1. CD8 T Cells Regulate Allergic Contact Dermatitis by Modulating CCR2–Dependent TNF/iNOS–Expressing Ly6C+CD11b+ Monocytic Cells 
Shu Zhen Chong, Kar Wai Tan, Fiona H.S. Wong, Yen Leong Chua, Yafang Tang, Lai Guan Ng, Veronique Angeli, David M. Kemeny 
Journal of Investigative Dermatology 
Volume 134, Issue 3, Pages (March 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
2,4-Dinitrochlorobenzene (DNCB)–elicited contact hypersensitivity (CHS) results in tumor necrosis factor-α/inducible nitric oxide synthase (TNF-α/iNOS) expression with neutrophil and monocytic cell infiltration. (a) Ear thickness of mice treated with DNCB (red circle), trimellitic anhydride (TMA) (blue square), and vehicle (black triangle). (b) Hematoxylin and eosin (H&E) staining of ear tissue sections. Bar=200 μm. (c) Dermal CD45+ TNF-α– and iNOS–expressing CD11b+ cells. Dermal CD45+CD11b+ cell numbers positive for (d) iNOS and (e) TNF-α. Cell numbers of (f) neutrophils (CD45+CD11b+Ly6G+Ly6C+) and (g) monocytic cells (CD45+CD11b+Ly6G−Ly6C+) quantified by flow cytometry. Results shown are mean±SD of six to eight mice per group and representative of one out of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 (compared with vehicle-treated mice), ++P<0.01, +++P<0.001 (compared with TMA–treated mice).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Characterization of tumor necrosis factor-α/inducible nitric oxide synthase (TNF-α/iNOS)–expressing cells in 2,4-dinitrochlorobenzene (DNCB)–treated mice. (a) TNF-α/iNOS expression of Ly6G+ cells gated from CD45+CD11b+ cells. (b) Ear thickness of mice depleted for neutrophils (anti-Ly6G) compared with control (IgG). ***P< (c) Percentage of CD11b+TNF-α/iNOS+ cells in each gate (R1=Ly6C−MHCII+; R2=Ly6C+MHCII+; R3=Ly6C+MHCII−; R4=Ly6C−MHCII−) among CD45+Ly6G− cells. (d) Percentage and median florescence index (MFI) of TNF-α/iNOS–expressing cells from each gate. (e) Comparative histograms and (f) MFI expression from each gate of DAF-FM-DA–expressing cells (measure of NO). *P<0.05, **P<0.01; ***P<0.001 (compared with R1 gate). (g) MFI of indicated markers on cells from each gate. Results shown are mean±SD of five to seven mice per group and representative of one out of three independent experiments. Inc., increase; MHCII, major histocompatibility complex II.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Impaired C–C chemokine receptor type 2 (CCR2) signaling abrogates Ly6C+CD11b+ monocytic cells and tumor necrosis factor-α/inducible nitric oxide synthase (TNF-α/iNOS) expression during contact hypersensitivity (CHS). (a) MHCII+/-–expressing Ly6C+CD11b+ dermal cells gated from CD45+Ly6G− cells were quantified by flow cytometry. (b) CD45+CD11b+ cells were gated and TNF-α– and iNOS–expressing cells quantified by flow cytometry. (c) Increase in ear thickness. (d) Hematoxylin and eosin (H&E) staining of ear tissue sections. Bar=200 μm. (e) CHS response, measured by increase in ear thickness, after adoptive transfer of 2,4-dinitrochlorobenzene (DNCB)–primed wild-type (WT) CD8 T cells into naive WT (red circle) or CCR2−/− (blue square) recipients. Results shown are mean±SD of six mice per group and representative of one out of four independent experiments. *P<0.05, **P<0.01, and ***P< Inc., increase; MHCII, major histocompatibility complex II.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Monocyte depletion abrogates tumor necrosis factor-α/inducible nitric oxide synthase (TNF/iNOS)–expressing Ly6C+CD11b+ monocytic cells and attenuates contact hypersensitivity (CHS) responses. (a) Percentage numbers of circulating blood monocytes and dermal monocytic cells after phosphate-buffered saline (PBS) or clodronate administration 24 hours after challenge. (b) MHCII+/-–expressing Ly6C+CD11b+ dermal cells gated from CD45+Ly6G− cells were quantified by flow cytometry. (c) CD45+CD11b+ cells were gated and TNF-α– and iNOS–expressing cells quantified by flow cytometry. (d) Increase in ear thickness. Results shown are mean±SD of six mice per group and representative of one out of three independent experiments. *P<0.05, **P<0.01, and ***P< Inc., increase; MHCII, major histocompatibility complex II.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
CD8 T cells regulate tumor necrosis factor-α/inducible nitric oxide synthase (TNF/iNOS)–expressing Ly6C+CD11b+ monocytic cells through IL-17 and IFN-γ. Wild-type (WT), β2m−/−, perforin−/−, IFN-γ−/−, and mice treated with anti-CD8 (CD8dep) or anti-IL-17 (IL-17neu) antibodies were 2,4-dinitrochlorobenzene (DNCB) treated. (a, b) MHCII+/- expressing Ly6C+CD11b+ cells and (c, d) TNF-α/iNOS+ CD45+CD11b+ cells quantified by flow cytometry. *P<0.05, **P<01, and ***P< (e) Increase in ear thickness and (f) Ly6C+CD11b+ cell numbers in naive recipients or recipients treated with anti-IL-17 antibodies (IL-17neu) after adoptive transfer of WT or IFN-γ−/− DNCB–primed CD8 T cells. ***P<0.001 (compared with IL-17neu CD8 transfer), +P<0.05, ++P<0.01 (compared with IFN-γ−/− CD8 transfer). Results shown are mean±SD of five to eight mice per group and representative of one out of three independent experiments. β2m, β2 Microglobulin; Inc., increase; MHCII, major histocompatibility complex II.
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions