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IL-6-mediated Environmental Conditioning of Defective Th1 Differentiation Dampens Antitumour Immune Responses in Old Age

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IL-6-mediated Environmental Conditioning of Defective Th1 Differentiation Dampens Antitumour Immune Responses in Old Age

Hirotake Tsukamoto et al. Nat Commun.

Abstract

Decline in immune function and inflammation concomitantly develop with ageing. Here we focus on the impact of this inflammatory environment on T cells, and demonstrate that in contrast to successful tumour elimination in young mice, replenishment of tumour-specific CD4(+) T cells fails to induce tumour regression in aged hosts. The impaired antitumour effect of CD4(+) T cells with their defective Th1 differentiation in an aged environment is restored by interleukin (IL)-6 blockade or IL-6 deficiency. IL-6 blockade also restores the impaired ability of CD4(+) T cells to promote CD8(+) T-cell-dependent tumour elimination in aged mice, which requires IFN-γ. Furthermore, IL-6-stimulated production of IL-4/IL-21 through c-Maf induction is responsible for impaired Th1 differentiation. IL-6 also contributes to IL-10 production from CD4(+) T cells in aged mice, causing attenuated responses of CD8(+) T cells. These findings suggest that IL-6 serves as an extrinsic factor counteracting CD4(+) T-cell-mediated immunity against tumour in old age.

Figures

Figure 1
Figure 1. Tumour-specific CD4+ T-cell-mediated protective antitumour immunity is impaired with age-dependent increases of IL-6 and sIL-6R.
(a) Young and aged mice were vaccinated with or without OVA-IIp emulsified in IFA. Seven days after immunization, mice were inoculated with MCA-OVA. (b) CD45.1+ young naive OT-II T cells were transferred into young or aged CD45.2+ mice. The mice were then immunized by transfer of DCs pulsed with or without OVA-IIp. Tumour inoculation was then performed. Each line represents the kinetics of tumour growth in an individual mouse (n=4–12 mice per group). *P<0.05, ***P<0.001, unpaired Student's t-test. (c) Quantitation of IL-6 (upper) and sIL-6R (lower) in sera from 3-, 6-, 18- or 24-month-old mice. Control or anti-Gr-1 Ab was injected 5 days before serum harvest. Each point represents an individual mouse. (d) OT-II cell transfer and immunization were performed as described in b. Mice were treated with control or anti-IL-6 Ab on day −1 and day 1. Six days after immunization, MCA-OVA were injected. Representative data from three independent experiments with similar results are shown (n=6–8 mice per group). *P<0.05, **P<0.01, ***P<0.001, analysis of variance followed by Tukey's post hoc test. NS, not significant.
Figure 2
Figure 2. IL-6 increased in the aged environment is responsible for the impairment of CD4+ T-cell-mediated antitumour immunity.
(a) OT-II cells were transferred into young or aged IL-6+/+ or IL-6−/− mice. Immunization was performed as in Fig. 1b. Five days after immunization, luciferase-expressing MO4 were intravenously injected for the pulmonary metastatic model. Luminescence images at day 32 (left) and kinetics of photon counts per mouse (right) are shown. (b) Young and aged mice were immunized by the transfer of EnvH13.3 peptide-pulsed DCs, and were treated with control Ab or anti-IL-6R Ab 1 day before and after immunization. On day 6 post immunization, RMA tumour cells were inoculated and their growth was monitored. (c,d) Young and aged mice were treated with anti-CD4 Ab. After 2 days, the mice were immunized with DCs pulsed with OVA-Ip (SIINFEKL), and then inoculated with MCA-OVA. Tumour progression was monitored with time (c). Five days after tumour inoculation, draining LNs were harvested and re-stimulated with OVA-Ip-pulsed DC ex vivo. IFN-γ production was determined by the ELISPOT assay (d). Representative from two or three experiments are shown as the mean±s.e.m. (n=5–10 mice per group). *P<0.05, **P<0.01, analysis of variance followed by Tukey's post hoc test. NS, not significant.
Figure 3
Figure 3. The aged environment dampens antigen-induced proliferation of CD4+ T cells.
(ac) OT-II cell transfer, immunization and Ab treatment were performed as described in Fig. 1d. The frequency of donor CD45.1+OT-II cells in PBL was monitored over time (a), and the total number of donor OT-II cells (b) and CD4+Foxp3+Treg cells (c) in spleen and LNs were determined 6 days after immunization. (d) The number of Gr-1+CD11b+ MDSC in spleen and LN cells from young or aged IL-6+/+ or IL-6−/− mice were determined. (eg) Anti-Gr-1 Ab was injected 5 days before transfer of the CFSE-labelled OT-II cells, followed by immunization. The total number at day 6 (e) and CFSE profile at day 4 (f) of donor OT-II cells under the indicated conditions are shown. Seven days after immunization, MCA-OVA were inoculated into the aged mice. The outgrowth in aged mice was measured over time (g). The values are mean±s.e.m. (n=6 per group); *P<0.05, **P<0.01, analysis of variance followed by Tukey's post hoc test. Representative data from two or three experiments with similar results are presented.
Figure 4
Figure 4. Age-related increase in IL-6 attenuates Th1 differentiation of antigen-primed CD4+ T cells.
(a) OT-II cell transfer, immunization and Ab treatment were performed as described in Fig. 1d. Six days after immunization, CD4+CD62Llo cells were sorted from the spleen and LNs of young and aged mice (details are described in Methods), and then were re-stimulated ex vivo with OVA-IIp-pulsed DCs for 36 h. The culture supernatant was harvested, and the concentrations of the indicated cytokines were measured. The values are mean±s.e.m. (n=4 per group). (bd) OT-II cell transfer, immunization and anti-IL-6 Ab injection were performed. IL-6+/+ or IL-6−/− mice were utilized as hosts in d. Donor OT-II cells were isolated 6 days after immunization, and re-stimulated with phorbol-12-myristate-13-acetate/ionomycin. Cytokine production was assessed by intracellular staining. Representative dot plots (b) and individual values along with the mean (c,d) in the indicated cytokine-positive fractions are shown. Representative data from 3–4 independent experiments with similar results are shown. *P<0.05, **P<0.01, ***P<0.001, analysis of variance followed by Tukey's post hoc test. NS, not significant.
Figure 5
Figure 5. IL-6 and the aged environment alter the expression profile of transcription factors in CD4+ T cells.
OT-II cell transfer, immunization and anti-IL-6 Ab injection were performed as in Fig. 1d. (a) After 5 days, total CD4+ T cells were sorted from spleen and LNs, and RNA was isolated from them. mRNA expression of indicated transcription factors was analysed by real-time quantitative PCR. Shown is relative value to Gapdh expression (mean±s.e.m. with n=4–6 per group). Y, young hosts; A, aged hosts. (b,c) Six days after immunization, donor OT-II cells were analysed for protein expression of indicated transcription factors. Individual values along with the mean from Ab-treated IL-6+/+ mice (b) and the representative histograms from IL-6+/+ or IL-6−/− mice (c) are shown. Representative from three independent experiments are shown. *P<0.05, **P<0.01, ***P<0.001, analysis of variance followed by Tukey's post hoc test. NS, not significant.
Figure 6
Figure 6. Tumour-specific CD4+ T-cell-derived IFN-γ is a prerequisite for the effect of IL-6 blockade on antitumour activity in aged mice.
OT-II cells from young wild-type (WT) or IFN-γ-deficient (IFN-γ knockout (KO)) mice were transferred into young and aged mice. Immunization and tumour inoculation were performed as described in Fig. 2a. (a) One week after tumour inoculation, IFN-γ concentration in serum was determined by an enzyme-linked immunosorbent assay. Individual values along with the mean are shown. (b) The in vivo growth of luciferase-expressing MO4 cells was monitored over time, and is shown as the photon counts in luminescence images. Data shown are representatives from two experiments with similar results (mean±s.e.m. with n=4–6 per group; *P<0.05, analysis of variance followed by Tukey's post hoc test). NS, not significant.
Figure 7
Figure 7. Th1-dependent helper activity for tumour-specific CD8+ T cells is diminished by IL-6 in aged mice.
(a) T-cell transfer, immunization and Ab treatment were performed as in Fig. 1d. Five days after immunization, the mice were injected with anti-CD8 or control Ab. MCA-OVA was inoculated on day 0. Data for tumour growth are mean±s.e.m. (n=8–10 mice per group). (be) MCA-OVA were inoculated 5 days after OT-II transfer and immunization. Another 6 days later, OVA-specific CD8+ T cells in tumour-draining LNs (c and d) and tumour-infiltrated lymphocytes (TILs) (e) were analysed. Representative plots (c) and the total number (d) of OVA-Ip/H-2Kb-tetramer+CD8+CD44hi cells are shown. (f,g) WT or IFN-γ-deficient (knockout (KO)) OT-II cells were transferred into young and aged mice. OVA-specific CD8+ T cells in young or aged hosts that were treated as described in b were determined by OVA-Ip/H-2Kb-tetramer staining (f). OVA-Ip-specific CD8+ T-cell response in tumour-draining LNs was also assessed by IFN-g ELISPOT (g). (h) Immunization with EnvH13.3-pulsed DC and RMA inoculation in young or aged mice were performed as described in Fig. 2b. Five days after tumour inoculation, indicated tumour-associated peptide-specific IFN-γ production in tumour-draining LNs were assessed by ELISPOT. Representative data from 2–3 experiments are shown (mean±s.e.m. with n=4–5 per group). *P<0.05, **P<0.01, ***P<0.001 analysis of variance followed by Tukey's post hoc test.
Figure 8
Figure 8. IL-6-induced IL-4/IL-21 production is responsible for the defect of Th1 differentiation.
Young naive CD4+ T cells were stimulated with anti-CD3 and anti-CD28 Abs plus exogenous IL-12 in vitro. (a) Four days after stimulation, expressions of the indicated transcription factors were analysed. (b,c) Naive polyclonal CD4+ T cells from homozygous c-Maf-mutant mice (c-Maf Mut) or littermate control mice (WT) were stimulated in the presence or absence of IL-6. Five days after stimulation, effector cells were re-stimulated with phorbol-12-myristate-13-acetate/ionomycin. Representative plots of cytokine-producing cells are shown (b). Indicated cytokine mRNA expression at day 3 was also assessed by real-time quantitative PCR (c). Results are shown as mean±s.e.m. with n=4–6 per group; *P<0.05, **P<0.01, ***P<0.001, analysis of variance followed by Tukey's post hoc test. NS, not significant. (d,e) Naive OT-II cells were stimulated in the presence of indicated cytokines or Abs. Five days after stimulation, effector cells were re-stimulated and analysed for the ability to produce IFN-γ, IL-2 and/or IL-21. Representative plots are shown. The data are representative of at least three independent experiments. NS, not significant; PBS, phosphate-buffered saline.
Figure 9
Figure 9. IL-6-induced IL-4/IL-21/IL-10 production is responsible for the defective CD8 help in aged mice.
(a,b) OT-II cells were primed in young or aged mice at day 0 as in Fig. 1b, and then were treated with control Ab, anti-IL-4 and anti-IL-21 Abs (a) or anti-IL-10 Ab (b) on days 3 and 4. Six days after immunization, the frequencies of IFN-γ+ cells in the donor OT-II cells were determined. (b) Representative plots. (c) OT-II transfer, immunization, Ab treatment and MCA-OVA inoculation were performed as in Fig. 1d. Four days after tumour inoculation, IL-10 concentration in serum was determined. (d,e) OT-II transfer, immunization and treatments of anti-IL-4 and anti-IL-21 Abs were performed as in a. Five days after immunization, mice were inoculated with MCA-OVA. Anti-IL-10 Ab was injected twice at 2 and 3 days after tumour inoculation. Five days after tumour inoculation, draining LNs were analysed for OVA-specific CD8+ T cells using OVA-Ip tetramer (d). OVA-Ip-specific CD8+ T-cell response was also evaluated by the IFN-γ ELISPOT assay (e). Data shown are mean±s.e.m. with n=4–5 per group. (f) CD62Llo effector OT-II cells primed in young or aged IL-6+/+ or IL-6−/− mice were sorted as in Fig. 4a and were co-cultured with CFSE-labelled CD8+ T cells in the presence or absence of anti-IL-10 Ab. After 3 days, CFSE profile and IFN-γ production in CD8+ T cells were determined using a flow cytometer. Representative data from two independent experiments are shown. *P<0.05, **P<0.01, ***P<0.001, analysis of variance followed by Tukey's post hoc test.

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