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Blood, 1 April 2005, Vol. 105, No. 7, pp. 2887-2890. Prepublished online as a Blood First Edition Paper on December 14, 2004; DOI 10.1182/blood-2004-06-2423.
IMMUNOBIOLOGY Abnormal interleukin-7 function in common variable immunodeficiencyFrom the Section of Clinical Immunology and Infectious Diseases, Medical Department; the Research Institute for Internal Medicine; and the Department of Microbiology, Rikshospitalet/The National Hospital, Oslo, Norway.
Common variable immunodeficiency (CVID) is characterized by low levels of circulating immunoglobulins, leading to frequent infections, particularly of the respiratory tract. Frequently, T-cell abnormalities are observed. Interleukin-7 (IL-7) is involved in the homeostasis of lymphocytes, and may be elevated in lymphopenia. Mutations of genes related to IL-7 may lead to severe immunodeficiency disorders. We report elevated plasma levels of circulating IL-7 in a subgroup of CVID. These patients have increased numbers of circulating CD8+ T cells with decreased apoptosis and a predominance of CC chemokine receptor 7- (CCR7-) effector-memory T cells. Moreover, in some of these patients there is impaired response to IL-7 as assessed by in vitro proliferation and secretion of interferon  and transforming growth factor  . These findings suggest novel pathogenic mechanisms and specific targets for further research in CVID.
Common variable immunodeficiency (CVID) is a heterogeneous group of immunodeficiency disorders,1 characterized by deficient B-cell function. However, about half the cases show signs of T-cell deficiency,2 possibly contributing to the defective antibody production.
Interleukin-7 (IL-7) plays a critical role in lymphocyte development and homeostasis.3 High plasma levels of IL-7 have been correlated with low CD4+ T-cell counts (eg, in HIV infection), reflecting a homeostatic response to T-cell depletion.4-6 However, high IL-7 levels may also lead to autoimmunity and ectopic lymphocyte infiltrates, as suggested in animal models examining the effects of exogenous IL-7 administration.7 Deficiencies of the IL-7 receptor Although IL-7 seems to be crucially involved in the function of human lymphocytes and defects in the IL-7 signaling system in humans have been described, the role of IL-7 in CVID has not been studied. Herein, we examine the expression of IL-7 and its role in T-cell function in CVID.
Patients Patients with CVID1 without clinically apparent infections were included (Table 1). Healthy donors were included as controls. Informed consent was obtained from all subjects before inclusion. The study was conducted according to the ethical guidelines at our hospital and the Helsinki declaration, and approved by the Rikshospitalet University Hospital's authorized representative.
Cell isolation and cultures
Negative selection of CD3+ T cells was performed as previously described.9 The CD3+ T cells were resuspended in RPMI 1640 with 2 mM L-glutamine and 25 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer (Gibco, Paisley, United Kingdom) supplemented with 10% fetal calf serum (FCS) and incubated in 96-well plates (106 cells/mL). The cells were stimulated with anti-CD3 (SpvT3b; final concentration, 40 ng/mL for optimal stimulation; suboptimal stimulation, 10 ng/mL; Zymed, San Francisco, CA), anti-CD28 (clone 15E8; CLB, Amsterdam, the Netherlands; final concentration 50 ng/mL for optimal stimulation; not added for suboptimal stimulation) with and without IL-7 (R&D Systems, Oxon, United Kingdom) and cross-linked.10 For cytokine and lactate-dehydrogenase (LDH) analyses, cell-free supernatants were harvested after 24 (interferon Flow cytometry
Absolute numbers of circulating lymphocyte subsets were assessed using TruCount and TriTEST (Becton Dickinson, San Diego, CA). Other staining for flow cytometry was performed using fluorescein isothiocyanate (FITC)-conjugated anti-CD4; phycoerythrin (PE)-conjugated anti-IL-7R Cytokine and LDH assay Cytokine levels were measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems). LDH activity was measured using Cytotoxicity Detection Kit (LDH; Roche-Applied-Science, Basel, Switzerland). Statistical methods Data are presented as median [25th-75th percentile]. The 2-tailed Mann-Whitney U test, Wilcoxon signed-rank test, chi-square test, and Spearman signed rank test were used as appropriate. Tests were considered significant when P is less than .05.
We found significantly higher plasma levels of IL-7 in CVID patients (n = 72) compared with healthy controls (n = 23), with particularly high levels in 13 patients (> 10 pg/mL; hereafter termed CVIDIL-7high; Figure 1A). Except for a higher proportion of males in the CVIDIL-7high group (P = .026), there was no association between IL-7 levels and clinical characteristics of the study group (Table 2). In contrast to previous reports in T-cell-depleted individuals, we did not find any correlation between IL-7 levels and CD3+ or CD4+CD3+ T-cell counts in CVID (Figure 1B). In fact, the 13 CVIDIL-7high patients had significantly higher CD8+ T-cell counts than the remaining patients (1043 [299-2040] vs 531 [353-722] CD3+CD8+ cells/mm3; P = .02). There were no different proportions of CD45RA+ T cells, but the predominance of CCR7- effector-memory T cells11,12 was significantly stronger in the CVIDIL-7high patients (88% [75%-90%] vs 68% [45%-90%] CCR7- of CD3+ cells; P = .01). There was no difference in the expression of the proliferation marker Ki67 between CVID and controls or between the CVIDIL-7high and the remaining patients, indicating that the increased numbers of CD8+ T cells were not due to increased proliferation in vivo. However, the proportion of apoptotic cells after in vitro isolation correlated inversely to IL-7 levels in the CVID patients (r = -0.080; P = .055), suggesting a protective effect of IL-7. There was no difference between CVID patients and controls in the expression of IL-7R  on T cells, and there was no association between in vitro responses of IL-7 in proliferation or cytokine assays and the expression of IL-7R.
In further in vitro analyses of 16 randomly selected CVID patients, we found an inverse correlation between IL-7 levels and in vitro T-cell proliferation in the CVIDIL-7high patients (r = -0.55, P = .034). While IL-7 had no effect on T-cell proliferation when given alone or in combination with optimal stimulation in patients or controls, it strongly enhanced proliferation in suboptimally stimulated T cells in healthy controls (Figure 1C). In contrast, while there was a heterogeneous response among CVID patients with normal IL-7 levels, none of the CVIDIL-7high patients reached similar levels (Figure 1C).
While the addition of IL-7 in vitro increased IFN
We found that T cells from CVID patients spontaneously secreted less TGF
Finally, the impaired responses to IL-7 in T-cell proliferation and IFN We describe a subgroup of CVID patients with high plasma levels of IL-7, associated with elevated numbers of CD8+ T cells and increased proportion of terminally differentiated effector-memory T cells11,12 that do not show signs of increased proliferation, but appear to have reduced apoptosis. Some of these patients also have reduced response to IL-7 in vitro. Our observations suggest abnormalities in IL-7-mediated lymphocyte homeostasis in some CVID patients, possibly involving mechanisms such as abnormal signaling pathways (similar to certain forms of SCID),8 or impaired feedback to IL-7-producing stroma cells due to primary T-cell defects.11,15-18 IL-7 also plays a role in APC-mediated T-cell stimulation.3 Thus, the IL-7 dysfunction may also be involved in the defective dendritic cell function recently described in CVID.19,20 Considering the role of IL-7 in both development and peripheral homeostasis of lymphocytes,3 it is conceivable that the abnormal IL-7 function as demonstrated in this study may be involved in the skewing of lymphocyte subsets and in the development of an impaired T- and B-cell function in some CVID patients.
Submitted June 28, 2004; accepted November 30, 2004.
Prepublished online as Blood First Edition Paper, December 14, 2004; DOI 10.1182/blood-2004-06-2423.
Supported by the Norwegian Research Council.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Are Martin Holm, Research Institute for Internal Medicine, The National Hospital, N-0027 Oslo, Norway; e-mail: a.m.holm{at}klinmed.uio.no.
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Abstract
Introduction
) and 23 healthy controls (
). Horizontal lines indicate median values (median (25%-75%) for CVID, 4.9 pg/mL [3.05-7.65 pg/mL] and for controls, 3.0 pg/mL [2.15-4.85 pg/mL]). (B) The absent correlation between plasma IL-7 and numbers of circulating T cells in CVID patients (n = 72). (C) The effects of IL-7 (10 ng/mL) on in vitro proliferation in suboptimally stimulated (anti-CD3 [10 ng/mL]) T cells from CVID patients with normal plasma levels of IL-7 (n = 10; 
2), there was a particularly strong response to IL-7 in suboptimally stimulated T cells in both CVID patients and controls (fold change, 
