Nur77 controls tolerance induction, terminal differentiation, and effector functions in semi-invariant natural killer T cells

Semi-invariant natural killer T (iNKT) cells are self-reactive lymphocytes, yet how this lineage attains self-tolerance remains unknown. iNKT cells constitutively express high levels of Nr4a1-encoded Nur77, a transcription factor that integrates signal strength downstream of the T cell receptor (TCR) within activated thymocytes and peripheral T cells. The function of Nur77 in iNKT cells is unknown. Here we report that sustained Nur77 overexpression (Nur77^(tg)) in mouse thymocytes abrogates iNKT cell development. Introgression of a rearranged Vα14-Jα18 TCR-α chain gene into the Nur77^(tg) (Nur77^(tg);Vα14^(tg)) mouse rescued iNKT cell development up to the early precursor stage, stage 0. iNKT cells in bone marrow chimeras that reconstituted thymic cellularity developed beyond stage 0 precursors and yielded IL-4–producing NKT2 cell subset but not IFN-γ–producing NKT1 cell subset. Nonetheless, the developing thymic iNKT cells that emerged in these chimeras expressed the exhaustion marker PD1 and responded poorly to a strong glycolipid agonist. Thus, Nur77 integrates signals emanating from the TCR to control thymic iNKT cell tolerance induction, terminal differentiation, and effector functions

Nur77 controls tolerance induction, terminal differentiation, and effector functions in semi-invariant natural killer T cells

Semi-invariant natural killer T (iNKT) cells are self-reactive lymphocytes, yet how this lineage attains self-tolerance remains unknown. iNKT cells constitutively express high levels of Nr4a1-encoded Nur77, a transcription factor that integrates signal strength downstream of the T cell receptor (TCR) within activated thymocytes and peripheral T cells. The function of Nur77 in iNKT cells is unknown. Here we report that sustained Nur77 overexpression (Nur77^(tg)) in mouse thymocytes abrogates iNKT cell development. Introgression of a rearranged Vα14-Jα18 TCR-α chain gene into the Nur77^(tg) (Nur77^(tg);Vα14^(tg)) mouse rescued iNKT cell development up to the early precursor stage, stage 0. iNKT cells in bone marrow chimeras that reconstituted thymic cellularity developed beyond stage 0 precursors and yielded IL-4–producing NKT2 cell subset but not IFN-γ–producing NKT1 cell subset. Nonetheless, the developing thymic iNKT cells that emerged in these chimeras expressed the exhaustion marker PD1 and responded poorly to a strong glycolipid agonist. Thus, Nur77 integrates signals emanating from the TCR to control thymic iNKT cell tolerance induction, terminal differentiation, and effector functions

Rgs2 Mediates Pro-Angiogenic Function of Myeloid Derived Suppressor Cells in the Tumor Microenvironment via Upregulation of MCP-1

Tumor growth is intimately linked with stromal interactions. Myeloid derived suppressor cells (MDSCs) are dramatically elevated in cancer patients and tumor bearing mice. MDSCs modulate the tumor microenvironment through attenuating host immune response and increasing vascularization.In searching for molecular mediators responsible for pro-tumor functions, we found that regulator of G protein signaling-2 (Rgs2) is highly increased in tumor-derived MDSCs compared to control MDSCs. We further demonstrate that hypoxia, a common feature associated with solid tumors, upregulates the gene expression. Genetic deletion of Rgs2 in mice resulted in a significant retardation of tumor growth, and the tumors exhibit decreased vascular density and increased cell death. Interestingly, deletion of Rgs2 in MDSCs completely abolished their tumor promoting function, suggesting that Rgs2 signaling in MDSCs is responsible for the tumor promoting function. Cytokine array profiling identified that Rgs2-/- tumor MDSCs produce less MCP-1, leading to decreased angiogenesis, which could be restored with addition of recombinant MCP-1.Our data reveal Rgs2 as a critical regulator of the pro-angiogenic function of MDSCs in the tumor microenvironment, through regulating MCP-1 production

Deletion of Rgs2 in MDSCs retards tumor growth.

<p>(A) Rgs2−/− and C57BL/6 wild type mice were injected with 5×10⁵ 3LL tumor cells subcutaneously in the hindlimb, and tumor size was measured by caliper over time (n = 12 mice per group). This experiment was repeated 3 times. (B) Wild type mice were injected subcutaneously in the hindlimb with 1×10⁵ 3LL cells alone, or 3LL cells combined with 1×10⁴ wild type or Rgs2−/− MDSCs sorted by flow cytometry (>95% purity; data not shown) from spleens of tumor-bearing mice. Tumor growth was measured by caliper over time. n = 8 mice per group. This experiment was performed twice. * p<0.05.</p

Tumors in Rgs2−/− mice exhibit decreased vascular density and increased cell death.

<p>(A), (C), (E) Sections from size matched 3LL tumors grown in wild type mice and Rgs2 null mice were stained for CD31, active caspase-3 and PCNA, respectively. Representative images are shown. (B), (D), (F) The numbers of CD31 positive vascular structures, active caspase-3 positive cells, and PCNA positive cells, respectively, were quantified in 10 randomly selected fields under microscopy. These experiments were repeated 3–4 times. ** p<0.005, * p<0.05.</p

Rgs2 deficiency has minimal effects on MDSC expansion and differentiation.

<p>(A) Wild type and Rgs2−/− mice were injected with 1×10⁵ 3LL cells in the hindlimb, and 20 days later, spleens were isolated and analyzed by flow cytometry for Gr-1+CD11b+ MDSCs. This experiment was performed at least 3 times and the graphs shown are results from pooling of 3 mice per group. (B) MDSCs were isolated from spleens of tumor bearing Rgs2−/− and wild type mice using the MACS system, spun onto slides using a cytospin centrifuge, and stained. The slides were read by a hematopathologist in a blind fashion, and cells were categorized by morphology. This experiment was performed 4 times. * p<0.01.</p