IL-21 expression in lymph nodes from CLL patients. (A) IL-21 expression was studied by qRT-PCR in RNA from PBMCs from 2 healthy donors and 2 CLL patients (samples 6A, 8), after activation with αCD3 or αCD28 antibodies or PMA + ionomycin, or in total frozen LN samples from CLL patients (samples 34 to 40). (B) IL-21 expression was analyzed in 2 paraffin-embedded CLL LN samples by IHC. Upper panels show nonspecific staining with control antibodies; lower panels show results with anti-IL-21 antibodies. Original magnification ×40, further digital magnification ×4. (C) Matched samples of PB and LN from 2 CLL patients (samples 11 and 14) were stimulated as indicated in the “Methods” section for 4 hours. They were then stained for expression of CD3, CD4, CXCR5, CD40L, and IL-21. Results depict the histograms for CXCR5 expression (unstimulated samples, left panels) and the expression of IL-21 and CD40L by CXCR5⁺ and CXCR5⁻ CD3⁺ CD4⁺ cells for PB (upper) and LN (lower) samples.