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Science 11 January 2008:
Vol. 319. no. 5860, pp. 215 - 220
DOI: 10.1126/science.1148886
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Reports

Recognition of a Ubiquitous Self Antigen by Prostate Cancer-Infiltrating CD8+ T Lymphocytes

Peter A. Savage1, Keith Vosseller2, Chulho Kang3, Kevin Larimore4, Elyn Riedel5, Kathleen Wojnoonski1, Achim A. Jungbluth6 and James P. Allison1*

1 Department of Immunology, Howard Hughes Medical Institute, and Ludwig Center for Cancer Immunotherapy, Memorial Sloan-Kettering Cancer Center (MSKCC), New York, NY 10021, USA.
2 Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, PA 19102, USA.
3 Cancer Research Laboratory, University of California, Berkeley, CA 94720, USA.
4 Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
5 Department of Epidemiology and Biostatistics, MSKCC, New York, NY 10065, USA.
6 New York Branch, Ludwig Institute for Cancer Research, New York, NY 10021, USA.


Figure 1 Fig. 1. CD8{alpha}+ T cells bearing Vβ8.3+ TCRs with a conserved CDR3 length are reproducibly overrepresented in the prostate of TRAMP+/+ mice. (A and B) TCR Vβ8.3 CDR3 size spectratyping analysis of TRAMP+/+ prostate. (A) cDNA from prostate tissue from 9 (21- to 36-week-old) B6 control mice, 10 (21-week-old) TRAMP+/+ mice, and 10 (27-week-old) TRAMP+/+ mice. (B) cDNA from CD4+ or CD8{alpha}+ T cells purified from the prostates of nine 27-week-old TRAMP+/+ mice by fluorescence-activated cell sorting. The "reference" spectra [bottom row in (A)] are derived from B6 female spleen samples. NP indicates no PCR product. (C) Flow cytometric analysis of thymocytes and splenocytes from B6 mice and Rag1–/– TCR{alpha}β transgenic mice expressing the conserved V{alpha}2+ and Vβ8.3+ TCR chains. [View Larger Version of this Image (32K GIF file)]
 

Figure 2 Fig. 2. T cells expressing the conserved TCR{alpha}β recognize a histone H4–derived peptide. (A to C) Reactivity of the clonotypic T cell hybridoma 6B1-6. Extracts or proteins were boiled in 10% acetic acid and resolved by reversed-phase HPLC. Fractions were then dried and cultured with 6B1-6 and Kb-expressing APCs, and 6B1-6 stimulation was assayed. (A) Reactivity of 6B1-6 to crude extracts from prostate (PR), spleen (SP), and liver (LIV) from 27-week-old male TRAMP+/+ mice and liver from male (M) and female (F) B6 mice. Where indicated, a cocktail of antibodies to Kb (anti-Kb) or control antibodies (ctrl Ab) was added to the culture. (B) Reactivity of 6B1-6 to extracts of subcellular fractions. B16 melanoma cells were fractionated according to Wysocka et al. (16). The indicated subcellular fractions were isolated, acid-treated, processed, and assayed. (C) Reactivity of 6B1-6 to histone H4. A mixture of histone proteins or the indicated individual histones were acid-treated, processed, and assayed. (D) Stimulation of clonotypic T cells with histone H4–derived peptides. T cells expressing the conserved TCR{alpha}β (either 6B1-6 or TCR{alpha}β transgenic T cells) were cultured with peptide and APCs [either primary dendritic cells (DCs) or L-Kb cells]. The histone H4–derived peptides assayed and the control SIINFEKL peptide are indicated. [View Larger Version of this Image (36K GIF file)]
 

Figure 3 Fig. 3. T cell recognition of histone H4 in vivo. (A) Identification of endogenous histone H4–reactive T cells with peptide/MHC tetramers. T cells from ≥24-week-old TRAMP+/+ and B6 mice were stained with H4/Kb tetramer and antibodies to cell-surface markers. (Top) Representative samples from SP, periaortic lymph nodes (pLN), brachial lymph nodes (bLN), and PR. (Bottom) Summary of tetramer staining results, pooled from five experiments. To determine which TRAMP samples were "tetramer+," we used data from B6 samples to establish a limit of detection (dashed horizontal bars), defined as the mean plus two SDs. For PR samples, solid and open circles denote samples in which ≥20 or ≤20 H4/Kb tetramer+ cells, respectively, were identified. (B) Division of HRC T cells transferred into TRAMP+/+ mice. CD45.1+ HRC T cells were labeled with CFSE, transferred into TRAMP+/+ and B6 mice, and analyzed 5 days (top) or 30 days (bottom) after transfer. Asterisks indicate statistically significant differences between TRAMP+/+ and B6 pLN values (P ≤ 0.0001 for day 5, P ≤ 0.006 for day 30). For 5 and 30 days after transfer, data are pooled from four and two experiments, respectively. Solid horizontal bars in (A) and (B) indicate median values. [View Larger Version of this Image (34K GIF file)]
 

Figure 4 Fig. 4. Trafficking and effector function of histone H4–reactive T cells. (A) Trafficking of HRC T cells to TRAMP+/+ prostate. CD45.1+ HRC T cells were transferred into ≥23-week-old TRAMP+/+ or B6 mice and analyzed 4 to 8 weeks after transfer. Solid and open circles denote samples in which ≥20 or ≤20 CD45.1+ cells, respectively, were identified. For three representative mice, flow plots are shown. (B) CD44 expression and IFN-{gamma} production by HRC T cells. CFSE-labeled CD45.1+ HRC T cells that had been transferred 30 days earlier into 23-week-old TRAMP+/+ mice were re-stimulated in vitro and analyzed. The positive control indicates cells from B6 mice that had previously received in vitro–activated CD45.1+ HRC cells. (C) Lack of detectable cytolytic activity of HRC T cells. CD45.1+ HRC cells were injected into ≥24-week-old TRAMP+/+ or B6 mice. Fourteen days later, mixtures of target cells labeled with either VVYAFKR (H4) or control SIINFEKL [ovalbumin (OVA)] peptide were injected, and the recovery of targets was quantified. The positive control (OVA vax) indicates B6 mice that had been vaccinated against OVA peptide. (D) Slight reduction in PR mass and in genitourinary tract (GU) mass in TRAMP+/– HRV mice. Organ masses were determined for 26- to 28-week-old TRAMP+/– HRV (n = 106 mice) and TRAMP+/– (n = 119 mice) animals. Mice that died before the analysis point (n = 9 for TRAMP+/– HRV and n = 10 for TRAMP+/–) were not included in the analysis. Median masses were 902 mg (GU) and 95 mg (PR) for TRAMP+/– HRV mice and 1042 mg (GU) and 112 mg (PR) for TRAMP+/– mice. Horizontal bars indicate median values. [View Larger Version of this Image (31K GIF file)]
 

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