This scene between John and Irene in ASiB is so crucial. Because, apart from the occasional offhand remark from Mrs Hudson, which can easily be brushed aside as his landlady having a joke, John has never been confronted about his feelings for Sherlock before. It’s in response to Irene’s suggestion that John and Sherlock are a couple, that John remarks “I’m not actually gay” (obviously, ‘not gay’ doesn’t equate to ‘straight’, but that’s a matter for another day, considering that discussing all the clues towards John’s bisexuality would probably warrant a 5000-word essay). Irene then counters “well, I am”, meaning, of course, that she considers herself to be gay. And there are some fairly obvious clues towards this as well; she seems to favour females in her work as a dominatrix, and also lives with someone called Kate - in their scenes together, they demonstrate a mutual attraction. Most tellingly, she then says “look at us both.” This line suggests that both John and Irene are in the same position. That is, they both like women, but have also fallen for Sherlock. And John doesn’t deny this! Going by his reaction in the final gif, he is clearly aware of what Irene is implying. And you can physically see him considering her words, and realising, I think, for the first time, that she is right.