When a contact unsusbcribes, they have their status on the AllSubscriber's List changed to Unsubscribe, so any messages sent to them will be held. In your case, even though you are using the same audience, they will not get any message as the system will check against the AllSubscriber's List.
Also as Markus Slabina has pointed out, this only holds Emails sent using the "Commercial Send Definition". Contacts will still continue to receive transaction emails (i.e receipts, password reminders etc).
There is also a slight nuance between a global unsubscribe (where a contact unsubscribes from all communications, registered in the All Subscribers) and a list unsubscribe (where a contact unsubscribes from a particular publication list)
Also as Adam Spriggs points out, If your preference center is logging an unsub event and the send is associated with a List or Publication, then the unsubscribe is isolated to that list membership. Also unsubscribes are localized to the business unit if designated in the BU configuration
For more information please visit: Unsubscribes