Effect of TH-lines and clones on the growth and differentiation of B cell clones in microculture

Mokcular Immunology, Vol. 25. No. 2, pp. 147-155, Printed in Great Britain.

EFFECT

0161.5890/88$3.00+ 0.00 Pergamon Journals Ltd

1988

OF T,-LINES AND CLONES ON THE GROWTH AND DIFFERENTIATION OF B CELL CLONES IN MICROCULTURE

DEBRA B. KOTLOFF and JOHN J. CEBRA Department of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018, U.S.A. (Firsf received 8 December

1986; accepted in revised form

11 September 1987)

Abstract-Antibody isotype expression by B cell clones was analyzed using in oifro microcultures containing low numbers of hapten-gelatin-enriched B cells and higher numbers of hemocyanin-specific helper T cell lines or clones. Twenty-eight to sixty-three percent of clones grown in microculture with haptenated hemocyanin and T cells from established lines expressed IgG and/or IgA isotypes in random mixtures, almost always accompanied by IgM. Helper T cells from hemocyanin-specific clones also supported the expression of non-IgM isotypes by the B cell clones, suggesting that a single specificity of T cell can provide sufficient growth and differentiation factors for the display of isotype switching. A positive correlation between the antibody output of clones and the expression of non-IgM isotypes indicated that the switching process may be associated with cell division. Although memory B cells that give clones expressing IgG and/or IgA in the absence of IgM are also enriched on haptenated gelatin, they are not stimulable under conditions of this microculture_assay.

INTRODUCTION Extensive studies in vitro using bulk cultures of B lymphocytes have indicated that T cells and their factors are involved in the activation of B cells and in their differentiation and maturation to antibodyforming cells. Experiments which led to operational definitions of isotype-specific T cells (Kishimoto and Ishizaka, 1973; Kiyono et al., 1982; Kawanishi et al., 1983; Vitetta et al., 1984) employed such bulk cultures, as did several other studies (Seman et al., 1982; Leclercq et al., 1985) which failed to support a requirement for such cells in the expression of isotypes other than IgM by B cells. Just as a definitive understanding of the mechanism of action of individual T cell factors such as BSFl on B cells is facilitated by their purification via recombinant DNA methodology (Vitetta et al., 1985; Lee et al., 1986), a resolution of the controversy over the existence and/or mechanism of action of isotype-specific T cells will require analysis of their interaction with clones in vitro. Bulk cultures, or the use of B cells at relatively high density, precludes the possibility of defining precursor B cell-product B cell relationships during growth in culture. In addition, these types of cultures are more likely to reflect complex B cell interactions and to suffer from contamination by T cells or other non-B cells that have not been deliberately added. We have studied the effects of antigen-specific T cells from lines and clones on B cell isotype expression using a modified splenic fragment assay (Kotloff et al., 1987) and found that while the antigen-specific T cells were necessary for the generation of the response, the isotype pattern reflected the B cell source, spleen or Peyer’s patches. In addition, a clone of antigen-specific helper T cells was found to be by

itself sufficient for the expression of multiple isotypes because it permitted such responses in splenic fragments from athymic recipients. Beyond the manipulation of cell inputs and recipient phenotypes, analyses of such cultures are limited by our inability to define and control the elements contained within a splenic fragment. As a result, we chose to pursue the question of the regulation of isotype expression by developing a clonal in vitro culture system consisting of dispersed cells rather than splenic fragments. The system consists of clonal microcultures of haptenspecific B cells co-cultured with the antigen-specific T cell lines or clones. Advantages over splenic fragment assays include a well defined cell input and microenvironment, and an increased cloning efficiency. Moreover, the problem of low and perhaps selective lodging of input B cells in recipient spleens is eliminated. Hapten-specific B cells have been used extensively in microculture by Pike and Nossal and their coworkers (1985) to test the action of various growth and differentiation factors on individual B cells, but the isotype potential as expressed by their clones grown in the presence or absence of either filler or helper T cells has not been previously reported. The accompanying paper presents our isotype analyses of single B cell clones grown in the absence of other cells, the majority of which expressed solely IgM. In the presence of filler xid/Y thymocytes, about 20% of the antibody-secreting clones made IgG and IgA isotypes together with IgM (Schweitzer and Cebra, 1988). In this paper we report the effects of co-culture of helper T cells with such clones. We find that hemocyanin-specific T cell lines support the clonal outgrowth of antigen-specific B cells in microculture such that up to 63% of the clones expressed non-IgM 147

148

DEBRAB. KOTLOFFand JOHN J. CEBRA

isotypes within a given experiment. To date, no other in vitro dispersed cell cultures have supported the clonal outgrowth of B cells that express an equivalent variety of isotypes. The majority of “switched” clones in microculture expressed IgM along with IgG and/or IgA. Clones that may have previously switched in vivo to the expression of non-IgM isotypes were scored only occasionally in this assay, while they are frequently detected in the splenic fragment assay. A portion of the response in microculture occurs in the absence of antigen, and thus reflects a bystander response probably induced by factors released by the T cell lines. The relationship between clonal output, or the concn of specific antibody secreted by a particular clone, and the expression of “switched” clones was analyzed. In general, clones with a higher output expressed non-IgM isotypes more frequently than did clones giving a lower output. Finally, evidence has been obtained in the microculture assay that a cloned T cell line representing a single specificity can provide sufficient help for the generation of B cell clones that switch to the expression of non-IgM isotypes. MATERIALS AND METHODS Mice Young adult BALB/c and CBA/N male mice were raised in our colony.

x BALB/c

F,

response to Hy or to Con A, but did not proliferate in the absence of antigen or in the presence of irrelevant antigen. of hapten-speciJic

Preparation

B celIs

Antigen-specific B cells were enriched by panning spleen or Peyer’s patch cells from unprimed donors on thin layers of haptenated gelatin coated Petri dishes (FLU-gelatin or PC-TGG-gelatin) as described previously (Haas and Layton, 1975; Nossal et aI., 1978; Schweitzer and Cebra, 1988). The binding population has been demonstrated to consist of >97% B cells and is approx. 200-fold enriched in B cells responsive to thymus-independent and thymusdependent antigens (Pike et al., 1983; Hebbard et al., 1984). Fragment

assay

From 6 to 13 x lo3 PC-enriched B cells were injected i.v. into Hy-primed, lethally irradiated (1600rds) recipients or together with 3 x 10” Hyspecific cells from a helper T cell line into unprimed, lethally irradiated recipients. At 18 hr following cell transfer, recipient spleens were removed, chopped into approx. 50 fragments, and plated at one fragment per well into 96-well microtiter plates. Fragments were cultured in vitro as described (Kotloff et al., 1987). Microcultures

Antigens Phosphocholine-tyrosyl-glycyl-glycyl-hemocyanin(PC-TGG-Hy) was prepared as described previously 1979). Fluorescein(Gearhart and Cebra, hemocyanin (FLU-Hy) was prepared as described (Nossal et al., 1978) with the substitution of Limulus hemocyanin for keyhole limpet hemocyanin. Antigen-speczjic

heIper T cell Iines and clones

A modification of the method described by Kimoto and Fathman (1980) was used for the generation and maintenance of the Hy-specific T cell lines. Culture media consisted of RPMI-1640 (GIBCO, Grand Island, NY) supplemented with 10% fetal bovine serum (K.C. Biologicals, Corning, NY), 2mM 1 x 10m5 M 2-mercaptoL-glutamine (GIBCO), ethanol, and 50pg/ml gentamicin (GIBCO). Hyspecific helper T cell clones were generated and maintained by the procedure of Sredni et al. (1981). Clones were restimulated every 7 days with antigen and fresh filler cells at 2 x lo6 cells/ml, and expanded from 96-well microtiter plates to 24-well plates and to tissues culture flasks (all from Costar, Cambridge, MA). After several months of growth, clones were subcloned by limiting dilution. The surface phenotype of the T cell lines and clones were analyzed by indirect immunofluorescence as measured on a cytofluorograph. These cells were Thy l+, Lyt I+, Lyt 2-, L3T4+, and Ia-. In a 3H-thymidine incorporation assay, the lines and clones proliferated in

About 15 to 30 PC- or FLU-specific B cells were cultured in 60-well Terasaki trays (Miles Laboratories, Naperville, IL) in 10 ~1 as described (Schweitzer and Cebra, 1988) except that FLU-Hy or PC-TGGHy (50 ng/ml) were also used as antigens. After 68 days of culture 5 ~1 of medium were added to each well, then 12~1 of each supernatant were removed and diluted into 50~1. Portions of 10~1 were analyzed for total antibody and isotypes by radioimmunoassay using the reagents of specificity previously described (Hurwitz et al., 1982). In general, all anti-isotypes have similar sensitivities and gave with 0.5 ng antibody of approx. signals 2.5 x background (20&300cpm). Signals greater than 0.5 ng were taken as positive. The total antibody secreted by clones grown in microculture with T, cells ranged from 0.5 to 75ng with most making l&20 ng. The determination of frequency of responding cells has been described in the accompanying paper (Schweitzer and Cebra, 1988). The low number of B cells cultured was chosen so that most positive supernatants statistically contained monoclonal antibodies. To support the clonality of most of the positive cultures, mixtures of FLU-specific cells from BALB/c (Ig”) and CB20 (Igb) congenic mice were plated as described (Schweitzer and Cebrd, 1988) except that they were co-cultured with 10’ xid/Y thymocytes, 3 x lo3 Tn cells specific for Hy, and 50 ng/ml FLU-Hy. A total of 94 of 240 cultures produced antibody (0.49/well) at a frequency of

149

Effect of T,-lines and clones on specific B cells responding cells of 2.5%. Using the pair of reagents specific for the IgM-isotype or the IgM-allotype (Ig”) described (Schweitzer and Cebra, 1988), 73 of the 94 positive supernatants contained a single allotype and 21 were mixtures. Random and independent assortment of responding B cells would have led to the expectation that about 11 of the cultures should contain antibodies from two or three clones that secreted different allotypes. Thus, allotyping generally supports the clonality of most of the antibody responses in limiting dilution cultures and does not indicate any interdependence or linkage of responding clones. RESULTS

The haptenated-gelatin fractionation technique enriches hapten -specific B cells that respond in the splenic fragment assay without altering their pattern of isotype expression Since we hoped to study the isotype potential of both virgin and primed (memory) hapten-specific B cells in microcultures, we first tested the potential of the enriched B cells with both the conventional and modified versions of the splenic fragment assay, which are known to support clonal outgrowth of both primed and unprimed individual B cells (Teale, 1983; Riley and Klinman, 1985). Splenic or Peyer’s patch B cells enriched by binding to PC-gelatin were injected into Hy-primed recipients or into unprimed recipients along with Hy-specific helper T cells from lines grown in vitro. Very low numbers (c-13 x 103) of PC-enriched B cells gave statistically clonal cultures in the splenic fragment assay as compared with the numbers of unfractionated B cells generally needed (6-12 x 106). Assuming a similar lodging incidence (4%) for unfractionated and enriched B cells, the enrichment for responding cells was approx. lOOO-fold. The isotype

Table 1. Isotype of anti-PC-producing

patterns expressed by the clones grown in splenic fragments from PC-enriched cells from spleen and Peyer’s patches were similar to those expressed from PC-responding cells within unfractionated populations from these tissues (see Table 1, and Gearhart and Cebra, 1979). For example, a large proportion of clones from PC-enriched B cells from Peyer’s patches expressed IgA antibody alone (Table 1). No antibody-producing clones were detected in splenic fragments from unprimed recipients given PCenriched B cells alone without Hy-specific helper T cells. This experiment demonstrated that B cells isolated by haptenated gelatin fractionation are highly enriched for T-dependent antigen reactivity and contain a high proportion of clones that switch in vitro to the expression of non-IgM isotypes. The hapten-enriched B cells were also shown to contain that population of cells which appears to have already switched in vivo and gives clones secreting only non-IgM isotypes, such as IgA, in vitro (Gearhart and Cebra, 1979). Response of hapten-speciJic B cells in clonal microcultures containing carrier -speciJic helper T cells Increasing numbers of Hy-specific helper T cells from lines were added to a small, constant number of PC-specific B cells. The frequency of responding B cells increased with the addition of increasing numbers of T cells, as depicted in Table 2. This same trend was observed in many subsequent experiments which also showed that the response plateaued at about the level of 3 x lo3 added T cells per well (data not shown). A variable bystander response was observed in microculture in the absence of antigen. Also, in the experiment summarized in Table 2, the response to FLU-Hy suggests that linked recognition of hapten attached to carrier is not a requirement for many of the clonal antibody responses in this system. In other experiments, no response was elicited in the absence

clones from PC-specific

B cells grown in splenic fragment

% Anti-PC positive fragments expressing as generated from the following:” PC-specific splenic B + Hy T cells Unprimed recipients IgM only Some IgM Some IgG Some IgA IgG only IgA only IgM + IgG + IgA IgG + IgA IgM + IgA IgM + IgG Number of positive fragments analyzed

PC-specific Hy-primed

splenic B recipients

culture

product

PC-specific Peyer’s patch B Hy-primed recipients

I7 79 64 72 4 9 45 9 II 6

2 84 9 98 0 II 5 5 77 0

0 36 I8 100 0 64 I8 0 I8 0

47

44

I7

“Six x IO’ to I3 x IO’ PC-specific B cells from spleen or Peyer’s patches were injected into Hy-primed recipients. Thirteen x IO3 PC-specific B cells and t&3 x IO6 Hy T cells were injected into unprimed recipients. No positive wells were detected when 0 Hy T cells were injected into unprimed recipients. Recipients were irradiated with 1600 r. The designation “some Igx” indicates the percentage of antibody-containing cultures that contain the IgX antibody isotype either alone or with any other isotype(s).

150

DEBRA B. KOTLOFF and JOHN J. CEBRA Table 2. Frequencies (%) of clones from PC-enriched cells as a function of antigen and of T, cell dose” Number of T, cells Antigen

None

NOW

0.1

0.8

PC-TGG-Hyh PC-LPS FLU-H+

0.8 0.8 0.1

1.9 4.4 1.9

“Approximately *Fifty ng/ml. ‘0.2 &ml. ‘Fifty ng/ml.

2.2 0.6

1.4 2.5 7.0 3.2

15 PC-enriched cells were cultured per mierccuiture well

of antigen, while occasionally the frequency of the response without antigen approached that in the presence of antigen (data not shown). Usually, this antigen-independent response induced by helper T cells was about one-third that of the response in the presence of antigen, as noted also by Hebbard et al. (1984) in their experiments. Approximately 4 x 10’ Hy-specific T cells were cultured with increasing numbers of either FLUenriched or non-enriched splenic B cells in the presence of either FLU-Hy or PC-TGG-Hy, respectively. Number of cells per well 30 40 Flu-enriched

50 B cells

: 0.2 1” x a

The number of responding wells increased as the number of B cells per culture increased. When the log of the fraction of non-responding cultures is plotted vs the B cell dose [Fig. l(A) and (B)], the response is linear in both cases, indicating that the B cell was the limiting element in these assays as routinely performed. The frequency of anti-FLU responsive B cells among the FLU-gelatin-enriched B cells, and PC responsive cells among unfractionated splenic B cells was determined by chi-square minimization and was found to be 2.3 x lo-’ and 9.4 x lo-‘, respectively. Of course, these frequencies may relate only to a particular subset of B cells responsive under these conditions and not to the total FLU- or PC-specific cells among those cultured. T cells were either used immediately following 4 days of stimulation with antigen (T cell blasts) or after culture without antigen for at least 1 week (resting T cells). The frequency of responding B cells was higher in the presence of T cell blasts than in the presence of resting T cells (Table 3). This finding may reflect variability in the concn of lymphokines secreted by the T cells depending upon their state of activation. I~~~y~es secreted by clones generated from

Number of cells per well

specl$c B cells microcultured

haptenwith helper T cells

The individual isotypes secreted by clones generated from PC- or FLU-specific B cells in microculture were analyzed by RIA. In one experiment, both PCand FLU-specific B cells were enriched on PC- or FLU-gelatin coated petri dishes and plated in microculture wells along with resting Hy-specific helper T

0.2 1s

Table 3. Hy-specific T cell blasts are more efhcient than resting KY-specific T cells for the generation of anti-FLU

Fig. 1. (A) Limiting dilution analysis of the frequency of FLU-Hy responsive cells in the FLU-gelatin-binding B cell population. FLU-binding B cells were cultured with approx. 5000 Hy-specific T cells per microculture well in the presence of 50ng/ml FLU-Hy and 5% Con A supernatant. The frequency of anti-FLU clonal precursors was calculated to be 2.3 x 10m2 by chi-square minimization. (B) Limiting dilution analysis of the frequency of splenic B cells responsive to PC-TGG-Hy when cultured in the presence of 3500 Hy-specific helper T cells from lines and 5~ng~rnl PC-TGG-Hy. The frequency of anti-PC clonal precursors was calculated to be 9.4 x 10d5 B cells by chi-square minimization.

secreting clones from FLU-specific in microculture’

T cells IO3 blasts 3 x IO’ blasts 10’ resting 3 x IO’ resting

B cells

Anti-FLU clones frequency (%) 0.6 2.5 0.1 1.8

“Approximately 30 FLU-gelatin binding B cells were cuitured pet microculture well in the presence of 50ng/ml of FLU-Hy and the indicated Hy-

specific T cells. Clones were detected by RIA. One hundred and twenty wells were assayed per T cell dose.

Effect of T,-lines

and clones

cells from lines and CBA/N x BALB/c F, male thy mocytes as filler cells. Table 4 illustrates the isotype analysis of 215 clones scored in this experiment. Thirty-seven percent of anti-PC clones and 28% of anti-FLU clones expressed non-IgM isotypes. IgG or IgA isotypes appeared in random mixtures, almost always along with IgM. IgA was not secreted by any of the anti-FLU clones in this experiment. The isotypic response of FLU-specific B cells in microculture was generally less varied than that of PCspecific B cells. The more varied response from PC-specific B cells may reflect prior environmental priming by the PC determinant. Thus, these microcultures do support the clonal outgrowth of B cells that can express multiple isotypes, but B cells that have presumably switched previously in vivo, and thus should give clones expressing only IgG and/or IgA isotypes, are scored infrequently if at all. As indicated above, secreting clones grown under these conditions made from 0.5 to 75 ng of antibody with most making about l&20 ng. When cultures contained IgM along with IgG and/or IgA isotypes, the IgG or IgA component usually predominated, often occurring at 2-IO-fold the amount of IgM. Thus, these cultures differed from those grown with haptenated-LPS and thymocytes alone in that IgM almost always greatly predominated in the latter when more than one isotype was detected (Schweitzer and Cebra, 1988). The presence of thymocyte filler cells usually increased the frequency of responding B cells but did not alter their isotype potential. Clones generated in the absence of thymocytes but with helper T cells (Table 5) expressed as diverse an isotypic pattern as those generated in the presence of thymocytes (see Table 4). In fact, the experiments summarized in Table 5 contained a higher percentage of “switched” clones than the experiment shown in Table 4, in which thymocyte filler cells were present along with helper T cells in the microculture wells. In general, the frequency of isotype switching observed within-individual experiments was somewhat variable. Table 5 also depicts the isotype profile of clones generated from unfractionated spleen cells in microculture. When clones from unfractionated spleen cells were compared with clones from hapten-specific cells within the same experiment, the hapten-specific cells tended to express a more diverse array of isotypes. Cloned, antigen-speciJic helper T cells can generate B cell clones in microculture that can switch to the expression of IgG or IgA isotypes Evidence we have obtained using a modified splenic fragment assay suggested that a single specificity of helper T cell can provide help to B cells for the expression of multiple isotypes (Kotloff et al., 1987). In order to corroborate this finding in a better defined system, we have tested Hy-specific cloned T cells in the microculture assay. By using clonal B cell

on specific

B cells

151

cultures and a cloned T cell line, we hoped to determine whether or not a particular specificity of T cell is sufficient for the generation of B cell clones that can switch to the expression of non-IgM isotypes. The isotype profile of PC-enriched B cell clones scored in the presence of either of two cloned T cell lines is illustrated in Table 6 (experiment 1). Although the isotypic response is not as diverse as that observed in previous experiments with the T cell lines, several switched clones were detected. In this experiment, the frequency of responding B cells increased with increasing concns of antigen (data not shown). In addition, the T cell clones required the presence of Con A supernatant in the medium for survival and function in this assay. These clones were also tested along with a third Hy-specific T cell clone in microculture with B cells enriched on FLU-gelatin (Table 6, experiment 2). The response was similar to that expressed by FLU-specific B cells cultured with Hy-

Table 4. Isotype

diversity

of anti-PC and anti-FLU in microculture”

rsotype IgM only Some IaM I&ii IgA, no IgM some IgC3 Some IgGl Some IgG2 Some IgA IgA only IgG only IgM + IgG IgM + IgG + IgA IgM + IgA IgG + IgA “Switched” Number of clones analyzed

clones grow”

% Of clones secreting isotype Anti-PC Anti-FLU 63 94 I 6 22 I I6 5
12 95 0 N.D.” N.D. N.D. 0 0 5 23 0 0 0 28 43

“Fifteen PC-specific and 30 FLU-specific B cells were cultured with 0.7s-3 x 101 resting Hy-specific T cells and IO’ CBA/N x BALB/c F, male thymocytes per microculture well. Fifty “g/ml of PC-TGG-Hy or FLU-Hy was present per well. “N.D. = not done.

Table 5. Isotype distribution of anti-PC clones grown in microculture in the absence of filler cells

Isotype IgM only Some IgM IgM + IgG IgM + IgA IgM + IgG + IgA IgG only Iga only IgG + IgA “Switched” Number of clones analyzed

% Of clones secreting isotype as generated from the following: PC-specific Unfractionated B cells“ spleen* 31 86 48 0 2 8 5 2 63 65

7297 I5 8 3 0 3 0 28 39

“Eight to twenty B cells enriched on PC-TGG-gelatin coated plates were cultured with Hy-specific helper T cells per microculture well in the presence of 50 ng/ml PC-TGG-Hy. The results represent clones from two separate experiments. ‘Clones analyzed were from wells which contained 3 x IO’-I.25 x IO4 unfractionated spleen cells. Wells with higher numbers of spleen cells were not analyzed for isotype because the response was not clonal at the higher cell doses. Fifty “g/ml PC-TGG-Hy was present in the medium.

152

DEBRA

B.

KOTLOFF and

presence of

Table 6. Response of bapten-specific B cells in the Hy-specific helper T cells

% Clones expressing isotype generated in the presence of the following Hy T clone: Experiment Experiment 1” Ell Ell c3 c3

from the data presented in Fig. 2 that, in general, as the total output of antibodies secreted by clones increases, so does the likelihood that non-IgM isotypes will be expressed. In Fig. 2, the incidence of clones with a given output of antibody, measured by RIA with anti-Fab and given as cpm in 1OOOcpm intervals over the range of 10’ to >30 x lo’, is plotted for supernatants from the same assay which only contained IgM or which also contained another isotype. Since almost all of these clones express some IgM, a similar correlation was sought and found based on the concn of IgM secreted (data not shown). Both correlation analyses show considerable overlap in clones with similar outputs expressing either IgM alone or IgM plus other isotypes. However, clones expressing a low output of only IgM were the most common. The same data plotted in Fig. 2 are also expressed in Table 7 after being resolved into three separate sets according to the helper T cell dose used in the cultures. Again, incidence of positive cultures with a given content of antibody, determined by RIA and expressed in intervals of lo3 cpm, was plotted and compared for clones only making IgM vs those expressing an additional isotype. The median signal determined by RIA (median clonal output) given by the two types of clones is shown in Table 7 for cultures grown with different doses of T, cells. Table 7 shows that the relationship between high antibody output and switching clones holds regardless of the helper T cell dose or frequency of the response.

cloned

2h E3

IgM only Some IgM IgM + IgG IgG only IgA only

86 91 5 0 9

82 100 18 0 0

81 100 I9 0 0

86 97 10 3 0

86 93 7 7 0

Number of clones analyzed

22

I1

16

29

I4

“Approximately 30 PC-specific B cells were cultured with 35OWOOO cloned Hy-specific T cells and 7 x IO4 CBA/N x BALB/c F, male thymocytes per microculture well. No response was detected in the absence of antigen or T cell clones. PC-TGG-Hy was used at 0.1-10 pg/ml. Ten percent Con A supernatant was present in the medium. 6Approximately eight FLU-specific B cells were cultured with 4000 resting cloned Hy-specific T cells and 10’ CBA/N x BALB/c F, male thymocytes per microculture well. FLU-Hy was used at 50 “g/ml. No response was detected in the absence of T cells. Five percent Con A supernatant was presented in the medium.

specific T cell lines (see Table 4). From 7 to 19% of the clones scored expressed IgG antibodies, with or without IgM. Consistent with previous findings from experiments with unprimed FLU-specific B cells, IgA was not expressed by any of the clones scored. Due to the relatively low numbers of switched clones scored, this evidence is considered preliminary, but does seem to support our contention that a single specificity of T cell, in this case a carrier-specific Tn cell, is sufficient to initiate B cell clones that switch to the expression of IgG or IgA.

DEXUSSION

High clonal output is associated with increased expression oJ’ switching

We have investigated helper T cell participation in the expression of antibody isotypes by clones of hapten-specific B cells in vitro. Our results supported previous findings from our laboratory made using a modification of the splenic fragment assay based on athymic recipients which suggested that a single specificity of T cell is capable of supporting the expression of non-IgM isotypes by B cells (Kotloff et

We compared the concn of antibody secreted by clones with the diversity of isotypes expressed and their frequency of occurrence. For this analysis, we used anti-PC clones from cultures containing PCspecific B cells, Hy-specific T cells from lines, and CBA/N x BALB/c F, male thymocytes. It appears

Median.

IgM

only

clones

.

.

12

0

JOHN J. CEBRA

Median.

5

10

clones exprmsing

15

20

IgG or IgA

25

CPM, 1000 CPM intervals

Fig. 2. Distribution of output of total antibody by clones only secreting IgM compared with that of clones also secreting IgG and/or IgA. The cpm given by l/6 of the total antibody secreted by each clone, when tested by RIA and developed using radiolabeled anti-mouse Fab, is plotted vs the number of clones within an output interval of 1000 cpm.

Effect Table 7. Influence

of

T,-lines and clones on specific B cells

of T,-dose on frequency, incidence of “switching”, ‘antibody output of anti-PC clones Median

T, cell dose + 10’ Thy 0.75 x 10’ T, 1.5 x lO”T, 3.0 x IO’T, “Defined

W) Frequency 1.8 2.6 3.5

based on the intervals

% Switching 17 (6/35) 23 (1 l/47) 57 (34/60) of IO’cpm

al., 1987). Since the interpretation of results from experiments which employ splenic fragments is restricted by our limited understanding of the microenvironment within a fragment, as well as by its resistance to regulation, we developed an assay in which the participating cell populations could be well controlled. By using B cells enriched for antigen specificity on haptenated gelatin coated plates, we are able to culture very small numbers of B cells with helper T cells from lines or clones and obtain reasonable frequencies of cells responding to both Tindependent and T-dependent antigens. We obtained T-dependent B cell responses that were clonal and limited by the B cell input, as demonstrated by limiting dilution analysis. These B cells generated a high percentage (2863%) of clones that switched in tiitro from IgM expression to the expression of nonIgM isotypes. A number of B cell clones that were initiated by a cloned helper T cell line also expressed IgG or IgA, usually along with IgM. Our results support those of Leclercq et al. (1985) who showed that a single, antigen-specific Thl type helper T cell clone, in the presence of high antigen concns, could polyclonally activate unprimed B cells in bulk culture to proliferate and secrete antibodies, including IgG and IgA. Although IgM antibodies comprised the majority of the response, IgG and IgA were also secreted, even when the input B cell population consisted of sIgGor sIgA_ cells. Actual isotype switching within a B cell clone was not demonstrated, but the occurrence of a switch was inferred from the detection of IgG or IgA secreted from sIgG_ or sIgA_ cell populations. Since bulk cultures were used, the percentage of B cell clones that actually switched to secretion of non-IgM isotypes could not be determined, nor could the patterns of isotype expression within individual clones. The microculture assay has the advantage of providing this type of information. For example, in our studies presented in this report, a high proportion of clones expressed non-IgM isotypes, but almost always accompanied by IgM. Clones which expressed IgG or IgA in the absence of IgM were rarely scored, even though the hapten-specific input B cell population did contain these cells, as evidenced by their response in the splenic fragment assay. Such memory cells have in fact not been scored in any clonal in vitro assay to date, other than in the splenic fragment assay. B cells were sorted based on their surface immunoglobulin

153 and clonal

clonal output’

cpm 10 /~1/60 pl scored with anti-Fab* (IO-‘) Switched IgM only 6.5-7.5 8.5-9.5 5.54.5

12.5-13.5 14.>-15.5 17.5-18.5

shown in Fig. 2.

isotype on a fluorescence activated cell sorter (FACS) and used as donor cells in the splenic fragment assay. The results revealed that precursors of clones which express IgG isotype, with or without IgA but in the absence of IgM, bear mostly sIgG (Gearhart and Cebra, 1981). Similarly, clones from sIgA-bearing cells secreted exclusively IgA (Cebra et al., 1986), while IgA negative B cell precursors could produce clones which expressed IgA or mixtures of other isotypes. We believe that the clones making IgG and/or IgA isotypes without IgM arise from memory B cells that result from antigen-driven clonal expansion and switching in oivo. The memory B cells generated in this fashion are restricted in isotype potential or committed to the expression of a particular isotype, and have the potential to expand clonally when restimulated in vitro by specific antigen (Cebra et al., 1986). We are currently attempting to determine the in vitro requirements for the stimulation of clonal growth and secretion by these cells in microculture. The response observed in microculture in the absence of antigen may reflect a variable population of pre-activated B cells which mature under the influence of T cell differentiation factors alone (Andersson et al., 1980). Of greater concern to us is the apparent lack of evidence for a requirement for linked recognition of hapten and carrier for the generation of a clonal antibody response in microculture. In reported bystander responses, the helper T cell, following activation by specific antigen, releases factors which stimulate B cells to respond, regardless of their specificities. Such polyclonal stimulation of B cells by T cell lines, clones, or hybridomas has been previously reported (Augustin and Coutinho, 1980; DeFranco et al., 1984). The activation of resting B cells and their induction to IgM secretion was observed in bulk cultures of cells in the presence of high (100 p g/ml) concns of antigen for which the T cells were specific. Our responses in microculture required very low concns of antigen, below that which would cause polyclonal activation in bulk culture (DeFranc0 et al., 1984) yet we still see this effect in our microcultures, as did Hebbard et al. (1984). The geometry of a microculture well may provide a microenvironment especially supportive of bystander B cell activation. Ordinarily antigen-specific B cells would be preferentially stimulated when B cell activation and maturation factors are at limiting concns

154

DEBRA B. KOTLOFF and JOHN J. CEBRA

because antigen bridging may increase the likelihood that they will be close to the site of factor release, the T cells themselves. The local concn of factors may be high enough in a microculture well containing 10 ~1 of media to activate a portion of B cells nonspecifically. Bulk cultures, in contrast, contain large vols (200 ~1) and high numbers of extraneous B cells which may be capable of absorbing excess factors, and as a result may be limited in the concns of factors present. It is likely that we can reduce or eliminate the bystander response in microculture by adjusting the numbers of both B and T cells, as well as by testing T cell clones with varying lymphokine outputs. These experiments are in progress. An analysis of our data suggested a positive correlation between clone size as inferred from antibody output and the likelihood of isotype switching. If switching is division-dependent, then the more a clone divides, the larger its size and the more likely it is to undergo isotype switching. Another possibility is that clonal daughters that switch to IgG or IgA production may originate from clonal precursors that are highly responsive to growth and differentiation factors. Low output would represent those with fewer members reaching the secretory stage. Thus, such clones may display only the part of their potential limited to IgM. This variability in responsiveness may reflect the numbers of receptors for factors present on the surface of the clonal precursors and their progeny. Although refinement of the system is warranted, the virtues of the microculture assay seem evident. Most importantly, it can provide much needed information about isotype expression by B cell clones in vitro. Secondly, B cell clones in microculture are well suited for in vitro manipulation, such as studies to test the effect of the addition of cell supernatants, purified factors or particular cell subpopulations on the clonal output and isotypes expressed by individual, antigenspecific B cells. Since very few B cells are necessary for a response, small numbers of cells can be tested, such as uncommon cells expressing a certain phenotype and recoverable by FACS in only small numbers. Acknowledgements-We thank Ethel Cebra and Joan Taylor for expert technical assistance. The work described here was supported by a grant AI-17997 from the National Institutes of Health. D.B.K. was a VMSTP Trainee supported by grant GM-07170 from the National Institutes of Health. REFERENCES

Andersson J., Schreier M. H. and Melchers F. (1980) T-cell dependent B-cell stimulation is H-2 restricted and antigen dependent only at the resting B cell level. Proc. natn. Acud. Sci. U.S.A. 77, 1612-1616. Augustin A. A. and Coutinho A. (1980) Specific T helper cells that activate B cells polvclonallv. J. Exe,. Med. 151, _ _ 587601. Cebra J. J., Cebra E. R., Kotloff D. B., Lebman D. A., Schweitzer P. A. and Shahin R. D. (1986) C, isotype

switching in B cell differentiation. Biochem. Sot. Symp. 51, 1599172. DeFranco A. L., Ashwell J. D., Schwartz R. H. and Paul W. E. (1984) Polyclonal stimulation of resting B lymphocytes by antigen-specific T lymphocytes. J. exp. Med. 159, 861-880. Gearhart P. J. and Cebra J. J. (1979) Differentiated B lymphocytes. Potential to express particular antibody variable and constant regions depends on site of lymphoid tissue and antigen load. J. exp. Med. 149, 216227. Gearhart P. J. and Cebra J. J. (1981) Most B cells that have switched surface immunoglobulin isotypes generate clones of cells that do not secrete IgM. J. Immun. 127, 103&1034. Haas W. and Layton J. E. (1975) Separation of antigenspecific lymphocytes. I. Enrichment of antigen-binding cells. J. exp. Med. 141, 1004-1014. Hebbard G. S., Pike B. L. and Nossal G. J. V. (1984) Single-cell studies on hapten-specific B cells: response to T-cell dependent antigens. Proc. natn. Acad. Sci. U.S.A. 81, 2479-2483. Hun&z J. L., Tagart V. B., Schweitzer P. A. and Cebra J. J. (1982) Patterns of isotype expression by B cell clones responding to thymus-dependent and thymusindependent antigens in vitro. Eur. J. Immun. 12,342-348. Kawanishi H., Saltzman L. E. and Strober W. (1983) Mechanisms regulating IgA class-specific immunoglobulin production in murine gut-associated lymphoid tissues. I. T cells derived from Peyer’s patches that switch sIgM B cells to sIgA B cells in virro. J. exp. Med. 157, 4333450. Kimoto M. and Fathman C. G. (1980) Antigen-reactive T cell clones. I. Transcomplementing hybrid I-A region gene products function effectively in antigen presentation. J. exp. Med. 152, 759-770. Kishimoto T. and Ishizaka K. (1973) Regulation of antibody response in vitro. VI. Carrier-specific helper cells for IgG-and_IgE antibody response. J. immun. 111, 72&732. Kivono H., McGhee J. R.. Mosteller L. M.. Eldridae J. H.. i(oopman W. J., Kearney J. F. and Michalek S. M. (1982) Murine Peyer’s patch T cell clones. Characterization of antigen-specific helper T cells for immunoglobulin A responses. J. exp. Med. 156, I1 15-l 130. Kotloff D. B., Zitron I. M. and Cebra J. J. (1987) Carrierspecific T cells sufficient for the expression of multiple isotypes in B cell cultures. Eur. J. Zmmun. 17, 21-26. Leclercq L., Chastagnier P., Mishal Z., Bismuth G. and Theze J. (1985) Antigen-specific major histocompatibility complex-restricted helper T cell clones are sufficient to induce unprimed B cells to switch and to secrete IgG and IgA in a primary in vitro polyclonal response. Eur. J. Immun. 15, 1060-1066. Lee F., Yokota T., Otsuka T., Meyerson P., Villaret D., Coffman R., Mosmann T., Rennick D., Roehm N., Smith L., Zlotnik A. and Arai K.-I. (1986) Isolation and characterization of a mouse interleukin sDNA clone that expresses B cell stimulatory factor 1 activities and T-cell and mast-cell stimulating factors. Proc. natn. Acad. Sri. U.S.A. 83, 2061-2065. Nossal G. J. V., Pike B. L. and Battye F. L. (1978) Sequential use of hapten-gelatin fractionation and fluorescence-activated cell sorting in the enrichment of hapten-specific B lymphocytes. Eur. J. Zmmun. 8, 15 I-1 57. Pike B. L. and Nossal G. J. V. (1985) A hiah-efficiencv cloning system for single hapten-specific B lymphocytes that is suitable for assay of putative growth and differentiation factors. Proc. natn. Acad. Sci. U.S.A. 82, 3395-3399. Pike B. L., Vaux D. L. and Nossal G. J. V. (1983) Single cell studies on hapten-specific B lymphocytes: differential cloning efficiency of cells of various sizes. J. Zmmun. 131, 55&560. Riley R. L. and Klinman N. R. (1985) Differences in

Effect of T,-lines

and clones

antibody repertoires for (4-hydroxy-3-nitrophenyl) acetyl (NP) in splenic vs immature bone marrow precursor cells. .I. Immun. 135, 305(t3055. Schweitzer P. A. and Cebra J. J. (1988) Quality of antibodies secreted by clones in microcultures from B cells enriched on haptenated gelatin: isotypes and avidities. M&c. Immun. (submitted). Seman M., Zilberfarb V., Gougeon M. and Theze J. (1982) Functional analysis of GAT-specific T cell clones: H-2 restricted monoclonal T helper cells do not regulate expression of antibody isotype. J. Immun. 129, 217-222. Sredni B., Tse H. Y., Chen C. and Schwartz R. (1981) Antigen-specific clones of proliferating T lymphocytes. I. Methodology, specificity, and MHC restriction. J. Immun. 126, 341-347.

on specific B cells

155

Teale J. M. (1983) The use of specific helper T cell clones to study the regulation of isotype expression by antigenstimulated B cell clones. J. Immun. 131, 2170-2177. Vitetta E. S., Brooks K., Chen Y.-W., Isakson P., Jones S., Layton J., Mishra G. C., Pure E., Weiss E., Word C., Yuan D., Tucker P., Uhr J. W. and Krammer P. H. (1984) T cell-derived lymphokines that induce IgM and Igd secretion in activated murine B cells. Immun. Rev. 78, 137-157. Vitetta E. S., O’Hara J., Myers C. D., Layton J. E., Krammer P. H. and Paul W. E. (1985) Serologic, biochemical, and functional identity of B cell-stimulatory factor 1 and B cell differentiation factor for IgGl. J. exp. Med. 162, 17261731.