*2.1. Decreased Severity and Incidence of rhG1-Induced Arthritis in Nkx2-3 Knock-Out Mice*

The spleen plays a critical role in the correct development and recirculation of B cell populations. The connection between the peritoneal B cell pool and the splenic B cells is also well established [26]. Recombinant human G1-induced arthritis is provoked by repeated intraperitoneal injections of the aggrecan G1 domain in the dimethyl-dioctadecyl-ammonium (DDA) adjuvant. By intraperitoneal immunization the first antigen encounter occurs in the peritoneal cavity, but soon the antigen is transported to other lymphoid tissues like the local lymph nodes through lymph vessels and the spleen through blood vessels. In GIA, both the local peritoneal activation of immune cells and the activation of the splenic cells are thought to be critical for the development of autoimmune arthritis [8].

Since Nkx2-3 knock-out mice were present with severe splenic developmental defects, we were curious to test whether rhG1-induced arthritis could be observed in them. To this end, we immunized Nkx2-3−/<sup>−</sup> and wild-type control BALB/c mice side-by-side. Nkx2-3−/<sup>−</sup> mice developed arthritis; however, to a lesser extent than the control BALB/c mice indicated by both the lower clinical severity scores (9.2 <sup>±</sup> 1.0 in Nkx2-3−/<sup>−</sup> versus 13.0 <sup>±</sup> 0.9 in BALB/c controls at Day 61) (Figure 1A) and the lower incidence (~70% in Nkx2-3−/<sup>−</sup> versus >90% in BALB/c controls after the third immunization) (Figure 1B). The clinical scores were supported by the limb thickness measurements, too. We measured the wrist (Figure 1(Ca)), leg (Figure 1(Cb)) and ankle (Figure 1(Cc)) thickness two weeks after the third immunization. We found that in arthritic Nkx2-3−/<sup>−</sup> mice, the limbs were significantly less swollen (Figure 1(Ca–Cc)) indicating a lower degree of inflammatory edema. Interestingly, in Nkx2-3−/<sup>−</sup> mice, the disease progression was also different from that seen in BALB/c mice: in GIA, typically, the arthritis develops progressively and once established it will not regress, however, in the case of Nkx2-3−/<sup>−</sup> mice we have seen the undulation of paw inflammation in some cases.
