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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Science. Author manuscript; available in PMC 2014 March 4.
Published in final edited form as:
PMCID: PMC3941467
NIHMSID: NIHMS532623

T Follicular Helper Cell Dynamics in Germinal Centers

Abstract

T follicular helper cells (TFH) are a specialized subset of effector T cells that provide help to and thereby select high-affinity B cells in germinal centers (GCs). To examine the dynamic behavior of TFH cells in GCs in mice we combined two-photon microscopy and optical highlighting using a photoactivatable fluorescent reporter. Unlike GC B cells, which are clonally restricted, TFH distributed among all GCs in lymph nodes and continually emigrated into the follicle and neighboring GCs. Moreover, newly activated TFH cells invaded pre-existing GCs, where they contributed to B cell selection and plasmablast differentiation. Our data suggest that dynamic exchange of TFH between GCs ensures maximal diversification of T cell help and their ability to enter ongoing GCs accommodates antigenic variation during the immune response.

T cells play a pivotal role in affinity maturation by selecting B cells to enter the GC, regulating GC positive selection, and directing B cell differentiation to plasma cells and memory B cells (16). These events are orchestrated by a specialized population of GC-resident T follicular helper (GC-TFH) cells that develop in concert with GC B cells (713). TFH cells express chemokine receptor CXCR5, CD28 family members ICOS and PD-1, transcription factor Bcl-6, and cytokines interleukin (IL)-4 and IL-21, many of which are required for GC-B cell survival and differentiation (7). Less is known about the dynamic properties of TFH cells and how these might affect GC B cell selection.

To document the kinetics of T cell expansion and localization during the GC reaction, we examined whole-mounted lymph nodes using two-photon laser scanning microscopy (TPSLM) (14, 15). In agreement with previous reports (16), large numbers of antigen-specific T cells were found in the T cell zone 3 days after immunization, which then spread throughout the lymph node, including B cell follicles and nascent GCs by day 5 (Fig. 1A). T cells only began to concentrate in GCs after 8–11 days (Fig. 1A) coinciding with the peak of T cell help to GC B cells (5). Thus, unlike B cell clones, which are thought to expand in a confined micro-anatomical region to produce pauciclonal GCs (17, 18), responding T cells are initially evenly distributed throughout the entire lymph node, and accumulate in GCs only after these have coalesced.

Figure 1
Expansion kinetics and anatomical distribution of TFH during germinal center formation

TFH and GC-TFH are commonly defined based on functional properties and the expression of cell surface markers (8, 19), rather than on anatomical localization. To verify the correspondence between surface phenotype and microanatomical location, we labeled cells within spatially restricted areas using photoactivatable GFP (PAGFP) (3). Flow-cytometric analysis of photoactivated OT-II T cells (Fig. S1A–C) showed that CXCR5 and PD-1 expression were highest among cells physically inside the GC and lowest among T cells in the paracortex, whereas T cells in the follicle outside the GC showed intermediate levels of expression of these molecules (Fig. 1B–C and fig. S1D). In contrast, ICOS expression was comparable in all three locations (Fig. 1B–C and fig. S1D). Similar results were obtained by photoactivation of endogenous polyclonal T cells (Fig. S2). Thus, although CXCR5 and PD-1 expression distinguish between GC-TFH and paracortical T cells, they cannot be used to definitively distinguish follicular TFH from either of these populations.

To determine whether GC-TFH cells are clonally restricted within individual GCs like their B cell counterparts (17, 18), we immunized mice that had received a mixture of OT-II T cells expressing one of three fluorescent proteins and visualized GCs 10 days after immunization (Fig. 2A). As suggested by our initial observations, the proportion of T cells of each color within individual GCs was constant across all GCs in the same lymph node (LN), indicating that T cells are not clonally restricted within GCs (Fig. 2A–B and fig. S3A).

Figure 2
GC-TFH cells in individual germinal centers are not clonally restricted

This pattern of T cell distribution might result from initial colonization by multiple clones, and/or from T cell exchange between GCs. To examine the first of these possibilities, we analyzed early GCs. GC-TFH distribution was homogeneous across GCs even at the earliest observable time point, suggesting that individual GCs are indeed colonized by several distinct T cell clones (Fig. 2C and Fig. S3B–C). We confirmed this by transferring a much smaller number of T cells (3×104 total T cells per mouse) at a 19:1 ratio of GFP:DsRed cells (Fig. S4), as well as by reconstituting TCRβ-deficient mice with mixtures of genetically-labeled polyclonal CD4+ T cells (Fig. S5). In both cases, T cells were evenly distributed across GCs after immunization (Fig. S4 and S5). Thus, we find no evidence that GC-TFH are clonally restricted to individual GCs; instead, GCs appear to be colonized by a heterogeneous population of T cells representing all responding T cell clones.

To determine whether GC-TFH cells can also exchange between GCs by migration, we imaged these cells by TPLSM. Short-term intravital movies showed that GC-TFH occasionally migrated out of GCs (Movie S1). Whether these emigrants are in fact leaving the GC rather than making a transient foray into the follicle could not be determined due to constraints in the duration of conventional intravital imaging movies (20). To observe GC-TFH migration for longer periods, we therefore photoactivated PAGFP-OT-II TFH within single GCs (Fig. S6A). Photoactivation does not alter the motility of PAGFP+ OT-II cells compared to controls (Movie S2), and, because GC-TFH are in G1 phase (Fig. S6B), activated PAGFP is not diluted by cell division, allowing for long-term cell tracking. Immediately after photoactivation, PAGFP+ OT-II cells were restricted to the targeted region (Fig. S6C). In contrast, ~20 hours after activation, 32% of photoactivated GC-TFH cells were found outside the original GC (Fig. 3A–B). Emigrating cells were found almost entirely within neighboring follicles and other GCs, and only very rarely (1 cell out of 487 analyzed) in the T cell zone (Fig. 3B and S6D, Movie S3). Consistent with their near absence from the T cell zone, we could detect only a very small number of photoactivated cells in the blood or pooled distal lymphoid organs of mice examined 36 hours after photoactivation (1–4 cells per mouse, 7 mice in three independent experiments, Fig. S6E). Transfer of PAGFP+ OT-II T cells before OVA priming did not substantially alter GC size or the ratio of T to B cells in the GC (Fig. S6F–G); thus, GC-TFH cell emigration to the follicle and neighboring GCs cannot be attributed to a non-physiological density of TFH cells.

Figure 3
GC-TFH exchange between neighboring germinal centers

To determine the phenotype of emigrant GC-TFH cells, we microdissected lymph nodes into two fragments, one of which contained the original photoactivated GC (Fig. 3C and S7A–B). Immediately after activation (0 hours), the lymph node fragment contralateral to the activated GC contain no photoactivated cells (Fig. S7C). In contrast, after 24 hours, photoactivated cells corresponding to GC-TFH that had emigrated from the original GC could also be found in the non-photoactivated half of the lymph node (Fig. 3C). In agreement with the lymph node scans (Fig. 3A), emigrant GC-TFH cells remained phenotypically similar to authentic GC-TFH, although expression of PD-1 and CXCR5 was slightly decreased (Fig. 3C and fig. S7D–E). We conclude that GC-TFH emigrate and redistribute throughout the follicles and to neighboring GCs, but only rarely leave the follicle to enter circulation in the timeframe analyzed.

The exchange of T cells between GCs suggested a hitherto unappreciated dynamic equilibrium between T cells in the follicles and in different GCs. We reasoned that, if GCs are in fact open to TFH exchange, then newly-activated T cells may also be able to join an ongoing GC reaction. To examine this possibility, we transferred GFP-expressing OT-II cells into WT recipients that were then primed with OVA to induce OT-II expansion. These mice then received CFP-expressing B1-8hi cells, and were immunized with NP-CGG in alum to produce GCs in which endogenous CGG specific TFH provide help for B1-8hi-CFP. The immunized mice were then boosted subcutaneously with soluble NP-OVA to trigger invasion of these GCs by OT-II cells (Fig. S8A). Before the boost, small numbers of GFP-OT-II cells were found scattered throughout the T cell zone, occasionally entering existing GCs for brief periods (Fig. 4A and fig. S8B; Movie S4). One day after immunization, GFP+ cells could be seen proliferating in foci of T and B cells at the follicle/T zone border (Fig. 4A, Movie S5). By day 3 after boost, GFP-OT-II cells were found throughout the lymph node follicles and within pre-existing GCs (Fig. 4A, Movie S6). All GCs evaluated contained invading GFP-OT-II cells, indicating that these cells were indeed entering existing GCs rather than producing GCs de novo. Accordingly, control mice boosted with NP-OVA without pre-immunization with NP-CGG had no GCs at this time point (Fig. S8C). Over time, GFP-OT-II cells began to accumulate progressively within GCs, so that by day 11 after boost, the newly activated T cells were mostly concentrated inside these structures (Fig. 4A and S8B; Movie S6), interacting actively with GC B cells (Fig. 4B; Movie S7). Therefore, ongoing GCs are open to invasion by newly-activated TFH.

Figure 4
Newly-activated TFH can enter ongoing germinal center reactions

To determine whether invading TFH contribute to the GC reaction, we measured their ability to provide help to B cells selectively expressing high levels of cognate peptide-MHC by targeting OVA to GC B cells using antibodies to DEC-205 (encoded by Ly75) (3, 4). OVA-specific GC-TFH cells were induced to invade a CGG-specific GC containing a mixture of 85% Ly75−/− CD45.1/2 and 15% Ly75+/+ CD45.1/1 B1-8hi B cells, and cognate MHC-peptide expression was artificially increased on the Ly75+/+ B cells by injection of anti-DEC205-OVA antibodies (Fig. S8D). Whereas PBS or control antibody had no effect, anti-DEC205-OVA injection resulted in selective and dramatic expansion of the Ly75+/+ but not Ly75−/− GC B cells, and a nearly 10-fold increase in plasmablast frequency (Fig. 4C–D). We conclude that T cells that invade ongoing GCs actively participate as helper T cells and positively select B cells expressing high levels of MHC-peptide.

Boosting ongoing NP-CGG-specific GCs with NP-OVA (as detailed in Fig. 4A) resulted in a five-fold increase in the proportion of GC B cells five days after boost (from 0.9% to 5.1%), while the proportion of transferred to endogenous B cells remained constant (Fig. 4E). Thus, GC invasion by newly-activated TFH cells can augment ongoing GC reactions.

Physical restriction of responding B cells to a single GC minimizes competition between B cells in different GCs, thereby preventing the immune response from converging on a single dominant clone (21). In contrast, free movement of TFH cells between GCs may be advantageous, in that it ensures diversified and robust support for B cell clonal expansion and affinity maturation (2224). Thus, diversity in the antibody response appears to be favored by restricting B cell clones to single GCs while exposing them to a multiplicity of different T cell clones that transit between different GCs.

In addition to polyclonal colonization and dynamic exchange between GCs, newly-activated T cells can also enter ongoing GCs. This raises the possibility that T cells that are activated late in the immune response can join and potentially prolong the GC reaction, a feature that may be a significant advantage in responding to chronic infections and/or pathogens that diversify their antigenic epitopes during infection, such as HIV.

Materials and Methods

Mice

Wild-type C57BL6, B6.SJL (CD45.1+), Tcrb−/−, and transgenic mice ubiquitously expressing CFP, DsRed, or GFP were purchased from Jackson Laboratories. Fluorescent strains were bred to OT-II TCR-transgenic (Y chromosome) mice in our laboratories. PAGFP-transgenic (3), B1-8hi knock-in (25) and Ly75−/− (DEC205-deficient) mice (26) were generated and maintained in our laboratories. MHC-II-GFP mice (27) were generated and maintained at the Whitehead Institute animal facility. Mice ubiquitously expressing tdTomato were generated by crossing ROSA26-Lox-Stop-Lox-tdTomato (Ai9) mice (28) (Jackson) to a strain expressing Cre recombinase in the early embryo (EIIA-Cre), which was subsequently bred out.

Cell transfer

Spleens and lymph nodes were forced through a 40 or 70 μm mesh into complete RPMI media (Gibco) with 10% serum. Resting B cells and CD4+ T cells were obtained by magnetic cell sorting (MACS) by using CD43 beads and the CD4 cell negative isolation kit (Miltenyi) respectively, according to the manufacturer’s instructions.

Immunizations

To generate primary GCs, 5–10-week-old C57BL/6 recipient mice were immunized subcutaneously with 10 μg/footpad of NP-OVA (NP11-OVA, NP14-OVA or NP18-OVA, used interchangeably), NP28-KLH or NP23-CGG (Biosearch Technologies) precipitated in alum (Imject® Alum, Thermo Scientific) at a 2:1 antigen(PBS):alum ratio in a 25 μl volume. For secondary GC generation (photoactivation experiments) and for T cell invasion experiments, mice were primed, i.p., with 50 μg of OVA (Grade V, Sigma) precipitated in alum at a 2:1 antigen(PBS):alum ratio in a 100 μl volume. 2–6 weeks later, mice were boosted with 25 μg of NP14- or NP18-OVA (Biosearch Technologies) in hind footpads, and popliteal LN were either imaged or harvested for flow cytometry. αDEC-OVA and αDEC-CS were produced as chimeric antibodies in 293T cells as described (29), and 3 μg of recombinant antibody were injected into the footpads of mice 6 days after induction of T cell invasion.

Intravital imaging

Intravital imaging was conducted essentially as described previously (3, 4). Mice were anaesthetized with 100 mg ketamine, 15 mg xylazine and 2.5 mg acepromazine per kg body weight and were kept anaesthetized by inhalation of 1.25% isofluorane in 100% oxygen. Hind legs were shaved using a double-edged razor blade, mice were restrained on a stage warmer at 37 °C (BioTherm Micro S37; Biogenics) and an incision was made behind the knee of one hind leg. The popliteal LN was exposed and restrained using a metal strap, and the mouse was placed under the microscope objective, fitted with an objective heater set at 40 °C. For follicular dendritic cell labeling, red fluorescent protein tdTomato and yellow fluorescent protein YPet were produced in E. coli and conjugated to NP as described (3), and mice were injected subcutaneously with 1 μg and 5 μg per footpad of NP-tdTomato and NP-YPet, respectively, one day prior to imaging. For photoactivation experiments, lymph nodes containing PAGFP cells were photoactivated by scanning with a femtosecond-pulsed multiphoton laser tuned to 820–830 nm wavelength, and then imaged at 940 nm wavelength, as described in reference 3. For intravital experiments, cell motility was monitored immediately after photoactivation and at later time points as a measure of viability. For long-term photoactivation experiments, the incision was closed after photoactivation by suturing, and mice were allowed to recover for 17–20 h. After this period, the photoactivated lymph node was explanted and imaged as described previously (15). The half-life of photoactivatable GFP in vivo in non-dividing cells is roughly 30 hours (3). For analysis of TFH markers or cell cycle stage by flow cytometry, we photoactivated PAGFP+ cells inside GCs (labeled with CFP+ B1-8hi cells or NP-tdTomato, respectively), in the T cell zone (labeled by injection of DsRed+ or tdTomato+ naïve T cells), or in B cell follicles (between GCs and T cell zone) in explanted LN. All imaging experiments were carried out using Olympus BX61 upright microscopes (Olympus 25× 1.05 NA Plan objective), fitted with either a Coherent Chameleon Vision II laser (Rockefeller University Bio-Imaging Resource Center) or a SpectraPhysics MaiTai DeepSee laser (Victora lab, Whitehead Institute).

Image acquisition and analysis

Images were acquired using 920–940 nm excitation and the following filters: a first pair of overlapping CFP (460–510) and GFP (495–540) filters, separated by a 505 dichroic mirror, which allowed maximal signal detection for GFP, PAGFP, CFP, and YPet; 2nd harmonics (collagen) were also detected in the CFP filter at 930–40 nm excitation; and a third filter (575–630) for the red signal (tdTomato, DsRed). For whole-mounted LN reconstructions, we acquired tiles of 250 μm-deep Z-stacks (corresponding to the cortical half of the LN) with 5 or 10 μm Z resolution, and an X-Y resolution of 320 × 320 or 512 × 512 pixels per tile (1.325 μm per pixel) (15). GFP OT-II cell density in whole-LN scans was quantified using Imaris 7.3.1 software (Bitplane AG). GC and LN boundaries were defined with the Surfaces feature, and GFP+ cells were counted using the Spots automatic detection feature. T cell density ratio between GCs and the remainder of the LN was calculated as (GC T cells/GC volume)/[(total T cells – GC T cells)/(LN volume – GC volume)]. The positions of photoactivated cells and of CFP/GFP/DsRed T cells in multiple color experiments were determined manually, slice by slice, by combining the Spots and Ortho-slicer tools in Imaris. Photoacitvated cells detected outside of the originally photoactivated area (defined as the GC with the highest density of photoactivated cells) either in the follicles or neighboring GCs were counted as emigrating T helper cells. Movies were acquired as 40 μm Z-stacks with 5 μm Z resolution and 512 × 512 X-Y resolution (0.4 to 0.8 μm/pixel, depending on magnification). Time resolution was ~30 s per frame. Movies are presented at approximately 300x real time.

Flow cytometry

Lymph nodes were forced through a 40 or 70 μm mesh into complete RPMI with 10% fetal calf serum. Single-cell suspensions thus obtained were pretreated for 5′ with 1 μg/ml of anti-CD16/32 (2.4G2, Bio-X-Cell) and then stained for 30 min at 4 °C in PBS supplemented with 2% FCS and 1mM EDTA using the antibodies indicated in Table S1. For cell cycle analysis, cells were fixed and permeabilized using the Cytofix/CytopermTM kit (BD) and stained with 10 μg/ml DAPI (Invitrogen) for 3 minutes. Samples were analyzed on BD Fortessa or BD LSR-I flow cytometers (BD). T cells were gated as CD4+ and CD45.1+, for PAGFP OT-II analysis, or CD4+, TCRβ+, CD44high, CD62Llow for polyclonal PAGFP T cell analysis. GC B cells were gated as live/single, B220+, FAShigh, CD38. Plasmablasts/plasma cells were gated as live/single, B220int, CD138+.

To test whether germinal center T cells egress entirely from lymph nodes, we adoptively transferred OT-II PAGFP+ CD45.1+ CD4 T cells and B1-8hi CD45.1+ B cells into OVA-primed wild type (CD45.2+) mice and boosted the hind footpads subcutaneously with NP-OVA. At day 6 of the GC response, 3–6 GCs in the draining lymph nodes were photoactivated in living mice. After 36 hours, mice were sacrificed and cells from blood, spleen and LN (excluding the photoactivated LN), were labeled with anti-CD45.1-PE followed by anti-PE magnetic beads. CD45.1+ cells from each individual mouse were then enriched by binding to magnetized (MACS) columns. Eluted cells were analyzed by flow cytometry for detection of photoactivated CD45.1+ T cells. T cells were pre-gated as B220, MHC-II, CD11C, CD11b, F4/80, CD3+, CD4+.

Lymph node microdissection

Popliteal lymph nodes were photoactivated in vivo as described above (Intravital imaging), with the exception that 3×106 PAGFP+B1-8hi B cells were transferred 24 hours prior to boost to increase signal intensity in the photoactivated GC. Lymph nodes were explanted either immediately or 24 hours after photoactivation. Explanted lymph nodes were mounted between 2 coverslips and scanned by TPLSM to identify the original photoactivated GC (defined as the GC with the highest density of photoactivated cells). The original GC was photoactivated once more at high laser power to increase visibility, and the LN was cut into two fragments using a #10 disposable scalpel (Miltex) under a Leica MZFLIII fluorescence stereomicroscope fitted with a PlanAPO 1.0 X objective. Both parts of the lymph node were processed and stained in separate, as described above (Flow cytometry).

Supplementary Material

Shulman video 1

Movie S1:

TFH cells can leave the germinal center. Collapsed 4D dataset showing motility of T cells in the light zone of a germinal center. Green, GFP-expressing OT-II cells; static red, NP-tdTomato (FDC labeling); motile red, naïve DsRed-expressing B cells; blue, collagen (2nd harmonics). T cells that leave the germinal center during the imaging season are marked with a circle.

Shulman video 2

Movie S2:

Photoactivation does not affect T cell viability and motility. Collapsed 4D dataset showing motility of both photoactivated PAGFP T cells and CFP-expressing OT-II cells in the light zone of a germinal center. Green, photoactivated OT-II cells; red, NP-tdTomato (FDC labeling); blue, CFP-expressing OT-II cells and collagen (2nd harmonics). Few T cells initially photoactivated in the light zone can be detected outside the germinal center.

Shulman video 3

Movie S3:

250 μm Z-series showing a whole-mounted popliteal lymph node 20 h after photoactivation of T cells in a single GC. Red, NP-tdTomato (FDC labeling); blue, collagen (2nd harmonics). Green spots were placed over photoactivated OT-II cells using Imaris software.

Shulman video 5

Movie S5:

Invasion of memory OVA-specific T cells into CGG-specific GCs initiates at the T-B border. Collapsed 4D datasets showing either CGG-specific germinal center or the T-B border, one day after boosting with NP-OVA. Green, OT-II cells; blue, B1-8hi cells.

Shulman video 6

Movie S6:

Kinetics of T cell invasion into pre-existing GCs. Collapsed 4D datasets showing motility and distribution of OVA-specific OT-II cells during invasion into CGG-specific GCs. Green, OT-II cells; blue, B1-8hi cells; red, NP-tdTomato (FDC labeling). Movies taken on days 3, 5, 8 and 11 after boost with NP-OVA are shown.

Shulman video 7

Movie S7. Invading T cells form contacts with GC B cells. Collapsed 4D datasets showing an invading OT-II cell in simultaneous contact with two GC B cells (arrowheads). Movie was taken 11 days after boosting with NP-OVA. Green, OT-II T cells; blue, B1-8hi cells.

Fig. S1. (A) Diagrammatic representation of the experimental protocol for cell transfer into wild-type mice prior to immunization with NP-OVA in alum and photoactivation (main figure 1B–C). (B) Examples of photoactivation of PAGFP+ OT-II cells in different areas of the LN. Scale bar, 40 mm. (C) Pre-gating strategy used for main figure 1B–C and S1D. (D) Expression of TFH markers on PAGFP+ OT-II cells photoactivated in different LN areas.

Fig. S2. (A) Diagrammatic representation of the experimental protocol for photoactivation of polyclonal T cells. (B) Examples of photoactivation of endogenous PAGFP+ cells in different LN areas. Scale bar, 40 mm. (C) Pre-gating strategy used for analysis of endogenous PAGFP+ T cells. (D–F) Expression of TFH markers on endogenous photoactivated cells, pre-gated on CD4+ CD44high CD62Llow T cells. (F) Each symbol represents one experiment.

Fig. S3. (A) Additional quantitation of experiments presented in main Figure 2A–B.

(B) Additional GC images, as presented in main Figure 2C. Scale bar, 100 μm.

(C) Additional quantitation of experiments presented in main Figure 2C. Numbers above the bars indicate the number of T cells counted in each GC.

Fig. S4. (A) A total of 3 × 104 OT-II cells expressing either GFP or DsRed (at a 19:1 ratio) and CFP-expressing B1-8hi B cells were transferred into WT recipients 1 day before subcutaneous immunization with NP11-OVA in alum. Draining LN were explanted 5 days later and imaged by TPLSM. Images are maximum intensity Z-projections of 15 slices 10 μm apart, and show 3 GCs from the same lymph node. Scale bar, 100 μm.

(B) Quantitation of data as in (A) across multiple LN from different mice in two experiments. Numbers above the bars indicate the number of T cells counted in each GC.

(C–D) as in (A–B), analyzed 10 days after immunization.

Fig. S5. (A) A total of 2 × 107 polyclonal T cells expressing CFP, GFP or DsRed (~6.7 × 106 cells of each color) and 5 × 106 non-fluorescent B1-8hi B cells were transferred into Tcrb−/− host mice. FDCs labeled as in main figure 2A. Whole-lymph node section (50 μm in depth, maximum intensity projection) and additional images of GCs and T cell zone (bottom-right panel), are shown. Scale bars, 50 μm (small panels) and 300 μm (large panel). (B) Quantitation of cell type proportions in multiple GCs in two experiments (as described in main Fig. 2B) are shown. TZ: proportion of T cells of each color in the T-zone of the same LN. Numbers above the bars indicate the number of T cells counted in each GC.

Fig. S6. (A) Diagrammatic representation of the experimental layout for panels (B–F) and main Figure 3. (B) Flow cytometry histograms showing DNA content (DAPI staining) among PAGFP+ OT-II T cells photoactivated inside GCs (top) and whole GC B cells (B220+ FAShigh CD38low) from the same lymph node. Representative of two independent experiments. (C–D) LN prepared as in (A) were scanned either immediately or 20 h after photoactivation. Images show collapsed 40 μm Z-stacks, 10 μm/slice. (C) Absence of photoactivated cells outside original GC immediately after photoactivation. Arrowhead indicates photoactivated GC. (D) Absence of photoactivated cells in T cell zone 20h after photoactivation. Photoactivated cells are circled in green. Dotted line indicates T cell zone (rich in DsRed+ resting B cells). Scale bars, 300 μm. (E) Detection of photoactivated cells 36 h after photoactivation. Mouse blood, spleen, and all non-photoactivated LN harvested and enriched for CD45.1+ (PAGFP+OT-II) cells using magnetic beads. Flow cytometry plots (gated on PAGFP+ OT-II T cells) show presence of photoactivated cells outside the original LN at 36h after photoactivation. Data representative of three independent experiments. (F–G) Ratio of B/T cells in presence or absence of transferred OT-II T cells. Mice prepared as in (A), with or without the initial OT-II transfer. Multiple GC light zones were photoactivated in each LN on day 7 post-boost. (F) Gating strategy for determining ratio of B to T cells within GC light zones. (G) Quantitation of B/T ratio and total GC size in three mice from two independent experiments.

Fig. S7. (A) Schematic representation of the microdissection protocol for experimental and control conditions. Experimental setup as described in main Figure 3. Transfer of B1-8hi B cells prior to boost and 2nd round of photoactivation are required to improve visualization of photoactivated GCs under the dissecting microscope. (B) Fluorescent images of photoactivated lymph nodes prior to and after dissection. Arrowheads indicate photoactivated GCs; dotted line indicates dissection plane. (C) FACS plots showing specificity of dissection (absence of photoactivated cells in non-photoactivated half immediately after photoactivation). Scale bar, 500 μm. (D) Flow cytometry histograms showing the phenotype of emigrant and stationary GC-TFH cells. (F) Summary of data from five mice, 3 independent experiments.

Fig. S8. (A) Diagrammatic representation of the experimental protocol presented in main figure 4A–B. (B) Quantitation of total T cell density in LN and relative T cell density (inside and outside GCs), based on data from two independent experiments as shown in main Fig. 4A. (C) An image of a LN from a mouse that did not receive NP-CGG at day -10 (as depicted in the experimental protocol in A) showing absence of B1-8hi GCs. The white box in the left panel indicates the area shown at higher-magnification in the right panel. Scale bars, 0.5 mm (left), 100 μm (right). (D) Diagrammatic representation of the experimental protocols presented in main Fig. 4C–D. (E) Diagrammatic representation of the experimental protocols presented in main Fig. 4E.

Table S1. Reagents used for flow cytometry

Shulmanvideo c4

Movie S4:

Collapsed 4D dataset showing motility of OVA-specific OT-II cells in proximity to CGG-specific germinal centers. Green, OT-II memory T cells; blue, B1-8hi cells; red, NP-tdTomato (FDC labeling). 3 different example movies are shown.

Acknowledgments

We thank H. Ploegh, K. Strijbis and H. Sive for mice and equipment use, and E. Browne for helpful discussion. The data presented in the paper are tabulated in the main paper and in the supplementary materials. ZS is a Human Frontiers of Science fellow. GP is a Swiss National Science Foundation fellow. ADG was supported by NIH Medical Scientist Training Program grant T32GM07739 to the Weill Cornell/Rockefeller/Sloan-Kettering Tri-Institutional MD-PhD Program. Support for the Rockefeller University multiphoton microscope was granted by the Empire State Stem Cell Fund through NYSDOH Contract #C023046. This work was supported in part by NIH grant 5DP5OD012146-02 and MOD Basil O’Connor Award to GDV and by NIH grants AI037526-19, AI072529-06 and AI100663-01 to MCN. MCN is an HHMI investigator.

Footnotes

References and Notes

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