Thomas Kerkau

Selective targeting of regulatory T cells with CD28 superagonists allows effective therapy of experimental autoimmune encephalomyelitis

CD4+CD25+ regulatory T cells (T reg cells) play a key role in controlling autoimmunity and inflammation. Therefore, therapeutic agents that are capable of elevating numbers or increasing effector functions of this T cell subset are highly desirable. In a previous report we showed that a superagonistic monoclonal antibody specific for rat CD28 (JJ316) expands and activates T reg cells in vivo and upon short-term in vitro culture. Here we demonstrate that application of very low dosages of the CD28 superagonist into normal Lewis rats is sufficient to induce T reg cell expansion in vivo without the generalized lymphocytosis observed with high dosages of JJ316. Single i.v. administration of a low dose of the CD28 superagonist into Dark Agouti (DA) rats or Lewis rats that suffered from experimental autoimmune encephalomyelitis (EAE) proved to be highly and equally efficacious as high-dose treatment. Finally, we show that T reg cells that were isolated from CD28-treated animals displayed enhanced suppressive activity toward myelin basic protein–specific T cells in vitro, and, upon adoptive transfer, protected recipients from EAE. Our data indicate that this class of CD28-specific monoclonal antibodies targets CD4+CD25+ T reg cells and provides a novel means for the effective treatment of multiple sclerosis and other autoimmune diseases.

Topological Requirements and Signaling Properties of T Cell–activating, Anti-CD28 Antibody Superagonists

Full activation of naive T cells requires both engagement of the T cell antigen receptor (TCR; signal 1) and costimulatory signaling by CD28 (signal 2). We previously identified two types of rat CD28-specific monoclonal antibodies (mAbs): “conventional,” TCR signaling–dependent costimulatory mAbs and “superagonistic” mAbs capable of inducing the full activation of primary resting T cells in the absence of TCR ligation both in vitro and in vivo. Using chimeric rat/mouse CD28 molecules, we show that the superagonists bind exclusively to the laterally exposed C′′D loop of the immunoglobulin-like domain of CD28 whereas conventional, costimulatory mAbs recognize an epitope close to the binding site for the natural CD80/CD86 ligands. Unexpectedly, the C′′D loop reactivity of a panel of new antibodies raised against human CD28 could be predicted solely on the basis of their superagonistic properties. Moreover, mouse CD28 molecules engineered to express the rat or human C′′D loop sequences activated T cell hybridomas without TCR ligation when cross-linked by superagonistic mAbs. Finally, biochemical analysis revealed that superagonistic CD28 signaling activates the nuclear factor κB pathway without inducing phosphorylation of either TCRζ or ZAP70. Our findings indicate that the topologically constrained interactions of anti-CD28 superagonists bypass the requirement for signal 1 in T cell activation. Antibodies with this property may prove useful for the development of T cell stimulatory drugs.

Activation of CD4+ T Lymphocytes Improves Wound Healing and Survival After Experimental Myocardial Infarction in Mice

Background— The role of adaptive immunity, especially CD4+ T-helper cells, has not yet been systematically investigated in wound healing and remodeling after myocardial infarction (MI). Therefore, we studied whether CD4+ T cells become activated and influence wound healing after experimental MI in mice. Methods and Results— When we compared sham versus MI in wild-type (WT) mice, T-cell receptor–dependent activation of both conventional Foxp3− and regulatory Foxp3+ CD4+ T cells could be demonstrated in heart-draining lymph nodes within the first week after MI. Concomitantly, we found infiltration of CD4+ T cells in infarcted myocardium. To study the role of CD4+ T cells in wound healing and remodeling, CD4+ T-cell–deficient mice (CD4 knockout [KO], MHCII&Dgr;/&Dgr;) and T-cell receptor–transgenic OT-II mice recognizing an irrelevant ovalbumin-derived peptide were studied. Serial echocardiography up to day 56 after MI revealed increased left ventricular dilation in CD4 KO compared with WT mice. Within the infarcted myocardium, CD4 KO mice displayed higher total numbers of leukocytes and proinflammatory monocytes (18.3±3.0 104/mg WT versus 75.7±17.0 104/mg CD4 KO, P<0.05). MHCII&Dgr;/&Dgr; and OT-II mice displayed significantly greater mortality (21% WT versus 48% OT-II, P<0.05, and WT 22% versus 52% MHCII&Dgr;/&Dgr;, P<0.05) and myocardial rupture rates than WT mice. Collagen matrix formation in the infarct zone was severely disturbed in CD4 KO and MHCII&Dgr;/&Dgr; mice, as well as in OT-II mice. Conclusions— The present study provides the first evidence that CD4+ T cells become activated after MI, presumably driven by recognition of cardiac autoantigens, and facilitate wound healing of the myocardium.

Foxp3+ CD4+ T Cells Improve Healing After Myocardial Infarction by Modulating Monocyte/Macrophage Differentiation

Rationale: An exaggerated or persistent inflammatory activation after myocardial infarction (MI) leads to maladaptive healing and subsequent remodeling of the left ventricle. Foxp3+ CD4+ regulatory T cells (Treg cells) contribute to inflammation resolution. Therefore, Treg cells might influence cardiac healing post-MI. Objective: Our aim was to study the functional role of Treg cells in wound healing post-MI in a mouse model of permanent left coronary artery ligation. Methods and Results: Using a model of genetic Treg-cell ablation (Foxp3DTR mice), we depleted the Treg-cell compartment before MI induction, resulting in aggravated cardiac inflammation and deteriorated clinical outcome. Mechanistically, Treg-cell depletion was associated with M1-like macrophage polarization, characterized by decreased expression of inflammation-resolving and healing-promoting factors. The phenotype of exacerbated cardiac inflammation and outcome in Treg-cell–ablated mice could be confirmed in a mouse model of anti-CD25 monoclonal antibody–mediated depletion. In contrast, therapeutic Treg-cell activation by superagonistic anti-CD28 monoclonal antibody administration 2 days after MI led to improved healing and survival. Compared with control animals, CD28-SA–treated mice showed increased collagen de novo expression within the scar, correlating with decreased rates of left ventricular ruptures. Therapeutic Treg-cell activation induced an M2-like macrophage differentiation within the healing myocardium, associated with myofibroblast activation and increased expression of monocyte/macrophage-derived proteins fostering wound healing. Conclusions: Our data indicate that Treg cells beneficially influence wound healing after MI by modulating monocyte/macrophage differentiation. Moreover, therapeutic activation of Treg cells constitutes a novel approach to improve healing post-MI.

An EF hand mutation in Stim1 causes premature platelet activation and bleeding in mice

Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.

Superagonistic anti-CD28 antibodies : potent activators of regulatory T cells for the therapy of autoimmune diseases

This paper reviews the existing evidence regarding the use of superagonistic anti-CD28 antibodies (CD28 superagonists) for therapeutic manipulation of regulatory T cells (Treg cells). The molecular properties of superagonistic anti-CD28 antibodies allow the generation of a strong activating signal in mature T cells, including Treg cells, without additional stimulation of the T cell receptor complex. CD28 superagonist administration in vivo leads to the preferential expansion and strong activation of naturally occurring CD4+CD25+CTLA-4+FoxP3+ Treg cells over conventional T cells. In animal models, both prophylactic and therapeutic administration of a CD28 superagonist prevented or at least greatly mitigated clinical symptoms and induced remission. Adoptive transfer experiments have further shown that CD28 superagonists mediate protection by expansion and activation of CD4+CD25+ Treg cells. Therefore, superagonistic anti-CD28 antibodies offer a promising novel treatment option for human autoimmune diseases and the first clinical trials are eagerly awaited.

STIM1-Independent T Cell Development and Effector Function In Vivo

Store-operated Ca2+ entry (SOCE) is believed to be of pivotal importance in T cell physiology. To test this hypothesis, we generated mice constitutively lacking the SOCE-regulating Ca2+ sensor stromal interaction molecule 1 (STIM1). In vitro analyses showed that SOCE and Ag receptor complex-triggered Ca2+ flux into STIM1-deficient T cells is virtually abolished. In vivo, STIM1-deficient mice developed a lymphoproliferative disease despite normal thymic T cell maturation and normal frequencies of CD4+Foxp3+ regulatory T cells. Unexpectedly, STIM1-deficient bone marrow chimeric mice mounted humoral immune responses after vaccination and STIM1-deficient T cells were capable of inducing acute graft-versus-host disease following adoptive transfer into allogeneic hosts. These results demonstrate that STIM1-dependent SOCE is crucial for homeostatic T cell proliferation, but of much lesser importance for thymic T cell differentiation or T cell effector functions.

Selective loss of regulatory T cells in thymomas.

Myasthenia gravis (MG) is the prime autoimmune manifestation of thymomas. We investigated the generation of T cells with a regulatory phenotype (TR) in thymomas with and without associated MG. In patients with MG(+) thymomas, maturation and export of TR cells but not of other T‐cell subsets was significantly reduced. We conclude that imbalance between effector and regulatory T cells in thymomas may be involved in modulation of onset and/or severity of MG. Ann Neurol 2004;56:901–904

CD28 superagonist-mediated boost of regulatory T cells increases thrombo-inflammation and ischemic neurodegeneration during the acute phase of experimental stroke.

While the detrimental role of non-regulatory T cells in ischemic stroke is meanwhile unequivocally recognized, there are controversies about the properties of regulatory T cells (Treg). The aim of this study was to elucidate the role of Treg by applying superagonistic anti-CD28 antibody expansion of Treg. Stroke outcome, thrombus formation, and brain-infiltrating cells were determined on day 1 after transient middle cerebral artery occlusion. Antibody-mediated expansion of Treg enhanced stroke size and worsened functional outcome. Mechanistically, Treg increased thrombus formation in the cerebral microvasculature. These findings confirm that Treg promote thrombo-inflammatory lesion growth during the acute stage of ischemic stroke.

Superagonistic CD28 stimulation of allogeneic T cells protects from acute graft-versus-host disease

Acute graft-versus-host disease (aGVHD) often precludes successful immunotherapy of hematologic malignancies with allogeneic T cells. Therefore, we investigated the effect of immunomodulatory superagonistic anti-CD28 monoclonal antibodies (CD28-SA) on the capacity of allogeneic T cells to mediate both aGVHD and the protective graft-versus-tumor (GVT) response. In vivo pretreatment of donor C57BL/6 mice or short-term in vitro culture of donor lymph node cells with a CD28-SA efficiently protected BALB/c recipient mice from aGVHD. This protection strongly relied on the presence of CD28-SA-activated CD4+ CD25+ Foxp3+ regulatory T cells in the donor T-cell inoculum. With respect to the GVT response, CD28-SA-prestimulated T cells were still as potent in clearing lymphoma cells as were T cells without CD28-SA preactivation. Taken together, our data suggest that CD28-SA stimulation of bulk leukocyte cultures in vitro markedly increases the therapeutic window for adoptive immunotherapy with allogeneic T cells in vivo.