Reduction of CD8 T Cell Functionality but Not Inhibitory Capacity by Integrase Inhibitors.

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, MIP1ß 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.

The effect of age on the magnitude and longevity of Th1-directed CD4 T-cell responses to SARS-CoV-2.

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.

IκB(NS) protein mediates regulatory T cell development via induction of the Foxp3 transcription factor.

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-ß (TGF-ß) 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.

Defective migration and dysmorphology of neutrophil granulocytes in atypical chronic myeloid leukemia treated with ruxolitinib.

BACKGROUND: The identification of pathologically altered neutrophil granulocyte migration patterns bears strong potential for surveillance and prognostic scoring of diseases. We recently identified a strong correlation between impaired neutrophil motility and the disease stage of myelodysplastic syndrome (MDS). Here, we apply this assay to study quantitively increased neutrophils of a patient suffering from a rare leukemia subtype, atypical chronic myeloid leukemia (aCML). METHODS: A 69-year-old male was analyzed in this study. Besides routine analyses, we purified the patient's neutrophils from peripheral whole blood and studied their migration behavior using time-lapse video microscopy in a standardized assay. These live cell migration analyses also allowed for the quantification of cell morphology. Furthermore, the cells were stained for the markers CD15, CD16, fMLPR, CXCR1 and CXCR2. RESULTS: Despite cytoreductive therapy with hydroxyurea, the patient's WBC and ANC were poorly controlled and severe dysgranulopoiesis with hypogranularity was observed. Neutrophils displayed strongly impaired migration when compared to healthy controls and migrating cells exhibited a more flattened-out morphology than control neutrophils. Because of a detected CSF3R (p.T618I) mutation and constitutional symptoms treatment with ruxolitinib was initiated. Within 1 week of ruxolitinib treatment, the cell shape normalized and remained indistinguishable from healthy control neutrophils. However, neutrophil migration did not improve over the course of ruxolitinib therapy but was strikingly altered shortly before a sinusitis with fever and bleeding from a gastric ulcer. Molecular work-up revealed that under ruxolitinib treatment, the CSF3R clone was depleted, yet the expansion of a NRAS mutated subclone was promoted. CONCLUSION: These results demonstrate the usefulness of neutrophil migration analyses to uncover corresponding alterations of neutrophil migration in rare myeloid neoplasms. Furthermore, in addition to monitoring migration the determination of morphological features of live neutrophils might represent a useful tool to monitor the effectiveness of therapeutic approaches.

Surveillance of Myelodysplastic Syndrome via Migration Analyses of Blood Neutrophils: A Potential Prognostic Tool.

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.

c-REL and IκBNS Govern Common and Independent Steps of Regulatory T Cell Development from Novel CD122-Expressing Pre-Precursors.

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.

Quantitative Analysis of Proteome Modulations in Alveolar Epithelial Type II Cells in Response to Pulmonary Aspergillus fumigatus Infection.

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.

IκBNS regulates murine Th17 differentiation during gut inflammation and infection.

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.

Autophagy in T-cell development, activation and differentiation.

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.

Cellular-FLIP, Raji isoform (c-FLIP R) modulates cell death induction upon T-cell activation and infection.

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.

Atypical IκB proteins - nuclear modulators of NF-κB signaling.

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-1ß 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.

Machine learning analysis of humoral and cellular responses to SARS-CoV-2 infection in young adults.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces B and T cell responses, contributing to virus neutralization. In a cohort of 2,911 young adults, we identified 65 individuals who had an asymptomatic or mildly symptomatic SARS-CoV-2 infection and characterized their humoral and T cell responses to the Spike (S), Nucleocapsid (N) and Membrane (M) proteins. We found that previous infection induced CD4 T cells that vigorously responded to pools of peptides derived from the S and N proteins. By using statistical and machine learning models, we observed that the T cell response highly correlated with a compound titer of antibodies against the Receptor Binding Domain (RBD), S and N. However, while serum antibodies decayed over time, the cellular phenotype of these individuals remained stable over four months. Our computational analysis demonstrates that in young adults, asymptomatic and paucisymptomatic SARS-CoV-2 infections can induce robust and long-lasting CD4 T cell responses that exhibit slower decays than antibody titers. These observations imply that next-generation COVID-19 vaccines should be designed to induce stronger cellular responses to sustain the generation of potent neutralizing antibodies.

Vaccines against the original strain of SARS-CoV-2 provide T cell memory to the B.1.1.529 variant.

BACKGROUND: The SARS-CoV-2 variant B.1.1.529 potentially escapes immunity from vaccination via a heavily mutated Spike protein. Here, we analyzed whether T cell memory towards the B.1.1.529 Spike protein is present in individuals who received two or three doses of vaccines designed against the original Wuhan strain of SARS-CoV-2. METHODS: PBMCs were isolated from two- and three-times vaccinated study participants and incubated in vitro with peptide pools of the Spike protein derived from sequences of the original Wuhan or the B.1.1.529 strains of SARS-CoV-2. Activated antigen-specific T cells were detected by flow cytometry. In silico analyses with NetMHCpan and NetMHCIIpan were used to determine differences in MHC class presentation between the original strain and the B.1.1.529 strain for the most common MHCs in the European-Caucasian population. RESULTS: Here we show, that both CD4 and CD8 responses to the B.1.1.529 Spike protein are marginally reduced compared to the ancestor protein and a robust T cell response is maintained. Epitope analyses reveal minor differences between the two SARS-CoV-2 strains in terms of MHC class presentations for the MHC-alleles being most common in the European-Caucasian population. CONCLUSIONS: The memory T cell response induced via first generation vaccination remains robust and is mostly unaffected by B.1.1.529 mutations. Correspondingly, in silico analyses of MHC presentation of epitopes derived from the B.1.1.529 Spike protein shows marginal differences compared to the ancestral SARS-CoV-2 strain.

Vaccination against SARS-CoV-2 results in the production of proteins called antibodies, that bind and inactivate the virus, and cells that help to eliminate it from the body in a future encounter, such as memory T cells. Both antibodies and memory T cells remain in the body after vaccination with memory T cells being present for longer than antibodies. Here, we determined that even though most of the first generation vaccines were created to prevent infection with the original SARS-CoV-2 virus, the memory T cells generated by this vaccination can also detect the omicron variant.

A Central Role for Atg5 in Microbiota-Dependent Foxp3+ RORγt+ Treg Cell Preservation to Maintain Intestinal Immune Homeostasis.

Autophagy is an evolutionary conserved catabolic pathway that ensures the degradation of intracellular components. The autophagic pathway is regulated by autophagy-related (Atg) proteins that govern formation of double-membraned vesicles called autophagosomes. Autophagy deficiency in regulatory T (Treg) cells leads to increased apoptosis of these cells and to the development of autoimmune disorders, predominantly characterized by intestinal inflammation. Recently, RORγt-expressing Treg cells have been identified as key regulators of gut homeostasis, preventing intestinal immunopathology. To study the role of autophagy in RORγt+ Foxp3+ Treg cells, we generated mice lacking the essential component of the core autophagy machinery Atg5 in Foxp3+ cells. Atg5 deficiency in Treg cells led to a predominant intestinal inflammation. While Atg5-deficient Treg cells were reduced in peripheral lymphoid organs, the intestinal RORγt+ Foxp3+ subpopulation of Treg cells was most severely affected. Our data indicated that autophagy is essential to maintain the intestinal RORγt+ Foxp3+ Treg population, thereby protecting the mice from gut inflammatory disorders.

Up-regulation of c-FLIP short by NFAT contributes to apoptosis resistance of short-term activated T cells.

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-FLIP(short), whereas expression of c-FLIP(long) was only marginally modulated. In contrast to the general view that c-FLIP transcription is controlled predominantly by nuclear factor-kappaB (NF-kappaB), induction of c-FLIP(short) 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.

Community standards for open cell migration data.

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.

Generation of Foxp3+CD25- Regulatory T-Cell Precursors Requires c-Rel and IκBNS.

Next to the classical developmental route, in which first CD25 and subsequently Foxp3 are induced to generate thymic regulatory T (Treg) cells, an alternative route has been described. This alternative route is characterized by reciprocal induction of Foxp3 and CD25, with CD25 induction being required to rescue developing Treg cells from Foxp3-induced apoptosis. NF-κB has been demonstrated to be crucial for the development of thymic Treg cells via the classical route. However, its impact on the alternative route is poorly characterized. Using single and double deficient mice for key regulators of the classical route, c-Rel and IκBNS, we here demonstrate that NF-κB is essential for the generation of alternative CD25-Foxp3+ precursors, as well. Thus, c-Rel and IκBNS govern both routes of thymic Treg cell development.

Weight statistics controls dynamics in recurrent neural networks.

Recurrent neural networks are complex non-linear systems, capable of ongoing activity in the absence of driving inputs. The dynamical properties of these systems, in particular their long-time attractor states, are determined on the microscopic level by the connection strengths wij between the individual neurons. However, little is known to which extent network dynamics is tunable on a more coarse-grained level by the statistical features of the weight matrix. In this work, we investigate the dynamics of recurrent networks of Boltzmann neurons. In particular we study the impact of three statistical parameters: density (the fraction of non-zero connections), balance (the ratio of excitatory to inhibitory connections), and symmetry (the fraction of neuron pairs with wij = wji). By computing a 'phase diagram' of network dynamics, we find that balance is the essential control parameter: Its gradual increase from negative to positive values drives the system from oscillatory behavior into a chaotic regime, and eventually into stationary fixed points. Only directly at the border of the chaotic regime do the neural networks display rich but regular dynamics, thus enabling actual information processing. These results suggest that the brain, too, is fine-tuned to the 'edge of chaos' by assuring a proper balance between excitatory and inhibitory neural connections.

Imaging of cytotoxic antiviral immunity while considering the 3R principle of animal research.

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. KEY MESSAGES: TCRtg CD8+ T cells are obtained repetitively from the blood samples of single donors. One thousand transferred TCRtg CD8+ T cells get activated, are functional, and proliferate. Several adoptive cell transfers from the same donor show reproducible results. One thousand transferred cells take part in the FV immune response without modifying it. Use of fluorescent transfer cells allows in vivo imaging and single cell tracking.

c-FLIP Expression in Foxp3-Expressing Cells Is Essential for Survival of Regulatory T Cells and Prevention of Autoimmunity.

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.