Dr. David Draper Scientist, Scientific Development

A DEEPER DIVE INTO THE TUMOR-INFILTRATING T CELL IMMUNOPHENOTYPE

Introducing the New MI Expanded CompT™ Multi-Color Flow Cytometry Panel

Flow cytometry using large antibody panels has the advantage of examining a greater number of cell subsets. This capability is particularly important when a comprehensive data set is required, but limited tumor material is available for analysis. Moreover, panels with a large number of antibodies can also be used to enable deep immunophenotypic insight into a few subsets, or an even deeper analysis into a single subset. In this Tech Spotlight, we demonstrate the latter capability by presenting data generated using the new MI-Expanded CompT™ panel, a 16-color panel that takes flow analysis of T cell activation and differentiation to a level above any other panel in the Covance service portfolio.

The MI-Expanded CompT™ panel builds upon the MI-CompT™ panel, our most popular standard panel to examine CD4+ and CD8+ T cells. This improved panel adds effector and memory T cell markers, plus four additional markers for analysis of T cell activation and exhaustion. Table 1 describes the components of the Expanded MI-CompT™ panel, and using untreated murine MC38 colon adenocarcinomas, Figure 1 illustrates its gating and analysis strategy.

Table 1. MI-Expanded CompT™ panel antibodies and description of their utility

Antibody/Dye Description

CD45 Pan immune cell marker

CD3 Pan-T cell marker

CD4 CD4+ T cell marker

CD8 CD8+ T cell marker

FoxP3 Regulatory T cell marker

CD25 Regulatory T cell marker/IL-2 receptor

CD44 Activation/Memory marker

CD62L Naïve T cell/Memory marker

Ki-67 Proliferation marker

CD69 T cell activation marker

PD-1 T cell activation/Exhaustion marker

LAG-3 T cell activation/Exhaustion marker

ICOS T cell activation marker

Granzyme B Anti-tumor cytotoxicity marker

Viability Dye Dead cell exclusion

The MI-Expanded CompT™ can be customized to include NK/NKT cell markers (CD49b/CD335) to enable granzyme B and activation marker expression in these subsets.

MI-Expanded CompT™ Panel Gating Strategy

As with all Covance T cell panels, analysis begins with dead cell exclusion and subsequent CD45+ immune cell delineation to gate on CD3+ T cells (not shown). Fig. 1A displays the downstream endpoints of CD4+ and CD8+ T cell analysis that are shared between the MI-CompT™ and MI-Expanded CompT™ panels. These include CD69 and PD-1, which become upregulated upon T cell activation. Their expression has been correlated with the exhausted T cell phenotype.1 Another shared endpoint is CD8+ T cell proliferation, provided by using Ki-67 expression as a surrogate marker. Finally, CD4+ T cells are examined to quantify helper T cells and regulatory T cells (Tregs). Fig. 1B and 1C illustrate the added endpoints used to expand upon the MI-CompT™ panel, which are further described below.

Figure 1. Analysis of T cells using the MI-Expanded CompT™ panel. Naïve MC38 tumors were harvested from C57BL/6 mice. (A) CD4+ helper, CD8+ T cell, and Treg quantitation, including measurements of proliferation (Ki-67 analysis) and CD69/PD-1 marker expression. (B) Effector/memory CD8+ T cell analysis to quantify naïve, Teff, Tem and Tcm subsets. (C) Expanded analysis of T cell activation/exhaustion markers including granzyme B. Red peaks represent target-stained cells. Blue peaks represent the unstained negative controls.

Effector/Memory T Cell Analysis

Analysis of effector and memory CD8+ T cell differentiation is shown in Fig. 1B. Conversion of T cells to a memory phenotype is important for the development of lasting immunological response to rechallenge in the context of both infection and cancer pathogenesis. CD44 and CD62L analysis enables the delineation of T cells into four differentiation states. These include naïve or inactivated T cells, activated effector T cells (Teff), effector memory (Tem) and central memory (Tcm) subsets. Tem and Tcm subsets can circulate but have tendencies to reside in non-lymphoid and lymphoid tissues, respectively.2 Recent reports have demonstrated that both of these subsets have distinct roles in the anti-tumor response. A third resident memory (Trm) population has more recently been described as playing an important role in controlling tumor growth in a variety of models and can be delineated using CD103, among other markers.3 The MI-Expanded CompT™ panel can be customized to include analysis of Trm cells.

T Cell Activation, Exhaustion and Granzyme B Analysis

The MI-Expanded CompT™ panel includes ICOS, LAG-3, TIM-3 and granzyme B analysis, which are four intensively investigated biomarkers for T cell functionality (Fig. 1C). The analysis of these targets alone and in combination can provide insight into the anti-tumor potential of CD8+ T cells. Evidence supports a co-stimulatory and anti-tumor role for ICOS receptor signaling, thus making ICOS an attractive therapeutic target.4 Granzyme B is often used as a biomarker for cytolytic activity and can correlate with CD8+ T cell anti-tumor responses. Conversely, PD-1, LAG-3 and TIM-3 are inhibitory receptors and while the expression of these three receptors has been linked to T cell exhaustion, a growing body of data suggests heterogeneity among sub-populations exists within the exhausted PD-1 expressing CD8+ T cells.5 This heterogeneity correlates with the expression pattern of these inhibitory receptors. This profile can help define different sub-populations that have distinct potential to be re-invigorated to proliferate and/or lyse tumor cells.6 Figure 2 illustrates how the MI-Expanded CompT™ panel can quantify cells with double and triple positive expression for inhibitory receptors and provide insight into the heterogeneous PD-1 expressing T cell subset and its functionality. Numerous other T cell activation and inhibitory receptors have been described and implicated in influencing tumor immune responses; these include TIGIT, OX-40, CD137, CTLA-4 and others. Covance has experience analyzing many of these markers in ex vivo tumor analysis. With minimal developmental efforts, the MI-Expanded CompT™ can be customized to meet your unique preclinical needs.

Figure 2. Multiplexed analysis of T cell activation/exhaustion markers in naïve MC38 tumor-derived cells. The Expanded MI-CompT™ panel measures co-expression of several T cell biomarkers for both exhaustion and anti-tumor activity simultaneously. In this example, PD1+ CD8+ T cells were first gated. Downstream analysis then quantifies cells with double and triple positive expression of PD-1, LAG-3 and TIM-3.

Customization - NK/NKT Cell Analysis and Further Options

Covance can configure custom panels with up to 18 colors, which creates options for the MI-Expanded CompT™ panel. In addition to substituting or adding different T cell activation/exhaustion markers as described in the previous section, NK/NKT cell analysis is a potentially valuable endpoint. This is enabled by the addition of CD49b/CD335 markers to the panel (Fig. 3).

NK and NKT cells are an important source of IFNγ, have indirect effects on enhancing CD8+ T cell anti-tumor responses, and can directly lyse tumor cells by releasing cytolytic granules such as granzyme B.7,8 Other options include IFNγ, TNFα, or other cytokine analyses for a more in depth profile of PD1+ and PD1- CD8+ T cells. Or add CD103 analysis to examine resident memory T cells for a deeper memory T cell profile in the tumor. The team at Covance has extensive experience developing custom flow cytometry services. To learn more about how the MI-Expanded CompT™ panel can be incorporated into your preclinical research, contact the scientists at Covance.

References

1. Jiang, Y., Y. Li and B. Zhu. “T-cell exhaustion in the tumor microenvironment.” Cell death & disease 6.6 (2015): e1792.

2. Klebanoff, Christopher A., Luca Gattinoni and Nicholas P. Restifo. “CD8+ T-cell memory in tumor immunology and immunotherapy.” Immunological reviews 211.1 (2006): 214-224

3. Mami-Chouaib, Fathia, et al. “Resident memory T cells, critical components in tumor immunology.” Journal for immunotherapy of cancer 6.1 (2018): 87.

4. Amatore, Florent, Laurent Gorvel and Daniel Olive. “Inducible Co-Stimulator (ICOS) as a potential therapeutic target for anti-cancer therapy.” Expert opinion on therapeutic targets 22.4 (2018): 343-351.

5. Miller, Brian C., et al. “Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade.” Nature immunology 20.3 (2019): 326.

6. Xiong, Huizhong, et al. “Coexpression of Inhibitory Receptors Enriches for Activated and Functional CD8+ T Cells in Murine Syngeneic Tumor Models.” Cancer immunology research 7.6 (2019): 963-976.

7. Zhu, Yanting, Bo Huang and Jue Shi. “Fas ligand and lytic granule differentially control cytotoxic dynamics of natural killer cell against cancer target.” Oncotarget 7.30 (2016): 47163.

8. Zhao, Jie, et al. “Polyclonal type II natural killer T cells require PLZF and SAP for their development and contribute to CpG-mediated antitumor response.” Proceedings of the National Academy of Sciences 111.7 (2014): 2674-2679.

Note: Studies were performed in accordance with applicable animal welfare regulations in an AAALAC-accredited facility.

Figure 3. MI-Expanded CompT™ panel customization to enables analysis NK and NKT cell subsets in naïve MC38 tumor-derived cells. CD3 expression was used to delineate CD49b/CD335+ NK cells and NKT cells. Granzyme B expression levels in these subsets was quantified by downstream analysis.

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