Combination CTLA4Ig and Anti-CD40 Ligand Treatment Modifies T and B Cell Metabolic Profiles and Promotes B Cell Receptor Remodeling in a Mouse Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus is a complex autoimmune disease with significant morbidity that demands further examination of tolerance-inducing treatments. Short-term treatment of lupus-prone NZB/WF1 mice with combination CTLA4Ig and anti-CD40 ligand, but not single treatment alone, suppresses disease for >6 mo via modulation of B and T cell function while maintaining immune responses to exogenous Ags. Three months after a 2-wk course of combination costimulatory blockade, we found a modest decrease in the number of activated T and B cells in both combination and single-treatment cohorts compared with untreated controls. However, only combination treatment mice showed a 50% decrease in spare respiratory capacity of splenic B and T cells. RNA sequencing and gene set enrichment analysis of germinal center (GC) B cells confirmed a reduction in the oxidative phosphorylation signature in the combination treatment cohort. This cohort also manifested increased expression of BCR-associated signaling molecules and increased phosphorylation of PLCγ in GC B cells after stimulation with anti-IgG and anti-CD40. GC B cells from combination treatment mice also displayed a signature involving remodeling of GPI-linked surface proteins. Accordingly, we found a decrease in cell surface expression of the inhibitory molecule CD24 on class-switched memory B cells from aged NZB/W mice that corrected in the combination treatment cohort. Because both a profound decrease in BCR signaling and remodeled immune cell metabolism enhance loss of tolerance in lupus-prone mice, our findings help to explain the restoration of tolerance observed after short-term combination costimulatory blockade.

Molecular Mechanisms of Lupus Susceptibility Allele PBX1D.

Pre-B cell leukemia homeobox 1 (PBX1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, PBX1D, which lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus than healthy control subjects. PBX1D overexpression in CD4+ T cells impaired regulatory T cell homeostasis and expanded inflammatory CD4+ T cells. In this study, we showed that PBX1 message expression is downregulated by activation in CD4+ T cells as well as in B cells. PBX1D protein was less stable than the normal isoform, PBX1B, and it is degraded through the ubiquitin-proteasome-dependent pathway. The DNA binding domain lacking in PBX1D has two putative ubiquitin binding sites, K292 and K293, that are predicted to be in direct contact with DNA. Mutation of K292-293 reduced PBX1B stability to a level similar to PBX1D and abrogated DNA binding. In addition, contrary to PBX1B, PBX1D is retained in the cytoplasm without the help of the cofactors MEIS or PREP1, indicating a different requirement for nuclear translocation. Overall, these findings suggest that multiple post-transcriptional mechanisms are responsible for PBX1D loss of function and induction of CD4+ T cell inflammatory phenotypes in systemic lupus erythematosus.

Glucose Requirement of Antigen-Specific Autoreactive B Cells and CD4+ T Cells.

The activation of lymphocytes in patients with lupus and in mouse models of the disease is coupled with an increased cellular metabolism in which glucose plays a major role. The pharmacological inhibition of glycolysis with 2-deoxy-d-glucose (2DG) reversed the expansion of follicular helper CD4+ T cells and germinal center B cells in lupus-prone mice, as well as the production of autoantibodies. The response of foreign Ags was however not affected by 2DG in these mice, suggesting that B and CD4+ T cell activation by autoantigens is uniquely sensitive to glycolysis. In this study, we tested this hypothesis with monoclonal B cells and CD4+ T cells specific for lupus-relevant autoantigens. AM14 Vκ8R (AM14) transgenic B cells are activated by IgG2a/chromatin immune complexes and they can receive cognate help from chromatin-specific 13C2 CD4+ T cells. We showed that activation of AM14 B cells by their cognate Ag PL2-3 induced glycolysis, and that the inhibition of glycolysis reduced their activation and differentiation into Ab-forming cells, in the absence or presence of T cell help. The dependency of autoreactive B cells on glycolysis is in sharp contrast with the previously reported dependency of 4-hydroxy-3-nitrophenyl acetyl-specific B cells on fatty acid oxidation. Contrary to AM14 B cells, the activation and differentiation of 13C2 T cells into follicular helper CD4+ T cells was not altered by 2DG, which differs from polyclonal CD4+ T cells from lupus-prone mice. These results further define the role of glycolysis in the production of lupus autoantibodies and demonstrate the need to evaluate the metabolic requirements of Ag-specific B and T cells.

Pharmacologically Inferred Glycolysis and Glutaminolysis Requirement of B Cells in Lupus-Prone Mice.

Several studies have shown an enhanced metabolism in the CD4+ T cells of lupus patients and lupus-prone mice. Little is known about the metabolism of B cells in lupus. In this study, we compared the metabolism of B cells between lupus-prone B6.Sle1.Sle2.Sle3 triple-congenic mice and C57BL/6 controls at steady state relative to autoantibody production, as well as during T cell-dependent (TD) and T cell-independent (TI) immunizations. Starting before the onset of autoimmunity, B cells from triple-congenic mice showed an elevated glycolysis and mitochondrial respiration, which were normalized in vivo by inhibiting glycolysis with a 2-deoxy-d-glucose (2DG) treatment. 2DG greatly reduced the production of TI-Ag-specific Abs, but showed minimal effect with TD-Ags. In contrast, the inhibition of glutaminolysis with 6-diazo-5-oxo-l-norleucine had a greater effect on TD than TI-Ag-specific Abs in both strains. Analysis of the TI and TD responses in purified B cells in vitro suggests, however, that the glutaminolysis requirement is not B cell-intrinsic. Thus, B cells have a greater requirement for glycolysis in TI than TD responses, as inferred from pharmacological interventions. B cells from lupus-prone and control mice have different intrinsic metabolic requirements or different responses toward 2DG and 6-diazo-5-oxo-l-norleucine, which mirrors our previous results obtained with follicular Th cells. Overall, these results predict that targeting glucose metabolism may provide an effective therapeutic approach for systemic autoimmunity by eliminating both autoreactive follicular Th and B cells, although it may also impair TI responses.

Metabolic determinants of lupus pathogenesis.

The metabolism of healthy murine and more recently human immune cells has been investigated with an increasing amount of details. These studies have revealed the challenges presented by immune cells to respond rapidly to a wide variety of triggers by adjusting the amount, type, and utilization of the nutrients they import. A concept has emerged that cellular metabolic programs regulate the size of the immune response and the plasticity of its effector functions. This has generated a lot of enthusiasm with the prediction that cellular metabolism could be manipulated to either enhance or limit an immune response. In support of this hypothesis, studies in animal models as well as human subjects have shown that the dysregulation of the immune system in autoimmune diseases is associated with a skewing of the immunometabolic programs. These studies have been mostly conducted on autoimmune CD4+ T cells, with the metabolism of other immune cells in autoimmune settings still being understudied. Here we discuss systemic metabolism as well as cellular immunometabolism as novel tools to decipher fundamental mechanisms of autoimmunity. We review the contribution of each major metabolic pathway to autoimmune diseases, with a focus on systemic lupus erythematosus (SLE), with the relevant translational opportunities, existing or predicted from results obtained with healthy immune cells. Finally, we review how targeting metabolic programs may present novel therapeutic venues.

Relative Contributions of B Cells and Dendritic Cells from Lupus-Prone Mice to CD4+ T Cell Polarization.

Mouse models of lupus have shown that multiple immune cell types contribute to autoimmune disease. This study sought to investigate the involvement of B cells and dendritic cells in supporting the expansion of inflammatory and regulatory CD4+ T cells that are critical for lupus pathogenesis. We used lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) and congenic C57BL/6J (B6) control mice to investigate how the genetic predisposition of these two cell types controls the activity of normal B6 T cells. Using an allogeneic in vitro assay, we showed that TC B1-a and conventional B cells expanded Th17 cells significantly more than their B6 counterparts. This expansion was dependent on CD86 and IL-6 expression and mapped to the Sle1 lupus-susceptibility locus. In vivo, TC B cells promoted greater differentiation of CD4+ T cells into Th1 and follicular helper T cells than did B6 B cells, but they limited the expansion of Foxp3 regulatory CD4+ T cells to a greater extent than did B6 B cells. Finally, when normal B6 CD4+ T cells were introduced into Rag1-/- mice, TC myeloid/stromal cells caused their heightened activation, decreased Foxp3 regulatory CD4+ T cell differentiation, and increased renal infiltration of Th1 and Th17 cells in comparison with B6 myeloid/stromal cells. The results show that B cells from lupus mice amplify inflammatory CD4+ T cells in a nonredundant manner with myeloid/stromal cells.

Glucose Oxidation Is Critical for CD4+ T Cell Activation in a Mouse Model of Systemic Lupus Erythematosus.

We have previously shown that CD4(+) T cells from B6.Sle1Sle2.Sle3 lupus mice and patients present a high cellular metabolism, and a treatment combining 2-deoxy-D-glucose, which inhibits glucose metabolism, and metformin, which inhibits oxygen consumption, normalized lupus T cell functions in vitro and reverted disease in mice. We obtained similar results with B6.lpr mice, another model of lupus, and showed that a continuous treatment is required to maintain the beneficial effect of metabolic inhibitors. Further, we investigated the relative roles of glucose oxidation and pyruvate reduction into lactate in this process. Treatments of B6.Sle1Sle2.Sle3 mice with either 2-deoxy-D-glucose or metformin were sufficient to prevent autoimmune activation, whereas their combination was necessary to reverse the process. Treatment of B6.Sle1Sle2.Sle3 mice with dichloroacetate, an inhibitor of lactate production, failed to effectively prevent or reverse autoimmune pathology. In vitro, CD4(+) T cell activation upregulated the expression of genes that favor oxidative phosphorylation. Blocking glucose oxidation inhibited both IFN-γ and IL-17 production, which could not be achieved by blocking pyruvate reduction. Overall, our data show that targeting glucose oxidation is required to prevent or reverse lupus development in mice, which cannot be achieved by simply targeting the pyruvate-lactate conversion.

The Lupus Susceptibility Gene Pbx1 Regulates the Balance between Follicular Helper T Cell and Regulatory T Cell Differentiation.

Pbx1 controls chromatin accessibility to a large number of genes and is entirely conserved between mice and humans. The Pbx1-d dominant-negative isoform is more frequent in CD4(+) T cells from lupus patients than from healthy controls. Pbx1-d is associated with the production of autoreactive T cells in mice carrying the Sle1a1 lupus-susceptibility locus. Transgenic (Tg) expression of Pbx1-d in CD4(+) T cells reproduced the phenotypes of Sle1a1 mice, with increased inflammatory functions of CD4(+) T cells and impaired Foxp3(+) regulatory T cell (Treg) homeostasis. Pbx1-d-Tg expression also expanded the number of follicular helper T cells (TFHs) in a cell-intrinsic and Ag-specific manner, which was enhanced in recall responses and resulted in Th1-biased Abs. Moreover, Pbx1-d-Tg CD4(+) T cells upregulated the expression of miR-10a, miR-21, and miR-155, which were implicated in Treg and follicular helper T cell homeostasis. Our results suggest that Pbx1-d impacts lupus development by regulating effector T cell differentiation and promoting TFHs at the expense of Tregs. In addition, our results identify Pbx1 as a novel regulator of CD4(+) T cell effector function.

Metabolic Factors that Contribute to Lupus Pathogenesis.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which organ damage is mediated by pathogenic autoantibodies directed against nucleic acids and protein complexes. Studies in SLE patients and in mouse models of lupus have implicated virtually every cell type in the immune system in the induction or amplification of the autoimmune response as well as the promotion of an inflammatory environment that aggravates tissue injury. Here, we review the contribution of CD4+ T cells, B cells, and myeloid cells to lupus pathogenesis and then discuss alterations in the metabolism of these cells that may contribute to disease, given the recent advances in the field of immunometabolism.

Immune Cell Metabolism in Systemic Lupus Erythematosus.

Cellular metabolism represents a newly identified checkpoint of effector functions in the immune system. A solid body of work has characterized the metabolic requirements of normal T cells during activation and differentiation into polarized effector subsets. Similar studies have been initiated to characterize the metabolic requirements for B cells and myeloid cells. Only a few studies though have characterized the metabolism of immune cells in the context of autoimmune diseases. Here, we review what is known on the altered metabolic patterns of CD4+ T cells, B cells, and myeloid cells in lupus patients and lupus-prone mice and how they contribute to lupus pathogenesis. We also discuss how defects in immune metabolism in lupus can be targeted therapeutically.

Activation of rheumatoid factor-specific B cells is antigen dependent and occurs preferentially outside of germinal centers in the lupus-prone NZM2410 mouse model.

AM14 rheumatoid factor (RF) B cells in the MRL/lpr mice are activated by dual BCR and TLR7/9 ligation and differentiate into plasmablasts via an extrafollicular (EF) route. It was not known whether this mechanism of activation of RF B cells applied to other lupus-prone mouse models. We investigated the mechanisms by which RF B cells break tolerance in the NZM2410-derived B6.Sle1.Sle2.Sle3 (TC) strain in comparison with C57BL/6 (B6) controls, each expressing the AM14 H chain transgene in the presence or absence of the IgG2a(a) autoantigen. The TC, but not B6, genetic background promotes the differentiation of RF B cells into Ab-forming cells (AFCs) in the presence of the autoantigen. Activated RF B cells preferentially differentiated into plasmablasts in EF zones. Contrary to the MRL/lpr strain, TC RF B cells were also located within germinal centers, but only the formation of EF foci was positively correlated with the production of RF AFCs. Immunization of young TC.AM14 H chain transgenic mice with IgG2a(a) anti-chromatin immune complexes (ICs) activated RF B cells in a BCR- and TLR9-dependent manner. However, these IC immunizations did not result in the production of RF AFCs. These results show that RF B cells break tolerance with the same general mechanisms in the TC and the MRL/lpr lupus-prone genetic backgrounds, namely the dual activation of the BCR and TLR9 pathways. There are also distinct differences, such as the presence of RF B cells in GCs and the requirement of chronic IgG2a(a) anti-chromatin ICs for full differentiation of RF AFCs.

Mouse IgM Fc receptor, FCMR, promotes B cell development and modulates antigen-driven immune responses.

FcR specific for pentameric IgM (FCMR) is expressed at high levels by B cells. Although circulating IgM has profound effects on responses to pathogens, autoimmunity, and B cell homeostasis, the biologic consequences of its binding to FCMR are poorly understood. We interrogated FCMR contributions to B cell function by studying mice that lack FCMR. FCMR transcripts are expressed at different levels by various B cell subsets. FCMR-deficient mice have reduced numbers of developing B cells, splenic follicular and peritoneal B-2 cells, but increased levels of peritoneal B-1a cells and autoantibodies. After immunization, germinal center B cell and plasma cell numbers are increased. FCMR-deficient B cells are sensitive to apoptosis induced by BCR ligation. Our studies demonstrate that FCMR is required for B cell differentiation and homeostasis, the prevention of autoreactive B cells, and responsiveness to antigenic challenge.

Human CD300a binds to phosphatidylethanolamine and phosphatidylserine, and modulates the phagocytosis of dead cells.

CD300a is an immunoreceptor tyrosine-based inhibitory motif (ITIM) containing molecule that belongs to the CD300 family of paired activating/inhibitory receptors. It has been shown that its ligation inhibits activation signals on cells of both myeloid and lymphoid lineages. The ligands for CD300a have not been identified. Here, we show that a CD300a-Ig fusion protein specifically binds to apoptotic cells that are evolutionary apart, such as human and insect cells, suggesting that the ligand has to be conserved. Using surface plasmon resonance, ultracentrifugation, ELISA, and reporter cell assays, we identified phosphatidylethanolamine (PE) and phosphatidylserine (PS), 2 phospholipids that translocate to the outer leaflet of the plasma membrane of dead cells, as the ligands for CD300a. Mutational and structural modeling studies identified residues that are involved in the binding of CD300a to PE and PS and that form a cavity where the hydrophilic heads of PE and PS, can penetrate. CD300a down-regulates the uptake of apoptotic cells by macrophages and its ectopic expression in CD300a-negative cell lines also decreased the engulfment of dead cells. Collectively, our results indicate that PE and PS are ligands for CD300a, and that this interaction plays an important role in regulating the removal of dead cells.

Leukocyte-associated Ig-like receptor-1-deficient mice have an altered immune cell phenotype.

Cross-linking of the collagen binding receptor leukocyte-associated Ig-like receptor-1 (LAIR-1) in vitro delivers an inhibitory signal that is able to downregulate activation-mediated signals. To study the in vivo function of LAIR-1, we generated LAIR-1(-/-) mice. They are healthy and fertile and have normal longevity; however, they show certain phenotypic characteristics distinct from wild-type mice, including increased numbers of splenic B, regulatory T, and dendritic cells. As LAIR-1(-/-) mice age, the splenic T cell population shows a higher frequency of activated and memory T cells. Because LAIR-1(+/+) and LAIR-1(-/-) T cells traffic with equal proficiency to peripheral lymphoid organs, this is not likely due to abnormal T lymphocyte trafficking. LAIR-1(-/-) mice have lower serum levels of IgG1 and, in response to T-dependent immunization with trinitrophenyl-OVA, switch less efficiently to Ag specific IgG2a and IgG2b, whereas switching to IgG1 is not affected. Several mouse disease models, including experimental autoimmune encephalitis and colitis, were used to examine the effect of LAIR-1 deficiency, and no differences in the responses of LAIR-1(-/-) and LAIR-1(+/+) mice were observed. Taken together, these observations indicate that LAIR-1 plays a role in regulating immune cells and suggest that any adverse effects of its absence may be balanced in vivo by other inhibitory receptors.

Regulation of the STAT3 pathway by lupus susceptibility gene Pbx1 in T cells.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease in which poorly characterized genetic factors lead to the production of proinflammatory or autoreactive T cells. Pre-B cell leukemia homeobox 1 (PBX1) is a transcription factor whose dominant negative isoform (PBX1-D) is overexpressed in the CD4+ T cells of SLE patients and lupus-prone mice. Pbx1-D overexpression favors the expansion of proinflammatory T cells and impairs regulatory T (Treg) cell development. Here we show that Pbx1 deficiency and Pbx1-D overexpression decreased STAT3 expression and activation in T cells. Accordingly, Pbx1 deficiency in T cells and Pbx1-D overexpression reduced STAT3-dependent TH17 cell polarization in vitro, but it had no effect in vivo at steady state. STAT3-dependent follicular helper T (TFH) cell polarization in vitro and splenic TFH cell frequency were not affected by either Pbx1 deficiency or Pbx1-D overexpression. Pbx1 deficiency also increased the expression of cell cycle arrest and pro-apoptotic genes, with an increased apoptosis in T cells. Our results suggest a complex interplay between PBX1 and STAT3, which may contribute to lupus pathogenesis through dysregulation of the cell cycle and apoptosis.

Lupus susceptibility gene Pbx1 controls the development, stability, and function of regulatory T cells via Rtkn2 expression.

The maintenance of regulatory T (Treg) cells critically prevents autoimmunity. Pre-B cell leukemia transcription factor 1 (Pbx1) variants are associated with lupus susceptibility, particularly through the expression of a dominant negative isoform Pbx1-d in CD4+ T cells. Pbx1-d overexpression impaired Treg cell homeostasis and promoted inflammatory CD4+ T cells. Here, we showed a high expression of Pbx1 in human and murine Treg cells, which is decreased in lupus patients and mice. Pbx1 deficiency or Pbx1-d overexpression reduced the number, stability, and suppressive activity of Treg cells, which increased murine responses to immunization and autoimmune induction. Mechanistically, Pbx1 deficiency altered the expression of genes implicated in cell cycle and apoptosis in Treg cells. Intriguingly, Rtkn2, a Rho-GTPase previously associated with Treg homeostasis, was directly transactivated by Pbx1. Our results suggest that the maintenance of Treg cell homeostasis and stability by Pbx1 through cell cycle progression prevent the expansion of inflammatory T cells that otherwise exacerbates lupus progression in the hosts.

Dietary tryptophan and genetic susceptibility expand gut microbiota that promote systemic autoimmune activation.

Tryptophan modulates disease activity and the composition of microbiota in the B6.Sle1.Sle2.Sle3 (TC) mouse model of lupus. To directly test the effect of tryptophan on the gut microbiome, we transplanted fecal samples from TC and B6 control mice into germ-free or antibiotic-treated non-autoimmune B6 mice that were fed with a high or low tryptophan diet. The recipient mice with TC microbiota and high tryptophan diet had higher levels of immune activation, autoantibody production and intestinal inflammation. A bloom of Ruminococcus gnavus (Rg), a bacterium associated with disease flares in lupus patients, only emerged in the recipients of TC microbiota fed with high tryptophan. Rg depletion in TC mice decreased autoantibody production and increased the frequency of regulatory T cells. Conversely, TC mice colonized with Rg showed higher autoimmune activation. Overall, these results suggest that the interplay of genetic and tryptophan can influence the pathogenesis of lupus through the gut microbiota.

Complex regulation of human NKG2D-DAP10 cell surface expression: opposing roles of the γc cytokines and TGF-ß1.

Natural killer (NK) cells help protect the host against viral infections and tumors. NKG2D is a vital activating receptor, also expressed on subsets of T cells, whose ligands are up-regulated by cells in stress. Ligation of NKG2D leads to phosphorylation of the associated DAP10 adaptor protein, thereby activating immune cells. Understanding how the expression of NKG2D-DAP10 is regulated has implications for immunotherapy. We show that IL-2 and TGF-ß1 oppositely regulate NKG2D-DAP10 expression by NK cells. IL-2 stimulation increases NKG2D surface expression despite a decrease in NKG2D mRNA levels. Stimulation with IL-2 results in a small increase of DAP10 mRNA and a large up-regulation of DAP10 protein synthesis, indicating that IL-2-mediated effects are mostly posttranscriptional. Newly synthesized DAP10 undergoes glycosylation that is required for DAP10 association with NKG2D and stabilization of NKG2D expression. TGF-ß1 has an opposite and dominant effect to IL-2. TGF-ß1 treatment decreases DAP10, as its presence inhibits the association of RNA polymerase II with the DAP10 promoter, but not NKG2D mRNA levels. This leads to the down-regulation of DAP10 expression and, as a consequence, NKG2D protein as well. Finally, we show that other γ(c) cytokines act similarly to IL-2 in up-regulating DAP10 expression and NKG2D-DAP10 surface expression.

Cutting edge: mouse CD300f (CMRF-35-like molecule-1) recognizes outer membrane-exposed phosphatidylserine and can promote phagocytosis.

Reportedly, CD300f negatively regulates interactions between dendritic and T cells and acts as an anti-inflammatory molecule in a multiple sclerosis mouse model. We found that a CD300f/Fc chimeric protein specifically binds to apoptotic/dead splenocytes and to apoptotic cells from starved or irradiated lymphocytic cell lines, an observation extended to insect cells. CD300f also binds PMA/ionomycin-activated splenocytes and Ag-stimulated T cells, an interaction inhibited by Annexin V. By ELISA, cosedimentation, and surface plasmon resonance using phospholipid-containing liposomes, we show that CD300f preferentially binds phosphatidylserine and requires a metal ion. Exogenous expression of CD300f in cell lines results in enhanced phagocytosis of apoptotic cells. We conclude that expression of CD300f conveys additional capacity to recognize phosphatidylserine to myeloid cells. The result of this recognition may vary with the overall qualitative and quantitative receptor content, as well as signaling capacity of the expressing effector cell, but enhanced phagocytosis is one measurable outcome.

Transcriptional and metabolic programs promote the expansion of follicular helper T cells in lupus-prone mice.

The expansion of follicular helper T (Tfh) cells, which is tightly associated with the development of lupus, is reversed by the inhibition of either glycolysis or glutaminolysis in mice. Here we analyzed the gene expression and metabolome of Tfh cells and naive CD4+ T (Tn) cells in the B6.Sle1.Sle2.Sle3 (triple congenic, TC) mouse model of lupus and its congenic B6 control. Lupus genetic susceptibility in TC mice drives a gene expression signature starting in Tn cells and expanding in Tfh cells with enhanced signaling and effector programs. Metabolically, TC Tn and Tfh cells showed multiple defective mitochondrial functions. TC Tfh cells also showed specific anabolic programs including enhanced glutamate metabolism, malate-aspartate shuttle, and ammonia recycling, as well as altered dynamics of amino acid content and their transporters. Thus, our study has revealed specific metabolic programs that can be targeted to specifically limit the expansion of pathogenic Tfh cells in lupus.