Small RNA Enhances Antitumor T-cell Therapy
miR-155 augments cytotoxic T cell antitumor activity by enhancing responsiveness to limited amounts of homeostatic cytokines. A, In the lymphoreplete host, homeostatic cytokine signaling is limited by the activity of multiple negative regulators, such as Ship1, Socs1, and Ptpn2. B, Lymphodepletion increases the availability of homeostatic cytokines, overcoming the inhibitory effects of Ship1, Socs1, and Ptpn2 and resulting in enhanced antitumor responses of adoptively transferred tumor-specific cytotoxic T cells. C, Overexpression of miR-155 in adoptively transferred tumor-specific cytotoxic T cells inhibits the expression of Ship1, Socs1, and Ptpn2, resulting in enhanced cytokine signaling and antitumor responses even in the presence of limited amounts of homeostatic cytokines.
Adoptive T-cell transfer is an effective form of anticancer immunotherapy in which patients receive infusions of cytotoxic T cells that seek out and destroy targeted cancer cells. This type of therapy is usually preceded by a lymphodepleting chemotherapy regimen and combined with high doses of the cytokine interleukin-2 (IL-2) to eliminate immunosuppressive and other immune cells and to enhance the survival and activity of the transplanted cells. Unfortunately, these high-intensity treatments often lead to severe side effects, such as a prolonged reduction of white blood cells, an increased risk of clotting events, or an accumulation of fluid in the tissues, which limit the pool of patients healthy enough to receive the treatment and can result in prolonged hospitalization and higher health care costs. New approaches that are less toxic but equally effective could allow for more widespread use of adoptive T-cell transfer.
Luca Gattinoni, M.D, of CCR’s Experimental Transplantation and Immunology Branch, and his colleagues, including Nicholas Restifo, M.D., of CCR’s Surgery Branch, and Thomas Waldmann, M.D., of CCR’s Lymphoid Malignancies Branch, decided to examine the activity of the microRNA miR-155 in adoptive T-cell transfer, given its essential role in cytotoxic T-cell responses against tumors. The researchers transduced cytotoxic T cells that recognize gp100, an antigen on the surface of some melanomas and normal melanocytes, with retroviruses expressing GFP and miR-155, GFP and a scrambled microRNA (scr), or GFP alone. They infused the modified cells into mice with intact immune systems bearing subcutaneous melanomas along with a recombinant vaccinia virus expressing gp100, which is necessary for tumor regression in this model system. Encouragingly, the investigators observed a significant reduction in tumor size in mice that received the GFP-miR-155 overexpressing cells but not the GFP or GFP-scr cells. The only acute adverse effect they saw in mice treated with the GFP-miR-155 cells was a reduction in skin pigmentation (vitiligo), a known toxicity of the gp100-targeted cells.
To understand how overexpression of miR-155 enhanced T-cell antitumor activity, the scientists measured the growth and function of GFP-miR-155 and GFP T cells in their mouse model. The GFP cells rapidly expanded, with growth peaking four days after virus infection, and then swiftly contracted so that almost no cells survived at day seven. The GFP-miR-155 cells underwent a similar expansion, but their contraction was significantly delayed. The cells also showed increased polyfunctionality and retained the ability to secrete cytokines, such as IL-2, longer than the GFP cells. These results suggest that miR-155 augments the T-cell anticancer response by prolonging cell survival and function.
The researchers then asked whether the miR-155-induced antitumor activity could be further increased by lymphodepletion. As expected, GFP-expressing T cells showed better antitumor efficacy, even prolonging survival, in mice exposed to total body irradiation. In contrast, GFP-miR-155 cells had similar anticancer activity in lymphodepleted and lymphoreplete mice.
Since one role of lymphodepletion in adoptive T-cell transfer is to reduce the number of endogenous cells competing for homeostatic cytokines, the investigators wondered whether miR-155 functioned by enhancing the T-cell response to limiting levels of these cytokines. In support of this idea, they found that giving high doses of the homeostatic cytokines IL-7 and IL-15 promoted the antitumor activity of GFP T cells but not of GFP-miR-155-expressing cells. To determine whether IL-7 and IL-15 are required for the enhanced T-cell activity of miR-155, the researchers infused GFP or GFP-miR-155 T cells and the gp100 vaccine into wild type mice, mice lacking the IL-15 gene, and mice lacking both the IL-15 and IL-7 genes. Loss of IL-15 resulted in a reduced number of functional GFP-miR-155 cells, which were further depleted in the double mutant mice. When they assessed the antitumor activity of the cells in the wild type and double mutant mice, the researchers saw a significant decrease in GFP-miR-155 cell efficacy in mice lacking IL-15 and IL-7, indicating that homeostatic cytokines are essential for the enhanced activity of these cells.
Finally, the investigators wanted to understand the molecular mechanism by which miR-155 promotes T-cell efficacy. They evaluated well-known signaling pathways downstream of the homeostatic cytokines and found increased Akt and Stat5 activity in miR-155-expressing cells. miR-155 is known to control the expression of the Akt negative regulator Ship1 and the Stat5 negative regulator Socs1, and in fact, the scientists showed reduced levels of both proteins in the T cells overexpressing miR-155. They also found that miR-155 suppressed the expression of Ptpn2, another inhibitor of Stat5 signaling. To determine how the two pathways contribute to miR-155-mediated T-cell function, the researchers expressed constitutively-active Stat5 or Akt in GFP or GFP-miR-155 T cells. They found that Stat5 was required for delaying T-cell contraction while Akt was necessary for prolonging T-cell function.
These studies demonstrate that overexpression of miR-155 in cytotoxic T cells was sufficient to replace immunodepletion and high dose cytokine administration to enhance the antitumor activity of adoptively transferred T cells. Though further studies will be required, expressing miR-155 in T cells may eliminate the need for highly toxic preparative therapies and make adoptive T-cell transfer available to a broader spectrum of cancer patients.Summary Posted: Thu, 01/01/2015
Ji Y, Wrzesinski C, Yu Z, Hu J, Gautam S, Hawk NV, Telford WG, Palmer DC, Franco Z, Sukumar M, Roychoudhuri R, Clever D, Klebanoff CA, Surh CD, Walmann TA, Restifo NP, and Gattinoni L. miR-155 Augments CD8+ T-cell Antitumor Activity in Lymphoreplete Hosts by Enhancing Responsiveness to Homeostatic γc Cytokines. PNAS. December 20, 2014 PubMed Link