The question what is the nature of the NKG2D-L involved was not addressed

in our study and has to be elucidated in future work. The data may be explained in the light of the two-step process of NK-cell activation. This model postulates that activation of resting NK cells requires engagement of at least two receptors that convey a priming and a triggering event 26. IL-2 and NCR have been defined as priming and triggering molecules, Enzalutamide chemical structure respectively 26. Tumors may evade lysis through the lack of either efficient priming (type 1) or triggering (type 2). In our model, NK-cell activation correlated with MHC class I reduction of early-stage λ-myc lymphomas but not with their NKG2D-L levels (Fig. 3C, D), and in vitro lysis as well as tumor rejection not only required an activated NK-cell phenotype but were additionally dependent on NKG2D-L (Table 1, Fig. 4B). Since up-regulation of activation markers was mediated by MHC class Ilow target LY294002 chemical structure cells, by IL-15 or by DC, but not by NKG2D-L in the absence of the former stimuli, we suggest that NKG2D-L only act as a triggering signal, whereas MHC class Ilow cells provide a priming signal for NK cells. This was also suggested by our previous studies where we showed that in normal mice, transplanted MHC class I-positive lymphomas are effectively controlled provided (i) NK cells

are previously activated in vivo by injecting DC or CpG-ODN and (ii) sufficient amounts of NKG2D-L are expressed by the tumor 22. Transplanted MHC class Ilow lymphomas with sufficient NKG2D-L levels

are rejected even without preceding NK-cell activation 6. Whereas the priming signal provides unspecific activation, the tumor specificity of the NK-cell response may be mediated by the second signal. Taken together, apart from IL-2, other effectors that provide priming signals may include MHClow cells, DC, CpG-ODN or IL-15. Of course, it cannot be precluded that in λ-myc mice, other mechanisms may also act as priming signals and may be instrumental in inducing the activated phenotype of NK cells, for example microenvironment-derived cytokines. It is also possible that a higher fraction of immature NK cells is recruited to the tumor sites. The requirement of NKG2D-L Tolmetin for NK-cell triggering and tumor rejection also argues for its role in immune evasion. A synergism between “missing self” and NKG2D-mediated signals was also suggested by a previous in vitro study, but its implications for tumor surveillance in vivo and its significance in the context of the sequential NK-cell activation model were not addressed in this report 25. In transplantation models, injection of tumor cells with NK cell-activating potential gave rise to NK-cell cytotoxicity and IFN-γ expression and, eventually, to CTL responses 6, 43.

Thus, infections caused by S. epidermidis biofilms are particularly hard to eradicate. Biofilm formation by S. epidermidis is a multistep process and involves (1) attachment of the bacterial cells to a polymer surface or to the host-derived matrix that has previously coated the polymeric device and (2) accumulation to form multilayered cell clusters with cell-to-cell

adherence mediated by the production of a slimy extracellular matrix (Vadyvaloo & Otto, 2005). Several genes have been identified to play important roles in the biofilm formation of S. epidermidis (Mack et al., 2007). The atlE gene encodes autolysin AtlE, which mediates the initial attachment of S. epidermidis to a polymer surface (Heilmann et al., 1997), and the ica gene locus (icaADBC) encodes the biosynthesis

of polysaccharide intercellular adhesion (PIA), which is essential in the accumulation process (Heilmann et al., 1996). A few regulatory PLX4032 solubility dmso genes of biofilm formation were also identified (Mack et al., 2007). For example, the icaR gene affects the ability of biofilm formation by repressing the icaADBC operon (Conlon et al., 2002). The sarA gene encodes an activator of the icaADBC operon and positively regulates the biofilm formation of S. epidermidis (Tormo et al., 2005). The rsbU gene, a positive regulator of the alternative sigma factor, σB, positively regulates the biofilm formation of S. epidermidis by repressing icaR (Knobloch

et al., buy Fulvestrant 2004). Besides, LuxS (Xu et al., 2006) and Agr (Kong et al., 2006), a quorum-sensing system, also mediate biofilm formation in S. epidermidis. Recent work indicates that the regulation of biofilm formation in S. epidermidis is a complex networking and may involve mechanisms other than the ica system. The sarZ gene encodes a regulator that activates the transcription of the icaADBC operon in an icaR-independent manner and positively regulates the biofilm formation of S. epidermidis (Wang et al., 2008) Additionally, it is not uncommon to find clinical isolates that accumulate biofilm in an ica-independent mode (Ruzicka et al., 2004; Hennig et al., 2007; Qin et al., 2007), which indicates that there may be other mechanisms mediating biofilm formation. Protein degradation is essential for cell viability and homeostasis, and this process is commonly Thymidylate synthase mediated by ATP-dependent proteases. One notable case is ClpXP proteases, which function in degrading SsrA-tagged misfolded proteins (Gottesman et al., 1998), controlling the RpoS concentration in Escherichia coli (Gottesman et al., 1998) and regulating bacterial adaptation to stress (Porankiewicz et al., 1999). ClpXP proteases also play a crucial role in the biofilm formation of Pseudomonas fluorescens (O’Toole & Kolter, 1998), Streptococcus mutans (Lemos & Burne, 2002), Staphylococcus aureus (Frees et al., 2004) and S. epidermidis (Wang et al., 2007).

tuberculosis, and tetanic toxoid. Analysis of the specific immune response to mycobacterial antigens in comparison to the NS culture revealed an increase in spot-forming cells both in RR and RR/HIV when cells were stimulated with ML [Fig. 2a,b; RR NS = 135 (30–260) versus ML = 830 (50–5380); P < 0·01; RR/HIV NS = 202·5 (40·0–2560) versus ML = 2260 (50·0–7380); P < 0·05]. The ML p38 peptide

did not modulate the frequency of IFN-γ-producing cells after 48 hr of culture in the PBMCs of the different groups tested. ML peptide p69, which induces a T CD8 response, increased the www.selleckchem.com/products/AZD2281(Olaparib).html frequency of IFN-γ-producing cells in the PBMCs of RR patients when compared with NS cells [Fig. 2a,b; RR NS = 140 (50–250) versus PD0332991 purchase p69 = 830 (390–1000); P < 0·05]. However, no significant differences were observed between the PBMCs of RR/HIV stimulated or not with p69 (Fig. 2a,b). In addition, an increase in IFN-γ production in both RR and RR/HIV cells stimulated in vitro with p69 was also observed in contrast to cells in the HC group under the same conditions [Fig. 2b; HC 370 (70–650) versus RR/HIV 830 (250–1960); P < 0·05]. Although M. tuberculosis stimulation induced spots in both RR and RR/HIV cells, there

were no significant differences when compared with unstimulated cells or the HC group. Tetanus toxoid induced an increase in IFN-γ production only in the HC group when compared with NS cells (Fig 2a,b). As expected, PHA stimulation induced a greater number of spots in the HC, RR and RR/HIV groups when compared with the NS cells (Fig. 2a,b). HIV infection induces OSBPL9 significant immunological impairment, resulting in the increased expression of activation markers such as CD38 and HLA-DR in CD8+ T cells. This increased expression has been associated with particular clinical outcomes.[24] The next step was to evaluate whether ML stimulation modulates the activation of the immune system in RR/HIV co-infected patients. For this purpose, cellular activation parameters were investigated by analysing the surface expression

markers CD25, CD69 and CD38 in both CD4 and CD8 T cells in the PBMC cell culture after stimulation with irradiated ML for 24 hr. As observed in Fig. 3(a), ML increased CD4+ CD69+ T-cell frequencies in the HC and RR groups but not in the RR/HIV patients that presented a greater percentage of CD4+ CD69+ cells in the NS cell culture regardless of ML stimulus [Fig. 3a,b; HC NS = 2·78 (1·57–5·42) versus ML = 9·33 (4·97–17·43), P < 0·01; RR NS = 2·27 (0·57–8·72) versus ML = 10·39 (7·27–18·87), P < 0·01]. Although ML did not affect the expression of CD4+ T-cell activation markers in RR/HIV patients, an increase in CD8+ CD69+ T-cell frequencies in ML-stimulated cells was observed in this group compared with the NS cells [Fig. 4a,b; NS = 13·90 (5·16–22·80) versus ML = 44·49 (21·69–56·90), P < 0·05].

When we analysed the cytokines induced by immunization with recombinant proteins, it was found that rTcSPA, rTcSPR and rTcSPC induced Th1- and Th2-type cytokines and rTcSP induced Th2-type cytokines, while the four proteins induced the proinflammatory cytokines IL-6 and TNF. When the mice were immunized with naked DNA, the cytokine levels were lower than those detected after

immunization with the recombinant proteins, and cytokines were not detected after immunization with pBKTcSPC. Immunization with the plasmids pBKTcSP or pBKTcSPA induced a mixed Th1/Th2 T-cell response, and immunization with pBKTcSPR induced IL-10 and IFN-γ. The proinflamatory cytokine IL-6 was induced by three plasmids. However, the survival rate of the immunized mice at 60 days was very low in the mice immunized with recombinant proteins and variable in Silmitasertib ic50 the mice immunized with naked DNA.

Combining the decreased parasitemia and increased survival rate, the plasmids protected parasite infected mice in the following order: pBKTcSPR > pBKTcSPC > pBKTcSP > pBKTcSPA. The mice immunized with pBKTcSPR showed induction of IL-10 and IFN-γ. IL-10 is a cytokine that stimulates NK cells and promotes the recruitment of macrophages and neutrophils [50], while Dolichyl-phosphate-mannose-protein mannosyltransferase IFN-γ is required to activate macrophages and indirectly constitutes an important source of protective proinflammatory cytokines, which can effectively kill intracellular parasites such as T. cruzi Roscovitine purchase by nitric oxide (NO) dependent mechanisms [51]. However, significantly higher levels of IFN-γ were detected in the groups immunized with pBKTcSP and pBKTcSPA, which showed no reduction in parasitemia. Therefore, other factors may be involved in the reduction

of parasitemia. One of these factors could be IL-10, as it can participate as an immunoregulatory cytokine in the Th1 response [52], thereby preventing collateral damage generated by a strong immune response against the parasite and suppressing the development of inflammatory cell infiltrate that otherwise would be exacerbated. Therefore, resolution of T. cruzi infections depends on the host’s ability to mount a protective immune response. It has been proposed that an exacerbated response to infections may result in deleterious lesions [53]. One of the main differences detected in the mice immunized with pBKTcSPR compared with the other mice that were immunized with DNA or protein is the low level of serum IL-10. It has been shown that IL-10 increases host susceptibility to intracellular and extracellular micro-organisms.

[44] Treatment of NZB/W F1 mice with soluble TACI-Ig fusion protein prevented the development

of proteinuria and prolonged the survival of the animals.[44] These findings underscored the involvement of CDK phosphorylation BLys and its receptors in the development of SLE and hence the TACI-Ig was proposed as a promising treatment for human autoimmune disease. Furthermore, mice treated with exogenous BLys showed increased numbers of anti-chromatin B cells and augmented anti-dsDNA production.[45] Deletion of either BLys or BR3 critically impaired B cell maturation beyond the transitional developmental stages.[37, 40, 44, 46] T cell-deficient BAFF transgenic (Tg) mice developed SLE similar to T cell-sufficient BAFF Tg mice, and such features were associated with innate B lymphocyte Ivacaftor manufacturer activation and pro-inflammatory autoantibodies release. These data suggest that a dysregulated innate activation of B cells alone can drive disease independently of the T cells.[47] In human lupus patients, the circulating BLys level was raised in human lupus and is correlated with the anti-dsDNA level.[48] In a survey which measured the serum BLys level and disease activities, healthy subjects universally exhibits a normal longitudinal serum BLys profile, whereas escalated BLys level was observed in SLE patients (persistent rise in 25% and intermittent increase in another 25% of patients).

Increased cerebrospinal fluid levels of a proliferation-inducing ligand (APRIL) are also observed SLE patients with neuropsychiatric manifestations. The antagonism of BLys has been one of the important progresses in the treatment of SLE. Recently, belimumab Unoprostone was approved by the Food and Drug Administration (FDA) for the treatment of SLE. The efficacy and safety of belimumab in active SLE had been evaluated by two large multicentre randomized control trials. Both studies have demonstrated that the use of belimumab is associated with significant improvement in the SLE Responder Index (defined as ≥4 points improvement in SLEDAI) at 52 weeks, reduced SLE activity

and severe flares, as well as a comparable tolerability profile to placebo.[33, 34] Analysis of the pooled data from these two large trials showed that belimumab treatment improved overall SLE disease activity mostly in the musculoskeletal and mucocutaneous organ domains and less deterioration occurred in the haematological, immunological and renal domains.[49] In a post-hoc analysis of the BLISS study, the rates of renal flare, renal remission, renal organ disease improvement, proteinuria reduction and serologic activity all favoured belimumab, although the between-group differences in most renal outcomes were not significant. Among the 267 patients with renal involvement at baseline, belimumab resulted in greater renal improvement among patients receiving mycophenolate mofetil or those with active serology at baseline when compared with placebo.

GraphPad Prism version 5·0 software was used for statistical analyses. Results are expressed as mean ± standard deviation (s.d.). Relationships between different values were examined by Pearson’s correlation coefficient. A proportion of cell subsets were compared using Student’s t-test for normally or non-normally distributed subsets as appropriate. Statistical significance GSK2126458 in vitro was expressed by a P-value of < 0·05. MSCs isolated from

SSc patients were characterized by expressing the surface molecules CD90, CD105 and CD73. They did not express CD45, CD34 and CD14, as assessed by flow cytometry analysis. Moreover, MSCs showed normal ability in differentiating into osteoblast, adipocytes and chondroblast ABT-263 order in vitro (data not shown). The cumulative population doublings for MSCs isolated from SSc patients, as markers of the replication

rate, was consistently lower than that of HC cells (HC–MSCs 3·07 ± 0·38 versus SSc–MSCs 2·42 ± 0·16, P < 0·0070; Fig. 1a). In order to assess whether this reduced proliferation of SSc–MSCs was due to a growth-arrested status and the different cell cycle distributions with respect to HC cells, both SSc and HC–MSCs were analysed by flow cytometry after DNA staining with PI. Of note, no significant differences were observed between HC– and SSc–MSC, as cell cycle analysis revealed that the large percentage of MSCs obtained from both HC and SSc were in G0/G1 phases [HC–MSCs 80·23 ± 1·79 versus SSc–MSCs 83·00 ± 3·33%, P = not significant (n.s.)]; on the contrary, only a small population of cells were engaged in active proliferation (S+G2/M phases: HC–MSCs 18·75 ± 2·09 versus SSc–MSCs 15·65 ± 3·41%, Loperamide P = n.s., Fig. 1b), although not significantly. Because the above method does not distinguish between actively growing (G1) and growth-arrested (G0) cells, to distinguish more effectively between proliferative and resting

cells we assessed Ki67 gene expression by qPCR analysis. We found that MSCs isolated from SSc patients showed a lower expression of Ki67 gene when compared to HC cells (HC–MSCs 3·44 ± 0·20 versus SSc–MSCs 1·57 ± 0·53 mRNA levels, P = 0·019), confirming that the majority of cells was in G0 phase (Fig. 1c). No differences were observed in the proliferative ability of SSc–MSCs between the two disease subsets. Given the functional implications of the in-vitro senescence of MSCs, we employed β-Gal as a senescence marker. We observed that the percentage of β-Gal-positive stained cells was significantly higher in SSc when compared to HC (HC–MSCs 7·67 ± 4·41% versus SSc–MSCs 26·00 ± 4·34%, P = 0·03, Fig. 2a). Furthermore, we cultured both HC and SSc cells for 24 h in the presence of 5 μg/ml of doxorubicin, which represents a well-accepted in-vitro model to recreate the premature ageing of stem cells [29].

Naïve CD4+ T cells were labeled with CFSE and co-cultured with primary Th17 clones, and naïve CD4+ T-cell proliferation was determined

by FACS analysis of CFSE dilutions. As shown in Fig. 1E, we observed that these Th17 clones increased the proliferation of naïve T cells with several cell generations in the presence of OKT3, suggesting that these Th17 clones had effector T-cell function. Furthermore, Th1-C1, a Th1 cell line derived from a melanoma TILs NVP-BEZ235 ic50 which served as an effector T-cell control, also increased the proliferation of naïve T cells. In contrast, a CD4+CD25+ Treg line, which served as a suppressive control, strongly inhibited the proliferation of naïve CD4+ T cells. We confirmed these data using Autophagy Compound Library in vitro 3H-thymidine incorporation assays and obtained

consistent results 27. Taken together, our studies show that we had established Th17 clones derived from TILs and that possessed characteristics of the Th17 lineage. Recent studies in humans and mice have shown that Th17 cells retain greater developmental plasticity than other types of T-cell lineages 7, 18–20. In order to maintain the cell line stability and to obtain the quantities of Th17 cells needed for future studies, we attempted to expand these Th17 clones in vitro with a standard protocol, using irradiated allogeneic PBMCs in the presence of soluble OKT3 (100 ng/mL) and IL-2. This strategy has been successfully used to expand tumor-reactive TILs for adoptive transfer immunotherapy in cancer patients 40. After each of three expansion cycles, the expanded Th17 cells were rested for 3–5 days and then analyzed for their phenotypes. We first determined IL-17, IL-4, IFN-γ-producing

cell populations and FOXP3 expression in the Th17 cells using flow cytometric analyses. Results from a representative Th17 clone were shown in Fig. 2A. We unexpectedly found that the percentages of IL-17-producing cells markedly dropped following each unbiased expansion, from over 95% before expansion (E0) to only 60% after the third expansion (E3). In contrast, the percentages of IFN-γ-producing and FOXP3+ cells were significantly Prostatic acid phosphatase increased in the Th17 clones after three rounds of expansion, from 3.7 to over 60% and from 2 to 57%, respectively (Fig. 2A). Furthermore, increased proportions of IL-17+IFN-γ+ and IL-17+FOXP3+ double-positive cell populations were observed following expansion (40 and 42%, respectively, after the third round of expansion) (Fig. 2A). In addition, the percentages of IL-4-producing T-cell populations were low (<2%) in all expanded Th17 clones, and this did not change with the expansion. In addition, we obtained similar results from the other Th17 clones shown in Supporting Information Fig. 1. Notably, these expanded Th17 clones (E1–E3) maintained the same TCR-Vβ gene expression patterns as did the original Th17 clones (E0) (Fig. 1B and data not shown), suggesting the preservation of homogeneous clonality with progressive expansion.

Present study aims to evaluate the effect of renal lipid metabolism in the extrarenal vascular injury. Methods: Eight to nine week old male L-FABP Tg and its wild-type littermates (WT) mice were used in this study. The left middle cerebral artery was obstructed, and was released after 60 min later. At 24 hr the reperfusion (MCAOR), histological changes, ischemic or oxidative stress and lipid-related mRNA expression

in kidneys were evaluated. Histological findings were examined by hematoxylin eosin (HE) staining. Ischemic and oxidative stress were evaluated by pimonidazole, Aloxistatin in vivo HO-1 stainings and urinary 8-OHdG. mRNA expression of lipid-related enzymes were also evaluated by real time PCR. Results: Increase of intra- or extra-renal oxidative stress was detected by pimonidazole, and HO-1 staining and urinary 8-OHdG became clear in WT mice with MCAOR, but not in WT with sham opertion. There were significant differences in the renal expression of mRNA related to synthesis of fatty acid and cholesterol between WT and L-FABP Tg mice. Conclusion: It appears that the extrarenal vascular injury like MCAOR may induce Selleckchem MLN0128 renal oxidative stress and alteration of renal lipid metabolism, suggesting one of basic mechanisms in brain-renal association.

HAO LI1, YAN JUN-FANG1, WANG DE-GUANG1, XIE SHENG-XUE2, YUAN LIANG1 1Nephrology Department, the Second Affiliated Hospital of Anhui Medical University, Hefei; 2General Surgery Department, the Second Affiliated Hospital of Anhui Medical University, Hefei Introduction: The study was conduct to investigate the expression of α-klotho and fibroblast growth factor receptor (FGFR) 1c in the parathyroid tissue obtained from parathyroidectomy in chronic kidney disease patients. Methods: Hyperplastic parathyroid

glands (n = 90) were obtained from 24 patients with renal secondary hyperparathyroidism and surgically resected at Second Affiliated Hospital of Anhui Medical Farnesyltransferase University. Normal parathyroid tissue was obtained from glands inadvertently removed in conjunction with thyroidectomy from patients (n = 6) with thyroid carcinoma. The expression levels of α-klotho and fibroblast growth factor receptor (FGFR)1c in parathyroid tissue were detected by immunohistochemical staining technique. Results: Compared with the normal parathyroid tissue, the levels of α-klotho and FGFR1c were significantly reduced in hyperplastic parathyroid, and with the progress of parathyroid pathological degree. A significant positive correlation was observed between α-klotho and FGFR1c (r = 0.38, p < 0.01). Both α-klotho (r = −0.42, p < 0.01) and FGFR1c (r = −0.21, p < 0.05) correlated negatively with the volume of hyperplastic parathyroid. Conclusion: The expressions of α-klotho and FGFR1c decreased in parathyroid glands from patients with renal secondary hyperparathyroidism. The results suggested a pathogenesis linkage of α-klotho and FGFR1c in renal secondary hyperparathyroidism.

We focused on Vβ13 and analysed the nucleotide sequences containing the CDR3 of TCR-β. cDNAs obtained by reverse transcription-PCR (RT-PCR) of CDR3 combined with Vβ13 in CD8+ CD122+ CD49dhigh cells, CD8+ CD122+ CD49dlow cells and CD8+ CD122− cells were cloned and compared with one another. In the clones analysed to determine the nucleotide sequences in each cell population, the most common CDR3 sequences are listed in Fig. 4. There was only one CDR3 sequence that appeared twice during DNA www.selleckchem.com/products/17-AAG(Geldanamycin).html sequence analysis of CD8+ CD122− cells (Fig. 4c). In comparison with the result obtained from CD8+ CD122− cells, three

different CDR3 sequences were found twice in CD8+ CD122+ CD49dlow cells (Fig. 4b), possibly suggesting a higher frequency of expanded clones in this cell population. In contrast with the reasonably divergent CDR3 sequences in CD8+ CD122− cells, identical CDR3 sequences were Dabrafenib frequently found in CD8+ CD122+CD49dhigh cells. In particular, one CDR3 sequence (ASSYRGAEQF) was found five times in the first experiment and six times in the second independent experiment, which suggests the expansion of T cells possessing one characteristic TCR β-chain (Fig. 4a). Exp. 1 and Exp. 2 in Figure 4 were totally independent experiments started from different mice, from which we obtained four common sequences. This result confirms that such cloning of

identical TCRs from different mice is the reflection of universal events occurring in every ADAM7 mouse, not the accidental events that occurred in some cloning step. These CDR3 sequence data are consistent with the data from the immunoscope analysis. The most frequent sequence observed in CD8+ CD122+ CD49dhigh cells (ASSYRGAEQF) and possibly by addition of sequences with the same length (e.g. ASSFRNTEVF) corresponded to the highest peak in the immunoscope analysis of Vβ13 left side peak of the red line in Fig. 3a), which was not observed in CD8+ CD122+ CD49dlow cells and CD8+ CD122− cells. We further analysed cDNA obtained from CD8+ CD122− cells, CD8+ CD122+ CD49dhigh cells, CD8+ CD122+CD49dlow

cells by immunoscope using primers for TCR Jβ combined with Vβ13, and some Vαs combined with Cα. The results of Vβ13-Jβ and Vα-Cα are shown in the Supplementary material, Fig. S1a and S1b, respectively. Although, the immunoscopic analysis using Jβ primers showed some skewed peaks as expected, it gave no further information than the analysis by Vβs-Cβ There was no clonal or oligoclonal enrichment of specific amplification of TCR clones, which would attract our attention to go into further analysis. By the analysis of α-chain by immunoscope of 11 different Vαs, we have not found any remarkable skewing of peaks in CD8+ CD122+ CD49dhigh cells or CD8+ CD122+ CD49dlow cells. We only analysed 11 different Vαs to represent all the Vαs, which are estimated to be around 100.

Indirect allorecognition (i.e. involving recipient APCs) and direct allorecognition (i.e. involving donor APCs) occur in chronic and acute rejection, respectively 15. Thus, to analyze allograft-derived donor APCs in acute rejection process, we transplanted WT and CalpTG skin allografts onto BALB/C mice and examined the skin allograft survival. The survival of the C57BL/6 skin allograft was not affected by the presence of the transgene under these conditions (10 d for allografts derived from both WT and CalpTG donors;

n=5 and 6, respectively). To further assess whether the defective recruitment of T cells in CalpTG recipients was explained by a direct effect of calpastatin transgene in T cells, we transplanted BALB/C skin allografts onto recipient mice lacking T cells (RAG-1−/− mice) and reconstituted

with either WT or CalpTG spleen lymphocytes. At BGB324 day 8, allograft infiltration by CD3+ cells was significantly reduced after adoptive transfer of lymphocytes from CalpTG as compared with WT mice (59.6±15.0 versus 508.8±102.6 cells/high power field (HPF); n=4; p<0.004). Thus, calpastatin transgene expression in lymphocytes is sufficient to limit markedly LY294002 order skin allograft infiltration by these cells. Prior to gain insight onto how calpastatin transgene might affect T-cell recruitment, we verified the ability of calpastatin transgene to limit calpain activity in T cells. As assessed by measuring the calpain-specific cleavage of fluorescent 7-Amino-4-methylcoumarin (AMC) (Fig. 3A) and by measuring the 145/150-kDa spectrin BDP expression by Western DNA ligase blotting (Fig. 3B), calpastatin excess had no effect on calpain activity in resting T cells, but limited TCR-dependent calpain activation in

T cells exposed to αCD3 mAb. These data are consistent with a model in which calpains and calpastatin are not co-localized within the cell at rest. Calpastatin diffusion after calcium-related cell stimulation allows calpastatin to interact with calpains, thereby modulating its activity 13. Given that the calpain activity is involved in the activation of NF-κB and NFATc1 6, 9, two pathways leading to the generation of effector T cells 16, the nuclear expression of these transcription factors was also determined in T cells from WT and CalpTG. As shown in Fig. 3C and D, αCD3 mAb-induced nuclear translocation of NF-κB and NFATc1 was not modified by calpastatin transgene expression. These data suggest that the activation of NF-κB and NFATc1 is not essential for the control of T-cell recruitment by calpastatin transgene. Failure of T-cell recruitment into skin allograft is potentially explained by sequestration of circulating T cells into secondary lymphoid tissues and/or impairment in T-cell adhesion, migration and proliferation. We first determined by flow cytometry the number of CD3+ cells in spleen and graft-draining lymph nodes, 8 days after skin transplantation.