The relationship between surface immunoglobulin isotype and immune function of murine B lymphocytes II. Surface immunoglobulin isotopes on unprimed B cells in the spleen

We investigated the ability of IgM-, IgD-, and IgG-bearing cells from the spleens of unprimed (BALB/c x C57BL/Ka)F1 mice to restore the adoptive primary anti-BSA and anti-DNP antibody responses. Purified populations of isotype-specific cells were prepared by immunofluorescent staining and sorting on the fluorescence activated cell sorter. Bright or dull cells were transferred to irradiated syngeneic recipients which were challenged with DNP-BSA in complete Freund's adjuvant. Unfractionated spleen cells as well as IgM- and IgD-bearing cells restored the adoptive primary IgM and IgG antibody response. IgG-bearing cells restored a vigorous adoptive response which was all IgG (2-mercaptoethanol resistant). Depletion of IgG-bearing cells markedly increased the adoptive IgM response, and depletion of IgM-bearing cells markedly increased the IgG response. However, depletion of IgD-bearing cells resulted in a considerable reduction in the IgG response. The latter finding indicates that there is a subpopulation of IgD-bearing cells which express little or no surface IgM and which make a considerable contribution to the adoptive primary IgG response.     

The relationship between surface immunoglobulin isotype and immune function of murine B lymphocytes. I. Surface immunoglobulin isotypes on primed B cells in the spleen

We investigated the ability of IgM-, IgD-, and IgG-bearing cells from the spleens of (BALB/c x C57BL/Ka)F1 mice primed to dinitrophenyl-bovine serum albumin (DNP-BSA) to restore the adoptive secondary anti-BSA and anti-DNP antibody responses. A rabbit anti-mouse IgD antiserum was prepared and the specificity documented by radioimmunoprecipitation, and cell surface staining. Purified populations of IgM-, IgD-, and IgG-bearing cells were prepared by immunofluorescent staining with isotype-specific reagents, and sorting on the fluorescence activated cell sorter. Bright or dull cells were transferred to irradiated syngeneic recipients which were challenged with DNP-BSA in saline. Unfractionated spleen cells restored an adoptive secondary serum antibody response which was all IgG (2-mercaptoethanol resistant). Purified IgM- or IgD-bearing cells restored both the secondary IgM and IgG antibody response. IgG-bearing cells restored only the IgG response. In addition, the IgG-bearing cells appear to suppress the adoptive secondary IgM response, since depletion of IgG-bearing cells from transferred spleen cells results in a marked increase in the adoptive IgM response.     

B-cell tolerance. IV. Differential role of surface IgM and IgD in determining tolerance susceptibility of murine B cells

During ontogeny IgD appears later than IgM on splenocytes of neonatal mice (1) and at a time when mice develop a markedly increased immune responsiveness (2). Based on these observations, it was suggested that IgD serves as a “triggering” isotype for induction of immune responses, whereas surface IgM functions as a tolerizing receptor (3). To test this hypothesis, the susceptibility of adult splenocytes (which are predominantly μ(+)δ(+)[4-6]) and neonatal splenocytes (which bear predominantly IgM [μp(+); 1, 4-6]) to tolerance induction were compared. The results indicate that neonatal splenic B cells responsive to thymus dependent (TD) antigens are exquisitely susceptible to tolerance induction compared with those from adult mice (7-9). However, cells from both adult and neonatal mice were highly susceptible to tolerance induction when thymus independent (TI) antigen was used as immunogen (8). These results suggest that the major precursor for the TD response is a μ(+)δ(+)-cell which appears late in ontogeny and is resistant to tolerance induction and that the μp(+)-cell is the major precursor for the TI response and is highly susceptible to tolerance induction. Other differences between responders for TI and TD antigens have been described previously (10-12). To test this concept, adult splenocytes were treated with papain under conditions in which IgD, but not five other surface molecules, was removed (13). Such treated splenocytes were shown to be markedly susceptible to tolerance induction, resembling TD responders from neonatal animals. This experiment was interpreted as indicating that IgD confers resistance to tolerance induction on μ(+)δ(+)-cells. To prove this interpretation, it is necessary to show that specific removal of IgD with anti-δ also results in increased susceptibility to tolerance induction and that treatment with anti-μ does not have a similar effect. In the present studies, we have removed surface IgM or IgD by antibody-induced capping and assessed the tolerance susceptibility of the treated cells. Our results demonstrate that removal of IgD, but no IgM, from TD responders increases their susceptibility to tolerance induction.     

IMMUNOLOGICAL MEMORY IN MICE : III. MEMORY TO HETEROLOGOUS ERYTHROCYTES IN BOTH T CELL AND B CELL POPULATIONS AND REQUIREMENT FOR T CELLS IN EXPRESSION OF B CELL MEMORY. EVIDENCE USING IMMUNOGLOBULIN ALLOTYPE AND MOUSE ALLOANTIGEN THETA MARKERS WITH CONGENIC MICE

Using anti-allotype sera and AKR anti θC3H sera, a requirement for two cell types has been demonstrated in the adoptive secondary response of mice to heterologous erythrocytes. The cell types have been designated B cells [precursors of plaque-forming cells (PFC)] and T cells (thymus-influenced cells, not providing precursors of detectable PFC). The in vivo indirect PFC response of spleen cells from primed mice is markedly reduced by in vitro treatment of the cells with a mixture of anti-θ serum and guinea pig serum (Anti θ + GPS). This B cell response is fully restored to control levels by thymus cells from normal mice which do not themselves provide precursors of indirect PFC. Thus memory is carried by the B cell lineage but the expression of this memory is dependent on the presence of a cell population which is sensitive to Anti θ + GPS and which is replaced functionally by unprimed T cells. When assayed for T cell activity, thoracic duct cells from specifically primed mice are better than cells from nonspecifically primed mice in restoring the B cell response of spleen cells from immunized mice. Moreover, the T cell activity of a reconstitutive cell population from primed mice is reduced by incubation with Anti θ + GPS. We conclude that memory to heterologous erythrocyte antigens is carried by the T cell lineage as well as the B cell lineage even though unprimed T cells are sufficient for expression of B cell memory.     

A dichotomy between the expression of IgD on B cells and its requirement for triggering such cells with two T-independent antigens

The majority of adult B lymphocytes in the mouse bear two immunoglobulin isotypes, IgM and IgD (μ(+)δ(+) cells) (1). A small population of IgM-bearing cells lacks, or expresses very low levels of IgD (μ- predominant [μp] cells) (1). These cells are believed to constitute a less mature subset of B cells analogous to neonatal B cells (2). Based on the time during ontogeny when responses to T-independent (TI) and T-dependent (TD) antigens appear (3, 4) and the ability to block in vitro responses with anti- μ or anti-δ (5, 6, D. Mosier, personal communication), it has been suggested that the precursors of two TI-1 responses, trinitrophenyl (TNP)- Brucella (TNP-BA) and TNP-lipopolysaccharide (TNP-LPS) are μp cells (5, 6), whereas the precursor for a TD response, TNP-sheep erythrocytes (TNP-SRBC), bears both IgM and IgD (6). However, the possibility cannot be excluded that IgD is present on some or all of the TI precursors, but that it is not obligatory for triggering. In the present experiments we have examined the phenotypes of TI and TD precursors by treating cells with C’ and either anti-μ or anti-δ before stimulation with antigen. Our results suggest that the majority of B cells that respond to TNP-BA, TNP-LPS, and TNP-SRBC bear IgD, even though in the case of the two TI antigens, IgD is not required for triggering.     

Expression of IgD by murine lymphocytes. Loss of surface IgD indicates maturation of memory B cells

B lymphocytes capable of generating primary IgM and IgG plaque-forming cells (PFC) responses to burro erythrocytes have surface IgD, as do primary IgM PFC. IgG memroy cells arising after one injection of antigen are divided into two groups, one of which expresses surface IgD while the other has no detectable membrane IgD. PFC generated from the IgG memory cells lacking surface IgD show a higher average avidity than those arising from IgD-positive IgG memory cells, indicating that mature IgG memory cells do not have surface IgD. After more than one injection of antigen, few, if any, IgG memory cells have surface IgD. IgG PFC arising in primary or secondary immune response lack membrane-bound IgD. These data provide the outlines for a B-cell maturation pathway in which IgD marks unprimed and early memory B cells and is lost in mature memory cells. Studies presented here were conducted by isolating IgD+ and IgD- cells with the fluorescence-activated cell sorter and functional testing of the isolated populations in adoptive transfer experiments.     

Cell surface immunoglobulin. XX. Antibody responsiveness of subpopulations of B lymphocytes bearing different isotypes

Murine spleen cells were depleted of specific B-cell subpopulations bearing different immunoglobulin isotypes by means of complement-mediated cytolysis after treatment with antisera specific for micron- and gamma-chains. The functional effect of this depletion was measured by assaying both the primary and secondary plaque-forming cell responses of the residual cells after transfer to carrier-primed lethally irradiated hosts. The results suggest that cells bearing IgM are the progenitors of plaque-forming cells in the primary response and cells bearing IgG are the major progenitors of IgG plaque-forming cells in the secondary response. The quantity of IgM on progenitors of secondary IgM plaque-forming cells decreases markedly as the interval between primary immunization and antigenic challenge increases. Long-term memory cells for the secondary IgM response bear small amounts of both IgM and IgG.     

Reduced isotype switching in splenic B cells from mice deficient in mismatch repair enzymes.

Mice deficient in various mismatch repair (MMR) enzymes were examined to determine whether this repair pathway is involved in antibody class switch recombination. Splenic B cells from mice deficient in Msh2, Mlh1, Pms2, or Mlh1 and Pms2 were stimulated in culture with lipopolysaccharide (LPS) to induce immunoglobulin (Ig)G2b and IgG3, LPS and interleukin (IL)-4 to induce IgG1, or LPS, anti-delta-dextran, IL-4, IL-5, and transforming growth factor (TGF)-beta1 to induce IgA. After 4 d in culture, cells were surface stained for IgM and non-IgM isotypes and analyzed by FACS((R)). B cells from MMR-deficient mice show a 35-75% reduction in isotype switching, depending on the isotype and on the particular MMR enzyme missing. IgG2b is the most affected, reduced by 75% in Mlh1-deficient animals. The switching defect is not due to a lack of maturation of the B cells, as purified IgM(+)IgD(+) B cells show the same reduction. MMR deficiency had no effect on cell proliferation, viability, or apoptosis, as detected by [(3)H]thymidine incorporation and by propidium iodide staining. The reduction in isotype switching was demonstrated to be at the level of DNA recombination by digestion-circularization polymerase chain reaction (DC-PCR). A model of the potential role for MMR enzymes in class switch recombination is presented.     

MATURATION OF B LYMPHOCYTES IN THE RAT : I. MIGRATION PATTERN, TISSUE DISTRIBUTION, AND TURNOVER RATE OF UNPRIMED AND PRIMED B LYMPHOCYTES INVOLVED IN THE ADOPTIVE ANTIDINITROPHENYL RESPONSE

The migration pattern, tissue distribution, and turnover rate of unprimed and primed B lymphocytes involved in the adoptive anti-DNP response was studied. The adoptive primary response restored by unprimed spleen or thoracic duct cells passaged through an intermediate host (intravenous injection and subsequent collection in the thoracic duct lymph) was markedly diminished as compared with that restored by unpassaged cells. On the other hand, the adoptive response restored by passaged spleen or thoracic duct cells from DNP-primed donors was greater than or the same as that restored with unpassaged cells, respectively. This suggests that unprimed B cells change from nonrecirculating to recirculating lymphocytes after exposure to antigen. Studies of the adoptive anti-DNP response restored by unprimed or primed bone marrow cells showed little change in the time-course or amplitude of the response restored by either population of cells. The relative inability of marrow cells to carry immunological memory was related to the inability of recirculating memory cells to penetrate the marrow. The turnover rate of unprimed and primed B cells was investigated by treating the cell donors with [³H]thymidine for 48 h before removal of thoracic duct or spleen cells. The adoptive anti-DNP response restored by unprimed or primed cells was not affected by [³H]thymidine treatment. This indicates that both populations of cells turn over slowly. However, our previous studies show that unprimed B cells involved in the adoptive antibody response to ferritin turn over rapidly. The different findings are discussed in the context of antigen-dependent B-cell maturation.     

Generation of Splenic Follicular Structure and B Cell Movement in Tumor Necrosis Factor–deficient Mice

Secondary lymphoid tissue organogenesis requires tumor necrosis factor (TNF) and lymphotoxin α (LTα). The role of TNF in B cell positioning and formation of follicular structure was studied by comparing the location of newly produced naive recirculating and antigen-stimulated B cells in TNF^−/− and TNF/LTα^−/− mice. By creating radiation bone marrow chimeras from wild-type and TNF^−/− mice, formation of normal splenic B cell follicles was shown to depend on TNF production by radiation-sensitive cells of hemopoietic origin. Reciprocal adoptive transfers of mature B cells between wild-type and knockout mice indicated that normal follicular tropism of recirculating naive B cells occurs independently of TNF derived from the recipient spleen. Moreover, soluble TNF receptor–IgG fusion protein administered in vivo failed to prevent B cell localization to the follicle or the germinal center reaction. Normal T zone tropism was observed when antigen-stimulated B cells were transferred into TNF^−/− recipients, but not into TNF/LTα^−/− recipients. This result appeared to account for the defect in isotype switching observed in intact TNF/LTα^−/− mice because TNF/LTα^−/− B cells, when stimulated in vitro, switched isotypes normally. Thus, TNF is necessary for creating the permissive environment for B cell movement and function, but is not itself responsible for these processes.     

CELL-TO-CELL INTERACTION IN THE IMMUNE RESPONSE : X. T-CELL-DEPENDENT SUPPRESSION IN TOLERANT MICE

Specific immunological tolerance was induced in CBA mice by a single injection of deaggregated fowl immunoglobulin G (FγG). The unresponsive state was stable on adoptive transfer and irreversible by pretreatment of tolerant cells with trypsin. Tolerant spleen cells could suppress the response of normal syngeneic recipients. They also suppressed the adoptive primary response of spleen cells to FγG in irradiated hosts. The inhibitory effect was on the indirect (7S) plaque-forming cell (PFC) response. Incubation of the tolerant cell population with anti-θ serum and complement reversed the suppressor effect. Furthermore, the addition of purified T cells from normal donors restored the capacity of the anti-θ serum-treated tolerant cells to transfer an adoptive response to FγG. The existence of FγG-reactive B cells was supported by the demonstration of normal numbers of antigen-binding cells in the spleen and thoracic duct lymph from tolerant animals. Moreover, the formation of caps by these cells implied that they could bind antigen normally. These experiments provided direct evidence for the existence of suppressor T cells in the tolerant population. Further evidence was derived from examination of the effect of antigen "suicide". Tolerant spleen cells were treated with radioactive FγG under conditions known to abrogate T-cell helper function. When these cells were transferred together with normal spleen cells into irradiated hosts, suppression of the primary adoptive response to FγG was no longer observed. Inhibition of an adoptive secondary response to FγG was obtained by transferring tolerant spleen cells with primed B cells provided high doses of tolerant cells were used. By contrast low doses exerted a helper rather than a suppressor effect in this system.     

Human B lymphocyte subsets. I. IgG-bearing B cell response to pokeweed mitogen

The subset of B lymphocytes having IgG on their surfaces was purified from human spleen and blood using a fluorescence-activated cell sorter (FACS). This subset constituted about 15% of B lymphocytes. The remaining non-IgG-bearing B cells were also obtained for study. These two populations were examined for (a) their expression of other surface immunoglobulin isotypes, (b) their ability to give rise to IgG- and IgM- secreting (plaque-forming) cells in a pokeweed mitogen (PWM)-driven culture system, and (c) their ability to proliferate in response to PWM stimulation. The results of these studies indicate that most IgG- bearing B cells also express surface IgM and IgD. Less than 15% had only IgG. The IgG-positive cell gave rise to both IgG and IgM plaque- forming cells when driven by PWM, and in fact were responsible for most of the total plaque response in both the IgG and IgM classes. The non- IgG-bearing B cells were depleted of both IgG and IgM responsiveness. The failure of the non-IgG-bearing B cells to give a strong response to PWM did not appear to be due to either depletion of accessory cells or to any suppressive influence. Finally, proliferation studies indicated that both the IgG-bearing and the non-IgG-bearing cells proliferated in the presence of PWM with a somewhat stronger proliferative response in the IgG-bearing cells. These results demonstrate that the IgG-bearing cell is not irreversibly committed to IgG production but can also give rise to IgM-secreting cells, and that human PWM-driven immunoglobulin secretory responses are predominantly due to a numerically small subset of B cells.     

Double isotype production by a neoplastic B cell line. I. Cellular and biochemical characterization of a variant of BCL1 that expresses and secretes both IgM and IgG1

We have subcloned the in vitro-adapted murine B cell leukemia, BCL1.B1, to obtain a variant that expresses both IgM and IgG1. By fluorescence analysis, radioiodination, and immunoprecipitation of cell surface Ig, and by RIA of medium from limiting dilution cultures, we have shown that: (a) all the cells express and secrete both isotypes. The heavy chains of both IgG1 and IgM have the apparent molecular weights of membrane mu and gamma 1 chains; (b) both isotypes bear the same idiotype as determined by immunoprecipitation with antiidiotypic antibody, and both use the same VDJ rearrangement as shown by Southern blotting; and (c) the cells express the membrane and secreted forms of mRNA for both mu and gamma 1 but not gamma 2b or gamma 3. Taken together, the data suggest that all the cells are synthesizing, expressing on their surface, and secreting two isotypes that use the same VDJ rearrangement in the DNA and express the same serologically-defined idiotype. The molecular basis responsible for the production of the two isotypes in a single cell is the subject of the accompanying paper.     

Human Dendritic Cells Skew Isotype Switching of CD40-activated Naive B Cells towards IgA1 and IgA2

Within T cell–rich areas of secondary lymphoid organs, interdigitating dendritic cells recruit antigen-specific T cells that then induce B cells to secrete Igs. This study investigates the possible role(s) of dendritic cells in the regulation of human B cell responses. In the absence of exogenous cytokines, in vitro generated dendritic cells (referred to as Dendritic Langerhans cells, D-Lc) induced surface IgA expression on ~10% of CD40-activated naive sIgD⁺ B cells. In the presence of IL-10 and TGF-β, a combination of cytokines previously identified for its capacity to induce IgA switch, D-Lc strongly potentiated the induction of sIgA on CD40-activated naive B cells from 5% to 40–50%. D-Lc alone did not induce the secretion of IgA by CD40-activated naive B cells, which required further addition of IL-10. Furthermore, D-Lc skewed towards the IgA isotype at the expense of IgG, the Ig production of CD40-activated naive B cells cultured in the presence of IL-10 and TGF-β. Importantly, under these culture conditions, both IgA₁ and IgA₂ were detected. In the presence of IL-10, secretion of IgA2 by CD40-activated naive B cells could be detected only in response to D-Lc and was further enhanced by TGF-β. Collectively, these results suggest that in addition to activating T cells in the extrafollicular areas of secondary lymphoid organs, human D-Lc also directly modulate T cell–dependent B cell growth and differentiation, by inducing the IgA isotype switch.     

SURFACE ANTIGENS OF IMMUNOCOMPETENT CELLS : I. EFFECT OF ϑ AND PC.1 ALLOANTISERA ON THE ABILITY OF SPLEEN CELLS TO TRANSFER IMMUNE RESPONSES

Spleen cell transfer studies were done in BALB/c strain mice in an attempt to define the role of θ-antigen-bearing lymphoid cells in immune responses to SE. Incubation with alloantiserum to θ-C3H and rabbit C' virtually completely abolished the ability of the cells to transfer both primary and secondary (IgM and IgG) responses to 650 R irradiated recipients. Normal thymus cells partially reconstituted the ability of such treated cells to transfer the primary but not the secondary response. The results are interpreted as showing immunological memory for SE in the θ-bearing thymus-derived cells. Incubation of the spleen cells with alloantiserum to the PC.1 antigen present on antibody-forming cells did not significantly affect the ability to transfer either primary or secondary response.     

Antigen specific receptors on murine B cell lineage. 2. Different effect of rosette-forming cell depletion on IgM and IgG antibody forming cell precursors in primary and secondary response

Spleen cells from unprimed mice or those primed with horse red blood cells (HRBC) were depleted of rosette-forming cells (RFC) with HRBC by the Ficoll-Hypaque density sedimentation, and the cells were examined in the adoptive transfer system whether they could raise IgM or IgG antibody-forming cells (AFC) after an immunization with HRBC. When spleen cells were pooled from unprimed mice, the response to HRBC of those depleted of RFC with HRBC (HRBC-RFC) was decreased to about a half in both IgM and IgG AFC. On the other hand, when spleen cells were from mice primed with HRBC, the response to HRBC of those depleted of HRBC-RFC was decreased dramatically to 1/20 of that of original cells in IgG AFC, but it was decreased to about a half in IgM AFC. In the time course of the response to HRBC of RFC-depleted spleen cells from mice primed with HRBC, an early IgG response was abolished but the late one was as high as that of untreated spleen cells. These results suggest that the depletion of RFC is most effective on the depletion of direct precursors of the secondary IgG AFC.     

B cell differentiation and isotype switching is related to division cycle number

The mature, resting immunoglobulin (Ig) M, IgD+ B lymphocyte can be induced by T cells to proliferate, switch isotype, and differentiate into Ig-secreting or memory cells. Furthermore, B cell activation results in the de novo expression or loss of a number of cell surface molecules that function in cell recirculation or further interaction with T cells. Here, a novel fluorescent technique reveals that T- dependent B cell activation induces cell surface changes that correlate with division cycle number. Furthermore, striking stepwise changes are often centered on a single round of cell division. Particularly marked was the consistent increase in IgG1+ B cells after the second division cycle, from an initial level of < 3% IgG1+ to a plateau of approximately 40% after six cell divisions. The relationship between the percentage of IgG1+ B cells and division number was independent of time after stimulation, indicating a requirement for cell division in isotype switching. IgD expression became negative after four divisions, and a number of changes centered on the sixth division, including the loss of IgM, CD23, and B220. The techniques used here should prove useful for tracking other differentiation pathways and for future analysis of the molecular events associated with stepwise differentiation at the single cell level.     

CELL-TO-CELL INTERACTION IN THE IMMUNE RESPONSE : VI. CONTRIBUTION OF THYMUS-DERIVED CELLS AND ANTIBODY-FORMING CELL PRECURSORS TO IMMUNOLOGICAL MEMORY

Collaboration between thymus-derived lymphocytes and nonthymus-derived antibody-forming cell precursors occurs in the primary antibody response of mice to heterologous erythrocytes and serum proteins. The purpose of the experiments reported here was to determine whether collaboration took place in an adoptive secondary antibody response. A chimeric population of lymphocytes was produced by reconstituting neonatally thymectomized CBA mice soon after birth with (CBA x C57BL)F₁ thymus lymphocytes. These mice could be effectively primed to fowl immunoglobulin G (FγG) and their thoracic duct lymphocytes adoptively transferred memory responses to irradiated mice. The activity of these cells was impaired markedly by preincubation with CBA anti-C57BL serum and to a lesser extent by anti-θ-serum. Reversal of this deficiency was obtained by adding T cells in the form of thoracic duct cells from normal CBA mice. Cells from FγG-primed mice were at least 10 times as effective as cells from normal mice or from CBA mice primed to horse erythrocytes. These results were considered to support the concept that memory resides in the T cell population and that collaboration between T and B cells is necessary for an optimal secondary antibody response. Poor antibody responses were obtained in irradiated mice given mixtures of thoracic duct cells from primed mice and of B cells from unprimed mice (in the form of spleen or thoracic duct cells from thymectomized donors). In contrast to the situation with T cells, the deficiency in the B cell population could not be reversed by adding B cells from unprimed mice. It was considered that memory resides in B cells as well as in T cells and that priming probably entails a change in the B cell population which is fundamentally different from that produced in the T cell population.     

Antiimmunoglobulin-treated B cells respond to a B cell differentiation factor for IgG1

We have determined whether B cells previously activated by anti-Ig (anti-Ig blasts) are responsive to lymphokines that induce isotype switching. Culture of anti-Ig blasts with a mixture of lymphokines, including BSF-1, resulted in marked secretion of IgM and IgG1, but not other IgG isotypes. The IgG1 response of anti-Ig blasts to lymphokines was 13-fold greater than was observed with splenic B cells. B cell blasts induced by 8-mercaptoguanosine or dextran sulfate did not secrete high levels of any IgG isotype in response to lymphokines alone. An mAb against BSF-1 suppressed the IgG1 response of anti-Ig blasts, but not the IgM response to lymphokines. These data suggest that anti-Ig-treated B cells respond to at least one of the effects of BSF-1.