1 Department of Immunology, The Wenner-Gren Institute, Arrhenius Laboratories for Natural Sciences, Stockholm University, 106 91 Stockholm, Sweden.
2 Division of Clinical Immunology, Karolinska Hospital, 171 76 Stockholm, Sweden
Correspondence to: C. Fernández
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Abstract |
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Keywords: dextran B512, Ig repertoire
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Introduction |
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Another mouse strain, the CBA/N mouse, was also found to be non-responder to dextran but the mechanism of unresponsiveness was different from the other non-responders. CBA/N mice are genetically identical to CBA mice with the exception of a mutation in their X chromosome. This mutation affects the response to dextran and to other thymus-independent (TI)-2 antigens but it is not supposed to affect the Ig genetic background of the mice. CBA/N were unable to respond to native dextran or to any hapten coupled to it. Our interpretation of these results was that these mice could not respond to the polyclonal activating properties of native dextran B512 (1). This hypothesis was reinforced by the finding that CBA/N mice could overcome the state of unresponsiveness and were able to respond when dextran was given as a thymus-dependent (TD) antigen (4). Thus, the unresponsive state of these mice was not due to a defect in the Ig repertoire but rather to their inability to respond to the activating signals of TI-2 antigens.
Restricted immune responses to a number of antigens have been described (for review, see 5 and References therein). Usually, the restriction is broken during the maturation of the immune response; however, for some antigens the restricted pattern remains. In C57BL mice the response to dextran is restricted to the preferential expression of the VHB512 and the VOX1 gene combination (6). BALB/c mice also respond to dextran B512 in a restricted manner but these mice use another gene of the VH J558 family, i.e. the 19.1.2 VH gene (7). Interestingly, the restricted response in BALB/c mice is also linked to the use of the V
OX1 gene characteristic of the response in C57BL mice. The V
OX1 gene was first described in antibodies produced in response to oxazolone (8) but it is apparently an ubiquitous gene found assembled with various VH genes. The direct contribution of V
OX1 to dextran binding has not been assessed.
All anti-dextran B512 mAb described to date, >200 (7, 911 and the authors unpublished results), use light chains. No anti-dextran antibody using
has ever been observed. The most trivial explanation of the lack of
light chain usage in dextran responses is that in mice
light chains contribute to only 510% of the antibodies in serum. However, it could also be that the use of the light chain is not random because
-bearing antibodies are not compatible to dextran binding or alternatively that the
light chains cannot assemble with the heavy chain variable domains used in response to dextran.
To ascertain whether the restriction of the use of V genes in response to dextran B512 was more stochastic or due to other factors, we have studied the response to dextran B512 in mice where the C
domain has been disrupted. These mice are unable to express
light chains and their humoral antibodies bear light chains of the
type. We found that the C
knockout mice are apparently normal regarding the expression of VH genes and are also perfectly able to mount an immune response to a number of antigens. However, these mice were unable to respond to dextran given under optimal immunogenic conditions. We discuss the possible mechanisms for this third type of unresponsiveness to dextran B512.
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Methods |
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Antigens and immunizations
Native dextran B512 (TI antigen) with a mol. wt of 540x106 was obtained from INC Pharmaceuticals (Cleveland, OH). A TD form of dextran was obtained by conjugating dextran with a mol. wt of 103 (35 glucose units) to the protein chicken serum albumin (CSA) (Sigma, St Louis, MO). Dextran was conjugated to hydrazide-CSA via its terminal aldehyde group using reductive amination and was kindly provided by Dr Christian Krog-Jensen. DNPdextran (DNPDx) was obtained by coupling DNP-lysine to CN-activated native dextran. Cholera toxin (CT) was obtained from List Biological Laboratories (Campbell, CA). Mice were immunized i.p. with 10 µg native dextran, 50 µg DNPDx or 100 µg CSADx. All antigens were administered in soluble form, alone or together with CT, 1 µg per mouse and dose both for primary and secondary immunizations. For studies of secondary responses, mice were immunized a second time after a resting period of 4 weeks. Mice were bled by retro-orbital puncture under light anesthesia at various points after immunization. Day 10 after primary immunization and day 7 after secondary immunization were shown to represent optimal splenic as well as humoral responses for all groups. Serum was prepared after centrifugation and tested in ELISA.
Detection of antibodies in serum with ELISA
ELISA was performed as described before (13). Briefly, ELISA plates (Costar, Cambridge, MA) were coated with 10 µg dextran T250, CSA or TNP-BSA. Sera was added at various dilutions starting with a 1/100 dilution. Bound Ig was detected with alkaline phosphatase-labeled goat anti-mouse IgM and IgG (Southern Biotechnology Associates, Birmingham, AL) and p-nitrophenyl phosphatase substrate (Sigma). OD values at 405 nm were determined using an Anthos Reader 2001 (Anthos Labtech Instruments, Saltzburg, Austria).
Preparation of spleen sections and in situ immunofluorescence
Spleens were removed and immediately frozen in liquid nitrogen and stored at 70°C. Spleens were embedded in Tissue Tek OTC compound (Miles, Naperville, IL) and cryostat sections (6 µm) were cut and mounted. The slides were air-dried for 3060 min and stored at 70°C until use. Cryostat sections were fixed for 15 min in ice-cold acetone. Subsequently, slides were rinsed in PBS and blocked with horse serum (5% in PBS) for 30 min. Sections were stained with FITC-conjugated dextran (Pharmacia, Uppsala, Sweden) and biotin-conjugated peanut agglutinin (PNA) developed with streptavidinTexas Red conjugate (Vector, Burlingame, CA). This double staining has been shown to specifically detect dextran-binding B cells by analysis of PNA/FITCDx double-stained spleen cells from dextran-immunized mice with multiparameter FACS (14). The sections were incubated with biotinylated reagents as indicated for 60 min, and then incubated with avidin-conjugated Texas Red and fluoresceinated reagents for 60 min. All stainings were performed in a humidified chamber protected from light.
RNA, cDNA and PCR preparations
Total RNA was extracted from spleen cells with TRIzol reagent (Gibco/BRL, Life Technologies, Stockholm, Sweden). Preparation of the first-strand cDNA was performed using the forwards Cµ primer 5'-GCTCCTGCAGGAGACGAG-3'and the reverse transcriptase Superscript II (Gibco/BRL, Life Technologies). PCR amplification was performed using the forwards Cµ primer described above and a universal backwards primer, VHM, 5'-AGGTGCAGCTCGAGG/CAGTCTGGA-3'. Primers were synthesized by Scandinavian Gene Synthesis (Köping, Sweden) and by Gibco/BRL, Life Technologies. The following PCR program was used: one hot start cycle at 99°C, 5 min for denaturation, addition of Taq DNA polymerase (Gibco/BRL, Life Technologies) at 85°C followed by 30 cycles for 1 min at 94°C, 2 min at 58°C and 1 min at 72°C.
Probe preparation
The VH J558 and the VH Q52 probes were prepared from the anti-dextran B512 hybridoma cell lines F4H2 and F12H3 respectively (6). For amplification of the VH J558 probe we used the J558 forwards 5'-ATTAGACTGCAGAGTCCT-3' and J558 backwards 5'-AGGTCCAGCTGCAGCCA-3' specific primers. For the VH Q52 probe we used the specific Q52 forwards 5'-TAGGCTCTTGGAGTTGTC-3' and the universal VHM backwards primers. The PCR products were run on an LMP agarose (Gibco/BRL, Life Technologies) gel, and the VH J558- and VH Q52-specific bands were cut out from the gel and labeled with [32P]dCTP (Amersham, Little Chalfont, UK) using a Megaprime DNA labeling system (Amersham).
VH gene family utilization analysis
RNA was obtained from lipopolysaccharide (LPS)-derived B cell blasts. Spleen cells from individual C57BL/6 and C57BL/6.CT mice were cultured in the presence of the polyclonal B cell activator LPS. The cells were put in culture at a concentration of 1x106 cells/ml together with 25 µg/ml LPS and harvested at day 3 after the initiation of the cultures. Induction of B cell blasts is a known method to obtain RNA enriched for Ig messenger of random specificities.
Aliquots of 30 µg RNA were separated on an 1% agarose gel and blotted onto a Hybond-XL nylon membrane (Amersham). Prehybridizations and hybridizations were done at 42°C as described (15). Washing was performed under low-stringency conditions in 2xSSC, 0.2% SDS solution at room temperature followed by 30 min at 42°C and 60 min at 60°C as described before (16). The membranes were exposed in a PhosphoImager (Pharmacia/Amersham Biotech, Uppsala, Sweden) and quantified with ImageQuant software (Molecular Dynamics AB, Uppsala, Sweden) or exposed to X-Omat L film (Kodak, Rochester, NY) and scanned in an LKB 2202 ultrascan densitometer (Kabi-Pharmacia, Uppsala, Sweden). Membranes were melted by washing in 0.1xSSC, 0.1% SDS at 90°C until no radioactivity signal was detected and were then hybridized to other VH DNA probes.
The intensity of the various individual bands is expressed as a relative value. After scanning, the value of the VH J558/ VH Q52 band intensity ratio in C57BL/6 was arbitrary set to 1.00 and all the other values were expressed in relation to this. We decided to express our values in this manner to avoid errors caused by variations in the amounts of RNA loaded, probe labeling and exposure from various experiments. For each value given, four membranes were used. SD were calculated from the different experiments.
VH J558 subfamily utilization analysis
This assay was done as described before (16). Briefly. PCR products from individual C57BL/6 and C57BL/6.CT were digested with PstI (Gibco/BRL, Life Technologies), separated on a 2% agarose and blotted onto a Hybond-N+ nylon membrane (Amersham). Prehybridizations, hybridizations and washings were done as described above. The presence of an internal PstI site defines the various VH J558 gene subfamilies in mice carrying the Ig CHb allotype (16,17). The estimated random expression of VH J558 genes according to the PstI restriction fragment length polymorphisms (RFLP) is as follows: 50% of the genes would display a 380 bp fragment which represents the VH subfamilies v186.12, v3 and v130; 25% of the genes would have a 200 bp fragment representing the vGAM 3.0, G4D11 and 13.2 subfamilies; and 25% would have the 260 fragment representing the 205.12 subfamily. This subfamily includes the VHB512 gene expressed preferentially in response to dextran in association with the V
OX1 gene (6).
To assess the relative proportions of the three major VH fragments of 380, 260 and 200 bp we used a LKB 2202 ultrascan densitometer (Kabi-Pharmacia). Although minor differences in the expression cannot be detected by this method, the RFLP indicates if major changes in fragment distribution (and consequently major differences in subfamily utilization) have occurred (16).
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Results |
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The primary and secondary immune response to native dextran, TI form, is dominated by the presence of IgM, therefore we show here only IgM (Fig. 1). For the TD responses we show both IgM and IgG production in secondary responses (Fig. 2A and B
). Our results show that the C57BL.C
T mice could not respond to dextran B512 under any of the immunization conditions used. Since these mice were put under the most favorable conditions of responsiveness and the antigen was given as a TI antigen (direct B cell activation) or TD highly immunogenic form, we concluded that the C
T mice may be immunologically incompetent or that they are unable to specifically respond to the dextran epitope.
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We addressed this question by testing the response to dextran in C57BL.CT mice where the lpr mutation had been introduced. The lpr mutation is known to affect the expression of the Fas protein which is involved in the delivery of apoptotic signals to the cell. One of the consequences of the lpr mutation is that cells from mutated animals have a prolonged life span due to a deficient apoptosis mechanism (21). We reasoned that a lpr-induced increase in the life span could contribute to the increase in the B cell clone size and, therefore, could make the response to dextran more easily detectable. However, this was not the case. We show in Figs 1 and 2
that the introduction of the lpr mutation was not sufficient to change the pattern of unresponsiveness in the C57BL.C
T mice.
C57BL.CT mice are immunologically competent to respond to other antigens
To ascertain whether the CT mice were immunologically incompetent in a general manner, we analyzed the humoral response of the mice immunized with CSADx but this time we looked at the response against the protein epitopes of the conjugate. The response against CSA in the C57BL.C
T mice was of the same magnitude as the response observed in the wild-type mice (Fig. 3
). Thus, the C57BL.C
T mice react differently to two epitopes on the same molecule. This supports two conclusions: (i) that the C57BL.C
T mice are immunocompetent, as it has also been shown by others (22), and (ii) that the CSADx conjugate was clearly immunogenic under the conditions of this study.
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C57BL.CT mice cannot mount a germinal center (GC) reaction against dextran
The immune response to dextran B512 is characterized by the appearance of dextran-specific B cells in the spleen where ~50% of them are localized inside the GC (14,19). We have described that splenic dextran-specific GC can be observed after immunization with both the TD and the TI forms of dextran, and that the GC reaction is dramatically enhanced by simultaneous administration of CT and the antigen (19). However, in CT mice immunized with native dextran and CT (Fig. 5
) and with CSADx (data not shown) GC areas were apparent but these GC were devoid of dextran-specific B cells. Dextran-negative GC were probably induced against CT or against the protein carrier in the TD immunized animals. Dextran deposits were found in the mutant and in the wild-type mice indicating that dextran was delivered to the immunocompetent areas in the spleen (Fig. 5
).
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Discussion |
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Independently on the validity of the various explanations discussed above, the importance in the immune response to dextran of the heavy chain and the VHB512 gene is suggested by the fact that mice with Ig CH allotype different from C57BL are low or non-responders to dextran. On the contrary, the VOX1 gene is a rather ubiquitous gene, frequently found not only in the response to dextran in mouse strains different from C57BL (7,10) but also in response to unrelated antigens such as 2-phenyloxazolone (24). However, it appears as if the light chain may also play a major role in the response to dextran since, as we describe here, mice with the C
locus disrupted by gene targeting are non-responders to dextran B512. These mice are apparently normal in the immune response to other antigens but are unable to mount an immune response to dextran administered not only as a TI but also as a TD antigen. The lack of responsiveness is specifically directed to the dextran epitopes since these mice are fully competent to respond to other antigenic structures present in the same immunogenic molecule. Furthermore, native dextran (TI form) can function as a carrier for the hapten DNP in the C57BL.C
T mice.
The restricted response against dextran B512 appears to be rather unique in this respect because in responses against other antigens, defined carbohydrate epitopes and haptens, the V light chain could reconstitute the response by utilizing different VH genes (22,25 and this study). Furthermore, the Ig repertoire for
-bearing antibodies has been found to be exceedingly diverse as it has been demonstrated by, for example, the enumeration of antigen specific
+ B cell colonies induced by LPS (26). Therefore, it is interesting that the C57BL.C
T mice are unable to compensate the repertoire with
-bearing antibodies and mount an immune response to dextran.
An interpretation of these results is that it may not be possible to generate dextran-specific -bearing antibodies and, in consequence, that the C57BL.C
T mice have a hole in the Ig repertoire. It has been suggested that the higher
/
ratio in normal mice could not only be caused by the limited diversity of the
locus alone but it may also be the result of a restricted ability of the V
to associate to various VH segments. Non-stochastic pairing between the dominant
subtype V
1 + VH are observed in the periphery of
-deficient and normal mice (2729).
An alternative explanation is that canonical antibody molecules raised against dextran in the high-responder mouse strains might be the optimal antibodies to be generated. Thus, any changes in the structure of these molecules such as mutations or modifications in the VH + VL combinations would lead to a decrease in the affinity for dextran and eventually end in the destruction of the binding capacity of the antibodies. This could even result in the creation of undesired specificities and these antibodies would, therefore, be negatively selected. Several groups such as Cohn and Langman favor this selection theory. These authors have argued that a biased murine :
ratio may represent the result of strong selection by antigen (30).
We have at present no satisfactory explanation for the inability of the CT mice to mount an immune response to dextran B512. A pure mechanistic explanation is conceptually difficult to understand, and we, therefore, favor a selection and specialization of the
and
repertoires as the cause for the unresponsiveness to dextran B512 in C57BL.C
T mice. However, one way of exploring the validity of the mechanistic theory could be to use phage expression libraries made by the combination of all VH genes with
light chains because under these circumstances no internal selection should operate.
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Abbreviations |
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CSA chicken serum albumin |
CT cholera toxin |
DNPDx DNP-conjugated dextran |
GC germinal center |
LPS lipopolysaccharide |
PNA peanut agglutinin |
RFLP restriction fragment length polymorphism |
TD thymus dependent |
TI thymus independent |
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Notes |
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Received 17 September 1999, accepted 29 November 1999.
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References |
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