BACKGROND:
Evidence is accumulating to indicate the potential roles of CD4+ cytotoxic lymphocytes (CTLs), a distinct subset from other CD4+ helper T cell subsets, in tumor immunity because they can degranulate massively cytolytic proteins such as glanzyme B and perforin upon activation. However, it remains elusive on their roles in chemotherapeutic-induced tumor eradication process. Hence, we delineated cyclophosphamide (CTX)-induced tumor eradication process in mice with a focus on CD4+ CTLs.
METHODS and RESULTS:
We inoculated a murine hepatoma cell line, BNL 1ME A.7R.1 (BNL), subcutaneously into naïve Balb/c mice and gave a single intraperitoneal injection of 150 mg/kg CTX after a tumor formed. CTX injection eradicated tumors in wild-type mice (6 / 9 heads), whereas none of the tumors disappeared in nude mice (p<0.05). Moreover, WT mice whose tumor disappeared after CTX treatment rejected re-challenged BNL, but not an unrelated murine colorectal cancer cell line, Col26. Furthermore, CD4+ but not CD8+ cell depletion abrogated CTX-induced tumor eradiation. Consistently, CTX treatment increased intratumoral CD4+ CTLs, which expressed a cytolytic granule molecule, CD107a, and granzyme B. When congenic CD45.1 splenocytes were transferred to tumor-bearing CD45.2 mice after CTX treatment, transferred CD4+ cells were recruited into tumors on day 3. Moreover, the recruited cells expressed CD107a without antigen presentation at draining lymph nodes and proliferation in tumor tissues. CTX administration enhanced mRNA expression of a CC chemokine, CCL3, in tumor tissues. CTX-mediated tumor regression was attenuated in mice deficient in CCR5, the receptor for CCL3. Consistently, CTX-induced accumulation of intratumoral CD107a-expressing CD4+ T cells was less in mice receiving CCR5-deficient mouse-derived splenocytes than those receiving WT mouse-derived splenocytes.
CONCLUSION:
CTX induces intratumoral expression of CCL3, which recruits CD4+ CTLs into tumor sites, thereby eradicating tumors and subsequently inducing specific tumor immunity.