Lauren A. Henderson

Pathologically expanded peripheral T helper cell subset drives B cells in rheumatoid arthritis

CD4+ T cells are central mediators of autoimmune pathology; however, defining their key effector functions in specific autoimmune diseases remains challenging. Pathogenic CD4+ T cells within affected tissues may be identified by expression of markers of recent activation. Here we use mass cytometry to analyse activated T cells in joint tissue from patients with rheumatoid arthritis, a chronic immune-mediated arthritis that affects up to 1% of the population. This approach revealed a markedly expanded population of PD-1hiCXCR5−CD4+ T cells in synovium of patients with rheumatoid arthritis. However, these cells are not exhausted, despite high PD-1 expression. Rather, using multidimensional cytometry, transcriptomics, and functional assays, we define a population of PD-1hiCXCR5− ‘peripheral helper’ T (TPH) cells that express factors enabling B-cell help, including IL-21, CXCL13, ICOS, and MAF. Like PD-1hiCXCR5+ T follicular helper cells, TPH cells induce plasma cell differentiation in vitro through IL-21 secretion and SLAMF5 interaction (refs 3, 4). However, global transcriptomics highlight differences between TPH cells and T follicular helper cells, including altered expression of BCL6 and BLIMP1 and unique expression of chemokine receptors that direct migration to inflamed sites, such as CCR2, CX3CR1, and CCR5, in TPH cells. TPH cells appear to be uniquely poised to promote B-cell responses and antibody production within pathologically inflamed non-lymphoid tissues.

First reported case of Omenn syndrome in a patient with reticular dysgenesis

To the Editor: Reticular dysgenesis (RD) is a rare form of autosomalrecessive severe combined immunodeficiency characterized by lack of circulating T lymphocytes, severe congenital neutropenia, and sensorineural deafness.1,2 The disease is caused by mutations in the gene encoding adenylate kinase 2 (AK2), a mitochondrial protein important for regulating intracellular levels of adenosine diphosphate and maintaining mitochondrial membrane potential. A similar function is also mediated by the cytoplasmic enzyme AK1. While most tissues express both AK1 and AK2 enzymes, neutrophils, T lymphocytes, and cells of the stria vascularis in the inner ear uniquely express AK2, thus explaining the RD phenotype.3,4 Omenn syndrome (OS) has been described in association with several genetic defects responsible for severe combined immunodeficiency and results from residual development of oligoclonal T lymphocytes that undergo peripheral expansion and infiltrate various tissues including the skin, gut, liver, and lymphoid organs.5 We report the first case of OS in a patient with RD. A male infant of Kuwaiti origin was born to consanguineous parents who had a previously affected daughter with RD due to a homozygous missense AK2 mutation (c.524 G>A, p.R175Q). The sister’s first hematopoietic stem cell transplant from cord blood with reduced intensity conditioning failed, but she later had a successful matched related transplant with conditioning consisting of busulfan and cyclophosphamide.6 At birth, the infant was vigorous with an otherwise unremarkable physical examination; however, laboratory studies demonstrated leukopenia, absence of neutrophils, and undetectable IgA and IgM levels (Table I), and a chest radiograph lacked a thymic shadow. Immunologic evaluation at our institution at 6 weeks of age revealed undetectable T-cell receptor excision circles with detectable RNAse P, near-absence of natural killer cells, and B-cell lymphopenia. The T-cell count was slightly reduced, composed entirely of CD45RA-cells. T-lymphocyte proliferation to mitogens was decreased (Table I). Auditory brain stem response-evoked potential testing documented profound hearing loss. A diagnosis of RD was presumed, and mutation analysis by using genomic DNA derived from fibroblasts confirmed homozygosity for the same AK2 mutation as the infant’s sister. The patient was started on prophylactic antibiotics and antifungal medications along with intravenous gammaglobulin. At 8 weeks, the infant developed desquamative erythroderma, pachydermia (see Fig E1 in this article’s Online Repository at www.jacionline.org), diarrhea, and generalized lymphadenopathy. The CD3+ T-cell count increased to 10,032 cells/μL (Table I). Treatment with methylprednisolone 1 mg/kg given twice daily and cyclosporine was started; improvement was noted clinically, with laboratory studies documenting decreased circulating T lymphocytes (Table I). The infant underwent a 9/10 HLA-A mismatched unrelated donor bone marrow transplant at 3 months of age, with conditioning consisting of 4 days of busulfan given every 6 hours with area under the curve dosing targeted to 800 to 1200 μmol*min/L. cyclophosphamide 50 mg/kg daily for 4 days, and equine antithymocyte globulin 30 mg/kg daily for 3 days. Cyclosporine and 1 mg/kg per day of methylprednisolone were continued for graft versus host prophylaxis. There was no evidence for liver involvement secondary to the OS as the pretransplant liver function test results were normal. Vitamin E and ursodiol were administered for veno-occlusive disease prophylaxis. He achieved neutrophil engraftment on day +16, with 100% donor chimerism in the peripheral blood documented on day +21. The child unfortunately developed severe veno-occlusive disease with portal venous thrombosis and was treated with defibrotide. He subsequently developed progressive multiorgan dysfunction with fevers, respiratory insufficiency, hemodynamic instability, renal insufficiency, and coagulopathy. Similar to published cases of infants transplanted for immunodeficiency treated with corticosteroids,10 he developed cardiomyopathy with new severe concentric left ventricular hypertrophy. Cardiac biopsy on day +25 showed a mild T lymphocyte and histiocyte infiltrate and lack of myocyte hypertrophy. Rectal biopsy on day +28 demonstrated severe colitis consistent with acute graft versus host disease. Methylprednisolone was increased to 3 mg/kg per day, but despite this therapy, the patient died on day +31 after developing asystole. Postmortem examination was not performed. TABLE I Hematologic and immunologic characteristics of the patient We sought to confirm the diagnosis of RD by assessing AK2 protein expression and function. We also examined the etiology of this infant’s rash, diarrhea, and lymphadenopathy in the setting of the expanded CD3+ T-lymphocyte population, considering maternal engraftment or OS as possible causes of the presentation. AK2 protein was detected at reduced levels in the patient’s CD3+ T cells and fibroblasts by Western blot (Fig 1, A). To determine whether this AK2 protein was derived from autologous or maternally engrafted T cells, we performed fluorescence in situ hybridization of peripheral blood cells (99% carried a Y chromosome), short tandem repeat analysis of purified CD3+ cells (0% maternal), and flow cytometry analysis (0.5% bearing the noninherited maternal HLA-A2 allele), thus ruling out maternal T-cell engraftment as the source of expanded T cells (Fig 1, B). Somatic reversion in T cells was ruled out by detecting the same AK2 mutation in purified CD3+ T lymphocytes (see Fig E2 in this article’s Online Repository at www.jacionline.org). OS is characterized by the production and expansion of oligoclonal T cells, and indeed, the infant’s CD3+, CD4+, and CD8+ T-cell receptor repertoire was highly oligoclonal, with only 5 of 24, 2 of 24, and 3 of 24 TCRs variable region families falling in the normal range, respectively (Fig 1, C). Purified CD3+ T lymphocytes showed susceptibility to apoptosis, evidenced by increased depolarization of the mitochondrial membrane potential at 3, 6, and 9 hours of incubation with staurosporin, indicating abnormal function of the mutant AK2 (Fig 1, D) (see additional information in this article’s Methods section in the Online Repository at www.jacionline.org). FIG 1 Characteristics of the infant’s presentation. A, Expression of AK2 protein detected in patient’s PBMC and fibroblasts (Pt) compared with normal control (Ctrl). Expression of β-actin is shown as a loading control. B, Maternal engraftment ... This infant’s presentation of desquamative erythroderma, diarrhea, and generalized lymphadenopathy in the setting of an expanded and oligoclonal autologous CD3+ T-cell population is consistent with OS. This case indicates that at least some missense AK2 mutations may result in residual T-lymphocyte development, oligoclonal expansion, and symptoms of OS. Thus, AK2 should be added to the list of severe combined immunodeficiency–causing genes that may manifest as OS.

Quantifying Temporomandibular Joint Synovitis in Children With Juvenile Idiopathic Arthritis

Juvenile idiopathic arthritis (JIA) frequently affects the temporomandibular joints (TMJs) and is often undetected by history, examination, and plain imaging. Qualitative assessment of gadolinium‐enhanced magnetic resonance images (MRIs) is currently the standard for diagnosis of TMJ synovitis associated with JIA. The purpose of this study is to apply a quantitative analysis of synovial enhancement to MRIs of patients with and without JIA to establish a disease threshold and sensitivity and specificity for the technique.

Shrinking Lung Syndrome as a Manifestation of Pleuritis: A New Model Based on Pulmonary Physiological Studies

Objective. The pathophysiology of shrinking lung syndrome (SLS) is poorly understood. We sought to define the structural basis for this condition through the study of pulmonary mechanics in affected patients. Methods. Since 2007, most patients evaluated for SLS at our institutions have undergone standardized respiratory testing including esophageal manometry. We analyzed these studies to define the physiological abnormalities driving respiratory restriction. Chest computed tomography data were post-processed to quantify lung volume and parenchymal density. Results. Six cases met criteria for SLS. All presented with dyspnea as well as pleurisy and/or transient pleural effusions. Chest imaging results were free of parenchymal disease and corrected diffusing capacities were normal. Total lung capacities were 39%–50% of predicted. Maximal inspiratory pressures were impaired at high lung volumes, but not low lung volumes, in 5 patients. Lung compliance was strikingly reduced in all patients, accompanied by increased parenchymal density. Conclusion. Patients with SLS exhibited symptomatic and/or radiographic pleuritis associated with 2 characteristic physiological abnormalities: (1) impaired respiratory force at high but not low lung volumes; and (2) markedly decreased pulmonary compliance in the absence of identifiable interstitial lung disease. These findings suggest a model in which pleural inflammation chronically impairs deep inspiration, for example through neural reflexes, leading to parenchymal reorganization that impairs lung compliance, a known complication of persistently low lung volumes. Together these processes could account for the association of SLS with pleuritis as well as the gradual symptomatic and functional progression that is a hallmark of this syndrome.

Multiple juvenile idiopathic arthritis subtypes demonstrate proinflammatory IgG glycosylation.

OBJECTIVE Rheumatoid arthritis is associated with an excess of agalactosylated (G0) IgG that is considered relatively proinflammatory. Assessment of this association in juvenile idiopathic arthritis (JIA) is complicated by age-dependent IgG glycan variation. The aim of this study was to conduct the first large-scale survey of IgG glycans in healthy children and patients with JIA, with a focus on early childhood, the time of peak JIA incidence. METHODS IgG glycans from healthy children and disease-modifying antirheumatic drug-naive patients with JIA were characterized using high-performance liquid chromatography. Agalactosylated glycans were quantitated with reference to monogalactosylated (G1) species. Associations were sought between the G0:G1 ratio and disease characteristics. RESULTS Among healthy children ages 9 months to 16 years (n = 165), the G0:G1 ratio was highly age dependent, with the ratio peaking to 1.19 in children younger than age 3 years and declining to a nadir of 0.83 after age 10 years (Spearmans ρ = 0.60, P < 0.0001). In patients with JIA (n = 141), the G0:G1 ratio was elevated compared with that in control subjects (1.32 versus 1.02; P < 0.0001). The G0:G1 ratio corrected for age was abnormally high in all JIA subtypes (enthesitis-related arthritis was not assessed), most strikingly in systemic JIA. Glycosylation aberrancy was comparable in patients with and those without antinuclear antibodies and in both early- and late-onset disease and exhibited at most a weak correlation with markers of inflammation. CONCLUSION IgG glycosylation is skewed toward proinflammatory G0 variants in healthy children, in particular during the first few years of life. This deviation is exaggerated in patients with JIA. The role for IgG glycan variation in immune function in children, including the predilection of JIA for early childhood, remains to be defined.

Characterization of T and B cell repertoire diversity in patients with RAG deficiency

Differences in B and T cell repertoires in patients with RAG deficiency associate with clinical severity. Taking SCID genetics to the clinic Mutations that lead to deficiencies in the recombination-activating genes RAG1 and RAG2 result in a spectrum of immunodeficiencies ranging from loss of T and/or B cell repertoire diversity to a complete lack of T and B cells—severe combined immunodeficiency (SCID). Here, Lee et al. perform next-generation B and T cell repertoire sequencing on 12 patients with RAG mutations who have immunodeficiencies of varying severity. They found that the level of repertoire skewing was associated with the severity of disease and that specific repertoire deficiencies were associated with particular phenotypes. These data support a genotype-phenotype connection for primary immunodeficiencies. Recombination-activating genes 1 and 2 (RAG1 and RAG2) play a critical role in T and B cell development by initiating the recombination process that controls the expression of T cell receptor (TCR) and immunoglobulin genes. Mutations in the RAG1 and RAG2 genes in humans cause a broad spectrum of phenotypes, including severe combined immunodeficiency (SCID) with lack of T and B cells, Omenn syndrome, leaky SCID, and combined immunodeficiency with granulomas or autoimmunity (CID-G/AI). Using next-generation sequencing, we analyzed the TCR and B cell receptor (BCR) repertoire in 12 patients with RAG mutations presenting with Omenn syndrome (n = 5), leaky SCID (n = 3), or CID-G/AI (n = 4). Restriction of repertoire diversity skewed usage of variable (V), diversity (D), and joining (J) segment genes, and abnormalities of CDR3 length distribution were progressively more prominent in patients with a more severe phenotype. Skewed usage of V, D, and J segment genes was present also within unique sequences, indicating a primary restriction of repertoire. Patients with Omenn syndrome had a high proportion of class-switched immunoglobulin heavy chain transcripts and increased somatic hypermutation rate, suggesting in vivo activation of these B cells. These data provide a framework to better understand the phenotypic heterogeneity of RAG deficiency.

Ligase-4 Deficiency Causes Distinctive Immune Abnormalities in Asymptomatic Individuals

PurposeDNA Ligase 4 (LIG4) is a key factor in the non-homologous end-joining (NHEJ) DNA double-strand break repair pathway needed for V(D)J recombination and the generation of the T cell receptor and immunoglobulin molecules. Defects in LIG4 result in a variable syndrome of growth retardation, pancytopenia, combined immunodeficiency, cellular radiosensitivity, and developmental delay.MethodsWe diagnosed a patient with LIG4 syndrome by radiosensitivity testing on peripheral blood cells, and established that two of her four healthy siblings carried the same compound heterozygous LIG4 mutations. An extensive analysis of the immune phenotype, cellular radiosensitivity, telomere length, and T and B cell antigen receptor repertoire was performed in all siblings.ResultsIn the three genotypically affected individuals, variable severities of radiosensitivity, alterations of T and B cell counts with an increased percentage of memory cells, and hypogammaglobulinemia, were noticed. Analysis of T and B cell antigen receptor repertoires demonstrated increased usage of alternative microhomology-mediated end-joining (MHMEJ) repair, leading to diminished N nucleotide addition and shorter CDR3 length. However, overall repertoire diversity was preserved.ConclusionsWe demonstrate that LIG4 syndrome presents with high clinical variability even within the same family, and that distinctive immunologic abnormalities may be observed also in yet asymptomatic individuals.

Next-Generation Sequencing Reveals Restriction and Clonotypic Expansion of Treg Cells in Juvenile Idiopathic Arthritis.

Treg cell–mediated suppression of Teff cells is impaired in juvenile idiopathic arthritis (JIA); however, the basis for this dysfunction is incompletely understood. Animal models of autoimmunity and immunodeficiency demonstrate that a diverse Treg cell repertoire is essential to maintain Treg cell function. The present study was undertaken to investigate the Treg and Teff cell repertoires in JIA.

Abnormalities of T-cell receptor repertoire in CD4+ regulatory and conventional T cells in patients with RAG mutations: Implications for autoimmunity

Jared H. Rowe, MD, Brian D. Stadinski, PhD, Lauren A. Henderson, MD, Lisa Ott de Bruin, MD, Ottavia Delmonte, MD, Yu Nee Lee, PhD, M. Teresa de la Morena, MD, Rakesh K. Goyal, MD, Anthony Hayward, MD, PhD, Huang Chiung-Hui, PhD, Maria Kanariou, MD, Alejandra King, MD, Taco W. Kuijpers, MD, Jian Yi Soh, MD, Benedicte Neven, MD, PhD, Jolan E. Walter, MD, PhD, Eric S. Huseby, PhD, Luigi D. Notarangelo, MD

A161: Novel 3‐Dimensional Explant Method Facilitates the Study of Lymphocyte Populations in the Synovium and Reveals a Large Population of Resident Memory T cells in Rheumatoid Arthritis

Traditionally, immunologic memory was thought to be maintained by populations of central (TCM) and effector (TEM) memory lymphocytes that circulate in the blood and lymphatics, only temporarily extravagating into peripheral tissue to execute immunologic responses. Recently, this theory of adaptive memory has been challenged by the discovery of long‐lived and stable populations of tissue‐resident memory T cells (TRM). While TRM have been implicated in the pathogenesis of skin, intestinal, and lung inflammatory diseases, little information is available about TRM in synovium. The study of TRM in arthritis has been impaired by the scarcity of synovium samples coupled with the meager yield of lymphocytes from this tissue using conventional tissue digestion protocols. We have employed novel culturing techniques, previously used to examine TRM in skin, to further characterize TRM in inflammatory arthritis.