Noa Simchoni

Microbiota regulate the ability of lung dendritic cells to induce IgA class-switch recombination and generate protective gastrointestinal immune responses.

Ruane et al. demonstrate a role for the microbiota in modulating protective immunity to intranasal vaccination via the ability of lung dendritic cells to induce B cell IgA class switching.

IRAK-4 and MyD88 deficiencies impair IgM responses against T-independent bacterial antigens

IRAK-4 and MyD88 deficiencies impair interleukin 1 receptor and Toll-like receptor (TLR) signaling and lead to heightened susceptibility to invasive bacterial infections. Individuals with these primary immunodeficiencies have fewer immunoglobulin M (IgM)(+)IgD(+)CD27(+) B cells, a population that resembles murine splenic marginal zone B cells that mount T-independent antibody responses against bacterial antigens. However, the significance of this B-cell subset in humans is poorly understood. Using both a 610 carbohydrate array and enzyme-linked immunosorbent assay, we found that patients with IRAK-4 and MyD88 deficiencies have reduced serum IgM, but not IgG antibody, recognizing T-independent bacterial antigens. Moreover, the quantity of specific IgM correlated with IgM(+)IgD(+)CD27(+) B-cell frequencies. As with mouse marginal zone B cells, human IgM(+)CD27(+) B cells activated by TLR7 or TLR9 agonists produced phosphorylcholine-specific IgM. Further linking splenic IgM(+)IgD(+)CD27(+) B cells with production of T-independent IgM, serum from splenectomized subjects, who also have few IgM(+)IgD(+)CD27(+) B cells, had reduced antibacterial IgM. IRAK-4 and MyD88 deficiencies impaired TLR-induced proliferation of this B-cell subset, suggesting a means by which loss of this activation pathway leads to reduced cell numbers. Thus, by bolstering the IgM(+)IgD(+)CD27(+) B-cell subset, IRAK-4 and MyD88 promote optimal T-independent IgM antibody responses against bacteria in humans.

Clonal and constricted T cell repertoire in Common Variable Immune Deficiency

We used high throughput sequencing to examine the structure and composition of the T cell receptor β chain in Common Variable Immune Deficiency (CVID). TCRβ CDR3 regions were amplified and sequenced from genomic DNA of 44 adult CVID subjects and 22 healthy adults, using a high-throughput multiplex PCR. CVID TCRs had significantly less junctional diversity, fewer n-nucleotide insertions and deletions, and completely lacked a population of highly modified TCRs, with 13 or more V-gene nucleotide deletions, seen in healthy controls. The CVID CDR3 sequences were significantly more clonal than control DNA, and displayed unique V gene usage. Despite reduced junctional diversity, increased clonality and similar infectious exposures, DNA of CVID subjects shared fewer TCR sequences as compared to controls. These abnormalities are pervasive, found in out-of-frame sequences and thus independent of selection and were not associated with specific clinical complications. These data support an inherent T cell defect in CVID.

Expansion of inflammatory innate lymphoid cells in patients with common variable immune deficiency.

BACKGROUND Common variable immunodeficiency (CVID) is an antibody deficiency treated with immunoglobulin; however, patients can have noninfectious inflammatory conditions that lead to heightened morbidity and mortality. OBJECTIVES Modular analyses of RNA transcripts in whole blood previously identified an upregulation of many interferon-responsive genes. In this study we sought the cell populations leading to this signature. METHODS Lymphoid cells were measured in peripheral blood of 55 patients with CVID (31 with and 24 without inflammatory/autoimmune complications) by using mass cytometry and flow cytometry. Surface markers, cytokines, and transcriptional characteristics of sorted innate lymphoid cells (ILCs) were defined by using quantitative PCR. Gastrointestinal and lung biopsy specimens of subjects with inflammatory disease were stained to seek ILCs in tissues. RESULTS The linage-negative, CD127(+), CD161(+) lymphoid population containing T-box transcription factor, retinoic acid-related orphan receptor (ROR) γt, IFN-γ, IL-17A, and IL-22, all hallmarks of type 3 innate lymphoid cells, were expanded in the blood of patients with CVID with inflammatory conditions (mean, 3.7% of PBMCs). ILCs contained detectable amounts of the transcription factors inhibitor of DNA binding 2, T-box transcription factor, and RORγt and increased mRNA transcripts for IL-23 receptor (IL-23R) and IL-26, demonstrating inflammatory potential. In gastrointestinal and lung biopsy tissues of patients with CVID, numerous IFN-γ(+)RORγt(+)CD3(-) cells were identified, suggesting a role in these mucosal inflammatory states. CONCLUSIONS An expansion of this highly inflammatory ILC population is a characteristic of patients with CVID with inflammatory disease; ILCs and the interferon signature are markers for the uncontrolled inflammatory state in these patients.

Toll‐like receptor signaling in primary immune deficiencies

Toll‐like receptors (TLRs) recognize common microbial or host‐derived macromolecules and have important roles in early activation of the immune system. Patients with primary immune deficiencies (PIDs) affecting TLR signaling can elucidate the importance of these proteins to the human immune system. Defects in interleukin‐1 receptor‐associated kinase‐4 and myeloid differentiation factor 88 (MyD88) lead to susceptibility to infections with bacteria, while mutations in nuclear factor‐κB essential modulator (NEMO) and other downstream mediators generally induce broader susceptibility to bacteria, viruses, and fungi. In contrast, TLR3 signaling defects are specific for susceptibility to herpes simplex virus type 1 encephalitis. Other PIDs induce functional alterations of TLR signaling pathways, such as common variable immunodeficiency in which plasmacytoid dendritic cell defects enhance defective responses of B cells to shared TLR agonists. Dampening of TLR responses is seen for TLRs 2 and 4 in chronic granulomatous disease (CGD) and X‐linked agammaglobulinemia (XLA). Enhanced TLR responses, meanwhile, are seen for TLRs 5 and 9 in CGD, TLRs 4, 7/8, and 9 in XLA, TLRs 2 and 4 in hyper IgE syndrome, and for most TLRs in adenosine deaminase deficiency.

IgH sequences in common variable immune deficiency reveal altered B cell development and selection

Deep sequence analysis of the IgH repertoires of common variable immune deficiency patients highlights phenotypic features of the disorder and potential disease mechanisms. Not immune to memory problems Although elderly patients typically lament loss of memory, Roskin et al. now highlight a different kind of memory problem plus developmental difficulties that bedevils the immune systems of children and young adults with common variable immune deficiency (CVID). These aberrations may drive a variety of outcomes, including impaired antibody responses to foreign antigens, generation of autoimmune responses, and lymphoid cancers. As the most common symptomatic primary immune deficiency, CVID affects nearly one in 25,000 persons, but the biological basis for the constellation of clinical phenotypes remains hazy. Genetic recombination in the V(D)J regions of immunoglobulin (Ig) genes, which occurs in developing lymphocytes during the early stages of B cell maturation, gives rise to the diverse repertoire of antibodies produced by activated B lymphocytes. This so-called Ig gene rearrangement—a seminal component of the adaptive immune system—gives rise to a broad range of amino acid sequences in the antigen-binding regions of Igs, which permits the recognition of antigens from many pathogens and abnormal cells (such as cancer cells) and subsequent activation of the immune response. The authors first used high-throughput genomic DNA sequencing to explore Ig heavy chain gene rearrangements in B cells from CVID patients versus control subjects. CVID patients displayed abnormal VDJ rearrangement and thus abnormal formation of complementarity determining region 3 (CDR3), which are part of the Ig variable chains and central to the ability of B cells to recognize a wide range of antigens. The authors then sorted B cell populations to retrieve, specifically, the naïve and memory B cells. They detected decreased selection against antibodies with long CDR3 regions in CVID memory repertoires and fewer of the antibody gene mutations that accompany the formation of immune memory. The unmutated naïve B cell pool displayed decreased diversity and abnormal clonal expansion. Together, these alterations could explain the immune deficiencies and autoimmune reactions detected in CVID patients, and suggest that CVID phenotypes stem from aberrant generation and selection of B cell repertoires. Common variable immune deficiency (CVID) is the most common symptomatic primary immune deficiency, affecting ~1 in 25,000 persons. These patients suffer from impaired antibody responses, autoimmunity, and susceptibility to lymphoid cancers. To explore the cellular basis for these clinical phenotypes, we conducted high-throughput DNA sequencing of immunoglobulin heavy chain gene rearrangements from 93 CVID patients and 105 control subjects and sorted naïve and memory B cells from 13 of the CVID patients and 10 of the control subjects. The CVID patients showed abnormal VDJ rearrangement and abnormal formation of complementarity-determining region 3 (CDR3). We observed a decreased selection against antibodies with long CDR3s in memory repertoires and decreased variable gene replacement, offering possible mechanisms for increased patient autoreactivity. Our data indicate that patient immunodeficiency might derive from both decreased diversity of the naïve B cell pool and decreased somatic hypermutation in memory repertoires. The CVID patients also exhibited an abnormal clonal expansion of unmutated B cells relative to the controls. Although impaired B cell germinal center activation is commonly viewed as causative in CVID, these data indicate that CVID B cells diverge from controls as early as the pro–B stage, cell and suggest possible explanations for the increased incidence of autoimmunity, immunodeficiency, and lymphoma CVID patients.

High-throughput sequencing reveals an altered T cell repertoire in X-linked agammaglobulinemia

To examine the T cell receptor structure in the absence of B cells, the TCR β CDR3 was sequenced from DNA of 15 X-linked agammaglobulinemia (XLA) subjects and 18 male controls, using the Illumina HiSeq platform and the ImmunoSEQ analyzer. V gene usage and the V-J combinations, derived from both productive and non-productive sequences, were significantly different between XLA samples and controls. Although the CDR3 length was similar for XLA and control samples, the CDR3 region of the XLA T cell receptor contained significantly fewer deletions and insertions in V, D, and J gene segments, differences intrinsic to the V(D)J recombination process and not due to peripheral T cell selection. XLA CDR3s demonstrated fewer charged amino acid residues, more sharing of CDR3 sequences, and almost completely lacked a population of highly modified Vβ gene segments found in control DNA, suggesting both a skewed and contracted T cell repertoire in XLA.

Biliary Epithelial Cells Are Not the Predominant Source of Hepatic CXCL12

Hepatic expression levels of CXCL12, a chemokine important in inflammatory and stem cell recruitment, and its receptor, C-X-C chemokine receptor 4, are increased during all forms of liver injury. CXCL12 is expressed by both parenchymal and nonparenchymal hepatic cells, and on the basis of immunohistochemistry, biliary epithelial cells (BECs) are thought to be a predominant source of hepatic CXCL12, thereby promoting periportal recruitment of C-X-C chemokine receptor 4-expressing lymphocytes. Our study aims to show that BECs may, in fact, not be the predominant source of hepatic CXCL12. We measured CXCL12 secretion and expression from human and murine BECs using enzyme-linked immunosorbent assay and Western blot analysis from cell culture supernatants and whole cell lysates, respectively, whereas CXCL12 expression in murine livers was analyzed in a Cxcl12-Gfp reporter mouse. Cell culture supernatants and whole cell lysates from BECs failed to demonstrate their expression of CXCL12. Furthermore, we confirmed these results with a Cxcl12-Gfp reporter mouse in which green fluorescent protein expression is notably absent from BECs. Interestingly, on the basis of green fluorescent protein expression, we demonstrate a population of CXCL12-expressing cells within the portal tract that are distinct, yet intimately associated with BECs. These findings indicate that BECs are not a predominant source of CXCL12.