Functional CD95 ligand and CD95 death-inducing signaling complex in activation-induced cell death and doxorubicin-induced apoptosis in leukemic T cells.

Activation-induced cell death (AICD) in T cells is mediated by CD95 ligand (CD95L)/receptor interaction, which has also been implicated in apoptosis induction by some anticancer agents. In this article we show that both anti-CD3-triggering (AICD) and doxorubicin treatment led to the production of a functionally active CD95L in the CD3+/T-cell receptor-positive (TCR+) T leukemia cell line H9. CD95L-expressing H9 cells killed CD95-sensitive J16 or CEM target cells, but not CD95-resistant CEM or J16 cells overexpressing dominant negative FADD (J16/FADD-DN). By immunoprecipitation, CD95L was physically bound to CD95, suggesting that AICD and doxorubicin-induced apoptosis involve CD95L-mediated CD95 aggregation, thereby triggering the CD95 death pathway. CD95 aggregation was associated with the recruitment of FADD and caspase-8 to the CD95 receptor to form the CD95 death-inducing signaling complex (DISC), resulting in caspase-8 activation and cleavage of the effector caspase-3 and PARP. Blocking of the CD95L/receptor interaction by antagonistic antibodies to CD95 or to CD95L also blocked AICD and inhibited the early phase of doxorubicin-induced apoptosis, though cell death induced by doxorubicin eventually proceeded in a CD95-independent manner. These findings may explain some conflicting data on the role of death receptor systems in drug-induced apoptosis. Thus, in cells with an inducible CD95 receptor/ligand system, drug-induced apoptosis may be mediated by CD95L-initiated DISC formation and activation of downstream effector programs similar to AICD in T cells. (Blood. 2000;95:301-308)

Anti-CD3 x anti-tumor F(ab')2 bifunctional antibody activates and retargets tumor-infiltrating lymphocytes.

Bifunctional Abs (BFA) with specificity for the TCR/CD3 complex of T cells and tumor Ag can bridge T lymphocytes and tumor cells and, thereby, trigger activation events. The ability of intact and F(ab')2 anti-CD3 (500A2) x anti-p97 (96.5) BFA to induce activation of T lymphocytes in the presence of murine melanoma tumor cells (CL-62) expressing human melanoma-associated Ag (p97) was investigated in vitro and in vivo. Intact and F(ab')2 BFA induced significant proliferation of T lymphocytes in the presence of p97+ tumor cells. Incubation of splenocytes with intact or F(ab')2 BFA and p97+ tumor cells increased BFA-mediated cytotoxicity against relevant tumor cells. Intact BFA, in contrast to F(ab')2 BFA, induced some activation of T cells in vitro even in the absence of p97+ target cells. In nontumor-bearing mice, administration of F(ab')2 BFA, in contrast to intact BFA, did not increase cytotoxic activity of lymph node (LN) cells and splenocytes. However, when F(ab')2 BFA was administrated into CL-62-bearing mice, an increase of BFA-mediated cytotoxicity of tumor-infiltrating lymphocytes, but not splenocytes nor LN cells, was observed. Moreover, in D-galactosamine-sensitized mice, injection of intact BFA (1 microgram/mice) induced 100% lethality, whereas the same dose of F(ab')2 BFA was not toxic. These results demonstrate that F(ab')2 BFA can induce activation of cytotoxic lymphocytes only in the presence of relevant tumor cells, both in vitro and in vivo. That these activated lymphocytes can be redirected to lyse relevant tumor cells by the same BFA has important implications for the clinical application of BFA anti-tumor therapies.

An anti-murine CD3 monoclonal antibody with a low affinity for Fc gamma receptors suppresses transplantation responses while minimizing acute toxicity and immunogenicity.

145-2C11, a hamster mAb directed against the mouse CD3 complex, is a potent immunosuppressive agent. Upon initial treatment, 145-2C11 triggers a systemic release of multiple cytokines that is responsible for the acute toxicity of the mAb. This cellular activation is a consequence of the cross-linking between T lymphocytes and Fc gamma R-bearing cells, mediated by the high affinity of the hamster mAb for murine Fc gamma Rs. Repeated mAb injections result in the onset of a neutralizing humoral response. Therefore, there has been an increased interest in developing nonmitogenic forms of anti-CD3 mAbs, although it is not clear whether these Abs will retain immunosuppressive properties. To determine whether the initial cytokine production is necessary for the immunosuppressive properties and the immunogenicity of anti-CD3 mAbs in vivo, we have generated chimeric (hamster 145-2C11 F(ab')2 region/mouse Fc gamma portion) mAbs using murine isotypes with different affinities for Fc gamma Rs. The 145-2C11 and a chimeric IgG2a isotype, both of which bind murine Fc gamma Rs avidly, had similar activating, immunogenic, and immunosuppressive properties in mice. The administration of a chimeric IgG3 isotype with a very low affinity for murine Fc gamma Rs did not result in cytokine production, a humoral response against the mAb, or TCR desensitization. Nevertheless, prolongation of skin graft survival was similar in the IgG3, IgG2a, and 145-2C11-treated mice, indicating that Fc gamma R nonbinding anti-CD3 mAbs retain potent immunosuppressive properties in vivo while not being immunogenic. This enhanced therapeutic to toxic profile may be beneficial in clinical transplantation.

Modification of the anti-CD3-induced cytokine release syndrome by anti-interferon-gamma or anti-interleukin-6 antibody treatment: protective effects and biphasic changes in blood cytokine levels.

Anti-interferon-gamma (IFN-gamma) antibodies were found to protect mice against pathological changes induced by injection of anti-CD3 antibody: incidence of diarrhea, severity of hypothermia and mortality rates were dramatically reduced. In anti-IFN-gamma antibody-treated mice, IFN-gamma blood levels were significantly reduced at 1.5 h post anti-CD3 challenge, but more elevated levels were found from 4 to 24 h. This rebound-like IFN-gamma response coincided with more profound hypoglycemia. Tumor necrosis factor and interleukin (IL)-6 levels were not affected by anti-IFN-gamma treatment. Exogenous IFN-gamma, administered within 3 h (but not later) of the anti-CD3 challenge made the syndrome worse. Furthermore, inter-mouse strain differences in sensitivity to the anti-CD3 syndrome correlated with the ability of the strain to produce IFN-gamma. Anti-IL-6 antibodies provided only marginal protection against hypothermia and mortality, but did markedly reduce hypoglycemia. Levels of biologically active IL-6 in serum were not influenced by anti-IL-6 antibody treatment during the first few hours after anti-CD3 challenge, but were significantly increased at later times. The data provide evidence that endogenous IFN-gamma is a critical element in the early phase of the anti-CD3 syndrome; endogenous IL-6, while possibly being involved in hypoglycemia, seems of lesser importance for the outcome of the syndrome.

General aspects of cytokine-release syndrome: timing and incidence of symptoms.

OKT3 has become a commonly employed immunosuppressant for transplantation both as a prophylactic agent and as a means of reversing rejection. It was first thought to have few side effects associated with its use, but many findings have become recognized as manifestations of cytokine release. The severity of some CRS symptoms can be reduced by careful patient management. Strategies to moderate CRS show promise. The potential for use of nonactivating anti-CD3 antibodies may obviate the symptom complex entirely.

Cellular basis of the resistance of newborn mice to the pathogenic effects of anti-CD3 treatment.

We have analysed the mechanisms underlying the differences in the susceptibilities of adult and newborn mice to the pathogenic effects of anti-CD3 mAbs. Our data show that the thymus cell number in adults is reduced by 93% 48 h after one single injection of 5 mg/kg of Ab whereas the same dose in newborns induces only a 30% decrease. In the adult, this effect is associated with a marked depletion of CD4+ CD8+ double positive (DP) cells and with the appearance of important areas of cell necrosis in the thymic cortex. In newborns, the DP cells are less affected and the thymic cortex does not present any cell necrosis even after an injection of 45 mg/kg of mAbs. Pre-treatment of adults with anti-CD4 and anti-CD8 Abs, while completely abolishing the toxic side-effects induced by anti-CD3 mAbs, does not protect the thymus from the depletion of DP cells. In vitro, anti-CD3 mAbs induce the proliferation of thymocytes and spleen cells from adults but not from newborns. Tumour necrosis factor-alpha (TNF alpha) is found in the serum of adults 90 min after injection of anti-CD3 but is never detected in the serum of anti-CD3 treated newborns. Taken together our data support the view that anti-CD3 mAbs act by two different mechanisms. The first one results from the binding of anti-CD3 on the CD3+ thymocytes which induces a direct toxicity for only the CD4+ CD8+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)