Intrinsically determined turnover underlies broad heterogeneity in plasma-cell lifespan.

Antibodies produced by antibody-secreting plasma cells (ASCs) underlie multiple forms of long-lasting immunity. Here we examined the mechanisms regulating ASC turnover and persistence using a genetic reporter to time-stamp ASCs. This approach revealed ASC lifespans as heterogeneous and falling on a continuum, with only a small fraction surviving for >60 days. ASC longevity past 60 days was independent of isotype but correlated with a phenotype that developed progressively and ultimately associated with an underlying "long-lived" ASC (LL ASC)-enriched transcriptional program. While some of the differences between LL ASCs and other ASCs appeared to be acquired with age, other features were shared with some younger ASCs, such as high CD138 and CD93. Turnover was unaffected by altered ASC production, arguing against competition for niches as a major driver of turnover. Thus, ASC turnover is set by intrinsic lifespan limits, with steady-state population dynamics governed by niche vacancy rather than displacement.

Long-lived plasma cells accumulate in the bone marrow at a constant rate from early in an immune response.

Vaccines work largely by generating long-lived plasma cells (LLPCs), but knowledge of how such cells are recruited is sparse. Although it is clear that LLPCs preferentially originate in germinal centers (GCs) and relocate to survival niches in bone marrow where they can persist for decades, the issues of the timing of LLPC recruitment and the basis of their retention remain uncertain. Here, using a genetic timestamping system in mice, we show that persistent PCs accrue in bone marrow at an approximately constant rate of one cell per hour over a period spanning several weeks after a single immunization with a model antigen. Affinity-based selection was evident in persisting PCs, reflecting a relative and dynamic rather than absolute affinity threshold as evidenced by the changing pattern of VH gene somatic mutations conveying increased affinity for antigen. We conclude that the life span of persistent, antigen-specific PCs is in part intrinsic, preprogrammed, and varied and that their final number is related to the duration of the response in a predictable way. This implies that modulating vaccines to extend the duration of the GC reaction will enhance antibody-mediated protective immunity.

CD137L and CD4 T cells limit BCL6-expressing pre-germinal center B cell expansion and BCL6-driven B cell malignancy.

Aberrant expression of the proto-oncogene BCL6 is a driver of tumorigenesis in diffuse large B cell lymphoma (DLBCL). Mice overexpressing BCL6 from the B cell-specific immunoglobulin heavy chain µ intron promoter (Iµ-Bcl6Tg/+ ) develop B cell lymphomas with features typical of human DLBCL. While the development of B cell lymphoma in these mice is tightly controlled by T cells, the mechanisms of this immune surveillance are poorly understood. Here we show that CD4 T cells contribute to the control of lymphoproliferative disease in lymphoma-prone Iµ-Bcl6Tg/+ mice. We reveal that this CD4 T cell immuno-surveillance requires signaling by the co-stimulatory molecule CD137 ligand (CD137L; also known as 4-1BBL), which may promote the transition of pre-malignant B cells with an activated phenotype into the germinal center stage via reverse signaling, preventing their hazardous accumulation. Thus, CD137L-mediated CD4 T cell immuno-surveillance adds another layer of protection against B cell malignancy to that provided by CD8 T cell cytotoxicity.

IL-21 has a critical role in establishing germinal centers by amplifying early B cell proliferation.

The proliferation and differentiation of antigen-specific B cells, including the generation of germinal centers (GC), are prerequisites for long-lasting, antibody-mediated immune protection. Affinity for antigen determines B cell recruitment, proliferation, differentiation, and competitiveness in the response, largely through determining access to T cell help. However, how T cell-derived signals contribute to these outcomes is incompletely understood. Here, we report how the signature cytokine of follicular helper T cells, IL-21, acts as a key regulator of the initial B cell response by accelerating cell cycle progression and the rate of cycle entry, increasing their contribution to the ensuing GC. This effect occurs over a wide range of initial B cell receptor affinities and correlates with elevated AKT and S6 phosphorylation. Moreover, the resultant increased proliferation can explain the IL-21-mediated promotion of plasma cell differentiation. Collectively, our data establish that IL-21 acts from the outset of a T cell-dependent immune response to increase cell cycle progression and fuel cyclic re-entry of B cells, thereby regulating the initial GC size and early plasma cell output.

Interleukin-21, acting beyond the immunological synapse, independently controls T follicular helper and germinal center B cells.

Germinal centers (GCs), transient structures within B cell follicles and central to affinity maturation, require the coordinated behavior of T and B cells. IL-21, a pleiotropic T cell-derived cytokine, is key to GC biology through incompletely understood mechanisms. By genetically restricting production and receipt of IL-21 in vivo, we reveal how its independent actions on T and B cells combine to regulate the GC. IL-21 established the magnitude of the GC B cell response by promoting CD4+ T cell expansion and differentiation in a dose-dependent manner and with paracrine activity. Within GC, IL-21 specifically promoted B cell centroblast identity and, when bioavailability was high, plasma cell differentiation. Critically, these actions may occur irrespective of cognate T-B interactions, making IL-21 a general promoter of growth as distinct to a mediator of affinity-driven selection via synaptic delivery. This promiscuous activity of IL-21 explains the consequences of IL-21 deficiency on antibody-based immunity.

The concerted change in the distribution of cell cycle phases and zone composition in germinal centers is regulated by IL-21.

Humoral immune responses require germinal centres (GC) for antibody affinity maturation. Within GC, B cell proliferation and mutation are segregated from affinity-based positive selection in the dark zone (DZ) and light zone (LZ) substructures, respectively. While IL-21 is known to be important in affinity maturation and GC maintenance, here we show it is required for both establishing normal zone representation and preventing the accumulation of cells in the G1 cell cycle stage in the GC LZ. Cell cycle progression of DZ B cells is unaffected by IL-21 availability, as is the zone phenotype of the most highly proliferative GC B cells. Collectively, this study characterises the development of GC zones as a function of time and B cell proliferation and identifies IL-21 as an important regulator of these processes. These data help explain the requirement for IL-21 in normal antibody affinity maturation.

B cell memory: understanding COVID-19.

Immunological memory is a mechanism to protect us against reinfection. Antibodies produced by B cells are integral to this defense strategy and underlie virtually all vaccine success. Here, we explain how B cells memory is generated by infection and vaccination, what influences its efficacy and its persistence, and how characterizing these parameters in the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will help achieve protective immunity through vaccination.

EBV renders B cells susceptible to HIV-1 in humanized mice.

HIV and EBV are human pathogens that cause a considerable burden to worldwide health. In combination, these viruses are linked to AIDS-associated lymphomas. We found that EBV, which transforms B cells, renders them susceptible to HIV-1 infection in a CXCR4 and CD4-dependent manner in vitro and that CXCR4-tropic HIV-1 integrates into the genome of these B cells with the same molecular profile as in autologous CD4+ T cells. In addition, we established a humanized mouse model to investigate the in vivo interactions of EBV and HIV-1 upon coinfection. The respective mice that reconstitute human immune system components upon transplantation with CD34+ human hematopoietic progenitor cells could recapitulate aspects of EBV and HIV immunobiology observed in dual-infected patients. Upon coinfection of humanized mice, EBV/HIV dual-infected B cells could be detected, but were susceptible to CD8+ T-cell-mediated immune control.

The Amount of BCL6 in B Cells Shortly after Antigen Engagement Determines Their Representation in Subsequent Germinal Centers.

It is unknown whether the incremental increases in BCL6 amounts in antigen-activated B cells influence the unfolding differentiation before germinal center (GC) formation. By comparing shortly after immunization the distribution of conventional B cells to those enforced to express BCL6 at the upper quartile of normal and those lacking BCL6 altogether, we determined that B cell representation in the stages before the GC compartment was related to BCL6 amounts. This was not by increased proliferation or suppression of early plasmablast differentiation, but rather by preferential recruitment and progression through these early stages of B cell activation, culminating in preferential transition into GC. Once established, this bias was stable in GC over several weeks; other BCL6-regulated GC B cell behaviors were unaffected. We propose that setting BCL6 amounts very early in activated B cells will be central in determining clonal representation in the GC and thus memory populations.

IVIG efficacy in CIDP patients is not associated with terminal complement inhibition.

Patients with acute and chronic inflammatory demyelinating neuropathies exhibit elevated serum and cerebrospinal fluid (CSF) levels of terminal complement activation products and therapeutic inhibition of complement activation is currently tested for its safety and efficacy in patients with Guillain-Barré syndrome (GBS). Here, we determined serum levels of the complement activation products C3a, C5a and the soluble terminal complement complex (sTCC) in 39 individuals with chronic inflammatory demyelinating polyneuropathy (CIDP) who participated in one of the largest ever conducted clinical trial in patients with CIDP (ICE trial) and received Intravenous Immunoglobulin (IVIG) or placebo (albumin) in 3 week intervals for up to 24 weeks. In placebo-treated patients with spontaneous disease remission, serum sTCC levels moderately decreased over time. Levels of complement activation products were, however, not modulated by IVIG and remained unchanged in patients with a beneficial response to IVIG therapy as compared to those with steady or worsened disease. These results suggest that the therapeutic efficacy of IVIG in CIDP is based on immunomodulatory mechanisms different from complement inhibition. Terminal complement activation merits further investigation as a surrogate marker for disease progression and therapeutic target in patients with CIDP.

ATG-dependent phagocytosis in dendritic cells drives myelin-specific CD4+ T cell pathogenicity during CNS inflammation.

Although reactivation and accumulation of autoreactive CD4+ T cells within the CNS are considered to play a key role in the pathogenesis of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), the mechanisms of how these cells recognize their target organ and induce sustained inflammation are incompletely understood. Here, we report that mice with conditional deletion of the essential autophagy protein ATG5 in classical dendritic cells (DCs), which are present at low frequencies in the nondiseased CNS, are completely resistant to EAE development following adoptive transfer of myelin-specific T cells and show substantially reduced in situ CD4+ T cell accumulation during the effector phase of the disease. Endogenous myelin peptide presentation to CD4+ T cells following phagocytosis of injured, phosphatidylserine-exposing oligodendroglial cells is abrogated in the absence of ATG5. Pharmacological inhibition of ATG-dependent phagocytosis by the cardiac glycoside neriifolin, an inhibitor of the Na+, K+-ATPase, delays the onset and reduces the clinical severity of EAE induced by myelin-specific CD4+ T cells. These findings link phagocytosis of injured oligodendrocytes, a pathological hallmark of MS lesions and during EAE, with myelin antigen processing and T cell pathogenicity, and identify ATG-dependent phagocytosis in DCs as a key regulator in driving autoimmune CD4+ T cell-mediated CNS damage.

Persistent KSHV Infection Increases EBV-Associated Tumor Formation In Vivo via Enhanced EBV Lytic Gene Expression.

The human tumor viruses Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) establish persistent infections in B cells. KSHV is linked to primary effusion lymphoma (PEL), and 90% of PELs also contain EBV. Studies on persistent KSHV infection in vivo and the role of EBV co-infection in PEL development have been hampered by the absence of small animal models. We developed mice reconstituted with human immune system components as a model for KSHV infection and find that EBV/KSHV dual infection enhanced KSHV persistence and tumorigenesis. Dual-infected cells displayed a plasma cell-like gene expression pattern similar to PELs. KSHV persisted in EBV-transformed B cells and was associated with lytic EBV gene expression, resulting in increased tumor formation. Evidence of elevated lytic EBV replication was also found in EBV/KSHV dually infected lymphoproliferative disorders in humans. Our data suggest that KSHV augments EBV-associated tumorigenesis via stimulation of lytic EBV replication.

Fc-Galactosylation of Human Immunoglobulin Gamma Isotypes Improves C1q Binding and Enhances Complement-Dependent Cytotoxicity.

Binding of the complement component C1q to the CH2 domain of antigen-bound immunoglobulin gamma (IgG) activates the classical complement pathway and depends on its close proximity to Fc fragments of neighboring antibodies. IgG subclasses contain a highly conserved asparagine 297 (N)-linked biantennary glycan within their CH2 domains, the core structure of which can be extended with terminal galactose and sialic acid residues. To investigate whether Fc-glycosylation regulates effector functions of human IgG subclasses, we cloned the antigen-binding region of the CD20-specific monoclonal antibody rituximab into IgG isotype expression vectors. We found that Fc-galactosylation enhances the efficacy of CD20-targeting complement-fixing antibodies for C1q binding and complement-mediated tumor cell lysis. Increased efficacies were restricted to IgG1 and IgG3 subclasses indicating that Fc-galactosylation alone is not sufficient for IgG2 and IgG4 to acquire complement-fixing properties. Addition of terminal galactose to the N-glycan specifically improved binding of C1q without changing antigen- and FcγRIIIa-binding affinities of IgG isotypes. These data indicate that Fc galactosylation can be harnessed to enhance the complement-activating properties of IgG1 and IgG3 antibodies.

IVIG regulates the survival of human but not mouse neutrophils.

Intravenous immunoglobulin (IVIG) are purified IgG preparations made from the pooled plasma from thousands of healthy donors and are being tested in preclinical mouse models. Inherent challenges, however, are the pluripotency of IVIG and its xenogeneicity in animals. IVIG can alter the viability of human neutrophils via agonistic antibodies to Fas and Siglec-9. In this study, we compared the effects of IVIG on human and mouse neutrophils using different death assays. Different commercial IVIG preparations similarly induced cytokine-dependent death in human neutrophils, whereas they had no effects on the survival of either peripheral blood or bone marrow neutrophils from C57BL/6 or BALB/c mice. F(ab')2 but not Fc fragments of IVIG induced death of human neutrophils, whereas neither of these IVIG fragments, nor agonistic monoclonal antibodies to human Fas or Siglec-9 affected the viability of mouse neutrophils. Pooled mouse IgG, which exhibited a different immunoprofile compared to IVIG, also had no effect on mouse cells. Together, these observations demonstrate that effects of IVIG on neutrophil survival are not adequately reflected in current mouse models, despite the key role of these cells in human inflammatory and autoimmune diseases.

The autophagy machinery restrains iNKT cell activation through CD1D1 internalization.

Invariant natural killer T (iNKT) cells are innate T cells with powerful immune regulatory functions that recognize glycolipid antigens presented by the CD1D protein. While iNKT cell-activating glycolipids are currently being explored for their efficacy to improve immunotherapy against infectious diseases and cancer, little is known about the mechanisms that control CD1D antigen presentation and iNKT cell activation in vivo. CD1D molecules survey endocytic pathways to bind lipid antigens in MHC class II-containing compartments (MIICs) before recycling to the plasma membrane. Autophagosomes intersect with MIICs and autophagy-related proteins are known to support antigen loading for increased CD4+ T cell immunity. Here, we report that mice with dendritic cell (DC)-specific deletion of the essential autophagy gene Atg5 showed better CD1D1-restricted glycolipid presentation in vivo. These effects led to enhanced iNKT cell cytokine production upon antigen recognition and lower bacterial loads during Sphingomonas paucimobilis infection. Enhanced iNKT cell activation was independent of receptor-mediated glycolipid uptake or costimulatory signals. Instead, loss of Atg5 in DCs impaired clathrin-dependent internalization of CD1D1 molecules via the adaptor protein complex 2 (AP2) and, thus, increased surface expression of stimulatory CD1D1-glycolipid complexes. These findings indicate that the autophagic machinery assists in the recruitment of AP2 to CD1D1 molecules resulting in attenuated iNKT cell activation, in contrast to the supporting role of macroautophagy in CD4+ T cell stimulation.

Regulation of antibody effector functions through IgG Fc N-glycosylation.

Immunoglobulin gamma (IgG) antibodies are key effector proteins of the immune system. They recognize antigens with high specificity and are indispensable for immunological memory following pathogen exposure or vaccination. The constant, crystallizable fragment (Fc) of IgG molecules mediates antibody effector functions such as complement-dependent cytotoxicity, antibody-mediated cellular cytotoxicity, and antibody-dependent cell-mediated phagocytosis. These functions are regulated by a single N-linked, biantennary glycan of the heavy chain, which resides just below the hinge region, and the presence of specific sugar moieties on the glycan has profound implications on IgG effector functions. Emerging knowledge of how Fc glycans contribute to IgG structure and functions has opened new avenues for the therapeutic exploitation of defined antibody glycoforms in the treatment of cancer and autoimmune diseases. Here, we review recent advances in understanding proinflammatory IgG effector functions and their regulation by Fc glycans.

Interleukins 12 and 15 induce cytotoxicity and early NK-cell differentiation in type 3 innate lymphoid cells.

Type 3 innate lymphoid cells (ILC3s) fulfill protective functions at mucosal surfaces via cytokine production. Although their plasticity to become ILC1s, the innate counterparts of type 1 helper T cells, has been described previously, we report that they can differentiate into cytotoxic lymphocytes with many characteristics of early differentiated natural killer (NK) cells. This transition is promoted by the proinflammatory cytokines interleukin 12 (IL-12) and IL-15, and correlates with expression of the master transcription factor of cytotoxicity, eomesodermin (Eomes). As revealed by transcriptome analysis and flow cytometric profiling, differentiated ILC3s express CD94, NKG2A, NKG2C, CD56, and CD16 among other NK-cell receptors, and possess all components of the cytotoxic machinery. These characteristics allow them to recognize and kill leukemic cells with perforin and granzymes. Therefore, ILC3s can be harnessed for cytotoxic responses via differentiation under the influence of proinflammatory cytokines.

Terminal complement activation is increased and associated with disease severity in CIDP.

Chronic inflammatory demyelinating polyneuropathy (CIDP) is the most common chronic autoimmune neuropathy. While both cell-mediated and humoral mechanisms contribute to its pathogenesis, the rapid clinical response to plasmapheresis implicates a circulating factor responsible for peripheral nerve injury. We report that treatment-naïve patients with CIDP show increased serum and CSF levels of the anaphylatoxin C5a and the soluble terminal complement complex (sTCC). Systemic terminal complement activation correlates with clinical disease severity as determined by the Inflammatory Neuropathy Cause and Treatment (INCAT) disability scale. These data indicate that complement activation contributes to peripheral nerve injury and suggest that complement inhibition should be explored for its potential therapeutic merit in CIDP.