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PresentationChain-Independent Role for H2-M in Antigen Cutting Edge: A Critical, Invariant

Martin, Luc Van Kaer and Steven L. ReinerKevin Swier, Daniel R. Brown2, Jennifer J. Bird, W. David

http://www.jimmunol.org/content/160/2/5401998; 160:540-544; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/160/2/540.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists All rights reserved.Copyright © 1998 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Cutting Edge: A Critical, Invariant Chain-Independent Role for H2-M in AntigenPresentation1

Kevin Swier,2*‡ Daniel R. Brown,2†‡ Jennifer J. Bird,*‡

W. David Martin,3§ Luc Van Kaer,§ and Steven L. Reiner4*†‡

Antigen presentation by MHC class II (class II) is facilitated bythe accessory molecules, invariant chain (Ii) and H2-M. Ii as-sociates with class II during biosynthesis and promotes trans-port of class II to Ag-loading compartments. One function ofH2-M is the removal of Ii fragments from MHC class II. Wehave previously demonstrated that Ii-deficient mice, unlikeclass II-deficient mice, are resistant to L. major infection. Inthe present study, we found that H2-M-deficient (H2-M0)mice were susceptible to progressive infection with L. major.The dispensability of Ii for control of L. major allowed geneticanalysis of whether H2-M functions by association with orindependently of Ii. In contrast to Ii-deficient (Ii0) mice,Ii0H2-M0 mice were as susceptible to L. major as H2-M0

mice. Thus, H2-M has an essential, Ii-independent functionduring presentation of microbial pathogens. The Journal ofImmunology, 1998, 160: 540–544.

T he generation of a CD41 T helper cell repertoire and ex-pansion of Ag-specific Th cells during infection requirepresentation of peptides by MHC class II (class II)5. Ii and

H2-M (HLA-DM in humans) are two accessory molecules neces-sary for efficient expression of peptide-bound class II molecules. Iiassociates with class II in the ER and directs class II to endosomalcompartments, where Ii is proteolytically cleaved (1). The finalportion of Ii to remain associated with class II is called CLIP (classII-associated invariant chain peptide), which occupies the peptide-binding groove (2). H2-M catalyzes the release of CLIP from pu-rified class II molecules (3–5). APCs from cell lines lacking

HLA-DM (6, 7) or from mice lacking H2-M (8–10) contain classII that is predominantly associated with CLIP. In vitro studies sug-gest H2-M can additionally function to stabilize empty class II (11,12) and remove suboptimal peptides (13–16).

Infection of inbred strains of mice withLeishmaniamajor is awell-established model for examining class II function.Leishma-nia invade macrophages and replicate within endosomal compart-ments that contain class II (17). Control of infection depends uponproduction of IFN-g by class II-restricted Th1 cells that activatemacrophages to a microbicidal state (18). Class II-deficient (classII0) mice are completely susceptible to infection (19–21), whereasMHC class I-deficient mice control infection (21, 22). Ii0 micehave reduced numbers of CD41 T cells, reduced class II expres-sion on APCs, and inefficient presentation of Ags in vitro and invivo (23–26). Despite their impaired ability to present parasiteAgs, Ii0 mice are highly resistant to infection withL. major (21).In the present study, we have used microbial immunity to definethe role of H2-M. By generating Ii0H2-M0 mice we have geneti-cally demonstrated an essential, Ii-independent role for H2-Min vivo.

Materials and Methods

Mice

H2-M0 mice (8) and TAP-1-deficient mice (27) (from aLeishmania-resis-tant C57BL/6X129 background) have been previously described. C57BL/6Class II0 (28) mice and Ii0 mice (24) were generously provided by DianeMathis and Christophe Benoist (INSERM, France). H2-M0 mice (H-2b)were mated with Ii0 mice (H-2d congenic) to produce double heterozygous(Ii1/2H2-M1/2, H-2dxb) mice. Double heterozygotes were backcrossed toparental Ii0 mice. Transmission of the mutant H2-M allele was detected bystaining peripheral blood lymphocytes using a mAb specific for the closelylinked Kb molecule (Caltag Laboratories, South San Francisco, CA) whileIi1/2H2-M1/2 and Ii0H2-M1/2 littermates were distinguished by levels ofMHC class II. Ii0H2-M1/2 mice were intercrossed and Ii0H2-M0 micewere identified by homozygosity for Kb. Wild-type C57BL/6 and BALB/cmice were obtained from The Jackson Laboratory (Bar Harbor, ME). Allmice were maintained in a specific pathogen-free environment before in-fection. All work was performed in accordance with the University ofChicago guidelines for animal use and care.

Syngeneic mixed lymphocyte reaction

CD41 T cells from lymph nodes were enriched by depleting B2201 andCD81 cells with mAbs and magnetic beads (PerSeptive Biosystems, Cam-bridge, MA). Enriched cells (1.53 105) were cultured in 200ml ofIscove’s complete medium with 53 105 irradiated splenocytes (2500 rads)from wild-type C57BL/6 mice or on plates coated with anti-CD3 mAb (5mg/ml). After 3 days, 1mCi of methyl-[3H]thymidine was added, and in-corporated radioactivity was measured 18 h later using a Betaplate 1205counter (Wallac, Turku, Finland).

*Department of Medicine, †Committee on Immunology, ‡Gwen Knapp Centerfor Lupus and Immunology Research, University of Chicago, Chicago, Illinois60637; §Howard Hughes Medical Institute, Department of Microbiology andImmunology, Vanderbilt University School of Medicine, Nashville, Tennessee372321 D.R.B. was supported by the University of Chicago Medical Scientist TrainingProgram and Immunology Training Grant (AI-07090). W.D.M. is an Associateand L.V.K. is an Assistant Investigator of the Howard Hughes Medical Institute.S.L.R. is supported by the Burroughs Wellcome Fund and the National Institutesof Health (AI-01309).2 These authors contributed equally to this work3 Present address: Purdue University, West Layfayette, Indiana, 47907.4 Address correspondence and reprint requests to Steven L. Reiner, Gwen KnappCenter, University of Chicago, 924 E. 57th Street, JFK R420, Chicago, IL 60637–5420. E-mail address: sreiner@midway.uchicago.edu5 Abbreviations used in this paper: Class II, MHC class II; Ii, Invariant chain; Ii0,invariant chain-deficient; H2-M0, H2-M-deficient; CLIP, class II-associated in-variant chain peptide; class II0, MHC class II-deficient.

Copyright © 1998 by The American Association of Immunologists 0022-1767/98/$02.00

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Flow cytometry

Single cell suspensions of lymph nodes were stained with designated mAbsspecific for MHC class II I-Ab (25–9–17-FITC) and CD44 (IM7-PE)(PharMingen, San Diego, CA) as well as B220 (RA3–6B2-PE), CD4 (CT-CD4-PE), CD8 (CT-CD8a-TC), and TCR-ab (H57–597-FITC) (CaltagLaboratories). Light scatter properties were used to gate on lymphocytes.

Leishmania infection

Leishmania major(WHOM/IR/-/173) metacyclic promastigotes (53 105)were injected into each hind footpad. Footpad diameter was measuredweekly with a metric caliper. At termination of infection, parasite burdensin feet and spleens were determined as previously described (21).

Assay of Ag-specific cytokine production

Five 3 105 popliteal lymph node cells were incubated with or withoutsoluble extracts from freeze-thawedL. majorpromastigotes (100mg/ml) inround-bottom 96-well plates. Designated cultures were supplemented with1 3 106 irradiated C57BL/6 splenocytes as a source of APCs. IFN-g wasmeasured by ELISA (PharMingen) from supernatants collected at 48 h.

Competitive RT-PCR analysis

RNA was extracted with Trizol Reagent (Life Technologies, Gaithersburg,MD) from unfractionated popliteal lymph node cells or purified CD41 Tcells from mice infected withL. major. RNA was reverse transcribed usingrandom hexamer primers (Pharmacia, Piscataway, NJ) for analysis by com-petitive PCR as previously described (29). In brief, a polycompetitor con-struct containing addition-mutations of authentic cDNA was amplified in

the same reaction as the experimental cDNA. When resolved on an agarosegel, the larger m.w. product served as an internal standard for comparisonof the relative amounts of lower m.w. experimental cDNA between groups.Amplification for the housekeeping gene, hypoxanthine-guanine phospho-ribosyl transferase (HPRT), was performed to confirm that the input cDNAwas equivalent between groups.

Results and DiscussionH2-M0 mice are susceptible to L. major infection

Control ofL. major infection requires MHC class II-restricted re-sponses (19–21). To determine the requirement for H2-M in theprocessing and presentation of parasite Ags, H2-M0 mice wereinfected withL. major. The course of disease was compared withclass II0 mice, Ii0 mice, genetically susceptible BALB/c mice, andgenetically resistant C57BL/6 mice. Extensive footpad lesiongrowth (greater than 4 mm) occurred between 4 to 6 wk in allBALB/c mice and class II0 mice (Fig. 1A). The onset of lesiongrowth was variable in H2-M0 mice, with some animals maintain-ing a footpad size less than 4 mm for several weeks longer thanBALB/c and class II0 mice (Fig. 1A). By 17 wk, however, allH2-M0 mice had developed large non-healing footpad lesions (Fig.1A). By contrast, no C57BL/6 wild-type or Ii0 mice developedlesions larger than 4 mm (Fig. 1A). Infected mice were killed at 5wk and 9 wk postinfection for quantitation of parasite burdens. Atthe earlier time point, cultures from feet and spleens revealed

FIGURE 1. H2-M0 mice are susceptible to L. major infection. A, Lesion development in mice infected with L. major. Ii0, H2-M0, MHC class II0

(CII0), BALB/c, and C57BL/6 mice were infected as described in Materials and Methods. The percentage of mice in each group with mean hindfootpad diameters less than 4 mm is depicted over time. Results are from 5 Ii0, 14 H2-M0, 3 class II0, 13 BALB/c, and 9 C57BL/6 mice infectedin six separate experiments. B, Parasite burdens of infected mice. Culture results from feet and spleens of mice infected for indicated amount oftime are depicted as numbers of parasites per organ from individual animals on a log10 scale. At 5 wk postinfection, 4 H2-M0, 1 class II0, and 1C57BL/6 (B6) mice were analyzed. At 9 wk postinfection, 5 H2-M0, 2 class II0, and 2 C57BL/6 mice were analyzed. C, Assessment of Th1 responsesin mice infected with L. major. Five weeks after infection, draining lymph node cells from wild-type C57BL/6 mice, infected H2-M0 mice, anduninfected H2-M0 (naive H2-M0) mice were cultured for 48 h with no additions (No Ag), L. major Ags (Ag), wild-type APCs (WT APCs), orwild-type APCs plus L. major Ags (WT APCs 1 Ag). IFN-g was measured from supernatants by ELISA after 48 h. Bars represent mean of triplicatecultures for individual animals with SDs as y-axis error bars. Results are representative of four separate experiments. D, Competitive PCR analysisof draining lymph node cells. RNA from unfractionated (total) or CD41 T cells (CD41) of draining lymph nodes of individual infected wild-typeC57BL/6 (WT), and H2-M0 mice was subjected to competitive RT-PCR as described in Materials and Methods. Upper bands correspond toamplification of competitor molecule while lower bands correspond to amplification of experimental cDNA. Amplification of HPRT was done toconfirm equivalent amounts of cDNA were used for amplification. Results are representative of three experiments using 4 C57BL/6 and 5 H2-M0

mice.

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higher parasite loads in class II0 mice compared with H2-M0 andC57BL/6 mice (Fig. 1B). At the later time point, however, H2-M0

mice had parasite loads comparable to class II0 mice (Fig. 1B).Thus, H2-M, unlike Ii, is required for efficient control ofL. majorinfection.

To determine whetherL. major-specific Th1 responses werepresent in infected H2-M0 mice, we measured IFN-g produced byT cells restimulated in vitro. Draining popliteal lymph node cellsfrom mice killed after 5 wk were cultured with or withoutL. majorAgs. Cells from H2-M0 mice cultured with parasite Ags producedvery little IFN-g (Fig. 1C). When wild-type APCs were added toaugment Ag presentation, cells from H2-M0 mice produced IFN-g(Fig. 1C). Although part of the response from the cells of H2-M0

mice was due to reactivity to syngeneic APCs (8–10 and Fig. 1C),a small but detectable portion of the response wasLeishmaniaAg-specific. The Ag-specific portion of the response was notpresent in previously uninfected H2-M0 mice (Fig. 1C). Additionof anti-class II Abs to cultures blocked all production of IFN-g,(data not shown) suggesting that the response was derived fromhelper T cells. Analyses of numerous mice over various timepoints revealed an inverse relationship between footpad size andIFN-g production. To assess the Th1 response directly ex vivo weperformed competitive RT-PCR on the lymph node cells of in-fected mice. H2-M0 mice produced discernibly less IFN-g tran-scripts than wild-type C57BL/6 mice (Fig. 1D). The IFN-g mRNAwas, however, enriched by the selection of CD41 T cells from bothwild-type and H2-M0 mice. Together, these results demonstratethat H2-M0 mice generate weak Ag-specific Th1 responses toL.major that cannot sustain control of the infectious challenge.

Why is the immune response of H2-M0 mice insufficient to con-trol infection? It is possible that the weak response cannot keeppace with the replicative capacity of the parasite because of aninability to generate sufficient IFN-g to activate infected macro-phages. Alternatively, H2-M0 APCs may present altered or insuf-ficient peptide/MHC complexes that either tolerize or fail to reac-tivate Ag-specific T cells. Finally, APCs may vary in theirdependence on H2-M for Ag presentation. Thus, the Th1 responseobserved in H2-M0 mice may be stimulated by a subset of H2-M-independent APCs while a subset of H2-M-dependent APCs sup-ports parasite growth due to an inability to redirect macrophage-activating T cells.

Analysis of lymphocytes from Ii0H2-M0 mice

The inability of H2-M0 mice to control infection withL. majormay result from a failure to remove CLIP from the binding cleft ofclass II. To analyze the function of H2-M in the absence of CLIP,we generated Ii0H2-M0 mice. We first examined the surface phe-notype of lymphocytes from mice lacking Ii and/or H2-M. Stainingwith anti-Ab mAb, 25–9–17, showed reduced levels of MHC classII on B cells from H2-M0 mice (Fig. 2). This was likely due to thesensitivity of the Ab to detect conformational changes since manyanti-class II mAbs stain H2-M1 and H2-M0 B cells equally well,while conformation-sensitive reagents stain H2-M0 B cells lessefficiently (8–10). We observed a characteristic reduction in classII staining on Ii0 cells (23–25 and Fig. 2) and a slightly greaterdefect in class II staining on the B cells of Ii0H2-M0 mice (Fig. 2).H2-M0 mice and Ii0 mice both have decreased numbers of periph-eral CD41 T cells (8–10, 23–26, and Fig. 2). Ii0H2-M0 mice,however, had a greater reduction in CD41 T cells than Ii0 mice(Fig. 2). CD41 T cells from Ii0 mice have an abnormal surfacephenotype (26), which has been attributed to inefficient positiveselection (30). The defect is characterized by low levels ofTCR-ab and high levels of CD44 expression. CD41 T cells fromIi0H2-M0 and Ii0 mice had similar abnormalities inab and CD44expression (Fig. 2).

The absence of H2-M markedly alters the specificity of CD41 Tcells. The predominant expression of CLIP-bearing MHC class IImolecules in the thymi of H2-M0 mice prevents the negative se-lection of T cells responsive to the normal array of self-peptidesdisplayed on syngeneic wild-type APCs (8–10). To test whetherthe reactivity of T cells from H2-M0 mice to syngeneic APCs isdue to selection on a monomorphic ligand (CLIP/Ab), we mea-sured the proliferative response of CD41 T cells from Ii0H2-M0

mice cultured with syngeneic wild-type APCs. Consistent withpublished results, T cells from H2-M0 mice proliferated strongly inresponse to syngeneic APCs from C57BL/6 mice (Fig. 3). By con-trast, CD41 T cells from Ii0H2-M0 mice, like those from wild-typemice (Fig. 3) and Ii0 mice (data not shown), did not proliferate inresponse to wild-type APCs. T cells from Ii0H2-M0 mice, how-ever, proliferated vigorously in response to ligation with anti-CD3(Fig. 3), suggesting their functional competence to respond toTCR-mediated stimuli. Thus, the absence of CLIP can correct the

FIGURE 2. Surface phenotype of lymphocytesfrom Ii0H2-M0 mice. Lymph node cells fromC57BL/6 (WT), H2-M0, Ii0, and Ii0H2-M0 micewere stained with fluorescence-conjugated mAbsand analyzed by flow cytometry. MHC class IIstaining (far left column) was performed on B2202

(thin line) and B2201 (thick line) lymphocytes. Themean fluorescence intensities of class II staining onB2201 cells were: WT, 39; H2-M0, 17.3; Ii0, 14.6;and Ii0H2-M0, 10.5. Lymphocytes were stainedwith mAbs specific for CD4 and CD8 (2nd columnfrom left). Numbers next to the boxes indicate thepercentage of CD41 and CD81 T cells. The expres-sion of TCR-ab (3rd column from left) and CD44(far right column) was examined among gatedCD41 T cells. Results are representative of threeseparate experiments.

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abnormal reactivity of T cells from H2-M0 mice, confirming anIi-dependent role of H2-M.

Ii0H2-M0 mice are susceptible to L. major infection

Ii 0 mice are resistant toL. major infection (Fig. 1A and Ref. 21)while H2-M0 mice are susceptible to infection (Fig. 1). If the majorfunction of H2-M is the removal of CLIP, then the absence of Iishould rescue resistance in H2-M0 mice. To test this hypothesis,we studied the response toL. major in H2-M0 mice that lack Ii. Ii0

mice were fully resistant to infection (Fig. 4A). This was not dueto a compensatory contribution from class I-restricted CD81 Tcells since Ii0TAP0 mice were equally resistant to infection (Fig.4A). Ii0H2-M0 mice, by contrast, developed progressive footpadlesions in response toL. major infection (Fig. 4A). The onset oflesion development was somewhat variable in Ii0H2-M0 mice (Fig.4A) and closely resembled the course of infection in H2-M0 micethat were infected simultaneously (Fig. 1Aand data not shown).Cultures of feet and spleens of Ii0H2-M0 mice confirmed extensivelocal growth and visceral dissemination of the parasite, respec-tively (data not shown). Thus, H2-M is required for control ofL.major even when class II is not occupied by Ii-derived peptides.

The later onset of susceptibility in Ii0H2-M0 mice comparedwith MHC class II0 mice prompted us to test whether Th1 re-sponses were primed in the absence of both Ii and H2-M. Lymphnode cells from Ii0H2-M0 mice yielded low levels of IFN-g whenrestimulated with parasite Ags, and this weak Th1 response wascompletely inhibited by the addition of anti-class II Abs (Fig. 4B).Some spontaneous production of IFN-g was observed from thecells of Ii0H2-M0 and wild-type mice, which was most likely dueto carry-over ofL. major Ags in the lymph node APCs. Ii0H2-M0

mice also had detectable IFN-g transcripts in the draining lymphnode, as assessed by competitive RT-PCR, although the levelswere lower than those of Ii0 mice (Fig. 4C). Thus, in the absenceof both Ii and H2-M, class II can still gain some access to endo-somal compartments and bind antigenic peptides, but this limitedfunction is insufficient for anti-parasitic immunity.

The susceptibility of H2-M0 and Ii0H2-M0 to L. major mayresult from an inability to present parasite Ags during infection orfrom an inability to generate normal T cells during thymic selec-tion. In the absence of H2-M, negative selection is altered such thatT cells are reactive to self-peptides presented by wild-type synge-neic APCs. This is not likely the reason H2-M0 mice are suscep-tible to L. major since susceptible Ii0H2-M0 mice are tolerant towild-type syngeneic APCs (Fig. 3). It is also unlikely that thereduced number of CD41 T cells in H2-M0 or Ii0H2-M0 miceresults in susceptibility since the resistance of Ii0 mice is unim-

peded by a relatively comparable reduction in the number of CD41

T cells (Fig. 2). We, therefore, favor the explanation that H2-M0

and Ii0H2-M0 mice are susceptible to infection due to inefficientAg presentation rather than abnormal T cell development. We arecurrently generating chimeric mice containing T cells from wild-type mice and APCs from Ii0H2-M0 mice to test this hypothesis.

Previous studies have revealed important functions for Ii andH2-M, two cofactors that have been evolutionarily conserved topotentiate class II function. One of the major functions of H2-M isthe removal of CLIP peptides from class II molecules. By gener-ating Ii0H2-M0 mice, we now show that there is an additional roleor at least a broader specificity for H2-M in vivo. In the absence ofIi, H2-M may enhance the presentation ofL. major Ags by stabi-lizing empty class II molecules (11, 12). In addition, H2-M may

FIGURE 3. CD41 T cells from Ii0H2-M0 mice do not react with wild-type syngeneic APCs. Purified CD41 T cells from the indicated animalswere cultured for 4 days with either no APCs (no stimulus), syngeneicAPCs, or on plates coated with anti-CD3 mAb (5 mg/ml). Methyl-[3H]thymidine was added during the last 18 h of culture. Bars depictmean c.p.m. from triplicate cultures with SDs expressed as y-axis errorbars.

FIGURE 4. Ii0H2-M0 mice are susceptible to L. major. A, Lesion de-velopment in mice infected with L. major. Ii0, Ii0TAP0, Ii0H2-M0, andclass II0 (CII0) mice were infected with L. major, and footpad lesionsize is depicted over time. Symbols represent the mean hind footpaddiameter of all mice within a group except for Ii0H2-M0 mice in whichsymbols represent the mean of two hind footpad measurements ofindividual animals. The SDs of the footpad size of Ii0 and Ii0TAP0 micewere less than 25% of the mean. Results are a compilation of threeexperiments using 3 Ii0, 2 Ii0TAP0, 5 Ii0H2-M0, and 1 class II0 mice. At6 wk after infection, wild-type BALB/c mice and C57BL/6 mice hadmean footpad diameters of 5.9 6 0.4 and 2.9 6 0.3 mm, respectively.B, Assessment of Th1 responses in mice infected with L. major. Drain-ing popliteal lymph node cells from individual C57BL/6 (B6), Ii0, andIi0H2-M0 were cultured with no additions (No Ag), or with L. majorAgs in the absence (Ag) or presence (aCII 1 Ag) of the anti-class II mAbM5/114. After 48 h, IFN-g was measured from supernatants by ELISA.C, Competitive PCR analysis of draining lymph node cells from miceinfected with L. major. IFN-g transcripts were amplified from RNAisolated from draining lymph node cells of individual wild-typeC57BL/6 (WT), H2-M0, Ii0, and Ii0H2-M0 mice using competitive RT-PCR as described in Figure 1.

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change the repertoire of Ags (31) by exchanging self-peptides withantigenic peptides (13–16). Further analysis will be required toclarify the mechanisms of H2-M function in vivo.

AcknowledgmentsWe are grateful to Michael Mahowald, Charles Brown, Elle Travis, MarisaNaujokas, and Jim Miller for helpful discussion and assistance.

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