J. Farrant

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+u2003CD3+ 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 16u2003h. 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+u2003CD28+ 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+u2003CD28− 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) (nu2003=u200355) and X‐linked agammaglobulinaemia (XLA) (nu2003=u200319) 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 (nu2003=u200360) (289/μl, range 56–640, Pu2003<u20030·001). Total lymphocyte concentrations were significantly lower in CVID (median 1587/μl, range 523–7519) compared with normal subjects (median 2019/μl, range 1124–3149, Pu2003=u20030·004), 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%, Pu2003<u20030·001). In XLA, absolute NK cell numbers (median 140/μl, range 32–551, Pu2003<u20030·001) 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.

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 (200–400u2003mg/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.

Raised serum levels of CD8, CD25 and β2-microglobulin in common variable immunodeficiency

Soluble CD8, soluble CD4, soluble CD25 (IL‐2 receptor), β2‐microglobulin and the cytokine tumour necrosis factor‐alpha (TNF‐α) were measured in sera from patients with common variable immunodeficiency (CVI). Levels of soluble CD8, soluble CD25 and β2‐microglobulin but not of soluble CD4 and TNF‐α were raised significantly above levels in normal sera. Sera from patients with X‐linked agammaglobulinaemia, who are also antibody deficient, did not show this marked elevation. The raised levels of soluble CD8, soluble CD25 and β2‐microglobulin in CVI, correlated with the extent of the defects in the B lymphocytes assessed in vitro, as well as with the clinical severity of the disease. The selective release of these molecules into sera may indicate that abnormal cellular activation occurs in most CVI patients. It is also possible that the raised levels of these soluble molecules play a part in the immunodeficiency.

Co-Stimulation with anti-CD28 (Kolt-2) enhances DNA synthesis by defective T cells in common variable immunodeficiency

In normal T cells, an ann‐CD28 MoAb (Kolt‐2) will synergize with the mitogenic stimuli Phytohaemagglutinin (PHA). anti‐CD3 (OKT3) or a combination of anti‐CD2 antibodies (OKT11 and GT2) in the induction of DNA synthesis. A subgroup of patients with common variable immunodeficiency (CVID) show a defect in DNA synthesis by T cells stimulated in vitro with the above mitogens. We have now investigated whether anti‐CD28 will correct the defect. This strategy partially restored DNA synthesis, providing evidence that the CD28 co‐stimulatory pathway in CVID T cells is normal. Ligation of CT28 acts through co‐stimulating IL‐2 secretion. The natural ligand (B7) for CD28 on antigen‐presenting cells from CVID patients is expressed normally. We conclude that the defect in CVID T cells lies in pathways that lead to transcription of the IL‐2 gene other than that induced by ligation of CD2K with Kolt‐2.

Role of interleukin-2 and interleukin-6 in the mitogen responsiveness of T cells from patients with ‘common-variable’ hypogammaglobulinaemia

We have assessed the ability of interleukin‐2 (IL‐2) and interleukin‐6 (IL‐6) to augment the proliferative response of T lymphocytes from ‘common‐variable’ hypogammaglobulinaemia (CVH) patients and from normal controls, to the mitogens phytohaemagglutinin (PHA) and OKT3. We show that with cells from the control group and from those patients whose T cells respond to PHA within the control range, both IL‐2 and IL‐6 will significantly augment the response to OKT3. However, in those patients with a T cell defect in which the PHA response is below the control range, neither IL‐2 nor IL‐6 could restore the PHA or OKT3 response to normal. Responses to IL‐2 or IL‐6 alone were always in or above the control range.