Naturally Acquired MAGE-A10- and SSX-2-Specific CD8+ T Cell Responses in Patients with Hepatocellular Carcinoma

Gabriel Bricard, Hanifa Bouzourene, Olivier Martinet, Donata Rimoldi, Nermin Halkic, Michel Gillet, Pascal Chaubert, H. Robson MacDonald, Pedro Romero, Jean-Charles Cerottini and Daniel E. Speiser
J Immunol February 1, 2005, 174 (3) 1709-1716; DOI: https://doi.org/10.4049/jimmunol.174.3.1709

Article Figures & Data

Figures

  • FIGURE 1.
    FIGURE 1.

    Expression of CT Ags in HCC but not normal liver tissue. cDNA was prepared by reverse-transcription using 1–2 μg of total RNA extracted from tumor (T) and nontumoral (N) liver tissue. In parallel, serially diluted (1/10) RNA from SK-MEL-37 (CT Ag-expressing line, positive control) and Na8-MEL (CT Ag nonexpressing line, negative control) melanoma cell lines were used. PCR was performed with equivalent amounts of cDNA to detect expression of the CT Ags NY-ESO-1, LAGE-1, MAGE-A10, and SSX-2, and β-actin as internal control. Open arrows indicate the weakly detectable long splice variant of MAGE-A10 in tumors of patients LAU 669 and LAU 766.

  • FIGURE 2.
    FIGURE 2.

    Immunohistochemical detection of CD3+, CD8+, and CD4+ cells in HCC and nontumoral liver tissue. Immunohistochemical analysis of HCC from patient LAU 748 (A to D) and patient LAU 812 (I to L) and nontumoral liver from the same patients (E to H and M to P, respectively). Samples were analyzed by HE staining (A, E, I, and M), CD3 staining (B, F, J, and N), CD8 staining (C, G, K, and O) and CD4 staining (D, H, L, and P). In HE-stained sections, infiltrating cells appear in dark blue. Cells labeled with Abs specific for CD3/CD4/CD8 are stained in brown. Note that the CD4-specific Ab stains CD4+ T and Kupffer cells because of their CD4 expression. Patient LAU 748 shows low tumor infiltration by lymphocytes in contrast to patient LAU 812 with strong intratumoral infiltration by lymphocytes.

  • FIGURE 3.
    FIGURE 3.

    CD8+ T cells specific for CT Ags in tumor but not normal liver tissue. Flow cytometric analysis of CT Ag-specific CD8+ T cells using MAGE-A10254–262 or SSX-241–49 peptide-loaded HLA-A*0201 fluorescent multimers. Results using IHL and TIL derived from patients LAU 669 (A) and LAU 812 (B) are shown. TIL were generated by incubation of thin tumor fragments in IL-2- and IL-7-supplemented medium during 3–4 wk and an additional 2-wk PHA stimulation. IHL cells were recovered after Percoll gradient separation of mechanically dissociated liver tissue, and were also stimulated with PHA before analysis. Percentage of tetramer+ cells among CD8+ cells is indicated. C, In addition a Flu matrix58–66/HLA-A*0201-specific CTL clone was used for negative control stainings.

  • FIGURE 4.
    FIGURE 4.

    CD8+ T cells specific for CT Ags in PBMC from HCC patients. Flow cytometric analysis of CT Ag-specific CD8+ T cells using NY-ESO-1157–165(C165A), CAMEL1–11, SSX-241–49, or MAGE-A10254–262 peptide-loaded HLA-A*0201 multimers. Results with peptide-stimulated PBMC derived from patients LAU 669 (left panel) and LAU 812 (right panel) are shown. CD8+ PBMC were stimulated with specific peptide at 10 μM using autologous CD8 depleted cells, restimulated 2 wk later using peptide-pulsed T2 cells and analyzed 2 wk later. Percentage of tetramer+ cells among CD8+ cells is indicated.

  • FIGURE 5.
    FIGURE 5.

    MAGE-A10254–262- and SSX-241–49-specific CD8+ T cell lines and clones recognize tumor Ag with high avidity. A, Flow cytometric analysis of CD8+/multimer+ sorted cells after expansion with PHA. Multimer+ MAGE-A10254–262-specific polyclonal CTL were derived from peptide-stimulated PBMC (PBMC line 4) or from TIL (TIL line 3). MAGE-A10254–262-specific clone 1F8 was isolated by limiting dilution from multimer+ sorted TIL. Multimer+ SSX-241–49-specific polyclonal CTL was derived from peptide-stimulated PBMC (line A). The percentage of tetramer/CD8 double-positive cells is indicated. B, Avidity of Ag recognition of MAGE-A10254–262 (left panel)- or SSX-241–49 (right panel)-specific CD8+ T cells was assessed in a 4-h chromium-release assay, using T2 cells pulsed with various peptide concentrations ranging from 10−6 to 10−15 M (−6 to −15) and controls without peptide (no peptide) or with 10−6 M of the irrelevant Flu matrix58–66 peptide (Flu MA). T2 cells were pulsed with MAGE-A10254–262 peptide (□, ○, and ⋄, left panel) or with SSX-241–49 (▴, right panel). E:T cell ratio was 30:1.

  • FIGURE 6.
    FIGURE 6.

    MAGE-A10254–262- and SSX-241–49-specific CD8+ T cells efficiently kill melanoma and HCC cell lines. Functional lytic activity of MAGE-A10254–262 (TIL line 3, clone 1F8 and PBMC line 4)- or SSX-241–49 (PBMC line A)-specific cells against two tumor cell lines in absence (○ and □) or presence (• and ▪) of synthetic peptide. Tumor cells were melanoma cell line Me 275 (▪ and □; HLA-A2+, MAGE-A10+, SSX-2+) and HCC cell line Hep-G2 (• and ○; HLA-A2+, MAGE-A10−, SSX-2−).

Tables

  • Table I.

    HCC patient characteristics

    Patient CodeAge/SexNumber of Tumor Lesions, SizeHistologyaSerologyHLA-A, -Bb
    LAU 66970/maleSix lesions, diameters 18, 5.5, 2, 1.8, 1.6, and 0.8 cm; infiltration of neighbouring organsLow differentiation; moderate lymphocytic infiltrationHBs negative, anti-HBc negative, HCV negative, anti-HCV negativeA*0201, A24(9), B62(15), Bx
    LAU 67468/maleThree lesions, largest lesion 3 × 2 × 2 cmModerate differentiation; slight lymphocytic inifiltration; liver cirrhosis.HBs negative, anti-HBc negative, anti-HCV positiveA*0201c, A3, B35, B51(5)
    LAU 69875/maleTwo lesions, diameters 5 and 3.5 cmModerate differentiation; slight lymphocytic infiltrationHBs negative, anti-HBc negative, HCV negative, anti-HCV negativeA1, A*0201, B7, B37
    LAU 74881/femaleOne lesion, diameter 13 × 6 × 5 cmLow differentiation; no lymphocytic infiltrationHBs negative, anti-HBc negative, anti-HCV negativeA*0201, A28, B18, B27
    LAU 76670/maleMultiple lesions, diameters between 1 and 2 cmModerate differentiation; no lymphocytic infiltration; liver cirrhosisHBs negative, anti-HBc negative, anti-HCV positiveA*0201, A*31d
    LAU 81268/femaleOne lesion, diameter 2 × 2 × 2 cmWell differentiated; strong lymphocytic infiltrationHBs negative, anti-HBc negative, anti-HCV negativeA*0201, A3, B13, B62(15)

    • a Tumor differentiation and cirrhosis was analyzed with H&E sections. Intratumoral T cell infiltration was analyzed with sections stained with CD3-specific Ab.

    • b HLA typing for HLA-A,-B was done by serology, and for A2 subtypes with HLA allele-sequence-specific primers.

    • c The rare alleles A*0259/60/64 cannot be excluded.

    • d The rare heterozygous combinations A*0258/59/60/64 and A*3109 cannot be excluded.

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