Age dependency and mutual relations in T and B lymphocyte abnormalities in common variable immunodeficiency patients

Authors


Mgr. Marcela Vlková, Department of Clinical Immunology and Allergology, St Anne's University Hospital, Masaryk University, Brno, Czech Republic.
E-mail: marcela.vlkova@fnusa.cz

Summary

Common variable immunodeficiency (CVID) is primary hypogammaglobulinaemia with an unknown aetiopathogenesis. Although various abnormalities of T and B cells have been described, their pathogenetic roles are unclear. We determined T and B lymphocyte subsets known to be abnormal in CVID in order to disclose possible relations between numerical abnormalities in those cells. Markers associated with B cell development (CD21, CD27, IgM, IgD) were determined on B lymphocytes (CD19+); T lymphocyte development (CD45RA, CD45RO, CD62L) and activation markers (CD25, CD27, CD28, CD29, CD38, CD57, HLA-DR) were determined on CD4+ and CD8+ T lymphocytes in 42 CVID patients and in 33 healthy controls. Abnormalities in CD4+ T lymphocyte activation markers (increase in CD29, HLA-DR, CD45RO, decrease in CD27, CD62L, CD45RA) were observed particularly in patients with a decreased number of memory (CD27+) and mature (CD21+) B cells (group Ia according to the Freiburg group's classification), while abnormalities observed in CD8+ cells (increase in CD27 and CD28 and decrease in HLA-DR, CD57 and CD38) did not depend upon grouping patients together according to B lymphocyte developmental subpopulations. We observed correlations between immature B cells (IgM+ CD21) and expression of CD27, CD62L, CD45RA, CD45RO and HLA-DR on CD4+ T cells in CVID patients but not in the control group. The expression of CD27 and CD45RA on CD4+ T lymphocytes, such as the percentage of IgD+ CD27 and IgD+ CD27+ cells in B lymphocytes, showed age dependency to be more significant than in the control group. Our study demonstrates that T and B lymphocyte abnormalities in CVID are partially related to each other. Some of those abnormalities are not definite, but may evolve with age of the patient.

Introduction

Common variable immunodeficiency (CVID) is a primary humoral immunodeficiency disease characterized by impaired antibody response; immunodeficiency manifests in people older than 2 years of age. Decreased levels of IgG, IgA and sometimes also IgM are observed in these patients [1]. They suffer from recurrent and complicated infections, specifically of the respiratory tract. Other frequent complications include autoimmune diseases (22% in one study) [2]. The incidence of malignancies is also increased with a standardized incidence ratio for all malignancies of 1·8 [3]. The aetiopathogenetic mechanisms leading to the disease are probably heterogeneous, causing variable clinical and laboratory patterns in affected individuals.

Previous studies have shown various abnormalities in both T and B lymphocytes. Defective T cell activation [4–6] and impaired cytokine production have been described [7–9]. A plethora of T subpopulation abnormalities, including reduced expression of CD45RA and CD62L or increased expression of CD45RO, HLA-DR and CD57, have been reported [10–13].

Determination of B lymphocyte subpopulations showed that the number of circulating B cells was normal or reduced. Recent results have shown that CVID patients have a reduced number of memory CD27+ IgDIgM B cells [14,15]. A new classification of CVID based on flow-cytometric analysis of B cell subpopulations was proposed by the Freiburg group [16], who divided CVID patients into four groups and subgroups: group I comprises patients with a decreased number of switched memory B cells [below 0·4% of total peripheral blood lymphocytes (PBLs)] and group II includes all patients with a normal number of switched memory B cells (> 0·4 of PBLs). Group I was subdivided into group Ia with an increased number of immature B cells (> 20% of CD19 number IgM+ CD21) and group Ib with a normal number of IgM+ CD21 immature B cells. Group III had less than 1% B cells of PBLs. This approach was accepted by other groups [17].

The above-mentioned abnormalities of both T and B lymphocytes are not present in all CVID patients. Very little is known about relations between T and B cell abnormalities in CVID. Only Piqueras et al. have shown that T lymphocyte markers associated with chronic stimulation of CD4+ cells (increase in HLA-DR, CD95 and decrease in CD45RA), such as expansion of HLA-DR and CD57 on CD8+ cells, were less up-regulated in CVID patients with a normal number of memory (IgDCD27+) cells compared to those with a decreased proportion of CD27+ cells (both IgD and IgD+) [18]. It is not clear which of the T and B cell abnormalities are possibly causative, which are secondary, and which are only epiphenomena. A more detailed analysis of mutual relations between numbers of T and B cells in various developmental stages, together with markers associated with different stages of activation, might help in elucidating mechanisms leading to T–B cells imbalance in CVID. For this purpose we determined those T and B cell subpopulations that were most frequently abnormal in CVID patients.

Patients and methods

Patients and controls

B and T cell subpopulations of 42 CVID patients (aged 10–75 years, mean 43·7 ± 15 years; 28 females, 13 males) and 33 healthy controls (aged 21–59 years, mean 37·5 ± 10·7; 7 males, 26 females) were examined. Twenty-three patients fulfilled the European Society for Immunodeficiencies (ESID) diagnostic criteria for CVID [1], including low isohaemagglutinin levels or decreased response to specific antigen stimulation. In 19 patients, namely those whose treatment was initiated before 1996 (the introduction of relevant tests in our laboratory), the diagnosis was made by low immunoglobulin levels, clinically significant immunodeficiency and exclusion of other causes of hypogammaglobulinaemia. The patients were on regular immunoglobulin substitution by intravenous immunoglobulin (i.v. IG) in 38 patients, dose 250–520 mg/kg/3–4 weeks; four patients were on intramuscular immunoglobulin at a dose of 1·2 g/1–2 weeks.

After informed consent was obtained, blood samples were collected before the i.v. IG infusion was given. Healthy donors were studied in parallel with the patients.

B cell analysis

Peripheral blood mononuclear cells (PBMC) were isolated from 10 ml of heparinized blood by density gradient centrifugation (Lymphoprep; Nycomed Pharma AS, Oslo, Norway), washed twice with phosphate-buffered saline and resuspended in RPMI-1640 (Sigma R0883) supplemented with 10% fetal calf serum.

T cell analysis

Five millilitres of peripheral blood were taken into ethylenediamine tetraacetic acid (EDTA) tubes and stained using a standard procedure by direct immunofluorescence. In brief, 50 µl of the blood was treated with 5–10 µl of the appropriate antibodies for 30 min at laboratory temperature in the dark. The stained blood was then processed by the Multi-Q-Prep workstation (Beckman Coulter, Miami, FL, USA) for automated lysis, stabilization and fixation of the sample.

Monoclonal antibodies and flow cytometry

Two commercial cocktails of monoclonal antibodies (mAb) for basic lymphocyte population analysis were used: (1) anti-CD45FITC, anti-CD4PE, anti-CD8ECD and anti-CD3PC5; and (2) anti-CD45FITC, anti-CD56PE, anti-CD19ECD, and anti-CD3PC5; (Cyto-Stat tetraChrome, Beckman Coulter). Monoclonal antibody anti-CD16PE (Immunotech, Marseille, France) was added to the second cocktail.

In addition, the following mAbs directed against leucocyte antigens were used: fluorescein isothiocyanate (FITC)-conjugated anti-CD25, CD27, CD28, CD29, CD57, CD62L, phycoerythrin (PE)-conjugated anti-CD38, CD45RA, CD45RO, R-Phycoerythrin-Cyanine 5 (PC5)-conjugated anti-CD3 (all from Dako A/S, Glostrup, Denmark); PC5-conjugated anti-CD27, ECD (phycoerythrin-Texas Red X)-conjugated anti-CD4, CD8, CD19 (all from Beckman Coulter); FITC-conjugated anti-CD38, PE-conjugated anti-IgD and anti-CD21, PC5-conjugated anti-IgM (all from Pharmingen International, San Diego, CA, USA) and PE-conjugated anti-HLA DR (Becton Dickinson, San Jose, CA, USA).

Phenotyping was performed by four-colour cytometry Epics MCL (Beckman Coulter).

Statistical analysis

The differences between the groups were evaluated by analysis of variance (anova) followed by Dunnet's (comparison between patients and control group) or Newman–Keul's (general comparison between groups of patients) contrasts. The results were confirmed using non-parametric Mann–Whitney test. P-values less than 0·05 were considered statistically significant in all statistical analyses. Student's t-test and Fisher's exact test were used when appropriate.

Correlations between variables were evaluated using Spearman's correlation coefficient (Rs) and the results were revised by Bonferroni's correction for each group of lymphocytes: CD4+, CD8+ and CD19+ [19]. The statistical package statistica (StatSoft, Inc., Tulsa, OK, USA) version 7 was used.

Results

Fluorescence-activated cell sorter (FACS)-based classification of CVID patients according to B cells

We have classified our patients into four groups in accordance with the Freiburg group's classification [17]. Six patients had B cells less than 1% of PBLs (group III). The remaining 36 patients were divided into the following groups: 11 patients belonged to group Ia, 15 to group Ib and 10 to group II (Table 1). The clinical characteristics of patients belonging to these subpopulations are given in Table 2. We observed a higher incidence of splenomegaly in groups Ia and III compared with group II (Ia versus II: P = 0·03, III versus II: P = 0·04, Fisher's exact test). We found significant differences in the patients’ age in groups Ia and Ib (P = 0·008, Student's t-test and P = 0·009, Mann–Whitney test) (Fig. 1). We did not find any significant differences in age between the remaining groups.

Table 1.  Percentage of B lymphocyte subpopulations in various subgroups of common variable immunodeficiency (CVID) patients; for definition of CVID subgroups see Introduction.
 CVID with B cells (mean ± s.d.)Ia (mean ± s.d.)Ib (mean ± s.d.)II (mean ± s.d.)IIIControl (mean ± s.d.)
  • §

    Statistically significant difference from the control group (P < 0·05) shown in bold type; n.a. = not applicable.

No. of patients38111510 633
%CD19 9·4 ± 5·6 6·7 ± 3·3 9·4 ± 5·012·9 ± 6·9< 1 9·5 ± 3·7
%IgD+ CD27of CD19+75·7 ± 18·0§81·0 ± 10·8§87·1 ± 7·2§56·4 ± 16·1n.a.57·4 ± 13·3
%IgD+ CD27+ of CD19+13·0 ± 13·1§10·5 ± 9·56·5 ± 4·1§25·5 ± 17·7n.a.18·1 ± 6·8
%IgDCD27+ of CD19+4·7 ± 7·2§1·6 ± 1·6§1·8 ± 2·0§ 9·0 ± 2·8§n.a.18·7 ± 6·9
%IgM+ CD21 of CD19+20·4 ± 15·2§35·8 ± 13·0§10·2 ± 4·220·4 ± 15·0§n.a. 6·4 ± 3·3
Table 2.  Clinical characteristics of common variable immunodeficiency patients.
 IaIbIIIII
Number of patients1115106
Male/female4/72/136/42/4
Age50·2 ± 11·435·8 ± 13·548·3 ± 18·044·7 ± 17·8
Age of diagnosis30·4 ± 12·922·0 ± 13·733·0 ± 18·631·0 ± 12·0
Splenomegaly10945
Lymphadenopathy4841
Autoimmunity2133
Figure 1.

Age distribution in common variable immunodeficiency subgroups.

Age-dependent variation of lymphocyte subsets in CVID

Because of the above-mentioned differences in the age of patients in groups Ia and Ib, correlations between T lymphocyte and B lymphocyte markers and the age in both patient and control groups were performed. The percentage of naive B cells (IgD+ CD27) and IgM memory B cells (IgD+ CD27+) was age-dependent in the CVID group, such as the percentage of CD27+, CD45RA+ and CD45RO+ in CD4+ cells. The only significant age-dependent correlation in the control group was the percentage of CD45RO+ in CD4+ cells. Figure 2 shows that although the regression line both in patients and controls always has the same direction, in CVID patients the slopes are greater.

Figure 2.

Plots of significant correlations between age and evaluated lymphocyte subpopulations in common variable immunodeficiency patients. Spearman's rank correlation (R) was used. Using Bonferroni's correction only values of P < 0·002 were considered to be significant. No significant correlations were observed for all antigens expressed on CD8+ cells and for CD4+ cells expressing markers CD28, CD29, CD62L CD25 and HLA-DR. The only positive correlation observed in the control group was between the age and percentage of CD45RO+ CD4+/CD4+. Using Bonferroni's correction only values of P < 0·0001 were considered to be significant for the control group.

T lymphocytes

T lymphocyte subpopulations in various CVID subgroups are shown in Table 3. When analysing significant differences in T lymphocyte subpopulations in CVID subgroups compared to controls, we found that the abnormalities in CD8+ subpopulations scarcely depended on the CVID subgroups, as abnormalities in the percentage of CD27+, CD28+, CD38+ and CD57+ cells in the CD8+ subpopulation were observed in almost all groups investigated.

Table 3.  T lymphocyte subpopulations and expression of activation markers in various subgroups of common variable immunodeficiency (CVID) patients; see Introduction for definition of CVID subgroups.
 CVID with B cells (mean ± s.d.)1a (mean ± s.d.)1b (mean ± s.d.)II (mean ± s.d.)III (mean ± s.d.)Controls (mean ± s.d.)
  • §

    Statistically significant difference from the control group (P < 0·05) shown in bold type.

CD4+37·2 ± 9·6§40·3 ± 11·036·8 ± 10·0§34·2 ± 6·5§36·4 ± 8·9§47·2 ± 7·7
 %CD27+ of CD4+81·0 ± 13·6§68·1 ± 15·2§88·0 ± 7·284·5 ± 9·080·0 ± 9·7§89·6 ± 6·3
 %CD28+ of CD4+93·4 ± 5·9§92·7 ± 5·993·6 ± 4·493·8 ± 8·192·1 ± 8·396·7 ± 4·7
 %CD29+ of CD4+64·0 ± 17·0§72·0 ± 21·7§58·4 ± 15·463·6 ± 10·058·0 ± 17·749·9 ± 11·0
 %CD62L+ of CD4+64·0 ± 19·545·8 ± 18·6§73·7 ± 14·669·5 ± 12·672·2 ± 17·574·2 ± 10·9
 %CD45RA+ of CD4+24·9 ± 17·2§ 8·1 ± 6·9§37·3 ± 15·724·7 ± 10·8§21·1 ± 17·2§46·2 ± 13·9
 %CD45RO+ of CD4+77·4 ± 16·7§92·5 ± 7·6§67·9 ± 16·7§75·1 ± 12·3§76·5 ± 18·5§55·7 ± 14·3
 %CD25+ of CD4+4·1 ± 2·4§ 3·9 ± 2·7 4·4 ± 2·6§ 3·8 ± 1·8§ 2·0 ± 1·0 1·9 ± 0·8
 %HLA DR+ of CD4+15·0 ± 9·3§23·3 ± 10·5§10·7 ± 5·912·4 ± 6·3§11·1 ± 6·0§ 5·3 ± 3·0
CD8+32·1 ± 10·1§36·8 ± 9·0§32·7 ± 10·2§25·9 ± 8·8§43·9 ± 11·4§18·3 ± 5·1
 %CD27+ of CD8+46·3 ± 21·7§31·3 ± 16·0§52·6 ± 22·1§53·4 ± 19·829·2 ± 16·7§68·9 ± 18·0
 %CD28+ of CD8+38·9 ± 20·5§24·4 ± 11·8§43·9 ± 21·7§47·6 ± 19·0§22·7 ± 12·8§68·0 ± 19·4
 %CD29+ of CD8+78·8 ± 23·2§90·8 ± 9·965·9 ± 26·7§85·0 ± 19·685·5 ± 21·795·5 ± 4·3
 %CD62L+ of CD8+37·7 ± 19·130·5 ± 15·441·1 ± 23·140·8 ± 15·524·4 ± 15·142·7 ± 16·7
 %CD45RA+ of CD8+53·4 ± 14·647·5 ± 14·561·2 ± 12·348·2 ± 13·550·6 ± 20·559·0 ± 14·1
 %CD45RO+ of CD8+43·8 ± 13·649·4 ± 12·036·7 ± 11·948·2 ± 13·946·5 ± 19·939·8 ± 13·3
 %HLA DR+ of CD8+21·3 ± 15·8§26·6 ± 7·9§19·0 ± 21·918·8 ± 10·5§23·6 ± 12·5§ 7·1 ± 6·9
 %CD38+ of CD8+29·8 ± 16·6§34·1 ± 14·6§30·6 ± 19·3§23·7 ± 13·7§31·8 ± 13·1§11·3 ± 6·1
 %CD57+ of CD8+32·8 ± 14·3§41·2 ± 8·2§28·5 ± 16·8§29·8 ± 12·7§49·1 ± 10·2§16·2 ± 12·9

Conversely, the CD4+ subpopulation abnormalities differed according to CVID subgroups. Although the increase in CD45RO+ activated/memory CD4+ T cells was documented in all CVID subgroups, abnormalities in other T lymphocyte activation markers (increased in CD29, HLA-DR, decreased in CD27, CD62L, CD45RA) were observed specifically in group Ia and also the differences from the control group were more significant in the Ia subgroup than in the remaining groups.

Comparing the frequency of T cell abnormalities in various CVID subgroups, no differences were observed in subpopulations CD4+, CD28 of CD4+ or CD25 of CD4+. Statistically significant differences were observed most frequently comparing groups Ia and Ib; comparing other groups, the differences were relatively rare (see Table 4).

Table 4.  Statistical comparison of differences between common variable immunodeficiency subgroups as determined by two-sided binomial testing expression of defined T lymphocyte activation markers. The numbers in the table represent P-values for the difference in proportion of patients with abnormal values of particular T lymphocytes. Statistically significant difference between groups is shown in bold type.
 Ia/IbIa/IIIa/IIIIb/IIIb/IIIII/III
%CD27 of CD4+<0·0010·0270·0610·140·1420·902
%CD29 of CD4+  0·0320·1360·0830·5720·8760·571
%CD62L of CD4+  0·0130·0560·0830·6430·8150·884
%CD45RA of CD4+  0·0020·0270·4950·3070·0230·171
%CD45RO of CD4+  0·0020·0100·0240·5720·5340·902
% HLA-DR of CD4+  0·0430·1370·1860·6260·6801·000

Relations between T and B lymphocyte subpopulations in CVID patients

No significant correlations were observed between T cell activation markers and the percentage of memory CD27+ IgD+ B cells. When analysing relations between immature IgM+ CD21 B cell and T lymphocyte activation markers, we showed a correlation between CD27+, CD62L+, CD45RA+, CD45RO+ and HLA-DR+ of the CD4+ cells. Concerning CD8+ T cells, we detected correlations between IgM+ CD21 B cell and CD57+, such as HLA-DR+ subpopulations (see Table 5).

Table 5.  Correlations between the expression of maturation/activation markers on B and T lymphocytes. Spearman's rank correlation (R) was used. Using Bonferroni's correction only the values P < 0·0028 and P < 0·004 for CD4+ and CD8+ subpopulations (subsequently) were considered as significant (shown in bold type).
 IgM+21low of CD19IgD27+ of CD19
RPRP
CD4−0·0635820·712574 0·0919120·610953
 %CD27 of CD4+−0·6909070·0000030·2132350·233467
 %CD28 of CD4+−0·3137910·062366−0·0655080·717207
 %CD29 of CD4+ 0·4983590·001979 0·1674470·351638
 %CD62L of CD4+−0·7354400·0000000·1079550·549846
 %CD45RA of CD4+−0·6430270·0000230·1875780·295874
 %CD45RO of CD4+ 0·6288690·000040 0·1910250·286926
 %CD25 of CD4+−0·1307680·447141−0·1803290·315266
 % HLA-DR of CD4+ 0·5459810·000572 0·0093580·958778
CD8 0·2205920·196068−0·0477940·791687
 %CD27 of CD8+−0·4867440·002610 0·1092910·544886
 %CD28 of CD8+−0·4041180·014509−0·0802140·657229
 %CD29 of CD8+ 0·3361650·045009 0·0083560·963191
 %CD62L of CD8+−0·2921490·083813−0·1159760·520408
 %CD45RA of CD8+−0·2455600·148860−0·2663770·134019
 %CD45RO of CD8+ 0·2169880·203660 0·2025400·258305
 % HLA-DR of CD8+ 0·5240670·001036 0·0274090·879654
 %CD38 of CD8+ 0·1855860·278514−0·0755350·676107
 %CD57 of CD8+ 0·5893560·0001560·0173800·923524

Discussion

The goal of our study was to disclose relations between T and B cell abnormalities in CVID patients. Some lymphocyte abnormalities were observed in the majority of CVID patients almost independently of B cell-based (Freiburg group) classification: the CD8+ cells were not only increased in number, but were also activated as seen by increased expression of CD38, CD57 and HLA-DR and decreased expression in CD27 and CD28. CD8+ cells expressing CD57 and CD38 without expression of CD27 and CD28 were described as effector cells in viral infections [20–22]. Although, with the exception of chronic enteroviral meningoencephalitis, no well-defined viral infection was proved to be increased in CVID [23], perhaps common respiratory viruses [24] cause this stimulation.

A different situation was found in CD4+ T cells. We observed that these cells were activated predominantly in patients with decreased activated memory (CD27+) and increased immature B cells (IgM+ CD21) (group Ia according to the Freiburg group classification) [17]. The patients from this subgroup also had the sharpest change in T lymphocyte subpopulations compared to controls: decreased in CD27, CD62L, CD45RA and increased in CD29, HLA-DR, CD45RO of CD4+ cells. The change in these CD4+ lymphocyte markers indicates a shift to activated and memory phenotypes. Our observation is in accordance with the study by Piqueras et al., who showed that patients with normal numbers of memory (IgDCD27+) cells up-regulate lymphocytes less with activation markers associated with chronic stimulation of CD4+ cells (increased in HLA-DR, CD95 and decreased in CD45RA) and CD8+ cells (increased in HLA-DR and CD57) compared to patients with a decreased proportion of CD27+ cells [18].

However, CD4+ T lymphocyte abnormalities were also seen in other subgroups of CVID patients. CD25 expression on T lymphocytes was increased in groups Ib and II, while there were no significant changes in CD62L and CD29 antigen expression. CD25 is an early activation marker whose expression begins 4 h after activation [25], while CD29 is positive mainly on memory T cells long after previous activation [26]. CD62L (l-selectin) initiates entry of T cells into the peripheral lymph nodes; it is shed from the cell surface following lymphocyte activation [27]. Taken together, the early activation pattern in particular is seen on CD4+ lymphocytes in groups Ib and II.

We observed several correlations between CD4+ cell activation markers and differentiating markers of B lymphocytes. They were limited to the IgM+ CD21 subpopulation of immature B cells, which correlated with the expression of several T cell markers (decreased in CD27, CD62L, CD45RA and increased in CD29, HLA-DR, CD45RO). This shows that the extent of activation of CD4+ T cells in group Ia may be related to the number of immature B cells. These results can be explained by a more significant regulatory defect or by secondary stimulation of the immune system by the antigen load in immunodeficient patients.

Surprisingly, when comparing T lymphocyte abnormalities in various CVID subgroups we observed most differences between subgroups Ia and Ib, both of which are characterized by decreased numbers of memory (CD27+) B cells; moreover, the subgroup Ia was more similar to subgroups II (normal B cell subpopulations) and III (decreased number of B cells) than to Ib. We also showed that subgroup Ib was more similar to normal donors than the other subgroups. Because the number of IgM+ CD21 (immature B cells) is also increased in subgroup II, it seems that increased T cell activation accompanies disturbed maturation of B cells from immature to mature cells in CVID patients.

We showed that the decrease in memory-switched cells in CVID patients may be age-dependent, because the number of CD27IgM+ naive B cells decreased with age, while CD27+ IgM+ non-switched memory cells increased. This phenomenon was not observed either in our control group or in healthy people in another study [28]. Evaluating T lymphocyte subpopulations, only an age-dependent shift from CD45RA into CD45RO in CD4+ populations was observed in both CVID patient and control groups, while the decrease in CD27 in CD4+ populations was seen in CVID patients but not in controls. In another study a similar decrease in the proportion of CD4+ CD27+ in CD4+ cells was observed in older people (75–84 years) compared to young people (19–28 years) [29]. In our study the proportion of CD27+ CD4+ cells in the control group was similar, but the proportion of CD27+ CD4+ cells in middle-aged CVID patients was comparable to healthy older patients [29]. The increase in CD45RO+ and decrease in CD45RA+ CD4+ cells was more extensive in our CVID patients than in older people [28]. However, in other studies we found that the defect in functional activation of T cells [5,7] and the cytokine release (e.g. interleukin-4) in CVID patients was different from the outcome of the study of older people [29]. This shows that the observed subpopulation changes in CVID may not only be the results of ageing of the immune system.

Future studies of T and B cell subpopulations might help to define more effectively subgroups of CVID patients and disclose aetiopathogenetic mechanisms leading to a blockade of antibody production in various CVID subgroups.

Acknowledgement

This work was supported by grant no. NI/7981-3 from the Czech Ministry of Health.

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