This is due to an oversight in _CallPythonObject in Modules/_ctypes/callbacks.c. The setfunc protocol is to return None if there's no object to keep alive. This isn't applicable to py_object (i.e. O_set in Modules/_ctypes/cfield.c). So the problem is the unconditional decref of None in the following snippet from _CallPythonObject:

    if (keep == NULL) /* Could not convert callback result. */
        PyErr_WriteUnraisable(callable);
    else if (keep == Py_None) /* Nothing to keep */
        Py_DECREF(keep);
    else if (setfunc != _ctypes_get_fielddesc("O")->setfunc) {
        if (-1 == PyErr_WarnEx(PyExc_RuntimeWarning,
                               "memory leak in callback function.",
                               1))
            PyErr_WriteUnraisable(callable);
    }

I'd rewrite it as follows:

    if (keep == NULL) { /* Could not convert callback result. */
        PyErr_WriteUnraisable(callable);
    } else if (setfunc != _ctypes_get_fielddesc("O")->setfunc) {
        if (keep == Py_None) { /* Nothing to keep */
            Py_DECREF(keep);
        } else if (PyErr_WarnEx(PyExc_RuntimeWarning, "memory leak "
                   "in callback function.", 1) == -1) {
            PyErr_WriteUnraisable(callable);
        }
    }