ARTICLE
Received 16 Jun 2014 | Accepted 20 Feb 2015 | Published 7 Apr 2015
Decline in immune function and inammation concomitantly develop with ageing. Here we focus on the impact of this inammatory environment on T cells, and demonstrate that in contrast to successful tumour elimination in young mice, replenishment of tumour-specic CD4 Tcells 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 deciency. 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-g. 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 ndings suggest that IL-6 serves as an extrinsic factor counteracting CD4 T-cell-mediated immunity against tumour in old age.
DOI: 10.1038/ncomms7702 OPEN
IL-6-mediated environmental conditioning of defective Th1 differentiation dampens antitumour immune responses in old age
Hirotake Tsukamoto1, Satoru Senju1, Keiko Matsumura1, Susan L. Swain2 & Yasuharu Nishimura1
1 Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Chuo-ku, Kumamoto 860-8556, Japan.
2 Department of Pathology, University of Massachusetts Medical School, 335 Plantation Street, Worcester, Massachusetts 01655, USA. Correspondence and requests for materials should be addressed to H.T. (email: mailto:[email protected]
Web End [email protected] ) or to Y.N. (email: mailto:[email protected]
Web End [email protected] ).
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7702
The emerging usefulness of tumour-specic T-cell-mediated cancer immunotherapies is increasingly appreciated. For a long time, antitumour responses of CD8 T cells have
been a main focus in the therapeutic effects. Currently, accumulating evidences have indicated that active immunotherapy inducing tumour-specic CD4 T cells is also potentially powerful and broadly applicable for tumour rejection14. CD4
T cells participate in tumour elimination by helping to activate other immune components such as CD8 T cells, natural killer cells and macrophages1,5,6, exhibiting direct cytotoxicity against tumour cells3, and driving tumour cells into senescence4. An increase in interferon (IFN)-g-producing T helper (Th)1 cells has been recognized as an antitumour immune signature in cancer patients5,7, because favourable prognosis is closely correlated with high expression of Th1-related genes, Ifng and Tbx21 (T-bet)5. In contrast, Th2 rather than Th1 cells are predominantly increased in patients with advanced cancer7 and aged individuals8,9. Therefore, it has been assumed that strategies to promote the activation of tumour-specic Th1 cells would be useful for effective cancer immunotherapy.
Immune-based approaches are potentially less toxic than chemo- or radiotherapy. From this perspective, immunotherapy may be suitable for older cancer patients. However, immune responses become compromised during ageing. Age-related defects including both the relatively low number and the dysfunction of aged T cells, appear to not only increase cancer incidence in later life, but also to decrease the effectiveness of immunotherapy to mount T-cell responses against cancers, which leads to high morbidity and mortality in the elderly population10. Our and other studies have demonstrated that the functions of CD4 T cells are profoundly altered by the ageing process1113.
The lower efcacy of CD4 T-cell-mediated immune responses in old age can be attributable to several mechanisms including
T-cell-intrinsic1113 and -extrinsic effects14. However, the inuences of age-related changes in CD4 T-cell-mediated immune responses on the effectiveness of cancer immunotherapy are obscure because much of our understanding about antitumour immunotherapy is based on studies with young animals. To design effective immunotherapeutic interventions specically tailored to older cancer patients, it is important to know why T-cell functions are diminished in old age, and how to potentiate the aged immune system.
It has been assumed that the chronic low-grade inammation that accompanies ageing plays a role in the pathogenesis of several age-associated diseases including cancer10,1517. For instance, increased levels of the pro-inammatory cytokine interleukin (IL)-6 are correlated with frailty in these patients15,18. In addition, various studies have revealed that IL-6 is one of the adverse prognostic factors for cancer progression and has tumour-promoting effects19. However, little attention has been paid to an inuence of excessive levels of IL-6 on T-cell-mediated antitumour responses in old age.
In the present study, we asked whether CD4 T-cell dysfunction in aged hosts could be reversed by complementation with young tumour-specic CD4 T cells. However, young tumour-specic CD4 T cells primed in aged mice did not mount protective immune responses against tumour. Thus, we focused on an altered cytokine milieu in aged animals, and evaluated the inuence of IL-6, which found to be abundantly present in aged mice and humans, on the poor CD4 T-cell-mediated antitumour responses. Although IL-6 did not diminish or promote in vivo expansion of CD4 T cells in response to vaccination, the age-associated increase in IL-6 dampened
Th1 differentiation of CD4 T cells and subsequent induction of tumour-specic CD8 T cells, and thereby promoted cancer progression in aged mice. Our ndings also suggest that
IL-6-induced c-Maf/IL-4/IL-21/IL-10 axis is a mechanistic feature of the aged environmental conditioning of CD4 T cells.
ResultsCD4 T-cell-mediated therapy is less effective in aged mice.
We examined the effect of CD4 T-cell-mediated antitumour vaccination using MCA205 tumour cells expressing ovalbumin (OVA) as a surrogate antigen (hereafter referred to as MCAOVA). As previously reported in cancer patients10, tumour masses grew more slowly in aged mice than in young mice (Fig. 1a, left). In young mice, tumour outgrowth was prevented by vaccination with OVA peptide recognized by major histocompatibility complex (MHC) class-II-restricted CD4 T cells (referred to as
OVA-IIp). In contrast, vaccinated aged mice did not eliminate the tumours (Fig. 1a, right). This might be due to the reduced number of aged CD4 T cells and their intrinsic defect in ability to expand in response to antigenic stimulation1113.
Therefore we asked whether transfer of young OT-II cells and immunization with OVA-IIp-pulsed young dendritic cells (DCs) overcome these defects in aged mice. When MCA-OVA were inoculated, OT-II cell transfer was strikingly effective for tumour rejection in young hosts, but had little therapeutic effect in aged mice (Fig. 1b). These results suggest that the antitumour immune responses initiated by tumour-specic CD4 T cells are dampened in aged mice, and that the age-associated dysfunction in CD4 T cells in vivo is partly due to the extrinsic factors surrounding CD4 T cells in aged hosts.
IL-6 is suppressive for CD4-mediated antitumour immunity. As previously reported in human18, serum IL-6 levels were elevated with increasing age (Fig. 1c). As well as IL-6, the levels of soluble IL-6 receptor (sIL-6R), which forms a complex with IL-6 and promotes to transduce intracellular signal through glycoprotein 130 (ref. 20), were also systemically increased with ageing. To determine whether circulating IL-6 inuences the ability of CD4 T cells to control tumours, we employed IL-6 blockade combined with OT-II cell transfer. Following tumour challenge, the growth of MCA-OVA was slightly faster in young mice than in aged mice receiving control antibody (Ab) singly (Fig. 1d, left). Young hosts exhibited marked tumour eradication regardless of IL-6 blockade when donor CD4 T cells were primed with peptide-pulsed DCs, whereas as expected the antitumour effect of CD4 T cells was impaired in control Ab-treated aged mice. On the other hand, the combination of vaccination with anti-IL-6 Ab treatment signicantly improved the antitumour effect of OT-II cells in aged mice (Fig. 1d, right).
The negative impact of the aged environment on CD4 T-cell responses might be manifested more prominently in the case of malignant cells with vigorous progression and metastasis. Therefore, employing a metastatic model of aggressive melanoma, we re-evaluated the CD4 T-cell-mediated antitumour effects in aged mice. As was observed with the MCA-OVA, OT-II cells primed with peptide-pulsed DCs were ineffective for metastatic melanoma in aged IL-6 / mice compared with those in young
IL-6 / or IL-6 / mice (Fig. 2a). However, when immunized in aged IL-6 / mice, their efcacy was signicantly improved, suggesting that increased IL-6 levels in aged hosts dampened the function of tumour-specic CD4 T cells.
We also investigated the suppressive effect of IL-6 on more physiological immune responses of endogenous CD4 T cells in protecting aged mice against the outgrowth of murine leukaemia virus (MuLV)-induced lymphoma, RMA21. When mice were immunized with DCs pulsed with EnvH13. 3 peptide, which was a natural viral epitope derived from MuLV and was recognized by I-Ab-restricted CD4 T cells, the vaccination delayed tumour
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25 IFA/peptide
Young
20 Aged
20 IFA/OVA-IIp +
120
240
180
120
60
0 3 6 18 24 18 18 (mo)
Cont
NS
NS
Tumour index (mm)
IL-6 (pg ml1 )
slL-6R (pg ml1 )
* *
**
****
*
Young
Aged
15 10
5 0
*
80
40
0
0 10 20 30
Days after tumour inoculation
Days after tumour inoculation
Days after tumour inoculation
OT-II/DC/peptide OT-II/DC/OVA-IIp +
30
20
Tumour index (mm)
Tumour index (mm)
15
10
5
25
20
15
10
5
0
Young
Aged *
Young
Aged ***
Anti-Gr-1 Ab
10
0 0 20 30 40
10
10
20
30 40
0
25 20 15 10
5 0
0 0 10 20 30
16
DC DC/OVA-IIp +
12
* *
*
8
4
0
0 20 30 40
Young + control Ab Aged + control Ab
Aged + anti-IL-6 Ab
Young + anti-IL-6 Ab
10
20 30 40
0
10
Figure 1 | Tumour-specic 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 emulsied in IFA. Seven days after immunization, mice were inoculated with MCA-OVA.
(b) CD45.1 young naive OT-II Tcells 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 412 mice per
group). *Po0.05, ***Po0.001, unpaired Students 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 68 mice per group). *Po0.05, **Po0.01, ***Po0.001,
analysis of variance followed by Tukeys post hoc test. NS, not signicant.
outgrowth in young mice in comparison with that in nonvaccinated young mice21 or in vaccinated aged mice (Fig. 2b). Moreover, in aged mice treated with DC vaccination together with anti-IL-6 Ab, progression of RMA tumour was signicantly repressed as observed in the MCA-OVA model.
Even when endogenous CD4 T cells were depleted and OT-II cells was not transferred, immunization with DCs pulsed with
SIINFEKL (OVA-Ip), which is recognized by MHC class-I-restricted OVA-specic CD8 T cells, induced moderate antitumour responses in young mice (Fig. 2c). However, in aged mice, immunization with DCs that primed the endogenous OVA-specic CD8 T cells without OT-II transfer was less effective than that observed in young mice. Moreover, IL-6 blockade neither improved nor diminished the antitumour immunity in CD4 T-cell-depleted and CD8 T-cell-primed aged mice.
These antitumour effects were closely correlated with IFN-g responses of OVA-Ip (tumour)-specic CD8 T cells, which was not affected by anti-IL-6 Ab administration (Fig. 2d), although
aged CD8 T-cell responses were decreased as compared with young ones because of their quantitative decline in CD8 T cells13. These results ruled out the possibility that the benecial effect of IL-6 blockade was exerted directly through CD8
T-cell-mediated antitumour immunity. Therefore, heightened IL-6 appeared to act predominantly on tumour-specic CD4 T cells in aged mice.
The aged environment dampens CD4 T-cell proliferation. It was possible that in the aged environment, CD4 T cells failed to expand efciently in response to antigenic stimulation, resulting in poor antitumour immunity. To test this possibility, we examined the proportion of young donor OT-II cells in aged mice after immunization. Vigorous increases of donor OT-II cells were observed in peripheral blood within 7 days in both control and anti-IL-6 Ab-treated young hosts, and the donor cells contracted afterwards (Fig. 3a). In contrast, donor T-cell expansion was less
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ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7702
Young Aged
(cps)
108
107
106
IL-6+/+
DC IL-6+/+
DC/OVA-IIp
IL-6/
DC/OVA-IIp
20,000 18,050 16,100 14,150 12,200 10,250
8,300 6,350 4,400 2,450
500
Bioluminescence (photon counts)
**
Hosts Treatment
DC
DC/OVA-IIp
DC/OVA-IIp
Young
Aged
IL-6+/+
IL-6+/+
IL-6+/+
IL-6+/+
IL-6/
IL-6/
DC
DC/OVA-IIp
DC/OVA-IIp
105
0
10 50
20
30
40
Days after tumour inoculation
Treatment
Hosts
DC
DC/H13.3 + control Ab
DC/H13.3 + anti-IL-6 Ab
DC/H13.3 + anti-IL-6 Ab
Young
CD4 depleted
DC/OVA-Ip +
Treatment
DC/OVA-Ip +
Control Ab
Anti-IL-6R Ab
Control Ab
CD4 depleted
hosts Anti-IL-6R Ab
DC
DC/H13.3 + control Ab
Aged
Young
DC Control Ab
Anti-IL-6R Ab
Aged
**
Tumour index (mm)
30
20
10
0
Tumour index (mm)
18
12
6
0
IFN- ELISPOT / 10 5 dLN cells
250 200 150 100
50
*
*
0
0
10 20 30 40
Days after tumour inoculation
0 10 50
NS
20
30
40
Young Aged
Days after tumour inoculation
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-g production was determined by the ELISPOT assay (d). Representative from two or three experiments are shown as the means.e.m. (n 510 mice per group). *Po0.05, **Po0.01, analysis of variance followed by Tukeys post hoc test. NS, not signicant.
prominent in aged hosts than in young counterparts in the blood, and in spleen and lymph nodes (LNs) when vaccinated with DCs (Fig. 3a,b), or when the homeostatic division was induced in irradiated mice (Supplementary Fig. 1). Moreover, anti-IL-6 Ab treatment did not improve the inadequate expansion of donor OT-II cells in aged mice.
CD4Foxp3 regulatory T cells (Treg) could potentially act as an extrinsic factor that affects the T-cell responses in aged hosts, because they were abundantly present in aged humans22 and mice (Fig. 3c)23. However, control and anti-IL-6 Ab-treated aged mice had similar number of Treg. Another T-cell-suppressive component was Gr-1CD11b myeloid-derived suppressor cells (MDSCs), which were also expanded during ageing (Supplementary Fig. 2)23. Interestingly, IL-6 deciency did not alter the age-related increase in MDSC proportion (Fig. 3d). To test their suppressive effect on antigen-induced expansion of CD4 T cells, we depleted Gr-1 cells including MDSC using anti-Gr-1 Ab. Although the total number and carboxyuorescein diacetate succinimidyl ester (CFSE) prole of donor OT-II cells revealed their reduced proliferation in aged mice, no measurable changes in their expansion were observed between anti-Gr-1 Ab-
treated and control aged hosts (Fig. 3e,f). Furthermore, unlike anti-IL-6 Ab, anti-Gr-1 Ab treatment had no effects on CD4
T-cell-mediated antitumour immunity in aged mice (Fig. 3g). These results suggest that the suppressive effects of accumulated Treg, MDSC or other Gr-1 cells such as neutrophils were independent of the improved efcacy of CD4 T-cell-mediated antitumour immunity in IL-6-neutralized aged mice.
IL-6 hampers Th1 development in an aged environment. It was hypothesized that IL-6 upregulated in aged mice altered the cytokine production by tumour-specic CD4 T cells to prevent them from attacking the tumours. Indeed, when CD62Llo effector CD4 T cells including OT-II cells primed in vivo with or without IL-6 blockade were re-stimulated with OVA-IIp ex vivo, we found that the levels of IFN-g, IL-4, IL-5,
IL-13 and IL-10, but not IL-2, IL-17A, tumour necrosis factor (TNF)-a or IL-9 produced from effector CD4 T cells, were remarkably affected by the aged environment and IL-6 blockade (Fig. 4a). To test whether these changes were due to altered differentiation of effector CD4 T cells in vivo, we assessed the
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Donor OT-II
**
Foxp3+ Treg
Young + control Ab Young + anti-IL-6 Ab Aged + control Ab Aged + anti-IL-6 Ab
% Of donor OT-II cells in PBL
100
101
102
103
30
20
10
* **
**
Absolute no. ( 105 )
25
20
15
10
5
0
**
0 Young
Aged Young Aged
0 10 20 30 40
Absolute no. ( 104 )
Days after immunization
DC Control Ab
+ DC/OVA-IIp
Gr-1+CD11b+ MDSC
**
Donor OT-II
*
Absolute no. ( 104 )
20
15
10
Control Ab Anti-Gr-1 Ab
Absolute no. ( 104 )
6
4
2
0
5
IL-6+/+ IL-6/
0
Young Aged
Young Aged
20
15
10
5
Day 4 Control Ab Control Ab
Anti-IL-6 Ab
Anti-Gr-1 Ab
% Of max
% Of max
100 80
40 20
0
100
Tumour index (mm)
60
80
40 20
0
10 10
10 10 10
10 10 10
FL1-H: CFSF
FL1-H: CFSF
10 10
Anti-Gr-1 Ab
60
Aged DC
Aged/DC/OVA-IIp
Young/DC/OVA-IIp
10
0 0 20
30
Days after tumour inoculation
CFSE
Figure 3 | The aged environment dampens antigen-induced proliferation of CD4 Tcells. (ac) OT-II cell transfer, immunization and Ab treatment were performed as described in Fig. 1d. The frequency of donor CD45.1OT-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 prole 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 means.e.m. (n 6 per group); *Po0.05, **Po0.01, analysis of variance followed by Tukeys post hoc test. Representative data from
two or three experiments with similar results are presented.
ability of donor OT-II cells to produce cytokines on a per-cell basis. The frequency of IFN-g-producing OT-II cells from aged hosts was signicantly lower than that from young hosts (Fig. 4b,c). Of note, this defective Th1 differentiation of donor OT-II cells was signicantly restored by IL-6 blockade in aged hosts. We found no difference between control and anti-IL-6 Ab-treated aged mice in their ability to produce IL-2, TNF-a and
IL-17A (Fig. 4b,c). Conversely, the proportions of IL-21- and IL-10-producing cells were increased in aged mice, and these were diminished by IL-6 blockade (Fig. 4c; Supplementary Fig. 3a). Similar trends were also observed in messenger RNA (mRNA) expression of these cytokines in primed CD4 T cells (Supplementary Fig. 3b). Furthermore, evaluation of cytokine production from donor OT-II cells primed in IL-6-decient aged
mice conrmed that the IL-6-dependent impairment of Th1 differentiation in aged hosts (Fig. 4d). In line with no signicant effect of anti-Gr-1 Ab on IL-6 production (Fig. 2a) and antitumour immune response in aged mice (Fig. 3g), depletion of Gr-1 cells including MDSC did not change the cytokine proles in donor OT-II cells primed in aged mice (Supplementary
Fig. 2b). These results suggest that the increased levels of IL-6 serve as a major mechanism underlying the impaired Th1 differentiation of donor CD4 T cells in aged mice. The impaired
Th1 response in donor OT-II cells in aged mice was consistent with their reduced expression of IFN-g-inducible chemokine receptor, CXCR3 (ref. 24), which was upregulated by IL-6 blockade (Supplementary Fig. 2c,d). Although CD25 expression was also decreased in donor cells primed in aged mice as
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IL-2
IL-9
IL-17A TNF- IFN-
300
200
100
**
(ng ml1)
(ng ml1)
3
2
1
0
30
20
10
4
3
2
1
0
**
DC/OVA-IIp +
Control Ab Anti-IL-6 Ab
0
0
IL-10
IL-4 IL-5 IL-13
10
8
6
4
2
0
**
50 ** ** ****
0.8
0.6
0.4
0.2
0
50
40
30
20
10
0
200
150
100
50
0
**
Young Aged Young Aged Young Aged Young Aged Young Aged
IFN-
IL-17A
NS
+ Control Ab + Anti-IL-6 Ab Young
Aged
104 2.34% 24.7% 1.68% 23.1% ** **
40
30
20
10
0
104
**
% Positive cells % Positive cells
103
103
102
102
2.0
1.5
1.0
0.5
0
101
10 101
101
10 101
22.2%
50.8% 19.7% 55.5%
102
103
104
102
103
104
104
IFN-
2.42% 13.3% 6.01%
41.4%
20.7%
103
103
IL-21
TNF-
40
30
20
10
0 0
90
60
30
102
102
* **
NS
101
10 101
101
10 101
40.4% 43.8% 31.9%
102
103
104
0
100
102
103
104
IL-2
IFN- IL-2
IL-6+/+
IL-6/
**
IL-10
Young Aged
30
20
10
0
100
80
60
40
20
0
*
NS
8 *** **
Control Ab
Anti-IL-6 Ab
% Positive cells
% Positive cells
Young Aged Young Aged
Young Aged
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, CD4CD62Llo 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 means.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 34 independent experiments with similar results are shown. *Po0.05, **Po0.01, ***Po0.001, analysis of variance followed by Tukeys post hoc test.
NS, not signicant.
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7702 ARTICLE
compared with those in young mice, IL-6 blockade did not alter its expression as with other surface markers. Increased expression of the markers for exhausted T cells such as PD-1 were not found in donor OT-II cells in aged mice (Supplementary Fig. 2d).
We also examined the effect of age-related increase of IL-6 on gene expression of key transcription factors that regulate T-cell differentiation. In total CD4 T cells isolated from OT-II-transferred and vaccinated mice, the expression levels of Tbx21, which encodes T-bet and regulates Th1 differentiation25, tended to be decreased in aged mice as compared with in young mice, but the difference was not signicant. Moreover, Gata3 expression that regulates Th2 differentiation was also lower in CD4 T cells from vaccinated aged mice than that in young mice. In contrast, Foxp3 and Th17-specic transcription factor
Rorc were upregulated, but those expressions were not altered by IL-6 blockade in either young or aged mice (Fig. 5a). As was observed with Rorc expression, c-maf expression in CD4 T cells
was markedly augmented in aged hosts, but was repressed by IL-6 blockade. To analyse the expression of transcription factors in antigen-specic CD4 T cells more precisely, their protein expressions in donor OT-II cells were examined at the single-cell level. As expected, similar patterns were observed in protein expression of donor OT-II cells primed in young or aged mice (Fig. 5b). Of note, only c-Maf expression was altered by both host age and IL-6 level, and this was conrmed in donor OT-II cells primed in IL-6-replete and -decient aged mice (Fig. 5c).
Defective IFN-c response in IL-6-mediated CD4 T-cell dysfunction. We found that the serum level of IFN-g was augmented in OT-II-transferred and vaccinated young mice in response to tumour inoculation (Fig. 6a). Furthermore, the relatively lower levels of IFN-g in vaccinated aged mice were improved by the treatment with anti-IL-6 Ab, which was
DC/OVA-IIp +
Control Ab
Anti-IL-6Ab
Tbx21 Gata3 Rorc c-maf Bcl6 Foxp3
** 0.0200.0150.0100.005 0
0.5
8 *
* * ***
*
Relative expression
(/Gapdhs)
0.4
0.3
0.2
0.1
0
0.05
* 0.04
0.03
0.02
0.01
0
10
8
6
4
*
6 4 2
2
0
0
Y A Y A Y A
4 3 2 1 0
Y A Y A Y A
T-bet GATA-3
NS
RORt c-Maf
** ** ** **
* * * *
% Positive
80 60 40 20
0
NS
in donor OT-II cells
60
40
20
0
25
20
15
10
5
0
60
40
20
0
NS
Young
Aged Young Aged Young Aged Young Aged
Donor OT-II gated
Young
Aged
IL-6+/+
IL-6/
100
80
60
% Of max
40
0 10 10 10 10 10
10 10 10FL2-H:: T-bet-PE FL2-H:: GATA3-PE FL4-H:: ROR-APC FL4-H:: c-Maf-APC
10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10
20
100
80
60
% Of max
40
0
20
0 10 10 10 10 10
0
10 10 10 10 10
0 10 10 10 10 10
0
0 0
T-bet GATA-3 RORt
c-Maf
Figure 5 | IL-6 and the aged environment alter the expression prole of transcription factors in CD4 Tcells. 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 (means.e.m. with n 46 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. *Po0.05, **Po0.01, ***Po0.001, analysis of variance followed by Tukeys post hoc test. NS, not signicant.
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Control Ab
IFN-
Anti-IL-6 Ab
(ng ml1 )
50
40
30
20
10
0
* *
NS
WT KO WT KO
Donor OT-II IFN- :
Immunization :
(DC/OVA-IIp)
+ + + + + +
Young + WT + Control Ab
Young + IFN- KO + Control Ab Aged + IFN- KO + Control Ab
Aged + WT + Control Ab
Aged + WT + Anti-IL6 Ab
Aged + IFN- KO + Anti-IL6 Ab
Young Aged
Host Donor OT-II Day 1
109
Bioluminescence (photon counts)
108
*
107
106
105
10
20 30 40
Days after tumour inoculation
Figure 6 | Tumour-specic CD4 T-cell-derived IFN-c 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-g-decient (IFN-g 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-g 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 photoncounts in luminescence images. Data shown are representatives fromtwo experiments with similar results (means.e.m. with n 46 per group;
*Po0.05, analysis of variance followed by Tukeys post hoc test). NS, not signicant.
consistent with more Th1 differentiation in donor OT-II cells. However, aged mice that were transferred with IFN-g-decient
OT-II cells did not evoke IFN-g induction even when anti-IL-6 Ab was administered.
Given that production of IFN-g, one of the most critical cytokines for antitumour immunity3,5,7, was defective in tumourspecic CD4 T cells in aged mice, we next examined whether
IL-6-induced impairment of Th1 differentiation indeed contributed to the age-associated dysfunction in antitumour responses of CD4 T cells. As shown in Fig. 6b, vigorous progression of pulmonary metastatic tumour was prevented in young mice when either IFN-g-sufcient or -decient CD4 T
cells were transferred, whereas vaccinated aged mice failed to suppress the tumour outgrowth. However, IL-6 blockade signicantly improved the therapeutic efcacy of wild-type (WT) donor OT-II cells in aged mice, as demonstrated before. In contrast, this improvement was not observed when aged mice received combined treatment with anti-IL-6 Ab and IFN-gdecient T cells. These results suggest that IL-6 blockade improves the antitumour effect of CD4 T cells through augmented IFN-g production in aged mice.
Decreased CD8 help by CD4 T cells in an aged environment. MCA-OVA neither express MHC-II nor present OVA peptide to
CD4 T cells26. The antitumour effects of CD4 T cells might be exerted through other host-derived components such as tumour-specic CD8 T cells. Therefore, we investigated whether aged hosts depleted of CD8 T cells could mount antitumour immunity when CD4 T-cell therapy was combined with IL-6 blockade. As demonstrated before, even in aged mice tumour outgrowth was substantially diminished by donor OT-II cells primed concomitantly with IL-6 blockade when endogenous CD8 T cells were retained (Fig. 7a). However, the depletion of
CD8 T cells abrogated this antitumour effect, suggesting that therapeutic effect of tumour-specic CD4 T cells in IL-6-neutralized aged mice was mediated through CD8 T cells.
Neutralization of IL-6 activity inuenced only at priming of donor CD4 T cells with DCs, but did not affect CD8 T-cell activation, because the DCs used in this vaccination were only pulsed with OVA-IIp, and tumour-specic CD8 T cells should be exposed to antigen only after the challenge with MCA-OVA (Fig. 7b). We thus analysed the ability of effector CD4 T cells primed in aged hosts to promote tumour-specic CD8 T-cell responses after tumour challenge. Six days after tumour challenge, young mice in which donor OT-II cells had been primed, induced OVA-specic CD8 T cells in tumour-draining
LNs and in tumour more efciently than mice possessing naive OT-II cells without DC vaccination, regardless of anti-IL-6 Ab treatment (Fig. 7ce; Supplementary Fig. 4). In contrast, fewer OVA-specic CD8 T cells were promoted by donor OT-II cells primed in aged mice. Notably, their helper activity for OVA-specic CD8 T cells was markedly improved in aged mice where OT-II cells were primed in the presence of anti-IL-6 Ab.
In the case of RMA tumour in aged mice, IFN-g response of tumour (MuLV EnvH13.3)-specic CD4 T cells was restored by
IL-6 blockade (Fig. 7f). On the other hand, EnvH13.3-specic IL-4 and IL-10 production were reciprocally downregulated by IL-6 blockade in aged mice (Supplementary Fig. 5). Furthermore, vaccination that primed the tumour-specic CD4 T cells enhanced the responses of tumour (Env/Kb and GagL/Db)21-specic CD8 T cells in aged mice only when anti-IL-6 Ab was administered at the CD4 T-cell-priming phase (Fig. 7f).
We also examined the requirement of donor CD4 T-cell-derived IFN-g for promoting the response of tumourspecic CD8 T cells. In agreement with the results of tumour growth (Fig. 6), no remarkable induction of OVA-specic CD8
T cells and their functional enhancement that was monitored by IFN-g response were observed in aged mice transferred with
IFN-g-decient OT-II cells, regardless of IL-6 blockade (Fig. 7g,h). These results suggest that CD4 T-cell-derived
IFN-g contribute to promoting the tumour-specic CD8 T-cell response in anti-IL-6 Ab-treated aged mice.
IL-6 dampens Th1 development through a c-Maf/IL-4/IL-21 axis. To further clarify the inhibitory effect of IL-6 on Th1 differentiation, young CD4 T cells were stimulated with anti-CD3/
CD28 Abs in the presence or absence of IL-6 in vitro. In the
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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7702 ARTICLE
Hosts Day 7
OVAtet+CD8+CD44hi
Day 2
Young + anti-IL-6 Ab + anti-CD8 Ab
Aged + anti-IL-6 Ab + anti-CD8 Ab
Aged + control Ab + control Ab
Aged + anti-IL-6 Ab + control Ab
Young + anti-IL-6 Ab + control Ab
Control Ab
Anti-IL-6 Ab
10
No immunization Young
Young Young
Aged Aged
0.138%
***
**
10
OVA
tetramer
2.5
1.5
1.0
0.5
10
2.0
**
30
20
10
10
10
10 10 10 10 10 10
Absolute no.
in dLNs ( 104 )
1.51.00.5 0
10
10
10
10
10
Tumour index (mm)
DC/OVA-IIp immunization
CD8
+ Control Ab + Anti-IL-6 Ab
10 10 10 10 10
0.430% 0.425%
0.367%
**
Immunization :
(DC/OVA-IIp)
+ + + +
Aged
Young Aged
**
**
10
10 10
10
10
10
10
10
10
10
*
0 0 10 20 30 40 50
Days after tumour inoculation
Anti-IL-6 Ab tumour inoculation OT-II
Day 1 0 1 5 11
OVAtet+CD8+CD44hi
10 10 10 10 10
% Positive in TIL
0.233%
10
10
10
OVA-IIp/DC CD8 in dLNs
10
10
10
10
10
10
10
10
10
10
0 + + + +
10 Immunization :
(DC/OVA-IIp)
Young
250 200 150 100
50
0
250 200 150 100
50
0
250 ***
*
***
*
H13.3/Ab
*** ***
*** **
* *
Control Ab
Anti-IL-6 Ab
Absolute no.
in dLNs ( 104 )
5 4 3 2 1 0
** *
***
Env/Kb
OT-II IFN- :
Immunization :
(DC/OVA-IIp)
Specific IFN-ELISPOT/105dLN cells
**
+ + + + + +
Aged
Young
500 400 300 200 100
0
WT KO
WT
KO
Control Ab
Anti-IL-6 Ab
GagL/Db
IFN-ELISPOT
/105dLN cells
200 150 100
50
Anti-IL-6 Ab
OT-II IFN- :
Immunization :
(DC/OVA-IIp)
WT KO
WT
KO
Young
+ + + + + +
Aged
Immunization :
(DC/H13.3)
0 + + + +
Aged
Control Ab
Young
Figure 7 | Th1-dependent helper activity for tumour-specic 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 means.e.m. (n 810 mice per group). (be) MCA-OVA were inoculated 5 days after OT-II transfer and
immunization. Another 6 days later, OVA-specic CD8 T cells in tumour-draining LNs (c and d) and tumour-inltrated lymphocytes (TILs) (e) were analysed. Representative plots (c) and the total number (d) of OVA-Ip/H-2Kb-tetramer CD8CD44hi cells are shown. (f,g) WT or IFN-g-decient (knockout (KO)) OT-II cells were transferred into young and aged mice. OVA-specic 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-specic 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-specic IFN-g production in tumour-draining LNs were assessed by ELISPOT.
Representative data from 23 experiments are shown (means.e.m. with n 45 per group). *Po0.05, **Po0.01, ***Po0.001 analysis of variance
followed by Tukeys post hoc test.
presence of IL-12, stimulated T cells upregulated T-bet expression (Fig. 8a). Simultaneous IL-6 stimulation did not change the levels of either T-bet or GATA-3, but upregulated c-Maf expression even in the Th1-skewed condition. To directly assess the requirement of c-Maf for IL-6-mediated Th1 suppression, we utilized T cells with a homozygous mutant in c-Maf that lacks
DNA-binding activity to its target genes27. c-Maf activity is dispensable for the development and peripheral homeostasis of T cells (Supplementary Fig. 6a,b). Proliferation of c-Maf-mutant naive CD4 T cells in response to antigenic stimulation was also comparable to that of WT cells (Supplementary Fig. 6c). As shown in Fig. 8b, stimulation of WT cells with IL-6, which
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OT-II stimulated with anti-CD3/28 Abs + IL-12 (day 4)
Naive
Control
Control
+ IL-12
+ IL-6
+ IL-6
WT c-Maf Mut
2.57% 12.4% 19.9%
14.0%
104
100
8.30%
26.5%
104
100
12.5% 50.1%
26.1%
IL-6
104
100
104
100
23.1%
16.5%
22.5%
21.2%
16.0%
24.4%
103
103
103
103
+ IL-6
102
102
c-Maf
102
102
101
101
101
101
42.2%
11.4%
15.2%
103 104
0 100 101 102 10
100 101 102
103 104
100 101 102
103 104
100 101 102
103 104
100 101 102
3 104
IFN-
104
100
104
100
17.0%
T-bet
GATA-3
103
103
Il4
Il21
Il10
Il17a
+
102
102
8 *** *** ***
***
***
3
0
*
IL-6
101
101
***
6
4
2
0
8
6
4
2
0
56.8%
103 104
100 101 102 103 104
100 101 102
2
**
Fold induction
IL-21
*
*
1
+ IL-12
NS
IL-6 Control Ab Anti-IL-4 Ab
104
100
27.0%
28.3%
32.5%
104
100
28.6% 32.8%
13.5%
c-Maf: WT WT
Mut Mut
WT Mut WT Mut
103
103
102
103 104
100 101 102 103 104
100 101 102
102
+ IL-12
IL-21
101
101
IL-21 PBS Anti-IL-21 Ab
12.2%
25.1%
104
100
104
100
104
100
IL-2
IL-6
12.1%
36.9%
16.4%
21.0%
41.2%
12.8%
24.3% 10.4%
11.4%
103
103
103
104
100
104
100
38.0%
18.9%
5.73%
40.7%
30.9%
102
102
102
103
103
101
101
101
+
102
103 104
100 101 102 103 104
100 101 102
102
34.6%
24.9%
53.8%
101
101
100 101 102
103 104
100 101 102
103 104
100 101 102
103 104
IFN-
37.3% 19.3% 9.12%
104
100
104
100
104
100
12.5% 9.62%
37.0%
5.89%
53.0%
7.25%
33.8%
12.4% 9.10%
103
103
103
IFN-
+
102
102
102
101
101
101
40.9%
54.3% 24.2%
103 104
100 101 102 103 104
100 101 102 103 104
100 101 102
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 means.e.m. with n 46 per group; *Po0.05, **Po0.01, ***Po0.001, analysis of variance followed by Tukeys post hoc test. NS, not
signicant. (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-g, IL-2 and/or IL-21. Representative plots are shown. The data are representative of at least three independent experiments. NS, not signicant; PBS, phosphate-buffered saline.
mimicked the aged environment, attenuated the development of IFN-g-producing cells in vitro. Under these conditions, there was only subtle inhibition of Th1 differentiation by IL-6 in c-Maf-mutant T cells, suggesting the importance of c-Maf in IL-6-mediated Th1 suppression.c-Maf is a critical transcription factor to induce IL-4 and IL-21 production25,28. Indeed, IL-6-stimulated CD4 T cells from both young C57BL/6 and Balb/c mice produced signicantly higher amounts of IL-4 immediately after primary T cell receptor (TCR) stimulation, and this was diminished in c-Maf-mutant T
cells (Supplementary Fig. 7a,b; Fig. 8c). IL-6 stimulation also induced IL-21 and IL-10 production in a c-Maf-dependent manner (Fig. 8b,c; Supplementary Fig. 7). The c-Maf-dependent-altered prole of cytokine expression raised the possibility that these cytokines contributed to the IL-6-mediated suppression of Th1 differentiation. To test this possibility, Th1 differentiation was assessed in IL-6-stimulated T cells when these cytokine activities were neutralized. Neutralization of IL-4 activity signicantly improved Th1 differentiation (Fig. 8d; Supplementary Fig. 7d). Blocking of IL-21 activity
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also mitigated IL-6-mediated Th1 suppression to a lesser extent, whereas exogenous IL-21 attenuated Th1 differentiation in the absence of IL-6 stimulation (Fig. 8e). Of note, these in vitro results were conrmed by the fact that neutralization of both IL-4 and IL-21 signicantly restored the Th1
differentiation in aged mice (Fig. 9a), while IL-10 blockade did not improve the attenuated Th1 differentiation (Fig. 9b). Collectively, these results suggest that IL-6-induced Th1 suppression was mediated through a c-Maf/IL-4/IL-21 axis in the aged environment.
Control Ab Control Ab
Anti-IL-21/4 Ab
Anti-IL-6 Ab
80 60 40
*
40
IFN- IL-10 OVAtet+CD8+ in dLNs
*** ***
*
**
Anti-IL-4 Ab
**
% Of positive cells
IL-10 (pg ml1 )
Immunization
(DC/OVA-IIp)
Absolute no. (x 104 )
8 6 4 2 0
*
30 20 10
20
0
0 + + + +
Young Young Aged
Aged
Young Aged
+ Control Ab + Anti-IL-10 Ab
Young
Aged
Control Ab
Anti-IL-4 Ab
Anti-IL-21/4 Ab
Anti-IL-10 Ab
** *
104
100
FL1-H::IFNg-FITC FL1-H::IFNg-FITC
104 48.4% 1.53% 43.2% 1.72%
49.1% 0.946% 54.1% 0.940%
25.7% 1.31% 22.2% 1.18%
71.0% 1.97% 74.0% 2.59%
103
103
102
103 104
100 101 102 103 104
100 101 102
102
101
101
IFN-
IFN-ELISPOT
/ 105 dLN cells
500
400
300
200
100
*
100
104
100
104
100
103
103
102
103 104
100 101 102 103 104
100 101 102
102
0
101
101
Young
Aged
FL2-H::IL-17A-PE FL2-H::IL-17A-PE
IL-17A
+ CD4+ T cells primed in
In vitro
treatment
Young IL-6+/+
26.1%
27.5%
Young IL-6/ Aged IL-6+/+ Aged IL-6/
104
100
104
100
FL2-H::IFNg-PE
26.7%
29.5%
104
100
15.4%
23.2%
104
100
22.9%
24.5%
Control Ab
103
103
103
102
14.9%
102
13.9%
102
26.4%
102
14.2%
101
101
101
101
100 101 102
103 104
100 101 102
103 104
100 101 102
103 104
100 101 102
103 104
IFN-
104
100
104
100
104
100
104
100
Anti-IL-10 Ab
FL2-H::IFNg-PE
103
103
103
103
103
102
13.6%
102
11.1%
102
15.7%
102
13.1%
101
101
101
101
100 101 102
103 104
100 101 102
103 104
100 101 102
103 104
100 101 102
103 104
FL1-H::CFSE
FL1-H::CFSE FL1-H::CFSE FL1-H::CFSE
CFSE (gated on CD8)
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-g 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-specic CD8 T cells using OVA-Ip tetramer (d). OVA-Ip-specic CD8 T-cell response was also evaluated by the IFN-g ELISPOT assay (e). Data shown are means.e.m. with n 45 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 prole and IFN-g production in CD8 T cells were determined using a ow cytometer. Representative data from two independent experiments are shown. *Po0.05, **Po0.01, ***Po0.001, analysis of variance followed by Tukeys post hoc test.
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IL-6-altered cytokine milieu modulates CD8 T-cell responses. Consistent with IL-6-dependent IL-10 production in OT-II cells (Fig. 4c), increased level of IL-10 in the serum was observed in aged mice after tumour inoculation (Fig. 9c), while this increase was not detected in aged mice with IL-6 blockade. Thus, to explore the effect of IL-10, in addition to IL-4 and IL-21, on induction of antitumour CD8 T-cell responses through CD4 helper activity, the number and function of CD8 T cells were assessed in aged mice treated with blocking Abs for these cytokines. In line with the results of Th1 differentiation (Fig. 9a), combined treatment of anti-IL-4 and anti-IL-21 Abs signicantly reversed the impaired induction and function of tumour-specic CD8 T cells in response to tumour inoculation in aged mice.
IL-10 blockade also improved the CD8 T-cell responses in aged mice although Th1 differentiation was not affected by IL-10 blockade, suggesting that in contrast to IL-4/IL-21, IL-6-mediated IL-10 production by CD4 T cells primed in the aged environment modulated CD8 T-cell response independently of Th1 differentiation.
To conrm the direct inhibitory role of IL-6-sensitized CD4 T cells, we performed the co-culture of CD8 T cells with effector
OT-II cells primed in young or aged IL-6 / or IL-6 / mice (Fig. 9f). Co-culture with CD4 T cells primed in aged IL-6 / mice markedly suppressed the expansion and IFN-g production of CD8 T cells, but these responses were substantially induced when co-cultured with effector CD4 T cells isolated from aged
IL-6 / mice, or with CD4 T cells from aged IL-6 / mice in the presence of anti-IL-10 Ab in vitro. These ndings suggest that excessive production of IL-4, IL-21 and IL-10 induced by IL-6 in aged mice have negative impacts on antitumour responses of CD8 T cells through (1) attenuating Th1 differentiation and their helper activity and (2) a direct inhibitory effect on the expansion and function of CD8 T cells.
DiscussionConsidering the broad effects of ageing on CD4 T cells, one of principal causes for the failure to mount the antitumour immune responses is the quantitative declines in number and TCR repertories of peripheral T cells due to thymic involution in old age12,13,29. A second risk factor is thought to be impairment of antigen-induced T-cell expansion, which is regulated by both intrinsic mechanisms in aged CD4 T cells11,12 and extrinsic factor(s) in the aged environment (Fig. 3)14. The inherent proliferative defect in aged CD4 T cells is largely due to reduced
IL-2 production11, whereas the molecular mechanism(s) by which the aged environment depresses young CD4 T-cell expansion have not yet been dened; therefore further analyses will be required.
Given that the balance between Th1 and Th2 cytokines is important for antitumour responses5,7, particular attention has been devoted to evaluating the age-related decline in IFN-g production. Although a shift from Th1 towards Th2 responses in elderly subjects has been suggested8,9, some reports are contradictory30. This discrepancy may be due to the experimental materials, in which the effector CD4 T cells primed at old age were not distinguished from the memory T cells that had been committed at young age. To rule out such possibilities, we established a model system that enabled us to assess the differentiation from naive to effector CD4 T cells in the aged environment. Using this system, we found that the excessive increase in IL-6 induced the dysregulation of Th1 differentiation, which was suggested to a third cause of impaired antitumour immunity in aged animals.
Young mice rejected the OVA-expressing tumour even when IFN-g-decient OT-II was transferred, because IFN-g deciency
in the transferred OT-II cells could be counterbalanced by abundant IFN-g-producing endogenous WT CD4 T cells in young mice, which might be sufcient to mount antitumour immunity. In this regard, in aged mice, the effect of IFN-g deciency in the transferred OT-II cells appeared to be underscored by signicantly decreased proportion of endogenous CD4 T cells13,29. Taken together, not only complementation with tumour-specic young CD4 T cells, but IL-6 blockade that augmented IFN-g production was also necessary for improvement of antitumour responses in aged mice.
IL-6-mediated Th1 suppression may be involved in a recent nding that the level of IL-6 is correlated with clinical outcome in melanoma patients receiving immunotherapy31. Therefore, the effect of IL-6 on endogenous or transferred T cells should be taken into account in aged individuals therapeutically infused with tumour-targeting T cells32,33. Age-related chronic inammation accompanied with cancer, smoking, subclinical disorder (atherosclerosis, sarcopenia and cognitive decline) and obesity seem to contribute to systemic increase of IL-6/sIL-6R in an elderly population15,17. Overall, we suggest that a rational design of cancer immunotherapy mediated by CD4 T cells in the elderly can be administered concurrently with temporal neutralization of IL-6 activity to restore the defective Th1 development without adversely affecting their antitumour activities. Theoretically, depletion of cells producing IL-6 or sIL-6R might represent viable options to ameliorate their activities. However, under inammatory conditions, diverse types of cells such as aged adipose tissue34, MDSC26,35, macrophages36, nonhematopoietic endothelial stromal cells and/ or cancer-associated broblasts37 can serve as the sources of IL-6 in tumour-bearing mice. For this reason, it has to be considered that the production of IL-6 and sIL-6R are not conned to one cell type and thus it is difcult to target their cellular sources that contribute to systemic control of Th1 suppression.
At present, IL-6 blockade using humanized anti-IL-6 Ab19, anti-IL-6R Ab tocilizumab or curcumin, a potent inhibitor of IL-6 production35,is a promising clinical application for improvement of the currently inefcient antitumour vaccination in aged individuals, and for controlling the age-dependent proinammatory status, called inammaging, responsible for tissue damage or systemic toxicities in aged animals16,17.
The detailed molecular mechanism(s) underlying the defect in Th1 development are still unclear, and several scenarios are worth considering. (1) It was intriguing that IL-4/IL-21 neutralization substantially relieved the suppressive effect of IL-6 on Th1 differentiation. This was supported by the nding that IL-6-mediated impairment of Th1 development was substantially restored by a loss of function in c-Maf. Diminished IL-4/IL-21 production in c-Maf-mutant T cells likely allowed them to differentiate into Th1 cells, since IL-4 inhibits IFN-g production and stimulates the production of other Th2 cytokines25. Furthermore, in addition to previous in vitro study38, we have rst demonstrated the IL-6-mediated regulation of c-Maf expression under in vivo situation of aged mice (Fig. 5). On the basis of these ndings, immunotherapy for aged individuals aimed at cancer regression may be accomplished by overcoming or redirecting Th2-prone responses. However, IL-4/IL-21 blockade did not completely rescue the impaired Th1 differentiation in aged mice, suggesting an additional requirement for optimal Th1 development.(2) In addition to the IL-4/IL-21-mediated Th1 suppression, direct engagement of c-Maf in repressing ifng transcription is also likely. This possibility is supported by a previous report that forced expression of c-Maf in Th1 cells inhibited IFN-g production via an
IL-4-independent mechanism39. Furthermore, in addition to increased c-Maf expression, expression of T-bet and GATA3
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were downregulated in donor OT-II cells primed in aged mice, implying that mutually balanced expression of several transcription factors within T cells contributes to attenuated Th1 differentiation in the aged environment. (3) Moreover, as an alternative mechanism of IL-6-mediated Th1 suppression, impaired Signal Transducer and Activator of Transcription 1-dependent IFN-g receptor signalling via IL-6-induced expression of Suppressor of Cytokine Signalling protein has been proposed40. These possible mechanisms should be investigated in greater detail.
Our nding provides the notion that less susceptibility to the antitumour vaccination in aged individuals should be carefully considered in terms of the complex effects of ageing on other components that interact with CD4 T cells. In addition to the quantitative declines in aged CD8 T cells, their qualitative defects have been also reported41. However, there are other conicting data suggesting that intrinsic function of CD8
T cells is preserved even in later life42,43. These seeming discrepancies can be explained in part by the defective ability of CD4 T cells to help cognate CD8 T cells in old age. Indeed, we found that CD4 T cells primed in aged mice were ineffective in supporting cognate CD8 T-cell responses, resulting in poor control of tumour progression in aged mice. Moreover, their helper activity for CD8 T cells was mediated through IFN-g, which was consistent with previous reports showing that CD4
T-cell-derived IFN-g enhanced the function and recruitment of CD8 T cells into draining LNs following viral or bacterial infection44,45. Importantly, when Th1 differentiation was restored by the blockade of IL-6 or subsequent IL-4/IL-21 activities, induction of tumour-specic aged CD8 T cells was enhanced as well, implying minimum intrinsic functional defects in aged
CD8 T cells.
It is noteworthy that IL-21 is known to have the ability to potentiate the expansion and function of CD8 T cells in a synergistic context with IL-15 in young mice46, which is apparently opposed to our ndings. One likely cause for this contradiction may be the reduced availability of IL-15 presentation in the aged environment, because the stimulatory effect of IL-21 is not exerted on CD8 T cells in the absence of
IL-15 (ref. 46). Indeed, previous studies reported that the levels of IL-15 and IL-15Ra decline with ageing47,48. These reports appear to be consistent with our hypothesis that insufcient cooperative action of IL-15 is responsible for the decreased stimulatory function of IL-21. However, it remains a matter of debate as to whether ageing alters levels of IL-15, because in contrast to these reports, a recent study demonstrated that IL-15 expression in splenic stromal cells was increased with ageing49.
Interestingly, our in vitro experiments demonstrated that IL-4/IL-21 blockade downregulated IL-10 production from IL-6-sensitized effector CD4 T cells (Supplementary Fig. 7c), suggesting that IL-21 stimulated IL-10 production in CD4
T cells, which in turn, constrained the optimal response of CD8 T cells in an IL-10-dependent manner. Taken together, in addition to the negative impact on Th1 differentiation, this cytokine circuit could be another mechanistic feature of IL-6 in converting the function of IL-21 from immunostimulatory to tolerogenic for tumour outgrowth in aged animals. On the basis of these ideas, strategies that bypass the requirement of CD4
T-cell-mediated help and compensate for apparently helpless CD8 T cells, such as treatment with agonistic anti-CD40L Ab (plus TNF-a blockade)16, or anti-PD-1 Ab50 may be the potential approaches to restore the ineffective cancer immunosurveillance in aged animals without distraction of defective CD4 T cells.
Methods
Mice. Two-month-old young male C57BL/6 mice were purchased from Nihon Clea, and were maintained until they reached 1824 months of age, which were
utilized as aged mice. Two-month-old Balb/c mice (Nihon Clea) were also utilized as a source of naive CD4 T cells. OVA-specic OT-II TCR-transgenic mice were crossed with B6.SJL-PtprcaPep3b/BoyJ (The Jackson Laboratory) or with IFN-gdecient mice51 to establish CD45.1 OT-II mice or IFN-g-decient OT-II mice, respectively. C3H/HeH F1 background MafO mice bearing a mutant c-Maf that lacks the ability to bind to its target genes27 were provided from the Medical Research Council. All mice including IL-6-decient mice were housed at the Center for Animal Resources and Development, Kumamoto University under specic pathogen-free conditions. All the experimental procedures were performed in accordance with the guidelines of the Institutional Animal Committee of Kumamoto University.
Tumour inoculation and Ab treatment in vivo. An MCA205 brosarcoma cell line expressing OVA protein was previously established21. RMA cells, a Rauscher MuLV-induced lymphoma21 was kindly provided by Dr Akira Shibuya (University of Tsukuba, Japan). Mice were inoculated subcutaneously with 8 105 MCA-OVA or
1 105 RMA, or were intravenously injected with 1 105 of the OVA/rey
luciferase-expressing melanoma cell line, MO4-Luc26,52, in the pulmonary metastatic model. Tumour size is expressed as a tumour index, the square root of(length width), as described previously53. To measure MO4 metastasis in the lung,
luminescence images were analysed using NightOWL II (Berthold Technologies)26. Two hundred mg of anti-IL-6R Ab, 15A7; anti-IL-6 Ab, MP520F3 (BioXCell); or control rat IgG Ab (Millipore) was injected 1 day before and after immunization. Daily intraperitoneal injections of 200 mg of anti-IL-4 Ab (BioXCell) and 100 mg of anti-IL-21 Ab (R&D Systems) were performed for 2 days after T-cell transfer. Two hundred mg of anti-IL-10 Ab (JES5-2A5; BioXCell) was injected every other day after tumour inoculation. For in vivo cell depletion, mice were injected with 200 mg of anti-CD4 Ab (GK1.5), anti-CD8 Ab (2.43) or anti-Gr-1 Ab (R5686; BioXCell) 45 days before T-cell transfer, immunization or tumour inoculation.
Adoptive transfer and immunization. Naive CD4 T cells (1 106) isolated
from OT-II TCR-transgenic mice using the naive CD4 T-cell isolation kit (Miltenyi Biotec) were injected intravenously into mice. Eight hours after the T-cell transfer, mice were immunized by an intravenous injection of 4 105 young bone
marrow-derived DCs26 pulsed with peptide ISQAVHAAHAEINEAGR, which is recognized by I-Ab-restricted OT-II cells, or peptide SIINFEKL, which is recognized by H2-Kb-restricted OVA-specic CD8 T cells (referred to as OVA
IIp or OVA-Ip, respectively). Alternatively, OVA-IIp emulsied in IFA was subcutaneously injected. Transfer of DCs pulsed with MuLV Env-gp70 H13.3 peptide SLTPRCNTAWNRL (EnvH13.3) was also performed for the vaccination against RMA tumour progression21. Labelling with CFSE (Dojindo) was performed according to the manufacturers instructions.
Flow cytometric analysis. Tumour-inltrated lymphocytes were prepared from tumours by enzymatic digestion with 2.5 mg ml 1 collagenase D (Roche) and0.1 mg ml 1 DNase I (Sigma) for 30 min at 37 C. Cell suspensions from blood, spleen, LNs and tumour were stained with the following antibodies for ow cytometric analysis: anti-CD11b (1:400), anti-Vb5 (1:200), anti-CD8 (1:200), anti-
CD4 (1:400), anti-CD27 (1:100), anti-CD25 (1:400), anti-CD69 (1:400) Abs and PerCP-streptavidin (1:400) were purchased from BD Biosciences. Anti-Ly6C, (Biolegend), anti-Gr-1 (1:100), anti-CD45.1 (1:200), anti-Ly6G (1:100), anti-CXCR3 (1:100), anti-CD127 (1:100), anti-PD-1 (1:100), anti-PD-L1 (1:200), anti-ICOS (1:200) and anti-OX40 (1:100) Abs were purchased from eBioscience. The H-2Kb/SIINFEKL-OVA tetramer-PE was purchased from MBL (1:50). For cytokine staining, CD4 T cells that were isolated from mice using anti-CD4 beads or from in vitro cultures were re-stimulated with OVA-IIp-pulsed DCs or Phorbol-12-Myristate-13-acetate/ionomycin, and then were stained with anti-IL-2 (1:400), anti-IL-17A (1:100), anti-IL-10 (1:100), anti-IFN-g (2:100), (eBioscience) or anti-
TNF-a Abs (1:400; Biolegend)54. Intracellular staining of IL-21 was performed by using IL-21R-Fc chimeric protein (R&D Systems) and uorescein-conjugated anti-human IgG (Jackson ImmunoResearch)28. Staining with anti-T-bet (1:200), anti-GATA-3(1:50), anti-RORgt (1:50), anti-Foxp3 (1:50) or anti-c-Maf Abs (1:100)
were performed using the Transcription factor buffer set (BD Biosciences). Immunouorescence images were analysed using the FACSVerse or FACSCalibur (Becton Dickinson). Data were analysed using FlowJo software (Treestar).
Real-time quantitative PCR. Total RNA from isolated CD4 T cells was extracted using the RNeasy Plus Mini Kit (Qiagen), and then was reverse-transcribed with the ReverTra Ace (TOYOBO). Real-time quantitative PCR was performed in triplicate on the ViiA7 Real-Time PCR System with TaqMan probes and Master Mix reagents (Applied Biosystems). Each gene expression was normalized to the expression of mouse Gapdh on the basis of the comparative 2[ DDCT]
method. TaqMan probes were as follows: c-maf (Mm02581355_s1), Tbx21 (Mm00450960_m1), Gata3 (Mm00484683_m1), Rorc (Mm01261022_m1), Foxp3 (Mm00475162_m1), Il10 (Mm01288386_m1), Bcl6 (Mm00477633_m1), Il17a (Mm00439618_m1), Il4 (Mm00445259_m1), Ifng (Mm01168134_m1), Il21 (Mm00517640_m1) and Gapdh (Mm99999915_g1).
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Homeostatic expansion. To analyze homeostatic division, young and aged mice were lethally irradiated with 9.5 Gy in two doses of 4.75 Gy each, with a 6-h interval. These irradiated mice were then intravenously transferred with CFSE-labelled young OT-II cells (1 106).
Proliferation assay. To assess the primary T-cell expansion, naive CD4 T cells(2.5 104 per well) were isolated from homozygous MafO/O mice or littermate
control mice, and then stimulated with plate-coated anti-CD3 Ab (2 mg ml 1) and anti-CD28 Ab (5 mg ml 1). After 48 h of in vitro culture, T cells were pulsed with [3H]-thymidine, and the incorporation of radiolabel was determined by counting radioactivity 18 h later.
Cytokine measurement. CD62Llo effector CD4 T cells were isolated using the CD4 T-cell isolation kit, followed by negative selection with the CD62L MACS beads (Miltenyi Biotec) from mice that were transferred OT-II cells and immunized with OVA-IIp-pulsed DCs, and then the T cells (3 104) were re-stimulated
with OVA-IIp-pulsed DCs for 36 h ex vivo. Cytokine levels in culture supernatants were measured using the custom Bio-Plex Pro assay kit and suspension array system (Bio-Rad). IL-6 and soluble IL-6R (sIL-6R) levels in serum, or IL-4 and IL-21 levels in supernatants were determined using the enzyme-linked immunosorbent assay (R&D systems).
For the mouse enzyme-linked immunospot (ELISPOT) assay (BD Biosciences), draining LN cells (1 105 cells per well) and bone marrow-derived DCs (3 104)
pulsed with OVA-Ip, OVA-IIp, Db-binding MuLV GagL peptide (LCCLCLTVFL) or Kb-binding Env peptide (SSWDFITV)21 were added to wells of ELISPOT plates in triplicate and incubated for 12 h. IFN-g spots were visualized with biotinconjugated IFN-g detection Ab, avidin-horseradish peroxidase and 3-amino-9-ethylcarbazole substrate reagent (BD Biosciences). ELISPOT plates were analysed using Eli photo (Minerva Tech, Tokyo, Japan).
In vitro Th1 differentiation. Naive T cells from young mice were stimulated with plate-bound anti-CD3 and anti-CD28 Abs (both eBioscience) in the presenceof IL-12 (8 ng ml 1; Wako) at 2.5 105 cells ml 1 for 5 days. IL-6 (10 ng ml 1;
Peprotech), IL-21 (20 ng ml 1; Wako), anti-IL-4 Ab (10 mg ml 1) and/or anti-IL-21 Ab (10 mg ml 1; R&D Systems) was added to the culture.
CD8 T-cell suppression assay. CD62Llo effector CD4 T cells primed in young or aged mice were isolated using CD4 isolation kit, followed by depletion of non-CD62Lhi cells with CD62L MACS beads (Miltenyi Biotec). Sorted CD4
T cells were co-cultured with CFSE-labelled naive CD8 T cells in anti-CD3 and anti-CD28 Abs-coated plates at CD4 /CD8 T-cell ratios of 4:1. After 3 days, proliferation and IFN-g production in CD8 T cells were determined by CFSE dilution and intracellular IFN-g staining, respectively. IL-10 activity was neutralized by the addition of anti-IL-10 Ab (10 mg ml 1).
Statistical analysis. All statistical analyses were performed using Prism 4.0 software (GraphPad). Multiple group comparisons were performed by one-way analysis of variance followed by Tukeys post hoc tests. Data were also analysed using an unpaired Students t-test when comparing two experimental groups.
P values less than 0.05 were considered signicant.
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Acknowledgements
We are grateful to Drs Youichiro Iwakura, Shigeo Koyasu and Akira Sibuya for the
generous supply of IL-6-decient mouse embryos, OT-II TCR Tg mouse embryos and
RMA cells, respectively. We also thank Ms Michiko Teramoto for helpful secretarial
assistance. This work was supported by Japanese Society for the Promotion of Science,
Grants-in-Aid for Young Scientists No. 24700994 (H.T.). H.T. was also supported by The
YASUDA Medical Foundation.
Author contributions
H.T. conceptualized the research, designed and performed the experiments, analysed the
data, and wrote the manuscript. S.S. analysed the data and edited the manuscript. K.M.
performed research. S.L.S. conceptualized the research, analysed the data and wrote the
manuscript. H.T. and Y.N. directed the study and edited the manuscript.
Additional information
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How to cite this article: Tsukamoto, H. et al. IL-6-mediated environmental conditioning
of defective Th1 differentiation dampens antitumour immune responses in old age. Nat.
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Copyright Nature Publishing Group Apr 2015
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.
You have requested "on-the-fly" machine translation of selected content from our databases. This functionality is provided solely for your convenience and is in no way intended to replace human translation. Show full disclaimer
Neither ProQuest nor its licensors make any representations or warranties with respect to the translations. The translations are automatically generated "AS IS" and "AS AVAILABLE" and are not retained in our systems. PROQUEST AND ITS LICENSORS SPECIFICALLY DISCLAIM ANY AND ALL EXPRESS OR IMPLIED WARRANTIES, INCLUDING WITHOUT LIMITATION, ANY WARRANTIES FOR AVAILABILITY, ACCURACY, TIMELINESS, COMPLETENESS, NON-INFRINGMENT, MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE. Your use of the translations is subject to all use restrictions contained in your Electronic Products License Agreement and by using the translation functionality you agree to forgo any and all claims against ProQuest or its licensors for your use of the translation functionality and any output derived there from. Hide full disclaimer