Article Figures & Data
Figures
- Fig. 1.
TCR VB families in PBL and TIL. PBL were gated on CD4+ or CD8+ T cells and tested for expression of individual TCR VB chains by flow cytometry. No major TCR VB expansion could be detected in PBL. In contrast, the majority of TIL (>98% CD4+) stained positive for the TCR VB16. +, detection of monoclonal TCR VB chains as listed in Table 2.
- Fig. 2.
Detection of TCR VB16+ T cells infiltrating into cervical cancer. Serial sections from tumor tissue were stained for CD3, CD4, CD8, and TCR VB16+ T cells. Note that CD4+ T cells represent the majority of T cells and that TCR VB16+ T cells directly infiltrate the tumor lesion.
- Fig. 3.
Titration of HPV33 E773–87 peptides onto autologous antigen-presenting cells. Peptides were serially diluted and pulsed onto antigen-presenting cells, incubated for 2 h at room temperature, and tested for CD4+VB16+ T-cell recognition, as determined by IFN-γ secretion. The peptide ASDLRTIQQLLMGTV represents the dominant epitope.
Tables
- Table 1.
TIL recognize autologous tumor cellsa
Target Blocking MAb IFN-γ (pg/ml/106 cells) Tumor None 0 TIL None 23 Tumor + TIL None 400 W6/32 400 L243 20 ↵a Autologous cervical cancer cells harboring HPV33 were tested for T-cell recognition as determined by IFN-γ and IL-2 secretion by ELISA. TIL secreted IFN-γ, but not IL-2 in response to tumor cells, which could be blocked with the anti-DR-directed MAb L243. Blocking with the anti-DR-directed MAb clone B8.12.2 showed a similar result in a parallel experiment.
- Table 2.
Deduced TCR amino acid sequencesa
T-cell population TCR chain Variable region sequence CDR3 sequence Sequence of joining region PBL-VA CD8+ VA2 DSQPSDSATYLCA VSG NDMRFGAG (AJ43) VA3* ADTASYFCA SRGGD NTDKLIFGTG (AJ34) VA4 TLRDAAVYYCI LSFAS AGGTSYGKLTFGQG (AJ52) VA9* FAQEEDSAMYYCA YGNNRLAFGKG (AJ7) VA16 SALVSDSALYFCA VRHRI DTGRRALTFGSG (AJ5) VA21 QPGDSAVYFCA ASES GTSYGKLTFGQG (AJ52) VA23 ASQPGDSATYLCA VRYLL SSGSARQLTFGSG (AJ22) VA25 STYLCA VGPS SSGSARQLTFGSG (AJ22) PBL-VB CD8+ VB2 SAHPEDSSFYIC SARGGGSG NQPQHFGDG (BJ1-5) VB3 SASTNQTSMYLCA SSLGVG YEQYFGPG (BJ2-7) VB9 LELGDSAVYFCA SSQLWTSGAPP STDTQYFGPG (BJ2-3) CD4+TIL-VA VA2 FNTSCGTDYLCA MSS DRGSTLGRLYFGRG (AJ18) VA3* ADTASYFCA SRGGD NTDKLIFGTG (AJ34) VA6 ASQLGDSAMYFCA MREGW AAGNKLTFGGG (AJ17) VA9* FAQEEDSAMYYCA YGNNRLAFGKG (AJ7) VA10 (a) AAQPGDTGLYLCA GG SDGQKLLFARG (AJ16) VA10 (b) AAQPGDTGLYLCA GGP MDSSYKLIFGSG (AJ12) VA21 PSQPGDSAVYFCA ASGG KLVFGKG (AJ57) VA26 IYLCA GG SDGQKLLFARG (AJ16) CD4+TIL-VB16 VB16 PAELDSGVYFCA SSRGTSGTLK QYFGPG (BJ2-7) TIL VB16 sorted VA10 AAQPGDTGLYLCA GGP MDSSYKLIFGSG (AJ12) VA21 PSQPGDSAVYFCA AS GG KLVFGKG (AJ57) VA22 AVYFCA LS YGCCRLAFGKG (AJ7) ↵a Monoclonal TCRs in CD8+ or CD4+ T-cell populations were identified by DNA sequence analysis as described previously (18). Exclusively the CDR3 region and the adjacent variable and joining TCR segments are listed. Note that the CD4+ VB16+ population represents 96% of all T cells in TIL (Fig. 1). Sorting of these VB16+T cells yields a TCR VB16+ VA10+VA21+ T-cell line. Note that in unsorted TIL, two TCR VA10 chains could be identified, but only one of these was present in the VB16-sorted T-cell population [TCR VA10(b)]. *, identical TCR sequences in PBL and TIL. TCR VA chains combined with VB16 exhibit a common CDR3 motif: TCR VA10 and TCR VA21 share glycine residues (bold), and TCR VA21 shares a serine residue (underlined) with VA22.
- Table 3.
HPV33 E773–87 represents the dominant epitope defined by CD4+VB16+ TILa
Peptide Sequence IFN-γ secretion (pg/ml per 106 cells per 24 h) Autologous cells (DR*0402) T2 T2-DR*0401 E7 0 0 0 E719–3 PTDLYCYEQLSDSSD 0 0 0 E764–78 TVRLCVNSTASDLRT 0 0 0 E773–87 ASDLRTIQQLLMGTV 4,000 0 4,800 E762–76 NTTVRLCVNSTASDL 0 0 0 E75–19 KPTLKEYVLDLYPEP 0 0 0 E79–23 KEYVLDLYPEPTDLY 0 0 0 E776–90 LRTIQQLLMGTVNIV 350 0 0 E779–93 IQQLLMGTVNIVVPT 0 0 0 TT QYIKANSKFIGITE 0 0 0 ↵a Peptides were pulsed onto autologous antigen-presenting cells (1 μM), T2 cells, or T2 cells expressing the DR*0401 molecule and tested for T-cell recognition by IFN-γ secretion determined by ELISA. The HPV33 E773–87 peptide could be presented by either autologous antigen-presenting cells or T2 cells expressing DR*0401. Nontransfected T2 cells served as a negative control. A tetanus toxin peptide (TT) (28) was not recognized.
- Table 4.
HLA-DR-restricted recognition of HPV33 E773–87 epitope by CD4+ TILa
Target cells Blocking MAb IFN-γ secretion (pg/ml/2 × 106 cells per 24 h) Autologous macrophages IgG 4,800 Anti-DR 400 T2-DR4 IgG 3,800 Anti-DR 0 ↵a The HPV33 E773–87 peptide was pulsed onto autologous antigen-presenting cells or T2 cells expressing HLA-DR*0401. Antigen-presenting cells were incubated with IgG or anti-DR-directed MAbs (5 μg/well), and an IFN-γ release assay was performed. Different anti-DR-directed MAbs (clone L243 and clone B8.12.2) exhibited similar results. Data are from a blocking experiment with the L243 MAb.
