Identification of Orch3, a Locus Controlling Dominant Resistance to Autoimmune Orchitis, as Kinesin Family Member 1C

Experimental autoimmune orchitis (EAO), the principal model of non-infectious testicular inflammatory disease, can be induced in susceptible mouse strains by immunization with autologous testicular homogenate and appropriate adjuvants. As previously established, the genome of DBA/2J mice encodes genes that are capable of conferring dominant resistance to EAO, while the genome of BALB/cByJ mice does not and they are therefore susceptible to EAO. In a genome scan, we previously identified Orch3 as the major quantitative trait locus controlling dominant resistance to EAO and mapped it to chromosome 11. Here, by utilizing a forward genetic approach, we identified kinesin family member 1C (Kif1c) as a positional candidate for Orch3 and, using a transgenic approach, demonstrated that Kif1c is Orch3. Mechanistically, we showed that the resistant Kif1c$^{D2}$ allele leads to a reduced antigen-specific T cell proliferative response as a consequence of decreased MHC class II expression by antigen presenting cells, and that the L$^{578}$→P$^{578}$ and S$^{1027}$→P$^{1027}$ polymorphisms distinguishing the BALB/cByJ and DBA/2J alleles, respectively, can play a role in transcriptional regulation. These findings may provide mechanistic insight into how polymorphism in other kinesins such as KIF21B and KIF5A influence susceptibility and resistance to human autoimmune diseases

Notch signaling expands a pre-malignant pool of T-cell acute lymphoblastic leukemia clones without affecting leukemia-propagating cell frequency

NOTCH1 pathway activation contributes to the pathogenesis of over 60% of T-cell acute lymphoblastic leukemia (T-ALL). While Notch is thought to exert the majority of its effects through transcriptional activation of Myc, it also likely has independent roles in T-ALL malignancy. Here, we utilized a zebrafish transgenic model of T-ALL, where Notch does not induce Myc transcription, to identify a novel Notch gene expression signature that is also found in human T-ALL and is regulated independently of Myc. Cross-species microarray comparisons between zebrafish and mammalian disease identified a common T-ALL gene signature, suggesting that conserved genetic pathways underlie T-ALL development. Functionally, Notch expression induced a significant expansion of pre-leukemic clones; however, a majority of these clones were not fully transformed and could not induce leukemia when transplanted into recipient animals. Limiting-dilution cell transplantation revealed that Notch signaling does not increase the overall frequency of leukemia-propagating cells (LPCs), either alone or in collaboration with Myc. Taken together, these data indicate that a primary role of Notch signaling in T-ALL is to expand a population of pre-malignant thymocytes, of which a subset acquire the necessary mutations to become fully transformed LPCs

Analysis of the Role of Bphs/Hrh1 in the Genetic Control of Responsiveness to Pertussis Toxin

In vivo intoxication with Bordetella pertussis toxin (PTX) elicits a variety of physiological responses including a marked leukocytosis, disruption of glucose regulation, adjuvant activity, alterations in vascular function, hypersensitivity to vasoactive agents, and death. We recently identified Bphs, the locus controlling PTX-induced hypersensitivity to the vasoactive amine histamine, as the histamine H(1) receptor (Hrh1). In this study Bphs congenic mice and mice with a disrupted Hrh1 gene were used to examine the role of Bphs/Hrh1 in the genetic control of susceptibility to a number of phenotypes elicited following in vivo intoxication. We report that the contribution of Bphs/Hrh1 to the overall genetic control of responsiveness to PTX is restricted to susceptibility to histamine hypersensitivity and enhancement of antigen-specific delayed-type hypersensitivity responses. Furthermore, the genetic contribution of Bphs/Hrh1 to vasoactive amine sensitization is specific for histamine, since hypersensitivity to serotonin was unaffected by Bphs/Hrh1. Bphs/Hrh1 also did not significantly influence susceptibility to the lethal effects, the leukocytosis response, disruption of glucose regulation, and histamine-independent increases in vascular permeability associated with in vivo intoxication. Nevertheless, significant interstrain differences in susceptibility to the lethal effects of PTX and leukocytosis response were observed. These results indicate that the phenotypic variation in responsiveness to PTX reflects the genetic control of distinct intermediate phenotypes rather than allelic variation in genes controlling overall susceptibility to intoxication

Susceptibility to Anthrax Lethal Toxin Is Controlled by Three Linked Quantitative Trait Loci

Anthrax lethal toxin (LT) is the principal virulence factor associated with lethal pathologies following infection with Bacillus anthracis. Macrophages are the primary effector cells mediating lethality since macrophage-depleted mice are resistant to LT challenge. Recently, Ltxs1, the gene controlling differential susceptibility of murine macrophages to cytolysis following in vitro exposure to LT, was identified as Kif1c. To directly assess the in vivo role of Kif1c alleles in mortality, we studied a panel of interval-specific recombinant congenic lines carrying various segments of central chromosome 11 derived from LT-resistant DBA/2 mice on the LT-susceptible BALB/c background. The results of this study reveal that mortality is controlled by three linked quantitative trait loci (QTL): Ltxs1/Kif1c (42–43 cM), Ltxs2 (35–37 cM), and Ltxs3 (45–47 cM). The Ltxs3 interval encompasses Nos2, which is an attractive candidate gene for Ltxs3. In this regard, we demonstrate that selective, pharmacologically based inhibition of Nos2 activity in vivo partially overrides genetic resistance to LT and that Nos2 expression as determined by reverse transcription-polymerase chain reaction differs significantly between DBA/2 and BALB/c macrophages. Additionally, to recapitulate dominant resistance to mortality as seen in (BALB/c × DBA/2) F1 hybrids, DBA/2 alleles are required at all three QTL

Structural polymorphisms at amino acid residues 578 and 1027 influence KIF1c function.

<p>Jurkat cells were co-transfected with a plasmid containing the (A) <i>Kif1c^D2</i> (open bar), <i>Kif1c^CByJ</i> (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 <i>Il2</i> 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.</p

Histopathology of autoimmune orchitis.

<p>(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).</p

Identification of <i>Orch3</i> as <i>Kif1c</i>.

<p>(CD2-3.2×CByJ)×CByJ backcross mice were screened from recombinants using microsatellite markers spanning the <i>Orch3</i> interval. Three sub-ISRC lines were identified, fixed and homozygous progeny studied for susceptibility to EAO (D = <i>D2</i> allele; C = <i>CByJ</i> allele). The significance of differences in EAO among CByJ, CD2-3.2a, CD2-3.2b, CD2-3.2c and Tg-<i>Kif1c^D2</i> transgenic mice was determined using the Kruskal-Wallis test (overall <i>p</i>-value<0.0001) followed by Dunn's multiple comparison test (**p<0.01). Region outlined in red reflects location of <i>Orch3</i> based on high resolution congenic mapping relative to the lower resolution mapping outlined in blue.</p

Evaluation of Ag-specific T cell stimulatory capacity of APCs.

<p>Ag-specific T cell proliferative responses were evaluated by [³H] thymidine incorporation. (A) OVA-specific CD4 T cells, and (B) PLP_180–199-specific CD4 T cells from NLC mice were co-cultured with T cell-depleted/mitomycin C-treated/OVA pulsed APCs (A) and PLP_180–199 pulsed APCs (B). Open bars are Tg-<i>Kif1c^D2</i>-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). PLP_180–199-specific response: effect of [PLP_180–199] (p<0.0001); effect of strain (p<0.0001); interaction (p = 0.08).</p