merism studies are important to document engraftment or rejection and to get an insight into the immune reconstitution. The origin of cells of the B-cell lineage ...
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1996 87: 818-826
The origin of IgG production and homogeneous IgG components after allogeneic bone marrow transplantation MJ van Tol, EJ Gerritsen, GG de Lange, AM van Leeuwen, CM Jol-van der Zijde, NJ Oudeman-Gruber, E de Vries, J Radl and JM Vossen
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The Origin of IgG Production and Homogeneous IgG Components After Allogeneic Bone Marrow Transplantation By M.J.D. van Tol, E.J.A. Gerritsen, G.G. de Lange, A.M. van Leeuwen, C.M. Jol-van der Zijde, N.J. Oudeman-Gruber, E. de Vries, J. Radl, and J.M. Vossen Pediatric recipients (n = 25) of an allogeneic bone marrow (BM) graft were selected on the basis of informative IgG allotype (Gm) differences between the BM donor and the recipient. To investigate the kinetics of the appearance of IgG of donor origin and the disappearance of IgG of recipient origin, G l m and G2m allotype levels were quantifiedin sera obtained at regular intervals between 3 months and 5 years after BM transplantation(BMT). For this quantification, a dot immunobinding assay (DIBA) has been developed. In 19 of 22 informative recipients, the Gm allotype distribution had reached the range of values expected on the basis of the Gm phenotype of the donor within 6 months after BMT. Remarkably, lgG of recipient origin persisted in 15 of 18 informative recipients until last follow up, ie, for several years after BMT. In addition to the originof total IgG production,
the origin of homogeneous IgG components (H-lgG) appearing after BMT was investigated. H-lgG of donor origin could be detected as early as 3 weeks after BMT, but also H-IgG of recipient origin werepresent in 8 of 13 informative recipients for a period of up t o 1 year after BMT. We conclude that host-type IgG-producing cells were not eradicated by the (myeloablative) conditioning regimen and persisted in a high number ofgraft recipients. It is our hypothesisthat lack of graft-versus-host disease (GVHD) in the majorityof these recipients results in the persistence of IgG-producing cells of host origin. These observations may be relevant for the evaluation of patients whoreceived allogeneic BMT for the treatment of multiple myeloma. 0 1996 by The American Society of Hematology.
A
months up to 5 years after BMT. In addition, H-Ig of the IgG isotype (H-IgG) were characterized for their IgG subclass and Gm allotype in sera obtained at 3 weeks after BMT and later. The results indicate that a minor but substantial part of serum IgG was still of recipient origin in 15 of 18 informative recipients at l year after BMT and, in 1 1 of 13 of these cases, at last follow up between 3 and 8 years after BMT, despite engraftment of the donor B-cell lineage. This IgG of recipient origin can be either heterogeneous or of restricted clonal diversity. In addition, Gm allotyping of HIg early after BMT was helpful in determining the origin of cells of the B-cell lineage, especially in cases where administration of Ig-containing blood products interfered with the quantification of Gm allotypes.
LLOGENEIC BONE marrow transplantation (BMT) is applied for the treatment of hematologic malignancies, severe aplastic anemia (SAA), and congenital diseases. Chimerism studies are important to document engraftment or rejection and to get an insight into the immune reconstitution. The origin of cells of the B-cell lineage can be investigated by analysis of polymorphic epitopes (allotypes) of immunoglobulins (Ig) in case the donor and the recipient of the graft express a different phenotype."6 Hemagglutination inhibition (HAI) assays, routinely used in this respect, allow only semiquantitative measurement ofIg allotype levels inserum.' The generation of monoclonal antibodies (MoAbs) specific for allotypes of IgG heavy chains (Gm allotypes) offered the possibility t o develop quantitative assays. Determination of the quantity of Gm allotypes in serum of recipients early after BMT is hampered by plasma transfusions and Ig suppletion. However, sera ofBM graft recipients frequently contain transient homogeneous Ig components (H-Ig).X~'" These H-Ig are mostly of low concentration, appear within weeks after BMT, and are absent from the administered Igcontaining blood products. The kinetics of the appearance of H-Ig suggests that they represent expansions of B-cell clones of donor origin. In the present retrospective study, levels of IgG subclasses and Gm allotypes were quantified in sera obtained from BM graft recipients at regular intervals within the period of 3 From the Department of Pediatrics, University Hospital, Leiden: the Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam: and the TNOPreventionandHealth, Leiden, The Netherlands. Submitted July 31, 1995; accepted September I , 1995. Address reprint requests to M.J.D. van Tol, PhD, Department of Pediatrics, Leiden University Hospital, Building I , P3-31, P.O. Box 9600, 2300 RC Leiden, The Netherlands. The publication costs of this article were defrayedin part by page chargepayment. This article must thereforebeherebymarked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this f a t . 0 1996 by The American Society of Hematology. 0006-4971/96/8702-0014$3.00/0 818
MATERIALS AND METHODS Patients. Twenty-fivepediatricrecipients of anallogeneicBM graft were included in this study. They were selected becauseof the existence of informative IgG allotype (Gm) differences between the BM donor and the recipient and the possibility for a follow-up period of at least 1 year after BMT. Relevant characteristics of the patients (ie, the original disease, the Gm phenotypes of the donor and the recipient as determined by HA1 assays, the pretreatment for BMT, the time period of plasma transfusions andor Ig suppletion, and the occurrence of acute graft-versus-host disease (GVHD) =grade 2 or extensive chronic GVHD) are given in Table 1. All patients received an unmodified BM graft while staying in the protective environment of alaminar flow isolatorandafterantimicrobialsuppressionof their intestinal microflora.I3 Sera were obtained from the donor before BMT and from the recipient before and at regular intervals (3. 6, and 9 weeks: 3 , 6, and 9 months;and I , 2, 3, 4, and 5 years) after BMT. The sera were stored at -20°C until investigation. Quant$cation. Serum levels of IgG subclasses were determined by enzyme-linked immunosorbent assay (ELISA) or dot immunobinding assay (DIBA), as described elsewhere.I4 Gm allotypes were quantified by DIBA using the MoAbslisted in Table 2. The specificity and usefulness of these MoAbs in (semi-) quantitative assays havebeenextensively reported."' Some of the MoAbs directed against Gm allotypes are known to show crossreactivity with other IgG subclasses and Gm allotypes, depending on the technique and the dilution in which they are applied. Consequently, for each of the MoAbs, conditions had to be defined to obtain optimal signalnoise ratios. In addition to serum samples under investigation, two Blood, Vol 87, No 2 (January 15). 1996: pp 818-826
From bloodjournal.hematologylibrary.org by guest on July 14, 2011. For personal use only. ORIGIN OF (H-)IGG AFTER ALLOGENEIC BMT
819
Table 1. Characteristics of the Graft Recipients and BMT Procedure GVHD Gm Phenotypes* Recipient No.
Diagnosis
Donor ~
AML
ALL ALL
1 2 3 4 5
SCID(N) CID
6
SAA
7 8 9 10 111 12 13 14 15 16
SAA
17n 18 19 20 21 22 23 24 25
SAA SAA SAA
SAA SAAll AML AML A ML AML AML AML AML ALL ALL ALL ALL ALL ALL
fb zafngb fnb zafgb zafng zaxfgb zaxfgb zaxfngb zaxfngb zaxffgb fb afnb zafngb zafngb zafgb fnb zaxg afnb fnb zag zanb fb fflb zag zaxg zaxfngb
Suppletion
Recipient
Pretreatment
fnb fnb zaxfngb zafngb b fnb fnb zaxg zaxg b!f zafnb fnb zafgb fnb za b zafel! zafngb zafnb zafigb fnb zaxfgb zxfgb zafgb zaxfigb fnb
ATG BUCY PAPAPACY PAPAPACY PAPAPACY
y-Globulin5 Plasmat
Acute (rgrade 2)
Chronic (extensive) -
~~~
PAPAPACY CY CY, TBI-4.0 PAPAPACY CY, TBI-7.5 CY, TBI-7.5
-
-
-
0-2 -
-
-
-
-
-
-
-
-
-
+ -
-
-
+ -
0-1
+ +
-
-
-
-
0-2 0-2
-
-
-
-
-
-
-
-
0-2
-
-
+
0-2 0-3$
-
-
1-2
+
-
0-2
CY, CY, CY, CY, CY,
TBI-7.5 TBI-8.0 TBI-8.0 TBI-8.0 TBI-8.0
0-2
CY, CY, CY, CY, CY, CY, CY, CY, CY,
TBI-8.0 TBI-7.0 TBI-7.5 TBI-7.5 TBI-7.5 TBI-8.0 TBI-8.0 TBI-8.0 TBI-8.0
0-2 0-2 0-2 0-2
0-2
-
0-4 -
-
+
-
-
-
-
-
-
+
-
0-2 0-3 0-2 0-2
0-2 0-3
-
-
+
+
-
-
-
-
-
-
0-2
-
-
-
Abbreviations: SCID(N), severe combined immunodeficiency, Nezelof-type (T-cell deficiency, presence of B cells, plasma cells, and lg, but absence of specific antibody production"); CID, low T-cell numbers, defective antibody production; AML, acute myeloblastic leukemia; ALL, acute lymphoblastic leukemia; ATG, rabbit antithymocyte globulins (2 mg/kg X 3 days); BU, busulphan 4 mgikg X 4 days; CY, cyclophosphamide 50 mgkg x 4 days (alone or in combination with BU) or 60 mg/kg x 2 days (in combination with TBI): PAPAPACY, see Storb et allz: TBI, total body irradiation dosage in Grays (single fraction, high dose rate). * Discriminative Gm allotypes, excluded Glm(x), are underscored. t 5 1 0 mUkg/wk during the indicated period (months after BMT). 10 mUkg 2 5 days per week between 2 and 3 months after BMT. 5 50 to 100 mg/kg/wk during the indicated period (months after BMT). 11 Patient with graft rejection and recovery of autologous hematopoiesis. 1 Children from Chinese ethnic origin.
*
reference sera with known amounts of the IgG subclasses were obtained from individuals homozygous for the Gm (za(x);..;g) and for theGm (f;n; b) haplotypes, respectively. Simultaneous investigation of these reference sera and serum samples of donors and recipients not only served as a specificity control, but also allowed the construction of a standard curve for the calculation of the level of a distinct Gm allotype in the samples to be analyzed. Quantification of G l m allotypes by DIBA was validated against a radial immunodiffusion (RID) assay, developed by one of us (A.M.v.L.). In both techniques, 45 sera obtained from recipients after allogeneic BMT were investigated. These sera were selected because they contained IgGl heterozygous for the G l m allotypes and because the concentration of the products of the various G l m alleles was quite variable. Linear regression analysis showed a good correlation between the data obtained by DIBA and RID: Glm(z): y = 0 . 9 1 ~+ 0.33 ( r = .93); Glm(a):y = 0 . 7 5 ~+ 0.34 ( r = .95); and Glm(f): y = 0 . 9 0 ~+ 0.33 ( r = .90). In addition, mixing experiments were performed in which a serum from an individual homozygous for the Gm (za(x);..;g) haplotype was titrated into a serum from an individual homozygous for the Gm (fin;b) haplotype. The absolute concentrations of the distinct G l m allotypes measured
by DIBA corresponded well with the anticipated values (data not shown). H-IgC. H-Ig of the IgG isotype were characterized for their IgG subclass and Gm allotype by immunoblotting after high-resolution agar gel electrophoresis of diluted serum samples according to Wieme" (WIBL), as previously described.'"." The MoAbs used are given in Table 2. The sensitivity limit of detection of H-Ig by WIBL is between 0.5 and 5.0 pg/mL." The WIBL patterns were independently interpreted by two investigators. Only H-IgG, which were detected in immunoblotting patterns for the IgG subclasses and which were also present at the same electrophoretic mobility in immunoblotting patterns for the corresponding Gm allotypes, were taken into consideration. This approach enabled us to apply the crossreactive MoAbs recognizing the G3m allotypes successfully in WIBL. For IgG2, this strategy could not be followed, because antibodies specific for the product of the G2m(..) allele are not available. RESULTS
Gm allotype levels in homozygous and heterozygous individuals. The specificity of the MoAbs directed against Gm
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VAN TOL ET AL
Table 2. MoAbs Used in DIBA and lrnrnunoblotting MoAb
IUISMIHO*
Specificity
Origint
NL 16 NI 6014 NI 86 NI 315 5A1 5E7 5F10 SH21 8D10 12D9
HP6012 HP6014 HP6080 HP6206 HP6189 HP6184 HP6185
a b b b
-
lgGl lgG2 lgG3 m(u) lgG4 Glm(z) Glm(a) Glm(f) G2m(n)
HP6191 HP6194
G3m(g) G3m(bl/u)
C
C C
d C C
Working Dilution
1:104 1:104 1:104 1:2.5 X 104 1~05 1:3 X lo5 1:105 1:105 1:106 1:106
* See Jefferis et al.'6.17 t a, Unipath, Bedford, UK;b,
Nordic Immunological Laboratories, Tilburg, The Netherlands; c,CLB, Amsterdam, The Netherlands; d, Bio-Makor, Rehovot, Israel.
allotypes and their applicability in DIBA was assessed by testing the MoAbs at various dilutions on sera obtained from individuals homozygous for the Gm (za(x); ..;g) and for the Gm (f;n; b) haplotype, respectively. For each of the MoAbs used at optimal dilutions (Table 2), a background signal was observed in sera negative for the relevant Gm allotype. A distinct Gm allotype was considered tobe absent from a serum sample when the Glm(z), Glm(a),and Glm(f) levels werebelow 5%, O S % , and 0.5% of the concentration of total IgG1, respectively, andwhen the G2m(n) levelwas below 5% of the concentration of total IgG2 (Table 3). For G3m(g) and G3m(b), these values were 25%and 45% of the concentration of total IgC3, respectively (Table 3). Because of these high cut-off levels for negative individuals, it was decided that quantification of the G3m allotypes by DIBA using these MoAbs was not feasible. It has been described that the Glm(a), G2m(n), and G3m(b) alleles contribute more to the production of the corresponding IgG subclass in heterozygotes than the Glm(f), G2m(..), and G3m(g) alleles, respectively.'".'' In sera of heterozygous individuals, the contribution of distinct Gm allotypes to the level of the corresponding IgG subclasses was defined (Table 3). Finally, in homozygous individuals, the concentration of the positive Gm allotypes was compared with the amount of the corresponding IgG subclasses, and the ranges are given in Table 3. Deviation from the expected 100% value must be ascribed to experimental variance of the independent determinations. AppearanceofIgGofdonororiginafterBMT. Determination of Gm allotypes to investigate B-cell engraftment is theoretically most informative when the BM donor expresses Gm allotypes, which are not produced by the recipient. Regarding Glrn allotypes, this was the case in 10 of the 25 BMT couples of our study group (Table 1). The distribution of the Glrn allotypes was determined longitudinally from 3 months up to 2 years after BMT in the sera from 9 of these I O BM graft recipients. For the two recipients with a homozygous donor (recipients 15 and 20), the proportion of IgGl of donor Glrn allotype versus total IgGl reached the range of homozygous individuals (Table 3) between 3 and12 months after BMT (Fig 1). It should be noted that in recipient
15 the proportion of donor-derived IgGl dropped below this range at 2 years after BMT because of partial recovery of IgGl production by cells of recipient origin (see below). In six of seven recipients with a heterozygous donor, the relative contribution of IgGl of donor Glm allotype to the level of total IgGl was within therange of heterozygous individuals at 3 (n = 3), 6 (n = 2), and12 (n = 1) monthsafter BMT, respectively, and later (Fig l ). The one except'Ion was recipient 9, with graft rejection and recovery of autologous hematopoiesis. IgGl of Glm(f) allotype, indicating donor origin, was almost completely undetectable in serum of this recipient at 6 months after BMT and later. In addition, 10 recipients who wereheterozygous for G 1 m allotypes received a BM graft from a Glrn homozygous donor. In 9 of these I O cases, the proportion of IgGl of donor Glm allotype reached the range of homozygous individuals at 6 months after BMT, and in one case, at 12 months after BMT (data not shown). With respect to IgG2, there were five G2m(..) recipients with a G2m(n) homozygous (n = 2) or heterozygous (n = 3) donor in our study group (Table 1). Furthermore, three G2m(n) heterozygous recipients had a G2m(n) homozygous donor. At 3 months after BMT, four of four recipients, longitudinally investigated after BMT, showed G2m allotype levels within the corresponding range of homozygous or heterozygous individuals. In recipient 9 with graft rejection and recovery of autologous hematopoiesis, the appearance of IgG2 of donor origin was only transient (data not shown). Persistence oj' IgG qf recipient origin after BMT. Twelve BM graft recipients expressed Glrn allotypes that werenotproducedby the donor (Table 1). In the first 6 months after BMT, nine of them showed a fast decrease of IgGl of recipient origin (Fig 2). However, as shown in Fig 2 and Table 4, IgG I of recipient G 1m allotype still persisted at 1 year after BMT in I O of 12 recipients at a proportion above the cut-off level (Table 3) of the corresponding Glrn allotype in negative individuals. Seven of these 10 recipients could be investigated later than I year after BMT, and all of them showed persistence of recipient-derived IgG 1 at last follow up at 3 to 8 years after BMT (Table 4).
Table 3. Gm Allotype Concentrations Relative to the Amount of the Corresponding IgG Subclass Homozygous Negative
Glm Glm Glm G2m G3m G3m
(z) (a) (f) (n) (g) (b)
Heterozygous
Homozygous Positive
%
n*
Yo t
n*
Yo t
nx
