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. Author manuscript; available in PMC: 2015 Aug 17.
Published in final edited form as: Immunity. 2015 Feb 10;42(2):344–355. doi: 10.1016/j.immuni.2015.01.010

Figure 5. Identification of Two Mutants Defective in Their Ability to Inhibit Killing and to Hemagglutinate Red Blood Cells.

Figure 5

(A) YTS hTIGIT cells were incubated with 35S-labeled 721.221 cells and with 721.221 cells that were pre-incubated with or without the wild-type F. nucleatum 726 (designated FN726) or with mutants generated in the F. nucleatum 726 (designated with numbers, the two non-inhibitory mutants K50 and D22 are designated with letters and number). The bacteria to cells ratio was 600:1. Incubation was performed for 5 hr at an E:T ratio of 5:1. Figure shows several randomly selected mutants. *p < 0.0005. The error bars are derived from triplicates.

(B) Fifty microliters of human red blood cells (2% in PBS) were incubated with 50 μl of two fold serial dilutions (in PBS) of the indicated bacteria (original O.D was 1). Hemagglutination was evaluated following 120 min incubation at room temperature.

(C) Flow cytometry of FITC-labeled wild-type 726 bacterium and mutants K50 and D22 binding to 721.221 cells. The bacteria to 721.221 ratio was 600:1. The left top dot plot shows 721.221 cells without bacteria. The percentages of binding are indicated in the figure. See also Figure S1. Figure shows one representative experiment out of three performed.

(D) NK cytotoxicity assays were performed on 35S-labeled 721.221 cells incubated without or with F. nucleatum 726 (designated 721221+FN726) and with the two mutants K50 and D22 (designated 721221+K50 and 721221+D22, respectively). The bacteria to cell ratio was 600:1. The various cells were then incubated with primary activated human NK cells for 5 hr and at various E:T ratios indicated on the x axis. Figure shows one representative experiment out of three performed. *p < 0.005. The error bars are derived from triplicates.

(E) BWhTIGIT cells were incubated with F. nucleatum 726 and with the two F. nucleatum mutants K50 and D22 at bacterium to BW ratio of 300:1. Mouse IL-2 in the supernatants was determined using ELISA. Figure shows one representative experiment out of three performed. *p < 0.0005. The error bars are derived from triplicates.