THE JOURNAL OF I:\,FECTIOUS DISEASES. VOL. 136, SUPPLEMENT. AUGUST 1977
© 1977 by the University of Chicago. All rights reserved.
Formation of Antibody in the Newborn Mouse: Study of T-Cell-Independent Antibody Response D. E. Mosier, N. M. Zaldivar, E. Goldings, J. Mond, I. Scher, and W. E. Paul
From the Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health; and the Department of Clinical and Experimental Immunology, Naval Medical Research Institute) National Naval Medical Center, Bethesda, Maryland
The ability of newborn animals to form antibody to antigens of potentially pathogenic organisms is important for their survival. The purpose of this discussion is to evaluate the functional maturity of each of the cellular components of the immune response in the neonatal mouse. Such data can be used to predict the functional development of the immune system in humans through the use of an age-equivalence scale. The level of antibody production in the mouse is determined by the interaction of three major cell lines. These lines are: bone marrow-derived (B-) cells, those immunoglobulin-bearing lymphocytes of bone marrow origin that form antibody; thymus-derived (T-) cells, lymphocytes that differentiate within the thymus and serve to regulate
antibody production by B-cells; and macrophages, the cells that seem to enhance interactions of both antigen and T-cells with B-cells. Within each of these broad classes of cells are several subpopulations, which contain cells either at a distinct stage of maturation or of a separate subline of differentiation. Many surface markers have been described in the mouse that allow us to identify these subpopulations to a degree not yet possible in other species and to follow their appearance in ontogeny [1-4]. The time of appearance in the spleen of most of the well-studied B- and T-cell markers, as well as their proportional representation in an eightweek-old mouse, are summarized in figure I. B-cells with surface IgM appear in the spleen within 24 hr of birth and have been detected in fetal liver at 16 days of gestation [1]. Small numbers of B-lymphocytes bearing IgM and IgG or Igl\1 and IgA can be detected within two weeks of birth, and, soon afterwards, B-cells bearing only IgG or IgA are found. The mouse equivalent of human IgD appears on large numbers of splenic B-lymphocytes at about four weeks of age, and, thereafter, cells bearing both IgM and IgD-like determinants are the predominant subclass of B-
This work was supported in part by work unit no. MR041.02.01.0020 from the United States Naval Medical Research and Development Command. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the U.S. Navy Department or the naval service at large. Please address requests for reprints to Dr. D. E. Mosier, Laboratory of Immunology, Building 10, Room llDl3, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014.
514
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The ontogeny of immune responsiveness, as assayed by antibody formation in vitro, of mouse spleen lymphocytes to thymus-independent antigens is reviewed. Responsiveness to trinitrophenyl (TNP)-lipopolysaccharide and TNP-Brucella abortus appear soon after birth and one to two weeks before TNP-Ficoll or capsular polysaccharide of Streptococcus pneumoniae (SSS-III) elicits significant antibody formation. This hierarchy of responsiveness to antigens is also apparent in the CBA/N mutant mouse strain, which has a bone marrow-derived (B-) cell maturation arrest and fails to respond to either TNP-Ficoll or SSS-IlI. These findings are interpreted to suggest sequential maturation of different populations or lines of B-Iymphocytes, each of which can respond to a defined class of thymus-independent antigens. The implication for vaccine use in humans is that a late-appearing subclass of B-cells may be req uired for adequate immune responses to polysaccharide antigens.
S15
T'50 % r a + ?
• AH'~
BIRTH
AGE IN WEEKS
Figure 1. Development of bone marrow-derived (E-), thymus-derived (T-), and macrophage cell lines in the mouse. sIg+ = positive for immunoglobulin; p8+ = positive for surface IgM and surface IgD; Fc+ = positive for the receptor for the Fe region of immunoglobulin; CRL+ = positive for the C3 receptor for activated complement; Ia+ = positive for I-region coded antigens; MIs+ = positive for the minor lymphocyte-stimulating locus product; T-I = thymus-independent; T-D == thymus-dependent; MLC = mixed lymphocyte culture; eLM = cellmediated lympholysis.
cells [5]. At the same time as the shift in class distribution of surface Ig is occurring, the relative amount of surface IgM per B-cdl seems to decrease. Most of the other B-cell surface markers appear within two weeks of birth; some, like the Fe receptor, are present at levels found in adult mice, and others, like the C3 receptor, are present
on a relatively small number of B-Iymphocytes. By six weeks of age (in normal strains of mice), adult levels of all of the B-cell markers listed have been attained. There is a similar ontogeny of appearance of T-cell markers in the neonatal mouse [6, 7]. Much of the differentiation of T-cells takes place within
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Mosier et al.
516
the thymus, and it is about seven days after birth before many cells bearing the Thy 1 (formerly 0) marker are detected in the spleen. The TL marker is a character"istic of T-Iymphocytes in the intrathymic environment and is not expressed on peripheral T-cells. The Ly series of antigens is expressed on the first T-cells found in the spleen, but the precise timing of the appearance of subsets Ly J+ and Ly 2,3+ is not yet known. Remarkably little is known about the ontogeny of the monocyte-macrophage line, although differentiated function is present by nine days of age [8].
Materials and Methods
Our studies have concentrated on the acquisition of functional immune competence by mice during the first few weeks of life. Spleen cells from mice of several strains (mainly BALB/c, [C57BL/6 X DBA/2]F 1 or BDF 1 , and AKR) have been stimulated with a variety of antigens in microculture, and the number of cells synthesizing specific antibody was enumerated after two to five days of culture by hemolysis in gel of antigen-coated erythrocytes. The techniques have been described in detail [9, 10].
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Figure 2. Relative in vitro plaque-forming cell (PFC) responses of spleen cells from AKR (.&.), BDF 1 (e), and BALB/c (0) mice to the thymus-independent antigens trinitrophenyl (TNP)-Brucella abortus, TNP-Ficoll, or TNP-lipopolysaccharide (LPS). Results are expressed as percentages of PFCs with antibody to TNP of syngeneic 12- to 16-week-old mice.
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The antigens used in these studies all have been conjugated with trinitrophenyl (TNP) groups, and antibody responses to TNP have been measured. In this way, the precursor pool of B-cells is potentially the same for all the antigens; that is, only TNP-binding B-cells should be capable of being activated. Five different carriers have been coupled with TNP: (1) Ficoll, an epichlorhydrin cross-linked polysucrose with a molecular weight of 400,000 daltons; (2) E. coli lipopolysaccharide (LPS); (3) Brucella abortus; (4) sheep red blood cells (SRBC); and (5) keyhole limpet hemocyanin (KLH). The latter two conjugates, TNP-SRBC and TNP-KLH, require T-cells for the antibody response of adult spleen cells and are functionally termed thymus-dependent antigens. The other three conjugates, TNP-aminoethylcarbamylmethyl (AECM)-Ficoll, TNP-lipopolysaccharide (LPS), and TNP-B. abortus) stimulate undiminished antibody responses in the adult after T-cell depletion and are functionally termed thymus- or Tindependent antigens. T-cells may be able to augment the suboptimal response of immature B-cells to antigens that are clearly T-cell-independent in the adult, so these classifications of antigens are ones of convenience and are not to be construed in absolute terms.
817
T-eell-Independent Antibody Formation
Results
Normal cells" BDFI Anti-Thy -1treated BDF 1 BDFI Anti-Thy-ltreated BDF 1 BDFI Anti-Thy -1treated BDF 1 None None
Lymph node cellst
Spleen cells:\:
Concanavalin A§
No. of PFCII
o o 20
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"Spleen cells from one-week-old BDF 1 mice were treated with either brain-absorbed or complete antiserum to Thy 1.2 and guinea pig complement. Control cells are referred to as BDFI and T-cell-depleted cultures as anti-Thy I-treated BDFI. tPeripheral lymph node cells (105) were added to 10 6 spleen cells from one-week-old mice. +Irradiated adult spleen cells (105) were added to 5 X 10 5 spleen cells from one-week-old mice. § Concanavalin A (1 IJ.gJml) was added to cultures 24 hr after initiation. II Direct count of PFCs with antibody to trinitrophenylconjugated antigen per 10 7 normal cells initially cultured.
antibody responses are suboptimal because of a lack of sufficient T-cell helpers and/or an excess of T-cell suppression. The suppressive effect of neonatal T-cells has been confirmed by purification of such cells by nylon wool filtration and demonstration that they will suppress the response of neonatal or adult B-cells [I OJ. Whether or not helper T-cells exist at all in the neonatal spleen is not yet clear since their putative function may be completely masked by an excess of suppressor T-cells. There is evidence to suggest that suppressor and helper T-cells belong to separate subsets [12], so they may well mature at different times. Discussion
These results suggest that there is a hierarchy of antigens to which neonatal animals can respond by production of antibody. Some T-independent antigens, like TNP-B. abortus and TNP-LPS, can stimulate B-cells early in their maturation. The response to such antigen appears first in ontogeny.
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The pooled results of many experiments are shown in figure 2. In vitro antibody responses of spleen cells from BALB/c, BDF 1 , or AKR mice one to four weeks old to TNP-AECM-Ficoll, TNPLPS, or TNP-B. abortus are plotted with the data expressed as a percentage of the appropriate adult (eight to 12 week old) response. Spleen cells from one-week-old mice responded to both TNP-B. abortus and TNP-LPS by producing 20%-30% as many anti-TNP antibody-forming cells as comparable cultures of adult cells. TNP-AECM-Ficoll did not stimulate antibody formation unless spleen cell donors were at least two weeks old. Both TNP-SRBC and TNP-KLH failed to stimulate significant numbers of antibody-forming cells until mice were older than four to five weeks (hence, no data for these responses is shown in figure I). The failure of these two antigens to induce an antibody response does not mean that B-cells responsive to such antigens are lacking, as will be shown below. The results presented so far were obtained by culture of unfractionated spleen cells. If neonatal B-cells were purified by the use of columns containing antibody to immunoglobulin and/or treatment with antiserum to Thy 1.2, somewhat different results were obtained. The time of onset of responses to TNp·B. abortus) TNP-LPS, and TNP-AECM-Ficoll was unchanged (one or two weeks of age). However, the magnitude of the antiTNP responses to these antigens was increased by 50%-100% [10]. The response of purified neonatal B-cells could be further augmented to adult levels by nonspecific T-cell help induced by the addition of adult T-cells or by irradiated adult cells which were further stimulated by concanavalin A after culture for 24 hr [11]. Such nonspecific T-cell help also allowed B-cells from one- to two-week-old BDF 1 mice to respond to TNP-SRBC at a level comparable to that of unfractionated adult spleen cells (table 1). Control experiments showed that all antibody-forming cells were derived from the neonatal spleen B-cells. In addition, separate experiments with fractionated, adherent macrophages from two-week-old mice showed that they adequately replace adherent cells from adult mice [10]. These experiments strongly suggest that the ability of neonatal B-cells to respond to some antigens has developed by two weeks of age but that
Table 1. Restoration of the plaque-forming cell (PFC) responses of neonatal bone marrow-derived (B-) cells by adult thymus-derived (T-) cells or nonspecific T-cell factors.
Mosier et al.
SIS
antibody formation by 24 weeks of gestation. The major criticism of this comparison is that it ignores possible suppressive effects of the intrauterine environment on development as well as the possible stimulating effects of antigens encountered by the newborn.
References 1. Lawton, A. R., Kincade, P. W., Cooper, M. D. Sequential expression of germ line gcnes in development of immunoglobulin class diversity . Federation Proc. 34:
33,1975. 2. Ahmed, A., Scher, I., Sell, K. W . Functional studies of the ontogeny of the M-Iocus product; a surface antigen of murine B lymphocytes, In V. Eijsvogel [ed.]. Proceedings of the Eleventh Leukocyte Culture Conference. Academic Press, New York, 1976, p. 703-709. 3. Gelfand, M. C., Elfenbeing, G. J., Frank, M. M., Paul, W. E. Ontogeny of B lymphocytes. II. Relative rates of appearance of lymphocytes bearing surface immunoglobulin and complement receptors. J. Exp. Med. 139:1I25,1974. 4. Hammerling, U., Chin, A. F., Abbot, J., Schied, M. The ontogeny of B lymphocytes. I. Induction of phenotypic conversion of Ia- to Ia+ lymphocytes. J. Immunol. ll5: 1425, 1975. 5. Vitetta, E. S., Melcher, U., McWilliams, M., Lamrn, E., Phillips-Quagliata, M., Uhr, J. W. Cell surface immunoglobulin. XI. The appearance of an IgD-like molecule on murine lymphoid during ontogeny. J. Exp. Med. 141:206, 1975. 6. Cantor, H., Boyse, E. A. Functional subclasses of T lymphocytes bearing different Ly antigens. I. The generation of functionally distinct T-cell subclasses is a differentiative process independent of antigen. J. Exp. Med. 141:1376,1975. 7. Weissman, I. L., Small, M., Fathman, C. G., Herzenberg, L. A. Differentiation of thymus cells. Fed. Proc. 34:141-144,1975. 8. Landahl, C., Auerbach, R. Murine adherent cell ontogeny: in vitro responscs to sheep erythrocytes and to alloantigens. Fed. Proc. 34:1031, 1975. 9. Mosier, D. E., Johnson, B. M., Paul, \V. E., McMaster, P. R. B. Cellular requirements for the primary in vitro antibody response to DNP-Ficoll. J. Exp. Med. 139:1354,1974. 10. Mosier, D. E., Johnson, B. M. Ontogeny of mouse lymphocyte function. II. Development of the ability to produce antibody is modulated by T lymphocytes. J. Exp. Med. 141:216, 1975. II. Dutton, R. W. Inhibition and stimulatory effects of concanavalin A on the response of mouse spleen cell suspensions to antigen. II. Evidence for separate stimulatory and inhibitory cells. J. Exp. Med. 138: 1496, 1973. 12. Cohen, P. L., Cross, S. S., Mosier, D. E. Immunological
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T-cells are not required for the response to TNPB. abortus or TNP-LPS even in the neonate; however, the addition of adult T-cells can augment the response to these antigens. Other T-independent antigens, of which TNP-AEClVI-Ficoll is the prototype, stimulate B-cells of a more advanced stage of maturation or, alternatively, of a later appearing subline of B-cells. This conclusion also is supported by study of the CBA/N/strain of mice, which appears to have an X chromosome-linked defect in B-cell maturation [13, 14]. Spleen cells from such mice respond to TNP-LPS but not to TNP-AECl\I-Ficoll [14, 15]. Finally, antibody responses to T-dependent antigens like TNP-SRBC and TNP-KLH appear last in ontogeny. B-cells potentially capable of responding to such antigens do exist in the spleens of neonatal mice, but they fail to respond because of some combination of defective T-cell help and/or excess T-cell suppression. The time of appearance of responsiveness to antigens as well as the relative effects of T-cells and macrophages are shown in juxtaposition to the ontogeny of surface markers in figure I. It can be seen that the B-cells from one-week-old mice, which respond to antigens like TNP-B. abortus and TNP-LPS, have different surface characteristics than do the majority of B-cells from adult mice. The amount of surface IglVI is greater on the neonatal B-cells, and a number of B-cell markers have yet to appear. The fact that these B-Iymphocytes can be triggered implies that the IgD homologue and the C3 receptor, for instance, are not required for the response to all antigens. Detailed analysis of the dose-response relationship of these early neonatal B-cells to antigen suggests that they require more antigen than do adult B-cells for optimal activation. Antigens like TNP-AECMFicoll may not be able to trigger such B-cells because either Ficoll does not have sufficient epitope (antigenic determinant) density or cannot effectively cross-link B-cell receptors. These results can be extrapolated to the human if we make use of an age-equivalence scale based on comparison of a large number of developmental milestones in mice and humans [16]. By this method of comparison, the ability to form antibody to T-independent antigens should appear by 16 weeks of human gestation, and even T-dependent antigens should be capable of stimulating
T-eell-Independent Antibody Formation
effects of neonatal infection with mouse thymic virus. J. Immunol. 115:706, 1975. 13. Scher, I., Ahmed, A., Strong, D. M., Steinberg. A. D., Paul, W. E. X-linked B-lymphocyte immune defect in CBAjHN mice. I. Studies of the function and composition of spleen cells. J. Exp. Med, 141:788, 1975. 14. Cohen, P. L., Scher, I., Mosier, D. E. In vitro studies of the genetically determined unresponsiveness to thymic-independent antigens in CBAjN mice. J. Immunol. 116:301, 1976.
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15. Mosier, D. E., Scher, I., Paul, W. E. In vitro responses of CRAjN mice: spleen cells of mice with an X-linked defect that precludes immune response to several thymus-independent antigens can respond to TNPlipopolysaccharide. J. Immunol. 117:1363,1976. 16. Solomon, J. B. Foetal and neonatal immunology. North Hollandj American Elsevier, New York, 1971, p. 343361.
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© 1977 by the University of Chicago. All rights reserved.
Formation of Antibody in the Newborn Mouse: Study of T-Cell-Independent Antibody Response D. E. Mosier, N. M. Zaldivar, E. Goldings, J. Mond, I. Scher, and W. E. Paul
From the Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health; and the Department of Clinical and Experimental Immunology, Naval Medical Research Institute) National Naval Medical Center, Bethesda, Maryland
The ability of newborn animals to form antibody to antigens of potentially pathogenic organisms is important for their survival. The purpose of this discussion is to evaluate the functional maturity of each of the cellular components of the immune response in the neonatal mouse. Such data can be used to predict the functional development of the immune system in humans through the use of an age-equivalence scale. The level of antibody production in the mouse is determined by the interaction of three major cell lines. These lines are: bone marrow-derived (B-) cells, those immunoglobulin-bearing lymphocytes of bone marrow origin that form antibody; thymus-derived (T-) cells, lymphocytes that differentiate within the thymus and serve to regulate
antibody production by B-cells; and macrophages, the cells that seem to enhance interactions of both antigen and T-cells with B-cells. Within each of these broad classes of cells are several subpopulations, which contain cells either at a distinct stage of maturation or of a separate subline of differentiation. Many surface markers have been described in the mouse that allow us to identify these subpopulations to a degree not yet possible in other species and to follow their appearance in ontogeny [1-4]. The time of appearance in the spleen of most of the well-studied B- and T-cell markers, as well as their proportional representation in an eightweek-old mouse, are summarized in figure I. B-cells with surface IgM appear in the spleen within 24 hr of birth and have been detected in fetal liver at 16 days of gestation [1]. Small numbers of B-lymphocytes bearing IgM and IgG or Igl\1 and IgA can be detected within two weeks of birth, and, soon afterwards, B-cells bearing only IgG or IgA are found. The mouse equivalent of human IgD appears on large numbers of splenic B-lymphocytes at about four weeks of age, and, thereafter, cells bearing both IgM and IgD-like determinants are the predominant subclass of B-
This work was supported in part by work unit no. MR041.02.01.0020 from the United States Naval Medical Research and Development Command. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as official or as reflecting the views of the U.S. Navy Department or the naval service at large. Please address requests for reprints to Dr. D. E. Mosier, Laboratory of Immunology, Building 10, Room llDl3, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20014.
514
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on July 25, 2015
The ontogeny of immune responsiveness, as assayed by antibody formation in vitro, of mouse spleen lymphocytes to thymus-independent antigens is reviewed. Responsiveness to trinitrophenyl (TNP)-lipopolysaccharide and TNP-Brucella abortus appear soon after birth and one to two weeks before TNP-Ficoll or capsular polysaccharide of Streptococcus pneumoniae (SSS-III) elicits significant antibody formation. This hierarchy of responsiveness to antigens is also apparent in the CBA/N mutant mouse strain, which has a bone marrow-derived (B-) cell maturation arrest and fails to respond to either TNP-Ficoll or SSS-IlI. These findings are interpreted to suggest sequential maturation of different populations or lines of B-Iymphocytes, each of which can respond to a defined class of thymus-independent antigens. The implication for vaccine use in humans is that a late-appearing subclass of B-cells may be req uired for adequate immune responses to polysaccharide antigens.
S15
T'50 % r a + ?
• AH'~
BIRTH
AGE IN WEEKS
Figure 1. Development of bone marrow-derived (E-), thymus-derived (T-), and macrophage cell lines in the mouse. sIg+ = positive for immunoglobulin; p8+ = positive for surface IgM and surface IgD; Fc+ = positive for the receptor for the Fe region of immunoglobulin; CRL+ = positive for the C3 receptor for activated complement; Ia+ = positive for I-region coded antigens; MIs+ = positive for the minor lymphocyte-stimulating locus product; T-I = thymus-independent; T-D == thymus-dependent; MLC = mixed lymphocyte culture; eLM = cellmediated lympholysis.
cells [5]. At the same time as the shift in class distribution of surface Ig is occurring, the relative amount of surface IgM per B-cdl seems to decrease. Most of the other B-cell surface markers appear within two weeks of birth; some, like the Fe receptor, are present at levels found in adult mice, and others, like the C3 receptor, are present
on a relatively small number of B-Iymphocytes. By six weeks of age (in normal strains of mice), adult levels of all of the B-cell markers listed have been attained. There is a similar ontogeny of appearance of T-cell markers in the neonatal mouse [6, 7]. Much of the differentiation of T-cells takes place within
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STE~
Mosier et al.
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the thymus, and it is about seven days after birth before many cells bearing the Thy 1 (formerly 0) marker are detected in the spleen. The TL marker is a character"istic of T-Iymphocytes in the intrathymic environment and is not expressed on peripheral T-cells. The Ly series of antigens is expressed on the first T-cells found in the spleen, but the precise timing of the appearance of subsets Ly J+ and Ly 2,3+ is not yet known. Remarkably little is known about the ontogeny of the monocyte-macrophage line, although differentiated function is present by nine days of age [8].
Materials and Methods
Our studies have concentrated on the acquisition of functional immune competence by mice during the first few weeks of life. Spleen cells from mice of several strains (mainly BALB/c, [C57BL/6 X DBA/2]F 1 or BDF 1 , and AKR) have been stimulated with a variety of antigens in microculture, and the number of cells synthesizing specific antibody was enumerated after two to five days of culture by hemolysis in gel of antigen-coated erythrocytes. The techniques have been described in detail [9, 10].
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Figure 2. Relative in vitro plaque-forming cell (PFC) responses of spleen cells from AKR (.&.), BDF 1 (e), and BALB/c (0) mice to the thymus-independent antigens trinitrophenyl (TNP)-Brucella abortus, TNP-Ficoll, or TNP-lipopolysaccharide (LPS). Results are expressed as percentages of PFCs with antibody to TNP of syngeneic 12- to 16-week-old mice.
Downloaded from http://jid.oxfordjournals.org/ at Florida Atlantic University on July 25, 2015
The antigens used in these studies all have been conjugated with trinitrophenyl (TNP) groups, and antibody responses to TNP have been measured. In this way, the precursor pool of B-cells is potentially the same for all the antigens; that is, only TNP-binding B-cells should be capable of being activated. Five different carriers have been coupled with TNP: (1) Ficoll, an epichlorhydrin cross-linked polysucrose with a molecular weight of 400,000 daltons; (2) E. coli lipopolysaccharide (LPS); (3) Brucella abortus; (4) sheep red blood cells (SRBC); and (5) keyhole limpet hemocyanin (KLH). The latter two conjugates, TNP-SRBC and TNP-KLH, require T-cells for the antibody response of adult spleen cells and are functionally termed thymus-dependent antigens. The other three conjugates, TNP-aminoethylcarbamylmethyl (AECM)-Ficoll, TNP-lipopolysaccharide (LPS), and TNP-B. abortus) stimulate undiminished antibody responses in the adult after T-cell depletion and are functionally termed thymus- or Tindependent antigens. T-cells may be able to augment the suboptimal response of immature B-cells to antigens that are clearly T-cell-independent in the adult, so these classifications of antigens are ones of convenience and are not to be construed in absolute terms.
817
T-eell-Independent Antibody Formation
Results
Normal cells" BDFI Anti-Thy -1treated BDF 1 BDFI Anti-Thy-ltreated BDF 1 BDFI Anti-Thy -1treated BDF 1 None None
Lymph node cellst
Spleen cells:\:
Concanavalin A§
No. of PFCII
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890 680
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"Spleen cells from one-week-old BDF 1 mice were treated with either brain-absorbed or complete antiserum to Thy 1.2 and guinea pig complement. Control cells are referred to as BDFI and T-cell-depleted cultures as anti-Thy I-treated BDFI. tPeripheral lymph node cells (105) were added to 10 6 spleen cells from one-week-old mice. +Irradiated adult spleen cells (105) were added to 5 X 10 5 spleen cells from one-week-old mice. § Concanavalin A (1 IJ.gJml) was added to cultures 24 hr after initiation. II Direct count of PFCs with antibody to trinitrophenylconjugated antigen per 10 7 normal cells initially cultured.
antibody responses are suboptimal because of a lack of sufficient T-cell helpers and/or an excess of T-cell suppression. The suppressive effect of neonatal T-cells has been confirmed by purification of such cells by nylon wool filtration and demonstration that they will suppress the response of neonatal or adult B-cells [I OJ. Whether or not helper T-cells exist at all in the neonatal spleen is not yet clear since their putative function may be completely masked by an excess of suppressor T-cells. There is evidence to suggest that suppressor and helper T-cells belong to separate subsets [12], so they may well mature at different times. Discussion
These results suggest that there is a hierarchy of antigens to which neonatal animals can respond by production of antibody. Some T-independent antigens, like TNP-B. abortus and TNP-LPS, can stimulate B-cells early in their maturation. The response to such antigen appears first in ontogeny.
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The pooled results of many experiments are shown in figure 2. In vitro antibody responses of spleen cells from BALB/c, BDF 1 , or AKR mice one to four weeks old to TNP-AECM-Ficoll, TNPLPS, or TNP-B. abortus are plotted with the data expressed as a percentage of the appropriate adult (eight to 12 week old) response. Spleen cells from one-week-old mice responded to both TNP-B. abortus and TNP-LPS by producing 20%-30% as many anti-TNP antibody-forming cells as comparable cultures of adult cells. TNP-AECM-Ficoll did not stimulate antibody formation unless spleen cell donors were at least two weeks old. Both TNP-SRBC and TNP-KLH failed to stimulate significant numbers of antibody-forming cells until mice were older than four to five weeks (hence, no data for these responses is shown in figure I). The failure of these two antigens to induce an antibody response does not mean that B-cells responsive to such antigens are lacking, as will be shown below. The results presented so far were obtained by culture of unfractionated spleen cells. If neonatal B-cells were purified by the use of columns containing antibody to immunoglobulin and/or treatment with antiserum to Thy 1.2, somewhat different results were obtained. The time of onset of responses to TNp·B. abortus) TNP-LPS, and TNP-AECM-Ficoll was unchanged (one or two weeks of age). However, the magnitude of the antiTNP responses to these antigens was increased by 50%-100% [10]. The response of purified neonatal B-cells could be further augmented to adult levels by nonspecific T-cell help induced by the addition of adult T-cells or by irradiated adult cells which were further stimulated by concanavalin A after culture for 24 hr [11]. Such nonspecific T-cell help also allowed B-cells from one- to two-week-old BDF 1 mice to respond to TNP-SRBC at a level comparable to that of unfractionated adult spleen cells (table 1). Control experiments showed that all antibody-forming cells were derived from the neonatal spleen B-cells. In addition, separate experiments with fractionated, adherent macrophages from two-week-old mice showed that they adequately replace adherent cells from adult mice [10]. These experiments strongly suggest that the ability of neonatal B-cells to respond to some antigens has developed by two weeks of age but that
Table 1. Restoration of the plaque-forming cell (PFC) responses of neonatal bone marrow-derived (B-) cells by adult thymus-derived (T-) cells or nonspecific T-cell factors.
Mosier et al.
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antibody formation by 24 weeks of gestation. The major criticism of this comparison is that it ignores possible suppressive effects of the intrauterine environment on development as well as the possible stimulating effects of antigens encountered by the newborn.
References 1. Lawton, A. R., Kincade, P. W., Cooper, M. D. Sequential expression of germ line gcnes in development of immunoglobulin class diversity . Federation Proc. 34:
33,1975. 2. Ahmed, A., Scher, I., Sell, K. W . Functional studies of the ontogeny of the M-Iocus product; a surface antigen of murine B lymphocytes, In V. Eijsvogel [ed.]. Proceedings of the Eleventh Leukocyte Culture Conference. Academic Press, New York, 1976, p. 703-709. 3. Gelfand, M. C., Elfenbeing, G. J., Frank, M. M., Paul, W. E. Ontogeny of B lymphocytes. II. Relative rates of appearance of lymphocytes bearing surface immunoglobulin and complement receptors. J. Exp. Med. 139:1I25,1974. 4. Hammerling, U., Chin, A. F., Abbot, J., Schied, M. The ontogeny of B lymphocytes. I. Induction of phenotypic conversion of Ia- to Ia+ lymphocytes. J. Immunol. ll5: 1425, 1975. 5. Vitetta, E. S., Melcher, U., McWilliams, M., Lamrn, E., Phillips-Quagliata, M., Uhr, J. W. Cell surface immunoglobulin. XI. The appearance of an IgD-like molecule on murine lymphoid during ontogeny. J. Exp. Med. 141:206, 1975. 6. Cantor, H., Boyse, E. A. Functional subclasses of T lymphocytes bearing different Ly antigens. I. The generation of functionally distinct T-cell subclasses is a differentiative process independent of antigen. J. Exp. Med. 141:1376,1975. 7. Weissman, I. L., Small, M., Fathman, C. G., Herzenberg, L. A. Differentiation of thymus cells. Fed. Proc. 34:141-144,1975. 8. Landahl, C., Auerbach, R. Murine adherent cell ontogeny: in vitro responscs to sheep erythrocytes and to alloantigens. Fed. Proc. 34:1031, 1975. 9. Mosier, D. E., Johnson, B. M., Paul, \V. E., McMaster, P. R. B. Cellular requirements for the primary in vitro antibody response to DNP-Ficoll. J. Exp. Med. 139:1354,1974. 10. Mosier, D. E., Johnson, B. M. Ontogeny of mouse lymphocyte function. II. Development of the ability to produce antibody is modulated by T lymphocytes. J. Exp. Med. 141:216, 1975. II. Dutton, R. W. Inhibition and stimulatory effects of concanavalin A on the response of mouse spleen cell suspensions to antigen. II. Evidence for separate stimulatory and inhibitory cells. J. Exp. Med. 138: 1496, 1973. 12. Cohen, P. L., Cross, S. S., Mosier, D. E. Immunological
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T-cells are not required for the response to TNPB. abortus or TNP-LPS even in the neonate; however, the addition of adult T-cells can augment the response to these antigens. Other T-independent antigens, of which TNP-AEClVI-Ficoll is the prototype, stimulate B-cells of a more advanced stage of maturation or, alternatively, of a later appearing subline of B-cells. This conclusion also is supported by study of the CBA/N/strain of mice, which appears to have an X chromosome-linked defect in B-cell maturation [13, 14]. Spleen cells from such mice respond to TNP-LPS but not to TNP-AECl\I-Ficoll [14, 15]. Finally, antibody responses to T-dependent antigens like TNP-SRBC and TNP-KLH appear last in ontogeny. B-cells potentially capable of responding to such antigens do exist in the spleens of neonatal mice, but they fail to respond because of some combination of defective T-cell help and/or excess T-cell suppression. The time of appearance of responsiveness to antigens as well as the relative effects of T-cells and macrophages are shown in juxtaposition to the ontogeny of surface markers in figure I. It can be seen that the B-cells from one-week-old mice, which respond to antigens like TNP-B. abortus and TNP-LPS, have different surface characteristics than do the majority of B-cells from adult mice. The amount of surface IglVI is greater on the neonatal B-cells, and a number of B-cell markers have yet to appear. The fact that these B-Iymphocytes can be triggered implies that the IgD homologue and the C3 receptor, for instance, are not required for the response to all antigens. Detailed analysis of the dose-response relationship of these early neonatal B-cells to antigen suggests that they require more antigen than do adult B-cells for optimal activation. Antigens like TNP-AECMFicoll may not be able to trigger such B-cells because either Ficoll does not have sufficient epitope (antigenic determinant) density or cannot effectively cross-link B-cell receptors. These results can be extrapolated to the human if we make use of an age-equivalence scale based on comparison of a large number of developmental milestones in mice and humans [16]. By this method of comparison, the ability to form antibody to T-independent antigens should appear by 16 weeks of human gestation, and even T-dependent antigens should be capable of stimulating
T-eell-Independent Antibody Formation
effects of neonatal infection with mouse thymic virus. J. Immunol. 115:706, 1975. 13. Scher, I., Ahmed, A., Strong, D. M., Steinberg. A. D., Paul, W. E. X-linked B-lymphocyte immune defect in CBAjHN mice. I. Studies of the function and composition of spleen cells. J. Exp. Med, 141:788, 1975. 14. Cohen, P. L., Scher, I., Mosier, D. E. In vitro studies of the genetically determined unresponsiveness to thymic-independent antigens in CBAjN mice. J. Immunol. 116:301, 1976.
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15. Mosier, D. E., Scher, I., Paul, W. E. In vitro responses of CRAjN mice: spleen cells of mice with an X-linked defect that precludes immune response to several thymus-independent antigens can respond to TNPlipopolysaccharide. J. Immunol. 117:1363,1976. 16. Solomon, J. B. Foetal and neonatal immunology. North Hollandj American Elsevier, New York, 1971, p. 343361.
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