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-Dependent CXC Chemokines in Syngeneic Mice1
* Dipartimento di Oncologia e Neuroscienze, Università di Chieti, Chieti, Italy;
Istituto Nazionale per la Ricerca sul Cancro, and
Istituto Giannina Gaslini, Genoa, Italy; and
Istituto Nazionale Tumori, Milan, Italy
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Abstract |
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helix cytokine produced by activated T cells. To study the in vivo antitumor activities of IL-21, TS/A murine mammary adenocarcinoma cells were genetically modified to secrete IL-21 (TS/A-IL-21). These cells developed small tumors that were subsequently rejected by 90% of s.c. injected syngeneic mice. Five days after injection, TS/A-IL-21 tumors showed numerous infiltrating granulocytes, NK cells, and to a lesser extent CD8+ T cells, along with the expression of TNF-, IFN-
, and endothelial adhesion molecules ICAM-1 and VCAM-1. At day 7, CD8+ and CD4+ T cells increased together with IFN-, and the CXC chemokines IFN--inducible protein 10, monokine induced by IFN-, and IFN-inducible T cell -chemoattractant. The TS/A-IL-21 tumor displayed a disrupted vascular network with abortive sprouting and signs of endothelial cell damage. In vivo depletion experiments by specific Abs showed that rejection of TS/A-IL-21 cells required CD8+ T lymphocytes and granulocytes. When injected in IFN--deficient mice, TS/A-IL-21 cells formed tumors that regressed in only 29% of animals, indicating a role for IFN- in IL-21-mediated antitumor response, but also the existence of IFN--independent effects. Most immunocompetent mice rejecting TS/A-IL-21 cells developed protective immunity against TS/A-pc (75%) and against the antigenically related C26 colon carcinoma cells (61%), as indicated by rechallenge experiments. A specific CTL response against the gp70-env protein of an endogenous murine retrovirus coexpressed by TS/A and C26 cells was detected in mice rejecting TS/A-IL-21 cells. These data suggest that IL-21 represents a suitable adjuvant in inducing specific CTL responses. |
Introduction |
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IL-21 is a recently discovered four helix bundle cytokine, produced by activated T cells, which belongs to the IL-2 cytokine family and shares structural similarities with other members of this family, particularly with IL-15 (4). The IL-21 biological activities are mediated through a specific IL-21R chain, structurally related to the IL-2R
chain (4, 5), which associates with the common chain, required for IL-21 complete signal transduction (6, 7). Early studies proposed that IL-21 is involved in the regulation of NK cell proliferation and differentiation from bone marrow precursors, in conjunction with IL-15, and that it also acts as a costimulus for mature T and B cell proliferation (4). More recent data, generated in IL-21R-/- mice (8), have challenged the role of IL-21 in NK cell development, because these mice, differently from IL-15-/- (9) or IL-15R-/- mice (10), display no defects in NK cell number and function. In addition, IL-21 limited the NK cell expansion triggered by IL-15, but it efficiently promoted proliferation, cytotoxic function, and IFN- production by murine CD8+ effector T cells, upon allogenic MLC stimulation (8). IL-21 has been also reported to support the clonal expansion of CMV-specific effector human CD8+ cells (11). In view of these functions, IL-21 acts as a key element in driving the transition from NK cell responses to specific CTL responses. In addition, IL-21 was shown to play an important role in the regulation of B cell responses, because it specifically inhibits IgE production (12, 13). Conflicting data have been reported concerning the role of IL-21 in the polarization (type 1 or 2) of the immune response. A report proposed that IL-21 is produced by Th2 cells and inhibits IFN- production by developing Th1 cells (14), whereas other authors reported that IL-21 induces expression of IFN-, and of other genes associated with innate immunity and Th1 response (15).
In previous studies, tumor cells genetically engineered to secrete IL-15, the IL-2-family member most similar to IL-21 (4), displayed reduced tumorigenicity in vivo (16, 17) and induced protective immunity, involving CD8+ T cells and/or NK cells and IFN- (17, 18, 19).
In this study, we used TS/A mammary adenocarcinoma cells (20) genetically modified to secrete IL-21 to investigate the functional activities of IL-21 and to test its potential adjuvant properties in vivo. The IL-21-modified tumor cells displayed a clear-cut reduction in their tumorigenic potential in syngeneic mice and primed a protective immune response. CD8+ CTLs, IFN-, and downstream third-order CXC chemokines, mediating antiangiogenic effects, were involved in the IL-21-driven immune reaction.
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Materials and Methods |
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TS/A murine breast adenocarcinoma (kindly provided by Prof. P. Lollini (University of Bologna, Bologna, Italy)) (20), C26 colon carcinoma, and F1F fibrosarcoma cells were cultured in DMEM supplemented with 2 mM L-glutamine, 1% PenStrepto (BioWhittaker, Verviers, Belgium), and 10% FCS (Seromed Biochrom, Berlin, Germany).
Plasmid assembly and TS/A cell transfection
The open reading frame of murine IL-21 was amplified by RT-PCR from total RNA extracted from Con-A-stimulated T lymphoblasts using the following primers: forward, 5'-CTGTCATCAGCTCCTGGAGA, and reverse, 5'-CTTCGGGTCCTATGTGTTCT (30 cycles: 30 s at 95°C, 30 s at 56°C, and 45 s at 72°C, with a final extension at 72°C for 7 min). cDNA was first subcloned in the PCR2.1 vector using the TOPO-TA cloning system (Invitrogen, Milan, Italy), sequenced, and then subcloned into the p-IRES1neo expression plasmid (Clontech, Palo Alto, CA). TS/A cells were transfected with 10 µg of pIL-21IRES1neo or of empty p-IRES1neo plasmid using the Fugene 6 transfection reagent (Roche, Milan, Italy). Stable transfectants were cloned by limiting dilution, in medium containing G418 (500 µg/ml; Roche) and were then screened for IL-21 mRNA expression by RT-PCR as described above.
Western blot analysis of IL-21 expression
For the evaluation of secreted IL-21, 2 x 106 aliquots of cells were cultured in serum-free DMEM containing 1x Nutridoma-HU (Roche) for 48 h. Cell supernatants were then collected, and the secreted proteins were precipitated with 10% TCA at 4°C for 12 h. Protein pellets were washed three times with cold acetone, dried, resuspended in 25 µl of water, and analyzed by SDS-PAGE on 14% polyacrylamide gels under reducing conditions. As positive control, rIL-21 (R&D Systems, Milan, Italy) was run in a parallel lane. The gel was blotted onto a Hybond-C membrane (Amersham, Milan, Italy) at 250 mA for 2 h at 4°C. The blot was saturated overnight in TBST containing 10% nonfat dry milk, and then incubated for 2 h in 2% BSA in PBS containing 2 µg/ml goat anti-mouse IL-21 Ab (R&D Systems). After washings with TBST, the blot was stained with 1/2000 dilution of HRP-conjugated anti-goat Ig antiserum (DAKO, Glostrup, Denmark) for 2 h. Bands were visualized by the ECL system (Amersham).
Tumorigenicity assay in syngeneic immunocompetent and IFN--deficient mice
Five- to 7-wk-old female BALB/cAnNCrlBR (BALB/c) mice were purchased from Harlan (Udine, Italy). IFN- knockout (GKO)4 mice (21) on a BALB/c background were purchased from The Jackson Laboratory (Bar Harbor, ME). Homozygous mice were bred and maintained in isolators in-house. Mice were allowed to rest for 1 wk before any treatment.
Animals (six to seven mice for each group) were injected s.c. with 105 TS/A parental cells (TS/A-pc) per mouse, TS/A cells transfected with empty pIRES1neo (TS/A-mock), or TS/A cells transfected with IL-21 gene (TS/A-IL-21) cells. Cells were mycoplasma free, as assessed by ELISA (Boehringer/Roche, Milan, Italy) or by PCR analysis before injection. Cells were washed three times in endotoxin-free RPMI 1640 medium without FCS and one time in endotoxin-free PBS before injection. Wider and smaller diameters of s.c. tumors were measured using a caliper at weekly intervals. The multiple of the wider and smaller diameter of each tumor was used as an estimate of the growth area, and data were displayed as mean ± SD for each group of animals at a given time point. Statistical analysis was performed by the Mann-Whitney test; values of p < 0.05 were considered as significant.
Depletion studies were performed by i.p. injections of rabbit anti-asialo-GM1 antiserum (Wako Chemicals, Dusseldorf, Germany) (0.2 ml per mouse of 1/10 diluted stock solution); anti-granulocyte rat RB6-8C5 mAb (0.2 ml of a 1/50 dilution of ascites) (kindly provided by Dr. R. Coffman (DNAX, Palo Alto, CA)), or anti-CD8 (24.3) or anti-CD4 (GK1.5) rat mAbs, both from American Type Culture Collection (Manassas, VA), as previously reported (18). Control animals received normal rabbit serum or an irrelevant rat mAb. Depletion efficiency (>90%) was assessed by immunofluorescence using the relevant FITC-labeled Ab (BD PharMingen, San Diego, CA) and FACS analysis on PBMC obtained from the retro-orbital sinus of viable mice or from spleen cell suspensions of euthanized mice.
Morphologic analysis of TS/A-pc or TS/A-IL-21 tumor growth or rejection area
Groups of three mice were sacrificed at 5, 7, and 12 days after TS/A-pc or TS/A-IL-21 tumor cell inoculation in BALB/c or GKO mice.
For histologic evaluation, tissue samples were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 4 µm, and stained with H&E or with the trichrome method (22).
For immunohistochemistry, acetone-fixed cryostat sections were immunostained with anti-CD11b/CD18 (clone M1/70.5), anti-CD8 (Ly/T2; clone YT5 169.4), and anti-CD4 (LT34; clone YT5.191.1.2) (all from Sera-lab, Crawley Down, Sussex, U.K.), or anti-GR1 (clone RB6-8C5; American Type Culture Collection), anti-VCAM-1 (CD106; clone 429 MVCAM.A) (BD PharMingen), or anti-NK (asialo GM1), anti-endothelial cells (mEC-13.324), and anti-endothelial leucocyte adhesion molecule-1 (E selectin; CD62E; clone 10.E9.6; both provided by Dr. A. Vecchi (Istituto Mario Negri, Milan, Italy)), anti-IFN--inducible protein 10 (IP-10) (5) (PeproTech, London, U.K.), anti-monokine induced by IFN- (MIG) (5) (R&D Systems), anti-TNF- (clone MP6-XT22; Immuno Kontact, Frankfurt, Germany), anti-IFN- (clone XMG1.2; provided by Dr. S. Landolfo (University of Turin, Turin, Italy)), anti-ICAM-1 (CD54; clone 3E2), and anti-IFN-inducible T cell -chemoattractant (I-TAC) (5) (clone A.15) (both from Santa Cruz, Biotechnology, Santa Cruz, CA) Abs. After washing, sections were overlaid with biotinylated goat anti-rat, anti-hamster, and anti-rabbit, and horse anti-goat Ig (Vector Laboratories, Burlingame, CA) for 30 min. Unbound Ig was removed by washing, and slides were incubated with ABC (avidin/biotin complex)/alkaline phosphatase (DAKO). Tetramethylrhodamine isothiocyanate-conjugated goat anti-rat IgG was used as secondary Ab for endothelial cell immunofluorescence. Quantitative studies of stained sections were performed independently by three pathologists in a blind fashion. Expression of cytokines and adhesion molecules was scored as absent (-), low (±), moderate (+), or frequent (++). Cell counts were obtained in 10 randomly chosen fields under a Leica DMLB light microscope (x400 field; 0.180 mm2/field; Leica, Deerfield, IL). Confocal microscopy was performed with Zeiss LSM 510 META (Zeiss, Oberkochen, Germany).
In vitro restimulation and CTL assay
Spleen cells from mice that had rejected TS/A-IL-21 cells and were further resistant to TS/A-pc were seeded at 106 cells/ml in culture medium and restimulated in vitro for 5 days at 37°C in presence of the gp70-derived AH1 peptide (23) (SPSYVYHQF; synthesized by Primm (Milan, Italy)) at a final concentration of 1 µg/ml. The capability of lymphoblasts to lyse F1F, C26, and TS/A-pc target cells was evaluated by a standard 51Cr release assay, and percentage of lysis was then calculated.
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Results |
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TS/A cells were transfected with the pIL-21IRES-1neo plasmid and selected in G418-containing medium. Two TS/A-IL-21 clones (nos. 19 and 20) showing RT-PCR positivity for IL-21 mRNA expression (Fig. 1a) and displaying growth kinetics in vitro similar to that of TS/A-pc (not shown), were selected for further studies. Western blot analysis of concentrated culture supernatants of these clones showed two bands of an apparent Mr of 
16 and 20 kDa, higher than the Mr of Escherichia coli-derived rIL-21 (14 kDa). These bands, undetectable in TS/A-mock cell supernatants, may represent two different glycosylation forms of IL-21 (Fig. 1b).
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Histological and immunohistochemical features of TS/A-IL-21 tumor and draining lymph nodes in syngeneic mice
The rejection pattern of the small s.c. TS/A-IL-21 no. 20 tumors in BALB/c mice was compared with the growth pattern of the TS/A-pc tumors at progressive times from cell injection. At day 5, TS/A-IL-21 cells had given rise to a small tumor surrounded by a slight fibrotic reaction (Fig. 2a) and heavily infiltrated by macrophages, which were also observed in the TS/A-pc tumor (Table I). Granulocytes and NK cells were significantly (p < 0.005) more numerous; CD8+ cells were slightly more represented (Table I and Fig. 2, d and g); and TNF-, IFN-, and the endothelial adhesion molecules ICAM-1 and VCAM-1 were distinctly expressed in the TS/A-IL-21 tumor (Table I).
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was increased together with that of the CXC chemokines IP-10, I-TAC, and particularly MIG (Table I).
The TS/A-pc tumor was well vascularized with evenly distributed blood vessels, whereas the vascular network of the TS/A-IL-21 tumor displayed abortive sprouting (single endothelial cells were frequent instead of well-developed endothelial walls and/or tubes) (Fig. 3, a and b) as well as signs of endothelial and vessel damage (c and d). The number of blood microvessels was significantly reduced (p 
0.005) in TS/A-IL-21 tumor (19 ± 2 vs 26 ± 4 in TS/A-pc tumor (x400 field)), and their lumens were lined by swollen endothelial cells expressing high levels of endothelial adhesion molecules (Fig. 3, e and f) and often obstructed by stacked reactive cells or thrombi (b and c).
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in the medullary sinuses close to the paracortical areas of the lymph nodes draining the TS/A-IL-21 tumor, and MIG and I-TAC were expressed by endothelial cells of high endothelial venules and splenocytes resembling APCs (Fig. 4).
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0.005) reduced as compared with the 7-day TS/A-IL-21 tumor, whereas the number of T cells was unchanged and consistent with the day 7 values (Table I and Fig. 2, f and i). Effect of CD8+, CD4+, NK cell, and granulocyte depletion on the tumorigenicity of TS/A-IL-21 cells
To gain further information on the role of different cell subsets in the rejection process, TS/A-IL-21 no. 20 cells were injected s.c. in BALB/c mice that had been treated with either anti-CD8, anti-CD4, anti-asialo GM1, or anti-granulocyte depleting Abs. As shown in Fig. 5, depletion of CD8+ lymphocytes completely restored tumorigenicity of TS/A-IL-21 cells (100% tumor take; p < 0.05 vs Ig control), indicating a role of CTLs in the rejection response. Anti-GR1 mAb depleted granulocytes temporarily (24), and accordingly, such treatment resulted in a delay of the rejection process. Depletion of NK or CD4+ cells and treatment with nonimmune control Ig was ineffective.
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in TS/A-IL-21 cell rejection
Data from immunohistochemical analysis indicated that IL-21 induced secondary cytokine production, particularly IFN- and IFN--inducible third-order antiangiogenic chemokines I-TAC, IP-10, and MIG. In addition, previous studies reported that IL-21 promotes IFN- production by CTLs and NK cells (8). Therefore, we further analyzed the tumorigenicity of the TS/A-IL-21 no. 20 cells in IFN--deficient (GKO) mice (21). As shown in Fig. 6, TS/A-mock cells showed a very rapid growth kinetics in 100% of GKO mice, and TS/A-IL-21 were also tumorigenic in most GKO mice (71% of tumor-bearing mice at 4 wk vs 5% in immunocompetent syngeneic mice), although the TS/A-IL-21 tumor growth kinetics was significantly reduced as compared with TS/A-mock. These data point to a relevant role of IFN- as a mediator of the immune response triggered by transduced IL-21 gene, although the IL-21 effects were not completely abrogated in the absence of IFN-.
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TS/A-IL-21 no. 20 tumors, developed 7 days after s.c. cell injection in GKO mice, showed no evident T lymphocyte infiltrate (CD8+ cells, 5 ± 2; CD4+ cells, 4 ± 2; x400 field). NK cells were well represented, although there were only half as many as in BALB/c mice (15 ± 3 vs 30 ± 6), whereas granulocytes and macrophages were not significantly increased compared with the TS/A-pc tumor (23 ± 4 vs 26 ± 4, and 27 ± 4 vs 24 ± 3, respectively). The expression of IP-10 was scanty, and that of MIG and I-TAC was absent. The TS/A-IL-21 tumor vasculature was significantly more evident and developed than in BALB/c mice (number of vessels, 29 ± 3 vs 19 ± 2).
The presence of a relevant number of infiltrating NK cells suggests a possible NK cell involvement in the IFN--independent antitumor effects of IL-21 in GKO mice. Indeed, depletion of NK cells by anti-asialo GM1 Ab completely restored tumorigenicity (100% take rate; p < 0.05) of TS/A-IL-21 in GKO mice (Fig. 7), whereas treatment with preimmune polyclonal rabbit Ig resulted in a 36% rejection rate.
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To verify whether immunocompetent syngeneic mice, which had rejected TS/A-IL-21 no. 20 cells, developed immunity against TS/A-pc, these mice were rechallenged s.c. with a fully tumorigenic dose of TS/A-pc. As shown in Fig. 8, a and b, most of these mice (75%) rejected TS/A-pc, while the same cells induced rapid tumor growth in all unprimed mice. In addition, most of the immune mice (61%) were also cross-protected to s.c. rechallenge with the antigenically related C26 colon adenocarcinoma cells, which show a very rapid tumor take (100%) in unprimed mice (Fig. 8b). By contrast, subsequent challenge of TS/A-pc-resistant mice with the unrelated syngeneic fibrosarcoma F1F cells showed no protection (100% take rate), indicating specificity of the response (not shown). In addition, mice rejecting TS/A-IL-21 cells showed no enhancement of systemic NK activity in the spleen, as detected by a cytotoxicity assay against YAC cells (data not shown), further suggesting that resistance to C26 or TS/A-pc rechallenge is related to a specific immune response.
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Discussion |
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The tumor inhibition mediated by IL-21 gene transfection in TS/A cells was superior to that obtained in the same experimental model using the cDNA of the structurally related cytokine IL-15, which resulted in a 50% rejection rate in syngeneic mice (18).
Morphological and immunohistochemical examination of TS/A-IL-21 cell injection area and of draining lymph nodes illustrated the dynamics of IL-21-induced tumor regression and revealed some pathways shared with that induced by IL-12 (26, 27, 28, 29, 30, 31), although remarkable differences were also evident.
When continuously released into the tumor microenvironment, IL-21 initially promotes a slight TNF- and IFN- production in the few tumor-infiltrating NK and T cells and induces endothelial cell activation (32), as revealed by leukocyte integrin ligand (ICAM-1 and VCAM-1) expression, and, hence, intratumoral transmigration of leukocytes. IL-21 has been reported to regulate NK cell activation and IFN- production by both NK cells and CTLs (4, 8, 33). Although NK cells infiltrated the TS/A-IL-21 tumor mass at early stages, they seemed not strictly required for rejection of a TS/A-IL-21 tumor in immunocompetent mice, as evidenced by the selective depletion experiments. Our data partially differ from those recently reported using IL-21 gene-transduced colon carcinoma cells (34), showing the involvement of both NK and T cells in the rejection process. Also, depletion of CD4+ cells was unable to prevent TS/A-IL-21 rejection, thus indicating that tumor-secreted IL-21 may directly act on CTLs (8), and bypass the requirement for Th cells. In contrast, the IFN- production appeared of leading importance in TS/A-IL-21 tumor rejection, because TS/A-IL-21 cells were significantly more tumorigenic in GKO than in immunocompetent mice.
Moreover, in immunocompetent mice, along with an increasing expression of IFN- close to the tumor microvessels, and chiefly in the draining lymph nodes, high levels of the CXC chemokines MIG, IP-10, and I-TAC (35, 36, 37), were found in the immune cells, endothelial cells of blood microvessels, and high endothelial venules. Through their interaction with CXCR-3, these chemokines are potent T cell chemoattractants and angiogenesis inhibitors (38) and are downstream mediators of IL-12-triggered antitumor effects (28, 29, 30, 31). In this study, we propose a role of IFN--induced CXC chemokines for IL-21-dependent antitumor reaction. The vascular network of TS/A-IL-21 tumor was greatly altered in the form of abortive-like branching, thrombi, and signs of endothelial cell damage. These events resulted in ischemic-necrotic events, and may promote a fibrotic postischemic response.
The finding that, in GKO mice, the number and the integrity of TS/A-IL-21 tumor microvessels was not compromised, and that IFN--inducible CXC chemokines were not expressed, strongly supports the concept that inhibition of tumor angiogenesis through the IFN-/CXC chemokine network plays an important role in the inhibition of TS/A tumor by IL-21. Nonetheless, the finding that TS/A-IL-21 tumors developed with a reduced kinetics in GKO mice, and were rejected by about one-third of these mice, indicates that IFN-/CXC chemokine-independent pathways also play a role in IL-21-mediated antitumor effects. In this context, it should be outlined that the antitumor reactions triggered by IL-12 or by IL-21 differ for their IFN- dependency in this tumor model, because TS/A cells transduced with IL-12 gene were almost completely tumorigenic in GKO mice (19, 25). Different from what was observed in immunocompetent mice, in GKO mice, IL-21 required NK cells for its antitumor activity. While we were submitting the present article, another report showed that IL-21 antitumor effects were predominantly IFN- independent and involved perforins, CTLs, and NK cells in the B16 melanoma model (39). Altogether these observations suggest that, depending on the experimental tumor model, IL-21 released in the microenvironment by transduced tumor cells may trigger different pathways leading to an effective immune response and tumor rejection.
In immunocompetent mice, also granulocytes appeared to be involved in TS/A-IL-21 tumor rejection, because they markedly infiltrated the tumor area, and their selective removal delayed tumor rejection. These observations suggest a cross-talk of granulocytes with endothelial cells and lymphocytes (2, 40) and their cooperative role in the IL-21-mediated antiangiogenic effect (31).
IL-21 secreted locally by tumor cells induced an early granulocyte and NK cell accumulation at the tumor site, which was followed by a progressive T cell influx, and by a late tumor rejection. This kinetics differs from that observed in mice injected with TS/A cells transduced with IL-12, where a rapid intratumoral CD8+ T cell recruitment and activation mediated tumor rejection (29, 41). The slow rejection response induced by IL-21, together with the ability of IL-21 to mediate the transition from innate to adaptive immunity (8), results in a strong priming effect and in the induction of an efficient immune memory. Thus, following TS/A-IL-21 cell rejection, 75% of mice were protected against a subsequent parental cell challenge, whereas only 32% of mice rejecting TS/A-IL-12 cells become immune to rechallenge (41).
The crucial role of Ag-specific CTLs in the TS/A-IL-21 model was evidenced by both CD8+ cell depletion experiments, which completely restored tumorigenicity of TS/A-IL-21 cells in vivo, and by the identification of gp70-specific CTLs in mice primed by TS/A-IL-21 cells. These data, together with the finding that most mice rejecting TS/A-IL-21 are cross-protected against rechallenge with C26 colon carcinoma, which shares with TS/A gp70 Ag expression, strongly support the concept that gp70 is an immunodominant Ag in the TS/A model (25). Although gp70-specific CTLs lysed C26 more efficiently than TS/A cells in vitro, mice rejecting TS/A-IL-21 cells appeared slightly more resistant to TS/A than to C26 cell rechallenge. This finding may reflect the higher tumorigenic potential of C26 cells in vivo as compared with TS/A (25), but a role of as-yet-unknown TS/A-specific Ags, different from gp70, and not shared by C26, cannot be excluded.
In conclusion, our data indicate that IL-21 acts in part as a Th1-related cytokine in vivo, based on its ability to stimulate IFN- production, the related antiangiogenic functions, and specific CTL responses. In view of these activities and of additional IFN--independent antitumor properties, IL-21 should be regarded as a suitable molecule for cancer immunoprevention or gene therapy.
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Acknowledgments |
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Footnotes |
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2 E.D.C. and A.C. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Silvano Ferrini at the current address: Laboratory of Immunopharmacology, c/o Centro di Biotecnologie Avanzate, Largo Rosanna Benzi no. 10, 16132 Genoa, Italy. E-mail address: silvano.ferrini{at}istge.it
4 Abbreviations used in this paper: GKO, IFN- knockout; IP-10, IFN--inducible protein 10; MIG, monokine induced by IFN-; I-TAC, IFN-inducible T cell -chemoattractant.
Received for publication July 14, 2003. Accepted for publication November 4, 2003.
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T. Onoda, M. Rahman, H. Nara, A. Araki, K. Makabe, K. Tsumoto, I. Kumagai, T. Kudo, N. Ishii, N. Tanaka, et al. Human CD4+ central and effector memory T cells produce IL-21: effect on cytokine-driven proliferation of CD4+ T cell subsets Int. Immunol., October 1, 2007; 19(10): 1191 - 1199. [Abstract] [Full Text] [PDF]
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 V. Ostiguy, E.-L. Allard, M. Marquis, J. Leignadier, and N. Labrecque IL-21 promotes T lymphocyte survival by activating the phosphatidylinositol-3 kinase signaling cascade J. Leukoc. Biol., September 1, 2007; 82(3): 645 - 656. [Abstract] [Full Text] [PDF]
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K. A. Casey and M. F. Mescher IL-21 Promotes Differentiation of Naive CD8 T Cells to a Unique Effector Phenotype J. Immunol., June 15, 2007; 178(12): 7640 - 7648. [Abstract] [Full Text] [PDF]
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 I. D. Davis, B. K. Skrumsager, J. Cebon, T. Nicholaou, J. W. Barlow, N. P. H. Moller, K. Skak, D. Lundsgaard, K. S. Frederiksen, P. Thygesen, et al. An Open-Label, Two-Arm, Phase I Trial of Recombinant Human Interleukin-21 in Patients with Metastatic Melanoma Clin. Cancer Res., June 15, 2007; 13(12): 3630 - 3636. [Abstract] [Full Text] [PDF]
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 S Vitale, B Cambien, B F Karimdjee, R Barthel, P Staccini, C Luci, V Breittmayer, F Anjuere, A Schmid-Alliana, and H Schmid-Antomarchi Tissue-specific differential antitumour effect of molecular forms of fractalkine in a mouse model of metastatic colon cancer Gut, March 1, 2007; 56(3): 365 - 372. [Abstract] [Full Text] [PDF]
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J. M. Coquet, K. Kyparissoudis, D. G. Pellicci, G. Besra, S. P. Berzins, M. J. Smyth, and D. I. Godfrey IL-21 Is Produced by NKT Cells and Modulates NKT Cell Activation and Cytokine Production J. Immunol., March 1, 2007; 178(5): 2827 - 2834. [Abstract] [Full Text] [PDF]
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D. Konforte and C. J. Paige Identification of Cellular Intermediates and Molecular Pathways Induced by IL-21 in Human B Cells J. Immunol., December 15, 2006; 177(12): 8381 - 8392. [Abstract] [Full Text] [PDF]
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R. Schirmbeck, P. Riedl, M. Kupferschmitt, U. Wegenka, H. Hauser, J. Rice, A. Kroger, and J. Reimann Priming Protective CD8 T Cell Immunity by DNA Vaccines Encoding Chimeric, Stress Protein-Capturing Tumor-Associated Antigen J. Immunol., August 1, 2006; 177(3): 1534 - 1542. [Abstract] [Full Text] [PDF]
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A. Cappuccio, M. Elishmereni, and Z. Agur Cancer immunotherapy by interleukin-21: potential treatment strategies evaluated in a mathematical model. Cancer Res., July 15, 2006; 66(14): 7293 - 7300. [Abstract] [Full Text] [PDF]
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M. Pelletier and D. Girard Differential Effects of IL-15 and IL-21 in Myeloid (CD11b+) and Lymphoid (CD11b-) Bone Marrow Cells J. Immunol., July 1, 2006; 177(1): 100 - 108. [Abstract] [Full Text] [PDF]
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J. M. Roda, R. Parihar, A. Lehman, A. Mani, S. Tridandapani, and W. E. Carson III Interleukin-21 Enhances NK Cell Activation in Response to Antibody-Coated Targets J. Immunol., July 1, 2006; 177(1): 120 - 129. [Abstract] [Full Text] [PDF]
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L. Mortara, P. Castellani, R. Meazza, G. Tosi, A. De Lerma Barbaro, F. A. Procopio, A. Comes, L. Zardi, S. Ferrini, and R. S. Accolla CIITA-Induced MHC Class II Expression in Mammary Adenocarcinoma Leads to a Th1 Polarization of the Tumor Microenvironment, Tumor Rejection, and Specific Antitumor Memory. Clin. Cancer Res., June 1, 2006; 12(11): 3435 - 3443. [Abstract] [Full Text] [PDF]
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M. J. Smyth, Y. Hayakawa, E. Cretney, N. Zerafa, P. Sivakumar, H. Yagita, and K. Takeda IL-21 Enhances Tumor-Specific CTL Induction by Anti-DR5 Antibody Therapy J. Immunol., May 15, 2006; 176(10): 6347 - 6355. [Abstract] [Full Text] [PDF]
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D. de Totero, R. Meazza, S. Zupo, G. Cutrona, S. Matis, M. Colombo, E. Balleari, I. Pierri, M. Fabbi, M. Capaia, et al. Interleukin-21 receptor (IL-21R) is up-regulated by CD40 triggering and mediates proapoptotic signals in chronic lymphocytic leukemia B cells Blood, May 1, 2006; 107(9): 3708 - 3715. [Abstract] [Full Text] [PDF]
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K. Kawakami, M. Terabe, M. Kawakami, J. A. Berzofsky, and R. K. Puri Characterization of a Novel Human Tumor Antigen Interleukin-13 Receptor {alpha}2 Chain. Cancer Res., April 15, 2006; 66(8): 4434 - 4442. [Abstract] [Full Text] [PDF]
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C. Liu, S. Yu, K. Zinn, J. Wang, L. Zhang, Y. Jia, J. C. Kappes, S. Barnes, R. P. Kimberly, W. E. Grizzle, et al. Murine Mammary Carcinoma Exosomes Promote Tumor Growth by Suppression of NK Cell Function J. Immunol., February 1, 2006; 176(3): 1375 - 1385. [Abstract] [Full Text] [PDF]
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S. J. Burgess, A. I. Marusina, I. Pathmanathan, F. Borrego, and J. E. Coligan IL-21 Down-Regulates NKG2D/DAP10 Expression on Human NK and CD8+ T Cells J. Immunol., February 1, 2006; 176(3): 1490 - 1497. [Abstract] [Full Text] [PDF]
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A. Comes, O. Rosso, A. M. Orengo, E. Di Carlo, C. Sorrentino, R. Meazza, T. Piazza, B. Valzasina, P. Nanni, M. P. Colombo, et al. CD25+ Regulatory T Cell Depletion Augments Immunotherapy of Micrometastases by an IL-21-Secreting Cellular Vaccine J. Immunol., February 1, 2006; 176(3): 1750 - 1758. [Abstract] [Full Text] [PDF]
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P. A. Antony, C. A. Piccirillo, A. Akpinarli, S. E. Finkelstein, P. J. Speiss, D. R. Surman, D. C. Palmer, C.-C. Chan, C. A. Klebanoff, W. W. Overwijk, et al. CD8+ T Cell Immunity Against a Tumor/Self-Antigen Is Augmented by CD4+ T Helper Cells and Hindered by Naturally Occurring T Regulatory Cells J. Immunol., March 1, 2005; 174(5): 2591 - 2601. [Abstract] [Full Text] [PDF]
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M. Pelletier, A. Bouchard, and D. Girard In Vivo and In Vitro Roles of IL-21 in Inflammation J. Immunol., December 15, 2004; 173(12): 7521 - 7530. [Abstract] [Full Text] [PDF]
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