The common gamma-chain cytokines IL-2, IL-7, IL-15, and IL-21 induce the expression of programmed death-1 and its ligands

The programmed death (PD)-1 molecule and its ligands (PD-L1 and PD-L2), negative regulatory members of the B7 family, play an important role in peripheral tolerance. Previous studies have demonstrated that PD-1 is up-regulated on T cells following TCR-mediated activation; however, little is known regarding PD-1 and Ag-independent, cytokine-induced T cell activation. The common gamma-chain (gamma c) cytokines IL-2, IL-7, IL-15, and IL-21, which play an important role in peripheral T cell expansion and survival, were found to up-regulate PD-1 and, with the exception of IL-21, PD-L1 on purified T cells in vitro. This effect was most prominent on memory T cells. Furthermore, these cytokines induced, indirectly, the expression of PD-L1 and PD-L2 on monocytes/macrophages in PBMC. The in vivo correlate of these observations was confirmed on PBMC isolated from HIV-infected individuals receiving IL-2 immunotherapy. Exposure of gamma c cytokine pretreated T cells to PD-1 ligand-IgG had no effect on STAT5 activation, T cell proliferation, or survival driven by gamma c cytokines. However, PD-1 ligand-IgG dramatically inhibited anti-CD3/CD28-driven proliferation and Lck activation. Furthermore, following restimulation with anti-CD3/CD28, cytokine secretion by both gamma c cytokine and anti-CD3/CD28 pretreated T cells was suppressed. These data suggest that gamma c cytokine-induced PD-1 does not interfere with cytokine-driven peripheral T cell expansion/survival, but may act to suppress certain effector functions of cytokine-stimulated cells upon TCR engagement, thereby minimizing immune-mediated damage to the host.     

CD80 (B7-1) and CD86 (B7-2) are functionally equivalent in the initiation and maintenance of CD4+ T-cell proliferation after activation with suboptimal doses of PHA

Effective activation of T cells requires engagement of two separate T-cell receptors. The antigen-specific T-cell receptor (TCR) binds foreign peptide antigen-MHC complexes, and the CD28 receptor binds to the B7 (CD80/CD86) costimulatory molecules expressed on the surface of antigen-presenting cells (APC). The simultaneous triggering of these T-cell surface receptors with their specific ligands results in an activation of this cell. In contrast, CTLA-4 (CD152) is a distinct T-cell receptor that, upon binding to B7 molecules, sends an inhibitory signal to T cell activation. Many in vitro and in vivo studies demonstrated that both CD80 and CD86 ligands have an identical role in the activation of T cells. Recently, functions of B7 costimulatory molecules in vivo have been investigated in B7-1 and/or B7-2 knockout mice, and the authors concluded that CD86 could be more important for initiating T-cell responses, while CD80 could be more significant for maintaining these immune responses. In this study, we directly compared the role of CD80 and CD86 in initiating and maintaining proliferation of resting CD4(+) T cells in an in vitro mode system that allowed to provide the first signal-to-effector cells through the use of suboptimal doses of PHA and the second costimulatory signal through cells expressing CD80 or CD86, but not any other costimulatory molecules. Using this experimental system we demonstrate that the CD80 and CD86 molecules can substitute for each other in the initial activation of resting CD4(+) T cells and in the maintenance of their proliferative response.     

CD4+CD25high regulatory cells in human peripheral blood

Thymectomy in mice on neonatal day 3 leads to the development of multiorgan autoimmune disease due to loss of a CD(+)CD25(+) T cell regulatory population in their peripheral lymphoid tissues. Here, we report the identification of a CD4(+) population of regulatory T cells in the circulation of humans expressing high levels of CD25 that exhibit in vitro characteristics identical with those of the CD4(+)CD25(+) regulatory cells isolated in mice. With TCR cross-linking, CD4(+)CD25(high) cells did not proliferate but instead totally inhibited proliferation and cytokine secretion by activated CD4(+)CD25(-) responder T cells in a contact-dependent manner. The CD4(+)CD25(high) regulatory T cells expressed high levels of CD45RO but not CD45RA, akin to the expression of CD45RB(low) on murine CD4(+)CD25(+) regulatory cells. Increasing the strength of signal by providing either costimulation with CD28 cross-linking or the addition of IL-2 to a maximal anti-CD3 stimulus resulted in a modest induction of proliferation and the loss of observable suppression in cocultures of CD4(+)CD25(high) regulatory cells and CD4(+)CD25(-) responder cells. Whereas higher ratios of CD4(+)CD25(high) T cells are required to suppress proliferation if the PD-L1 receptor is blocked, regulatory cell function is shown to persist in the absence of the PD-1/PD-L1 or CTLA-4/B7 pathway. Thus, regulatory CD4 T cells expressing high levels of the IL-2 receptor are present in humans, providing the opportunity to determine whether alterations of these populations of T cells are involved in the induction of human autoimmune disorders.     

Naive CD8+ T cells do not require costimulation for proliferation and differentiation into cytotoxic effector cells

Most current models of T cell activation postulate a requirement for two distinct signals. One signal is delivered through the TCR by engagement with peptide/MHC complexes, and the second is delivered by interaction between costimulatory molecules such as CD28 and its ligands CD80 and CD86. Soluble peptide/MHC tetramers provide an opportunity to test whether naive CD8+ T cells can be activated via the signal generated through the TCR-alphabeta in the absence of any potential costimulatory molecules. Using T cells from two different TCR transgenic mice in vitro, we find that TCR engagement by peptide/MHC tetramers is sufficient for the activation of naive CD8+ T cells. Furthermore, these T cells proliferate, produce cytokines, and differentiate into cytolytic effectors. Under the conditions where anti-CD28 is able to enhance proliferation of normal B6 CD4+, CD8+, and TCR transgenic CD8+ T cells with anti-CD3, we see no effect of anti-CD28 on proliferation induced by tetramers. The results of this experiment argue that given a strong signal delivered through the TCR by an authentic ligand, no costimulation is required.     

Chimeric co-stimulatory molecules that selectively act through CD28 or CTLA-4 on human T cells

CD28 and CTLA-4 (CD152) play a pivotal role in the regulation of T cell activation. Upon ligation by CD80 (B7-1) or CD86 (B7-2), CD28 induces T cell proliferation, cytokine production, and effector functions, whereas CTLA-4 signaling inhibits expansion of activated T cells and induces tolerance. Therefore, we hypothesized that co-stimulatory molecules that preferentially bind CD28 or CTLA-4 would have dramatically altered biological properties. We describe directed molecular evolution of CD80 genes derived from human, orangutan, rhesus monkey, baboon, cat, cow, and rabbit by DNA shuffling and screening. In contrast to wild-type CD80, the evolved co-stimulatory molecules, termed CD28-binding protein (CD28BP) and CTLA-4-binding protein (CTLA-4BP), selectively bind to CD28 or CTLA-4, respectively. Furthermore, CD28BP has improved capacity to induce human T cell proliferation and interferon-gamma production compared with wild-type CD80. In contrast, CTLA-4BP inhibited human mixed leukocyte reaction (MLR) and enhanced interleukin 10 production in MLR, supporting a role for CTLA-4BP in inducing T cell anergy and tolerance. In addition, co-stimulation of purified human T cells was significantly suppressed when CTLA-4BP was cotransfected with either CD80 or CD28BP. The amino acid sequences of CD28BP and CTLA-4BP were 61 and 96% identical with that of human CD80 and provide insight into the residues that are critical in the ligand binding. These molecules provide a new approach to characterization of CD28 and CTLA-4 signals and to manipulation of the T cell response.     

CD80 and CD86 Differentially Regulate Mechanical Interactions of T-Cells with Antigen-Presenting Dendritic Cells and B-Cells

Functional T-cell responses are initiated by physical interactions between T-cells and antigen-presenting cells (APCs), including dendritic cells (DCs) and B-cells. T-cells are activated more effectively by DCs than by B-cells, but little is known about the key molecular mechanisms that underpin the particular potency of DC in triggering T-cell responses. To better understand the influence of physical intercellular interactions on APC efficacy in activating T-cells, we used single cell force spectroscopy to characterize and compare the mechanical forces of interactions between DC:T-cells and B:T-cells. Following antigen stimulation, intercellular interactions of DC:T-cell conjugates were stronger than B:T-cell interactions. DCs induced higher levels of T-cell calcium mobilization and production of IL-2 and IFNγ than were elicited by B-cells, thus suggesting that tight intercellular contacts are important in providing mechanically stable environment to initiate T-cell activation. Blocking antibodies targeting surface co-stimulatory molecules CD80 or CD86 weakened intercellular interactions and dampen T-cell activation, highlighting the amplificatory roles of CD80/86 in regulating APC:T-cell interactions and T-cell functional activation. The variable strength of mechanical forces between DC:T-cells and B:T-cell interactions were not solely dependent on differential APC expression of CD80/86, since DCs were superior to B-cells in promoting strong interactions with T-cells even when CD80 and CD86 were inhibited. These data provide mechanical insights into the effects of co-stimulatory molecules in regulating APC:T-cell interactions.     

Blockade of B7-H1 on macrophages suppresses CD4+ T cell proliferation by augmenting IFN-gamma-induced nitric oxide production

PD-1 is an immunoinhibitory receptor that belongs to the CD28/CTLA-4 family. B7-H1 (PD-L1) and B7-DC (PD-L2), which belong to the B7 family, have been identified as ligands for PD-1. Paradoxically, it has been reported that both B7-H1 and B7-DC co-stimulate or inhibit T cell proliferation and cytokine production. To determine the role of B7-H1 and B7-DC in T cell-APC interactions, we examined the contribution of B7-H1 and B7-DC to CD4+ T cell activation by B cells, dendritic cells, and macrophages using anti-B7-H1, anti-B7-DC, and anti-PD-1 blocking mAbs. Anti-B7-H1 mAb and its Fab markedly inhibited the proliferation of anti-CD3-stimulated naive CD4+ T cells, but enhanced IL-2 and IFN-gamma production in the presence of macrophages. The inhibition of T cell proliferation by anti-B7-H1 mAb was abolished by neutralizing anti-IFN-gamma mAb. Coculture of CD4+ T cells and macrophages from IFN-gamma-deficient or wild-type mice showed that CD4+ T cell-derived IFN-gamma was mainly responsible for the inhibition of CD4+ T cell proliferation. Anti-B7-H1 mAb induced IFN-gamma-mediated production of NO by macrophages, and inducible NO synthase inhibitors abrogated the inhibition of CD4+ T cell proliferation by anti-B7-H1 mAb. These results indicated that the inhibition of T cell proliferation by anti-B7-H1 mAb was due to enhanced IFN-gamma production, which augmented NO production by macrophages, suggesting a critical role for B7-H1 on macrophages in regulating IFN-gamma production by naive CD4+ T cells and, hence, NO production by macrophages.     

Distinct role of CD80 and CD86 in the regulation of the activation of B cell and B cell lymphoma.

To date, not much has been known regarding the role of CD80 and CD86 molecules in signaling of B cells. The CD28/CTLA4 ligands, CD80 (B7-1) and CD86 (B7-2), are expressed on the surface of freshly isolated splenic B cells, and their expression is up-regulated by lipopolysaccharides. In the present study, we have investigated whether signaling via CD80/CD86 could alter the proliferation and immunoglobulin synthesis of B cells. Splenic B cells were stimulated with lipopolysaccharides in the presence of anti-B7-1 (16-10A1) and anti-B7-2 (GL1) monoclonal antibodies (mAbs). Exciting features observed during the study were that cross-linking of CD86 with GL1 enhanced the proliferation and production of IgG1 and IgG2a isotypes. In contrast, anti-B7-1 (16-10A1) mAb could efficiently block the proliferation and production of IgG1 and IgG2a. Furthermore, GL1 mAb could also induce the secretion of IgG isotypes from B cell lymphomas. Importantly, 16-10A1 could retard the growth of lymphomas and favored the up-regulation of pro-apoptotic molecules caspase-3, caspase-8, Fas, FasL, Bak, and Bax and down-regulation of anti-apoptotic molecule Bcl-x(L). In contrast, GL1 augmented the level of anti-apoptotic molecules Bcl-w and Bcl-x(L) and decreased the levels of pro-apoptotic molecule caspase-8, thereby providing a novel insight into the mechanism whereby triggering through CD80 and CD86 could deliver regulatory signals. Thus, this study is the first demonstration of a distinct signaling event induced by CD80 and CD86 molecules in B cell lymphoma. Finally, the significance of the finding is that CD80 provided negative signal for the proliferation and IgG secretion of normal B cells and B cell lymphomas. In contrast, CD86 encouraged the activity of B cells.     

Dietary eicosapentaenoic acid modulates CTLA-4 expression in murine CD4+ T-cells

We have demonstrated that downregulation of proliferation by CD4(+) T-cells in mice fed n-3 PUFA diets is dependent on the involvement of CD28. Therefore, we hypothesized that the balance of co-stimulatory and downregulatory properties of CD28 and CTLA-4, respectively, would be altered by diet. Mice were fed a control corn oil (CO)-enriched diet devoid of n-3 PUFA, or diets enriched with either docosahexaenoic acid (DHA) or eicosapentaenoic acid (EPA) for 14d. The proliferation of splenic CD4(+) T-cells was suppressed by DHA and EPA following stimulation with anti-CD3 and anti-CD28. Surprisingly, the number of surface CD28 molecules was not reduced in activated CD4(+) T-cells from either group of n-3 PUFA-fed mice. However, in mice fed EPA, CTLA-4 protein levels were enhanced significantly 72 h post-activation (P<0.01). Therefore, we conclude that dietary EPA may suppress CD4(+) T-cell activation by enhancing the downregulatory co-receptor CTLA-4, while not altering the levels of CD28.     

Costimulation light: activation of CD4+ T cells with CD80 or CD86 rather than anti-CD28 leads to a Th2 cytokine profile

To examine the role of CD28 and CTLA-4 in Th cell differentiation, we used a novel microsphere-based system to compare the effects of CD28 ligation by Ab or CD80/CD86. One set of beads was prepared by coating with anti-CD3 and anti-CD28 Ab. Another set of beads was prepared by immobilizing anti-CD3 and murine CD80-Ig fusion protein or murine CD86-Ig fusion protein on the beads. The three sets of beads were compared in their effects on the ability to activate and differentiate splenic CD4 T cells. When purified naive CD4(+) cells were stimulated in vitro, robust proliferation of similar magnitude was induced by all three sets of beads. When cytokine secretion was examined, all bead preparations induced an equivalent accumulation of IL-2. In contrast, there was a marked difference in the cytokine secretion pattern of the Th2 cytokines IL-4, IL-10, and IL-13. The B7-Ig-stimulated cultures had high concentrations of Th2 cytokines, whereas there were low or undetectable concentrations in the anti-CD28-stimulated cultures. Addition of anti-CTLA-4 Fab augmented B7-mediated IL-4 secretion. These studies demonstrate that B7 is a critical and potent stimulator of Th2 differentiation, and that anti-CD28 prevents this effect.     

CD152 ligation by CD80 on T cells is required for the induction of unresponsiveness by costimulation-deficient antigen presentation

Two apparently contradictory observations have been made concerning peripheral T cell tolerance; costimulation-deficient Ag presentation leads to unresponsiveness, and CTLA4 (CD152) ligation is required for unresponsiveness to be induced. This issue was addressed using a CD80- CD86low B cell line to present Ag to DO.11.10 naive CD4+ T cells. Proliferation was substantially enhanced by anti-CD80 or anti-CD152, but was inhibited by anti-CD86. Furthermore, anti-CD80 partially, and anti-CD152 totally protected cloned DO.11.10 T cells from the induction of unresponsiveness following culture with peptide and Chinese hamster ovary H2-Ad+ CD80- CD86- cells. Fab of anti-CD80 caused similar enhancement, and coimmobilized anti-CD80 failed to costimulate the anti-CD3 response of purified T cells, indicating that direct signaling by anti-CD80 was not responsible for these effects. The possibility that anti-CD80 liberated CD28 molecules that were sequestered by the T cell-expressed CD80, enabling them to coaggregate with TCR:CD3 complexes was excluded by finding that anti-CD80 and anti-CD152 individually caused maximal enhancement, rather than having additive effects. These data suggest that T cell-expressed CD80 has a regulatory function and plays a key role in the induction of unresponsiveness due to costimulation-deficient Ag presentation by the ligation of CD152 on neighboring, or even the same, T cell.     

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.     

Deleterious effect of HIV-1 plasma viremia on B cell costimulatory function

HIV infection leads to numerous immunologic defects, including impaired B cell function. An effective humoral response requires bidirectional interactions between B cells and CD4(+) T cells, critical of which are interactions between CD80/CD86 expressed on activated B cells and CD28 expressed on responder CD4(+) T cells. In the present study, we examined the effect of active HIV replication on B cell costimulatory function. Induction of CD80/CD86 on B cells following B cell receptor and CD40 triggering and responsiveness of CD4(+) T cells to activated B cells were investigated in a system where B cells of HIV-infected patients were compared concurrently to B cells of HIV-negative donors. In contrast to HIV-aviremic patients, B cells of HIV-viremic patients were ineffective at stimulating CD4(+) T cells, as measured by the induction of activation markers and proliferation. The importance of interactions of CD80/CD86 and CD28 in activating CD4(+) T cells was clear; the ablation of a normal response following the addition of neutralizing anti-CD86/CD80 Abs mirrored the response of CD4(+) T cells to B cells of HIV-viremic patients, while the addition of exogenous CD28 ligands partially restored the poor CD4(+) T cell response to the B cells of HIV-viremic patients. Ineffective B cell costimulatory function in HIV-viremic patients was associated with low induction of CD80/CD86 expression on B cells. Our findings further delineate the scope of defects associated with cognate B cell-CD4(+) T cell interactions in HIV infection and suggest that therapeutic interventions designed to enhance CD28-dependent costimulatory pathways may help restore immune functions.     

A novel bispecific tetravalent antibody fusion protein to target costimulatory activity for T-cell activation to tumor cells overexpressing ErbB2/HER2

Persistent activation of T-lymphocytes requires two signals: one is initiated by T-cell receptor binding to antigenic peptide presented by MHC molecules. In addition, binding of the B7 family members CD80 or CD86 on professional antigen presenting cells to CD28 on T cells is considered to provide an important costimulatory signal. Activation without costimulation induces T-cell unresponsiveness or anergy. To selectively localize costimulatory activity to the surface of tumor cells and enhance activation of tumor-specific T cells, we have developed a novel molecular design for bispecific costimulatory proteins with antibody-like structure. Within a single polypeptide chain we have assembled the IgV-like, CD28-binding domain of human CD86 (CD86(111)) together with hinge, CH2 and CH3 domains of human IgG1, and the scFv(FRP5) antibody fragment which recognizes the ErbB2 (HER2) protooncogene present at high levels on the surface of many human tumor cells. Upon expression in the yeast Pichia pastoris, the resulting CD86(111)-IgG-scFv(FRP5) protein could be purified as a homodimeric, tetravalent molecule from culture supernatants using single-step affinity chromatography. Bispecific binding of the molecule to ErbB2 on the surface of tumor cells and to the B7 counter receptor CTLA-4 was demonstrated by FACS analysis. Potent costimulatory activity of chimeric CD86(111)-IgG-scFv(FRP5) was confirmed by its ability to stimulate the proliferation of primary human lymphocytes pre-activated by low concentrations of anti-CD3 antibody. Our results suggest that such multivalent soluble proteins which combine specific targeting to tumor cells with costimulatory activity may become useful tools to elicit and/or improve T-cell mediated, tumor-specific immune responses.     

Ligation of CD28 in vivo induces CD40 ligand expression and promotes B cell survival.

Functional activation of T cells requires ligation of Ag receptors with specific peptides presented by MHC molecules on APCs concurrent with appropriate contacts of cell surface accessory molecules. Among these accessory molecules, interactions between CD28/CTLA-4 with B7 family members (CD80 and CD86) and CD40 with CD40 ligand (CD40L) play a decisive role in regulating the progression of balanced immune responses. However, most information regarding the role of accessory molecules in immune responses has been derived in the context of signals from the TCRs. Little understanding has been achieved regarding the consequence of ligation of costimulation molecules in absence of signals from the TCR. By employing an in vivo murine system, we show, herein, that ligation of CD28 alone with anti-CD28 Abs leads to a dramatic enlargement of the peripheral lymphoid organs characterized primarily by the expansion of B cells. B cells from anti-CD28-treated mice are resistant to spontaneous and anti-IgM-induced apoptosis. These cells are also unsusceptible to FasL-mediated apoptosis. Interestingly, this in vivo effect of CD28 on B cells is largely mediated by inducing the expression of CD40L, since coadministration of a blocking Ab against CD40L inhibited CD28-mediated B cell survival and expansion. Therefore, CD28-mediated expression of CD40L may play an important role in the regulation of lymphocyte homeostasis.     

Targeting CD28, CTLA-4 and PD-L1 Costimulation Differentially Controls Immune Synapses and Function of Human Regulatory and Conventional T-Cells

CD28, CTLA-4 and PD-L1, the three identified ligands for CD80/86, are pivotal positive and negative costimulatory molecules that, among other functions, control T cell motility and formation of immune synapse between T cells and antigen-presenting cells (APCs). What remains incompletely understood is how CD28 leads to the activation of effector T cells (Teff) but inhibition of suppression by regulatory T cells (Tregs), while CTLA-4 and PD-L1 inhibit Teff function but are crucial for the suppressive function of Tregs. Using alloreactive human T cells and blocking antibodies, we show here by live cell dynamic microscopy that CD28, CTLA-4, and PD-L1 differentially control velocity, motility and immune synapse formation in activated Teff versus Tregs. Selectively antagonizing CD28 costimulation increased Treg dwell time with APCs and induced calcium mobilization which translated in increased Treg suppressive activity, in contrast with the dampening effect on Teff responses. The increase in Treg suppressive activity after CD28 blockade was also confirmed with polyclonal Tregs. Whereas CTLA-4 played a critical role in Teff by reversing TCR-induced STOP signals, it failed to affect motility in Tregs but was essential for formation of the Treg immune synapse. Furthermore, we identified a novel role for PD-L1-CD80 interactions in suppressing motility specifically in Tregs. Thus, our findings reveal that the three identified ligands of CD80/86, CD28, CTLA-4 and PD-L1, differentially control immune synapse formation and function of the human Teff and Treg cells analyzed here. Individually targeting CD28, CTLA-4 and PD-L1 might therefore represent a valuable therapeutic strategy to treat immune disorders where effector and regulatory T cell functions need to be differentially targeted.     

Antagonist properties of monoclonal antibodies targeting human CD28

Antagonist antibodies targeting CD28 have been proposed as an alternative to the use of CD80/86 antagonists to modulate T cell responses in autoimmunity and transplantation. Advantages would be the blockade of CD28-mediated co-stimulatory signals without impeding the co-inhibitory signals dependent on CD80 interactions with CTLA-4 and PD-L1 that are important for the control of immune responses and for the function of regulatory T cells. Anti-CD28 antibodies are candidate antagonists only if they prevent access to the CD80/86 ligands without simultaneously stimulating CD28 itself, a process that is believed to depend on receptor multimerization. In this study, we evaluated the impact of different formats of a potentially antagonist anti-human CD28 antibody on T cell activation. In particular, we examined the role of valency and of the presence of an Fc domain, two components that might affect receptor multimerization either directly or in the presence of accessory cells expressing Fc receptors. Among monovalent (Fab’, scFv), divalent (Fab’2), monovalent-Fc (Fv-Fc) and divalent-Fc (IgG) formats, only the monovalent formats showed consistent absence of induced CD28 multimerization and absence of associated activation of phosphoinositol-3-kinase, and clear antagonist properties in T cell stimulation assays. In contrast, divalent antibodies showed agonist properties that resulted in cell proliferation and cytokine release in an Fc-independent manner. Conjugation of monovalent antibodies with polyethylene glycol, α-1-antitrypsin or an Fc domain significantly extended their in vivo half-life without modifying their antagonist properties. In conclusion, these data indicate that monovalency is mandatory for maintaining the antagonistic activity of anti-CD28 monoclonal antibodies.     

CD80+Gr-1+ myeloid cells inhibit development of antifungal Th1 immunity in mice with candidiasis

To find out whether polymorphonuclear neutrophils (PMN), abundantly recruited in disseminated Candida albicans infection, could directly affect the activation of Th cells we addressed the issues as to whether murine PMN, like their human counterparts, express costimulatory molecules and the functional consequence of this expression in terms of antifungal immune resistance. To this purpose, we assessed 1) the expression of CD80 (B7-1) and CD86 (B7-2) molecules on peripheral, splenic, and inflammatory murine Gr-1+ PMN; 2) its modulation upon interaction with C. albicans in vitro, in vivo, and in human PMN; 3) the effect of Candida exposure on the ability of murine PMN to affect CD4+ Th1 cell proliferation and cytokine production; and 4) the mechanism responsible for this effect. Murine PMN constitutively expressed CD80 molecules on both the surface and intracellularly; however, in both murine and human PMN, CD80 expression was differentially modulated upon interaction with Candida yeasts or hyphae in vitro as well as in infected mice. The expression of the CD86 molecule was neither constitutive nor inducible upon exposure to the fungus. In vitro, Gr-1+ PMN were found to inhibit the activation of IFN-gamma-producing CD4+ T cells and to induce apoptosis through a CD80/CD28-dependent mechanism. A population of CD80+Gr-1+ myeloid cells was found to be expanded in conventional as well as in bone marrow-transplanted mice with disseminated candidiasis, but its depletion increased the IFN-gamma-mediated antifungal resistance. These data indicate that alternatively activated PMN expressing CD80 may adversely affect Th1-dependent resistance in fungal infections.     

Stimulation via the CD3 and CD28 molecules induces responsiveness to IL-4 in CD4+CD29+CD45R- memory T lymphocytes

Although both IL-2 and IL-4 can promote the growth of activated T cells, IL-4 appears to selectively promote the growth of those helper/inducer and cytolytic T cells which have been activated via their CD3/TCR complex. The present study examines the participation of CD28 and certain other T cell-surface molecules in inducing T cell responsiveness to IL-4. Purified small high density T cells were cultured in the absence of accessory cells with various soluble anti-human T cell mAb with or without soluble anti-CD3 mAb and their responsiveness to IL-4 was studied. None of the soluble anti-T cell mAb alone was able to induce T cell proliferation in response to IL-4. A combination of soluble anti-CD3 with anti-CD28 mAb but not with mAb directed at the CD2, CD5, CD7, CD11a/CD18, or class I MHC molecules induced T cell proliferation in response to IL-4. Anti-CD2 and anti-CD5 mAb enhanced and anti-CD18 mAb inhibited this anti-CD3 + anti-CD28 mAb-induced T cell response to IL-4. In addition, anti-CD2 in combination with anti-CD3 and anti-CD28 mAb induced modest levels of T cell proliferation even in the absence of exogenous cytokines. IL-1, IL-6, and TNF were each unable to replace either anti-CD3 or anti-CD28 mAb in the induction of T cell responsiveness to IL-4, but both IL-1 and TNF enhanced this response. The anti-CD3 + anti-CD28 mAb-induced response to IL-4 was exhibited only by cells within the CD4+CD29+CD45R- memory T subpopulation, and not by CD8+ or CD4+CD45R+ naive T cells. When individually cross-linked with goat anti-mouse IgG antibody immobilized on plastic surface, only anti-CD3 and anti-CD28 mAb were able to induce T cell proliferation. These results indicate that the CD3 and CD28 molecules play a crucial role in inducing T cell responsiveness to IL-4 and that the CD2, CD5, and CD11a/CD18 molecules influence this process.