Research ArticleALLERGY

Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite–derived phospholipase

See allHide authors and affiliations

Science Translational Medicine  10 Feb 2016:
Vol. 8, Issue 325, pp. 325ra18
DOI: 10.1126/scitranslmed.aad6833
  • Fig. 1. Circulating CD1a-reactive HDM-responsive T cells produce IFNγ, GM-CSF, and IL-13.

    T cells were isolated by CD3 magnetic cell sorter (MACS) beads from donor peripheral blood mononuclear cells (PBMCs) (R9500) and incubated overnight with CD1a-transfected K562 (CD1a) or untransfected K562 cells pulsed with HDM extract. (A to D) IFNγ (A), GM-CSF (B, left), and IL-13 (B, right) productions were measured by enzyme-linked immunospot (ELISpot) in the absence or presence of anti-CD1a antibody (C) and at different HDM concentrations (D). (E) CD1a-expressing K562 (left), in vitro–derived mDC (middle), or LC-like cells (right) were pulsed with HDM extract overnight and incubated with autologous peripheral blood T cells from donor R2. IFNγ production was measured by ELISpot in the presence or absence of anti-CD1a antibody. Data are representative of at least three donors for each experiment. Bars represent SE. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Student’s t test.

  • Fig. 2. HDM-responsive CD1a-reactive T cells are enriched in blood and skin of AD patients.

    (A and B) T cells derived from the peripheral blood of healthy controls (HC), patients with AD, or from cord blood were incubated with CD1a-transfected K562 or untransfected K562 cells in the presence or absence of HDM extract. IFNγ (A) and IL-13 (B) productions were measured by ELISpot and expressed as percentage of responding T cells (A: n = 30 healthy controls, 24 AD patients, 10 cord blood; B: n = 20 healthy controls, 17 AD patients, 10 cord blood). Autoreactive is the response to CD1a-K562 in the absence of HDM. (C) Skin blister T cells from donor R229 were incubated with CD1a-transfected K562 or untransfected K562 cells in the presence or absence of HDM extract. IFNγ (left), GM-CSF (middle), and IL-13 (right) productions were measured by ELISpot. Data are representative of at least three separate donors for each experiment. (D) Example of skin suction blister raised after 60 min of 200 mmHg negative pressure. (E) Skin blister T cells were isolated from unchallenged skin of healthy controls (n = 5) or patients with AD (n = 4) and incubated with CD1a-transfected or untransfected K562 cells in the presence or absence of HDM extract. IL-13 production was measured by ELISpot and expressed as percentage of CD1a-reactive T cells. Bars represent SE. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Student’s t test.

  • Fig. 3. HDM-responsive CD1a-reactive T cells infiltrate skin after HDM challenge.

    Skin blister T cells were isolated from donor R4 24 hours after HDM skin challenge, expanded, and incubated with CD1a-transfected or untransfected K562 cells in the presence or absence of HDM extract. (A) IFNγ (left), GM-CSF (middle), and IL-13 (right) production by donor R4 cells were measured by ELISpot. Data are representative of at least three separate donors for each experiment. (B) (Left panel) Overall frequencies of HDM-responsive CD1a-reactive IFNγ-producing T cells infiltrating human skin after saline or HDM skin challenge were compared between healthy controls (n = 4) and AD patients (n = 8). Autoreactive refers to responses to unpulsed K562-CD1a cells. (Right panel) Skin blister cells derived from HDM-challenged or unchallenged skin were incubated with live attenuated VZV, and IFNγ production was measured by ELISpot. (C) Concentrations of type 2 cytokines were measured in skin blister fluid by multiplex bead array after saline (nil) or HDM skin challenge in healthy controls (n = 8) or atopic (n = 16) individuals. Bars represent SE. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Student’s t test.

  • Fig. 4. HDM extract contains PLA2 activity in vivo and in vitro.

    (A) T cells were isolated by CD3 MACS beads from AD and healthy donor PBMCs and incubated overnight with CD1a-transfected K562 or untransfected K562 cells pulsed with HDM total extract, either aqueous protein phase or lipid phase. IFNγ production was measured by ELISpot. (B and C) PLA2 activity in saline or HDM-challenged skin blister fluid was detected by measuring free thiol release in the presence of the diheptanoyl thio-PC substrate in the absence (B) or presence (C) of 10 μM of the PLA2 inhibitor manoalide. (D) PLA2 activity in HDM extract in vitro was detected by measuring free thiol release in the presence of the diheptanoyl thio-PC substrate and manoalide and after heat inactivation. Data are representative of at least three separate experiments. Bars represent SE. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Student’s t test.

  • Fig. 5. HDM-derived PLA2 generates neolipid antigens for presentation by CD1a to blood and skin T cells.

    T cells were isolated by CD3 MACS beads from AD and healthy donor PBMCs and incubated overnight with CD1a-transfected K562 or untransfected K562 cells pulsed with HDM total extract. (A) IFNγ production was measured by ELISpot after HDM heat inactivation (hiHDM) (left) or overnight incubation with a dose titration of the PLA2 inhibitor manoalide (right). Skin blister T cells were isolated 24 hours after HDM skin challenge, expanded, and incubated with CD1a-transfected or untransfected K562 cells in the presence or absence of HDM extract that had been heat-inactivated or incubated with manoalide. (B) IFNγ (left) and GM-CSF (right) productions were measured by ELISpot. (C) Skin blister T cells were isolated 24 hours after HDM skin challenge, expanded, and incubated with CD1a-transfected or untransfected K562 cells in the presence or absence of HDM extract or purified bee venom PLA2 (1 μg/ml). IFNγ production was measured by ELISpot. Data are representative of at least three separate experiments. Bars represent SE. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Student’s t test.

  • Fig. 6. Filaggrin inhibits HDM PLA2 activity and inhibits responses of HDM-responsive CD1a-reactive T cells isolated from blood and skin.

    (A) PLA2 activity in HDM extract in vitro was detected by measuring free thiol release in the presence of the diheptanoyl thio-PC substrate, 10 μM PLA2 inhibitor manoalide, and human recombinant filaggrin (1 μg/ml). (B) PLA2 activity in HDM-challenged skin blister fluid was detected by measuring free thiol release diheptanoyl thio-PC substrate in the absence or presence of filaggrin. (C) T cells were isolated by CD3 MACS beads from AD and healthy donor PBMCs and incubated overnight with CD1a-transfected K562 or untransfected K562 cells pulsed with HDM total extract. GM-CSF, IL-13, and IFNγ productions were measured by ELISpot in the presence or absence of filaggrin. (D) Skin blister T cells were isolated 24 hours after HDM skin challenge and incubated with CD1a-transfected or untransfected K562 cells in the presence or absence of HDM extract, filaggrin, or anti-CD1a antibody. Data are representative of at least three separate experiments. Bars represent SE. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001, Student’s t test.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/325/325ra18/DC1

    Fig. S1. mDC and LC-like cell expression of CD1a and langerin.

    Fig. S2. HDM-responsive CD1a-reactive T cells can produce IFNγ and/or IL-13 and are not enriched in patients with psoriasis.

    Fig. S3. HDM-responsive CD1a-reactive T cells associate with FLG mutations.

    Fig. S4. Viral-specific T cell responses in the presence of manoalide and filaggrin.

    Table S1. Source data.

  • Supplementary Material for:

    Filaggrin inhibits generation of CD1a neolipid antigens by house dust mite–derived phospholipase

    Rachael Jarrett, Mariolina Salio, Antonia Lloyd-Lavery, Sumithra Subramaniam, Elvire Bourgeois, Charles Archer, Ka Lun Cheung, Clare Hardman, David Chandler, Maryam Salimi, Danuta Gutowska-Owsiak, Jorge Bernardino de la Serna, Padraic G. Fallon, Helen Jolin, Andrew Mckenzie, Andrzej Dziembowski, Ewa Izabela Podobas, Wojciech Bal, David Johnson, D. Branch Moody, Vincenzo Cerundolo, Graham Ogg*

    *Corresponding author. E-mail: graham.ogg{at}ndm.ox.ac.uk

    Published 10 February 2016, Sci. Transl. Med. 8, 325ra18 (2016)
    DOI: 10.1126/scitranslmed.aad6833

    This PDF file includes:

    • Fig. S1. mDC and LC-like cell expression of CD1a and langerin.
    • Fig. S2. HDM-responsive CD1a-reactive T cells can produce IFNγ and/or IL-13 and are not enriched in patients with psoriasis.
    • Fig. S3. HDM-responsive CD1a-reactive T cells associate with FLG mutations.
    • Fig. S4. Viral-specific T cell responses in the presence of manoalide and filaggrin.
    • Table S1. Source data.

    [Download PDF]

Navigate This Article