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Antigen-induced plaque-forming cell responses in cultures of peripheral blood mononuclear cells of human neonates and infants
Antigen-induced plaque-forming cell responses in cultures of peripheral blood mononuclear cells of human neonates and infants
Human cord blood mononuelear cells (CBMC) were stimulated in vitro with a number of T cell-dependent antigens. Antigen-induced B cell activation was measured applying a plaque-forming cell assay for the detection of antigen-specific lgM-secreting B cells. With the exception of diphtheria toxoid, the antigens ovalbumin, sheep red blood cells, Helix pomatia hemocyanin, burro red blood cells, and tetanus toxoid elicited an IgM-plaque-forming cell response in cultures of CBMC to levels obtained for peripheral blood mononuclear cells (PBMC) from adult controls. However, for each antigen used, the antigen dose optimal for the induction o f a response was consistently found to be a hundred to a thousand times lower than the concentration of the corresponding antigen optimal for adult PBMC. Longitudinal studies on PBMC obtained from infants between 2 and 30 months of age revealed that a shift of the antigen dose toward concentrations needed to induce plaque-forming cells in cultures of adult PBMC occurs at approximately age 8 months. Our data indicate that various antigens can be used for the in vitro analysis o f antigen-specific B cell activation and regulatory T cell functions in studies concerning the ontogeny of the humoral immune response in humans. (J Ps
105:738, 1984)
Maarten J. D. van Tol, M.Sc., Jitske Zijlstra, Ben J. M. Zegers, Ph.D., and Rudy E. Ballieux, Ph.D. Utrecht, The Netherlands
AT BIRTH the capacity to mount a humoral immune response is not fully developed in a number of mammalian species. In humans antibody production is mainly restricted to the IgM class of immunoglobulins after intrauterine infection and after active immunization during the first 6 months of life.~ Because lymphocyte subpopulations belonging to the B and T cell lineage, respectively, as well as monocytes appear early in gestation,2-4 it might be inferred that a dichotomy exists between the development From University Children's Hospital Her Wilhelmina Kinderziekenhuis, and University Hospital. Supported by the Foundation for Medical Research (Fungo), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). Submitted for publication Jan. 31, 1984; accepted April 13, 1984. Reprint requests: Maarten J. D. van Tol, University Children's Hospital Het Wilhelmina Kinderziekenhuis, Nieuwe Gracht I37, 3512 LK Utrecht, The Netherlands.
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of the cellular constituents of the humoral immune response and their functional activity. However, in the human fetus and newborn infant, relatively high numbers BRBC CBMC HPH MNC OA PBMC PFC SRBC TN P-PAA
Burro red blood cells Cord blood mononuclear cells Helix pomat~a hemocyanin Mononuclear cells Ovalbumin Peripheral blood mononuclear cells Plaque-forming cells Sheep red blood cells Trinitrophenylated polyacrylamide beads
of B and T cells have an immature surface phenotype.Z, 3, 5, 6 The cellular basis of the defi6ient humoral immune response in the human newborn infant has usually been analyzed by in vitro activation of B cells with polyclonal stimulators.7-9 Only a few reports have focused on the
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ontogeny of the antigen-specific B cell function and the regulation of the antibody response after antigenic stimulation?0. ~ Human cord blood mononuclear cells are able to mount a primary hapten-specific IgM plaque-forming cell response after in vitro stimulation with the hapten-carrier complex TNP-PAA? ~ However, only four of 42 newborn infants responded, and the level of the response was substantially lower than that induced in cultures of adult peripheral blood mononuclear cells. Applying the T celldependent antigens sheep red blood cells and ovalbumin, a different picture has emerged from studies in our laboratory. Consistently, an IgM-PFC response was obtained in cultures of CBMC to a level comparable to that found in adult PBMC. H However, the dose of antigen optimal for the induction of a response of CBMC appeared to be a hundred times lower compared with that optimal for adult PBMC. This observation could be related to a difference in antigen handling between monocytes in neonates and adults that partly determines the antigen dose dependency for the activation of regulatory T cell subsets? 2 Early observations in the mouse, rat, and sheep have indicated that in vivo responsiveness to various antigens develops in a stepwise fashion during fetal or neonatal life] T M Antigens can be placed in a hierarchical sequence according to their capacity to induce an antibody response "early" or "late" in gestation or in the first postnatal period. The cellular basis of this phenomenon is not fully established. The aim of our study was to extend the investigations on the capacity of human CBMC to mount an in vitro PFC response by using antigens described to be "late" antigens in other mammalian species. Furthermore, the development of the antigen dose dependency for the induction of PFCs from the "neonatal type" to the "adult type" was studied by culturing P B M C from children between 2 and 30 months of age. METHODS Donors of mononnclear cells. Mononuclear cells were isolated from cord blood collected immediately after birth. In all cases the mothers had had uncomplicated and full-term pregnancies. The PBMC were obtained from peripheral blood of healthy adult volunteers ("adult PBMC"). For the experiments concerning the longitudinal study, blood was obtained from children between 2 and 30 months of age after written and informed consent of their parents. These children were seen at the Children's Hospital for surgery or because of clinical symptoms not associated with disorders affecting the immune system. Isolation of mononuclear cells. Lymphocytes and monocytes were prepared by centrifugation of heparinized blood
Antigen-specific plaque-forming cell response
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diluted once in minimal essential medium (MEM-Tris, Gibco, Grand Island, N.Y.) on Ficoll-Isopaque density gradients (p = 1.077 gm/cm 3) at 700 X g for 20 minutes. The CBMC were usually contaminated with erythrocytes, which were removed by NH4Cl-induced lysis followed by two washings with cold MEM-Tris containing 0.5% (wt/ vol) bovine serum albumin (Sigma, St. Louis, Mo.). When monocytes were present in the MNC-suspension in amounts exceeding 15% of the total cell number, MNC were partly depleted of monocytes by adherence to plastic flasks (Falcon, Oxnard, Calif.) at 37 ~ C in MEM-Tris supplemented with 20% (vol/vol) fetal calf serum. After incubation for 45 to 90 minutes the nonadherent cell fraction containing lymphocytes and nonadherent monocytes was decanted and washed twice with MEM-Tris. The adherent cells (>90% nonspecific esterase-positive cells) were harvested by gentle scraping with a rubber policeman and washed twice with MEM-Tris. Adherent cells (monocytes) were returned to the nonadherent cell fraction to establish a final proportion of 10% monocytes. Antigenic stimulation of mononuclear cells. A total of 4 to 5 x 106 M N C were cultured in tissue culture tubes (Falcon, 17 X 100 mm) in 10 ml R P M I 1640 (Gibco) containing 10% (vol/vol) heat-inactivated and SRBCabsorbed human AB serum, fungizone (25/~g/ml), penicillin (100 ~g/ml), streptomycin (100 IU/ml), and Lglutamine (2 mM). When BRBC were applied as antigen, SRBC-absorbed AB serum was replaced by BRBCabsorbed AB serum. The cells were stimulated with the antigens SRBC, ovalbumin, BRBC, Helix pomatia hemocyanin, diphtheria toxoid, and tetanus toxoid, respectively, which were present in the concentrations indicated. After culture for 6 to 7 days at 37 ~ C in an atmosphere of 5% CO2 the cells were collected, washed twice in MEM-Tris at 4 ~ C, and assayed for the presence of PFCs. When SRBC or BRBC were used for antigenic stimulation, the cultured MNC were freed of the erythrocytes by NH4Cl-induced lysis before the PFC-assay. H P H was kindly provided by Dr. T. H. Th6 (University Hospital, Groningen, The Netherlands). Diphtheria toxoid and tetanus toxoid were supplied by Dr. J. Nagel (National Institute of Public Health, Bilthoven, The Netherlands), and OA (grade VI) was obtained from Sigma. Plaque-forming call assay. The PFCs were determined by the method originally described by Kennedy and Axelrad 16 according to the modifications introduced by Dosch and Gelfand? 7. Briefly, SRBC, antigen-coated SRBC, or BRBC were centrifuged for 5 minutes at 1000 x g on the bottom of Falcor~ microtiter plates (No. 3040) precoated with poly-L-lysine (Sigma, Mr --<100,000) in a concentration of 100 #g/ml distilled water by incubation for 60 minutes at 37 ~ C. Viable cultured cells in
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PFC / 1 0 6
The Journal o f Pediatrics November 1984
ly.
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Fig. 1. Antigen dose-response relationship for induction of plaque-forming cells in cultures of adult peripheral blood mononuclear cells (PBMC). A, sheep red blood cells (n = 5) or burro red blood ceils (n = 3). B, Ovalbumin (n = 5) or Hefix pomatia hemocyanin (n = 5). C, Tetanus toxoid (n = 3) or diphtheria toxoid (n = 2). Results shown for each antigen are mean _+ SD of experiments using different peripheral blood mononuclear cell donors. Data are expressed as PFC/106 lymphocytes.
various dilutions were i n c u b a t e d for 60 to 90 minutes in the presence of S R B C - or B R B C - a b s o r b e d guinea pig complement, respectively. A f t e r stimulation of M N C with OA, H P H , diphtheria toxoid, or tetanus toxoid the level of the induced P F C response was analyzed using monolayers of
102
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Fig. 2. Antigen dose-response relationship for induction of plaque-forming cells in cultures of cord blood mononuclear cells (CBMC). A, sheep red blood ceils (n = 5) or burro red blood cells (n = 3). B, Ovalbumin (n = 5) or Helix pomatia hemocyanin (n = 3). C, Tetanus toxoid (n ~- 3) or diphtheria toxoid (n = 4). Results shown for each antigefi are mean + SD of experiments using different CBMC donors. Data are expressed as PFC/10 ~ lymphocytes.
S R B C coated with the relevant antigen by the method described by Goding. ~8 In the P F C assay only IgMsecreting cells were detected. T h e antigen specificity of the P F C response of C B M C as well as~'PBMC from adults was ascertained in experiments published earlier. H, ~9.20In addition, stimulation of M N C with B R B C or H P H only gave rise to the detection of P F C s in monolayers of S R B C at
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background levels ( ~ 2 0 0 PFC/106 lymphocytes). The same observation was true for the stimulation of M N C with diphtheria toxoid or tetanus toxoid followed by the performance of the PFC assay using monolayers of tetanus toxoid- or diphtheria toxoid-coated SRBC. These results indicated that none of the applied antigens in the range of concentrations used for stimulation caused polyclonal B cell activation.
Antigen-specific plaque-forming cell response
PFC / 106 ly.
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RESULTS Ovalbumin and SRBC-induced PFC response of adult PBMC and CBMC. Adult PBMC and CBMC were cultured in the presence of variable doses of the corpuscular antigen SRBC or the soluble antigen OA. A PFC resPonse was observed after stimulation of adult PBMC (Fig. i) as well as CBMC (Fig. 2) with either antigen. The level Of the response induced in cultures of adult PBMC was comparable to that obtained for CBMC. However, an optimal response in cuitures of CBMC was elicited at an antigen concentration a hundred times lower than that optimal for adult PBMC (0.03 vs 3.0 /~g O A / m l and 5 x 104 vs 5 X 106 SRBC per culture, respectively): None of the newborn infants failed to mount an IgM-PFC response to SRBC and OA, and all of the neonates tested Showed the same antigen dose-response relationship . PFC response of adult PBMC and CBMC toward other antigens. To investigate the repertoire of functionally active antigen-specific lymphocytes present at birth, CBMC and adult PBMC were stimulated with various doses of HPH, tetanus toxoid, diphtheria toxoid, and BRBC. A PFC response could be obtained in cultures of CBMC as well as adult PBMC with HPH, tetanus toxoid, and BRBC (Figs. 1 and 2). Irrespective to the antigen applied, the dose optimal for the induction of PFCs in cultures of CBMC was shifted to a hundredfold to thousandfold lower antigen concentration in comparison with the conditions optimal for the stimulation of adult PBMC (HPH, 1 vs 100 #g H P H / m l ; tetanus to'old, 0.1 vs 100 ng TT/ml; BRBC, 5 x 104 v s 5 X 106 BRBC per culture). Regarding the stimulation of adult PBMC with tetanus toxoid, the donors used in this study we)e immunized with this antigen during childhood. However, ` despite a higher level of the PFC response, the antigen dose-response relationship for immunized donors did not differfrom that obtained with nonimmunized adults (data not shown). Diphtheria toxoid failed to induce a PFC respons e in cultures of CBMC, whereas a response was established in cultures of adult PBMC from immunized donors. It is interesting that an anti-diphtheria toxoid PFC response Mso appeared to be absent in cultures of PBMC derived from a nonimmunized adult (data not shown). Development of dose-response relationship with age.
74 1
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age ( m o n t h s ) Fig. 3. Induction of plaque-forming cells (PFC) in cultures of peripheral blood mononuclear cells (PBMC) obtained from infants ranging from 2 to 30 months of age after stimulation with low dose of antigen. PBMC from individual donors were stimulated with ovalbumin 0.03 ~g/mi (U) and 5 X 104 sheep red blood cells per culiure (F'I),respectively. Level of PFC response induced is expressed as PFC/10 ~ lymphocytes and plotted against age of infant.
Experiments were performed to analyze whether the antigen dose dependency for the induction of PFCs shifts during infancy from a "neonatal type" of dose-response relationship observed in CBMC to an "adult type" of response established in cultures of adult PBMC. To this end, PBMC obtained from Children between 2 and 30 months of age were cultured in the presence of variable doses of SRBC and OA, respectively. The levels of the responses induced in cultures of PBMC indicate that a correlation exists between the dose of antigen optimal for the induction of a PFC response and the age of the child (Figs. 3 and 4). From the age of 8 months, 11 of 12 infants studied mounted an optimal PFC response after stimulation with 3.0 #g O A / m l (i.e., an "adult type" of antigen dose-response relationship). Stimulation of PBMC obtained from nine donors of this group of 11 infants with SRBC indicated that this observat~bn was not restricted to the antigen OA. However, at younger than 8 months a completely different picture emerged: five of six infants showed a "neonatal type" of antigen dose-response rela-
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The Journal of Pediatrics November 1984
/1061y.
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age (months) Fig. 4. Induction of plaque-forming cells (PFC) in cultures of peripheral blood mononuclear cells (PBMC) obtained from infants ranging 2 to 30 months of age after stimulation with high dose of antigen. PBMC from individual donors Were stimulated with ovalbumin 3.0 ~zg/ml (0) and 5 X 106 sheep red blood cells per culture (0), respectively. Level of PFC response induced is expressed as PFC/106 lymphocytes and plotted against age of infant.
ship for the induction of PFCs, with an antigen dose oPtimum of 0.03 #g OA/ml. The same observation held for the two infants who could also be analyzed using SRBC as the stimulus. In comparison with the older group, the optimal antigen dose was found to be shifted from 5 • 106 to 5 • 104 SRBC. ~ DISCUSSION Recently it was demonstrated that human CBMC mount an antigen-specific PFC response after in vitro stimulation with OA and SRBC. H In accordance with observations from studies using adult PBMC, 2~ the presence of T cells and monocytes in cultures of CBMC was required for the effective induction of IgM-secreting PFCs with these antigens. In addition, the decline of the response at supraoptimal antigen levels was caused by the generation of antigen-specific T suppressor cells. H These findings not only indicate that OA- and SRBC-specific PFC precursors are already in the circulation at birth, but also suggest that functionally active antigen-specific T
helper cells, T suppressor cells, and antigen-presenting monocytes are present. Various studies in a number of mammalian species have revealed that the acquisition of the competence to develop a humoral immune response in vivo is a gradual process, which differs in time for different antigens. Consequently, antigens can be arranged in a hierarchical sequence. ~3-~5 From studies in sheep, Silverstein ~5 concluded that the duration of immaturity at the level of the B cell or antigen-processing cell may determine the onset of immune reactivity toward a distinct antigen. Sherwin and Rowlands t3 presented preliminary data that indicate that B cells with different antigen specificity appear at different times during ontogeny in mice. On the contrary, Blaese and Lawrence! 4 have obtained strong evidence for the decisive role of the macrophage in the determination of the sequential development of responsiveness to different antigens in the rat. We used two corpuscular antigens (SRBC and BRBC) and four antigens of a protein nature (OA, HPH, diphtheria toxoid, and tetanus toxoid); each has been described to be T cell~lependent in the culture systems used here or by other investigators. ~92~-24H P H is widely applied for the in vivo analysis of the capacity to mount a primary immune response in patients with immunologic disorders. 25 Diphtheria toxoid has been described to be a "late" antigen in sheep, which do not acquire immunologic competence to this antigen until 40 days after birth. 15 BRBC were incorporated in this study because of the observed delay in the development of responsiveness to this antigen in the rat2" Except for diphtheria toxoid, a PFC response could be induced in cultures of CBMC after stimulation with each of these antigens. In comparison, adult PBMC mount a PFC response to all of the antigens used for stimulation. This observation may suggest that diphtheria toxoidreactive cells are absent among CBMC. However, in contrast to tetanus toxoid, diphtheria toxoid failed also to induce a response in cultures of adult PBMC derived from a nonimmunized donor (dat~ not shown). This result may indicate that the poor immunogenicity of diphtheria toxoid as a primary antigen in this culture system, rather than the possible absence of diphtheria toxoid-reactive cells at birth, causes the failure of CBMC to respond to this antigen. It can be concluded that functionally active antigenspecific B cells, T helper cells, T suppressor cells, and antigen-presenting, monocytes with reactivity to a variety of antigens are present in human's at least from birth. However, this conclusion does not exclude the possibility of a stepwise development of immune reactivity for distinct antigens during gestation. Gill et al. 26 showed recently that
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IgM anti-tetanus toxoid antibodies are detectable in the serum of newborn infants after immunization of their mothers with tetanus toxoid between the fifth and eighth months of pregnancy. The level of those antibodies in the maternal and neonatal sera seemed to be comparable. The ability to induce an anti-tetanus response of the fetus by transplacental immunization indicates that tetanus toxoidreactive cells are already developed during fetal life. Although the magnitude of the PFC response elicited in cultures of CBMC resembles that of adult PBMC, the antigen dose optimal for the induction of PFCs in PBMC is consistently found to be much lower compared to that optimal for the stimulation of adult PBMC, irrespective of the nature of the antigen applied. With the exception of the response to diphtheria toxoid, a PFC response was always induced in cultures of CBMC with the same characteristic antigen dose dependency. This observation is in contrast to data published by Pascal et al. t~ concerning the induction of a primary IgM anti-TNP response in CBMC using TNP-PAA as stimulus. It appears that an "adult type" of antigen dose-response relationship is reached at about the age of 8 months. PrevioUs investigations have revealed that antigen handling by the monocyte population of the human newborn infant plays a key role in the determination of the particular antigen dose optimal for the induction of PFCs in CBMC. t2' 27This was concluded from experiments Showing that parental PBMC, after depletion of monocytes and reconstitution with semi-allogeneic neonatal monocytes, mount a PFC response with an antigen dose dependency characteristic for CBMC. An "adult type" of PFC response was obtained by culturing CBMC after replacement of the neonatal monocytes by parental monocytes. Therefore an altered handling of antigen by monocytes at this stage of infancy most probably underlies this shift in the antigen dose optimum. The observations from these in vitro experiments may have important implications for the effective triggering of the immune system in young children by environmental antigens. When mothers are confronted with such an antigen during pregnancy, maternal antibodies of the IgG class will pass through the placenta arid be found in the infant's circulation until a few months after birth. These antibodies may interfere with the induction of a humoral immune response by the corresponding antigen, for instance by neutralizing antigen, thus lowering the effective antigen concentration for the triggering of antigenreactive cells. 28 It might be beneficial to young individuals if their immune system were less sensitive to this immune suppressive effect of maternal antibodies. This could be achieved if less antigen is needed to trigger effectively the cells participating in the humoral immune response (i.e.,
Antigen-specific plaque-forming cell response
743
through a different antigen-handling capacity of monocytes during this period), At the age of about 8 months, when the maternally derived antibodies have disappeared from the infant's circulation, cells belonging to the monocyte population have matured, leading to a shift in the immunogenic dose of antigen to higher concentrations. It may be concluded from this study that in vitro antigenic stimulation of lymphocytes and the evaluation of the B cell response using an antigen-specific PFC assay may be valuable for the analysis of the ontogeny of the hum0ral immune system in humans and for the delineation of the B cell function and regulatory T cell and monocyte activity in health and disease. We thank the medical staffs of the Departments of Gynaecology, university Hospital and Overvecht Hospital, for their cooperation in collectingcord blood; the medical staff of the Department of Surgery, Children's Hospital Het Wilhelmina Kinderziekenhuis for the support in obt~tining blood from infants; Mrs. Marianne Smit for assistance in preparation of the manuscript. REFERENCES
1. Pabst HF, Kreth HW: Ontogeny of the immune response as a basis of childhood disease. J PEDIATR97:519, 1980. 2. Gathings WE, Kubagawa H, Cooper MD: A distinctive pattern of B cell immaturity in perinatal humans, lmmunol Rev 57:107, 1981. 3. Asma GEM, Langlois van den Bergh R, Vossen JM: Use of monoclonal antibodies in a study of the development of T-lymphocytes in the human fetus. Clin Exp Immunol 53:429, 1983. 4. Rosenthal P, Rimm IJ, Umiel T, Griffin JD, Osathanondh R, Schlossman SF, Nadler LM: Ontogeny of human hematopoietic cells: Analysis utilizing monoclonal antibodies. J lmmunol 131:232, 1983. 5. Maccario R, Nespoli L, Mingrat G, Vitiello A, Ugazio AG, Burgio GR: Lymphocyte subpopulations in the neonate: Identification of an immature subset of OKT8-positive, OKT3-negative cells. J lmmunol 130:1I29, 1983. 6. Gerli R, Rambotti P, Cernetti C, Velardi A, Spinozzi F: Evidence for phenotypic T precursor cells in human cord blood. Br J Haematol 53:685, 1983. 7. Andersson U, Bird AG, Britton S, Palacios R: Humoral and cellular immunity in humans studied at the cell level from birth to two years of age. lmmunol Rev 57:5, 198 l. 8. Hayward AR: Development of lymphocyte responses and interactions in the human fetus and newborn, lmmunol Rev 57:39, 1981. 9. Miyawaki T, Moriya N, Nagaoki T, Taniguchi N: Maturation of B cell differentiation ability and T cell regulatory function in infancy and childhood. Immunol Rev 57:61, 198t. t0. Pascal C, Galanaud P, Dormont J, Wallon C: Primary in vitro antibody response in human cord blood lymphocytes. J Reprod Immunol 1:275, 1980. 11. Van Tol MJD, Zijlstra J, Heijnen CJ, Kuis W, Zegers BJM, Ballieux RE: Antigen-specificplaqtle-formingcell response of human cord blood lymphocytesafter in vitro stimulation by T cell-dependent antigens. Eur J Immunol 13:390, 1983.
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12. Van Tol MJD, Zijlstra J, Thomas CMG, Zegers BJM, Ballieux RE: Distinct role Of neonatal and adult monocytes in the regulation of the in vitro antigen-induced plaque-forming cell response in man. J lmmunol (In press.) 13. Sherwin WK, Rowlands DT: Determinants of the hierarchy of humoral immune responsiveness during ontogeny. J Immunol 115:1549, 1975. 14. Btaese RM, Lawrence EC: Development of macrophage function and the expression of immunocompetence. In Cooper MD, Dayton DH, editors: Development of host defenses. New York, 1977, Raven Press, p 201. 15. Silverstein AM: Ontogeny of the immune response: A perspective, In Cooper MD, Dayton DH, editors: Development of host defenses. New York, 1977, Raven Press, p 1. 16. Kennedy JC, Axelrad MA: An improved assay for haemolytic plaque-forming cells. Immunology 20:253, 1971. 17. Dosch HM, Gelfand EW: In vitro induction and measurement of hemolytic plaque-forming cells in man. J Immunol Methods 11:107, 1976. 18. Goding JW: The chromic chloride method of coupling antigens to erythrocytes: Definition of some important parameters. J Immunol Methods 10:61, 1976. 19. Ballieux RE, Heijnen CJ, UytdeHaag F, Zegers BJM: Regulation of B cell activity in man: Role ofT cells, lmmunol Rev 45:3, 1979. 20. Heijnen C J, UytdeHaag F, Gmelig-Meyling FHJ, Ballieux RE: Human B cell activation in vitro: Localization of antigenspecific helper and suppressor function in distinct T cell subpopulafions. Cell lmmunol 43:282, 1979. 21. Heijnen C J, UytdeHaag F, Pot KH, Ballieux RE: Antigenspecific human T cell factors. I. T cell helper factor: Biologic properties. J Immunol 126:497, 1981.
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22. Platts-Mills T, Oldham G, de la Concha EG, Snajdr M J: T-B collaboration in the in vitro response to diphtheria toxoid: Evidence favouring a role for B cells in the generation of T cell help. In Fauci AS, Ballieux RE, editors: Antibody production in man: In vitro synthesis and clinical implications. New York, 1979, Academic Press, p 173. 23. Mori T, Kano K, Merrick JM, Milgrom F: Formation of heterophile antibodies by human tonsilar lymphocytes. I. In vitro stimulation with heterologous antigens. Cell lmmunol 40:28, 1978. 24. Volkman D J, Allyn SP, Fauci AS: Antigen-induced in vitro antibody production !n humans: Tetanus toxoid-specific antibody synthesis. J lmmunol 129:107, 1982. 25. Kallenberg CGM, Limburg PC, van Slochteren C, van der Woude F J, The TH: B cell activity in systemic lupus erythematosus: Depressed in vivo humoral immune response to a primary antigen (haemocyanin) and increased in vitro spontaneous immunoglobulin synthesis. Clin Exp Immunol 53:371, 1983. 26. Gill TJ lII, Repetti CF, Metlay LA, Rabin BS, Taylor FH, Thompson DS, Cortese AL: Transplacental immunization of the human fetus to tetanus by immunization of the mother. J Clin Invest 72:987, 1983. 27. Van Tol MJD, Zijlstra J, Zegers BJM, Ballieux RE: The regulatory role of human neonatal monocytes in the in vitro antigen-specific plaque-forming cell response. In Wedgwood R J, Rosen FS, Paul NW, editors: Primary immunodeficiency diseases. New York, 1983, Alan R. Liss, p 51. 28. Taylor RB: Regulation of antibody responses by antibody toward the immunogen, lmmunol Today 3:47, 1982.
Human cord blood mononuelear cells (CBMC) were stimulated in vitro with a number of T cell-dependent antigens. Antigen-induced B cell activation was measured applying a plaque-forming cell assay for the detection of antigen-specific lgM-secreting B cells. With the exception of diphtheria toxoid, the antigens ovalbumin, sheep red blood cells, Helix pomatia hemocyanin, burro red blood cells, and tetanus toxoid elicited an IgM-plaque-forming cell response in cultures of CBMC to levels obtained for peripheral blood mononuclear cells (PBMC) from adult controls. However, for each antigen used, the antigen dose optimal for the induction o f a response was consistently found to be a hundred to a thousand times lower than the concentration of the corresponding antigen optimal for adult PBMC. Longitudinal studies on PBMC obtained from infants between 2 and 30 months of age revealed that a shift of the antigen dose toward concentrations needed to induce plaque-forming cells in cultures of adult PBMC occurs at approximately age 8 months. Our data indicate that various antigens can be used for the in vitro analysis o f antigen-specific B cell activation and regulatory T cell functions in studies concerning the ontogeny of the humoral immune response in humans. (J Ps
105:738, 1984)
Maarten J. D. van Tol, M.Sc., Jitske Zijlstra, Ben J. M. Zegers, Ph.D., and Rudy E. Ballieux, Ph.D. Utrecht, The Netherlands
AT BIRTH the capacity to mount a humoral immune response is not fully developed in a number of mammalian species. In humans antibody production is mainly restricted to the IgM class of immunoglobulins after intrauterine infection and after active immunization during the first 6 months of life.~ Because lymphocyte subpopulations belonging to the B and T cell lineage, respectively, as well as monocytes appear early in gestation,2-4 it might be inferred that a dichotomy exists between the development From University Children's Hospital Her Wilhelmina Kinderziekenhuis, and University Hospital. Supported by the Foundation for Medical Research (Fungo), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (ZWO). Submitted for publication Jan. 31, 1984; accepted April 13, 1984. Reprint requests: Maarten J. D. van Tol, University Children's Hospital Het Wilhelmina Kinderziekenhuis, Nieuwe Gracht I37, 3512 LK Utrecht, The Netherlands.
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of the cellular constituents of the humoral immune response and their functional activity. However, in the human fetus and newborn infant, relatively high numbers BRBC CBMC HPH MNC OA PBMC PFC SRBC TN P-PAA
Burro red blood cells Cord blood mononuclear cells Helix pomat~a hemocyanin Mononuclear cells Ovalbumin Peripheral blood mononuclear cells Plaque-forming cells Sheep red blood cells Trinitrophenylated polyacrylamide beads
of B and T cells have an immature surface phenotype.Z, 3, 5, 6 The cellular basis of the defi6ient humoral immune response in the human newborn infant has usually been analyzed by in vitro activation of B cells with polyclonal stimulators.7-9 Only a few reports have focused on the
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ontogeny of the antigen-specific B cell function and the regulation of the antibody response after antigenic stimulation?0. ~ Human cord blood mononuclear cells are able to mount a primary hapten-specific IgM plaque-forming cell response after in vitro stimulation with the hapten-carrier complex TNP-PAA? ~ However, only four of 42 newborn infants responded, and the level of the response was substantially lower than that induced in cultures of adult peripheral blood mononuclear cells. Applying the T celldependent antigens sheep red blood cells and ovalbumin, a different picture has emerged from studies in our laboratory. Consistently, an IgM-PFC response was obtained in cultures of CBMC to a level comparable to that found in adult PBMC. H However, the dose of antigen optimal for the induction of a response of CBMC appeared to be a hundred times lower compared with that optimal for adult PBMC. This observation could be related to a difference in antigen handling between monocytes in neonates and adults that partly determines the antigen dose dependency for the activation of regulatory T cell subsets? 2 Early observations in the mouse, rat, and sheep have indicated that in vivo responsiveness to various antigens develops in a stepwise fashion during fetal or neonatal life] T M Antigens can be placed in a hierarchical sequence according to their capacity to induce an antibody response "early" or "late" in gestation or in the first postnatal period. The cellular basis of this phenomenon is not fully established. The aim of our study was to extend the investigations on the capacity of human CBMC to mount an in vitro PFC response by using antigens described to be "late" antigens in other mammalian species. Furthermore, the development of the antigen dose dependency for the induction of PFCs from the "neonatal type" to the "adult type" was studied by culturing P B M C from children between 2 and 30 months of age. METHODS Donors of mononnclear cells. Mononuclear cells were isolated from cord blood collected immediately after birth. In all cases the mothers had had uncomplicated and full-term pregnancies. The PBMC were obtained from peripheral blood of healthy adult volunteers ("adult PBMC"). For the experiments concerning the longitudinal study, blood was obtained from children between 2 and 30 months of age after written and informed consent of their parents. These children were seen at the Children's Hospital for surgery or because of clinical symptoms not associated with disorders affecting the immune system. Isolation of mononuclear cells. Lymphocytes and monocytes were prepared by centrifugation of heparinized blood
Antigen-specific plaque-forming cell response
739
diluted once in minimal essential medium (MEM-Tris, Gibco, Grand Island, N.Y.) on Ficoll-Isopaque density gradients (p = 1.077 gm/cm 3) at 700 X g for 20 minutes. The CBMC were usually contaminated with erythrocytes, which were removed by NH4Cl-induced lysis followed by two washings with cold MEM-Tris containing 0.5% (wt/ vol) bovine serum albumin (Sigma, St. Louis, Mo.). When monocytes were present in the MNC-suspension in amounts exceeding 15% of the total cell number, MNC were partly depleted of monocytes by adherence to plastic flasks (Falcon, Oxnard, Calif.) at 37 ~ C in MEM-Tris supplemented with 20% (vol/vol) fetal calf serum. After incubation for 45 to 90 minutes the nonadherent cell fraction containing lymphocytes and nonadherent monocytes was decanted and washed twice with MEM-Tris. The adherent cells (>90% nonspecific esterase-positive cells) were harvested by gentle scraping with a rubber policeman and washed twice with MEM-Tris. Adherent cells (monocytes) were returned to the nonadherent cell fraction to establish a final proportion of 10% monocytes. Antigenic stimulation of mononuclear cells. A total of 4 to 5 x 106 M N C were cultured in tissue culture tubes (Falcon, 17 X 100 mm) in 10 ml R P M I 1640 (Gibco) containing 10% (vol/vol) heat-inactivated and SRBCabsorbed human AB serum, fungizone (25/~g/ml), penicillin (100 ~g/ml), streptomycin (100 IU/ml), and Lglutamine (2 mM). When BRBC were applied as antigen, SRBC-absorbed AB serum was replaced by BRBCabsorbed AB serum. The cells were stimulated with the antigens SRBC, ovalbumin, BRBC, Helix pomatia hemocyanin, diphtheria toxoid, and tetanus toxoid, respectively, which were present in the concentrations indicated. After culture for 6 to 7 days at 37 ~ C in an atmosphere of 5% CO2 the cells were collected, washed twice in MEM-Tris at 4 ~ C, and assayed for the presence of PFCs. When SRBC or BRBC were used for antigenic stimulation, the cultured MNC were freed of the erythrocytes by NH4Cl-induced lysis before the PFC-assay. H P H was kindly provided by Dr. T. H. Th6 (University Hospital, Groningen, The Netherlands). Diphtheria toxoid and tetanus toxoid were supplied by Dr. J. Nagel (National Institute of Public Health, Bilthoven, The Netherlands), and OA (grade VI) was obtained from Sigma. Plaque-forming call assay. The PFCs were determined by the method originally described by Kennedy and Axelrad 16 according to the modifications introduced by Dosch and Gelfand? 7. Briefly, SRBC, antigen-coated SRBC, or BRBC were centrifuged for 5 minutes at 1000 x g on the bottom of Falcor~ microtiter plates (No. 3040) precoated with poly-L-lysine (Sigma, Mr --<100,000) in a concentration of 100 #g/ml distilled water by incubation for 60 minutes at 37 ~ C. Viable cultured cells in
740
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PFC / 1 0 6
The Journal o f Pediatrics November 1984
ly.
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BRBC SRBC
A
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A
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1500-
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Fig. 1. Antigen dose-response relationship for induction of plaque-forming cells in cultures of adult peripheral blood mononuclear cells (PBMC). A, sheep red blood cells (n = 5) or burro red blood ceils (n = 3). B, Ovalbumin (n = 5) or Hefix pomatia hemocyanin (n = 5). C, Tetanus toxoid (n = 3) or diphtheria toxoid (n = 2). Results shown for each antigen are mean _+ SD of experiments using different peripheral blood mononuclear cell donors. Data are expressed as PFC/106 lymphocytes.
various dilutions were i n c u b a t e d for 60 to 90 minutes in the presence of S R B C - or B R B C - a b s o r b e d guinea pig complement, respectively. A f t e r stimulation of M N C with OA, H P H , diphtheria toxoid, or tetanus toxoid the level of the induced P F C response was analyzed using monolayers of
102
Jug t o x o i d /ml
Fig. 2. Antigen dose-response relationship for induction of plaque-forming cells in cultures of cord blood mononuclear cells (CBMC). A, sheep red blood ceils (n = 5) or burro red blood cells (n = 3). B, Ovalbumin (n = 5) or Helix pomatia hemocyanin (n = 3). C, Tetanus toxoid (n ~- 3) or diphtheria toxoid (n = 4). Results shown for each antigefi are mean + SD of experiments using different CBMC donors. Data are expressed as PFC/10 ~ lymphocytes.
S R B C coated with the relevant antigen by the method described by Goding. ~8 In the P F C assay only IgMsecreting cells were detected. T h e antigen specificity of the P F C response of C B M C as well as~'PBMC from adults was ascertained in experiments published earlier. H, ~9.20In addition, stimulation of M N C with B R B C or H P H only gave rise to the detection of P F C s in monolayers of S R B C at
Volume 105 Number 5
background levels ( ~ 2 0 0 PFC/106 lymphocytes). The same observation was true for the stimulation of M N C with diphtheria toxoid or tetanus toxoid followed by the performance of the PFC assay using monolayers of tetanus toxoid- or diphtheria toxoid-coated SRBC. These results indicated that none of the applied antigens in the range of concentrations used for stimulation caused polyclonal B cell activation.
Antigen-specific plaque-forming cell response
PFC / 106 ly.
1500
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RESULTS Ovalbumin and SRBC-induced PFC response of adult PBMC and CBMC. Adult PBMC and CBMC were cultured in the presence of variable doses of the corpuscular antigen SRBC or the soluble antigen OA. A PFC resPonse was observed after stimulation of adult PBMC (Fig. i) as well as CBMC (Fig. 2) with either antigen. The level Of the response induced in cultures of adult PBMC was comparable to that obtained for CBMC. However, an optimal response in cuitures of CBMC was elicited at an antigen concentration a hundred times lower than that optimal for adult PBMC (0.03 vs 3.0 /~g O A / m l and 5 x 104 vs 5 X 106 SRBC per culture, respectively): None of the newborn infants failed to mount an IgM-PFC response to SRBC and OA, and all of the neonates tested Showed the same antigen dose-response relationship . PFC response of adult PBMC and CBMC toward other antigens. To investigate the repertoire of functionally active antigen-specific lymphocytes present at birth, CBMC and adult PBMC were stimulated with various doses of HPH, tetanus toxoid, diphtheria toxoid, and BRBC. A PFC response could be obtained in cultures of CBMC as well as adult PBMC with HPH, tetanus toxoid, and BRBC (Figs. 1 and 2). Irrespective to the antigen applied, the dose optimal for the induction of PFCs in cultures of CBMC was shifted to a hundredfold to thousandfold lower antigen concentration in comparison with the conditions optimal for the stimulation of adult PBMC (HPH, 1 vs 100 #g H P H / m l ; tetanus to'old, 0.1 vs 100 ng TT/ml; BRBC, 5 x 104 v s 5 X 106 BRBC per culture). Regarding the stimulation of adult PBMC with tetanus toxoid, the donors used in this study we)e immunized with this antigen during childhood. However, ` despite a higher level of the PFC response, the antigen dose-response relationship for immunized donors did not differfrom that obtained with nonimmunized adults (data not shown). Diphtheria toxoid failed to induce a PFC respons e in cultures of CBMC, whereas a response was established in cultures of adult PBMC from immunized donors. It is interesting that an anti-diphtheria toxoid PFC response Mso appeared to be absent in cultures of PBMC derived from a nonimmunized adult (data not shown). Development of dose-response relationship with age.
74 1
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age ( m o n t h s ) Fig. 3. Induction of plaque-forming cells (PFC) in cultures of peripheral blood mononuclear cells (PBMC) obtained from infants ranging from 2 to 30 months of age after stimulation with low dose of antigen. PBMC from individual donors were stimulated with ovalbumin 0.03 ~g/mi (U) and 5 X 104 sheep red blood cells per culiure (F'I),respectively. Level of PFC response induced is expressed as PFC/10 ~ lymphocytes and plotted against age of infant.
Experiments were performed to analyze whether the antigen dose dependency for the induction of PFCs shifts during infancy from a "neonatal type" of dose-response relationship observed in CBMC to an "adult type" of response established in cultures of adult PBMC. To this end, PBMC obtained from Children between 2 and 30 months of age were cultured in the presence of variable doses of SRBC and OA, respectively. The levels of the responses induced in cultures of PBMC indicate that a correlation exists between the dose of antigen optimal for the induction of a PFC response and the age of the child (Figs. 3 and 4). From the age of 8 months, 11 of 12 infants studied mounted an optimal PFC response after stimulation with 3.0 #g O A / m l (i.e., an "adult type" of antigen dose-response relationship). Stimulation of PBMC obtained from nine donors of this group of 11 infants with SRBC indicated that this observat~bn was not restricted to the antigen OA. However, at younger than 8 months a completely different picture emerged: five of six infants showed a "neonatal type" of antigen dose-response rela-
742
van Tol et al.
PFC
The Journal of Pediatrics November 1984
/1061y.
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age (months) Fig. 4. Induction of plaque-forming cells (PFC) in cultures of peripheral blood mononuclear cells (PBMC) obtained from infants ranging 2 to 30 months of age after stimulation with high dose of antigen. PBMC from individual donors Were stimulated with ovalbumin 3.0 ~zg/ml (0) and 5 X 106 sheep red blood cells per culture (0), respectively. Level of PFC response induced is expressed as PFC/106 lymphocytes and plotted against age of infant.
ship for the induction of PFCs, with an antigen dose oPtimum of 0.03 #g OA/ml. The same observation held for the two infants who could also be analyzed using SRBC as the stimulus. In comparison with the older group, the optimal antigen dose was found to be shifted from 5 • 106 to 5 • 104 SRBC. ~ DISCUSSION Recently it was demonstrated that human CBMC mount an antigen-specific PFC response after in vitro stimulation with OA and SRBC. H In accordance with observations from studies using adult PBMC, 2~ the presence of T cells and monocytes in cultures of CBMC was required for the effective induction of IgM-secreting PFCs with these antigens. In addition, the decline of the response at supraoptimal antigen levels was caused by the generation of antigen-specific T suppressor cells. H These findings not only indicate that OA- and SRBC-specific PFC precursors are already in the circulation at birth, but also suggest that functionally active antigen-specific T
helper cells, T suppressor cells, and antigen-presenting monocytes are present. Various studies in a number of mammalian species have revealed that the acquisition of the competence to develop a humoral immune response in vivo is a gradual process, which differs in time for different antigens. Consequently, antigens can be arranged in a hierarchical sequence. ~3-~5 From studies in sheep, Silverstein ~5 concluded that the duration of immaturity at the level of the B cell or antigen-processing cell may determine the onset of immune reactivity toward a distinct antigen. Sherwin and Rowlands t3 presented preliminary data that indicate that B cells with different antigen specificity appear at different times during ontogeny in mice. On the contrary, Blaese and Lawrence! 4 have obtained strong evidence for the decisive role of the macrophage in the determination of the sequential development of responsiveness to different antigens in the rat. We used two corpuscular antigens (SRBC and BRBC) and four antigens of a protein nature (OA, HPH, diphtheria toxoid, and tetanus toxoid); each has been described to be T cell~lependent in the culture systems used here or by other investigators. ~92~-24H P H is widely applied for the in vivo analysis of the capacity to mount a primary immune response in patients with immunologic disorders. 25 Diphtheria toxoid has been described to be a "late" antigen in sheep, which do not acquire immunologic competence to this antigen until 40 days after birth. 15 BRBC were incorporated in this study because of the observed delay in the development of responsiveness to this antigen in the rat2" Except for diphtheria toxoid, a PFC response could be induced in cultures of CBMC after stimulation with each of these antigens. In comparison, adult PBMC mount a PFC response to all of the antigens used for stimulation. This observation may suggest that diphtheria toxoidreactive cells are absent among CBMC. However, in contrast to tetanus toxoid, diphtheria toxoid failed also to induce a response in cultures of adult PBMC derived from a nonimmunized donor (dat~ not shown). This result may indicate that the poor immunogenicity of diphtheria toxoid as a primary antigen in this culture system, rather than the possible absence of diphtheria toxoid-reactive cells at birth, causes the failure of CBMC to respond to this antigen. It can be concluded that functionally active antigenspecific B cells, T helper cells, T suppressor cells, and antigen-presenting, monocytes with reactivity to a variety of antigens are present in human's at least from birth. However, this conclusion does not exclude the possibility of a stepwise development of immune reactivity for distinct antigens during gestation. Gill et al. 26 showed recently that
Volume 105 Number 5
IgM anti-tetanus toxoid antibodies are detectable in the serum of newborn infants after immunization of their mothers with tetanus toxoid between the fifth and eighth months of pregnancy. The level of those antibodies in the maternal and neonatal sera seemed to be comparable. The ability to induce an anti-tetanus response of the fetus by transplacental immunization indicates that tetanus toxoidreactive cells are already developed during fetal life. Although the magnitude of the PFC response elicited in cultures of CBMC resembles that of adult PBMC, the antigen dose optimal for the induction of PFCs in PBMC is consistently found to be much lower compared to that optimal for the stimulation of adult PBMC, irrespective of the nature of the antigen applied. With the exception of the response to diphtheria toxoid, a PFC response was always induced in cultures of CBMC with the same characteristic antigen dose dependency. This observation is in contrast to data published by Pascal et al. t~ concerning the induction of a primary IgM anti-TNP response in CBMC using TNP-PAA as stimulus. It appears that an "adult type" of antigen dose-response relationship is reached at about the age of 8 months. PrevioUs investigations have revealed that antigen handling by the monocyte population of the human newborn infant plays a key role in the determination of the particular antigen dose optimal for the induction of PFCs in CBMC. t2' 27This was concluded from experiments Showing that parental PBMC, after depletion of monocytes and reconstitution with semi-allogeneic neonatal monocytes, mount a PFC response with an antigen dose dependency characteristic for CBMC. An "adult type" of PFC response was obtained by culturing CBMC after replacement of the neonatal monocytes by parental monocytes. Therefore an altered handling of antigen by monocytes at this stage of infancy most probably underlies this shift in the antigen dose optimum. The observations from these in vitro experiments may have important implications for the effective triggering of the immune system in young children by environmental antigens. When mothers are confronted with such an antigen during pregnancy, maternal antibodies of the IgG class will pass through the placenta arid be found in the infant's circulation until a few months after birth. These antibodies may interfere with the induction of a humoral immune response by the corresponding antigen, for instance by neutralizing antigen, thus lowering the effective antigen concentration for the triggering of antigenreactive cells. 28 It might be beneficial to young individuals if their immune system were less sensitive to this immune suppressive effect of maternal antibodies. This could be achieved if less antigen is needed to trigger effectively the cells participating in the humoral immune response (i.e.,
Antigen-specific plaque-forming cell response
743
through a different antigen-handling capacity of monocytes during this period), At the age of about 8 months, when the maternally derived antibodies have disappeared from the infant's circulation, cells belonging to the monocyte population have matured, leading to a shift in the immunogenic dose of antigen to higher concentrations. It may be concluded from this study that in vitro antigenic stimulation of lymphocytes and the evaluation of the B cell response using an antigen-specific PFC assay may be valuable for the analysis of the ontogeny of the hum0ral immune system in humans and for the delineation of the B cell function and regulatory T cell and monocyte activity in health and disease. We thank the medical staffs of the Departments of Gynaecology, university Hospital and Overvecht Hospital, for their cooperation in collectingcord blood; the medical staff of the Department of Surgery, Children's Hospital Het Wilhelmina Kinderziekenhuis for the support in obt~tining blood from infants; Mrs. Marianne Smit for assistance in preparation of the manuscript. REFERENCES
1. Pabst HF, Kreth HW: Ontogeny of the immune response as a basis of childhood disease. J PEDIATR97:519, 1980. 2. Gathings WE, Kubagawa H, Cooper MD: A distinctive pattern of B cell immaturity in perinatal humans, lmmunol Rev 57:107, 1981. 3. Asma GEM, Langlois van den Bergh R, Vossen JM: Use of monoclonal antibodies in a study of the development of T-lymphocytes in the human fetus. Clin Exp Immunol 53:429, 1983. 4. Rosenthal P, Rimm IJ, Umiel T, Griffin JD, Osathanondh R, Schlossman SF, Nadler LM: Ontogeny of human hematopoietic cells: Analysis utilizing monoclonal antibodies. J lmmunol 131:232, 1983. 5. Maccario R, Nespoli L, Mingrat G, Vitiello A, Ugazio AG, Burgio GR: Lymphocyte subpopulations in the neonate: Identification of an immature subset of OKT8-positive, OKT3-negative cells. J lmmunol 130:1I29, 1983. 6. Gerli R, Rambotti P, Cernetti C, Velardi A, Spinozzi F: Evidence for phenotypic T precursor cells in human cord blood. Br J Haematol 53:685, 1983. 7. Andersson U, Bird AG, Britton S, Palacios R: Humoral and cellular immunity in humans studied at the cell level from birth to two years of age. lmmunol Rev 57:5, 198 l. 8. Hayward AR: Development of lymphocyte responses and interactions in the human fetus and newborn, lmmunol Rev 57:39, 1981. 9. Miyawaki T, Moriya N, Nagaoki T, Taniguchi N: Maturation of B cell differentiation ability and T cell regulatory function in infancy and childhood. Immunol Rev 57:61, 198t. t0. Pascal C, Galanaud P, Dormont J, Wallon C: Primary in vitro antibody response in human cord blood lymphocytes. J Reprod Immunol 1:275, 1980. 11. Van Tol MJD, Zijlstra J, Heijnen CJ, Kuis W, Zegers BJM, Ballieux RE: Antigen-specificplaqtle-formingcell response of human cord blood lymphocytesafter in vitro stimulation by T cell-dependent antigens. Eur J Immunol 13:390, 1983.
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12. Van Tol MJD, Zijlstra J, Thomas CMG, Zegers BJM, Ballieux RE: Distinct role Of neonatal and adult monocytes in the regulation of the in vitro antigen-induced plaque-forming cell response in man. J lmmunol (In press.) 13. Sherwin WK, Rowlands DT: Determinants of the hierarchy of humoral immune responsiveness during ontogeny. J Immunol 115:1549, 1975. 14. Btaese RM, Lawrence EC: Development of macrophage function and the expression of immunocompetence. In Cooper MD, Dayton DH, editors: Development of host defenses. New York, 1977, Raven Press, p 201. 15. Silverstein AM: Ontogeny of the immune response: A perspective, In Cooper MD, Dayton DH, editors: Development of host defenses. New York, 1977, Raven Press, p 1. 16. Kennedy JC, Axelrad MA: An improved assay for haemolytic plaque-forming cells. Immunology 20:253, 1971. 17. Dosch HM, Gelfand EW: In vitro induction and measurement of hemolytic plaque-forming cells in man. J Immunol Methods 11:107, 1976. 18. Goding JW: The chromic chloride method of coupling antigens to erythrocytes: Definition of some important parameters. J Immunol Methods 10:61, 1976. 19. Ballieux RE, Heijnen CJ, UytdeHaag F, Zegers BJM: Regulation of B cell activity in man: Role ofT cells, lmmunol Rev 45:3, 1979. 20. Heijnen C J, UytdeHaag F, Gmelig-Meyling FHJ, Ballieux RE: Human B cell activation in vitro: Localization of antigenspecific helper and suppressor function in distinct T cell subpopulafions. Cell lmmunol 43:282, 1979. 21. Heijnen C J, UytdeHaag F, Pot KH, Ballieux RE: Antigenspecific human T cell factors. I. T cell helper factor: Biologic properties. J Immunol 126:497, 1981.
The Journal o f Pediatrics November 1984
22. Platts-Mills T, Oldham G, de la Concha EG, Snajdr M J: T-B collaboration in the in vitro response to diphtheria toxoid: Evidence favouring a role for B cells in the generation of T cell help. In Fauci AS, Ballieux RE, editors: Antibody production in man: In vitro synthesis and clinical implications. New York, 1979, Academic Press, p 173. 23. Mori T, Kano K, Merrick JM, Milgrom F: Formation of heterophile antibodies by human tonsilar lymphocytes. I. In vitro stimulation with heterologous antigens. Cell lmmunol 40:28, 1978. 24. Volkman D J, Allyn SP, Fauci AS: Antigen-induced in vitro antibody production !n humans: Tetanus toxoid-specific antibody synthesis. J lmmunol 129:107, 1982. 25. Kallenberg CGM, Limburg PC, van Slochteren C, van der Woude F J, The TH: B cell activity in systemic lupus erythematosus: Depressed in vivo humoral immune response to a primary antigen (haemocyanin) and increased in vitro spontaneous immunoglobulin synthesis. Clin Exp Immunol 53:371, 1983. 26. Gill TJ lII, Repetti CF, Metlay LA, Rabin BS, Taylor FH, Thompson DS, Cortese AL: Transplacental immunization of the human fetus to tetanus by immunization of the mother. J Clin Invest 72:987, 1983. 27. Van Tol MJD, Zijlstra J, Zegers BJM, Ballieux RE: The regulatory role of human neonatal monocytes in the in vitro antigen-specific plaque-forming cell response. In Wedgwood R J, Rosen FS, Paul NW, editors: Primary immunodeficiency diseases. New York, 1983, Alan R. Liss, p 51. 28. Taylor RB: Regulation of antibody responses by antibody toward the immunogen, lmmunol Today 3:47, 1982.