Low numbers of regulatory T cells in common variable immunodeficiency: association with chronic inflammation in vivo

Abstract

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by defective immunoglobulin production and high frequency of bacterial infections, autoimmunity and manifestations of chronic inflammation. Abnormalities of CD4⁺CD25^highforkhead box P3 (FoxP3)⁺ regulatory T cells (T_reg) have been associated with autoimmune and inflammatory disorders, and we hypothesized that CVID might be characterized by T_reg abnormalities. CD3⁺ cells from patients and controls were analysed for the expression of FoxP3 mRNA by real time reverse transcription–polymerase chain reaction (RT–PCR). Peripheral blood mononuclear cells from CVID patients and controls were stained for T_reg markers, analysed by flow cytometry and compared to clinical characteristics. The main findings were: (i) CVID patients had significantly decreased expression of FoxP3 mRNA and decreased proportions of CD4⁺CD25^highFoxP3⁺ cells compared to controls; (ii) CVID patients with splenomegaly had even lower proportions of T_reg compared to other patients and controls; (iii) serum levels of the inflammatory marker neopterin were correlated negatively with the proportions of T_reg within the CVID population, while there was no significant association with bronchiectasis. We have demonstrated decreased proportions of T_reg in CVID patients, particularly in those with signs of chronic inflammation. Decreased proportions of T_Reg are suggested to be pathogenetically important in autoimmunity, and our results suggest that T_Reg may have a similar role in CVID.

Introduction

Common variable immunodeficiency (CVID) is a heterogeneous syndrome characterized by failure of B cell differentiation and defective immunoglobulin (Ig) production leading to recurrent bacterial infections, particularly in the respiratory tract. Although reduced Ig secretion from B cells is the hallmark of CVID, T cell abnormalities such as dysregulated cytokine production, impaired T cell proliferation in vitro and altered distribution of T cell subsets are seen in a considerable proportion of patients. These abnormalities may be of importance for both the B cell deficiency and for some of the clinical manifestations in these patients, which include autoimmunity and other manifestations of inflammatory and immunological hyperactivity such as splenomegaly and raised levels of inflammatory markers [1–3].

Several subgroups of T cells mediate regulatory mechanisms in the T cell immune response. The so-called regulatory T cells (T_reg) are a subgroup of CD4⁺ T cells characterized by the up-regulation of the interleukin (IL)-2 receptor α-chain (CD25) and expression of the transcription factor protein forkhead box P3 (FoxP3) [4,5]. The biological role of T_reg is thought to dampen immune responses through mechanisms which are not fully elucidated [4,6,7]. T_reg are now characterized in both mice and humans, and their role in various clinical conditions such as transplant rejection reactions, cancer, autoimmunity and chronic infectious diseases is a subject of active investigation [8–11]. We hypothesized that CVID, with its high frequency of autoimmunity and signs of chronic inflammation, might be characterized by T_reg abnormalities.

Materials and methods

Patients

Twenty-six patients with CVID, according to the criteria of the World Health Organization expert group for primary immunodeficiencies [1], attending our department were included consecutively in the study (Table 1). The patients did not have any clinically apparent infection when recruited, and did not receive treatment with antibiotics or corticosteroids. The diagnosis of splenomegaly was defined as spleen length > 13 cm on ultrasonographic or computed tomography (CT) scan examination and the diagnosis of bronchiectasis was based on typical findings on high-resolution CT scan of the thorax [12]. Eleven sex- and age-matched healthy individuals were included as controls. Informed consent for blood sampling was obtained from all subjects. The study was conducted according to the ethical guidelines at our hospital, which comply with the Helsinki declaration, and was approved by the hospital's authorized representative.

Table 1.

Characteristics of the study group.

	Controls (n = 11)	CVID (n = 26)	P-value
Demographics
Gender (male/female, %)	45/55	50/50	0·8
Age (years, median and 25th −75th percentiles)	45 (38–65)	49 (39–58)	0·92
Clinical characteristics
Bronchiectasis (%)		69
Splenomegaly (%)		58
Idiopathic thrombocytopenic purpura (%)*		34
Granulomatous disease (%)**		15
Therapy
SCIG/IVIG (%)***		62/38

^*Cases include any history of idiopathic thrombocytopenic purpura.

^**Cases include granulomatous disease as confirmed by biopsy.

^***Some patients on IVIG received supplementary SCIG. IVIG: intravenous immunoglobulin therapy; SCIG: subcutaneous immunoglobulin therapy.

Isolation of cells

Peripheral blood mononuclear cells (PBMC) were obtained from heparinized blood by Isopaque-Ficoll (Lymphoprep; Nycomed Pharma, Oslo, Norway) gradient centrifugation. For flow cytometry, peripheral blood mononuclear cells (PBMC) were cryopreserved in liquid nitrogen. Further separation of CD3⁺ T cells (negative selection by monodisperse immunomagnetic beads; Dynal, Oslo, Norway) was performed as described elsewhere [13,14]. The negatively selected T cells consisted of > 90% CD3⁺ cells.

Flow cytometry

Cryopreserved PBMC were thawed and stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4, phycoerythrin (PE)-conjugated anti-FoxP3 and allophycocyanin (APC)-conjugated anti-CD25 antibodies with appropriate isotype controls (all from eBiosciences, San Diego, CA, USA). Flow cytometry was performed using a fluorescence activated cell sorter (FACS)Calibur instrument with CellQuest software (Becton Dickinson, San Diego, CA, USA). List mode files were collected for 200 000 cells from each sample. CD25^high cells were defined as described elsewhere [15]. Cells from the lymphocyte gate was used for analysis and prior to permeabilization this gate contained less than 5% dead cells as measured by staining with propidium iodide.

Real-time quantitative reverse transcription–polymerase chain reaction (RT–PCR)

Total RNA was extracted from T cells using RNeasy columns (Qiagen, Hilden, Germany), subjected to DNase I treatment (RQI DNase; Promega, Madison, WI, USA) and stored at −80°C. Primers were designed using the Primer Express software, version 2·0 (Applied Biosystems, Foster City, CA, USA) for FoxP3 (forward primer: 5′-ACTGCCAGGCGGACCAT-3′, reverse primer: 5′-CTTCTCCAGCACCAGCTGCT-3′). Quantification of mRNA was performed using the ABI Prism 7000 (Applied Biosystems). Gene expression of the housekeeping gene α-actin (forward primer: 5′-AGGCACCAGGGCGTGAT-3′, reverse primer: 5′-TCGTCCCAGTTGGTGACGAT-3′) was used for normalization.

Cell cultures

Cell cultures were performed as described elsewhere [16]. Briefly, negatively selected T cells (10⁶ cells/ml, 0·2 ml/well; Costar, Cambridge, MA, USA) were stimulated with anti-CD3 (clone SpvT3b, 40 ng/ml; Dynal) and anti-CD28 (clone 15E8, 50 ng/ml; CLB, Amsterdam, the Netherlands) for 48 h and supernatants were analysed for interleukin (IL)-10 levels by enzyme immunoassay (R&D Systems, Minneapolis, MN, USA).

Statistical methods

For comparison of two groups of individuals, the Mann–Whitney U-test was used. When more than two groups of individuals were compared, the Kruskal–Wallis test was used, and if significant a Mann–Whitney U-test was performed comparing differences between each pair of groups. Coefficients of correlation were calculated by the Spearman's rank test. P-values are two-sided and considered significant when < 0·05.

Results

The proportion of T_teg in CVID patients and healthy controls

As shown in Fig. 1a, CVID patients (n = 25) had significantly decreased gene expression of the transcriptional factor FoxP3 in CD3⁺ cells compared to healthy controls (n = 11). This reduction of FoxP3 was extended and confirmed by flow cytometry, showing decreased proportions of CD4⁺CD25^highFoxP3⁺ cells in the CD4⁺ cell population of the CVID patients (n = 24)(Figs 1b and 2).

Fig. 1.

Expression of forkhead box P3 (FoxP3) mRNA in T cells (a) and proportions of CD4⁺CD25^highFoxP3⁺ cells in the CD4⁺ cell population (b) among healthy controls and common variable immunodeficiency patients.

Fig. 2.

CD4⁺CD25^high and CD4⁺CD25^highforkhead box P3 (FoxP3)⁺ cells in one representative healthy control (a, b, respectively) and in one representative common variable immunodeficiency (CVID) patient with splenomegaly (c, d, respectively) as measured by flow cytometry. Boxes (a, c) indicate cells in the CD25^high population. Quadrants (b, d) indicate CD25^highversus CD25^{low/intermediate} cells and FoxP3⁺versus FoxP3^– cells.

The proportion of T_reg in relation to clinical and immunological parameters in CVID

As shown in Fig. 3a, patients with splenomegaly had markedly decreased proportions of CD4⁺CD25^highFoxP3⁺ cells compared to both healthy controls and other CVID patients. Moreover, CVID patients with idiopathic thrombocytopenic purpura (ITP) had a decrease in the CD4⁺CD25^highFoxP3⁺ cell populations compared to other CVID patients, although the difference did not reach statistical significance (P = 0·086). CVID patients with granulomas as confirmed by biopsy also had a non-significant decrease in T_reg compared to patients without confirmed granulomatous disease (P = 0·373). In contrast, there was no association between the proportions of CD4⁺CD25^highFoxP3⁺ cells and the occurrence of bronchiectasis in the CVID population. Serum neopterin level is a reliable marker of monocyte/macrophage activation [17] previously shown to be elevated in CVID [18–20]. Herein we found that serum levels of neopterin were correlated negatively to the proportions of CD4⁺CD25^highFoxP3⁺ cells within the CVID group (Fig. 3b).

Fig. 3.

Proportions of CD4⁺CD25^highforkhead box P3 (FoxP3)⁺ cells in the CD4⁺ cell population among healthy controls and common variable immunodeficiency (CVID) patients with and without splenomegaly (a). Correlation between proportions of CD4⁺CD25^highFoxP3⁺ cells and serum levels of neopterin in CVID patients (b).

We have described previously impaired IL-10 release by T cells from CVID patients [16], and IL-10 levels in T cell supernatants of these patients correlated significantly with the proportion of CD4⁺CD25^highFoxP3⁺ cells found in the same patients in our study (r = 0·730, P = 0·046).

Discussion

In the present study we found decreased proportions of CD4⁺CD25^highFoxP3⁺ cells in the CVID group, particularly in patients with splenomegaly and raised levels of serum neopterin, indicating low numbers of T_reg in these patients.

While the co-expression of CD4 and CD25^high has been used widely as a phenotype marker for T_reg, the identification of FoxP3 now allows for a more strict and valid classification of T_reg even in clinical studies [21]. Previous studies on T_reg may have been confounded by a failure to separate regulatory cells that highly express CD25 from other CD4⁺CD25⁺ cells [22]. We believe that using the combination of CD4⁺CD25^high with FoxP3 as a marker of T_reg will reflect more accurately the true numbers of T_reg. In this study, the decreased proportions of CD4⁺CD25^highFoxP3⁺ cells in CVID patients compared to controls is supported by low expression of FoxP3 mRNA in CD3⁺ cells in the same patients.

CVID is a heterogeneous group of disorders with a variety of clinical and immunological characteristics, including signs of chronic inflammation. Lymphoid hyperplasia in the form of splenomegaly is common in CVID [2], particularly in those subgroups of CVID characterized by low numbers of memory B cells [23,24], and often in association with raised levels of inflammatory cytokines [25,26]. Interestingly, we find even lower proportions of T_reg in CVID patients with splenomegaly compared to both healthy controls and other CVID patients. Moreover, the negative correlation between levels of neopterin, reflecting the degree of monocyte/macrophage activation [17], and proportions of T_reg further supports a link between chronic inflammation and a decrease of T_reg in these patients. We have described previously a subgroup of CVID patients with chronic inflammation in vivo characterized by splenomegaly, signs of T cell dysfunction and raised neopterin levels [19,25,27,28] and our findings in this study suggest that low numbers of T_reg could be added to the list of immunological features characterizing this group of CVID patients.

The reason for the low numbers of T_reg in CVID patients with splenomegaly is unclear; while low numbers of circulatory CD4⁺CD25^high cells have been observed in some other diseases [10], even without splenomegaly, this is less well documented for CD4⁺CD25^highFoxP3⁺ cells [29,30]. The homeostasis of T_reg is probably controlled by several factors, among them the differentiation of T_reg in the thymus, induction and/or expansion of T_reg in the periphery, half-life of T_reg in the circulation and possible redistribution of T_reg to extra-vascular tissue [31]. We can only speculate which factors contribute to our finding, but redistribution of T_reg to the spleen is one of several possible explanations.

Both ITP and granulomatous disease are common complications of CVID, and although the decrease of T_reg in these groups of patients did not reach statistical significance we cannot exclude a possible association. Interestingly, we found no correlation between the numbers of T_reg and the occurrence of bronchiectasis, suggesting that the inflammatory burden imposed by the recurrent bacterial pulmonary infections associated with this condition is different from the chronic inflammation associated with splenomegaly.

The effector mechanisms of T_reg are not clear, but cell contact appears to be of major importance. However, although the role of soluble cytokines has not been established firmly, IL-10 and transforming growth factor-α have been suggested to be involved in the T_reg-mediated regulation of T cell responses in vivo [4,6,7]. We have reported previously decreased secretion of IL-10 in T cell cultures from CVID patients [16], and our finding in the present study with a correlation between secreted IL-10 levels in T cell cultures and T_reg numbers in CVID may further support a possible link between T_reg and IL-10.

The present study is, to our knowledge, the first demonstration of decreased proportions of T_reg in CVID patients, showing a particularly low percentage in those with signs of chronic inflammation in vivo. Decreased proportions of T_reg and a FoxP3 defect have been suggested to be pathogenetically important in autoimmunity [10]. Our findings in the present study, showing markedly decreased proportions of CD4⁺CD25^highFoxP3⁺ cells in CVID patients with splenomegaly and a negative correlation to neopterin levels, suggest that T_reg may have a similar role in subgroups of this immunodeficiency. However, further studies are needed to clarify whether this T_reg abnormality contributes to the pathogenesis of CVID.