could be at least in part driven by the gain of T cell effector function, the loss of suppression and tissue healing functions, or by other mechanisms. Importantly, these changes could occur concurrently and across different T cell subsets highlighting the need to holistically interrogate the T cell compartment. To facilitate such comprehensive analyses, we have developed a 37-color spectral flow cytometry panel. We use this to interrogate phenotype (ex vivo) and function (upon stimulation) from surgically discarded oral mucosa tissue from routine periodontic procedures, ranging from mildly inflamed (gingivitis), to severely inflamed (periodontitis). We found that T cell subset frequencies and functional abilities remain largely consistent between mucosal tissues of various states of inflammation. This includes regulatory CD4 T cells, indicating that pathology is not driven by a lack of suppression. Notably, markers of tissue healing ability are maintained during severe inflammation. Overall, our data suggest that human mucosal T cells preserve the functional capacity to mediate tissue repair even in severely inflamed tissues, indicating that repair functions are intact and could be elicited by therapeutic targeting. 

Author: Nevada Reinhardt-Slaughter

Abstract:

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterized by deficits in social, language, and locomotor functions. Inflammatory pathways in the central nervous system (CNS) are implicated in the pathology of ASD; however, the mechanisms by which pharmacological treatments modulate these pathways remain poorly characterized. Previous research by our group has shown that treatment with cannabidiol (CBD), a non-intoxicating phytocannabinoid, improves sociability and reduces anxiety in the BTBR mouse model of ASD. While neuroinflammatory pathways are a strong candidate mechanism through which CBD may improve ASD-related symptoms, the effects of CBD administration on neuroinflammatory phenotypes in BTBR mice have not yet been explored.

We therefore sought to test how daily oral acute CBD (25 mg/kg/day; 48-hour) or chronic administration (14-day) influences the infiltration and activation of immune cells within the CNS of BTBR mice. Flow cytometric analysis revealed several changes in both innate and adaptive immune cell populations infiltrating the CNS. Within the myeloid compartment, CBD administration resulted in a decrease in CNS-infiltrating neutrophils and monocytes. Increased expression of MERTK, an immunoregulatory receptor, on infiltrating monocytes and macrophages was also observed. Within the lymphoid compartment, CBD-treated mice exhibited increased expression of the immunosuppressive markers PD-1 and TIGIT on CNS-infiltrating regulatory T cells (Treg). The overall number of infiltrating Treg remained the same between treatment groups. Collectively, these results show that CBD treatment in the BTBR model of ASD is associated with decreased neuroinflammation across both innate and adaptive immune cell subsets.

Our next steps will involve RNA sequencing to characterize transcriptional profiles of CNS-infiltrating granulocytes and Treg, along with antibody depletion of individual immune cell subsets to determine their contributions to behavioral symptoms of ASD. This work establishes a novel correlation between CBD administration and decreased neuroinflammation, providing a potential underlying mechanism for the observed therapeutic effects of CBD on behavioral symptoms of ASD in BTBR mice.

Author: Sivasankaran M Ponnan

Abstract:

CD19 CAR T cells have shown significant efficacy against autoreactive B cells and have yielded promising results in systemic lupus erythematosus (SLE). To investigate mechanisms of tolerance re‑established after B-cell depletion, we developed and optimized a 46‑colour spectral flow cytometry panel enabling comprehensive analysis of the regenerated immune system, with a focus on T and B cells in peripheral blood. This approach maximizes detection of rare autoreactive B and T cells in SLE while evaluating restoration of tolerance in patients enrolled in the phase 1 REACT‑01 trial, an open-label, non-randomized study of CD19 CAR T cells in pediatric and young adult patients with treatment‑refractory childhood‑onset SLE (cSLE).

The spectral panel was designed with minimally overlapping fluorochromes to reduce spectral spread and enhance resolution, thereby supporting simultaneous assessment of numerous markers and detailed immune profiling. The panel includes lineage markers for B- and T-cell subsets and an extensive set of phenotyping markers targeting activation and differentiation states of both lymphocyte compartments. We will perform comprehensive immunophenotyping of PBMC samples from trial participants at baseline, post–CAR T-cell treatment, and long-term follow-up. Although clinical benefit of CD19 CAR T cells in SLE was anticipated, durable remission despite B-cell reconstitution was unexpected. We hypothesize that, in addition to restoring B-cell tolerance, B-cell depletion alters phenotypes of T-cell subsets implicated in lupus pathogenesis. Our 46‑colour spectral flow panel will dissect immune responses to anti‑CD19 CAR T treatment and identify phenotypic differences within B- and T-cell compartments associated with treatment response.

Author: Hang Yin

Abstract:

B cells are essential components of the adaptive immune response, capable of uniquely ‘recognizing’ foreign antigens through their B cell receptor (BCR). The combination of BCR-antigen binding and other activating signals, such as CD40-CD40L binding, mediated by T cell help, can induce memory and antibody-secreting cell (ASC) differentiation. The early changes in human B cell surface molecule expression that occur upon BCR- and CD40-mediated activation are not fully understood. Identifying unique surface markers of activation may open new avenues for the isolation and study of antigen-reactive B cell populations.    

​To comprehensively assess surface expression changes after ex vivo BCR and CD40 activation, we screened the expression of 354 molecules on activated B cells from three healthy peripheral blood donors by spectral flow cytometry. In this screen, we employed a unique ex vivo stimulation approach that directly activated IgK-isotype B cells, thus enabling assessment of bystander effects on IgL-isotype B cells in the same cultures. We identified 6 surface molecules that were consistently upregulated on BCR- and CD40-stimulated cells and showed minimal changes among bystander cells. To reduce background cell activation due to culture media components in follow-up assays, we assessed B cell viability and activation levels across several culture media conditions and identified a chemically defined serum free-based culture condition that maintained high cell viability while minimizing cellular activation. Finally, we defined the expression dynamics of the 6 activation-induced surface molecules through spectral flow cytometry analysis of B cells at rest (unstimulated), after BCR/CD40 activation, and BCR activation alone for 3, 20 and 72 hours. Collectively, our findings revealed surface marker expression changes induced by BCR and CD40 engagement among both antigen-inexperienced and -experienced human B cells.  Further, our results suggest potentially novel approaches for the enrichment of antigen-reactive B cell that can be applied to studies in the context of autoimmunity and cancer.  

Author: Quinn Peters

Abstract:

The steroid sex hormone progesterone is thought to be broadly immunosuppressive. Tumor studies have shown that increases in progesterone are associated with greater immune dysfunction. Previous studies have relied on synthetic progestins used in contraception, which signal through additional steroid receptors and do not confer the same immunological profile as endogenous progesterone (P4). To better define effects of P4 on tissue and circulating immunity, we studied T cell responses across menstruation at the P4 low follicular phase and the P4 dominant luteal phase in a cohort of 20 healthy participants. Of note, systemic P4 levels are typically more than 40 times higher in the luteal than the follicular phase, which we confirmed in our participants by quantifying P4 in serum at both cycle phases. At each study visit, blood was drawn and skin, vaginal tract, and rectal tissues were biopsied. We first tested the feasibility of high parameter spectral flow cytometry to assess both ex vivo phenotype and functional capacity (in response to ex vivo stimulation) in donor-matched tissues. We assessed tissue resident CD8+ T cells, non-resident CD8+ T cells, conventional CD4+ T cells, regulatory CD4+ T cells, and TCRgd+ T cells for pro-inflammatory Th1, Th2, and Th17 cytokines, granzyme B, and tissue repair factors. We found that the biopsies yielded a sufficient number of T cells for these assays, which allowed us to detect inter-tissue differences across T cell subsets. Our unique sample set will enable us to characterize changes in T cell responses between P4 low and high timepoints across multiple tissues in a donor-matched manner. Ongoing ex vivo experiments will help assess direct and indirect effects of P4 on T cell responses, and control for other variables such as changes in estradiol.

Author: Valerie Woodings

Abstract:

Natural killer (NK) cells are a subset of lymphocytes that are essential in innate immune response against pathogens and tumor cells. They are defined as CD3- lymphocytes with expression of CD16 and/or CD56, and are broadly categorized in two main subsets: naive cytokine-producing CD56brightCD16dim and differentiated cytotoxic CD56dimCD16bright. Flow cytometric diagnosis of NK cell neoplasms if often challenging due to difficulties in assessment of clonality. Monotypic expression of various NK cell receptors (such as Killer Immunoglobulin-like Receptors (KIRs or CD158), NKG2A and CD94) have been used as a surrogate for defining clonality. We developed a flow cytometry assay that includes these NK cell receptors and tested its utility in differentiating reactive and neoplastic/clonal NK cell populations. Antibodies against the following markers were included in the panel: NKG2A, CD8, CD57, CD56, CD7, CD158a, CD158e, CD94, CD3, CD158b, CD16, and CD45. Under normal conditions, naive NK cells uniformly express CD94 and NKG2A with no expression of KIRs, while differentiated NK cells show polytypic expression of KIRs with variable expression of CD94 and NKG2A. In this study, we analyzed 46 normal (peripheral blood, bone marrow, fluid, and lymph node) samples and 52 abnormal samples. The distribution of these NK cell subsets varies based on sample type with CD56brightCD16dim NK cells being dominant in lymph node and fluid samples, while CD56dimCD16bright NK cells are dominant in peripheral blood samples. Reference ranges for the expression of various NK cell markers were established. Abnormal cases included EBV+ NK/T cell lymphoma, NK-cell large granular lymphocytic leukemia (NK-LGLL), and NK-cell clone of undetermined significance (NK-CUS). Importantly, EBV+ NK/T cell lymphoma cases were uniformly positive for CD94 and NKG2A and negative for KIRs. In contrast, cases of NK-LGLL and NK-CUS showed variable expression of CD94 and NKG2A and included cases with KIR restriction. Our data indicates that NK cell receptor immunophenotyping is valuable in differentiating reactive and clonal/neoplastic NK cell populations and will be helpful in the diagnosis and follow up of NK cell neoplasms in our patient population.