Immune competency of a hairless mouse strain for improved preclinical studies in genetically engineered mice - PubMed (original) (raw)
doi: 10.1158/1535-7163.MCT-10-0207. Epub 2010 Jul 27.
Marcia H Grayson, Joy M Wortham, Courtney B Kubicek, Amanda T McCleish, Suresh I Prajapati, Laura D Nelon, Michelle M Brady, Inkyung Jung, Tohru Hosoyama, Leslea M Sarro, Martha A Hanes, Brian P Rubin, Joel E Michalek, Charles B Clifford, Anthony J Infante, Charles Keller
Affiliations
- PMID: 20663932
- PMCID: PMC2921575
- DOI: 10.1158/1535-7163.MCT-10-0207
Immune competency of a hairless mouse strain for improved preclinical studies in genetically engineered mice
Beverly S Schaffer et al. Mol Cancer Ther. 2010 Aug.
Abstract
Genetically engineered mouse models (GEMM) of cancer are of increasing value to preclinical therapeutics. Optical imaging is a cost-effective method of assessing deep-seated tumor growth in GEMMs whose tumors can be encoded to express luminescent or fluorescent reporters, although reporter signal attenuation would be improved if animals were fur-free. In this study, we sought to determine whether hereditable furlessness resulting from a hypomorphic mutation in the Hairless gene would or would not also affect immune competence. By assessing humoral and cellular immunity of the SKH1 mouse line bearing the hypomorphic Hairless mutation, we determined that blood counts, immunoglobulin levels, and CD4+ and CD8+ T cells were comparable between SKH1 and the C57Bl/6 strain. On examination of T-cell subsets, statistically significant differences in naïve T cells (1.7 versus 3.4 x 10(5) cells/spleen in SKH1 versus C57Bl/6, P = 0.008) and memory T cells (1.4 versus 0.13 x 10(6) cells/spleen in SKH1 versus C57Bl/6, P = 0.008) were detected. However, the numerical differences did not result in altered T-cell functional response to antigen rechallenge (keyhole limpet hemocyanin) in a lymph node cell in vitro proliferative assay. Furthermore, interbreeding the SKH1 mouse line to a rhabdomyosarcoma GEMM showed preserved antitumor responses of CD56+ natural killer cells and CD163+ macrophages, without any differences in tumor pathology. The fur-free GEMM was also especially amenable to multiplex optical imaging. Thus, SKH1 represents an immune competent, fur-free mouse strain that may be of use for interbreeding to other genetically engineered mouse models of cancer for improved preclinical studies.
(c) 2010 AACR.
Figures
Figure 1. Genetic and Morphological Features of the _Hairless_SKH1 Mouse
A, Genomic structure of the Hairless gene as related to viral insertion, genotyping primers (am05, am06, am07) and RT-PCR primers (th009 and th010). While _pmv_43 has not been yet sequenced by any group, the size of _pmv_43 is estimated at 9.5 Kb based upon the largest clone (MX40A) having extended 3′ from a BamHI restriction site in pol (1), which is then assumed to be approximately 4.1 Kb from the 5′ end of the prototypic pmv. Note, also, that labeling of exons given here is consistent with the published literature, but that databases now predict Hairless to have 22 instead of 19 exons (Mouse Genome Informatics ID U015825,
http://lgsun.grc.nia.nih.gov/geneindex/mm9/bin/giU.cgi?genename=U015825
). According to the database schema, _pmv_43 would be inserted between exons 7 and 8, instead of 6 and 7. In this figure, the locus is to scale, but the virus is not to scale. Chr, chromosome. B, Genotyping PCR, demonstrating a 350 bp band for wildtype Hairless allele and a 250 bp band for the SKH1 allele. Het, herterozygous. Hom, homozygous. C, Quantitative RT-PCR showing residual full-length Hr transcript in skin of SKH1 mice (left). The PCR product for SKH1 and B6 Hr are a singlet band of predicted size. D, Progressive rostral to caudal alopecia in 7, 13, 18 and 24 day old mouse pups (left, top to bottom) as well as a 3 month old adult female SKH1 mouse (right).
Figure 2. Humoral Immunity of SKH1 vs. B6 mice
A to C, Serum immunoglobin levels of IgM, IgG and IgA, respectively (B6 n=12, 11, 12; SKH1 n=15, 15, 15, respectively). D, CD19 positive B cell counts from spleen (B6 n=5; SKH1 n=5). E to F, Pre- and Post-immunization anti-KLH antibody IgM and IgG levels, respectively. IgM levels (B6 n=8; SKH1 n=10) were measured 5 days after immunization, and IgG levels (B6 n=11; SKH1 n=10) were measured 2 weeks after immunization.
Figure 3. Cellular Immunity of SKH1 vs. B6 mice
A, Thymus cell counts for T cell subpopulations. No statistical differences were found. B, Peripheral (splenic) cell counts for CD3 T cells, CD8 T cells, CD49+ Natural Killer (NK) cells, CD4+CD25+ regulatory T cells, CD44Lo CD62LHI naïve T cells and CD44HI CD62LLo memory T cells. C, ex vivo leukocyte proliferation following KLH immunization. D, in vivo immune response following KLH immunization (no significant difference was seen between B6 and SKH1 mice). Data presented in (A) or (B) are absolute numbers of cells per thymus or spleen, respectively. For all experiments, B6 n=5; SKH1 n=5
Figure 4. A Hairless Rhabdomyosarcoma Model
A, Histology (Hematoxylin and eosin, H&E) of rhabdomyosarcoma tumors for _Myf6_ICNm/WT _Pax3_P3Fm/P3Fm _Trp53_F2-10/F2-10 _Rosa26_Lusapm/WT mice harboring wildtype Hr alleles (Control, left) or homozygous SKH1 mutation (SKH1, right). B, Tumors were positive for myogenin (arrows) in both cohorts. C to D, Tumors in both cohorts also harbored infiltrating CD56+ NK cells and CD163+ macrophages, respectively (arrows). All panels represent 400×.
Figure 5. Multiplex Optical Imaging of a Hairless Rhabdomyosarcoma Model
A, From left to right: photograph, luciferase luminescence (scale 2.8×105 to 8.0×108 p/sec/cm2/sr), IR-820 fluorescence (scale 3.2×105 to 1.6×109 p/sec/cm2/sr) and MMPsense fluorescence (scale 0 to 2.8×109 p/sec/cm2/sr) for a mouse bearing a rhabdomyosarcoma of the left foot. Viable tumor is shown with arrowheads. A metastatic lesion is shown by an asterisk (*). MMPsense activity is shown by an arrow at the advancing front of the tumor. Apparent MMPsense signal in the abdomen is artifact attributable to alfalfa in mouse chow. B, repeat imaging for a more restricted field of view. Scales: luminescence (2.8×104 to 8.6×108 p/sec/cm2/sr), IR-820 fluorescence (2.1×107 to 3.4×109 p/sec/cm2/sr) and MMPsense fluorescence (0 to 4.9×109 p/sec/cm2/sr). C, necropsy photomicrograph demonstrating an enlarged subcutaneous lymph node that overlied the spleen. D, H&E of primary tumor consistent with rhabdomyosarcoma (400×). E to F, Immunohistochemistry positive for nuclear myogenin and cytoplasmic desmin, respectively (400×). G, H&E of lymph node shown in A, B and C (20×). H, Close-up of G (200×). I, Myogenin positive rhabdomyosarcoma cells in the lymph node (400×).
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