Oxidative stress, caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses, is one of the characteristic features of cancer. Here, we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo, when compared with blood or normal subcutaneous fluid. Therefore, we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells, and by comparing T, B, and NK cells' sensitivities to redox stress and their antioxidant capacities, we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However, the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore, we genetically modified NK cells to stably overexpress PRDX1, which led to increased survival and NK cell activity in redox stress conditions. Finally, we generated PD-L1–CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide, at concentrations detected in the TME, suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors.

Immunotherapy revolutionized cancer treatment in the last decade. Both NK cells and T cells are key components of host immunity against malignant cells that are being extensively investigated in the field of cancer immunotherapy. While approaches have been developed to improve the antitumor activity of NK and T cells, the tumor microenvironment remains an obstacle to effective NK and T-cell–based therapies. Here, we demonstrated that cancer-conditioned medium suppresses the antitumor activity of NK cells. Ammonia, a by-product of cancer cell metabolism, accumulated in cancer-conditioned medium and in the tumor microenvironment, and impaired the cytotoxicity of NK cells as well as the efficacy of antibody-based and chimeric antigen receptor (CAR) NK and CAR T-cell–based therapies in vitro. Ammonia induced NK and T-cell dysfunction by decreasing the amount of mature perforin in secretory lysosomes, which was dependent on its lysosomotropic features and ability to increase pH in acidic compartments. These findings demonstrate that, in addition to its previously described role in promoting tumor growth as a source of nitrogen for tumor biomass, ammonia promotes tumor immune escape by inhibiting both NK and CAR T-cell cytotoxicity. Significance: Ammonia is elevated in the tumor microenvironment and functions as an immunoinhibitory metabolite in cancer by reducing perforin levels, inhibiting NK and T-cell–mediated immunity and limiting the efficacy of immunotherapies.

Chimeric antigen receptor (CAR)-T cell therapy is undeniably a promising tool in combating various types of hematological malignancies. However, it is not yet optimal and a significant number of patients experience a lack of response or relapse after the treatment. Therapy improvement requires careful analysis of the occurring problems and a deeper understanding of the reasons that stand behind them. In this review, we summarize the recent knowledge about CAR-T products’ clinical performance and discuss diversified approaches taken to improve the major shortcomings of this therapy. Especially, we prioritize the challenges faced by CD19 CAR-T cell-based treatment of B cell-derived malignancies and revise the latest insights about mechanisms mediating therapy resistance. Since the loss of CD19 is one of the major obstacles to the success of CAR-T cell therapy, we present antigens that could be alternatively used for the treatment of various types of B cell-derived cancers.

Supplementary Fig. 8 shows effects of ammonia on perforin gene expression at the transcriptional level

Supplementary Fig. 4 shows that ammonia accumulates in NK cells, affects their cytotoxic potential but not their viability.

Ammonia concentration is increased in cancer cell–conditioned medium and TIF. Ammonia concentration in the tumor-conditioned medium, collected after 48 hours of incubation, was measured using a Dimension Ammonia assay (Siemens; n = 3). Cells were cultured at different densities: lymphoma (0.5 × 106, 1.0 × 106, 1.5 × 106/mL; A), multiple myeloma (1 × 106, 2 × 106, 4 × 106/mL; B), and breast cancer cell lines (0.5 × 106, 1 × 106, 1.5 × 106/mL; C). In A–C, empty medium incubated for 48 hours (without the cells) at 4°C or 37°C is presented as a control. D, Schematic presentation of the TIF and SCF isolation from mice. TIF was collected from tumors not exceeding 1,500 mm3. SCF was isolated at the same time from the contralateral tight as a control tissue fluid. E–G, The concentration of ammonia in TIF and SCF isolated from Raji tumor–bearing NSG (n = 5; E), MM.1s tumor–bearing SCID mice (n = 5; F), and breast cancer–bearing mice (EMT6 in BALB/c mice, n = 5; E0771 in BALB/c mice, n = 5; 4T1 in BALB/c mice; n = 5; MCF7 in NSG mice, n = 4; MDA-MB-231 in NSG mice, n = 3; G). P values were calculated using a paired t test. Data show individual values and means ± SEM. n values are the number of biological replicates in in vitro experiments or the number of mice used to obtain the data. D, Created with BioRender.com. Winiarska, M. (2025) https://BioRender.com/x56a982.