Adjuvant IL-7 or IL-15 overcomes immunodominance and improves survival of the CD8+ memory cell pool

Abstract

Current models of T cell memory implicate a critical role for IL-7 in the effector-to-memory transition, raising the possibility that IL-7 therapy might enhance vaccine responses. IL-7 has not been studied, to our knowledge, before now for adjuvant activity. We administered recombinant human IL-7 (rhIL-7) to mice during immunization against the male antigen HY and compared these results with those obtained from mice immunized with rhIL-2 and rhIL-15. Administration of rhIL-7 or rhIL-15, but not rhIL-2, increased effector cells directed against these dominant antigens and dramatically enhanced CD8(+) effectors to subdominant antigens. The mechanisms by which the cytokines augmented effector pool generation were multifactorial and included rhIL-7-mediated costimulation and rhIL-15-mediated augmentation of the proliferative burst. The contraction phase of the antigen-specific response was exaggerated in cytokine-treated mice; however, CD8(+) memory pools in rhIL-7- or rhIL-15-treated groups demonstrated superior long-term survival resulting in quantitative advantages that remained long after the cytokines were discontinued, as demonstrated by improved survival after challenge with an HY-expressing tumor undertaken several weeks after cytokine cessation. These results confirm the adjuvant activity of rhIL-15 and demonstrate that rhIL-7 also serves as a potent vaccine adjuvant that broadens immunity by augmenting responses to subdominant antigens and improving the survival of the CD8(+) T cell memory pool.

Figures

Figure 1

The γc cytokines expand splenic T cell populations. (A–C) C57BL/6 mice received rhIL-2 (n = 9), rhIL-7 (n = 9), rhIL-15 (n = 9), or carrier alone (n = 9) daily i.p. from day 0 (d 0) to day 27 as described in Methods. Data from 1 representative experiment are shown; results were confirmed in 3 separate experiments. (A) Serum levels of hIL-7 (left panel) and hIL-15 (right panel) were measured 24 hours after injection. Levels were stable throughout therapy and returned to baseline upon cessation of therapy. (B) All cytokine groups showed significant increases in splenocyte number on day 28. (C) CD8+ (P < 0.0001) and CD4+ (P < 0.0001) splenocyte populations were expanded equally (1.5–to 2.0–fold) by rhIL-7, with persistent CD4+ expansion for 14 days despite serum clearance of the cytokine. CD8+ cell populations were expanded (4–fold; P < 0.0001) but CD4+ T cell numbers were reduced by 30% by rhIL-15. Increases of less than 2-fold were induced by rhIL-2 in CD8+ (P < 0.0001) and CD4+ (P = 0.008) splenocytes.

Figure 2

The cytokine rhIL-7 expands both naive and memory/activated CD8+ T cell populations, whereas rhIL-15 preferentially expands memory/activated CD8+ T cell populations. (A–D) Mice received rhIL-7 (n = 7), rhIL-15 (n = 8), or carrier alone (n = 8), as described in Figure 1. Data from 1 representative experiment are shown; results were confirmed in 3 separate experiments. (A and C) CD8+CD122+ and CD8+CD44+ splenic T cells are increased in both rhIL-7– and rhIL-15–treated animals on day 28. (B and D) Mice treated with rhIL-7 (red lines) show no difference compared with sham-treated mice (gray filled histograms) in the frequency of CD8+ cells that were CD122+ or CD44+ at any time point studied. The increased frequency of CD8+CD122+ and CD8+CD44+ T cells in rhIL-15–treated mice (blue line) on day 28 persists through day 120.

Figure 3

T cell populations from cytokine-treated and control mice undergo equivalent homeostatic expansion. (A and B) LN T cells (3 × 106) harvested on day 120 after therapy with carrier, rhIL-7, or rhIL-15 as described in Figure 1 were adoptively transferred into lymphopenic SCID recipients. At 22 days after transfer, splenocytes (A) and LN cells (B) in SCID mice were enumerated. Significant differences between control SCID mice versus recipients of adoptive transfer from the various groups are shown. There were no significant differences in the degrees of expansion observed using inocula harvested from rhIL-7–, rhIL-15–, or sham-treated mice.

Figure 4

Both rhIL-7 and rhIL-15 increase CD8+ effectors specific for the immunodominant antigen. (A–E) Mice were treated as described in Figure 1. Uty responses on day 28 are shown from 3 pooled experiments with n = 25 (sham, rhIL-7, and rhIL-15) and n = 17 (rhIL-2). (A) Splenocytes binding Uty tetramers, with binding to irrelevant E7 tetramers subtracted. (B) FACS plots illustrating Uty tetramer binding in representative sham-, rhIL-7–, and rhIL-15–treated mice and E7 tetramer background staining. (C) Absolute number (left) and frequency (right) of splenocytes producing IFN-γ after in vitro stimulation with Uty peptide. (D) LN cells from cytokine- or sham-treated immunized mice were harvested on day 26, labeled with CFSE, and transferred to congenic recipients, and cytokine therapy was continued for 96 hours. CFSE expression on antigen-specific cells (shaded histograms) and non–antigen-specific cells (white histograms) are shown from 4 representative animals per group. Blue brackets on each histogram designate gates used to generate the percentages shown above each panel. Mean percents in the far left gates were 20 ± 2.4 for rhIL-7 versus 39 ± 3.5 for rhIL-15 versus 26.7 ± 4.8 for sham (P = 0.008, rhIL-15 versus sham; n = 6 per group). (E) Day 28 tetramer-binding cells from sham- and cytokine-treated mice were analyzed for MFI. Each colored histogram displays tetramer binding on gated CD8+CD44+ cells from individual mice within each group. RhIL-7–treated animals had significantly reduced tetramer binding, as assessed by MFI, compared to sham-treated animals (P = 0.04).

Figure 5

Both rhIL-7 and rhIL-15 increase effectors specific for subdominant and MHC class II–restricted antigens. (A–C) Results were pooled from the groups of animals described in Figure 4. Splenocytes were analyzed for IFN-γ production using ELISPOT. Data are shown as absolute number of responding splenocytes (left panels) and frequency of responding splenocytes (right panels) to the antigen designated on the y axis. (A and B) Net IFN-γ–producing cells were calculated by enumeration of the ELISPOT IFN-γ response to the Smcy peptide (A) and Dby peptide (B) and subtraction of the number of cells responding to the control peptides, as described in Methods. (C) Net male-reactive cells were calculated by enumeration of responses to male splenocytes and subtraction of responses to female splenocytes. P values are shown where results for sham- and cytokine-treated mice are significantly different.

Figure 6

Administration of rhIL-7 or rhIL-15 with immunization results in long-term expansion of the memory pool. (A–F) Animals (n = 8 per group) were immunized (days 0, 14, 28, and 42) and treated with cytokines or carrier from days 0–27, then allowed to rest until day 120. Frequency of splenocytes producing IFN-γ in response to Uty (A) and binding Uty tetramers (B). (C) Uty tetramer–binding cells were electronically gated as described in Figure 4D. Each panel shows colored histograms from 9 individual mice in each group. No significant differences in tetramer binding, as measured by MFI, were seen between groups. Frequency of splenocytes producing IFN-γ in response to Smcy (D) and binding Smcy tetramers (E). (F) Frequency of cells producing IFN-γ in response to Dby. (G and H) Animals were immunized on day 0 and treated with cytokines from days 0–27. On day 60, groups were challenged with 3 × 106 MB49 tumor cells, then were euthanized when tumor progression met NCI Animal Care and Use guidelines. (G) Mean tumor volume 21 days after tumor challenge. (H) Overall survival is shown. There is significantly prolonged survival for rhIL-7 compared with all groups (versus vaccine alone, P = 0.0029; versus IL-2, P = 0.002; versus no vaccine, P = 0.008; versus rhIL-15, P = 0.02).

Figure 7

Mice treated with rhIL-7 or rhIL-15 show exaggerated contraction of the effector pool but improved survival of the memory pool. (A–C) Animals received cytokines as described in Figure 1 (horizontal black bars), were immunized as described in Methods (arrows), and then were sacrificed for analysis at day 21 (n = 16), day 28 (n = 24), day 42 (n = 9), day 56 (n = 24), and day 120 (n = 8). Data are pooled from 2 to 3 experiments. (A) The frequency of Uty-specific cells in cytokine-treated groups dramatically drops from day 28 to day 42, indicating a greater death rate for antigen-specific than for non–antigen-specific cells during cytokine withdrawal. Compared with controls, effectors generated with cytokines show a steeper slope, indicating increased contraction from day 28 to day 42, but a diminished slope from day 56 to day 120, indicating improved survival. (B) Ratio of spot-forming unit (SFU) frequencies in cytokine-treated versus sham-treated animals is shown as the SFU frequency ratio. Therapy with rhIL-7 and rhIL-15 expands the effector pools, which undergo exaggerated contraction from day 28 to day 42, resulting in a downward slope. From day 56 onward, antigen-specific CD8+ cells in rhIL-7– and rhIL-15–treated hosts have improved survival, resulting in an upward slope. Survival of CD4+ memory cells is not improved in cytokine-treated groups. (C) IL-15Tg mice (6 per time point) and littermate controls (9 per time point) were immunized on day 0 and day 14. Data represent ratio of the mean frequency of IFN-γ producers/106 splenocytes in IL-15Tg mice to that in control mice at designated time points.