Processing and release of IL-16 from CD4+ but not CD8+ T cells is activation dependent

IL-16 is synthesized as a precursor molecule of 68 kDa (pro-IL-16) that is processed by caspase-3, a member of the IL-1 converting enzyme (ICE) family. This cleavage results in a 13-kDa carboxy terminal peptide, which constitutes the bioactive secreted form of IL-16. We have previously reported constitutive IL-16 mRNA expression and pro-IL-16 protein in CD4+ and CD8+ T cells. Although bioactive IL-16 protein is present in unstimulated CD8+ T cells, there is no bioactive IL-16 present in CD4+ T cells. Along these lines, unstimulated CD8+ T cells contain active caspase-3. In the current studies we investigated the regulation of IL-16 protein and mRNA expression in CD4+ T cells and determined the kinetics of secretion following stimulation of the TCR. CD4+ T cells release IL-16 protein following antigenic stimulation, and this release is accelerated in time by costimulation via CD28. However, CD3/CD28 costimulation did not alter IL-16 mRNA appearance or stability in either CD4+ or CD8+ T cells. The secretion of bioactive IL-16 from CD4+ T cells correlated with the appearance of cleavage of pro-caspase-3 into its 20-kDa active form. Thus, resting CD8+ T cells contain active caspase-3 that is capable of cleaving pro-IL-16, whereas CD4+ T cells require activation for the appearance of active caspase-3. The mechanism of release or secretion of bioactive IL-16 is currently unknown, but does not correlate with cellular apoptosis.     

Selective migration of highly differentiated primed T cells, defined by low expression of CD45RB, across human umbilical vein endothelial cells: effects of viral infection on transmigration

Low expression of CD45RB on CD45RO+ T lymphocytes defines a subset of highly differentiated T lymphocytes that accumulate in vivo within the affected joints of patients with rheumatoid arthritis (RA). Although it is known that CD45RO+ T lymphocytes migrate to sites of inflammation in vivo, it is not clear whether within this subset the CD45RBlo cells are selectively recruited or develop in situ within the joint. Using a transwell system we show that a small proportion of resting T lymphocytes migrated across unactivated human umbilical vein endothelial cells (HUVEC). These migrating cells were CD45RO+ and enriched for low CD45RB expression. In addition, both the CD45RO+CD45RBlo subset and migrating cells expressed increased levels of beta 1 and beta 2 integrins and CD44. The percentage of CD45RO+CD45RBlo T lymphocytes was increased in the circulation of patients with acute Epstein-Barr virus (EBV) infection. These in vivo activated cells also expressed increased levels beta 1 and beta 2 integrins and CD44, and showed an enhanced rate of transmigration compared with resting T lymphocytes. Transmigration of T lymphocytes was increased using the chemokines RANTES and lymphotactin and the cytokine interleukin-15 (IL-15). In addition, infection of the HUVEC with cytomegalovirus (CMV) led to an enhanced movement of T lymphocytes. In all of these cases the selective migration of the CD45RBlo subset was maintained. Thus although the rate of T-lymphocyte transmigration could be influenced by a number factors, the CD45RO+CD45RBlo subset has a migratory advantage suggesting that more differentiated CD45RO+CD45RBlo T lymphocytes are selectively recruited to sites of inflammation.     

Beta-galactoside-binding protein secreted by activated T cells inhibits antigen-induced proliferation of T cells

We have used mRNA differential display PCR to search for genes induced in activated T cells and have found the LGALS1 (lectin, galactoside-binding, soluble) gene to be strongly up-regulated in effector T cells. The protein coded by the LGALS1 gene is a beta-galactoside-binding protein (betaGBP), which is released by cells as a monomeric negative growth factor but which can also associate into homodimers (galectin-1) with lectin properties. Northern blot analysis revealed that ex vivo isolated CD8+ effector T cells induced by a viral infection expressed high amounts of LGALS1 mRNA, whereas LGALS1 expression was almost absent in resting CD8+ T cells. LGALS1 expression could be induced in CD4+ and CD8+ T cells upon activation with the cognate peptide antigen and high levels of LGALS1 expression were found in concanavalin A-activated T cells but not in lipopolysaccharide-activated B cells. Gel filtration and Western blot analysis revealed that only monomeric betaGBP was released by activated CD8+ T cells and in vitro experiments further showed that recombinant betaGBP was able to inhibit antigen-induced proliferation of naive and antigen-experienced CD8+ T cells. Thus, these data indicate a role of betaGBP as an autocrine negative growth factor for CD8+ T cells.     

Absence of bcl-2 expression by activated CD45RO+ T lymphocytes in acute infectious mononucleosis supporting their susceptibility to programmed cell death

bcl-2 proto-oncogene encodes an inner mitochondrial membrane protein that blocks programmed cell death (apoptosis). There is now increasing evidence that regulation of bcl-2 expression is a determinant of life or death in normal lymphocytes. We have recently described that activated (CD45RO+) CD4+ and CD8+ T cells in acute infectious mononucleosis (IM) undergo apoptotic cell death on culturing, indicating an activation-driven cell death of mature T cells. In this work, we examine bcl-2 expression by activated T cells in acute IM using a flow-cytometric analysis with an anti-bcl-2 monoclonal antibody (MoAb). It was consistently observed that most T cells from acute IM patients displayed only much less bcl-2, while normal T cells expressed bcl-2 relatively strongly. Multicolor analysis showed that bcl-2-lacking T cells in acute IM were restricted to the CD45RO+ (activated) populations of CD4+, as well as CD8+ T cells. In contrast, the relatively intense levels of bcl-2 were expressed in both CD45RO+ and CD45RO- T-cell populations from normal subjects. This marked difference in bcl-2 expression of CD45RO+ T cells between acute IM and normal controls was also confirmed by Western blot analysis. Activated (CD45RO+) T cells with low bcl-2 expression, but not bcl-2-expressing CD45RO- T cells, in acute IM patients were found to die easily when cultured without added growth factors. However, in normal individuals, both CD45RO+ and CD45RO- T cells were relatively stable on culturing. These findings suggest that lack of bcl-2 expression by activated (CD45RO+) T cells in acute IM might be associated with their susceptibility to programmed cell death.     

Interleukin-7 modulates CD23 and HLA-DR expression on CD4+ T cells and promotes a Th-2 type citokine profile

The expression of CD23 on PHA-activated human PBT (peripheral blood T) cells of healthy donors was investigated. It appears that CD23 is expressed solely on activated CD4+ T cells. Cytofluorotometric analysis revealed that 6% of PHA-activated CD4+ T cells expressed CD23, while unstimulated CD4+ T cells express no detectable CD23. The addition of IL-7 (1000 U/ml) to activated CD4+ T cells resulted in a marked augmentation of CD23 expression (29%). CD23 expression was blocked by M20 and M26 mAbs, but no reduction was detected by anti-IL-2R (CD25) mAb. This suggests that IL-7 has a specific regulatory effect on CD23 expression independent of IL-2. Northern Blot analysis showed a marked increase of CD23 mRNA detected in PHA-activated CD4+ T cells plus IL-7. IL-7 was also able to upregulate the expression of HLA-DR on activated CD4+ T cells. Optimal HLA-DR and CD23 induction by IL-7 occurred at 48 and 72 h of culture. The addition of CHX revealed that the induction of CD23 and HLA-DR by IL-7 required intact protein synthesis. Furthermore, PHA activated CD4+ T cells cultured in the presence of IL-7 are polarized to a Th-2 pattern of cytokine production.     

Increased TNF-alpha-induced apoptosis in lymphocytes from aged humans: changes in TNF-alpha receptor expression and activation of caspases

Aging is characterized by increased T cell lymphopenia, T cell dysfunction, and increased serum TNF levels. In this study, we have examined the role of TNF-induced apoptosis in T cell deficiency in lymphocytes from aged humans. The constitutive expression of TNF receptors (TNFRI and TNFRII) and the adapter molecules, including TNFR-associated death domain protein (TRADD), TNFR-associated factor 2 (TRAF-2), and receptor interacting protein (RIP), were analyzed both at the protein level by flow cytometry or Western blotting, and at the mRNA level using quantitative PCR or Northern blotting in lymphocytes from aged and young subjects. The susceptibility of T cells to undergo TNF-induced apoptosis was analyzed using terminal deoxynucleotidyltransferase-mediated UTP-end-labeling (TUNEL) and DNA ladder assays. Caspase (caspase-8 and caspase-3) activation was compared between aged and young subjects using Western blotting and colorimetric assays. In lymphocytes from aged humans, there was an increased susceptibility of CD4+ and CD8+ T cells to undergo TNF-alpha-induced apoptosis, as observed by TUNEL assay and DNA fragmentation ladder assay. Increased TNF-alpha-induced apoptosis was also observed in both CD45RA+ and CD45RO+ T cells from aging subjects. An increased constitutive expression of TNFRI and TRADD and decreased expression of TNFRII and TRAF-2 were observed in lymphocytes from aged as compared with young controls. In addition, there was an early and increased activation of caspases (caspase-8 and caspase-3) involved in TNFR/TNF signaling pathway, as evident by early cleavage of caspase-8, poly(ADP-ribose) polymerase (PARP), and caspase-3 substrate DEVD-p-nitroamilide NA. These data suggest that an increased TNF-alpha-induced apoptosis may play a role in T cell deficiency associated with human aging.     

Differential localization of Th1 and Th2 cells in autoimmune gastritis

The vast majority of CD4+ T cells infiltrating into gastric mucosa (GM) and in the draining (gastric) lymph node (GLN) shows an activated/memory phenotype, CD45RB(low) L-selectin(low) CD44(high), in neonataly thymectomized BALB/c mice bearing autoimmune gastritis (AIG), indicating that these cells are actively involved in this disease. CD4+ T cells sort-purified from GLN expressed mRNAs encoding for both IFN-gamma and IL-4. However, those infiltrating into GM expressed very low levels of IL-4 mRNA, even though they strongly expressed IFN-gamma mRNA. Among CD4+ T cells separated from AIG mice expressing detectable levels of either IFN-gamma or IL-4 by intracellular staining, less than one-seventh expressed IL-4 and thus most of them expressed IFN-gamma in GM, whereas roughly half and one-third expressed IL-4 in GLN and spleen respectively. These findings indicate that the Th1 cells predominantly infiltrate into autoimmune lesions and Th2 cells are mainly resident in the regional LN. We further set up an in vitro model system of transendothelial migration using a murine endothelial cell line, F-2, and found that Th1 cells in CD4+ T cells separated from lymphoid tissues of AIG mice preferentially passed through the monolayer of endothelial cells while only a small portion of Th2 cells did so. This differing ability of transendothelial migration and localization might explain the dominance of Th1 cells destroying the tissue in focal lesions without inhibition by the Th2 cells, in spite of both subsets being simultaneously activated in AIG mice, and the functions of each T cell subset seems to be mutually exclusive.     

Evidence for the presence of a kininogen-like species in a case of total deficiency of low and high molecular weight kininogens

EBI1-ligand chemokine (ELC) is a CC chemokine constitutively expressed in various lymphoid tissues and a high-affinity functional ligand for EBI1/CCR7, a seven transmembrane G-protein-coupled receptor originally identified as an Epstein-Barr virus (EBV)-inducible gene. Here we examined chemotactic activity of ELC on peripheral blood leukocytes. ELC attracted both CD4+ and CD8+ T cells, particularly efficiently after activation with IL-2 or with phytohemagglutinin (PHA) plus IL-2, as well as CD19+ B cells, but not CD16+ NK cells, CD14+ monocytes or neutrophils. Among CD3+ T cells, ELC attracted both CD45RO- naive and CD45RO+ memory subsets. ELC also induced vigorous calcium mobilization in T cells stimulated with IL-2 with an ED50 of 3 nM. ELC fused with the secreted form of alkaline phosphatase (ELC-SEAP) specifically bound to lymphocytes and this binding was blocked only by ELC among 10 CC chemokines so far tested. Notably, lymphocytes stimulated with IL-2 or T cells expanded by PHA plus IL-2 showed much higher levels of binding than fresh lymphocytes. Consistently, CCR7 mRNA was detected in CD4+ and CD8+ T cells as well as B cells, but not in NK cells, monocytes or neutrophils, and was dramatically increased in T cells upon treatment with IL-2 or with PHA plus IL-2. Like ELC mRNA, CCR7 mRNA was expressed in various lymphoid tissues. By in situ hybridization, ELC and CCR7 mRNA were detected in the parafollicular and inner cortical regions of a lymph node, and in the parafollicular regions of an appendix. Collectively, ELC and CCR7 may be involved in the trafficking of a broad spectrum of lymphocytes, especially activated T cells, into and within various lymphoid tissues.     

IL-12 up-regulates CD40 ligand (CD154) expression on human T cells

IL-12 is a heterodimeric cytokine produced by APC that promotes the development of CD4+ Th1 cells and their IFN-gamma production after TCR/CD3 triggering. We here investigated the capacity of IL-12 to modify the expression on T cells of CD40 ligand (CD40L or CD154), a molecule transiently expressed on activated T cells and known to be of utmost importance for cognate interaction with B cells and for activation of dendritic cells and macrophages. Our data demonstrate that IL-12 up-regulates CD40L expression on anti-CD3-activated human peripheral blood T cells. For optimal induction of CD40L, IL-12 synergizes with IL-2 as well as with other costimulatory interactions, such as B7/CD28. The effect of IL-12 was observed at both the protein and the mRNA level. T cells costimulated by IL-12 provided more efficient help for IL-4-dependent B cell proliferation and for IgG production than when activated in the absence of IL-12. This helper activity was blocked by an mAb against CD40L, indicating that the effect of IL-12 on B cells is mediated indirectly through CD40L. The data thus suggest that the effects of IL-12 on cellular and humoral immune responses are partly mediated through CD40L induction.     

CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28- T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor-associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase-4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.     

A comparison of analytical methods for the study of fractional Brownian motion

Interleukin-3 (IL-3) is expressed in T lymphocytes and stimulates the growth of multipotent hematopoietic progenitors. Little is known, however, about the stimuli that lead to IL-3 protein release. We examined IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNA expression and protein secretion in human T lymphocytes following activation via the TCR/CD3 complex, the CD2 receptor, and the IL-2 receptor. GM-CSF mRNA expression and protein release were found in CD3 and CD2 activated T cells with maximum GM-CSF release following stimulation with IL-2. IL-3 protein release is regulated via the CD2 receptor with virtually no IL-3 release after T cell stimulation via CD3. In contrast, IL-3 mRNA accumulation is more pronounced after CD3 activation than after CD2 activation. This suggests that upregulation of IL-3 protein release following T cell stimulation via CD-2 occurs largely at the translational or posttranslational level. These data also indicate that differential control of cytokine production can occur in response to activation of the alternative T cell receptor. Interaction of the T cell CD2-receptor with its natural ligand LFA-3 expressed on stromal cells might represent a regulatory mechanism for rapid release of IL-3, facilitating proliferation of multipotent hematopoietic cells.     

Coexpression of GD3 ganglioside with CD45RO in resting and activated human T lymphocytes

The ganglioside GD3 is preferentially expressed on the surface of malignant T cell lymphoblasts and on resting T cells which express the memory cell phenotype, CD45RA-CD29+. However, GD3 expression in activated T cells and its potential function in proliferating normal and malignant T cells are unclear. Utilizing three-color immunostaining and flow cytometry, we examined changes in the expression of GD3 in conjunction with the RA and RO isoforms of CD45 during in vitro T cell activation. GD3 was equally expressed in resting CD4 and CD8 cells and was specifically found in the CD45RO+RA population. Activation of T cells with PHA resulted in an increased percentage of GD3+ cells. This increase was evident by the first day and was observed in the CD45RO (naive cell) population; by 2 days, GD3 was expressed heterogeneously in a large population of CD45RO+RA+ cells. Further activation of T cells with PHA or anti-CD3 monoclonal antibody (OKT3) resulted in a further increase in GD3-expressing cells, and the increase in GD3 density correlated with increased CD45RO and loss of CD45RA. In contrast, increases in GD3 and interleukin-2 receptor (CD25) expression in response to PHA or OKT3 occurred independently, indicating that the GD3/ CD45RO coexpression observed was not a general consequence of cell activation. The results provide evidence for specific comodulation of GD3 and CD45RO during T cell mitogenesis, and thus suggest that these molecules may colocalize on the T cell surface.     

The monoclonal antibody CZ-1 identifies a mouse CD45-associated epitope expressed on interleukin-2-responsive cells

We have previously described a monoclonal antibody (mAb), CZ-1, which reacts with an epitope expressed on most peripheral basophils, natural killer cells, B cells, and CD8+ T cells, but not with most thymocytes or peripheral CD4+ T cells. Here we show that mAb CZ-1 defines a sialic acid-dependent epitope associated with a subpopulation of CD45 molecules. This conclusion is based on the ability to block binding of mAb CZ-1 by sialic acid, neuramin-lactose, neuraminidase, and mAb to CD45RB, and by expression of the epitope on transfected psi 2 cells expressing exon B of CD45. The results suggest that the CZ-1 epitope is a post-translational modification expressed on a subpopulation of the CD45 molecules also expressing the B exon. Expression of the CZ-1 epitope was required for freshly isolated lymphocytes to respond to interleukin-2 (IL-2). Depletion of CZ-1+ cells by C' or by cell sorting of thymocytes or splenocytes eliminated the IL-2 responsive cells. The subpopulations of thymocytes and CD4+ splenocytes responding to IL-2 were exclusively within the small CZ-1+ subpopulation. mAb CZ-1 was also used to subdivide CD45+ and CD45RB+ splenocytes into IL-2-responsive and -nonresponsive subpopulations. The CZ-1 epitope was also expressed on virtually all lymphokine-activated killer cell precursors. These data, thus, indicate that cells responsive to IL-2 express this sialated modification of CD45.     

Collaborative interactions between MEF-2 and Sp1 in muscle-specific gene regulation

We recently reported that previously activated T cells, irrespective of the nature of the first stimulus they encountered, are unable to respond to Staphylococcal enterotoxin B (SEB), nor to soluble anti-CD3 monoclonal antibody (mAb) presented by splenic antigen-presenting cells (APC). Such previously activated T cells are, however, fully capable of responding to plate-bound anti-CD3 plus splenic APC. These data suggest differential integration of the T-cell receptor (TCR) and co-stimulatory signalling pathways in naive versus antigen-experienced T cells. Consistent with this hypothesis, anti-CD28 mAb restores the proliferative capacity of resting ex vivo CD45RBlo CD4+ T cells (representing previously activated T cells) to both soluble anti-CD3 mAb and SEB. Interestingly, mAb-mediated engagement of cytotoxic T-lymphocyte antigen-4 (CTLA-4) completely negates the rescue effects mediated by anti-CD28 mAb in CD45RBlo cells. Nevertheless, the non-responsiveness of CD45RBlo CD4+ T cells cannot be reversed by anti-CTLA-4 Fab fragments, indicating that it is not related to negative regulatory effects of CTLA-4 engagement itself. Interestingly, the addition of interleukin-2 (IL-2) restores the proliferative capacity of CD45RBlo CD4+ T cells to SEB and soluble anti-CD3 mAb. Moreover, when rescued by IL-2, the cells are less susceptible to the negative regulatory effects of CTLA-4 engagement. Together, these findings suggest that the non-responsiveness of CD45RBlo CD4+ T cells to certain stimuli may be related to inadequate TCR signalling, primarily affecting IL-2 production.     

Regulation of IL-17, IFN-gamma and IL-10 in human CD8(+) T cells by cyclic AMP-dependent signal transduction pathway

In the present study, the expression of interleukin 17 (IL-17) by human CD8(+) T lymphocytes and its regulation following PKA activation was determined and compared with that of interferon gamma (IFN-gamma) and IL-10. IL-17 mRNA was highly expressed in human CD8(+) T lymphocytes at least at the same level than in CD4(+) T cells that were isolated from peripheral blood mononuclear cells (PBMC). Expression of IL-17 mRNA in CD8(+) T cell was induced by prior activation of PBMC for 18 h with Ca2+ ionophore and phorbol myristate acetate (PMA). Furthermore, our results clearly showed that CD8(+) T cells are sensitive to elevation of cAMP and PKA activation pathway. Data demonstrated a significant inhibition of IL-17 as well as of IFN-gamma mRNA expression in CD8(+) T cells isolated from activated PBMC cultured in the presence of either dibutyryl cAMP (db-cAMP) or PGE2. In contrast, IL-10 mRNA expression was strongly enhanced in the same experimental conditions. The differential expression of IL-10 and IFN-gamma production in CD8(+) T cells was also observed at the protein level as it was measured by a double immunofluorescence technique and flow cytometry analysis. Taken together, these results provide evidence that human CD8(+) T cells are also the source of massive expression of IL-17, and that PKA plays a prominent role in the switch of CD8(+) T cells to a Th2 like profile and an inhibition of IL-17 expression, thus suggesting that the activation of cAMP signal transduction pathway may have consequences for the relative role of CD8(+) T cells in the immune and inflammatory process.     

IL-4 regulation of perforin gene expression and BLT-esterase production in alpha CD3-induced activated killer cells

This present study examines Il-4 regulation of perforin gene expression and cytolytic granule production in alpha CD3-induced activated killer cells CD3-AK. After stimulation of resting T cells with alpha CD3, proliferative response could be detected at 1 day after activation. The expression of perforin mRNA and production of cytolytic granules (using BLT-E as indicator) was detected on days 2-4, and this time course correlated with the generation of lytic CD3-AK cells. These findings indicate that killer cells generation is a late event during the course of alpha CD3 activation. Generation of CD3-AK cells is primarily PKC dependent and is blocked by the depletion or inhibition of PKC by PMA or SSP. These changes are accompanied by the suppression of perforin gene expression (mRNA) and BLT-E production. However, adding IL-4 into the cultures restored the perforin mRNA expression and BLT-E production, and also the cytolytic activity of the CD3-AK cells. Furthermore, for preactivated CD3-AK cells cultured in IL-2, SSP also suppressed the perforin mRNA and BLT-E with the concomitant reduction of cytolytic activity. Similar to the resting T cells, in the SSP-maintained preactivated CD3-AK cells, switching the cytokine from IL-2 to IL-4/IL-2 restored perforin mRNA expression and BLT-E production, with concomitant restoration of the cytolytic activity. In contrast, switching from IL-4/IL-2 gave the opposite effect. These results could be reproduced by using amiloride which also inhibited PKC activity but did not affect the growth of preactivated CD3-AK cells. These findings indicate that IL-4 may play a role in the late stage of alpha CD3 activation to regulate the expression of perforin gene and probably the translation process during the generation of activated killer cells.     

Human effector memory T cells express CD86: a functional role in naive T cell priming

The glycoprotein CD86 expressed on APCs provides a costimulatory signal necessary for an efficient activation of naive T cells. In contrast, there is controversy about the condition of expression and the function of CD86 on T cells. In this study, we have analyzed the phenotype and the biological activity of CD86+ T cells generated from human PBMC. Results show that CD86 expression on T cells is induced by long term stimulation via CD3 and IL-2R and is down-regulated as the cells become quiescent. The CD86-expressing cells are memory effector T cells: 1) they express CD45RO and high levels of the activation markers CD25, CD54, and HLA-Dr; 2) they selectively express CD30, CD40-ligand, and CD70; and 3) in response to stimulation, most of them produce IFN-gamma before dying by apoptosis. We then analyzed whether CD86 expressed on T cells is functional. Results show that paraformaldehyde-fixed CD86+ T cells enhance the proliferation and production of IFN-gamma by anti-CD3 mAb-stimulated naive T cells and induce proliferation of resting allogenic T cells. All these effects are prevented by neutralizing anti-CD86 mAbs. In contrast, we report no autocrine effect of CD86 in CD86+ T cell activation. In conclusion, these data show that human memory effector T cells express a functional form of CD86 that can costimulate naive T cell responses.     

Galectin-1 specifically modulates TCR signals to enhance TCR apoptosis but inhibit IL-2 production and proliferation

Galectin-1 is an endogenous lectin expressed by thymic and lymph node stromal cells at sites of Ag presentation and T cell death during normal development. It is known to have immunomodulatory activity in vivo and can induce apoptosis in thymocytes and activated T cells (1-3). Here we demonstrate that galectin-1 stimulation cooperates with TCR engagement to induce apoptosis, but antagonizes TCR-induced IL-2 production and proliferation in a murine T cell hybridoma and freshly isolated mouse thymocytes, respectively. Although CD4+ CD8+ double positive cells are the primary thymic subpopulation susceptible to galectin-1 treatment alone, concomitant CD3 engagement and galectin-1 stimulation broaden susceptible thymocyte subpopulations to include a subset of each CD4- CD8-, CD4+ CD8+, CD4- CD8+, and CD4+ CD8- subpopulations. Furthermore, CD3 engagement cooperates with suboptimal galectin-1 stimulation to enhance cell death in the CD4+ CD8+ subpopulation. Galectin-1 stimulation is shown to synergize with TCR engagement to dramatically and specifically enhance extracellular signal-regulated kinase-2 (ERK-2) activation, though it does not uniformly enhance TCR-induced tyrosine phosphorylation. Unlike TCR-induced IL-2 production, TCR/galectin-1-induced apoptosis is not modulated by the expression of kinase inactive or constitutively activated Lck. These data support a role for galectin-1 as a potent modulator of TCR signals and functions and indicate that individual TCR-induced signals can be independently modulated to specifically affect distinct TCR functions.     

HLA class I-specific inhibitory receptors in human T lymphocytes: interleukin 15-induced expression of CD94/NKG2A in superantigen- or alloantigen-activated CD8+ T cells

A fraction of human T lymphocytes, predominantly CD8+, express receptors for HLA class I molecules typical of natural killer cells (natural killer receptors or NKRs) that inhibit T cell receptor-mediated functions. Herein, we analyzed possible mechanism(s) leading to the expression of NKRs by T cells responding to superantigens or allogeneic cells in vitro. We show that, in the presence of interleukin 15 (IL-15), T cells (depleted of NKR+ cells) responding to toxic shock syndrome toxin 1 de novo express CD94, a molecule that is part of a heterodimeric NKR with a broad specificity for different HLA class I alleles. Maximal CD94 expression occurred when IL-15 was added shortly after the cells were placed into culture, and CD94 expression started 4-6 days after addition of IL-15. Although both CD4+ and CD8+ cells expressed CD94, the simultaneous expression of NKG2A (i.e., the other component of the CD94/NKG2A inhibitory NKR) was confined to CD8+ cells. Similar data were obtained in T cell populations activated in mixed lymphocyte cultures in the presence of IL-15. The expression of CD94/NKG2A led to an impairment of allo-specific cytolytic activity by mixed lymphocyte culture-derived T cell populations or clones. Remarkably, cytolysis could be restored by the addition of anti-CD94 mAb, i.e., by masking the inhibitory NKRs.     

Bcl-2 is expressed in human natural killer cells and is regulated by interleukin-2

Bcl-2 is a major anti-apoptotic protein expressed in many normal and malignant cells. Recently, low to absent expression was reported in human natural killer (NK) cells cultured in serum-free media which could be induced with stem cell factor. We investigated the expression of bcl-2 protein of NK cells in normal blood donors and compared the bcl-2 expression in CD56+ NK cells with CD3+ T cells. To determine bcl-2 reactivity, a three-color flow-cytometric technique was used. CD56+ CD3- NK cells had an average bcl-2 expression of 83% compared with CD3+ T cells. CD56 and CD3 double positive T cells had an average content of 111% compared with all peripheral CD3+ T lymphocytes. When peripheral mononuclear cells were cultured with interleukin-2 (IL-2), bcl-2 could be upregulated by IL-2 in all cell populations studied. The induction of bcl-2 in these cell populations paralleled the induction in CD56- T lymphocytes cultured under identical conditions. The induction of bcl-2 by IL-2 was confirmed by Western blotting. The maximum induction of bcl-2 by IL-2 was observed at an IL-2 dose of 100-1,000 U/ml. Our data confirm the anti-apoptotic protein bcl-2 as an activation- or proliferation-associated marker of normal NK cells which can be induced by IL-2.