Persistent complement dysregulation with signs of thromboinflammation in active Long Covid.

Long Covid is a debilitating condition of unknown etiology. We performed multimodal proteomics analyses of blood serum from COVID-19 patients followed up to 12 months after confirmed severe acute respiratory syndrome coronavirus 2 infection. Analysis of >6500 proteins in 268 longitudinal samples revealed dysregulated activation of the complement system, an innate immune protection and homeostasis mechanism, in individuals experiencing Long Covid. Thus, active Long Covid was characterized by terminal complement system dysregulation and ongoing activation of the alternative and classical complement pathways, the latter associated with increased antibody titers against several herpesviruses possibly stimulating this pathway. Moreover, markers of hemolysis, tissue injury, platelet activation, and monocyte-platelet aggregates were increased in Long Covid. Machine learning confirmed complement and thromboinflammatory proteins as top biomarkers, warranting diagnostic and therapeutic interrogation of these systems.

A systematic review of interleukin-2-based immunotherapies in clinical trials for cancer and autoimmune diseases.

BACKGROUND: The cytokine interleukin-2 (IL-2) can stimulate both effector immune cells and regulatory T (Treg) cells. The ability of selectively engaging either of these effects has spurred interest in using IL-2 for immunotherapy of cancer and autoimmune diseases. Thus, numerous IL-2-based biologic agents with improved bias or delivery towards effector immune cells or Treg cells have been developed. This study systematically reviews clinical results of improved IL-2-based compounds. METHODS: We searched the ClinicalTrials.gov database for registered trials using improved IL-2-based agents and different databases for available results of these studies. FINDINGS: From 576 registered clinical trials we extracted 36 studies on different improved IL-2-based compounds. Adding another nine agents reported in recent literature reviews and based on our knowledge totalled in 45 compounds. A secondary search for registered clinical trials of each of these 45 compounds resulted in 141 clinical trials included in this review, with 41 trials reporting results. INTERPRETATION: So far, none of the improved IL-2-based compounds has gained regulatory approval for the treatment of cancer or autoimmune diseases. NKTR-214 is the only compound completing phase 3 studies. The PIVOT IO-001 trial testing the combination of NKTR-214 plus Pembrolizumab compared to Pembrolizumab monotherapy in metastatic melanoma missed its primary endpoints. Also the PIVOT-09 study, combining NKTR-214 with Nivolumab compared to Sunitinib or Cabozantinib in advanced renal cell carcinoma, missed its primary endpoint. Trials in autoimmune diseases are currently in early stages, thus not allowing definite conclusions on efficacy. FUNDING: This work was supported by public funding agencies.

Human memory B cells show plasticity and adopt multiple fates upon recall response to SARS-CoV-2.

The B cell response to different pathogens uses tailored effector mechanisms and results in functionally specialized memory B (Bm) cell subsets, including CD21+ resting, CD21-CD27+ activated and CD21-CD27- Bm cells. The interrelatedness between these Bm cell subsets remains unknown. Here we showed that single severe acute respiratory syndrome coronavirus 2-specific Bm cell clones showed plasticity upon antigen rechallenge in previously exposed individuals. CD21- Bm cells were the predominant subsets during acute infection and early after severe acute respiratory syndrome coronavirus 2-specific immunization. At months 6 and 12 post-infection, CD21+ resting Bm cells were the major Bm cell subset in the circulation and were also detected in peripheral lymphoid organs, where they carried tissue residency markers. Tracking of individual B cell clones by B cell receptor sequencing revealed that previously fated Bm cell clones could redifferentiate upon antigen rechallenge into other Bm cell subsets, including CD21-CD27- Bm cells, demonstrating that single Bm cell clones can adopt functionally different trajectories.

Adaptive Immunosuppression in Lung Transplant Recipients Applying Complementary Biomarkers: The Zurich Protocol.

Achieving adequate immunosuppression for lung transplant recipients in the first year after lung transplantation is a key challenge. Prophylaxis of allograft rejection must be balanced with the adverse events associated with immunosuppressive drugs, for example infection, renal failure, and diabetes. A triple immunosuppressive combination is standard, including a steroid, a calcineurin inhibitor, and an antiproliferative compound beginning with the highest levels of immunosuppression and a subsequent tapering of the dose, usually guided by therapeutic drug monitoring and considering clinical results, bronchoscopy sampling results, and additional biomarkers such as serum viral replication or donor-specific antibodies. Balancing the net immunosuppression level required to prevent rejection without overly increasing the risk of infection and other complications during the tapering phase is not well standardized and requires repeated assessments for dose-adjustments. In our adaptive immunosuppression approach, we additionally consider results from the white blood cell counts, in particular lymphocytes and eosinophils, as biomarkers for monitoring the level of immunosuppression and additionally use them as therapeutic targets to fine-tune the immunosuppressive strategy over time. The concept and its rationale are outlined, and areas of future research mentioned.

Interleukin-2-based therapies in cancer.

Molecular insights into the mechanism of beneficial and adverse effects of interleukin-2 (IL-2) have resulted in the development of improved IL-2 formulations with IL-2 receptor bias and tissue-targeting properties. Several of these compounds are currently in clinical development and are ushering IL-2 therapy into the current era of cancer immunotherapy.

T-cell recovery and evidence of persistent immune activation 12 months after severe COVID-19.

BACKGROUND: T-cell lymphopenia and functional impairment is a hallmark of severe acute coronavirus disease 2019 (COVID-19). How T-cell numbers and function evolve at later timepoints after clinical recovery remains poorly investigated. METHODS: We prospectively enrolled and longitudinally sampled 173 individuals with asymptomatic to critical COVID-19 and analyzed phenotypic and functional characteristics of T cells using flow cytometry, 40-parameter mass cytometry, targeted proteomics, and functional assays. RESULTS: The extensive T-cell lymphopenia observed particularly in patients with severe COVID-19 during acute infection had recovered 6 months after infection, which was accompanied by a normalization of functional T-cell responses to common viral antigens. We detected persisting CD4+ and CD8+ T-cell activation up to 12 months after infection, in patients with mild and severe COVID-19, as measured by increased HLA-DR and CD38 expression on these cells. Persistent T-cell activation after COVID-19 was independent of administration of a COVID-19 vaccine post-infection. Furthermore, we identified a subgroup of patients with severe COVID-19 that presented with persistently low CD8+ T-cell counts at follow-up and exhibited a distinct phenotype during acute infection consisting of a dysfunctional T-cell response and signs of excessive pro-inflammatory cytokine production. CONCLUSION: Our study suggests that T-cell numbers and function recover in most patients after COVID-19. However, we find evidence of persistent T-cell activation up to 12 months after infection and describe a subgroup of severe COVID-19 patients with persistently low CD8+ T-cell counts exhibiting a dysregulated immune response during acute infection.

Autoantibodies in COVID-19 correlate with antiviral humoral responses and distinct immune signatures.

BACKGROUND: Several autoimmune features occur during coronavirus disease 2019 (COVID-19), with possible implications for disease course, immunity, and autoimmune pathology. In this study, we longitudinally screened for clinically relevant systemic autoantibodies to assess their prevalence, temporal trajectory, and association with immunity, comorbidities, and severity of COVID-19. METHODS: We performed highly sensitive indirect immunofluorescence assays to detect antinuclear antibodies (ANA) and antineutrophil cytoplasmic antibodies (ANCA), along with serum proteomics and virome-wide serological profiling in a multicentric cohort of 175 COVID-19 patients followed up to 1 year after infection, eleven vaccinated individuals, and 41 unexposed controls. RESULTS: Compared with healthy controls, similar prevalence and patterns of ANA were present in patients during acute COVID-19 and recovery. However, the paired analysis revealed a subgroup of patients with transient presence of certain ANA patterns during acute COVID-19. Furthermore, patients with severe COVID-19 exhibited a high prevalence of ANCA during acute disease. These autoantibodies were quantitatively associated with higher SARS-CoV-2-specific antibody titers in COVID-19 patients and in vaccinated individuals, thus linking autoantibody production to increased antigen-specific humoral responses. Notably, the qualitative breadth of antibodies cross-reactive with other coronaviruses was comparable in ANA-positive and ANA-negative individuals during acute COVID-19. In autoantibody-positive patients, multiparametric characterization demonstrated an inflammatory signature during acute COVID-19 and alterations of the B-cell compartment after recovery. CONCLUSION: Highly sensitive indirect immunofluorescence assays revealed transient autoantibody production during acute SARS-CoV-2 infection, while the presence of autoantibodies in COVID-19 patients correlated with increased antiviral humoral immune responses and inflammatory immune signatures.

Immunoglobulin signature predicts risk of post-acute COVID-19 syndrome.

Following acute infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) a significant proportion of individuals develop prolonged symptoms, a serious condition termed post-acute coronavirus disease 2019 (COVID-19) syndrome (PACS) or long COVID. Predictors of PACS are needed. In a prospective multicentric cohort study of 215 individuals, we study COVID-19 patients during primary infection and up to one year later, compared to healthy subjects. We discover an immunoglobulin (Ig) signature, based on total IgM and IgG3 levels, which - combined with age, history of asthma bronchiale, and five symptoms during primary infection - is able to predict the risk of PACS independently of timepoint of blood sampling. We validate the score in an independent cohort of 395 individuals with COVID-19. Our results highlight the benefit of measuring Igs for the early identification of patients at high risk for PACS, which facilitates the study of targeted treatment and pathomechanisms of PACS.

Signature of long-lived memory CD8+ T cells in acute SARS-CoV-2 infection.

Immunological memory is a hallmark of adaptive immunity and facilitates an accelerated and enhanced immune response upon re-infection with the same pathogen1,2. Since the outbreak of the ongoing COVID-19 pandemic, a key question has focused on which SARS-CoV-2-specific T cells stimulated during acute infection give rise to long-lived memory T cells3. Here, using spectral flow cytometry combined with cellular indexing of transcriptomes and T cell receptor sequencing, we longitudinally characterized individual SARS-CoV-2-specific CD8+ T cells of patients with COVID-19 from acute infection to 1 year into recovery and found a distinct signature identifying long-lived memory CD8+ T cells. SARS-CoV-2-specific memory CD8+ T cells persisting 1 year after acute infection express CD45RA, IL-7 receptor-α and T cell factor 1, but they maintain low expression of CCR7, thus resembling CD45RA+ effector memory T cells. Tracking individual clones of SARS-CoV-2-specific CD8+ T cells, we reveal that an interferon signature marks clones that give rise to long-lived cells, whereas prolonged proliferation and mechanistic target of rapamycin signalling are associated with clonal disappearance from the blood. Collectively, we describe a transcriptional signature that marks long-lived, circulating human memory CD8+ T cells following an acute viral infection.

Profound dysregulation of T cell homeostasis and function in patients with severe COVID-19.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and shows a broad clinical presentation ranging from asymptomatic infection to fatal disease. A very prominent feature associated with severe COVID-19 is T cell lymphopenia. However, homeostatic and functional properties of T cells are ill-defined in COVID-19. METHODS: We prospectively enrolled individuals with mild and severe COVID-19 into our multicenter cohort and performed a cross-sectional analysis of phenotypic and functional characteristics of T cells using 40-parameter mass cytometry, flow cytometry, targeted proteomics, and functional assays. RESULTS: Compared with mild disease, we observed strong perturbations of peripheral T cell homeostasis and function in severe COVID-19. Individuals with severe COVID-19 showed T cell lymphopenia and redistribution of T cell populations, including loss of naïve T cells, skewing toward CD4+ T follicular helper cells and cytotoxic CD4+ T cells, and expansion of activated and exhausted T cells. Extensive T cell apoptosis was particularly evident with severe disease and T cell lymphopenia, which in turn was accompanied by impaired T cell responses to several common viral antigens. Patients with severe disease showed elevated interleukin-7 and increased T cell proliferation. Furthermore, patients sampled at late time points after symptom onset had higher T cell counts and improved antiviral T cell responses. CONCLUSION: Our study suggests that severe COVID-19 is characterized by extensive T cell dysfunction and T cell apoptosis, which is associated with signs of homeostatic T cell proliferation and T cell recovery.

A distinct innate immune signature marks progression from mild to severe COVID-19.

Coronavirus disease 2019 (COVID-19) manifests with a range of severities, but immune signatures of mild and severe disease are still not fully understood. Here, we use mass cytometry and targeted proteomics to profile the innate immune response of patients with mild or severe COVID-19 and of healthy individuals. Sampling at different stages allows us to reconstruct a pseudo-temporal trajectory of the innate response. A surge of CD169+ monocytes associated with an IFN-γ+MCP-2+ signature rapidly follows symptom onset. At later stages, we observe a persistent inflammatory phenotype in patients with severe disease, dominated by high CCL3 and CCL4 abundance correlating with the re-appearance of CD16+ monocytes, whereas the response of mild COVID-19 patients normalizes. Our data provide insights into the dynamic nature of inflammatory responses in COVID-19 patients and identify sustained innate immune responses as a likely mechanism in severe patients, thus supporting the investigation of targeted interventions in severe COVID-19.

Systemic and mucosal antibody responses specific to SARS-CoV-2 during mild versus severe COVID-19.

BACKGROUND: Whereas severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody tests are increasingly being used to estimate the prevalence of SARS-CoV-2 infection, the determinants of these antibody responses remain unclear. OBJECTIVES: Our aim was to evaluate systemic and mucosal antibody responses toward SARS-CoV-2 in mild versus severe coronavirus disease 2019 (COVID-19) cases. METHODS: Using immunoassays specific for SARS-CoV-2 spike proteins, we determined SARS-CoV-2-specific IgA and IgG in sera and mucosal fluids of 2 cohorts, including SARS-CoV-2 PCR-positive patients (n = 64) and PCR-positive and PCR-negtive health care workers (n = 109). RESULTS: SARS-CoV-2-specific serum IgA titers in patients with mild COVID-19 were often transiently positive, whereas serum IgG titers remained negative or became positive 12 to 14 days after symptom onset. Conversely, patients with severe COVID-19 showed a highly significant increase of SARS-CoV-2-specific serum IgA and IgG titers after symptom onset. Very high titers of SARS-CoV-2-specific serum IgA were correlated with severe acute respiratory distress syndrome. Interestingly, some health care workers with negative SARS-CoV-2-specific serum antibody titers showed SARS-CoV-2-specific IgA in mucosal fluids with virus-neutralizing capacity in some cases. SARS-CoV-2-specific IgA titers in nasal fluids were inversely correlated with age. CONCLUSIONS: Systemic antibody production against SARS-CoV-2 develops mainly in patients with severe COVID-19, with very high IgA titers seen in patients with severe acute respiratory distress syndrome, whereas mild disease may be associated with transient production of SARS-CoV-2-specific antibodies but may stimulate mucosal SARS-CoV-2-specific IgA secretion.

Interleukin-2 signals converge in a lymphoid-dendritic cell pathway that promotes anticancer immunity.

Tumor-infiltrating dendritic cells (DCs) correlate with effective anticancer immunity and improved responsiveness to anti-PD-1 checkpoint immunotherapy. However, the drivers of DC expansion and intratumoral accumulation are ill-defined. We found that interleukin-2 (IL-2) stimulated DC formation through innate and adaptive lymphoid cells in mice and humans, and this increase in DCs improved anticancer immunity. Administration of IL-2 to humans within a clinical trial and of IL-2 receptor (IL-2R)-biased IL-2 to mice resulted in pronounced expansion of type 1 DCs, including migratory and cross-presenting subsets, and type 2 DCs, although neither DC precursors nor mature DCs had functional IL-2Rs. In mechanistic studies, IL-2 signals stimulated innate lymphoid cells, natural killer cells, and T cells to synthesize the cytokines FLT3L, CSF-2, and TNF. These cytokines redundantly caused DC expansion and activation, which resulted in improved antigen processing and correlated with favorable anticancer responses in mice and patients. Thus, IL-2 immunotherapy-mediated stimulation of DCs contributes to anticancer immunity by rendering tumors more immunogenic.

Rapid expansion of Treg cells protects from collateral colitis following a viral trigger.

Foxp3+ regulatory T (Treg) cells are essential for maintaining peripheral tolerance and preventing autoimmunity. While genetic factors may predispose for autoimmunity, additional environmental triggers, such as viral infections, are usually required to initiate the onset of disease. Here, we show that viral infection with LCMV results in type I IFN-dependent Treg cell loss that is rapidly compensated by the conversion and expansion of Vß5+ conventional T cells into iTreg cells. Using Vß5-deficient mice, we show that these Vß5+ iTreg cells are dispensable for limiting anti-viral immunity. Rather, the delayed replenishment of Treg cells in Vß5-deficient mice compromises suppression of microbiota-dependent activation of CD8+ T cells, resulting in colitis. Importantly, recovery from clinical symptoms in IBD patients is marked by expansion of the corresponding Vß2+ Treg population in humans. Collectively, we provide a link between a viral trigger and an impaired Treg cell compartment resulting in the initiation of immune pathology.

IL-4 receptor engagement in human neutrophils impairs their migration and extracellular trap formation.

BACKGROUND: Type 2 immunity serves to resist parasitic helminths, venoms, and toxins, but the role and regulation of neutrophils during type 2 immune responses are controversial. Helminth models suggested a contribution of neutrophils to type 2 immunity, whereas neutrophils are associated with increased disease severity during type 2 inflammatory disorders, such as asthma. OBJECTIVE: We sought to evaluate the effect of the prototypic type 2 cytokines IL-4 and IL-13 on human neutrophils. METHODS: Human neutrophils from peripheral blood were assessed without or with IL-4 or IL-13 for (1) expression of IL-4 receptor subunits, (2) neutrophil extracellular trap (NET) formation, (3) migration toward CXCL8 in vitro and in humanized mice, and (4) CXCR1, CXCR2, and CXCR4 expression, as well as (5) in nonallergic versus allergic subjects. RESULTS: Human neutrophils expressed both types of IL-4 receptors, and their stimulation through IL-4 or IL-13 diminished their ability to form NETs and migrate toward CXCL8 in vitro. Likewise, in vivo chemotaxis in NOD-scid-Il2rg-/- mice was reduced in IL-4-stimulated human neutrophils compared with control values. These effects were accompanied by downregulation of the CXCL8-binding chemokine receptors CXCR1 and CXCR2 on human neutrophils on IL-4 or IL-13 stimulation in vitro. Ex vivo analysis of neutrophils from allergic patients or exposure of neutrophils from nonallergic subjects to allergic donor serum in vitro impaired their NET formation and migration toward CXCL8, thereby mirroring IL-4/IL-13-stimulated neutrophils. CONCLUSION: IL-4 receptor signaling in human neutrophils affects several neutrophil effector functions, which bears important implications for immunity in type 2 inflammatory disorders.

The transcription factor Rfx7 limits metabolism of NK cells and promotes their maintenance and immunity.

Regulatory factor X 7 (Rfx7) is an uncharacterized transcription factor belonging to a family involved in ciliogenesis and immunity. Here, we found that deletion of Rfx7 leads to a decrease in natural killer (NK) cell maintenance and immunity in vivo. Genomic approaches showed that Rfx7 coordinated a transcriptional network controlling cell metabolism. Rfx7-/- NK lymphocytes presented increased size, granularity, proliferation, and energetic state, whereas genetic reduction of mTOR activity mitigated those defects. Notably, Rfx7-deficient NK lymphocytes were rescued by interleukin 15 through engagement of the Janus kinase (Jak) pathway, thus revealing the importance of this signaling for maintenance of such spontaneously activated NK cells. Rfx7 therefore emerges as a novel transcriptional regulator of NK cell homeostasis and metabolic quiescence.

The role of cytokines in T-cell memory in health and disease.

Upon stimulation with their cognate antigen, naive T cells undergo proliferation and differentiation into effector cells, followed by apoptosis or survival as precursors of long-lived memory cells. These phases of a T-cell response and the ensuing maintenance of memory T cells are shaped by cytokines, most notably interleukin-2 (IL-2), IL-7, and IL-15 that share the common γ chain (γc ) cytokine receptor. Steady-state production of IL-7 and IL-15 is necessary for background proliferation and homeostatic survival of CD4+ and CD8+ memory T cells. During immune responses, augmented levels of IL-2, IL-15, IL-21, IL-12, IL-18, and type-I interferons determine the memory potential of antigen-specific effector CD8+ cells, while increased IL-2 and IL-15 cause bystander proliferation of heterologous CD4+ and CD8+ memory T cells. Limiting availability of γc cytokines, reduction in regulatory T cells or IL-10, and persistence of inflammation or cognate antigen can result in memory T cells, which fail to become cytokine-dependent long-lived cells. Conversely, increased IL-7 and IL-15 can expand memory T cells, including pathogenic tissue-resident memory T cells, as seen in lymphopenia and certain chronic-inflammatory disorders and malignancies. These abovementioned factors impact immunotherapy and vaccines directed at memory T cells in cancer and chronic infection.

Modulation of T cell responses by IL-2 and IL-2 complexes.

Interleukin-2 (IL-2) is a cytokine centrally involved in the regulation of immune tolerance and activation by its effects on CD4+ T regulatory (Treg) cells and cytotoxic effector lymphocytes, respectively. Due to these properties IL-2 immunotherapy has been used, as low-dose IL-2, in the treatment of autoimmune and chronic-inflammatory disorders; conversely, at high doses, IL-2 has shown efficacy in a subset of patients with metastatic cancer. Recent advances have highlighted the possibility of using improved IL-2-based therapies, such IL-2-antibody complexes (IL-2 complexes), able to selectively and potently stimulate either Treg cells or cytotoxic effector cells. This article discusses the properties and clinical implications of IL-2 and IL-2 complexes.

Use of enhanced interleukin-2 formulations for improved immunotherapy against cancer.

The use of interleukin-2 (IL-2) for the stimulation of an effector immune response against metastatic cancer dates back to the early 1980s. Administration of unmodified IL-2, either alone or together with antigen-specific approaches, has resulted in remarkably long-term survival of some patients suffering from metastatic melanoma. However, such treatment is usually hampered by the appearance of toxic adverse effects, which has motivated the engineering of modified IL-2 formulations showing reduced toxicity while being more potent at stimulating anti-tumor effector immune cells. In this review we summarize and discuss the features and biological relevance of several enhanced IL-2 formulations, compare these to IL-15-based therapeutics, and try to foreshadow their potential in immunological research and immunotherapy.