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Targeted knockdown of Kv1.3 channels in T lymphocytes corrects the disease manifestations associated with systemic lupus erythematosus

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Science Advances  18 Nov 2020:
Vol. 6, no. 47, eabd1471
DOI: 10.1126/sciadv.abd1471
  • Fig. 1 LN kidneys show increased infiltration with CD8+ T cells with a predominantly memory phenotype.

    (A) IHC of CD8 (brown signal) and CD4 (pink signal) expression in representative kidney biopsies from LN, DN, and NK individuals. Scale bar, 100 μm. Magnified tissue from the highlighted area (yellow rectangles) is presented in the bottom panels. (B to D) Number of CD8+ T cells (B) and CD4+ T cells (C) in LN, DN, and NK were quantitated as described in Materials and Methods. (D) The ratio of CD8+ T cells to CD4+ T cells measured in LN kidneys, DN kidneys, and NKs. (E) Representative confocal images of kidney biopsy sections stained for CD8 (green) and CD45RO channels (magenta) in patients with LN and DN. Scale bar, 50 μm. (F and G) Number of CD45RO+ cells (F) and CD8+CD45RO+ cells (G) were quantitated as described in Materials and Methods. In (B) to (D), data are presented as box and whisker plots. The data are reported as the median (horizontal line), first (top box), and third (bottom box) quartiles in biopsy samples from 10 LN kidneys, 10 DN kidneys, and 10 NKs. In (F) and (G), the data are presented as box and whisker plots in biopsy samples from four LN kidneys and four DN kidneys where at least five fields were imaged per patient. Data in (B) to (D) were analyzed by one-way analysis of variance (ANOVA) (P < 0.001), and post hoc testing was performed by Tukey’s test, while data in (F) and (G) were analyzed by Student’s t test.

  • Fig. 2 Immune cell profiling of kidney biopsies from LN, DN, and healthy individuals (NK) with NanoString nCounter Autoimmune Profiling panel.

    Shown here is the pairwise comparison of the abundance of the total tissue-infiltrating leukocytes (TILs) and the individual immune cell types between for (A) LN (n = 4 patients) and NK (n = 7 individuals) samples and (B) DN (n = 7 patients) and NK (n = 7 individuals) samples. The abundance of the different immune cell types (at the RNA level) in the kidney biopsies was calculated as log2 cell type scores (see Materials and Methods) and is presented as box and whisker plots. The data are reported as the median (horizontal line), first (top box), and third (bottom box) quartiles, and each symbol represents a single LN, DN, and NK individual. Statistical significance for the comparative cell type abundance was calculated using two-tailed Student’s t test. The cell scores for a specific cell type can only be compared between two groups (such as NK and LN) but do not support claims that a cell type is more abundant than another cell type within the same group.

  • Fig. 3 CD8+ T cells in LN kidneys show increased cytotoxicity and proliferation.

    (A) Representative merged confocal images of kidney biopsy sections stained for CD8 (green), Kv1.3 (magenta), GrB (cytotoxicity marker; orange,), and Ki-67 (proliferation marker; blue) in LN, DN, and NK individuals. Scale bar, 25 μm. (B to D) Fluorescence intensities (measured as mean gray values) of Kv1.3 (B), GrB (C), and Ki-67 (D) in CD8+ T cells in kidney biopsies from LN, DN, and NK individuals. In (B) to (D), data are presented as box and whisker plots. The data are reported as the median (horizontal line), first (top box), and third (bottom box) quartiles for 675 CD8+ T cells from 10 LN kidneys, 520 cells from 10 DN kidneys, and 36 cells from 10 NK kidney biopsies. Data were analyzed by one-way ANOVA [P < 0.001 for (B) to (D)]. Post hoc testing was performed by Dunn’s test.

  • Fig. 4 In vitro treatment with Kv1.3-NPs decreases CD40L expression and IFNγ production in CD45RO+ T cells from patients with LN.

    (A) Schematic representation of the structure of a lipid NP used to deliver siRNA against Kv1.3 (Kv1.3-NPs) or scramble sequence RNA (scr-NP). PE-PEG-biotin, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000]; Ab, antibody. (B) Representative flow cytometry plots gated on SLE CD3+ T cells incubated overnight with either scr-NPs or Kv1.3-NPs showing CD40L expression after stimulation with thapsigargin (TG) for 3 hours. CD45RO+ population was identified by gating on the live CD3+ T cell population expressing NP fluorescence [Alexa Fluor 488 streptavidin (SAV) was used for functionalization of the NPs]. (C) Quantification of CD40L abundance in activated, scr-NP– or Kv1.3-NP–treated CD3+ T cells in five patients with SLE. (D) Representative histogram showing CD40L expression in activated NP+ CD3+ T cells from a patient with SLE incubated with either scr-NPs or Kv1.3-NPs. (E) Average geometric MFI (gMFI) of CD40L expression in activated, scr-NP– or Kv1.3-NP–treated CD3+ T cells in five patients with SLE. Data were normalized to MFI of scr-NPs. (F) Representative flow cytometry plots gated on activated NP+ CD3+ T cells from a patient with SLE incubated with either scr-NPs or Kv1.3-NPs showing IFNγ production. (G) Quantification of IFNγ induction in activated, scr-NP– or Kv1.3-NP–treated CD3+ T cells in three patients with SLE. (H) Representative histogram showing IFNγ production in activated NP+ CD3+ T cells from a patient with SLE incubated with either scr-NPs or Kv1.3-NPs. (I) gMFI of IFNγ abundance in activated, scr-NP– or Kv1.3-NP–treated CD3+ T cells in three patients with SLE. (J) Representative flow cytometry plots gated on SLE CD3+ T cells incubated overnight with either scr-NPs or Kv1.3-NPs showing Ki-67 expression. (K) Quantification of Ki-67 in activated, scr-NP– or Kv1.3-NP–treated CD3+ T cells in three patients with SLE. Bars represent means ± SEM. Data were analyzed by paired Student’s t test.

  • Fig. 5 Humanized mouse generated by engraftment of LN PBMCs exhibits LN-like phenotype.

    (A) Schematic of humanized mouse model generation by engraftment of a NSG mouse with PBMCs from either a patient with LN or an HDs. (B) Left: Splenic T cell abundance in LN and HDs engrafted, as well as NE, mice measured by flow cytometry 6 to 8 weeks after engraftment. Human-specific antibodies were used to detect the T lymphocyte markers presented in the figure. (C) The ratio of CD8+ T cells to CD4+ T cells measured in splenocytes from LN and HDs mice. (D) Serum IgG levels (human) measured 6 weeks after engraftment in LN, HDs, and NE mice. (E) IHC of CD3 and CD8 expression (brown signal) in kidney tissues harvested from LN, HDs, and NE mice 6 weeks after engraftment. Representative image is shown here. Scale bar, 100 μm. (F) Urine protein measured in LN and HDs engrafted, as well as NE, mice. (G) Survival in LN, HDs, and NE mice presented as Kaplan-Meier Survival curve. Significance was evaluated by a log-rank test. All experiments were performed 6 to 8 weeks after engraftment. PBMCs from three SLE/LN individuals and two HDs were used for engrafting 4 to 12 mice per group. Bars represent means ± SEM, and each symbol represents an individual mouse. In (B), the CD4, CD8, CD3, and CD45 abundances were compared between the NE, HDs, and LN groups by one-way ANOVA (P < 0.001 for all groups). Data in (C) were analyzed by Student’s t test, while data in (D) and (F) were analyzed by one-way ANOVA (P < 0.05) and post hoc testing was performed by Holm-Sidak method.

  • Fig. 6 The ability of LN T cells to produce increased IFNγ is conserved in splenocytes of LN engrafted mice.

    (A) Experimental protocol to measure IFNγ levels in peripheral blood T cells from patients with LN. (B to D) IFNγ levels in unstimulated (B), Phytohemagglutinin (PHA)–stimulated (C), and anti-CD3/CD28–stimulated (D) peripheral blood CD3+ T cells from four patients with LN (SLE). (E) Experimental protocol for ex vivo measurement of IFNγ levels in LN mice. Single cells from the bone marrow, lungs, and spleens of the LN mice were stimulated ex vivo, and IFNγ levels were measured. (F to H) IFNγ levels in unstimulated (F), PHA-stimulated (G), and anti-CD3/CD28–stimulated (H) single cells from the bone marrows, lungs, and spleens from four LN mice on day 7 after engraftment. The in vitro and ex vivo cell activations were performed for 48 hours. Bars represent means ± SEM, and each symbol represents an individual patient/mouse. Data in (B) to (D) were analyzed by Student’s t test, while data in (F) to (H) were analyzed by one-way ANOVA [P < 0.001 for (F), P < 0.001 for (G), and P < 0.001 for (H)] and post hoc testing was performed by Holm-Sidak method.

  • Fig. 7 CD8+ T cells in the kidneys of LN mice show increased Kv1.3 expression.

    (A) Representative confocal images of kidney and spleen tissues harvested 6 weeks after engraftment from LN mice that were stained for CD8 (yellow), Kv1.3 (magenta), and nuclei [4′,6-diamidino-2-phenylindole (DAPI); cyan]. Scale bar, 50 μm. (B) Left: Merged images of CD8 and Kv1.3 channels in the kidney and spleen from LN mice showing Kv1.3 staining in the CD8+ T cells. Right: Fluorescence intensities (measured as mean gray values) of Kv1.3 in CD8+ T cells in the kidneys and spleens from LN mice. Data are presented as box and whisker plots. The data are reported as the median (horizontal line), first (top box), and third (bottom box) quartiles for 134 CD8+ T cells from two LN mice kidneys and 400 CD8+ T cells from two LN mice spleens. Significance was determined by Mann-Whitney rank sum test.

  • Fig. 8 Pretreatment of PBMCs from patients with LN with Kv1.3-NPs before engraftment decreases CD40L and IFNγ production and increases survival in the humanized LN mice.

    (A) PBMCs from patients with LN were preincubated overnight with either scr-NPs or Kv1.3-NPs and engrafted in NSG mice. Ex vivo flow cytometry experiments were performed on these mice, and survival was recorded in a separate set of experiments. (B to D) Fluorescence intensities of CD40L (B), IFNγ (C), and Ki-67 (D) in splenocytes of LN mice pretreated with either scr-NPs or Kv1.3-NPs before engraftment, as shown in (A). Cells were stimulated with TG for 3 hours. Shown here are fluorescence intensities measured on days 2 and 7 after engraftment. Four to six mice were engrafted per group with PBMCs from two to three patients with LN. Each symbol represents an individual mouse. Horizontal red lines represent the means for each group. Significance was calculated by Student’s t test. (E) Survival in LN mice: PBMCs from patients with LN were treated with either scr-NPs or Kv1.3-NPs before engraftment. Kaplan-Meier survival curve is shown. Data are from 10 to 11 mice per group engrafted with PBMCs from two patients with LN. Significance was calculated by a log rank test.

  • Table 1 Immune cell population in LN mice on days 2 and 7 after engraftment.

    PBMCs from two patients with LN were engrafted in four NSG mice, and immune cell populations were profiled on days 2 and 7 by flow cytometry and are presented as percentages of total live cells. Naïve T cells were defined as CD3+CD45ROCD38FSCintermediate; Tm cells were defined as CD3+CD45RO+CD38FScintermediate; plasma cells were defined as CD3CD38+. Data were analyzed by Student’s t test.

    % of total live cells
    Day 2Day 7PN
    SpleenCD4+ Tnaive0.17 ± 0.0022.18 ± 0.001<0.0014
    CD4+ Tm0.04 ± 0.00011.24 ± 0.001<0.0014
    CD8+ Tnaive0.21 ± 0.0011.84 ± 0.0001<0.0014
    CD8+ Tm0.01 ± 0.00020.8 ± 0.011<0.0014
    CD38+ plasma0.87 ± 0.00070.27 ± 0.001<0.0014
    Bone marrowCD4+ T0.23 ± 0.0010.17 ± 0.003<0.0014
    CD4+ Tm0.1 ± 0.0010.04 ± 0.0001<0.0014
    CD8+ T0.23 ± 0.0010.14 ± 0.002<0.0014
    CD8+ Tm0.03 ± 0.00020.001 ± 0.0001<0.0014
    CD38+ plasma0.06 ± 0.00010.07 ± 0.0030.0164
    LungCD4+ T1.16 ± 0.0071.4 ± 0.002<0.0014
    CD4+ Tm0.25 ± 0.0080.24 ± 0.0009<0.0014
    CD8+ T0.35 ± 0.00120.32 ± 0.001<0.0014
    CD8+ Tm0.11 ± 0.0010.11 ± 0.0021.0004
    CD38+ plasma0.12 ± 0.00090.22 ± 0.002<0.0014

Supplementary Materials

  • Supplementary Materials

    Targeted knockdown of Kv1.3 channels in T lymphocytes corrects the disease manifestations associated with systemic lupus erythematosus

    Marat Khodoun, Ameet A. Chimote, Farhan Z. Ilyas, Heather J. Duncan, Halima Moncrieffe, K. Shashi Kant, Laura Conforti

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