Lymph node stromal cells negatively regulate antigen-specific CD4+ T cell responses

Lymph node (LN) stromal cells (LNSCs) form the functional structure of LNs and play an important role in lymphocyte survival and the maintenance of immune tolerance. Despite their broad spectrum of function, little is known about LNSC responses during microbial infection. In this study, we demonstrate that LNSC subsets display distinct kinetics following vaccinia virus infection. In particular, compared with the expansion of other LNSC subsets and the total LN cell population, the expansion of fibroblastic reticular cells (FRCs) was delayed and sustained by noncirculating progenitor cells. Notably, newly generated FRCs were preferentially located in perivascular areas. Viral clearance in reactive LNs preceded the onset of FRC expansion, raising the possibility that viral infection in LNs may have a negative impact on the differentiation of FRCs. We also found that MHC class II expression was upregulated in all LNSC subsets until day 10 postinfection. Genetic ablation of radioresistant stromal cell-mediated Ag presentation resulted in slower contraction of Ag-specific CD4(+) T cells. We propose that activated LNSCs acquire enhanced Ag-presentation capacity, serving as an extrinsic brake system for CD4(+) T cell responses. Disrupted function and homeostasis of LNSCs may contribute to immune deregulation in the context of chronic viral infection, autoimmunity, and graft-versus-host disease.

The immunology of the porcine skin and its value as a model for human skin.

The porcine skin has striking similarities to the human skin in terms of general structure, thickness, hair follicle content, pigmentation, collagen and lipid composition. This has been the basis for numerous studies using the pig as a model for wound healing, transdermal delivery, dermal toxicology, radiation and UVB effects. Considering that the skin also represents an immune organ of utmost importance for health, immune cells present in the skin of the pig will be reviewed. The focus of this review is on dendritic cells, which play a central role in the skin immune system as they serve as sentinels in the skin, which offers a large surface area exposed to the environment. Based on a literature review and original data we propose a classification of porcine dendritic cell subsets in the skin corresponding to the subsets described in the human skin. The equivalent of the human CD141(+) DC subset is CD1a(-)CD4(-)CD172a(-)CADM1(high), that of the CD1c(+) subset is CD1a(+)CD4(-)CD172a(+)CADM1(+/low), and porcine plasmacytoid dendritic cells are CD1a(-)CD4(+)CD172a(+)CADM1(-). CD209 and CD14 could represent markers of inflammatory monocyte-derived cells, either dendritic cells or macrophages. Future studies for example using transriptomic analysis of sorted populations are required to confirm the identity of these cells.

EAACI IG Biologicals task force paper on the use of biologic agents in allergic disorders.

Biologic agents (also termed biologicals or biologics) are therapeutics that are synthesized by living organisms and directed against a specific determinant, for example, a cytokine or receptor. In inflammatory and autoimmune diseases, biologicals have revolutionized the treatment of several immune-mediated disorders. Biologicals have also been tested in allergic disorders. These include agents targeting IgE; T helper 2 (Th2)-type and Th2-promoting cytokines, including interleukin-4 (IL-4), IL-5, IL-9, IL-13, IL-31, and thymic stromal lymphopoietin (TSLP); pro-inflammatory cytokines, such as IL-1β, IL-12, IL-17A, IL-17F, IL-23, and tumor necrosis factor (TNF); chemokine receptor CCR4; and lymphocyte surface and adhesion molecules, including CD2, CD11a, CD20, CD25, CD52, and OX40 ligand. In this task force paper of the Interest Group on Biologicals of the European Academy of Allergy and Clinical Immunology, we review biologicals that are currently available or tested for the use in various allergic and urticarial pathologies, by providing an overview on their state of development, area of use, adverse events, and future research directions.

T Cell Motility as Modulator of Interactions with Dendritic Cells

It is well established that the balance of costimulatory and inhibitory signals during interactions with dendritic cells (DCs) determines T cell transition from a naïve to an activated or tolerant/anergic status. Although many of these molecular interactions are well reproduced in reductionist in vitro assays, the highly dynamic motility of naïve T cells in lymphoid tissue acts as an additional lever to fine-tune their activation threshold. T cell detachment from DCs providing suboptimal stimulation allows them to search for DCs with higher levels of stimulatory signals, while storing a transient memory of short encounters. In turn, adhesion of weakly reactive T cells to DCs presenting peptides presented on major histocompatibility complex with low affinity is prevented by lipid mediators. Finally, controlled recruitment of CD8(+) T cells to cognate DC-CD4(+) T cell clusters shapes memory T cell formation and the quality of the immune response. Dynamic physiological lymphocyte motility therefore constitutes a mechanism to mitigate low avidity T cell activation and to improve the search for "optimal" DCs, while contributing to peripheral tolerance induction in the absence of inflammation.

The kinases NDR1/2 act downstream of the Hippo homolog MST1 to mediate both egress of thymocytes from the thymus and lymphocyte motility

The serine and threonine kinase MST1 is the mammalian homolog of Hippo. MST1 is a critical mediator of the migration, adhesion, and survival of T cells; however, these functions of MST1 are independent of signaling by its typical effectors, the kinase LATS and the transcriptional coactivator YAP. The kinase NDR1, a member of the same family of kinases as LATS, functions as a tumor suppressor by preventing T cell lymphomagenesis, which suggests that it may play a role in T cell homeostasis. We generated and characterized mice with a T cell-specific double knockout of Ndr1 and Ndr2 (Ndr DKO). Compared with control mice, Ndr DKO mice exhibited a substantial reduction in the number of naïve T cells in their secondary lymphoid organs. Mature single-positive thymocytes accumulated in the thymus in Ndr DKO mice. We also found that NDRs acted downstream of MST1 to mediate the egress of mature thymocytes from the thymus, as well as the interstitial migration of naïve T cells within popliteal lymph nodes. Together, our findings indicate that the kinases NDR1 and NDR2 function as downstream effectors of MST1 to mediate thymocyte egress and T cell migration.

Longitudinal Telomere Erosion in Lymphocyte Subsets of Patients with Atherosclerotic Peripheral Arterial Disease (PAD).

Telomere attrition has been linked to accelerate vascular ageing and seems to predispose for vascular disease. Our aim was to study the telomere length dynamics over time and in subsets of leukocytes from 15 patients with peripheral arterial disease (PAD). The mean telomere length in subsets of leukocytes of patients with PAD was in the normal range of age-related telomere length values from healthy individuals. However, we found significant higher telomere attrition for T-cells from patients with PAD over a time period of six months when compared to the controls. The higher telomere loss in T-cells of patients with PAD most likely reflects a higher cell turnover of this leukocyte subset, which is involved in the process of chronic inflammatory disease underlying vascular disease. Further studies are needed to confirm these data and to assess how far this T-cell telomere attrition will correlate to the extent of the disease.

Identification of two Th1 cell epitopes on the Babesia bovis-encoded 77-kilodalton merozoite protein (Bb-1) by use of truncated recombinant fusion proteins

Previous studies have demonstrated the serologic and T-cell immunogenicity for cattle of a recombinant form of the apical complex-associated 77-kDa merozite protein of Babesia bovis, designated Bb-1. The present study characterizes the immunogenic epitopes of the Bb-1 protein. A series of recombinant truncated fusion proteins spanning the majority of the Bb-1 protein were expressed in Escherichia coli, and their reactivities with bovine peripheral blood mononuclear cells and T-cell clones derived from B. bovis-immune cattle and with rabbit antibodies were determined. Lymphocytes from two immune cattle were preferentially stimulated by the N-terminal half of the Bb-1 protein (amino acids 23 to 266, termed Bb-1A), localizing the T-cell epitopes to the Bb-1A portion of the molecule. CD4+ T-cell clones derived by stimulation with the intact Bb-1 fusion protein were used to identify two T-cell epitopes in the Bb-1A protein, consisting of amino acids SVVLLSAFSGN VWANEAEVSQVVK and FSDVDKTKSTEKT (residues 23 to 46 and 82 to 94). In contrast, rabbit antiserum raised against the intact fusion protein reacted only with the C-terminal half of the protein (amino acids 267 to 499, termed Bb-1B), which contained 28 tandem repeats of the tetrapeptide PAEK or PAET. Biological assays and Northern (RNA) blot analyses for cytokines revealed that following activation with concanavalin A, T-cell clones reactive against the two Bb-1A epitopes produced interleukin-2, gamma interferon, and tumor necrosis factors beta and alpha, but not interleukin-4, suggesting that the Bb-1 antigen preferentially stimulates the Th1 subset of CD4+ T cells in cattle. The studies described here report for the first time the characterization, by cytokine production, of the Th1 subset of bovine T cells and show that, as in mice, protozoal antigens can induce Th1 cells in ruminants. This first demonstration of B. bovis-encoded Th1 cell epitopes provides a rationale for incorporation of all or part of the Bb-1 protein into a recombinant vaccine.

PHENOTYPIC CHARACTERIZATION OF SPONTANEOUS AND IN VITRO-MAINTAINED AKR LYMPHOMAS

T-cell lymphomas from AKR mice were studied to determine their potential as a model of T-cell differentiation. Homogeneous tumor cell lines have been used as model to study normal lymphocyte subpopulations, including differentiation lineages, functional properties, and the inducibility to maturation. The underlying concept is that each lymphoid tumor represents a monoclonal neoplastic proliferation of a discrete lymphoid subpopulation arrested at a particular differentiation stage.^ Individual tumors were analyzed to determine the extent of intertumor heterogeneity, and to determine whether lymphomas represented different thymocyte subsets, by determining the cell-surface antigenic phenotype, PNA-binding capacity, and terminal deoxynucleotidyl transferase (TdT) activity. Splenic and thymic tumor cells were compared to determine if the particular lymphoid microenvironment influenced T-cell marker expression. Several of the lymphomas were passaged in syngeneic hosts to verify the original tumor phenotype and to assess the stability of the cell surface and TdT phenotype after transplantation.^ Lymphomas were adapted to in vitro culture to determine whether the T-cell phenotype was maintained in the absence of the host microenvironment. Clonal progeny were analyzed and compared with each other and with parent cell lines to determine the extent of intratumor heterogeneity in this lymphoma system. Parent and cloned cell lines were passaged in vivo to determine whether alterations in surface phenotype occurred after transplantation.^ Our investigation has verified that most spontaneous AKR lymphomas phenotypically resemble known T-cell subsets, including both immature and mature thymic subpopulations. The in vitro lines, however, expressed a highly unstable phenotype in culture that included loss of Ly-1 and Ly-2 antigen expression. After transplantation in vivo, the in vitro lines exhibited alterations in phenotype, including re-expression of Ly antigen on some lymphomas. The inducibility of T-cell antigen markers on tumor cell lines passaged in vivo suggests that the in vitro lines may serve as a possible model system to study the molecular events involved in gene expression in the T-cell system. ^

IN VITRO INDUCTION OF XENOIMMUNE RESPONSES TO LIPOSOMES CONTAINING HUMAN COLON TUMOR ANTIGENS

Liposomes prepared with human LS174T colon tumor cell membranes induce specific primary and secondary xenogeneic immune responses in BALB/c splenocytes in vitro. The multilamellar vesicular liposomes were prepared by adding sonicated membrane fragments in 8 mM CaCl(,2) to a dried lipid film. Cytoxic splenocytes generated in vivo exhibited specificity for the LS174T cell; liposomes elicited higher levels of cytotoxicity than did membranes (P < 0.01). Secondary blastogenic responses elicited in in vivo-primed spleen cells by liposomes also produced a significantly greater (P < 0.005) response than membranes. Subsequently, in vitro induction of primary blastogenic and cytotoxic responses by liposomes were accomplished and revealed similar kinetics to that of whole LS174T cell immunogens. Specificity of the in vitro-primed spleen cells was clearly demonstrated (P < 0.01) on a variety of human tumor cells using both the primed lymphocyte and cell-mediated cytotoxicity assays. The results of competitive inhibition tests with autologous lymphoblasts demonstrated that 30% of the cytotoxic activity was directed against lymphocyte antigens.^ The adjuvant effect of liposomes was shown to be mediated primarily by tumor antigens exposed on the outer surface of liposomes. Trypsinization of the liposomes which eliminated 96% of the surface protein reduced the ability of liposomes to induce cytotoxic splenocytes. The generation of cytolytic activity with liposomes of increasing protein concentration showed that while 10 (mu)g protein was threshold, 100 (mu)g protein induced maximal responses. In addition, membrane fluidity studies revealed that rigid liposomes were significantly (P < 0.05) more efficacious than fluid liposomes in inducing cytotoxicity.^ The induction of the primary response required the presence of nonadherent splenocytes bearing the Thy-1, Lyt-1, and Lyt-2 surface markers. The role of a Lyt-123 subpopulation was suggested by the inability of both the Lyt-1 and Lyt-2 depleted populations to completely restore the cytolytic levels to normal. In addition, the interaction of I-A('+) spleen adherent cells with liposomes for at least 8 hours was required to generate maximal cytotoxic activity. The phenotype of the cytotoxic effector was Thy-1('+), Lyt-2('+), and I-A('d-).^ Incorporation of tumor antigens into liposomes has thus enabled primary immunization in vitro to human colon cancer antigens and may afford an adaptable means to evaluate and to select specific immune responses, as well as to identify colon tumor-specific determinants.^

Glucocorticoid alterations in the human type-1/type-2 cytokine balance and the role of CD28/B7 costimulation

We have recently reported that psychological stress is associated with a shift in the human type-1/type-2 cytokine balance toward a type-2 cytokine response. The mechanisms of these cytokine alterations are unknown, but likely involve glucocorticoid (GC) modulation of cytokine production. Therefore we sought to characterize the effects of GC on the in vitro human type-1/type-2 cytokine balance. We hypothesized that GC induce a type-2 cytokine shift through modulation of critical regulatory cytokines and alterations in the CD28/B7 costimulatory pathway. ^ We first sought to characterize the effect of the GC, dexamethasone (DEX), on type-1 (IFN-γ, IL-12) and type-2 (IL-4, IL-10) cytokine production by human peripheral blood mononuclear blood cells (pBMC) stimulated with a variety of T-lymphocyte and monocyte stimuli. DEX, at concentrations mimicking stress and supraphysiologic levels of cortisol, decreased IFN-γ and IL-12 production and increased IL-4 and IL-10 production, indicating a shift in the type-1/type-2 cytokine balance toward a type-2 response. Furthermore, both CD4+ and CD8+ T-lymphocytes were susceptible to the cytokine modulating effects of DEX. Furthermore, in the absence of the monocyte, the DEX-induced alterations in T-lymphocyte cytokine production were reduced, indicating that the interaction between the monocyte and T-lymphocyte plays a significant role. ^ We next determined the role of regulatory cytokines, known to modulate the type-1/type-2 cytokine balance, in the DEX-induced cytokine alterations. The addition of the recombinant IL-12p70 and IFN-γ, but not the neutralization of IL-4, IL-10 or IL-13 using monoclonal antibodies, attenuated the DEX-induced type-1/type-2 cytokine alterations. These data suggest that the DEX-induced cytokine alterations are mediated, at least in part, through the initial inhibition type-1 cytokines. Lastly, we investigated the role of the CD28/B7 costimulatory pathway in these cytokine alterations. DEX decreased the expression of CD80 and CD86 on THP-1 cells, a monocyte cell line, and the expression of CD28 and CTLA-4 on PHA-stimulated pBMC. The DEX-induced decrease in CD28 and CTLA-4 expression was attenuated by rhIL-12. Finally, CD28 activation attenuated the DEX-induced decrease in IFN-γ production, suggesting that modulation of the CD28/B7 costimulatory pathway may contribute to the DEX-induced type-1/type-2 cytokine alterations. ^

Functional and physical associations of beta1 integrins in the activation of human T lymphocytes

Adhesion involves interactions between cells or cells with extracellular matrix components and is a fundamental process for all multicellular organisms as well as many pathogenic microbes. Integrins are heterodimeric transmembrane proteins that function as adhesion molecules and transduce signals between the extracellular environment and the intracellular cytoskeletal machinery. β1 integrin subfamily is highly expressed on T lymphocytes and mediates cell spreading, adhesion and coactivation. T lymphocytes have an important role in the regulation and homeostasis of the immune system therefore, the goals of this study were to first to investigate β1 integrin interaction with fibronectin binding protein A (FnbpA), a surface protein expressed on gram-negative bacteria Staphylococcus aureus. Second, characterize the association and function of a non-integrin surface protein, CD98, with β1 integrins on T lymphocytes. ^ FnbpA binds to fibronectin (FN), also a ligand for α5β1 and α4β1 integrins on T lymphocytes. Since both bacterial proteins FnbpA and T cell integrins utilize FN, it was of interest to determine the effects FnbpA on T cell activation. Results demonstrated that recombinant FnbpA (rFnbpA) coimmobilized with OKT3 mediated T cell coactivation in a soluble FN-dependent manner. Integrin α5β1 was identified as the main integrin utilized by Staphylococcus aureus FnbpA from studies using soluble antibodies to inhibit T cell proliferation and parallel plate flow chamber assays. The mechanism of rFnbpA-mediated coactivation was one that used soluble FN as a bridge between rFnbpA and integrin α5β1 on the T lymphocyte. ^ Since integrins are utilized by T lymphocytes and bacterial proteins, it was of interest to identify proteins involved in integrin regulation. Anti-CD98 mAb 80A10 was identified and characterized from a screen to identify surface proteins involved in integrin signaling and functions. CD98 is a non-integrin protein that was sensitive to integrin inhibition in human T lymphocyte aggregation and activation, thus suggested that CD98 shared a common signaling pathway with integrins. These results led to the question of whether CD98 physically associates with β1 integrins. Fluorescence microscopy and biochemical analysis determined that CD98 is specifically associated with β1 integrin on human T lymphocytes and may be part of a larger multimolecular signaling complex. ^

17-beta estradiol and progesterone-induced alterations in human type-1/type-2 cytokine balance and the role of costimulatory and apoptotic mechanisms

Evidence suggests that sex-based differences in immune function may predispose women to numerous hypersensitivity conditions such as Systemic lupus erythematosus (SLE), Hashimoto's thyroiditis and asthma. To date, the exact mechanisms of sexual dimorphism in immunity are not fully characterized but sex hormones such as 17-β estradiol (E2) and progesterone (PR) are believed to be involved. Since E2 and PR may modulate the production of critical regulatory cytokines, we sought to characterize their effects on the in vitro human type-1/type-2 cytokine balance. We hypothesized that E2 and/or PR vary cytokine production and influence costimulatory molecule expression and apoptosis. We first described the effect of E2 and/or PR on type-1 (IFN-γ and IL-12) and type-2 (IL-4 and IL-10) cytokine production by human peripheral blood mononuclear cells (PBMC) treated with various T-lymphocyte and monocyte stimuli. E2 and/or PR were each used at concentrations similar to those found at the maternal-fetal interface during pregnancy. At this dose, E2 increased IFN-γ and IL-12 production and PR decreased IFN-γ production and tended to increase IL-4 production. Furthermore, the combination of E2+PR decreased IL-12 production. This suggests that E2 shifts the type-1/type-2 cytokine balance towards a type-1 response and that PR and E2+PR shift the balance towards a type-2 response. Next, we used intracellular cytokine detection to demonstrate that E2 and/or PR are capable of altering cytokine production of CD3+ T-cells and the CD3+CD4+ and CD3+CD8+ subsets. In addition, we used the H9 T-lymphocyte cell line and the THP-1 monocyte cell line to show that E2 and/or PR can induce cytokine effects in both T-cells and monocytes independent of their interaction. Lastly, we determined the effect of E2 and/or PR on costimulatory molecule expression and apoptosis as potential mechanisms for the cytokine-induced alterations. E2 increased and PR decreased CD80 expression on THP-1 cells and PR and E2+PR decreased CD28 expression in PBMC and Jurkat cells. Furthermore, E2, PR and E2+PR increased Fas-mediated apoptosis in Jurkat cells and E2 increased FasL expression on THP-1 cells. Thus, E2 and/or PR may alter the cytokine balance by modulating the CD28/CD80 costimulatory pathway and apoptosis. ^

The immune response to the VlsE antigenic variation protein of Borrelia burgdorferi

The spirochete Borrelia burgdorferi (Bb) is the causative agent of Lyme disease. During infection, a strong immune response is elicited towards Bb by its host; however, the organism is able to persist and to disseminate to many different tissues. The vls locus is located on the linear plasmid lp28-1, a plasmid shown to be important for virulence in the mouse model. During infection, vlsE undergoes antigenic variation through a series of gene conversions, which results in the insertion of sequences from the silent, unexpressed cassettes into the vlsE cassette. We hypothesize that this antigenic variation is important in the spirochete's ability to persist within mammals by allowing it to evade the immune system. To define the role of vls in immune evasion, the immune response against VlsE was determined by using a recombinant form of VlsE (VlsE1-His) as an antigen to screen patient sera. Lyme patients produce antibodies that recognize VlsE, and these antibodies are present throughout the course of disease. Immunization with the VlsE1-His protein provided protection against infection with Bb expressing the same variant of VlsE (VlsE1), but was only partially protective when mice were infected with organisms expressing VlsE variants; however, subsequent VlsE immunization studies yielded inconsistent protection. Successful immunizations produced different antibody reactivities to VlsE epitopes than non-protective immunizations, but the reason for this variable response is unclear. In the process of developing genetic approaches to transform infectious Bb, it was determined that the transformation barrier posed by plasmids lp25 and lp56 could be circumvented by replacing the required lp25 gene pncA. To characterize the role of vlsE in infectivity, Bb lacking lp28-1 were complemented with a shuttle plasmid containing the lp25 encoded virulence determinant pncA and vlsE. Complemented spirochetes express VlsE, but the gene does not undergo antigenic variation and infectivity in the mouse model was not restored, indicating that either antigenic variation of vlsE is necessary for survival in the mouse model or that other genes on lp28-1 are important for virulence. ^

B cells in ADA deficiency

Deficiency of the enzyme adenosine deaminase (ADA) results in severe lymphopenia in humans. Mice with an inactivating mutation in the ADA gene also exhibit profound lymphopenia, as well as pulmonary insufficiency and ribcage abnormalities. In fact, the mouse model has a phenotype that is remarkably similar to that of the human disease, making the mice valuable tools for unraveling the mechanism of lymphocyte destruction in absence of this housekeeping gene. T cell deficiency in ADA deficiency has been extensively studied by others, revealing a block in early thymocyte development. In contrast, our studies revealed that early B cell development in the bone marrow is normal. ADA-deficient mice, however, exhibit profound defects in germinal center formation, preventing antigen-dependent B cell maturation in the spleen. ADA-deficient spleen B cells display significant defects in proliferation and activation signaling, and produce more IgM than their normal counterparts, suggesting that extrafollicular plasmablasts are overrepresented. B cells from ADA-deficient mouse spleens undergo apoptosis more readily than those from normal mouse spleens. Levels of ADA's substrates, adenosine and 2′-deoxyadenosine, are elevated in both bone marrow and spleen in ADA-deficient mice. S ′-adenosyihomoeysteine hydrolase (SAH hydrolase) activity is significantly inhibited in both locales, as well. dATP levels, though, are only elevated in spleen, where B cell development is impaired, and not in bone marrow, where B cell ontogeny is normal. This finding points to dATP as the causative agent of lymphocyte death in ADA deficiency. ADA deficiency results in inhibition of the enzyme ribonucleotide reductase, thereby depleting nucleoside pools needed for DNA repair. Another mouse model that lacks a functional gene encoding a protein involved in DNA repair and/or cell cycle checkpoint regulation, p53-binding protein 1, exhibits blocks in T and B cell development that are similar to those seen in ADA-deficient mice. Unraveling the mechanisms of lymphocyte destruction in ADA deficiency may further understanding of lymphocyte biology, facilitate better chemotherapeutic treatment for lymphoproliferative diseases, and improve gene and enzyme therapy regimens attempted for ADA deficiency. ^

T cell adhesion regulation from clustering GM1 lipid rafts

Lipid rafts are small laterally mobile cell membrane structures that are highly enriched in lymphocyte signaling molecules. Lipid rafts can form from the assembly of specialized lipids and proteins through hydrophobic associations from saturated acyl chains. GM1 gangliosides are a common lipid raft component and have been shown to be essential in many T cell functions. Current lipid raft theory hypothesizes that certain aspects of T cell signaling can be initiated from the coalescence of these signaling-enriched lipid rafts to sites of receptor engagement. We have described how the specific aggregation of GM1 lipid rafts can cause a reorganization of cell surface molecular associations which include dynamic associations of β1 integrins with GM1 lipid rafts. These associations had pronounced effects on T cell adhesive and migratory states. We show that GM1 lipid raft aggregation can dramatically inhibit T cell migration and chemotaxis on the extracellular matrix constituent fibronectin. This inhibition of migration function was shown to be dependent on the src kinase Lck and PKC-regulated F-actin polymerization to extending pseudopods. Furthermore, GM1 lipid raft clustering could activate T cell adhesion-strengthening mechanisms. These include an increase in cellular rigidity, the creation of polymerized cortical F-actin structures, the induction of high affinity integrin states, an increase in surface area and symmetry of the contact plane, and resistance to shear flow detachment while adherent to fibronectin. This indicates that GM1 lipid raft aggregation defines a novel stimulus to regulate lymphocyte motility and cellular adhesion which could have important implications in T cell homing mechanisms. ^