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. 2011 Sep 1;187(5):2193-201.
doi: 10.4049/jimmunol.1100152. Epub 2011 Jul 25.

Presentation of Type B peptide-MHC Complexes From Hen Egg White Lysozyme by TLR Ligands and Type I IFNs Independent of H2-DM Regulation

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Free PMC article

Presentation of Type B peptide-MHC Complexes From Hen Egg White Lysozyme by TLR Ligands and Type I IFNs Independent of H2-DM Regulation

Beverly S I Strong et al. J Immunol. .
Free PMC article

Abstract

In APCs, presentation by MHC II molecules of the chemically dominant peptide from the protein hen egg white lysozyme (HEL) generates different conformational isomers of the peptide-MHC II complexes (pMHC). Type B pMHCs are formed in early endosomes from exogenous peptides in the absence of H2-DM, whereas in contrast, type A pMHC complexes are formed from HEL protein in late vesicles after editing by H2-DM. Thus, H2-DM edits off the more unstable pMHC complexes, which are not presented from HEL. In this study, we show that type B pMHC complexes were presented from HEL protein only after stimulation of dendritic cells (DC) with TLR ligands or type I IFN. Type I IFN contributed to most TLR ligand-induced type B pMHC generation, as presentation decreased in DC lacking the receptor for type I IFNs (IFNAR1(-/-)). In contrast, presentation of type A pMHC from HEL and from peptide was minimally affected by TLR ligands. The relative effectiveness of CD8α(+) DC or CD8α(-) DC in presenting type B pMHC complexes varied depending on the TLR ligand used. The mechanisms of generation of type B pMHC from HEL protein with TLR stimulation did not involve H2-DM or release of peptides. DC from H2-DM-deficient mice in the presence of TLR ligands presented type B pMHC. Such DC showed a slight enhancement of HEL catabolism, but peptide release was not evident. Thus, TLR ligands and type I IFN alter the pathways of presentation by MHC II molecules of DC such that type B pMHCs are generated from protein Ag.

Conflict of interest statement

The authors have no financial conflicts of interest.

Figures

Figure 1
Figure 1. TLR Ligand-Induced Presentation of Type B pMHC from HEL
Presentation by DC incubated with HEL (A, B) or HEL:48-61 peptide (C, D) to type B 11A10 (A, C) or type A 3A9 (B, D) T cell hybridomas, in the presence or absence of TLR-ligands. Representative of at least four independent experiments. Error bars represent SD.
Figure 2
Figure 2. Dependence on MyD88-/- DC for TLR Ligand-Induced Presentation of Type B pMHC from HEL
Presentation to type B 11A10 (A, C) or type A 3A9 (B, D) T cell hybridomas by DC from wild-type (A, B) or MyD88-/- mice (C, D) incubated with HEL, with or without TLR ligands. Error bars represent SD. Representative of three independent experiments.
Figure 3
Figure 3. Presentation by Sorted CD8α+ and CD8α- DC
(A) Representative flow cytometry assay on sorted cells showing CD11c and CD8α expression on unsorted and sorted DC populations. (B, C). Presentation to type B 11A10 T cell hybridoma by sorted CD8α+ (B) and CD8α- (C) DC incubated with HEL protein, with or without CpG. (D, E). Presentation to type B 11A10 T cell hybridoma by sorted DC incubated with HEL protein (D) or peptide (E) with or without TLR stimulants. Error bars represent SD. B and C representative of eleven independent experiments; D and E representative of four independent experiments.
Figure 4
Figure 4. Type I IFN-Induced Presentation of Type B pMHC from HEL and Production of Type I IFN by TLR Stimulation
(A-D) Presentation by DC incubated with (A, B) HEL or (C, D) HEL peptide 48-62 with or without CpG or recombinant IFN-α or IFN-β to (A, C) type B 11A10 or (B, D) type A 3A9 T cell hybridomas. Representative of twelve independent experiments. (E) Sorted cDC were incubated with or without stimulants for 2 or 6 hours before RNA was isolated. qRT-PCR was performed to assess transcript levels of IFN-beta. ΔΔCΤ calculations were performed using 18S as the standard.
Figure 5
Figure 5. Role of Type I IFN Signaling in CpG-Induced pMHC Presentation from HEL
(A, B) Presentation to type B 11A10 T cell hybridoma by sorted CD11chiCD8a+ (A) versus CD11chiCD8a- (B) cells pre-treated with MAR1-5A3 (anti-IFNAR1) or control mouse IgG before adding antigen and stimulants. (C) Presentation to type B 11A10 T cell hybridoma by sorted cDC from B10.BR or IFNAR1-/- mice incubated with HEL protein with or without stimulants. (D) Presentation to 11A10 by cDC treated as in B. Results shown for 10 μM HEL protein dose. Complete antigen titrations are shown in Figure S1. Error bars represent SD. All graphs are representative of two independent experiments.
Figure 6
Figure 6. Role of Type I IFN Signaling in TLR-Induced Changes in Costimulatory Molecule Expression
DC from wild type B10.BR or IFNAR1-/- mice were incubated alone or with (A) Zymosan, (B) Poly (I:C), (C) LPS, (D) Gardiquimod, (E) CpG, (F) IFN-alpha, or (G) IFN-beta for 18 hours. DC were stained for CD40, CD80, CD86, or I-Ak and analyzed by flow cytometry. Data represented as fold change MFI of stimulated over un-stimulated DC. Data gated on live CD11chigh Siglec H- cells. Data are representative of two independent experiments. Full histograms are shown in Figure S2.
Figure 7
Figure 7. Role of H2-DM Regulation in TLR-Induced Type B pMHC Presentation from HEL and Accelerated Catabolism in CpG-B-Activated DC
(A) qRT-PCR analysis of RNA isolated from sorted cDC (CD11chighSiglecH-CD19-) were incubated with stimulants for 2, 6, or 18 hours. (B) Intracellular flow-cytometry analysis of H2-DM and H2-DO protein levels in sorted cDC after 18 hours of stimulation. (C-F) Presentation to (A, E) type B 11A10 or (B, D) type A 3A9 by sorted (C, D) wild-type B10.BR or (E, F) H2-DM-/- cDC incubated with HEL protein with or without stimulants. (G, H) DC were incubated for 18 hours with or without CpG-B, washed, incubated with I125-HEL for two hours at room temperature, washed extensively, then chased for indicated times at 37°C. TCA-Soluble and TCA-Precipitable fractions were collected from the (G) intracellular and (H) supernatant fractions. Data plotted as percentage of total cpm at each time point.

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