Author:

David Draper, PhD | Associate Director, Scientific Development

Date:

February 2019

T cell-mediated anti-tumor responses hinge on the activity of antigen-presenting cells (APC) that present tumor-associated antigen (TAA) and provide co-stimulatory signals to T cells. This in turn activates tumor-specific T cells, triggering their expansion and recruitment to the tumor where they exert their effects. Dendritic cells (DC) are professional APCs that are instrumental in this process. DCs can be activated to express numerous costimulatory ligands and can internalize antigen and present immunodominant peptides to both CD4+ and CD8+ T cells. The development of novel therapeutics that harness DC function is an area of intensive investigation. To facilitate these efforts and address the growing need for robust comprehensive DC immunophenotyping in murine pre-clinical models, MI Bioresearch has configured a new standard panel, MI-CompDC™. In this Tech Spotlight, we will demonstrate how various DC subsets in tumor and other tissue-derived cell samples can be analyzed using this panel.

For a general introduction to the different DC subsets and their function in cancer biology, read our recent blog “An Introduction to Dendritic Cell Biology and Analysis Using Syngeneic Immuno-Oncology Models”

The MI-CompDC™ panel allows investigators to determine if in vivo therapy affects the abundance and activation state of different DC subsets in the tumor, tumor-draining lymph nodes (LN), or other host compartments. It is a 12-color panel that combines a set of antibodies that when properly examined, delineates distinct DC subsets, and provides insight into the T cell activation potential of the DCs (Table 1). Through a meticulous gating strategy, the panel first facilitates DC analysis with precision by excluding macrophages, granulocytes, and B cells using an exclusion gate. This process minimizes the risk that DC analysis is compromised due to contamination by irrelevant or autofluorescent cells. CD24 expression is then used help delineate conventional DC subsets, which are generally classified by medium to high expression levels of CD11c and MHC class II. These conventional DCs include the CD103+/CD8+ DC1 subset and the CD11b+ DC2 subset (Figure 1A), which have been demonstrated to drive CD8+ and CD4+ T cell anti-tumor responses respectively.[1] XCR1 expression has been documented by multiple groups as a marker for DCs with cross-presenting activity, a phenotype required for DC-mediated activation of CD8+ T cells.[2] Figures 1B and 1C demonstrate XCR1+ DC analysis in B16-F10 tumor-derived cells and draining lymph nodes. XCR1 expression is generally associated with the DC1 subset, as shown. Finally, the expression of maturation markers CD80 and CD86 is measured to assess the T cell co-stimulatory potential of the DCs. Figure 1D shows that the expression of these markers is relatively low on DCs in B16-F10 melanoma tumors, indicative of the immunosuppression imparted on the DCs by the tumor microenvironment (TME).

Table 1: MI-CompDC™ Panel Antibodies and Description of Their Utility

Antibody/Dye Description
CD45 Pan immune cell marker

F4/80, Ly-6G, CD19

Exclusion gate for macrophages, granulocytes, and B cells
CD11c Dendritic cell marker
CD24 Dendritic cell marker
CD8 DC1 marker
MHC Class II Dendritic cell marker/maturation marker
CD103 DC1 marker
CD11b DC2 marker
XCR1 Cross presenting/migratory dendritic cell marker
CD80 Maturation marker
CD86 Maturation marker
Viability Dye Dead cell exclusion
The MI-CompDC™ can be customized to analyze inflammatory DC (InfDC) and plasmacytoid DC subsets by substituting in Ly-6C/CD206 and Siglec-H antibodies respectively.

 

Image for the 2019_0205 Tech Spotlight: CompDC Fig. 1: Analysis of DC Subsets Using the MI-CompDC™ Panel
Fig. 1: Analysis of DC subsets using the MI-CompDC™ panel. B16-F10 melanoma tumors were harvested from mice. DC1 and DC2 subset analysis in tumor-derived cells (A), XCR1 expression in CD103+ DC1 cells in the tumor (B), and lymph nodes (LN) from B16-F10 tumor bearing mice (C). Expression of co-stimulatory markers CD80 and CD86 in total tumor-derived DCs (D). The red peak represents target-stained cells. The blue peak represents the unstained negative control.

The MI-CompDC™ can also be customized for analysis of two additional DC subsets. This includes inflammatory DCs (infDCs), which is performed by substituting in anti-CD206 and anti-Ly-6C antibodies. InfDCs have been demonstrated to promote anti-tumor activity and can be delineated out of the monocytic myeloid-derived suppressor cell (M-MDSC) gate as CD11c+MHCII+CD11b+CD206+ cells (Figure 2A).[3] Plasmacytoid DCs (pDCs) can also be quantified by substituting in an anti-Siglec-H antibody. While pDC function in the TME has not been fully characterized, many reports have demonstrated that these pDCs can have impaired function and can even have an immunosuppressive effect on anti-tumor responses.[4] pDCs generally have low levels of CD11c expression and are positive for Siglec-H, as shown in Figure 2B.

 

019_0205_TS_Fig. 2: Analysis of infDC and pDC Subsets is Enabled by Customization of the MI-CompDC™ Panel
Fig. 2: Analysis of infDC and pDC subsets is enabled by customization of the MI-CompDC™ panel. (A) Analysis of infDC in B16-F10 tumor-derived cells. (B) Analysis of pDC in CT26 colorectal carcinoma tumor-derived cells.

To learn more about the MI-CompDC™ panel and how to incorporate it into your research, contact the scientists at MI Bioresearch.

1Gardner, A., & Ruffell, B. (2016). Dendritic cells and cancer immunity. Trends in immunology37(12), 855-865.

2Wylie, B., Seppanen, E., Xiao, K., Zemek, R., Zanker, D., Prato, S., … & Waithman, J. (2015). Cross-presentation of cutaneous melanoma antigen by migratory XCR1+ CD103− and XCR1+ CD103+ dendritic cells. Oncoimmunology4(8), e1019198.

3Kuhn, S., Yang, J., & Ronchese, F. (2015). Monocyte-derived dendritic cells are essential for CD8+ T cell activation and antitumor responses after local immunotherapy. Frontiers in immunology6, 584.

4Mitchell, D., Chintala, S., & Dey, M. (2018). Plasmacytoid dendritic cell in immunity and cancer. Journal of neuroimmunology.

 

David Draper, PhDAbout the Author: Dr. David Draper is an immunologist and a member of the Scientific Development Group. He has been employed at MI Bioresearch since 2015. Dr. Draper holds a Ph.D. in Microbiology from North Carolina State University. His post-doctoral work at Duke University and the National Institutes of Health focused on uncovering the mechanisms of the host pulmonary immune response to bacterial, viral, and allergen challenge using genetically engineered animal models. This body of work provided the foundation of Dr. Draper’s technical expertise in the area of immune cell immunophenotypic and functional characterization.

 

Comprehensive Dendritic Cell Analysis by Flow Cytometry Using the New MI-CompDC™ Panel (PDF Version)

Comprehensive Dendritic Cell Analysis by Flow Cytometry Using the New MI-CompDC™ Panel (PDF Version)