A Leishmania major response locus identified by interval-specific congenic mapping of a T helper type 2 cell bias-controlling quantitative trait locus

Abstract

The propensity of naive CD4 T cells to become T helper (Th) type 2 cells correlates with susceptibility to infection by the protozoal parasite Leishmania major. Using genetic linkage analysis, we earlier identified Dice1 as a Th2 cell bias-controlling quantitative trait locus on chromosome 16. Using interval-specific congenic mapping, we now resolve Dice1 into two independent genetic loci, Dice1.1 and Dice1.2, which control Il4 expression from naive Th cells and thereby indirectly control Th2 cell bias. Interestingly, only one of the two congenic intervals containing Dice1.1 and Dice1.2, respectively, also contained an L. major response locus, indicating that L. major responsiveness can be insensitive to determinants that influence Th2 cell bias by controlling naive T cell Il4 expression. These results lay the groundwork for identifying the Dice1.1 and Dice1.2 genes controlling naive T cell Il4 expression and L. major responses, and for testing whether these control other Th2 cell-dependent processes such as worm expulsion, allergic asthma, and dermatitis.

Figure 1.

Physical location of C.16D2 congenic intervals on chromosome 16. Shown for each C.16D2 strain (labeled at the top) are the portions of chromosome 16 inherited from B10.D2 (shaded) and BALB/c (unshaded). Locations where genotypes were determined are indicated with black diamonds. Genotyping markers used and their locations along chromosome 16 are indicated on the right axis. Double-headed arrows and horizontal lines indicate the inferred locations for Dice1.1 (closed arrowheads) and Dice1.2 (open arrowheads).

Figure 2.

Representative raw Th2 cell bias and nIL-4 data. CD4-enriched splenocyte cultures stimulated with plate-bound anti-TCR and IL-2 were harvested (A) at 5 d for cells or (B) at 16 h for RNA. Cells harvested at 5 d were washed, counted, and restimulated at equivalent cell numbers for an additional 2 d, followed by analysis of IL-4 content in the media by ELISA. RNA analysis was by real-time RT-PCR with data normalized to HPRT and expressed relative to expression in NIH3T3 fibroblasts. Red, black, and open bars represent BALB/c, B10.D2, and C.16D2 strain mice (–9), respectively. Where bars are not visible, value was below the limit of detection.

Figure 3.

Concordance between Th2 cell bias and nIL-4 traits among C.16D2 congenic strains. Shown are scatter plots of Th2 cell bias (top) and nIL-4 index values (bottom) of individual BALB/c (open circles) and C.16D2 congenic animals (gray triangles). Data shown are from experiments in which control BALB/c and B10.D2 differed significantly. For Th2 cell bias experiments involving C16.D2 strains 1–5, 7, and 8, P < 0.09; for C16.D2 strains 6 and 9, P < 0.2; and for all nIL-4 experiments, P ≤ 0.1. C16.D2 strains with phenotypes significantly lower than control BALB/c are indicated with an asterisk. Multiple comparison tests of significance were performed using the Mann-Whitney nonparametric test. P-values (Th2 cell bias, nIL-4) are as follows: C16D2/1 (0.5000, 0.500), C16D2/2 (0.7357, 0.8689), C16D2/3 (<0.0001, <0.0001), C16D2/4 (0.0002, 0.0008), C16D2/5 (0.0018, <0.0001), C16D2/6 (0.7884, 0.5000), C16D2/7 (0.0163, 0.003), C16D2/8 (0.0286, 0.0271), and C16D2/9 (0.1304, 0.5000).

Figure 4.

Response to L. major infection of mice harboring Dice1.1 and Dice1.2 congenic intervals. Groups of five to six mice were infected with metacyclic L. major parasites. The disease course was monitored by weekly measurement of footpad lesion size (A and C) and, at termination, footpad parasite burdens (B and D) in BALB/c (red fill), B10.D2 (black fill), and C16.D2 (white fill) strain mice. Error bars show SEM. Similar results for C.16D2/8 were obtained in three independent experiments.