A. D. B. Webster

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.

Intracellular cytokine production by human CD4+ and CD8+ T cells from normal and immunodeficient donors using directly conjugated anti-cytokine antibodies and three-colour flow cytometry

Using three‐colour flow cytometry, we have measured intracellular IL‐2, interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α) induced in human CD4+ and CD8+ T cells from normal donors and patients with common variable immunodeficiency (CVID). Since a new range of directly FITC‐conjugated anti‐cytokine antibodies was used, conditions were optimized for the concentration of antibody, for cell permeabilization and fixation, and for the time of exposure to monensin to retain the cytokines within the cell. Kinetics of intracellular cytokine production were measured for up to 20h in culture with phorbol myristate acetate (PMA) and ionomycin, or with phytohaemagglutinin (PHA). Kinetic studies of activation with PMA and ionomycin show that a higher proportion of normal CD4+ cells can make IL‐2 than the other two cytokines, and that there are more TNF‐α‐positive CD4+ cells than cells with IFN‐γ. For normal CD8+ cells the highest production of cytokine is of IFN‐γ (up to 50% of the cells) especially at longer times (10–20h) of stimulation. For CD8+ cells, IL‐2‐positive cells exceed those with TNF‐α. The other mitogenic stimulus used (PHA) was grossly inferior to PMA and ionomycin in its ability to induce intracellular cytokines. The time of exposure to monensin was also examined. Its continuous presence in the cultures (up to a maximum of 20h) increased the detection of IL‐2‐positive cells without apparently reducing the percentage of cytokine‐positive CD4+ or CD8+ cells. Finally, using optimal conditions, we compared cytokine production in cells from patients with the disease CVID and showed normal cellular levels of ability to produce IL‐2 and TNF‐α but significantly raised levels of production of IFN‐γ in both CD4+ and CD8+ lymphocytes. This suggests that the pathology of this disease may involve an excessive Th1‐type response.

Failure in antigen responses by T ceils from patients with common variable immunodeficiency (CVID)

Antigen‐driven responses by T cells from patients with CVID and normal subjects have been assessed. Low‐density cells enriched for antigen‐presenting dendritic cells were cultured with T cells using a 20‐μl hanging drop system, T cells from all subgroups of CVID patients showed markedly reduced responses lo the recall antigens purified protein derivative (PPD) or tetanus toxoid. whereas responses by cells from patients with X‐linked agammaglobulinaemia, used as a disease control, were in the normal range. However, primary allo‐stimulation of CVID T cells was normal, CVID cells from two patients failed lo respond to stimulation with a neoantigen, an HIV env peptide. under conditions where normal T ceils did respond. These data illustrate a profound defect in antigen‐stimulated T cell proliferation in vitro in all groups of CVID patients, but do not distinguish whether the defect is in the presenting cell or in the T lymphocyte. In vitro, germinal centre B cells are thought lo present antigen to primed T cells lo obtain essential signals (e.g. CD40 ligand and IL‐2) for B cell survival and progression to immunoglobulin secretion. A failure of antigen‐specific T cell function in vivo in CVID would thus not provide the primed T cells needed for B cell rescue, and could be the primary defect leading to the low immunoglobulin production in this condition.

Primary defect in CD8+ lymphocytes in the antibody deficiency disease (common variable immunodeficiency): abnormalities in intracellular production of interferon-gamma (IFN-γ) in CD28+ (‘cytotoxic’) and CD28− (‘suppressor’) CD8+ subsets

We have measured by flow cytometry the ability of subsets of CD8+ CD3+ lymphocytes within mononuclear cell preparations to make intracellular cytokines (IL‐2, tumour necrosis factor‐alpha (TNF‐α) and IFN‐γ) on stimulation in vitro with phorbol myristate acetate (PMA) and ionomycin for 16 h. These CD8+ subsets were defined by the presence or absence of CD28 or HLA‐DR. Subsets of normal CD8+ cells were compared with cells from the antibody deficiency disease common variable immunodeficiency (CVID). In CVID there was a significant increase in the production of IFN‐γ in the CD8+ CD28+ subset (‘cytotoxic’). This reflects a shift in this disease towards an excessive Th1 response away from B cell help. Paradoxically, some CVID patients also showed a reduction in IFN‐γ production in the CD8+ CD28− subset (‘suppressor’) which was associated with a failure of these cells to maintain a state of activation after a stimulus in vitro. The B cell problem in this disease is known to be related to a failure of T cell help shown by an inability to produce the antigen‐specific CD4+ memory T cells needed for successful B cell maturation. The two pathological CD28 subsets of CD8+ cells we have found in CVID may both be detrimental to a normal CD4‐dependent immune response. The CD28− suppressor subset expands and is unable to maintain activation and cytokine secretion, and the CD28+ cytotoxic subset is over‐producing the Th1 cytokine IFN‐γ.

Deficiency in circulating natural killer (NK) cell subsets in common variable immunodeficiency and X‐linked agammaglobulinaemia

Absolute and relative NK cell numbers were determined in peripheral whole blood by flow cytometry in patients with common variable immunodeficiency (CVID) (n = 55) and X‐linked agammaglobulinaemia (XLA) (n = 19) on regular immunoglobulin (IVIG) therapy. Absolute CD3−CD16+ NK cell numbers were significantly reduced in CVID patients (median 108/μl, range 23–815), compared with normal subjects (n = 60) (289/μl, range 56–640, P < 0·001). Total lymphocyte concentrations were significantly lower in CVID (median 1587/μl, range 523–7519) compared with normal subjects (median 2019/μl, range 1124–3149, P = 0·004), with the percentage of NK cells also being significantly decreased (median 7·5%, range 3·0–33·0%, compared with 14·2%, range 2·6–30·8%, P < 0·001). In XLA, absolute NK cell numbers (median 140/μl, range 32–551, P < 0·001) but not relative numbers were significantly reduced compared with normal controls. We excluded the possibility that IVIG interferes with in vitro binding of CD16 MoAbs. Further analysis of NK cell subsets showed a deficiency of both CD16+ and CD56+ cells in CVID, most marked in the CD3−CD8dim subpopulation, which may be due to increased homing of these cells to the gut. Serial studies on a small number of patients suggest that IVIG therapy has no short‐term effect on NK cells, although we cannot exclude an effect with prolonged use. Although there are no obvious clinical effects of the NK depletion in CVID and XLA, this may be a factor in their predisposition to cancer.

Prevalence of SAP gene defects in male patients diagnosed with common variable immunodeficiency

The molecular basis of common variable immunodeficiency (CVID) is undefined, and diagnosis requires exclusion of other diseases including X‐linked lymphoproliferative disease (XLP). This rare disorder of immunedysregulation presents typically after Epstein–Barr virus infection and results from defects in the SAP (SLAM associated protein) gene. SAP mutations have been found in a few patients diagnosed previously as CVID, suggesting that XLP may mimic CVID, but no large‐scale analysis of CVID patients has been undertaken. We therefore analysed 60 male CVID and hypogammaglobulinaemic patients for abnormalities in SAP protein expression and for mutations in the SAP gene. In this study only one individual, who was found later to have an X‐linked family history, was found to have a genomic mutation leading to abnormal SAP cDNA and protein expression. These results demonstrate that SAP defects are rarely observed in CVID patients. We suggest that routine screening of SAP may only be necessary in patients with other suggestive clinical features.

In vivo modulation of cytokine synthesis by intravenous immunoglobulin

We examined the effects of intravenous immunoglobulin (IVIG) on cytokine regulation in vivo using samples taken before and after replacement‐dose (200–400 mg/kg) IVIG in a group of patients with common variable immunodeficiency (CVID) and X‐linked agammaglobulinaemia (XLA). The intracellular cytokine content of CD4+ and CD8+ lymphocytes, and their CD28+/− subsets, were measured following in vitro activation with phorbol myristate acetate (PMA) and ionomycin. The cytokines IL‐2, interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α), and the early activation marker CD69, were assessed by four‐colour flow cytometry of whole blood cultures taken before and after IVIG infusion. There was a significant increase in IL‐2 expression in CD4+ (and CD4+28−) cells and an increase in TNF‐α expression in CD8+28− cells following IVIG in CVID, but not in XLA patients. IFN‐γ and CD69 expression were not affected by IVIG infusion. This increase in TNF‐α and IL‐2, combined with unchanged IFN‐γ expression, is evidence against the putative ‘anti‐inflammatory’ role of IVIG, and may explain the failure of resolution of granulomata in CVID patients treated with IVIG alone.

Defects in proliferative responses of T cells from patients with common variable immunodeficiency on direct activation of protein kinase C.

DNA synthesis in response to mitogens has been studied in T cells from nine patients with common variable immunodeficiency (CVI) and seven normal individuals. Five out of the nine patients had cells with subnormal responses to the mitogen phytohaemagglutinin (PHA). As PHA‐induced responses are largely mediated through activation of Ca2+‐dependent protein kinase C, we studied whether the defective response was still present on direct activation of protein kinase C. This was done using combinations of concentrations of phorbol 12,13,‐dibutyrate and the calcium ionophore ionomycin which induced proliferation in normal T cells. We found that in CVI patients with T cells which had normal responses to PHA, responses to phorbol ester and ionomycin were at the same level as in normal T cells. However, with this treatment, in which the linkage between the membrane receptor and protein kinase C is bypassed, the level of DNA synthesis was still depressed in the patient group whose T cells had subnormal responses to PHA. IL‐2 failed to restore the DNA synthesis to normal levels when added with the phorbol ester and ionomycin to T cells from one patient in this group. These data suggest that in a group of CVI patients there are defects in T cell activation pathways at or down‐stream of protein kinase C.

Defects in antigen‐driven lymphocyte responses in common variable immunodeficiency (CVID) are due to a reduction in the number of antigen‐specific CD4+ T cells

T cells from patients with CVID have defects that may relate to the failure in vivo of B cell production of antibodies. Antigen‐driven responses of T cells from CVID patients and normal subjects have been assessed by measuring DNA synthesis in vitro. Low density cells enriched for antigen‐presenting dendritic cells were pulsed with purified protein derivative (PPD) and cultured with autologous T cells. Overall, T cells from CVID patients showed a significantly low mean response to PPD, although non‐specific DNA synthesis induced in CVID T cells by IL‐2 was within the normal range. However, mean PPD‐specific T cell responses in CVID were not restored by IL‐2 irrespective of the presence of monocytes. Depletion of CD8+ cells also failed to restore the mean PPD response of CVID CD4+ T cells. Limiting dilution analysis showed that in CVID there was a reduced frequency of antigen‐specific cells within the T cell preparations. The mean frequency of the PPD‐specific T cells in cultures from patients vaccinated with bacille Calmette‐Guérin (BCG) was reduced to 1 in 109000 T cells compared with 1 in 18 600 T cells in BCG‐vaccinated normal donors. These data show that the reduced PPD‐specific response in CVID is due to a partial peripheral loss of antigen‐specific cells.

Evidence for increased expression of regulatory cytokine receptors interleukin-12R and interleukin-18R in common variable immunodeficiency

We investigated the expression of T helper (Th)1/Th2 regulatory cytokine receptors on lymphocytes from patients with common variable immunodeficiency (CVID), a disorder associated with raised Th1 cytokine production, comparing the results with those from healthy individuals and atopic asthmatics, the latter generally considered to have a Th2‐driven disease. We proposed that alterations in some of the relevant receptors might be related to the observed imbalances in Th1/Th2 cytokines. Cells from CVID patients showed an increase in the percentages of CD212 [interleukin (IL)‐12Rβ1] cells within the CD4+ CD45RA+ and CD8+ CD45RA+ subsets (24% and 41%, respectively), as compared to CD4+ CD45RA+ and CD8+ CD45RA+ in healthy subjects (6% and 23%, respectivey). Approximately 21% of the CD4+ CD45RA+ naïve cells expressed IL‐18Rα, compared with 11% in healthy subjects. In contrast, the cytokine‐receptor expression in asthmatics was similar to that of controls. In spite of the above differences, after 72 h of stimulation with anti‐CD3 and anti‐CD28, cytokine receptor up‐regulation was similar in all three groups, with up to 80% of both CD45RA+ and CD45RO+ lymphocytes expressing CD212 (IL‐12Rβ1) and IL‐18Rα. Approximately 50% of the ‘naïve’, and 25% of the ‘memory’ subpopulations up‐regulated IL‐12Rβ2. These findings provide further evidence of a polarization towards a Th1 immune response in CVID, the mechanism possibly involving up‐regulation of IL‐12‐mediated pathways.