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Expression of Ia antigens on hapten-specific B cells. I. Delineation of B-cell subpopulations.

The nonimmune adult spleen contains at least two B-cell subpopulations. The majority of primary B cells express cell surface Ia determinants and have the capacity to give rise to IgG antibody-producing clones after T-cell dependent antigenic stimulation. There is also a small subpopulation of primary B cells which are, by definition, Ia negative, since their activity is not eliminated by negative selection with anti-Ia serum and complement. The Ia-negative B cells give rise to clones that produce only IgM antibody. These B-cell subsets may form a continuum in B-cell maturation, or they may exist as discrete B-cell lineages. Since the cellular expression of Ia antigens appears to correlate with the ability of the B cell to generate IgG-producing clones, it is speculated that Ia molecules may have a role in the IgM to IgG B-cell switch mechanism.

antigen, then these determinants may function as antigen recognition units (29).
The studies reported in this communication were initiated (a) to determine whether Ia antigens are expressed on all hapten-specific primary and secondary B cells; (b) to determine whether the expression of Ia antigens can be used as a marker to delineate primary B-cell subpopulations; and (c) to determine whether Ia antigens play a role in the antigenic stimulation of B cells, particularly with regard to T-cell-B-cell interactions. Spleen cells from nonimmune (primary) and previously immunized (secondary) donor mice were negatively selected by pretreatment with an anti-Ia antiserum and complement and then analyzed for precursor cell activity by an in vitro splenic focus technique for Bcell cloning (30) which maximizes carrier help for B-cell stimulation. The results of these experiments indicate that (a) the majority of primary precursor cells and almost every secondary precursor cell is Ia positive; (b) a finite subpopulation of Ia-"negative" primary precursor cells exists in the spleens of nonimmune mice; and (c) the Ia-negative primary B-cell subpopulation contains those precursor cells which give rise solely to IgM antibody production, whereas the Ia-positive B-cell subpopulation contains precursor cells capable of giving rise to IgG antibody production, or both IgG as well as IgM antibody, under appropriate stimulatory conditions. It has been demonstrated recently that a significant proportion of primary precursor cells can be stimulated in the carrier-primed allogeneic environment, but that only IgM antibody production is elicited. This effect has been mapped to the I-A-I-B subregions (11). Since the studies reported here demonstrate that the majority of primary precursor cells are Ia positive and have the capacity to give rise to IgG antibody production upon syngeneic T-cell interaction, these results suggest that Ia antigens may play a role in, or correlate with, the ability of the B cell to switch from IgM to IgG antibody production. The results also suggest that Ia recognitive interactions between T cells and B cells are not required for stimulation to IgM antibody production, even when B-cell stimulation is T-cell dependent.

Materials and Methods
Animals and Immunization. Breeding pairs of the congenic line BALB.K (H-2 k) were provided by Dr. Frank Lilly. This congenic line was maintained by brother-sister mating. Mice were carrier primed with 0.1 mg of Limulus polyphemus hemocyanin (Hyp in complete Freund's adjuvant (CFA) 2-3 mo before use as recipients for cell transfer. Donor spleen cells were obtained either from nonimmune mice (primary precursor cell analysis) or from mice immunized 2-4 mo previously with 0.1 mg of 2,4-dinitrophenylated Hy (DNP-Hy; i0 mol of DNP per 100,000 g of Hy) (31) in CFA (secondary precursor cell analysis).
Irradiation. Carrier-primed (Hy) recipient mice were lethally irradiated 6 h before cell transfer with 1,100-1,300 R from a Phillips X-ray generator, at 15 mA and 250 kV, with a corrected 0.25 mm Cu/1.0 mm Al filter, half value layer (HVL)-I.3 mm Cu, delivering 62 R/rain, at a distance of 64 cm to midmouse.
Fresh frozen rabbit serum was used as the complement (C') source. Rabbit serum was adsorbed with agarose, and the complement activity and nonspecific toxicity screened before use by methods described previously (12,13).
100 × 106 spleen cells were pelleted in plastic Falcon tubes (Falcon Plastics, Oxnard, Calif.), resuspended in 1 ml of NMS or anti-Ia antiserum diluted 1/5 in PBS-FCS, and incubated for 25 rain at 4°C. After washing in PBS-FCS, the pretreated spleen cells were then incubated in 3-4 ml of adsorbed rabbit complement, diluted 1/8-1/12 in Dulbecco's minimal essential medium (MEM) (GIBCO) for 45 rain in a 37~C, 95% air-5% COs aerated incubator. After C' incubation, the cell suspensions were immediately placed on ice, and the percent cytotoxicity was measured by dye exclusion. The cells were washed three times in MEM and adjusted to a cell concentration of 1-2 × 10 ~ viable cells/ml. Lethally irradiated, carrier-primed recipients received 1-2 × 10 ~ viable, pretreated cells intravenously (i.v.). The number of total donor cells injected was calculated on the basis of the final cell count measured by dye exclusion before cell injection.
Fragment Cell Culture and Radioimmunoassay. The frequency of hapten-specific clonal precursor cells was measured by an in vitro splenic focus technique for B-cell cloning. The methodology of this technique and the detection of positive (anti-hapten antibody producing) fragment cultures have been described previously (30). 14-16 h after cell transfer, recipient spleens were sliced into l-ram cubes, placed in vitro in organ fragment culture, and stimulated for 3 days with 1 × 10 -8 M DNP-Hy. Culture fluids were changed and collected at 2-to 3-day intervals thereafter.
The production of anti-DNP monofocal antibody by the splenic foci was determined by a sensitive, relatively affinity-independent radioimmunoassay using a DNP-lysine-conjugated-bromoacetyl cellulose immunoadsorbent and l~SI-labeled purified rabbit anti-mouse Fab antibody (30,31). The heavy chain class of anti-DNP monofocal antibody produced by each clone was also determined by the radioimmunoassay using iodinated, purified goat antibodies against either the mouse ~-or ~l-heavy chain class (30,33).

Results
The frequency of DNP-specific precursor cells in the spleens of nonimmune and DNP-Hy immunized adult BALB.K mice was quantitated after pretreatment either with A.TH N'MS or with A.TH anti-A.TL antiserum (anti-Ia k) and complement. Limiting numbers (1-2 × 106 viable cells per recipient) of pretreated spleen cells were injected into lethally irradiated, carrier (Hy)-primed, syngeneic recipients. No primary foci were detected after transfer of either NMS or anti-Ia k and complement-pretreated cells into lethally irradiated, normal syngeneic recipients. This is consistent with previous findings that carrierprimed recipients are necessary for the induction of primary B-cell clones by DNP-Hy (30,34).
After pretreatment with NMS and complement, the average frequency of BALB.K primary precursor cells specific for DNP ranged from 6-10 foci per 10 ~ total injected cells, with a mean frequency of 7.64 foci per 10 ~ cells (Table I). This frequency is similar to that obtained with untreated, nonimmune spleen cells (data not shown). When the spleen cells were pretreated with anti-Ia k and complement, however, the mean precursor cell frequency was lowered to 0.94 foci per 106 total injected cells, a reduction of 88%. While the majority of DNPspecific primary B cells are thus Ia positive, a small amount of primary precursor cell activity does remain after negative selection. Pretreatment of nonimmune spleen cells with the reciprocal antiserum, A.TL anti-A.TH (anti-IaS), does not reduce the precursor cell frequency (Table II); this is expected, since the H-2 k and H-2 s haplotypes do not share cross-reacting Ia specificities when antisera produced by reciprocal immunization are used (2,14). Table II also demonstrates that this negative selection procedure is complement dependent.
The results of three experiments in which spleen cells from BALB.K mice previously immunized to DNP-Hy in CFA were treated with anti-Ia k and complement, and subsequently analyzed for precursor cell activity, are presented in Table III. The mean frequency of NMS-treated DNP-specific secondary precursor cells is 13.46 foci per 106 injected cells. This frequency was reduced to 0.59 per 106 cells after negative selection, a reduction of 96%. Hence, secondary precursor cells can be almost totally eliminated.
The monofocal anti-DNP antibodies produced by the primary and secondary clones presented in Tables I and HI were analyzed for their isotype (heavy chain class) by radioimmunoassay. The distribution of primary and secondary clones producing IgM, IgG~, or beth IgM and IgG~ anti-DNP antibody is shown in Table  IV. After pretreatment of the donor spleen cells with NMS and complement, an average of 72% of the clones produced only IgG1 antibody; 14% produced only IgM antibody; and 14% produced both IgM and IgG1 antibody (double producers). Less than 10% of the clones dete~ed by 125I-anti-Fab antibody produced anti-DNP antibody which was neither of the /~-or y~-heavy chain class. This isotype distribution of anti-DNP monofocal antibodies produced by BALB.K primary clones is similar to that observed for BALB/c clones (33). Primary clones derived from spleen cells pretreated with anti-Ia k and complement gave an isotype distribution markedly altered from that of the NMS and complement control group. An average of 78% of the clones generated from the remaining primary B cells produced solely IgM anti-DNP antibody; 18% produced IgG1 antibody; and 4% produced both IgM and IgG1 antibody. The secondary monofocal antibody isotype distribution is distinct from that of primary monofocal antibodies: an average of 2% of secondary clones produced solely IgM anti-DNP antibody; the other clones produced either IgG1 or both IgM and IgG~ antibody. After negative selection, however, the majority of clones derived from the secondary spleen cell population produced solely IgM antibody.
The selective survival of purcursor cells which give rise to solely IgM antibody-producing clones is further illustrated in Fig. 1 and 2. From the data presented in Tables I, HI, and IV, the frequency per 106 total injected cells of primary and secondary precursor cells giving rise to clones producing antibody of the/~, ~1, or beth/~ and Y~ heavy chain class was calculated. It is apparent that the reduction in the total primary precursor cell frequency is correlated with the marked reduction (>95%) in the mean frequency of primary B cells giving rise to IgG~ antibody-producing clones (Fig. 1). In contrast, on a total cell basis, the mean frequency of primary precursor cells which give rise to solely IgM antibody-producing clones is only slightly lowered (20-30%). The precursor cell contribution to the total primary IgM response is reduced by pretreatment with anti-Ia k and complement, since the contribution to total IgM antibody by clones producing both IgM and IgG1 antibody has been depleted (>90%) as well. Pretreatment of secondary spleen cells reduced the frequency of secondary B cells giving rise to IgG1 antibody-producing clones by 95% and the frequency of secondary B cells giving rise to both IgG1 and IgM antibody-producing clones by 100% (Fig. 2). The low frequency of precursor cells in the secondary spleen cell population giving rise to only IgM antibody-producing clones was not affected.
Two "restricted" Ia antisera were also analyzed for their ability to reduce the DNP-specific primary precursor cell response. Spleen cells from BALB.

The studies presented here were conducted to determine whether Ia antigens are expressed on DNP-specific B cells and whether the expression of Ia determinants can be used to delineate B-cell subpopulations in the spleens of nonimmune and previously immunized mice. To measure the relevance of Ia expression to specific precursor cell stimulation on a cell to cell basis, and under conditions of T-cell excess, the experimental approach employed negative selection with specific anti-Ia serum and complement and an in vitro splenic focus technique (30) for B-cell cloning. The splenic focus technique allows the enumeration of clonal precursor cells in the population with and without negative selection and permits an analysis of effects of such selection at the level of the individual B cell and its clonal progeny.
It has been demonstrated using this splenic focus technique that the limiting cell for focus formation is the B cell, and that the monofocal antibody produced is derived from the clonal progeny of a single stimulated precursor cell (30,34).   Tables I and III for details.  DNP-specific clones from Tables I and III analyzed for heavy chain class by radioimmunoaseay. § Fab refers to clones detected by 125I rabbit anti-mouse Fab which are not detected by either iodinated anti-~ or anti-T1 antibody; in general, such clones represent <10% of the total clones.
The majority of splenic foci produce antibody that appears homogeneous by the criteria of heavy and light chain recombination analysis, equilibrium dialysis, idiotypic analysis, and isoelectric focusing (30,34,35). The only exception to the evidence for the homogeneity of the monofocal antibody produced by these clones has been the demonstration that a significantly greater than random frequency of individual clones produce monospecific anti-DNP antibody in beth the ft-and 7rheavy chain classes (33). The occurrence of double producers and the implication that the clonal progeny of a single stimulated precursor cell may produce antibody in more than one heavy chain class specific for a single determinant has recently been confirmed by the studies of Gearhart et al. (35), who demonstrated that monospecific, antiphosphorylcholine antibodies from the same clone were of more than one heavy chain class, yet share the same specificity region by idiotypic analysis.    The expression of Ia antigens thus appears to define two subsets of primary B cells. The majority of primary B cells are Ia positive and have the potential to generate IgG-producing progeny. There is also a small subpepulation of B cells which is by definition Ia negative. These B cells generate only IgMoproducing clones under the conditions employed in these studies. Ia-positive and Ianegative B cells can therefore be demarcated by their clonal phenotype. Two other pieces of evidence indicate that Ia-negative B cells exist as a discrete subset: (a) when nonimmune spleen cells are separated by immunofluorescence on the cell sorter, significant precursor cell activity can be detected in the Ianegative cell population (J. Press, unpublished observations); (b) analysis by size fractionation on 1 g velocity sedimentation gradients indicates that the Ianegative B cells which give rise to only IgM-producing clones are found predominantly in the large cell fractions, whereas both the large and small cell fractions contain Ia-positive primary B cells (37).

Expression of la Antigens on B-Cell
The interrelationship of the Ia-positive and Ia-negative B-cell subsets is not clear. A continuum may exist within the nonimmune adult spleen, where Ianegative (and possibly Ig negative) B cells differentiate into Ia-pesitive cells. In this single pathway model, the B cell would acquire the capacity to switch to IgG production concomitant with the expression of Ia, and the expression of Ia molecules could be used as a marker to follow B-cell maturation. Alternatively, the Ia-positive and negative B-cell subsets may constitute separate, parallel lineages. The Ia-negative B-cell subset would exist in low frequency in the adult spleen and would not diferentiate into Ia-positive cells. Preliminary findings indicate that the IgM-producing clonal progeny of Ia-negative B cells do not express Ia antigens during the in vitro culture period (J. Press and N. Klinman, unpublished observations). It is not clear whether Ia-negative B cells are capable of making IgG antibody but do not due to the lack of Ia determinant expression, or whether such cells are intrinsically capable of producing only IgM antibody upon stimulation. It will be of interest to determine whether the Iapositive and Ia-negative B-cell subsets share the same repertoire of antibody clonotypes and express the same requirements for stimulation, particularly with regard to T-cell dependent versus T-cell independent antigens.
Inasmuch as the expression of Ia antigens on the B cell correlates with the ability of that cell to give rise to IgG production, Ia molecules may have a role in the specific induction of the B-cell switch mechanism from IgM to IgG production. Indirect support for this speculation comes from recent findings of Pierce and Klinman (11) that 60-70% of primary B cells can be stimulated in allogeneic, carrier-primed recipients. This stimulation does not occur if the allogeneic recipient is not carrier primed. When the donor and recipient haplotype combinations were selected such that there was genetic nonidentity only in the I-A and I-B subregions, then antigen-specific stimulation yielded clones producing solely IgM anti-DNP antibody. In contrast, if there were background differences only, or differences in the H-2D end, then IgG antibody-producing clones were obtained (11). These findings have been confirmed using several congenic and recombinant strain combinations, but there may be preferred H-2 haplotype combinations for optimal stimulation (J. Press and N. Klinman, unpublished observations).
Thus, gene products affecting the ability of B-cell-T-cell interactions to lead to IgG production map in the I region (7)(8)(9)(10)(11). However, Ia recognitive interactions between B cells and T cells do not appear obligatory for B-cell triggering per se, since Ia-negative cells can be specifically stimulated to IgM antibody production. These data suggest that the Ia-positive primary B cell can give rise either to IgM or to IgG production, depending on whether there is/-region identity between the B cell and T cell. This is compatible with the hypothesis that Ia antigens are involved in the B-cell induction to IgG production. The requisites for/-region identity between B cells and T cells for IgG production may be more stringent for primary B cells than for secondary B cells (38; J. Press, unpublished observations). In situations where IgG antibody is elicited in histoincompatible T-cell-B-cell interactions, e.g. tetraparental mice (5) or bone marrow chimeras (39), this could be due either to tolerance of alloreactive T cells or to the learned recognition of both parental haplotype cell surface/-region antigens as a consequence of in situ immunization.
It is clear that the expression of Ia molecules on B cells can be used to classify at least two DNP-specific primary B-cell subpopulations in the spleens of adult mice. At issue is whether Ia expression is a B-ceU differentiation event in a common lineage pathway and whether Ia antigens per se have any functional role in the IgM to IgG switch mechanism. The cellular expression of Ia molecules may constitute an inherent molecular mechanism for the regulation of Bcell stimulation. This regulatory role of Ia determinants could be quite distinct from the mechanism of action and antigen specificity of Ir genes. Experiments are in progress to resolve these issues.

Summary
The nonimmune adult spleen contains at least two B-cell subpopulations. The majority of primary B cells express cell surface Ia determinants and have the capacity to give rise to IgG antibody-producing clones afLer T-cell dependent antigenic stimulation. There is also a small subpepulation of primary B cells which are, by definition, Ia negative, since their activity is not eliminated by negative selection with anti-Ia serum and complement. The Ia-negative B cells give rise to clones that produce only IgM antibody. These B-cell subsets may form a continuum in B-cell maturation, or they may exist as discrete B-cell lineages. Since the cellular expression of Ia antigens appears to correlate with the ability of the B cell to generate IgG-producing clones, it is speculated that Ia molecules may have a role in the IgM to IgG B-cell switch mechanism.