In vivo analysis of CRISPR-edited germinal center murine B cells.

The germinal center (GC) reaction is crucial for somatic hypermutation, affinity maturation, and the selection of high-affinity B cells, all of which are hallmarks of the humoral immune response. Understanding the distinct roles of various B cell genes is essential for elucidating the selection mechanisms within the GC reaction. Traditionally, studying B cell gene function in the GC reaction involved generating knock-out mice, a highly time-consuming method that necessitates complex vectors. The advent of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has simplified the creation of knock-out mice. However, even with CRISPR, the generation of knock-out mice still faces challenges, including being time-consuming, costly, having low knock-out efficiency, and raising ethical concerns regarding animal use. To address these challenges, we developed an alternative method to traditional knock-out mouse generation. Our approach entails the ex vivo CRISPR editing of B cells from transgenic donor mice with different B cell receptor affinities followed by their adoptive transfer into recipient mice. We present a cost-effective, rapid, versatile, and adaptable CRISPR-Cas9 method for in vivo loss-of-function studies of individual murine B cell genes within the context of the GC reaction. This method provides a valuable tool for investigating the complex roles of different B cell genes in the GC selection process. As proof of concept, we validated our approach by examining the role of the pro-apoptotic gene Fas in the GC selection process. We adoptively transferred a mix of Fas knock-out (FasKO) low-affinity B cells, Fas wild-type (FasWT) low-affinity B cells, and FasWT high-affinity B cells into recipient mice. From our results, FasKO low-affinity B cells were still outcompeted by the FasWT high-affinity B cells for selection in the GC. An important observation was the accumulation of FasKO low-affinity GC B cells when compared to the FasWT low-affinity B cells, which suggested a role of Fas in the GC selection process.

Oligoclonal CD4+CXCR5+ T cells with a cytotoxic phenotype appear in tonsils and blood.

In clinical situations, peripheral blood accessible CD3+CD4+CXCR5+ T-follicular helper (TFH) cells may have to serve as a surrogate indicator for dysregulated germinal center responses in tissues. To determine the heterogeneity of TFH cells in peripheral blood versus tonsils, CD3+CD4+CD45RA-CXCR5+ cells of both origins were sorted. Transcriptomes, TCR repertoires and cell-surface protein expression were analysed by single-cell RNA sequencing, flow cytometry and immunohistochemistry. Reassuringly, all blood-circulating CD3+CD4+CXCR5+ T-cell subpopulations also appear in tonsils, there with some supplementary TFH characteristics, while peripheral blood-derived TFH cells display markers of proliferation and migration. Three further subsets of TFH cells, however, with bona fide T-follicular gene expression patterns, are exclusively found in tonsils. One additional, distinct and oligoclonal CD4+CXCR5+ subpopulation presents pronounced cytotoxic properties. Those 'killer TFH (TFK) cells' can be discovered in peripheral blood as well as among tonsillar cells but are located predominantly outside of germinal centers. They appear terminally differentiated and can be distinguished from all other TFH subsets by expression of NKG7 (TIA-1), granzymes, perforin, CCL5, CCR5, EOMES, CRTAM and CX3CR1. All in all, this study provides data for detailed CD4+CXCR5+ T-cell assessment of clinically available blood samples and extrapolation possibilities to their tonsil counterparts.

Class switch toward noninflammatory, spike-specific IgG4 antibodies after repeated SARS-CoV-2 mRNA vaccination.

RNA vaccines are efficient preventive measures to combat the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. High levels of neutralizing SARS-CoV-2 antibodies are an important component of vaccine-induced immunity. Shortly after the initial two mRNA vaccine doses, the immunoglobulin G (IgG) response mainly consists of the proinflammatory subclasses IgG1 and IgG3. Here, we report that several months after the second vaccination, SARS-CoV-2-specific antibodies were increasingly composed of noninflammatory IgG4, which were further boosted by a third mRNA vaccination and/or SARS-CoV-2 variant breakthrough infections. IgG4 antibodies among all spike-specific IgG antibodies rose, on average, from 0.04% shortly after the second vaccination to 19.27% late after the third vaccination. This induction of IgG4 antibodies was not observed after homologous or heterologous SARS-CoV-2 vaccination with adenoviral vectors. Single-cell sequencing and flow cytometry revealed substantial frequencies of IgG4-switched B cells within the spike-binding memory B cell population [median of 14.4%; interquartile range (IQR) of 6.7 to 18.1%] compared with the overall memory B cell repertoire (median of 1.3%; IQR of 0.9 to 2.2%) after three immunizations. This class switch was associated with a reduced capacity of the spike-specific antibodies to mediate antibody-dependent cellular phagocytosis and complement deposition. Because Fc-mediated effector functions are critical for antiviral immunity, these findings may have consequences for the choice and timing of vaccination regimens using mRNA vaccines, including future booster immunizations against SARS-CoV-2.

Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a life-threatening autoimmune disease characterized by adaptive immune system activation, formation of double-stranded DNA autoantibodies and organ inflammation. Five patients with SLE (four women and one man) with a median (range) age of 22 (6) years, median (range) disease duration of 4 (8) years and active disease (median (range) SLE disease activity index Systemic Lupus Erythematosus Disease Activity Index: 16 (8)) refractory to several immunosuppressive drug treatments were enrolled in a compassionate-use chimeric antigen receptor (CAR) T cell program. Autologous T cells from patients with SLE were transduced with a lentiviral anti-CD19 CAR vector, expanded and reinfused at a dose of 1 × 106 CAR T cells per kg body weight into the patients after lymphodepletion with fludarabine and cyclophosphamide. CAR T cells expanded in vivo, led to deep depletion of B cells, improvement of clinical symptoms and normalization of laboratory parameters including seroconversion of anti-double-stranded DNA antibodies. Remission of SLE according to DORIS criteria was achieved in all five patients after 3 months and the median (range) Systemic Lupus Erythematosus Disease Activity Index score after 3 months was 0 (2). Drug-free remission was maintained during longer follow-up (median (range) of 8 (12) months after CAR T cell administration) and even after the reappearance of B cells, which was observed after a mean (±s.d.) of 110 ± 32 d after CAR T cell treatment. Reappearing B cells were naïve and showed non-class-switched B cell receptors. CAR T cell treatment was well tolerated with only mild cytokine-release syndrome. These data suggest that CD19 CAR T cell transfer is feasible, tolerable and highly effective in SLE.

A pair of noncompeting neutralizing human monoclonal antibodies protecting from disease in a SARS-CoV-2 infection model.

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.

SARS-CoV-2 in severe COVID-19 induces a TGF-ß-dominated chronic immune response that does not target itself.

The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-ß, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-ß. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-ß, and is distracted from itself.

Staphylococcus aureus Protein A Induces Human Regulatory T Cells Through Interaction With Antigen-Presenting Cells.

Despite continuous exposure and development of specific immunity, Staphylococcus aureus (Sa) remains one of the leading causes of severe infections worldwide. Although innate immune defense mechanisms are well understood, the role of the T cell response has not been fully elucidated. Here, we demonstrate that Sa and one of its major virulence factors protein A (SpA) induce human regulatory T cells (Tregs), key players in immune tolerance. In human PBMC and MoDC/T cell cocultures CD4+CD25+CD127dim Tregs were induced upon stimulation with Sa and to a lower extent with SpA alone. Treg induction was strongly, but not exclusively, dependent on SpA, and independent of antigen presentation or T cell epitope recognition. Lastly, soluble factors in the supernatant of SpA-stimulated MoDC were sufficient to trigger Treg formation, while supernatants of MoDC/T cell cocultures containing Sa-triggered Tregs displayed T cell suppressive activity. In summary, our findings identify a new immunosuppressory function of SpA, which leads to release of soluble, Treg-inducing factors and might be relevant to establish colonization.