SUMMARY Differentiation of naïve CD4+ T cells into T helper (Th) cells is a defining event in adaptive immunity. The cytokines and transcription factors that control Th cell differentiation are understood, however it is not known how this process is orchestrated within lymph nodes (LNs). Here we have shown that the CXCR3 chemokine receptor was required for optimal generation of interferon (IFN)-γ secreting Th1 cells in vivo. Using a CXCR3 ligand reporter mouse, we found that stromal cells predominately expressed the chemokine ligand CXCL9 while hematopoietic cells expressed CXCL10 in LNs. Dendritic cell (DC)-derived CXCL10 facilitated T cell-DC interactions in LNs during T cell priming while both chemokines guided intranodal positioning of CD4+ T cells to interfollicular and medullary zones. Thus, different chemokines acting on the same receptor can function locally to facilitate DC-T cell interactions and globally to influence intranodal positioning, and that both functions contribute to Th1 cell differentiation.

Alterations to the tumor microenvironment following localized irradiation may influence the effectiveness of subsequent immunotherapy. The objective of this study was to determine how IFN-γ influences the inflammatory response within this dynamic environment following radiotherapy. B16/OVA melanoma cells were implanted into C57BL/6 (wild-type (WT)) and IFN-γ-deficient (IFN-γ−/−) mice. Seven days after implantation, mice received 15 Gy of localized tumor irradiation and were assessed 7 days later. Irradiation up-regulated the expression of VCAM-1 on the vasculature of tumors grown in WT but not in IFN-γ−/− mice. Levels of the IFN-γ-inducible chemokines MIG and IFN-γ-inducible protein 10 were decreased in irradiated tumors from IFN-γ−/− mice compared with WT. In addition to inducing molecular cues necessary for T cell infiltration, surface MHC class I expression is also up-regulated in response to IFN-γ produced after irradiation. The role of IFN-γ signaling in tumor cells on class I expression was tested using B16/OVA cells engineered to overexpress a dominant negative mutant IFN-γ receptor (B16/OVA/DNM). Following implantation and treatment, expression of surface class I on tumor cells in vivo was increased in B16/OVA, but not in B16/OVA/DNM tumors, suggesting IFN-γ acts directly on tumor cells to induce class I up-regulation. These increases in MHC class I expression correlated with greater levels of activated STAT1. Thus, IFN-γ is instrumental in creating a tumor microenvironment conducive for T cell infiltration and tumor cell target recognition.

Messenger RNA (mRNA) represents an attractive therapeutic modality for potentially a wide range of clinical indications but requires uridine chemistry modification and/or tuning of the production process to prevent activation of cellular innate immune sensors and a concomitant reduction in protein expression. To decipher the relative contributions of these factors on immune activation, here, we compared, in multiple cell and in vivo models, mRNA that encodes human erythropoietin incorporating either canonical uridine or N1-methyl-pseudouridine (1mΨ), synthesized by either a standard process shown to have double-stranded RNA (dsRNA) impurities or a modified process that yields a highly purified mRNA preparation. Our data demonstrate that the lowest stimulation of immune endpoints was with 1mΨ made by the modified process, while mRNA containing canonical uridine was immunostimulatory regardless of process. These findings confirm that uridine modification and the reduction of dsRNA impurities are both necessary and sufficient at controlling the immune-activating profile of therapeutic mRNA.

Highlights d CXCR2 controls neutrophil recruitment into the brain in HSV-1 encephalitis d Cxcr2 À/À mice have decreased BBB permeability and improved outcome in HSV-1 infection d CXCL1 is produced by both astrocytes and neurons in response to IL-1a d CXCL1-CXCR2 is required for neutrophil transendothelial migration into the brain