The effects of folate-conjugated gold nanorods in combination with plasmonic photothermal therapy on mouth epidermal carcinoma cells.

The use of lasers has emerged to be highly promising for cancer therapy modalities, most commonly, the photothermal therapy method. Unfortunately, the most common disadvantage of laser therapy is its nonselectivity and requirement of high power density. The use of plasmonic nanoparticles as highly enhanced photoabsorbing agents has thus introduced a much more selective and efficient cancer therapy strategy. In this study, we aimed to demonstrate the selective targeting and destruction of mouth epidermal carcinoma cells (KB cells) using the photothermal therapy of folate-conjugated gold nanorods (F-GNRs). Considering the beneficial characteristics of GNRs and overexpression of the folate receptor by KB cells, we selected F-GNRs as a targeted photothermal therapy agent. Cell viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by flow cytometry using an annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit. No cell damage or cytotoxicity from the individual treatment of laser light or F-GNRs was observed. However, a 56% cell lethality was achieved for KB cells using combined plasmonic photothermal therapy of 20 µM F-GNRs with seven pulses of laser light and 6-h incubation periods. Cell lethality strongly depends on the concentration of F-GNRs and the incubation period that is mainly due to the induction of apoptosis. This targeted damage is due to the F-GNRs present in the cancer cells strongly absorbing near-infrared laser light and rapidly converting it to heat. This new therapeutic avenue for cancer therapy merits further investigation using in vivo models for application in humans.

4-1BBL costimulation retrieves CD28 expression in activated T cells.

Binding of CD80/86 to CD28 is regarded as the main T cell costimulatory interaction. However, CD28 downregulates soon after T cell activation. To investigate potential cross-interaction between CD137 (4-1BB) and CD28, we stimulated T cells with anti-CD3 in the presence of A549 lung carcinoma cells expressing CD80/CD86 and 4-1BBL molecules, transduced into the cells using recombinant non-replicating adenoviruses. Following initial T cell proliferation, the proportion of CD28(+) cells in both CD4(+) and CD8(+) populations was rapidly reduced by CD80/86 costimulation, whereas cultures costimulated with just 4-1BBL continued to express CD28. CD28 was also downregulated in cultures costimulated with both CD80/86 and 4-1BBL. Interestingly, in cells costimulated with CD80/86 that had downregulated CD28 expression and ceased to proliferate, reactivation of proliferation by 4-1BBL costimulation also restored their CD28 expression. These findings show a positive effect of CD137 signalling on CD28 expression, similar to the effect of CD28 engagement on 4-1BB expression during the initial phases of T cell activation. Moreover, they point to the importance of signals through 4-1BB for the purposes of ex-vivo T cell activation and expansion.

Preferential T cell expansion by artificial antigen presenting cells expressing 4-1BBL alone or in combination with CD80 or CD86.

BACKGROUND: Varieties of artificial antigen presenting cells (aAPCs) with different efficiencies have been introduced to expand whole T cell population or antigen specific ones for the purpose of T cell therapy. From antibody coated beads to gene modified dendritic cells each has some advantages and disadvantages. However, no one can ignore the importance and the necessity of costimulation interaction during T cell activation. OBJECTIVE: This study was designed to compare the effectiveness of CD80/CD86 and 4-1BBL, two major costimulatory families, in costimulation of autologous T cell responses. METHODS: We used recombinant non-replicative adenoviral vectors and transferred genes of these ligands to autologous blood monocytes and skin fibroblasts to create aAPCs system. T cell response to anti-CD3 pan stimulation and some viral peptide Ags, in co-culture with gene modified monocytes and fibroblasts were studied using CFSE and HLA tetramers, respectively. RESULTS: Over-expression of ligands was able to expand the T cell population significantly higher than normal cells with no interference with antigen stimulation. Presence of 4-1BBL alone or in combination with B7 members enhanced T cell expansion and promoted more Ag-specific cells to accumulate in these culture systems. CONCLUSION: Considering the inhibitory proportion of B7 costimulation route, 4-1BBL, as an alternative signaling pathway, in combination with B7 will promote T cell proliferation and expansion.

Co-stimulation with 4-1BB ligand allows extended T-cell proliferation, synergizes with CD80/CD86 and can reactivate anergic T cells.

Activation of T cells requires co-stimulation, in addition to signals through the antigen-receptor complex. Antigen encounter without adequate co-stimulation results in T-cell desensitization or anergy, a mechanism of peripheral tolerance and an apparent obstacle to cancer immunotherapy. One important co-stimulatory pathway involves CD28 engagement by CD80 or CD86. However, other ligand-receptor pairs can also provide co-stimulation and may have important functions modulating the immune response. Previous reports indicated that co-stimulation using 4-1BB ligand (4-1BBL) or agonistic anti-4-1BB antibodies could prolong T-cell responses, avoid activation-induced cell death and promote anti-tumour responses in mice. To further investigate the potential for cancer immunotherapy, we studied the effects of CD80/CD86 and 4-1BBL in repeated stimulation of human T cells and asked whether 4-1BBL might be capable of reversing anergy. We expressed CD80, CD86 and 4-1BBL in A549 lung carcinoma cells using adenovirus vectors and co-cultured these with human T cells stimulated with anti-CD3 antibody. Proliferation co-stimulated by CD80 or CD86 was transient; however, 4-1BBL-co-stimulated cultures continued to proliferate for up to 5 weeks, with repeated stimulation. Combined co-stimulation with CD80/CD86 and 4-1BBL also allowed continuous proliferation at a faster rate than either signal alone. Co-stimulation with 4-1BBL did not suppress expression of the inducible, inhibitory CD80/CD86R, CTLA-4. Significantly, we show that T cells that had become non-responsive to anti-CD3, either alone or together with CD80/CD86 co-stimulation, and thus were anergic, could be reactivated to proliferate when costimulated with 4-1BBL, either alone or combined with CD80/CD86.