Local and systemic XAGE-1b-specific immunity in patients with lung adenocarcinoma

XAGE-1b is a cancer/testis antigen aberrantly expressed in pulmonary adenocarcinoma. Systemic antibody and T cell responses have been demonstrated in adenocarcinoma patients, but so far, local antigen-specific immunity has not been reported. In this study, XAGE-1b expression by tumor cells as well as the presence of systemic and/or local XAGE-1b-specific immunity was assessed in peripheral blood, tumor tissue and tumor-draining lymph nodes of Caucasian patients with pulmonary adenocarcinoma. XAGE-1b protein expression was detected in 43.6 % (17 of 39) of patients when at least two different parts of a resected tumor were assessed. In 20 patients, analysis of T cells isolated and expanded from the primary tumor and its draining lymph node demonstrated XAGE-1b-specific responses in two patients. XAGE-1b-specific immunoglobulin G antibodies were found in 3 of 40 patients. These three antibody-positive patients had also mounted a systemic T cell response to XAGE-1b, measured by proliferation, cytokine production and expression of T cell activation markers on peripheral blood mononuclear cells. The population of XAGE-1b-specific T cells comprised both CD4+ and CD8+ T cells secreting both type I and II cytokines. Epitope mapping showed that T cells predominantly targeted the N-terminal part of the XAGE-1b protein, while the B cell response was directed against the C-terminal domain. Our study for the first time provides evidence for the presence of XAGE-1b-specific T cells within adenocarcinoma tissue, which supports the concept that XAGE-1b acts as a genuine tumor antigen and, therefore, might form an attractive target for a vaccine-based approach of immunotherapy. Electronic supplementary material The online version of this article (doi:10.1007/s00262-015-1716-2) contains supplementary material, which is available to authorized users.

ABSTrACT XAGE-1b is a cancer-testis antigen aberrantly expressed in pulmonary adenocarcinoma. Systemic antibody and T-cell responses have been demonstrated in adenocarcinoma patients, but so far local antigen-specific immunity has not been reported. In this study, XAGE-1b expression by tumor cells as well as the presence of systemic and/or local XAGE-1b specific immunity was assessed in peripheral blood, tumor tissue and tumor-draining lymph nodes of Caucasian patients with pulmonary adenocarcinoma. XAGE-1b protein expression was detected in 43.6% (17 of 39) of patients when at least two different parts of a resected tumor were assessed. In 20 patients, analysis of T cells isolated and expanded from the primary tumor and its draining lymph node demonstrated XAGE-1b specific responses in 2 patients. XAGE-1b-specific immunoglobulin G antibodies were found in 3 of 40 patients. These 3 antibody positive patients had also mounted a systemic T-cell response to XAGE-1b, measured by proliferation, cytokine production and expression of T-cell activation markers on peripheral blood mononuclear cells (PBMCs). The population of XAGE-1b-specific T cells comprised both CD4+ and CD8+ T cells secreting both type I and II cytokines. Epitope mapping showed that T cells predominantly targeted the N-terminal part of the XAGE-1b protein while the B-cell response was directed against the C-terminal domain. Our study for the first time provides evidence for the presence of XAGE-1b-specific T cells within adenocarcinoma tissue, which supports the concept that XAGE-1b acts as a genuine tumor antigen and, therefore, might form an attractive target for a vaccine-based approach of immunotherapy.

INTrOduCTION
Lung cancer is the most common cause of cancer mortality in men in the developed world and one of the leading causes in women 1 . Non-small cell lung cancer (NSCLC) comprises about 80 % of all lung cancers 2 . The 5-year survival rates rapidly drops with increased stage at diagnosis 3 . The current treatment modalities include surgery, radiotherapy combined with chemotherapy or palliative chemotherapy 4 . Active immunotherapy, focusing on the reinforcement of the tumor-specific T cell response, has emerged as a new modality to treat cancer 5 . NSCLC is characterized by infiltration of different types of immune cells. Infiltration with M1-macrophages and T cells is positively associated with clinical outcomes, suggesting a protective role for the immune system in NSCLC 6 . This is supported by the recent finding that infusion of antibodies blocking programmed cell death protein 1 (PD1) on T cells has clinical impact in advanced NSCLC 7 . Peptidebased therapeutic vaccines aim at the induction of tumor-specific T-cell responses 5 . This approach is highly dependent on the identification of suitable tumor antigens 8 . An important group of tumor antigens is encoded by the cancer-testis (CT) genes. These CT antigens are present in a significant subset of tumors, including NSCLC 9 , and comprise XAGE-1. The XAGE-1 protein has 4 transcripts (a, b, c and d), of which XAGE-1b (81 amino acids) is the mainly expressed isoform 10,11 . Nuclear staining has been observed in 53% of pulmonary adenocarcinomas, a subtype that accounts for 40% of NSCLC, but not in adjacent normal tissues indicating its preferential expression by cancer cells 12 . A positive association between the expression of XAGE-1b and HLA class I with prolonged survival was reported 10 , although no link with XAGE-1b specific immunity was made. A recent study revealed the presence of XAGE-1b-specific antibodies in 10% of all NSCLC patients and in 19% of stage IIIb/IV adenocarcinoma patients. More than half of the patients with a XAGE-1b antibody response displayed a concomitant systemic CD4+ and CD8+ T-cell response 13 .
To date, studies on XAGE-1b have been performed in Asian populations but not in Caucasian subjects. Furthermore, no data exist on the presence of XAGE-1b specific T cells within the tumor or its draining lymph node. To this end, we have conducted an explorative study in which a European cohort of patients with pulmonary adenocarcinoma was studied with respect to XAGE-1b expression and the presence of systemic and local XAGE-1b mediated immunity.

XAgE-1b immunohistochemistry (IhC)
Tumor blocks were cut in 4 µm sections and deparaffinized in xylene. Endogenous peroxidase activity was blocked by incubation in 0.3% hydrogen peroxide/methanol for 10 minutes at room temperature (RT). Antigen retrieval was performed by heating the samples to 97 o C for 30 minutes in citrate buffer (pH 6.0, DAKO, Glostrup, Denmark), cooled on ice and incubated at RT for 1 h with 2 µg/ml XAGE-1 mouse monoclonal antibody LX199#5 (kindly provided by the Ludwig Institute for Cancer Research, LICR) in phosphate buffered saline (PBS, Fresenius Kabi Bad Homburg, Germany) with 1% bovine serum albumin (BSA). After washing, the slides were incubated with horseradish peroxidase-conjugated anti-mouse IgG (DAKO envision) for 30 minutes at RT. NovaRed (Vector, Burlingame, USA) was applied as a chromagen and sections were counterstained with Mayer's hematoxylin (Klinipath). All slides were mounted with Pertex mounting medium (HistoLab, Sweden). All washing steps were done with PBS. XAGE-1b expression was scored according to a previously described method 11 as negative (<5% cancer cells positive), focal (5-10% positive), intermediate (11-50% positive) or diffuse (>50% positive). From each slide, 10 random tumor fields (magnification 20x, approximately the size of a biopsy) were scored for XAGE-1b expression. When available, tissue blocks from multiple tumor sections were assessed.

XAgE-1b protein and overlapping peptides
E. coli produced recombinant XAGE-1b protein (81 amino acids) was obtained using a XAGE-1 plasmid DNA, kindly provided by the LICR. Five synthetic overlapping peptides covering the entire sequence of the XAGE-1b protein were synthesized at the LUMC by solid-phase strategies on an automated peptide synthesizer (Abimed AMS 422, Germany) using Fmoc-chemistry. Peptides were analyzed by reverse-phase HPLC; dissolved in DMSO at 50 mg/ml, aliquoted and stored at -80°C until use . The amino acid sequences  of the 5 peptides are:  p1, amino acid 1-32, MESPKKKNQQLKVGILHLGSRQKKIRIQLRSQ;  p2 amino acid
Lymph node (LN) mononuclear cells were isolated from tumor-draining lymph nodes and cultured for a week in medium alone (LN neg) or supplemented with a mix of 5 XAGE-1b overlapping peptides (LN XAGE, 2.5 µg/ml/per peptide), after which the T-cells were expanded with recombinant human IL-2 (150 IU/ml, refreshed 3 times per week) for 2-3 weeks. After harvesting, the T cells were evaluated for presence of XAGE-1b-specific CD4+CD25+ Foxp3 high T cells, XAGE-1b-specific proliferation and cytokine production.

Analysis of XAgE-1b-specific T cells by proliferation assay and cytokine profile
T cells (50,000/well) were stimulated with autologous monocytes pulsed with XAGE-1b peptides and/or protein (5 μg/ml) in triplicate wells in a three day proliferation assay 14 with phytohaemagglutinin (PHA) (Remel, Germany) used as positive control. Supernatants collected after 2 days were analyzed for Th1/Th2 cytokines (IFNγ, TNF-α, IL-10, IL-5,IL-4, IL-2) by cytometric bead array (CBA, BD Biosciences). Proliferation was measured by 3 H-thymidine incorporation (0.5 μCi/well) during the last 18 hours of the assay 18 . The stimulation index (SI) was calculated by taking mean counts of stimulated wells divided by mean counts of the medium control wells. A XAGE-1b-specific T cell response was defined by either a SI index of >3 or by XAGE-1b-specific cytokine production, which was defined as a cytokine concentration above the cut-off value (20 pg/ml, except for IFNγ for which the cut-off was 100 pg/ml) and more than twice the concentration of medium control 19 .

ELISPOT
Details of the 4-days IFNγ ELISPOT assay have been reported previously 17 . Spots were counted with a fully automated computer-assisted-video-imaging analysis system (BioSys 5000). Specific spots were calculated by subtracting the mean number of spots in quadruplicate wells + 2xSD of the medium only control from the mean number of spots in test wells. Antigen-specific T-cell frequencies were considered to be increased compared to medium control when specific T-cell frequencies were ≥ 1/10,000 17 .

Analysis of XAgE-1b-specific T cells by multiparameter flow cytometry
T cells were stimulated overnight with autologous monocytes pulsed with individual XAGE-1b peptides (5 μg/mL), a XAGE-1b peptide mix (5 μg per peptide/mL) and XAGE-1b protein (10 μg/mL). E7 protein of the human papillomavirus type 16 (HPV16 E7) was used as negative control. The percentage and polarization of XAGE-1b-specific T-cells was measured by simultaneous staining for T-cell markers (CD3, CD4 and CD8), T cell activation markers (CD137 and CD154) and cytokines (IFNγ, IL-2) according to standard operating procedures (SOPs) 14,16,20 . XAGE-1b-specific T cells were detected when the percentage of XAGE-1b-specific CD4+CD154+CD137+, CD8+CD137+ or cytokine producing T cells was at least twice the percentage detected in the medium control. The responding cells were visible as a clearly distinguishable population in the flow cytometry contour plot. An example of a gating strategy is provided in Supplementary Figure 1.

Isolation of XAgE-1b-specific T-cell clones
T-cell clones were isolated from PBMC using limiting dilution as described earlier 15 . Specificity of T-cell clones for XAGE-1b was tested by proliferation assay on peptide or protein loaded irradiated autologous Epstein Bar virus (EBV) transformed B cell lines (B-LCL) or autologous monocytes. Furthermore, clones were tested by flow cytometry for phenotype (CD4/CD8) and T cell receptor Vβ (TCRVβ) expression using a TCRVβ-kit (Beckman Coulter) comprising eight sets of antibodies, each consisting of three differently labeled antibodies specific for three different TCRVβ families, ultimately covering about 70% of the normal human TCRVβ repertoire. A TCRVβ was considered dominant (>10%), subdominant (3-10%), or minor (<3%) based on the percentage of T cells using the same TCRVβ 14,21 .

rESuLTS
An overview of patient characteristics is presented in Table 1. The mean age was 65.9 years (range 45-82 years) and the male/female ratio was 20/20.

XAgE-1b expression in lung adenocarcinoma
In our patient cohort the whole primary tumor was available for XAGE-1b staining from 28 patients who underwent surgical resection (stage I/II). A biopsy of the primary tumor was available in 9 cases (2 stage I/II patients and 7 stage III/IV patients). In 3 cases, XAGE-1b expression was assessed on metastasized tissue only. In one patient (X-4) the biopsy of the primary tumor could not be retrieved and hence XAGE-1b status was not assessed. Overall, in 17 of 39 evaluable cases (43.6%), XAGE-1b expression was observed: focal (n=5), intermediate (n=6) and diffuse (n=6). Positive staining was found in the primary tumor (n=15), metastatic tissue (n=1) or both (n=1) ( Table 1). The XAGE-1b staining pattern was always nuclear and occasionally, also cytoplasmic staining was observed. An example of XAGE-1b staining is presented in Supplementary Figure 2. While 54% (15 of 28) of the resected tumor specimens stained positive for XAGE-1b, this was only the case for 1 out of 8 biopsies evaluated. To assess whether the XAGE-1b status in biopsies truly reflects the XAGE-1b status of the primary tumor, 10 random tumor fields

XAgE-1b-specific T cells are present in the primary lung tumor and its draining lymph nodes
Fresh samples of tumor and lymph node tissue were collected from 24 of the 28 stage I/ II adenocarcinoma patients. In 20 cases, we successfully expanded TILs. Tumor-draining lymph node (LN) mononuclear cells were expanded in vitro in the presence (LN XAGE) or absence (LN neg) of exogenous XAGE-1b peptides. The phenotype (CD4, CD8, Foxp3) and activation status (i.e. CD25 expression) of these T cells is presented in Supplementary Figure 3.
Next, the presence of local (TIL/LN neg/LN XAGE) as well as circulating XAGE-1bspecific T cells (PBMC) was investigated by analysis of XAGE-1b-specific proliferation and cytokine secretion in these 20 patients. In one patient (X-14) with diffuse (>50%) XAGE-1b staining in the primary tumor, a T helper 1 (Th1) response to XAGE-1b was detected (Figure 1). XAGE-1b-specific secretion of IFNγ and TNFα was detected in both TIL and LN XAGE cells after co-culture with autologous monocytes pulsed with a mix of XAGE-1b overlapping peptides. In addition, the T helper type 2 (Th2) cytokines IL-5 and IL-10 were secreted (Supplementary Figure 4). Despite the XAGE-1b-specific high production of cytokines, the proliferative response was low in TIL (SI index 0.7) and LN XAGE (SI 2.1), indicating that these T cells had poor proliferative capacity after their initial expansion (data not shown).
Another patient (X-20), without XAGE-1b expression in the primary tumor, showed XAGE-1b-specific T cell reactivity in its draining LN. XAGE-1b-specific IFNγ and TNFα production was detected in the LN cells only expanded with IL-2 (LN neg) (Figure 1), and these T cells also produced IL-5 but not IL-10 (Supplementary Figure 4). A XAGE-1b-specific response in the LN cells cultured with XAGE-1b peptides (LN XAGE) was not demonstrated potentially due to the high background cytokine production in these activated T cells after culture (Figure 1). Again, despite high cytokine release in the LN neg cells, the XAGE-1b-specific proliferative response was low (SI 1.2, data not shown). Overall, we found local XAGE-1b mediated Th1/Th2-cell immunity in two of 20 patients tested indicating that XAGE-1b acts as a genuine tumor antigen.

Identification of XAgE-1b B cell epitopes
The ELISA for measuring XAGE-1b-specific IgG antibodies was developed with a high IgG titer serum sample (KLU 187) 13 as positive control. A relatively strong XAGE-1b-specific IgG response was detected in patient X-14 (Figure 2). In total, 3 out of 40 evaluated patients (7.5%) displayed a XAGE-1b-specific IgG response when tested against a mix of the 5 XAGE-1b overlapping peptides. These three serum samples were also tested against the individual XAGE-1b peptides ( Figure 2C). Although peptide p5 showed the strongest response, the sera showed a response to multiple epitopes in XAGE-1b.
Of these three patients, one patient (X-14) expressed XAGE-1b in the tumor and mounted a local XAGE-1b-specific T cell response. Surprisingly, the second patient (X-27) did not show XAGE-1b expression in the tumor, despite the analysis of 6 separate tumor sections. For the last patient (X-4) the XAGE-1b tumor status could not be assessed due to unavailability of the tumor sample.

XAgE-1b-specific T cells are present in peripheral blood both direct ex-vivo and after in vitro expansion
Since the presence of IgG antibodies indicate an underlying T cell response 13 , the three patients (X-4, X-14, X-27) with XAGE-1b-specific IgG were analyzed for circulating XAGE-1b-specific T cells. First, PBMC were stimulated with the pool of XAGE-1b peptides or medium only and examined in a direct ex-vivo IFNg-ELISPOT. In one patient (X-4) a strong XAGE-1b-specific IFNg response was demonstrated (Supplementary Figure 5) indicative for the presence of circulating XAGE-1b-specific T cells. PBMC of the other patients (X-14, X-27) did not show a direct ex-vivo XAGE-1b-specific T-cell response (data not shown). Subsequently, PBMC samples of these three patients were stimulated with the mix of XAGE-1b overlapping peptides in vitro for 10 days, and then tested for XAGE-1b-specific proliferation and Th1/Th2 cytokine production (Figure 3). Patient X-4 showed a Th1 response to XAGE-1b reflected by the production of high amounts of IFNγ and low levels of IL-5 and IL-10 to peptide p2 and to the mix of 5 peptides. Similar to our previous observation, the proliferative response of the T cells was modest at best to the peptide mix (SI 2.5) and undetectable to individual peptides. The PBMC culture of patient X-14 produced large amounts of IFNγ when stimulated with peptide p1, p2 or the peptide mix as well as low amounts of TNFα and IL-5 upon stimulation with p1 and the peptide mix. The PBMCs of this patient proliferated when stimulated with p1 (SI 8.5) and peptide mix (SI 8.4) but not when stimulated with p2.
The third patient (X-27) neither showed cytokine production nor a proliferative response specific for XAGE-1b (Figure 3). However, we observed an unusually high concentration of CD14+ myeloid cells in the peripheral blood of this patient. Myeloid derived suppressor cells have been shown to be elevated and to have a suppressive effect on T cells in NSCLC 22,23 . Therefore, the CD14 + cells were removed from the PBMC by magnetic-activated cell sorting (MACS) and the remaining cells were stimulated with the XAGE-1b peptide mix. As a result, the 10-day cultured PBMCs produced IFNγ, TNFα and IL-5 and proliferated upon stimulation with p2 (SI 3.9) and the XAGE-1b peptide mix (SI 4.1) (Supplementary Figure 6). Thus XAGE-1b-specific T cells are present in the peripheral blood of XAGE-1b IgG positive patients but their reactivity can be obscured by a CD14+ myeloid population.

Type and specificity of the XAgE-1b-specific T-cell response
To type and enumerate XAGE-1b-specific T cells in 8 weeks cultured PBMCs, we assessed the frequency of CD4+ and CD8+ T cells with increased expression of the T-cell activation markers CD137 and CD154 after stimulation with antigen-pulsed autologous monocytes ( Figure 4A). CD4+ T cells of patient X-4 showed a strong response towards stimulation with p1, p2 and the peptide mix. A weak response was observed to p3, p4 and XAGE-1b protein. The type I cytokines IFNγ and/or IL-2 were produced by the double positive (CD137+CD154+) T cells (Figure 4B) upon stimulation with p1, p2, p3, p4 and the peptide mix. Unfortunately, the number of expanded CD8+ T cells in this culture was too low to thoroughly assess XAGE-1b-specific CD8+ T-cell reactivity.  Figure 3. Circulating XAGE-1b specific T cells: proliferation and release of type I/II cytokines A) PBMC samples from three patients (X-4, X-14, X-27) with XAGE-1b-specific IgG antibodies were stimulated with XAGE-1b overlapping peptides in a 10 day culture and subsequently tested for XAGE-1b-specific proliferation. A positive response (indicated with asterisk) was defined as a SI index of ≥ 3. Patient X-14 showed a response specific for peptide p1 and XAGE-1b peptide mix, X-4 showed a modest proliferative response to the XAGE-1b peptide mix, whereas X-27 showed no proliferative response at all. B) Release of Th1 cytokines (IFNγ, TNFα) was observed in two patients (X-4, X14) specific for peptide p1, p2 and peptide mix. C) Release of Th2 cytokines (IL-10, IL-5) was observed in two patients (X-4, X14) and was specific for peptide p1, p2 and peptide mix. The third patient (X-27) showed no detectable response (B,C).   Figure  1). Subsequently, CD4+ double positive (CD137+CD154+) T cells were gated. Finally, the intracellular IFNγ/ IL-2 production within this population was plotted. Upon stimulation with XAGE-1b peptide mix and XAGE-1b a specific upregulation of the T-cell activation markers CD137/CD154 and the intracellular expression of IFNγ and IL-2 is demonstrated as compared to medium control and the negative control protein (HPV16 E7). B) The specificity of CD4+CD137+CD154+ T cells for individual peptides and XAGE-1b protein and type I intracellular cytokine profile are shown for 2 patients (X-4 and X- 14). Asterisks indicate positive responses (at least twice the percentage detected in the medium control). For patient X-4, a CD4+ T-cell response was observed specific for peptide p1, p2 and the peptide mix, as well as a weak response to p3, p4 and p5. Patient X-14 displayed a strong CD4+ T-cell response when stimulated with p1, peptide mix and XAGE-1b protein as well as a moderate response to p2.
The cultured PBMC of patient X-14 showed a strong CD4+ T-cell mediated response when stimulated with p1, peptide mix and XAGE-1b protein as well as a moderate response to p2, corroborating the earlier data ( Figure 4B). The cytokine profile of double positive T cells mainly showed high IFNγ production specific for p1 and the peptide mix. Notably, the CD8+ T cells within this culture reacted to p1, the peptide mix and to XAGE-1b protein (Supplementary Figure 7). Surprisingly, the peptide and protein specific IFNγ production in these CD8 T cells is not accompanied by an increased expression of the activation marker CD137 (Supplementary Figure 8).
Subsequently, T cell clones were isolated from the bulk cultured PBMCs from patients X-4 and X-14. During the cloning procedure the expanded PBMCs were kept in culture and, subsequently the established clones and bulk cultured PBMCs were characterized with respect to TCR-Vβ usage and antigen-specific proliferation. Analysis of TCR-Vβ usage showed the presence of at least 11 different TCR-Vβ families of CD4+ T cells in the bulk cultured PBMCs of X-4 (Supplementary Figure 9A). Upon stimulation with peptide p1, p2, the peptide mix and XAGE-1b protein, the bulk cultured PBMCs of this patient showed a proliferative response (Supplementary Figure 9B). Only one XAGE-1bspecific CD4+ T-cell clone could be established (Supplementary Figure 9C). This clone (X-4.6) responded to p1, p2 and the peptide mix. The TCR-Vβ analysis did not lead to the identification of a specific TCR-Vβ family usage (data not shown), indicating that the TCR-Vβ used was outside the range of families covered by the eight sets of antibodies.
The CD4+ T cells in the bulk cultured PBMC of patient X-14 displayed the use of at least 16 different TCR-Vβ-families (Figure 5A), of which one was considered dominant (Vβ5.1) and 5 were considered subdominant (Vβ2/3/8/14/21.3). The bulk cultured PBMCs showed a broad response to all 5 overlapping XAGE-1b peptides, the peptide mix and to XAGE-1b protein ( Figure 5B). A total of 10 XAGE-1b-specific CD4+ T cell clones were obtained of which 8 were analyzed for TCR-Vβ usage. Staining for the dominant TCR-Vβ 5.1 was demonstrated for 4 clones, whereas the subdominant TCR-Vβ 21.3 was expressed by one clone (Figure 5C). The established clones showed XAGE-1b specific proliferation when stimulated with p1, p2, the peptide mix and XAGE-1b protein, indicating that CD4+ T-cell clones isolated from this culture recognized their naturally processed cognate antigen. Flow cytometric analysis of intracellular Foxp3 expression by the isolated clones did not reveal the presence of this transcription factor (data not shown).
Taken together, we conclude that T-cell reactivity to XAGE-1b involves polyclonal CD4+ and CD8+ T-cell populations with the capacity to recognize the naturally processed antigen and reactive to peptide epitopes different from those recognized by the IgG antibodies.

dISCuSSION
This is the first study on the immunogenicity of XAGE-1b in a Caucasian study cohort of 40 pulmonary adenocarcinoma patients. We found XAGE-1b protein expression to be present in 43.6% of cases, which is within the frequency range (33-53%) reported for Asian patients 10,11,24 . XAGE-1b was expressed both in primary and metastatic tumor specimens. However, XAGE-1b positive tumors do not show protein overexpression in all tumor samples nor in all fields covering an area that approximates the size of a bronchial biopsy. Hence, the frequency of XAGE-1b overexpressing tumors can be underestimated when only assessing a single section of the tumor and most likely when a biopsy is analyzed. Based on our findings, showing that sometimes only 2 out of 10 randomly analyzed tumor fields stain positive for XAGE-1b, its status is probably best assessed by the analysis of at least two sections of the primary tumor or 5 biopsies. Moreover, our study reveals for the first time the presence of XAGE-1b specific T cells in the primary lung tumor and the tumor-draining lymph nodes from 2 out of 20 evaluated patients. The XAGE-1b reactive T cells displayed both a Th1 (IFNγ/TNFα) and Th2 (IL-5/IL-10) cytokine polarization. These results indicate that XAGE-1b-specific T cells can contribute to the anti-tumor response but the low response rate among patients also implies that the spontaneous activation of T cells to XAGE-1b is limited. In our cohort, we observed XAGE-1b-specific IgG antibodies in 3 of 40 patients (7.5%). Two previous studies with Asian adenocarcinoma patients also assessed anti-XAGE-1b antibody frequency. The first study 11 found a XAGE-1b IgG frequency of 8.9% in a similar (mainly stage I/II) patient group. The second study 13 found a higher frequency (19%) of XAGE-1b IgG responses but this was in stage IIIb/IV patients. Possibly, XAGE-1b-specific antibodies are more prevalent in patients with a more advanced tumor stage, as also observed for antibodies against p53, NY-ESO-1 and survivin 25 .
The presence of XAGE-1b specific IgG antibodies was accompanied by an antigenspecific T cell response in the peripheral blood of all 3 antibody positive patients. In one patient (X-4), an ex-vivo IFNγ response to XAGE-1b peptide mix was found by ELISPOT assay, whereas in two patients (X-4 and X-14) Th1 and Th2 cytokine responses were found after one round of in vitro stimulation of PBMC with XAGE-1b overlapping peptides. In depth analysis revealed that the XAGE-1b-specific T-cell population comprised both CD4 and CD8 T cells producing both Th1 and Th2 cytokines. The T cells reacted mainly to the overlapping peptides p1 and p2. Recently this N-terminal part of XAGE-1b was shown to comprise a CD4+ T-cell epitope (aa 18-31) and an overlapping CD8+ T-cell epitope (aa 21-29) 13 . Our study confirms this part of XAGE-1b as a T-cell epitope containing domain. Interestingly, while the T-cell response was predominantly directed against sequences present in p1 and p2, the dominant B-cell response was targeted to p5 of XAGE-1b within the small group of patients positive in this study.
In one patient (X-27, Supplementary Figure 6), an underlying XAGE-1b specific T-cell response was only detected after removal of a myeloid CD14 + population from the PBMC sample. Immature myeloid cell populations have been shown to inhibit T cell activation 26,27 , and their suppressive activity has been specifically reported for NSCLC 22,23,28 . Therefore, removal of the potentially suppressive myeloid cell populations from the peripheral blood of adenocarcinoma patients before analysis of T cell responses might reveal a higher percentage of XAGE-1b specific T-cell responders among Caucasian patients. In addition, based on the current success with co-inhibitory receptor blocking in patients with NSCLC 7 one could consider to test the response in assays where known co-inhibitory receptors are blocked.
In conclusion, our study demonstrates that XAGE-1b acts as a genuine tumor antigen eliciting integrated systemic and/or tumor-infiltrating antigen-specific humoral and cellular immune responses in Caucasian patients with pulmonary adenocarcinoma. As such, this tumor antigen forms an attractive target for active immunotherapy in lung cancer using XAGE-1b based therapeutic vaccines. Based on the presence of these integrated XAGE-1b-specific responses in non-vaccinated patients, it is not to be expected that the induction of such a response by XAGE-1b vaccination will result in safety problems. Based on these results, we have recently started a phase 1 clinical trial with XAGE-1b synthetic long peptides.   Figure 4. Local XAGE-1b mediated immunity, Th2 response Day 2 supernatants from PBMC, TIL and LN cells tested in a proliferation assay were analyzed for Th2 cytokine release (IL-10, IL-5). A positive response (indicated with asterisks) was defined by a cytokine concentration above the cut-off value (above 20 pg/mL) and more than twice the concentration of medium control. PHA was used as positive control. Results from two positive (X-14, X-20) and one negative (X-18) patients are shown. Ex-vivo XAGE-1b specifi c IFNγ response by ELISPOT assay PBMC samples of patient X-4 were evaluated for XAGE-1b reactivity in a direct ex-vivo ELISPOT assay as described in the Methods section. XAGE-1b specifi c T-cell frequencies were considered to be increased compared to medium control when frequencies are ≥ 1/10,000 cells.