Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model

B cells have been reported to promote graft rejection through alloantibody production. However, there is growing evidence that B cells can contribute to the maintenance of tolerance. Here, we used a mouse model of MHC-class I mismatched skin transplantation to investigate the contribution of B cells to graft survival. We demonstrate that adoptive transfer of B cells prolongs skin graft survival but only when the B cells were isolated from mice housed in low sterility "conventional" (CV) facilities and not from mice housed in pathogen free facilities (SPF). However, prolongation of skin graft survival was lost when B cells were isolated from IL-10 deficient mice housed in CV facilities. The suppressive function of B cells isolated from mice housed in CV facilities correlated with an anti-inflammatory environment and with the presence of a different gut microflora compared to mice maintained in SPF facilities. Treatment of mice in the CV facility with antibiotics abrogated the regulatory capacity of B cells. Finally, we identified transitional B cells isolated from CV facilities as possessing the regulatory function. These findings demonstrate that B cells, and in particular transitional B cells, can promote prolongation of graft survival, a function dependent on licensing by gut microflora

[89Zr]Oxinate4 for long-term in vivo cell tracking by positron emission tomography

Purpose 111In (typically as [111In]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 89Zr PET tracer for cell labelling and compare it with [111In]oxinate3 single photon emission computed tomography (SPECT). Methods [89Zr]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 [89Zr]oxinate4 or [111In]oxinate3 was monitored for up to 14 days. 89Zr 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 111In labelling. Retention of 89Zr in cells in vitro after 24 h was significantly better (range 71 to >90 %) than 111In (43–52 %). eGFP-5T33 cells in vivo showed the same early biodistribution whether labelled with 111In or 89Zr (initial pulmonary accumulation followed by migration to liver, spleen and bone marrow), but later translocation of radioactivity to kidneys was much greater for 111In. In liver, spleen and bone marrow at least 92 % of 89Zr remained associated with eGFP-positive cells after 7 days in vivo. Conclusion [89Zr]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

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

Diversity of gut microflora is required for the generation of B cell with regulatory properties in a skin graft model

B cells have been reported to promote grafft rejectfion through alloantfibody productfion. However, there fis growfing evfidence that B cells can contrfibute to the mafintenance off tolerance. Here, we used a mouse model off MHC-class I mfismatched skfin transplantatfion to finvestfigate the contrfibutfion off B cells to grafft survfival. We demonstrate that adoptfive transffer off B cells prolongs skfin grafft survfival but only when the B cells were fisolated ffrom mfice housed fin low sterfilfity “conventfional” (CV) ffacfilfitfies and not ffrom mfice housed fin pathogen ffree ffacfilfitfies (SPF). However, prolongatfion off skfin grafft survfival was lost when B cells were fisolated ffrom IL-10 deficfient mfice housed fin CV ffacfilfitfies. The suppressfive ffunctfion off B cells fisolated ffrom mfice housed fin CV ffacfilfitfies correlated wfith an antfi-finlammatory envfironment and wfith the presence off a dfifferent gut mficrolora compared to mfice mafintafined fin SPF ffacfilfitfies. Treatment off mfice fin the CV ffacfilfity wfith antfibfiotfics abrogated the regulatory capacfity off B cells. Ffinally, we fidentfified transfitfional B cells fisolated ffrom CV ffacfilfitfies as possessfing the regulatory ffunctfion. These findfings demonstrate that B cells, and fin partficular transfitfional B cells, can promote prolongatfion off grafft survfival, a ffunctfion dependent on lficensfing by gut mficrolora

Noninvasive Imaging of Activated Complement in Ischemia-Reperfusion Injury Post–Cardiac Transplant

Ischemia‐reperfusion injury (IRI) is inevitable in solid organ transplantation, due to the transplanted organ being ischemic for prolonged periods prior to transplantation followed by reperfusion. The complement molecule C3 is present in the circulation and is also synthesized by tissue parenchyma in early response to IRI and the final stable fragment of activated C3, C3d, can be detected on injured tissue for several days post‐IRI. Complement activation post‐IRI was monitored noninvasively by single photon emission computed tomography (SPECT) and CT using (99m)Tc‐recombinant complement receptor 2 ((99m)Tc‐rCR2) in murine models of cardiac transplantation following the induction of IRI and compared to (99m)Tc‐rCR2 in C3(−/−) mice or with the irrelevant protein (99m)Tc‐prostate–specific membrane antigen antibody fragment (PSMA). Significant uptake with (99m)Tc‐rCR2 was observed as compared to C3(−/−) or (99m)Tc‐PSMA. In addition, the transplanted heart to muscle ratio of (99m)Tc‐rCR2 was significantly higher than (99m)Tc‐PSMA or C3(−/−). The results were confirmed by histology and autoradiography. (99m)Tc‐rCR2 can be used for noninvasive detection of activated complement and in future may be used to quantify the severity of transplant damage due to complement activation postreperfusion

Whole-body imaging of adoptively transferred T cells using magnetic resonance imaging, single photon emission computed tomography and positron emission tomography techniques, with a focus on regulatory T cells

Cell-based therapies using natural or genetically modified regulatory T cells (T(regs)) have shown significant promise as immune-based therapies. One of the main difficulties facing the further advancement of these therapies is that the fate and localization of adoptively transferred T(regs) is largely unknown. The ability to dissect the migratory pathway of these cells in a non-invasive manner is of vital importance for the further development of in-vivo cell-based immunotherapies, as this technology allows the fate of the therapeutically administered cell to be imaged in real time. In this review we will provide an overview of the current clinical imaging techniques used to track T cells and T(regs) in vivo, including magnetic resonance imaging (MRI) and positron emission tomography (PET)/single photon emission computed tomography (SPECT). In addition, we will discuss how the finding of these studies can be used, in the context of transplantation, to define the most appropriate T(reg) subset required for cellular therapy