Role for the Abi/wave protein complex in T cell receptor-mediated proliferation and cytoskeletal remodeling

BACKGROUND: The molecular reorganization of signaling molecules after T cell receptor (TCR) activation is accompanied by polymerization of actin at the site of contact between a T cell and an antigen-presenting cell (APC), as well as extension of actin-rich lamellipodia around the APC. Actin polymerization is critical for the fidelity and efficiency of the T cell response to antigen. The ability of T cells to polymerize actin is critical for several steps in T cell activation including TCR clustering, mature immunological synapse formation, calcium flux, IL-2 production, and proliferation. Activation of the Rac GTPase has been linked to regulation of actin polymerization after TCR stimulation. However, the molecules required for TCR-mediated actin polymerization downstream of activated Rac have remained elusive. Here we identify a novel role for the Abi/Wave protein complex, which signals downstream of activated Rac, in the regulation of actin polymerization and T cell activation in response to TCR stimulation. RESULTS: Here we show that Abi and Wave rapidly translocate from the T cell cytoplasm to the T cell:B cell contact site in the presence of antigen. Abi and Wave colocalize with actin at the T cell:B cell conjugation site. Moreover, Wave and Abi are necessary for actin polymerization after T cell activation, and loss of Abi proteins in mice impairs TCR-induced cell proliferation and IL-2 production in primary T cells. Significantly, the impairment in actin polymerization in cells lacking Abi proteins is due to the inability of Wave proteins to localize to the T cell:B cell contact site in the presence of antigen, rather than the destabilization of the components of the Wave protein complex. CONCLUSIONS: The Abi/Wave complex is a novel regulator of TCR-mediated actin dynamics, IL-2 production, and proliferation.     

Immune complex and Fc receptor-mediated augmentation of antigen presentation for in vivo Th cell responses

It has recently been established that FcRs are involved in the triggering of type II and III inflammatory responses. Although FcR is not believed to be involved in the regulation of T cell function, the in vivo contribution of FcRs to T cell function still remains unclear. We analyzed in vivo responses of delayed-type hypersensitivity and proliferation of CD4+ T cells to Ags in FcRgamma-/- mice lacking the expression and function of FcgammaRI, FcgammaRIII, and FcepsilonRI. We found that the delayed-type hypersensitivity response in FcRgamma-/- mice is significantly decreased compared with that in wild-type mice. Moreover, the secondary responses of proliferation and cytokine production as well as the Ab formation by CD4+ T cells from FcRgamma-/- mice to Ag and normal APCs were also reduced. In contrast, in vitro primary T cell proliferative responses upon stimulation with anti-TCR Ab or MLR as well as in vivo primary response against staphylococcus enterotoxin B administration were not different between T cells from FcRgamma-/- and wild-type mice. In addition, the Ag presentation function of APCs from unimmunized FcRgamma-/- mice was normal. On the other hand, Ab-deficient mice also revealed impaired T cell responses. These results demonstrate that the defective T cell responses in FcRgamma-/- mice were due to impaired Ag presentation during in vivo priming not to a defect in T cells. Therefore, they suggest that the FcRs on APCs mediate efficient priming of Th cell responses in vivo in an immune complex-dependent manner.     

Age-related defects in moesin/ezrin cytoskeletal signals in mouse CD4 T cells

Cytoskeletal proteins of the ezrin-radixin-moesin (ERM) family contribute to T cell activation in response to Ag, and also to T cell polarization in response to connective tissue matrix proteins and chemokine gradients. Previous work has shown that T cells from aged mice are defective in their ability to develop molecular linkages between surface macromolecules and the underlying cytoskeletal framework, both for proteins that move to the synapse and those that are excluded from the site of T cell-APC interaction. T cells from aged mice also show defective cytoskeletal rearrangements and lamellipodia formation when placed in contact with slides coated with Abs to the TCR/CD3 complex. In this study, we show that old CD4 T cells differ from young CD4 T cells in several aspects of ERM biochemistry, including ERM phosphorylation and ERM associations with CD44, CD43, and EBP50. In addition, CD4 T cells from aged mice show defects in the Rho GTPase activities known to control ERM function.     

Beta8 integrin binds Rho GDP dissociation inhibitor-1 and activates Rac1 to inhibit mesangial cell myofibroblast differentiation

Alpha(v)beta8 integrin expression is restricted primarily to kidney, brain, and placenta. Targeted alpha(v) or beta8 deletion is embryonic lethal due to defective placenta and brain angiogenesis, precluding investigation of kidney alpha(v)beta8 function. We find that kidney beta8 is localized to glomerular mesangial cells, and expression is decreased in mouse models of glomerulosclerosis, suggesting that beta8 regulates normal mesangial cell differentiation. To interrogate beta8 signaling pathways, yeast two-hybrid and co-precipitation studies demonstrated beta8 interaction with Rho guanine nucleotide dissociation inhibitor-1 (GDI). Selective beta8 stimulation enhanced beta8-GDI interaction as well as Rac1 (but not RhoA) activation and lamellipodia formation. Mesangial cells from itgb8-/- mice backcrossed to a genetic background that permitted survival, or gdi-/- mice, which develop glomerulosclerosis, demonstrated RhoA (but not Rac1) activity and alpha-smooth muscle actin assembly, which characterizes mesangial cell myofibroblast transformation in renal disease. To determine whether Rac1 directly modulates RhoA-associated myofibroblast differentiation, mesangial cells were transduced with inhibitory Rac peptide fused to human immunodeficiency virus-Tat, resulting in enhanced alpha-smooth muscle actin organization. We conclude that the beta8 cytosolic tail in mesangial cells organizes a signaling complex that culminates in Rac1 activation to mediate wild-type differentiation, whereas decreased beta8 activation shifts mesangial cells toward a RhoA-dependent myofibroblast phenotype.     

Cell-surface receptor for complement component C1q (gC1qR) is a key regulator for lamellipodia formation and cancer metastasis

We previously demonstrated that the receptor for the complement component C1q (gC1qR) is a lipid raft protein that is indispensable for adipogenesis and insulin signaling. Here, we provide the first report that gC1qR is an essential component of lamellipodia in human lung carcinoma A549 cells. Cell-surface gC1qR was concentrated in the lamellipodia along with CD44, monosialoganglioside, actin, and phosphorylated focal adhesion kinase in cells stimulated with insulin, IGF-1, EGF, or serum. The growth factor-induced lamellipodia formation and cell migration were significantly decreased in gC1qR-depleted cells, with a concomitant blunt activation of the focal adhesion kinase and the respective receptor tyrosine kinases. Moreover, the gC1qR-depleted cells exhibited a reduced proliferation rate in culture as well as diminished tumorigenic and metastatic activities in grafted mice. We therefore conclude that cell-surface gC1qR regulates lamellipodia formation and metastasis via receptor tyrosine kinase activation.     

The Transmembrane Adaptor Protein LIME Is Essential for Chemokine-Mediated Migration of Effector T Cells to Inflammatiory Sites

Lck-interacting transmembrane adaptor 1 (LIME) has been previously identified as a raft-associated transmembrane protein expressed predominantly in T and B lymphocytes. Although LIME is shown to transduce the immunoreceptor signaling and immunological synapse formation via its tyrosine phosphorylation by Lck, a Src-family kinase, the in vivo function of LIME has remained elusive in the previous studies. Here we report that LIME is preferentially expressed in effector T cells and mediates chemokine-mediated T cell migration. Interestingly, in LIME^-/- mice, while T cell receptor stimulation-dependent proliferation, differentiation to effector T cells, cytotoxic T lymphocyte (CTL) function and regulatory T lymphocyte (Treg) function were normal, only T cell-mediated inflammatory response was significantly defective. The reduced inflammation was accompanied by the impaired infiltration of leukocytes and T cells to the inflammatory sites of LIME^-/- mice. More specifically, the absence of LIME in effector T cells resulted in the reduced migration and defective morphological polarization in response to inflammatory chemokines such as CCL5 and CXCL10. Consistently, LIME^-/- effector T cells were found to be defective in chemokine-mediated activation of Rac1 and Rap1, and dysregulated phosphorylation of Pyk2 and Cas. Taken together, the present findings show that LIME is a critical regulator of inflammatory chemokine-mediated signaling and the subsequent migration of effector T cells to inflammatory sites.     

Itk functions to control actin polymerization at the immune synapse through localized activation of Cdc42 and WASP

Actin polymerization at the immune synapse is required for T cell activation and effector function; however, the relevant regulatory pathways remain poorly understood. We showed previously that binding to antigen presenting cells (APCs) induces localized activation of Cdc42 and Wiskott-Aldrich Syndrome protein (WASP) at the immune synapse. Several lines of evidence suggest that Tec kinases could interact with WASP-dependent actin regulatory processes. Since T cells from Rlk-/-, Itk-/-, and Rlk-/- x Itk-/- mice have defects in signaling and development, we asked whether Itk or Rlk function in actin polymerization at the immune synapse. We find that Itk-/- and Rlk-/- x Itk-/- T cells are defective in actin polymerization and conjugate formation in response to antigen-pulsed APCs. Itk functions downstream of the TCR, since similar defects were observed upon TCR engagement alone. Using conformation-specific probes, we show that although the recruitment of WASP and Arp2/3 complex to the immune synapse proceeds normally, the localized activation of Cdc42 and WASP is defective. Finally, we find that the defect in Cdc42 activation likely stems from a requirement for Itk in the recruitment of Vav to the immune synapse. Our results identify Itk as a key element of the pathway leading to localized actin polymerization at the immune synapse.     

Condensation of the plasma membrane at the site of T lymphocyte activation

After activation, T lymphocytes restructure their cell surface to form membrane domains at T cell receptor (TCR)-signaling foci and immunological synapses (ISs). To address whether these rearrangements involve alteration in the structure of the plasma membrane bilayer, we used the fluorescent probe Laurdan to visualize its lipid order. We observed a condensation of the plasma membrane at TCR activation sites. The formation of ordered domains depends on the presence of the transmembrane protein linker for the activation of T cells and Src kinase activity. Moreover, these ordered domains are stabilized by the actin cytoskeleton. Membrane condensation occurs upon TCR stimulation alone but is prolonged by CD28 costimulation with TCR. In ISs, which are formed by conjugates of TCR transgenic T lymphocytes and cognate antigen-presenting cells, similar condensed membrane phases form first in central regions and later at the periphery of synapses. The formation of condensed membrane domains at T cell activation sites biophysically reflects membrane raft accumulation, which has potential implications for signaling at ISs.     

CD100 enhances dendritic cell and CD4+ cell activation leading to pathogenetic humoral responses and immune complex glomerulonephritis

CD100, a member of the semaphorin family, is a costimulatory molecule in adaptive immune responses by switching off CD72's negative signals. However, CD100's potential pathogenetic effects in damaging immune responses remain largely unexplored. We tested the hypothesis that CD100 plays a pathogenetic role in experimental immune complex glomerulonephritis. Daily injection of horse apoferritin for 14 days induced immune complex formation, mesangial proliferative glomerulonephritis and proteinuria in CD100-intact (CD100+/+) BALB/c mice. CD100-deficient (CD100-/-) mice were protected from histological and functional glomerular injury. They exhibited reduced deposition of Igs and C3 in glomeruli, reduced MCP-1 and MIP-2 intrarenal mRNA expression, and diminished glomerular macrophage accumulation. Attenuated glomerular injury was associated with decreased Ag-specific Ig production, reduced CD4+ cell activation and cytokine production. Following Ag injection, CD4+ cell CD100 expression was enhanced and dendritic cell CD86 expression was up-regulated. However, in CD100-/- mice, dendritic cell CD86 (but not CD80) up-regulation was significantly attenuated. Following i.p. immunization, CD86, but not CD80, promotes early Ag-specific TCR-transgenic DO11.10 CD4+ cell proliferation and IFN-gamma production, suggesting that CD100 expression enables full expression of CD86 and consequent CD4+ cell activation. Transfer of CD100+/+ DO11.10 cells into CD100-/- mice resulted in decreased proliferation demonstrating that CD100 from other sources in addition to CD100 from Ag-specific CD4+ cells plays a role in initial T cell proliferation. Although T cell-B cell interactions also may be relevant, these studies demonstrate that CD100 enhances pathogenetic humoral immune responses and promotes the activation of APCs by up-regulating CD86 expression.     

Requirement for Tec kinases in chemokine-induced migration and activation of Cdc42 and Rac

Cell polarization and migration in response to chemokines is essential for proper development of the immune system and activation of immune responses. Recent studies of chemokine signaling have revealed a critical role for PI3-Kinase, which is required for polarized membrane association of pleckstrin homology (PH) domain-containing proteins and activation of Rho family GTPases that are essential for cell polarization and actin reorganization. Additional data argue that tyrosine kinases are also important for chemokine-induced Rac activation. However, how and which kinases participate in these pathways remain unclear. We demonstrate here that the Tec kinases Itk and Rlk play an important role in chemokine signaling in T lymphocytes. Chemokine stimulation induced transient membrane association of Itk and phosphorylation of both Itk and Rlk, and purified T cells from Rlk(-/-)Itk(-/-) mice exhibited defective migration to multiple chemokines in vitro and decreased homing to lymph nodes upon transfer to wt mice. Expression of a dominant-negative Itk impaired SDF-1alpha-induced migration, cell polarization, and activation of Rac and Cdc42. Thus, Tec kinases are critical components of signaling pathways required for actin polarization downstream from both antigen and chemokine receptors in T cells.     

Cutting edge: T lymphocyte activation by repeated immunological synapse formation and intermittent signaling

The activation of biological T cell responses requires prolonged contact with APCs and sustained signaling. We investigated whether signaling must be uninterrupted to commit T cells to cytokine production or whether T cell activation may also result from summation of interrupted signals. Upon periodic addition and removal of a src kinase inhibitor, human CD4(+) T cells destroyed and re-formed immunological synapses while aborting and restarting signal transduction. Remarkably, under these conditions, T cells were eventually activated to IFN-gamma production and the amount of IFN-gamma produced was directly related to the total signaling time despite the repeated interruptions. Our results illustrate that T cell activation does not require a stable immunological synapse and can be achieved by interrupted signaling. It is implied that T cells can add activation signals, possibly collected on multiple APCs.     

The actin-bundling protein L-plastin is essential for marginal zone B cell development

B cell development is exquisitely sensitive to location within specialized niches in the bone marrow and spleen. Location within these niches is carefully orchestrated through chemotactic and adhesive cues. In this article, we demonstrate the requirement for the actin-bundling protein L-plastin (LPL) in B cell motility toward the chemokines CXCL12 and CXCL13 and the lipid chemoattractant sphingosine-1-phosphate, which guide normal B cell development. Impaired motility of B cells in LPL(-/-) mice correlated with diminished splenic maturation of B cells, with a moderate (40%) loss of follicular B cells and a profound (>80%) loss of marginal zone B cells. Entry of LPL(-/-) B cells into the lymph nodes and bone marrow of mice was also impaired. Furthermore, LPL was required for the integrin-mediated enhancement of Transwell migration but was dispensable for integrin-mediated lymphocyte adhesion. These results suggest that LPL may participate in signaling that enables lymphocyte transmigration. In support of this hypothesis, the phosphorylation of Pyk-2, a tyrosine kinase that integrates chemotactic and adhesive cues, is diminished in LPL(-/-) B cells stimulated with chemokine. Finally, a well-characterized role of marginal zone B cells is the generation of a rapid humoral response to polysaccharide Ags. LPL(-/-) mice exhibited a defective Ab response to Streptococcus pneumoniae, indicating a functional consequence of defective marginal zone B cell development in LPL(-/-) mice.     

MyD88-dependent activation of dendritic cells and CD4(+) T lymphocytes mediates symptoms, but is not required for the immunological control of parasites during rodent malaria

We investigated the role of different TLRs and MyD88 in host resistance to infection and malaria pathogenesis. TLR2(-/-), TLR4(-/-), TLR6(-/-), TLR9(-/-) or CD14(-/-) mice showed no change in phenotypes (parasitemia, body weight and temperature) when infected with Plasmodium chabaudi chabaudi (AS). MyD88(-/-) mice displayed comparable ability to wild type animals in controlling and clearing parasitemia. Importantly, MyD88(-/-) mice exhibited impaired production of TNF-alpha and IFN-gamma as well as attenuated symptoms, as indicated by changes in body weight and temperature during parasitemia. Consistently, CD11b(+) monocytes and CD11c(+) dendritic cells from infected MyD88(-/-) mice were shown impaired for production of pro-inflammatory cytokines, and in initiating CD4(+) T cell responses. Importantly, the inhibition of T cell activation with anti-CD134L, mostly inhibited IFN-gamma, partially inhibited TNF-alpha production, and protected the animals from malaria symptoms. Our findings suggest that MyD88 and possibly its associated TLRs expressed by dendritic cells play an important role in pro-inflammatory responses, T cell activation, and pathogenesis of malaria, but are not critical for the immunological control of the erythrocytic stage of P. chabaudi.     

Cutting edge: murine CD59a modulates antiviral CD4+ T cell activity in a complement-independent manner

CD59 blocks formation of the membrane attack complex of complement by inhibiting binding of C9 to the C5b-8 complex. To investigate a role for CD59 in promoting T cell responses, we compared T cell activation in CD59a-deficient (Cd59a-/-) and wild-type (WT) mice after in vitro stimulation and after infection with rVV. Virus-specific CD4+ T cell responses were significantly enhanced in Cd59a-/- mice compared with WT mice. Similarly, Cd59a-/- T cells responded more vigorously to in vitro stimulation with CD3-specific Abs compared with WT mice. This effect of CD59a on T cell proliferation was found to be complement-independent. Collectively, these results demonstrate that CD59a down-modulates CD4+ T cell activity in vitro and in vivo, thereby revealing another link between complement regulators and T cell activation.     

Distinct roles of Cdc42 in thymopoiesis and effector and memory T cell differentiation

Cdc42 of the Rho GTPase family has been implicated in cell actin organization, proliferation, survival, and migration but its physiological role is likely cell-type specific. By a T cell-specific deletion of Cdc42 in mouse, we have recently shown that Cdc42 maintains naïve T cell homeostasis through promoting cell survival and suppressing T cell activation. Here we have further investigated the involvement of Cdc42 in multiple stages of T cell differentiation. We found that in Cdc42^−/− thymus, positive selection of CD4⁺CD8⁺ double-positive thymocytes was defective, CD4⁺ and CD8⁺ single-positive thymocytes were impaired in migration and showed an increase in cell apoptosis triggered by anti-CD3/-CD28 antibodies, and thymocytes were hyporesponsive to anti-CD3/-CD28-induced cell proliferation and hyperresponsive to anti-CD3/-CD28-stimulated MAP kinase activation. At the periphery, Cdc42-deficient naive T cells displayed an impaired actin polymerization and TCR clustering during the formation of mature immunological synapse, and showed an enhanced differentiation to Th1 and CD8⁺ effector and memory cells in vitro and in vivo. Finally, Cdc42^−/− mice exhibited exacerbated liver damage in an induced autoimmune disease model. Collectively, these data establish that Cdc42 is critically involved in thymopoiesis and plays a restrictive role in effector and memory T cell differentiation and autoimmunity.     

The myeloid differentiation factor 88 (MyD88) is required for CD4+ T cell effector function in a murine model of inflammatory bowel disease

Abnormal T cell responses to commensal bacteria are involved in the pathogenesis of inflammatory bowel disease. MyD88 is an essential signal transducer for TLRs in response to the microflora. We hypothesized that TLR signaling via MyD88 was important for effector T cell responses in the intestine. TLR expression on murine T cells was examined by flow cytometry. CD4(+)CD45Rb(high) T cells and/or CD4(+)CD45Rb(low)CD25(+) regulatory T cells were isolated and adoptively transferred to RAG1(-/-) mice. Colitis was assessed by changes in body weight and histology score. Cytokine production was assessed by ELISA. In vitro proliferation of T cells was assessed by [(3)H]thymidine assay. In vivo proliferation of T cells was assessed by BrdU and CFSE labeling. CD4(+)CD45Rb(high) T cells expressed TLR2, TLR4, TLR9, and TLR3, and TLR ligands could act as costimulatory molecules. MyD88(-/-) CD4(+) T cells showed decreased proliferation compared with WT CD4(+) T cells both in vivo and in vitro. CD4(+)CD45Rb(high) T cells from MyD88(-/-) mice did not induce wasting disease when transferred into RAG1(-/-) recipients. Lamina propria CD4(+) T cell expression of IL-2 and IL-17 and colonic expression of IL-6 and IL-23 were significantly lower in mice receiving MyD88(-/-) cells than mice receiving WT cells. In vitro, MyD88(-/-) T cells were blunted in their ability to secrete IL-17 but not IFN-gamma. Absence of MyD88 in CD4(+)CD45Rb(high) cells results in defective T cell function, especially Th17 differentiation. These results suggest a role for TLR signaling by T cells in the development of inflammatory bowel disease.