Recognition of E-cadherin on epithelial cells by the mucosal T cell integrin αM290β7 (αEβ7)

The integrin α_M290β7 on the surface of a T cell hybridoma, MTC-1, mediated adhesion of these cells to the mouse epithelial cell line CMT93. This interaction was critically dependent on the presence of divalent cations; Mn²⁺ strongly promoted adhesion, Ca²⁺ was ineffective and Mg²⁺ gave intermediate results. Antibodies to molecules on the surface of CMT93 cells were tested for inhibition of adhesion. One monoclonal antibody (mAb) against E-cadherin, ECCD-2, was found to have significant inhibitory activity. Other mAb to E-cadherin and antibodies to other molecules had no effect. To show that inhibition by ECCD-2 was specific for adhesion mediated by α_M290β7, MTC-1 cells were induced to adhere to CMT93 via the LFA-1/ICAM-1 pathway. For this purpose, the epithelial cells were treated with interferon-γ and tumor necrosis factor-α to induce ICAM-1 expression and, in addition, α_M290β7 on MTC-1 cells was down-regulated by culturing the cells in the absence of transforming growth factor β. Under these circumstances adhesion of MTC-1 cells to CMT93 was inhibited by an antibody to LFA-1 but not by ECCD-2. Transfection of mouse L cells with cDNA for mouse E-cadherin enabled MTC-1 cells to adhere to them through the α_M290β7 integrin; this interaction was inhibited both by ECCD-2 and by blocking antibody against the integrin. These data strongly suggest that E-cadherin is a principal ligand for α_M290β7.     

Expression of the mucosal T cell integrin αM290β7 by a major subpopulation of dendritic cells in mice

Dendritic interdigitating cells isolated from lymph nodes, Langerhans' cells from skin and dendritic cells from the spleen have been tested for expression of the mucosal T cell integrin αM290β7. Almost all interdigitating cells isolated from mesenteric lymph nodes expressed the integrin at high levels; 50-60% of those from lymph nodes draining non-mucosal sites also expressed this adhesion molecule. In contrast, α_M290β7 was not detected on either Langerhans' cells or splenic dendritic cells. Because this integrin has recently been shown to recognize a ligand that seems to be expressed mainly or exclusively by epithelial cells, it is proposed that this adhesion pathway may be important for antigen sampling by dendritic cells in mucosal tissues.     

Expression and regulation of adhesion molecules by γδ T cells from lymphoid tissues and intestinal epithelium

T cells bearing the γδ T cell receptor localize largely in epithelial tissues, but are also present at low frequency in organized secondary lymphoid organs. To assess the role of cell surface adhesion molecules in the traffic and tissue localization of γδ T cells, we compared the expression of these molecules on both αβ and γδ T cells in several lymphoid and non-lymphoid organs. In the gut epithelium, γδ cells express less LFA-1 (CD11a), Pgp-1 (CD44), and α4 integrin than the corresponding αβ cells. In lymph nodes (LN) and Peyer's patches (PP), adhesion molecule expression by γδ cells is heterogeneous, with some of the cells having a phenotype similar to that of intraepithelial γδ cells and the rest expressing high levels of CD44 and L-selectin (CD62L) but lower β7 and α_{M 290}, a phenotype more like lymph node αβ cells. Therefore, the particular set of adhesion molecules expressed by a T cell is dependent, in part, on its anatomic location. Superimposed upon this, however, are differences in expression that are based on the type of T cell; LN and PP γδ T cells express less CD44 but much more β7, α_{M 290} and ICAM-1 (CD54) than αβ T cells in the same organ. The differences in adhesion molecules between αβ and γδ cells are not due simply to differences in their activation status, because these molecules are regulated differently after activation through the T cell receptor (TcR)/CD3 complex. The differential expression of adhesion molecules on cells bearing a particular TcR V region suggests that distinct adhesion phenotypes may arise from prior contact with specific antigen and resultant cell activation in vivo. Lastly, the presence of high level expression of α4β7 and α_{M 290} on L-selectin^lo γδ cells in lymph nodes suggests that these γδ cells may be uniquely capable of migrating to the gut. The differences in adhesion molecule expression and regulation between γδ and αβ T cells could explain, in part, the distinct homing and tissue localization of these T cell subsets in vivo.     

α6 integrins participate in pro-T cell homing to the thymus

During embryogenesis, colonization of the thymic rudiment by hemopoietic progenitor cells depends on the adhesion of these cells to the jugular endothelium. Previously, we showed that progenitor T cells (pro-T cells) interact with α₆ integrins present on vascular endothelium. Here, we demonstrate that anti-α₆ integrin antibodies reduced the number of thymocytes up to 80 % in a congenic mouse model for thymus colonization by pro-T cells. In organotypic thymus cultures, the anti-α₆ integrin antibodies did not influence T cell development and proliferation. From this, we conclude that α₆ integrin participates in thymus homing. During mouse thymus ontogeny, α₆ integrin mRNA and protein expression was found as early as day 10 of development; at day 11, perithymic endothelial cells were α₆ integrin positive. Two α₆ integrin mRNA exist which are produced by alternative exon usage. The longer form, α₆, integrin, predominates during early embryonic stages, while the shorter α_6A form was present later during development. Although α₆, integrins can be displayed by immature thymocytes, strongest expression was found on intra- and perithymic vascular endothelium. These data suggest that α₆ integrins are involved in the homing of pro-T cells to the developing thymus by mediating adhesion of pro-T cells to the vascular endothelium.     

Distinct structural and functional epitopes of the αEβ7 integrin

Intestinal intraepithelial lymphocytes (iIEL) are predominantly CD3⁺, CD8⁺ T lymphocytes located above or adjacent to the mucosal basement membrane. Although they are positioned to interact with intercellular luminal antigen or with enterocytes, the function of iIEL remains unknown. Most (> 85%) of the iIEL express the α^Eβ₇ integrin which appears to be involved in the adhesion of lymphocytes to epithelial cells. We report the characterization of three monoclonal antibodies (mAb) termed αE7-1, αE7-2, and αE7-3, that react with the α^Eβ₇ integrin recognized by the previously described mAb HML-1 as demonstrated by identical sodium dodecyl sulfate – polyacrylamide gel electrophoresis mobility and charge. Flow cytometric analysis of antibody cross-blocking indicated that these mAb recognize distinct epitopes of α^Eβ₇. While all of the mAb were capable of blocking the adhesion of cultured iIEL to a breast epithelial cell line, only HML-1 and αE7-1 (which recognize an identical or closely related epitope) were co-stimulatory with suboptimal concentrations of anti-CD3 mAb in inducing proliferation of cultured iIEL. Thus, these mAb appear to recognize functionally distinct epitopes of α^Eβ₇ and will be useful to study relationships between the structure and function of this integrin.     

Functional analysis of α1β1 integrin in human natural killer cells

Upon activation with interleukin (IL)-2 human natural killer (NK) cells acquire on their surface the α1β1 and α2β1 integrins and down-regulate the expression of α6β1. By employing α1β1-specific monoclonal antibody (mAb) HP-2B6, characterized in our laboratory, we examined the functional role of the α1β1 integrin in NK cells. Treatment with HP-2B6 mAb partially interfered with attachment of cultured NK cells to type I collagen, and combined with an anti-α2β1 (TEA 1/41) mAb, it completely abrogated cell adhesion to this extracelular matrix protein. In contrast, NK cell attachment to laminin was completely blocked by the anti-β1 LIA 1/2 mAb, but was unaffected by α1 and α2-specific mAb; as α3β1 and α6β1 were undetectable, the data indicate that the α1β1 integrin binding sites for type I collagen and laminin are different. Incubation with anti-α1 HP-2B6 or its F(ab')₂ fragments specifically induced a rapid homotypic aggregation of NK cells that was dependent on active metabolism, an intact cytoskeleton and the presence of divalent cations (Ca²⁺ and Mg²⁺); homotypic cell adhesion was selectively blocked by anti-CD18, CD11a or CD54 mAb. In addition, stimulation of cultured NK cells with the anti-α1 HP-2B6 enhanced TNF-α production and induced tyrosine phosphorylation of a 110-kDa protein. Pretreatment with specific inhibitors of protein tyrosine kinase (PTK) activity (tyrphostin 25 and herbimycin A) completely abrogated the functional effects induced by the anti-α1 HP-2B6 mAb. Our data show that ligation of the α1β1 integrin positively modulates IL-2-activated NK cell function via a PTK-dependent pathway.     

Developmental expression of the αIELβ7 integrin on T cell receptor γδ and T cell receptor αβ T cells

A novel monoclonal antibody, 2E7, was shown by immunoprecipitation to be reactive with the α_IELβ7 integrin and was employed to analyze the expression of this integrin in lymphocyte subsets and during T cell ontogeny. In adult lymph nodes, α_IEL was expressed at low levels by 40–70% of CD8⁺ T cells and < 5% of CD4⁺ T cells. However, virtually all intestinal intraepithelial lymphocytes and ?20% of lamina propria CD4⁺ T cells were 2E7⁺, indicating a preferential expression of this integrin on mucosal T cells. Examination of α_IEL integrin expression during thymus ontogeny revealed that ?3–5% of fetal or adult thymocytes were 2E7⁺. Interestingly, early in fetal thymus ontogeny, ?40% of 2E7⁺ cells expressed T cell receptor (TcR)-γδ and this subset persisted through birth. A developmental switch occurred such that 2E7⁺ TcR^? CD4^?8⁺ cells detected on fetal day 19 were followed by 2E7⁺ TcR-αβ CD4^?8⁺ cells in the neonatal thymus. The latter population persisted throughout thymus ontogeny into adulthood. Interestingly, a subset of TcR-γδ Vγ3⁺ day 16 fetal thymocyte dendritic epidermal cell (DEC) precursors were 2E7⁺, but all mature DEC expressed high levels of α_IEL integrin, suggesting that the α_IEL integrin was acquired late in DEC maturation. This possibility was strenghthened by immunohistochemical localization of the majority of 2E7⁺ γδ and αβ T cells to the medullary regions of the thymus. Overall, the results demonstrate a developmentally ordered expression pattern of the α_IELβ₇ integrin that suggests a common function for this integrin during TcR-γδ and -αβ CD4^?8⁺ T cell thymocyte development or perhaps in effector functions for these subsets.     

Homotypic aggregation of CD103 (αEβ7)+ lymphocytes by an anti-CD103 antibody,HML-4

One monoclonal antibody, HML-4, directed against the αEβ7 integrin (CD103), an integrin preferentially expressed on human intestinal intraepithelial lymphocytes (IEL), induced the homotypic aggregation of IEL and of a CD103⁺ MOLT16 cell line. Aggregation was an active adhesion event dependent on an intact cytoskeleton, on tyrosine phosphorylation but not on activation of protein kinase C. It was blocked by four other anti-CD103 antibodies but by none of the antibodies blocking known adhesion lymphocyte pathways. It was associated with a redistribution of the CD103 integrin in the areas of cell-cell contacts. These results indicated that HML-4zx-induced homotypic adhesion was mediated via CD103 and resulted from the binding of the integrin to an as yet undefined ligand expressed by CD103⁺ cells. This ligand was distinct from the epithelial ligand of CD103: in contrast with homotypic adhesion, heterotypic adhesion of CD103⁺ MOLT16 cells on two epithelial intestinal cell lines (DLD1 and HT29) was dependent on the presence of divalent cations, was not enhanced by HML-4, was inhibited by HML-1 but not by the three other antibodies with an inhibitory effect on homotypic adhesion. Finally, the study of conjugates between CD103⁺ and CD103^- sublines derived from the MOLT16 cell line suggested that HML-4-induced homotypic aggregation resulted from homophilic CD103-CD103 interactions.     

Developmental relationship between cytotoxic α/β T cell receptor-positive intraepithelial lymphocytes and Peyer's patch lymphocytes

Following intraduodenal priming of mice with reovirus, precursor cytotoxic T lymphocytes (pCTL) rapidly appear in intraepithelial lymphocytes (IEL) and Peyer's patches. These cells express CTL activity after secondary in vitro stimulation with reovirus-infected cells. Adoptive transfer of Peyer's patch lymphocytes from normal BALB/c mice into reovirus-infected CB.17 severe combined immunodeficiency mice results in the infection-dependent appearance of large numbers of both CD8⁺Thy-1⁺ and CD8^?Thy-1⁺, IEL that express the α/β T cell receptor (TcR). Phenotypic and functional characterization of IEL derived from conventionally reared, reovirus-infected mice also points to extensive similarities in the pCTL derived from Peyer's patches and IEL. As in the Peyer's patches, pCTL are persistent in the IEL compartment for up to 4 weeks after infection. A large percentage of IEL that are recovered from reovirus-primed mice after in vitro culture are CD8⁺Thy-1⁺ cells that express α/β TcR. Furthermore, depletion experiments demonstrate that the CD8⁺Thy-1⁺ population mediates the virus-specific CTL activity. Using limiting dilution analyses, it was estimated that 7 days after intraduodenal infection the average frequency of virus-specific pCTL was 197/10⁶ CD8⁺Thy-1+ IEL and 190/10⁶ CD8⁺Thy-1⁺ Peyer's patch lymphocytes. Taken together, these observations provide evidence that specific cellular immunity to reovirus in IEL is mediated, at least in part, by conventional cytotoxic T lymphocytes and that these cells are functionally and phenotypically similar to the pCTL derived from the Peyer's patches.     

Sequence analysis of rat integrin αE1 and αE2 subunits: Tissue expression reveals phenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph

The integrin α_OX-62 subunit is defined by the OX-62 monoclonal antibody that was raised against rat dendritic cells in lymph (veiled cells) and shows properties similar to those of human α_E2 that is predominantly expressed on intraepithelial lymphocytes. To clone α_OX-62, rat probes generated using primers specific for the human α_E sequence were used to screen rat T cell cDNA libraries. cDNA clones encoding two similar but not identical α subunits that are closely related to but distinct from human α_E were isolated. α_E1 is predicted to be the rat homolog of mouse α_M290 and α_E2 corresponds to rat α_OX-62. Immunofluorescence analysis revealed that mouse α_E1 and rat α_E2 are expressed in dendritic epidermal T cells in the skin, intraepithelial lymphocytes in the small intestine and in cells with a dendritic morphology present at sites where γδ T cells occur in lymphoid organs. Unexpectedly, α_E2 is co-expressed with intracellular CD3-δ and a 33-kDa CD3 chain but not the T cell receptor in veiled cells. These findings suggest that veiled cells may be derived from a lymphoid precursor. Furthermore, veiled cells show phenotypic similarities to intraepithelial lymphocytes.     

Integrin α chain cytoplasmic tails regulate “antibody-redirected” cell adhesion,independently of ligand binding

Here we describe a novel “antibody-redirected cell adhesion” (ARCA) assay. This assay measures heterotypic cell-cell adhesion, resulting from antibody bridging between Fcγ receptors type II (CD32) on leukocytes, and clustered intergrins on adherent cell monolayers. This ARCA activity, facilitated by integrins αβ₁ or α⁴β₁, required an intact cytoskeleton, but did not involve typical integrin ligand binding sites or divalent cations. Furthermore, deletion of the α⁴ cytoplasmic tail almost completely abrogated integrin ARCA activity, suggesting an alteration of integrin recruitment into adhesive sites. If two or more tail residues were present after the conserved GFFKR motif, then ARCA activity was largely restored. Although α⁴ tail deletion caused loss of ARCA activity, it had no effect on the binding of VCAM-1 to intact α⁴-transfected K562 cells. In conclusion, the integrin α chain tail can positively regulate integrin-dependent cell adhesion by a receptor recruitment/clustering mechanism independent of conventional integrin ligand-binding considerations.     

Pyk2 is differentially regulated by β 1 integrin- and CD28-mediated co-stimulation in human CD4+ T lymphocytes

β₁ integrins can provide T cell co-stimulation, but little is known concerning their downstream signaling pathways. We found that Pyk2, a focal adhesion kinase-related tyrosine kinase, is regulated by β₁ integrin signaling in human T cells. Stimulation of Jurkat T cells with the α₄β₁ integrin ligand VCAM-1 results in Pyk2 tyrosine phosphorylation, and combined stimulation with VCAM-1 and anti-CD3 mAb induces rapid and sustained synergistic Pyk2 phosphorylation. Studies with mAb suggest that in synergistic CD3- and α₄β₁ integrin-mediated Pyk2 tyrosine phosphorylation, a major contribution of CD3-derived signals is independent of their effects on regulating integrin adhesion. Analysis of resting human CD4⁺ T cells confirmed the ability of CD3-derived signals to synergize with β₁ integrin-dependent signals in the induction of Pyk2 tyrosine phosphorylation. In addition, although CD28-mediated co-stimulatory signals were able to synergize with CD3-mediated signals in inducing ERK and JNK activation and secretion of IL-2 in the primary T cells, they did not contribute to the induction of Pyk2 phosphorylation. Taken together, these results indicate a potential role for Pyk2 in T cell costimulation mediated specifically by β₁ integrins.     

MAdCAM-1 costimulates T cell proliferation exclusively through integrin α4β7, whereas VCAM-1 and CS-1 peptide use α4β1: evidence for “remote” costimulation and induction of hyperresponsiveness to B7 molecules

We have analyzed the effects of the α⁴ integrin ligands mucosal addressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1), and the fibronectin CS-1 splice variant on T cell activation. Immobilized MAdCAM-1 and VCAM-1 IgG-Fc chimeras and a fibronectin CS-1 peptide efficiently costimulate T cell proliferation when antigen presentation is mimicked by anti-CD3 antibody. VCAM-1-Fc and fibronectin CS-1, which are adhesive ligands for both the α⁴ β₁ and α⁴ β₇ integrins, medicate T cell costimulation exclusively through integrin α⁴ β₁, but not through α⁴ β₇. The inability of VCAM-1-Fc to costimulate via α⁴ β₇ suggests that cell adhesion per se is insufficient, and that exquisite recognition and activation events must be triggered. MAdCAM-1-Fc mediates costimulation exclusively via α⁴ β₇, and can both synergize with and induce hyperresponsiveness to the classical costimulator B7-2. MAdCAM-1-Fc and VCAM-1-Fc, but not B7-2, effectively costimulate when immobilized on sites spatially distant from the anti-CD3 antibody (“remote” costimulation). In vitro, the relative potencies of the CAM were VCAM-1-Fc > ICAM-1-Fc > MAdCAM-1-Fc > B7-Fc, except at high concentrations where ICAM-1 was the most potent. Features of costimulatory CAM revealed by this study have important implications for the design of immunotherapeutic vaccine strategies to combat cancer and infection.     

Expression of functionally active α4β1 integrin by thymic epithelial cells

We have investigated the expression and function of the VLA-4 heterodimer α₄β₁, a member of the β₁ integrin subfamily, on human thymic epithelial cells (TEC) derived from cortical epithelium. The expression of the α₄ integrin chain was studied in four different cloned TEC lines derived from either fetal or post-natal human thymus by both flow cytometry and immunoprecipitation techniques with anti-α₄ MoAbs. All different cell lines assayed expressed significant levels of α₄, as revealed by their reactivity with MoAbs specific for distinct α₄ epitopes. The α₄ subunit expressed by TEC was associated to β₁ but not to β₇ chain, and displayed the characteristic 80/70 kD pattern of proteolytic cleavage. The VLA-4 integrin in these cells was constitutively active in terms of adhesiveness to both fibronectin and vascular cell adhesion molecule-1 (VCAM-1). In addition, this heterodimer localized to punctate regions of the cell in the area of contact with the substratum, named point contacts assessed by staining with the anti-β₁ activation epitope 15/7 MoAb. According to the cortical origin of the TEC lines expressing VLA-4, human thymus sections stained with different anti-α₄ antibodies revealed the presence of cortical, and in smaller numbers medullary epithelial cells bearing α₄ integrin. The expression of α₄ in the thymus was also found in both adult and fetal rats, in which epithelial cells were also specifically stained. Altogether, our data show that VLA-4 is an additional component of the integrin repertoire of TEC, and suggest that it could have an important role in thymus epithelial cell–thymocyte interactions.     

Activated T lymphocytes in the celiac lesion: Non-proliferative activation (CD25) of CD4+ α/β cells in the lamina propria but proliferation (Ki-67) of α/β and γ/δ cells in the epithelium

In order to identify any dominating subset of activated T cells in the celiac lesion, we examined CD3⁺, CD4⁺, CD8⁺ and T cell receptor (TcR) γ/δ⁺ Lymphocytes in jejunal cryosections from 25 patients with celiac disease and 10 controls by three-color immunofluorescence staining for expression of the nuclear proliferation marker detected by monoclonal antibody (mAb) Ki-67 and the p55 α chain of interleukin-2 receptor (CD25). mAb Ki-67⁺ intraepithelial lymphocytes (IEL) were exclusively observed in celiac patients. The median proportion of CD3⁺ IEL positive for Ki-67 increased from nil in controls to 4.5% in partly treated (range 0-19.0%; n = 10; p = 0.05) and 12.8% in untreated celiac disease (range 4.0-30.7%; n = 15; p 0.005). Only 1.5% of CD3⁺ subepithelial T cells expressed the Ki-67 marker in celiac disease (range 0-9.5%). Two- and three-color staining combining mAb to CD3 and Ki-67 with mAb to CD4, CD8 or TcR5 showed that both TcR α/β⁺ CD8⁺ and TcR γδ⁺ (but not CD4⁺) mucosal T cells proliferated in the epithelium. By contrast, CD25 were almost exclusively expressed on CD4⁺ T cells in the lamina propria. The percentage of CD25⁺ T cells increased significantly from 1.7% in controls (range 0-2.9%) to 7.5% in partly treated (range 0.8-17.8%, p 0.002), and to 14.65% in untreated celiac disease (range 3.9-21%, p 0.002). These results suggest that gluten ingestion in celiac disease induces proliferative activation of TcR α/β⁺ CD8⁺ and TcR γδ⁺ IEL but non-proliferative activation (lymphokine production?) of lamina propria CD4⁺ T cells.     

Cross talk between αvβ3 and α4β1 integrins regulates lymphocyte migration on vascular cell adhesion molecule 1

Local inflammation leads to increased expression of the vascular cell adhesion molecule (VCAM)-1 on vascular endothelium which contributes to the encapture of leukocytes from the circulating blood through the leukocyte ligand α4β1 integrin. Inflammatory vascular endothelium expresses VCAM-1 at high density. We found that the speed of locomotion of activated lymphocytes migrating along surfaces coated with recombinant VCAM-1 at a comparable density to that found on inflammatory endothelium was slow. However, lymphocytes do migrate and extravasate rapidly under inflammatory conditions, indicating that there must be mechanisms that regulate the interaction between α4β1 and VCAM-1 in vivo. Here we show that the lymphocyte αβ3 integrin and integrin-associated protein (IAP) is able to regulate this interaction. The occupancy of lymphocyte αvβ3 integrin by platelet cell adhesion molecule-1 or vitronectin regulated the speed of α4β3 integrin-dependent locomotion of lymphocytes on recombinant VCAM-1. This allowed rapid lymphocyte migration at VCAM-1 densities which are typical of inflammatory vessels. This αvβ3-mediated enhanced migration of lymphocytes via α4β1 is likely to depend on the interaction of αvβ3 integrin with the IAP. Furthermore, this motile process correlates with polarization of the actin cytoskeleton in lymphocytes. Our results suggest that cross talk between αvβ3 integrin and α4β1 integrin is a mechanism in the regulation of lymphocyte locomotion along inflammatory endothelium and subsequent transendothelial migration. This can explain how lymphocytes overcome tight adhesion to the vascular endothelium and start rapid migration along and through the endothelial lining of blood vessels into inflammatory tissue.     

Sphingosine‐1‐phosphate activates chemokine‐promoted myeloma cell adhesion and migration involving α4β1 integrin function

Myeloma cell adhesion dependent on α4β1 integrin is crucial for the progression of multiple myeloma (MM). The α4β1‐dependent myeloma cell adhesion is up‐regulated by the chemokine CXCL12, and pharmacological blockade of the CXCL12 receptor CXCR4 leads to defective myeloma cell homing to bone marrow (BM). Sphingosine‐1‐phosphate (S1P) regulates immune cell trafficking upon binding to G‐protein‐coupled receptors. Here we show that myeloma cells express S1P1, a receptor for S1P. We found that S1P up‐regulated the α4β1‐mediated myeloma cell adhesion and transendothelial migration stimulated by CXCL12. S1P promoted generation of high‐affinity α4β1 that efficiently bound the α4β1 ligand VCAM‐1, a finding that was associated with S1P‐triggered increase in talin‐β1 integrin association. Furthermore, S1P cooperated with CXCL12 for enhancement of α4β1‐dependent adhesion strengthening and spreading. CXCL12 and S1P activated the DOCK2‐Rac1 pathway, which was required for stimulation of myeloma cell adhesion involving α4β1. Moreover, in vivo analyses indicated that S1P contributes to optimizing the interactions of MM cells with the BM microvasculture and for their lodging inside the bone marrow. The regulation of α4β1‐dependent adhesion and migration of myeloma cells by CXCL12‐S1P combined activities might have important consequences for myeloma disease progression. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Coordinate expression of β1 and β2 integrin “activation” epitopes during T cell responses in secondary lymphoid tissue

The monoclonal antibodies (mAb) 15/7 and 24 recognize unique activation-dependent, conformational epitopes on β1 and β 2-integrins, respectively. The expression of both of these epitopes closely correlates with the ligand binding ability of their respective integrins, and thus serves as indicators of functional integrin “activation”. Here, we have used six-parameter flow cytometry to examine the expression of these epitopes and conventional β1- and β2-integrin epitopes during human T cell activation in secondary lymphoid tissues in vivo, focusing particularly on the virgin to memory/effector cell transition. Fresh tonsil lymphocytes were stained with mAb against conventional or activation-dependent integrin epitopes, followed by staining with mAb against CD3, CD45RA, and CD45RO, thus allowing the determination of integrin epitope expression on virgin (CD3⁺) T cells (CD45RA⁺/RO^?to±), memory/effector (CD45RA^?/RO⁺⁺) T cells, and T cells undergoing the virgin to memory/effector transition: transition region-1 (T1; CD45RA^+to++/RO⁺); -2 (T2; CD45RA⁺⁺/RO⁺⁺); and -3 (T3; CD45RA⁺/RO⁺⁺). Conventional β1- and β2-integrin epitopes progressively increase during the virgin to T3 stages of the transition in tonsil, in keeping with the generally higher levels of these adhesion molecules on memory/effector vs. virgin T cells. Expression of both the β1 (15/7)-and β2 (24)-integrin activation epitopes first appears on transitional T cells, and is maintained on a relatively constant number of cells (averaging 25-30%) throughout the T1-T3 stages. These epitopes are also noted on a subset of activated memory/effector T cells. Importantly, on both transitional and activated memory/effector T cell subsets, the expression patterns of the 15/7 and 24 epitopes vs. a variety of T cell activation antigens are identical, and the expression of these epitopes relative to each other is linearly correlated, findings strongly supporting the coordinate activation of β1 and β2 integrins duringT cell activation in vivo. These results provide the first evidence of integrin activation during an in vivo immunologic response, and demonstrate the usefulness of mAb recognizing conformational epitopes and multiparameter flow cytometry in delineating the dynamic interplay of adhesion molecules during complex physiologic processes.     

Migration of encephalitogenic CD8 T cells into the central nervous system is dependent on the α4β1‐integrin

Although CD8 T cells are key players in neuroinflammation, little is known about their trafficking cues into the central nervous system (CNS). We used a murine model of CNS autoimmunity to define the molecules involved in cytotoxic CD8 T‐cell migration into the CNS. Using a panel of mAbs, we here show that the α4β1‐integrin is essential for CD8 T‐cell interaction with CNS endothelium. We also investigated which α4β1‐integrin ligands expressed by endothelial cells are implicated. The blockade of VCAM‐1 did not protect against autoimmune encephalomyelitis, and only partly decreased the CD8⁺ T‐cell infiltration into the CNS. In addition, inhibition of junctional adhesion molecule‐B expressed by CNS endothelial cells also decreases CD8 T‐cell infiltration. CD8 T cells may use additional and possibly unidentified adhesion molecules to gain access to the CNS.     

Interaction of integrin α6β4 with ErbB3 and implication in heregulin‐induced ErbB3/ErbB2‐mediated DNA synthesis

Integrin α₆β₄ is abundantly expressed in normal epithelial cells and forms hemidesmosomes, one of cell–extracellular matrix junctions. In many types of cancer cells, integrin α₆β₄ is up‐regulated, laminin, an integrin α₆β₄‐binding extracellular matrix protein, is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and a facilitation of their invasion. It was previously shown that integrin α₆β₄ interacts with ErbB1 and ErbB2 and enhances cell proliferation and motility. Here we show that integrin α₆β₄ interacts with ErbB3 but not with ErbB1, ErbB2 or ErbB4, and enhances the heregulin‐induced, ErbB3/ErbB2 heterodimer‐mediated DNA synthesis, but not cell motility, in A549 cells.