Radiolabelling of Polyclonally Expanded Human Regulatory T Cells (Treg) with 89Zr-oxine for Medium-Term In Vivo Cell Tracking.

Regulatory T cells (Tregs) are a promising candidate cell therapy to treat autoimmune diseases and aid the longevity of transplanted solid organs. Despite increasing numbers of clinical trials using human Treg therapy, important questions pertaining to their in vivo fate, distribution, and function remain unanswered. Treg accumulation in relevant tissues was found to be crucial for Treg therapy efficacy, but existing blood-borne biomarkers are unlikely to accurately reflect the tissue state. Non-invasive Treg tracking by whole-body imaging is a promising alternative and can be achieved by direct radiolabelling of Tregs and following the radiolabelled cells with positron emission tomography (PET). Our goal was to evaluate the radiolabelling of polyclonal Tregs with 89Zr to permit their in vivo tracking by PET/CT for longer than one week with current preclinical PET instrumentation. We used [89Zr]Zr(oxinate)4 as the cell-labelling agent and achieved successful radiolabelling efficiency of human Tregs spanning 0.1-11.1 Bq 89Zr/Treg cell, which would be compatible with PET tracking beyond one week. We characterized the 89Zr-Tregs, assessing their phenotypes, and found that they were not tolerating these intracellular 89Zr amounts, as they failed to survive or expand in a 89Zr-dose-dependent manner. Even at 0.1 Bq 89Zr per Treg cell, while 89Zr-Tregs remained functional as determined by a five-day-long effector T cell suppression assay, they failed to expand beyond day 3 in vitro. Moreover, PET imaging revealed signs of 89Zr-Treg death after adoptive transfer in vivo. In summary, 89Zr labelling of Tregs at intracellular radioisotope amounts compatible with cell tracking over several weeks did not achieve the desired outcomes, as 89Zr-Tregs failed to expand and survive. Consequently, we conclude that indirect Treg labelling is likely to be the most effective alternative method to satisfy the requirements of this cell tracking scenario.

B lymphocytes contribute to indirect pathway T cell sensitization via acquisition of extracellular vesicles.

B cells have been implicated in transplant rejection via antibody-mediated mechanisms and more recently by presenting donor antigens to T cells. We have shown in patients with chronic antibody-mediated rejection that B cells control the indirect T cell alloresponses. To understand more about the role of B cells as antigen-presenting cells for CD4+ T cell with indirect allospecificity, B cells were depleted in C57BL/6 mice, using an anti-CD20 antibody, prior to receiving MHC class I-mismatched (Kd ) skin. The absence of B cells at the time of transplantation prolonged skin graft survival. To study the mechanisms behind this observation, T cells with indirect allospecificity were transferred in mice receiving a Kd skin transplant. T cell proliferation was markedly inhibited in the absence of recipient B cells, suggesting that B cells contribute to indirect pathway sensitization. Furthermore, we have shown that a possible way in which B cells present alloantigens is via acquisition of MHC-peptide complexes. Finally, we demonstrate that the addition of B cell depletion to the transfer of regulatory T cells (Tregs) with indirect alloresponse further prolonged skin graft survival. This study supports an important role for B cells in indirect T cell priming and further emphasizes the advantage of combination therapies in prolonging transplant survival.

MicroRNAs affect dendritic cell function and phenotype.

MicroRNA (miRNA) are small, non-coding RNA molecules that have been linked with immunity through regulating/modulating gene expression. A role for these molecules in T-cell and B-cell development and function has been well established. An increasing body of literature now highlights the importance of specific miRNA in dendritic cell (DC) development as well as their maturation process, antigen presentation capacity and cytokine release. Given the unique role of DC within the immune system, linking the innate and adaptive immune responses, understanding how specific miRNA affect DC function is of importance for understanding disease. In this review we summarize recent developments in miRNA and DC research, highlighting the requirement of miRNA in DC lineage commitment from bone marrow progenitors and for the development of subsets such as plasmacytoid DC and conventional DC. In addition, we discuss how infections and tumours modulate miRNA expression and consequently DC function.

Monitoring the efficacy of dendritic cell vaccination by early detection of (99m) Tc-HMPAO-labelled CD4(+) T cells.

DC vaccines have been used to induce tumour-specific cytotoxic T cells . However, this approach to cancer immunotherapy has had limited success. To be successful, injected DCs need to migrate to the LNs where they can stimulate effector T cells . We and others have previously demonstrated by MRI that tumour antigen-pulsed-DCs labelled ex vivo with superparamagnetic iron oxide nanoparticles migrated to the draining LNs and are capable of activating antigen-specific T cells . The results from our study demonstrated that ex vivo superparamagnetic iron oxide nanoparticles-labelled and OVA-pulsed DCs prime cytotoxic CD8(+) T-cell responses to protect against a B16-OVA tumour challenge. In the clinic, a possible noninvasive surrogate marker for efficacy of DC vaccination is to image the specific migration and accumulation of T cells following DC vaccination.

Tolerogenic Donor-Derived Dendritic Cells Risk Sensitization In Vivo owing to Processing and Presentation by Recipient APCs.

Modification of allogeneic dendritic cells (DCs) through drug treatment results in DCs with in vitro hallmarks of tolerogenicity. Despite these observations, using murine MHC-mismatched skin and heart transplant models, donor-derived drug-modified DCs not only failed to induce tolerance but also accelerated graft rejection. The latter was inhibited by injecting the recipient with anti-CD8 Ab, which removed both CD8(+) T cells and CD8(+) DCs. The discrepancy between in vitro and in vivo data could be explained, partly, by the presentation of drug-modified donor DC MHC alloantigens by recipient APCs and activation of recipient T cells with indirect allospecificity, leading to the induction of alloantibodies. Furthermore, allogeneic MHC molecules expressed by drug-treated DCs were rapidly processed and presented in peptide form by recipient APCs in vivo within hours of DC injection. Using TCR-transgenic T cells, Ag presentation of injected OVA-pulsed DCs was detectable for ≤ 3 d, whereas indirect presentation of MHC alloantigen by recipient APCs led to activation of T cells within 14 h and was partially inhibited by reducing the numbers of CD8(+) DCs in vivo. In support of this observation when mice lacking CD8(+) DCs were pretreated with drug-modified DCs prior to transplantation, skin graft rejection kinetics were similar to those in non-DC-treated controls. Of interest, when the same mice were treated with anti-CD40L blockade plus drug-modified DCs, skin graft survival was prolonged, suggesting endogenous DCs were responsible for T cell priming. Altogether, these findings highlight the risks and limitations of negative vaccination using alloantigen-bearing "tolerogenic" DCs.

Acquisition of MHC:peptide complexes by dendritic cells contributes to the generation of antiviral CD8+ T cell immunity in vivo.

There is an increasing body of evidence suggesting that the transfer of preformed MHC class I:peptide complexes between a virus-infected cell and an uninfected APC, termed cross-dressing, represents an important mechanism of Ag presentation to CD8+ T cells in host defense. However, although it has been shown that memory CD8+ T cells can be activated by uninfected dendritic cells (DCs) cross-dressed by Ag from virus-infected parenchymal cells, it is unknown whether conditions exist during virus infection in which naive CD8+ T cells are primed and differentiate to cytolytic effectors through cross-dressing, and indeed which DC subset would be responsible. In this study, we determine whether the transfer of MHC class I:peptide complexes between infected and uninfected murine DC plays a role in CD8+ T cell priming to viral Ags in vivo. We show that MHC class I:peptide complexes from peptide-pulsed or virus-infected DCs are indeed acquired by splenic CD8α⁻ DCs in vivo. Furthermore, the acquired MHC class I:peptide complexes are functional in that they induced Ag-specific CD8+ T cell effectors with cytolytic function. As CD8α⁻ DCs are poor cross-presenters, this may represent the main mechanism by which CD8α⁻ DCs present exogenously encountered Ag to CD8+ T cells. The sharing of Ag as preformed MHC class I:peptide complexes between infected and uninfected DCs without the restraints of Ag processing may have evolved to accurately amplify the response and also engage multiple DC subsets critical in the generation of strong antiviral immunity.

C3a and C5a promote renal ischemia-reperfusion injury.

Renal ischemia reperfusion injury triggers complement activation, but whether and how the small proinflammatory fragments C3a and C5a contribute to the pathogenesis of this injury remains to be elucidated. Using C3aR-, C5aR-, or C3aR/C5aR-deficient mice and models of renal ischemia-reperfusion injury, we found that deficiency of either or both of these receptors protected mice from injury, but the C3aR/C5aR- and C5aR-deficient mice were most protected. Protection from injury was associated with less cellular infiltration and lower mRNA levels of kidney injury molecule-1, proinflammatory mediators, and adhesion molecules in postischemic kidneys. Furthermore, chimera studies showed that the absence of C3aR and C5aR on renal tubular epithelial cells or circulating leukocytes attenuated renal ischemia-reperfusion injury. In vitro, C3a and C5a stimulation induced inflammatory mediators from both renal tubular epithelial cells and macrophages after hypoxia/reoxygenation. In conclusion, although both C3a and C5a contribute to renal ischemia-reperfusion injury, the pathogenic role of C5a in this injury predominates. These data also suggest that expression of C3aR and C5aR on both renal and circulating leukocytes contributes to the pathogenesis of renal ischemia-reperfusion injury.

Inhibition of thrombin on endothelium enhances recruitment of regulatory T cells during IRI and when combined with adoptive Treg transfer, significantly protects against acute tissue injury and prolongs allograft survival.

Ischemia-reperfusion injury (IRI) amplifies T cell alloimmune responses after transplantation with thrombin playing a key pro-inflammatory role. To explore the influence of thrombin on regulatory T cell recruitment and efficacy we used a well-established model of IRI in the native murine kidney. Administration of the cytotopic thrombin inhibitor PTL060 inhibited IRI, and by skewing expression of chemokines (reducing CCL2 and CCL3 but increasing CCL17 and CCL22) increased the infiltration of M2 macrophages and Tregs. When PTL060 was combined with infusion of additional Tregs, these effects were further amplified. To test the benefits of thrombin inhibition in a transplant model, BALB/c hearts were transplanted into B6 mice with or without perfusion with PTL060 in combination with Tregs. Thrombin inhibition or Treg infusion alone led to small increments in allograft survival. However, the combined therapy led to modest graft prolongation by the same mechanisms as in renal IRI; graft survival was accompanied by increased numbers of Tregs and anti-inflammatory macrophages, and reduced expression of pro-inflammatory cytokines. While the grafts succumbed to rejection associated with the emergence of alloantibody, these data suggest that thrombin inhibition within the transplant vasculature enhances the efficacy of Treg infusion, a therapy that is currently entering the clinic to promote transplant tolerance.

Spatiotemporal in vivo tracking of polyclonal human regulatory T cells (Tregs) reveals a role for innate immune cells in Treg transplant recruitment.

Regulatory T cells (Tregs) are emerging as a new cell-based therapy in solid organ transplantation. Adoptive transfer of Tregs has been shown preclinically to protect from graft rejection, and the safety of Treg therapy has been demonstrated in clinical trials. Despite these successes, the in vivo distribution and persistence of adoptively transferred Tregs remained elusive, which hampers clinical translation. Here we isolated human Tregs using a GMP-compatible protocol and lentivirally transduced them with the human sodium iodide symporter to render them traceable in vivo by radionuclide imaging. Engineered human Tregs were characterized for phenotype, survival, suppressive capacity, and reporter function. To study their trafficking behavior, they were subsequently administered to humanized mice with human skin transplants. Traceable Tregs were quantified in skin grafts by non-invasive nano-single-photon emission computed tomography (nanoSPECT)/computed tomography (CT) for up to 40 days, and the results were validated ex vivo. Using this approach, we demonstrated that Treg trafficking to skin grafts was regulated by the presence of recipient Gr-1+ innate immune cells. We demonstrated the utility of radionuclide reporter gene-afforded quantitative Treg in vivo tracking, addressing a fundamental need in Treg therapy development and offering a clinically compatible methodology for future Treg therapy imaging in humans.

Vav1 transduces T cell receptor signals to the activation of phospholipase C-gamma1 via phosphoinositide 3-kinase-dependent and -independent pathways.

Vav1 is a signal transducing protein required for T cell receptor (TCR) signals that drive positive and negative selection in the thymus. Furthermore, Vav1-deficient thymocytes show greatly reduced TCR-induced intracellular calcium flux. Using a novel genetic system which allows the study of signaling in highly enriched populations of CD4(+)CD8(+) double positive thymocytes, we have studied the mechanism by which Vav1 regulates TCR-induced calcium flux. We show that in Vav1-deficient double positive thymocytes, phosphorylation, and activation of phospholipase C-gamma1 (PLCgamma1) is defective. Furthermore, we demonstrate that Vav1 regulates PLCgamma1 phosphorylation by at least two distinct pathways. First, in the absence of Vav1 the Tec-family kinases Itk and Tec are no longer activated, most likely as a result of a defect in phosphoinositide 3-kinase (PI3K) activation. Second, Vav1-deficient thymocytes show defective assembly of a signaling complex containing PLCgamma1 and the adaptor molecule Src homology 2 domain-containing leukocyte phosphoprotein 76. We show that this latter function is independent of PI3K.

Application of carbon nanotubes in cancer vaccines: Achievements, challenges and chances.

Tumour-specific, immuno-based therapeutic interventions can be considered as safe and effective approaches for cancer therapy. Exploitation of nano-vaccinology to intensify the cancer vaccine potency may overcome the need for administration of high vaccine doses or additional adjuvants and therefore could be a more efficient approach. Carbon nanotube (CNT) can be described as carbon sheet(s) rolled up into a cylinder that is nanometers wide and nanometers to micrometers long. Stemming from the observed capacities of CNTs to enter various types of cells via diversified mechanisms utilising energy-dependent and/or passive routes of cell uptake, the use of CNTs for the delivery of therapeutic agents has drawn increasing interests over the last decade. Here we review the previous studies that demonstrated the possible benefits of these cylindrical nano-vectors as cancer vaccine delivery systems as well as the obstacles their clinical application is facing.

Protease Activated Receptor 4 as a Novel Modulator of Regulatory T Cell Function.

Regulatory T cells (Tregs) are a subpopulation of T cells that maintain immunological tolerance. In inflammatory responses the function of Tregs is tightly controlled by several factors including signaling through innate receptors such as Toll like receptors and anaphylatoxin receptors allowing an effective immune response to be generated. Protease-activated receptors (PARs) are another family of innate receptors expressed on multiple cell types and involved in the pathogenesis of autoimmune disorders. Whether proteases are able to directly modulate Treg function is unknown. Here, we show using two complimentary approaches that signaling through PAR-4 influences the expression of CD25, CD62L, and CD73, the suppressive capacity, and the stability of Tregs, via phosphorylation of FoxO1 and negative regulation of PTEN and FoxP3. Taken together, our results demonstrate an important role of PAR4 in tuning the function of Tregs and open the possibility of targeting PAR4 to modulate immune responses.

Membrane Radiolabelling of Exosomes for Comparative Biodistribution Analysis in Immunocompetent and Immunodeficient Mice - A Novel and Universal Approach.

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin and inherent intercellular biomolecule delivery capability. An in-depth knowledge of their in vivo biodistribution is therefore essential. This work aimed to develop a novel, reliable and universal method to radiolabel exosomes to study their in vivo biodistribution. Methods: Melanoma (B16F10) cells were cultured in bioreactor flasks to increase exosome yield. B16F10-derived exosomes (ExoB16) were isolated using ultracentrfugation onto a single sucrose cushion, and were characterised for size, yield, purity, exosomal markers and morphology using Nanoparticle Tracking Analysis (NTA), protein measurements, flow cytometry and electron microscopy. ExoB16 were radiolabelled using 2 different approaches - intraluminal labelling (entrapment of 111Indium via tropolone shuttling); and membrane labelling (chelation of 111Indium via covalently attached bifunctional chelator DTPA-anhydride). Labelling efficiency and stability was assessed using gel filtration and thin layer chromatography. Melanoma-bearing immunocompetent (C57BL/6) and immunodeficient (NSG) mice were injected intravenously with radiolabelled ExoB16 (1x1011 particles/mouse) followed by metabolic cages study, whole body SPECT-CT imaging and ex vivo gamma counting at 1, 4 and 24 h post-injection. Results: Membrane-labelled ExoB16 showed superior radiolabelling efficiency and radiochemical stability (19.2 ± 4.53 % and 80.4 ± 1.6 % respectively) compared to the intraluminal-labelled exosomes (4.73 ± 0.39 % and 14.21 ± 2.76 % respectively). Using the membrane-labelling approach, the in vivo biodistribution of ExoB16 in melanoma-bearing C57Bl/6 mice was carried out, and was found to accumulate primarily in the liver and spleen (~56% and ~38% ID/gT respectively), followed by the kidneys (~3% ID/gT). ExoB16 showed minimal tumour i.e. self-tissue accumulation (~0.7% ID/gT). The membrane-labelling approach was also used to study ExoB16 biodistribution in melanoma-bearing immunocompromised (NSG) mice, to compare with that in the immunocompetent C57Bl/6 mice. Similar biodistribution profile was observed in both C57BL/6 and NSG mice, where prominent accumulation was seen in liver and spleen, apart from the significantly lower tumour accumulation observed in the NSG mice (~0.3% ID/gT). Conclusion: Membrane radiolabelling of exosomes is a reliable approach that allows for accurate live imaging and quantitative biodistribution studies to be performed on potentially all exosome types without engineering parent cells.

Regulatory T cell-derived extracellular vesicles modify dendritic cell function.

Regulatory T cells (Treg) are a subpopulation of T cells that maintain tolerance to self and limit other immune responses. They achieve this through different mechanisms including the release of extracellular vesicles (EVs) such as exosomes as shown by us, and others. One of the ways that Treg derived EVs inhibit target cells such as effector T cells is via the transfer of miRNA. Another key target for the immunoregulatory function of Tregs is the dendritic cells (DCs). In this study we demonstrate directly, and for the first time, that miRNAs are transferred from Tregs to DCs via Treg derived EVs. In particular two miRNAs, namely miR-150-5p and miR-142-3p, were increased in DCs following their interaction with Tregs and Treg derived exosomes. One of the consequences for DCs following the acquisition of miRNAs contained in Treg derived EVs was the induction of a tolerogenic phenotype in these cells, with increased IL-10 and decreased IL-6 production being observed following LPS stimulation. Altogether our findings provide data to support the idea that intercellular transfer of miRNAs via EVs may be a novel mechanism by which Tregs regulate DC function and could represent a mechanism to inhibit immune reactions in tissues.

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-induced (Ang II-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 Ang II-induced effector T cell (Teff) 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 Ang II-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 antibody depletion of Tregs. Mechanistically, Nox2-/y Tregs showed higher in vitro suppression of Teff 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 Ang II-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.

What Is Direct Allorecognition?

Direct allorecognition is the process by which donor-derived major histocompatibility complex (MHC)-peptide complexes, typically presented by donor-derived 'passenger' dendritic cells, are recognised directly by recipient T cells. In this review, we discuss the two principle theories which have been proposed to explain why individuals possess a high-precursor frequency of T cells with direct allospecificity and how self-restricted T cells recognise allogeneic MHC-peptide complexes. These theories, both of which are supported by functional and structural data, suggest that T cells recognising allogeneic MHC-peptide complexes focus either on the allopeptides bound to the allo-MHC molecules or the allo-MHC molecules themselves. We discuss how direct alloimmune responses may be sustained long term, the consequences of this for graft outcome and highlight novel strategies which are currently being investigated as a potential means of reducing rejection mediated through this pathway.

Regulatory T cell-derived exosomes: possible therapeutic and diagnostic tools in transplantation.

Exosomes are extracellular vesicles released by many cells of the body. These small vesicles play an important part in intercellular communication both in the local environment and systemically, facilitating in the transfer of proteins, cytokines as well as miRNA between cells. The observation that exosomes isolated from immune cells such as dendritic cells (DCs) modulate the immune response has paved the way for these structures to be considered as potential immunotherapeutic reagents. Indeed, clinical trials using DC derived exosomes to facilitate immune responses to specific cancer antigens are now underway. Exosomes can also have a negative effect on the immune response and exosomes isolated from regulatory T cells (Tregs) and other subsets of T cells have been shown to have immune suppressive capacities. Here, we review what is currently known about Treg derived exosomes and their contribution to immune regulation, as well as highlighting their possible therapeutic potential for preventing graft rejection, and use as diagnostic tools to assess transplant outcome.

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 ((99m)TcO(4)(-)) 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 (99m)TcO(4)(-). 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.

Inefficient clustering of tyrosine-phosphorylated proteins at the immunological synapse in response to an antagonist peptide.

Interactions of T cells with MHC plus peptide in the peripheral lymphoid system are important for their survival. In this study we investigated further the molecular consequences of such interactions using F5 TCR transgenic mice and peptides previously shown to induce either negative or positive selection in the thymus. Following TCR ligation with the negatively selecting agonist peptide, mature CD8(+) cells proliferated and up-regulated the activation marker CD69. Interestingly, ligation of this TCR with MHC molecules loaded with high concentrations of the positively selecting peptide also resulted in the aforementioned changes, but with slower kinetics. Analysis of the biochemical changes that occur following stimulation with these peptides showed that phosphorylation of key signaling molecules, such as ZAP-70, CD3zeta, Vav, SLP-76, LAT, and ERK-1 and 2, could be detected after exposure to agonist but not antagonist peptide. Confocal microscopy, however, revealed infrequent phosphorylation 'patches' at the site of contact between T cells and APC presenting the antagonist peptide. Our data suggest that peptides capable of inducing positive selection in the thymus can be recognized by mature T cells and cause proliferation, up-regulation of CD69 and accumulation of phosphorylated proteins at the immunological synapse with low efficiency; however no phosphorylation of signaling molecules can be detected using conventional biochemical assays.