A Restricted Role for FcγR in the Regulation of Adaptive Immunity.

By their interaction with IgG immune complexes, FcγR and complement link innate and adaptive immunity, showing functional redundancy. In complement-deficient mice, IgG downstream effector functions are often impaired, as well as adaptive immunity. Based on a variety of model systems using FcγR-knockout mice, it has been concluded that FcγRs are also key regulators of innate and adaptive immunity; however, several of the model systems underpinning these conclusions suffer from flawed experimental design. To address this issue, we generated a novel mouse model deficient for all FcγRs (FcγRI/II/III/IV-/- mice). These mice displayed normal development and lymphoid and myeloid ontogeny. Although IgG effector pathways were impaired, adaptive immune responses to a variety of challenges, including bacterial infection and IgG immune complexes, were not. Like FcγRIIb-deficient mice, FcγRI/II/III/IV-/- mice developed higher Ab titers but no autoantibodies. These observations indicate a redundant role for activating FcγRs in the modulation of the adaptive immune response in vivo. We conclude that FcγRs are downstream IgG effector molecules with a restricted role in the ontogeny and maintenance of the immune system, as well as the regulation of adaptive immunity

Mice Deficient for All PIM Kinases Display Reduced Body Size and Impaired Responses to Hematopoietic Growth Factors

The Pim family of proto-oncogenes encodes a distinct class of serine/threonine kinases consisting of PIM1, PIM2, and PIM3. Although the Pim genes are evolutionarily highly conserved, the contribution of PIM proteins to mammalian development is unclear. PIM1-deficient mice were previously described but showed only minor phenotypic aberrations. To assess the role of PIM proteins in mammalian physiology, compound Pim knockout mice were generated. Mice lacking expression of Pim1, Pim2, and Pim3 are viable and fertile. However, PIM-deficient mice show a profound reduction in body size at birth and throughout postnatal life. In addition, the in vitro response of distinct hematopoietic cell populations to growth factors is severely impaired. In particular, PIM proteins are required for the efficient proliferation of peripheral T lymphocytes mediated by synergistic T-cell receptor and interleukin-2 signaling. These results indicate that members of the PIM family of proteins are important but dispensable factors for growth factor signaling

Involvement of Virus-Induced Interferon Production in IgG Autoantibody-Mediated Anemia

Infection with viruses, such as the lactate dehydrogenase-elevating virus (LDV), is known to trigger the onset of autoimmune anemia through the enhancement of the phagocytosis of autoantibody-opsonized erythrocytes by activated macrophages. Type I interferon receptor-deficient mice show enhanced anemia, which suggests a protective effect of these cytokines, partly through the control of type II interferon production. The development of anemia requires the expression of Fcγ receptors (FcγR) I, III, and IV. Whereas LDV infection decreases FcγR III expression, it enhances FcγR I and IV expression in wild-type animals. The LDV-associated increase in the expression of FcγR I and IV is largely reduced in type I interferon receptor-deficient mice, through both type II interferon-dependent and -independent mechanisms. Thus, the regulation of the expression of FcγR I and IV, but not III, by interferons may partly explain the exacerbating effect of LDV infection on anemia that results from the enhanced phagocytosis of IgG autoantibody-opsonized erythrocytes

Immunogenicity of rat-neu+ mouse mammary tumours determines the T cell-dependent therapeutic efficacy of anti-neu monoclonal antibody treatment

The use of Trastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2/neu, results in an increased median survival in Her2+ breast cancer patients. The tumour mutational burden and the presence of tumour infiltrating lymphocytes (TILs) clearly correlate with response to trastuzumab. Here, we investigated if the immunogenicity of the transplantable rat-neu+ tumour cell line (TUBO) derived from a BALB/c-NeuT primary tumour is associated with the response to anti-neu mAb therapy. We compared the TUBO tumour outgrowth and tumour infiltrating T cells in isogenic (BALB/c-NeuT) and non-isogenic (WT BALB/c) recipient mice. Furthermore, therapeutic efficacy of anti-neu mAb and the contribution of T cells were examined in both mouse strains. The outgrowth of untreated tumours was significantly better in BALB/c-NeuT than WT BALB/c mice. Moreover, tumour infiltrating T cells were more abundantly present in WT BALB/c than BALB/c-NeuT mice, showing that the TUBO tumour was more immunogenic in WT BALB/c mice. In TUBO tumour bearing WT BALB/c mice, anti-neu mAb therapy resulted in an increase of tumour infiltrating T cells and long-term survival. When T cells were depleted, this strong anti-tumour effect was reduced to an outgrowth delay. In contrast, in TUBO tumour bearing BALB/c-NeuT mice, treatment with anti-neu mAb resulted only in tumour outgrowth delay, both in the presence and absence of T cells. We concluded that in immunogenic tumours the response to anti-neu mAb therapy is enhanced by additional T cell involvement compared to the response to anti-neu mAb in non-immunogenic tumours

Dystrophin expression pattern and morphological examination of del52hDMD/<i>mdx</i> mouse lines.

<p><b>A</b>) Western blot analyses of heart and quadriceps, incubated with either GTX (human and mouse specific) or Mandys106 (human specific). Wild type expression levels of human dystrophin were observed in hDMD/<i>mdx</i> mice. Notably, del52hDMD/<i>mdx</i>#37 mice expressed traces of human dystrophin, in both cardiac and skeletal muscle, while this was not observed in del52hDMD/<i>mdx</i>#35 and <i>mdx</i>(BL6) mice. <b>B</b>) Sections of the heart and quadriceps stained with human specific dystrophin antibodies. Expression of human dystrophin is at wild type level in hDMD/<i>mdx</i> mice as anticipated. Both C57BL/6J, <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice did not express human dystrophin. Interestingly, in most fibers of del52hDMD/<i>mdx</i>#37 mice, human dystrophin was expressed at low levels. Haematoxylin and eosin staining revealed signs of degeneration and regeneration in the quadriceps of both del52hDMD/<i>mdx</i> strains, as evident by variation in fiber size, centralized nuclei and patches of fibrosis and inflammation. Overall pathology appeared to be slightly less extensive in del52hDMD/<i>mdx</i>#37 mice compared to <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice. <b>C</b>) Almost no centralized nuclei were found in wild type mice, while half of the myofibers in <i>mdx</i>(BL6) and del52hDMD/<i>mdx</i>#35 mice had centrally located nuclei. The percentage in del52hDMD/<i>mdx</i>#37 mice was with 26% significantly lower. Data were based on manual counts of 5 randomly taken pictures of 2 males and 2 females per genotype. Asterisks indicate <i>P</i><0.01.</p

PCR analysis of targeted clones and confirmation of deletion exon 52 on RNA level.

<p>Single ES clones were cultured in 96-well plates and DNA was isolated and used as template in a multiplex PCR. Here the exons 46, 51 and 52 of the <i>hDMD</i> gene were analysed where exon 46 and 51 are positive controls and exon 52 the target to be deleted. <b>A</b>) An example is shown where candidate samples 2 and 5 are of interest because they are negative for exon 52 but positive for the control exons. <b>B</b>) For a large number of clones additional fragments were found for exon 52, suggesting non-homologues end joining (NHEJ) of TALEN induced double stranded breaks <b>C</b>) Representative image of LR-PCR performed on DNA of sub-clones of four exon 52 negative clones (9B4, 10H2, 11C9 and 11E7). LR-PCR was performed with primers targeting intron 51 (outside the targeting arm) and blasticidin (only present after homologous recombination), to rule out loss of PCR primer recognition sites by NHEJ and to confirm true targeting. <b>D</b>) RT-PCR was performed for RNA isolated from embryoid bodies of selected clones. The different fragments were isolated, purified and Sanger sequence analysed. In the wild type situation exon 52 was present, whereas in the properly targeted clones exon 52 was not present. This confirmed the exon 52 deletion on RNA level.</p

Confirmational analysis of offspring from blastocyst transplanted mice.

<p>Pups were analysed for chimerism by multiplex PCR and MCA. <b>A</b>) Four of the pups derived after transplantation of blastocysts injected with ES cells of clone 9B4 showed presence of the exon 52 deleted <i>hDMD</i> gene (lines 1, 4, 8 and 11). <b>B</b>) Melting curve analysis revealed that all male pups were also chimeric for the <i>mdx</i> point mutation.</p