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Prognostic impact of circulating Her-2-reactive T-cells producing pro- and/or anti-inflammatory cytokines in elderly breast cancer patients

Prognostic impact of circulating Her-2-reactive T-cells producing pro- and/or anti-inflammatory... Background: Treating elderly breast cancer patients remains a challenge but the increasing availability of immunotherapeutic approaches instills optimism that these tumours may also be susceptible to immune control. Because aging leads to a number of alterations in the immune system (“immunosenescence”) reflecting potential exhaustion which could compromise immunomodulatory antibody therapy, here we have assessed the immunocompetence of elderly breast cancer patients compared with a group of younger patients, and related this to the 5-year survival of the former. Methods: T-cell responses to Her-2 peptide pools in vitro were assessed by analyzing pro- and anti-inflammatory cytokine production by CD4+ and CD8+ T-cells in 40 elderly and 35 younger breast cancer patients. Results: The proportions of older and younger patients whose peripheral T-cells responded to Her-2 peptides in vitro were found to be similar, although a significantly higher fraction of younger patients possessed IL-2-producing CD4+ Her-2-reactive T-cells than in the elderly (p = 0.03). However, IL-2 production did not impart a survival benefit to the latter. In contrast, there was a survival benefit of possessing Her-2-reactive CD8+ T-cells, but this was abrogated in patients if they also had CD4+ Her-2-responsive T-cells that producedIL-5 and/or IL-17 (p = 0.01). This resulted in a 5-yr survival rate of only 29 % compared to 76 % for patients whose her-2-reactive CD4+ T-cells did not produceIL-5 and/or IL-17. Additionally, patients whose CD8+ T-cells produced TNF had a significantly better survival than those that did not (93 % compared to 52 %, p = 0.01), whereas no survival benefit was attributable to possessing IFN-γ-producing cells. Conclusions: Elderly breast cancer patients appear perfectly immunocompetent to respond to Her-2 peptide pools in vitro, with response patterns very similar to younger patients. The nature of this response is associated with 5-year survival of these elderly patients, suggesting that boosting anti-tumor responses and modulating the nature of the T-cell response is likely to be effective even in potentially immunosenescent elderly breast cancer patients, and might be useful for predicting which patients are most likely to benefit from such treatments. Keywords: T-cells, Breast cancer, Her-2, Elderly, Tumour-associated antigens, Cytokines, Immunosenescence Background challenge. The elderly are underrepresented in clinical The prevalence of most types of cancer increases with trials, of which there are hardly any that have specifically age. One reason for this may be that aging is associated considered elderly cohorts. Generally, the same treatment with dysregulated immunity, which could contribute to regimens are applied to both younger and older cancer increased susceptibility to infections and cancer [1]. patients, but the latter are not able to tolerate chemother- Treating elderly breast cancer patients remains a major apy or radiotherapy to the same extent and may thus be under-treated [2, 3]. Although there are many suggestions as to how to treat elderly breast cancer patients [4-6], and * Correspondence: jithendra.kini@gmail.com Department of Internal Medicine II, Centre for Medical Research, University considering the recent breakthroughs in immunotherapy of Tuebingen, Waldhoernlestr 22, 72072 Tuebingen, Germany 5 of melanoma and other cancer types, more investigations Present Address: Yale Cancer Center, Yale University School of Medicine, need to be conducted to understand the immune system New Haven, CT, USA Full list of author information is available at the end of the article © 2015 Bailur et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 2 of 9 of these patients and the potential role of ageing and survival, where we were able to show the association of immunosenescence in treatment outcome. The hallmarks certain pro- and anti-inflammatory cytokines produced by of immunosenescence may be observed chronologically CD4+ and CD8+ T-cells with overall survival, analogous earlier in chronic viral infections and cancer [7, 8]. There to similar findings in melanoma [20]. is a number of alterations that are known to occur with aging [9, 10], which include changes in the number and Results distribution of T and B lymphocytes and their differenti- T-cell responses to Her-2 ation states [8, 11]. As T-cells play a crucial role in adaptive A majority of the elderly(97 %, n =38) and all younger immunity, their efficient activation to defend against cancer (100 %, n = 35) breast cancer patients analyzed had in vitro or viral infection is important. Dendritic cells (DCs) are T cell responses to mixtures of Her-2 peptides. FACS plots necessary for activating the T-cells efficiently. Aging may from a representative donor are shown in Additional file 1: also result in alterations of important co-receptors on DCs Table S2. CD4+ T-cell responses to Her-2 were observed in that could result in weakened T-cell response, possibly most individuals in the case of both older (32/38, 87 %) and reducing anti-cancer immunity [12, 13]. Generally, higher younger (33/35, 94 %) patients, whereas only 18 of the levels of serum inflammatory markers are also observed in former (47 %) and 21 of the latter (60 %) possessed CD8+ the elderly, which could play a role in the activation of T-cells responding to Her-2 peptides. ThisCD8+ T-cell immunosuppressive cell subsets making it more difficult to response was present irrespective of whether the patients resist cancer [14, 15]. Thus, there are several loci at which also had a CD4+ T-cell response to Her-2. Taking advan- immunosenescence could interfere with anti-cancer im- tage of our ability to analyze 6 different cytokines simultan- munity and accordingly compromise immunotherapies. eously by intra-cellular staining of individual T-cells by flow The recent striking clinical success of employing immuno- cytometry, we grouped the Her-2 responders according to modulatory antibodies such as anti-CTLA-4, PD-1 or PDL- the cytokines produced by their CD4+ and CD8+ T-cells. 1which “take the brakes off” immunity and are showing extremely promising results in some cancers in a propor- CD8+ T-cell responses to Her-2 tion of treated patients makes it even more important to As described above, a CD8+ T-cell response to Her-2, determine the effect of age on the immune status of cancer defined as the production of any one of the 6 tested patients [16-18]. Such therapies are likely to be effective cytokines, was observed in 18/38 (47 %) older and 21/35 only when the patient remains capable of mounting an (60 %) younger patients. In a high proportion of these anti-cancer immune response. In elderly patients, immuno- patients, CD8+ T-cells responding to Her-2produced the senescence could compromise these responses. pro-inflammatory cytokines TNF (14/18, 78 % in old; Earlier, we showed that the presence of peripheral T-cells 16/21, 76 % in young) and IFN-γ (13/18, 72 % in old; responding to certain tumor-associated antigens (TAA) in 18/21, 86 % in young).Only a small proportion of CD8+ stage IV melanoma was associated with longer survival and T-cells produced IL-2 and IL-10 in either old or young that the pro- or anti-inflammatory nature of the T cell re- patients (Tables 1 and 2). sponse influenced the strength of this association [19, 20]. Thus, T-cell responsiveness to TAA is an important prog- CD4+ T-cell responses to Her-2 nostic biomarker of survival. Melanoma patients tend to be Analyzing the nature of the CD4+ T-cell responses to Her- relatively young compared to most other solid cancer pa- 2 peptides, we observed that these cells mainly produced tients, and so we asked whether this type of biomarker the pro-inflammatory cytokines TNF (27/32, 84 % in old; reflecting T-cell functional integrity would also be inform- 27/33, 82 % in young), IFN-γ (23/32, 72 % in old; 28/33, ative in older patients. We therefore elected to examine this 85 % in young) and, unlike for CD8+ T-cells, also IL-2 (13/ issue in a cohort of some of the oldest such patients, 32, 41 % in old; 22/33, 67 % in young). The higher fraction namely in elderly female breast cancer patients. We had of younger relative to older patients whose CD4+ T-cells previously reported that elderly patients with in vitro CD8+ produced IL-2 in response to Her-2 was statistically signifi- T-cell responses against pooled Her-2 peptides survived cantly different (p = 0.03). As for CD8+ T-cell responses, longer than those who did not [21], suggesting that immu- IL10,IL-5and IL-17wererarelyproducedbyeitheryoung nosenescence had not compromised responsiveness and or old patients (Tables 1 and 2). that immunomodulatory therapies should also be effective in these patients. Here, we compared the immunocompe- The T-cell response to Her-2 correlates with overall tence of these elderly patients with a group of younger survival patients and found that they were indeed similar in this Previously, we had shown that elderly patients who had a respect. We have continued these first studies on the CD8+ T-cell response to Her-2 had a survival advantage elderly to dissect the nature of their CD4+ and CD8+ T- over those who did not(p = 0.03). The overall (5-year) cell responses to Her-2 peptides in relation to their overall survival rate was 52 % for patients without a CD8+ T-cell Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 3 of 9 Table 1 Cytokines produced by Her-2 responders in old breast cancer patients Old (n = 38) CD4 Response No. of patients % Dead 5-year survival rate P value TNF Yes 27 37 60 0.1 No 11 18 81 IFN-γ Yes 23 43 57 0.1 No 15 13 84 IL-5 Yes 7 57 43 0.1 No 31 23 73 IL-10 Yes 3 33 67 0.8 No 35 31 65 IL-2 Yes 13 46 54 0.3 No 25 24 74 IL-17 Yes 3 67 33 0.3 No 35 29 70 IL-5 and or IL-17 Yes 10 50 29 0.01 No 28 25 76 CD8 TNF Yes 14 7 93 0.01 No 24 46 52 IFN- γ Yes 13 23 76 0.3 No 25 36 62 IL-10 Yes 2 50 50 0.7 No 36 31 68 IL-2 Yes 1 0 100 0.5 No 37 32 66 Results of survival analysis according to Kaplan Meier method and p values from Mantel-Cox (log-rank) test response to Her-2, compared with 82 % for those had a younger patients because only two had died. Nonetheless, CD8+ T-cell response. There was only 17 % mortality in these two had both CD4+ and CD8+ T-cell responses to the group of patients with a CD8+ T-cell response to Her-2 Her-2. whereas this was 45 % for patients without a CD8+ T-cell response. In the present study, we stratified patients ac- Impact of the presence of CD4+ T-cells producing IL-5 cording to whether they had only a CD8+ T-cell response, and IL-17 on survival only a CD4+ T-cell response or both CD4+ and CD8+ T- Patients were stratified according to the cytokines produced cell responses to Her-2 peptides, and performed a Kaplan- by their CD4+ or CD8+ T-cells. Considering the elderly Meier analysis (Table 3). We confirmed a survival benefit breast cancer patients, Kaplan-Meier survival analysis for patients with Her-2-reactive CD8+ T-cells as observed showed that patients whose CD4+ T-cells produced IL-5 in our earlier study, but there was no survival advantage for tended to have poorer outcomes (p=0.1,Fig.2a) with a patients with a CD4+ T-cell response to Her-2. Moreover, survival rate of 43 % compared to 73 % for patients whose those patients with a CD8+ T-cell response to Her-2 who CD4+ T-cells did not produce IL-5 when stimulated with also had a CD4+ T-cell response had poorer survival than Her-2 peptides. A weaker trend towards a similar impact thosewithonlyaCD8+T-cellresponse. A significant early on survival was observed for patients whose CD4+ T-cells negative impact on survival can also be observed for produced IL-17 (p=0.3,Fig.2b).Here, thosewhose CD4+ patients with no CD8+ T-cell response to Her-2 (Gehan T-cells produced IL-17 had a 33 % 5-year survival com- Breslow test: p = 0.02) (Fig. 1). The 5-year survival rate for pared to 70 % for those who did not produce IL-17. Despite patients with a solely CD4+ T-cell response was 49 % com- the lack of statistical significance when considering IL 5 pared to 77 % and 100 % for those with combined CD4+ and IL 17 production separately, when we analyzed and CD8+ T-cell responses or only a CD8+ T-cell response, patients whose CD4+ T-cells produced IL-5 and/or IL-17, respectively. Survival analyses were not possible in the we did observe a significant impact on the overall survival Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 4 of 9 Table 2 Cytokines produced by Her-2 responders in young breast cancer patients Young (n = 35) CD4 Response No. of patients % Dead P value TNF Yes 27 7 0.5 No 8 0 IFN-γ Yes 28 7 0.6 No 7 0 IL-5 Yes 8 25 0.04 No 27 0 IL-10 Yes 4 0 0.7 No 31 6 IL-2 Yes 22 9 0.4 No 13 0 Fig. 1 Survival analysis of elderly breast cancer patients according to IL-17 Yes 6 17 0.6 CD8+ T-cell responses to Her-2peptides in vitro. Survival analysis of No 29 3 elderly patients (n = 38) grouped according to CD8+ T-cell responses to Her-2 (CD8 response), CD4+ and CD8+ T-cell responses (CD4 & IL-5 and or IL-17 Yes 12 17 0.1 CD8response) and CD4+ T-cell responses to Her-2 (CD4 response) No 23 0 CD8 TNF Yes 16 13 0.3 patients whose CD4+ T-cells produced IL-5 and/or IL-17 No 19 0 (p = 0.02, Fig. 3b). Here, the survival rate was only 33 % IFN-γ Yes 18 11 0.3 compared to 80 % for patients whose CD4+ T-cells did not produce these cytokines. No 17 0 Analogous observations were also made anecdotally in IL-10 Yes 2 0 0.3 the younger patients, where those whose CD4+ T-cells No 33 6 produced IL-5 had poor survival, with 25 % (2/8) of the IL-2 Yes 2 0 0.8 IL-5-producing patients dying, compared to none whose No 33 6 CD4+ T-cells did not produce IL-5. Results of survival analysis according to Kaplan Meier method and p values from Mantel-Cox (log-rank) test Impact of TNF-producing CD8+ T-cells on survival We previously reported the beneficial effects of having a (p = 0.01, Fig. 3a) in these elderly breast cancer patients. CD8+ T-cell response to Her-2. Here, we investigated the Thus, the survival rate for patients whose CD4+ T-cells function of these Her-2 specific CD8+ T-cells to identify produced IL-5 and/or IL-17 was 29 % compared to 76 % the factors associating with survival benefit in this cohort. for the patients without these cytokines. From the Kaplan- We observed that patients whose CD8+ T-cell produced Meier analysis of different parameters in the elderly we TNF had a significantly better survival than those whose observed the expected impact of metastasis on survival CD8+ T-cells did not produce TNF, when all elderly (Additional file 2: Table S1). Limiting the analysis to non- patients were included in the analysis (p = 0.01, Fig. 4a). metastatic elderly breast cancer patients only, we still Thesurvivalratefor thepatients whoseCD8+ T-cellspro- observed a similar negative impact on survival for the duced TNF was 93 % compared to 52 %, for those with no Table 3 Phenotype of Her-2 responders in older patients Response to Her-2 No. of patients Percentage dead p value CD4 response Yes 33 36 % 0.1 No 4 0 % CD8 response Yes 18 13 % 0.03 No 19 47 % CD4 and CD8 response Yes 14 21 % 0.2 No 23 39 % Results of survival analysis according to Kaplan Meier method and p values from Mantel-Cox (log-rank) test Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 5 of 9 Fig. 3 Survival analysis of elderly breast cancer patients according to IL-5 Fig. 2 Survival analysis of elderly breast cancer patients according to and/or IL-17 production by Her-2-stimulated CD4+ T-cells. a patients IL-5 or IL-17 production by Her-2-stimulated CD4+ T-cells. a Patients grouped according to IL-5 and/or IL-17 production in all patients and b grouped according to IL-5 production; b IL-17 production in non-metastatic patients only TNF-producing CD8+ T-cells. This survival advantage was Discussion still observed when only non-metastatic elderly patients In the present study, we investigated associations of the were considered (p = 0.05, Fig. 4b). In the case of non- presence of circulating Her-2-reactive CD4+ and CD8+ T- metastatic patients, these figures were 92 % and 57 %, cells with survival in older breast cancer patients relative to respectively. No survival associations could be observed in their pro- and anti-inflammatory cytokine production on young. stimulation, and compared the immunocompetence of the elderly, as assessed in this manner, with that of younger patients. Previously, we had reported a survival benefit ac- Impact of IFN-γ producing CD8+ T-cell on survival cruing to patients having a CD8+ T-cell response to Her-2 Here, we grouped the patients according to CD8+ T- [21]. To test the hypothesis that the immune system in the cell production of IFN-γ. Unlike the findings with elderly may be compromised due to aging, here we investi- TNF above, unexpectedly, Kaplan-Meier survival ana- gated differences in T-cell responses to Her-2 in younger lysis indicated no significant differences between IFN- and older breast cancer patients. To the best of our know- producers and non-producers both in young and old ledge, this is the first study to investigate the phenotype (p = 0.3,Fig.4c). and function of Her-2 reactive T-cells, or those reactive to Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 6 of 9 any tumor-associated antigen, in young and old breast can- cer patients. In the present study, stratifying the patients according to those having a CD8+ T-cell response alone (positive impact on survival as previously shown), or both CD4+ and CD8+ T-cell responses, or only a CD4+ T-cell response revealed a trend towards a negative general survival impact for CD4+ T-cell reactivity in older patients. These results clearly con- firmed the importance of having a CD8+ T-cell response to Her-2 but also showed that there was reduced survival when a CD4+ T-cell response to Her-2 was also present in the same patient. This implies that at least in terms of responses to Her-2, CD4+ T-cell reactivity to this TAA is likely to have unanticipated negative effects. CD4+ regula- tory T cells would be the obvious candidates for the cells mediating these negative effects. The lack of CD8+ T-cell responses in patients with poorer survival could also be due to the presence of increased levels of Tregs. This would be consistent with other reportedstudies showingthatTregs could control the T-cell response against certain TAAs in an antigen-specific manner [22, 23]. We were not able to test for classic Treg phenotypes in the current study due to the limitations of the flow cytometric technique, but will focusonthisquestioninfuture studies.We can only say at this juncture that one cytokine commonly implicated in Treg function, IL-10, does not seem to be responsible for these results, whereas two other cytokines not generally implicated in Treg function, namely IL-5 and IL-17, do seem to play important roles. Thus, functional analysis of 6 different cytokines allowed us to group patients according to the cytokines produced by the CD4+ and CD8+ T-cells responding to Her-2. Survival analysis showed that the presence of CD4+ T-cells producing IL-5 and/or IL-17 had a negative impact on survival in the elderly (p = 0.01). Com- pared to other anti-inflammatory cytokines, the role of IL-5 in peripheral cells seems to be under-investigated. Studies have indicated that IL-5 may facilitate lung metastasis, and high expression of IL-5 in the tumor was also negatively associated with survival [24, 25]. A potential role in im- mune surveillance has been proposed [26]. Similarly, there are few data on IL-17 in this context. Although IL-17 is primarily considered a pro-inflammatory cytokine, its role in cancer is still unclear. Somestudies have shownanti- tumor effects of IL-17, so that it has been proposed for use as a cancer immunotherapeutic [27, 28]. However, tumor- Fig. 4 Survival analysis of elderly breast cancer patients according to infiltrating lymphocytes with a high level of IL-17-positivity CD8+ T-cell responses to Her-2 peptides in vitro. a patients grouped correlate with poor prognosis in some studies [29, 24], and according to TNF production in all patients and b in non-metastatic an association of IL-17 with Tregs in the tumor has been patients only; c patients grouped according to IFN-γ production reported [29]. We also need to bear in mind that not only (all patients) Tregs, but also myeloid-derived suppressor cells (MDSCs) may play a role in suppressing T-cell activity. We previously reported the possible suppressive role of MDSCs and Tregs in relation to T-cell responses to Her-2 and also the pos- sible indirect role of these immunosuppressive subsets in Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 7 of 9 breast cancer [21]. MDSCs are also associated with IL-17, present in the circulation is important and has an impact as they are able to mediate IL-17 production by naïve T- on patient survival. Considering the results presented in cells, depending on the cytokines produced by the MDSCs this study, depletion of Her-2-specific IL-5- and IL-17- themselves [30]. Consistent with the present report, our producing CD4+ T-cells and enrichment of TNF- previous study investigating the role of IL-17 in the context producing CD8+ T-cells for adoptive T-cell therapy of CD4+ T-cell responses to TAAs documented a negative would be important in breast cancer. Thus, considering impact ofIL-17-producing T-cells in late-stage melanoma our previous results, it is not only immunosuppressive [20]. There are several other cytokines such as IL-4 and IL- cells like Tregs and MDSCs that might have to be targeted 13 that are known to have a negative impact on survival but it is also important to consider the specific cytokines [31, 32] and which would have been interesting to examine produced by the CD4+ and CD8+ T-cell that are reactive here, especially as we previously found that IL 4 was rele- to, in this case, Her-2. This study may also provide some vant in melanoma. IL-13 is known to regulate cancer inva- mechanistic insights for a better understanding of the im- sion and metastasis in different cancers [31, 32]. In our mune system in elderly and younger breast cancer patients. previous study on melanoma, we observed a negative In any event, the findings that elderly breast cancer patients impact on survival for the patients who produced IL-4 are not immunosenescent according to the assays con- reactive to Melan-A [20]. IL-4 is also known to trigger the ducted in this work augur well for the efficacy of immuno- cancer proliferation mechanism [33]. Not only these but modulatory antibodies even in older patients. also IL-6 and IL-1 have shown a negative impact on survival and tumor progression [34, 35]. However, the Methods number of different variables that could be tested at the Patients same time was limited and we were unable to include The study included 40 elderly and 35 younger patients these other cytokines in the present analysis. with breast cancer (Additional file 3: Figure S1). Blood The role of the pro-inflammatory cytokines IL-2, IFN-γ samples from patients with different TNM stage were and TNF in cancer is usually considered to be well- collected between March and November 2009, at the established. They play an important role in inducing a University Hospital Tuebingen. Blood was drawn prior protective immune response [36] and IL-2 has long been to any treatment or surgery. Standard Ficoll-Hypaque used as an immunotherapeutic drug which can result in a gradient centrifugation was used to isolate the peripheral small minority of very durable responses [37]. In the blood mononuclear cells (PBMCs) before they were present study, one of the few differences that we observed cryopreserved for experimental purposes. The study between younger and older patients was that significantly was approved by the Institutional Ethics Committee of lower proportion of the latter possessed Her-2-reactive University Hospital Tuebingen (71/2009BO2) and a IL-2 producing CD4+ T-cells, possibly contributing to bet- waiver of informed consent was granted for this study. ter survival of the former. However, whether or not an elderly patient possessed IL-2-producing CD4+ T-cells Detection of tumour-associated antigen-reactiveT-cells had no impact on survival. To assess T-cell reactivity to Her-2, a 12-day in vitro culture IFN-γ is known to play an important role in anti-tumor was performed as described previously [21]. First, after activity [38, 39] and has also been considered as an immu- thawing carefully, washing extensively, and assessing viabil- notherapeutic drug to treat cancer patients [40, 41]. In our ity, PBMCs (1x10 )were culturedinX-Vivo15defined study, however, we could discern no significant survival medium (Lonza) supplemented with IL-4 (5 ng/ml: Sandoz, advantage for patients with Her-2-reactive CD8+ T-cells Basel, Switzerland) and IL-7 (5 ng/ml: Sterling-Winthrop, producing IFN-γ, although there was a survival advantage US), on day 0. On day 1, the PBMCs were stimulated with for patients who had Her-2-reactive TNF-producing CD8+ mixtures of Her-2 15-mer overlapping peptides (with an T-cells. TNF is involved in a number of important cellular overlap of 11 amino acids) (PepMix, JPT Technologies, functions including cell proliferation, survival and death Berlin, Germany) at a concentration of 1 μg/ml. The cells and has been used in patients with locally advanced tumors were supplemented with IL-2 (40U/ml: Chiron Behring [42]. In the present study, we observed that patients whose GmbH,Marburg,Germany)on day3.On day12, after CD8+ T-cells produced TNF had a significant survival ad- harvesting and washing, the cultured T-cells were re- vantage over those who did not, indicating the importance stimulated (0.4-0.5 x 10 cells/well) with Her-2 PepMix at of Her-2-reactive TNF-producing CD8+ T-cells for these a concentration of 1 μg/ml or left unstimulated as a nega- elderly breast cancer patients. tive control for 12 hours. Golgi-plug (BD Biosciences) was added at 1 μl/ml to all cultures. Patients’ cells were stimu- Conclusions lated with influenza nucleoprotein (NP) and membrane From our results, we propose that the nature of the anti- protein (M1) peptides as a positive control, as all subjects tumor T-cell response and the TAA targeted by T-cells have been exposed to influenza during their lives and all Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 8 of 9 possess T cells responsive to these peptides. After harvest- manuscript. All authors read and approved the final version of the manuscript. ing and washing, the cells were incubated with Gamunex (Talecris) to block Fc receptors, and with ethidium mono- Acknowledgements azide (EMA,MoBiTec GmbH, Goettingen, Germany), a This project was supported by the European Commission (FP7 259679 “IDEAL”) marker for dead cells. Intracellular cytokine staining was and the Bundesministerium für Bildung und Forschung (ISPE-BREAST, FKZ 01EI1401). performed after fixing and permeabilizing the cells with Cytofix/Cytoperm (BD Biosciences). Cells were simultan- Author details eously stained with CD3-PO (Invitrogen), CD4-Pacific Blue, Department of Internal Medicine II, Centre for Medical Research, University of Tuebingen, Waldhoernlestr 22, 72072 Tuebingen, Germany. Radiology TNF-FITC, IL-2-Alexa Fluor-700, IL-5-PE (Bio Legend), Clinic, Diagnostic and Interventional Radiology, University Hospital CD8-APC-Cy7, IFN-γ-PE-Cy7 (BD Biosciences), IL- Tuebingen, Tuebingen, Germany. Present Address: BioNTech AG, Mainz, 10-APC (Miltenyi Biotech) and IL-17-PerCP-Cy5.5 Germany. School of Science and Technology, College of Arts and Science, Nottingham Trent University, Nottingham, UK. Present Address: Yale Cancer (eBioscience). Following the staining, cells were mea- Center, Yale University School of Medicine, New Haven, CT, USA. sured on a BD-LSR-II flow cytometer using the FACS- Diva software (BD-Biosciences). Received: 7 June 2015 Accepted: 28 August 2015 Flow cytometry data analysis References Data were analyzed using FlowJo software (Tree Star Inc.) 1. Fulop T, Larbi A, Witkowski JM, Kotb R, Hirokawa K, Pawelec G. as shown earlier [21]. To detect cytokine-producing cells, Immunosenescence and cancer. Crit Rev Oncog. 2013;18(6):489–513. 2. Du XL, Osborne C, Goodwin JS. Population-based assessment of the stimulated samples were compared with unstimulated hospitalizations for toxicity from chemotherapy in older women with breast (negative) control and the response was considered positive cancer. J Clin Oncol. 2002;20(24):4636–42. when at least one cytokine was produced by the stimulated 3. Pallis AG, Hatse S, Brouwers B, Pawelec G, Falandry C, Wedding U, et al. 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Proc Natl Acad Sci U S A. 1998;95(13):7556–61. www.biomedcentral.com/submit http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal for ImmunoTherapy of Cancer Springer Journals

Prognostic impact of circulating Her-2-reactive T-cells producing pro- and/or anti-inflammatory cytokines in elderly breast cancer patients

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Springer Journals
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Copyright © 2015 by Bailur et al.
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Medicine & Public Health; Oncology
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Abstract

Background: Treating elderly breast cancer patients remains a challenge but the increasing availability of immunotherapeutic approaches instills optimism that these tumours may also be susceptible to immune control. Because aging leads to a number of alterations in the immune system (“immunosenescence”) reflecting potential exhaustion which could compromise immunomodulatory antibody therapy, here we have assessed the immunocompetence of elderly breast cancer patients compared with a group of younger patients, and related this to the 5-year survival of the former. Methods: T-cell responses to Her-2 peptide pools in vitro were assessed by analyzing pro- and anti-inflammatory cytokine production by CD4+ and CD8+ T-cells in 40 elderly and 35 younger breast cancer patients. Results: The proportions of older and younger patients whose peripheral T-cells responded to Her-2 peptides in vitro were found to be similar, although a significantly higher fraction of younger patients possessed IL-2-producing CD4+ Her-2-reactive T-cells than in the elderly (p = 0.03). However, IL-2 production did not impart a survival benefit to the latter. In contrast, there was a survival benefit of possessing Her-2-reactive CD8+ T-cells, but this was abrogated in patients if they also had CD4+ Her-2-responsive T-cells that producedIL-5 and/or IL-17 (p = 0.01). This resulted in a 5-yr survival rate of only 29 % compared to 76 % for patients whose her-2-reactive CD4+ T-cells did not produceIL-5 and/or IL-17. Additionally, patients whose CD8+ T-cells produced TNF had a significantly better survival than those that did not (93 % compared to 52 %, p = 0.01), whereas no survival benefit was attributable to possessing IFN-γ-producing cells. Conclusions: Elderly breast cancer patients appear perfectly immunocompetent to respond to Her-2 peptide pools in vitro, with response patterns very similar to younger patients. The nature of this response is associated with 5-year survival of these elderly patients, suggesting that boosting anti-tumor responses and modulating the nature of the T-cell response is likely to be effective even in potentially immunosenescent elderly breast cancer patients, and might be useful for predicting which patients are most likely to benefit from such treatments. Keywords: T-cells, Breast cancer, Her-2, Elderly, Tumour-associated antigens, Cytokines, Immunosenescence Background challenge. The elderly are underrepresented in clinical The prevalence of most types of cancer increases with trials, of which there are hardly any that have specifically age. One reason for this may be that aging is associated considered elderly cohorts. Generally, the same treatment with dysregulated immunity, which could contribute to regimens are applied to both younger and older cancer increased susceptibility to infections and cancer [1]. patients, but the latter are not able to tolerate chemother- Treating elderly breast cancer patients remains a major apy or radiotherapy to the same extent and may thus be under-treated [2, 3]. Although there are many suggestions as to how to treat elderly breast cancer patients [4-6], and * Correspondence: jithendra.kini@gmail.com Department of Internal Medicine II, Centre for Medical Research, University considering the recent breakthroughs in immunotherapy of Tuebingen, Waldhoernlestr 22, 72072 Tuebingen, Germany 5 of melanoma and other cancer types, more investigations Present Address: Yale Cancer Center, Yale University School of Medicine, need to be conducted to understand the immune system New Haven, CT, USA Full list of author information is available at the end of the article © 2015 Bailur et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 2 of 9 of these patients and the potential role of ageing and survival, where we were able to show the association of immunosenescence in treatment outcome. The hallmarks certain pro- and anti-inflammatory cytokines produced by of immunosenescence may be observed chronologically CD4+ and CD8+ T-cells with overall survival, analogous earlier in chronic viral infections and cancer [7, 8]. There to similar findings in melanoma [20]. is a number of alterations that are known to occur with aging [9, 10], which include changes in the number and Results distribution of T and B lymphocytes and their differenti- T-cell responses to Her-2 ation states [8, 11]. As T-cells play a crucial role in adaptive A majority of the elderly(97 %, n =38) and all younger immunity, their efficient activation to defend against cancer (100 %, n = 35) breast cancer patients analyzed had in vitro or viral infection is important. Dendritic cells (DCs) are T cell responses to mixtures of Her-2 peptides. FACS plots necessary for activating the T-cells efficiently. Aging may from a representative donor are shown in Additional file 1: also result in alterations of important co-receptors on DCs Table S2. CD4+ T-cell responses to Her-2 were observed in that could result in weakened T-cell response, possibly most individuals in the case of both older (32/38, 87 %) and reducing anti-cancer immunity [12, 13]. Generally, higher younger (33/35, 94 %) patients, whereas only 18 of the levels of serum inflammatory markers are also observed in former (47 %) and 21 of the latter (60 %) possessed CD8+ the elderly, which could play a role in the activation of T-cells responding to Her-2 peptides. ThisCD8+ T-cell immunosuppressive cell subsets making it more difficult to response was present irrespective of whether the patients resist cancer [14, 15]. Thus, there are several loci at which also had a CD4+ T-cell response to Her-2. Taking advan- immunosenescence could interfere with anti-cancer im- tage of our ability to analyze 6 different cytokines simultan- munity and accordingly compromise immunotherapies. eously by intra-cellular staining of individual T-cells by flow The recent striking clinical success of employing immuno- cytometry, we grouped the Her-2 responders according to modulatory antibodies such as anti-CTLA-4, PD-1 or PDL- the cytokines produced by their CD4+ and CD8+ T-cells. 1which “take the brakes off” immunity and are showing extremely promising results in some cancers in a propor- CD8+ T-cell responses to Her-2 tion of treated patients makes it even more important to As described above, a CD8+ T-cell response to Her-2, determine the effect of age on the immune status of cancer defined as the production of any one of the 6 tested patients [16-18]. Such therapies are likely to be effective cytokines, was observed in 18/38 (47 %) older and 21/35 only when the patient remains capable of mounting an (60 %) younger patients. In a high proportion of these anti-cancer immune response. In elderly patients, immuno- patients, CD8+ T-cells responding to Her-2produced the senescence could compromise these responses. pro-inflammatory cytokines TNF (14/18, 78 % in old; Earlier, we showed that the presence of peripheral T-cells 16/21, 76 % in young) and IFN-γ (13/18, 72 % in old; responding to certain tumor-associated antigens (TAA) in 18/21, 86 % in young).Only a small proportion of CD8+ stage IV melanoma was associated with longer survival and T-cells produced IL-2 and IL-10 in either old or young that the pro- or anti-inflammatory nature of the T cell re- patients (Tables 1 and 2). sponse influenced the strength of this association [19, 20]. Thus, T-cell responsiveness to TAA is an important prog- CD4+ T-cell responses to Her-2 nostic biomarker of survival. Melanoma patients tend to be Analyzing the nature of the CD4+ T-cell responses to Her- relatively young compared to most other solid cancer pa- 2 peptides, we observed that these cells mainly produced tients, and so we asked whether this type of biomarker the pro-inflammatory cytokines TNF (27/32, 84 % in old; reflecting T-cell functional integrity would also be inform- 27/33, 82 % in young), IFN-γ (23/32, 72 % in old; 28/33, ative in older patients. We therefore elected to examine this 85 % in young) and, unlike for CD8+ T-cells, also IL-2 (13/ issue in a cohort of some of the oldest such patients, 32, 41 % in old; 22/33, 67 % in young). The higher fraction namely in elderly female breast cancer patients. We had of younger relative to older patients whose CD4+ T-cells previously reported that elderly patients with in vitro CD8+ produced IL-2 in response to Her-2 was statistically signifi- T-cell responses against pooled Her-2 peptides survived cantly different (p = 0.03). As for CD8+ T-cell responses, longer than those who did not [21], suggesting that immu- IL10,IL-5and IL-17wererarelyproducedbyeitheryoung nosenescence had not compromised responsiveness and or old patients (Tables 1 and 2). that immunomodulatory therapies should also be effective in these patients. Here, we compared the immunocompe- The T-cell response to Her-2 correlates with overall tence of these elderly patients with a group of younger survival patients and found that they were indeed similar in this Previously, we had shown that elderly patients who had a respect. We have continued these first studies on the CD8+ T-cell response to Her-2 had a survival advantage elderly to dissect the nature of their CD4+ and CD8+ T- over those who did not(p = 0.03). The overall (5-year) cell responses to Her-2 peptides in relation to their overall survival rate was 52 % for patients without a CD8+ T-cell Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 3 of 9 Table 1 Cytokines produced by Her-2 responders in old breast cancer patients Old (n = 38) CD4 Response No. of patients % Dead 5-year survival rate P value TNF Yes 27 37 60 0.1 No 11 18 81 IFN-γ Yes 23 43 57 0.1 No 15 13 84 IL-5 Yes 7 57 43 0.1 No 31 23 73 IL-10 Yes 3 33 67 0.8 No 35 31 65 IL-2 Yes 13 46 54 0.3 No 25 24 74 IL-17 Yes 3 67 33 0.3 No 35 29 70 IL-5 and or IL-17 Yes 10 50 29 0.01 No 28 25 76 CD8 TNF Yes 14 7 93 0.01 No 24 46 52 IFN- γ Yes 13 23 76 0.3 No 25 36 62 IL-10 Yes 2 50 50 0.7 No 36 31 68 IL-2 Yes 1 0 100 0.5 No 37 32 66 Results of survival analysis according to Kaplan Meier method and p values from Mantel-Cox (log-rank) test response to Her-2, compared with 82 % for those had a younger patients because only two had died. Nonetheless, CD8+ T-cell response. There was only 17 % mortality in these two had both CD4+ and CD8+ T-cell responses to the group of patients with a CD8+ T-cell response to Her-2 Her-2. whereas this was 45 % for patients without a CD8+ T-cell response. In the present study, we stratified patients ac- Impact of the presence of CD4+ T-cells producing IL-5 cording to whether they had only a CD8+ T-cell response, and IL-17 on survival only a CD4+ T-cell response or both CD4+ and CD8+ T- Patients were stratified according to the cytokines produced cell responses to Her-2 peptides, and performed a Kaplan- by their CD4+ or CD8+ T-cells. Considering the elderly Meier analysis (Table 3). We confirmed a survival benefit breast cancer patients, Kaplan-Meier survival analysis for patients with Her-2-reactive CD8+ T-cells as observed showed that patients whose CD4+ T-cells produced IL-5 in our earlier study, but there was no survival advantage for tended to have poorer outcomes (p=0.1,Fig.2a) with a patients with a CD4+ T-cell response to Her-2. Moreover, survival rate of 43 % compared to 73 % for patients whose those patients with a CD8+ T-cell response to Her-2 who CD4+ T-cells did not produce IL-5 when stimulated with also had a CD4+ T-cell response had poorer survival than Her-2 peptides. A weaker trend towards a similar impact thosewithonlyaCD8+T-cellresponse. A significant early on survival was observed for patients whose CD4+ T-cells negative impact on survival can also be observed for produced IL-17 (p=0.3,Fig.2b).Here, thosewhose CD4+ patients with no CD8+ T-cell response to Her-2 (Gehan T-cells produced IL-17 had a 33 % 5-year survival com- Breslow test: p = 0.02) (Fig. 1). The 5-year survival rate for pared to 70 % for those who did not produce IL-17. Despite patients with a solely CD4+ T-cell response was 49 % com- the lack of statistical significance when considering IL 5 pared to 77 % and 100 % for those with combined CD4+ and IL 17 production separately, when we analyzed and CD8+ T-cell responses or only a CD8+ T-cell response, patients whose CD4+ T-cells produced IL-5 and/or IL-17, respectively. Survival analyses were not possible in the we did observe a significant impact on the overall survival Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 4 of 9 Table 2 Cytokines produced by Her-2 responders in young breast cancer patients Young (n = 35) CD4 Response No. of patients % Dead P value TNF Yes 27 7 0.5 No 8 0 IFN-γ Yes 28 7 0.6 No 7 0 IL-5 Yes 8 25 0.04 No 27 0 IL-10 Yes 4 0 0.7 No 31 6 IL-2 Yes 22 9 0.4 No 13 0 Fig. 1 Survival analysis of elderly breast cancer patients according to IL-17 Yes 6 17 0.6 CD8+ T-cell responses to Her-2peptides in vitro. Survival analysis of No 29 3 elderly patients (n = 38) grouped according to CD8+ T-cell responses to Her-2 (CD8 response), CD4+ and CD8+ T-cell responses (CD4 & IL-5 and or IL-17 Yes 12 17 0.1 CD8response) and CD4+ T-cell responses to Her-2 (CD4 response) No 23 0 CD8 TNF Yes 16 13 0.3 patients whose CD4+ T-cells produced IL-5 and/or IL-17 No 19 0 (p = 0.02, Fig. 3b). Here, the survival rate was only 33 % IFN-γ Yes 18 11 0.3 compared to 80 % for patients whose CD4+ T-cells did not produce these cytokines. No 17 0 Analogous observations were also made anecdotally in IL-10 Yes 2 0 0.3 the younger patients, where those whose CD4+ T-cells No 33 6 produced IL-5 had poor survival, with 25 % (2/8) of the IL-2 Yes 2 0 0.8 IL-5-producing patients dying, compared to none whose No 33 6 CD4+ T-cells did not produce IL-5. Results of survival analysis according to Kaplan Meier method and p values from Mantel-Cox (log-rank) test Impact of TNF-producing CD8+ T-cells on survival We previously reported the beneficial effects of having a (p = 0.01, Fig. 3a) in these elderly breast cancer patients. CD8+ T-cell response to Her-2. Here, we investigated the Thus, the survival rate for patients whose CD4+ T-cells function of these Her-2 specific CD8+ T-cells to identify produced IL-5 and/or IL-17 was 29 % compared to 76 % the factors associating with survival benefit in this cohort. for the patients without these cytokines. From the Kaplan- We observed that patients whose CD8+ T-cell produced Meier analysis of different parameters in the elderly we TNF had a significantly better survival than those whose observed the expected impact of metastasis on survival CD8+ T-cells did not produce TNF, when all elderly (Additional file 2: Table S1). Limiting the analysis to non- patients were included in the analysis (p = 0.01, Fig. 4a). metastatic elderly breast cancer patients only, we still Thesurvivalratefor thepatients whoseCD8+ T-cellspro- observed a similar negative impact on survival for the duced TNF was 93 % compared to 52 %, for those with no Table 3 Phenotype of Her-2 responders in older patients Response to Her-2 No. of patients Percentage dead p value CD4 response Yes 33 36 % 0.1 No 4 0 % CD8 response Yes 18 13 % 0.03 No 19 47 % CD4 and CD8 response Yes 14 21 % 0.2 No 23 39 % Results of survival analysis according to Kaplan Meier method and p values from Mantel-Cox (log-rank) test Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 5 of 9 Fig. 3 Survival analysis of elderly breast cancer patients according to IL-5 Fig. 2 Survival analysis of elderly breast cancer patients according to and/or IL-17 production by Her-2-stimulated CD4+ T-cells. a patients IL-5 or IL-17 production by Her-2-stimulated CD4+ T-cells. a Patients grouped according to IL-5 and/or IL-17 production in all patients and b grouped according to IL-5 production; b IL-17 production in non-metastatic patients only TNF-producing CD8+ T-cells. This survival advantage was Discussion still observed when only non-metastatic elderly patients In the present study, we investigated associations of the were considered (p = 0.05, Fig. 4b). In the case of non- presence of circulating Her-2-reactive CD4+ and CD8+ T- metastatic patients, these figures were 92 % and 57 %, cells with survival in older breast cancer patients relative to respectively. No survival associations could be observed in their pro- and anti-inflammatory cytokine production on young. stimulation, and compared the immunocompetence of the elderly, as assessed in this manner, with that of younger patients. Previously, we had reported a survival benefit ac- Impact of IFN-γ producing CD8+ T-cell on survival cruing to patients having a CD8+ T-cell response to Her-2 Here, we grouped the patients according to CD8+ T- [21]. To test the hypothesis that the immune system in the cell production of IFN-γ. Unlike the findings with elderly may be compromised due to aging, here we investi- TNF above, unexpectedly, Kaplan-Meier survival ana- gated differences in T-cell responses to Her-2 in younger lysis indicated no significant differences between IFN- and older breast cancer patients. To the best of our know- producers and non-producers both in young and old ledge, this is the first study to investigate the phenotype (p = 0.3,Fig.4c). and function of Her-2 reactive T-cells, or those reactive to Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 6 of 9 any tumor-associated antigen, in young and old breast can- cer patients. In the present study, stratifying the patients according to those having a CD8+ T-cell response alone (positive impact on survival as previously shown), or both CD4+ and CD8+ T-cell responses, or only a CD4+ T-cell response revealed a trend towards a negative general survival impact for CD4+ T-cell reactivity in older patients. These results clearly con- firmed the importance of having a CD8+ T-cell response to Her-2 but also showed that there was reduced survival when a CD4+ T-cell response to Her-2 was also present in the same patient. This implies that at least in terms of responses to Her-2, CD4+ T-cell reactivity to this TAA is likely to have unanticipated negative effects. CD4+ regula- tory T cells would be the obvious candidates for the cells mediating these negative effects. The lack of CD8+ T-cell responses in patients with poorer survival could also be due to the presence of increased levels of Tregs. This would be consistent with other reportedstudies showingthatTregs could control the T-cell response against certain TAAs in an antigen-specific manner [22, 23]. We were not able to test for classic Treg phenotypes in the current study due to the limitations of the flow cytometric technique, but will focusonthisquestioninfuture studies.We can only say at this juncture that one cytokine commonly implicated in Treg function, IL-10, does not seem to be responsible for these results, whereas two other cytokines not generally implicated in Treg function, namely IL-5 and IL-17, do seem to play important roles. Thus, functional analysis of 6 different cytokines allowed us to group patients according to the cytokines produced by the CD4+ and CD8+ T-cells responding to Her-2. Survival analysis showed that the presence of CD4+ T-cells producing IL-5 and/or IL-17 had a negative impact on survival in the elderly (p = 0.01). Com- pared to other anti-inflammatory cytokines, the role of IL-5 in peripheral cells seems to be under-investigated. Studies have indicated that IL-5 may facilitate lung metastasis, and high expression of IL-5 in the tumor was also negatively associated with survival [24, 25]. A potential role in im- mune surveillance has been proposed [26]. Similarly, there are few data on IL-17 in this context. Although IL-17 is primarily considered a pro-inflammatory cytokine, its role in cancer is still unclear. Somestudies have shownanti- tumor effects of IL-17, so that it has been proposed for use as a cancer immunotherapeutic [27, 28]. However, tumor- Fig. 4 Survival analysis of elderly breast cancer patients according to infiltrating lymphocytes with a high level of IL-17-positivity CD8+ T-cell responses to Her-2 peptides in vitro. a patients grouped correlate with poor prognosis in some studies [29, 24], and according to TNF production in all patients and b in non-metastatic an association of IL-17 with Tregs in the tumor has been patients only; c patients grouped according to IFN-γ production reported [29]. We also need to bear in mind that not only (all patients) Tregs, but also myeloid-derived suppressor cells (MDSCs) may play a role in suppressing T-cell activity. We previously reported the possible suppressive role of MDSCs and Tregs in relation to T-cell responses to Her-2 and also the pos- sible indirect role of these immunosuppressive subsets in Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 7 of 9 breast cancer [21]. MDSCs are also associated with IL-17, present in the circulation is important and has an impact as they are able to mediate IL-17 production by naïve T- on patient survival. Considering the results presented in cells, depending on the cytokines produced by the MDSCs this study, depletion of Her-2-specific IL-5- and IL-17- themselves [30]. Consistent with the present report, our producing CD4+ T-cells and enrichment of TNF- previous study investigating the role of IL-17 in the context producing CD8+ T-cells for adoptive T-cell therapy of CD4+ T-cell responses to TAAs documented a negative would be important in breast cancer. Thus, considering impact ofIL-17-producing T-cells in late-stage melanoma our previous results, it is not only immunosuppressive [20]. There are several other cytokines such as IL-4 and IL- cells like Tregs and MDSCs that might have to be targeted 13 that are known to have a negative impact on survival but it is also important to consider the specific cytokines [31, 32] and which would have been interesting to examine produced by the CD4+ and CD8+ T-cell that are reactive here, especially as we previously found that IL 4 was rele- to, in this case, Her-2. This study may also provide some vant in melanoma. IL-13 is known to regulate cancer inva- mechanistic insights for a better understanding of the im- sion and metastasis in different cancers [31, 32]. In our mune system in elderly and younger breast cancer patients. previous study on melanoma, we observed a negative In any event, the findings that elderly breast cancer patients impact on survival for the patients who produced IL-4 are not immunosenescent according to the assays con- reactive to Melan-A [20]. IL-4 is also known to trigger the ducted in this work augur well for the efficacy of immuno- cancer proliferation mechanism [33]. Not only these but modulatory antibodies even in older patients. also IL-6 and IL-1 have shown a negative impact on survival and tumor progression [34, 35]. However, the Methods number of different variables that could be tested at the Patients same time was limited and we were unable to include The study included 40 elderly and 35 younger patients these other cytokines in the present analysis. with breast cancer (Additional file 3: Figure S1). Blood The role of the pro-inflammatory cytokines IL-2, IFN-γ samples from patients with different TNM stage were and TNF in cancer is usually considered to be well- collected between March and November 2009, at the established. They play an important role in inducing a University Hospital Tuebingen. Blood was drawn prior protective immune response [36] and IL-2 has long been to any treatment or surgery. Standard Ficoll-Hypaque used as an immunotherapeutic drug which can result in a gradient centrifugation was used to isolate the peripheral small minority of very durable responses [37]. In the blood mononuclear cells (PBMCs) before they were present study, one of the few differences that we observed cryopreserved for experimental purposes. The study between younger and older patients was that significantly was approved by the Institutional Ethics Committee of lower proportion of the latter possessed Her-2-reactive University Hospital Tuebingen (71/2009BO2) and a IL-2 producing CD4+ T-cells, possibly contributing to bet- waiver of informed consent was granted for this study. ter survival of the former. However, whether or not an elderly patient possessed IL-2-producing CD4+ T-cells Detection of tumour-associated antigen-reactiveT-cells had no impact on survival. To assess T-cell reactivity to Her-2, a 12-day in vitro culture IFN-γ is known to play an important role in anti-tumor was performed as described previously [21]. First, after activity [38, 39] and has also been considered as an immu- thawing carefully, washing extensively, and assessing viabil- notherapeutic drug to treat cancer patients [40, 41]. In our ity, PBMCs (1x10 )were culturedinX-Vivo15defined study, however, we could discern no significant survival medium (Lonza) supplemented with IL-4 (5 ng/ml: Sandoz, advantage for patients with Her-2-reactive CD8+ T-cells Basel, Switzerland) and IL-7 (5 ng/ml: Sterling-Winthrop, producing IFN-γ, although there was a survival advantage US), on day 0. On day 1, the PBMCs were stimulated with for patients who had Her-2-reactive TNF-producing CD8+ mixtures of Her-2 15-mer overlapping peptides (with an T-cells. TNF is involved in a number of important cellular overlap of 11 amino acids) (PepMix, JPT Technologies, functions including cell proliferation, survival and death Berlin, Germany) at a concentration of 1 μg/ml. The cells and has been used in patients with locally advanced tumors were supplemented with IL-2 (40U/ml: Chiron Behring [42]. In the present study, we observed that patients whose GmbH,Marburg,Germany)on day3.On day12, after CD8+ T-cells produced TNF had a significant survival ad- harvesting and washing, the cultured T-cells were re- vantage over those who did not, indicating the importance stimulated (0.4-0.5 x 10 cells/well) with Her-2 PepMix at of Her-2-reactive TNF-producing CD8+ T-cells for these a concentration of 1 μg/ml or left unstimulated as a nega- elderly breast cancer patients. tive control for 12 hours. Golgi-plug (BD Biosciences) was added at 1 μl/ml to all cultures. Patients’ cells were stimu- Conclusions lated with influenza nucleoprotein (NP) and membrane From our results, we propose that the nature of the anti- protein (M1) peptides as a positive control, as all subjects tumor T-cell response and the TAA targeted by T-cells have been exposed to influenza during their lives and all Bailur et al. Journal for ImmunoTherapy of Cancer (2015) 3:45 Page 8 of 9 possess T cells responsive to these peptides. After harvest- manuscript. All authors read and approved the final version of the manuscript. ing and washing, the cells were incubated with Gamunex (Talecris) to block Fc receptors, and with ethidium mono- Acknowledgements azide (EMA,MoBiTec GmbH, Goettingen, Germany), a This project was supported by the European Commission (FP7 259679 “IDEAL”) marker for dead cells. Intracellular cytokine staining was and the Bundesministerium für Bildung und Forschung (ISPE-BREAST, FKZ 01EI1401). performed after fixing and permeabilizing the cells with Cytofix/Cytoperm (BD Biosciences). Cells were simultan- Author details eously stained with CD3-PO (Invitrogen), CD4-Pacific Blue, Department of Internal Medicine II, Centre for Medical Research, University of Tuebingen, Waldhoernlestr 22, 72072 Tuebingen, Germany. Radiology TNF-FITC, IL-2-Alexa Fluor-700, IL-5-PE (Bio Legend), Clinic, Diagnostic and Interventional Radiology, University Hospital CD8-APC-Cy7, IFN-γ-PE-Cy7 (BD Biosciences), IL- Tuebingen, Tuebingen, Germany. Present Address: BioNTech AG, Mainz, 10-APC (Miltenyi Biotech) and IL-17-PerCP-Cy5.5 Germany. School of Science and Technology, College of Arts and Science, Nottingham Trent University, Nottingham, UK. Present Address: Yale Cancer (eBioscience). 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