Defective Self-Reactive Antibody Repertoire of Serum IgM in Patients with Hyper-IgM Syndrome

This information is current as Sébastien Lacroix-Desmazes, Igor Resnick, Dorothea Stahl, of September 25, 2021. Luc Mouthon, Teresa Espanol, Jacov Levy, Srini V. Kaveri, Luigi Notarangelo, Martha Eibl, Alain Fischer, Hans Ochs and Michel D. Kazatchkine J Immunol 1999; 162:5601-5608; ;

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Defective Self-Reactive Antibody Repertoire of Serum IgM in Patients with Hyper-IgM Syndrome1

Se´bastien Lacroix-Desmazes,* Igor Resnick,† Dorothea Stahl,* Luc Mouthon,* Teresa Espanol,‡ Jacov Levy,§ Srini V. Kaveri,* Luigi Notarangelo,¶ Martha Eibl,ʈ Alain Fischer,** Hans Ochs,†† and Michel D. Kazatchkine2*

We have analyzed the self-reactive repertoires of IgM and IgG Abs in the serum of 19 patients with hyper-IgM syndrome (HIM) by means of a quantitative immunoblotting technique that allows for a quantitative comparison of Ab repertoires in health and disease by multiparametric statistical analysis. Normal tissue extracts of liver, lung, stomach, and kidney were used as sources of self Ags. Extracts of Pseudomonas aeruginosa and Staphylococcus epidermidis were used as sources of nonself Ags. We demonstrate a significant bias in repertoires of reactivities of IgM of patients with HIM with self Ags. Ab repertoires of IgM toward nonself Downloaded from Ags did not differ, however, between patients and controls. No difference was found between IgM repertoires of untreated patients and those of patients receiving substitutive treatment with i.v. IgG. IgG in the serum of HIM patients lacked reactivity with self Ags, although it exhibited a pattern of reactivity with nonself Ags that was similar to that of IgG of healthy controls. The data demonstrate that functional CD40-CD40 ligand interactions are essential for the selection of natural self-reactive B cell repertoires. The Journal of Immunology, 1999, 162: 5601–5608.

mmunodeficiency with hyper-IgM syndrome (HIM)3 is a rare thrombocytopenia or other autoimmune manifestations such as he- http://www.jimmunol.org/ disease characterized by normal or increased serum concen- molytic anemia and nephritis (1, 19). An increased incidence of I trations of IgM with decreased or absent IgG, IgA, and IgE autoantibodies, including anti-erythrocyte, anti-platelet, anti- (1, 2). HIM results from defective interactions between CD40 li- thyroid, anti-nuclear, anti-cardiolipin, and anti-smooth muscle gand (CD40L) on activated T cells and CD40 on B cells. The Abs, has been reported in patients with HIM (19–23). X-linked form of HIM (X-HIM) is characterized by defective Natural Abs of the IgM, IgG, and IgA isotypes that are reactive CD40L due to deletions/insertions, point mutations, or truncation with a broad range of self Ags are present in normal serum (24– in the CD40L-encoding gene (3–12). Several in vivo and in vitro 26). Ab repertoires of natural IgM and IgG toward self Ags are

studies have documented that T cell-dependent isotype switch is highly homogeneous among healthy individuals and remain invari- by guest on September 25, 2021 strictly dependent on cognate interactions involving CD40 and ant with aging (27–30). Evidence obtained in mice suggests that CD40L and that impaired CD40-CD40L interactions inhibit the the selection of autoreactive B cells requires the presence of CD4ϩ development of germinal centers and the generation of B memory T lymphocytes under conditions of both pathological and physio- cells (13–17). Patients with HIM lack germinal centers in second- logical autoimmunity (31–34). ary lymphoid organs (18). The patients suffer from recurrent upper In the present study, we analyzed the Ab repertoires of IgM and and lower respiratory tract infections and also present with an un- IgG in the serum of patients with HIM by means of a quantitative usual susceptibility to Pneumocystis carinii pneumonia and Cryp- immunoblotting technique that allows for multiparametric statisti- tosporidium infection, suggesting impaired T cell functions. HIM cal analysis of Ab reactivities with self and nonself Ags. We dem- patients often present with persistent neutropenia and may develop onstrate that Ab repertoires of IgM toward self Ags are skewed in patients with HIM, whereas repertoires directed toward bacterial Ags do not differ between patients and healthy controls. In addi- *Institut National de la Sante´et de la Recherche Me´dicale (INSERM), Unit 430 and Universite´Pierre et Marie Curie, Hoˆpital Broussais, Paris, France; †Research Institute tion, little reactivity with self Ags of IgG in the serum of patients of Hematology, Moscow, Russia; ‡Immunology Unit, Vall d’Hebron, Barcelona, with HIM was detected. Our observations demonstrate that the Spain; §Department of Pediatrics, Soroka Medical Center, Beer Sheva, Israel; ¶De- ʈ lack of functional CD40-CD40L interactions and/or impaired T/B partment of Pediatrics, University of Brescia, Brescia, Italy; Institute of Immunology, University of Vienna, Vienna, Austria; **INSERM, Unit 429, Hoˆpital des Enfants- cell cooperation in HIM affect the selection processes of natural Malades, Paris, France; and ††Department of Pediatrics, University of Washington self-reactive B cell repertoires. Medical School, Seattle, WA 98195 Received for publication September 30, 1998. Accepted for publication February 12, 1999. Patients and Methods Patients The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance EDTA-plasma was obtained from 19 children (18 boys and 1 girl), between with 18 U.S.C. Section 1734 solely to indicate this fact. 3 and 14 years old, diagnosed with immunodeficiency with HIM. Twenty 1 This work was supported by Institut National de la Sante´et de la Recherche Me´di- healthy young adult male blood donors with a mean age of 34 Ϯ 5 years cale, France, and the Central Laboratory of the Swiss Red Cross (Bern, Switzerland). were used as normal controls. Previous studies from our laboratory had S.L.D. is a recipient of a grant from the Ministe`re de l’Education Nationale et de la demonstrated that self-reactive Ab repertoires in serum remain highly ho- Recherche, France. I.R. is a recipient of a grant from the European Union (PECO). mogeneous and invariant from early childhood to adulthood (29, 35). 2 Address correspondence and reprint requests to Dr. Michel D. Kazatchkine, IN- Sixteen of the male patients had X-HIM. Two male patients presented SERM U430, Hoˆpital Broussais, 96, rue Didot, 75014 Paris, France. with autosomal recessive HIM, and one female patient had secondary HIM 3 Abbreviations used in this paper: HIM, hyper-IgM syndrome; X-HIM, X-linked associated with congenital rubella. Activated PBMC totally lacked expres- HIM; CD40L, CD40 ligand; PCA, principal component analysis; LDA, linear dis- sion of the CD40L Ag in 11 X-HIM patients, whereas expression of the Ag criminant analysis; LDL, low density lipoprotein. was low in 3 patients (2 patients with X-HIM and 1 patient with autosomal

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 5602 SELF-REACTIVE REPERTOIRES OF IgM IN THE HYPER-IgM SYNDROME

recessive HIM) and unknown in the remaining 5 cases. All patients had corresponding to peaks of reactivity for concentrations of IgG between 50 suffered from severe and/or recurrent infections before diagnosis, including and 400 ␮g/ml and of IgM between 5 and 50 ␮g/ml. Saturation was upper respiratory tract infections (10 of 16), P. carinii pneumonia (2 of 16), achieved for concentrations of IgG and IgM above 400 and 50 ␮g/ml, and episodic or chronic infectious diarrhea (6 of 16 including Cryptospo- respectively. The reproducibility of the assay was 10% (variation coeffi- ridium infection in 3 cases). Hematological abnormalities included neutro- cient). The 95% confidence interval of the mean area under the curve penia in 11 patients and pure RBC aplasia in 2 patients due to parvovirus corresponding to each peak of immunoreactivity was 30% in the case of B19 infection, as previously reported (36). Nephritis was present in 1 pa- IgM and 25% in the case of IgG, as calculated by Student’s t test (27, 28). tient and arthritis in 2 patients (described in Ref. 1). Autosomal recessive HIM was diagnosed in 2 patients with low or normal expression of the Statistical analysis CD40L Ag, respectively, and suffering from upper respiratory tract infec- tions. One patient had secondary HIM associated with congenital rubella. Densitometric profiles of immunoblots were divided into sections corre- CD40L expression in this patient was normal, and the patient suffered sponding to individual peaks of immunoreactivity. Respective peak areas upper respiratory tract infections. Twelve of the 19 patients received sub- were calculated in the case of each tissue extract. To discriminate between stitutive therapy with i.v. Ig, in amounts ranging from 200 mg/kg/body individual repertoires, peak areas corresponding to sections obtained with weight every 5 wk to 600 mg/kg every 2 wk. Seven of the 19 patients had all self Ags were submitted simultaneously to principal component analysis not been treated with i.v. Ig at the time of serum sampling. (PCA) (38), using Mathematica (Wolfram Research, Champaign, IL) soft- The mean serum IgM concentration in HIM patients was 3.8 Ϯ ware. The repertoire of reactivities of each individual in a given sample 3.5 mg/ml (mean Ϯ SD, ranging from 0.4 to 14) at the time of diagnosis, was represented as a single symbol in a two-dimensional linear subspace. as determined by nephelometry. At the time of sampling, the mean IgM Discrimination between repertoires was assessed by submitting PCA data and IgG concentrations were 2.2 Ϯ 2.2 and 0.7 Ϯ 0.4 mg/ml, respectively, to linear discriminant analysis (LDA) and by subsequently comparing fac- in the serum of the 7 patients who were not substituted with i.v. Ig. These tors 1 of the LDA by Mann-Whitney U test. The statistical comparison concentrations were 2.3 Ϯ 2.1 and 2.6 Ϯ 2.1, respectively, in the serum of required that factor 1 of the LDA accounted for Ͼ70% of the variance of the 12 i.v. Ig-treated patients. The concentrations of serum IgM and IgG in the data. PCA of repertoires of Ab reactivities performed individually for Downloaded from healthy controls were 1.4 Ϯ 0.5 and 11.8 Ϯ 3.1 mg/ml, respectively. San- each group of individuals further allowed the calculation of respective doglobulin (a gift of the Central Laboratory of the Swiss Red Cross, Bern, variances. Variances were compared by F test. We also quantitated total Switzerland) was used as a standard for normal IgG. A reference prepa- immunoreactivities of serum IgM with each source of Ags by computing ration of normal IgM (i.v. IgM) was obtained by submitting Pentaglobin the total area under the curves of the respective densitometric profiles. (Biotest Pharma, Dreieich, Germany), an IgM-enriched therapeutic prep- Mean values of total reactivities obtained with each source of Ag were aration of pooled normal human IgG, to size exclusion chromatography on compared between groups of individuals in a global analysis by Fisher’s test.

Sephacryl HR S-300 (Pharmacia, Uppsala, Sweden). http://www.jimmunol.org/

Analysis of Ab repertoires by quantitative immunoblotting Analysis of IgM reactivity by ELISA For the study of Ab repertoires, we have used a quantitative immunoblot- Ninety-six-well ELISA plates (Nunc, Roskilde, Denmark) were coated ting technique that allows the simultaneous assessment of reactivities of with human Ag H (a gift from Prof. J.-P. Cartron, Institut National de la different sources of Abs with a large number of Ags in total tissue extracts Sante´ et de la Recherche Me´dicale (INSERM), Unit 76, Paris, France), (28, 37). The method has been described in detail elsewhere (27, 28). human laminin (Sigma), human thyroglobulin (Biogenesis, Poole, U.K.), Extracts of histologically normal human kidney, lung, liver, and stomach human transferrin (Sigma), calf actin, and calf thymus DNA (Sigma) at ␮ were used as sources of self Ags. Sources of bacterial Ags were extracts of 10 g/ml in PBS, pH 7.4, and with human low density lipoprotein (LDL, ␮ the commensal and pathogenic bacteria P. aeruginosa (Collection de a gift from J. Chevalier, INSERM, Unit 430, Paris, France) at 10 g/ml in ␮ l’Institut Pasteur, CIP A22) and Staphylococcus epidermidis (CIP 8155). PBS, 2.7 mM EDTA, and 20 M butylated hydroxytoluene (Sigma) over- by guest on September 25, 2021 ␮ Proteins were extracted from tissues and bacteria in 2.0% SDS, 1.45 M night at 4°C. Plates coated with DNA had been pretreated with 10 g/ml 2-ME, 125 mM Tris-HCl (pH 6.8), containing 1.0 ␮g/ml aprotinin, poly-L-lysine (Sigma). Plates were saturated with 1.0% BSA (Sigma) in 1.0 ␮g/ml pepstatin, and 1.0 mM EDTA on ice. Samples were sonicated 4 PBS for 30 min at 37°C. After a washing with PBS, the plates were incu- times for 30 s to disrupt DNA and boiled for 5 min. Protein concentrations bated with decreasing concentrations of serum IgM to be tested for1hat were determined by means of a Folin assay. The amount of tissue proteins 37°C before extensive washing with PBS and addition of goat anti-human subjected to electrophoresis and transfer ranged between 100 and 600 ␮g/ IgM Abs coupled to alkaline phosphatase (Southern Biotechnology Asso- gel, depending on the tissue extract, and was maintained constant for a ciates). The background reactivity of serum IgM scored with nonsensitized given tissue in all experiments. Low amounts of carbohydrates could be plates, or with poly-L-lysine-treated plates in the case of DNA, was sub- detected in solubilized protein samples. Proteins were subjected to prepar- tracted from the reactivity of IgM with the respective Ags. The mean val- ative SDS-PAGE in 10% polyacrylamide and transferred onto nitrocellu- ues of the corrected IgM reactivities were compared between groups for lose (Schleicher & Schuell, Dassel, Germany) for 60 min at 0.8 mA/cm2 each Ag by means of an one-sided Student t test. using a SemiDry Electroblotter A (Ancos, Denmark). Membranes were blocked with PBS-Tween. The source of Abs to be tested was then incu- Results bated with the membranes following the addition of one sample per slot in a Cassette Miniblot System (Immunetics, Cambridge, MA) overnight at Reactivity with self Ags of serum IgM of patients with the hyper- 4°C. The membranes were washed and incubated with ␮-chain-specific IgM syndrome secondary goat anti-human IgM Ab (Southern Biotechnology Associates, The reactivity with self Ags of IgM in the serum of healthy blood Birmingham, AL) or secondary goat anti-human Fc␥ Ab coupled to alka- line phosphatase (Sigma, St. Louis, MO). Immunoreactivities were re- donors and patients with HIM was analyzed by immunoblotting vealed using the nitroblue tetrazolium/bromochloroindolyl phosphate sub- using extracts of normal homologous human kidney, lung, liver, strate (Promega, Madison, WI). The dependency on variable regions of the and stomach as sources of self Ags. From 20 to 30 major peaks of recognition of blotted Ags by IgM and IgG has been documented previ- reactivity were scored after blotting of IgM of healthy individuals ously (27, 28). Quantitation of immunoreactivities was performed by den- sitometry in reflective mode using a high resolution CCD camera system with Ags in the extracts (Fig. 1 and data not shown). As previously (Masterscan, Scanalytics, Billerica, MA). Blotted proteins were then reported (28, 35), the densitometric profiles of reactivity of IgM of stained using colloidal gold (Protogold) (Biocell, Cardiff, U.K.) and sub- healthy donors exhibited a strong homogeneity between individu- jected to a second densitometric analysis to score the protein profile and to als with regard to the nature of the protein bands recognized in all quantitate transferred proteins. Data were analyzed using a Quadra 650 tissue extracts that were tested. From 15 to 20 major peaks of computer (Apple Computer, Cupertino, CA) and IGOR software (Wave- metrics, Lake Oswego, OR). Densitometric profiles of immunoreactivity reactivity were also detected after blotting of IgM of patients with were compared by referring to their corresponding protein profile, follow- HIM with self Ags (Fig. 1 and data not shown). The densitometric ing correction of the migration defects by superimposition of protein peaks. profiles of IgM reactivities exhibited a strong homogeneity be- A sample of the reference IgM or IgG preparations was included in each tween patients with regard to the nature of the protein bands rec- blot, to rescale the different membranes transferred with a given protein extract and to adjust for the intensity of staining of different membranes. ognized and were also homogeneous with regard to the intensity of Analysis of the densitometric profiles obtained with serial dilutions of immunoreactivities in lung and stomach Ags, but not in kidney and i.v. Ig demonstrated a dose-dependent decrease in the area under the curve liver Ags (Fig. 1 and data not shown). The Journal of Immunology 5603

FIGURE 1. Densitometric profiles of the reactiv- ity of serum IgM of 20 healthy donors (A and C) and of 19 patients with hyper-IgM syndrome (B and D) with Ags in extracts of normal human stomach (A and B) and liver (C and D). Sera were diluted to an IgM concentration of 20 ␮g/ml. The densitometric pattern of reactivity of IgM of each individual is depicted as a full line curve. Gray areas depict the densitometric pattern observed in the presence of the secondary anti-Fc␮ Ab alone. Migration distances and optical densities are expressed as arbitrary units (A. U.). Downloaded from http://www.jimmunol.org/

To compare the densitometric profiles of reactivities of IgM of differ significantly between healthy donors and HIM patients (Ta- healthy donors and patients, we computed the arithmetic mean ble II), demonstrating that self-reactive repertoires of IgM were reactivity profiles with self Ags of IgM of the 20 healthy donors homogeneous in both groups of individuals. Taken together, these and 19 patients (Fig. 2). Most of the protein bands detected by IgM data document that self-reactive repertoires of IgM of patients with in the serum of healthy donors were also recognized by HIM IgM HIM differ significantly from those of healthy donors. with lower intensity. Several of the IgM reactivities expressed by We then analyzed the reactivity of IgM in the serum of healthy healthy subjects were not present in the repertoire of reactivities of blood donors and patients with HIM in the case of several indi- by guest on September 25, 2021 IgM of HIM patients. The overall reactivity with self Ags of IgM vidual self Ags by ELISA. The reactivity of serum IgM of HIM of the HIM patients, as calculated by computing the mean area patients with DNA and with protein autoantigens, i.e., actin, lami- under the curve of the densitometric profiles obtained with each nin, thyroglobulin, and transferrin, was significantly lower than tissue extract, was significantly lower than that of healthy individ- that of healthy blood donors (Fig. 3). The reactivity of IgM from uals in kidney, liver, and stomach Ags, but not in lung Ags patients with HIM was, however, identical with that of healthy (Table I). subjects in the case of Ag H and LDL (Fig. 3), suggesting that the To further compare the self-reactive repertoires of IgM of pa- nature of the autoantigen determines whether T cell help is re- tients and healthy controls, we calculated the area of the peaks of quired for the selection of autoreactive B cell clones. immunoreactivity in densitometric profiles of IgM of each indi- vidual in each tissue extract. The data obtained with IgM of healthy donors were compared with those obtained in patients by Reactivity with nonself Ags of serum IgM of patients with the PCA within a 31- to 56-dimension vector space, depending on the hyper-IgM syndrome tissue extract, and fitted into the two-dimensional linear subspace To investigate Ab repertoires against nonself Ags, we assessed the that accounted for 55.6 to 69.4% of the variance and allowed the reactivity of IgM of patients with HIM with extracts of P. aerugi- most powerful discrimination of the individuals (Fig. 2). The data nosa and S. epidermidis. The densitometric profiles of reactivity of obtained by PCA were then submitted to LDA. Factors 1 of LDA IgM of healthy donors and HIM patients exhibited between 10 and were then compared by means of the Mann-Whitney U test. PCA 20 and between 5 and 10 major peaks of reactivity with the 2 discriminated between repertoires of Ab reactivities with self Ags sources of bacterial Ags, respectively (data not shown). The den- of healthy donors and patients with HIM ( p Ͻ 0.001 in the case of sitometric profiles of IgM of healthy donors exhibited a strong all tissue extracts). PCA did not discriminate between self-reactive homogeneity between individuals with regard to the nature of the repertoires of HIM patients undergoing substitutive therapy with protein bands recognized and the intensity of the peaks, whereas i.v. Ig and untreated patients (data not shown), indicating that the densitometric profiles of IgM of patients were homogeneous in the administration of normal IgG to HIM patients does not restore a case of P. aeruginosa extracts and heterogeneous in that of extracts physiological pattern of self-reactivity of IgM. PCA did not dis- of S. epidermidis (data not shown). The total reactivity of IgM of criminate between self-reactive IgM Ab repertoires of patients HIM patients with the bacterial extracts did not differ significantly with X-HIM and the patients with autosomal recessive HIM (data from that of healthy individuals (Table III). We then computed the not shown). We then assessed the relative homogeneity of Ab rep- areas of the peaks of immunoreactivity in the densitometric pro- ertoires of the 19 patients and 20 controls, by calculating the in- files to allow for multiparametric analysis of the data. The data dividual variances of the repertoires of self-reactivities in the case obtained in healthy donors and patients were compared by means of each protein extract, by means of PCA. The variances did not of PCA within a 46- to 58-dimension vector space, depending on 5604 SELF-REACTIVE REPERTOIRES OF IgM IN THE HYPER-IgM SYNDROME

Table I. Total reactivity with self-Ags of IgM in the serum of healthy individuals and patients with hyper-IgM syndrome a

Total Reactivity of IgM Repertoire

Tissue Extract Healthy donors HIM patients

Kidney 41.7 Ϯ 5.0 28.3 Ϯ 7.6 † Lung 48.3 Ϯ 9.7 41.8 Ϯ 9.0 NS Liver 39.1 Ϯ 8.2 33.1 Ϯ 10.9 ‡ Stomach 37.3 Ϯ 6.2 25.1 Ϯ 5.5 †

a Total areas under the curves of densitometric profiles of immunoreactivity of IgM with self-Ags in extracts of kidney, lung, liver, and stomach were calculated. Results are the mean Ϯ SD of the total reactivity within each group of individuals. Total reactivity is expressed as arbitrary units. Significant differences are indicated, as assessed by Fisher’s test (‡, p Ͻ 0.05; †, p Ͻ 0.0001).

patients with HIM appeared to generate an IgM Ab repertoire to commensal bacteria similar to that of healthy individuals, contrast- ing with impaired Ab repertoires to self Ags. Downloaded from Reactivity with self and nonself Ags of IgG in serum of patients with the hyper-IgM syndrome We analyzed the repertoires of reactivity of IgG in the serum of 7 patients who had not been substituted with i.v. Ig and of 5 control healthy subjects. The concentration of IgG in patients’ serum was Ͻ0.4 mg/ml in 3 patients and ranged between 0.6 and 1.2 mg/ml http://www.jimmunol.org/ in the 4 remaining patients. Reactivities of IgG were tested in serum diluted to achieve a final IgG concentration of 200 ␮g/ml. IgG of healthy donors strongly reacted with 20–30 protein bands, depending on the tissue extract (data not shown). As previously FIGURE 2. Comparative analysis of self-reactive IgM Abs in the serum described (27), the patterns of reactivities were heterogeneous of healthy donors and of patients with hyper-IgM syndrome. Depicted are among healthy individuals with regard to both the nature of the the mean densitometric profiles of self-reactive IgM (i.e., the arithmetic protein bands recognized and the intensity of reactivities. No re-

mean of the 800 recorded intensities constitutive of the reactivity profile of activity with self Ags and nonself Ags was seen with serum of the by guest on September 25, 2021 each individual) in the serum of 20 healthy donors (black areas and dotted 3 patients who exhibited the lowest concentrations of serum IgG. lines) and of 19 patients with HIM (white areas), with Ags in extracts of IgG of the remaining 4 HIM patients exhibited weak reactivity normal human liver (A) and stomach (B). IgM was tested at a concentration with 10 to 15 protein bands in the kidney extract (data not shown). of 20 ␮g/ml. Gray areas depict the densitometric pattern observed in the In contrast, a strong reactivity was observed with Ags in the S. presence of the secondary anti-Fc␮ Ab alone. Migration distances and optical densities are expressed as arbitrary units (A. U.). Insets depict the epidermidis extract (data not shown). The total reactivity of IgG of results of PCA of the repertoires of reactivities of IgM in the serum of HIM patients was significantly lower than that of healthy blood healthy donors (F) and patients (Ⅺ). For each donor, the densitometric donors in the case of kidney Ags ( p Ͻ 0.001), but not in that of profile of reactivity with a given tissue extract was divided into sections S. epidermidis Ags ( p ϭ 0.66) (Fig. 5 and Table V), indicating a corresponding to peaks of reactivity so as to calculate the height of each selective bias in the repertoire of reactivities of IgG toward self peak. The data were subjected to PCA within a 31- to 56-dimension vector Ags. space, depending on the tissue extract, and fitted within two-dimensional The data obtained upon immunoblotting of IgG were compared linear subspaces (factors 1 and 2). Percentages of variance accounted for by by means of PCA within 44- and 42-dimension vector spaces, in factors 1 and 2 are indicated on the abscissa and ordinate, respectively. the case of kidney and S. epidermidis extracts, respectively, and Each symbol represents the reactivity of IgM of a single individual. In both fitted into 2-dimensional linear subspaces that accounted for 91.4 liver and stomach extracts, PCA discriminated between repertoires of pa- tients and controls (p Ͻ 0.001 by the Mann-Whitney test). and 70.0% of the variance, respectively. PCA discriminated be- tween healthy donors and patients in the case of kidney Ags ( p Ͻ the bacterial extract, and fitted into the 2-dimensional linear sub- Table II. Total variances of the self-reactive repertoires of IgM of space that accounted for 69.5 to 70.3% of the variance and allowed healthy individuals and of patients with hyper-IgM syndrome a for the most powerful discrimination between individuals. PCA did not discriminate between repertoires of reactivities with bac- Total Variance of Repertoires of Reactivity of IgM terial Ags of patients and healthy donors (Fig. 4). PCA also did not discriminate between autoantibody repertoires of patients with X- Tissue Extract Healthy donors HIM patients HIM and patients with autosomal recessive HIM (data not shown). Kidney 1071.6 1582.8 NS When calculating the individual variances of repertoires of reac- Lung 2927.2 4633.7 NS tivity of IgM with bacterial Ags by PCA (Table IV), variances of Liver 1768.7 2621 NS Stomach 2806.4 2171.9 NS repertoires of healthy donors were found not to differ significantly from those of repertoires of HIM patients, indicating that the rel- a Total variances of repertoires of reactivities of IgM in whole serum with self- Ags in extracts of kidney, lung, liver, and stomach were calculated separately in a 31- ative homogeneity of the repertoires of reactivities of IgM to ex- to 56-dimension vector space, depending on the tissue extract. Differences were not ternal Ags was similar in the two groups of individuals. Thus, significant, as assessed by F test. The Journal of Immunology 5605

FIGURE 3. Reactivity of serum IgM of 12 healthy donors (f) and of 12 patients with HIM (Ⅺ) with a panel of self Ags, i.e., actin (Act), laminin (Lam), thyroglobulin (Tg), transferrin (Tf), DNA, Ag H, and LDL. Mean reactivities of IgM, at a concentration of 33 ␮g/ml as assessed by ELISA, Downloaded from are expressed as arbitrary units (A. U.). Mean IgM reactivities were com- p Ͻ 0.05; †, p Ͻ ,ء) pared between groups using an one-sided Student t test 0.01).

0.05), whereas it did not discriminate between repertoires of reac- FIGURE 4. Comparative analysis of reactivity of IgM Abs in the serum http://www.jimmunol.org/ tivity with S. epidermidis of healthy individuals and HIM patients of 20 healthy donors (F) and of 20 patients with hyper-IgM syndrome with ϭ ( p 0.22) (Fig. 5). Individual variances of the repertoires of re- foreign Ags (Ⅺ). Depicted are the results of PCA of the repertoires of activity of IgG with both kidney and S. epidermidis Ags, calcu- reactivities of IgM with Ags in extracts of P. aeruginosa (top) and S. lated by PCA, differed significantly between healthy donors and epidermidis (bottom). For each donor, the densitometric profile of reactiv- patients (Table VI), indicating a higher degree of heterogeneity of ity with a given tissue extract was divided into sections corresponding to the repertoires of reactivity of IgG in healthy subjects than in HIM peaks of reactivity so as to calculate the height of each peak. The data were patients. subjected to PCA within a 46- to 58-dimension vector space, depending on the bacterial extract, and fitted within two-dimensional linear subspaces Discussion (factors 1 and 2). Percentages of variance accounted for by factors 1 and 2 are indicated on the abscissa and the ordinate, respectively. Each symbol by guest on September 25, 2021 In the present study, we have characterized the Ab repertoires of represents the reactivity of Igs of a single individual. In both P. aeruginosa IgM and IgG in the serum of patients with the hyper-IgM syn- and S. epidermidis extracts, PCA did not discriminate between repertoires drome. We demonstrate a significant bias in repertoires of reac- of patients and controls. tivities of IgM of patients with HIM with self Ags. Furthermore, IgG of HIM patients lacked reactivity with self Ags, in contrast with IgG of healthy controls, when tested at similar concentrations. young adults and between males and females with regard to reac- Repertoires of reactivities of HIM IgM with foreign Ags did not tivity with self Ags (29, 35, 40). Significant alterations in self- differ from those of IgM in the serum of healthy individuals. These reactive Ab repertoires have been observed in autoimmune condi- results indicate that the lack of functional CD40-CD40L interac- tions, such as myasthenia gravis and systemic lupus erythematosus tions or defective T cell/B cell cooperation impact on the selection (41, 42). of physiological self-reactive Ab repertoires. Here we investigated Ab repertoires in the serum of 19 patients Natural autoreactive IgM and IgG Abs are found in the serum of with HIM, 16 of whom had X-HIM. We observed that the densi- healthy individuals (24, 39). The normal repertoire of Ab reactiv- tometric patterns of reactivity with self Ags of IgM in the serum of ities with self Ags is homogeneous between individuals and re- patients with HIM were homogeneous among the patients but dif- stricted to a limited set of immunodominant Ags that is conserved fered significantly from those of healthy donors. Several peaks of among healthy individuals and stable throughout life (27–29, 35). self-reactivity of IgM in normal serum were lacking in the serum Repertoires of serum IgM and IgG remain invariant in children and

Table IV. Total variances of the repertoires of reactivity of IgM of Table III. Total reactivity with foreign Ags of IgM in the serum of healthy subjects and of patients with hyper-IgM syndrome with Ags in healthy individuals and patients with hyper-IgM syndrome a bacterial extracts a

Total Reactivity of IgM Repertoire Total Variance of Repertoires of Reactivity of IgM

Tissue Extract Healthy donors HIM patients Tissue Extract Healthy donors HIM patients

P. aeruginosa 36.7 Ϯ 6.3 32.3 Ϯ 9.7 NS P. aeruginosa 54.6 64.5 NS S. epidermidis 32.8 Ϯ 7.7 28.3 Ϯ 11.5 NS S. epidermidis 64.5 130.6 NS

a Total areas under the curves of densitometric profiles of immunoreactivity of a Total variances of the repertoires of reactivity of IgM in whole serum with IgM with Ags in extracts of P. aeruginosa and S. epidermidis were calculated. Results extracts of P. aeruginosa and S. epidermidis were calculated by separate PCA of the are the mean Ϯ SD of the total reactivity within each group of individuals. Total data corresponding to each group. The data were analyzed in a 46- to 58-dimension reactivity is expressed as arbitrary units. Differences were not significant, as assessed vector space, depending on the bacterial extract. Differences were not significant, as by Fisher’s test. assessed by F test. 5606 SELF-REACTIVE REPERTOIRES OF IgM IN THE HYPER-IgM SYNDROME

Table V. Total reactivity of IgG in the serum of healthy individuals and patients with hyper-IgM syndrome a

Total Reactivity of IgM Repertoire

Tissue Extract Healthy donors HIM patients

ء Kidney 101.3 Ϯ 24.1 20.3 Ϯ 14.2 S. epidermidis 147.9 Ϯ 30.2 155.3 Ϯ 9.7 NS

a Total areas under the curves of densitometric profiles of immunoreactivity of IgM with Ags in extracts of kidney and S. epidermidis were calculated. Results are the mean Ϯ SD of the total reactivity within each group of individuals. Total reactivity is expressed as arbitrary units. Significant differences are indicated, as assessed by Fish- .(p Ͻ 0.001 ,ء) er’s test

mic mice (34). The transfer of syngeneic T cells to nude mice was also shown to restore normal titers of serum IgG (33, 34). A number of studies document a role for IgG in the selection of IgM repertoires. Thus, maternal IgG decreases the concentration

and alters the pattern of reactivity of serum IgM in newborn mice Downloaded from (34, 43). Treatment with i.v. IgG of autoimmune patients, is fol- lowed by altered titers of specific IgM and IgG autoantibodies (44–46). In patients with HIM, the administration of i.v. Ig often results in a dramatic and long-lasting decrease of serum IgM levels (21, 23, 47–49). The latter decrease may reflect the effects of i.v. Ig on B cell repertoires or, alternatively, be a consequence of a http://www.jimmunol.org/ better control of recurrent infections. In our study, however, no difference in the serum concentration of IgM was observed be- tween patients substituted with i.v. Ig and untreated patients (1). In addition, PCA did not discriminate between self-reactive Ab rep- ertoires of IgM of i.v. Ig-treated and untreated HIM patients. Since the concentration of IgG in the serum of the 12 patients treated FIGURE 5. Comparative analysis of repertoires of IgG Abs in the se- with i.v. Ig was significantly lower than that in the serum of rum of healthy donors and patients with HIM. Depicted are the mean den- healthy individuals (2.6 Ϯ 2.1 mg/ml), the possibility remains that sitometric profiles of IgG in the serum of 7 healthy donors (black areas and dotted lines) and 4 patients with HIM who had not received i.v. Ig (white IgG was at too low a concentration in plasma to efficiently influ- by guest on September 25, 2021 areas), with Ags in extracts of normal human kidney (top) and S. epider- ence the selection of B cell repertoires. Thus, it is unclear at midis (bottom). IgG was tested at a concentration of 200 ␮g/ml. Gray areas present whether alterations in the self-reactive repertoires of IgM depict the densitometric pattern observed in the presence of the secondary in patients with HIM are strictly dependent on defective T cell/B anti-Fc␥ Ab alone. Migration distances and optical densities are expressed cell signaling or whether these alterations are also a consequence as arbitrary units (A. U.). Insets depict the results of PCA of the repertoires of decreased levels of autologous IgG. of reactivities of IgG in the serum of healthy donors (F) and patients (Ⅺ). Increased concentrations of IgM in the serum of patients with For each donor, the densitometric profile of reactivity with a given tissue HIM were not associated with an increase in the overall reactivity extract was divided into sections corresponding to peaks of reactivity so as of IgM with self Ags. In fact, the total autoreactivity of IgM of to calculate the height of each peak. The data were subjected to PCA within patients was significantly lower than that of controls in the case of a 42- to 44-dimension vector space, depending on the tissue extract, and fitted within 2-dimensional linear subspaces (factors 1 and 2). Percentages three of the four tissue extracts that we tested as sources for self of variance accounted for by factors 1 and 2 are indicated on the abscissa Ags. Our results agree with a previous report on HIM patients who and ordinate, respectively. Each symbol represents the reactivity of IgG of were devoid of anti-Gal␣1–3Gal Abs as compared with healthy a single individual. PCA discriminated between repertoires of patients and individuals (50). However, in our study, the bias in IgM autore- controls in kidney extract (p Ͻ 0.01 by the Mann-Whitney test), whereas activity was restricted to protein Ags, suggesting that the role of T it did not discriminate between patients and controls in S. epidermidis. cell help in the selection of self-reactive B cell clones depends on the nature of the autoantigens. It may be speculated that autoim- mune manifestations which were reported to occur in patients with of HIM patients. Principal component analysis discriminated be- tween self-reactive IgM repertoires of patients and healthy donors. The variances of individual Ab repertoires were of a similar order Table VI. Total variances of the repertoires of reactivity of IgG of of magnitude in both groups, indicating that healthy donors and healthy subjects and of patients with hyper-IgM syndrome a patients with HIM represent homogenous groups of individuals characterized by distinct patterns of recognition of self Ags. These Total Variance of Repertoires of Reactivity of IgM observations emphasize the key role of T lymphocytes for estab- Tissue Extract Healthy donors HIM patients lishing natural self-reactive repertoires of IgM Abs, as suggested by previous studies in mice. Thus, it has been shown that euthymic Kidney 134.7 32.6 ‡ and athymic (nu/nu) BALB/c mice present with distinct self-reac- S. epidermidis 368.8 33.7 ‡ tive repertoires of IgM (34). The transfer of syngeneic T cells to a Total variances of the repertoires of reactivity of IgM in whole serum with athymic mice restored high frequencies of autoreactive precursor extracts of P. aeruginosa and S. epidermidis were calculated by separate PCA of the data corresponding to each group. The data were analyzed in a 42- to 44-dimension B cell clones (33) and altered repertoires of reactivities of natural vector space, depending on the extract. Significant differences are indicated, as as- autoantibodies, with patterns almost identical with those of euthy- sessed by F test (‡, p Ͻ 0.05). The Journal of Immunology 5607

HIM (2, 19) may be dependent on an impaired control of autore- 12. Razanajaona, D., C. van Kooten, S. Lebecque, J.-M. Bridon, S. Ho, S. Smith, activity, secondary to defective T cell-B cell interactions. In- R. Callard, J. Banchereau, and F. Briere. 1996. Somatic mutations in human Ig variable genes correlate with a partially functional CD40-ligand in the X-linked creased titers of IgM in patients with HIM have been suggested to hyper-IgM syndrome. J. Immunol. 157:1492. reflect a compensatory mechanism in individuals confronted with 13. Gray, D., P. Dullforce, and S. Jainandunsing. 1994. Memory B cell development but not germinal center formation is impaired by in vivo blockade of CD40-CD40 chronic stimulation of the immune system by infectious agents (1, ligand interaction. J. Exp. Med. 180:141. 2). If that were the case, one would have expected a bias in the 14. Kawabe, T., T. Naka, K. Yoshida, S. Suematsu, N. Yoshida, T. Kishimito, and repertoire of reactivities of IgM toward bacterial Ags. However, H. Kikutani. 1994. The immune response in CD40-deficient mice: impaired im- munoglobulin class switching and germinal center formation. Immunity 1:167. we did not observe qualitative or quantitative differences in the 15. Xu, J., T. Foy, J. Laman, T. Waldschmidt, J. Elsemore, R. Noelle, and R. Flawell. repertoires of IgM toward Ags of P. aeruginosa and S. epidermi- 1994. Mice deficient for the CD40 ligand. Immunity 1:423. dis. These observations suggest that IgM immune responses 16. Foy, T., J. Laman, J. Ledbetter, A. Aruffo, E. Claassen, and R. Noelle. 1994. gp39-CD40 interactions are essential for germinal center formation and the de- against foreign Ags similar to those of healthy individuals may velopment of B cell memory. J. Exp. Med. 180:157. occur in the absence of normal autologous T cells. In this respect, 17. Lane, P. 1997. Molecular mechanisms involved in T-B interactions. In Human B it is noteworthy that a proportion of B cells in patients with X-HIM Cell Populations, M. F. A. F. Caligaris-Cappio, ed. Karger, Basel, p. 1. 18. Facchetti, F., C. Appiani, L. Salvi, J. Levy, and L. D. Notarangelo. 1995. Im- may be induced to undergo somatic mutation (12). munohistological analysis of ineffective CD40-CD40 ligand interaction in lym- There was almost no reactivity toward self Ags of IgG in the phoid tissues from patients with X-linked immunodeficiency with hyper-IgM: serum of the seven untreated patients with X-HIM whom we abortive germinal center cell reaction and severe depletion of follicular dendritic cells. J. Immunol. 154:6624. tested. In contrast, when tested at a similar concentration, IgG in 19. Hinz, C. F. J., and J. T. Boyer. 1963. Dysgammaglobulinemia in the adult man- the serum of healthy individuals reacted with several protein Ags ifested as autoimmune hemolytic anemia. N. Engl. J. Med. 269. in homologous tissue extracts. 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