Immune Elimination of Leishmania major in Mice: Implications for Immune Memory, Vaccination, and Reactivation Disease 1 Jude E. Uzonna, Guojian Wei, Dean Yurkowski, and Peter Bretscher 2 Infection of susceptible BALB/c mice with a large, moderate, or low number of Leishmania major parasites respectively results in progressive disease, the formation of substantial but stable lesions, denoted as borderline disease, and the absence of a visible lesion. Infection with a low number of parasites results over the long term in either subclinical infections or an asymptomatic state. Subclinical mice produce a predominant Th1 response and are resistant to challenge, in contrast to their asymptomatic coun- terparts. Statistical and other evidence suggest that the asymptomatic state can arise from a subclinical state following parasite clearance, with consequent loss of resistance. Cell transfer studies demonstrate unequivocally that immune cells from subclinical mice can protect naive mice against a pathogenic challenge and can clear the parasite, leaving the mice susceptible to a rechallenge infection. This susceptibility is associated with the disappearance of both parasite-specific effector and memory T cells from secondary lymphoid organs. These findings have implications for vaccination, maintenance of memory, and prevention of reac- tivation disease. The Journal of Immunology, 2001, 167: 6967– 6974. I nfection of humans by the Leishmania species, which is able to cause cutaneous leishmaniasis, results in a spectrum of clinical manifestations, ranging from fatal diffuse disease to self-healing lesions (1, 2). The murine model of cutaneous leish- maniasis is extensively studied because different mouse strains mount immune responses of different Th1/Th2 phenotype upon challenge with a million parasites, associated with different clinical outcomes. The prototypical resistant C57BL/6 and CBA strains mount a predominant Th1 response and contain the infection, while susceptible BALB/c mice mount a predominant Th2 re- sponse and suffer progressive disease. The Th1/Th2 nature of the immune response determines resistance/susceptibility (3–5). Resistance/susceptibility to L. major depends, among other fac- tors, upon the number of parasites employed for infection (6 – 8). Susceptible BALB/c mice resist a low dose infection, and resistant CBA mice develop lesions when infected with very high numbers (8). We undertook a detailed investigation of the relationship be- tween the number of parasites employed for infection and the en- suing pathophysiological state in BALB/c mice. Our incidental observations over a period of several years following infection with low numbers of parasites suggested that immune elimination of L. major may occur, contrary to conventional views (9 –14). Given the potential importance of this tentative inference for an- alyzing the requirements for maintenance of immunological mem- ory, for vaccination, and for the control of reactivation disease, we attempted to develop a more incisive and reproducible system to examine whether parasite clearance can occur and to determine what the consequences are of such clearance for immunological resistance and memory. Materials and Methods Mice BALB/c mice, 8 –10 wk of age and bred at the animal facility of the De- partment of Microbiology and Immunology, University of Saskatchewan, were used. All mice were maintained according to the guidelines of the Canadian Council of Animal Care. Parasites and parasite Ags Mice were infected with stationary phase promastigotes of L. major MHOM (WHO MHOM/IL/80/Friedlin) strain as described previously (7). Soluble leishmanial Ag (SLA) 3 was prepared, and protein content was determined by the Bradford method (15). SLA was used at 30 g/ml for in vitro stimulation of splenocytes and lymph node cells for the detection of L. major Ag-specific cytokine-producing cells. Measurement of footpad lesions and detection of lymph node (LN) swellings The size of footpad lesions was measured with dial calipers (Oditest, Lan- genmesstechnik, Kroeplin, Germany), using the contralateral uninfected foot as a control. Mice were designated LN positive (LN + ) if the draining popliteal lymph nodes were enlarged, as assessed by palpation. Estimation of parasite burden The parasite burden in the footpad, draining popliteal lymph node, spleen, and bone marrow of infected mice was estimated by limiting dilution anal- ysis as previously described (16). To enhance sensitivity, 2 -fold dilutions of undiluted samples (up to 1/100) were used. Thereafter, the conventional 10-fold dilutions were used. Limiting dilution assay for the frequency of L. major-specific cells The frequency of L. major-specific precursor T cells able to produce IL-2-, IL-4-, and IFN--reactive cells was measured by limiting dilution as pre- viously described (17) with minor modifications. Briefly, single-cell sus- pensions of spleens and draining popliteal lymph nodes from age-matched Department of Microbiology and Immunology, University of Saskatchewan, Saska- toon, Saskatchewan, Canada Received for publication June 25, 2001. Accepted for publication October 9, 2001. 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 Grant MOP 37868 from the Medical Research Council of Canada (to P.A.B.) and the Health Services Utilization Research Council of Saskatchewan PDF fund (to J.E.U.). 2 Address correspondence and reprint requests to Dr. Peter Bretscher, Department of Microbiology and Immunology, University of Saskatchewan, 107 Wiggins Road, Saskatoon, Saskatchewan, Canada S7N 5E5. E-mail address: bretschr@duke.usask.ca 3 Abbreviations used in this paper: SLA, soluble leishmanial antigen; LN, lymph node. Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00