on lymphocytes from aged mice

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spleen cells bearing class I or class II major histocompatibility complex ... mice of less than 1 month to more than 2 years of age with a variety of ... cence (phycoerythrin and propidium iodide) values were collected for 20,000 cells. Live cells were ..... indicated: C57BL/6J (B6), young (Y, 2 months) or old (0, 2 years);. C3H/HeJ ...

Proc. Natl. Acad. Sci. USA Vol. 84, pp. 7624-7628, November 1987 Immunology

Increased expression of major histocompatibility complex antigens on lymphocytes from aged mice CHARLES L. SIDMAN*, EDGAR A. LUTHER, JAN D. MARSHALL, KIM-ANH NGUYEN, DERRY C. ROOPENIAN, WORTHEN

AND SHERRI M.

The Jackson Laboratory, Bar Harbor, ME 04609

Communicated by Elizabeth S. Russell, June 22, 1987 (received for review April 13, 1987)

ABSTRACT Many studies have reported age-related changes in immune responses that could be due to alterations in lymphoid cell numbers or functions. Here we report the results of studies using immunofluorescent staining and in vitro assays of cellular function to compare the expression of cell surface antigens on lymphocytes from mice up to 2 years of age, No significant changes were observed in the frequencies of spleen cells bearing class I or class II major histocompatibility complex (MHC) antigens, surface immunoglobulin, or Thy-1, Ly-1, Ly-2, or L3T4 antigens. However, the densities (per cell) of both class I and class H MHC antigens were increased significantly on cells from aged as compared to young mice, whereas the densities of the other cell surface antigens studied were unchanged or slightly decreased. The increased levels of MHC antigen expression in old relative to young mice were shown to be functionally significant regarding immunological stimulation. These data suggest that T-cell clones silent in young individuals may be activated in comparable situations in older animals, leading to immunological aterations perhaps including increased autoreactivity.

production colonies of The Jackson Laboratory. Spleens were removed and teased into single-cell suspensions, depleted of erythrocytes by treatment with Gey's solution (8), and washed. All cell manipulations were done in Earle's balanced salts solution containing 10 mM Hepes (pH 7.2) and 1% bovine serum albumin at 40C. Immunofluorescent Staining. Cells were stained for immunofluorescence analysis by direct or indirect labeling procedures. For direct labeling, antibodies were conjugated with fluorescein isothiocyanate (FITC); for indirect staining, unconjugated or biotin-conjugated antibodies were used as the first reagent, and FITC-labeled xenogeneic polyspecific or IgG class-specific antibodies, or FITC- or phycoerythrinconjugated avidin, were used as second reagent. For all indirect staining procedures, controls of only the second reagent without the first were done routinely. The following monoclonal antibodies were used in these studies: 331-12 (9) (anti-,u), 11-6.3 (10) (anti-8b), 30-H12 (11) and HO-13.4.9 (12) (anti-Thy-1.2), 28-13.3 (13) (anti-Kb), H141-30 (14) and 28-14.8 (13) (anti-Db), 25-9.17 (13) (anti-I-Ab), 53-7.3 (11)

METHODS Mice and Cells. C57BL/6J mice without external or internal macroscopically visible tumors were killed by CO2 asphyxiation at the ages indicated. A complete description of this aging colony has been published (7). Additionally, C3H/HeJ, DBA/2J, and various F1 hybrid mice were obtained from the

(anti-Ly-1), 53-6.7 (11) (anti-Ly-2), and GK1.5 (15) (antiL3T4). Polyspecific goat anti-mouse and anti-rat (not crossreactive with mouse immunoglobulins) antibodies (Southern Biotechnology Associates, Birmingham, AL) to total immunoglobulins, and to mouse IgG subclasses, were also used. All reagents were used at concentrations previously determined to give maximal staining, and the specificities of the antibodies were confirmed using spleen cells from mice of different strains, or with the "sandwich" reagents used without first-layer antibodies (data not shown). After the final washes, cells were counterstained with propidium iodide to allow exclusion of dead (brightly stained with propidium iodide) cells from the immunofluorescence analyses (16). FACS Analysis. Cells were examined in an Ortho model 50H cytofluorograph (FACS) using the 488-nm emission line of an argon laser. For each stained cell sample, forward scatter (related to cell size), right-angle scatter (related to granularity), green fluorescence (FITC), and red fluorescence (phycoerythrin and propidium iodide) values were collected for 20,000 cells. Live cells were those without bright propidium iodide staining and with sufficient forward scatter to exclude remaining erythrocytes or other cellular debris. In some experiments, lymphocytes were further distinguished and separately analyzed by virtue of their characteristic combination offorward and right-angle scatter. Control cell samples not stained with specific antibody allowed determination of the proper gates to distinguish positive from negative cells in each staining procedure. The percentages of fluorescence-positive live cells, and the mean fluorescence and scatter values of the fluorescence-positive cells, were determined by an Ortho model 2140 computer system. In sandwich staining procedures, the percentage of

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Abbreviations: MHC, major histocompatibility complex; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate. *To whom reprint requests should be addressed.

Many studies have examined the status of the immune system in aged as opposed to young animals and have reported alterations with age especially in T- but also in B-lymphocyte function (1, 2). Such alterations could be due to changing numbers of cells belonging to specific cell subsets or due to aspects of regulation either internal or external to these cells. The numbers of B and T cells in young adult and aged mice have been reported (3-6); these studies, however, did not enumerate specific B- and T-cell subsets, nor did they utilize the sensitive and quantitative methods of immunofluorescence analysis that are currently available. To determine whether the lymphoid cell populations in aged mice differ from those in younger animals, we stained splenocytes from mice of less than 1 month to more than 2 years of age with a variety of antibodies against cell surface antigens and examined the stained cells using a fluorescence-activated cell sorter (FACS). We thus determined both the numbers of cells bearing specific antigens and the density distributions of each antigen on the positive cells. The results suggest that agerelated alterations in murine immune function may be due to changes in major histocompatibility complex (MHC)-dependent stimulation or regulation of lymphoid cells rather than to changing levels of specific cell subsets.

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Immunology: Sidman et al. specifically stained cells was determined by subtracting the percentage of positive cells after control (second layer only) staining from that obtained after staining with both first- and second-layer antibodies. Histograms were plotted on an IBM PC-AT computer system with attached peripheral equipment. T-Cell Proliferation. The basic procedures for the T-cell proliferation assays were as described (17), with the following modifications. For the mixed lymphocyte cultures, 2 x 105 C3H/HeJ splenocytes (responders) were cultured in roundbottom 96-well plates with 5 x 105 irradiated (3300 rads; 1 rad = 0.01 Gy) spleen cells (stimulators) from the mice indicated. [methyl-3H]Thymidine (1 ,uCi per well; 1 p.Ci = 37 kBq) was added and its incorporation into DNA from 72 to 96 hr of culture was determined. For stimulation of the long-term I-Ab_(auto)reactive T-cell line SM/B1OHSP, 104 responder cells were cultured in round-bottom wells with the indicated numbers of irradiated (2000 rads) stimulator splenocytes, and [3H]thymidine incorporation was determined from 48 to 60 hr of culture.

RESULTS The percentages of spleen cells from young adult (1-2 months) and aged (.18 months) C57BL/6J mice bearing the various antigens investigated are shown in Fig. 1 [One to two months was chosen as the reference age because at this age the thymus is at its maximum cellularity (18) and the mice have become responsive to classes of antigens to which they were earlier unresponsive (19).] Only minor differences in the levels of different lymphocyte subsets were observed in young vs. old spleens. In both young and old mice, approximately 90%o of the splenocytes had easily detectable class I MHC antigens (K or D). About 50% of young, and 60o of aged, splenocytes were B cells (surface immunoglobulinpositive). The numbers of class II MHC (I-A)- and IgM (,u)-positive cells in both populations were comparable to or only slightly less than the number of total immunoglobulinbearing cells, whereas only one-half to two-thirds of the immunoglobulin-bearing cells were detected as IgD (8)positive. T cells (Thy-1 or Ly-1-positive) accounted for about one-third of the splenocytes in mice of both ages. The percentages of cells that were L3T4-positive plus those that were Ly-2-positive added up to almost the total T-cell population, with cells expressing L3T4 being slightly more 100 I

Proc. Natl. Acad. Sci. USA 84 (1987)

numerous than those with Ly-2. These percentages agree with previous reports on the levels of these cell subsets in young adult mice (20, 21). Although the percentages of spleen cells bearing various antigens were fairly constant, the amount of expression per cell of some antigens did change significantly with age. Fig. 2 shows the relative intensities of the various antigens on antigen-positive spleen cells of old (.18 months) mice, expressed relative to the staining intensities of these same antigens on cells from young (1-2 months) animals. (These numbers are not in absolute units, but were obtained by dividing the mean fluorescence channel of the positive cells from aged mice by the mean fluorescence channel of the positive cells from young adult animals, both as determined by the 2140 computer attached to the FACS. This procedure of expressing the amount of fluorescence on cells from mice of various ages was adopted in order to control for day-to-day variations in staining intensity and allow the comparison of data obtained in experiments done on different days.) Cells from old mice consistently showed both more total staining and higher antigen density for both class I (K and D) and class II (I-A) MHC antigens than cells from young mice, whereas they stained equally or less intensely for the other, cell-typespecific, antigens. Specifically, spleen cells from -18-monthold mice showed 142% ± 6%, 134% ± 4%, and 118% ± 5% as much K, D, and I-A staining, respectively, as cells from 1to 2-month-old control mice, and 139% ± 6%, 131% ± 4%, and 115% ± 6% as much staining density for these antigens (calculated by dividing the staining intensity by the amount of forward scatter, a measure of cell size). The same patterns of antigen expression were seen when only lymphocytes, the major cell population in spleen, were analyzed (data not shown). The decreased expression of Thy-1 per cell is in agreement with earlier reports (5, 6). Representative histograms of MHC staining of young and old mouse spleen cells are shown in Fig. 3. With both classes of MHC antigens, there was substantial overlap between the intensity distributions of cells from young and old mice, with a decreased number of cells with low amounts and an increased number of cells with high amounts of these antigens in old relative to young mouse spleens. In Fig. 3, the intervals marked A, B, and C show the regions containing all (A) or the brightest 25% (B) or 10% (C) of cells from the young mouse spleen stained with the respective antibodies. For the class I antigen Db, the older mouse showed 2.8 and 2.1 times the number of splenocytes with antigen density equal to the brightest 10% and 25% of young cells, respectively. Similarv

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Immunology: Sidman et al.

Proc. Natl. Acad. Sci. USA 84 (1987)

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animals at various stimulator concentrations. Similar data (not shown) were obtained for class I-reactive T-cell lines.

DISCUSSION

Fluorescence (linear) FIG. 3. Representative MHC staining histograms (intensity distributions, on a linear scale) from spleen cells from young (2 months) and old (18 months) C57BL/6J mice. (Upper) Db staining of young (light solid line) and old (heavy solid line) spleen cells. (Lower) I-Ab staining; young spleen cells without (light broken line) and with (light solid line) and old spleen cells without (heavy broken line) and with (heavy solid line) anti-I-Ab first layer (see Methods). Regions A, B, and C indicate all, the brightest 25%, and the brightest 10%o, respectively, of young spleen cells stained with the various antibodies. ly, for the class II antigen I-Ab, the older mouse spleen contained 2.3 and 1.9 times as many cells with antigen density equal to the brightest 10% and 25% of young cells. The ontogeny of MHC antigen staining intensity was determined, with immunoglobulin staining as a nonchanging marker for comparison. As shown in Fig. 4, the levels of MHC antigen staining rose sharply up to 2 months of age and then continued to rise more gradually throughout life. In contrast, the level of immunoglobulin staining was fairly constant on splenocytes from all mice assayed. Finally, to determine whether these changes in MHC antigen expression with age could be functionally significant, spleen cells from young and old mice were compared with respect to their stimulating capacities in anti-MHC proliferative responses in vitro. As shown in Fig. 5 (Upper), irradiated splenocytes from old C57BL/6J mice stimulated 1.7 times as great an anti-class II response by C3H/HeJ cells as did cells from young C57BL/6J animals. As controls, three types of (young) F1 stimulator cells [C57BL/6J x C3H/HeJ (BC), C57BL/6J x DBA/2J (BD), and C3H/HeJ x DBA/2J (CD)] were shown to provoke proliferation by the same C3H/HeJ responders at levels approximately halfway between the levels stimulated by their respective parental strains [C57BL/6J (B6), C3H/HeJ (C3), and DBA/2J (D2)]. When young and old splenocytes were used to stimulate a long-term T-cell line reactive to (self) I-Ab, cells from old mice were approximately 4 times as effective as those from young mice, with the old stimulators inducing 2-9 times as much [3H]thymidine incorporation as those from young

Many investigators have reported changes in the immune competence of aged as opposed to young adult animals, in general concluding that alterations in T-lymphocyte function were more significant than those of B lymphocytes (1, 2). The 12 9 6 x

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Stimulator cells, no. x 0-5 FIG. 5. Stimulation of MHC class II-dependent T-cell proliferation. (Upper) Responses, in mixed-lymphocyte cultures, of C3H/HeJ (2-month-old) spleen cells against the following irradiated stimulators, all from 2- to 3-month-old mice unless otherwise indicated: C57BL/6J (B6), young (Y, 2 months) or old (0, 2 years); C3H/HeJ (C3); DBA/2J (D2); (C57BL/6J x C3H/HeJ)Fl (BC); (C57BL/6J x DBA/2J)F1 (BD); (C3H/HeJ x DBA/2J)F1 (CD). (Lower) Stimulation of long-term T-cell line 5M/B1OHSP. with varying numbers of stimulator cells from young (Y, 2 months) or old (0, 2 years) C57BL/6J mice.

Immunology: Sidman et al. data presented here are significant regarding these earlier studies in two major ways. First, our data establish that the frequencies of total murine splenic B and T cells, or of particular subsets defined by the antibodies used here, do not change as a necessary and direct consequence of aging, at least through 2 years of age. The second major finding reported here regarding the immunology of aging is that the expression of MHC antigens on lymphocytes continues to increase with age. Although the distributions of MHC antigens on young and old spleen cells overlap substantially, spleens from aged mice contain 2-3 times the number of cells with high amounts of MHC antigen expression per cell as spleens from young adult animals. Mond et al. (22) observed that the amount of class II MHC antigen expressed on B cells increases severalfold from birth to several months of age. Our results confirm this and extend the finding by showing that it applies to both class I and II MHC antigens, although not to other cell surface molecules, and that MHC antigen expression continues to increase essentially throughout life. It is appropriate to speak here of the actual amount of cell surface antigen expression per cell, since the FACS measures the amount of antibody bound to individual cells, which is generally proportional to the amount of antigen expressed. In contrast, measurements of the amount of antibody bound to populations of cells yield only an average figure for antigen expression by the entire population, and antibody- and complement-mediated cytotoxicity reflects both the amount of antigen present and the susceptibility of cells to lysis by the particular antigen/antibody/complement combination (3, 5, 6). The data presented here suggest that the increased expression of class I MHC antigens occurs on the entire population of splenocytes, and double-label experiments (data not shown) indicate that it occurs on both B and T lymphocytes. In contrast, these studies have mainly measured the expression of class II MHC antigen on B cells, since we did not employ a marker to distinguish macrophages, a minor population of spleen cells also expressing these antigens. It is also important to point out that the animals used here were from an exceptionally clean, barrier-maintained colony (7) that is regularly monitored for microbiological infection, and even so, we did not utilize animals with gross visible abnormalities (2 of 16 total animals sacrificed at -18 months of age) in these studies. Possible changes occurring as a result of advanced pathological conditions rather than aging per se would thus not have been seen in this work. These findings imply that changes in immunological function with age do not occur via major alterations in the levels of the cell subsets identified by the antibodies used here but might be due to changes in T-lymphocyte regulation resulting from altered levels of MHC antigen expression. Essentially all antigen recognition by T cells [by helper (23), suppressor (24), and cytotoxic (25) T cells] occurs in association with ("is restricted by") MHC antigens on the surfaces of other cells. A number of investigators (26-29) have emphasized the importance of the quantitative level of expression of particular MHC antigens for specific immune responses. Here, we suggest the possibility of a kind of polyclonal immune response (30) phenomenon, based on quantitative changes in MHC antigen expression during aging. While MHC antigen expression was only about 1.5 times as great on old as opposed to young mouse splenocytes, the old cells were 3-4 times as effective stimulators (in mixed-lymphocyte cultures) as young cells, and in certain situations the old cells induced almost order-of-magnitude-greater responses than the young cells. This greater functional capacity may be due to a sharp threshold in T-cell stimulation by MHC antigens (26-29) and to the 2-3 times higher level of cells with high levels of MHC antigen expression in old as opposed to young splenocyte populations. Thus, foreign-antigen presentation and autoreactivity to MHC or MHC plus other self antigens may be

Proc. Natl. Acad. Sci. USA 84 (1987)

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much more efficient in aged as compared to young adult animals, leading to the activation in older individuals ofT-cell clones that would be inactive in younger animals. Although measured mainly on B lymphocytes here, the changing levels of class II MHC antigen expression may be significant, since antigen presentation by B cells is well established (31, 32). The possible relevance to autoimmunity is particularly intriguing because MHC antigen expression in the periphery of aged mice is substantially greater than that in the periphery of neonatal animals (where the self repertoire is assessed and immunological tolerance is largely determined) and in the thymus of aged mice (18) (where T cells continue to be educated). In this regard, cells from young animals are known to react to those from older individuals in vitro in a reaction now known as the "autologous mixed-leukocyte reaction" (33). Autoimmune strains of mice have also been reported to show abnormally high levels of class II MHC antigen expression (34, 35) and to have their autoimmunity greatly lessened by in vivo treatment with antibodies to these antigens (36). Several possible mechanisms might contribute to the specifically increased expression of MHC antigens on aged as opposed to young mouse lymphocytes. One might be a positive feedback cycle in which antigen recognition by the immune system leads to the elaboration of lymphokines such as ry-interferon (37) or B-cell-stimulatory factor 1 (interleukin 4) (38), which are known to directly increase MHC antigen expression by several cell types, and which may in turn allow greater antigen presentation and recognition, etc. An interesting question in this context would be the relationship between MHC antigen expression and the external antigenic load to which an animal is exposed. Other mechanisms perhaps involved in the differential MHC antigen expression on cells in young vs. aged mice might be changes in the levels of 1,25-dihydroxyvitamin D3 (39), glucocorticosteroid hormones (40), or prostaglandins (41), all of which have been shown to regulate MHC antigen expression in vitro. Finally, the altered expression of these antigens could be determined by factors intrinsic to the cells, rather than as a result of extracellular regulation. This possibility could be addressed by cell-transfer experiments between animals of different ages. We thank Dr. D. Harrison for providing the aged mice used, A. P. Davis for excellent technical assistance, and Drs. D. Harrison and G. Carlson for critical reading and comments on the manuscript. 1. Makinodan, T. & Kay, M. M. B. (1980) Adv. Immunol. 29, 287-330. 2. Gottesman, S. R. S. & Walford, R. L. (1982) in Testing the Theories of Aging, eds. Adelman, R. C. & Roth, G. S. (CRC, Boca Raton, FL), pp. 233-279. 3. Stutman, 0. (1972) J. Immunol. 109, 602-611. 4. Collard, R. E., Basten, A. & Waters, L. K. (1977) Cell. Immunol. 31, 26-36. 5. Olsson, L. & Claesson, M. H. (1973) Nature (London) New Biol. 244, 50-51. 6. Brennan, P. C. & Jaroslow, B. N. (1975) Cell. Immunol. 15, 51-56. 7. Harrison, D. E. & Archer, J. A. (1983) Exp. Aging Res. 9, 245-251. 8. Julius, M. H. & Herzenberg, L. A. (1974) J. Exp. Med. 140, 904-920. 9. Kincade, P. W., Lee, G., Sun, L. & Watanabe, T. (1981) J. Immunol. Methods 42, 17-26. 10. Oi, V. T., Jones, P. P., Goding, J. W., Herzenberg, L. A. & Herzenberg, L. A. (1978) Curr. Top. Microbiol. Immunol. 81, 115-129. 11. Ledbetter, J. A. & Herzenberg, L. A. (1979) Immunol. Rev. 47, 63-91. 12. Marshak-Rothstein, A., Fink, P., Gridley, T., Raulet, D. H., Bevan, M. J. & Gefter, M. L. (1979) J. Immunol. 122, 2491-2497. 13. Ozato, K. & Sachs, D. H. (1981) J. Immunol. 126, 317-321.

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14. Lemke, H., Hammerling, G. J. & Hammerling, U. (1979) Immunol. Rev. 47, 175-206. 15. Dialynas, D. P., Wilde, D. B., Marrack, P., Pierres, A., Wall, K. A., Havran, W., Otten, G., Loken, M. R., Pierres, M., Kappler, J. & Fitch, F. W. (1983) Immunol. Rev. 74, 29-51. 16. Parks, D. R. & Herzenberg, L. A. (1984) Methods Enzymol. 108, 197-241. 17. Roopenian, D. C., Orosz, C. G. & Bach, F. H. (1984) J. Immunol. 132, 1080-1084. 18. Farr, A. G. & Sidman, C. L. (1984) J. Immunol. 133, 98-103. 19. Mosier, D. E., Zaldivar, N. M., Goldings, E., Mond, J., Scher, I. & Paul, W. E. (1977) J. Infect. Dis. 136 (Suppl.), 14-19. 20. Hardy, R. R., Hayakawa, K., Haaijman, J. & Herzenberg, L. A. (1982) Nature (London) 297, 589-591. 21. Ledbetter, J. A., Rouse, R. H., Miklem, H. S. & Herzenberg, L. A. (1980) J. Exp. Med. 152, 280-295. 22. Mond, J. J., Kessler, S., Finkelman, F. D., Paul, W. E. & Scher, I. (1980) J. Immunol. 124, 1675-1682. 23. Schwartz, R. H. (1985) Annu. Rev. Immunol. 3, 237-261. 24. Dorf, M. E. & Benacerraf, B. (1984) Annu. Rev. Immunol. 2, 127-158. 25. Nabholz, M. & MacDonald, H. R. (1983) Annu. Rev. Immunol. 1, 273-306. 26. Heber-Katz, E., Schwartz, R. H., Matis, L. A., Hannum, C., Fairwell, T., Appella, E. & Hansburg, D. (1982) J. Exp. Med. 155, 1086-1099. 27. Conrad, P. J., Lerner, E. A., Murphy, D. B., Jones, P. P. & Janeway, C. A., Jr. (1982) J. Immunol. 129, 2616-2620.

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28. Janeway, C. A., Jr., Conrad, P. J., Tite, J., Jones, B. & Murphy, D. (1983) Nature (London) 306, 80-82. 29. Goldstein, S. A. N. & Mescher, M. F. (1987) J. Immunol. 138, 2034-2043. 30. McDevitt, H. 0. & Benacerraf, B. (1969) Adv. Immunol. 11, 31-74. 31. Chesnut, R. W. & Grey, H. M. (1981) J. Immunol. 126, 1075-1079. 32. Rock, K. L., Benacerraf, B. & Abbas, A. K. (1984) J. Exp. Med. 160, 1102-1113. 33. Howe, M. L., Goldstein, A. L. & Battisto, J. R. (1970) Proc. Nati. Acad. Sci. USA 67, 613-619. 34. Lu, C. Y. & Unanue, E. R. (1982) Cell. Immunol. Immunopathol. 25, 213-222. 35. Cronin, P. S., Sing, A. P., Glimcher, L. H., Kelley, V. E. & Reinisch, C. L. (1984) J. Immunol. 133, 822-829. 36. Adelman, N. E., Watling, D. L. & McDevitt, H. D. (1983) J. Exp. Med. 158, 1350-1355. 37. King, D. P. & Jones, P. P. (1983) J. Immunol. 131, 315-318. 38. Noelle, R., Krammer, P. H., Ohara, J., Uhr, J. W. & Vitetta, E. S. (1984) Proc. Nati. Acad. Sci. USA 81, 6149-6153. 39. Morel, P. A., Manolagas, S. C., Provvedini, D. M., Wegmann, D. R. & Chiller, J. M. (1986) J. Immunol. 136, 2181-2186. 40. Dennis, G. L. & Mond, J. J. (1986) J. Immunol. 136, 1600-1604. 41. Snyder, D. S., Beller, D. I. & Unanue, E. R. (1982) Nature (London) 299, 163-165.

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