Mice Lacking NK Cells Develop an Efficient Th1 Response and ...

Mice Lacking NK Cells Develop an Efficient Th1 Response and Control Cutaneous Leishmania major Infection1 Abhay R. Satoskar,2* Luisa M. Stamm,* Xingmin Zhang,† Anjali A. Satoskar,‡ Mitsuhiro Okano,* Cox Terhorst,† John R. David,* and Baoping Wang† NK cells are believed to play a critical role in the development of immunity against Leishmania major. We recently found that transplantation of wild-type bone marrow cells into neonatal tge 26 mice, which are deficient in T and NK cells, resulted in normal T cell development, but no or poor NK cell development. Using this novel model we analyzed the role of NK cells in the development of Th1 response and control of cutaneous L. major infection. Mice selectively lacking NK cells (NK2T1) developed an efficient Th1-like response, produced significant amounts of IL-12 and IFN-g, and controlled cutaneous L. major infection. Administration of neutralizing IL-12 Abs to NK2T1 mice during L. major infection resulted in exacerbation of the disease. These results demonstrate that NK cells are not critical for development of protective immunity against L. major. Furthermore, they indicate that IL-12 can induce development of Th1 response independent of NK cells in NK2T1 mice following L.major infection. The Journal of Immunology, 1999, 162: 6747– 6754.

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he leishmaniases comprising a number of diseases caused by the intracellular protozoan parasite Leishmania have a wide spectrum of clinical manifestations (1). While susceptible BALB/c mice develop large nonhealing lesions following L. major infection, most other mouse strains, including C3H, CBA/J, and C57BL/6, are resistant and develop small lesions that heal spontaneously. It is widely accepted that protective immunity against cutaneous L. major infection is associated with the development of a Th1-like response and the production of cytokines such as IL-12, IL-2, and IFN-g (2– 4), whereas susceptibility to L. major is associated with the development of a Th2-like response and the production of cytokines such as IL-4 and IL-10 (5). NK cells are a subpopulation of bone marrow-derived large, granular lymphocytes that lack T cell- and B cell-specific subset markers (TCR-, CD4-, CD8-, CD3gde-, and Ig-), but express some specific markers, such as NK 1.1 and ASGM1 (6). NK cells have been shown to play a critical role in innate immunity against a variety of viruses, bacteria, fungi, and parasites (7). The protective role of NK cells has been attributed to their ability to secrete immunoregulatory cytokines, such as IFN- g (8), lyse host cells infected with the intracellular pathogens, and directly inhibit growth of microorganisms (9, 10). Previous studies have demonstrated that depletion of NK cells using anti-asialo GM1 antiserum significantly reduces early IFN-g production in resistant C3H/HeN mice and renders them susceptible to cutaneous L. major infection, suggesting that NK cells are *Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston MA 02115; and †Department of Medicine, Division of Immunology, and ‡Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02115 Received for publication December 1, 1998. Accepted for publication March 17, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants A122532-13 (to J.R.D.), AI17651 (to C.T.), and HD35562-01(to B.W.). B.W. is the recipient of a Basil O’Connor Starter Scholar Research Award. 2

Address correspondence and reprint requests to Dr. Abhay R. Satoskar, Department of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Huntington Avenue, Boston, MA 02115. E-mail address: [email protected] Copyright © 1999 by The American Association of Immunologists

involved in host defense against this parasite (11). Furthermore, a recent study indicated that NK cells are involved in protection and healing of cutaneous leishmaniasis in humans (12). Therefore, we examined the development of Th1 response and growth of L. major in mice specifically lacking NK cells. Our results show that NK cells are not essential for the development of Th1 response and immunity to L. major infection in these mice.

Materials and Methods Mice Tge26 mice were maintained through sib breeding in the animal facility of the Beth Israel Deaconess Medical Center (Boston, MA) (13, 14). Although the tge26 transgenic founder was a (C57BL/6 3 CBA/J)F2, all tge26 mice used in this study were H-2k. Mice lacking NK cells (NK2T1) were generated by transplanting fetal liver or bone marrow cells from the (C57BL/6 3 CBA/J)F1 mice into neonatal tge26 mice, as described recently.3 Age- and sex-matched wild-type CBA/J (H-2k), C57BL/6 (H-2b), and BALB/c (H-2d) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Two types of immunocompetent mice with functionally competent NK and T cells were used as wild-type controls in this study. One type was (C57BL/6 3 CBA/J)F1 (H-2b/k) generated through breeding of C57BL/6 3 CBA/J. The other type was tge26 mice reconstituted with (C57BL/6 3 CBA/J)F1 (H-2b/k) bone marrow or fetal liver cells at 2–3 wk of age, instead of neonatally as in the generation of NK2T1 mice. These immunocompetent mice were termed as NK1T1(tge26Y) mice. Five weeks after the transplantation, NK1T1(tge26Y) mice develop functionally competent NK and T cells, and their levels are comparable to those observed in wild-type (C57BL/6 3 CBA/J)F1 mice. Furthermore, NK1T1 (tge26Y) and NK2T1 mice have similar levels of CD41 and CD81 T cells. Of note, all the NK1T1, NK1T1(tge26Y), and NK2T1 mice used in this study were analyzed by flow cytometry of PBL before the infection, and the lymph node and spleen cells upon sacrificing animals to confirm the lack or presence of NK cells and the presence of T cells.

Parasites and infection protocols L. major. LV39 was maintained by serial passage of amastigotes inoculated s.c. into the shaven rumps of BALB/c mice. Amastigotes isolated from the lesions of infected mice were grown to stationary-phase promastigotes as described previously (15). Mice were injected in the hind footpad with 2 3 106 L. major stationary-phase promastigotes. Disease progression 3 B. Wang, K. Nguyen, X. Zhang, A. Nichogiannopoulou, S. J. Simpson, J. Guimond, B. A. Croy, J.-C. Gutierrez Ramos, G. A. Hollander, C. A. Biron, and C. Terhorst. 1999. Distinct homing of engrafted hematopoietic stem cells in neonatal mice differentially affects T lymphocyte and NK cell development. Submitted for publication.

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NK CELL-DEFICIENT MICE CONTROL CUTANEOUS LEISHMANIASIS

was monitored by measuring the increase in thickness of the infected footpad using a dial-gauge micrometer (Mitutoyo, Kanagawa, Japan) at weekly intervals up to 10 wk after infection and comparing this to the thickness of the contralateral uninfected footpad.

Quantitation of parasite loads Parasite burdens in the infected footpad were determined by homogenizing footpads of individual mice and carrying out limiting dilution analysis as described previously (15). The results were expressed as reciprocal log parasite titers.

Ab ELISA Peripheral blood was collected at 2-wk intervals from tail snips of all experimental animals infected with L. major. Blood was centrifuged at 200 3 g, and serum was collected to determine titers of Th1-associated IgG2a and Th2-associated IgG1 Leishmania-specific Abs by ELISA as described before (15).

Flow cytometric analysis The lymph node cells, spleen cells, and PBL were analyzed by three-color flow cytometry as described previously (15). Briefly, 0.5–1 3 106 cells in 50 ml were incubated with prestaining buffer (PBS, 4% BSA, 0.5% sodium azide, 15% mixture of normal hamster, normal rat, and normal mouse sera, anti-Fc receptor Ab) for 5 min. The cells were then stained with biotinylated Ab for 30 min, washed once, followed by staining with a mixture of streptavidivin-RED670 (0.4 ml/sample; Life Technologies, Rockville, MD), PE- and FITC- conjugated Abs (0.5 mg/sample) for 30 min. The cells were washed twice, fixed in 1% formaldehyde, and analyzed with a FACScan using CellQuest software (Becton Dickinson, Mountain View, CA). All procedures were performed on ice until analysis. The following Abs were used: 145-2C11 (CD3-e, H57-597 (TCR-ab), RM4-5 (CD4), 53-5.8 (CD8a), 53-2.1 (Thy 1.2), RA3-6B2 (B220), PK136 (NK1.1), and DX5 (all purchased from PharMingen, San Diego, CA).

Allogenic T cell proliferation and cytotoxicity assays Allogenic T cell proliferation and cytotoxicity assays were performed by stimulating responsive spleen cells (H-2k or H-2k3b) with irradiated allogenic BALB/c (H-2d) spleen cells as described previously (14). NK cell cytotoxicity assay was performed using yeast artificial chromosome (YAC)4 cells as described before (13).

T cell proliferation and cytokine assays The draining popliteal lymph nodes were removed from L. major-infected mice at week 10 postinfection, and T cell proliferation assays were performed as previously described (15). Briefly, 3 3 105 lymph node cells were added in triplicate to the wells of 96-well flat-bottom tissue culture plates and stimulated with either 20 mg/ml L. major Ag (prepared from stationary-phase promastigotes by six cycles of freezing at 270°C and thawing at 37°C) or 1 mg/ml of Con A. Culture supernatants from these assays were analyzed for production of IL-4 (reagents purchased from Endogen, Cambridge, MA; detection limit, 5 pg/ml) and IFN-g (reagents purchased from PharMingen; detection limit, 20 pg/ml) by capture ELISA as described previously (15).

Histopathology Infected foot pads from L. major-infected NK2T1, NK1T1, and NK2T2 mice were excised and fixed in decalcifying solution F (Stephens Lab, Riverdale, NJ) for 7 days. The tissues were processed and embedded in paraffin, and 4- to 8-mm sections were cut. The sections were hydrated and stained by routine hematoxylin eosin staining.

Anti-IL-12 neutralizing Ab treatment Rat anti-mouse IL-12 (p40/p70) (clone: C17.8) neutralizing mAb was kindly provided by Dr. T. Veldman (Genetics Institute, Cambridge, MA). NK2T1 mice were treated by i.p. administration of 0.5 mg anti-IL-12 neutralizing Ab or control Ab 1 day before L. major infection and weekly dose of 0.5 mg/mouse thereafter until 7 wk.

Statistical analyses Student’s unpaired t test was used to determine significance of values obtained. Differences in Ab endpoint titers were determined using MannWhitney U prime test. 4

Abbreviation used in this paper: YAC, yeast artificial chromosome.

Results

NK2T1 mice have functionally normal CD41 and CD81 T cells but lack cytotoxic NK cells Recently, we demonstrated that transplantation of wild-type bone marrow or fetal liver cells into neonatal tge26 mice results in normal development of T cells, but poor NK cell development.3 All the neonatal transplanted tge26 mice used in this study had a normal number of CD41 and CD81 T cells as confirmed by flow cytometry. The NK1.11CD32TCR-ab2, which represent 2– 4% of wild-type splenocytes, however, were markedly diminished in the neonatal transplanted mice (Fig. 1, A and B). Peripheral lymph nodes from NK2T1 mice also showed markedly diminished levels of NK1.11CD32TCR-ab2 cells than those from NK1T1 mice (1–2% in NK1T1 mice and background levels (,0.3%) in NK2T1 mice). In contrast, NK1.11TCR-ab1 T cells, which represent 0.4 –1% of wild-type splenocytes, were present in the neonatal transplanted mice (Fig. 1B). The selective NK cell-deficient mice were termed as NK2T1 mice. Both CD41 and CD81 T cells from NK2T1 mice were functionally competent as assessed by MLR and CTL assays, respectively (Fig. 1, C and D). NK cell lytic function, as measured by splenocyte cytotoxicity against NK cellsensitive YAC-1 cells, however, was generally nondetectable or ,10% of wild-type control levels (Fig. 1E). When young tge26 mice were reconstituted with F1 bone marrow or fetal liver cells at 2–3 wk of age (tge26Y), these mice had comparable wild-type (F1) T (Fig. 2A) and NK cell (2.5 6 0.63%, 1.3 6 0.05%, and 0.3 6 0.1% of NK 1.11TCR-ab2 cells in NK1T1, NK1T1(tge26Y), and NK2T1 mice, respectively) levels. Furthermore, NK cells from NK1T1(tge26Y) mice were functionally as competent as those from NK1T1 mice (Fig. 2B). Therefore, these mice were termed as NK1T1(tge26Y). Since they have identical background as the NK2T1 mice, the NK1T1(tge26Y) mice were also used as NK cell competent controls, in addition to the wild-type mice (NK1T1). NK2T1 mice control cutaneous L. major infection Following infection with L. major, NK2T1, NK1T1, and NK1T1(tge26Y) mice developed lesions, which resolved spontaneously within 60 –70 days (Figs. 2C and 3A). The course of L. major infection was similar in NK2T1, NK1T1, and NK1T1(tge26Y) mice (Figs. 2C and 3A). In contrast, concomitantly infected tge26 (NK2T2) and BALB/c mice developed large nonhealing lesions and did not control the infection (Figs. 2C and 3A). The lesion grew significantly faster in NK2T2 mice than in BALB/c mice (Fig. 3A). Examination of the histopathology of the infected footpads from NK2T2 and BALB/c mice revealed ulceration and extensive s.c. tissue destruction with a diffuse inflammatory infiltrate consisting of heavily parasitized macrophages, eosinophils, and neutrophils (Fig. 3C). On the other hand, infected foot pads from NK2T1 and NK1T1 mice displayed inflammatory infiltrate comprised predominantly of lymphocytes and macrophages with few parasites (Fig. 3, D and E). There were no significant differences in the parasite burdens in footpads of NK1T1 and NK2T1 mice. The lesions from L. major-infected NK2T2 and BALB/c mice, however, contained significantly more parasites (at least 10 logs more) than NK2T1 and NK1T1 mice (Fig. 3B). These results indicate that, although NK cells may play a role in innate immunity to L. major as reported in previous studies using SCID and RAG-22/2 mice, they suggest that NK cells are not essential for control of L. major infection when immunocompetent T cells are present.

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FIGURE 1. NK2T1 mice lack NK1.11CD32TCR-ab2 cells but have normal T cell development. A, CD4 and CD8 profile of lymph node cells from a wild-type (NK1T1) and an NK2T1 mouse. B, NK1.1 and TCR-ab profile of spleen cells, indicating that NK2T1 mice have markedly diminished NK1.11ab2 cells, but have a relatively normal number of NK 1.11ab1 cells. C, NK2T1 mice have functionally normal CD41 T cells as assessed by MLR assay. D, NK2T1 mice have functionally normal CD81 T cells as assessed by CTL assay. E, NK2T1 cell mice lack NK cell cytotoxicity against YAC-1 cells. Data for C–E represents the analysis of the spleen cells obtained from a single NK1T1 and two NK2T1 mice.

NK2T1 mice develop efficient Th1-like response following L. major infection NK cells have been shown to be a major source of IFN-g, a cytokine critical for development of the Th1 lymphocyte subset of the CD41 T cell population in resistant mice following L. major infection (11). Therefore, we compared IL-12 and IFN-g production by Leishmania Ag-stimulated draining lymph node cells from L. major-infected NK2T1 and NK1T1 mice. On day 60 postinfection, the draining lymph node cells from L. major-infected NK1T1 and NK2T1 mice contained a similar number of lymphocytes (1.02 6 0.13 3 107 and 1.35 6 0.4 3 107 in NK1T1 and NK2T1 mice, respectively; p , 0.375). Furthermore, there were no significant differences in proportions of B2201 (43.7 6 5.4% and 41.6 6 1.9% in NK1T1 and NK2T1 mice, respectively), CD41 (28.6 6 4.2% and 32.9 6 4.7% in NK1T1 and NK2T1 mice, respectively), and CD81 (10.1 6 1.6% and 11.1 6 1.1% in

NK1T1 and NK2T1 mice, respectively). NK 1.11 cells, however, were markedly reduced in the lymph nodes from NK2T1 mice (2.1 6 0.31% and 0.4 6 0.06% in NK1T1 and NK2T1 mice, respectively). At this time, lymphocytes from NK1T1, NK2T1, and BALB/c mice displayed greater Ag-specific proliferative responses than those derived from NK2T2 mice. At this time, LmAg-stimulated lymph node cells from both NK2T1 and NK1T1 mice produced IL-12 and IFN-g, although IL-12 levels were significantly higher in NK2T1 mice (Fig. 4, A and B; p , 0.05). Ag-stimulated lymph node cells from concomitantly infected BALB/c mice produced significant amounts of IL-4, but no IL-4 was detected in culture supernatants from NK2T1 and NK1T1 mice (Fig. 4C). Neither IL-4 nor IFN-g was detectable in the lymph node cell culture supernatants from NK2T2 mice, which contained basal levels of IL-12. (Fig. 4, A–C). All groups produced comparable levels of IL-10 (data not shown). Similarly,

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FIGURE 2. NK1T1(tge26Y) mice develop Th1 response and control L. major infection as efficiently as NK1T1 mice. A, CD4 and CD8 profile of lymph node cells from a wild-type (NK1T1) and NK1T1(tge26Y) mice. B, NK1T1(tge26Y) mice have normal NK cell cytotoxicity against YAC-1 cells similar to that observed in NK1T1 mice. C, Course of L. major infection in NK1T1, NK1T1(tge26Y), NK2T1, and NK2T2 mice. Disease progression was monitored by measuring increase in the thickness of infected footpad, as described before. D, In vitro LmAg-induced IFN-g production by the lymph node cells. Data expressed as mean 6 SE. Asterisk indicates statistically significant difference between two groups. ND, not detectable.

at an earlier time point on day 36 postinfection, both NK2T1 and NK1T1 mice produced IL-12 (0.114 6 0.4 ng/ml and 1.3 6 0.17 ng/ml in NK1T1 and NK2T1 mice, respectively) and IFN-g (1.068 6 0.6 ng/ml and 2.9 6 0.25 ng/ml in NK1T1 and NK2T1 mice, respectively), but no IL-4. In additional experiments, NK1T1(tge26Y) mice were also examined. NK1T1(tge26Y) mice controlled L. major infection as efficiently as NK1T1 mice (Fig. 2C). Furthermore, there was no significant difference in IFN-g production by LmAg-stimulated lymph node cells from NK1T1 and NK1T1(tge26Y) mice (Fig. 2D). NK2T1 mice fail to produce Leishmania-specific IgG2a, despite development of Th1 response Ab responses in L. major-infected NK2T1, NK1T1, NK2T2, and BALB/c mice were analyzed by measuring titers of Leishmaniaspecific Th1-dependent IgG2a and Th2-dependent IgG1 Abs on days 30, 45, and 60 postinfection. On day 30 and thereafter, NK1T1 and BALB/c mice developed significant levels of Leishmania-specific IgG1 and IgG2a, although BALB/c mice produced significantly more (Fig. 5A). On the other hand, L. major-infected NK2T1 mice displayed high titers of Leishmania-specific IgG1, but failed to produce any measurable quantities of Leishmaniaspecific IgG2a throughout the course of infection (Fig. 5B). Both NK1T1 and NK1T1(tge26Y) mice produced significant titers of LmAg-specific IgG2a (2666.2 6 533 and 16200 6 12049 in

NK1T1 and NK1T1(tge26Y) mice, respectively). Similar results were observed on days 30 and 45 postinfection (data not shown). IL-12 is critical for development of Th1 response and controls L. major infection in NK2T1 mice To determine whether IL-12 is critical for development of Th1 response in NK2T1 mice during L. major infection, we treated L. major-infected NK2T1 with i.p. injections of IL-12 neutralizing Ab or control Ab 1 day before infection and weekly thereafter for 7 wk. Anti-IL-12 mAb-treated NK2T1 mice developed significantly larger lesions than control animals following L. major infection (Fig. 6). At wk 8 postinfection, Ag-stimulated lymph node cells from control NK2T1 mice produced significantly higher amounts of IFN-g (mean levels, 1.34 6 0.4 ng/ml) than those from anti-IL-12-treated NK2T1 mice, which produced only basal levels (,0.05 ng/ml; p , 0.05).

Discussion The results presented here indicate that although NK1.11CD32TCRab2 (NK) cells play a role in innate immunity to L. major, they are not required for development of Th1-like response and control of L. major infection in resistant mice. Furthermore, they also demonstrate that in the absence of NK cells, IL-12 can directly induce development of a Th1 response during L. major infection.


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FIGURE 3. NK2T1 mice control cutaneous L. major infection. A, Course of L. major infection following infection with 2 3 106 stationary-phase promastigotes in NK2T1, NK1T1, BALB/c, and tge26 (NK2T2) mice. Progress of lesion growth was monitored by measuring the increase in thickness of the infected footpad and comparing this to the thickness of the contralateral uninfected footpad. NK2T2 were sacrificed on day 30 after the infection, due to the development of large lesions. All other mice were monitored up to 60 days. B, Footpad parasite burdens in L. major-infected NK1T1, NK2T1, and NK2T2 mice. Data expressed as log parasite titer 6 SE. C–E, Hematoxylin-eosin stained skin lesions from L. major-infected NK2T2, NK1T1, and NK2T1 mice. Lesions from NK2T2 mice showed ulceration and extensive tissue destruction with inflammatory infiltrate comprising of parasitized macrophages, neutrophils, and eosinophils. C and D, Similarly stained skin from the inoculation sites of NK1T1 and NK2T1 mice displayed a more preserved skin structure with lymphocytes and some macrophages with few intracellular parasites (original magnification, 340). Results are representative of three experiments with four to five animals per group. Data expressed as mean 6 SE.

NK cells have been demonstrated to be involved in the first line of defense against viruses, bacteria, and parasites (7, 16). The importance of NK cells in early antibacterial immunity has been demonstrated by a study showing that SCID mice that lack ab and gd T cells but have NK cells develop activated macrophages and partially control Listeria monocytogenes (17–19). Later, it was demonstrated that spleen cells from naive SCID and nude mice produced significant levels of IFN-g following incubation with heatkilled L. monocytogenes (18). Furthermore, administration of neutralizing anti-IFN-g mAb or NK cell depletion before infection abolished macrophage activation in SCID mice. Together, these data indicate that NK cell-derived IFN-g is involved in vivo macrophage activation following L. monocytogenes infection (19). Similarly, many studies have demonstrated that NK cells also play a critical role in immunity against viruses such as murine CMV, Coxsackievirus B4, and influenza virus (20 –22). This has been attributed to their cytolytic capacity and ability to produce type I IFNs that have antiviral activity (23). Although some studies using NK cell-deficient beige mice had suggested that NK cells were required for the control of visceral leishmaniasis (24), others using SCID mice indicated that NK cellderived IFN-g was unlikely to participate in the early regulation of visceral leishmaniasis caused by L. donovani (25). Similarly, some studies in murine cutaneous leishmaniasis indicate that the NK cell-derived IFN-g plays an important role in early resistance and development of a Th1 response following L. major infection (11, 26). Others, however, using beige mice have demonstrated that NK

cells are not required for the control of cutaneous L. tropica infection, which supports our observations in the present study. These differences are probably due to the different experimental models of NK cell deficiency used and the differences in the experimental approaches. For example, although poly(I:C) activates NK cells and significantly reduces the parasite burdens in the early course of L. major infection in BALB/c mice (26), it also induces production of type I (IFN-a/b) IFN from NK cells, which has been shown to induce expression of nitric oxide synthase 2 (NOS2) in vivo and regulate innate immunity to L. major (27). Conversely, depletion of NK cells prior to L. major infection using antiAsGM1 Ab significantly decreased early IFN-g production and exacerbated the infection in resistant C3H/HeN, but had no effect on the ultimate disease outcome (11, 26). Systemic depletion of cells using Ab treatment is efficient but not absolute (28). Furthermore, in a recent study, NK cell depletion using anti-NK1.1 as well as anti-AsGM-1 Abs failed to alter the Th1/Th2 balance of Ag-driven cytokine synthesis (29). The ability of NK-depleted C3H/HeN mice to heal L. major infection, however, could be attributed to the repopulation of NK cells in these mice following cessation of anti-AsGM-1 treatment (11, 26). Finally, beige mice, which controlled L. tropica (30), exhibit normal numbers of NK 1.11CD32 cells and normal NK cell cytotoxicity against viruses, although they have very low cytotoxicity against YAC-1 cells (31, 32). By our use of a novel model of specific murine NK cell deficiency, these possibilities have been excluded. Unlike beige mice, the NK2T1 mice lack NK cell cytotoxicity

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FIGURE 5. L. major-specific IgG1 and IgG2a production in NK1T1 and NK2T1 mice on day 60 postinfection presented as reciprocal endpoint titers on a log scale. Similar results were observed in three independent experiments. NK2T2 mice were sacrificed on day 30 postinfection, due to the development of large lesions. ND, not detectable.

FIGURE 4. NK2T1 mice develop a Th1-like response. In vitro LmAginduced (20 mg/ml) IL-12 (A), IFN-g (B), and IL-4 (C) production by the lymph node cells from NK1T1, NK2T1, and BALB/c mice measured on day 60 postinfection. NK2T2 (tge26) mice were sacrificed on day 30 postinfection for proliferation assays and cytokine production, due to the development of large lesions. Data expressed as mean 6 SE. Asterisks indicate statistically significant differences between NK1T1 and NK2T1 mice. Similar results were observed in two independent experiments. ND, not detectable.

against lymphocytic choriomeningitis virus and YAC-1 cells.3 Therefore, these mice are truly deficient in NK cell lytic function, and the number of NK1.11CD32 cells are either absent or markedly diminished in these mice.3 We have reported previously that transplantation of wild-type bone marrow into adult tge26 mice results in aberrant NK cell development, which is caused by the generation of aberrant T cells that leads to high levels of TNF-a in

vivo (33). This mechanism, however, could not account for NK cell deficiency in the neonatal tge26 model described in this study. First, in the neonatal tge26 model, NK cell deficiency is independent of T cell development,3 and there was no overproduction of TNF-a in sera from L. major-infected NK1T1 and NK2T1 mice (97 6 28 pg/ml and 71 6 40 pg/ml in NK1T1 and NK2T1 mice, respectively). Second, we demonstrated that the lack of cytotoxic NK cells in the neonatally transplanted tge26 mice was due to the failure of transplanted hematopoietic stem cells to home to bone marrow, whereas the T cell development in the same mice was due to the migration of the hematopoietic stem cells during neonatal period to the thymus.3 We have previously demonstrated that TCR-ab2/2 mice lacking T cells on genetically resistant background (15) and RAG-22/2 mice that have innately high levels of IFN-g and IL-12 control early lesion growth of L. major but later succumb to the disease (our unpublished observations). Our findings in the present study that tge26 mice (NK2T2) are susceptible to L. major and develop lesions significantly faster than similarly infected RAG22/2 or TCR-ab2/2 mice (NK1T2) suggest that NK cells may be involved in controlling early resistance to L. major. However, the ability of NK2T1 mice to control cutaneous L. major infection as


FIGURE 6. Administration of IL-12 neutralizing Ab to NK2T1 mice inhibits Th1 development and exacerbates cutaneous L. major infection. Data expressed as mean lesion size 6 SE.

efficiently as wild-type (NK1T1) mice indicates that, in the presence of functional T cells, NK cells are not essential for the control of L. major lesion growth. Furthermore, they also indicate that NK cell-derived IFN-g is not essential for the Th1 differentiation of CD41 T cell following cutaneous L. major infection. Acquired protective immunity against cutaneous L. major infection in genetically resistant mice is dependent on the ability to develop a CD4 Th1-type lymphocyte response and produce cytokines such as IL-2 and IFN-g (2, 34). Many recent observations indicate that IL-12 plays a critical role in the development of a Th1-like response in resistant mice following L. major infection (3, 4, 35). Thus, for example, IL-12 administered simultaneously at the time of vaccination with Leishmania Ag facilitated Th1 development (35). Furthermore, in two independent studies, susceptible BALB/c mice treated with IL-12 during L. major infection developed a significant Th1-like response and healed (3, 4). In contrast, administration of IL-12 neutralizing Ab to resistant C3H/HeN mice rendered them susceptible to L. major (36). Similarly, genetically resistant mice lacking the IL-12 gene failed to mount a Th1like response and developed large nonhealing lesions following L. major infection (37). Neutralization of IL-12 in vivo has been shown to abrogate NK cell cytotoxicity and decrease early IFN-g production by lymph node cells from L. major-infected C3H mice (36). This result suggested that the protective role of IL-12 was mediated by its ability to activate NK cells to produce IFN-g critical for the subsequent development of the Th1 subset of the CD41 T cells (36). On the other hand, a recent study demonstrated that IFN-g derived from CD41 T cells is sufficient to mediate Th1 development following L. major infection (38). In the present study, we found that L. major-infected NK2T1 mice produced significantly higher amounts of IL-12 and IFN-g than similarly infected NK1T1 mice and efficiently controlled the infection. Conversely, administration of anti-IL-12 neutralizing Abs to NK2T1 mice during L. major infection significantly reduced IFN-g production and resulted in the development of large rapidly growing lesions. Possibly, the increased IL-12 production in NK2T1 mice is due to the development of compensatory mechanisms or loss of NK cell-mediated negative feedback regulating IL-12 production by macrophages. In fact, recent studies have demonstrated that NK cell-derived type 1 IFN (IFN-a/b), which has been shown to regulate innate responses in L. major infection (27), can down-regulate IL-12 production (39). Nevertheless, taken together, these results suggest that in the absence of NK cells, IL-12 can directly induce Th1 development in vivo following L. major infection.

6753 NK cells have been shown to induce Ab production from activated murine B cells (40) as well as resting human B cells (41). A recent study, however, has demonstrated that IL-12 enhances Ab responses and increase levels of IgG2a to T-independent polysaccharide vaccines in the absence of T and NK cells (42). Interestingly, despite the production of IL-12 and development of Th1-like response, L. major-infected NK2T1 mice failed to produce Th1associated Leishmania-specific IgG2a Ab throughout the course of infection. Similarly, in our ongoing studies, NK2T1 produced significant amounts of IFN-g but displayed baseline levels of Agspecific IgG2a following immunization with keyhole limpet hemocyanin (KLH) or OVA in CFA, which induces Th1-biased response (our unpublished observations). Furthermore, there was no difference in levels of Th2-type cytokines IL-4 and IL-10 produced by Ag-stimulated spleen cells from these mice (our unpublished observations). This data has been reproduced in four independent studies conducted so far in our laboratory using KLH and OVA. These results suggest that NK cells may play a critical role in production of IgG2a against T-dependent Ags. Recently, a population of CD4 T cells that express NK1.1 and TCR-ab (NKT cells) has been shown to produce IL-4 (43) as well as IFN-g (44). Although early studies had indicated that NKT cells may be the initial source of IL-4 that induces Th2 development (43), recent studies indicate that in a susceptible mouse, L. major induces rapid IL-4 production by CD41 cells that are NK 1.1negative (45). Recent studies have demonstrated that IL-12-stimulated NK1.11 T cells produce high levels of IFN-g (46, 47), as well as exhibit cytotoxicity against tumor cells (46). Furthermore, endogenous IL-12 has been shown to down-regulate IL-4-producing NK 1.11 T cells in liver and improve protective immunity against listeriosis (48). NK2T1 mice used in the present study have a normal number of NK1.11CD41 T cells. Additionally, administration of anti-IL-12 Ab L. major-infected NK2T1 mice inhibited Th1 development and rendered them susceptible to infection. Therefore, one may speculate that NK 1.11 T cell-derived IFN-g initiates Th1 development in these mice. A recent study, however, demonstrated that RAG-2/IFN-g2/2 (double mutant) mice reconstituted with the wild-type CD41 NK 1.12 T cells develop Th1 response and control cutaneous L. major infection (38). Taken together, these results indicate that neither NK cell-derived IFN-g nor NK 1.11CD41 T cells are critical for the development of protective Th1 response following L. major infection. In conclusion, L. major-infected NK2T1 mice on genetically resistant background develop an efficient Th1-like response as measured by significant IFN-g production by the lymph node cells following stimulation with Leishmania Ag and control cutaneous L. major infection. Furthermore, administration of neutralizing antiIL-12 Abs to NK2T1 mice during L. major infection inhibits development of Th1-like response and enhances cutaneous lesion growth.

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