IL-13 determines specific IgE responses and SARS-CoV-2 immunity after mild COVID-19 and novel mRNA vaccination.

After recovery, mild and severe COVID-19 diseases are associated with long-term effects on the host immune system, such as prolonged T-cell activation or accumulation of autoantibodies. In this study, we show that mild SARS-CoV-2 infections, but not SARS-CoV-2 spike mRNA vaccinations, cause durable atopic risk factors such as a systemic Th2- and Th17-type environment as well as activation of B cells responsive of IgE against aeroallergens from house dust mite and mold. At an average of 100 days post mild SARS-CoV-2 infections, anti-mold responses were associated with low IL-13 levels and increased pro-inflammatory IL-6 titers. Acutely severely ill COVID-19 patients instead showed no evidence of atopic reactions. Considering convalescents of mild COVID-19 courses and mRNA-vaccinated individuals together, IL-13 was the predominant significantly upregulated factor, likely shaping SARS-CoV-2 immunity. Application of multiple regression analysis revealed that the IL-13 levels of both groups were determined by the Th17-type cytokines IL-17A and IL-22. Taken together, these results implicate a critical role for IL-13 in the aftermath of SARS-CoV-2 mild infections and mRNA vaccinations, conferring protection against airway directed, atopic side reactions that occur in mildly experienced COVID-19.

CTLA-4 (CD152): A versatile receptor for immune-based therapy.

Coreceptor-based immunotherapy is a rapidly developing approach to treat cancer patients. Among those targeted receptors, CTLA-4 is the primary attenuator of adaptive immune responses and the most prominent and extensively investigated molecule in this field. CTLA-4 is involved in broad range of mechanisms that regulate and control immune cell functions and therefore provides versatile strategies for therapeutic interventions. Despite being successfully harnessed in clinical treatments the different facets of CTLA-4 biology still remain incompletely understood. Here, we review the various aspects of CTLA-4 functions and CTLA-4-based immunotherapies and discuss challenges to improve current approaches.

PD-1/PD-L1 Control of Antigen-Specifically Activated CD4 T-Cells of Neonates.

Newborns are highly susceptible to infections; however, the underlying mechanisms that regulate the anti-microbial T-helper cells shortly after birth remain incompletely understood. To address neonatal antigen-specific human T-cell responses against bacteria, Staphylococcus aureus (S. aureus) was used as a model pathogen and comparatively analyzed in terms of the polyclonal staphylococcal enterotoxin B (SEB) superantigen responses. Here, we report that neonatal CD4 T-cells perform activation-induced events upon S. aureus/APC-encounter including the expression of CD40L and PD-1, as well as the production of Th1 cytokines, concomitant to T-cell proliferation. The application of a multiple regression analysis revealed that the proliferation of neonatal T-helper cells was determined by sex, IL-2 receptor expression and the impact of the PD-1/PD-L1 blockade. Indeed, the treatment of S. aureus-activated neonatal T-helper cells with PD-1 and PD-L1 blocking antibodies revealed the specific regulation of the immediate neonatal T-cell responses with respect to the proliferation and frequencies of IFNγ producers, which resembled in part the response of adults' memory T-cells. Intriguingly, the generation of multifunctional T-helper cells was regulated by the PD-1/PD-L1 axis exclusively in the neonatal CD4 T-cell lineage. Together, albeit missing memory T-cells in neonates, their unexperienced CD4 T-cells are well adapted to mount immediate and strong anti-bacterial responses that are tightly controlled by the PD-1/PD-L1 axis, thereby resembling the regulation of recalled memory T-cells of adults.

Unique autoantibody prevalence in long-term recovered SARS-CoV-2-infected individuals.

The variability in resolution of SARS-CoV-2-infections between individuals neither is comprehended, nor are the long-term immunological consequences. To assess the long-term impact of a SARS-CoV-2-infection on the immune system, we conducted a prospective study of 80 acute and former SARS-CoV-2 infected individuals and 39 unexposed donors to evaluate autoantibody responses and immune composition. Autoantibody levels against cyclic citrullinated peptide (CCP), a specific predictor for rheumatoid arthritis (RA), were significantly (p = 0.035) elevated in convalescents only, whereas both acute COVID-19 patients and long-term convalescents showed critically increased levels of anti-tissue transglutaminase (TG), a specific predictor of celiac disease (CD) (p = 0.002). Both, anti-CCP and anti-TG antibody levels were still detectable after 4-8 months post infection. Anti-TG antibodies occurred predominantly in aged patients in a context of a post-SARS-CoV-2-specific immune composition (R2 = 0.31; p = 0.044). This study shows that increased anti-CCP and anti-TG autoantibody levels can remain long-term after recovering even from mildly experienced COVID-19. The inter-relationship of the lung as viral entry side and RA- and CD-associated autoimmunity indicates that a SARS-CoV-2-infection could be a relevant environmental factor in their pathogenesis.

Early changes in the metabolic profile of activated CD8(+) T cells.

BACKGROUND: Antigenic stimulation of the T cell receptor (TCR) initiates a change from a resting state into an activated one, which ultimately results in proliferation and the acquisition of effector functions. To accomplish this task, T cells require dramatic changes in metabolism. Therefore, we investigated changes of metabolic intermediates indicating for crucial metabolic pathways reflecting the status of T cells. Moreover we analyzed possible regulatory molecules required for the initiation of the metabolic changes. RESULTS: We found that proliferation inducing conditions result in an increase in key glycolytic metabolites, whereas the citric acid cycle remains unaffected. The upregulation of glycolysis led to a strong lactate production, which depends upon AKT/PKB, but not mTOR. The observed upregulation of lactate dehydrogenase results in increased lactate production, which we found to be dependent on IL-2 and to be required for proliferation. Additionally we observed upregulation of Glucose-transporter 1 (GLUT1) and glucose uptake upon stimulation, which were surprisingly not influenced by AKT inhibition. CONCLUSIONS: Our findings suggest that AKT plays a central role in upregulating glycolysis via induction of lactate dehydrogenase expression, but has no impact on glucose uptake of T cells. Furthermore, under apoptosis inducing conditions, T cells are not able to upregulate glycolysis and induce lactate production. In addition maintaining high glycolytic rates strongly depends on IL-2 production.

CTLA-4 (CD152) enhances the Tc17 differentiation program.

Although CD8(+) T cells that produce IL-17 (Tc17 cells) have been linked to host defense, Tc17 cells show reduced cytotoxic activity, which is the characteristic function of CD8(+) T cells. Here, we show that CTLA-4 enhances the frequency of IL-17 in CD8(+) T cells, indicating that CTLA-4 (CD152) specifically promotes Tc17 differentiation. Simultaneous stimulation of CTLA-4(+/+) and CTLA-4(-/-) T cells in cocultures and agonistic CTLA-4 stimulation unambiguously revealed a cell-intrinsic mechanism for IL-17 control by CTLA-4. The quality of CTLA-4-induced Tc17 cells was tested in vivo, utilizing infection with the facultative intracellular bacterium Listeria monocytogenes (LM). Unlike CTLA-4(+/+) Tc17 cells, CTLA-4(-/-) were nearly as efficient as Tc1 CTLA-4(+/+) cells in LM clearance. Additionally, adoptively transferred CTLA-4(-/-) Tc17 cells expressed granzyme B after rechallenge, and produced Tc1 cytokines such as IFN-γ and TNF-α, which strongly correlate with bacterial clearance. CTLA-4(+/+) Tc17 cells demonstrated a high-quality Tc17 differentiation program ex vivo, which was also evident in isolated IL-17-secreting Tc17 cells, with CTLA-4-mediated enhanced upregulation of Tc17-related molecules such as IL-17A, RORγt, and IRF-4. Our results show that CTLA-4 promotes Tc17 differentiation that results in robust Tc17 responses. Its inactivation might therefore represent a central therapeutic target to enhance clearance of infection.

Activation of the Hippo pathway by CTLA-4 regulates the expression of Blimp-1 in the CD8+ T cell.

During the primary response, the commitment of the CD8(+) T cell to Blimp-1 expression and the terminal differentiation that Blimp-1 induces must be timed so as not to impair the process of clonal expansion. We determined whether the Hippo pathway, which links cell-cell contact to differentiation in other cell lineages, controls Blimp-1 expression. Activating the CD8(+) T cell with antigen and IL-2 causes expression of the core Hippo pathway components, including the pivotal transcriptional cofactor Yap. Contact between activated CD8(+) T cells induces Hippo pathway-mediated Yap degradation and Blimp-1 expression; a Hippo-resistant, stable form of Yap suppresses Blimp-1 expression. Cytotoxic T lymphocyte antigen 4 (CTLA-4) and CD80 comprise the receptor-ligand pair that mediates contact-dependent Hippo pathway activation. In vivo, CD8(+) T cells expressing Hippo resistant-Yap or lacking CTLA-4 have diminished expression of the senescence marker, KLRG1, during a viral infection. The CTLA-4/Hippo pathway/Blimp-1 system may couple terminal differentiation of CD8(+) T cell with the magnitude of clonal expansion.

PAG/Cbp suppression reveals a contribution of CTLA-4 to setting the activation threshold in T cells.

BACKGROUND: PAG/Cbp represents a ubiquitous mechanism for regulating Src family kinases by recruiting Csk to the plasma membrane, thereby controlling cellular activation. Since Src kinases are known oncogenes, we used RNA interference in primary human T cells to test whether the loss of PAG resulted in lymphocyte transformation. RESULTS: PAG-depletion enhanced Src kinase activity and augmented proximal T-cell receptor signaling; exactly the phenotype expected for loss of this negative regulator. Surprisingly, rather than becoming hyper-proliferative, PAG-suppressed T cells became unresponsive. This was mediated by a Fyn-dependent hyper-phosphorylation of the inhibitory receptor CTLA-4, which recruited the protein tyrosine phosphatase Shp-1 to lipid rafts. Co-suppression of CTLA-4 abrogates this inhibition and restores proliferation to T cells. CONCLUSION: We have identified a fail-safe mechanism as well as a novel contribution of CTLA-4 to setting the activation threshold in T cells.

PD-1 limits differentiation and plasticity of Tc17 cells.

Blockade of surface co-inhibitory receptor programmed cell death-1 (PD-1; CD279) has been established as an important immunotherapeutic approach to treat malignancies. On a cellular level, PD-1 is demonstrated to be of particular importance in inhibiting differentiation and effector function of cytotoxic Tc1 cells (CTLs). Nevertheless, the role of PD-1 in modulating interleukin (IL)-17-producing CD8+ T-cells (Tc17 cells), which generally display suppressed cytotoxic nature, is not well understood. To evaluate the impact of PD-1 in Tc17 responses, we examined its functioning using different in vitro and in vivo models. Upon activation of CD8+ T-cells in Tc17 environment, we found that PD-1 was rapidly expressed on the surface of CD8+ T-cells and triggered a T-cell-internal mechanism that inhibited the expression of IL-17 and Tc17-supporting transcription factors pSTAT3 and RORγt. Expression of type17-polarising cytokine IL-21 and the receptor for IL-23 were also suppressed. Intriguingly, adoptively transferred, PD-1-/- Tc17 cells were highly efficient in rejection of established B16 melanoma in vivo and displayed Tc1 like characteristics ex vivo. When using IL-17A-eGFP reporter mice for in vitro fate tracking, IL-17A-eGFP expressing cells lacking PD-1 signaling upon re-stimulation with IL-12 quickly acquired Tc1 characteristics such as IFN-γ, and granzyme B expression, implicating lineage independent upregulation of CTL-characteristics that are needed for tumor control. In line with plasticity characteristics, absence of PD-1 signaling in Tc17 cells increased the expression of the stemness and persistence-associated molecules TCF1 and BCL6. Thus, PD-1 plays a central role in the specific suppression of Tc17 differentiation and its plasticity in relation to CTL-driven tumor rejection, which provides further explanation as to why the blockade of PD-1 is such an efficient therapeutic target for inducing tumor rejection.

Age-related Differences in Immune Reactions to SARS-CoV-2 Spike and Nucleocapsid Antigens.

BACKGROUND/AIM: The manifestation and severity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections show a clear correlation to the age of a patient. The younger a person, the less likely the infection results in significant illness. To explore the immunological characteristics behind this phenomenon, we studied the course of SARS-CoV-2 infections in 11 households, including 8 children and 6 infants/neonates of women who got infected with SARS-CoV-2 during pregnancy. MATERIALS AND METHODS: We investigated the immune responses of peripheral blood mononuclear cells (PBMCs), umbilical cord blood mononuclear cells (UCBCs), and T cells against spike and nucleocapsid antigens of SARS-COV-2 by flow cytometry and cytokine secretion assays. RESULTS: Upon peptide stimulation, UCBC from neonates showed a strongly reduced IFN-γ production, as well as lower levels of IL-5, IL-13, and TNF-α alongside with decreased frequencies of surface CD137/PD-1 co-expressing CD4+ and CD+8 T cells compared with adult PBMCs. The PBMC response of older children instead was characterized by elevated frequencies of IFN-γ+ CD4+ T cells, but significantly lower levels of multiple cytokines (IL-5, IL-6, IL-9, IL-10, IL-17A, and TNF-α) and a marked shift of the CD4+/CD8+ T-cell ratio towards CD8+ T cells in comparison to adults. CONCLUSION: The increased severity of SARS-CoV-2 infections in adults could result from the strong cytokine production and lower potential to immunomodulate the excessive inflammation, while the limited IFN-γ production of responding T cells in infants/neonates and the additional higher frequencies of CD8+ T cells in older children may provide advantages during the course of a SARS-CoV-2 infection.

An integrated proteome and transcriptome of B cell maturation defines poised activation states of transitional and mature B cells.

During B cell maturation, transitional and mature B cells acquire cell-intrinsic features that determine their ability to exit quiescence and mount effective immune responses. Here we use label-free proteomics to quantify the proteome of B cell subsets from the mouse spleen and map the differential expression of environmental sensing, transcription, and translation initiation factors that define cellular identity and function. Cross-examination of the full-length transcriptome and proteome identifies mRNAs related to B cell activation and antibody secretion that are not accompanied by detection of the encoded proteins. In addition, proteomic data further suggests that the translational repressor PDCD4 restrains B cell responses, in particular those from marginal zone B cells, to a T-cell independent antigen. In summary, our molecular characterization of B cell maturation presents a valuable resource to further explore the mechanisms underpinning the specialized functions of B cell subsets, and suggest the presence of 'poised' mRNAs that enable expedited B cell responses.

Bifidobacteria shape antimicrobial T-helper cell responses during infancy and adulthood.

Microbial infections early in life are challenging for the unexperienced immune system. The SARS-CoV-2 pandemic again has highlighted that neonatal, infant, child, and adult T-helper(Th)-cells respond differently to infections, and requires further understanding. This study investigates anti-bacterial T-cell responses against Staphylococcus aureus aureus, Staphylococcus epidermidis and Bifidobacterium longum infantis in early stages of life and adults and shows age and pathogen-dependent mechanisms. Beside activation-induced clustering, T-cells stimulated with Staphylococci become Th1-type cells; however, this differentiation is mitigated in Bifidobacterium-stimulated T-cells. Strikingly, prestimulation of T-cells with Bifidobacterium suppresses the activation of Staphylococcus-specific T-helper cells in a cell-cell dependent manner by inducing FoxP3+CD4+ T-cells, increasing IL-10 and galectin-1 secretion and showing a CTLA-4-dependent inhibitory capacity. Furthermore Bifidobacterium dampens Th responses of severely ill COVID-19 patients likely contributing to resolution of harmful overreactions of the immune system. Targeted, age-specific interventions may enhance infection defence, and specific immune features may have potential cross-age utilization.

Immune-checkpoint blockade of CTLA-4 (CD152) in antigen-specific human T-cell responses differs profoundly between neonates, children, and adults.

The monoclonal antibody against CTLA-4, Ipilimumab, is a first-in-class immune-checkpoint inhibitor approved for treatment of advanced melanoma in adults but not extensively studied in children. In light of the fact that the immune response early in life differs from that of adults, we have applied a human in vitro model stimulating CD4+ T-cells from neonates, children (1-5 years), and adults antigen-specifically with Staphylococcus aureus (S. aureus) for assessment of CTLA-4 blockade early in life. We show that T-cell proliferation as well as frequencies of antigen-specific T-cells (CD40L+CD4+) were enhanced in neonatal T-cells upon CTLA-4 blockade showing a larger variance within the group (F-test p < .0001). Using machine learning algorithm Random Forest, adult and neonatal T-cell responses can be unambiguously categorized (F1 score-0.75) on the basis of their cytokine (co-)expression. Blockade of CTLA-4 enhanced frequencies of IL-8, IFNγ, and IL-10 producers among CD40L+ T-cells. Of note, antigen-specific T-cells from neonates displayed higher cytokine coproduction at baseline, while T-cells from children caught up to neonates, and adults to baseline of children upon CTLA-4 blockade. These findings reveal that in neonatal T-cells blockade of CTLA-4 mainly unleashes the antigen-specific capacity by increasing the numbers of responding T-cells, whereas in children and adults it promotes the coexpression of cytokines by individual T-cells. Thus, CTLA-4 blockade boosts antitumor immunity through different mechanisms depending on the patients' age. These data implicate a strong impact of the developmental stage of the T-cell compartment on the effects of immune-checkpoint therapy.

Cell Survival Failure in Effector T Cells From Patients With Systemic Lupus Erythematosus Following Insufficient Up-Regulation of Cold-Shock Y-Box Binding Protein 1.

OBJECTIVE: The importance of cold-shock Y-box binding protein 1 (YB-1) for cell homeostasis is well-documented based on prior observations of its association with certain cancer entities. This study was undertaken to explore the role of YB-1 in T cell homeostasis and survival and the potential contribution of YB-1 to the pathogenesis of systemic lupus erythematosus (SLE). METHODS: In the peripheral blood from 25 SLE patients and 25 healthy donors, the expression of YB-1 and frequency of T cell apoptosis was analyzed by quantitative polymerase chain reaction (qPCR) and fluorescence-activated cell sorting of CD4+ T cells ex vivo and also analyzed in T cells in vitro after 6 days of stimulation with anti-CD3-coupled or anti-CD3/anti-CD28-coupled microspheres. YB-1 was overexpressed using lentiviral transduction with wild-type green fluorescent protein (wtGFP) YB-1, and knockdown of YB-1 was achieved using specific short hairpin RNA (shRNA) (3-fold reduction; P < 0.0001). RESULTS: YB-1 expression was significantly lower in apoptosis-prone T cells and in activated T cells from SLE patients compared to YB-1 expression in nonapoptotic T cells and activated T cells from healthy donors (P = 0.001). Knockdown of YB-1 in T cells consequently led to expression of proapoptotic molecules and caspase 3 activation (1.6-fold), and subsequently, to apoptosis. Furthermore, YB-1 promoted survival pathways involving enhanced protein expression of the kinase Akt (2-fold) and Bcl-2 (3-fold), even when Fas/CD95 was triggered. YB-1-mediated T cell survival was reversed by Akt and phosphatidylinositol 3-kinase (PI3K) inactivation. In SLE patients, rescue of YB-1 expression strongly promoted survival of T cells and even prevented cell death in T cells that were extremely apoptosis-prone. CONCLUSION: Our data show that failure of YB-1 up-regulation in T cells from SLE patients led to enhanced apoptosis. These findings imply that YB-1 plays a crucial role in the disturbed homeostasis of activated T cells leading to hematopoietic alterations in SLE. These insights may help facilitate the development of new treatment strategies for SLE.

CTLA-4-mediated posttranslational modifications direct cytotoxic T-lymphocyte differentiation.

The blockade of inhibitory receptors such as CTLA-4 (CD152) is being used as immune-checkpoint therapy, offering a powerful strategy to restore effective immune responses against tumors. To determine signal components that are induced under the control of CTLA-4 we analyzed activated murine CD8+ T cells by quantitative proteomics. Accurate mass spectrometry revealed that CTLA-4 engagement led to central changes in the phosphorylation of proteins involved in T-cell differentiation. Beside other targets, we discovered a CTLA-4-mediated induction of the translational inhibitor programmed cell death-4 (PDCD4) as a result of FoxO1 nuclear re-localization. PDCD4 further bound a distinct set of mRNAs including Glutaminase, which points out a critical role for CTLA-4 in CD8+ T-cell metabolism. Consequently, PDCD4-deficient cytotoxic T-lymphocytes (CTLs) expressed increased amounts of otherwise repressed effector molecules and ultimately led to superior control of tumor growth in vivo. These findings reveal a novel CTLA-4-mediated pathway to attenuate CTLs and indicate the importance of post-transcriptional mechanisms in the regulation of anti-tumor immune responses.

The differentiation and plasticity of Tc17 cells are regulated by CTLA-4-mediated effects on STATs.

As the blockade of inhibitory surface-molecules such as CTLA-4 on T cells has led to recent advances in antitumor immune therapy, there is great interest in identifying novel mechanisms of action of CD8+ T cells to evoke effective cytotoxic antitumor responses. Using in vitro and in vivo models, we investigated the molecular pathways underlying the CTLA-4-mediated differentiation of IL-17-producing CD8+ T cells (Tc17 cells) that strongly impairs cytotoxicity. Our studies demonstrate that Tc17 cells lacking CTLA-4 signaling have limited production of STAT3-target gene products such as IL-17, IL-21, IL-23R and RORγt. Upon re-stimulation with IL-12, these cells display fast downregulation of Tc17 hallmarks and acquire Tc1 characteristics such as IFNγ and TNF-α co-expression, which is known to correlate with tumor control. Indeed, upon adoptive transfer, these cells were highly efficient in the antigen-specific rejection of established OVA-expressing B16 melanoma in vivo. Mechanistically, in primary and re-stimulated Tc17 cells, STAT3 binding to the IL-17 promoter was strongly augmented by CTLA-4, associated with less binding of STAT5 and reduced relative activation of STAT1 which is known to block STAT3 activity. Inhibiting CTLA-4-induced STAT3 activity reverses enhancement of signature Tc17 gene products, rendering Tc17 cells susceptible to conversion to Tc1-like cells with enhanced cytotoxic potential. Thus, CTLA-4 critically shapes the characteristics of Tc17 cells by regulating relative STAT3 activation, which provides new perspectives to enhance cytotoxicity of antitumor responses.

Migration of Th1 lymphocytes is regulated by CD152 (CTLA-4)-mediated signaling via PI3 kinase-dependent Akt activation.

Efficient adaptive immune responses require the localization of T lymphocytes in secondary lymphoid organs and inflamed tissues. To achieve correct localization of T lymphocytes, the migration of these cells is initiated and directed by adhesion molecules and chemokines. It has recently been shown that the inhibitory surface molecule CD152 (CTLA-4) initiates Th cell migration, but the molecular mechanism underlying this effect remains to be elucidated. Using CD4 T lymphocytes derived from OVA-specific TCR transgenic CD152-deficient and CD152-competent mice, we demonstrate that chemokine-triggered signal transduction is differentially regulated by CD152 via phosphoinositide 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt). In the presence of CD152 signaling, the chemoattractant CCL4 selectively induces the full activation of Akt via phosphorylation at threonine 308 and serine 473 in pro-inflammatory Th lymphocytes expressing the cognate chemokine receptor CCR5. Akt signals lead to cytoskeleton rearrangements, which are indispensable for migration. Therefore, this novel Akt-modulating function of CD152 signals affecting T cell migration demonstrates that boosting CD152 or its down-stream signal transduction could aid therapies aimed at sensitizing T lymphocytes for optimal migration, thus contributing to a precise and effective immune response.