Thymus-independent induction and antigen-dependent recovery of idiotype-specific suppression

BALB/c nude (nu/nu) mice and euthymic (nu/+) littermates were treated as neonates with anti-T15 antibody and challenged at various ages with either a thymus-independent, PC-Brucella abortus (PC-BA), or thymus-dependent, PC-keyhole limpet hemocyanin (PC-KLH), form of phosphorylcholine (PC). Nu/nu mice challenged with PC-KLH received KLH-primed splenic T cells prior to immunization. Neither neonatally anti-idiotype-treated nu/+ nor nu/nu mice responded with the production of T15-positive anti-PC antibodies after challenge with either form of PC antigen. It is concluded that neither induction nor maintenance of a state of T15-specific suppression requires thymus-matured T cells. Recovery of anti-PC responsiveness in suppressed nu/+ or nu/nu mice was similar and was found to be related to the form of antigen used to elicit the response. Immunization with PC-KLH revealed a long-lasting unresponsiveness (up to 16 weeks). In contrast, immunization with PC-BA elicited a full anti-PC response as early as at 6.5 weeks of age.     

Resistance to Streptococcus pneumoniae is induced by a phosphocholine-protein conjugate

Previous studies demonstrated that naturally occurring antibodies to the pneumococcal cell wall hapten phosphocholine (PC) are important for the survival of mice against infection with Streptococcus pneumoniae, and that passively administered hybridoma antibody to PC results in added resistance. To determine if a PC-protein conjugate could elicit protective levels of anti-PC antibody, mice were immunized with PC-keyhole limpet hemocyanin (KLH) and tested for their ability to resist challenge with virulent S. pneumoniae. PC-KLH-immunized mice were observed to be resistant to 10- to 1000-fold more organisms than unimmunized control animals. The levels of protection were comparable to those induced with capsular polysaccharide antigens, but had the advantage of not being type-specific; immunization with PC-KLH protected mice against both type 1 and type 3 organisms. The induced immunity appeared to be antibody-mediated; it could be passively transferred with immune serum, and absorption of the immune serum with PC-Sepharose removed its protective capacity. Anti-PC antibodies in the serum of immunized mice were primarily IgM and IgG3 and possessed predominantly the T15 idiotype. Antibodies with these particular isotypes and this idiotype also arise after immunization with heat-killed rough pneumococci and recently were shown to be important in the resistance of mice to pneumococcal infection.     

CD36 is differentially expressed on B cell subsets during development and in responses to antigen

Of a number of mAbs made by immunization with sort-purified marginal zone (MZ) B cells, one was shown to recognize the mouse scavenger receptor CD36. Although CD36 is expressed by most resting MZ B cells and not by follicular and B1 B cells, it is rapidly induced on follicular B cells in vitro following TLR and CD40 stimulation. In response to T-independent and T-dependent Ag challenge, we found that CD36 was expressed on IgM+ plasma cells, but down-regulated on isotype-switched plasma cells in vivo. Although development, localization, and phenotype of MZ B cells in CD36-/- mice appeared normal, there was a minor block in the transitional stages of mature B cell development. In both primary and secondary Ab responses to heat-killed Streptococcus pneumoniae (R36A strain), both phosphoryl choline (PC)-specific IgM and IgG levels in CD36-/- mice were slightly reduced compared with wild-type mice. In addition, mice deficient in both TLR2 and CD36 produced significantly reduced levels of anti-PC IgG titers than those of single gene-deficient mice, suggesting that they may cooperate in an anti-PC Ab response. Collectively, these results show that CD36 does not affect the development of B cells, but modulates both primary and secondary anti-PC Ab responses during S. pneumoniae infection similarly to TLR2.     

Immune response to phosphorylcholine. I.V. Comparison of homologous and isologous anti-idiotypic antibody.

Homologous and isologous anti-idiotypic antibodies against the PC-binding BALB/c myeloma protein HOPC-8 were raised in A/He and BALB/c mice, respectively. Isologous and homologous anti-HOPC-8 serum suppressed the response to PC in vitro specifically. The antibodies were purified by using HOPC-8 immunoabsorbent. Purified isologous and homologous anti-idiotypic antibody were labeled with 125I and compared for Ig class composition and idiotype-binding specificity. Both kinds of anti-idiotypic antibodies were predominantly IgG1, were highly specific for the HOPC-8 idiotype, and had a smiliar affinities for the PC-binding site. These findings demonstrate that anti-HOPC-8 antibodies raised in A/He and BALB/c mice are very similar in their biologic and immunochemical poperties.     

Perturbation of the idiotypic network. II. Transfer of suppression to neonatal mice

The cellular mechanisms which are responsible for idiotype perturbation induced by repeated stimulation with either allogeneic mixed lymphocyte culture-generated syngeneic blasts or allogeneically stimulated syngeneic spleen cells were investigated as described in the preceding article. Using the splenic fragment culture system, the precursor frequencies of T and B cells for anti-phosphorylcholine (PC) antibodies and T15 idiotypic antibodies were determined in allogeneically challenged mice. Adoptive transfers of T cells to neonatal BALB/c mice induced suppression of the T15+ anti-PC responses. In addition, the effect of immunization with internal image-bearing anti-idiotopes on the level of anti-PC antibodies and T15 idiotype was determined. Results from this study demonstrated (i) a decrease in the precursor frequency in the PC-specific and idiotype-specific B cell repertoire; (ii) a decrease in the precursor frequency of T helper cells, which recognize idiotypes and anti-idiotypes; (iii) the possibility to transfer T15 suppression to neonatal mice; and (iv) the possibility to restore T15 dominance by anti-idiotypic antibody immunization. These data indicate that both the T and B compartments are involved in the maintenance of suppression induced by repeated exposure to alloantigen-sensitized syngeneic cells. Collectively these findings show that a nonspecific general suppression induced by allohyperimmunization can perturb the T15+ anti-PC response.     

Helper T lymphocytes from xid and normal mice support anti-phosphocholine antibody responses with equivalent T15, 511, and 603 idiotypic composition

We have examined the idiotypic composition of secondary adoptive transfer antibody responses to phosphocholine (PC) supported by KLH-primed helper T cells derived from normal mice or xid mice. CBA/N x BALB/c F1 male xid mice have diminished anti-PC responses and virtually undetectable levels of the T15 idiotype; xid mice do express the 511 and 603 idiotypes. Nonetheless, we find helper T cells derived from such mice are indistinguishable from T cells primed in a normal environment in their ability to cooperate with B cells producing anti-PC antibody bearing the T15, 511, or 603 idiotype markers. This result is in contrast to a previously published report from this laboratory. T cells from xid mice did support more IgG PFC than normal T cells, but serum IgG anti-PC antibody levels were similar in both groups. The IgM anti-PC response was predominantly of the T15 idiotype, whereas the 511 idiotype was associated with a minor fraction of IgG1 antibodies. The majority of the secondary IgG "anti-PC" antibody response bore none of the idiotypic markers associated with PC-binding myeloma or hybridoma antibodies, and was directed against phenyl-PC rather than PC. The phenomenon of T15 clonal dominance in the anti-PC response therefore is largely confined to the IgM response. We would conclude that the idiotype levels in the T cell priming environment do not influence the subsequent ability of such primed T cells to support anti-PC antibody responses.     

Alteration of the B cell surface phenotype, immune response to phosphocholine and the B cell repertoire in M167 mu plus kappa transgenic mice

M167, mu plus kappa, transgenic mice have been analyzed for the expression of the transgene product as a cell surface, Ag-specific receptor and for their ability to respond to Ag. The vast majority of B cells in these H + L transgenics (97 to 99%) express large amounts of the transgene product on their surface and are capable of binding phosphocholine. A total of 4 to 30% of the B cells also express endogenous IgM and IgD H chain products. After immunization with phosphocholine (PC)-conjugated keyhole limpet hemocyanin, more than 1000 micrograms/ml of anti-PC antibody bearing the transgene IgMa allotype marker are produced. Surprisingly, significant amounts of anti-PC antibodies that express the endogenous, IgMb allotype, are also produced; however, these antibodies lack the T15-idiotype which dominates the anti-PC response in their nontransgenic littermate controls. The B cells producing these endogenous anti-PC antibodies also fail to switch to IgG anti-PC synthesis, whereas B cells producing anti-keyhole limpet hemocyanin antibodies readily undergo class switching. These last two observations may be due to the fact that the endogenous anti-PC antibody actually results from mixed mu a + mu b molecules in which the transgene encoded H and L chains are most likely responsible for the binding of PC. Thus, a switch of the endogenous isotype from mu b to IgG would result in a loss of specificity for PC in the IgG molecules produced using the endogenous VH-gene product(s), and mu a + gamma b hybrid molecules are not likely to be formed. This hypothesis is supported by the fact that the majority of (mu a + mu b) hybridomas have the mu b-allotype joined with a VH region other than the VH1 gene which is required for PC-binding and T15 idiotype expression.     

Mechanism of neonatal idiotype suppression. II. Alterations in the T cell compartment suppress the maturation of B cell precursors

The cellular mechanism in neonatally suppressed BALB/c mice, which maintains the chronic suppressed state of the TEPC-15 idiotype in the antibody response to phosphorylcholine (PC), was investigated. Cells taken from these suppressed mice cannot transfer suppression to adult BALB/c or affect the in vitro response to PC of adult BALB/c spleen cells. However, spleen cells or T cells from neonatally suppressed mice given to neonatal animals induce chronic suppression of the TEPC-15 idiotype in the anti-PC response. Co-transfer of T cells from neonatally suppressed cells with normal T cells prevented the induction of suppression in neonates. Transfer of T cells from normal or keyhole limpet hemocyanin-primed BALB/c increased the expression of TEPC-15 idiotype in chronically suppressed mice, whereas T cells from neonatally suppressed were ineffective. These findings show that T cells in neonatally suppressed mice can affect the development of immature but not mature cells. The restoration of TEPC-15 expression in neonatally suppressed animals by normal T cells and the failure to induce suppression in neonates by co-transfers of T cells from normal and chronically suppressed mice demonstrate the profound role of an altered T cell compartment in sustaining chronic idiotype suppression.     

Intrapulmonary BCG cell wall vaccination in mice

Mice of two inbred strains, Balb/c and C3H/He, were given three dosages of mycobacterium bovis BCG (5 X 10(4), 5 X 10(2) and 5 colony forming units) by either the intravenous route or by a direct intratracheal (non-aerosol) route. The magnitude of the infectivity of these inoculae given by these routes was assessed by measurement of weight changes and mycobacterial multiplication in the spleen and lung. As expected, the Balb/c strain was more susceptible to infection than the C3H/He strain. However, for both strains, infection by the intratracheal route resulted in mycobacterial counts in the lungs which were more than seven-fold higher than mycobacterial counts after intravenous challenge. Naive Balb/c mice were immunized with BCG cell wall vaccine by the intratracheal route, by the intravenous route or by subcutaneous immunization. Four weeks later mice were challenged with live BCG by the intratracheal route. Following challenge, mycobacterial counts in the lungs of mice immunized by the intratracheal route, but not in the lungs of the mice immunized by the intravenous and subcutaneous routes, were significantly lower compared to controls. These results suggest that immunization with killed BCG by the intratracheal route imparts more effective mycobacterial intrapulmonary immunity than immunization by systemic routes.     

Regulation of T15 idiotype dominance. II. Genes unlinked to the Igh locus regulate T15 dominance of the secondary adoptive transfer response to phosphocholine

We have studied the idiotype and fine specificity of the secondary immune response to phosphocholine (PC) in C57BL (B10, B10.D2, and B.C8) and BALB (BALB/c, BAB-14, and C.B20) congenic strains of mice. In vivo IgM responses of mice from these two genetic backgrounds differed in their T15 idiotypic representation. BALB strains expressed the T15 idiotype on greater than 90% of their IgM, PC-specific plaque-forming cells (PFC), whereas C57BL strains expressed the T15 idiotype on approximately 50% of their IgM PFC. All strains examined expressed greater than 75% PC-inhibitable, VHPC idiotype-positive, IgM PFC. The IgG3 and IgA memory responses were similar to the IgM memory response; BALB strains produced a higher proportion of T15+ PFC than C57BL strains; however, the majority of IgG3 and IgA PFC in all strains were VHPC+, and PC-inhibitable. In contrast, the IgG1 memory response was not dominated by T15+, VHPC+, PC-inhibitable PFC in any of the strains tested. The IgG1 PFC required nitrophenylphosphocholine (NPPC) for efficient inhibition. The IgG2 memory response generally mimicked the IgG1 response with respect to idiotype and specificity. These data demonstrate that the representation of the T15 idiotype in the anti-PC immune response is determined by genes outside both the MHC and Igh genetic loci. Control of T15 expression in secondary IgM, IgG3, and IgA anti-PC responses was examined by using a cell-mixing protocol with primed T and B cells from BALB/c and B10.D2 mice. T15 representation in these responses was determined by the genotype of the B cell, not by the genotype of the helper T cell. Similarly, the B cell genotype was responsible for the idiotypic profile of a primary, in vitro, T-dependent, anti-PC response.     

Perturbation of the idiotypic network. I. Induction with multiple alloantigen stimulation

The effect of hyperimmunization on the immune network with allostimulated syngeneic lymphocytes responding to different haplotypes was analyzed. Ten different haplotypes were used to stimulate syngeneic donor mice. Control mice were multiply immunized with incomplete Freund's adjuvant alone or with syngeneic mixed lymphocyte culture-generated lymphocytes. BALB/c mice were immunized consecutively with alloreactive blasts or allogeneically stimulated spleen cells at 10-day intervals. After a rest period of 2 months, the ratio of T helpers to T suppressors was determined by immunofluorescent staining. The functional network was probed by immunizing the mice with phosphorylcholine (PC) coupled to hemocyanin. The sera were analyzed for anti-PC antibodies and TEPC15 (T15) idiotypic expression. The results demonstrated (i) a decrease in the level of anti-PC antibody titer and T15 idiotypic expression; (ii) a decrease in the number of T helper cells and an increase in the number of T suppressor cells; (iii) a loss of PC epitope specificity; (iv) an increase of IgM antibodies expressing T15 without anti-PC specificity; and (v) an elevated level of preimmune lymphocyte proliferation and Ig secretion. These results reveal a functional network linkage in the regulation of alloreactivity and antigen response and show how repeated exposure to alloantigens can induce a perturbation of the idiotypic network controlling the response of a non-alloantigen-related BALB/c strain dominant idiotype (T15).     

B7 requirements for primary and secondary protein- and polysaccharide-specific Ig isotype responses to Streptococcus pneumoniae

The requirements for B7 costimulation during an in vivo humoral response to an intact extracellular bacteria have not been reported. In this study we immunized mice with Streptococcus pneumoniae (R36A) to determine the B7 requirements for induction of Ig, specific for two determinants on R36A, the phosphorylcholine (PC) determinant of C-polysaccharide and pneumococcal surface protein A (PspA). We show that the primary anti-PspA response, the development of PspA-specific memory, and the induction of the secondary anti-PspA response in primed mice were completely dependent upon B7 costimulation. Of note, costimulation was required only briefly after the secondary immunization compared with after the primary immunization for optimal induction of Ig. Blockade of B7 costimulation at the time of secondary immunization also completely abrogated the established state of memory, but did not induce tolerance. In contrast to the anti-PspA response, the primary anti-PC response involved only a very short period of B7 costimulation. Whereas B7-2 alone was required for induction of the primary anti-PspA and anti-PC responses, a redundant role for B7-1 and B7-2 was noted for the PspA-specific secondary response. CTLA4Ig blocked both the anti-PC and anti-PspA responses equally well over a wide range of bacterial doses. These studies demonstrate a critical, but variable, role for B7-dependent costimulation during an Ig response to an extracellular bacteria.     

Relation of a cross-reactive idiotype to genetic control of the immune response.

Antibodies elicited in strain A mice by immunization with keyhole limpet hemocyanin-p-azophenylarsonate (KLH-Ar) produce anti-Ar antibodies, some of which share a cross-reactive idiotype (CRI); in general, 20 to 70% of the antihapten antibody population carries the idiotype. Large amounts of antibody can be produced by the induction of ascitic fluids, using a 9:1 ratio of complete Freund's adjuvant (CFA) to antigen. Antibodies with the CRI can be isolated by isoelectric focusing from selected mice that have produced a high concentration of the CRI. The H chains exhibit a single homogeneous sequence through the first hypervariable region and, when isolated from a large number of individual mice, appear to be invariant in the first framework region. These findings indicate that somatic mutation is not a significant factor in the determination of framework sequences. Appearance of the CRI can be suppressed in adult A/J mice by administration of rabbit anti-idiotypic antiserum prior to immunization. Such suppressed mice produce normal concentrations of anti-Ar antibodies lacking the CRI. Anti-idiotypic antibodies produced against such antibodies failed to show cross-reactivity with anti-Ar antibodies arising in idiotypically suppressed or nonsuppressed A/J mice. The great sensitivity of the assay indicates that the number of such "private" idiotypes, all present on anti-Ar antibodies of a single strain, must be extremely large; this supports a somatic mechanism for the generation of diversity. The "private" idiotypes arising in suppressed, hyperimmunized mice can be adoptively transferred into multiple, irradiated (200 R) recipients by injections of spleen cells or of cells from ascitic fluids. The use of ascitic fluids permits the rapid production of a colony of mice bearing the idiotype. This should facilitate structural studies of a variety of idiotypically different molecules sharing the same (anti-Ar) specificity, as well as studies of the mechanism of suppression.     

Isoelectric focus analysis of rat anti-phosphocholine antibodies.

Anti-phosphocholine (PC) antibodies in sera from four strains of rats were examined before and afterimmunization with either Streptococcus pneumoniae R36A, which contains PC as a cell wall component, or with PC-coupled keyhole limpet hemocyanin (PC-KLH). PC-specific protein was purified from pooled immune sera and shown by a combination of isoelectric focus (IEF) in acrylamide and crossed immunoelectrophoresis, as well as by molecular weight determination in NaDodSO4-acrylamide, to be immunoglobulin. An additional, small molecular weight, nonimmunoglobulin protein (pI = 7.1-7.3) was present in sera from normal and germ-free rats which had the ability to bind the C-carbohydrate of S. pneumoniae R36A, but without specificity for PC. The IEF profile of normal and immune sera showed marked sharing of bands of anti-PC antibody between individual rats as well as between strains. In addition, other anti-PC antibodies which focused between pH 8.5 and 9.5 were less regularly shared. The uniformity of IEF profile of the bulk of anti-PC antibodies in rats is most consistent with their being the products of germ line genes.     

Regulation of clones responding to α 1 → 3 dextran: I. Individual variation in the expression of idiotypes

Balb/c mice were immunized with dextran B1355S and assayed for serum antibodies and direct plaque-forming cells. The earliest detectable anti-dextran response occurred in 2-week-old animals. Anti-idiotypic antisera against MOPC-104E and J558 were raised in A/He mice and rendered individual idiotype specific by cross-absorption. When the amounts of MOPC-104E and J558 idiotypes in immune sera and the PFC response of individual mice were analyzed, the ratio of both idiotypes were found highly variable in all tested animals. This observed individual variability in the expression of two major idiotypes in the Balb/c response to dextran B1355S provides an experimental basis for investigating the mechanisms regulating idiotype expression.     

Interference between Neisseria meningitidis and PC-KLH Induced Anti-Phosphorylcholine PFC Responses in NZB/W Autoimmune Mice

In this work, we demonstrate that the simultaneous injection of PC-KLH and Neisseria meningitidis-derived antigens [NMB or PC-(NMB)HI] induced in old NZB/W mice defective responses as does PC-KLH challenge. On the other hand, the simultaneous injection of both immunogenic preparations of N. meningitidis evoked responses similar to those shown by old mice challenged with NMB alone. Alteration in PC-specific PFC responses also affected hapten-free inhibition profiles and their heterogeneities. The increase in PC₅₀s of anti-phosphorylcholine PFC responses and their heterogeneities induced by certain antigens with aging is correlated with a decrease in T15 idiotype expression, suggesting that after the T15 dominant clone disappears no other clone takes control of the anti-PC response. These results suggest that the mechanism(s) involved in the regulation of T15 marker expression play an important role in the inability of old NZB/W mice to mount good anti-PC responses and suggest that regulatory mechanisms induced by PC-KLH dominate those elicited by NMB.     

Vaccination of BALB/c mice with Escherichia coli-expressed vaccinia virus proteins A27L, B5R, and D8L protects mice from lethal vaccinia virus challenge

The potential threat of smallpox use in a bioterrorist attack has heightened the need to develop an effective smallpox vaccine for immunization of the general public. Vaccination with the current smallpox vaccine, Dryvax, produces protective immunity but may result in adverse reactions for some vaccinees. A subunit vaccine composed of protective vaccinia virus proteins should avoid the complications arising from live-virus vaccination and thus provide a safer alternative smallpox vaccine. In this study, we assessed the protective efficacy and immunogenicity of a multisubunit vaccine composed of the A27L and D8L proteins from the intracellular mature virus (IMV) form and the B5R protein from the extracellular enveloped virus (EEV) form of vaccinia virus. BALB/c mice were immunized with Escherichia coli-produced A27L, D8L, and B5R proteins in an adjuvant consisting of monophosphoryl lipid A and trehalose dicorynomycolate or in TiterMax Gold adjuvant. Following immunization, mice were either sacrificed for analysis of immune responses or lethally challenged by intranasal inoculation with vaccinia virus strain Western Reserve. We observed that three immunizations either with A27L, D8L, and B5R or with the A27L and B5R proteins alone induced potent neutralizing antibody responses and provided complete protection against lethal vaccinia virus challenge. Several linear B-cell epitopes within the three proteins were recognized by sera from the immunized mice. In addition, protein-specific cellular responses were detected in spleens of immunized mice by a gamma interferon enzyme-linked immunospot assay using peptides derived from each protein. Our data suggest that a subunit vaccine incorporating bacterially expressed IMV- and EEV-specific proteins can be effective in stimulating anti-vaccinia virus immune responses and providing protection against lethal virus challenge.     

Amino acid sequence of a phosphocholine-binding antibody from an immune defective CBA/N mouse employing the T15 VH region associated with unusual DH, JH, and V kappa segments

Mice expressing the xid gene exhibit an altered immune response to phosphocholine (PC)-conjugated keyhole limpet hemocyanin (KLH). Less than 25% of their anti-PC-KLH response is PC specific, and most of these antibodies lack the normally predominant T15 idiotype. These findings suggested that immune defective mice might employ different variable region genes than normal mice in their anti-PC response. To examine this possibility, we characterized by Southern blot analysis the gene family encoding PC-VH regions and determined the amino acid sequence and fine specificity of binding of a T15-, IgG2, PC-specific hybridoma (1B8E5) produced by fusion of the SP2/O cell line and PC-KLH immune CBA/N spleen cells. Southern blot analysis of DNA from CBA/N mice by using a PC-VH probe (S107 VH) revealed a hybridization pattern virtually identical to that of DNA from normal CBA/J mice, indicating that CBA/N mice do not suffer from a gross deletion of PC-VH genes. Analysis of the 1B8E5 antibody reveals that both the binding specificity and relative affinity of this antibody are different from the anti-PC antibodies of the T15, M167-M511, and M603 families. The complete amino acid sequence of the heavy (H) chain variable region shows that 1B8E5 uses a VH segment identical to the allelic form of T15 (C3) but has a unique D region of three amino acids and use the JH1 joining segment. Both the DH and JH regions are unusual when compared to PC-specific antibodies from normal mice, which have a D region composed of five to eight amino acids and use the JH1 joining segment. The amino terminal sequence of the 1B8E5 light (L) chain demonstrates that this anti-PC antibody carries a Vk3 subgroup L chain. Chains from this subgroup have not previously been found in association with PC-binding antibodies. Thus, the Vk, DH, and JH segments expressed in 1B8E5 make this hybridoma unique in terms of the anti-PC antibodies studied to date, and suggests that additional PC-specific antibodies exist in inbred mice that employ "unusual" V gene segments.     

Immunologic memory to phosphocholine. VII. Lack of T15 V1 gene utilization in Xid anti-PC hybridomas

CBA/N mice carrying the Xid defect fail to make antibodies expressing the T15 idiotype in response to immunization with PC-KLH. Antibodies predominating in the Xid response have binding properties characteristic of group II antibodies that emerge in the memory response in BALB/c; the prototype group II antibody utilizes a VH gene product distinct from the V1 gene product expressed by T15 idiotype-positive antibodies. To examine VH gene usage in the anti-PC response of Xid B cells, hybridomas were produced from Xid mice immune to PC-KLH. Four hybridomas possessing properties typical of the predominant group II antibody response in Xid mice and two representing minor components of the response were studied. Analysis of DNA by Southern blot hybridization revealed that none of the hybridomas utilized the T15 V1 gene segment, nor did they share use of a common VDJ gene product. These results indicate that Xid group II antibodies either make use of different VH gene segments or use the same VH in combination with various D and JH segments.     

In vivo polysaccharide-specific IgG isotype responses to intact Streptococcus pneumoniae are T cell dependent and require CD40- and B7-ligand interactions.

In vivo Ig responses to soluble, haptenated polysaccharide (PS) Ags are T cell independent and do not require CD40 ligand (CD40L). However, little is known regarding the regulation of in vivo PS-specific Ig responses to intact bacteria. We immunized mice with a nonencapsulated, type 2 Streptococcus pneumoniae (R36A) and compared the parameters that regulated in vivo Ig isotype responses to the bacterial cell wall C-PS determinant, phosphorylcholine (PC), relative to Ig responses to the cell wall protein, pneumococcal surface protein A. Consistent with previous reports using soluble PS and protein Ags, the anti-PC and anti-pneumococcal surface protein A responses differed in that the anti-PC response was induced more rapidly, had a distinctive Ig isotype profile, and failed to demonstrate boosting upon secondary challenge with R36A. However, in contrast to previous studies, the IgG anti-PC response was TCR-alphabeta+ T cell dependent, required CD40L, and was blocked by administration of CTLA4 Ig. The nature of the T cell help for the anti-PC response had distinct features in that it was only partially blocked by CTLA4 Ig and was dependent upon both CD4+ and CD8+ T cells. Surprisingly, whereas the IgM anti-PC response was largely T cell independent, a strong requirement for CD40L was still observed, suggesting the possibility of an in vivo T cell-independent source for CD40L-dependent help. These data suggest that the regulatory parameters that govern in vivo Ig responses to purified, soluble PS Ags may not adequately account for PS-specific Ig responses to intact bacteria.