Methods for Establishing a Renal Cell Carcinoma Tumor Spheroid Model With Immune Infiltration for Immunotherapeutic Studies

Tumor spheroids play an increasingly important role in cancer research. Their ability to recapitulate crucial features of tumor biology that are lost in the classically used 2D models along with their relative simplicity and handiness have made them the most studied 3D tumor model. Their application as a theranostic tool or as a means to study tumor-host interaction is now well-established in various cancers. However, their use in the field of Renal Cell Carcinoma (RCC) remains very limited. The aim of this work is to present methods to implement a basic RCC spheroid model. These methods cover the steps from RCC tumor dissociation to spheroid infiltration by immune cells. We present a protocol for RCC dissociation using Liberase TM and introduce a culture medium containing Epithelial Growth Factor and Hydrocortisone allowing for faster growth of RCC primary cells. We show that the liquid overlay technique allows for the formation of spheroids from cell lines and from primary cultures. We present a method using morphological criteria to select a homogeneous spheroid population based on a Fiji macro. We then show that spheroids can be infiltrated by PBMCs after activation with OKT3 or IL-15. Finally, we provide an example of application by implementing an immune spheroid killing assay allowing observing increased spheroid destruction after treatment with PD-1 inhibitors. Thus the straightforward methods presented here allow for efficient spheroid formation for a simple RCC 3D model that can be standardized and infused with immune cells to study immunotherapies

Tolerogenic Function of Dimeric Forms of HLA-G Recombinant Proteins: A Comparative Study In Vivo

HLA-G is a natural tolerogenic molecule involved in the best example of tolerance to foreign tissues there is: the maternal-fetal tolerance. The further involvement of HLA-G in the tolerance of allogeneic transplants has also been demonstrated and some of its mechanisms of action have been elucidated. For these reasons, therapeutic HLA-G molecules for tolerance induction in transplantation are actively investigated. In the present study, we studied the tolerogenic functions of three different HLA-G recombinant proteins: HLA-G heavy chain fused to β2-microglobulin (B2M), HLA-G heavy chain fused to B2M and to the Fc portion of an immunoglobulin, and HLA-G alpha-1 domain either fused to the Fc part of an immunoglobulin or as a synthetic peptide. Our results demonstrate the tolerogenic function of B2M-HLA-G fusion proteins, and especially of B2M-HLA-G5, which were capable of significantly delaying allogeneic skin graft rejection in a murine in vivo transplantation model. The results from our studies suggest that HLA-G recombinant proteins are relevant candidates for tolerance induction in human transplantation

HLA-G-mediated Immune Tolerance: Past and New Outlooks

The non-classical HLA class I molecule HLA-G is different from classical HLA class I molecules because of the low polymorphism in the coding region, the fact that HLA-G primary transcript is alternatively spliced in seven isoforms, and the inhibitory action on immune cells. Although HLA-G is low polymorphic, variants in both promoter and 3’ un-translated region (UTR) of HLA-G locus regulate its expression. In healthy conditions, a basal level of HLA-G gene transcription is observed in most cells and tissues; however, translation into HLA-G protein is restricted to trophoblasts in the placenta, where it participates in promoting tolerance at the fetal-maternal interface. HLA-G is also expressed by thymic epitelial, cornea, mesenchymal stem cells, nail matrix, pancreatic beta cells, erythroid, and endothelial precursors. HLA-G can be neo-expressed in adult tissues in pathological conditions, and its expression has been documented autoimmune disorders, viral infections, and cancer. In the latter setting de novo HLA-G expression is associated with the capability of tumor cells to evade the immune control. In the last decade it has become evident that HLA-G expression on T cells and antigenpresenting cells confers to these cells tolerogenic properties. This Research Topic focused on i) summarizing updated clinical and immunological evidences that HLA-G expression is associate with beneficial or detrimental tolerance, ii) gathering new insights into the mechanisms governing the expression of HLA-G in healthy and pathological conditions, such as pre-eclampsia, and iii) examining the mechanisms underlying HLA-G mediated tolerance

Pseudomonas aeruginosa quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine-lactone induces HLA-G expression in human immune cells

HLA-G is a nonclassical class I human leukocyte antigen (HLA) involved in mechanisms of immune tolerance. The objective of this study was to determine whether N-(3-oxododecanoyl)-l-homoserine lactone (3O-C12-HSL), a quorum sensing molecule produced by Pseudomonas aeruginosa, could modify HLA-G expression to control the host immune response. We evaluated the ability of 3O-C12-HSL to induce HLA-G expression in primary immune cells, monocytes (U937 and THP1), and T-cell lines (Jurkat) in vitro and analyzed the cellular pathway responsible for HLA-G expression. We studied the HLA-G promoter with a luciferase assay and interleukin-10 (IL-10) and p38/CREB signaling with enzyme-linked immunosorbent assay and immunofluorescence, respectively. We observed that 3O-C12-HSL is able to induce HLA-G expression in human monocytes and T cells. We showed that the induction of HLA-G by 3O-C12-HSL is p38/CREB and IL-10 dependent. 3O-C12-HSL treatment is able to arrest only the U937 cell cycle, possibly due to the peculiar expression of the ILT2 receptor in the U937 cell line. Our observations suggest HLA-G as a mechanism to create a protected niche for the bacterial reservoir, similar to the role of HLA-G molecules during viral infections

The Dual Role of HLA-G in Cancer

We here review the current data on the role of HLA-G in cancer based on recent findings of an unexpected antitumor activity of HLA-G in hematological malignancies. For the past decade, HLA-G has been described as a tumor-escape mechanism favoring cancer progression, and blocking strategies have been proposed to counteract it. Aside from these numerous studies on solid tumors, recent data showed that HLA-G inhibits the proliferation of malignant B cells due to the interaction between HLA-G and its receptor ILT2, which mediates negative signaling on B cell proliferation. These results led to the conjecture that, according to the malignant cell type, HLA-G should be blocked or conversely induced to counteract tumor progression. In this context, we will here present (i) the dual role of HLA-G in solid and liquid tumors with special emphasis on (ii) the HLA-G active structures and their related ILT2 and ILT4 receptors and (iii) the current knowledge on regulatory mechanisms of HLA-G expression in tumors