Bruno Lucas

Divergent Changes in the Sensitivity of Maturing T Cells to Structurally Related Ligands Underlies Formation of a Useful T Cell Repertoire

CD4+ CD8+ thymocyte differentiation requires TCR signaling induced by self-peptide/MHC ligands. Nevertheless, the resulting mature T cells are not activated by these self-complexes, whereas foreign ligands can be potent stimuli. Here, we show that the signaling properties of TCR change during thymocyte maturation, differentially affecting responses to related peptide/MHC molecule complexes and contributing to this discrimination. Weak agonists for CD4+ CD8+ thymocytes lose potency during development, accompanied by a change in TCR-associated phosphorylation from an agonist to a partial agonist/antagonist pattern. In contrast, sensitivity to strong agonists is maintained, along with full signaling. This yields a mature T cell pool highly responsive to foreign antigen while possessing a wide margin of safety against activation by self-ligands.

Th2 Lymphoproliferative Disorder of LatY136F Mutant Mice Unfolds Independently of TCR-MHC Engagement and Is Insensitive to the Action of Foxp3+ Regulatory T Cells

Mutant mice where tyrosine 136 of linker for activation of T cells (LAT) was replaced with a phenylalanine (LatY136F mice) develop a fast-onset lymphoproliferative disorder involving polyclonal CD4 T cells that produce massive amounts of Th2 cytokines and trigger severe inflammation and autoantibodies. We analyzed whether the LatY136F pathology constitutes a bona fide autoimmune disorder dependent on TCR specificity. Using adoptive transfer experiments, we demonstrated that the expansion and uncontrolled Th2-effector function of LatY136F CD4 cells are not triggered by an MHC class II-driven, autoreactive process. Using Foxp3EGFP reporter mice, we further showed that nonfunctional Foxp3+ regulatory T cells are present in LatY136F mice and that pathogenic LatY136F CD4 T cells were capable of escaping the control of infused wild-type Foxp3+ regulatory T cells. These results argue against a scenario where the LatY136F pathology is primarily due to a lack of functional Foxp3+ regulatory T cells and suggest that a defect intrinsic to LatY136F CD4 T cells leads to a state of TCR-independent hyperactivity. This abnormal status confers LatY136F CD4 T cells with the ability to trigger the production of Abs and of autoantibodies in a TCR-independent, quasi-mitogenic fashion. Therefore, despite the presence of autoantibodies causative of severe systemic disease, the pathological conditions observed in LatY136F mice unfold in an Ag-independent manner and thus do not qualify as a genuine autoimmune disorder.

Naive T cells proliferate strongly in neonatal mice in response to self-peptide/self-MHC complexes

Adult naive T cells, which are at rest in normal conditions, proliferate strongly when transferred to lymphopenic hosts. In neonates, the first mature thymocytes to migrate to the periphery reach a compartment devoid of preexisting T cells. We have extensively analyzed the proliferation rate and phenotype of peripheral T cells from normal C57BL/6 and T cell antigen receptor transgenic mice as a function of age. We show that, like adult naive T cells transferred to lymphopenic mice, neonatal naive T cells proliferate strongly. By using bone-marrow transfer and thymic-graft models, we demonstrate that the proliferation of the first thymic emigrants reaching the periphery requires T cell antigen receptor-self-peptide/self-MHC interactions and is regulated by the size of the peripheral T cell pool.

Suppression of CD4+ T Lymphocyte Effector Functions by CD4+CD25+ Cells In Vivo

CD4+CD25+ regulatory T cells have been extensively studied during the last decade, but how these cells exert their regulatory function on pathogenic effector T cells remains to be elucidated. Naive CD4+ T cells transferred into T cell-deficient mice strongly expand and rapidly induce inflammatory bowel disease (IBD). Onset of this inflammatory disorder depends on IFN-γ production by expanding CD4+ T cells. Coinjection of CD4+CD25+ regulatory T cells protects recipient mice from IBD. In this study, we show that CD4+CD25+ regulatory T cells do not affect the initial activation/proliferation of injected naive T cells as well as their differentiation into Th1 effectors. Moreover, naive T cells injected together with CD4+CD25+ regulatory T cells into lymphopenic hosts are still able to respond to stimuli in vitro when regulatory T cells are removed. In these conditions, they produce as much IFN-γ as before injection or when injected alone. Finally, when purified, they are able to induce IBD upon reinjection into lymphopenic hosts. Thus, prevention of IBD by CD4+CD25+ regulatory T cells is not due to deletion of pathogenic T cells, induction of a non reactive state (anergy) among pathogenic effector T cells, or preferential induction of Th2 effectors rather than Th1 effectors; rather, it results from suppression of T lymphocyte effector functions, leading to regulated responses to self.

Peripheral CD8+CD25+ T Lymphocytes from MHC Class II-Deficient Mice Exhibit Regulatory Activity

We characterized CD8+ T cells constitutively expressing CD25 in mice lacking the expression of MHC class II molecules. We showed that these cells are present not only in the periphery but also in the thymus. Like CD4+CD25+ T cells, CD8+CD25+ T cells appear late in the periphery during ontogeny. Peripheral CD8+CD25+ T cells from MHC class II-deficient mice also share phenotypic and functional features with regulatory CD4+CD25+ T cells: in particular, they strongly express glucocorticoid-induced TNFR family-related gene, CTLA-4 and Foxp3, produce IL-10, and inhibit CD25− T cell responses to anti-CD3 stimulation through cell contacts with similar efficiency to CD4+CD25+ T cells. However, unlike CD4+CD25+ T cells CD8+CD25+ T cells from MHC class II-deficient mice strongly proliferate and produce IFN-γ in vitro in response to stimulation in the absence of exogenous IL-2.

Conversion of Naive T Cells to a Memory-Like Phenotype in Lymphopenic Hosts Is Not Related to a Homeostatic Mechanism That Fills the Peripheral Naive T Cell Pool

To examine directly whether a limited number of naive T cells transferred to lymphopenic hosts can truly fill the peripheral naive T cell pool, we compared the expansion and phenotype of naive T cells transferred to three different hosts, namely recombination-activating gene-deficient mice, CD3ε-deficient mice, and irradiated normal mice. In all three recipients, the absolute number of recovered cells was much smaller than in normal mice. In addition, transferred naive T cells acquired a memory-like phenotype that remained stable with time. Finally, injected cells were rapidly replaced by host thymic migrants in irradiated normal mice. Only continuous output of naive T cells by the thymus can generate a full compartment of truly naive T cells. Thus, conversion of naive T cells to a memory-like phenotype in lymphopenic hosts is not related to a homeostatic mechanism that fills the peripheral naive T cell pool.

Lymphopenia-induced spontaneous T-cell proliferation as a cofactor for autoimmune disease development

Lymphopenia is thought to be a major cause of tolerance breakdown. In a lymphopenic environment, self-recognition events induce some T cells to expand strongly (a mechanism known as spontaneous proliferation). In this study, we show that in C57BL/6 mice, the repertoire resulting from lymphopenia-induced spontaneous CD4(+) T-cell proliferation included a proportion of regulatory T cells as large as that observed in a normal mouse, and no autoimmune disorder was observed. By contrast, in nonobese diabetic mice, differences in the ability of conventional and regulatory T cells to expand in response to lymphopenia led to an unbalance between these 2 T-cell compartments at the expense of regulatory T cells, resulting in the onset of autoimmune diseases. Notably, this accounted for the rapid transfer of diabetes with small numbers of BDC2.5 CD4(+) T cells. Thus, lymphopenia does not itself induce autoimmunity, but it should be considered as a cofactor for the development of autoimmune disorders.

On the role of MHC class II molecules in the survival and lymphopenia-induced proliferation of peripheral CD4+ T cells.

CD4+ T cells expand after transfer into lymphopenic H-2b Aβ−/− mice (I-Aβ-, I-Eα-deficient mice) but not after transfer into lymphopenic MHC IIΔ/Δ mice (I-Aα-, I-Aβ-, I-Eα-, and I-Eβ-deficient mice), implying that in H-2b Aβ−/− mice, Aα chain and Eβ chain associate to form a hybrid AαEβ MHC class II molecule. In light of this unexpected result, we reexamined the MHC class II requirement in the survival and lymphopenia-induced proliferation of CD4+ T cells. Here we show that expansion, but not short-term survival, of CD4+ T cells depends on interactions with MHC class II molecules in lymphopenic mice. Nevertheless, interactions with classical MHC class II molecules are required for CD4+ T cells to survive in CD8+ T-cell-containing mice.

Long-term reversal of established autoimmunity upon transient blockade of the LFA-1/intercellular adhesion molecule-1 pathway.

Transgenic models and administration of mAbs directed against the LFA-1/intercellular adhesion molecule 1 (ICAM-1) pathway have shown that these costimulatory molecules play a key role in generating effector cells mediating inflammatory responses. In this report, durable remission of recent diabetes in nonobese diabetic (NOD) mice was induced by transient expression of an immunoadhesin gene encoding the soluble form of ICAM-1 (sICAM-1/Ig). A single i.v. injection of an adenovirus vector encoding the immunoadhesin gene led to 70% diabetes remission as opposed to 0% in mice injected with a control adenovirus vector. Despite the rapid decline of sICAM-1/Ig serum levels, diabetes remission remained stable in 50% of NOD mice for >6 mo. sICAM-1/Ig expression also led to long-term protection against diabetes in prediabetic NOD mice. sICAM-1/Ig in vitro induced an agonistic effect of T cell activation in a TCR-transgenic model, increasing T cell proliferation and IL-2 secretion. Importantly, protected mice were not immunosuppressed because they rejected skin allografts normally and developed immunity against the adenovirus vector. Rather, sICAM-1/Ig induced active tolerance, as assessed by the persistence of diabetogenic T cells in protected mice and the reversal of protection by immunosuppression with cyclophosphamide.

Opening a Window on Thymic Positive Selection: Developmental Changes in the Influence of Cosignaling by Integrins and CD28 on Selection Events Induced by TCR Engagement

How TCR and non-TCR signals are integrated by thymocytes to generate a decision to undergo either positive or negative selection remains incompletely understood. Recent evidence suggests that TCR signal transduction changes its quality during thymocyte maturation, but whether the contributions of various cosignaling or costimulatory pathways to thymocyte selection also are modified during development is unclear. Questions also remain about the possible selective roles of specific costimulatory pathways in induction of differentiation vs death among thymocytes at any given stage of maturity. To address these issues, a quantitative in vitro analysis of initiation of CD4+CD8+ thymocyte differentiation as measured by CD69 up-regulation/coreceptor down-modulation was conducted in parallel with an analysis of induction of death. Using transfected cells varying in their surface display of ICAM-1 or B7.1 along with antibody blocking experiments, we demonstrate here that ICAM-1 provides a selective boost to signaling for differentiation without substantially affecting induction of death among CD4+CD8+ cells, a property that is lost as thymocytes mature further. In contrast, B7 engagement enhances both cell activation and death in parallel. Based on these data, we propose that the high level of ICAM-1 on cortical epithelial cells plays a special role in opening a window between TCR signaling for differentiation vs death, permitting efficient initiation of positive selection on epithelial ligands. In contrast, late CD28-dependent cosignaling on hemopoietic cells in the medulla would help enforce negative selection by augmenting the effects of TCR engagement by low levels of high affinity ligands.