CD28 T cell costimulatory receptor function is negatively regulated by N-linked carbohydrates

CD28 is a cell surface glycoprotein expressed on T cells that modulates immune responses through its ability to transduce costimulatory signals. Even though nearly 50% of the molecular mass of CD28 is N-glycan, the physiological significance of CD28 glycosylation is at present unknown. In this report, we have investigated the function of hypoglycosylated wildtype CD28 and its splice variant, CD28i. When N-glycosylation was prevented through point mutations in N-glycosylation sites in CD28, or reduced by glycosidase inhibitors, the binding of CD28 to CD80 significantly increased. Stimulation of hypoglycosylated CD28 induced IL-2 promoter activity greater than that induced through the stimulation of wildtype CD28. Unlike hypoglycosylated wildtype CD28, hypoglycosylation of CD28i did not alter CD28i functions. Our data indicate that N-glycans of CD28 negatively regulate CD28/CD80 interactions, resulting in diminished CD28 signaling. It is also suggested that N-glycans regulate the density of CD28 clustering upon ligation with CD80/CD86. The results support the hypothesis that the N-glycosylation negatively regulates CD28-mediated T cell adhesion and costimulation.     

Antigen-presenting cells containing multiple costimulatory molecules promote activation and expansion of human antigen-specific memory CD8<Superscript>+</Superscript> T cells

We have previously demonstrated that multiple immunizations with vector-based vaccines containing transgenes for tumor Ags and a triad of costimulatory molecules (TRICOM) enhance the expansion and functional avidity of Ag-specific memory CD8⁺ T cells in a mouse model. However, the effect of enhanced costimulation on human memory CD8⁺ T cells is still unclear. The study reported here was an in vitro investigation of the proliferation and function of CEA-specific human memory CD8⁺ T cells following enhanced costimulation. Our results demonstrated that TRICOM costimulation enhanced production of multiple cytokines and expansion of CEA-specific memory CD8⁺ T cells. The lytic capacity of memory CTLs toward CEA⁺ tumors was also significantly enhanced. IL-2Rα (CD25) was upregulated dramatically following APC-TRICOM stimulation, suggesting that the enhanced expansion of memory CD8⁺ T cells may be mediated by increased expression of IL-2R on memory T cells. The enhanced cytokine production and proliferation following TRICOM signaling was completely blocked by the combination of neutralizing Abs against B7-1, ICAM-1, and LFA-3, the costimulatory molecules comprising TRICOM. No difference in T-cell apoptosis was observed between APC-TRICOM and APC-wild-type groups, as determined by annexin V, Bcl-2, and active caspase-3 staining. Results indicated that enhanced costimulation greatly expanded human CEA-specific CD8⁺ T cells and enhanced T-cell function, without inducing increased apoptosis of CEA-specific memory CD8⁺ T cells.     

Quantitative interplay between activating and pro-apoptotic signals dictates T cell responses

Antigen-presenting cells (APC) can express surface ligands with both T cell activating and inhibitory capacities, prompting the question of how responding T cells integrate opposing trans signals concurrently delivered by APC. To address this question in a quantitative fashion, we turned to protein transfer as a unique experimental approach that is well-suited for addressing such questions from a quantitative standpoint. Costimulatory (either B7-1*Fc(gamma1) or Fc(gamma1)*4-1BBL) and pro-apoptotic (Fc(gamma1)*FasL) Fc fusion proteins were quantitatively "painted" in varying ratios onto surrogate APC pre-coated with palmitated-protein A, the latter serving as a surface anchor. Evaluating the signaling potential of these various painted cells in a standard in vitro T cell proliferation assay, we demonstrated that at a given level of TCR triggering, the quantitative balance between costimulator (B7-1 or 4-1BBL) and FasL dictates the magnitude of the proliferative T cell response. Furthermore, when the costimulator density is kept constant, there is also a quantitative balance between TCR-directed and FasL signals. Interesting species-specific nai;ve versus memory T cell subset differences emerged with regard to susceptibility to Fas-mediated apoptosis and costimulator:FasL opposition. Taken together, these data demonstrate for the first time a quantitative interplay between activating and pro-apoptotic trans signals that dictates the magnitude of T cell responses.     

Age-related changes in lck-Vav signaling pathways in mouse CD4 T cells

Activation of lck-fyn kinases during T cell receptor signaling leads to Vav phosphorylation, activation of downstream targets including Rac1, and a transient decline in ezrin and moesin phosphorylation. We have shown that age increases Rac1 activity and lowers ezrin and moesin phosphorylation in resting mouse CD4 cells, changes that could be the results of alterations in lck-Vav signaling. Analysis of Vav in CD4 cells from old mice shows increases in the phosphorylation of two key regulatory residues, Tyr160 and Tyr174, suggesting enhancement of Vav GTPase activity. In addition, analysis of lck status also shows age-related increases in phosphorylation of two key residues, Tyr394 and Tyr505, which have opposite effects on lck function. These changes in lck-Vav signals in resting CD4 cells may contribute in turn to age-related increases in Rac1 activity and declines in phosphorylation of cytoskeletal proteins including Ezrin and Moesin.     

Effect of fluvastatin on apoptosis in human CD4+ T cells

Statins are lipid-lowering agents with pleiotropic effects. We investigated the apoptotic effects of fluvastatin on peripheral CD4+ T cells from healthy subjects. Fluvastatin induced apoptosis in resting CD4+ T cells but not in CD4+ T cells strongly activated with a high concentration of PMA plus ionomycin (PMA/I) analyzed with annexin V and propidium iodide staining. However, CD4+ T cells activated with a low concentration of PMA/I or with anti-CD3 antibodies were apoptotic after treatment with fluvastatin. Activities of caspases-8, -9, and -3 were increased in resting CD4+ T cells treated with fluvastatin (10 microM). In strongly activated CD4+ T cells, fluvastatin inhibited the activation of caspase-8 induced by PMA/I and increased caspase-9 activity. The caspase-3 activity did not differ between untreated and fluvastatin-treated strongly activated CD4+ T cells. Treatment with fluvastatin (10 microM) enhanced cytochrome c release and increased the Bax/Bcl-2 ratio in both resting and strongly activated CD4+ T cells. Although the in vitro concentration of fluvastatin used in this study is higher than in vivo, other factors may sensitize apoptotic cell death of CD4+ T cells in vivo. In conclusion, fluvastatin induces apoptosis in resting T cells but not in strongly activated T cells, a difference that might be due to the interaction between caspase-8 and caspase-9.     

Differential regulation of soluble and membrane CD40L proteins in T cells

CD40 ligand is an important immunoregulatory protein expressed by T cells. This protein exists as two isoforms, a membrane glycoprotein and a truncated soluble form. Here we demonstrate that membrane and soluble CD40L (sCD40L) are differentially regulated depending upon the activation stimulus. In T cell receptor activated cells, both membrane and sCD40L proteins are expressed and CD28 costimulation further increases their expression. The dissection of TCR generated signals into calcium and PKC-dependent pathways demonstrates that calcium is sufficient to induce membrane CD40L yet insufficient for sCD40L. In contrast, sCD40L is preferentially induced by PKC. Moreover, sCD40L production is blocked by Zn(2+)-dependent metalloproteinase inhibitors while membrane CD40L is concurrently increased. This profile suggests the potential involvement of the ADAM-10 protease which was subsequently shown to cleave membrane CD40L to generate sCD40L. Given the role of sCD40L in numerous disease pathologies and its ability to activate proximal and distal immune responses, the regulated cleavage of CD40L may likely contribute to disease mechanisms.     

Impaired antigen-induced CD8+ T cell clonal expansion in aging is due to defects in antigen presenting cell function

CD8+ T cell activation depends on interaction with antigen-presenting cells (APCs) and this interaction leads to the expansion of T cells with the capacity to control infection. Using professional APCs, we demonstrate that with age, the duration of APC-T cell contact time required to achieve clonal expansion increases. Na?ve CD8+ T cells from aged mice showed no defect in antigen-induced proliferation when stimulated with APC from young mice. In contrast, CD8+ T cells from young mice exhibited reduced clonal expansion and secreted significantly lower amounts of IFN-gamma when stimulated by APCs from aged mice. The aged APCs were defective in costimulatory molecule expression and cytokine and chemokine secretion. These data indicate that defects in APC function lead to poor T cell clonal expansion and function in aging.     

Upregulation of ICOS on CD43+ CD4+ murine small intestinal intraepithelial lymphocytes during acute reovirus infection

Murine intestinal intraepithelial lymphocytes (IELs) can be classified according to expression of a CD43 glycoform recognized by the S7 monoclonal antibody. In this study, we examined the response of S7+ and S7- IELs in mice during acute reovirus serotype 3 (Dearing strain) infection, which was confirmed by virus-specific real-time PCR. In vivo proliferation increased significantly for both S7- and S7+ IELs on day 4 post-infection as determined by BrdU incorporation; however, expression of the inducible costimulatory (ICOS) molecule, which peaked on day 7 post-infection, was upregulated on S7+ CD4+ T cells, most of which were CD4+8- IELs. In vitro ICOS stimulation by syngeneic peritoneal macrophages induced IFN-gamma secretion from IELs from day 7 infected mice, and was suppressed by treatment with anti-ICOS mAb. Additionally, IFN-gamma mRNA increased in CD4+ IELs on day 6 post-infection. These findings indicate that S7- and S7+ IELs are differentially mobilized during the immune response to reovirus infection; that the regulated expression of ICOS is associated with S7+ IELs; and that stimulation of IELs through ICOS enhances IFN-gamma synthesis during infection.     

Interleukin-10 is upregulated by nanomolar rosiglitazone treatment of mature dendritic cells and human CD4+ T cells

Activators of peroxisome proliferator-activated receptor (PPAR)-gamma are anti-inflammatory and have been proposed as therapeutic agents for the treatment of Th1-type inflammatory diseases. We report that nanomolar concentrations of rosiglitazone enhance the production of IL-10 from activated human mature monocyte-derived dendritic cells. Also, rosiglitazone specifically induces the production of IL-10 from TCR-activated human CD4+ T cells and that this effect is PPAR-gamma-dependent. We also demonstrate for the first time the presence of a functional PPAR response element (PPRE) in the human IL-10 promoter region. Finally we show that rosiglitazone can induce IL-10 in combination with 1,25 alpha-dihydroxyvitamin D3 to a greater extent than each treatment alone. In summary our findings demonstrate that IL-10 is upregulated by nanomolar TZDs in immune cells, and this may, in part, be responsible for the potential anti-inflammatory effects of PPAR-gamma in humans.     

CD4+CD25+ regulatory T cells control the progression from periinsulitis to destructive insulitis in murine autoimmune diabetes

Non-obese diabetic (NOD) mice develop spontaneous T-cell responses against pancreatic beta-cells, leading to islet cell destruction and diabetes. Despite high genetic similarity, non-obese resistant (NOR) mice do not develop diabetes. We show here that spleen cells of both NOD and NOR mice respond to the islet cell antigen glutamic acid decarboxylase-65 in IFN-gamma-ELISPOT assays. Moreover, NOR-T cells induce periinsulitis in NOD SCID recipient mice. Thus, a potentially pathogenic islet cell-specific T-cell response arises in NOR and NOD mice alike; the mechanism that prevents the autoimmune progression of self-reactive T cells in NOR mice presumably acts at the level of effector function. Consistent with this hypothesis, CD4+CD25+ cell-depleted spleen cells from NOR mice mediated islet cell destruction and overt diabetes in NOD SCID mice. Therefore, islet cell-specific effector cells in NOR mice appear to be under the control of CD4+CD25+ regulatory T cells, confirming the importance of regulatory cells in the control of autoimmune diabetes.     

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.     

CD28 is required for induction and maintenance of immunological memory in toxin-reactive CD4+ T cells in vivo

We previously reported that Vbeta3+ CD4+ T cells maintained a protracted expansion, with the phenotypes of memory Th2 cells, for 30 days in C57BL/6 (B6) mice implanted with SEA-containing mini-osmotic pumps. In the present study, we followed the fate of Vbeta3+ CD4+ T cells in CD28-/- mice. Vbeta3+ CD4+ T cells increased to a degree similar to that of B6 Vbeta3+ CD4+ T cells until day 10 after implantation, then declined rapidly reaching the control level by 28 days. Remaining Vbeta3+ CD4+ T cells at that time did not exhibit memory phenotypes nor Th2-deviated responses. The rapid drop in Vbeta3+ CD4+ T cells in CD28-/- mice was attributable to upregulated induction of apoptosis owing to marginal inductions of Bcl-2 and Bcl-xL. Collectively, these data indicate CD28 to play critical roles in the generation and maintenance of SEA-reactive CD4+ T cells in vivo.     

Localized expression of GITR-L in the tumor microenvironment promotes CD8<Superscript>+</Superscript> T cell dependent anti-tumor immunity

The systemic administration of an agonist antibody against glucocorticoid-induced tumor necrosis factor receptor related (GITR) protein has been shown to be effective in overcoming immune tolerance and promoting tumor rejection in a variety of murine tumor models. However, little is known regarding the functional consequence of ligation of GITR with its natural ligand (GITR-L) in the context of regulatory T cell (Treg) suppression in vivo. To determine the mechanism of GITR-L action in vivo, we generated a panel of tumor cell clones that express varying levels of GITR-L. The ectopic expression of GITR-L on the tumor cell surface was sufficient to enhance anti-tumor immunity and delay tumor growth in syngeneic BALB/c mice. Within the range examined, the extent of anti-tumor activity in vivo did not correlate with the level of GITR-L expression, as all clones tested exhibited a similar delay in tumor growth. The localized expression of GITR-L on tumor cells led to a significant increase in CD8⁺ T cell infiltration compared to the levels seen in control tumors. The increased proportion of CD8⁺ T cells was only observed locally at the tumor site and was not seen in the tumor draining lymph node. Depletion studies showed that CD8⁺ T cells, but not CD4⁺ T cells, were required for GITR-L mediated protection against tumor growth. These studies demonstrate that signaling between GITR-L and GITR in the tumor microenvironment promotes the infiltration of CD8⁺ T cells, which are essential for controlling tumor growth. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cell cycle block in anergic T cells during tolerance induction

The induction of anergy, or T cell unresponsiveness to antigen, is preceded by T cell activation and cell division in response to fed antigens. These events parallel the activation observed in T cells following sensitization with antigen and adjuvant. The events that distinguish eventual sensitization versus tolerance remain unclear. Using a T lymphocyte transfer model specific to OVA, we demonstrated previously that oral encounter with antigen leads to functional anergy. Antigen-specific CD4+ T cells nevertheless become activated and cycle briefly after encounter with antigen. In this study, we measured the extent of cell cycling of antigen-specific T cells after oral encounter with their antigen. Whereas T cells cycle on the average of eight times in 4 days after conventional immunization, an abortive proliferation was observed in the draining LN T cells after oral encounter with antigen; OVA-specific T cells divided fewer times after exposure to fed OVA, compared to T cells in mice immunized with OVA. This abortive proliferation is antigen specific and not due to bystander suppression, as coadministration of an unrelated antigen that was previously used as a tolerogen does not alter the degree of abortive proliferation. Measurement of BrdU incorporation in mice that were previously fed ovalbumin indicates that up to 3 days following feeding, OVA-specific cells are actively cycling in vivo. However, by day 4, they have stopped cycling while identical T cells in OVA-sensitized mice continue to cycle. Our results indicate either that tolerance is a default pathway and a secondary stimulus is required at day 3 to progress to sensitization, or that elements that limit cell cycle progression are provided for tolerance induction.     

Mycobacterium tuberculosis-specific CD4+, IFNgamma+, and TNFalpha+ multifunctional memory T cells coexpress GM-CSF

Multifunctional T cells expressing several cytokines in parallel are thought to play a crucial role in protection against different infections. To characterize T cell cytokine patterns associated with disease and protection in Mycobacterium tuberculosis infection we determined the expression of IFNγ, IL-2, TNFα, and GM-CSF in T cell subpopulations from children with tuberculosis (TB) and healthy latently M. tuberculosis-infected children (LTBI) after short-term in vitro restimulation. We identified CD4⁺ effector memory T cells (T_EM) as the major source of all measured cytokines after antigen-specific restimulation. T_EM from children with TB expressed higher proportions of IFNγ, TNFα, and IL-2 after Mtb restimulation while no differences were detected for GM-CSF between both study groups. GM-CSF secretion strongly depended on antigen-specific stimulation. Analyses of multiple cytokine patterns revealed that the majority of GM-CSF-positive M. tuberculosis-specific memory T cells coexpressed IFNγ and TNFα therefore showing a characteristic feature of multifunctional T cells. We conclude that children with active TB possess higher proportions of IFNγ-, TNFα-, and/or IL-2-positive T_EM than children with LTBI while GM-CSF coexpression reveals a novel subpopulation within CD4⁺ memory T cells not increased in children with active TB.     

PD-1 ligands, negative regulators for activation of naive, memory, and recently activated human CD4+ T cells

We examined the role of the PD-1 pathway on the activation of naive, memory, and recently activated human CD4+ T cells to test whether they responded differently. PD-1 ligand blockade modestly enhanced the percentage of responding T cells and production of IFN-gamma in a primary response to myelin basic protein (MBP) in normal donors. PD-1 ligand blockade strongly enhanced proliferation and cytokine production by memory or recently activated T cells (tetanus toxoid and MBP). Blockade of PD-L1 alone had more effect than PD-L2, consistent with its higher expression on ex vivo dendritic cells; furthermore, anti-PD-L1 plus anti-PD-L2 resulted in the greatest enhancement. Moreover, PD-L1-Ig inhibited anti-CD3 induced activation of naive, memory, and recently activated CD4+ T cells. Together, our data demonstrated PD-1 functioned as a negative regulatory pathway on naive T cells during a primary response, and more potently, on memory or recently activated T cells during a secondary response.     

Cloning,expression, and characterization of fugu CD4, the first ectothermic animal CD4

We have cloned and sequenced the first ectothermic animal CD4 gene from fugu, Takifugu rubripes, using a public database of the third draft sequence of the fugu genome. The fugu CD4 gene encodes a predicted protein of 463 amino acids containing four extracellular immunoglobulin (Ig)-like domains, a transmembrane region, and a cytoplasmic tail. Fugu CD4 shares low identity of about 15–20% with avian and mammalian CD4 proteins. Unlike avian and mammalian CD4, fugu CD4 lacks the Cys pair of the first Ig-like domain, but has a unique possible disulfide bond in the third domain. These differences suggest that fugu CD4 may have a different structure that could affect binding of major histocompatibility complex class II molecules and subsequent T-cell activation. In the putative fugu cytoplasmic region, the protein tyrosine kinase p56^lck binding motif is conserved. The predicted fugu CD4 gene is composed of 12 exons, differing from other CD4 genes, but showing conserved synteny and many conserved sequence motifs in the promoter region. RT-PCR analysis demonstrated that the fugu CD4 gene is expressed predominantly in lymphoid tissues. We also show that fugu CD4 can be expressed on the surface of cells via transfection. Molecular characterization of CD4 in fish provides insights into the evolution of both the CD4 molecule and the immune system.     

4-1BB costimulation enhances HSV-1-specific CD8+ T cell responses by the induction of CD11c+CD8+ T cells

Since 4-1BB plays a predominant role in CD8+ T cell responses, we investigated the effects of 4-1BB triggering on the primary and memory CD8+ T responses to HSV-1 infection. 4-1BB was detected on 10-15% of CD4+ and CD8+ T cells following the infection. 4-1BB-positive T cells were in the proliferative mode and showed the enhanced expression of anti-apoptotic proteins. Agonistic anti-4-1BB treatment exerted preferential expansion of CD8+ T cells and gB/H-2Kb-positive CD8+ T cells, and enhanced cytotoxicity against HSV-1 that was mainly mediated by CD11c+CD8+ T cells. CD11c+CD8+ T cells were re-expanded following re-challenge with HSV-1 at post-infection day 50, indicating that CD11c+CD8+ phenotype was maintained in memory CD8+ T cell pool. Our studies demonstrated that 4-1BB stimulation enhanced both primary and memory anti-HSV-1 CD8+ T cell responses, which was mediated by a massive expansion of antigen-specific CD11c+CD8+ T cells.     

Interplay between T cell receptor binding kinetics and the level of cognate peptide presented by major histocompatibility complexes governs CD8+ T cell responsiveness

Through a rational design approach, we generated a panel of HLA-A*0201/NY-ESO-1(157-165)-specific T cell receptors (TCR) with increasing affinities of up to 150-fold from the wild-type TCR. Using these TCR variants which extend just beyond the natural affinity range, along with an extreme supraphysiologic one having 1400-fold enhanced affinity, and a low-binding one, we sought to determine the effect of TCR binding properties along with cognate peptide concentration on CD8(+) T cell responsiveness. Major histocompatibility complexes (MHC) expressed on the surface of various antigen presenting cells were peptide-pulsed and used to stimulate human CD8(+) T cells expressing the different TCR via lentiviral transduction. At intermediate peptide concentration we measured maximum cytokine/chemokine secretion, cytotoxicity, and Ca(2+) flux for CD8(+) T cells expressing TCR within a dissociation constant (K(D)) range of ～1-5 μM. Under these same conditions there was a gradual attenuation in activity for supraphysiologic affinity TCR with K(D) < ～1 μM, irrespective of CD8 co-engagement and of half-life (t(1/2) = ln 2/k(off)) values. With increased peptide concentration, however, the activity levels of CD8(+) T cells expressing supraphysiologic affinity TCR were gradually restored. Together our data support the productive hit rate model of T cell activation arguing that it is not the absolute number of TCR/pMHC complexes formed at equilibrium, but rather their productive turnover, that controls levels of biological activity. Our findings have important implications for various immunotherapies under development such as adoptive cell transfer of TCR-engineered CD8(+) T cells, as well as for peptide vaccination strategies.