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

Dual stimulation of antigen presenting cells using carbon nanotube-based vaccine delivery system for cancer immunotherapy

Although anti−cancer immuno−based combinatorial therapeutic approaches have shown promising results, efficient tumour eradication demands further intensification of anti−tumour immune response. With the emerging field of nanovaccinology, multi−walled carbon nanotubes (MWNTs) have manifested prominent potentials as tumour antigen nanocarriers. Nevertheless, the utilization of MWNTs in co−delivering antigen along with different types of immunoadjuvants to antigen presenting cells (APCs) has not been investigated yet. We hypothesized that harnessing MWNT for concurrent delivery of cytosine−phosphate−guanine oligodeoxynucleotide (CpG) and anti-CD40 Ig (αCD40), as immunoadjuvants, along with the model antigen ovalbumin (OVA) could potentiate immune response induced against OVA−expressing tumour cells. We initially investigated the effective method to co−deliver OVA and CpG using MWNT to the APC. Covalent conjugation of OVA and CpG prior to loading onto MWNTs markedly augmented the CpG−mediated adjuvanticity, as demonstrated by the significantly increased OVA−specific T cell responses in vitro and in C57BL/6 mice. αCD40 was then included as a second immunoadjuvant to further intensify the immune response. Immune response elicited in vitro and in vivo by OVA, CpG and αCD40 was significantly potentiated by their co−incorporation onto the MWNTs. Furthermore, MWNT remarkably improved the ability of co−loaded OVA, CpG and αCD40 in inhibiting the growth of OVA−expressing B16F10 melanoma cells in subcutaneous or lung pseudo−metastatic tumour models. Therefore, this study suggests that the utilization of MWNTs for the co−delivery of tumour−derived antigen, CpG and αCD40 could be a competent approach for efficient tumours eradication

Carbon nanotubes' surface chemistry determines their potency as vaccine nanocarriers in vitro and in vivo

Carbon nanotubes (CNTs) have shown marked capabilities in enhancing antigen delivery to antigen presenting cells. However, proper understanding of how altering the physical properties of CNTs may influence antigen uptake by antigen presenting cells, such as dendritic cells (DCs), has not been established yet.Wehypothesized that altering the physical properties of multi-walled CNTs (MWNTs)-antigen conjugates, e.g. length and surface charge, can affect the internalization of MWNT-antigen by DCs, hence the induced immune response potency. For this purpose, pristineMWNTs (p-MWNTs) were exposed to various chemical reactions to modify their physical properties then conjugated to ovalbumin (OVA), a model antigen. The yieldedMWNTs-OVA conjugateswere longMWNT-OVA (~386 nm), bearing net positive charge (5.8mV), or shortMWNTs-OVA (~122 nm) of increasing negative charges (−23.4, −35.8 or −39 mV). Compared to the short MWNTs-OVA bearing high negative charges, short MWNT-OVA with the lowest negative charge demonstrated better cellular uptake and OVAspecific immune response both in vitro and in vivo. However, long positively-chargedMWNT-OVA showed limited cellular uptake and OVA specific immune response in contrast to shortMWNT-OVA displaying the least negative charge. We suggest that reduction in charge negativity of MWNT-antigen conjugate enhances cellular uptake and thus the elicited immune response intensity. Nevertheless, length of MWNT-antigen conjugate might also affect the cellular uptake and immune response potency; highlighting the importance of physical properties as a consideration in designing a MWNT-based vaccine delivery system

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 accelerated graft rejection. The latter was inhibited by recipient injection with anti-CD8 antibody, 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 antigen presenting cells (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 T cell receptor-transgenic T cells, antigen presentation of injected OVA-pulsed DCs was detectable for ≤3 days whilst indirect presentation of MHC alloantigen by recipient APCs led to activation of T cells within 14 hours 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 non-DC treated controls. Interestingly, 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