Chimeric antigen receptor (CAR)-modified natural killer cell-based immunotherapy and immunological synapse formation in cancer and HIV

ABSTRACT Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells contribute to the body’s immune defenses. Current chimeric antigen receptor (CAR)-modified T cell immunotherapy shows strong promise for treating various cancers and infectious diseases. Although CAR-modified NK cell immunotherapy is rapidly gaining attention, its clinical applications are mainly focused on preclinical investigations using the NK92 cell line. Despite recent advances in CAR-modified T cell immunotherapy, cost and severe toxicity have hindered its widespread use. To alleviate these disadvantages of CAR-modified T cell immunotherapy, additional cytotoxic cell-mediated immunotherapies are urgently needed. The unique biology of NK cells allows them to serve as a safe, effective, alternative immunotherapeutic strategy to CAR-modified T cells in the clinic. While the fundamental mechanisms underlying the cytotoxicity and side effects of CAR-modified T and NK cell immunotherapies remain poorly understood, the formation of the immunological synapse (IS) between CAR-modified T or NK cells and their susceptible target cells is known to be essential. The role of the IS in CAR T and NK cell immunotherapies will allow scientists to harness the power of CAR-modified T and NK cells to treat cancer and infectious diseases. In this review, we highlight the potential applications of CAR-modified NK cells to treat cancer and human immunodeficiency virus (HIV), and discuss the challenges and possible future directions of CAR-modified NK cell immunotherapy, as well as the importance of understanding the molecular mechanisms of CAR-modified T cell- or NK cell-mediated cytotoxicity and side effects, with a focus on the CAR-modified NK cell IS

The Ebola Interferon Inhibiting Domains Attenuate and Dysregulate Cell-Mediated Immune Responses

<div><p>Ebola virus (EBOV) infections are characterized by deficient T-lymphocyte responses, T-lymphocyte apoptosis and lymphopenia. We previously showed that disabling of interferon-inhibiting domains (IIDs) in the VP24 and VP35 proteins effectively unblocks maturation of dendritic cells (DCs) and increases the secretion of cytokines and chemokines. Here, we investigated the role of IIDs in adaptive and innate cell-mediated responses using recombinant viruses carrying point mutations, which disabled IIDs in VP24 (EBOV/VP24m), VP35 (EBOV/VP35m) or both (EBOV/VP35m/VP24m). Peripheral blood mononuclear cells (PBMCs) from cytomegalovirus (CMV)-seropositive donors were inoculated with the panel of viruses and stimulated with CMV pp65 peptides. Disabling of the VP35 IID resulted in increased proliferation and higher percentages of CD4⁺ T cells secreting IFNγ and/or TNFα. To address the role of aberrant DC maturation in the IID-mediated suppression of T cell responses, CMV-stimulated DCs were infected with the panel of viruses and co-cultured with autologous T-lymphocytes. Infection with EBOV/VP35m infection resulted in a significant increase, as compared to wt EBOV, in proliferating CD4⁺ cells secreting IFNγ, TNFα and IL-2. Experiments with expanded CMV-specific T cells demonstrated their increased activation following co-cultivation with CMV-pulsed DCs pre-infected with EBOV/VP24m, EBOV/VP35m and EBOV/VP35m/VP24m, as compared to wt EBOV. Both IIDs were found to block phosphorylation of TCR complex-associated adaptors and downstream signaling molecules. Next, we examined the effects of IIDs on the function of B cells in infected PBMC. Infection with EBOV/VP35m and EBOV/VP35m/VP24m resulted in significant increases in the percentages of phenotypically distinct B-cell subsets and plasma cells, as compared to wt EBOV, suggesting inhibition of B cell function and differentiation by VP35 IID. Finally, infection with EBOV/VP35m increased activation of NK cells, as compared to wt EBOV. These results demonstrate a global suppression of cell-mediated responses by EBOV IIDs and identify the role of DCs in suppression of T-cell responses.</p></div

Disabling of IIDs changes cytokine and chemokine expression in co-cultures of CD4⁺ T cells with infected DCs.

<p><b>A, B.</b> CMV-pulsed, EBOV-infected DCs were co-cultured with expanded CMV-specific responder T-lymphocytes for 24 hours, and concentrations of cytokines (<b>A</b>) and chemokines (<b>B</b>) in supernatants were determined using a bead-based multiplex analysis. Heatmap showing the relative fold change following normalization to mock-infected samples. Results from 3 individual donors are shown. <b>C</b>, <b>D.</b> Flow cytometry analysis of IFNγ⁺ CD4⁺ T cells in DC-T cell co-cultures after transfer of conditioned media from DCs infected with the indicated viruses: representative primary data (<b>C</b>) and mean values with SE based on triplicate samples (<b>D</b>). Black asterisks indicate significant differences to mock, and red asterisks indicate significant differences to wt EBOV infection (p<0.05). The experiment was performed two times with different donors, with essentially same results.</p

Suppression of IFN-I signaling and the prevention of IFN-I release by EBOV play only a limited role in inhibition of DC maturation.

<p><b>A.</b> Effect of IFNAR2 blockade on expression of CD86. <b>B.</b> Effect of exogenously added IFNα and IFNβ on expression of CD86. Mean fluorescent intensity (MFI) for CD86⁺ mock-treated DC (black), or DC treated with IFNAR2 antibodies, IFNα, or IFNβ (colors) are indicated in the top left corners. The experiment was performed two times with different donors, with essentially similar results.</p

The VP35 IID reduces Th1 responses.

<p><b>A.</b> Schematic representation of the genomes of wt EBOV and mutant EBOVs used to infect cells; mutations R312A in VP35 and K142A in VP24 are indicated by arrows, and the inserted GFP gene is indicated by green rectangles. <b>B.</b> Schematic representation of the cultivation experiment: PBMCs from CMV-positive donors were labeled with CFSE, inoculated with the indicated viruses at MOI of 2 PFU/cell or SEB, and stimulated with CMV pp65 peptides for 4 hours. PBMCs were washed and cultured for 7 days. Thereafter, cells were re-stimulated for 6 hours in culture medium containing CMV pp65 peptides in presence of brefeldin A, monensin, anti-CD28 mab, anti-CD49d mab and DNase. As a positive control, cells were stimulated with SEB and treated with phorbol-12-myristate-13 acetate (PMA) and ionomycin. Cells were stained intracellularly for the indicated cytokines followed by multicolor flow cytometry. <b>C.</b> Percentages of total and proliferating (CFSE^-) CD4⁺ T cells secreting total IFNγ, IL-2 or TNFα normalized to wt EBOV (100%, indicated by the red horizontal lines). Percentages of proliferating and total CD4⁺ T cells were individually normalized to wt EBOV values in the same donor (100%, indicated by the red horizontal lines). Cells from each individual donor are represented by the same symbol across the panels; mean values are indicated by horizontal bars. Red asterisks indicate significant differences to wt EBOV (p<0.05). <b>D.</b> Analysis of total and proliferating (CFSE-) CD4⁺ T cells secreting combinations of multiple cytokines using Boolean gating: percentages of CD4⁺ T cells positive for the indicated cytokines and negative for the other cytokines included in the analysis; otherwise the designations are same as in Panel C. <b>E.</b> Concentrations of IL-4, IL-5 and IL-13 in supernatants of infected DCs co-cultured with expanded CMV-specific responder T-lymphocytes for 24 hours. Mean values with SE based on three donors. Black asterisks indicate significant differences to mock, and red asterisks indicate significant differences to wt EBOV infection (p<0.05). The experiment was performed 3 times with different donors, with essentially same results.</p

Ebola Virus Binding to Tim-1 on T Lymphocytes Induces a Cytokine Storm

Ebola virus (EBOV) disease (EVD) results from an exacerbated immunological response that is highlighted by a burst in the production of inflammatory mediators known as a “cytokine storm.” Previous reports have suggested that nonspecific activation of T lymphocytes may play a central role in this phenomenon. T-cell immunoglobulin and mucin domain-containing protein 1 (Tim-1) has recently been shown to interact with virion-associated phosphatidylserine to promote infection. Here, we demonstrate the central role of Tim-1 in EBOV pathogenesis, as Tim-1−/− mice exhibited increased survival rates and reduced disease severity; surprisingly, only a limited decrease in viremia was detected. Tim-1−/− mice exhibited a modified inflammatory response as evidenced by changes in serum cytokines and activation of T helper subsets. A series of in vitro assays based on the Tim-1 expression profile on T cells demonstrated that despite the apparent absence of detectable viral replication in T lymphocytes, EBOV directly binds to isolated T lymphocytes in a phosphatidylserine–Tim-1-dependent manner. Exposure to EBOV resulted in the rapid development of a CD4Hi CD3Low population, non-antigen-specific activation, and cytokine production. Transcriptome and Western blot analysis of EBOV-stimulated CD4+ T cells confirmed the induction of the Tim-1 signaling pathway. Furthermore, comparative analysis of transcriptome data and cytokine/chemokine analysis of supernatants highlight the similarities associated with EBOV-stimulated T cells and the onset of a cytokine storm. Flow cytometry revealed virtually exclusive binding and activation of central memory CD4+ T cells. These findings provide evidence for the role of Tim-1 in the induction of a cytokine storm phenomenon and the pathogenesis of EVD

VP35 and VP24 IID block phosphorylation of TCR complex-associated adapters and downstream signal molecules.

<p>CD4⁺ T cells were co-cultured with DCs infected with wt EBOV or the mutated viruses for 4 days, and TCR signal transduction was analyzed by Western blotting of phosphorylated adapter molecules and downstream signaling molecules (<b>A</b>) and anti-apoptotic Bcl-2 family members and the SRC molecule (<b>B</b>). <b>C</b>. Map of signaling cascades associated with MHC-TCR engagement that promotes transduction of survival signals in the absence of antigen-dependent proliferation according reference [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006031#ppat.1006031.ref095" target="_blank">95</a>]; the map itself is adapted from Cell Signaling Technology web site (<a href="http://www.cellsignal.com" target="_blank">http://www.cellsignal.com</a>). As shown in panel A, basal phosphorylation of adapter molecules was observed following culture of autologous CD4⁺ T-cells with mock-DCs. The identified VP24 and VP35 IID-mediated blocks and stimulations of signal transduction are shown; note that block of signal transduction is associated with altered DCs function.</p

The VP35-mediated reduction of Th1 response results from the deficient maturation of DC.

<p><b>A.</b> Schematic representation of the cultivation experiment: immature DCs from CMV-positive donors were inoculated with the indicated viruses and stimulated with CMV pp65 peptides for 4 hours, washed and co-cultured with CFSE-labeled autologous purified CD4⁺ T cells for 7 days. Thereafter, cells were processed and analyzed as described in the legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006031#ppat.1006031.g002" target="_blank">Fig 2B</a>. <b>B.</b> Percentages of total and proliferating (CFSE^-) CD4⁺ T cells secreting total IFNγ, IL-2 or TNFα individually normalized to wt EBOV in the same donor; see legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006031#ppat.1006031.g002" target="_blank">Fig 2C</a> for details. <b>C</b>. Analysis of total and proliferating (CFSE-) CD4⁺ T cells secreting combinations of multiple cytokines using Boolean gating; see legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006031#ppat.1006031.g002" target="_blank">Fig 2D</a> for details. <b>D.</b> Schematic of T-lymphocyte responders assay. PBMCs were stimulated with CMV peptides for 48-hours and used for positive selection of CD137⁺ T-lymphocyte. Isolated CMV responders were expanded using dynabeads human T-cell activator beads for 8 days. In parallel, positively selected CD14⁺ monocyte were differentiated into DC for 7 days, stimulated with CMV peptides and infected with the panel of viruses. After 24 hours, expanded CMV-responders were added to stimulated/infected DCs. Following a 24 hour co-culture, supernatants were collected and cells were analyzed by flow cytometry. <b>E.</b> Percentages of CD4⁺ T-cells staining positive for intracellular IFNγ, IL-2 and TNFα determined by flow cytometry following a 24 hour co-culture of expanded CMV-specific T-lymphocyte responders with CMV-stimulated DCs infected with the indicated viruses. Black asterisks indicate significant differences to mock, and red asterisks indicate significant differences to wt EBOV infection (p<0.05). The experiment was performed 3 times with different donors, with essentially same results.</p

VP35 and VP24 IID suppress activation of NK cells.

<p>PBMCs were infected with wt EBOV or the mutated viruses, and gated CD56⁺CD3^- NK cells were analyzed for the indicated markers of activation, inhibition or dead cell markers: <b>A</b>, CD38; <b>B</b>, NKp46; <b>C</b>, CD27; <b>D</b>, CD158b; <b>E</b> KLRG1; <b>F</b>, Live/Dead. Mean values based on 3 donors with SE are shown. Statistically significant differences (P<0.05) for wt EBOV as compared to mock are indicated with black asterisks, and for the mutated viruses as compared to wt EBOV are indicated with red asterisks. Percentages of cell populations are indicated next to each histogram.</p

VP35 IID impairs the formation of immunological synapses.

<p>CMV-pulsed DCs infected with wt EBOV or EBOV/VP35m incubated with autologous CD4⁺ T cells. <b>A</b>. Confocal microscopy data showing co-localization of DC-expressed HLA-DR (green) and CD4⁺ T cell-expressed CD3 (red). Scale bar = 1 μm. Immunological synapses are shown by white arrows. Representative images from one of three donors analyzed. Note, a representative image of CD4⁺ T-cells co-cultured with wt EBOV infected DCs demonstrates one of a very limited number of dimly fluorescent HLA-DR⁺ cells co-localized with CD3⁺ T cells. Since HLA-DR staining of DCs infected with wt EBOV is dim, an inlet with increased brightness to visualize HLA-DR is shown. <b>B</b>. Numbers of CD4⁺ T cell—DC immunological synapses counted in 13 mm diameter cover slips. Results are expressed as numbers of immunological synapses per 100 cells. <b>C</b>. Percentages of HLA-DR⁺ DCs in infected or mock-infected DCs determined by flow cytometry. Mean values with SE based on triplicate samples from one of two independent experiments performed with different donors, which resulted in essentially same results. B, C, black asterisks indicate significant differences to mock, and red asterisks indicate significant differences to wt EBOV infection (p<0.05).</p