Eur. J. Immunol. 2011. 41: 1410–1419 Immunomodulation 1413 Figure 2. Thymic development declines after P9 in Rac1N/Rac3KO mice. (A) WT, Rac1N, Rac3KO, and Rac1N/Rac3KO mice were sacriﬁced at the indicated postnatal days, and (A) thymocyte and (B) splenocyte numbers were determined by ﬂow cytometry. Total numbers of viable cells are depicted. Each symbol represents data from an individual mouse; horizontal bar represents the mean. analyse this possibility we investigated whether Rac was expressed in the thymus and spleen by immunoblots. While Rac3 was readily immunoprecipitable from brain extracts, it remained undetectable in thymus and spleen extracts of both WT and Rac1N mice (Fig. 5A and B). Furthermore, Rac levels were unchanged in the thymus of Rac1N with respect to WT mice (Fig. 5B), supporting preserved Rac representation in the lymphoid compartment of Rac1N/Rac3KO mutant mice. Thus, the ﬁnding that Rac3 is not expressed in the thymus and spleen, together with the observation that lymphoid organ aplasia was only found after full deletion of Rac3 and neuronal deletion of Rac1 strengthens the notion that rather than a defect intrinsic to the lymphoid tissues, an extrinsic mechanism was responsible for the observed impairment of the immunocompetence. To further understand whether neuronal depletion of Rac1 and Rac3 had an impact on mature T-cell function, we char- acterized their surface and functional phenotype. In spite of the observed peripheral lymphopenia, central (CD44highCD62Lhigh) and effector (CD44highCD62Llow) memory cells were equally represented among CD41 (Fig. 6A) and CD81 (data not shown) T cells derived from Rac1N/Rac3KO and Rac3KO littermates. Most T lymphocytes maintained a na¨ıve CD44lowCD62Lhigh phenotype (Fig. 5A), proper expression of CD127 (Supporting Information Fig. 4B), and a low rate of cell division in short BrdU-pulse & 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim experiments in vivo (Supporting Information Fig. 4D). Further- more, ﬂow cytometry CFSE dilution assays revealed that CD41 (Fig. 6B) and CD81 (data not shown) T cells from Rac1N/Rac3KO and Rac3KO littermates proliferated to a similar extent after polyclonal CD3/CD28-driven stimulation in vitro. These results support the existence of extrinsic rather than cell autonomous defects. Axonal thymic innervation is suboptimal in Rac1N/ Rac3KO double KO mice at P13 Progressive loss in cellularity in the thymus and the spleen of Rac1N/Rac3KO mice was accompanied by an increased rate of apoptosis, revealed by immunohistochemistry with anti-cleaved caspase 3 mAb, both in the thymus cortex and medulla (Fig. 7), and in the spleen (Supporting Information Fig. 5C). As progressive loss of immunocompetence parallels the onset of neurological manifestations, we investigated whether defective organ innervation might account for the observed phenotype. Innervation of the thymus by non-myelinated sympathetic and parasympathetic nerves has been shown to be important for thymus function and to maintain T-cell maturation. We analysed the expression in the thymus of tyrosine hydroxylase (TH), a key enzyme in the synthesis of norepinephrine, which has been used previously to study axonal endings from sympathetic motor neurons in the thymic cortex, and associated with thymic involution in Twitcher mice [16]. We found that at P13, the density of sympathetic innervation of the thymus was comparable in Rac1N/Rac3KO double mutants and control Rac3KO mutants, indicating that a defect at this level might not account for the observed phenotype (Fig. 8A and Supporting Information Fig. 7). We also studied cholinergic innervation by immunohistochem- istry. Unfortunately, while antibodies to choline acetyltransfer- ase, a marker of cholinergic innervation, revealed speciﬁc signals on control brain sections, they failed to provide a reliable signal on thymic sections (data not shown). In the case of neuroﬁlament, expression in the thymus of double KO mice was comparable to that of control tissue (Supporting Information Fig. 8). Antibodies for other neuronal markers also failed to provide a speciﬁc signal or revealed high non-speciﬁc background on lymphoid tissue sections (Supporting Information Fig. 9A and data not shown). In the case of the Tuj1 mAb for b3 tubulin it has been found to be also expressed in lymphoid cells [17], and only detectable in neuronal terminals in immunologically mature mice by confocal microscopy [18]. We thus attempted to address the quantitative expression of neuronal markers in the thymus by immunoblotting of tissue extracts. Quantiﬁcation of b3 tubulin did not show any signiﬁcant differ- ences between the thymi of double KO and control mice (Supporting Information Fig. 9B). On the other hand, we found that the microtubule-associated protein tau, a speciﬁc axonal marker [19], was detectable in the thymus of P13 mice (Fig. 8B). Speciﬁcally, we observed a signiﬁcant reduction (E40%) of the major intermediate band (tau-m) recognized by the anti-tau www.eji-journal.eu