Addition Of Exogenous IL-2 Research Articles

Expression of Mad1 in T cells leads to reduced thymic cellularity and impaired mitogen-induced proliferation.

To investigate Mad1 function in vivo, transgenic mice were generated that express a Mad1 transgene in T lineage cells under the control of the proximal lck promoter. Thymus size in lck-Mad1 transgenic mice is drastically reduced although representation of the various thymocyte sub populations appears normal. To investigate more closely any effects of Mad1 expression on thymocytes, we examined thymic selection using MHC class I-restricted H-Y-TCR transgenic mice. Mad1 expression in vivo reduces the efficiency of positive selection. Furthermore, thymocytes and splenic T cells from lck-Mad1 transgenic mice display a profound proliferative defect in response to activation with either PMA/Ionomycin or immobilized anti-CD3/CD28 antibody. This proliferative defect is not reversed by addition of exogenous IL-2 and is p53-independent. The growth inhibition caused by Mad1 is overcome by expression of active c-Myc.

Analysis of the costimulatory requirements for generating human virus-specific in vitro T helper and effector responses.

The present study analyzes the role of CD28-B7-mediated costimulation during in vitro human peripheral blood memory T cell activation by influenza A virus. Inhibition studies using the B7-binding fusion protein CTLA4Ig and antibodies against CD80 and CD86 demonstrate that CTLA4Ig and anti-CD86 inhibited influenza-specific T cell proliferation, interleukin (IL)-2 and interferon (IFN)-gamma production, and generation of influenza-specific CD8+ CTL. The production of IL-10 and IL-18, which are known to modulate T cell immune responses, were not affected by blocking the CD28-B7 costimulatory pathway. Inhibition of diverse influenza-specific T cell functions could be reversed by the addition of exogenous IL-2 or IL-12 but not by the addition of IFN-gamma or IL-18. Although IL-2 is known to overcome CD28-B7 costimulatory requirements, this is the first report showing that exogenous IL-12 is able to bypass CD28-B7 costimulatory blockade induced by CTLA4Ig in vitro. The induction of IFN-gamma production with the recently described IFN-gamma inducing cytokine IL-18 was not detected. In conclusion, these results demonstrate that CD86 represents a major costimulatory signal for the activation of resting peripheral blood memory T cells with recall antigens. These observations may have important implications for the development of immunotherapeutic strategies in diverse immunodeficiency diseases as well as in tumor immunotherapy.

Interleukin-12 can replace CD28-dependent T-cell costimulation during nonspecific cytotoxic T lymphocyte induction by anti-CD3 antibody

Cytotoxic T lymphocyte (CTL) development is regulated closely by an intricate series of signals provided by the T-cell receptor/CD3 complex, cytokines, and costimulatory ligand/receptor systems. In this study, we have explored the role of interleukin (IL)-12 and CD28 in mouse CTL development. Activation of T cells with anti-CD3 monoclonal antibody (mAb) in the presence of anti-CD86 mAb, which prevents CD28-CD86 interaction, led to decreased production of type 1 (IL-2, interferon-gamma) and type 2 (IL-4, IL-6, IL-10) cytokines, as well as diminished expression of granzyme B (Gzm B) and reduced cytotoxic effector function. Cytolytic activity in T-cell cultures that were activated in the presence of anti-CD86-blocking mAb alone or in combination with anti-CD80 mAb could be restored by the addition of exogenous IL-12 at initiation of culture. The ability of IL-12 to substitute for CD28-costimulatory signaling during CTL development was found to be dependent on the presence of IL-2 rather than interferon-gamma. IL-2 is required for IL-12Rbeta2 expression by T cells activated in the presence of anti-CD86 mAb. Moreover, IL-12Rbeta2 expression by T cells activated in the presence of anti-CD86 mAb is enhanced by IL-12. We, therefore, conclude that the ability of IL-12 to substitute for CD28-costimulatory signaling during CTL development is a result of the interaction of IL-12 with IL-12Rbeta2 induced by low levels of IL-2 synthesized by T cells activated in a CD28-independent manner.

ChlamydiapneumoniaeInhibits Apoptosis in Human Peripheral Blood Mononuclear Cells Through Induction of IL-10

Chlamydia pneumoniae is a common cause of pulmonary infection, with serum positivity in at least 50% of the general population. In this study, we report that human PBMCs exposed to C. pneumoniae are resistant to apoptosis induced by the potent photoactivated chemotherapeutic agents 8-methoxypsoralen and hypericin. In contrast, PBMCs treated with a heat-inactivated inoculum exhibit normal susceptibility to apoptosis. We also observed that human PBMCs are responsive to C. pneumoniae infection by secretion of key immune regulatory cytokines, including IL-12 and IL-10. While IL-12 may play an important role in limiting C. pneumoniae proliferation within cells, IL-10 serves an anti-inflammatory function by down-regulating proinflammatory cytokines such as IL-12 and TNF-alpha. Depletion of endogenous IL-10, but not of IL-12, abolished the apoptosis resistance of C. pneumoniae-infected PBMCs. Furthermore, addition of exogenous IL-10, but not IL-12, significantly increased the resistance of control inoculum-treated PBMCs to photoactivated 8-methoxypsoralen- and hypericin-induced apoptosis. Therefore, we conclude that C. pneumoniae possesses an antiapoptotic mechanism. The resistance to apoptosis observed in PBMCs exposed to C. pneumoniae is due, at least partially, to the IL-10 induced during C. pneumoniae infection.

ANERGIC T CELLS GENERATED IN VITRO SUPPRESS REJECTION RESPONSE TO ISLET ALLOGRAFTS

Induction of antigen-specific unresponsiveness to grafts is the ultimate goal for organ transplantation. It has been shown that anergic T cells generated in vivo can be transferred as suppressor cells. Anergic cells generated in vitro have never been successfully used to prevent allograft rejection in vivo. We examined whether anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway can suppress allograft rejection in vivo. Anergic T cells were generated in vitro by the addition of anti-B7-1 and anti-B7-2 monoclonal antibodies (mAbs) to primary mixed lymphocyte reaction (MLR) consisting of C57BL/6 (B6) splenocytes as responder and irradiated BALB/c splenocytes as stimulator. We tested the ability of these cells to respond to various stimuli and to suppress alloreactive T-cell responses in vitro. For in vivo studies, 4x10(7) anergic cells were injected intravenously immediately after transplantation of BALB/c islets under the renal subcapsular space of streptozotocin-induced diabetic and 2.5-Gy X-irradiated B6 mice. Anergic cells treated with both mAbs in the primary MLR did not proliferate in secondary MLR against BALB/c and third-party C3H/He stimulators. The cells also failed to respond to immobilized anti-CD3 mAb, although they proliferated in response to concanavalin A or phorbol myristate acetate + ionomycin. The anergic state was reversed by the addition of exogenous IL-2. Furthermore, these cells suppressed the proliferation of naive B6 T cells against either the same (BALB/c) or third-party (C3H/He) stimulator cells. In in vivo studies, irradiated B6 mice rejected BALB/c islet allografts acutely with a mean survival time of 27.0+/-8.3 days, whereas two of six animals injected with the anergic cells accepted the allografts indefinitely (>100 days) with a mean survival time of 52.0+/-38.2 days. Anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway suppress islet allograft rejection after adoptive transfer. This procedure might be clinically useful for promoting allograft survival.

Induction of xenoreactive CD4+ T-cell anergy by suppressor CD8+CD28- T cells.

The underlying mechanism of immune suppression mediated by regulatory T cells is not completely understood. In previous studies we have shown that antigen-specific human T suppressor cells (Ts) can be generated in vitro by multiple rounds of stimulation with allogeneic, xenogeneic, or antigen-pulsed autologous antigen-presenting cells (APC). Human Ts express the CD8+CD28- phenotype and require specific recognition of MHC class I/peptide complexes on the surface of APC to block proliferation of T helper cells (Th). The aim of the present study was to explore the activation requirements of Ts as well as the nature of Th unresponsiveness to xenogeneic (swine) antigens induced by Ts. We investigated whether specific antigenic stimulation of Ts is required for their ability to inhibit early activation of xenoreactive Th (up-regulation of CD40 ligand). Flow cytometry studies indicated that Ts function required specific recognition of MHC class I on the surface of the stimulating APC. However, neither proliferation nor protein synthesis was required for the ability of Ts to inhibit Th. Ts drastically reduced the capacity of xenoreactive Th cells to produce interleukin (IL)-2 in response to the specific APC, without affecting their surface expression of IL-2 receptor. The suppressor effect that Ts exerted on Th proliferation could not be circumvented by CD40 ligation on the surface of the APC but could be reversed by the addition of exogenous IL-2. These data indicate that Ts induce anergy of xenoreactive human Th cells upon specific recognition of MHC class I antigens. Hence, Ts may prevent the activation of T cell-mediated immune responses against xenogeneic transplants.

The defects in effector generation associated with aging can be reversed by addition of IL-2 but not other related γc-receptor binding cytokines

Aged naive CD4 T cells produce low levels of IL-2, leading to inefficient generation of effectors. The cells expand poorly, giving rise to few effectors with less activated phenotypes and reduced ability to produce cytokines. The aged cells also respond less vigorously in vivo. Addition of exogenous IL-2 or other γc receptor-signaling cytokines, restores expansion. However, only effectors generated in the presence of IL-2, are able to produce IL-2 in normal amounts and to become polarized to secrete Th2 cytokines. The defect in IL-2 production may be the only critical deficiency of aged naive CD4 T cells. Importantly, memory CD4 T cells generated from the IL-2 “restored” effectors are also deficient in IL-2 production, suggesting that a heritable change occurs during aging which effects production of IL-2 by resting naive and memory CD4 T cells, but not by optimally generated effectors.

Activation of lymphocytes by anti-CD3 single-chain antibody dimers expressed on the plasma membrane of tumor cells.

Activation of cytotoxic T cells without MHC restriction was attempted by expressing single-chain antibodies (scFv) against CD3 on the surface of tumor cells. A chimeric protein consisting of a scFv of mAb 145.2C11, the hinge-CH2-CH3 region of human IgG1, and the transmembrane and cytosolic domains of murine CD80 formed disulfide-linked dimers on the plasma membrane of cells and specifically bound lymphocytes. Anti-CD3 scFv dimers expressed on the cell surface induced CD25 (IL-2 receptor alpha-chain) expression and proliferation of splenocytes. CT26 tumor cells engineered to express surface scFv dimers (CT26/2C11) also induced potent lymphocyte cytotoxicity with or without addition of exogenous IL-2. Splenocytes activated by CT26/2C11 cells also killed wild-type CT26 cells, indicating that activated splenocytes could kill bystander tumor cells. Immunization of BALB/c mice with irradiated CT26/2C11 cells did not protect against a lethal challenge of CT26 cells, suggesting that systemic immunity was not induced. However, the growth of CT26 tumors containing 50% CT26/2C11 cells was significantly retarded compared with CT26 tumors, whereas CT26/2C11 tumors did not grow in syngeneic mice. These results suggest that expression of anti-CD3 scFv dimers on tumors may form the basis for a novel therapeutic strategy for tumors that exhibit defects in antigen processing or presentation. Gene Therapy (2000) 7, 339-347.

Inhibition of IL-4 responses after T cell priming in the context of LFA-1 costimulation is not reversed by restimulation in the presence of CD28 costimulation.

Costimulation is one of several factors that influence the differentiation of CD4+ Th cell responses. Previously, we have shown that Ag presentation in the context of LFA-1 costimulation by fibroblasts transfected with class II and ICAM-1 (ProAd-ICAM) can drive naive CD4-positive T cells into cell cycle, but these T cells die by apoptosis 4-5 days after stimulation. In this report we show that the death of these cells can be prevented by the addition of exogenous IL-2 (20 U/ml) or by restimulation with Ag presented in the context of CD28 costimulation. Under these conditions, T cells go through extensive cell division and normal cell expansion. However, when T cells that have been primed by Ag presented in the context of LFA-1 costimulation are restimulated, they secrete IL-2 and IFN-gamma, but little or no IL-4. The inability of ProAd-ICAM-primed T cells to produce IL-4 was restored by the addition of IL-4 to the priming culture. However, IL-4 responses were not restored by representation of Ag in the context of CD28 costimulation, even as early as 24 h after priming with Ag presented by ProAd-ICAM cells. These findings suggest that differential expression of B7-1 and ICAM-1 by APCs during the initiation of immune responses may alter the differentiation of Th populations.

Defective CD8+ T Cell Activation and Cytolytic Function in the Absence of LFA-1 Cannot Be Restored by Increased TCR Signaling

Signaling through the TCR as well as engagement of costimulatory molecules are required for efficient T cell activation and progression into differentiated effector cells. The beta2 integrin LFA-1 (CD11a/CD18) has been implicated in TCR costimulation as well as in cell-cell adhesion function, but its exact role is still ambiguous. The present study focuses on the requirement for LFA-1 in CD8+ T cell activation and effector function using LFA-1-deficient cells expressing the 2C transgenic TCR as a model system. The lack of LFA-1 expression in 2C T cells resulted in severely diminished proliferative response toward allogeneic BALB/c splenocytes. Increase in TCR signaling alone by pulsing stimulators with high affinity peptides, p2Ca or QL9, had minimal effects in restoring proliferation. Addition of exogenous IL-2, however, enhanced the effect of peptide pulsing on proliferation of LFA-1-deficient 2C T cells. LFA-1-deficient 2C CTLs generated from alloantigen stimulation exhibited a defective cytotoxic activity when tested on a variety of target cells. Cytolysis could be improved, but not fully rectified by peptide pulsing of target cells. Thus, in the 2C TCR model, LFA-1 has a requisite role for optimal CD8+ T cell activation and effector function, which cannot be overcome by increasing peptide/MHC density on either the APCs or target cells, respectively.

Monocyte-Driven Activation-Induced Apoptotic Cell Death of Human T-Lymphotropic Virus Type I-Infected T Cells

We attempted apoptotic cell death induction of T cells infected with human T lymphotropic virus type I (HTLV-I) which induces HTLV-I-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. T cells acutely infected and expressing HTLV-Igag Ags were killed by cross-linking their TCR with anti-CD3 mAb. Cells in apoptotic process were found by staining with annexin V. The apoptosis was not affected by costimulation through CD28 molecules and was resistant to ligation of Fas molecules. Whereas the virus-infected T cells expressed higher levels of HLA-DR, CD25, CD80, and CD86 Ags than apoptosis-resistant PHA-blasts, the T cell apoptosis was enhanced by addition of exogenous IL-2. Furthermore, in this apoptosis, monocytes played an important role because T cells infected in the absence of monocytes were resistant to the death signals. The apoptosis-sensitive T cells responded to TCR signaling more strongly by proliferating than those apoptosis-resistant cells. Monocytes weakly affected the expression levels of viral Ags on T cells. However, HTLV-I-infected monocytes primed T cells to die by subsequent TCR signaling. T cells primed with the monocytes, subsequently infected in the absence of monocytes, were killed by TCR signaling. These observations suggest that primed and infected T cells could be killed by activation-induced cell death.

Simultaneous Activation of T Cells and Accessory Cells by a New Class of Intact Bispecific Antibody Results in Efficient Tumor Cell Killing

Bispecific Abs (bsAb) are promising immunological tools for the elimination of tumor cells in minimal residual disease situations. In principle, they target an Ag on tumor cells and recruit one class of effector cell. Because immune reactions in vivo are more complex and are mediated by different classes of effector cell, we argue that conventional bsAb might not yield optimal immune responses at the tumor site. We therefore constructed a bsAb that combines the two potent effector subclasses mouse IgG2a and rat IgG2b. This bispecific molecule not only recruits T cells via its one binding arm, but simultaneously activates FcgammaR+ accessory cells via its Fc region. We demonstrate here that the activation of both T lymphocytes and accessory cells leads to production of immunomodulating cytokines like IL-1beta, IL-2, IL-6, IL-12, and DC-CK1. Thus this new class of bsAb elicits excellent antitumor activity in vitro even without the addition of exogenous IL-2, and therefore represents a totally self-supporting system.

Autoregulatory effect of interleukin-10 on proinflammatory cytokine production by Porphyromonas gingivalis lipopolysaccharide-tolerant human monocytes.

Pretreatment of human peripheral blood monocytes with a very low concentration (0.1 ng/ml) of Porphyromonas gingivalis lipopolysaccharides (LPS) resulted in a significant decrease of interleukin-6 (IL-6) production, but not IL-8 production, by restimulation of a high concentration (1 microg/ml) of the same LPS. In contrast, the same pretreatment with Escherichia coli LPS resulted in the enhanced production of both IL-6 and IL-8 after restimulation. The selective induction by P. gingivalis LPS tolerance of IL-6 production developed in a time-dependent manner during the primary culture. P. gingivalis LPS-pretreated cells were also refractory to a high-dose E. coli LPS restimulation in terms of IL-6 production. The expression of IL-6 mRNA decreased 10 h after restimulation of P. gingivalis LPS-pretreated monocytes. Furthermore, an up-regulation of anti-inflammatory cytokine IL-10 upon a second high-dose LPS rechallenge occurred at the same time point in the pretreated cells. We studied the role of IL-10 in the process of IL-6 down-regulation. Neutralization by an anti-IL-10 polyclonal antibody prevented IL-6 down-regulation in P. gingivalis LPS-pretreated monocytes, whereas IL-8 production was not affected. Addition of exogenous IL-10 during the high-dose LPS stimulation of untreated cells substituted for the LPS pretreatment and resulted in the inhibition of IL-6 production in a dose-dependent manner. A higher dose of IL-10 was required to suppress IL-8 synthesis from monocytes. Our data suggest that IL-10 mediates IL-6 down-regulation in P. gingivalis LPS-tolerant monocytes in an autocrine manner.

Anergic cells generated in vitro suppress rejection response to islet allografts

Background. Induction of antigen-specific unresponsiveness to grafts is the ultimate goal for organ transplantation. It has been shown that anergic T cells generated in vivo can be transferred as suppresser cells. Anergic cells generated in vitro have never been successfully used to prevent allograft rejection in vivo. We examined whether anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway can suppress allograft rejection in vivo. Methods. Anergic T cells were generated in vitro by the addition of anti-B7-1 and anti-B7-2 monoclonal antibodies (mAbs) to primary mixed lymphocyte reaction (MLR) consisting of C57BL/6 (B6) splenocytes as responder and irradiated BALB/c splenocytes as stimulator. We tested the ability of these cells to respond to various stimuli and to suppress alloreactive T-cell responses in vitro. For in vivo studies, 4310 7 anergic cells were injected intravenously immediately after transplantation of BALB/c islets under the renal subcapsular space of streptozotocin-induced diabetic and 2.5-Gy X-irradiated B6 mice. Results. Anergic cells treated with both mAbs in the primary MLR did not proliferate in secondary MLR against BALB/c and third-party C3H/He stimulators. The cells also failed to respond to immobilized antiCD3 mAb, although they proliferated in response to concanavalin A or phorbol myristate acetate 1 ionomycin. The anergic state was reversed by the addition of exogenous IL-2. Furthermore, these cells suppressed the proliferation of naive B6 T cells against either the same (BALB/c) or third-party (C3H/He) stimulator cells. In in vivo studies, irradiated B6 mice rejected BALB/c islet allografts acutely with a mean survival time of 27.068.3 days, whereas two of six animals injected with the anergic cells accepted the allografts indefinitely (>100 days) with a mean survival time of 52.0638.2 days. Conclusions. Anergic cells generated in vitro by blocking CD28/B7 costimulatory pathway suppress islet allograft rejection after adoptive transfer. This procedure might be clinically useful for promoting allograft survival.

CD4-Mediated Inhibiton of IL-2 Production in Activated T Cells

The role of CD4 in T cell activation has been attributed to its capacity to increase the avidity of interaction with APC and to shuttle associated Lck to the TCR/CD3 activation complex. The results presented in this study demonstrate that ligation of CD4 inhibits ongoing responses of preactivated T cells. Specifically, delayed addition of CD4-specific mAb is shown to inhibit Ag- or mAb-induced responses of both primary T cells and T cell clonal variants. The Ag responses of the latter are independent of the adhesion provided by CD4; thus the observed inhibition is not due to blocking CD4-MHC interactions. Further, analysis of the clonal variants demonstrates that CD4-associated Lck is not essential for the inhibition observed, as anti-CD4 inhibits responses of clonal variants, expressing a form of CD4 unable to associate with Lck (double cysteine-mutated CD4). The inhibition is counteracted by the addition of exogenous IL-2, demonstrating that the block is not due to a lesion in IL-2 utilization, rather its production. It is demonstrated that the delayed addition of anti-CD4 results in a rapid reduction in steady-state levels of IL-2 mRNA in both primary T cells and clonal variants.

Dexamethasone induces apoptosis in human T cell clones expressing low levels of Bcl-2.

Previous results of ours have demonstrated that the same clonotype can express both a sensitive and a resistant phenotype to Dex-mediated PCD induction depending on its cell cycle phase. In particular, we demonstrated that human T lymphocytes, arrested in the G0/G1 phase of the cell cycle, are susceptible, while proliferating T cells are resistant to Dex-mediated apoptosis. In this paper, we have further characterized the sensitive and resistant phenotypes and investigated whether a different expression of the apoptotic genes Fas, FasL, Bcl-2, Bcl-x and Bax is involved in the regulation of Dex-mediated apoptosis. The results show that the amount of Bcl-2 expression, that changes during cell cycle phases, determines susceptibility or resistance to apoptosis induced by Dex. In fact, undetectable expression of Bcl-2 in sensitive cells favors Dex-mediated apoptosis while high expression of Bcl-2 in proliferating cells counterbalances apoptosis induction. Moreover, the addition of exogenous IL-2, in the presence of Dex, fails to up-regulate Bcl-2 expression and to revert Dex-mediated apoptotic phenomena.

Characterization of a mutant T-cell hybridoma line with defects in the TCR-mediated apoptotic pathway.

A mutant T-cell hybridoma line named mutant 51 was developed that, unlike the parental line, did not die after T-cell receptor (TCR) engagement and demonstrated reduced death in response to dexamethasone. Intracellular calcium measurements showed that available calcium stores were markedly reduced in the mutant cell line. Unlike control cells, secretion of IL-2 from mutant cells was also greatly reduced, although addition of exogenous IL-2 did not facilitate increased apoptosis. Although levels of the cell death gene product Nur77 were equivalent, additional studies showed that mutant cells expressed Nur77 predominantly in the cytoplasm following TCR engagement, while parental cells displayed a nuclear translocalization of Nur77. In addition, Fas levels and Fas ligand dependant killing were both markedly reduced in the mutant clone. From these data we hypothesize a role for available calcium stores and Nur77 nuclear localization in TCR-mediated apoptosis in T-cell hybridomas.

Berbamine inhibits IL-2 receptor expression, but spares IL-2 production and its mRNA expression

The purpose of this paper was to elucidate the mechanism of action of berbamine on the T-lymphocyte mediated immune responses. Effects of berbamine on IL-2 production, IL-2 receptor expression and the interaction between IL-2 and IL-2 receptor were observed. The results clearly demonstrated that berbamine suppressed the expression of IL-2 receptors on PHA induced T-blast cells, but did not alter the production of IL-2 of mouse splenic cells and human tonsil lymphocytes. Futhermore, the results also showed that berbamine at higher concentrations had a slight inhibitory effect on the interaction between IL-2 and IL-2 receptors, and addition of exogenous IL-2 did not reverse the inhibitory action of berbamine on T-cell proliferation.

Resistance to Apoptosis and Elevated Expression of Bcl-2 in Clonally Expanded CD4+CD28− T Cells from Rheumatoid Arthritis Patients

AbstractPatients with rheumatoid arthritis have a subset of CD4+ T lymphocytes that are characterized by a defect in CD28 expression. CD4+CD28− T cells frequently undergo clonal expansion in vivo. These clonotypes include autoreactive cells and persist over many years. The clonogenic potential and longevity of these T cells could be related to an altered response to apoptosis-inducing signals. To explore this possibility, CD4+CD28− T cell lines and clones were examined for their response pattern to stimuli inducing physiologic cell death. CD4+CD28− T cells were found to be resistant to apoptosis upon withdrawal of the growth factor, IL-2. To examine whether the altered sensitivity to this apoptotic signal was correlated with the expression of proteins of the bcl-2 family, the expression of bcl-2, bcl-x, and bax proteins was determined. CD28+ and CD28−CD4+ T cells could not be distinguished by the levels of bax or bcl-xL protein; however, CD4+CD28− T cells expressed higher amounts of bcl-2 protein than did CD4+CD28+ T cells. The increased bcl-2 expression in CD4+CD28− T cells was relatively independent of signals provided by exogenous IL-2. In CD28-deficient CD4+ T cells, bcl-2 was not significantly up-regulated by the addition of exogenous IL-2 and was maintained despite IL-2 withdrawal, as opposed to CD28-expressing CD4+ T cells. We propose that CD4+CD28− T cells are characterized by a dysregulation of the survival protein, bcl-2, which may favor the clonal outgrowth of autoreactive T cells and thus contribute to the pathogenesis of rheumatoid arthritis.

A Humanised Therapeutic CD4 mAb Inhibits TCR-Induced IL-2, IL-4, and IL-10 Secretion and Expression of CD25, CD40L, and CD69

The actions of a humanised therapeutic CD4 mAb YHB.46 on T cell activation were investigatedin vitro.Soluble YHB.46 IgG or YHB.46-derived F(ab′)2fragments caused inhibitions of up to 100% of the proliferation of purified CD4+T cells activated with immobilised CD3 mAb. The inhibitory effects of the CD4 mAb were equally potent in both CD45RA+and CD45RO+T cell subset proliferation assays. Inhibitory effects on DNA synthesis were not explicable by increased T cell apoptosis. YHB.46 was inhibitory even when added 70 h after exposure of cells to immobilised CD3 mAb, but it had little effect on IL-2 receptor-driven proliferation signals. The CD4 mAb inhibited the CD3-induced expression of the CD25 and CD69 activation markers on the T cell surface and suppressed CD40 ligand expression, but not that of CD25 and CD69, when their expression was induced by phorbol ester plus ionomycin. YHB.46 also exerted a profound inhibitory effect on the production of IL-2, IL-4, and IL-10, irrespective of whether T cells were activated with CD3 mAb or with phorbol ester plus ionomycin. The inhibitory effects of YHB.46 on CD4+T cell proliferation were partially prevented by the addition of exogenous IL-2 or autologous monocytes and were completely prevented by activating T cells with a novel CD3–CD28 bivalent F(ab′)2reagent. However, the inhibitory effects of YHB.46 on T cell proliferation were equipotent in the presence or the absence of CTLA-4Ig, showing that the CD4 mAb was not acting on CD28-induced activation signals per se. Our results show that the inhibitory effects of YHB.46 on T cell activation do not involve CD28 or IL-2 receptor signalling, but are directed at the TCR-mediated G0-G1 transition. These findingsin vitropredict that YHB.46 may act as a potent immunosuppressant in the clinical context.