Additional file 2: Figure S2. of Analyzing pathogenic (double-stranded (ds) DNA-specific) plasma cells via immunofluorescence microscopy

Correlation between dsDNA-specific plasma cells numbers in spleen and bone marrow with the anti-dsDNA antibody titer in the serum. (JPEG 1553 kb

Effects of short-term depletion treatments on plasma cell numbers in bone marrow and spleen.

<p><b>(A)</b> Representative FACS histogram of bone marrow and splenic CD138⁺ intracellular κ⁺ BrdU⁺ short-lived plasma cells (SLPCs), and CD138⁺ intracellular κ⁺ BrdU^- long-lived plasma cells (LLPCs) from each treatment group. Percentage of remaining cell numbers relative to the control mean of (<b>B)</b> bone marrow and (<b>C)</b> splenic CD138⁺ intracellular κ⁺ total plasma cells (PCs), SLPCs, and LLPCs in mice treated with PBS, anti-CD20, anti-CD20 plus integrin-blocking antibodies (Int; anti-LFA1 and anti-VLA4 antibodies), anti-CD20 plus bortezomib (Bz) and anti-CD20 plus Int and Bz. Total PCs, SLPCs and LLPCs were enumerated by flow cytometry 7 days after the start of treatment (<i>n</i> = 5–6 mice per each group). Values are mean±SEM; ns, non-significant; P>0.05, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, post-hoc test. Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; FMO, Fluorescence-minus-one; Int, Integrin blocking antibodies; anti-LFA1 and anti-VLA4 antibodies.</p

B cell depletion (BCD) maintenance therapy after short-term depletion (STD) of B and plasma cells with ant-CD20 and bortezomib improves the disease in NZB/W F1 mice.

<p>Mice (n = 4) were treated with anti-CD20 and bortezomib (STD) alone or continuous B cell depletion without bortezomib (BCD, n = 5) or treated as STD followed by BCD maintenance therapy with anti-CD20 (STD+BCD, n = 4). (<b>A)</b> Serum IgM and IgG anti-dsDNA antibody levels in treated and untreated mice (n = 9), as measured by ELISA. (<b>B)</b> Proteinuria in treated and untreated mice. Statistical differences between treated and untreated mice were analyzed using the post-hoc test (ns, non-significant, P>0.05, *<i>P</i><0.05; **<i>P</i><0.01, ***<i>P</i><0.001). (<b>C)</b> Survival curves for treated and untreated NZB/W F1 mice (Kaplan—Meier log-rank test). Abbreviations: STD, Short-term depletion (anti-CD20 and bortezomib); BCD; B cell depletion (anti-CD20).</p

Effects of short-term depletion treatments on the numbers of different B-cell subsets in bone marrow and spleen.

<p>Percentage of remaining B cell subsets in the bone marrow and spleen in ratio to the mean of control. (<b>A)</b> Bone marrow B-cell subsets identified by flow cytometry: total B cells (BCs) (CD19⁺), bone marrow pro-B cells (CD93⁺CD117⁺), pre-B cells (CD24⁺IgM^-IgD^-), immature B cells (CD24⁺IgM⁺IgD^-), and mature B cells (CD24^-IgM⁺IgD⁺). (<b>B)</b> Splenic B-cell subsets identified by flow cytometry: follicular (FO) B cells (CD23⁺CD21⁺IgM⁺), marginal zone (MZ) B cells (CD23^- CD21⁺IgM⁺), germinal center (GC) B cells (IgD^-GL7⁺), and B1 B cells (CD23^-CD21^-IgM⁺). Values are mean±SEM; ns, non-significant, P>0.05, *<i>P</i><0.05; **<i>P</i><0.01, ***<i>P</i><0.001, post-hoc test (<i>n</i> = 5–6 mice per group). Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; Int, Integrin blocking antibodies; anti-LFA1 and anti-VLA4 antibodies.</p

Effects of short-term depletion treatments on bone marrow and splenic T cells.

<p>Percentage of remaining CD3⁺ T cells, CD4⁺ T-helper cells, and CD8⁺ T-cytotoxic cells after one week of treatment in ratio to the mean of control in (<b>A)</b> the bone marrow, and (<b>B)</b> spleen. Values are mean±SEM; ns, non-significant, P>0.05, *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, post-hoc test (<i>n</i> = 5–6 mice per group). Abbreviations: Bz, bortezomib; CD20, anti-mouse CD20 antibody; Int, Integrin blocking antibodies, anti-LFA1 and anti-VLA4 antibodies.</p

For the validation of 165 monocyte- (a, b and c) and 94 T helper lymphocyte-specific (d, e and f) common-IFN signature genes, their expression were followed in PBMC's of an independent cohort of yellow-fever vaccinated individuals.

<p>Expression profiles were generated from PBMC's at day 3 (a and d), 7 (b and e) and 21 (c and f) after vaccination and compared to baseline levels at d0. Both, the common type I interferon signatures of monocytes and CD4 lymphocytes allowed the monitoring of the induction of interferon responses in PBMCs at d3, peaking at d7 and almost declining at d21.</p

Cluster diagrams of SLE patients and healthy donors before and after immunisation with the yellow fever vaccine.

<p>Healthy donors (ND) immunised with the yellow fever vaccine are designated as “ND_YF_day7”, and ND before immunisation are designated as “ND_YF_day0”. (A) IFN signature in CD4⁺ T cells. The 94 probe-sets of “common” IFN signatures observed both in the SLE patients and immunised ND and the 86 probe-sets of “autoimmune-specific” IFN signatures observed only in the SLE patients distinguish the SLE patients from the immunised ND. (B) IFN signature in CD16⁻ monocytes. The 165 probe-sets of “common” IFN signatures, 164 probe-sets of “autoimmune-specific” IFN signatures and 8 probe-sets of “immunisation-specific” IFN signatures distinguish the SLE patients from the immunised ND. (C) IFN signature in CD16⁺ monocytes. The 173 probe-sets of “common” IFN signatures, 120 probe-sets of “autoimmune-specific” IFN signatures and 5 probe-sets of “immunisation-specific” IFN signatures distinguish the SLE patients from the immunised ND.</p

Cell-Specific Type I IFN Signatures in Autoimmunity and Viral Infection: What Makes the Difference?

<div><p>Gene expression profiling of peripheral blood mononuclear cells (PBMCs) has revealed a crucial role for type I interferon (IFN) in the pathogenesis of systemic lupus erythematosus (SLE). However, it is unclear how particular leucocyte subsets contribute to the overall type I IFN signature of PBMCs and whole blood samples.Furthermore, a detailed analysis describing the differences in the IFN signature in autoimmune diseases from that observed after viral infection has not been performed to date. Therefore, in this study, the transcriptional responses in peripheral T helper cells (CD4⁺) and monocyte subsets (CD16⁻ inflammatory and CD16⁺ resident monocytes) isolated from patients with SLE, healthy donors (ND) immunised with the yellow fever vaccine YFV-17Dand untreated controls were compared by global gene expression profiling.It was striking that all of the transcripts that were regulated in response to viral exposure were also found to be differentially regulated in SLE, albeit with markedly lower fold-change values. In addition to this common IFN signature, a pathogenic IFN-associated gene signature was detected in the CD4⁺ T cells and monocytes from the lupus patients. IL-10, IL-9 and IL-15-mediated JAK/STAT signalling was shown to be involved in the pathological amplification of IFN responses observed in SLE. Type I IFN signatures identified were successfully applied for the monitoring of interferon responses in PBMCs of an independent cohort of SLE patients and virus-infected individuals. Moreover, these cell-type specific gene signatures allowed a correct classification of PBMCs independent from their heterogenic cellular composition. In conclusion, our data show for the first time that monocytes and CD4 cells are sensitive biosensors to monitor type I interferon response signatures in autoimmunity and viral infection and how these transriptional responses are modulated in a cell- and disease-specific manner.</p></div

Number of probe-sets differentially expressed in SLE patients and immunized healthy donors with yellow fever vaccine.

<p>This table summarizes differentially expressed probe sets as obtained by comparing arrays of the experimental group versus baseline group. Indicated are the total number of differentially expressed probe sets, the number of overlapping IFN-associated genes (absolute number and percentage of total number of differentially expressed probe sets) and the number of IFN-associated genes with fold changes ≥2 or ≤−2.</p><p>^a 2.442 interferon (IFN) signature genes were extracted from previous publications by Romos PS et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083776#pone.0083776-Ramos1" target="_blank">[17]</a> and Smiljanovic B et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083776#pone.0083776-Smiljanovic1" target="_blank">[11]</a>.</p><p>^b FC: fold change.</p><p>^c Expression data of healthy donors (ND) before immunization with yellow fever vaccine (YFV) was used as baseline for all comparisons.</p><p>^d ND 7 days after immunization with YFV.</p

Distribution of “common” and “autoimmune-specific” IFN signature probe-sets in SLE patients and immunised healthy donors (ND).

<p>Red circles (described as “Autoimmunity”) indicate the number of IFN signature gene probes observed in the SLE patient samples. Blue circles (described as “Viral infection”) indicate the number of IFN signature gene probes observed in ND immunised with the yellow fever vaccine. The overlaps of the red and blue circles indicate “common” IFN signatures that were detected in both SLE and viral infection. When genes had several probe-sets that categorised them into multiple groups, they were excluded from the “autoimmune-/immunisation-specific” groups and only included in the “common” group. There were 11/1 (SLE/immunised ND) of these probe sets in the CD4⁺ T cells, 13/5 in the CD16⁻ monocytes and 19/1 in the CD16⁺ monocytes. Two different numbers in the area of overlaps, for example 94/79 in CD4⁺ T cells, are shown because only probe-sets that meet the cutoff of fold-change values > = 2 or < = −2 were counted in this figure.</p