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.

A Phosphorus-centred, Zirconocene-bridged Tetraradical: Synthesis, Structure and Application as Molecular Double Switch.

Biradicals are important intermediates in the formation and breaking of a chemical bond. Their use as molecular switches is of particular interest. Much less is known about tetraradicals, which can, for example, consist of two biradical(oid) units. Here we report the synthesis of the first persistent phosphorus-centred tetraradical bound to a transition metal fragment. Starting from a zirconocene complex, rac-(ebthi)ZrCl2 (rac-(ebthi) = 1,2-ethylene-1,10-bis(h5-tetrahydroin-denyl), two cyclo-1,3-diphospha-pentane-1,3-diyls were successfully introduced, which finally led to the isolation of a deep green zircon-cene-bridged bis(biradicaloid) complex (5) that can act as a double molecular switch. Under the influence of light (570 nm), this tetra-radical forms a transannular bond in each of the two five-membered biradical units, leading to the formation of housane 5h. Upon irradiation at 415 nm, the reverse reaction is observed, fully reco-vering tetraradical 5. Through single-crystal-to-single-crystal trans-formation, both stable species of the molecular switch could be structurally characterised using SCXRD. The switching under the influence of light and the activation of molecular hydrogen were analysed in solution using NMR and UV spectroscopy. It was found that the addition of one or two equivalents of molecular hydrogen can be switched on and off by light.

Harmonized analysis of PBMC by mass cytometry.

Mass cytometry permits the high dimensional analysis of cellular systems at single-cell resolution with high throughput in various areas of biomedical research. Here, we provide a state-of-the-art protocol for the analysis of human peripheral blood mononuclear cells (PBMC) by mass cytometry. We focus on the implementation of measures promoting the harmonization of large and complex studies to aid robustness and reproducibility of immune phenotyping data.

Single-cell insights into immune dysregulation in rheumatoid arthritis flare versus drug-free remission.

Immune-mediated inflammatory diseases (IMIDs) are typically characterised by relapsing and remitting flares of inflammation. However, the unpredictability of disease flares impedes their study. Addressing this critical knowledge gap, we use the experimental medicine approach of immunomodulatory drug withdrawal in rheumatoid arthritis (RA) remission to synchronise flare processes allowing detailed characterisation. Exploratory mass cytometry analyses reveal three circulating cellular subsets heralding the onset of arthritis flare - CD45RO+PD1hi CD4+ and CD8+ T cells, and CD27+CD86+CD21- B cells - further characterised by single-cell sequencing. Distinct lymphocyte subsets including cytotoxic and exhausted CD4+ memory T cells, memory CD8+CXCR5+ T cells, and IGHA1+ plasma cells are primed for activation in flare patients. Regulatory memory CD4+ T cells (Treg cells) increase at flare onset, but with dysfunctional regulatory marker expression compared to drug-free remission. Significant clonal expansion is observed in T cells, but not B cells, after drug cessation; this is widespread throughout memory CD8+ T cell subsets but limited to the granzyme-expressing cytotoxic subset within CD4+ memory T cells. Based on our observations, we suggest a model of immune dysregulation for understanding RA flare, with potential for further translational research towards novel avenues for its treatment and prevention.

Immune signatures of checkpoint inhibitor-induced autoimmunity-A focus on neurotoxicity.

BACKGROUND: Neurologic immune-related adverse events (irAE-n) are rare but severe toxicities of immune checkpoint inhibitor (ICI) treatment. To overcome diagnostic and therapeutic challenges, a better mechanistic understanding of irAE-n is paramount. METHODS: In this observational cohort study, we collected serum and peripheral blood samples from 34 consecutive cancer patients with irAE-n (during acute illness) and 49 cancer control patients without irAE-n (pre- and on-ICI treatment, n = 44 without high-grade irAEs, n = 5 with high-grade nonneurologic irAEs). Patients received either anti-programmed cell death protein (PD)-1 or anti-PD ligand-1 monotherapy or anti-PD-1/anti-cytotoxic T-lymphocyte-associated protein-4 combination therapy. Most common cancers were melanoma, lung cancer, and hepatocellular carcinoma. Peripheral blood immune profiling was performed using 48-marker single-cell mass cytometry and a multiplex cytokine assay. RESULTS: During acute illness, patients with irAE-n presented higher frequencies of cluster of differentiation (CD)8+ effector memory type (EM-)1 and central memory (CM) T cells compared to controls without irAEs. Multiorgan immunotoxicities (neurologic + nonneurologic) were associated with higher CD8+ EM1 T cell counts. While there were no B cell changes in the overall cohort, we detected a marked decrease of IgD- CD11c+ CD21low and IgD- CD24+ CD21high B cells in a subgroup of patients with autoantibody-positive irAE-n. We further identified signatures indicative of enhanced chemotaxis and inflammation in irAE-n patients and discovered C-X-C motif chemokine ligand (CXCL)10 as a promising marker to diagnose high-grade immunotoxicities such as irAE-n. CONCLUSIONS: We demonstrate profound and partly subgroup-specific immune cell dysregulation in irAE-n patients, which may guide future biomarker development and targeted treatment approaches.

Rational Design of a Phosphorus-Centered Disbiradical.

Phosphorus-centered disbiradicals, in which the radical sites exist as individual spin doublets with weak spin-spin interaction have not been known so far. Starting from monoradicals of the type [⋅P(µ-NTer)2 P-R], we have now succeeded in linking two such monoradical phosphorus centers by appropriate choice of a linker. To this end, biradical [⋅P(µ-NTer)2 P⋅] (1) was treated with 1,6-dibromohexane, affording the brominated species {Br[P(µ-NTer)]2 }2 C6 H12 (3). Subsequent reduction with KC8 led to the formation of the disbiradical {⋅[P(µ-NTer)]2 }2 C6 H12 (4) featuring a large distance between the radical phosphorus sites in the solid state and formally the highest biradical character observed in a P-centered biradical so far, approaching 100 %. EPR spectroscopy revealed a three-line signal in solution with a considerably larger exchange interaction than would be expected from the molecular structure of the single crystal. Quantum chemical calculations revealed a highly dynamic conformational space; thus, the two radical sites can approach each other with a much smaller distance in solution. Further reduction of 4 resulted in the formation of a potassium salt featuring the first structurally characterized P-centered distonic radical anion (5- ). Moreover, 4 could be used in small molecule activation.

Heteroatom-Based Diradical(oid)s.

Heteroatom-centered diradical(oid)s have been in the focus of molecular main group chemistry for nearly 30 years. During this time, the diradical concept has evolved and the focus has shifted to the rational design of diradical(oid)s for specific applications. This review article begins with some important theoretical considerations of the diradical and tetraradical concept. Based on these theoretical considerations, the design of diradical(oid)s in terms of ligand choice, steric, symmetry, electronic situation, element choice, and reactivity is highlighted with examples. In particular, heteroatom-centered diradical reactions are discussed and compared with closed-shell reactions such as pericyclic additions. The comparison between closed-shell reactivity, which proceeds in a concerted manner, and open-shell reactivity, which proceeds in a stepwise fashion, along with considerations of diradical(oid) design, provides a rational understanding of this interesting and unusual class of compounds. The application of diradical(oid)s, for example in small molecule activation or as molecular switches, is also highlighted. The final part of this review begins with application-related details of the spectroscopy of diradical(oid)s, followed by an update of the heteroatom-centered diradical(oid)s and tetraradical(oid)s published in the last 10 years since 2013.

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.

Peripheral Blood Immune Cell Composition After Autologous MSC Infusion in Kidney Transplantation Recipients.

Tacrolimus is the backbone of immunosuppressive agents to prevent transplant rejection. Paradoxically, tacrolimus is nephrotoxic, causing irreversible tubulointerstitial damage. Therefore, infusion of mesenchymal stromal cells (MSC) 6 and 7 weeks post-transplantation was assessed to facilitate withdrawal of tacrolimus in the randomized phase II TRITON trial. Here, we performed detailed analysis of the peripheral blood immune composition using mass cytometry to assess potential effects of MSC therapy on the immune system. We developed two metal-conjugated antibody panels containing 40 antibodies each. PBMC samples from 21 MSC-treated patients and 13 controls, obtained pre-transplant and at 24 and 52 weeks post-transplantation, were analyzed. In the MSC group at 24 weeks, 17 CD4+ T cell clusters were increased of which 14 Th2-like clusters and three Th1/Th2-like clusters, as well as CD4+FoxP3+ Tregs. Additionally, five B cell clusters were increased, representing either class switched memory B cells or proliferating B cells. At 52 weeks, CCR7+CD38+ mature B cells were decreased. Finally, eight Tc1 (effector) memory cytotoxic T cell clusters were increased. Our work provides a comprehensive account of the peripheral blood immune cell composition in kidney transplant recipients after MSC therapy and tacrolimus withdrawal. These results may help improving therapeutic strategies using MSCs with the aim to reduce the use of calcineurin inhibitors. Clinical Trial Registration: ClinicalTrials.gov, identifier NCT02057965.

Rational Design of Persistent Phosphorus-Centered Singlet Tetraradicals and Their Use in Small-Molecule Activation.

Biradicals are important intermediates in the process of bond formation and breaking. While main-group-element-centered biradicals have been thoroughly studied, much less is known about tetraradicals, as their very low stability has hampered their isolation and use in small-molecule activation. Herein, we describe the search for persistent phosphorus-centered tetraradicals. Starting from an s-hydrindacenyl skeleton, we investigated the introduction of four phosphorus-based radical sites linked by an N-R unit and bridged by a benzene moiety. By varying the size of the substituent R, we finally succeeded in isolating a persistent P-centered singlet tetraradical, 2,6-diaza-1,3,5,7-tetraphospha-s-hydrindacene-1,3,5,7-tetrayl (1), in good yields. Furthermore, it was demonstrated that tetraradical 1 can be utilized for the activation of small molecules such as molecular hydrogen or alkynes. In addition to the synthesis of P-centered tetraradicals, the comparison with other known tetraradicals as well as biradicals is described on the basis of quantum mechanical calculations with respect to its multireference character, coupling of radical electrons, and aromaticity. The strong coupling of radical electrons enables selective discrimination between the first and the second activations of small molecules, which is shown by the example of H2 addition. The mechanism of hydrogen addition is investigated with parahydrogen-induced hyperpolarization NMR studies and DFT calculations.

Spatially Resolved Multi-Omics Single-Cell Analyses Inform Mechanisms of Immune Dysfunction in Pancreatic Cancer.

BACKGROUND & AIMS: As pancreatic ductal adenocarcinoma (PDAC) continues to be recalcitrant to therapeutic interventions, including poor response to immunotherapy, albeit effective in other solid malignancies, a more nuanced understanding of the immune microenvironment in PDAC is urgently needed. We aimed to unveil a detailed view of the immune micromilieu in PDAC using a spatially resolved multimodal single-cell approach. METHODS: We applied single-cell RNA sequencing, spatial transcriptomics, multiplex immunohistochemistry, and mass cytometry to profile the immune compartment in treatment-naïve PDAC tumors and matched adjacent normal pancreatic tissue, as well as in the systemic circulation. We determined prognostic associations of immune signatures and performed a meta-analysis of the immune microenvironment in PDAC and lung adenocarcinoma on single-cell level. RESULTS: We provided a spatially resolved fine map of the immune landscape in PDAC. We substantiated the exhausted phenotype of CD8 T cells and immunosuppressive features of myeloid cells, and highlighted immune subsets with potentially underappreciated roles in PDAC that diverged from immune populations within adjacent normal areas, particularly CD4 T cell subsets and natural killer T cells that are terminally exhausted and acquire a regulatory phenotype. Differential analysis of immune phenotypes in PDAC and lung adenocarcinoma revealed the presence of extraordinarily immunosuppressive subtypes in PDAC, along with a distinctive immune checkpoint composition. CONCLUSIONS: Our study sheds light on the multilayered immune dysfunction in PDAC and presents a holistic view of the immune landscape in PDAC and lung adenocarcinoma, providing a comprehensive resource for functional studies and the exploration of therapeutically actionable targets in PDAC.

Synthesis of Stable Potassium Phosphinophosphides and Reaction with Organosilyl Halides and Chlorophosphanes.

The synthesis of sterically demanding 2,6-bis(2,4,6-trimethylphenyl)phenyl (Ter)-stabilized and H-substituted diphosphanes TerHP-PR2 (4a-4c) via conversion of the phosphide TerPHK (2) with secondary chlorophosphanes ClPR2 (3a-3c, where R = iPr, Ph, and tBu, respectively) is described. The diphosphanes 4a-4c were deprotonated using KH in tetrahydrofuran, selectively yielding the potassium phosphinophosphides K[TerP-PR2] (5a-5c). These phosphinophosphides are stable in solution as well as in the solid state and can be further functionalized via salt-metathesis reactions. Reaction with organosilyl halides selectively yields the silylated diphosphanes Ter(SiR12R2)P-P(iPr)2 (6a and 6b, where R1 = R2 = CH3 and R1 = CH3, R2 = Ph, respectively), whereas conversion with chlorophosphanes selectively yields the triphosphanes R12P-P(Ter)-P(iPr)2 (7a and 7b, where R = iPr and Ph, respectively).

Coinage metal complexes of multidentate Pacman phosphane ligands.

We present the extension of Pacman ligands to bidentate phosphane ligands enabling them to bind metals in their sterically protected cavity. The coordination of coinage metals shows the ability of the ligand to adopt different coordination modes by distortion, of which some additionally include the imine nitrogen atoms. Besides the coordinated metal, the substitution on the phosphorus atoms influences the type of coordination.

Synthesis of Benzene Derivatives with Multiple Dichlorophosphino Groups.

The chlorination of 1,2-diphosphinobenzene with PCl5 to 1,2-bis(dichlorophosphino)benzene was performed with high yields (93 %) despite the high number of P-H functions. The method was further applied to other phosphanes, enabling the first synthesis and complete characterization of 1,2,4-tris(dichlorophosphino)benzene (89 % yield) and 1,2,4,5-tetrakis(dichlorophosphino)benzene (91 % yield), valuable precursors for example for binuclear complexes, coordination polymers, organic wires, or metal-organic frameworks. The application of the chlorophosphanes in base induced ring closure reactions with primary amines is demonstrated.

On the Dynamic Behavior of Pacman Phosphanes─A Case of Cooperativity and Redox Isomerism.

In solution, the Pacman chlorophosphane (2Cl) shows fast exchange of the endo/exo-orientation of the two P-Cl bonds in the molecule featuring cooperativity. Experimental and quantum mechanical investigations of the inversion on the phosphorus(III) centers reveal a crucial role of chloride ions in the dynamic process. To confirm the results, the homologous Pacman halogen-phosphanes 2X were prepared by halogen exchange reactions (X = F, Br, and I). Besides accelerated dynamic behavior for the heavier analogues, significant differences in the molecular structure are caused by the halogen exchange reactions, including the formation of an endo-endo substituted Pacman fluorophosphane as well as dicationic species by phosphorus halogen bond dissociation. The latter process can be regarded as redox isomerism since two PIII atoms in 2X become PV centers in the dications.

Access to Benzo- and Naphtho-Azaphospholes via C-H Bond Activation of Aryl-Substituted Isonitriles.

Differently substituted phenyl isonitriles (with C-H bonds in ortho-position) and naphthyl isonitriles were reacted with the cyclic biradical [⋅P(µ-N-Ter)2 P⋅] (1). Insertion of the isonitrile formed a cyclic five-membered biradical [⋅P(NTer)2 C(R)P⋅] (2R, R=phenyl, naphthyl) in the first step, followed by C-H activation at the aryl substituent, resulting in novel azaphospholes (5R), which could be isolated and fully characterized. The formation of the azaphospholes can be prevented by the addition of a second equivalent of isonitrile, which causes the blocking of the radical centers in 2R by adduct formation (3R). Quantum mechanical calculations showed that a significant increase in the aromaticity of the benzo- and naphtho-azaphospholes is one of the driving forces for the activation process leading to the formation of thermodynamically favored azaphospholes. Targeted activation of C-H bonds using biradical systems represents a new synthetic approach to generate benzo- and naphtho-azaphospholes.

A Lewis Acid Stabilized Ketenimine in an Unusual Variant of the Electrophilic Aromatic Substitution.

Electrophilic aromatic substitution (EAS) can provide a straightforward approach to the efficient synthesis of functionalized complex aromatic molecules. In general, Lewis acids serve as a beneficial stimulus for the formation of a Wheland complex, the intermediate in the classical SE Ar mechanism of EAS, which is responsible for H/E (E=electrophile) substitution under formal H+ elimination. Herein, we report an unusual variant of EAS, in which a complex molecule such as the tricyanomethane, HC(CN)3 , is activated with a strong Lewis acid (B(C6 F5 )3 ) to the point where it can finally be used in an EAS. However, the Lewis acid here causes the isomerization of the tricyanomethane to the ketenimine, HN=C=C(CN)2 , which in turn directly attacks the aromatic species in the EAS, with simultaneous proton migration of the aromatic proton to the imino group, so that no elimination occurs that is otherwise observed in the SE Ar mechanism. By this method, it is possible to build up amino-malononitrile-substituted aromatic compounds in one step.

Concerted addition of aldehydes to the singlet biradical [P(µ-NTer)]2.

The reaction of the singlet biradical [P(µ-NTer)]2 with various aldehydes selectively yielded the corresponding [2.1.1]-bicyclic addition products in a very fast reaction. All products were fully characterized, including by NMR and vibrational spectroscopy as well as single-crystal X-ray diffraction. The mechanism of the addition was investigated theoretically using high-level ab initio methods (CCSD(T) with triple- and quadruple-zeta basis sets) and corresponds to a concerted cycloaddition reaction with a very low activation barrier. For comparison, the mechanisms of the literature-known cycloadditions of H2, alkenes, and alkynes were also studied, indicating a similar reaction profile for all unsaturated reactants.

Radical Reactivity of the Biradical [⋠P(µ-NTer)2 P⋠] and Isolation of a Persistent Phosphorus-Cantered Monoradical [⋠P(µ-NTer)2 P-Et].

The activation of C-Br bonds in various bromoalkanes by the biradical [⋅P(µ-NTer)2 P⋅] (1) (Ter=2,6-bis-(2,4,6-trimethylphenyl)-phenyl) is reported, yielding trans-addition products of the type [Br-P(µ-NTer)2 P-R] (2), so-called 1,3-substituted cyclo-1,3-diphospha-2,4-diazanes. This addition reaction, which represents a new easy approach to asymmetrically substituted cyclo-1,3-diphospha-2,4-diazanes, was investigated mechanistically by different spectroscopic methods (NMR, EPR, IR, Raman); the results suggested a stepwise radical reaction mechanism, as evidenced by the in-situ detection of the phosphorus-centered monoradical [⋅P(µ-NTer)2 P-R].< To provide further evidence for the radical mechanism, [⋅P(µ-NTer)2 P-Et] (3Et⋅) was synthesized directly by reduction of the bromoethane addition product [Br-P(µ-NTer)2 P-Et] (2 a) with magnesium, resulting in the formation of the persistent phosphorus-centered monoradical [⋅P(µ-NTer)2 P-Et], which could be isolated and fully characterized, including single-crystal X-ray diffraction. Comparison of the EPR spectrum of the radical intermediate in the addition reaction with that of the synthesized new [⋅P(µ-NTer)2 P-Et] radical clearly proves the existence of radicals over the course of the reaction of biradical [⋅P(µ-NTer)2 P⋅] (1) with bromoethane. Extensive DFT and coupled cluster calculations corroborate the experimental data for a radical mechanism in the reaction of biradical [⋅P(µ-NTer)2 P⋅] with EtBr. In the field of hetero-cyclobutane-1,3-diyls, the demonstration of a stepwise radical reaction represents a new aspect and closes the gap between P-centered biradicals and P-centered monoradicals in terms of radical reactivity.