Imaging Inflammation and Tumorigenesis in vivo
TAX-LUC mice are doubly transgenic mice in which i) the Tax gene from HTLV-1 is restricted to activated NK and T cells by the granzyme B promoter and ii) luciferase, under the control of the HTLV-1 LTR, is activated by Tax [25]. In principle, luciferase, which catalyzes a light emitting reaction in the presence of its substrate D-luciferin, serves as an indirect biomarker for activated NK and T cells in TAX-LUC mice. Alternatively, upon activation of leukocytes during inflammation, neutrophil myeloperoxidases are expressed that catalyze the production of hypochlorous acid from hydrogen peroxide and chloride ions [27]. Luminol emits light when exposed to oxidizing agents and can be used to sensitively and non-invasively detect leukocyte activity during inflammation in vivo. We have shown that administration of either luminol or D-luciferin produces bioluminescence in primary TAX-LUC tumors and that microscopic bioluminescent lesions precede tumorigenesis. We sought to determine the effects of inflammation on bioluminescence and tumorigenesis in this model.
We first asked whether wounding was sufficient to result in a luciferase-mediated bioluminescent signature in TAX-LUC mice. We found that minor incisions on the ear, tail or foot (Fig. 1) were sufficient to produce a significant bioluminescent signature and that introduction of adjuvant in the wound increased the intensity and duration of the signal. These data confirmed a close correlation between wounding and reporter expression in vivo.
Generalized T Cell Activation is Associated with Tumorigenesis
While Tax is activated in malignant LGL cells of inflamed tumors, the granzyme B promoter is also inducible in T and NK cells by T-cell receptor (TCR)-dependent, TCR-independent, and cytokine-mediated stimuli [28]. A number of direct and indirect inducers of generalized T cell activation were utilized to locally activate this promoter and image Tax activity during inflammation. These included phorbol 12-myristyl 13-acetate (PMA), which when administered topically, promotes T lymphocyte infiltration and activation mediated by protein kinase C, and has been shown to stimulate the human granzyme B promoter in transgenic mice [29, 30]. Topical administration of PMA to the ear resulted in luciferase based bioluminescence in TAX-LUC mice, but not LTR-LUC mice (Fig. 2A, top panels) even though a massive inflammatory infiltrate was seen in all PMA treated ears (Fig. 2B). Luminol based bioluminescence emanating from the PMA treated ears compared to the vehicle treated contralateral ears (Fig. 2A, bottom panels) served as a reporter for inflammation. The intensity of luminol BLI after PMA treatment was greater in TAX-LUC mice than LTR-LUC littermates that lack the Tax transgene (fold flux increase 11.5 vs. 7.4; p = 0.018). These findings serve as proof of principle for the appropriate regulation of the transgenes in TAX-LUC mice, confirm that acute inflammation is sufficient to produce bioluminescence in this model, and suggest that Tax expression exacerbates the inflammatory response in vivo.
Con A, a potent lectin with broad activity towards T lymphocytes, is also known to activate the granzyme B promoter. To determine whether induction of inflammation affected tumorigenesis in this model, we examined 5 TAX-LUC mice and 5 LTR-LUC in each group given tail vein injections of con A or saline (Fig. 2C). While TAX-LUC mice develop peripheral tumors most frequently on the tail, this method of con A inoculation is known to preferentially target T cell activation in the liver [31, 32]. All 5 con A treated mice developed liver bioluminescence, and two died within 1 week of acute hepatitis. The other 3 con A treated mice developed lymphoma initiated in the liver with spread to the gastrointestinal tract, spleen, and cervical nodes, as detected by BLI and histological analysis at necropsy (Fig. 2C). While the 5 saline injected TAX-LUC mice developed tail tumors, none developed a similar form of aggressive lymphoma, characterized by massive visceral infiltration. LTR-LUC animals did not develop tumors. This experiment suggested that con A-induced inflammation and T cell activation in TAX-LUC mice were sufficient to modify the presentation of lymphoma from peripheral and indolent to visceral and aggressive.
We also utilized CFA a mixture of paraffin oil, surfactant, and heat-killed mycobacteria that leads to TH1 lymphocyte activation [33]. In addition, we examined inducers of T cell activation through effects on TLRs on antigen-presenting cells (APCs). These inducers included poly I:C, a mimic of double stranded RNA that activates the interferon response, and LPS, found in the cell wall of gram negative bacteria, that rapidly activates pyrogenic cytokines and cells involved in innate immunity [34]. In the tumors that arise in TAX-LUC animals, the malignant cells are rarely T cells, but instead are CD16Hi LGLs that lack TCRs. Primary TAX-LUC tumors also contain a large population of CD16Lo cells which are predominantly neutrophils and CD16- cells which include tumor infiltrating T cells. We next sought to determine if bioluminescence resulting from acute inflammation correlates with the recruitment or proliferation of CD16Hi LGLs. The representative results of intraperitoneal injections into 3 mice each of saline, con A, CFA, poly I:C, and LPS are shown in Fig. 3. Mice were imaged 0.5 hour prior to injections and then at 2 and 6 hours after injection, then sacrificed and examined. BLI performed prior to injection exhibited very low background levels of activity primarily within the gastrointestinal tract TAX-LUC mice. Con A treatment resulted in increased numbers of CD16lo cells and BrdU+ cells in the spleen and liver compared to saline treated animals (Fig. 3A, B), whereas the number of CD16Hi cells increased in spleen but not liver. After con A injection BLI was increased in the gastrointestinal tract and liver as compared to saline injected animals (Fig. 3C). Intraperitoneal injection of CFA was similar to the effects of con A. The number of BrdU positive cells in the spleen and liver was increased after CFA treatment, and infiltrates of lymphoid cells in the liver were apparent. Two hours after CFA injection, bioluminescence localized primarily to the liver (Fig. 3C). Intraperitoneal injection of poly(I:C) and LPS also resulted in increased numbers of CD16lo cells and BrdU+ cells in spleen and liver compared to animals injected with saline. Unlike Con A and CFA, bioluminescence in TAX-LUC mice after treatment with poly(I:C) and LPS was more evident in the spleen and gastrointestinal tract than liver.
Taken together, these studies indicated that bioluminescence in TAX-LUC mice serves as a sensitive indicator of acute inflammation in vivo. However, the bioluminescence profile does not correlate with CD16Hi cells nor proliferating cells, suggesting the light emitting cells during inflammation are not identical to the population of cells that subsequently undergo malignant transformation. While malignant LGLs in TAX-LUC tumors are bioluminescent, these results demonstrated that during acute inflammation other luciferase-expressing cell types predominate, possibly activated T cells. Based on these findings, we sought to use a genetic approach to determine if activated T cells promote tumorigenesis in TAX-LUC mice.
Specific T-Cell Receptor Activation Accelerates Tax-Mediated Tumorigenesis
DO11.10 mice carry a transgenic MHC class II restricted rearranged T cell receptor which reacts with a specific ovalbumin (OA) peptide antigen [6, 7]. IP administration of OA results in deletion of immature CD4+ CD8+ TCRlo thymocytes and expansion of CD4+ TCRHi thymocytes. Within 3 days post injection all of the immature non-OVA reactive thymocytes are removed and OA reactive CD4+ T cells represent approximately 70% of T cells in these mice. In order to examine the specific effects of TCR activation, triple transgenic mice were utilized, resulting from breeding TAX-LUC mice with DO mice (Fig. 4). In one experiment, 5 TAX-LUC-DO mice were inoculated with OA in CFA, and 2 control TAX-LUC-DO mice were inoculated with CFA alone. Double transgenic LUC-DO and TAX-LUC mice were also inoculated with OA in CFA to serve as controls. The immune response to OA in CFA could be observed non-invasively in these mice using BLI (Fig. 4A-B) which served as an internal control to ensure each immunization produced a response. Bioluminescence was detectable 7 hours after injection and by day 3 predominantly localized to the spleen (Fig. 4A). Subsequent injections in primed animals produced a bioluminescent response of increased intensity and duration (Fig. 4B). Interestingly, bioluminescence was also detected in LUC-DO animals, although it was more intense in Tax transgenic animals (Fig. 4C). These results demonstrate that OA in CFA is sufficient to activate basal HTLV LTR transcriptional activity, which is further activated by induction of Tax expression in TAX-LUC-DO mice. Over the course of 1 year, 4-10 tail tumors arose in each of the TAX-LUC-DO mice inoculated with OA in CFA, and 2-3 tail tumors arose in each of the TAX-LUC mice (Fig. 4C, numbers at bottom of panels). No tumors arose in mice lacking the Tax transgene (LUC-DO), nor in the two TAX-LUC-DO controls that received no OA.
These findings were confirmed and extended in additional experiments (Fig. 5A). Significantly more tumors were noted in triple transgenic TAX-LUC-DO mice inoculated with OA in CFA compared to those inoculated with CFA alone (6.5 vs 3.1, p = 0.0014) (Fig. 5A, panel 1). Moreover, survival was significantly shorter in TAX-LUC-DO and TAX-DO mice treated with OA in CFA compared to those administered CFA alone (Fig. 5B). No tumors developed in the absence of the Tax transgene in LUC-DO mice, DO mice, or LUC mice (Fig. 5A, panels 2, 3, and 4, respectively). Doubly transgenic TAX-LUC mice lacking the specific TCR had fewer tumors in the presence than absence of OA (1.5 vs. 4.3 p = 0.0083). Since the average tumor onset in Tax mice occurs within 200-300 days and many animals do not develop tumors until the second year of life, some Tax positive animals did not develop tumors during the time course of this experiment [18].
While the OA-restricted TCR in TAX-LUC-DO animals is expressed on CD4+ lymphocytes, the presence of TCRova cells in tumors was variable. Typically, the malignant LGL population in tumors that spontaneously arise in TAX-LUC mice is TCR-, and tumor infiltrating lymphocytes are TCR+. This is consistent with what we observed in tumors arising on the tails in TAX-LUC-DO mice which included both TCR- and TCR+ cells (Fig. 5C). In contrast, tumors arising in the gastrointestinal (GI) tract, which were only found in TAX-LUC-DO animals treated with OA, were composed of TCR+ cells with a minor population of cells expressing exceptionally high levels of TCRova (Fig. 5C). Alternatively, tumors arising in the ears contained very few TCR+ cells and were primarily composed of malignant LGLs. Representative histology (Fig. 5D) for tumors arising in OA stimulated TAX-LUC-DO mice, includes examples of tumors invading spleen, lung, and liver as well as primary tumors arising in intestine and peripheral tissues. In each case, a proliferation of lymphoid cells is evident, however, the size, morphology, and expression profiles of CD16 and TCRova indicated that tumors arising in the gut were distinct from peripheral tumors that typically arise on TAX-LUC mice. Unlike peripheral tumors arising in the tail or ear, gut tumors include very few if any CD16 expressing cells but an abundance of TCRova + and Tax expressing cells (Fig. 5E). Taken together, these results indicate that T cell activation in TCR transgenic TAX-LUC mice resulted in increased peripheral tumor burden, decreased survival, and the presentation of a novel form of visceral lymphoma composed of CD16- TCRova lymphocytes similar to tumors that arose in con A treated TAX-LUC mice.