Functional Analysis of Tumor Infiltrating Lymphocytes by Flow Cytometry Using the Cytokine/Cytotoxicity™ Panel

AUTHOR:

David Draper, PhD

DATE:

August 2020

Pro-inflammatory cytokine responses coupled with the expression of granzyme B and CD107a (LAMP-1), are valuable biomarkers in the immuno-oncology space1-3. Cell-based assays that can quantify the expression of these targets in tumor-derived effector lymphocytes provide insights into the mechanism of action of drug candidates. Covance’s Preclinical Oncology Group offers a new off-the-shelf assay that utilizes the Cytokine/Cytotoxicity standard flow cytometry panel to quantify these endpoints in T cells, natural killer (NK) and natural killer T cell (NKT) subsets. This technology spotlight demonstrates how the Cytokine/Cytotoxicity™ panel can be incorporated into in vivo studies and, combined with conventional immunophenotyping, help build robust data sets during preclinical drug development.

Cytokines including IFNγ, TNFα, and IL-2 have anti-tumor activity by promoting innate and adaptive immune cell activation and T cell proliferation in the tumor microenvironment. In vivo therapies that inhibit tumor growth by way of T cell activation, through direct or indirect mechanisms, often increase the frequency of tumor-infiltrating T cells that are primed to respond vigorously to ex vivo stimulation. Similar effects have been demonstrated to occur in NK and NKT cells with respect to priming and subsequent IFNγ/TNFα production. In addition to cytokine production, in vivo treatment can induce effector lymphocytes to acquire heightened cytotoxic properties that enable tumor cell lysis by direct cell-to-cell contact mechanisms. This process is mediated by the expression and release of perforins and granzymes from cytolytic granules stored inside the immune cells. These toxins disrupt the cell membrane and induce apoptosis in the target tumor cell. Furthermore, degranulation coincides with the expression of CD107a on the surface of the effector cell and can thus be used as a marker for cytotoxic activity.

Together, the signatures described above can be profiled to examine the activation state of lymphocyte subsets in the tumor microenvironment. The Cytokine/Cytotoxicity panel enables these measurements by combining cell surface immunophenotyping using T cell, NK, and NKT cell-specific antibodies with intracellular staining for cytokines and granzyme B expression (Table 1).

Table 1: Cytokine/Cytotoxicity™ Panel

Antibody/Dye

Description

CD45

Pan-hematopoietic cell marker

CD3

Pan-T cell marker

CD4

CD4+ T cell marker

CD8

CD8+ T cell marker

CD49b/CD335

Natural killer/Natural killer T cell marker

IFNγ

Pro-inflammatory cytokine

TNFα

Pro-inflammatory cytokine

IL-2*

T cell activator

Granzyme B

Cytotoxicity marker

CD107a

Degranulation marker

Viability Dye

Dead cell exclusion

*IL-2 can be substituted with other cytokines including IL-4 and IL-17a

 

Analysis of Cytokines and Granzyme B in 4T1-luc Tumor Derived Effector Cells Following Radiation and Anti-mCTLA-4 Therapy

The utility of the Cytokine/ Cytotoxicity panel to measure cytokines and granzyme B is demonstrated in this data set using the murine 4T1-luc mammary carcinoma model. Figure 1 shows the gating strategy used to delineate T cell and NK cell subsets. As with all of our preclinical oncology flow cytometry panels, analysis begins with dead cell exclusion and subsequent delineation of CD45+ immune cells to identify immune subsets of interest (not shown). Representative profiles of IFNγ, TNFα, IL-2 and granzyme B expression following PMA/ionomycin stimulation are shown. 

Figure 1: Gating of pro-inflammatory cytokines and granzyme B in lymphocyte subsets by flow cytometry
Figure 1: Gating of pro-inflammatory cytokines and granzyme B in lymphocyte subsets by flow cytometry

The 4T1-luc tumor microenvironment is highly immunosuppressive and is characterized by the presence of a large granulocytic myeloid-derived suppressor cell infiltrate (not shown). 4T1-luc is a model known to be resistant to checkpoint inhibition therapies. However, when combined with radiation, delivered by the Small Animal Radiation Research Platform (SARRP, Xstrahl), anti-mCTLA-4 improves tumor growth inhibition of 4T1-luc tumors (Figure 2A, left). Tumor growth inhibition is likely not due to a treatment-induced increase in effector cell recruitment as flow cytometry does not demonstrate increased T cell or NK cell counts in the tumor following any in vivo treatment condition, when compared to the isotype control group (Figure 2A, right). However, ex vivo stimulation of tumor-derived cells with PMA/ionomycin reveal that CD8+ T cells have an enhanced capacity to produce IFNγ. In addition, NK cells express higher levels of granzyme B, compared to controls (Figure 2B). These data suggest that combined treatment with radiation and anti-mCTLA-4 enhance the activation state of CD8+ T cells and NK cells. This demonstrates the value of including mechanistic markers in studies where efficacy and immunophenotyping are being pursued.

Figure 2 - Cytokine-Cytotoxicity Panel
Figure 2: Analysis of the pro-inflammatory response in effector cells in the 4T1-luc Model. BALB/c mice with established 4T1-luc tumors (n=6/group) were dosed with anti-mCTLA-4 (clone 9D9), focal radiation (“RAD”; SARRP, Xstrahl Inc.), a combination of the two, or isotype control antibodies. Tumors were dissociated into single cell suspensions (gentleMACS™, Miltenyi Biotec), and labeled with fluorescent antibodies prior to data acquisition. For cytokine and granzyme B analysis, tumor-derived cells were stimulated ex vivo with PMA/ionomycin for 5 hours in the presence of brefeldin A. Following incubation, cells were collected and immunostained for intracellular targets. Data was acquired on an Attune™ NxT (ThermoFisher Scientific) flow cytometer and then analyzed using Flowjo software (BD).

 

Analysis of Degranulation in CT26 Tumor Derived Effector Cells Following Anti-mCTLA-4 Therapy

CD107a becomes expressed on the cell surface during degranulation and is a marker commonly used to measure treatment-induced effects on cytotoxic activity. The Cytokine/Cytotoxicity™ panel was used to examine the effects of anti-mCTLA-4 on lymphocyte cytotoxicity and tumor growth in the murine CT26 colorectal carcinoma model. CT26 tumor-bearing mice treated with anti-mCTLA-4 respond with a 74% tumor growth inhibition (Figure 3A). To examine cytotoxicity, CD107a and granzyme B expression was measured in CD8+ T cells, NK, and NKT cells following ex vivo stimulation of tumor-derived cells with PMA/ionomycin. Representative expression profiles are shown in Figure 3B. Analysis reveals that anti-mCTLA-4 treatment triggers an increase in CD107a expression in all three subsets compared to isotype control treated animals. This coincides with a decrease in granzyme B expression levels in NK and NKT cells. The data suggests that granzyme B stores in NK and NKT cells are depleted during degranulation. Note that CD8+ T cell granzyme B expression was not different between groups, perhaps because of an equilibrium in the de novo expression of granzyme B triggered by treatment and the rate of degranulation. Taken together this phenotype is consistent with a treatment-induced enhancement in cytotoxicity and potential for direct tumor cell killing activity.

Figure 3 - Cytokine-Cytotoxicity Panels
Figure 3: Analysis of Cytotoxicity in the CT26 Model. BALB/c mice with established CT26 tumors (n=10/group) were dosed with anti-mPD-1 (clone RMP1-14) or isotype control antibodies. Ex vivo stimulation and immunophenotyping were performed as described above.

 

Customization – Panel Configuration to Customize Cytokine Responses

The Cytokine/Cytotoxicity™ panel is pre-configured to measure pro-inflammatory cytokines. The panel can also be customized to examine other cytokine responses. For example, helper CD4+ T cells differentiate into different CD4+ effector phenotypes, which include Th1, Th2, and Th17. These subsets have different roles in tumor pathogenesis. Th1 cells are characterized by the release of IFNγ, whereas Th2 and Th17 can be characterized by the production of IL-4 and IL-17, respectively. The Cytokine/Cytotoxicity™ panel can be customized to include IL-4 and IL-17A to facilitate mechanistic analysis of treatment-induced effects on helper T cell differentiation in the tumor microenvironment.

To learn more about how the Cytokine/Cytotoxicity™ panel can be incorporated into your preclinical oncology research, contact the scientists at Covance.

Please note that all animal care and use was conducted according to animal welfare regulations in an AAALAC-accredited facility with IACUC protocol review and approval.

 

1. Clynes, R. A., & Desjarlais, J. R. (2019). Redirected T cell cytotoxicity in cancer therapy. Annual Review of Medicine70, 437-450.

2. Miller, J. S., & Lanier, L. L. (2019). Natural killer cells in cancer immunotherapy. Annual Review of Cancer Biology3, 77-103.

3. Bae, E. A., Seo, H., Kim, I. K., Jeon, I., & Kang, C. Y. (2019). Roles of NKT cells in cancer immunotherapy. Archives of Pharmacal Research, 1-6.