Supporting Information

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Fig. 6. Viral load and LCMV-specific CD8⁺ T cell kinetics after l.d. or h.d. infection. (A) C57BL/6 mice were infected with 200 pfu (l.d.) or with 10⁶ pfu (h.d.) LCMV Docile, and LCMV titers were determined in blood and spleen at the indicated time points. (B) LCMV gp33-specific of np396-specific CD8⁺ T cell frequencies or numbers were determined by tetramer staining at the indicated time points in spleen. Symbols represent the average of at least three mice per group. One of three similar experiments is shown.

Fig. 7. Viral load kinetics after l.d. or h.d. infection with or without previous adoptive transfer of TCR tg CD8⁺ T cells. 10⁴ naïve Ly5.1⁺ TCR tg CD8⁺ T cells were adoptively transferred into naïve C57BL/6 mice (closed circles) and were infected the next day with 200 pfu (l.d.) or with 10⁶ pfu (h.d.) LCMV Docile. Similarly, naive C57BL/6 mice were infected with 200 pfu (l.d.) or with 10⁶ pfu (h.d.) LCMV Docile (open symbols). LCMV titers were determined in blood and spleen at the indicated time points. Symbols represent the average of at least three mice per group. One of two similar experiments is shown.

Fig. 8. In vivo BrdU uptake of TCR tg CD8⁺ T cells. 10⁴ naïve Ly5.1⁺ TCR tg CD8⁺ T cells were adoptively transferred into naïve C57BL/6 mice and infected the next day with 200 pfu (l.d.) or with 10⁶ pfu (h.d.) LCMV Docile. Sixteen days later, mice were given BrdU in the drinking water for 5 consecutive days before spleen cells were harvested and stained for CD8, Ly5.1, and BrdU. While >40% of TCR tg CD8⁺ T cells were cycling in the 5-day period in h.d. infected mice, only 2.1% of TCR tg CD8⁺ T cells were cycling in l.d. infected mice, indicating the continued presence of cognate antigen in vivo in h.d. infected mice. The left and the middle plots are gated on CD8⁺ Ly5.1⁺ T cells; the right plot (naive C57BL/6 mouse) is gated on total CD8⁺ T cells. One of three representative stainings is shown of three independent experiments.

Fig. 9. In vivo activation of TCR tg CD8⁺ T cells in chronically infected mice with or without previous adoptive transfer of TCR tg CD8⁺ T cells. 10⁴ naïve Ly5.2⁺ TCR tg CD8⁺ T cells were adoptively transferred into naïve C57BL/6 mice and were infected the next day with 10⁶ pfu (TCR tg and h.d.) LCMV Docile. Alternatively, naive C57BL/6 mice were infected with 10⁶ pfu (h.d.) LCMV Docile. 22 days later, 4 ´ 10⁶ naïve CFSE-labeled Ly5.1 TCR tg CD8⁺ T cells were transferred into these infected recipients as well as into naïve C57BL/6 mice and 3 days after transfer proliferation of Ly5.1⁺ CD8⁺ T cells was assessed in spleen and LN by flow cytometry. There was no difference in proliferation of TCR tg CD8⁺ T cells in h.d. infected recipients irrespective of whether they had or had not received 10⁴ TCR tg CD8⁺ T cells before infection, indicating that the in vivo persisting virus had not escaped recognition by TCR tg CD8⁺ T cells. One representative staining of three mice per group is shown.

Fig. 10. Intracellular p-ERK staining. 10⁴ naïve Ly5.1⁺ TCR tg CD8⁺ T cells were adoptively transferred into naïve C57BL/6 mice and mice were infected the next day with 200 pfu (l.d.) or with 10⁶ pfu (h.d.) LCMV Docile. Twenty days later, spleen cells were harvested and stimulated for 20 min ± PMA. Cells were stained extracellularly for CD8 and Ly5.1 and intracellularly for phosphorylated ERK. Plots are gated on Ly5.1⁺CD8⁺ T cells. Some cells were pretreated with 100 mM PD98059 (Middle) or 10 mM calphostin C (Bottom) for 2 h before stimulation. TCR tg CD8⁺ T cells from both l.d. and h.d. infected mice showed ERK phosphorylation upon stimulation, which was blocked by PD98059 or calphostin C, indicating that ERK phosphorylation is functional in cells from chronically infected mice and that both drugs were effectively interfering with ERK phosphorylation. One representative staining of three mice per group and two independent experiments is shown.

Fig. 11. Degranulation and IFN-g production of HIV- and CMV-specific CD8⁺ T cells. PBMC from 12 HLA-A2⁺ individuals with untreated advanced HIV infection were stained with anti-CD8 and HLA-A2 tetramers containing the following HLA-A2 restricted peptides: HIV Gag P17 peptide amino acid 77-85 (SLY, SLYNTVATL), HIV RT peptide amino acid 464-472 (ILK, ILKEPVHGV) and CMV pp65 peptide (pp65, NLVPMVATVQ). Alternatively, cells were stimulated with the indicated peptides (pp65, SLY or ILK) or with a pool of overlapping peptides spanning the HIV Gag p24 protein (Gag) and analyzed for degranulation and IFNg production. (A) Representative staining for CD107 and IFN-g after mock, staphylococcal enterotoxin B (SEB), pp65 or Gag stimulation. Cells are gated on CD8⁺ T cells. (B) Percentage of degranulating or IFN-g-producing CD8⁺ T cells upon Gag or pp65 stimulation. (C) Ratio of CD107⁺:tet⁺ CD8⁺ T cells or ratio of IFN-g⁺:tet⁺ CD8⁺ T cells specific for pp65, SLY or ILK. In case of the CMV-specific cells, an average of 55% and 60% of the pp65 tet⁺ CD8⁺ T cells were able to produce IFN-g or to degranulate, respectively. In contrast, only 20% of the tet⁺ HIV-specific CD8⁺ T cells were able to produce IFN-g, whereas 60-70% were able to degranulate.

Fig. 12. Purity of nuclear and cytoplasmic protein extracts. Nuclear and cytoplasmic extracts were prepared from 6 ´ 10⁵ CD8⁺ T cells according to the instructions of the supplier (NE-PER, Perbio, Basel, Switzerland). Purity of nuclear and cytoplasmic separation was analyzed by Western blot analysis using an anti-tubulin specific antibody or an anti RCC1-specific antibody. RCC1 is a guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin.

SI Materials and Methods

BrdU staining was essentially performed as described (1). Briefly, BrdU was added to the drinking water (0.8 mg/ml) for 5 days before spleen cells were harvested. Cells were first surface stained for Ly5.1 and CD8 and thereafter fixed /permeabilized for 10 min at room temperature by using 500 ml of FIX/perm solution (FIX/perm solution: FACSLyse (BD) diluted to 2´ concentration with H₂0 and 0.05% Tween 20 (Sigma). Cells were washed once with ice-cold PBS and resuspended in 500 ml of ice-cold 0.15 M NaCl. 1.2 ml of ice-cold 95% ethanol was added drop wise while gently agitating the cells followed by 30 min incubation on ice. Cells were washed with ice-cold PBS, resuspended in 1 ml of PBS, 1% (wt/vol) paraformaldehyde, 0.01% (vol/vol) Tween-20 and incubated for 30 min at room temperature. Cells were centrifuged directly at 4°C, resuspended in 1 ml of DNaseI solution (50 Kunitz units/ml), and incubated for 10 min at room temperature. Cells were washed with ice-cold PBS, resuspended in 0.1 ml of PBS containing anti-BrdU-FITC, and incubated for 30 min at room temperature. Cells were washed with ice-cold PBS and resuspended in PBS/0.1% PFA.

Intracellular staining for phosphorylated ERK was performed as described (2). Briefly, spleen cells were stimulated for 20 min with PMA (50 ng/ml), washed at 4°C, and stained anti Ly5.1 antibodies for 20 min at 4°C. Cells were washed with FACS buffer and fixed with 1.5% formaldehyde for 10 min at room temperature. Cells were pelleted by centrifugation, resuspended in 1 ml of 100% methanol, and incubated for 10 h at 4°C. Cells were pelleted and washed twice with FACS buffer, followed by incubation with anti-phospho-ERK antibodies (BS Biosciences, Basel, Switzerland) in FACS buffer for 30 min at room temperature. Cells were washed with FACS buffer and analyzed by flow cytometry.

Twelve untreated chronically HIV-1 infected HLA-A2-positive patients (SI Table 1) were recruited at the University Hospital Zurich. Written informed consent according to the guidelines of the local Ethical Committee was obtained from all patients.

PBMC were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation and cryopreserved until use. For stimulation of human CD8⁺ T cells, cryopreserved PBMC were thawed and recovered over night in medium. Cells were stimulated for 6 h analogous to mouse cells.

The HLA-A2-restricted HIV Gag P17 peptide amino acid 77-85 (SLY, SLYNTVATL), the HLA-A2-restricted HIV RT peptide amino acid 464-472 (ILK, ILKEPVHGV), and the HLA-A2-restricted CMV pp65 peptide (pp65, NLVPMVATVQ) were purchased from NeoMPS (Strasbourg, France); overlapping peptides spanning the HIV p24 protein (15-mers, overlapping by 11 aa, clade B consensus sequence) were provided by the AIDS Research and Reference Reagent Program, National Institutes of Health.

1. Tough DF, Sprent J (1994) J Exp Med 179:1127-1135.

2. Perez OD, Nolan GP (2002) Nat Biotechnol 20:155-162.