Homing of activated T cells to sites where they are most needed (where pathogens are present) or where they are most harmful (in autoimmunity) are likely to be controlled by similar mechanisms. Using T cells that recognize an antigen uniquely produced by a specialized tissue, the islets of Langerhans in the pancreas, we were able to analyze the interactions required for the islet-specific homing of CD8+
T cells. Homing is a multistep process that allows T cells to cross into tissues through the endothelium of microcapillary vessels. Each of these steps (initial tethering, slow rolling, activation-dependent arrest, and diapedesis) is controlled by different interactions of surface molecules and multiple signaling pathways triggered by such interactions (30
Some aspects of T cell trafficking to the pancreas, such as the role of integrins and their ligands, have been addressed previously. It has been shown that LFA-1/ICAM-1, VLA-1/VCAM-1, and α4 integrin/MadCAM interactions are important for the homing of diabetogenic T cells to the pancreas (21
). It is widely believed (and rightly so) that inflammation that induces many adhesion molecules facilitates the homing of T cells. However, when we introduced IS-CD8+
T cells into mice with no preexistent inflammation, the mice rapidly developed diabetes, and labeled IS-CD8+
cells were found in their islets (). This was taken as evidence that no inducible molecules are required for the initiation of homing of IS-CD8+
to the islets. Interestingly, IS-CD8+
cells were never found in salivary glands of NOD mice (), although NOD mice typically develop inflammation of salivary glands. Thus, inflammation by itself cannot determine the homing of IS-CD8+
To explain the inflammation-independent organ-specific homing of IS-CD8+ cells, we put forward a hypothesis that endothelium can cross-present the antigen and thus participate in T cell adhesion in an antigen-specific fashion. We found experimental evidence to support this hypothesis: first, homing to the pancreas but not to other organs depended on expression of MHC class I and of the specific peptide ( and ); second, IS-CD8+ cells up-regulated itegrin avidity upon stimulation of their TCR (online supplemental Fig. S4) and demonstrated adhesion to endothelial cells under shear stress in the presence of specific peptide (); third, endothelial cells from pancreatic islets could be directly recognized in in vitro cytotoxicity assay (). This direct recognition did not occur due to the presence of insulin in the tissue culture medium, as aortal endothelium cultivated in the same medium was not recognized by IS-CD8+ cells ( B, b). Thus, pancreatic endothelial cells have a necessary machinery to acquire insulin from β cells and produce the cognate peptide. The mode of acquisition of the Kd-InsB15–23 complexes by endothelial cells is not yet established. However, the observation that IS-CD8+ cells have a reduced ability to home into the islets of NOD.B6Akita/+ mice () suggested that the peptide was generated by endothelial cells from secreted insulin rather than acquired as a peptide or an MHC–peptide complex produced by β cells. It is possible, however, that in NOD.B6Akita/+ mice other properties of endothelium are affected by insulin deficiency reducing T cell adhesion.
The need in secreted insulin for peptide presentation suggests that the peptide is generated by endosomal rather than proteosomal degradation. This scenario is not unprecedented, as evidence in favor of alternatives to the conventional MHC class I peptide processing and presentation pathway is accumulating (46
). These studies have shown that MHC class I molecules could be found in classical MHC class II compartments, and that the TAP-independent and proteosome-independent pathways can lead to presentation of peptides by MHC class I. The fact that endothelial cells can serve as APC has been established previously (10
). Hepatic endothelial cells were found capable of presenting complexes of MHC class I molecules with peptides derived from an injected foreign soluble protein (50
). However, cross-presentation of peptides derived from endogenously produced protein has not been described. Local concentration of insulin is very high amounting to 7% of total islet protein (51
). Insulin produced locally in such high concentrations may be broken down into peptides by endothelial cells. We do not know yet whether endothelium is destroyed by IS-CD8+
cells in vivo, or it is activated to facilitate T cell adhesion and diapedesis. This is an important issue and a matter of future studies. Our own data ( B) showed that secondary activation of endothelium is occurring with time, leading to an increase of the numbers of IS-CD8+
cells found in the isthmus.
Firm adhesion of IS-CD8+
cells is also controlled by G-protein–coupled chemokine receptors. We have found that SLC is the chemokine responsible for homing of IS-CD8+
T cells ( C). Its expression was detected in the pancreas by immunostaining ( D), but it was present not only in NOD mice, which may have an ongoing inflammation, but also in DBA/2J mice (with no preexistent inflammation) and in C3D2 mice, in which IS-CD8+
T cells do not home to the islets. It is very likely that at advanced stages of diabetes development, SLC expression in the pancreas of NOD mice increases enhancing the infiltration by T cells (52
). However, our data showed that constitutive presence of SLC in the pancreas in diabetes-free mice is required for IS-CD8+
homing, but by itself is not sufficient for successful homing. Similarly, in the in vitro adhesion under shear stress assay, SLC treatment of T cells slightly increased their adhesion to the endothelium without the cognate peptide. Nevertheless, the best adhesion was achieved when MHC/peptide complexes and SLC worked in concert ( B). A model describing additive effects of antigen recognition and activation of the chemokine receptor CCR7 on the surface of IS-CD8+
cells is shown in . CCR7 expression by IS-CD8+
cells was confirmed by staining with ELC-Ig fusion protein, and by real-time RT-PCR (not shown). Thus, SLC-triggered integrin activation may be aided by TCR-mediated activation of integrins (26
). We found that TCR-mediated integrin activation was also a property of IS-CD8+
cells, as the avidity of VLA-4 toward fibronectin increased after stimulation with anti-TCR antibody (online supplemental Fig. S4). Other integrins (e.g., LFA-1) are likely to be activated in IS-CD8+
cells in a similar fashion after TCR engagement. Homing specificity is maintained by fulfillment of both requirements (presence of appropriate peptide and an appropriate chemokine receptor): a CCR7-positive T cell clone LPa/2R-1 with different specificity did not show any significant accumulation in the pancreatic islets 2 or 24 h after injection (online supplemental Fig. S2). The results show that pancreatic endothelium is not randomly attracting any T cells that are capable of sensing SLC, but only those that have appropriate specificity ().
Figure 8. A model showing how endothelial cells in the pancreas contribute to islet-specific homing of diabetogenic T cells. (A) The vast microcapillary network within the islet of Langerhans revealed by corrosion casting technique suggests an immense surface of (more ...)
Our study suggests that endothelial cells may play a more prominent role in the trafficking of activated T cells than is currently appreciated, providing antigen-driven specificity of homing. It remains to be established how general this principle is. While insulin is a secreted protein, it is unclear whether similar mechanisms may work for nonsecreted antigens. However, two recent studies (49
) suggest that endothelium (at least in transplantation models) can cross-present nonsecreted antigens. It is difficult to underestimate the significance of such a mechanism for the development of autoimmune diabetes. Because IS-CD8+
cells were found to be a predominant fraction of the islet-infiltrating cell population in NOD mice very early in the pathogenesis of diabetes (5
) they may provide assistance to other T cells in the penetration of islets through activation of the local endothelium. The presence of SLC and activating signals from IS-CD8+
cells may lead to the initial accumulation of lymphocytes at the vascular entrance (known as periinsulitis), which later develops into accumulation of lymphocytes within the islets (insulitis) and damage to the insulin-producing cells (diabetes).