Derrick J. Todd

The endoplasmic reticulum stress response in immunity and autoimmunity

Many exogenous sources of stress can lead to cell death. In recent years, endogenous cellular sources of stress have also been identified, including the stress that arises from the accumulation of unfolded proteins within a cells endoplasmic reticulum (ER). To counterbalance this type of ER stress, higher eukaryotic cells possess a three-pronged signal-transduction pathway termed the unfolded-protein response (UPR). This Review focuses on the role of the UPR in the mammalian immune system and how manipulation of this complex signalling pathway may be of therapeutic benefit in human disease.

BAX Inhibitor-1 Is a Negative Regulator of the ER Stress Sensor IRE1α

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). Bax inhibitor-1 (BI-1) is an evolutionarily conserved ER-resident protein that suppresses cell death. Here we have investigated the role of BI-1 in the UPR. BI-1 expression suppressed IRE1alpha activity in fly and mouse models of ER stress. BI-1-deficient cells displayed hyperactivation of the ER stress sensor IRE1alpha, leading to increased levels of its downstream target X-box-binding protein-1 (XBP-1) and upregulation of UPR target genes. This phenotype was associated with the formation of a stable protein complex between BI-1 and IRE1alpha, decreasing its ribonuclease activity. Finally, BI-1 deficiency increased the secretory activity of primary B cells, a phenomenon regulated by XBP-1. Our results suggest a role for BI-1 in early adaptive responses against ER stress that contrasts with its known downstream function in apoptosis.

XBP1 governs late events in plasma cell differentiation and is not required for antigen-specific memory B cell development

The unfolded protein response (UPR) is a stress response pathway that is driven by the increased load of unfolded proteins in the endoplasmic reticulum of highly secretory cells such as plasma cells (PCs). X box binding protein 1 (XBP1) is a transcription factor that mediates one branch of the UPR and is crucial for the development of antibody-secreting PCs. PCs represent only one class of terminally differentiated B cells, however, and little is known about the role for XBP1 in the other class: memory B cells. We have developed an XBP1fl/fl CD19+/cre conditional knockout (XBP1CD19) mouse to build upon our current understanding of the function of XBP1 in PC differentiation as well as to explore the role of XBP1 in memory cell development. Using this model, we show that XBP1CD19 mice are protected from disease in an autoantibody-mediated mouse lupus model. We also identify a novel developmental stage at which B cells express the traditional PC marker CD138 (syndecan-1) but have yet to undergo XBP1-dependent functional and morphological differentiation into antibody-secreting cells. Finally, we show that memory B cells develop normally in XBP1CD19 mice, demonstrating that XBP1-mediated functions occur independently of any memory cell lineage commitment.

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.

Erroneous augmentation of multiplex assay measurements in patients with rheumatoid arthritis due to heterophilic binding by serum rheumatoid factor

OBJECTIVE Serum rheumatoid factor (RF) and other heterophilic antibodies potentially interfere with antibody-based immunoassays by nonspecifically binding detection reagents. The purpose of this study was to assess whether these factors confound multiplex-based immunoassays, which are used with increasing frequency to measure cytokine and chemokine analytes in patients with rheumatoid arthritis (RA). METHODS We performed multiplex immunoassays using different platforms to measure analyte concentrations in RA patient samples. Samples were depleted of RF by column-based affinity absorption or were exposed to agents that block heterophilic binding activity. RESULTS In RA patients with high-titer RF, 69% of analytes demonstrated at least a 2-fold stronger multiplex signal in non-RF-depleted samples as compared to RF-depleted samples. This degree of erroneous signal amplification was less frequent in low-titer RF samples (17% of analytes; P < 0.0000001). Signal amplification by heterophilic antibodies was blocked effectively by HeteroBlock (≥ 150 μg/ml). In 35 RA patients, multiplex signals for 14 of 22 analytes were amplified erroneously in unblocked samples as compared to blocked samples (some >100-fold), but only in patients with high-titer RF (P < 0.002). Two other blocking agents, heterophilic blocking reagent and immunoglobulin-inhibiting reagent, also blocked heterophilic activity. CONCLUSION All multiplex protein detection platforms we tested exhibited significant confounding by RF or other heterophilic antibodies. These findings have broad-reaching implications in the acquisition and interpretation of data derived from multiplex immunoassay testing of RA patient serum and possibly also in other conditions in which RF or other heterophilic antibodies may be present. Several available blocking agents effectively suppressed this erroneous signal amplification in the multiplex platforms tested.

Drug-associated polymyalgia rheumatica/giant cell arteritis occurring in two patients after treatment with ipilimumab, an antagonist of ctla-4.

Here we describe 2 cases of polymyalgia rheumatica and giant cell arteritis (PMR/GCA) occurring in patients with malignant melanoma who received ipilimumab, an immunopotentiating antagonist of CTLA-4. Metastatic melanoma is a devastating malignancy with a median survival of 1 year and for which few effective treatment options are available (1). In March 2011, the biologic monoclonal antibody ipilimumab was approved for the treatment of unresectable or metastatic melanoma. Ipilimumab is directed against CTLA-4 expressed on the surface of activated T cells (2). CTLA-4 binds CD80 and CD86 on the surface of antigen-presenting cells. This interaction dampens the costimulatory “second signal” of T cell activation mediated via CD80/CD86 interaction with CD28 on the surface of T cells (3). Thus, by blocking CTLA-4, ipilimumab disinhibits T cells and potentiates immune responses. What results is an augmented antitumor immunologic response that has shown promise in clinical trials in the treatment of unresectable stage III or IV melanoma (4). As might be expected of any immunopotentiating therapy, immune-related adverse events (AEs) occur as autoimmune complications of anti–CTLA-4 treatment. The most common immune-related AEs include noninfectious enterocolitis, dermatitis, endocrinopathies, and hepatitis (4). Reports of the occurrence of autoimmune arthritis, lupus nephritis, and antinuclear antibody seroconversion in patients receiving anti– CTLA-4 treatment are of relevance to rheumatologists (5–7). The mechanisms of immune-related AEs have been hypothesized to involve a breakdown of peripheral tolerance and induction of organ-specific inflammatory processes. We diagnosed and treated 2 cases of possible ipilimumab-associated PMR/GCA at our hospital. The first patient, a 62-year-old man, was admitted with a 6-week history of occipital headache, scalp tenderness, jaw claudication, shoulder and neck myalgias, and transient diplopia with a single episode of amaurosis fugax. He received ipilimumab at a dosage of 10 mg/kg every 3 weeks for treatment of stage IV melanoma. His last dose of ipilimumab was administered 1 week prior to the presentation of his symptoms; at that time, he had received a total of 5 treatment cycles and experienced noninfectious colitis and autoimmune hypophysitis, both of which were considered to be immune-related AEs associated with ipilimumab. The results of laboratory studies were notable for leukocytosis, normocytic anemia, an erythrocyte sedimentation rate (ESR) of 97 mm/hour, and a C-reactive protein (CRP) level of 296 mg/liter. The possibility of GCA was considered because of the patient’s symptoms of cranial arteritis and the finding of increased expression of inflammatory markers. The patient declined empiric high-dose corticosteroid therapy, and a biopsy of the right temporal artery was performed. Histologic analysis revealed active arteritis, intimal proliferation, and disruption of the internal elastic lamina (Figures 1A and B). GCA was diagnosed, and the patient was treated with oral prednisone at a dosage of 60 mg daily. His symptoms of cranial arteritis resolved within 2 days of the initiation of prednisone. Six weeks later, the ESR was 26 mm/hour, and the CRP level was 21 mg/liter. At month 6, he had not experienced recurrent symptoms of cranial arteritis, and both the ESR and the CRP level remained low (20 mm/hour and 9.6 mg/liter, respectively). The second patient, another 62-year-old man, was admitted to our hospital with a 2-week history of arthralgias, trismus, and left-sided facial swelling. He reported experiencing morning stiffness in his proximal shoulder joints. He received ipilimumab for stage IV melanoma that was diagnosed 18 years prior and was complicated by sequential metastases to the cervical lymph nodes, lungs, and brain. He received a total of 4 cycles of ipilimumab treatment over 3 months, with the most recent dose administered 10 weeks prior to the onset of symptoms. The results of laboratory studies were notable for normocytic anemia and an elevated CRP level of 71 mg/liter. He was initially treated with dexamethasone at a dosage of 4 mg twice daily because of concern of worsening metastatic disease, but magnetic resonance imaging did not support this possibility. The possibility of GCA was then considered because of the PMR symptoms, facial swelling, trismus, and an elevated CRP level. Corticosteroids were changed to prednisone at a dosage of 50 mg daily, and a biopsy of the right temporal artery was performed 5 days later. Histopathologic analysis revealed

Drug-Associated Dermatomyositis Following Ipilimumab Therapy: A Novel Immune-Mediated Adverse Event Associated With Cytotoxic T-Lymphocyte Antigen 4 Blockade

IMPORTANCE Ipilimumab, a human monoclonal antibody targeted against cytotoxic T-lymphocyte antigen 4, has shown promise in the treatment of metastatic melanoma. However, given its mechanism of action, immune-related adverse effects have been reported with this therapy. Despite increasing reports of immune-related adverse effects related to ipilimumab therapy, dermatomyositis associated with this agent has not previously been reported. OBSERVATIONS We describe a woman undergoing treatment with ipilimumab for metastatic melanoma who developed classic cutaneous findings of dermatomyositis along with proximal muscle weakness and elevated muscle enzymes. CONCLUSIONS AND RELEVANCE This case adds to the expanding literature regarding immune-related adverse events associated with ipilimumab. To our knowledge, drug-induced dermatomyositis from ipilimumab has not previously been reported. Physicians should be aware of these potential immune-related adverse events and consider drug-associated dermatomyositis in the differential diagnosis in patients receiving ipilimumab who present with a cutaneous eruption or muscle weakness.

A new isolation method for rat intraepithelial lymphocytes.

Abstract Intraepithelial lymphocytes (IELs) play critical roles in gut immunity. In mice, γδ T cells are a large component of the IEL population. In the rat, γδ IELs are reportedly much less common, but technical issues suggest that previous analyses should be interpreted cautiously. The study of IELs in rats has been impeded by isolation procedures that are lengthy and complex, leading to small cell yields. For this reason, it is possible that rat IELs analyzed in previous studies have not been representative of the entire IEL compartment. We report a new method for the isolation of rat IELs that is based on the selective removal of intestinal epithelial cells under conditions that leave the basement membrane undisturbed. The method is rapid and requires neither enzymatic digestion, nor surgical removal of Peyers patches, nor vigorous mechanical manipulation of the intestine. The yield of rat IELs using this method is 5- to 10-fold greater than that reported for other methods. Morphological and phenotypic analyses demonstrated that the purified cell population is comprised of IELs and is not contaminated with lamina propria or Peyers patch lymphocytes. Phenotypic analysis revealed five major subsets of IELs based on differential cell surface expression of CD4, CD8, and αβ T cell receptor (TcR). Among the αβTcR− cells was a population of γδ T cells present at levels not previously detected. The isolation of IEL sub-populations using this methodology should facilitate studies of the function of these cells in gut immunity.

Dyspnoea in a young woman with active systemic lupus erythematosus

Diffuse alveolar haemorrhage (DAH) is a rare but life-threatening complication of systemic lupus erythematosus (SLE). We present the case of a 24-year-old Cambodian woman with SLE followed in the Brigham and Women’s Hospital Lupus Center in Boston, Massachusetts. She presented with dyspnoea and chest pain and was found to have DAH that required a prolonged hospitalization that was complicated by recurrent DAH episodes and multiple infections. We discuss the diagnostic approach and management of patients with SLE-associated DAH as well as treatment options for refractory disease. Emerging therapies include plasmapheresis, the anti-CD20 monoclonal antibody rituximab and recombinant activated Factor VII therapy. In addition, we review the literature to date and compile what is known about the epidemiology, presenting features, diagnostic findings, management and outcomes in this condition. We found that DAH has been reported in 1.9% of patients with SLE. These patients were mostly female (88%) and young (mean age 30.2 years). Common presenting features included dyspnoea (94%), anaemia (97%) and new radiographic chest infiltrate (99%). Bronchoscopy, when performed, identified DAH in 90% of cases. Corticosteroids were the mainstay of care, and usage of cyclophosphamide varied by report. Despite recent advances in therapy, mortality has not improved substantially (48% overall survival versus 53% survival in reports published since 1993).

Deficiencies in Gut NK Cell Number and Function Precede Diabetes Onset in BB Rats

Defects in the intestinal immune system may contribute to the pathogenesis of autoimmune diseases. Intraepithelial lymphocytes represent a substantial fraction of gut-associated lymphocytes, but their function in mucosal immunity is unclear. A newly described population of NK cells that spontaneously secrete IL-4 and IFN-γ is present in the intraepithelial lymphocyte compartment of the rat. We hypothesized that defects in the number or function of these cells would be present in rats susceptible to autoimmunity. We report that the number of NKR-P1A+CD3− intraepithelial NK (IENK) cells is deficient before onset of spontaneous autoimmune diabetes in diabetes-prone BB (BBDP) rats. The absolute number of recoverable IENK cells was only ∼8% of that observed in WF rats. Bone marrow transplantation from histocompatible WF donors reversed the IENK cell deficiency (and prevented diabetes) in these animals, suggesting a hemopoietic origin for their IENK cell defect. Analysis of diabetes-resistant BB rats, which develop autoimmune diabetes only after perturbation of the immune system, revealed IENK cell numbers intermediate between that of BBDP and WF rats. IENK cells were selectively depleted during treatment to induce diabetes. Prediabetic BBDP and diabetes-resistant BB animals also exhibited defective IENK cell function, including decreased NK cell cytotoxicity and reduced secretion of IL-4 and IFN-γ. IENK functional defects were also observed in LEW and BN rats, which are susceptible to induced autoimmunity, but not in WF, DA, or F344 rats, which are resistant. Defects in IENK cell number and function may contribute to the pathogenesis of autoimmune diseases including type 1 diabetes.