1 Université Libre de Bruxelles, Laboratory of Animal Physiology, rue des Chevaux 67, 1640 Rhode-Saint-Genèse, Belgium
2 Schering-Plough, Laboratory for Immunological Research, 27 chemin des Peupliers, BP11, 69571 Dardilly, France
3 Université de Liège, Unité d'Immunologie, rue des Pitteurs 20, 4020 Liège, Belgium
Correspondence to: P. J. Urbain
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Abstract |
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high-affinity antibodies. By contrast, in partially shielded and irradiated A/J mice (autologous reconstitution) immunized with Arskeyhole limpet hemocyanin (KLH), both the dominance of the CRIA idiotype and the affinity maturation are lost, whereas the anti-Ars antibody titer is not affected. To understand these alterations, we have analyzed a collection of 27 different anti-Ars hybridomas from nine partially shielded and irradiated A/J mice that had been immunized twice with ArsKLH. Sequence analysis of the productively rearranged heavy chain variable region genes from those hybridomas revealed that (i) the canonical V(D)J combination was rare, (ii) the pattern of V(D)J gene usage rather corresponded to a primary repertoire with multiple gene combinations and (iii) the frequency of somatic mutations was low when compared to a normal secondary response to Ars. In addition, immunohistological analysis has shown a delay of 2 weeks in the appearance of full blown splenic germinal centers in autoreconstituting mice, as compared to controls. Such a model could be useful to understand the immunological defects found in patients transplanted with bone marrow.
Keywords: bone marrow, idiotypes, lymphoid organization, memory, repertoire development
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Introduction |
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One particular response exhibiting all these characteristics is the anti-Ars response of A/J mice immunized with p-azophenylarsonate coupled to keyhole limpet hemocyanin (ArsKLH) (2). Early after a primary immunization, anti-Ars antibodies are encoded by several genetic combinations, but as the response proceeds, a major canonical combination emerges. This dominant gene combination (CRIA idiotype) is made up of a V segment belonging to a VH J558 subfamily (VHidcr11), associated with DFL16.1 and JH2 segments. The light chain corresponds to the association of V10 and J
1. The secondary anti-Ars antibodies which display higher affinity for the antigen contain mainly somatic variants of this canonical combination (39).
We previously reported that in A/J mice either irradiated with protected limbs and autologously reconstituting, or lethally irradiated and reconstituted with naive syngeneic bone marrow or naive spleen cells, the secondary response to ArsKLH was altered (10). Indeed, despite normal titers of antibodies against Ars, the dominance of the CRIA idiotype was lost. Moreover, no affinity maturation was observed during the anti-Ars response. Interestingly, KLH-primed T cells or naive peritoneal B cells co-transfer could restore neither the idiotype expression nor the affinity maturation (10).
To understand the molecular basis of this altered secondary response, we sequenced the rearranged VDJ genes from a collection of hybridomas obtained during a secondary anti-Ars response of irradiated A/J mice with autologous reconstitution.
In agreement with the serological data, sequencing analysis showed that the anti-Ars mAb from those mice displayed features of a primary rather than of a secondary response: multiple gene segment combinations, low frequency of the canonical combination, predominance of the IgM isotype and a low frequency of somatic mutations (0.95% as compared to 3% in the usual anti-Ars secondary response).
In addition immunohistological studies revealed that the appearance of mature GC is delayed in irradiated autologously reconstituting mice. A 2 week delay is observed when compared to normal A/J mice immunized with antigen. Nevertheless, at the day of fusion both the size and the number of GC were normal. The different steps of this T cell-dependent immune response which might be affected by irradiation, and possibly responsible for this altered response, are discussed.
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Methods |
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Reagents
mAb CRIA+ anti-Ars 93G7 (11) and 3665 are of A/J origin. mAb 3665 is encoded by the unmutated canonical combination (VHidcr11DFL16.1JH2 and V10J
1) (8). The 2D3 idiotope was located on the second complementarity-determining region from VHidcr11; E3 and E4 have been associated with the DFL16.1JH2 segments. The idiotopes are recognized by a panel of mAb2 of BALB/C origin named as the target idiotope (1113).
KLH (Calbiochem Berhing, La Jolla, CA) and BSA (Sigma, St Louis, MO) were conjugated to arsanilic acid (Sigma) as described by Nisonoff (14). L-Tyrosine (Sigma) was coupled to arsanilic acid as described (15).
Fusion and screening of hybridomas
Nine partially shielded and irradiated A/J mice were immunized with Ars coupled to KLH 1 day after irradiation by an i.p. injection of 100 µg of antigen emulsified in complete Freund's adjuvant (v/v) and post-challenged 2 weeks later with the immunogen emulsified in incomplete Freund's adjuvant. Two weeks after the second immunization, mice received 100 µg of ArsKLH in saline and were sacrificed 3 days later. Spleen cells were fused with the SP2/0 myeloma cells according to standard methods.
Hybridomas were first tested for binding to ArsBSA in a standard solid-phase ELISA. Positive hybridomas were cloned 3 times by limiting dilution. Ascites were produced in CAF1 mice for hybridomas isolated from S and G mice. These ascitic mAb were purified on DEAE columns, and tested for the expression of CRIA determinants and for Ars affinity in a competition immunoassay.
Ars-binding assays
Microtiter plates were coated with 2 µg/ml of ArsBSA overnight at 4°C, blocked with 1% BSA for 2 h at room temperature and incubated overnight at 4°C with serial dilutions of sera, supernatants or purified mAb. The assay was developed using sheep anti-mouse-POD Fab fragments (Boehringer-Mannheim, Mannheim, Germany) and revealed by a peroxidase substrate (OPDA; Sigma). Binding of serial dilutions of the purified mAb 3665 (CRIA+) provides a standard curve to estimate the anti-Ars antibody titers.
The isotypes were determined as described above using anti-mouse isotypes IgG2a-POD, IgGl-POD and IgM-POD (Sigma).
Expression of CRIA determinants
Idiotypic analysis was performed using microtiter plates coated with 50 µl of a predetermined amount of 2D3 (1.62 µg/ml), E3 (1.21.6 µg/ml) or E4 (0.61 µg/ml) mAb2 anti-CRIA antibodies. Binding of biotinylated 3665 to the various mAb2 was inhibited by prior incubation with serial dilutions of sera, supernatants or mAb overnight at 4°C. Inhibition of the binding of biotinylated 3665 on each mAb2 by serial dilutions of unlabeled 3665 provides standard curves to calculate the concentrations of idiotopes in µg of 3665 equivalents/ml.
Relative affinity determinations
Relative affinities were determined as previously described (16). Briefly, microtiter plates were coated with differentially substituted ArsBSA (2 µg/ml) and serial dilutions of samples were incubated overnight at 4°C [volumes of test samples (µl) giving 50% binding are determined]. Relative affinities of anti-Ars serum antibodies are expressed as a ratio of the serum volume giving 50% of maximal binding to ArsBSA highly substituted (40 Ars per carrier) divided by the volume required for 50% binding to ArsBSA lightly substituted (7 Ars per carrier).
Measurement of apparent affinity (Ka) was performed using hapten-inhibition enzymatic assays, as described elsewhere (15). Briefly, microtiter plates were coated with highly arsanylated (40 Ars molecules per carrier protein) or lightly arsanylated (12, 7 or 2 Ars molecules per carrier protein) BSA (2 µg/ml). Then 25 µl of serial dilutions of haptenic competitor ArsTyr (102 to 107 M) were dispensed on microtiter plates and a constant amount of purified mIgG1, predetermined to allow 70% of Ars binding, was added. Determination of Ka was performed as previously described and using computer assisted curve fitting (15,17).
RNA isolation and PCR amplification of heavy chain transcripts
Total RNA was extracted from 12x106 hybridoma cells by the single-step method using guanidium thiocyanate phenolchloroform (18). The total RNA yield was reverse transcribed into cDNA using a random hexamer primer (Pharmacia LKB, Bromma, Sweden) and SuperScript RNAse H reverse transcriptase (Gibco/BRL, Life Technologies, Eggenstein, Germany), in a total volume of 20 µl. First-strand cDNA (3 µl) was used for PCR amplification. The PCR reaction was carried out for 35 cycles under standard conditions (preheating 3 min at 80°C, denaturation 1 min at 94°C, annealing 2 min at 65°C, extension 3 min at 72°C) in a final volume of 100 µl containing 10 mM TrisHCl, pH 8.3, at room temperature, 50 mM KCl, 1.5 mM MgCl2, 0.001% (w/v) gelatin, 200 µM of each dNTPs, 2.5U Taq polymerase (GeneAmp PCR reagents kit; Perkin-Elmer Cetus, Norwalk, CT), 5% DMSO and 100 ng of each amplification primers (Appligen, Illkirch, France).
Oligonucleotide sequences correspond to the murine
VH consensus (5'-AGGT(C/G)(A/C)(A/G)CTGCAG(C/G)AGTC(A/T)GG-3'), Cµ (5'-GAGAATTCCAGGAGACGAGGGGGAAGAC-3') and C (5'-GAGAATTCCAGGGGCCAGTGGATAGAC-3').
Purification and cloning of the PCR products
The PCR products were size-fractionated by electrophoresis in 1% low-melting point agarose gel (FMC Bioproducts, Rockland, ME). Specific bands were excised and digested by gelase (Epicentre Technologies, Madison, WI). The purified PCR products were ligated into an EcoRV digested and ddT-tailed BlueScript vector (pBlueScript was purchased from Stratagene, La Jolla, CA). The ligation mixtures were used to transform MAX efficiency DH5 competent cells (Gibco/BRL, Life Technologies). Bacteria were grown in LB medium supplemented with 50 µg/ml X-Gal, 1 mM IPTG and 100 µg/ml ampicillin. Positive colonies were screened by white/blue coloration and plasmids were extracted using a Plasmid Mini-kit (Qiagen, Chatsworth, CA). Purified plasmids were further screened by restriction analysis and the recombinant plasmids were sequenced in both directions using Taq dye-deoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, CA; Sequencer ABI).
Analysis of DNA sequences
To exclude Taq polymerase nucleotide misincorporations from somatic mutation analysis, two independent PCR reactions were performed for each hybridoma and three plasmids derived from the cloning of each PCR product were sequenced on both strands. Sequences shown correspond to the consensus of six plasmids for each hybridoma.
Clonal relatedness were determined according to the CDR3 sequences. Among different clonally related sequences, only independent mutations were counted to calculate the percentage of mutation.
For hybridoma cells expressing V(D)J rearrangements different from the canonical combination (VHidCR11DFL16. 1JH2), the nature of D and J segments was determined using the Wu and Kabat database (19), whereas the putative corresponding germline VH gene segments were determined by a search in EMBL (release 53, 12/97) and GenBank (release 104, 12/97) data banks, using the Wisconsin package program (version 8.1, March 96). The sequences reported in this paper have been deposited in the EMBL database (accession nos AJ229149AJ229175).
Immunohistochemistry
Spleens from irradiated and control mice were removed and embedded in Tissue-Tek OCT compound (Sakura, Finetek, Ewap, Netherlands) by flash-freezing with liquid N2. The frozen tissues were stored at 80°C. Sections (810 µm) were cut with a cryostat (Reichert frigocut 2800) and mounted onto gelatin-coated slides. They were stored at 20°C.
For staining, spleen sections were allowed to air-dry for 10 min, fixed in ice-cold acetone for 10 min and rehydrated with PBS (0.01 M sodium phosphate, 0.9% NaCl, pH 7.3). Endogenous peroxidase activity was neutralized by a 10 min incubation with 0.3% H2O2 before staining.
Sections were stained with biotinylated peanut agglutinin (PNA; Vector, Burlingame, CA) or biotinylated rat Ig anti-mouse IgD (LOMD6, a generous gift from Professor H. Bazin).
Peroxidase-conjugated streptavidin labeling was performed using an ABC kit (Zymed, San Francisco, CA) according to the supplier's protocol. Peroxidase was developed by using the aminoethyl carbazole substrate kit (Zymed). Finally, sections were counterstained with hematoxylin, dehydrated and mounted.
In double staining, the binding of biotinylated anti-IgD was visualized by using streptavidinalkaline phosphatase (ABC kit, Vectastain; Vector) followed by enzymatic detections with vector blue kit III (Vector) in 0.1 M TrisHCl, pH 8.5, according to the supplier's protocol, in the presence of levamisole (Vector) inhibiting endogenous phosphatases. In double staining, sections were not counterstained.
GC were counted on different spaced sections with an optical microscope (detailed in results). At day 11 of the immune response, the spleen sections of irradiated mice were smaller as compared to controls and the ratio (spleen section of irradiated mice/controls) was 0.8.
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Results |
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Generation and characterization of secondary anti-Ars mAb from irradiated A/J mice
Hybridomas were generated from spleen cells of nine immunized mice and screened using high-density haptenated carrier protein, in order to reduce the selection bias for high-affinity antibodies. Twenty-seven hybridomas were studied and each mAb was characterized. Each mouse is designated by a letter and each clone by a number. The results are depicted in Table 1. Surprisingly 14 mAb exhibit the IgM class, the other 13 are characterized by the
1 isotype. The high number of IgM antibodies is not the result of a biased selection, but corresponds to the high concentration of IgM antibodies in the serum on day 10 of the secondary response (data not shown). Eighteen mAb do not express the CRIA idiotopes 2D3, E3 and E4, while nine still express some idiotopes, albeit weakly. Apparent affinities could not be measured for IgM mAb. For IgG1 antibodies, two of them (S25 and S32) had significantly higher affinities than 3665, which is the germline canonical combination (Table 2
). It will be shown below that these two antibodies are produced by clonally related subclones.
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Six independent clones are encoded by genes belonging either to a J558 family different from VHidcr11 or to the 7183 family. The three other subclones use rearranged VH found in the anti-influenza response or the anti-DNP response (20,21). They belong to the SB3.2 family (22), which corresponds, with the exception of one insertion, to the VH of a minor recurrent idiotype CRIC (90% identity with VH 3660) (23). The D segment from this mAb is unusual. This D segment does not correspond to any known D. However, the first part of this D (TGGGAC) corresponds to the 5' end of DQ52, while the 3' end of the D segment (GTAGG) is present in the non-coding strand of DSP2.6. Therefore we propose, in agreement with the data of Meek, that the D of this antibody stems from a DD fusion (24). We cannot exclude the existence of undiscovered D segments in the A/J genome.
Obviously this repertoire is much more diversified than the repertoire of normal immune A/J mice and looks in secondary, more like the normal primary response to Ars.
The shift towards the predominance of the canonical combination does not occur in irradiated recipients with autologous reconstitution during the secondary or even the tertiary response (10).
Somatic mutations in the VH repertoire from the secondary anti-Ars response in irradiated A/J mice
The different VH segments identified as VHidcr11 have been used to study somatic mutation rates (see Table 3). Mutation rates were derived from the number of base substitutions between every VH and the germline sequence of VHidcr11. However, when several VH had been shown to be clonally related only independent substitutions were taken into account.
In the 17 subclones using a rearranged VHidcr11, two were unmutated, six slightly mutated (<3 substitutions) and nine subclones were more heavily mutated (310 substitutions). The overall mutation rate is close to 0.95% (a total of 45 independent base substitutions for 4739 positions) significantly higher than the PCR rate error (ranging from 0.15 to 0.2%).
Two subclones, S25 and S32, were highly mutated (respectively 5 and 10 substitutions resulting from 11 independent mutations) and give rise to antibodies with a significantly higher affinity for Ars than the germline 3665. Furthermore these subclones exhibit some substitutions known to be recurrently associated with the affinity maturation of A/J mice in the Ars system: in S18 Asn52 is substituted by His, while Asn54 and Lys58 are respectively substituted by Lys and His in S32 (25).
GC in irradiated and control mice
A kinetic analysis of the GC development was performed in irradiated and control groups, at various times during the primary and secondary responses. GC were defined as groups of PNA+ B cells surrounded by IgD+ B lymphocytes. During the early response in irradiated mice (the first 2 weeks), white pulp is largely reduced (Fig. 3a) as compared to controls (Fig. 3b
). GC in irradiated mice are rare and atypical (small size clusters) as shown in Table 4
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Discussion |
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We previously reported that the secondary anti-Ars response in partially shielded and irradiated A/J mice was altered, despite the production of high titers of Ars-specific antibodies. Indeed, the expression of the recurrent and dominant CRIA idiotype is lost, and a lack of affinity maturation is observed (10). In those mice immunized with ArsKLH, the co-transfer of KLH-primed T cells or naive peritoneal B cells failed to restore the expression of the dominant idiotype (10).
To determine the molecular basis of this phenomenon, we analyzed the anti-Ars secondary response of partially shielded and irradiated A/J mice, and a collection of hybridomas was prepared. Serological data confirmed the loss of CRIA and the absence of affinity maturation described above.
From nine mice, 27 different hybridomas were analyzed. It was estimated that 20 belong to independent clones. Whereas two mAb expressed almost all the CRIA idiotopes, the vast majority of anti-Ars mAb did not exhibit CRIA idiotopes. Despite the inherent bias in hybridomas selection, these mAb seem to reflect the overall serological response of irradiated mice.
In strong contrast with the normal anti-Ars secondary response of A/J mice, the canonical combination was only found in three independent clones out of 20. The heavy chain repertoire from the anti-Ars response is very large, and includes eight D segments and all four JH segments. The VHidcr11 is used 17 times. The other VH genes belong to the J558, 7183 or SB3.2 (3660) families.
The anti-Ars mAb were next analyzed for the presence of somatic mutations in their VH genes. This study included 13 independent clones for which the germline counterpart was clearly identified (VHidcr11). The overall frequency of somatic mutations was low (0.95%) when compared to normal secondary response (3%) (7). While eight subclones were almost unmutated (02 mutations per sequence), several subclones (G15, S25, S32 and B30) displayed a low but significant number of nucleotide changes. The subclones S25 and S32, which have rearranged JH1 instead of JH2, when referred to the canonical combination, are the only ones to display the features of a normal secondary response (affinity maturation, somatic mutations). Moreover the subclone S32 exhibits two recurrent mutations involved in affinity maturation (Asn54 Lys and Lys58
His). The recurrent mutations usually found in normal secondary anti-Ars responses and leading to affinity maturation are absent in the majority of the sequences. Thus, the low frequency of somatic mutations and the nature of the substitutions may partially explain the lack of affinity maturation.
The `antigen drift' and the `repertoire shift' which take place during the GC reaction give rise to high-affinity somatic variants during the normal anti-Ars response of A/J mice (8,27,28). The importance of the GC formation in the Ars system of normal A/J mice is stressed by the fact that the injection of soluble antigen (ArsBGG 1µg) 6 days after immunization with ArsKLH, a treatment known to impair the function of GC, leads to a reduced anti-Ars response with an almost absence of the idiotype (unpublished data). While some GC could develop without hypermutation (29) our data likely result from a defective GC formation due to irradiation. We performed an immunohistological analysis of the spleen of the irradiated mice. Two major points emerge. First there is, in agreement with others, a significant delay (23 weeks) in the appearance of full blown GC. Second, at the fusion day, typical GC were present. The delay in GC formation can explain partially the altered immune response. The low but significant mutation frequency (0.95% as compared to 0.15% for the PCR error) and the abundance of IgM in the secondary response correspond to a shorter time window for somatic mutation and selection. The results also suggest that the GC observed at the day of fusion are functional.
The irradiation could affect each of the cell types involved in the development of GC: T cells, FDC, GC dendritic cells (GCDC) and the GC precursor B cells. As co-transfer of either thoracic duct T lymphocytes (TDL) (30) or carrier-primed T cells (10) failed to restore a normal response, it is unlikely that the altered response results from a T cell defect. Although FDC are resistant to high doses of radiation, functional properties could be affected, resulting in a failure to up-regulate the antigen-presenting cell function of GC B cells (31) and to rescue centroblasts from apoptosis. However, the restoration of normal GC formation in irradiated rats reconstituted with TDL (but not with bone marrow) suggests that the defect is rather related to circulating cells than to cells resident in the secondary lymphoid organs (30). In this respect, the recently isolated GCDC (32) could play a key role in the initiation of the GC reaction, through the introduction of the antigen in the primary follicle. It can be argued that the reconstitution of bone marrow-derived functional GCDC in the periphery would require a few weeks after irradiation.
In addition, irradiation and reconstitution could have altered the balance between different B cell subsets. It has been proposed that the B cell precursors of primary and secondary B responses belong to different lineages both established before antigen arrival. These two subsets express high and low membrane density of the heat stable antigen (HSA) molecule respectively. Data from our group strongly suggest that CRIA+ antibodies are synthesized by the secondary B cell lineage (HSAlow) (33), in agreement with the typical memory idiotype features of CRIA. A detailed PCR analysis of the HSAlow and the HSAhigh subsets of naive A/J mice shows that the CRIA precursors which have rearranged the canonical combination are detected almost exclusively in the HSAlow subset (Masungi et al., in preparation). Furthermore, Klinman has shown that only the secondary B cell lineage displays a very significant rate of somatic mutations and favors the formation of GC (34). Therefore, a temporary depletion of the HSAlow subset could explain both the loss of CRIA dominance and the defect in GC formation in irradiated mice. However, during the tertiary response to Ars in autologously reconstituted mice, the affinity maturation is partially recovered, while the CRIA expression is not restored (10). Those observations suggest that distinct mechanisms are responsible for the selection of high-affinity variants and idiotype expression. Indeed, we have shown that depletion of auto-anti-idiotypic B cells at birth leads to the loss of CRIA idiotype dominance in A/J mice subsequently immunized with Ars, despite normal titers of specific antibodies (35).
These different explanations (impairment of FDC functional properties, lack of GCDC, depletion or inactivation of the HSAlow subset) are not mutually exclusive but could strengthen each other to explain the unusual properties of the anti-Ars response following irradiation.
In conclusion, irradiated animals with autologous reconstitution display a prolonged primary response to ArsKLH, with clonal expansion and elevated anti-Ars titers, but delayed affinity maturation and GC formation. This is reminiscent of the immune response in aged mice, where the rate of somatic mutations is very low, while some GC are still present (36). Similarly, the affinity maturation is low in Xenopus, where GC are absent (37).
Our results might have practical implications to improve the recovery of normal immune response in patients after bone marrow transplantation, since patients grafted with bone marrow exhibit a deficiency in somatic mutations as compared to healthy controls (38,39). The identification of the cell subsets affected by irradiation and responsible for the alteration of repertoire and affinity selection is currently assessed by transfer experiments.
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Acknowledgments |
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Abbreviations |
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Ars | p-azophenylarsonate |
KLH | keyhole limpet hemocyanin |
CRI | cross-reactive idiotype |
FDC | follicular dendritic cell |
GC | germinal center |
GCDC | GC dendritic cell |
HSA | heat stable antigen |
PNA | peanut agglutinin |
TDL | thoracic duct lymphocyte |
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Notes |
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Received 3 December 1998, accepted 7 April 1999.
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References |
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