T cell phenotypes in patients with common variable immunodeficiency disorders: associations with clinical phenotypes in comparison with other groups with recurrent infections.

Common variable immunodeficiency disorders (CVID) are a group of heterogeneous conditions that have in common primary failure of B cell function, although numerous T cell abnormalities have been described, including reduced proliferative response and reduced regulatory T cells. This study compared the T cell phenotype of CVID patients subdivided into clinical phenotypes as well as patients with partial antibody deficiencies [immunoglobulin (Ig)G subclass deficiency and selective IgA deficiency], X-linked agammaglobulinaemia (XLA) and healthy and disease controls. Absolute numbers of T cell subpopulations were measured by four-colour flow cytometry: naive T cells, central and effector memory and terminally differentiated (TEM) T cells, using CD45RA and CCR7 expression. Early, intermediate and late differentiation status of T cells was measured by CD27/CD28 expression. Putative follicular T cells, recent thymic emigrants and regulatory T cells were also assessed. Significant reduction in naive CD4 T cells, with reduced total CD4 and recent thymic emigrant numbers, was observed in CVID patients, most pronounced in those with autoimmune cytopenias or polyclonal lymphoproliferation. These findings suggest a lack of replenishment by new thymically derived cells. CD8 naive T cells were reduced in CVID patients, most significantly in the autoimmune cytopenia subgroup. There was a reduction in early differentiated CD4 and CD8 T cells and increased CD8 TEM in the CVID patients, particularly autoimmune cytopenia and polyclonal lymphoproliferation subgroups, suggesting a more activated T cell phenotype, due perhaps to an antigen-driven process. XLA patients had significantly reduced putative follicular T cells, which may depend on B cells for survival, while no significant alterations were observed in the T cells of those with IgG subclass deficiency or selective IgA deficiency.

NOD2 polymorphisms in clinical phenotypes of common variable immunodeficiency disorders.

Common variable immunodeficiency disorders (CVIDs) are a heterogeneous group of diseases characterized by hypogammaglobulinaemia and consequent susceptibility to infection. CVID patients commonly develop a variety of additional manifestations for which the causative factors are not fully understood. Two such manifestations are granulomatous disease and enteropathy. Because the ability to predict complications would aid clinical management, we continue to search for possible disease modifier genes. NOD2 acts a microbial sensor and is involved in proinflammatory signalling. Particular mutations of the NOD2 gene are associated with Crohn's disease including gly908arg, leu1007finsc and arg702trp polymorphisms. We hypothesized that NOD2 polymorphisms may be a disease modifier gene towards an enteropathic or granulomatous phenotype within CVIDs. Sequence-specific primers returned genotypes for 285 CVID patients from centres across the United Kingdom and Europe. We present the frequencies of the different phenotypes of patients within our international cohort. Arg702trp polymorphisms were significantly less frequent than wild-type (WT) (P = 0·038) among international CVID patients with splenomegaly. Gly908arg polymorphisms were more prevalent than WT in UK patients with autoimmune disorders (P = 0·049) or enteropathy (P = 0·049). NOD2 polymorphisms were not more prevalent than WT in CVID patients with clinical phenotypes of granulomata. UK allele frequencies of 0·014, 0·056 and 0·026 were found for gly908arg, arg702trp and leu1007finsc NOD2 polymorphisms, respectively. These do not differ significantly from UK immunocompetent controls confirming, as expected, that in addition these NOD2 polymorphisms do not confer susceptibility to CVIDs per se.

Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans.

The functional interaction of BAFF and APRIL with TNF receptor superfamily members BAFFR, TACI and BCMA is crucial for development and maintenance of humoral immunity in mice and humans. Using a candidate gene approach, we identified homozygous and heterozygous mutations in TNFRSF13B, encoding TACI, in 13 individuals with common variable immunodeficiency. Homozygosity with respect to mutations causing the amino acid substitutions S144X and C104R abrogated APRIL binding and resulted in loss of TACI function, as evidenced by impaired proliferative response to IgM-APRIL costimulation and defective class switch recombination induced by IL-10 and APRIL or BAFF. Family members heterozygous with respect to the C104R mutation and individuals with sporadic common variable immunodeficiency who were heterozygous with respect to the amino acid substitutions A181E, S194X and R202H had humoral immunodeficiency. Although signs of autoimmunity and lymphoproliferation are evident, the human phenotype differs from that of the Tnfrsf13b-/- mouse model.

Measurement of peripheral B cell subpopulations in common variable immunodeficiency (CVID) using a whole blood method.

Recent reports have described reduced populations of CD27+ memory B cells and increased percentages of undifferentiated B cells in peripheral blood of patients with common variable immunodeficiency (CVID). This work has prompted two attempts to classify CVID based on rapid flow cytometric quantification of peripheral blood memory B cells and immature B cells. Evidence to support the hypothesis that such in vitro B cell classification systems correlate with clinical subtypes of CVID is being sought. For the classification to be useful in routine diagnosis, it is important that the flow cytometric method can be used without prior separation of peripheral blood mononuclear cells (PBMC). We have examined 23 CVID patients and 24 controls, using both PBMC and whole blood, and find an excellent correlation between these methods. The reproducibility of the method was excellent. We classified the CVID patients by all three of the existing classifications, including secretion of immunoglobulin by B cells in vitro as described by Bryant, as well as the more recent flow cytometric classification methods. Only one patient changed classification as a result of using whole blood.

Immunogenicity of routine vaccination against diphtheria, tetanus, and Haemophilus influenzae type b in Asian infants born in the United Kingdom.

AIM: To determine the immunogenicity of routine vaccination against diphtheria, tetanus, and Haemophilus influenzae type b (Hib) in Asian infants born in the UK, and whether maternal antibody suppression occurs. METHODS: A cohort study with 80% power, within 95% confidence limits, to show that 80% or fewer Asian infants would respond with an anti-PRP antibody concentration >0.15 microg/ml. Infants of South Asian origin born in Berkshire were enrolled at two general practices in Reading: 41 Asian families sequentially asked to participate within 2 weeks of birth; 36 infants were enrolled and 34 completed the study. Main outcome measures were: antibody concentration against diphtheria, tetanus, and Hib expressed as geometric mean titres (GMT) and proportion of infants about a threshold protective antibody concentration. RESULTS: Median age for completing primary vaccination course was 5 months. All 34 achieved anti-PRP antibody concentration of >0.15 microg/ml, 33 were >1.0 microg/ml, and the GMT was 15.0 microg/ml. All infants developed protective antibody concentration >0.1 IU/ml for tetanus and diphtheria; the respective GMTs were 1.94 and 5.57 IU/ml. Infants with high (>0.25 IU/ml) antibody concentrations against diphtheria and tetanus at 2 months achieved lower antibody concentrations after their three dose course than those with low concentrations (<0.1 IU/ml) (p = 0.06 and 0.03, respectively). CONCLUSIONS: Despite evidence for maternal antibody suppression of the response to tetanus and diphtheria vaccination, excellent antibody responses were achieved by routine vaccination according to the accelerated schedule. High vaccine coverage should be encouraged to provide protection against the possibility of imported infection.

Development of an anti-Salmonella typhi Vi ELISA: assessment of immunocompetence in healthy donors.

We have developed a solid-phase enzyme-linked immunosorbent assay (ELISA) to study the vaccination responses to Vi capsular polysaccharide of Salmonella typhi (S. typhi Vi) vaccine. Purified S. typhi Vi polysaccharide was biotinylated and bound to streptavidin coated microtitre plates. Reproducibility was determined across a range of IgG antibody levels: mean interassay coefficients of variation (CVs) were <11.9% for non-vaccinated sera with low levels and <11.1% for sera with very high levels of anti-S. typhi Vi IgG. Specificity was assessed by inhibition studies using salmonella antigen. We have developed the ELISA based on normal adult serum responses to test immunization with S. typhi Vi vaccine. We also report here anti-S. typhi Vi IgG levels in a group of healthy preschool children. In non-vaccinated adult sera (n = 104), the median value of anti-S. typhi Vi IgG, expressed in S. typhi Vi arbitrary units (AU/ml), was 5.3 AU/ml and in non-vaccinated sera from children (n = 44) the median value was 1.4 AU/ml. The data from immunization of healthy volunteers (n = 23) show that geometric mean levels of anti-S. typhi Vi IgG were significantly higher (P < 0.0001) for post-vaccination subjects (39.2 AU/ml) compared to paired prevaccination (3.9 AU/ml) values. A total of 21/23 vaccine recipients had <8 AU/ml S. typhi Vi IgG in their sera prior to vaccination and of these 20/21 (95%) exhibited threefold increases and 14/21 (67%) fourfold increases in their S. typhi Vi IgG following vaccination. Based on the data in this study, we propose a threefold increase in anti-S. typhi Vi IgG post-vaccination to be considered a positive vaccination response. The ability to demonstrate clearly an antibody rise in response to immunization with S. typhi Vi capsular polysaccharide vaccine suggests that this is likely to be a useful vaccine for the assessment of B cell function in patients with suspected immune deficiency.

Lymphoproliferative disease in antibody deficiency: a multi-centre study.

We have undertaken a retrospective study of antibody deficient patients, with and without lymphoma, and assessed the ability of specific polymerase chain reaction (PCR) primers to determine if the detection of clonal lymphocyte populations correlates with clinical and immunohistochemical diagnosis of lymphoma. We identified 158 cases with antibody deficiency presenting during the past 20 years. Paraffin-embedded biopsy specimens or slides were available for analysis in a cohort of 34 patients. Of these patients, 29 had common variable immunodeficiency, one X-linked agammaglobulinaemia, one X-linked immunoglobulin deficiency of uncertain cause and three isolated IgG subclass deficiency. We have confirmed that lymphoma in antibody deficiency is predominantly B cell in origin. Clonal lymphocyte populations were demonstrated in biopsies irrespective of histology (16/19 with lymphoma and 11/15 without). Isolated evidence of clonality in biopsy material is therefore an insufficient diagnostic criterion to determine malignancy. Furthermore, our data suggest that clonal expansions are rarely the result of Epstein-Barr virus-driven disease.

Prospective audit of adverse reactions occurring in 459 primary antibody-deficient patients receiving intravenous immunoglobulin.

Intravenous immunoglobulin (IVIG) is used as the standard replacement therapy for patients with primary antibody deficiencies. A previous study of adverse reactions in patients self-infusing at home over 1 year showed an overall reaction rate of 0.7%. A larger prospective study is reported here, involving a greater number of immunology centres and including children and adults who received infusions from medical or nursing staff as well as those self-infusing. Four hundred and fifty-nine patients were entered into this study and 13 508 infusions were given. The study showed that no severe reactions occurred and the reaction rate was low at 0.8%. This figure could have been lower, 0.5%, if predisposing factors responsible for some reactions had been considered before infusion. In conclusion, the study shows the importance of ongoing training for patients and staff to recognize the predisposing factors to prevent avoidable reactions. Because none of these reactions were graded as severe, the present guidance to prescribe self-injectable adrenaline for patients infusing outside hospital should be reviewed.

A preliminary assessment of alpha-1 antitrypsin S and Z deficiency allele frequencies in common variable immunodeficiency patients with and without bronchiectasis.

Common variable immunodeficiency (CVID) is the name given to a clinically heterogeneous group of hypogammaglobulinaemic immunodeficiency states. Bronchiectasis is a feature of this disease and is believed to be the result of recurrent bacterial infection affecting the respiratory tract. Bronchiectasis is also a feature associated with emphysematous changes of the lung in alpha-1 antitrypsin (AAT) deficiency, a serious and relatively common disease, affecting 1 : 2000 in the United Kingdom. This has been demonstrated to result from possession of deficiency alleles, the most clinically important alleles being PI*Z and PI*S. Isolated reports of families with antibody deficiency and AAT deficiency have been published but to date no study has been performed to specifically investigate if AAT deficiency is associated with the lung damage seen in CVID patients. We have developed a PCR genotyping assay that identifies S and Z deficiency alleles and we have used this assay in a preliminary study to investigate the occurrence of these deficiency alleles of AAT in 43 CVID patients. Results of this preliminary study suggest that CVID patients did not have an altered distribution of AAT genes when compared to 70 normal controls. Subgrouping of CVID patients into those with and without bronchiectasis demonstrated a Z allele frequency of 0.077 in those patients with bronchiectasis, which is higher than found in normal controls, namely 0.029 (P < 0.15). Due to the relatively small numbers studied, these results are inconclusive in determining whether AAT deficiency may exacerbate lung damage in some CVID patient, the data does however, indicate that a larger multi-centre study involving many more CVID patients may be useful.

Five-year follow-up of patients with primary antibody deficiencies following an outbreak of acute hepatitis C.

This report is the 5-year follow-up of those 25 UK patients with primary antibody deficiencies infected with hepatitis C virus (HCV), type 1a, from one batch of contaminated anti-HCV-screened intravenous immunoglobulin in 1993-1994. Of these patients, who were reported previously (1, 2), 2 cleared HCV spontaneously, 18 received early interferon-alpha (IFN) treatment for 6 months, and 5 declined treatment or treatment was contraindicated. The clinical course of this cohort was followed prospectively using serial standardized questionnaires. Seven patients (54% of those who had completed therapy) had a sustained response (normal transaminase levels, negative serum HCV RNA) for 5 years posttreatment. Eight patients died: 3 from decompensated cirrhosis, 2 from pneumonia but with evidence of liver failure, and 3 from unrelated causes. One further patient developed decompensated cirrhosis but was successfully transplanted. Seven patients remain chronically infected; only 1 patient is symptomatic but 1 further patient has evidence of progressive fibrosis on liver histology. In conclusion, within 5 years, rapid end-stage HCV liver disease has been seen in 6/25 (24%) patients. Seven patients, (54% of those fully treated) remain well after treatment, making 9/25 (36% of the cohort) clear of virus after 5 years. Those who completed early treatment with IFN had a relatively high sustained response rate compared to previous studies in both immunodeficient and immunocompetent patients.

A rapid flow cytometric assay to detect CD4+ and CD8+ T-helper (Th) 0, Th1 and Th2 cells in whole blood and its application to study cytokine levels in atopic dermatitis before and after cyclosporin therapy.

BACKGROUND: The immune response in atopic dermatitis (AD) is thought to be driven by T-helper (Th) 2 cytokines. Using flow cytometry, higher frequencies of peripheral blood CD4+ and CD8+ T cells producing interleukin (IL)-4 and correspondingly lower frequencies of CD4+ T cells producing interferon (IFN)-gamma have been found in patients with AD compared with healthy controls. It would be of interest to know whether other Th1 and Th2 cytokines such as IL-5, IL-13 and tumour necrosis factor (TNF)-alpha are similarly skewed in patients with AD and whether this immune skewing, detected via a simple blood assay, can be correlated with other clinical measurements or treatments in AD. OBJECTIVES: To use a rapid (4-h) flow cytometric assay to study a wide range of Th1 and Th2 cytokine patterns in peripheral blood lymphocytes from patients with AD, comparing them with non-atopic healthy controls. To correlate cytokine patterns with the degree of eosinophilia observed and in the case of one patient with severe disease, to observe the effect of cyclosporin therapy on peripheral blood cytokine patterns. METHODS: Peripheral blood from eight patients with AD and 23 healthy controls was examined for the frequencies of CD4+ and CD8+ T cells expressing IL-2, IL-4, IL-5, IL-13, IFN-gamma and TNF-alpha using flow cytometry. RESULTS: Significantly higher frequencies of CD4+/IL-4+ (P < 0.005) and CD4+/IL-13+ (P < 0.0001) and lower frequencies of CD4+/IFN-gamma+ (P < 0.002) and CD8+/TNF-alpha+ (P < 0.05) T lymphocytes were found in patients with AD compared with controls. There were significant positive correlations with the increased percentages of CD4+/IL-4+ and CD4+/IL-13+ T lymphocytes and the degree of eosinophilia observed (P < 0.05, P < 0.001) and a negative correlation between the percentage of CD4+/IFN-gamma+ T lymphocytes and eosinophilia (P < 0.05). In one patient examined before and 8 days after cyclosporin therapy, 50% or greater reductions were observed in percentages of peripheral blood CD8+/IL-5+, CD8+/IL-13+, CD4+/IL-4+ and CD4+/IL-5+ T lymphocytes following cyclosporin therapy. A smaller reduction of 15% after cyclosporin therapy was found in percentages of CD4+/IL-13+ T cells. CONCLUSIONS: These data strongly support a Th2 predominance in the peripheral blood of AD. The results suggest that administration of cyclosporin therapy in patients with AD may help to restore the Th2 cytokine imbalance seen in these patients.

The comparison of the efficacy and safety of intravenous versus subcutaneous immunoglobulin replacement therapy.

To compare the efficacy of immunoglobulin replacement therapy given intravenously versus subcutaneously to prevent infections in patients with primary antibody deficiency syndromes, an international, multicenter, open label, crossover study was designed. Forty patients were randomized to receive either subcutaneous or intravenous immunoglobulin replacement therapy for 1 year. In the second year, patients were switched to the alternative treatment, enabling patients to act as their own controls. Equivalent doses were given by both routes. Ethical approval was obtained from the review boards of the hospitals in which the patients were seen and written consent obtained from each patient. Patients with a primary antibody deficiency syndrome, either common variable immunodeficiency or IgG subclass deficiency or specific antibody deficiency, who required immunoglobulin replacement therapy were included in the study. Patients were excluded if they had significant thrombocytopenia (defined as platelets less than 50 x 10(9)/liter), had high levels of anti-IgA antibodies (defined as greater than 1:8192), or had severe adverse reactions to a blood product within the last 2 years. The primary end point was the number of infections and their severity (moderate and major) during the two treatment periods. Secondary end points were adverse reactions, length of infections, days lost from school or work due to infections, and acceptability of treatment regimens to the patients. Based on the assumption that it was difficult to prove equivalence of therapies statistically in crossover studies, an arbitrary number of 40 patients was selected on the basis that this might be achievable in 2 years. There are no significant differences in efficacy or adverse reaction rates between immunoglobulin replacement therapy given subcutaneously or intravenously.

Lymphoid interstitial pneumonitis associated with common variable hypogammaglobulinaemia treated with cyclosporin A.

Lymphoid interstitial pneumonitis (LIP) is a rare clinicopathological entity that may be associated with common variable immune deficiency (CVID) and may lead to respiratory failure and death. Some patients may respond to prolonged corticosteroid treatment. We hypothesised that, in view of the predominant T cell nature of LIP, cyclosporin A would be a more appropriate choice of immunosuppressive agent and report the first case of its successful use in a woman with LIP associated with CVID.

The use of self infused intravenous immunoglobulin home therapy in the treatment of acquired chronic demyelinating neuropathies.

High dose intravenous immunoglobulin therapy is becoming the established maintenance treatment for several neurological conditions. Therapeutic immunoglobulin is expensive and its administration is time consuming. Efficacy is variable and must be closely monitored. Here, we show that self infused immunoglobulin at home, combined with daily functional strength diaries to predict dose and dosage intervals, is effective, time saving, and convenient for both patients and medical staff.

Antipolysaccharide antibodies in 450 children with otitis media.

We have measured antibodies to pneumococcal and Haemophilus polysaccharides in a prospective study of 450 children aged 2-16 years with otitis media requiring grommets (ear tubes). Pneumococcal antibody levels were significantly higher in the 2-6 year (P < 0.004) and 7-10 year (P < 0.04) study groups in comparison with age-matched controls. There was no difference in Haemophilus antibody levels between the study and control group children for the age groups 2-6 years and 11-16 years. Haemophilus antibody levels were significantly lower in the 7-10 year (P < 0.003) group in comparison with age-matched controls. Eighty-eight out of 450 (19.6%) children had pneumococcal antibody levels below the 25th percentile. Nineteen out of 88 (21.6%) children with pneumococcal antibody levels below the 25th centile were test immunized with 23 valent Pneumococcal polysaccharide and unconjugated Haemophilus type b capsular polysaccharide. Of these 19 children (aged 4-11 years), five mounted suboptimal responses to both polysaccharide antigens, whilst one child failed to respond to Haemophilus polysaccharide alone. There was no significant difference in the prevalence of IgG subclass deficiency between the normal responders and poor responders to immunization (P = 0.12). We found no evidence of specific polysaccharide antibody deficiency in the vast majority of the 450 children studied. However, the significance of poor antibody responses to test immunization in a small minority of children with otitis media is unclear. Long-term follow up of these children is required to determine whether poor immunization responses herald the development of frank antibody deficiency.

TNF and lymphotoxin-alpha polymorphisms associated with common variable immunodeficiency: role in the pathogenesis of granulomatous disease.

A subgroup of common variable immunodeficiency (CVID) patients have distinct clinical features, particularly granulomata splenomegaly, characteristic blood lymphocyte phenotype, and elevated circulating TNF levels. To investigate the genetic basis for this phenotype, 150 CVID patients and 200 controls were genotyped for six biallelic TNF and lymphotoxin-alpha (LT alpha) polymorphisms and eight class I and II HLA loci using PCR and sequence specific primers (PCR-SSP) sequence-specific primers. Clinical and immunophenotypic data were collected for 90 patients to examine associations with CVID patient subgroups. The presence of granulomata (22% of patients) was strongly associated with splenomegaly, T and B lymphopenia, reduced CD4+ CD45RA+ T cells, and CD8+ CD57+ lymphocytosis, confirming the concept of a subgroup of patients with distinct clinical and laboratory features. The uncommon TNF +488A allele was strongly associated with this subgroup (p = 0.0005). The association between "granulomatous" CVID and TNF +488A was independent of HLA class I and II associations. We postulate that the presence of the TNF +488A allele, or alleles in linkage disequilibrium with it, contributes to the high levels of TNF and granulomatous complications characteristic of this subgroup of patients.