Research Article

Differential IL-2 expression defines developmental fates of follicular versus nonfollicular helper T cells

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Science  14 Sep 2018:
Vol. 361, Issue 6407, eaao2933
DOI: 10.1126/science.aao2933

(IL-)2 be or not to be?

Immunological T follicular helper (TFH) cells are a subpopulation of CD4+ T cells that support B cell antibody production and the establishment of B cell memory. By contrast, non-TFH cells orchestrate enhanced innate immune cell functions at sites of pathogen encounter. The factors underlying differentiation into a TFH or non-TFH cell remain poorly understood, though there is evidence to suggest that the T cell growth factor interleukin-2 (IL-2) may play a role. Using IL-2 reporter mice, DiToro et al. show that naïve CD4+ T cells that produce IL-2 are fated to become TFH cells, whereas nonproducers, which receive IL-2, become non-TFH cells. The CD4+ T cell–fate decision was linked to T cell receptor strength—only those naïve CD4+ T cells that received the highest T cell receptor signals were able to produce IL-2.

Science, this issue p. eaao2933

Structured Abstract

INTRODUCTION

The adaptive immune system has evolved to mount different types of responses that are matched to the type of invading pathogen. For CD4+ T cells, this is predicated on the multipotentiality of clonally restricted naïve T cells, which differentiate into distinct subsets of effector T cells contingent on recognition of cognate antigen and cytokine cues from cells of the innate immune system. There are two broad divisions of effector CD4+ T cells: T follicular helper (TFH) cells, which are programmed to interact with B cells within lymphoid tissues to support production of high-affinity, class-switched antibodies, and non-TFH effector cells, including T helper 1 (TH1), TH2, and TH17 cells, which are programmed to egress from lymphoid tissues to orchestrate heightened innate immune cell function at sites of pathogen entry. The mechanisms controlling bifurcation into TFH versus non-TFH effector cell pathways are incompletely understood.

RATIONALE

An impediment to understanding mechanisms controlling TFH–non-TFH cell divergence is an absence of early markers to define cells destined for these alternative fates. Unlike effector CD4+ T cells, which produce a diversity of cytokines that define their phenotype and function, naïve CD4+ T cells are largely limited to the rapid production of interleukin-2 (IL-2) when activated by antigen. IL-2 is only produced by a subset of activated naïve T cells, suggesting a possible relationship between IL-2 production and effector cell fate determination. To explore this, we developed two IL-2 reporter mice strains with complementary features that enabled the tracking and deletion of T cells on the basis of differential IL-2 expression. This allowed us to determine whether naïve T cells that do, or do not, produce IL-2 are biased in their developmental programming and, if so, how.

RESULTS

RNA sequencing of naïve T cells sorted on the basis of IL-2 reporter expression identified cosegregation of transcripts encoding IL-2 and Bcl6—the signature transcription factor of TFH cells. Conversely, IL-2–negative (IL-2) cells preferentially expressed the gene Prdm1, which encodes the transcriptional repressor Blimp1. Blimp1, in turn, antagonizes Bcl6 and the TFH developmental program. This suggested that IL-2 producers give rise to TFH cells, whereas IL-2 nonproducers give rise to non-TFH effector cells. Moreover, the fact that IL-2 receptor signaling induces expression of Prdm1 via Stat5 suggested that IL-2 producers resisted IL-2 signaling and activated IL-2 signaling in nonproducers in trans. Indeed, in vivo studies established that IL-2 signaling was mostly paracrine and that depletion of IL-2–producing cells selectively impaired TFH cell development. Finally, IL-2 expression was limited to a subset of naïve T cells that received the strongest T cell receptor (TCR) signals, establishing a link between TCR signal strength, IL-2 production, and TFH versus non-TFH differentiation.

CONCLUSION

This study provides new insights into the mechanisms that control early bifurcation of CD4+ T cells into TFH and non-TFH effectors. Naïve T cells that receive differing strengths of TCR signals stratify into those that exceed a threshold predisposing them to IL-2 production and early TFH commitment and those that do not express IL-2 yet receive IL-2 signaling, which reinforces non-TFH effector commitment.

IL-2–producing CD4+ T cells become TFH cells, whereas IL-2 nonproducers become non-TFH cells.

(Left) Strong TCR signaling via an antigen presenting cell induces Il2 and Bcl6 gene expression (red pathway); weaker signaling induces expression of non-TFH genes (blue pathway), including Prdm1 and S1pr1, which encodes the S1P receptor S1PR1. Bcl6+ cells (red) secrete IL-2 in trans to T regulatory (Treg) cells (yellow) and recently activated IL-2 cells (blue), up-regulating IL-2 receptor IL2rα and reinforcing Prdm1. (Top right) Bcl6+ cells engage cognate B cells (green) and migrate to germinal centers (GCs); Bcl6+ TFH cells mature into GC-TFH cells. (Bottom right) Prdm1+ cells migrate to efferent lymphatics and mature into non-TFH effectors in nonlymphoid tissues. MHCII, major histocompatibility complex class II; ICOS, inducible costimulator; CXCR5, a receptor for chemokine CXCL13.

Abstract

In response to infection, naïve CD4+ T cells differentiate into two subpopulations: T follicular helper (TFH) cells, which support B cell antibody production, and non-TFH cells, which enhance innate immune cell functions. Interleukin-2 (IL-2), the major cytokine produced by naïve T cells, plays an important role in the developmental divergence of these populations. However, the relationship between IL-2 production and fate determination remains unclear. Using reporter mice, we found that differential production of IL-2 by naïve CD4+ T cells defined precursors fated for different immune functions. IL-2 producers, which were fated to become TFH cells, delivered IL-2 to nonproducers destined to become non-TFH cells. Because IL-2 production was limited to cells receiving the strongest T cell receptor (TCR) signals, a direct link between TCR-signal strength, IL-2 production, and T cell fate determination has been established.

Naïve CD4+ T cells are multipotent precursors that differentiate into functionally distinct effector subsets to coordinate different aspects of immunity. T helper 1 (TH1), TH2, and TH17 cells are products of developmental pathways induced by different classes of pathogens. They are programmed to egress from T cell zones of secondary lymphoid tissues soon after induction to orchestrate heightened innate immune cell function at sites of pathogen entry. T follicular helper (TFH) cells develop concurrently with TH1, TH2, and TH17 cells but are programmed to migrate to B cell zones within secondary lymphoid tissues. They provide help to B cells to support the production of high-affinity, class-switched antibodies. TFH- and non-TFH effector cell development diverges early in evolving adaptive responses. However, the type of immune response (type 1, 2, or 3) is linked such that pathogen-clearance mechanisms mediated by innate immune cells are amplified by coordinated help from non-TFH cell effectors and the antibodies that result from TFH cell–mediated B cell help. Cytokines elicited from innate immune cells by pathogens appear to be dominant in determining the type of adaptive response (1), whereas the intensity of T cell receptor (TCR) signaling appears to contribute to TFH–non-TFH cell specification (2) by mechanisms that are incompletely understood.

An impediment to understanding the mechanisms controlling TFH–non-TFH cell divergence is the absence of reliable early markers to define cells destined for these alternative fates. Unlike effector CD4+ T cells, which are distinguished by a diversity of cytokines that define their phenotype and function, naïve CD4+ T cells are largely limited to the production of interleukin-2 (IL-2), which is produced rapidly by a subset of antigen-activated cells (3). Through the activation of Stat5 and induction of Blimp1 (4, 5), IL-2 suppresses Bcl6—a central TFH transcription factor—and, consequently, TFH development (6). This implies a direct relationship between the production of IL-2 by naïve CD4+ T cells and their development into either non-TFH or TFH effector cells. We explored this relationship using transgenic mice engineered to report the expression of IL-2.

IL-2 and Bcl6 expression cosegregate within hours of naïve T cell activation

IL-2.eGFP reporter mice were generated by the targeted insertion of an IRES-eGFP (internal ribosome entry site–enhanced green fluorescent protein) expression cassette into the fourth exon of the endogenous Il2 gene (Fig. 1A). Naïve CD4+ T cells from IL-2.eGFP mice stimulated under nonpolarizing conditions in vitro diverged into CD69+IL-2+ (GFP+) and CD69+IL-2 (GFP) subpopulations within hours of activation and before cell division (Fig. 1, B to E). Reporter expression was rapidly detectable and peaked at approximately 24 hours before declining. This decline lagged production of IL-2 because of the relatively long half-life of the reporter. To define genes differentially expressed by IL-2 producers and nonproducers, CD69+IL-2+ and CD69+IL-2 cells were analyzed by RNA-sequencing (RNA-seq) (Fig. 1C). Among the 151 genes that were preferentially expressed by IL-2+ cells were Bcl6 and the tumor necrosis factor (TNF) superfamily member Cd40l, which are important in TFH cell development or function, respectively. Also enriched in IL-2+ cells was Zbtb32, which, like Bcl6, encodes a member of the POK/ZBTB family of transcription factors and has been shown to restrict the expression of TH1 and TH2 cell cytokines (7). By contrast, among the 210 genes preferentially expressed by IL-2 cells were multiple genes characteristic of non-TFH effector cell differentiation, including Prdm1, which encodes Blimp1, as well as S1pr1 and Klf2. Similar results were obtained from an analysis of naïve SMARTA TCR-transgenic IL-2.eGFP CD4+ T cells stimulated with antigen (fig. S1). S1pr1 is required for the egress of non-TFH effector CD4+ T cells from secondary lymphoid tissues (8), and its expression inhibits TFH development in vivo (9, 10). Klf2 suppresses TFH differentiation while promoting non-TFH effector cell differentiation, at least in part via the induction of Blimp1 (9). These findings, which were independently validated by quantitative polymerase chain reaction (qPCR) (Fig. 1, D and E), suggested that IL-2 producers may be fated to become TFH cells, whereas IL-2 nonproducers may be fated to become non-TFH effector cells. Akin to findings in CD4+ T cells, the differential expression of Bcl6 and Blimp1 was found in IL-2+ and IL-2 subsets isolated from activated naïve CD8+ T cells (fig. S2). This suggests that, despite their lower production of IL-2 relative to that of CD4+ T cells, early divergence of CD8+ T cells destined to become Blimp1+ short-lived effector cells or Bcl6+ memory precursor effector cells (11) may be similarly linked to the differential expression of IL-2.

Fig. 1 Differential expression of Bcl6 and Blimp1 by IL-2+ and IL-2 T cells

(A) Gene-targeting strategy for the generation of IL-2.eGFP knockin reporter mice. The loxP-flanked neomycin resistance cassette was deleted by crossing founders to EIIa-Cre transgenic mice. (B) Sorted naïve (GFPCD44CD62L+) IL-2.eGFP CD4+ T cells were labeled with CellTrace Violet (CTV), stimulated in vitro with soluble anti-CD3 (5 μg/ml) and irradiated CD4-depleted feeder cells, and then examined for expression of CD69 and IL-2.eGFP by flow cytometry at the indicated time points. Data are representative of four experiments with at least three replicates per condition. CTV staining was performed in two of four experiments. (C) Total RNA isolated from naïve IL-2.eGFP CD4+ T cells stimulated for 18 to 24 hours as in (B) and fluorescence-activated cell sorter (FACS)–purified into CD69+GFP (IL-2) or CD69+GFP+ (IL-2+) fractions was analyzed by comparative expression profiling with RNA-seq. Data depict two biological replicates per condition. padj, adjusted P value. (D) RNA isolated from IL-2.eGFP CD4+ T cells stimulated and FACS-purified as in (C) was analyzed by qPCR for expression of Il2, Bcl6, and Prdm1 at the indicated time points. Error bars represent SEM of three technical replicates per sample. Data are representative of four experiments. (E) Validation of selected transcript expression with RNA isolated from IL-2.eGFP CD4+ T cells stimulated and FACS-purified as in (C). Three technical replicates per sample are shown. Data were analyzed by using Student’s t tests and are representative of two experiments. **P < 0.01; ***P < 0.001; ****P < 0.0001; error bars depict SEM.

The differential expression kinetics of Bcl6 and Prdm1 by CD4+ T cells were discordant (Fig. 1D). Bcl6 expression tracked with Il2 expression and decayed to background levels as Prdm1 expression increased. Indeed, at the peak of differential Bcl6 expression (8 hours), Prdm1 expression remained at background levels in both IL-2+ and IL-2 cells. Thus, although these transcription factors are believed to be directly antagonistic in the specification of TFH versus non-TFH effectors (12), the rapid, reciprocal expression of Bcl6 in IL-2+ and IL-2 fractions was not controlled by Blimp1. Instead, we found differential expression of the gene encoding Mxd1 (also known as Mad1) (Fig. 1C and fig. S3), which has been shown to directly bind and down-regulate Bcl6 during the differentiation of germinal center B cells into plasma cells (13). The contemporaneous, reciprocal expression of Mxd1 and Bcl6 antecedent to the expression of Prdm1 suggests that repression of Bcl6 by Mxd1, rather than by Blimp1, may contribute to the early bifurcation of TFH and non-TFH effectors (Fig. 1D and fig. S3).

Although Bcl6, like Blimp1, often acts as a transcriptional repressor, the parallel kinetics of Il2 and Bcl6 expression suggested that Bcl6 may positively regulate Il2 expression. Thus, we performed chromatin immunoprecipitation (ChIP) analysis of conserved noncoding sequences in the Il2 promoter and 35 kb upstream that were identified by ATAC-seq (assay for transposase-accessible chromatin–sequencing) analysis as accessible in IL-2+ cells compared with naïve and IL-2 cells (Fig. 2A). Bcl6 preferentially bound these sites in IL-2–producing cells relative to IL-2 nonproducers, at a time point (20 hours) when the expression of Bcl6 and Blimp1 overlapped (Fig. 2B). Blimp1 preferentially bound these sites in IL-2 cells, as did Foxo1, which was recently shown to suppress TFH differentiation (14). The permissive histone modification trimethylated histone H3 lysine 4 (H3K4me3) was significantly enriched in IL-2+ cells at the sites of Bcl6 binding, whereas repressive H3K27me3 histone marks were reduced in both IL-2+ and IL-2 cells relative to naïve cells. Thus, the expression of Il2 correlated positively with Bcl6 binding at sites of induced chromatin accessibility in the Il2 gene locus and negatively with binding of Blimp1 (and Foxo1) at the same sites. Because the expression of Prdm1 significantly trailed the peak of differential Il2 expression (Fig. 1D), occupancy of these sites by Blimp1 did not appear to be required for the repression of Il2 early in IL-2 cells. Rather, Blimp1 appeared to act primarily to reinforce the lack of Il2 expression in the IL-2 fraction of activated naïve T cells at later time points. This is consistent with Blimp1’s reported role as a feedback inhibitor of IL-2 (15, 16). These findings indicate that, in addition to its predictive value in defining early precursors of TFH and non-TFH effector cells, expression of IL-2 may be directly regulated by the antagonistic actions of Bcl6 versus Blimp1 and Foxo1 at conserved cis-regulatory elements in the Il2 gene locus.

Fig. 2 Differential chromatin accessibility and transcription factor binding at the Il2 locus in IL-2+ and IL-2 T cells.

(A) ATAC-seq was performed on nuclei isolated from naïve (GFPCD44CD62L+) IL-2.eGFP CD4+ T cells and FACS-purified CD69+GFP+ (IL-2+) and CD69+GFP (IL-2) fractions treated as inFig. 1C. Chromatin accessibility peaks were visualized by using Integrated Genome Browser (IGB) and are shown aligned against a VISTA plot of syntenic regions of mouse and human chromosomes corresponding to Il2-Il21 and IL2-IL21 gene loci, respectively. Data are representative of two experiments. (B) Naïve IL-2.eGFP CD4+ T cells were treated as in Fig. 1C, and the IL-2 promoter region (Il2p) and conserved noncoding sequence 35 kb upstream of the Il2 transcription start site (CNS-35) of CD69+GFP+ (IL-2+) and CD69+GFP (IL-2) fractions were analyzed by quantitative ChIP-PCR for the presence of Bcl6, Blimp1, and Foxo1 binding or H3K4me3 and H3K427me3 histone modifications, normalized to total DNA input. Three technical replicates per group are shown. Data for each region were analyzed separately by one-way analysis of variance (ANOVA). Not significant (ns), P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; error bars depict SEM.

Because TFH cell development occurs concurrently with each of the CD4+ T effector cell pathways, we determined if the correlation between reciprocal expression of Bcl6-Blimp1 and IL-2 occurred under TH1, TH2, and TH17 cell polarizing conditions (Fig. 3A), as it did for TH0 cells (Figs. 1 and 3). Under each of these activation conditions, the expression of the IL-2.eGFP reporter was limited to a subset of cells expressing the highest amounts of CD69 (Fig. 3A). GFP expression also correlated positively with Bcl6 expression and negatively with Prdm1 expression (Fig. 3B). Cells activated under TH17 cell conditions expressed the highest frequency and single-cell levels of IL-2, despite the reported suppression of TH17 cell differentiation by IL-2 signaling (17). Thus, Il2 and Bcl6 expression mirrored one another under each of the conditions examined (Fig. 3B), and the highest amounts of Il2 and Bcl6 were found in IL-2+ cells activated under TH17 cell conditions. This may reflect the shared requirement for IL-6 in both TH17 and TFH cell developmental programs.

Fig. 3 Bcl6 and IL-2 cosegregate early in each T effector cell developmental program.

(A) Naïve (GFPCD44CD62L+CD69CD25) IL-2.eGFP CD4+ T cells were stimulated in vitro under TH0, TH1, TH2, and TH17 cell conditions for 20 hours and examined by flow cytometry for CD69 and IL-2.eGFP expression. Data are representative of two experiments. Flow plots depict cell number–controlled concatenated averages of three samples per group. Error bars depict SD. gMFI, geometric mean fluorescence intensity. (B) Experiment performed as in (A), with CD69+ IL-2.eGFP+ and IL-2.eGFP CD4+ T cells sorted 20 hours after activation. RNA was isolated and analyzed by qPCR for expression of Il2, Bcl6, and Prdm1. Three technical replicates per condition are shown. N, naïve T cells. Error bars depict SEM. Data for (A) and (B) are representative of two experiments each.

IL-2 signaling is predominantly paracrine

Although the foregoing studies suggested a link between Il2 gene expression and TFH–non-TFH cell fate determination, the differentiation of physiologic TFH cells ex vivo has not yet been established. Thus, we examined this relationship in vivo, in the context of infection with ActA-deficient Listeria monocytogenes (ActA-Lm) (2, 18). This attenuated type 1 bacterial pathogen was engineered to express peptide antigens, which enabled the tracking of endogenous antigen-specific CD4+ T cell responses with peptide-loaded major histocompatibility complex class II (MHCII) (p:MHC) tetramers or transferred TCR-transgenic T cells (2, 19). Naïve CD4+ T cells from CD45.2+ IL-2.eGFP-SMARTA TCR-transgenic mice were transferred into wild-type (WT) CD45.1+ mice and infected with ActA-Lm that express ovalbumin (OVA) peptide and the gp66 peptide recognized by the SMARTA TCR (ActA-Lm-OVA-gp66). Antigen-activated SMARTA T cells were recovered near the peak of IL-2 expression and sorted into IL-2+ and IL-2 fractions for differential gene expression analysis by RNA-seq (Fig. 4A). In agreement with our in vitro findings (Fig. 1C), IL-2+ cells were significantly enriched for expression of Il2, Bcl6, Zbtb32, and CD40l, whereas IL-2 cells were significantly enriched for Prdm1, S1pr1, and Klf2. Multiple TH1 cell–associated transcripts (e.g., Ifng, Il12rb2, Ltb, and Gzmb) were identified in IL-2 cells, consistent with the induction of type 1 immunity by ActA-Lm. Gene set enrichment analysis (GSEA) identified enhanced activity of multiple effector signaling pathways in IL-2 cells. Of these, two of the most significant were interferon- and inflammatory-signaling gene sets (Fig. 4B). By contrast, the most significantly enhanced gene sets in IL-2+ cells were those of Myc and the E2F family of transcription factors (Fig. 4B and fig. S4, A and B). Both are involved in cell-cycle regulation and are suppressed by Blimp1 in germinal center B cells (20, 21). Mxd1, the transcript for which was again one of the most highly enriched in IL-2 cells, antagonizes Myc by competing for binding to a shared dimerization partner, Max (22). Myc expression by T cells correlates directly with strength of activation (23, 24), implicating a role for Mxd1 in restraining the actions of Myc in less strongly activated T cells and consistent with expression of IL-2 by more strongly activated naïve CD4+ T cells.

Fig. 4 IL-2+ T cells activate IL-2 T cells via paracrine IL-2 signaling to drive differential gene expression in vivo.

(A) Sorted naïve (GFPCD44CD62L+) IL-2.eGFP CD45.2+ SMARTA CD4+ T cells were transferred into CD45.1+ WT mice infected with ActA-Lm-gp66 24 hours before transfer. Total RNA was isolated by FACS-purified CD45.2+ CD69+GFP+ (IL-2+) and CD69+GFP (IL-2) CD4+ T cells 20 to 24 hours after transfer and analyzed by RNA-seq. Data depict three biological replicates per condition from three separate experiments. Sac, sacrificed. (B) Hallmark GSEA of IL-2+ and IL-2 T cells from (A). For each pathway, mean and 95% confidence intervals are plotted and then color coded to indicate false discovery rate–corrected P values. FPKM, fragments per kilobase million. (C) Schematic of targeting strategy to generate IL-2.BAC-in Thy1.1 (2BiT) transgenic reporter mice. BAC, bacterial artificial chromosome. (D) Sorted naïve (Thy1.1CD44CD62L+) 2BiT CD4+ T cells were stimulated in vitro with soluble anti-CD3 (5 μg/ml) and irradiated CD4-depleted feeder cells for 24 hours and then examined by flow cytometry for expression of CD69 and Thy1.1. RNA isolated from CD69+Thy1.1+ (IL-2+) and CD69+Thy1.1 (IL-2) CD4+ T cells was analyzed by qPCR for expression of Il2 mRNA. Error bars represent SEM of three technical replicates per sample. Data are representative of two experiments. (E) 2BiT mice were infected with ActA-Lm. After 18 hours, mice were sacrificed and splenic CD4+ T cells were analyzed by flow cytometry for the expression of IL-2.Thy1.1, CD25, and tyrosine phosphorylation of Stat5 (p-Stat5). Data are representative of two experiments. (F) Congenic CD45.1+ WT mice were infected with ActA-Lm-gp66. After 24 hours, naïve CD45.2+ SMARTA 2BiT CD4+ T cells were transferred into infected CD45.1+ recipients. Mice were sacrificed at the indicated times, and splenic CD4+ T cells were analyzed for expression of Thy1.1, Foxp3, and p-Stat5. Data are representative of two experiments.

Notably, Il2ra, which encodes the inducible, high-affinity component of the IL-2 receptor (IL-2rα or CD25) that is up-regulated on activated T cells, was enriched in IL-2 cells (Fig. 4A). Accordingly, the hallmark IL-2–Stat5 signaling gene set was significantly enriched in IL-2 cells (Fig. 4B). Among a manually curated consensus list of 23 gene sets modulated by IL-2 signaling (fig. S4C), those up-regulated in response to IL-2 were enriched in IL-2 cells, many of which include Il2ra (fig. S4, D and E). By contrast, genes down-regulated in response to IL-2 were enriched in IL-2+ cells.

Collectively, these findings support a model in which highly activated naïve T cells up-regulate Bcl6, produce IL-2, and are fated to become TFH effectors. IL-2 producers deliver IL-2 to nonproducers, inducing the latter’s up-regulation of Blimp1 and differentiation into non-TFH effectors. To examine the relationship between IL-2 production and utilization in vivo and directly address the fate of IL-2 producers and nonproducers, we generated a second transgenic IL-2 reporter mouse line with features complementary to those of the IL-2.eGFP mice (Fig. 4C). IL-2.BAC-in Thy1.1 (2BiT) reporter mice were engineered to express high amounts of the surface molecule Thy1.1 under control of the Il2 gene locus to facilitate intracellular costaining by flow cytometry and enable the in vivo deletion of IL-2–producing cells (25). As with T cells from IL-2.eGFP mice, activated (CD69+) 2BiT T cells rapidly bifurcated into Thy1.1+ (IL-2+) and Thy1.1 (IL-2) fractions (Fig. 4D and fig. S5). To determine whether IL-2 production and signaling segregate in antigen-activated naïve CD4+ T cells, 2BiT mice were infected with ActA-Lm and analyzed for the expression of IL-2 versus intracellular phospho-Stat5 (p-Stat5) at the peak of IL-2 expression (Fig. 4E). Reciprocal IL-2 expression and IL-2 signaling were observed; Thy1.1 (IL-2) was almost exclusively expressed by p-Stat5 CD4+ T cells (Fig. 4E), whereas p-Stat5 was limited to Thy1.1 cells. Consistent with gene expression results (Fig. 4A), nearly all Thy1.1+ cells were CD25 at this time point, whereas nearly all p-Stat5+ cells were CD25+. Thus, IL-2 signals predominantly in a paracrine, not autocrine, manner (26). Moreover, IL-2 producers are initially resistant to IL-2 signaling, in accord with their lack of CD25 up-regulation.

Most endogenous p-Stat5+ CD4+ T cells immediately after infection are Foxp3+ regulatory T (Treg) cells (26), owing to their constitutive expression of CD25 and relative abundance compared with that of naïve clonal precursors. To examine IL-2–induced Stat5 signaling in naïve pathogen-specific non-Treg cells, naïve CD45.2+ 2BiT-SMARTA T cells were transferred into CD45.1+ WT mice infected with ActA-Lm expressing the gp66 peptide (ActA-Lm-OVA-gp66) (Fig. 4F). Analysis of transferred 2BiT-SMARTA (clonotypic) and endogenous CD4+ T cells showed that the majority of endogenous p-Stat5+ cells were Foxp3+, whereas p-Stat5+ clonotypic T cells were Foxp3. Thus, the paracrine model of IL-2 signaling applies to both “bystander” Treg cells as well as naïve CD4+ T cells responding to infection.

TFH cells are derived from IL-2 producers

To examine the fate of antigen-activated IL-2–producing and –nonproducing T cells in vivo, 2BiT mice were treated with a depleting anti-Thy1.1 or nondepleting control antibody (25) immediately before infection with ActA-Lm coexpressing OVA and the antigenic peptides gp66, 2W1S, Cbir1, or FliC (Fig. 5 and fig. S6). MHCII-tetramer analysis of endogenous CD4+ T cells specific for each of these peptides showed that IL-2 (Thy1.1) expression was restricted to CXCR5+ cells and that the depletion of IL-2–expressing cells preferentially eliminated TFH cells and spared non-TFH (TH1) effectors, as defined by expression of CXCR5 and PD-1 (Fig. 5A and fig. S6) or CXCR5 and Bcl6 (Fig. 5B). Notably, the number of non-TFH effectors was not compromised by the depletion of IL-2 producers (Fig. 5C). This suggested that IL-2 was not required for the clonal expansion of non-TFH effectors. However, this likely reflects the discordant kinetics of IL-2 secretion relative to reporter expression and antibody-mediated cell depletion, because IL-2 reporter expression and Stat5 phosphorylation were only partially decreased at the peak of IL-2 expression (fig. S7). Thus, TFH effector cells developed from IL-2–expressing precursors, whereas non-TFH effectors did not. Accordingly, IL-2 was a reliable marker with which to distinguish precursors fated to become TFH or non-TFH effector cells.

Fig. 5 IL-2 producers are precursors of TFH cells.

(A to C) 2BiT mice were injected with 250 μg of anti-Thy1.1 or isotype control (Ctrl) monoclonal antibody (mAb), then infected 1 day later with ActALm-gp66. Endogenous CD4+ T cells specific for IAb-gp66 were enriched from lymph nodes and spleens 3 days after infection by using tetramer-based magnetic sorting and analyzed by flow cytometry for IAb-gp66 tetramer binding (tet+) and expression of Ly6C, CXCR5, IL-2.Thy1.1, and PD-1 (A) or Bcl6 (B). Flow plots depict cell number–controlled concatenated averages of all samples within a group. Data for (A) and (B) are representative of two experiments each. (C) Data from the experiments depicted in (A) and fig. S6 were analyzed by two-way ANOVA. A total of eight control and eight treatment animals from two separate experiments are shown. (D) 2BiT mice were injected with 250 μg of anti-Thy1.1 or isotype control mAb and immunized with 2 × 1010 colony-forming units (CFU) of HKLm. Mice were bled every 6 days for 24 days, and serum anti-Lm IgG was measured by enzyme-linked immunosorbent assay (ELISA). n = 7 mice per group. Data are representative of two experiments. (E) Magnetically enriched WT CD45.1+ and 2BiT CD45.2+ CD4+ T cells were transferred into Tcrb–/– mice. After 24 hours, mice were immunized with 2 × 1010 CFU HKLm and injected with 250 μg anti-Thy1.1 or isotype control mAb. Mice were sacrificed 5 days after immunization, and splenic CD4+ T cells were analyzed by flow cytometry for expression of CD44, CD45.1, CD45.2, PD-1, and CXCR5. Results were analyzed by two-way ANOVA. n = 3 mice per group. Data are representative of three experiments. (F) CD4+ T cells magnetically enriched from WT CAG-eGFP (CD45.2) mice and CD45.1+ 2BiT mice were adoptively transferred into TCRβ-deficient recipients. After 24 hours, the mice were immunized with 2 × 1010 CFU HKLm and injected with 250 μg of anti-Thy1.1 or isotype control mAb. Two weeks after immunization, spleens were collected and analyzed by confocal microscopy for the expression of GFP (WT), CD45.1 (2BiT), Ki67, and IgD. Quantitation of WT (GFP+), 2BiT (CD45.1+), and total T cell numbers in germinal centers (GCs) was performed by computer-assisted counting. Splenic B cells were analyzed by flow cytometry for the expression of IgD, B220, GL7, and Fas in an IgDlo B cell gate. n = 3 mice per group. Data are representative of three experiments. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; error bars depict SEM.

Although surface markers define TFH cells capable of providing B cell help, TFH cell function is predicated on a subset of TFH cells that localize to the germinal center after productive interactions with cognate B cells. Referred to as germinal center (GC-TFH) T cells, these cells express high amounts of PD-1, Bcl6, and CXCR5 and support the germinal center response and production of high-affinity, class-switched antibodies (12). To examine the effects of depletion of IL-2–expressing precursors on the development and function of this TFH cell subset, we characterized the effects of anti-Thy1.1 depletion on antibody responses and the generation of GC-TFH cells. 2BiT mice were immunized with heat-killed Lm (HKLm) (Fig. 5, D to F), because infection with live Lm does not induce good antibody responses (27). Anti-Thy1.1 depletion of IL-2–producing T cells reduced the production of Lm-specific immunoglobulin G (IgG) by more than 90% compared with treatment with an isotype control antibody (Fig. 5D). Similarly, anti-Thy1.1 treatment of 2BiT mice immunized with OVA under type 1 conditions markedly impaired the anti-OVA IgG response, in association with the depletion of endogenous OVA-specific TFH cells and reduction of germinal center B cells (fig. S8).

To examine the effects on GC-TFH cell differentiation, we transferred equivalent numbers of congenically marked 2BiT and GFP-expressing WT CD4+ T cells into T cell–deficient (Tcrb–/–) mice, which were immunized with HKLm (Fig. 5, E and F). Anti-Thy1.1 treatment selectively depleted CD45.1+ 2BiT PD-1+CXCR5+ T cells and had no significant effect on CD45.2+ GFP+ control T cells (Fig. 5E). The magnitude of reduction was highest among PD-1hiCXCR5hi cells. Immunohistology revealed that Thy1.1 depletion dramatically reduced the number of 2BiT CD4+ T cells found within germinal centers, with a compensatory increase in the numbers of WT GFP+ T cells, resulting in no change in total numbers of germinal center T cells or B cells (Fig. 5F). These data establish that functional TFH effectors that populate germinal centers and provide help for class-switched antibody responses in response to type 1 pathogens develop from IL-2–producing precursors.

To extend these findings, we determined whether the in vivo depletion of IL-2+ cells also selectively targeted TFH cells under conditions of type 2 (TH2) and type 3 (TH17) immune induction (Fig. 6). Anti-Thy1.1 treatment of 2BiT mice immunized with OVA by using the TH2-inducing adjuvant alum resulted in the specific depletion of OVA-specific TFH cells and ablation of germinal center B cell and anti-OVA antibody responses, while sparing OVA-specific non-TFH effectors (Fig. 6A). Similarly, anti-Thy1.1 depletion of 2BiT mice challenged with the TH17 cell–inducing enteric pathogen Citrobacter rodentium (28) resulted in a loss of bacterial clearance, the kinetics of which were characteristic of an impaired anti-Citrobacter antibody response (29, 30). This was associated with depletion of TFH cells specific for the immunodominant Citrobacter antigen intimin, as well as a markedly impaired germinal center B cell response (Fig. 6B). There was no significant decrease in IL-17A– or interferon-γ (IFNγ)–producing cells in infected spleens (Fig. 6C), both of which are characteristic of the T cell response against Citrobacter (28). However, a modest, but significant, decrease in TH17 cells, but not TH1 cells, was observed in the mesenteric lymph nodes of mice depleted of IL-2 producers (Fig. 6D). These findings indicate that, in the context of type 3 responses, there is some overlap in the developmental fate of IL-2–producing precursors of TFH and TH17 effector cells. They also establish that IL-2+ T cells are precursors for TFH cells in the context of type1, type 2, and type 3 effector responses.

Fig. 6 IL-2 producers are fated to become TFH cells in type 2 and type 3 immune responses.

(A) 2BiT mice were injected with 250 μg of anti-Thy1.1 or isotype control. After 24 hours, they were immunized with OVA emulsified in alum. Mice were bled and sacrificed on day 12. Splenic IAb-OVA tetramer-specific CD4+ T cells were analyzed by flow cytometry for the expression of CD44, PD-1, and CXCR5. Splenic B cells were analyzed for the expression of B220, IgD, GL7, and Fas. Serum anti-OVA IgG was measured by ELISA. n = 5 or 6 mice per group. (B) 2BiT mice were injected with 250 μg of anti-Thy1.1 or isotype control. After 24 hours, they were orally gavaged with 1 × 109 to 2 × 109 CFU C. rodentium strain DBS100 (ATCC 51459) or the bioluminescent ICC180 derivative. Whole-body bioluminescence was tracked and quantified after infection. Splenic IAb-Int884C tetramer-specific CD4+ T cells harvested on day 14 were analyzed by flow cytometry for the expression of CD44, PD-1, and CXCR5. Splenic B cells were analyzed for the expression of B220, IgD, GL7, and Fas. n = 5 or 6 mice per group. Flow plots depict cell number–controlled concatenated averages. Data are representative of two experiments. (C and D) Splenic (C) and mesenteric lymph node (MLN) (D) CD4+ T cells harvested from mice treated as in (B) were isolated, restimulated with phorbol myristate acetate (PMA) and ionomycin and then analyzed by flow cytometry for the expression of CD44, Foxp3, IFNγ, and IL-17A. Flow plots and bar graphs are gated on CD4+CD44+Foxp3 cells. Flow plots depict cell number–controlled concatenated averages. n = 5 or 6 mice per group. Data were analyzed by using Student’s t tests. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; error bars depict SEM.

IL-2 production and TFH cell differentiation correlate with TCR signal strength

The developmental divergence of TFH and non-TFH cells is influenced by a combination of cell-intrinsic factors, including TCR affinity, and cell-extrinsic factors, such as antigen availability, strength of costimulation, and cytokine milieu. Given its direct correlation with TFH–non-TFH cell fate determination, we examined whether IL-2 expression shared similar mechanistic underpinnings. To examine the relationship between antigen dose, IL-2 expression, and TFH–non-TFH cell specification on T cells of uniform TCR affinity, naïve CD45.2+ IL-2.eGFP-SMARTA T cells were transferred into WT CD45.1+ recipients, which were infected with various doses of ActA-Lm expressing the gp66 peptide (ActA-Lm-OVA-gp66; condition Lm-gp66). Nonspecific effects of Lm-induced inflammatory signals were excluded by keeping the total dose of ActA-Lm constant via coinfection with ActA-Lm expressing an irrelevant specificity (ActA-Lm-OVA-2W1S; condition Lm-2W1S) (Fig. 7, A and B). Splenic CD45.2+ cells were analyzed for CD69 and GFP (IL-2) expression around the peak of IL-2 expression (Fig. 7A). The frequencies and absolute numbers of endogenous gp66-specific TFH and non-TFH cells were quantified near the peak of the effector T cell response (Fig. 7B). The expression of both CD69 and GFP correlated tightly with pathogen-expressed antigen dose, as did the magnitude of clonal expansion and reciprocal TFH versus non-TFH differentiation. There was a similar correlation over a broader dose range of ActA-Lm-OVA-gp66 administered alone (fig. S9). Thus, the frequencies of CD69+ and IL-2+ cells correlated positively with Lm-gp66 dose, as did the generation of TFH cells. This indicates that antigen dose—and consequently TCR signal strength—is a major determinant of the fraction of clonal precursors that express IL-2 and are fated to become TFH cells.

Fig. 7 IL-2 production and TFH differentiation correlate with TCR signal strength.

(A) WT CD45.1+ recipient mice were infected with ActA-Lm-OVA-gp66 and/or ActA-Lm-OVA-2WIS at the indicated doses. After 24 hours, 106 naïve (GFPCD44CD62L+) SMARTA IL-2.eGFP CD4+ T cells were adoptively transferred into infected hosts. Splenic CD4+ T cells were harvested 15 hours after transfer and analyzed for expression of CD69 and IL-2.eGFP by flow cytometry. Values in the larger boxes of flow cytometric plots represent percentages of CD69+ cells, and values in the smaller boxes represent percentages IL-2.eGFP+ cells within the CD69+ fraction. n = 4 mice per group. Data are representative of two experiments. (B) WT mice were infected with ActA-Lm-OVA-gp66 and/or ActA-Lm-OVA-2WIS at the indicated doses. After 5 days, magnetically enriched endogenous splenic CD4+ T cells were analyzed by flow cytometry for binding of IAb-gp66 tetramer and expression of CD44, CXCR5, and PD-1. n = 3 mice per group. Data are representative of two separate experiments. (C) 2D affinity measurements were performed on splenic TCR-transgenic CD4+ T cells via micropipette adhesion frequency assays with biotinylated p:MHC IAb-gp66 and IAb-OVA3C monomers. Log-normalized data are shown. WT CD45.1+ recipient mice were infected with ActA-Lm-OVA-gp66. After 24 hours, 0.5 × 106 naïve (GFPCD44CD62L+CD69CD25) SMARTA IL-2.eGFP and OTII IL-2.eGFP CD4+ T cells were pooled and adoptively transferred into infected hosts. Splenic CD4+ T cells were harvested 18 hours after transfer and analyzed for expression of CD45.1, CD45.2, Vβ5, CD69, and IL-2.eGFP by flow cytometry. Values in the larger boxes of flow cytometric plots depicting CD69 and IL-2.eGFP represent percentages of CD69+ cells, and values in the smaller boxes represent the percentages of IL-2.eGFP+ cells within the CD69+ fraction. n = 3 mice per group. Data are representative of two experiments. Ac Ka, is the 2D affinity (see methods for details). (D) WT mice were infected with ActA-Lm-OVA-gp66. Five days after infection, magnetically enriched endogenous splenic CD4+ T cells were costained with IAb-gp66 and IAb-OVA3C tetramers and analyzed by flow cytometry for expression of CD44, CXCR5, and PD-1. Data are representative of four experiments. (E) WT mice were infected with 2.5 × 107 CFU ActA-Lm-OVA-gp66. Enriched splenic CD4+ T cells harvested 5 days after infection were stained for IAb-gp66, CD44, TCRβ, PD-1, and CXCR5. Log-normalized 2D affinity measurements were performed on FACS-purified IAb-gp66 tetramer-positive splenic TFH and non-TFH cells pooled from three to five animals. TCRβ quantifications were performed on unsorted aliquots stained separately. Data from two experiments are shown. (F) Naïve (GFPCD44CD62L+) SMARTA IL-2.eGFP CD4+ T cells were stimulated for 16 hours with irradiated CD4-depleted feeder cells and 1 μg/ml of gp66 and analyzed by flow cytometry for expression of CD69, Vα2, and IL-2.eGFP. Data are representative of two experiments. Data were analyzed by using Student’s t tests. ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; error bars depict SEM.

The relative development of TFH and non-TFH effectors is also influenced by TCR affinity, which is invariant on individual T cell clones but varies between different clones within the repertoire (2). In agreement with the strong correlation between differential IL-2 expression and TFH–non-TFH cell bifurcation, we found that two clonal populations of the same precursor frequency, which had differing TCR specificities (OVA versus gp66) and affinities, produced significantly different frequencies of IL-2–expressing T cells in response to the same antigen dose despite no difference in the frequency of cells expressing CD69 (Fig. 7C). Similarly, the relative frequencies of endogenous TFH and non-TFH cells generated by two TCR specificities of differing TCR affinities diverged in response to the same antigen dose (Fig. 7D). In accord with these results, TFH cells were characterized by a significantly greater two-dimensional (2D) TCR affinity than non-TFH cells that developed in a polyclonal T cell response to the same antigen (gp66) (Fig. 7E). Together with our antigen-dose results, these findings support a deterministic function of TCR signal strength in driving TFH versus non-TFH cell development (2), such that higher TCR signaling favors TFH cell development.

To calculate 2D affinity values, normalized adhesion-bond measurements must be adjusted to control for TCR density (31). Although equivalent in terms of cell size, gp66-specific TFH cells expressed more TCR molecules per cell than non-TFH cells (Fig. 7E). Similar results were found for OVA-specific and total TFH cells. Accordingly, the difference in the normalized adhesion bond was accounted for by differences in both TCR affinity and TCR number per cell. Thus, we examined the influence of variation in TCR number on T cell activation and IL-2 production (fig. S10A). As expected, there was no difference in 2D TCR affinity of CD69+IL-2+ SMARTA IL-2.eGFP T cells compared with that of CD69+IL-2 cells. However, when naïve SMARTA IL-2.eGFP T cells were stimulated with limiting concentrations of gp66 peptide, only cells expressing the highest amounts of the TCR component Vα2 up-regulated CD69 and IL-2 (Fig. 7F). Moreover, when naïve T cells were sorted on the basis of high or low TCRβ expression and stimulated with a range of anti-CD3 concentrations, cells with higher TCR numbers showed higher CD69 expression across the full range of anti-CD3 concentrations and expressed increased IL-2 (fig. S10B). Thus, in addition to intrinsic TCR affinity differences between T cell clones, variation in TCR number within a clonal population may influence the probability that a given cell will exceed a threshold for activation and expression of IL-2 and, therefore, TFH versus non-TFH cell differentiation.

The expression of CD69 by activated naïve T cells has been shown to correlate linearly with the expression of Myc and Nur77, providing an indicator of graded TCR signal intensity (23, 32). Yet, on the basis of the observed limitation of IL-2 expression to a subset of the highest CD69 expressors (Figs. 3A and 7A), our findings suggested that only those T cells that exceeded a minimum TCR signaling intensity produced IL-2, linking TFH–non-TFH cell bifurcation to a threshold of TCR signaling.

To explore this further, the relative expression of CD69 and IL-2 by naïve T cells was assessed under conditions in vitro for which only the intensity of TCR stimulation was varied (Fig. 8A). The percentage of cells expressing CD69 and IL-2 correlated positively with the dose of anti-CD3, as did expression amounts of CD69 and IL-2, consistent with our in vivo antigen-dose experiments (Fig. 7A and fig. S8A). However, although the distribution and mean expression of CD69 varied with the intensity of TCR stimulation, CD69 expression among IL-2+ cells was constant, and only cells that exceeded an invariant, high magnitude of CD69 expressed IL-2. A similar effect was seen with the induction of inducible costimulator (ICOS) expression (fig. S11A), which is a functional marker of TFH cell differentiation (33). ICOS expression was highest among IL-2+ cells across a range of stimulation conditions. Although the mean expression of ICOS varied with the intensity of TCR stimulation among IL-2 cells, its expression by IL-2+ cells was constant. The expression of ICOS ligand by antigen-presenting cells was not required for the induction of IL-2 (fig. S11B). Thus, under conditions for which only TCR signaling strength is varied, there is a minimum threshold for IL-2 expression and, by extension, TFH cell differentiation.

Fig. 8 IL-2 producers and TFH exhibit enhanced cell-cycle progression.

(A) Naïve (GFPCD44CD62L+CD69CD25) IL-2.eGFP CD4+ T cells were stimulated for 18 hours with indicated concentrations of plate-bound anti-CD3 and 1 μg/ml of soluble anti-CD28. They were then analyzed for the expression of CD69 and IL-2.eGFP by flow cytometry. The MFI of CD69 expression within the CD69+GFP and CD69+GFP+ gates was quantitated for the indicated concentrations of anti-CD3 (right). Three technical replicates per condition are shown. This experiment was performed three times. (B) Naïve (GFPCD44CD62L+CD69CD25) IL-2.eGFP CD4+ T cells were stimulated in vitro with soluble anti-CD3 (2.5 μg/ml), soluble anti-CD28 (0.5 μg/ml), and irradiated CD4-depleted feeder cells. qPCR was performed on CD69+ IL-2.eGFP+ and IL-2.eGFP CD4+ T cells sorted 20 hours after activation. Three technical replicates per condition are shown. Data are representative of two experiments and were analyzed by one-way ANOVA. (C) WT CD45.1+ recipient mice were infected with ActA-Lm-OVA-gp66. After 24 hours, 5 × 104 CTV-labeled naïve (GFPCD44CD62L+CD69CD25) SMARTA CD4+ T cells were adoptively transferred into infected hosts. Splenic CD4+ T cells harvested 3 days after transfer were analyzed for expression of CD44, PD-1, and CXCR5 by flow cytometry. n = 4 mice per experiment. This experiment was performed three times. ns, P > 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; error bars depict SEM.

The expression of Myc, which has been directly correlated with the number of cell divisions that T cells are fated to undergo (24), was tightly correlated with CD69 expression and thus TCR signal intensity and IL-2 expression (Fig. 8B). The early expression of E2F family members, which are associated with cell-cycle entry, was limited to cells that exceeded a threshold for IL-2 expression. This was consistent with the GSEA data (Fig. 4B and fig. S4), which indicated that E2F family targets are strongly enriched in IL-2+ cells compared with IL-2 cells. This suggests that IL-2+ cells enter the cell cycle more rapidly than IL-2 cells and are likely to undergo a greater number of cell divisions before exiting the cell cycle. Accordingly, TFH effectors demonstrated an increase in the average number of cell divisions relative to non-TFH effectors in vivo (Fig. 8C). Thus, although IL-2 has traditionally been viewed as a T cell growth factor, precursors of TFH cells, which do not respond to IL-2 despite producing it, appear programmed for earlier cell-cycle entry and a greater number of cell divisions than precursors of non-TFH effectors, which do respond to IL-2.

Our results support a model whereby cell-intrinsic and -extrinsic variables that influence TCR signal strength contribute to a threshold that is tightly associated with IL-2 production and TFH cell differentiation. Although TCR affinity and antigen dose clearly contribute to the probability that a cell will attain this threshold, costimulation and cytokine signals can also contribute. It has been shown that transforming growth factor–β (TGFβ) signaling can attenuate TCR signaling (34) and limit T cell responses to high-affinity antigens (35). Indeed, the addition of TGFβ significantly reduced the mean expression of CD69 by activated naïve T cells, the proportion of CD69+ cells that produced IL-2, and the mean expression of IL-2, whereas TGFβ blockade had the opposite effect (fig. S12). However, the percentage of cells that expressed CD69 was unaltered by either intervention. Thus, in addition to modulating TCR-independent signaling cascades that control effector T cell specification, TGFβ may also influence IL-2 production by limiting TCR-signal strength without limiting the frequency of naïve T cells that receive activating TCR signals (36). In this regard, it is notable that the addition of IL-6 overrode the repressive effects of TGFβ (TH17 cell conditions; Fig. 3A), resulting in significantly higher IL-2 expression. Indeed, this was greater than any other T effector–polarizing condition. Because the major effects of IL-6 are independent of TCR-signal strength and its actions contribute to both TFH and TH17 cell development, clearly, TCR-independent factors that modulate IL-2 production may affect TFH–non-TFH cell developmental decisions. Furthermore, in view of the shared requirement for IL-6–induced Stat3 signaling and high production of IL-21 by both TH17 and TFH cells, these data suggest a considerable overlap in the developmental programming of these two subsets and, perhaps, shared regulation of the tightly syntenic Il2 and Il21 loci (Fig. 2A).

Discussion

The findings in this study provide new insights into the mechanics that control the early bifurcation of CD4+ T cells into TFH and non-TFH effectors, placing reciprocal production and utilization of IL-2 at the center of this key developmental decision. Because divergent IL-2 signaling and Bcl6 expression have been linked to effector versus central memory, respectively (37), these results may also have implications for alternate programming of CD4+ T cell memory. Our findings predict that IL-2 nonproducers are fated for effector memory, whereas IL-2 producers are fated for central memory.

It has been proposed that asymmetric cell division results in the partitioning of factors that guide the divergent development of progeny of activated naïve T cells (38). Although the observation herein that TFH–non-TFH cell fate determination is initially encoded well before cell division does not preclude a role for asymmetric cell division, it does suggest that signaling between T cells that receive differing activation signals likely plays a dominant role. It has been shown that homotypic T cell conjugation mediated by LFA-1–ICAM interactions between activated T cells facilitates directional, paracrine delivery of IL-2 via multifocal synapses (39). The expression of Icam1 was enhanced on IL-2 cells in the current study (Figs. 1C and 4A). Because the kinetics of IL-2 production are within the average dwell time of T cells that form long-lived interactions on an activating dendritic cell (4042), our findings suggest that T cell–T cell interactions that result in directional IL-2 signaling between IL-2 producers and nonproducers may occur on the same dendritic cell, although this will necessitate further study. The role of Treg cells in buffering IL-2 availability to non–IL-2 producers, owing to the constitutive expression of the high-affinity IL-2 receptor and high-avidity LFA-1 by Treg cells will also require additional inquiry.

Results in this report indicate that naïve CD4+ T cells that receive differing strengths of TCR signals—whether the result of intrinsic TCR affinity or expression differences or receipt of contemporaneous non-TCR signals that augment or repress TCR signal strength—stratify into those that exceed a threshold that predisposes to IL-2 production and early TFH cell commitment and those that fail to express IL-2 yet are programmed to receive IL-2 signaling that reinforces non-TFH effector commitment. However, although the exceedance of this threshold appears necessary, it is not always sufficient, because some cells that express comparable levels of CD69 do not express IL-2, implying the contribution of additional factors yet to be defined. Moreover, although in a primary response, IL-2 expression strongly correlates with the development of TFH versus non-TFH cells, this correlation is not fixed for subsequent responses (fig. S13). Thus, IL-2 expression by TFH cell precursors does not ensure IL-2 expression by TFH effectors in a recall response nor does lack of IL-2 expression by non-TFH cell precursors preclude IL-2 expression by non-TFH cell effectors. Nevertheless, the utility of IL-2 as an early marker for cells fated to these different effector programs is established herein. This will offer the opportunity to discover new factors that determine the bifurcation into TFH and non-TFH effectors, as exemplified by the finding of a possible Mxd-Myc-Max axis in controlling the early differential expression of Bcl6. We propose that this should provide a basis for strategies to modulate the balance of effector T cell responses for therapeutic ends.

Materials and methods

Mice

B6.Cg-Tg-IL2tm1(eGFP)Weav (IL-2.eGFP) and B6.IL-2.BAC-inThy1.1 (2BiT) were generated using strategies previously described (25) and bred at the University of Alabama at Birmingham (UAB) animal facility. B6N-Tyrc-Brd/BrdCrCrl (albino B6) and B6-LY5.2/Cr (congenic B6 CD45.1) were purchased from Frederick Cancer Center and intercrossed to produce albino B6.CD45.1. C57BL/6 (WT B6), Tcrb–/– (B6.129P2-Tcrbtm1Mom/J), OT-II (B6.Cg-Tg(TcraTcrb)425Cbn/J) mice and mice transgenic for constitutive eGFP expression (C57BL/6-Tg(CAG-EGFP)1310sb/LeySop/J) were purchased from The Jackson Laboratory. SMARTA Tg [Tg(TcrLCMV)1Aox] (43) on a B6 background were a generous gift from A. Zajac (Department of Microbiology, UAB). All intercrosses to generate additional strains, such as SMARTA IL-2.eGFP, SMARTA 2BiT, SMARTA IL-2.eGFP Thy1.1+, OT-II IL-2.eGFP, and 2BiT CD45.1 were generated by crosses in UAB’s breeding facility. Animals were bred and maintained under specific pathogen-free conditions in accordance with institutional animal care and use committee regulations.

Tissue processing and flow cytometric analysis

Mice were sacrificed by isoflurane euthanisia before removal of spleen and/or lymph nodes. Secondary lymphoid tissues were disrupted by mashing with a syringe in complete RPMI-1640 over a 70-μm filter. Surface staining was performed in PBS with 2% FBS and 0.1% sodium azide. T cells from 2BiT animals were directly stained for surface Thy1.1 (clone HIS5-1) without secondary stimulation. For identification of TFH, cells were incubated with biotinylated anti-CXCR5 for 1 hour at room temperature, then washed and incubated with streptavidin-APC or PE-Cy7 and additional surface markers for 20 min at 4°C. Intracellular staining for transcription factors was performed using either BD Fix/Perm or eBioscience Foxp3 staining kits. For ex vivo phospho-Stat staining, freshly harvested splenocytes were fixed for 10 min at 37°C in 4% PFA in PBS, stained with eFluor450- or PacBlue-conjugated anti-Thy1.1, refixed with 4% PFA in PBS and permeabilized in 90% MeOH for 30 min on ice. Following this, cells were stained for phosphorylated Stat5 and additional markers at room temperature for 1 hour. Alternatively, cells were fixed in 4% PFA in PBS for 10 min at 37°C, permeabilized in 90% MeOH for 30 min on ice, and then stained for 1 hour at room temperature.

Absolute T and B cell numbers were calculated using PKH reference beads (Sigma- Aldrich) or concentration values (events per microliter) obtained on the Attune NxT. Absolute TCRβ numbers were calculated using BD Quantibrite Beads Fluorescence Quantification Kits. Briefly, cells were stained at saturating concentrations of PE-labeled anti-TCRβ (clone H57-597). A standard curve generated from PE Quantibrite Beads was then used to transform TCRβ MFI measurements into absolute quantifications.

All flow cytometric data were acquired on an Attune NxT (Thermo Fisher Scientific), LSRII, or an Aria II (BD Immunocytometry Systems, San Jose, CA) and analyzed with FlowJo software (Tree Star, Eugene, OR).

T cell isolation

Naïve T cell isolation

Polyclonal CD62LhiCD25CD44loCD4+ and CD8+ T cells were purified from single-cell suspensions of secondary lymphoid tissues (spleen with or without axillary, brachial, cervical, mesenteric, inguinal, and medial iliac lymph nodes) in two stages. First, CD8+ or CD4+ T cells were isolated using Dynabeads (ThermoFisher 11445D), MACS Cell Separation (Miltyeni Biotec 130-104-454) or prepared by negative selection against CD8 or CD4, MHC class II, CD11b, B220, and CD25 (all Abs labeled with FITC) using anti-FITC BioMag particles (Polysciences, Warrington, PA). Second, cells were purified by sorting on a FACSAria II, gating on the CD4+CD25CD62LhiCD44lo (and in some cases IL-2.Thy1.1/IL-2.eGFP) fraction. Naïve SMARTA TCR Tg cells were sorted directly as above from lymph node and splenic tissue.

Activated T cell isolation

Cells cultured in vitro were harvested at various time points, resuspended in labeling buffer (2% FBS in PBS), and FAC-sorted a second time as CD4+ or CD8α+ CD69+ and either IL-2.eGFP+/Thy1.1+ or as IL-2.eGFP/Thy1.1. SMARTA IL-2.eGFP T cells isolated ex vivo from acutely activated recipient mice were processed from tissue by negative selection with biotinylated antibodies to CD11b, CD11c, and B220, streptavidin-conjugated microbeads, and LS columns (Miltenyi Biotech). Column flow-through fractions were then stained for a congenic marker (Thy1.1, Thy1.2, or CD45.2) before sorting as above.

In vitro T cell activation

Sorted naïve T cells were activated in complete RPMI-1640 (RPMI medium containing 10% FBS, 100 IU/ml penicillin, 100 μg/ml streptomycin, 1 mM sodium pyruvate, nonessential amino acids, 50 μM β-mercaptoethanol and 2 mM l-glutamine) for 4 to 36 hours with anti-CD3 (2.5 μg ml−1) or 1 μg ml−1 LCMV glycoprotein peptide 66-77, anti-CD28 (1 μg ml−1), and irradiated splenocytes at a 5:1 ratio of splenocytes to T cells under nonpolarizing conditions (i.e., without additional cytokines or antibodies). In some experiments, sorted naïve T cells were activated with a range of plate-bound anti-CD3 concentrations and 0.5 μg/ml anti-CD28 or a range of soluble anti-CD3 concentrations and irradiated feeders at a CD4:feeder ratio of 1:5.

For restimulation of splenic TFH and non-TFH cells, magnetically enriched splenic CD4+ T cells were stained simultaneously with tetramer and biotin-labeled anti-CXCR5 (see table above) for 1 hour at room temperature. Cells were then washed and stained with fluorophore-labeled streptavidin and PD1 for 20 min at 4°C. Labeled cells were then incubated for 4 hours in complete RPMI with 2 μg/ml anti-CD28 on flat-bottom 96-well plates precoated with 5 μg/ml anti-CD3. Following restimulation, cells were stained for CD69, CD44, CD4, additional surface markers, and viability dye for 15 to 20 min at 4°C.

Adoptive transfer and Ab-mediated in vivo depletion

For adoptive transfer experiments examining IL-2.eGFP or IL-2.Thy1.1 expression at early time points, 1 × 106 to 2.5 × 106 naïve cells were injected i.v. into congenic recipient mice infected 24 hours before transfer unless otherwise indicated. For experiments involving cotransfer of Smarta.IL-2.eGFP and OT-II.IL-2.eGFP donor cells, 5 × 105 sorted naïve cells of each donor strain were injected retro-orbitally (RO) into mice infected 24 hours before transfer. For adoptive transfer experiments examining TFH differentiation three or more days after transfer, 5 × 104 sorted naive donor cells were injected RO into congenic recipients infected 24 hours before transfer unless otherwise indicated. For cotransfer into TCRβ KO recipients, 1 × 106 magnetically enriched bulk CD4+ T cells from wild-type B6 or transgenic eGFP and CD45.1 2BiT congenic mice were injected RO into TCRβ-deficient mice followed by infection 1 day after transfer. For depletion of IL-2.Thy1.1 (2BiT) cells in vivo, mice were given a single intraperitoneal injection of 250 μg of anti-Thy1.1 or isotype monoclonal antibody 24 hours before infection or immunization.

Infections and protein immunizations

Lm

Mice were immunized i.v. with 200 μl of PBS containing live (dose as indicated) or heat-killed Actin A–deficient Listeria monocytogenes (ActALm; 2 × 109 to 2 × 1010) (18). All Lm strains used were transformed by a plasmid containing OVA250-387 and one of four different IAb–specific “foreign” peptides: (i) a mutant epitope of I-Ea (“2W1S”); (ii) flagellin peptide 456-475 from Clostridium (“Cbir1”); (iii) glycoprotein 66-77 peptide of LCMV (“gp66”); or (iv) flagellin peptide 427-441 from Salmonella typhimurium (“FliC”) all expressed under the control of the hly (listeriolysin O) promoter. All Lm strains were produced in the laboratory of S.-S. Way as previously described (44). Bacteria were grown in brain-heart infusion (BHI) medium with 15 μg/ml chloramphenicol to an absorbance of >0.1 at 600 nm, and doses varied as indicated. The actual number of live bacteria injected was confirmed by dilution and growth on BHI agar plates containing chloramphenicol.

OVA/CFA

Mice were immunized i.p. with 100 μl of a 100-μg chicken egg ovalbumin emulsion in CFA.

OVA/Alum

Mice were injected i.p. with 200 μl of a 0.5-mg/ml emulsion of chicken egg ovalbumin in alum. The emulsion was prepared by mixing 1 mg/ml ovalbumin dissolved in water (Invivogen vac-pova) 1:1 with alum (FisherScientific Imject Alum 77161).

Citrobacter rodentium

Mice were orally gavaged with 1 × 109 to 2 × 109 CFU of Citrobacter rodentium strain DBS100 (ATCC 51459) or the bioluminescent ICC180 derivative (generously provided by G. Frankel and S. Wiles, Imperial College London). Mice infected with the ICC180 derivative were shaved and imaged with an IVIS 100 Imaging System (Xenogen, Inc.) as previously described (28).

RNA-seq and analysis

For sample preparation and hybridization, total RNA was isolated from purified naïve (CD4+ or CD8a+, CD25CD69CD44loCD62L+ IL-2.eGFP) and activated (CD4+ or CD8a+, CD69+ IL-2.eGFP+ or IL-2.eGFP) T cells with Qiazol and miRNeasy micro kits according to manufacturer’s recommendations (Qiagen). Library preparation was performed using Illumina TruSeq techonology. Samples were processed at UAB Heflin Center for Genomic Science for Next Generation Sequencing (NGS) or La Jolla Institute (LJI) using the Illumina HiSeq 2000 Sequencing System. Reads were mapped to the mm10 genome using TopHat (version 2.0.12) (45). BAM files were sorted using SAMtools (version 0.1.19) (46), and reads were counted for each gene using HTSeq (version 0.6.1) (47) and NCBI Mus musculus Annotation Release 106 (GRCm38.p4). RNA expression was normalized using the rlog function from the DEseq2 R package (version 1.8.2) (48). Differential gene expression analysis was performed using DEseq2, and P values were corrected with the Benjamini-Hochburg procedure. Volcano plots were created using the ggrepel R package. To calculate gene set enrichment, a differential expression probability density function (PDF) was determined for each gene using Quantitative Set Analysis for Gene Expression (QuSAGE) (49). PDFs were combined for each gene set to calculate gene set activity after correcting for gene-gene correlation. Gene set PDFs were compared using Welch’s t test, and P values were adjusted using the Benjamini-Hochberg procedure.

Statistical analysis

Experimental P values were calculated using unpaired Student’s t tests, Welch’s t tests, or one-way or two-way ANOVA tests with Tukey’s post hoc multiple comparisons analysis. A P value of <0.05 was considered significant. See figure legends for details.

Supplementary Materials

www.sciencemag.org/content/361/6407/eaao2933/suppl/DC1

Materials and Methods

Figs. S1 to S13

References (5057)

References and Notes

Acknowledgments: The authors thank members of the Weaver Lab, L. Harrington, H. Hu, S. Kaech, A. Weinmann, and A. Zajac for helpful discussions. We thank D. Wright and B. Dale for mouse breeding and genotyping. Funding: This work was supported by NIH grants R01 AI035783 and R01 DK113739 (C.T.W.), R01 AI110113 (B.D.E.), R01 AI107120 (J.J.M.), P30 DK04335 (J.J.M.), R21 AI124143 (J.J.M.), and DP1 AI131080 (S.S.W.). Trainee support was provided by NIH T32 AI007051 to C.J.W., D.D., D.P., and D.J.S. and by NIH F30 DK105680 (J.R.S.). Additional support was provided by UAB Institutional Funds (C.T.W.), March of Dimes Foundation (S.S.W.), HHMI Scholar’s Program (S.S.W.), Burroughs Wellcome Fund (S.S.W.), and the Milton Fund (J.J.M.). D.D., S.W., J.R.S., and C.G.W. are members of the UAB Medical Scientist Training Program (MSTP), supported by NIH T32 GM008361. We acknowledge the UAB Epitope Recognition and Immunoreagent Core Facility for provision of some antibodies used in this study. Author contributions: Author contributions are as follows: project conception, experimental design, and data interpretation by D.D., C.J.W., and C.T.W.; reporter mouse design, construction, and validation by C.J.W., D.D., R.J.L., H.T., and C.T.W.; RNA-seq studies and data analysis by C.J.W., S.W., R.D.H., and B.T.W.; ChIP-PCR and ATAC-seq experiments and data analysis by D.P. and R.D.H.; in vivo Listeria, Citrobacter, and immunization data collection, analysis, and interpretation by C.J.W., D.D., D.J.S., C.G.W., J.R.S., and C.T.W.; confocal microscopy by C.L.Z.; TCR-affinity and stimulation-strength experiments and data interpretation by D.D., R.A., E.M.K., R.J.M., and B.D.E.; additional ex vivo experiments by D.D., C.J.W., C.G.W., and J.R.S.; design, construction, and validation of MHCII tetramers and data interpretation by J.J.M.; design, construction, and validation of ActA Listeria strains and data interpretation by S.S.W.; and manuscript preparation and editing by D.D., C.J.W., and C.T.W. Competing interests: The authors declare no competing interests. Data and materials availability: RNA-seq data are deposited in the NCBI Gene Expression Omnibus under accession number GSE116608. Any additional data needed to evaluate the conclusions in this paper are present either in the main text or the supplementary materials.
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