Frédéric Triebel

The negative regulatory function of the lymphocyte-activation gene-3 co-receptor (CD223) on human T cells

Accumulating evidence indicates that the CD4 homologue lymphocyte activation gene‐3 (LAG‐3) plays a down‐regulatory role on T‐cell responses. However, the role of LAG‐3/major histocompatibility complex (MHC) class II interactions on primary human T‐cell responses, as well as the mechanism by which down‐regulation occurs, are not clear. Here, we show that LAG‐3 colocalized with CD3, CD4 or CD8 in areas of cholesterol‐rich raft aggregation during this primary response, as well as in the clustered raft region formed between T cells and antibody‐coated beads. Addition of a blocking LAG‐3‐specific monoclonal antibody to both CD4 and CD8 primary resting T cells activated under conditions of antigen‐presenting cell‐driven stimulation and low antigen concentrations augments CD69 activation antigen expression, T‐cell expansion and T helper 1 (Th1, but not Th2) cytokine production. Blocking LAG‐3/MHC class II interactions leads to an increase in the number of cells entering division at these low concentrations of antigen and to more rounds of divisions with an accumulation of cells in the S‐phase of the cell cycle. These results indicate that LAG‐3 signalling inhibits early events in primary activation of human CD4 and CD8 T cells and further support a role for LAG‐3 signalling in regulating the expansion of activated effector or memory T cells, either directly or indirectly through Treg suppressor activity.

Maturation and Activation of Dendritic Cells Induced by Lymphocyte Activation Gene-3 (CD223)

Lymphocyte activation gene-3 (LAG-3) is an MHC class II ligand expressed on activated T and NK cells. A LAG-3Ig fusion protein has been used in mice as an adjuvant protein to induce antitumor responses and specific CD8 and CD4 Th1 responses to nominal Ags. In this work we report on the effect of LAG-3Ig on the maturation and activation of human monocyte-derived dendritic cells (DC). LAG-3Ig binds MHC class II molecules expressed in plasma membrane lipid rafts on immature human DC and induces rapid morphological changes, including the formation of dendritic projections. LAG-3Ig markedly up-regulates the expression of costimulatory molecules and the production of IL-12 and TNF-α. Consistent with this effect on DC maturation, LAG-3Ig disables DC in their capacity to capture soluble Ags. These events are associated with the acquisition of professional APC function, because LAG-3Ig increases the capacity of DC to stimulate the proliferation and IFN-γ response by allogeneic T cells. These effects were not observed when using ligation of MHC class II by specific mAb. Class II-mediated signals induced by a natural ligand, LAG-3, lead to complete maturation of DC, which acquire the capacity to trigger naive T cells and drive polarized Th1 responses.

Human Dendritic Cells Acquire a Semimature Phenotype and Lymph Node Homing Potential through Interaction with CD4+CD25+ Regulatory T Cells

Interactions between dendritic cells (DC) and T cells are known to involve the delivery of signals in both directions. We sought to characterize the effects on human DC of contact with different subsets of activated CD4+ T cells. The results showed that interaction with CD25highCD4+ regulatory T cells (Tregs) caused DC to take on very different properties than contact with naive or memory phenotype T cells. Whereas non-Tregs stimulated DC maturation, culture with Tregs produced DC with a mixed phenotype. By many criteria, Tregs inhibited DC maturation, inducing down-regulation of costimulatory molecules and T cell stimulatory activity. However, DC exposed to Tregs also showed some changes typically associated with DC maturation, namely, increased expression of CCR7 and MHC class II molecules, and gained the ability to migrate in response to the CCR7 ligand CCL19. Both soluble factors and cell-associated molecules were shown to be involved in Treg modulation of DC, with lymphocyte activation gene 3 (LAG-3) playing a predominant role in driving maturation-associated changes. The data show that Tregs induce the generation of semimature DC with the potential to migrate into lymphoid organs, suggesting a possible mechanism by which Tregs down-modulate immune responses.

Soluble Human LAG-3 Molecule Amplifies the In vitro Generation of Type 1 Tumor-Specific Immunity

The adjuvant activities of the human lymphocyte activation gene-3 (LAG-3) molecule have been evaluated in a human setting by investigating the ability of a soluble recombinant human LAG-3 protein (hLAG-3Ig) to enhance the in vitro induction of viral- and tumor-specific CTLs. We found that soluble human LAG-3 significantly sustained the generation and expansion of influenza matrix protein Melan-A/MART-1 and survivin-specific CD8+ T lymphocytes in peripheral blood mononuclear cells (PBMC) of both cancer patients and healthy donors, showing its ability to boost CD8+ T-cell memory response or to prime naive T cells in vitro. The peptide-specific T cells generated in the presence of hLAG-3Ig were endowed with cytotoxic activity and enhanced release of type 1 cytotoxic T (Tc1) cytokines and were able to recognize tumor cells expressing their nominal antigen. Phenotype and cytokine/chemokines produced by antigen-presenting cells (APC) of PBMCs exposed in vitro for 2 days to peptide and hLAG-3Ig indicate that the LAG-3-mediated adjuvant effect may depend on a direct activation of circulating APCs. Our data revealed the activity of hLAG-3Ig in inducing tumor-associated, antigen-specific CD8+ T-cell responses in a human setting and strongly support the conclusion that this recombinant protein is a potential candidate adjuvant for cancer vaccines.

MHC class II engagement by its ligand LAG-3 (CD223) leads to a distinct pattern of chemokine and chemokine receptor expression by human dendritic cells.

Upon stimulation by infectious agent products, dendritic cells (DC) become activated, express high levels of class I and class II antigens, CD80, CD86 and CD83 and migrate to secondary lymphoid organs where they can prime naive CD4-helper and CD8-cytotoxic T-cells. Cognate CD4(+) T-cell help mediated by CD40L along with DC stimulation with another T-cell effector molecule, termed lymphocyte activated gene-3 (LAG-3 or CD223, a ligand for MHC class II) have been shown to induce this maturation process. Both CD40L and LAG-3 have been used as vaccine adjuvants to induce CTL and CD4 Th1 responses. Here, we studied the effect of a soluble LAG-3Ig molecule on the chemokine and chemokine receptor profile of human immature monocyte-derived DC. LAG-3Ig, unlike CD40L, induced an inflammatory signal in terms of IL-8 and MIP-1alpha/CCL3 production and, in contrast to LPS, induced production of chemokines (MDC/CCL22 and TARC/CCL17) known to direct the migration of maturing DC to lymph nodes. In LAG-3-matured DC, surface expression of CCR5 (a receptor for MIP-1alpha/CCL3) was down-regulated and CCR7 (a receptor for MIP-3beta and SLC) was up-regulated. However, LAG-3-matured, but not LPS- or CD40L-matured DC retained their capacity to migrate in chemotaxis chambers and to respond to MIP-1alpha. Altogether, these data represent the first evidence that MHC class II signaling may affect DC migration to secondary lymphoid tissues.

KIR down-regulation on NK cells is associated with down-regulation of activating receptors and NK cell inactivation

We previously reported that killer cell immunoglobulin‐like receptors (KIR) could be down‐regulated from the surface of T cells. Here, we show that KIR down‐regulation is also induced on the surface of natural killer (NK) cells upon ligand binding. Common down‐regulation characteristics are found on these two cell types: a slow kinetics and a phenomenon observed for long inhibitory formsonly. Importantly, KIR down‐regulation on NK cells is associated with a down‐regulation of activating receptors (CD16, CD2 and 2B4) as well as with a lack of cell responsiveness (antibody‐dependentand natural killing activities). This unresponsive state was not observed for MHC‐restricted T cells. Our data implicate that, in addition to prevention of the immediate target cell lysis, KIR‐MHC class I interactions may also regulate the subsequent NK cell cytotoxic activity. This observation opens new perspectives in the understanding of NK cell regulation.

IMP321 (sLAG-3), an immunopotentiator for T cell responses against a HBsAg antigen in healthy adults: a single blind randomised controlled phase I study

BackgroundLAG-3 (CD223) is a natural high affinity ligand for MHC class II. The soluble form (sLAG-3) induces maturation of monocyte-derived dendritic cells in vitro and is used as a potent Th1-like immune enhancer with many antigens in animal models. To extend this observation to human, a proof of concept study was conducted with a clinical-grade sLAG-3, termed IMP321, coinjected with alum-non-absorbed recombinant hepatitis B surface antigen.MethodsIn a randomised, single blind controlled phase I dose escalation study, 48 seronegative healthy volunteers aged 18–55 years were vaccinated at 0, 4 and 8 weeks by subcutaneous injection with 10 μg HBsAg mixed with saline (control) or with IMP321 at one of four doses (3, 10, 30 and 100 μg). To evaluate the efficacy of this three injections over 2 months immunization protocol, an additional control group was injected with the commercial vaccine Engerix-B®.ResultsIMP321 was very well tolerated. Indeed, a lower incidence of adverse events was reported from the HBsAg plus IMP321 groups than from the Engerix-B® group. HBsAg-specific antibody responses (anti-HBs) appeared sooner and were higher at 8 and 12 weeks in IMP321 recipients compared to HBsAg control subjects. More importantly, increased numbers of responders to HBsAg were found in IMP321 recipients compared HBsAg group, as revealed by higher post-vaccination frequencies of CD4 Th1 or CD8 Tc1 antigen specific T cells. IMP321 induced CD4 Th1 antigen-specific T cells in some of these naïve individuals after only one injection, especially in the 10 and 30 μg dose groups.ConclusionIMP321 as an adjuvant to HBsAg was well-tolerated and enhanced T cell response vaccine immunogenicity (i.e. induced both CD4 Th1 and CD8 Tc1 antigen-specific T cells). This latter property has allowed the development of IMP321 as an immunopotentiator for therapeutic vaccines.

LAG-3 (CD223) reduces macrophage and dendritic cell differentiation from monocyte precursors

Major histocompatibility complex (MHC) class II molecules expressed on monocytes may play a role in the control of differentiation of antigen‐presenting cells. A soluble LAG‐3 (CD223) molecule (sLAG‐3) is a natural, high‐affinity ligand for MHC class II. It is known to induce maturation of monocyte‐derived dendritic cells in vitro and is used as a vaccine adjuvant to induce CD4 T helper type 1 responses and CD8 T‐cell responses in vivo. Here, we demonstrate that sLAG‐3 (but not an MHC class II‐specific monoclonal antibody) reduces the differentiation of monocytes into macrophages in the presence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF) as well as their differentiation into dendritic cells in the presence of GM‐CSF and interleukin‐4, as shown by a decrease in CD14 and CD1a expression, respectively. Dendritic cells derived from monocytes in the presence of sLAG‐3 showed impaired antigen‐presentation function, as assessed by the reduced capability to induce proliferation of T cells. Our results suggest that activated LAG‐3+ lymphocytes present at sites of inflammation may reduce the differentiation of monocytes into macrophages or fully competent antigen‐presenting dendritic cells, thus limiting the magnitude of the ongoing T‐cell immune responses.