Immunogenetics 39: 404-411, 1994 _ / / /H / / / / / /O - genetics

© Springer-Verlag 1994

The IgG2a antibody response to thyroglobulin is linked to the Igh locus in mouse

Rudolf C. Kuppers 1, Leonardo D. Epstein2, Ingrid M. Outschoorn3, Noel R. RosO

1 Department of Immunology and Infectious Disease, The Johns Hopkins School of Hygiene and PuNic Health, 615 N. Wolfe St., Baltimore, MD 21205, USA

2 Department of Biostatistics, The Johns Hopkins School of Hygiene and Public Health, 615 N. Wolfe St., Baltimore, MD 21205, USA 3 Centre Nacional de Biologia Celular y Retrovirus, Institute Carlos III, Majadahonda, Madrid 28220, Spain

Received December 7, 1993/Revised version received January 10, 1994

Abstraetl The IgG-subclass usage by several strains of mice in the response to immunizat ion with mouse thy- roglobulin (mTg) was examined in the experimental autoimmune thyroidit is model. Whi le the subclass usage by most mouse strains was similar, the Igh b al lotype- bearing mice consistently produced lower IgG2a levels to mTg. Using CBA-Igh b congenic and recombinant inbred strains of mice, the lower level of IgG2a in the Igh b mouse was mapped to the Igh locus. The regulat ion of IgG2a appeared to be cis controlled, as the CBA x C57BL/6F1 mouse also produced reduced IgG2a of the Igh b (B6) al lotype but not of the IghJ (CBA) al- lotype.

Introduction

In the mouse model of experimental autoimmune thy- roiditis (EAT), induced by thyroglobulin, genes within and without the major his tocompat ibi l i ty complex (MHC) locus have been implicated in the regulat ion of the disease process (Kuppers et al. 1988 a; Vladutiu and Rose, 1971; Tomazic et al. 1974; Ben-Nun et al., 1980; Beisel et al. 1982a, 1982b). Genetic loci outside the MHC locus that influence EAT have not been mapped. The immunoglobul in heavy chain gene locus, Igh, is of part icular interest, since it has been shown to influence

Correspondence to: R. Kuppers, SAIC, 5340 Spectrum Dr., Suite N, Frederick, MD 21701-7337, USA.

antibody responses to several antigens (Seman and Zilberfarb 1979; Lifshitz et al. 1980; Brown et al. 1981; Ju et al. 1981; Nutt et al. 1981; Van Snick 1981; Mongini and Paul 1982; B6naroch et al. 1993; Van Snick et al. 1983). In this study, we report the mapping of the control of serum Tg-specif ic IgG2a concentra- tions to the Igh locus.

Materials and methods

Mice and immunization. All mice were obtained from the Jackson Laboratories (Bar Harbor, ME), except the CBA/Tufts and CBA- Ighb/Tufts strains which were bred in our facility, using breeding stock generously provided by H. Wortis, Tufts School of Medicine (Boston, MA). Mouse Tg was prepared as described previously (Kuppers et al. 1988b). Mice were immunized once with 50 gg of mTg in complete Freund's adjuvant. Animals were bled between days 28-34, and these samples were used for mTg-specific titer measurements.

ELISA assays for total mTg-specific and mTg subclass-specific IgG subclass titer. ELISA for total mTg-specific antibody. The ELISA assay for total mTg-specific antibody has been described pre- viously (Kuppers et al. 1988b). An alkaline phosphatase-con- jugated goat anti-mouse IgG (H+L) antibody (Sigma, St. Louis, MO; Jackson Immunochemical, PA) was used for detection. Sub- strate (p-nitrophenol phosphate, 1 mg/ml; Sigma) was added and the reaction stopped after a control serum reached a set OD value. The latter was used to standardized the end point of the reaction between plates and assays. The relative titer is defined as the log2 serum dilution giving an OD value of 0.100.

ELISA assay for IgG subclasses. The relative IgG subclass dis- tribution was measured, using an ELISA assay. MTg (5 gg/ml, overnight at 4 ° C) was used to coat the plates. After blocking with 1% bovine serum albumin (BSA), duplicate serial dilutions of the sera, normally starting at 1/40, were made for each subclass to be assayed. After a further incubation overnight at 4 ° C, plates were washed and an alkaline phosphatase-conjugated goat anti-mouse IgG subclass-specific antibody (Southern Biotechnological As-

R. C. Kuppers et al.: Linkage of IgG2a usage to the Igh locus in EAT

sociates, Birmingham, AL) was added at the appropriate con- centration. The dilutions were normally 1/1000 for anti-IgG1 and anti-IgG2b, and 1/300 for anti-IgG2a and IgG3. This IgG2a- specific reagent will be referred to as pan-IgG2a-specific, as it recognizes all IgG2a allotypes examined. After 2 h, the plates were washed and substrate added. The reaction was stopped after a control serum reached a standard OD value. The titer was defined as above. It should be noted that this is a relative titer and not an absolute quantity of antibody. Comparisons can be made within a subclass, but not between subclasses.

The specificity of the polyclonal IgGl- , IgG2a-, and IgG2b- specific reagents were confirmed, using monoclonal antibodies (mAb) from CBA/J (Igh-lJ) and BALB/cJ(Igh-1 a) mice. We had no IgG3 mAb to verify this reagent; however, the reagent was active, as we could detect IgG3 in serum. We also tested the relative allotype specificity of these reagents, using CBA/J, BALB/c, and C57BL/6/J (B6), (Igh-1 b) mAbs of the IgG1, IgG2a, and/or IgG2b isotypes.

In addition, we used an allotype-specific monoclonal mouse anti-mouse IgG2ab-alkaline phosphate conjugate (PharMingen, San Diego, CA) at a 1/1000 dilution. The specificity of the latter was confirmed by using B6 and C3H antibodies.

Statistical analysis of recombinant inbred strains. A statistical model was developed to test whether the IgG2a subclass titers differed between the Igh b and IghJ allotype-bearing recombinant inbred strains of mice. The data were analyzed using a model based on the plot of the IgG2a subclass titer versus total titer of the Ighb and IghJ-bearing RI strains.

The data were organized into two groups according to allotype, where a (a = j, b) is the index for the two allotypes IghJ and Igh b, respectively. Let na denote the number of mice of allotype a (a = j, b) and let i (i = 1 ..... na) index the individual mice within allotype group a (a = j, b). Further, denote by zi, a the average of the two total titer determinations made on individual i in group a (i = 1,..., ha; a = j, b), and denote by Ca, i, k (k = 1, 2) the two subclass titer determinations made on individual i in group a. The data for this analysis were then the pairs (za. i; Ca, i, k). A plot of the data showed that in each group the pairs (za. i; ~a, i, k) were scattered about straight lines. Therefore, it appeared appropriate to model these data using the equation:

Ca, i, k = IXa 4- [~a~a, i 4- Ca, i. k, a = j, b; i = 1 ..... na; k = 1, 2, (1)

405

where Cta and ~a are the intercepts and slopes, respectively, of the lines corresponding to groups a : j, b, and a is the error in the individual measurements.

The model was further simplified, as the lines generated by the data, corresponding to each of the allotypes, were shown to be essentially parallel: that is, a model where their corresponding s l o p e s , [~b and [~j, are the same, i.e., Ca, i, k = O~a 4- ~'l~a, i 4- Ea, i, k, where [3 is the common slope of the two lines. A model with a common slope for the two allotypes allowed the use of the intercept of each line, c~j for allotype j and I~b for allotype b, as a measure of overall IgG2a response level for an allotype group, and therefore permitted using their difference cq-c~b as a measure of the overall IgG2a subclass titer difference between the allotypes. The parallel line analysis of covariance model ANCOVA (Seber 1977) allows an assessment of the hypothesis that the IgG2a subclass titer differs for allotype j and b by obtaining a point estimate and a confidence interval for cq-ab .

A more detailed inference was obtained using the ANCOVA model, by analyzing the 16 strains as independent groups. The resulting model is entirely analogous to equation (1), but the allotype index a (a = a, j), is replaced with a strain index s (s = 1 ..... 16), Ca, i, k : O~a 4- ~'~a, i 4- Ea, i, k. The strain-specific intercepts txs (s = 1 .. . . . 16), represent a measure of overall IgG2a response level of each strain. The separation between allotype j and b can be examined using da : A J - A b, where AJ is the average of the intercepts c~ for strains of allotype j and Ab is the average of the intercepts C(s for strains of allotype b.

Results

Subclass composition of mTg-specific antibodies. T h e r e l a t i v e I g G s u b c l a s s d i s t r i b u t i o n o f t h e a n t i b o d i e s to

m T g w a s i n v e s t i g a t e d in s e v e r a l s t r a ins o f m i c e d i f f e r -

i ng in Igh a n d H-2 h a p l o t y p e s a f t e r i m m u n i z a t i o n w i t h

m T g in C F A . T a b l e 1 s h o w s the r e l a t i v e s u b c l a s s d i s -

t r i b u t i o n fo r s e v e r a l m o u s e s t ra ins . I t c a n b e s e e n tha t

t h e t i t e r s v a r i e d c o n s i d e r a b l y a m o n g d i f f e r e n t s t ra ins .

T h e C 5 7 L , C 3 H . S W , B6 , H-2b; a n d D B A / 2 , H - 2 d an i -

m a l s a re p o o r - r e s p o n d e r m i c e , w h e r e a s t h e B 1 0 . B R ,

Table 1. IgG subclass distribution of anti-mTg antibodies in mice of varying Igh haplotypes.

Strain Haplotype* Relative IgG titer (logz)t

Igh-1 Igh- 3 Igh-4 IgG1 IgG2a lgG2b

IgG2a/IgG2b Ratio

C57L a - a 8.5 4.4 4.9 0.90 C57BL/6 b b b 9.0 < 4.0 7.8 < 0.50 B10.BR b b b 14.8 10.1 13.9 0.72 DBA/J c a a 7.9 5.8 6.2 0.94 SWR/J c - a 15.5 14.7 15.8 0.93 AKR/J d d a 13.1 12.3 11.6 1.06 A/J e e a 14.3 11.5 11.2 1.03 C3H/HeSn j - a 13.3 12.7 12.3 1.03 C3H.SW j - a 10.8 10.9 12.1 0.90 CBA/J j a a 10.8 10.9 14.0 0.94 C3H x B6 F1 j/b 12.9 12.9 14.0 0.92

* Igh haplotype based on Handbook on Genetically Standardized JAX Mice, 3rd edn. (Suppl 1), The Jackson Laboratories, Bar Harbor, 1982; (-) = not typed.

t The relative titer of each strain was determined by using an ELISA assay as described in the Materials and Methods.

406 R.C. Kuppers et al.: Linkage of IgG2a usage to the Igh locus in EAT

A

O

° ~

I--

O ° ~

t~

o v e

20

18

16

14

12

10

8

6

4 CBA-Tu

S t r a i n

[ ] IgG1 [ ] IgG2a [ ] IgG2b

CBA.Igh b

Fig. 1. The distribution of the IgG subclass response of CBA/Tu and CBA/Tu-Igh b mice to mouse thy- roglobulin. CBA/Tu and CBA/Tu-Ighb mice were immunized once with mTg and bled on day 21. The subclass titer to mTg was measured as described in the methods. The results are the mean titer of a group of five animals.

CBA, AKR, H-2k; A/J, H-2a; and SWR, H-2q mice are high-responder strains as previously reported (Vladutiu and Rose 1971). The overall antibody titer reflected the influence of the MHC haplotype.

IgG1 was the major subclass used in the mTg-spe- cific response. The IgG3 subclass was generally present in very low amounts and the data are not presented. IgG2b antibody was found in all strains and, in general, IgG2b levels were similar in these animals. In contrast, the IgG2a response of the Igh b allotype B10.BR (H-2 ~) high-responder and the low-responder B6 (H-2b) mice was significantly lower relative to the other subclasses. This is clearly shown by the IgG2a/IgG2b ratio. The IgG2a levels in the B6 were in most cases below the sensitivity level of the assay. These results indicate that, as in the response to certain other antigens, the Igh b allotype-bearing animals show a skewed utilization of the IgG2a subclass in their response to mTg.

Subclass distribution of the antibody response to mTg in the CBA and CBA-Igh b strains. The reduced IgG2a re- sponse in Black background animals might be attributed to the fact that these animals carried the Igh b locus. To verify this, we examined the CBA and CBA-Igh b strains.

The subclass distribution of mTg-specific antibodies was determined in the CBA (IghJ) and CBA-Igh b strains. Both these H-2k strains are good responders, differing at the Igh region. The results of these experiments are shown in Figure 1. It can be seen that the CBA-Igh b strain failed to produce a significant IgG2a response to mTg. While the CBA-Igh b strain has a very much re- duced IgG2a response, its overall response to mTg in

these experiments was slightly, but statistically (P = .01), higher than the CBA mouse, with titers of 16.4 and 15.4, respectively. These results further confirm that the IgG2a response to mTg is down-regulated in Igh b- bearing animals.

To test this possibility, CBA and CBA-Igh b mice were immunized with TNP-modified mTg. It has been shown that TNP can elicit an IgG2a response (Mongini and Paul 1982). Therefore, a strong IgG2a response to TNP as well as to mTg would be expected in the same mouse. As can be seen in Figure 2, an IgG2a response to mTg was not found. However, the IgG2a response to TNP was high. Immunization with KLH, shown pre- viously to induce an IgG2a response in the Igh b animal (L~iszl6 et al. 1985), also induced an IgG2a response to KLH (data not shown). The failure to mount an IgG2a response to mTg appears linked to the Igh locus, but can not be explained by an overall failure of an IgG2a re- sponse in these animals, since an IgG2a response is seen against TNR

Mapping of the mTg-specific IgG2a responsiveness to the Igh region. To confirm the mapping of this effect to the Igh region, BXH recombinant inbred mouse strains were used. Twelve BXH recombinant inbred mouse strains were examined, in addition to the two progenitor strains, C3H and B6, and the congenic C3H.SW (H-2b, IghJ) and the B10.BR (H-2 ~, Igh b) strains. Mice were immunized with mTg as described in the Methods and materials. Serum was taken on day 28 and analyzed by ELISA for total titer and relative subclass titer to mTg.

On the basis of preliminary studies, we determined that when the IgG2a mTg-specific subclass titer was

R. C. Kuppers et al.: Linkage of IgG2a usage to the lgh locus in EAT 407

MTg

CBA-Ig 6

0 IgG1 IgG2a IgG2b

B TNP-BSA

11

7

IgG1 IgG2a

[] CBA b [ ] CBA-Igh

IgG2b

Subclass

Fig. 2. The distribution of the IgG subclass response of CBA/Tu and CBA/Tu-Igh b mice to TNP and mTg after immunization with TNP-mTg. Mice were immunized once with TNP-conju- gated mTg in CFA. Mice were bled on day 21 and the subclass response to TNP and mTg measured, using TNP-BSA and mTg- coated wells, respectively, as described in the Methods. The results are the mean titer of five animals in each group.

plotted against total titer for each animal separately the data points for all the Igh3 and all the Igh b strains were each distributed more or less around straight, but dif- ferent lines. The results of this analysis (P-value of 0.988) supported the fact that the slopes of the plots of IgG2a subclass versus the total titer for each Igh allotype were the same, and therefore allowed the use of the ANCOVA model (see Methods). Figure 3 presents the calculated intercept values of the sixteen strains and their corresponding 95% confidence intervals. There is a vertical line for each strain. The abscissa of the vertical lines are the estimated intercepts, and the hortizonal line

represents the confidence interval of the corresponding intercept. In addition, H-2 and Igh genotypes are shown on this figure.

From Figure 3, one immediately sees that the IghJ strains, as a group, have higher intercepts than the Igh b strains. Statistically, the difference in separation be- tween the two allotype groups was obtained by using the average of the intercepts. The estimate of this difference is da = 4.62 and a 95% confidence interval for da is [4.35; 4.89], which excludes zero. This strongly sup- ports the conclusion that the IgG2a subclass titer of the IghJ strains is higher than the Igh b strains, and argues

408 R.C. Kuppers et al.: Linkage of IgG2a usage to the Igh locus in EAT

AIIotype I b b b bb b b j j j j j j j j j ] MHC b k b bb k k b b bk b k k k k

, 1 , "r I

BXH-2

I 1 1 , ,, I I , , , BXH-9 C3H.SW

, 1 1 , ,. I I ,, I

BXH-4 BXH- 12 , , 1 1 , , 1 , I I ~' I I I T I I 1F I

C3H BXH-6 BXH-7

, 1 , I V I

BXH-8 I 1 , I" i

BXH- 10 ., .-1 , ,' . 1 ,' I

B6 BXH-11 I 1 1 , ,, I I ,. ,

BXH-3 BXH-14

1 I ' I "

BIO.BR , 1 I T BXH- 1 9

I I I I I I i -6 -4 -2 0 2 4 6

Intercept and 95% confidence intervals

Fig. 3. Estimated intercept values of each BXH and progenitor strain. The intercept of each strain was calculated based on the assumption that the data was linear and the slopes of all groups were identical (see text). The intercepts are represented by vertical lines with horizontal lines showing the corresponding 95% confidence intervals. Different heights for the confidence intervals were chosen for the sake of clarity. Above the lines are the Igh allotype, and H-2 haplotype where high responders are H-2k animals and low responders are H-2b.

that the Igh locus influences IgG2a subclass titers in the response to mTg.

The fact that within an Igh allotype one sees a wide spread of intercept values suggests that other genes further influence the IgG2a response. Certain strains such as the B6 have much lower IgG2a antibody levels to mTg than most other Igh b strains. In contrast, the Igh k strain, BXH7, appears to cluster above other Igh k strains even though H-2 and Igh are shared (Fig. 3). While these differences may reflect the influence of other genes on the antibody response to mTg, we do not feel that the data provide strong enough evidence to further group the strains in term of IgG2a titers. Clearly, how- ever, the Igh b locus appears to influence the IgG2a re- sponse to mTg, but not necessarily other antigens such as the haptenic TNP group nor KLH.

The control o f IgG2a b response is cis acting. We ex- amined IgG2a allotype expression in the antibody re- sponse to mTg in the C3H x B6 F1 mouse. Using an IgG2ab-specific reagent and the pan-IgG2a reagent used in previous experiments, we determined the proportion of IgG2a b and IgG2aJ antibody in the F1 mouse. The total mTg-specific, the pan-IgG2a, and the IgG2a b endpoint titers were determined as described in the Methods. In order to determine the proportion of each allotype utilized in the mTg response of the F1, we needed to normalize the pan-IgG2a and IgG2a b titers. To do this, the ratio of the pan-IgG2a and IgG2a b OD reading of the B10.BR and CBA-Igh b mice, both high responders, was determined. As the B 10.BR and CBA- Igh b mice are both Igh b, the pan-IgG2a value and the

Ighb-2a represent the same amount of antibody. A cor- rection, therefore, can be made that would adjust for differences in reagent activity. The average of these ratios for individual mice was used to adjust the OD value of all pan-IgG2a OD values.

The proportion of the IgG2a response relative to the total mTg-specific titer in each animal was determined by dividing the IgG2a value by the total mTg-specific OD value. The mean of these data grouped by sex for the F1 animals are presented in Table 2. It can be seen from the B10.BR and CBA.Igh b animals that the nor- malized values give very similar results for the IgG2a/ mTg, IgG2ab/mTg, and 2ab/2a ratios, the latter differing. no more than 0.05, i.e., 1.04 and 0.99, respectively for these strains. The data clearly show that the IgG2a b allotype is reduced in the F1 animal as compared with the IgG2aJ response, i. e., the 2ab/2a ratio is about 0.3, as opposed to the expected 0.5 if the Igh loci are equipo- tent. This is shown even more clearly by the 2ab/2aJ ratio of about 0.4, where a ratio of 1 would be expected if the two allotypes are equally expressed. The overall IgG2a response was also lower in the F1 animal as compared with the high responder C3H parent, i.e., the IgG2a/mTg ratio. This reduction in the combined IgG2a b and IgG2aJ levels probably reflects a gene dose effect, the animals being hemizygous at these alleles. However, one might argue that the IgG2aJ response in the F1 is in fact over-represented, as the ratio of the IgG2aJ/mTg response is greater than half the response of the C3H parent, i.e., 0.286 versus 0.488. The slight difference between the sexes in the Ft was not sig- nificant.

R. C. Kuppers et al.: Linkage of IgG2a usage to the Igh locus in EAT

Table 2. The IgG2a response in the C3H x B6 FI.*

409

Proportion of IgG2a*

Strain IgG2a/mTg IgG2ab/mTg IgG2aJ/mTg 2ab/2a 2aJ/2a 2ab/2aJ

BXH-F1 ~ 0.423 0.134 0.289 0.31 0.69 0.45 BXH-F~ ~ 0.382 0.099 0.283 0.27 0.73 0.39

B 10.BR 0.222 0.220 n/a* 1.04 n/a n/a CBA-Ighb 0.127 0.130 n/a 0.99 n/a n/a C3H 0.488 n/a 0.488 rda 1.00 rda

* Relative IgG2a concentrations of the strains above were determined using a pan-IgG2a-specific (pan-IgG2a OD) and a monoclonal IgG2ab-specific (IgG2a b OD) reagent as described in the Methods. The pan-IgG2a and IgG2a b readings were normalized by calculating the ratio of pan-IgG2a/mAb-IgG2a b of the B 10.BR and CBA-Igh b (both Igh b) animals and using this ratio to correct the pan-IgG2a value of the F1 and C3H data. IgG2a/mTg = corrected pan-IgG2a OD/mTg-specific OD; IgG2ab/mTg = IgG2a b OD/mTg-specific OD; IgG2aJ/mTg = (corrected pan-IgG2a OD-IgG2a b OD)/mTg-specific OD; 2a = corrected pan-IgG2a OD value; 2a b = IgG2a b OD value; 2aJ = corrected pan-IgG2a OD-IgG2a u OD for F1, or pan-IgG2a OD for C3H.

* rda = Not applicable.

Overall, the results of this experiment would argue that the reduced IgG2a b level is controlled by the Igh b locus. The C3H background does not override the under-utilization of the IgG2a subclass of the Igh b locus in the FI mouse, nor does there appear to be an influence of other genes on IgG2a usage, suggesting the effect is cis controlled within the Igh locus.

Discussion

The antibody response to mTg is controlled by multiple genes within the H-2 locus, the major controlling ele- ments being the I-A region (Kuppers et al. 1988 a). The present study demonstrates that the magnitude of the IgG2a response to mTg is linked to elements in or near the Igh locus. The effect was shown to map to the Igh locus, using congenic CBA-Igh b mice and recombinant inbred mouse strains.

The effect of the Igh locus is not a total suppression of the IgG2a response. The influence of the Igh locus appears to be modulated by the overall response, which is primarily controlled by the H-2 locus. However, the broad distribution of intercepts seen in Figure 3 argues for additional genetic influences on the antibody re- sponse. Therefore, IgG2a levels are possibly controlled by multiple genetic factors, one being the Igh locus.

Igh-linked, variable region structural genes have been shown to influence the antibody response to sev- eral antigens through changes in fine specificity, affi- nity, and idiotypic expression (Lifshitz et al. 1980; Brown et al. 1981; Ju et al. 1981). Lfiszl6 and co- workers (1985) have shown that the IgG subclass dis- tribution in the contact-sensitivity response to ox- azolone (Ox) is similarly linked to the Igh-1 region. The IgG2a response after contact-sensitization with Ox is

reduced in animals of the Igh b haplotype. However, the secondary Ox-IgG2a response of both the CBA and CBA-Igh b strains was low when mouse serum albumin was the carrier, but high when BSA was used, sug- gesting that IgG2a usage is influenced by the carrier. However, this is genetically linked to the Igh b locus.

It is possible that mTg, like mouse serum albumin, perhaps because they are self antigens, are in some way recognized differently from other antigens. However, Halpern and co-workers (1992) have recently reported that the MRL x B6-1pr mouse response to anti-ssDNA, chromatin, and histones is predominantly of the Igh b allotype. In contrast, no allotype skewing in the anti- TNP or dsDNA-specific responses was seen. These authors also suggest that the VH region is not involved in the preferential use of the b allotype in their system. On the other hand, Lfiszl6 and co-workers (1985) have suggested that a limited rearrangement of the V genes to the Igh-1 b gene segment might occur in the Ox antibody response, possibly because of a restricted idiotypic re- sponse to Ox in the animals of the Igh b allotype. While we have not looked at the B6 mouse, we did not find the mTg-specific response in CBA mice to be restricted. Numerous VH, Vk, and J region segments are used in the antibody response to mTg (Gleason et al. 1990).

The carrier effect reported by Lgzsl6 and co-workers (1985) suggests that the regulation of the IgG2a re- sponse occurs at the T-cell level. There have been sev- eral reports suggesting that the Igh locus influences the selection of the T-cell repertoire (Nutt et al. 1981; Seman and Zilberfarb 1979; Monguini and Paul 1982; B6naroch et al. 1993). T cells can recognize B-cell idiotypes, especially those that are highly cross-reactive (Hetzelberger and Eichmann 1978; Gleason and K6hler 1982). These regulatory idiotypes may allow for T-B cell interaction and B-cell regulation. The phenomenon of

410 R. C. Kuppers et al.: Linkage of IgG2a usage to the Igh locus in EAT

isotype regulat ion has been described by Herzenberg and co-workers (1982). The subclass response to SRBC has been shown to be influenced by the Igh locus gene products (Nutt et al. 1981), and in the C57B10 mouse may be media ted by suppressor T cells (Seman and Zilberfarb 1979). It has also been suggested that isotype switching can be influenced by cytokine production by non-T cells (Snapper and Mond 1993).

Interferon gamma has been shown to be an inducer of IgG2a antibodies in vitro (Snapper et al. 1988) and in vivo (Finkelman et al. 1988). However, the results from the TNP-mTg study, showing a normal IgG2a response to TNR would argue that overall lymphokine levels do not p lay a role in this phenomenon. It is difficult to explain the reduced IgG2a b response to mTg by any of the above mechanisms. However, the consistent role of the I gh b locus in such phenomena indicates this locus may have some unique regulatory propert ies that other al lotypes may not possess.

Al lo type-associa ted subclass regulat ion has also been shown in humans. The response to bacterial anti- gens being pr imari ly of the IgG2 subclass is one ex- ample of restricted subclass usage (Yount et al. 1968; Riesen et al. 1976; Barrett and Ayoub 1986). Numerous associat ions of the Gm and Km allotypes, and al lotypes and H L A with a variety of autoantibodies in human autoimmune diseases, have been reported (Whit t ingham and Propert 1986). Therefore, an understanding of the mechanism and importance of the I gh locus on the se- lection of part icular isotypes in response to part icular antigens may provide insight into certain autoimmune disease processes.

Acknowledgments. The authors thank Ms. Denise Patrick and Ms. Rose Thornton for help in the preparation of this manuscript, and Dr. A. Scott for his helpful suggestions. I. M. O. would like to acknowledge the Universidad Autonoma de Madrid (Faculty of Medicine) and the Instituto Carlos III for providing sabbatical leave and/or support for this study. Supported in part by PHS grants R01AR31632 and U01AI35043, and a Johns Hopkins School of Hygiene Faculty Development Award to R. C. K.

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