Identification of a tumor-associated antigen of the guinea pig L2C leukemia by using syngeneic antisera.

Inbred strain 2 guinea pigs immunized with L2C leukemia cells produced antibodies to L2C cells detected by 125I-protein A assay. L2C-associated tumor antigens were reacted with syngeneic antisera and analyzed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. These sera recognized idiotypic determinants on surface IgM molecules of L2C cells but did not recognize any determinants on normal strain 2 spleen cells. Thus, determinants on IgM molecular act as tumor-associated antigens in the L2C system and can be detected by syngeneic sera.     

Strain differences in susceptibility to experimental allergic encephalomyelitis and the immune response to the encephalitogenic determinant in inbred guinea pigs.

Strain differences in susceptibility to experimental allergic encephalomyelitis (EAE) in guinea pigs were correlated with the cellular immune response to the basic encephalitogenic protein (BE). The response to BE was determined in strains 2 and 13 guinea pigs in vivo by the delayed hypersensitivity skin test and in vitro by the lymphocyte transformation technique. The response to the intact BE of both heterologous (bovine) and homologous (guinea pig) origins was indistinguishable between the two strains. Guinea pigs sensitized with the guinea pig BE showed complete cross-reaction when tested with the bovine BE. On the other hand, there appears to be significant differences in the response to specific determinants on the molecule. Thus, only strain 13 and F₁ hybrids which are susceptible to EAE responded to the encephalitogenic nonapeptide (residue 114–122 of the BE molecule), whereas strain 2 guinea pigs which are resistant to EAE did not respond to this determinant.     

Immune responses of inbred guinea pigs to the sequential polymer poly(L-Tyr-L-Ala-Gly): studies with the oligomers of the polymers.

Synthetic known sequence polypeptides poly(Tyrosine-Glutamic acid-Alanine-Glycine) T-G-A-Gly), were found to be very immunogenic in responder inbred guinea pigs. Two and one-half micrograms were enough to elicit both humoral and cellular responses. Only the alpha-helical oligomers were immunogenic and were able to inhibit the homologous antigen-antibody reactions. The random polymers of comparable amino acid composition, i.e., poly(glutamic acid60alanine40) (GA), poly(glutamic acid50 tyrosin50) (GT), poly(glutamic acid60alanine30tyrosine10)(GAT10), did not inhibit. The antibodies against (T-G-A-Gly)n did not bind to the closely related known sequence polymer poly tyrosine-alanine-glutamic acid-glycine) (T-A-G-Gly)n or to the above random polymers. It is thus concluded that antibodies against (T-G-A-Gly)n are directed against conformational determinats.     

Nonrandom catabolism of proteins in the formation of antigenic peptide fragments

To examine the role of protein catabolism in the formation of antigenic peptide fragments, human fibrinopeptide-immune guinea pig T cells were stimulated with the large native molecule, human fibrinogen. Two different systems were tested. In the first, we determined responses by human fibrinopeptide B (hFPB)-immune T cells, to which strain (St.) 2 guinea pigs are responders and St. 13 are nonresponders, and by human fibrinopeptide A (hFPA)-immune T cells to which St. 13 are responders and St. 2 are nonresponders. Of interest in this comparison is that both hFPA and hFPB are amino terminal peptides on the A and B chain of fibrinogen, respectively, and are readily cleaved by thrombin during fibrin formation and by other trypsin-like enzymes, leaving a carboxyl terminal Arg. Thus, if fibrinogen catabolism occurred, both antigenic peptides should be equally represented for availability in T cell responses. It was found that hFPB-immune St. 2 T cells responded to fibrinogen, but no response was observed with hPFA-immune St. 13 T cells cultured with fibrinogen. To rule out that there was a general catabolic defect in St. 13 antigen-presenting cells, fibrinogen was presented by (2 X 13)F1 macrophages to fibrinopeptide-immune parental T cells. Again it was found that F1 macrophages could present fibrinogen to hFPB-immune T cells but failed to present hFPA. In another comparison, responses with fibrinogen were also determined with des-ARg-hFPB, which lacks the carboxyl terminal Arg of hFPB, to which St. 13 are responders and St. 2 are nonresponders. The advantage of this comparison is that both antigenic determinants are contained within the same small peptide. St. 13 des-Arg-hFPB-immune T cells failed to respond in vitro by culture with human fibrinogen, suggesting that these antigenic determinants are not produced from larger peptides or proteins containing those determinants. To rule out the possibility that this was only an in vitro phenomenon, guinea pigs were immunized with the larger protein, the B chain of fibrinogen, and the immune T cells were examined for responses to fibrinopeptides derived from the B chain. Immune St. 2 T cells responded to hFPB but not to des-Arg-hFPB, whereas St. 13 T cells remained unresponsive with both peptides. These results indicate that proteolysis of larger proteins to form small antigenic peptides is not a random event and that not all potential antigenic determinants contained in a protein are produced during antigen processing.     

Genetically restricted immune responses in guinea pigs primed in vivo with antigen-bearing macrophages.

Guinea pigs injected intradermally with antigen pulsed macrophages generate a population of immune T cells that proliferate in vitro on second exposure to antigen. T cells from F1 (2 X 13) guinea pigs immunized with DNP-OVA on one parental macrophage respond in vitro only to DNP-OVA on macrophages identical to those used for immunization and not to DNP-OVA associated with the other parental macrophages. These results demonstrate that the immunogenicity of antigen is dependent upon the macrophages used for priming in that, with this approach, strain 2 or 13 guinea pigs immunized with allogeneic macrophages pulsed with antigen do not respond to either allogeneic or syngeneic antigen-bearing macrophages. However, lysates of antigen-pulsed macrophages can still immunize either allogeneic or syngeneic recipient via their own macrophages. F1 (2 X 13) guinea pigs are immunized by insulin B chain pulsed strain 13 macrophages (responder) but not by strain 2 macrophages (nonresponder) suggesting that whether a F1 (nonresponder X responder) guinea pig recognizes antigen bound to a parental macrophage is genetically restricted before immunization to the same extent as the donor parental macrophages used for immunization.     

Genetic mapping of lymphocytic choriomeningitis virus pathogenicity: virulence in guinea pigs is associated with the L RNA segment.

The Armstrong CA 1371 (ARM) and WE strains of lymphocytic choriomeningitis virus (LCMV) differ in the ability to produce disease in adult guinea pigs. Infection with the ARM strain is not lethal, even at high virus doses (greater than 10,000 PFU), whereas the WE strain causes 100% mortality even at low doses (less than 10 PFU). To determine the genetic basis of this virulence, intertypic reassortants were made between the ARM and WE strains of LCMV. The two reassortants with the genotypes WE/ARM (L segment of WE and S segment of ARM) and ARM/WE (L segment of ARM and S segment of WE) were tested for their pathogenicity in guinea pigs. The ARM/WE reassortant was avirulent like the ARM/ARM parental strain. Minimal viral replication was observed in organs of guinea pigs inoculated with 10(2) or 10(5) PFU of ARM/ARM or ARM/WE, and all animals survived. In contrast, the WE/ARM reassortant was highly virulent like the WE/WE parental strain and killed all of the infected animals. High levels of viral replication were observed in guinea pigs infected with the latter two strains. In contrast to these in vivo observations, both the parental strains and the ARM/WE or WE/ARM reassortants had similar growth potential in cultured guinea pig fibroblasts. Thus, the L RNA segment of LCMV WE is important for viral replication in vivo and is associated with fatal acute disease after infection of adult guinea pigs.     

Synergistic effects of active specific immunotherapy and chemotherapy in guinea pigs with disseminated cancer

Sewall Wright strain 2 guinea pigs bearing pulmonary metastases of the syngeneic line 10 (L10) hepatocarcinoma were treated with a vaccine composed of 10(7) bacillus Calmette-Guérin admixed with 10(7) x-irradiated L10 tumor cells beginning 10 days after tumor inoculation. Although this treatment failed to cure most of the guinea pigs of their metastatic disease, histologic examination of the pulmonary tumors in the vaccinated guinea pigs provided evidence of a cell-mediated hypersensitivity response that disrupted the normally compact architecture seen in control tumors. When a monoclonal antibody against the L10 tumor was injected i.v. to evaluate the vascular permeability of the tumors, significantly more antibody localized in tumors of vaccinated guinea pigs than in tumors of untreated controls. These results suggested that blood-borne substances could be delivered more efficiently to L10 metastases after the tumor-bearing guinea pigs had been treated with vaccine. To determine whether such increased vascular permeability would enhance the antitumor effects of chemotherapeutic agents, combined immunotherapy and chemotherapy studies were performed. Although cyclophosphamide treatment by itself did not cure L10-bearing guinea pigs, cyclophosphamide used in conjunction with prior immunotherapy increased the survival rate of animals to more than twice that of animals treated with immunotherapy alone (74 vs 33%). These results suggest that one mechanism by which active specific immunotherapy enhances chemotherapy of disseminated tumors is by rendering tumor foci more permeable to subsequently administered cytotoxic drugs.     

Solubilization and partial characterization of a tumor-associated transplantation antigen of the guinea pig L2C leukemia.

A tumor-associated transplantation antigen (TATA) from guinea pig L2C leukemia cells was solubilized by different methods. It was found that the 3 M KCl extraction yielded the most immunogenic TATA of L2C cells. Immunization of normal strain 2 guinea pigs with this extract in complete Freund's adjuvant gave complete protection against a subsequent challenge with tumor cells. Further fractionation of the KCl extract of L2C cells by Sephadex G-200 chromatography suggested that the immunogenic activity was present in the fraction containing materials with estimated m.w. of less than 20,000 daltons.     

Isolation and characterization of an I-A-restricted T cell clone with dual specificity for poly(Glu60Ala30Tyr10) (GAT) and Mlsa,dl

We have isolated a BALB/c (H-2d, Mlsb) T cell clone (JTL-G12) specific for the synthetic polypeptide antigen poly(Glu60Ala30Tyr10) (GAT) in the context of self I-A determinants and for Mlsa,d antigens in the absence of GAT. JTL-G12 proliferation in response to GAT was mapped to the Kd, I-Ad subregions by using inbred H-2 congenic and recombinant strains. In addition, monoclonal antibody directed against I-Ad but not Kd or I-As determinants blocked JTL-G12 proliferation in response to GAT presented by syngeneic splenocytes, indicating I-A restriction. The Mls cross-reactivity of this clone was verified by using a panel of inbred strains bearing the Mlsa,b,c,d alleles and by using BXD recombinant inbred strains bearing the Mlsa allele or the Mlsb allele. All of the Mlsa BXD strains of the H-2d or H-2b haplotypes stimulated JTL-G12 in the absence of GAT, whereas all of the Mlsb BXD strains were nonstimulatory. This response pattern is in complete accordance with recognition of the Mlsa determinant encoded by Mls or closely linked loci on chromosome 1. JTL-G12 proliferation in response to GAT/I-Ad and Mlsa,d determinants could be blocked with a monoclonal antibody (GK1.5) directed against L3T4, a structure involved in class II major histocompatibility complex antigen recognition. These results suggest that antigen/class II responsiveness, Mls reactivity, and expression of L3T4 can be properties of a single T cell population.     

Alveolar macrophages in pulmonary immune responses. I. Role in the initiation of primary immune responses and in the selective recruitment of T lymphocytes to the lung

Antigen inoculated intratracheally (IT) into animals can induce primary immune responses and selectively recruit specific T cells to the lung. In the current study, the role of alveolar macrophages (AM) in these two responses was investigated. Antigen-pulsed bronchoalveolar cells (BAC) inoculated IT into guinea pigs generated a population of immune T cells that proliferated in vitro on reexposure to antigen-pulsed macrophages (M?). The possibility that antigen-pulsed donor BAC shed antigen that was subsequently processed and presented by host M? was ruled out by genetic experiments. Thus, peritoneal exudate lymphocytes (PEL) from (2 X 13)F1 guinea pigs primed with antigen-pulsed BAC from strain 2 animals responded preferentially to antigen-pulsed strain 2 M? rather than to antigen-pulsed strain 13 M?. In a second set of studies, antigen-pulsed BAC inoculated IT into guinea pigs selectively recruited antigen-specific T cells to the lung. Genetic experiments verified that inoculated BAC were the source of the antigen-presenting cells responsible for selective recruitment. Thus, antigen-pulsed strain 2 BAC inoculated IT recruited a greater proportion of (2 X 13)F1 T cells that recognized antigen in the context of strain 2 M? than F1 T cells that recognized antigen on strain 13 M?. Taken together, these studies suggest that AM contribute to the regulation of pulmonary immunity by both inducing T lymphocyte immunity and selectively recruiting specific T cells to the lung.     

Allogeneic manipulation of the GAT idiotypic cascade. Immunization of C57BL/6 mice by BALB/c anti-idiotypes stimulates similar strain-specific V genes as the original antigen

Antibodies specific for the immunizing Ag (Ab1) (Id+ Ag+) and Ab3 (Id+ Ag+ or Id+ Ag-) of the (Glu60 Tyr10 Ala30) (GAT) idiotypic cascade express similar pGAT public determinants in BALB/c and C57BL/6 strains. These determinants have been shown to be dependent upon both VH and Vkappa encoded segments. The VH of the BALB/c Ab1 (germ-line gene H10) and that of the C57BL/6 Ab1 (germ-line gene V186-2) are only 75% homologous, whereas VK are much more conserved. C57BL/6 mice were immunized with BALB/c Ab2 (anti-idiotypic) antibodies and monoclonal Ab3 were derived after fusion of immunized spleen cells with the nonsecreting hybridoma cell line Sp/2.0-Ag. From 13 cell lines, five clones (four Id+ Ag- and one Id+ Ag+) were isolated and the mRNA V regions sequenced. Immunization with BALB/c anti-idiotypes elicits expression of the same or closely related C57BL/6 VH and Vkappa genes as when C57BL/6 mice were immunized with GAT, although functional VH BALB/c equivalents have been isolated in the B6 strain. Our results suggest that manipulation of the repertoire via antigenic or idiotypic stimulation both lead to the expression of different genes in different strains. They further confirm that the immune system is largely degenerate, for both idiotype expression and Ag recognition.     

Immune response gene control of antibody specificity

The expression of the histocompatibility-linked PLL Ir gene was investigated in guinea pig B cells. Strain 2 and F₁ (2 × 13) guinea pigs, immunized with the αDnp-Lys₉, produce both T cells and antibody which are equally discriminatory for αDnp-Lys₉. In contrast strain 13 (PLL Ir gene negative) guinea pigs immunized with αDnp-Lys₉ do not develop specific T-cell responses and the antibody produced while restricted in heterogeneity cannot differentiate the immunizing antigen from Dnp-OH. However, if in a F₁ (2 × 13) environment, PLL Ir gene-negative B cells are provided with F₁ (2 × 13) T cells they express the ability to make antibody as specific and discriminatory as the antibody produced by PLL Ir gene-positive B cells. These findings strongly suggest that in the guinea pigs the PLL Ir gene defect is localized to the T cells and that the repertoire of specificity of B cells is similar if not identical in both responder and nonresponder animals. In addition these observations support the notion that the cellular locus for the PLL Ir gene expression in the guinea pigs is limited to T cells and not to macrophages and B lymphocytes.     

Major histocompatibility complex restriction of T-cell responses to varicella-zoster virus in guinea pigs.

Varicella-zoster virus (VZV), adapted to grow in guinea pig fibroblasts, was injected subcutaneously into Hartley, strain 2, and strain 13 guinea pigs. Serum immunoglobulin G antibodies were detected 2 weeks later, and T-cell proliferative responses by blood lymphocytes were found 3 weeks after injection. The proliferating cells bound the 155 antibody, which defines a CD4-like subset of guinea pig T lymphocytes. VZV-infected fibroblasts of human, Hartley, and strain 13 origin elicited equivalent amounts of proliferation, which was quantitatively greater than that obtained with an extracted VZV antigen. Uninfected (control) human or guinea pig fibroblasts did not elicit T-cell proliferation. The proliferative response to VZV required the presence of autologous (strain 2 or 13) antigen-presenting cells and was blocked by the addition of an anti-class II major histocompatibility complex antibody. Effector cells obtained from in vitro cultures mediated class II-restricted cytotoxicity to L2C cells incubated with VZV. Class I-restricted responses were obtained only by cross-priming strain 2 animals with strain 13 peritoneal exudate cells which had been preincubated with VZV. The data indicate that guinea pigs resemble humans in that class II-restricted T cells with specificity for VZV are more readily cultured from blood than are class I-restricted cells.     

Serological studies on the major histocompatibility complex of new inbred strains of the guinea pig.

New inbred strains of guinea pigs, JY 1, JY 2, JY 3, JY 6, JY 9 and JY 10 have been established in this Institute. Serologic studies of guinea pig leukocyte antigens (GPLA antigens) were carried out in order to examine their major histocompatibility complex (GPLA complex). Antisera specific for Ia antigens were raised by cross-immunization of NIH strain 2 (NIH 2) and NIH strain 13 (NIH 13) guinea pigs, well known inbred guinea pigs. The sera identified four distinct Ia specificities, which were designated as Ia.2a, Ia2b, Ia.13a and Ia.13b. Six antigenic specificities different from the above Ia specificities were identified by sera obtained by appropriate immunization of the inbred guinea pigs and were designated as P.1, P.2, P.3, P.4, P.5 and P.6. Antigenic specificities of GPLA antigens recognized in inbred guinea pigs were : NIH 2 (Ia.2a, Ia.2b, P.1, P.2, P.O, P.4), NIH 13 (Ia.13a, Ia.13b, P.1, P.2, P.3, P.4), JY 1 (Ia.13a, Ia.13b, P.5), JY 2 (Ia.2b, Ia.13b, P.3, P.4, P.6), JY 3 (Ia.13a, Ia.13b, P.2, P.4, P.5), JY 6 (Ia.2b, Ia.13b, P.3, P.6), JY 9 (Ia.13a, Ia.13b, P.4, P.5), JY 10 (Ia.13a, Ia.13b, P.2, P.3, P.4, P.6), JY 9 (Ia.13a, Ia.13b, P.4, P.5), JY 10 (Ia.13a, Ia.13b, P.2, P.3, P.4, P.6). The correspondence of these specificities to those already reported was discussed and the P.2 or P.4 was considered to be an additional specificity of GPLA antigens that have not been reported yet. As the new inbred strains of guinea pigs were thus found to possess characteristic GPLA complex, which differ from each other and from those of NIH 2 and NIH 13 strain, they should be useful for studies of roles of the major histocompatibility complex in the immune system.     

Idiotypic analysis of anti-GAT antibodies. III. Determinant specificity and immunoglobulin class distribution of CGAT idiotype.

The humoral response to poly-(L-glutamic acid60, L-alanine 30, L-tyrosine10), GAT, in mice is further characterized by both idiotype and fine specificity analyses. The common idiotype on murine anti-GAT antibodies (CGAT) was identified in anti-GAT antisera from seven additional strains of mice. These data confirm that the CGAT idiotype can be induced in all inbred strains of mice. Using a partially inbred strain of mice selected for their ability to respond to poly-(L-glutamic acid50, L-tyrosine50), GT, we demonstrated that the GT copolymer is capable of inducing antibodies that express the CGAT idiotype. In contrast, antisera directed against poly-(L-glutamic acid60, L-alanine40), GA, bind GAT but lack CGAT idiotype. These results indicate that GAT molecules contain determinants either similar or identical to those on GT molecules, which are responsible for the induction of CGAT idiotypic antibodies. We also demonstrated that both GAT responder and nonresponder strains of mice can produce anti-GAT antibodies with similar fine specificity patterns and CGAT idiotype. In addition, we demonstrate that antibodies of the IgM, IgG1, and IgG2 immunoglobulin classes express the CGAT idiotype.     

Characterization of the oncornavirus particles in the plasma of guinea pigs with L2C leukemia.

The inoculation of L2C guinea pig leukemia cells into strain 2 guinea pigs results in the death of the animals within 12 to 15 days. Death is preceded by the simultaneous appearance in the plasma of (i) elevated leukocyte levels, (ii) extracellular virus particles, and (iii) a particle-associated RNA-directed DNA polymerase. This enzyme activity has a cation preference identical to that of the type B bromodeoxyuridine-induced guinea pig virus, i.e., an Mg2+ optimum at 20 mM and no activity using Mn2+. Competitive molecular hybridization studies also revealed that the plasma of leukemic guinea pigs contained approximately 2 X 10(9) genome equivalents per ml of an RNA that is homologous to the RNA of the bromodeoxyuridine-induced guinea pig virus. Morphological observations indicate that most, but not all, of the extracellular particles observed in leukemia plasma are derived from the intracisternal particles seen in the L2C tumor cells. The possibilities that either two viral populations are present or that the in vivo morphogenesis of the type B bromodexoyuridine-inducible guinea pig virus is markedly different from its in vitro morphogenesis are discussed.     

Genetic control of immune response against random copolymers of glutamic acid and alanine (GA) and tyrosine (GT) in inbred mice.

An immune response (lr) gene(s) controlling responses to synthetic random copolymers of two amino acids of glutamic acid and alanine (GA) in mice has been well documented. This specific lr gene is linked to the major histocompatibility complex of the species. Mice of the H-2a,b,d,f,k,r and s haplotypes produce high titer antibodies and thus are high responders; whereas mice of the H-2 haplotypes j, ja, p and q produce no detectable antibody and are non responders. The response is "all or none" and high response is dominant. Attempts to detect an Ir-GT gene (glutamic acid and tyrosine) after immunization with two GT random copolymers were unsuccessful. However, GT-antibody could be produced after immunization with GT-methylated bovine serum albumin aggregates.     

RNA extracts of lymphoid cells sensitized to DNP-oligolysines convert nonresponder lymphoid cells to responder cells which release migration inhibition factor

Strain 13 nonresponder peritoneal exudate cells were converted to responder status to α or ?,DNP-oligolysines after incubation of the cells with RNA extracts prepared from responder guinea pigs skin test sensitive to these synthetic antigens. The conversion of nonresponder strain 13 cells was assessed by the direct cell migration inhibition correlate of delayed hypersensitivity. Nonresponder cells were not converted by RNA extracts prepared from unimmunized responder guinea pigs or from non-responder strain 13 guinea pigs previously injected with DNP-oligolysines. Thus, it seems possible to correct immunological unresponsiveness in vitro in spite of a specific genetically determined deficiency of the immune response related to the Ir gene.     

Evolutionary Divergence in the Structure of Myelin Basic Protein: Comparison of Chondrichthye Basic Proteins with Those from Higher Vertebrates

Abstract: A basic protein has been purified from the CNS myelin of the gummy shark (Mustelus antarticus). Electroblotting was used to examine the capacity of rabbit antisera raised against this electrophoretically pure protein to recognize myelin basic protein from higher vertebrates. The antisera bound to two shark proteins including the original polypeptide antigen and to chicken, bovine, and human myelin basic proteins. Thus, the shark protein appeared to possess antigenic determinants that have been retained through evolutionary divergence of these proteins. Whereas bovine basic protein caused experimental allergic encephalomyelitis in guinea pigs, animals that received injections of the shark protein showed neither clinical nor histological signs of this disease. However, tests for delayed-type hypersensitivity and for Arthus reaction following injection with the shark protein revealed a T-cell-mediated response to this antigen and substantial cross-reactivity with higher vertebrate basic proteins. Analysis of the amino acid composition of the shark protein, and comparison of its tryptic peptide map with that of the bovine protein, revealed substantial changes in the amino acid sequence. Although the shark protein has some antigenic determinants in common with the proteins from higher vertebrates, it appears that much of the structure differs.