Background: Exosome vesicles can transfer molecular information previously shown to stimulate tumor development; however, the mechanism of exosome uptake is unknown.Results: Mammalian cells internalize exosomes through lipid raft-mediated endocytosis negatively regulated by caveolin-1.Conclusion: Our findings provide novel insights into cellular uptake of exosomes.Significance: Our data provide potential strategies for how the exosome uptake pathway may be targeted.

Integrins are the major adhesion receptors of leukocytes and platelets. β 1 and β 2 integrin function on leukocytes is crucial for a successful immune response and the platelet integrin α IIb β 3 initiates the process of blood clotting through binding fibrinogen1-3. Integrins on circulating cells bind poorly to their ligands but become active after 'inside-out' signaling through other membrane receptors4,5. Subjects with leukocyte adhesion deficiency-1 (LAD-I) do not express β 2 integrins because of mutations in the gene specifying the β 2 subunit, and they suffer recurrent bacterial infections6,7. Mutations affecting α IIb β 3 integrin cause the bleeding disorder termed Glanzmann's thrombasthenia3. Subjects with LAD-III show symptoms of both LAD-I and Glanzmann's thrombasthenia. Their hematopoietically-derived cells express β 1 , β 2 and β 3 integrins, but defective inside-out signaling causes immune deficiency and bleeding problems8. The LAD-III lesion has been attributed to a C→A mutation in the gene encoding calcium and diacylglycerol guanine nucleotide exchange factor (CALDAGGEF1; official symbol RASGRP2) specifying the CALDAG-GEF1 protein9, but we show that this change is not responsible for the LAD-III disorder. Instead, we identify mutations in the KINDLIN3 (official symbol FERMT3) gene specifying the KINDLIN-3 protein as the cause of LAD-III in Maltese and Turkish subjects. Two independent mutations result in decreased KINDLIN3 messenger RNA levels and loss of protein expression. Notably, transfection of the subjects' lymphocytes with KINDLIN3 complementary DNA but not CALDAGGEF1 cDNA reverses the LAD-III defect, restoring integrin-mediated adhesion and migration.

A successful immune response depends on the capacity of immune cells to travel from one location in the body to another–these cells are rapid migrators, travelling at speeds of μm/minute. Their ability to penetrate into tissues and to make contacts with other cells depends chiefly on the β2 integrin known as LFA-1. For this reason, we describe the control of its activity in some detail. For the non-immunologist, the fine details of an immune response often seem difficult to fathom. However, the behaviour of immune cells, known as leukocytes (Box 1), is subject to the same biological rules as many other cell types, and this holds true particularly for the functioning of the integrins on these cells. In this Commentary, we highlight, from a cell-biology point of view, the integrin-mediated immune-cell migration and cell-cell interactions that occur during the course of an immune response.

The cytoplasmic phosphatase PTPN22 (protein tyrosine phosphatase non-receptor type 22) plays a key role in regulating lymphocyte homeostasis, which ensures that the total number of lymphocytes in the periphery is kept more or less constant. Mutations in PTPN22 confer an increased risk of developing autoimmune diseases. The precise function of PTPN22 and how mutations contribute to autoimmunity is controversial. Loss of function mutations in PTPN22 are associated with increased numbers of effector T cells and autoreactive B cells in humans and mice; however, the complete absence of PTPN22 in mice does not result in spontaneous autoimmunity.We found that PTPN22 was a key regulator of regulatory T cell (Treg) function by fine-tuning the functions of the T cell receptor (TCR) and integrins. PTPN22 -/-Tregs were more potent suppressors than were wild-type Tregs, and they suppressed the activity of PTPN22 -/-effector T cells and maintained tolerance. Mechanistically, PTPN22 -/-Tregs showed increased IL-10 production and elevated LFA-1 mediated adhesion, processes critical for Treg function. This previously undiscovered role of PTPN22 in regulating integrin signaling and Treg function could prove to be a useful therapeutic target for manipulating Treg function in human disease. ‡

a b s t r a c tThe PTPN22 locus is one of the strongest risk factors outside of the major histocompatability complex that associates with autoimmune diseases. PTPN22 encodes lymphoid protein tyrosine phosphatase (Lyp) which is expressed exclusively in immune cells. A single base change in the coding region of this gene resulting in an arginine to tryptophan amino acid substitution within a polyproline binding motif associates with type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosis, Hashimotos thyroiditis, Graves disease, Addison's disease, Myasthenia Gravis, vitiligo, systemic sclerosis juvenile idiopathic arthritis and psoriatic arthritis. Here, we review the current understanding of the PTPN22 locus from a genetic, geographical, biochemical and functional perspective. Crown

A successful immune response depends on the migration of lymphocytes into lymph nodes or inflamed tissues where they make contact with antigen-presenting cells. We are interested in how one member of the integrin family, leukocyte function-associated antigen-1 (LFA-1), controls the function and, in particular, the migration of immune cells. We find that this integrin operates not only as an adhesion receptor for T lymphoblasts (T cells) but also induces their migration in vitro at approximately 15 microm/min. Migration requires active myosin light chain kinase at the leading edge and Rho kinase at the trailing edge of the cell. Two active conformations of LFA-1 are differently distributed on the T-cell membrane and regulate independent aspects of migration. High-affinity LFA-1 is located in a midcell 'focal zone' and influences the speed of migration, whereas intermediate affinity LFA-1 controls leading edge adhesions. Manipulating LFA-1 conformation in vivo can be performed, for example, by creating the active conformation in a transgenic mouse, and this model gives further insight into the role of LFA-1 in migration. In humans, the beneficial effect of functioning CD18 integrins in combating infections in vivo is illustrated by rare patients displaying two forms of leukocyte adhesion deficiency. In summary, we speculate that T cells have evolved a mode of rapid migration that is of paramount importance in achieving the high-speed immune surveillance upon which depends the body's protection against diverse invaders from pathogens to cancer cells.

Key Points CLL cells induce defects in T-cell LFA-1–mediated migration by altering Rho GTPase activation signaling, downregulating RhoA and Rac1, and upregulating Cdc42. Lenalidomide repairs these T-cell defects by restoring normal Rho GTPase activation signaling.

The integrin lymphocyte functionassociated antigen 1 (LFA-1) controls many functions of T lymphocytes and is particularly essential during lymphocyte migration from blood into tissues. LFA-1 is considered to initiate "outside-in" signaling when bound to ligand intercellular adhesion molecule 1 (ICAM-1), but little is known about the proteins involved or where in the cell such LFA-1-mediated signaling might be operating. Here we show that LFA-1 is constitutively associ- IntroductionA successful immune response is critically dependent on the ability of leukocytes to leave the circulation by migrating across the vasculature into a lymph node or infected tissue site. [1][2][3] The blood-to-tissue transition occurs through a well-defined sequence of adhesive events along the vessel wall involving selectins and integrins during which the leukocyte gradually slows down, arrests, and transmigrates into a tissue space. In this context, the rapid rolling phase is mediated by selectin engagement and the arrest and migration phase by integrins. The binding of E-selectin to its ligands on the neutrophil provides signals to activate integrin, converting fast to slow rolling 4,5 or, alternatively, signaling through receptors such as chemokine or T-cell receptors can induce rapid integrin-mediated attachment of T cells. 6 Whether the core signaling components of these individual "inside-out" pathways are identical for each mode of stimulation leading to active integrin is not yet certain. 7 For a leukocyte such as a T cell, the ␤2 integrin lymphocyte function-associated antigen-1 (LFA-1) is the major integrin participating in these events. 3,8 Inside-out signaling causes the bent, inactive form of LFA-1 to extend, making its intercellular adhesion molecule 1 (ICAM-1)-binding I domain accessible. 9 This conformation represents intermediate-affinity LFA-1. Further change to the I domain region generates a third form of LFA-1 with higher affinity for ligand. Our previous work shows that migrating T cells express both intermediate-and high-affinity LFA-1, with each form localized to a distinct region of the T cell. Intermediate-affinity LFA-1 is associated with the lamellipodium at the dynamic leading edge, whereas high-affinity LFA-1 is clustered in a highly adhesive mid-cell focal zone corresponding to the lamellar region. 10,11 When exposed to shear force resembling that experienced by leukocytes in the circulation, T cells use LFA-1 to attach with subsecond timing to ICAM-1. 6,12 Integrins are allosteric receptors and it is considered that exposure to shear force contributes to formation of a high-affinity conformation. 13,14 Such a switch is, however, insufficient on its own to generate cell spreading and motility. 15 For these activities, signaling that leads to turnover in the cell's actin cytoskeleton and motor machinery is essential and, for T cells, this is a feature of LFA-1-mediated signaling. [16][17][18] Integrins can signal into the leukocytes on which they are expressed but little is known about the "outside-in" si...