Yumi Yashiro-Ohtani

Non-CD28 costimulatory molecules present in T cell rafts induce T cell costimulation by enhancing the association of TCR with rafts.

While CD28 functions as the major T cell costimulatory receptor, a number of other T cell molecules have also been described to induce T cell costimulation. Here, we investigated the mechanisms by which costimulatory molecules other than CD28 contribute to T cell activation. Non-CD28 costimulatory molecules such as CD5, CD9, CD2, and CD44 were present in the detergent-insoluble glycolipid-enriched (DIG) fraction/raft of the T cell surface, which is rich in TCR signaling molecules and generates a TCR signal upon recruitment of the TCR complex. Compared with CD3 ligation, coligation of CD3 and CD5 as an example of DIG-resident costimulatory molecules led to an enhanced association of CD3 and DIG. Such a DIG redistribution markedly up-regulated TCR signaling as observed by ZAP-70/LAT activation and Ca2+ influx. Disruption of DIG structure using an agent capable of altering cholesterol organization potently diminished Ca2+ mobilization induced by the coligation of CD3 and CD5. This was associated with the inhibition of the redistribution of DIG although the association of CD3 and CD5 was not affected. Thus, the DIG-resident costimulatory molecules exert their costimulatory effects by contributing to an enhanced association of TCR/CD3 and DIG.

The unique target specificity of a nonpeptide chemokine receptor antagonist: selective blockade of two Th1 chemokine receptors CCR5 and CXCR3.

CC chemokine receptor (CCR) 5 and CXC chemokine receptor (CXCR)3 are expressed on T helper cell type 1 cells and have been implicated in their migration to sites of inflammation. Our preceding study demonstrated that a nonpeptide synthetic CCR5 antagonist, TAK‐779 {N, N‐dimethyl‐N‐[4‐[[[2‐(4‐methylphenyl)‐6, 7‐dihydro‐5H‐benzocyclohepten‐8‐yl]carbon‐yl]amino]benzyl]‐tetrahydro‐2H‐pyran4‐aminium chloride, inhibits the development of experimentally induced arthritis by modulating the migration of CCR5+/CXCR3+ T cells to joints. The present study investigated the functional properties of TAK‐779, including the effect of this antagonist on CXCR3 function. For this purpose, transfectants expressing mouse CCR5 (mCCR5) or mCXCR3 and expressing mCCR4 or mCXCR4 as controls were established by introducing each relevant gene into 2B4 T cells and were subjected to the following assays. First, the ligand binding to chemokine receptors was assayed by incubating transfectants with [125I]‐labeled relevant ligand or with the unlabeled relevant ligand followed by staining with anti‐ligand antibody. Second, chemokine‐induced lymphocyte function‐associated antigen‐1 (LFA‐1) activation was assayed by measuring the adhesion of cells to microculture plates coated with purified intercellular adhesion molecule‐1. Third, chemokine‐stimulated chemotaxis was assayed by observing the cell migration through transwells. In these assays, TAK‐779 blocked the ligand binding as well as LFA‐1 up‐regulating and chemotactic function of mCXCR3 and mCCR5 but did not elicit a biologically significant inhibition of those functions of mCCR4 and mCXCR4. These observations indicate the unique target specificity of TAK‐779 and explain why this antagonist efficiently blocks the migration of T cells expressing CCR5 and CXCR3 to sites of inflammation.

Fusogenic liposomes efficiently deliver exogenous antigen through the cytoplasm into the MHC class I processing pathway

Exogenous soluble proteins enter the endosomal pathway by endocytosis and are presented in association with MHC class II rather than class I. In contrast, the delivery of exogenous protein antigens (Ag) into the cytosol generates MHC class I‐restricted cytotoxic T lymphocytes (CTL) responses. Although several immunization approaches, such as the utilization of liposomes, have induced the in vivo priming of MHC class I‐restricted CTL responses to protein Ag, it remains unclear whether this priming results from the direct delivery of protein Ag to the cytosol. Here we report that fusogenic liposomes (FL), which are prepared by fusing simple liposomes with Sendai virus particles, can deliver the encapsulated soluble protein directly into the cytosol of cells cultured concurrently and introduce it into the conventional MHC class I Ag presentation pathway. Moreover, a single immunization with ovalbumin (OVA) encapsulated in FL but not in simple liposomes results in the potent priming of OVA‐specific CTL. Thus, FL function as an efficient tool for the delivery of CTL vaccines.

Molecular Mechanisms Underlying Differential Contribution of CD28 Versus Non-CD28 Costimulatory Molecules to IL-2 Promoter Activation

T cell costimulation via CD28 and other (non-CD28) costimulatory molecules induces comparable levels of [3H]TdR incorporation, but fundamentally differs in the contribution to IL-2 production. In this study, we investigated the molecular basis underlying the difference between CD28 and non-CD28 costimulation for IL-2 gene expression. Resting T cells from a mutant mouse strain generated by replacing the IL-2 gene with a cDNA encoding green fluorescent protein were stimulated with a low dose of anti-CD3 plus anti-CD28 or anti-non-CD28 (CD5 or CD9) mAbs. CD28 and non-CD28 costimulation capable of inducing potent [3H]TdR uptake resulted in high and marginal levels of green fluorescent protein expression, respectively, indicating their differential IL-2 promoter activation. CD28 costimulation exhibited a time-dependent increase in the binding of transcription factors to the NF-AT and NF-κB binding sites and the CD28-responsive element of the IL-2 promoter, whereas non-CD28 costimulation did not. Particularly, a striking difference was observed for the binding of NF-κB to CD28-responsive element and the NF-κB binding site. Decreased NF-κB activation in non-CD28 costimulation resulted from the failure to translocate a critical NF-κB member, c-Rel, to the nuclear compartment due to the lack of IκBβ inactivation. These observations suggest that unlike CD28 costimulation, non-CD28 costimulation fails to sustain IL-2 promoter activation and that such a failure is ascribed largely to the defect in the activation of c-Rel/NF-κB.

IL-12 plays a pivotal role in LFA-1-mediated T cell adhesiveness by up-regulation of CCR5 expression

The chemokine receptor CCR5 has been implicated in the recruitment of T cells to inflammatory sites. However, the regulation of CCR5 induction on T cells and its contribution to T cell adhesiveness are poorly understood. Using a Th1 clone, 2D6, that can be maintained with interleukin (IL)‐12 or IL‐2 alone (designated 2D6IL‐12 or 2D6IL‐2, respectively), we investigated how CCR5 is induced on T cells and whether CCR5 is responsible for up‐regulating the function of adhesion molecules. 2D6IL‐12 grew, forming cell aggregates, in culture containing IL‐12. This was due to lymphocyte function‐associated antigen (LFA)‐1–intercellular adhesion molecule (ICAM)‐1 interaction, because 2D6IL‐12 expressed both LFA‐1 and ICAM‐1 and cell aggregation was inhibited by anti‐ICAM‐1 monoclonal antibody. Despite comparable levels of LFA‐1 and ICAM‐1 expression, 2D6IL‐2 cells did not aggregate in culture with IL‐2. It is important that there was a critical difference in CCR5 expression between 2D6IL‐12 and 2D6IL‐2; the former expressed high levels of CCR5, and the latter expressed only marginal levels. Both types of cells expressed detectable albeit low levels of RANTES (regulated on activation, normal T expressed and secreted) mRNA. Unlike IL‐12 or IL‐2, IL‐18 induced high levels of RANTES mRNA expression without modulating CCR5 expression. Therefore, combined stimulation with IL‐12 and IL‐18 strikingly up‐regulated 2D6 cell aggregation. Notably, LFA‐1‐mediated aggregation of 2D6IL‐12 cells was suppressed by anti‐CCR5 antibody. These results indicate that IL‐12 plays a critical role in CCR5 expression on Th1 cells and consequently contributes to CCR5‐mediated activation of LFA‐1 molecules.

CD5 Costimulation Up-Regulates the Signaling to Extracellular Signal-Regulated Kinase Activation in CD4+CD8+ Thymocytes and Supports Their Differentiation to the CD4 Lineage

CD5 positively costimulates TCR-stimulated mature T cells, whereas this molecule has been suggested to negatively regulate the activation of TCR-triggered thymocytes. We investigated the effect of CD5 costimulation on the differentiation of CD4+CD8+ thymocytes. Coligation of thymocytes with anti-CD3 and anti-CD5 induced enhanced tyrosine phosphorylation of LAT (linker for activation of T cells) and phospholipase C-γ (PLC-γ) compared with ligation with anti-CD3 alone. Despite increased phosphorylation of PLC-γ, this treatment down-regulated Ca2+ influx. In contrast, the phosphorylation of LAT and enhanced association with Grb2 led to activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. When CD3 and CD5 on CD4+CD8+ thymocytes in culture were coligated, they lost CD8, down-regulated CD4 expression, and induced CD69 expression, yielding a CD4+(dull)CD8−CD69+ population. An ERK inhibitor, PD98059, inhibited the generation of this population. The reduction of generation of CD4+CD8− cells resulted from decreased survival of these differentiating thymocytes. Consistent with this, PD98059 inhibited the anti-CD3/CD5-mediated Bcl-2 induction. These results indicate that CD5 down-regulates a branch of TCR signaling, whereas this molecule functions to support the differentiation of CD4+CD8+ thymocytes by up-regulating another branch of TCR signaling that leads to ERK activation.

Induction of surface CCR4 and its functionality in mouse Th2 cells is regulated differently during Th2 development.

T helper cell type 1 (Th1) and Th2 cells express distinct sets of chemokine receptors. In contrast to Th1 chemokine receptors, it is largely unknown how Th2 chemokine receptors such as CC chemokine receptor 4 (CCR4) are induced during Th2 differentiation. Here, we investigated the induction of CCR4 surface expression and ligand responsiveness evaluated by functional assays such as chemokine binding and chemotaxis. This was done in comparison with those of a Th1 chemokine receptor, CXC chemokine receptor 3 (CXCR3). Resting T cells expressed neither CXCR3 nor CCR4. CXCR3 expression and ligand responsiveness were observed when resting T cells were stimulated with anti‐CD3 plus anti‐CD28 in the presence of [interleukin (IL)‐12+anti‐IL‐4] and then recultured without T cell receptor (TCR) stimulation. Unlike CXCR3, CCR4 was induced immediately after anti‐CD3/anti‐CD28 stimulation in the presence of (IL‐4+anti‐interferon‐γ+anti‐IL‐12). However, these CCR4‐positive cells failed to exhibit chemokine binding and chemotaxis. Although the levels of surface CCR4 expression were not increased after the subsequent reculture in the absence of TCR stimulation, CCR4 responsiveness was induced in this stage of Th2 cells. The induction of CCR4 expression and the acquisition of CCR4 responsiveness did not occur in IL‐4‐deficient (IL‐4–/–) and signal transducer and activator of transcription (STAT)6–/– T cells. CCR4 expression and functionality were regained in IL‐4–/– but not in STAT6–/– T cells by the addition of recombinant IL‐4. Although surface expression and functionality of CCR4 are induced depending on the IL‐4/STAT6 signaling pathway, the present results indicate that the functionality of CCR4 does not correlate with CCR4 expression but emerges at later stages of Th2 differentiation.