Rap1-GTP-interacting adaptor molecule (RIAM), an adaptor molecule of the Mig-10/RIAM/ Lamellipodin (MRL) family, plays a critical role in actin reorganization and inside-out activation of integrins in lymphocytes and platelets. We investigated the role of RIAM in T cell receptor (TCR)-mediated signaling. Elimination of endogenous RIAM by short hairpin RNA (shRNA) resulted in impaired generation of inositol 1,4,5-trisphosphate (IP 3 ) and mobilization of intracellular Ca 2+ , whereas phosphorylation of ζ chain-associated protein kinase of 70 kD (ZAP-70) and formation of the linker of activated T cells (LAT) signalosome were unaffected. Knockdown of RIAM also resulted in defective nuclear translocation of the transcription factor nuclear factor of activated T cells (NFAT) and activation of Ras guanine nucleotide-releasing protein 1 (RasGRP)1, which led to the diminished transcription of Il2. These events were associated with the impaired translocation of phosphorylated phospholipase C γ1 (PLC-γ1) to the actin cytoskeleton, which was required for the recruitment of PLC-γ1 to the immediate proximity of its substrate phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5)P 2 ], and were reversed by reconstitution of cells with RIAM. Thus, by regulating the activation of PLC-γ1, RIAM has a central role in the activation of T cells and the transcription of target genes.

Cell cycle re-entry of quiescent T lymphocytes regulated by cdk2 is required for antigen-specific clonal expansion and generation of productive T cell responses. Recently, we determined that induction of antigen-specific T cell tolerance results in impaired cdk2 activity leading to enhanced Smad3 transactivation, upregulation of p15 and blockade of cell cycle progression. Here we report that pharmacologic inhibition of cdk2 with (R)-roscovitine blocked expansion of alloreactive T cells in vitro and in vivo and protected from lethal acute GvHD. In addition to inhibiting alloreactive T cell responses, (R)-roscovitine prevented TNFα-mediated activation of NFκB pathway, which is involved in the inflammatory process leading to the development of GvHD. The combined anti-proliferative and anti-inflammatory properties of (R)-roscovitine make it an attractive treatment modality toward control of GvHD.

Myeloid-derived suppressor cells (MDSC) are induced by and accumulate within many histologically distinct solid tumors, where they promote disease by secreting angiogenic and immunosuppressive molecules. Although IL1b can drive the generation, accumulation, and functional capacity of MDSCs, the specific IL1b-induced inflammatory mediators contributing to these activities remain incompletely defined. Here, we identified IL1b-induced molecules that expand, mobilize, and modulate the accumulation and angiogenic and immunosuppressive potencies of polymorphonuclear (PMN)-MDSCs. Unlike parental CT26 tumors, which recruited primarily monocytic (M)-MDSCs by constitutively expressing GM-CSF-and CCR2-directed chemokines, IL1btransfected CT26 produced higher G-CSF, multiple CXC chemokines, and vascular adhesion molecules required for mediating infiltration of PMN-MDSCs with increased angiogenic and immunosuppressive properties. Conversely, CT26 tumors transfected with IL1b-inducible molecules could mobilize PMN-MDSCs, but because they lacked the ability to upregulate IL1b-inducible CXCR2-directed chemokines or vascular adhesion molecules, additional PMN-MDSCs could not infiltrate tumors. IL1b-expressing CT26 increased angiogenic and immunosuppressive factors of tumor-infiltrating MDSCs, as did CT26 tumors individually transfected with G-CSF, Bv8, CXCL1, or CXCL5, demonstrating that mediators downstream of IL1b could also modulate MDSC functional activity. Translational relevance was indicated by the finding that the same growth factors, cytokines, chemokines, and adhesion molecules responsible for the mobilization and recruitment of PMN-MDSCs into inflammatory CT26 murine tumors were also coordinately upregulated with increasing IL1b expression in human renal cell carcinoma tumors. These studies demonstrated that IL1b stimulated the components of a multifaceted inflammatory program that produces, mobilizes, chemoattracts, activates, and mediates the infiltration of PMN-MDSCs into inflammatory tumors to promote tumor progression.

Anisakiasis is an infectious parasitic disease contracted by eating third stage larvae of Anisakis simplex (L3) carried by marine fishes. Human anisakiasis was researched for specific IgG with L3 excretory secretory products (ESP). L3ESP were prepared by daily harvesting of culture supernatant from day 2 to day 5, using two kinds culture media of RPMI-1640 and phosphate buffered saline (PBS). When the sera from persons diagnosed with anisakiasis by means of endoscopy were analyzed using indirect ELISA and Western blot, the sera was classified into four groups depending on specific antigen recognition patterns. In addition, the presence of a new antigen for L3, located at less than 13 kDa (AgLT13) was demonstrated in L3ESP with a modified Western blot condition. The production of AgLT13 was mainly found in L3ESP harvested both on day 2 and day 3, and that in PBS was predominant over that in RPMI-1640. Among those sera, the high reactive sera to L3ESP-day two prepared with phosphate buffer in indirect ELISA recognized AgLT13, and 33% of the low reactive sera did so. These results indicate that the binding pattern of human L3 specific antibody is diverse depending on L3ESP preparations and that AgLT13 demonstrated with a Western blot condition is a specific antigen for L3.