Subclass restriction of murine antibodies

CELLULAR

IMMUNOLOGY

68, 139- 145 (1982)

Subclass

Restriction

of Murine Antibodies

V. The IgG Plaque-Forming Cell Response to Thymus-Independent Thymus-Dependent Antigens in Athymic and Euthymic Mice JOHN Department

of Microbiology

and

H. SLACK’ AND JOSEPH M. DAVIE and Immunology, Washington University St. Louis. Missouri 631 IO

Received November

2.5, 1981; accepted

January

School of Medicine,

24, I982

Antigens differ in their abilities to stimulate antibodies of various isotypes. Many thymusindependent (TI) polysaccharide antigens stimulate largely IgG3 and IgM antibodies while thymus-dependent (TD) protein antigens stimulate predominantly IgGl and smaller amounts of other isotypes. Here we determine whether thymus dependenceor independence is a property of antigens which is expressed equally by all isotypes. To do this nu/+ and nu/nu mice were immunized with several TI and TD antigens and antibody responsesof IgM and the four IgG subclasses measured. We found that, within the conditions of these experiments, all IgG isotypes were influenced equally by the presence or absence of T lymphocytes. Second, in agreement with J. L. Press (J. Zmmunol. 126, 1234, 1981), we propose a division of TD antigens into two types based upon the ability to stimulate responsesin the CBA/N mouse.

INTRODUCTION Antigens are currently classified on the basis of thymus dependence, by response patterns in the B-cell-deficient CBA/N mouse, and by the isotypes of antibodies elicited in normal animals. Thymus-independent type 1 (TI-1)’ and TI-2 antigens are distinguished by their abilities to stimulate the immunodefective CBA/N mouse; TI-1 antigens such as TNP-lipopolysaccharide (LPS) stimulate this mouse strain, but TI-2 antigens such as TNP-Ficoll, the C-polysaccharide of pneumococcus, and (Y(1 + 3) dextran fail to stimulate CBA/N ( l-7). Further, these antigen categories differ in the IgG subclasses they stimulate: TI-1 antigens elicit similar amounts of IgG2 and IgG3 while TI-2 antigens stimulate chiefly IgG3 (8, 9). However, group A streptococcal carbohydrate [(GAC), given as a bacterial vaccine], although similar to TI-2 antigens in that it is a carbohydrate that elicits an IgG3 response (8) and fails to stimulate CBA/N mice, nonetheless is a thymusdependent (TD) antigen (10). Furthermore, Press has found other TD antigens ’ Present address: Scripps Clinic and Research Foundation, LaJolla, Calif. 92101. 2 Abbreviations used: TI, thymus independent; TD, thymus dependent; LPS, lipopolysaccharide; DNP, dinitrophenyl; TNP, trinitrophenyl; BA, Brucella abortus; BSA, bovine serum albumin; GA-vaccine, group A streptococcal vaccine; GAC, group A streptococcal carbohydrate; SRBC, sheep red blood cell; PFC, plaque-forming cell; PC, phosphocholine; KLH, keyhole limpet hemocyanin; ABA, p-azobenzenearsonate. 139 OOOS-8749/82/050139-07802.00/O Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any fom mmvd.

140

SLACK

AND

DAVIE

that fail to stimulate antibody responses in CBA/N mice and has classified them as TD-2 antigens ( 11). Therefore, thymus dependence or independence may not be critical in determining isotype preference. Here we analyze the IgG response to several TI-1, TI-2, TD-1, and TD-2 antigens in athymic mice to determine whether differences exist among IgG isotypes with regard to thymus dependence. MATERIALS AND METHODS Animals and mouse cell lines. Seven-week-old female (nu/nu) nude mice (BALB/c Born with the nu gene backcrossed five generations) and normal heterozygotes (nu/+) were obtained from Gibco Animal Resources Laboratories, Madison, Wisconsin. Both nude and heterozygous mice were raised in a pathogenfree environment and then maintained in our animal facility under clean conditions. The BALB/c plasmacytomas used in this study were obtained from Litton Bionetics, Inc., Kensington, Maryland, under NC1 Contract NOl-CB-94326 and the somatic cell hybrids were provided by Dr. Brian Clevinger of Washington University School of Dental Medicine. Antigen preparation and immunizations. Trinitrophenyl-N-(aminoethyl)-carbamylmethyl-Ficoll (TNP-Ficoll) with an average of 26 TNP groups per Ficoll molecule (MW 400,000; Pharmacia Fine Chemicals, Inc., Uppsala, Sweden) was made by the method of Inman (12). TNP-Brucella abortus (BA) was prepared with BA obtained from the U.S. Department of Agriculture Animal and Plant Health Inspection Service, Ames, Iowa (13), and TNP,,-LPS (Escherichia coli 011 l:B4 LPS, Difco Laboratories, Detroit, Mich.) was prepared as described previously ( 14). TNP-bovine serum albumin (BSA) was prepared as before ( 15) and had an average of 17 TNP groups per BSA molecule. Mice in groups of five were immunized intraperitoneally (ip) with 100 pg TNP-Ficoll, 100 pg TNP-LPS, or lo9 TNP-BA organisms. Plaque-forming cell (PFC) responses were measured 7 days later. Mice were hyperimmunized with group A streptococcal vaccine (GAvaccine) as described previously (16) and the PFC responses determined 5 days after the last injection. Responses to TNP-BSA were induced by ip injections of 100 pg antigen in complete Fruend’s adjuvant (Difco), followed in 1 month by the same dose in Freund’s incomplete adjuvant; responseswere measured 5 days later. Zsotype-speciJic plaque assay. Spleen cells secreting antibody of IgM, IgGl, IgG2a, IgG2b, and IgG3 subclasseswere detected in antigen-specific plaque assay systems with the use of subclass-specific facilitating antisera. SRBC coated with GAC ( 16) and TNP ( 17) by the previously described methods were used as indicator cells. The subclass-specific facilitating antisera were prepared in rabbits as originally described (9). Specificities and efficiencies of these facilitating antisera have been verified using a panel of mouse plasmacytomas and/or hybridomas secreting antibody of the major mouse immunoglobulin isotypes. To detect only IgG-secreting spleen cells in the antigen-specific system, rabbit anti-p (anti-Ml04 absorbed with J558Sepharose to remove anti-idiotype and anti-X) was included in the agarose to suppress direct (IgM) PFC. RESULTS Zsotype Distribution of TZ-I, TZ-2, TD-I, and TD-2 Responses in Heterozygous (nu/+) Mice To test whether differences existed among the IgG subclasses with regard to thymus dependence, antigens representative of the four major classes, TI-1, TI-2,

IgG RESPONSES IMMUNOGEN

IgG20

WI

TNP-Ficdl

OF NUDE

141

MICE

IqG2b

Ifi3

P

GA-VACCINE I+

TNP-LPS

i

TNP-BA

I

P

TNP-BSA I I I I I I I I I 1 50 75 25 50 25 25 50 75 25 M 75 SUBCLASS REPRESENTATION (% OF TOTAL IgG)

I

I

75

FIG. 1. Immunoglobulin isotypc distributions in the normal heterozygous (nu/+) mouse generated in response to TI-1, TI-2, and TD antigens. Shown are the geometric means and standard errors of the antigen-specific PFC facilitated with anti-yl, y2a, y2b, and 73 antisera, normalized to the total indirect PFC response.

TD-1, and TD-2 were used to stimulate antibody responses among all isotypes. Although previous studies have described the cellular requirements for responses to these antigens, most have concentrated on the IgM response (10, 18-20). In fact, not until recently has the major IgG3 response of the TI-1 and TI-2 antigens been appreciated (9). Figure 1 shows the subclass distribution generated by these antigens in the heterozygous (nu/+) mouse. The isotype responses of the (nu/+) mouse are indistinguishable from those obtained earlier in the conventionally raised BALB/c mouse (9). The response to TNP-Ficoll and GA-vaccine is dominated by IgG3, the antigens TNP-LPS and TNP-BA stimulate IgG2b and IgG3, and the IgG response to TNP-BSA is dominated by IgGl with relatively little IgG3 and IgG2. In order to generate significant IgG responses to the various antigens, different immunization protocols were required. Primary responses to TNP-Ficoll, TNP-LPS, and TNP-BA, hyperimmune responses to GAC, and secondary responses to TNP-BSA in adjuvant were compared. Although the influences of adjuvants and time after immunization on isotype responsesto most of these antigens have not been systematically evaluated, a more extensive survey of antigens showed no correlation of isotype with the use of adjuvant (21). Comparison Mouse

of TI-1, TI-2, and TD Responses in the Heterozygous

and Nude

The responses in the heterozygous mice confirmed that the major antigen categories stimulate distinct IgG subclasses. It was of interest to determine whether the subclass distributions were altered in mice lacking the thymus. Table 1 shows the total IgG and IgM responsesto TI and TD antigens in both nude and heterozygous mice. Both the IgG and IgM responses to TNP-Ficoll and TNP-LPS are conserved in the nude mouse. Figure 2 shows further that the isotype distributions generated by these two antigens remain unchanged in the nude mouse with TNPFicoll stimulating IgG3 and TNP-LPS generating both IgG3 and IgG2b. Thus, for these antigens, IgM, IgG3, and IgG2b antibody responses are independent of

142

SLACK AND DAVIE TABLE 1 Antigen-Specific Responses in Nude (W/W) and Heterozygous (nu/+) Mice” Direct PFC/spleen

Antigen

nulnu

Flu/+

TNP-Ficoll TNP-LPS GA-Vaccine TNP-BA TNP-BSA

18,930 (1.2) 10,800 (2.0) 3,930 (1.4) 1,367 (1.4) 6,574 (1.2)

21,810 (1.3) 12,260 (1.2) 824,900 (2.1) 37,640 (1.2) 25,290 (1.3)

Indirect PFC/spleen nulnu m/-l0.87 0.88
nulnu 1553 (1.3) 6361 (1.4)
nul+ 2,366 (1.4) 6,299 (1.3) 20,260 (1.8) 38,940 (1.2) 14,720 (1.6)

0.65 1.01 10.01
a Shown are the geometric means and standard error factors in parentheses of the direct (IgM) and indirect (totals of the individual IgG subclasses)plaque responsesobtained from five mice. See Materials and Methods for immunization protocol.

T-cell influences. Simlarly, Mond et al. and Mongini et al. have found that the responses to TNP-Ficoll in nu/nu and nu/+ BALB/c and C57BLIO mice were equivalent ( 19) except in the minor IgG2a component (22). Unexpectedly, we found the responseto TNP-BA decreased in nu/nu mice: the IgG3 and IgG2b components were absent and the IgM response was decreased by 25fold. Several different antigen preparations were tested covering a broad range of coupling ratios, including the preparation shown to stimulate CBA/N mice (9), but similar results were obtained with each. The TD nature of TNP-BA is at variance with results of other studies, but only IgM responses were measured previously (2, 18, 19). Regardless of the basis of these differences, they demonstrate that TNP-BA, which shares several features with TI-1 antigens, namely, stimulation of IgG2b and IgG3 isotypes and ability to stimulate a partial response in CBA/N mice, can nonetheless be a TD antigen. Similarly, GA-vaccine, which resembles a TI-2 antigen in failing to stimulate antibodies in CBA/N mice and generating predominantly IgG3 responses in normal mice, is clearly a TD antigen [Table 1, Fig. 2, (lo)]. Thus, IgG3 and IgG2b isotypes are thymus dependent with some antigens and thymus independent with others. Finally, all three IgG isotypes stimulated by TNP-BSA, IgGl, IgG2b, and IgG3, are equally thymus dependent. DISCUSSION This study has two major conclusions: first, that the present system of grouping antigens does not describe functionally uniform categories and second, that T-cell influences at least do not play the only role in determining isotype preference. There are presently three functional characteristics used to categorize antigens: thymus dependence or independence, the ability to stimulate the CBA/N mouse, and the predominant isotypes stimulated in normal animals. We have shown before that the ability to stimulate the CBA/N mouse and the predominant isotypes elicited are related traits in that those antigens that generate predominantly IgG3 responses fail to stimulate the CBA/N mouse. In this paper we show that antigens that are uniform with regard to CBA/N and isotype characteristics can differ with regard to thymus dependence. For example, GA-vaccine and TNP-Ficoll generate the same isotype distributions but differ drastically in form and thymus dependence.

IgG RESPONSES

OF NUDE

143

MICE

Similar differences exist between the two antigens which elicit IgG3 and IgG2 responses, TNP-LPS, and TNP-BA. Thus, IgG3 and IgG2b can be generated in an antigen-specific system in both a TI and TD manner by either soluble or particulate antigens. Therefore, the former categories of TD, TI-1, and TI-2 are inadequate. Instead, we would agree with revisions to this system proposed by Press that subdivide TD antigens into those that stimulate responses in CBA/N mice (TD-1) and those that don’t (TD-2) (Table 2). Thus, GA-vaccine is a TD-2 antigen and TNP-BA is a TD-1 antigen. Other TD-1 antigens are hapten-protein conjugates, several of which have been shown to stimulate large amounts of IgGl, in contrast to TNP-BA, which stimulates little IgGl. However, all TD-1 antigens tested so far stimulate responses in CBA/N mice which show a selective decrease in IgG3 components. Depending on the relative amount of IgG3 produced to the particular TD-1 antigen, the deficiency in CBA/N can be minor or substantial with regard to total antibody production. On the other hand, TD-2 antigens are those in which all isotypes are reduced in CBA/N mice. For GAC this involves reduced levels of IgG3 and IgM. Press, however, has described other TD-2 antigens, Ir gene-controlled oligopeptides, that stimulate IgG3, IgM, and IgGl in normal animals (11). This is the first instance in which the CBA/N deficiency has been found to involve subclasses other than IgG3 and IgM. Whether the inability of CBA/N to produce IgGl to these antigens reflects a new defect, possibly involving specific regulatory elements, or is an additional facet of the B-cell deficiency needs clarification. Nonetheless, all antigens tested to date regardless of category fail to stimulate normal IgG3 responses in CBA/N mice. The second conclusion we can reach from this study is that T cells seem not to PFC/106 I

0

SPLEEN CELLS I00

1000

RELATIVE IgG3 0.6

RESPONSE I TU nu /nu +)

IgG2b

w

w

-

TNP-Fiioll

1.3

~

I 0.007

j

006 006 0006 023

FIG. 2. The IgG PFC response to the major antigen categories in normal heterozygous (nu/+) mice (solid bar) and the thymusless nude (nu/nu) mouse (open bar). Mice in groups of five were immunized as described under Materials and Methods. Five or seven days later IgM, IgGl, IgG2, and IgG3 antigenspecific plaques were determined in the spleen; shown are the geometric means and standard errors. The minor IgG2a component found in these antigen-specific responses (Fig. 1) could not be monitored accurately.

144

SLACK AND DAVIE TABLE 2 Revised System of Categorizing Antigens Functionally

Antigen category

Examples

Old nomenclature

Thymus dependence

Predominant isotvpe

Response in CBA/N mice

TI-1

TNP-LPS

TI-1

No

IgM, IgGZb, IgG3

Normal except for IgG3

TI-2

TNP-Ficoll phosphocholine dextran

TI-2

No ’

IgM, IgG3 >>others

All isotypes decreased

TD-1

TNP-BSA TNP-Hy TNP-KLH TNP-BA

TD TD

(T, G)-A-L

TD

GAC

(TI-2)

TD-2

gl)

Yes IgM, IgGl % IgG2, IgG3 Normal except for IgG3 (all isotypes) IgM, IgGl % IgG2, IgG3 IgM, IgGl B IgG2, IgG3 IgM, IgG2, IgG3 Yes IgM, IgGl % IgG2, IgG3 All isotypes decreased (all isotypcs) IgM, IgG3

be the sole regulators of IgG isotype expression. That is, stimulation of IgG3 and IgG2 isotypes sometimes requires T cells and other times not, depending on the immunogen. Furthermore, our results demonstrated no major differences among the isotypes of antibodies elicited by TD and TI antigens in athymic mice. Thus, within the limits of our experimental approach, all isotypes stimulated by a single immunogen were equally influenced or not by the absence of normal numbers of T cells. Mongini er al., however, did detect differences in T-cell dependence among the isotypes in anti-TNP-Ficoll response, but the difference was found only in the minor IgG2a component (22). It is possible that more detailed examination of this question in vitro may disclose more subtle distinction. However, involvement of T cells at the isotype level is certainly not ruled out by these studies. For example, Mond et al. showed that careful removal of residual T cells from m/m splenocytes in vitro depleted responsivenessto TNP-Ficoll but had less affect on TNP-BA responses (19). Thus, antigens may vary in the degree of thymus dependence and may therefore be relatively thymus independent or dependent. Nude mice provide a convenient model for measuring relative thymus independence. Further, IgGI is generated in antigen-specific or polyclonal systems only by TD antigens or mitogens (M&earn et al., manuscript in preparation). Hapten-protein conjugates, sheep erythrocytes, and pokeweed mitogen have been the major stimulants of IgGl secretion. Further, an enriched T-helper-cell population shows isotype preference for IgGl and IgG2 (23). Nonetheless, major questions exist with regard to isotype preference to haptenprotein antigens. Recent studies from this laboratory on rat isotype preferences to various antigens showed that isotype preference was related to the hapten specificity of the antibodies. Immunization of rats with DNP-KLH- or ABA-KLH-stimulated IgG2a antibodies predominantly while immunization with PC-KLH-stimulated almost entirely IgG2c and no IgG2a (21). [IgG2c is thought to be the rat analog of mouse IgG3 (24).] Once again, isotype preference cannot be attributed easily to general characteristics of antigen nor to T-cell function. Cambier et al. (25) feel

IgG RESPONSES

OF NUDE

MICE

145

that intermediate stages of a differentiating B cell are responsive to a particular antigen form and/or an accessory helper component. Thus, isotype-specific responses could be generated from cells varying in competency to express the IgG subclasses.Alternatively, distinct B-cell subpopulations may exist with precursors committed to different IgG subclasses. We maintain that the B cell plays the major role in determining isotype preference. T-Cell influences, although clearly important for many responses, cannot explain the tendency of different antigen groups to select particular IgG isotypes. Rather, this implies the existence of distinct B-cell populations responsive to different antigen groups. ACKNOWLEDGMENTS We are grateful to Dr. Brian L. Clevinger for the production of the hybridoma cell lines used in this work. This work was supported by U.S. Public Health Service Grants CA-091 18, AI-15926, and Al11635.

REFERENCES 1. 2. 3. 4. 5. 6.

7. 8.

Mosier, D. E., Scher, I., and Paul, W. E., J. Immunof. 117, 1363, 1976. Mond, J. J., Scher, I., Mosier, D. E., Blaese, M., and Paul, W. E., Eur. J. fmmunol. 8, 459, 1978. Cohen, P. L., Scher, I., and Mosier, D. E., J. Immunol. 116, 301, 1976. Mond, J. J., Lieberman, R., Inman, J. K., Mosier, D. E., and Paul, W. E., J. Exp. Med. 146, 1138, 1977. Fernandez, C., and Moller, G., J. Exp. Med. 146, 1663, 1977. Paul, W. E., Subbarao, B., Mond, J. J., Sieckmann, D. G., Zitron, I., Ahmed, A., Mosier, D. E. and Scher, I., In “Cells of Immunoglobulin Synthesis” (B. Pernis and H. J. Vogel, Eds.), p. 383. Academic Press, New York, 1979. Mosier, D. E., Zitron, I., Mond, J. J., Ahmed, A., Scher, I., and Paul, W. E., Immunol. Rev. 37, 89, 1977. Perlmutter, R. M., Hansburg, D., Briles, D. E., Nicolotti, R. A., and Davie, J. M., J. Immunol. 121,566,

9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

1978.

Slack, J., Der-Balian, G. P., Nahm, M.. and Davie, J. M., J. Exp. Med. 151, 853, 1980. Braun, D. G., Kindred, B., and Jacobson, E. B., Eur. J. Immunol. 2, 138, 1972. Press, J. L., J. Immunol. 126, 1234, 1981. Inman, J. K., J. Immunol. 114, 704, 1975. Mosier, D. E., J. Immunol. 121, 1453, 1978. Jacobs, D. M., and Morrison, D. C., J. Immunol. 114, 360, 1975. Little, J. R., and Eisen, H. E., Methods Immunol. tmmunochem. 1, 128, 1967. Briles, D. E., and Davie, J. M., J. Exp. Med. 141, 1291, 1975. Chesebro, B., and Metzger, H., Biochemistry 11, 766, 1972. Takahashi, T., Mond, J. J., Carswell, E. A., and Thorbecke, G. J., J. Immunol. 107, 1520, 1971. Mond, J. J., Mongini, P. K. A., Sieckmann, D., and Paul, W. E., J. Immunol. 125, 1066, 1980. Klaus, G. G. B., Phillips, J. M., Humphrey, J. H., Dresser, D. W., and Cross, A. M., Eur. J. Immunol. 6,429, 1976. Der-Balian, G. P., Slack, J., Clevinger, B. L., Bazin, H., and Davie, J. M., J. Exp. Med. 152, 209, 1980. Mongini, P. K., Stein, K. E., and Paul, W. E., J. Exp. Med. 153, 1, 1981. Augustin, A. M., and Coutinho, A., J. Exp. Med. 151, 587, 1980. Nahm, M., Der-Balian, G. P., Venturini, D., Bazin, H., and Davie, J. M., Immunogenetics (NY) 11, 199, 1980. Cambier, J. C., Vitetta, E. S., Uhr, J. W., and Kettman, J. R., J. Exp. Med. 145, 778, 1977.