Cutting Edge: miR-17-92 Is Required for Both CD4 Th1 and T Follicular Helper Cell Responses during Viral Infection

Tuoqi Wu, Andreas Wieland, Judong Lee, J. Scott Hale, Jin-Hwan Han, Xiaojin Xu and Rafi Ahmed
J Immunol September 15, 2015, 195 (6) 2515-2519; DOI: https://doi.org/10.4049/jimmunol.1500317

Abstract

Viral infections induce the differentiation of naive CD4 T cells into two distinct lineages, Th1 cells and T follicular helper (TFH) cells. Two recent studies demonstrated that the microRNA cluster miR-17-92 selectively promotes CD4 TFH responses. However, we show in this study that miR-17-92 expression is required for the clonal expansion of both virus-specific Th1 and TFH cells. Upon viral infection, miR-17-92–deficient CD4 T cells showed impaired clonal expansion and subsequent memory formation. Although miR-17-92 deficiency impaired the clonal expansion of both Th1 and TFH cells, the expansion of Th1 cells was more affected. Overexpression of miR-17-92 in CD4 T cells resulted in increased expansion of both virus-specific Th1 and TFH cells but selectively enhanced the Th1 response. Taken together, our data suggest that miR-17-92 is necessary for both Th1 and TFH cells to respond efficiently to viral infections and that the Th1 response is more sensitive to the level of miR-17-92 expression.

Introduction

Effector CD4 T cells regulate immune responses by communicating with other immune cells via cytokines and direct engagement of cell surface molecules. Upon viral infection, naive Ag-specific CD4 T cells differentiate into two subsets of effector CD4 T cells: Th1 cells and T follicular helper (TFH) cells (1). Th1 cells contribute to the control of viral infections by producing cytokines such as IFN-γ and TNF-α (2). TFH cells, characterized by their expression of CXCR5, are necessary for the initiation and maintenance of the germinal center (GC) reaction and, therefore, are crucial for Ab affinity maturation and the generation of memory B cells and long-lived plasma cells (3). Bcl-6, a transcriptional repressor, promotes the differentiation of TFH cells, whereas Blimp-1 antagonizes Bcl-6 and enforces the differentiation into non-TFH cells (4–6). After Ag clearance, the majority of effector CD4 T cells undergo apoptosis, whereas a fraction survives and differentiates into memory cells, maintaining their commitment to the Th1 or TFH lineage (1, 7, 8).

MicroRNAs are a family of small regulatory RNAs ∼22 nt that bind to the 3′-untranslated region of target transcripts and suppress gene expression by blocking translation and/or degrading target transcripts (9, 10). Our group (11) showed a critical role for the miR-17-92 cluster in regulating virus-specific CD8 T cell differentiation. Two recent publications showed that miR-17-92 is also necessary for TFH cell differentiation and the humoral immune response (12, 13). However, our current study shows that miR-17-92 positively regulates the expansion of both Th1 and TFH cells during viral infection. The impaired humoral response observed in miR-17-92 conditional-knockout mice is caused by a defective expansion of CD4 T cells rather than a selective defect in the differentiation of TFH cells. In fact, our data show that Th1 cells are more dependent on the expression of miR-17-92 for their clonal expansion, as well as the production of effector cytokines, than are TFH cells.

Materials and Methods

Mice and infection

SMARTA mice expressing a transgenic TCR specific for the GP61–80 epitope of lymphocytic choriomeningitis virus (LCMV) were backcrossed to B6.CD45.1+ mice (The Jackson Laboratory) (14). C57BL/6 (CD45.1−) mice, purchased from The Jackson Laboratory, were used as recipients of SMARTA CD4 T cells. miR-17-92 conditional-knockout and miR-17-92 conditional-overexpressing mice were purchased from The Jackson Laboratory and bred to a CD4-cre–transgenic strain (Taconic) (15–17). For infection, mice were injected i.p. with 2 × 105 PFU LCMV Armstrong. Animal experiments were conducted in accordance with Emory University Institutional Animal Care and Use Committee protocols.

In vitro T cell–proliferation assay

Purified CD4 T cells were labeled with CFSE and stimulated with plate-bound anti-CD3 and soluble anti-CD28 Abs (BD Biosciences) for 48 h. Cell division was determined by flow cytometry analysis of CFSE dilution.

Flow cytometry

I-AbGP66–77–specific CD4 T cells were labeled with I-AbGP66–77 tetramers (National Institutes of Health Tetramer Core Facility, Atlanta, GA), as previously described (1). A three-step CXCR5 staining was performed, as previously described (4). Phospho-ribosomal protein S6 was detected using anti–phospho-S6 Ab (Cell Signaling Technology) (11).

ELISA and ELISPOT assays

ELISA and ELISPOT assays to detect LCMV-specific Abs and Ab-secreting cells, respectively, were performed as previously described (18).

Retroviral transduction

MSCV-PGK-GFP plasmid with a miR-17-92 insert was described previously (11). Activated SMARTA CD4 T cells were purified and infected by retroviruses, with or without the miR-17-92 insert. Cells were cultured with 10 ng/ml IL-2, and GFP+ cells were sorted 2 d later. A total of 2 × 104 GFP+ cells was transferred into each recipient.

Statistical analysis

All data analysis was performed using GraphPad Prism v5. The p values were determined by a two-tailed, unpaired Student t test and were considered significant at p < 0.05.

Results and Discussion

miR-17-92 is required for the generation of virus-specific Th1 and TFH cells

To study the role of miR-17-92 in the CD4 T cell response to viral infections, we bred miR-17-92 loxP/loxP mice to a CD4-cre transgenic strain, resulting in a T cell–specific miR-17-92 deficiency (17). miR-17-92−/− (miR-17-92 loxP/loxP; CD4-cre) mice and littermate controls (miR-17-92 loxP/loxP) were infected with LCMV Armstrong. On day 8 postinfection (p.i.), there was an ∼10-fold decrease in the number of I-AbGP66–77 tetramer+ CD4 T cells, as well as a reduction in the total CD44high effector CD4 T cell population, in miR-17-92−/− mice compared with littermate controls (Fig. 1A). Moreover, CFSE-labeled miR-17-92−/− CD4 T cells divided less than did their wild-type (WT) counterparts after in vitro stimulation with anti-CD3 and anti-CD28 Abs (Supplemental Fig. 1A). Therefore, the diminished clonal expansion of virus-specific miR-17-92−/− effector cells was likely caused by the reduced proliferative capacity of these cells after antigenic stimulation.

FIGURE 1.
FIGURE 1.

miR-17-92 is critical to the CD4 T cell response during LCMV infection. Splenocytes of miR-17-92−/− mice and littermate controls were collected on day 8 p.i. (A) FACS plots of I-AbGP66–77 tetramer staining (gated on CD4 T cells) (left panels) and number of I-AbGP66–77 tetramer+ CD4 T cells/spleen (right panel). (B) FACS plots of intracellular IFN-γ staining (gated on CD4 T cells) (left panels) and number of IFN-γ+ CD4 T cells/spleen (right panel). (C) FACS analysis of CXCR5 and SLAM expression on I-AbGP66–77 tetramer+ CD4 T cells (left panels) and number of Th1 or TFH I-AbGP66–77 tetramer+ CD4 T cells/spleen. Results are representative of at least three independent experiments with n ≥ 3. ***p < 0.001.

Like many antiviral CD4 T cell responses, the CD4 T cell response to LCMV infection exhibits a strong Th1 polarization and expansion of effector CD4 T cells producing the Th1 signature cytokine IFN-γ (2, 19). A recent study suggested that the Th1 compartment and IFN-γ–producing CD4 T cells are largely unaffected by the loss of miR-17-92 after LCMV infection (13). However, considering the substantial reduction in the number of LCMV-specific CD4 effectors in miR-17-92−/− mice, as indicated by both tetramer and CD44 staining, it is highly unlikely that the number of Th1 cells remains unchanged. To examine whether miR-17-92 is necessary for the generation of LCMV-specific IFN-γ–producing CD4 T cells, we restimulated splenocytes from miR-17-92−/− and control mice with GP61–80 peptide and quantified IFN-γ+ CD4 T cells on day 8 p.i. Strikingly, miR-17-92−/− mice showed an ∼16-fold decrease in the number of IFN-γ+ CD4 T cells, as well as a reduction in the IFN-γ protein level among IFN-γ+ cells, compared with littermate controls (Fig. 1B, Supplemental Fig. 1B). In fact, the decrease in IFN-γ+ CD4 T cells was larger than the decrease in tetramer+ cells, which suggests that the Th1 response is more susceptible to the loss of miR-17-92.

An important function of effector CD4 T cells is to support the GC reaction, which is mediated by CD4 TFH cells (3). To determine whether miR-17-92 regulates Th1/TFH cell differentiation, we analyzed the expression of the TFH marker CXCR5 and the Th1 marker SLAM on LCMV-specific CD4 T cells (4). On day 8 p.i., the frequency of TFH (CXCR5+SLAMint) cells within I-AbGP66–77 tetramer+ CD4 T cells in miR-17-92−/− mice was not reduced compared with control mice (Fig. 1C). We quantified I-AbGP66–77 tetramer+ Th1 and TFH cells and found that both populations were severely reduced in the knockout mice (Fig. 1C). The reduced numbers of TFH cells in miR-17-92−/− mice coincided with reduced numbers of GC B cells and anti-LCMV IgG-secreting cells (Supplemental Fig. 1D). Taken together, our results suggest that the loss of miR-17-92, which is upregulated upon T cell activation in virus-specific Th1 and TFH cells, causes a general defect in the expansion of virus-specific CD4 T cells but does not specifically impact the development of TFH cells (Supplemental Fig. 1H).

miR-17-92 deficiency reduces the number of virus-specific memory CD4 T cells

To investigate how miR-17-92 deficiency impacts CD4 memory differentiation, we analyzed knockout and control mice on day 108 p.i. There were ∼4-fold fewer I-AbGP66–77 tetramer+ CD4 T cells and ∼7-fold fewer IFN-γ+ CD4 T cells after GP61–80 peptide stimulation in the knockout mice than in the littermate controls (Fig. 2A, 2B). Consistent with our results at the effector time points, the more profound loss of IFN-γ+ cells in miR-17-92−/− mice confirms that the generation of IFN-γ+ Th1 cells is more dependent on miR-17-92. Moreover, production of IFN-γ was significantly reduced in miR-17-92−/− memory CD4 T cells, suggesting that miR-17-92 is important for the functionality of virus-specific CD4 T cells (Supplemental Fig. 1C). We showed recently that LCMV-specific memory CD4 T cells can be segregated into three populations: CXCR5−Ly6C+ memory Th1 cells, CXCR5+Ly6C+ memory TFH cells, and CXCR5+Ly6C− memory TFH cells (1). Both Ly6C+ memory CD4 T cell subsets were strikingly reduced in miR-17-92−/− mice compared with littermate controls (Fig. 2C, 2D). In addition, miR-17-92−/− mice had significantly lower numbers of I-AbGP66–77 tetramer+ memory CD4 T cells in liver and lung (Fig. 2E), which are primarily occupied by Th1 memory cells rather than TFH memory cells (1). Therefore, miR-17-92−/− mice mounted compromised effector CD4 T cell responses but also showed significant defects in the number of virus-specific memory CD4 T cells.

FIGURE 2.
FIGURE 2.

miR-17-92 deficiency reduces the number of virus-specific memory CD4 T cells generated after the primary immune response. Splenocytes of miR-17-92−/− mice and littermate controls were collected on day 108 p.i. Number of I-AbGP66–77 tetramer+ CD4 T cells (A) and IFN-γ+ CD4 T cells (B) per spleen. FACS plots of CXCR5 and Ly6C (C) and the number of I-AbGP66–77 specific Th1, Ly6C+ TFH, and TFH memory CD4 T cells (D). (E) Number of I-AbGP66–77 tetramer+ CD4 T cells in liver and lung on day 244 p.i. Results are representative of at least three independent experiments with n ≥ 3. *p < 0.05, **p < 0.01, ***p < 0.001.

miR-17-92 overexpression enhances the virus-specific CD4 T cell response

We next sought to determine whether increasing miR-17-92 expression can enhance CD4 T cell responses and generate more IFN-γ+ effectors. We bred a previously described conditional miR-17-92–overexpressing strain to CD4-cre–transgenic mice, resulting in a T cell–specific overexpression of miR-17-92 (15). Cre+ mice bearing two copies of the miR-17-92 transgene (Tg) are designated as miR-17-92 Tg/Tg, and their cre− littermates were used as controls. Tg/Tg mice and their littermate controls were infected with LCMV Armstrong and analyzed on day 10 p.i. We found that miR-17-92 Tg/Tg mice had almost twice as many I-AbGP66–77 tetramer+ CD4 T cells as control mice, indicating an enhanced CD4 T cell response (Fig. 3A). Moreover, there were ∼3-fold more IFN-γ+ CD4 T cells and more IFN-γ expression on a per-cell basis in miR-17-92 Tg/Tg mice than in control mice on day 10 p.i. (Fig. 3B, Supplemental Fig. 1E). Therefore, these results confirm that miR-17-92 positively regulates the antiviral immune response mediated by CD4 T cells and exerts a stronger effect on IFN-γ+ CD4 T cells. The frequency of CXCR5+SLAMint TFH cells was reduced by almost half in miR-17-92 Tg/Tg tetramer+ CD4 T cells relative to their WT counterparts (Fig. 3C). Accordingly, the frequencies of CXCR5−SLAMhigh Th1 cells increased upon overexpression of miR-17-92. However, the decrease in TFH cell frequency was due to an increased Th1 response rather than a decreased TFH response (Fig. 3D). Accordingly, the number of GC B cells in Tg/Tg mice was largely unaffected (Supplemental Fig. 1G). Thus, miR-17-92 overexpression enhances the generation of IFN-γ+ CD4 T cells and promotes the expansion of Th1 cells.

FIGURE 3.
FIGURE 3.

miR-17-92 overexpression preferentially enhances the Th1 effector response. (A–D) Splenocytes from miR-17-92 Tg/Tg and control mice were collected on day 10 p.i. (A) FACS plots of I-AbGP66–77 tetramer staining (gated on CD4 T cells) (left panels) and number of I-AbGP66–77 tetramer+ CD4 T cells/spleen (right panel). (B) FACS plots of intracellular IFN-γ staining (gated on CD4 T cells) (left panels) and the number of IFN-γ+ CD4 T cells/spleen (right panel). (C) FACS plots of CXCR5 and SLAM staining (gated on I-AbGP66–77 tetramer+ CD4 T cells). (D) Number of I-AbGP66–77 tetramer+ Th1 and TFH CD4 T cells/spleen. (E–G) Splenocytes of miR-17-92 Tg/Tg and control mice were collected on day 115 p.i. (E) Number of I-AbGP66–77 tetramer+ CD4 T cells (left panel) and IFN-γ+ CD4 T cells (right panel) per spleen. FACS plots of CXCR5 and Ly6C (F) and the number of I-AbGP66–77–specific Th1, Ly6C+ TFH, and TFH memory CD4 T cells (G). Results are representative of at least three independent experiments with n ≥ 3. **p < 0.01, ***p < 0.001. N.S., not significant.

We next sought to determine whether excessive miR-17-92 expression disrupts memory CD4 T cell differentiation. On day 115 p.i., Tg/Tg mice showed comparable or even slightly more I-AbGP66–77 tetramer+ CD4 T cells, as well as IFN-γ+ CD4 T cells, than their WT counterparts, indicating that miR-17-92 overexpression does not compromise the generation of memory CD4 T cells (Fig. 3E, Supplemental Fig. 1F). Consistent with our observation that miR-17-92 deficiency causes a reduction in Ly6C+ memory CD4 T cells, miR-17-92 overexpression seems to favor the formation of Ly6C+ memory CD4 T cell subsets (Fig. 3F, 3G).

To confirm that the effect of miR-17-92 overexpression on effector CD4 T cell differentiation and expansion is cell autonomous, we adoptively transferred SMARTA CD4 T cells retrovirally transduced with an MSCV construct, with or without an miR-17-92 insert, into WT recipients and infected the chimeras with LCMV (Supplemental Fig. 2A). On day 8 p.i., SMARTA cells overexpressing miR-17-92 expanded significantly more and produced more IFN-γ than did those transduced with the empty MSCV (Fig. 4A, Supplemental Fig. 2B), which supports the fact that miR-17-92 enhances Th1 differentiation in a CD4 T cell–intrinsic manner. However, retroviral miR-17-92 overexpression also increased the number of TFH cells (∼3-fold increase for TFH cells versus ∼12-fold increase for Th1 cells) (Fig. 4B, 4C). Comparable to our previous results in LCMV-specific CD8 T cells, miR-17-92–overexpressing SMARTA cells contained more phosphorylated ribosomal protein S6 (Supplemental Fig. 2C), suggesting increased mTOR signaling (11).

FIGURE 4.
FIGURE 4.

The effect of miR-17-92 overexpression on LCMV-specific CD4 T cell expansion and differentiation is cell autonomous. SMARTA cells were transduced with the MSCV construct, with or without the miR-17-92 insert, and transferred to C57BL/6 recipients. On day 8 p.i., splenocytes were collected for analysis. (A) FACS plots of SMARTA cells (gated on total lymphocytes) (left panels) and number of SMARTA cells/spleen (right panel). (B) FACS plots of CXCR5 and SLAM staining (gated on SMARTA cells). (C) Number of Th1 and TFH SMARTA cells/spleen. Results are representative of at least three independent experiments with n ≥ 3. *p < 0.05, **p < 0.01.

In summary, by using loss-of-function and gain-of-function approaches, we demonstrated an essential role for miR-17-92 in regulating the CD4 T cell response to viral infection. miR-17-92 is critical for the clonal expansion of CD4 T cells during acute LCMV infection. Moreover, the Th1 polarization and secretion of effector cytokine IFN-γ also requires miR-17-92 expression. Although both Th1 and TFH responses are positively regulated by miR-17-92, the degree of Th1 expansion is more sensitive to the level of miR-17-92 expression.

Our results show that miR-17-92 enhances mTOR signaling and is required for antiviral Th1 and TFH responses. It is likely that miR-17-92 suppresses the expression of phosphatase and tensin homolog, a negative regulator of the PI3K-mTOR signaling pathway and a well-documented target of miR-17-92, in both subsets to achieve optimal PI3K-mTOR signaling for their clonal expansion (15). Overexpression of miR-17-92 in Tg/Tg mice increased the Th1 response without affecting the TFH response, whereas retroviral overexpression of miR-17-92 enhanced the Th1 response and, to a lesser extent, the TFH response. We cannot exclude that excessive expression of miR-17-92 driven by the retroviral construct might cause potential off-target effects, but given that the retroviral LTR promoter drove a more robust expression of miR-17-92 than did the CAG promoter in Tg/Tg mice, our data suggest that, although miR-17-92 positively regulates both Th1 and TFH cell expansion, Th1 cells are more sensitive to the dose of miR-17-92. The greater sensitivity of Th1 expansion could be due to lineage-specific differences in the expression of miR-17-92 targets.

Two recent studies concluded that miR-17-92 selectively regulates TFH cell differentiation after LCMV infection (12, 13). In contrast, our results demonstrate that miR-17-92 is required for both Th1 and TFH cells to respond effectively to viral infections. These studies identified effector TFH cells by staining total CD4 T cells for CXCR5 and PD-1. Although this staining strategy can be used as a surrogate to quantitate Ag-specific TFH effector cells, it does not provide any information regarding the number of Th1 effector cells. It is difficult to judge whether miR-17-92 deficiency selectively affects TFH cell differentiation without comparing the numbers of Ag-specific TFH and Th1 cells between miR-17-92–knockout and control mice. We used MHC class II tetramer staining to identify Ag-specific CD4 T cells and distinguished TFH and Th1 lineages within the Ag-specific CD4 T cells based on the differential expression of various surface markers, such as CXCR5 and SLAM. This approach demonstrates that the numbers of virus-specific TFH and Th1 cells are substantially reduced in miR-17-92–knockout mice p.i. Moreover, given that the decrease in the GC response was comparable to, if not less than, the decrease in the effector CD4 T cell response (Fig. 1A, 1F), a compromised B cell helper function of miR-17-92−/− CD4 T cells seems implausible. Our results also show a significant reduction in IFN-γ producing CD4 T cells in the absence of miR-17-92, which is in contrast to a recent study (13). This discrepancy might be due to different promoters controlling Cre expression. In our study, naive CD4 T cells already lacked miR-17-92 (CD4-cre), whereas in the other study, miR-17-92 deficiency was only achieved ∼3 d after T cell activation (OX40-cre). These results suggest that miR-17-92 may function in different stages of the Th1 and TFH response: miR-17-92 might be required in Th1 cells during initial activation, whereas it might be required in TFH cells after initial activation. Thus, our study underlines the importance of miR-17-92 to the antiviral immune response of both CD4 Th1 and TFH cells. A better understanding of the regulatory pathways involved in CD4 T cell differentiation will provide the framework for the design of rational vaccination strategies.

Disclosures

The authors have no financial conflicts of interest.

Footnotes

  • This work was supported by National Institutes of Health Grant R01-AI030048 (to R.A.) and by the Cancer Research Insitute’s Irvington Institute Fellowship Program (to J.-H.H.).

  • The online version of this article contains supplemental material.

  • Abbreviations used in this article:

    GC
    germinal center
    LCMV
    lymphocytic choriomeningitis virus
    p.i.
    postinfection
    TFH
    follicular helper
    Tg
    transgene
    WT
    wild-type.

  • Received February 19, 2015.
  • Accepted June 20, 2015.

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