The Leishmania major LACK Antigen with an Immunodominant ...

INFECTION AND IMMUNITY, Dec. 2004, p. 6924–6931 0019-9567/04/$08.00⫹0 DOI: 10.1128/IAI.72.12.6924–6931.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Vol. 72, No. 12

The Leishmania major LACK Antigen with an Immunodominant Epitope at Amino Acids 156 to 173 Is Not Required for Early Th2 Development in BALB/c Mice Ben L. Kelly and Richard M. Locksley* Departments of Medicine and Microbiology/Immunology, Howard Hughes Medical Institute, University of California—San Francisco, San Francisco, California Received 13 July 2004/Returned for modification 22 August 2004/Accepted 5 September 2004

The Leishmania major LACK antigen contains an immunodominant epitope at amino acids 156 to 173 (LACK156-173) that is believed to nucleate the pathological Th2 immune response in susceptible BALB/c mice. To test this hypothesis, we generated L. major parasites that express a mutated LACK that fails to activate V␤4/V␣8 T-cell receptor transgenic T cells specific for this epitope. Although mutant parasites attenuated the expansion of endogenous LACK-specific, interleukin-4 (IL-4)-expressing, CD4 T cells compared to wild-type parasites in vivo, the overall frequency of IL-4 and gamma interferon-secreting lymphocytes was similar to that elicited by wild-type L. major. Mutant parasites demonstrated diminished amastigote viability and delayed lesion development in mice, although parasites could be recovered over 200 days after infection. Complementation with a wild-type lack fusion construct partially rescued these defects, indicating a role for endogenous LACK in parasitism. Mice inoculated with mutant parasites were not protected against subsequent infection with wild-type L. major. Leishmania major is a clinically important intracellular protozoan pathogen that has been used extensively in inbred strains of mice to examine the relationship between T helper (Th) cell subset differentiation and disease susceptibility. Immunologic control of the intracellular amastigotes requires activation of phagocytic cells, particularly macrophages, in which the organisms live and divide in the vertebrate host. The protective response is dependent on gamma interferon (IFN-␥) elaborated by CD4 Th1 cells, which orchestrate immunity in resistant strains, such as C57BL/6 and B.10.D2 mice. Conversely, susceptible BALB/c mice develop an aberrant Th2 response to the parasite, which results in failure to restrict the growth of intracellular amastigotes with fatal dissemination (1, 15). A striking observation remains the ability of interleukin-4 (IL-4) antibody, when administered at the time of infection, to reverse the phenotype in BALB/c mice by a process associated with the differentiation of protective Th1 cells (19). Expression cloning was used to identify the immunodominant focus of the early BALB/c CD4 Th2 cell response as LACK, an L. major RACK-like homolog of the WD protein family (12), and peptide mapping was used to establish a dominant epitope comprising amino acids 156 to 173 as the major LACK peptide (LACK156-173) presented by I-Ad major histocompatibility complex (MHC) molecules (9). Corroboration of these findings included the generation of resistant BALB/c mice by expression of LACK in the thymus, thus leading to the deletion of the LACK-reactive repertoire (6), and the use of antagonist LACK peptides to attenuate the course of disease in BALB/c mice (13). Furthermore, deletion of the few CD4 T cells identified by using LACK156-173/I-Ad tetramers from the endog-

enous BALB/c T-cell repertoire resulted in attenuation, although not cure, when transferred into immunodeficient recipients (23). Moreover, deletion of the LACK-specific repertoire in resistant B10.D2 mice further facilitated parasite clearance, suggesting that this immune response is deleterious in I-Ad-expressing mice, even on a resistant genotype (5). The LACK156-173 epitope is highly conserved among all Leishmania species (24). In recent studies, we provided evidence that at least one lack copy is required for parasite viability and that at least two lack copies appear to be required to maintain virulence in the vertebrate host (7). We mutated the LACK156-173 epitope to create a peptide that did not activate transgenic T cells specific for the wild-type sequence. Although mutant promastigotes were generally comparable to wild-type parasites, amastigote forms grew poorly within macrophages and were less virulent in BALB/c mice. Immunologic studies, however, showed that the overall numbers of CD4 T cells that secreted IL-4 and gamma interferon (IFN-␥) were unchanged early after infection with the mutant parasites and further that prior infection with mutant parasites did not protect from subsequent challenge with wild-type organisms. MATERIALS AND METHODS Construction of plasmids for LACK gene targeting and complementation. To replace LACK1 with the mutagenized LACK coding region LACKH164N (amino acid residue His 164 substituted for Asn), designated LACK-N164, the construct pL1SD-LKN-L1 (Fig. 1B) was made by ligating the 1.3-kb 5⬘ LACK1 fragment (fragment a, Fig. 1) 5⬘ to the SAT⫺ DHFRTS drug selection cassette (7), followed by LACK-N164 (13) ligated downstream of SAT⫺ DHFRTS. A 3.3-kb fragment (fragment b) from immediately downstream of the LACK1 stop codon was then ligated 3⬘ to the LACK-N164 sequence (Fig. 1). L. major LACK-N164 mutants were complemented with pXGLACK. This plasmid consisted of the LACK coding region downstream of sequence encoding a hemagglutinin epitope, six histidine residues, and a factor X cleavage site (13) inserted into pXG (3; S. Pingel, unpublished data). Transfection and selection of LACK-deficient L. major clones. Replacement of the remaining LACK1 gene of lack⫹⫺/⫺⫺ parasites (7) with LACK-N164 was

* Corresponding author. Mailing address: UCSF Medical Center, Room C-443, 521 Parnassus Ave., San Francisco, CA 94143-0654. Phone: (415) 476-5859. Fax: (415) 476-9364. E-mail: locksley@medicine .ucsf.edu. 6924

VOL. 72, 2004

L. MAJOR LACK AND EARLY Th2 DEVELOPMENT IN MICE

6925

FIG. 1. Targeting the L. major lack genes. (A) Physical map of the L. major lack genes with restriction enzyme sites indicated: B, BamHI; H, HindIII; K, KpnI; N, NsiI; S, StuI; Sp, SphI. The gray box denotes the LACK1 coding region. Fragments a and b (heavy lines) were used to make the targeting construct. (B) Diagram of the targeting construct. DHFR, dihydrofolate reductase; LACKN, LACK-N164. (C) Southern blot hybridizations of L. major transfectant genomic DNAs. Genomic DNAs (10 ␮g/lane) from L. major lack⫹⫺/⫺⫺ parasites (lane 1), L. major lack⫹⫺/⫺⫺ parasites transfected with L1SD-LKN-L1 (L. major LACK-N164; lane 2), and L. major LACK-N164 parasites transfected with pXGLACK (L. major LACK-N164/pXGLACK; lanes 3 and 4) were triple digested with StuI, BamHI, and SpeI. Genomic DNAs were size fractionated, blotted, and hybridized with 32P-labeled lack coding sequence (lined bar). (D) Lysates from 2 ⫻107 LACK-deficient L. major promastigotes were loaded as follows: lane a, lack⫹⫹/⫺⫺; lane b, LACK-N164; lane c, LACK-N164/pXGLACK. After size fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteins were blotted and incubated with rabbit polyclonal antiserum (␣-) raised against recombinant LACK protein (upper panel). The blot was then stripped and reprobed with goat polyclonal antiserum against eukaryotic 40S ribosomal protein S6 (lower panel).

performed by electroporation with 20 ␮g of purified pL1SD-LKN-L1 insert, followed by selection with nourseothricin as described previously (7) to give rise to L. major LACK-N164 mutant lines. L. major LACK-N164 mutants were complemented by transfection with 40 ␮g of pXGLACK followed by selection in G418. Targeting and transfections were verified by sequencing and Southern and Western blotting for LACK protein as described previously (7). Parasites and mice. Parasites were cultured in medium 199 (M199) supplemented with 10% heat-inactivated fetal calf serum (FCS) and antibiotics as described previously (7). BALB/c mice were purchased from Jackson Laboratories (Bar Harbor, Maine). BALB/c T-cell receptor (TCR)-C␣⫺/⫺ mice (7), BALB/c 4get mice (10), and LACK TCR transgenic ABLE mice (14) were each backcrossed at least 9 generations onto BALB/c. Prior to inoculation into mice, infectious metacyclic forms were purified from stationary-phase promastigote cultures by using peanut agglutinin (PNA), which specifically binds terminal ␤linked galactose moieties on surface lipophosphoglycan side chains from noninfective promastigotes (18), as previously described (13). For infections, designated mice were inoculated subcutaneously in the hind footpad with 4 ⫻ 105 or 2 ⫻ 106 metacyclic promastigotes. Lesions were monitored weekly with a Vernier caliper. Animal experiments were performed in compliance with federal guidelines. Flow cytometric analysis. Where designated, naïve T cells were purified from ABLE ⫻ TCR-C␣⫺/⫺ mice. These cells were labeled with CFSE (5,6-carboxyfluorescein diacetate succinimidyl ester) as described previously (2), and 106 cells

were transferred into recipient BALB/c mice. After infection with L. major, popliteal lymph node cells were collected at 24, 72, and 96 h to assess the early CD4 T-cell response. Cells from ABLE ⫻ TCR-C␣⫺/⫺ BALB/c mice were resuspended at 2 ⫻ 108/ml in 5% FCS in phosphate-buffered saline (PBS), and 15-␮l aliquots were stained for 30 min on ice by mixing 1:1 with the following antibody cocktail: 1/50 fluorescein isothiocyanate (FITC)–anti-CD69; 1/50 phycoerythrin (PE)–anti-TCR V␤4; 1/50 Tricolor (TC)–anti-CD8; 1/50 TC–antiB220 and 1/50 antigen-presenting cell (APC)–anti-CD4 (Pharmingen, San Diego, Calif.; Caltag, Burlingame, Calif.) in 5% FCS–PBS. The cells were washed with 5% FCS–PBS and resuspended at 107/ml in 5% FCS–PBS prior to analysis with a Facscalibur flow cytometer (Becton Dickinson). Acquired data were analyzed with FlowJo software (Tree Star, San Carlos, Calif.). Staining with recombinant MHC II I-Ad/LACK tetramers was used to assess endogenous LACK-specific T cells in infected wild-type and 4get BALB/c mice with a MoFlo cytometer (DakoCytomation, Fort Collins, Colo.) as described previously (23), except that staining for CD4 was performed with APC-Cy7-antiCD4. To control for nonspecific binding of the tetramer reagent to activated CD4 T cells, activated CD4 T cells were isolated from the popliteal lymph nodes of ovalbumin-specific D011.10 BALB/c TCR transgenic mice that had been injected in the footpad 4 days previously with 250 ␮g of ovalbumin in incomplete Freund’s adjuvant. These cells were stained with I-Ad/LACK tetramers and used as a negative control to set gates defining LACK-specific CD4 T-cell staining.

6926

KELLY AND LOCKSLEY

Immunologic analyses. Lymph node suspensions from 4-day-infected mice were cultured as described earlier (21) and restimulated overnight with 50 ␮g of soluble Leishmania antigen extracts per ml (20) prepared by sonication of stationary-phase, wild-type L. major parasites in PBS at 4°C. The number of IL-4and IFN-␥-producing cells per 106 lymphocytes was determined as described previously (21). Parasite enumeration. Viable parasites were enumerated by culturing dispersed popliteal lymph nodes from infected mice in 5 ml of M199 plus 10% FCS. Suspensions were serially diluted in microtiter plates. After 7 days of culture at 27°C, dilutions containing 1 or more viable parasites were scored as positive. Macrophage infections. Bone marrow-derived macrophages from BALB/c mice were cultured on glass chamber slides as described previously (7). Promastigotes were added at a ratio of approximately 10 per macrophage and incubated for 16 h before repeated washing with PBS to remove extracellular organisms. Monolayers were stained after 16 and 96 h with Diff-Quik (DADE Behring, Dudingen, Switzerland), and the numbers of amastigotes per cell were determined microscopically.

RESULTS Generation of LACK-N164-deficient L. major by gene targeting. Transfection of L. major lack⫹⫺/⫺⫺ parasites with a pL1SD-LKN-L1 insert (Fig. 1A and B) resulted in a 3-kb StuI/ BamHI genomic DNA fragment (Fig. 1C, lane 2) that hybridized to lack, which is thus consistent with replacement of the remaining wild-type LACK coding region with LACK-N164. The intensity of this band was approximately threefold greater than that of the lack-hybridizing band observed in L. major lack⫹⫺/⫺⫺ parasites (Fig. 1C, lane 1), indicating that insertion of several copies of LACK-N164 had occurred. DNA sequencing of PCR products amplified from these parasite clones, designated L. major LACK-N164, using lack-specific primers, confirmed replacement of the wild-type His 164 codon for Asn (B.K., data not shown). L. major LACK-N164 parasites were complemented by transfection of the Leishmania expression construct pXGLACK that expresses a LACK fusion protein previously shown to activate LACK-specific TCR transgenic T cells (13) (Fig. 1C, lanes 3 and 4). As predicted by successful transfection, triple digestion of genomic DNA from the transfectants with StuI, BamHI, and SpeI yielded lack-hybridizing genomic DNA fragments of 3 and 1 kb, corresponding to the integrated StuI/ BamHI LACK-N164 fragment and the SpeI/BamHI lack coding fragment from pXGLACK, respectively. These complemented parasite lines were designated L. major LACK-N164/ pXGLACK. LACK protein levels in L. major LACK-N164 promastigotes were determined by Western blotting using anti-LACK antiserum (Fig. 1D) as described previously (7). Taking into account the S6 ribosomal protein loading control (Fig. 1D, lower panel), the L. major LACK-N164 promastigotes showed moderately reduced levels of LACK protein (Fig. 1D, upper panel, lane b) compared to L. major lack⫹⫹/⫺⫺ parasites (Fig. 1D, upper panel, lane a), despite possessing several copies of LACK-N164. Unexpectedly, L. major LACK-N164/pXG LACK organisms showed markedly reduced levels of LACKN164 protein compared to L. major LACK-N164 (Fig. 1D, upper panel, lane c, lower band) and high levels of the wildtype LACK fusion protein expressed from pXG (Fig. 1D, upper panel, lane c, upper band). Fluorescence-activated cell sorting analysis of the mutant parasites after incubation with labeled PNA indicated that the

INFECT. IMMUN.

ability of these mutant parasites to display determinants consistent with metacyclogenesis was comparable to that of wildtype organisms (B.K., data not shown). L. major LACK-N164 parasites do not activate LACK TCR transgenic T cells. ABLE mice express a V␤4/V␣8 TCR specific for the immunodominant LACK peptide in I-Ad (9, 14). Following inoculation of L. major LACK-N164 into the hind footpads of ABLE ⫻ TCR-C␣⫺/⫺ BALB/c mice, the frequency of V␤4⫹ LACK-specific T cells that became activated (Fig. 2A), as indicated by enhanced CD69 expression at 20 h postinfection, was comparable to that of naïve controls, ranging from 1.2 to 1.25%. In contrast, inoculation of L. major lack⫹⫺/⫺⫺ parasites resulted in robust T-cell activation that was comparable to inoculation of wild-type L. major and L. major lack⫹⫹/⫺⫺ parasites (7). Inoculation of L. major LACK-N164/ pXGLACK also caused readily apparent T-cell activation (23.8% CD69-positive cells). The lesser response in LACKN164/pXGLACK as compared to lack⫹⫺/⫺⫺ parasites may reflect partial antagonism by the LACK-N165 epitope, but also the loss of the pXGLACK plasmid from parasites as they escape the selecting drug pressure in vivo. The ability of L. major LACK-N164 parasites to elicit proliferation of LACK-specific T cells was also tested. As shown in Fig. 2B, when mice that had been previously injected with CFSE-labeled, ABLE T cells were challenged with parasites, cell division of I-Ad/LACK-positive T cells, as shown by CFSE dilution at 72 h postinfection, occurred only in mice that received wild-type (lack⫹⫹/⫹⫹) or lack⫹⫺/⫺⫺ parasites. Robust ABLE T-cell division was also observed with L. major LACKN164/pXG LACK (B.K., data not shown). In contrast, no significant proliferation was observed in mice that received an equal dose of L. major LACK-N164. Thus, the mutant LACK epitope has abrogated T-cell recognition by these LACK-specific transgenic T cells. L. major LACK-N164 parasites elicit reduced expansion of endogenous LACK-specific T cells. Having established that L. major LACK-N164 cells do not activate LACK-specific TCR transgenic T cells, activation of endogenous LACK-specific CD4 T cells was investigated with recombinant MHC class II/LACK peptide tetramers and IL-4 reporter mice (4get), as described in Materials and Methods (Fig. 3A). Consistent with the diminished LACK TCR-transgenic T-cell response to LACK-N164 parasites, these organisms elicited reduced expansion compared to LACK wild-type-expressing L. major, with averages of 38, 77, and 132 tetramer-positive cells per lymph node for L. major LACK-N164, LACK-N164/ pXGLACK, and lack⫹⫹/⫺⫺ parasites, respectively, at the peak of the response. The numbers of tetramer-positive cells that were also expressing IL-4 (i.e., green fluorescent protein positive) were 19, 34, and 75 cells per lymph node for LACKN164, LACK-N164/pXGLACK, and lack⫹⫹/⫺⫺, respectively. These represent fourfold- and twofold-higher numbers of IL4-expressing LACK-specific cells for L. major lack⫹⫹/⫺⫺ and LACK-N164/pXGLACK, respectively, as compared to the L. major LACK-N164 mutant. L. major LACK-N164 parasites elicit Th2 cells and fail to protect from challenge with wild-type L. major. To determine whether or not the reduced number of IL-4-expressing LACKspecific CD4 T cells observed following inoculation of L. major LACK-N164 influenced Th cytokine protein expression pro-

VOL. 72, 2004


6927

FIG. 2. Activation and expansion of LACK-specific TCR-transgenic T cells in vivo by L. major LACK-N164. (A) LACK-specific TCRtransgenic (ABLE ⫻ TCR-C␣⫺/⫺) BALB/c mice were inoculated in the hind footpads with 2 ⫻ 106 metacyclic LACK-deficient promastigotes or left uninfected, as indicated. After 20 h, the T cells were analyzed for CD69 expression by flow cytometry. Frequencies (percentages) of V␤4 T cells located within the CD69-positive gates (bar on each panel) are indicated. Data are representative of three experiments. (B) LACK-specific TCR-transgenic T cells from ABLE ⫻ TCR-C␣⫺/⫺ BALB/c mice were isolated, labeled with CFSE, and adoptively transferred into wild-type BALB/c mice. The next day, 2 ⫻ 106 wild-type (lack⫹⫹/⫹⫹) or LACK mutant L. major parasites were inoculated into the hind footpads. After 72 h, the popliteal lymphocytes were isolated and stained with PE-labeled I-Ad/LACK-peptide MHC class II tetramers before analysis with flow cytometry. Each panel is representative of three mice per treatment.

files in the draining lymph nodes, enzyme-linked immunospot assays were performed on popliteal lymphocytes from BALB/c mice infected 96 h previously with either L. major lack⫹⫹/⫺⫺, L. major LACK-N164, or L. major LACK-N164/pXGLACK parasites (Fig. 3B). Unexpectedly, after restimulation in vitro with soluble Leishmania extract, both L. major LACK-N164 and LACK-N164/pXGLACK parasites induced numbers of IL-4-producing and IFN-␥-producing cells that were similar to L. major lack⫹⫹/⫺⫺ parasites, with 120 to 145 IL-4-producing cells and 40 to 55 IFN-␥-producing cells per 106 lymphocytes.

Since L. major LACK-N164 elicited T-helper cytokine profiles in draining lymph nodes that were similar to wild-type L. major, despite the reduced expansion of IL-4-expressing, tetramer-specific cells in 4get mice, the host response to these mutants was further clarified by determining whether or not L. major LACK-N164 could mediate protection in BALB/c mice against subsequent challenge with virulent wild-type L. major (Fig. 3C). The mutant parasites were injected into the left-hind footpad, and 30 days later, the mice were challenged with virulent wild-type L. major in the right footpad, which was

6928

KELLY AND LOCKSLEY

INFECT. IMMUN.

subsequently monitored for lesion development. None of the mutant L. major strains conferred protection on subsequent wild-type parasite infection in BALB/c mice. Taken together, these data indicate that the BALB/c Th2 response remains unperturbed in response to infection with parasites lacking the LACK156-173 epitope. BALB/c mice fail to resolve infection with LACK-N164 and LACK-N164/pXGLACK parasites despite delayed lesion development. The ultimate course of infection of BALB/c mice with L. major lack⫹⫹/⫺⫺, LACK-N164, or LACK-N164/ pXGLACK parasites was also determined (Fig. 4). Infection with L. major lack⫹⫹/⫺⫺ parasites resulted in progressive infection that necessitated sacrificing the mice after 80 days. In contrast, infections by both L. major LACK-N164 and LACK-N164/pXGLACK parasites were significantly attenuated and comparable to previous observations of single-copy lack⫹⫺/⫺⫺ mutants (7). Despite the attenuated course of disease, small ulcerations were present on the primary lesions on most of the mice, consistent with parasite persistence. This was confirmed by terminal cultures of the footpads that revealed low numbers of the LACK-N164 mutants that were somewhat compensated for by the presence of the pXGLACK rescue plasmid (Fig. 4B). LACK-N164 and LACK-N164/pXGLACK parasites are attenuated for macrophage growth in vitro. The attenuated growth of these mutant LACK parasites likely reflects parasiteintrinsic, rather than immunologic, differences, as lesion development was similarly attenuated in BALB/b mice (B.K., data not shown), which do not express I-Ad and do not present the LACK156-173 epitope. To assess this in vitro, bone marrow-derived macrophages demonstrated comparable uptake of each of the mutant organisms, but completely constrained replication of the LACK-N164 mutants at 96 h as compared to either lack⫹⫹/⫺⫺ or LACKN-164/pXGLACK parasites (Fig. 5). DISCUSSION Earlier findings suggested a critical role for LACK156-173specific V␤4⫹ CD4 T cells in the pathological Th2 response to L. major (6, 9, 13). These observations were based on modifications of the host immune response. To clarify these findings, we sought to modify the parasite in a complementary fashion by removing the LACK156-173 epitope from the parasite genome as a stringent test for its requirement in the BALB/c immune response. We used gene targeting to generate L. major cells that express a variant of the LACK protein, LACKN164, which does not activate these T cells (13). As predicted, the LACK-N164 mutant parasites failed to activate transgenic LACK156-173-specific V␤4⫹ T cells and attenuated the expanFIG. 3. CD4 T-cell responses and disease progression in BALB/c mice inoculated with L. major LACK-N164. (A) BALB/c 4get mice were inoculated with 2 ⫻ 106 LACK-deficient L. major parasites or left uninfected, as indicated. After 96 h, the popliteal lymph nodes were dispersed and stained with labeled I-Ad/LACK MHC class II tetramers to determine the numbers of CD4 T cells that were tetramer positive (bars) and, of those, the number that were fluorescent, indicating activation of the IL-4 gene (gray shading). These data represent the averages of two experiments. (B) BALB/c mice were inoculated with 2 ⫻ 106 LACK-deficient L. major parasites, and after 96 h, draining popliteal lymph node cells were isolated and restimulated with L. major

lysates and assayed for the frequencies of IL-4 (open bars)- and IFN-␥ (solid bars)-producing cells with enzyme-linked immunospot assays. These data represent the averages of three experiments. (C) A total of 2 ⫻ 106 metacyclic LACK-deficient parasites were inoculated into the left hind footpad of BALB/c mice as indicated. After 30 days, the mice were inoculated in the right footpad with 4 ⫻105 lesion-derived wildtype metacyclic L. major parasites. Lesion progression in the right footpad was monitored, with the data representing averages of five mice per group.

VOL. 72, 2004


6929

their failure to activate a protective anti-Leishmania immune response. A major conclusion of this study is that the aberrant BALB/c response to L. major proceeds independent from the presence or absence of the dominant LACK epitope. In spite of this finding, while the LACK-N164 peptide is clearly antagonistic in vitro and in vivo (13), the failure of LACK-N164 to antagonize the response to wild-type LACK in the context of live parasites is intriguing. One possible explanation is that the LACK-N164 peptide may not be processed and presented efficiently from the mutant parasites. Indeed, the mutant LACK protein was clearly regulated differently from the wild-type copy, consistent with our previous studies identifying LACK as critical to the intracellular survival of amastigotes (7). It seems unlikely that the response was driven solely by replacement with LACK-N164-specific CD4 T cells that nucleated the pathology, however, since restimulation with wild-type L. major extracts lacking this mutant epitope revealed comparable numbers of IL-4-producing CD4 T cells from mice infected with LACK-N164 parasites (Fig. 3B). Rather, these data corroborate multiple reports that the levels of induction of IL-4-producing CD4 T cells in the draining lymph nodes early after infection (0.5 to 1 week) are comparable in both resistant and susceptible infected animals (11, 16, 22, 23), and thus induction is not linked to the eventual outgrowth of protective Th1 or nonprotective Th2 cells. Furthermore, multiple nonoverlapping approaches have clearly demonstrated that ablation of the LACK-specific CD4 T-cell response prior to infection enhances resistance to the parasite (6, 9, 13, 23). Taken together, the data suggest that ablating a

FIG. 4. Course of infection in BALB/c mice inoculated with mutant L. major. (A) BALB/c mice were inoculated in the hind footpad with 2 ⫻ 106 metacyclic L. major lack⫹⫹/⫺⫺, LACK-N164, or LACK-N164/ pXGLACK parasites. Lesion size and the standard error of the mean are shown (averages of five mice per group). (B) Suspensions of popliteal lymph nodes isolated from mice inoculated in panel A were collected at 90 days postinfection for L. major lack⫹⫹/⫺⫺ (open bar) parasites or 220 days postinfection for L. major LACK-N164 (hatched bar) and L. major LACK-N164/pXGLACK (dotted bar) parasites for culture for viable parasites. The data indicate the last dilution that contained viable L. major for the individual mice: circles, L. major lack⫹⫹/⫺⫺; diamonds, L. major LACK-N164; triangles, L. major LACKN164/pXGLACK.

sion of endogenous LACK156-173-specific CD4 T cells in vivo. Despite the clear demonstration that these parasites eluded activation of the dominant epitope-specific response to LACK, the overall activation of IL-4-producing and IFN-␥-producing cells in the draining lymph nodes remained unaltered as compared to wild-type L. major. These parasites, although highly attenuated for virulence in vivo, failed to protect against subsequent infection with wild-type organisms, consistent with

FIG. 5. Infection of mouse bone marrow-derived macrophages with LACK-deficient L. major. Bone marrow-derived macrophages were infected with metacyclic LACK-deficient L. major promastigotes. Monolayers were washed after 16 h to remove noninternalized parasites and then stained immediately (open bars) or incubated for an additional 3 days and then stained (dark bars). The numbers of amastigotes per 100 macrophages represent averages from four experiments.

6930

KELLY AND LOCKSLEY

restricted set of epitope-specific T cells achieves a different outcome from ablating the epitope of those T cells in the pathogen. Although it remains unclear why the outcomes of these two experimental approaches differ, several variables, including aberrant expression of alternate parasite antigens, abnormal trafficking of mutant parasites in host cells, and effects on T-cell responses after manipulation of the host repertoire, may affect variables in the host response in unknown ways. Indeed, administration of anti-IL-4 or deletion of the LACK-specific T-cell response was associated with a decrease in the overall numbers of early IL-4-producing cells, whereas mutation of the epitope, as shown here, did not decrease the magnitude of the early IL-4 response. Despite the ability of LACK-N164 and LACK-N164/ pXGLACK L. major parasites to elicit a robust Th2 response in BALB/c mice, the attenuation of amastigote growth in vivo suggested parasite-intrinsic defects in these mutants. Parasite recovery at the termination of the experiments suggested that pXGLACK did rescue some of the phenotype, although this was partial (Fig. 4B). The deficiency in these parasites undoubtedly reflects the crucial role for LACK protein in amastigote growth in vivo. Indeed, inoculation of 10-fold-higher parasite doses of LACK-N164 could not overcome the growth attenuation in vivo (B.K., data not shown). Although the ability of LACK-N164 and LACK-N164/pXGLACK parasites to gain entry into macrophages was comparable to that of wild-type parasites, their subsequent proliferation as intracellular amastigotes was impaired (Fig. 5). Similar defects were seen in single-copy lack⫹⫺/⫺⫺ parasites (7), which suggests that LACK -N164 does not function as efficiently as the wild-type protein. Indeed, His 164 is conserved across all Leishmania species studied (24), even when expressed from several copies. We were only able to obtain mutants that contained three copies of LACK-N164, suggesting a process of dose compensation driven by suboptimal function of the mutant protein. Although the 41-kDa LACK fusion protein was efficiently overexpressed in L. major LACK-N164/pXGLACK (Fig. 1D), the reduced abundance of the 36-kDa band encoded by the integrated LACK-N164 gene additionally suggests tight control of LACK, such that elevated levels of wild-type LACK protein may be deleterious in these parasites. This possibility is further supported by failure of LACK overexpression to restore virulence in L. major LACK-N164/pXGLACK. In mammalian cells, the ability of the LACK homolog, RACK1, to suppress certain signaling pathways by diverting and/or sequestering ␤IIPKC has been suggested (8), implying that overexpression of these proteins may have deleterious physiological consequences. Furthermore, in fission yeast, overexpression of the WD repeat adaptor protein, Sum 1, inhibits cell cycle responses to stress (4). Taken together, these data suggest that stringent qualitative and quantitative restrictions may have been placed upon LACK during the evolution of macrophage parasitization by these trypanosomatids. Our observations support further elucidation of the biochemical and physiologic roles of LACK in these parasites as a means to the development of potential drug targeting strategies. Although it is clear that robust Th1 and Th2 responses lead to resistance and susceptibility to L. major infection, respectively, the precise mechanisms by which these disease outcomes occur remain complex (17). These findings

INFECT. IMMUN.

emphasize the need for further study of the interplay between host and parasite determinants in this disease. ACKNOWLEDGMENTS We thank D. B. Stetson for assistance with CFSE labeling, adoptive transfers, and flow cytometry advice; the University of California San Francisco Biomolecular Resource Center for DNA sequencing; C. McArthur for flow cytometry; and N. Flores-Wilson for animal management. This work was supported by NIAID 26918 and the Ellison Medical Foundation. R.M.L. is a Senior Scholar of the Ellison Medical Foundation for Global Infectious Disease. REFERENCES 1. Fowell, D. J., and R. M. Locksley. 1999. Leishmania major infection of inbred mice: unmasking genetic determinants of infectious diseases. BioEssays 21:510–518. 2. Fowell, D. J., K. Shinkai, X. C. Liao, A. M. Beebe, R. L. Coffman, D. R. Littman, and R. M. Locksley. 1999. Impaired NFATc translocation and failure of Th2 development in Itk-deficient CD4⫹ T cells. Immunity 11:399– 409. 3. Ha, D. S., J. K. Schwartz, S. J. Turco, and S. M. Beverley. 1996. Use of green fluorescent protein as a marker in transfected Leishmania. Mol. Biochem. Parasitol. 77:57–64. 4. Humphrey, T., and T. Enoch. 1998. Sum 1, a highly conserved WD-repeat protein, suppresses S-M checkpoint mutants and inhibits the osmotic stress cell cycle response in fission yeast. Genetics 148:1731–1742. 5. Julia, V., and N. Glaichenhaus. 1999. CD4⫹ T cells which react to the Leishmania major LACK antigen rapidly secrete interleukin-4 and are detrimental to the host in resistant B10.D2 mice. Infect. Immun. 67:3641– 3644. 6. Julia, V., M. Rassoulzadegan, and N. Glaichenhaus. 1996. Resistance to Leishmania induced by tolerance to a single antigen. Science 274:421– 423. 7. Kelly, B. L., D. B. Stetson, and R. M. Locksley. 2003. Leishmania major LACK antigen is required for efficient vertebrate parasitization. J. Exp. Med. 198:1689–1698. 8. Korchak, H. M., and L. E. Kilpatrick. 2001. Roles for ␤II-protein kinase C and RACK1 in positive and negative signaling for superoxide anion generation in differentiated HL60 cells. J. Biol. Chem. 276:8910–8917. 9. Launois, P., I. Maillard, S. Pingel, K. G. Swihart, I. Xenarios, H. AchaOrbea, H. Diggelmann, R. M. Locksley, H. R. MacDonald, and J. A. Louis. 1997. IL-4 rapidly produced by V␤4 V␣8 CD4⫹ T cells instructs Th2 development and susceptibility to Leishmania major in BALB/c mice. Immunity 6:541–549. 10. Mohrs, M., K. Shinkai, K. Mohrs, and R. M. Locksley. 2001. Analysis of type 2 immunity in vivo with a bicistronic IL-4 reporter. Immunity 15: 303–331. 11. Morris, L., A. B. Troutt, E. Handman, and A. Kelso. Changes in precursor frequencies of IL-4 and IFN-gamma secreting CD4 T cells correlate with resolution of lesions in murine cutaneous leishmaniasis. J. Immunol. 149: 2715–2721. 12. Mougneau, E., F. Altare, A. E. Wakil, S. Zheng, T. Coppola, Z. E. Wang, R. Waldmann, R. M. Locksley, and N. Glaichenhaus. 1995. Expression cloning of a protective Leishmania antigen. Science 268:563–566. 13. Pingel, S., P. Launois, D. J. Fowell, C. W. Turck, S. Southwood, A. Sette, N. Glaichenhaus, J. A. Louis, and R. M. Locksley. 1999. Altered ligands reveal limited plasticity in the T cell response to a pathogenic epitope. J. Exp. Med. 189:1111–1120. 14. Reiner, S. L., D. J. Fowell, N. H. Moskowitz, K. Swier, D. R. Brown, C. R. Brown, C. W. Turck, P. A. Scott, N. Killeen, and R. M. Locksley. 1998. Control of Leishmania major by a monoclonal alpha beta T cell repertoire. J. Immunol. 160:884–889. 15. Reiner, S. L., and R. M. Locksley. 1995. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13:151–177. 16. Reiner, S. L., S. Zheng, Z. E. Wang, L. Stowring, and R. M. Locksley. 1994. Leishmania promastigotes evade interleukin 12 (IL-12) induction by macrophages and stimulate a broad range of cytokines from CD4⫹ T cells during initiation of infection. J. Exp. Med. 179:447–456. 17. Sacks, D., and N. Noben-Trauth. 2002. The immunology of susceptibility and resistance to Leishmania major in mice. Nat. Rev. Immunol. 2:845– 858. 18. Sacks, D. L., S. Hieny, and A. Sher. 1985. Identification of cell surface carbohydrate and antigenic changes between noninfective and infective developmental stages of Leishmania major promastigotes. J. Immunol. 135: 564–569. 19. Sadick, M. D., F. P. Heinzel, B. J. Holaday, R. T. Pu, R. S. Dawkins, and R. M. Locksley. 1990. Cure of murine leishmaniasis with anti-interleukin 4 monoclonal antibody. J. Exp. Med. 171:115–127.

VOL. 72, 2004


20. Scott, P., E. Pearce, P. Natovitz, and A. Sher. 1987. Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes. J. Immunol. 139:221–227. 21. Shinkai, K., M. Mohrs, and R. M. Locksley. 2002. Helper T cells regulate type-2 innate immunity in vivo. Nature 420:825–829. 22. Sommer, F., M. Meixner, M. Mannherz, A. L. Ogilvie, M. Rollinghoff, and M. Lohoff. 1998. Analysis of cytokine patterns produced by individual CD4⫹ lymph node cells during experimental murine leishmaniasis in resistant and

Editor: J. D. Clements

6931

susceptible mice. Int. Immunol. 10:1853–1861. 23. Stetson, D. B., M. Mohrs, V. Mallet-Designe, L. Teyton, and R. M. Locksley. 2002. Rapid expansion and IL-4 expression by Leishmania-specific naïve helper T cells in vivo. Immunity 17:191–200. 24. Taladriz, S., G. Gonzalez-Aseguinolaza, A. Marquet, and V. Larraga. 1999. Cloning, molecular analysis and differential cell localization of the p36 RACK analogue antigen from the parasite protozoon Crithidia fasciculata. FEBS Lett. 443:375–380.