Figure 1.
Histopathology of autoimmune orchitis.
(A, C) Cross section of normal testis histology in an immunized C.D2-3 mouse: (A) Seminiferous tubules appear normal; (C) A seminiferous tubule (ST) containing normal meiotic spermatocytes and spermatids, with intact tubular boundary (arrows). (B, D) CByJ mouse with sever and diffuse orchitis: (B) All seminiferous tubules are necrotic and have lost cell nuclear staining; (D) Sever orchitis in one seminiferous tubule (arrows) that contains numerous neutrophils and occasional multinuclear giant macrophages (double arrow); the tubular boundary (arrows) is poorly defined. (H&E; A and B, ×4; C and D, ×40).
Figure 2.
Congenic mapping places Orch3 within the Kif1c/Nlrp1a/b/c interval.
For convenience, D2 alleles have been shaded. The significance of differences in severity of EAO among CByJ, CD2F1 hybrids and CD2-ISRC lines was determined using the Kruskal-Wallis test (overall p-value<0.0001) followed by Dunn's multiple comparison test. Region outlined in blue depicts the location of Orch3.
Figure 3.
Identification of Orch3 as Kif1c.
(CD2-3.2×CByJ)×CByJ backcross mice were screened from recombinants using microsatellite markers spanning the Orch3 interval. Three sub-ISRC lines were identified, fixed and homozygous progeny studied for susceptibility to EAO (D = D2 allele; C = CByJ allele). The significance of differences in EAO among CByJ, CD2-3.2a, CD2-3.2b, CD2-3.2c and Tg-Kif1cD2 transgenic mice was determined using the Kruskal-Wallis test (overall p-value<0.0001) followed by Dunn's multiple comparison test (**p<0.01). Region outlined in red reflects location of Orch3 based on high resolution congenic mapping relative to the lower resolution mapping outlined in blue.
Figure 4.
Generation of BALB/cByJ-CD11B-Kif1cD2 transgenic (Tg-Kif1cD2) mice.
(A) Schematic representation of the Kif1c gene used to generate the transgenic mice showing the promoter (CD11B/ITGAM), and the Kif1c gene, followed by the hGH/polyA signal sequence. Arrows indicate PCR-primers for screening. (B and C) Percentage of splenic F4/80+ (B) and CD11b+ (C) cells of Tg-Kif1cD2 and NLC. The analysis was performed on gated live cells according to their FSC vs. SSC profile. Statistical significance was determined using the Mann-Whitney U test. Data represent the mean ± SEM of at least 5 individual mice. (D) Kif1c expression in thioglycolate-induced adherent cells by Western blotting using whole-cell extracts and the anti-Kif1c mAb. Actin was used as a loading marker. (E) mRNA expression of Kif1c was measured from sorted TCRβ−CD19−CD11b+ myeloid cells of CByJ mice and compared with TCRβ−CD19−CD11b+ myeloid cells of D2 mice. β2-microglobulin and GAPDH were used as an endogenous control. Data represent the mean ± SEM of two experiments (pool of 5 animals/each).
Figure 5.
Analysis of MHC II expression on splenic CD11b+ myeloid cells.
(A) Scatterplot of genes differentially expressed in splenic TCRβ−CD19−CD11b+ myeloid cells of Tg-Kif1cD2 and NLC mice as determined by microarray. There were 164 genes differentially expressed (FDR≤0.05) and for each gene, the log2 fold change was plotted on the ordinate against the -log10 p-value, plotted on the abscissa. Each data point represents the log2 fold change (Tg-Kif1cD2 minus NLC) for each gene. Dark blue data points indicate H2 genes that were downregulated in splenic CD11b+ cells of Tg-Kif1cD2 mice. (B) Flow cytometric analysis of the frequency of TCRβ−CD19−CD11b+-myeloid cells expressing MHC II in the spleen of Tg-Kif1cD2, D2, D.2-C3.2, and NTC. Statistical significance was determined using the Kruskal-Wallis test (overall ***p-value<0.0001) followed by Dunn's multiple comparison test (**p<0.01, *p<0.05). Data represent the mean ± SEM of at least 5 individual mice.
Figure 6.
Evaluation of Ag-specific T cell stimulatory capacity of APCs.
Ag-specific T cell proliferative responses were evaluated by [3H] thymidine incorporation. (A) OVA-specific CD4 T cells, and (B) PLP180–199-specific CD4 T cells from NLC mice were co-cultured with T cell-depleted/mitomycin C-treated/OVA pulsed APCs (A) and PLP180–199 pulsed APCs (B). Open bars are Tg-Kif1cD2-APCs, and closed bars are NLC-APCs. Each bar represents the mean cpm ± SEM of 3 independent experiments. The significance of the differences was determined by two-way ANOVA. OVA-specific response: effect of [OVA] (p<0.0001); effect of strain (p<0.0001); interaction (p = 0.08). PLP180–199-specific response: effect of [PLP180–199] (p<0.0001); effect of strain (p<0.0001); interaction (p = 0.08).
Figure 7.
Structural polymorphisms at amino acid residues 578 and 1027 influence KIF1c function.
Jurkat cells were co-transfected with a plasmid containing the (A) Kif1cD2 (open bar), Kif1cCByJ (grey bar) alleles, or control plasmid (black bar), or (B) Kif1cD2 (PPY; open bar), mutant 578 (LPY; left striped bar), or mutant 1027 (PSY; right striped bar) plasmids, and Il2 promoter luciferase reporter. Cells were stimulated for 3 hours with PMA and calcimycin, and the luciferase activity was quantified. Data are representative of two independent experiments.