IL-2 is a pleotropic cytokine with potent pro- and anti-inflammatory effects. These divergent impacts can be directed in vivo by forming complexes of IL-2 and anti–IL-2 mAbs (IL-2C) to target IL-2 to distinct subsets of cells based on their expression of subunits of the IL-2R. In this study, we show that treatment of mice with a prototypical anti-inflammatory IL-2C, JES6-1–IL-2C, best known to induce CD25+ regulatory CD4 T cell expansion, surprisingly causes robust induction of a suite of inflammatory factors. However, treating mice infected with influenza A virus with this IL-2C reduces lung immunopathology. We compare the spectrum of inflammatory proteins upregulated by pro- and anti-inflammatory IL-2C treatment and uncover a pattern of expression that reveals potentially beneficial versus detrimental aspects of the influenza-associated cytokine storm. Moreover, we show that anti-inflammatory IL-2C can deliver survival signals to CD4 T cells responding to influenza A virus that improve their memory fitness, indicating a novel application of IL-2 to boost pathogen-specific T cell memory while simultaneously reducing immunopathology.

Interleukin-2 is a critical cytokine for orchestrating optimal immune responses. IL-2 acts as an autocrine T cell growth factor (1, 2) and can signal in a paracrine manner to promote the activation of other leukocyte subsets, most notably NK cells and CD8 T cells (3, 4). However, IL-2 is also central to the maintenance and function of regulatory CD4 T cells (Tregs) that constrain immune responses and limit immunopathology (5, 6). These divergent activities of IL-2 have been shown in diverse models and have been exploited clinically (6, 7). Many strategies are being developed to specifically engage the pro- versus anti-inflammatory properties of IL-2 in context-dependent situations. For example, exogenously administered IL-2 can be targeted to either the α- (CD25) or β (CD122)-chain of the IL-2R by using IL-2 and anti–IL-2 Ab complexes (IL-2C) made with different mAbs (710). In the mouse, the Ab clone S4B6 forms proinflammatory IL-2C that preferentially signal cells expressing high CD122, predominantly CD8 T and NK cells, whereas anti-inflammatory IL-2C made with Ab clone JES6-1A12 (JES6) targets IL-2 to CD25 expressing cells, most notably Tregs (7) in the steady-state.

We recently showed that IL-2 secreted by memory CD4 T cells responding to influenza A virus (IAV) can promote disease symptoms by increasing the production of inflammatory cytokines and chemokines in the lung (11). As part of these studies we treated naive mice or mice infected intranasally (i.n.) with a sublethal 0.2 LD50 dose of the mouse-adapted IAV strain A/PuertoRico/8/1934 (A/PR8) for 3 d with S4B6–IL-2C and found that such treatment induced a remarkably broad inflammatory response that synergizes with IAV infection to exacerbate disease (11). We used this regimen of IL-2C treatment as it delivers physiologically relevant IL-2 signals to IL-2R–expressing CD4 T cells that promote memory formation during IAV infection (12), and similar protocols are widely employed in many different murine models.

How JES6–IL-2Cs that target CD25-expressing cells affect inflammatory cytokine and chemokine production systemically as well as in tissues such as the lung is not well characterized. In this study, we determine the impact of JES6 -IL-2C on acute inflammation when given to naive mice and to mice challenged with IAV. We confirmed the treatment boosted Treg numbers and innate lymphoid cell (ILC) populations (1316) in the spleen as well as in the lung. However, JES6 -IL-2C treatment drove an acute systemic inflammatory response defined by elevated levels of a diverse suite of cytokines and chemokines detected in serum and in lungs. JES6 -IL-2C given to mice also challenged with low-dose IAV enhanced levels of IFN-γ paradoxically at the same time as several Th2-associated factors to levels above those detected in mice receiving either IAV or IL-2C alone. Although our previous studies found that treatment of IAV-infected mice with S4B6–IL-2C containing 2 μg of IL-2 results in acute death of all treated mice (11), IAV-infected mice treated with JES6 -IL-2C all survived infection. Furthermore, JES6–IL-2C treatment reduced the extent of lung immunopathology associated with IAV infection.

Given the differential outcome of JES6– versus S4B6–IL-2C treatment, we directly compared the inflammatory response induced by each in uninfected as well as in IAV-infected mice. Our results clearly show shared elements and unique patterns in the inflammatory milieu induced by JES6–IL-2C in the absence and presence of infection, demonstrating a complex governance of cytokine and chemokine expression, especially during IAV infection.

Finally, we asked if JES6–IL-2C could be used to deliver physiological IL-2 signals that are required for memory establishment to conventional CD25–expressing antiviral CD4 T effector cells responding to infection. We thus tested if JES6–IL-2C could rescue memory formation by IL-2–deficient (Il2−/−) CD4 T cells responding to IAV that fail to survive long-term without receipt of IL-2 signals during 5–7 d postinfection (dpi) (12). JES6–IL-2C rescued Il2−/− CD4 T cell memory formation to a similar degree as that observed with S4B6–IL-2C (12). Our results thus demonstrate that CD25-targeted IL-2C can deliver physiologically relevant IL-2 signals that promote antiviral memory CD4 T cell formation while simultaneously promoting tissue integrity during pathogen challenge.

Experimental animal procedures were conducted in accordance with guidelines outlined by the Office of Laboratory Animal Welfare, National Institutes of Health. Protocols were approved by the Animal Care and Use Committee at Trudeau Institute (Saranac Lake, NY), the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School (Worcester, MA), and the University of Central Florida (Orlando, FL).

BALB/c Thy1.2 or BALB/c Thy1.1 mice were used in experiments when 8–12 wk old. Naive CD4 T cells were obtained from 5- to 8-wk-old male or female Il2−/− D011.10 Thy1.2 or Thy1.2/Thy1.1 mice originally provided by A. Abbas (University of California, San Francisco). BALB/c and D011.10 mice were bred in the vivarium of the Trudeau Institute, the University of Massachusetts Medical School, or the University of Central Florida.

Mice were treated for the indicated days with IL-2C that consisted of 2 μg per day of rIL-2 (Thermo Fisher Scientific) premixed with 20 μg of anti-mouse IL-2 mAb clone JES6-1A12 (JES6) (Thermo Fisher Scientific), or IL-2C premixed with IL-2 and the anti–IL-2 Ab clone S4B6 (BD Biosciences). In certain experiments, the amount of IL-2 in the complexes was varied, as indicated. Complexes were incubated at room temperature for 20 min before i.p. injection in 200 μl of PBS. Control mice received 200 μl of PBS alone.

For some experiments, mice were treated as indicated with 0.25 mg of anti-CD25 (IL-2 Rα) Ab (clone PC-61.5.3; BioXCell) to block IL-2 signaling 1 d prior to initiation of IL-2C treatment. Ab was delivered by i.p. injection in 200 μl of PBS.

Influenza PR8 (H1N1), originating from stocks prepared at the Trudeau Institute and in use in experiments since 1997, and A/PR8-OVAII (H1N1), from stock obtained from P. Doherty at St Jude’s Children’s Hospital (17), were produced in the allantoic cavity of embryonated hen eggs at the Trudeau Institute, and the lethal dose (LD50), egg-infective dose, or tissue culture–infective dose were characterized. Mice were infected i.n. under light isoflurane anesthesia (Webster Veterinary Supply) with a sublethal 0.2 LD50 dose of virus in 50 μl of PBS, and morbidity and mortality were monitored.

At different time points after IL-2C treatment and/or virus infection, blood and lungs were obtained from euthanized animals for Luminex multiplex analysis. Lungs were harvested and homogenized in RPMI 1640 media supplemented with 2 mM l-glutamine, 100 IU of penicillin, 100 μg/ml streptomycin (Invitrogen), 10 mM HEPES (Research Organics), 50 μM 2-ME (Sigma-Aldrich), and 7.5% FBS (HyClone), and serum was collected from blood.

Alternatively, for flow cytometry, mice were euthanized by cervical dislocation followed by exsanguination by perforation of the abdominal aorta. Lungs were perfused by injecting 10 ml of PBS in the left ventricle of the heart. Lungs and spleen were prepared into single-cell suspensions by mechanical disruption of organs and passage through a nylon membrane. Flow cytometry was performed as previously described (18), using fluorochrome-labeled Abs at the manufacturer’s recommended dilutions for surface staining, including anti-Thy1.1 (OX-7), anti-Thy1.2 (53-2.1), anti-CD4 (RM4.5 and GK1.5), anti-CD8 (53-6.7), anti-CD45.2 (104), anti–γδ TcR (GL3), anti-CD3 (17A2), anti-CD25 (PC61), anti-CD11b (M1/70), anti–Gr-1 (RB6-8C5), anti-CD127 (A7R34), and anti-CD49b (DX5), as well as murine hematopoietic lineage Ab mixture containing anti-CD3 (17A2), anti–CD45R/B220 (RA3-6B2), anti-CD11b (M1/70), anti–TER-119 (TER-119), and anti–Ly-G6/Gr-1 (RB6-8C5). ILCs were identified as CD45+, Lineage, CD3, CD90+, and CD127+ (IL-7R) lymphocytes.

Intracellular staining for FOXP3 and Ki-67 was performed as per the manufacturer’s instructions with the FOXP3 Transcription Factor Fixation/Permeabilization Concentrate and Diluent (Life Technologies, eBioscience) and fluorochrome-labeled anti-FOXP3 (FJK-16s) and Ki-67 (SolA15) Abs. Analysis was performed using FACS Canto II and LSRII instruments (BD Biosciences) and FlowJo (Tree Star) analysis software.

Levels of cytokines and chemokines in lung homogenates or serum were determined using mouse multiplex kits (Invitrogen and Millipore) read on a Bio-Plex Multiplex 200 Luminex reader (Bio-Rad Diagnostics), as per manufactures’ instructions. The assay sensitivity for 12 of the 14 the analytes presented is below 1 pg/ml, ranging from 0.03 to 0.69 pg/ml, and is 1.16 and 3.43 pg/ml for the remaining analytes CXCL2 and CCL2, respectively.

For assessment of immunopathology following viral infection and IL-2C treatment, lungs lobes were isolated and immediately fixed in 10% neutral buffered formalin. Lung samples were subsequently processed, embedded in paraffin, sectioned, placed on l-lysine–coated slides, and stained with H&E, using standard histological techniques at the Morphology Core at University of Massachusetts Medical School. Triplicate nonserial sections were graded blindly from 0 to 4, for the extent of inflammatory cell infiltration and damage of bronchi, arteries, or alveoli by a certified pathologist (S. Sell) as described previously (19).

Noninvasive whole-body plethysmography (Buxco) was employed to measure respiratory rates (breaths per minute), minute volumes (milliliter per minute), and enhanced pause, on conscious, unrestrained animals following IL-2C treatment. The minute volume is defined as the volume of air exchanged during a 1 min interval and is calculated as follows (respiratory rate × tidal volume).

Naive CD4+ T cells were obtained from pooled spleen and peripheral lymph nodes as previously described (18). Briefly, cells were purified by nylon wool and Percoll density gradient separation. CD4 T cells were isolated by positive CD4 MACS selection (Miltenyi). Resulting CD4 cells routinely expressed a characteristic naive phenotype (small size, CD62Lhi, CD44lo, and CD25lo) >97% TcR+. TH1-polarized effectors were generated in vitro as described (20). Briefly, naive Il2−/− CD4 T cells were cultured with an equal number of irradiated APC (2 × 105 per ml) in the presence of exogenous IL-2 (20 ng per ml), 2 ng per ml IL-12 (PeproTech), 10 μg per ml anti–IL-4 Ab (11B11; BioXCell), and 5 μM OVAII peptide. In vitro–primed memory cells were obtained by thoroughly washing effector cultures at 4 d and reculturing the cells in fresh media for at least 3 d in the absence of Ag and exogenous cytokines. Live cells were isolated by Lympholyte Separation (Cedar Lane Laboratories). All donor CD4 T cells were adoptively transferred in 200 μl of PBS by i.v. injection. A number of donor cells previously determined to be detectable at the memory phase, 2 × 106, was transferred. Donor cell injection and viral infection occurred on the same day.

Group sizes of n = 3 to 6 were employed for all experiments. For unpaired Student t tests, ∝ = 0.05 was used to assess whether the means of two normally distributed groups differed significantly. One-way ANOVA with the appropriate multiple comparison posttest, Bonferroni or Tukey, was employed to compare multiple means. All error bars represent the SD. Significance is indicated as *p < 0.05, **p < 0.005, ***p < 0.001, and ****p < 0.0001.

We first delivered JES6–IL-2C containing 2 μg of recombinant murine IL-2 to naive mice by i.p. injection for 3 consecutive d. This is the same treatment regimen we used to test the impact of S4B6–IL-2C during IAV infection in our previous study (11). The mice were analyzed on the fourth day after initiation of treatment and were compared with control mice receiving PBS. First, we confirmed the expected activity of JES6–IL-2C in dramatically increasing the number of CD25+ FOXP3+ CD4 Tregs in the spleen (Fig. 1A, 1B). JES6–IL-2C treatment significantly increased the mean expression of CD25 on FOXP3+ CD4 T cells (Fig. 1C). We found the upregulated CD25 expression on FOXP3+ Tregs in JES6–IL-2C–treated mice to associate with roughly a 2-fold increase in the frequency of FOXP3+ cells high for the proliferation marker Ki-67 (Fig. 1D). In JES6 -IL-2C–treated mice, we also found small but significant increases in total CD4 T cells but not CD8 T cells, a 2-fold increase in NK cells, and a 4-fold increase in γδ T cell numbers (Fig. 1E). These results for CD8 T cells and NK cells are not as marked as those observed previously in which CD8 T cells and NK cells expand more than 4- and 16-fold, respectively, following S4B6–IL-2C administration (11). Given published observations of IL-2–dependent expansion of ILC (1316), we also assessed whether ILC were impacted by IL-2C treatment. JES6–IL-2C administration significantly increased ILCs in a manner consistent with previous findings (1315). The gating strategies used are shown in Supplemental Fig. 1.

FIGURE 1.

JES6–IL-2C treatment increases diverse lymphocyte subsets. Mice were treated for 3 d with JES6–IL-2C, and on the fourth day, spleens were analyzed by flow cytometry. (A) The total number of Tregs (CD25+ FOXP3+) and (B) representative staining from untreated and treated mice of CD25 and FOXP3 costaining. (C) CD25 mean fluorescence intensity on FOXP3+ CD4 T cells and (D) frequency of Ki-67+ FOXP3+ cells with representative staining (dark shaded histograms are total CD4 T cells). (E) Total numbers of CD4 T cells, CD8 T cells, NK cells, ILCs, and γδ T cells in mice treated only with PBS (white, n = 6) or with JES6–IL-2C (gray, n = 4). Representative results from one of three replicate experiments following Student t test analysis. *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 1.

JES6–IL-2C treatment increases diverse lymphocyte subsets. Mice were treated for 3 d with JES6–IL-2C, and on the fourth day, spleens were analyzed by flow cytometry. (A) The total number of Tregs (CD25+ FOXP3+) and (B) representative staining from untreated and treated mice of CD25 and FOXP3 costaining. (C) CD25 mean fluorescence intensity on FOXP3+ CD4 T cells and (D) frequency of Ki-67+ FOXP3+ cells with representative staining (dark shaded histograms are total CD4 T cells). (E) Total numbers of CD4 T cells, CD8 T cells, NK cells, ILCs, and γδ T cells in mice treated only with PBS (white, n = 6) or with JES6–IL-2C (gray, n = 4). Representative results from one of three replicate experiments following Student t test analysis. *p < 0.05, ***p < 0.001, ****p < 0.0001.

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We next assessed protein levels of a broad array of cytokines and chemokines in the serum on the fourth day following JES6–IL-2C treatment and compared levels to those detected in mice receiving PBS alone. Surprisingly, despite its widespread use as an anti-inflammatory agent, JES6–IL-2C treatment significantly increased levels of a number of prototypical proinflammatory factors, including TNF, IL-1, IL-6, and IFN-γ (Fig. 2A). Treatment also enhanced levels of cytokines typically associated with Th2 and ILC responses, including IL-4, IL-5, IL-13, as well as IL-10 (Fig. 2B).

FIGURE 2.

JES6–IL-2C treatment induces widespread systemic expression of inflammatory cytokines and chemokines. Mice were treated with JES6–IL-2C or with PBS alone for 3 consecutive d. On the fourth day, serum was harvested and was analyzed by Luminex for protein levels of (A) inflammatory cytokines and chemokines typically associated with Th1 responses and (B) cytokines associated with Th2 responses (n = 4 mice per group). Results from one of three replicate experiments following Student t test analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

JES6–IL-2C treatment induces widespread systemic expression of inflammatory cytokines and chemokines. Mice were treated with JES6–IL-2C or with PBS alone for 3 consecutive d. On the fourth day, serum was harvested and was analyzed by Luminex for protein levels of (A) inflammatory cytokines and chemokines typically associated with Th1 responses and (B) cytokines associated with Th2 responses (n = 4 mice per group). Results from one of three replicate experiments following Student t test analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

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Given that the lung is particularly sensitive to IL-2–driven inflammation (11), we analyzed the impact of JES6–IL-2C treatment on cellular subsets and inflammatory mediators in the lung. As compared with the spleen, which increases in total cellularity following IL-2C treatment by 2- to 3-fold, significant increases in total cellularity in the lungs were not observed (data not shown). However, similar to the expansion of CD25+ FOXP3+ CD4 T cells (Tregs) in the spleen following JES6–IL-2C treatment, a robust increase in CD25+ FOXP3+ CD4 T cells was observed in the lung (Fig. 3A, 3B). A small but significant increase in lung γδ T cells and a 3-fold expansion of ILC were also detected, whereas total CD4 and CD8 T cells, and NK cells numbers were not impacted (Fig. 3C). Nevertheless, most of the inflammatory factors that were found to be elevated in the serum in Fig. 1 were also detected at higher levels in the lungs of mice treated with JES6–IL-2C versus in control animals (Fig. 3D, 3E).

FIGURE 3.

Systemic JES6–IL-2C treatment induces inflammation in the lung. Mice treated systemically with IL-2C or PBS alone were analyzed for changes in lymphocyte populations and inflammation in the lungs. Shown in (A), the number of Tregs with (B) representative staining from treated and untreated mice. (C) Total numbers of CD4 T cells, CD8 T cells, NK cells, γδ T cells, and ILC (n = 4 mice per group). Lung homogenates from separate groups of JES6–IL-2C–treated or control mice (n = 4 per group) were analyzed for protein levels of inflammatory factors associated with (D) Th1 or (E) Th2 responses. Results from one of three replicate experiments following Student t test analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3.

Systemic JES6–IL-2C treatment induces inflammation in the lung. Mice treated systemically with IL-2C or PBS alone were analyzed for changes in lymphocyte populations and inflammation in the lungs. Shown in (A), the number of Tregs with (B) representative staining from treated and untreated mice. (C) Total numbers of CD4 T cells, CD8 T cells, NK cells, γδ T cells, and ILC (n = 4 mice per group). Lung homogenates from separate groups of JES6–IL-2C–treated or control mice (n = 4 per group) were analyzed for protein levels of inflammatory factors associated with (D) Th1 or (E) Th2 responses. Results from one of three replicate experiments following Student t test analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

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We titrated the amount of IL-2 delivered during IL-2C treatment and found that although some factors were significantly enhanced when less IL-2 was used, the 2 μg dose was required to see robust, widespread effects in the lung (Supplemental Fig. 2). To confirm that the IL-2C promote inflammatory responses by binding to CD25+ cells, we pretreated mice with blocking Ab against CD25 by i.p. injection and the next day administered JES6–IL-2C for 3 consecutive d. Blocking CD25 abrogated the impact of the JES6–IL-2C (Supplemental Fig. 2), confirming that IL-2C binding to the CD25 receptor is required and ruling out that contaminants in reagents or unexpected binding of IL-2 to the CD122 component of the IL-2R, or other receptors, is responsible for the proinflammatory impacts observed.

The factors detected in the lungs in Fig. 3 could arise from local impacts of JES6–IL-2C or may have originated from systemic cellular sources. We thus delivered the IL-2C to mice by i.n. administration to analyze the local versus systemic impact. We observed markedly higher levels of inflammation at 4 d posttreatment in the lung versus in the serum following i.n. IL-2C administration, strongly supporting that CD25+ cells in the lungs can respond to IL-2 and promote strong local inflammation in the absence of pathogen infection (Supplemental Fig. 3). These results demonstrate the acute induction of proinflammatory mediators in the lung by the JES6–IL-2C and reinforce that the lung environment is particularly sensitive to IL-2–induced inflammatory signals (9, 11).

Given the unexpected ability of JES6–IL-2C to concurrently boost Tregs and lung inflammation, we infected mice with a low dose of IAV and on the same day, initiated treatment with IL-2C for 3 d and assessed infection outcomes. Only one inflammatory cytokine, IFN-γ was detected at markedly higher levels in the lungs of IAV-infected mice treated with JES6–IL-2C than in mice infected with IAV or treated with JES6–IL-2C alone (Fig. 4A). In contrast, treatment of IAV-infected mice with S4B6–IL-2C i.p. in this model drives heightened lung levels of many inflammatory cytokines and chemokines (IL-6, IFN-γ, IL-17, CCL2, CCL3, and G-CSF) beyond those seen in mice treated with IL-2C alone or in mice only infected with IAV (11). We also observed significant but more restrained increases in the levels of IL-1α, IL-4, IL-5, IL-13, and IL-10 in the lungs of IAV-infected mice treated with JES6–IL-2C (Fig. 4B).

FIGURE 4.

JES6–IL-2C treatment improves outcomes of IAV infection. Groups of mice were infected with a sublethal 0.2 LD50 dose of IAV and treated i.p. with either PBS alone or with JES6–IL-2C. On day 4, levels of stated cytokines and chemokines detected by Luminex from lung homogenates and associated with either (A) Th1 or (B) Th2 responses from four mice per group were determined. The average level of analytes detected following JES6–IL-2C administration alone is depicted as a dashed line in each graph. Separate mice were infected with IAV and treated with either PBS alone (white circle) or with JES6–IL-2C for 3 d. (C) Shown is the percent survival of four mice per group. (D) Mice treated as in (C) were harvested at 7 dpi and assessed for histopathological changes. Shown is the cumulative histopathology score broken down by alveolar inflammation (AV; black), bronchial inflammation (BR; gray), and perivascular inflammation (PV; white). (E) Groups of five mice were treated with either PBS or JES6–IL-2C i.p. for 3 consecutive d and were assessed on stated days for respiratory rate (RR; in breaths per minute), minute volume (MV; in cubic centimeter per minute), and enhanced pause (PenH). All results representative of at least two replicate experiments following Student t test (A and B) or one-way ANOVA (D). *p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 4.

JES6–IL-2C treatment improves outcomes of IAV infection. Groups of mice were infected with a sublethal 0.2 LD50 dose of IAV and treated i.p. with either PBS alone or with JES6–IL-2C. On day 4, levels of stated cytokines and chemokines detected by Luminex from lung homogenates and associated with either (A) Th1 or (B) Th2 responses from four mice per group were determined. The average level of analytes detected following JES6–IL-2C administration alone is depicted as a dashed line in each graph. Separate mice were infected with IAV and treated with either PBS alone (white circle) or with JES6–IL-2C for 3 d. (C) Shown is the percent survival of four mice per group. (D) Mice treated as in (C) were harvested at 7 dpi and assessed for histopathological changes. Shown is the cumulative histopathology score broken down by alveolar inflammation (AV; black), bronchial inflammation (BR; gray), and perivascular inflammation (PV; white). (E) Groups of five mice were treated with either PBS or JES6–IL-2C i.p. for 3 consecutive d and were assessed on stated days for respiratory rate (RR; in breaths per minute), minute volume (MV; in cubic centimeter per minute), and enhanced pause (PenH). All results representative of at least two replicate experiments following Student t test (A and B) or one-way ANOVA (D). *p < 0.05, ***p < 0.001, ****p < 0.0001.

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We thus next tested whether IL-2 targeted to CD25+ cells would impact survival following sublethal IAV challenge. We previously found that S4B6–IL-2C administration for 4 instead of 3 d resulted in the acute death of all mice challenged with a normally sublethal dose of IAV without altering immunopathology in the lungs (11). When the same treatment regimen was employed, no mice that received JES6–IL-2C and sublethal IAV infection succumbed to infection (Fig. 4C). Remarkably, despite the proinflammatory impact of the JES6–IL-2C in terms of induction of soluble factors, there was a striking decrease in the extent of histological changes observed in the lungs of mice treated with JES6–IL-2C and IAV versus mice infected with IAV alone. This was most apparent in terms of reduced bronchial inflammation, which was virtually absent in IAV-challenged mice treated with JES6–IL-2C versus in mice infected with IAV alone (Fig. 4D, Supplemental Fig. 4). These results reveal a surprising disconnect in terms of levels of soluble factors in the lung typically associated with damaging inflammation and the degree of immunopathology observed during acute pulmonary viral infection.

Even in the absence of IAV infection, S4B6–IL-2C–induced inflammation correlated with acute impairment of respiratory mechanics (11). We therefore tested the extent to which JES6–IL-2C treatment in the absence of IAV infection impacted lung function of mice. Despite the induction of inflammation summarized in Fig. 2, JES6–IL-2C did not affect breathing as measured by several parameters (Fig. 4E), which we found to be significantly altered by S4B6–IL-2C administration (11).

Our results in this study and in our previous work (11) demonstrate that S4B6– and JES6–IL-2C both enhance the production of soluble mediators of inflammation but have dramatically different impacts on respiratory functions and immunopathology. We thus asked if we could identify patterns within the inflammatory responses induced by treatment with these IL-2C. We first compared the relative impact of S4B6– and JES6–IL-2C treatment on cytokines and chemokines detected in the lung in the absence of infection within the same experiment. We present heatmaps based on average protein expression to better visualize and compare the scope of the two distinct inflammatory responses. Fig. 5A summarizes the expression patterns of analytes significantly upregulated in the lung by either JES6– or S4B6–IL-2C treatment. A complex pattern of overlapping and unique induction of distinct factors is evident. Both complexes induce the upregulation of the majority of mediators assessed and in general, the upregulation of analytes impacted by both treatments was higher with S4B6– versus JES6–IL-2C. In the absence of infection, JES6–IL-2C uniquely induced higher levels of IL-1α, IL-4, and IL-13. These results support that although S4B6- and JES6-based IL-2C have some overlapping impacts on acute inflammation, each IL-2C also induces a distinct suite as well as different levels of proinflammatory factors.

FIGURE 5.

Distinct and overlapping patterns in IL-2C–induced inflammation. Separate groups of uninfected or IAV-infected mice were treated with PBS, JES6–IL-2C, or S4B6–IL-2C for 3 consecutive d. On the fourth day, lung homogenates were harvested and analyzed by Luminex for protein levels of inflammatory cytokines and chemokines. A heat map of the average amount of analytes significantly induced with IL-2C complex treatment in (A) uninfected mice and (B) sublethally 0.2 LD50 IAV–infected mice (three mice per group; one of three experiments). (C) Venn diagram depicting analytes significantly induced by both IL-2C or uniquely induced by JES6–IL-2C or S4B6–IL-2C during IAV infection.

FIGURE 5.

Distinct and overlapping patterns in IL-2C–induced inflammation. Separate groups of uninfected or IAV-infected mice were treated with PBS, JES6–IL-2C, or S4B6–IL-2C for 3 consecutive d. On the fourth day, lung homogenates were harvested and analyzed by Luminex for protein levels of inflammatory cytokines and chemokines. A heat map of the average amount of analytes significantly induced with IL-2C complex treatment in (A) uninfected mice and (B) sublethally 0.2 LD50 IAV–infected mice (three mice per group; one of three experiments). (C) Venn diagram depicting analytes significantly induced by both IL-2C or uniquely induced by JES6–IL-2C or S4B6–IL-2C during IAV infection.

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We reasoned that this approach may provide insight into the beneficial versus most deleterious elements of the “cytokine storm” associated with detrimental outcomes of severe IAV infection (21). We thus compared levels of proinflammatory factors in lung homogenates of groups of mice on day 4 after either infection with low-dose IAV alone or infection with IAV and treatment for 3 d with either S4B6– or JES6–IL-2C. A heatmap summarizing those inflammatory factors upregulated by either IL-2C treatment versus levels detected in mice only challenged with IAV is shown in Fig. 5B. Improved outcomes associated with reduced immunopathology following treatment with JES6–IL-2C during IAV infection correlate with increased expression of IL-4, GM-CSF, and CXCL2. Conversely, detrimental outcomes in IAV-infected and S4B6-treated mice was associated with higher levels of many classic proinflammatory factors, including TNF, IL-6, CCL2, CCL3, and IL-1β. Thus, although there is a considerable overlap in the inflammatory signatures in IAV-infected mice that are treated with S4B6– and JES6–IL-2C, unique patterns in the response are evident (Fig. 5C).

In addition to driving inflammation and modulating diverse lymphocyte populations, IL-2 is a key signal in promoting T cell memory (22, 23). We previously showed that the late IL-2–dependent signals required for IAV-specific CD4 T cells responding to IAV to form memory could be delivered to Il2−/− CD4 T cells by treating mice with S4B6–IL-2C from 5 to 7 dpi (12). IL-2 signals promote upregulation of the IL-7R (CD127) on the surface of effector CD4 T cells at 7 dpi, thus increasing their memory fitness versus cells expressing less IL-7R (24, 25). Given that JES6–IL-2C reduces immunopathology associated with IAV infection, whereas S4B6–IL-2C instead promotes immunopathology (11), we asked if JES6–IL-2C could be employed to rescue memory formation from Il2−/− CD4 T cells responding to IAV. We thus treated recipients of Il2−/− DO11.10 cells that were challenged with PR8-OVAII with JES6– or S4B6–IL-2C between 5 and 7 dpi and assessed donor cell recovery at 7 dpi, the peak of expansion, and, at 28 dpi, a memory timepoint.

S4B6– and JES6–IL-2C both increased CD25 expression on the surface of Il2−/− donor CD4 T cells at the effector phase of the response, although the upregulation associated with JES6–IL-2C was not significantly enhanced compared with untreated mice, perhaps because of binding of the JES6–IL-2C to CD25 (Fig. 6A, 6B). Nevertheless, S4B6– and JES6–IL-2C similarly upregulated CD127 expression versus expression on donor cells in mice receiving PBS alone (Fig. 6C, 6B). At 28 dpi, we observed near-identical rescue of memory formation from the Il2−/− donor cells in the spleen, draining lymph node, and especially the lung, in which the number of donor cells was ∼2 logs increased in mice receiving either IL-2C versus in mice not receiving IL-2C (Fig. 6C). These results indicate that JES6–IL-2C delivered systemically can efficiently deliver promemory IL-2 signals to CD4 T cells responding in vivo.

FIGURE 6.

JES6–IL-2C deliver promemory signals to CD4 T cells responding to IAV. BALB/c mice received congenically marked Il2−/− DO11.10 CD4 T cells followed by priming with low-dose 0.2 LD50 PR8-OVAII. Groups of mice were either treated with S4B6–IL-2C (black line), JES6–IL-2C (gray line), or PBS alone (filled histogram) from 5 to 7 dpi. At 7 dpi, donor cells gated as in (A) were analyzed for expression of CD25 and CD127, with representative staining and summary mean fluorescence intensity (MFI) analysis from three mice per group shown in (B) and (C), respectively. (D) At 28 dpi, the total number of donor cells was enumerated in the spleen, draining lymph nodes (dLN), and lung from groups of three to four mice treated with either IL-2C or with PBS alone. Summary of two replicate experiments and determined by one-way ANOVA. *p < 0.05, **p < 0.01.

FIGURE 6.

JES6–IL-2C deliver promemory signals to CD4 T cells responding to IAV. BALB/c mice received congenically marked Il2−/− DO11.10 CD4 T cells followed by priming with low-dose 0.2 LD50 PR8-OVAII. Groups of mice were either treated with S4B6–IL-2C (black line), JES6–IL-2C (gray line), or PBS alone (filled histogram) from 5 to 7 dpi. At 7 dpi, donor cells gated as in (A) were analyzed for expression of CD25 and CD127, with representative staining and summary mean fluorescence intensity (MFI) analysis from three mice per group shown in (B) and (C), respectively. (D) At 28 dpi, the total number of donor cells was enumerated in the spleen, draining lymph nodes (dLN), and lung from groups of three to four mice treated with either IL-2C or with PBS alone. Summary of two replicate experiments and determined by one-way ANOVA. *p < 0.05, **p < 0.01.

Close modal

Although using IL-2 to promote or inhibit immune responses in clinical settings is gaining momentum, how IL-2 acts to shape inflammatory responses is still not well-understood. Furthermore, given that IL-2 available for paracrine consumption during immune responses can signal cells expressing high CD122 and/or high CD25, an analysis of how CD25-targeted IL-2 impacts inflammatory responses is required. This is also an important consideration for the myriad of experimental models that use IL-2C administration as a tool for the targeted expansion of specific subsets of lymphocytes, as this strategy could also have off target effects that impact the results observed. Indeed, we showed recently that IL-2 produced by CD4 T cells responding to IAV, or S4B6–IL-2C given to IAV-challenged mice, markedly enhances a broad spectrum of inflammatory cytokines and chemokines both systemically and in the infected lung (11). This IL-2–induced inflammatory response correlated with reduced lung function, less-efficient viral clearance, and enhanced weight loss. We show in this study using the same experimental system, that administration of JES6-based IL-2C also drives a strong, acute inflammatory response under steady-state conditions and during viral infection. However, in contrast to results observed with S4B6–IL-2C, JES6–IL-2C administration correlates with improved outcomes after IAV infection.

We provide several novel, to our knowledge, findings demonstrating that CD25-targeted IL-2 complexes delivered systemically induce a broad range of inflammatory factors systemically and in the lung in otherwise naive mice. An even stronger local response in the lung is generated upon i.n. JES6–IL-2C administration. These observations are surprising for two reasons. The first being that expression of CD25 is most-often tied to lymphocytes in activated states, most notably on T cells in which high CD25 levels are maintained only short-term following Ag stimulation, and relatively few highly activated cells are expected to populate the steady-state. The second is the well-known ability of JES6–IL-2C to selectively promote FOXP3+ Treg expansion, as this subset constitutively expresses CD25, an outcome commonly associated with anti-inflammatory impacts.

JES6–IL-2C have recently been used to expand ILCs in vivo (1315, 26). We speculate that ILC contribute to the “Th2”-associated cytokines (IL-4, IL-5, and IL-13) induced by JES6–IL-2C administration. In addition, as ILC have been implicated as key players in lung repair following IAV challenge, the activation of ILC by JES6–IL-2C may contribute to the reduced immunopathology seen in our studies (27, 28). Our own results (unpublished data) as well as other reports (29) indicate that FOXP3+ Tregs have a minimal impact on outcomes of primary IAV infection and play a greater role during secondary infection, in which stronger T cell responses in the lung have a greater capacity to cause immunopathology. Interestingly, in addition to CD4 T cell–derived IL-2, ILC3–derived IL-2 has recently been shown to promote Treg homeostasis in the small intestine through an inflammatory axis dependent upon the production of the inflammatory cytokine IL-1 (30). Lymphoid tissue inducer–like ILC1s and lung ILC3s are also capable of producing IL-2 (26, 31), and whether they similarly promote Treg homeostasis remains to be determined. JES6–IL-2C administration also induces a marked expansion of γδ T cells in the spleen and a significant but smaller response in the lung. In contrast to the beneficial outcomes reported in this study, IL-2 stimulation of γδ T cells and the subsequent production of IL-1 has recently been reported to compromise lung integrity (32). The opposing actions of IL-2 signals and the outcome of inflammatory cytokine production are perplexing, and further experiments are thus needed to address the precise roles of ILC, Tregs, and γδ T cells in the responses summarized in this study.

Our results highlight an unexpected disconnect between the detection of inflammatory factors at a site of infection and the degree of histological changes observed. This finding prompted us to probe whether patterns could be identified in the inflammatory milieu induced by S4B6–IL-2C, which correlates with worsened outcomes (11), and with JES6–IL-2C, which correlates with improved outcomes. Indeed, we found striking patterns of inflammation driven by IL-2 that correlated with improved outcomes (higher IL-4, GM-CSF, and CXCL2) or acute death (IL-1β, IL-6, TNF, CCL2, CCL3, and CXCL10). Our analysis may provide a roadmap to begin to determine positive versus negative elements of the “cytokine storm” associated with severe IAV infection, and thus an approach to develop novel therapeutic interventions to improve clinical outcomes.

Finally, we show that JES6–IL-2C that targets CD25 can be used to deliver promemory IL-2 signals to CD4 T cells responding to infection in vivo. This may, at first glance, be surprising given that the CD25 subunit of the IL-2R lacks cytoplasmic signaling capacity. However, given that JES6–IL-2C are well-known to stimulate proliferation of Tregs, which is supported in this study by the observation that the majority of Tregs in treated mice are high for the proliferation marker Ki-67, and given that we show that pretreatment of mice with anti-CD25 Ab abrogates the impacts of JES6–IL-2C, we surmise that the CD25-dependent binding of the IL-2C is able to stimulate similar signaling as S4B6–IL-2C. We stress that induction of different patterns of inflammatory cytokine and chemokine production by JES6– and S4B6–IL-2C but similar rescue of memory formation from Il2−/− CD4 T cells suggests that it is the direct impact of IL-2 signals on the CD4 T cells, and not other aspects of IL-2–induced inflammation, that is responsible. This agrees with findings using an adoptive transfer model that show that the inflammatory milieu associated with IAV has a minimal effect in promoting CD4 T cell memory (33). CD25-targeted IL-2 signals may thus be developed into powerful clinical approaches to simultaneously improve T cell memory while protecting tissue integrity.

In summary, we provide data indicating that prototypical anti-inflammatory IL-2C made with rIL-2 and the anti–IL-2 Ab clone JES6-1A12 are capable of driving a robust, acute systemic inflammatory response when administered i.p., and a local inflammatory response in the lungs when delivered i.n. Surprisingly, this inflammatory response improves rather than worsens immunopathology in the lung during IAV infection. Comparing the patterns of cytokine and chemokine upregulation by JES6- and S4B6-based IL-2C demonstrates remarkably stable hallmarks in both the steady-state and during infection. Our analysis provides an avenue for determining the most detrimental elements of the “cytokine storm” induced by influenza versus those aspects that may help to counter tissue damage or that correlate with this activity. Our studies also suggest that properly timed IL-2 signals can be used to simultaneously protect against tissue damage and promote robust CD4 T cell memory during IAV infection.

This work was supported by the National Institute of Allergy and Infectious Diseases, National Institutes of Health (R21AI117457 [to T.M.S.] and U10AI109858 Project 2 [to S.L.S.]), the National Institute of Child Health and Human Development, National Institutes of Health (R21HD093948 [to T.M.S.]), the American Heart Association (14SDG18600020 [to K.K.M.]), the State of Florida Crohn’s Funding Appropriation, the University of Massachusetts Medical School, and the University of Central Florida College of Medicine Burnett School of Biomedical Sciences.

The online version of this article contains supplemental material.

Abbreviations used in this article:

A/PR8

A/PuertoRico/8/1934

dpi

day postinfection

IAV

influenza A virus

ILC

innate lymphoid cell

IL-2C

IL-2 and anti–IL-2 Ab complex

i.n.

intranasal(ly)

Treg

regulatory CD4 T cell.

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The authors have no financial conflicts of interest.

Supplementary data