Janet E. Buhlmann

Regulation of PD‐1, PD‐L1, and PD‐L2 expression during normal and autoimmune responses

Newer members of the B7‐CD28 superfamily include the receptor PD‐1 and its two ligands, PD‐L1 and PD‐L2. Here, we characterize the expression of PD‐1, PD‐L1, and PD‐L2 in tissues of naive miceand in target organs from two models of autoimmunity, the pancreas from non‐obese diabetic (NOD) mice and brain from mice with experimental autoimmune encephalomyelitis (EAE). In naive mice, proteiexpression of PD‐1, PD‐L1, and PD‐L2 was detected in the thymus, while PD‐1 and PD‐L1 were detected in the spleen. PD‐L1, but not PD‐L2, was also detected at low levels on cardiac endothelium, pancreatic islets, and syncyciotrophoblasts in the placenta. In pre‐diabetic NOD mice, PD‐1 and PD‐L1 were expressed on infiltrating cells in the pancreatic islets. Furthermore, PD‐L1 was markedly up‐regulated on islet cells. In brains from mice with EAE, PD‐1, PD‐L1, and PD‐L2 were expressed on infiltrating inflammatory cells, and PD‐L1 was up‐regulated on endothelium within EAE brain. The distinct expression patterns of PD‐L1 and PD‐L2 led us to compare their transcriptional regulation in STAT4–/–, STAT6–/–, or NF‐κB p50–/–p65+/– dendritic cells (DC).PD‐L2, but not PD‐L1, expression was dramatically reduced in p50–/–p65+/– DC. Thus, PD‐L1 and PD‐L2 exhibit distinct expression patterns and are differentially regulated on the transcriptional level.

Ox40-ligand has a critical costimulatory role in dendritic cell: T cell interactions

The tumor necrosis factor family molecule Ox40-ligand (Ox40L) has been identified as a potential costimulatory molecule and also has been implicated in T cell homing and B cell activation. To ascertain the essential functions of Ox40L, we generated and characterized Ox40L-deficient mice. Mice lacking Ox40L exhibit an impaired contact hypersensitivity response, a dendritic cell-dependent T cell-mediated response, due to defects in T cell priming and cytokine production. In contrast, Ox40L-deficient mice do not have defects in T cell homing or humoral immune responses. In vitro, Ox40L-deficient dendritic cells are defective in costimulating T cell cytokine production. Thus, Ox40L has a critical costimulatory function in vitro and in vivo for dendritic cell:T cell interactions.

Mutations in the WRN gene in mice accelerate mortality in a p53-null background.

ABSTRACT Werners syndrome (WS) is a human disease with manifestations resembling premature aging. The gene defective in WS, WRN, encodes a DNA helicase. Here, we describe the generation of mice bearing a mutation that eliminates expression of the C terminus of the helicase domain of the WRN protein. Mutant mice are born at the expected Mendelian frequency and do not show any overt histological signs of accelerated senescence. These mice are capable of living beyond 2 years of age. Cells from these animals do not show elevated susceptibility to the genotoxins camptothecin or 4-NQO. However, mutant fibroblasts senesce approximately one passage earlier than controls. Importantly,WRN−/− ;p53−/− mice show an increased mortality rate relative toWRN+/− ;p53−/− animals. We consider possible models for the synergy betweenp53 and WRN mutations for the determination of life span.

A Role for the B7-1/B7-2:CD28/CTLA-4 Pathway During Negative Selection

Although costimulation plays an important role in activating naive T cells, its role in negative selection is controversial. By following thymocyte deletion induced by endogenous superantigens in mice lacking B7-1 and/or B7-2, we have identified a role for both B7-1 and B7-2 in negative selection. Studies using CD28-deficient and CD28/CTLA-4-double-deficient mice have revealed that either CD28 or another as yet undefined coreceptor can mediate these B7-dependent signals that promote negative selection. Finally, CTLA-4 delivers signals that inhibit selection, suggesting that CTLA-4 and CD28 have opposing functions in thymic development. Combined, the data demonstrate that B7-1/B7-2-dependent signals help shape the T cell repertoire.

Conservation of CD1 Intracellular Trafficking Patterns Between Mammalian Species

Dendritic cells (DC) are potent APCs that sample Ags from the surrounding environment and present them to naive T cells using cell surface Ag-presenting molecules. The DC in both lymphoid and nonlymphoid tissues express high levels of CD1, a cell surface glycoprotein capable of presenting lipids and glycolipids to T cells. Distinct group 1 CD1 isoforms (CD1a, -b, -c) in man are known to traffic to different parts of the endocytic system where microbial Ags may be sampled. Guinea pigs are the only known rodent species that express the group 1 CD1 proteins. Therefore, we examined the expression and trafficking of guinea pig CD1 (gpCD1) isoforms on isolated DC. Confocal microscopy using mAbs specific for individual gpCD1 isoforms revealed differential trafficking of two distinct CD1b isoforms within DC. Colocalization of MHC class II was observed with the gpCD1b1 isoform, consistent with localization in the late endosomes of DC. In contrast, the gpCD1b3 isoform lacks an endosomal sorting motif and remains on the cell surface. Following incubation with Mycobacterium tuberculosis lipoarabinomannan, colocalization of endocytosed lipoarabinomannan with the gpCD1b1 isoform was observed but not with the gpCD1b3 isoform, which remained primarily on the cell surface. These data demonstrate that guinea pig DC express CD1 isoforms with unique trafficking patterns that recapitulate the patterns seen for human CD1 isoforms. This suggests evolutionary pressure for a conserved mechanism in mammals that allows CD1 to sample lipid Ags from various subcompartments of the endocytic system.

Multiple Checkpoint Breach of B Cell Tolerance in Rasgrp1-Deficient Mice

Lymphopenic hosts offer propitious microenvironments for expansion of autoreactive B and T cells. Despite this, many lymphopenic hosts do not develop autoimmune disease, suggesting that additional factors are required for breaching self-tolerance in the setting of lymphopenia. Mice deficient in guanine nucleotide exchange factor Rasgrp1 develop a lymphoproliferative disorder with features of human systemic lupus erythematosus. Early in life, Rasgrp1-deficient mice have normal B cell numbers but are T lymphopenic, leading to defective homeostatic expansion of CD4 T cells. To investigate whether B cell–intrinsic mechanisms also contribute to autoimmunity, Rasgrp1-deficient mice were bred to mice containing a knockin autoreactive BCR transgene (564Igi), thereby allowing the fate of autoreactive B cells to be assessed. During B cell development, the frequency of receptor-edited 564Igi B cells was reduced in Rasrp1-deficient mice compared with Rasgrp1-sufficient littermate control mice, suggesting that tolerance was impaired. In addition, the number of 564Igi transitional B cells was increased in Rasgrp1-deficient mice compared with control mice. Immature 564Igi B cells in bone marrow and spleen lacking RasGRP1 expressed lower levels of Bim mRNA and protein, suggesting that autoreactive B cells elude clonal deletion during development. Concomitant with increased serum autoantibodies, Rasgrp1-deficient mice developed spontaneous germinal centers at 8–10 wk of age. The frequency and number of 564Igi B cells within these germinal centers were significantly increased in Rasgrp1-deficient mice relative to control mice. Taken together, these studies suggest that autoreactive B cells lacking Rasgrp1 break central and peripheral tolerance through both T cell–independent and –dependent mechanisms.

Characterization of Rho-GDIγ and Rho-GDIα mRNA in the developing and mature brain with an analysis of mice with targeted deletions of Rho-GDIγ

Abstract Rho-GDIs are a family of Rho GDP-dissociation inhibitors that are critical in modulating the activity of the small GTPases, Cdc42 and RhoA. Two Rho-GDI isoforms are expressed in the brain, Rho-GDIγ and Rho-GDIα. Here, we describe the expression of both of these isoforms in the developing and mature brain. The mRNA expression patterns of Rho-GDIγ and Rho-GDIα were almost identical in the brain with expression in the developing and mature cerebral cortex, striatum, and hippocampus. In addition, we generated mice with targeted deletions of Rho-GDIγ that are viable and fertile and have no obvious phenotypic abnormalities. Mutant brains looked histologically normal and demonstrated normal patterns of dendritogenesis and neuronal layering as determined by Golgi staining. Mutant mice had normal sleep/wake patterns and sleep EEGs and showed normal hippocampal-dependent learning as assayed by the Morris water maze task. Based on the co-expression of Rho-GDIα and Rho-GDIγ in identical populations of cells in the brain, the lack of phenotype caused by targeted deletion of Rho-GDIγ may not be surprising given that Rho-GDIα may compensate for the loss of Rho-GDIγ. Whether deletion of both Rho-GDIα and Rho-GDIγ, thereby eliminating all GDI activity in the brain, would produce an observable phenotype remains to be determined.

CD275-Independent IL-17–Producing T Follicular Helper–like Cells in Lymphopenic Autoimmune-Prone Mice

T cells undergo homeostatic expansion and acquire an activated phenotype in lymphopenic microenvironments. Restoration of normal lymphocyte numbers typically re-establishes normal homeostasis, and proinflammatory cytokine production returns to baseline. Mice deficient in guanine nucleotide exchange factor RasGRP1 exhibit dysregulated homeostatic expansion, which manifests as lymphoproliferative disease with autoantibody production. Our previous work revealed that autoreactive B cells lacking RasGRP1 break tolerance early during development, as well as during germinal center responses, suggesting that T cell–independent and T cell–dependent mechanisms are responsible. Examination of whether a particular T cell subset is involved in the breach of B cell tolerance revealed increased Th17 cells in Rasgrp1-deficient mice relative to control mice. Rasgrp1-deficient mice lacking IL-17R had fewer germinal centers, and germinal centers that formed contained fewer autoreactive B cells, suggesting that IL-17 signaling is required for a break in B cell tolerance in germinal centers. Interestingly, a fraction of Th17 cells from Rasgrp1-deficient mice were CXCR5+ and upregulated levels of CD278 coordinate with their appearance in germinal centers, all attributes of T follicular helper cells (Tfh17). To determine whether CD278–CD275 interactions were required for the development of Tfh17 cells and for autoantibody, Rasgrp1-deficient mice were crossed with CD275-deficient mice. Surprisingly, mice deficient in RasGRP1 and CD275 formed Tfh17 cells and germinal centers and produced similar titers of autoantibodies as mice deficient in only RasGRP1. Therefore, these studies suggest that requirements for Tfh cell development change in lymphopenia-associated autoimmune settings.

The Role of Costimulation in T Cell Differentiation

The last 15 years has seen great advances in our understanding of the events necessary for the activation of T cells. From this work, what has come to be accepted as the “two-signal hypothesis of T cell activation” has evolved. This hypothesis was adapted from studies orginally done by Bretscher and Cohn investigating the activation of B cells (1). According to this hypothesis, the first required activation signal for T cells is received after recognition of specific pepted antigens complexed to class I or class II major histocompatibility molecules (MHC) by the T cells receptor (TCR). If this is the only signal received by the T cells a state of unresponsiveness or anergy may be induced (2). The second signal, which can prevent the induction of anergy, comes from an antigen-nonspecific receptor, the costimulatory molecule. This costimulatory signal can have multiple effects on the responding T cells including enhanced proliferation, cytokine production, T cell-effector differentiation and survival (3). Although there are many receptor-ligand pairs that have been implicated in costimulation, this chapter will focus on two major pathways, the B-7CD28/CTLA-4 pathway and the CD40–CD154 pathway, and their effects on T helper (Th) differentiation.