IL-4-Secreting secondary Tfh cells arise from memory T cells, not persisting Tfh cells, through a B cell dependent mechanism

Abstract

Humoral immunity requires crosstalk between T follicular helper (Tfh) and B cells. Nevertheless, a detailed understanding of this intercellular interaction during secondary immune responses is lacking. We examined this by focusing on the response to a soluble, unadjuvanted, pathogen-derived Ag (SEA) that induces type 2 immunity. We found that activated Tfh cells persisted for long periods within germinal centers following primary immunization. However, the magnitude of the secondary response appeared not to depend on pre-existing Tfh cells. Instead, Tfh cell populations expanded through a process dependent on memory T cells recruited into the reactive LN, and the participation of B cells. We found that during the secondary response, IL-4 was critical for the expansion of a population of plasmablasts that correlated with increased SEA-specific IgG1 titers. Additionally, following immunization with SEA (but not with an Ag that induced type 1 immunity), IL-4 and IL-21 were co-produced by individual Tfh cells, revealing a potential mechanism through which appropriate class-switching can be coupled to plasmablast proliferation to enforce type 2 immunity. Our findings demonstrate a pivotal role for IL-4 in the interplay between T and B cells during a secondary Th2 response and have significant implications for vaccine design.

Introduction

T follicular helper cells (Tfh cells) are a critical subset of CD4⁺ T cells that are specialized to provide cognate help to B cells (1). Tfh cells express CXCR5, allowing them to access B cell follicles, where they participate in germinal center (GC) development and secrete cytokines such as IL-21, IL-4, and IFNγ, that drive both B cell proliferation and immunoglobulin (Ig) class switching to allow the production of IgG1/IgE (IL-4) and IgG2a (IFNγ) (2-4). Tfh cell and GC B cell numbers are tightly correlated and the two cell types appear to be able to support each other's prolonged persistence as long as antigen (Ag) is available (5). Developmental studies have revealed that Tfh cells express a distinct repertoire of genes, and can develop in vivo where conditions for Th1, Th2, or Th17 cell development are impaired (6, 7). These types of study have led to the conclusion that Tfh cells are a distinct lineage. Other studies, including our own, suggest that in type 2 immunity, Tfh cells emerge from cells that are already committed to the Th2 lineage, and therefore can be regarded as a specialized subset of these cells (8, 9). However, the relatedness of Tfh cells to Th2 cells in type immunity has been questioned especially in light of the fact that IL-4, a key marker of Th2 cells, has also been defined as a marker of Tfh cells (10). It is has been unclear how this situation could be compatible with the preferential induction of IgG2a during type 1 immune responses. On a related issue, while the role of IL-4 in the primary type 2 response is well documented (11, 12), its role if any in a secondary type 2 response, which presumably involves the reactivation of memory B cells that are already class-switched, remains unclear.

As is the case with other helminth parasites, infections with the parasite Schistosoma mansoni leads to strong type 2 immunity; much of this response is induced by, and directed towards Ag secreted by the egg stage of the parasite (13, 14). Type 2 immunity in this infection involves the development of Th2 cells, IL-4-producing Tfh cells and IgG1-producing B cells, which together play important protective roles during infection (15, 16). Intriguingly, a soluble extract of S. mansoni eggs (SEA) is able to induce strong Th2 and Tfh responses in the absence of additional adjuvant (8), allowing us to study natural immune responses without the confounding factors of infection.

There has been considerable interest lately in the nature of secondary Tfh cell responses. Recent work revealed that, following Ag clearance, Tfh cells do possess the capacity to further differentiate into a resting memory CD4⁺ T cell pool. The properties of these memory cells remain unclear, since some reports have shown that upon re-challenge they retain their Tfh lineage commitment (17), while others have shown that, depending on the nature of the secondary response, they possess the ability to differentiate into Th effector cells (18). The situation is complicated by the fact that in a few reports Tfh cells have been shown to persist following primary immunization, and it has been suggested that these cells serve as lymphoid reservoirs of antigen-specific memory Tfh cells (19). However, whether these cells truly are memory cells or not is debatable, since it is now clear that maintenance of the Tfh cell phenotype requires GC B cells and persistent Ag (5), suggesting that if Tfh cells are detected late after immunization it is because they are continuing to be stimulated by Ag. The possibility that Tfh cells arise from memory T cells following secondary immunization raises the question of whether B cells play a role in this process as they do in the generation of primary Tfh cell responses (1).

Here we have explored the development of Tfh cells during a secondary response to unadjuvanted SEA, focusing on the role of persistent Tfh cells vs. committed memory cells in this process. We have further asked whether B cells play a role in secondary Tfh cell responses, and explored the function of IL-4 during the secondary type 2 response. Our data suggest that in the course of a secondary type 2 response, Tfh cells arise largely from memory T cells, and not persisting Tfh cells, and that this process is dependent on non–GC B cells. Additionally, we found that IL-4 production is coupled to IL-21 production in Tfh cells in secondary type 2, but not type 1 responses, and that IL-4 plays a critical role in supporting the rapid outgrowth of plasmablasts that characterize this response.

Materials and Methods

Mice and Parasites

C57Bl/6, Balb/c 4get/KN2, and C57BL/6 IL-4^-/-mice, were bred in-house. All experimental procedures with mice were approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis. Snails infected with S. mansoni (strain NMRI, NR-21962) were provided by the Schistosome Research Reagent Resource Center for distribution by BEI Resources, NIAID NIH. Soluble egg Antigen (SEA) was prepared from isolated schistosome eggs as previously described (16, 20). Stag was made from culture-derived Toxoplasma gondiii tachyzoites (Strain PTG, a clone of the ME49 line) according to a previously described method (21). Omega 1, IPSE/alpha, and Kappa 5 were purified from SEA as described (22, 23). For all immunizations 30μg of extract were injected s.c. into rear footpads of mice.

In vivo treatments

For B cell depletion experiments Balb/c 4get/KN2 at 90 days post SEA injection were injected i.p. with 50μg of Anti-CD20 (a generous gift from Genentech) every 7 days for 4 weeks. At the time of the 4th injection, 25μg of mAb was also injected into the footpad that had previously been immunized. For IL-4R blockade experiments Balb/c 4get/KN2 at 60-75 days post SEA injection were injected i.p. with 250μg of anti IL-4R antibody (M1) at days -1 and 1 post secondary SEA immunization. An additional 50μg of anti IL-4R mAb was injected s.c into the footpad at day 1 post secondary immunization. For BrdU labeling of CD4 T cells, mice were injected i.p. with 0.5 mg BrdU (BD Biosciences or Sigma-Aldrich) and sacrificed 16 hours later. For FTY720 experiments mice were injected i.p. with 70 μg of FTY720 (Caymen) every 12-14 hours for 6 injections.

Flow cytometric analysis

Popliteal LN were removed and dissociated into single cell suspensions as previously described (8). Surface staining with monoclonal antibodies, acquisition, and analyses were performed essentially as described previously (8). Samples were acquired using a FACSCanto II flow cytometer (BD) and analyzed with FlowJo software (Tree Star, Inc.). Cytokine production by T cells was determined by intracellular staining (antibody identification below) after re-stimulation for 5 h with PMA (phorbol 12-myristate 13-acetate; 50ng/ml) and ionomycin (1ug/ml) in the presence of brefeldin A (10ug/ml) (24). BrdU incorporation was detected with a BrdU Flow kit (BD Pharmagen) according to the manufacturers instructions. The following mAb (BD, eBioscience, BioLegend, R&D, or Invitrogen) against mouse antigens were used as PE, PE-Cy5, PE-Cy7, allophycocyanin (APC), APC-Cy7, Pacific blue, or biotin conjugates: BrdU (MoBU-1), CD4 (RM4-5), CD19 (1D3), CD138 (281-2), IgG1 (A85-1), IgD (11-26), IgM (11/41), HuCD2 (RPA-2.10), PD-1 (J43), CXCR5 (2G8), and IFNγ (XMG1.2). IL-21R/FC chimera (R&D systems 596-MR-100) was used to detect IL-21 producing cells, followed by detection with PE conjugated anti-human Ab (BD) . Biotinylated Abs were secondarily stained with APC-Cy7-conjugated streptavidin. For identification of GFP positive fixed cells, cells were permeabilized and stained with unconjugated anti-GFP (ebioscience), washed, and then stained with Fitc conjugated Goat anti Rabbit IgG secondary antibody (Jackson Immunoresearch). Fc-block (anti mouse CD16/32 clone 93) was used in all experiments to minimize non-specific signals. Plots shown are on a Logicle scale.

ELISA and ELISPOT

SEA specific serum IgG1 and endpoint titers were determined by ELISA using the IgG1-specific mAb X56 (BD). Immulon 4HBX plates (Thermo Fisher Scientific) were coated overnight at 4°C with 2 μg/ml of SEA or Stag, blocked with 1% milk, and incubated with serial dilutions of sera, followed by a peroxidase coupled anti-mouse IgG1 and ABTS substrate. Single antigen titers were determined as above except that plates were coated with 500ng/ml of antigen. For ELISPOTs, single-cell bone marrow suspensions were cultured in RPMI 1640 supplemented with FCS for 24h in MultiScreen-HA plates (Millipore, Billerica, MA) coated with 2 μg/ml of SEA. Bound Ab was detected with HRP labeled anti-mouse IgG1 (SouthernBiotech), using the AEC Chromogen Kit (Sigma) per the manufacturer's instructions, and spots were counted using an Immunospot analyzer (v4.1, C.T.L, Cellular Technology Limited).

Statistical analyses

Data were analyzed with ANOVA followed by the unpaired Student' t test, or Pearson's Correlation test via Prism 6.0 (GraphPad Software); p values ≤ 0.05 were considered statistically significant.

Results

IL-4 and IL-21 production are coupled in Tfh cells responding to secondary immunization with Type 2 response-inducing soluble pathogen derived Ag

We used 4get/KN2 mice, in which cells that have expressed IL-4 are marked by GFP, and cells which are producing IL-4 express surface HuCD2 (25), to examine Tfh cell and GC development in popliteal LN draining sites of s.c. injection with unadjuvanted SEA. As shown previously (8), SEA immunization resulted in a robust CD4⁺CXCR5⁺PD-1⁺ Tfh cell response by 8 days after primary immunization (Figure 1A). Within this population the majority of cells were GFP⁺HuCD2⁺ (Figure 1B). The strong Tfh response was accompanied by GC B cell (defined as CD19⁺FAS⁺PNA⁺), plasmablast (defined as CD19⁺CD138⁺IgD^- cells) and plasma cell (defined as CD19^-CD138⁺IgD^- cells) responses (Figure 1C).

Figure 1. Tfh cell and B cell responses following primary or secondary immunization with SEA.

Balb/c 4get/KN2 mice were immunized s.c. in the footpad with SEA and lymphocytes were isolated from the draining popliteal LN at days 8 and 60 post primary, or day 3 post secondary immunization. Cells were identified by staining for the indicated markers (here and in subsequent figures) and analyzed by flow cytometry. A) Tfh cells defined, as CD4⁺CXCR5⁺PD-1⁺, in immunized mice compared to naïve mice. B) Expression of GFP and HuCD2, as markers of IL-4 transcription and protein production respectively, in the Tfh cells gated as in A. C) GC B cell responses, defined as CD19⁺PNA⁺Fas⁺ cells, and plasmablasts and plasma cells, defined as IgD^-CD19⁺CD138⁺ and IgD^-CD19^-CD138⁺ respectively, in immunized compared to naïve mice. D) IL-21, IFN and HuCD2 expression in Tfh cells. Cells were re-stimulated as described in the methods prior to staining for intracellular IL-21 and IFNγ. E-G) Balb/c 4get/KN2 mice were immunized s.c. in the footpad with Stag and lymphocytes were isolated from the draining popliteal LN at days sixty post primary and day 3 post secondary immunization. E) Tfh cells, defined as CD4⁺CXCR5⁺PD-1⁺, in immunized mice. F) Expression of GFP and HuCD2 in the Tfh cells gated as in E. G) IL-21, IFNγ and HuCD2 expression in Tfh cells. Cells were re-stimulated as described in the methods prior to staining for intracellular IL-21 and IFNγ. H) The ratio of IL-4:IFNγ secreting Tfh cells at day 3 post secondary. I) The ratio of IgG1 to IgG2a positive cells in response to SEA or Stag at day 3 post secondary injection. J) The correlation between the ratio of IgG1:IgG2a and the ration of IL-4:IFNγ producing cells combined r square =0.9422 and p=0.0104 for Stag p= 0.0478 for SEA. FACS data shown are concatenated from 3-5 animals per group. Experiments were performed 2-4 times. Numbers in A-G represent frequencies of cells in quadrants or indicated gates. Pearson's Correlation test and Student's t test were used to determine statistical significance. K,L, Frozen popliteal LN sections from the indicated time-points were stained for B220 (red), GL7 (white), and CD4 (blue) and imaged with a laser scanning confocal microscope. Cells positive for both CD4 and GL7 appear purplish blue (*). Images are representative of 3-5 individual animals.

In previously published work, Tfh cells that developed in response to immunization with a model Ag in adjuvant were found to be unexpectedly persistent in reactive LN (19). We asked whether Tfh cells were long-lived following immunization with SEA. We were able to detect distinct Tfh cell populations in mice that were immunized with SEA 60 days earlier (Figure 1A, and data not shown). These populations were detectable in reactive LN for prolonged periods (>180 days, data not shown). Notably, persistent Tfh cells remained active, as measured by the fact that they continued to produce IL-4, as reported by HuCD2 expression (Figure 1B). This prolonged responsiveness was associated with persisting GC B cells (Figure 1C).

Next we examined Tfh cell responses following secondary immunization. We found that secondary immunization resulted in marked and rapid (within 3 days) expansion of the Tfh cell pool that was apparent both in the frequency of these cells, and in overall numbers (Figure 1A, data not shown). The majority of Tfh cells were GFP⁺ and approximately half of these cells were actively making IL-4 (Figure 1B). Interestingly, secondary immunization did not result in increased GC B cell frequencies, although overall numbers increased due to LN expansion (this point will be addressed further in Figure 4). Moreover there was a notable increase in the frequency of plasmablasts within the B cell compartment (Figure 1C). Plasma cell production is strongly supported by Tfh cells through the production of IL-21 (26). Consistent with previous reports about the frequency of IL-21 producing cells within the Tfh cell compartment (18) we found that IL-21 was made by approximately 20% of Tfh cells in mice immunized and challenged with SEA (Figure 1D). As might be anticipated, given the type 2 bias of the SEA-induced response, relatively few Tfh cells made IFNγ, and moreover production of this cytokine was uncoupled from that of IL-4 (Figure 1D). Interestingly however, production of IL-21 was tightly coupled to IL-4, but not IFNγ production (Figure 1D).

Figure 4. Secondary immunization induces a rapid expansion of Tfh cells, GC B cells and plasma cells, with a corresponding increase in IL-4 and Ab production.

Balb/c 4get/KN2 mice were immunized once or twice with SEA and lymphocytes from reactive LN following primary immunization (at day 8 “Primary”, or Day 90), or at day 4 post secondary immunization (“Secondary”, and “Contralateral Secondary”, as described in the text), were analyzed by flow cytometry for expression of the markers indicated. Mice were also bled for serum collection, and B cells in bone marrow were plated for ELISPOT detection of SEA-specific IgG1 secreting cells. A.) Frequencies of CD4⁺ T cells that are competent to make IL-4 (GFP⁺; first column), and of GFP⁺ Tfh cells and Th2 cells (GFP⁺PD-1^-CXCR5^-) cells (second column), and of Th2 cells and Tfh cells that are expressing HuCD2 as a marker of IL-4 production (third and fourth columns respectively). B-D, F) Total numbers of Th2 (B), Tfh (C) HuCD2⁺ cells (D) and IgG1⁺FAS⁺ GC B cells (F), in draining LN of mice immunized as shown in the key for B, at days 0 - 14 post SEA injection. E) Frequencies of FAS^-IgG1⁺ memory B cells and IgG1⁺FAS⁺ GC B cells in reactive LN. G) Time-course of SEA specific IgG1 titers following primary and secondary SEA immunizations, as indicated in the key for B. H) Numbers of SEA-specific IgG1 secreting cells (“spots”), as measured by ELISPOT, at day 4 (Primary) or day 90 post primary immunization, or at day 4 post secondary or contralateral secondary immunization. I, J) Serum IgG1 titers for the indicated purified SEA proteins at day 90 post primary (I) and 17 post secondary (J) SEA immunization. Numbers in A and E represent frequencies of cells in quadrants or indicated gates. Data shown are concatenated from 3-5 animals per group and all experiments were performed 3 times. Graphed data represent mean ± SEM. N.D. indicates Not detected.

The coupling of IL-4 to IL-21 production in Tfh cells in a type 2 response raised the question of the nature of the Tfh cytokine response in the context of a type 1 response. To examine this, we immunized mice with Stag, an unadjuvanted soluble Ag from the protozoan parasite Toxoplasma gondii: Stag has previously been utilized for studying type 1 immunity (27) and as such is a good counterpart to SEA for our studies. The overall intensity of the primary Tfh response, the persistence of Tfh cells following immunization, and the expansion of the response following secondary immunization, were all very similar to what was observed in SEA-immunized animals (Figure 1E, and data not shown). However, we were surprised to find that the frequencies of Tfh cells that were GFP⁺ were greater than 60% in Stag-immunized mice, and that within the GFP⁺ population in the Stag-immunized mice a substantial number of Tfh cells were actually making IL-4 (Figure 1F). While these frequencies are lower than those elicited by SEA, they do indicate that the majority of Tfh cells elicited in response to a Th1 antigen open the IL-4 locus. This finding is consistent with conclusions from other studies that regardless of the overall bias of the immune response, IL-4 production is a signature of all Tfh cells (10, 28, 29). From previous work it is clear that the major T cell cytokine associated with Stag stimulation is IFNγ (21), a cytokine that is critical for class switching to IgG2a/IgG2c (30, 31). We therefore asked whether Tfh cells from Stag-immunized mice make IFNγ. We found a substantially higher frequency of IFNγ-positive Tfh cells in the Stag- compared to SEA-immunized mice (Figure 1G); in both cases there was relatively little overlap in IFNγ and IL-4 secretion (Figure 1D,G), suggesting that there are distinct Tfh cell populations that secrete either IL-4 or IFNγ. The ratios of IL-4:IFNγ producing T cells in the SEA- vs. Stag-immunized mice strongly correlated with the final bias in IgG1:IgG2a in the induced responses (Figure 1J). Finally, it was notable in these studies that in contrast to Tfh cells from SEA-immunized mice, IL-21 and IL-4 production were largely uncoupled in Tfh cells from mice immunized with Stag, whereas approximately half of the Tfh cells making IL-21 were also making IFNγ in Stag immunized mice (Figure 1G).

To confirm that persisting activated Tfh cells and GC cells identified via flow cytometry were indeed in organized GC structures, we imaged B cells and CD4⁺ T cells in GCs on fresh frozen sections of draining and non-draining LNs at days 14 and 80 post immunization. At Day 14 post immunization, LNs were enlarged and contained multiple clusters of B220⁺GL7⁺ cells with infiltrating CD4⁺GL7⁺ cells, and were bordered by CD4⁺ cells (Figure 1K). The CD4⁺GL7⁺ cells are likely to be Tfh cells, which have been reported to express GL7 in addition to PD-1 and CXCR5 (10). Late after immunization, GCs were smaller and located peripherally within the LN, but remained organized with a ring of CD4⁺GL7⁺ Tfh cells (Figure 1L, delineated with *) at the outer edges of the GC B220⁺GL7⁺ cells. GL7+CD4+ Tfh cells were also occasionally found in the center of the GC clusters (delineated with *).

IL-4 plays critical roles in primary and secondary responses to SEA

Given the importance of IL-4 in type 2 immunity (32, 33), we focused on the role of IL-4 in the primary and secondary Tfh/B cell response. Initially we compared the outcome of the response to SEA in WT vs. IL-4^-/- mice. We found that the absence of IL-4 resulted in a diminished frequency of Tfh cells at day 8, the peak of the primary response (Figure 2A), and a reduction in the frequency of GC B cells (Figure 2B). Consistent with the known function of IL-4 as a class switch factor for IgG1, there was a markedly lower frequency of Fas⁺IgG1⁺ GC B cells in SEA immunized IL-4^-/- mice than in WT mice (Figure 2C). This was accompanied by only a small increase in the frequency of IgG2a⁺ cells (Figure 2C). This reduction in IgG1⁺ B cells correlated with a reduction in the frequency of plasmablasts (Figure 2D). Thus, as anticipated, IL-4 is essential for the primary IgG1 response to SEA, an Ag noted for its ability to induce type 2 immunity.

Figure 2. IL-4 is required for optimal Tfh cell and GC development in a primary type 2 response, and for expansion of the plasmablast population that characterizes the secondary type 2 response.

B6 IL-4 ^-/- or WT mice (A-D) or Balb/c 4get/KN2 mice (E-H) were immunized once or twice s.c. with SEA and lymphocytes were isolated from the draining popliteal LN 8 or 90 days post primary immunization (A-E), or at day 3 following secondary immunization (E-H). Cells were stained for the indicated markers and analyzed by flow cytometry. A) Tfh cells B) GC B cells. C) IgG1⁺ and IgG2a⁺ GC B cells. D, E) Plasmablasts and plasma cells. F-H) At day 90 post primary immunization, mice were given anti-IL-4R mAb or isotype control mAb and a secondary immunization with SEA. Three days later cells from the draining LN or blood were analyzed by flow cytometry. F) GFP⁺ Tfh cells. G) Plasmablasts and plasma cells. H) Total numbers of plasmablasts per LN (left panel) and frequency of plasmablasts in the blood. Data shown are concatenated from 4-5 animals per group for A-D and 5-6 mice per group for E-H. Experiments were repeated twice for A-D and 3 times for E-H. Numbers in A-G represent frequencies of cells in quadrants or indicated gates. Statistical significance was determined using Student's t test. Data points in H represent individual mice.

Next we assessed the role of IL-4 in the secondary response to SEA. We noted that secondary immunization with SEA resulted in the appearance, within 4 days, of a population of plasmablasts in responding LN (Figure 2E). The kinetics of this B cell response paralleled the expansion of the IL-4-producing Tfh cell pool following secondary immunization (data not shown). To test the role of IL-4 in this process, WT mice primed with SEA 60 days earlier were given a secondary immunization with or without blocking anti-IL-4Rα mAb, (34). We found that IL-4Rα blockade had no effect on Tfh cell expansion (Figure 3F), but substantially reduced the plasmablast pool not only in the draining LN but also in the blood (Figure 3G,H). Taken together, our data indicate that IL-4 production plays a critical role in the response to SEA not only in IgG1 class switching but also in the expansion of the plasma cell pool that underlies the enhanced Ab response that characterizes secondary immunization.

Figure 3. B cells are required for increases in Tfh cells, IL-4 production by Tfh cells, and numbers of plasmablasts that occur following secondary immunization.

Balb/c 4get/KN2 mice were immunized with SEA and 90 days later were treated with either anti-CD20 mAb or isotype control mAb. Forty five days later, the mice were injected with PBS (A-D) or received a secondary immunization with SEA (E-H) and then, 3 days later, popliteal LN cells were isolated, stained for the indicated markers and analyzed by flow cytometry. A, E) GFP+ Tfh cells and HuCD2 expression in gated GFP+ Tfh cells. B, F) Frequencies of B cells, Fas^-IgG1⁺ memory B cells, FAS⁺IgG1⁺ GC B cells and plasmablasts and plasma cells. C, D) Total numbers of GFP⁺ T cells, Tfh cells, FAS^-IgG1⁺ memory B cells and Fas⁺IgG1⁺ GC B cells at day 45 after anti-CD20 mAb treatment of mice that had received a primary immunization with SEA. G) Total numbers of mature plasma cells and plasmablasts at day 3 post secondary immunization in anti-CD20 mAb-treated or control mice. H) SEA specific IgG1 titers at day 3 post secondary challenge of anti-CD20 mAb or control treated mice. Data in A-C are concatenated from 3-4 mice, experiments were performed twice. Numbers in A, B, E, F represent frequencies of cells in quadrants or indicated gates. Data shown are concatenated from 3-5 mice per group. Data in C, D, and G represent mean ± SEM. Data points in H are from individual mice. Statistical significance was determined with a Student's t test.

Reciprocal Tfh cell/B cell interaction is critical for the secondary Tfh cell and plasmablast response

B cells have been shown to be necessary for the generation and maintenance of the Tfh cell compartment early following primary immunization (5, 8, 35-38). In light of these reports we evaluated the importance of B cells for the maintenance of persistent Tfh cells late after immunization, and for the expansion of the Tfh cell population following secondary immunization. For these experiments, mice immunized with SEA 90 days earlier were treated with anti-CD20 mAb to deplete B cells (39, 40), or isotype control Ig. This led to the rapid depletion of >95% of naïve and memory B cells, although GC B cells remained in the draining LN (data not shown). We assessed CD4⁺ T cells and B cells in the reactive LN 45 days following initiation of anti-CD20 mAb treatment. We found that the frequency of Tfh cells, the percentages of these cells that continued to express HuCD2, and the overall numbers of Th2 cells and Tfh cells, were unaffected by anti-CD20 mAb treatment (Figure 3A,C). We did however measure a decreased frequency of B cells (Figure 3B) that reflected lasting effects of mAb treatment on the numbers of naive B cells and Fas^-IgG1⁺ memory B cells (Figure 3B,D). Due to the loss of other B cell populations, the frequency of GC B cells increased in anti-CD20 mAb treated mice (Figure 3B), although treated and control mice had equivalent total numbers of these cells at the time of analysis (Figure 3D). We then gave these mice a secondary immunization and analyzed the subsequent response. We found that the expansion of Tfh cells was substantially diminished in mice that had been treated with anti-CD20 mAb, and that substantially fewer cells were expressing the HuCD2 marker of IL-4 production within the Tfh population than was the case in the control mice (Figure 3E). Taken together, these data indicate that the emergence of an enlarged, activated Tfh population following secondary immunization is dependent on naïve and/or memory B cells, but that the persistence of the active Tfh population for prolonged periods following primary immunization is not (rather, Tfh cell persistence is reported to depend on GC B cells (5)). Our data indicate that either naïve, and/or memory B cells are necessary for both the expansion of the Tfh cell compartment, and for the stimulation of IL-4 production from newly differentiated Tfh cells.

Since SEA-immunized mice treated with anti-CD20 mAb displayed a significant loss of IgG1⁺ memory B cells but not IgG1⁺ GC cells (Figure 3B), we were able to examine the contributions of each of these populations to the enlarged plasmablast pool following secondary challenge (Figure 2E). We considered it likely that the expanded population of plasmablasts that characterizes the secondary response to SEA was derived from memory B cells being recruited into the secondary response. We found that frequencies of B cells within reacting LN increased as a result of secondary immunization in control mice and in anti-CD20 treated mice (Figure 3F compared to 3B), but that while there was a greater frequency of GC B cells, there was a reduction in both frequency (Figure 3F) and number (Figure 3G) of plasmablasts in the animals treated with anti-CD20 mAb. This reduction in plasmablast numbers was accompanied by a significant reduction in anti-SEA IgG1 titers as compared to control mice (Figure 3H). Our data suggest that memory B cells are responsible for generating at least 50% of the plasmablasts that rapidly emerge in a reacting LN following secondary challenge, and that this process is critical for the enhanced secondary response to SEA.

Tfh, Th2 cells and plasma cell numbers expand rapidly and equivalently following secondary immunizations that target previously reactive LNs or non-reactive LNs

Our data indicate that immunization with SEA induces a persistent, active Tfh cell compartment within reactive LN, and that there is a potent secondary Tfh response following secondary immunization. We wondered whether the persistent Tfh cells contribute to the nature of a secondary immune response, and began to address this experimentally by comparing secondary responses in mice immunized either in the same footpad that received the primary SEA injection in order to engage the persistently active popliteal LN (a “secondary” immunization, as performed in prior experiments herein), or in the contralateral site to engage the previously non-reactive LN (a “contralateral secondary” immunization) (Figure 4A). We found that secondary immunization led to an accelerated and marked increase in GFP⁺ T cells that included both non-Tfh cells (defined as CD4⁺GFP⁺CXCR5^-PD-1^-, which we previously classified as Th2 cells (8)), as well as Tfh cells in the draining LN, that substantially exceeded the magnitude of a primary response of similar duration (Figure 4A,B,C). This was recapitulated, albeit at slightly reduced intensity, following contralateral secondary immunization (Figure 4B,C). In both cases the secondary T cell responses peaked at day 4 post challenge, which preceded the day 8 peak of the primary response (Figure 4B,C), and was marked by substantially increased overall numbers of IL-4-secreting cells at this time compared to at the peak of the primary response (Figure 4D). The secondary B cell response largely mirrored the secondary T cell response, with more IgG1⁺ class-switched B cells being generated by day 4 post challenge than at any point during the primary response, regardless of which footpad was immunized (Figure 4E,F). Overall SEA-specific IgG1 titers were indistinguishable between mice that had received secondary or contralateral secondary immunizations (Figure 4G), a finding that was consistent with the fact that bone marrow from mice immunized in these two ways contained equivalent numbers of plasma cells, enumerated as SEA-specific IgG1 antibody-secreting cells (ASCs) (Figure 4H). SEA-specific ASCs were undetectable in the bone marrow (or draining LN) of mice that had received a primary immunization with SEA 4 days earlier, and were detectable, but only in low numbers in the bone marrow of mice immunized 90 days previously (Figure 4H). As expected given these findings, Ab titers following secondary immunization and contralateral secondary immunization were comparable, but both were significantly higher than those measured early or late following primary immunization (Figure 4G).

We were interested in the specific target Ag of the type 2 response induced by SEA immunization. Previous work has implicated three glycoproteins as major target Ag within SEA: Omega-1, IPSE/Alpha-1, and Kappa-5 (41, 42). Therefore we used ELISA to measure Ab against these Ag, and found that while only IPSE/alpha-1 was strongly targeted during the primary response, all three became targets for strong IgG1 responses following secondary immunization with SEA (Figure 4I,J).

The enhanced Tfh response during secondary immunization results from the recruitment of memory CD4⁺ T cells into the Tfh pool

The conventional view of CD4⁺ T cell responses to acute Ag exposure is that Ag-specific cells proliferate to a maximal number, and thereafter the population contracts to leave quiescent long-lived IL-7R⁺ memory cells that are able to respond strongly upon challenge with the same Ag (43, 44). Consistent with this, in our system, where few CD4⁺ cells are either IL-7R⁺or GFP⁺ Tfh cells in naïve LN (Figure 5C), the majority of CD4⁺ T cells that responded to immunization, and which were marked by GFP expression, were IL-7R^- at the peak of the primary response (Figure 5A), whereas at later times the majority of GFP⁺ cells were IL-7R⁺ (Figure 5B). When we focused on Tfh cells within the overall GFP⁺ population, we found that at early times post immunization the majority of them were BrdU⁺ following a 16 h label, expressed HuCD2 as a marker for IL-4 production, and were IL-7R^- (Figure 5A). At later time points, the persisting Tfh cells remained HuCD2⁺ but were no longer proliferating and moreover differed from the remainder of the GFP⁺ population in not expressing IL-7R (Figure 5B). Thus, Tfh cells continue to make cytokine and fail to express IL-7R even at times when they have stopped proliferating. Interestingly, the Tfh response was largely restricted to the initially reactive LN, and we noted no increase in Tfh cells in distal LN, although there was an increased frequency of GFP⁺ cells in these LN compared to completely naïve mice (Figure 5C).

Figure 5. Primary Tfh cells are long-lived producers of IL-4 in the absence of proliferation or IL-7R expression.

Balb/c 4get/KN2 mice were immunized with SEA and either sacrificed at days 8 or 85 for analysis of cells in LN draining sites of immunization, or distal sites. Cells were analyzed by flow cytometry for expression of the markers indicated. A, B) Expression of IL-7Rα in GFP^- vs GFP⁺CD4⁺ T cells (first panel), GFP⁺ Tfh cells (second panel), BrdU incorporation in Tfh cells following a 16 h in vivo pulse (third panel), HuCD2 expression in Tfh cells, and IL-7Rα expression in Tfh cells, on the days indicated post primary immunization. Data shown concatenated from 3-5 animals per group and experiments were performed 3 times. C) Expression of IL-7Rα in GFP^- vs GFP⁺CD4⁺ T cells (first panel) and GFP⁺ Tfh cells (second panel) in non-draining LN in mice immunized for 85 days, vs. naïve mice, as indicated. Numbers represent frequencies of cells in quadrants or indicated gates.

Next we asked whether secondary Tfh responses differ depending on whether the challenge immunization was delivered to the same site as the primary, or to a distal site. Within 4 days of secondary immunization or contralateral secondary immunization we observed an approximately 5-10 fold increase in the frequency of IL-7R^-CD4⁺ T cells in reactive LN (Figure 6A,B, compared to Figure 5B,C). Within these populations, approximately 60-70% of cells incorporated BrdU within a 16 h time period, and of these proliferating cells, 22-28% were Tfh cells (Figure 6A,B). We reasoned that since, prior to secondary immunization, Tfh cells were IL-7R^- and non-proliferative in the face of ongoing Ag presentation (which presumably underpins ongoing IL-4 production by these cells), it was unlikely that Tfh cells themselves accounted for the emergence of a proliferating population of IL-7R⁺ Tfh cells following the additional exposure to Ag provided by secondary immunization. This led us to speculate that the secondary response Tfh cells were derived from the proliferation and differentiation of long-lived non-Tfh CD4⁺ T cells. The fact that Tfh cell and GC B cell numbers in LNs responding to contralateral secondary immunization, where Tfh cells persisting from the primary immunization were absent (Figure 5C), were greatly increased following secondary immunization (Figure 4C), supported this view. Moreover, contralateral secondary immunization led to reactivation of CD4⁺ cells in the originally reactive LN and the emergence of a proliferating population of GFP⁺IL-7R⁺CXCR5⁺PD-1^-CD4⁺ cells (Figure 5C) that are phenotypically consistent with reactivated central memory cells (45). We were also able to detect significant populations of these cells in draining LNs following secondary or contralateral secondary immunization (Figure 5A,B). Thus we reasoned that the recruitment of these cells into the Tfh cell pool could underpin the expansion of the Tfh cell populations observed following secondary immunization. To test this directly, we examined Tfh responses following contralateral secondary immunizations in mice treated with FTY720, which inhibits T cell egress from lymphoid organs (46-48), and therefore the recruitment of cells from other sites into responding LNs. At the time of peak responsiveness following contralateral secondary immunization, responding LNs in FTY720-treated mice had a higher frequency of CD4⁺ T cells than in control mice, but reduced expansion of the CD4⁺GFP⁺ population, within which the frequency of Tfh cells was 60% reduced compared to controls (Figure 6D); FTY720 treatment resulted in an overall reduction in the Tfh cell population of greater than 10 fold (Figure 6E). Consistent with this, we found significantly reduced numbers, compared to controls, of IgG1⁺ B cells in FTY720 treated mice following contralateral secondary immunization (Figure 6F). We interpret these data as being consistent with the development of Tfh cells from a population of SEA-specific memory CD4⁺ T cells recruited from the circulation into the reacting LN during the secondary response. In this context it is interesting that most of the cells in the Tfh population following contralateral secondary immunization were IL-7R⁺ (Figure 6D), which was in contrast to early or late during the primary response (Figure 5 A,B), and may suggest that they recently arose from IL-7R⁺ memory cell precursors and had yet to down-regulate expression of this receptor (Figure 6C). The IL-7R⁺CD4⁺GFP⁺CXCR5^-PD1^- cells evident in LN-draining sites of primary immunization (Figure 6B), and in non-draining distal LN (Figure 6C), are candidates for memory T cells that could support this process. Taken together, these data support the view that during secondary immunization with SEA, Tfh cells develop from a population of SEA-specific GFP⁺IL-7R⁺CXCR5⁺PD-1^- memory CD4⁺ T cells recruited into the reacting LN.

Figure 6. Recruitment of IL-7R⁺ memory T cells is required for rapid and enhanced Tfh cell and plasmablast development following secondary immunization.

Balb/c 4get/KN2 mice were immunized twice with SEA and lymphocytes from the reactive or non reactive LN at day 4 post secondary immunization were analyzed by flow cytometry for expression of the markers indicated. A-C) Frequencies of CD4⁺ T cells that are co-express GFP and IL-7R; (first column), frequency of BrdU incorporation in GFP⁺IL-7R⁺ cells (second column), the percentages of BrdU⁺ cells that express CXCR5 and PD-1 (third column). Data shown are concatenated from 3-5 mice per group and experiments were performed 3 times. D-F) Mice were immunized with SEA and 85 days later were given FTY720 or PBS (Control) prior to receiving a secondary immunization in a distal site with SEA. Lymphocytes from draining LN were analyzed 3 days later. CD4⁺ T cells (first panel), GFP⁺CD4⁺ T cells (second panel), GFP⁺ Tfh cells (third panel), and IL-7R⁺ Tfh cells (fourth panel). Data shown are concatenated from 4 mice per group and experiments were performed twice. E, F) Total numbers of Tfh and CD19⁺IgG1⁺ B cells cells in draining LN at day 3 post secondary immunization in mice that received FTY720 or PBS prior to challenge. Data represent mean ± SEM of results from 4 mice per group. Numbers in A-D represent frequencies of cells in quadrants or indicated gates. Statistical significance was determined by Student's t test.

Discussion

We examined secondary responses to the unadjuvanted, soluble parasite-derived Ag SEA. We found that in this setting co-production of IL-4 and IL-21 are coupled in individual Tfh cells, and that activated Tfh cells persist for remarkably long periods (>60 days). Nevertheless, our data suggest that memory CD4⁺ T cells rather than persistent Tfh cells are necessary for Tfh cell population expansion during a secondary response to SEA. We found that this expansion of the Tfh cell population following secondary immunization required B cells, and moreover that memory B cells required IL-4 for efficient differentiation into plasmablasts, indicating that secondary type 2 immune responses depend on an intimate IL-4-centered crosstalk between B cells and Tfh cells.

Our findings support earlier work that expression of the IL-4 gene is a characteristic of Tfh cells (10, 28, 29, 49), since we observed this to be true not only in SEA-induced type 2 immunity, but also in a type 1 biased response induced by Stag, a soluble extract of the parasitic protist T. gondii. When first reported, the fact that all Tfh cells express IL-4 was somewhat perplexing in that IL-4 is a known class-switch factor for IgG1 and IgE, but not for IgG2a or IgG2c, which rather are induced by IFNγ, and are characteristic of type 1 immunity. Therefore it has been difficult to understand how production of IL-4 by all Tfh cells would be compatible with this distinction. However, we have been able to show that not only is the frequency of Tfh cells producing IL-4 in the context of type 2 immunity induced by SEA greater than in a type 1 immune response induced by Stag, but also that the frequency of IFNγ producing cells is greater in Stag immunized mice than in SEA immunized mice. Moreover, in SEA-immunized mice IL-4 production was coupled to the production of the critical Tfh cell cytokine IL-21 whereas IL-4 and IL-21 production were uncoupled in Stag immunized mice. These data support a model in which Tfh cells coordinate the production of class switch inducing cytokines with IL-21 to ensure that the emerging Ig isotype is functionally linked to effector functions induced in parallel by effector Th cells. The role, if any, of Tfh cell derived IL-4 produced in type 1 immunity is currently under investigation in our laboratory. In addition to providing a class switch signal, IL-4 is able to support B cell survival (50), so it is feasible that IL-4-producing Tfh cells are contributing to humoral responses in type 1 immunity in capacities other than Ig class switching.

Using SEA we have found that activated, IL-7R^- cytokine-producing Tfh cells persist for extended periods (>60 days) following primary immunization. These Tfh cells remain associated with active GCs in which the proliferation of class-switched B cells is ongoing (data not shown). Our data show that persistent Tfh cells are discretely localized to the LN that reacted to the primary immunization. The ability of activated Tfh cells to persist within active GCs for prolonged periods following primary immunization is a striking feature of the biology of these cells. This characteristic has been reported before in the context of responses induced by adjuvanted Ag (19) and acute viral infection (Hale 2013), but has not been previously noted in responses induced by injected pathogen-derived Ag in the absence of exogenous adjuvant. We reason that prolonged GC activity reflects the capture and retention of immune complexes (ICs) containing these Ags on follicular dendritic cells and that this represents the Ag depot that is sampled by GC B cells and used in cognate interactions to activate Tfh cells. The length of time over which Tfh cells and related GCs persist following immunization with SEA implies that ICs containing these Ag are both stable and present at high concentrations. At present we have little idea of the specificity of the Tfh cells or B cells that are responding to SEA. This Ag is in fact a complex mixture of soluble molecules released when the parasite eggs are mechanically disrupted, but nevertheless within this mixture three glycoproteins Omega-1, IPSE/alpha-1 and Kappa-5, have been defined as major Ab targets (42, 51). Of these, Omega1 has notable effects on dendritic cells and can directly affect the maturation state of these cells, conditioning them to drive type 2 responses, through its ability to interact with Mannose Receptor (22, 52, 53). Nevertheless, the IgG1 responses to IPSE/alpha-1 and Kappa-5 were stronger than those against Omega-1 in mice immunized with SEA, such that the titer against these Ag represented ∼20% of the overall response to SEA. We speculate, based on this, that these two Ag would be found trapped in GC in mice immunized with SEA, and that Tfh cells with specificity against these Ag would be well-represented within the Tfh population that develops in these animals. IPSE/alpha-1 has previously been shown to possess non-specific immunoglobulin binding properties (54, 55), so it is feasible that it binds GC immune complexes in addition to being represented in the FDC antigen pool, increasing potential exposure to IPSE/alpha-1.

The mechanisms underlying the biology of prolonged Tfh cell responsiveness are unclear. We have found that Tfh cells that persist long after primary immunization continue to produce cytokines but are no longer proliferating. A possible explanation for this is that these cells are being constrained due to their defining expression of PD-1, a known inhibitory receptor that is expressed on exhausted effector T cells in chronic infections and in tumors (56, 57). In other systems, lack of PD1, or blockade of the interaction of this receptor with its ligand PD-L1, expressed by GC B cells, results in increases in the number of these cells (58, 59). The physiologic effects of this appear to be situation dependent in that in some systems PD1/PD-L1 blockade results in increased Ag-specific Ab responses, whereas on others it leads to diminished plasma cell and GC responses (58-61).

Our experiments using anti-CD20 mAb to deplete B cells revealed a role for these cells in the expansion of the Tfh cell population following secondary immunization. Anti-CD20 mAb has been reported to inefficiently deplete GC B cells (5), and indeed this was also our finding. Thus our experiments did not directly address the role of GC B cells in the maintenance of Tfh cells in SEA-immunized mice. Nevertheless, based on previous work on the effects of blocking ICOS/ICOS-L or CD40/CD40L interactions on Tfh cells (5, 62) it seems reasonable to assume that intimate Tfh cell/GC B cell interactions are required for persistent GCs in SEA-immunized mice. Our experimental approach did reveal that the loss of non-GC B cells, which include memory and naïve B cells, following primary immunization significantly limits the rapid expansion of the Tfh cell population following secondary immunization. Thus, as during primary immunization (8, 36, 63, 64), B cells play a significant role in Tfh cell development following secondary immunization.

In previous work, BAFF has been shown to enhance survival of ASCs that are generated from memory B cells during a recall response (65), and IL-6, IL-10, and IL-21 have been implicated in the generation of plasma cells following primary immunization (66-71), but the role of IL-4 in humoral immunity following secondary immunization has been unclear. Mice deficient in IL-4 signaling have been shown to have reduced serum IgG1 titers during a primary immune response (72), but a role for IL-4 signaling in the control of plasmablast generation during a secondary response has not been reported previously. We consistently observed increased IL-4 secretion during secondary responses to SEA as compared to primary, as well as a correlation between Tfh cell IL-4 secretion and the magnitude of plasmablast generation. In light of the fact that we observed a significant reduction in both blood and LN plasma cells when IL-4Rα signaling was blocked during the secondary response, we believe that there is a requirement for IL-4Rα engagement on IgG1⁺ memory B cells in order to induce differentiation into plasmablasts during a secondary response. This requirement has implications for vaccine development, as immunization strategies that maximize the development of the IL-7R⁺ memory cells that we believe are able to rapidly differentiate into Tfh cells and secrete IL-4 during re-exposure to Ag should lead to a more robust development of Ag specific immunoglobulin.

There has been great interest recently in the idea of committed memory Tfh cells. The available evidence indicates that Tfh cells that become disengaged from Ag stimulation have the potential to down-regulate expression of Tfh markers such as Bcl-6 and PD-1, and persist as memory cells (73). However, the fate of these memory cells remains in debate. Some studies have shown that these cells are capable of being recruited into effector T cell or Tfh cell pools upon secondary immunization (18, 45), while others indicate that they are committed to the Tfh cell lineage (17). Our experiments did not address this issue directly, but rather attempted to determine whether persisting Tfh cells play a role in the generation of an enhanced Tfh response following secondary immunization. Our data show that as a reactive LN continues to support persistent active Tfh cells and ongoing GCs, it becomes home to a population of CD4⁺ T cells that are GFP⁺IL-7R⁺CXCR5⁺PD-1^-, which begin to proliferate following secondary immunization; cells with this phenotype can also be found in LN draining sites of contralateral secondary immunization. Whether these reflect Tfh cells that have disengaged from Ag stimulation and entered the memory pool, or memory T cells of non-Tfh origins is unclear. Nevertheless, early during the development of a secondary response at distal sites, Tfh cells are IL-7R⁺, and fail to develop in FTY-720-treated mice, suggesting that they are emerging from the activation of recruited memory-like T cells even as the original population of Tfh cells continues to engage B cells in the persisting GCs of the originally reactive LN.

The function of persisting Tfh cells and GCs remains unclear. If the expansion of Tfh cell populations and the dramatic increases in Ab titers that accompany them can occur independently of persisting active Tfh cells, the role of these cells presumably lies in the ongoing maintenance of Ab titers rather than in a memory response per se. It seems reasonable to conclude that Ag depots play an important role in this process by continuing to nucleate cognate Tfh cell /B cell interactions in GCs, allowing the ongoing production of plasma cells long after the initial exposure to Ag (74). Presumably this type of response confers significant benefits in terms of maintaining Ab titers following prior exposure to Ag, which in terms of immunity to infection would be considered beneficial. However, these types of persistent Tfh/GC responses may also play detrimental roles in autoimmune settings in which Ab plays a pathologic role, and it is feasible that their resistance to anti-CD20 mAb explains the inability of anti-CD20 based therapies in the treatment of diseases such as systemic lupus erythematosus (75). Greater understanding of the role of persistent Tfh cells in resistance to infection or the development of autoimmunity may lead to rational approaches to promote or inhibit these responses.