Plxnd1 Expression in Thymocytes Regulates Their Intrathymic Migration While That in Thymic Endothelium Impacts Medullary Topology

An important role for plexinD1 in thymic development is inferred from studies of germline Plxnd1 knockout (KO) mice where mislocalized CD69+ thymocytes as well as ectopic thymic subcapsular medullary structures were observed. Given embryonic lethality of the Plxnd1−/− genotype, fetal liver transplantation was employed in these prior analyses. Such embryonic hematopoietic reconstitution may have transferred Plxnd1 KO endothelial and/or epithelial stem cells in addition to Plxnd1 KO lymphoid progenitors, thereby contributing to that phenotype. Here we use Plxnd1flox/flox mice crossed to pLck-Cre, pKeratin14-Cre, or pTek-Cre transgenic animals to create cell-type specific conditional knockout (CKO) lines involving thymocytes (D1ThyCKO), thymic epithelium (D1EpCKO), and thymic endothelium (D1EnCKO), respectively. These CKOs allowed us to directly assess the role of plexinD1 in each lineage. Loss of plexinD1 expression on double positive (DP) thymocytes leads to their aberrant migration and cortical retention after TCR-mediated positive selection. In contrast, ectopic medulla formation is a consequence of loss of plexinD1 expression on endothelial cells, in turn linked to dysregulation of thymic angiogenesis. D1EpCKO thymi manifest neither abnormality. Collectively, our findings underscore the non-redundant roles for plexinD1 on thymocytes and endothelium, including the dynamic nature of medulla formation resulting from crosstalk between these thymic cellular components

RGMb is a novel binding partner for PD-L2 and its engagement with PD-L2 promotes respiratory tolerance

We report that programmed death ligand 2 (PD-L2), a known ligand of PD-1, also binds to repulsive guidance molecule b (RGMb), which was originally identified in the nervous system as a co-receptor for bone morphogenetic proteins (BMPs). PD-L2 and BMP-2/4 bind to distinct sites on RGMb. Normal resting lung interstitial macrophages and alveolar epithelial cells express high levels of RGMb mRNA, whereas lung dendritic cells express PD-L2. Blockade of the RGMb–PD-L2 interaction markedly impaired the development of respiratory tolerance by interfering with the initial T cell expansion required for respiratory tolerance. Experiments with PD-L2–deficient mice showed that PD-L2 expression on non–T cells was critical for respiratory tolerance, but expression on T cells was not required. Because PD-L2 binds to both PD-1, which inhibits antitumor immunity, and to RGMb, which regulates respiratory immunity, targeting the PD-L2 pathway has therapeutic potential for asthma, cancer, and other immune-mediated disorders. Understanding this pathway may provide insights into how to optimally modulate the PD-1 pathway in cancer immunotherapy while minimizing adverse events

CD40L-Tri, a novel formulation of recombinant human CD40L that effectively activates B cells

CD40L has a well-established role in enhancing the immunostimulatory capacity of normal and malignant B cells, but a formulation suitable for clinical use has not been widely available. Like other TNF family members, in vivo and in vitro activity of CD40L requires a homotrimeric configuration, and growing evidence suggests that bioactivity depends on higher-order clustering of CD40. We generated a novel formulation of human recombinant CD40L (CD40L-Tri) in which the CD40L extracellular domain and a trimerization motif are connected by a long flexible peptide linker. We demonstrate that CD40L-Tri significantly expands normal CD19+ B cells by over 20- to 30-fold over 14 days and induces B cells to become highly immunostimulatory antigen-presenting cells (APCs). Consistent with these results, CD40L-Tri-activated B cells could effectively stimulate antigen-specific T responses (against the influenza M1 peptide) from normal volunteers. In addition, CD40L-Tri could induce malignant B cells to become effective APCs, such that tumor-directed immune responses could be probed. Together, our studies demonstrate the potent immune-stimulatory effects of CD40L-Tri on B cells that enable their expansion of antigen-specific human T cells. The potent bioactivity of CD40L-Tri is related to its ability to self-multimerize, which may be facilitated by its long peptide linker. Electronic supplementary material The online version of this article (doi:10.1007/s00262-012-1331-4) contains supplementary material, which is available to authorized users

Molecular design of the γδT cell receptor ectodomain encodes biologically fit ligand recognition in the absence of mechanosensing

High-acuity αβT cell receptor (TCR) recognition of peptides bound to major histocompatibility complex molecules (pMHCs) requires mechanosensing, a process whereby piconewton (pN) bioforces exert physical load on αβTCR–pMHC bonds to dynamically alter their lifetimes and foster digital sensitivity cellular signaling. While mechanotransduction is operative for both αβTCRs and pre-TCRs within the αβT lineage, its role in γδT cells is unknown. Here, we show that the human DP10.7 γδTCR specific for the sulfoglycolipid sulfatide bound to CD1d only sustains a significant load and undergoes force-induced structural transitions when the binding interface-distal γδ constant domain (C) module is replaced with that of αβ. The chimeric γδ–αβTCR also signals more robustly than does the wild-type (WT) γδTCR, as revealed by RNA-sequencing (RNA-seq) analysis of TCR-transduced Rag2−/− thymocytes, consistent with structural, single-molecule, and molecular dynamics studies reflective of γδTCRs as mediating recognition via a more canonical immunoglobulin-like receptor interaction. Absence of robust, force-related catch bonds, as well as γδTCR structural transitions, implies that γδT cells do not use mechanosensing for ligand recognition. This distinction is consonant with the fact that their innate-type ligands, including markers of cellular stress, are expressed at a high copy number relative to the sparse pMHC ligands of αβT cells arrayed on activating target cells. We posit that mechanosensing emerged over ∼200 million years of vertebrate evolution to fulfill indispensable adaptive immune recognition requirements for pMHC in the αβT cell lineage that are unnecessary for the γδT cell lineage mechanism of non-pMHC ligand detection

Plxnd1 expression in thymocytes regulates their intrathymic migration while that in thymic endothelium impacts medullary topology

An important role for plexinD1 in thymic development is inferred from studies of germline Plxnd1 knockout (KO) mice where mislocalized CD69+ thymocytes as well as ectopic thymic subcapsular medullary structures were observed. Given embryonic lethality of the Plxnd1-/- genotype, fetal liver transplantation was employed in these prior analyses. Such embryonic hematopoietic reconstitution may have transferred Plxnd1 KO endothelial and/or epithelial stem cells in addition to Plxnd1 KO lymphoid progenitors, thereby contributing to that phenotype. Here we use Plxnd1flox/flox mice crossed to pLck-Cre, pKeratin14-Cre or pTek-Cre transgenic animals to create cell-type specific conditional knockout (CKO) lines involving thymocytes (D1ThyCKO), thymic epithelium (D1EpCKO) and thymic endothelium (D1EnCKO), respectively. These CKOs allowed us to directly assess the role of plexinD1 in each lineage. Loss of plexinD1 expression on double positive (DP) thymocytes leads to their aberrant migration and cortical retention after TCR-mediated positive selection. In contrast, ectopic medulla formation is a consequence of loss of plexinD1 expression on endothelial cells, in turn linked to dysregulation of thymic angiogenesis. D1EpCKO thymi manifest neither abnormality. Collectively, our findings underscore the non-redundant roles for plexinD1 on thymocytes and endothelium, including the dynamic nature of medulla formation resulting from crosstalk between these thymic cellular components

SH3 domain recognition of a proline-independent tyrosine-based RKxxYxxY motif in immune cell adaptor SKAP55

Src-homology 3 (SH3) domains recognize PXXP core motif preceded or followed by positively charged residue(s). Whether SH3 domains recognize motifs other than proline-based sequences is unclear. In this study, we report SH3 domain binding to a novel proline-independent motif in immune cell adaptor SKAP55, which is comprised of two N-terminal lysine and arginine residues followed by two tyrosines (i.e. RKxxYxxY). Domains capable of binding to class I proline motifs bound to the motif, while the class II domains failed to bind. Peptide precipitation, alanine scanning and in vivo co-expression studies demonstrated a requirement for the arginine, lysine and tandem tyrosines of the motif. Two-dimensional NMR analysis of the peptide bound FYN-SH3 domain showed overlap with the binding site of a proline-rich peptide on the charged surface of the SH3 domain, while resonance signals for other residues (W119, W120, Y137) were not perturbed by the RKGDYASY based peptide. Expression of the RKGDYASY peptide potently inhibited TcRζ/CD3-mediated NF-AT transcription in T cells. Our findings extend the repertoire of SH3 domain binding motifs to include a tyrosine-based motif and demonstrate a regulatory role for this motif in receptor signaling

Downregulation of semaphorin 3E promotes hallmarks of experimental chronic allergic asthma

Guidance cues such as semaphorins are attractive novel therapeutic targets for allergic disorders. We have previously described an inhibitory effect of semaphorin 3E (Sema3E) on human airway smooth muscle cell function. We have further addressed a canonical role for Sema3E in acute model of allergic asthma in vivo. Considering the chronic nature of the disease, the potential implication of Sema3E to alleviate long-lasting deficits should be investigated. Expression of Sema3E in a chronic model of allergic asthma was assessed after exposure to house dust mite (HDM) as a clinically relevant allergen. Chronic features of allergic asthma including airway hyper-responsiveness (AHR), inflammation, and remodeling were studied in Sema3E-deficient mice. Additionally, the effect of exogenous Sema3E treatment was evaluated in prophylactic and therapeutic experimental models. We have demonstrated that expression of Sema3E is robustly suppressed in the airways upon chronic HDM exposure. Chronic allergic airway disease was significantly augmented in Sema3E-deficient mouse model which was associated with an increased AHR, remodeling, and Th2/Th17 inflammation. Intranasal Sema3E administration restored chronic deficits of allergic asthma in mice. Data from this study unveil a key regulatory role of Sema3E in chronic course of asthma via orchestration of impaired inflammatory and remodeling responses