Recruitment of plasma cells to the bone marrow in primary and secondary immune reactions (preprint)

Bone marrow plasma cells (BMPC) emerge as a consequence of immune reactions and are considered the source of antibodies that protect against recurrent infectious diseases throughout life. Despite their importance, it remains unclear if these cells reflect different activation environments or the differentiation/maturation stages of their precursors. Here we track the recruitment of plasma cells, generated in primary and secondary immune reactions to SARS-CoV-2 spike protein vaccines, to the human bone marrow. Trajectories based on single cell transcriptomes and antigen-receptor clonotypes of antibody-secreting cells exiting the immune reaction and of those residing in the bone marrow, allow to follow the evolution of the immune response to these vaccines, leading to sequential colonization of these cells to different compartments (clans) of BMPC, and their establishment as long-lived (memory) plasma cells. In primary immune reactions, both CD19low (clans 1 and 4) and CD19high (clan 0) BMPC are generated. In secondary immune reactions, mostly CD19high BMPC of the largest compartment (clan 0) are generated, resulting from the reactivation of memory B lymphocytes. The latter is also observed in vaccinated convalescent individuals and upon recall vaccination against diphtheria/tetanus/pertussis (DTP). Thus, humoral immunological memory, i.e. serum antibodies secreted by long-lived memory BMPC, is generated already in the primary immune response, more so in the secondary, and it represents the evolution of the immune response.

Differential durability of humoral and T cell immunity after two and three BNT162b2 vaccinations in adults aged >80 years (preprint)

A third mRNA-based “booster” vaccination is the favored strategy to maintain protection against SARS-CoV-2 infection. Yet, significant waning of specific immunity within six months after 2nd vaccination, along with higher incidence of breakthrough infections associated with the time elapsed since 2nd vaccination raises concerns regarding the durability of immunity also after 3rd vaccination. We assessed virus-specific serum antibody and T cell response in the blood after vaccination with the mRNA vaccine BNT162b2 in more than 50 individuals older than 80 years. All old adults demonstrated a strong humoral response to 3rd vaccination which was at average higher and waned slower than the response to 2nd vaccination, indicative of enhanced humoral immunity. In contrast, their respective T cell response quantitatively limited to the level obtained after 2nd vaccination, with similar waning over time and no evidence for enhanced IFNg production. Because BNT162b2-mediated protection from the Omicron variant relies more on T cells than antibodies, our findings raise concern on the durability of protection from the Omicron variant by BNT162b2 in the senior population.

Induction of robust cellular and humoral immunity against SARS-CoV-2 after a third dose of BNT162b2 vaccine in previously unresponsive elderly (preprint)

Herein, we compared SARS-CoV-2-specific antibody and T-cell responses to two doses of BNT162b2 mRNA in 51 vaccinees > 80 years old and 46 (20–53 years old) controls. The responses of the elderly were much lower and 10% non-responders were identified. Importantly, in four of them, a third vaccination raised the immune response to levels seen in responders after two vaccinations, thus implying that non-response is not fateful even in the elderly.

Complement Activation Induces Excessive T Cell Cytotoxicity in Severe COVID-19 (preprint)

Severe COVID-19 is linked to both dysfunctional immune response and unrestrained immunopathogenesis, and it remains unclear if T cells contribute to disease pathology. Here, we combined single-cell transcriptomics and proteomics with mechanistic studies assessing pathogenic T cell functions and inducing signals. We identified activated, CD16+ T cells with increased cytotoxic functions in severe COVID-19. CD16 expression enabled immune complex-mediated, T cell receptor-independent degranulation and cytotoxicity not found in other diseases. CD16+ T cells from COVID-19 patients promoted microvascular endothelial cell injury and release of neutrophil and monocyte chemoattractants. CD16+ T cell clones persisted beyond acute disease maintaining their cytotoxic phenotype. Age-dependent generation of C3a in severe COVID-19 induced activated CD16 + cytotoxic T cells. The proportion of activated CD16+ T cells and plasma levels of complement proteins upstream of C3a correlated with clinical outcome, supporting a pathological role of exacerbated cytotoxicity and complement activation in COVID-19.Funding: This work was supported by the German Research Foundation (DFG): SA1383/3-1 to B.S.; SFB-TR84 114933180 to L.E.S., S.B., P.G., S.H. and W.M.K. INST 37/1049-1, INST 216/981- 1, INST 257/605-1, INST 269/768-1, INST 217/988-1, INST 217/577-1, and EXC2151- 390873048 to J.L.S.; GRK 2168 – 272482170, ERA CVD (00160389 to J.L.S.; SFB 1454 – 432325352 to A.C.A. and J.L.S.; SFB TR57 and SPP1937 to J.N.; GRK2157 to A.-E.S.; and ME 3644/5-1 to H.E.M.; RTG2424 to N.B.; SFB-TRR219 322900939, BO3755/13-1 Project- ID 454024652 to P.B.; the Berlin University Alliance (BUA) (PreEP-Corona grant to L.E.S. and V.M.C.); the Berlin Institute of Health (BIH) (to L.E.S., V.M.C.,B.S. and W.M.K.); Helmholtz- Gemeinschaft Deutscher Forschungszentren, Germany (sparse2big to J.L.S.), EU projects SYSCID (733100 to J.L.S.); European Research Council Horizon 2020 (grant agreement No 101001791 to P.B.); the DZIF, Germany (TTU 04.816 and 04.817 to J.N.); the Hector Foundation (M89 to J.N.); the EU projects ONE STUDY (260687), BIO-DrIM (305147) and INsTRuCT (860003) to B.S.); German Registry of COVID-19 Autopsies through Federal Ministry of Health (ZMVI1-2520COR201 to P.B.); Federal Ministry of Education and Research (DEFEAT PANDEMICs, 01KX2021 and STOP-FSGS-01GM1901A to P.B.); the Berlin Senate to German Rheumatism Research Centre (DRFZ); the Berlin Brandenburg School for regenerative Therapies (BSRT) to C.B.; the German Federal Ministry of Education and Research (BMBF) projects RECAST (01KI20337) to B.S., V.M.C., L.E.S and M.R.; VARIPath (01KI2021) to V.M.C.; NUM COVIM (01KX2021) to L.E.S., V.M.C., F.K., J.L.S., J.N. and B.S.; RAPID to and S.H.,; SYMPATH to N.S. and W.M.K.; PROVID to S.H. and W.M.K.; ZissTrans (02NUK047E) to N.B; National Research Node ‘Mass spectrometry in Systems Medicine (MSCoresys) (031L0220A) to M.R. and N.B.; Diet–Body–Brain (DietBB) (01EA1809A) to J.L.S.; the UKRI/NIHR through the UK Coronavirus Immunology Consortium (UK-CIC), the Francis Crick Institute through the Cancer Research UK (FC001134), the UK Medical Research Council (FC001134), the Wellcome Trust (FC001134 and IA 200829/Z/16/Z) to M.R.; a Charité 3R project (to B.S., S.H., W.M.K.); and an intramural grant from the Department of Genomics & Immunoregulation at the LIMES Institute to A.C.A. We are grateful to the patients and donors volunteering to participate in this study making this research possible in the first place and wish for a speedy and full recovery.Conflict of Interest: V.M.C. is named together with Euroimmun GmbH on a patent application filed recently regarding SARS-CoV-2 diagnostics via antibody testing. A.R.S. and H.E.M. are listed asinventors on a patent application by the DRFZ Berlin in the field of mass cytometry.Ethical Approval: The study was approved by the Institutional Review board of Charité(EA2/066/20).

Deep Spatial Profiling of COVID-19 Brains Reveals Neuroinflammation by Compartmentalized Local Immune Cell Interactions and Targets for Intervention (preprint)

COVID-19 can cause acute and chronic neurological symptoms. The underlying pathophysiological mechanisms, the involved immune cells, their spatial distribution, cellular interactions and the role of virus tropism remain largely unclear. Here, we deeply interrogated the brain stem and olfactory bulb in COVID-19 patients with imaging mass cytometry to understand the local immune response at a spatially resolved, high-dimensional single-cell level. We observed significant immune activation in the CNS and identified distinct phenotypes of T cells and microglial clusters, their presence in specific anatomical regions and context-specific cellular interactions. Microglial nodules and perivascular immune cell clusters constitute key sites of the local immune response, with viral antigen present in ACE2-expressing cells in the perivascular compartment. Disease-associated neuroinflammation is associated with astrogliosis and severe axonal damage as a structural basis for the neurologic deficits. Finally, we identify compartment- and cluster-specific immune checkpoints that can be targeted for future therapeutic interventions.Funding: This project was supported by grants from the Deutsche Forschungsgemeinschaft (DFG, GermanResearch Foundation) (SFB 992, SFB1160, SFB/TRR167, SFB/TRR179, German Excellency strategyCIBSS - EXC-2189– Project ID390939984) and special research funds from the Ministry for Science, Research and Art of Baden-Wuerttemberg dedicated to “COVID-19 research” and “Neuroinflammation”.B.B. was further supported by DFG grant BE-5496/5-1 and M.P. was further supported by the Sobek Foundation, the Ernst-Jung Foundation, the Reinhart-Koselleck-Grant and Gottfried Wilhelm Leibniz-Prize.H.E.M. was supported by DFG ME-3644/5-1.Ethical Approval: The analyses were performed with the approval of the Institutional Review Boards (Ethic Committee of the Albert-Ludwigs-University, Freiburg: 322/20, 10008/09; Ethics Committee of the Hamburg Chamber of Physicians: WF-051/20, PV7311). The study was performed in agreement with the principles expressed in the Declaration of Helsinki (2013).Conflict of Interest: None to declare.

Deep spatial profiling of COVID19 brains reveals neuroinflammation by compartmentalized local immune cell interactions and targets for intervention (preprint)

COVID-19 causes neurological symptoms that can be potentially life-threatening in up to 67 % of the patients. The underlying pathophysiological mechanisms of COVID-19 associated encephalopathy, the involved immune cells, their spatial distribution and their cellular interactions during disease remain largely unclear. In this study, we performed a 38-biomarker imaging mass cytometry analysis of the brain stem from 25 patients and additional controls to understand the local immune response during SARS-CoV-2 infection at a spatially resolved, high-dimensional single-cell level. Importantly, utilizing an unbiased image segmentation and cell classification pipeline, we observed a significant immune activation in the central nervous system (CNS) and identified novel context-specific CD8 T cell and microglial clusters. Spatially resolved single-cell analysis identified distinct phenotypes of T cells and microglial clusters, their presence in specific anatomical regions and their cellular interactions. Our analysis further highlights microglial nodules and perivascular immune cell clusters as key sites of the local immune response. It also demonstrates that disease-associated neuroinflammation is associated with severe axonal damage as a structural basis for the neurologic deficits. Finally, we identified compartment- and cluster-specific immune checkpoints that can be used for future therapeutic interventions.

Ex vivo detection of SARS-CoV-2-specific CD8+ T cells: rapid induction, prolonged contraction, and formation of functional memory (preprint)

CD8+ T cells are critical for the elimination and long-lasting protection of many viral infections, but their role in the current SARS-CoV-2 pandemic is unclear. Emerging data indicates that SARS-CoV-2-specific CD8+ T cells are detectable in the majority of individuals recovering from SARS-CoV-2 infection. However, optimal virus-specific epitopes, the role of pre-existing heterologous immunity as well as their kinetics and differentiation program during disease control have not been defined in detail. Here, we show that both pre-existing and newly induced SARS-CoV-2-specific CD8+ T-cell responses are potentially important determinants of immune protection in mild SARS-CoV-2 infection. In particular, our results can be summarized as follows: First, immunodominant SARS-CoV-2-specific CD8+ T-cell epitopes are targeted in the majority of individuals with convalescent SARS-CoV-2 infection. Second, MHC class I tetramer analyses revealed the emergence of phenotypically diverse and functionally competent pre-existing and newly induced SARS-CoV-2-specific memory CD8+ T cells that showed similar characteristics compared to influenza-specific CD8+ T cells. Third, SARS-CoV-2-specific CD8+ T-cell responses are more robustly detectable than antibodies against the SARS-CoV-2-spike protein. This was confirmed in a longitudinal analysis of acute-resolving infection that demonstrated rapid induction of the SARS-CoV-2-specific CD8+ T cells within a week followed by a prolonged contraction phase that outlasted the waning humoral immune response indicating that CD8+ T-cell responses might serve as a more precise correlate of antiviral immunity than antibody measurements after convalescence. Collectively, these data provide new insights into the fine specificity, heterogeneity, and dynamics of SARS-CoV-2-specific memory CD8+ T cells, potentially informing the rational development of a protective vaccine against SARS-CoV-2.