Protein kinase C signaling during T cell activation induces the endoplasmic reticulum stress response

T cell receptor (TCR) ligation (signal one) in the presence of co-stimulation (signal two) results in downstream signals that increase protein production enabling naïve T cells to fully activate and gain effector function. Enhanced production of proteins by a cell requires an increase in endoplasmic reticulum (ER) chaperone expression, which is accomplished through activation of a cellular mechanism known as the ER stress response. The ER stress response is initiated during the cascade of events that occur for the activation of many cells; however, this process has not been comprehensively studied for T cell function. In this study, we used primary T cells and mice circulating TCR transgenic CD8⁺ T cells to investigate ER chaperone expression in which TCR signaling was initiated in the presence or absence of co-stimulation. In the presence of both signals, in vitro and in vivo analyses demonstrated induction of the ER stress response, as evidenced by elevated expression of GRP78 and other ER chaperones. Unexpectedly, ER chaperones were also increased in T cells exposed only to signal one, a treatment known to cause T cells to enter the ‘nonresponsive’ states of anergy and tolerance. Treatment of T cells with an inhibitor to protein kinase C (PKC), a serine/threonine protein kinase found downstream of TCR signaling, indicated PKC is involved in the induction of the ER stress response during the T cell activation process, thus revealing a previously unknown role for this signaling protein in T cells. Collectively, these data suggest that induction of the ER stress response through PKC signaling is an important component for the preparation of a T cell response to antigen.     

Generation of HSP60-specific regulatory T cell and effect on atherosclerosis

Although CD4(+)CD25(+) regulatory T cells are pivotal in the suppression of autoimmunity, little is known about the effect of antigen-specific regulatory T cells on the formation of atheromatous plaques. Here, we describe the induction of heat-shock protein 60 (HSP60)-specific CD4(+)CD25(high) T cells by rapamycin (RPM)-treated immature dendritic cells in vitro and explore their effect on plaques in apolipoprotein E-deficient mice. Rapamycin-treated bone marrow-derived dendritic cells (DC) were immature, expressing a low level of co-stimulation factors CD86 and CD80. Naive CD4(+) T cells expressed high levels of CD25 and forkhead box P3 (Foxp3) after incubation with rapamycin-treated and HSP60-loaded DC and displayed moderate antigen-specific, IL-10-independent inhibitory function in vitro. After adoptive transfer, HSP60-specific CD4(+)CD25(high) T cells inhibited the formation of plaques, while ovalbumin-specific cells did not. These findings suggest that RPM-treated DC can induce antigen-specific CD4(+)CD25(high) Treg cells that have inhibitory activity in vitro and prevent the development of plaques in vivo.     

Contrasting effects of T cell growth factors on T cell responses to melanoma in vitro

 Determinants of T cell responses to tumor cells remain largely unknown. In the present study we have used long-term cultures of human melanoma cells and autologous peripheral blood lymphocytes to examine the influence of cytokines with T cell growth activity on the phenotype and cytotoxic and proliferative response of T cells to melanoma. It was found that addition of interleukin-4 (IL-4) inhibited the response of CD8⁺ T cells and promoted the response of the CD4 subset. IL-2 or IL-7 was effective in increasing melanoma-specific cytotoxic T lymphocyte (CTL) activity in cultures where CD8 T cells were predominant, whereas IL-4 followed by IL-2 was most effective in cultures where CD4 T cells predominated. IL-10 or IL-12 inhibited proliferation and CTL activity against melanoma in long-term cultures. The effects of IL-12 were reproduced in long-term cultures of T cells stimulated with mAb against CD3 and were shown to depend on prior exposure of T cells to IL-12 before IL-2. As yet unidentified factors, such as co-factor expression on melanoma, appear to be as important as exogenous cytokines in determining the nature of T cell responses to melanoma. These results suggest that analysis of responses in long-term culture may assist in defining the role of key cytokines and other determinants of immune responses to melanoma. Received: 4 June 1996 / Accepted: 12 November 1996     

Synergistic and additive interactions between receptor signaling networks drive the regulatory T cell versus T helper 17 cell fate choice

CD4+ T cells differentiate into subsets that promote immunity or minimize damage to the host. T helper 17 cells (Th17) are effector cells that function in inflammatory responses. T regulatory cells (Tregs) maintain tolerance and prevent autoimmunity by secreting immunosuppressive cytokines and expressing check point receptors. While the functions of Th17 and Treg cells are different, both cell fate trajectories require T cell receptor (TCR) and TGF-β receptor (TGF-βR) signals, and Th17 polarization requires an additional IL-6 receptor (IL-6R) signal. Utilizing high-resolution phosphoproteomics, we identified that both synergistic and additive interactions between TCR, TGF-βR, and IL-6R shape kinase signaling networks to differentially regulate key pathways during the early phase of Treg versus Th17 induction. Quantitative biochemical analysis revealed that CD4+ T cells integrate receptor signals via SMAD3, which is a mediator of TGF-βR signaling. Treg induction potentiates the formation of the canonical SMAD3/4 trimer to activate a negative feedback loop through kinases PKA and CSK to suppress TCR signaling, phosphatidylinositol metabolism, and mTOR signaling. IL-6R signaling activates STAT3 to bind SMAD3 and block formation of the SMAD3/4 trimer during the early phase of Th17 induction, which leads to elevated TCR and PI3K signaling. These data provide a biochemical mechanism by which CD4+ T cells integrate TCR, TGF-β, and IL-6 signals via generation of alternate SMAD3 complexes that control the development of early signaling networks to potentiate the choice of Treg versus Th17 cell fate.     

PKCtheta is required for the activation of human T lymphocytes induced by CD43 engagement

The turnover of phosphoinositides leading to PKC activation constitutes one of the principal axes of intracellular signaling. In T lymphocytes, the enhanced and prolonged PKC activation resulting from the engagement of the TcR and co-receptor molecules ensures a productive T cell response. The CD43 co-receptor promotes activation and proliferation, by inducing IL-2 secretion and CD69 expression. CD43 engagement has been shown to promote phosphoinositide turnover and DAG production. Moreover, PKC activation was found to be required for the activation of the MAP kinase pathway in response to CD43 ligation. Here we show that CD43 engagement led to the membrane translocation and enzymatic activity of specific PKC isoenzymes: cPKC (alpha/beta), nPKC (epsilon and theta;), aPKC (zeta) and PKCmu. We also show that activation of PKCtheta; resulting from CD43 ligation induced CD69 expression through an ERK-dependent pathway leading to AP-1, NF-kappaB activation and an ERK independent pathway promoting NFAT activation. Together, these data suggest that PKCtheta; plays a critical role in the co-stimulatory functions of CD43 in human T cells.     

The hyperfluidization of mammalian cell membranes acts as a signal to initiate the heat shock protein response

The concentrations of two structurally distinct membrane fluidizers, the local anesthetic benzyl alcohol (BA) and heptanol (HE), were used at concentrations so that their addition to K562 cells caused identical increases in the level of plasma membrane fluidity as tested by 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy. The level of membrane fluidization induced by the chemical agents on isolated membranes at such concentrations corresponded to the membrane fluidity increase seen during a thermal shift up to 42 degrees C. The formation of isofluid membrane states in response to the administration of BA or HE resulted in almost identical downshifts in the temperature thresholds of the heat shock response, accompanied by increases in the expression of genes for stress proteins such as heat shock protein (HSP)-70 at the physiological temperature. Similarly to thermal stress, the exposure of the cells to these membrane fluidizers elicited nearly identical increases of cytosolic Ca2+ concentration in both Ca2+-containing and Ca2+-free media and also closely similar extents of increase in mitochondrial hyperpolarization. We obtained no evidence that the activation of heat shock protein expression by membrane fluidizers is induced by a protein-unfolding signal. We suggest, that the increase of fluidity in specific membrane domains, together with subsequent alterations in key cellular events are converted into signal(s) leading to activation of heat shock genes.     

TL1A Increases Expression of CD25, LFA-1, CD134 and CD154, and Induces IL-22 and GM-CSF Production from Effector CD4 T-Cells

Elevated levels of the cytokine TL1A is associated with several autoimmune diseases e.g. rheumatoid arthritis and inflammatory bowel disease. However, the exact role of TL1A remains elusive. In this study, we investigated the function of TL1A in a pro-inflammatory setting. We show that TL1A together with IL-12, IL-15 and IL-18 increases expression of the co-stimulatory molecules CD154 (CD40 ligand) and CD134 (OX40) on previously activated CD4⁺ T cells. This indicates that TL1A functions as a co-stimulatory molecule, decreasing the activation threshold of T-cells. We have previously shown that TL1A co-stimulation strongly induces IL-6 in human healthy leukocytes. Interestingly, the cytokine-activated effector T-cells did not produce IL-6 in response to TL1A, indicating distinct effects of TL1A on different cell populations. We further show that this co-stimulation increases the expression of CD25 (IL-2Rα) and CD11a (α-chain of LFA-1) on CD4 T-cells, likely governing increased IL-2/IL-15 sensitivity and cell-cell contact. Along with this, TL1A co-stimulation caused a specific induction of IL-22 and GM-CSF from the activated T-cells. These results substantially contribute to the explanation of TL1A''s role in inflammation. Our results suggest that TL1A should be considered as a target for immunotherapeutic treatment of rheumatoid arthritis and inflammatory bowel disease.     

Preferential effects of leptin on CD4 T cells in central and peripheral immune system are critically linked to the expression of leptin receptor

Leptin can enhance thymopoiesis and modulate the T-cell immune response. However, it remains controversial whether these effects correlate with the expression of leptin receptor, ObR. We herein addressed this issue by using in vivo animal models and in vitro culture systems. Leptin treatment in both ob/ob mice and normal young mice induced increases of CD4 SP thymocytes in thymus and CD4 T cells in the periphery. Interestingly, expression of the long form ObR was significantly restricted to DN, DP and CD4 SP, but not CD8 SP thymocytes. Moreover, in the reaggregated DP thymocyte cultures with leptin plus TSCs, leptin profoundly induced differentiation of CD4 SP but not CD8 SP thymocytes, suggesting that the effects of leptin on thymocyte differentiation might be closely related to the expression of leptin receptor in developing thymocytes. Surprisingly, ObR expression was markedly higher in peripheral CD4 T cells than that in CD8 T cells. Furthermore, leptin treatment with or without IL-2 and PHA had preferential effects on cell proliferation of CD4 T cells compared to that of CD8 T cells. Collectively, these data provide evidence that the effects of leptin on differentiation and proliferation of CD4 T cells might be closely related to the expression of leptin receptor.     

Effect of growth temperature,surface type and incubation time on the resistance of Staphylococcus aureus biofilms to disinfectants

The goal of this study was to investigate the effect of the environmental conditions such as the temperature change, incubation time and surface type on the resistance of Staphylococcus aureus biofilms to disinfectants. The antibiofilm assays were performed against biofilms grown at 20 °C, 30 °C and 37 °C, on the stainless steel and polycarbonate, during 24 and 48 h. The involvement of the biofilm matrix and the bacterial membrane fluidity in the resistance of sessile cells were investigated. Our results show that the efficiency of disinfectants was dependent on the growth temperature, the surface type and the disinfectant product. The increase of growth temperature from 20 °C to 37 °C, with an incubation time of 24 h, increased the resistance of biofilms to cationic antimicrobials. This change of growth temperature did not affect the major content of the biofilm matrix, but it decreased the membrane fluidity of sessile cells through the increase of the anteiso-C19 relative amount. The increase of the biofilm resistance to disinfectants, with the rise of the incubation time, was dependent on both growth temperature and disinfectant product. The increase of the biofilm age also promoted increases in the matrix production and the membrane fluidity of sessile cells. The resistance of S. aureus biofilm seems to depend on the environment of the biofilm formation and involves both extracellular matrix and membrane fluidity of sessile cells. Our study represents the first report describing the impact of environmental conditions on the matrix production, sessile cells membrane fluidity and resistance of S. aureus biofilms to disinfectants.     

The CD7(-) subset of CD4(+) memory T cells is prone to accelerated apoptosis that is prevented by interleukin-15 (IL-15)

The CD7(-) subset of CD4(+) T cells reflects a stable differentiation state of post-thymic helper T cells with CD45R0(+)CD45RA(-) 'memory' phenotype. Here we report that CD4(+)CD7(-) T cells are prone to increased spontaneous apoptosis in vitro compared to CD4(+)CD7(+) T cells. Spontaneous apoptosis is prevented by IL-15, but not by IL-2. Moreover, IL-15 increases Bcl-2 and decreases CD95/Fas expression of CD7(-), but not of CD7(+) T cells. Because IL-15 is physiologically not secreted but expressed in a membrane-bound form, we cocultured T cells with TNF-alpha stimulated fibroblasts that expose membrane IL-15. TNF-alpha stimulated fibroblasts rescue CD4(+)CD7(-) T cells from apoptosis whereas unstimulated fibroblasts do not. Rescue from apoptosis requires cell-cell contact and is abolished by addition of neutralizing antibodies to IL-15. We conclude that membrane IL-15 prevents accelerated apoptosis of CD4(+)CD7(-) T cells. This mechanism may contribute to accumulation of CD7(-) T cells in chronic inflammatory skin lesions.     

The effect of membrane-fluidizing agents on the adhesion of CHO cells

Treatment of CHO cells with drugs which are known to increase membrane lipid fluidity reduced the cells' ability to adhere to protein coated substrates, The concentrations of local anesthetics, nonionic detergents or aliphatic alcohols required to reduce CHO cell adhesion by 50% were similar to those reported to block nerve conduction, indicating that these drugs can affect the membrane at physiologically significant concentrations. Nonionic detergents and aliphatic alcohols, but not local anesthetics, caused increases in the fluidity of CHO plasma membranes (measured by fluorescence polarization) at concentrations which inhibited cell adhesion. The adhesion versus temperature profile had a sigmoidal shape, suggesting that a temperature dependent cooperative process such as a lipid phase transition, might be involved. However, the temperature profile for CHO membrane fluidity manifested no discontinuities, indicating the absence of any discrete phase transitions of the lipid matrix. This observation, coupled with the result that the inhibition of CHO cell adhesion produced by low temperatures was not relieved by drugs which can increase membrane fluidity, suggests that the reduced adhesion seen at low temperature is probably not due to reduced lipid fluidity.     

Zinc and immunity

Nutritional deficiency of zinc is widespread throughout the developing countries and a conditioned deficiency of zinc is known to occur in many diseased states. Zinc is known to play an important role in the immune system and zinc deficient subjects may experience increased susceptibility to a variety of pathogens. We have studied the effects of a mild deficiency of zinc on T cells in an experimental model of human zinc deficiency. We showed that T cell functions were affected adversely even when the deficiency of zinc was mild in humans. Characteristically during zinc deficiency, the serum thymulin activity (a thymic hormone) was decreased which was restored following zinc supplementation. Our studies also showed that zinc deficiency caused an imbalance between TH1 and TH2 functions. The production of IFN-g, IL-2, TNF-a (products of TH1 cells) were decreased, whereas the production of IL-4, IL-6 and IL-10 (products of TH2) were not affected during zinc deficiency. T cell subpopulation studies revealed that the CD4+ CD45RA+ to CD4+ CD45RO+ ratio was decreased as a result of zinc deficiency, suggesting that zinc may be required for the regeneration of new CD4+ T cells. We further documented that zinc deficiency decreased NK cell lytic activity and caused a decrease in the percentage of CD8+ CD73+ T cells which are known to be predominantly precursors of cytotoxic T cells. In a suitable cell culture model our studies revealed that the gene expression of a DNA synthesizing enzyme TK was affected adversely which resulted in delayed cell cycle and decreased cell growth. The above immunological consequences of zinc deficiency may be responsible for decreased cell mediated immune functions in zinc deficient subjects.     

Baculovirus activates murine dendritic cells and induces non-specific NK cell and T cell immune responses

We previously reported that the baculovirus induced a strong host immune response against infections and malignancies. Among the immune cells, the dendritic cells were most strongly infected and activated by the baculovirus, although the exact mechanism remained unclear. Here, we evaluated the non-specific immune responses of bone marrow-derived dendritic cells (BMDCs) after infection by a wild-type baculovirus. MHC class I and II molecules and co-stimulation molecules (CD40, CD80, and CD86) on BMDCs were up-regulated by baculovirus infection. At the same time, the BMDCs produced pre-inflammatory cytokines (IL-6, IL12p70, and TNF-α) and IFN-α. NK cells showed IFN-γ production, CD69 up-regulation, and enhanced cytotoxicity when they were co-cultured with baculovirus-infected BMDCs. T cells showed IFN-γ production, CD69 up-regulation, and cell proliferation. Ex vivo analysis performed in vitro produced similar results. These findings suggested that baculovirus-infected dendritic cells induce non-specific immune responses and can be used as an immunotherapeutic agent against viral infections and malignancies, together with present therapeutic drug regimens.     

NSOM/QD-Based Direct Visualization of CD3-Induced and CD28-Enhanced Nanospatial Coclustering of TCR and Coreceptor in Nanodomains in T Cell Activation

Direct molecular imaging of nano-spatial relationship between T cell receptor (TCR)/CD3 and CD4 or CD8 co-receptor before and after activation of a primary T cell has not been reported. We have recently innovated application of near-field scanning optical microscopy (NSOM) and immune-labeling quantum dots (QD) to image Ag-specific TCR response during in vivo clonal expansion, and now up-graded the NSOM/QD-based nanotechnology through dipole-polarization and dual-color imaging. Using this imaging system scanning cell-membrane molecules at a best-optical lateral resolution, we demonstrated that CD3, CD4 or CD8 molecules were distinctly distributed as single QD-bound molecules or nano-clusters equivalent to 2–4 QD fluorescence-intensity/size on cell-membrane of un-stimulated primary T cells, and ∼6–10% of CD3 were co-clustering with CD4 or CD8 as 70–110 nm nano-clusters without forming nano-domains. The ligation of TCR/CD3 on CD4 or CD8 T cells led to CD3 nanoscale co-clustering or interaction with CD4 or CD8 co-receptors forming 200–500 nm nano-domains or >500 nm micro-domains. Such nano-spatial co-clustering of CD3 and CD4 or CD3 and CD8 appeared to be an intrinsic event of TCR/CD3 ligation, not purely limited to MHC engagement, and be driven by Lck phosphorylation. Importantly, CD28 co-stimulation remarkably enhanced TCR/CD3 nanoscale co-clustering or interaction with CD4 co-receptor within nano- or micro-domains on the membrane. In contrast, CD28 co-stimulation did not enhance CD8 clustering or CD3–CD8 co-clustering in nano-domains although it increased molecular number and density of CD3 clustering in the enlarged nano-domains. These nanoscale findings provide new insights into TCR/CD3 interaction with CD4 or CD8 co-receptor in T-cell activation.     

CD28 regulates glucocorticoid-induced TNF receptor family-related gene expression on CD4+ T cells via IL-2-dependent mechanisms

The glucocorticoid-induced TNF-related gene receptor (GITR) is the newest member of the costimulatory molecule family and is expressed on both resting CD4+CD25+ regulatory T (T(R)) cells and activated CD4+ T cells. We investigated the endogenous mechanisms that regulate GITR expression on both T(R) and CD4+ T cells, as well as the functional interaction between GITR and other costimulatory molecules. CD28 stimulation increased GITR expression on both T(R) and CD4+ T cells via IL-2-dependent mechanisms. In addition, ligation of GITR and/or CD28 increased the level of CD4+ T cell proliferation and effector function under both APC-dependent and -independent conditions, suggesting that these costimulatory molecules cooperate to regulate CD4+ T cell activation and function by directly signaling to the CD4+ T cell. Thus, GITR may serve opposing functional roles on CD4+ T(R) and effector cells and alterations in GITR expression and/or function may tip the balance between immune tolerance and effector function.     

OX40 ligand regulates splenic CD8 dendritic cell-induced Th2 responses in vivo

In mice, splenic conventional dendritic cells (cDCs) can be separated, based on their expression of CD8α into CD8⁻ and CD8⁺ cDCs. Although previous experiments demonstrated that injection of antigen (Ag)-pulsed CD8⁻ cDCs into mice induced CD4 T cell differentiation toward Th2 cells, the mechanism involved is unclear. In the current study, we investigated whether OX40 ligand (OX40L) on CD8⁻ cDCs contributes to the induction of Th2 responses by Ag-pulsed CD8⁻ cDCs in vivo, because OX40–OX40L interactions may play a preferential role in Th2 cell development. When unseparated Ag-pulsed OX40L-deficient cDCs were injected into syngeneic BALB/c mice, Th2 cytokine (IL-4, IL-5, and IL-10) production in lymph node cells was significantly reduced. Splenic cDCs were separated to CD8⁻ and CD8⁺ cDCs. OX40L expression was not observed on freshly isolated CD8⁻ cDCs, but was induced by anti-CD40 mAb stimulation for 24 h. Administration of neutralizing anti-OX40L mAb significantly inhibited IL-4, IL-5, and IL-10 production induced by Ag-pulsed CD8⁻ cDC injection. Moreover, administration of anti-OX40L mAb with Ag-pulsed CD8⁻ cDCs during a secondary response also significantly inhibited Th2 cytokine production. Thus, OX40L on CD8⁻ cDCs physiologically contributes to the development of Th2 cells and secondary Th2 responses induced by Ag-pulsed CD8⁻ cDCs in vivo.