Impairment in a negative regulatory system for TCR signaling in CD4+ T cells from old mice

To examine the involvement of lipid rafts in an age-associated decline in T cell function, we analyzed the effect of aging on the constituents of lipid rafts in resting mouse CD4(+) T cells. We found a pronounced, age-dependent reduction in PAG/Cbp, which is involved in the regulation of Src family kinases (SFKs) by recruiting Csk (a negative regulator of SFKs) to lipid rafts. This reduction is specific for T cells and is attributed, at least in part, to the reduction in its mRNA level. The reduction of PAG accompanies marked impairment in recruiting Csk to lipid rafts and a concomitant decrease in the inactive forms of SFKs. These findings indicate that old mouse CD4(+) T cells have a defect in a negative SFK regulatory system.     

Age-related changes in mature CD4+ T cells: cell cycle analysis

T cell proliferative responses decrease with age, but the mechanisms responsible are unknown. We examined the impact of age on memory and naive CD4(+) T cell entry and progression through the cell cycle using acridine orange to identify cell cycle stage. For both subsets, fewer stimulated cells from old donors were able to enter and progress through the first cell cycle, with an increased number of cells arrested in G(0) and fewer cells in post G(0) phases. The number of dead cells as assessed by sub-G(0) DNA was also significantly greater in the old group. CD4(+) T cells from old mice also exhibited a significant reduction in clonal history as assessed by CFSE staining. This was associated with a significant decline in cyclin D2 mRNA and protein. We propose that decreases in cyclin D2 are at least partially responsible for the proliferative decline found in aged CD4(+) T cells.     

Regulatory T cells constrain the TCR repertoire of antigen‐stimulated conventional CD4 T cells

To analyze the potential role of Tregs in controlling the TCR repertoire breadth to a non‐self‐antigen, a TCRβ transgenic mouse model (EF4.1) expressing a limited, yet polyclonal naïve T‐cell repertoire was used. The response of EF4.1 mice to an I‐Ab‐associated epitope of the F‐MuLV envelope protein is dominated by clones expressing a Vα2 gene segment, thus allowing a comprehensive analysis of the TCRα repertoire in a relatively large cohort of mice. Control and Treg‐depleted EF4.1 mice were immunized, and the extent of the Vα2‐bearing, antigen‐specific TCR repertoire was characterized by high‐throughput sequencing and spectratyping analysis. In addition to increased clonal expansion and acquisition of effector functions, Treg depletion led to the expression of a more diverse TCR repertoire comprising several private clonotypes rarely observed in control mice or in the pre‐immune repertoire. Injection of anti‐CD86 antibodies in vivo led to a strong reduction in TCR diversity, suggesting that Tregs may influence TCR repertoire diversity by modulating costimulatory molecule availability. Collectively, these studies illustrate an additional mechanism whereby Tregs control the immune response to non‐self‐antigens.     

Defective T cell receptor-mediated signal transduction in memory CD4 T lymphocytes exposed to superantigen or anti-T cell receptor antibodies

Lymphocytes must promote protective immune responses while still maintaining self-tolerance. Stimulation through the T cell receptor (TCR) can lead to distinct responses in naive and memory CD4 T cells. Whereas peptide antigen stimulates both naive and memory T cells, soluble anti-CD3 antibodies and bacterial superantigens stimulate only naive T cells to proliferate and secrete cytokines. Further, superantigens, like staphylococcal enterotoxin B (SEB), cause memory T cells to become anergic while soluble anti-CD3 does not. In the present report, we show that signal transduction through the TCR is impaired in memory cells exposed to either anti-CD3 or SEB. A block in signaling leads to impaired activation of the kinase ZAP-70 so that downstream signals and cell proliferation do not occur. We further show that the signaling defect is unique to each agent. In anti-CD3-treated memory T cells, the src kinase Lck is only transiently activated and does not phosphorylate and activate ZAP-70. In SEB-treated memory T cells, ZAP-70 does not interact with the TCR/CD3 complex to become accessible to Lck. Finally, we provide evidence that alternative signaling pathways are initiated in SEB-treated memory cells. Altered signaling, indicated by an elevation in activity of the src kinase Fyn, may be responsible for memory cell anergy caused by SEB. Thus, differentiation of naive T cells into memory cells is accompanied by alterations in TCR-mediated signaling that can promote heightened recall immunity or specific tolerance.     

Phosphorylation of the N-terminal and C-terminal CD3-epsilon-ITAM tyrosines is differentially regulated in T cells

Tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within CD3 chains is crucial for the recruitment of protein tyrosine kinases and effector molecules into the T cell receptor. Thus, phenylalanine substitution at the N-terminal tyrosine residue of the CD3-epsilon-ITAM abolished signal transduction functions of this ITAM, including phosphorylation at the C-terminal ITAM tyrosine, and its association with ZAP-70. In contrast, mutation at the C-terminal tyrosine of CD3-epsilon-ITAM did not prevent phosphorylation at the N-terminal tyrosine, nor its association with Lck, or p85 PI 3-K regulatory subunit. In contrast to the ZAP-70/diphosphorylated CD8-epsilon-ITAM interaction, the Lck/monophosphorylated CD8-epsilon-ITAM interaction was sensitive to octylglucoside, an agent that disrupts Lck interaction with membrane rafts. Therefore, association of Lck with membrane rafts seems to be essential for stabilization of Lck/CD3-epsilon protein-protein interactions. Overall, the data suggest that the sequential and coordinated phosphorylation of CD3-epsilon-ITAM tyrosines provides to CD3-epsilon the potential to interact with multiple downstream effectors and signaling pathways.     

Antigen-specific CD4 effector T cells: Analysis of factors regulating clonal expansion and cytokine production: Clonal expansion and cytokine production by CD4 effector T cells

In order to fully understand T cell-mediated immunity, the mechanisms that regulate clonal expansion and cytokine production by CD4⁺ antigen-specific effector T cells in response to a wide range of antigenic stimulation needs clarification. For this purpose, panels of antigen-specific CD4⁺ T cell clones with different thresholds for antigen-induced proliferation were generated by repeated stimulation with high- or low-dose antigen. Differences in antigen sensitivities did not correlate with expression of TCR, CD4, adhesion or costimulatory molecules. There was no significant difference in antigen-dependent cytokine production by TG40 cells transfected with TCR obtained from either high- or low-dose-responding T cell clones, suggesting that the affinity of TCRs for their ligands is not primary determinant of T cell antigen reactivity. The proliferative responses of all T cell clones to both peptide stimulation and to TCRβ crosslinking revealed parallel dose-response curves. These results suggest that the TCR signal strength of effector T cells and threshold of antigen reactivity is determined by an intrinsic property, such as the TCR signalosome and/or intracellular signaling machinery. Finally, the antigen responses of high- and low-peptide-responding T cell clones reveal that clonal expansion and cytokine production of effector T cells occur independently of antigen concentration. Based on these results, the mechanisms underlying selection of high “avidity” effector and memory T cells in response to pathogen are discussed.     

Silencing OCILRP2 leads to intrinsic defects in T cells in response to antigenic stimulation

We have previously demonstrated that OCILRP2 interaction with its ligand NKRP1f provides a co-stimulatory signal for optimal T cell proliferation and IL-2 production. Here, using RNA interference technology, we will demonstrate that silencing OCILRP2 in vivo leads to intrinsic impairment in T cell response to CD3- and CD28-cross-linking as well as antigenic stimulation. OCILRP2-silenced T cells have reduced cell proliferation and IL-2 production, which can be bypassed by PMA and ionomycin treatment. OCILRP2-silenced T cells also failed to undergo TCR capping and had impaired cytoskeleton reorganization. Moreover, in OCILRP2-silenced T cells, tyrosine phosphorylation of Lck was diminished, while tyrosine phosphorylation of linkers for activation of T cells was unchanged. Interestingly, NF-kappaB activation was also impaired as the result of OCILRP2 silencing. Together, our data strongly support a novel role for OCILRP2 C-type lectin in TCR-mediated signal transduction. The observation that OCILRP2 is involved in TCR capping and cytoskeletal organization suggests that OCILRP2-NKRP1f may facilitate lipid rafts and immunological synapse formation during T cell interaction with antigen presenting cells.     

Orally tolerant CD4 T cells respond poorly to antigenic stimulation but strongly to direct stimulation of intracellular signaling pathways

The response of splenic CD4 T cells from ovalbumin (OVA)-specific T cell receptor (TCR) transgenic mice after long-term feeding of a diet containing this antigen was examined. These CD4 T cells exhibited a decreased response to OVA peptide stimulation, in terms of proliferation, interleukin-2 secretion, and CD40 ligand expression, compared to those from mice fed a control diet lacking OVA, demonstrating that oral tolerance of T cells had been induced through oral intake of the antigen. We investigated the intracellular signaling pathways, which were Ca/CN cascade and Ras/MAPK cascade, of these tolerant CD4 T cells using phorbol-12-myristate-13-acetate (PMA) and ionomycin, which are known to directly stimulate these pathways. In contrast to the decreased response to TCR stimulation by OVA peptide, it was shown that the response of splenic CD4 T cells to these reagents in the state of oral tolerance was stronger. These results suggest that splenic CD4 T cells in the state of oral tolerance have an impairment in signaling, in which signals are not transmitted from the TCR to downstream signaling pathways, and have impairments in the vicinity of TCR. This revised version was published online in July 2006 with corrections to the Cover Date.     

Functional characteristics of human peripheral blood alpha/betaTCR+, CD4- and CD8- double-negative (DN) T cells

The function of human peripheral blood alpha/betaTCR positive, CD4- and CD8- double-negative T lymphocytes (DN cells) in vivo is not completely understood. The response of immunomagnetically isolated DN cells to PHA and anti-CD3 was compared to the response of single-positive (SP) CD4 and CD8 subsets. Proliferation of DN cells in response to PHA was largely independent of APC. This suggests activation requirements for DN cells that are different from SP cells. Upon activation, HLA-DR was found to be upregulated early on DN cells, and IL-4 and IL-10 were detected in the supernatants of DN cells. These observations in vitro could correspond with an immunoregulatory role of human DN cells in vivo.     

Intact T cell receptor signaling by CD4+ T cells cultured in the rotating wall‐vessel bioreactor

T lymphocytes fail to proliferate or secrete cytokines in response to T cell receptor (TCR) agonists during culture in spaceflight or ground‐based microgravity analogs such as rotating wall‐vessel (RWV) bioreactors. In RWVs, these responses can be rescued by co‐stimulation with sub‐mitogenic doses of the diacyl glycerol (DAG) mimetic phorbol myristate acetate. Based on this result we hypothesized that TCR activation is abrogated in the RWV due to impaired DAG signaling downstream of the TCR. To test this hypothesis we compared TCR‐induced signal transduction by primary, human, CD4⁺ T cells in RWV, and static culture. Surprisingly, we found little evidence of impaired DAG signaling in the RWV. Upstream of DAG, the tyrosine phosphorylation of several key components of the TCR‐proximal signal was not affected by culture in the RWV. Similarly, the phosphorylation and compartmentalization of ERK and the degradation of IκB were unchanged by culture in the RWV indicating that RAS‐ and PKC‐mediated signaling downstream of DAG are also unaffected by simulated microgravity. We conclude from these data that TCR signaling through DAG remains intact during culture in the RWV, and that the loss of functional T cell activation in this venue derives from the affect of simulated microgravity on cellular processes that are independent of the canonical TCR pathway. J. Cell. Biochem. 109: 1201–1209, 2010. © 2010 Wiley‐Liss, Inc.     

Differential Ras signaling via the antigen receptor and IL-2 receptor in primary T lymphocytes

Ras can become activated via multiple distinct receptors in T lymphocytes. However, mechanistic studies of Ras signaling in normal T cells have been hampered by the lack of an efficient technology for gene transfer into resting post-thymic cells. We have overcome this limitation by utilizing adenoviral transduction of T cells from Coxsackie/adenovirus receptor transgenic mice. Unexpectedly, dominant negative Ras17N blocked activation of Ras and ERK in response to IL-2R engagement but not TCR/CD3 ligation. However, TCR-induced ERK activation was suppressed by inhibitors of PKC and PLC-gamma. This first biochemical study of DN Ras in normal quiescent T cells reveals a striking contrast in Ras signaling via two receptors, and suggests that the principal mechanism of TCR-induced Ras activation in normal T cells may be distinct from that utilized in T-lineage tumor cell lines.     

胰腺星状细胞活化相关的信号转导通路

胰腺纤维化是慢性胰腺炎（chronic pancreatitis,CP）和胰腺癌主要的病理学特征,活化的胰腺星状细胞（pancreatic stellate cells,PSCs）是公认的致胰腺纤维化的主要效应细胞。PSCs的活化涉及到几个重要的信号转导通路：有丝分裂原活化蛋白激酶（mitogen-activated protein kinases,MAPK）、磷酯酰肌醇3激酶（phosphatidylinositol 3-kinase,PI3K）=、Smad信号转导蛋白、过氧化物酶体增生物激活受体-γ（PPAR-γ）、Rho-ROCK等细胞内信号途径。探讨这些信号通路在胰腺纤维化中所起的作用对慢性胰腺炎、胰腺癌及糖尿病的治疗有重要意义。现就与PSCs激活有关的信号通路的研究结合最新进展作一综述。     

Responses to T cell receptor/CD3 and interleukin-2 receptor stimulation are altered in T cells from B cell non-hodgkin's lymphomas

T cells infiltrating (T-TIL) B cell non-Hodgkin's lymphomas (NHL) are thought to represent a local host response to the tumor. However, tumor progression in the presence of this T cell infiltrate suggests that the T-TIL may be functionally impaired. To address this issue we determined whether response to stimulation of T-TIL from 25 patients with NHL through the T cell receptor (TCR/CD3) and the interleukin-2 (IL-2) receptor (IL-2R) was intact, since activation of these receptors is important for proliferation and cytokine production. Our results demonstrate defects in response to stimulation via TCR/CD3 and the IL-2R in T-TIL cells from patients with NHL that were not observed with T cells from the peripheral blood. T-TIL showed minimal proliferation to anti-CD3 and only modest proliferation to IL-2 alone or when combined with anti-CD3. Moreover, cytokine production in T-TIL was impaired since stimulation through the TCR/CD3 complex did not induce mRNA for interferon (IFN), IL-2, IL-4 or IL-10. The functional unresponsiveness of these cells may be linked to altered signalling through the TCR/CD3 since an abnormal tyrosine phosphorylation pattern was detected in T-TIL after stimulation with anti-CD3.     

Staphylococcal enterotoxin B induces anergy to conventional peptide in memory T cells

Microbial superantigens can alter host immunity through aberrant activation and subsequent anergy of responding naive T cells. We show here that the superantigen, staphylococcal enterotoxin B (SEB), directly induces tolerance in memory CD4 T cells. Murine naive and memory CD4(+) T cells were labeled with the fluorescent dye CFSE and the cells were exposed to SEB before they were cultured with specific peptide antigen. Memory, but not naive, T cells became anergic and did not respond to their cognate peptide antigen. The extent and duration of T cell receptor (TCR) clustering was similar to promote naive T cell activation and memory T cell anergy, suggesting similar TCR-SEB interactions led to distinct intracellular signaling processes in the two cell types. Like SEB, soluble anti-CD3 mAb does not stimulate memory cell proliferation. However, unlike SEB, soluble anti-CD3 mAbs did not induce anergy to cognate peptide. Anergy was directly visualized in vivo. CD4(+) memory T cells were identified in mice that had been administered SEB. The cells failed to proliferate in response to subsequent immunization with their cognate recall antigen. Hence, one mode of pathogen survival is the modulation of host immunity through selective elimination of memory T cell responses.     

Ex vivo activation of tumor-draining lymph node T cells reverses defects in signal transduction molecules

 The adoptive transfer of tumor-draining lymph node (LN) T cells activated ex vivo with anti-CD3 and interleukin 2 (IL-2) mediates the regression of the poorly immunogenic murine melanoma D5. The efficacy of the activated LN cells is augmented when the sensitizing tumor is a genetically modified variant (designated D5G6) that secretes granulocyte/macrophage-colony-stimulating factor. In contrast to anti-CD3/IL-2-activated LN cells, adoptive transfer of freshly isolated tumor-draining LN T cells has no therapeutic activity. To determine whether the acquisition of antitumor function during ex vivo activation is associated with modifications in signal transduction capacity, the protein tyrosine kinases p56 ^lck and p59 ^fyn and proteins of the NF-κB family were analyzed in tumor-draining LN T cells. The levels of p56 ^lck and p59 ^fyn were lower in tumor-draining than in normal LN T cells and production of tyrosine-phosphorylated substrates was markedly depressed following anti-CD3 stimulation. After 5-day anti-CD3/IL-2 activation, levels of p56 ^lck and p59 ^fyn and protein tyrosine kinase activity increased. Interestingly, the levels of p56 ^lck , p59 ^fyn , and tyrosine kinase activity were higher in activated T cells derived from LN that drained D5G6 than they were in those from D5 tumors. In contrast, the cytoplasmic levels of c-Rel and Rel A were normal in freshly isolated tumor-draining LN, as was nuclear κB DNA-binding activity induced by anti-CD3 mAb or phorbol myristate acetate. Stimulation of activated LN cells with D5 tumor cells induced the nuclear translocation of NF-κB. These findings indicate that the recovery of proteins mediating signal transduction through the T cell receptor/CD3 complex in LN T cells activated ex vivo was associated with the acquisition of antitumor function. Received: 28 August 1997 / Accepted: 23 February 1998     

The role of ABA and MAPK signaling pathways in plant abiotic stress responses

As sessile organisms, plants have developed specific mechanisms that allow them to rapidly perceive and respond to stresses in the environment. Among the evolutionarily conserved pathways, the ABA (abscisic acid) signaling pathway has been identified as a central regulator of abiotic stress response in plants, triggering major changes in gene expression and adaptive physiological responses. ABA induces protein kinases of the SnRK family to mediate a number of its responses. Recently, MAPK (mitogen activated protein kinase) cascades have also been shown to be implicated in ABA signaling. Therefore, besides discussing the role of ABA in abiotic stress signaling, we will also summarize the evidence for a role of MAPKs in the context of abiotic stress and ABA signaling.     

Differential activation of MAP kinase family members triggered by CD99 engagement

The molecular basis for the modulatory properties of CD99 is not well understood. Treatment of human Jurkat T lymphocytes with anti-CD99 antibody led to activation of three mitogen-activated protein kinase (MAPK) members, ERK, JNK, and p38 MAPK, along with homotypic aggregation. While phosphorylation of ERK and JNK was inhibited by the pretreatment of a PKC inhibitor, bisindolylmaleimide I, activation of p38 MAPK was upregulated by the same pretreatment. The signaling pathways to MAPKs by CD99 engagement were independent of PI-3 kinase, distinguishing from those by CD3 engagement. Among MAPKs, ERK pathway was essential for homotypic aggregation together with intracytoplasmic Ca(2+).