Date:November 20, 2018
Productive immunotherapy driven anti-tumor responses rely on the activation of T cells that target tumor-associated antigens (TAA). The dendritic cell (DC) plays an essential role in the activation of T cell antigen-specific responses, which occurs naturally in the context of infection. However, this same process supports tumor immune surveillance as well as active anti-tumor responses. Research into new approaches that harness DC TAA presentation to T cells to boost anti-tumor immunity has received much attention. In this blog, we will discuss DC subsets in the tumor-microenvironment and mechanisms that tumors use to suppress their function. We will explore how pre-clinical models can be used to investigate DC responses to immunotherapy. Furthermore, we will highlight recent advances in the field of DC immunotherapy, including different approaches to upregulate DC activity, such as STING pathway activators.
Dendritic Cell Subsets and the Tumor Microenvironment
The tumor microenvironment (TME) promotes DC dysfunctionality by the release of factors that either inhibit or reverse DC maturation and function. A crucial function of the tumor-infiltrating DC is to internalize and present immunogenic antigens to T cells, particularly to CD8+ T cells through a process called antigen cross-presentation. Combined with DC maturation events that upregulate MHC and costimulatory molecules CD80, CD86, and CD40, this process drives the T cell antigen-specific response. Due to the elimination of T cells that target host cell antigens during thymic development, immunodominant T cell clones in the tumor are generally reactive to what are classified as either shared antigens (common across multiple tumor types) that are often “altered-self,” or neo-antigens that arise from spontaneous mutations that occur during malignant transformation. To further dampen T cell activation by DCs, there are multiple suppressive signals produced in the TME. These include secreted anti-inflammatory mediators such as VEGF, CSF-1, IL-6 and IL-10. Furthermore, the hypoxic nature of the TME is inhibitory to DC function. Together, these cues combine to upregulate PD-L1 expression, prevent maturation, inhibit pro-inflammatory cytokine production, and block antigen cross-presentation in DCs.
In mice, research has uncovered the different tumor infiltrating DC subsets and their function, which has opened new therapeutic options to enhance DC activity (reviewed in ). DCs are a heterogeneous family and are broadly classified as CD11c+MHCII+ cells. Except for inflammatory DCs (infDC), which differentiate from monocytes, most tumor-infiltrating DCs arise from a common DC precursor (CDP). The majority of these DCs are CD24+ and classified as conventional cDC1 and cDC2 subsets, which express CD103 or CD11b respectively (Figure 1). Plasmacytoid DCs (pDC) are a subset characterized by low expression of CD11c and are positive for Siglec-H, PDCA1, and B220 expression. Finally, infDC are Ly-6C, CD11b, and CD206 positive. The infDC monocyte precursors initially take on an immunosuppressive phenotype upon entering the tumor, which aids the tumor in evading the immune system. However, these monocytic myeloid-derived suppressor cells (M-MDSC) can give rise to infDC under certain inflammatory environmental conditions and promote anti-tumor activity. While, these signals that drive infDC differentiation have not yet been clearly elucidated, reports have indicated a role for GM-CSF, toll-like receptor (TLR) activation, and T cell-derived factors.
Strategies for Analysis of Tumor-Infiltrating Dendritic Cells
The use of syngeneic models can be helpful to address novel hypotheses and test new therapies aimed at augmenting DC function in the TME. Areas of interest include:
- Treatment-induced expansion of DC subsets
- Enhancement of DC maturation
- DC trafficking of TAA to draining lymph node (LN)
- DC-mediated TAA specific T cell activation
Covance has assembled custom immunophenotyping panels to help address these research aims and Figure 1 illustrates data that can be gained using these panels. Antigen presentation to T cells can occur inside the TME. cDC1 activate CD8+ T cells directly whereas cDC2 preferentially present antigen to CD4+ T cells that in turn boost CD8+ T cell function (Figure 1A). Expansion of these two subsets can indicate that treatment is shifting the balance toward a more anti-tumor response. As mentioned earlier, upregulation of CD80 and CD86 that engage CD28 on T cells, is required to trigger a productive T cell response. Figure 1B shows that the expression of these markers is low on DCs in B16-F10 melanoma tumors, indicative of the immunosuppression imparted on the DCs by the TME. Drugs that have an adjuvant-like activity are intended to upregulate these surface molecules to increase the magnitude of T cell responses upon DC engagement. The CD103+ cDC1 cells in the TME, have been shown by many groups to contain a migratory DC population that is responsible for trafficking TAA to draining LN where they encounter naïve T cells. These migratory DCs are characterized by XCR1 expression (Figure 1C). Likewise, when the draining LNs are analyzed, CD103+ DCs can be largely XCR1+ (Figure 1D). An expansion in this population of CD11c+MHCII+CD103+CD11b–XCR1+ cells following treatment can indicate that DC migration as well as cross-presentation of TAA to CD8+ T cells in the LN is enhanced.
InfDC are another subset documented to have the ability to cross-present TAA and also promote CD4+ T cell responses. This subset differentiates from M-MDSCs if the appropriate inflammatory signals are received. Figure 1E illustrates how infDC can be delineated from the M-MDSC gate by flow cytometry. A gating strategy for pDC delineation in mouse splenocytes is shown in Figure 1F. pDCs are another subset that infiltrates the tumor and have been demonstrated to take on immunosuppressive functions. In the clinic, increased pDC numbers can correlate with a poor patient prognosis. However, pDCs are also a potent source of type I interferon, which is a powerful inducer of cross-presentation and the induction of anti-tumor T cell activity. This response is triggered by the Stimulator of Interferon Genes (STING) pathway, which has recently become the focus of a new class of STING activator immunotherapies.
Analysis of DC subsets in the LN can be a challenge. While migration of DCs to LNs where they present TAA is a critical component of effective T cell expansion, detection of this rare subset in disaggregated LN tissue requires careful technique. DCs typically represent less than 1% of total LN cells. To add an additional layer of complexity, DCs strongly adhere to the LN stroma. Standard mechanical dissociation techniques fail to effectively release DCs into suspension, therefore necessitating the use of enzymatic dissociation to enable robust analysis of LN DC subsets. To illustrate this, Figure 2 demonstrates that incorporating enzymatic dissociation increases the DC yield from tumor-draining LN.
Immunotherapeutic Approaches to Boost Host Dendritic Cell Activity
Several drug candidates that enhance the activity of DCs are currently under intensive preclinical investigation, with some advancing to clinical trials. Early research examined the efficacy of compounds that expand DC numbers in vivo. These include GM-CSF and FLT3-L, which are produced naturally by the host. While both have been shown to expand multiple DC subsets, they have non-redundant activities, specifically regarding monocyte-derived DC development. Both GM-CSF and FLT3-L drug candidates have advanced into clinical trials with encouraging results.[8,9] Other drug candidates act as adjuvants to trigger DC maturation and increase T cell activation. These typically target innate immune receptors on DC such as TLRs, which results in the upregulation of costimulatory molecules and increase pro-inflammatory cytokine production. An example is Poly (I:C), which is a synthetic TLR3 agonist. Early clinical studies using Poly (I:C)-based immunotherapies demonstrated toxic side effects. However recent preclinical data demonstrated efficacious results using a modified Poly (I:C) derivative in a melanoma model without host toxicity, renewing interest in this approach. As mentioned above, another DC immunotherapy that is receiving much attention are the stimulator of interferon genes (STING) agonists. In nature, activation of this pathway is triggered by cytosolic DNA, which results in the production of type I interferon. It has been recently demonstrated that this response has a multifaceted effect on DCs to promote T cell activation, which includes upregulation of costimulatory molecules and increased cross-presentation activity. Preclinical results have demonstrated that STING agonists inhibit tumor growth in melanoma, colon cancer, and lymphoma, and clinical trials are currently underway.
To learn more about customizable dendritic cell flow cytometry panels and how to incorporate them into your research, contact the scientists at Covance.
1Dhodapkar, K., & Dhodapkar, M. (2016). Harnessing shared antigens and T-cell receptors in cancer: Opportunities and challenges. Proceedings of the National Academy of Sciences, 113(29), 7944-7945.
2 Munn, D. H., & Bronte, V. (2016). Immune suppressive mechanisms in the tumor microenvironment. Current opinion in immunology, 39, 1-6.
3Lee, C. T., Mace, T., & Repasky, E. A. (2010). Hypoxia-driven immunosuppression: a new reason to use thermal therapy in the treatment of cancer?. International Journal of Hyperthermia, 26(3), 232-246.
4Veglia, F., & Gabrilovich, D. I. (2017). Dendritic cells in cancer: the role revisited. Current opinion in immunology, 45, 43-51.
5Wylie, 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. Oncoimmunology, 4(8), e1019198.
6Demoulin, S., Herfs, M., Delvenne, P., & Hubert, P. (2013). Tumor microenvironment converts plasmacytoid dendritic cells into immunosuppressive/tolerogenic cells: insight into the molecular mechanisms. Journal of leukocyte biology, 93(3), 343-352
7Treilleux, I., Blay, J. Y., Bendriss-Vermare, N., Ray-Coquard, I., Bachelot, T., Guastalla, J. P., … & Lebecque, S. (2004). Dendritic cell infiltration and prognosis of early stage breast cancer. Clinical Cancer Research, 10(22), 7466-7474.
8Kwek, S. S., Kahn, J., Greaney, S. K., Lewis, J., Cha, E., Zhang, L., … & Spitler, L. E. (2016). GM-CSF and ipilimumab therapy in metastatic melanoma: Clinical outcomes and immunologic responses. Oncoimmunology, 5(4), e1101204.
9Bhardwaj, N., Pavlick, A. C., Ernstoff, M. S., Hanks, B. A., Albertini, M. R., Luke, J. J., … & Vitale, L. (2016). A Phase II Randomized Study of CDX-1401, a Dendritic Cell Targeting NY-ESO-1 Vaccine, in Patients with Malignant Melanoma Pre-Treated with Recombinant CDX-301, a Recombinant Human Flt3 Ligand.
10Krown, S.E. & Kerr, D & Stewart, W.E. & Field, A.K. & Oettgen, H.F.. (1986). Phase I trials of poly(I, C) complexes in advanced cancer. Journal of biological response modifiers. 4. 640-9
11Rajendrakumar, S., Mohapatra, A., Singh, B., Revuri, V., Lee, Y. K., Kim, C., … & Park, I. K. (2018). Self-Assembled, Adjuvant/Antigen-Based Nanovaccine Mediates Anti-Tumor Immune Response against Melanoma Tumor. Polymers, 10(10), 1063.
About 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.