Eur. J. Immunol. 2011. 41: 1410–1419 ﬁbres within lymphoid organs, which release norepinephrine that can bind to adrenergic receptors expressed by various cell types in the lymphoid organs [2]. The close proximity of sympathetic nerve terminals to immune cells provides a mechanism for the speciﬁc targeting of catecholamines to immune cells [3, 4]. On the other hand, little is known about the role of, and the molecular mechanisms underlying, the innervation of the immune system. Rac GTPases are members of the Rho family, which positively regulate the dynamics of the actin cytoskeleton, thus affecting cell morphology and development [5]. It is known that the endogenous Rac GTPases speciﬁcally expressed in each organ play important functions in the development of the neural and immune system respectively [6–8]. Speciﬁcally, two of the three mammalian Rac GTPases are expressed in the immune system: the ubiquitous Rac1 and the hematopoietic-speciﬁc Rac2 proteins [9, 10]. On the other hand, the neural-speciﬁc Rac3/Rac1B GTPase is co-expressed with Rac1 in the nervous system [11, 12], while it is undetectable in the lymphoid organs [13]. We have generated a number of KO mice in which either one or both Rac1 and Rac3 genes have been deleted during neuronal development [14]. These mice include the full deletion of the neural-speciﬁc gene for Rac3 (Rac3KO), and the conditional neuronal deletion of the gene for Rac1 by the Synapsin-Cre transgene (Rac1N). While single Rac3KO or Rac1N mutant mice do not show evident phenotypes and develop normally, Rac1N/Rac3KO double mutant mice are neuro- logically impaired, and die around P13 (postnatal day 13), with identiﬁable speciﬁc defects in the development of the brain [14]. In this study, we have addressed the indirect effects of the deletion of the neuronal Rac proteins on the development of the immune system. We found that the simultaneous inactivation of neuronal Rac1 and Rac3 causes speciﬁc defects in the development of the spleen and thymus of the Rac1N/Rac3KO double KO mice, and a concomitant defect in the innervation of the thymus of Rac1N/Rac3KO mice. Our results show for the ﬁrst time that the impairment of speciﬁc molecular mechanisms underlying neuronal development has striking effects on the development of the immune system, and support the notion of a direct important implication of innervation on the development of the lymphoid organs. Results Neuronal deletion of Rac1 and Rac3 impacts on lymphoid organ development Rac1N/Rac3KO double-deﬁcient mice have abnormal postnatal development with severe runting and death by 2 wk of age [14]. By P13 Rac1N/Rac3KO double KO mice show clear neurological symptoms [14], are smaller than control littermates (data not shown), and reveal a lower body weight (Fig. 1A). We noticed that by this time, the size (Fig. 1B and D) and the cellularity (Fig. 1C and E) of the thymus and the spleen were severely reduced in the double-mutant mice with respect to Rac3KO littermates (Fig. 1B and D) and to Rac1N and WT mice (data not shown). Accordingly, the total numbers of thymocytes and & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Immunomodulation 1411 splenocytes in Rac1N/Rac3KO double KO mice were also decreased when compared with WT, Rac1N, and Rac3KO littermates (Fig. 1C and E). In contrast, the cellularity of peripheral lymph nodes was comparable in double KO and control littermates (Supporting Information Fig. 1). To better understand this phenomenon we performed longitudinal analyses. Mice were sacriﬁced at P4, P7, P9, and P13 and organ cellularity was assessed by viable counts. While thymic growth was comparable up to P9 in all the mice and continued to grow in WT, Rac1N, and Rac3KO littermates, thymic involution was found by P13 in Rac1N/Rac3KO double-mutant mice (Fig. 2A). In the case of the spleen (Fig. 2B), differences were already evident by P9, a time at which both neurological manifestation and reduced body weight are already evident [14]. With the aim of deﬁning the immunophenotype of Rac1N/ Rac3KO mice, primary and secondary lymphoid organs were examined by ﬂow cytometric analysis using lineage-speciﬁc markers during disease progression at P13, a time at which the cellularity of both thymus and spleen is decreased in Rac1N/ Rac3KO mice. The relative distribution of CD4 CD8 double negative (DN) cells and subsets (identiﬁed by the relative expression of CD44 and CD25, data not shown), CD41CD81 double positive, and CD41 or CD81 single-positive thymocytes was comparable in WT, Rac1N, Rac3KO, and double-mutant mice at P13 (Fig. 3). Similar results were obtained at P9 (Supporting Information Table 1). In spite of the fact that the relative repre- sentation of thymocyte precursor was mostly preserved, as a consequence of overall decreased cellularity (Fig. 1C and E), the total numbers of all major subsets were reduced in Rac1N/Rac3KO double KO mice when compared with WT, Rac1N, and Rac3KO littermates (data not shown). This might suggest that thymic defects could not be ascribed to a speciﬁc developmental arrest but rather to a general defect of organ function. At P13, CD4 and CD8 mature T cells were found to be slightly enriched in frequencies in the spleen of double-mutant mice when compared with WT mice, Rac1N, and Rac3KO littermates (Fig. 4A–C), although reduced in total number as a consequence of defective cellularity of the organ at this time (Fig. 1E). Also the frequency of mature B2201 cells was found to be normal (Fig. 4D), while their total numbers were lower in Rac1N/Rac3KO double KO mice when compared with WT, Rac1N, and Rac3KO littermates (data not shown). This was in spite of their normal representation in the BM, which revealed comparable frequencies of eritroid, myeloid, and hematopoietic cells in WT and mutant mice (Supporting Information Fig. 2). We next investigated the histological appearance of cryo-preserved P13 tissue sections. The general architecture of the thymus was preserved in Rac1N/ Rac3KO with proper cortico-medullary differentiation. Immuno- staining of stromal components indicated that both mTEC (cytokeratin 5; CK51) and cTEC (CK81) were correctly posi- tioned. Similarly, immunoreactivity for claudin-4, detecting mature mTEC, was comparable (Supporting Information Fig. 3). Immature thymocytes, identiﬁed by the TdT, were also found to be correctly positioned and mostly distributed within cortical region with only rare TdT1 cells present within the medulla of www.eji-journal.eu