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Genetic primary immunodeficiency diseases are increasingly recognized, with pathogenic mutations changing the composition of circulating leukocyte subsets measured by flow cytometry (FCM). Discerning changes in multiple subpopulations is challenging, and subtle trends might be missed if traditional reference ranges derived from a control population are applied. We developed an algorithm where centiles were allocated using non-parametric comparison to controls, generating multiparameter heat maps to simultaneously represent all leukocyte subpopulations for inspection of trends within a cohort or segregation with a putative genetic mutation. To illustrate this method, we analyzed patients with Primary Antibody Deficiency (PAD) and kindreds harboring mutations in TNFRSF13B (encoding TACI), CTLA4, and CARD11. In PAD, loss of switched memory B cells (B-SM) was readily demonstrated, but as a continuous, not dichotomous, variable. Expansion of CXCR5+/CD45RA- CD4+ T cells (X5-Th cells) was a prominent feature in PAD, particularly in TACI mutants, and patients with expansion in CD21-lo B cells or transitional B cells were readily apparent. We observed differences between unaffected and affected TACI mutants (increased B cells and CD8+ T-effector memory cells, loss of B-SM cells and non-classical monocytes), cellular signatures that distinguished CTLA4 haploinsufficiency itself (expansion of plasmablasts, activated CD4+ T cells, regulatory T cells, and X5-Th cells) from its clinical expression (B-cell depletion), and those that were associated with CARD11 gain-of-function mutation (decreased CD8+ T effector memory cells, B cells, CD21-lo B cells, B-SM cells, and NK cells). Co-efficients of variation exceeded 30% for 36/54 FCM parameters, but by comparing inter-assay variation with disease-related variation, we ranked each parameter in terms of laboratory precision vs. disease variability, identifying X5-Th cells (and derivatives), naïve, activated, and central memory CD8+ T cells, transitional B cells, memory and SM-B cells, plasmablasts, activated CD4 cells, and total T cells as the 10 most useful cellular parameters. Applying these to cluster analysis of our PAD cohort, we could detect subgroups with the potential to reflect underlying genotypes. Heat mapping of normalized FCM data reveals cellular trends missed by standard reference ranges, identifies changes associating with a phenotype or genotype, and could inform hypotheses regarding pathogenesis of genetic immunodeficiency.

Systemic lupus erythematosus (SLE) is the prototypic systemic autoimmune disease. It is thought that many common variant gene loci of weak effect act additively to predispose to common autoimmune diseases, while the contribution of rare variants remains unclear. Here we describe that rare coding variants in lupus-risk genes are present in most SLE patients and healthy controls. We demonstrate the functional consequences of rare and low frequency missense variants in the interacting proteins BLK and BANK1, which are present alone, or in combination, in a substantial proportion of lupus patients. The rare variants found in patients, but not those found exclusively in controls, impair suppression of IRF5 and type-I IFN in human B cell lines and increase pathogenic lymphocytes in lupus-prone mice. Thus, rare gene variants are common in SLE and likely contribute to genetic risk.

Hyperinflammatory syndromes are life-threatening disorders caused by overzealous immune cell activation and cytokine release, often resulting from defects in negative feedback mechanisms. In the quintessential hyperinflammatory syndrome familial hemophagocytic lymphohistiocytosis (HLH), inborn errors of cytotoxicity result in effector cell accumulation, immune dysregulation and, if untreated, tissue damage and death. Here, we describe a human case with a homozygous nonsense R688* RC3H1 mutation suffering from hyperinflammation, presenting as relapsing HLH. RC3H1 encodes Roquin-1, a posttranscriptional repressor of immune-regulatory proteins such as ICOS, OX40 and TNF. Comparing the R688* variant with the murine M199R variant reveals a phenotypic resemblance, both in immune cell activation, hypercytokinemia and disease development. Mechanistically, R688* Roquin-1 fails to localize to P-bodies and interact with the CCR4-NOT deadenylation complex, impeding mRNA decay and dysregulating cytokine production. The results from this unique case suggest that impaired Roquin-1 function provokes hyperinflammation by a failure to quench immune activation., Roquin-1 is a posttranscriptional regulator that controls the expression of many immune-related genes such as ICOS and TNFA. Here, the authors report a homozygous R688* loss of function mutation in Roquin-1 in a patient with syndromic uncontrolled hyperinflammation associated with immune cell activation and hypercytokinemia.

Class-switch recombination (CSR) is a DNA recombination process that replaces the immunoglobulin (Ig) constant region for the isotype that can best protect against the pathogen. Dysregulation of CSR can cause self-reactive BCRs and B cell lymphomas; understanding the timing and location of CSR is therefore important. Although CSR commences upon T cell priming, it is generally considered a hallmark of germinal centers (GCs). Here, we have used multiple approaches to show that CSR is triggered prior to differentiation into GC B cells or plasmablasts and is greatly diminished in GCs. Despite finding a small percentage of GC B cells expressing germline transcripts, phylogenetic trees of GC BCRs from secondary lymphoid organs revealed that the vast majority of CSR events occurred prior to the onset of somatic hypermutation. As such, we have demonstrated the existence of IgM-dominated GCs, which are unlikely to occur under the assumption of ongoing switching.

Activation-induced deaminase (AID) is a B lymphocyte-specific DNA deaminase that triggers Ig class-switch recombination (CSR) and somatic hypermutation. It shuttles between cytoplasm and nucleus, containing a nuclear export sequence (NES) at its carboxyterminus. Intriguingly, the precise nature of this NES is critical to AID's function in CSR, though not in somatic hypermutation. Many alterations to the NES, while preserving its nuclear export function, destroy CSR ability. We have previously speculated that AID's ability to potentiate CSR may critically depend on the affinity of interaction between its NES and Crm1 exportin. Here, however, by comparing multiple AID NES mutants, we find that – beyond a requirement for threshold Crm1 binding – there is little correlation between CSR and Crm1 binding affinity. The results suggest that CSR, as well as the stabilisation of AID, depend on an interaction between the AID C-terminal decapeptide and factor(s) additional to Crm1.

The concept that the immune system can recognise tumour cells and either eliminate them (tumour immune surveillance) or select for immunologically resistant variants (immunoediting) is gaining general acceptance by immunologists. In terms of an adaptive immune response to cancer, however, much of the research has focused on the response of cytotoxic CD8+ T lymphocytes to tumour-specific antigens and the production of Th1 cytokines by CD4+ and CD8+ T cells. In contrast, Th2-mediated immunity has traditionally been viewed as favouring tumour growth, both by promoting angiogenesis and by inhibiting cell-mediated immunity and subsequent tumour cell killing. While there is evidence that components of type 2 inflammation, such as B cells and interleukin-10, do promote tumour growth, there are also many studies demonstrating the anti-tumour activity of CD4+ Th2 cells, particularly in collaboration with tumour-infiltrating granulocytes, such as eosinophils. In this review, we examine all the components of type 2 immunity and their effects on tumour growth. Collectively, from this analysis, we conclude that there is a great potential for the development of Th2-mediated immunotherapies that harness the cytotoxic activity of eosinophils.

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. DOI: http://dx.doi.org/10.7554/eLife.08698.001, eLife digest The immune system protects the body from invading microbes like bacteria and viruses. Upon recognizing the presence of these microbes, cells in the immune system are activated to destroy the foreign threat and clear it from the body. A type of immune cell called T follicular helper cells (or Tfh for short) are formed during an infection and are essential for coordinating other immune cells to produce high-quality antibody proteins that attack the microbes. Without Tfh cells, life-long production of these protective antibodies is severely crippled, which can cause common variable immune deficiency and other serious immunodeficiency diseases. On the other hand, the body must also avoid generating excessive numbers of Tfh cells, which can lead to the production of antibodies that attack healthy cells of the body. ROQUIN is a protein that inhibits the formation of Tfh cells and other types of active T cells. A region on the protein called the ROQ domain destabilizes particular molecules of ribonucleic acid (RNA) that are required for these specialist T cells to form and work properly. ROQUIN belongs to a large family of enzymes that have a so-called RING domain, which is a feature that enables these enzymes to attach tags onto specific target proteins to modify their activity or stability. However, it was not known whether the RING domain of ROQUIN was active. Ramiscal et al. now address this question in mice. Unexpectedly, the experiments show that the RING domain is required to promote the formation of Tfh cells, but not other types of active T cells. This domain allows ROQUIN to repress an enzyme called AMPK, which normally blocks cell growth by regulating cell metabolism. The findings suggest that the different roles of the ROQ and RING domains allow ROQUIN to fine-tune the numbers of Tfh cells so that they remain within a safe range. In the future, these findings may aid the development of vaccines that are more efficient at generating protective Tfh cells to prevent infectious diseases. DOI: http://dx.doi.org/10.7554/eLife.08698.002

Plasma cells (PCs) represent the final stage of B-cell differentiation and are devoted to the production of immunoglobulin (Ig). Perturbations to their development can result in human disorders characterized by PC expansion and hypergammaglobulinemia. Ig-secreting cells (ISCs) have been identified in secondary lymphoid tissues and bone marrow (BM). Most ISCs in lymphoid tissue are short-lived; in contrast, ISCs that migrate to the BM become long-lived PCs and continue to secrete immunoglobulin for extended periods. However, a small population of long-lived PCs has been identified in rodent spleen, suggesting that PCs may persist in secondary lymphoid tissue and that the spleen, as well as the BM, plays an important role in maintaining long-term humoral immunity. For these reasons, we examined ISCs in human spleen and identified a population that appears analogous to long-lived rodent splenic PCs. Human splenic ISCs shared morphologic, cellular, molecular, and functional characteristics with long-lived PCs in BM, demonstrating their commitment to the PC lineage. Furthermore, the detection of highly mutated immunoglobulin V region genes in splenic ISCs suggested they are likely to be antigen-selected and to secrete high-affinity immunoglobulin. Thus, our results suggest that splenic ISCs have an important role in humoral immunity and may represent the affected cell type in some B-cell dyscrasias.

Angioimmunoblastic T-cell lymphoma (AITL) is the second most common peripheral T-cell lymphoma with unusual clinical and pathologic features and a poor prognosis despite intensive chemotherapy. Recent studies have suggested AITL derives from follicular helper T (TFH) cells, but the causative molecular pathways remain largely unknown. Here we show that approximately 50% of mice heterozygous for the “san” allele of Roquin develop tumors accompanied by hypergammaglobulinemia by 6 months of age. Affected lymph nodes displayed the histologic features diagnostic of AITL, except for the presence of expanded FDC networks. Accumulation of TFH cells preceded tumor development, and clonal rearrangements in the TCR-β genes were present in most tumors. Furthermore, TFH cells exhibited increased clonality compared with non-TFH cells from the same lymph nodes, even in the absence of tumors. Genetic manipulations that prevent TFH development, such as deletion of ICOS, CD28, and SAP, partially or completely abrogated tumor development, confirming a TFH-derived origin. Roquinsan/+ mice emerge as a useful model to investigate the molecular pathogenesis of AITL and for preclinical testing of therapies aimed at targeting dysregulated TFH cells or their consequences.

Earlier studies have shown that the adoptive transfer of Th2-polarized CD4 T cells can clear established tumors from mice in an antigen-specific manner. Although eosinophils were implicated in this process, the exact mechanism of tumor clearance and which immune effector cells were involved, remain to be defined. Consequently, experiments were undertaken to elucidate the mechanism of Th2-mediated destruction of B16-F1 melanoma cells by examining the in vitro antitumor activity of leukocytes within a type-2 inflammatory infiltrate. The experimental data show that activation of alternatively activated macrophages (aaMacs) within type-2 infiltrates by IL-4 or IL-13 can inhibit B16-F1 melanoma cell proliferation through a mechanism that is dependent on arginase-1 depletion of L-arginine within the tumor cell microenvironment. Interestingly, whilst at higher E:T ratios aaMac exhibited antitumor activity, at lower E:T ratios aaMacs were observed to enhance rather than inhibit B16-F1 melanoma cell growth. This highlights the fine balance between stimulating the antitumorigenic and protumorigenic properties of aaMacs in tumor immunotherapy protocols.

The concept that the immune system has the potential to recognize tumor cells and either eliminate them (tumor immune surveillance) or select for immune-resistant variants (immunoediting) has gained a resurgence of interest by the scientific community in the last decade. To date, much of the research on the immune response to cancer has focused on the response of cytotoxic CD8+ T lymphocytes to tumor-specific antigens and the production of Th1 cytokines by CD4+ and CD8+ T cells. In contrast, Th2-mediated immunity has traditionally been viewed as enhancing tumor growth, both by promoting angiogenesis and by inhibiting cell-mediated immunity and subsequent tumor cell killing. Although components of Th2-mediated immunity have been shown to promote tumor growth, there is also an expanding body of evidence demonstrating the anti-tumor activity of CD4+ Th2 cells, particularly in collaboration with tumor-infiltrating granulocytes, such as eosinophils. In this chapter we examine all the key components of type 2 immunity and their effects on tumor growth. Based on this collective data, there exists great potential for the development of Th2-mediated immunotherapies that harness the anti-tumor activity of eosinophils, alternatively activated macrophages and the antigen–IgE–receptor axis.

The role of the immune system in the surveillance of transformed cells has seen a resurgence of interest in the last 10 years, with a substantial body of data in mice and humans supporting a role for the immune system in host protection from tumor development and in shaping tumor immunogenicity. A number of earlier studies have demonstrated that eosinophils, when recruited into tumors, can very effectively eradicate transplantable tumors. In this study, we investigated whether eosinophils also play a role in tumor immune surveillance by determining the incidence of methylcholanthrene (MCA)-induced fibrosarcomas in IL-5 transgenic mice that have greatly enhanced levels of circulating eosinophils, CCL11 (eotaxin-1)-deficient mice that lack a key chemokine that recruits eosinophils into tissues, and the eosinophil-deficient mouse strains, IL-5/CCL11−/− and ΔdblGATA. It was found that MCA-induced tumor incidence and growth were significantly attenuated in IL-5 transgenic mice of both the BALB/c and C57BL/6 backgrounds. Histological examination revealed that the protective effect of IL-5 was associated with massively enhanced numbers of eosinophils within and surrounding tumors. Conversely, there was a higher tumor incidence in CCL11−/− BALB/c mice, which was associated with a reduced eosinophil influx into tumors. This correlation was confirmed in the eosinophil-deficient IL-5/CCL11−/− and ΔdblGATA mouse strains, where tumor incidence was greatly increased in the total absence of eosinophils. In addition, subsequent in vitro studies found that eosinophils could directly kill MCA-induced fibrosarcoma cells. Collectively, our data support a potential role for the eosinophil as an effector cell in tumor immune surveillance.

Plasma cells (PC) localize to discrete areas of secondary lymphoid tissue and bone marrow (BM). The positioning of PC in different sites is believed to be regulated by chemokines and adhesion molecules expressed by accessory cells in the lymphoid tissue microenvironment. However, the mechanisms responsible for the positioning of PC within the red pulp (RP) of human spleen have not been elucidated. Therefore, we examined the contribution of human splenic stromal cells to the migration and function of human PC. Splenic PC expressed the chemokine receptor CXCR4 and responded to its ligand CXCL12. In contrast, PC lacked CXCR5 and CCR7, and consequently exhibited minimal migration towards CXCL13 and CCL21. Splenic stromal cells proved to be a rich source of CXCL12, and could induce the migration of human B cells. Furthermore, they supported Ig production by splenic PC mainly by secreting IL-6. Lastly, a striking difference between splenic and BM PC was the constitutive expression of CD11a by only splenic PC. Notably, splenic stromal cells expressed high levels of CD54, the counter-structure of CD11a, and splenic PC were positioned adjacent to stromal cells in the RP. Thus, we propose that stromal cells attract PC to the RP and contribute to their retention and function through the combined expression of CXCL12, CD54 and IL-6.

The transcription factor BATF directly regulates key components of the formation and function of follicular helper T cells and antibody class switching in B cells.

Follicular helper T (Tfh) cells provide selection signals to germinal center B cells, which is essential for long-lived antibody responses. High CXCR5 and low CCR7 expression facilitates their homing to B cell follicles and distinguishes them from T helper 1 (Th1), Th2, and Th17 cells. Here, we showed that Bcl-6 directs Tfh cell differentiation: Bcl-6-deficient T cells failed to develop into Tfh cells and could not sustain germinal center responses, whereas forced expression of Bcl-6 in CD4(+) T cells promoted expression of the hallmark Tfh cell molecules CXCR5, CXCR4, and PD-1. Bcl-6 bound to the promoters of the Th1 and Th17 cell transcriptional regulators T-bet and RORgammat and repressed IFN-gamma and IL-17 production. Bcl-6 also repressed expression of many microRNAs (miRNAs) predicted to control the Tfh cell signature, including miR-17-92, which repressed CXCR5 expression. Thus, Bcl-6 positively directs Tfh cell differentiation, through combined repression of miRNAs and transcription factors.