_:b6001389 _:b6001395 . _:b6001389 _:b6001396 . _:b413576330 "2"^^ . _:b6001389 _:b6001397 . _:b6001389 _:b6001398 . . . _:b6001374 "allows us to monitor the anti-tumor T cell response [5], [15], [22], but it does not impact on tumor immunogenicity, as evidenced by the fact that AB1-HA cured mice are also protected against re-challenge with the parental AB1 line [>>23<<]." . _:b6001389 _:b6001399 . _:b6001389 _:b6001400 . _:b413576341 "2"^^ . _:b6001389 _:b6001401 . _:b6001345 . _:b6001387 . _:b6001389 _:b6001402 . _:b6001389 _:b6001403 . _:b6001389 _:b6001404 . _:b6001346 . _:b6001389 _:b6001405 . _:b413576340 "2"^^ . _:b6001389 _:b6001406 . _:b6001389 _:b6001407 . . _:b6001440 . _:b6001347 . _:b413576343 "2"^^ . . _:b6001432 "It is not entirely clear how CY has such a strong effect on proliferating T cells, although several studies suggest that it may be linked to increased numbers of NO-producing myeloid-derived suppressor cells [42], [>>43<<]. Whereas depletion of proliferating regulatory T cell is beneficial [18], [19], [44], the impact of CY on the CD8 T cell response could be negative and seems inconsistent with the key role of CD8 T cells in tumor resolution. Our" . _:b6001356 . _:b413576342 "2"^^ . _:b6001388 . . _:b413576341 . _:b6001357 . _:b413576337 "2"^^ . _:b6001381 . _:b6001358 . _:b6001415 . _:b413576336 "2"^^ . _:b6001389 _:b6001390 . _:b413576306 . _:b6001389 _:b6001391 . _:b6001359 . _:b413576339 "2"^^ . _:b413576280 . _:b6001404 . _:b6001400 . _:b6001352 . _:b413576338 "2"^^ . . . . _:b6001353 . _:b413576349 "2"^^ . . _:b6001423 "There is direct evidence for the former hypothesis [>>29<<], but the fact that cells exposed to the CY-related drug melphalan activate phagocytosing DCs [40] indicates that the latter may also occur." . . _:b6001377 . _:b6001354 . _:b413576348 "2"^^ . _:b6001355 . . . _:b6001364 . _:b413576350 "2"^^ . . . _:b6001365 . _:b413576345 "2"^^ . _:b413576343 . _:b6001392 . _:b6001366 . _:b6001414 . _:b413576344 "2"^^ . _:b413576350 . _:b6001375 "In the 1980s, it was shown that CY depleted cycling suppressor T cells, now known as regulatory T cells, and thereby activated anti-tumor CD8 T cells [>>24<<]. However, we have recently shown that the anti-tumor efficacy of CY in the AB1-HA model cannot be explained by regulatory T cell depletion alone [18]. Here, we show that CY kills tumor cells by apoptosis and that it has a CD8 T cell- and" . _:b413576347 "2"^^ . _:b6001367 . _:b413576276 . _:b6001360 . _:b413576346 "2"^^ . . _:b6001393 . _:b6001361 . _:b413576320 . _:b6001362 . _:b6001440 "TRAIL sensitization of tumor cells, including human mesothelioma cells, by chemotherapeutic drugs has been well characterized [47], [>>48<<] and has been translated into therapeutic approaches involving soluble TRAIL or other TRAIL-receptor agonists [35], [49], [50]." . _:b6001363 . _:b413576336 . _:b6001372 . . . _:b6001373 "The tumor-expressed HA protein allows us to monitor the anti-tumor T cell response [5], [15], [>>22<<], but it does not impact on tumor immunogenicity, as evidenced by the fact that AB1-HA cured mice are also protected against re-challenge with the parental AB1 line [23]." . _:b6001373 . _:b6001374 . _:b6001359 "As a result, chemo- and immunotherapy are no longer considered to be a priori antagonistic [11] and the concept of combined chemo-immuno therapy is receiving more attention [>>11<<], [12], [13]." . _:b6001408 "Ki-67 is a nuclear marker that is upregulated in cycling cells [18], [31], [>>32<<]. Interestingly, CY treatment selectively depleted cycling cells, as shown by the almost complete absence of Ki-67hi CD8 T cells three days after a single CY injection ( Figure 4A ). We have recently reported similar findings for CD4 T" . _:b6001448 . . _:b6001375 . . _:b6001368 . _:b413576267 . _:b6001355 . _:b6001369 . _:b6001434 . _:b413576330 . _:b6001370 . _:b6001349 . _:b6001371 . _:b6001446 "Similar in vitro data were shown earlier with cycloheximide and sensitivity to TRAIL apoptosis [>>52<<]. TRAIL sensitivity can also be increased through the proteasome inhibitor bortezomib [53]. The liver toxicity that we observed in our current study seems consistent with recent work showing that the drug 5-azacytidine sensitizes" . . _:b6001380 . _:b6001381 . . _:b6001347 "Tumor antigens are cross-presented to the immune system [1], [>>2<<], [3]. However, the ensuing anti-tumor CD8 T cell response is usually not effective and fails to control tumor growth." . _:b6001382 . _:b6001383 . _:b6001376 . _:b6001377 . _:b413576332 . . . _:b6001378 . . _:b413576258 . _:b6001379 . _:b413576319 . _:b6001408 . _:b6001388 . _:b413576335 . _:b6001428 . . _:b6001389 . _:b6001365 . _:b413576328 . _:b6001389 "results" . _:b6001390 . _:b6001391 . _:b6001391 . _:b6001353 . _:b6001384 . . . _:b6001385 . _:b413576284 . _:b6001386 . _:b6001432 . _:b6001387 . . _:b6001400 "Therefore, the combined data suggest that CY-injection in tumor-bearing mice results in systemic immune activation, consistent with the concept of immunogenic cell death [>>7<<], [18] and consistent with previously reported data [17], [29]." . _:b413576311 . _:b6001396 . . _:b6001397 . _:b6001395 "Ly6AE upregulation on T cells is a very early non-antigen-specific (bystander) response of T cells to IFN-exposure [27], [>>28<<] and can therefore serve as a surrogate for type-I IFN (IFN-\u03B1/\u03B2) production." . _:b6001398 . _:b413576348 . _:b6001399 . _:b6001382 . _:b6001362 . _:b6001392 . _:b6001393 . "10.1371%2Fjournal.pone.0006982" . _:b6001450 "The role of IFN-\u03B3 could be related to TRAIL, since both type-I and type-II IFNs have the capacity to induce TRAIL expression [>>56<<], [57] and anti-tumor function [58]." . _:b6001394 . _:b413576291 . _:b6001381 "Generation and maintenance of the BALB/c-derived mouse mesothelioma cell line AB1 and transfection with the HA gene (AB1-HA) has been described [>>15<<], [16]. AB1-HA cells (1\u00D7106 in PBS) were injected s.c. into the right flank of recipient mice and tumor growth monitored using microcalipers." . _:b6001441 . _:b6001395 . _:b6001404 . _:b6001405 . _:b413576340 . _:b6001406 . _:b413576290 . _:b6001407 . . _:b413576324 . _:b6001400 . _:b413576257 . . _:b6001401 . _:b413576271 . . _:b6001402 . _:b6001403 . _:b6001420 "CY kills cells by inducing apoptosis and this seems to result in a systemic type-I IFN response, confirming earlier studies [17], [>>29<<], [39] and consistent with the notion of \u2018immunogenic cell death\u2019. Similar data have been reported by Schiavoni and coworkers who used expression of Ly6C on T cells (similar to Ly6AE) and PCR to detect type-I IFN production [17]." . _:b6001418 "This could suggest that T cells and NK cells are required to prevent a small number of chemotherapy-surviving tumor cells from re-establishing a new tumor [>>38<<]. Second, the CY analogue maphosphamide induces apoptosis in tumor cells, consistent with its direct cytotoxic effects on AB1-HA tumor cells [18]. In vivo, the result of chemotherapy is a systemic and long-lived type-I IFN response. Third," . . . . . _:b6001348 "Tumor antigens are cross-presented to the immune system [1], [2], [>>3<<]. However, the ensuing anti-tumor CD8 T cell response is usually not effective and fails to control tumor growth." . . . _:b6001401 "Therefore, the combined data suggest that CY-injection in tumor-bearing mice results in systemic immune activation, consistent with the concept of immunogenic cell death [7], [18] and consistent with previously reported data [>>17<<], [29]." . . _:b413576337 . . . . _:b6001383 "The IFN-\u03B1/\u03B2 neutralizing sheep Ig and matching normal sheep Ig [>>17<<], [61] were used as described [5]. Mice were i.v. injected with 0.2 ml of immunoglobulins on day \u20131, +2, and +4 with respect to CY administration." . _:b413576304 . . _:b413576347 . _:b6001423 . _:b6001421 "Similar data have been reported by Schiavoni and coworkers who used expression of Ly6C on T cells (similar to Ly6AE) and PCR to detect type-I IFN production [>>17<<]. Thus, we now confirm and extend these observations. There may be two possible sources for IFN production: the tumor cells themselves [29] and the monocytes that phagocytose apoptotic tumor cells. There is direct evidence for the former" . _:b6001422 . . _:b6001441 "of tumor cells, including human mesothelioma cells, by chemotherapeutic drugs has been well characterized [47], [48] and has been translated into therapeutic approaches involving soluble TRAIL or other TRAIL-receptor agonists [>>35<<], [49], [50]. It has been proposed that the synergy of TRAIL-ligation and chemotherapy can be explained by integration of the intrinsic (through chemotherapy) and alternative (death receptor) pathways of apoptosis [47]." . _:b6001452 "The role of IFN-\u03B3 could be related to TRAIL, since both type-I and type-II IFNs have the capacity to induce TRAIL expression [56], [57] and anti-tumor function [>>58<<]. The importance of TRAIL suggests that TRAIL production by T cells or NK cells could be an important readout to predict the efficacy of cancer vaccines or immunotherapies, possibly equally important as perforin-mediated cell apoptosis and" . _:b6001360 "As a result, chemo- and immunotherapy are no longer considered to be a priori antagonistic [11] and the concept of combined chemo-immuno therapy is receiving more attention [11], [>>12<<], [13]. However, the notion that chemotherapy and anti-tumor T cell responses can be synergistic must be reconciled with the fact that many chemotherapeutic drugs deplete lymphocytes [14]. In fact, lymphodepletion after chemotherapy was" . . _:b6001420 . . _:b413576334 . _:b413576286 . _:b6001430 . . _:b6001371 "The tumor-expressed HA protein allows us to monitor the anti-tumor T cell response [>>5<<], [15], [22], but it does not impact on tumor immunogenicity, as evidenced by the fact that AB1-HA cured mice are also protected against re-challenge with the parental AB1 line [23]." . _:b413576299 . . _:b6001390 . _:b6001346 . . . . _:b6001349 "Altering that context is an important goal for anti-cancer immunotherapy [>>4<<], [5], [6]." . _:b6001353 "Cytotoxic chemotherapy can play a role in this process since apoptotic tumor cell death can be an immunostimulatory event (\u2018immunogenic cell death\u2019) [7], [>>8<<], [9], potentially adding an immunostimulatory signal to cross-presented antigens." . . . _:b413576308 . . _:b413576272 . _:b6001380 "BALB/c perforin-deficient mice were originally generated at the Peter MacCallum Cancer Centre [>>60<<] and bred at the Animal Care Unit at UWA (Perth, Australia)." . _:b6001407 . _:b6001352 "Cytotoxic chemotherapy can play a role in this process since apoptotic tumor cell death can be an immunostimulatory event (\u2018immunogenic cell death\u2019) [>>7<<], [8], [9], potentially adding an immunostimulatory signal to cross-presented antigens." . _:b6001363 "To study this, we have used a mouse model of malignant mesothelioma (AB1-HA), which is sensitive to both chemotherapy and immunotherapy [4], [>>15<<], [16], in combination with the chemotherapeutic drug cyclophosphamide (CY), since CY treatment is associated with innate and adaptive immune activation [17], [18], [19], [20], [21]." . _:b6001416 "Unfortunately, the CY/anti-DR5 combination treatment was also associated with liver toxicity, preventing us from studying long-term control of tumor growth [>>37<<]. TRAIL blockade alone did not significantly affect the efficacy of CY in nude mice, suggesting that the transient anti-tumor effect did not depend on this mechanism (Figure 7B)." . _:b413576303 . _:b6001354 "Cytotoxic chemotherapy can play a role in this process since apoptotic tumor cell death can be an immunostimulatory event (\u2018immunogenic cell death\u2019) [7], [8], [>>9<<], potentially adding an immunostimulatory signal to cross-presented antigens." . . _:b6001409 . _:b6001434 "Whereas depletion of proliferating regulatory T cell is beneficial [18], [19], [>>44<<], the impact of CY on the CD8 T cell response could be negative and seems inconsistent with the key role of CD8 T cells in tumor resolution." . _:b6001367 "is sensitive to both chemotherapy and immunotherapy [4], [15], [16], in combination with the chemotherapeutic drug cyclophosphamide (CY), since CY treatment is associated with innate and adaptive immune activation [17], [18], [19], [>>20<<], [21]. The AB1-HA tumor cell line was generated by transfection of the asbestos-induced AB1 tumor cell line [16] with the influenza virus HA gene [15]." . . _:b6001364 . _:b6001417 . _:b413576336 . _:b413576337 . _:b413576338 . _:b413576339 . _:b413576340 . _:b6001409 "This specific loss of Ki-67hi T cells was not seen with a different cytotoxic drug, gemcitabine (GEM) ( Figure 4A ), despite the fact that GEM causes lymphodepletion in mice [>>14<<]. Since both drugs are cytotoxic, the difference between CY and GEM may be one of kinetics, in which the CY-mediated T cell proliferation arrest is more long-lived. Second, to assess to the impact of CY on the tumor-specific CD8 T cell" . _:b413576341 . _:b413576342 . _:b413576343 . _:b413576344 . _:b413576345 . _:b413576346 . _:b413576347 . _:b413576348 . _:b413576349 . _:b413576350 . _:b413576320 . _:b413576321 . _:b6001431 . _:b413576322 . _:b413576323 . _:b413576324 . _:b413576325 . _:b413576326 . _:b6001448 "study seems consistent with recent work showing that the drug 5-azacytidine sensitizes hepatocytes for TRAIL apoptosis [54] and with a recent study showing that DR5-mediated apoptosis of cholangiocytes contributes to liver disease [>>37<<]." . _:b413576327 . _:b413576328 . _:b413576329 . _:b413576330 . _:b413576331 . _:b413576332 . _:b413576292 . _:b413576333 . _:b413576334 . _:b413576335 . . _:b413576316 . . _:b6001369 "The AB1-HA tumor cell line was generated by transfection of the asbestos-induced AB1 tumor cell line [>>16<<] with the influenza virus HA gene [15]." . . _:b6001359 . _:b6001369 . _:b6001358 . . _:b413576274 . _:b6001366 . _:b413576349 . _:b6001379 "TRAIL-deficient mice were originally generated at Immunex Corporation [35], [>>59<<] and were backcrossed to BALB/c at the Peter MacCallum Cancer Centre (Melbourne, Australia) (n\u200A=\u200A12)." . _:b6001445 "Recently, Johnstone and colleagues showed that the histone diacetylase inhibitor vorinostat augmented the anti-tumor effects of anti-DR5 antibodies in vivo and in vitro through downregulation of the apoptosis inhibitor cellular-FLIP [>>51<<]. Similar in vitro data were shown earlier with cycloheximide and sensitivity to TRAIL apoptosis [52]." . . _:b413576269 . . . _:b413576296 . _:b413576259 . . . _:b6001447 . . _:b6001358 "As a result, chemo- and immunotherapy are no longer considered to be a priori antagonistic [>>11<<] and the concept of combined chemo-immuno therapy is receiving more attention [11], [12], [13]." . . . . _:b6001406 "Thus, our data suggest that the CY-induced anti-tumor T cell response is type-I IFN independent and may therefore not be a de novo response because such a response would be type-I IFN-dependent [>>8<<]. Instead, we reasoned that CY could make existing responses more effective, either by expanding tumor-specific CD8 T cells or by increasing the sensitivity of tumor cells to T cell-mediated apoptosis. To test this, we further studied" . . _:b6001445 . _:b6001410 . . _:b6001437 . _:b6001427 "However, CY-induced anti-tumor responses in our model are clearly less dependent on type-I IFNs, in contrast to the anti-tumor effects of poly-I:C and imiquimod in the same model [>>5<<], [23]. This indicates that CY mobilizes anti-tumor CD8 T cells in a way that does not involve type-I IFN dependent activation and/or expansion [30], [41]." . _:b413576287 . _:b6001430 "This indicates that CY mobilizes anti-tumor CD8 T cells in a way that does not involve type-I IFN dependent activation and/or expansion [30], [>>41<<]." . _:b413576282 . _:b6001412 . _:b6001426 "Irrespective of the mechanism, type-I IFNs are essential to generate de novo CD8 T cell responses to apoptotic cells [8] and to cross-presented antigens [>>30<<] and we hypothesized that CY-associated IFN responses might augment potential anti-tumor CD8 T cell responses." . . . _:b6001451 "The role of IFN-\u03B3 could be related to TRAIL, since both type-I and type-II IFNs have the capacity to induce TRAIL expression [56], [>>57<<] and anti-tumor function [58]." . . _:b413576344 . . . _:b6001367 . . _:b413576304 . _:b413576305 . _:b413576306 . _:b413576307 . _:b413576308 . _:b413576309 . . _:b413576310 . _:b413576311 . _:b413576312 . _:b413576313 . _:b6001433 . . _:b413576314 . _:b413576315 . _:b413576316 . _:b6001417 _:b6001418 . _:b413576317 . . _:b6001417 _:b6001419 . _:b413576318 . _:b6001417 _:b6001420 . _:b413576319 . _:b6001411 . . _:b6001417 _:b6001421 . _:b413576288 . _:b6001372 "The tumor-expressed HA protein allows us to monitor the anti-tumor T cell response [5], [>>15<<], [22], but it does not impact on tumor immunogenicity, as evidenced by the fact that AB1-HA cured mice are also protected against re-challenge with the parental AB1 line [23]." . _:b6001417 _:b6001422 . _:b413576289 . _:b6001435 . _:b6001412 . _:b6001370 "The AB1-HA tumor cell line was generated by transfection of the asbestos-induced AB1 tumor cell line [16] with the influenza virus HA gene [>>15<<]. The tumor-expressed HA protein allows us to monitor the anti-tumor T cell response [5], [15], [22], but it does not impact on tumor immunogenicity, as evidenced by the fact that AB1-HA cured mice are also protected against re-challenge" . _:b6001417 _:b6001423 . _:b413576290 . _:b6001417 _:b6001424 . _:b413576291 . _:b6001417 _:b6001425 . _:b413576266 . _:b6001417 _:b6001426 . _:b413576292 . _:b413576346 . _:b6001446 . _:b6001413 . _:b6001417 _:b6001427 . _:b413576293 . _:b413576294 . _:b6001417 _:b6001428 . _:b413576295 . _:b6001438 "A tumor cell sensitization model [>>34<<] is supported by the key role of TRAIL. Since no curative responses were ever observed in athymic nude mice, it is evident that CY does not kill all tumor cells." . _:b6001417 _:b6001429 . _:b413576296 . _:b6001417 _:b6001430 . _:b413576297 . _:b6001449 "Our data suggest that CD8 T cells are a major source of TRAIL, since these cells are essential for tumor resolution. NK cells may also be required [>>55<<]. A more precise determination of the relative roles of NK cells and CD8 T cells and of the source(s) of TRAIL will be the subject of further studies, which should include measuring the in vitro sensitivity of maphosphamide-treated tumor" . _:b6001414 . _:b6001417 _:b6001431 . _:b413576298 . _:b6001417 _:b6001432 . _:b413576299 . _:b6001417 _:b6001433 . _:b413576300 . _:b6001417 _:b6001434 . _:b413576301 . _:b6001415 . _:b6001417 _:b6001435 . _:b413576302 . _:b6001417 _:b6001436 . _:b413576303 . _:b6001417 _:b6001437 . _:b413576272 . _:b6001417 _:b6001438 . _:b413576273 . _:b6001408 . _:b6001417 _:b6001439 . _:b413576274 . _:b6001417 _:b6001440 . . _:b6001417 _:b6001441 . _:b413576275 . _:b413576276 . _:b6001417 _:b6001442 . _:b413576277 . _:b6001409 . _:b6001417 _:b6001443 . _:b413576278 . _:b6001417 _:b6001444 . _:b413576279 . _:b6001417 _:b6001445 . _:b413576280 . _:b6001417 _:b6001446 . _:b413576281 . _:b6001410 . _:b6001388 "at days -1, 3 and 7 relative to CY treatment (50 \u00B5g/injection). Neutralizing TRAIL antibodies (clone N2B2) [>>36<<] were injected i.p." . _:b6001417 _:b6001447 . _:b6001357 "An immune priming effect has now been shown for several chemotherapeutic drugs, including gemcitabine [4], [10] and doxorubicin [>>7<<]. As a result, chemo- and immunotherapy are no longer considered to be a priori antagonistic [11] and the concept of combined chemo-immuno therapy is receiving more attention [11], [12], [13]. However, the notion that chemotherapy and" . _:b6001417 _:b6001448 . _:b413576282 . _:b413576261 . _:b6001417 _:b6001449 . _:b413576283 . _:b413576284 . _:b6001361 "However, the notion that chemotherapy and anti-tumor T cell responses can be synergistic must be reconciled with the fact that many chemotherapeutic drugs deplete lymphocytes [>>14<<]. In fact, lymphodepletion after chemotherapy was the main reason why chemo- and immunotherapy were seen as antagonistic. The growing insight that chemotherapy can be immunostimulatory presents a paradox: how are effective anti-tumor T" . _:b6001417 _:b6001450 . _:b413576285 . _:b6001411 . _:b6001417 _:b6001451 . _:b413576286 . _:b6001417 _:b6001452 . _:b413576260 . _:b6001390 "The objective of this study was to investigate the potential discrepancy between lymphodepleting anti-tumor chemotherapy and the anti-tumor immune responses that are linked to successful chemotherapy [>>11<<], [25]. To test this, we used a murine mesothelioma model (AB1-HA) that is sensitive to both chemotherapy and immunotherapy [1], [4], [18], in combination with the DNA cross-linker cyclophosphamide (CY)." . _:b413576287 . _:b413576257 . _:b6001420 . _:b413576258 . _:b413576263 . _:b413576259 . _:b413576260 . _:b413576261 . _:b6001421 . _:b413576262 . _:b413576262 . _:b413576263 . _:b413576264 . _:b413576265 . _:b6001422 . _:b413576266 . _:b413576257 . _:b413576267 . _:b6001346 "Tumor antigens are cross-presented to the immune system [>>1<<], [2], [3]. However, the ensuing anti-tumor CD8 T cell response is usually not effective and fails to control tumor growth." . _:b413576268 . _:b6001348 . _:b413576269 . _:b6001423 . _:b413576270 . . _:b413576271 . . _:b6001416 . _:b413576259 . _:b6001417 . . _:b413576258 . _:b6001418 . _:b413576269 . . . . _:b6001419 . _:b6001419 . _:b413576268 . _:b6001428 . _:b6001438 . _:b413576271 . _:b6001429 . _:b6001421 . _:b413576270 . _:b413576313 . _:b413576275 . _:b6001430 . _:b413576265 . _:b6001431 . _:b413576309 . _:b6001356 . _:b413576264 . _:b413576317 . _:b6001425 . _:b6001424 . _:b413576267 . . _:b6001425 . _:b6001439 . _:b413576266 . . _:b6001426 . _:b413576277 . _:b6001427 . _:b413576276 . _:b6001403 "Since CY treatment is associated with an IFN-\u03B1/\u03B2 response and because primary CD8 T cell responses to apoptotic cells and cross-presented antigens are type-I IFN-dependent [>>8<<], [30], we hypothesized that the type-I IFN response could be responsible for priming the anti-tumor CD8 T cell response." . _:b6001436 . _:b413576279 . _:b6001376 . _:b6001437 . _:b413576278 . _:b6001438 . _:b6001428 "However, CY-induced anti-tumor responses in our model are clearly less dependent on type-I IFNs, in contrast to the anti-tumor effects of poly-I:C and imiquimod in the same model [5], [>>23<<]. This indicates that CY mobilizes anti-tumor CD8 T cells in a way that does not involve type-I IFN dependent activation and/or expansion [30], [41]." . _:b413576273 . _:b6001447 "TRAIL sensitivity can also be increased through the proteasome inhibitor bortezomib [>>53<<]. The liver toxicity that we observed in our current study seems consistent with recent work showing that the drug 5-azacytidine sensitizes hepatocytes for TRAIL apoptosis [54] and with a recent study showing that DR5-mediated apoptosis of" . . _:b6001368 "sensitive to both chemotherapy and immunotherapy [4], [15], [16], in combination with the chemotherapeutic drug cyclophosphamide (CY), since CY treatment is associated with innate and adaptive immune activation [17], [18], [19], [20], [>>21<<]. The AB1-HA tumor cell line was generated by transfection of the asbestos-induced AB1 tumor cell line [16] with the influenza virus HA gene [15]." . _:b6001439 . _:b413576272 . _:b6001424 . _:b6001432 . _:b6001389 . _:b413576275 . _:b6001402 "Therefore, the combined data suggest that CY-injection in tumor-bearing mice results in systemic immune activation, consistent with the concept of immunogenic cell death [7], [18] and consistent with previously reported data [17], [>>29<<]." . . _:b6001433 . _:b413576264 . _:b413576274 . _:b6001373 . _:b6001443 . _:b6001434 . _:b413576285 . _:b6001452 . _:b6001435 . _:b413576284 . _:b413576333 . . _:b6001444 . _:b413576287 . _:b6001361 . _:b6001345 "introduction" . _:b6001354 . _:b6001445 . _:b6001345 . _:b413576286 . _:b6001446 . _:b413576281 . _:b413576261 . _:b413576327 . _:b6001447 . . _:b413576280 . _:b6001440 . _:b413576283 . _:b413576302 . _:b6001441 . _:b6001393 "Apoptotic tumor cell death can be immune-stimulatory [>>7<<]. Thus, to evaluate the immune effects of CY-induced tumor cell apoptosis, we analyzed changes in gene expression within CY-treated tumors using real time PCR SuperArrays, allowing simultaneous analysis of 84 cytokine genes using real-time" . _:b6001405 . _:b413576282 . _:b6001442 . _:b413576326 . . _:b6001397 "Indeed, we have recently confirmed that Ly6AE-upregulation on CD8 T cells occurs in a strictly IFN-\u03B1/\u03B2-dependent manner after AB1-HA tumor treatment with the TLR3 and TLR7 agonists poly-I:C and imiquimod [5], [>>23<<]. Therefore, to assess IFN-\u03B1/\u03B2 production, we measured Ly6AE expression on peripheral blood CD8 T cells harvested three days after administration of CY (150 mg/kg) in AB1-HA tumor-bearing mice. We found that CY treatment resulted in an" . _:b413576293 . _:b6001351 . _:b6001443 . _:b413576292 . . _:b6001452 . _:b413576295 . _:b6001385 "The IFN-\u03B1/\u03B2 neutralizing sheep Ig and matching normal sheep Ig [17], [61] were used as described [>>5<<]. Mice were i.v. injected with 0.2 ml of immunoglobulins on day \u20131, +2, and +4 with respect to CY administration." . _:b6001413 . _:b413576300 . _:b6001392 "To test this, we used a murine mesothelioma model (AB1-HA) that is sensitive to both chemotherapy and immunotherapy [1], [>>4<<], [18], in combination with the DNA cross-linker cyclophosphamide (CY)." . . _:b413576294 . _:b6001391 "To test this, we used a murine mesothelioma model (AB1-HA) that is sensitive to both chemotherapy and immunotherapy [>>1<<], [4], [18], in combination with the DNA cross-linker cyclophosphamide (CY)." . _:b6001444 . _:b413576289 . _:b6001436 . _:b413576301 . _:b6001355 "An immune priming effect has now been shown for several chemotherapeutic drugs, including gemcitabine [>>4<<], [10] and doxorubicin [7]." . _:b6001399 "serum, completely inhibited Ly6AE upregulation on peripheral blood CD8 T cells ( Figure 2A ), confirming our previous finding that upregulation of Ly6AE expression is a bona fide marker for systemic type-I IFN release [5], [>>23<<]. To evaluate the longevity of the IFN-\u03B1/\u03B2 response, we analyzed Ly6AE expression on day 10 after CY injection in the tumor draining and non-draining lymph nodes." . _:b413576288 . _:b6001422 "There may be two possible sources for IFN production: the tumor cells themselves [>>29<<] and the monocytes that phagocytose apoptotic tumor cells." . _:b6001448 . . _:b6001396 . _:b413576291 . _:b6001394 "Ly6AE upregulation on T cells is a very early non-antigen-specific (bystander) response of T cells to IFN-exposure [>>27<<], [28] and can therefore serve as a surrogate for type-I IFN (IFN-\u03B1/\u03B2) production." . _:b413576312 . _:b6001449 . _:b6001402 . . _:b413576290 . _:b6001450 . _:b413576260 . _:b6001398 . _:b413576301 . _:b413576281 . _:b413576263 . _:b6001451 . _:b413576300 . _:b6001431 "It is not entirely clear how CY has such a strong effect on proliferating T cells, although several studies suggest that it may be linked to increased numbers of NO-producing myeloid-derived suppressor cells [>>42<<], [43]. Whereas depletion of proliferating regulatory T cell is beneficial [18], [19], [44], the impact of CY on the CD8 T cell response could be negative and seems inconsistent with the key role of CD8 T cells in tumor resolution. Our" . _:b413576331 . _:b413576329 . _:b413576303 . . _:b413576278 . _:b413576302 . _:b6001451 . _:b6001387 "CD8 depletion (>95%) was verified by FACS analysis of PBL. Agonistic anti-DR5 antibody (clone MD5-1) [>>36<<] was injected i.p." . _:b6001412 "Phenotypic characterization of pentamer+ cells using the cytotoxicity marker CD43 [>>33<<] revealed that in untreated tumor-bearing mice, both CD43hi and CD43lo pentamer+ tumor specific CD8 T cells were present ( Figure 4D ), whereas the small populations of pentamer+ CD8 T cells in CY-treated mice appeared to be CD43lo." . _:b413576297 . _:b413576296 . . _:b413576299 . . _:b413576298 . _:b413576309 . _:b413576318 . . _:b413576308 . _:b6001399 . . _:b6001398 "an anti-IFN-\u03B1/\u03B2 serum, completely inhibited Ly6AE upregulation on peripheral blood CD8 T cells ( Figure 2A ), confirming our previous finding that upregulation of Ly6AE expression is a bona fide marker for systemic type-I IFN release [>>5<<], [23]. To evaluate the longevity of the IFN-\u03B1/\u03B2 response, we analyzed Ly6AE expression on day 10 after CY injection in the tumor draining and non-draining lymph nodes." . _:b413576262 . . . _:b413576342 . . _:b413576311 . _:b6001385 . _:b413576310 . _:b6001397 . _:b413576283 . _:b6001377 "TCR transgenic CL4 mice, expressing a TCR specific for the H-2d-restricted peptide IYSTVASSL (residues 518\u2013526) from the influenza virus A/PR8/8/34 HA protein, were generated and screened as described [>>15<<]. TRAIL-deficient mice were originally generated at Immunex Corporation [35], [59] and were backcrossed to BALB/c at the Peter MacCallum Cancer Centre (Melbourne, Australia) (n\u200A=\u200A12). Perforin-IFN-\u03B3 double-deficient mice were bred at the" . _:b6001386 . _:b413576305 . _:b6001427 . _:b6001365 "(AB1-HA), which is sensitive to both chemotherapy and immunotherapy [4], [15], [16], in combination with the chemotherapeutic drug cyclophosphamide (CY), since CY treatment is associated with innate and adaptive immune activation [>>17<<], [18], [19], [20], [21]. The AB1-HA tumor cell line was generated by transfection of the asbestos-induced AB1 tumor cell line [16] with the influenza virus HA gene [15]." . _:b6001429 . _:b413576304 . _:b6001351 "Altering that context is an important goal for anti-cancer immunotherapy [4], [5], [>>6<<]. Cytotoxic chemotherapy can play a role in this process since apoptotic tumor cell death can be an immunostimulatory event (\u2018immunogenic cell death\u2019) [7], [8], [9], potentially adding an immunostimulatory signal to cross-presented" . _:b413576307 . _:b413576265 . _:b6001449 . _:b413576306 . _:b6001366 "which is sensitive to both chemotherapy and immunotherapy [4], [15], [16], in combination with the chemotherapeutic drug cyclophosphamide (CY), since CY treatment is associated with innate and adaptive immune activation [17], [18], [>>19<<], [20], [21]. The AB1-HA tumor cell line was generated by transfection of the asbestos-induced AB1 tumor cell line [16] with the influenza virus HA gene [15]." . _:b413576317 . _:b413576307 . _:b413576316 . _:b413576319 . _:b413576318 . _:b6001407 "Ki-67 is a nuclear marker that is upregulated in cycling cells [18], [>>31<<], [32]. Interestingly, CY treatment selectively depleted cycling cells, as shown by the almost complete absence of Ki-67hi CD8 T cells three days after a single CY injection ( Figure 4A ). We have recently reported similar findings for CD4" . _:b6001426 . _:b413576313 . _:b413576312 . _:b413576325 . _:b413576315 . _:b413576305 . _:b6001435 "The benefit of regulatory T cell depletion [18], [>>19<<], [45], [46] may be that existing anti-tumor CD8 T cells are liberated from suppression, allowing them to kill the sensitized tumor cells." . _:b413576314 . . _:b413576325 . _:b6001350 "Altering that context is an important goal for anti-cancer immunotherapy [4], [>>5<<], [6]. Cytotoxic chemotherapy can play a role in this process since apoptotic tumor cell death can be an immunostimulatory event (\u2018immunogenic cell death\u2019) [7], [8], [9], potentially adding an immunostimulatory signal to cross-presented" . _:b413576324 . _:b6001389 _:b6001408 . _:b6001360 . _:b6001375 . _:b6001389 _:b6001409 . _:b6001389 _:b6001410 . _:b6001389 _:b6001411 . _:b6001425 "Irrespective of the mechanism, type-I IFNs are essential to generate de novo CD8 T cell responses to apoptotic cells [>>8<<] and to cross-presented antigens [30] and we hypothesized that CY-associated IFN responses might augment potential anti-tumor CD8 T cell responses." . _:b413576327 . _:b6001389 _:b6001412 . _:b6001389 _:b6001413 . . _:b6001389 _:b6001414 . _:b6001389 _:b6001415 . _:b413576326 . _:b6001389 _:b6001416 . . . _:b413576321 . . _:b6001371 . . _:b413576320 . _:b6001384 . . _:b413576323 . _:b6001437 "The benefit of regulatory T cell depletion [18], [19], [45], [>>46<<] may be that existing anti-tumor CD8 T cells are liberated from suppression, allowing them to kill the sensitized tumor cells." . _:b413576322 . . _:b413576268 . _:b413576261 "4"^^ . . _:b413576333 . _:b413576332 . _:b413576260 "5"^^ . _:b413576277 . _:b413576263 "4"^^ . _:b413576335 . _:b413576298 . _:b6001386 "169 monoclonal antibody (Dr Kathy Davern, Monoclonal Antibody Facility, Western Australian Institute for Medical Research) as previously described [>>5<<]. CD8 depletion (>95%) was verified by FACS analysis of PBL. Agonistic anti-DR5 antibody (clone MD5-1) [36] was injected i.p. at days -1, 3 and 7 relative to CY treatment (50 \u00B5g/injection). Neutralizing TRAIL antibodies (clone N2B2) [36]" . _:b6001404 "Since CY treatment is associated with an IFN-\u03B1/\u03B2 response and because primary CD8 T cell responses to apoptotic cells and cross-presented antigens are type-I IFN-dependent [8], [>>30<<], we hypothesized that the type-I IFN response could be responsible for priming the anti-tumor CD8 T cell response." . _:b413576262 "4"^^ . _:b413576334 . _:b413576329 . _:b6001442 "of tumor cells, including human mesothelioma cells, by chemotherapeutic drugs has been well characterized [47], [48] and has been translated into therapeutic approaches involving soluble TRAIL or other TRAIL-receptor agonists [35], [>>49<<], [50]. It has been proposed that the synergy of TRAIL-ligation and chemotherapy can be explained by integration of the intrinsic (through chemotherapy) and alternative (death receptor) pathways of apoptosis [47]." . _:b413576328 . _:b6001424 "There is direct evidence for the former hypothesis [29], but the fact that cells exposed to the CY-related drug melphalan activate phagocytosing DCs [>>40<<] indicates that the latter may also occur." . _:b413576331 . _:b413576259 "5"^^ . . _:b413576257 "7"^^ . _:b413576321 . _:b413576330 . _:b413576258 "5"^^ . _:b413576270 . _:b413576269 "3"^^ . _:b413576341 . _:b413576268 "3"^^ . _:b6001436 "The benefit of regulatory T cell depletion [18], [19], [>>45<<], [46] may be that existing anti-tumor CD8 T cells are liberated from suppression, allowing them to kill the sensitized tumor cells." . _:b413576340 . _:b413576271 "3"^^ . _:b413576343 . _:b413576270 "3"^^ . _:b413576342 . . _:b6001376 "materials and methods" . _:b413576265 "4"^^ . _:b413576337 . _:b6001362 "To study this, we have used a mouse model of malignant mesothelioma (AB1-HA), which is sensitive to both chemotherapy and immunotherapy [>>4<<], [15], [16], in combination with the chemotherapeutic drug cyclophosphamide (CY), since CY treatment is associated with innate and adaptive immune activation [17], [18], [19], [20], [21]." . _:b413576264 "4"^^ . . _:b413576336 . _:b413576267 "3"^^ . _:b6001411 "Confirming previous work employing this same assay [>>1<<], we observed robust proliferation of tumor-specific CD8 T cells in the tumor-draining lymph nodes of untreated control tumor-bearing mice, as can be inferred from the serial dilution of CFSE dye intensity ( Figure 4B )." . . _:b413576339 . _:b6001443 "tumor cells, including human mesothelioma cells, by chemotherapeutic drugs has been well characterized [47], [48] and has been translated into therapeutic approaches involving soluble TRAIL or other TRAIL-receptor agonists [35], [49], [>>50<<]. It has been proposed that the synergy of TRAIL-ligation and chemotherapy can be explained by integration of the intrinsic (through chemotherapy) and alternative (death receptor) pathways of apoptosis [47]." . . _:b413576266 "3"^^ . . . _:b413576338 . _:b413576277 "3"^^ . _:b6001356 "An immune priming effect has now been shown for several chemotherapeutic drugs, including gemcitabine [4], [>>10<<] and doxorubicin [7]." . _:b6001350 . _:b413576349 . _:b6001442 . _:b413576276 "3"^^ . _:b413576348 . _:b413576279 "2"^^ . . _:b6001374 . _:b413576278 "2"^^ . . "PMC0" . _:b413576350 . _:b413576273 "3"^^ . . _:b413576345 . _:b413576272 "3"^^ . . _:b413576323 . _:b413576344 . . _:b6001410 "Clone 4 transgenic CD8 T cells recognize a Kd-restricted epitope from the influenza virus HA protein which is expressed as a tumor neo-antigen in AB1-HA cells [>>15<<]. Ten million CFSE-labeled tumor-specific transgenic CD8 T cells were adoptively transferred into mice at day 7 after tumor cell inoculation. Mice were treated with CY (150 mg/kg) at day 9 and lymphocytes were isolated from the" . _:b413576275 "3"^^ . _:b413576347 . _:b413576339 . _:b413576274 "3"^^ . _:b413576346 . _:b6001405 "As a positive control for the efficiency of in vivo IFN-\u03B1/\u03B2-neutralization, we have shown that poly-I:C-mediated anti-tumor responses were abrogated using this anti-IFN-\u03B1/\u03B2 treatment [>>5<<]. Thus, our data suggest that the CY-induced anti-tumor T cell response is type-I IFN independent and may therefore not be a de novo response because such a response would be type-I IFN-dependent [8]. Instead, we reasoned that CY could" . _:b413576285 "2"^^ . _:b6001450 . _:b413576284 "2"^^ . _:b413576315 . _:b413576287 "2"^^ . . _:b413576286 "2"^^ . _:b413576273 . . _:b413576281 "2"^^ . _:b413576280 "2"^^ . _:b413576297 . _:b413576283 "2"^^ . _:b413576345 . . _:b413576282 "2"^^ . . _:b413576293 "2"^^ . . _:b6001368 . . . _:b413576292 "2"^^ . _:b413576295 "2"^^ . _:b413576294 "2"^^ . _:b6001415 "We tested this by combining CY-treatment in nude mice with agonistic anti-DR5 antibodies (clone MD5-1) [35], [>>36<<], reasoning that CY-sensitized tumor cells would then be killed by antibody-mediated DR5 ligation, compensating for the lack of T cell derived TRAIL." . _:b6001414 "We tested this by combining CY-treatment in nude mice with agonistic anti-DR5 antibodies (clone MD5-1) [>>35<<], [36], reasoning that CY-sensitized tumor cells would then be killed by antibody-mediated DR5 ligation, compensating for the lack of T cell derived TRAIL." . _:b413576289 "2"^^ . . _:b413576288 "2"^^ . . _:b413576291 "2"^^ . _:b413576285 . _:b413576290 "2"^^ . _:b413576301 "2"^^ . _:b413576322 . _:b413576300 "2"^^ . . _:b6001394 . _:b413576303 "2"^^ . _:b413576314 . _:b413576302 "2"^^ . _:b6001379 . _:b413576297 "2"^^ . . _:b413576338 . _:b413576296 "2"^^ . . . _:b413576299 "2"^^ . _:b6001345 _:b6001346 . _:b6001345 _:b6001347 . _:b6001345 _:b6001348 . _:b413576298 "2"^^ . _:b6001345 _:b6001349 . _:b6001345 _:b6001350 . _:b6001382 "Generation and maintenance of the BALB/c-derived mouse mesothelioma cell line AB1 and transfection with the HA gene (AB1-HA) has been described [15], [>>16<<]. AB1-HA cells (1\u00D7106 in PBS) were injected s.c. into the right flank of recipient mice and tumor growth monitored using microcalipers." . _:b6001378 "TRAIL-deficient mice were originally generated at Immunex Corporation [>>35<<], [59] and were backcrossed to BALB/c at the Peter MacCallum Cancer Centre (Melbourne, Australia) (n\u200A=\u200A12)." . _:b6001345 _:b6001351 . _:b413576279 . _:b6001417 "discussion" . _:b6001345 _:b6001352 . _:b413576309 "2"^^ . _:b6001345 _:b6001353 . _:b413576295 . _:b6001345 _:b6001354 . _:b6001345 _:b6001355 . _:b6001345 _:b6001356 . _:b413576308 "2"^^ . _:b6001345 _:b6001357 . _:b6001345 _:b6001358 . _:b6001384 "The IFN-\u03B1/\u03B2 neutralizing sheep Ig and matching normal sheep Ig [17], [>>61<<] were used as described [5]. Mice were i.v. injected with 0.2 ml of immunoglobulins on day \u20131, +2, and +4 with respect to CY administration." . _:b413576294 . _:b6001345 _:b6001359 . _:b6001345 _:b6001360 . _:b413576311 "2"^^ . _:b6001345 _:b6001361 . _:b6001345 _:b6001362 . _:b6001345 _:b6001363 . _:b6001345 _:b6001364 . _:b413576310 "2"^^ . _:b6001396 "Indeed, we have recently confirmed that Ly6AE-upregulation on CD8 T cells occurs in a strictly IFN-\u03B1/\u03B2-dependent manner after AB1-HA tumor treatment with the TLR3 and TLR7 agonists poly-I:C and imiquimod [>>5<<], [23]. Therefore, to assess IFN-\u03B1/\u03B2 production, we measured Ly6AE expression on peripheral blood CD8 T cells harvested three days after administration of CY (150 mg/kg) in AB1-HA tumor-bearing mice. We found that CY treatment resulted in" . _:b6001345 _:b6001365 . _:b6001345 _:b6001366 . _:b6001345 _:b6001367 . _:b6001345 _:b6001368 . _:b413576305 "2"^^ . _:b6001345 _:b6001369 . _:b6001345 _:b6001370 . _:b6001345 _:b6001371 . _:b6001345 _:b6001372 . _:b6001364 "To study this, we have used a mouse model of malignant mesothelioma (AB1-HA), which is sensitive to both chemotherapy and immunotherapy [4], [15], [>>16<<], in combination with the chemotherapeutic drug cyclophosphamide (CY), since CY treatment is associated with innate and adaptive immune activation [17], [18], [19], [20], [21]." . _:b413576304 "2"^^ . . _:b6001345 _:b6001373 . _:b6001345 _:b6001374 . _:b6001345 _:b6001375 . _:b413576288 . _:b413576307 "2"^^ . _:b413576306 "2"^^ . _:b413576293 . . _:b413576317 "2"^^ . _:b413576316 "2"^^ . . _:b6001413 "As several chemotherapeutic drugs sensitize tumor cells for death ligand-mediated cell apoptosis (TRAIL) [>>34<<], we analyzed expression of the TRAIL receptor DR5 on AB1-HA tumor cells. We could detect expression of DR5 on tumor cells ( Figure 6A ) suggesting that TRAIL-mediated apoptosis could be involved in the anti-tumor effects of CY." . . _:b413576319 "2"^^ . _:b413576318 "2"^^ . _:b413576289 . _:b6001372 . _:b413576313 "2"^^ . . _:b6001439 "TRAIL sensitization of tumor cells, including human mesothelioma cells, by chemotherapeutic drugs has been well characterized [>>47<<], [48] and has been translated into therapeutic approaches involving soluble TRAIL or other TRAIL-receptor agonists [35], [49], [50]." . _:b413576312 "2"^^ . . _:b6001370 . _:b413576315 "2"^^ . _:b413576314 "2"^^ . _:b6001444 "It has been proposed that the synergy of TRAIL-ligation and chemotherapy can be explained by integration of the intrinsic (through chemotherapy) and alternative (death receptor) pathways of apoptosis [>>47<<]. Recently, Johnstone and colleagues showed that the histone diacetylase inhibitor vorinostat augmented the anti-tumor effects of anti-DR5 antibodies in vivo and in vitro through downregulation of the apoptosis inhibitor cellular-FLIP [51]." . _:b413576325 "2"^^ . _:b413576324 "2"^^ . _:b6001378 . . . _:b413576327 "2"^^ . _:b413576326 "2"^^ . . . _:b6001347 . _:b6001418 . . _:b413576321 "2"^^ . . _:b6001429 "This indicates that CY mobilizes anti-tumor CD8 T cells in a way that does not involve type-I IFN dependent activation and/or expansion [>>30<<], [41]." . _:b413576320 "2"^^ . _:b6001416 . _:b6001376 _:b6001380 . _:b413576323 "2"^^ . _:b6001376 _:b6001381 . _:b6001376 _:b6001382 . _:b6001383 . _:b6001376 _:b6001383 . _:b6001352 . _:b6001363 . _:b413576322 "2"^^ . _:b6001376 _:b6001377 . _:b6001376 _:b6001378 . _:b6001376 _:b6001379 . _:b6001376 _:b6001388 . _:b413576333 "2"^^ . _:b6001433 "Whereas depletion of proliferating regulatory T cell is beneficial [18], [>>19<<], [44], the impact of CY on the CD8 T cell response could be negative and seems inconsistent with the key role of CD8 T cells in tumor resolution." . _:b6001401 . _:b6001376 _:b6001384 . _:b6001395 . _:b413576332 "2"^^ . _:b6001403 . _:b6001376 _:b6001385 . _:b6001376 _:b6001386 . _:b413576310 . _:b6001376 _:b6001387 . _:b413576335 "2"^^ . . . _:b413576334 "2"^^ . _:b6001348 . _:b6001357 . _:b6001349 . . _:b413576329 "2"^^ . _:b6001350 . _:b6001380 . _:b413576328 "2"^^ . _:b6001419 "CY kills cells by inducing apoptosis and this seems to result in a systemic type-I IFN response, confirming earlier studies [>>17<<], [29], [39] and consistent with the notion of \u2018immunogenic cell death\u2019. Similar data have been reported by Schiavoni and coworkers who used expression of Ly6C on T cells (similar to Ly6AE) and PCR to detect type-I IFN production [17]." . _:b6001389 _:b6001392 . _:b6001351 . _:b6001406 . _:b6001389 _:b6001393 . _:b413576331 "2"^^ . _:b6001389 _:b6001394 .