Interleukin-2 (IL-2) is a lymphocytotropic hormone which is thought to have a key role in the immune response of mammalian cells. It is produced by a subpopulation of activated T-lymphocytes and acts in vitro as the principal auto- and paracrine T-cell growth factor (for reviews see refs 1-3). IL-2 is, however, not the sole T-cell growth factor, nor does it act exclusively on T cells, also promoting growth of NK cells and differentiation of B cells. A role for IL-2 in T-cell development has been postulated but remains controversial. Here we test the requirement for IL-2 in vivo using IL-2-deficient mice generated by targeted recombination. We find that mice homozygous for the IL-2 gene mutation are normal with regard to thymocyte and peripheral T-cell subset composition, but that a dysregulation of the immune system is manifested by reduced polyclonal in vitro T-cell responses and by dramatic changes in the isotype levels of serum immunoglobulins.

Activation of naive CD8 + T cells with antigen in the absence of skewing cytokines triggers their differentiation into effector CTL, which induces death of target cells. We show that CD8 + T cells activated in the presence of the cytokines IL-6 or IL-21 plus TGF-b similar to CD4 + T cells, develop into IL-17-producing (Tc17) cells. These cells display greatly suppressed cytotoxic function along with low levels of the CTL markers: T-box transcription factor Eomesodermin, granzyme B and IFN-c. Instead, these cells express hallmark molecules of Th17 program including retinoic acid receptor-related orphan receptor (ROR)ct, RORa, IL-21 and IL-23R. The expression of the type 17 master regulator RORct is causally linked to Tc17 generation, because its overexpression stimulates production of IL-17 in the presence of IL-6 or IL-21. Both, upregulation of the type 17 program as well as suppression of CTL differentiation are STAT3 dependent. Furthermore, Tc17 cells producing IL-17 but not granzyme B are also detectable in EAE, a mouse model for multiple sclerosis. Our data point to the existence of mutually exclusive CTL and Tc17 developmental pathways in vitro and in vivo.Key words: CTL . EAE . RORgt . STAT3 . Tc17Supporting Information available online Introduction CTL are important effector cells in the immune response to intracellular pathogens and tumors. They differentiate from naive CD8 + T cells following activation by antigen in the absence of skewing cytokines, and during this process they acquire the ability to destroy their targets by releasing cytotoxic molecules such as perforin and granzymes, from granules into the immunological synapse. In addition, CTL secrete cytokines, mostly IFN-g and TNF-a, which function to induce or augment inflammation [1][2][3].Two T-box transcription factors, Eomesodermin (Eomes) and T-bet, are important for the development of effector and memory CTL [4][5][6]. Studies using deletion, overexpression or dominant negative analogs of these factors have suggested that both of them are involved in the regulation of expression of granzyme B and perforin [4,5,7]. Consistent with these data, CD8 + T cells with combined deletion of the Eomes and Tbx21 (encoding T-bet) genes differentiate into cells with highly impaired cytotoxic activity and IFN-g production [8]. Instead, these cells produce Th17 type cytokines and express the IL-23 receptor (IL-23R) as well as the transcription factor retinoic acid receptor-related orphan receptor (ROR)gt, both of 1716Frontline which are characteristic for the type 17 differentiation program [8]. Thus, the phenotype of CD8 + T cells deficient for both Eomes and T-bet is reminiscent of the newly described Th17-cell subset.Th17 cells produce IL-17A, IL-17F, IL-21 and IL-22, which are highly pro-inflammatory and induce severe autoimmunity, e.g. during EAE, the mouse model for multiple sclerosis [9]. The differentiation of these cells requires TGF-b in combination with 11]. Two additional cytokines, IL-21 and IL-23, are also critically involved in the diffe...

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.

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.

Naive T cell activation requires signaling by the T cell receptor and by nonclonotypic cell surface receptors. The most important costimulatory protein is the monovalent homodimer CD28, which interacts with CD80 and CD86 expressed on antigen-presenting cells. Here we present the crystal structure of a soluble form of CD28 in complex with the Fab fragment of a mitogenic antibody. Structural comparisons redefine the evolutionary relationships of CD28-related proteins, antigen receptors and adhesion molecules and account for the distinct ligand-binding and stoichiometric properties of CD28 and the related, inhibitory homodimer CTLA-4. Cryo-electron microscopy-based comparisons of complexes of CD28 with mitogenic and nonmitogenic antibodies place new constraints on models of antibody-induced receptor triggering. This work completes the initial structural characterization of the CD28-CTLA-4-CD80-CD86 signaling system.

CD4 + CD25 + T cells play a central role in the suppression of autoimmunity and inflammation, making their in vivo expansion a highly attractive therapeutic target. By phenotyping with a novel rat CTL antigen-4 (CTLA-4)-specific monoclonal antibody (mAb) and functional in vitro assays, we here first establish that rat CD4 + CD25 + T cells correspond to the regulatory T cells (Treg cells) described in mice and humans: they constitutively express CTLA-4, produce IL-10 but not IL-2, and are able to suppress the proliferation of costimulated CD25-negative indicator cells. Furthermore, we show that rat Treg cells respond less well than CD25 -T cells to conventional costimulation, but are readily expanded in vitro with "superagonistic" CD28-specific mAb which are potent mitogens for all T cells without the need for TCR engagement. In vivo, functional Treg cells are preferentially expanded by CD28 stimulation over other T cell subsets, leading to a 20-fold increase within 3 days in response to a single antibody dose. These data suggest that CD28-driven activation of Treg cells may be highly effective in the treatment of inflammatory and autoimmune diseases.

Human volunteers receiving TGN1412, a humanized CD28-specific monoclonal antibody, experienced a life-threatening cytokine release syndrome during a recent trial. Preclinical tests using human PBMCs had failed to announce the rapid release of TNF, IFN-␥, and other toxic cytokines in response to this CD28 "superagonist" (CD28SA). CD28SA activate T-lymphocytes by ligating CD28 without overt engagement of the TCR. They do, however, depend on "tonic" TCR signals, which they amplify. Here we show that short-term preculture of PBMCs at high, but not at low, cell density results in massive cytokine release during subsequent stimulation with soluble TGN1412. Restoration of reactivity was cell-contact dependent, involved functional maturation of both monocytes and T cells, was sensitive to blockade by HLA-specific mAb, and was associated with TCR polarization and tyrosine phosphorylation. CD4 effector memory T cells were identified as the main source of proinflammatory cytokines. Importantly, responses to other T-cell activating agents, including microbial antigens, were also enhanced if PBMCs were first allowed to interact under tissue-like conditions. We provide a protocol, which strongly improves reactivity of circulating T cells to soluble stimulants, thereby allowing for more reliable preclinical testing of both activating and inhibitory immunomodulatory drugs. (Blood. 2011;118(26):6772-6782)

The TCR has been identified in several species as a heterodimer of two variable chains (reviewed in reference 1) that is associated with a set of invariant polypeptides collectively referred to as CD3 . MHC-restricted antigen recognition by both cytotoxic (mostly CD8') and helper (mostly CD4') T cells is mediated by clonotypic heterodimers of the a/ß type, while a second type of CD3-associated TCR, termed y/S, has recently been discovered on a small subpopulation of human (2) and mouse (3) T cells . mAbs to the TCR and CD3 molecules have been instrumental to the discovery and analysis of the TCR complex . Recent work in the mouse system has especially profited from the generation of mAbs to Vß segments expressed at a frequency detectable in unimmunized T cell populations (4-7), and to the invariant CD3E chain (8) . No mAb to . a constant determinant of the mouse TCR-a/ß is available, however, that could be used to discriminate TCR-a/ß and TCR-,y/8 expressing T cells and thymocytes. In the rat system, analysis of T cell maturation and activation has been hampered by a complete lack of TCR-and CD3-specific monoclonal reagents, despite an otherwise excellent collection of mAbs to cell surface molecules .Here, we describe a new mAb, termed R73, that detects a rat pan T cell surface antigen with the predicted properties of the TCR-a/ß on mature and immature cells of the T cell lineage and reports its functional effects on resting T lymphocytes . Materials and MethodsAnimals. Young adult Wistar and Lewis rats ofboth sexes were obtained from the animal quarters of the Max Planck Institute for Biochemistry, Martinsried, FRG, or from the Zentralinstitut für Versuchstierzucht, Medizinische Hochschule Hannover, FRG. Results obtained did not vary significantly between both strains .Immunization and Cell Fusion . Spleen cells from a BALB/c mouse alternately immunized intraperitoneally with rat T blasts and rat erythrocytes (it was also intended to generate an mAb to rat LFA-3) were fused 3 d after an intravenous injection of 10' rat erythrocytes with This work was funded by a grant from the Bundesministerium für Forschung und Technologie . Generation of the R73 cell line was funded by Genzentrum e.V. J . Exp. MED.