Apolipoprotein C3 induces inflammation and organ damage by alternative inflammasome activation.

NLRP3-inflammasome-driven inflammation is involved in the pathogenesis of a variety of diseases. Identification of endogenous inflammasome activators is essential for the development of new anti-inflammatory treatment strategies. Here, we identified that apolipoprotein C3 (ApoC3) activates the NLRP3 inflammasome in human monocytes by inducing an alternative NLRP3 inflammasome via caspase-8 and dimerization of Toll-like receptors 2 and 4. Alternative inflammasome activation in human monocytes is mediated by the Toll-like receptor adapter protein SCIMP. This triggers Lyn/Syk-dependent calcium entry and the production of reactive oxygen species, leading to activation of caspase-8. In humanized mouse models, ApoC3 activated human monocytes in vivo to impede endothelial regeneration and promote kidney injury in an NLRP3- and caspase-8-dependent manner. These data provide new insights into the regulation of the NLRP3 inflammasome and the pathophysiological role of triglyceride-rich lipoproteins containing ApoC3. Targeting ApoC3 might prevent organ damage and provide an anti-inflammatory treatment for vascular and kidney diseases.

Alterations in pathogen-specific cellular and humoral immunity associated with acute peripheral facial palsy of infectious origin.

BACKGROUND: Peripheral facial palsy (PFP) is a common neurologic symptom which can be triggered by pathogens, autoimmunity, trauma, tumors, cholesteatoma or further local conditions disturbing the peripheral section of the nerve. In general, its cause is often difficult to identify, remaining unknown in over two thirds of cases. As we have previously shown that the quantity and quality of pathogen-specific T cells change during active infections, we hypothesized that such changes may also help to identify the causative pathogen in PFPs of unknown origin. METHODS: In this observational study, pathogen-specific T cells were quantified in blood samples of 55 patients with PFP and 23 healthy controls after stimulation with antigens from varicella-zoster virus (VZV), herpes-simplex viruses (HSV) or borrelia. T cells were further characterized by expression of the inhibitory surface molecule CTLA-4, as well as markers for differentiation (CD27) and proliferation (Ki67). Pathogen-specific antibody responses were analyzed using ELISA. Results were compared with conventional diagnostics. RESULTS: Patients with PFP were more often HSV-seropositive than controls (p = 0.0003), whereas VZV- and borrelia-specific antibodies did not differ between groups. Although the quantity and general phenotypical characteristics of antigen-specific T cells did not differ either, expression of CTLA-4 and Ki67 was highly increased in VZV-specific T cells of 9 PFP patients, of which 5 showed typical signs of cutaneous zoster. In the remaining 4 patients, a causal relationship with VZV was possible but remained unclear by clinical standard diagnostics. A similar CTLA-4- and Ki67-expression profile of borrelia-specific T cells was also found in a patient with acute neuroborreliosis. DISCUSSION: In conclusion, the high prevalence of HSV-seropositivity among PFP-patients may indicate an underestimation of HSV-involvement in PFP, even though HSV-specific T cell characteristics seem insufficient to identify HSV as a causative agent. In contrast, striking alterations in VZV- and borrelia-specific T cell phenotype and function may allow identification of VZV- and borrelia-triggered PFPs. If confirmed in larger studies, antigen-specific immune-phenotyping may have the potential to improve specificity of the clinical diagnosis.

Preventing infections in immunocompromised patients with kidney diseases: vaccines and antimicrobial prophylaxis.

The coronavirus disease 2019 (COVID-19) pandemic revealed that our understanding of infectious complications and strategies to mitigate severe infections in patients with glomerular diseases is limited. Beyond COVID-19, there are several infections that specifically impact care of patients receiving immunosuppressive measures. This review will provide an overview of six different infectious complications frequently encountered in patients with glomerular diseases, and will focus on recent achievements in terms of vaccine developments and understanding of the use of specific antimicrobial prophylaxis. These include influenza virus, Streptococcus pneumoniae, reactivation of a chronic or past infection with hepatitis B virus in cases receiving B-cell depletion, reactivation of cytomegalovirus, and cases of Pneumocystis jirovecii pneumonia in patients with anti-neutrophil cytoplasmic antibody-associated vasculitis. Varicella zoster virus infections are particularly frequent in patients with systemic lupus erythematosus and an inactivated vaccine is available to use as an alternative to the attenuated vaccine in patients receiving immunosuppressants. As with COVID-19 vaccines, vaccine responses are generally impaired in older patients, and after recent administration of B-cell depleting agents, and high doses of mycophenolate mofetil and other immunosuppressants. Strategies to curb infectious complications are manifold and will be outlined in this review.

Immunogenicity and reactogenicity of homologous mRNA-based and vector-based SARS-CoV-2 vaccine regimens in patients receiving maintenance dialysis.

Patients receiving maintenance dialysis (MD) are vulnerable to COVID-19-related morbidity and mortality. Currently, data on SARS-CoV-2-specific cellular and humoral immunity post-vaccination in this population are scarce. We conducted a prospective single-center study exploring the specific cellular (interferon-γ and interleukin-2 ELISpot assays) and humoral immune responses (dot plot array and chemiluminescent microparticle immunoassay [CMIA]) at 4 weeks and 6 weeks following a single dose or a complete homologous dual dose SARS-CoV-2 vaccine regimen in 60 MD patients (six with a history of COVID-19). Our results show that MD patients exhibit a high seroconversion rate (91.7%) but the anti-spike IgG antibodies (CMIA) tend to wane rapidly after full immunization. Only 51.7% of the patients developed T cell immune response. High anti-spike IgG antibodies may predict a better cellular immunity. While patients with prior COVID-19 showed the best response after one, SARS-CoV-2-naïve patients may benefit from a third vaccine injection.

Effect of everolimus-based drug regimens on CMV-specific T-cell functionality after renal transplantation: 12-month ATHENA subcohort-study results.

Post-transplant cytomegalovirus (CMV) infections and increased viral replication are associated with CMV-specific T-cell anergy. In the ATHENA-study, de-novo everolimus (EVR) with reduced-exposure tacrolimus (TAC) or cyclosporine (CyA) showed significant benefit in preventing CMV infections in renal transplant recipients as compared to standard TAC + mycophenolic acid (MPA). However, immunomodulatory mechanisms for this effect remain largely unknown. Ninety patients from the ATHENA-study completing the 12-month visit on-treatment (EVR + TAC n = 28; EVR + CyA n = 19; MPA + TAC n = 43) were included in a posthoc analysis. Total lymphocyte subpopulations were quantified. CMV-specific CD4 T cells were determined after stimulation with CMV-antigen, and cytokine-profiles and various T-cell anergy markers were analyzed using flow cytometry. While 25.6% of MPA + TAC-treated patients had CMV-infections, no such events were reported in EVR-treated patients. Absolute numbers of lymphocyte subpopulations were comparable between arms, whereas the percentage of regulatory T cells was significantly higher with EVR + CyA versus MPA + TAC (p = 0.019). Despite similar percentages of CMV-specific T cells, their median expression of CTLA-4 and PD-1 was lower with EVR + TAC (p < 0.05 for both) or EVR + CyA (p = 0.045 for CTLA-4) compared with MPA + TAC. Moreover, mean percentages of multifunctional CMV-specific T cells were higher with EVR + TAC (27.2%) and EVR + CyA (29.4%) than with MPA + TAC (19.0%). In conclusion, EVR-treated patients retained CMV-specific T-cell functionality, which may contribute to enhanced protection against CMV infections.

Cytomegalovirus in the transplant setting: Where are we now and what happens next? A report from the International CMV Symposium 2021.

The CMV Symposium in September 2021 was an international conference dedicated to cytomegalovirus (CMV) infection after solid organ or hematopoietic stem cell transplantation. This review provides an overview of the presentations given by the expert faculty, supplemented with educational clinical cases. Topics discussed include CMV epidemiology and diagnosis, the burden of CMV infection and disease, CMV-specific immunity and management of CMV in transplant settings. Major advances in the prevention and treatment of CMV in the past decade and increased understanding of CMV immunity have led to improved patient outcomes. In the future, management algorithms may be individualized based on the transplant recipient's immune profile, which will mark the start of a new era for patients with CMV.

Quantitative and time-resolved miRNA pattern of early human T cell activation.

T cells are central to the immune response against various pathogens and cancer cells. Complex networks of transcriptional and post-transcriptional regulators, including microRNAs (miRNAs), coordinate the T cell activation process. Available miRNA datasets, however, do not sufficiently dissolve the dynamic changes of miRNA controlled networks upon T cell activation. Here, we established a quantitative and time-resolved expression pattern for the entire miRNome over a period of 24 h upon human T-cell activation. Based on our time-resolved datasets, we identified central miRNAs and specified common miRNA expression profiles. We found the most prominent quantitative expression changes for miR-155-5p with a range from initially 40 molecules/cell to 1600 molecules/cell upon T-cell activation. We established a comprehensive dynamic regulatory network of both the up- and downstream regulation of miR-155. Upstream, we highlight IRF4 and its complexes with SPI1 and BATF as central for the transcriptional regulation of miR-155. Downstream of miR-155-5p, we verified 17 of its target genes by the time-resolved data recorded after T cell activation. Our data provide comprehensive insights into the range of stimulus induced miRNA abundance changes and lay the ground to identify efficient points of intervention for modifying the T cell response.

Impact of COVID-19 in solid organ transplant recipients.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exploded onto the world stage in early 2020. The impact on solid organ transplantation (SOT) has been profound affecting potential donors, candidates, and recipients. Importantly, decreased donations and the pressure of limited resources placed on health care by the pandemic also disrupted transplant systems. We address the impact of COVID-19 on organ transplantation globally and review current understanding of the epidemiology, outcomes, diagnosis, and treatment of COVID-19 in SOT recipients.

Cellular immunity predominates over humoral immunity after homologous and heterologous mRNA and vector-based COVID-19 vaccine regimens in solid organ transplant recipients.

Knowledge on the immunogenicity of vector-based and mRNA-vaccines in solid organ transplant recipients is limited. Therefore, SARS-CoV-2-specific T cells and antibodies were analyzed in 40 transplant recipients and 70 controls after homologous or heterologous vaccine-regimens. Plasmablasts and SARS-CoV-2-specific CD4 and CD8 T cells were quantified using flow cytometry. Specific antibodies were analyzed by ELISA and neutralization assay. The two vaccine types differed after the first vaccination, as IgG and neutralizing activity were more pronounced after mRNA priming (p = .0001 each), whereas CD4 and CD8 T cell levels were higher after vector priming (p = .009; p = .0001). All regimens were well tolerated, and SARS-CoV-2-specific antibodies and/or T cells after second vaccination were induced in 100% of controls and 70.6% of transplant recipients. Although antibody and T cell levels were lower in patients, heterologous vaccination led to the most pronounced induction of antibodies and CD4 T cells. Plasmablast numbers were significantly higher in controls and correlated with SARS-CoV-2-specific IgG- and T cell levels. While antibodies were only detected in 35.3% of patients, cellular immunity was more frequently found (64.7%) indicating that assessment of antibodies is insufficient to identify COVID-19-vaccine responders. In conclusion, heterologous vaccination seems promising in transplant recipients, and combined analysis of humoral and cellular immunity improves the identification of responders among immunocompromised individuals.

High-dose intranasal application of titanium dioxide nanoparticles induces the systemic uptakes and allergic airway inflammation in asthmatic mice.

BACKGROUND: Titanium dioxide nanoparticles (TiO2 NPs) have a wide range of applications in several industrial and biomedical domains. Based on the evidence, the workers exposed to inhaled nanosized TiO2 powder are more susceptible to the risks of developing respiratory diseases. Accordingly, this issue has increasingly attracted the researchers' interest in understanding the consequences of TiO2 NPs exposure. Regarding this, the present study was conducted to analyze the local effects of TiO2 NPs on allergic airway inflammation and their uptake in a mouse model of ovalbumin (OVA)-induced allergic airway inflammation. METHODS: For the purpose of the study, female BALB/c mice with or without asthma were intranasally administered with TiO2 NPs. The mice were subjected to histological assessment, lung function testing, scanning electron microscopy (SEM), inductively coupled plasma mass spectrometry (ICP-MS), and NP uptake measurement. In addition, T helper (Th) 1/Th2 cytokines were evaluated in the lung homogenate using the enzyme-linked immunosorbent assay. RESULTS: According to the results, the mice receiving OVA alone or OVA plus TiO2 NPs showed eosinophilic infiltrates and mucus overproduction in the lung tissues, compared to the controls. Furthermore, a significant elevation was observed in the circulating Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13 after NP exposure. The TiO2 NPs were taken up by alveolar macrophages at different time points. As the results of the SEM and ICP-MS indicated, TiO2 NPs were present in most of the organs in both asthmatic and non-asthmatic mice. CONCLUSION: Based on the findings of the current study, intranasally or inhalation exposure to high-dose nanosized TiO2 particles appears to exacerbate the allergic airway inflammation and lead to systemic uptake in extrapulmonary organs. These results indicate the very important need to investigate the upper limit of intranasally or inhalation exposure to nanosized TiO2 particles in occupational and environmental health policy.

Elite athletes on regular training show more pronounced induction of vaccine-specific T-cells and antibodies after tetravalent influenza vaccination than controls.

Compliance of elite athletes with vaccination recommendations is low mainly based on concerns about side-effects and perceived poor vaccine efficacy due to continued physical training. We therefore employed seasonal influenza vaccination to investigate the effect of regular physical training on vaccine-induced cellular and humoral immunity in elite athletes and controls. Lymphocyte subpopulations and vaccine-specific T-cells were quantified and functionally characterized from 45 athletes and 25 controls before, and 1, 2 and 26 weeks after vaccination. Moreover, influenza-specific antibodies and their neutralizing function were quantified. Both groups showed a significant increase in vaccine-reactive CD4 T-cell levels which peaked one week after vaccination (p < 0.0001). The increase was significantly more pronounced in athletes (4.1-fold) compared to controls (2.3-fold; p = 0.0007). The cytokine profile changed from multifunctional T-cells co-producing IFNγ, IL-2 and TNFα to cells with restricted cytokine expression. This change in functionality was associated with a significant increase in CTLA-4 expression (p < 0.0001), which again was more pronounced in athletes. Likewise, the increase in neutralizing antibodies was stronger in athletes (p = 0.004 for H1N1; p = 0.032 for H3N2). In conclusion, both groups mounted a strong vaccine-specific cellular and humoral immunity after standard vaccination. The more pronounced increase in specific T-cells and neutralizing antibodies indicates that high frequency and intensity of training enhance vaccine-responses in elite athletes.

CTLA-4-expression on VZV-specific T cells in CSF and blood is specifically increased in patients with VZV related central nervous system infections.

VZV-reactivation may lead to symptomatic central nervous system (CNS) diseases, but identification of VZV as causative pathogen of CNS-diseases is challenging. This study was performed to characterize VZV-specific T cells from cerebrospinal fluid (CSF) and blood of patients with active CNS-disease and to determine whether this may improve differential diagnosis. 27 patients with pleocytosis in the CSF were recruited and classified into three groups (10 VZV-related, 10 non-VZV-related, 7 unclear). VZV-specific CD4+ T cells were quantified in CSF and blood after simultaneous stimulation with a VZV-antigen lysate and detection of cytokines (IFN-γ, IL-2, TNF-α) and CTLA-4. Polyclonal stimulation served as positive control. VZV-specific CD4+ T-cell frequencies were highest in both CSF (p = 0.0001) and blood (p = 0.011) of patients with VZV-infection, and were enriched at the site of infection (p = 0.002). While cytokine-expression profiles only showed minor differences between the groups, CTLA-4-expression levels on VZV-specific T cells from CSF and blood were significantly increased in VZV-related CNS-infections (p = 0.0002 and p<0.0001) and clearly identified VZV-related CNS-diseases (100% sensitivity and 100% specificity). Polyclonally stimulated T cells did not show any quantitative and phenotypical differences between the groups. Increased frequency and CTLA-4-expression of VZV-specific T cells from CSF or blood are specifically found in patients with VZV-related CNS-infection.

Assay for improved detection of antigen-specific immune cells from extrasanguinous fluids.

In this approach, pre-stained cells from extrasanguinous fluids (ESFs) are stimulated in the presence of blood from the same individual. Thus, blood-derived antigen-presenting cells enable stimulation of both ESF- and blood T cells. Pre-staining allows distinction of T cells from ESF and blood, and simultaneous analysis of antigen-specific T cells in both compartments.

Assigning Cytomegalovirus Status in Children Awaiting Organ Transplant: Viral Shedding, CMV-Specific T Cells, and CD27-CD28-CD4+ T Cells.

Passive antibodies, maternal or transfusion-acquired, make serologic determination of pretransplant cytomegalovirus (CMV) status unreliable. We evaluated 3 assays unaffected by passive antibodies, in assignment of CMV infection status in children awaiting solid organ transplant and in controls: (1) CMV nucleic acid amplification testing (NAAT), (2) quantification of CMV-specific CD4+ T cells, and (3) quantification of CD27-CD28-CD4+ T cells. Our results highlight that CMV NAAT, from urine and oropharynx, is useful in confirming positive CMV status. Detection of CMV-specific CD4+ T cells was sensitive and specific in children >18 months but was less sensitive in children <12 months. CD27-CD28-CD4+ T cells are not likely useful in CMV risk stratification in children.

Donor-specific alloreactive T cells can be quantified from whole blood, and may predict cellular rejection after renal transplantation.

Preformed cellular alloreactivity can exist prior to transplantation and may contribute to rejection. Here, we used a rapid flow-cytometric whole-blood assay to characterize the extent of alloreactive T cells among 1491 stimulatory reactions from 61 renal transplant candidates and 75 controls. The role of preformed donor-specific alloreactive T cells in cellular rejection was prospectively analyzed in 21 renal transplant recipients. Alloreactive CD8+ T cells were more frequent than respective CD4+ T cells, and these levels were stable over time. CD8+ T cells were effector-memory T cells largely negative for expression of CD27, CD62L, and CCR7, and were susceptible to steroid and calcineurin inhibitor inhibition. Alloreactivity was more frequent in samples with higher number of HLA mismatches. Moreover, the percentage of individuals with alloreactive T cells was higher in transplant candidates than in controls. Among transplant candidates, 5/61 exhibited alloreactive CD8+ T cells against most stimulators, 23/61 toward a limited number of stimulators, and 33/61 did not show any alloreactivity. Among 21 renal transplant recipients followed prospectively, one had donor-specific preformed T-cell alloreactivity. She was the only patient who developed cellular rejection posttransplantation. In conclusion, donor-specific alloreactive T cells may be rapidly quantified from whole blood, and may predict cellular rejection after transplantation.

Quantity, quality, and functionality of peripheral blood cells derived from residual blood of different apheresis kits.

BACKGROUND: Research with primary human white blood cell (WBC) subpopulations requires high quantity, quality, and functionality of peripheral blood mononuclear cells (PBMCs) as a source to further characterize cellular subpopulations such as T and B lymphocytes, monocytes, or natural killer cells. Apart from buffy coats derived from whole blood, residual blood from preparative hemapheresis kits are used as a source for PBMCs, but knowledge on the yield and functionality of cells from different devices is limited. STUDY DESIGN AND METHODS: We evaluated quantity and quality of PBMCs isolated from apheresis kits of two apheresis devices (AMICUS, Fenwal; and Trima Accel, Terumo BCT), the latter being our standard source for many years. PBMCs derived from Trima or AMICUS were tested for yield and subtype composition by flow cytometry. Functionality was assessed by cytokine induction of CD4+ and CD8+ T cells and by degranulation. Moreover, cytotoxic activity of natural killer cells was quantified by a real-time killing assay. RESULTS: Mean numbers of isolated cells were 5.5 ± 2.4 × 108 for AMICUS, and 10.3 ± 6.4 × 108 for Trima Accel, respectively. The proportion of WBC subtypes corresponded to well-known numbers from whole blood, with minor differences between the two apheresis systems. Likewise, minor differences in cytokine induction were found in stimulated CD4+ or CD8+ T cells. Finally, PBMCs derived from the two systems showed comparable cytotoxic activity. CONCLUSION: PBMC derived from residual blood of the AMICUS and Trima Accel apheresis devices serve as an economic and easily accessible source for functional PBMCs with comparable quantity and quality to PBMCs derived from whole blood.

Rapid reconstitution of CMV-specific T-cells after stem-cell transplantation.

OBJECTIVE: As reconstitution of virus-specific T-cells is critical to control cytomegalovirus (CMV)-viremia following stem-cell transplantation (SCT), we characterized the dynamics in CMV-specific T-cell reconstitution after SCT. METHODS: Cytomegalovirus-specific T-cells from 51 SCT-recipients were prospectively quantified and phenotypically characterised by intracellular cytokine-staining after specific stimulation and HLA class-I-specific pentamers using flow cytometry. RESULTS: Cytomegalovirus-specific CD4 T-cells reconstituted after a median of 2.3 (IQR, 2.0-3.0) weeks following autografting, and 4.0 (IQR, 3.0-5.6) weeks after allografting, with CMV-specific T-cells originating from donors and/or recipients. The time for reconstitution of CMV-specific CD4 and CD8 T-cells did not differ (P = .58). Factors delaying the time to initial reconstitution of CMV-specific CD4 T-cells included a negative recipient serostatus (P = .016) and CMV-viremia (P = .026). Percentages of CMV-specific CD4 T-cells significantly increased over time and reached a plateau after 90 days (P = .043). Relative CMV-specific CD4 T-cell levels remained higher in long-term transplant recipients compared with those in controls (P < .0001). However, due to persisting lymphopenia, absolute numbers of CMV-specific T-cells were similar as in controls. CONCLUSION: Cytomegalovirus-specific T-cells rapidly reconstitute after SCT and their percentages remain high in the long term. In the face of persistent lymphopenia, this results in similar absolute numbers of CMV-specific T-cells as in controls to ensure sufficient pathogen control.