Supporting Architectural Composition at Runtime with π -ADL.NET
Supporting Information...2012/10/11 · Supporting Information Mandl et al. 10.1073/pnas.1211717109...
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Supporting Information Mandl et al. 10.1073/pnas.1211717109 SI Materials and Methods Mice. C57BL/6, splenectomised B6, Thy1.1 + B6, and C57BL/10 mice were purchased from Jackson Laboratories. C57BL/10.A and CD45.1 + H2-Aβ −/− (MHCII −/− ) B6 were purchased from Taconic Farms. β 2 M −/− (MHCI −/− ) B6 were purchased either from Jackson Laboratories or from Taconic Farms. CD45.1 + B6, K b D b−/− (MHCI −/− ) B6, Lck-Cre B6, and CD45.1 + B10.A were obtained from the National Institute of Allergy and Infectious Diseases contract colony at Taconic Farms. Flow Cytometry. Anti-Thy1.1 (HIS51 or OX-7), CD4 (RM4-5), CD8 (53-6.7), CD45.1 (A20), CD45.2 (104), CD44 (IM7), CD5 (53-7.3), CD11a (2D7), chemokine (C-X-C motif) receptor 4 (CXCR4) (2B11), and P-selectin glycoprotein ligand-1 (PSGL-1) (2PH1) antibodies were purchased from eBioscience or BD Pharmingen. Anti-CCR7 (4B12) and CD29 (HMβ1-1) antibodies were purchased from BioLegend. UV-live/dead fixable dead cell stain kits were used according to the manufacturer’s protocol. All samples were acquired on an LSRII flow cytometer (Becton Dickinson), and data were analyzed using FlowJo (Tree Star). T-Cell Recirculation Block. To inhibit trafficking of T cells from blood into lymph nodes (LNs) and back into the blood, thus trapping T cells in either blood or LNs, splenectomized C57BL/6 mice were given 100 μg anti-αL and anti-α4 to block LN entry, in addition to 0.5 μg/g FTY720 i.p. to block LN egress, as previously described (1). Transwell Migration Assays. To assess the fraction of cells capable of migrating toward a chemokine (C-C motif) ligand 21 (CCL21) or sphingosine-1-phosphate (S1P) gradient, 4 × 10 6 cells were added to 5-μm transwells (Corning Costar) whose bottom wells contained media alone, CCL21 (R&D Systems) or S1P (Cayman Chemical) and incubated for 3 h at 37 °C. For CCL21 migration assays, cells and chemokines were diluted in 5% (vol/vol) FCS- containing RPMI. For S1P migration assays, cells were rested for 30 min in serum-free RPMI at 37 °C and transwells incubated with 10% (vol/vol) FCS RPMI at 37 °C for 2 h, before washing the bottom wells with serum-free RPMI and adding cells to transwells. All wells were set up in duplicate. The percentage migrated cells was enumerated by running cells on a flow cy- tometer for a defined length of time and comparing acquired cell counts to premigration cell samples run in the same volume. Dendritic Cell Cultures. WT and MHCII KO bone marrow-derived dendritic cells (DCs) were cultured in complete medium RPMI/ 10% (vol/vol) FCS with murine GM-CSF (5 ng/mL) and murine IL-4 (10 ng/mL) (Peprotech) as previously described (2). DCs were harvested and pulsed with LPS (10 ng/mL) at 37 °C for 1 h before fluorescent labeling and s.c. injection into recipient mice for imaging. Two-Photon Imaging and Manual Analyses. After activating WT and MHCII KO DCs with LPS, DCs were differentially labeled with 25 μM Cell Tracker Blue, 5 μM Cell Tracker Orange, or 2.5 μM Carboxyfluorescein succinimidyl ester (CFSE) (all from In- vitrogen), and an equal number (∼1 × 10 6 cells per mouse) of each population were mixed and injected s.c. into the right dorsal footpad of MHCII KO recipient mice. Eighteen to 48 h later, CD4 + and CD8 + T cells were isolated from B6 mice as de- scribed and labeled with either 5 μM SNARF-1 or 2.5 μM CFSE (Invitrogen). Equal numbers (∼1 × 10 7 cells per mouse) of each cell type were then coinjected i.v. into the same recipients. Two to 24 h after T cell injection, recipient mice were anesthetized with nebulized isoflurane (2% induction, 1% maintenance) in 30% O2/70% air, with body temperatures maintained at 37 °C via a temperature-controlled environmental chamber and heating pads. The right popliteal LN was then surgically exposed and imaged through a 20× water immersion lens (N.A. 1.0) using a Leica SP5 on a DM6000 stage fitted with a 16W IR laser (Chameleon; Coherent) tuned to 800 nm. Imaging planes (760 × 760 μm) collected at 5-μm z-intervals were repeated at 15- to 30-s intervals for up to 2 h to yield xyzt data sets for processing and analysis by Imaris (BitPlane). The processed imaging series were converted to tiff series and subjected to either computational or manual analyses of contact frequency and duration. For manual analyses, individual DC–T-cell contacts were inspected using Imaris on xy images through z stacks. The number of sequential images in which a particular DC–T-cell contact was visually confirmed was multiplied by the fixed time interval between xyz imaging stacks to derive the duration of contact [contact dura- tion = (frame number of continual DC:T-cell contact) × (time interval between xyz imaging frames)]. Automated Image Analyses. To be certain that there were no biases inadvertently introduced by our manual analysis of the imaging data, we also developed image analysis software to track the length of cell–cell interactions in an automated manner and used this software to analyze the same data sets. Image segmentation and analysis of imaging data were based on the image analysis software 2PISA that the authors developed previously (3) and that was extended by an automated cell-tracking feature. After the initial (static) interaction analysis was performed according to the protocol (3), the segmentation result was reprocessed using the novel cell-tracking module based on cell proximity and velocity information. This allows for a fully automated tracking of interacting pairs of cells and computation of dynamic in- terface properties and interaction statistics. Modeling T-Cell Transit Dynamics. Because for both CD4 + and CD8 + T-cell populations the mean and SD of DC contact time were much smaller than mean transit time, a model of naïve T- cell transit through LN consistent with the exponential distri- bution of LN residence times is that T cells have a constant and small egress probability, q, after a DC encounter. In this model, a good approximation to the expected number of contacts, C, is simply C = 1/q = 1/rτ, where r is the LN egress rate (h −1 ) and τ is the mean DC contact time (h). In other words, C= (mean transit time)/(mean duration of a single DC–T-cell contact). This is an upper bound on C because the calculation assumes that the time spent between DC is much smaller than the time spent in contact with DC. If this assumption does not hold, C = 1/[r(τ + β)], where β is the mean time spent moving from one DC to another. We make the simplest (most parsimonious) assumption re- garding egress, that the probability of a T cell leaving an LN after any given DC contact is a constant q, independent of the length of time that T cell has been resident in the LN, and independent of the total number of T cells in the LN. The probability of egress after exactly n contacts is then geometrically distributed as q(1 − q) n−1 , and the mean number of contacts a cell has while transiting is 1/q. The mean number of contacts is large—on the order of hundreds (Fig. 5A)—and so q is much smaller than 1. In this case the number of contacts (n) is approximately exponen- tially distributed, with P(n) ∼ exp(−nq). If interactions with DC Mandl et al. www.pnas.org/cgi/content/short/1211717109 1 of 7
Transcript of Supporting Information...2012/10/11 · Supporting Information Mandl et al. 10.1073/pnas.1211717109...
Supporting InformationMandl et al. 10.1073/pnas.1211717109SI Materials and MethodsMice. C57BL/6, splenectomised B6, Thy1.1+ B6, and C57BL/10mice were purchased from Jackson Laboratories. C57BL/10.Aand CD45.1+ H2-Aβ−/− (MHCII−/−) B6 were purchased fromTaconic Farms. β2M−/− (MHCI−/−) B6 were purchased eitherfrom Jackson Laboratories or from Taconic Farms. CD45.1+ B6,KbDb−/− (MHCI−/−) B6, Lck-Cre B6, and CD45.1+ B10.A wereobtained from the National Institute of Allergy and InfectiousDiseases contract colony at Taconic Farms.
Flow Cytometry. Anti-Thy1.1 (HIS51 or OX-7), CD4 (RM4-5),CD8 (53-6.7), CD45.1 (A20), CD45.2 (104), CD44 (IM7), CD5(53-7.3), CD11a (2D7), chemokine (C-X-C motif) receptor 4(CXCR4) (2B11), and P-selectin glycoprotein ligand-1 (PSGL-1)(2PH1) antibodies were purchased from eBioscience or BDPharmingen. Anti-CCR7 (4B12) and CD29 (HMβ1-1) antibodieswere purchased from BioLegend. UV-live/dead fixable dead cellstain kits were used according to the manufacturer’s protocol.All samples were acquired on an LSRII flow cytometer (BectonDickinson), and data were analyzed using FlowJo (Tree Star).
T-Cell Recirculation Block. To inhibit trafficking of T cells fromblood into lymph nodes (LNs) and back into the blood, thustrapping T cells in either blood or LNs, splenectomized C57BL/6mice were given 100 μg anti-αL and anti-α4 to block LN entry, inaddition to 0.5 μg/g FTY720 i.p. to block LN egress, as previouslydescribed (1).
Transwell Migration Assays. To assess the fraction of cells capableof migrating toward a chemokine (C-C motif) ligand 21 (CCL21)or sphingosine-1-phosphate (S1P) gradient, 4 × 106 cells wereadded to 5-μm transwells (Corning Costar) whose bottom wellscontained media alone, CCL21 (R&D Systems) or S1P (CaymanChemical) and incubated for 3 h at 37 °C. For CCL21 migrationassays, cells and chemokines were diluted in 5% (vol/vol) FCS-containing RPMI. For S1P migration assays, cells were rested for30 min in serum-free RPMI at 37 °C and transwells incubatedwith 10% (vol/vol) FCS RPMI at 37 °C for 2 h, before washingthe bottom wells with serum-free RPMI and adding cells totranswells. All wells were set up in duplicate. The percentagemigrated cells was enumerated by running cells on a flow cy-tometer for a defined length of time and comparing acquired cellcounts to premigration cell samples run in the same volume.
Dendritic Cell Cultures. WT and MHCII KO bone marrow-deriveddendritic cells (DCs) were cultured in complete medium RPMI/10% (vol/vol) FCS with murine GM-CSF (5 ng/mL) and murineIL-4 (10 ng/mL) (Peprotech) as previously described (2). DCswere harvested and pulsed with LPS (10 ng/mL) at 37 °C for1 h before fluorescent labeling and s.c. injection into recipientmice for imaging.
Two-Photon Imaging and Manual Analyses.After activating WT andMHCII KO DCs with LPS, DCs were differentially labeled with25 μM Cell Tracker Blue, 5 μM Cell Tracker Orange, or 2.5 μMCarboxyfluorescein succinimidyl ester (CFSE) (all from In-vitrogen), and an equal number (∼1 × 106 cells per mouse) ofeach population were mixed and injected s.c. into the right dorsalfootpad of MHCII KO recipient mice. Eighteen to 48 h later,CD4+ and CD8+ T cells were isolated from B6 mice as de-scribed and labeled with either 5 μM SNARF-1 or 2.5 μM CFSE(Invitrogen). Equal numbers (∼1 × 107 cells per mouse) of each
cell type were then coinjected i.v. into the same recipients. Twoto 24 h after T cell injection, recipient mice were anesthetizedwith nebulized isoflurane (2% induction, 1% maintenance) in30% O2/70% air, with body temperatures maintained at 37 °C viaa temperature-controlled environmental chamber and heatingpads. The right popliteal LN was then surgically exposed andimaged through a 20× water immersion lens (N.A. 1.0) usinga Leica SP5 on a DM6000 stage fitted with a 16W IR laser(Chameleon; Coherent) tuned to 800 nm. Imaging planes (760 ×760 μm) collected at 5-μm z-intervals were repeated at 15- to 30-sintervals for up to 2 h to yield xyzt data sets for processing andanalysis by Imaris (BitPlane). The processed imaging series wereconverted to tiff series and subjected to either computational ormanual analyses of contact frequency and duration. For manualanalyses, individual DC–T-cell contacts were inspected usingImaris on xy images through z stacks. The number of sequentialimages in which a particular DC–T-cell contact was visuallyconfirmed was multiplied by the fixed time interval between xyzimaging stacks to derive the duration of contact [contact dura-tion = (frame number of continual DC:T-cell contact) × (timeinterval between xyz imaging frames)].
Automated Image Analyses.To be certain that there were no biasesinadvertently introduced by our manual analysis of the imagingdata, we also developed image analysis software to track thelength of cell–cell interactions in an automated manner and usedthis software to analyze the same data sets. Image segmentationand analysis of imaging data were based on the image analysissoftware 2PISA that the authors developed previously (3) andthat was extended by an automated cell-tracking feature. Afterthe initial (static) interaction analysis was performed accordingto the protocol (3), the segmentation result was reprocessedusing the novel cell-tracking module based on cell proximity andvelocity information. This allows for a fully automated trackingof interacting pairs of cells and computation of dynamic in-terface properties and interaction statistics.
Modeling T-Cell Transit Dynamics. Because for both CD4+ andCD8+ T-cell populations the mean and SD of DC contact timewere much smaller than mean transit time, a model of naïve T-cell transit through LN consistent with the exponential distri-bution of LN residence times is that T cells have a constant andsmall egress probability, q, after a DC encounter. In this model,a good approximation to the expected number of contacts, C, issimply C = 1/q = 1/rτ, where r is the LN egress rate (h−1) and τ isthe mean DC contact time (h). In other words, C = (mean transittime)/(mean duration of a single DC–T-cell contact). This is anupper bound on C because the calculation assumes that the timespent between DC is much smaller than the time spent in contactwith DC. If this assumption does not hold, C = 1/[r(τ + β)], whereβ is the mean time spent moving from one DC to another.We make the simplest (most parsimonious) assumption re-
garding egress, that the probability of a T cell leaving an LN afterany given DC contact is a constant q, independent of the lengthof time that T cell has been resident in the LN, and independentof the total number of T cells in the LN. The probability ofegress after exactly n contacts is then geometrically distributed asq(1 − q)n−1, and the mean number of contacts a cell has whiletransiting is 1/q. The mean number of contacts is large—on theorder of hundreds (Fig. 5A)—and so q is much smaller than 1. Inthis case the number of contacts (n) is approximately exponen-tially distributed, with P(n) ∼ exp(−nq). If interactions with DC
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have mean duration τ, and the time between DC contacts ismuch smaller than τ, then the probability of a cell remaining inthe lymph node a time T after entry is P(T = nt) = P(n) ∼ exp(−Tq/τ). (If cells spend a mean time β between DC contacts, τ isreplaced with τ + β). This model predicts exponentially distrib-uted LN residence times, in agreement with our data, andP(T) = exp(−Tq/τ), which is by definition: exp(−r T), where r isthe egress rate. We measure both r and τ, so the probability ofegress per DC interaction is q = τ.r, or the mean contact dura-tion (τ) divided by the mean transit time (1/r) (Fig. 5B).It has been described that T cells reexpress surface S1PR1 to
maximum levels only by 2–4 h after LN entry (4). The need toreacquire S1P sensitivity may impose a minimum LN dwell time.A more refined model of transit might divide populations intocells that are not yet competent to egress, and cells that egresswith constant probability q per DC encounter. By using thesimplest single-population model, we will slightly underestimateq and thus slightly overestimate the expected number of contactseach T cell makes with DCs. Our estimate of C is therefore anupper bound. However, even if the mean time of recently en-
tered cells in which they are unable to egress is as long as 4 h, thisdoes not substantially alter our conclusions.
Confocal Analysis of CD4+ and CD8+ T-Cell Localization. Total CD4+
and CD8+ T cells (∼90% naïve, CD44lo) were purified by negativeselection using MACS (Miltenyi Biotec) and differentially dye la-beled with 3 μM CellTracker Red (CMTPX) and 100 μM Cell-Tracker Blue (CMF2HC), respectively, in PBS for 15 min at 37 °C.A total of 1.3 × 106 labeled CD4+ and CD8+ T cells were mixed1:1 and injected i.v. into WT recipients. Inguinal LNs were har-vested 24 h later, fixed, and stained as described with rabbit anti–LYVE-1 and rat anti–ERTR-7 (Novus Biologicals), then stainedwith anti-rabbit IgG Alexa Fluor 647 and anti-rat IgG Alexa Fluor488 (Invitrogen) (5). Images were acquired using a Zeiss 710confocal microscope with a motorized stage for tiled imaging.
Statistical Analyses. We analyzed data using GraphPad Prism orR Project version 2.10.1 (R Development Core Team, 2011).A criterion level of P < 0.05 (two-tailed) was used in all statisticalanalyses.
1. Matloubian M, et al. (2004) Lymphocyte egress from thymus and peripheral lymphoidorgans is dependent on S1P receptor 1. Nature 427(6972):355–360.
2. Castellino F, et al. (2006) Chemokines enhance immunity by guiding naive CD8+ T cellsto sites of CD4+ T cell-dendritic cell interaction. Nature 440(7086):890–895.
3. Klauschen F, Qi H, Egen JG, Germain RN, Meier-Schellersheim M (2009) Computationalreconstruction of cell and tissue surfaces for modeling and data analysis. Nat Protoc 4(7):1006–1012.
4. Lo CG, Xu Y, Proia RL, Cyster JG (2005) Cyclical modulation of sphingosine-1-phosphatereceptor 1 surface expression during lymphocyte recirculation and relationship tolymphoid organ transit. J Exp Med 201(2):291–301.
5. Gerner MY, Kastenmuller W, Ifrim I, Kabat J, Germain RN (2012) Histo-cytometry:A method for highly multiplex quantitative tissue imaging analysis applied to dendriticcell subset microanatomy in lymph nodes. Immunity 37(2):364–376.
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Fig. S1. Interactions of T cells with self-pMHC are ongoing and take place in secondary lymphoid organs. (A) Flow cytometric analysis of CD5 surface ex-pression on CD4+ and CD8+ T cells in inguinal and brachial LNs 2 and 20 h after adoptive transfer into MHCI−/−, MHCII−/−, or WT recipients. RFI, relativefluorescent intensity, calculated as percentage of 0 h CD5 mean expression. (B) Expression of CD5 on CD4+ and CD8+ T cells in LNs or blood isolated fromsplenectomized mice 24 h after injection of PBS or anti-α4/αL and FTY720 to block both entry and egress from LNs. RFI calculated as percentage of CD4+ or CD8+
T cells in control group for LN or blood. ns, not significant; ***P < 0.0001; **P < 0.005; *P < 0.05. Data are pooled from two independent experiments each withthree mice per group. Circles denote individual animals, lines represent means.
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Fig. S2. CD4+ T cells have faster LN egress rates than CD8+ T cells. LN transit times of T cells were estimated after treatment of mice with integrin-blockingantibodies to prevent further entry of lymphocytes. (A) Egress rate estimates for transferred naïve CD4+ and CD8+ T cells in peripheral and mesenteric LNs. (B)Egress rate and mean dwell time estimates for endogenous naïve CD4+ and CD8+ T cells. Data points denote egress estimates from one experiment (as shown inFig. 2C). Lines represent calculated means. Closed symbols, iLN; open symbols, bLN; ●, B6 mice; ▲, B10.A mice; ▼, B10 mice; egress estimates were obtainedfrom 8 to 19 independent experiments.
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Fig. S3. Similar CD4+ and CD8+ T-cell localization and dynamic behavior in the LN. (A) A total of 1.3 × 106 CMTPX-labeled CD4+ T cells and CMF2HC-labeledCD8+ T cells were transferred i.v. into WT recipients. Twenty-four hours later, an inguinal LN was harvested, fixed, cut into 20-μm sections, and stained withERTR-7 and LYVE-1. (Right) Image is magnified from boxed region at Left. (B) A total of 4 × 106 CMFDA+ CD8+ T cells and CMTPX+ CD4+ T cells were transferredand the inguinal LN imaged using two-proton (2P) intravital microscopy 18 h later. (Left) Snapshot of T cells moving (Movie S2); (Right) Individual CD4+ andCD8+ T-cell tracks during a period of 44 min. (Scale bar, 40 μm.) (C) Quantification of mean and maximum velocity, fraction of cells arrested, and total dis-placement for CD4+ and CD8+ T cells. Circles represent means calculated from one 2P microscopy movie. Data are shown from nine independent movies. (D)Responsiveness of naïve CD4+ and CD8+ T cells to indicated concentrations of S1P (in nM) as assessed by transwell migration assays. A representative examplefrom one mouse is shown (assays were performed in duplicate).
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Fig. S4. Expression of LN homing and adhesion receptors on naïve CD4+ and CD8+ T cells. (A) Surface expression of CD11a, CXCR4, CD29, PSGL-1, and CD62L onnaïve CD4+ and CD8+ T cells. Example fluorescent intensity histograms from representative mice are shown. (B) CCR7 surface expression on a cohort ofadoptively transferred naïve CD4+ and CD8+ T cells at different time points after anti-α4/αL administration to block LN entry measured in the inguinal LN. (Left)Geometric mean fluorescent intensity (MFI); (Right) Relative fluorescent intensity (RFI) normalized to the mean level in transferred CD4+ and CD8+ T cells at 0 h.Data are from four to six mice per time point.
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Fig. S5. Faster LN egress rates of CD4+ T cells in the absence of MHCII but not of CD8+ T cells in the absence of MHCI. LN egress rate estimates for transferrednaïve CD4+ and CD8+ T cells in WT and MHCII−/− (a), or WT and MHCI−/− (b). Circles denote transit time estimates from one experiment. Lines represent groupmeans obtained from five to seven independent experiments. Closed symbols, iLN; open symbols, bLN. In (b):●, β2M−/−- or WT-matched recipients; ▲, KbDb−/−- orWT-matched recipients.
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Fig. S6. DC scanning dynamics of CD4+ T cells in LNs is altered when DCs do not express MHCII. Estimates of the mean number of WT or MHCII KO DCs scannedby an individual CD4+ T cell during steady-state LN transit. Means (points) with bootstrapped joint 95% confidence intervals (shaded areas) are shown for DC–T-cell contacts obtained from both the manual and automated estimates of the distributions of DC contact times.
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Movie S1. Contacts between CD4+ T cells and MHCII+/+ or MHCII−/− DCs in the lymph node in the absence of antigen. CD4+ T cells (red) interacting with WT(blue) or MHCII−/− (green) DCs in vivo. (Scale bar, 20 μm.) Time is shown in min:s.
Movie S1
Movie S2. Dynamic behavior of naïve CD4+ and CD8+ T cells in the lymph node. CD4+ T cells (red) and CD8+ T cells (green) in the inguinal LN (from Fig. S2B).(Scale bar, 50 μm.) Time is shown in min:s.
Movie S2
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