Tetramer-blocking assay for defining antigen-specific cytotoxic T lymphocytes using peptide-MHC tetramer

Abstract

Peptide-MHC tetramers have been engineered to allow accurate detection of antigen-specific cytotoxic C lymphocytes (CTL) by flow cytometry. Here, we propose a novel use for peptide-MHC tetramers in the specific and sensitive analysis of the cytotoxic function of antigen-specific CTL by blocking MHC-restricted antigen-specific cytotoxicity. We found that pretreatment of ovalbumin (OVA)-specific CD8(+) CTL (OT-1 CTL), derived from OT-1 T-cell receptor (TCR)-transgenic mice, with OVA(257-264) peptide-H-2K(b) tetramer caused a marked inhibition of the cytotoxicity against OVA-expressing EG-7 tumor cells. OVA(257-264) peptide-H-2K(b) tetramer did not block the cytotoxicity mediated by 2C mouse (H-2(b))-derived CD8(+) CTL, which recognize allo (H-2L(d)) antigens. Moreover, OT-I CTL activity was not inhibited by an irrelevant HBV(208-216) peptide-H-2K(b) tetramer. These results indicate that the blocking of CTL activity with peptide-MHC tetramer was caused by interference with the interaction between the TCR and H-2K(b)-OVA(257-264) peptide complex, but not with the CD8-MHC class I interaction. The blocking activity of OVA(257-264) peptide-H-2K(b) tetramer was reversible because OT-I CTL pretreated with the tetramer recovered their cytotoxicity after culturing with interleukin-2 for 24 h. The same results were also demonstrated in freshly isolated, in vivo-primed OT-1 CTL sorted by the tetramer. These results demonstrate that peptide-MHC tetramer is a useful tool for defining MHC-restricted antigen-specific CTL function. Moreover, our finding implies that the measurement of CTL activity immediately after tetramer-guided sorting is not a suitable method for evaluating the function of in vivo-induced tetramer-positive CTL. We believe that the tetramer-blocking assay presented here will be useful for functionally monitor the induction of MHC-restricted antigen-specific CTL during vaccination therapy against tumor and infectious diseases.

Figure 1

Cytotoxicity of OVA‐specific CTL is inhibited by OVA‐MHC tetramer. OT‐I CTL were generated in vitro with MMC‐treated C57BL/6 splenocytes and the necessary cytokines. (a) FCM analysis of in vitro‐generated non‐specific CD8⁺ T cells from wild‐type C57BL/6 mice was carried out using PE‐labeled SIINFEKL/H‐2K^b tetramer (OVA‐Tet‐PE) and FITC‐labeled anti‐CD8 mAb. (b) FCM analysis of the generated OT‐I CTL was carried out using PE‐labeled OVA‐MHC tetramer and FITC‐labeled anti‐CD8 mAb. The indicated number shows the percentage of the gated population within the total cell population. OT‐I CTL were added to produce the indicated effector : target (E:T) ratios, and then a standard 4‐h ⁵¹Cr release assay was carried out. (c) Cytotoxicity against E.G7 (•), EL‐4 (▴) and MBL‐2 (▵) cells mediated by OT‐I CTL without tetramer labeling. (○), Cytotoxicity against E.G7 cells mediated by OT‐I CTL prelabeled with OVA‐MHC tetramers. (d) 1 × 10⁶ OT‐1 CTL were labeled with 0.025 µg/mL, 0.175 µg/mL and 3.3 µg/mL OVA‐MHC tetramer and 3.3 µg/mL HBV‐MHC tetramer. The cytotoxicity of OT‐I CTL with or without tetramer was examined against E.G7 or EL‐4 cells (E:T = 4:1). The bars represent the mean ± SE of duplicate samples.

Figure 2

OVA‐specific cytotoxicity of OT‐1 CTL is not affected by irrelevant peptide‐loaded cognate MHC tetramers, and OVA‐MHC tetramer does not inhibit allo‐specific cytotoxicity. (a,b) OT‐1 CTL were labeled with (○) or without (•) OVA‐MHC tetramers, or with PE‐labeled HBV peptide (ILSPFLPLL)/H‐2K^b tetramers (HBV‐MHC tetramers) (▵), added to produce the indicated effector : target (E : T) ratios, and then a standard 4‐h ⁵¹Cr release assay was carried out against (a) LLC‐OVA and (b) E.G.7 cells. Cytotoxicity mediated by OT‐I CTL without tetramer binding against (a) LLC (▵) and (b) EL‐4 (▵) cells. (c) H‐2L^d‐specific 2C cells were prepared by culturing with MMC‐treated BALB/c splenocytes and the necessary cytokines. On day 7, the generated 2C cells were stained with or without OVA‐MHC tetramers, adjusted to give the indicated E:T ratios and then standard 4‐h ⁵¹Cr release assays were carried out. Cytotoxicity against P815 (H‐2^d) cells mediated by 2C cells with (○) or without (•) tetramers is indicated. Cytotoxicity mediated by 2C cells in the absence of tetramer labeling against MBL‐2 (H‐2^b) cells (▴) and E.G7 (H‐2^b) cells (▵). The bars represent the mean ± SE of duplicate samples.

Figure 3

OVA‐specific cytotoxicity of OT‐I CTL prelabeled with OVA‐MHC tetramer is reversible if they are cultured for 24 h in the presence of IL‐2 before assay. (a,c) Cytotoxicity of OT‐I CTL was detected by a 4‐h ⁵¹Cr release assay against (a) LLC‐OVA and (c) E.G7 cells with (○) or without (•) OVA‐MHC tetramer. The OT‐I CTL pretreated with OVA‐MHC tetramer were cultured with IL‐2 for 24 h, and subjected to 4‐h ⁵¹Cr release assay. (b) Cytotoxicity against LLC‐OVA (○) or LLC (▵) cells mediated by the 24 h‐incubated OT‐I CTL pretreated with OVA‐MHC tetramer. (d) Cytotoxicity against E.G7 (○) or EL‐4 (▴) cells mediated by the 24 h‐incubated OT‐I CTL pretreated with OVA‐MHC tetramer. (e) FCM analysis of OT‐I CTL labeled with OVA‐MHC tetramer. (f) FCM analysis of 24 h‐incubated OT‐I CTL prelabeled with OVA‐MHC tetramer. The indicated number shows the percentage of the gated cell populations in the total cell population. The bars represent the mean ± SE of duplicate samples.

Figure 4

The cytotoxicity of freshly isolated OT‐1 CTL is inhibited by OVA‐MHC tetramer. (a) The cytotoxiciy of OT‐1 CTL isolated by OVA‐MHC tetramer (○) or by anti‐CD8 monoclonal antibody (•, ▴) was determined by 4‐h ⁵¹Cr release assay against E.G7 (•, ○) or against EL‐4 (▴) cells. (b) Upon 24 h incubation with IL‐2 after tetramer treatment, the cytotoxicity of the OT‐1 CTL was determined against E.G7 (○) and EL‐4 (▵) cells. (c) FCM analysis of freshly isolated‐OT‐I CTL labeled with OVA‐MHC tetramer. (d) FCM analysis of 24 h‐incubated OT‐I CTL prelabeled with OVA‐MHC tetramer. The bars represent the mean ± SE of duplicate samples. Indicated numbers stand for the percentages of the gated populations within the total cell population.