Induction of transplantation tolerance in guinea pigs by spleen allografts. II. Responses in the mixed leukocyte reaction correlate with the tolerant state

We have developed a model for the induction of transplantation tolerance in the guinea pig by vascularized spleen allografts. Spleen allografts from strain 13 to strain 2 hosts frequently survived in healthy recipients without clinical GVHD or induced clinical GVHD. (2 x 13)F1 to strain 2 spleen allografts survived indefinitely without inducing GVHD. In contrast, strain 2 spleen allografts were rejected by strain 13 hosts. An excellent correlation was observed between the clinical course and the degree of reactivity to donor strain stimulator cells in the MLR. Animals that had rejected their grafts had normal or enhanced proliferative responses in the MLR. Strain 2 hosts with long-term surviving strain 13 or (2 x 13)F1 grafts had markedly suppressed anti-13 responses. Animals with GVHD had a suppressed MLR toward donor strain stimulator cells with simultaneous reactivity to host strain stimulator cells. Cells capable of suppressing the response of normal host strain cells to donor strain stimulators were present in some long-term surviving animals and may be responsible in part for the maintenance of the tolerant state.     

The effect of alloantisera on antigen-induced T cell proliferation.

In the guinea pig, alloantisera raised by cross-immunization of strain 2 and strain 13 animals are capable of specifically inhibiting the in vitro proliferative response of (2 X 13)F1 T lymphocytes to those antigens the response to which is controlled by Ir genes linked to the genes controlling the alloantigens against which the serum is directed. However, in similar studies performed in the two parental strains, the responses to antigens not known to be under unigenic control were also markedly inhibited by the appropriate alloantisera. We have extended our studies of this "nonspecific" inhibitory effect of alloantisera on T cell proliferation and have demonstrated that the proliferative response of strain 2 and strain 13 T cells to a large number of antigens is markedly inhibited by anti-2 and anti-13 sera, respectively. Antisera to the B alloantigen, the product of a linked but distinct histocompatibility locus, which is present in both strain 2 and strain 13 animals, also produced a marked inhibition of T-lymphocyte proliferation. A number of possible explanations for the generalized inhibitory effect of alloantisera on T cell proliferation are discussed.     

The role of the macrophage in genetic control of the immune response.

The ability of an animal to mount an immune response is controlled by a number of autosomal dominant immune response (Ir) genes that are linked to the major histocompatibility complex of the species. In the guinea pig, alloantiserums raised by cross-immunization of inbred strain 2 and strain 13 animals specifically inhibited the in vitro proliferative responses of (2 X 13) F1 lymphocytes to those antigens the response to which is controlled by Ir genes linked to the alloantigens against which the serums are directed. A genetic analysis indicated that the inhibitory activity of the alloantiserums was directed against the alloantigens rather than the products of specific Ir genes. The interaction of antigen-pulsed macrophages with immune T lymphocytes is also mediated by the 2/13 alloantigens and alloantiserums are capable of inhibiting macrophage-T lymphocyte interaction. Studies involving combinations of macrophages and lymphocytes that differed at alloantigens or Ir gene products or both raised the possibility of the expression of the Ir gene product in the macrophage.     

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.     

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.     

Nature of the antigenic complex recognized by T lymphocytes. V. Genetic predisposition of independent F1 T cell subpopulations responsive to antigen-pulsed parental macrophages.

Nonimmune (2 x 13)F1 guinea pig T lymphocytes initially stimulated with trinitrophenyl- (TNP) modified macrophages from one parental strain and then treated with bromodeoxyuridrine (BUdR) and light are unable to be primed subsequently with TNP-modified macrophages of the same parental strain. In contrast, these BUdR and light treated F1 T cells can be primed with TNP-modified macrophages of the other parental strain. These results demonstrate that in a nonimmune F1 animal two T cell subpopulations exist before priming that are genetically predisposed to respond to antigen associated with macrophages derived from one or the other parent.     

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.     

The immunopathology of experimental allergic encephalomyelitis (EAE). III. Differential in situ expression of strain 13 Ia on endothelial and inflammatory cells of (strain 2 x strain 13)F1 guinea pigs with EAE

This study was undertaken to determine if there is differential expression of the relevant strain-specific Ia antigen on endothelial and parenchymal inflammatory cells in the central nervous systems (CNS) of guinea pigs (GP) with acute experimental allergic encephalomyelitis (EAE). Adult inbred GP were sensitized with GP spinal cord homogenate and complete Freund's adjuvant. Strain 13 GP and (2 x 13)F1 hybrids developed clinical disease within 2 to 3 wk after sensitization, whereas strain 2 GP did not, although all sensitized GP had CNS mononuclear inflammatory infiltrates. By using monoclonal antibodies to strain-specific and framework Ia epitopes with an immunoperoxidase technique, the distribution and amount of the strain 2 and strain 13 Ia were analyzed. Equivalent strain 2 and strain 13 Ia expression was found in normal tissues from F1 animals. Strain 2 and strain 13 GP sensitized for EAE had increased strain-specific Ia staining of CNS vessels and inflammatory cells over controls. However, F1 GP with EAE had markedly increased strain 13, but not strain 2, Ia on CNS parenchymal vessels and mononuclear inflammatory cells (p less than 0.001, for both). These results suggest for the first time that specific major histocompatibility complex gene products are selectively expressed on endothelial and inflammatory cells in situ in immune reactions in the target organ of individuals of heterogeneous immunogenetic composition.     

Specific absorption of T lymphocytes committed to soluble protein antigens by incubation on antigen-pulsed macrophage monolayers.

Monolayers of macrophages (Mphi) pulsed with antigen were used as immunosorbents for T lymphocytes from guinea pigs primed to soluble protein antigens. T lymphocytes were cultured on the Mphi monolayers for 4 hr, then aspirated and reincubated on a fresh monolayer pulsed with the same antigen for a second and a third step. T lymphocytes so treated were selectively deprived of cells responding in assay for antigen-dependent proliferation against the antigen used for pulsing the absorbing monolayer, but maintained their response to other antigens. The lymphocytes adhering to the Mphi of the absorbing monolayer were capable of giving a full response to the antigen used for pulsing the Mphi of the monolyers. The proliferative response of F1 T lymphocytes to antigen in association with Mphi of either parental strain could be absorbed leaving the response to antigen in association with Mphi of the other parental strain. The absorption of the proliferative response was not inhibited by addition of excess soluble antigen to the medium of the absorption culture. Our results indicate that specific guinea pig T lymphocytes responding by proliferation to soluble protein antigens recognize and bind specifically to a complex of Ia antigen and protein antigen at the surface of the Mphi.     

In vitro studies of the genetically determined unresponsiveness to thymus-independent antigens in CBA/N mice.

The X-chromosome-linked B lymphocyte defect of CBA/N mice has been studied in vitro by comparing the ability of (CBA/N X DBA/2)F1 (X-/X- X X+/Y) male (X-/Y) and female (X-/X+) spleen cells to respond to the thymus-independent antigen DNP (or TNP)-AECM-Ficoll. (CBA/N X DBA/2)F1 male spleen cells failed to generate significant in vitro anti-TNP antibody responses to DNP- or TNP-AECM-Ficoll, in contrast to spleen cells from F1 female (X-/X+) mice which responded normally to these T-independent antigens. Spleen cells from male F1 mice responded almost as well as F1 female cells to the thymus-dependent antigen, TNP-sheep red blood cells (TNP-SRBC) in vitro. Adding F1 male cells to F1 female cells failed to reduce the response of the latter to DNP-AECM-Ficoll, suggesting that the inability of F1 male cells to respond was not due to active suppression. The response of F1 male spleen cells to TNP-SRBC was not impaired by adding high concentrations of TNP-AECM-Ficoll indicating that the mechanism of unresponsiveness was not tolerance induction in all TNP-specific precursors. Lymphocytes from F1 male mice were capable of forming anti-TNP antibody after stimulation with lipopolysaccharide (LPS) in high concentrations; DNP-AECM-Ficoll had no effect on this polyclonal response. B lymphocytes from mice bearing only the X-chromosome of the CBA/N strain thus display a profound defect in B cell activation. This functional defect may represent either an inability of the defective B cells to be activated by thymus-independent antigens or the absence of a sub-class of B cells which respond to thymus-independent antigens.     

Suppression of experimental autoimmune thyroiditis in guinea pigs by pretreatment with thyroglobulin-coupled spleen cells

Pretreatment of Strain 2 and Strain 13 guinea pigs with guinea pig thyroglobulin (GPTG) coupled to syngeneic spleen cells (GPTG-SC) suppressed the development of experimental autoimmune thyroiditis (EAT) induced by immunization with GPTG in complete Freund's adjuvant (CFA). Antibody titers to GPTG were only minimally suppressed in GPTG-SC pretreated animals. GPTG-SC also suppressed the sensitization of periotneal exudate T lymphocytes which proliferate in vitro in the presence of GPTG.     

Suppressors of the graft vs graft reaction in tolerance to alloantigens

Immune response and suppressor cell activity of CBA (H-2k) mice made tolerant to allogeneic C57B1/6 (H-2b) heart graft were studied in graft-versus-graft reaction (GvGR). Intact CBA spleen cells inhibited response of (CBA X C57B1/6)F1 cells to antigenic stimulus (sheep red blood cells--SRBC), when injected together into lethally irradiated (CBA X C57B1/6)F1 mice. Spleen cells of tolerant mice were unable to decrease immune response of (CBA X C57B1/6F1 lymphocytes to SRBC and suppressed specifically the inhibition induced by intact CBA spleen cells. Spleen cells from tolerant mice were also capable of suppressing GvGR induced by CBA lymphocytes immune to C57B1/6 cells. Pretreatment of tolerant spleen cells with rabbit antithymocyte globulin and complement before adoptive transfer diminished markedly the suppression. The results obtained in the study suggest that suppression of transplantation immunity in this model is mostly due to T suppressor cells.     

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.     

Swine refusal factors elaborated by Fusarium strains and identified as trichothecenes.

Fusarium poae (Peck) Wollenw. NRRL 3287, F. nivale (Fr.) Ces. NRRL 3289, and F. moniliforme Sheldon NRRL 3197, each grown on cracked corn (13 days at 28 degrees C), produced refusal factors in pig bioassays. Substantial quantities of trichothecenes were detected in the refused corn: T-2 toxin (30 micrograms/g) was detected in corn fermented with the F. poae strain; the level of vomitoxin (1 microgram/g) in corn cultured with F. nivale did not account for the 48% refusal response in the pigs tested. The F. moniliforme concomitantly produced T-2 toxin (33 micrograms/g) and vomitoxin (1.5 micrograms/g). This strain's taxonomic position was reexamined, and it is shown to be a cultural variant of the species F. tricinctum (Cda.) Sacc.     

Normal tissue alloantigens and genetic control of susceptibility to tumors: microcytotoxicity studies on resistant C3Hf and susceptible (A X C3Hf) F1 mice inoculated with transplacentally induced C3Hf lung tumor.

The immune response of mice to a transplacentally induced lung tumor was investigated with the microcytotoxicity (MC) assay. The tumor, originally induced in C3Hf mice, does not grow readily when transplanted to normal syngeneic C3Hf recipients. It grows readily, however, in (A C3Hf)F1 hybrids and in strain C3H mice, which express in their normal lung tissue a component which constitutes a strong lung tumor-associated transplantation antigen (TATA) in C3Hf mice. Both lung tumor-immunized C3Hf and tumor-bearing (A X C3Hf)F1 and C3H mice possessed lymphoid cells reactive against cultured lung tumor cells in the MC assay. Reactivity was also observed against cells cultured from normal lungs of (A X C3Hf)F1 and C3H mice, but not against cells similarly cultured from C3Hf of C57BL/6 mice. Anti-tumor MC was inhibited by serum-blocking factors present in some but not all tumor-bearing and tumor-immunized mice. The MC assay and detection by it of serum-blocking factors does not distinguish the effective anti-C3Hf lung tumor immune response of immunized C3Hf mice from the ineffective immune response of tumor-bearing (A X C3Hf)F1 and C3H mice. Furthermore, in lung tumor-bearing mice cells reactive in the MC assay may be directed against a normal tissue antigen rather than a tumor-associated antigen.     

AKR/J gene(s) unlinked to H-2 determines dominant inheritance of lymphocyte hyporesponsiveness to acetylcholine receptor

Mice with the H-2b major histocompatibility complex haplotype are high immune responders to nicotinic acetylcholine receptors (AChR), whereas mice with the H-2k haplotype are generally low responders. F1 progeny of C57BL/6 (H-2b) mice crossed with mice of most H-2k strains are high responders to AChR in standard conditions of testing helper T cell proliferation in vitro (4 X 10(5) lymph node cells/microwell, 1 wk after primary challenge in vivo). In contrast, the F1 progeny of AKR/J (H-2k) crossed with high responder (H-2b) strains (B6, A.BY, or C3H.SW) were all hyporesponsive to AChR when lymphocytes were tested at 4 X 10(5) cells/well. However, at a density of 1 X 10(6) or greater/well, a high level of antigen-specific responsiveness was demonstrable in the F1 hybrid lymphocytes. A shift from low to high responsiveness to AChR at high cell densities was observed also in the H-2b strain AKR.B6. Other strains previously demonstrated to be low responders to AChR did not become responsive to AChR when lymphocyte numbers were increased to 1.4 X 10(6)/well. The N2 generation yielded by backcrossing (AKR X B6)F1 mice to AKR/J were all low responders, whereas N2 progeny derived by backcrossing F1 to B6 were high or low responders in a ratio of approximately 1:1 (independent of their H-2 phenotype). Results consistent with this observation were obtained in (AKR X B6) F2 mice. These data suggest that at least one AKR/J gene outside of the H-2 complex exerts a hyporesponsive influence on the I-A-dependent helper T cell response to AChR in H-2b mice.     

Stimulation of mouse lymphocytes by a mitogen derived from Mycoplasma arthritidis. VII. Responsiveness is associated with expression of a product(s) of the V beta 8 gene family present on the T cell receptor alpha/beta for antigen

Mycoplasma arthritidis produces a potent soluble T lymphocyte mitogen (MAM) which is dependent upon accessory cells bearing the alpha-chain of the I-E molecule (E alpha). Lymphocytes from the RIIIS mouse strain which possess E alpha yet whose T cells fail to recognize the MAM-E alpha complex were shown not to express V beta 8.1, 8.2 or 8.3 gene products present on the TCR-alpha/beta by virtue of their lack of reactivity with the F23.1 mAb. Because lymphocytes from the congenic B10.RIII mouse strain reacted positively with F23.1, we examined the progeny from (RIIIS x B10.RIII)F1 x RIIIS backcross mice for cosegregation of lymphocytes expressing F23.1-reactive sites and ability to proliferate in response to MAM. Whereas lymphocytes of all mice responded to Con A, only lymphocytes from progeny expressing F23.1-reactive cells responded to MAM. Similar studies were conducted on progeny from the F23.1- SWR mouse which was backcrossed to (SWR x B10.RIII)F1 mice. Of the E alpha-bearing progeny, there was a direct correlation between lymphocyte expression of F23.1 determinants and ability to respond to MAM. These results established that MAM reactivity was dependent upon a product(s) of the V beta locus of the TCR-alpha/beta. Nodal and thymic lymphocytes cultured for 3 days in vitro with MAM exhibited clonal expansion of F23.1 and F23.2-reactive cells as compared with cultures treated with Con A. We also demonstrated that F23.1 and F23.2 mAb inhibited the ability of lymphocytes to proliferate in response to MAM but had little effect on responses to Con A. The combined data suggest that the MAM-E alpha complex can utilize a V beta 8 gene product(s) on the TCR-alpha/beta.     

A novel guinea pig macrophage-specific polymorphic molecule. II. Biochemical analysis of the polymorphism

We have identified a macrophage-specific molecule, termed gp98, which has a m.w. of 98,000, is encoded by a gene not linked to the guinea pig lymphocyte antigen complex, is highly immunogenic, and displays a serologic polymorphism among several inbred guinea pig strains. The gp98 molecule was biochemically analyzed to identify a basis for the serologically detected polymorphism. The molecule was demonstrated to be a glycoprotein containing N-linked oligosaccharides. The strain 2 serologic variant, gp98-2, migrated with an apparent m.w. approximately 2500 more than did the strain 13 variant gp98-13. This differential migration was observed in a (strain 2 X strain 13) F1 animal, and persisted after neuraminidase and endoglycosidase F treatment, and after reduction. Trypsin and endoproteinase Lys-C digestion localized the biochemical basis of the polymorphism to the peptide portion of the molecule. Biochemical analysis of the gp98 molecules from five different inbred strains indicated that only two biochemical variants correlating with the serologic variants existed among the five strains.     

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.     

Strain differences in aryl hydrocarbon hydroxylase induction by 3-methylcholanthrene in rabbits.

We determined both basal and induced AHH activity in livers of six partially inbred strains of rabbits. Strain III rabbits had the highest enzyme activity upon induction by 3-MCA, i.e., four to five times that in strain WH (noninducible), which has the lowest enzyme activity. AHH induction was also "low" in strains X, OS, ACEP, and AC. F1 hybrids between strains III and WH revealed a differential response to the induction of liver AHH activity by MCA: the levels of induced hydroxylase activity were consistently higher in (III X WH)F1 rabbits than in the reciprocal (WH X III)F1 hybrids. All possible crosses between these two "extreme" strains are now being analyzed to estimate the number of genes involved in their response difference to MCA.