of March 17, 2014.This information is current as

with Mycobacterial Lipid AntigensResponses in Guinea Pigs by Immunization Induction of CD1-Restricted Immune

Brenner and Steven A. PorcelliLeClair, Masahiko Sugita, Stephen T. Furlong, Michael B. Kenji Hiromatsu, Christopher C. Dascher, Kenneth P.

http://www.jimmunol.org/content/169/1/3302002; 169:330-339; ;J Immunol 

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Induction of CD1-Restricted Immune Responses in GuineaPigs by Immunization with Mycobacterial Lipid Antigens 1

Kenji Hiromatsu, 2* Christopher C. Dascher,* Kenneth P. LeClair,† Masahiko Sugita,*Stephen T. Furlong,3* Michael B. Brenner,* and Steven A. Porcelli4*

Group 1 CD1 molecules have been shown to present lipid and glycolipid Ags of mycobacteria to human T cells. However, a suitableanimal model for the investigation of this component of antimycobacterial immunity has not yet been established. Previously, wefound that guinea pigs express multiple isoforms of group 1 CD1 proteins that are homologous to human CD1b and CD1c. In thisstudy, we show that CD1-restricted T cell responses can be generated in guinea pigs following immunization with lipid Ags fromMycobacterium tuberculosis. Splenic T cells from lipid Ag-immunized guinea pigs showed strong proliferative responses to totallipid Ags and partially purified glycolipid fractions from M. tuberculosis. These lipid Ag-reactive T cells were enriched in CD4-negative T cell fractions and showed cytotoxic activity against CD1-expressing guinea pig bone marrow-derived dendritic cellspulsed with M. tuberculosis lipid Ags. Using guinea pig cell lines transfected with individual CD1 isoforms as target cells incytotoxic T cell assays, we found that guinea pig CD1b and CD1c molecules presentedM. tuberculosis glycolipid Ags to T cellsraised by mycobacterial lipid immunization. These results were confirmed using a T cell line derived fromM. tuberculosis lipidAg-immunized guinea pigs, which also showed CD1-restricted responses and cytolytic activity. Our results demonstrate thatCD1-restricted responses against microbial glycolipid Ags can be generated in vivo by specific immunization and provide supportfor the use of the guinea pig as a relevant small animal model for the study of CD1-restricted immune responses to mycobacterialpathogens. The Journal of Immunology, 2002, 169: 330–339.

T he human CD1 gene family consists of five nonpolymor-phic genes, CD1A, B, C, D, and E, which map to a locuson chromosome 1 (1). The proteins encoded by this gene

family are transmembrane glycoproteins expressed in associationwith �2-microglobulin on the surface of dendritic cells and otherAPCs. The CD1 proteins show limited but significant homology toMHC class I and II Ag-presenting molecules that present peptideAgs to T cells (2). This homology of structure provided a clue tothe possible function of CD1 proteins in the presentation of foreignAgs to T cells. However, CD1 proteins are distinct from MHC-encoded molecules by the marked abundance of hydrophobicamino acids in the membrane distal domains. This finding sug-gested that CD1 proteins might have a different type of Ag bindingsite than the peptide binding groove found in MHC molecules (2).This prediction has been confirmed by the crystal structure of amouse CD1 protein, which shows a deep internal cavity formed by

the �1 and �2 domains which is lined almost entirely by hydro-phobic amino acid side chains (3)

The unique structure of the Ag binding groove of CD1 sug-gested that CD1 binds and presents something other than peptides.Direct evidence now confirms this hypothesis, as several studieshave shown that the human group 1 CD1 proteins (i.e., the groupcomposed of CD1a, CD1b, and CD1c) mediate specific T cellrecognition of mycobacterial lipid and glycolipid Ags (4). Agsidentified to date include mycolic acid, glucose monomycolate,lipoarabinomannan, and other phosphoglycolipids (5–8). T cellsrecognizing mycobacterial lipid Ags presented by CD1 can releaseIFN-� and show cytolytic activity upon contact with lipid Ag-pulsed target cells and mycobacteria-infected monocytes. Further-more, CD8� CD1-restricted T cells have also been recently shownto mediate a direct microbicidal effect on intracellular microorgan-isms by granulysin-mediated killing (9). These activities suggest aproinflammatory role for these T cells in type 1 cellular immuneresponses that are critical for the elimination of intracellular patho-gens such as Mycobacterium tuberculosis (MTb)5 and Mycobac-terium leprae (10).

Whereas previous work establishes the function of CD1 in Agrecognition by T cells, the critical question of the role of this sys-tem in host defense against infection in vivo has not been exam-ined. Evaluation of the in vivo role of the CD1 system in theprotective immune response to pathogens such as mycobacteriawill require the development of a suitable animal model in whichsuch responses are generated and can be studied. In the case ofMTb infection, such an animal model would ideally be one thataccurately replicates the important immunopathological and clin-ical features of human tuberculosis and also must have a CD1system comparable to that which is present in humans. Mice and

*Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hos-pital, and Harvard Medical School, Boston, MA 02115; and †Antigenics Inc.,Woburn, MA 01801

Received for publication February 26, 2002. Accepted for publication April 26, 2002.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported by grants from the Irene Diamond Foundation and Na-tional Institutes of Health/National Institute of Allergy and Infectious Diseases(AI48933 and AI45889) to S.A.P. and National Institutes of Health/Small BusinessInnovation Research (AI40798) to K.P.L. K.H. was partially supported by a fellow-ship from the Medical Foundation.2 Current address: Department of Microbiology and Immunology, Albert EinsteinCollege of Medicine, Bronx, NY 10461.3 Current address: Astra-Zeneca Pharmaceuticals, 1800 Concord Pike, Wilmington,DE 19850.4 Address correspondence and reprint requests to Dr. Steven A. Porcelli at the currentaddress: Department of Microbiology and Immunology, Albert Einstein College ofMedicine, Forchheimer Building, Room 416, 1300 Morris Park Avenue, Bronx, NY10461. E-mail address: porcelli@aecom.yu.edu

5 Abbreviations used in this paper: MTb, Mycobacterium tuberculosis; MPL, mono-phosphoryl lipid A; BM-DC, bone marrow-derived dendritic cell; DN, doublenegative.

The Journal of Immunology

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rats are probably not suitable animal models in this case becausethey express only CD1d-like molecules and lack obvious ho-mologs of the human group1 CD1 proteins (i.e., CD1a, b, and c)which are all known to present microbial lipid Ags (4). In contrast,our previous studies suggested that the guinea pig may prove to bean excellent animal model for studying group1 CD1-dependentimmune responses in MTb infection (11).

We have already reported that the guinea pig genome containsan extended family of CD1 genes, with at least eight distinct CD1homologs (11). Based on analyses of predicted amino acid se-quences, it was possible to classify each guinea pig CD1 gene asa homolog of one specific human CD1 isoform. This revealed thatthe guinea pig has genes that encode multiple homologs of humanCD1b (i.e., guinea pig CD1B1, B2, B3, and B4) and CD1c(CD1C1, C2, and C3), in addition to at least one gene encoding aCD1e-like protein. In the current study, we demonstrate the exis-tence of CD1-restricted nonpeptide lipid Ag-reactive T cells inguinea pigs. These CD1-restricted T cell responses were shown tobe induced in vivo following immunization with MTb lipid Agsand led to the generation of cytotoxic T cell responses restricted byseveral different members of the guinea pig group 1 CD1 family.These findings provide further justification for the use of theguinea pig as a relevant small animal model for study of the in vivorole of CD1-mediated immune responses to lipid Ags in host de-fense against MTb and other microbial pathogens.

Materials and MethodsAnimals

Outbred female Hartley guinea pigs were purchased from Charles RiverBreeding Laboratories (Wilmington, MA). Inbred strain 2 guinea pigs wereobtained from Tenovus Research Laboratory (Southampton, U.K.) andbred in our animal facility at the Dana-Farber Cancer Institute. Animalswere housed under specific pathogen-free conditions and all animal exper-iments were performed according to the Guidelines on Animal Welfareapproved by the committee on animal use at Harvard Medical School andthe Brigham and Women’s Hospital.

Monoclonal Abs

CT5 (anti-pan guinea pig T cell), CT6 (anti-guinea pig CD8), and CT7 (anti-guinea pig CD4) were obtained from Serotec (Raleigh, NC). CD1F2/6B5(mouse IgG1), reactive with all of the currently known isoforms of guineapig group 1 CD1, was produced in our laboratories from a hybridoma cellline generated from splenocytes of a mouse hyperimmunized with guineapig CD1 proteins (12). A hybridoma line producing mAb IVA12 (anti-human MHC class II monomorphic determinant and cross-reactive toguinea pig MHC class II) was obtained from the American Type CultureCollection (Manassas, VA).

Purification of lipid Ags from MTb

Lipid Ags were purified as previously described (13). Briefly, total soni-cates (10 mg dry weight bacteria/ml) of MTb (strain H37Ra) in PBS wereextracted with four volumes of chloroform/methanol (2:1, v/v) according tothe method of Folch et al. (14). After phase separation, the organic phasewas collected, dried by rotary evaporation, and resuspended in the originalstarting volume in chloroform for storage. For further subfractionation ofMTb total lipids, silica column chromatography was used (5). After evap-oration of the solvent from the MTb total lipid extract, the lipids wereresuspended in hexane and passed over open silicic acid columns to whichall major lipid classes bind. The columns were then eluted with solvents ofincreasing polarity (chloroform followed by acetone and finally methanol)to elute the major lipid fractions in order of increasing polarity (neutrallipids followed by glycolipids and phospholipids in sequential fashion).After elution from silicic acid columns, all lipid fractions were dried undera nitrogen stream and then redissolved in chloroform for storage. Mycolicacids were released from dried MTb cell walls by saponification and pu-rified using C18 reverse-phase HPLC as previously described (5). The com-position of lipid preparations was assessed using analytical TLC with de-velopment of TLC plates by charring in sulfuric acid to reveal all lipidspecies. Further characterization of lipid preparations was conducted usingspecial stains of TLC plates that revealed the presence of carbohydrate(orcinol), amino groups (ninhydrin), and phosphorous (molybdenum blue).

In addition, to assess the possibility of protein contaminants, amino acidcomposition using standard methods of acid hydrolysis followed by re-verse-phase HPLC was conducted by the Harvard Microchemistry Facility(Cambridge, MA). Based on this analysis, the maximum possible levels ofprotein contamination in lipid Ag fractions used in this study were asfollows: MTb total lipid, 4.05%; neutral lipid fraction, 0.06%; glycolipidfraction, 0.19%; phospholipid fraction, 6.41%; and mycolic acid, 0.33%.These values most likely represent overestimates of protein content sincethe majority of amino acids detected (�70%) were alanine and glutamate/glutamine, probably originating from mycobacterial peptidoglycan ratherthan proteins (15). Consistent with this, silver staining of SDS-PAGE gels(sensitivity of �10 ng for a single protein band) revealed no visible bandin a 100-�g sample of the MTb total lipid Ag preparation, indicating pro-tein contamination of �0.01% for any single protein constituent (data notshown). Each of the lipid fractions was dried under a stream of nitrogenand then resuspended by sonication into complete medium (RPMI 1640;Life Technologies, Grand Island, NY) with 10% FCS (HyClone Labora-tories, Logan, UT) and additional supplements as previously described forin vitro use in T cell recognition assays (16).

Liposome preparation for immunization of guinea pigs withMTb lipid Ags

Liposomes containing MTb lipid Ags with or without adjuvants were pre-pared using the dehydration/rehydration method (17, 18). Briefly, 50 mg of1,2-distearoyl-sn-glycero-3-phosphocholine (Avanti, Alabaster, AL) and25 mg of cholesterol (Avanti) were dissolved in 5 ml of chloroform andadded to a clean round-bottom vacuum flask. For those preparations thatcontained monophosphoryl lipid A (MPL) as adjuvant, 1 ml of chloroformcontaining 1 mg of MPL from Salmonella minnesota R595 (Ribi, Hamil-ton, MT) was added (19). Approximately 2 mg of each of the MTb lipid Agfractions (glycolipids, neutral lipids, phospholipids, and mycolic acids asindicated) dissolved in chloroform were added, and the entire lipid mixturewas dried to a thin film using a Rotovap flash-evaporator (Buchler Instru-ments, Fort Lee, NJ). Five milliliters of distilled water was then added tothe flask, and the lipid was suspended into the solution as liposomes bysonicating in a water bath sonicator (Fisher Scientific, Pittsburgh, PA). Forthose preparations that contained QS-21 as adjuvant, 150 �g of QS-21(Antigenics, Woburn, MA) was added during the rehydration step. In someexperiments, liposomes containing OVA were made by adding solubleOVA (900 �g) during the rehydration step. Liposome suspensions werelyophilized for storage at �20°C and immediately before use were resus-pended by adding an appropriate volume of PBS and passing three timesthrough a 25-gauge needle.

Immunization of guinea pigs with lipid Ags

Specific pathogen-free outbred female Hartley strain guinea pigs (250–300g) or inbred strain 2 guinea pigs (250–300 g) were immunized s.c. withpurified lipid Ags of MTb in liposomes with or without adjuvant (QS-21 orMPL). For each immunization, animals received liposomes containing amixture of some or all of the MTb lipid Ags (glycolipids, neutral lipids,phospholipids, and mycolic acids) as indicated. In each case, the amount ofimmunogen used per animal contained 500–600 �g of each lipid compo-nent, and the dose of QS-21 or MPL applied as adjuvant was 50 or 300 �gper immunization, respectively (19, 20). In some experiments, guinea pigswere immunized with bone marrow-derived dendritic cells (BM-DCs)pulsed with MTb total lipid Ags. BM-DCs were obtained from bone mar-row cultures supplemented with human GM-CSF (300 U/ml; PeproTech,Rocky Hill, NJ) as described elsewhere (12). BM-DCs were pulsed over-night with MTb total lipid extract (30 �g/ml) and, after washing three timeswith PBS, Ag-pulsed BM-DCs (2 � 107 cells total per immunization peranimal) were injected s.c. into two or three sites on the abdomen. ForCD1-restricted CTL induction (see Figs. 3 and 4), guinea pigs were im-munized one, two, or three times, as specified, with a mixture of liposomesreconstituted with MTb lipid Ags (600 �g each of glycolipids, neutrallipids, and phospholipids) along with QS-21 (50 �g) and MPL (300 �g).Animals were sacrificed at the indicated time points, and primary ex vivoCTL responses were analyzed as described below.

T cell proliferation assays

T cells were enriched from the spleens of immunized guinea pigs by pas-sage through nylon wool fiber columns. T cells (5–10 � 104/well) werecultured in complete medium as described in triplicate in 96-well flat-bottom microtiter plates with or without irradiated (5000 rad) CD1� BM-DCs or autologous nylon wool-adherent spleen cells (1–5 � 104/well) inthe presence or absence of Ag (16). To assess CD1 restriction, purifiedmAb CD1F2/6B5 (reactive with guinea pig CD1b1, b2, b3, b4, c1, c2, and

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c3) or isotype-matched nonbinding control mAb (P3, mouse IgG1) wasadded at a final concentration of 20 �g/ml. Cultures were incubated for 3–4days at 37°C in a 5% CO2 incubator, pulsed with [3H]thymidine (1 �Ci/well, 6.7 Ci/mmol; NEN Life Science Products, Boston, MA), and incu-bated for an additional 5–6 h. The plates were harvested on a Tomtec96-well plate harvester (Wallac, Turku, Finland) and thymidine incorpo-ration was measured with a Betaplate liquid scintillation counter (Wallac).

Cytotoxic assays

Guinea pig cell lines transfected with expression vector constructs encod-ing the guinea pig CD1b1, b2, b3, b4, c2, or c3 proteins (12) or mock-transfected cells (i.e., stably transfected with empty vector) were labeledwith 51Cr for 2 h. The target cells were cultured overnight at 37°C in a 5%CO2 incubator in complete medium with or without MTb total lipid extract(10 or 20 �g/ml), and then harvested and extensively washed. The labeledtarget cells were plated in quadruplicate in 96-well V-bottom microtiterplates with effector T cells at a range of different E:T ratios in a totalvolume of 150 �l of complete medium and incubated at 37oC for 4 h.Samples of supernatant (25 �l) were harvested and counted in a gammacounter. Total release of 51Cr was determined by addition of 1% TritonX-100 to wells containing target cells, and spontaneous release was deter-mined from wells in which Ag-pulsed or Ag-unpulsed target cells werecultured without effector T cells. The data were calculated as percent spe-cific lysis according to the following formula: (100 � (cpm release fromexperimental sample � cpm spontaneous release)/(cpm maximum re-lease � cpm spontaneous release)).

For the source of splenic effector T cells for primary CTL responses,guinea pigs (strain 2 or Hartley as specified) were immunized with MTblipid Ag mixture (600 �g each of glycolipids, neutral lipids, and phospho-lipids) reconstituted in liposomes along with 50 �g of QS-21 and 300 �gof MPL as adjuvants. Strain 2 guinea pigs immunized once were sacrificedat 2 wk after the primary immunization (S2-005, Fig. 3A) or sacrificed at4 wk after the primary immunization (S2-007/8, Fig. 3C). In some exper-iments, strain 2 guinea pigs were immunized twice (primary and boosted atweek 4) and sacrificed 12 wk after the second immunization (S2-003, Fig.3C); Hartley strain guinea pigs were boosted twice after the primary im-munization at 4 and 8 wk and sacrificed 24 wk after the last immunization(H0408, Fig. 3C). To prepare effector T cells, spleen T cells from MTblipid Ag-immunized guinea pigs were enriched by nylon wool columns andthen cultured for 6 days in complete medium containing a mixture of MTbtotal lipid (10 �g/ml) and glycolipid fraction (30 �g/ml) in the presence ofmitomycin C (Sigma-Aldrich, St. Louis, MO)-treated splenic adherent cellsas APCs and 0.5 nM human rIL-2. In preliminary experiments, we foundthat in vitro stimulation with MTb glycolipid Ag in combination with MTbtotal lipid Ags induced better CTL responses than stimulation with glyco-lipid Ag alone, possibly due to enhancement of bystander help from CD4�

T cells activated by the broader antigenic challenge or some other adjuvantactivity of the total lipid preparation. At the end of the 6-day culture period,viable T cells were purified by Ficoll-Hypaque density centrifugation (spe-cific gravity, 1.107) and used as effector T cells in CTL assays with MTblipid Ag-pulsed CD1-expressing target cells.

Generation of CD1-restricted T cell lines

An inbred strain 2 guinea pig was s.c. immunized twice with MTb lipidAgs (mixture of 600 �g each of glycolipids, neutral lipids, and phospho-lipids) in liposomes along with QS-21 (50 �g) and MPL (300 �g) at 4-wkintervals. The animal was further boosted with 2 � 107 syngeneic BM-DCsthat had been pulsed overnight with MTb total lipid extract (30 �g/ml). Thespleen was harvested 2 wk after the last immunization and splenic T cellswere first enriched using a nylon wool column (Polysciences, Warrington,PA). CD4� T cells were then depleted by staining with anti-CD4 mAb(CT7) followed by magnetic separation using goat anti-mouse Ig-coupledDynabeads (Dynal Biotech, Lake Success, NY) according to the manufac-turer’s instructions. The resulting CD4-depleted T cells were cultured withMTb total lipid extract (20 �g/ml) in the presence of irradiated (5000 rad)syngeneic BM-DCs in 1 ml of complete medium (3 � 106 T cells with 3 �105 BM-DCs in 1 ml of medium/well of a 24-well plate). Four days afterthe start of culture, half of the medium was replaced with fresh completemedium containing 0.25 nM human rIL-2 (PeproTech). Subsequently, cul-tures were fed every 3–4 days by replacing half of the medium with freshcomplete medium containing 0.25 nM IL-2. Live cells were collected after�2 wk of expansion and restimulated using BM-DCs plus MTb total lipidextract. Cultures were carried through four similar cycles of Ag stimulation(in some cases preceded by immunomagnetic depletion of residual CD4�

T cells) followed by expansion in medium containing IL-2 to generate lineS2-1031, which showed CD1-restricted reactivity to MTb lipids.

Statistical analyses

Data were analyzed by unpaired t test using GraphPad Prism (GraphPadSoftware, San Diego, CA).

ResultsInduction of ex vivo T cell responses in guinea pigs immunizedwith mycobacterial lipid Ags

Our initial experiments focused on the induction of specific T cellproliferative responses following the immunization of guinea pigswith suitable preparations of MTb lipid Ags. To produce uniformsuspensions of the lipid Ags in a form that was likely to be im-munogenic, we incorporated a mixture of three MTb lipid Ag frac-tions (glycolipids, neutral lipids, and mycolic acids) into lipo-somes. To provide a suitable adjuvant, we incorporated eitherMPL or QS-21 into the immunogen. Four weeks after immuniza-tion with the liposome-entrapped MTb lipid Ag mixture with orwithout adjuvant, guinea pigs were sacrificed and T cells werepurified from their spleens by passage over nylon wool columns.The resulting T cell-enriched populations were �95% T cellsbased on FACS analysis using mAb CT5 and were �70% CD4�

(mAb CT7) and 20% CD8� (mAb CT6); data not shown). Thesecells were tested for proliferative responses to the MTb lipid Agfractions using irradiated nylon wool column-adherent spleen cellsas APCs (60% positive by FACS for CD1 expression with mAbCD1F2/6B5; data not shown). As shown in Fig. 1A, nylon woolcolumn-passed splenic T cells from guinea pigs immunized twicewith liposome-entrapped MTb lipid Ags without adjuvant showedvery weak proliferative responses to total MTb lipid Ags and noresponses to the glycolipid, neutral lipid, or mycolic acid fractions.In contrast, immunization of guinea pigs with liposomes contain-ing MTb lipids plus either QS-21 (Fig. 1B) or MPL (Fig. 1, C andD) as an adjuvant induced significant proliferative responses byspleen T cells to the MTb total lipid and glycolipid preparations.Splenic T cells purified from naive unimmunized guinea pigsshowed only minimal, if any, responses to mycobacterial lipid Agsin vitro (Fig. 1D and additional data not shown). We could notraise significant T cell responses to neutral lipids or mycolic acidsfrom these guinea pigs using these immunization protocols, sug-gesting either that these lipid Ag fractions are less immunogenic orthat the frequency of T cells specific to them is very low.

In vivo immunization with BM-DCs pulsed with MTb lipid Ags

As an alternative method for inducing T cell responses againstMTb lipid Ags in vivo, we immunized guinea pigs with lipid Ag-pulsed DCs without the addition of chemical adjuvants. DCs ex-pressing CD1 molecules were generated from guinea pig bonemarrow cells using in vitro cultivation with human rGM-CSF (12).Fig. 2A shows a representative FACS profile of guinea pig BM-DCs at day 10 after culture of bone marrow cells. When gated onthe high forward scatter population, nearly 90% of the BM in thesecultures expressed high levels of MHC class II and CD1 proteinson their surfaces. Given the known properties of DCs in the stim-ulation of primary T cell responses in vivo in humans and mice, wetested the ability of guinea pig BM-DCs to prime lipid Ag-specificresponses following MTb lipid Ag-loading ex vivo and injectionback into animals (21).

Naive guinea pigs received two s.c. injections 2 wk apart ofBM-DCs pulsed with MTb total lipid Ag extract. At 2 wk after thelast immunization, proliferative responses of purified splenic Tcells against different MTb lipid Ag fractions in the presence ofirradiated BM-DCs as APCs were examined (Fig. 2, B and C). Asshown in Fig. 2B, an outbred Hartley guinea pig that was immu-nized with BM-DCs pulsed with MTb lipid Ag showed strong

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proliferative responses to MTb glycolipid and phospholipid frac-tions. The high level of background proliferation observed in thiscase (medium alone) was most likely due to the simultaneous sen-sitization of alloreactive T cells, since Hartley guinea pigs are notfully inbred. To rectify this potential problem, we conducted anal-ogous experiments using inbred strain 2 guinea pigs as the BM-DCdonor and as the recipient immunization with BM-DCs pulsedwith MTb lipid Ag. As shown in Fig. 2C, in vivo immunization ofan inbred strain 2 guinea pig with MTb lipid Ag-pulsed BM-DCsof strain 2 origin also induced significant proliferative responses ofsplenic T cells to total MTb-extractable lipids in vitro, as well asa strong response to the phospholipid fraction and a weak butsignificant response to the glycolipid fraction. In vivo sensitizationof strain 2 guinea pigs using MTb lipid Ag-pulsed BM-DCs wasrepeated three times, showing consistent levels of T cell prolifer-ation to total MTb lipid Ags and phospholipids (stimulation indexof 3.2–5.5 compared with medium alone) and a lesser level ofproliferation to glycolipids (stimulation index, 1.7–2.6), with min-imal or no responses to neutral lipids. In similar experiments con-ducted in parallel, we also observed that guinea pigs immunizedwith splenic DCs pulsed with the total MTb lipid Ag extractshowed proliferative responses to MTb lipid Ags upon in vitrochallenge (data not shown). These results indicated that BM-DCsand splenic DCs expressing CD1 could present mycobacterial lipid

Ags to T cells in vivo, thus leading to lipid Ag-specific recallresponses that could be detected by T cell proliferation ex vivo.

Ex vivo CTL responses from guinea pigs immunized withmycobacterial lipid Ags

In humans, the majority of group 1 CD1-restricted T cell linesreactive with mycobacterial lipid Ags that have been studied todate exhibit cytotoxic activity against lipid Ag-pulsed DCs orCD1-expressing transfectant cells along with proliferative re-sponses and IFN-� production. Therefore, we next examinedwhether the MTb lipid Ag-reactive T cells arising after immuni-zation of guinea pigs showed cytotoxic activity against MTb lipidAg-pulsed target cells. Based on the results described in Fig. 1, weimmunized naive strain 2 or Hartley guinea pigs with MTb lipidAgs (mixture of glycolipid, neutral lipid, and phospholipid frac-tions) entrapped in liposomes with the combination of QS-21 andMPL as adjuvants. Effector T cells were prepared from immunizedguinea pigs by in vitro stimulation of splenic T cells with MTbtotal lipid extract (10 �g/ml) and glycolipid-enriched fraction (30�g/ml) for 6 days. We first tested CTL responses against lipidAg-pulsed syngeneic strain 2 BM-DCs, which we have shown toexpress high levels of multiple different guinea pig CD1 proteins(12). As shown in Fig. 3A, the effector T cells derived from theMTb lipid Ag-immunized guinea pigs lysed the target BM-DCs

FIGURE 1. Primary ex vivo proliferative T cell responses from guinea pigs immunized with liposome containing MTb lipid Ags. Proliferative responsesof splenic T cells from Hartley strain guinea pigs immunized with MTb lipid Ags (mixture of glycolipids, neutral lipids, and mycolic acids) entrapped inliposomes without adjuvant (animal A025, A), with QS-21 (animal A029, B), or MPL (animal A032, C and D). Four weeks after immunization, guinea pigswere sacrificed and spleen T cells were purified and tested for proliferative responses to the indicated lipid Ags in the presence of irradiated autologoussplenic nylon wool fiber-adherent cells as APCs. A range of concentrations was tested for each of the lipid Ag fractions, and the results shown in A–Crepresent the optimal dose for each lipid Ag preparation shown (12.5 �g/ml for MTb total lipids, 25 �g/ml for all others). D, One representative exampleof a dose titration of the lipid Ag fractions is shown for the proliferative responses of T cells from a guinea pig immunized with MTb lipid Ags plus MPLin liposomes (animal A032, f) and for T cells from an age-matched naive guinea pig (Œ). Asterisks indicate statistically significant difference comparedwith medium only control (�, p � 0.01; ��, p � 0.001).

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pulsed with MTb glycolipids or total lipids significantly above thebackground level of target cells that were not pulsed with MTblipids. Similar to the proliferative responses observed after MTblipid Ag immunization using the liposome vehicle with QS-21 orMPL as adjuvant (Fig. 1), effector T cells in cytolytic assaysshowed the strongest reactivity to the MTb total lipid Ag prepa-ration and the glycolipid-enriched fraction, but also showed a trendtoward weak recognition of BM-DCs pulsed with the neutral lipid-enriched fraction.

Next, we examined these cytotoxic T cell responses for theirdependence on the expression of particular CD1 molecules by thetarget cells. As described elsewhere, we generated a new mAb(CD1F2/6B5) against guinea pig CD1 proteins which recognizedall seven of the currently identified isoforms of guinea pig group 1CD1 proteins (12). As shown in Fig. 3B, the cytolytic activity ofeffector T cells obtained from lipid Ag-immunized guinea pigsagainst BM-DCs pulsed with glycolipids or total lipids were sig-nificantly inhibited by anti-CD1 mAb, but not by isotype-matchedcontrol mAb (P3, mouse IgG1). These results strongly suggestedthat mycobacterial lipid Ag-reactive cytotoxic T cells elicited inguinea pigs by immunization with MTb lipid Ags were at leastpartially CD1 restricted.

To address which of the multiple different group 1 CD1 proteinsof the guinea pig were involved in CTL responses evoked by im-munization with MTb lipid Ags, we used a panel of guinea pig celllines stably transfected with one of six different forms of guineapig CD1 as target cells in CTL assays. Subclones of the guinea pigcell line 104C1 each transfected with a construct encoding one ofsix different guinea pig CD1 proteins (i.e., guinea pig CD1b1, b2,b3, b4, c2, or c3) or with empty vector alone (mock transfectantcells) (12) were pulsed with the MTb glycolipid fraction and usedas target cells for CTL assay (note that guinea pig CD1c1 was notincluded in this analysis because we were unable to achieve stablehigh levels of expression of this isoform by transfecting 104C1cells). The MTb glycolipid fraction was used for this analysis be-cause this fraction was consistently active in preliminary CTL ex-

periments (Fig. 3A and additional data not shown) and representeda more purified form of lipid Ag without significant protein orpeptide contamination. As shown in Fig. 3C, the CTL responses ofeither inbred strain 2 or Hartley guinea pigs immunized with MTblipid Ags in liposomes plus MPL and QS-21 showed consistentrecognition of MTb glycolipid-pulsed transfectants expressing ei-ther guinea pig CD1b1 or guinea pig CD1c2. Note that one animal(Hartley guinea pig H0408) was immunized with lipid Ag threetimes and sacrificed 6 mo after the final immunization to investi-gate the long-term memory responses of lipid Ag-reactive CD1-restricted CTLs in this system. The appearance of similar levels ofCTL activity in this animal that was rested for an extended periodof time compared with the animals that had been more recentlyboosted strongly suggested that a long-term CD1-restricted mem-ory CTL response can be induced following MTb lipid Agimmunization.

A more detailed analysis of the effects of key parameters of theCTL reaction (glycolipid Ag concentration used for pulsing tar-gets, and E:T ratio) was conducted for CTL recognition of targetsexpressing guinea pig CD1b1 and guinea pig CD1c2 (Fig. 4). Asshown in Fig. 4, guinea pig CD1b1 and c2 but not mock-trans-fected 104C1 were recognized by effector T cells from strain 2guinea pigs immunized twice with lipid Ags in liposomes withQS-21 plus MPL, and the level of lysis varied directly with theMTb glycolipid Ag concentration and the E:T ratio. Furthermore,to exclude the possibility of random variations between different104C1 transfectant clones accounting for the observed CTL lysis,we analyzed the CTL responses using multiple independently de-rived subclones of 104C1-expressing guinea pig CD1b1 (twoclones: clone 16 and clone 18) or guinea pig CD1c2 (three clones:clone 5, clone 11, and clone 12). All of these subclones behavedsimilarly as target cells in these assays, yielding specific lysis val-ues that were significantly greater than the background level ob-tained with the mock transfectant cells (data not shown). Takentogether, these results indicated that immunization with MTb lipidAgs elicited MTb glycolipid-specific CTL responses in vivo that

FIGURE 2. Primary ex vivo proliferative T cell responses from guinea pigs immunized with MTb lipid Ag-pulsed BM-DCs. A, FACS analysis ofBM-DCs obtained from bone marrow cells cultured in the presence of human GM-CSF. Anti-guinea pig MHC class II (mAb IVA12), anti-pan guinea pigCD1 (mAb CD1F2/6B5), and isotype-matched, nonbinding control mAb (mAb P3) were used as primary Abs and detected with a FITC-conjugated goatanti-mouse IgG/M F(ab�)2. The histograms shown are representative of FACS profiles obtained on more than 10 preparations of BM-DCs. B, BM-DCs froma naive Hartley guinea pig were pulsed overnight with MTb total lipid extract (30 �g/ml). After extensive washing with PBS, the Ag-pulsed BM-DCs wereinjected s.c. into a naive Hartley guinea pig (day 0). On day 14, the guinea pig was boosted with total MTb lipid Ag-pulsed BM-DCs and on day 28 theanimal was sacrificed and T cell proliferative responses to lipid Ag were tested in the presence of the same irradiated BM-DC preparation that was usedfor the immunizations. C, BM-DCs obtained from naive inbred strain 2 guinea pigs were pulsed with MTb total lipid Ags overnight and injected s.c. intonaive strain 2 guinea pigs. The animals were boosted with a second injection of Ag-pulsed syngeneic BM-DCs 2 wk later and then sacrificed after anadditional 2 wk. Purified splenic T cells were tested for proliferative responses to MTb lipid Ags in the presence of syngeneic BM-DCs as APCs. Asterisksindicate statistically significant difference compared with medium-only control (�, p � 0.01; ��, p � 0.001).

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were restricted by at least two different group 1 guinea pig CD1proteins.

Phenotype of MTb lipid Ag-specific T cells elicited in immunizedguinea pigs

To gain further insight into the types of CD1-restricted T cells thatarise following MTb lipid Ag immunization in vivo, we examinedthe phenotype of the lipid-reactive T cells with respect to expres-sion of CD4 and CD8 coreceptor molecules. Since the T cellsshowed CTL activity, we reasoned that at least a fraction of themwould belong to the CD8 single-positive fraction or possibly to theCD4�CD8� double-negative (DN) fraction which has also beenassociated with CTL effector activity (13, 22). Thus, to confirmthat MTb lipid Ag-reactive T cells reside in the CD4-negativefraction (i.e., among CD8 single-positive or DN T cells), we per-formed the following experiments. Hartley guinea pigs were s.c.immunized with OVA and a mixture of MTb lipid Ags (glycolip-

ids, neutral lipids, and phospholipids) incorporated into liposomesalong with the QS-21 and MPL. OVA was added concomitantly asa control, representing a foreign protein Ag that should elicit pep-tide-specific CD4� T cell responses that could be used to monitorthe effectiveness of the immunization. At 4 wk after immunization,T cells were enriched from the spleens of sacrificed animals bynylon wool column (purity �90%) and then further fractionated byimmunomagnetic depletion of CD4� T cells (residual CD4� T cellcontamination �5%). The proliferative responses to MTb lipidAgs and OVA of the T cell-enriched fraction and the CD4-de-pleted T cell-enriched fraction were then examined in the presenceof irradiated autologous BM-DCs as APC. As shown in Fig. 5A,splenic T cells isolated from an immunized guinea pig 4 wk afterMTb lipid Ag priming showed significant dose-dependent prolif-eration to MTb total lipid Ags and to OVA. Depletion of CD4�

T cells led to a marked reduction in the OVA-specific T cellproliferation, but had little effect or even slightly increased the

FIGURE 3. Primary ex vivo cytotoxic T cell responses to mycobacterial lipid Ags. A and B, CTL activity against MTb lipid Ag-pulsed BM-DCs. A strain2 guinea pig (S2-005) was immunized with MTb lipid Ags (mixture of glycolipid, neutral lipid, and phospholipid fractions) entrapped in liposomes withQS-21 and MPL. Two weeks after the immunization, splenic T cells were purified and further stimulated with MTb total lipids (10 �g/ml) and glycolipid-enriched fraction (30 �g/ml) for 6 days in the presence of irradiated splenic adherent cells as APCs and subsequently tested for cytotoxic activity againstlipid-pulsed BM-DCs as target cells. In the experiment shown in B, mAb CD1F2/6B5 against guinea pig CD1 proteins or isotype-matched control mAb(P3) were tested for inhibition of the CTL responses. Asterisks indicate statistically significant difference compared with medium-only control (A) or toisotype-matched control mAb (B) (�, p � 0.035; ��, p � 0.001). C, CTL responses of guinea pig T cells from MTb lipid Ag-immunized guinea pigs againstguinea pig CD1 transfectant cells. Guinea pigs were immunized with MTb lipid Ag (mixture of glycolipid, neutral lipid, and phospholipid fractions) inliposomes along with QS-21 and MPL. Guinea pig fibroblast cell line 104C1 transfected with each isoform of guinea pig CD1 gene were labeled with 51Cr,pulsed with glycolipids, and used as target cells in CTL assay. Effector T cells were prepared by 6 days of in vitro stimulation of splenic T cells with amixture of MTb glycolipids (30 �g/ml) and total lipids (10 �g/ml) in the presence of autologous APCs. Effector splenic T cells were from strain 2 guineapigs immunized once (animal S2007/8, sacrificed 4 wk after a single immunization) or twice with MTb lipids (animal S2-003, sacrificed12 wk after thesecond immunization) and from a Hartley guinea pig immunized three times (animal H0408, sacrificed 6 mo after the third immunization). Asterisksindicate statistically significant difference compared with lysis of mock transfectant (�, p � 0.001).

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proliferative responses to the MTb total lipid Ag (Fig. 5B). Thissuggested that the T cell fraction responding to MTb lipid Ags wasmainly or exclusively composed of CD4�CD8� and/or CD8� Tcells. This finding was confirmed by preliminary experiments de-signed to determine optimal conditions for establishing CD1-re-stricted MTb lipid Ag-specific T cell lines using CD4-depletedsplenic T cells from inbred strain 2 animals similarly immunizedwith MTb lipid Ags. As shown in Fig. 5C, CD4-depleted T cellfractions from immunized strain 2 guinea pigs again responded toMTb whole lipid extract and to the glycolipid-enriched Ag frac-tion. These results provided further support for the conclusion thatCD4-negative T cells were involved in the proliferative responsesto these Ags.

Establishment of a CD1-restricted MTb lipid Ag-reactive T cellline from an immunized guinea pig

The ability to detect proliferative and cytotoxic T cell responses toMTb lipid Ags prompted us to establish CD1-restricted MTb lipidAg-specific T cell lines to facilitate more detailed in vitro analyses.Since the CD4 depletion studies indicated that CD1-restricted Tcells primed by MTb lipid immunization of guinea pigs may bepredominantly CD4 negative, we used CD4-depleted splenic Tcells from strain 2 guinea pigs immunized with liposome-en-trapped purified MTb lipid Ags along with QS-21 and MPL andboosted with lipid Ag-pulsed BM-DCs to initiate cultured T celllines. These T cells were cultivated in the presence of CD1� BM-DCs from inbred strain 2 guinea pigs and pulsed with an MTbwhole lipid extract. To prevent possible overgrowth of any residualCD4� T cells during the culture, the CD4 depletion was repeatedthree times during the course of culture. After two stimulationsusing syngeneic CD1� BM-DCs, the third and fourth stimulationswere conducted with CD1� BM-DCs from allogeneic outbredHartley guinea pigs to favor presentation of lipid Ags by nonpoly-morphic Ag-presenting molecules such as CD1. After the sixth re-stimulation, one T cell line was obtained (designated as S2-1031) thathad expanded sufficiently to permit detailed analysis. FACS analysisrevealed that line S2-1031 was composed predominantly ofCD4�CD8� and CD8� T cells, with only 5.2% CD4� cells (Fig. 6A).

The specificity and restriction of T cell line S2-1031 was ex-amined by [3H]thymidine incorporation assay and mAb blocking.The T cell line proliferated strongly to CD1� BM-DCs pulsed withMTb total lipid Ags. Attempts to characterize the specific lipid Agrecognized by line S2-1031 were unsuccessful, as this T cell line

responded to MTb total lipid extract but not to any of the standardsubfractions derived from it (i.e., purified mycolic acids or silicicacid column fractions enriched in MTb glycolipids, neutral lipids,or phospholipids; data not shown). The CD1 restriction of thisproliferative response to MTb lipids was shown by adding theCD1-specific blocking mAb (CD1F2/6B5.1) to the proliferationassay. The mAb (CD1F2/6B5.1) blocked the MTb lipid Ag-spe-cific response of line S2-1031 by �90%, whereas the same mAbdid not affect the proliferative responses of an alloreactive CD4�

guinea pig T cell line (Fig. 6B). Isotype-matched control mAb(mAb P3, mouse IgG1) had no effect on these proliferative re-sponses (data not shown).

To further dissect the CD1 restriction of this MTb lipid Ag-reactive T cell line, CTL responses were determined using trans-fectants of cell line 104C1 that selectively expressed each of thecloned guinea pig CD1 isoforms. T cell line S2-1031 lysed MTblipid Ag-pulsed 104C1 cells stably transfected with guinea pigCD1b1, -b2, and -c3, but not mock transfectants in an Ag dose-dependent manner, as shown in Fig. 7. Consistent with the findingsobtained in the proliferation assays with this T cell line, we couldnot clearly detect significant CTL responses (i.e., �10% specificlysis) to more highly purified lipid Ag fractions (purified mycolates orthe MTb glycolipid, neutral lipid, and phospholipid-enriched fraction;data not shown). Thus, although the precise lipid Ag specificity ofline S2-1031 cannot yet be determined, the results provided clearevidence for the restriction of its MTb-specific CTL activity by atleast three members of the guinea pig group 1 CD1 family.

DiscussionTo test the possible role of group 1 CD1 molecules in host defense,it is necessary to develop an appropriate animal model for assess-ment of the functions of group 1 CD1-restricted T cell responses invivo. Mice and rats are inadequate for this purpose since the ge-nomes of these rodents lack all group 1 CD1 genes. As previouslyreported, we have analyzed the guinea pig CD1 gene family andproposed that guinea pigs could serve as a suitable small animalmodel to test the role of group 1 CD1 proteins in host defense (11).In the current study, we have extended our previous findings onthis animal model by demonstrating that guinea pigs can mountCD1-restricted T cell responses following in vivo immunizationwith MTb lipid Ags. This is the first demonstration to our knowl-edge of group 1 CD1-restricted lipid Ag-specific T cell responsesin a nonhuman species and also the first successful demonstration

FIGURE 4. Guinea pig CD1b1 and CD1c2 proteins present glycolipid Ags to T cells from MTb lipid-immunized guinea pigs. Strain 2 guinea pigsimmunized as described in Fig. 3 were sacrificed 3 wk after the second immunization and splenic T cells were further stimulated in vitro with total lipidand glycolipid as described. Effector cells were pooled from two identically treated animals (animals S2-009 and S2-010). Two guinea pig CD1b1transfectant clones and three guinea pig CD1c2 transfectant clones along with mock transfectant cells were used as target cells for CTL assay. Repre-sentative results obtained with one clone of each transfectant are shown. Target cells were pulsed with two different concentrations of MTb glycolipid-enriched fraction in these CTL assays (50 �g/ml (f) or 25 �g/ml (Œ)) or with medium only (F).

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of CD1-restricted recall responses following in vivo immunizationwith lipid Ags.

Evidence supporting the hypothesis that MTb lipid Ag-specificCD1-restricted T cells play a role in host defense against microbialpathogens comes from the recent finding that human CD1c-re-stricted T cells specific for a mycobacterial isoprenoid glycolipidAg are seen in individuals previously infected with MTb, but notin naive donors (8). CD1-restricted T cells specific for mycobac-terial Ags have also been derived from the active skin lesions ofleprosy patients, and effective cell-mediated immunity is corre-lated with expression of group 1 CD1 molecules by the DCs inleprosy skin lesions (23, 24). Furthermore, these CD1-restrictedmycobacterial lipid Ag-specific T cells have been found to be bi-ased toward secretion of Th1 type cytokines and can kill CD1�

target cells pulsed with lipid Ags while concurrently producing thebactericidal protein granulysin (25). Together, these data providestrong circumstantial evidence that CD1-restricted T cells specificfor foreign lipid Ags may play a role in host defense against bac-terial infections.

In humans, CD1-restricted responses were initially found amongCD4�CD8� DN T cells, although subsequent reports also con-

firmed the existence of CD1-restricted T cells that were CD4 (23)or CD8 single positive (13). In our analysis of MTb lipid-immu-nized guinea pigs, we found that CD1-restricted lipid Ag-specificT cells were prominent in the CD4-negative fraction of splenic Tcells, consistent with CD1 restriction being mainly a feature ofCD8� or DN T cells in this experimental setting. These CD4-negative CD1-restricted T cells were associated with both prolif-erative and cytotoxic activities, similar to what has been describedfor human CD1-restricted T cells reactive with mycobacterial lipidAgs. In the case of human MTb lipid-reactive T cells, Ag recog-nition has typically been associated with production of IFN-�, akey cytokine in the immune response to pathogenic mycobacteria.We are not yet able to test whether this is a property of the CD1-restricted T cells that develop after MTb lipid immunization in theguinea pig because of the current lack of reagents for the specificdetection of IFN-� or other cytokines in this species.

Although our current study established the priming of Ag-spe-cific T cell responses following immunization with suitably adju-vanted and reconstituted mycobacterial lipid preparations, it is alsoof interest to determine whether such responses develop during

FIGURE 5. Enrichment of MTb lipid Ag-specific cells in CD4-depleted T cell fractions. A, Proliferative responses over a range of Ag doses to OVA(Œ) or MTb total lipids (f) of unfractionated splenic T cells from a Hartley guinea pig immunized with a mixture of MTb lipid Ags and OVA in liposomeswith QS-21 plus MPL. APCs were irradiated autologous nylon wool-adherent splenocytes. B, Effect of CD4 T cell depletion on reactivity to MTb lipid Agsand OVA. A Hartley strain guinea pig was immunized with MTb lipid Ags (mixture of glycolipid, neutral lipid, and phospholipid fractions) with OVA inliposomes along with QS-21 and MPL. The animal was sacrificed 4 wk after immunization, and splenic T cells were purified and used as responders inproliferation assays with irradiated autologous splenic nylon wool-adherent cells as APCs. Some portions of splenic T cells were further treated withanti-CD4 mAb, followed by depletion of CD4� cells using magnetic beads. Whole splenic T cells and CD4-depleted splenic T cells were compared forreactivity to 12.5 �g/ml MTb total lipid Ag extract or OVA. C, CD4-depleted splenic T cells obtained from inbred strain 2 guinea pigs immunized as inB were tested for proliferative responses to MTb total lipid extract (12.5 �g/ml) or glycolipid-enriched fraction (12.5 �g/ml) using irradiated syngeneicsplenic nylon wool-adherent cells as APCs. Two representative experiments obtained from separate individual guinea pigs are shown. Asterisks indicatestatistically significant difference compared with medium-only control (�, p � 0.001).

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actual infections with mycobacterial pathogens. Our previous stud-ies on PBL from human donors with previous mycobacterial in-fection strongly support the view that CD1-restricted T cells re-sponses to specific lipid Ags do develop as a result of naturalinfections with MTb (8). In the guinea pig model, preliminary

experiments to determine whether such T cell responses areprimed in animals infected with MTb strain H37Ra or bacillusCalmette-Guerin have demonstrated lymphocyte proliferative re-sponses to mycobacterial lipid Ags similar to those elicited uponimmunization with MTb lipids (K. Hiromatsu, unpublished data).However, we have thus far failed to detect CD1-restricted CTLactivity in lymphocytes isolated from guinea pigs infected withMTb strain H37Ra. This raises the possibility that active myco-bacterial infection may be associated with suppression of certainaspects of the immune response that can be elicited by isolatedlipid vaccination, possibly reflecting a significant immune evasionstrategy of the pathogen, and suggests that vaccination by liveattenuated mycobacteria could actually be less effective than vac-cination with purified subunit formulations in some cases. This isa potentially important issue and its resolution will require exten-sive further studies using the guinea pig model and other animalmodels of MTb infection.

The conservation of multiple homologs of human CD1b andCD1c in the guinea pig strongly suggests that the group 1 CD1molecules may have evolved specialized functions early in thecourse of mammalian evolution (11). The guinea pig model mayalso provide additional insights into the selective pressures thatmay have led to the diversification of CD1 into its multiple distinctisoforms. From studies of the subcellular localization and Ag-pre-senting properties of human CD1a, b, and c molecules, it has beenpostulated that these separate CD1 isoforms may each be special-ized to efficiently survey different intracellular compartments forlipid Ags (26–28). If this is correct, then it would appear that thedifferent isoforms of group 1 CD1 are not functionally redundant,but are specialized to complement each other in providing detec-tion of foreign or abnormal self-lipids. Interestingly, in our studiesof the subcellular localization of the various guinea pig CD1 iso-forms, we have found that guinea pig CD1 proteins show at least

FIGURE 6. Generation of a CD1-restricted T cell line specific for MTb lipid Ag from an immunized guinea pig. A, FACS analysis showing phenotypeof lipid Ag-specific guinea pig T cell line S2-1031. The T cell line was stained with anti-guinea pig pan-T cell (mAb CT5), anti-guinea pig CD4 (mAbCT7), anti-guinea pig CD8 (mAb CT6), or isotype-matched control Ab (P3). B, CD1 restriction of T cell line S2-1031. T cells were stimulated with MTbtotal lipid Ags in the presence of syngeneic BM-DCs. Anti-CD1 mAb (CD1F2/6B5) was added to a final concentration of 20 �g/ml. An alloreactive CD4�

T cell line derived from a Hartley guinea pig by stimulating with BM-DCs from a strain 2 guinea pig was also stimulated with strain 2 BM-DCs with orwithout anti-CD1 mAb. Results shown are representative of three independent experiments. Asterisk indicates statistically significant difference (�, p �0.0066).

FIGURE 7. Restriction of MTb lipid Ag-specific T cell line by individ-ual guinea pig CD1 molecules. Guinea pig fibroblast cell line 104C1 cellstransfected with different isoforms of guinea pig CD1 were used as targetcells in CTL assay. Transfectant cells were first labeled with 51Cr andpulsed for 18 h with either 10 or 20 �g/ml MTb total lipid Ags or inmedium alone. The CD1-restricted lipid Ag-specific T cell line S2-1031was used for the effector cells. CTL activity at an E:T ratio of 40:1 isshown. Asterisks indicate statistically significant difference compared withlysis of cells not pulsed with MTb lipid Ag (�, p � 0.05; ��, p � 0.01).

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three distinct patterns of intracellular localization (12). Given ourcurrent findings on the ability of several guinea pig CD1 proteinsto present MTb lipid Ags to T cells, it will be of interest to deter-mine in future studies whether the different guinea pig CD1 mol-ecules involved in these responses acquire their lipid Ags in dis-tinct subcellular compartments of APCs.

Considerable work remains to be done to identify the specificMTb lipids that are recognized by guinea pig CD1-restricted Tcells. As shown in the current study, MTb lipid Ag-immunizedguinea pigs frequently gave positive T cell responses to the MTbglycolipid-enriched fraction after immunization with a broad mix-ture of mycobacterial lipid Ags, whereas highly purified mycolicacids and a neutral lipid-enriched fraction appeared not to be wellrecognized in most cases. Analysis of amino acid content of thevarious lipid preparations that we have used does indicate a pos-sibility of low levels of residual protein contamination in some ofthese, since up to 6.4% amino acid content on a weight basis wasdetected in the total lipid extract and phospholipid-enriched frac-tions. However, analysis by SDS-PAGE and ultrasensitive silverstaining failed to reveal any detectable protein contaminants in ourlipid Ag preparations, and the profile of amino acids present sug-gest that most of these are derived from peptidoglycan rather thanfrom proteins. Thus, although we cannot completely rule out at thispoint the possibility that trace amounts of highly immunogenicprotein Ags in our lipid preparations could contribute to some ofthe T cell responses we have observed, this appears to be unlikely.It is also noteworthy that in several cases we demonstrated guineapig CD1-restricted recognition of the MTb glycolipid-enrichedfraction, which represents material eluted with acetone from a si-licic acid column and contains only trace amounts of detectableamino acids. Finally, the restriction of these responses by guineapig CD1 proteins provides further evidence in favor of lipid ratherthan protein Ag targets, since work on human and murine CD1 hasstrongly linked these proteins to the binding and presentation oflipids rather than proteins or peptides.

Our current data provide further support for the possibility ofusing the guinea pig as an animal model in which to test the hy-pothesis that augmentation of group 1 CD1-restricted mycobacte-rial lipid Ag-specific T cell responses can be used as a componentof an effective vaccination strategy against MTb infection (29).This appears to us to be an appealing strategy, given that CD1genes are largely nonpolymorphic and that the CD1 target ligands(i.e, complex lipids and glycolipids) are essential components ofbacterial cells that may be less variable in their structures than themore highly mutable peptide Ags that are targets of MHC-re-stricted T cells. Currently, Mycobacterium bovis bacillusCalmette-Guerin is the only available vaccine against tuberculosis,and its efficacy had been disputed despite extensive use in humansand testing in the field. CD1-mediated presentation of lipid Agsmay provide a completely new vaccination strategy that could offeradvantages to new protein subunit vaccines or live attenuated my-cobacterial vaccines that are currently under development. Thus,the guinea pig model may provide valuable opportunities for test-ing the efficacy of lipid Ag vaccination in a small animal modelthat includes the potential for a prominent group 1 CD1-restrictedT cell response.

AcknowledgmentsWe thank Daphney Frederique for technical assistance in preparing lipo-somes, Gerald Watts for purification of mAb, and William Lane at theHarvard Microchemistry Facility for performing analyses of amino acidcontent of lipid preparations.

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