Dendritic cells in transplantation - Friend or foe?

The existence of two sets of dendritic cells in transplantation (of donor and recipient origin) poses unique problems in alloimmunity and tolerance. Donor DCs are potent stimulators of the direct pathway, in which recipient T cells respond to peptides presented on donor MHC products or to the donor MHC molecules themselves. Reduced DC function in the direct pathway is used to explain the acceptance of certain allografts that have been depleted of passenger leukocytes. Reciprocally, purified donor DCs powerfully stimulate the rejection of these grafts as well as the mixed leukocyte reaction by purified allogeneic T cells in culture. Donor DCs also can act as specialized antigen transport vehicles in cross-priming for the indirect pathway. Here, recipient DCs process MHC molecules from the donor. The indirect pathway of rejection is actually the rule for passenger leukocyte-depleted grafts. Furthermore, the indirect pathway seems to be the dominant alloresponse detected in long-term graft recipients, both in experimental models and clinical transplantation, particularly in those with chronic rejection. The stimulatory function of DCs in both direct and indirect pathways is regulated by their maturation in response to inflammatory stimuli, especially those delivered via IL-1, TNF, and Toll receptor families. Since the normal function of DCs is to generate immunity against invading pathogens, the indirect response to peptides continually derived from the allogeneic MHC molecules in a tissue allograft (a surgically introduced pathogen because of the associated inflammation and necrosis) may be more difficult to silence. However, in addition to their allostimulatory role, immature or in vitro-manipulated DCs also have the potential to downregulate direct and indirect antidonor responses through a variety of mechanisms. Intriguing new evidence shows that immature DCs can actively induce regulatory T cells. Given the probable role of the indirect pathway in driving chronic rejection, the induction of T cell tolerance (deletion, anergy, and regulation), especially in those T cells with indirect antidonor allospecificity, by the injection of immature DCs pretransplant, could serve as a critical therapeutic strategy

Detection of donor-specific hyporesponsiveness following late failure of human renal allografts

Detection of donor-specific hyporesponsiveness following late failure of human renal allografts. Limiting dilution assays to measure the frequency of interleukin-2-secreting peripheral blood T cells were carried out in patients, whose renal allografts had failed due to acute rejection (9 patients) and in patients whose grafts failed more than two years after transplantation without any recent evidence of acute rejection. Using a modified form of the assay we demonstrate that nearly half of 18 patients whose renal transplants had failed after more than two years have low or undetectable HTLp frequencies against donor, but not third-party DR antigens. No such difference was observed in any of the nine patients studied whose transplants were lost from early acute rejection. These results provide the first indication that, as in rodent models of transplantation, T cell unresponsiveness towards donor MHC antigens can occur following prolonged residence of an allograft in humans. Furthermore, the results suggest that chronic rejection may be driven by mechanisms other than direct allorecognition. The assay may be a valuable tool to study the evolution of donor-specific direct T cell alloresponsiveness in patients with well-functioning grafts

Inhibition of Thrombin Receptor Signaling on alpha-Smooth Muscle Actin(+) CD34(+) Progenitors Leads to Repair After Murine Immune Vascular Injury

OBJECTIVE: The goal of this study was to use mice expressing human tissue factor pathway inhibitor (TFPI) on α-smooth muscle actin (α-SMA)(+) cells as recipients of allogeneic aortas to gain insights into the cellular mechanisms of intimal hyperplasia (IH). METHODS AND RESULTS: BALB/c aortas (H-2(d)) transplanted into α-TFPI-transgenic (Tg) mice (H-2(b)) regenerated a quiescent endothelium in contrast to progressive IH seen in C57BL/6 wild-type (WT) mice even though both developed aggressive anti-H-2(d) alloresponses, indicating similar vascular injuries. Adoptively transferred Tg CD34(+) (but not CD34(-)) cells inhibited IH in WT recipients, indicating the phenotype of α-TFPI-Tg mice was due to these cells. Compared with syngeneic controls, endogenous CD34(+) cells were mobilized in significant numbers after allogeneic transplantation, the majority showing sustained expression of tissue factor and protease-activated receptor-1 (PAR-1). In WT, most were CD45(+) myeloid progenitors coexpressing CD31, vascular endothelial growth factor receptor-2 and E-selectin; 10% of these cells coexpressed α-SMA and were recruited to the neointima. In contrast, the α-SMA(+) human TFPI(+) CD34(+) cells recruited in Tg recipients were from a CD45(-) lineage. WT CD34(+) cells incubated with a PAR-1 antagonist or taken from PAR-1-deficient mice inhibited IH as Tg cells did. CONCLUSIONS: Specific inhibition of thrombin generation or PAR-1 signaling on α-SMA(+) CD34(+) cells inhibits IH and promotes regenerative repair despite ongoing immune-mediated damage

Monitoring of In Vivo Function of Superparamagnetic Iron Oxide Labelled Murine Dendritic Cells during Anti-Tumour Vaccination

Dendritic cells (DCs) generated in vitro to present tumour antigens have been injected in cancer patients to boost in vivo anti-tumour immune responses. This approach to cancer immunotherapy has had limited success. For anti-tumour therapy, delivery and subsequent migration of DCs to lymph nodes leading to effective stimulation of effector T cells is thought to be essential. The ability to non-invasively monitor the fate of adoptively transferred DCs in vivo using magnetic resonance imaging (MRI) is an important clinical tool to correlate their in vivo behavior with response to treatment. Previous reports of superparamagnetic iron oxides (SPIOs) labelling of different cell types, including DCs, have indicated varying detrimental effects on cell viability, migration, differentiation and immune function. Here we describe an optimised labelling procedure using a short incubation time and low concentration of clinically used SPIO Endorem to successfully track murine DC migration in vivo using MRI in a mouse tumour model. First, intracellular labelling of bone marrow derived DCs was monitored in vitro using electron microscopy and MRI relaxometry. Second, the in vitro characterisation of SPIO labelled DCs demonstrated that viability, phenotype and functions were comparable to unlabelled DCs. Third, ex vivo SPIO labelled DCs, when injected subcutaneously, allowed for the longitudinal monitoring by MR imaging of their migration in vivo. Fourth, the SPIO DCs induced the proliferation of adoptively transferred CD4+ T cells but, most importantly, they primed cytotoxic CD8+ T cell responses to protect against a B16-Ova tumour challenge. Finally, using anatomical information from the MR images, the immigration of DCs was confirmed by the increase in lymph node size post-DC injection. These results demonstrate that the SPIO labelling protocol developed in this study is not detrimental for DC function in vitro and in vivo has potential clinical application in monitoring therapeutic DCs in patients with cancer

In Vivo SPECT Reporter Gene Imaging of Regulatory T Cells

Regulatory T cells (Tregs) were identified several years ago and are key in controlling autoimmune diseases and limiting immune responses to foreign antigens, including alloantigens. In vivo imaging techniques including intravital microscopy as well as whole body imaging using bioluminescence probes have contributed to the understanding of in vivo Treg function, their mechanisms of action and target cells. Imaging of the human sodium/iodide symporter via Single Photon Emission Computed Tomography (SPECT) has been used to image various cell types in vivo. It has several advantages over the aforementioned imaging techniques including high sensitivity, it allows non-invasive whole body studies of viable cell migration and localisation of cells over time and lastly it may offer the possibility to be translated to the clinic. This study addresses whether SPECT/CT imaging can be used to visualise the migratory pattern of Tregs in vivo. Treg lines derived from CD4+CD25+FoxP3+ cells were retrovirally transduced with a construct encoding for the human Sodium Iodide Symporter (NIS) and the fluorescent protein mCherry and stimulated with autologous DCs. NIS expressing self-specific Tregs were specifically radiolabelled in vitro with Technetium-99m pertechnetate (99mTcO4−) and exposure of these cells to radioactivity did not affect cell viability, phenotype or function. In addition adoptively transferred Treg-NIS cells were imaged in vivo in C57BL/6 (BL/6) mice by SPECT/CT using 99mTcO4−. After 24 hours NIS expressing Tregs were observed in the spleen and their localisation was further confirmed by organ biodistribution studies and flow cytometry analysis. The data presented here suggests that SPECT/CT imaging can be utilised in preclinical imaging studies of adoptively transferred Tregs without affecting Treg function and viability thereby allowing longitudinal studies within disease models

A new method for measuring and calibrating cinema audio systems for optimal sound quality

The aim of this research is to utilize new methodologies and technology in order to gain insight into how the modern cinema audio system could be calibrated to provide improved audio performance. To this end, both objective and subjective measurements were developed to better understand the audio preferences of listeners, the requirements of the audio systems inclusive of the acoustic environment, and how the two are related. Part of the data for this research was derived from a survey of re-recording mixers regarding their use and opinion of the current SMPTE standard. The survey confirmed anecdotal information suggesting that re-recording mixers use high-end pre-emphasis to compensate for the severe roll-off induced by the SMPTE X-curve. It is also noted that the re-recording mixers' opinions of how well their mix translates from dub-stage to cinema is correlated to how many years they have spent in the industry. The aim of this research is to utilize new methodologies and technology in order to gain insight into how the modern cinema audio system could be calibrated to provide improved audio performance. To this end, both objective and subjective measurements were developed to better understand the audio preferences of listeners, the requirements of the audio systems inclusive of the acoustic environment, and how the two are related. Part of the data for this research was derived from a survey of re-recording mixers regarding their use and opinion of the current SMPTE standard. The survey confirmed anecdotal information suggesting that re-recording mixers use high-end pre-emphasis to compensate for the severe roll-off induced by the SMPTE X-curve. It is also noted that the re-recording mixers' opinions of how well their mix translates from dub-stage to cinema is correlated to how many years they have spent in the industry. To further understand listener preference to in-room responses curves, a series of listening tests utilizing the BRS system were conducted using various sized cinemas, seating positions within the cinemas, audio tracks (including those mixed on a SMPTE calibrated dub-stage) and target curves. The overwhelming outcome was that regardless of cinema size, seating position or audio track utilized; the "curve" that listeners preferred is a relatively flat 0.9dB/octave slope with a 6.5dB bass boost below 105Hz and a -2.5dB roll off above 2.5kHz. Of the 5 target curves presented, the SMPTE X-curve place fourth with scores very near the low-rated perceptual anchor. This calls into question the notion of the X-curve providing "ideal" translation between dub-stage and cinema and in fact, challenges the concept of translation all together. Research was completed in an effort to identifying the number of microphone positions required, along with their placement, in order to accurately capture a cinema's response for calibration purposes. A novel experiment utilizing anechoic loudspeaker data as a guideline for xxi analysis demonstrated that, with proper data, the number of microphones and their positions plays a less critical factor in determining the room response. The collected data shows that even with as few as 4 microphones at varied positions, the resultant room response will trend towards the anechoic data above 1kHz. From around 300Hz to 1kHz, there is evidence of seat effects that may be resolved through randomizing the microphone heights. Below 300Hz, the room becomes the dominating factor and more than 5 microphone positions will be required to properly identify any problems

Nox2 in regulatory T cells promotes angiotensin II–induced cardiovascular remodeling

The superoxide-generating enzyme Nox2 contributes to hypertension and cardiovascular remodeling triggered by activation of the renin-angiotensin system. Multiple Nox2-expressing cells are implicated in angiotensin II (AngII)-induced pathophysiology, but the importance of Nox2 in leukocyte subsets is poorly understood. Here, we investigated the role of Nox2 in T cells, particularly Tregs. Mice globally deficient in Nox2 displayed increased numbers of Tregs in the heart at baseline whereas AngII-induced T-effector cell (Teffs) infiltration was inhibited. To investigate the role of Treg Nox2, we generated a mouse line with CD4-targeted Nox2 deficiency (Nox2fl/flCD4Cre+). These animals showed inhibition of AngII-induced hypertension and cardiac remodeling related to increased tissue-resident Tregs and reduction in infiltrating Teffs, including Th17 cells. The protection in Nox2fl/flCD4Cre+ mice was reversed by anti-CD25 Ab-depletion of Tregs. Mechanistically, Nox2-/y Tregs showed higher in vitro suppression of Teffs proliferation than WT Tregs, increased nuclear levels of FoxP3 and NF-κB, and enhanced transcription of CD25, CD39, and CD73. Adoptive transfer of Tregs confirmed that Nox2-deficient cells had greater inhibitory effects on AngII-induced heart remodeling than WT cells. These results identify a previously unrecognized role of Nox2 in modulating suppression of Tregs, which acts to enhance hypertension and cardiac remodeling

Leptin protects mice from starvation-induced lymphoid atrophy and increases thymic cellularity in ob/ob mice.

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