Analyses of Idiotypes on Anti-α-1, 6-Dextran Antibodies in Mice

When mice of strains C57BL/6, C3H/He, and BALB/c were immunized with native dextran B512, only a small amount of IgM antibody was produced, but a substantial amount of anti-dextran antibody of IgG class was produced after immunization with a conjugate of dextran T10 and keyhole limpet hemocyanin regardless of the mouse strain used. Isoelectric focusing (IEF) spectra revealed limited heterogeneity of anti-dextran antibody of IgG class with strict consistency in all individual sera from C57BL/6 mice, even after secondary immunization, whereas antibodies from C3H/He and BALB/c mice showed more heterogeneous IEF spectra with some individual variations. Rabbit anti-idiotypic (Id) antibodies were raised by immunization with a subfraction of anti-dextran antibody of IgG class from C57BL/6 mice, which showed major bands focused at around pH 7.7 upon IEF. It was found by using the anti-Id antibodies that virtually all anti-dextran antibody molecules of both IgG and IgM classes from C57BL/6 mice possessed common Id determinants which can be classified into two specificities, one specific for antibody from C57BL/6 mice and the other cross-reactive with antibodies from BALB/c and C3H/He mice. About 80% of the antibody molecules from BALB/c and less than 20% of those from C3H/He mice were positive for the interstrain cross-reactive Id. Both Id determinants seemed to be closely related to the antigen binding sites, or at least to reside in the vicinity of the antigen binding sites of anti-dextran antibody.     

In vivo-induced suppression of T cell proliferation: the relationship between the specificity of induction and control

We have previously shown that sc immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and to peptide 8. In contrast, immunization of B10.BR (H-2k) mice either with TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous Ag. In the present article , we report that in the B10.BR (H-2k) strain, ip prepriming with (TMVP) 7 days prior to sc immunization with peptide 8 causes a drastic reduction in the in vitro proliferative response of peptide 8-specific T cells while no such effect is seen in the congenic C57BL/10 (H-2b) strain. This suppression of T cell responsiveness can be transferred with TMVP-primed spleen cells. Moreover, deleting T cells from the transferred spleen cells abrogates the suppressive effect. In both H-2 haplotypes, ip prepriming with peptide 8 causes suppression of the proliferative T cell response induced by subsequent immunization with peptide 8. This prepriming has no effect on the response to TMVP immunization of B10.BR mice but does effect the response of C57BL/10 mice. Using various synthetic peptides to analyze the specificity of the suppression, we have determined that (1) T cells involved in the suppression of the proliferative T cell response to a single peptide determinant do not suppress the proliferative T cell response to other determinants on the protein antigen and (2) these T cells with suppressor function, and proliferating T cells which are ultimately regulated, can exhibit specificity for the same epitope. These studies suggest that there may exist fundamental differences as to how T cells which participate in suppression an proliferating T cells (which include mainly T helper cells) recognize protein antigens.     

Comparative study of the T cell response to two allelic forms of a malarial vaccine candidate protein.

T cell responses to two allelic forms of the merozoite surface Ag 2 (MSA2) of Plasmodium falciparum were mapped in mice using the rMSA2 proteins, Ag 1609 which has the sequence of the FCQ27/PNG strain and Ag 1615 which has the sequence of the Indochina 1 strain. Lymph node cells of BL/10 and B10.BR mice immunized with either Ag 1609 or Ag 1615 responded to both Ag in in vitro proliferation assays. Lymph node cells of BALB/c mice did not respond. The T cell determinants recognized by the responder strains were mapped to conserved and variant regions of these Ag using overlapping synthetic peptides. The determinants recognized by each mouse strain were distinct. Marked difference in sequence between the central regions of the two rMSA2 proteins did not affect antigenic processing of the conserved N and C terminal regions. Hence lymph node cells of BL/10 mice immunized with either Ag 1615 or Ag 1609 recognized an immunodominant T cell determinant at the highly conserved N terminal end within the sequence YSNTFINNAYNMSIR (peptide 3b) and B10.BR mice similarly immunized recognized an immunodominant determinant at the highly conserved C terminal within the sequence CTDGNKENCGAATSL (peptide 23). Several peptides identified as containing immunodominant T cell determinants specific to BL/10 mice induced peptide-specific T cells in both BL/10 and B10.BR mouse strains when used as immunogens. However, the ability of the peptide-primed T cells to proliferate in response to the rMSA2 proteins was confined to BL/10 mice. An example of this was observed with peptides 3b and N (KNESKYSNTFINNAYNMSIRRSMAN). Peptide N was able to prime B10.BR and BL/10 mice for an enhanced antibody response when these mice were subsequently immunized with Ag 1615 even though Ag 1615-specific T cell proliferation was not detected in B10.BR mice primed with N. The study concluded that 1) conserved sequences such as peptide N when used in vaccines may give rise to MSA2-specific memory Th cells amenable to boosting by subsequent exposure to all parasite strains and 2) peptide priming may be a useful pathway for inducing defined memory Th cells in a wider population and for preferentially inducing T dependent over T independent responses to some malarial Ag.     

The regulatory role of sialic acids in the response of class II reactive T cell hybridomas to allogeneic B cells

Two different kinds of alloreactive T cell hybridomas were established in previous experiments. One is reactive and the other is nonreactive to allogeneic I-A region-associated membrane antigen (mIa) on B cells. In the present experiments the difference between these hybridomas were analyzed by using representative clones, B cell mIa-reactive clone CB-11.4, and nonreactive clone HTB-9.3. Unresponsiveness of HTB-9.3 clone to allogeneic B cells could not be due to the inability of B cells in interleukin 1 production or the density of mIa molecules on B cells. HTB-9.3 clone could respond to C57BL/6 mouse B cells treated with neuraminidase (Nase), and Nase-treated HTB-9.3 clone could respond to normal B cells from C57BL/6 mouse, indicating that sialic acid on both B cells and HTB-9.3 clone plays a regulatory role in the alloreactivity of the clone. In response to B cells from C57BL/6 mouse, T cells from C3H/He mouse spleen showed similar reactivity to HTB-9.3 clone; that is, T cells could respond to Nase-treated B cells, and Nase-treated T cells to B cells, and T cells primed with C57BL/6 spleen cells in vitro showed similar reactivity to CB-11.4 clone. These results suggest that HTB-9.3 clone represents virgin T cells and CB-11.4 clone-primed T cells at least in alloreactivity. Anti-L3T4a was shown to block alloreactivities of both T cell hybridomas and splenic T cells against B cells more efficiently than against splenic adherent cells. These results suggest that L3T4a on T cell plays more important role in allogeneic response to B cells than to splenic adherent cells.     

Synthetic Plasmodium falciparum circumsporozoite peptides elicit heterogenous L3T4+ T cell proliferative responses in H-2b mice

The ability of synthetic P. falciparum (NANP)n circumsporozoite peptides to elicit murine T cell proliferative responses was studied. When C57BL/6, C3H, and DBA/2 mice were injected with (NANP)40, only C57BL/6 (H-2b)-immune lymph node cells proliferated on restimulation in vitro with the same peptide. By using anti-I-A monoclonal antibodies or spleen cells from congenic H-2b mice as a source of antigen-presenting cells, the T cell proliferative response was shown to be restricted to the I-Ab region of the C57BL/6 haplotype. These results are in agreement with previous experiments which demonstrated that the anti-(NANP)40 antibody response was uniquely restricted to C57BL/6 (H-2b) mice. Several C57BL/6 long-term (NANP)n-specific T cell lines and clones were derived. All of the clones exhibited the L3T4 helper T cell phenotype. A considerable heterogeneity of T cell responses was observed when the lines and clones were stimulated with different concentrations of the various peptides studied. The results, together with the observed genetic restriction for both antibody and T cell responses, suggest that perhaps not all individuals who receive a similar repetitive tetrapeptide sporozoite malaria vaccine will develop T cell and or antibody responses.     

The role of class II MHC molecules in the activation of class I-reactive T cell hybridomas

To examine the role of Ia molecules in T cell responses to allo-class I major histocompatibility antigens, a series of allo-class I-reactive T cell hybridomas was established. Of 134 T cell hybridomas obtained from the fusion of C3H/HeJm or B10.HTT T cells stimulated with C57BL/6 splenocytes, nine T cell hybridomas were reactive to class I antigens and 126 T cell hybridomas were reactive to class II antigens. Six of the nine IL 2-producing T cell hybridomas were further analyzed: five mapped to H-2Kb and the other mapped to H-2Db. Three of these T cell hybridomas, HTB-157.7, HTB-176.10, and HTB-177.2, could react to the EL-4 cell line that expresses H-2Kb and H-2Db class I antigens but lacks class II I-Ab molecules. Furthermore, the activation of these three T cell hybridomas with C57BL/6-derived splenocytes was not blocked by either anti-I-A or anti-L3T4 antibody. In contrast, the other three T cell hybridomas, CB-127.6, CB-221.7, and HTB-102.7, failed to react with EL-4 but reacted with the LB cell line which expresses class I (H-2Kb, H-2Db) and class II (I-Ab) molecules. Although class II molecules were required for activation of the latter clones, there was no apparent I-A allele specificity, suggesting that a relatively nonpolymorphic Ia determinant was involved. The activation of the three latter T cell hybridoma clones with C57BL/6 splenocytes could be blocked completely by either anti-I-A or anti-L3T4 antibody. The data are interpreted in terms of possible T cell receptor models for recognition of class I with nonpolymorphic class II determinants.     

Influenza viruses as lymphocyte mitogens. I. B cell mitogenesis by influenza A viruses of the H2 and H6 subtypes is controlled by the I-E/C subregion of the major histocompatibility complex

Influenza A viruses of the H2, H3, and H6 subtypes function as T cell-independent B cell mitogens for lymphocytes from BALB/c mice. Lymphocytes from C57BL/10 mice, however, undergo mitogenesis only in response to H3 viruses. The failure of C57BL/10 lymphocytes to respond to H2 and H6 viruses was shown not to reflect a difference in dose-response profile or kinetics of the response, nor was it due to the activity of suppressor T cells. Experiments with congenic and recombinant strains of mice established that mitogenic responsiveness to H2 and H6 viruses is linked to the major histocompatibility complex, and is controlled by a gene located in the I-E/C subregion. Furthermore, responsiveness was shown to correlate with the expression of surface I-E antigen, being positive for mouse strains that express I-E antigen (haplotypes a, d, k, p, r) and negative for strains that do not (haplotypes b, f, q, s). The data suggest that influenza A viruses of the H2 and H6 subtypes may interact directly with I-E molecules on the surface of B cells or possibly on an accessory cell. Because mitogenesis by H3 viruses is not I-E dependent, it appears that influenza A viruses stimulate B cell mitogenesis by at least two different mechanisms.     

Ultraviolet light induces tumors with both unique and host-associated antigenic specificities

Murine tumors induced by ultraviolet light (UV) are immunogenic in syngeneic and semi-syngeneic hosts, evoking antibody of several different specificities. Cytotoxic antibody specific for the immunizing syngeneic tumor (tumor-specific antigen) comprises the early response and a minor portion of the later response of C3H and C3H.SW mice. It is the primary specificity to which C57BL/6 and C57BL/10 hosts respond. The major portion of the antibody produced by C3H and C3H.SW against syngeneic tumors cross-reacts strongly with other tumors, both UV and chemically induced, arising in C3H and C3H.SW but not in B6.H2k, C57BL/6, C57BL/10, or (C3H X B6)F1. Normal adult cells or embryonic fibroblasts do not cross-react with the antisera. These results are interpreted as evidence for the involvement of a host component (non-MHC) in this tumor-associated antigen (TAA). (C3H X B6)F1 and (C3H X BALB/c)F1 hosts respond to C3H tumors with antibody with cross-reactive specificities identical to those of the C3H and C3H.SW hosts. thus detecting the TAA(C3H) specificity. (C3H X B6)F1 hosts respond to syngeneic F1 tumors, however, with a totally cross-reactive antibody that is interpreted as evidence for the existence of a common antigen in addition to the evident immune response control. An undetected TAA (B6) specificity in the (C3H X B6)F1 tumors is speculatively proposed.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Antibody response against hamster red blood cells (H-RBC) was examined in inbred strains of C57BL/6, AKR, C3H/He, DDD and SL mice, and outbred CF1 mice. 1) There were strain differences in antibody response after a primary intravenous injection of H-RBC. DDD, SL and CF1 mice belonged to high-responder strains, while C57BL/6, AKR and C3H/He to low-responder strains. In the spleens of immunized CF1 and SL, 40 to 70 times as many plaque-forming cells (PFC) as those in C57BL/6 mice were detected. The magnitudes of the response were: CF1 ≒ SL>DDD>>C3H/He ? AKR>C57BL/6. 2) 2-mercaptoethanol resistant (MER) antibody was detected in neither low- nor high-responders after a primary intravenous antigen-injection. 3) After a secondary intravenous antigen-injection, MER antibody was detected in all the SL mice, but only in 30 to 50% of AKR and C57BL/6 mice. 4) A subcutaneous injection of H-RBC in Freund's complete adjuvant (FCA) did not elicit antibody production within 10 days. When mice pre-sensitized 7 days in advance w^Tith H-RBC in FCA were intravenously injected with H-RBC, enhanced antibody production of the primary type was observed in all the mouse strains. 5) In pre-sensitized mice, the extent of the enhancement of antibody production was the highest in low-responder C57BL/6 mice and the lowest in high-responder SL and CF1 strains. Thus, there was no strain difference in antibody titers or the numbers of PFC after the booster.     

An antigen cross-reactive with gp52 of mammary tumor virus is expressed on a B cell subpopulation of mice

The reactivity of murine lymphoid tissue with biotinylated F(ab')2 fragments of monoclonal antibody VE7 (BIOT VE7), which reacts with gp52 of murine mammary tumor virus (MuMTV), was tested with fluoresceinated avidin in an indirect fluorescent antibody assay on live splenocytes. A small percentage of splenic lymphocytes in C3H/He mice infected exogenously with MuMTV, and in C57BL/6 mice that, like C3H/He, harbor several endogenous MuMTV proviruses in their genomes, were reactive with the monoclonal antibody. The antigen-positive splenocytes were shown to represent a subpopulation of B cells. The possible nature of the B cell-associated antigen recognized by monoclonal antibody VE7 is discussed.     

Determinant selection in murine experimental autoimmune myasthenia gravis. Effect of the bm12 mutation on T cell recognition of acetylcholine receptor epitopes.

C57BL/6 (B6) mice respond to immunization with acetylcholine receptor (AChR) from Torpedo californica as measured by T cell proliferation, antibody production, and the development of muscle weakness resembling human myasthenia gravis. The congenic strain B6.C-H-2bm12 (bm12), which differs from B6 by three amino acid substitutions in the beta-chain of the MHC class II molecule I-A, develops a T cell proliferative response but does not produce antibody or develop muscle weakness. By examining the fine specificity of the B6 and bm12 T cell responses to AChR by using T cell clones and synthetic AChR peptides, we found key differences between the two strains in T cell epitope recognition. B6 T cells responded predominantly to the peptide representing alpha-subunit residues 146-162; this response was cross-reactive at the clonal level to peptide 111-126. Based on the sequence homology between these peptides and the T cell response to a set of truncated peptides, the major B6 T cell epitope was determined to be residues 148-152. The cross-reactivity of peptides 146-162 and 111-126 could also be demonstrated in vivo. Immunization of B6 mice with either peptide primed for T cell responses to both peptides. In contrast, immunization of bm12 mice with peptide 111-126 primed for an anti-peptide response, which did not cross-react with 146-162. Peptide-reactive T cells were not elicited after immunization of bm12 mice with 146-162. These results define a major T cell fine specificity in experimental autoimmune myasthenia gravis-susceptible B6 mice to be directed at alpha-subunit residues 148-152. T cells from disease-resistant bm12 mice fail to recognize this epitope but do recognize other portions of AChR. We postulate that alpha-148-152 is a disease-related epitope in murine experimental autoimmune myasthenia gravis. In this informative strain combination, MHC class II-associated determinant selection, rather than Ag responsiveness per se, may play a major role in determining disease susceptibility.     

Mapping of theLyb-4 gene to different chromosomes in DBA/2J and C3H/HeJ mice

Linkage has been established between the Lyb-4 alloantigen locus and the chromosome 4 markersLyb- 2 andMup- 1 using recombinant inbred (RI) strains. Only 2 of 24 BXD RI strains possess recombinant genotypes with respect to the B cell alloantigen lociLyb- 4 andLyb- 2, for an estimated recombination frequency of 0.024 ±0.019. One additional BXD RI strain was a recombinant with respect toLyb- 4 andMup- 1 (major urinary protein locus) for an estimated recombination frequency of 0.039 ± 0.026. These linkages were confirmed and further quantitated in a (C57BL/6J × DBA/2J)F₁ × C57BL/6J backcross population, in which the recombination frequency betweenLyb- 4 andMup- 1 was 0.049 ± 0.019. No recombination between the expression of Lyb-4.1 antigen and the ability of anti-Lyb-4.1 serum to suppress MLC reactivity was found, indicating that the genes controlling the antigenic determinant which is recognized with cytotoxic antibodies in anti-Lyb-4.1 serum is the same as, or is very closely linked to, the gene which is responsible for augmentation of the MLC response. In contrast, no linkage was observed between the gene controlling the Lyb-4.1 determinant andMup- 1 in RI strain and backcross mice derived from the cross of C3H/HeJ and C57BL/6J. Again, there was complete concordance between the serologically recognized determinant and the ability of anti-Lyb-4.1 serum to suppress the MLC response. Absorption of anti-Lyb-4.1 serum with C3H/HeJ, DBA/2J, and C57BL/6J lymphocytes, followed by the cytotoxic assay of the absorbed sera on lymphocytes of each of these three strains showed that serologically the Lyb-4.1 antigenic determinant on DBA/2 mice was indistinguishable from that on C3H/HeJ mice. Thus, both traits appear to be under the control of single genes in both DBA/2J and C3H/HeJ, but the C3H/HeJ gene appears to be nonallelic and unlinked to the DBA/2J gene.Abbreviations used in this paper LAD lymphocyte activating determinants - LPS lipopolysaccharide - MLC mixed lymphocyte culture - RI recombinant inbred     

Changes in cell populations and immunoglobulin-producing cells in the spleens of mice infected with Trypanosoma cruzi: correlations with parasite-specific antibody response

Infection of mice with Trypanosoma cruzi elicits the production of parasite-specific antibodies which reach high levels and remain elevated for at least 105 days of infection. The more susceptible C3H(He) mouse actually has a higher level of "natural" antibodies for T. cruzi but may show a greater lag time in the production of antibodies in response to infection than the more resistant C57BL/6 mouse. Comparison of the kinetics of antibody production against T. cruzi and the numbers of immunoglobulin-producing cells in the spleen during the course of infection suggests that a large number of the immunoglobulin-producing cells are probably producing antibodies directed against the parasite and are not the result of an exhaustive polyclonal B-cell activation. Cell numbers in the spleen change dramatically both in total numbers and in the percentage of different cell types during infection with T. cruzi. The percentage of T cells in the spleen remains relatively unchanged throughout infection in both mouse strains tested but numbers of Ig-positive cells decrease markedly during the acute phase of infection while macrophage numbers increase up to sixfold. Cell numbers and proportions of B cells, T cells, and macrophages return to near normal values by 105 days of infection in the C57BL/6 mouse.     

The occurrence of anti-3-fucosyllactosamine antibodies and their cross-reactive idiotopes in preimmune and immune mouse sera

The carbohydrate determinant 3-fucosyllactosamine (3-FL), Gal(beta 1-4)[Fuc alpha 1-3]GlcNAc-R, which is also known as SSEA-1 or as the X determinant, is very immunogenic in BALB/c mice. Many monoclonal antibodies directed against this structure have been obtained by immunization of mice with murine teratocarcinomas and human carcinomas and myeloid cells. We have undertaken an analysis of the regulation of these antibodies and of their idiotypic, structural, and genetic diversity. We have described previously the preparation of syngeneic monoclonal anti-idiotypic antibodies (6C4 and 6B1) that reacted with 50% of a panel of monoclonal anti-3-FL antibodies. In this study, we have examined the occurrence of anti-3-FL antibodies and their cross-reactive idiotopes in the sera of unimmunized and immunized mice. All BALB/c sera examined had more naturally occurring antibodies against 3-FL than against four other glycolipid antigens, and the 6C4 and 6B1 idiotopes were also detected in these sera. Approximately 25% of the anti-3-FL antibodies could be removed from a pool of BALB/c sera by a 6C4 affinity column, and the eluate from this column exhibited strong binding to 3-FL antigens. After a single i.v. injection of a 3-FL-positive glycolipid coated on Salmonella minnesota, anti-3-FL titers rose in BALB/c mice. The level of 6B1 idiotope rose in most mice, but the idiotope level showed no correlation with anti-3-FL titers. Naturally occurring antibodies against 3-FL were also noted in the sera of AKR, C3H/He, DBA/2, BALB/c-nu/nu, and CBA/CaHN mice but not in C57BL/6, SM, or CBA/N mice. A single i.v. injection of antigen elicited an antibody response in C3H/He mice but not in C57BL/6, SM, or DBA/2 mice. These data indicate that several strains of mice have more naturally occurring IgM antibodies against the 3-FL structure than against other glycolipids, and that this response may be genetically regulated. The 6C4 and 6B1 cross-reactive idiotopes that we have identified previously on monoclonal antibodies are also present in preimmune and immune sera. The existence of a population of B lymphocytes that are primed against the 3-FL determinant accounts in part for the immunogenicity of this structure.     

Trypanosoma cruzi: Effect on B-cell-responsive and -responding clones

During the course of Trypanosoma cruzi infection in C57BL/6 mice, which are relatively resistant to the parasite, the hosts developed antibody activity against previously unencountered antigens. The anti-sheep erythrocyte and antitrinitrophenyl antibody levels increased rapidly from Day 7 of infection, reached a peak by the 21st day, and were maintained at this level through 120 days postinfection in these mice. In contrast, highly susceptible C3H(He) mice did not have demonstrable antibody responses to SRBC or TNP during the 24-day infection period. Autoantibody activity against the selfantigens presented on isologous erythrocytes or thymocytes, however, were reduced in infected C57BL/6 mice. No significant reduction in autoreactivity to the self-antigens on erythrocytes or thymocytes was observed in C3H(He) mice infected with T. cruzi although a trend of reduced autoresponsiveness toward erythrocytes appeared to be developing by the time of death. C57BL/6 mice immunized with sheep erythrocytes as neonates and infected with T. cruzi as adults, or adult mice primed with low doses of sheep erythrocytes prior to infection, had elevated antibody responses to sheep erythrocytes unless the mice were immunized with sheep erythrocytes during the course of infection, in which case suppression of the response against sheep erythrocytes resulted. The nonspecific synthesis of immunoglobulins in infected C57BL/6 mice was, in part, a result of the lymphocyteactivating properties of T. cruzi-associated antigens. The T. cruzi-associated antigens induced proliferative and differentiative responses in spleen cells in vitro. It is proposed that the T. cruzi-associated antigens differentially affect lymphocytes capable of responding to antigen and those lymphocytes previously stimulated by antigen.     

The effect of aging on the induction of humoral and cellular immunity and tolerance in two long-lived mouse strains.

Aging is a complex process that adversely affects most if not all components of the immune system. In this report, two long-lived mouse strains have been compared in ability to generate both antigen-specific immunity and tolerance. Although CBA/CaJ mice produced high levels of antibody following injection of aqueous preparations of aggregated human gamma-globulin (AHGG), C57BL/6 mice made only meager antibody responses to such preparations. Age dramatically affects the humoral anti-HGG response to aqueous AHGG in both strains, but the meager response of young C57BL/6 mice was at insignificant levels in aged C57BL/6 mice. Conversely, both mouse strains generated good responses following injection of HGG in complete Freund's adjuvant at both the T and B cell level as evidenced by in vitro antigen-specific T cell proliferation and anti-HGG antibody production. Aged mice of both strains showed a marked decrease in the production of serum anti-HGG antibody in comparison to young mice. Although the antigen-specific T cell proliferative response was significantly decreased in aged CBA/CaJ mice, such proliferation was not affected in aged mice of the C57BL/6 strain. Removal of CD8+ cells from lymph node T cells of either young or aged C57BL/6 mice did not increase the antigen-specific proliferative response, suggesting that loss of CD8+ suppressors during the aging process is not responsible for the high level of antigen-specific T cell proliferation in aged C57BL/6 mice. Tolerance to HGG was readily induced in both young and aged C57BL/6 and CBA/CaJ mice although aged mice demonstrate a modest resistance to tolerance induction when compared to their young counterparts. This resistance was observed in both antibody production and antigen-specific T cell proliferation.     

Strain Differences in the Immunogenicity of Aggregated Human IgG and the Adjuvant Action of Lipopolysaccharide on the Low-Responder Strain of Mice

Strain differences in the antibody response to human IgG (HGG) were observed when aggregated HGG was injected intravenously. Lipopolysaccharide (LPS) administered subsequently markedly enhanced the antibody response to HGG in low responder C57BL/6 mice as compared with that in high responder DDD, C3H/He or (C57BL/6 × DDD)F₁ mice. Aggregate-free preparation of HGG at a dose of 0.5 mg induced immunological tolerance in all strains of mice tested. LPS injected subsequently converted tolerogenic, aggregate-free HGG into immunogen in DDD mice but not in C57BL/6 mice. To determine the correlation between adjuvanticity and mitogenicity of LPS, spleen cells from normal mice were cultured in the presence of LPS and ³H-thymidine uptake was measured. Spleen cells of DDD mice incorporated three times as much ³H-thymidine as those of C57BL/6 mice. There seems no strong correlation between both activities of LPS. The data obtained are discussed in terms of strain differences in the macrophage function for processing the antigen.     

A synthetic peptide to the E glycoprotein of Murray Valley encephalitis virus defines multiple virus-reactive T- and B-cell epitopes.

Synthetic peptides from the envelope glycoprotein sequence of Murray Valley encephalitis (MVE) virus were previously evaluated in various strains of mice for both the induction of antibody and the in vitro proliferation of peptide-primed T-helper (Th) cells. MVE peptide 6 (amino acids 230 to 251) elicited reciprocal Th- and B-cell reactivity with native MVE virus after primary inoculation of C57BL/6 mice. In this study, we prepared overlapping subunit peptides of MVE peptide 6 and evaluated their immunogenicity. Analysis of these peptides delineated at least two B-cell epitopes that induced antibody reactive with MVE and other Japanese encephalitis serocomplex viruses. This antibody at low titer neutralized MVE virus. Genetic restriction of the antibody response to various T-cell elements within peptide 6 was observed in C3H, BALB/c, C57BL/6, and B10 congenic mice. One element demonstrable after primary immunization, located in the carboxy terminus, associated only with major histocompatibility complex class II IAb and IAbiEk glycoproteins. Functional stimulation with the peptides in association with IAkIEk and IAdIEd molecules was observed only after in vivo secondary stimulation. Peptide 6-1 (amino acids 230 to 241) was nonimmunogenic but could be recognized by Th cells from peptide 6-immunized mice. Further association of peptide 6 with the IAkIEk and IAdIEd subregions was demonstrated by the finding that T cells from MVE peptide 6-inoculated C3H and BALB/c mice primed for an antibody response to MVE virus. These results suggest that the peptide 6 sequence, which is relatively conserved among a number of flaviviruses, should be given consideration when synthetic immunogens for vaccine purposes are designed.     

Structural aspects of a protein epitope and their role in the major histocompatibility complex control of T cell responsiveness.

We have previously shown that immunization of C57BL/10 (H-2b) mice with the tobacco mosaic virus protein (TMVP) or with its tryptic peptide number 8, representing residues 93-112 of TMVP, induces T cells which proliferate in vitro in response to TMVP and peptide 8. In contrast, immunization of congenic B10.BR (H-2k) mice with either TMVP or with peptide 8 induces T cells which respond in vitro to the homologous but not the heterologous antigen. The capacity to exhibit cross-reactivity between TMVP and peptide 8 on the T cell level has been shown to be under major histocompatibility complex (MHC)-linked genetic control. The lack of cross-reactivity has been attributed to the inability of the H-2k APC to present the appropriate epitope to T cells. In the present paper, we report results of a comparative analysis of the role of structural aspects of the epitope on the proliferative T cell responses from TMVP and peptide 8-immune C57BL/10 (H-2b) and B10.BR (H-2k) mice. Utilizing a panel of synthetic peptides representing portions of peptide 8 and a panel of peptide-protein conjugates, we have determined that peptide 8-immune T cells of the H-2k strain appear to recognize a single epitope within peptide 8, located at its N-terminus. In contrast, in the H-2b strain, both TMVP and peptide 8-immune T cells appear to recognize two overlapping epitopes within peptide 8; one located in the middle region and the other toward the N-terminus. Experiments with H-2b T cells revealed that random amino acids added to the carboxyl or amino-terminus of nonstimulatory peptides can confer activity to these peptides, demonstrating limited specificity of interaction between antigen and Iab. Results of experiments dealing with fixation of antigen-presenting cells suggest that TMVP requires processing in order to be recognized by peptide 8-immune H-2b proliferative T cells whereas peptide 8 does not. Taken together the results suggest that the T cell responsiveness to TMVP and peptide 8 exhibited by these two congenic strains H-2b and H-2k is not only controlled by the strains MHC but is also influenced by antigen processing. Antigen processing may eliminate a potential epitope for the primary induction and the secondary stimulation of B10.BR T cells.