Treatment with Interleukin-7 Restores Host Defense against Pneumocystis in CD4+ T-Lymphocyte-Depleted Mice

Abstract

Pneumocystis pneumonia (PCP) is a major cause of morbidity and mortality in patients with HIV infection. CD4(+) T lymphocytes are critical for host defense against this infection, but in the absence of CD4(+) T lymphocytes, CD8(+) T lymphocytes may provide limited host defense. The cytokine interleukin-7 (IL-7) functions to enhance lymphocyte proliferation, survival, and recruitment of immune cells to sites of infection. However, there is little known about the role of IL-7 in PCP or its potential use as an immunotherapeutic agent. We hypothesized that treatment with recombinant human IL-7 (rhIL-7) would augment host defense against Pneumocystis and accelerate pathogen clearance in CD4-depleted mice. Control and CD4-depleted mice were infected with Pneumocystis, and rhIL-7 was administered via intraperitoneal injection. Our studies indicate that endogenous murine IL-7 is part of the normal host response to Pneumocystis murina and that administration of rhIL-7 markedly enhanced clearance of Pneumocystis in CD4-depleted mice. Additionally, we observed increased recruitment of CD8(+) T lymphocytes to the lungs and decreased apoptosis of pulmonary CD8(+) T lymphocytes in rhIL-7-treated animals compared to those in untreated mice. The antiapoptotic effect of rhIL-7 was associated with increased levels of Bcl-2 protein in T lymphocytes. rhIL-7 immunotherapy in CD4-depleted mice also increased the number of gamma interferon (IFN-γ)-positive CD8(+) central memory T lymphocytes in the lungs. We conclude that rhIL-7 has a potent therapeutic effect in the treatment of murine Pneumocystis pneumonia in CD4-depleted mice. This therapeutic effect is mediated through enhanced recruitment of CD8(+) T cells and decreased apoptosis of lung T lymphocytes, with a preferential action on central memory CD8(+) T lymphocytes.

FIG 1

IL-7 secretion is part of the normal host defense against P. murina infection. Wild-type BALB/c mice and CD4-depleted BALB/c mice were administered 5 μg of rhIL-7 via i.p. injection. CD4-intact animals were given rhIL-7 every 3 days with the first dose, coinciding with P. murina infection. CD4-depleted animals were infected with P. murina, and infection was allowed to establish for 2 weeks. CD4-depleted animals were then continuously treated with rhIL-7 every 3 days. rhIL-7 treatments were given continuously throughout the experiment. CD4-intact and CD4-depleted mice were sacrificed, and rhIL-7 and endogenous IL-7 levels in the serum were assayed. (A) CD4-intact mice exhibit a significant increase in the levels of rhIL-7 at 3, 7, and 14 days postinfection (dpi) as determined by ELISA. (B) CD4-depleted animals had significant increases in the levels of rhIL-7 at 28 and 42 dpi compared to the values for untreated mice. (C) CD4-intact mice exhibit significant increases in the levels of endogenous IL-7 at 3, 7, and 14 dpi. (D) CD4-depleted animals had significant increases in the levels of endogenous IL-7 at 28 and 42 dpi compared to the values for untreated mice. Data are reported as means ± SEM; n = 6. *, P < 0.01 compared to the value for mice treated with PBS, as determined by repeated-measures two-way ANOVA with post hoc comparison of the means using Bonferroni's multiple-comparison test. N.D., not detected.

FIG 2

Treatment with rhIL-7 significantly decreases P. murina lung burden. Wild-type BALB/c mice and CD4-depleted BALB/c mice were administered 5 μg of rhIL-7 via i.p. injection, as described in Materials and Methods. P. murina lung burden was assessed for CD4-intact and CD4-depleted mice following treatment with rIL-7. (A) CD4-intact mice exhibit a significant decrease in fungal 14 dpi as determined by quantitative PCR. (B) CD4-depleted animals had significant decreases in P. murina lung burden at both 28 and 42 dpi compared to the values for untreated mice. P. murina lung burden is expressed as copy numbers per right lung. Data are reported as means ± SEM; n = 5. *, P < 0.01 compared to the values for mice treated with PBS, as determined by repeated measures two-way ANOVA with post hoc comparison of the means using Bonferroni's multiple-comparison test.

FIG 3

Treatment with rhIL-7 significantly increases the number of CD4 central memory cells. CD4-intact mice were treated with rIL-7, and the numbers of CD4⁺ T cells and CD4⁺ T-cell subsets in the BAL fluid at 3, 7, and 14 dpi were determined via flow cytometry. Treatment of CD4-intact mice with rhIL-7 increased the absolute numbers of CD4⁺ T cells (A) and effector memory CD4⁺ T cells (B). rhIL-7 treatment significantly increased the number of central memory CD4⁺ T cells (C) 7 dpi compared to the value for mice that did not receive rhIL-7. Data are reported as means ± SEM; n = 6. *, P < 0.01 compared to the value for mice treated with PBS, determined by repeated-measures two-way ANOVA with post hoc comparison of the means using Bonferroni's multiple-comparison test. EM, effector memory; CM, central memory.

FIG 4

Treatment with rhIL-7 significantly increases the number of CD8 central memory cells. CD4-intact mice were treated with rIL-7, and the numbers of CD8⁺ T cells and CD8⁺ T-cell subsets in the BAL fluid at 3, 7, and 14 dpi were determined via flow cytometry. Treatment of CD4-intact mice with rhIL-7 increased the absolute numbers of CD8⁺ T cells (A) and effector memory CD8⁺ T cells (B). rhIL-7 treatment significantly increased the number of central memory CD8⁺ T cells (C) 7 dpi compared to the value for mice that did not receive rhIL-7. Data are reported as means ± SEM; n = 6. *, P < 0.01 compared to the value for mice treated with PBS, determined by repeated-measures two-way ANOVA with post hoc comparison of the means using Bonferroni's multiple-comparison test.

FIG 5

Treatment of CD4-depleted mice with rIL-7 significantly increases the numbers of CD8⁺ T cells and CD8⁺ T-cell subsets. CD4-depleted mice were treated with rIL-7, and the numbers of CD8⁺ T cells and CD8⁺ T-cell subsets in the BAL fluid at 28 and 42 dpi were determined via flow cytometry. Treatment of CD4-depleted mice with rhIL-7 significantly increased the absolute numbers of CD8⁺ T cells (A), effector memory CD8⁺ T cells (B), and central memory CD8⁺ T cells (C) at both 28 and 42 dpi compared to the values for mice that did not receive rhIL-7. Data are reported as means ± SEM; n = 6. *, P < 0.01 compared to the value for mice treated with PBS, determined by repeated-measures two-way ANOVA with post hoc comparison of the means using Bonferroni's multiple-comparison test.

FIG 6

Treatment of CD4-depleted mice with rIL-7 increases the numbers IL-7R^hi (CD127) CD8⁺ T cells and CD8⁺ T-cell subsets. Mice were administered rhIL-7 as described in Materials and Methods. CD4-depleted mice were then sacrificed 6 weeks after infection with P. murina. The absolute numbers of BAL fluid (A to D) and spleen (E to H) CD8⁺ T cells and CD8⁺ T-cell subsets were assessed via flow cytometry. CD8⁺ T cells and CD8⁺ T-cell subsets from the BAL fluid of CD4-depleted mice treated with rhIL-7 exhibited 7.9-, 6.13-, 9.69-, and 11.57-fold increases in IL-7R expression on CD8⁺ T cells (A), naive CD8⁺ T cells (B), effector memory CD8⁺ T cells (C), and central memory CD8⁺ T cells (D), respectively. CD8⁺ T cells and CD8⁺ T-cell subsets from the spleens of CD4-depleted mice treated with rhIL-7 exhibited 6.12-, 6.7-, 3.35-, and 4.77-fold increases in IL-7R expression in CD8⁺ T cells (E), naive CD8⁺ T cells (F), effector memory CD8⁺ T cells (G), and central memory CD8⁺ T cells (H), respectively. Data are reported as means ± SEM; n = 6. *, P < 0.01 compared to the value for mice treated with PBS, as determined by unpaired Student's t test analysis.

FIG 7

CD8⁺ T cell and CD8⁺ T-cell subset proliferation is enhanced by rhIL-7 treatment. CD4-depleted mice were treated with 5 μg of rhIL-7 or PBS, as described previously. Six weeks (42 days) after infection with P. murina BAL fluid, cells were harvested and intracellular staining of the proliferation marker Ki67 was assessed for CD8⁺ T cells and each CD8⁺ T-cell subset via flow cytometry. Treatment with rIL-7 significantly increased absolute numbers of CD8⁺ T cells (A), naive CD8⁺ T cells (B), effector memory CD8⁺ T cells (C), and central memory CD8⁺ T cells (D). Data are reported as means ± SEM; n = 6. *, P < 0.01 compared to the value for mice treated with PBS, as determined by unpaired Student's t test analysis.

FIG 8

rhIL-7 treatment significantly decreases camptothecin-induced apoptosis of lung CD8⁺ T cells and CD8⁺ T-cell subsets. P. murina-inoculated CD4-depleted mice were treated with 5 μg of rIL-7 every 3 days beginning 2 weeks after P. murina inoculation. At 6 weeks, mice were sacrificed and lung BAL fluid cells were harvested. Apoptosis was determined by the cellular poly(ADP)-ribose polymerase (PARP) apoptosis assay coupled with flow cytometry. Mice treated with rhIL-7 exhibited significant decreases in the percentages of CD8⁺ T cells (A), naive CD8⁺ T cells (B), effector memory CD8⁺ T cells (C), and central memory CD8⁺ T cells (D). Data are reported as means ± SEM; n = 6. *, P < 0.01 compared to the value for mice treated with PBS, as determined by unpaired Student's t test analysis.

FIG 9

Mice treated with rhIL-7 exhibit increased levels of Bcl-2 in CD8⁺ T cells and CD8⁺ T-cell subsets. CD4-depleted mice were inoculated with P. murina. Two weeks postinoculation, mice were treated with 5 μg of rhIL-7. BAL fluid cells were harvested 6 weeks later, and intracellular levels of Bcl-2 were determined for CD8⁺ T cells and CD8⁺ T-cell subsets by flow cytometry. Mice treated with rhIL-7 exhibited significantly increased absolute numbers of cells expressing Bcl-2 in CD8⁺ T cells (A), naive CD8⁺ T cells (B), effector memory CD8⁺ T cells (C), and central memory CD8⁺ T cells (D). Data are reported as means ± SEM; n = 5. *, P < 0.01 compared to the value for mice treated with PBS, as determined by unpaired Student's t test analysis.

FIG 10

CD4-depleted mice treated with rh-IL-7 exhibit a marked increase in intracellular IFN-γ levels. CD4-depleted mice were treated with either 5 μg of rhIL-7 or PBS every 3 days beginning 2 weeks after P. murina inoculation. Mice were sacrificed, and BAL fluid cells were harvested. A total of 1 × 10⁶ cells were plated and stimulated for 12 h with P. murina antigen, followed by treatment with Golgistop for an additional 4 h. Cells were stained with CD3, CD8, CD62L, and CD44 antibody, followed by intracellular IFN-γ staining. Cells were assayed via flow cytometry. Treatment of mice with rIL-7 resulted in significantly higher numbers of cells expressing IFN-γ in CD8⁺ T cells (A), naive CD8⁺ T cells (B), effector memory CD8⁺ T cells (C), and central memory CD8⁺ T cells (D). Data are reported as means ± SEM; n = 6. *, P < 0.01 compared to the value for mice treated with PBS, as determined by unpaired Student's t test analysis.