Figure S1. Kv1.3 and Kca3.1 Are Expressed in CLL Cells

Figure S1. Kv1.3 and KCa3.1 are expressed in CLL cells

(A) Isolated CLL cells from patients (N = 9) identified by immunofluorescence (IF) labeling of CD19 were patch-clamped in the whole cell mode (left: brightfield image; right: fluorescence image). (B) Delivery of ten identical depolarizing voltage steps from -80 to +40 mV separated by 1 s showed use-dependent inactivation indicative of Kv1.3. 1– 10 label the first to tenth pulse. (C) Consecutive voltage ramps from -120 to +40 mV and 1 µM free intracellular Ca2+visualized KCa3.1 currents. PAP-1 [200 nM] eliminated all interfering Kv-currents and TRAM-34 [500nM] erased residual Ca2+ activated currents.

Figure S2. PMA and ionomycin activates CLL cells and healthy B cells accompanied by changes in K+ channel expression

(A) CLL cells from patients (N = 28) and (B) B cells from healthy donors (N = 4) isolated from PBMCs were cultured with PMA and ionomycin for 19 h.Expression of the activation marker CD69 was analyzed via flow cytometry (FC)(exemplary FC plots)(Ai, Bi); “iso” depicts isotype controls; (Aii) qRT-PCR of CLL cells demonstrated Kv1.3 levels were down-regulated following activation (P < 0.0001), while KCa3.1 mRNA levels remained stable (P = 0.5401), creating a predominance of KCa3.1 (P=0.0104). (Bii) In activated B cells, KCa3.1 mRNA was up-regulated (P = 0.0114) and Kv1.3 down-regulated (P = 0.1975), revealing a similar predominance of KCa3.1 (P=0.0047). (C) qRT-PCR of freshly isolated versus PMA and ionomycin treated CLL cells (N = 28) and healthy B cells (N = 4) showed minimal levels of herg1 mRNA in resting CLL cells and B cells. Activated cells down-regulated herg1 mRNA (CLL: P < 0.0001); B cells: P= 0.0215).

Figure S3.Co-culture of CLL cells and activated CD4+ T cells activates CLL cells and induces cell division after five to six days accompanied by the up-regulation of KCa3.1

(A) CLL cells in co-culture with α-CD3/CD28-activated T4 cells exhibited enhanced expression of the co-stimulatory molecules CD80 and CD86 after three (P = 0.0372) and after five to six days (P = 0.0325). (B) These culture conditions induced cell divisionin CLL cells; (Bi) representative FC plotfor CFSE dilution in dividing CLL cells (N =5); (Bii) median percentage of dividing cells was enhanced from 1.4% (CLL only; N=3) and 2.9% (CLL + T4; N=5) to 9.3% (CLL + T4 + α-CD3/CD28 beads; N=5) (P = 0.0078). (C) qRT-PCR revealed up-regulation of KCa3.1 mRNA levels in dividing samples (CLL + T4 + αCD3/CD28 beads) (N =5) compared to non-activated CLL cells (+ T4 cells)(N =5)P=0.1097) resulting in a predominance of KCa3.1 over Kv1.3 in dividing CLL cells (P=0.0430).

Figure S4. CD19, Ki-67 and KCa3.1 expression pattern in CLL and normal lymphoid tissue

Immunohistochemistry (IHC) staining was performed on sequential CLL formalin-fixed paraffin-embedded (FFPE)(A) -lymph node (N=2), (B) -tonsil (N = 1) and (C) -bone marrow (N= 7) samples from patients (N = 6) as well as on (D) -lymph node (N = 3) and (E) -tonsil (N = 1) samples from healthy donors (N = 4). Brownish color depict CD19, Ki-67 and KCa3.1 expression pattern, respectively. Blue counterstains mark cell nuclei.(A-B) Overview images were taken with a 4x objective, small images in the left lower corner were taken with a 20x objective. (A)Representative CLL lymph node with diffuse CLL cell infiltration (CD19+) depictsdiffuseKi-67 as well as KCa3.1 expression. (B) CLL tonsil with nodular CLL infiltration (CD19+); areas of high Ki-67 expression overlap with CD19+ areas and high KCa3.1 expressing areas; (C) Bone marrow images were taken with a 10x objective. The areas of nodular CD19+ CLL infiltrate also exhibited the highest KCa3.1 expression and Ki67 density. (D) Representative normal lymph node exhibiting germinal center structures; (E) active germinal center structures (Ki-67high) in a human tonsilshows high B cell (CD19+) proliferation (Ki-67+) coincides with high KCa3.1 expression.

Figure S5.Effects of co-culture system on cells

(A) CLL cells cultivated on a CD40L-expressing fibroblast layer for 24 h (Ai) up-regulated the late activation markers CD80 and CD86 (P = 0.0803) coinciding with (Aii) increased expression of KCa3.1 mRNA (P = 0.0070). (B) Effects of three day TRAM-34 [10µM] exposure on (Bi) growth and viability of CD40L-fibroblasts (P = 0.5981), determined by manual counting of viable cells (trypan blue-negative), and (Bii) CD40L-expression (P =0.1169), analyzed by FC. (C) Effects of TRAM-34 [10µM] on (Ci) viability (P = 0.7021) and (Cii) Ki-67 expression (P =0.2171) of α-CD3/CD28 activated T cells co-cultured with CLL cells on a CD40L-fibroblast layer, measured by FC (N = 6).

Figure S6.Absolute percentages of proliferating and viable CLL cells in K+ channel blocker treatment studies

(A) CLL cells, 24 h pre-activated on CD40L-expressing fibroblasts, were treated with TRAM-34 [10 µM] or clotrimazole [10 µM] and PAP-1 [10 µM] or Psora-4 [10 µM], respectively, followed by α-CD3/CD28 T cell activation. Ki67 expression was determined after another 48 h by FC. (Ai) PAP-1 and Psora-4 did not decrease Ki-67 expression (PAP-1: P=0.1875; Psora-4: P = 0.6250), whereas TRAM-34 (P = 0.0002) and clotrimazole (P=0.0625) did. (Aii) Clotrimazole reduced cell viability (AnnexinV-7-AAD- cells) (P= 0.0088), TRAM-34, PAP-1 and Psora-4 did not (TRAM: P=0.8973; PAP-1: P=0.8426; Psora-4: P = 0.7294). (B) Dose-escalation of TRAM-34 using 1, 5 or 10 µM showed (Bi)concentration-dependentreduction of Ki-67 expression on CLL cells: 1 µM: P = 0.1132; 5µM: P= 0.0015; 10 µM: P= 0.0002,(Bii) without changing viability of CLL cells. (C) 24 h CpG pre-stimulated CLL cells were treated with escalating doses of TRAM-34 prior to supplementation with IL-2. Ki-67 expression of CLL cells was reduced in a dose-dependent manner (10µM: P = 0.0395).

Table S1: KCa3.1 channels are up-regulated in activated CLL cells

Values were calculated from whole cell patch clamp measurements, representing mean±SEM

Table S2: List of antibodies used for flow cytometry, immunohistochemistry, immune fluorescence microscopy and electrophysiology

Table S3: List of patients involved in the project, including parameters of prognostic relevance and detailed experimental contribution