the unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via toll-like...

The Unc93b1 mutation 3d disrupts exogenousantigen presentation and signaling via Toll-likereceptors 3, 7 and 9

Koichi Tabeta1, Kasper Hoebe1, Edith M Janssen2, Xin Du1, Philippe Georgel1, Karine Crozat1,Suzanne Mudd1, Navjiwan Mann1, Sosathya Sovath1, Jason Goode1, Louis Shamel1, Anat A Herskovits3,Daniel A Portnoy3, Michael Cooke4, Lisa M Tarantino4, Tim Wiltshire4, Benjamin E Steinberg5,Sergio Grinstein5 & Bruce Beutler1

Here we have identified ‘triple D’ (3d), a recessive N-ethyl-N-nitrosourea-induced mutation and phenotype in which no

signaling occurs via the intracellular Toll-like receptors 3, 7 and 9 (sensors for double-stranded RNA, single-stranded RNA and

unmethylated DNA, respectively). The 3d mutation also prevented cross-presentation and diminished major histocompatibility

complex class II presentation of exogenous antigen; it also caused hypersusceptibility to infection by mouse cytomegalovirus and

other microbes. By positional identification, we found 3d to be a missense allele of Unc93b1, which encodes the 12-membrane-

spanning protein UNC-93B, a highly conserved molecule found in the endoplasmic reticulum with multiple paralogs in

mammals. Innate responses to nucleic acids and exogenous antigen presentation, which both initiate in endosomes, thus seem

to depend on an endoplasmic reticulum–resident protein, which suggests communication between these organellar systems.

The mammalian Toll-like receptors (TLRs) sense conserved mole-cules of microbial origin, including bacterial lipopolysaccharide(LPS)1, lipopeptide2,3, glucan4, flagellin5 and nucleic acids6–10.Whereas TLR1, TLR2, TLR4 and TLR6 are at least partly representedon the surface of mammalian cells, TLR3, TLR7 and TLR9, whichdetect double-stranded RNA7, single-stranded RNA8–10 and un-methylated DNA, respectively6, are mainly or entirely intra-cellular receptors, residing in the endoplasmic reticulum and/orendosomes11–16. As the key receptors required for host awarenessof microbial infection, TLRs mediate most phenomena associatedwith microbial infections17, including the upregulation of costimula-tory molecules18 that are needed to initiate adaptive immuneresponses to antigens processed and presented by antigen-presentingcells of the host19–22.

However, TLR signaling is not required for adaptive immuneresponses themselves. For example, apoptotic cells exert TLR-independent adaptive immune activation (E.M.J. et al., unpub-lished data). Moreover, antigen processing and antigen presenta-tion do not depend on TLR signaling at all. In contrast, bothendogenous and exogenous antigen presentation occur constitutivelyin antigen-presenting cells, in the absence of infection and in the

absence of TLR signaling (K.H. et al., unpublished data, and E.M.J.et al., unpublished data). Where exogenous antigens are concerned,processing for major histocompatibility complex (MHC) class Ipresentation (cross-presentation) involves a series of biochemicalevents very different from those associated with processing forMHC class II presentation23,24.

Immunodeficiency states in which TLR signaling is altered orabolished have been systematically created and identified using therandom germline mutagen N-ethyl-N-nitrosourea25–28. We nowdescribe a single-nucleotide substitution that abrogated signaling viaTLR3, TLR7 and TLR9 (but not other TLRs) and also markedlyimpaired MHC class I and class II presentation of exogenous antigens.This mutation demonstrates a point of intersection between thecellular events required for exogenous antigen presentation andthose required for signaling by the intracellular TLRs. Both processesdepend on a single, highly conserved protein, the function of whichwas previously unknown. The protein seems to be an intrinsiccomponent of the endoplasmic reticulum, which suggests that com-munication between the endoplasmic reticulum and endosomes isrequired for both exogenous antigen presentation and TLR3, TLR7and TLR9 signaling.

Received 11 August 2005; accepted 1 December 2005; published online 15 January 2006; doi:10.1038/ni1297

1Department of Immunology, The Scripps Research Institute, La Jolla, California 92037, USA. 2Division of Cellular Immunology, The La Jolla Institute for Allergy andImmunology, San Diego, California 92121, USA. 3Department of Molecular and Cell Biology and School of Public Health, Berkeley, California 94720-3202, USA.4Genomics Institute of the Novartis Research Foundation, San Diego, California 92121, USA. 5Division of Cell Biology, Hospital for Sick Children, Toronto M5G 1X8,Canada. Correspondence should be addressed to B.B. ([email protected]).

156 VOLUME 7 NUMBER 2 FEBRUARY 2006 NATURE IMMUNOLOGY

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RESULTS

Generation of the 3d phenotype

By screening macrophages derived from C57BL/6 mice homozygousfor N-ethyl-N-nitrosourea-induced germline mutations, we identifiedtwo G3 mice of a single kindred that failed to produce normalquantities of tumor necrosis factor (TNF) in response topoly(I)�poly(C) (a TLR3 stimulus), resiquimod (a TLR7 stimulus)and unmethylated DNA oligonucleotides bearing CpG motifs (a TLR9stimulus). We bred these mice to produce a homozygous mutantstock, which permitted phenotypic characterization of a larger num-ber of homozygous mice and obligate heterozygous mice. The mutantphenotype, called ‘triple D’ (3d) to denote a triple defect in nucleicacid sensing, was strictly recessive, 100% penetrant and specific for thedetection of nucleic acids (Fig. 1a). There was normal sensing of LPS,lipoteichoic acid and both di- and tri-acylated bacterial lipopeptides.Not only TNF production but also the induction of costimulatorymolecule expression was abolished by the 3d mutation (Fig. 1b).However, some residual response to poly(I)�poly(C) was evident inthe last assay, consistent with the published observation that C57BL/6mice have a TLR-independent pathway for sensing double-strandedRNA18. There were normal quantities of TLR3, TLR7 and TLR9 byimmunoblot of whole-macrophage extracts (data not shown), con-sistent with the interpretation that the mutation causes a defect in

TLR function rather than a defect in the expression of TLR3, TLR7and TLR9.

Mice homozygous for the 3d mutation had normal developmentof lymphoid organs and normal numbers of peripheral CD4 and CD8T cells, B cells, natural killer cells and natural killer T cells. They hadnormal expression of MHC class I and class II antigens on the surfacesof antigen-presenting cells. Moreover, they showed no obvious devel-opmental defects of any kind and demonstrated normal cage activityand fertility.

The 3d mutation does not prevent endosome acidification

We ‘mimicked’ the 3d sensing defect by treating cells with chloroquineor bafilomycin before stimulating them with nucleic acids (data notshown). These agents are known to dissipate the pH gradient acrossacidic compartments. Therefore, we considered that the 3d defectmight produce an inability to properly acidify endomembrane com-partments. However, phagosome acidification in 3d macrophages wascomparable to that of C57BL/6 control cells when estimated usingLysoTracker Red after internalization of 3.1-mm latex beads (Fig. 1c,d).Moreover, careful comparison of the lumenal pH of several intra-cellular compartments by dual-excitation fluorescence ratio imaging in3d and normal (wild-type) macrophages showed no substantialdifferences. Specifically, the pH of the Golgi complex (wild-type,

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Figure 1 TLR3, TLR7 and TLR9 signaling are prevented by the 3d mutation,

which has no effect on endosomal pH. (a) TNF production by wild-typeand mutant macrophages. Rsq, resiquimod; LP, bacterial lipopeptide

PAM3CysSer(Lys)4; PGN-LTA, peptidoglycan and lipoteichoic acid. Data represent

means ± s.e.m (n ¼ 6 mice). (b) TLR3-, TLR7- and TLR9-induced costimulatory

molecule expression is inhibited in mice homozygous for the 3d mutation. Flow

cytometry of CD40 and CD86 expression of peritoneal macrophages incubated

for 24 h with inducers (CpG, resiquimod and poly(I)�poly(C)) or left uninduced

(Control). Each symbol represents measurement of macrophages from a single

mouse: ovals, 3d/3d; inverted triangles, 3d/+. (c,d) Acidification of phagosomes

in C57BL/6 control macrophages (c) and 3d macrophages (d). Cells were allowed

to internalize IgG-opsonized 3.1-mm latex beads and after 45 min were labeled

for 5 min with 1 mM LysoTracker Red. Arrows indicate representative phagosomes. Top insets (c,d), acidic compartments labeled with LysoTracker Red before

phagocytosis; bottom inset (c), macrophage treated with 200 nM concanamycin and 10 mM nigericin before the addition of LysoTracker Red. Scale bars,

5 mm. (e,f) Acidification of intracellular compartments is comparable in C57BL/6 control and 3d/3d macrophages. Data are means ± s.e.m. (n ¼ 3).

(f) Representative calibration curve for lysosomes loaded with Oregon 514–dextran.

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6.47 ± 0.14; 3d, 6.49 ± 0.07), endosomes (wild-type, 6.45 ± 0.12; 3d,6.26 ± 0.23), lysosomes (wild-type, 4.64 ± 0.12; 3d, 4.58 ± 0.20) andphagosomes (wild-type, 5.39 ± 0.33; 3d, 4.99 ± 0.24) was similarregardless of homozygosity for the mutation (Fig. 1e,f).

The 3d mutation enhances susceptibility to diverse microbes

As mutations that affect signaling via the TLR3 and TLR9 path-ways27,28 are known to enhance susceptibility to mouse cytomegalo-virus (MCMV), a b-herpes virus to which C57BL/6 mice normallydemonstrate robust resistance, we assessed the MCMV susceptibilityof mice homozygous for the 3d mutation. All mice inoculated with5 � 105 plaque-forming units of MCMV died within 5 d and had titersof the virus in spleen homogenates approximately 10,000-fold higherthan those in C57BL/6 control mice (comparable to BALB/c mice,which are susceptible to MCMV). The short time frame in which micehomozygous for the 3d mutation died after MCMV infection wasconsistent with an innate immunodeficiency state. Moreover, micehomozygous for the 3d mutation did not mount an adequate cytokineresponse to MCMV in vivo (Fig. 2a–f), which suggested a sensingdefect as the cause of susceptibility. Innate responses to other microbeswere also impaired. For example, production of TNF and IL-12p40mRNA in response to Listeria monocytogenes, processes known to bedependent on the adaptor molecule MyD88 (refs. 29–31), werediminished, most notably when organisms incapable of escapingfrom the endosome were used to infect macrophages (the hly mutant,which lacks the listerolysin protein; Fig. 2g,h). Notably, when macro-phages were infected by hly mutant L. monocytogenes, they producedfar more IL-12p40 mRNA than did macrophages infected by wild-typeL. monocytogenes organisms that efficiently escape from the endosome,indicating that a stronger signal was generated as a result of endosomalconfinement. But this signal was nullified when the host was

homozygous for the 3d mutation. Mice homozygous for the 3dmutation were also less capable of eliminating a Staphylococcus aureusinfection (Supplementary Fig. 1 online).

A defect in exogenous antigen processing

Apart from the innate sensing defects described above, mice homo-zygous for the 3d mutation also failed to process antigen normally.When we injected mice homozygous for the 3d mutation withCFSE (carboxyfluorescein diacetate succinimidyl diester)–labeledOT-II cells (CD4 T cells from a C57BL/6 mouse transgenic for arearranged T cell receptor that recognizes ovalbumin (OVA)) andsubsequently challenged them with very pure OVA, we noted adiminished OT-II mitogenic response in vivo. Hence, MHC class IIantigen presentation was much reduced, although not entirelyabolished, in the 3d environment (Fig. 3a). Cross-presentation ofOVA for class I activation was even more strongly inhibited by the 3dmutation. We immunized mice homozygous for the 3d mutationand C57BL/6 control mice with ultraviolet irradiation–treatedC57BL/6 splenocytes expressing OVA32,33. This form of immunizationelicited a strong adaptive immune response that was entirely inde-pendent of TLR signaling (E.M.J. et al., unpublished data). Wecollected spleens 7 d after immunization and restimulated splenocyteswith MHC class I–specific OVA peptide in vitro. Mice homozygousfor the 3d mutation produced no OVA peptide–specific CD8+ inter-feron-g (IFN-g)–positive cells, and their cells showed no cytolyticactivity against peptide antigen–loaded targets (Fig. 3b,c). Moreover,the 3d mutation prevented expression of OVA peptide on thesurface of antigen-presenting cells in the context of MHC class Iprotein, whether soluble OVA or OVA associated with cellstreated with ultraviolet irradiation was provided to these cells as asource of antigen (Fig. 3d,e). Therefore, we concluded that the

BALB/c C57BL/6 C57BL/63d/3d 3d/3d C57BL/6

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Figure 2 Susceptibility to infection of mice homozygous for the 3d mutation. (a) Splenic viral titers determined by plaque-forming assay 4 d afterinoculation with 5 � 105 plaque-forming units (PFU) of MCMV. n, number of mice in each group. BALB/c mice serve as a positive control for susceptibility.

(b) Mortality after intraperitoneal inoculation with 5 � 105 plaque-forming units of MCMV. (c–f) Concentration of interferon-g (c), IL-12 (d) and TNF (e)

and type I interferon (f) in plasma 36 h after inoculation with MCMV (5 � 105 plaque-forming units, intraperitoneally), measured by enzyme-linked

immunosorbent assay (c–e) and by biological assay (inhibition of VSV plaque formation; f). Error bars indicate s.e.m.; n ¼ 6 mice. P values (a–f), 3d/3d

versus wild-type. (g,h) Primary bone marrow–derived macrophages from 3d/3d mutant mice show decreased expression of IL-12p40 and TNF after infection

with L. monocytogenes. Real-time PCR of IL-12p40 and TNF mRNA in macrophages from C57BL/6 or 3d/3d mutant mice 6 h after infection with wild-type

(WT LM) or hly mutant (hly) L. monocytogenes or stimulation with Pam3CysSer(Lys)4 (PAM3CSK4). Results are presented as a mean ± s.d. of three

independent experiments, relative to expression of glyceraldehyde phosphate dehydrogenase (GAPDH).


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mutation confers a defect of exogenous antigen processing and/orcross-presentation.

Positional cloning of 3d

We genetically mapped the 3d locus by outcrossing the mutant stock toC3H/HeN mice and backcrossing and intercrossing the offspring. On45 meioses, the locus was assigned to proximal chromosome 19 with apeak logarithm-of-odds score of 8.6 (Supplementary Fig. 2 online),and on 5131 meioses, the locus was confined to a chromosomalinterval 0.46 megabases in length (between 3.43 and 3.89 megabasesfrom the centromere (Ensembl distances, release 27.33c.1)). Onecrossover separated the mutation from the proximal markerD19Mit68 and two crossovers separated the mutation from the distalmarker D19SNP3 (Supplementary Figs. 3 and 4 online). We con-firmed each of these three crossover events in a subsequent generationof mice. The critical region (Supplementary Fig. 5 online) contained20 annotated genes. We sequenced all candidates at the cDNA level andgenomic DNA level using primer pairs that amplified all annotatedexons and splice junctions. We visualized only one mutation, a singlebase pair transversion, in both genomic and cDNA templates (Supple-mentary Fig. 6 online). This mutation was located in exon 9 of the 11-exon gene Unc93b1 (Mouse Genome Informatics accession number,1859307), encoding UNC-93B (unc-93 homolog B1 (Caenorhabditiselegans)) and was predicted to alter the 598–amino acid UNC-93B byintroducing a positively charged residue into the ninth of twelvetransmembrane domains (H412R). The residue in question wasfound to be invariant among all known vertebrate UNC-93B orthologs,including those of mice, rats, humans, chickens and the green puffer-fish and in Drosophila melanogaster (Supplementary Fig. 7 online).

After transfection of bone marrow–derived dendritic cells frommice homozygous for the 3d mutation to express the wild-type

UNC-93B cDNA including a C-terminal green fluorescent protein(GFP) tag, the phenotypic defect (impaired nucleic acid sensing) wascorrected in vitro. When a cDNA encoding a GFP-tagged UNC-93Bbearing the 3d substitution H412R was overexpressed, a moderatecodominant suppressive effect was evident. Neither the normal northe mutant form of the protein affected TLR4 signaling (Fig. 4a,b).These studies confirmed the Unc93b1 mutation as the cause ofdefective nucleic acid sensing.

To be certain that the Unc93b1 mutation was also responsiblefor the observed defects of antigen presentation (which wereless useful as endpoints in high-throughput mapping based onphenotype), we intercrossed heterozygous mice and examined atotal of 26 F2 mice (6 homozygous for the mutation, 7 wild-typeand 13 heterozygous for the mutation). We loaded granulocyte-monocyte colony-stimulating factor–derived antigen-presenting cellsfrom each mouse with OVA expressed by cells treated with ultra-violet irradiation (E.M.J. et al., unpublished data) and measured theirability to prime or cross-prime OT-I or OT-II cells. There wasperfect concordance between genotype and phenotype. With regardto cross-presentation, the 3d mutation exerted a definite codominanteffect (Fig. 4c,d). Because we studied 52 meioses without findinga single instance of discordance between genotype and phenotype,we calculated with 95% confidence that both the 3d mutation andthe mutational source of the antigen presentation defect were inthe same 6.6-centimorgan region of the genome, encompassing0.4% of genomic DNA. Moreover, as already noted, many of thecoding exons in this interval were excluded from consideration byDNA sequencing. On that basis, we concluded that the nucleic acid–sensing and antigen-presentation phenotypes almost certainlyemanated from the same mutation, located in the coding regionof Unc93b1.

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Figure 3 The 3d/3d mice are defective in presentation of exogenous antigen. (a) MHC class II presentation is impaired in 3d/3d mice in vivo. After 3d/3d

mice received CFSE-labeled Va2+CD4+ OT-II cells intravenously, they were challenged with 0.25 mg chicken OVA freshly prepared from egg whites and

free of microbial contaminants. Division of antigen-specific Va2+CD4+ cells was measured in the spleen 48 h and 72 h later. Wedges indicate increasing

CFSE fluorescence intensity. Results are from experiments using two 3d/3d mice and two wild-type (WT) mice. (b,c) Cross-presentation of OVA for MHC class

I activation is inhibited by the 3d mutation. First, 3d/3d and C57BL/6 control mice were immunized with ultraviolet irradiation–treated C57BL/6 splenocytes

expressing OVA; then, 7 d later, splenocytes were restimulated with MHC class I–specific OVA peptide in vitro (OVA(257–264)) or were left unstimulated

(control (C)). Cross-presentation was evaluated by determination of the number of OVA peptide–specific CD8+IFN-g+ cells and the cytolytic activity against

OVA peptide–loaded targets (effector/target ratio, 100:1). (d,e) The 3d/3d mice have defective MHC class I antigen presentation compared with that of wild-

type mice. Dendritic cells (DC) were exposed for 24 h to apoptotic cells expressing membrane-associated OVA under the influence of an actin promoter

(actm-OVA cells) or to soluble ovalbumin, after which SIINFEKL (H-2Kb) complex formation was measured with a specific monoclonal antibody.


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Structure, expression and homologs of Unc93b1

Bioinformatic analyses showed that Unc93b1 was highly expressedmouse B cells and that its human homolog (UNC93B1) was highlyexpressed in dendritic cells (Genomics Institute of the NovartisResearch Foundation expression anatomy database), consistent withinvolvement in microbial sensing and antigen presentation and withthe phenotypic effects noted in mice homozygous for the 3d mutation.In humans, a total of five proteins homologous to UNC-93B werepredicted by Ensembl. Three of these (including one encoded by a very

closely linked gene and two genes located on other chromosomes)resembled truncations of UNC-93B with nearly complete sequenceidentity, but their expression has yet to be verified experimentally. Inboth humans and in mice, UNC-93A (National Center for Biotech-nology Information accession number, CAD19523) was found to bethe nearest paralog of UNC-93B with a distinctly divergent sequencealong its entire length (Supplementary Fig. 8 online). Its function hasnot been determined. In mice, UNC-93A is encoded by a gene onchromosome 17 (Unc93a; Mouse Genome Informatics accession

Figure 5 Subcellular localization of UNC-93B

and the distribution of histocompatibility

complexes and TLR9 in wild-type andUnc93b13d/3d macrophages. (a,b) Distribution of

UNC-93B and the endoplasmic reticulum marker

GFP-KDEL. Cells were cotransfected to express

Myc-tagged UNC-93B and GFP-KDEL. The

epitope was detected by immunostaining (a, main

image); the green fluorescence of GFP-KDEL was

visualized directly (b, main image). Bottom right

inset in b, scatter plot correlating green (GFP-

KDEL) and red (UNC-93B) fluorescence intensity

in individual pixels of the area defined by the

rectangles (dashed outlines) in the main images.

The scattergram was used to calculate Pearson’s

colocalization coefficient: R ¼ 0.88 (P o 0.01).

Bottom left insets (a,b), distribution of UNC-93B

(a) and LAMP-1 (b). Original magnification,

�200 (main images) and �400 (bottom left

insets). (c–i) Images of peritoneal macrophages

from wild-type and Unc93b13d/3d mice. Cellswere fixed and permeabilized and then stained

with monoclonal anti–MHC class I (MHC I; c,d),

anti–MHC class II (MHC II; e,f) or anti-TLR9 (red

in g–i). MHC class I distribution was confirmed with two different monoclonal antibodies (clones 28-14-8 and 34-4-20S), and two separate antibodies were

used to verify the distribution of MHC class II (clones M5/115.15.2 and Y3P). Insets (c,d), staining with a mouse IgG2a isotype control appropriate for

mouse anti–MHC class I and anti–MHC class II staining (c–f). In g–i, plasma membranes are stained with fluorescein isothiocyanate–conjugated B subunit of

cholera toxin (green). (i) Staining of primary macrophages from TLR9-knockout mice with the same antibody as in g,h, demonstrating the specificity of the

anti-TLR9. Original magnification, �200 (main images) and �400 (insets).

UNC-93b

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the 3d phenotype and the effect of Unc93b1

genotype on antigen presentation. (a,b) Bone

marrow–derived dendritic cells from mutant mice

(Unc93b13d/3d) or wild-type mice (Unc93b1+/+)

were transfected with an expression vector

containing C-terminally GFP-tagged versions of

UNC-93B (wild-type or H412R). TNF+ cells wereassessed with (+) or without (�) stimulation using

10 mg/ml of poly(I)�poly(C) (PIC; a) or 10 ng/ml of

LPS (b). Mock, transfected with vector DNA

(vector control). Error bars indicate s.e.m. of three

independent experiments. (c,d) Cross-priming of

CD8 responses (c) and priming of CD4 responses

(d) in mice with various Unc93b1 genotypes.

Dendritic cells from wild-type mice (Unc93b1+/+),

heterozygous mutant mice (Unc93b1+/3d) or

homozygous mutant mice (Unc93b13d/3d),

derived using the ligand for the cytokine receptor

Flt3, were exposed to g-irradiated (1,500) cells

expressing membrane-associated OVA under the

influence of an actin promoter. After 24 h, CFSE-

labeled OT-I (c) or OT-II (d) cells were added and

priming (cross-priming) ability was assessed as

the percentage of responding cells, measured by

proliferation of OT-I and OT-II cells. Each dot

represents a single mouse.


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number, 1933250). UNC-93A is homologous to a still more distantand still un-named paralog, also of unknown function (MouseGenome Informatics accession number, 1917150)34, encoded by agene on chromosome 11. We did not definitively exclude the existenceof still other, more distant family members. Analysis using the SimpleModular Architecture Research Tool disclosed no domains in UNC-93B that could be assigned functions with high confidence. Betweenresidues 124 and 189, we identified a pFAM (protein families databaseof alignments and hidden Markov models) domain of unknownfunction (DUF895) and also found it to be represented in UNC-93A as well as the more distant paralog mentioned above.

The subcellular location of UNC-93B

Because TLR3, TLR7 and TLR9 signaling, MHC class II loadingand acquisition of antigens destined for cross-presentation all occurin the endosomal-lysosomal compartment, we anticipated thatUNC-93B would be a component of this organellar system. Unex-pectedly, however, when expressed in RAW 264.7 macrophages asa C-terminally GFP-tagged or Myc-tagged species, UNC-93B waswidely distributed, with a reticular pattern suggestive of endo-plasmic reticulum localization. This was confirmed by the extensivecolocalization of Myc-tagged UNC-93B with the endoplasmic reticu-lum marker GFP-KDEL (Fig. 5a,b; Pearson’s colocalization coefficient¼ 0.88; P o 0.01). In contrast, the distribution of UNC-93B wasdistinct from that of LAMP-1 (Fig. 5a,b, insets), a marker of lateendosomes and lysosomes, and from the Golgi or plasma membrane(data not shown).

We then considered that the 3d mutation might exert its phenotypiceffect by altering the subcellular distribution of MHC class I and classII proteins as well as TLR3, TLR7 and TLR9 through an effect on thetrafficking of these proteins. However, detailed immunolocalizationstudies excluded that hypothesis (Fig. 5c–i). Direct immunostainingof TLR9 showed it resided in a punctate sub-plasmalemmal vesicularcompartment (Fig. 5g–i). There was no redistribution of GFP-taggedUNC-93B in response to TLR9 or TLR3 ligands, nor was there adifference in ligand uptake or distribution on comparisons ofhomozygous 3d versus wild-type cells (data not shown). Ultrastruc-tural analysis of macrophages collected from mice homozygous forthe 3d mutation and wild-type mice provided no added insight intothe mechanism of action of UNC-93B, as the cells seemed identical inall respects.

DISCUSSION

By intentionally searching for previously unknown immunodeficiencyphenotypes among randomly generated germline mutant mice, wehave identified a fundamentally new genetic defect that disruptsseveral immune processes. The 3d phenotype was initially found bysystematic monitoring of TLR signal transduction. Mice homozygousfor the 3d mutation were then also found to have a unique defect ofexogenous antigen presentation (absence of cross-presentation andmarkedly diminished MHC class II presentation). Although miceseemed normal in the absence of microbial challenge, they wereunable to cope with experimental infections caused by diversemicrobes and were particularly susceptible to MCMV, which isknown to stimulate an innate response mediated by the nucleicacid–sensitive TLR3-TRIF (an adaptor molecule)27,28 and TLR9-MyD8827,35 axes. All the defects noted in mice homozygous for the3d mutation have been ascribed to a single nonconservative aminoacid substitution that is very likely to disrupt the conformation ofthe affected protein, UNC-93B. UNC-93B has no structural featuresthat would suggest involvement in either TLR signaling or exogenous

antigen presentation and has had no function previously assignedin mammals.

In C. elegans, the prototypic UNC-93 protein36 is a regulatorysubunit of a tripartite two-pore potassium channel36–38 and mutationsof the gene encoding UNC-93 cause a motility defect. The otherchannel components are encoded by the genes sup-9 and sup-10. Thesup-9 product is a 329–amino acid protein with sequence similarity tothe mammalian TASK (two-pore acid-sensitive potassium channel)family of proteins39. Based on genetic and biochemical arguments,sup-10 and unc-93 are believed to encode essential regulatory compo-nents of the potassium channel38. In the mammalian context, how-ever, we consider it likely that UNC-93B has assumed an entirely newfunction unrelated to potassium transport. The mammalian proteinhas only 18% amino acid identity with its C. elegans homolog and nosup-10 equivalent is known in mammals. Mice homozygous for the 3dallele of Unc93b1 show no defects of neuromuscular function. TheUNC-93B protein seems to be an integral component of the endo-plasmic reticulum, and by applying a null-point titration methodbased on the use of K+-H+-exchanging ionophores40, we found theendoplasmic reticulum potassium concentration to be similar to thatof the cytosol in cells from both mice homozygous for the 3dmutation and wild-type mice (data not shown).

TLR3, TLR7 and TLR9 signaling and exogenous antigen presenta-tion are fundamentally separate processes. For example, in mice homo-zygous for the compound mutations MyD88–/– and Trif Lps2/Lps2,which cannot signal via TLR3, TLR7 and TLR9, exogenous antigenpresentation occurs normally (K.H. et al., unpublished data, andE.M.J. et al., unpublished data). Conversely, mice deficient in eithertransporter associated with antigen processing 1 or MHC class II havenormal TLR3, TLR7 and TLR9 signaling (K.H. et al., unpublisheddata). It is therefore illuminating to find that a single protein isrequired for both processes to occur normally and that mutationalmodification of the protein does not have more generalized effects inthe organism as a whole. The challenge now is to understand preciselyhow UNC-93B functions. Many possibilities have been excluded, asdescribed below.

We first note that exogenous antigen presentation and TLR3, TLR7and TLR9 signaling all depend on events in the endosomal-lysosomalpathway; hence, UNC-93B must be presumed to influence theseevents. TLR3, TLR7 and TLR9 are believed to be anchored inendosomal membranes and to detect nucleic acids in the lumens ofendosomes after acidification10,41–45, after which they recruit TRIF (inthe case of TLR3) or MyD88 (in the case of TLR7 and TLR9),initiating signaling events that have been described before17,46,47.MHC class II loading is believed to depend on hydrolysis of theinvariant chain and hydrolysis of antigen, which also occur in anacidified endosomal compartment24. Cross-presentation of antigen onMHC class I molecules entails the translocation of proteins from theendosomes to the cytosol, where they are believed to be processed viaproteasomes and then admitted to the endoplasmic reticulum throughthe transporter associated with antigen processing channel in the formof proteolytic fragments23.

Because acidification of the endosomes may be essential to eachof these processes, we suspected that UNC-93B might be requiredfor acidification. However, at least the endosomal-lysosomalcompartments probed using internalized cholera toxin B subunitand dextran were equally acidic in wild-type and 3d cells (we cannotexclude the possibility that specialized subcompartments not accessedby these probes may have been altered by the mutation). We none-theless considered that UNC-93B might be an intrinsic componentof the endosomal-lysosomal pathway. This hypothesis would seem


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particularly likely given that L. monocytogenes mutants incapable ofescaping from phagosome elicited especially strong TLR-mediatedresponses, which were nullified by the 3d mutation. However, with thecaveat that GFP-tagged proteins overexpressed under the influence ofa foreign promoter do not always mimic the distribution of theendogenous protein, UNC-93B seems to reside in the endoplasmicreticulum. It is not detected much in components of the endosomal-lysosomal pathway.

We further considered that UNC-93B might be required for normaltrafficking of the nucleic acid–sensing TLRs and/or MHC class IIproteins, permitting them to access the endosomes. However, the 3dmutation had no effect on the subcellular location of these moleculesor on the subcellular location of MHC class I protein. UNC-93B isnecessary for TLR signaling (although not for the presence of TLRs inendosomes) and for the presentation and cross-presentation ofexogenous antigens.

If endogenous UNC-93B, like the tagged versions used here, provesto be expressed mainly in the endoplasmic reticulum, we wouldconclude that TLR3, TLR7 and TLR9 signaling, as well as exogenousantigen presentation, depend on direct or indirect communicationbetween the endoplasmic reticulum and components of the endoso-mal-lysosomal pathway. Such communication, which might involvedirect contact between the endoplasmic reticulum and the endosomesor the transfer of specific (and unknown) proteins from the endo-plasmic reticulum to the endosomes to support the processes beingdiscussed here, would seem to depend on UNC-93B. Because UNC-93B is a highly conserved protein required for innate responses(signaling via the nucleic acid–sensing TLR3, TLR7 and TLR9) andfor exogenous antigen presentation, we speculate that both processesrely on a common evolutionary substratum. Further mechanisticunderstanding of UNC-93B may therefore help to elucidate theevolution of exogenous antigen presentation, which presumablyacquired its essential function in adaptive immunity after nucleicacid sensing became established in vertebrates and certainly after theevolution of UNC-93B.

In conclusion, a single protein is necessary to support signalingfrom the nucleic acid–sensing TLRs as well as exogenous antigenpresentation. Evidence now suggests that the protein is required onlyfor normal immune function and not for development, fertility orneuromuscular function. As an amino acid substitution in UNC-93Bhad very clear and specific immunosuppressive effects, this proteinmay be regarded a likely target for pharmacological intervention inautoimmune and/or inflammatory disease states, in which nucleicacid sensing and presentation of exogenous self antigens may bothcontribute to pathogenesis48–50.

METHODSReagents. Monensin was from Calbiochem; chloroquine was from Sigma.

LysoTracker Red DND-99 was from Invitrogen. Monoclonal mouse antibody to

MHC class I (anti–MHC class I; clone 28-14-8) and anti–MHC class II (clone

M5/115.15.2) were obtained from eBioscience; monoclonal mouse anti–MHC

class I (clone 34-4-20S) and anti–MHC class II (clone Y3P) were gifts from

T. Watts (University of Toronto, Toronto, Canada). Mouse anti–human TLR9

(clone 26C593.2) was obtained from Imgenex, and indocarbocyanine-

conjugated donkey anti-mouse IgG was purchased from Jackson Immuno-

Research Laboratories.

Germline mutagenesis. N-ethyl-N-nitrosourea was obtained from Sigma and

germline mutagenesis was done as described28,51.

Animals and peritoneal macrophage response assays. C57BL/6 mice (germ-

line mutants or controls) were injected intraperitoneally with 3% thioglycolate;

3 d later, macrophages were isolated. For assay of TLR signaling activity, cells

were cultured at a density of 5 � 105 cells/well with varying concentrations of

the TLR-dependent inducers poly(I)�poly(C) (5 mg/ml), resiquimod (100 ng/ml),

CpG oligodeoxynucleotide (2 mM), LPS (10 ng/ml) and Pam3CysSer(Lys)4

(100 ng/ml) and a preparation of peptidoglycan known to contain lipoteichoic

acid (10 mg/ml; Sigma). Cells were incubated for 4 h at 37 1C, and media were

collected for TNF bioassay. For induction of costimulatory molecule expres-

sion, cells were induced for 24 h with CpG oligodeoxynucleotide (2 mM/ml),

resiquimod (100 ng/ml) or poly(I)�poly(C) (5 mg/ml) or were left uninduced.

Expression of CD40 or CD86 was measured by flow cytometry.

Innate immune activators and antibodies. Salmonella minnesota Re595 LPS

was obtained from Alexis. Peptidoglycan was purchased from Fluka. Double-

stranded RNA (poly(I)�poly(C)) was obtained from Amersham Pharmacia

Biotech. The bacterial lipopeptide Pam3CysSer(Lys)4 was purchased from EMC

Microcollections. Phosphorothioate-stabilized unmethylated DNA oligonucleo-

tide bearing CpG motifs (5¢-TCCATGACGTTCCTGATGCT-3¢) was obtained

from Integrated DNA Technologies. Fluorescein isothiocyanate–labeled anti-

CD40 and anti-CD86 were purchased from BD Biosciences. Enzyme-linked

immunosorbent assay kits for IL-12 and IFN-g measurements were obtained

from R&D systems.

TNF and type I interferon assays. TNF assays were done as described28,51.

Type I interferon assays were also done as described52. Purified IFN-a (R&D

systems) was used as standard.

Immunofluorescence. For induction of expression of MHC class II, wild-type

and 3d macrophages were stimulated with 100 ng/ml of LPS for 24 h before

staining. For MHC class I and class II staining, macrophages were fixed for

60 min with 4% paraformaldehyde in PBS. Cells were washed with 100 mM

glycine and were made permeable with 0.1% Triton X-100 in PBS. For TLR9

staining, macrophages were fixed and made permeable in 100% methanol at

–20 1C for 20 min. The cells were incubated for 60 min with 10 mg/ml of the

appropriate primary antibody, followed by incubation for 60 min with

indocarbocyanine-conjugated secondary antibody. Coverslips were mounted

onto slides with Fluorescent Mounting Medium (Dako Cytomation). Fluor-

escent images were acquired with a spinning disk laser confocal microscope

(Quorum) with a 100� oil-immersion objective. Acquisition was controlled by

the Volocity Improvision software and images were prepared with Adobe

Photoshop CS and Adobe Illustrator CS.

Live-cell imaging. Mouse embryonic fibroblasts from wild-type or 3d mice

were seeded on 25-mm glass coverslips and were transfected with various GFP-

tagged plasmids using FuGene 6 according to the manufacturer’s instructions

(Roche Diagnostics). All cells were used for experiments 20–48 h after

transfection. Acidic organelles were visualized by staining with 1 mM

LysoTracker Red (Molecular Probes) added 5 min before imaging. TLR3 or

TLR9 signaling was stimulated by incubation of cells for 60 min with 10 mg/ml

of poly(I)�poly(C) or 1 mM CpG OGN, respectively. Where indicated, organellar

acidification was impaired by the addition of 1 mM chloroquine or 10 mg/ml

of monensin. For imaging, coverslips were washed twice with PBS and were

mounted in a Leiden chamber holder maintained at a constant temperature

of 37 1C on the stage of the spinning disk microscope described above in

bicarbonate-free RPMI 1640 medium.

Intracompartmental pH assay. The pH of endomembrane compartments was

measured by fluorescence ratio imaging as described53. The pH of phagosomes

was determined after engulfment of fluorescein isothiocyanate–conjugated

zymosan (Molecular Probes). Lysosomes were loaded with 1 mg/ml of Oregon

green 514–dextran (Molecular Probes) by a 10-hour pulse followed by a 1-hour

chase. Endosomal and Golgi compartments were labeled by incubation with

fluorescein isothiocyanate–conjugated recombinant cholera toxin B subunit

(List Biological Laboratories) for 10 min and more than 45 min, respectively.

Coverslips with labeled cells were placed in a Leiden chamber holder main-

tained at a constant temperature of 37 1C on the stage of a Leica DM IRB

microscope. A filter wheel (Sutter Instruments) was used to alternate between

two excitation filters (440 and 490 nm). Light was directed at the sample with a

505-nm dichroic mirror, with emitted light filtered with a 510-nm filter placed

in front of a Hamamatsu Orca AG cooled charge-coupled device camera under


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the control of Metamorph/Metafluor software (Universal Imaging). After

acquisition of baseline data, in situ calibration was done by sequential bathing

of the cells with isotonic K+ solution (145 mM KCl, 20 mM HEPES, 10 mM

glucose, 1 mM MgCl2 and 1 mM CaCl2) buffered to predetermined pH values

and containing 1 mM nigericin. In these conditions, the intracompartmental

pH equilibrates with that of the bathing solution and a calibration curve can be

constructed by plotting of the fluorescence intensity ratio (490 nm/440 nm)

versus pH.

Determination of endoplasmic reticulum potassium by null-point titration.

Determination of the free K+ concentration in the endoplasmic reticulum was

done by the null-point method described54. This procedure involves measure-

ment of the direction and magnitude of the pH change induced by the

electroneutral K+-H+ exchanger nigericin, which is proportional to the trans-

membrane K+ gradient. For measurement of the pH of the endoplasmic

reticulum lumen, wild-type and 3d mouse embryonic fibroblasts were trans-

fected with a pH-sensitive fluorescent protein targeted to the endoplasmic

reticulum by fusion of the KDEL sequence to its C terminus. Cells were bathed

in an isotonic K+ solution to eliminate the K+ gradient across the plasma-

lemma, and the vacuolar-type ATPase inhibitor concanamycin (250 nM) was

added to preclude release of acid equivalents from acidic compartments after

the addition of nigericin. Fluorescence was then determined before and after

the addition of nigericin at various cytosolic K+ concentrations with a Leica

DM IRB microscope adapted with a Hamamatsu Orca AG cooled charge-

coupled device camera under the control of Metamorph/Metafluor software

(Universal Imaging). Finally, pH calibration was done in situ as described54.

‘Rescue’ studies and expression of GFP-tagged Unc93b1 for localization.

‘Rescue’ studies with bone marrow cells were done as described28,51. UNC-93B

and UNC-93B (H412R) were expressed using the vector pGFP-N (BD

Biosciences). Transfected cells were identified by GFP fluorescence and intra-

cellular TNF staining was done in this population with rat monoclonal

antibody MP6-XT22 labeled with phycoerythrin (BD Biosciences). RAW

264.7 cells were stably transfected with C-terminally GFP-tagged versions of

UNC-93B and were used for colocalization studies with LAMP-1. LAMP-1 was

visualized with rat monoclonal antibody 1D4B (Developmental Hybridoma

Bank) followed by indocarbocyanine-conjugated anti-rat. Where indicated,

Myc-tagged UNC-93B was expressed together with the endoplasmic reticulum

marker GFP-KDEL. The Myc epitope was visualized with monoclonal antibody

9E10 (Santa Cruz) followed by indocarbocyanine-conjugated anti-mouse.

Images were acquired with a spinning disk laser confocal microscope

(Quorum) and were analyzed with OpenLab Improvision software.

MCMV infection study. MCMV infection studies were done as described27.

Infection of bone marrow–derived macrophages by L. monocytogenes.

Primary bone marrow macrophages prepared from wild-type C57BL/6 mice

and C57BL/6 Unc93b13d/3d mice were incubated with 100 U/ml of IFN-g(Biosource) for 36 h before infection with L. monocytogenes wild-type strain

(10403S at a multiplicity of infection of about 5:1), isogenic Dhly strain (DP-

L2161 at a multiplicity of infection of about 100:1) or by stimulation with

Pam3CysSer(Lys)4 (300 ng/ml; Invivogen). Then, 6 h after infection, total RNA

was extracted and was reverse-transcribed with random hexamers. IL-12p40

and TNF mRNA were measured by SYBR Green real-time quantitative PCR

with specific primers and were normalized to glyceraldehyde phosphate

dehydrogenase mRNA.

MHC class II antigen presentation. CFSE-labeled Va2+CD4+ cells (1 � 107)

obtained from OT-2 mice were transferred intravenously into 3d/3d mice. After

immunization with 0.25 mg OVA, MHC class II-dependent priming was

analyzed based on the CFSE intensity of dividing Va2+CD4+ cells after 48 h

and 72 h.

Cross-presentation. C57BL/6 Unc93b13d/3d and C57BL/6 control mice were

immunized with 1 � 107 ultraviolet irradiation–treated transgenic C57BL/6

splenocytes expressing membrane-associated OVA under the influence of an

actin promoter. Then, 7 d later, splenocytes were collected from the immunized

mice and were restimulated in vitro with MHC class I–specific OVA peptide

(SIINFEKL). Cross-priming was evaluated by measurement of the percentage

of OVA peptide–specific CD8+IFN+ cells as well as cytolytic activity against

OVA peptide–loaded target cells at an effector/target ratio of 100:1.

Immunofluorescence staining of SIINFEKL (H-2Kb) complex. Bone

marrow–derived, granulocyte-monocyte colony-stimulating factor–treated

dendritic cells from wild-type and C57BL/6 Unc93b13d/3d mice were either

cultured together in presence of g-irradiated splenocytes expressing membrane-

associated OVA under the influence of an actin promoter (at dendritic cell/

OVA-expressing cell ratios of 10:1, 1:1, 1:2 and 1:5) or in presence of various

concentrations of OVA (10, 1, 0.1, 0.01 and 0.001 mg/ml). After 24 h, cells were

stained with Alexa Fluor–coupled monoclonal antibody 25-D1.16 specifically

recognizing the SIINFEKL (H-2Kb) complex55.

Note: Supplementary information is available on the Nature Immunology website.

ACKNOWLEDGMENTSWe thank C.C. Scott (HSC), L. Yu (HSC) and A. Kiemer (University ofCalifornia, San Diego) for help and advice. Supported by the NationalInstitutes of Health, Canadian Institutes of Health Research, Uehara MemorialFoundation (K.T.) and Leukemia and Lymphoma Society (3248-05 to E.J.).

COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests.

Published online at http://www.nature.com/natureimmunology/

Reprints and permissions information is available online at http://npg.nature.com/

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the unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via toll-like...

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