Fig 2: Nicotine induces HSC quiescence after transplantation.a Scheme illustrating experimental design. Irradiated WT CD45.2 recipients were transplanted with 2 × 106 BM nucleated cells from CD45.1 mice and treated with 65 µg/ml nicotine or vehicle in their drinking water over 7 weeks. b, c Circulating donor-derived (CD45.1+) haematopoietic cells (b, P = 0.03) and white blood cells (c, P = 0.006,0.04) at specified time after transplantation (N = 3). d Donor-derived B cells (P = 0.007), T cells (P < 0.0001) and myeloid cells (P = 0.009) from mice treated with nicotine or vehicle at 7 weeks (N = 3). e Cell cycle profiles of donor-derived lin-sca1+ckit+ (LSK) CD34-flt3- HSCs isolated from the endosteal or central BM 7 weeks after transplantation and chronic treatment with nicotine or vehicle (N = 3;P = 0.005,0.04). (b–e) Data are mean of biological replicates ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. (b–e) ANOVA and Sidak’s comparisons.

Fig 3: tDCs Display Lymphoid Characteristics Associated with pDCs(A) TF expression in sorted mouse splenic DC subsets measured by qPCR (n = 3 in 3 exp). Expression represents ??Cq relative to the internal control gene Rpl13a and cDC2s.(B) Sorted mouse splenic DC subsets analyzed by PCR assay for IgH D-J rearrangement. Actin and IgH germline (GL) are also shown (1 representative of 6 exp).(C) EGFP expression in splenic DC subsets from PTCRA-EGFP mice compared with wild-type (WT) mice. Two mice for each condition shown. Numbers indicate frequency of EGFP+ cells.(D) tSNE map of Lin- events from negatively enriched (anti-CD3 and anti-CD19) mouse splenocytes analyzed by CyTOF. Left: manual annotation; right: colored by protein expression. Bottom panels show splenic pre-DCs gated as CD3- CD19- CD335- B220- and CD11c+ MHC class II- CD135+ CD172a- as described (Liu et al., 2009). Pre-DCs were overlaid in the tSNE map (pink dots). See Figure S5 for a comparison between spleen tDCs and BM pre-DCs.(E) Total numbers of tDCs and pre-DCs (n = 5 in 3 exp). The frequency of pre-DCs that overlap tDCs (white) or other cells (black) is shown.(F) Frequency of tDCs that correspond to pre-DCs (average of n = 5 in 3 exp).(G) Surface marker expression in spleen pre-DCs and tDCs representative of 3 exp. Numbers indicate frequency of positive cells.Bar graphs indicate mean ± SD.