Clin. exp. Immunol.
ADONIS 009910491001553
(I991) 84, 373-375 EDITORIAL REVIEW
Human B lymphocytes mature J. GORDON Division of Immunology, School of Medical Science, The University of Birmingham, Birmingham, England (Acceptedfor publication 7 March 1991)
INTRODUCTION Functional immaturity in the immune system of newborns is highlighted by an inability to mount humoral responses to TI-2 antigens such as bacterial capsular polysaccharides. A lack of 'immune experience' is not sufficient to account for this deficit as it persists for the first 2 years of life even where there is repeated exposure to exogenous antigen (Kahty et al., 1984). It has been tempting to suggest that neonatal B lymphocytes are inherently immature, a notion given some credence by the finding that most cord blood B cells express the CD5 marker (Gadol & Ault, 1986). Moreover, studies using polyclonal activators to assess their capacity for differentiation in vitro have supported the thesis that neonatal B cells are in some way functionally naive. In this issue, an important paper by Tucci et al. (1991) reexamines the question of functional competence among cord blood lymphocytes; the conclusions reached will be discussed later. Their study exploits one of several systems which have recently been described for driving potent B cell growth and differentiation in vitro. The main purpose ofthis brief article is to review those systems. THE ZUBLER SYSTEM In 1985, Zubler et al. reported that a mutant subclone of the mouse thymoma line EL4 activates both human and murine B cells via direct cell-to-cell contact in a polyclonal, antigen- and lectin-independent manner (Zubler et al., 1985). Two years later, with some modifications to the system, it was shown that some 90% of B lymphocytes from human blood or spleen could be driven to proliferate and differentiate to immunoglobulinsecreting cells (ISC) (Wen et al., 1987). In addition to the irradiated thymoma cells, of which 5 x 104 are added in a final volume of 200 p1, cultures are supplemented with T cell/ macrophage supernatants. These are produced by stimulating mixtures of T cells and macrophages, which have been isolated from peripheral blood, with phytohaemagglutinin (PHA) and phorbol ester for 36 h; supernatants are added at a final concentration of 5-10%. Over 10 days, under conditions of limiting dilution, single B cells could generate clones numbering on average 380 cytoplasmic immunoglobulin positive (cIg+) cells; the mean amount of immunoglobulin secreted per clone was 20 ng. In addition to its application in the study of Tucci et al. (1991), this system has been used to determine the frequency
of B cells producing antibody to Plasmodium falciparum in malaria-infected individuals (Wen et al., 1987). THE LIPSKY SYSTEM In 1988 Peter Lipsky's group described a method for generating high-rate immunoglobulin production from human B cells by co-culturing them with T cells that had been stimulated with immobilized antibody to CD3 (Hirohata, Jelinek & Lipsky, 1988). More recently, they have estimated the frequency of B cells responding in this system and also determined the factors which modify the response (Amoroso & Lipsky, 1990). Under conditions of limiting dilution, the proportion of B cells responding to anti-CD3-stimulated T cells was found to be in the region of 10%. When comparing the efficacy of different cells stimulated through CD3, T cell clones were seen to provide a more potent system than freshly isolated, mitomycin C-treated T cells. However, with the addition of IL-2 and accessory cells, B cell responses to anti-CD3-activated fresh T cells now reached a precursor frequency of 60-80%. IL-6 was also found to augment the number of B cells responding; IL-4 inhibited the IL-2-dependent increase while promoting no change by itself. Multiple immunoglobulin class secretion was observed in wells where single B cells had been seeded indicating that class switching was occurring in these cultures. THE BANCHEREAU SYSTEM A report published in 1987 noted that a combination of IL-4 and an antibody to CD40 provided optimal signals for maintaining the cell cycle in prestimulated human B cells (Gordon et al., 1987). However, in the absence of any additional support, the cultures succumbed to cell death within 9-12 days. At the beginning of this year Banchereau et al. (1991) reported greater success using the two activities, describing for the first time the ability to sustain active proliferation of non-transformed B cells for many weeks. In addition to IL-4 and CD40 antibody, their culture system utilizes a feeder layer of mouse L cells into which the CDw32 gene encoding FcyR has been transfected and expressed. This allows the fibroblast to 'grab' the CD40 antibody and present it to the B cell in a fashion that is presumably optimal for long-term stimulation. Simple immobilization or cross-linking of the antibody with a second reagent cannot substitute for the transfected L cells; this implies either that the orientation and dynamics of presenting the antibody in a fluid membrane is particularly appropriate, or that the fibroblasts are themselves providing additional signals. IgM, IgG, and IgE production are found to accompany the growth of
Correspondence: Dr J. Gordon, Division of Immunology, School of Medical Science, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
373
374
J. Gordon
B cells stimulated with IL-4 and CD40 antibody while the additional presence of IL-2 further increases the level of IgM and results in large amounts of IgA being secreted (Rousset, Garcia & Banchereau, 1991). Interestingly, as has recently been found in more conventional in vitro systems, CD40 antibody not only enhances IL-4-dependent IgE production but also prevents IFN-)' from exerting its normal inhibitory influence on this change.
COMPARISON OF THE SYSTEMS All three of the novel systems designed to generate high-rate immunoglobulin production from human B cells in vitro are clearly a huge improvement on those based upon conventional polyclonal activators. For pokeweed mitogen (PWM) and Epstein-Barr virus (EBV), the number of B cells responding by immunoglobulin secretion is between 0 2%Yo and 3%, (MartinezMaza & Britton, 1983); Staphylococcus aureus Cowan (SAC) plus T cell supernatants encourage small amounts of IgM production (Saiki & Ralph, 1981). The conventional mitogens also suffer from their target selectively: SAC stimulates only those 300% of B cells expressing VH3 determinants (Shokri et al., 1991); PWM has a preference for large, IgD, post-switched B cells (Kuritani & Cooper, 1982); EBV shows a predilection for small resting B cells (Aman, Ehlin-Henriksson & Klein, 1984). Thus, quite a false impression of functional responsivity could be given when using any one of these polyclonal activators in situations where a particular B cell subset might be under- or over-represented (as in ontogeny or disease, for example). Probably the most 'unphysiological' of the recently described high-efficiency systems is that of Zubler's, relying as it does on xenogeneic tumour cells and supernatants containing lectin and phorbol ester. Nevertheless, it undoubtedly works and appears to develop aggressively the full functional potential of any given B cells. By contrast, Lipsky's approach could be considered a model par excellance for examining the parameters of B cell responses to T-dependent antigens; already, it has been used to show the CDl Ia/CD54 dependency of T cell-driven immunoglobulin production (Tomha, Hirohata & Lipsky, 1991). Banchereau's system differs from the other two both in its T cell-independence and in the ability to sustain growth over a much longer period (perhaps inter-related phenomena?). It is clear that, particularly when culturing under conditions of limiting dilution, the feeder layer employed in each of the high-potency systems supplies essential signals for the optimal growth and differentiation of the B cell. There is mounting evidence that T cell membranes can themselves be stimulatory for B cells but the molecules involved remain to be defined (Brian, 1988); recent data indicate that fibroblast-derived factors may provide survival signals to activated lymphocytes (Scott, Pandolfi & Kurnick, 1990). One outstanding question relates to the nature of the CD40 counterstructure: so far, all studies on this intriguing molecule have used presumed agonistic antibodies to elaborate functional change. CD40 has homology with receptors for tumour necrosis factor (TNF) and nerve growth factor (NGF) (Smith et al., 1990), both of which are cytokines that act optimally as trimers and regulate cell survival (negatively and positively, respectively). In addition to its capacity to maintain the growth of cycling B cells, CD40 ligation also promotes the survival of germinal centre cells and their neoplastic equivalents (Liu et al., 1989; Gregory et al., 1991),
and may be instrumental in the establishment of B cell memory (Liu et al., 1991). Several groups of investigators are now hotly in pursuit of the natural ligand for CD40 and its future availability may revolutionize studies of human B cell function.
BACK TO THE WOMB The paper of Tucci et al. (1991) in this issue of Clinical and E.xperimental Immunology, the catalyst for this mini-review, uses the Zubler system to delineate the functional maturity of cord blood B cells. Lipsky's group recently approached the same question in their system which uses T cells stimulated through CD3 to drive B cell responses (Splawski, Jelinek & Lipsky, 1991). The study reported in this issue concludes that cord blood B cells, if provided with the extremely potent signals supplied by the mutant EL4 thymoma cells and T cell supernatants, do reveal a potential for differentiation which is both qualitatively and quantitatively indistinct from that of adult B cells. Using the 'more physiological' system of Lipsky's, it was seen that neonatal B cells do indeed have a greater functional potential than previously realized: the production of both IgG and IgA, in addition to the usual IgM, was observed but the amounts of the switched isotypes produced were small by comparison to adult B cells. The concensus view from the two studies seems to be that while neonatal B cells are probably functionally naive, their inherent potential for stimulation approaches that of adult B cell: this can be realized as long as sufficiently strong help is available. With these examples, and the promise of what is soon to come, it can be confidently stated that the study of human B cells has finally matured. REFERENCES AMAN, P., EHLIN-HENRIKSSON, B. & KLEIN, G. (1984) Epstein-Barr virus susceptibility of normal B cell populations. J. exp. Med. 159, 208. AMOROSO, K. & LIPSKY, P.E. (1990) Frequency of human B cells that differentiate in response to anti-CD3-activated T cells. J. Immunol. 145, 3155. BANCHEREAU, J., DE PAOLI, P., ALLE, A., GARCIA, E. & RouSSET, F. (1991) Long term human B cell lines dependent on interleukin 4 and antibody to CD40. Science, 251, 70. BRIAN, A. (1988) Stimulation of B-cell proliferation by membraneassociated molecules from activated T cells. J. Immunol. 85, 564. GADOL, N. & AULT, K.A. (1986). Phenotypic and functional characteristics of human Leu-l/CD5 B cells. Immunol. Ret. 93, 23. GORDON, J., MILLSUM, M.J., Guy, G.R. & LEDBETTER, J.A. (1987) Synergistic interaction between interleukin 4 and anti-Bp5O (CDw4O) revealed in a novel B cell restimulation assay. Eur. J. Immunol. 17, 153. GREGORY, C.D., DIVE, C., HENDERSON, S., SMITH, C.A., WILLIAMS, G.T., GORDON, J. & RICKINSON, A.B. (1991) Activation of EpsteinBarr virus latent genes protects luna B cells from death by apoptosis. Nature, 349, 612. HIROHATA, S., JELINEK, D.F. & LIPSKY, P.E. (1988) T cell dependent activation of B cell proliferation and differentiation by immobilized antibodies to CD3. J. Immunol. 240, 3736. KAHTY, H., KARANKO, V., PELTOLA, H. & MAKELA P.H. (1984) Serum antibodies after vaccination with Haemophilus influenza type b capsular polysaccharide vaccine. Pediatrics, 74, 857. KURITANI, T. & COOPER, M.D. (1982) Human B cell differentiation. II. Pokeweed mitogen-responsive B cells belong to a surface immunoglobulin D-negative subpopulation. J. exp. Med. 155, 1561. Liu, Y.J., CAIRNS, J.A., HOLDER, M.J., ABBOT, S.D., JANSEN, K.U., BONNEFOY, J.Y., GORDON, J. & MACLENNAN, I.C.M. (1991) Recom-
Human B lymphocytes mature binant 25-kDa CD23 and interleukin 1 a promote the survival of germinal center B cells: evidence for bifurcation in the development of centrocytes rescued from apoptosis. Eur. J. Immunol. (In press). Liu, Y.J., JOSHUA, D.E., WILLIAMS, G.T., SMITH, C.A., GORDON, J. & MACLENNAN, I.C.M. (1989) Mechanism of antigen-driven selection in germinal centres. Nature, 342, 929. MARTINEZ-MAZA, 0. & BRITTON, S. (1983) Frequencies of human B cell subsets activatable to Ig secretion by Epstein-Barr virus and pokeweed mitogen. J. exp. Med. 157, 1808. ROUSSET, F., GARCIA, E. & BANCHEREAU, J. (1991) Cytokine induced proliferation and Ig production of human B lymphocytes triggered through their CD40 antigen. J. exp. Med. (In press). SAIKI, 0. & RALPH, P. (1981) Induction of human immunoglobulin secretion: synergistic effect of human B cell mitogen Cowan I plus T cell mitogens or factors. J. Immunol. 127, 1044. SCO-r, S., PANDOLFI, F. & KURNICK, J. (1990) Fibroblasts mediate T cell survival: a proposed mechanism for retention of primed T cells. J. exp. Med. 172, 1873. SHOKRI, F., MAGEED, R.A., MAZIAK, B.R. & JEFFERIS, R. (1991) Expression of VHIII associated cross-reactive idiotope on human B lymphocytes: association with Staphyloccal protein A binding and Staphylococcal aureus Cowan I stimulation. J. Immunol. 146, 936.
375
SMITH, C.A., DAVIS, T., ANDERSON, D., SOLAM, L., BECKMANN, M.P., JERZY, R., DOWER, S.K., COSMAN, D. & GOODWIN, R.G. (1990) A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins. Science, 248, 1019. SPLAWSKI, J.D., JELINEK, D.F. & LIPSKY, P.E. (1991) Delineation of the functional capacity of human neonatal lymphocytes. J. clin. Invest. 87, 54. TOMHA, S., HIROHATA, S. & LIPSKY, P.E. (1991) The role of CDI la/ CD54 interactions in human T cell-dependent B cell activation. J. Immunol. 146, 576. TuccI, A., MOUZAKI, A., JAMES, H., BONNEFOY, J.Y. & ZUBLER, R.H. (1991) Are cord blood B cells functionally mature? Clin. exp. Immunol. 84, 389. WEN, L., HANVANICH, M., WERNER-FAVRE, C., BROUWERS, N., PERRIN, L.H. & ZUBLER, R.H. (1987) Limiting dilution assay for human B cells based on their activation by mutant EL4 thymoma cells: total antimalaria responder B cell frequencies. Eur. J. Immunol. 17, 887. ZUBLER, R.H., ERARD, F., LEES, R.K., VON LAER, M., MINGARI, C., MORETTA, L. & MACDONALD, H.R. (1985) Mutant EL-4 thymoma cells polyclonally activate murine and human B cells via direct cell interaction. J. Immunol. 134, 3662.
ADONIS 009910491001553
(I991) 84, 373-375 EDITORIAL REVIEW
Human B lymphocytes mature J. GORDON Division of Immunology, School of Medical Science, The University of Birmingham, Birmingham, England (Acceptedfor publication 7 March 1991)
INTRODUCTION Functional immaturity in the immune system of newborns is highlighted by an inability to mount humoral responses to TI-2 antigens such as bacterial capsular polysaccharides. A lack of 'immune experience' is not sufficient to account for this deficit as it persists for the first 2 years of life even where there is repeated exposure to exogenous antigen (Kahty et al., 1984). It has been tempting to suggest that neonatal B lymphocytes are inherently immature, a notion given some credence by the finding that most cord blood B cells express the CD5 marker (Gadol & Ault, 1986). Moreover, studies using polyclonal activators to assess their capacity for differentiation in vitro have supported the thesis that neonatal B cells are in some way functionally naive. In this issue, an important paper by Tucci et al. (1991) reexamines the question of functional competence among cord blood lymphocytes; the conclusions reached will be discussed later. Their study exploits one of several systems which have recently been described for driving potent B cell growth and differentiation in vitro. The main purpose ofthis brief article is to review those systems. THE ZUBLER SYSTEM In 1985, Zubler et al. reported that a mutant subclone of the mouse thymoma line EL4 activates both human and murine B cells via direct cell-to-cell contact in a polyclonal, antigen- and lectin-independent manner (Zubler et al., 1985). Two years later, with some modifications to the system, it was shown that some 90% of B lymphocytes from human blood or spleen could be driven to proliferate and differentiate to immunoglobulinsecreting cells (ISC) (Wen et al., 1987). In addition to the irradiated thymoma cells, of which 5 x 104 are added in a final volume of 200 p1, cultures are supplemented with T cell/ macrophage supernatants. These are produced by stimulating mixtures of T cells and macrophages, which have been isolated from peripheral blood, with phytohaemagglutinin (PHA) and phorbol ester for 36 h; supernatants are added at a final concentration of 5-10%. Over 10 days, under conditions of limiting dilution, single B cells could generate clones numbering on average 380 cytoplasmic immunoglobulin positive (cIg+) cells; the mean amount of immunoglobulin secreted per clone was 20 ng. In addition to its application in the study of Tucci et al. (1991), this system has been used to determine the frequency
of B cells producing antibody to Plasmodium falciparum in malaria-infected individuals (Wen et al., 1987). THE LIPSKY SYSTEM In 1988 Peter Lipsky's group described a method for generating high-rate immunoglobulin production from human B cells by co-culturing them with T cells that had been stimulated with immobilized antibody to CD3 (Hirohata, Jelinek & Lipsky, 1988). More recently, they have estimated the frequency of B cells responding in this system and also determined the factors which modify the response (Amoroso & Lipsky, 1990). Under conditions of limiting dilution, the proportion of B cells responding to anti-CD3-stimulated T cells was found to be in the region of 10%. When comparing the efficacy of different cells stimulated through CD3, T cell clones were seen to provide a more potent system than freshly isolated, mitomycin C-treated T cells. However, with the addition of IL-2 and accessory cells, B cell responses to anti-CD3-activated fresh T cells now reached a precursor frequency of 60-80%. IL-6 was also found to augment the number of B cells responding; IL-4 inhibited the IL-2-dependent increase while promoting no change by itself. Multiple immunoglobulin class secretion was observed in wells where single B cells had been seeded indicating that class switching was occurring in these cultures. THE BANCHEREAU SYSTEM A report published in 1987 noted that a combination of IL-4 and an antibody to CD40 provided optimal signals for maintaining the cell cycle in prestimulated human B cells (Gordon et al., 1987). However, in the absence of any additional support, the cultures succumbed to cell death within 9-12 days. At the beginning of this year Banchereau et al. (1991) reported greater success using the two activities, describing for the first time the ability to sustain active proliferation of non-transformed B cells for many weeks. In addition to IL-4 and CD40 antibody, their culture system utilizes a feeder layer of mouse L cells into which the CDw32 gene encoding FcyR has been transfected and expressed. This allows the fibroblast to 'grab' the CD40 antibody and present it to the B cell in a fashion that is presumably optimal for long-term stimulation. Simple immobilization or cross-linking of the antibody with a second reagent cannot substitute for the transfected L cells; this implies either that the orientation and dynamics of presenting the antibody in a fluid membrane is particularly appropriate, or that the fibroblasts are themselves providing additional signals. IgM, IgG, and IgE production are found to accompany the growth of
Correspondence: Dr J. Gordon, Division of Immunology, School of Medical Science, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
373
374
J. Gordon
B cells stimulated with IL-4 and CD40 antibody while the additional presence of IL-2 further increases the level of IgM and results in large amounts of IgA being secreted (Rousset, Garcia & Banchereau, 1991). Interestingly, as has recently been found in more conventional in vitro systems, CD40 antibody not only enhances IL-4-dependent IgE production but also prevents IFN-)' from exerting its normal inhibitory influence on this change.
COMPARISON OF THE SYSTEMS All three of the novel systems designed to generate high-rate immunoglobulin production from human B cells in vitro are clearly a huge improvement on those based upon conventional polyclonal activators. For pokeweed mitogen (PWM) and Epstein-Barr virus (EBV), the number of B cells responding by immunoglobulin secretion is between 0 2%Yo and 3%, (MartinezMaza & Britton, 1983); Staphylococcus aureus Cowan (SAC) plus T cell supernatants encourage small amounts of IgM production (Saiki & Ralph, 1981). The conventional mitogens also suffer from their target selectively: SAC stimulates only those 300% of B cells expressing VH3 determinants (Shokri et al., 1991); PWM has a preference for large, IgD, post-switched B cells (Kuritani & Cooper, 1982); EBV shows a predilection for small resting B cells (Aman, Ehlin-Henriksson & Klein, 1984). Thus, quite a false impression of functional responsivity could be given when using any one of these polyclonal activators in situations where a particular B cell subset might be under- or over-represented (as in ontogeny or disease, for example). Probably the most 'unphysiological' of the recently described high-efficiency systems is that of Zubler's, relying as it does on xenogeneic tumour cells and supernatants containing lectin and phorbol ester. Nevertheless, it undoubtedly works and appears to develop aggressively the full functional potential of any given B cells. By contrast, Lipsky's approach could be considered a model par excellance for examining the parameters of B cell responses to T-dependent antigens; already, it has been used to show the CDl Ia/CD54 dependency of T cell-driven immunoglobulin production (Tomha, Hirohata & Lipsky, 1991). Banchereau's system differs from the other two both in its T cell-independence and in the ability to sustain growth over a much longer period (perhaps inter-related phenomena?). It is clear that, particularly when culturing under conditions of limiting dilution, the feeder layer employed in each of the high-potency systems supplies essential signals for the optimal growth and differentiation of the B cell. There is mounting evidence that T cell membranes can themselves be stimulatory for B cells but the molecules involved remain to be defined (Brian, 1988); recent data indicate that fibroblast-derived factors may provide survival signals to activated lymphocytes (Scott, Pandolfi & Kurnick, 1990). One outstanding question relates to the nature of the CD40 counterstructure: so far, all studies on this intriguing molecule have used presumed agonistic antibodies to elaborate functional change. CD40 has homology with receptors for tumour necrosis factor (TNF) and nerve growth factor (NGF) (Smith et al., 1990), both of which are cytokines that act optimally as trimers and regulate cell survival (negatively and positively, respectively). In addition to its capacity to maintain the growth of cycling B cells, CD40 ligation also promotes the survival of germinal centre cells and their neoplastic equivalents (Liu et al., 1989; Gregory et al., 1991),
and may be instrumental in the establishment of B cell memory (Liu et al., 1991). Several groups of investigators are now hotly in pursuit of the natural ligand for CD40 and its future availability may revolutionize studies of human B cell function.
BACK TO THE WOMB The paper of Tucci et al. (1991) in this issue of Clinical and E.xperimental Immunology, the catalyst for this mini-review, uses the Zubler system to delineate the functional maturity of cord blood B cells. Lipsky's group recently approached the same question in their system which uses T cells stimulated through CD3 to drive B cell responses (Splawski, Jelinek & Lipsky, 1991). The study reported in this issue concludes that cord blood B cells, if provided with the extremely potent signals supplied by the mutant EL4 thymoma cells and T cell supernatants, do reveal a potential for differentiation which is both qualitatively and quantitatively indistinct from that of adult B cells. Using the 'more physiological' system of Lipsky's, it was seen that neonatal B cells do indeed have a greater functional potential than previously realized: the production of both IgG and IgA, in addition to the usual IgM, was observed but the amounts of the switched isotypes produced were small by comparison to adult B cells. The concensus view from the two studies seems to be that while neonatal B cells are probably functionally naive, their inherent potential for stimulation approaches that of adult B cell: this can be realized as long as sufficiently strong help is available. With these examples, and the promise of what is soon to come, it can be confidently stated that the study of human B cells has finally matured. REFERENCES AMAN, P., EHLIN-HENRIKSSON, B. & KLEIN, G. (1984) Epstein-Barr virus susceptibility of normal B cell populations. J. exp. Med. 159, 208. AMOROSO, K. & LIPSKY, P.E. (1990) Frequency of human B cells that differentiate in response to anti-CD3-activated T cells. J. Immunol. 145, 3155. BANCHEREAU, J., DE PAOLI, P., ALLE, A., GARCIA, E. & RouSSET, F. (1991) Long term human B cell lines dependent on interleukin 4 and antibody to CD40. Science, 251, 70. BRIAN, A. (1988) Stimulation of B-cell proliferation by membraneassociated molecules from activated T cells. J. Immunol. 85, 564. GADOL, N. & AULT, K.A. (1986). Phenotypic and functional characteristics of human Leu-l/CD5 B cells. Immunol. Ret. 93, 23. GORDON, J., MILLSUM, M.J., Guy, G.R. & LEDBETTER, J.A. (1987) Synergistic interaction between interleukin 4 and anti-Bp5O (CDw4O) revealed in a novel B cell restimulation assay. Eur. J. Immunol. 17, 153. GREGORY, C.D., DIVE, C., HENDERSON, S., SMITH, C.A., WILLIAMS, G.T., GORDON, J. & RICKINSON, A.B. (1991) Activation of EpsteinBarr virus latent genes protects luna B cells from death by apoptosis. Nature, 349, 612. HIROHATA, S., JELINEK, D.F. & LIPSKY, P.E. (1988) T cell dependent activation of B cell proliferation and differentiation by immobilized antibodies to CD3. J. Immunol. 240, 3736. KAHTY, H., KARANKO, V., PELTOLA, H. & MAKELA P.H. (1984) Serum antibodies after vaccination with Haemophilus influenza type b capsular polysaccharide vaccine. Pediatrics, 74, 857. KURITANI, T. & COOPER, M.D. (1982) Human B cell differentiation. II. Pokeweed mitogen-responsive B cells belong to a surface immunoglobulin D-negative subpopulation. J. exp. Med. 155, 1561. Liu, Y.J., CAIRNS, J.A., HOLDER, M.J., ABBOT, S.D., JANSEN, K.U., BONNEFOY, J.Y., GORDON, J. & MACLENNAN, I.C.M. (1991) Recom-
Human B lymphocytes mature binant 25-kDa CD23 and interleukin 1 a promote the survival of germinal center B cells: evidence for bifurcation in the development of centrocytes rescued from apoptosis. Eur. J. Immunol. (In press). Liu, Y.J., JOSHUA, D.E., WILLIAMS, G.T., SMITH, C.A., GORDON, J. & MACLENNAN, I.C.M. (1989) Mechanism of antigen-driven selection in germinal centres. Nature, 342, 929. MARTINEZ-MAZA, 0. & BRITTON, S. (1983) Frequencies of human B cell subsets activatable to Ig secretion by Epstein-Barr virus and pokeweed mitogen. J. exp. Med. 157, 1808. ROUSSET, F., GARCIA, E. & BANCHEREAU, J. (1991) Cytokine induced proliferation and Ig production of human B lymphocytes triggered through their CD40 antigen. J. exp. Med. (In press). SAIKI, 0. & RALPH, P. (1981) Induction of human immunoglobulin secretion: synergistic effect of human B cell mitogen Cowan I plus T cell mitogens or factors. J. Immunol. 127, 1044. SCO-r, S., PANDOLFI, F. & KURNICK, J. (1990) Fibroblasts mediate T cell survival: a proposed mechanism for retention of primed T cells. J. exp. Med. 172, 1873. SHOKRI, F., MAGEED, R.A., MAZIAK, B.R. & JEFFERIS, R. (1991) Expression of VHIII associated cross-reactive idiotope on human B lymphocytes: association with Staphyloccal protein A binding and Staphylococcal aureus Cowan I stimulation. J. Immunol. 146, 936.
375
SMITH, C.A., DAVIS, T., ANDERSON, D., SOLAM, L., BECKMANN, M.P., JERZY, R., DOWER, S.K., COSMAN, D. & GOODWIN, R.G. (1990) A receptor for tumor necrosis factor defines an unusual family of cellular and viral proteins. Science, 248, 1019. SPLAWSKI, J.D., JELINEK, D.F. & LIPSKY, P.E. (1991) Delineation of the functional capacity of human neonatal lymphocytes. J. clin. Invest. 87, 54. TOMHA, S., HIROHATA, S. & LIPSKY, P.E. (1991) The role of CDI la/ CD54 interactions in human T cell-dependent B cell activation. J. Immunol. 146, 576. TuccI, A., MOUZAKI, A., JAMES, H., BONNEFOY, J.Y. & ZUBLER, R.H. (1991) Are cord blood B cells functionally mature? Clin. exp. Immunol. 84, 389. WEN, L., HANVANICH, M., WERNER-FAVRE, C., BROUWERS, N., PERRIN, L.H. & ZUBLER, R.H. (1987) Limiting dilution assay for human B cells based on their activation by mutant EL4 thymoma cells: total antimalaria responder B cell frequencies. Eur. J. Immunol. 17, 887. ZUBLER, R.H., ERARD, F., LEES, R.K., VON LAER, M., MINGARI, C., MORETTA, L. & MACDONALD, H.R. (1985) Mutant EL-4 thymoma cells polyclonally activate murine and human B cells via direct cell interaction. J. Immunol. 134, 3662.
