Heat shock protein 70 binds caspase-activated DNase and enhances its activity in TCR-stimulated T cells

AbstractDNA fragmentation is a hallmark of cells undergoing apoptosis and is mediated mainly by the caspase-activated DNase (CAD or DNA-fragmentation factor 40 [DFF40]), which is activated when released from its inhibitor protein (ICAD or DFF45) upon apoptosis signals. Here we analyzed the effect of heat shock protein 70 (Hsp70) on CAD activity in T-cell receptor (TCR)–induced apoptosis using a T-cell line (TAg-Jurkat). Overexpression of Hsp70 significantly augmented the apoptotic cell death as well as DNA fragmentation in CD3/CD28- or staurosporine-stimulated cells. Following stimulation of cells with CD3/CD28 or staurosporine, Hsp70 was coprecipitated with free CAD, but not with CAD associated with ICAD. Furthermore, the purified Hsp70 dose-dependently augmented DNA-fragmentation activity of caspase-3–activated CAD in a cell-free system. Peptide-binding domain–deleted Hsp70 could neither bind nor augment its activity, while adenosine triphosphate (ATP)–binding domain–deleted Hsp70 or the peptide-binding domain itself bound CAD and augmented its activity. These results indicate that the the binding of Hsp70 to the activated CAD via the peptide-binding domain augments its activity. Although CAD lost its activity in an hour after being released from ICAD in vitro, its activity was retained after an hour of incubation in the presence of Hsp70, suggesting that Hsp70 may be involved in stabilization of CAD activity. Finally, CAD that had been coprecipitated with Hsp70 from the cell lysate of staurosporine-activated 293T cells induced chromatin DNA fragmentation and its activity was not inhibited by ICAD. These results suggest that Hsp70 binds free CAD in TCR-stimulated T cells to stabilize and augment its activity.

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Peripheral T-Cell Reactivity to Heat Shock Protein 70 and Its Cofactor GrpE from Tropheryma whipplei Is Reduced in Patients with Classical Whipple's Disease

Infection and Immunity ◽

10.1128/iai.00363-17 ◽

2017 ◽

Vol 85 (8) ◽

Cited By ~ 3

Author(s):

Lucia Trotta ◽

Kathleen Weigt ◽

Katina Schinnerling ◽

Anika Geelhaar-Karsch ◽

Gerrit Oelkers ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Protein 70 ◽

T Cell Responses ◽

Tropheryma Whipplei ◽

Content Type ◽

Cell Responses ◽

Ifn Γ

ABSTRACT Classical Whipple's disease (CWD) is characterized by the lack of specific Th1 response toward Tropheryma whipplei in genetically predisposed individuals. The cofactor GrpE of heat shock protein 70 (Hsp70) from T. whipplei was previously identified as a B-cell antigen. We tested the capacity of Hsp70 and GrpE to elicit specific proinflammatory T-cell responses. Peripheral mononuclear cells from CWD patients and healthy donors were stimulated with T. whipplei lysate or recombinant GrpE or Hsp70 before levels of CD40L, CD69, perforin, granzyme B, CD107a, and gamma interferon (IFN-γ) were determined in T cells by flow cytometry. Upon stimulation with total bacterial lysate or recombinant GrpE or Hsp70 of T. whipplei, the proportions of activated effector CD4+ T cells, determined as CD40L+ IFN-γ+, were significantly lower in patients with CWD than in healthy controls; CD8+ T cells of untreated CWD patients revealed an enhanced activation toward unspecific stimulation and T. whipplei-specific degranulation, although CD69+ IFN-γ+ CD8+ T cells were reduced upon stimulation with T. whipplei lysate and recombinant T. whipplei-derived proteins. Hsp70 and its cofactor GrpE are immunogenic in healthy individuals, eliciting effective responses against T. whipplei to control bacterial spreading. The lack of specific T-cell responses against these T. whipplei-derived proteins may contribute to the pathogenesis of CWD.

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Inducible Heat Shock Protein 70 Reduces T Cell Responses and Stimulatory Capacity of Monocyte-derived Dendritic Cells

Journal of Biological Chemistry ◽

10.1074/jbc.m111.307579 ◽

2012 ◽

Vol 287 (15) ◽

pp. 12387-12394 ◽

Cited By ~ 40

Author(s):

Pawel Stocki ◽

Xiao N. Wang ◽

Anne M. Dickinson

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Protein 70 ◽

T Cell Responses ◽

Inflammatory Reactions ◽

Cell Responses ◽

Immature Dendritic Cells

Heat shock protein 70 (Hsp70) has gained a lot of attention in the past decade due to its potential immunoregulatory functions. Some of the described proinflammatory functions of Hsp70 became controversial as they were based on recombinant Hsp70 proteins specimens, which were later shown to be endotoxin-contaminated. In this study we used low endotoxin inducible Hsp70 (also known as Hsp72, HSPA1A), and we observed that after a 24-h incubation of monocyte-derived immature dendritic cells (mo-iDCs) with 20 μg/ml of low endotoxin Hsp70, their ability to stimulate allogenic T cells was reduced. Interestingly, low endotoxin Hsp70 also significantly reduced T cell responses when they were simulated with either IL-2 or phytohemagglutinin, therefore showing that Hsp70 could alter T cell responses independently from its effect on mo-iDCs. We also reported a greater response of Hsp70 treatment when activated versus nonactivated T cells were used. This effect of Hsp70 was similar for all tested populations of T cells that included CD3+, CD4+, or CD8+. Taken together, our observations strongly suggest that Hsp70 might dampen, rather than provoke, T cell-mediated inflammatory reactions in many clinical conditions where up-regulation of Hsp70 is observed.

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The Immunosuppressive Activity of Heat Shock Protein 70

Autoimmune Diseases ◽

10.1155/2012/617213 ◽

2012 ◽

Vol 2012 ◽

pp. 1-6 ◽

Cited By ~ 12

Author(s):

Pawel Stocki ◽

Anne M. Dickinson

Keyword(s):

T Cells ◽

T Cell ◽

Heat Shock ◽

Heat Shock Protein ◽

Heat Shock Protein 70 ◽

Immune Reactivity ◽

Antigenic Peptides ◽

Endotoxin Contamination ◽

Stimulatory Capacity ◽

Clinical Conditions

Heat shock protein 70 (HSP70) has previously been described as a potent antitumour vaccine. The mechanism relied on the ability of tumour derived HSP70 to associate with antigenic peptides, which, when cross presented, elicited a T cell mediated antitumour response. Subsequently, HSP70 was incorrectly described as a potent adjuvant of innate immunity, and although mistakes in the experimental approaches were exposed and associated with endotoxin contamination in the recombinant HSP70 specimen, questions still remain regarding this matter. Here we review only publications that have cautiously addressed the endotoxin contamination problem in HSP70 in order to reveal the real immunological function of the protein. Accordingly, “endotoxin free” HSP70 stimulates macrophages and delivers antigenic peptides to APCs, which effectively prime T cells mediating an antitumour reaction. Conversely, HSP70 has potent anti-inflammatory functions as follows: regulating T cell responses, reducing stimulatory capacity of DCs, and inducing development of immunosuppressive regulatory T cells. These activities were further associated with the immune evasive mechanism of tumours and implicated in the modulation of immune reactivity in autoimmune diseases and transplant-related clinical conditions. Consequently, the role of HSP70 in immune regulation is newly emerging and contrary to what was previously anticipated.

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T-Cell Receptor Variable γ Chain Gene Expression in the Interaction between Rat γδ-Type T Cells and Heat-Shock Protein 70-Like Molecule

Microbiology and Immunology ◽

10.1111/j.1348-0421.2003.tb03406.x ◽

2003 ◽

Vol 47 (5) ◽

pp. 351-357 ◽

Cited By ~ 5

Author(s):

Takashi Ichinohe ◽

Shingo Ichimiya ◽

Akihiko Kishi ◽

Yasuaki Tamura ◽

Nobuhiko Kondo ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

T Cell ◽

Heat Shock ◽

Heat Shock Protein ◽

T Cell Receptor ◽

Heat Shock Protein 70 ◽

Cell Receptor ◽

Chain Gene

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Efficient Activation of Human T Cells of Both CD4 and CD8 Subsets by Urease-Deficient Recombinant Mycobacterium bovis BCG That Produced a Heat Shock Protein 70-M. tuberculosis-Derived Major Membrane Protein II Fusion Protein

Clinical and Vaccine Immunology ◽

10.1128/cvi.00564-13 ◽

2013 ◽

Vol 21 (1) ◽

pp. 1-11 ◽

Cited By ~ 4

Author(s):

Tetsu Mukai ◽

Yumiko Tsukamoto ◽

Yumi Maeda ◽

Toshiki Tamura ◽

Masahiko Makino

Keyword(s):

T Cells ◽

T Cell ◽

Heat Shock ◽

Membrane Protein ◽

Heat Shock Protein ◽

Fusion Protein ◽

T Cell Activation ◽

Heat Shock Protein 70 ◽

Cell Activation ◽

Content Type

ABSTRACTFor the purpose of obtainingMycobacterium bovisbacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed ofMycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8+T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8+T cells and perforin-producing effector CD8+T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive toin vitrosecondary stimulation with HSP70, MMP-II, andM. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challengedM. tuberculosismore efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.

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Mutations in the substrate binding site of human heat-shock protein 70 indicate specific interaction with HLA-DR outside the peptide binding groove

Immunology ◽

10.1111/imm.12249 ◽

2014 ◽

Vol 142 (2) ◽

pp. 237-247 ◽

Cited By ~ 6

Author(s):

Karin M. Rohrer ◽

Markus Haug ◽

Daniela Schwörer ◽

Hubert Kalbacher ◽

Ursula Holzer

Keyword(s):

Heat Shock ◽

Heat Shock Protein ◽

Binding Site ◽

Heat Shock Protein 70 ◽

Specific Interaction ◽

Peptide Binding ◽

Hla Dr ◽

Peptide Binding Groove ◽

Binding Groove ◽

Human Heat Shock Protein

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Heat shock protein 70 (Hsp70) interacts with the Notch1 intracellular domain and contributes to the activity of Notch signaling in myelin-reactive CD4 T cells

Journal of Neuroimmunology ◽

10.1016/j.jneuroim.2015.08.007 ◽

2015 ◽

Vol 287 ◽

pp. 19-26 ◽

Cited By ~ 4

Author(s):

Maciej Juryńczyk ◽

Przemysław Lewkowicz ◽

Małgorzata Domowicz ◽

Marcin P. Mycko ◽

Krzysztof W. Selmaj

Keyword(s):

T Cells ◽

Heat Shock ◽

Heat Shock Protein ◽

Notch Signaling ◽

Cd4 T Cells ◽

Heat Shock Protein 70 ◽

Intracellular Domain

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Interaction of human heat shock protein 70 with tumor-associated peptides

Biological Chemistry ◽

10.1515/bc.2009.038 ◽

2009 ◽

Vol 390 (4) ◽

Cited By ~ 8

Author(s):

Maya J. Pandya ◽

Henriette Bendz ◽

Florian Manzenrieder ◽

Elfriede Noessner ◽

Horst Kessler ◽

...

Keyword(s):

T Cell ◽

Heat Shock ◽

Heat Shock Protein ◽

T Cell Activation ◽

Heat Shock Protein 70 ◽

Cell Activation ◽

Peptide Sequence ◽

Antigenic Peptides ◽

Human Hsp70

Abstract Molecular chaperones of the heat shock protein 70 (Hsp70) family play a crucial role in the presentation of exogenous antigenic peptides by antigen-presenting cells (APCs). In a combined biochemical and immunological approach, we characterize the biochemical interaction of tumor-associated peptides with human Hsp70 and show that the strength of this interaction determines the efficacy of immunological cross-presentation of the antigenic sequences by APCs. A fluorescein-labeled cytosolic mammalian Hsc70 binding peptide is shown to interact with human Hsp70 molecules with high affinity (Kd=0.58 μm at 25°C). Competition experiments demonstrate weaker binding by Hsp70 of antigenic peptides derived from the tumor-associated proteins tyrosinase (Kd=32 μm) and melanoma antigen recognized by T cells (MART-1) (Kd=2.4 μm). Adding a peptide sequence (pep70) with high Hsp70 binding affinity (Kd=0.04 μm) to the tumor-associated peptides enables them to strongly interact with Hsp70. Presentation of tumor-associated peptides by B cells resulting in T cell activation in vitro is enhanced by Hsp70 when the tumor-associated peptides contain the Hsp70 binding sequence. This observation has relevance for vaccine design, as augmented transfer of tumor-associated antigens to APCs is closely linked to the vaccine's efficacy of T cell stimulation.

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