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Effector memory T-cell phenotype is not sufficient to confer sensitivity to ShK-mediated inhibition. ( a ) Expression of <t>Kv1.3</t> and KCa3.1 on sorted CD4 + and CD8 + naive, T CM and T EM subsets. CD45RO and CCR7 as markers to define naive, T CM and T EM subsets as shown in dot plots. Expression was determined on ex vivo cell subsets or on sorted T-cell subsets after 4 days stimulation with anti-CD3 and anti-CD28. Kv1.3 and KCa3.1 expression were normalized to RPL19 expression and shown relative to expression in ex vivo naive cells. ( b ) Effect of ShK on naive, T CM and T EM CD4 + subsets. Sorted cells were stimulated with anti-CD3 and either vehicle or 1 μM ShK. Proliferation was determined at day 4 of stimulation; IFN-γ was measured after 3 days. ( c ) Bulk PBMC from healthy donors were stimulated with anti-CD3 and anti-CD28 in the absence or presence of 10 nM ShK or 1 μM TRAM-34. IFN-γ production was measured after 3 days stimulation. Proliferation responses were determined at day 7 by CFSE dilution of CD4 + - or CD8 + -gated cells. Representative dot plots from one donor are shown. % inhibition of proliferation was determined by comparing proliferation in the presence of inhibitor to proliferation of cells in the presence of vehicle control. Data are shown as individual data points with mean±s.d. ( n =15 biological replicates). ( d ) CD4 + T cells repeatedly stimulated with anti-CD3 and anti-CD28. Purified CD4 + T cells from healthy donor were stimulated with 5 μg ml −1 plate-bound anti-CD3 and 2 μg ml −1 soluble anti-CD28 for 4 days. Cells were then harvested, washed, and rested for 3 days. In subsequent stimulations, cells were reactivated with 1 μg ml −1 soluble anti-CD3 and 1 μg ml −1 anti-CD28. In the fourth round of stimulation, cells were cultured with 10-fold increases in ShK concentrations, starting at 10 − 5 nM. Cell supernatants were harvested 3 days after initiation of last round of stimulation for determination of IFN-γ. Proliferation responses were determined at day 4 by 3 H-thymidine incorporation. ( e ) Purified memory CD4 + T cells were stimulated for six rounds with anti-CD3 and anti-CD28, as described in d .
Fitc Conjugated Kv1 3 Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions"

Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

Journal: Nature Communications

doi: 10.1038/ncomms14644

Effector memory T-cell phenotype is not sufficient to confer sensitivity to ShK-mediated inhibition. ( a ) Expression of Kv1.3 and KCa3.1 on sorted CD4 + and CD8 + naive, T CM and T EM subsets. CD45RO and CCR7 as markers to define naive, T CM and T EM subsets as shown in dot plots. Expression was determined on ex vivo cell subsets or on sorted T-cell subsets after 4 days stimulation with anti-CD3 and anti-CD28. Kv1.3 and KCa3.1 expression were normalized to RPL19 expression and shown relative to expression in ex vivo naive cells. ( b ) Effect of ShK on naive, T CM and T EM CD4 + subsets. Sorted cells were stimulated with anti-CD3 and either vehicle or 1 μM ShK. Proliferation was determined at day 4 of stimulation; IFN-γ was measured after 3 days. ( c ) Bulk PBMC from healthy donors were stimulated with anti-CD3 and anti-CD28 in the absence or presence of 10 nM ShK or 1 μM TRAM-34. IFN-γ production was measured after 3 days stimulation. Proliferation responses were determined at day 7 by CFSE dilution of CD4 + - or CD8 + -gated cells. Representative dot plots from one donor are shown. % inhibition of proliferation was determined by comparing proliferation in the presence of inhibitor to proliferation of cells in the presence of vehicle control. Data are shown as individual data points with mean±s.d. ( n =15 biological replicates). ( d ) CD4 + T cells repeatedly stimulated with anti-CD3 and anti-CD28. Purified CD4 + T cells from healthy donor were stimulated with 5 μg ml −1 plate-bound anti-CD3 and 2 μg ml −1 soluble anti-CD28 for 4 days. Cells were then harvested, washed, and rested for 3 days. In subsequent stimulations, cells were reactivated with 1 μg ml −1 soluble anti-CD3 and 1 μg ml −1 anti-CD28. In the fourth round of stimulation, cells were cultured with 10-fold increases in ShK concentrations, starting at 10 − 5 nM. Cell supernatants were harvested 3 days after initiation of last round of stimulation for determination of IFN-γ. Proliferation responses were determined at day 4 by 3 H-thymidine incorporation. ( e ) Purified memory CD4 + T cells were stimulated for six rounds with anti-CD3 and anti-CD28, as described in d .
Figure Legend Snippet: Effector memory T-cell phenotype is not sufficient to confer sensitivity to ShK-mediated inhibition. ( a ) Expression of Kv1.3 and KCa3.1 on sorted CD4 + and CD8 + naive, T CM and T EM subsets. CD45RO and CCR7 as markers to define naive, T CM and T EM subsets as shown in dot plots. Expression was determined on ex vivo cell subsets or on sorted T-cell subsets after 4 days stimulation with anti-CD3 and anti-CD28. Kv1.3 and KCa3.1 expression were normalized to RPL19 expression and shown relative to expression in ex vivo naive cells. ( b ) Effect of ShK on naive, T CM and T EM CD4 + subsets. Sorted cells were stimulated with anti-CD3 and either vehicle or 1 μM ShK. Proliferation was determined at day 4 of stimulation; IFN-γ was measured after 3 days. ( c ) Bulk PBMC from healthy donors were stimulated with anti-CD3 and anti-CD28 in the absence or presence of 10 nM ShK or 1 μM TRAM-34. IFN-γ production was measured after 3 days stimulation. Proliferation responses were determined at day 7 by CFSE dilution of CD4 + - or CD8 + -gated cells. Representative dot plots from one donor are shown. % inhibition of proliferation was determined by comparing proliferation in the presence of inhibitor to proliferation of cells in the presence of vehicle control. Data are shown as individual data points with mean±s.d. ( n =15 biological replicates). ( d ) CD4 + T cells repeatedly stimulated with anti-CD3 and anti-CD28. Purified CD4 + T cells from healthy donor were stimulated with 5 μg ml −1 plate-bound anti-CD3 and 2 μg ml −1 soluble anti-CD28 for 4 days. Cells were then harvested, washed, and rested for 3 days. In subsequent stimulations, cells were reactivated with 1 μg ml −1 soluble anti-CD3 and 1 μg ml −1 anti-CD28. In the fourth round of stimulation, cells were cultured with 10-fold increases in ShK concentrations, starting at 10 − 5 nM. Cell supernatants were harvested 3 days after initiation of last round of stimulation for determination of IFN-γ. Proliferation responses were determined at day 4 by 3 H-thymidine incorporation. ( e ) Purified memory CD4 + T cells were stimulated for six rounds with anti-CD3 and anti-CD28, as described in d .

Techniques Used: Inhibition, Expressing, Ex Vivo, Purification, Cell Culture

AIA and DTH response in Kcna3 − / − rats. ( a ) WT (blue) or Kcna3 − / − (green) rats were given a single injection of CFA to induce AIA (filled symbols, n =5 biological replicates per group) or were untreated (open symbols, naive, n =2 per group) and clinical score assessed at day 21. Individual animals are represented by discrete symbols and mean±s.d. are shown. Delayed-type hypersensitivity. ( b ) WT (blue) or Kcna3 − / − (green) OVA-immunized rats were subsequently challenged with OVA (filled symbols, n =6 biological replicates per group) or PBS (open symbols, n =4 per group) and ear swelling was measured 24 h later. Individual animals are represented by discrete symbols and mean±s.d. are shown. Experiment was performed twice, with each experiment delineated by the dotted line. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Effect of Kv1.3 blockade on DTH inflammatory responses. WT rats were immunized with OVA then subsequently challenged 1 week later with either PBS or OVA. OVA-rechallenged animals were treated with control anti-ragweed (aRGW) antibody (pink), CTLA4-Ig (orange) or ShK (blue). Ear swelling was measured 24 h later. Individual biological replicates (n=6 per group) are shown with mean±s.d. Experiment was performed three times, with each experiment delineated by dotted lines. Statistically significant differences between ShK and anti-ragweed control groups are denoted with P values; CTLA4-Ig inhibition was statistically significant in all experiments ( P
Figure Legend Snippet: AIA and DTH response in Kcna3 − / − rats. ( a ) WT (blue) or Kcna3 − / − (green) rats were given a single injection of CFA to induce AIA (filled symbols, n =5 biological replicates per group) or were untreated (open symbols, naive, n =2 per group) and clinical score assessed at day 21. Individual animals are represented by discrete symbols and mean±s.d. are shown. Delayed-type hypersensitivity. ( b ) WT (blue) or Kcna3 − / − (green) OVA-immunized rats were subsequently challenged with OVA (filled symbols, n =6 biological replicates per group) or PBS (open symbols, n =4 per group) and ear swelling was measured 24 h later. Individual animals are represented by discrete symbols and mean±s.d. are shown. Experiment was performed twice, with each experiment delineated by the dotted line. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Effect of Kv1.3 blockade on DTH inflammatory responses. WT rats were immunized with OVA then subsequently challenged 1 week later with either PBS or OVA. OVA-rechallenged animals were treated with control anti-ragweed (aRGW) antibody (pink), CTLA4-Ig (orange) or ShK (blue). Ear swelling was measured 24 h later. Individual biological replicates (n=6 per group) are shown with mean±s.d. Experiment was performed three times, with each experiment delineated by dotted lines. Statistically significant differences between ShK and anti-ragweed control groups are denoted with P values; CTLA4-Ig inhibition was statistically significant in all experiments ( P

Techniques Used: Injection, Inhibition

Effects of siRNA knockdown of Kv1.3 or KCa3.1 on TT-specific human T-cell sensitivity to inhibitors. ( a ) Kv1.3 (left) and KCa3.1 (right) expression was measured in primary TT-stimulated T cells (1° TT) or T cells that underwent four rounds of TT stimulation (4° TT) that were transfected with Kv1.3 siRNA (blue bars), KCa3.1 siRNA (green bars), a combination of both siRNA (orange bars) or scramble control siRNA (grey bars). Relative expression of targeted genes is shown in comparison with scramble siRNA transfected cells. Data are shown as mean±s.d. of triplicate measurements from one representative experiment. ( b ) Kv1.3 surface protein expression on 1° TT or 4° TT cells following siRNA transfection. Primary (left) or repeatedly stimulated (4°, right) TT cells were transfected with Kv1.3 siRNA (red histograms) or scramble siRNA (blue histograms) and then stained for Kv1.3 48 h later. Grey filled histogram represents isotype control. T-cell responses of 1° TT-stimulated T cells ( c , d ) or 4° TT-stimulated T cells ( e , f ) transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green), a combination of both (orange) or scramble control siRNA (black). Cells were then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( c , e ) or TRAM-34 ( d , f ) at the indicated concentrations. Proliferation responses were determined at day 4 of culture by 3 H-thymidine incorporation; IFN-γ concentration was measured in culture supernatants harvested 3 days after restimulation. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.
Figure Legend Snippet: Effects of siRNA knockdown of Kv1.3 or KCa3.1 on TT-specific human T-cell sensitivity to inhibitors. ( a ) Kv1.3 (left) and KCa3.1 (right) expression was measured in primary TT-stimulated T cells (1° TT) or T cells that underwent four rounds of TT stimulation (4° TT) that were transfected with Kv1.3 siRNA (blue bars), KCa3.1 siRNA (green bars), a combination of both siRNA (orange bars) or scramble control siRNA (grey bars). Relative expression of targeted genes is shown in comparison with scramble siRNA transfected cells. Data are shown as mean±s.d. of triplicate measurements from one representative experiment. ( b ) Kv1.3 surface protein expression on 1° TT or 4° TT cells following siRNA transfection. Primary (left) or repeatedly stimulated (4°, right) TT cells were transfected with Kv1.3 siRNA (red histograms) or scramble siRNA (blue histograms) and then stained for Kv1.3 48 h later. Grey filled histogram represents isotype control. T-cell responses of 1° TT-stimulated T cells ( c , d ) or 4° TT-stimulated T cells ( e , f ) transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green), a combination of both (orange) or scramble control siRNA (black). Cells were then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( c , e ) or TRAM-34 ( d , f ) at the indicated concentrations. Proliferation responses were determined at day 4 of culture by 3 H-thymidine incorporation; IFN-γ concentration was measured in culture supernatants harvested 3 days after restimulation. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.

Techniques Used: Expressing, Transfection, Staining, Concentration Assay

Full inhibition of antigen-specific human T cells requires blockade of both Kv1.3 and KCa3.1. ( a , b ) Inhibition of CD4 + ( a ) or CD8 + ( b ) T-cell proliferation response to TT stimulation as determined by CFSE dilution. One representative donor is shown. Percentages indicated in red denote % reduction in proliferation relative to vehicle-treated cells. Primary (1°) T-cell response to TT was determined in the absence or presence of either 10 nM ShK ( n =8 biological replicates) or 1 μM TRAM-34 ( n =9). ( c ) Proliferation responses were determined at day 4; IFN-γ concentrations were measured at day 3. % inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( d ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates TT-specific T cell responses. PBMC from T1D donors were stimulated with TT in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes absence of TT. Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. ( e ) Proliferation and IFN-γ production of TT-specific T cells following four rounds of stimulation (4°). After three rounds of TT stimulation, cells were rested and then restimulated in the presence of irradiated autologous PBMC with TT in the absence or presence of either 10 nM ShK ( n =11 biological replicates) or 1 μM TRAM-34 ( n =4). ( f ) Effect of ShK or TRAM-34 on proliferation or IFN-γ production of TT-specific T cells after increasing rounds of stimulation. Data shown are representative of one donor. ( g ) Gene expression of KCNA3 and KCNN4 in purified T cells after 1° or 4° TT stimulation. Expression of each channel is presented relative to expression in naive T cells. Data shown are representative of three donors. ( h ) Kv1.3 surface protein expression on 1° TT or 4° TT cells. Blue histogram represents Kv1.3 staining; grey filled histogram represents isotype control.
Figure Legend Snippet: Full inhibition of antigen-specific human T cells requires blockade of both Kv1.3 and KCa3.1. ( a , b ) Inhibition of CD4 + ( a ) or CD8 + ( b ) T-cell proliferation response to TT stimulation as determined by CFSE dilution. One representative donor is shown. Percentages indicated in red denote % reduction in proliferation relative to vehicle-treated cells. Primary (1°) T-cell response to TT was determined in the absence or presence of either 10 nM ShK ( n =8 biological replicates) or 1 μM TRAM-34 ( n =9). ( c ) Proliferation responses were determined at day 4; IFN-γ concentrations were measured at day 3. % inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( d ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates TT-specific T cell responses. PBMC from T1D donors were stimulated with TT in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes absence of TT. Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. ( e ) Proliferation and IFN-γ production of TT-specific T cells following four rounds of stimulation (4°). After three rounds of TT stimulation, cells were rested and then restimulated in the presence of irradiated autologous PBMC with TT in the absence or presence of either 10 nM ShK ( n =11 biological replicates) or 1 μM TRAM-34 ( n =4). ( f ) Effect of ShK or TRAM-34 on proliferation or IFN-γ production of TT-specific T cells after increasing rounds of stimulation. Data shown are representative of one donor. ( g ) Gene expression of KCNA3 and KCNN4 in purified T cells after 1° or 4° TT stimulation. Expression of each channel is presented relative to expression in naive T cells. Data shown are representative of three donors. ( h ) Kv1.3 surface protein expression on 1° TT or 4° TT cells. Blue histogram represents Kv1.3 staining; grey filled histogram represents isotype control.

Techniques Used: Inhibition, Concentration Assay, Irradiation, Expressing, Purification, Staining

Kv1.3 and KCa3.1 are both required for human autoreactive T-cell responses. ( a ) PBMC from HLA-DR4 + T1D donors ( n =10 biological replicates) were stimulated with a combination of HLA-DR4-restricted GAD65 peptides in the absence or presence of either 10 nM ShK or 1 μM TRAM-34. ( b ) PBMC from non-HLA- or HLA-typed T1D donors were stimulated with GAD65 protein ( n =13 biological replicates). Proliferation responses (left) were determined at day 4. IFN-γ concentrations (right) were measured 3 days after stimulation. %inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates autologous autoreactive T cell responses. PBMC from T1D donors were stimulated with GAD65 protein in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes unstimulated cell conditions (absence of GAD65 protein). Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. Gene expression for Kv1.3 ( KCNA3 , d ) and KCa3.1 ( KCNN4, e ) in GAD65-stimulated purified T cells following siRNA transfection with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey). ( f , g ) Effects of siRNA knockdown of Kv1.3 or KCa3.1 on sensitivity to inhibitors. PBMC from T1D donor was stimulated with GAD65 protein for 7 days. After 3 days rest, T cells were purified and transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey) and then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( f ) or TRAM-34 ( g ) at the indicated concentrations. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.
Figure Legend Snippet: Kv1.3 and KCa3.1 are both required for human autoreactive T-cell responses. ( a ) PBMC from HLA-DR4 + T1D donors ( n =10 biological replicates) were stimulated with a combination of HLA-DR4-restricted GAD65 peptides in the absence or presence of either 10 nM ShK or 1 μM TRAM-34. ( b ) PBMC from non-HLA- or HLA-typed T1D donors were stimulated with GAD65 protein ( n =13 biological replicates). Proliferation responses (left) were determined at day 4. IFN-γ concentrations (right) were measured 3 days after stimulation. %inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates autologous autoreactive T cell responses. PBMC from T1D donors were stimulated with GAD65 protein in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes unstimulated cell conditions (absence of GAD65 protein). Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. Gene expression for Kv1.3 ( KCNA3 , d ) and KCa3.1 ( KCNN4, e ) in GAD65-stimulated purified T cells following siRNA transfection with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey). ( f , g ) Effects of siRNA knockdown of Kv1.3 or KCa3.1 on sensitivity to inhibitors. PBMC from T1D donor was stimulated with GAD65 protein for 7 days. After 3 days rest, T cells were purified and transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey) and then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( f ) or TRAM-34 ( g ) at the indicated concentrations. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.

Techniques Used: Inhibition, Concentration Assay, Expressing, Purification, Transfection

Characterization of Kcna3 − / − T cells. ( a ) K + -channel expression in human, rat and mouse T cells. Gene expression of Kv1 family members and KCa3.1 in naive CD4 + T cells from human (left), rat (centre) or mouse (right). Relative expression was determined by normalizing to housekeeping gene RPL19. Electrophysiological and pharmacological tests show null Kv1.3 channel in Kcna3 − / − T cells. ( b ) Representative voltage-currents from WT and Kcna3 − / − T cells. Currents were elicited by depolarizing voltage steps from −60 to +40 mV (10 mV increments every 30 s, with −80 mV membrane-holding potential). ( c ) Kv1.3 channel number in WT ( n =40) and Kcna3 − / − ( n =50) T cells after 48 h activation. The mean Kv1.3 channel numbers were 1667±187 in WT and undetectable in Kcna3 − / − T cells. ( d ) Normalized WT and Kcna3 − / − T cell K + currents before and after Shk inhibition. 89% WT T cell K + current was blocked by 1 nM Shk, but no Shk-sensitive current was detected in Kcna3 − / − T cells. Kcna3 − / − T-cell responses to activation. ( e ) Proliferation (left) and IFN-γ (right) responses to anti-CD3 and anti-CD28 stimulation. Spleen cells from WT or Kcna3 − / − rats were stimulated for 3 days. Individual biological replicates ( n =4 per group) are shown with mean±s.d. ( f ) Effects of ShK and TRAM-34 on polyclonal T-cell activation. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( g ) OVA-specific T-cell proliferation responses. Draining lymph node and spleen cells from OVA-immunized WT and Kcna3 − / − rats were plated at a 1:10 lymph node:spleen cell ratio and stimulated in vitro with OVA at various concentrations. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( h , i ) Kcna3 − / − rat dendritic cell competency. CD4 + ( h ) or CD8 + . ( i ) T cells isolated from DLN of OVA-immunized WT (blue) and Kcna3 − / − (green) rats were co-cultured with APCs from WT (filled circles) or Kcna3 − / − (open circles) rats and stimulated with OVA. Proliferation responses were determined at day 3 of culture. Individual biological replicates ( n =4 per group) are shown with mean±s.d.
Figure Legend Snippet: Characterization of Kcna3 − / − T cells. ( a ) K + -channel expression in human, rat and mouse T cells. Gene expression of Kv1 family members and KCa3.1 in naive CD4 + T cells from human (left), rat (centre) or mouse (right). Relative expression was determined by normalizing to housekeeping gene RPL19. Electrophysiological and pharmacological tests show null Kv1.3 channel in Kcna3 − / − T cells. ( b ) Representative voltage-currents from WT and Kcna3 − / − T cells. Currents were elicited by depolarizing voltage steps from −60 to +40 mV (10 mV increments every 30 s, with −80 mV membrane-holding potential). ( c ) Kv1.3 channel number in WT ( n =40) and Kcna3 − / − ( n =50) T cells after 48 h activation. The mean Kv1.3 channel numbers were 1667±187 in WT and undetectable in Kcna3 − / − T cells. ( d ) Normalized WT and Kcna3 − / − T cell K + currents before and after Shk inhibition. 89% WT T cell K + current was blocked by 1 nM Shk, but no Shk-sensitive current was detected in Kcna3 − / − T cells. Kcna3 − / − T-cell responses to activation. ( e ) Proliferation (left) and IFN-γ (right) responses to anti-CD3 and anti-CD28 stimulation. Spleen cells from WT or Kcna3 − / − rats were stimulated for 3 days. Individual biological replicates ( n =4 per group) are shown with mean±s.d. ( f ) Effects of ShK and TRAM-34 on polyclonal T-cell activation. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( g ) OVA-specific T-cell proliferation responses. Draining lymph node and spleen cells from OVA-immunized WT and Kcna3 − / − rats were plated at a 1:10 lymph node:spleen cell ratio and stimulated in vitro with OVA at various concentrations. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( h , i ) Kcna3 − / − rat dendritic cell competency. CD4 + ( h ) or CD8 + . ( i ) T cells isolated from DLN of OVA-immunized WT (blue) and Kcna3 − / − (green) rats were co-cultured with APCs from WT (filled circles) or Kcna3 − / − (open circles) rats and stimulated with OVA. Proliferation responses were determined at day 3 of culture. Individual biological replicates ( n =4 per group) are shown with mean±s.d.

Techniques Used: Expressing, Activation Assay, Inhibition, In Vitro, Isolation, Cell Culture

Autoreactive but not autologous pathogen-specific T cells are sensitive to Kv1.3 blockers. ( a , b ) ShK inhibits GAD65-specific T1D T cells but not autologous TT-specific T cells. PBMC from T1D donors ( n =6 biological replicates) were stimulated with a combination of four HLA-DR4-restricted GAD65 peptides. Proliferation and IFN-γ responses were compared with TT stimulation in the absence or presence of either 10 nM ShK ( a ) or 1 μM TRAM-34 ( b ). Data shown are % inhibition of proliferation response as determined by 3 H-thymidine incorporation after 4 days primary in vitro stimulation and % inhibition of IFN-γ production after 3 days stimulation. Coloured symbols and corresponding lines represent each individual donor.
Figure Legend Snippet: Autoreactive but not autologous pathogen-specific T cells are sensitive to Kv1.3 blockers. ( a , b ) ShK inhibits GAD65-specific T1D T cells but not autologous TT-specific T cells. PBMC from T1D donors ( n =6 biological replicates) were stimulated with a combination of four HLA-DR4-restricted GAD65 peptides. Proliferation and IFN-γ responses were compared with TT stimulation in the absence or presence of either 10 nM ShK ( a ) or 1 μM TRAM-34 ( b ). Data shown are % inhibition of proliferation response as determined by 3 H-thymidine incorporation after 4 days primary in vitro stimulation and % inhibition of IFN-γ production after 3 days stimulation. Coloured symbols and corresponding lines represent each individual donor.

Techniques Used: Inhibition, In Vitro

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    Millipore fitc conjugated kv1 3 ab
    Effector memory T-cell phenotype is not sufficient to confer sensitivity to ShK-mediated inhibition. ( a ) Expression of <t>Kv1.3</t> and KCa3.1 on sorted CD4 + and CD8 + naive, T CM and T EM subsets. CD45RO and CCR7 as markers to define naive, T CM and T EM subsets as shown in dot plots. Expression was determined on ex vivo cell subsets or on sorted T-cell subsets after 4 days stimulation with anti-CD3 and anti-CD28. Kv1.3 and KCa3.1 expression were normalized to RPL19 expression and shown relative to expression in ex vivo naive cells. ( b ) Effect of ShK on naive, T CM and T EM CD4 + subsets. Sorted cells were stimulated with anti-CD3 and either vehicle or 1 μM ShK. Proliferation was determined at day 4 of stimulation; IFN-γ was measured after 3 days. ( c ) Bulk PBMC from healthy donors were stimulated with anti-CD3 and anti-CD28 in the absence or presence of 10 nM ShK or 1 μM TRAM-34. IFN-γ production was measured after 3 days stimulation. Proliferation responses were determined at day 7 by CFSE dilution of CD4 + - or CD8 + -gated cells. Representative dot plots from one donor are shown. % inhibition of proliferation was determined by comparing proliferation in the presence of inhibitor to proliferation of cells in the presence of vehicle control. Data are shown as individual data points with mean±s.d. ( n =15 biological replicates). ( d ) CD4 + T cells repeatedly stimulated with anti-CD3 and anti-CD28. Purified CD4 + T cells from healthy donor were stimulated with 5 μg ml −1 plate-bound anti-CD3 and 2 μg ml −1 soluble anti-CD28 for 4 days. Cells were then harvested, washed, and rested for 3 days. In subsequent stimulations, cells were reactivated with 1 μg ml −1 soluble anti-CD3 and 1 μg ml −1 anti-CD28. In the fourth round of stimulation, cells were cultured with 10-fold increases in ShK concentrations, starting at 10 − 5 nM. Cell supernatants were harvested 3 days after initiation of last round of stimulation for determination of IFN-γ. Proliferation responses were determined at day 4 by 3 H-thymidine incorporation. ( e ) Purified memory CD4 + T cells were stimulated for six rounds with anti-CD3 and anti-CD28, as described in d .
    Fitc Conjugated Kv1 3 Ab, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effector memory T-cell phenotype is not sufficient to confer sensitivity to ShK-mediated inhibition. ( a ) Expression of Kv1.3 and KCa3.1 on sorted CD4 + and CD8 + naive, T CM and T EM subsets. CD45RO and CCR7 as markers to define naive, T CM and T EM subsets as shown in dot plots. Expression was determined on ex vivo cell subsets or on sorted T-cell subsets after 4 days stimulation with anti-CD3 and anti-CD28. Kv1.3 and KCa3.1 expression were normalized to RPL19 expression and shown relative to expression in ex vivo naive cells. ( b ) Effect of ShK on naive, T CM and T EM CD4 + subsets. Sorted cells were stimulated with anti-CD3 and either vehicle or 1 μM ShK. Proliferation was determined at day 4 of stimulation; IFN-γ was measured after 3 days. ( c ) Bulk PBMC from healthy donors were stimulated with anti-CD3 and anti-CD28 in the absence or presence of 10 nM ShK or 1 μM TRAM-34. IFN-γ production was measured after 3 days stimulation. Proliferation responses were determined at day 7 by CFSE dilution of CD4 + - or CD8 + -gated cells. Representative dot plots from one donor are shown. % inhibition of proliferation was determined by comparing proliferation in the presence of inhibitor to proliferation of cells in the presence of vehicle control. Data are shown as individual data points with mean±s.d. ( n =15 biological replicates). ( d ) CD4 + T cells repeatedly stimulated with anti-CD3 and anti-CD28. Purified CD4 + T cells from healthy donor were stimulated with 5 μg ml −1 plate-bound anti-CD3 and 2 μg ml −1 soluble anti-CD28 for 4 days. Cells were then harvested, washed, and rested for 3 days. In subsequent stimulations, cells were reactivated with 1 μg ml −1 soluble anti-CD3 and 1 μg ml −1 anti-CD28. In the fourth round of stimulation, cells were cultured with 10-fold increases in ShK concentrations, starting at 10 − 5 nM. Cell supernatants were harvested 3 days after initiation of last round of stimulation for determination of IFN-γ. Proliferation responses were determined at day 4 by 3 H-thymidine incorporation. ( e ) Purified memory CD4 + T cells were stimulated for six rounds with anti-CD3 and anti-CD28, as described in d .

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Effector memory T-cell phenotype is not sufficient to confer sensitivity to ShK-mediated inhibition. ( a ) Expression of Kv1.3 and KCa3.1 on sorted CD4 + and CD8 + naive, T CM and T EM subsets. CD45RO and CCR7 as markers to define naive, T CM and T EM subsets as shown in dot plots. Expression was determined on ex vivo cell subsets or on sorted T-cell subsets after 4 days stimulation with anti-CD3 and anti-CD28. Kv1.3 and KCa3.1 expression were normalized to RPL19 expression and shown relative to expression in ex vivo naive cells. ( b ) Effect of ShK on naive, T CM and T EM CD4 + subsets. Sorted cells were stimulated with anti-CD3 and either vehicle or 1 μM ShK. Proliferation was determined at day 4 of stimulation; IFN-γ was measured after 3 days. ( c ) Bulk PBMC from healthy donors were stimulated with anti-CD3 and anti-CD28 in the absence or presence of 10 nM ShK or 1 μM TRAM-34. IFN-γ production was measured after 3 days stimulation. Proliferation responses were determined at day 7 by CFSE dilution of CD4 + - or CD8 + -gated cells. Representative dot plots from one donor are shown. % inhibition of proliferation was determined by comparing proliferation in the presence of inhibitor to proliferation of cells in the presence of vehicle control. Data are shown as individual data points with mean±s.d. ( n =15 biological replicates). ( d ) CD4 + T cells repeatedly stimulated with anti-CD3 and anti-CD28. Purified CD4 + T cells from healthy donor were stimulated with 5 μg ml −1 plate-bound anti-CD3 and 2 μg ml −1 soluble anti-CD28 for 4 days. Cells were then harvested, washed, and rested for 3 days. In subsequent stimulations, cells were reactivated with 1 μg ml −1 soluble anti-CD3 and 1 μg ml −1 anti-CD28. In the fourth round of stimulation, cells were cultured with 10-fold increases in ShK concentrations, starting at 10 − 5 nM. Cell supernatants were harvested 3 days after initiation of last round of stimulation for determination of IFN-γ. Proliferation responses were determined at day 4 by 3 H-thymidine incorporation. ( e ) Purified memory CD4 + T cells were stimulated for six rounds with anti-CD3 and anti-CD28, as described in d .

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Inhibition, Expressing, Ex Vivo, Purification, Cell Culture

    AIA and DTH response in Kcna3 − / − rats. ( a ) WT (blue) or Kcna3 − / − (green) rats were given a single injection of CFA to induce AIA (filled symbols, n =5 biological replicates per group) or were untreated (open symbols, naive, n =2 per group) and clinical score assessed at day 21. Individual animals are represented by discrete symbols and mean±s.d. are shown. Delayed-type hypersensitivity. ( b ) WT (blue) or Kcna3 − / − (green) OVA-immunized rats were subsequently challenged with OVA (filled symbols, n =6 biological replicates per group) or PBS (open symbols, n =4 per group) and ear swelling was measured 24 h later. Individual animals are represented by discrete symbols and mean±s.d. are shown. Experiment was performed twice, with each experiment delineated by the dotted line. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Effect of Kv1.3 blockade on DTH inflammatory responses. WT rats were immunized with OVA then subsequently challenged 1 week later with either PBS or OVA. OVA-rechallenged animals were treated with control anti-ragweed (aRGW) antibody (pink), CTLA4-Ig (orange) or ShK (blue). Ear swelling was measured 24 h later. Individual biological replicates (n=6 per group) are shown with mean±s.d. Experiment was performed three times, with each experiment delineated by dotted lines. Statistically significant differences between ShK and anti-ragweed control groups are denoted with P values; CTLA4-Ig inhibition was statistically significant in all experiments ( P

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: AIA and DTH response in Kcna3 − / − rats. ( a ) WT (blue) or Kcna3 − / − (green) rats were given a single injection of CFA to induce AIA (filled symbols, n =5 biological replicates per group) or were untreated (open symbols, naive, n =2 per group) and clinical score assessed at day 21. Individual animals are represented by discrete symbols and mean±s.d. are shown. Delayed-type hypersensitivity. ( b ) WT (blue) or Kcna3 − / − (green) OVA-immunized rats were subsequently challenged with OVA (filled symbols, n =6 biological replicates per group) or PBS (open symbols, n =4 per group) and ear swelling was measured 24 h later. Individual animals are represented by discrete symbols and mean±s.d. are shown. Experiment was performed twice, with each experiment delineated by the dotted line. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Effect of Kv1.3 blockade on DTH inflammatory responses. WT rats were immunized with OVA then subsequently challenged 1 week later with either PBS or OVA. OVA-rechallenged animals were treated with control anti-ragweed (aRGW) antibody (pink), CTLA4-Ig (orange) or ShK (blue). Ear swelling was measured 24 h later. Individual biological replicates (n=6 per group) are shown with mean±s.d. Experiment was performed three times, with each experiment delineated by dotted lines. Statistically significant differences between ShK and anti-ragweed control groups are denoted with P values; CTLA4-Ig inhibition was statistically significant in all experiments ( P

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Injection, Inhibition

    Effects of siRNA knockdown of Kv1.3 or KCa3.1 on TT-specific human T-cell sensitivity to inhibitors. ( a ) Kv1.3 (left) and KCa3.1 (right) expression was measured in primary TT-stimulated T cells (1° TT) or T cells that underwent four rounds of TT stimulation (4° TT) that were transfected with Kv1.3 siRNA (blue bars), KCa3.1 siRNA (green bars), a combination of both siRNA (orange bars) or scramble control siRNA (grey bars). Relative expression of targeted genes is shown in comparison with scramble siRNA transfected cells. Data are shown as mean±s.d. of triplicate measurements from one representative experiment. ( b ) Kv1.3 surface protein expression on 1° TT or 4° TT cells following siRNA transfection. Primary (left) or repeatedly stimulated (4°, right) TT cells were transfected with Kv1.3 siRNA (red histograms) or scramble siRNA (blue histograms) and then stained for Kv1.3 48 h later. Grey filled histogram represents isotype control. T-cell responses of 1° TT-stimulated T cells ( c , d ) or 4° TT-stimulated T cells ( e , f ) transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green), a combination of both (orange) or scramble control siRNA (black). Cells were then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( c , e ) or TRAM-34 ( d , f ) at the indicated concentrations. Proliferation responses were determined at day 4 of culture by 3 H-thymidine incorporation; IFN-γ concentration was measured in culture supernatants harvested 3 days after restimulation. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Effects of siRNA knockdown of Kv1.3 or KCa3.1 on TT-specific human T-cell sensitivity to inhibitors. ( a ) Kv1.3 (left) and KCa3.1 (right) expression was measured in primary TT-stimulated T cells (1° TT) or T cells that underwent four rounds of TT stimulation (4° TT) that were transfected with Kv1.3 siRNA (blue bars), KCa3.1 siRNA (green bars), a combination of both siRNA (orange bars) or scramble control siRNA (grey bars). Relative expression of targeted genes is shown in comparison with scramble siRNA transfected cells. Data are shown as mean±s.d. of triplicate measurements from one representative experiment. ( b ) Kv1.3 surface protein expression on 1° TT or 4° TT cells following siRNA transfection. Primary (left) or repeatedly stimulated (4°, right) TT cells were transfected with Kv1.3 siRNA (red histograms) or scramble siRNA (blue histograms) and then stained for Kv1.3 48 h later. Grey filled histogram represents isotype control. T-cell responses of 1° TT-stimulated T cells ( c , d ) or 4° TT-stimulated T cells ( e , f ) transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green), a combination of both (orange) or scramble control siRNA (black). Cells were then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( c , e ) or TRAM-34 ( d , f ) at the indicated concentrations. Proliferation responses were determined at day 4 of culture by 3 H-thymidine incorporation; IFN-γ concentration was measured in culture supernatants harvested 3 days after restimulation. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Expressing, Transfection, Staining, Concentration Assay

    Full inhibition of antigen-specific human T cells requires blockade of both Kv1.3 and KCa3.1. ( a , b ) Inhibition of CD4 + ( a ) or CD8 + ( b ) T-cell proliferation response to TT stimulation as determined by CFSE dilution. One representative donor is shown. Percentages indicated in red denote % reduction in proliferation relative to vehicle-treated cells. Primary (1°) T-cell response to TT was determined in the absence or presence of either 10 nM ShK ( n =8 biological replicates) or 1 μM TRAM-34 ( n =9). ( c ) Proliferation responses were determined at day 4; IFN-γ concentrations were measured at day 3. % inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( d ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates TT-specific T cell responses. PBMC from T1D donors were stimulated with TT in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes absence of TT. Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. ( e ) Proliferation and IFN-γ production of TT-specific T cells following four rounds of stimulation (4°). After three rounds of TT stimulation, cells were rested and then restimulated in the presence of irradiated autologous PBMC with TT in the absence or presence of either 10 nM ShK ( n =11 biological replicates) or 1 μM TRAM-34 ( n =4). ( f ) Effect of ShK or TRAM-34 on proliferation or IFN-γ production of TT-specific T cells after increasing rounds of stimulation. Data shown are representative of one donor. ( g ) Gene expression of KCNA3 and KCNN4 in purified T cells after 1° or 4° TT stimulation. Expression of each channel is presented relative to expression in naive T cells. Data shown are representative of three donors. ( h ) Kv1.3 surface protein expression on 1° TT or 4° TT cells. Blue histogram represents Kv1.3 staining; grey filled histogram represents isotype control.

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Full inhibition of antigen-specific human T cells requires blockade of both Kv1.3 and KCa3.1. ( a , b ) Inhibition of CD4 + ( a ) or CD8 + ( b ) T-cell proliferation response to TT stimulation as determined by CFSE dilution. One representative donor is shown. Percentages indicated in red denote % reduction in proliferation relative to vehicle-treated cells. Primary (1°) T-cell response to TT was determined in the absence or presence of either 10 nM ShK ( n =8 biological replicates) or 1 μM TRAM-34 ( n =9). ( c ) Proliferation responses were determined at day 4; IFN-γ concentrations were measured at day 3. % inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( d ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates TT-specific T cell responses. PBMC from T1D donors were stimulated with TT in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes absence of TT. Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. ( e ) Proliferation and IFN-γ production of TT-specific T cells following four rounds of stimulation (4°). After three rounds of TT stimulation, cells were rested and then restimulated in the presence of irradiated autologous PBMC with TT in the absence or presence of either 10 nM ShK ( n =11 biological replicates) or 1 μM TRAM-34 ( n =4). ( f ) Effect of ShK or TRAM-34 on proliferation or IFN-γ production of TT-specific T cells after increasing rounds of stimulation. Data shown are representative of one donor. ( g ) Gene expression of KCNA3 and KCNN4 in purified T cells after 1° or 4° TT stimulation. Expression of each channel is presented relative to expression in naive T cells. Data shown are representative of three donors. ( h ) Kv1.3 surface protein expression on 1° TT or 4° TT cells. Blue histogram represents Kv1.3 staining; grey filled histogram represents isotype control.

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Inhibition, Concentration Assay, Irradiation, Expressing, Purification, Staining

    Kv1.3 and KCa3.1 are both required for human autoreactive T-cell responses. ( a ) PBMC from HLA-DR4 + T1D donors ( n =10 biological replicates) were stimulated with a combination of HLA-DR4-restricted GAD65 peptides in the absence or presence of either 10 nM ShK or 1 μM TRAM-34. ( b ) PBMC from non-HLA- or HLA-typed T1D donors were stimulated with GAD65 protein ( n =13 biological replicates). Proliferation responses (left) were determined at day 4. IFN-γ concentrations (right) were measured 3 days after stimulation. %inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates autologous autoreactive T cell responses. PBMC from T1D donors were stimulated with GAD65 protein in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes unstimulated cell conditions (absence of GAD65 protein). Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. Gene expression for Kv1.3 ( KCNA3 , d ) and KCa3.1 ( KCNN4, e ) in GAD65-stimulated purified T cells following siRNA transfection with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey). ( f , g ) Effects of siRNA knockdown of Kv1.3 or KCa3.1 on sensitivity to inhibitors. PBMC from T1D donor was stimulated with GAD65 protein for 7 days. After 3 days rest, T cells were purified and transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey) and then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( f ) or TRAM-34 ( g ) at the indicated concentrations. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Kv1.3 and KCa3.1 are both required for human autoreactive T-cell responses. ( a ) PBMC from HLA-DR4 + T1D donors ( n =10 biological replicates) were stimulated with a combination of HLA-DR4-restricted GAD65 peptides in the absence or presence of either 10 nM ShK or 1 μM TRAM-34. ( b ) PBMC from non-HLA- or HLA-typed T1D donors were stimulated with GAD65 protein ( n =13 biological replicates). Proliferation responses (left) were determined at day 4. IFN-γ concentrations (right) were measured 3 days after stimulation. %inhibition was determined by comparing proliferation or IFN-γ concentration in the presence of inhibitor to vehicle control. Data are shown as individual data points with mean±s.d. Statistically significant differences are denoted with P values as determined by Student's t- test. ( c ) Inhibition of both Kv1.3 and KCa3.1 fully abrogates autologous autoreactive T cell responses. PBMC from T1D donors were stimulated with GAD65 protein in the absence or presence of 10 nM ShK, 1 μM TRAM-34 or a combination of both (combo). ‘No stim' denotes unstimulated cell conditions (absence of GAD65 protein). Data are shown as mean±s.d. of replicate wells and are representative of three independent experiments. Gene expression for Kv1.3 ( KCNA3 , d ) and KCa3.1 ( KCNN4, e ) in GAD65-stimulated purified T cells following siRNA transfection with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey). ( f , g ) Effects of siRNA knockdown of Kv1.3 or KCa3.1 on sensitivity to inhibitors. PBMC from T1D donor was stimulated with GAD65 protein for 7 days. After 3 days rest, T cells were purified and transfected with Kv1.3 siRNA (blue), KCa3.1 siRNA (green) or scramble control siRNA (grey) and then restimulated with anti-CD3 (0.5 μg ml −1 ) in the absence or presence of ShK ( f ) or TRAM-34 ( g ) at the indicated concentrations. Data are shown as mean±s.d. of replicate wells and are representative of at least two independent experiments.

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Inhibition, Concentration Assay, Expressing, Purification, Transfection

    Characterization of Kcna3 − / − T cells. ( a ) K + -channel expression in human, rat and mouse T cells. Gene expression of Kv1 family members and KCa3.1 in naive CD4 + T cells from human (left), rat (centre) or mouse (right). Relative expression was determined by normalizing to housekeeping gene RPL19. Electrophysiological and pharmacological tests show null Kv1.3 channel in Kcna3 − / − T cells. ( b ) Representative voltage-currents from WT and Kcna3 − / − T cells. Currents were elicited by depolarizing voltage steps from −60 to +40 mV (10 mV increments every 30 s, with −80 mV membrane-holding potential). ( c ) Kv1.3 channel number in WT ( n =40) and Kcna3 − / − ( n =50) T cells after 48 h activation. The mean Kv1.3 channel numbers were 1667±187 in WT and undetectable in Kcna3 − / − T cells. ( d ) Normalized WT and Kcna3 − / − T cell K + currents before and after Shk inhibition. 89% WT T cell K + current was blocked by 1 nM Shk, but no Shk-sensitive current was detected in Kcna3 − / − T cells. Kcna3 − / − T-cell responses to activation. ( e ) Proliferation (left) and IFN-γ (right) responses to anti-CD3 and anti-CD28 stimulation. Spleen cells from WT or Kcna3 − / − rats were stimulated for 3 days. Individual biological replicates ( n =4 per group) are shown with mean±s.d. ( f ) Effects of ShK and TRAM-34 on polyclonal T-cell activation. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( g ) OVA-specific T-cell proliferation responses. Draining lymph node and spleen cells from OVA-immunized WT and Kcna3 − / − rats were plated at a 1:10 lymph node:spleen cell ratio and stimulated in vitro with OVA at various concentrations. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( h , i ) Kcna3 − / − rat dendritic cell competency. CD4 + ( h ) or CD8 + . ( i ) T cells isolated from DLN of OVA-immunized WT (blue) and Kcna3 − / − (green) rats were co-cultured with APCs from WT (filled circles) or Kcna3 − / − (open circles) rats and stimulated with OVA. Proliferation responses were determined at day 3 of culture. Individual biological replicates ( n =4 per group) are shown with mean±s.d.

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Characterization of Kcna3 − / − T cells. ( a ) K + -channel expression in human, rat and mouse T cells. Gene expression of Kv1 family members and KCa3.1 in naive CD4 + T cells from human (left), rat (centre) or mouse (right). Relative expression was determined by normalizing to housekeeping gene RPL19. Electrophysiological and pharmacological tests show null Kv1.3 channel in Kcna3 − / − T cells. ( b ) Representative voltage-currents from WT and Kcna3 − / − T cells. Currents were elicited by depolarizing voltage steps from −60 to +40 mV (10 mV increments every 30 s, with −80 mV membrane-holding potential). ( c ) Kv1.3 channel number in WT ( n =40) and Kcna3 − / − ( n =50) T cells after 48 h activation. The mean Kv1.3 channel numbers were 1667±187 in WT and undetectable in Kcna3 − / − T cells. ( d ) Normalized WT and Kcna3 − / − T cell K + currents before and after Shk inhibition. 89% WT T cell K + current was blocked by 1 nM Shk, but no Shk-sensitive current was detected in Kcna3 − / − T cells. Kcna3 − / − T-cell responses to activation. ( e ) Proliferation (left) and IFN-γ (right) responses to anti-CD3 and anti-CD28 stimulation. Spleen cells from WT or Kcna3 − / − rats were stimulated for 3 days. Individual biological replicates ( n =4 per group) are shown with mean±s.d. ( f ) Effects of ShK and TRAM-34 on polyclonal T-cell activation. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( g ) OVA-specific T-cell proliferation responses. Draining lymph node and spleen cells from OVA-immunized WT and Kcna3 − / − rats were plated at a 1:10 lymph node:spleen cell ratio and stimulated in vitro with OVA at various concentrations. Data are shown as mean±s.d. ( n =4 biological replicates per group). ( h , i ) Kcna3 − / − rat dendritic cell competency. CD4 + ( h ) or CD8 + . ( i ) T cells isolated from DLN of OVA-immunized WT (blue) and Kcna3 − / − (green) rats were co-cultured with APCs from WT (filled circles) or Kcna3 − / − (open circles) rats and stimulated with OVA. Proliferation responses were determined at day 3 of culture. Individual biological replicates ( n =4 per group) are shown with mean±s.d.

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Expressing, Activation Assay, Inhibition, In Vitro, Isolation, Cell Culture

    Autoreactive but not autologous pathogen-specific T cells are sensitive to Kv1.3 blockers. ( a , b ) ShK inhibits GAD65-specific T1D T cells but not autologous TT-specific T cells. PBMC from T1D donors ( n =6 biological replicates) were stimulated with a combination of four HLA-DR4-restricted GAD65 peptides. Proliferation and IFN-γ responses were compared with TT stimulation in the absence or presence of either 10 nM ShK ( a ) or 1 μM TRAM-34 ( b ). Data shown are % inhibition of proliferation response as determined by 3 H-thymidine incorporation after 4 days primary in vitro stimulation and % inhibition of IFN-γ production after 3 days stimulation. Coloured symbols and corresponding lines represent each individual donor.

    Journal: Nature Communications

    Article Title: Potassium channels Kv1.3 and KCa3.1 cooperatively and compensatorily regulate antigen-specific memory T cell functions

    doi: 10.1038/ncomms14644

    Figure Lengend Snippet: Autoreactive but not autologous pathogen-specific T cells are sensitive to Kv1.3 blockers. ( a , b ) ShK inhibits GAD65-specific T1D T cells but not autologous TT-specific T cells. PBMC from T1D donors ( n =6 biological replicates) were stimulated with a combination of four HLA-DR4-restricted GAD65 peptides. Proliferation and IFN-γ responses were compared with TT stimulation in the absence or presence of either 10 nM ShK ( a ) or 1 μM TRAM-34 ( b ). Data shown are % inhibition of proliferation response as determined by 3 H-thymidine incorporation after 4 days primary in vitro stimulation and % inhibition of IFN-γ production after 3 days stimulation. Coloured symbols and corresponding lines represent each individual donor.

    Article Snippet: FITC-conjugated Kv1.3 Ab was purchased from Sigma-Aldrich.

    Techniques: Inhibition, In Vitro