Generation of an HLA-DQ2.5 Knock-In Mouse

Alisa E. Dewan, Frank Koentgen, Marie K. Johannesen, M. Fleur du Pre and Ludvig M. Sollid

http://www.immunohorizons.org/content/5/1/25https://doi.org/10.4049/immunohorizons.2000107doi:

2021, 5 (1) 25-32ImmunoHorizons 

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Generation of an HLA-DQ2.5 Knock-In Mouse

Alisa E. Dewan,*,† Frank Koentgen,‡ Marie K. Johannesen,*,† M. Fleur du Pre,*,† and Ludvig M. Sollid*,†

*KG Jebsen Coeliac Disease Research Centre, Institute of Clinical Medicine, University of Oslo, 0372 Oslo, Norway; †Department of Immunology,

Oslo University Hospital-Rikshospitalet, 0372 Oslo, Norway; and ‡Ozgene Pty. Ltd., Bentley, Western Australia 6983, Australia

ABSTRACT

The human MHC class II molecule HLA-DQ2.5 is implicated in multiple autoimmune disorders, including celiac disease, type 1

diabetes, and systemic lupus erythematosus. The pathogenic contribution of HLA-DQ2.5 in many of these disorders is not fully

understood. There is thus a need for an HLA-DQ2.5 humanized mouse model with physiological expression of this MHC molecule

that can be integrated into disease models. In this article, we report the generation of an HLA-DQ2.5 knock-in mouse strain on a

C57BL/6 background in which sequences encoding the extracellular moieties of mouse MHC class II H2-IAa and H2-IAb1 have been

replaced with those of HLA-DQA1*05:01 and HLA-DQB1*02:01. In heterozygous knock-in mice, the expression of HLA-DQ2.5 is

superimposable with the expression of H2-IA. This was not the case in a regular untargeted HLA-DQ2.5 transgenic mouse. HLA-

DQ2.5 in the knock-in animals is functional for T cell development and for Ag presentation to HLA-DQ2.5–restricted and gluten-

specific T cells. Because C57BL/6 mice do not express H2-IEa, the only functional MHC class II molecule in homozygous HLA-DQ2.5

knock-in mice is the knock-in gene product. This alleviates the need for crossing with MHC class II knockout mice to study the

isolated function of the MHC transgene. Our novel mouse strain provides an important tool to study the involvement of HLA-DQ2.5

in models of diseases with association to this HLA allotype. ImmunoHorizons, 2021, 5: 25–32.

INTRODUCTION

Autoimmune diseases are typically associated with certain MHCclass II (MHC II) variants. The DR3DQ2 haplotype is over-represented in many diseases, including type 1 diabetes, myasthe-nia gravis, Sjogren syndrome, systemic lupus erythematous, andceliac disease (CeD) (1, 2). Although the tight linkage disequilib-rium of this haplotype makes it difficult to pinpoint the disease-associated HLA genes, in many instances HLA-DQ2 encoded byDQA1*05:01 and DQB1*02:01 (i.e., HLA-DQ2.5) is a candidate fordisease involvement in these diseases. In CeD, the strong diseaseassociation of HLA-DQ2.5 has been explained by this HLA

allotype’s preferential binding of negatively charged, posttransla-tionally modified gluten residues that are defining Ags of thisdisease (3).

Autoimmune diseases with MHC II associations are typicallyhallmarked by the production of autoantibodies and pathogenicinvolvement of B cells, underscoring the crucial role for T cell–B cell interactions in disease pathogenesis (4). In studies of suchdiseases, there is a quest forgood invivomodels enabling studies todecipher key pathogenic events.

Many mouse strains transgenic (tg) for various human HLAclass II allotypes have been made over the years, including forHLA-DQ2.5 (5–9), but most of these mice have been made by

Received for publication December 1, 2020. Accepted for publication December 14, 2020.

Address correspondence and reprint requests to: Prof. Ludvig M. Sollid, Department of Immunology, University of Oslo, Oslo University Hospital-Rikshospitalet,N-0372 Oslo, Norway. E-mail address: l.m.sollid@medisin.uio.no

ORCIDs: 0000-0001-7314-5778 (A.E.D.); 0000-0002-5660-4028 (F.K.); 0000-0003-4501-4222 (M.K.J.); 0000-0001-9231-643X (M.F.d.P.); 0000-0001-8860-704X (L.M.S.).

This work was supported by grants from the Research Council of Norway (Project 179573/V40, Centre of Excellence funding scheme, and Project 275053) and theSouth-Eastern Norway Regional Health Authority (Project 2010048).

A.E.D. designed and performed experiments, analyzed data, and wrote the manuscript; F.K. designed and generated knock-in mice; M.K.J. performed experiments andanalyzed data; M.F.d.P. designed and performed experiments, analyzed data, and edited the manuscript; L.M.S. designed knock-in mice, conceptualized and supervisedthe study, analyzed data, and wrote the manuscript.

Abbreviations used in this article: CeD, celiac disease; ES, embryonic stem; FRT, flippase recognition target; KI, knock-in; MHC II, MHC class II; PGK, phosphoglyceratekinase I; tg, transgenic(ally); WT, wild type.

The online version of this article contains supplemental material.

This article is distributed under the terms of the CC BY-NC 4.0 Unported license.

Copyright © 2021 The Authors

https://doi.org/10.4049/immunohorizons.2000107 25

RESEARCH ARTICLE

Adaptive Immunity

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untargeted insertion of either cDNA or fragments of genomichuman DNA. Untargeted insertion is an effective method of in-troducing transgenes in mice, but it carries the risk of unintendedconsequences that canaffect thediseasemodel (10).Thecommonlyobserved insertion of multiple gene copies can negatively impactexpression of the transgene (11) or lead to otherwise inauthenticexpression levels. Targeted insertion of transgenes into thehomologous locus of the model organism avoids many of theissues with untargeted insertion.

In this article, we report on the generation of an HLA-DQ2.5knock-in (KI) mouse in which the extracellular domains ofH2-IAb have been replaced by the corresponding parts of HLA-DQ2.5. Untargeted HLA class II tg mice are usually crossedonto MHC II knockout mice to study the effect of the tg HLAmolecule. Our HLA-DQ2.5 KI is generated in C57BL/6 mice,

which lack expression of H2-IE because of a deletion of thepromoter and first exon of the H2-Ea gene (12). When bred tohomozygosity for the KI gene, the only MHC II moleculeexpressed by these mice is HLA-DQ2.5 without the need forfurther crossing. This novel mouse fills a need for an HLA-DQ2.5 humanizedmodel for studies of HLA-associated diseasepathogenesis with physiological transgene expression.

MATERIALS AND METHODS

Generation of HLA-DQ2.5 KI miceThe HLA-DQ2.5 a- and b-chain (DQA1*05:01, DQB1*02:02) KImice were generated by Ozgene (Perth, WA, Australia). Briefly,CHORI-502humanBACCOXcell line librarycloneXXbac-254C11

FIGURE 1. Generation of HLA-DQ2.5 KI mice.

(A) Introduction of HLA-DQA1*05:01 sequences into H2-Aa. Top, Targeting construct with human exons 2 and 3 (gray) and FRT-flanked PGK-

hygromycin (HygroR) selection cassette (white). Middle, Mouse H2-Aa locus with indication of replaced segments. Dashed lines indicate mouse-

human sequence transition sites. Bottom, Mouse H2-Aa locus with integrated transgene with human exons in gray and mouse exons in black after

flippase-mediated excision of the FRT-flanked selection cassette. (B) Introduction of HLA-DQB1*0201 sequences into H2-Ab1. Top, Targeting

construct with human sequences for part of exon 1 and exons 2 and 3 (gray) and FRT-flanked PGK-neomycin (Neo) selection cassette (white).

Middle, Mouse H2-Ab1 locus with indication of replaced segments. Dashed lines indicate mouse-human sequence transition sites. Bottom, Mouse

H2-Ab1 locus with integrated transgene with human exons in gray and mouse exons in black after flippase-mediated excision of the FRT-flanked

selection cassette. (C) Detection of HLA-DQ2.5 surface expression. Splenocytes from WT, HLA-DQ2.5 KI heterozygous, and HLA-DQ2.5 KI ho-

mozygous mice were stained using anti-mouse MHC II (H2-IA/IE) and anti-human HLA-DQ2.5. Data are representative of more than three different

experiments (n = 1 mouse per experiment).

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(AL731683; BACPAC Resource Center) (13) was used as PCRtemplate for generation of amplicons for targeting vectorswithHLA-DQA1*05:01 andHLA-DQB1*02:01 sequences.MouseBACs RP2313B17 and RP23444J20 (Genomics Shared Resource,Roswell Park Comprehensive Cancer Center) (14) were used asPCR templates for amplicons of murine sequences for targetingvectors. Mouse sequences used as reference transcripts formurine genes were ENSMUST00000040655 (H2-Aa) andENSMUST00000040828 (H2-Ab1). The targeting constructswere verified by DNA sequencing. The HLA-DQA1*05:01 target-ing construct used the human sequence for exons 2 and3 downstream of a flippase recognition target (FRT)–flankedphosphoglycerate kinase I (PGK) hygromycin resistance cassette(Fig. 1A). The first, unsuccessful, targeting construct for in-troduction of HLA-DQB1*02:01 used the human sequence forexons 2 and 3 downstream of an FRT-flanked PGK neomycinresistance cassette (Supplemental Fig. 1A). The second targetingconstruct for introduction of HLA-DQB1*02:01 was similar tothefirst,with the FRT-flanked resistance cassettemoved furtherupstream of exon 2 (Supplemental Fig. 1B). The mouse-humansequence transitions for both these constructs were in the 1–2intronic sequence and in the 3–4 intronic sequence. The thirdand successful targeting construct for introduction of HLA-DQB1*02:01 used the human sequence from HLA-DQB1*02:01for exon 1 from the initial conserved alanine in the leadersequence, through exons 2 and 3, upstream of the FRT-flankedPGK neomycin resistance cassette in intron 3–4 (Fig. 1B).C57BL/6 Bruce4 embryonic stem (ES) cells were used forinsertion of linearized target constructs by electroporation.Southern blotting was used to screen for correct integration oftarget constructs, and ES cells were injected into blastocyststhat were transferred into pseudopregnant females to generatechimeric offspring. For the first two attempts, the HLA-DQ2.5b-chain tgmicewere generatedfirst, andEScells for humanizationof H2-Aa were generated from animals with confirmed germlinetransmissionof tgb-chain sequences.For thesuccessfulHLA-DQ2.5KI, ES cells from a-chain offspring with germline transmissionwereused for insertionofb-chain targetingconstructs. goGermline(15) technology was implemented for the selection of mice withgermline transmission. Offspring with double KI a- and b-chaincosegregation and germline transmission were bred to B6J-Gt(ROSA)26Sortm1(UbiC2FLPe)Ozg/Ozg flippase-expressing miceto delete the neomycin and hygromycin selection cassettes.

Sequence analysis of nonfunctional HLA-DQ2.5 KI miceSingle-cell suspensions from spleens of HLA-DQ2.5 KI foundermice were prepared by carefully grinding and filtering tissuethrough a 70 mm diameter nylon mesh (BD Biosciences). TotalRNAwas isolated using the RNeasyMini kit (Qiagen) and reversetranscribed intocDNAintwostepsusingamixof 50ng/ml randomhexamers, 20 nM oligoDT, and 1 mM dNTPs and incubation at65°C for 5 min, then placed on ice for at least 1 min, before adding5 mM MgCl2, 10 mM DTT, RNasin (Promega), and SuperscriptIII (Invitrogen) and incubating at 25°C for 10 min, 50°C for

50min, and85°C for5min. cDNAwasamplifiedbyPCR(a-fwd59-GCAGCAGAGCTCTGATTCTG-39, a-rev 59-TGTGGATCAGGGTCTCTGA-39, b-fwd 59-CAGAACTTTGCTTTCTGAAGGG-39,b-rev- 59-GTGCGGCTGTACACAGATG-39). PCR products wereeitherdirectly sequencedor cloned intopGEMEasyVectorSystemI (Promega) cloning vector. Sanger sequencing was performed byEurofins Scientific/GATC using PCR primers for directly se-quenced PCR products or for vector SP6 (59-ATTTAGGTGACACTATAAA-39) and T7 (59-TAATACGACTCACTATAGGG-39).

MiceHomozygous HLA-DQ2.5 KI mice were obtained by crossingheterozygous founders. Resulting litters were genotyped by PCR(H2-IAb fwd 59-TCGTGTACCAGTTCATGGGC-39, H2-IAbrev 59-GTAGTTGTGTCTGCACACCG-39, HLA-DQ2.5 KIb-fwd 59-ACCTGAGGCACCCAGAGGG-39, HLA-DQ2.5 KIb-rev 59-CTCCGGGGATCCGCAAAGCC-39). Non-tg litter-mates from heterozygous breedings were used as wild-type(WT) controls. Experiments were performed on mice of bothsexes indiscriminately. HLA-DQ2.5 tg mice (7) were crossed tohomozygosity, and genotype was confirmed by backcrossing,then maintained as homozygous crossings. HeterozygousHLA-DQ2.5 tgmicewere obtained fromhomozygous crossingsto WT mice. Homozygous and heterozygous HLA-DQ2.5 KImice were interbred with TCR-glia-a2 (16) mice to obtainHLA-DQ2.5 KI TCR-glia double tg mice. Mice were maintainedon gluten-free chow (RDI OpenStandard Diet; Research Diets)and bred under specific-pathogen-free conditions at the De-partment of Comparative Medicine, Oslo University Hospital,Rikshospitalet, Oslo, Norway. All experiments were approved bythe Norwegian Food Safety Authority.

Flow cytometrySingle-cell suspensions from spleens were prepared as describedabove. Bone marrow was flushed from the femur. Erythrocyteswere removed by treatment with ammonium-chloride-potassiumlysis buffer. Cells were resuspended in PBS supplemented with2% (vol/vol) FCS and incubated with optimal amounts of Absagainst B220 (RA3-6B2; BioLegend; Invitrogen), CD3 (145-2C11;BioLegend, eBioscience), CD4 (GK1.5; BioLegend), CD8a (53-6.7;BioLegend), CD11b (M1/70; BioLegend), CD11c (N418; Bio-Legend), CD19 (6D5; BioLegend, 1D3; eBioscience), CD21 (7G6;BDPharmingen), CD23 (B3B4; eBioscience), HLA-DQ2.5 [2.12.E11(17)], IA/IE (M5/114.15.2; eBioscience), IgD (1126c; eBioscience),IgM (11/41; eBioscience), SiglecH (440c; eBioscience), and Vb3/R(Ancell).Forallflowcytometryassays,FcblockagainstmouseCD16/32 (93; BioLegend) was used. Cells were analyzed on FACSCalibur(BD Biosciences) or Attune NxT Flow Cytometer (ThermoFisher Scientific). Data analysis was performed using FlowJosoftware (BD).

T cell proliferation assaysCD4+ T cells were isolated from pooled spleens and lymph nodesfrom HLA-DQ2.5 tg TCR-glia-a2 tg mice by negative selection

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FIGURE 2. H2-IA and HLA-DQ2.5 expression in B cell and dendritic cell subsets in WT, heterozygous HLA-DQ2.5 tg, heterozygous HLA-DQ2.5

KI, and homozygous HLA-DQ2.5 KI mice.

(A) Surface expression of HLA-DQ2.5 and H2-IA in CD19+ B220+ B cells and CD11chi dendritic cells from HLA-DQ2.5 KI and HLA-DQ2.5 tg animals

is different. (B) Representative plots showing H2-IA and HLA-DQ2.5 expression on gated splenic follicular/T2 (B220+ CD19+ CD23+ CD21+) B cells.

(C) H2-IA and HLA-DQ2.5 expression on splenic dendritic cells. Splenic CD192 B2202 CD11chi dendritic cells were stained for CD8a, CD11b, and

SiglecH to identify cDC1 (CD8a+ CD11b2), cDC2 (CD8a2 CD11b+), and pDC (SiglecH+) subsets. (D) H2-IA and HLA-DQ2.5 expression on (Continued)

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(EasySepMouseCD4+Tcell isolationkit; StemCellTechnologies).Purity of the isolated T cells was assessed by flow cytometry andwas typically .80%. T cell purity was taken into considerationwhen calculating T cell numbers. Isolated CD4+ T cells werelabeled with Cell Trace Violet (Molecular Probes). Splenocytesfrom heterozygous and homozygous HLA-DQ2.5 KI and HLA-DQ2.5 tg mice were irradiated at 30 Gy and used as APC. CD4+

T cells (7 3 105 cells/ml, corrected for purity) were coculturedwith irradiated splenocytes (3.3 3 107 cells/ml) and deamidateda2-gluten 33mer (33merEEE) (LQLQPFPQPELPYPQPELPYPQPELPYPQPQPF; Genscript) at indicated concentrations inRPMI 1640 supplementedwith 10% (vol/vol) FCS, 1%penicillin/streptomycin, 1% HEPES, 1% nonessential amino acids,1% sodium-pyruvate, and 100 mM 2-ME for 72 h at 37°C.

RESULTS

Generation of an HLA-DQ2.5 KI mouse strainWe generated HLA-DQ2.5 KI mice that express a chimeric MHCII surface protein in which the extracellular domains derive fromHLA-DQA1*05:01 and HLA-DQB1*02:01. The introduction ofHLA-DQA1*05:01 andHLA-DQB1*02:01 sequences into themousegenome was done by means of homologous recombination withtargeting vectors in two separate and consecutive handlings ofC57BL/6 Bruce4 ES cells.

For introduction ofHLA-DQA1*05:01, exons 2 and 3 ofH2-Aawere replaced by the corresponding HLA-DQA1*05:01 exonsequences, and an FRT-flanked hygromycin selection cassettewas introduced upstream of exon 2 (Fig. 1A). For introduction ofHLA-DQB1*02:01, altogether three attempts were made to obtaina functional KI mouse, the first two being unsuccessful. Inthe successful attempt, H2-Ab1 was humanized from the firstconserved alanine residue in exon 1, and the neomycin selectionmarker was placed in intron 3–4 of H2-Ab1 (Fig. 1B). Surfaceexpression of HLA-DQ2.5 was detected by flow cytometry ofspleen cells from double tg mice (Fig. 1C). The failed attemptsused essentially the same strategy as for HLA-DQA1*05:01 withpositioning of an FRT-flanked neomycin selection cassette intwo different locations upstream of exon 2 (Supplemental Fig. 1A,1B). With this strategy, no surface expression of HLA-DQ2.5 wasobserved indoubleKImice, and improper exon–intron splicingofH2-Ab1/DQB1*02:01was identified as the problem (SupplementalFig. 2).

The generated HLA-DQ2.5 KI mouse expresses a chimerichuman-mouse MHC II molecule with mouse sequences in thetransmembrane and intracellular domains. The extracellular

part of the chimeric b-chain is identical to that of HLA-DQ2.5,and the chimeric a-chain differs from HLA-DQ2.5 at two exon1–encoded residues in the very N-terminal part (KI chimericprotein sequence: EDDIEAD; HLA-DQ2.5 protein sequence:E-DIVAD).

Targeted integration of the transgene is important forphysiological MHC II expressionWe next analyzed expression of HLA-DQ2.5 and H2-IA indifferent cell subsets, comparing HLA-DQ2.5 KI mice withHLA-DQ2.5 tg animals that have had the transgene introducedin an untargeted manner (7). As expected, CD11chi dendritic cellshad higher surface expression levels of H2-IA than B220+ B cells.Importantly, HLA-DQ2.5 expression in HLA-DQ2.5 KI animalsmatched this pattern, whereas in HLA-DQ2.5 tg animals, a lowerrelative expression of HLA-DQ2.5 was seen on dendritic cellscompared with B cells (Fig. 2A).

To further assess expression patterns in our tg andKI animals,we examined splenic APC. B cell subsets defined by CD21/35 andCD23 staining were present in normal proportions and coex-pressed HLA-DQ2.5 and H2-IA in heterozygous HLA-DQ2.5 KIanimals. In theCD21/35+ CD23+ follicular B cell subset fromHLA-DQ2.5 tg animals, we identified a small population of cells thatexpressed only H2-IA (Fig. 2B). Dendritic cell subsets defined bycoexpression of CD11c and CD8, CD11b, or SiglecH likewisecoexpressed HLA-DQ2.5 andH2-IA in heterozygous HLA-DQ2.5KI animals, but not in HLA-DQ2.5 tg animals, in which apopulation of cells in each subset expressed endogenous H2-IAbut not the transgene (Fig. 2C).

The disparate expression of HLA-DQ2.5 and H2-IA asobserved in untargeted tg animals was even more pronouncedduring cellular development. We examined expression of H2-IAand HLA-DQ2.5 during B cell developmental stages in the bonemarrow as identified by IgD and IgM staining (Fig. 2D). In HLA-DQ2.5 tg mice, we found H2-IA surface expression in immatureB cells that did not coexpress HLA-DQ2.5, and a significantproportion of translational B cells displayed the same surfaceMHC II expression patterns. In mature recirculating B cells inthe bone marrow, this population made up a small percentage oftotal B cells, confirming our observations with mature follicularB cells in the spleen. HLA-DQ2.5 KI mice coexpressed surfaceH2-IA and HLA-DQ2.5 at all B cell developmental stages.

We also saw that in HLA-DQ2.5 KI heterozygous animalsthere was lower surface expression of H2-IA compared withWT and HLA-DQ2.5 tg mice (Fig. 2E). HLA-DQ2.5 surfaceexpression was also lower in KI animals compared withuntargeted tg, even when comparing homozygous KI animals

developing B cells in the bone marrow. Representative plots of bone marrow CD19+ B220+ B cells stained with IgD and IgM to identify preB (IgD2 IgM2),

immature (IgD2 IgM+), transitional (IgDlo IgM+), and mature recirculating (IgDhi, IgM+) B cell developmental subsets. Cells stained with anti-IA/IE and

anti–HLA-DQ2.5 show differential expression of H2-IA and HLA-DQ2.5 between assayed genotypes. (E) Surface expression of H2-IA and HLA-DQ2.5 in

CD19+, B220+ splenic B cells compared between WT, HLA-DQ2.5 tg, and HLA-DQ2.5 KI mice. All plots are representative of at least three independent

experiments (n = 1 mouse per experiment).

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with heterozygous untargeted tg. Untargeted tg mice usuallycarry an unknown number of transgene inserts, which likelyaccounts for these observations.

Taken together, our data imply nonnatural expression of thetransgene in HLA-DQ2.5 tg animals in several cell types andappears to be most marked in early developmental stages. Thissuggests that the use of the endogenous mouse promotor for ourchimeric HLA-DQ2.5 KI protein ensures normal cell surfaceexpression patterns of the gene product.

HLA-DQ2.5 KI is functional for T cell presentationTo evaluate whether knock-in HLA-DQ2.5 was functional andable to present Ag to CD4+ T cells, we took advantage of anHLA-DQ2.5-restricted gluten-specific TCR tg mouse strainthat recognizes the DQ2.5-glia-a2 epitope (16), which wecrossed to HLA-DQ2.5 KI mice to obtain TCR-glia-a2 tg HLA-DQ2.5 KI heterozygous or homozygous animals. CD4+ T cellswere present in the spleens of these and non-TCR tg HLA-DQ2.5 KI animals, showing that KI HLA-DQ2.5 is able tosupport T cell differentiation (Fig. 3A). Splenic cellularityremained consistent across genotpyes, but we observed adecrease in the frequency of CD4+ T cells in the spleens ofHLA-DQ2.5 KI homozygous animals compared with hetero-zygous animals, which may suggest the absence of a populationof CD4+ T cells that are selected on H2-IA.

We isolated gluten-specific CD4+ T cells from HLA-DQ2.5TCR-glia-a2 double tg mice and cocultured them with irradiatedsplenocytes from WT, HLA-DQ2.5 KI heterozygous, or HLA-DQ2.5 KI homozygous mice and cognate gluten peptides for72 h. We found that gluten-specific CD4+ T cells proliferatewhen cocultured with HLA-DQ2.5+ APCs but not withWT APCs(Fig. 3B).

We compared gluten-specific CD4+ T cell proliferation after 72 hof coculture with APCs that were heterozygous or homozygousfor the targeted HLA-DQ2.5 KI or for the above-mentioneduntargeted HLA-DQ2.5 tg and found no difference in T cellproliferation betweenCD4+T cells culturedwith differentHLA-DQ2.5 APCs (Fig. 3C). Of note, in vivo proliferation of adoptivelytransferred gluten-specific TCR tg CD4+ T cells in response toorally administered deamidated gluten digest has been observedin HLA-DQ2.5 KI mice (18).

FIGURE 3. KI HLA-DQ2.5 is functional for T cell development and

gluten peptide presentation.

(A) CD4+ T cells are present in spleens of HLA-DQ2.5 KI heterozygous

and HLA-DQ2.5 KI homozygous mice. Flow cytometric analysis of

spleen cells from HLA-DQ2.5 KI mice stained with Abs recognizing

CD4. Data are mean 6 SEM (n = 3) and representative of at least two

independent experiments. *p , 0.05, ***p , 0.0005 as determined by

Student t test. (B) In vitro functionality of HLA-DQ2.5 KI protein. CD4+

T cells isolated from spleens and lymph nodes of HLA-DQ2.5 KI TCR-

glia-a2 tg mice were labeled with Cell Trace Violet (CTV) proliferation

tracking dye and cultured with irradiated splenocytes from WT, hetero-

zygous, or homozygous HLA-DQ2.5 KI mice and indicated concentrations

of the deamidated 33mer gluten peptide (33merEEE: LQLQPFPQPEL

PYPQPELPYPQPELPYPQPQPF). The percentage of CD3+ CD4+ T cells

that had proliferated at least once after 72 h of culture is shown. Data

are mean 6 SEM (n = 2) and are representative of more than three in-

dependent experiments. (C) Induction of proliferation of gluten-specific

CD4+ T cells did not differ between APCs from targeted HLA-DQ2.5 KI

and untargeted HLA-DQ2.5 tg mice. CTV-labeled CD4+ T cells were

cocultured with irradiated splenocytes as described in (A) in the presence

of 0.1 mM 33merEEE peptide. Data are pooled from three independent

experiments. Bars represent mean 6 95% confidence interval.

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DISCUSSION

Most autoimmune disorders display associations to certain HLAallotypes. MHC II molecules play important roles in CD4+ T cellselection in the thymus and in Ag presentation to mature cells inthe periphery. To study the involvement of human disease-associated HLA allotypes in disease processes in a humanizedmouse, tg introduced humanMHC II should optimally be presentand operate in a physiological way. In this project, our aimwas tocreate a physiological HLA-DQ2.5 tg mouse by employing KItechnology.

For the generation of tg mice, targeted gene KI has severalbenefits over untargeted transgenes. Untargeted gene insertioncarries a risk of off-target effects, like silencing of genes in whichthe transgene might have inserted (19, 20), large-scale chromo-somal rearrangements (21, 22), or effects on surrounding genes(23). Untargeted tg organisms also have an unknown gene copynumber, which can affect studies in which gene dose is important.Targeted gene insertion avoids these issues and utilizes naturalmouse promoter/enhancer gene regulation, resulting in physio-logical expression of the transgene. When comparing our novelHLA-DQ2.5 KI mouse to the existing HLA-DQ2.5 tg mouse, weobserved some of these effects. We observed differences inquantitative expression of the transgene between the two mice,most likely due to the integration of multiple copies of thetransgene into the genome of the untargeted tgmice. Interestingly,we also observed qualitative differences in the expression of H2-IAand HLA-DQ2.5 in the untargeted tg mice, which importantlywere not observed in the KI mice. The differences in expressionof HLA-DQ2.5 and H2-IA were particularly notable for B cellsduring their maturation in the bonemarrow. Althoughwe did notobserve any marked differences related to HLA-DQ2.5 functionbetween the untargeted tg andKI animals in the settingswe tested,such differences may well occur in more complex autoimmunedisease models. Thus, we believe that the HLA-DQ2.5 KI miceoffer advantages over the untargeted HLA-DQ2.5 tg mice. Inaddition to providingmicewith physiological expression of theintroduced transgene, the KImice have the advantage of avoidingcrossing to MHC II null mice when breeding the HLA tg miceonto genetically modified mouse strains, which may be requiredfor dissection of immune mechanisms.

In C57BL/6mice, there is no expression of theH2-IE a-chain,leaving the H2-IE b-chain free to potentially pair with the H2-IAa-chain, or in this KI model, the HLA-DQ2.5 a-chain (12). Giventhe differences between the intracellular and transmembraneregions of both the DQa- and the DQb-chains and a two residuedifference in the extracellular region of the DQa-chain in theHLA-DQ2.5 KI and HLA-DQ2.5 tg mice, it is theoreticallyconceivable that there would be disparate interactions of thetwo distinct DQa-chains with H2-IEb. However, we find thispossibility unlikely because whereas MHC II ab-chain pairingpreference is dictated by sequence variation in the extracellularN-terminal domains (24), the veryN-terminal part of the a-chainis not a region known to affect chain pairing (25). We find it evenless likely that the structural differences between HLA-DQ2.5

and the chimeric KI protein are responsible for the observeddifferences in MHC II expression between the HLA-DQ2.5 KIand HLA-DQ2.5 tg animals.

Potential trans-species effects on molecular interactions be-tween the introduced human molecules and endogenous mousemolecules like CD4 and the invariant chain (CD74) are a generalchallenge faced by MHC II tg. Previous in vivo studies on theinteraction between mouse CD4 and HLA-DQ or HLA-DRtransgenes have shown that TCR tg T cell selection proceedswithout requiring species-matched CD4, but less efficiently (26,27). In this study, we observed positive selection of HLA-DQ2.5–restricted gluten-specific TCR tg T cells, which also madevigorous responses in the periphery when stimulated with Ag. Aswe have not made a side-by-side comparison of these T cells withand without human CD4, we are unable to conclude whether thelack of human CD4 in our system leads to inferior T cell selectionor peripheral responses.

In the generation of theHLA-DQ2.5 KImice, the introductionof HLA-DQB1*02:01 was not straightforward. The two firstattempts were unsuccessful because of improper exon–intronsplicing of the KI gene. We have no obvious explanation for thisfaulty behavior, as the two initial designs of the targeting constructfor HLA-DQB1*02:01 were very similar to that of HLA-DQA1*05:01, and the DQA1 construct was working properly.

Taken together, we present the making of a novel HLA-DQ2.5KImouse,which shouldbeuseful for the studyof thepathogenesisof CeD and other HLA-DQ2.5–associated disorders.

DISCLOSURES

F.K. is the Chief Executive Officer of Ozgene Pty. Ltd., whichprovided KO mice under commercial contracts with OsloUniversity Hospital and the University of Oslo. The other authorshave no financial conflicts of interest.

ACKNOWLEDGMENTS

We are grateful to Liv Kleppa for technical assistance and the staff of theDepartment of Comparative Medicine, Oslo University Hospital for animalhusbandry. We thank Lars Fugger for sharing the HLA-DQ2.5 tg mouse thatwas generated as part of the EU-Biomed 2 project (BMH4-CT98-3087).

REFERENCES

1. Lechler, R., and A. N. Warrens. 2000. HLA in Health and Disease.Academic Press, San Diego, CA.

2. Price, P., C. Witt, R. Allcock, D. Sayer, M. Garlepp, C. C. Kok, M. French,S. Mallal, and F. Christiansen. 1999. The genetic basis for the associationof the 8.1 ancestral haplotype (A1, B8, DR3) with multiple immuno-pathological diseases. Immunol. Rev. 167: 257–274.

3. Sollid, L. M. 2000. Molecular basis of celiac disease. Annu. Rev.Immunol. 18: 53–81.

4. Sollid, L. M., W. Pos, and K. W. Wucherpfennig. 2014. Molecularmechanisms for contribution of MHC molecules to autoimmunediseases. Curr. Opin. Immunol. 31: 24–30.

https://doi.org/10.4049/immunohorizons.2000107

ImmunoHorizons GENERATION OF AN HLA-DQ2.5 KNOCK-IN MOUSE 31

by guest on February 4, 2022http://w

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5. Black, K. E., J. A. Murray, and C. S. David. 2002. HLA-DQ determinesthe response to exogenous wheat proteins: a model of gluten sensi-tivity in transgenic knockout mice. J. Immunol. 169: 5595–5600.

6. Chen, Z., N. Dudek, O. Wijburg, R. Strugnell, L. Brown, G. Deliyannis,D. Jackson, F. Koentgen, T. Gordon, and J. McCluskey. 2002. A 320-kilobase artificial chromosome encoding the human HLA DR3-DQ2MHC haplotype confers HLA restriction in transgenic mice. J.Immunol. 168: 3050–3056.

7. Du Pré, M. F., A. E. Kozijn, L. A. van Berkel, M. N. ter Borg,D. Lindenbergh-Kortleve, L. T. Jensen, Y. Kooy-Winkelaar, F. Koning,L. Boon, E. E. Nieuwenhuis, et al. 2011. Tolerance to ingesteddeamidated gliadin in mice is maintained by splenic, type 1 regulatoryT cells. Gastroenterology 141: 610–620, 620.e1–e2.

8. Madsen, L. S., E. C. Andersson, L. Jansson, M. krogsgaard,C. B. Andersen, J. Engberg, J. L. Strominger, A. Svejgaard, J. P. Hjorth,R. Holmdahl, et al. 1999. A humanized model for multiple sclerosisusing HLA-DR2 and a human T-cell receptor. Nat. Genet. 23: 343–347.

9. Rashtak, S., E. Marietta, S. Cheng, M. Camilleri, M. Pittelkow, C. David,J. Grande, and J. Murray. 2010. Spontaneous lupus-like syndrome inHLA-DQ2 transgenic mice with a mixed genetic background. Lupus 19:815–829.

10. Rijkers, T., A. Peetz, and U. Ruther. 1994. Insertional mutagenesis intransgenic mice. Transgenic Res. 3: 203–215.

11. Garrick, D., S. Fiering, D. I. Martin, and E. Whitelaw. 1998. Repeat-induced gene silencing in mammals. Nat. Genet. 18: 56–59.

12. Mathis, D. J., C. Benoist, V. E. Williams II., M. Kanter, and H. O.McDevitt. 1983. Several mechanisms can account for defective E alphagene expression in different mouse haplotypes. Proc. Natl. Acad. Sci.USA 80: 273–277.

13. Stewart, C. A., R. Horton, R. J. Allcock, J. L. Ashurst, A. M. Atrazhev,P. Coggill, I. Dunham, S. Forbes, K. Halls, J. M. Howson, et al. 2004.Complete MHC haplotype sequencing for common disease genemapping. Genome Res. 14: 1176–1187.

14. Osoegawa, K., M. Tateno, P. Y. Woon, E. Frengen, A. G. Mammoser,J. J. Catanese, Y. Hayashizaki, and P. J. de Jong. 2000. Bacterialartificial chromosome libraries for mouse sequencing and functionalanalysis. Genome Res. 10: 116–128.

15. Koentgen, F., J. Lin, M. Katidou, I. Chang, M. Khan, J. Watts, andP. Mombaerts. 2016. Exclusive transmission of the embryonic stemcell-derived genome through the mouse germline. Genesis 54: 326–333.

16. du Pré, M. F., J. Blazevski, A. E. Dewan, J. Stamnaes, C. Kanduri,G. K. Sandve, M. K. Johannesen, C. B. Lindstad, K. Hnida, L. Fugger, et al.

2020. B cell tolerance and antibody production to the celiac diseaseautoantigen transglutaminase 2. J. Exp. Med. 217: e20190860.

17. Viken, H. D., G. Paulsen, L. M. Sollid, K. E. Lundin, G. E. Tjønnfjord,E. Thorsby, and G. Gaudernack. 1995. Characterization of an HLA-DQ2-specific monoclonal antibody. Influence of amino acid substitu-tions in DQ beta 1*0202. Hum. Immunol. 42: 319–327.

18. Lindstad, C. B., S. W. Qiao, M. K. Johannesen, L. Fugger, L. M. Sollid,and M. F. du Pre. 2021. Characterization of T-cell receptor transgenicmice recognizing immunodominant HLA-DQ2.5-restricted glutenepitopes. Eur. J. Immunol. DOI: 10.1002/eji.202048859.

19. Krakowsky, J. M., R. E. Boissy, J. C. Neumann, and J. B. Lingrel. 1993.A DNA insertional mutation results in microphthalmia in transgenicmice. Transgenic Res. 2: 14–20.

20. McNeish, J. D., W. J. Scott Jr., and S. S. Potter. 1988. Legless, a novelmutation found in PHT1-1 transgenic mice. Science 241: 837–839.

21. Covarrubias, L., Y. Nishida, M. Terao, P. D’Eustachio, and B. Mintz.1987. Cellular DNA rearrangements and early developmental arrestcaused by DNA insertion in transgenic mouse embryos.Mol. Cell. Biol.7: 2243–2247.

22. Mahon, K. A., P. A. Overbeek, and H. Westphal. 1988. Prenatal le-thality in a transgenic mouse line is the result of a chromosomaltranslocation. Proc. Natl. Acad. Sci. USA 85: 1165–1168.

23. Olson, E. N., H. H. Arnold, P. W. Rigby, and B. J. Wold. 1996. Knowyour neighbors: three phenotypes in null mutants of the myogenicbHLH gene MRF4. Cell 85: 1–4.

24. Sant, A. J., N. S. Braunstein, and R. N. Germain. 1987. Predominantrole of amino-terminal sequences in dictating efficiency of class IImajor histocompatibility complex alpha beta dimer expression. Proc.Natl. Acad. Sci. USA 84: 8065–8069.

25. Braunstein, N. S., R. N. Germain, K. Loney, and N. Berkowitz. 1990.Structurally interdependent and independent regions of allelic poly-morphism in class II MHC molecules. Implications for Ia functionand evolution. J. Immunol. 145: 1635–1645.

26. Altmann, D. M., D. C. Douek, A. J. Frater, C. M. Hetherington,H. Inoko, and J. I. Elliott. 1995. The T cell response of HLA-DR trans-genic mice to human myelin basic protein and other antigens in thepresence and absence of human CD4. J. Exp. Med. 181: 867–875.

27. Yamamoto, K., Y. Fukui, Y. Esaki, T. Inamitsu, T. Sudo, K. Yamane,N. Kamikawaji, A. Kimura, and T. Sasazuki. 1994. Functional interactionbetween human histocompatibility leukocyte antigen (HLA) class IIand mouse CD4 molecule in antigen recognition by T cells in HLA-DRand DQ transgenic mice. J. Exp. Med. 180: 165–171.

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