Nature of T lymphocyte recognition of macrophage-associated antigens. III. Definition of the antigenic regions of human fibrinopeptide B involved in guinea pig T-cell responses

The clonal diversity of guinea pig T-lymphocyte responses to the 14-amino-acid peptide antigen human fibrinopeptide B (hFPB, Bβ1–14) and sequential hFPB³ homologs (Bβ5–14 and 7–14) was examined using bromodeoxyuridine (BUdR) and light elimination of T cell responses. PPD and hFPB-immune strain 2 guinea pig T cells and macrophages were stimulated in a first culture with PPD, Bβ1–14, 5–14, or 7–14; BUdR was added on the second day and the cultures exposed to light on the third day. The BUdR and light-treated T cells recovered from the first culture were restimulated in a second culture containing fresh stimulator macrophages and PPD, Bβ1–14, 5–14, and 7–14. BUdR and light-treated T cells initially stimulated with Bβ1–14 in the first culture showed no responsiveness to Bβ1–14, 5–14, or 7–14 in the second culture. BUdR and light-treatment of T cells initially stimulated with Bβ5–14 eliminated 70 to 80% of the subsequent response to Bβ1–14 and all of the responsiveness to Bβ7–14. Similar treatment of T cells stimulated with Bβ7–14 reduced responsiveness to Bβ1–14 by 50 to 60% and to Bβ5–14 by 60 to 70%. These observations indicate that T-cell responses are directed against three antigenic regions in the hFPB molecule; the major region defined by the carboxy-terminal sequence including residues 7 to 14, a second minor antigenic region including residues 5 and 6, and a third minor region including the amino terminal residues 1 to 4. Results are discussed with respect to the regions of the hFPB molecules that are recognized by antigen-binding T-cell receptors and the regions which interact with stimulator macrophages.     

Nature of T lymphocyte recognition of macrophage-associated antigens. IV. Inhibition of peptide antigen presentation by brief treatment of macrophages with anti-Ia serum

To examine the role of macrophage la antigens in T-lymphocyte stimulation, guinea pig macrophages were briefly treated with anti-Ia serum before or after antigen pulsing with the peptide antigen human fibrinopeptide B (hFPB). To assess their antigen-specific stimulatory capacity, the variously treated macrophages were added to culture with hFPB-immune guinea pig T cells and stimulation was determined by the incorporation of [³H]thymidine. Macrophages treated with anti-Ia serum before antigen pulsing stimulated T-cell responses equivalent to those observed with antigen-pulsed macrophages treated with normal serum. By contrast, brief anti-Ia treatment of macrophages immediately following a 2-hr antigen pulse reduced subsequent T-cell responses by 45 to 70%. Similar treatment of macrophages pulsed with antigen for only 1 hr produced only modest inhibition of T-cell responses. However, if macrophages pulsed for 1 hr with antigen were incubated several hours before brief anti-Ia serum treatment, the subsequent T-cell responses were reduced by 40 to 60%. This inhibition was specific for antiserum directed against Ia antigens of the guinea pig MHC, since brief macrophage treatment with antiserum directed against B.1 antigens, the guinea pig equivalent of murine H-2K and H-2D antigens, produced no inhibition of their T-cell stimulatory capacity. These results are discussed with respect to the formation of the immunogen presented by macrophages for T-cell recognition.     

Expression of human T lymphocyte antigens by killer cells.

K cells, the effectors of antibody-dependent cell-mediated cytotoxicity, were found to express human T but not B lymphocyte antigens detected by rabbit anti-HTLA and anti-HBLA. Pretreatment of effector cells with anti-HTLA+C inhibited ADCC by specifically lysing K cells: no inhibition of ADCC by anti-HTLA occurred when deltaC was substituted for C. By contrast, pretreatment of effector cells with anti-HBLA nonspecifically inhibited ADCC, probably for forming antigen-antibody complexes with HBLA+ cells in effector suspensions: a) treatment with anti-HBLA deltaC was more inhibitory of ADCC than treatment with anti-HBLA+C, and b) the inhibitory effect of anti-HBLA on ADCC was either eliminated or markedly reduced if effector suspensions were first passed through a nylon fiber column, a procedure that removed most HBLA+ cells without affecting K cell activity. HTLA antigens expressed by K cells and NK cells are the same as HTLA antigens expressed by thymocytes since thymocytes completely absorb the anti-K cell and NK cell reactivity of anti-HTLA.     

Functional differentiation in the genetic control of murine T lymphocyte responses to human fibrinopeptide B

The ability to generate proliferative and helper T lymphocyte responses in mice was compared by using the 14 amino acid peptide, human fibrinopeptide B (hFPB). Lymph node or peritoneal exudate T cells from mice immunized with hFPB were assessed for in vitro proliferation to soluble hFPB as determined by the uptake of 3H-thymidine. The T cell proliferative response to hFPB was found to be under MHC-linked Ir gene control; mice possessing the H-2a,k haplotypes were responders, whereas H-2b,d,q,s mice were nonresponders. The influence of non-H-2 genes on these responses was not investigated, so exclusive regulation by H-2 is provisional. The absence of a detectable lymph node and peritoneal exudate T cell proliferative response persisted in H-2b,d,q,s mice after immunization and boosting with several doses of hFPB. In addition, the capacity to produce a T cell proliferative response was inherited in an autosomal dominant manner and gene(s) controlling responsiveness to hFPB mapped to the I-A subregion of the H-2 complex. To measure peptide-specific helper T cell activity, an in vitro microculture assay in which hFPB-primed lymph node T cells and normal spleen B cells and macrophages were used was developed measuring anti-fluorescein isothiocyanate (FITC) IgM and IgG plaque-forming cell (PFC) responses after culture with FITC-conjugated peptide. Immunization of B10.BR, C57BL/10, B10.D2, and B6AF mice with hFPB primed for significant helper T cell activity as assessed by the ability to augment a primary in vitro IgM response to FITC. The normal B cell IgM responses were completely dependent on hFPB-primed T cells and required that hapten (FITC) and carrier (peptide) be linked. In addition, immunization with FITC-conjugated peptide elicited positive in vivo PFC responses to FITC in B10.BR and C57BL/10 mice, indicating similar genetic control of helper activity in both the intact animals and the in vitro microcultures. Thus, B10.BR mice show both T help and T proliferative responses to hFPB, whereas C57BL/10 mice show only T help and no T proliferative responses. In contrast to B10.BR mice, C3H and CBA mice immunized with hFPB were completely unresponsive when assayed for helper T cell activity in vitro despite their ability to generate positive lymph node T cell proliferative responses. These results indicate responsiveness to hFPB by T helper and proliferating cells is different and is under separate genetic control.     

The function of macrophages in antigen recognition by guinea pig T lymphocytes. III. Genetic analysis of the antigens mediating macrophage-T lymphocyte interaction.

Activation of immune T lymphocytes by antigen-pulsed macrophages is mediated by the Ia antigens of the guinea pig MHC or the products of closely linked genes. Studies using combinations of macrophages and T cells derived from outbred animals with different Ia antigens and/or Ir gene products have demonstrated that sharing of Ir gene products between macrophage and T cells is not sufficient for effective macrophage-T cell interaction. The role of the Ia antigens in the absence of the linked Ir gene products could not be directly examined because we were unable to identify an animal which bore the full complement of Ia antigens in the absence of the Ir gene that is normally associated with them. The results of these studies support the concept of the functional expression of the Ir gene product in the macrophage.     

Viral-restricted cytolytic T lymphocyte recognition of hybrid human-murine class I histocompatibility antigens

Hybrid human-murine major histocompatibility antigens have been constructed and expressed on the surface of both human RD and murine L cell lines after DNA mediated gene transfer. These antigens linked the polymorphic domains (alpha 1 and alpha 2) of H-2Kb and the carboxy-terminal domains (alpha 3, transmembrane, and intracellular) of HLA-A2. Previously we demonstrated that these antigens were serologically intact and were recognized by allospecific cytolytic T lymphocytes. However, the cell lines expressing the hybrid antigen were less well lysed than the native H-2Kb expressing cell lines. In this study, we extend these observations and demonstrate that virally restricted cytolytic T lymphocytes specific for vesicular stomatitis virus and for Sendai virus can recognize cell lines expressing the hybrid antigen, whether expressed on murine (L cell) or human (RD cell) lines. Furthermore, the data show a profound influence by the carboxy-terminal domains upon the polymorphic T-cell restricting epitopes.     

Defining cytolytic T lymphocyte recognition of chemically modified self. I. Response to trinitrophenyl-H-2Kk

We used purified class I antigen incorporated into liposomes to examine the response of secondary cytolytic T lymphocytes (CTL) to chemically modified self. By generating the secondary response in the presence of T cell helper factor, the level of CTL response was limited by CTL recognition of added antigen rather than by helper cell generation of lymphokines. We found a strong secondary response against chemically modified self when spleen cells from trinitrophenyl (TNP)-primed C3H/HeJ mice were stimulated with a) TNP-modified liposomes containing H-2Kk, b) liposomes containing H-2Kk purified from TNP-modified RDM-4 (H-2k) cells, or c) liposomes containing the limited trypsin proteolysis product of H-2Kk that had been directly modified with TNP. In contrast, we were not able to generate a significant CTL response with unmodified H-2Kk incorporated into vesicles along with TNP-modified membrane components lacking H-2Kk. These results suggest that TNP-modified H-2Kk is a major antigenic site recognized by CTL from C3H/HeJ mice after priming against TNP-modified self.     

T lymphocyte-dependent B lymphocyte proliferative response to antigen. II. Induction of polyclonal B lymphocyte activation of normal B cells and of Lyb-5- B cells from xid mice via recognition of self-IA antigens

We extended our investigations into the genetic requirements and antigen dependence for the induction of polyclonal B lymphocyte proliferation by primed T lymphocytes. By using recombinant inbred mouse strains and antigen-specific T lymphocyte clones that lack alloreactivity, the genetic requirement was mapped to the IA subregion of the MHC. Furthermore, approaches that prevented or limited the accessibility of antigens to the B lymphocyte surface demonstrated that antigen binding onto the B lymphocyte surface was probably not necessary for induction of B lymphocyte proliferation. These experiments suggest strongly that T lymphocyte recognition of B lymphocyte Ia molecules in the absence of sIg cross-linking or in the absence of antigen bound nonspecifically to B lymphocytes can cause cellular activation. Similar T lymphocyte-dependent B lymphocyte activation was seen when Lyb-5- cells from CBA/N mice with the xid defect were cultured. Increases in the number of cells secreting immunoglobulins could be detected in the proliferating B lymphocyte cultures, suggesting that the culture conditions had fulfilled the requirements for B lymphocyte differentiation into antibody-producing cells. Although anti-Ig did not interfere with the B lymphocyte proliferative responses, it did diminish the number of cells secreting immunoglobulins. The implications of these experiments in extending our understanding of the activation pathway of Lyb-5- and Lyb-5+ B lymphocytes are discussed.     

Cytotoxic T lymphocyte recognition of HLA-A/B antigens introduced into EL4 cells by cell-liposome fusion

HLA-A2 and -B7 antigens were introduced into EL4 (H-2b) cells by cell-liposome fusion and were used as targets or stimulators for cytotoxic T lymphocytes (CTL) generated in C57B1/6 (H-2b) mice. It was found that such EL4-HLA cells were not recognized by CTL that had been raised against either a human cell line bearing these HLA antigens or the purified HLA-A2 and -B7 antigens reconstituted into liposomes. In addition, EL4-HLA cells were not capable of inducing CTL that could recognize a human cell line bearing HLA-A2 and -B7 antigens. Instead, EL4-HLA cells induced CTL that specifically lysed EL4-HLA cells and not human cells expressing HLA-A2 and -B7. CTL recognition required the presence of HLA antigens on the EL4 cell surface and was inhibited by antibodies against either H-2b or HLA-A/B. Monoclonal antibody binding studies showed that the expected polymorphic determinants of the HLA-A2 and -B7 antigens were still present on EL4-HLA cells. However, the specificity of CTL or their precursors that are capable of recognizing HLA-A2 or -B7 was altered after these antigens became associated with the EL4 surface. Possible explanations for these results are discussed.     

Cytotoxic T lymphocyte recognition of class I H-2 antigens after DNA-mediated gene transfer

Cytotoxic T lymphocyte (CTL) recognition sites on class I major histocompatibility complex molecules have been investigated by several laboratories by using cloned genes expressed on mouse L cells by DNA-mediated gene transfer. Recombinant genes, constructed by restriction endonuclease treatment of cloned H-2Dd and Ld genes and exchange of the N and C1 exons (exon shuffling) have provided an additional tool. These hybrid H-2 molecules expressed on L cells have been used as targets to achieve more precise localization of site(s) recognized by allospecific and virus-specific CTLs. CTL systems were chosen that limit recognition to either the Dd or Ld alloantigen or to virus and Dd or Ld complexes. Using this approach, we were able to map essential restricting site(s) to the N and/or C1 domains. Additional evidence is presented that the cytoplasmic tail of H-2 may be involved in interactions with some viral antigens and effect the formation of an immunogenic complex.     

On the mechanism of binding of human lymphocyte products to guinea pig macrophages.

When lymphocytes from a majority of patients with cancer are incubated with encephalitogenic factor, a lymphocyte product is released that reduces the anodic electrophoretic mobilities of guinea pig macrophages and fixed, tanned sheep erythrocytes. Although these reactions are not specific for cancer, it is distinctly possible that in patients with cancer, products from stimulated lymphocytes are capable of altering the surfaces of the patients' own macrophages, thereby modifying the course of their disease. In this paper, we attempt to elucidate some mechanisms for the binding of lymphocyte products to macrophages, such as occurs in the macrophage electrophoretic mobility (MEM) test, since this may be of general interest. Binding of lymphocyte product to macrophages has been monitored by measurements of their electrophoretic mobilities and by electron microscopic determination of the density of binding of electron-dense, cationic colloidal iron hydroxide particles to their surfaces. The results show that the lymphocyte products reduce the net surface negativity of the macrophages by (coulombic) binding of this net positively charged material to sialic acids at the macrophage surface. Product-binding can be prevented by prior treatment of the macrophages with neuraminidase. It appears that only a minority of sialic acids are involved in the binding process, which occurs without demonstrable blocking of adjacent sialic acids or redistribution of such sites over the macrophage surface. Parallel experiments with fixed tanned erythrocytes also suggest that binding of lymphocyte product is not solely determined by surface sialic acids, although it cannot occur without them.     

Characterization of an antiserum to guinea pig antithrombin III (AT III). I. Reactivity with guinea pig T lymphocytes

In the course of studies investigating the effects of antisera prepared against a variety of guinea pig proteins on lymphocyte function, a goat antiserum prepared against a guinea pig gamma-globulin preparation was found to react with guinea pig T lymphocytes. This antiserum, serum 592, contained a significant titer of antibodies that were cytotoxic for a subpopulation of lymph node cells and thymocytes, and mitogenic for lymph node T cells. Immunoelectrophoretic analysis and selective absorptions of the antiserum demonstrated that the antigen recognized on thymocytes was also present on an alpha 2 globulin in guinea pig plasma, which, on the basis of physiochemical characteristics and heparin-binding affinity, appeared to be guinea pig antithrombin III (AT III). Although the antiserum was shown to contain antibodies to both protein and carbohydrate determinants on the AT III molecule, studies comparing the effects of 7 M guanidine and periodate oxidation on the antigenicity of the AT III determinant also recognized on the thymocytes indicated that this shared antigenic determinant was carbohydrate in nature. The thymocyte membrane molecule bearing this determinant was also isolated and was found to be a 210,000-dalton macromolecule that was very sensitive to proteolytic and/or autolytic degradation. These data raise the interesting possibility that guinea pig lymphoid cells may have a membrane-associated protease inhibitor related to plasma AT III.     

Mouse B and T lymphocyte responses to purified timothy pollen antigens in vitro.

Purified populations of splenic B and T lymphocytes from LAF mice immunized with a crude extract of timothy pollen (WST) responded specifically to pollen antigens in an in vitro lymphocyte transformation system. The peak lymphocyte transformation response occurred 5 days after a secondary immunization and was the result of T-B cell cooperation in vitro. Wtih two purified pollen antigens as in vitro stimulants we were able to define at least two antigen-specific population of B cells and one population of T cells. These results were confirmed by inhibition studies with a monovalent hapten from WST, Antigen D.     

Antigen-specific regulation of T lymphocyte proliferative responses to contact-sensitizing chemicals in the guinea pig

Skin painting of guinea pigs with either 4-ethoxymethylene-2-phenyloxazol-5-one or 2,4-dinitrofluorobenzene induced not only a primary proliferative response in the draining lymph node but also the systemic suppression of subsequent proliferative responses to topically applied hapten. The inhibition of lymphocyte proliferation, as assessed by the incorporation of [3H]-thymidine and the presence of large pyroninophilic cells in the paracortex, was hapten-specific and long-lasting. This study demonstrates that, in common with the mouse, the sensitization of guinea pigs results in the induction of a hapten-specific suppressor mechanism, which serves to control the proliferative response following reexposure to hapten. However, the antigen-nonspecific suppression of proliferation observed in the mouse following exposure to some potent contact sensitizers was not, under the conditions employed, detectable in the guinea pig.