Dendritic cells are a heterogeneous group of specialized antigen-presenting cells (APCs) that play critical roles in innate and adaptive immune responses. These cells are distributed throughout all mammalian organs and tissues and dynamically navigate between non-lymphoid and lymphoid tissues. Their high expression of major histocompatibility complex molecules, MHC class I and II, enables them to present engulfed and processed antigen peptides to T cells. Through their specialized antigen presentation functions and multiple secreted factors, dendritic cells are major players in several immune processes, such as naïve T cell activation, development of T cell memory, establishment and maintenance of immune tolerance, and stimulation of B cell antibody responses.
The origin of dendritic cells can be traced to the bone marrow, where hematopoietic stem cells (HSCs) give rise to a diverse group of progenitor cells known as lymphoid-primed multipotent progenitors or LMPPs. First, the differentiation of LMPPs into common myeloid progenitors (CMPs) leads to a macrophage and dendritic cell progenitor (MDP) from which common dendritic cell progenitors (CDPs) are produced. Next, under the influence of the growth factor Fms-like tyrosine kinase 3 ligand (Flt3L), CDPs differentiate into pre-conventional dendritic cells (Pre-cDCs). Ultimately, Pre-cDCs exit the bone marrow and give rise to the two classical dendritic cell groups; conventional type I DCs (cDC1) and conventional type II DCs (cDC2). Conventional dendritic cells remain resting and continually monitor the organs and tissues where they reside. Once activated, typically by a pathogen or damage-associated molecular patterns (PAMPs or DAMPs), cDCs travel to secondary lymphoid organs to prime T cells through the presentation of processed antigens (Cabeza-Cabrerizo et al. 2021, Khan et al. 2022).
Beyond conventional subtypes, other functionally and phenotypically similar subsets include plasmacytoid DCs (pDCs), monocyte-derived DCs (MoDCs), and Langerhans cells (LCs). Nevertheless, these subsets differ from conventional dendritic cells in ontogeny and gene expression (Cabeza-Cabrerizo et al. 2021).
Dendritic cell ontogeny. Two subsets of conventional dendritic cells (cDCs) are derived from Pre-cDC progenitors and identified by their gene expression profile: cDC1-XCR1, DNGR-1, CD141, and CADM1; cDC2-CD1c, SIRPa , –XCR1, and –DNGR-1) “MDP, macrophage and DC progenitor; Pre-pDC, pre-plasmacytoid DC; Pre-cDC, pre-conventional DC; cDC1, conventional type I DC; cDC2, conventional type II DC; GM-CSF, granulocyte-macrophage colony-stimulating factor; M-CSF, macrophage colony-stimulating factor; FLT3-L, Fms-like tyrosine kinase 3 ligand.” The Diagram and a portion of the legend for Figure 1 were retrieved from Khan et al. 2022. https://creativecommons.org/licenses/by/4.0/
Anti-tumor T cell responses, especially the activation of cytotoxic tumor cell killing by CD8+ T lymphocytes, have been critically linked to positive outcomes in cancer treatment. Significantly, dendritic cells can activate innate immune cells and lymphocytes, including CD4+ and CD8+ T cells, to target tumor cells. Therefore, several cancer immunotherapy strategies have been tested leveraging the specialized functions of dendritic cells, such as the adoptive transfer of anti-tumor dendritic cells (i.e., DC vaccine) and their in vivo expansion (Cabeza-Cabrerizo et al. 2021). However, a factor limiting the success of such strategies is the immunosuppression commonly found within the tumor microenvironment, which reduces the infiltration and function of dendritic cells and their anti-tumor potential.
Dendritic cells and the tumor microenvironment. The tumor microenvironment exerts both positive and negative influences on dendritic cell functions. For instance, NK cells secrete several cytokines and chemokines that stimulate their recruitment and function. In contrast, macrophages and tumor cell-derived immunosuppressive factors limit dendritic cell functions. Retrieved from (Gardner et al. 2020) https://creativecommons.org/licenses/by/4.0
Given the limitations imposed by the immunosuppressive tumor microenvironment on dendritic cell function, several combined immunotherapies are being tested. For instance, strategies leveraging immune checkpoint blockers (e.g., anti-PD1 antibody) in combination with intratumoral administration of Flt3L and Poly-ICLC aim at releasing the breaks on immune responses while recruiting and activating dendritic cells. Additionally, radiotherapy is implemented to help release tumor antigens, making them more accessible. This approach is under current clinical investigation for non-Hodgkin’s lymphoma, metastatic breast cancer, and head and neck squamous cell carcinoma (NCT03789097).
Another strategy to improve dendritic cell performance within the tumor microenvironment leverages oncolytic viruses as immunotherapy. Oncolytic virus-based cancer therapy takes advantage of the natural ability of these viruses to kill tumor cells and disrupt the immunosuppressive tumor microenvironment. Oncolysis improves immune-cell tumor infiltration and provides access to tumor-specific antigens for cross-presentation and activation of T cells (Kim et al. 2015).
A new study by scientists at the Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, led by Dr. Joshua Brody, MD, leverages combined vaccination with Flt3L and the oncolytic Newcastle Disease Virus (NDV) in cancer immunotherapy (Svensson-Arvelund et al. 2022). Such an approach would provide a less-personalized or off-the-shelf cancer immunotherapy to benefit a larger patient population.
Dendritic cells play a critical role in establishing immune memory against tumors. Svensson-Arvelund and colleagues recognized that cancer immunotherapies often fail at engaging these cells and therefore fall short of providing long-lasting benefits. However, current approaches to harness the benefits of dendritic cells in cancer are technically challenging and costly. In one approach, dendritic cells are loaded ex vivo with tumor-derived antigen peptides for adoptive cell transfer into cancer patients. The success of such a strategy depends on identifying neoantigens, tumor-specific antigens derived from new proteins formed through genomic instability typical of tumorigenesis processes. While these new antigens provide great opportunities as candidates for cancer immunotherapy, the workflow involved in developing such personalized vaccines is complex. Commonly neoantigen discovery involves acquiring sufficient tumor tissue, mass spectral analysis, and extensive in vitro testing with synthetic peptides to ensure that the identified sequences are indeed immunogenic.
Therefore, Svensson-Arvelund and colleagues have developed a new strategy allowing in vivo recruitment and activation of dendritic cells to present tumor-specific antigens. Their approach leverages intratumoral administration of Flt3L to expand and recruit dendritic cells and the oncolytic virus, Newcastle disease virus (NDV), to expose tumor antigens, promote antigen uptake, and stimulate their activation.
Having first demonstrated in vitro NDV’s capacity to kill tumor cells in patient-derived lymphoma samples, the team found consequent induction of inflammatory signals, such as IFN-γ and molecules relevant for antigen cross-presentation. Significantly, the team demonstrated that NDV’s oncolytic activity could induce T cell proliferation and targeted tumor cell killing, findings replicated in the in vivo setting. Yet, Svensson-Arvelund and colleagues found that while NDV infection led to significant CD8+ T cell activation, anti-tumor activity in lymphoma-bearing mice was not long-lasting, highlighting the shortcomings of oncolysis as a single immunotherapy approach and the need for stimulating dendritic cell contribution.
Therefore, the team proposed combined Flt3L administration and NDV infection within tumors to recruit dendritic cells and effectively potentiate T cell cross-priming. This approach would enable the expansion of immature cells available for activation under conditions of NDV-mediated tumor cell killing
First, in vitro studies with NDV-infected tumor cells and splenocytes derived from Flt3L-treated mice enabled Svensson-Arvelund et al. to demonstrate the effective activation of dendritic cells (i.e., cDC1 and cDC2 subsets). Similarly, NDV tumor cell killing could drive the activation of conventional subsets in peripheral blood derived from lymphoma patients previously treated with Flt3L. Furthermore, dendritic activation was partly associated with increased type I interferons and evidenced by induced expression of damage-associated receptors (e.g., Axl), which correlated well with enhanced antigen uptake activity.
Next, having demonstrated the desired effects of NDV and Flt3L on activating dendritic cells, Svensson-Arvelund and colleagues developed in vitro assays that directly connected tumor-antigen presentation to the expansion of anti-tumor CD8+ T cells. Ultimately, the effectiveness of the combined NDV/Flt3L immunotherapy was tested in vivo following intratumoral administration in A20 tumor-bearing mice. Compared to NDV alone, combined NDV/Flt3L use led to significant improvements in long-term tumor control and survival. In agreement with these outcomes, the team confirmed increased numbers of activated cDCs and pDCs at the tumor site and tumor-draining lymph nodes.
Long-term tumor control relies on tumor-specific T cell responses. To uncover whether the dual NDV/Flt3L treatment elicited desirable tumor-antigen specific T cell responses, Svensson-Arvelund and colleagues leveraged synthetic peptides for in vitro assays.
“Peptides (25-mers) used for initial screening were synthesized and purified using PepPower Peptide Synthesis Platform (GenScript). Peptide quality was assessed by mass spectrometry and HPLC to guaranty >75% purity and solubility of individual peptide was tested to determine adequate solvents. 8-11-mer Lrrk1mut peptides were ordered from Genscript” Svensson-Arvelund et al., 2022.
Assaying Neoantigen Specific T cell Responses.“k–m TdLN cells from Flt3L+NDV-treated A20 or GFP+ A20-tumor-bearing mice were co-cultured with DCs pulsed with pooled or individual neoepitope peptides identified by exome and RNA sequencing, or GFP-peptide. l, m IFN-γ production after 24 h; data pooled from 2 independent experiments, n = 4 mice per tumor type, one-way ANOVA with Dunnett’s multiple comparisons test (l) and representative (n = 4) contour plots of CD44+PD1+CD8+ T cells reactive to peptide pool 2 and Lrrk1mut (m). Data show mean ± SD.” Retrieved with modification from Svensson-Arvelund et al. 2022, only diagrams and legends for portions of Figure 6 (k to m) are shown. https://creativecommons.org/licenses/by/4.0/
First, the team identified somatic mutations expressed in the A20 lymphoma B cell line through whole-exome and RNA sequencing, procuring the synthesis of over 80 peptides containing potential neoantigens from GenScript. Next, cells derived from tumor-draining lymph nodes, isolated from NDV/Flt3L treated A20-tumor bearing mice, were co-cultured with synthetic peptide-loaded dendritic cells. Lastly, the extent of T cell activation induced by various synthetic peptide pools was determined through IFN-γ analysis. Svensson-Arvelund and colleagues narrowed the positive reactivity elicited by “Pool 2,” which contains a mutated sequence corresponding to Leucine-rich repeat kinase 1 (Lrrk1mut peptide).
