CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4+ T cells.

As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection.

Acute T-Cell-Driven Inflammation Requires the Endoglycosidase Heparanase-1 from Multiple Cell Types.

It has been accepted for decades that T lymphocytes and metastasising tumour cells traverse basement membranes (BM) by deploying a battery of degradative enzymes, particularly proteases. However, since many redundant proteases can solubilise BM it has been difficult to prove that proteases aid cell migration, particularly in vivo. Recent studies also suggest that other mechanisms allow BM passage of cells. To resolve this issue we exploited heparanase-1 (HPSE-1), the only endoglycosidase in mammals that digests heparan sulfate (HS), a major constituent of BM. Initially we examined the effect of HPSE-1 deficiency on a well-characterised adoptive transfer model of T-cell-mediated inflammation. We found that total elimination of HPSE-1 from this system resulted in a drastic reduction in tissue injury and loss of target HS. Subsequent studies showed that the source of HPSE-1 in the transferred T cells was predominantly activated CD4+ T cells. Based on bone marrow chimeras, two cellular sources of HPSE-1 were identified in T cell recipients, one being haematopoiesis dependent and the other radiation resistant. Collectively our findings unequivocally demonstrate that an acute T-cell-initiated inflammatory response is HPSE-1 dependent and is reliant on HPSE-1 from at least three different cell types.

Regulatory roles of IL-10-producing human follicular T cells.

Mucosal lymphoid tissues such as human tonsil are colonized by bacteria and exposed to ingested and inhaled antigens, requiring tight regulation of immune responses. Antibody responses are regulated by follicular helper T (TFH) cells and FOXP3+ follicular regulatory T (TFR) cells. Here we describe a subset of human tonsillar follicular T cells identified by expression of TFH markers and CD25 that are the main source of follicular T (TF) cell-derived IL-10. Despite lack of FOXP3 expression, CD25+ TF cells resemble T reg cells in high CTLA4 expression, low IL-2 production, and their ability to repress T cell proliferation. CD25+ TF cell-derived IL-10 dampens induction of B cell class-switching to IgE. In children, circulating total IgE titers were inversely correlated with the frequencies of tonsil CD25+ TF cells and IL-10-producing TF cells but not with total T reg cells, TFR, or IL-10-producing T cells. Thus, CD25+ TF cells emerge as a subset with unique T and B cell regulatory activities that may help prevent atopy.

TFH-derived dopamine accelerates productive synapses in germinal centres.

Protective high-affinity antibody responses depend on competitive selection of B cells carrying somatically mutated B-cell receptors by follicular helper T (TFH) cells in germinal centres. The rapid T-B-cell interactions that occur during this process are reminiscent of neural synaptic transmission pathways. Here we show that a proportion of human TFH cells contain dense-core granules marked by chromogranin B, which are normally found in neuronal presynaptic terminals storing catecholamines such as dopamine. TFH cells produce high amounts of dopamine and release it upon cognate interaction with B cells. Dopamine causes rapid translocation of intracellular ICOSL (inducible T-cell co-stimulator ligand, also known as ICOSLG) to the B-cell surface, which enhances accumulation of CD40L and chromogranin B granules at the human TFH cell synapse and increases the synapse area. Mathematical modelling suggests that faster dopamine-induced T-B-cell interactions increase total germinal centre output and accelerate it by days. Delivery of neurotransmitters across the T-B-cell synapse may be advantageous in the face of infection.

B Cell-Extrinsic Myd88 and Fcer1g Negatively Regulate Autoreactive and Normal B Cell Immune Responses.

MyD88 and FcR common γ-chain (Fcer1g, FcRγ) elicit proinflammatory responses to exogenous Ags. Deletion of these receptors in autoimmune models has generally led to reduced overall disease. In B cells, Myd88 is required for anti-DNA and anti-RNA autoantibody responses, whereas Fcer1g is not expressed in these cells. The roles of these receptors in myeloid cells during B cell autoimmune activation remain less clear. To investigate the roles of Myd88 and Fcer1g in non-B cells, we transferred anti-self-IgG (rheumatoid factor) B cells and their physiologic target Ag, anti-chromatin Ab, into mice lacking Fcer1g, Myd88, or both and studied the extrafollicular plasmablast response. Surprisingly, we found a markedly higher and more prolonged response in the absence of either molecule; this effect was accentuated in doubly deficient recipients, with a 40-fold increase compared with wild-type recipients at day 10. This enhancement was dependent on CD40L, indicating that Myd88 and FcRγ, presumably on myeloid APCs, were required to downregulate T cell help for the extrafollicular response. To extend the generality, we then investigated a classic T cell-dependent response to (4-hydroxy-3-nitrophenyl)acetyl conjugated to chicken γ globulin and found a similar effect. Thus, these results reveal novel regulatory roles in the B cell response for receptors that are typically proinflammatory.

Attenuation of AMPK signaling by ROQUIN promotes T follicular helper cell formation.

T follicular helper cells (Tfh) are critical for the longevity and quality of antibody-mediated protection against infection. Yet few signaling pathways have been identified to be unique solely to Tfh development. ROQUIN is a post-transcriptional repressor of T cells, acting through its ROQ domain to destabilize mRNA targets important for Th1, Th17, and Tfh biology. Here, we report that ROQUIN has a paradoxical function on Tfh differentiation mediated by its RING domain: mice with a T cell-specific deletion of the ROQUIN RING domain have unchanged Th1, Th2, Th17, and Tregs during a T-dependent response but show a profoundly defective antigen-specific Tfh compartment. ROQUIN RING signaling directly antagonized the catalytic α1 subunit of adenosine monophosphate-activated protein kinase (AMPK), a central stress-responsive regulator of cellular metabolism and mTOR signaling, which is known to facilitate T-dependent humoral immunity. We therefore unexpectedly uncover a ROQUIN-AMPK metabolic signaling nexus essential for selectively promoting Tfh responses.

Rheumatoid factor B cell memory leads to rapid, switched antibody-forming cell responses.

B cells are critical in the initiation and maintenance of lupus. Autoreactive B cells clonally expand, isotype switch, and mutate--properties associated with memory B cells (MBCs), which are typically generated via germinal centers. The development and functions of autoreactive MBCs in lupus are poorly understood. Moreover, mounting evidence implicates the extrafollicular (EF) response in the generation of switched and mutated autoantibodies that are driven by BCR and TLR corecognition, raising the question of whether MBCs are generated in this context. In this study, we investigated autoreactive MBC generation associated with this type of response. We transferred B cells from AM14 site-directed BCR transgenic mice into nontransgenic normal recipients and elicited an EF response with anti-chromatin Ab, as in prior studies. By following the fate of the stimulated cells at late time points, we found that AM14 B cells persisted at increased frequency for up to 7 wk. Furthermore, these cells had divided in response to Ag but were subsequently quiescent, with a subset expressing the memory marker CD73. These cells engendered rapid, isotype-switched secondary plasmablast responses upon restimulation. Both memory and rapid secondary responses required T cell help to develop, emphasizing the need for T-B collaboration for long-term self-reactivity. Thus, using this model system, we show that the EF response generated persistent and functional MBCs that share some, but not all, of the characteristics of traditional MBCs. Such cells could play a role in chronic or flaring autoimmune disease.

Developing connections amongst key cytokines and dysregulated germinal centers in autoimmunity.

Systemic autoimmunity owing to overactivity of Tfh and dysregulated germinal centers has been described in mice and humans. Cytokines such as IL-21, IFN-γ, IL-6 and IL-17 are elevated in the plasma of mouse models of lupus, arthritis, and multiple sclerosis, and in subsets of patients with autoimmune disease. Monoclonal antibodies targeting these cytokines are entering clinical trials. While these cytokines exert pleiotropic effects on immune cells and organs, it is becoming clear that each and all of them can profoundly regulate Tfh numbers and/or function and induce or maintain the aberrant germinal center reactions that lead to pathogenic autoantibody formation. Here we review recent discoveries into the roles of IL-21, IFN-γ, IL-6, and IL-17 in germinal center responses and antibody-driven autoimmunity. These new insights used in conjunction with biomarkers of an overactive Tfh pathway may help stratify patients to rationalize the use of emerging monoclonal anti-cytokine antibody therapies.

Facultative role for T cells in extrafollicular Toll-like receptor-dependent autoreactive B-cell responses in vivo.

Extrafollicular (EF) B-cell responses are increasingly being recognized as an alternative pathway of B-cell activation, particularly in autoimmunity. Critical cellular interactions required for the EF B-cell response are unclear. A key question in autoimmunity, in which Toll-like receptor (TLR) signals are costimulatory and could be sufficient for B-cell activation, is whether T cells are required for the response. This is pivotal, because autoreactive B cells are considered antigen-presenting cells for autoreactive T cells, but where such interactions occur has not been identified. Here, using AM14 site-directed transgenic rheumatoid factor (RF) mice, we report that B cells can be activated, differentiate, and isotype-switch independent of antigen-specific T-cell help, αß T cells, CD40L signaling, and IL-21 signaling to B cells. However, T cells do dramatically enhance the response, and this occurs via CD40L and IL-21 signals. Surprisingly, the response is completely inducible T-cell costimulator ligand independent. These results establish that, although not required, T cells substantially amplify EF autoantibody production and thereby implicate T-independent autoreactive B cells as a potential vector for breaking T-cell tolerance. We suggest that these findings explain why autoreactivity first focuses on self-components for which B cells carry TLR ligands, because these will uniquely be able to activate B cells independently of T cells, with subsequent T-B interactions activating autoreactive T cells, resulting in chronic autoimmunity.

A new site-directed transgenic rheumatoid factor mouse model demonstrates extrafollicular class switch and plasmablast formation.

The AM14 rheumatoid factor (RF) transgenic (Tg) mouse has been valuable for studying how self-reactive B cells are regulated beyond central tolerance, because they remain ignorant in normal mice. AM14 B-cell activation can be studied on autoimmune-prone strains or by inducing activation with IgG2a anti-chromatin antibodies (Abs). Despite the utility of conventional Ig-Tg mice, site-directed Ig-Tg (sd-Tg) mice provide a more physiological model for B-cell responses, allowing class switch and somatic hypermutation. We report here the creation of an AM14 sd-Tg mouse and describe its phenotype on both normal and autoimmune-prone backgrounds. AM14 sd-Tg B cells develop normally but remain unactivated in the BALB/c background, even after significant aging. In contrast, in the autoimmune-prone strain MRL/lpr, AM14 sd-Tg B cells become activated and secrete large amounts of IgG RF Ab into the serum. Class-switched Ab-forming cells were found in the spleen and bone marrow. IgG RF plasmablasts were also observed in extrafollicular clusters in the spleens of aged AM14 sd-Tg MRL/lpr mice. Class switch and Ab secretion were observed additionally in AM14 sd-Tg BALB/c B cells activated in vivo using IgG2a anti-chromatin Abs. Development of IgG auto-Abs is a hallmark of severe autoimmunity and is related to pathogenesis. Using the AM14 sd-Tg, we now show that switched auto-Ab-forming cells develop robustly outside germinal centers, further confirming the extrafollicular expression of activation induced cytidine deaminase (AID). This model will allow more physiological studies of B-cell biology in the future, including memory responses marked by class switch.

T cell-independent and toll-like receptor-dependent antigen-driven activation of autoreactive B cells.

On the lupus-prone MRL-lpr/lpr (MRL-lpr) background, AM14 rheumatoid factor (RF) B cells are activated, differentiate into plasmablasts, and undergo somatic hypermutation outside of follicles. Using multiple strategies to impair T cells, we found that such AM14 B cell activation did not require T cells but could be modulated by them. In vitro, the signaling adaptor MyD88 is required for IgG anti-chromatin to stimulate AM14 B cell proliferation when T cells are absent. However, the roles of Toll-like receptors (TLRs) in AM14 B cell activation in vivo have not been investigated. We found that activation, expansion, and differentiation of AM14 B cells depended on MyD88; however, mice lacking either TLR7 or TLR9 displayed partial defects, indicating complex roles for these receptors. T cell-independent activation of certain autoreactive B cells, which gain stimuli via endogenous TLR ligands instead of T cells, may be the initial step in the generation of canonical autoantibodies.

Pathogenic myelin oligodendrocyte glycoprotein antibodies recognize glycosylated epitopes and perturb oligodendrocyte physiology.

Antibodies to myelin components are routinely detected in multiple sclerosis patients. However, their presence in some control subjects has made it difficult to determine their contribution to disease pathogenesis. Immunization of C57BL/6 mice with either rat or human myelin oligodendrocyte glycoprotein (MOG) leads to experimental autoimmune encephalomyelitis (EAE) and comparable titers of anti-MOG antibodies as detected by ELISA. However, only immunization with human (but not rat) MOG results in a B cell-dependent EAE. In this study, we demonstrate that these pathogenic and nonpathogenic anti-MOG antibodies have a consistent array of differences in their recognition of antigenic determinants and biological effects. Specifically, substituting proline at position 42 with serine in human MOG (as in rat MOG) eliminates the B cell requirement for EAE. All MOG proteins analyzed induced high titers of anti-MOG (tested by ELISA), but only antisera from mice immunized with unmodified human MOG were encephalitogenic in primed B cell-deficient mice. Nonpathogenic IgGs bound recombinant mouse MOG and deglycosylated MOG in myelin (tested by Western blot), but only pathogenic IgGs bound glycosylated MOG. Only purified IgG to human MOG bound to live rodent oligodendrocytes in culture and, after cross-linking, induced repartitioning of MOG into lipid rafts, followed by dramatic changes in cell morphology. The data provide a strong link between in vivo and in vitro observations regarding demyelinating disease, further indicate a biochemical mechanism for anti-MOG-induced demyelination, and suggest in vitro tools for determining autoimmune antibody pathogenicity in multiple sclerosis patients.