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

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 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.

Fig. 1

Local XAGE-1b-mediated immunity, Th1 response. Day 2 supernatants from PBMCs, TILs and LN cells, co-cultured with XAGE-1b pulsed monocytes, were analyzed for Th1 (IFNγ and TNF-α) and Th 2 (IL-5 and IL-10) cytokine release. A positive response (indicated with asterisks) was defined by a cytokine concentration above the cutoff value (20 pg/ml, except for IFNγ, 100 pg/ml) and more than twice the concentration of medium control. Depicted here are the Th1 cytokines. XAGE-1b-specific Th1 cytokine release was observed in TILs (X-14) and LN cells (X-14, X-20). Included are the results from one negative (X-18) patient. PHA was used as positive control

Fig. 2

XAGE-1b-specific IgG antibodies in patient sera. a Example of a XAGE-1b-specific IgG response (X-14) to XAGE-1b peptide mix. KLU 187 represents a serum sample previously shown to have high IgG antibody titers [13] and was used as positive control to set up the ELISA. Serial dilutions are shown. b Example of a patient (X-18) with no XAGE-1b IgG response to XAGE-1b peptide mix. X-14 was used as positive control. c IgG response (serum 1:100 diluted) to individual XAGE-1b peptides in 3 of the 40 tested patients shows broad recognition. A positive response was defined as at least a twofold increase compared to background (no antigen) and is indicated with asterisks

Fig. 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)

Fig. 4

Type and specificity of XAGE-1b-specific CD4 T cell response. a The CD4+ T cells in the 8-week cultured PBMCs of patient X-14 were gated (see Supplementary 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 are 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 two 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

Fig. 5

TCR-Vβ expression and XAGE-1b-specific proliferation of bulk-cultured PBMCs of patient X-14. a Sixteen different TCR-Vβ families were discovered in the bulk-cultured PBMCs of patient X-14. b XAGE-1b-specific proliferation (conducted in two separate assays) demonstrated a broad response to all 5 overlapping XAGE-1b peptides, the peptide mix and to XAGE-1b protein. c In total, 10 clones were isolated from the expanded PBMCs; XAGE-1b-specific proliferation and TCR-Vβ usage are shown for three clones with different patterns of antigen recognition (p1, p2, the peptide mix and XAGE-1b protein) and TCR-Vβ expression (Vβ 21.3 and 5.1)