ADONIS 0009910491001588

Clin. exp. Immunol. (1991) 84, 389-394

Are cord blood B cells functionally mature? A. TUCCI*, A. MOUZAKIt, H. JAMES*, J.-Y. BONNEFOYt & R. H. ZUBLER* *Division of Haematology, Department of Medicine, Geneva University Hospital, Geneva, tDepartment of Molecular Biology, Sciences II, University of Geneva, Geneva, and tGlaxo Institute for Molecular Biology, Geneva, Switzerland

(Acceptedfor publication 12 February 1991)

SUMMARY Very low immunoglobulin secretion occurs in pokeweed mitogen (PWM) stimulated cord blood mononuclear cells (MNC) and has been attributed to an 'immaturity' of both T and B cells of the newborn. The cord blood T cells are phenotypically 'naive' cells, in which suppressor activity for B cell function appears to dominate over helper activity. The cord blood B cells, in spite of their expression of different membrane immunoglobulin isotypes, secrete almost no IgG and IgA in the various B cell assays so far compared. We found that cord blood B cells are as competent as B cells from adults to generate clonal IgM, IgG and IgA responses in a culture system in which a cell contact with mutant EL-4 thymoma cells in conjunction with T cell supernatant leads to strong B cell activation. The supernatants derived from adult or cord blood T cells had the same B cell stimulating activity. As regarding the possible causes of the low cord blood PWM response, we studied the role of transforming growth factor-beta 1 (TGF-l1), a potent inhibitor of lymphocyte functions. TGF-fl sensitivity of B cells and TGF-fll mRNA levels in MNC were found to be similar for adult and cord blood cells. A neutralizing anti-TGF-fil antibody enhanced the adult PWM response, but the immunoglobulin secretion in cord MNC remained very low. We conclude that suppression by endogenous TGF-PfI occurs in the PWM system but is not responsible for the low immunoglobulin response of cord blood MNC, and that the newborn's B cell 'immaturity' can be overcome with potent T cell signals in vitro. This is consistent with the newborn's capacity to generate a T-dependent B cell response in vivo. Keywords cord blood B lymphocytes immunoglobulins transforming growth factor-beta levels in vivo are only reached several years after birth, and IgA levels not before puberty (Cooper, 1987). Anti-polysaccharide antibody responses are poor during the first 2 years of life (Timens et al., 1989). However, evidence was obtained for IgG and IgA expression early in fetal life (Gathings, Kubagawa & Cooper, 1981; Dosch et al., 1989); and normal proportions of cells with membrane IgM, IgG and IgA exist in cord blood (Durandy et al., 1990). The functional 'abnormalities' of T cells from newborns, as revealed in the PWM system (with adult B cells), can be summarized as dominance of suppression over helper activity (Moller, 1981; Andersson et al., 1983). Recently, surface markers have been identified that seem to distinguish between 'naive' versus 'memory' T cells (Budd et al., 1987; Sanders, Makgoba & Shaw, 1988). More than 90% of CD4+ T cells in cord blood express the naive phenotype, which includes low levels of CD45RO and various cell adhesion receptors (Sanders et al., 1988). This could explain low helper activity. Alternatively, suppression could be increased. The naive CD4+ T cells from adults were found to activate CD8+ suppressor cells (Morimoto et al., 1985), whereas the corresponding cells from cord blood were found to inhibit B cells directly (Clement, Vink

INTRODUCTION Very low immunoglobulin secretion in pokeweed mitogen (PWM) stimulated cord blood mononuclear cells (MNC) is a hallmark of what appears to be an immune system 'immaturity' of the human newborn (Wu et al., 1976; Hayward & Lawton, 1977; Moller, 1981). The newborn's B and T cells seem to behave differently from the lymphocytes of adults. Low immunoglobulin secretion (IgM with few IgG and almost no IgA) is also found in B cell assays which, unlike the PWM system, do not depend on a cell contact-mediated helper T cell activity. This concerns activation of isolated cord blood B cells with Staphylococcus aureus Cowan I (SAC) or anti-p antibodies plus various cytokines (Mbller, 1981; Durandy et al., 1990), or with EpsteinBarr virus (Tosato et al., 1980; Moller, 1981; Miyawaki et al., 1988). Most B cells from cord blood but only few from adult peripheral blood express the CD5 molecule (Antin et al., 1986), and such CD5+ B cells were proposed to be different from normal B cells (Hayakawa & Hardy, 1988). Adult-like IgG Correspondence: Rudolf H. Zubler, MD, Division of Haematology, University Hospital, 1211 Geneva 4, Switzerland.

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& Bradley, 1990). The suppressor mechanism involved is not known. Transforming growth factor-beta I (TGF-fil), which can be made by T and B (as well as other) cells, is a potent inhibitor cytokine for human lymphocytes; in particular, immunoglobulin secretion is strongly inhibited (Kehrl et al., 1986a, 1986b). It is not known whether increased TGF-#1 suppression is responsible for the low PWM immunoglobulin response with cord blood MNC. However, because so far no in vitro B cell system could produce adult-level IgG and IgA responses with newborn B cells, it is not clear whether these are mature enough to differentiate during short-term cultures into high-rate IgG- and IgA-secreting cells. Therefore, we analysed isotype secretion in another culture system in which a direct cell contact with mutant EL-4 thymoma cells in conjunction with human T cell supernatant leads to B cell activation (Wen et al., 1987; Zubler et al., 1987). In addition, the possible role of TGF-f31 in the EL-4 and PWM systems was investigated.

MATERIALS AND METHODS Cord blood and adult peripheral blood MNC: cell separations Heparinized cord blood was obtained from healthy newborns at the time of delivery (Department of Gynaecology and Obstetrics, University Hospital, Geneva) and heparinized venous blood was collected from healthy adult donors (Transfusion Center, Division of Haematology, University Hospital, Geneva). MNC were prepared by centrifugation over a FicollPaque gradient (Pharmacia, Uppsala, Sweden). T cell-enriched (E+ cells, >95% CD3+) and B cell-enriched (E- cells, about 80% CD19+) populations were obtained by rosetting with neuraminidase-treated sheep erythrocytes as described (Wen et al., 1987). B lymphocytes were then isolated with an EPICS V cell sorter (Coulter Electronics, Hialeah, FL; sorter of the Geneva Medical Center operated by D. Wohlwend) from Ecells stained with FITC-conjugated anti Leul2 (CD19) monoclonal antibody (MoAb) (Becton Dickinson, Mountain View, CA). Single B cells were added to EL-4 cultures with the AutocloneTm apparatus of the cell sorter as described (Zhang, Hauser & Zubler, 1990). FITC-anti-CD4 MoAb (Coulter) was used to isolate helper T cells from E+ cells.

General culture conditions All cultures were performed at 370C, 5% CO2 in RPMI 1640 medium (Gibco, Basel, Switzerland) supplemented with 10% heat-inactivated (560C, 40 min) fetal calf serum (Seromed, Munich, Germany), 2 mM L-glutamine, 25 mm HEPES buffer, 5 x 10-5 M 2-mercaptoethanol, 100 U/ml penicillin, 100 pg/ml

streptomycin (GIBCO). P WM culture system MNC were added into 96-well flat-bottomed plates (2 x 105/200 p1 culture) in the absence or presence of 1 pg/ml PWM (Sigma Chemical Co., St Louis, MO). The cells were either cultured in the same medium for 9 days or centrifuged and resuspended in the same volume of fresh medium on day 3 of culture.

EL-4 culture system, T cell supernatant B cells (500 cells/well for bulk cultures, or autocloned B cells) were added into 96-well flat-bottomed plates (200 pl cultures) together with 5 x 104 irradiated (50 Gy) murine thymoma cells

(mutant EL-4 cells, clone 6.1.5.5) plus human T cell supernatant as described (Wen et al., 1987). B cell activation in this system is lectin-independent (Zubler et al., 1987). T cell supernatant was prepared by culturing adult or cord E+ cells (106/ml) for 36 h in the presence of 10 ng/ml PMA and/or 5 ,g/ml phytohaemagglutinin (PHA) as indicated below.

Cell proliferation, immunoglobulin assays B cell proliferation in the EL-4 system was measured by thymidine incorporation; the mutant EL-4 cells are thymidine kinase-negative (Wen et al., 1987). The cultures were pulsed with I pCi of 3H-thymidine (Radiochemical Centre, Amersham, UK) overnight between day 6 and day 7 of the cultures, harvested and counted as described (Zhang et al., 1990). IgM, IgG and IgA levels in culture supernatants (day 9 for PWM cultures, day 10 for EL-4 cultures) were quantified with isotypespecific ELISAs (Zhang et al., 1990).

TGF-flJ, anti-TGF-f31 Human platelet-derived, purified TGF-P1 and purified rabbit IgG anti-TGF-,Bl with neutralizing activity were purchased from R&D Systems, Minneapolis MN. Non-specific control rabbit IgG was from Cappel Laboratories, Cochraneville, PA. RNA extraction, Northern blot analysis Total RNA was extracted from 5 x 106 -2 x 107 MNC. The cells were lysed with Tris/SDS in the presence of Vanadyl Ribonuclease complex (GIBCO, BRL) and proteinase K (Merck, Zurich, Switzerland), followed by phenol extraction and ethanol precipitation. The pellet was treated with DNAse (grade 1, Boehringer, Mannheim, Germany) and proteinase K, followed by reextraction with phenol and ethanol precipitation. The dried pellets were resuspended in 1 mm EDTA in water for OD determination. Aliquots of 5- 10 pg total RNA were subjected to Northern blotting on gene screen nylon membranes followed by u.v. irradiation, as described (Khandjian, 1986). A Shimadzu dual-wavelength flying spot scanner was used for densitometry of autoradiograms. The probes included human TGF-,B1, 300 bp EcoRI/HindIII fragment cut from the riboprobe plasmid PGH TGF-,B27, and human IL-2, 347 bp EcoRI/HindIII fragment cut from riboprobe plasmid PGH IL-2 (provided by Dr E. Kawashima, Glaxo Institute for Molecular Biology, Geneva, Switzerland).

RESULTS IgM, IgG and IgA responses by cord blood B cells in the EL-4 culture system The cord blood B cells' competence for immunoglobulin isotype secretion was analysed in the EL-4 system, in experiments which included parallel cultures with cord or adult B cells. CDl9+ B cells were added with the autoclone apparatus of the cell sorter at a mean number of I cell/well into cultures set up with 50 000 irradiated mutant EL-4 thymoma cells and 5% T cell supernatant from PHA+PMA-induced adult T cells. By means of staining B cells with carboxy fluorescein diacetate (which gives very bright cells) and then analysing with the fluorescence microscope 670 microdroplets from the autoclone apparatus collected onto glass plates, we found 93% droplets with a single cell, 5-5% with no cell, and 1-5% with more than one cell or unclear interpretation (unpublished results). Thus, the auto-

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Maturity of cord blood B cells Table 1. Clonal B cell analysis in the EL-4 culture system: B cells from neonates and adults produce similar IgM, IgG and IgA responses

Cord blood B cells (236 positive wells/480 tested wells)t

Adult peripheral blood B cells (225 positive wells/480 tested wells)*

Isotope pattern

Positive wells (%)

IgM IgG IgA IgM+IgG IgM+IgA IgG+IgA IgM + IgG + IgA

18 7 11-6 80 17 3 9-8 62 28-4

Total isotype distribution (%)

Immunoglobulin (ng) produced per clone IgM

IgG

IgA

Total

Positive wells (%)

42 0 0 42 32 0 70

0 33 0 29 0 17 36

0 0 28 0

42 33 28 71 67 30 137

24-1 11 0 55 11-4 13 6 3-4 30 9

IgM 51 9

IgG 27-4

IgA 20 8

35 13 31

Immunoglobulin (ng) produced per clone

IgM

IgG

IgA

Total

47 0 0 48 76 0 96

0 23 0 46 0 20 23

0 0 17 0 25 17 33

47 23 17 94 101 37 152

IgM 56-7

IgG 23-6

IgA 19-7

Four different experiments with cells from cord and adult blood; CDl 9+ B cells were cloned with the cell sorter, cultures were scored positive if the immunoglobulin level was > 3 s.d. above the non-specific ELISA background in control cultures (15 for each experiment) without B cells. * 34-63% positive wells (responder B cell frequency) in four experiments, total mean of 74 ng immunoglobulin/positive well. t25-88% responder B cell frequency, total mean of 87 ng immunoglobulin/positive well.

clone cultures represent mostly single B clone responses. However, the point for this study is that there appeared no significant differences between adult and cord B cell responses with regard to either (i) the total responder B cell frequency; (ii) the relative proportions of cultures producing IgM, IgG, IgA or more than one isotype; and (iii) the mean amounts of isotypes produced per well (Table 1). It has been shown previously that one B cell can generate a mean of 380 plasma cells in this system and that the response does not continue after 10 days of culture (Wen et al., 1987). The present data show competence for IgM, IgG, IgA secretion of the cord blood B cells. P WM responses by cord and adult MNC Different modalities of the PWM system were carried out in order to possibly increase the response by cord blood B cells. For the experiments shown below, it is relevant that with MNC

from adults, the IgM, IgG and IgA levels at day 9 of PWM cultures became five to 12 times higher, respectively, if the cells were not continuously cultured in the same medium, but were centrifuged and resuspended in the same volume of fresh medium on day 3 (Table 2). The immunoglobulin response by cord blood MNC was very low (total immunoglobulin < 5% of adult response) with or without such a medium change. However, this finding suggested that soluble inhibitors could be involved. Various other experiments essentially confirmed previous reports (Mdller, 1981; Morimoto et al., 1985; Clement et al., 1990) and these data are therefore not shown in detail; we observed no increase of the PWM-dependent cord blood B cell immunoglobulin response by (i) stimulation of isolated cord CD4+ T and B (E-) cells instead of total MNC (cord CD4+ cells were > 90% CD45RO-); (ii) removal of plastic adherent

Table 2. PWM-induced immunoglobulin secretion in blood mononuclear cells from adults and neonates: effect of medium change at day 3 of culture

Adult mononuclear cells immunoglobulin secretion (ng/ml)

IgM No PWM

95

(0-09) PWM

PWM and medium change at day 3

676 (025) 7,956 (0.16)

Cord mononuclear cells immunoglobulin secretion (ng/ml)

IgG

IgA

Total

IgM

IgG

IgA

Total

429 (0-11) 862 (0-12) 4,594 (0.15)

250 (0-09) 425 (0 16) 5,267 (0 23)

774 (008) 1,963 (0-16) 17,817 (0 18)

8 (030) 52 (0 17) 14 (0-37)

37 (022) 26 (009) 11 (0 04)

6 (017) 12 (0 14) 5 (0.14)

51 (013) 90 (0 13) 30 (0.14)

Triplicate cultures of 200 p1 were set up with 2 x 105 MNC and lasted 9 days. Resuspension of PWM cultures in their old medium on day 3 gave no increased response. Geometric means of six different experiments, and log values of 1 s.e.m. in parentheses.

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A. Tucci et al. TGF-0 mRNA

-a r a)

IL-2 mRNA

E3000

1--

33

Adult

fresh

2

Adult

fresh

32

3

Adult

fresh

5

4

Adult

24 h rested

5

Adult

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Cb)

0 0

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6

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activ. a h

55

at

7

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61

62

8 Adult

octlv. 24 h

60

76

9 Adult

Activ. 48 h

100

0

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2000

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125

TGF-/3

E

1000

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00

IgM

Adult

fresh

53

0

II

Cord

fresh

6

0

12 Cord

fresh

20

0

13 Cord

fresh

68

0

14 Cord

activ. 16h

34

21

15 Cord

activ. 24h

31

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16 Adult

aetlv. 24h

60

s76

5

500C250 1000 ~~~~~~~~~62

CP

Cd)

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B cell proliferation in the EL-4 system 100- (a) 50

:0 so

~ ~ ~ ~ Ot c

IgG Adult PBMC

(pg/mI)

b)ml

0 13 1030

Anti-TGF-/3

(1.tg/mI)

IgM IgG IgA Cord blood PBMC

IgA

Fig. 2. Immunoglobulin secretion by cord versus adult blood MNC in PWM cultures in the presence or absence of anti-TGF-fil neutralizing antibody or control IgG. These are 9-day cultures without medium change; the bars represent 1 s.e.m. values from 12 experiments. The antiTGF-,B1 antibody effect was significant (P< 0-01) for total immunoglobulin, IgM and IgA, but not for IgG. 0, Control; anti-TGF-f, 30 pg/ml; *, non-specific IgG, 30 pg/ml. Inset: (a) the inhibitory effect of added TGF-#l on thymidine incorporation by B cells in the EL-4 culture system (500 B cells, 5% T cell supernatant); (b) the neutralizing activity of the anti-TGF-/3I antibody tested in the presence of 300 pg/ml U,

TGF-fil. Fig. 1. TGF-#fI and IL-2 mRNA levels in cord versus adult blood MNC. Fresh, MNC from freshly collected blood samples; the MNC shown on line 4 were derived from the cells of line 3 by culturing 5 x 105 cells/ml (total of 6 x 107 cells) for 24 h in medium without mitogens, and aliquots of such cells were thereafter activated for various times with PHA (5 Pg/ ml) and PMA (10 ng/ml). The cord MNC activated with PHA+PMA for 16 h or 24 h, were derived from the cells on line 13. Aliquots of 5 x 106 to 2 x 107 cells were processed for Northern blotting. Sequential hybridization with TGF-flI and IL-2 probes was performed on the same gene screen membranes. Columns a/b and c/d represent two membranes: 10 and 5 pg total RNA per line were used in a/b and c/d, respectively. Autoradiography was longer for c, and a second TGF-fJ mRNA (Akhurst, Fee & Balmain, 1988) in addition to the major 2 5-kb species was only detected in c. The same RNA samples were blotted in lines 3 and 10, and lines 8 and 16, respectively, for normalization of the densitometric values (represented by the numbers).

(a) E

C: .t0

a) 0

0)

In

0

C)

macrophages and/or addition of adherent cells from adults; and (iii) coculture of cord B and adult blood CD4+CD45RO- T cells (which gave a three to five times higher immunoglobulin response with adult B cells than did CD4+CD45RO+ T cells). When PWM was replaced with PMA and ionomycin, at concentrations which induced high proliferative responses, no immunoglobulin secretion occurred with either cord or adult MNC. Role of TGF-#1 Since TGF-flI is a potent inhibitor of lymphocytes (Kehrl et al., 1986a, 1986b), we studied whether suppression by endogenous TGF-fll is increased in the cord blood PWM response and whether cord blood B cells are more sensitive than adult B cells to this factor. The TGF-#1 mRNA levels as analysed by Northern blots (Fig. 1) were found to exhibit considerable variation in three cord blood MNC samples, but the mean mRNA level (relative value of 31, obtained by densitometry)

E E

lo,

0

3

10

30

30

Tcel supernotant (%)

1

0

62

125 250 500

TGF-)1 (pg/ml)

Fig. 3. (a) Cord and adult blood T cell supernatants have similar activities in the EL-4 culture system. Shown are the dose-response effects on total immunoglobulin secretion (ng/ml) by PHA-induced cord or adult T cell supernatants in triplicate cultures with 50 000 irradiated EL-4 cells and 500 cord or adult B cells, as indicated. The proportions of IgM, IgG and IgA were very similar to those in Table 1 for all T cell supernatants/B cell combinations and no immunoglobulin response above 50 ng/ml occurred in the absence of EL-4 cells; (b) cord and adult blood B cells show similar TGF-fI responsiveness. EL-4 cultures were set up as above, with 20% PHA-T cell supernatants in addition to the indicated concentrations of TGF- Il. 0, Adult T cell supernatants/cord B cells; 0, adult T cell supernatants/adult B cells; 0, cord T cell supernatants/cord B cells; cord T cell supernatants/adult B cells. U,

Maturity of cord blood B cells was not higher than in three adult blood MNC samples (value of 39). When MNC from one sample of adult and one of cord blood cells were activated for various times with PHA + PMA, relatively small changes of TGF-fI mRNA levels occurred. In previous studies TGF-#l message translation was activationdependent in T cells but not in B cells (Kehrl et al., (1986a, 1986b) or macrophages (Assoian et al., 1987). As shown in Fig. 1, IL-2 mRNA was only detected in the stimulated cells. As in other studies, this message was present at 4 h (McCrady et al., 1988) and disappeared at 48 h (Kronke et al., 1985). To obtain information on TGF-/31 function, which does not necessarily correlate with mRNA levels, a neutralizing antiTGF-f1 antibody was added to 9-day PWM cultures. With adult MNC this resulted in a significant increase of the total immunoglobulin response (mean of 2-3 times, 12 experiments) (Fig. 2). Control rabbit IgG had no significant effect. The antibody concentration (30 pg/ml) was 10 times in excess of the amount required to reverse in the EL-4 system a 96% suppressive effect of 300 pg/ml exogenous TGF-fll (see inset of Fig. 2). However, in PWM cultures with cord MNC the immunoglobulin response remained very low in spite of the presence of such antibody. T cell supernatant derived from adult or cord blood T cells were also compared in the EL-4 system. Since addition of PMA into this system can overcome the requirement for T cell supernatant whereas PHA has no effect (Zubler et al., 1987; Zhang et al., 1990), supernatants were obtained from T cells stimulated for 36 h with PHA only. It was found that cord and adult T cell supernatants had very similar stimulating activity for both cord and adult B cell immunoglobulin responses (Fig. 3a). Moreover, the sensitivity of cord B cells to exogenous TGFf1 in the EL-4 system was identical to the sensitivity of adult B cells (Fig. 3b). For both types of B cells the inhibitory effect of exogenous TGF-PI was reversed by anti-TGF-fll antibody, but the stimulatory effects of adult or cord T cell supernatants were not enhanced by the antibody. The kinetics of the proliferative and immunoglobulin responses in the EL-4 system were also not influenced by anti-TGF-P1 (data not shown).

DISCUSSION This study shows that newborn B cells are competent to differentiate into high-rate IgM, IgG and IgA secreting cells during a 10-day culture period in the presence of mutant EL-4 thymoma cells and T cell supernatants derived from either adult or cord blood T cells. The short-term cloning efficiency, the proportions of clones producing one or more isotypes, and the amount of immunoglobulin produced per clone did not differ significantly for cord versus adult blood B cells. Thus, the immaturity of B cells from neonates can be overcome in vitro by some potent activation signals involving cell contact (Zhang et al., 1990). This is consistent with the observed capacity to express isotypes during fetal life (Gathings et al., 1981; Dosch et al., 1989) and the presence of adult-like proportions of membrane IgG- or IgA-expressing B cells in cord blood (Durandy et al., 1990). The IgG or IgA committed clones in the EL-4 system are derived from membrane IgG+ or IgA+ cells (X. Zhang & Ch. Werner-Favre, R.Z., personal communication), but it is not

393

known whether these are memory B cells. Their proportion in cord blood may not be representative for the whole organism. It takes several years for IgG, and until the time of puberty for IgA, to reach adult-like serum levels (Cooper, 1987). Most likely this time is required for the antigen-driven development of lymphoid micro-environments which produce such immunoglobulins effectively. The large proportion of B cells with activation markers already present in cord blood (Durandy et al., 1990), suggests an early start of this process, perhaps by involving physiologic autoimmunity (Guilbert, Dighiero & Avrameas, 1982). The CD5 molecule was recently found to behave as an activation marker; it becomes transiently expressed on CD5- B cells when they respond in the EL-4 system (Werner-Favre et al., 1989). Thus, the CD5 + cord blood B cells do not necessarily belong to a separate B cell lineage (Hayakawa & Hardy, 1988). At the present time it is not clear why the cord blood PWM response is so low. With MNC from adults we found that a change of culture medium on day 3 of PWM cultures had a strong enhancing effect on the immunoglobulin response, and this could be mimicked in part by adding neutralizing antiTGF-fil antibody. In accordance with other studies (Kehrl et al., 1986b; Assoian et al., 1987), TGF-f I mRNA was constitutively expressed in MNC. Enhancement of the SAC-induced adult B cell response with anti-TGF-Pl antibody has recently been reported (Kehrl et al., 1989). However, TGF-/31 mRNA was not increased in cord blood MNC, the big difference between cord and adult PWM responses persisted in the presence of anti-TGF-/31 antibody, and the sensitivity to exogenous TGF-P I as measured in the EL-4 system was also the same for adult and cord blood B cells. Since in addition the supernatants derived from cord or adult T cells had the same stimulatory effect in the EL-4 system, it seems that neither TGFfll nor in fact other soluble inhibitors are primarily responsible for the low cord blood PWM response. On the one hand, there exists now evidence for a reduced helper activity in vitro of naive T cells which show low expression of various cell adhesion receptors involved in T-B contact formation (Morimoto et al., 1985; Sanders et al., 1988; Kupfer & Singer, 1989; Clement et al., 1990; Springer, 1990) and which obviously predominate in the neonate. On the other hand, the cord blood B cells generate weak IgG and IgA responses in the presence of normal memory T cells from adults in the PWM system. Recently it was found that recombinant IL-4 induces IgE secretion in cord blood MNC, without stimulating IgG and IgA responses (Peleman & Delespesse, 1990). Cord blood B cells also generate low IgG and IgA responses in B cell assays which do not depend on direct T-B contact, such as stimulation with SAC or anti-s antibodies together with various cytokines (Moller, 1981; Durandy et al., 1990). Whereas the primary T cell-dependent B response is not deficient in vivo in the newborn, possibly because of interaction of B cells with already activated T cells and various accessory cells in lymphoid follicles, the capacity to generate an apparently T-independent anti-polysaccharide response appears relatively late in infants (Timens et al., 1989). Taken together, these observations suggest that some maturation, analogous to the naive-memory cell conversion of T cells, may also have to occur in B cells, before they can respond by IgG and IgA secretion in standard in vitro human B cell assays, i.e. that naive B cells could require stronger activation signals than memory B cells.

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A. Tucci et al. ACKNOWLEDGMENTS

We thank V. von Fliedner, Ludwig Institute for Cancer Research, Epalinges, Switzerland, for two-colour FACS analysis and separation (CD4/CD45RO) of T cells. This work was supported in part by a grant from the Swiss National Science Foundation (No 31-26502.89) to R.H.Z.

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Are cord blood B cells functionally mature?

Very low immunoglobulin secretion occurs in pokeweed mitogen (PWM) stimulated cord blood mononuclear cells (MNC) and has been attributed to an 'immatu...
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