Therapeutic blockade of transforming growth factor beta fails to promote clearance of a persistent viral infection

Abstract

Persistent viral infections often overburden the immune system and are a major cause of disease in humans. During many persistent infections, antiviral T cells are maintained in a state of immune exhaustion characterized by diminished effector and helper functions. In mammalian systems, an extensive immune regulatory network exists to limit unwanted, potentially fatal immunopathology by inducing T cell exhaustion. However, this regulatory network at times overprotects the host and fosters viral persistence by severely dampening adaptive immune responsiveness. Importantly, recent studies have shown that T cell exhaustion is mediated in part by host immunoregulatory pathways (e.g., programmed death 1 [PD-1], interleukin 10 [IL-10]) and that therapeutic blockade of these pathways either before or during persistent infection can promote viral clearance. Transforming growth factor beta (TGF-β) is another immunosuppressive cytokine known to impede both self- and tumor-specific T cells, but its role in regulating antiviral immunity is not entirely understood. In this study, we inhibited TGF-β with three potent antagonists to determine whether neutralization of this regulatory molecule is a viable approach to control a persistent viral infection. Our results revealed that these inhibitors modestly elevate the number of antiviral T cells following infection with a persistent variant of lymphocytic choriomeningitis virus (LCMV) but have no impact on viral clearance. These data suggest that therapeutic neutralization of TGF-β is not an efficacious means to promote clearance of a persistent viral infection.

Fig 1

dnTGF-β RII mice rapidly control a persistent LCMV infection. (A) TGF-β1, -2, and -3 expression was quantified by qPCR using splenic RNA extracted from naïve, day 7 Arm-infected, and day 7 CL13-infected mice (n = 4 mice per group). All TGF isoforms were significantly upregulated in LCMV-infected mice. Asterisks denote a statistically significant increase from the naïve control group as determined by one-way ANOVA (P < 0.05). (B) The geometric mean fluorescent intensities of pSmad2/3 (less the isotype control signal) was quantified in splenic D^bGP_33–41-specific Thy1.1⁺ CD8⁺ T cells (P14) and I-A^bGP_61–80 CD45.1⁺ CD4⁺ T cells (SMARTA) 7 days following infection with Arm or CL13 (n = 5 mice per group). No statistically significant differences between the groups were observed. (C, D) Frequencies of D^b-GP_33–41 tetramer⁺ CD8⁺ T cells (B) and I-A^b-GP_61–80 tetramer⁺ CD4⁺ T cells (C) in the blood were determined in CL13-infected dnTGF-β RII⁺ and dnTGF-β RII⁻ mice at the denoted time points postinfection (n = 7 mice per group). Tetramer⁺ T cells were significantly increased (asterisks, P < 0.05) in dnTGF-β RII⁺ mice relative to in wild-type control mice. (E) At selected time points postinfection, serum viral titers were measured by plaque assay in CL13-infected dnTGF-β RII⁺ and dnTGF-β RII⁻ mice. dnTGF-β RII⁺ mice cleared virus significantly faster (asterisks, P < 0.05) than wild-type controls. All data in this figure are plotted as means ± standard deviations (SD) and are representative of at least two independent experiments.

Fig 2

LCMV CL13-infected dnTGF-β RII mice mount an increased antiviral T cell response. (A, B) The frequency (A) and absolute number (B) of MHC-I (D^bGP_33–41 and D^b-NP_396–404) and MHC-II (I-A^bGP_61–80) tetramer⁺ T cells were calculated in the spleen 10 days following CL13 infection of dnTGF-β RII⁺ and dnTGF-β RII⁻ mice (n = 5 mice per group). Representative dot plots are gated on CD8⁺ T cells for MHC-I tetramers and CD4⁺ T cells for MHC-II tetramers. Boxes denote the mean frequency of tetramer-positive cells in each group. Absolute numbers are plotted as means ± SD. (C) On day 10 postinfection, virus-specific CD8⁺ and CD4⁺ T cell responses were assessed by ex vivo peptide stimulation using splenocytes from wild-type and dnTGF-β RII⁺ mice (n = 5 mice per group). The absolute number of IFN-γ- and TNF-α-producing CD8⁺ (stimulated with GP_33–41 or NP_396–404) and CD4⁺ (stimulated with GP_61–80) T cells is plotted as mean ± SD. (D) Geometric mean fluorescent intensities (GMFI) for IFN-γ and TNF-α production from panel C are plotted (n = 5 mice per group). Asterisks on all plots and graphs denote a statistically significant increase (P < 0.05) in dnTGF-β RII⁺ mice relative to wild-type controls. Data are representative of at least two independent experiments.

Fig 3

Therapeutic blockade of TGF-β increases antiviral T cells but does not impact viral clearance. To block TGF-β signaling, mice were injected with IN1233 (25 mg/kg/day), anti-TGF-β antibody (1 mg every other day), or TGF-β RII-Fc (50 μg every other day) (see Materials and Methods). (A, B) Graphs illustrate the frequency (A) and absolute number (B) of D^b-GP_33–41 tetramer⁺ CD8⁺ T cells and I-A^b-GP_61–80 tetramer⁺ CD4⁺ T cells in the spleen of treated mice 10 days following infection (mean ± SD) (n = 5 mice per group). (C) The functionality of LCMV-specific CD4⁺ and CD8⁺ T cells in the spleen was evaluated by ex vivo peptide stimulation with GP_61–80 and NP_396–404, respectively. The graph shows the absolute number (mean ± SD) of IFN-γ- and TNF-α-producing T cells that respond to peptide (n = 5 mice per group). (D) Nine days postinfection, serum viral titers were determined by plaque assay (n = 5 mice per group). Data are expressed as PFU/ml of serum sampled (mean ± SD). All data are representative of at least two independent experiments. Asterisks denote a statistically significant increase (P < 0.05) relative to the PBS control group.

Fig 4

Administration of anti-TGF-β antibodies does not promote clearance of a persistent viral infection. To determine the efficiency of TGF-β neutralization, serum concentrations of the cytokine were quantified by ELISA in day 8 CL13-infected mice treated with anti-TGF-β or isotype control antibodies (1.5 mg every other day). Naïve untreated mice served as a control (n = 5 mice per group). Anti-TGF-β antibodies reduced the cytokine to an undetectable level (mean ± SD = 0 ± 0) in all CL13-infected mice. (B, C) MHC-I (D^bGP_33–41) and MHC-II (I-A^bGP_61–80) tetramers were used to calculate frequencies (A) and absolute numbers (B) of virus-specific T cells in the spleens of isotype versus anti-TGF-β antibody (1.5 mg every other day)-treated mice at day 9 postinfection. Representative dot plots (A) are gated on CD8⁺ T cells for MHC-I tetramers and CD4⁺ T cells for MHC-II tetramers. Boxes denote the mean frequency of tetramer-positive cells in each group. The absolute number of splenic tetramer-positive T cells was calculated from the frequency data shown in panel A (n = 5 mice per group). (D) On day 10 postinfection, virus-specific CD8⁺ and CD4⁺ T cell responses were assessed by ex vivo peptide stimulation of splenocytes from both isotype and anti-TGF-β antibody-treated mice. Boxes denote the absolute number (mean ± SD) of cytokine-producing cells for each group (n = 5 mice per group). (E) Average geometric mean fluorescent intensities (GMFI) from panel C were graphed for all cytokines. (F) At selected time points, serum viral titers were determined by plaque assay and plotted as means ± SD (n = 5 mice per group). Asterisks on all graphs denote a statistically significance increase (P < 0.05) relative to the control group, and data are representative of at least two independent experiments.

Fig 5

Adoptively transferred dnTGF-β RII memory T cells do not accelerate clearance of a persistent viral infection. A total of 10⁴ wild-type or dnTGF-β RII Thy1.1⁺ CD8⁺ P14 cells was transferred into naïve C57BL/6J mice and then infected intravenously with LCMV CL13 (n = 5 mice per group). (A) Representative dot plots gated on CD8⁺ T cells show the mean frequency of Thy1.1⁺ CD8⁺ T cells (black boxes) in the blood of mice at day 8 and day 13 following LCMV infection. (B) The frequencies of Thy1.1⁺ CD8⁺ T cells in the blood are plotted over time (mean ± SD). Note that dnTGF-β RII Thy1.1⁺ CD8⁺ P14 cells are rejected from CL13-infected mice by day 13 postinfection. Asterisks denote a statistically significant difference between the groups (P < 0.05). (C) At 45 days following i.p. infection with LCMV Arm, memory splenocytes were extracted from wild-type and dnTGF-β RII mice and then adoptively transferred i.p. into wild-type and dnTGF-β RII LCMV carrier mice, respectively (n = 4 mice per group). The correction factor for dnTGF-β RII splenocytes was calculated by staining pooled memory cells with anti-CD8 antibody and a D^bGP_33–41 tetramer prior to injection. A correction factor was introduced into each experiment to ensure that comparable numbers of wild-type and dnTGF-β RII memory CD8⁺ T cells were transferred into LCMV carrier mice (see Materials & Methods). MHC-I (D^bGP_33–41) and MHC-II (I-A^bGP_61–80) tetramers were used to calculate the absolute number of transferred virus-specific T cells in the spleens of wild-type and dnTGF-β RII carrier mice at day 11 posttransfer. Data are plotted as means ± SD. (D) At selected time points following adoptive immunotherapy, serum viral titers in recipient carrier mice were determined by plaque assay (n = 4 mice per group). Data are expressed as PFU/ml of serum sampled (mean ± SD) and are representative of three independent experiments.

Fig 6

dnTGF-β RII mice contain a large repertoire of activated T cells prior to infection. (A) Heterozygous dnTGF-β RII mice were crossed with Thy1.1⁺ P14 mice. Splenic Thy1.1⁺ CD8⁺ T cells in the resultant F1 mice were screened at 8 weeks for expression of CD44 and CD122. Histograms and dot plots are gated on Thy1.1⁺ CD8⁺ P14 T cells. Note that CD44 and CD122 expressions are elevated on CD8⁺ T cells from dnTGF-β RII⁺ mice relative to those of wild-type littermate controls (n = 3 mice per group). Boxes denote the mean frequency of CD122⁺ Thy1.1⁺ CD8⁺ T cells in each group. (B) Naïve 8-week-old Thy1.2⁺ P14 mice were injected every other day i.p. for 3 weeks with 1 mg of anti-TGF-β or isotype control antibodies. CD44 and CD122 expression was then examined on splenic Thy1.2⁺ CD8⁺ T cells as described in panel A. No significant differences were observed between the groups (n = 3 mice per group). (C, D) dnTGF-β RII⁺ or dnTGF-β RII⁻ mice were seeded with 10⁴ naïve Thy1.1⁺ CD8⁺ P14 cells and then infected i.v. with LCMV CL13. The frequency of Thy1.1⁺CD8⁺ P14 cells was determined in the blood at day 7 postinfection, and the absolute number of cells was calculated in the spleen at day 10 postinfection (n = 5 mice per group). Asterisks denote a statistically significance increase (P < 0.05) in dnTGF-β RII⁺ recipients relative to wild-type controls. All data are representative of at least two independent experiments.