Infection Leishmania Major Th1 Response and Control Cutaneous

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. Ad- ministration 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. he leishmaniases comprising a number of diseases caused involved in host defense against this parasite (11). Furthermore, a by the intracellular protozoan parasite Leishmania have a recent study indicated that NK cells are involved in protection and T wide spectrum of clinical manifestations (1). While sus- healing of cutaneous leishmaniasis in humans (12). Therefore, we ceptible BALB/c mice develop large nonhealing lesions following examined the development of Th1 response and growth of L. ma- L. major infection, most other mouse strains, including C3H, jor in mice specifically lacking NK cells. Our results show that NK CBA/J, and C57BL/6, are resistant and develop small lesions that cells are not essential for the development of Th1 response and heal spontaneously. It is widely accepted that protective immunity immunity to L. major infection in these mice. against cutaneous L. major infection is associated with the development of a Th1-like response and the production of cytokines Materials and Methods such as IL-12, IL-2, and IFN-g (2–4), whereas susceptibility to L. Mice major is associated with the development of a Th2-like response Tge26 mice were maintained through sib breeding in the animal facility of and the production of cytokines such as IL-4 and IL-10 (5). the Beth Israel Deaconess Medical Center (Boston, MA) (13, 14). Al- e 3 NK cells are a subpopulation of bone marrow-derived large, though the tg 26 transgenic founder was a (C57BL/6 CBA/J)F2, all 2 1 granular lymphocytes that lack T cell- and B cell-specific subset tge26 mice used in this study were H-2k. Mice lacking NK cells (NK T ) gde were generated by transplanting fetal liver or bone marrow cells from the markers (TCR-, CD4-, CD8-, CD3 -, and Ig-), but express some 3 e (C57BL/6 CBA/J)F1 mice into neonatal tg 26 mice, as described re- specific markers, such as NK 1.1 and ASGM1 (6). NK cells have cently.3 Age- and sex-matched wild-type CBA/J (H-2k), C57BL/6 (H-2b), been shown to play a critical role in innate immunity against a and BALB/c (H-2d) mice were purchased from The Jackson Laboratory variety of viruses, bacteria, fungi, and parasites (7). The protective (Bar Harbor, ME). Two types of immunocompetent mice with functionally role of NK cells has been attributed to their ability to secrete im- competent NK and T cells were used as wild-type controls in this study. 3 b/k g One type was (C57BL/6 CBA/J)F1 (H-2 ) generated through breeding munoregulatory cytokines, such as IFN- (8), lyse host cells in- of C57BL/6 3 CBA/J. The other type was tge26 mice reconstituted with fected with the intracellular pathogens, and directly inhibit growth 3 b/k (C57BL/6 CBA/J)F1 (H-2 ) bone marrow or fetal liver cells at 2–3 wk of microorganisms (9, 10). of age, instead of neonatally as in the generation of NK2T1 mice. These 1 1 Previous studies have demonstrated that depletion of NK cells immunocompetent mice were termed as NK T (tge26Y) mice. Five 1 1 e using anti-asialo GM1 antiserum significantly reduces early IFN-g weeks after the transplantation, NK T (tg 26Y) mice develop functionally competent NK and T cells, and their levels are comparable to those production in resistant C3H/HeN mice and renders them suscep- 3 1 1 observed in wild-type (C57BL/6 CBA/J)F1 mice. Furthermore, NK T tible to cutaneous L. major infection, suggesting that NK cells are (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 *Department of Immunology and Infectious Diseases, Harvard School of Public † the lymph node and spleen cells upon sacrificing animals to confirm the Health, Boston MA 02115; and Department of Medicine, Division of Immunology, lack or presence of NK cells and the presence of T cells. and ‡Department of Pathology, Beth Israel Deaconess Medical Center, Boston, MA 02115 Parasites and infection protocols Received for publication December 1, 1998. Accepted for publication March 17, 1999. L. major. LV39 was maintained by serial passage of amastigotes inocu- lated s.c. into the shaven rumps of BALB/c mice. Amastigotes isolated 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 from the lesions of infected mice were grown to stationary-phase promas- with 18 U.S.C. Section 1734 solely to indicate this fact. tigotes as described previously (15). Mice were injected in the hind footpad with 2 3 106 L. major stationary-phase promastigotes. Disease progression 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. 3 B. Wang, K. Nguyen, X. Zhang, A. Nichogiannopoulou, S. J. Simpson, J. Guimond, 2 Address correspondence and reprint requests to Dr. Abhay R. Satoskar, Department B. A. Croy, J.-C. Gutierrez Ramos, G. A. Hollander, C. A. Biron, and C. Terhorst. of Immunology and Infectious Diseases, Harvard School of Public Health, 665 Hun- 1999. Distinct homing of engrafted hematopoietic stem cells in neonatal mice differ- tington Avenue, Boston, MA 02115. E-mail address: [email protected] entially affects T lymphocyte and NK cell development. Submitted for publication. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 6748 NK CELL-DEFICIENT MICE CONTROL CUTANEOUS LEISHMANIASIS was monitored by measuring the increase in thickness of the infected foot- Results pad using a dial-gauge micrometer (Mitutoyo, Kanagawa, Japan) at weekly NK2T1 mice have functionally normal CD41 and CD81 T cells intervals up to 10 wk after infection and comparing this to the thickness of the contralateral uninfected footpad. but lack cytotoxic NK cells Quantitation of parasite loads Recently, we demonstrated that transplantation of wild-type bone marrow or fetal liver cells into neonatal tge26 mice results in nor- Parasite burdens in the infected footpad were determined by homogenizing mal development of T cells, but poor NK cell development.3 All footpads of individual mice and carrying out limiting dilution analysis as described previously (15). The results were expressed as reciprocal log the neonatal transplanted tge26 mice used in this study had a nor- 1 1 parasite titers. mal number of CD4 and CD8 T cells as confirmed by flow 1 2 ab2 Ab ELISA cytometry. The NK1.1 CD3 TCR- , which represent 2–4% of wild-type splenocytes, however, were markedly diminished in Peripheral blood was collected at 2-wk intervals from tail snips of all the neonatal transplanted mice (Fig. 1, A and B). Peripheral lymph experimental animals infected with L. major. Blood was centrifuged at 2 1 3 nodes from NK T mice also showed markedly diminished levels 200 g, and serum was collected to determine titers of Th1-associated 1 2 2 1 1 IgG2a and Th2-associated IgG1 Leishmania-specific Abs by ELISA as of NK1.1 CD3 TCR-ab cells than those from NK T mice described before (15). (1–2% in NK1T1 mice and background levels (,0.3%) in 2 1 1 ab1 Flow cytometric analysis NK T mice). In contrast, NK1.1 TCR- T cells, which represent 0.4–1% of wild-type splenocytes, were present in the neo- The lymph node cells, spleen cells, and PBL were analyzed by three-color 6 natal transplanted mice (Fig. 1B). The selective NK cell-deficient flow cytometry as described previously (15). Briefly, 0.5–1 3 10 cells in 2 1 1 1 50 ml were incubated with prestaining buffer (PBS, 4% BSA, 0.5% sodium mice were termed as NK T mice. Both CD4 and CD8 T cells 2 1 azide, 15% mixture of normal hamster, normal rat, and normal mouse sera, from NK T mice were functionally competent as assessed by anti-Fc receptor Ab) for 5 min. The cells were then stained with biotinyl- MLR and CTL assays, respectively (Fig. 1, C and D). NK cell lytic ated Ab for 30 min, washed once, followed by staining with a mixture of function, as measured by splenocyte cytotoxicity against NK cell- streptavidivin-RED670 (0.4 ml/sample; Life Technologies, Rockville, MD), PE- and FITC- conjugated Abs (0.5 mg/sample) for 30 min.

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