SUMMARY

INTRODUCTION

The generation of long-lived plasma cells that secrete high affinity antibodies is critical for the establishment of protective immunity to influenza and other pathogens. The selection of long-lived B cells with high affinity BCRs is dependent on events that occur in the germinal center (GC) (Allen et al., 2007; Cyster, 2010; Kelsoe, 1996). GCs are structures found in the B cell follicles of reactive lymphoid tissues and support intense B cell clonal expansion, somatic hypermutation of immunoglobulin genes, affinity maturation of the B cell response and ultimately, the differentiation of both memory B cells and long-lived plasma cells (Allen et al., 2007; Cyster, 2010; Vinuesa et al., 2010). Given that the goal of most vaccination strategies is the generation of long-lived antibody responses (Cox et al., 2004), it is essential that we understand the factors that regulate GC formation and maintenance.

GCs are dependent on help provided by CD4⁺ T follicular helper (Tfh) cells - a recently defined CD4⁺ T cell subset with a unique ability to help B cell responses (Crotty, 2011; Yu and Vinuesa, 2010). B cells and Tfh cells provide each other with reciprocal differentiation and survival signals (King, 2011). Tfh cells provide CD40 ligand (Goodnow et al., 2010; Han et al., 1995) and IL-21 to B cells (Bryant et al., 2007; Linterman et al., 2010; Vogelzang et al., 2008) and B cells present antigen and provide ICOS ligand to Tfh cells (Choi et al., 2011; Deenick et al., 2011; Johnston et al., 2009; Nurieva et al., 2009). As a result, all three of these molecules are essential for germinal center formation and long-lived antibody responses (Goodnow et al., 2010). Importantly, the differentiation of Tfh cells depends on the expression of the transcription factor Bcl6, which represses the expression of other lineage-specific transcription factors and promotes the expression of CXCR5 (Johnston et al., 2009; Nurieva et al., 2009; Yu et al., 2009). In turn, CXCR5 facilitates homing to the B cell follicle and germinal center (Hardtke et al., 2005; Haynes et al., 2007). As a result, Bcl6-deficient CD4⁺ T cells do not properly express CXCR5, fail to migrate into B cell follicles and do not to support GC reactions. Thus, Bcl6 is considered the master regulator of Tfh differentiation (Johnston et al., 2009; Nurieva et al., 2009; Yu et al., 2009). However, the molecular mechanisms that regulate Bcl6 expression and the differentiation of Tfh cells in vivo are incompletely defined (Crotty, 2011; Yu and Vinuesa, 2010).

Recent results demonstrate that CD4⁺ T cells responding to LCMV can be separated based on the expression of the interleukin-2 receptor α (CD25) into CD25^hi and CD25^lo subsets (Choi et al., 2011). Importantly, CD25^hi T cells do not express Bcl6, suggesting a possible role for IL-2 in the segregation of effector and Tfh cells. IL-2 was originally defined as an essential T cell growth factor that promotes expansion of effector T cells and facilitates CD8⁺ memory T cell programming (Blattman et al., 2003; Malek, 2008; Malek and Castro, 2010; Williams et al., 2006). However, IL-2 signaling also controls the contraction of T cell responses by enhancing susceptibility of responding T cells to activation-induced cell death (Lenardo, 1991). IL-2 signaling through CD25 enhances CD25 expression and sensitivity to IL-2 and also controls the expression of key transcription factors, such as eomesodermin (Eomes), Blimp1 and Bcl6 in CD8 T cells (Pipkin et al., 2010).

The generation and maintenance of FoxP3-expressing CD4⁺ regulatory T cells (Tregs) are also dependent on IL-2 (Almeida et al., 2002; de la Rosa et al., 2004; Malek and Castro, 2010; Malek et al., 2002). Tregs suppress the activation of autoreactive T cells, prevent the development autoimmune disease (Campbell and Koch, 2011; Malek et al., 2002) and play an important role in the control of B cell responses (Alexander et al., 2011; Chung et al., 2011; Lim et al., 2005; Lim et al., 2004; Linterman et al., 2011). Given the essential role for IL-2 in the maintenance of Tregs, some investigators are using IL-2 therapy to treat autoimmune disease in mouse models (Grinberg-Bleyer et al., 2010; Humrich et al., 2010). In humans, IL-2 therapy has been used to enhance antitumor immunity (Kovacs et al., 2001; Rosenberg et al., 1985a; Rosenberg et al., 1985b), particularly in combination with B cell depletion in the treatment of non-Hodgkin lymphoma (Friedberg et al., 2002; Gillies et al., 2005; Gluck et al., 2004; Lopes de Menezes et al., 2007). Thus, IL-2 may be used to treat a variety of diseases.

Although IL-2 can modulate effector and Treg cell function by a variety of mechanisms, the role of IL-2 in the control of B cell and Tfh responses remain unclear. Here we tested the role of IL-2 in the GC B cell response to influenza. We found that IL-2 impaired the influenza-specific GC response, reduced the influenza-specific IgG response and prevented the accumulation of influenza-specific Tfh cells. We demonstrated that IL-2 did not directly inhibit GCs, but instead directly suppressed Tfh cell responses. We found that CD25 signaling triggered by IL-2 negatively modulated Bcl6 expression and Tfh cell development. These data demonstrate that the physiological availability of IL-2 is a critical factor that regulates successful Tfh and B cell responses in vivo.

RESULTS

IL-2 treatment impairs GC B cell formation and antibody responses following influenza infection

To determine the role of IL-2 in the B cell response to influenza, we infected C57BL/6 (B6) mice with influenza virus A/PR8/34 (PR8) and treated infected mice with 30,000 U of human recombinant IL-2 (rIL-2) or PBS twice a day starting three days after infection. On day 21 post-infection, we collected sera from rIL-2 treated and control mice and determined the titers of influenza-specific IgG by ELISA (Fig. 1A). We observed a substantial reduction in influenza-specific IgG1 titers in the rIL-2 treated group compared to control mice. To determine whether rIL-2 treatment specifically blocked the generation of influenza-specific IgG-secreting plasma cells that home to the bone marrow (BM) and provide long-lasting immunity to influenza, we enumerated influenza-specific IgG-secreting plasma cells in the BM on day 21 post-infection by ELISPOT. In agreement with the antibody titers, we found that the number of IgG-secreting influenza-specific plasma cells in the bone marrow was reduced in the BM of rIL-2-treated mice relative to control mice (Fig. 1B). Thus, our results indicate that rIL-2 treatment compromised influenza-specific humoral immune responses.

Open in a separate window

Figure 1

IL-2 inhibits B cell responses to influenza

B6 mice were infected with PR8 and treated with 30,000 U of human rIL-2 or PBS daily starting 3 days after infection. (A) Serum was obtained on day 21 after infection and titers of influenza-specific IgG1 were determined by ELISA. (B) Influenza-specific IgG-secreting cells in the BM were enumerated by ELISPOT on day 21 after infection. Data are representative of two independent experiments (mean ± s.d of 5 mice per group).

Given that GC formation is required for the differentiation of long-lived plasma cells (Johnston et al., 2009; Nurieva et al., 2009; Vinuesa et al., 2010; Yu et al., 2009), we hypothesized that rIL-2 treatment may inhibit the formation of GCs following influenza infection. To test this hypothesis, we infected B6 mice with influenza and treated them with either rIL-2 or PBS. On day 10 post-infection, we probed cryosections of mediastinal lymph nodes (mLNs) with fluorochrome-labeled PNA and anti-B220 to detect GCs. As expected, PNA⁺ GC structures were easily detected in the B cell follicles of control mice. However, although B cell follicles were present in rIL-2-treated mice, PNA⁺ GC structures were virtually undetectable after rIL-2 treatment (Fig. 2A). Consistent with the histological results, the frequency (Fig. 2B) and number (Fig. 2C) of CD19⁺PNA^hiFAS⁺ GC B cells were reduced in rIL-2-treated mice.

Open in a separate window

Figure 2

IL-2 impairs GC B cell responses to influenza

(A) B6 mice were infected with PR8 and treated with 30,000 U of human recombinant rIL-2 or PBS twice a day starting 3 days after infection. mLNs were obtained on day 10 after infection and cryosections were stained with anti-B220 (blue) and PNA (red) and analyzed by fluorescent microscopy. (B–C) Cells from the mLNs of mice treated with IL-2 or PBS were obtained on day 10 and the percentage of CD19⁺ B cells with a PNA⁺FAS⁺ GC phenotype was determined (B), and the number of GC B cells was calculated (C). Data are representative of four independent experiments (mean ± s.d of 5 mice per group). (D–E) Cells from mLN were obtained on day 10 after infection and GC B cells were identified by flow cytometry using an NP-tetramer. The percentage of NP-specific B cells with a PNA⁺FAS⁺ GC phenotype was determined (D) and the number of NP-specific PNA⁺FAS⁺ CD19⁺ B cells was calculated (E). Data are representative of three independent experiments (mean ± s.d of 5 mice per group). All P values were determined using a two-tailed Student´s t-test. See also Supp. Fig. 1.

To determine whether the IL-2 treatment impaired the development of influenza-specific GC B cell responses, we used fluorochrome-labeled recombinant influenza nucleoprotein (NP) tetramers to identify NP-specific PNA^hiFAS⁺ GC B cells. We found that rIL-2 treatment reduced the frequency of NP-specific PNA^hiFAS⁺ GC B cells by nearly 80% compared to control mice (Fig. 2D). In a control experiment, we demonstrated that the NP B cell tetramer only bound B cells from influenza-infected mice and not B cells from mice infected with other pathogens (Supp Fig. 1). Collectively, these data show that the influenza-specific GC B cell response was severely compromised in the presence of excess IL-2.

IL-2 indirectly suppresses the GC B cell response to influenza

To determine whether rIL-2 treatment acted directly on GC B cells, we generated 50:50 mixed BM chimeras using B6.CD45.1 bone marrow (BM) and Cd25^−/− BM (CD45.2). As expected, the cells that repopulated the chimeric mice were derived equally from both donors (Fig. 3A). We subsequently infected the chimeric mice with influenza, administered either rIL-2 or PBS, and determined the frequency of PNA^hiFAS⁺ GC cells within the B6 and Cd25^−/− CD19⁺ B cells on day 10. We found that B6 (CD45.1) and Cd25^−/− (CD45.2) B cells contained equal frequencies of GC B cells in both PBS-treated (Fig. 3B), and IL-2-treated mice (Fig. 3C). Although, the GC B cell response was diminished in the rIL-2 treated chimeras relative to that in the PBS-treated mice (Fig. 3B and C), the ratio of B6 to Cd25^−/− GC B cells was close to 1.0 in both groups (Fig. 5D). We observed similar results when we examined NP-specific GC B cell response of B6 and Cd25^−/− donors (Fig. 3E–G). These results suggest that IL-2 inhibited the development of GC B cells indirectly and not through a B cell intrinsic mechanism.

Open in a separate window

Figure 3

rIL-2 indirectly inhibits GC B cell response to influenza

(A–H) B6 mice were irradiated and reconstituted with a 50:50 mix of BM from wild type CD45.1 donors and Cd25^−/− donors (CD45.2) and the percentage of leukocytes that expressed either CD45.1 or CD45.2 was determined in the mLN 8 weeks after reconstitution (A). (B–D) Reconstituted mice were infected with PR8, treated daily with 30,000 U of PBS (B) or rIL-2 (C) starting 3 days after infection and the percentage of CD45.1⁺ or CD45.2⁺ CD19⁺ B cells with a PNA⁺FAS⁺ GC phenotype cells was determined on day 10. (D) The ratio of B6 to Cd25^−/− PNA⁺FAS⁺ CD19⁺ B cells was calculated. (E–G) Reconstituted mice were infected with PR8, treated daily with 30,000 U of rIL-2 or PBS starting 3 days after infection and on day 10 the percentage of CD45.1⁺ or CD45.2⁺ NP-specific CD19⁺ B cells with a PNA⁺FAS⁺ GC phenotype was determined in the mLN of mice treated with PBS (E) or with rIL-2 (F). (G) The ratio of B6 to Cd25^−/− PNA⁺FAS⁺ NP-specific CD19⁺ B cells was determined. Data are representative of three independent experiments (mean ± s.d of 3–5 mice five mice per group). P values were determined using a two-tailed Student´s t-test.

Open in a separate window

Figure 5

rIL-2 impairs the Tfh cell response to influenza

(A) B6 mice were infected with PR8 and the expression of Bcl6 and ICOS was evaluated on PD-1^hiCXCR5^hiCD4⁺ T cells and PD-1^loCXCR5^loCD4⁺ T cells on day 10. (B–D) B6 mice were infected with PR8, treated with 30,000 U of human rIL-2 or PBS twice a day starting 3 days after infection and cells from the mLNs were analyzed by flow cytometry on day 10. (B) The expression of Bcl6 in CD4⁺ T cells was evaluated. (C) The percentage of CD4⁺ T cells with a CXCR5^hiPD-1^hi Tfh phenotype was determined. (D) The number of CXCR5^hiPD-1^hi Tfh cells was calculated. (E–F) B6 mice were infected with PR8, treated with 30,000 U of human rIL-2 or PBS twice a day starting 3 days after infection and cells from the mLNs were analyzed by flow cytometry on day 6. (E) The percentage of CD4⁺ T cells with a CXCR5^hiPD-1^hi Tfh phenotype was determined. (F) The number of CXCR5^hiPD-1^hi Tfh cells was calculated. (G) Bcl6, ICOS and CD25 expression was evaluated on PD-1^hiCXCR5^hi NP-specific CD4⁺ T cells and PD-1^loCXCR5^lo NP-specific CD4⁺ T cells. (H) The expression of Bcl6 in NP-specific CD4⁺ T cells was evaluated. (I) The percentage of NP-specific CD4⁺ T cells with a CXCR5^hiPD-1^hi Tfh phenotype was determined. (J) The number of NP-specific CXCR5^hiPD-1^hi Tfh cells was calculated. (K) The number of NP-specific CXCR5^loPD-1^lo effector CD4⁺ T cells was calculated. (L) The expression of Bcl6 on CD4⁺CXCR5⁺PD-1^hi cells and NP-specific CD4⁺CXCR5⁺PD-1^hi cells from IL-2-treated and control mice was evaluated by flow cytometry. Data are representative of four independent experiments (mean ± s.d of 5 mice per group). P values were determined using a two-tailed Student´s t-test.

rIL-2 suppresses the GC B cell response to influenza by a Treg independent mechanism

IL-2 signaling is crucial for the generation and maintenance of FoxP3-expressing Tregs (Almeida et al., 2002; de la Rosa et al., 2004; Malek and Castro, 2010; Malek et al., 2002). Corresponding with this idea, we found more Tregs in influenza-infected mice that were treated with rIL-2 than in control mice (Fig. 4A). Since Tregs suppress B cell immune responses, we next tested whether the inhibitory effect of rIL-2 treatment on GC formation was mediated by enhanced Treg function. Therefore, we treated FoxP3-DTR mice (Kim et al., 2007) with diptheria toxin (DT), infected them with influenza virus and treated them with either rIL-2 or PBS. Treatment of Foxp3-DTR mice with DT efficiently depleted FoxP3 expressing cells even after rIL-2 administration (Fig. 4A). Infected mice were then administered rIL-2 on a daily basis, and we determined the frequency of PNA^hiFAS⁺ GC cells within CD19⁺ B cells on day 10 after infection. As expected, FoxP3-DTR treated with rIL-2 alone had a decreased frequency (Fig. 4B), and number (Fig. 4C) of GC B cells compared to untreated FoxP3-DTR mice. Importantly, the frequency (Fig. 4B), and number (Fig. 4C) of GC B cells were even more reduced in Treg-depleted mice after rIL-2 treatment. As a control, DT administration to B6 mice did not affect the accumulation of GC B cell after influenza infection (Data not shown). Thus, these results indicate that rIL-2 inhibited GC B cell development by a Treg independent mechanism.

Open in a separate window

Figure 4

Inhibition of the GC B cells response after rIL-2 treatment do not required FoxP3⁺ Tregs

(A) FoxP3-DTR mice were infected with PR8, administered PBS, or DT on days 0, 4 and 7 after infection, or received DT on days 0, 4 and 7 after infection together with 30,000 U of human recombinant rIL-2 twice a day starting 3 days after infection. Cells from the mLN were analyzed on day 10. The percentage of CD4⁺ T cells that expressed FoxP3 (A), and the percentage of CD19⁺ B cells with a FAS⁺PNA⁺ GC phenotype (B) were determined by flow cytometry. (C) The number of FAS⁺PNA⁺ GC B cells was calculated. Data are representative of two independent experiments (mean ± s.d of 5 mice per time point). P values were determined using a two-tailed Student´s t-test.

IL-2 directly suppresses the Tfh response to influenza

Since rIL-2 compromised the accumulation of Tfh cells, we next considered the possibility that rIL-2 acted directly on CD4⁺ T cells and inhibited Tfh development. To test this possibility, we generated mixed BM chimeras that contained a 50:50 mix of B6 and Cd25^−/− cells. Two months after reconstitution, we infected the chimeric mice with influenza, administered rIL-2 or PBS, and determined the frequency of Tfh cells within the B6 and Cd25^−/− compartments on day 10 after infection. We found a higher frequency of Tfh cells in the Cd25^−/− compartment than in the B6 compartment (Fig. 6A). Similar results were obtained when we analyzed the frequency of NP-specific Tfh cells (Fig. 6B). Thus, these results indicated that rIL-2 acts directly on T cells to inhibit Tfh development, and that the loss of Tfh cells is the likely cause of poor GC and antibody responses in rIL-2-treated mice.

Open in a separate window

Figure 6

IL-2 signaling directly inhibits Tfh responses to influenza

(A–B) B6 mice were irradiated and reconstituted with a 50:50 mix of BM from wild type CD45.1 donors and Cd25^−/− donors (CD45.2). Reconstituted mice were infected with PR8, treated daily with 30,000 U of rIL-2 starting 3 days after infection and cells from the mLN were analyzed on day 10. The percentage of CD45.1⁺ or CD45.2⁺ CD4⁺CXCR5⁺PD-1^hi Tfh cells (A) and NP-specific CD45.1⁺ or CD45.2⁺ CD4⁺CXCR5⁺PD-1^hi Tfh cells (B) was determined. Data were pooled from three independent experiments (mean ± s.d ). Representative plots gated on CD4+ T cells are shown. (C–D) B6 mice were irradiated and reconstituted with a 50:50 mix of BM from wild type CD45.1 donors and Cd25^−/− donors (CD45.2). Reconstituted mice were infected with PR8, and cells from the mLN were analyzed on day 10. The percentage of CD45.1⁺ or CD45.2⁺ CD4⁺CXCR5⁺PD-1^hi Tfh cells (C) and NP-specific CD45.1⁺ or CD45.2⁺ CD4⁺CXCR5⁺PD-1^hi Tfh cells (D) was determined. (E) The percentage of CD45.1⁺ or CD45.2⁺ total CD4+ T cells was determined. Plot is gated on CD4⁺ T cells. Data were pooled from three independent experiments (mean ± s.d ). Representative plots gated on CD4+ T cells are shown. P values were determined using a two-tailed Student´s t-test.

The previous results suggested that CD25 signaling negatively regulates Tfh development. If this was true, then we would predict a higher frequency of Tfh cells in the Cd25^−/− CD4⁺ T cell population compared to the B6 counterparts - even in untreated mice. To test this hypothesis, we infected B6:Cd25^−/− mixed BM chimeras with influenza and calculated the frequency of Tfh cells within the the B6 and Cd25^−/− compartments 10 days post infection. We found more Cd25^−/− Tfh cells than B6 Tfh cells when we looked at total Tfh cells (Fig. 6C) and NP-specific Tfh cells (Fig 6D). As a control, we also determined the relative frequencies of CD45.1 and CD45.2 cells in the total CD4⁺ T cell compartment. We found that the frequencies of B6 and Cd25^−/− T cells in the 50:50 chimeras were essentially identical (Fig. 6E). Thus, our results indicated that lack of CD25 did not impair CD4⁺ T cell development, but did promote the differentiation of Tfh cells.

DISCUSSION

Our data shows that the availability of IL-2 regulates the differentiation of Tfh cells and thus controls GC formation and long-lived antibody responses. In conditions when excess IL-2 is available, Tfh cells are suppressed, GCs do not form and influenza-specific IgG responses are reduced. Importantly, the lack of germinal center B cells in IL-2-treated mice is not a consequence of IL-2 signaling in B cells, but is the result of IL-2 signaling in T cells, which impairs Tfh differentiation. Moreover, although IL-2 signaling promotes the accumulation of Tregs, which could suppress Tfh and GCs, Tregs are not required for the ability of IL-2 to repress Tfh differentiation. Thus, our results demonstrate that IL-2 fine-tunes the balance of T effector and B cell helper aspects of the immune response to influenza.

An important functional consequence of impaired Tfh differentiation is the reduced number of GC B cells and influenza-specific IgG-secreting cells. Although we cannot easily measure the affinity of the influenza-specific antibody produced in IL-2-treated mice, we anticipate that the antibodies that are produced would be of low affinity due to lack of selection in the GC. Moreover, in addition to reductions in long-lived, influenza-specific IgG-secreting cells in the BM, we would also anticipate that there would be defects in the generation of influenza-specific memory B cells, as both these cell types are also thought to depend on events that occur in the GC. Thus, the ability of IL-2 to repress Tfh differentiation has multiple effects on the humoral immune response to influenza.

IL-2 is often perceived as a T cell growth factor for both CD4⁺ and CD8⁺ T cells (Malek, 2008). However, both CD4⁺ and CD8⁺ T cells respond to antigen and proliferate in mice lacking IL-2 or its receptor (Bachmann et al., 1995; Malek and Bayer, 2004; Williams et al., 2006). In fact, IL-2-deficient mice develop autoimmunity due to excessive effector T cell responses (Sadlack et al., 1995; Sadlack et al., 1993; Suzuki et al., 1995). This phenomenon can be attributed, in part, to poor Treg survival in the absence of IL-2 signaling (de la Rosa et al., 2004; Malek et al., 2002). Although Tregs clearly have the ability to impair CD4 T cell responses and even B cell responses (Alexander et al., 2011; Chung et al., 2011; Linterman et al., 2011), the role of IL-2 in preventing Tfh differentiation and GC formation does not appear to be dependent on Treg. Despite the presence of higher numbers of Tregs in IL-2-treated mice, IL-2 still suppresses GC formation when Tregs are eliminated. Moreover, only Cd25^+/+ Tfh are suppressed by the addition of IL-2 in B6:Cd25^−/− chimeric mice, despite the equal opportunity of Tregs to interact with both B6 and Cd25^−/− T cells. Thus, IL-2 acts directly on differentiating Tfh precursor cells, rather than indirectly via Treg-mediated suppression.

The transcription factor Blimp1 controls the terminal differentiation of B cells into antibody secreting plasma cells. As part of this pathway, Blimp1 represses the expression of Bcl6 (Johnston et al., 2009). Similarly, Blimp1 represses Bcl6 expression in T cells and promotes their terminal differentiation into effector cells (Crotty, 2011; Fazilleau et al., 2009; Poholek et al., 2010). Of course, impaired Bcl6 expression in T cells would also preclude the differentiation of Tfh cells (Johnston et al., 2009; Nurieva et al., 2009; Yu et al., 2009). Conversely, Bcl6 over-expression represses Blimp1 and favors Tfh cell development (Johnston et al., 2009). Thus, the balance between Blimp1 and Bcl6 expression is thought to control the relative commitment of CD4 T cells to effector or Tfh pathways (Crotty, 2011) (Choi et al., 2011).

Interestingly, Blimp1 expression is increased in response to IL-2 (Gong and Malek, 2007). Thus, we speculate that one way in which IL-2 signaling impairs Tfh responses to influenza is by altering the Blimp1:Bcl6 ratio. This view is in agreement our results, and previous results demonstrating that Blimp1 and Bcl6 are reciprocally expressed in CD25^hi and CD25^lo CD4⁺ T cells responding to LCMV (Choi et al., 2011). Therefore, we suggest that prolonged IL-2 signaling leads to increased Blimp1 expression and represses Bcl6 expression and thus impairs the differentiation or maintenance of Tfh cells. Interestingly, our data show that although excess IL-2 suppresses Tfh cells, there is not a corresponding increase in the number of effector CD4⁺ T cells. These results suggest that IL-2 signaling compromises the maintenance or survival of T cells committed to the Tfh pathway, rather than diverting influenza-specific CD4⁺ T cells from the Tfh pathway into the Th1 effector pathway.

Published results show that IL-2 controls the differentiation of T cells by regulating the expression of a variety of transcription factors (Pipkin et al., 2010). For example, IL-2 represses RORγt in CD4 T cells and prevents their differentiation into Th17 cells (Chen et al., 2011; Laurence et al., 2007; Pandiyan et al., 2011). IL-2 and STAT5 are also negative regulators of BCL6 in CD8⁺ T cells and consequently, IL-2 signaling promotes the expansion of effector CD8⁺ T cells at the expense of memory CD8⁺ T cells (Kalia et al., 2010; Pipkin et al., 2010). Similarly, STAT5 inhibits the differentiation of Tfh cells via the BLIMP-1-dependent repression of BCL6 (Johnston et al., 2012). Consistent with this idea, we suggest that IL-2 signaling impairs or prevents Bcl6 expression in primed CD4⁺ T cells, possibly via a STAT5-dependent mechanism (Johnston et al., 2012), and prevents their subsequent differentiation into Tfh cells. In the absence of Bcl6, Tfh precursors do not express CXCR5, do not home to the T cell area and do not receive the necessary signals from B cells to be maintained as Tfh cells (Choi et al., 2011; Deenick et al., 2011; Good-Jacobson et al., 2010). In contrast, once CD4⁺ T cells have differentiated into Tfh cells, they lose CD25 expression and the addition of IL-2 does not affect Bcl6 or CXCR5 expression. Thus, we believe that IL-2 acts early in the Tfh differentiation pathway.

One of the hallmarks of Tfh cells is the expression of high levels of PD-1 (Yu and Vinuesa, 2010). PD-1 is an inhibitory receptor that is often associated with T cell exhaustion (Sharpe et al., 2007). However, PD-1 is also required for Tfh cell function and for germinal center formation (Good-Jacobson et al., 2010). However, it is not clear how PD-1 contributes to Tfh differentiation or maintenance. Interestingly, PD-1 signaling inhibits IL-2 production (Chikuma et al., 2009). Based on our observations showing that IL-2 signaling is deleterious to Tfh responses, we speculate that PD-1 signaling may promote the maintenance of Tfh cells by blocking autocrine IL-2 production.

Collectively, our results demonstrate that IL-2 plays a central role in the control of the Tfh cell response to influenza. Our findings also offer a new perspective for how Tfh cell development is regulated, and demonstrate that the physiological availability of IL-2 is a critical factor that regulates successful B cell responses in vivo.

EXPERIMENTAL PROCEDURES

Supplementary Material

ACKNOWLEDGEMENTS

Footnotes

REFERENCES