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Ca2+ release from the endoplasmic reticulum of NY-ESO-1-specific T cells is modulated by the affinity of TCR and by the use of the CD8 coreceptor.

ABSTRACT: Although several cancer immunotherapy strategies are based on the use of analog peptides and on the modulation of the TCR affinity of adoptively transferred T cells, it remains unclear whether tumor-specific T cell activation by strong and weak TCR stimuli evoke different Ca(2+) signatures from the Ca(2+) intracellular stores and whether the amplitude of Ca(2+) release from the endoplasmic reticulum (ER) can be further modulated by coreceptor binding to peptide/MHC. In this study, we combined functional, structural, and kinetic measurements to correlate the intensity of Ca(2+) signals triggered by the stimulation of the 1G4 T cell clone specific to the tumor epitope NY-ESO-1(157-165). Two analogs of the NY-ESO-1(157-165) peptide, having similar affinity to HLA-A2 molecules, but a 6-fold difference in binding affinity for the 1G4 TCR, resulted in different Ca(2+) signals and T cell activation. 1G4 stimulation by the stronger stimulus emptied the ER of stored Ca(2+), even in the absence of CD8 binding, resulting in sustained Ca(2+) influx. In contrast, the weaker stimulus induced only partial emptying of stored Ca(2+), resulting in significantly diminished and oscillatory Ca(2+) signals, which were enhanced by CD8 binding. Our data define the range of TCR/peptide MHC affinities required to induce depletion of Ca(2+) from intracellular stores and provide insights into the ability of T cells to tailor the use of the CD8 coreceptor to enhance Ca(2+) release from the ER. This, in turn, modulates Ca(2+) influx from the extracellular environment, ultimately controlling T cell activation.

SUBMITTER: Chen JL

PROVIDER: S-EPMC4222200 | biostudies-literature | 2010 Feb

REPOSITORIES: biostudies-literature

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Ca2+ release from the endoplasmic reticulum of NY-ESO-1-specific T cells is modulated by the affinity of TCR and by the use of the CD8 coreceptor.

Chen Ji-Li JL Morgan Anthony J AJ Stewart-Jones Guillaume G Shepherd Dawn D Bossi Giovanna G Wooldridge Linda L Hutchinson Sarah L SL Sewell Andrew K AK Griffiths Gillian M GM van der Merwe P Anton PA Jones E Yvonne EY Galione Antony A Cerundolo Vincenzo V

Journal of immunology (Baltimore, Md. : 1950) 20100106 4

Although several cancer immunotherapy strategies are based on the use of analog peptides and on the modulation of the TCR affinity of adoptively transferred T cells, it remains unclear whether tumor-specific T cell activation by strong and weak TCR stimuli evoke different Ca(2+) signatures from the Ca(2+) intracellular stores and whether the amplitude of Ca(2+) release from the endoplasmic reticulum (ER) can be further modulated by coreceptor binding to peptide/MHC. In this study, we combined fu ...[more]

PMID: 20053942

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Project description:Dendritic cells (DCs) are crucial for the efficacy of cancer vaccines, but current vaccines do not harness the key cDC1 subtype required for effective CD8+ T-cell-mediated tumor immune responses. Vaccine immunogenicity could be enhanced by specific delivery of immunogenic tumor antigens to CD141+ DCs, the human cDC1 equivalent. CD141+ DCs exclusively express the C-type-lectin-like receptor CLEC9A, which is important for the regulation of CD8+ T cell responses. This study developed a new vaccine that harnesses a human anti-CLEC9A antibody to specifically deliver the immunogenic tumor antigen, NY-ESO-1 (New York esophageal squamous cell carcinoma 1), to human CD141+ DCs. The ability of the CLEC9A-NY-ESO-1 antibody to activate NY-ESO-1-specific naïve and memory CD8+ T cells was examined and compared with a vaccine comprised of a human DEC-205-NY-ESO-1 antibody that targets all human DCs. Human anti-CLEC9A, anti-DEC-205 and isotype control IgG4 antibodies were genetically fused to NY-ESO-1 polypeptide. Cross-presentation to NY-ESO-1-epitope-specific CD8+ T cells and reactivity of T cell responses in patients with melanoma were assessed by interferon γ (IFNγ) production following incubation of CD141+ DCs and patient peripheral blood mononuclear cells with targeting antibodies. Humanized mice containing human DC subsets and a repertoire of naïve NY-ESO-1-specific CD8+ T cells were used to investigate naïve T cell priming. T cell effector function was measured by expression of IFNγ, MIP-1β, tumor necrosis factor and CD107a and by lysis of target tumor cells. CLEC9A-NY-ESO-1 antibodies (Abs) were effective at mediating delivery and cross-presentation of multiple NY-ESO-1 epitopes by CD141+ DCs for activation of NY-ESO-1-specific CD8+ T cells. When benchmarked to NY-ESO-1 conjugated to an untargeted control antibody or to anti-human DEC-205, CLEC9A-NY-ESO-1 was superior at ex vivo reactivation of NY-ESO-1-specific T cell responses in patients with melanoma. Moreover, CLEC9A-NY-ESO-1 induced priming of naïve NY-ESO-1-specific CD8+ T cells with polyclonal effector function and potent tumor killing capacity in vitro. These data advocate human CLEC9A-NY-ESO-1 Ab as an attractive strategy for specific targeting of CD141+ DCs to enhance tumor immunogenicity in NY-ESO-1-expressing malignancies.

Project description:BackgroundBecause of the shortage of ideal cell surface antigens, the development of T-cell receptor (TCR)-engineered T cells (TCR-T) that target intracellular antigens such as NY-ESO-1 is a promising approach for treating patients with solid tumors. However, endogenous TCRs in vector-transduced T cells have been suggested to impair cell-surface expression of transduced TCR while generating mispaired TCRs that can become self-reactive.MethodsWe conducted a first-in-human phase I clinical trial with the TCR-transduced T-cell product (TBI-1301) in patients with NY-ESO-1-expressing solid tumors. In manufacturing TCR-T cells, we used a novel affinity-enhanced NY-ESO-1-specific TCR that was transduced by a retroviral vector that enables siRNA (small interfering RNA)-mediated silencing of endogenous TCR. The patients were divided into two cohorts. Cohort 1 was given a dose of 5×108 cells (whole cells including TCR-T cells) preconditioned with 1500 mg/m2 cyclophosphamide. Cohort 2 was given 5× 109 cells preconditioned with 1500 mg/m2 cyclophosphamide.ResultsIn vitro study showed that both the CD8+ and CD4+ T fractions of TCR-T cells exhibited cytotoxic effects against NY-ESO-1-expressing tumor cells. Three patients and six patients were allocated to cohort 1 and cohort 2, respectively. Three of the six patients who received 5×109 cells showed tumor response, while three patients developed early-onset cytokine release syndrome (CRS). One of the patients developed a grade 3 lung injury associated with the infiltration of the TCR-T cells. No siRNA-related adverse events other than CRS were observed. Cytokines including interleukin 6 I and monocyte chemotactic protein-1/chemokine (C-C motif) ligand (CCL2)increased in the sera of patients with CRS. In vitro analysis showed these cytokines were not secreted from the T cells infused. A significant fraction of the manufactured T cells in patients with CRS was found to express either CD244, CD39, or both at high levels.ConclusionsThe trial showed that endogenous TCR-silenced and affinity-enhanced NY-ESO-1 TCR-T cells were safely administered except for grade 3 lung injury. The TCR-T cell infusion exhibited significant tumor response and early-onset CRS in patients with tumors that express NY-ESO-1 at high levels. The differentiation properties of the manufactured T cells may be prognostic for TCR-T-related CRS.Trial registration numberNCT02366546.

Project description:Phototransduction in Drosophila is mediated by phospholipase C (PLC) and Ca2+-permeable TRP channels, but the function of endoplasmic reticulum (ER) Ca2+ stores in this important model for Ca2+ signaling remains obscure. We therefore expressed a low affinity Ca2+ indicator (ER-GCaMP6-150) in the ER, and measured its fluorescence both in dissociated ommatidia and in vivo from intact flies of both sexes. Blue excitation light induced a rapid (tau ∼0.8 s), PLC-dependent decrease in fluorescence, representing depletion of ER Ca2+ stores, followed by a slower decay, typically reaching ∼50% of initial dark-adapted levels, with significant depletion occurring under natural levels of illumination. The ER stores refilled in the dark within 100-200 s. Both rapid and slow store depletion were largely unaffected in InsP3 receptor mutants, but were much reduced in trp mutants. Strikingly, rapid (but not slow) depletion of ER stores was blocked by removing external Na+ and in mutants of the Na+/Ca2+ exchanger, CalX, which we immuno-localized to ER membranes in addition to its established localization in the plasma membrane. Conversely, overexpression of calx greatly enhanced rapid depletion. These results indicate that rapid store depletion is mediated by Na+/Ca2+ exchange across the ER membrane induced by Na+ influx via the light-sensitive channels. Although too slow to be involved in channel activation, this Na+/Ca2+ exchange-dependent release explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors exposed to Ca2+-free solutions.SIGNIFICANCE STATEMENT Phototransduction in Drosophila is mediated by phospholipase C, which activates TRP cation channels by an unknown mechanism. Despite much speculation, it is unknown whether endoplasmic reticulum (ER) Ca2+ stores play any role. We therefore engineered flies expressing a genetically encoded Ca2+ indicator in the photoreceptor ER. Although NCX Na+/Ca2+ exchangers are classically believed to operate only at the plasma membrane, we demonstrate a rapid light-induced depletion of ER Ca2+ stores mediated by Na+/Ca2+ exchange across the ER membrane. This NCX-dependent release was too slow to be involved in channel activation, but explains the decades-old observation of a light-induced rise in cytosolic Ca2+ in photoreceptors bathed in Ca2+-free solutions.

Dataset Information

Ca2+ release from the endoplasmic reticulum of NY-ESO-1-specific T cells is modulated by the affinity of TCR and by the use of the CD8 coreceptor.

Publications

Ca2+ release from the endoplasmic reticulum of NY-ESO-1-specific T cells is modulated by the affinity of TCR and by the use of the CD8 coreceptor.

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