IL-10–dependent macrophage polarization and macrophage-polarizing cytokines in TCLs. (A) Monocytes were cultured in media alone or in media supplemented with either IL-10 (20 ng/mL) or conditioned-media (50% v/v) obtained from the TCL cells indicated. An isotype control or neutralizing IL-10 monoclonal antibody was included, as indicated, and STAT3 phosphorylation (Y705) determined by flow cytometry. (Left) Representative histograms (open, isotype control; gray, pSTAT3). (Right) Change in mean fluorescent intensity (ΔMFI) of pSTAT3 from triplicate wells (±standard error). (B) Monocyte-derived macrophages (MDMs) were generated and polarized in TCL-conditioned media with an isotype control or IL-10 neutralizing antibody. CD163, CD16, and HLA-DR expression (gray histogram) were determined (isotype control, open histogram). MDMs generated in the presence of H9-conditioned media are shown in (left) representative histograms and (right) change in mean fluorescent intensity (ΔMFI) from triplicate wells (±standard error). (C) MDMs were generated and functionally polarized in TCL-conditioned media (TCL-CM) supplemented with an isotype control (closed markers) or IL-10 neutralizing monoclonal antibody (open markers) in triplicate in a flat-bottom 96-well plate. Adherent macrophages were gently washed with fresh media, and lipopolysaccharide was added. IL-10 production was determined 24 hours later. The mean (±standard error) from 10 individual normal donors is shown (note logarithmic scale). (D) MDMs were polarized with TCL-CM in triplicate wells of a 96-well plate. CFSE-labeled allogeneic CD3+ T cells were added, and T-cell proliferation determined by CFSE dilution. (Upper) Representative histograms using MDMs generated in H9-CM. (Lower) Mean proliferation (% CFSElo) (±standard error). All experiments shown are representative of ≥3 similarly performed experiments. (E) The abundance of cytokine transcripts (IFN-γ, IL-10, IL-4, IL-13) were quantified by Nanostring nCounter technology from FFPE reactive lymph node (n = 7), CTCL (n = 11), and 48 PTCL (PTCL, NOS, n = 31; AITL, n = 10, ALCL, n = 7) specimens. (F) Immunofluorescent double staining for CD163 and pSTAT3 (Y705) with nuclei stained by 4′6 diamidino-2-phenylindole was performed in PTCL, NOS specimens (n = 80, representative examples shown). *P < .05 and **P < .01 in unpaired 2-sided Student t test.
