1414 Veronica Basso et al. Eur. J. Immunol. 2011. 41: 1410–1419 Figure 3. Thymocyte subset composition is preserved in Rac1N/Rac3KO double KO mice. WT, Rac1N, Rac3KO, and Rac1N/Rac3KO mice were sacriﬁced at P13. Thymi were explanted, reduced to single-cell suspensions, and analysed by ﬂow cytometry after staining with anti-CD4, CD8 mAbs. (A) Representative dot plots after gating on viable cells. (B–E) Frequency of (B) CD4/CD8 DN, (C) CD4/CD8 double positive, and (D) CD4 or (E) CD8 single positive (SP) cells of individual mice. Each symbol represents data from an individual mouse; horizontal bar represents the mean. Data were not statistically different by one-way ANOVA with Bonferroni correction. antibody in the thymus of Rac1N/Rac3KO double KO mice with respect to control Rac3KO littermates (Fig. 8C). No signiﬁcant differences were detected between Rac1N and WT P13 littermates when quantiﬁed in independent experiments (Fig. 8B and C). The decrease of tau in the thymi of double KO mice with respect to control Rac3KO mice was evident already at P9 (Fig. 8D and E). No differences were detectable for either tau or b3 tubulin in the spleen from P13 double KO mice with respect to controls (Supporting Information Fig. 10). These results indicate that axonal endings within the thymus are compromised in Rac1N/Rac3KO double-mutant mice, providing a possible causa- tive link between neuronal perturbation and the observed thymic deﬁcits. Discussion The results from this study show that the speciﬁc neuronal deletion of the genes for the two GTPases Rac1 and Rac3 hinders the acquisition of immune-competence, and this can be explained, at least in part, by defective thymus innervations by tau-positive neurons. Rac1N/Rac3KO double-deﬁcient mice die by 2 wk of age [14]. By P13 Rac1N/Rac3KO double KO mice show clear neurological & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim symptoms [14] and reduced body weight when compared with control littermates. While brain, lungs, and lymph nodes of Rac1N/Rac3KO double KO were comparable to those of Rac3KO mice, kidney, heart, liver, thymus, and the spleen were found to be smaller in size. Behavioural analysis of the mice (observation) indicated the absence of nutritional problems. Furthermore, histological examination of tissue sections revealed normal for most of the organs, except for the spleen, suggesting that the loss of immune-competence is not the result of a general metabolic stress. Results rather indicate that neurological manifestations might impact the function of several body organs to different extents, possibly with different kinetics and mechanisms, to which the lymphoid organs might be most sensitive. In longitudinal analysis we observed that the thymus and the spleen progressively enlarged during postnatal life, doubling in size between P4 and P13 in normal WT mice, in mice lacking Rac3, or in mice with neuronal speciﬁc inactivation of Rac1. In contrast, in Rac1N/Rac3KO double-mutant mice thymic growth was normal up to P9 but severely hindered at later times. In the case of the spleen, but not the peripheral lymph nodes, differ- ences between Rac1N/Rac3KO and control littermates were evident even at P9. Of note, in addition to T cells, also B cells were reduced in numbers in Rac1N/Rac3KO mice. This was in spite of proper representation of BM precursors, and rather reﬂected a peripheral defect evidenced by the disrupted archi- tecture of the spleen. Reduced thymic output, together with hindered homeostasis of mature T (and B) cells caused a profound peripheral lymphopenia in the double KO animals. Of note, although cells of maternal origin might contribute to the circulating pool of mature T cells, their frequency in offspring is generally very low (o0.1%) [20]. Since double-mutant mice are indistinguishable from controls up to P7, it seems unlikely that maternal lympho- cytes account for all circulating cells. In spite of the lymphopenic environment, most of the CD41 and CD81 T cells in the periphery maintained a na¨ıve phenotype (CD44low, CD62Lhigh, CD127high), and a very low frequency of cells incorporated BrdU in short pulse experiments in both double KO and control littermates. These ﬁndings suggest that proliferation of mature T-cell precursors, commonly observed in lymphopenic conditions and known to cause na¨ıve T cells to acquire a memory phenotype, does not play a relevant role by this early age of postnatal life. Thus, the lower T-cell numbers observed in double KO mice have to be ascribed to extrinsic mechanisms rather than to defects intrinsic to the lymphocytes. Accordingly, (i) T cells do not express Rac3, but rely on Rac1 and Rac2 [9, 10] that were not affected in the lymphoid organs of Rac1N/Rac3KO mice; (ii) T cells derived from Rac1N/Rac3KO double-mutant mice were able to respond to TCR/CD28 stimulation, and to proliferate to extents comparable to those of WT cells, supporting a preserved functionality. Accordingly, previous reports indicated that dele- tion of the Rac1 and Rac2 genes within the lymphoid cells evoked immune phenotypes that differed from those described here. For instance, Rac2-deﬁcient mice revealed normal thymocyte development or positive/negative selection, and only impaired www.eji-journal.eu