Group “Mobilization of dendritic cells” – Project details

Directed by Jenny VALLADEAU-GUILEMOND, PhD, HDR

T lymphocyte (LT) activation is mediated by antigen-presenting cells, especially dendritic cells (DC). In humans, different DC subsets are well characterized, including plasmacytoid DC (pDC) secreting large amounts of interferon alpha (IFN-a) and rather dysfunctional and with a poor prognosis in breast cancer^1–3. There are also 2 subpopulations of conventional DC (cDC) called cDC1 (cDC1) and cDC2, inflammatory and unconventional DCs from monocytes and finally Langerhans cells (LC) whose prognostic impacts are controversial³. cDC1 is specialized in activating cytotoxic LT (CTL) by Ag cross-presentation compared to other DC^4–7. They are also characterized by the expression of toll-like receptor 3 (TLR3) whose activation by poly-I:C induces a high production of interferon type III (IFN-III)⁴.

IFN-III, also known as interferon lambda (IFN-λ, or IL-28A and B and IL-29), characterizes a new subtype of interferon that shares many characteristics with Type I IFNs (IFN-a and –b). Among them, we find i) the same intracellular signaling pathway with STAT1 phosphorylation, ii) same genes induction (Interferon Inducible gene, ISG) and iii) a strong impact on antiviral responses. In this context, several studies have shown that IFN-I responses are critical in the early stages of anti-tumor immune surveillance^8,9 as well as in the effectiveness of radiotherapy¹⁰, or certain chemotherapy¹¹. However, very little was known about the impact of cDC1 and IFN-λ in human cancers.

In this context, we showed the functional superiority of cDC1 to cross-present tumor Ag to CD8+ LT and showed the helper function of Natural killer cells (NK) to induce this cross-presentation. Then, we showed for the first time the infiltration of primary breast tumors by cDC1 (and other subpopulations of DC) as well as the preponderant role of type III interferon in inducing an anti-tumor immune response. In addition, we have demonstrated the ability of cDC1, present in the tumor environment, to naturally produce IFN-III in 20% of patients. Interestingly, we found no or little type I IFN secreted in breast tumors unlike IFN-III again suggesting a major role of IFN-III in breast cancer¹².

Although we have demonstrated through an in silico approach that cDC1 represents the subpopulation of DC most frequently associated with prolonged overall survival in most solid tumors, the prognostic and predictive value of response to Immunotherapies of cDC1 remains largely unknown. To date, the only study that suggested a correlation between cDC1 enrichment and a better response to anti-PD1 treatment was conducted in silico in a small cohort of melanoma patients¹³. In addition, the signals that lead to the recruitment of cDC1 within the tumor, and the signals/cytokines selectively produced by cDC1 and delivered to CD8 LTs to activate them are poorly described. Few data are available in humans on the role of IFN-III in anti-tumor immunity and its ability to induce responses to immunotherapies. It therefore seems important to better characterize the predictive impact of IFN-III among other factors in the tumor environment including cDC1. Indeed, a better understanding of its role will allow us to propose new potential therapeutic targets in monotherapy or in combination with current therapeutic strategies in cancers.

In addition, immune evasion mechanisms in established tumors have been well characterized over the past decade, culminating in the clinical approval of several drugs targeting immune checkpoint blockers (ICBs) in T cells. However, the very early events that allow the detection of pre-neoplastic cells by immune cells remain poorly understood. Indeed, although the concept of immune surveillance is well accepted and various innate immune cells probably contribute to the detection of tumors, only NK cells have been described to date as capable of detecting cellular stress (via the NKG2D/NKG2DL interaction). Due to the key role of antigen presenting cells, we believe that the presence of DC subsets and the activation of IFN-I/IFN-III pathways in these DCs are essential for initiating anti-tumor immune surveillance at an early stage of tumor development. In addition, we are convinced that these immune surveillance processes contribute to the response to treatment with current ICBs, as demonstrated for the cDC1 population and IFN-I in mice^8,9,14,15.

Thus, in order to answer the many questions that arise on the role of DC subsets and IFN-III in the response to IT or in the cancer immune surveillance, my group is developing today three complementary research axes:

1. DCs and IFN-III functions in breast cancer immune surveillance
To this end, we are performing a deep transcriptomic characterization of tumor-infiltrating myeloid cell subpopulations in humans and mice at the single cell level (scRNAseq). We will identify the signaling pathways activated in DC subpopulations at the early tumor stage (and neutralized at the invasive stage) by bioinformatic and system biology approaches. It will then be important to confirm and extend our main results obtained in scRNAseq by using immunofluorescence on sections to measure the expression of identified targets. Our partnership with OSE Immunotherapeutics will allow us to validate our hypotheses in vitro and in vivo with the development of drug candidates (monoclonal antibodies for example).

2. Identification of IFN-λ effects on tumor infiltrating cells and in the blood.
Indeed, if we have observed that IFN-III is associated with a good prognosis, the cells that respond to this IFN and the precise role of this IFN within the tumor microenvironment and in general in other non-infectious pathophysiological contexts remains unknown. Our preliminary data have already shown that IFNL-Receptor is well expressed by B lymphocytes, in agreement with the literature and even more strongly by pDCs. Thus, through RNAseq approaches and in vitro models, we are currently characterizing the effect of type III IFN on pDCs.

3. Clinical impact of the different DC populations in correlation to the response to IT by in situ analyses
The presence of different DC populations and their spatial organization in breast cancer have never been studied in response to IT. Therefore, we developed spectral microscopy protocols using OPAL® multiparametric immunofluorescence technology (7 colors) to identify DC subsets, macrophages, CD8 TL, tumor cells (cytokeratin), NKs (NKp46), different macrophages (CD68, CD163) and Tregs (FOXP3). We have defined an analysis pipeline that allows us to quantify in whole slide each population and to integrate in the same expression matrix the spatial data that allows us to analyze the frequency, the localization (tumor/stroma), and the distances between the cell subtypes and the nearest neighbors (KNN). In order to go further in the analysis of cellular partners present in each specific area of the tumor, we use the 10X Visium Spatial Gene expression technology (available and validated at the CRCL) which allows to analyze the transcriptome in a tissue context with a resolution of 5-10 cells on average per spot.

For all those purposes, we have access to a number of patient cohorts treated with IT: 1) the Breast-Immun-3 cohort (post-NAC TNBC patients, treated with anti-PD-1 + anti-CTLA-4 + radiotherapy (RT) vs chemotherapy (CT) + RT. N=60); 2); the MELPREDICT cohort (stage IV melanoma patients, n=40, first line IT) and the LUNG-PREDICT cohort (NSCLC, Pembrolizumab; first line n=90, IT vs. chemo) that we set up with the help of Dr. Elisa GOBBINI (MD, PhD in my group) and the Groupe Français de Pneumo Cancérologie (GFPC).

The ultimate impact of our projects is the development of new pharmacological tools or vaccine strategies to induce adaptive anti-tumor immunity in breast cancer patients resistant to current immunotherapies.

References

1. Sisirak, V. et al.  Impaired IFN-α production by plasmacytoid dendritic cells favors regulatory T-cell expansion that may contribute to breast cancer progression.  Cancer Res.  72, 5188–5197 (2012).
2. Treilleux, I. et al.  Dendritic cell infiltration and prognosis of early stage breast cancer.  10, 7466–74 (2004).
3. Hubert, M., Gobbini, E., Bendriss-Vermare, N., Caux, C. & Valladeau-Guilemond, J. Human Tumor-Infiltrating Dendritic Cells: From in Situ Visualization to High-Dimensional Analyses. Cancers (Basel) 11, (2019).
4. Lauterbach, H. et al.  Mouse CD8alpha+ DCs and human BDCA3+ DCs are major producers of IFN-lambda in response to poly IC.  J. Exp. Med.  207, 2703–2717 (2010).
5. Crozat, K. et al.  The XC chemokine receptor 1 is a conserved selective marker of mammalian cells homologous to mouse CD8α+ dendritic cells.  Journal of Experimental Medicine 207, 1283–1292 (2010).
6. L Jongbloed, S. et al.  Human CD141+ (BDCA-3)+ dendritic cells (DCs) represent a unique myeloid DC subset that cross-presents necrotic cell antigens.  The Journal of experimental medicine 207, 1247–60 (2010).
7. Bachem, A. et al.  Superior antigen cross-presentation and XCR1 expression define human CD11c+ CD141+ cells as homologues of mouse CD8+ dendritic cells.  Journal of Experimental Medicine jem. 20100348 (2010).
8. Diamond, M. S. et al.  Type I interferon is selectively required by dendritic cells for immune rejection of tumors.  208, 1989–2003 (2011).
9. Woo, S.-R. et al.  STING-Dependent Cytosolic DNA Sensing Mediates Innate Immune Recognition of Immunogenic Tumors.  Immunity 41, 830–842 (2014).
10. Deng, L. et al.  STING-Dependent Cytosolic DNA Sensing Promotes Radiation-Induced Type I Interferon-Dependent Antitumor Immunity in Immunogenic Tumors.  Immunity 41, 843–852 (2014).
11. Zitvogel, L., Galluzzi, L., Kepp, O., Smyth, M. J. & Kroemer, G. Type I interferons in anticancer immunity.             Nature Reviews Immunology 15, nri3845 (2015).
12. Hubert, M. et al.  IFN-III is selectively produced by cDC1 and predicts good clinical outcome in breast cancer.  Sci Immunol 5, (2020).
13. Barry, K. C. et al.  A natural killer-dendritic cell axis defines checkpoint therapy-responsive tumor microenvironments.  Nat. Med.  24, 1178–1191 (2018).
14. Dunn, G. P. et al.  A critical function for type I interferons in cancer immunoediting.  Nat Immunol 6, 722–729 (2005).
15. Fuertes, M. B., Woo, S.-R., Burnett, B., Fu, Y.-X. & Gajewski, T. F. Type I interferon response and innate immune sensing of cancer. Trends Immunol.  34, 67–73 (2013).