Leishmania Major by a Monoclonal ␣␤ T Cell Repertoire1

Control of Leishmania major by a Monoclonal ␣␤ T Cell Repertoire1

Steven L. Reiner,2* Deborah J. Fowell,†‡ Naomi H. Moskowitz,* Kevin Swier,* Daniel R. Brown,* Charles R. Brown,* Christoph W. Turck,†§ Phillip A. Scott,2¶ Nigel Killeen,‡ and Richard M. Locksley3†‡§

Little is known regarding the diversity of the host T cell response that is required to maintain immunologic control of microbial pathogens. Leishmania major persist as obligate intracellular parasites within macrophages of the mammalian host. Immunity is dependent upon activation of MHC class II-restricted T cells to an effector state capable of restricting growth and dissemination of the organisms. We generated ␣-␤ Leishmania-specific (ABLE) TCR transgenic mice with MHC class II-restricted T cells that recognized an immunodominant Leishmania Ag designated LACK. Naive T cells from ABLE mice proliferated in vitro after incubation with recombinant LACK or with Leishmania-parasitized macrophages and in vivo after injection into infected mice. Infected ABLE mice controlled Leishmania infection almost as well as wild-type mice despite a drastic reduction in the T cell repertoire. ABLE mice were crossed to mice with disruption of the TCR constant region ␣ gene to create animals with a single ␣␤ T cell repertoire. Although mice deficient in all ␣␤ T cells (TCR-C␣o mice) failed to control L. major, mice with a monoclonal ␣␤ T cell repertoire (ABLE TCR-C␣o mice) displayed substantial control. The immune system is capable of remarkable efficiency even when constrained to recognition of a single epitope from a complex organism. The Journal of Immunology, 1998, 160: 884–889.

eishmania major is an obligate intracellular parasite of lesser or better, but generally incomplete, protection when used as macrophages within mammalian hosts; control of infec- vaccines against subsequent challenge with virulent Leishmania L tion is dependent upon the development of effector T lym- (reviewed in Ref. 13). It is unclear whether optimal protection phocytes capable of activating macrophages to a microbicidal state requires a diverse response against numerous parasite Ags or an (1). Much has been learned regarding the host immune response in optimal response against single, dominant, determinants. Such un- inbred mice using mAb depletion or, more recently, gene targeting. derstanding may have important implications for vaccine design. Whereas MHC class II-restricted ␣␤ T cells are required for re- We have created TCR transgenic mice with MHC class II-resistance (2–4), MHC class I-restricted T cells are dispensable (5). stricted T cells that recognize the LACK4 Ag of Leishmania major by guest on September 30, 2021. Copyright 1998 Pageant Media Ltd. IL-12 (6), IFN-␥ (7, 8), and inducible (macrophage) nitric oxide (14). LACK is highly conserved among Leishmania species, and synthase (9) are required for host immunity, perhaps reflecting constitutes the immunodominant focus of the early CD4ϩ T cell activities mediated by CD40 ligand-CD40 interactions between T response in vivo (15). Crossing these mice to animals with dis- cells and APC (10–12). These numerous experiments suggest that ruption of the TCR C␣ gene (TCR-C␣o) (16) created mice with a MHC class II-restricted Th1 effector cells and activated macro- single ␣␤ T cell repertoire. These mice were used to assess the phages are necessary and sufficient for control of this intracellular requirements for a diverse pathogen-specific T cell repertoire in a infection. disease controlled by MHC class II-restricted T cells. Although these host determinants have been established, the complexity of the Ags presented by the parasite that are necessary https://www.jimmunol.org Materials and Methods for control of disease remains unknown. Numerous Ags provide Transgenic mice The rearranged ␣ and ␤ TCR genes were isolated from a CD4ϩ Th1 clone, *Department of Medicine, Committee on Immunology and Gwen Knapp Center 9.1-2, that was isolated from a BALB/c mouse that had been immunized for Lupus and Immunology Research, University of Chicago, Chicago, IL 60637; with soluble Ags of L. major (17). Clone 9.1-2 expresses rearranged TCR Departments of †Medicine and ‡Microbiology and Immunology, and §Howard gene segments V␣8.2A-J␣TA72 and V␤4-D␤1-J␤1.6 (15). Primers spe- Hughes Medical Institute, University of California, San Francisco, CA 94143; cific for the 5Ј region of V␣8.2A and V␤4 were used in conjunction with ¶ Downloaded from and Department of Pathobiology, University of Pennsylvania School of Veter- antisense primers from the introns beyond J␣TA72 and J␤1.7, respectively, inary Medicine, Philadelphia, PA 19104 to amplify the rearranged V(D)J portions of genomic DNA extracted from Received for publication July 23, 1997. Accepted for publication October clone 9.1-2. The 5Ј portion of each TCR transgene construct containing the 1, 1997. 5Ј untranslated and initial coding region of each V gene was subcloned The costs of publication of this article were defrayed in part by the payment of from cosmids obtained from a BALB/c genomic library (provided by K. page charges. This article must therefore be hereby marked advertisement in Wang and L. Hood, California Institute of Technology, Pasadena, CA). accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The 5Ј portions were spliced into the V(D)J PCR products using unique 1 sites within the V genes of the TCR ␣- and ␤-chains (PstI and SalI, re- This work was supported by National Institutes of Health Grants AI30663 and ␣ AI01309, a fellowship from the Juvenile Diabetes Foundation International (to spectively). The TCR -chain transgene was completed by appending a D.J.F.), and a Burroughs Wellcome Fund Scholarship in Molecular Parasitology 9-kb BamHI fragment of the C␣ locus (provided by D. Loh, Washington (to R.M.L.). 2 Burroughs Wellcome Fund New Investigator in Molecular Parasitology. 4 Abbreviations used in this paper: LACK, Leishmania homologue of receptor for 3 Address correspondence and reprint requests to Dr. R. M. Locksley, University activated C kinase; ABLE, ␣/␤ Leishmania-specific T cell receptor for antigen of California, C-443, 521 Parnassus Ave., San Francisco, CA 94143–0654. transgenic mice; WD, tryptophan-aspartic acid; dull, low level; DN, double neg- E-mail address: [email protected] ative; PE, phycoerythrin.

University, St. Louis, MO) containing 0.8 kb of upstream intron, the entire coding region, and the downstream enhancer to the 3Ј end of the promoter- V-J-intron construct. The TCR ␤-chain transgene was completed by appending a C␤1 and downstream ␤-chain enhancer construct (provided by S. Hedrick, University of California-San Diego) to the 3Ј end of the promoter-V-D-J-intron construct at the J␤-C␤ intron using a naturally occur- ring NsiI site. Equimolar concentrations of gel-purified constructs were injected into ϫ the pronuclei of [C57BL/6 DBA/2] F1 eggs that had been fertilized by BALB/c males before implantation in pseudopregnant recipients. One of three founders that expressed V␤4 on all peripheral blood T cells was used as the progenitor of the ABLE transgenic line. Progeny were backcrossed to B10.D2 mice. Simultaneously, TCR-C␣o mice (16), which lack all ␣␤ T cells, were backcrossed to B10.D2 mice. After five generations, mice were intercrossed to yield TCR-C␣ϩ, TCR-C␣o, and ABLE TCR-C␣o mice on the B10.D2 background. ABLE mice were crossed to the MHC H-2k haplotype as a source of naive donor cells in designated experiments. Reagents Directly fluorescence-conjugated mAbs against CD4, CD8, CD3, V␤4, and CD69 were used for surface immunofluorescent labeling (Caltag, South San Francisco, CA). Anti-MHC class II Abs used in designated experiments included mAb M5/114 (rat IgG2b, anti-Ab,d and anti-Ed,k), mAb MKD6 (mouse IgG2a, anti-Ad), or mAb 10–2–16 (mouse IgG2b, anti-Ak).

The LACK156–173 peptide from the fourth WD domain of the LACK protein (ICFSPSLEHPIVVSGSWD) (14) was synthesized using an Advanced Chemtech Multiple Peptide Synthesizer (Louisville, KY). Peptides were FIGURE 1. T cell development in ABLE TCR-C␣o mice. Six-week- purified by reverse phase HPLC, and their identities were confirmed by old H-2d control (wild-type; left panels) and ABLE TCR-C␣o mice analysis with an LCQ mass spectrometer (Finnigan MAT, San Jose, CA). (right panels) were used to evaluate surface expression of T cell mark- Recombinant LACK protein was expressed in Escherichia coli and purified as previously described (14). ers. Thymocytes were stained with anti-CD4-FITC and anti-CD8-PE before flow cytometric analysis (upper panels). Peripheral lymph Parasites and infections nodes were stained with either anti-V␤4-FITC and anti-CD3-PE (mid- L. major (strain WHOM/IR/-/173) was passaged and maintained as previ- dle panels) or anti-CD4-FITC, anti-CD3-PE, and anti-CD8-Tricolor (lower panels). Ten thousand thymocyte-gated (upper panels), lym- ously described (18). Leishmania amazonensis (strain LV-78) was pro- ϩ vided by K. P. Chang (Chicago Medical School, Chicago, IL). For the phocyte-gated (middle panels), or CD3 lymphocyte-gated (lower infection of macrophages in vitro, the designated numbers of stationary panels) events are displayed. In the lower panels, numbers next to phase promastigotes were cultured for 24 h with bone marrow-derived boxes represent the percentage of CD3ϩ cells expressing CD4 or CD8. macrophages, prepared as previously described (18), in 96-well, flat-bot- Results represent an individual mouse from each group and are rep- ϫ 4 tom, microtiter plates containing 5 10 macrophages/well. The mono- resentative of more than eight mice per group. layers were washed extensively to remove extracellular parasites before

by guest on September 30, 2021. Copyright 1998 Pageant Media Ltd. addition of T cells (see below). For in vivo infections, designated cohorts of four to eight mice were inoculated in the hind footpads with 5 ϫ 105 metacyclic promastigotes of mented tissue culture medium (RPMI 1640 with 2 mM L-glutamine, 0.1 L. major prepared as previously described (18). The sizes of the footpad mM sodium pyruvate, 50 ␮M ␤2-ME, antibiotics, and 10% heat-inacti- ␮ vated FCS) in the presence or the absence of 5 M LACK156–173 peptide. lesions were measured weekly using a metric caliper. Mice were killed ϩ Ϫ after 6 to 10 wk and analyzed as individual animals. The footpad parasite In some experiments, the CD4 and CD4 ABLE T cells were enriched burdens were quantitated by homogenizing tissue in 3 ml of medium 199 into two populations using anti-CD4 mAb coupled to ferrous beads (Ad- supplemented with 20% FCS. Aliquots were diluted serially across 96-well vanced Magnetics, Inc., Cambridge, MA) and separation in a magnetic plates and scored at 1 wk for the presence of motile promastigotes. ﬁeld before incubation with the LACK peptide and irradiated spleen cells Amastigotes were puriﬁed from the spleens and lymph nodes of TCR- at a ratio of 1 T cell:10 spleen cells. After 48 h, supernatants were analyzed C␣o mice that had been infected 10 wk previously with L. major as pre- for IFN-␥ by ELISA (PharMingen).

https://www.jimmunol.org viously described (19). Brieﬂy, tissues were dispersed by repeated forceful expression through a syringe and 27-gauge needle in PBS supplemented Stimulation of ABLE T cells in vivo with 10 mM glucose and 2 mM EDTA until free amastigotes were visible Cohorts of TCR-C␣o mice that had been infected with promastigotes or by light microscopic analysis of smeared aliquots. The organisms were amastigotes of L. major were reconstituted i.v. with 107 V␤4ϩ T cells that recovered after centrifugation from a 45 to 100% Percoll interface, washed were enriched from ABLE mice by complement-mediated lysis of CD8Ϫ, ␣o extensively, counted, and injected into the footpads of naive TCR-C class II- and B220-bearing cells as previously described (20). The puriﬁed ϫ 5 ϩ mice using 5 10 organisms/footpad. cells were Ͼ88% V␤4 T cells. At designated periods, the popliteal lymph

Downloaded from Stimulation of ABLE T cells in vitro nodes were harvested, and cells were stained with fluorescent mAbs against CD4, V␤4, and the activation marker, CD69, for cell surface analysis Naive ABLE T cells were isolated from lymph nodes of MHC H-2k mice (FACScan, Becton Dickinson, Mountain View, CA). crossed to ABLE transgenic mice. Positive selection of the transgenic T cells occurs in MHC H-2k mice, although H-2k APCs neither present the Results LACK Ag or peptide in vitro nor stimulate alloreactivity by ABLE T cells (see Results). T cells (4 ϫ 105/well) were placed in triplicate wells con- Characterization of ABLE transgenic mice ϩ taining H-2d (I-Ad ) bone marrow-derived macrophages that had either ABLE transgenic mice were normal in size and appearance and been infected with Leishmania promastigotes or been incubated with the had normal reproductive capacity (data not shown). Homozygous designated concentrations of the recombinant LACK protein for 24 h. Where designated, irradiated (2000 rad) H-2d spleen cells were used as TCR transgenic animals developed and lived normal life-spans, APCs, using 4 ϫ 105 cells/well. After 48 h, supernatants were collected indicating that the transgene insertion site did not disrupt an es- and analyzed for IL-2 production by ELISA (PharMingen, San Diego, CA). sential gene (data not shown). Lymphocyte surface markers were 3 Alternatively, 1 ␮Ci of [ H]thymidine was added to each well, and the examined on cells from H-2d, age-matched, wild-type, ABLE and radioactivity incorporated was assessed after 18-h additional incubation as ␣o an index of T cell proliferation. ABLE TCR-C mice using fluorescence-conjugated mAbs and ABLE T cells from the popliteal lymph nodes of infected mice were flow cytometry. Results were similar using ABLE and ABLE cultured in triplicate in 96-well round-bottom microtiter plates in supple- TCR-C␣o mice (despite the use of endogenous TCR ␣-chains in 886 LEISHMANIA-SPECIFIC TCR TRANSGENIC MICE

the former), and only the latter are shown (Fig. 1). Compared with control mice, ABLE TCR-C␣o mice had an increase in CD4ϩ single-positive and a reduction in CD8ϩ single-positive thymocytes, consistent with the class II restriction of the TCR. In the peripheral lymph nodes, control mice had only a small population of CD3ϩ cells that expressed V␤4 TCR. In contrast, essentially all CD3ϩ cells from the lymph nodes of ABLE and ABLE TCR-C␣o mice expressed V␤4 TCR. Analysis of the CD3ϩ cells revealed the expected distribution of CD4 and CD8 coreceptors in control mice. ABLE TCR-C␣o mice had marked reduction in CD8ϩ T cells, as anticipated, but unexpectedly, had a reduced number of CD4ϩ T cells and a majority population of CD3ϩ cells that expressed a CD4ϪCD8Ϫ or a CD4ϪCD8dull double-negative (DN) phenotype (Fig. 1). When bred to the MHC H-2k MHC haplotype, ABLE T cells developed comparably and displayed both CD4ϩ and DN phenotypes (data not shown).

ABLE T cells are activated by recombinant LACK Ag and parasitized macrophages ABLE T cells were isolated from ABLE-H-2k mice, which support positive thymic selection and peripheral localization of the ABLE transgenic T cells. H-2d macrophages that had been infected with either L. major or L. amazonensis or had been incubated with the recombinant LACK Ag stimulated IL-2 production from ABLE T cells in a dose-dependent fashion (Fig. 2A). T cells from wild-type B10.D2 mice produced no IL-2 in response to LACK peptide or parasitized macrophages under these conditions (data not shown). Lymph node cells from the ABLE-H2k mice did not by themselves support IL-2 production by the peptide, demonstrating the inability of H-2k to present the LACK epitope, and the requirement of LACK Ag for the production of IL-2 was consistent with the absence of alloreactivity (Fig. 2B). The speciﬁcity of the restriction was conﬁrmed by the capacity of anti-MHC class II I-Ad mAb to abrogate IL-2 production (Fig. 2B) and by Ag-dependent prolif- FIGURE 2. Activation of ABLE T cells in vitro. A, H-2d bone mar- d row-derived macrophages were incubated with L. major (closed cir-

by guest on September 30, 2021. Copyright 1998 Pageant Media Ltd. eration in response to ﬁbroblasts transfected with I-A (data not cles), L. amazonensis (closed squares), or increasing amounts of re- shown). Similar results were obtained if T cell proliferation was combinant LACK Ag (open circles with dose in micrograms per 3 assayed using the incorporation of [ H]thymidine: the mean uptake milliliters in parentheses) for 24 h and washed extensively before the using ABLE lymph node cells incubated with the LACK peptide addition of H-2k ABLE lymph node cells. After 48 h, supernatants were was 46,100 Ϯ 2,300 cpm, and this decreased to 1,100 Ϯ 880 cpm collected and analyzed for IL-2 production by ELISA. B, Lymph node in the presence of blocking anti-I-Ad mAb and to 1,250 Ϯ 740 cpm cells from H-2k ABLE mice were incubated with (ϩ) and without (Ϫ)

in the absence of the LACK peptide. The LACK-dependent pro- irradiated H-2d spleen cells (APCs), LACK153–176 peptide (peptide), or liferation, production of IL-2, and I-Ad restriction could be dem- blocking anti-MHC class II mAb (anti-class II). Data represent the onstrated using both puriﬁed CD4ϩ and DN T cells isolated from means and SDs of triplicate determinations. Results are representative o of three separate experiments. https://www.jimmunol.org ABLE TCR-C␣ mice (data not shown).

ABLE T cells are activated by Leishmania Ags expressed in vivo Prior studies using LACK-reactive hybridomas have suggested number and CD69 expression increased in relation to the duration that amastigotes within macrophages cease to present the LACK of the infection. Thus, ABLE T cells became activated in response

Downloaded from epitope with surface MHC class II molecules (21). To assess this to infected macrophages in vitro and in vivo. possibility using ABLE T cells, TCR-C␣o mice were infected with amastigotes puriﬁed from the lymphoid tissues of previously in- ABLE mice control infection with L. major fected TCR-C␣o mice, thus establishing infection only with the B10.D2 mice are resistant to L. major and control infection with intracellular form of the parasite. A similar cohort was infected the development of small lesions at the site of inoculation of or- with metacyclic promastigotes. Either the same day or 4 or 28 days ganisms. After infection, B10.D2 ABLE mice also displayed con- later, animals were reconstituted with 107 V␤4ϩ CD4ϩ T cells trol markedly different from that shown by concurrently inoculated from ABLE mice. After varying periods, the popliteal lymph node susceptible BALB/c mice (Fig. 3A). Occasional ABLE mice had cells were collected, and the total numbers of cells were enumer- some enlargement of the lesions over 10 to 12 wk compared with ated and stained with mAbs to V␤4, CD4, and CD69 (Table I). By B10.D2 controls, but the vast majority had a completely wild-type this analysis, large numbers of the transferred ABLE TCR trans- phenotype and controlled parasite replication in both the footpad genic T cells expressed the activation Ag CD69, which was ex- and spleen (Fig. 3B). Infected ABLE mice developed type 1 im- pressed by 5 to 8% of the cells after transfer into uninfected TCR- mune responses, as assessed by the production of IFN-␥, but not C␣o mice. The acquisition of CD69 expression occurred after IL-4, by peripheral lymph node cells in culture (data not shown). infection with both promastigotes and amastigotes, and both T cell Thus, restriction of the T cell repertoire to V␤4-expressing cells, The Journal of Immunology 887

Table I. Activation of ABLE T cells after transfer into infected TCR-C␣0 mice a

Promastigote infected Amastigote infected

Cells Cells Total T Cell No. Total T Cell No. Transferred Analyzed (ϫ105) % CD69ϩ (ϫ105) % CD69ϩ

Day 0 Day 5 4.3 15.8 2.2 13.5 Day 14 12.8 38.3 11.1 27.4 Day 28 52.8 47.8 69.0 70.0 Day 4 Day 14 21.5 25.6 34.2 35.7 Day 28 Day 40 92.0 62.0 86.0 56.7 a Cohorts of three to four B10.D2 TCR-C␣0 mice were infected with metacyclic promastigotes or tissue-derived amastigotes of L. major in the hind footpads and then reconstituted with 107 puriﬁed V␤4ϩ T cells from B10.D2 ABLE mice on the day of infection (day 0), or 4 (day 4) or 28 (day 28) days later. The popliteal lymph nodes were harvested on the days designated after T cell reconstitution for analysis (cells analyzed), and the total number of T cells was counted (total T cell number ϫ 105) and the V␤4ϩ CD4ϩ T cells were analyzed for their expression of CD69, which is depicted as a percentage. Results varied within 10% among different mice in each cohort. Reconstituted T cells recovered from uninfected mice were 5 to 8% positive for CD69 expression at the various time points analyzed.

FIGURE 4. L. major infection in ABLE TCR-C␣o mice. A, Cohorts of B10.D2 (open circles), TCR-C␣o (open squares), and ABLE TCR-C␣o (closed circles) mice were infected with L. major promastigotes, and the footpad lesions were measured weekly to assess disease progres- sion. Two experiments are graphed separately. The numbers of animals in each group are indicated in parentheses. Data points represent means and SDs. B, Footpad tissues from the designated mice were collected at the end of the experiments, homogenized, and cultured in vitro to assess the recovery of viable parasites using limiting dilution. Results represent the mean of the two animals that were analyzed in each group and were comparable in separate experiments. C, CD4ϩ by guest on September 30, 2021. Copyright 1998 Pageant Media Ltd. and DN T cells were enriched from infected ABLE TCR-C␣o mice and restimulated in vitro using irradiated I-Ad spleen cells with and without ␥ LACK156–173 peptide. After 48 h, supernatants were analyzed for IFN- by ELISA. Results represent means and SDs from triplicate determina- FIGURE 3. L. major infection in ABLE mice. A, Cohorts of five re- tions. IFN-␥ production in the absence of added peptide was below the sistant B10.D2, susceptible BALB/c, and ABLE mice were infected with detection limit of the assay. L. major promastigotes, and the footpad lesions were monitored over 10 wk. Data represent means and SDs. Results are representative of five comparable experiments. B, Footpad tissues from the designated o mice were collected at the end of the experiment, homogenized, and B). In contrast, ABLE TCR-C␣ B10.D2 mice more closely re- https://www.jimmunol.org cultured in vitro for the recovery of viable parasites after limiting di- sembled wild-type mice, with normal weight gain and activity, but lution. Results represent means of the log final dilutions after evalua- with small, nonulcerating, footpad lesions that were approximately tion of each mouse individually. 1 mm larger than those in infected wild-type mice. Further, the number of recovered parasites from ABLE TCR-C␣o mice was 1,000-fold greater than that in wild-type animals, although this was ␣o ␣ ␤ over 10,000-fold fewer than that in TCR-C mice. Popliteal Downloaded from many of which express the clonotypic V 8/V 4 LACK-specific lymph node cells isolated from infected mice and restimulated in receptor, did not compromise immunologic control of Leishmania. vitro with the LACK peptide produced large amounts of IFN-␥ and Due to incomplete allelic exclusion of TCR ␣-chains in TCR negligible quantities of IL-4, consistent with the development of transgenic mice and our inability to create a clonotypic mAb, we Th1 effector cells. When CD4ϩ and DN cells from the ABLE could not assess the contributions to host defense by the nonclo- TCR-C␣o mice were enriched, both populations were capable of notypic T cell repertoire, e.g., cells that expressed the V␤4 trans- generating IFN-␥ in an Ag-dependent manner (Fig. 4C). Thus, gene together with endogenous V␣-chains. To exclude a role for substantial disease control was maintained by a monoclonal rep- endogenous TCR ␣ gene products, ABLE mice were crossed to ertoire consisting of a single parasite-specific TCR-␣␤. TCR-C␣o mice, thus creating animals with a single T cell repertoire that recognized the LACK determinant. Unlike B10.D2 mice, ␣␤ T cell-deficient B10.D2 mice (TCR-C␣o) were highly suscep- Discussion tible to L. major. These mice developed large footpad lesions, We describe the production of TCR transgenic mice that express displayed loss of weight associated with systemic dissemination, an MHC class II-restricted TCR specific for the immunodominant and had significantly greater numbers of parasites recovered from Ag of L. major and demonstrate the capacity of mice that express the footpads and spleens than did wild-type animals (Fig. 4, A and only this TCR to affect substantial control over the replication of 888 LEISHMANIA-SPECIFIC TCR TRANSGENIC MICE

this intracellular protozoan parasite. Leishmania are complex or- these cells from the thymus before transit through the usual ganisms with an estimated 35.5-megabase genome expressing CD4ϩCD8ϩ, double-positive, stage (20, 26, 27). Despite their un- some 10,000 proteins (22). As best determined by protein frac- usual lineage, such cells accumulate in the periphery as fully Ag- tionation and expression screening, the 18-amino acid determinant reactive T cells (26, 27). CD4ϩ and DN ABLE T cells proliferated in the LACK Ag presented by I-Ad represents the only Leishma- in response to the LACK Ag and differentiated to competent ef- nia-derived Ag recognized by the ABLE T cells (14, 17). The fector type 1 cells during infection with L. major, consistent with remarkable capacity to establish a healer phenotype despite such prior observations regarding the capacity of CD4-deficient Th cells drastic reduction in the Th cell repertoire emphasizes the sensitiv- to function as Th1-like cells (4, 20, 28). ity of the adaptive immune response to single foreign determinants One intriguing aspect of the LACK Ag regards the finding that within pathogens and, further, the inability of Leishmania to es- deletion of the LACK-reactive T cell repertoire, either by thymic cape immune surveillance of this epitope, at least under the con- expression of LACK (29) or by superantigen-mediated deletion ditions used in these experiments. Although control of infection after infection with mouse mammary tumor virus (SIM) (30), ab- was incomplete, as shown by both lesion development and the rogated the exquisite susceptibility of BALB/c mice to L. major, numbers of recovered parasites, it should be emphasized that im- thus enabling infected mice to heal. Thus, recognition of the munity against Leishmania is never sterile, even in resistant inbred LACK epitope was not required to effect a healer phenotype. mouse strains (23), and that the mice were visibly indistinguish- CD4ϩ T cells from BALB/c mice have a genetic propensity to able from healer mice. produce more IL-4 during priming than do T cells from B10.D2 Extensive prior studies have documented the requirements for mice (31), a finding that may reflect extinction of IL-12-mediated class II-restricted T cells in mediating host protection against these signaling in committed T cells (32). The immunodominant re- parasites (1). Contributions from the innate immune system or ␥␦ sponse to LACK coupled with a genetic tendency to overproduce T cells in providing host defense in ABLE TCR-C␣o mice were IL-4 may combine to mediate susceptibility of BALB/c mice to L. unlikely, as established by the inability of infected TCR-C␣o mice major. Abrogation of the early IL-4 produced in these mice in to contain disease. The substantial protection mediated by ABLE response to LACK was sufficient to reverse susceptibility (30), T cells may reflect the dominant nature of the LACK epitope. The implying that additional Ags from Leishmania are capable of fo- V␤4/V␣8 TCR expressed by ABLE T cells represents an oligo- cusing a protective Th1 response. The absence of reactivity to clonal T cell response that occurred in the lymph nodes of mice these additional Ags may explain the greater parasite burdens that early after infection with L. major (15). This restricted response occurred in infected ABLE TCR-C␣o compared with those in may account for the isolation of clone 9.1-2, a Th1 clone estab- wild-type, mice. Reconstitution of ABLE TCR-C␣o mice with ␣␤ lished from an immunized BALB/c mouse, that mediated protec- T cells with additional parasite specificities will be useful in de- tion after transfer into lightly irradiated recipients (17). Impor- termining the minimal requirements for a fully competent immune tantly, these data establish the capacity of ABLE T cells to alone response to complex organisms. Such insights will be important in mediate protection by differentiation to effector cells in situ, in considering vaccine approaches using recombinant proteins. contrast to the protection mediated by the adoptive transfer of The observation that ABLE mice can sustain protective immune clone 9.1-2, a fully committed Th1 effector cell, in the setting of a responses to L. major illustrates the ability to use TCR transgenic

by guest on September 30, 2021. Copyright 1998 Pageant Media Ltd. diverse T cell repertoire (17). The dominant nature of the LACK mice to examine effector T cell differentiation in vivo. To the best epitope could be due to a combination of factors, including Ag of our knowledge, these mice remain the only MHC class II-re- abundance, processing, and stability; MHC affinity; and/or T cell stricted TCR transgenic mice expressing pathogen-specific recep- precursor frequency. tors. Although studies employing peptide Ags have questioned the The LACK Ag is highly conserved among different species of fidelity of T cell reactivity in TCR transgenic mice, as assessed in

Leishmania (14). The immunogenic LACK156–173 epitope was vivo (33), organisms and/or their biologic products often engender completely conserved, and 9.1-2 cells proliferated after incubation responses quite different from those induced by isolated protein with APCs that had been primed with soluble extracts from all reagents (34–36). The requirements for activation or generation of species of Leishmania tested (14, 17). As demonstrated here, this polarized Th subset responses in vivo may be more readily re- https://www.jimmunol.org epitope is presented by viable organisms during infection by dif- vealed in the presence of chronic stimulation achieved by persis- ferent Leishmania species in vitro and in vivo, as demonstrated by tent infection. The ABLE mice should provide insights into the the activation of ABLE T cells after transfer into mice infected homing, activation, expansion, maturation, and death of Th cells in with amastigotes and by the control of disease in ABLE TCR-C␣o response to biologic agents in vivo, much as prior experiments mice. LACK represents the Leishmania homologue of RACK1, a using MHC class I, virus-speciﬁc, TCR transgenic mice have pro- membrane-associated protein to which activated PKC translocates vided information regarding cytotoxic T cells (37).

Downloaded from from the cytosol, presumably to establish an enzymatically active complex (24). In turn, these proteins are members of the highly Acknowledgments conserved WD motif proteins that perform similar scaffold-like functions critical for numerous intracellular activities (25). The The authors thank members of the laboratory for helpful discussions, and requirements for binding substrates by distinct domains of these N. Glaichenhaus (University of Nice, Nice, France) for the kind gift of recombinant LACK Ag. molecules may underlie both their conserved nature and their req- uisite biologic functions. LACK may be an indispensable compo- nent of the organisms’ cell biology that has evolved to provide an References invariant target in the mammalian host. 1. Reiner, S. L., and R. M. Locksley. 1995. The regulation of immunity to Leish- One peculiar aspect of the ABLE mice was the large numbers of mania major. Annu. Rev. Immunol. 13:151. 2. Moll, H., R. Scollay, and G. F. Mitchell. 1988. Resistance to cutaneous leish- TCR transgene-positive DN cells that occurred in both thymic and maniasis in nude mice injected with L3T4ϩ T cells but not with Ly-2ϩ T cells. peripheral compartments. These cells, which have been noted pre- Immunol. Cell Biol. 66:57. viously in TCR transgenic mice (discussed in Ref. 26), may arise 3. Varkila, K., R. Chatelain, L. M. Leal, and R. L. Coffman. 1993. Reconstitution of C.B-17 scid mice with BALB/c T cells initiates a T helper type-1 response and by an accelerated developmental process in response to premature renders them capable of healing Leishmania major infection. Eur. J. Immunol. expression of the rearranged transgene that supports the release of 23:262. The Journal of Immunology 889

4. Locksley, R. M., S. L. Reiner, F. Hatam, D. R. Littman, and N. Killeen. 1993. 20. Fowell, D. J., J. J. Magram, C. W. Turck, N. Killeen, and R. M. Locksley. 1997. Helper T cells without CD4: control of leishmaniasis in CD4-deficient mice. Impaired Th2 subset development in the absence of CD4. Immunity 6:559. Science 261:1448. 21. Prina, E., T. Lang, N. Glaichenhaus, and J.-C. Antoine. 1996. Presentation of the 5. Wang, Z.-E., S. L. Reiner, F. Hatam, F. P. Heinzel, J. Bouvier, C. W. Turck, and protective parasite antigen LACK by Leishmania-infected macrophages. J. Im- ␤ R. M. Locksley. 1993. Targeted activation of CD8 cells and infection of 2- munol. 156:4318. microglobulin-deficient mice fail to confirm a primary protective role for CD8 22. Ivens, A. C., and J. M. Blackwell. 1996. Unraveling the Leishmania genome. cells in experimental leishmaniasis. J. Immunol. 151:2077. Curr. Opin. Genet. Dev. 6:704. 6. Mattner, F., J. Magram, J. Ferrante, P. Launois, K. Di Padova, R. Behin, M. K. Gately, J. A. Louis, and G. Alber. 1996. Genetically resistant mice lacking 23. Aebischer, T., S. F. Moody, and E. Handman. 1993. Persistence of virulent Leish- interleukin-12 are susceptible to infection with Leishmania major and mount a mania major in murine cutaneous leishmaniasis: a possible hazard for the host. polarized Th2 cell response. Eur. J. Immunol. 26:1553. Infect. Immun. 61:220. 7. Belosevic, M., D. S. Finbloom, P. H. van der Meide, M. V. Slayter, and 24. Ron, D., C.-H. Chen, J. Caldwell, L. Jamieson, E. Orr, and D. Mochly-Rosen. C. A. Nacy. 1989. Administration of monoclonal anti-IFN-␥ antibodies in vivo 1994. Cloning of an intracellular receptor for protein kinase C: a homolog of the abrogates natural resistance of C3H/HeN mice to infection with Leishmania ma- ␤ subunit of G proteins. Proc. Natl. Acad. Sci. USA 91:839. jor. J. Immunol. 143:266. 25. Neer, E. J., C. J. Schmidt, R. Nambudripad, and T. F. Smith. 1994. The ancient 8. Wang, Z.-E., S. L. Reiner, S. Zheng, D. K. Dalton, and R. M. Locksley. 1994. regulatory-protein family of WD-repeat proteins. Nature 371:297. ϩ ␥ CD4 effector cells default to the Th2 pathway in interferon- -deficient mice 26. Capone, M., J. Curnow, G. Bouvier, P. Ferrier, and B. Horvat. 1995. T cell infected with Leishmania major. J. Exp. Med. 179:1367. development in TCR-␣␤ transgenic mice: analysis using V(D)J recombination 9. Wei, X.-Q., I. G. Charles, A. Smith, J. Ure, G.-J. Feng, F.-P. Huang, D. Xu, substrates. J. Immunol. 154:5165. W. Muller, S. Moncada, and F. Y. Liew. 1995. Altered immune responses in mice 27. Liu, C.-P., J. W. Kappler, and P. Marrack. 1996. Thymocytes can become mature lacking inducible nitric oxide synthase. Nature 375:408. ϩ ϩ 10. Soong, L., J.-C. Xu, I. S. Grewal, P. Kima, J. Sun, B. J. Longley, Jr., T cells without passing through the CD4 CD8 , double-positive stage. J. Exp. N. H. Ruddle, D. McMahon-Pratt, and R. A. Flavell. 1996. Disruption of CD40- Med. 184:1619. CD40 ligand interactions results in an enhanced susceptibility to Leishmania 28. Rahemtulla, A., T. M. Kundig, A. Narendran, M. F. Bachmann, M. Julius, amazonensis infection. Immunity 4:263. C. J. Paige, P. S. Ohashi, R. M. Zinkernagel, and T. W. Mak. 1994. Class II major 11. Campbell, K. A., P. J. Ovendale, M. K. Kennedy, W. C. Fanslow, S. G. Reed, and histocompatibility complex-restricted T cell function in CD4-deficient mice. Eur. C. R. Maliszewski. 1996. CD40 ligand is required for protective cell-mediated J. Immunol. 24:2213. immunity to Leishmania major. Immunity 4:283. 29. Julia, V., M. Rassoulzadegan, and N. Glaichenhaus. 1996. Resistance to Leish- 12. Kamanaka, M., P. Yu, T. Yasui, K. Yoshida, T. Kawabe, T. Horii, T. Kishimoto, mania major induced by tolerance to a single antigen. Science 274:421. and H. Kikutani. 1996. Protective role of CD40 in Leishmania major infection at 30. Launois, P., I. Maillard, S. Pingel, K. G. Swihart, I. Xenarios, H. Acha-Orbea, two distinct phases of cell-mediated immunity. Immunity 4:275. H. Diggelmann, R. M. Locksley, H. R. MacDonald, and J. A. Louis. 1997. IL-4 13. Bogdan, C., and M. Rollinghoff. 1996. The impact of the type 1 and type 2 T produced by V␤4V␣8 CD4ϩ T cells instructs Th2 development and the suscep- helper cell concept on novel vaccine design with emphasis on protection against tibility to Leishmania major in BALB/c mice. Immunity 6:541. Leishmania parasites. In Concepts in Vaccine Development, S. H. E. Kaufmann, ed. Walter de Gruyter & Co., Berlin, pp. 205–242. 31. Hsieh, C.-S., S. E. Macatonia, A. O’Garra, and K. M. Murphy. 1995. T cell 14. Mougneau, E., F. Altare, A. E. Wakil, S. Zheng, T. Coppola, Z.-E. Wang, genetic background determines default T helper phenotype development in vitro. R. Waldmann, R. M. Locksley, and N. Glaichenhaus. 1995. Expression cloning J. Exp. Med. 181:713. of a protective Leishmania antigen. Science 268:563. 32. Guler, M. L., J. D. Gorham, C.-S. Hsieh, A. J. Mackey, R. G. Steen, 15. Reiner, S. L., Z.-E. Wang, F. Hatam, P. Scott, and R. M. Locksley. 1993. Th1 and W. F. Dietrich, and K. M. Murphy. 1996. Genetic susceptibility to Leishmania: Th2 cell antigen receptors in experimental leishmaniasis. Science 259:1457. IL-12 responsiveness in Th1 cell development. Science 271:984. 16. Mombaerts, P., A. R. Clarke, M. A. Rudnicki, J. Iacomini, S. Itohara, 33. Kearney, E. R., K. A. Pape, D. Y. Loh, and M. K. Jenkins. 1994. Visualization J. J. Lafaille, L. Wang, Y. Ichikawa, R. Jaenisch, M. L. Hooper, and of peptide-specific T cell immunity and peripheral tolerance induction in vivo. S. Tonegawa. 1992. Mutations in T-cell antigen receptor genes ␣ and ␤ block Immunity 1:327. thymocyte development at different stages. Nature 360:225. 34. Vella, A. T., J. E. McCormack, P. S. Linsley, J. W. Kappler, and P. Marrack. 17. Scott, P., P. Caspar, and A. Sher. 1990. Protection against Leishmania major in 1995. Lipopolysaccharide interferes with the induction of peripheral T cell death. BALB/c mice by adoptive transfer of a T cell clone recognizing a low molecular Immunity 2:261. weight antigen released by promastigotes. J. Immunol. 144:1075. 35. Kundig, T. M., A. Shahinian, K. Kawai, H.-W. Mittrucker, E. Sebzda,

by guest on September 30, 2021. Copyright 1998 Pageant Media Ltd. 18. Reiner, S. L., S. Zheng, Z.-E. Wang, L. Stowring, and R. M. Locksley. 1994. M. F. Bachmann, T. W. Mak, and P. S. Ohashi. 1996. Duration of TCR stimu- Leishmania promastigotes evade interleukin 12 (IL-12) induction by macro- ϩ lation determines costimulatory requirement of T cells. Immunity 5:41. phages and stimulate a broad range of cytokines from CD4 T cells during initiation of infection. J. Exp. Med. 179:447. 36. Brown, D. R., J. M. Green, N. H. Moskowitz, M. Davis, C. B. Thompson, and 19. Sadick, M. D., R. M. Locksley, C. Tubbs, and H. V. Raff. 1986. Murine cuta- S. L. Reiner. 1996. Limited role of CD28-mediated signals in T helper subset neous leishmaniasis: resistance correlates with the capacity to generate interfer- differentiation. J. Exp. Med. 184:803. on-␥ in response to Leishmania antigens in vitro. J. Immunol. 136:655. 37. Zinkernagel, R. M. 1996. Immunology taught by viruses. Science 271:173. https://www.jimmunol.org Downloaded from