Effect of stromal factors on TLR7-signaling responses. (A) Morphology of CLL splenic stromal cells are shown by direct light microscopy or (B) following May-Grünwald/Giemsa-staining of a cytospin preparation. Scale bars, 1 mm. (C) Flow cytometry indicate the cells do not express the hematopoietic marker CD45. (D) 104 CLL cells were cultured alone or with IL-2 and resiquimod in the indicated concentrations of supernatant from confluent stromal cells cultured in AIM-V for 48 hours. Viable cells were counted daily for 9 days by trypan-blue exclusion. Averages and standard errors of 3 separate measurements are shown for 4 different patient samples. (E-F) CLL cells were cultured overnight in the presence or absence of 30% conditioned media with or without the IL-6 receptor antibody actemra (15 μg/ml) and then stimulated with resiquimod (1 μg/mL). Membrane TNF-α and CD83 expression were measured 4 hours later by flow cytometry. An example for patient #22 is shown (E) with similar results obtained for 8 other patient samples. Numbers in the top right quadrants indicate percentages of mTNF-α+CD83+ cells. (F) Before resiquimod activation, TLR7 (left panel) and TNFA (right panel) transcripts were measured by PCR and normalized to HPRT. Averages and standard errors of results from 3 independent measurements for 4 different patient samples are shown. **P < .01; ***P < .001. Act, actemra; Pt, patient; R, resiquimod; SS, stromal supernatant.
