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Skin immune homeostasis is a multi-faceted process where dermal dendritic cells (DDCs) are key in orchestrating responses to environmental stressors. We have previously identified CD141 + CD14 + DDCs as a skin-resident immunoregulatory population that is vitamin-D 3 (VitD3) inducible from monocyte-derived DCs (moDCs), termed CD141 hi VitD3 moDCs. We demonstrate that CD141 + DDCs and CD141 hi VitD3 moDCs share key immunological features including cell surface markers, reduced T cell stimulation, IL-10 production, and a common transcriptomic signature. Bioinformatic analysis identified the neuroactive ligand receptor pathway and the neuropeptide, urocortin 2 (UCN2), as a potential immunoregulatory candidate molecule. Incubation with VitD3 upregulated UCN2 in CD141 + DCs and UVB irradiation induced UCN2 in CD141 + DCs in healthy skin in vivo. Notably, CD141 + DDC generation of suppressive Tregs was dependent upon the UCN2 pathway as in vivo administration of UCN2 reversed skin inflammation in humanized mice. We propose the neuropeptide UCN2 as a novel skin DC-derived immunoregulatory mediator with a potential role in UVB and VitD3-dependent skin immune homeostasis., Competing Interests: F.O.N. is an employee of Sanofi, USA, and C.C.C. is an employee of Unilever, China., (© 2023 The Authors.)

Anesthesia and analgesia are major components of many interventional studies on laboratory animals. However, various studies have shown improper reporting or use of anesthetics/analgesics in research proposals and published articles. In many cases, it seems "anesthesia" and "analgesia" are used interchangeably, while they are referring to two different concepts. Not only this is an unethical practice, but also it may be one of the reasons for the proven suboptimal quality of many animal researches. This is a widespread problem among investigations on various species of animals. However, it could be imagined that it may be more prevalent for the most common species of laboratory animals, such as the laboratory mice. In this review, proper anesthetic/analgesic methods for routine procedures on laboratory mice are discussed. We considered the available literature and critically reviewed their anesthetic/analgesic methods. Detailed dosing and pharmacological information for the relevant drugs are provided and some of the drugs' side effects are discussed. This paper provides the necessary data for an informed choice of anesthetic/analgesic methods in some routine procedures on laboratory mice., (© 2022. The Author(s).)

Although mesenchymal stem cells (MSCs) have exhibited promising immunomodulatory potential in preclinical studies, clinical studies have revealed variable results. These results often depend on environmental cues. Pre-conditioning MSCs with cytokines is one of the methods used to enhance their immunomodulatory effects. In this study, we harvested adipose-derived MSCs from mice and cultured them with different doses of the cytokine, IFN-γ, and the corticosteroid drug, dexamethasone, in order to investigate their effects on MSC immunosuppressive function. We found the co-culture or supernatant of MSCs, pre-conditioned with IFN-γ, together with spleen mononuclear cells resulted in a significant reduction of mononuclear cell proliferation. Although the supernatant of MSCs, pre-conditioned with dexamethasone, showed similar results, dexamethasone pre-conditioning of co-cultured MSCs increased mononuclear cell proliferation. The results further our understanding of immune-related effects of MSCs which may provide a basis for further in vivo studies to achieve better clinical results. We propose that pre-conditioning with cytokines might be an effective method to boost the immunomodulatory effects of MSCs.

Background: Mathematical modeling offers the possibility to select the optimal dose of a drug or vaccine. Considerable evidence show that many bacterial components can activate dendritic cells (DCs). Our previous report showed that multiple doses of DCs matured with Listeria monocytogenes led to tumor regression whereas multiple doses of CpG-matured DCs affected tumor reversely., Objective: To assess a combined pattern of DC vaccination proposed by a mathematical model for tumor regression., Method: WEHI164 cells were inoculated subcutaneously in the right flank of BALB/c mice. Bone marrow-derived DCs were matured by Listeria monocytogenes and CpG motifs. DCs were injected using specific patterns and doses predicted by mathematical modeling. Effector cell-mediated cytotoxicity, gene expression of T cell-related transcription factors, as well as tumor growth and survival rate, were assessed in different groups., Results: Our study indicated that the proposed mathematical model could simulate the tumor and immune system interaction, and it was verified by decreasing tumor size in the List+CpG group. However, comparing the effect of different treatment modalities on Th1/Treg transcription factor expression or cytotoxic responses revealed no advantage for combined therapy over other treatment modalities., Conclusions: These results suggest that finding new combinations of DC vaccines for the treatment of tumors will be promising in the future. The results of this study support the mathematical modelling for DC vaccine design. However, some parameters in this model must be modified to provide a more optimized therapy approach.

Regulatory T cells (Tregs) are an immunosuppressive subgroup of CD4 + T cells which are identified by the expression of forkhead box protein P3 (Foxp3). The modulation capacity of these immune cells holds an important role in both transplantation and the development of autoimmune diseases. These cells are the main mediators of self-tolerance and are essential for avoiding excessive immune reactions. Tregs play a key role in the induction of peripheral tolerance that can prevent autoimmunity, by protecting self-reactive lymphocytes from the immune reaction. In contrast to autoimmune responses, tumor cells exploit Tregs in order to prevent immune cell recognition and anti-tumor immune response during the carcinogenesis process. Recently, numerous studies have focused on unraveling the biological functions and principles of Tregs and their primary suppressive mechanisms. Due to the promising and outstanding results, Tregs have been widely investigated as an alternative tool in preventing graft rejection and treating autoimmune diseases. On the other hand, targeting Tregs for the purpose of improving cancer immunotherapy is being intensively evaluated as a desirable and effective method. The purpose of this review is to point out the characteristic function and therapeutic potential of Tregs in regulatory immune mechanisms in transplantation tolerance, autoimmune diseases, cancer therapy, and also to discuss that how the manipulation of these mechanisms may increase the therapeutic options., Competing Interests: FB and SJ were employed at Aryogen Pharmed. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Bayati, Mohammadi, Valadi, Jamshidi, Foma and Sharif-Paghaleh.)

Previous studies have demonstrated that maturation of dendritic cells (DCs) by pathogenic components through pathogen-associated molecular patterns (PAMPs) such as Listeria monocytogenes lysate (LML) or CpG DNA can improve cancer vaccination in experimental models. In this study, a mathematical model based on an artificial neural network (ANN) was used to predict several patterns and dosage of matured DC administration for improved vaccination. The ANN model predicted that repeated co-injection of tumor antigen (TA)-loaded DCs matured with CpG (CpG-DC) and LML (List-DC) results in improved antitumor immune response as well as a reduction of immunosuppression in the tumor microenvironment. In the present study, we evaluated the ANN prediction accuracy about DC-based cancer vaccines pattern in the treatment of Wehi164 fibrosarcoma cancer-bearing mice. Our results showed that the administration of the DC vaccine according to ANN predicted pattern, leads to a decrease in the rate of tumor growth and size and augments CTL effector function. Furthermore, gene expression analysis confirmed an augmented immune response in the tumor microenvironment. Experimentations justified the validity of the ANN model forecast in the tumor growth and novel optimal dosage that led to more effective treatment.

In vivo tracking and monitoring of adoptive cell transfer has a distinct importance in cell-based therapy. There are many imaging modalities for in vivo monitoring of biodistribution, viability and effectiveness of transferred cells. Some of these procedures are not applicable in the human body because of low sensitivity and high possibility of tissue damages. Shortwave infrared region (SWIR) imaging is a relatively new technique by which deep biological tissues can be potentially visualized with high resolution at cellular level. Indeed, scanning of the electromagnetic spectrum (beyond 1000 nm) of SWIR has a great potential to increase sensitivity and resolution of in vivo imaging for various human tissues. In this review, molecular imaging modalities used for monitoring of biodistribution and fate of administered cells with focusing on the application of non-invasive optical imaging at shortwave infrared region are discussed in detail., (© 2019 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Mesenchymal stem cells (MSCs) show considerable promise as a cellular immunotherapy for the treatment of a number of autoimmune and inflammatory disorders. However, the precise physiologically and therapeutically relevant mechanism(s) by which MSCs mediate immune modulation remains elusive. Dental pulp stem cells are a readily available source of MSCs that have been reported to show similar immune modulation in vitro as bone marrow MSCs. To test their potential in vivo, we used a clinically relevant humanized mouse model of GvHD in which only human T cells engraft. In this model, we found no effects on either T-cell proliferation, T-cell phenotype or disease progression. To determine if this lack of efficacy was related to a failure of engraftment or persistence of the cells, we used viability dependent radioactive cell tracking and showed that no cells were detectable after 24-h postinjection. Given the apparent failure of MSC to survive following intravenous injection, we hypothesized that their apoptosis may account for the widely reported therapeutic effect in numerous experimental models in vivo. To address this, we employed a well-established model of allergic airway inflammation to compare the efficacy of live and apoptotic MSCs in a fully immunocompetent model. In this model, both live and apoptotic dental pulp MSCs induced a robust immune suppressive reaction that was substantially greater with apoptotic cells. We propose that the mechanism of immune modulation following systemic application of MSCs is a result of cell entrapment and apoptosis occurring in the lungs.

Objectives: Toll-like receptors play an important role in innate and adaptive immune responses and can induce acute graft rejection, especially in the early phase after transplant. The aim of this study was to evaluate the possible association between TLR2, TLR4, and CD14 polymorphisms and acute renal rejection., Materials and Methods: Our study included 239 patients seen between 2013 and 2015. Patients were classified into 3 groups: acute rejection group (71 patients), stable graft function group (71 patients), and healthy control group (97 patients). Polymorphisms in TLR2 (Arg753Gln, rs5743708), TLR4 (Asp299Gly, rs4986790; Thr399Ile, rs4986791), and CD14 (-159C/T, rs2569190) were determined by the TaqMan allelic discrimination assay for detection of single-nucleotide polymorphisms., Results: The genotype distribution of CD14 rs2569190C/T was found to be significantly different among the acute rejection, stable graft function, and healthy control groups (P < .05). Interestingly, based on logistic regression, CD14 genotype (rs2569190) in patients with acute rejection was still significant after including risk factors. The adjusted odds ratio for CD14 CT+TT over CC genotype was calculated as 3.172 (95% confidence interval, 1.397-7.200; P = .006). Moreover, incidence of acute rejection and graft loss were significantly more frequent in recipients carrying CD14 TT (95% confidence interval, 2.81-27.16; P ≤ .001). In contrast to CD14, no significant differences were observed in the single-nucleotide polymorphisms of TLR2 and TLR4 genes in the acute rejection group versus the stable graft function and healthy control groups. The presence of CD14 T allele was associated with a significantly lower rejection-free survival compared with the CD14 CT and CC genotypes (P ≤ .001)., Conclusions: Renal transplant recipients carrying the CD14-159 TT genotype have significantly higher risk of acute rejection and reduced transplant survival rate than patients with heterozygous or wild-type genotypes.

Complement activation is a recognised mediator of myocardial ischaemia-reperfusion-injury (IRI) and cardiomyocytes are a known source of complement proteins including the central component C3, whose activation products can mediate tissue inflammation, cell death and profibrotic signalling. We investigated the potential to detect and quantify the stable covalently bound product C3d by external body imaging, as a marker of complement activation in heart muscle in a murine model of myocardial IRI. We used single-photon-emission-computed-tomography (SPECT) in conjunction with 99m Technecium-labelled recombinant complement receptor 2 ( 99m Tc-rCR2), which specifically detects C3d at the site of complement activation. Compared to control imaging with an inactive CR2 mutant ( 99m Tc-K41E CR2) or an irrelevant protein ( 99m Tc-PSMA) or using 99m Tc-rCR2 in C3-deficient mice, the use of 99m Tc-rCR2 in complement-intact mice gave specific uptake in the reperfused myocardium. The heart to skeletal muscle ratio of 99m Tc-rCR2 was significantly higher than in the three control groups. Histological analysis confirmed specific uptake of 99m Tc-rCR2. Following therapeutic inhibition of complement C3 activation, we found reduced myocardial uptake of 99m Tc-rCR2. We conclude, therefore that 99m Tc-rCR2 imaging can be used for non-invasive detection of activated complement and in future could be exploited to quantify the severity of myocardial damage due to complement activation.

Background: MicroRNAs (miRNAs) regulate most of encoding genes and protein. In this study, we aimed to investigate the expression levels of miR-142-5p, miR-142-3p, miR-155 and miR-223 in paired biopsy and peripheral blood mononuclear cell (PBMC) samples of renal allograft recipients with acute T-cell mediated rejection (ATCMR), compared with normal allografts (NA)., Methods: In this study, the expression levels of individual miRNAs were determined in biopsy and PBMC samples of 17 recipients with ATCMR and 18 recipients with NA., Results: Our results showed that the intragraft expression levels of all studied miRNAs were significantly higher in ATCMR than NA. However, regarding the PBMC samples, miR-142-3p and miR-223 were significantly increased in ATCMR than NA. Receiver operating characteristic (ROC) analysis showed that miR-142-5p, miR-142-3p, miR-155 and miR-223 in biopsy samples and miR-142-3p and miR-223 in PBMC samples could discriminate ATCMR from NA recipients., Conclusion: It has been reported that high intragraft expressions of miRNAs have a profound role in the pathogenesis of ATCMR process. Our results showed that high expression of all the studied miRNAs in biopsies and miR-142-3p and miR-223 in PBMC samples could be used as suggestive diagnostic tools to discriminate ATCMR patients from NA., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Purpose: (111)In (typically as [(111)In]oxinate3) is a gold standard radiolabel for cell tracking in humans by scintigraphy. A long half-life positron-emitting radiolabel to serve the same purpose using positron emission tomography (PET) has long been sought. We aimed to develop an (89)Zr PET tracer for cell labelling and compare it with [(111)In]oxinate3 single photon emission computed tomography (SPECT)., Methods: [(89)Zr]Oxinate4 was synthesised and its uptake and efflux were measured in vitro in three cell lines and in human leukocytes. The in vivo biodistribution of eGFP-5T33 murine myeloma cells labelled using [(89)Zr]oxinate4 or [(111)In]oxinate3 was monitored for up to 14 days. (89)Zr retention by living radiolabelled eGFP-positive cells in vivo was monitored by FACS sorting of liver, spleen and bone marrow cells followed by gamma counting., Results: Zr labelling was effective in all cell types with yields comparable with (111)In labelling. Retention of (89)Zr in cells in vitro after 24 h was significantly better (range 71 to >90%) than (111)In (43-52%). eGFP-5T33 cells in vivo showed the same early biodistribution whether labelled with (111)In or (89)Zr (initial pulmonary accumulation followed by migration to liver, spleen and bone marrow), but later translocation of radioactivity to kidneys was much greater for (111)In. In liver, spleen and bone marrow at least 92% of (89)Zr remained associated with eGFP-positive cells after 7 days in vivo., Conclusion: [(89)Zr]Oxinate4 offers a potential solution to the emerging need for a long half-life PET tracer for cell tracking in vivo and deserves further evaluation of its effects on survival and behaviour of different cell types.

Background: It is important to study differential inflammatory cellular migration, particularly of eosinophils and neutrophils, in asthma and how this is influenced by environmental stimuli such as allergen exposure and the effects of anti asthma therapy., Methods: We isolated blood neutrophils and eosinophils from 12 atopic asthmatic human volunteers (Group 1 - four Early Allergic Responders unchallenged (EAR); Group 2 - four Early and Late Allergic Responders (LAR) challenged; Group 3 - four EAR and LAR challenged and treated with systemic corticosteroids) using cGMP CD16 CliniMACS. Cells were isolated prior to allergen challenge where applicable, labelled with (99m)Tc-HMPAO and then re-infused intravenously. The kinetics of cellular influx/efflux into the lungs and other organs were imaged via scintigraphy over 4 h, starting at 5 to 6 h following allergen challenge where applicable., Results: Neutrophils and eosinophils were isolated to a mean (SD) purity of 98.36% (1.09) and 96.31% (3.0), respectively. Asthmatic neutrophils were activated at baseline, mean (SD) CD11b(High) cells 46 (10.50) %. Isolation and radiolabelling significantly increased their activation to > 98%. Eosinophils were not activated at baseline, CD69(+) cells 1.9 (0.6) %, increasing to 38 (3.46) % following isolation and labelling. Analysis of the kinetics of net eosinophil and neutrophil lung influx/efflux conformed to a net exponential clearance with respective mean half times of clearance 6.98 (2.18) and 14.01 (2.63) minutes for Group 1, 6.03 (0.72) and 16.04 (2.0) minutes for Group 2 and 5.63 (1.20) and 14.56 (3.36) minutes for Group 3. These did not significantly differ between the three asthma groups (p > 0.05)., Conclusions: Isolation and radiolabelling significantly increased activation of eosinophils (CD69) and completely activated neutrophils (CD11b(High)) in all asthma groups. Net lung neutrophil efflux was significantly slower than that of eosinophils in all asthma study groups. There was a trend for pre-treatment with systemic corticosteroids to reduce lung retention of eosinophils following allergen challenge.

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., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background: Hitherto, in vivo studies of human granulocyte migration have been based on indiscriminate labeling of total granulocyte populations. We hypothesized that the kinetics of isolated human neutrophil and eosinophil migration through major organs in vivo are fundamentally different, with the corollary that studying unseparated populations distorts measurement of both., Methods: Blood neutrophils and eosinophils were isolated on 2 separate occasions from human volunteers by using Current Good Manufacturing Practice CD16 CliniMACS isolation, labeled with technetium 99m-hexamethylpropyleneamine oxime, and then reinfused intravenously. The kinetics of cellular efflux were imaged over 4 hours., Results: Neutrophils and eosinophils were isolated to a mean purity of greater than 97% and greater than 95%, respectively. Activation of neutrophils measured as an increase in their CD11b mean fluorescence intensity in whole blood and after isolation and radiolabeling was 25.98 ± 7.59 and 51.82 ± 17.44, respectively, and was not significant (P = .052), but the mean fluorescence intensity of CD69 increased significantly on eosinophils. Analysis of the scintigraphic profile of lung efflux revealed exponential clearance of eosinophils, with a mean half-life of 4.16 ± 0.11 minutes. Neutrophil efflux was at a significantly slower half-life of 13.72 ± 4.14 minutes (P = .009). The migration of neutrophils and eosinophils was significantly different in the spleen at all time points (P = .014), in the liver at 15 minutes (P = .001), and in the bone marrow at 4 hours (P = .003)., Conclusions: The kinetics of migration of neutrophils and eosinophils through the lung, spleen, and bone marrow of human volunteers are significantly different. Study of mixed populations might be misleading., (Copyright © 2013 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Human skin immune homeostasis, and its regulation by specialized subsets of tissue-residing immune sentinels, is poorly understood. In this study, we identify an immunoregulatory tissue-resident dendritic cell (DC) in the dermis of human skin that is characterized by surface expression of CD141, CD14, and constitutive IL-10 secretion (CD141(+) DDCs). CD141(+) DDCs possess lymph node migratory capacity, induce T cell hyporesponsiveness, cross-present self-antigens to autoreactive T cells, and induce potent regulatory T cells that inhibit skin inflammation. Vitamin D(3) (VitD3) promotes certain phenotypic and functional properties of tissue-resident CD141(+) DDCs from human blood DCs. These CD141(+) DDC-like cells can be generated in vitro and, once transferred in vivo, have the capacity to inhibit xeno-graft versus host disease and tumor alloimmunity. These findings suggest that CD141(+) DDCs play an essential role in the maintenance of skin homeostasis and in the regulation of both systemic and tumor alloimmunity. Finally, VitD3-induced CD141(+) DDC-like cells have potential clinical use for their capacity to induce immune tolerance.

Introduction: Corneal transplantation is a surgical procedure in which damaged or diseased cornea is replaced by cadaveric corneal tissue. It is the most common form of solid-tissue transplantation in humans but its pharmacotherapy (in relation to graft rejection) has changed little for several decades. The mainstay of treatment of corneal graft rejection remains corticosteroids but these are variably effective and are associated with potentially serious adverse effects. Newer immunosuppressive drugs are increasingly being employed to manage high-risk grafts. However, these drugs are also not without side-effects, some of which can be severe and life-threatening., Areas Covered: This review outlines the corneal transplant procedure and the treatment options available in the management of transplant rejection., Expert Opinion: The surgical technique of corneal lamellar grafting has allowed for transplantation of smaller quantities of donor tissue to the recipient, thereby reducing the antigen load as a means of preventing a rejection episode. With greater understanding of the underlying molecular mechanisms involved in corneal transplant rejection pathology, potentially newer medications that will target specific cytokines or cells involved in rejection, whilst minimizing the potential side effects to the graft recipient, will be made available.

The occurrence of Graft-versus-Host Disease (GvHD) is a prevalent and potentially lethal complication that develops following hematopoietic stem cell transplantation. Humanized mouse models of xenogeneic-GvHD based upon immunodeficient strains injected with human peripheral blood mononuclear cells (PBMC; "Hu-PBMC mice") are important tools to study human immune function in vivo. The recent introduction of targeted deletions at the interleukin-2 common gamma chain (IL-2Rγ(null)), notably the NOD-scid IL-2Rγ(null) (NSG) and BALB/c-Rag2(null) IL-2Rγ(null) (BRG) mice, has led to improved human cell engraftment. Despite their widespread use, a comprehensive characterisation of engraftment and GvHD development in the Hu-PBMC NSG and BRG models has never been performed in parallel. We compared engrafted human lymphocyte populations in the peripheral blood, spleens, lymph nodes and bone marrow of these mice. Kinetics of engraftment differed between the two strains, in particular a significantly faster expansion of the human CD45(+) compartment and higher engraftment levels of CD3(+) T-cells were observed in NSG mice, which may explain the faster rate of GvHD development in this model. The pathogenesis of human GvHD involves anti-host effector cell reactivity and cutaneous tissue infiltration. Despite this, the presence of T-cell subsets and tissue homing markers has only recently been characterised in the peripheral blood of patients and has never been properly defined in Hu-PBMC models of GvHD. Engrafted human cells in NSG mice shows a prevalence of tissue homing cells with a T-effector memory (T(EM)) phenotype and high levels of cutaneous lymphocyte antigen (CLA) expression. Characterization of Hu-PBMC mice provides a strong preclinical platform for the application of novel immunotherapies targeting T(EM)-cell driven GvHD.

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.