Vanessa L. Bryant

Cytokine-Mediated Regulation of Human B Cell Differentiation into Ig-Secreting Cells: Predominant Role of IL-21 Produced by CXCR5+ T Follicular Helper Cells

Differentiation of B cells into Ig-secreting cells (ISC) is critical for the generation of protective humoral immune responses. Because of the important role played by secreted Ig in host protection against infection, it is necessary to identify molecules that control B cell differentiation. Recently, IL-21 was reported to generate ISC from activated human B cells. In this study, we examined the effects of IL-21 on the differentiation of all human mature B cell subsets—neonatal, transitional, naive, germinal center, IgM-memory, and isotype-switched memory cells—into ISC and compared its efficacy to that of IL-10, a well-known mediator of human B cell differentiation. IL-21 rapidly induced the generation of ISC and the secretion of vast quantities IgM, IgG and IgA from all of these B cell subsets. Its effect exceeded that of IL-10 by up to 100-fold, highlighting the potency of IL-21 as a B cell differentiation factor. Strikingly, IL-4 suppressed the stimulatory effects of IL-21 on naive B cells by reducing the expression of B-lymphocyte induced maturation protein-1 (Blimp-1). In contrast, memory B cells were resistant to the inhibitory effects of IL-4. Finally, the ability of human tonsillar CD4+CXCR5+CCR7− T follicular helper (TFH) cells, known to be a rich source of IL-21, to induce the differentiation of autologous B cells into ISC was mediated by the production of IL-21. These findings suggest that IL-21 produced by TFH cells during the primary as well as the subsequent responses to T cell-dependent Ag makes a major contribution to eliciting and maintaining long-lived humoral immunity.

Mycobacterial disease and impaired IFN-γ immunity in humans with inherited ISG15 deficiency.

Tuberculosis Vaccine Conundrum Some children experience severe clinical disease when they are vaccinated against tuberculosis, an attenuated live vaccine that is normally innocuous in humans. Several germline mutations have been identified that account for this susceptibility, and now Bogunovic et al. (p. 1684, published online 2 August) add another to the list—ISG15. Uncovering this mutation, which is inherited in an autosomal recessive manner, was a surprise because studies with mice deficient in ISG15 showed enhanced susceptibility to some viral, but not bacterial, infections. Nevertheless, patients lacking ISG15 were not able to produce adequate amounts of interferon-γ, a cytokine critical for clearance of the bacteria. A mutation that accounts for adverse reactions to the Bacille Calmette-Guérin vaccine against tuberculosis is identified. ISG15 is an interferon (IFN)-α/β–inducible, ubiquitin-like intracellular protein. Its conjugation to various proteins (ISGylation) contributes to antiviral immunity in mice. Here, we describe human patients with inherited ISG15 deficiency and mycobacterial, but not viral, diseases. The lack of intracellular ISG15 production and protein ISGylation was not associated with cellular susceptibility to any viruses that we tested, consistent with the lack of viral diseases in these patients. By contrast, the lack of mycobacterium-induced ISG15 secretion by leukocytes—granulocyte, in particular—reduced the production of IFN-γ by lymphocytes, including natural killer cells, probably accounting for the enhanced susceptibility to mycobacterial disease. This experiment of nature shows that human ISGylation is largely redundant for antiviral immunity, but that ISG15 plays an essential role as an IFN-γ–inducing secreted molecule for optimal antimycobacterial immunity.

Kinetics of Human B Cell Behavior and Amplification of Proliferative Responses following Stimulation with IL-21

Although recent studies indicated that IL-21 is an important regulator of human B cell activation, detailed comparison of the effects of IL-21 on distinct B cell subsets have not been performed. Our studies revealed that IL-21R is expressed by naive and germinal center B cells, but not memory or plasma cells. IL-21R was increased on naive and memory B cells following in vitro activation. Investigation into the kinetics and magnitude of responses of human B cells to IL-21 revealed that IL-21 potently augmented proliferation of CD40L-stimulated neonatal, splenic naive, and memory and tonsil germinal center B cells. This response exceeded that induced by IL-4, IL-10, and IL-13, cytokines that also induce B cell proliferation. Remarkably, CD40L/IL-21-stimulated naive B cells underwent the same number of divisions as memory cells and exhibited a greater enhancement in their response compared with CD40L alone than memory B cells. Therefore, IL-21 is a powerful growth factor for naive B cells. This may result from the higher expression of IL-21R on naive, compared with memory, B cells. Stimulation of human B cells with CD40L/IL-21 also induced IL-10 production and activation of STAT3. We propose that IL-21 may have therapeutic application in conditions of immunodeficiency where it could expand naive B cells, the predominant B cell subset in such patients. Conversely, because IL-21 is increased in murine models of lupus, dysregulated IL-21 production may contribute to perturbed B cell homeostasis observed in systemic lupus erythematosus. Thus, antagonizing IL-21 may be a novel strategy for treating Ab-mediated autoimmune diseases.

IL-21-Induced Isotype Switching to IgG and IgA by Human Naive B Cells Is Differentially Regulated by IL-4

Naive B cells can alter the effector function of their Ig molecule by isotype switching, thereby allowing them to secrete not only IgM, but also the switched isotypes IgG, IgA, and IgE. Different isotypes are elicited in response to specific pathogens. Similarly, dysregulated production of switched isotypes underlies the development of various diseases, such as autoimmunity and immunodeficiency. Thus, it is important to characterize mediators controlling isotype switching, as well as their contribution to the overall B cell response. Isotype switching in human naive B cells can be induced by CD40L together with IL-4, IL-10, IL-13, and/or TGF-β. Recently, IL-21 was identified as a switch factor for IgG1 and IgG3. However, the effect of IL-21 on switching to IgA, as well as the interplay between IL-21 and other switch factors, remains unknown. We found that IL-4 and IL-21 individually induced CD40L-stimulated human naive B cells to undergo switching to IgG, with IL-4 predominantly inducing IgG1+ cells and IL-21 inducing IgG3. Culture of naive B cells with CD40L and IL-21, but not IL-4, also yielded IgA+ cells. Combining IL-4 and IL-21 had divergent effects on isotype switching. Specifically, while IL-4 and IL-21 synergistically increased the generation of IgG1+ cells from CD40L-stimulated B cells, IL-4 concomitantly abolished IL-21-induced switching to IgA. Our findings demonstrate the dynamic interplay between IL-4 and IL-21 in regulating the production of IgG subclasses and IgA, and suggest temporal roles for these cytokines in humoral immune responses to specific pathogens.

STAT3 is required for IL-21–induced secretion of IgE from human naive B cells

The production of immunoglobulin E (IgE) is tightly regulated. This is evidenced by the fact that it comprises less than 0.0001% of serum Ig, and aberrant production causes atopic conditions, including allergy, rhinitis, and anaphylaxis. Interleukin-4 (IL-4) is a well-characterized inducer of IgE by human and murine B cells, whereas interferon-gamma can antagonize this effect. IL-21 has also been recognized for its ability to suppress IL-4-induced IgE production by murine B cells. Here, we identified IL-21 as an inducer of IgE production by CD40L-stimulated human naive B cells. Furthermore, there was a striking synergy between IL-4 and IL-21 on inducing IgE secretion by CD40L-stimulated human B cells, such that the levels detected under these conditions exceeded those induced by IL-4 or IL-21 alone by more than 10-fold. IL-21 induced activation of STAT3 and analysis of B cells from patients with loss-of-function STAT3 mutations revealed that the ability of IL-21 to induce IgE secretion, and augment that driven by IL-4, was STAT3-dependent. These findings highlight a fundamental difference between the regulation of IgE production by human and murine B cells and have implications for the dysregulated production of IgE in conditions characterized by extremely high levels of serum IgE.

Haploinsufficiency of the NF-κB1 Subunit p50 in Common Variable Immunodeficiency

Common variable immunodeficiency (CVID), characterized by recurrent infections, is the most prevalent symptomatic antibody deficiency. In ∼90% of CVID-affected individuals, no genetic cause of the disease has been identified. In a Dutch-Australian CVID-affected family, we identified a NFKB1 heterozygous splice-donor-site mutation (c.730+4A>G), causing in-frame skipping of exon 8. NFKB1 encodes the transcription-factor precursor p105, which is processed to p50 (canonical NF-κB pathway). The altered protein bearing an internal deletion (p.Asp191_Lys244delinsGlu; p105ΔEx8) is degraded, but is not processed to p50ΔEx8. Altered NF-κB1 proteins were also undetectable in a German CVID-affected family with a heterozygous in-frame exon 9 skipping mutation (c.835+2T>G) and in a CVID-affected family from New Zealand with a heterozygous frameshift mutation (c.465dupA) in exon 7. Given that residual p105 and p50—translated from the non-mutated alleles—were normal, and altered p50 proteins were absent, we conclude that the CVID phenotype in these families is caused by NF-κB1 p50 haploinsufficiency.

A novel form of human STAT1 deficiency impairing early but not late responses to interferons

Autosomal recessive STAT1 deficiency is associated with impaired cellular responses to interferons and susceptibility to intracellular bacterial and viral infections. We report here a new form of partial STAT1 deficiency in 2 siblings presenting mycobacterial and viral diseases. Both carried a homozygous missense mutation replacing a lysine with an asparagine residue at position 201 (K201N) of STAT1. This mutation causes the abnormal splicing out of exon 8 from most STAT1 mRNAs, thereby decreasing (by ~ 70%) STAT1 protein levels. The mutant STAT1 proteins are not intrinsically deleterious, in terms of tyrosine phosphorylation, dephosphorylation, homodimerization into γ-activating factor and heterotrimerization into ISGF-3, binding to specific DNA elements, and activation of the transcription. Interestingly, the activation of γ-activating factor and ISGF3 was impaired only at early time points in the various cells from patient (within 1 hour of stimulation), whereas sustained impairment occurs in other known forms of complete and partial recessive STAT1 deficiency. Consequently, delayed responses were normal; however, the early induction of interferon-stimulated genes was selectively and severely impaired. Thus, the early cellular responses to human interferons are critically dependent on the amount of STAT1 and are essential for the appropriate control of mycobacterial and viral infections.

Simple diagnosis of STAT1 gain-of-function alleles in patients with chronic mucocutaneous candidiasis

CMCD is a rare congenital disorder characterized by persistent or recurrent skin, nail, and mucosal membrane infections caused by Candida albicans. Heterozygous GOF STAT1 mutations have been shown to confer AD CMCD as a result of impaired dephosphorylation of STAT1. We aimed to identify and characterize STAT1 mutations in CMCD patients and to develop a simple diagnostic assay of CMCD. Genetic analysis of STAT1 was performed in patients and their relatives. The mutations identified were characterized by immunoblot and reporter assay using transient gene expression experiments. Patientsˈ leukocytes are investigated by flow cytometry and immunoblot. Six GOF mutations were identified, three of which are reported for the first time, that affect the CCD and DBD of STAT1 in two sporadic and four multiplex cases in 10 CMCD patients from Japan. Two of the 10 patients presented with clinical symptoms atypical to CMCD, including other fungal and viral infections, and three patients developed bronchiectasis. Immunoblot analyses of patientsˈ leukocytes showed abnormally high levels of pSTAT1 following IFN‐γ stimulation. Based on this finding, we performed a flow cytometry‐based functional analysis of STAT1 GOF alleles using IFN‐γ stimulation and the tyrosine kinase inhibitor, staurosporine. The higher levels of pSTAT1 observed in primary CD14+ cells from patients compared with control cells persisted and were amplified by the presence of staurosporine. We developed a flow cytometry‐based STAT1 functional screening method that would greatly facilitate the diagnosis of CMCD patients with GOF STAT1 mutations.

Haploinsufficiency at the human IFNGR2 locus contributes to mycobacterial disease

Mendelian susceptibility to mycobacterial diseases (MSMD) is a rare syndrome, the known genetic etiologies of which impair the production of, or the response to interferon-gamma (IFN-γ). We report here a patient (P1) with MSMD whose cells display mildly impaired responses to IFN-γ, at levels, however, similar to those from MSMD patients with autosomal recessive (AR) partial IFN-γR2 or STAT1 deficiency. Whole-exome sequencing (WES) and Sanger sequencing revealed only one candidate variation for both MSMD-causing and IFN-γ-related genes. P1 carried a heterozygous frame-shift IFNGR2 mutation inherited from her father. We show that the mutant allele is intrinsically loss-of-function and not dominant-negative, suggesting haploinsufficiency at the IFNGR2 locus. We also show that Epstein-Barr virus transformed B lymphocyte cells from 10 heterozygous relatives of patients with AR complete IFN-γR2 deficiency respond poorly to IFN-γ, in some cases as poorly as the cells of P1. Naive CD4(+) T cells and memory IL-4-producing T cells from these individuals also responded poorly to IFN-γ, whereas monocytes and monocyte-derived macrophages (MDMs) did not. This is consistent with the lower levels of expression of IFN-γR2 in lymphoid than in myeloid cells. Overall, MSMD in this patient is probably due to autosomal dominant (AD) IFN-γR2 deficiency, resulting from haploinsufficiency, at least in lymphoid cells. The clinical penetrance of AD IFN-γR2 deficiency is incomplete, possibly due, at least partly, to the variability of cellular responses to IFN-γ in these individuals.

Chemokines, their receptors and human disease: the good, the bad and the itchy

Chemokines are a highly specialized group of cytokines that coordinate trafficking and homing of leucocytes between bone marrow, lymphoid organs and sites of infection or inflammation. They are also responsible for structural organization within lymphoid organs. Aberrant expression or function of these molecules, or their receptors, has been linked to protection or susceptibility to specific infectious diseases, as well as the risk of autoimmune disease and malignancy, revealing critical roles of chemokines and their receptors in human health, disease and therapeutics. In this review, we focus on human diseases that provide lessons regarding the critical role of these specialized and complex cytokines.