Diminished T cell numbers in patients with chronic granulomatous disease

Chronic granulomatous disease is a neutrophil disorder in which phagocytic cells fail to produce a respiratory burst. Five genetic types of chronic granulomatous disease have been described and in each case the clinical manifestations relate to the inability to effectively kill catalase-positive organisms. It is classically described as a pure disorder of intracellular killing, with preservation of other aspects of phagocytic function such as migration and phagocytosis and normal function of nonmyeloid cells. This article describes a heretofore unrecognized feature of chronic granulomatous disease. Fifty-three patients with chronic granulomatous disease and 42 age-matched controls were studied by flow cytometry. Total T cell numbers and CD4 and CD8 T cell numbers were compared between patients and controls. Patients with chronic granulomatous disease had diminished T cell numbers compared to controls after 3 years of age. The difference increased with age. It is not known whether diminished T cell numbers influence the susceptibility to infections in these patients, but T cell effects could represent a significant cofactor for infection.     

Regulatory T cell features in chronic granulomatous disease

Chronic granulomatous disease (CGD) is a primary immunodeficiency caused by mutations in any of the genes encoding the phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase system, responsible for the production of reactive oxygen species (ROS). CGD is marked by invasive bacterial and fungal infections and by autoinflammation/autoimmunity, of which the exact pathophysiology remains elusive. Contributing factors include decreased neutrophil apoptosis, impaired apoptotic neutrophil clearance, increased proinflammatory protein expression and reduced ROS-mediated inflammasome dampening. We have explored a fundamentally different potential mechanism: it has been reported that macrophage-mediated induction of regulatory T cells (T_regs) depends on ROS production. We have investigated whether numerical or functional deficiencies exist in T_regs of CGD patients. As the prevalence of autoinflammation/autoimmunity differs between CGD subtypes, we have also investigated T_regs from gp91^phox-, p47^phox- and p40^phox-deficient CGD patients separately. Results show that T_reg numbers and suppressive capacities are not different in CGD patients compared to healthy controls, with the exception that in gp91^phox-deficiency effector T_reg (eT_reg) numbers are decreased. Expression of T_reg markers CD25, inducible T cell co-stimulator (ICOS), Helios, cytotoxic T lymphocyte antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR) did not provide any clue for differences in T_reg functionality or activation state. No correlation was seen between eT_reg numbers and patients' clinical phenotype. To conclude, the only difference between T_regs from CGD patients and healthy controls is a decrease in circulating eT_regs in gp91^phox-deficiency. In terms of autoinflammation/autoimmunity, this group is the most affected. However, upon culture, patient-derived T_regs showed a normal phenotype and normal functional suppressor activity. No other findings pointed towards a role for T_regs in CGD-related autoinflammation/autoimmunity.     

Histopathological features of chronic granulomatous disease (CGD) in childhood

AIMS: To describe the spectrum of histopathological features encountered in children with chronic granulomatous disease (CGD) at a specialist centre. METHODS AND RESULTS: The histopathological findings of 88 surgical pathology requests from a range of organ systems including upper and lower gastrointestinal tract biopsy series, liver, bladder, bone, lung, skin, soft tissue, bone marrow and lymph node biopsy specimens, in 32 patients aged 4 months to 18 years (median 7 years) with CGD were reviewed. In most tissues the features were those of active chronic inflammation, with or without abscess or granuloma formation, often associated with fungal infection. In some tissues, more characteristic findings were identified, including the presence of pigmented macrophages, especially in hepatic sinusoids and colonic mucosa, where active chronic eosinophil predominant colitis was also observed. CONCLUSIONS: Chronic granulomatous disease may present to histopathologists in a wide range of tissue specimens most often demonstrating features of active chronic inflammation with or without granuloma formation. The presence of numerous pigmented macrophages in association with such inflammation should raise suspicion of the diagnosis. In addition, diffuse granulomatous inflammation of the lung and hepatic abscess formation should be regarded as suggestive of the diagnosis.     

Late diagnosis of chronic granulomatous disease

Modern era advancements in medical care, with improved treatment of infections, can result in delayed diagnosis of congenital immunodeficiencies. In this study we present a retrospective cohort of 16 patients diagnosed with Chronic Granulomatous Disease (CGD) at adulthood. Some of the patients had a milder clinical phenotype, but others had a classic phenotype with severe infectious and inflammatory complications reflecting a profoundly impaired neutrophil function. It is therefore of great importance to investigate the individual journey of each patient through different misdiagnoses and the threads which led to the correct diagnosis. Currently the recommended definitive treatment for CGD is hematopoietic stem cell transplantation (HSCT). Although survival of our patients to adulthood might argue against the need for early HSCT during infancy, we claim that the opposite is correct, as most of them grew to be severely ill and diagnosed at a stage when HSCT is debatable with potentially an unfavorable outcome. This cohort stresses the need to increase awareness of this severe congenital immunodeficiency among clinicians of different specialties who might be treating undiagnosed adult patients with CGD.     

CYBB mutation analysis in X-linked chronic granulomatous disease

Chronic granulomatous disease (CGD) results from mutations of phagocyte NADPH oxidase. Seventy percent are X-linked (X-)CGD with absent or defective gp91(phox) protein encoded by the CYBB gene. A subset of X-CGD patients demonstrates partial oxidase activity and/or varied levels of the gp91(phox) protein. Definitive genotypic diagnosis in these unusual patients requires mutation analysis. Typically, CYBB mutation analysis has relied on initial screening of cDNA by single-stranded conformation polymorphism analysis, followed by selective sequencing. We report a fluorescent, automated method for CYBB mutation analysis using genomic DNA that provides more rapid and reliable results. Moreover, the use of genomic DNA in this approach allows mutation detection in the mRNA coding region, promoter/enhancer region, and intronic sequences flanking splice junctions and does not require mRNA preparation. The PCR conditions were optimized for each exon, including those with A+T-rich regions. We analyzed DNA from two unusual X-CGD patients and established the genetic basis for their phenotype. We also sequenced 100 normal X chromosomes to establish wild-type consensus sequences and identify polymorphisms.     

T cell targeted immunotherapy for autoimmune disease

Much emphasis has been placed on the so-called “biologics” in the treatment of immune disorders within the last few years. Here we discuss the expanding horizon of potential strategies for immunotherapies targeting T lymphocytes as key effectors and regulators of autoimmunity. We review emerging reagents in a variety of animal models and human disorders that may offer new therapeutic options in current or modified iterations.     

T cell responses induced by allergen-specific immunotherapy

Allergen‐specific immunotherapy is recognized as a highly effective practice in the treatment of patients with severe allergic rhinitis and/or asthma and is recommended by World Health Organization as an integrated part of allergy management strategy. Several studies have shown that allergen‐specific immunotherapy, based on the administration of increasing doses of allergen, achieves a hyposensitization and reduces both early and late responses occurring during the natural exposure to the allergen itself. This is the unique antigen‐specific immunomodulatory treatment in current use for human diseases. Successful immunotherapy is associated with reductions in symptoms and medication scores and improved quality of life. After interruption it usually confers long‐term remission of symptoms and prevents the onset of new sensitizations in children up to a number of years. Subcutaneous immunotherapy usually suppresses the allergen‐induced late response in target organs, likely due to the reduction of the infiltration of T cells, eosinophils, basophils, mast cells and neutrophils. In addition to the reduction of cells of allergic inflammation, immunotherapy also decreases inflammatory mediators at the site of allergen exposure. This review provides an update on the immunological T cell responses induced by conventional subcutaneous and sublingual immunotherapy, and gives a unifying view to reconciling the old dualism between immunoredirecting and immunoregulating mechanisms.     

Characterization of 11 novel mutations in the X‐linked chronic granulomatous disease (CYBB gene)

The most frequent form of chronic granulomatous disease (CGD) is caused by inactivation of the CYBB gene, which encodes the gp91‐phox subunit of phagocyte NADPH oxidase. This defect prevents phagocytes from producing reactive oxygen species and thus from eradicating bacterial and fungal infections. We investigated 16 unrelated male patients with suspected X‐linked CGD and gp91‐phox deficiency. A mutation was found in the CYBB gene of all 16 patients, and 11 of these mutations were novel. Eleven patients (69%) had a point mutation (84G>A in two unrelated patients, and 177C>G, 217C>T, 388C>T, 676C>T, 691C>T, 868C>T, 919A>C, 1384G>T and T1514G in one case each, yielding W28X, C59W, R73X, R130X, R226X, Q231X, R290X, T307P, E462X, L505R gp‐91phox). One patient had an in‐frame deletion removing two amino acids (R54 and A55). Finally, insertions or duplications were found in four patients (from +1 to +31 bases). Overall, 12 (75%) of the mutations led to the production of a truncated protein. No clear correlation was found between clinical manifestations and genomic/biochemical alterations. Thirteen mothers could be tested, and all were carriers. Hum Mutat 18:163, 2001. © 2001 Wiley‐Liss, Inc.     

Pulmonary manifestations of chronic granulomatous disease

Chronic granulomatous disease (CGD) is an inherited disorder, characterized by defects in superoxide-generating NADPH oxidase of phagocytes. The genetic defects in CGD induce failure to activate the respiratory burst in the phagocytes, leading to severe recurrent infections and unexplained prolonged inflammatory reactions that may produce granulomatous lesions. A noble advance in curative therapy for CGD is hematopoietic stem cell transplantation. Since the most common site of involvement in CGD is the lung, the pulmonologists (pediatrics or adult) may be among the first to recognize the pattern of infection, inflammation and granuloma formation, leading to diagnosis of CGD. Pulmonologists need to be aware of different lung manifestations of CGD.     

CD3ζ‐based chimeric antigen receptors mediate T cell activation via cis‐ and trans‐signalling mechanisms: implications for optimization of receptor structure for adoptive cell therapy

Chimeric antigen receptors (CARs) can mediate redirected lysis of tumour cells in a major histocompatibility complex (MHC)‐independent manner, thereby enabling autologous adoptive T cell therapy for a variety of malignant neoplasms. Currently, most CARs incorporate the T cell receptor (TCR) CD3ζ signalling chain; however, the precise mechanisms responsible for CAR‐mediated T cell activation are unclear. In this study, we used a series of immunoreceptor tyrosine‐based activation motif (ITAM)‐mutant and transmembrane‐modified receptors to demonstrate that CARs activate T cells both directly via the antigen‐ligated signalling chain and indirectly via associated chains within the TCR complex. These observations allowed us to generate new receptors capable of eliciting polyfunctional responses in primary human T cells. This work increases our understanding of CAR function and identifies new avenues for the optimization of CAR‐based therapeutic interventions.     

Expansion of T helper type 17 lymphocytes in patients with chronic granulomatous disease

Hyper-immunoglobulin (Ig)E syndrome (HIES) is a primary immunodeficiency associated with mutations in STAT3 resulting in impaired development of T helper type 17 (Th17) lymphocytes. HIES patients with a reduced frequency of Th17 cells present with infections caused by Staphylococcus aureus and/or Candida strains. The same spectrum of pathogens is present in patients with chronic granulomatous disease (CGD).We analysed the characteristics of the Th17 compartment in HIES and CGD. HIES patients showed very low numbers of Th17 cells. By contrast, the frequency of Th17 cells and production of Th17-derived cytokines was significantly higher among CGD patients when compared to both control samples and HIES. Naive CD4(+) cells in CGD patients had a normal capacity to differentiate into IL-17-producing cells and the numbers of Th17 cells in the CGD patients normalized following successful bone marrow transplantation. Our findings complement recent data on the importance of Th17 cells for elimination of infections with C. albicans and S. aureus.     

Harnessing proteases for T regulatory cell immunotherapy

The immune system is tightly regulated by a subset of T cells defined as regulatory T cells (Tregs). Tregs maintain immune homeostasis by restraining unwarranted immune cell activation and effector function. Here, we discuss an important but underappreciated role of proteases in controlling Treg function. Proteases regulate a number of vital processes that determine T cell immune responses and some of them such as furin, ADAM (through regulating LAG receptor), MALT, and asparaginyl endopeptidase are implicated in Treg immunobiology. Targeted protease inhibition, using either small molecule inhibitors or gene deficient mice has demonstrated their specificity in modulating Treg function in experimental murine models. These data further highlight the ability of proteases to specifically regulate Tregs but no other T effector lineages. Taken together, it is apparent that incorporating proteases as targets within Treg cell engineering protocols may enable generation of robust Treg cellular therapeutics. These engineered Tregs may possess enhanced regulatory function along with resistance to lineage deviation in inflammatory disease such as colitis and graft versus host disease. Within this review, we summarize research on the role of proteases in regulating Treg function and discuss the translational potential of harnessing Treg function by targeting protease driven regulatory pathways.     

Point mutations in the promoter region of the CYBB gene leading to mild chronic granulomatous disease

Chronic granulomatous disease (CGD) is a clinical syndrome of recurrent bacterial and fungal infections caused by a rare disorder of phagocytic cells. In CGD, the phagocytes are unable to generate oxygen radicals after stimulation of these cells, due to a defect in the NADPH oxidase system. This NADPH oxidase is a multicomponent enzyme of at least four subunits, of which the beta-subunit of cytochrome b558, gp91-phox, is encoded by an X-linked gene (called CYBB). We report here five patients from two families; in each family we found a different mutation in the promoter region of CYBB. Both mutations prevented the expression of gp91-phox in the patients' neutrophils and thus caused inability of these cells to generate oxygen radicals. However, the mutations left the gp91-phox expression and the function of the NADPH oxidase in the patients' eosinophils intact. The relatively mild course of the CGD in these patients can probably be attributed to the fact that the eosinophils have retained their oxidative capacity. Furthermore, our results indicate that neutrophils and eosinophils differ in their regulation of gp91-phox expression.     

Accelerated calcium influx and hyperactivation of neutrophils in chronic granulomatous disease

The relationship between activation of NADPH-oxidase, alterations in membrane potential and triggering of Ca2+ fluxes in human phagocytes has been investigated using neutrophils from four subjects with chronic granulomatous disease (CGD). Cytosolic Ca2+ and membrane potential were measured by spectrofluorimetry, and net efflux and influx of Ca2+ by radiometric procedures. Exposure of normal neutrophils to the chemotactic tripeptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP; 1 microM) was accompanied by an abrupt increase in cytosolic Ca2+ coincident with membrane depolarization and efflux of the cation. These events terminated at around 30 s after the addition of FMLP and were followed by membrane repolarization and store-operated influx of Ca2+, both of which were superimposable and complete after about 5 min. Activation of CGD neutrophils was also accompanied by an increase in cytosolic Ca2+, which, in spite of an efficient efflux response, was prolonged in relation to that observed in normal cells. This prolonged increase in cytosolic Ca2+ in activated CGD neutrophils occurred in the setting of trivial membrane depolarization and accelerated influx of Ca2+, and was associated with hyperactivity of the cells according to excessive release of elastase and increased activity of phospholipase A2. Treatment of CGD neutrophils with the type 4 phosphodiesterase inhibitor, rolipram (1 microM) restored Ca2+ homeostasis and attenuated the increase in elastase release. These findings support the involvement of NADPH-oxidase in regulating membrane potential and Ca2+ influx in activated neutrophils, and may explain the disordered inflammatory responses and granuloma formation which are characteristic of CGD.     

Chronic granulomatous disease of childhood: Respiratory cytology

Chronic granulomatous disease (CGD) of childhood is a rare inherited disease in which phagocytic cells fail to produce the normal respiratory burst in response to infectious stimuli, leaving the patient particularly susceptible to infections with bacteria and fungi that produce catalase. Between 1988 and 1993 at the NIH, 58 pulmonary cytology specimens were obtained on 24 CGD patients. The number of specimens per patient ranged from one to 13 with an average of 2.4. The 58 specimens included: 33 bronchoalveolar lavages; one bronchial brushing; 20 lung or pleural mass fine-needle aspirates; three pleural fluids, and one sputum. Two lung aspirates with insufficient material, five bronchoalveolar lavages performed post-treatment to confirm clinical resolution of disease, and two bronchoalveolar lavages not submitted for culture were excluded from further analysis. Of the 49 remaining specimens obtained from patients clinically suspected of having a pulmonary infection, cytology demonstrated a pathogenic organism in nine (18%). Microbiologic cultures were positive in 19/49 (39%). Cytology identified fungus in 8/13 (62%) of documented fungal infections, including four cases where microbiology was negative. Bacterial and mixed bacterial/fungal infections were usually not detected by cytology even with appropriate stains. No organisms were identified by cytology in the four cases of nocardia or the three cases of pseudomonas infection. The combination of cytology and microbiology provided the greatest diagnostic sensitivity, yielding a diagnosis in 22/49 cases (45%). Of the 27 cases with negative cytology and microbiology, an infectious agent was identified in eight upon submission of additional material: three cytology specimens and five tissue specimens. In the remaining 19 cases, no organisms were identified, however, the patients were treated presumptively. Characteristic pathologic features of granulomatous inflammation, necrosis, and giant cells were present in fine-needle aspirates, often when no organisms could be identified, but were not seen in other types of respiratory specimens. Diagn Cytopathol 1996;15:98–102. © 1996 Wiley-Liss, Inc.     

Follow up of patients with chronic granulomatous disease diagnosed since 1990

Outcomes for children with chronic granulomatous disease (CGD) have historically been poor, but significant improvements have occurred with the use of effective antibacterial prophylaxis. The present study aimed to document the clinical course of a cohort of children diagnosed with CGD since 1990 in a single centre. Twenty-one patients were identified, with a median age at last assessment of 4 years and 5 months. A third of these children were diagnosed in infancy because of a positive family history. Of the remaining, there was a median delay between the onset of symptoms and diagnosis of 13 months. No invasive or fungal infections were documented after diagnosis, nor were there any deaths in this cohort. A variety of non-infectious complications were noted, which responded well to steroids. As a group, these children were thriving and weight and height distributions fell within the population norm. All patients were receiving antibacterial prophylaxis, 90% with co-trimoxazole, and all but one patient were receiving a prophylactic anti-fungal agent (itraconazole). Both drugs were well tolerated. In conclusion, this cohort of patients, diagnosed in the last decade, tolerated antibacterial and anti-fungal prophylaxis well and on this regimen have a significantly decreased incidence of infection when compared with historical cohorts. Careful follow up of patients who have had aggressive antibacterial and anti-fungal prophylaxis should continue. The data reported on this cohort of patients should inform the debate about the use of more aggressive treatments, such as bone marrow transplantation, in this disease.     

Increased CTLA‐4+ T cells may contribute to impaired T helper type 1 immune responses in patients with chronic obstructive pulmonary disease

Impaired T helper type 1 (Th1) function is implicated in the susceptibility of patients with chronic obstructive pulmonary disease (COPD) to respiratory infections, which are common causes of acute exacerbations of COPD (AECOPD). To understand the underlying mechanisms, we assessed regulatory T (Treg) cells and the expression of an inhibitory T‐cell receptor, cytotoxic T‐lymphocyte‐associated antigen 4 (CTLA‐4). Cryopreserved peripheral blood mononuclear cells (PBMC) from patients with AECOPD (n = 17), patients with stable COPD (sCOPD; n = 24) and age‐matched healthy non‐smoking controls (n = 26) were cultured for 24 hr with brefeldin‐A or monensin to detect intracellular or surface CTLA‐4 (respectively) by flow cytometry. T cells in PBMC from AECOPD (n = 9), sCOPD (n = 14) and controls (n = 12) were stimulated with anti‐CD3 with and without anti‐CTLA‐4 blocking antibodies and cytokines were quantified by ELISA. Frequencies of circulating T cells expressing intracellular CTLA‐4 were higher in sCOPD (P = 0·01), whereas patients with AECOPD had more T cells expressing surface CTLA‐4 than healthy controls (P = 0·03). Increased frequencies of surface CTLA‐4⁺ CD4⁺ T cells and CTLA‐4⁺ Treg cells paralleled increases in plasma soluble tumour necrosis factor receptor‐1 levels (r = 0·32, P = 0·01 and r = 0·29, P = 0·02, respectively) in all subjects. Interferon‐γ responses to anti‐CD3 stimulation were inversely proportional to frequencies of CD4⁺ T cells expressing intracellular CTLA‐4 (r = −0·43, P = 0·01). Moreover, CTLA‐4 blockade increased the induction of interferon‐γ, tumour necrosis factor‐α and interleukin‐6 in PBMC stimulated with anti‐CD3. Overall, chronic inflammation may expand sub‐populations of T cells expressing CTLA‐4 in COPD patients and therefore impair T‐cell function. CTLA‐4 blockade may restore Th1 function in patients with COPD and so aid the clearance of bacterial pathogens responsible for AECOPD.     

The in vitro generation of multi-tumor antigen-specific cytotoxic T cell clones: Candidates for leukemia adoptive immunotherapy following allogeneic stem cell transplantation

Adoptive T-cell immunotherapy is a promising approach to manage and maintain relapse-free survival of leukemia patients, especially following allogeneic stem cell transplantation. Post-transplant adoptive immunotherapy using cytotoxic T lymphocytes (CTLs) of the donor origin provide graft-versus-tumor effects, with or without graft–versus-host disease. Myeloid leukemias express immunogenic leukemia associated antigens (LAAs); such as WT-1, PRAME, MAGE, h-TERT and others, most of them are able to induce specific T cell responses whenever associated with the proper co-stimulation. We investigated the ability of a LAA-expressing hybridoma cell line to induce CTL clones in PBMCs of HLA-matched healthy donors in vitro. The CTL clones were induced by repetitive co-culture with LAAs-expressing, HLA-A*0201⁺ hybrid cell line, generated by fusion of leukemia blasts to human immortalized APC (EBV-sensitized B-lymphoblastoid cell line; HMy2). The induced cytotoxic T cell clones were phenotypically and functionally characterized by pentamer analysis, IFN-γ release ELISPOT and cellular cytotoxicity assays. All T cell lines showed robust peptide recognition and functional activity when sensitized with HLA-A*0201-restricted WT-1_235–243, hTERT_615–624 or PRAME_100–108 peptides-pulsed T2 cells, in addition to partially HLA-matched leukemia blasts. This study demonstrates the feasibility of developing multi-tumor antigen-specific T cell lines in allogeneic PBMCs in vitro, using LAA-expressing tumor/HMy2 hybrid cell line model, for potential use in leukemia adoptive immunotherapy in partially matched donor-recipient setting.