The immunoregulatory role of bone marrow. I. Suppression of the induction of antibody responses to T-dependent and T-independent antigens by cells in the bone marrow.

Bone marrow cells (BMC) from normal mice suppressed the in vitro IgM, but not the IgG, antibody (Ab) response of spleen cells. BMC were inhibitory only when added during the first 24 hr of culture, and inhibition was not due to an induced shift in the kinetics of the response. Addition of specifically activated T cells or nonspecific T-cell-replacing factors to normal or T-depleted spleen cell cultures did not abrogate suppression while the response to the T-independent antigen DNP-polymerized flagellin or lipopolysaccharide was also suppressed. BMC did not inhibit background Ab synthesis by normal or primed cells in the absence of antigen and did not inhibit, but stimulated, DNA synthesis in normal spleen cell cultures. In addition, high-avidity Ab synthesis was preferentially suppressed. A possible role for the bone marrow suppressor cell in the induction of B cell tolerance is discussed.     

C-Reactive protein (CRP)-mediated inhibition of the induction of in vitro antibody formation. I. T-cell dependence of the inhibition.

Purified human C-reactive protein (CRP) inhibited the in vitro anti-hapten antibody plaque-forming cells (PFC) response of both carrier keyhole limpet hemocyanin (KLH)-primed and unimmunized Balb/c spleen cells to TNP-KLH. The inhibitory effect was neutralized by the CRP-substrate, C-polysaccharide. The response to the T-independent antigens, TNP-T4 and DNP-lys-Ficoll, was not inhibited by CRP. A cell population that was suppressive for the in vitro PFC response was generated by incubating normal spleen cells with CRP. These cells suppressed the PFC response of syngeneic KLH-primed cells to TNP-KLH in proportion to the number of added lymphoid cells with bound CRP. Selective depletion of B cells, T cells or macrophages before incubation with CRP revealed that T cells were required for the induction of suppressive cells. Treatment of spleen cells after incubation with CRP, with T cell-specific antisera and C abolished suppressor-cell activity. Mitomycin-C treatment of the CRP-binding cells did not alter their suppressive activity. These results indicated that CRP mediates suppression of antibody induction to T-dependent antigens by interacting with T cells and generating a suppressive T-cell population.     

Effects of neonatal anti-delta antibody treatment on the murine immune system. II. Functional capacity of a stable sIgM+sIa+sIgD- B cell population

Mice were injected from birth with rabbit anti-mouse IgD (RaM delta). Studies in the accompanying paper indicated that the B cells from these mice have a stable sIgM+sIa+sIgD- B cell population. In the studies presented herein the in vivo and in vitro antibody responses of these mice were examined as well as their responsiveness to various B cell mitogens. The results indicate that splenic B cells from RaM delta-suppressed mice differ from normal adult murine splenic B cells by failure to express increased sIa antigen after in vitro stimulation with soluble anti-mu antibodies and failure to proliferate in response to in vitro stimulation with either soluble or Sepharose-bound anti-mu antibody. Nevertheless, these mice generate relatively normal in vivo IgM and IgG antibody responses to TI-2 and to both high and low epitope density forms of TD antigens as well as secondary IgG antibody responses to a TD antigen. In addition, B cells from RaM delta-treated mice generate relatively normal primary in vitro IgM antibody responses to TI-1, TI-2, and TD antigens. These data suggest that sIgD- B cells can produce antibody responses to the majority of antigenic signals even though they appear to lack one or more differentiative pathways.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Thymus-dependent and thymus-independent effector functions of mouse lymphoid cells. Comparison of cytotoxicity and primary antibody formation in vitro

We have compared two effector functions, antibody formation and cytotoxic capacity in vitro, of mouse cells of various origin with special reference to the T lymphocyte dependence of these processes. We have used addition of PHA and coating of target chicken erythrocytes (CRBC) with antibody as the two means of inducing cytotoxicity. Antibody formation in vitro has been studied both against thymus-dependent sheep erythrocytes (SRBC) and thymus-independent (E. coli) antigens. Spleen cells from thymectomized, lethally irradiated bone marrow-, or fetal liver-repopulated mice were deprived of phagocytic cells by uptake of colloidal iron. They did perform better than normal spleen cells in the antibody-induced cytotoxicity and were also induced to cytotoxicity by PHA. PHA did not induce increased DNA synthesis in these T cell-deprived spleen cell preparations, which could not make primary antibodies to SRBC but were able to do so against E. coli antigens. Fresh bone marrow and fetal liver cells, deprived of phagocytic cells, were also induced into a highly efficient cytotoxicity by anti-CRBC as well as by PHA. Pretreatment of spleen cells with an alloantiserum (θ) against T lymphocytes reduced but did not abolish the PHA-induced cytotoxicity. In contrast, it did not affect the antibody-induced cytotoxicity. Such treated cells could not make antibodies to SRBC but could do so against E. coli. Pretreatment of spleen cells with a heteroantiserum (MBLA) against mouse B lymphocytes completely abolished all cytotoxic- and antibody-forming abilities of the cells, although experiments with combinations of θ-treated and MBLA-treated cells suggested that the MBLA treatment had left behind a significant portion of helper T cells needed for the in vitro antibody response. From these data we conclude, as have others, that the antibody-induced cytotoxicity is independent of T lymphocytes. It can be induced in immature precursor cells from fetal liver or bone marrow, and these cells may also become cytotoxic on interaction with PHA. However, in normal spleen cells, at least part of the PHA-induced cytotoxicity is T cell dependent. Some preliminary data suggest that this PHA-induced cytotoxicity of normal spleen cells may be a joint process between T lymphocytes and other cells.     

Transfer of agammaglobulinemia in the chicken. I. Generation of suppressor activity by injection of bursa cells

Spleen cells from adult agammaglobulinemic (bursectomized) chickens taken 1 to 3 weeks after an injection of histocompatible bursa cells can inhibit the adoptive antibody response to B. abortus of normal spleen or bursa cells in irradiated recipients. Spleen cells from Aγ chickens not injected with bursa cells generally do not. Moreover, bursectomized chickens which have been reconstituted with spleen cells within the first week after hatching do not respond with suppressor cell formation upon bursa cell injection. This apparent “autoimmunization” with bursa cells induces suppressor T cells which are only minimally sensitive to treatment with mitomycin C or to 5000 R γ irradiation. The suppressor activity is neither induced nor potentiated by concanavalin A in vivo. It is much stronger in spleen than in thymus cells and appears to be macrophage independent and to require intact cells. The cell component which stimulates the suppressor activity is more pronounced on bursa than on spleen cells, and is at most present to a very limited extent on bone marrow, thymus, or peritoneal exudate cells. It is better represented in comparable cell numbers of Day 17 than of Day 14 embryonic bursa. The inducing cell component is present in the membrane fraction of disrupted bursa cells. Immunization with bursa cells from B locus histoincompatible chickens leads to suppressor activity against histocompatible bursa cells. Although the removal of Ig-bearing cells from bursa greatly diminishes its immunizing capacity, injection of serum IgM and IgG does not induce suppressor cells. It is suggested that tolerance to a B-cell antigen is lacking in adult Aγ chickens, resulting in an autoimmune response upon exposure to B cells. The B-cell antigen may be a cell surface-specific form of Ig, a complex of Ig and a membrane component, or a differentiation antigen which appears simultaneously with Ig during ontogeny.     

Regulation of immune responses. II. The cellular basis of cyclic AMP effects on humoral immunity.

DbcAMP, when added at 10^?3M for the first 12 hr, can increase the number of AFC to SRBC (a TD antigen) and POL (a TI antigen) in antigen-stimulated CBA/ J spleen cell cultures. The cellular basis of dbcAMP action was therefore investigated. It was found that dbcAMP does not act by a direct B cell effect. It also does not stimulate the activity of T helper cells, and it inhibits the function of macrophages. The stimulatory activity of dbcAMP to anti-SRBC and anti-POL responses is through inhibition of a θ-bearing regulator (or suppressor) cell. Removal of T cells by anti-θ treatment has the same effect on the anti-POL response as treatment with dbcAMP. Furthermore, in the absence of T cells, the enhanced anti-POL response was insensitive of dbcAMP treatment. The data also support the hypothesis that the number of anti-SRBC AFC formed is regulated by the ratio of T helper to T regulator cells.     

Activation of suppressor T cells by low-molecular-weight factors secreted by spleen cells from tumor-bearing mice

The spleens of mice bearing large M-1 fibrosarcomas have been shown to contain several populations of cells which nonspecifically suppress antibody synthesis by cocultured normal spleen cells. It has now been shown that the spleens of tumor-bearing mice also contain inducer cells which secrete soluble factors capable of activating suppressor T cells from unprimed precursor cells. The activated suppressor cells are Thy 1+, Lyt 1+2+ and secrete a soluble suppressive factor. They inhibit the in vitro generation of antibody-forming cells by cocultured normal spleen cells stimulated by T-cell-dependent antigens. They do not, however, suppress the antibody response to T-cell-independent antigens and do not inhibit antibody synthesis by cocultured nude mouse spleen cells cultured with T-cell-dependent antigens and exogenous helper factors. In addition, suppression is blocked if conditioned medium containing T-cell growth factors is added to the suppressor cell assays. These data suggest that cells in the spleens of tumor-bearing mice secrete inducing factors which activate suppressor cells. These activated suppressor cells in turn secrete soluble suppressor factors which inhibit antibody synthesis, possibly by interfering with the synthesis or release of T-cell growth factors.     

Subclass restriction of murine antibodies: V. The IgG plaque-forming cell response to thymus-independent and thymus-dependent antigens in athymic and euthymic mice

Antigens differ in their abilities to stimulate antibodies of various isotypes. Many thymus-independent (TI) polysaccharide antigens stimulate largely IgG3 and IgM antibodies while thymus-dependent (TD) protein antigens stimulate predominantly IgG1 and smaller amounts of other isotypes. Here we determine whether thymus dependence or independence is a property of antigens which is expressed equally by all isotypes. To do this nu/+ and nu/nu mice were immunized with several TI and TD antigens and antibody responses of IgM and the four IgG subclasses measured. We found that, within the conditions of these experiments, all IgG isotypes were influenced equally by the presence or absence of T lymphocytes. Second, in agreement with J. L. Press (J. Immunol.126, 1234, 1981), we propose a division of TD antigens into two types based upon the ability to stimulate responses in the CBA/N mouse.     

Induction of immunity and specific unresponsiveness in vitro by cell-bound antigen: I. Symmetry in enhancement of both responses

Pulse treatment of lymphoid cells from rabbits with solubilized antigens from T2 phage results in the firm binding of small but highly active amounts of antigen. Binding of phage antigens to viable, nonviable, or disrupted cells enhances their ability to evoke antibody formation or specific unresponsiveness in the primary in vitro response of rabbit spleen cells. Transfer of sonicate containing the equivalent of 10₂ to 10₃ antigen-pulsed cells carrying 10^?8 to 10^?7 μg phage protein nitrogen into spleen cell cultures regularly evokes antibody formation, while introduction to such cultures of 10^?3 μg phage protein nitrogen in cell-bound form evokes unresponsiveness. These findings indicate a 10- to 100-fold amplification of tolerogenic and immunogenic activities of cell-bound over soluble T2 antigen.     

Effects of carrageenan on immune responses. Studies on the macrophage dependency of various antigens after treatment with carrageenan.

Carrageenan, a sulfated polygalactose having macrophage toxic properties, elicited a marked suppression of IgM response to T cell-dependent antigens such as sheep red blood cells (SRBC), dinitrophenylated bovine serum gamma-globulin (DNP-BGG), and trinitrophenylated concanavalin A (TNP-Con A). In contrast, carrageenan did not inhibit antibody responses to such T cell-independent antigens as trinitrophenylated DEAE-dextran (TNP-DEAE-dextran), trinitrophenylated polyvinyl pyrrolidone(TNP-PVP), and trinitrophenylated Ficoll (TNP-Ficoll). Compared to total spleen cells, spleen cells from which macrophages had been removed by adhesion to plastic Petri dishes had less effect on the production of antibody against T cell-dependent antigens, but no change or a rather stimulated effect was observed in in vitro antibiody synthesis against T cell-independent antigens. These results strongly suggest that macrophages are involved in antibody responses to T cell-dependent antigens but not in those to T cell-independent antigens. However, the antibody response to trinitrophenylated lipopolysaccharide (TNP-LPS), a T cell-independent antigen, was inhibited by carrageenan treatment, suggesting that the response is macrophage dependent. Moreover, antibody response to higher doses of dinitrophenylated phytohemagglutinin (DNP-PHA), a T cell-dependent antigen, was shown to be macrophage independent by carrageenan treatment, although the antibody response to low doses of the antigen was macrophage dependent. Considering all these results, carrageenan treatment seems to be a very useful method to determine whether immune response to various antigens are macrophage dependent or not.     

T cell control of the antibody response to the T-independent antigen, DAGG-Ficoll

C57BL/6J nu/nu mice respond to the type 2 TI antigen DAGG-Ficoll, but not to the TD antigen SRC. A comparable difference can also be seen in vitro, but only at high spleen cell density and in the presence of selected batches of FBS. At low spleen cell density and in the absence of FBS, the DAGG-Ficoll-induced B cell response is strictly dependent on soluble helper factors or cloned specific helper T cells. The B cell response so induced requires that the T cell-depleted spleen cells be compatible in the I-A subregion of the H-2 complex. These helper factors, induced by antigen in an I-A-restricted T cell-macrophage interaction, provide helper for T cell-depleted spleen cells irrespective of their H-2 haplotype. Under conventional culture conditions, the stringent requirement for helper factors in the in vitro response to DAGG-Ficoll is obscured by FBS. In vitro culture of low numbers of spleen cells, in serum-free medium instead of FBS, provides a sensitive assay for helper factors. We have compared the helper activity for a B cell response to SRC or DAGG-Ficoll as provided by antigen-induced supernatants of various individual EA-specific T cell clones. There was a remarkable and consistent heterogeneity among individual T cell clones: their helper activity in the response to TI and TD antigens did not correlate, nor was there any correlation between helper activity and antigen-induced TCGF (interleukin 2) activity.     

Functional heterogeneity of memory B lymphocytes: in vivo analysis of TD-primed B cells responsive to secondary stimulation with TD and TI antigens

The functional heterogeneity of memory B cells induced by a single determinant, consisting of a decapeptide representing amino acid residues 103-112 of tobacco mosaic virus protein (TMVP), was analyzed. Decapeptide specific antibodies were elicited in mice adoptively transferred with TMVP-immune spleen cells when challenged with TMVP, decapeptide conjugated to succinylated human gamma-globulin (SHGG), or decapeptide conjugated to Brucella abortus (BA). Whereas secondary stimulation by either TMVP or decapeptide-SHGG was dependent on appropriately primed T cells, stimulation by decapeptide-BA was independent of conventional T cell help. Furthermore, memory B cells responsive to TMVP (TD), decapeptide-SHGG (TD), or decapeptide-BA (TI. 1 prototype) were shown to consist of overlapping populations because adoptive recipients of TMVP-primed cells challenged simultaneously with TD and TI decapeptide antigens did not result in a higher antibody response than that elicited by one of the TD antigens injected alone. However, decapeptide-BA consistently induced a smaller antidecapeptide response than either TMVP or decapeptide-SHGG. This suggested that only a fraction of the memory B cell population which was activated by the original priming antigen (thymus-dependent) was also responsive to secondary in vivo stimulation by the priming hapten conjugated to Brucella abortus. Detailed analyses of the antibodies induced in the recipients of TMVP-immune spleen cells after secondary challenge with either TMVP, decapeptide-SHGG, or decapeptide-BA failed to distinguish between the responsive memory B cells; the antidecapeptide antibodies induced by all three immunogens shared the same fine specificities and immunoglobulin isotype composition. These data are viewed as further evidence that subsets of TD-primed B cells, which may display differential sensitivity to cross-stimulation with TD and TI forms of the antigen, represent distinct stages of memory B cell maturation within a common B cell lineage. In support of this conclusion, we establish a developmental relationship between TI and/or TD responsive decapeptide memory B cell in the following communication.     

Activation of antigen-enriched B cells. II. Role of linked recognition in B cell proliferation to thymus-dependent antigens

The responsiveness to T-dependent (TD) and T-independent (TI) TNP-antigens of murine splenic B cells previously enriched for antigen-binding cells (ABC) was examined. TNP-TI antigens induced B cell proliferation. TNP-TD antigens did not induce a proliferative response regardless of the physical form or nature of the TNP-TD antigen (e.g., soluble vs particulate, low or high haptenation of carrier, TNP on various insoluble matrices, etc.). TNP-TD antigens were effective in enhancing the response of the TNP-ABC to all concentrations of lipopolysaccharide (LPS) tested, indicating that binding of antigen to surface immunoglobulin alters the LPS responsiveness of the cell. Irradiated, keyhole limpet hemocyanin- (KLH) primed T cells induced a threefold to fourfold greater B cell proliferative response with TNP-KLH than with fluoresceinated KLH (FLU-KLH) or FLU-KLH together with TNP-human serum albumin (TNP-HSA). Therefore, linked recognition appears essential for optimal T cell-mediated B cell proliferation, whereas the induction of B cell proliferation via nonlinked, carrier-activated T cells is a minor component of the response.     

Synergistic effects of the xid gene in X chromosome congenic mice. I. Inability of C3.CBA/N mice to respond to thymus-dependent antigens in adoptive transfer assays

The xid gene, which causes a B lymphocyte immune defect in CBA/N mice, has been bred onto the C3H/HeN background. The resulting X chromosome congenic mice (C3.CBA/N) exhibit immunologic defects that are much more profound than the defect exhibited by CBA/N mice; thus, the B cells from C3.CBA/N mice not only fail to respond to thymus-independent (TI) type 2 antigens such as TNP-Ficoll, but they fail to respond in vitro to TI-type 1 antigens such as TNP-Brucella abortus (BA) and B cell mitogens such as LPS and Nocardia water-soluble mitogen. In this paper we show that the synergistic defect seen in C3.CBA/N B cells is also elicited in adoptive transfer assays to thymus-dependent (TD) antigens such as TNP-KLH and PC-KLH, antigens to which both parental strains respond. Thus, the secondary adoptive transfer response of C3.CBA/N spleen cells is generally less than 5% of the immune response produced by CBA/N or C3H/HeN spleen cells. This synergistic defect is restricted to the C3.CBA/N B cells, since C3.CBA/N T cells can provide help to CBA/N B cells that is equivalent to the help obtained with CBA/N T cells. The low responsiveness of C3.CBA/N spleen cells to TD antigens, which is elicited in adoptive transfer assays, is not seen when the intact animal is immunized with antigen in CFA; this, intact C3.CBA/N mice produce anti-PC-KLH and anti-TNP-KLH responses only slightly lower than the responses of CBA/N mice to these same antigens. In contrast, when these mice are immunized with phenol-extracted LPS, a TI-type 1 antigen, their antibody responses are severely depressed. These data suggest that under conditions in which T cell help may be limiting or in which the intact physiology of the T and B cells has been disrupted, C3.CBA/N B cells demonstrate profound immunologic impairment; however, when adequate T cell help is available and the splenic architecture is not disrupted, their immune responses appear to progress in a normal fashion.     

Antigenic markers on mouse lymphoid cells: origin of cells mediating immunologic memory

Specific antisera were used for the purification of thymus dependent and thymus independent or bursa equivalent lymphoid cells in the mouse. Spleen cells from mice immune to sheep erythrocytes, a thymus dependent antigen, or to E. coli 055:B5 lipopolysaccharide, a thymus independent antigen, were treated with anti-θ (C3H) serum or anti-MBLA serum and complement prior to their adoptive transfer into lethally irradiated syngeneic recipients. Syngeneic thymocytes, bone marrow cells, or spleen cells from nonimmune donors were appropriately added to antiserum treated cells prior to transfer. The secondary response to these antigens was assayed in recipient spleens six days after cell transfer. The kinetics of the primary response to SRBC was investigated as to its effect on origin of specific hyper-reactive T or B lymphoid cells.The adoptive response to CPS originated in the B lymphoid cell population. Immunologic memory to CPS was demonstrated in recipients of immune cells, compared to recipients of normal cells, by a five fold increase in antibody forming cells.The IgM and IgG adoptive immune response to high doses of SRBC depended upon an increased number of specifically hyper-reactive T-lymphoid cells to facilitate cooperation between T and B lymphocytes. High doses of SRBC initially stimulated T cell memory but at 42 days after priming an increased number of specifically hyper-reactive B lymphoid cells were present.     

Inhibition of the in vitro 19S and 7S antibody response by immunoglobulin-binding factor (IBF) from alloantigen-activated T cells

A soluble factor secreted by alloantigen-activated mouse T cells which binds to the Fc fragment of IgG and inhibits complement activation by IgG (immunoglobulin-binding factor, IBF) suppressed the in vitro 19S and 7S antibody response by mouse spleen cells to T-dependent as well as T-independent antigens. IBF inhibited the 19S plaque response best when it was added late during PFC generation (between 48 and 72 hr). On the other hand, when it was left in cultures for up to 60 hr and then removed, antibody synthesis was not inhibited. However, its presence for only 2 hr starting after 72 hr of incubation was sufficient to inhibit PFC formation. The suppressive activity of IFB could be neutralized by adding aggregated mouse IgG prior to the critical stage around 72 hr. These data favour the view that IBF could be a suppressive T cell factor and point to the possibility that IBF may act on already triggered B cells during their final differentiation to active PFC.     

Regulation of antibody secretion by hybridoma cells. I. Suppression of antibody secretion by coculture of hybridoma cells with idiotype-induced suppressor cells

In this study, we have demonstrated that antibody secretion by hybridoma cell lines can be down-regulated by idiotype-specific immune spleen cells or by nylon wool nonadherent spleen cells. This suppression of antibody secretion can be abolished by treating the idiotype-specific immune spleen cells with anti-Thy 1.2 plus complement. The hybridoma we used for most of our experiments secretes IgM specific for the cross-reacting haptens 2,4,6-trinitrophenyl (TNP) and 2,4-dinitrophenyl (DNP). Suppression was achieved by direct coculture of hybridoma cells with immune cells from animals which were injected with affinity-purified hybridoma antibody-coupled syngeneic spleen cells. The suppressed and control cultures contained similar numbers of viable hybridoma cells, suggesting that a simple cytotoxic effect is not responsible. Idiotype specificity was established in experiments showing that two idiotype immune animals immunized with antibody from two different IgM anti-TNP hybridomas could suppress the hybridoma to which they were immunized but could not affect the other hybridoma. Immune spleen cells required 3-4 days of coculture with hybridoma cells before maximum suppression was achieved. The kinetics of the response suggest that the final effector suppressor cell is generated during the coculture period and that a second signal, perhaps a product of the hybridoma cells, may be required.     

Activation of T cells by glutaraldehyde-fixed erythrocyte antigens: radiosensitivity of cells mediating delayed-type hypersensitivity reactions in the mouse.

Mice immunized with glutaraldehyde-fixed sheep red blood cells (G-SRBC) show delayed-type hypersensitivity (DTH) reactions to G-SRBC or SRBC. The specificity of the DTH reaction of mice sensitized with glutaraldehyde-fixed antigens is similar to that found after sensitization with unfixed antigens. The dose-response curve for sensitization by glutaraldehyde-fixed SRBC was very different from the curve for normal SRBC. At low doses, both antigens were effective in sensitizing to show DTH but neither induced an antibody response. However, at high antigen doses, only the glutaraldehyde-fixed antigen was efficient in sensitizing to show DTH and it failed to raise an antibody titer. Spleen cells of mice sensitized with fixed RBC can transfer DTH locally but if the donor cells are irradiated (500 R), the transfer is abrogated. In contrast, the transfer of DTH by spleen cells of mice immunized with unfixed antigen is not affected by 500 R. The transfer of DTH by spleen cells of mice immunized with fixed antigen can be blocked by “in vitro desensitization” while the transfer of DTH by spleen cells from mice primed with normal antigen is resistant to “in vitro desensitization.” These results suggest that immunization of mice with different physical states of the same antigen can result in the activation of antigen-specific T cells which exhibit markedly different properties.     

T-Cell-Independent Immunoglobulin G Responses In Vivo Are Elicited by Live-Virus Infection but Not by Immunization with Viral Proteins or Virus-Like Particles

Immunoglobulin G (IgG) responses to viruses are generally assumed to be T-cell dependent (TD). Recently, however, polyomavirus (PyV) infection of T-cell-deficient (T-cell receptor β chain [TCR-β] −/− or TCR-β×δ −/−) mice was shown to elicit a protective, T-cell-independent (TI) antiviral IgM and IgG response. A repetitive, highly organized antigenic structure common to many TI antigens is postulated to be important in the induction of antibody responses in the absence of helper T cells. To test whether the repetitive structure of viral antigens is essential and/or sufficient for the induction of TI antibodies, we compared the abilities of three forms of PyV antigens to induce IgM and IgG responses in T-cell-deficient mice: soluble capsid antigens (VP1), repetitive virus-like particles (VLPs), and live PyV. Immunization with each of the viral antigens resulted in IgM production. VLPs and PyV elicited 10-fold-higher IgM titers than VP1, indicating that the highly organized, repetitive antigens are more efficient in IgM induction. Antigen-specific TI IgG responses, however, were detected only in mice infected with live PyV, not in VP1- or VLP-immunized mice. These results suggest that the highly organized, repetitive nature of the viral antigens is insufficient to account for their ability to elicit TI IgG response and that signals generated by live-virus infection may be essential for the switch to IgG production in the absence of T cells. Germinal centers were not observed in T-cell-deficient PyV-infected mice, indicating that the germinal center pathway of B-cell differentiation is TD even in the context of a virus infection.