In this study we have described a novel approach in the retroviral transduction of activated human lymphocytes with a CO IL-15 gene. The codon optimization process improved IL-15 expression in transduced cells. Transduced human lymphocytes produced IL-15 in quantities with a measurable biological impact, persisting in vitro for up to 180 days in the absence of exogenous cytokine and resisting cytokine withdrawal-induced apoptosis. Transduction with IL-15 did not perturb lymphocyte Ag recognition or specificity. Furthermore, IL-15-transduced lymphocytes retained the ability to recognize Ag and secrete IFN-γ after withdrawal from exogenous cytokine support.
Codon optimization is a term that has been applied to a variety of approaches in which codons are systematically altered to enhance gene expression. Highly expressed genes evolve to use codons that are highly represented in the genome (28
). In some instances, gene expression can be augmented by replacing rare codons with codons favored by highly expressed genes. This strategy has been applied to enhancing murine or human expression of parasitic and viral genes, which often contain codons infrequently used in mammalian genes (29
). Conversely, altered codon usage has been applied to systems in which production of mammalian proteins by bacterial systems is desired (32
). In addition to codon bias considerations, RNA secondary structure formation and stability clearly impact protein expression and can be influenced by alternate codon usage (34
). This approach continues to be refined, and its use is expanding. To our knowledge, codon optimization of cytokine expression by human cells has not been previously demonstrated.
Inefficient expression of IL-15 has been well established. The 5′-untranslated region, which contains multiple AUGs, has been implicated in the obstruction of mRNA translation (15
). Furthermore, an inhibitory effect of the secreted isoform of the IL-15 signal peptide and mature protein C terminus was demonstrated (16
). The expression of IL-15 was markedly increased when the native leader sequence was replaced with either the mouse IL-2 signal peptide or the bovine PPL sequence (6
). We generated a construct containing the PPL sequence and further modified the coding sequence through alternate codon usage, thereby minimizing the usage of rare codons and minimizing the free energy (and thus potential folding) of the mRNA transcript.
Interestingly, cells transfected with either the wild-type or CO IL-15 constructs produced similar amounts of protein (). This may be explained by the fact that transfected cells receive hundreds of copies of the plasmid DNA, possibly saturating the protein production machinery. However, retrovirally transduced NIH-3T3 cells receiving the CO gene demonstrated an ~2.5-fold increase in protein expression compared with cells receiving the wild-type gene (). The NIH-3T3 cells were transduced with retrovirus of comparable titer, and assessment of retroviral integration demonstrated similar efficiency of transduction (). Thus, we conclude that the codon optimization improved gene expression at the level of mRNA stability and/or efficiency of translation.
Having established a means to engineer human lymphocytes to efficiently express IL-15, we were able to make several observations. We detected IL-15Rα on the surface of activated CD4+
T cells, contrary to a previous report in which activated human CD8+
, but not CD4+
, T cells, expressed this receptor (36
). The IL-15Rα Ab used in our studies did not bind to lymphocytes cultured in exogenous IL-15 or transduced with the IL-15 gene, implying an interaction between IL-15 and the high affinity α receptor (). In these OKT3-activated T cells, we speculate that the Ab-binding site on the receptor is altered or blocked upon binding IL-15. Alternatively, IL-15Rα may be internalized after capturing IL-15. Based on the observed expression of IL-15Rα on activated T cells, it is possible that IL-15 gene expression in individual cells led to their persistence via an autocrine mechanism or that paracrine effects from a fraction of lymphocytes excreting IL-15 were sufficient to sustain the entire lymphocyte culture.
Common γ-chain cytokines promote the survival of activated T lymphocytes through common signaling pathways that induce the expression of antiapoptotic Bcl-2 family proteins (37
). We have demonstrated that IL-15-transduced lymphocytes, withdrawn from exogenous cytokine support, exhibited continued proliferation as well as resistance to apoptosis; this coincided with the maintenance of Bcl-2 and Bcl-xL
(–). In vitro, this resulted in prolonged survival in IL-15-transduced lymphocytes (). Furthermore, peptide-stimulated, IL-15-transduced lymphocytes retained Ag recognition and specificity even after withdrawal from exogenous IL-2 (). In most respects, IL-15-transduced lymphocytes behaved similarly to control cell populations maintained in the presence of IL-2. This is not entirely unexpected given that IL-2Rs and IL-15Rs share β and γ subunits as well as signaling through common Jak/Stat pathways (26
). Our studies corroborate previous reports in which IL-2 and IL-15 shared the ability to stimulate activated T cells in vitro (1
In several preclinical models, the addition of IL-15 to cell transfer therapy regimens significantly improved antitumor activity (13
). Briefly, these studies have demonstrated that culturing antitumor lymphocytes ex vivo in the presence of IL-15, delivering lymphocytes bearing an IL-15 transgene, or administering adjuvant IL-15 during the course of adoptive cell transfer resulted in superior antitumor activity. These models also demonstrated superior treatment effects of IL-15 compared with IL-2 when both cytokines were used in a similar fashion. The fact that IL-2 and IL-15 seem to have similar functions in vitro, but distinct and often opposing actions in vivo, is at least partially explained by murine models demonstrating that IL-15 is captured by APCs and stromal cells expressing high levels of the high affinity IL-15Rα and subsequently presented to lymphocytes and NK cells (42
). It seems likely that the complex interactions between IL-15-presenting cells and responding lymphocytes powerfully influence the differentiation of effector T cells. This is strongly supported by the finding that IL-15Rα-deficient memory CD8+
T cells could be sustained by other cells expressing IL-15Rα (46
). Furthermore, IL-15 antitumor activity in vivo may also be enhanced by the effects of IL-15 on cells other than the transferred lymphocyte, such as APCs or NK cells. To determine whether results reported in murine models, demonstrating superiority of IL-15 in adoptive cell transfer cancer models, would be recapitulated in humans would require clinical trials in which patients receive treatment with IL-15.
The use of IL-15-expressing T cells in adoptive transfer studies involving human patients has potential drawbacks. IL-15-transgenic mice develop autoimmunity and can ultimately succumb to lymphocytic leukemia (47
). In humans, abnormal expression of IL-15 has been associated with rheumatoid arthritis, inflammatory bowel disease, adult T cell leukemia, and tropical spastic paresis (48
). In our experience with IL-15-transduced human lymphocytes, one of 17 cultures evolved into a clonal population that exhibited growth for >1 year in the absence of exogenous cytokine (data not shown); it remains uncertain whether this is a consequence of IL-15 overexpression or vector integration at a site critical to cell cycle regulation. With these considerations, the clinical application of gene-engineered T cells constitutively expressing IL-15 would probably require a reliable way to terminate the response should adverse effects of the treatment arise. HSV thymidine kinase-engineered lymphocytes have proven effective in controlling graft-vs-host disease in human bone marrow transplant recipients (50
). Thus, the development of an IL-15 retroviral vector carrying HSV thymidine kinase might permit the safe administration of IL-15-transduced, tumor-reactive lymphocytes to patients with metastatic cancer.