The cathepsin B inhibitor, z-FA-FMK, inhibits human T cell proliferation in vitro and modulates host response to pneumococcal infection in vivo

The cathepsin B inhibitor, benzyloxycarbonyl-phenyl-alanyl-fluoromethylketone (z-FA-FMK) at nontoxic doses was found to be immunosuppressive and repressed human T cell proliferation induced by mitogens and IL-2 in vitro. We showed that z-FA-FMK suppresses the secretion of IL-2 and IFN-gamma as well as the expression of IL-2R alpha-chain (CD25) in activated T cells, whereas the expression of the early activated T cell marker, CD69, was unaffected. Furthermore, z-FA-FMK blocks NF-kappaB activation, inhibits T cell blast formation, and prevents cells from entering and leaving the cell cycle. z-FA-FMK inhibits the processing of caspase-8 and caspase-3 to their respective subunits in resting T cells stimulated through the Ag receptor, but has no effect on the activation of these caspases during Fas-induced apoptosis in proliferating T cells. When administered in vivo, z-FA-FMK significantly increased pneumococcal growth in both lungs and blood, compared with controls, in a mouse model of intranasal pneumococcal infection. Because host response to bronchopneumonia in mice is T cell dependent, our collective results demonstrated that z-FA-FMK is immunosuppressive in vitro and in vivo.     

Glutathione modulates activation-dependent proliferation of human peripheral blood lymphocyte populations without regulating their activated function

L-Buthionine-(S,R)-sulfoximine (BSO) specifically depletes GSH synthesis by inactivating gamma-glutamylcysteine synthetase, whereas 2-ME augments intracellular GSH concentration. These reagents were used to examine GSH regulation of the proliferation and function of human PBL in response to IL-2 or OKT-3 mAb directed at the CD3 T cell Ag. 2-ME enhanced both IL-2-induced proliferation of PBL and CD3- large granular lymphocytes (LGL) and OKT-3 mAb-induced proliferation of CD3+ T cells. BSO partially suppressed activation-induced proliferation in CD3- LGL and CD3+ T cells and totally inhibited the positive co-proliferative regulation by 2-ME in these cells. By contrast, neither BSO nor 2-ME appeared to affect the activation-dependent differentiation of cytotoxic lymphocytes. The absence of effect of 2-ME or BSO on activation-induced PBL NK activity and T cell cytotoxic potential was supported by their negligible effect on the induction of two different markers of activated cytotoxic lymphocytes, namely pore-forming protein gene expression and benzoyloxycarbonyl-1-L-lysine thiobenzylester-esterase activity. BSO inhibition of CD3- LGL proliferation accounted for the inhibitory effects of BSO on both IFN-gamma production in IL-2-stimulated PBL cultures and IL-2-induced PBL lymphokine activated killer activity. The modulatory effects of 2-ME and BSO on lymphocyte proliferation regardless of phenotype (LGL vs T cell) or stimulation (IL-2, via CD3, lectin, etc.) and the functional differentiation of cytotoxic lymphocytes independent of proliferation suggests that these cells share a common site of GSH regulation close to or at the level of DNA synthesis.     

Monocytes stimulated by 110-kDa fibronectin fragments suppress proliferation of anti-CD3-activated T cells

One hundred ten to 120-kDa fragments of fibronectin (FNf), generated by proteases released in the course of tissue injury and inflammation, stimulate monocytes to produce proinflammatory cytokines, promote mononuclear leukocytes (MNL) transendothelial migration, up-regulate monocyte CD11b and CD86 expression, and induce monocyte-derived dendritic cell differentiation. To investigate whether the proinflammatory consequences of FNf are offset by responses that can suppress proliferation of activated T lymphocytes, we investigated the effect of FNf-treated MNL on autologous T lymphocytes induced to proliferate by substrate-immobilized anti-CD3. FNf-stimulated MNL suppressed anti-CD3-induced T cell proliferation through both contact-dependent and contact-independent mechanisms. Contact-independent suppression was mediated, at least in part, by IL-10 and TGF-beta released by the FNf-stimulated MNL. After 24-48 h exposure to FNf, activated T cells and monocytes formed clusters displaying CD25, CD14, CD3, and CD4 that were not dissociable by chelation of divalent cations. Killing monocytes with l-leucine methyl ester abolished these T cell-monocyte clusters and the ability of the FNf-stimulated MNL to suppress anti-CD3 induced T cell proliferation. Thus, in addition to activating MNL and causing them to migrate to sites of injury, FNf appears to induce suppressor monocytes.     

Differential immunomodulation by anti-CD2 monoclonal antibodies of anti-CD3-induced T cell activation: dependence upon the individual anti-CD3 monoclonal antibody used for activation

Two monoclonal antibodies (mAb) recognizing different CD2 epitopes each inhibited anti-CD3-induced proliferation and anti-CD3-induced increase in surface CD2 expression. The magnitude of inhibition by either anti-CD2 mAb was dependent upon which anti-CD3 mAb was used as the stimulus, being more pronounced when the anti-CD3 mAb 454 was used as the stimulus than when either anti-CD3 mAb 147 or 446 was the stimulus. The effects of neuraminidase-treated sheep erythrocytes (which bind to CD2) were also more pronounced on mAb 454-induced proliferation than on mAb 147- or 446-induced proliferation. Furthermore, the effects of preincubation with anti-CD2 mAb depended upon the responder status of the donor to IgG1 anti-CD3 mAb. Preincubation of high-responder cells with anti-CD2 mAb had little effect on subsequent IgG1 anti-CD3-induced proliferation. In contrast, preincubation of low-responder cells with anti-CD2 mAb usually augmented the otherwise small proliferative response to IgG1 anti-CD3 mAb. Taken together, these observations suggest that interaction of surface CD2 with ligand alters the response of T cells to anti-CD3 mAb, but these effects depend upon the individual anti-CD3 mAb used for stimulation. These studies raise the possibility that perturbation of different parts of the CD3-T cell antigen receptor complex may lead to different sequelae, and, as a result, the T cell may respond to a given immunomodulator in different ways.     

Inhibition of Neutral Sphingomyelinase Activation and Ceramide Formation by Glutathione in Hypoxic PC12 Cell Death

Reduced glutathione (GSH) and N-acetylcysteine (NAC), but not other antioxidative or reducing agents, were found to inhibit cell death, both apoptosis and necrosis, induced by hypoxia in naive and nerve growth factor-differentiated PC12 cells. The level of intracellular total GSH decreased time-dependently during hypoxia, but exogenously added GSH prevented such a decrease in GSH. Pretreatment of cells with exogenous GSH or NAC resulted in inhibition of both neutral sphingomyelinase (SMase) activation and ceramide formation during hypoxia. In the in vitro assay system, neutral SMase activity was inhibited dose-dependently by GSH and NAC. Activation of caspase-3 induced by hypoxia was also inhibited by either GSH or NAC. NAC but not GSH inhibited caspase-3 activation induced by C2-ceramide. These results suggest that GSH protects cells from hypoxic injury by direct inhibition of neutral SMase activity and ceramide formation, resulting in inhibition of caspase-3 activation, and that NAC exerts an additional inhibitory effect(s) downstream of ceramide.     

CD7 augments T cell proliferation via the interleukin-2 autocrine pathway.

The role of CD7, a T cell differentiation antigen, in T cell function is not known at present; this study evaluates the effect of anti-CD7 mAb in PMBC cultures activated with suboptimal concentrations of lectins, antigens, and anti-CD3 mAb. We found that the inclusion of anti-CD7 resulted in increased IL-2 production and IL-2R-alpha expression in these cultures. H-7, a protein kinase C (PKC) inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, significantly suppressed the proliferation of T cells in comitogenic assays. This suggested that the comitogenic effect mediated by CD7 molecule involved both the PKC and the PTK pathways of T cell activation. These drugs appeared to affect the CD7-mediated effects by inhibiting the IL-2 autocrine pathway, especially the up-regulation of IL-2R-alpha since inhibition was not relieved with exogenous rIL-2. Taken together, our results suggest that CD7 augments T cell function by up-regulating IL-2R-alpha expression and IL-2 production via multiple pathways of protein phosphorylation.     

CD28-independent costimulation of T cells by OX40 ligand and CD70 on activated B cells.

OX40 and its ligand (OX40L) have been implicated in T cell-dependent humoral immune responses. To further characterize the role of OX40/OX40L in T-B cell interaction, we newly generated an anti-mouse OX40L mAb (RM134L) that can inhibit the costimulatory activity of OX40L transfectants for anti-CD3-stimulated T cell proliferation. Flow cytometric analyses using RM134L and an anti-mouse OX40 mAb indicated that OX40 was inducible on splenic T cells by stimulation with immobilized anti-CD3 mAb in a CD28-independent manner, while OX40L was not expressed on resting or activated T cells. OX40L was inducible on splenic B cells by stimulation with anti-IgM Ab plus anti-CD40 mAb, but not by either alone. These activated B cells exhibited a potent costimulatory activity for anti-CD3-stimulated T cell proliferation and IL-2 production. Anti-CD80 and anti-CD86 mAbs partially inhibited the costimulatory activity, and further inhibition was obtained by their combination with RM134L and/or anti-CD70 mAb. We also found the anti-IgM Ab- plus anti-CD40 mAb-stimulated B cells exhibited a potent costimulatory activity for proliferation of and IL-2 production by anti-CD3-stimulated CD28- T cells from CD28-deficient mice, which was substantially inhibited by RM134L and/or anti-CD70 mAb. These results indicated that OX40L and CD70 expressed on surface Ig- and CD40-stimulated B cells can provide CD28-independent costimulatory signals to T cells.     

Down-Regulation of T-Cell Proliferation in Response to Soluble Anti-CD3 Antibodies through Development of Redirected Cytolytic Activity Eliminating Costimulatory Cells

CD4⁺ T-depleted spleen cells (CD8⁺ T cells) activated by anti-CD3 antibodies (aCD3) suppressed proliferation of CD8⁺ T-depleted spleen cells (CD4⁺ T cells) and fresh normal T cells in response to aCD3. Antigen-nonspecific cytolytic activity was induced in splenic CD8⁺ T cells by stimulation with aCD3 and showed the peak level on day 3, whereas cytolytic activity induced in CD4⁺ T cells was weak. Intact Ig but not F(ab')₂ of aCD3 induced and mediated cytolytic activity. Correspondingly, the cytolytic activity induced by aCD3 was directed against target cells bearing Ig-binding Fc-receptor activity and cytolysis was inhibited by the addition of free Ig into the assay system. We showed that aCD3-activated T cells carried a high level of aCD3 on their surface at the time after the peak proliferation when they attained high cytolytic activity. This raised the possibility that the anti-CD3-induced aCD3-redirected cytolytic activity eliminated Fc-receptor-bearing costimulatory cells in the culture for down-regulation of the T-cell proliferation. This view was supported by partial restoration of anti-CD3-induced low responsiveness of CD8⁺ T cells by the addition of fresh costimulatory cells. These results suggested a new pathway of down-regulation of T-cell proliferation by aCD3-activated cytolytic CD8⁺ T cells.     

Induction of CD4(+)CD25(+)Foxp3(-) regulatory T cells by Thy-1 stimulation of CD4(+) T cells

Thy-1 (CD90) on mouse T cells has been reported to have both T-cell activating and regulatory roles. In this study, we show that monoclonal antibody (mAb)-mediated crosslinking of Thy-1 on CD4(+) mouse T-cells-induced regulatory T (T(reg)) cells that expressed CD25, CD39 and glucocorticoid-induced tumor necrosis factor receptor family-related gene, but not CD73, CD122 or Foxp3. The proliferation of CD4(+) T(responder) cells in response to anti-CD3/anti-CD28mAb-coated T-cell expander beads or syngeneic dendritic cells and soluble anti-CD3mAb was inhibited by Thy-1-induced T(reg) cells, in spite of elevated IL-2 levels in the co-cultures. Interestingly, stimulation with T-cell expander beads caused Thy-1-induced T(reg) cells to synthesize large amounts of interleukin-2 (IL-2). IL-10 was also elevated in co-cultures of activated T(responder) cells and Thy-1-induced T(reg) cells. However, mAb-mediated neutralization of IL-10 did not restore T(responder)-cell proliferation to control levels, which excluded IL-10 as a potential mediator of Thy-1-induced T(reg)-cell suppressor function. In addition, Thy-1-induced T(reg) cells did not inhibit IL-2-dependent proliferation of CTLL-2 cells, suggesting that IL-2 receptor signaling remained intact in the presence of Thy-1-induced T(reg) cells. We suggest that T(reg) cells induced by Thy-1 ligation in vivo may contribute to the maintenance of T-cell homeostasis.     

Kinetics of anti-CD4-induced T helper cell depletion and inhibition of function. Activation of T cells by the CD3 pathway inhibits anti-CD4-mediated T cell elimination and down-regulation of cell surface CD4.

In vivo treatment with anti-CD4 antibody profoundly suppresses a number of T cell-dependent responses and is clinically useful in the treatment of certain mouse models of autoimmune disease. Treatment with anti-CD4 antibody will inactivate and can deplete CD4 T cells, but the mechanisms responsible for these effects are incompletely understood. When mouse spleen cells were exposed in vitro to both SRBC and monoclonal anti-CD4, there was 55% reduction of the anti-SRBC response. If cultures were preincubated with anti-CD4 for 48 h before in vitro challenge, the reduction was greater than 80%. When unfractionated spleen cells were cultured with anti-CD4 for 96 h, there was actual elimination of CD4 cells in these cultures since virtually all CD3+ cells were CD8+. Activation of T cells by exposure to anti-CD3 rendered them resistant to antibody-mediated CD4 depletion. This resistance to CD4 depletion was seen even in cultures that were pretreated with anti-CD4 for as long as 24 h before anti-CD3 exposure. In cultures of purified T cells, anti-CD4 did not eliminate CD4 T cells. However, culture of T cells with macrophage-rich adherent cells and anti-CD4 resulted in elimination of CD4 T cells. Thus, it appears that macrophages play a role in anti-CD4-induced T cell elimination. While anti-CD4 did not eliminate CD4 cells from a population of purified T cells, there was profound down-regulation of cell surface CD4. Activating T cells with immobilized anti-CD3 before addition of anti-CD4 prevented down-regulation of CD4. These experiments demonstrate that T cell activation by anti-CD3 renders the activated cells resistant to antibody-induced CD4 down-regulation and to antibody-induced CD4 T cell depletion. These findings may have relevance to the application of anti-CD4 therapy in human diseases that are mediated by activated Th cells.     

Inhibitory effects of anti-CD2 monoclonal antibodies on interleukin 2 production and interleukin 2 receptor expression in anti-CD3-induced T cell activation

The effects of anti-CD2 monoclonal antibodies (mAb) on anti-CD3-driven interleukin 2 (IL2) production and IL2 receptor (IL2R) expression were investigated. Two anti-CD2 mAb, which had previously been shown to inhibit in vitro anti-CD3-induced T cell proliferation, also inhibited anti-CD3-induced IL2 production. However, it seemed unlikely that this was the crucial mechanism in the inhibition of anti-CD3-driven proliferation, since anti-CD2 mAb also partially inhibited T cell proliferation induced by the anti-CD3 mAb 446 which does not induce detectable IL2 levels. Anti-CD2 mAb also inhibited anti-CD3-induced surface IL2R expression as measured by immunofluorescence staining with an anti-IL2R mAb against the p55 chain. Inhibition of IL2R expression paralleled inhibition of proliferation. This anti-CD2-mediated inhibition involved a block in the generation of normal numbers of IL2R+ cells rather than a direct inhibitory effect on the IL2R+ cells themselves, since IL2R+ cells isolated from anti-CD2-containing cultures responded normally to IL2. Exogenous IL2 and IL4, singly or in combination, could reverse neither the anti-CD2-mediated inhibition of anti-CD3-induced proliferation nor the anti-CD2-mediated inhibition of anti-CD3-induced IL2R expression. Taken together, these observations suggest that anti-CD2 mAb inhibit anti-CD3-driven proliferation by inhibiting the generation of IL2R+ cells at a maturational stage proximal to their expression of surface IL2R. This inhibition cannot be overcome by exogenous IL2 or IL4, suggesting that the underlying biochemical mechanism involves an IL2- and IL4-independent pathway.     

5-Fluorouracil enhances apoptosis sensitivity of T lymphocytes mediated by CD3 epsilon

Previous studies by our laboratory have reported that the T cell receptor (TCR) TCR/CD3 complex could mediate activation as well as apoptosis of T lymphocytes. Two tyrosine residues in the ITAM (immuno-receptor tyrosine-based activation motifs) of CD3 epsilon were required for apoptosis signalling of Jurkat T lymphocytes. Stable cell lines TJK and T3JK produced from CD8(-) Jurkat T lymphocytes by transfection with wild-type and mutant CD8 epsilon (fusion of the extracellular and transmembrane domains of human CD8 alpha to the intracellular domain of mouse CD3 epsilon), were used with CD8(-) Jurkat T lymphocytes for studying the role of single intact CD3 epsilon. 5-Fluorouracil (5-FU), a chemotherapeutic drug can induce cell death of many tumour cell lines. In the present experiments, we examined the expression of caspase-3, p53 and Bid in the three cell lines induced by 5-FU and/or anti-CD8 antibody. We found high expression of p53 during activation-induced cell death of TJK cells mediated by anti-CD8 antibody and apoptosis of TJK and T3JK induced by 5-FU, implicating p53 involvement in apoptosis of leukemia cells induced by anti-CD8 antibody and 5-FU. We also detected the active form of caspase-3 and Bid in apoptotic leukemia cells after treatment with 5-FU and/or anti-CD8 antibody, indicating that the drug and antibody induced cell death through caspase-3 and the signal pathway may involve the Bcl-2 protein family. Our results showed that combined treatment with 5-FU and anti-CD8 antibody could enhance the rate of apoptosis induced by 5-FU or anti-CD8 antibody through increased expression of p53 and by promoting activation of caspase-3 and Bid. This suggests that the combination of 5-FU and anti-CD8 antibody may play an important role in inducing apoptosis of leukemia cells.     

Fibronectin promotes proliferation of naive and memory T cells by signaling through both the VLA-4 and VLA-5 integrin molecules.

The capacity of purified fibronectin to costimulate human T cell DNA synthesis was examined. Low concentrations of immobilized fibronectin, but not soluble fibronectin, augmented anti-CD3-induced proliferation of highly purified human T cells. In the absence of anti-CD3 stimulation, immobilized fibronectin did not induce T cell proliferation alone or in the presence of IL-2 or phorbol dibutyrate. Although fibronectin is present in high concentrations in the serum, immobilized fibronectin was able to costimulate T cell proliferation when cells were cultured in serum-containing medium. Immobilized collagen type I did not enhance anti-CD3 stimulated T cell responses, whereas gelatin (denatured collagen) and laminin were able to enhance anti-CD3 stimulated T cell responses modestly. The effects of gelatin, however, appeared to be indirect, because it could not enhance responses in medium devoid of fibronectin. Immobilized fibronectin enhanced anti-CD3 induced proliferation of both CD45RA dim and CD45RA bright subsets within both the CD4+ and CD8+ subpopulations of T cells, although cells with the CD45RA dim phenotype were costimulated by lower concentrations of immobilized fibronectin. Enhancement of anti-CD3 induced proliferation by immobilized fibronectin was completely inhibited by a mAb to CD29, the integrin beta 1-chain (4B4) and not by a variety of other mAb. In contrast to its effects on proliferation, 4B4 only partially blocked T cell binding to anti-CD3 and fibronectin-coated macrowells. These findings suggested that the interaction between fibronectin and its receptor transduced a signal to the T cell and did not merely stabilize the interaction between anti-CD3 and the CD3 complex. Further experiments confirmed this observation. Thus fibronectin could enhance anti-CD3 responses when it was immobilized to a separate surface. The augmentation of anti-CD3 stimulated proliferation induced by immobilized fibronectin was also inhibited partially by mAb to either VLA-4 or VLA-5 and completely by a combination of the two mAb. The mAb to VLA-4 not only blocked the capacity of immobilized fibronectin to enhance anti-CD3-induced T cell proliferation but also directly costimulated T cell responses. Thus, at least two fibronectin receptors are involved in fibronectin-mediated costimulation of T cell proliferation. These studies indicate that signals are transduced through the fibronectin receptors, VLA-4 and VLA-5, that augment T cell responses and therefore implicate the extracellular matrix protein fibronectin as an important influence regulating T cell responsiveness in vivo.     

Human T cell activation by OKT3 is inhibited by a monoclonal antibody to CD44.

The CD44 molecule, also known as Hermes lymphocyte homing receptor, human Pgp-1, and extracellular matrix receptor III, has been shown to play a role in T cell adhesion and activation. Specifically, anti-CD44 mAb block binding of lymphocytes to high endothelial venules, inhibit T cell-E rosetting, and augment T cell proliferation induced by the CD2 or CD3-TCR pathways. We have characterized an anti-CD44 mAb (212.3) which immunoprecipitates a 90-kDa protein and is specific for CD44 as shown by peptide mapping and antibody competition studies. Interestingly, our studies with 212.3 demonstrate that this CD44-specific mAb completely inhibits T cell proliferation stimulated by the anti-CD3 mAb, OKT3. Inhibition is not a result of reduced cell viability, but is associated with 1) inhibition of IL-2 production, 2) inhibition of IL-2R expression, and 3) inhibition of OKT3-mediated increases in intracellular Ca2+ levels. In addition, 212.3 does not inhibit proliferation by the T cell mitogens PHA or PWM nor does it inhibit proliferation in a mixed lymphocyte reaction. Similar to other anti-CD44 mAb, 212.3 also augments T cell proliferation induced by mAb directed against the T11(2) and T11(3) epitopes of CD2. Thus, these studies describe a novel CD44-specific mAb (212.3) that inhibits T cell activation by OKT3 by blocking early signal transduction. Furthermore, these studies suggest that "receptor cross-talk" between the CD3-TCR complex and CD44 may regulate T cell activation.     

Inhibition of TCR-induced CD8 T cell death by IL-12: regulation of Fas ligand and cellular FLIP expression and caspase activation by IL-12

In this study we demonstrate the anti-apoptotic effect of IL-12 and its underlying mechanism in CD8 T cells. The prolonged stimulation of CD8 T cells with anti-CD3 alone caused apoptosis mediated by Fas and the caspase signaling pathway. However, costimulation with IL-12 significantly prevented anti-CD3-induced apoptosis of CD8 T cells. IL-12 decreased the number of Fas ligand-positive CD8 T cells and inhibited the activation of caspase-8 and caspase-3. In addition, IL-12 up-regulated cellular FLIPs but not Bcl-2 family proteins or cellular inhibitor of apoptosis proteins. These data suggest that IL-12 provides survival signals to CD8 T cells by down-regulating Fas ligand and up-regulating cellular FLIPs, followed by inhibiting caspase activation, which implies a role for IL-12 in peripheral responses of CD8 T cells in vivo.     

Antibody blockade of Thy-1 (CD90) impairs mouse cytotoxic T lymphocyte induction by anti-CD3 monoclonal antibody

Thy-1 (CD90) expressed by mouse T cells is known to have signal transducing properties, but the ability of Thy-1 to enhance cytotoxic T lymphocyte (CTL) development is not well understood. Here we show that stimulation of mouse T cells with monoclonal antibodies (mAb) to CD3, CD28 and Thy-1 (clone G7), which were coimmobilized on polystyrene microbeads, resulted in a greater proliferative response than stimulation with only anti-CD3 and anti-CD28 mAb, indicating that Thy-1 cross-linking enhanced T cell receptor/CD28-driven T cell activation. Consistent with this finding, Thy-1 blockade with a soluble nonactivating anti-Thy-1 mAb (clone 30-H12) inhibited anti-CD3-induced proliferation of CD4+ and CD8+ T cells, and the induction of cytotoxic effector cells in a dose-dependent fashion. Interleukin-2 synthesis and CD25 expression were also impaired by Thy-1 blockade. The inhibitory effect involved a defect at or before the level of protein kinase C activation because the addition of phorbol ester ablated the anti-Thy-1-mediated inhibition of anti-CD3-induced T cell activation. The CTL that were induced in the presence of blocking anti-Thy-1 mAb adhered to target cells but showed reduced expression of granzyme B and perforin. In contrast, Fas ligand expression and function was not affected by Thy-1 blockade. We conclude that Thy-1 signalling promotes the in vitro generation of CTL that kill in a granule-dependent fashion.     

IL-2-R beta expression and function within resting CD8+ T cells preferentially segregate with the CD45R0+ subset.

Human peripheral blood CD8+ T cells constitutively express a low level of IL-2-R beta chains which were shown in this study to be preferentially carried by the CD45R0+ subset. Such receptors can transduce signals for in vitro IL-2-induced cytolytic function and for the initiation of soluble anti-CD3 and IL-2-induced cell proliferation. Using these stimulation models, a comparison was made between the responsiveness of resting, small CD45R0+ and CD45RA+ subpopulations of CD8+ T cells, both of them being isolated by negative selection and rigorously depleted of monocytes and of IL-2-inducible non-MHC-restricted CTL. Strong proliferation was induced in CD8+/CD45R0+ cells in response to IL-2 and soluble anti-CD3 (each of these stimuli being by itself ineffective), while in contrast, CD8+/CD45RA+ cells manifested, in this system, little reactivity. Accordingly, no conversion to the CD45R0 phenotype occurred in single stained CD45RA+ T cells following their incubation with the stimuli. A similar restriction of reactivity to CD8+/CD45R0+ T cells was observed with respect to IL-2-induced targetable T cell cytotoxicity. The CTL activity induced by IL-2 alone occurred without cell division. In contrast, the additional increase in CTL activity occurring upon the synergistic actions of anti-CD3 mAb and IL-2 coincided with intense cell proliferation, with no generation of LAK activity. The inhibition exerted by anti-IL-2-R beta mAb in the cytolytic and the proliferative activities induced by these stimuli in resting CD8+/CD45R0+ T cells emphasizes the importance of constitutive IL-2-R beta chains in the biology of these cells.     

Polymorphonuclear neutrophils enhance anti-CD3-induced T cell activation: the role of polymorphonuclear FC-receptors.

We investigated the effect of polymorphonuclear neutrophils (PMN) on anti-CD3 mAb (OKT3 and anti-Leu4)-mediated T cell activation. In the absence of monocytes, purified E-rosette-positive cells (further referred to as "T cells") require either solid-phase bound anti-CD3 or the combination of both a high concentration of soluble anti-CD3 and exogenous recombinant interleukin 2 (rIL-2) to proliferate. PMN cannot sustain T cell proliferation with soluble anti-CD3, but they markedly boost proliferation in the presence of soluble anti-CD3 and rIL-2. When PMN were added to T cell cultures stimulated with anti-CD3, this resulted in IL-2 receptor (IL-2R) expression and CD3 modulation. The mechanism of enhancement of anti-CD3-induced IL-2-responsiveness by PMN was further analyzed. A cellular T cell-PMN interaction was found to play a critical role and this was mediated through PMN Fc receptors (FcR). PMN bear two types of low-affinity FcR (FcRII and FcRIII). FcRII is known to bind mIgG1 (e.g., anti-Leu4) and FcRIII binds mIgG2a (e.g., OKT3). FcR involvement was demonstrated by two observations. Anti-FcRII mAb IV.3 inhibited the PMN signal for T cell activation with anti-Leu4. PMN bearing the second variant of FcRII which is unable to bind mIgG1 failed to promote anti-Leu4/IL-2-mediated T cell proliferation. Thus, PMN potentiate T cell responsiveness to IL-2 in the presence of anti-CD3 mAb and this potentiation by PMN requires interaction of anti-CD3 with PMN-FcR.