Immuno-oncology (IO) focuses on exploiting the immune system to elicit appropriate anti-tumor responses and to block the progression of cancer. The immune system is naturally equipped with defense mechanisms to prevent invasion by pathogens. Immunological checkpoints, such as programmed cell death protein-1 (PD-1) and CTLA-4, are an integral part of the repertoire of checks and balances available in the body for preventing attacks on its own immune cells. Such an ability to discriminate between self and non-self is critical for preventing uncontrolled immune responses. Cancer cells and the tumor microenvironment, however, can adopt these immunological checkpoints to pose as “self” molecules and evade the natural defense of the body’s own immune system. Tumor cells can express the PD-1 ligand (PD-L1) on their surface to bind with PD-1 and activate this brake or off-switch of the immune system. Immuno-oncology approaches involve breaking this manipulation of immune checkpoint proteins by using inhibitors (e.g., anti-PD-1/PD-L1 agents) to block PD-1/PD-L1 interaction and allow the recognition of tumor cells by the immune system.¹ Combining immunotherapy with other standard of care options, such as surgery, radiotherapy and chemotherapy, has also been adopted recently.

Sample collection

The BD Vacutainer^® family of products can be used for blood cell and biomarker preservation.

Sample preparation tools

The BD Horizon™ Dri Tumor and Tissue Dissociation Reagent (TTDR) offers gentle and effective dissociation with superior epitope preservation. TTDR maximizes cell yields, while minimizing cell death, which allows effective dissociation of a variety of tumor types to enable single-cell studies.

Tumor types evaluated by BD or external investigators include lung, breast, colon, lymphoma, melanoma/skin, pancreatic, esophageal, kidney, sarcoma and brain.

Cell Sorters

The BD FACSMelody™ Cell Sorter provides, simple, fast and quality cell sorting ideal for enrichment of rare populations.

Fluorescence activated cell sorting of hematopoietic stem and progenitor cells for downstream single-cell multiomics analysis to study tumor-driven perturbations in hematopoiesis.

Isolation of hematopoietic stem and progenitor cells from healthy and tumor-burdened mice for downstream single-cell multiomics analysis.

Right: C57BL/6 mice were injected subcutaneously with either B16-F10 melanoma cells (tumor-burdened mice, n = 4) or control media (healthy mice, n = 4). Bone marrow and spleen tissues were harvested after 21 days. Bone marrow and spleen cells from healthy or tumor-burdened mice were mechanically separated and enriched using the BD IMag™ Mouse Hematopoietic Progenitor (Stem) Cell Enrichment Set.

Cell analyzers

Multicolor flow cytometry with the BD FACSCelesta™ Flow Cytometer enables comprehensive immunophenotypic analysis of exhausted T cells.

Right: Co-expression patterns of inhibitory receptors in unstimulated and in vitro stimulated CD8+ and CD4+ T cells. The use of bivariate plots enabled the identification of complex co-expression patterns of inhibitory receptors and highlights the heterogeneous phenotype of in vitro persistently stimulated T cells and immunophenotypic analysis of exhausted T cells.

Plot analysis was performed to identify subsets of total CD8+ T and CD4+ cells co-expressing inhibitory receptors within fresh, unstimulated PBMCs and T cells persistently stimulated in vitro with Dynabeads™ Human Activator CD3/CD28 and human recombinant IL-2 for 9 days.

A. Bivariate plot analysis provided information on the heterogeneity of CD8+ T cells co-expressing inhibitory receptors. For example, distinct subsets of cells expressing only TIGIT, only PD-1 or co-expressing both inhibitory receptors were detected. B. More complex patterns of expression were observed upon persistent stimulation in vitro that resulted in differential regulation of the inhibitory receptors tested. For example, while the overall percentage of CD8+TIGIT+ cells decreased, an increase in cells co-expressing PD-1 and TIGIT was observed. Interestingly, only a small, discrete subset of CD8+ cells upregulated CTLA-4 expression, thus confirming the heterogeneity of cells persistently stimulated. (C–D) Similar observations were made for CD4+ T cell subsets.

The BD FACSLyric™ Flow Cytometry System helps in driving reproducibility and standardization. The BD FACSLyric™ Flow Cytometry System solution combines simplicity, speed and automation to ease workflow and improve productivity. This next-generation flow cytometer enables standardization and collaboration through consistent results and assay portability capabilities.

Multiomics

BD offers a wide range of multiomic solutions to help empower and streamline your research. The BD Rhapsody™ Single-Cell Analysis System with BD^® AbSeq Oligonucleotides provides a comprehensive single-cell workflow solution to increase the experimental power for your research. BD^® AbSeq Oligonucleotides enables synergies with high-parameter flow cytometry, allowing discoveries with BD^® AbSeq Olionucleotides to transfer to panel design and WTA discoveries transfer to targeted panels.

BD^® AbSeq Oligonucleotides characterizing CAR CD19 T cells (right). Participants from a trial at Westmead Hospital (Sydney, Australia) using CAR T cells based on 4-1BB coreceptor (instead of CD28) and the piggyBAC system for gene modification. Development of a single-cell multiomics approach was used to study the molecular, functional and transcriptomic profile of CAR T cells in the pre-infusion product and the blood of patients following adoptive transfer.

This was to test the hypothesis that survival of CAR T cells post-infusion is driven by long-term stem memory T cells (TSCMs). TSCMs are identified as a minor subset within the CAR T cell product and these are found expanded in the blood of patients up to 100 days post-CAR T cell post-infusion (data not shown).

The BD Rhapsody™ Single-Cell Analysis System enables high-throughput single cell multiomic analyses and includes a microwell-based instrument platform equipped with quality monitoring features; an array of RNA and protein assays such as the whole transcriptome assay, targeted RNA panels and TCR/BCR profiling assays; BD^® AbSeq Antibody-Oligo’s; and software tools. The BD Rhapsody™ System works seamlessly with other upstream BD cell enrichment technologies such as fluorescence-activated cell sorting enabling users to deep dive into their select cells of interest. The BD Rhapsody™ System can be used effectively in several single-cell multiomics applications, including immuno-oncology research.