Eur. J. Immunol. 2011. 41: 1410–1419 Immunomodulation 1415 Figure 4. Normal frequencies of mature T and B cells in double KO mice. WT, Rac1N, Rac3KO, and Rac1N/Rac3KO mice were sacriﬁced at P13. Spleens were explanted, reduced to single-cell suspensions, and analysed by ﬂow cytometry after staining with anti-CD4, CD8 and B220 mAbs. (A) Representative dot plots after gating on viable cells. (B–D) Frequency of (B) CD4-, (C) CD8- and (D) B220-expressing cells expressed as a percentage of total viable cells. Each symbol represents data from an individual mouse; horizontal bar represents the mean. po0.05; po0.005; po0.001 by one-way ANOVA with Bonferroni correction. Figure 5. Rac3 is not expressed in lymphoid tissues of WT and Rac1N mice. WT and Rac1N mice were sacriﬁced at P7. Protein lysates of brain, thymus (Thy), and spleen (Spl) from (A and B) WT and (B) Rac1N mice were immunoprecipitated with control (IP-C) or anti-Rac3 antibodies (IP-Rac3). Immunoprecipitates and lysates (200 mg) were blotted with an anti-Rac mAb recognizing both Rac1 and Rac3. Numbers on the left indicate molecular weight. Data are representative of three indepen- dent determinations. actin polymerization, Ca21 generation, and Erk and p38 activa- tion during T-cell stimulation and Th1 differentiation [21]. Furthermore, mice with hematopoietic-speciﬁc inactivation of Rac2 developed normally with weight and cellularity of spleens and thymi similar to those of control mice, and only revealed defective chemotaxis and superoxide production by neutrophils [22]. Whereas the absence of either Rac1 or Rac2 alone had no & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim effect on thymic development, their simultaneous deletion resulted in a developmental arrest, defective positive selection, and enrichment of CD4–CD8– DN thymocytes [23, 24]. B cell- lineage-speciﬁc inactivation of Rac1 in a Rac2-deﬁcient back- ground [25] showed no signiﬁcant changes in the number of pro-, pre-, and immature B cells, but profound defects in the number of splenic transitional types 1 and 2 and mature B cells [26]. Thus, while lymphoid-speciﬁc inactivation of Rac family members appears to interfere with proper central development and peripheral mature lymphocyte functions, our data rather indicate that the effects observed on the immune system are due to the ablation of Rac GTPases in the neuronal cells. Indeed, in the case of Rac1N/Rac3KO mice, the neuronal deletion of Rac1 and Rac3 did not cause a speciﬁc arrest at selected stages during thymic development and did not inﬂuence the ability of T lymphocytes to proliferate upon receptor engagement, thus supporting the lack of a T-cell intrinsic defect and rather the existence of a neuronal deﬁciency, impacting on organ innerva- tion. Indeed the thymus of double KO mice revealed a decreased level of the microtubule-stabilizing protein tau, a speciﬁc axonal marker [19]. Whether decreased innervation by tau1neurons might also explain the reduction in size of the spleen and even- tually of the kidney, heart, and liver with consequences that might become histologically visible at different times remains to be determined. The mechanisms that underlie the possible inﬂuence of neuronal function on the development of the immune system are poorly understood. Previous results suggested that the thymus receives parasympathetic innervations relatively early in onto- geny, and that nerve ﬁbres could be involved in the regulation of the organ activity through action upon the thymocytes and/or by modulation of the thymic vasculature or epithelial cell activity [27]. Direct innervation [1, 28, 29] as well as the release of hormones such as somatotrophin [30] have also been previously shown to exert an inﬂuence on primary and secondary immune organs, which are highly innervated by non-myelinated sympa- thetic projections from central motor neurons [31, 32]. We have shown here that perturbation of the function of neuronal Rac GTPases during neuronal development has a profound effect on the development of a functional immune system. To the best of our knowledge, this is the ﬁrst indication that perturbation of the machinery directly involved in neurite extension affects the development of the immune system. Moreover, our ﬁnding that the thymus of double Rac1/Rac3 mice showed decreased expression of the axonal protein tau provides support to a neuronal contribution to the homeostasis of primary lymphoid organs. In this direction, it has recently been shown that the degeneration of the autonomic innervation observed in the Twitcher mouse model for Krabbe disease, a neurological disor- der characterized by progressive demyelination, is also associated to thymic atrophy and loss of immune-competence [16], supporting a crucial role for neuronal function in the regulation of the functions of lymphoid organs. Progressive involution of the thymus starting at P9 in Rac1N/Rac3KO double-mutant mice closely resembles that found after P15 in the Twitcher mouse. As www.eji-journal.eu