Fig. 1.
Fig. 1. (A) Initial reactivity of polyclonal CD8+ T cells to HCMV nucleoproteins. After two rounds of stimulation on HCMV-infected autologous fibroblasts, PBMCs were depleted of CD4+ T cells. The population of PBMCs before depletion was tested in a CRA for recognition against autologous (diamonds) or mismatched (squares) EBVLCL targets infected with vaccinia viruses for 16 hours expressing the HCMV proteins written above each panel. Methods for conducting the experiments can be found in Materials and Methods. Spontaneous lysis did not exceed 30% for any of the vaccinia cell lines tested. Ten thousand targets were assayed for each condition at the E:T indicated. (B) Recognition of HCMV nucleoproteins by clonal CD8+ CTL. CD4+ depleted PBMCs were cloned in 96-well plates as described in Materials and Methods. Wells were restimulated every 14 days. Colonies visible to the eye were expanded in successively larger wells, and aliquots of cells were tested against the recombinant vaccinia viruses containing HCMV proteins pp28, pp65, and pp150.11 Conditions for the CRA can be found in Materials and Methods and the legend for (a). Six representative CD8+ T-cell clones, 1G6, 3F6, 1H2, 3G2, 2H3, and 3-3F4, were tested at two different E:Ts against autologous and mismatched EBVLCL targets infected with the vaccinia viruses indicated at the bottom of the figure. Data from the E:T = 10 series are shown. / (C) Specific recognition of HCMV-infected fibroblasts by CD8+ CTL clone 3-3F4. Autologous (▨) and mismatched () fibroblasts were treated with IFN-γ for 2 days before infection with the AD169 strain of HCMV as described in Materials and Methods. Mock-infected fibroblasts were treated with medium alone. Two thousand fibroblasts were incubated with 3-3F4 T cells at an E:T of 10 in a standard 4-hour CRA. (D) MHC restriction of the 3-3F4 T-cell clone response to HCMV pp65. EBVLCL targets from individuals HLA-typed at the COH were infected with vaccinia viruses with (pp65Vac) or without (wtVac) recombinant HCMV pp65, or mock-infected. A standard CRA was performed using 10,000 targets at an E:T of 10. Cell lines expressing single allele matches from the autologous EBVLCL (A2 A28 B51 B60) were tested for recognition by the T-cell clone 3-3F4, as well as a completely matched (autologous) and mismatched EBVLCL targets. / (E) T-cell clone 3-3F4 recognizes HCMV pp65 with HLA A*0201. EBVLCL JY (▨) or Jurkat cells transfected with the HLA A*0201 genomic DNA clone (), or the untransfected parental Jurkat cells (▥) were tested for recognition by T-cell clone 3-3F4 in a standard CRA using 10,000 targets at an E:T of 10.

(A) Initial reactivity of polyclonal CD8+ T cells to HCMV nucleoproteins. After two rounds of stimulation on HCMV-infected autologous fibroblasts, PBMCs were depleted of CD4+ T cells. The population of PBMCs before depletion was tested in a CRA for recognition against autologous (diamonds) or mismatched (squares) EBVLCL targets infected with vaccinia viruses for 16 hours expressing the HCMV proteins written above each panel. Methods for conducting the experiments can be found in Materials and Methods. Spontaneous lysis did not exceed 30% for any of the vaccinia cell lines tested. Ten thousand targets were assayed for each condition at the E:T indicated. (B) Recognition of HCMV nucleoproteins by clonal CD8+ CTL. CD4+ depleted PBMCs were cloned in 96-well plates as described in Materials and Methods. Wells were restimulated every 14 days. Colonies visible to the eye were expanded in successively larger wells, and aliquots of cells were tested against the recombinant vaccinia viruses containing HCMV proteins pp28, pp65, and pp150.11 Conditions for the CRA can be found in Materials and Methods and the legend for (a). Six representative CD8+ T-cell clones, 1G6, 3F6, 1H2, 3G2, 2H3, and 3-3F4, were tested at two different E:Ts against autologous and mismatched EBVLCL targets infected with the vaccinia viruses indicated at the bottom of the figure. Data from the E:T = 10 series are shown.

(C) Specific recognition of HCMV-infected fibroblasts by CD8+ CTL clone 3-3F4. Autologous (▨) and mismatched () fibroblasts were treated with IFN-γ for 2 days before infection with the AD169 strain of HCMV as described in Materials and Methods. Mock-infected fibroblasts were treated with medium alone. Two thousand fibroblasts were incubated with 3-3F4 T cells at an E:T of 10 in a standard 4-hour CRA. (D) MHC restriction of the 3-3F4 T-cell clone response to HCMV pp65. EBVLCL targets from individuals HLA-typed at the COH were infected with vaccinia viruses with (pp65Vac) or without (wtVac) recombinant HCMV pp65, or mock-infected. A standard CRA was performed using 10,000 targets at an E:T of 10. Cell lines expressing single allele matches from the autologous EBVLCL (A2 A28 B51 B60) were tested for recognition by the T-cell clone 3-3F4, as well as a completely matched (autologous) and mismatched EBVLCL targets.

(E) T-cell clone 3-3F4 recognizes HCMV pp65 with HLA A*0201. EBVLCL JY (▨) or Jurkat cells transfected with the HLA A*0201 genomic DNA clone (), or the untransfected parental Jurkat cells (▥) were tested for recognition by T-cell clone 3-3F4 in a standard CRA using 10,000 targets at an E:T of 10.

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