Preformed CD40L Is Stored in Th1, Th2, Th17, and T Follicular Helper Cells as Well as CD4+8− Thymocytes and Invariant NKT Cells but Not in Treg Cells

CD40L is essential for the development of adaptive immune responses. It is generally thought that CD40L expression in CD4+ T cells is regulated transcriptionally and made from new mRNA following antigen recognition. However, imaging studies show that the majority of cognate interactions between effector CD4+ T cells and APCs in vivo are too short to allow de novo CD40L synthesis. We previously showed that Th1 effector and memory cells store preformed CD40L (pCD40L) in lysosomal compartments and mobilize it onto the plasma membrane immediately after antigenic stimulation, suggesting that primed CD4+ T cells may use pCD40L to activate APCs during brief encounters. Indeed, our recent study showed that pCD40L is sufficient to mediate selective activation of cognate B cells and trigger DC activation in vitro. In this study, we show that pCD40L is present in Th1 and follicular helper T cells developed during infection with lymphocytic choriomeningitis virus, Th2 cells in the airway of asthmatic mice, and Th17 cells from the CNS of animals with experimental autoimmune encephalitis (EAE). pCD40L is nearly absent in both natural and induced Treg cells, even in the presence of intense inflammation such as occurs in EAE. We also found pCD40L expression in CD4 single positive thymocytes and invariant NKT cells. Together, these results suggest that pCD40L may function in T cell development as well as an unexpectedly broad spectrum of innate and adaptive immune responses, while its expression in Treg cells is repressed to avoid compromising their suppressive activity

Despite disorganized synapse structure, Th2 cells maintain directional delivery of CD40L to antigen-presenting B cells.

Upon recognition of peptide displayed on MHC molecules, Th1 and Th2 cells form distinct immunological synapse structures. Th1 cells have a bull's eye synapse structure with TCR/ MHC-peptide interactions occurring central to a ring of adhesion molecules, while Th2 cells have a multifocal synapse with small clusters of TCR/MHC interactions throughout the area of T cell/antigen-presenting cell interaction. In this study, we investigated whether this structural difference in the immunological synapse affects delivery of T cell help. The immunological synapse is thought to ensure antigen-specific delivery of cytolytic granules and killing of target cells by NK cells and cytolytic T cells. In helper T cells, it has been proposed that the immunological synapse may direct delivery of other effector molecules including cytokines. CD40 ligand (CD40L) is a membrane-bound cytokine essential for antigen-specific T cell help for B cells in the antibody response. We incubated Th1 and Th2 cells overnight with a mixture of antigen-presenting and bystander B cells, and the delivery of CD40L to B cells and subsequent B cell responses were compared. Despite distinct immunological synapse structures, Th1 and Th2 cell do not differ in their ability to deliver CD40L and T cell help in an antigen-specific fashion, or in their susceptibility to inhibition of help by a blocking anti-CD40L antibody

Human macrophages engineered to secrete a bispecific T cell engager support antigen-dependent T cell responses to glioblastoma

Background Targeted and effective treatment options are needed for solid tumors, including glioblastoma (GBM), where survival rates with standard treatments are typically less than 2 years from diagnosis. Solid tumors pose many barriers to immunotherapies, including therapy half-life and persistence, tumor penetrance, and targeting. Therapeutics delivered systemically may not traffic to the tumor site. If cellular therapies or drugs are able to access the tumor site, or can be delivered directly within the tumor, treatments may not persist for the duration necessary to reduce or eliminate tumor burden. An approach that allows durable and titratable local therapeutic protein delivery could improve antitumor efficacy while minimizing toxicities or unwanted on-target, off-tissue effects.Methods In this study, human monocyte-derived macrophages were genetically engineered to secrete a bispecific T cell engager (BiTE) specific to the mutated epidermal growth factor variant III (EGFRvIII) expressed by some GBM tumors. We investigated the ability of lentivirally modified macrophages to secrete a functional BiTE that can bind target tumor antigen and activate T cells. Secreted BiTE protein was assayed in a range of T cell functional assays in vitro and in subcutaneous and intracranial GBM xenograft models. Finally, we tested genetically engineered macrophages (GEMs) secreting BiTE and the proinflammatory cytokine interleukin (IL)-12 to amplify T cell responses in vitro and in vivo.Results Transduced human macrophages secreted a lentivirally encoded functional EGFRvIII-targeted BiTE protein capable of inducing T cell activation, proliferation, degranulation, and killing of antigen-specific tumor cells. Furthermore, BiTE secreting macrophages reduced early tumor burden in both subcutaneous and intracranial mouse models of GBM, a response which was enhanced using macrophages that were dual transduced to secrete both the BiTE protein and single chain IL-12, preventing tumor growth in an aggressive GBM model.Conclusions The ability of macrophages to infiltrate and persist in solid tumor tissue could overcome many of the obstacles associated with systemic delivery of immunotherapies. We have found that human GEMs can locally and constitutively express one or more therapeutic proteins, which may help recruit T cells and transform the immunosuppressive tumor microenvironment to better support antitumor immunity

CD40L transfer by Th2 cells is antigen-specific and largely CD40 dependent.

<p>Antigen-pulsed B cells (Ag+, red) were mixed with unpulsed, bystander B cells (Ag-, blue) and cultured overnight with Th2 cells in the presence of 1 μg/ml fluorescent anti-CD40L. (<b>A</b>) CD40L knockout (CD40LKO) T cells do not transfer CD40L to antigen-pulsed B cells or bystanders. (<b>B</b>) CD40L transfer by Th2 cells to CD40 knock-out antigen-pulsed B cells (CD40KO Ag+) and CD40 sufficient bystander B cells. (<b>C</b>) CD40L transfer by Th2 cells to antigen-pulsed (Ag+) and bystander (Ag-) CD40 sufficient B cells. This experiment is representative of 3 independent experiments.</p

Anti-IL-4 treated Th2 cells retain a pre-formed compartment of CD40L and maintain a multifocal synapse structure.

<p>(<b>A</b>) CD40L mobilization to the cell surface in 5CC7 Th1, 5CC7 Th2, and anti-IL-4 treated 5CC7 Th2 cells following 30 minutes (left histogram) or 2 hours (right histogram) of PMA and ionomycin stimulation as detected by inclusion of PE-labeled anti-CD40L antibody during stimulation. Control, unstimulated Th2 cells stained with PE-labeled anti-CD40L is shown in gray. (<b>B</b>) Three representative 5CC7 Th1, AD10 Th2, and anti-IL-4 treated AD10 Th2 immunological synapse structures are shown. ICAM-Cy5 is shown in red. Unlabeled peptide-bound I-E^k was included in the lipid bilayer. Th2 cells and anti-IL-4-treated Th2 cells were scored for formation of a bull’s eye ring of ICAM-1 in three independent experiments: 82 Th2 cells and 50 anti-IL-4-treated Th2 cells were scored in Experiment 1, 650 Th2 and 278 anti-IL-4-treated Th2 in Experiment 2, and 255 Th2 and 237 anti-IL-4 treated Th2 in Experiment 3.</p

In the absence of anti-CD40L or CsA, Th2 deliver help preferentially to antigen-presenting B cells, and inhibition of Th2 help by anti-CD40L is comparable to that of Th1 cells.

<p>ICAM-1 was measured on antigen-pulsed (Ag+) and bystander (Ag-) B cells mixed and incubated overnight with Th1 (<b>A</b>) or Th2 cells (<b>B</b>). The inhibition of Th2 help by addition of anti-CD40L or isotype IgG control at 10 μg/ml is shown in (<b>B</b>). Results are shown for three independent experiments. (<b>C</b>) Inhibition of help for antigen-presenting B cells by Th1 and Th2 cells is shown for a range of concentrations of anti-CD40L. The graph shows the percentage of maximum ICAM-1 fluorescent intensity in the presence of anti-CD40L blocking antibody. Results of three independent experiments are shown.</p

In vitro-generated Th1 and Th17, but not Th2 or iTreg cells mobilize pCD40L.

<p><i>A</i> and <i>B</i>, Mobilization assay. In vitro-generated Th1, Th2, Th17, and iTreg cells were stimulated with PMA plus ionomycin or left unstimulated in the presence of PE-isotype Ab, PE-anti-CD40L or PE-anti-CTLA-4 at 37°C for 30 minutes. The levels of CD40L (<i>A</i>) and CTLA-4 (<i>B</i>) are shown. <i>C</i> and <i>D</i>, Intracellular staining. Cells were fixed without stimulation, permeabilized, and stained with PE-isotype Ab, PE-anti-CD40L or PE-anti-CTLA-4. The levels of CD40L (<i>C</i>) and CTLA-4 (<i>D</i>) are shown. Data are representative of five independent experiments.</p

Severe inflammation does not compromise the lack of expression of pCD40L by Treg cells.

<p>CNS infiltrating leukocytes and splenocytes were obtained from EAE animals induced as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031296#pone-0031296-g005" target="_blank">Fig. 5<i>A</i></a>. <i>A</i>, Gating strategy for cells from CNS and spleen. <i>B & C</i>, Intracellular CD40L levels for effector CD4⁺ T cells (<i>B</i>) and Treg cells (<i>C</i>). Data are representative of two independent experiments.</p