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Age is associated with changes in the immune system which increase the risk for severe COVID-19. Here, we investigate SARS-CoV-2-reactive CD4 T cells from individuals recovered from SARS-CoV-2 infection with mild COVID-19 symptoms after 3, 6 and 9 months using incubation with SARS-CoV-2 S1, S2 and N-peptide pools, followed by flow cytometry for a Th1-activation profile or proliferation analyses. We found that SARS-CoV-2-reactive CD4 T cells are decreasing on average after 9 months but highly polyfunctional CD4 T cells can peak after 6-month recovery. We show that individuals older than 60 years of age have significantly more SARS-CoV-2-reactive T cells in their blood after 3 months of recovery compared to younger individuals and that the percentage of SARS-CoV-2-reactive Th1-directed CD4 T cells in the blood of mild-COVID-19-recovered individuals correlates with age. Finally, we show that individuals over the age of 40 have significantly increased the amounts of highly polyfunctional SARS-CoV-2-S-peptide-reactive CD4 T cells, compared to SARS-CoV-2 naïve individuals, than those under the age of 40. These findings suggest that in individuals recovered from mild COVID-19, increased age is associated with significantly more highly polyfunctional SARS-CoV-2-reactive CD4 T cells with a Th1-profile and that these responses persist over time.

Although HIV-specific CD8 T cells are effective in controlling HIV infection, they fail to clear infection even in the presence of antiretroviral therapy (ART) and cure strategies such as “shock-and-kill.” Little is known how ART is contributing to HIV-specific CD8 T cell function and the ability to clear HIV infection. Therefore, we first assessed the cytokine polyfunctionality and proliferation of CD8 T cells from ART-treated HIV+ individuals directly ex vivo and observed a decline in the multifunctional response as well as proliferation indices of these cells in individuals treated with integrase inhibitor (INSTI) based ART regimens compared to both protease inhibitor (PI) and nonnucleoside reverse transcriptase inhibitor (NNRTI) based regimens. We next cocultured CD8 T cells with different drugs individually and were able to observe reduced functional properties with significantly decreased ability of CD8 T cells to express IFN-γ, MIP1β and TNF-α only after treatment with INSTI-based regimens. Furthermore, previously activated and INSTI-treated CD8 T cells demonstrated reduced capacity to express perforin and granzyme B compared to PI and NNRTI treated cells. Unexpectedly, CD8 T cells treated with dolutegravir showed a similar killing ability 7 dpi compared to emtricitabine or rilpivirine treated cells. We next used a live cell imaging assay to determine the migratory capacity of CD8 T cells. Only INSTI-treated cells showed less migratory activity after SDF-1α stimulation compared to NRTI regimens. Our data show that the choice of ART can have a significant impact on CD8 T cell effector functions, but the importance for potential eradication attempts is unknown. IMPORTANCE Integrase Strand Transfer Inhibitors (INSTI) are recommended by national and international guidelines as a key component of ART in the treatment of HIV infected patients. In particular, their efficacy, tolerability and low drug-drug interaction profile have made them to the preferred choice as part of the first-line regimen in treatment-naive individuals. Here, we demonstrate that the choice of ART can have a significant impact on function and metabolism of CD8 T cells. In summary, our study provides first evidence on a significant, negative impact on CD8 T cell effector functions in the presence of two INSTIs, dolutegravir and elvitegravir, which may contribute to the limited success of eradicating HIV-infected cells through “shock-and-kill” strategies. Although our findings are coherent with recent studies highlighting a possible role of dolutegravir in weight gain, further investigations are necessary to fully understand the impact of INSTI–based regimens on the health of the individual during antiretroviral therapy.

As of November 2021, the viral pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), has infected more than 250 million individuals worldwide and killed over 5 million people. In addition to known risk groups, also cancer patients are at increased risk for severe disease progression. Therefore, understanding the nature of the accompanying immune response is essential, especially the role of T cells for successful virus defense but also during immunopathology leading to severe disease outcomes. Furthermore, as neutralizing antibodies are reported to decay within months post infection or vaccination or are even absent due to cancer treatment, SARS-CoV-2-specific T cell persistence is discussed as an indicator of long-term protective immunity. Especially, in immunocompromised cancer patients, levels of SARS-CoV-2-specifc antibodies may not be efficiently detected. Therefore, we developed a rapid and easy flow cytometric assay for the analysis of SARS-CoV-2-reactive CD4+ and CD8+ T cells directly from whole blood. Citation Format: Lorenz Fülle, Philipp David Gert, Verena Traska, Sofia Chatzopoulou, Christian Dose, Anne Richter, Marc Schuster. Rapid flow cytometric whole blood assay for assessment of SARS-CoV-2-reactive T cell immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 436.

Autonomous migration is a central characteristic of immune cells, and changes in this function have been correlated to the progression and severity of diseases. Hence, the identification of pathologically altered leukocyte migration patterns might be a promising approach for disease surveillance and prognostic scoring. However, because of the lack of standardized and robust assays, migration patterns have not been clinically exploited so far. In this study, we introduce an easy-to-use and cross-laboratory, standardized two-dimensional migration assay for neutrophil granulocytes from peripheral blood. By combining time-lapse video microscopy and automated cell tracking, we calculated the average migration of neutrophils from 111 individual participants of the German Heinz Nixdorf Recall MultiGeneration study under steady-state, formyl-methionyl-leucyl-phenylalanine–, CXCL1-, and CXCL8-stimulated conditions. Comparable values were obtained in an independent laboratory from a cohort in Belgium, demonstrating the robustness and transferability of the assay. In a double-blinded retrospective clinical analysis, we found that neutrophil migration strongly correlated with the Revised International Prognostic Scoring System scoring and risk category of myelodysplastic syndrome (MDS) patients. In fact, patients suffering from high-risk subtypes MDS with excess blasts I or II displayed highly significantly reduced neutrophil migration. Hence, the determination of neutrophil migration patterns might represent a useful tool in the surveillance of MDS. Taken together, we suggest that standardized migration assays of neutrophils and other leukocyte subtypes might be broadly applicable as prognostic and surveillance tools for MDS and potentially for other diseases.

Contains fulltext : 220755.pdf (Publisher’s version ) (Open Access) Cell migration research has become a high-content field. However, the quantitative information encapsulated in these complex and high-dimensional datasets is not fully exploited owing to the diversity of experimental protocols and non-standardized output formats. In addition, typically the datasets are not open for reuse. Making the data open and Findable, Accessible, Interoperable, and Reusable (FAIR) will enable meta-analysis, data integration, and data mining. Standardized data formats and controlled vocabularies are essential for building a suitable infrastructure for that purpose but are not available in the cell migration domain. We here present standardization efforts by the Cell Migration Standardisation Organisation (CMSO), an open community-driven organization to facilitate the development of standards for cell migration data. This work will foster the development of improved algorithms and tools and enable secondary analysis of public datasets, ultimately unlocking new knowledge of the complex biological process of cell migration.

Introduction: The emergence of SARS-CoV-2 virus, which causes COVID-19, is a major global health hazard. Therefore, a comprehensive characterization of the humoral and cellular immune responses to this virus is essential to combat the COVID-19 pandemic. Our goal was to develop reliable methods and tools for the analysis of humoral and cellular B- and T- cell responses, which will facilitate scientific research for prediction of disease progression, long-term immunity and will support vaccine development. Methods: Plasma samples and PBMCs of COVID-19 convalescent and healthy donors were obtained. For the detection of SARS-CoV-2 specific antibodies and identification of antigen-specific B cells, we manufactured recombinant mono-biotinylated protein variants of the Spike (S), Receptor Binding Domain (RBD) and Nucleoprotein (N). To identify antigen-reactive T cells, SARS-CoV-2 peptide pools were synthetized for the S, N and Membrane (M) antigens and used for stimulation. The peptide pools consist of mainly 15-mer peptides having an 11-mer amino acid overlap and thereby overspan a whole protein sequence. Results: To determine the presence of SARS-CoV-2 reactive antibodies a flow-based bead assay using recombinant, mono-biotinylated SARS-CoV-2 antigens loaded onto Streptavidin (SAV)-coated-PMMA beads was set up. The beads were incubated with plasma samples and fluorochrome conjugated anti-human isotype specific antibodies for flow cytometric analysis. All the antigens tested were shown to be suitable for the detection of antibodies to SARS-CoV-2 in COVID-19 convalescent plasma. To assess the feasibility of recombinant antigens for the detection and isolation of antigen-specific B cells, the mono-biotinylated Spike and RBD antigens were tetramerized on fluorochrome-conjugated SAV. These tetramers were used for staining, magnetic enrichment and flow cytometric sorting of B cells specific to SARS-CoV-2 antigens. We were able to demonstrate that our recombinant antigens can be used to assess the presence and enable the phenotyping and isolation of rare antigen-specific B cells. For further characterization of the SARS-CoV-2 reactive T cell immunity PBMCs were short term stimulated with the S, M and N peptide pools. After intracellular staining of IFNg, TNFa, IL-2 and CD154, reactive T cells were detected using flow cytometry. We could demonstrate T cell reactivity towards each peptide pool. However, strengths of T cell responses towards the S, M and N peptide pools were heterogeneous between different COVID-19 convalescent individuals. Conclusion: To support and improve current research activities for the identification and characterization of SARS-CoV-2 reactive humoral and cellular B- and T- cell responses, potent tools and assays were developed. Described here research solutions offer the opportunity to successfully address and contribute to the investigation on healthy and dysfunctional immune reactions towards SARS-CoV-2. Citation Format: Anna Baranska, Marc Schuster, Marek Wieczorek, Sebastian Flade, Lennart Wessels, Burhan Karaca, Verena Traska, Mario Assenmacher, Gregor Winkels. Detection of humoral and cellular responses to SARS-CoV-2 in COVID-19 convalescents [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2021 Feb 3-5. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(6_Suppl):Abstract nr P23.

Regulatory T (Treg) cells are critical for the shutdown of immune responses and have emerged as valuable targets of immunotherapies. Treg cells can rapidly proliferate; however, the homeostatic processes that limit excessive Treg cell numbers are poorly understood. Here, we show that, compared to conventional T cells, Treg cells have a high apoptosis rate ex vivo correlating with low c-FLIP expression. Treg-specific deletion of c-FLIP in mice resulted in fatal autoimmune disease of a scurfy-like phenotype characterized by absent peripheral Treg cells, activation of effector cells, multi-organ immune cell infiltration, and premature death. Surprisingly, blocking CD95L did not rescue Treg survival in vivo, suggesting additional survival functions of c-FLIP in Treg cells in addition to its classical role in the inhibition of death receptor signaling. Thus, our data reveal a central role for c-FLIP in Treg cell homeostasis and prevention of autoimmunity.

IL-17–producing Th17 cells mediate immune responses against a variety of fungal and bacterial infections. Signaling via NF-κB has been linked to the development and maintenance of Th17 cells. We analyzed the role of the unusual inhibitor of NF-κB, IκBNS, in the proliferation and effector cytokine production of murine Th17 cells. Our study demonstrates that nuclear IκBNS is crucial for murine Th17 cell generation. IκBNS is highly expressed in Th17 cells; in the absence of IκBNS, the frequencies of IL-17A–producing cells are drastically reduced. This was measured in vitro under Th17-polarizing conditions and confirmed in two colitis models. Mechanistically, murine IκBNS−/− Th17 cells were less proliferative and expressed markedly reduced levels of IL-2, IL-10, MIP-1α, and GM-CSF. Citrobacter rodentium was used as a Th17-inducing infection model, in which IκBNS−/− mice displayed an increased bacterial burden and diminished tissue damage. These results demonstrate the important function of Th17 cells in pathogen clearance, as well as in inflammation-associated pathology. We identified IκBNS to be crucial for the generation and function of murine Th17 cells upon inflammation and infection. Our findings may have implications for the therapy of autoimmune conditions, such as inflammatory bowel disease, and for the treatment of gut-tropic infections.

Adoptive cell transfer approaches for antigen-specific CD8+ T cells are used widely to study their effector potential during infections or cancer. However, contemporary methodological adaptations regarding transferred cell numbers, advanced imaging, and the 3R principle of animal research have been largely omitted. Here, we introduce an improved cell transfer method that reduces the number of donor animals substantially and fulfills the requirements for intravital imaging under physiological conditions. For this, we analyzed the well-established Friend retrovirus (FV) mouse model. Donor mice that expressed a FV-specific T cell receptor (TCRtg) and the fluorescent protein tdTomato were used as source of antigen-specific CD8+ T cells. Only a few drops of peripheral blood were sufficient to isolate ~ 150,000 naive reporter cells from which 1000 were adoptively transferred into recently FV-infected recipients. The cells became activated and functional and expanded strongly in the spleen and bone marrow within 10 days post infection. Transferred CD8+ T cells participated in the antiviral host response within a natural range and developed an effector phenotype indistinguishable from endogenous effector CD8+ T cells. Additionally, the generated reporter cell frequency allowed single cell visualization and tracking of a physiological antiretroviral CD8+ T cell response by intravital two-photon microscopy. Highly reproducible results were obtained in independent experiments by reusing the same donors repetitively for multiple transfers. Our approach allows a strong reduction of experimental animals required for studies on antigen-specific CD8+ T cell function and should be applicable to other transfer models.

The ubiquitous mold Aspergillus fumigatus threatens immunosuppressed patients as inducer of lethal invasive aspergillosis. A. fumigatus conidia are airborne and reach the alveoli, where they encounter alveolar epithelial cells (AEC). Previous studies reported the importance of the surfactant-producing AEC II during A. fumigatus infection via in vitro experiments using cell lines. We established a negative isolation protocol yielding untouched primary murine AEC II with a purity >90%, allowing ex vivo analyses of the cells, which encountered the mold in vivo. By label-free proteome analysis of AEC II isolated from mice 24h after A. fumigatus or mock infection we quantified 2256 proteins and found 154 proteins to be significantly differentially abundant between both groups (ANOVA p value ≤ 0.01, ratio of means ≥1.5 or ≤0.67, quantified with ≥2 peptides). Most of these proteins were higher abundant in the infected condition and reflected a comprehensive activation of AEC II on interaction with A. fumigatus. This was especially represented by proteins related to oxidative phosphorylation, hence energy production. However, the most strongly induced protein was the l-amino acid oxidase (LAAO) Interleukin 4 induced 1 (IL4I1) with a 42.9 fold higher abundance (ANOVA p value 2.91−10). IL4I1 has previously been found in B cells, macrophages, dendritic cells and rare neurons. Increased IL4I1 abundance in AEC II was confirmed by qPCR, Western blot and immunohistology. Furthermore, A. fumigatus infected lungs showed high levels of IL4I1 metabolic products. Importantly, higher IL4I1 abundance was also confirmed in lung tissue from human aspergilloma. Because LAAO are key enzymes for bactericidal product generation, AEC II might actively participate in pathogen defense. We provide insights into proteome changes of primary AEC II thereby opening new avenues to analyze the molecular changes of this central lung cell on infectious threats. Data are available via ProteomeXchange with identifier PXD005834.

Dysregulation of apoptosis caused by an imbalance of pro- and anti-apoptotic protein expression can lead to cancer, neurodegenerative, and autoimmune diseases. Cellular-FLIP (c-FLIP) proteins inhibit apoptosis directly at the death-inducing signaling complex of death receptors, such as CD95, and have been linked to apoptosis regulation during immune responses. While the isoforms c-FLIPL and c-FLIPS are well characterized, the function of c-FLIPR remains poorly understood. Here, we demonstrate the induction of endogenous murine c-FLIPR in activated lymphocytes for the first time. To analyze c-FLIPR function in vivo, we generated transgenic mice expressing murine c-FLIPR specifically in hematopoietic cells. As expected, lymphocytes from c-FLIPR transgenic mice were protected against CD95-induced apoptosis in vitro. In the steady state, transgenic mice had normal cell numbers and unaltered frequencies of B cells and T-cell subsets in lymphoid organs. However, when challenged with Listeria monocytogenes, c-FLIPR transgenic mice showed less liver necrosis and better bacterial clearance compared with infected wild-type mice. We conclude that c-FLIPR expression in hematopoietic cells supports an efficient immune response against bacterial infections.

Foxp3-expressing regulatory T cells (Tregs) are essential regulators of immune homeostasis and, thus, are prime targets for therapeutic interventions of diseases such as cancer and autoimmunity. c-REL and IκBNS are important regulators of Foxp3 induction in Treg precursors upon γ-chain cytokine stimulation. In c-REL/IκBNS double-deficient mice, Treg numbers were dramatically reduced, indicating that together, c-REL and IκBNS are pivotal for Treg development. However, despite the highly reduced Treg compartment, double-deficient mice did not develop autoimmunity even when aged to more than 1 y, suggesting that c-REL and IκBNS are required for T cell effector function as well. Analyzing Treg development in more detail, we identified a CD122+ subset within the CD25−Foxp3− precursor population, which gave rise to classical CD25+Foxp3− Treg precursors. Importantly, c-REL, but not IκBNS, controlled the generation of classical CD25+Foxp3− precursors via direct binding to the Cd25 locus. Thus, we propose that CD4+GITR+CD122+CD25−Foxp3− cells represent a Treg pre-precursor population, whose transition into Treg precursors is mediated via c-REL.

Foxp3-expressing regulatory T cells (Tregs) are essential regulators of immune homeostasis and, thus, are prime targets for therapeutic interventions of diseases such as cancer and autoimmunity. c-REL and IκB

Summary Forkhead box P3 positive (Foxp3 + ) regulatory T (Treg) cells suppress immune responses and regulate peripheral tolerance. Here we show that the atypical inhibitor of NFκB (IκB) IκB NS drives Foxp3 expression via association with the promoter and the conserved noncoding sequence 3 (CNS3) of the Foxp3 locus. Consequently, IκB NS deficiency leads to a substantial reduction of Foxp3 + Treg cells in vivo and impaired Foxp3 induction upon transforming growth factor-β (TGF-β) treatment in vitro. Moreover, fewer Foxp3 + Treg cells developed from IκB NS -deficient CD25 − CD4 + T cells adoptively transferred into immunodeficient recipients. Importantly, IκB NS was required for the transition of immature GITR + CD25 + Foxp3 − thymic Treg cell precursors into Foxp3 + cells. In contrast to mice lacking c-Rel or Carma1, IκB NS -deficient mice do not show reduced Treg precursor cells. Our results demonstrate that IκB NS critically regulates Treg cell development in the thymus and during gut inflammation, indicating that strategies targeting IκB NS could modulate the Treg cell compartment.

The adapter protein SLy2 (SH3 protein expressed in lymphocytes 2), also named HACS1, NASH1 or SAMSN1, is expressed in hematopoietic tissues, muscle, heart, brain, lung, pancreas, endothelial cells and myelomas. Endogenous SLy2 expression was shown to be upregulated in primary B cells upon differentiation and proliferation-inducing stimuli, and transduction experiments suggest a stimulatory role for SLy2 in B cell differentiation to plasma cells. However the signalling pathways regulated by SLy2 remain unknown. In this study we identify novel interaction partners of SLy2 providing a molecular framework for its function. We show that phosphorylated SLy2 directly interacts with 14-3-3 proteins via a previously unrecognized phosphorylation site. Furthermore, we demonstrate that 14-3-3 proteins control nucleo-cytoplasmic shuttling of SLy2 by retaining phosphorylated SLy2 in the cytoplasm. In the nucleus, SLy2 interacts with the SAP30/HDAC1 complex and regulates the activity of HDAC1. Thus, our findings unravel a novel mechanism how SLy2 localization is controlled and implicate SLy2 in the epigenetic control of gene expression.

Upon encounter with pathogens, T cells activate several defense mechanisms, one of which is the up-regulation of CD95 ligand (CD95L/FasL) which induces apoptosis in sensitive target cells. Despite expression of the CD95 receptor, however, recently activated T cells are resistant to CD95L, presumably due to an increased expression of antiapoptotic molecules. We show here that, in contrast to naive or long-term activated T cells, short-term activated T cells strongly up-regulate the caspase-8 inhibitor, cellular FLICE-inhibitory protein (c-FLIP). Intriguingly, upon activation, T cells highly induced the short splice variant c-FLIPshort, whereas expression of c-FLIPlong was only marginally modulated. In contrast to the general view that c-FLIP transcription is controlled predominantly by nuclear factor-κB (NF-κB), induction of c-FLIPshort in T cells was primarily mediated by the calcineurin-nuclear factor of activated T cells (NFAT) pathway. Importantly, blockage of NFAT-mediated c-FLIP expression by RNA interference or inhibition of calcineurin rendered T cells sensitive toward CD95L, as well as activation-induced apoptosis. Thus, the resistance of recently activated T cells depends crucially on induction of c-FLIP expression by the calcineurin/NFAT pathway. Our findings imply that preventing autocrine CD95L signaling by c-FLIP facilitates T-cell effector function and an efficient immune response.

The NF-κB/Rel signalling pathway plays a crucial role in numerous biological processes, including innate and adaptive immunity. NF-κB is a family of transcription factors, whose activity is regulated by the inhibitors of NF-κB (IκB). The IκB proteins comprise two distinct groups, the classical (cytoplasmic) and the atypical (nuclear) IκB proteins. Although the cytoplasmic regulation of NF-κB is well characterised, its nuclear regulation mechanisms remain marginally elucidated. However, work from recent years indicated that nuclear IκBs contribute significantly to the modulation of NF-κB-mediated transcription in the immune system. Here, we discuss the role of the atypical IκB proteins Bcl-3, IκBζ, IκBNS, IκBη and IκBL for the regulation of gene expression and effector functions in immune cells.

Autophagy is a vital catabolic process for degrading bulky cytosolic contents, which cannot be resorbed via the proteasome. First described as a survival mechanism during nutrient starvation conditions, recent reports have demonstrated that autophagy supports metabolic functions of T cells at various stages of maturation and effector function. Autophagy is crucial for T-cell development at the precursor stage as self-renewability and quiescence of hematopoietic stem cells depend on autophagy of the mitochondria and the endoplasmic reticulum. Later, during development in the thymus, autophagy regulates peptide presentation in stromal cells and professional antigen-presenting cells, which mediate thymocyte selection. Furthermore, the metabolic changes when mature T cells enter the periphery and when they are activated are both dependent on autophagy. Lastly, autophagy prevents early aging and, thus, ensures maintenance of memory T cells.

Nuclear factor κB (NF-κB) controls a multitude of physiological processes such as cell differentiation, cytokine expression, survival and proliferation. Since NF-κB governs embryogenesis, tissue homeostasis and the functions of innate and adaptive immune cells it represents one of the most important and versatile signaling networks known. Its activity is regulated via the inhibitors of NF-κB signaling, the IκB proteins. Classical IκBs, like the prototypical protein IκBα, sequester NF-κB transcription factors in the cytoplasm by masking of their nuclear localization signals (NLS). Thus, binding of NF-κB to the DNA is inhibited. The accessibility of the NLS is controlled via the degradation of IκBα. Phosphorylation of the conserved serine residues 32 and 36 leads to polyubiquitination and subsequent proteasomal degradation. This process marks the central event of canonical NF-κB activation. Once their NLS is accessible, NF-κB transcription factors translocate into the nucleus, bind to the DNA and regulate the transcription of their respective target genes. Several studies described a distinct group of atypical IκB proteins, referred to as the BCL-3 subfamily. Those atypical IκBs show entirely different sub-cellular localizations, activation kinetics and an unexpected functional diversity. First of all, their interaction with NF-κB transcription factors takes place in the nucleus in contrast to classical IκBs, whose binding to NF-κB predominantly occurs in the cytoplasm. Secondly, atypical IκBs are strongly induced after NF-κB activation, for example by LPS and IL-1β stimulation or triggering of B cell and T cell antigen receptors, but are not degraded in the first place like their conventional relatives. Finally, the interaction of atypical IκBs with DNA-associated NF-κB transcription factors can further enhance or diminish their transcriptional activity. Thus, they do not exclusively act as inhibitors of NF-κB activity. The capacity to modulate NF-κB transcription either positively or negatively, represents their most important and unique mechanistic difference to classical IκBs. Several reports revealed the importance of atypical IκB proteins for immune homeostasis and the severe consequences following their loss of function. This review summarizes insights into the physiological processes regulated by this protein class and the relevance of atypical IκB functioning.

Dysregulation of apoptosis caused by an imbalance of pro- and anti-apoptotic protein expression can lead to cancer, neurodegenerative, and autoimmune diseases. Cellular-FLIP (c-FLIP) proteins inhibit apoptosis directly at the death-inducing signaling complex of death receptors, such as CD95, and have been linked to apoptosis regulation during immune responses. While the isoforms c-FLIPL and c-FLIPS are well characterized, the function of c-FLIPR remains poorly understood. Here, we demonstrate the induction of endogenous murine c-FLIPR in activated lymphocytes for the first time. To analyze c-FLIPR function in vivo, we generated transgenic mice expressing murine c-FLIPR specifically in hematopoietic cells. As expected, lymphocytes from c-FLIPR transgenic mice were protected against CD95-induced apoptosis in vitro. In the steady state, transgenic mice had normal cell numbers and unaltered frequencies of B cells and T-cell subsets in lymphoid organs. However, when challenged with Listeria monocytogenes, c-FLIPR transgenic mice showed less liver necrosis and better bacterial clearance compared with infected wild-type mice. We conclude that c-FLIPR expression in hematopoietic cells supports an efficient immune response against bacterial infections.

Deregulation of apoptosis is common in cancer and is often caused by overexpression of anti-apoptotic proteins in tumour cells. One important regulator of apoptosis is the cellular FLICE-inhibitory protein (c-FLIP), which is overexpressed, for example, in melanoma and Hodgkin's lymphoma cells. Here, we addressed the question whether deregulated c-FLIP expression in urothelial carcinoma impinges on the ability of death ligands to induce apoptosis. In particular, we investigated the role of the c-FLIP splice variants c-FLIP(long) (c-FLIP(L)) and c-FLIP(short) (c-FLIP(S)), which can have opposing functions. We observed diminished expression of the c-FLIP(L) isoform in urothelial carcinoma tissues as well as in established carcinoma cell lines compared with normal urothelial tissues and cells, whereas c-FLIP(S) was unchanged. Overexpression and RNA interference studies in urothelial cell lines nevertheless demonstrated that c-FLIP remained a crucial factor conferring resistance towards induction of apoptosis by death ligands CD95L and TRAIL. Isoform-specific RNA interference showed c-FLIP(L) to be of particular importance. Thus, urothelial carcinoma cells appear to fine-tune c-FLIP expression to a level sufficient for protection against activation of apoptosis by the extrinsic pathway. Therefore, targeting c-FLIP, and especially the c-FLIP(L) isoform, may facilitate apoptosis-based therapies of bladder cancer in otherwise resistant tumours.