Human breast tumors are infiltrated by memory CD4+ T cells along with increased numbers of regulatory T cells (Treg) and plasmacytoid dendritic cells (pDC) that facilitate immune escape and correlate with poor prognosis. Here, we report that inducible costimulatory molecule (ICOS), a T cell costimulatory molecule of the CTLA4/PD1/CD28 family, is expressed mostly by tumor-associated Treg in primary breast tumors. A large proportion of these ICOS+ Treg were Ki67+ and this evident proliferative expansion was found to rely on interactions with tumor-associated pDC. Indeed, tumor-associated Treg highly expanded in presence of pDC but failed to proliferate under CD3/CD28 signal. In vitro experiments revealed that the addition of a neutralizing anti-ICOS antibody blocked pDC-induced Treg expansion and interleukin-10 secretion by memory CD4+ T cells, establishing a pivotal role for ICOS in this process. Supporting these findings, the presence of ICOS+ cells in clinical specimens of breast cancer correlated with a poor prognosis. Together, our results highlight an important relationship between Treg and pDC in breast tumors, and show that ICOS/ICOS-L interaction is a central event in immunosuppression of tumor-associated memory CD4+ T cells. These findings strongly rationalize antibody-mediated ICOS blockade as a powerful clinical strategy to correct immune escape and promote therapeutic responses in breast cancer. Cancer Res; 72(23); 6130–41. ©2012 AACR.

In several cancers, tumor growth and dissemination are associated with perversion of the immune system (1). We and others have shown that primary breast tumor-associated T cells (TA-T), are highly enriched in CD25hiCD127lowFoxP3hiCD4+ regulatory T cells (Treg; ref. 2) and that high infiltration of TA-Treg in breast tumor, and more particularly within lymphoid aggregates surrounding tumor correlates with poor prognosis (2). In the past decade, several studies highlighted the negative impact of TA-Treg on Th1 and cytotoxic T lymphocytes mediated antitumor immunity (3). Therapeutic targeting of Treg is clearly beneficial in mice as shown in multiple models based on anti-CD25 depletion (for review refs. 3, 4) or FoxP3 deletion (3, 5). A similar approach developed in human using interleukin (IL)-2-toxin (Denileukin diftitox; ref. 6) showed low efficacy possibly because of its limited specificity and the existence of toxicity. In the objective to increase T cell antitumor immunity, early clinical trial with the anti-CTLA4 mAb, recently approved in the treatment of melanoma, and anti-PD-1/PD-L1 showed remarkable long-lasting clinical benefit (7, 8). However, contribution of Treg neutralization in the clinical efficacy of anti-CTLA4 mAb remains uncertain (9, 10). Thus, there is an urgent need of alternative therapeutic strategies to selectively neutralize TA-Treg. A first step along this objective is the understanding of the immunosuppressive networks operating in the breast tumor microenvironment. We therefore undertook to characterize the mechanisms controlling TA-Treg enrichment within breast tumor.

We recently published that human TA-Treg recruitment in breast tumor from blood occurred through CCR4/CCL22 axis (2, 11). Breast tumor TA-Treg are strongly activated, proliferate, and express high levels of the inducible costimulatory (ICOS) molecule (2), suggesting the local recognition of a tumor-associated antigen, likely through presentation by a dendritic cell (DC) population. The presence of TA-plasmacytoid DC (pDC) within tumors has been correlated with poor prognosis in breast tumor (12), ovarian carcinoma (13), and melanoma (14). Interestingly, pDC have been reported to strongly favor Treg proliferation in human (15) and rodent models (16). Furthermore, TA-pDC are impaired in their major function of high IFNα secretion levels in response to TLR7/9-L in ovarian (13) and breast cancer (17), and we recently showed that such functional defect strongly favors TA-pDC ability to induce TA-Treg proliferation and increases T-CD4+ IL-10 secretion (17), leading to the establishment of an immunosuppressive Tr1-like response.

Identified in 1999 (18), ICOS, a T cell costimulatory molecule of the CTLA4/PD1/CD28 family, plays a nonoverlapping function with CD28 (19) on CD4+ T cells. In particular, ICOS is critical in the regulation of humoral response (20) through its role on T follicular helper cell activation as illustrated in ICOS KO mice (21) and deficient patients (22). ICOS expression has also been linked with Treg maintenance in mice (23) and mucosal tolerance (24). In human ICOS plays a predominant role in pDC/T-cell interaction (25) and participates in naive CD4+ T cell polarization into IL-10-secreting Tr1-like cells (26). ICOS was also associated with Treg homeostasis in nonobese diabetic (NOD) mice (27) and with increased Treg proliferative capacity and immunosuppressive functions (28). All these observations led us to decipher the contribution of ICOS in the establishment of tolerance through TA-Treg amplification in breast tumor mediated by TA-pDC.

Using a new blocking mAb against ICOS (clone 314.8), we showed that ICOS blockade inhibited pDC-induced TA-Treg proliferation and reduced IL-10 secretion by memory TA-TCD4+ without interfering with mDC-induced TA-TCD4+ activation. Finally, we showed that the presence of ICOS+ cells within primary breast tumor correlated with poor patients' survival. Thus, supported by the clinical efficacy of anti-CTLA4 and anti-PD-1/PD-L1 (7, 8, 29), a neutralizing anti-ICOS mAb would represent a potent therapeutic drug to neutralize Treg in breast cancers.

Tumor single cell suspensions, CD4+ T cells and pDC isolation

Primary breast tumor samples and blood in anticoagulant (CTAD)-coated tubes were obtained from non-pretreated patients diagnosed for primary breast carcinoma provided by the Centre Léon Bérard (CLB) tissue bank after written informed consent in accordance with the Declaration of Helsinki. The study was reviewed and approved by the Institutional Review Board of CLB. Human tonsils were obtained anonymously according to the institutional regulations and healthy donor (HD) human blood was purchased anonymously from the EFS.

Isolation of T cells and DC from tumor and healthy donor tissues

Tumor and tonsil single cell suspensions were generated as previously described (2). Healthy donor or patients' blood mononuclear cells were obtained by Ficoll density gradient. Viable HD-pDC (LinCD4+CD11c) and HD-mDC (LinCD4+CD11c+) were fluorescence-activated cell sorting (FACS) sorted from tonsil samples as described previously (13).

TA-TCD4+ were positively isolated based on their CD4 expression (Life Technologies). HD-TCD4+ were obtained as untouched memory CD4+ T cells by depletion. Treg and Tconv (defined as non-Treg memory T-CD4+) from blood and primary breast tumor were obtained using the CD25+ isolation kit (Life Technologies) or by FACS-sorting on the basis of a DAPI, CD4,CD25,CD127 stainings on memory HD-TCD4+.

Allogeneic DC/T-cell cocultures and anti-CD3/anti-CD28-coated beads T cells activation

pDC and mDC were preactivated for 24 hours in presence of IL-3 (20 ng/mL; Peprotech) ± granulocyte macrophage colony-stimulating factor (GM-CSF; 10 ng/mL; Schering Plough) with R848 (5 μg/mL; Invivogen) in RPMI medium supplemented with antibiotics, L-glutamin (Life Technologies), and 10% fetal calf serum (PAA). Activated DC were washed before coculture with T cells.

Cocultures were conducted at a DC:T cells ratio (1:5) for 5 days with or without exogenous rhIL-2 (Pro-Immune) in presence of IL-3 (20 ng/mL) ± GM-CSF (10 ng/mL) in RPMI containing 5% AB human pooled serum (EFS). In some experiments, TCD4+ were labeled with carboxyfluorescein diacetate succinidyl ester (CFSE; 1 μmol/L) or CellTrace violet (10 μmol/L; Life Technologies) before coculture. In blocking experiment mAbs anti-ICOS (clone 314.8), anti-CD28 (clone CD28.2), or isotype control were used at a concentration of 10 μg/mL.

Patient cohort

For the retrospective immunohistochemistry (IHC) study, tumors from 120 patients with invasive nonmetastatic breast tumors whose clinical and biologic data were available from the regularly updated CLB database were analyzed. Patients' characteristics are presented in Supplementary Table S1 (column 1). The median follow-up was 11 years [95% confidence interval (CI) 10.6–11.1]. Majority of the patients (96%) were treated with postoperative radiotherapy. Note that 80% patients received adjuvant hormonotherapy [tamoxifen, (91.7%), antiaromatase (2.1%) or combination (6.3%)]. Adjuvant chemotherapy, consisting in anthracyclins (69.4%), anthracyclin+Taxan (26.4%), or other treatments (4.2%), was administered to 59.2% patients (71/120).

IHC

Frozen breast tumor sections (5 μm) were subjected to sequential double IHC analyses with mAbs against BDCA-2 (10 μg/mL), FoxP3 (10 μg/mL), and cytokeratin (1/50) using ImmPRESS Anti-Mouse Ig peroxidase kit (Abcys), as previously described (2).

After antigenic retrieval by CC1 buffer pH8 (Ventana), breast tumor paraffin-embedded tissue microarray (TMA) were incubated for 30 minutes with Anti-ICOS mAb (1:50, Spring Biosciences), then revealed using Ultra View Kit and Hematoxylin-counterstained (Ventana). Each breast tumor sample was analyzed independently by 2 pathologists according to the guidelines for HER2/neu amplification, ER/PgR positivity, Scarff-Bloom-Richardson (SBR) grade, and TNM grade. ICOS+ cells were enumerated on 6 different spots on each tumor sample to obtain ICOS+ cell count (average). Cohort was equally separated in 2 groups according to the median of their ICOS+ cells number [ICOSlow/neg (<1.7 cells/spot) and ICOS+ (≥1.7 cells/spot)] to have sufficient number of patients in each group for statistical analyses.

Flow cytometry and cytokine quantification

All flow cytometry acquisitions were done using Cyan ADP cytometer (Beckman Coulter) and Summit 4.3 Software and analyses were conducted on FlowJo 7 Software. FACS-cell sorting had been conducted with the Aria III cell-sorter (Becton Dickinson) and DIVA Software. See Supplementary Table S2 for antibody list.

Cytokines (IL-10, IFNγ, and IL-2) secretions were quantified in supernatants by specific ELISA according to manufacturers' instructions (Bender MedSystems).

Statistical analysis

All statistical analyses were done using the Statistical SAS V9.2 package (Cay) 12.0 software. Correlations between clinico-biologic data and ICOS+ cell content in the lymphoid infiltrates were determined using a χ2 test or a Fisher exact test. Survival curves were plotted using the Kaplan–Meier method and compared using the Log-rank test.

CD4+FoxP3hi TA-Treg is the major T-cell subset in breast tumor expressing ICOS and proliferating in situ

Within breast tumor, a large majority of TA-Treg (defined as CD25hiCD127) expressed high ICOS levels (69.9%) compared with TA-Tconv (23.4%) or TA-TCD8+ (2%; Fig. 1A). As TA-TCD4+ are of memory phenotype (2), all the present study has been conducted on CD45RO+ T cells (TCD4+) isolated from breast tumor or healthy donors. Only weak or no ICOS expression was observed on healthy donor and patients' blood Treg (21.3% and 16.6%, respectively), Tconv (3.9% and 1.5%, respectively), or T-CD8+ (0.3% and no expression, respectively; Fig. 1A), suggesting that high expression of ICOS on TA-Treg is dependent on tumor environment. Statistical analysis on 11 breast tumor samples showed a higher percentage of ICOS+ cells and a higher mean fluorescence intensity (MFI) of ICOS expression on TA-Treg compared with TA-Tconv (%: P < 0.001 and MFI: P < 0.01). The percentage of ICOS+ cells was lower in TA-TCD8+ than in TA-Tconv (P < 0.05; Fig. 1B). To evaluate a potential association of ICOS with TA-Treg proliferation, we analyzed the coexpression of ICOS and Ki67 on TA-Tconv and TA-Treg but also on patients' and healthy donor blood. Within TA-TCD4+ containing 11.1% of FoxP3hi TA-Treg, 27.6% TA-Treg expressed Ki67 and 80% of them were ICOS+ (22.1% of ICOS+Ki67+ cells) whereas only 6.8% TA-Tconv were found Ki67+ containing 57% of ICOS+ cells (3.9% of ICOS+Ki67+ cells; Fig. 1C). Only 5.7% Ki67+ cells that weakly or did not express ICOS and FoxP3 were detected in TCD8+ compartment (Supplementary Fig. S1A). Interestingly, the percentage of Ki67+ cells was higher (P < 0.001) among FoxP3hi TA-Treg than other TA-TCD4+ or TA-TCD8+ on 11 different breast tumor samples (Fig. 1D). Thus, ICOS is selectively expressed on FoxP3hi Treg in tumor and is associated with their proliferation. The very low expression of Ki67 observed on TA-Tconv suggests that most of them are in an anergic state consistent with their low expression of activation marker and probably linked to in situ Treg-mediated immunosuppression.

Figure 1.

TA-Treg but neither TA-Tconv nor Treg and Tconv from blood strongly coexpress ICOS and Ki67. A, ICOS expression was analyzed on viable Treg (CD3+CD4+CD8CD25hiCD127low), Tconv (CD3+CD4+CD8CD25low/neg), and TCD8+ (CD3+CD4CD8+) in healthy donor blood, patients' blood, and breast tumor single cells suspensions, 1 representative experiment out of 5. B, dot plots represent percentage of ICOS+ cells among TA-Treg, TA-Tconv, or TA-TCD8+ (top) and ICOS MFI of the total population (bottom). C, Ki67 and ICOS expression were analyzed together by multicolor flow cytometry on breast tumor single cell suspensions. T-cell subpopulations were identified as in B. B and D, each symbol represents an independent donor (—, median). Statistical analysis was conducted using a Mann-Whitney U test. **, P < 0.01; *, P < 0.05.

Figure 1.

TA-Treg but neither TA-Tconv nor Treg and Tconv from blood strongly coexpress ICOS and Ki67. A, ICOS expression was analyzed on viable Treg (CD3+CD4+CD8CD25hiCD127low), Tconv (CD3+CD4+CD8CD25low/neg), and TCD8+ (CD3+CD4CD8+) in healthy donor blood, patients' blood, and breast tumor single cells suspensions, 1 representative experiment out of 5. B, dot plots represent percentage of ICOS+ cells among TA-Treg, TA-Tconv, or TA-TCD8+ (top) and ICOS MFI of the total population (bottom). C, Ki67 and ICOS expression were analyzed together by multicolor flow cytometry on breast tumor single cell suspensions. T-cell subpopulations were identified as in B. B and D, each symbol represents an independent donor (—, median). Statistical analysis was conducted using a Mann-Whitney U test. **, P < 0.01; *, P < 0.05.

Close modal

TA-pDC interacting with TA-Treg in situ in breast tumor induce a strong TA-Treg enrichment among TA-TCD4+ex vivo

To decipher the mechanisms controlling the strong proliferation of TA-Treg in vivo, we first compared the proliferative capacity of purified Treg and Tconv from healthy donor blood and breast tumor patients in response to costimulation using anti-CD3/anti-CD28-coated beads in the presence of high doses of rhIL-2 (Fig. 2A). Contrasting to the in vivo observations, TA-Tconv proliferated as well as healthy donor blood Tconv or Treg, whereas isolated TA-Treg did not proliferate, suggesting that other signals from the breast tumor microenvironment are involved in TA-Treg in vivo proliferation.

Figure 2.

TA-Treg are amplified in response to pDC but not under CD3/CD28 activation ex vivo. A, FACS sorted CD4+CD25hiCD127 Treg and CD4+CD25CD127+ Tconv from healthy donor blood or breast tumor samples were cultured with anti-CD3/anti-CD28-coated beads in presence of rhIL-2 (500 IU/mL). Numbers and viability of cells were quantified every 3 days. Results are representative of 3 independent healthy donor blood and breast tumor samples. B, TA-pDC stained in brown (anti-BDCA2 mAb) are localized in lymphoid infiltrate in breast tumor and not in tumor mass (anticytokeratin mAb) revealed in green (left). BDCA2+ cells (green) are in close contact with FoxP3+ Treg (brown) in breast tumor (right). IHC stainings were done on breast tumor frozen sections and observed with a 10- or 20-fold magnification (and ×40 fold in insets). C, purified and CFSE-labeled TA-TCD4+ encompassing 7.5% of FoxP3hi TA-Treg after purification were cocultured 5 days with R848-preactivated HD-pDC or anti-CD3/anti-CD28-coated beads without exogenous rhIL-2. FoxP3 detection and CFSE dilution were assessed on CD3+ viable cells by flow cytometry. Results are representative of 3 patient samples.

Figure 2.

TA-Treg are amplified in response to pDC but not under CD3/CD28 activation ex vivo. A, FACS sorted CD4+CD25hiCD127 Treg and CD4+CD25CD127+ Tconv from healthy donor blood or breast tumor samples were cultured with anti-CD3/anti-CD28-coated beads in presence of rhIL-2 (500 IU/mL). Numbers and viability of cells were quantified every 3 days. Results are representative of 3 independent healthy donor blood and breast tumor samples. B, TA-pDC stained in brown (anti-BDCA2 mAb) are localized in lymphoid infiltrate in breast tumor and not in tumor mass (anticytokeratin mAb) revealed in green (left). BDCA2+ cells (green) are in close contact with FoxP3+ Treg (brown) in breast tumor (right). IHC stainings were done on breast tumor frozen sections and observed with a 10- or 20-fold magnification (and ×40 fold in insets). C, purified and CFSE-labeled TA-TCD4+ encompassing 7.5% of FoxP3hi TA-Treg after purification were cocultured 5 days with R848-preactivated HD-pDC or anti-CD3/anti-CD28-coated beads without exogenous rhIL-2. FoxP3 detection and CFSE dilution were assessed on CD3+ viable cells by flow cytometry. Results are representative of 3 patient samples.

Close modal

Through IHC stainings conducted on breast tumor frozen sections, we observed FoxP3+ TA-Treg localized in close contact with BDCA2+ TA-pDC within lymphoid aggregates but not in cytokeratin+ tumor area (Fig. 2B), in agreement with our previous work demonstrating only scarce Treg within tumor area (2). We thus investigated the impact of pDC on TA-Treg proliferation among isolated TA-TCD4+, in absence of exogenous cytokines as TA-Tconv can secrete IL-2 favoring TA-Treg proliferation. Whereas anti-CD3/anti-CD28 stimulation was unable to favor TA-Treg maintenance and proliferation (only 1% FoxP3hi cells after CD3/CD28 activation although starting TA-TCD4+ population contained 7.5% FoxP3hi cells; Fig. 2C), 10% FoxP3hi TA-Treg were detected among TA-TCD4+ cultured with allogeneic TLR7-L-activated pDC. These results showed that activated pDC favored TA-Treg enrichment and maintenance among TA-TCD4+. Importantly, CFSE staining of TA-TCD4+ showed a higher proliferation of FoxP3hi and FoxP3int cells in pDC/TA-TCD4+ coculture than under anti-CD3/anti-CD28 stimulation (Fig. 2C). In contrast, a reduced TA-Tconv proliferation was observed with pDC compared with anti-CD3/anti-CD28 stimulation.

FoxP3hi Treg amplified in HD-pDC/HD-TCD4+ coculture do not produce cytokines as shown by IFNγ, and IL-10 staining on PMA/ionomycine reactivated HD-TCD4+ after 5 days of coculture (Supplementary Fig. S2A). Furthermore, 10 days culture of FACS-sorted HD-Treg with pDC gave increase to immunosuppressive cells reducing by 40% proliferation of allogeneic HD-TCD4+ at the ratio 1:1 (Supplementary Fig. S2B) and a strongly reduced IL-2 concentration in culture supernatants, directly dependent on the Treg proportion (Supplementary Fig. S2C). Altogether, these results are consistent with the ability of pDC to sustain Treg immunosuppressive functions.

Collectively these data showed that in contrast to CD3/CD28 costimulation, TLR7-L-activated HD-pDC induced and maintained TA-Treg enrichment among TA-TCD4+. Such enrichment resulted at least in part from a higher proliferation of FoxP3hi TA-Treg, rising the hypothesis that another costimulatory signal different from CD28 required for TA-Treg expansion is delivered during pDC/TA-TCD4+ interaction.

TA-pDC and TA-Treg are found in close vicinity in tumor mass consistent with ICOS/ICOS-L interaction

Activated pDC are known to express high ICOS-L levels (25, 26) and we previously described a partly activated status of TA-pDC within breast tumor environment (17). Consistently, TA-pDC expressed higher CD80 and CD40 levels than blood pDC and a similar upregulation was observed on TA-mDC. Contrasting to such activated phenotype, ICOS-L was undetectable on freshly isolated TA-pDC whereas healthy donor or patients' blood pDC expressed ICOS-L upon isolation and upregulated levels upon activation (Fig. 3A and Supplementary Fig. S3A). ICOS-L may be downregulated on TA-pDC membrane after engagement with ICOS highly expressed by TA-Treg. In line with this, ICOS-L expression on TLR7-L activated HD-pDC was prevented in the presence of high numbers of allogeneic-activated blood HD-TCD4+ and this blockade was reverted in presence of a neutralizing anti-ICOS mAb (Fig. 3B). To confirm the relevance in breast tumor, ICOS-L expression was analyzed, on TA-pDC and TA-mDC, after 48 hours culture of whole tumor cell suspension in IL-3 favoring TA-pDC survival and the impact of blocking ICOS was assessed. Interestingly, TA-pDC highly expressed CD86 in all culture conditions (Supplementary Fig. S3B) but ICOS-L was detectable at high level on TA-pDC only after culture with neutralizing anti-ICOS mAb and not with anti-CD28 mAb (Fig. 3C). As reported previously (26), high expression of ICOS-L was restricted to HD-pDC as ICOS-L was only marginally detected on mDC from healthy donor blood or breast tumor (Fig. 3A), even in the presence of TLR7-L (Supplementary Fig. S3A) or anti-ICOS mAb (Fig. 3C) although they strongly expressed CD86 (Supplementary Fig. S3B).

Figure 3.

ICOS-L engagement by ICOS during TA-pDC and TA-Treg interaction leads to ICOS-L downregulation on pDC membrane. A, breast tumor single cell suspension or PBMC from healthy donor blood were stained with Lin, CD4, CD11c, and CD123 mAbs to identify TA-pDC (LinCD4+CD11cCD123hi) and TA-mDC (LinCD4+CD11chiCD123neg). ICOS-L, CD40, HLA-DR, or appropriate controls were used to assess expression of activation markers. Results are representative of 4 independent healthy donor blood and tumor samples. B, freshly isolated and R848+IL-3-preactivated HD-pDC were cultured for 48 hours with allogeneic HD-TCD4+ from blood at different pDC:T cells ratio in the presence of control or anti-ICOS mAb (314.8) and analyzed for their expression of ICOS-L by flow cytometry. C, After 48-hour culture period of breast tumor single cell suspensions in presence of IL-3 (20 ng/mL) and anti-ICOS 314.8, anti-hCD28 (CD28.2) mAb, or isotype control, cells were then stained for ICOS-L to assess expression on LinCD4+CD11cnegCD123hi BDCA2+ TA-pDC or LinCD4+CD11chiCD123neg TA-mDC. Ctrl, control.

Figure 3.

ICOS-L engagement by ICOS during TA-pDC and TA-Treg interaction leads to ICOS-L downregulation on pDC membrane. A, breast tumor single cell suspension or PBMC from healthy donor blood were stained with Lin, CD4, CD11c, and CD123 mAbs to identify TA-pDC (LinCD4+CD11cCD123hi) and TA-mDC (LinCD4+CD11chiCD123neg). ICOS-L, CD40, HLA-DR, or appropriate controls were used to assess expression of activation markers. Results are representative of 4 independent healthy donor blood and tumor samples. B, freshly isolated and R848+IL-3-preactivated HD-pDC were cultured for 48 hours with allogeneic HD-TCD4+ from blood at different pDC:T cells ratio in the presence of control or anti-ICOS mAb (314.8) and analyzed for their expression of ICOS-L by flow cytometry. C, After 48-hour culture period of breast tumor single cell suspensions in presence of IL-3 (20 ng/mL) and anti-ICOS 314.8, anti-hCD28 (CD28.2) mAb, or isotype control, cells were then stained for ICOS-L to assess expression on LinCD4+CD11cnegCD123hi BDCA2+ TA-pDC or LinCD4+CD11chiCD123neg TA-mDC. Ctrl, control.

Close modal

Collectively, these observations strongly suggest that ICOS/ICOS-L is involved in TA-Treg and TA-pDC interaction in breast tumor leading to the downregulation of ICOS-L expression on TA-pDC.

pDC are strong inducers of Treg enrichment among CD4+ T cells through ICOS/ICOS-L costimulation

We analyzed the impact of ICOS blockade in HD-TCD4+ allogeneic reactions either with HD-pDC or HD-mDC. HD-Treg enrichment occurred with IL3+TLR7-L-preactivated HD-pDC (13.8 ± 2.4% of FoxP3hi HD-TCD4+, compared with 4.5 ± 0.25% at day 5 with HD-mDC; Fig. 4A and B). In HD-TCD4+/HD-pDC cocultures in absence of exogenous rhIL-2, ICOS inhibition reduced by 42% FoxP3hi Treg proliferation induced by HD-pDC (division index = 2.29 ± 0.21 and 1.33 ± 0.24, respectively, in presence of control and anti-ICOS mAb) and did not impact HD-Tconv proliferation. HD-Treg and HD-Tconv proliferation induced by mDC was also not affected by anti-ICOS mAb (Supplementary Fig. S4A and S4B). To assess the impact of ICOS/ICOS-L neutralization on isolated Treg proliferation, CFSE labeled HD-Treg or HD-Tconv purified from blood were cultured with TLR7-L+IL-3-preactivated HD-pDC purified from tonsil with exogenous rhIL-2 and neutralizing anti-ICOS mAb. HD-pDC induced proliferation of allogeneic FoxP3hi cells (23.3% of diluted CFSE FoxP3hi cells in presence of control mAb) that is almost completely blocked by anti-ICOS mAb (2.8%). In contrast, the anti-ICOS mAb decreased only moderately the proportion of proliferating HD-Tconv (78.1% and 52.2% in control and anti-ICOS mAb, respectively; Fig. 4C). Interestingly, ICOS neutralization in HD-Treg/HD-pDC coculture decreased the proportion of FoxP3hi Treg from 19 ± 2.4% to 3.3 ± 1% (6-fold, Fig. 4D) although the proliferation of HD-Tconv was only moderately affected.

Figure 4.

HD-pDC induce a strong Treg enrichment among HD-TCD4+ compared with mDC under the dependence of ICOS signaling. A and B, overnight activated (IL-3+GM-CSF+R848) FACS-sorted tonsil HD-pDC and HD-mDC were cocultured for 5 days with purified healthy donor CD4+CD45RO+ T cells (HD-TCD4+; containing 1.3% of FoxP3+ Treg at day 0). C and D, CFSE-labeled CD25hi HD-Treg and CD25neg HD-Tconv from blood were cocultured with R848+IL-3-preactivated HD-pDC in presence of rhIL-2 (100 IU/mL) with anti-ICOS or control mAbs. At day 5, CFSE dilution and FoxP3 expression were analyzed by flow cytometry after gating on CD3+ viable cells. B and D, histograms represent the frequency of FoxP3hi Treg among viable HD-TCD3+ of 1 representative experiment from 4 blood samples.

Figure 4.

HD-pDC induce a strong Treg enrichment among HD-TCD4+ compared with mDC under the dependence of ICOS signaling. A and B, overnight activated (IL-3+GM-CSF+R848) FACS-sorted tonsil HD-pDC and HD-mDC were cocultured for 5 days with purified healthy donor CD4+CD45RO+ T cells (HD-TCD4+; containing 1.3% of FoxP3+ Treg at day 0). C and D, CFSE-labeled CD25hi HD-Treg and CD25neg HD-Tconv from blood were cocultured with R848+IL-3-preactivated HD-pDC in presence of rhIL-2 (100 IU/mL) with anti-ICOS or control mAbs. At day 5, CFSE dilution and FoxP3 expression were analyzed by flow cytometry after gating on CD3+ viable cells. B and D, histograms represent the frequency of FoxP3hi Treg among viable HD-TCD3+ of 1 representative experiment from 4 blood samples.

Close modal

The efficacy of anti-ICOS mAb to reduce Treg proportion was evaluated on TA-TCD4+, even in the presence of exogenous rhIL-2. In accordance with results on HD-TCD4+, the FoxP3hi subpopulation induced by HD-pDC decreased in the presence of anti-ICOS mAb (13.5 ± 2% vs. 4 ± 1% in control and anti-ICOS mAb, respectively; Fig. 5A and B). In reverse experiments, purified IL-3±TLR7-L preactivated TA-pDC favored a strong Treg enrichment among HD-TCD4+ (containing initially 1.7% of FoxP3hi Treg) at day 5 (7.1- and 6.4-fold increase with IL-3 and IL-3+TLR7-L-preactivated TA-pDC, respectively). As expected, such Treg enrichment was strongly reduced by ICOS neutralization (57%–83% reduction in FoxP3hi cells compared with control condition; Fig. 5C and D). Taken together, these results showed the critical role of ICOS/ICOS-L interaction in TA-pDC-mediated TA-Treg amplification in breast tumor.

Figure 5.

ICOS is predominant in Treg enrichment during pDC/CD4+ T-cell coculture with cells from breast tumor origin. A and B, R848+IL-3-preactivated HD-pDC were cocultured with purified TA-TCD4+ cells (encompassing initially 11% FoxP3hi TA-Treg). C and D, healthy donor CD4+CD45RO+ T cells (HD-TCD4+) containing 1.7% FoxP3hi Treg after isolation were cocultured with IL-3±R848 preactivated TA-pDC. In A and B, cocultures were conducted during 5 days in presence of rhIL-2 (100 IU/mL) and anti-ICOS or control mAbs. Foxp3 expression was analyzed by flow cytomery on viable CD3+ cells. B and D, histograms represent 1 out of 3 experiments for B and 2 experiments for D carried out in triplicate, error bars represent standard deviation.

Figure 5.

ICOS is predominant in Treg enrichment during pDC/CD4+ T-cell coculture with cells from breast tumor origin. A and B, R848+IL-3-preactivated HD-pDC were cocultured with purified TA-TCD4+ cells (encompassing initially 11% FoxP3hi TA-Treg). C and D, healthy donor CD4+CD45RO+ T cells (HD-TCD4+) containing 1.7% FoxP3hi Treg after isolation were cocultured with IL-3±R848 preactivated TA-pDC. In A and B, cocultures were conducted during 5 days in presence of rhIL-2 (100 IU/mL) and anti-ICOS or control mAbs. Foxp3 expression was analyzed by flow cytomery on viable CD3+ cells. B and D, histograms represent 1 out of 3 experiments for B and 2 experiments for D carried out in triplicate, error bars represent standard deviation.

Close modal

ICOS neutralization inhibits pDC-induced IL-10 secretion by T-CD4+

We evaluated IL-10 and IFNγ production by HD-Tconv and HD-Treg cocultured with preactivated pDC. In HD-pDC/HD-Tconv coculture with exogenous rhIL-2, ICOS blockade strongly inhibited IL-10 (by 94%) and in a smaller proportion, IFNγ secretion (by 33%; Fig. 6A) whereas no IL-10 and IFNγ were detectable in cocultures with HD-Treg (Fig. 6A). ICOS blockade strongly reduced IL-10 production in TA-TCD4+/HD-pDC (90% inhibition; Fig. 6B, left panel) as well as in HD-TCD4+/TA-pDC (83%–72% inhibition) cocultures (Fig. 6B, right panel). IFNγ secretion was also significantly, but to a lesser extent, inhibited upon ICOS neutralization in all culture conditions (59%–63%). Consistent with the strong IL-10 secretion induced by pDC, we detected high IL-10 levels in 48-hour-culture supernatants of breast tumor single cell suspensions (Supplementary Fig. S5) in 7/13 tumors tested [median = 646.8 pg/mL, range (3.2–6915) pg/ml] while IL-2, IL-17, and IFNγ were never observed.

Figure 6.

pDC but not mDC enhance IL-10 secretion and IL-2 consumption by memory TCD4+ although ICOS engagement. A, IL-10 and IFNγ were quantified by ELISA in 5 days coculture supernatants of IL-3+R848-preactivated HD-pDC with FACS-sorted memory HD-Treg or HD-Tconv in presence of IL-2 (100 IU/mL), anti-ICOS, or control mAbs; 1 representative out of 3 independent experiments. B, IL-10 and IFNγ were measured by ELISA in 5 days coculture supernatants of purified TA-TCD4+ cells with IL-3+R848-preactivated-HD-pDC (n = 3; left) or purified HD-TCD4+ with IL-3±R848-preactivated TA-pDC in presence of rhIL-2 (100 IU/mL) and anti-ICOS or control mAbs (n = 2; right). C, HD-pDC and HD-mDC were preactivated as in Fig. 4A and cocultured with HD-TCD4+. When pDC and mDC were mixed at a ratio 1:1, the same number of total DC was cocultured with T cells in presence of anti-ICOS or control mAbs, ±rhIL-2 (100 IU/mL). After 5 days, supernatants were harvested and were quantified for IL-10 and IFNγ by ELISA (n = 4 independent experiments). D, IL-2 secretion was measured by ELISA in supernatant of HD-TCD4+/HD-pDC cocultures and anti-ICOS or control mAbs. Histograms represent the results of 6 independent experiments. IgG, immunoglobulin G.

Figure 6.

pDC but not mDC enhance IL-10 secretion and IL-2 consumption by memory TCD4+ although ICOS engagement. A, IL-10 and IFNγ were quantified by ELISA in 5 days coculture supernatants of IL-3+R848-preactivated HD-pDC with FACS-sorted memory HD-Treg or HD-Tconv in presence of IL-2 (100 IU/mL), anti-ICOS, or control mAbs; 1 representative out of 3 independent experiments. B, IL-10 and IFNγ were measured by ELISA in 5 days coculture supernatants of purified TA-TCD4+ cells with IL-3+R848-preactivated-HD-pDC (n = 3; left) or purified HD-TCD4+ with IL-3±R848-preactivated TA-pDC in presence of rhIL-2 (100 IU/mL) and anti-ICOS or control mAbs (n = 2; right). C, HD-pDC and HD-mDC were preactivated as in Fig. 4A and cocultured with HD-TCD4+. When pDC and mDC were mixed at a ratio 1:1, the same number of total DC was cocultured with T cells in presence of anti-ICOS or control mAbs, ±rhIL-2 (100 IU/mL). After 5 days, supernatants were harvested and were quantified for IL-10 and IFNγ by ELISA (n = 4 independent experiments). D, IL-2 secretion was measured by ELISA in supernatant of HD-TCD4+/HD-pDC cocultures and anti-ICOS or control mAbs. Histograms represent the results of 6 independent experiments. IgG, immunoglobulin G.

Close modal

Finally, we investigated the impact of ICOS blockade on mDC, pDC, or mDC+pDC/T cell cocultures. In presence of exogenous rhIL-2 HD-TCD4+ cocultured with HD-pDC produced more IL-10 but less IFNγ than with HD-mDC (respectively, 596 ± 103 pg/mL and 181 ± 28 pg/mL for IL-10 and 279 ± 52 pg/mL and 641 ± 9 pg/mL for IFNγ; Fig. 6C). Culture of HD-TCD4+ with mixed DC subsets resulted in lower IL-10 (232 ± 9 pg/mL) but higher IFNγ secretion (574 ± 80 pg/mL) compared with [pDC+TCD4+] cocultures. ICOS neutralization strongly reduced IL-10 levels (82% for pDC, 50% for mDC, and 62% for pDC+mDC) without significantly interfering with IFNγ secretion (+28% for pDC, −19% for mDC, and −24% for pDC+mDC) contrasting with observations done with cells from breast tumor origin (Fig. 6C). In absence of exogenous rhIL-2, except the lower levels of IL-10 and IFNγ, similar effects of ICOS neutralization were observed. Furthermore, ICOS blockade increased IL-2 concentration in coculture with HD-pDC (P = 0.035, Fig. 6D).

Overall these results showed that ICOS blockade leads to a strong inhibition of pDC-induced IL-10 secretion by HD-TCD4+ that is accompanied by increased levels of IL-2, without significantly affecting IFNγ production, in particular, in response to mDC.

Detection of ICOS+ cells within primary breast tumor correlates with poor prognosis

IHC analyses were conducted with anti-ICOS mAb on paraffin-embedded tumor section using TMA cores specific for tumor area or lymphoid infiltrates. High numbers (the median defined as cutoff = 1.7 cells/spot) of ICOS+ cells were detectable within lymphoid aggregates but not within tumor bed (Fig. 7A–C). The presence of ICOS+ cells within lymphoid infiltrates (≥median ICOS+ cells/spot) was significantly correlated with high SBR grade (P = 0.001), luminal A/B molecular subtypes (P < 0.001), absence of ER expression (P = 0.025) and Her2/neu overexpression (P = 0.017) by tumor cells, triple negative status (P = 0.02), and lymphatic emboles (P < 0.001; Supplementary Table S1). Importantly, in univariate analysis the presence of ICOS+ cells in lymphoid-enriched areas was associated with an increased risk of relapse (progression-free survival, Log-rank P value = 0.0285; Fig. 7D) and death (overall survival, Log-rank P value = 0.0465; Fig. 7E). However, when introduced in a multivariate analysis together with other significant clinical and biologic parameters, high ICOS expression was no more significant.

Figure 7.

High number of ICOS+ cells within primary breast tumor correlates with reduced patients' survival. A, 120 paraffin-embedded primary breast tumor samples with more than 10 years clinical follow-up were stained with anti-ICOS mAb and counterstained with hematoxylin. The number of positive cells was manually enumerated on 6 different cores for each tumor sample. Representative pictures of tumors noninfiltrated (A), weakly infiltrated (B), and highly infiltrated (C) by ICOS+ cells are shown (magnification, ×10). C, log-rank analysis of progression-free (PFS) and overall (OS) survival of patients from the cohort according to the presence of ICOS+ cells (the median was chosen as cutoff ≥1.7 positive cells).

Figure 7.

High number of ICOS+ cells within primary breast tumor correlates with reduced patients' survival. A, 120 paraffin-embedded primary breast tumor samples with more than 10 years clinical follow-up were stained with anti-ICOS mAb and counterstained with hematoxylin. The number of positive cells was manually enumerated on 6 different cores for each tumor sample. Representative pictures of tumors noninfiltrated (A), weakly infiltrated (B), and highly infiltrated (C) by ICOS+ cells are shown (magnification, ×10). C, log-rank analysis of progression-free (PFS) and overall (OS) survival of patients from the cohort according to the presence of ICOS+ cells (the median was chosen as cutoff ≥1.7 positive cells).

Close modal

In addition to our previous reports demonstrating that TA-Treg frequency within tumor lymphoid infiltrates correlates with poor prognosis in primary breast tumor (2, 30), we identify herein ICOS engagement as a major pathway contributing to their local accumulation through direct interaction with TA-pDC.

We confirm that TA-Treg within primary breast tumor express high ICOS and Ki67 levels in a specific manner compared with other TA-TCD3+ subsets or blood Treg. Interestingly, TA-Tconv and TA-TCD8+ contain a low minority of Ki67+ cells consistent with observations that Treg suppress Th1 and CD8+ T-cell cytotoxic activity in the context of human tumors, such as colon carcinoma (31), melanoma (9), and ovarian carcinoma (32). Early clinical trial with mAbs neutralizing CTLA4 or PD-1/PDL1 inhibitory receptors are showing promising clinical activity in melanoma, renal, and lung carcinoma (20%–30% of objective responses; refs. 7–9, 33).

Herein we report the major role of ICOS, a member of the CTLA4/PD1/CD28 family, in TA-Treg proliferation and accumulation in breast tumor. First, we accumulate evidences that strongly support the hypothesis of ICOS+ TA-Treg and TA-pDC interaction in situ interaction by IHC on primary breast tumor frozen sections and ICOS/ICOS-L engagement in breast tumor as illustrated by reversion of ICOS-L downregulation on TA-pDC upon ex vivo culture of whole tumor cell suspension in presence of blocking anti-ICOS mAb. Second, we also show that activated-pDC expressing ICOS-L, but not mDC or CD3/CD28 costimulation, favor allogeneic FoxP3hi TA-Treg enrichment among TA-TCD4+ that is abolished by ICOS blockade in agreement with data recently published in ovarian carcinoma (34). In agreement with this, thymic ICOS+ Treg need ICOS/ICOS-L costimulation to proliferate (15). Nevertheless, others molecules present in tumor environment such as IDO, OX40-L (35), TNFα (36), or TGFβ (37) could also participate to TA-Treg enrichment within breast tumor.

TA-pDC-induced FoxP3hi Treg amplification explains the positive correlation observed between TA-pDC and TA-Treg in breast tumor (Supplementary Fig. S6A) as well as their negative impact on patients' survival in breast tumor (Supplementary Fig. S6B; ref. 17) and ovarian carcinoma (13, 38). Furthermore, in contrast to recent works in melanoma (39) and glioblastoma (40), ICOS+ TA-Treg expansion in breast tumor is not mediated through direct interaction with tumor cells as (i) ICOS-L is not expressed either on primary breast tumor cells or on breast tumor cell lines (not shown) and (ii) Ki67+ proliferating Treg are only detected within lymphoid aggregates and not within tumor nests (2).

To date TA-Treg origin is not fully elucidated. pDC are known to favor immunosuppressive T-cell induction on both TCD4+ (15, 41) and TCD8+ (42) subsets and we cannot formally exclude the differentiation of Treg from naive TCD4+, as previously reported for both human and mouse pDC (41, 43, 44). However, we recently showed (i) a specific recruitment of CCR4+ Treg from the blood to breast tumor through the tumor cells CCL22 production (2, 11), and (ii) that all of TA-TCD4+ are of memory phenotype, consistent with a recruitment of TA-Treg from the periphery followed by their local expansion through ICOS/ICOS-L interaction with pDC in breast tumor environment.

In accordance with results in NOD type 1 diabetes murine model (27), ICOS+ TA-Treg display high immunosuppressive function (2) through not yet characterized mechanisms that may involve FASL, CD39/Adenosine, perforin, CTLA4, or PD-1 known to participate in Treg immunosuppressive functions (for review 4).

High IL-10 levels are detected in breast tumor environment and TA-TCD4+ secrete large amounts of IL-10 in coculture with pDC but not with mDC. ICOS costimulation of naive CD4+ T cells is already known to induce the differentiation of IL-10-secreting cells (26). Of importance, our results show that pDC preferentially activate preexisting IL-10 secreting cells among memory HD-Tconv. Furthermore, mDC-induced T-cell responses are not affected by ICOS blockade as they do not overexpress ICOS-L after activation. Interestingly, we note that ICOS blockade leads to IL-2 accumulation in cocultures with pDC suggesting that ICOS engagement favors the enrichment of TCD4+ subpopulation that do not secrete and/or consume high amounts of IL-2 in culture. This correlates with previous results (45) showing that T-cell responses under ICOS stimulation depend on exogenous rhIL-2.

Of most importance, IHC staining on 120 primary breast tumors with more than 10 years clinical follow-up allows to show that presence of high numbers of ICOS+ cells infiltrating primary breast tumors is associated with poor prognosis in univariate analysis but is no more significant when introduced in the multivariate analysis together with other significant clinical and biologic parameters.

Collectively our results suggest that abrogation of ICOS/ICOS-L interaction using neutralizing anti-ICOS mAb may reduce Treg expansion, IL-10 secretion, and IL-2 consumption by TA-TCD4+ that can favor antitumor immunity through ICOS-independent TCD8+ and TCD4+ activation.

This role of ICOS in TA-Treg biology is supported by the fact that (i) ICOS+ Treg have a stronger suppressive function in melanoma (46) and murine models (27, 28), and (ii) ICOS deletion in human (22) and mice (23) correlates to a decreased Treg proportion.

On the other hand ICOS could be expressed on activated T cells (47) and ICOS expression is upregulated on IFNγ secreting T cells during anti-CTLA4 treatment in phase III trials in melanoma patients (for review 33). However, there is no evidence in these clinical trials that ICOS contributes to antitumor immunity and ICOS may simply represent a T-cell effector marker. This could suggest that treatment with neutralizing anti-ICOS mAb needs to be restricted to a short time period to abrogate Treg amplification without impacting on potential restoration of effector cells expressing ICOS.

The treatment by the anti-ICOS 314.8 mAb may be particularly relevant in neo-adjuvant settings combined to therapies inducing antitumor immunity by favoring tumor cell death such as therapies targeting tumor molecular alterations (Herceptin, lapatinib; ref. 48) or immunogenic chemotherapies (anthracyclins; ref. 49).

No potential conflicts of interest were disclosed.

Conception and design: J. Faget, M. Gobert, J.-Y. Blay, C. Caux, C. Menetrier-Caux

Development of methodology: J. Faget, M. Gobert, D. Olive, C. Biota, J.-Y. Blay, C. Menetrier-Caux

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): J. Faget, N. Bendriss-Vermare, C. Biota, T. Bachelot, I. Treilleux, S. Goddard-Leon

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Faget, N. Bendriss-Vermare, D. Olive, E. Lavergne, S. Chabaud, J.-Y. Blay, C. Caux, C. Menetrier-Caux

Writing, review, and/or revision of the manuscript: J. Faget, N. Bendriss-Vermare, J.-Y. Blay, C. Caux, C. Menetrier-Caux

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T. Bachelot, C. Menetrier-Caux

Study supervision: J.-Y. Blay, C. Caux, C. Menetrier-Caux

Flow cytometry and cell sorting: I. Durand

The authors are grateful to the breast cancer patients who accepted to participate in this study and to the institutional Biological Research Center from the Centre Léon Bérard who provided us with breast tumor specimens. The authors thank Dr Rosenbusch, D. Giroux, and colleagues from clinics who provided us with tonsils samples. The authors thank Anthony Besse for statistical analyses.

This work was supported by institutional grants from Breast Cancer Research Fundation, Association pour la Recherche sur le Cancer (grant n 7896), Comité départemental du Rhône de la Ligue contre le cancer, ACI 2007–2009, ANR11-EMMA-045 VICIT-01, INCa-PLBio 2011 (2011-1-PL-Bio-12-IC-1), and FUI Project PLATINE. J. Faget is a recipient of a grant from la Ligue Nationale contre le Cancer. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Dunn
GP
,
Old
LJ
,
Schreiber
RD
. 
The immunobiology of cancer immunosurveillance and immunoediting
.
Immunity
2004
;
21
:
137
48
.
2.
Gobert
M
,
Treilleux
I
,
Bendriss-Vermare
N
,
Bachelot
T
,
Goddard-Leon
S
,
Arfi
V
, et al
Regulatory T cells recruited through CCL22/CCR4 are selectively activated in lymphoid infiltrates surrounding primary breast tumors and lead to an adverse clinical outcome
.
Cancer Res
2009
;
69
:
2000
9
.
3.
Tan
W
,
Zhang
W
,
Strasner
A
,
Grivennikov
S
,
Cheng
JQ
,
Hoffman
RM
, et al
Tumour-infiltrating regulatory T cells stimulate mammary cancer metastasis through RANKL-RANK signalling
.
Nature
2011
;
470
:
548
53
.
4.
Ménétrier-Caux
C
,
Curiel
T
,
Faget
J
,
Manuel
M
,
Caux
C
,
Zou
W
. 
Targeting regulatory T cells
.
Target Oncol
2012
;
7
:
15
28
.
5.
Klages
K
,
Mayer
CT
,
Lahl
K
,
Loddenkemper
C
,
Teng
MW
,
Ngiow
SF
, et al
Selective depletion of Foxp3+ regulatory T cells improves effective therapeutic vaccination against established melanoma
.
Cancer Res
2010
;
70
:
7788
99
.
6.
Morse
MA
,
Hobeika
AC
,
Osada
T
,
Serra
D
,
Niedzwiecki
D
,
Lyerly
HK
, et al
Depletion of human regulatory T cells specifically enhances antigen-specific immune responses to cancer vaccines
.
Blood
2008
;
112
:
610
8
.
7.
Brahmer
JR
,
Tykodi
SS
,
Chow
LQ
,
Hwu
WJ
,
Topalian
SL
,
Hwu
P
, et al
Safety and activity of anti-PD-L1 antibody in patients with advanced cancer
.
N Engl J Med
2012
;
366
:
2455
65
.
8.
Topalian
SL
,
Hodi
FS
,
Brahmer
JR
,
Gettinger
SN
,
Smith
DC
,
McDermott
DF
, et al
Safety, activity, and immune correlates of anti-PD-1 antibody in cancer
.
N Engl J Med
2012
;
366
:
2443
54
.
9.
Menard
C
,
Ghiringhelli
F
,
Roux
S
,
Chaput
N
,
Mateus
C
,
Grohmann
U
, et al
Ctla-4 blockade confers lymphocyte resistance to regulatory T-cells in advanced melanoma: surrogate marker of efficacy of tremelimumab?
Clin Cancer Res
2008
;
14
:
5242
9
.
10.
Vonderheide
RH
,
LoRusso
PM
,
Khalil
M
,
Gartner
EM
,
Khaira
D
,
Soulieres
D
, et al
Tremelimumab in combination with exemestane in patients with advanced breast cancer and treatment-associated modulation of inducible costimulator expression on patient T cells
.
Clin Cancer Res
2010
;
16
:
3485
94
.
11.
Faget
J
,
Biota
C
,
Bachelot
T
,
Gobert
M
,
Treilleux
I
,
Goutagny
N
, et al
Early detection of tumor cells by innate immune cells leads to Treg recruitment through CCL22 production by tumor cells
.
Cancer Res
2011
;
171
:
6143
52
.
12.
Treilleux
I
,
Blay
JY
,
Bendriss-Vermare
N
,
Ray-Coquard
I
,
Bachelot
T
,
Guastalla
JP
, et al
Dendritic cell infiltration and prognosis of early stage breast cancer
.
Clin Cancer Res
2004
;
10
:
7466
74
.
13.
Labidi-Galy
SI
,
Sisirak
V
,
Meeus
P
,
Gobert
M
,
Treilleux
I
,
Bajard
A
, et al
Quantitative and functional alterations of plasmacytoid dendritic cells contribute to immune tolerance in ovarian cancer
.
Cancer Res
2011
;
71
:
5423
34
.
14.
Jensen
TO
,
Schmidt
H
,
Moller
HJ
,
Donskov
F
,
Hoyer
M
,
Sjoegren
P
, et al
Intratumoral neutrophils and plasmacytoid dendritic cells indicate poor prognosis and are associated with pSTAT3 expression in AJCC stage I/II melanoma
.
Cancer
2012
;
118
:
2476
85
.
15.
Ito
T
,
Hanabuchi
S
,
Wang
YH
,
Park
WR
,
Arima
K
,
Bover
L
, et al
Two functional subsets of FOXP3(+) regulatory T cells in human thymus and periphery
.
Immunity
2008
;
28
:
870
80
.
16.
Ouabed
A
,
Hubert
FX
,
Chabannes
D
,
Gautreau
L
,
Heslan
M
,
Josien
R
. 
Differential control of T regulatory cell proliferation and suppressive activity by mature plasmacytoid versus conventional spleen dendritic cells
.
J Immunol
2008
;
180
:
5862
70
.
17.
Sisirak
V
,
Faget
J
,
Gobert
M
,
Goutagny
N
,
Vey
N
,
Treilleux
I
, et al
Impaired IFN-α production by plasmacytoid dendritic cells favors regulatory T cel expansion and may contribute to breast cancer progression
.
Cancer Res
2012
.
[Epub ahead of print]
.
18.
Hutloff
A
,
Dittrich
AM
,
Beier
KC
,
Eljaschewitsch
B
,
Kraft
R
,
Anagnostopoulos
I
, et al
ICOS is an inducible T-cell co-stimulator structurally and functionally related to CD28
.
Nature
1999
;
397
:
263
6
.
19.
Dong
C
,
Juedes
AE
,
Temann
UA
,
Shresta
S
,
Allison
JP
,
Ruddle
NH
, et al
ICOS co-stimulatory receptor is essential for T-cell activation and function
.
Nature
2001
;
409
:
97
101
.
20.
McAdam
AJ
,
Greenwald
RJ
,
Levin
MA
,
Chernova
T
,
Malenkovich
N
,
Ling
V
, et al
ICOS is critical for CD40-mediated antibody class switching
.
Nature
2001
;
409
:
102
5
.
21.
Tafuri
A
,
Shahinian
A
,
Bladt
F
,
Yoshinaga
SK
,
Jordana
M
,
Wakeham
A
, et al
ICOS is essential for effective T-helper-cell responses
.
Nature
2001
;
409
:
105
9
.
22.
Takahashi
N
,
Matsumoto
K
,
Saito
H
,
Nanki
T
,
Miyasaka
N
,
Kobata
T
, et al
Impaired CD4 and CD8 effector function and decreased memory T cell populations in ICOS-deficient patients
.
J Immunol
2009
;
182
:
5515
27
.
23.
Burmeister
Y
,
Lischke
T
,
Dahler
AC
,
Mages
HW
,
Lam
KP
,
Coyle
AJ
, et al
ICOS controls the pool size of effector-memory and regulatory T cells
.
J Immunol
2008
;
180
:
774
82
.
24.
Watanabe
M
,
Watanabe
S
,
Hara
Y
,
Harada
Y
,
Kubo
M
,
Tanabe
K
, et al
ICOS-mediated costimulation on Th2 differentiation is achieved by the enhancement of IL-4 receptor-mediated signaling
.
J Immunol
2005
;
174
:
1989
96
.
25.
Janke
M
,
Witsch
EJ
,
Mages
HW
,
Hutloff
A
,
Kroczek
RA
. 
Eminent role of ICOS costimulation for T cells interacting with plasmacytoid dendritic cells
.
Immunology
2006
;
118
:
353
60
.
26.
Ito
T
,
Yang
M
,
Wang
YH
,
Lande
R
,
Gregorio
J
,
Perng
OA
, et al
Plasmacytoid dendritic cells prime IL-10-producing T regulatory cells by inducible costimulator ligand
.
J Exp Med
2007
;
204
:
105
15
.
27.
Kornete
M
,
Sgouroudis
E
,
Piccirillo
CA
. 
ICOS-dependent homeostasis and function of Foxp3+ regulatory T cells in islets of nonobese diabetic mice
.
J Immunol
2012
;
188
:
1064
74
.
28.
Chen
Y
,
Shen
S
,
Gorentla
BK
,
Gao
J
,
Zhong
XP
. 
Murine regulatory T cells contain hyperproliferative and death-prone subsets with differential ICOS expression
.
J Immunol
2012
;
188
:
1698
707
.
29.
Fong
L
,
Small
EJ
. 
Anti-cytotoxic T-lymphocyte antigen-4 antibody: the first in an emerging class of immunomodulatory antibodies for cancer treatment
.
J Clin Oncol
2008
;
26
:
5275
83
.
30.
Ménétrier-Caux
C
,
Gobert
M
,
Caux
C
. 
Differences in tumor regulatory T-cell localization and activation status impact patient outcome
.
Cancer Res
2009
;
69
:
7895
8
.
31.
Svensson
H
,
Olofsson
V
,
Lundin
S
,
Yakkala
C
,
Bjorck
S
,
Borjesson
L
, et al
Accumulation of CCR4(+)CTLA-4 FOXP3(+)CD25(hi) regulatory T cells in colon adenocarcinomas correlate to reduced activation of conventional T cells
.
PLoS One
2012
;
7
:
e30695
.
32.
Curiel
TJ
,
Coukos
G
,
Zou
L
,
Alvarez
X
,
Cheng
P
,
Mottram
P
, et al
Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival
.
Nat Med
2004
;
10
:
942
9
.
33.
Verschraegen
C
. 
The monoclonal antibody to cytotoxic T lymphocyte antigen 4, ipilimumab, in the treatment of melanoma
.
Cancer Manag Res
2012
;
4
:
1
8
.
34.
Conrad
C
,
Gregorio
J
,
Wang
YH
,
Ito
T
,
Meller
S
,
Hanabuchi
S
, et al
Plasmacytoid dendritic cells promote immunosuppression in ovarian cancer via ICOS co-stimulation of Foxp3+ T regulatory cells
.
Cancer Res
2012
.
[Epub ahead of print]
.
35.
Xiao
X
,
Gong
W
,
Demirci
G
,
Liu
W
,
Spoerl
S
,
Chu
X
, et al
New insights on OX40 in the control of T cell immunity and immune tolerance in vivo
.
J Immunol
2012
;
188
:
892
901
.
36.
Grinberg-Bleyer
Y
,
Saadoun
D
,
Baeyens
A
,
Billiard
F
,
Goldstein
JD
,
Gregoire
S
, et al
Pathogenic T cells have a paradoxical protective effect in murine autoimmune diabetes by boosting Tregs
.
J Clin Invest
2010
;
120
:
4558
68
.
37.
Patel
SA
,
Meyer
JR
,
Greco
SJ
,
Corcoran
KE
,
Bryan
M
,
Rameshwar
P
. 
Mesenchymal stem cells protect breast cancer cells through regulatory T cells: role of mesenchymal stem cell-derived TGF-beta
.
J Immunol
2010
;
184
:
5885
94
.
38.
Labidi-Galy
SI
,
Treilleux
I
,
Goddard-Leon
S
,
Combes
JD
,
Blay
JY
,
Ray-Coquard
I
, et al
Plasmacytoid dendritic cells infiltrating ovarian cancer are associated with poor prognosis
.
OncoImmunology
2012
;
1
:
380
2
.
39.
Martin-Orozco
N
,
Li
Y
,
Wang
Y
,
Liu
S
,
Hwu
P
,
Liu
YJ
, et al
Melanoma cells express ICOS ligand to promote the activation and expansion of T-regulatory cells
.
Cancer Res
2010
;
70
:
9581
90
.
40.
Schreiner
B
,
Wischhusen
J
,
Mitsdoerffer
M
,
Schneider
D
,
Bornemann
A
,
Melms
A
, et al
Expression of the B7-related molecule ICOSL by human glioma cells in vitro and in vivo
.
Glia
2003
;
44
:
296
301
.
41.
Martin-Gayo
E
,
Sierra-Filardi
E
,
Corbi
AL
,
Toribio
ML
. 
Plasmacytoid dendritic cells resident in human thymus drive natural Treg cell development
.
Blood
2010
;
115
:
5366
75
.
42.
Gilliet
M
,
Liu
YJ
. 
Generation of human CD8 T regulatory cells by CD40 ligand-activated plasmacytoid dendritic cells
.
J Exp Med
2002
;
195
:
695
704
.
43.
Hadeiba
H
,
Sato
T
,
Habtezion
A
,
Oderup
C
,
Pan
J
,
Butcher
EC
. 
CCR9 expression defines tolerogenic plasmacytoid dendritic cells able to suppress acute graft-versus-host disease
.
Nat Immunol
2008
;
9
:
1253
60
.
44.
Ochando
JC
,
Homma
C
,
Yang
Y
,
Hidalgo
A
,
Garin
A
,
Tacke
F
, et al
Alloantigen-presenting plasmacytoid dendritic cells mediate tolerance to vascularized grafts
.
Nat Immunol
2006
;
7
:
652
62
.
45.
Riley
JL
,
Blair
PJ
,
Musser
JT
,
Abe
R
,
Tezuka
K
,
Tsuji
T
, et al
ICOS costimulation requires IL-2 and can be prevented by CTLA-4 engagement
.
J Immunol
2001
;
166
:
4943
8
.
46.
Strauss
L
,
Bergmann
C
,
Szczepanski
MJ
,
Lang
S
,
Kirkwood
JM
,
Whiteside
TL
. 
Expression of ICOS on human melanoma-infiltrating CD4+CD25highFoxp3+ T regulatory cells: implications and impact on tumor-mediated immune suppression
.
J Immunol
2008
;
180
:
2967
80
.
47.
Xu
F
,
Li
D
,
Zhang
Q
,
Fu
Z
,
Zhang
J
,
Yuan
W
, et al
ICOS gene polymorphisms are associated with sporadic breast cancer: a case-control study
.
BMC Cancer
2011
;
11
:
392
.
48.
Guarneri
V
,
Barbieri
E
,
Conte
P
. 
Biomarkers predicting clinical benefit: fact or fiction?
J Natl Cancer Inst Monogr
2011
;
2011
:
63
6
.
49.
Ghiringhelli
F
,
Apetoh
L
,
Tesniere
A
,
Aymeric
L
,
Ma
Y
,
Ortiz
C
, et al
Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors
.
Nat Med
2009
;
15
:
1170
8
.