Joshua M. Farber

Noninvasive monitoring of immune responses is important for increasing the efficacy of cancer immunotherapy. Although several approaches exist, few methods directly report on T-cell activation. We aimed to develop a novel PET probe targeting C-X-C motif chemokine ligand 9 (CXCL9), a chemokine specifically induced by interferon gamma (IFN-γ), a cytokine that is produced by activated T cells and group 1 innate lymphoid cells. CXCL9 binds to glycosaminoglycans, which are expressed on cell surfaces and the extracellular matrix, to recruit or position cells in the tumor microenvironment. In cancers, expression of CXCL9 has been associated with improved survival and predicts and reflects responses to immunotherapy. The pronounced upregulation of CXCL9 by IFN-γ and CXCL9's extracellular accessibility and site-specific accumulation make it a compelling biomarker for detecting T-cell activation. Methods: We developed a PET tracer targeting CXCL9 based on a high-affinity, antihuman CXCL9 nanobody (h2A12), which was isolated from a llama-derived phage display library and labeled with ¹⁸F. The tracer was evaluated in cell culture, a subcutaneous xenograft model, and a humanized mouse model of T-cell engager therapy. Results: The h2A12 nanobody demonstrated high specificity for human CXCL9 (K _d, 4.07 ± 0.44 nM) with no cross-reactivity to related chemokines. In xenograft models, [¹⁸F]F-h2A12 showed significant uptake in CXCL9-expressing tumors (10.33 ± 1.23 %IA/g) compared with control tumors (0.25 ± 0.04 %IA/g) at 2 h after injection, with excellent tumor-to-background ratios. In the humanized model, T-cell engager therapy induced CXCL9 expression that peaked at day 7, corresponding with increased [¹⁸F]F-h2A12 uptake in treated tumors (2.61 ± 0.50 %IA/g) versus controls (0.67 ± 0.12 %IA/g). Conclusion: [¹⁸F]F-h2A12 PET imaging enables noninvasive visualization of CXCL9 expression as a biomarker of immune activation. This approach offers potential applications in monitoring immunotherapy responses and studying immune-mediated diseases.

Human Th17/type 17 cells express the chemokine receptor CCR6, but the functions of CCR6 and other chemokine receptors in human type 17 Th cell extravasation have not been fully delineated. Here we show that human peripheral blood CD4⁺CCR6⁺ T cells co-expressing CCR2 have a pathogenic Th17 signature, can produce inflammatory cytokines without T cell receptor activation, and show enhanced expression of pathogenicity-associated and activation-associated genes in the cerebrospinal fluid of patients with multiple sclerosis as compared to controls. In flow chambers with activated endothelial cell (EC) monolayers, CD4⁺CCR6⁺CCR2⁺ T cells are efficient at transendothelial migration (TEM). Ligands for CCR5, CCR6 and CXCR3 localize to EC surfaces and mediate only arrest, whereas CCR2 ligands fail to bind well to ECs and mediate only TEM. Conversely, expressing a chimeric CCR2 ligand engineered to bind glycosaminoglycans on ECs results in CCR2-mediated arrest but blocks TEM induction. Our results from human pathogenic-like type 17 cells thus suggest that T cell migration arrest requires chemokine bound to EC surfaces, whereas TEM requires a transendothelial chemokine gradient.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of the coronavirus disease 2019 (COVID-19) pandemic, remains endemic worldwide ∼5 years since the first documented case. Severe COVID-19 is widely considered to be caused by a dysregulated immune response to SARS-CoV-2 within the respiratory tract. Circulating levels of the chemokine CXCL10 are strongly positively associated with poor outcome; however, its precise role in pathogenesis and its suitability as a therapeutic target have remained undefined. Here, we challenged 4-6 month old C57BL/6 mice genetically deficient in Cxcl10 with a mouse-adapted strain of SARS-CoV-2. Infected male, but not female, Cxcl10 ^-/- mice displayed increased mortality compared to wild type controls. Histopathological damage, inflammatory gene induction and virus load in the lungs of male mice 4 days post infection and before death were not broadly influenced by Cxcl10 deficiency. However, accumulation of B cells and both CD4 ⁺ and CD8 ⁺ T cells in the lung parenchyma of infected mice was reduced in the absence of Cxcl10. Thus, during acute SARS-CoV-2 infection, Cxcl10 regulates lymphocyte infiltration in the lung and confers protection against mortality. Our preclinical model results do not support targeting CXCL10 therapeutically in severe COVID-19.

Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related Th cells (type 17 cells) and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo. By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The phenotypes and epigenomes of CCR6+ cells are stable across cell divisions under noninflammatory conditions. Nonetheless, activation in polarizing and nonpolarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the type 17 continuum to yield the unusual plasticity ascribed to type 17 cells.

Pro-inflammatory T cells co-express multiple chemokine receptors, but the distinct functions of individual receptors on these cells are largely unknown. Human Th17 cells uniformly express the chemokine receptor CCR6, and we discovered that the subgroup of CD4 ⁺ CCR6 ⁺ cells that co-express CCR2 possess a pathogenic Th17 signature, can produce inflammatory cytokines independent of TCR activation, and are unusually efficient at transendothelial migration (TEM). The ligand for CCR6, CCL20, was capable of binding to activated endothelial cells (ECs) and inducing firm arrest of CCR6 ⁺ CCR2 ⁺ cells under conditions of flow - but CCR6 could not mediate TEM. By contrast, CCL2 and other ligands for CCR2, despite being secreted from both luminal and basal sides of ECs, failed to bind to the EC surfaces - and CCR2 could not mediate arrest. Nonetheless, CCR2 was required for TEM. To understand if CCR2’s inability to mediate arrest was due solely to an absence of EC-bound ligands, we generated a CCL2-CXCL9 chimeric chemokine that could bind to the EC surface. Although display of CCL2 on the ECs did indeed lead to CCR2-mediated arrest of CCR6 ⁺ CCR2 ⁺ cells, activating CCR2 with surface-bound CCL2 blocked TEM. We conclude that mediating arrest and TEM are mutually exclusive activities of chemokine receptors and/or their ligands that depend, respectively, on chemokines that bind to the EC luminal surfaces versus non-binding chemokines that form transendothelial gradients under conditions of flow. Our findings provide fundamental insights into mechanisms of lymphocyte extravasation and may lead to novel strategies to block or enhance their migration into tissue.

Th17 cells have been investigated in mice primarily for their contributions to autoimmune diseases. However, the pathways of differentiation of Th17 and related (type 17) cells and the structure of the type 17 memory population in humans are not well understood; such understanding is critical for manipulating these cells in vivo . By exploiting differences in levels of surface CCR6, we found that human type 17 memory cells, including individual T cell clonotypes, form an elongated continuum of type 17 character along which cells can be driven by increasing RORγt. This continuum includes cells preserved within the memory pool with potentials that reflect the early preferential activation of multiple over single lineages. The CCR6 ⁺ cells’ phenotypes and epigenomes are stable across cell divisions under homeostatic conditions. Nonetheless, activation in polarizing and non-polarizing conditions can yield additional functionalities, revealing, respectively, both environmentally induced and imprinted mechanisms that contribute differentially across the continuum to yield the unusual plasticity ascribed to type 17 cells.

Human MAIT cells show little expression of the selectin CD62L and the chemokine receptor CCR7, which are important for entering lymph nodes, and high expression of selectin ligands and chemokine receptors that mediate trafficking into inflamed tissue. Extravasation of leukocytes into tissue requires sequential steps including rolling, firm arrest, crawling, and transendothelial migration, and can be modeled using endothelial cell monolayers in flow chambers that approximate the sheer stress found in post-capillary venules. Using MAIT cells purified from elutriated lymphocytes by fluorescence-activated cell sorting, we have used flow chambers to demonstrate roles for individual chemokine receptors in specific steps required for extravasation. These methods provide a general way to study the molecular mechanisms underlying MAIT cell trafficking from blood into tissue.

Genome‐wide association studies have found that polymorphisms in genes for IL‐23 and its receptor are important in psoriasis, and blocking IL‐23 is an effective therapy in the disease. The use of Aldara^™, a cream that contains the TLR7 and TLR8 agonist imiquimod (IMQ), was found to exacerbate psoriasis in some patients with pre‐existing disease. Intradermal injections of IL‐23 and topical application of Aldara/IMQ induce skin inflammation in mice with features similar to psoriasis—including epidermal hyperplasia and accumulation of inflammatory cells in epidermis and dermis—which is mediated by IL‐17A, IL‐22, and other factors implicated in the human disease. Consequently, these models can be used in preclinical studies to investigate the molecular and cellular pathogenesis of psoriasis, as well as in the evaluation of potential therapies. This article provides detailed methodologies for creating and evaluating the IL‐23‐ and Aldara/IMQ‐induced mouse models of psoriasis. The article also provides a protocol for analyzing skin leukocytes by flow cytometry. © 2019 by John Wiley & Sons, Inc.

Many mediators and regulators of extravasation by bona fide human memory-phenotype T cells remain undefined. Mucosal-associated invariant T (MAIT) cells are innate-like, antibacterial cells that we found excelled at crossing inflamed endothelium. They displayed abundant selectin ligands, with high expression of FUT7 and ST3GAL4, and expressed CCR6, CCR5, and CCR2, which played non-redundant roles in trafficking on activated endothelial cells. MAIT cells selectively expressed CCAAT/enhancer-binding protein delta (C/EBPδ). Knockdown of C/EBPδ diminished expression of FUT7, ST3GAL4 and CCR6, decreasing MAIT cell rolling and arrest, and consequently the cells’ ability to cross an endothelial monolayer in vitro and extravasate in mice. Nonetheless, knockdown of C/EBPδ did not affect CCR2, which was important for the step of transendothelial migration. Thus, MAIT cells demonstrate a program for extravasastion that includes, in part, C/EBPδ and C/EBPδ-regulated genes, and that could be used to enhance, or targeted to inhibit T cell recruitment into inflamed tissue.