Gertrud Schwär

Natural killer (NK) cells are critical components of the first‐line immune defense, responsible for eliminating tumorigenic cells. NK cell‐based adoptive immunotherapy has gained increasing attention; however, cryopreservation, a standard technique for NK cell storage, significantly impairs NK cell cytotoxicity, particularly in physiological 3D environments. Here, we demonstrate that short‐term co‐culture with effector T cells markedly enhances NK cell motility and killing functionality. Notably, a brief 1‐day co‐culture is sufficient to restore cryopreservation‐impaired NK cell functionality in 3D environments. This enhancement requires direct contact between T cells and NK cells, which facilitates localized high concentrations of IL‐2 at the cell contact sites. To develop a controled, donor‐independent solution, we demonstrate that synthetic T cells with surface‐bound IL‐2 exhibit superior efficiency in revitalizing cryopreserved NK cells. These findings uncover a previously unrecognized role for physical contact‐mediated local IL‐2 signaling and provide an efficient, cost‐effective, and tunable strategy to rescue NK cell functionality post‐cryopreservation, paving the way for more scalable, potent, and clinically viable NK cell‐based immunotherapies.

Assessing the prognosis of patients with aggressive non-Hodgkin B cell lymphoma mainly relies on a clinical risk score (IPI). Standard first-line therapies are based on a chemo-immunotherapy with rituximab, which mediates CD16-dependent antibody-dependent cellular cytotoxicity (ADCC). We phenotypically and functionally analyzed blood samples from 46 patients focusing on CD16+ NK cells, CD16+ T cells and CD16+ monocytes. Kaplan-Meier survival curves show a superior progression-free survival (PFS) for patients having more than 1.6% CD16+ T cells (p = 0.02; HR = 0.13 (0.007–0.67)) but an inferior PFS having more than 10.0% CD16+ monocytes (p = 0.0003; HR = 16.0 (3.1-291.9)) at diagnosis. Surprisingly, no correlation with NK cells was found. The increased risk of relapse in the presence of > 10.0% CD16+ monocytes is reversed by the simultaneous occurrence of > 1.6% CD16+ T cells. The unexpectedly strong protective function of CD16+ T cells could be explained by their high antibody-dependent cellular cytotoxicity as quantified by real-time killing assays and single-cell imaging. The combined analysis of CD16+ monocytes (> 10%) and CD16+ T cells (< 1.6%) provided a strong model with a Harrell’s C index of 0.80 and a very strong power of 0.996 even with our sample size of 46 patients. CD16 assessment in the initial blood analysis is thus a precise marker for early relapse prediction. High CD16+ T cell counts have a positive correlation with PFS in aggressive NHL/DLBCL patients (p = 0.02; HR = 0.13, 0.01–0.7). High CD16+ monocyte counts have a negative correlation with PFS in aggressive NHL/DLBCL patients (p = 0.0003; HR = 16.0, 3-292). The combined assessment of CD16+ T cells and CD16+ monocytes accurately predicts PFS in aggressive NHL/DLBCL patients. The strong protective function of CD16+ T cells could be explained by their high antibody-dependent cellular cytotoxicity.

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. image Key points: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. Both CTL and NK cells attack the same targets, but most studies address them individually. In a sequential cytotoxicity model, the interdependence of antigen‐specific CTL and NK cell cytotoxicity against melanoma is quantified. High numbers of antigen‐specific CTL and NK cells eliminate all melanoma cells. However, lower numbers induce resistance if secondary CTL or NK cell exposure follows initial CTL exposure or if secondary NK cell exposure follows initial NK cell exposure. On the contrary, if secondary CTL exposure follows initial NK cell exposure, cytotoxicity is enhanced. Alterations in human leucocyte antigen class I expression and interleukin‐6 levels are correlated with resistance to NK cells, whereas a reduction in antigen expression is correlated with reduced CTL cytotoxicity; CTL cytotoxicity is rescued beyond control levels by exogenous antigen. This assay and the results on interdependencies will help us to understand and optimize immune therapies against cancer.

Immunological memory is an important concept to protect humans against recurring diseases. Memory CD8 ⁺ T cells are required for quick expansion into effector cells but also provide immediate cytotoxicity against their targets. Whereas many functions of the two main cytotoxic subtypes, effector memory CD8 ⁺ T cells (T _EM ) and central memory CD8 ⁺ T cells (T _CM ), are relatively well defined, single T _EM and T _CM cell cytotoxicity has not been quantified. Here, we analyze human CD8 ⁺ subtype distribution following SEA stimulation and quantify the expression of death mediators, including perforin, granzyme B, FasL and TRAIL. We find higher perforin, granzyme B and FasL expression in T _EM and compared to T _CM . To quantify single T _EM and T _CM cytotoxicity, we develop a FRET-based fluorescent assay with NALM6 target cells stably transfected with a GFP-RFP FRET construct based on a caspase-cleavage sequence (apoptosis sensor Casper-GR). Applying this assay, T _EM or T _CM induced target cell apoptosis or necrosis can be quantified. We find that single T _EM are much more effective than T _CM in killing their targets mainly by apoptosis and secondary necrosis. The reason for this is the higher perforin expression and on a more efficient lytic immunological synapse during T _EM -target contact compared to T _CM -target contact. Defining and quantifying single T _EM and T _CM cytotoxicity and the respective mechanism should be helpful to optimize future subset-based immune therapies.

Cytotoxic CD8 ⁺ T lymphocytes (CTL) eliminate infected cells or transformed tumour cells by releasing perforin-containing cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca ²⁺ -influx through store operated Ca ²⁺ channels, formed by STIM-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca ²⁺ -dependent target cell killing. Here, we isolated total RNA from natural killer (NK) cells, non-stimulated CD8 ⁺ T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8 ⁺ T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca ²⁺ -homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8 ⁺ T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca ²⁺ dependent cytotoxicity, candidates were also analyzed under Ca ²⁺ -limiting conditions. Overall, this strategy led to the identification of KCNN4, RCAN3, CCR5 and BCL2 as potential candidates to regulate the efficiency of Ca ²⁺ -dependent target cell killing.

Ca2+ is a crucial second messenger for proper T cell function. Considering the relevance of Ca2+ signals for T cell functionality it is surprising that no mechanistic insights into T cell Ca2+ signals from elderly individuals are reported. The main Ca2+ entry mechanism in T cells are STIM-activated Orai channels. Their role during lymphocyte aging is completely unknown. Here, we report not only reduced Ca2+ signals in untouched and stimulated, but also in central and effector memory CD8+ T cells from elderly (18-24 months) compared to adult (3-6 months) mice. Two mechanisms contribute to the overall reduction in Ca2+ signals of CD8+ T cells of elderly mice: 1) Reduced Ca2+ currents through Orai channels due to decreased expressions of STIMs and Orais. 2) A faster extrusion of Ca2+ owing to an increased expression of PMCA4. The reduced Ca2+ signals correlated with a resistance of the cytotoxic efficiency of CD8+ T cells to varying free [Ca2+]ext with age. In summary, reduced STIM/Orai expression and increased Ca2+ clearing rates following enhanced PMCA4 expression contribute to reduced Ca2+ signals in CD8+ T cells of elderly mice. These changes are apparently relevant to immune function as they reduce the Ca2+ dependency of CTL cytotoxicity.

Key points: Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to eliminate cancer cells. We analysed the Ca²⁺ dependence of CTL and NK cell cytotoxicity and found that in particular CTLs have a very low optimum of [Ca²⁺]_i (between 122 and 334 nm) and [Ca²⁺]_o (between 23 and 625 μm) for efficient cancer cell elimination, well below blood plasma Ca²⁺ levels. As predicted from these results, partial down‐regulation of the Ca²⁺ channel Orai1 in CTLs paradoxically increases perforin‐dependent cancer cell killing. Lytic granule release at the immune synapse between CTLs and cancer cells has a Ca²⁺ optimum compatible with this low Ca²⁺ optimum for efficient cancer cell killing, whereas the Ca²⁺ optimum for CTL migration is slightly higher and proliferation increases monotonously with increasing [Ca²⁺]_o. We propose that a partial inhibition of Ca²⁺ signals by specific Orai1 blockers at submaximal concentrations could contribute to tumour elimination. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to protect the human body against cancer. Ca²⁺ is a key metabolic factor for lymphocyte function and cancer homeostasis. We analysed the Ca²⁺ dependence of CTL and NK cell cytotoxicity against cancer cells and found that CTLs have a bell‐shaped Ca²⁺ dependence with an optimum for cancer cell elimination at rather low [Ca²⁺]_o (23–625 μm) and [Ca²⁺]_i (122–334 nm). This finding predicts that a partial inhibition of Orai1 should increase (rather than decrease) cytotoxicity of CTLs at [Ca²⁺]_o higher than 625 μm. We tested this hypothesis in CTLs and indeed found that partial down‐regulation of Orai1 by siRNA increases the efficiency of cancer cell killing. We found two mechanisms that may account for the Ca²⁺ optimum of cancer cell killing: (1) migration velocity and persistence have a moderate optimum between 500 and 1000 μm [Ca²⁺]_o in CTLs, and (2) lytic granule release at the immune synapse between CTLs and cancer cells is increased at 146 μm compared to 3 or 800 μm, compatible with the Ca²⁺ optimum for cancer cell killing. It has been demonstrated in many cancer cell types that Orai1‐dependent Ca²⁺ signals enhance proliferation. We propose that a decrease of [Ca²⁺]_o or partial inhibition of Orai1 activity by selective blockers in the tumour microenvironment could efficiently reduce cancer growth by simultaneously increasing CTL and NK cell cytotoxicity and decreasing cancer cell proliferation. image

The actin‐binding protein profilin1 (PFN1) plays a central role in actin dynamics, which is essential for cytotoxic T lymphocyte (CTL) functions. The functional role of PFN1 in CTLs, however still remains elusive. Here, we identify PFN1 as the only member of the profilin family expressed in primary human CD8⁺ T cells. Using in vitro assays, we find that PFN1 is a negative regulator of CTL‐mediated elimination of target cells. Furthermore, PFN1 is involved in activation‐induced lytic granule (LG) release, CTL migration and modulation of actin structures at the immunological synapse (IS). During CTL migration, PFN1 modulates the velocity, protrusion formation patterns and protrusion sustainability. In contrast, PFN1 does not significantly affect migration persistence and the rates of protrusion emergence and retraction. Under in vitro conditions mimicking a tumor microenvironment, we show that PFN1 downregulation promotes CTL invasion into a 3D matrix, without affecting the viability of CTLs in a hydrogen peroxide‐enriched microenvironment. Highlighting its potential relevance in cancer, we find that in pancreatic cancer patients, PFN1 expression is substantially decreased in peripheral CD8⁺ T cells. Taken together, we conclude that PFN1 is a negative regulator for CTL‐mediated cytotoxicity and may have an impact on CTL functionality in a tumor‐related context.

N-glycosylation of cell surface proteins affects protein function, stability, and interaction with other proteins. Orai channels, which mediate store-operated Ca²⁺ entry (SOCE), are composed of N-glycosylated subunits. Upon activation by Ca²⁺ sensor proteins (stromal interaction molecules STIM1 or STIM2) in the endoplasmic reticulum, Orai Ca²⁺ channels in the plasma membrane mediate Ca²⁺ influx. Lectins are carbohydrate-binding proteins, and Siglecs are a family of sialic acid–binding lectins with immunoglobulin-like repeats. Using Western blot analysis and lectin-binding assays from various primary human cells and cancer cell lines, we found that glycosylation of Orai1 is cell type–specific. Ca²⁺ imaging experiments and patch-clamp experiments revealed that mutation of the only glycosylation site of Orai1 (Orai1N223A) enhanced SOCE in Jurkat T cells. Knockdown of the sialyltransferase ST6GAL1 reduced α-2,6–linked sialic acids in the glycan structure of Orai1 and was associated with increased Ca²⁺ entry in Jurkat T cells. In human mast cells, inhibition of sialyl sulfation altered the N-glycan of Orai1 (and other proteins) and increased SOCE. These data suggest that cell type–specific glycosylation influences the interaction of Orai1 with specific lectins, such as Siglecs, which then attenuates SOCE. In summary, the glycosylation state of Orai1 influences SOCE-mediated Ca²⁺ signaling and, thus, may contribute to pathophysiological Ca²⁺ signaling observed in immune disease and cancer.

A systematic understanding of different factors influencing cell type specific microRNA profiles is essential for state-of-the art biomarker research. We carried out a comprehensive analysis of the biological variability and changes in cell type pattern over time for different cell types and different isolation approaches in technical replicates. All combinations of the parameters mentioned above have been measured, resulting in 108 miRNA profiles that were evaluated by next-generation-sequencing. The largest miRNA variability was due to inter-individual differences (34 %), followed by the cell types (23.4 %) and the isolation technique (17.2 %). The change over time in cell miRNA composition was moderate (<3 %) being close to the technical variations (<1 %). Largest variability (including technical and biological variance) was observed for CD8 cells while CD3 and CD4 cells showed significantly lower variations. ANOVA highlighted that 51.5 % of all miRNAs were significantly influenced by the purification technique. While CD4 cells were least affected, especially miRNA profiles of CD8 cells were fluctuating depending on the cell purification approach. To provide researchers access to the profiles and to allow further analyses of the tested conditions we implemented a dynamic web resource.