The immune system is composed of a portion of T cells that express an invariant T cell receptor (TCR) alpha chain known as V alpha 24 J alpha 18. These highly conserved populations are referred to as iNKT populations and have the ability to rapidly produce cytokines following activation, making them hot targets for therapeutic research initiatives. In this article, we will review van derVilet et al’s study of the effects of administering high dose interleukin-2 to immunoregulatory cell subsets in patients presenting advanced melanoma and renal cell cancer. This group uses antibodies in a variety of applications to determine whether these cells can rescue skin and kidney malignancies. In this paper, dendritic cell subsets (DC), CD1d-reactive invariant natural killer T cells (iNKT) and CD4+CD25+ regulatory-type T cells are exposed to high-dose IL-2 therapy.
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Flow (Cell Surface): V alpha 24 J alpha 18 TCR Antibody (6B11) [NBP2-00267] - A cell surface stain was performed on CD3+ hPBMCs with V alpha 24 J alpha 18 TCR antibody (6B11) NBP2-00267PE (top image) and a matched isotype control NBP2-27287 (bottom image). Cells were incubated in an antibody dilution of 1 ug/mL for 20 minutes at room temperature. Both antibodies were conjugated to Phycoerythrin. A co-stain was also performed using CD3 antibody NBP2-24867APC.
This research was conducted using a variety of experimental methods including flow cytometry, the generation of iNKT cell lines for testing, FACS and statistical analysis to measure most results. Patients that had a mean age range of 53 years that had been diagnosed with advanced melanoma or renal cell carcinoma were treated with high dose IL-2. In addition, healthy adult volunteers donated peripheral blood mononuclear cells, which helped to enrich iNKT cells, and were subsequently expanded and purified. Peripheral blood mononuclear cells were stained with antibodies to CD3, CD25, CD56, CD1d, CD86, CD19, CD14 and V alpha 24 J alpha 18 (6B11) and flow cytometry was performed using a Cytomics FC 500 machine. Lastly, CD25+ cells were isolated via magnetic-activated cell sorting. In the end, the supernatant from these cells were put through an ELISA assay to detect IFN gamma and IL-4 antibodies.
Through these experimental methods, this group found that high dose IL-2 treatment in peripheral blood lymphocyte subsets decreased levels of both CD4+ and CD8+ T cells at first, however these numbers gradually returned to normal. Similar results were found when IL-2 treatment was applied circulating myeloid DC cells. When this experiment was applied to iNKT cells, it was observed that there was a significant increase of double-negative iNKT cells after the first week of high dose IL-2 treatment. Moving on to CD4+CD25+ cells, results were similar for levels of these cells during and after IL-2 treatment. However, the treatment did induce changes in the cell surface expression of many molecules required for their function (GITR and CD30).
Overall, this data concludes that treatment of high dose IL-2 results in alterations of immunoregulatory cell subsets (specifically for that of CD4+CD25+ cells), which may hinder our innate immune response. Specifically, suppression of antitumor immune responses through CTLA-4 and CD30 were witnessed. Furthermore, while iNKT cells presented a flux in levels during IL-2 treatment, the increase in proinflammatory double-negative iNKT cells is still a point of interest. Using the V alpha 24 J alpha 18 (6B11) monoclonal antibody proved to be a useful tool in identifying these cell subtypes.
