Aldara is a cream used for topical treatment of non-melanoma skin cancer, and is thought to act through stimulation of anti-tumour immunity. The active ingredient, imiquimod, has been shown to stimulate toll-like receptor 7. Aldara also induces psoriasis-like lesions when applied to naive murine skin, and as such is used as a mouse model for psoriasis. Here we find that in naive murine skin, Aldara induces inflammation largely independently of toll-like receptor 7. Surprisingly, inflammasome activation, keratinocyte death and interleukin 1 release also occur in response to the vehicle cream in the absence of imiquimod. We show that isostearic acid, a major component of the vehicle, promotes inflammasome activation in cultured keratinocytes, and so may contribute to the observed effects of Aldara on murine skin. Aldara therefore stimulates at least two immune pathways independently, and both imiquimod and vehicle are required for a full inflammatory response. Although it remains to be tested, it is possible that imiquimod-independent effects also contribute to the therapeutic efficacy of Aldara.

Highlights d cGAS in cancer and STING in host cells are minimal requirements to activate CD8 + T cells d Cancer cells transfer cGAMP to myeloid cells in the TME that make STING-dependent IFN-I d Cancer-cell-intrinsic cGAS improves tumor immunogenicity and response to therapy

Radiotherapy induces DNA damage and cell death, but recent data suggest that concomitant immune stimulation is an integral part of the therapeutic action of ionizing radiation. It is poorly understood how radiotherapy supports tumor-specific immunity. Here we report that radiotherapy induced tumor cell death and transiently activated complement both in murine and human tumors. The local production of pro-inflammatory anaphylatoxins C3a and C5a was crucial to the tumor response to radiotherapy and concomitant stimulation of tumor-specific immunity. Dexamethasone, a drug frequently given during radiotherapy, limited complement activation and the anti-tumor effects of the immune system. Overall, our findings indicate that anaphylatoxins are key players in radiotherapy-induced tumor-specific immunity and the ensuing clinical responses.

Although malignant cells can be recognized and controlled by the immune system, in patients with clinically apparent cancer immunosurveillance has failed. To better understand local immunoregulatory processes that impact on cancer progression, we correlated intratumoral immunological profiles with the survival of patients affected by primary clear cell renal cell carcinoma (ccRCC). A retrospective analysis of 54 primary ccRCC samples for 31 different immune response-related transcripts, revealed a negative correlation of CD68 (a marker of tumor-associated macrophages, TAMs) and FOXP3 (a marker of regulatory T cells, Tregs) with survival. The subsequent analysis of 12 TAM-related transcripts revealed an association between the genes coding for CD163, interferon regulatory factor 4 (IRF4) and fibronectin 1 (FN1), all of which have been linked to the M2 TAM phenotype, with reduced survival and increased tumor stage, whereas the opposite was the case for the M1-associated gene coding for inducible nitric oxide synthetase (iNOS). The M2 signature of (CD68+) TAMs was found to correlate with CD163 expression, as determined in prospectively collected fresh ccRCC tissue samples. Upon co-culture with autologous tumor cells, CD11b+ cells isolated from paired blood samples expressed CD163 and other M2-associated proteins, suggesting that the malignant cells promote the accumulation of M2 TAMs. Furthermore, the tumor-associated milieu as well as isolated TAMs induced the skewing of autologous, blood-derived CD4+ T cells toward a more immunosuppressive phenotype, as shown by decreased production of effector cytokines, increased production of interleukin-10 (IL-10) and enhanced expression of the co-inhibitory molecules programmed death 1 (PD-1) and T-cell immunoglobulin mucin 3 (TIM-3). Taken together, our data suggest that ccRCC progressively attracts macrophages and induces their skewing into M2 TAMs, in turn subverting tumor-infiltrating T cells such that immunoregulatory functions are increased at the expense of effector functions.

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MHC-class I tetramers technology enabled the characterization of peptide-specific T cells at the single cell level in a variety of studies. Several laboratories have also developed MHC-class II multimers to characterize Ag-specific CD4 1 T cells. However, the generation and use of MHC-class II multimers seems more problematic than that of MHC-I multimers. We have generated HLA-DR*1101 tetramers in a versatile empty form, which can be loaded after purification with peptides of interest. We discuss the impact of critical biological and structural parameters for the optimal staining of Agspecific CD41 T cells using HLA-DR*1101 tetramers, such as: (i) activation state of CD41 T cells; (ii) membrane trafficking in the target CD41 T cells; (iii) binding characteristics of the loaded CD4 epitope. Our data indicate that reorganization of TCR on the plasma membrane upon CD41 T cell activation, as well as an homogenous binding frame of the CD4 epitopes to the soluble HLA-DR monomer, are critical for a stable TCR/MHC-class II tetramer interaction. These factors, together with the low frequencies and affinities of specific CD41 T cells, explain the need for in vitro expansion or ex vivo enrichment of specific T cells for the optimal visualization with MHC-class II tetramers. ' International Society for Advancement of Cytometry Key terms CD4+ T cells; tetramers; HLA-DR1101THE MHC tetramer technology was developed to investigate the dynamic of T cell response at the single cell level. Until the advent of these reagents (1), identification of antigen-specific T cells was only possible using either limiting dilution analysis or by tracking the TCR V repertoire at the molecular level. However, none of these techniques allow the direct phenotypic and functional characterization of the antigenspecific T cells. In this scenario, MHC class I tetramers appeared immediately powerful, enabling the direct visualization of CD8 1 T cells and clarifying relevant aspects of antigen-specific response in viral infections and cancer (2-7) (Chattopadhyay et al., Cytometry, in press [this issue]). By contrast, the use of MHC class II tetramers turned out to be more problematic, both for the possibility to obtain stable peptide-MHC class II complexes and the ability to detect specific CD4 1 T cells directly ex vivo.We will try to highlight the advantages and potentials, as well as the limitations and problems that are still under solution, of MHC class II tetramer technology.

Some breast tumors metastasize aggressively whereas others remain dormant for years. The mechanism governing metastatic dormancy remains largely unknown. Through high-parametric single-cell mapping in mice, we identify a discrete population of CD39+PD-1+CD8+ T cells in primary tumors and in dormant metastasis, which is hardly found in aggressively metastasizing tumors. Using blocking antibodies, we find that dormancy depends on TNFα and IFNγ. Immunotherapy reduces the number of dormant cancer cells in the lungs. Adoptive transfer of purified CD39+PD-1+CD8+ T cells prevents metastatic outgrowth. In human breast cancer, the frequency of CD39+PD-1+CD8+ but not total CD8+ T cells correlates with delayed metastatic relapse after resection (disease-free survival), thus underlining the biological relevance of CD39+PD-1+CD8+ T cells for controlling experimental and human breast cancer. Thus, we suggest that a primary breast tumor could prime a systemic, CD39+PD-1+CD8+ T cell response that favors metastatic dormancy in the lungs.

Combination of immune checkpoint inhibitors with chemotherapy is under investigation for cancer treatment. We studied the rationale of such a combination for treating mesothelioma, a disease with limited treatment options. The combination of gemcitabine and immune checkpoint inhibitors outperformed immunotherapy alone with regard to tumor control and survival in a preclinical mesothelioma model; however, the addition of dexamethasone to gemcitabine and immune checkpoint inhibitors nullified the synergistic clinical response. Furthermore, treatment with gemcitabine plus anti-PD-1 resulted in an objective clinical response in two patients with mesothelioma, who were resistant to gemcitabine or anti-PD-1 as monotherapy. Thus, treatment of mesothelioma with a combination of gemcitabine with immune checkpoint inhibitors is feasible and results in synergistic clinical response compared with single treatment in the absence of steroids.