1416 Veronica Basso et al. Eur. J. Immunol. 2011. 41: 1410–1419 in Rac1N/Rac3KO double-mutant mice, Twitcher mutants also revealed a normal development of thymi and peripheral lymphoid organs during the initial postnatal life. Thus, progres- Figure 6. Mature T cells from double KO mice reveal a naı¨ve phenotype and normal proliferative capacities. (A) Rac3KO and Rac1N/Rac3KO mice were sacriﬁced at P13. Splenocytes were recovered and analysed by ﬂow cytometry following staining with anti-CD4, CD44, and CD62L mAbs. The plots are gated on viable CD41 T cells. Data from one representative mouse out of ten analysed are depicted. The frequency of naı¨ve (CD44low, CD62Lhigh), central memory (CD44high, CD62Lhigh), and effector (CD44high, CD62Llow) cells is depicted. (B) Splenocytes were labelled with CFSE and cultured in the absence (Nil) or presence of CD3/CD28 mAbs. After 5 days cells were stained with anti-CD4 and CD8 mAb and analysed by ﬂow cytometry. Representative histograms depict viable CD41 cells. Similar results were obtained for CD81 cells (data not shown). sive loss of immune-competence, best explained by thymic invo- lution, found at P13 and P30 characterize both the Rac1N/ Rac3KO and Twitcher mice, respectively, which substantially differ in causes and manifestation of neurological dysfunctions but recapitulate similar immunological phenotypes. Of note, while there is a predominate sympathetic (catecholamine) input to all components of the immune system, afferent innervation might be limited to lymph nodes and BM. Thus, differences in organ innervations, in the relative contributions of Rac1 and Rac3 to the development of nerve ﬁbres serving distinct organs, or in innervation of the organ-associated vasculature, might account for the fact that functionality of spleen and thymus but not of BM or lymph nodes are impeded during initial postnatal life. Similarly, the ﬁnding that both the thymus and spleen mainly beneﬁt from sympathetic nerves, which, however, arise from different ganglia (from the upper paravertebral ganglia of the sympathetic chain and the superior mesenteric and celiac gang- lionic plexuses respectively [33]), might explain the difference in the timing of symptom manifestation. Hypoplasia and abnormal maturation of lymphoid organs have been documented in other diseases, including the wasted mouse and the MND2 mouse models of motor neuron disease [34], or the staggered mouse [35], and after experimental axonal loss [36–38]. Our data support this notion and further indicate that neuronal defects impede immune-competence regardless of the time of occurrence. Given the crucial role for neuronal function in the regulation of central and peripheral competence, it would be important to deﬁne appro- priate monitoring and therapeutic options for patients with neurological dysfunctions likely at risk for immune-incompetence development. Figure 7. Thymic involution in double KO mice reﬂects increased apoptosis. Rac3KO and Rac1N/Rac3KO were sacriﬁced at P13. Cryosections from the thymus were analysed by hematoxylin and eosin staining, and immunohistochemistry with anti-cleaved caspase 3 (c-Casp3) antibody. Bar: 50 mm. & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu