Macrophage migration inhibitory factor blockade reprograms macrophages and disrupts prosurvival signaling in acute myeloid leukemia.

The malignant microenvironment plays a major role in the development of resistance to therapies and the occurrence of relapses in acute myeloid leukemia (AML). We previously showed that interactions of AML blasts with bone marrow macrophages (MΦ) shift their polarization towards a protumoral (M2-like) phenotype, promoting drug resistance; we demonstrated that inhibiting the colony-stimulating factor-1 receptor (CSF1R) repolarizes MΦ towards an antitumoral (M1-like) phenotype and that other factors may be involved. We investigated here macrophage migration inhibitory factor (MIF) as a target in AML blast survival and protumoral interactions with MΦ. We show that pharmacologically inhibiting MIF secreted by AML blasts results in their apoptosis. However, this effect is abrogated when blasts are co-cultured in close contact with M2-like MΦ. We next demonstrate that pharmacological inhibition of MIF secreted by MΦ, in the presence of granulocyte macrophage-colony stimulating factor (GM-CSF), efficiently reprograms MΦ to an M1-like phenotype that triggers apoptosis of interacting blasts. Furthermore, contact with reprogrammed MΦ relieves blast resistance to venetoclax and midostaurin acquired in contact with CD163+ protumoral MΦ. Using intravital imaging in mice, we also show that treatment with MIF inhibitor 4-IPP and GM-CSF profoundly affects the tumor microenvironment in vivo: it strikingly inhibits tumor vasculature, reduces protumoral MΦ, and slows down leukemia progression. Thus, our data demonstrate that MIF plays a crucial role in AML MΦ M2-like protumoral phenotype that can be reversed by inhibiting its activity and suggest the therapeutic targeting of MIF as an avenue towards improved AML treatment outcomes.

Identification of a Kupffer cell subset capable of reverting the T cell dysfunction induced by hepatocellular priming.

Kupffer cells (KCs) are highly abundant, intravascular, liver-resident macrophages known for their scavenger and phagocytic functions. KCs can also present antigens to CD8+ T cells and promote either tolerance or effector differentiation, but the mechanisms underlying these discrepant outcomes are poorly understood. Here, we used a mouse model of hepatitis B virus (HBV) infection, in which HBV-specific naive CD8+ T cells recognizing hepatocellular antigens are driven into a state of immune dysfunction, to identify a subset of KCs (referred to as KC2) that cross-presents hepatocellular antigens upon interleukin-2 (IL-2) administration, thus improving the antiviral function of T cells. Removing MHC-I from all KCs, including KC2, or selectively depleting KC2 impaired the capacity of IL-2 to revert the T cell dysfunction induced by intrahepatic priming. In summary, by sensing IL-2 and cross-presenting hepatocellular antigens, KC2 overcome the tolerogenic potential of the hepatic microenvironment, suggesting new strategies for boosting hepatic T cell immunity.

Cross-dressing of CD8α+ Dendritic Cells with Antigens from Live Mouse Tumor Cells Is a Major Mechanism of Cross-priming.

Live cells are the most abundant sources of antigen in a tumor-bearing host. Here, we used live tumor cells as source of antigens to investigate the mechanism underlying their immunogenicity in murine tumor models. The live tumor cells were significantly more immunogenic than irradiated or apoptotic tumor cells. We examined the interaction of live and apoptotic tumor cells with major subsets of antigen-presenting cells, i.e., CD8α+ dendritic cells (DC), CD8α- DCs, plasmacytoid DCs, and CD169+ macrophages at skin draining lymph nodes. The CD8α+ DCs captured cell-associated antigens from both live and apoptotic tumor cells, whereas CD169+ macrophages picked up cell-associated antigens mostly from apoptotic tumor cells. Trogocytosis and cross-dressing of membrane-associated antigenic material from live tumor cells to CD8α+ DCs was the primary mechanism for cross-priming of tumor antigens upon immunization with live cells. Phagocytosis of apoptotic tumor cells was the primary mechanism for cross-priming of tumor antigens upon immunization with apoptotic or irradiated cells. These findings clarify the mechanism of cross-priming of cancer antigens by DCs, allowing for a greater understanding of antitumor immune responses.

Dynamics and genomic landscape of CD8+ T cells undergoing hepatic priming.

The responses of CD8+ T cells to hepatotropic viruses such as hepatitis B range from dysfunction to differentiation into effector cells, but the mechanisms that underlie these distinct outcomes remain poorly understood. Here we show that priming by Kupffer cells, which are not natural targets of hepatitis B, leads to differentiation of CD8+ T cells into effector cells that form dense, extravascular clusters of immotile cells scattered throughout the liver. By contrast, priming by hepatocytes, which are natural targets of hepatitis B, leads to local activation and proliferation of CD8+ T cells but not to differentiation into effector cells; these cells form loose, intravascular clusters of motile cells that coalesce around portal tracts. Transcriptomic and chromatin accessibility analyses reveal unique features of these dysfunctional CD8+ T cells, with limited overlap with those of exhausted or tolerant T cells; accordingly, CD8+ T cells primed by hepatocytes cannot be rescued by treatment with anti-PD-L1, but instead respond to IL-2. These findings suggest immunotherapeutic strategies against chronic hepatitis B infection.

Visualizing Endogenous Effector T Cell Egress from the Lymph Nodes.

Local anatomy of lymphoid tissues during infection has emerged as a critical regulator of immunity; thus, studying the cellular choreography in the context of an intact tissue environment in situ is crucial. Following an infection, the local pathogen-specific T cell migration and the subsequent egress of effector T cells from the draining lymph nodes are important and complex biological processes. The mechanisms that regulate this complex process can now be investigated by directly visualizing T cell dynamics in vivo using intravital two-photon (2P) microscopy. In addition, static whole-mount imaging technique can provide us with a comprehensive assessment of global changes in the distribution of cellular populations within an intact tissue. Thus, in this chapter, we detail methods to visualize the migration and egress of endogenous antigen-specific CD8 T cells following viral infection using two methods-intravital 2P microscopy and multicolor whole-mount in situ tetramer staining.

T cell-intrinsic S1PR1 regulates endogenous effector T-cell egress dynamics from lymph nodes during infection.

Viral clearance requires effector T-cell egress from the draining lymph node (dLN). The mechanisms that regulate the complex process of effector T-cell egress from the dLN after infection are poorly understood. Here, we visualized endogenous pathogen-specific effector T-cell migration within, and from, the dLN. We used an inducible mouse model with a temporally disrupted sphingosine-1-phosphate receptor-1 (S1PR1) gene specifically in endogenous effector T cells. Early after infection, WT and S1PR1(-/-) effector T cells localized exclusively within the paracortex. This localization in the paracortex by CD8 T cells was followed by intranodal migration by both WT and S1PR1(-/-) T cells to positions adjacent to both cortical and medullary lymphatic sinuses where the T cells exhibited intense probing behavior. However, in contrast to WT, S1PR1(-/-) effector T cells failed to enter the sinuses. We demonstrate that, even when LN retention signals such as CC chemokine receptor 7 (CCR7) are down-regulated, T cell intrinsic S1PR1 is the master regulator of effector T-cell emigration from the dLN.

CD8 T Cells Enter the Splenic T Cell Zones Independently of CCR7, but the Subsequent Expansion and Trafficking Patterns of Effector T Cells after Infection Are Dysregulated in the Absence of CCR7 Migratory Cues.

CCR7 is an important chemokine receptor that regulates T cell trafficking and compartmentalization within secondary lymphoid organs. However, the T cell-intrinsic role of CCR7 during infection in the spleen is not well understood. This study was designed to understand how CCR7-dependent localization and migration of CD8(+) T cells in different compartments of the spleen affected the primary and recall responses after infection. To this end, we used adoptive transfer of naive Ag-specific CD8 T cells (OT-I) that either lacked CCR7 or constitutively expressed CCR7 (CD2-CCR7) in mice that were subsequently infected i.v. with Listeria monocytogenes. We show that naive CCR7(-/-)CD8(+) T cells failed to enter the T cell zone, whereas CD2-CCR7 OT-I cells were exclusively confined to the T cell zones of the spleen. Surprisingly, however, CCR7(-/-) OT-I cells entered the T cell zones after infection, but the entry and egress migratory pattern of these cells was dysregulated and very distinct compared with wild-type OT-I cells. Moreover, CCR7-deficient OT-I cells failed to expand robustly when compared with wild-type OT-I cells and were preferentially skewed toward a short-lived effector cell differentiation pattern. Interestingly, CCR7(-/-), CD2-CCR7, and wild-type OT-I memory cells responded equally well to rechallenge infection. These results highlight a novel role of CCR7 in regulating effector CD8 T cell migration in the spleen and demonstrate differential requirement of CCR7 for primary and secondary CD8 T cell responses to infection.

Visualizing T Cell Migration in situ.

Mounting a protective immune response is critically dependent on the orchestrated movement of cells within lymphoid tissues. The structure of secondary lymphoid organs regulates immune responses by promoting optimal cell-cell and cell-extracellular matrix interactions. Naïve T cells are initially activated by antigen presenting cells in secondary lymphoid organs. Following priming, effector T cells migrate to the site of infection to exert their functions. Majority of the effector cells die while a small population of antigen-specific T cells persists as memory cells in distinct anatomical locations. The persistence and location of memory cells in lymphoid and non-lymphoid tissues is critical to protect the host from re-infection. The localization of memory T cells is carefully regulated by several factors including the highly organized secondary lymphoid structure, the cellular expression of chemokine receptors and compartmentalized secretion of their cognate ligands. This balance between the anatomy and the ordered expression of cell surface and soluble proteins regulates the subtle choreography of T cell migration. In recent years, our understanding of cellular dynamics of T cells has been advanced by the development of new imaging techniques allowing in situ visualization of T cell responses. Here, we review the past and more recent studies that have utilized sophisticated imaging technologies to investigate the migration dynamics of naïve, effector, and memory T cells.

Symbiotic bacterial metabolites regulate gastrointestinal barrier function via the xenobiotic sensor PXR and Toll-like receptor 4.

Intestinal microbial metabolites are conjectured to affect mucosal integrity through an incompletely characterized mechanism. Here we showed that microbial-specific indoles regulated intestinal barrier function through the xenobiotic sensor, pregnane X receptor (PXR). Indole 3-propionic acid (IPA), in the context of indole, is a ligand for PXR in vivo, and IPA downregulated enterocyte TNF-α while it upregulated junctional protein-coding mRNAs. PXR-deficient (Nr1i2(-/-)) mice showed a distinctly "leaky" gut physiology coupled with upregulation of the Toll-like receptor (TLR) signaling pathway. These defects in the epithelial barrier were corrected in Nr1i2(-/-)Tlr4(-/-) mice. Our results demonstrate that a direct chemical communication between the intestinal symbionts and PXR regulates mucosal integrity through a pathway that involves luminal sensing and signaling by TLR4.