BALB/c mice develop aberrant T helper 2 (Th2) responses and suffer progressive disease after infection with Leishmania major. These outcomes depend on the production of interleukin-4 (IL-4) early after infection. Here we demonstrate that the burst of IL-4 mRNA, peaking in draining lymph nodes of BALB/c mice 16 hr after infection, occurs within CD4+ T cells that express V beta 4 V alpha 8 T cell receptors. In contrast to control and V beta 6-deficient BALB/c mice, V beta 4-deficient BALB/c mice were resistant to infection, demonstrating the role of these cells in Th2 development. The early IL-4 response was absent in these mice, and T helper 1 responses occurred following infection. Recombinant LACK antigen from L. major induced comparable IL-4 production in V beta 4 V alpha 8 CD4+ cells. Thus, the IL-4 required for Th2 development and susceptibility to L. major is produced by a restricted population of V beta 4 V alpha 8 CD4+ T cells after cognate interaction with a single antigen from this complex organism.

Susceptibility of BALB/c mice to infection with Leishmania major is associated with a T helper type 2 (Th2) response. Since interleukin-4 (IL-4) is critically required early for Th2 cell development, the kinetics of IL-4 mRNA expression was compared in susceptible and resistant mice during the first days of infection. In contrast to resistant mice, susceptible mice exhibited a peak of IL-4 mRNA in their spleens 90 min after i.v. injection of parasites and in lymph nodes 16 h after s.c. injection. IL-12 and interferon-gamma (IFN-gamma) down-regulated this early peak of IL-4 mRNA; the effect of IL-12 was IFN-gamma dependent. Treatment of resistant C57BL/6 mice with anti-IFN-gamma allowed the expression of this early IL-4 response to L. major. The increased IL-4 mRNA expression occurred in V beta 8, 7, 2- CD4+ cells in BALB/c mice and NK1.1- CD4+ cells in anti-IFN-gamma treated C57BL/6 mice. These results show that the NK1.1+ CD4+ cells, responsible for the rapid burst of IL-4 production after i.v. injection of anti-CD3, do not contribute to the early IL-4 response to L. major.

SummaryMice with homologous disruption of the gene coding for the ligand-binding chain of the interferon (IFN) 3' receptor and derived from a strain genetically resistant to infection with Leishmania major have been used to study further the role of this cytokine in the differentiation of functional CD4 + T cell subsets in vivo and resistance to infection. Wild-type 129/Sv/Ev mice are resistant to infection with this parasite, developing only small lesions, which resolve spontaneously within 6 wk. In contrast, mice lacking the IFN-3, receptor develop large, progressing lesions. After infection, lymph nodes (LN) and spleens from both wild-type and knockout mice showed an expansion of CD4 § cells producing IFN-3, as revealed by measuring IFN-3, in supernatants of specifically stimulated CD4 + T cells, by enumerating IFN-3,-producing T cells, and by Northern blot analysis of IFN-v transcripts. No biologically active interleukin (IL) 4 was detected in supernatants of in vitro-stimulated LN or spleen cells from infected wild-type or deficient mice. Reverse transcription polymerase chain reaction analysis with primers specific for IL-4 showed similar IL-4 message levels in LN from both types of mice. The IL-4 message levels observed were comparable to those found in similarly infected C57BL/6 mice and significantly lower than the levels found in BALB/c mice. Anti-IFN-3" treatment of both types of mice failed to alter the pattern of cytokines produced after infection. These data show that even in the absence of IFN-v receptors, T helper cell (Th) 1-type responses still develop in genetically resistant mice with no evidence for the expansion of Th2 cells.

The major surface protease, gp63, of Leishmania chagasi is encoded by 18 or more tandem msp genes that can be grouped into three classes on the basis of their unique 3'-untranslated sequences (3'-UTRs) and their differential expression. RNAs from the mspLs occur predominantly during the logarithmic phase of promastigote growth in vitro, RNAs from the mspSs are present mainly in stationary phase, and RNAs from mspCs occur throughout growth in culture. All three classes of gp63 genes are constitutively transcribed during all growth phases, indicating that their expression is post-transcriptionally regulated. Chimeric plasmids containing the three different 3'-UTRs and downstream intergenic regions (IRs) fused downstream of the beta-galactosidase (beta-gal) coding region were transfected into L. chagasi, and their effects on beta-gal RNA processing and enzymatic activity were examined. The presence of the 3'-UTRs by themselves had no substantive effect on beta-gal expression. However, the 3'-UTR from a mspS plus its IR resulted in about 20-fold more beta-gal activity and RNA in stationary phase relative to logarithmic phase cells. In contrast, the 3'-UTRs plus IRs of mspL and mspC had either no or little effect, respectively, on beta-gal expression. Thus, differential expression of the mspLs and mspSs is post-transcriptionally controlled by different mechanisms.

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Previous results have documented a burst of IL-4 mRNA that peaks in draining lymph nodes of susceptible BALB/c mice 16 h after infection with Leishmania major. The importance of this early IL-4 response in subsequent Th2 cell maturation is supported by observations showing that 1) neutralization of IL-4 at the initiation of infection or 2) administration of IL-12, which results in an inhibition of the 16 h IL-4 mRNA burst, inhibits Th2 cell development. However, both treatments are effective in hampering Th2 cell development only if given at a time when IL-4 has been produced for <48 h. At this time after infection, lymph node CD4+ T cells from BALB/c mice no longer respond to IL-12. This IL-12 unresponsiveness is prevented in mice treated with anti-IL-4 Abs at the initiation of infection. Finally, the inhibition of Th2 development in BALB/c mice treated with anti-IL-4 Abs at the onset of infection results from maintenance of IL-12 responsiveness, since it requires IL-12. Together, these results reveal a narrow window of time, between 16 h and <48 h after infection, during which IL-4 produced rapidly in BALB/c mice renders T cells unresponsive to IL-12, allowing their differentiation toward the Th2 phenotype.