Since mid-July 2016, the comment depth limit on Explosm.net has been six: a top-level comment and five layers of replies. Deeply nested comments lack a "Reply" link. This was instituted after between Morse-coded roleplays between Cteklite and Noodle caused the page to become unreadably narrow and long.
For a while, it was traditional to make a reply chain to the earliest post:
First
- Gay
- First
- Pansexual
- Blue shell
- Last
- Blue shell
- Pansexual
- First
The first three are from #3074. It was extended in a fan comic posted to Meme Genius Wannabe's microblog in September 2016. The blue shell is the Spiny Shell from Nintendo's Mario Kart video games, which seeks out the driver in first place and brings his vehicle to a dead stop.
This limit is enforced solely by the HTML rendering of the comment section, not by the form that adds a comment.