A comparison of B16-F10-derived tumors in WT and Ccl21a-KO mice revealed that the Ccl21a-KO mice had significantly reduced tumor weight (Fig. 1A). In contrast, there was no significant difference in LLC-derived tumor weight in WT and Ccl21a-KO mice (Supplementary Fig. 1), suggesting thatCCL21-Ser selectively affected the tumorigenicity of the melanoma cell line B16-F10.In vivo bioluminescence imaging of melanoma proliferation showed that B16-F10 cells proliferated in WT mice until day 11 (Fig. 1B), whereas the luminescence in Ccl21a-KO mice was already lower than that in WT by day 4, suggesting that tumor growth was suppressed relatively at early phase in the Ccl21a-KO mice.The expression of Ccl21a and CCR7 are under detectable levels in B16F10 cells (Supplementary Fig. 2), suggesting the contribution of host-derived CCL21-Ser and CCR7-positive cells to B16F10 tumor growth.
To decipher the mechanisms underlying decreased tumorigenicity in Ccl21a-KO mice, we hypothesized that a stronger antitumor immunity was induced in the KO mice. A flow cytometric analysis of CD3, CD4, and CD8 T cell populations within the intratumoral cells (Fig.2A) revealed that the proportion of tumor infiltrating T cells, especially CD8 T cells, was significantly increased in the KO mice when compared with WT (Figs. 2B and 2C). In addition, a significant increase in activated T cells and decrease in naïve T cells was observed in theKO mice (Fig. 2D). To investigate whether depletion of CD8 T cells in vivo affected melanoma growth, we administered anti-CD8 mAb or isotype control immunoglobulin into Ccl21a-KO mice. We observed that a low proportion of CD8 T cells in the tumor showed a higher tumor weight (Supplementary Fig. 3), indicating that the decrease in melanoma growth is CD8 T cell-dependent. These results suggest that antitumor immunity by CD8 T cells is more efficiently induced in the absence of CCL21-Ser.
Ccl21a-KO mice develop autoimmune disease-like symptoms due to an incomplete negative selection of autoreactive T cells during lymphocyte differentiation in the thymus [4]. We hypothesized that the KO mice have an elevated tumor-specific immune response. We analyzed the number and proportion of T cell subsets in regional LNs associated with melanoma growth (Fig. 3A). In WT mice, we observed no significant difference in CD4 or CD8 T cell counts before and after tumorigenesis (Fig. 3A). In contrast, the T cell count after tumor formation significantly increased in Ccl21a-KO LNs. Although the ratio of CD4 to CD8 was not significantly different between the WT and the KO (Fig. 3B), a decrease in naïve T cells and an increase in effector memory T cells was observed in the LNs of KO mice (Fig. 3C). These results suggest that a greater number of antigen-specific effector T cells are generated in the LNs of mice deficient in CCL21-Ser.
Regulatory T cells (Tregs) control the differentiation of naïve CD8 T cells into memory T cells and suppress melanoma proliferation [17, 18]. The number of CD4+CD25+CD127lo Tregs was significantly lower in KO LNs without tumor, but increased in both KO and WT LNs as the tumor grew (Figs.4A and 4B). The increase was more prominent in the KO LNs (about 6-fold) compared with the WT LNs (about 1.7-fold). Treg frequency was also higher in KO tumor tissues, although the proportion of CD4 T cells was comparable between WT and KO (Fig.4C), suggesting that Treg proliferation was more efficiently induced with tumor formation in KO mice. On the other hand, the ratio of Tregs to activated CD8 T cells in tumor tissues was well correlated with tumor weight in WT, but not in KO mice (Fig.4D). These findings suggest that although Tregs are efficiently recruited to the tumor tissues, their immune suppressive function is impaired in KO mice.CCL21-Ser-induced signaling is critical for Treg activation in vivo, and Ccr7-KO mice showed increased infiltration of inflamed tissues with inactive Tregs [19]. Since the predominant Treg subset in human and mouse tumors is the thymus-derived nTreg [20], a lack of CCL21-Ser expression in the thymus of Ccl21a-KO mice may have caused intrinsic dysfunction of nTregs and accelerated CD8-mediated tumor rejection. Alternatively, considering that Tregs in tumor tissues are functionally activated in peritumoral LN-like structures with CCL21-Ser expression [15], the tumor-infiltrated Tregs may not be properly activated in the tumor microenvironment lacking CCL21-Ser. Further study on the expression of Treg activation markers is required to determine the functional competence of Tregs in Ccl21a-KO mice.