In mLNs, available MHC class II presented antigen may also comprise considerable proportions of intestinal antigen derived from food and bacterial flora. Therefore, we investigated the TCR sequence overlap of re-isolated donor Treg cells from spleen, pLN, mLN, and LPL (lamina propria lymphocytes) 9 wk after adoptive transfer of WT Treg cells as described for Fig. 2. We were able to analyze several thousands of

recovered Treg cells and revealed strikingly overlapping Tcra rearrangements in mLN and intestinal LPL (Fig. 5A). Comparing the 25 most abundant CDR3 sequences from each tissue, we found that mLN and LPL samples shared 14 out of 25 identical AA sequences, whereas only one was similar between pLN and mLN or pLN and LPL CCR antagonist (Fig. 5B and Table 1). Next, we asked to what extent such organ-specific expansion would be specific for Treg cells as compared with Foxp3− T cells. Therefore, we performed adoptive transfers of either pLN or mLN whole lymphocyte suspensions from CD45.1− WT mice into CD45.1+ TCR-Tg recipients (Fig. 6A). The percentage of input Foxp3+ Treg cells among all CD4+-gated T cells was similar in both cell suspensions. Nine wks after transfer of pLN cells, the frequency of Treg cells among all CD45.1−CD4+ input T cells was assessed. It had increased in spleen, pLN, and mLN (Fig. 6A and B), which is in line

with the Treg-cell expansion after transfer of purified Treg cells shown above. A decreased proportion among LPL may reflect antigen-specific expansion of Foxp3−CD4+ T cells. At the same time, transfer of mLN cells resulted in stable proportions of Treg cells in LPL and elevated frequencies in both mLN and Staurosporine pLN (Fig. 6A and B). Interestingly, expansion of mLN-derived Treg cells was similar in pLN and mLN, although lower than the expansion after transfer of pLN suspensions

(Fig. 6B). In conclusion, these results suggested that, besides homing receptor cues, organ-specific TCR shaping created distinct, highly before diverse but still overlapping TCR repertoires in pLNs and mLNs. After transfer, such locally optimized TCR repertoires supported the maintenance of donor Treg cells in their respective organs of origin. Next, we investigated the impact of Treg-cell repertoire diversity on their genuine function, i.e. their capacity to suppress T-cell activation. In an in vitro system based on T-cell activation with anti-CD3 mAb, Treg cells from TCR-Tg mice were equally efficient as Treg cells from WT mice in suppressing the proliferation of CD8+ and of CD4+ T cells (Fig. 7A and B). In contrast, in an experimental model of acute GvHD 35 less diverse Treg cells were less efficient than WT Treg cells in preventing the lethal disease (Fig. 7C and D). Co-transfer of allogeneic Treg cells derived from OT-II TCR-Tg mice showed only alleviation of the disease but not protection from GvHD (Fig. 7C and D). Taken together, these results suggest that the impact of TCR diversity on Treg-cell function is context dependent.

To distinguish between these two possibilities, we directly investigated whether constitutive activation of Btk had the capacity to change the B-cell Cabozantinib cost fate in the 3-83μδ Tg system. The 3-83μδ Tg encodes an antibody specific for MHC class I of the H-2Kk,b haplotype 29. On a non-autoreactive background, the expression of the 3-83μδ BCR commits B cells to the follicular or MZ subsets in the spleen. In these 3-83μδ BCR Tg mice only B cells that have edited their BCR are able to differentiate into

CD5+ B-1 B cells: all peritoneal B220lowCD5+ B-1 B cells have lost the 3-83μδ BCR specificity detected by the 54-1 anti-idiotypic antibody (Fig. 4A). We generated 3-83μδ Tg E-Btk-2 and EY-Btk-5 mice on the non-autoreactive H2-Kd background. As expected, in the spleen of these mice all conventional B220highCD5− B cells had high 54.1 reactivity. However, B220lowCD5+ B cells had lost their 54.1 reactivity (Fig. 4B), while surface Ig μ and κ expression levels were similar (data not shown). These results indicate that 3-83μδ Tg E-Btk-2 and EY-Btk-5 B220lowCD5+ B-1 B cells in the spleen had undergone receptor editing. We therefore conclude that the presence of the E-Btk-2

or EY-Btk-5 Tg did not change the follicular versus selleckchem B-1 B-cell subset choice. Instead, the increased proportions of splenic CD5+ B cells in E-Btk-2 and EY-Btk-5 mice most likely resulted from increased expansion or survival of B-1 B cells. The presence of the E-Btk-2 and EY-Btk-5 Tg also did not change the MZ B-cell subset choice in VH81X Tg mice, which carried a VH81X Tg encoding an Ig heavy chain favoring MZ B-cell development 30. As shown in Fig. 5A and B cells recognized by the 35-1 anti-idiotypic antibody are efficiently selected into the MZ B-cell compartment

in VH81X WT, but not in VH81X Tg Btk-deficient spleens (Fig. 5B). Splenic 35-1+ CD19+ B cells from VH81X E-Btk-2 Tg mice expressed similar CD5 levels as those from VH81X WT, lacked the B220low phenotype characteristic for CD5+ B cells and, importantly, had a CD21high/CD23low MZ phenotype similar to those of VH81X WT mice (Fig. 5A). ID-8 Moreover, in contrast to E-Btk-2 mice (which had few MOMA-1+ macrophages and no MZ B cells in the spleen), in E-Btk-2 mice that carried a VH81X Tg splenic architecture was corrected: EY-Btk-2 VH81X double Tg spleens contained IgM+ B cells within and outside rims of brightly staining MOMA-1+ macrophages (Fig. 5A; right panels). Collectively, these findings show that MZ cell fate was maintained in the presence of constitutive active Btk, indicating that the VH81X BCR specificity is dominant over the increased BCR signal strength generated by the E-Btk-2 Tg.

However, the effect of

human DN T cells on resting CD4+ and CD8+ T cells, their potential immunomodulatory Selleckchem Roxadustat role, and the mechanism of suppression are still rather unclear. In the present study, we demonstrate that human DN T cells can strongly suppress proliferation of CD4+ and CD8+ T cells. Moreover, DN T cells are also able to downregulate proliferation and cytokine production of highly activated effector T cells. In contrast to their murine counterparts, human DN T cells do not eliminate effector T cells by Fas/FasL-mediated apoptosis but suppress by an active cell contact-dependent mechanism. Together, these data suggest that human DN T cells might regulate proliferation and effector function of T cells and thereby contribute to peripheral tolerance. To determine the role of human DN T cells in suppressing immune responses, DN T cells were isolated and stimulated with allogeneic

mature DC as described in Materials and methods. In contrast to freshly isolated DN T cells, DC-stimulated DN T cells expressed activation markers and revealed an effector-memory phenotype (Fig. 1A). However, both resting and stimulated DN T cells lacked expression selleck chemicals llc of Foxp3 or the cytotoxic T lymphocyte antigen 4 (CTLA-4). First, we asked whether prestimulated DN T cells are able to inhibit proliferation of CD4+ and CD8+ T cells that are autologous to the DN T cells. To address this question, CFSE-labeled CD4+

or CD8+ T cells were cocultured with allogeneic DC in the presence or Ergoloid absence of DN T cells and proliferation of CD4+ and CD8+ T cells was measured by flow cytometry. After 5 days, CD4+ and CD8+ T cells revealed a strong proliferation, which was completely abrogated by addition of DN T cells (Fig. 1A). The data obtained by CFSE staining were confirmed by [3H]thymidine incorporation demonstrating a strong suppressive activity of DN T cells (Supporting Information Fig. 1A). Of interest, DN T cells were able to suppress proliferation of both CD45RA+ naive as well as CD45RO+ memory T cells (Supporting Information Fig. 1B). We also examined the efficacy of DN T-cell-mediated suppression by titration of increasing numbers of suppressor to responder cells (Fig. 1C). Notably, DN T cells significantly suppressed proliferation of responder cells up to a ratio of 1:10. To exclude that the suppressive effect of DN T cells relates to in vitro expansion, we used expanded CD4+ or CD8+ T cells as suppressor cells in the MLR. Of importance, both expanded T-cell lines failed to suppress proliferation of responder cells (Supporting Information Fig. 1C). Since T-cell responses in autoimmune diseases and during allograft rejection are known to be very strong, we aimed to determine whether DN T cells are capable to suppress highly activated T-cell lines. Thus, CD4+ and CD8+ T cells were stimulated weekly with allogeneic DC.

To clarify this question, we depleted mice of NK cells in vivo prior to and during infection with different influenza virus

titers. Furthermore, anti-NK1.1 was employed as an Palbociclib additional approach to deplete NK cells in these experiments since anti-asialo-GM1 can deplete subsets of cells from other lineages. Flow cytometric analysis confirmed depletion of CD3−NK1.1+ cells in lung and spleen by anti-NK1.1 (Fig. 4A). Depletion of NK cells improved the survival rate and recovery of body weight (Fig. 4B) in high-dose (5 hemagglutination unit (HAU)) influenza infection. Interestingly, the reverse results were found with medium dose (0.5 HAU) influenza infection, that is, depletion of NK cells increased morbidity and mortality in influenza infection (Fig. 4C). In low-dose (0.0625 HAU) influenza infection, compared to PBS control mice, depletion Volasertib in vitro of NK cells did not influence survival rate and recovery of body weight (Fig. 4D). These results indicate that NK cells can be deleterious, beneficial, or inconsequential, depending on the dose of virus

that the mice are exposed to. Results from NK-cell depletion experiments suggested that NK cells were deleterious during a high-dose pulmonary influenza infection. To further address this issue, we adoptively transferred lung NK cells isolated from high-dose influenza infected or uninfected mice to naive mice, or mice undergoing Rutecarpine a primary influenza infection. We purified NK cells from lungs by negative selection before transfer. Flow cytometric analysis confirmed that the purity of adoptively transferred NK cells was greater than 70%, with no contamination by CD8+ T cells in the transferred cells (data not shown). Transferred NK cells were detected in lung and spleen (Fig. 5A). Transferred lung NK cells from influenza-infected mice were not harmful to uninfected recipient mice (v-NK only). By contrast, lung NK cells from

high-dose influenza infected mice transferred to recipient mice infected with high-dose influenza virus significantly increased mortality and accelerated body weight loss (Fig. 5B and C). Transfer of lung NK cells from uninfected mice (normal NK) did not alter survival rate or weight loss and recovery kinetics compared to otherwise unmanipulated virus infected recipients. It is possible that influenza virus-induced NK cells enhanced pathology in lung and possibly systemically as well, and either or both contributions may have resulted in the more severe outcome from influenza infection observed. These results are consistent with the NK-cell depletion experiments, and support the conclusion that in high-dose lung influenza infection, NK cells are activated and can enhance mortality.

In the kidney, abundant mercury deposits were demonstrated in the epithelial cells of proximal convoluted tubules, although there were no noticeable pathological changes. In the liver, mercury deposits were detected in hepatocytes as well as Kupffer cells, but tissue

damage was not substantial. In contrast, Me-Hg-induced damage to the nervous system can be devastating. However, it never affects the system evenly: as a rule, the damage was the severest in the cerebral and cerebellar cortices, in which some parts were affected more severely than others. The brain stem was affected to a lesser extent, and the spinal cord was least affected. On the other hand, the pathology of peripheral nerves is RG7204 order unique in that it appears to be associated with prolonged duration of the disease: the nerves are affected only in cases other than those of acute and subacute types. The sensory nerves are damaged selectively buy Doxorubicin with regeneration in prolonged cases. This patient was a 64-year-old fisherman who lived in Minamata City in the southern part of Minamata Bay, which was found to be polluted with mercury from the nearby Chisso Co. Onset of disease was marked by numbness of the feet and disturbance in speech in the Spring of 1959. The patient was treated at Minamata City Hospital for pulmonary

tuberculosis during the period from May 1965 until July 1968. Neurological examination in October 1968 and December 1969 revealed slight constriction of visual fields on the temporal side, muscle rigidity, increased tendon reflexes, tremor of the fingers, dysgraphia, and adiadochokinesis. Other clinical findings included labyrinthine deafness, hyperesthesia, and hypalgesia as well as dysesthesia in the hands and regions below the knees, elevated blood pressure of 170–192 mmHg, a mask-like face, and dyskinesia. The patient died of massive hemorrhage from a gastroduodenal ulcer in January

1970. Autopsy materials from the cerebrum, cerebellum, brain stem, spinal cord, and peripheral nerves were embedded in paraffin Amoxicillin and stained with HE, and with KB and Bodian staining methods. Frozen sections were made from peripheral nerves including ventral and dorsal root nerve fibers, sciatic nerve, radial nerve and sural nerve, and stained by the Sugamo myelin and Suzuki’s axon staining methods. The Sugamo myelin stain was modified for use on frozen sections from Kultschiky’s method. Inorganic mercury was detected by photo-emulsion. The gyri of both hemispheres were atrophic and the sulci were widened. This was particularly remarkable in the calcarine cortex and pre- and postcentral gyri. The surface of the calcarine cortex showed moderate atrophy, with widening of the calcarine fissure on the coronal section. Gennari’s band on the calcarine cortex was stained pale with the KB staining method.