Jesse A. Green

Immune complex relay by subcapsular sinus macrophages and noncognate B cells drives antibody affinity maturation

Subcapsular sinus (SCS) macrophages capture antigens from lymph and present them intact for B cell encounter and follicular delivery. However, the properties of SCS macrophages are poorly defined. Here we show SCS macrophage development depended on lymphotoxin-α1β2, and the cells had low lysosomal enzyme expression and retained opsonized antigens on their surface. Intravital imaging revealed immune complexes moving along macrophage processes into the follicle. Moreover, noncognate B cells relayed antigen opsonized by newly produced antibodies from the subcapsular region to the germinal center, and affinity maturation was impaired when this transport process was disrupted. Thus, we characterize SCS macrophages as specialized antigen-presenting cells functioning at the apex of an antigen transport chain that promotes humoral immunity.

A Distinct Function of Regulatory T Cells in Tissue Protection

Regulatory T (Treg) cells suppress immune responses to a broad range of non-microbial and microbial antigens and indirectly limit immune inflammation-inflicted tissue damage by employing multiple mechanisms of suppression. Here, we demonstrate that selective Treg cell deficiency in amphiregulin leads to severe acute lung damage and decreased blood oxygen concentration during influenza virus infection without any measureable alterations in Treg cell suppressor function, antiviral immune responses, or viral load. This tissue repair modality is mobilized in Treg cells in response to inflammatory mediator IL-18 or alarmin IL-33, but not by TCR signaling that is required for suppressor function. These results suggest that, during infectious lung injury, Treg cells have a major direct and non-redundant role in tissue repair and maintenance-distinct from their role in suppression of immune responses and inflammation-and that these two essential Treg cell functions are invoked by separable cues.

Follicular dendritic cells help establish follicle identity and promote B cell retention in germinal centers

Selective ablation of follicular dendritic cells in mice results in disorganization of primary follicles and dispersal of B cells out of splenic germinal centers.

The sphingosine 1-phosphate receptor S1P2 maintains the homeostasis of germinal center B cells and promotes niche confinement

Mice deficient in sphingosine 1-phosphate receptor type 2 (S1P2) develop diffuse large B cell lymphoma. However, the role of S1P2 in normal germinal center (GC) physiology is unknown. Here we show that S1P2-deficient GC B cells outgrew their wild-type counterparts in chronically established GCs. We found that antagonism of the kinase Akt mediated by S1P2 and its downstream mediators Gα12, Gα13 and p115RhoGEF regulated cell viability and was required for growth control in chronically proliferating GCs. Moreover, S1P2 inhibited GC B cell responses to follicular chemoattractants and helped confine cells to the GC. In addition, S1P2 overexpression promoted the centering of activated B cells in the follicle. We suggest that by inhibiting Akt activation and migration, S1P2 helps restrict GC B cell survival and localization to an S1P-low niche at the follicle center.

Loss of signalling via G α 13 in germinal centre B-cell-derived lymphoma

Germinal centre B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) is a common malignancy, yet the signalling pathways that are deregulated and the factors leading to its systemic dissemination are poorly defined. Work in mice showed that sphingosine-1-phosphate receptor-2 (S1PR2), a Gα12 and Gα13 coupled receptor, promotes growth regulation and local confinement of germinal centre B cells. Recent deep sequencing studies of GCB-DLBCL have revealed mutations in many genes in this cancer, including in GNA13 (encoding Gα13) and S1PR2 (refs 5,6, 7). Here we show, using in vitro and in vivo assays, that GCB-DLBCL-associated mutations occurring in S1PR2 frequently disrupt the receptor’s Akt and migration inhibitory functions. Gα13-deficient mouse germinal centre B cells and human GCB-DLBCL cells were unable to suppress pAkt and migration in response to S1P, and Gα13-deficient mice developed germinal centre B-cell-derived lymphoma. Germinal centre B cells, unlike most lymphocytes, are tightly confined in lymphoid organs and do not recirculate. Remarkably, deficiency in Gα13, but not S1PR2, led to germinal centre B-cell dissemination into lymph and blood. GCB-DLBCL cell lines frequently carried mutations in the Gα13 effector ARHGEF1, and Arhgef1 deficiency also led to germinal centre B-cell dissemination. The incomplete phenocopy of Gα13- and S1PR2 deficiency led us to discover that P2RY8, an orphan receptor that is mutated in GCB-DLBCL and another germinal centre B-cell-derived malignancy, Burkitt’s lymphoma, also represses germinal centre B-cell growth and promotes confinement via Gα13. These findings identify a Gα13-dependent pathway that exerts dual actions in suppressing growth and blocking dissemination of germinal centre B cells that is frequently disrupted in germinal centre B-cell-derived lymphoma.

Sphingosine-1-phosphate receptor 2 is critical for follicular helper T cell retention in germinal centers

S1PR2 and CXCR5 cooperatively regulate localization of Tfh cells in GCs to support GC responses.

S1PR2 links germinal center confinement and growth regulation

Summary:  Germinal centers (GCs) are sites of rapid B‐cell proliferation and somatic mutation. These ovoid structures develop within the center of follicles and grow to a stereotypic size. The cell migration and interaction dynamics underlying GC B‐cell selection events are currently under intense scrutiny. In recent study, we identified a role for a migration inhibitory receptor, S1PR2, in promoting GC B‐cell confinement to GCs. S1PR2 also dampens Akt activation and deficiency in S1PR2 or components of its signaling pathway result in a loss of growth control in chronically stimulated mucosal GCs. Herein, we detail present understanding of S1PR2 and S1P biology as it pertains to GC B cells and place this information in the context of a current model of GC function.

The Eph-related tyrosine kinase ligand Ephrin-B1 marks germinal center and memory precursor B cells.

Identification of germinal center (GC) B cells is typically reliant on the use of surface activation markers that exhibit a wide range of expression. Here, we identify Ephrin-B1, a ligand for Eph-related receptor tyrosine kinases, as a specific marker of mature GC B cells. The number of Ephrin-B1+ GC B cells increases during the course of an immune response with Ephrin-B1+ GC B cells displaying elevated levels of Bcl6, S1pr2, and Aicda relative to their Ephrin-B1– counterparts. We further identified a small proportion of recently dividing, somatically mutated Ephrin-B1+ GC B cells that have begun to down-regulate Bcl6 and S1pr2 and express markers associated with memory B cells, such as CD38 and EBI2. Transcriptional analysis indicates that these cells are developmentally related to memory B cells, and likely represent a population of GC memory precursor (PreMem) B cells. GC PreMem cells display enhanced survival relative to bulk GC B cells, localize near the edge of the GC, and are predominantly found within the light zone. These findings offer insight into the significant heterogeneity that exists within the GC B cell population and provide tools to further dissect signals regulating the differentiation of GC B cells.

A nonimmune function of T cells in promoting lung tumor progression

The involvement of effector T cells and regulatory T (T reg) cells in opposing and promoting solid organ carcinogenesis, respectively, is viewed as a shifting balance between a breach versus establishment of tolerance to tumor or self-antigens. We considered that tumor-associated T cells might promote malignancy via distinct mechanisms used by T cells in nonlymphoid organs to assist in their maintenance upon injury or stress. Recent studies suggest that T reg cells can participate in tissue repair in a manner separable from their immunosuppressive capacity. Using transplantable models of lung tumors in mice, we found that amphiregulin, a member of the epidermal growth factor family, was prominently up-regulated in intratumoral T reg cells. Furthermore, T cell–restricted amphiregulin deficiency resulted in markedly delayed lung tumor progression. This observed deterrence in tumor progression was not associated with detectable changes in T cell immune responsiveness or T reg and effector T cell numbers. These observations suggest a novel “nonimmune” modality for intratumoral T reg and effector T cells in promoting tumor growth through the production of factors normally involved in tissue repair and maintenance.

Abstract A072: Glucose metabolism and O-linked GlcNAcylation in the tissue repair function of Treg cells

Regulatory T (Treg) cells dampen immune responses to avoid excessive, deleterious inflammation in the context of autoimmunity, allergy, and infection. Treg cells also impede anti-tumor immunity. Therefore, modulation of Treg cell activity is of great therapeutic potential for treating many debilitating inflammatory diseases and cancer. Recent studies have suggested that in addition to their prominent immunosuppressive function, Treg cells have a distinct and non-redundant role in tissue maintenance and remodeling. These Treg cells exhibit a unique transcriptional signature represented by high expression of the EGF family member amphiregulin (Areg). Importantly, tumor infiltrating Treg cells highly express Areg. Since Areg is known to exert pleiotropic oncogenic activities, Areg producing Treg cells may be crucial for establishing the tumor microenvironment. It has been increasingly appreciated that lymphocyte activation and differentiation are coupled to specific metabolic pathways. Metabolic reprogramming is dictated by cytokine and growth factor signals, as well as the availability of nutrients. In addition, metabolic products provide substrates that can alter the functional status of a cell through posttranslational modifications (PTMs). Among the various nutrients, glucose is a key metabolic substrate for T cells. Aerobic glycolysis in activated T cells is required for the acquisition of full effector function. Treg cells in the secondary lymphoid organs and the tissue have distinct glucose availability and metabolic needs. We thus hypothesized that glucose sensing and glucose metabolism play essential roles in shaping the unique phenotype and function of tissue resident Treg cells. O-linked GlcNAcylation is a PTM dynamically regulated by glucose levels and controls the function of many proteins, including transcription factors that play key roles in T cell activation such as NF-κB, CREB, and NFAT. To understand the mechanism by which glucose metabolism controls the function of tissue-resident Treg cells, we began to characterize protein O-GlcNAcylation in Treg cells, and found that both human and mouse Foxp3, the master regulator transcription factor of Treg cells, undergoes O-GlcNAcylation. We are in the process of mapping the site of O-GlcNAcylation of Foxp3, and understanding the physiological significance of this PTM. Citation Format: Wei Hu, Nicholas Arpaia, Jesse A. Green, Ronald C. Hendrickson, Alexander Y. Rudensky. Glucose metabolism and O-linked GlcNAcylation in the tissue repair function of Treg cells [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A072.