Genes and Immunity (2010) 11, 523–530 & 2010 Macmillan Publishers Limited All rights reserved 1466-4879/10 www.nature.com/gene
ORIGINAL ARTICLE
B-cell maturation and antibody responses in individuals carrying a mutated CD19 allele H Artac1, I Reisli1, R Kara1, I Pico-Knijnenburg2, S Adin-C ¸ inar3, S Pekcan1, CM Jol-van der Zijde4, 4 2 2 MJD van Tol , LE Bakker-Jonges , JJM van Dongen , M van der Burg2 and MC van Zelm2 Department of Pediatric Immunology and Allergy, Meram Medical Faculty, Selc¸uk University, Konya, Turkey; 2Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherlands; 3Department of Immunology, Institute for Experimental Medicine (DETAE), Istanbul University, Istanbul, Turkey and 4Department of Pediatrics, Leiden University Medical Center, Leiden, The Netherlands 1
Homozygous CD19 mutations lead to an antibody deficiency due to disruption of the CD19 complex and consequent impaired signaling by the B-cell antigen receptor. We studied the effects of heterozygous CD19 mutations on peripheral B-cell development and antibody responses in a large family with multiple consanguineous marriages. Sequence analysis of 96 family members revealed 30 carriers of the CD19 mutation. Lymphocyte subset counts were not significantly different between carriers and noncarriers in three different age groups (0–10 years; 11–18 years; adults). B cells of carriers had reduced CD19 and CD21 median expression levels, and had reduced proportions of transitional (0–10 years) and CD5 þ B cells (adults). CD19 carriers did not show clinical signs of immunodeficiency; they were well capable to produce normal serum Ig levels and had normal responses to primary and booster vaccinations. The frequency of mutated Vk alleles was not affected. Heterozygous loss of CD19 causes some changes in the naive B-cell compartment, but overall in vivo B-cell maturation or humoral immunity is not affected. Many antibody deficiencies are not monogenetic, but likely caused by a combination of multiple genetic variations. Therefore, functional analyses of immune cell function should be carried out to show whether heterozygous mutations contribute to disease. Genes and Immunity (2010) 11, 523–530; doi:10.1038/gene.2010.22; published online 6 May 2010 Keywords: CD19; CD21; B cell; antibody deficiency; vaccination response; heterozygous mutation
Introduction Primary antibody deficiency syndromes are a heterogeneous group of disorders in which the fundamental defect is an inability to produce effective antibody responses to pathogens.1,2 Consequently, the patients are highly susceptible to infections with encapsulated bacteria, and can have a high incidence of bronchitis and pneumonia, which often lead to progressive and irreversible lung damage. Initial diagnosis and subdivision into three categories is based on the reduction of serum immunoglobulin (Ig) levels in combination with the number of B cells in peripheral blood:1,2 (1) patients with strongly reduced B-cell numbers and serum Ig levels are defined as agammaglobulinemic; (2) patients with normal B-cell numbers, normal to high IgM, but severely reduced IgG and IgA suffer from a Hyper-IgM syndrome; and (3) patients with low to normal B-cell numbers and strongly reduced IgG, and IgA or IgM Correspondence: Dr MC van Zelm, Unit Molecular Immunology, Department of Immunology, Erasmus MC, University Medical Center, Dr Molewaterplein 50, 3015 GE Rotterdam, The Netherlands. E-mail:
[email protected] Received 6 January 2010; revised and accepted 16 March 2010; published online 6 May 2010
are diagnosed with a ‘common variable immunodeficiency disorder’ (CVID). In the last two decades, multiple gene defects have been identified that underlie these types of antibody deficiencies.2,3 In about 90% of patients diagnosed with agammaglobulinemia and about 75% of cases with a Hyper-IgM syndrome, the underlying genetic defect has been identified.2 Whereas mutations have been described in patients diagnosed with CVID,4–12 in over 90% of these patients no associated genetic defect has been found. In fact, in most CVID patients a complex genetic trade rather than a single affected gene is likely to contribute to development of the disease. For a full understanding, it will be essential to determine the functional effects of each associated genetic variant. Homozygous CD19 mutations can underlie an antibody-deficiency syndrome.5,6 CD19 forms a complex with CD21, CD81 and CD225, and functions to lower the threshold for B-cell antigen receptor (BCR) signaling following antigen engagement.13 Consequently, CD19deficient and CD81-deficient patients were found to be defective in BCR stimulation and showed poor responses to vaccination.5,12 We identified a Turkish immunodeficient boy who is a cousin of the first identified Turkish CD19-deficient patient, and was homozygous for the same CD19 gene mutation (c.972insA). Because the two patients were
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born in large families with consanguineous marriages, we assumed that many of the family members would be carriers of the CD19 mutation, and consequently have reduced protein expression.5 We hypothesized that CD19 expression levels regulate BCR signaling and, therefore, we set out to study peripheral B-cell development and antibody responses in carriers of the CD19 mutation.
Results CD19 mutation analysis and clinical evaluation We identified a CD19 deficiency in a 12-year-old Turkish boy with an antibody deficiency (patient ID558). CD19 mutation analysis showed homozygosity for the c.972insA mutation, which was found previously in a Turkish girl (patient ID037).5 Subsequent analysis of the family history revealed that both children were related and born in large families with consanguineous marriages (Figure 1). To study the inheritance of the mutation, we selected 96 family members: 47 children (29 girls, mean age 10.3±4.3 years) and 49 adults (26 women, mean age 34±11.9 years). None of the subjects were homozygous for the mutation; 30 were heterozygous for the mutation (15 children, 15 adults). There were no significant differences in age and gender between the carriers and noncarriers. The 30 subjects who inherited the c.972insA mutation belonged to different branches of the large family tree (Figure 1). Therefore, it is likely that multiple family members in generation III carried the mutation, and potentially even several in generation I. We concluded that the mutation originated before the indicated generation I and has been propagated over time throughout a large family in multiple individuals. Clinical evaluation of all 96 individuals revealed recurrent upper and lower respiratory infections in 22 subjects (18 noncarriers and 4 carriers), but none of the 22 individuals were finally diagnosed with an immunodeficiency. Two carriers had asthma and one had experienced autoimmune hemolytic anemia. On the basis of the clinical evaluation of 30 carriers, it was concluded that the heterozygous CD19 mutation is not directly associated with clinical signs of an antibody deficiency. Blood B-cell compartment Flow cytometric immunophenotyping of blood samples showed that total leukocyte, total lymphocyte, CD4 þ T-cell, CD8 þ T-cell, NK cell and B-cell counts of all 96 subjects were within the normal ranges for age (data not shown). To compare leukocyte and lymphocyte subset numbers between carriers and noncarriers, we divided all subjects into three age groups (Supplementary Table 1): 0–10 years (group 1), 11–18 years (group 2) and adults (group 3). The total leukocyte, total lymphocyte, CD4 þ T-cell, CD8 þ T-cell, NK cell and B-cell counts were not significantly different between carriers and noncarriers for all three age groups. Therefore, similar to homozygous loss of CD19, heterozygous loss of CD19 does not appear to grossly affect lymphocyte subset numbers. The effects of heterozygous loss of CD19 on the CD19 complex were studied by flow cytometric analysis of CD19 and CD21 expression levels on B cells in all 96 subjects (Figure 2a). CD19 median expression levels were significantly reduced in carriers (2.4-fold; Po0.0001) as Genes and Immunity
compared with noncarriers as determined with two different monoclonal CD19 antibodies. Furthermore, CD21 median expression levels were 1.3-fold reduced in carriers as compared with noncarriers (Po0.0001). These results clearly show that two normal CD19 alleles are required to generate normal CD19 protein levels and subsequent CD19 complex formation with normal CD21 expression. CD19-deficient individuals were shown to have altered blood B-cell compartments. Specifically, CD5 þ , IgD þ memory and IgD memory B-cell subsets were found to be reduced.5 In addition, flow cytometric immunophenotyping was carried out to study the relative frequencies of transitional, naive mature, natural effector, memory B cells and CD5 þ B cells (Figure 2 and Supplementary Table 1). No significant differences were found between carriers and noncarriers for natural effector and memory B cells. Apparently, efficient memory B-cell formation can take place despite one mutated CD19 allele. Within the naive B-cell compartment, several differences were observed. Both transitional and CD5 þ B cells showed a trend to be reduced in carriers of all three age groups (Figure 2). These differences were significant for transitional B cells in group 1 (0–10 years) and CD5 þ B cells in group 3 (adults). All transitional B cells are CD5 þ . Therefore, these results suggest that both homozygous and heterozygous CD19 mutations affect the transitional B-cell homeostasis. Considering that similar trends were observed in all three age groups, these effects appear to be age independent. Serum Ig levels To study whether the reduced CD19 expression on blood B cells from carriers affected serum Ig levels, we compared the serum IgG, IgA and IgM levels between carriers and noncarriers for the three different age groups (Figure 3). The median IgG levels were slightly lower for carriers as compared with noncarriers in all three age groups, but this difference was not significant. IgA and IgM serum levels were not different between carriers and noncarriers in age groups 1 and 3. However, carriers of group 2 had significantly increased serum IgA (2.3 g l1) as compared with noncarriers (1.6 g l1; P ¼ 0.017), whereas IgM serum levels were slightly reduced in carriers of group 2 (1.1 g l1) as compared with noncarriers (1.6 g l1; P ¼ 0.057). Despite the reduced CD19 expression levels, carriers of CD19 gene mutations are well capable of serum Ig production. Somatic hypermutation analysis Despite differences in the B-cell compartment, carriers of CD19 mutations are seemingly as capable as noncarriers in generation of memory B cells and serum Ig levels. To study whether the molecular processes initiated upon antigen recognition by B cells are normal, we studied somatic hypermutation (SHM) levels. The frequency of mutated Vk alleles was studied in the blood of nine noncarriers and six carriers (age range 11–17 years) using the Igk restriction enzyme hot-spot mutation assay.12 In all individuals, a large proportion of the expressed Vk3–20 alleles was mutated and these values were within the range of healthy controls as described by Andersen et al.12 with a median of 70% for noncarriers and 76% for carriers (Figure 4). The frequency of mutated
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Figure 1 Pedigrees of the family. (a) Common ancestry of family members to whom the CD19 c972insA could be traced back to. (b–k) Individual family trees of pedigrees from individuals of generation III. All individuals of generation III in panel (a) are identified by numbers, which are consistently used in all other panels. Solid symbols denote CD19-deficient patients, half-solid symbols denote known carriers of the mutation and N indicates family members who are shown not to be carriers.
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Humoral immunity in carriers of a CD19 mutation H Artac et al 10000 p
