Heterozygous OT-I mice reveal that antigen-specific CD8+ T cells shift from apoptotic to necrotic killers in the elderly

Numerous alterations in CD8+ T cells contribute to impaired immune responses in elderly individuals. However, the discrimination between cell-intrinsic dysfunctions and microenvironmental changes is challenging. TCR transgenic OT-I mice are utilized to investigate CD8+ T-cell immunity, but their immunodeficient phenotype hampers their use especially in aging. Here, we demonstrate that using a heterozygous OT-I model minimizes the current limitations and provides a valuable tool to assess antigen-specific T-cell responses even at old age. We analyzed phenotypic and functional characteristics of CD8+ T cells from OT-I +/+ and OT-I +/− mice to prove the applicability of the heterozygous system. Our data reveal that OVA-activated CD8+ T cells from adult OT-I +/− mice proliferate, differentiate, and exert cytolytic activity equally to their homozygous counterparts. Moreover, common age-related alterations in CD8+ T cells, including naive T-cell deterioration and decreased proliferative capacity, also occur in elderly OT-I +/− mice, indicating the wide range of applications for in vivo and in vitro aging studies. We used the OT-I +/− model to investigate cell-intrinsic alterations affecting the cytotoxic behavior of aged CD8+ T cells after antigen-specific in vitro activation. Time-resolved analysis of antigen-directed target cell lysis confirmed previous observations that the cytotoxic capacity of CD8+ T cells increases with age. Surprisingly, detailed single cell analysis revealed that transcriptional upregulation of perforin in aged CD8+ T cells shifts the mode of target cell death from granzymemediated apoptosis to rapid induction of necrosis. This unexpected capability might be beneficial or detrimental for the aging host and requires detailed evaluation

Faster cytotoxicity with age : Increased perforin and granzyme levels in cytotoxic CD8+ T cells boost cancer cell elimination

A variety of intrinsic and extrinsic factors contribute to the altered efficiency of CTLs in elderly organisms. In particular, the efficacy of antiviral CD8+ T cells responses in the elderly has come back into focus since the COVID-19 pandemic outbreak. However, the exact molecular mechanisms leading to alterations in T cell function and the origin of the observed impairments have not been fully explored. Therefore, we investigated whether intrinsic changes affect the cytotoxic ability of CD8+ T cells in aging. We focused on the different subpopulations and time-resolved quantification of cytotoxicity during tumor cell elimination. We report a surprising result: Killing kinetics of CD8+ T cells from elderly mice are much faster than those of CD8+ T cells from adult mice. This is true not only in the total CD8+ T cell population but also for their effector (TEM) and central memory (TCM) T cell subpopulations. TIRF experiments reveal that CD8+ T cells from elderly mice possess comparable numbers of fusion events per cell, but significantly increased numbers of cells with granule fusion. Analysis of the cytotoxic granule (CG) content shows significantly increased perforin and granzyme levels and turns CD8+ T cells of elderly mice into very efficient killers. This highlights the importance of distinguishing between cell-intrinsic alterations and microenvironmental changes in elderly individuals. Our results also stress the importance of analyzing the dynamics of CTL cytotoxicity against cancer cells because, with a simple endpoint lysis analysis, cytotoxic differences could have easily been overlooked

Interdependence of sequential cytotoxic T lymphocyte and natural killer cell cytotoxicity against melanoma cells

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24 h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL–NK cell anti-melanoma therapies

Model-based identification of TNF alpha-induced IKK beta-mediated and I kappa B alpha-mediated regulation of NF kappa B signal transduction as a tool to quantify the impact of drug-induced liver injury compounds

Drug-induced liver injury: mathematical model quantifies impact of liver-damaging drugs Drug-induced liver injury (DILI) is one of the most important obstacles during drug development. More than 1000 drugs have been identified to damage the liver, but the current test systems are poor in predicting DILI. A team of cell biologists, theoretical physicists, and clinical pharmacologists combined experimental data generated in cultured liver cells with mathematical modeling to quantify the impact of the anti-inflammatory drug diclofenac. The analysis demonstrated that diclofenac induces multiple changes in the signal transduction network activated by the tumor necrosis factor alpha (TNFα), one of the known factors to amplify liver toxicity. Data of other liver injury-causing compounds were integrated into the mathematical model and their impact was quantified, thereby demonstrating the potential use of the mathematical model for the further analysis of other compounds in order to improve DILI test systems

The B-cell Receptor Autoantigen LRPAP1 Can Replace Variable Antibody Regions to Target Mantle Cell Lymphoma Cells

Mantle cell lymphoma (MCL) accounts for 5%–10% of all lymphomas. The disease’s genetic hallmark is the t(11; 14)(q13; q32) translocation. In younger patients, the first-line treatment is chemoimmunotherapy followed by autologous stem cell transplantation. Upon disease progression, novel and targeted agents such as the BTK inhibitor ibrutinib, the BCL-2 inhibitor venetoclax, or the combination of both are increasingly used, but even after allogeneic stem cell transplantation or CAR T-cell therapy, MCL remains incurable for most patients. Chronic antigenic stimulation of the B-cell receptor (BCR) is thought to be essential for the pathogenesis of many B-cell lymphomas. LRPAP1 has been identified as the autoantigenic BCR target in about 1/3 of all MCLs. Thus, LRPAP1 could be used to target MCL cells, however, there is currently no optimal therapeutic format to integrate LRPAP1. We have therefore integrated LRPAP1 into a concept termed BAR, for B-cell receptor antigens for reverse targeting. A bispecific BAR body was synthesized consisting of the lymphoma-BCR binding epitope of LRPAP1 and a single chain fragment targeting CD3 or CD16 to recruit/engage T or NK cells. In addition, a BAR body consisting of an IgG1 antibody and the lymphoma-BCR binding epitope of LRPAP1 replacing the variable regions was synthesized. Both BAR bodies mediated highly specific cytotoxic effects against MCL cells in a dose-dependent manner at 1–20 µg/mL. In conclusion, LRPAP1 can substitute variable antibody regions in different formats to function in a new therapeutic approach to treat MCL

Model-based identification of TNFα-induced IKKβ-mediated and IκBα-mediated regulation of NFκB signal transduction as a tool to quantify the impact of drug-induced liver injury compounds

Drug-induced liver injury (DILI) has become a major problem for patients and for clinicians, academics and the pharmaceutical industry. To date, existing hepatotoxicity test systems are only poorly predictive and the underlying mechanisms are still unclear. One of the factors known to amplify hepatotoxicity is the tumor necrosis factor alpha (TNFα), especially due to its synergy with commonly used drugs such as diclofenac. However, the exact mechanism of how diclofenac in combination with TNFα induces liver injury remains elusive. Here, we combined time-resolved immunoblotting and live-cell imaging data of HepG2 cells and primary human hepatocytes (PHH) with dynamic pathway modeling using ordinary differential equations (ODEs) to describe the complex structure of TNFα-induced NFκB signal transduction and integrated the perturbations of the pathway caused by diclofenac. The resulting mathematical model was used to systematically identify parameters affected by diclofenac. These analyses showed that more than one regulatory module of TNFα-induced NFκB signal transduction is affected by diclofenac, suggesting that hepatotoxicity is the integrated consequence of multiple changes in hepatocytes and that multiple factors define toxicity thresholds. Applying our mathematical modeling approach to other DILI-causing compounds representing different putative DILI mechanism classes enabled us to quantify their impact on pathway activation, highlighting the potential of the dynamic pathway model as a quantitative tool for the analysis of DILI compounds