CX 3 CL1, a chemokine with transmembrane and soluble species, plays a key role in inflammation by acting as both chemoattractant and adhesion molecule. CX 3 CL1 is the only chemokine known to undergo constitutive internalization, raising the possibility that dynamic equilibrium between the endocytic compartment and the plasma membrane critically regulates the availability and processing of CX 3 CL1 at the cell surface. We therefore investigated how transmembrane CX 3 CL1 is internalized. Inhibition of dynamin using a nonfunctional allele or of clathrin using specific small interfering RNA prevented endocytosis of the chemokine in CX 3 CL1-expressing human ECV-304 cells. Perusal of the cytoplasmic domain of CX 3 CL1 revealed two putative adaptor protein-2 (AP-2)-binding motifs. Accordingly, CX 3 CL1 co-localized with AP-2 at the plasma membrane. We generated a mutant allele of CX 3 CL1 lacking the cytoplasmic tail. Deletion of the cytosolic tail precluded internalization of the chemokine. We used site-directed mutagenesis to disrupt AP-2-binding motifs, singly or in combination, which resulted in diminished internalization of CX 3 CL1. Although CX 3 CL1 was present in both superficial and endomembrane compartments, ADAM10 (a disintegrin and metalloprotease 10) and tumor necrosis factor-converting enzyme, the two metalloproteases that cleave CX 3 CL1, localized predominantly to the plasmalemma. Inhibition of endocytosis using the dynamin inhibitor, Dynasore, promoted rapid metalloprotease-dependent shedding of CX 3 CL1 from the cell surface into the surrounding medium. These findings indicate that the cytoplasmic tail of CX 3 CL1 facilitates its constitutive clathrin-mediated endocytosis. Such regulation enables intracellular storage of a sizable pool of presynthesized CX 3 CL1 that protects the chemokine from degradation by metalloproteases at the plasma membrane.Inflammation is marked by the migration of circulating leukocytes into sites of injury, a process that occurs via a series of coordinated interactions between leukocytes and endothelial or epithelial cells. Central to this process are chemokines, a family of low molecular weight proteins that can attract leukocytes bearing the complementary receptors. When engagement of the chemokine receptor occurs, the leukocyte becomes activated and is induced to firmly adhere to the inflamed endothelium. These initial steps culminate in diapedesis of the leukocyte across the endothelium and migration into the injured tissue. The local complement of chemokines elaborated is organ-specific and varies with the type of inflammation present. In addition, specific leukocyte subsets also bear distinct chemokine receptors. In this way, chemokines and chemokine receptors confer organ specificity to leukocyte migration and help to "fine-tune" the nature of the observed inflammatory response.Among the 40 chemokines identified so far, CX 3 CL1 is one of only two that have a transmembrane structure (1, 2). The chemokine domain of CX 3 CL1 binds to its complementary receptor, CX 3...

Adoptive transfer of patient-derived T cells modified to express chimeric antigen receptors (CARTs) has demonstrated dramatic success in relapsed/refractory pre-B-cell acute lymphoblastic leukemia (ALL), but response and durability of remission requires exponential CART expansion and persistence. Tumors are known to affect T-cell function, but this has not been well studied in ALL and in the context of chimeric antigen receptor (CAR) expression. Using TCF3/PBX1 and MLL-AF4-driven murine ALL models, we assessed the impact of progressive ALL on T-cell function in vivo. Vaccines protect against TCF3/PBX1.3 but were ineffective when administered after leukemia injection, suggesting immunosuppression induced early during ALL progression. T cells from leukemia-bearing mice exhibited increased expression of inhibitory receptors, including PD1, Tim3, and LAG3, and were dysfunctional following adoptive transfer in a model of T-cell receptor (TCR)-dependent leukemia clearance. Although expression of inhibitory receptors has been linked to TCR signaling, pre-B-cell ALL induced inhibitory receptor expression, at least in part, in a TCR-independent manner. Finally, introduction of a CAR into T cells generated from leukemia-bearing mice failed to fully reverse poor in vivo function.