Spontaneous Tumors Advanced her-2/neu Transgenic Mice Bearing T Effectors in + Tumor-Specific CD8 Suppressor Activity and a Concurrent Loss of Progressive Increase in Intratumoral T Chronic Immune Therapy Induces a

A single intratumoral injection of IL-12 and GM-CSF-encapsulated microspheres induces the complete regression of advanced spontaneous tumors in her-2/neu transgenic mice. However, tumor regression in this model is transient and long-term cure is not achieved due to recurrence. Posttherapy molecular analysis of immune activation/suppression markers within the tumor microenvironment demonstrated a dramatic up-regulation of IFN- (cid:2) and a concomitant down-regulation of Forkhead/winged-helix protein 3 (Foxp3), TGF (cid:3) , and IL-10 expression. Therapy-induced reversion of immune suppression was transient since all three markers of suppression recovered rapidly and surpassed pretherapy levels by day 7 after treatment, resulting in tumor resurgence. Repeated treatment enhanced short-term tumor regression, but did not augment long-term survival. Serial long-term analysis demonstrated that although chronic stimulation enhanced the IFN- (cid:2) response, this was countered by a parallel increase in Foxp3, TGF (cid:3) , and IL-10 expression. Analysis of tumor-inﬁltrating T lymphocyte populations showed that the expression of Foxp3 and IL-10 was associated with CD4 (cid:1) CD25 (cid:1) T cells. Repeated treatment resulted in a progressive increase in tumor-inﬁltrating CD4 (cid:1) CD25 (cid:1) Foxp3 (cid:1) T suppressor cells establishing their role in long-term neutralization of antitumor activity. Analysis of tumor-inﬁltrating CD8 (cid:1) T cells demonstrated that although treatment enhanced IFN- (cid:2) production, antitumor cytotoxicity was diminished. Monitoring of CD8 (cid:1) T cells that speciﬁcally recognized a dominant MHC class I her-2/neu peptide showed a dramatic increase in tetramer-speciﬁc CD8 (cid:1) T cells after the ﬁrst treatment; however, continuous therapy resulted in the loss of this population. These results demonstrate that both enhanced suppressor activity and deletion of tumor-speciﬁc T cells are responsible for the progressive loss of efﬁcacy that is associated with chronic immune therapy. The Journal of Immunology, 2006, 176: 7325–7334. augmentation T suppressor cell activity in the tumor microenvironment. To this end, quantitative monitoring of T effector cell activity and the ac-companying T suppressor cell responses in tumors has not been performed during chronic therapy. To determine whether repeated treatment of advanced spontaneous mammary tumors in her-2/neu transgenic mice resulted in such a response-counterresponse pro-cess, we monitored the quantitative and qualitative changes in immune activation and suppression markers in posttherapy tumors. The results establish that intratumoral delivery of IL-12 and GM-CSF promotes a dramatic reversal of immune suppression in the tumor microenvironment but that this reversion is transient because intratumoral T suppressor cell activity recovers rapidly. Im-portantly, our data demonstrate that whereas repeated treatment resurrects antitumor immune activity, reactivation is countered by a concurrent and progressive increase in the intensity of T suppressor cell inﬁltration into tumors, resulting in reduced T effector cytotoxicity and eventual deletion of tumor-speciﬁc CTL.