Person:

Sage, Peter

The ability for CD4 T cells to efficiently search for and subsequently respond to microbial pathogens is essential for protective immunity, but mechanisms controlling these responses are not completely understood. In this thesis I study the regulation of CD4 T cell responses at two different stages during an immune response. First, I analyze one of the most basic mechanisms by which T cells search for and become activated by an antigenic stimulus during the initial events in an adaptive immune response. Using human memory CD4 T cells in vitro I have identified a novel role for actin-rich invadapodia-like protrusions (ILPs) in overcoming the energy barrier required for the T cell receptor (TCR) to send signals into T cells when interacting with peptide-loaded MHC II. My studies show that ILPs, which are used during migration, are also essential for surveying the surface of other cells during cellular communication. Secondly, I explore the costimulatory requirements and function of T follicular regulatory ((T_{FR})) cells, a newly identified subset of regulatory T ((T_{REG})) cells. Using mouse models, I have discovered that the costimulatory receptor PD-1 inhibits the differentiation and function of (T_{FR}) cells in vivo. My work also has revealed that (T_{FR}) cells can circulate within the blood and that blood TFR cells can potently inhibit B cell mediated antibody production in vivo. Taken together, the studies presented here not only provide insights into the very initial events leading to adaptive immunity, but also demonstrate how adaptive immunity is controlled during the effector phase of an immune reaction.

Basic mechanisms by which cellular barriers sense and respond to integrity disruptions remain poorly understood. Despite its tenuous structure and constitutive exposure to disruptive strains, the vascular endothelium exhibits robust barrier function. We show that in response to micrometer-scale disruptions induced by transmigrating leukocytes, endothelial cells generate unique ventral lamellipodia that propagate via integrins toward and across these “micro-wounds” to close them. This novel actin remodeling activity progressively healed multiple micro-wounds in succession and changed direction during this process. Mechanical probe-induced micro-wounding of both endothelia and epithelia suggests that ventral lamellipodia formed as a response to force imbalance and specifically loss of isometric tension. Ventral lamellipodia were enriched in the Rac1 effectors cortactin, IQGAP, and p47Phox and exhibited localized production of hydrogen peroxide. Together with Apr2/3, these were functionally required for effective micro-wound healing. We propose that barrier disruptions are detected as local release of isometric tension/force unloading, which is directly coupled to reactive oxygen species–dependent self-restorative actin remodeling dynamics.

Foxp3+ regulatory T cells (Tregs) are required for immune homeostasis. One notable distinction between conventional T cells (Tconv) and Tregs is differential phosphatidylinositol 3-kinase (PI3K) activity: only Tconv downregulate PTEN, the primary negative regulator of PI3K, upon activation. Here, we show that control of PI3K in Tregs is essential for lineage homeostasis and stability. Mice lacking Pten in Tregs developed an autoimmune-lymphoproliferative disease characterized by excessive TH1 responses and B cell activation. Diminished control of PI3K activity in Tregs led to reduced CD25 expression, accumulation of Foxp3+CD25− cells and ultimately, loss of Foxp3 expression in these cells. Collectively, these data demonstrate that control of PI3K signaling by PTEN in Tregs is critical to maintain their homeostasis, function and stability.

Whereas the SLAMF-associated protein (SAP) is involved in differentiation of T follicular helper (Tfh) cells and antibody responses, the precise requirements of SLAMF receptors in humoral immune responses are incompletely understood. By analyzing mice with targeted disruptions of the Slamf1, Slamf5, and Slamf6 genes, we found that both T-dependent and T-independent antibody responses were twofold higher compared to those in single knockout mice. These data suggest a suppressive synergy of SLAMF1, SLAMF5, and SLAMF6 in humoral immunity, which contrasts the decreased antibody responses resulting from a defective GC reaction in the absence of the adapter SAP. In adoptive co-transfer assays, both [Slamf1 + 5 + 6]−/− B and T cells were capable of inducing enhanced antibody responses, but more pronounced enhancement was observed after adoptive transfer of [Slamf1 + 5 + 6]−/− B cells compared to that of [Slamf1 + 5 + 6]−/− T cells. In support of [Slamf1 + 5 + 6]−/− B cell intrinsic activity, [Slamf1 + 5 + 6]−/− mice also mounted significantly higher antibody responses to T-independent type 2 antigen. Furthermore, treatment of mice with anti-SLAMF6 monoclonal antibody results in severe inhibition of the development of Tfh cells and GC B cells, confirming a suppressive effect of SLAMF6. Taken together, these results establish SLAMF1, SLAMF5, and SLAMF6 as important negative regulators of humoral immune response, consistent with the notion that SLAM family receptors have dual functions in immune responses.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential negative regulator of T cell responses. Germline Ctla4 deficiency is lethal, making investigation of the function of CTLA-4 on mature T cells challenging. To elucidate the function of CTLA-4 on mature T cells, we have conditionally ablated Ctla4 in adult mice. We show that, in contrast to germline knockout mice, deletion of Ctla4 during adulthood does not precipitate systemic autoimmunity, but surprisingly confers protection from experimental autoimmune encephalomyelitis (EAE) and does not lead to increased resistance to MC38 tumors. Deletion of Ctla4 during adulthood was accompanied by activation and expansion of both conventional CD4+Foxp3− (T conv) and regulatory Foxp3+ (T reg cells) T cell subsets; however, deletion of CTLA-4 on T reg cells was necessary and sufficient for protection from EAE. CTLA-4 deleted T reg cells remained functionally suppressive. Deletion of Ctla4 on T reg cells alone or on all adult T cells led to major changes in the Ctla4 sufficient T conv cell compartment, including up-regulation of immunoinhibitory molecules IL-10, LAG-3 and PD-1, thereby providing a compensatory immunosuppressive mechanism. Collectively, our findings point to a profound role for CTLA-4 on T reg cells in limiting their peripheral expansion and activation, thereby regulating the phenotype and function of T conv cells.