T cells are critical to fight pathogenic microbes and combat malignantly transformed cells in the fight against cancer. To exert their effector function, T cells produce effector molecules, such as the pro-inflammatory cytokines IFN-γ, TNF-α and IL-2. Tumors possess many inhibitory mechanisms that dampen T cell effector function, limiting the secretion of cytotoxic molecules. As a result, the control and elimination of tumors is impaired. Through recent advances in genomic editing, T cells can now be successfully modified via CRISPR/Cas9 technology. For instance, engaging (post-)transcriptional mechanisms to enhance T cell cytokine production, the retargeting of T cell antigen specificity or rendering T cells refractive to inhibitory receptor signaling can augment T cell effector function. Therefore, CRISPR/Cas9-mediated genome editing might provide novel strategies for cancer immunotherapy. Recently, the first-in-patient clinical trial was successfully performed with CRISPR/Cas9-modified human T cell therapy. In this review, a brief overview of currently available techniques is provided, and recent advances in T cell genomic engineering for the enhancement of T cell effector function for therapeutic purposes are discussed.

The genetic alterations associated with cell transformation are in large measure expressed in the metabolic phenotype as cancer cells proliferate and change their local environment, and prepare for metastasis. Qualitatively, the fundamental biochemistry of cancer cells is generally the same as in the untransformed cells, but the cancer cells produce a local environment, the TME, that is hostile to the stromal cells, and compete for nutrients. In order to proliferate, cells need sufficient nutrients, either those that cannot be made by the cells themselves, or must be made from simpler precursors. However, in solid tumors, the nutrient supply is often limiting given the potential for rapid proliferation, and the poor quality of the vasculature. Thus, cancer cells may employ a variety of strategies to obtain nutrients for survival, growth and metastasis. Although much has been learned using established cell lines in standard culture conditions, it is becoming clear from in vivo metabolic studies that this can also be misleading, and which nutrients are used for energy production versus building blocks for synthesis of macromolecules can vary greatly from tumor to tumor, and even within the same tumor. Here we review the operation of metabolic networks, and how recent understanding of nutrient supply in the TME and utilization are being revealed using stable isotope tracers in vivo as well as in vitro.

In human tumors of distinct origin including renal cell carcinoma (RCC), the non-classical human leukocyte antigen G (HLA-G) is frequently expressed, thereby inhibiting the cytotoxic activity of T and natural killer (NK) cells. Recent studies demonstrated a strong post-transcriptional gene regulation of the HLA-G by miR-152, -148A, -148B and -133A. Standard methods were applied to characterize the expression and function of HLA-G, HLA-G-regulatory microRNAs (miRs) and the immune cell infiltration in 453 RCC lesions using a tissue microarray and five RCC cell lines linking these results to clinical parameters. Direct interactions with HLA-G regulatory miRs and the HLA-G 3\' untranslated region (UTR) were detected and the affinities of these different miRs to the HLA-G 3\'-UTR compared. qPCR analyses and immunohistochemical staining revealed an inverse expression of miR-148A and -133A with the HLA-G protein in situ and in vitro. Stable miR overexpression caused a downregulation of HLA-G protein enhancing the NK and LAK cell-mediated cytotoxicity in in vitro CD107a activation assays revealing a HLA-G-dependent cytotoxic activity of immune effector cells. A significant higher frequency of CD3+/CD8+ T cell lymphocytes, but no differences in the activation markers CD69, CD25 or in the presence of CD56+, FoxP3+ and CD4+ immune cells were detected in HLA-G+ compared to HLA-G- RCC lesions. This could be associated with higher WHO grade, but not with a disease-specific survival. These data suggest a miR-mediated control of HLA-G expression in RCC, which is associated with a distinct pattern of immune cell infiltration.

Toll-like receptor 9 (TLR9) is a cellular DNA-receptor of the innate immune system that is widely expressed in cancers. We demonstrated that low tumor TLR9 expression predicts poor disease-specific survival in triple negative breast cancer (TNBC) and renal cell carcinoma (RCC). We hypothesized that this is because TLR9 expression affects tumor immunophenotype. To begin to test this, we compared the number of tumor infiltrating CD8+ T lymphocytes with TLR9 expression in treatment naïve breast cancer (n = 197) and RCC (n = 94) cohorts with known TLR9 expression status. CD8+ T lymphocyte counts were assayed with image analysis after immunohistochemistry (IHC). Tumor TLR9 expression was not correlated with CD8+ T cell counts in breast cancer or RCC. CD8+ T cell counts were significantly associated with tumor proliferation index in TNBC, but not in non-TNBC. CD8+ T cell counts were also significantly associated with tumor grade in non-TNBC, but not in TNBC. In RCC, CD8+ T cell counts were significantly associated with tumor stage. CD8+ T cell counts were significantly associated with prognosis in TNBC and RCC, but the presence of CD8+ T cells in these tumors had opposite effects on disease-specific survival: High CD8+ counts were associated with better prognosis in TNBC and worse prognosis in RCC. Among TNBC patients, those with low tumor TLR9 and low CD8+ T cell counts had the poorest prognosis (log-rank p = 0.0002 vs. high tumor TLR9 and high CD8+ T cell count). In conclusion, pre-treatment tumor TLR9 status is not associated with tumor infiltrating CD8+ T lymphocytes in TNBC or RCC. The combination of TLR9 and CD8+ TIL count might be a novel composite prognostic marker in TNBC.

Osteosarcoma is the most common primary bone tumor characterized by juvenile onset, tumor heterogeneity, and early pulmonary metastasis. Therapeutic improvement stagnates since more than two decades. Unlike major malignancies, biomarkers as prognostic factors at time of diagnosis are missing. Disease rareness hampers study recruitment of patient numbers sufficient to outweigh tumor heterogeneity. Here, we analyzed in a multicenter cohort the osteosarcoma microenvironment to reduce effects of tumor cell heterogeneity. We hypothesized that quantitative ratios of intratumoral CD8+T-cells to FOXP3+T-cells (CD8+/FOXP3+-ratios) provide strong prognostic information when analyzed by whole-slide imaging in diagnostic biopsies. We followed recommendations-for-tumor-marker-prognostic-studies (REMARK). From 150 included cases, patients with complete treatment were identified and assigned to the discovery (diagnosis before 2004) or the validation cohort (diagnosis 2004-2012). Highly standardized immunohistochemistry of CD8+ and FOXP3+, which was validated by methylation-specific gene analysis, was performed followed by whole-slide analysis and clinical outcome correlations. We observed improved estimated survival in patients with CD8+/FOXP3+-ratios above the median (3.08) compared to patients with lower CD8+/FOXP3+-ratios (p = 0.000001). No patients with a CD8+/FOXP3+-ratio above the third quartile died within the observation period (median follow-up 69 mo). Multivariate analysis demonstrated independence from current prognostic factors including metastasis and response to neoadjuvant chemotherapy. Data from an independent validation cohort confirmed improved survival (p = 0.001) in patients with CD8+/FOXP3+-ratios above 3.08. Multivariate analysis proofed that this observation was also independent from prognostic factors at diagnosis within the validation cohort. Intratumoral CD8+/FOXP3+-ratio in pretreatment biopsies separates patients with prolonged survival from non-survivors in osteosarcoma.

The accumulation of tumor infiltrating lymphocytes (TILs) in ovarian cancer is prognostic for increased survival while increases in immunosuppressive regulatory T-cells (Tregs) are associated with poor outcomes. Approaches that bolster tumor-reactive TILs may limit tumor progression. However, identifying tumor-reactive TILs in ovarian cancer has been challenging, though adoptive TIL therapy in patients has been encouraging. Other forms of TIL immunomodulation remain under investigation including Treg depletion, antibody-based checkpoint modification, activation and amplification using dendritic cells, antigen presenting cells or IL-2 cytokine culture, adjuvant cytokine injections, and gene-engineered T-cells. Many approaches to TIL manipulation inhibit ovarian cancer progression in preclinical or clinical studies as monotherapy. Here, we review the impact of TILs in ovarian cancer and attempts to mobilize TILs to halt tumor progression. We conclude that effective TIL therapy for ovarian cancer is at the brink of translation and optimal TIL activity may require combined methodologies to deliver clinically-relevant treatment.

Melanoma is the most serious form of skin cancer. Metastatic melanoma historically carries a poor prognosis and until recently there have been few effective agents available to treat widely disseminated disease. Recognition of the immunogenic nature of melanoma has resulted in the development of various immunotherapeutic approaches, especially with regards to the programmed cell death 1 (PD-1) receptor and its ligand (PD-L1). Antibodies targeting the PD-1 axis have shown enormous potential in the treatment of metastatic melanoma. Here, we will review the immune basis for the disease and discuss approved immunotherapeutic options for advanced melanoma, as well as the current state of development of PD-1 and PD-L1 antibodies and their importance in shaping the future of melanoma treatment.

T cell adoptive therapies for immune-mediated regression of cancers have attracted a great deal of recent attention. Clinical results are glamorous, yet much remains to be uncovered behind the basic science that allows us to engineer T cells and T cell receptors (TCRs) for clinical use. We discuss the development of TCRs for therapeutic use in the context of thymic selection toward central tolerance and we review therapies based on tumor infiltrating lymphocytes (TILs), endogenous antigen specific TCRs, and engineered TCRs. Further we discuss the development of low and high affinity TCRs and the extent to which each challenges central tolerance. Current results suggest that adaptation of TCR engineering of moderate affinity TCRs coupled with co-regulatory and stimulatory molecules may be the safest and most efficacious road for TCR development aimed at tumor abolition.