Deconvolution of Blood Microarray Data Identifies Cellular Activation Patterns in Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is a systemic autoimmune disease with a complex spectrum of cellular and molecular characteristics including several dramatic changes in the populations of peripheral leukocytes. These changes include general leukopenia, activation of B and T cells, and maturation of granulocytes. The manifestation of SLE in peripheral blood is central to the disease but is incompletely understood. A technique for rigorously characterizing changes in mixed populations of cells, microarray expression deconvolution, has been applied to several areas of biology but not to SLE or to blood. Here we demonstrate that microarray expression deconvolution accurately quantifies the constituents of real blood samples and mixtures of immune-derived cell lines. We characterize a broad spectrum of peripheral leukocyte cell types and states in SLE to uncover novel patterns including: specific activation of NK and T helper lymphocytes, relationships of these patterns to each other, and correlations to clinical variables and measures. The expansion and activation of monocytes, NK cells, and T helper cells in SLE at least partly underlie this disease's prominent interferon signature. These and other patterns of leukocyte dynamics uncovered here correlate with disease severity and treatment, suggest potential new treatments, and extend our understanding of lupus pathology as a complex autoimmune disease involving many arms of the immune system

Analysis of the effect of promoter type and skin pretreatment on antigen expression and antibody response after gene gun-based immunization.

Monoclonal antibodies (mAbs) have enabled numerous basic research discoveries and therapeutic approaches for many protein classes. However, there still exist a number of target classes, such as multi-pass membrane proteins, for which antibody discovery is difficult, due in part to lack of high quality, recombinant protein. Here we describe the impact of several parameters on antigen expression and the development of mAbs against human claudin 4 (CLDN4), a potential multi-indication cancer target. Using gene gun-based DNA delivery and bioluminescence imaging, we optimize promoter type by comparing expression profiles of four robust in vivo promoters. In addition, we observe that most vectors rapidly lose expression, ultimately reaching almost background levels by three days post-delivery. Recognizing this limitation, we next explored skin pretreatment strategies as an orthogonal method to further boost the efficiency of mAb generation. We show that SDS pretreatment can boost antigen expression, but fails to significantly increase mAb discovery efficiency. In contrast, we find that sandpaper pretreatment yields 5-fold more FACS+ anti-CLDN4 hybridomas, without impacting antigen expression. Our findings coupled with other strategies to improve DNA immunizations should improve the success of mAb discovery against other challenging targets and enable the generation of critical research tools and therapeutic candidates

Comparison of <i>in vitro</i> and <i>in vivo</i> expression profiles of different promoters.

<p>(A) Luciferase expression levels in HaCat keratinocyte cells shows that CAG promoter greatly outperforms SFFV, EF1α, and UbC promoters (n = 4). (B) Luciferase expression levels in HEK293 cells similar expression from CAG and EF1α promoters (n = 4). (C) Luciferase expression levels in Balb/c mice shows that CAG promoter greatly outperforms SFFV, EF1α, and UbC promoters (n = 6; two shots in three mice). (D) Expression profile of each construct reveals a rapid decline in expression each day after delivery and ultimately, reaching background levels by day 3. Overall, the CAG promoter yields the highest expression at day 1 and day 2 (****: p<0.01, independent t-test).</p

Luciferase expression profiles after gene gun-based DNA delivery.

<p>Luciferase expression profile in Balb/c (A) and C57BL6 (B) mice for three different gas pressures (150, 300, and 400 psi) at days 1, 2, and 3 post-delivery (n = 5 mice).</p

Antibody discovery efficiency using different pretreatments.

<p>Antibody discovery efficiency using different pretreatments.</p

Effect of different skin pretreatment strategies on anti-huCLDN4 Ab response.

<p>(A) Sandpaper treatment results in a significant increase in MFI against HEK293 expressing huCLDN4 compared to other treatments and mock treated control (n = 5; p < 0.001, independent t-test). Samples were analyzed at a 1:100 dilution. (B) A comparison of FACS- and Mirrorball-based screen of hybridoma supernatants on HEK293 huCLDN4 and parental HEK293 cells demonstrates an 89% (51/57) overlap between the two methods.</p

Effect of different skin pretreatment strategies on luciferase expression.

<p>(A) 1% SDS treatment leads to an increase in luciferase expression one day after delivery compared to other treatments and mock treated control (**, ***: p<0.01, independent t-test) (n = 6; two shots in three mice).</p

Comparison of luciferase expression profiles in TNFα, IFNγ, and IFNα receptor knockout (KO) mice.

<p>(A-C) A rapid decline in luciferase expression for the CAG promoter is observed in all three KO strains as well as the wild-type C57BL6 strain (n = 5).</p

APRIL-Deficient Mice Have Normal Immune System Development

APRIL (a proliferation-inducing ligand) is a member of the tumor necrosis factor (TNF) superfamily. APRIL mRNA shows high levels of expression in tumors of different origin and a low level of expression in normal cells. APRIL shares two TNF receptor family members, TACI and BCMA, with another TNF homolog, BLyS/BAFF. BLyS is involved in regulation of B-cell activation and survival and also binds to a third receptor, BR3/BAFF-R, which is not shared with APRIL. Recombinant APRIL and BLyS induce accumulation of B cells in mice, while BLyS deficiency results in severe B-cell dysfunction. To investigate the physiological role of APRIL, we generated mice that are deficient in its encoding gene. APRIL(−/−) mice were viable and fertile and lacked any gross abnormality. Detailed histological analysis did not reveal any defects in major tissues and organs, including the primary and secondary immune organs. T- and B-cell development and in vitro function were normal as well, as were T-cell-dependent and -independent in vivo humoral responses to antigenic challenge. These data indicate that APRIL is dispensable in the mouse for proper development. Thus, BLyS may be capable of fulfilling APRIL's main functions