Roberto A. Maldonado

How tolerogenic dendritic cells induce regulatory T cells.

Since their discovery by Steinman and Cohn in 1973, dendritic cells (DCs) have become increasingly recognized for their crucial role as regulators of innate and adaptive immunity. DCs are exquisitely adept at acquiring, processing, and presenting antigens to T cells. They also adjust the context (and hence the outcome) of antigen presentation in response to a plethora of environmental inputs that signal the occurrence of pathogens or tissue damage. Such signals generally boost DC maturation, which promotes their migration from peripheral tissues into and within secondary lymphoid organs and their capacity to induce and regulate effector T cell responses. Conversely, more recent observations indicate that DCs are also crucial to ensure immunological peace. Indeed, DCs constantly present innocuous self- and nonself-antigens in a fashion that promotes tolerance, at least in part, through the control of regulatory T cells (Tregs). Tregs are specialized T cells that exert their immunosuppressive function through a variety of mechanisms affecting both DCs and effector cells. Here, we review recent advances in our understanding of the relationship between tolerogenic DCs and Tregs.

A role for the immunological synapse in lineage commitment of CD4 lymphocytes

Activation of the naive T-helper lymphocyte (Thp) directs it down one of two major developmental pathways called Th1 and Th2. Signals transmitted by T cell, co-stimulatory and cytokine receptors control Thp lineage commitment but the mechanism by which these signals are integrated remains a mystery. The interferon-γ (IFNGR) and interleukin 4 (IL-4R) cytokine receptors, in particular, direct the earliest stages of T-helper commitment. Here we report that on engagement of the T-cell receptor (TCR) on Thp cells, rapid co-polarization of IFNGR with the TCR occurs within the developing immunological synapse. Thp cells from the intrinsically Th1-like C57BL/6 mouse strain have significantly more receptor co-polarization than Th2-prone BALB/c Thp cells. Remarkably, in the presence of IL-4, a cytokine required for Th2 differentiation, IFNGR co-polarization with TCR is prevented. This inhibition depends on Stat6, the transcription factor downstream of IL-4R that is required for Th2 differentiation. This cytokine receptor crossregulation provides an explanation for the effect of IL-4 in inhibiting Th1 differentiation. These observations suggest a scenario in which physical co-polarization of critical receptors directs the fate of the naive Thp, and offer a novel function for the immunological synapse in directing cell differentiation. They further suggest a new mechanism of membrane-bound signalling control by the physical disruption of large receptor-rich domains on signalling through a functionally antagonistic receptor.

Polymeric synthetic nanoparticles for the induction of antigen-specific immunological tolerance.

Significance Synthetic nanoparticles containing either protein or peptide antigen and the immunosuppressant rapamycin are capable of inducing durable and specific resistance to mounting immune responses toward the antigen. This immunological tolerance operates on lymphocytes even after multiple immunogenic challenges with the antigen and adding enhancers of immune responses (adjuvants). As a result, the animals treated with these tolerogenic nanoparticles (tNPs) show reduced allergic hypersensitivity disorders, protection from disease relapse in a model of multiple sclerosis, and prevention of inhibitory antidrug antibody responses in an animal model of hemophilia A. These results show the potential for nanocarriers to modify the immunoreactivity of a given molecule by providing tolerogenic instructions to the immune system, thereby preventing or reversing pathological and neutralizing immune responses. Current treatments to control pathological or unwanted immune responses often use broadly immunosuppressive drugs. New approaches to induce antigen-specific immunological tolerance that control both cellular and humoral immune responses are desirable. Here we describe the use of synthetic, biodegradable nanoparticles carrying either protein or peptide antigens and a tolerogenic immunomodulator, rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor agonists. Treatment with tolerogenic nanoparticles results in the inhibition of CD4+ and CD8+ T-cell activation, an increase in regulatory cells, durable B-cell tolerance resistant to multiple immunogenic challenges, and the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis, and antibody responses against coagulation factor VIII in hemophilia A mice, even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. Tolerogenic nanoparticle therapy represents a potential novel approach for the treatment of allergies, autoimmune diseases, and prevention of antidrug antibodies against biologic therapies.

Improving the efficacy and safety of biologic drugs with tolerogenic nanoparticles.

The development of antidrug antibodies (ADAs) is a common cause for the failure of biotherapeutic treatments and adverse hypersensitivity reactions. Here we demonstrate that poly(lactic-co-glycolic acid) (PLGA) nanoparticles carrying rapamycin, but not free rapamycin, are capable of inducing durable immunological tolerance to co-administered proteins that is characterized by the induction of tolerogenic dendritic cells, an increase in regulatory T cells, a reduction in B cell activation and germinal centre formation, and the inhibition of antigen-specific hypersensitivity reactions. Intravenous co-administration of tolerogenic nanoparticles with pegylated uricase inhibited the formation of ADAs in mice and non-human primates and normalized serum uric acid levels in uricase-deficient mice. Similarly, the subcutaneous co-administration of nanoparticles with adalimumab resulted in the durable inhibition of ADAs, leading to normalized pharmacokinetics of the anti-TNFα antibody and protection against arthritis in TNFα transgenic mice. Adjunct therapy with tolerogenic nanoparticles represents a novel and broadly applicable approach to prevent the formation of ADAs against biologic therapies.

Rapamycin-treated human endothelial cells preferentially activate allogeneic regulatory T cells.

Human graft endothelial cells (ECs) can act as antigen-presenting cells to initiate allograft rejection by host memory T cells. Rapamycin, an mTOR inhibitor used clinically to suppress T cell responses, also acts on DCs, rendering them tolerogenic. Here, we report the effects of rapamycin on EC alloimmunogenicity. Compared with mock-treated cells, rapamycin-pretreated human ECs (rapa-ECs) stimulated less proliferation and cytokine secretion from allogeneic CD4+ memory cells, an effect mimicked by shRNA knockdown of mTOR or raptor in ECs. The effects of rapamycin persisted for several days and were linked to upregulation of the inhibitory molecules PD-L1 and PD-L2 on rapa-ECs. Additionally, rapa-ECs produced lower levels of the inflammatory cytokine IL-6. CD4+ memory cells activated by allogeneic rapa-ECs became hyporesponsive to restimulation in an alloantigen-specific manner and contained higher percentages of suppressive CD4+CD25(hi)CD127(lo)FoxP3+ cells that did not produce effector cytokines. In a human-mouse chimeric model of allograft rejection, rapamycin pretreatment of human arterial allografts increased graft EC expression of PD-L1 and PD-L2 and reduced subsequent infiltration of allogeneic effector T cells into the artery intima and intimal expansion. Preoperative conditioning of allograft ECs with rapamycin could potentially reduce immune-mediated rejection.

Nanoparticles for the Induction of Antigen-Specific Immunological Tolerance

Antigen-specific immune tolerance has been a long-standing goal for immunotherapy for the treatment of autoimmune diseases and allergies and for the prevention of allograft rejection and anti-drug antibodies directed against biologic therapies. Nanoparticles have emerged as powerful tools to initiate and modulate immune responses due to their inherent capacity to target antigen-presenting cells (APCs) and deliver coordinated signals that can elicit an antigen-specific immune response. A wide range of strategies have been described to create tolerogenic nanoparticles (tNPs) that fall into three broad categories. One strategy includes tNPs that provide antigen alone to harness natural tolerogenic processes and environments, such as presentation of antigen in the absence of costimulatory signals, oral tolerance, the tolerogenic environment of the liver, and apoptotic cell death. A second strategy includes tNPs that carry antigen and simultaneously target tolerogenic receptors, such as pro-tolerogenic cytokine receptors, aryl hydrocarbon receptor, FAS receptor, and the CD22 inhibitory receptor. A third strategy includes tNPs that carry a payload of tolerogenic pharmacological agents that can “lock” APCs into a developmental or metabolic state that favors tolerogenic presentation of antigens. These diverse strategies have led to the development of tNPs that are capable of inducing antigen-specific immunological tolerance, not just immunosuppression, in animal models. These novel tNP technologies herald a promising approach to specifically prevent and treat unwanted immune reactions in humans. The first tNP, SEL-212, a biodegradable synthetic vaccine particle encapsulating rapamycin, has reached the clinic and is currently in Phase 2 clinical trials.

Tolerogenic Nanoparticles Induce Antigen-Specific Regulatory T Cells and Provide Therapeutic Efficacy and Transferrable Tolerance against Experimental Autoimmune Encephalomyelitis

T cells reacting to self-components can promote tissue damage when escaping tolerogenic control mechanisms which may result in autoimmune disease. The current treatments for these disorders are not antigen (Ag) specific and can compromise host immunity through chronic suppression. We have previously demonstrated that co-administration of encapsulated or free Ag with tolerogenic nanoparticles (tNPs) comprised of biodegradable polymers that encapsulate rapamycin are capable of inhibiting Ag-specific transgenic T cell proliferation and inducing Ag-specific regulatory T cells (Tregs). Here, we further show that tNPs can trigger the expansion of endogenous Tregs specific to a target Ag. The proportion of Ag-specific Treg to total Ag-specific T cells remains constant even after subsequent Ag challenge in combination with a potent TLR7/8 agonist or complete Freund’s adjuvant. tNP-treated mice do not develop experimental autoimmune encephalomyelitis (EAE) after adoptive transfer of encephalitogenic T cells; furthermore, tNP treatment provided therapeutic protection in relapsing EAE that was transferred to naïve animals. These findings describe a potent therapy to expand Ag-specific Tregs in vivo and suppress T cell-mediated autoimmunity.

Unexpected role of clathrin adaptor AP-1 in MHC-dependent positive selection of T cells

Trafficking of transmembrane receptors to a specific intracellular compartment is conducted by adaptor molecules that bind to target motifs within the cytoplasmic domains of cargo proteins. We generated mice containing a lymphoid-specific deficiency of AP-1 using RNAi knockdown technology. Inhibition of AP-1 expression in thymocytes blocks progression from double-positive immature thymocytes, resulting in complete absence of CD4+ single-positive thymocytes and severe reduction of CD3+CD8+ single-positive thymocytes. Analysis of the contribution of AP-1 deficiency on the interaction between mature CD4+ T cells and antigen-presenting cells revealed that AP-1 is essential to efficient immune synapse formation and associated T cell activation, suggesting a possible mechanism of AP-1 function in thymocyte development.