Prophylactic antibodies inhibit spike-specific T and B cell responses after COVID-19 vaccination.

Active and passive immunization is used in high-risk patients to prevent severe courses of COVID-19, but the impact of prophylactic neutralizing antibodies on the immune reaction to the mRNA vaccines has remained enigmatic. Here we show that CD4 T and B cell responses to Spikevax booster immunization are suppressed by the therapeutic antibodies Casirivimab and Imdevimab. B cell and T cell responses were significantly induced in controls but not in antibody-treated patients. The data indicates that humoral immunity, i. e. high levels of antibodies, negatively impacts reactive immunity, resulting in blunted cellular responses upon boosting. This argues for temporal separation of vaccination efforts; with active vaccination preferably applied before prophylactic therapeutic antibody treatment.

Osmium-Labeled Microspheres for Bead-Based Assays in Mass Cytometry.

Polystyrene beads are broadly applied in flow cytometry. Implementing bead-based assays in mass cytometry is desired but hampered by the lack of an elemental label required for their detection. In this study, we introduce stable osmium tetroxide labeling as a universal approach for generating functionalized beads readily detectable by mass cytometry. We demonstrate the utility of osmium-labeled beads for signal spillover compensation in mass cytometry, and, strikingly, their application in quantitative Ab-binding capacity assays combined with high-dimensional profiling of human PBMC enabled the systematic assessment of receptor expression profiles across large numbers of cellular phenotypes. This analysis confirmed increased monocytic Siglec-1 expression in active systemic lupus erythematosus patients and, additionally, revealed interrelated reductions of CD4 expression by regulatory and memory CD4 T cells and HLA-DR expression by myeloid dendritic cells, pointing toward defective cross-talk at the immunological synapse that may limit immune responses in systemic lupus erythematosus. By converting conventional flow cytometry beads into beads suitable for mass cytometry, our approach paves the way toward the broad implementation of bead-based assays in high-dimensional cell profiling studies by mass cytometry in biomedical research.

Identification of cross-reactive antibodies for the detection of lymphocytes, myeloid cells and haematopoietic precursors in the naked mole rat.

The naked mole rat (Heterocephalus glaber, NMR) is a rodent with exceptional longevity, low rates of age-related diseases and spontaneous carcinogenesis. The NMR represents an attractive animal model in longevity and cancer research, but there are no NMR-specific antibodies available to study its immune system with respect to age- and cancer-related questions. Substantial homology of major NMR immune cell markers with those of Guinea pig, human and, to a lesser extent, mouse and rat origin are implicated for the existence of immunological cross-reactivity. We identified 10 antibodies recognising eight immunophenotypic markers expressed on the NMR's T and B lymphocytes, macrophages/monocytes and putative haematopoietic precursors and used them for an immunophenotyping of leukocyte subsets of peripheral blood, spleen and bone marrow samples. Overall, we found that the leukocyte composition of NMR peripheral blood is comparable to that of mice. Notably, the frequency of cytotoxic T cells was found to be lower in the NMR compared to corresponding mouse tissues and human blood. Antibodies used in the present paper are available either commercially or from the scientific community and will provide new opportunities for the NMR as a model system in ageing- and cancer-related research areas.

Dual-labelled antibodies for flow and mass cytometry: A new tool for cross-platform comparison and enrichment of target cells for mass cytometry.

Antibody conjugates applicable in both conventional flow and mass cytometry would offer interesting options for cross-platform comparison, as well as the enrichment of rare target cells by conventional flow cytometry (FC) sorting prior to deep phenotyping by mass cytometry (MC). Here, we introduce a simple method to generate dual fluorochrome/metal-labelled antibodies by consecutive orthogonal labelling. First, we compared different fluorochrome-conjugated antibodies specific for CD4, such as FITC, Vio667, VioGreen or VioBlue for their compatibility with the conventional secondary MAXPAR® labelling protocol. After labelling with 141 Pr, the fluorescence emission spectra of all fluorochromes investigated retained their characteristics, and CD4 dual conjugates (DCs) provided consistent results in immune phenotyping assays performed by FC and MC. The phenotypical composition of CD4+ T-cells was maintained after enrichment by FC sorting using different CD4 DCs. Finally, magnetic cell depletion was combined with FC sorting using CD19-VioBlue-142 Nd, CD20-VioGreen-147 Sm, CD27-Cy5-167 Er and CD38-Alexa488-143 Nd DC to enrich rare human plasmablasts to purities >80%, which allowed a subsequent deep phenotyping by MC. In conclusion, DCs have been successfully established for direct assay comparison between FC and MC, and help to minimise MC data acquisition time for deep phenotyping of rare cell subsets.

IFNα and its response proteins, IP-10 and SIGLEC-1, are biomarkers of disease activity in systemic lupus erythematosus.

OBJECTIVES: To evaluate and compare the clinical efficacy of three biomarkers for interferon (IFN) activity (measured directly and indirectly) and six traditional biomarkers in indicating current and prospective disease activity (DA) in systemic lupus erythematosus (SLE). METHODS: IFNα (dissociation-enhanced lanthanide fluorescent immunoassay), IFNγ-inducible protein 10 (IP-10) (ELISA) and sialic acid-binding Ig-like lectin 1 (SIGLEC-1) (flow cytometry) were measured in 79 accurately characterised patients with lupus and compared with serum titres of Anti-dsDNA (ELISA and radioimmunoassay), Anti-dsDNA-NcX ELISA, Anti-Nuc ELISA, and complement C3 and C4. DA was evaluated using the British Isles Lupus Assessment Group 2004 Index (BILAG-2004) and a modified SLE Disease Activity Index-2000 (mSLEDAI-2K). In addition, 31 clinically quiescent patients were monitored for flares over the course of 180 days. RESULTS: Increased levels of IFNα, IP-10 and SIGLEC-1 were found in 32%, 50% and 86%, respectively, of 66 patients with active SLE. IFNα (r=0.45; p<0.0001) and SIGLEC-1 (r=0.54; p<0.0001) correlated better with BILAG-2004 than did IP-10 (r=0.38; p=0.0002), Farr assay (r=0.40; p=0.0001), Anti-dsDNA-NcX ELISA (r=0.28; p=0.0061), Anti-dsDNA ELISA (r=0.31; p=0.0025), Anti-Nuc ELISA (r=0.25; p=0.0121), C3 (r=-0.43; p<0.0001) and C4 (r=-0.33; p=0.0013). Predictors of SLE flares were disease duration ≤92 months, mild clinical activity (in contrast with no activity), complement C3≤89 mg/dl and IFNα≥20 pg/ml, while only lymphocyte count and age were independent predictors in multivariate analysis. CONCLUSIONS: IFNα, IP-10 and SIGLEC-1 emerged as beneficial biomarkers of DA in patients with SLE. Therefore the implementation of IFN biomarkers in standard lupus diagnostics should be reappraised, especially in view of emerging anti-IFN-directed therapies.

SARS-CoV-2 specific plasma cells acquire long-lived phenotypes in human bone marrow.

BACKGROUND: SARS-CoV-2 specific antibody-secreting plasma cells (PC) mediating specific humoral immunity have been identified in the human bone marrow (BM) after COVID-19 or vaccination against SARS-CoV-2. However, it remained unclear whether or not they acquire phenotypes of human memory plasma cells. METHODS: SARS-CoV-2-specific human bone marrow plasma cells (BMPC) were characterised by tetramer-based, antigen-specific flow cytometry and FluoroSpot assay. FINDINGS: SARS-CoV-2 spike-S1-specific PC were detectable in all tested BM samples of previously vaccinated individuals, representing 0.22% of total BMPC. The majority of SARS-CoV-2-specific BMPC expressed IgG and their specificity for the spike S1 protein indicated emergence from a systemic vaccination response. Of note, one-fifth of SARS-CoV-2-specific BMPC showed the phenotype of memory plasma cells, i.e., downregulated CD19 and present or absent CD45 expression. INTERPRETATION: Our data indicate the establishment of phenotypically diverse SARS-CoV-2-specific PC in the human BM after basic mRNA immunization, including the formation of memory phenotypes. These results suggest the induction of durable humoral immunity after basic mRNA vaccination against SARS-CoV-2. FUNDING: The study was supported by funding by the DFG grants TRR130 TP24, ME 3644/8-1, and the Berlin Senate. SR received funding from DFGSFB-1444 C01 Central Service Project.

Induction of robust cellular and humoral immunity against SARS-CoV-2 after a third dose of BNT162b2 vaccine in previously unresponsive older adults.

Here we compared SARS-CoV-2-specific antibody and T-cell responses between older adults (>80 years old, n = 51) and a younger control group (20-53 years old, n = 46) after receiving two doses of BNT162b2. We found that responses in older adults were generally lower, and we identified 10% low-/non-responders. After receiving a third vaccination with BNT162b2, 4 out of 5 low-/non-responders showed antibody and T-cell responses similar to those of responders after two vaccinations.

Surface CD152 (CTLA-4) expression and signaling dictates longevity of CD28null T cells.

CD28(null) T cells are a highly enriched subset of proinflammatory T cells in patients with autoimmune diseases that are oligoclonal and autoreactive. In this study, we analyzed the role of CD152 signaling on the longevity of human CD28(null) T cells. Using a sensitive staining method for CD152, we show that human CD4(+)CD28(null) and CD8(+)CD28(null) T cells rapidly express surface CD152. Serological inactivation of CD152 using specific Fab or blockade of CD152 ligands using CTLA-4Ig in CD4(+)CD28(null) and CD8(+)CD28(null) T cells enhances apoptosis in a Fas/FasL-dependent manner. CD152 cross-linking on activated CD28(null) cells prevents activation-induced cell death as a result of reduced caspase activity. Apoptosis protection conferred by CD152 is mediated by phosphatidylinositol 3'-kinase-dependent activation of the kinase Akt, resulting in enhanced phosphorylation and thereby inhibition of the proapoptotic molecule Bad. We show that signals triggered by CD152 act directly on activated CD28(null) T lymphocytes and, due to its exclusive expression as a receptor for CD80/CD86 on CD28(null) T cells, prevention of CD152-mediated signaling is likely a target mechanism taking place during therapy with CTLA-4Ig. Our data imply strongly that antagonistic approaches using CD152 signals for chronic immune responses might be beneficial.

An explorative study on deep profiling of peripheral leukocytes to identify predictors for responsiveness to anti-tumour necrosis factor alpha therapies in ankylosing spondylitis: natural killer cells in focus.

BACKGROUND: Therapeutic targeting of tumour necrosis factor (TNF)-α is highly effective in ankylosing spondylitis (AS) patients. However, since one-third of anti-TNF-treated AS patients do not show an adequate clinical response there is an urgent need for new biomarkers that would aid clinicians in their decision-making to select appropriate therapeutic options. Thus, the aim of this explorative study was to identify cell-based biomarkers in peripheral blood that could be used for a pre-treatment stratification of AS patients. METHODS: A high-dimensional, multi-parametric flow cytometric approach was applied to identify baseline predictors in 31 AS patients before treatment with the TNF blockers adalimumab (TNF-neutralisation) and etanercept (soluble TNF receptor). RESULTS: As the major result, the frequencies of natural killer (NK) cells, and in particular CD8-positive (CD8+) NK cell subsets, were most predictive for therapeutic outcome in AS patients. While an inverse correlation between classical CD56+/CD16+ NK cells and reduction of disease activity was observed, the CD8+ NK cell subset behaved in the opposite direction. At baseline, responders showed significantly increased frequencies of CD8+ NK cells compared with non-responders. CONCLUSIONS: This is the first study demonstrating that the composition of the NK cell compartment has predictive power for prediction of therapeutic outcome for anti-TNF-α blockers, and we identified CD8+ NK cells as a potential new player in the TNF-α-driven chronic inflammatory immune response of AS.