Faculty Bibliography

BACKGROUND: Patients with heterozygous germline mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) experience autoimmunity and lymphoid hyperplasia. OBJECTIVES: Because regulation of the phosphoinositide 3-kinase (PI3K) pathway is critical for maintaining regulatory T (Treg) cell functions, we investigate Treg cells in patients with heterozygous germline PTEN mutations (PTEN hamartoma tumor syndrome [PHTS]). METHODS: Patients with PHTS were assessed for immunologic conditions, lymphocyte subsets, forkhead box P3 (FOXP3)+ Treg cell levels, and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway, we assessed Treg cell induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunologic synapse. RESULTS: Autoimmunity and peripheral lymphoid hyperplasia were found in 43% of 79 patients with PHTS. Immune dysregulation in patients with PHTS included lymphopenia, CD4+ T-cell reduction, and changes in T- and B-cell subsets. Although total CD4+FOXP3+ Treg cell numbers are reduced, frequencies are maintained in the blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PH domain leucine-rich repeat protein phosphatase (PHLPP) downstream of PTEN is highly expressed in normal human Treg cells and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Treg cells in vitro and Treg cell mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunologic synapse. CONCLUSION: Heterozygous loss of function of PTEN in human subjects has a significant effect on T- and B-cell immunity. Assembly of the PTEN-PHLPP phosphatase network allows coordinated phosphatase activities at the site of T-cell receptor activation, which is important for limiting PI3K hyperactivation in Treg cells despite PTEN haploinsufficiency.

One of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry, its exact role during the first steps of HIV-1 infection is not well characterized. Here, we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall, our data suggest that dynamin-2, as a tetramer, might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion.

Most immune cell communication takes place by intercellular transfer of cytokines or the contact-dependent interaction of surface receptors in immunological synapses. In this issue of the European Journal of Immunology, Gardell and Parker (Eur. J. Immunol. 2017, 47, 41-50) point to a new, hybrid mechanism for Th1-cell delivery of help to B cells, based on contact-dependent CD40L transfer. The transfer process and its specificity are both cell contact dependent and antigen dependent. CD40 expression is also required on the B-cell surface to capture the CD40L presented by the Th1 cell. While further studies are needed to confirm the phenomenon in vivo and to test the role of transferred CD40L in other aspects of T-cell help, this study provides an exceptional take-off point and makes excellent use of mouse genetics to work out some possible rules for B cells being able to order help 'to go'.

In this chapter, we present techniques, based on molecular-scale nanofabrication and selective self-assembly, for the presentation of biomolecules of interest (ligands, receptors, etc.) on a surface with precise spatial control and arbitrary geometry at the single-molecule level. Metallic nanodot arrays are created on glass coverslips and are then used as anchors for the immobilization of biological ligands via thiol linking chemistry. The nanodot size is controlled by both lithography and metallization. The reagent concentration in self-assembly can be adjusted to ensure single-molecule occupancy for a given dot size. The surrounding glass is backfilled by a protein-repellent layer to prevent nonspecific adsorption. Moreover, bifunctional surfaces are created, whereby a second ligand is presented on the background, which is frequently a requirement for simulating complex cellular functions involving more than one key ligand. This platform serves as a novel and powerful tool for molecular and cellular biology, e.g., to study the fundamental mechanisms of receptor-mediated signaling.

Supported lipid bilayers (SLB) formed on glass substrates have been a useful tool for study of immune cell signaling since the early 1980s. The mobility of lipid-anchored proteins in the system, first described for antibodies binding to synthetic phospholipid head groups, allows for the measurement of two-dimensional binding reactions and signaling processes in a single imaging plane over time or for fixed samples. The fragility of SLB and the challenges of building and validating individual substrates limit most experimenters to ~10 samples per day, perhaps increasing this few-fold when examining fixed samples. Successful experiments might then require further days to fully analyze. We present methods for automation of many steps in SLB formation, imaging in 96-well glass bottom plates, and analysis that enables >100-fold increase in throughput for fixed samples and wide-field fluorescence. This increased throughput will allow better coverage of relevant parameters and more comprehensive analysis of aspects of the immunological synapse that are well reconstituted by SLB.

Immunological synapses are specialized cell-cell junctions characterized by (1) close apposition of the immune cell membrane with the membrane of another cell driven by adaptive or innate immune recognition, (2) adhesion, (3) stability, and (4) directed secretion. This phenomenon was first recognized in the 1970s and the early 1980s through electron microscopy of ex vivo functioning immune cells. Progressive advances in fluorescence microscopy and molecular immunology in the past 20 years have led to rapid progress on understanding the modes of cell-cell interaction and underlying molecular events. This volume contains a diverse range of protocols that can be applied to the study of the immunological synapses and related immune cell junctions both in vitro and in vivo; and in disease settings in animal models and humans. We have also included chapters on critical molecular tools such as protein expression and mRNA electroporation that underpin or expand imaging approaches, although they are not specific to the study of immune synapses. We hope that these chapters will be of use to people entering the field as well as seasoned practitioners looking to expand their repertoire of methods.

HIV is transmitted most efficiently from cell to cell and productive infection occurs mainly in activated CD4 T cells. It is postulated that HIV exploits immunological synapses formed between CD4 T cells and antigen-presenting cells to facilitate the targeting and infection of activated CD4 T cells. This study sought to evaluate how the presence of the HIV envelope (Env) in the CD4 T cell immunological synapse affects synapse formation and intracellular signaling to impact the downstream T cell activation events. CD4 T cells were applied onto supported lipid bilayers (SLBs) that were reconstituted with HIV Env gp120, anti-TCR antibody OKT3 and ICAM-1 to represent the surface of HIV Env-bearing antigen-presenting cells. The results showed that the HIV Env did not disrupt immunological synapse formation. Instead, the HIV Env accumulated with TCR at the center of the synapse, altered the kinetics of TCR recruitment to the synapse, and affected the synapse morphology over time. The HIV Env also prolonged Lck phosphorylation at the synapse and enhanced TCR-induced CD69 upregulation, IL-2 secretion, and proliferation to promote virus infection. These results suggest that HIV uses the immunological synapse as a conduit not only for selective virus transmission to activated CD4 T cells, but also for boosting the T cell activation state, thereby increasing its likelihood for undergoing productive replication in the targeted CD4 T cells. IMPORTANCE: There are about two million new HIV infections every year. A better understanding on how HIV is transmitted to the susceptible cells is critical to devise effective strategies to prevent HIV infection. Activated CD4 T cells are preferentially infected by HIV, although how this is accomplished is not fully understood. This study examined whether HIV co-opts the normal T-cell activation process through the so-called immunological synapse. We found that the HIV envelope is recruited to the center of the immunological synapse together with the T-cell receptor and enhances the T cell receptor-induced activation of the CD4 T cells. The heightened cellular activation promotes the capacity of the CD4 T cells to support productive HIV replication. This study provides evidence for the exploitation of the normal immunological synapse and T-cell activation process by HIV to boost the activation state of the targeted CD4 T cells and promote infection of these cells.

The immunological synapse formed between a T-cell and an antigen-presenting cell is important for cell-cell communication during T-cell-mediated immune responses. Immunological synapse formation begins with stimulation of the T-cell receptor (TCR). TCR microclusters are assembled and transported to the center of the immunological synapse in an actin polymerization-dependent process. However, the physical link between TCR and actin remains elusive. Here we show that lymphocyte-specific Crk-associated substrate (Cas-L), a member of a force sensing protein family, is required for transport of TCR microclusters and for establishing synapse stability. We found that Cas-L is phosphorylated at TCR microclusters in an actin polymerization-dependent fashion. Furthermore, Cas-L participates in a positive feedback loop leading to amplification of Ca²⁺ signaling, inside-out integrin activation, and actomyosin contraction. We propose a new role for Cas-L in T-cell activation as a mechanical transducer linking TCR microclusters to the underlying actin network and coordinating multiple actin-dependent structures in the immunological synapse. Our studies highlight the importance of mechanotransduction processes in T-cell-mediated immune responses.

Myeloid cells make extensive use of the complement system in the context of recruitment, phagocytosis, and other effector functions. There are several types of complement receptors on myeloid cells, including G protein-coupled receptors for localizing the source of complement activation, and three sets of type I transmembrane proteins that link complement to phagocytosis: complement receptor 1, having an extracellular domain with tandem complement regulatory repeats; complement receptors 3 and 4, which are integrin family receptors comprising heterodimers of type I transmembrane subunits; and VSIG4, a member of the Ig superfamily. This review will focus on the role of the different classes of complement receptors and how their activities are integrated in the setting of immune tolerance and inflammatory responses.

Pancreatic ductal adenocarcinoma (PDA) is marked by an abundant fibroinflammatory microenvironment. Regulatory T (Treg) cell infiltration constitutes a prominent feature of PDA. However, the immunomodulatory function of Treg cells in PDA remains poorly understood. Using orthotopic and autochthonous mouse models of PDA we have found that Treg cell ablation is sufficient to evoke effective anti-tumor immune response in early and advanced pancreatic neoplasia. This response is dependent on IFN-gamma producing cytotoxic CD8 T cells. We show that Treg cells engage in extended interactions with tumor-associated CD11c dendritic cells (DCs) and restrain their immunogenic function by suppressing the expression of costimulatory ligands necessary for CD8 T cell activation. Consequently, tumor-associated CD8 T cells fail to display effector activities when Treg cell ablation is combined with DC depletion. We propose that tumor-infiltrating Treg cells promote immune-tolerance by suppressing DC immunogenicity. Therapeutic manipulation this axis might provide an effective approach for the targeting of PDA