Florentina Marches

Development and function of human innate immune cells in a humanized mouse model

Mice repopulated with human hematopoietic cells are a powerful tool for the study of human hematopoiesis and immune function in vivo. However, existing humanized mouse models cannot support development of human innate immune cells, including myeloid cells and natural killer (NK) cells. Here we describe two mouse strains called MITRG and MISTRG, in which human versions of four genes encoding cytokines important for innate immune cell development are knocked into their respective mouse loci. The human cytokines support the development and function of monocytes, macrophages and NK cells derived from human fetal liver or adult CD34+ progenitor cells injected into the mice. Human macrophages infiltrated a human tumor xenograft in MITRG and MISTRG mice in a manner resembling that observed in tumors obtained from human patients. This humanized mouse model may be used to model the human immune system in scenarios of health and pathology, and may enable evaluation of therapeutic candidates in an in vivo setting relevant to human physiology.

Breast cancer instructs dendritic cells to prime interleukin 13–secreting CD4+ T cells that facilitate tumor development

We previously reported (Bell, D., P. Chomarat, D. Broyles, G. Netto, G.M. Harb, S. Lebecque, J. Valladeau, J. Davoust, K.A. Palucka, and J. Banchereau. 1999. J. Exp. Med. 190: 1417–1426) that breast cancer tumors are infiltrated with mature dendritic cells (DCs), which cluster with CD4+ T cells. We now show that CD4+ T cells infiltrating breast cancer tumors secrete type 1 (interferon γ) as well as high levels of type 2 (interleukin [IL] 4 and IL-13) cytokines. Immunofluorescence staining of tissue sections revealed intense IL-13 staining on breast cancer cells. The expression of phosphorylated signal transducer and activator of transcription 6 in breast cancer cells suggests that IL-13 actually delivers signals to cancer cells. To determine the link between breast cancer, DCs, and CD4+ T cells, we implanted human breast cancer cell lines in nonobese diabetic/LtSz-scid/scid β2 microglobulin–deficient mice engrafted with human CD34+ hematopoietic progenitor cells and autologous T cells. There, CD4+ T cells promote early tumor development. This is dependent on DCs and can be partially prevented by administration of IL-13 antagonists. Thus, breast cancer targets DCs to facilitate its development.

Thymic stromal lymphopoietin fosters human breast tumor growth by promoting type 2 inflammation

TSLP released from human breast cancer cells promotes OX40L expression on DCs, and these OX40L-expressing DCs drive development of inflammatory Th2 cells which promote breast tumor development.

Macrophage-Derived Dendritic Cells Have Strong Th1-Polarizing Potential Mediated by β-Chemokines Rather Than IL-12

Monocyte-derived dendritic cells (MDDCs) activate naive T lymphocytes to induce adaptive immunity, effecting Th1 polarization through IL-12. However, little is known about other potential DC Th1 polarizing mechanisms, or how T cell polarization may be affected by DCs differentiating in, or exposed to, a proinflammatory environment. Macrophages (MΦs) are DC precursors abundant in inflamed tissues, lymph nodes, and tumors. Thus we studied the T cell-activating and -polarizing properties of MΦ-derived DCs (ΦDCs). Monocytes were cultured in MΦ-CSF (M-CSF) to produce MΦs, which were then differentiated into DCs following culture with GM-CSF plus IL-4. ΦDCs activated a significant allogeneic MLR and were significantly better than MDDCs in activating T cells with superantigen. Most strikingly, ΦDCs elicited up to 9-fold more IFN-γ from naive or Ag-specific T cells compared with MDDCs (with equivalent IL-4 secretion), despite producing up to 9-fold less IL-12. Neutralization of MDDC, but not ΦDC IL-12 significantly inhibited T cell IFN-γ induction. ΦDCs produced up to 12-fold more β-chemokines (macrophage-inflammatory protein-1α, -1β, and RANTES) than MDDCs. Ab blockade of CCR5, but not CXC chemokine receptor 4, inhibited T cell IFN-γ induction by ΦDCs significantly greater than by MDDCs. Thus DCs differentiating from MΦs induce T cell IFN-γ through β-chemokines with little or no requirement for IL-12. Myeloid DCs arising from distinct precursor cells may have differing properties, including different mechanisms of Th1 polarization. These data are the first reports of IFN-γ induction through chemokines by DCs.

Broad influenza-specific CD8+ T-cell responses in humanized mice vaccinated with influenza virus vaccines

The development of novel human vaccines would be greatly facilitated by the development of in vivo models that permit preclinical analysis of human immune responses. Here, we show that nonobese diabetic severe combined immunodeficiency (NOD/SCID) beta(2) microglobulin(-/-) mice, engrafted with human CD34+ hematopoietic progenitors and further reconstituted with T cells, can mount specific immune responses against influenza virus vaccines. Live attenuated trivalent influenza virus vaccine induces expansion of CD8+ T cells specific to influenza matrix protein (FluM1) and nonstructural protein 1 in blood, spleen, and lungs. On ex vivo exposure to influenza antigens, antigen-specific CD8+ T cells produce IFN-gamma and express cell-surface CD107a. FluM1-specific CD8+ T cells can be also expanded in mice vaccinated with inactivated trivalent influenza virus vaccine. Expansion of antigen-specific CD8+ T cells is dependent on reconstitution of the human myeloid compartment. Thus, this humanized mouse model permits preclinical testing of vaccines designed to induce cellular immunity, including those against influenza virus. Furthermore, this work sets the stage for systematic analysis of the in vivo functions of human DCs. This, in turn, will allow a new approach to the rational design and preclinical testing of vaccines that cannot be tested in human volunteers.

Pyridinylimidazole p38 mitogen-activated protein kinase inhibitors block intracellular Toxoplasma gondii replication

Toxoplasma gondii is a medically important, obligate intracellular parasite. Little is known regarding factors that regulate its replication within cells. Such knowledge would further understanding of T. gondii pathogenesis, and might lead to novel therapeutic strategies. Mitogen-activated protein kinases (MAPKs) govern diverse cellular processes including proliferation and differentiation. We now show that treatment of T. gondii-infected cells with SB203580 or SB202190, substituted pyridinylimidazoles that are potent inhibitors of human p38 MAPK, inhibits intracellular T. gondii replication. Several independent experimental approaches suggest that the anti-proliferative effects of pyridinylimidazoles depend on direct action on tachyzoites, not the host cell: (i) selective inhibition of host p38 MAPK using recombinant adenoviruses had little effect on tachyzoite replication, (ii) pyridinylimidazole-treated tachyzoites developed abnormal morphology suggesting defective parasite division, and (iii) pyridinylimidazole-resistant mutant tachyzoites were developed through culture in progressively higher drug concentrations. We hypothesise that pyridinylimidazoles target a human p38 MAPK homologue in tachyzoites that regulates their replication. Phylogenetic data suggest that T. gondii likely encodes a p38 MAPK homologue, but such a homologue is absent from the incomplete Toxoplasma genomic data base. As all eukaryotic pathogens, including agents of malaria, leishmaniasis and trypanosomiasis encode endogenous MAPKs, drugs inhibiting endogenous MAPK activation may represent a novel, potentially broadly-acting class of anti-parasitic agents. Pyridinylimidazoles also represent tools to elucidate factors governing intracellular tachyzoite replication.

Human CD141+ Dendritic Cells Induce CD4+ T Cells To Produce Type 2 Cytokines

Dendritic cells (DCs) play the central role in the priming of naive T cells and the differentiation of unique effector T cells. In this study, using lung tissues and blood from both humans and humanized mice, we analyzed the response of human CD1c+ and CD141+ DC subsets to live-attenuated influenza virus. Specifically, we analyzed the type of CD4+ T cell immunity elicited by live-attenuated influenza virus–exposed DCs. Both DC subsets induce proliferation of allogeneic naive CD4+ T cells with the capacity to secrete IFN-γ. However, CD141+ DCs are uniquely able to induce the differentiation of IL-4– and IL-13–producing CD4+ T cells. CD141+ DCs induce IL-4– and IL-13–secreting CD4+ T cells through OX40 ligand. Thus, CD141+ DCs demonstrate remarkable plasticity in guiding adaptive immune responses.

Reprogramming Tumor-Infiltrating Dendritic Cells for CD103+CD8+ Mucosal T-cell Differentiation and Breast Cancer Rejection.

Wu and colleagues show that intratumoral delivery of dectin-1 ligand curdlan in a humanized mouse model of breast cancer reprograms dendritic cells to induce Th1 cytokine production as well as expansion and accumulation of CD103+CD8+ mucosal T cells in tumors, leading to cancer rejection. Our studies showed that tumor-infiltrating dendritic cells (DC) in breast cancer drive inflammatory Th2 (iTh2) cells and protumor inflammation. Here, we show that intratumoral delivery of the β-glucan curdlan, a ligand of dectin-1, blocks the generation of iTh2 cells and prevents breast cancer progression in vivo. Curdlan reprograms tumor-infiltrating DCs via the ligation of dectin-1, enabling the DCs to become resistant to cancer-derived thymic stromal lymphopoietin (TSLP), to produce IL-12p70, and to favor the generation of Th1 cells. DCs activated via dectin-1, but not those activated with TLR-7/8 ligand or poly I:C, induce CD8+ T cells to express CD103 (αE integrin), a ligand for cancer cells, E-cadherin. Generation of these mucosal CD8+ T cells is regulated by DC-derived integrin αvβ8 and TGF-β activation in a dectin-1–dependent fashion. These CD103+CD8+ mucosal T cells accumulate in the tumors, thereby increasing cancer necrosis and inhibiting cancer progression in vivo in a humanized mouse model of breast cancer. Importantly, CD103+CD8+ mucosal T cells elicited by reprogrammed DCs can reject established cancer. Thus, reprogramming tumor-infiltrating DCs represents a new strategy for cancer rejection. Cancer Immunol Res; 2(5); 487–500. ©2014 AACR.

CHAPTER 7 – Isolation, culture and propagation of dendritic cells

Monocyte-derived dendritic cells (MDDCs) are the most commonly used type of human DC, but are only rarely prepared from mice largely because the yield is small, and there are other easier methods available to produce mouse DCs. Blood is obtained by cardiac puncture. PBMCs are separated by density gradient centrifugation and plated at 8–10 million cells/well in a six-well plate. The mouse peritoneal cavity harbors a small resident population of myeloid cells that are generally regarded as being macrophages. Peritoneal contents are recovered by gentle lavage. Mature mouse DCs will differentiate from fetal liver progenitor cells using a stromal cell feeder layer, GM-CSF, Flt-3L, and SCF to induce differentiation, followed by GM-CSF plus TNFα to induce maturation. DCs isolated from mouse myeloma or GM-CSF and CD40L-transduced colon carcinoma cells will activate tumor-specific T cells without exogenous antigen loading.

IL1 Receptor Antagonist Controls Transcriptional Signature of Inflammation in Patients with Metastatic Breast Cancer

Inflammation affects tumor immune surveillance and resistance to therapy. Here, we show that production of IL1β in primary breast cancer tumors is linked with advanced disease and originates from tumor-infiltrating CD11c+ myeloid cells. IL1β production is triggered by cancer cell membrane-derived TGFβ. Neutralizing TGFβ or IL1 receptor prevents breast cancer progression in humanized mouse model. Patients with metastatic HER2- breast cancer display a transcriptional signature of inflammation in the blood leukocytes, which is attenuated after IL1 blockade. When present in primary breast cancer tumors, this signature discriminates patients with poor clinical outcomes in two independent public datasets (TCGA and METABRIC).Significance: IL1β orchestrates tumor-promoting inflammation in breast cancer and can be targeted in patients using an IL1 receptor antagonist. Cancer Res; 78(18); 5243-58. ©2018 AACRSee related commentary by Dinarello, p. 5200.