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. Author manuscript; available in PMC: 2010 Sep 1.

Published in final edited form as: J Immunother. 2009 Sep;32(7):677–688. doi: 10.1097/CJI.0b013e3181ab1824

T cells from TRP1^-/-//DR4⁺ Tg mice react more strongly to TRP-1 than wild-type controls. (A) TRP-1^B-w (TRP-1^-/-) mice were bred with DR4 Tg mice to generate TRP1^-/-//DR4⁺ and control TRP1^+/-//DR4⁺ littermates. (B) Flow cytometry analysis: LN populations obtained after vaccination are phenotypically equivalent. TRP1^-/-//DR4⁺ (KO) and TRP1^+/-//DR4⁺ (WT) littermates were immunized twice with recombinant hTRP-1. 14 days after the second immunization, LN cells (WT, panels 1-3; KO, panels 4-6) were harvested and stained with antibodies specific for murine CD4, CD8, CD3, CD25, and Foxp3. The panels are representative of three distinct experiments. (C) LN cells obtained from TRP-1 KO mice are more potent than WT controls. Following immunization, LN cells (4×10⁵ per well) were stimulated ex vivo in ELISPOT plates with soluble anti-CD3 (2 μg/ml) or DR4⁺ EBV-B cells (1×10⁵ per well; 1088 EBV-B) pulsed with peptide (100 □M) or protein (50 μg/ml) or lysate (10⁵ cell equivalents). LN cells from both groups produce IFN-γ to anti-CD3 and EBV-B cells pulsed with mTRP-1_277-297 or hTRP-1_277-297 peptide, hTRP-1 or mTRP-1 protein, and to B16 and SK23 Mel lysate. Specific reactivity from both groups was blocked with anti-DR antibody HB55, but not with anti-MHC class I antibody W6/32. No reactivity observed to either the control peptide (HA306-318), protein (OVA) or lysates (MC-38, or 1102 Mel, both negative for TRP-1). (D) Ex Vivo ELISPOT titration assay: hTRP-1_277-297-specific T cells from TRP1^-/-//DR4⁺ mice are present at a higher frequency. TRP1^-/-//DR4⁺ and TRP1^+/-//DR4⁺ Tg littermates were immunized with hTRP-1 protein using the same immunization regimen. LN cells were harvested and stimulated (4×10⁵ per well in triplicates) ex vivo with hTRP-1_277-297 pulsed onto DR4⁺ EBV-B cells (1×10⁵ per well; 1088 EBV-B) at titrating (100 to 0.0001 mM) peptide concentrations. All experiments were performed 2-3 times with similar results.

T cells from TRP1^-/-//DR4⁺ Tg mice react more strongly to TRP-1 than wild-type controls. (A) TRP-1^B-w (TRP-1^-/-) mice were bred with DR4 Tg mice to generate TRP1^-/-//DR4⁺ and control TRP1^+/-//DR4⁺ littermates. (B) Flow cytometry analysis: LN populations obtained after vaccination are phenotypically equivalent. TRP1^-/-//DR4⁺ (KO) and TRP1^+/-//DR4⁺ (WT) littermates were immunized twice with recombinant hTRP-1. 14 days after the second immunization, LN cells (WT, panels 1-3; KO, panels 4-6) were harvested and stained with antibodies specific for murine CD4, CD8, CD3, CD25, and Foxp3. The panels are representative of three distinct experiments. (C) LN cells obtained from TRP-1 KO mice are more potent than WT controls. Following immunization, LN cells (4×10⁵ per well) were stimulated ex vivo in ELISPOT plates with soluble anti-CD3 (2 μg/ml) or DR4⁺ EBV-B cells (1×10⁵ per well; 1088 EBV-B) pulsed with peptide (100 □M) or protein (50 μg/ml) or lysate (10⁵ cell equivalents). LN cells from both groups produce IFN-γ to anti-CD3 and EBV-B cells pulsed with mTRP-1_277-297 or hTRP-1_277-297 peptide, hTRP-1 or mTRP-1 protein, and to B16 and SK23 Mel lysate. Specific reactivity from both groups was blocked with anti-DR antibody HB55, but not with anti-MHC class I antibody W6/32. No reactivity observed to either the control peptide (HA306-318), protein (OVA) or lysates (MC-38, or 1102 Mel, both negative for TRP-1). (D) Ex Vivo ELISPOT titration assay: hTRP-1_277-297-specific T cells from TRP1^-/-//DR4⁺ mice are present at a higher frequency. TRP1^-/-//DR4⁺ and TRP1^+/-//DR4⁺ Tg littermates were immunized with hTRP-1 protein using the same immunization regimen. LN cells were harvested and stimulated (4×10⁵ per well in triplicates) ex vivo with hTRP-1_277-297 pulsed onto DR4⁺ EBV-B cells (1×10⁵ per well; 1088 EBV-B) at titrating (100 to 0.0001 mM) peptide concentrations. All experiments were performed 2-3 times with similar results.