Presentation of theLeishmania antigen LACK by infected macrophages is dependent upon the virulence of the phagocytosed parasites

Abstract

We have previously demonstrated that murine macrophages (Mphi) infected with Leishmania promastigotes, in contrast to Mphi infected with the amastigote stage of these parasites, are able to present the Leishmania antigen LACK (Leishmania homologue of receptors for activated C kinase) to specific, I-Ad-restricted T cell hybrids and to the T cell clone 9.1-2. These T cells react with the LACK (158-173) peptide, which is immunodominant in BALB/c mice. Here, we show that the level of stimulation of the LACK-specific T cell hybridoma OD12 by promastigote-infected Mphi is clearly dependent upon the differentiation state of the internalized parasites. Thus, shortly after infection with log-phase or stationary-phase promastigotes of L. major or of L. amazonensis, Mphi strongly activated OD12. The activity was transient and rapidly lost. However, under the same conditions, activation of OD12 by Mphi infected with metacyclic promastigotes of L. major or of L. amazonensis was barely detectable. At the extreme, Mphi infected with amastigotes were incapable to stimulate OD12. Thus, the presentation of LACK by infected Mphi correlates with the degree of virulence of the phagocytosed parasites, the less virulent being the best for the generation/expression of LACK (158-173)-I-Ad complexes. While the intracellular killing of the parasites appears to be an important condition for the presentation of LACK, it is not the only requisite. The partial or total destruction of intracellular L. amazonensis amastigotes does not allow the presentation of LACK to OD12. A preferential interaction of LACK (158-173) with recycling rather than newly synthesized MHC class II molecules does not explain the transient presentation of LACK by Mphi infected with log-phase or stationary-phase promastigotes because brefeldin A strongly inhibited the presentation of LACK to OD12. Taken together, these results suggest that virulent stages of Leishmania, namely metacyclics and amastigotes, have evolved strategies to avoid or minimize their recognition by CD4+ T lymphocytes.

Proteome and morphological analysis show unexpected differences between promastigotes of Leishmania amazonensis PH8 and LV79 strains

“…Purification of L . amazonensis metacyclics is usually performed using antibody mAb 3A1 directed to procyclic LPG [ 66 ] or Ficoll gradients. Ficoll separation was developed for L .…”

LFR1 Ferric Iron Reductase of Leishmania amazonensis Is Essential for the Generation of Infective Parasite Forms

“…To generate L. amazonensis axenic amastigotes, log phase promastigotes were inoculated at 1 ϫ 10 6 parasites/ml into amastigote media (M199, 40 mM sodium succinate, pH 4.5, 20% heat-inactivated FBS, 5% penicillin/streptomycin, 0.1% hemin, 10 mM adenine, 5 mM L-glutamine, 0.25% glucose, and 0.5% trypticase) and incubated at 32°C. L. amazonensis metacyclic forms were purified from 7-day-old promastigote stationary phase cultures using the 3A.1 mAb, which specifically agglutinates L. amazonensis procyclic promastigotes but not the metacyclic forms (10,11). Parasites were washed twice with PBS, resuspended at 1.0 ϫ 10 8 parasites/ml in 1.0 ml of PBS containing a 1:30 dilution of 3A.1 culture supernatant for 30 min, and centrifuged at 250 ϫ g for 5 min.…”

“…In Vivo Virulence and Persistence Assays-Female BALB/c mice were injected in the right hind footpad with 1.0 ϫ 10 6 mAb 3A.1-purified metacyclic promastigotes of L. amazonensis (10,11), and lesion progression was followed by blinded weekly measurements with a caliper. The parasite tissue load of wild type and ⌬lfr1 L. amazonensis was determined after 9 or 11 weeks, respectively, using a limiting dilution assay (7,25).…”

Leishmania spp. Proteome Data Sets: A Comprehensive Resource for Vaccine Development to Target Visceral Leishmaniasis

“…Macrophages need to be activated, e.g., through cytokines such as IFN-γ to express MHC class II molecules, a prerequisite to present antigens to CD4 T cells (48, 49). …”