Multiplex immunofluorescence-guided laser capture microdissection for spatial transcriptomics of metastatic melanoma tissues.

We describe a pipeline for optimized and streamlined multiplexed immunofluorescence-guided laser capture microdissection allowing the harvest of individual cells based on their phenotype and tissue localization for transcriptomic analysis with next-generation RNA sequencing. Here, we analyze transcriptomes of CD3+ T cells, CD14+ monocytes/macrophages, and melanoma cells in non-dissociated metastatic melanoma tissue. While this protocol is described for melanoma tissues, we successfully applied it to human tonsil, skin, and breast cancer tissues as well as mouse lung tissues. For complete details on the use and execution of this protocol, please refer to Martinek et al. (2022)

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

Protocol for establishing primary human lung organoid-derived air-liquid interface cultures from cryopreserved human lung tissue.

Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coronavirus 2 infection in these cultures as an example of their utility. For complete details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (2023)

Human KIT+ myeloid cells facilitate visceral metastasis by melanoma.

Metastasis of melanoma significantly worsens prognosis; thus, therapeutic interventions that prevent metastasis could improve patient outcomes. Here, we show using humanized mice that colonization of distant visceral organs with melanoma is dependent upon a human CD33+CD11b+CD117+ progenitor cell subset comprising \u3c4% of the human CD45+ leukocytes. Metastatic tumor-infiltrating CD33+ cells from patients and humanized (h)NSG-SGM3 mice showed converging transcriptional profiles. Single-cell RNA-seq analysis identified a gene signature of a KIT/CD117-expressing CD33+ subset that correlated with decreased overall survival in a TCGA melanoma cohort. Thus, human CD33+CD11b+CD117+ myeloid cells represent a novel candidate biomarker as well as a therapeutic target for metastatic melanoma

Transcriptional profiling of macrophages in situ in metastatic melanoma reveals localization-dependent phenotypes and function.

Modulation of immune function at the tumor site could improve patient outcomes. Here, we analyze patient samples of metastatic melanoma, a tumor responsive to T cell-based therapies, and find that tumor-infiltrating T cells are primarily juxtaposed to CD14+ monocytes/macrophages rather than melanoma cells. Using immunofluorescence-guided laser capture microdissection, we analyze transcriptomes of CD3+ T cells, CD14 + monocytes/macrophages, and melanoma cells in non-dissociated tissue. Stromal CD14+ cells display a specific transcriptional signature distinct from CD14+ cells within tumor nests. This signature contains LY75, a gene linked with antigen capture and regulation of tolerance and immunity in dendritic cells (DCs). When applied to TCGA cohorts, this gene set can distinguish patients with significantly prolonged survival in metastatic cutaneous melanoma and other cancers. Thus, the stromal CD14+ cell signature represents a candidate biomarker and suggests that reprogramming of stromal macrophages to acquire DC function may offer a therapeutic opportunity for metastatic cancers

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

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. J Immunol 2014 Nov 1; 193(9):4335-43

Toxoplasma gondii-Infected Human Myeloid Dendritic Cells Induce T-Lymphocyte Dysfunction and Contact-Dependent Apoptosis

Dendritic cells ignite adaptive immunity by priming naïve T lymphocytes. Human monocyte-derived dendritic cells (MDDCs) infected with Toxoplasma gondii induce T-lymphocyte gamma interferon production and may thus activate T. gondii-specific immunity. However, we now demonstrate that T. gondii-infected MDDCs are poor at activating T lymphocytes and are unable to induce specific cytotoxic T lymphocytes. On the other hand, MDDCs acquiring nonviable T. gondii antigens directly, or indirectly through captured apoptotic or necrotic cell bodies, induce potent T-lymphocyte activation. T lymphocytes exposed to infected MDDCs are significantly impaired in upregulation of CD69 and CD28, are refractory to activation, and die through contact-dependent apoptosis mediated by an as-yet-unidentified mechanism not requiring Fas, tumor necrosis factor-related apoptosis-inducing ligand, leukocyte function antigen 1, intercellular adhesion molecule 1, tumor necrosis factor alpha, interleukin 10, alpha interferon, gamma interferon, prostaglandins, or reactive nitrogen intermediates. Bystander T lymphocytes that were neither infected nor apoptotic were refractory to activation, suggesting global dysfunction. Immunosuppression and T-lymphocyte unresponsiveness and apoptosis are typical of acute T. gondii infection. Our data suggest that infected dendritic cells contribute to these processes. On the other hand, host cells infected with T. gondii are resistant to multiple inducers of apoptosis. Thus, regulation of host cell and bystander cell apoptosis by viable T. gondii may be significant components of a strategy to evade immunity and enhance intracellular parasite survival