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SLC/CCL21-mediated anti-tumor responses require IFNgamma, MIG/CXCL9 and IP-10/CXCL10.

Sharma S, Yang SC, Hillinger S, Zhu LX, Huang M, Batra RK, Lin JF, Burdick MD, Strieter RM, Dubinett SM - Mol. Cancer (2003)

Bottom Line: We have previously shown that SLC/CCL21-mediated anti-tumor responses are accompanied by significant induction of IFNgamma and the CXC chemokines, monokine induced by IFNgamma (MIG/CXCL9) and IFNgamma-inducible protein-10 (IP-10/CXCL10).In vivo depletion of IP-10/CXCL10, MIG/CXCL9 or IFNgamma significantly reduced the anti-tumor efficacy of SLC/CCL21.These findings indicate that the full potency of SLC/CCL21-mediated anti-tumor responses require in part the induction of IFNgamma, MIG/CXCL9 and IP-10/CXCL10.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, UCLA Lung Cancer Research Program, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. sharmasp@ucla.edu

ABSTRACT

Background: SLC/CCL21, normally expressed in high endothelial venules and in T cell zones of spleen and lymph nodes, strongly attracts T cells and dendritic cells (DC). We have previously shown that SLC/CCL21-mediated anti-tumor responses are accompanied by significant induction of IFNgamma and the CXC chemokines, monokine induced by IFNgamma (MIG/CXCL9) and IFNgamma-inducible protein-10 (IP-10/CXCL10).

Results: We assessed the importance of IFNgamma, IP-10/CXCL10 and MIG/CXCL9 in SLC/CCL21 therapy. In vivo depletion of IP-10/CXCL10, MIG/CXCL9 or IFNgamma significantly reduced the anti-tumor efficacy of SLC/CCL21. Assessment of cytokine production at the tumor site showed an interdependence of IFNgamma, MIG/CXCL9 and IP-10/CXCL10; neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines. Similarly, neutralization of any one of these cytokines led to a decrease in the frequency of CXCR3+ve T cells and CD11c+ve DC at the tumor site.

Conclusion: These findings indicate that the full potency of SLC/CCL21-mediated anti-tumor responses require in part the induction of IFNgamma, MIG/CXCL9 and IP-10/CXCL10.

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SLC/CCL21-treated mice had a significant induction in IFNγ, MIG/CXCL9 and IP-10/CXCL10 at the tumor site compared to diluent treated control tumor bearing mice (p < 0.001). Assessment of cytokine production at the tumor site of SLC/CCL21 treated mice receiving anti-IFNγ, anti MIG/CXCL9 and anti IP-10/CXCL10 showed an interdependence of IFNγ, MIG/CXCL9 and IP-10/CXCL10: neutralization of any one of these cytokines in vivo caused a concomitant decrease in all three cytokines (*p < 0.01 compared to the control antibody treated group). Results are expressed as pg/mg of total protein. Total protein was determined by the Bradford assay. n= 8 mice per group
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Figure 2: SLC/CCL21-treated mice had a significant induction in IFNγ, MIG/CXCL9 and IP-10/CXCL10 at the tumor site compared to diluent treated control tumor bearing mice (p < 0.001). Assessment of cytokine production at the tumor site of SLC/CCL21 treated mice receiving anti-IFNγ, anti MIG/CXCL9 and anti IP-10/CXCL10 showed an interdependence of IFNγ, MIG/CXCL9 and IP-10/CXCL10: neutralization of any one of these cytokines in vivo caused a concomitant decrease in all three cytokines (*p < 0.01 compared to the control antibody treated group). Results are expressed as pg/mg of total protein. Total protein was determined by the Bradford assay. n= 8 mice per group

Mentions: To determine the importance of MIG/CXCL9, IP-10/CXCL10 and IFNγ in the SLC/CCL21 mediated antitumor response, these cytokines were depleted in SLC/CCL21-treated mice. Anti-IP-10/CXCL10, MIG/CXCL9 and IFNγ antibodies each partially but yet significantly inhibited the anti-tumor efficacy of SLC/CCL21 (Figure 1 * p < 0.01 compared to the control antibody group). Neutralization of IFNγ caused a significant decrease in both MIG/CXCL9 and IP-10/CXCL10 indicating that these chemokines are largely IFNγ dependent. Thus, an increase in IFNγ at the tumor site of SLC/CCL21-treated mice could explain the relative increases in IP-10/CXCL10 and MIG/CXCL9. The converse was also observed; IFNγ production at the tumor site was found to be MIG/CXCL9- and IP-10/CXCL10-dependent as indicated by the fact that neutralization of these cytokines caused a significant decrease in IFNγ(Figure 2). Neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines, thus indicating that IFNγ, MIG/CXCL9 and IP-10/CXCL10 are interdependent in the SLC/CCL21-mediated anti-tumor responses. Based on these findings, we speculated that the decrease of IP-10/CXCL10 and MIG/CXCL9 led to a decrease in IFNγ by limiting the influx of T cells producing IFNγ at the tumor site. Because MIG/CXCL9 and IP-10/CXCL10 are chemotactic for stimulated CXCR3 expressing activated T lymphocytes, we determined if neutralizing these chemokines in vivo in the SLC/CCL21-treated mice would decrease the frequency of CXCR3 expressing T cells at the tumor sites. Compared to SLC/CCL21-treated mice receiving control antibody, flow cytometric evaluation of single-cell suspensions of non-necrotic tumors from SLC/CCL21-treated mice receiving neutralizing antibodies to MIG/CXCL9, IP-10/CXCL10 and IFNγ showed 10, 12 and 31 % decreases, respectively, in the gated CD3 CXCR3+ve T cell populations (Table 1). The fact that neutralization of IFNγ was the most efficient at decreasing the total number of CXCR3-activated T cells may be due to the decrease in the IFNγ-dependent CXCR3 ligands MIG/CXCL9 and IP-10/CXCL10. Individually, neutralizing MIG/CXCL9 and/or IP-10/CXCL10 only led to a partial decrease in the total number of CXCR3-activated T cells. Because MIG/CXCL9 and IP-10/CXCL10 share the same receptor (CXCR3), one possible explanation of a partial decrease in CXCR3-activated T cells is that neutralization of one ligand may overexpose the receptor to the other ligand. Furthermore, because murine SLC/CCL21 binds CXCR3 receptor, SLC/CCL21 may be recruiting CXCR3+ve T cells directly. An alternative explanation is that residual cytokines such as IFN-inducible T cell α chemoattractant (ITAC/CXCL11) present after in vivo neutralization of IFNγ, MIG, IP-10 may have remaining activity and attract CXCR3+ve T cells. Our results imply that all 3 cytokines, IFNγ, MIG and IP-10 play interrelated roles in the recruitment of CXCR3-activated T cells in SLC/CCL21-mediated anti-tumor responses. While the number of CD4 and CD8 cells remained unaltered, compared to SLC/CCL21-treated mice receiving control antibody, mice administered neutralizing antibodies to MIG/CXCL9, IP-10/CXCL10 and IFNγ showed respective decreases of 38, 28 and 16% in the number of CD11c+ DEC205+ DC infiltrating the tumor (Table 1). At present it is not clear why there is a decrease in DC following neutralization of MIG/CXCL9, IP-10/CXCL10 and IFNγ. A plausible explanation is that these cytokines may be involved in complex interactions with other molecules that are chemotactic for DC. Further studies are required to delineate the mechanisms responsible for MIG/CXCL9, IP-10/CXCL10 and IFNγ mediated localization of DC at the tumor site.


SLC/CCL21-mediated anti-tumor responses require IFNgamma, MIG/CXCL9 and IP-10/CXCL10.

Sharma S, Yang SC, Hillinger S, Zhu LX, Huang M, Batra RK, Lin JF, Burdick MD, Strieter RM, Dubinett SM - Mol. Cancer (2003)

SLC/CCL21-treated mice had a significant induction in IFNγ, MIG/CXCL9 and IP-10/CXCL10 at the tumor site compared to diluent treated control tumor bearing mice (p < 0.001). Assessment of cytokine production at the tumor site of SLC/CCL21 treated mice receiving anti-IFNγ, anti MIG/CXCL9 and anti IP-10/CXCL10 showed an interdependence of IFNγ, MIG/CXCL9 and IP-10/CXCL10: neutralization of any one of these cytokines in vivo caused a concomitant decrease in all three cytokines (*p < 0.01 compared to the control antibody treated group). Results are expressed as pg/mg of total protein. Total protein was determined by the Bradford assay. n= 8 mice per group
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Figure 2: SLC/CCL21-treated mice had a significant induction in IFNγ, MIG/CXCL9 and IP-10/CXCL10 at the tumor site compared to diluent treated control tumor bearing mice (p < 0.001). Assessment of cytokine production at the tumor site of SLC/CCL21 treated mice receiving anti-IFNγ, anti MIG/CXCL9 and anti IP-10/CXCL10 showed an interdependence of IFNγ, MIG/CXCL9 and IP-10/CXCL10: neutralization of any one of these cytokines in vivo caused a concomitant decrease in all three cytokines (*p < 0.01 compared to the control antibody treated group). Results are expressed as pg/mg of total protein. Total protein was determined by the Bradford assay. n= 8 mice per group
Mentions: To determine the importance of MIG/CXCL9, IP-10/CXCL10 and IFNγ in the SLC/CCL21 mediated antitumor response, these cytokines were depleted in SLC/CCL21-treated mice. Anti-IP-10/CXCL10, MIG/CXCL9 and IFNγ antibodies each partially but yet significantly inhibited the anti-tumor efficacy of SLC/CCL21 (Figure 1 * p < 0.01 compared to the control antibody group). Neutralization of IFNγ caused a significant decrease in both MIG/CXCL9 and IP-10/CXCL10 indicating that these chemokines are largely IFNγ dependent. Thus, an increase in IFNγ at the tumor site of SLC/CCL21-treated mice could explain the relative increases in IP-10/CXCL10 and MIG/CXCL9. The converse was also observed; IFNγ production at the tumor site was found to be MIG/CXCL9- and IP-10/CXCL10-dependent as indicated by the fact that neutralization of these cytokines caused a significant decrease in IFNγ(Figure 2). Neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines, thus indicating that IFNγ, MIG/CXCL9 and IP-10/CXCL10 are interdependent in the SLC/CCL21-mediated anti-tumor responses. Based on these findings, we speculated that the decrease of IP-10/CXCL10 and MIG/CXCL9 led to a decrease in IFNγ by limiting the influx of T cells producing IFNγ at the tumor site. Because MIG/CXCL9 and IP-10/CXCL10 are chemotactic for stimulated CXCR3 expressing activated T lymphocytes, we determined if neutralizing these chemokines in vivo in the SLC/CCL21-treated mice would decrease the frequency of CXCR3 expressing T cells at the tumor sites. Compared to SLC/CCL21-treated mice receiving control antibody, flow cytometric evaluation of single-cell suspensions of non-necrotic tumors from SLC/CCL21-treated mice receiving neutralizing antibodies to MIG/CXCL9, IP-10/CXCL10 and IFNγ showed 10, 12 and 31 % decreases, respectively, in the gated CD3 CXCR3+ve T cell populations (Table 1). The fact that neutralization of IFNγ was the most efficient at decreasing the total number of CXCR3-activated T cells may be due to the decrease in the IFNγ-dependent CXCR3 ligands MIG/CXCL9 and IP-10/CXCL10. Individually, neutralizing MIG/CXCL9 and/or IP-10/CXCL10 only led to a partial decrease in the total number of CXCR3-activated T cells. Because MIG/CXCL9 and IP-10/CXCL10 share the same receptor (CXCR3), one possible explanation of a partial decrease in CXCR3-activated T cells is that neutralization of one ligand may overexpose the receptor to the other ligand. Furthermore, because murine SLC/CCL21 binds CXCR3 receptor, SLC/CCL21 may be recruiting CXCR3+ve T cells directly. An alternative explanation is that residual cytokines such as IFN-inducible T cell α chemoattractant (ITAC/CXCL11) present after in vivo neutralization of IFNγ, MIG, IP-10 may have remaining activity and attract CXCR3+ve T cells. Our results imply that all 3 cytokines, IFNγ, MIG and IP-10 play interrelated roles in the recruitment of CXCR3-activated T cells in SLC/CCL21-mediated anti-tumor responses. While the number of CD4 and CD8 cells remained unaltered, compared to SLC/CCL21-treated mice receiving control antibody, mice administered neutralizing antibodies to MIG/CXCL9, IP-10/CXCL10 and IFNγ showed respective decreases of 38, 28 and 16% in the number of CD11c+ DEC205+ DC infiltrating the tumor (Table 1). At present it is not clear why there is a decrease in DC following neutralization of MIG/CXCL9, IP-10/CXCL10 and IFNγ. A plausible explanation is that these cytokines may be involved in complex interactions with other molecules that are chemotactic for DC. Further studies are required to delineate the mechanisms responsible for MIG/CXCL9, IP-10/CXCL10 and IFNγ mediated localization of DC at the tumor site.

Bottom Line: We have previously shown that SLC/CCL21-mediated anti-tumor responses are accompanied by significant induction of IFNgamma and the CXC chemokines, monokine induced by IFNgamma (MIG/CXCL9) and IFNgamma-inducible protein-10 (IP-10/CXCL10).In vivo depletion of IP-10/CXCL10, MIG/CXCL9 or IFNgamma significantly reduced the anti-tumor efficacy of SLC/CCL21.These findings indicate that the full potency of SLC/CCL21-mediated anti-tumor responses require in part the induction of IFNgamma, MIG/CXCL9 and IP-10/CXCL10.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Medicine, UCLA Lung Cancer Research Program, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA. sharmasp@ucla.edu

ABSTRACT

Background: SLC/CCL21, normally expressed in high endothelial venules and in T cell zones of spleen and lymph nodes, strongly attracts T cells and dendritic cells (DC). We have previously shown that SLC/CCL21-mediated anti-tumor responses are accompanied by significant induction of IFNgamma and the CXC chemokines, monokine induced by IFNgamma (MIG/CXCL9) and IFNgamma-inducible protein-10 (IP-10/CXCL10).

Results: We assessed the importance of IFNgamma, IP-10/CXCL10 and MIG/CXCL9 in SLC/CCL21 therapy. In vivo depletion of IP-10/CXCL10, MIG/CXCL9 or IFNgamma significantly reduced the anti-tumor efficacy of SLC/CCL21. Assessment of cytokine production at the tumor site showed an interdependence of IFNgamma, MIG/CXCL9 and IP-10/CXCL10; neutralization of any one of these cytokines caused a concomitant decrease in all three cytokines. Similarly, neutralization of any one of these cytokines led to a decrease in the frequency of CXCR3+ve T cells and CD11c+ve DC at the tumor site.

Conclusion: These findings indicate that the full potency of SLC/CCL21-mediated anti-tumor responses require in part the induction of IFNgamma, MIG/CXCL9 and IP-10/CXCL10.

Show MeSH
Related in: MedlinePlus