Tumor-associated eosinophilia is frequently observed in cancer. However, despite numerous studies of patients with cancer and mouse models of cancer, it has remained uncertain if eosinophils contribute to tumor immunity or are mere bystander cells. Here we report that activated eosinophils were essential for tumor rejection in the presence of tumor-specific CD8(+) T cells. Tumor-homing eosinophils secreted chemoattractants that guided T cells into the tumor, which resulted in tumor eradication and survival. Activated eosinophils initiated substantial changes in the tumor microenvironment, including macrophage polarization and normalization of the tumor vasculature, which are known to promote tumor rejection. Thus, our study presents a new concept for eosinophils in cancer that may lead to novel therapeutic strategies.

Elevated numbers of regulatory T cells (Treg) in patient tumors are known to inhibit efficient antitumor T-cell responses. To study the mechanisms controlling tumor rejection, we assessed different mouse models for Treg depletion. In Foxp3DTR knock-in mice, about 99% Treg depletion was achieved, resulting in complete rejection of transplanted HCmel12 melanomas in a CD8 T-cell-dependent way. In contrast, about 90% Treg depletion obtained in BAC transgenic Foxp3.LuciDTR4 mice failed to induce complete rejection of HCmel12 melanomas, demonstrating that residual Tregs were able to control CD8 T-cell responses against the tumor. Ninety-nine percent of Treg depletion provoked drastic changes in the tumor microenvironment, such as strong infiltration of CD8 T cells and basophils. Intratumoral basophils enhanced CD8 T-cell infiltration via production of chemokines CCL3 and CCL4; antibody-based blocking of these chemokines inhibited CD8 T-cell infiltration. Therapeutic induction of basophilia by IL3/anti-IL3 antibody complexes, combined with transfer of CD8 T cells, resulted in enhanced T-cell infiltration and tumor rejection. Our study identifies a critical role basophils play in tumor rejection and that this role can be exploited for therapeutic intervention. Cancer Res; 77(2); 291-302. ©2016 AACR.

Effector T cells equipped with engineered antigen receptors specific for cancer targets have proven to be very efficient. Two methods have emerged: the Chimeric Antigen Receptors (CARs) and T-cell Receptor (TCR) redirection. Although very potent, CAR recognition is limited to membrane antigens which represent around 1% of the total proteins expressed, whereas TCRs have the advantage of targeting any peptide resulting from cellular protein degradation. However, TCRs depend on heavy signalling machinery only present in T cells which restricts the type of eligible therapeutic cells. Hence, an introduced therapeutic TCR will compete with the endogenous TCR for the signalling proteins and carries the potential risk of mixed dimer formation giving rise to a new TCR with unpredictable specificity. We have fused a soluble TCR construct to a CAR-signalling tail and named the final product TCR-CAR. We here show that, if expressed, the TCR-CAR conserved the specificity and the functionality of the original TCR. In addition, we demonstrate that TCR-CAR redirection was not restricted to T cells. Indeed, after transduction, the NK cell line NK-92 became TCR positive and reacted against pMHC target. This opens therapeutic avenues combing the killing efficiency of NK cells with the diversified target recognition of TCRs.

In tumor biology, nitric oxide (NO) is generally regarded as an immunosuppressive molecule that impedes T-cell functions and activation of endothelial cells. Contrasting with this view, we here describe a critical role for NO derived from inducible nitric oxide (iNOS)-expressing tumor macrophages in T-cell infiltration and tumor rejection as shown by iNOS gene deletion, inhibition of iNOS, or NO donors. Specifically, macrophage-derived NO was found to induce on tumor vessels adhesion molecules that were required for T-cell extravasation. Experiments with human endothelial cells revealed a bimodal dose-dependent effect of NO. High doses of NO donors were indeed suppressive but lower, more physiological concentrations, induced adhesion molecules in an NFkB-dependent pathway and preferentially activated transcription of genes involved in lymphocyte diapedesis. iNOS C macrophages in tumors appear to generate precisely the amount of NO that promotes endothelial activation and T-cell infiltration. These results will be valuable for the development of strategies designed to overcome the paucity of T-cell infiltration into tumors that is a major obstacle in clinical cancer immunotherapy.

In the version of this article initially published online, in the third and fourth paragraphs of the Discussion section, the mice are incorrectly identified as Rorc gfb/+ . The correct genotype is Rorc gfp/+ . The error has been corrected for the print, PDF and HTML versions of this article.

Efficient regulatory T cell (Treg) depletion leads to tumor rejection in mice. The goal of this study was to understand the mechanisms involved in this rejection process. For the selective and efficient depletion of Tregs, we used the FoxP3.LuciDTR-4 mouse model where Tregs express, among others, the human diphtheria toxin receptor. Our previous studies show that the depletion of more than 90% of Tregs in FoxP3.LuciDTR-4 mice promotes the infiltration of immune cells and subsequent rejection of ovalbumin (OVA)-expressing B16 melanomas. To unravel the mechanisms involved, we characterized the tumor-infiltrating immune populations and performed a cytokine profile on tumors at the onset of rejection. The analysis revealed a change in the population composition of the tumor-infiltrating leucocytes. There was an increase in T cell numbers, especially CD8+ T cells, and dendritic cells in tumors in rejection. Moreover, the amount of B cells was drastically reduced in tumors being rejected while they were enriched in tumors in progression. Tumor-infiltrating myeloid cells also differed phenotypically between tumors in rejection and tumors in progression. Inflammatory cytokines related to the type 1 response such as IFN-γ, TNF and IL-12 were significantly more expressed in tumors in rejection as measured at mRNA and protein levels. On the other hand, the expression of some cytokines related to the type 2 response such as IL-5 and IL-10 and of the inhibitory cytokine TGF-β did not change between tumors in rejection and in progression. We also determined that the concentration of molecules related to the M1 macrophage phenotype such as inducible nitric oxide synthase (iNOS), interferon regulatory factor 5 (IRF5), CXCL9 and CXCL10 was significantly higher in tumors in rejection; however, the concentration of most of the molecules related to the M2 macrophage phenotype such as CD163, macrophage mannose receptor 1 (MRC1), Fizz-1 and PPAR-γ was not different between tumors in rejection and tumors in progression. Interestingly, angiogenesis drivers like VEGF and angiotensin 1 (ANG1) were found to be significantly less expressed in tumors in rejection, a fact that correlates with the normalization of the tumor vasculature previously described to be promoted by Treg depletion. We conclude that Treg depletion provokes a strong change in the tumor microenvironment, not only in terms of the elevated number of immune infiltrating cells but also in terms of the enrichment of certain subpopulations. Furthermore, this change is associated with a striking alteration of the cytokine and chemokine profile that becomes proinflammatory, supports mainly a Type 1-related response and inhibits angiogenesis pathways. Citation Format: Oscar C. Salgado, Rafael Carretero, Xingrui Li, Ibrahim M. Sektioglu, Natalio Garbi, Günter J. Hämmerling. Tumor rejection driven by efficient Treg depletion is associated with a strong change in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B79.

<div>Abstract<p>Elevated numbers of regulatory T cells (Treg) in patient tumors are known to inhibit efficient antitumor T-cell responses. To study the mechanisms controlling tumor rejection, we assessed different mouse models for Treg depletion. In Foxp3DTR knock-in mice, about 99% Treg depletion was achieved, resulting in complete rejection of transplanted HCmel12 melanomas in a CD8⁺ T-cell–dependent way. In contrast, about 90% Treg depletion obtained in BAC transgenic Foxp3.LuciDTR4 mice failed to induce complete rejection of HCmel12 melanomas, demonstrating that residual Tregs were able to control CD8⁺ T-cell responses against the tumor. Ninety-nine percent of Treg depletion provoked drastic changes in the tumor microenvironment, such as strong infiltration of CD8⁺ T cells and basophils. Intratumoral basophils enhanced CD8⁺ T-cell infiltration via production of chemokines CCL3 and CCL4; antibody-based blocking of these chemokines inhibited CD8⁺ T-cell infiltration. Therapeutic induction of basophilia by IL3/anti-IL3 antibody complexes, combined with transfer of CD8⁺ T cells, resulted in enhanced T-cell infiltration and tumor rejection. Our study identifies a critical role basophils play in tumor rejection and that this role can be exploited for therapeutic intervention. <i>Cancer Res; 77(2); 291–302. ©2016 AACR</i>.</p></div>

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