Type II monocytes modulate T cell-mediated central nervous system autoimmunity
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1 Type II monocytes modulte T cell-medited centrl nervous system utoimmunity Mrtin S. Weer, Thoms Prod homme, Swsn Youssef, Shnnon E. Dunn, Cynthi D. Rundle, Lind Lee, Jun C. Ptrroyo, Olf Stüve, Rymond A. Soel, Lwrence Steinmn nd Scott S. Zmvil Prolifertion 6 4 OVA OVA lone Intct MBP Recominnt MOG Prolifertion Vehicle-treted monocytes - OVA Vehicle-treted monocytes - -treted monocytes- OVA treted monocytes- 1 Antigen concentrtion (µg/ml) 1 Antigen concentrtion (µg/ml) Supplementry Figure 1. OVA specific T cells do not cross-rect with or myelin ntigens. () Splenocytes from OT-II mice were cultured with, intct MBP or recominnt MOG. Antigen-specific T cell rectivity ws evluted y [ 3 H] thymidine incorportion. () Following co-culture with -treted or vehicle (PBS)-treted monocytes, resting OVA specific T cells did not proliferte in response to.
2 -treted monocytes 2 -treted monocytes Prolifertion 2 1 Vehicle-treted monocytes No monocytes IFN-γ (pg/ml) 1 Vehicle-treted monocytes No monocytes (µg/ml) (µg/ml) Supplementry Figure 2. -induced type II monocytes used for doptive trnsfer do not stimulte -specific T cells without exogenous. - or vehicle (PBS)-treted monocytes were co-cultured with CD4 + -rective T cell line to determine the possiility of functionl crry-over of ound to -treted monocytes. -treted monocytes, s vehicle-treted monocytes did not stimulte -rective T cells without exogenous s mesured y prolifertion nd secretion of IFN-γ. CD4 + -rective T cells without monocytes were cultured s negtive control.
3 Lymph node, dy 2 CD4.1-PE CD4.1-PE treted CD4.1 monocytes 16.8% 14.% 1.4% CD11-FITC % % % Vehicle-treted CD4.1 monocytes CD11-FITC No doptive trnsfer CD11-FITC Spinl cord, dy CD4.1-PE treted CD4.1 monocytes 9.1% CD11-FITC Vehicle-treted CD4.1 monocytes 8.3% CD11-FITC No doptive trnsfer.6% CD11-FITC Supplementry Figure 3. Identifiction of -treted nd vehicle-treted monocytes in recipient mice. () CD4.1 + monocytes were identified 2 dys fter doptive trnsfer in lymph nodes of CD4.2 recipient mice with EAE y CD4.1 + CD11 + doule stining (indicted is the percentge mong ll CD11 + cells). Donor monocytes remined CD11 +, CD11c - nd CD8 - (not shown). CD4.1 + CD3 + T cells were not detected. () Similr percentges of -treted nd vehicle-treted CD4.1 + monocytes could e identified in the CNS of recipient mice dys fter doptive trnsfer (indicted is the percentge of CD4.1 + CD11 + mong ll CD11 + CNS infiltrting cells).
4 % Vehicle-treted MHC II -/- monocytes % % -treted MHC II -/- monocytes 3.1% IFN-γ (pg/ml) 1,8 1,6 1,4 1, 1, MHC II -/- vehicle MHC II -/- WT vehicle WT 1 MOG p3- (mg/ml) Vehicle-treted WT monocytes -treted WT monocytes % 2.6% % % IL-4 (pg/ml) 2 1 MHC II -/- vehicle MHC II -/- WT vehicle WT 1 MOG p3- (mg/ml) Supplementry Figure 4. Type II monocytes from MHC clss II-deficient mice did not direct T cell immune devition in vivo. -treted type II monocytes from wild-type mice (WT), ut not from MHC clss II-deficient mice (MHC II -/- ), inhiited Th1 differentition nd induced Th2 cells nd Treg in recipient mice with estlished EAE. Splenic CD4 + T cells were isolted 12 dys fter doptive trnsfer of monocytes nd evluted for () intrcellulr expression of FoxP3 nd IL-4 (indicted is the percentge of FoxP3 + Treg or IL-4 producing Th2 cells mong ll CD4 + T cells (pregted)) or () secretion of IFN-γ or IL-4 upon restimultion with MOG p3-.
5 IFN-γ (pg/m) 1,8 1,6 1,4 1, 1, Intct MBP Antigen concentrtion (µg/ml) IL-4 (pg/ml)l Intct MBP Antigen concentrtion (µg/ml) Prolifertion 2 1 Intct MBP Antigen concentrtion (µg/ml) Supplementry Figure. Long-term -rective T cell lines secrete low levels of cytokines in response to intct MBP. -rective T cell lines were generted from (PL/J x SJL/J)F1 mice immunized with in IFA nd tested for prolifertion nd cytokine secretion in response to intct MBP nd in prllel with ech stimultion. During the first cycles of in vitro stimultion no prolifertion or cytokine secretion could e detected in response to intct MBP or (not shown). Strting from the 6 th stimultion cycle, low levels of IFN-γ nd IL-4 were secreted in response to intct MBP. Cross-rectivity mesured y prolifertion ws not detected.
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