An approach to the unification of suppressor T cell circuits: a simplified assay for the induction of suppression by T cell-derived, antigen-binding molecules (T-ABM)

A system is presented in which the in vitro response to sheep red blood cells (SRBC) can be regulated using antigenic determinants coupled to SRBC and T cell-derived antigen-binding molecules (T-ABM) directed against the coupled determinants. T suppressor-inducer factors (TsiF's) are composed of two molecules, one of which is a T-ABM and one which bears I-J determinants (I-J+ molecule). Using two purified T-ABM which have not previously been shown to have in vitro activity, we produced antigen-specific TsiF's which were capable of inducing the suppression of the anti-SRBC response. Suppression was found to require both the T-ABM and the I-J+ molecule, SRBC conjugated with the antigen for which the T-ABM was specific, and a population of Ly-2+ T cells in the culture. Two monoclonal TsiF (or TsF1) were demonstrated to induce suppression of the anti-SRBC response in this system, provided the relevant antigen was coupled to the SRBC in culture. The results are discussed in terms of the general functions of T-ABM in the immune system. This model will be useful in direct, experimental comparisons of the function of T-ABM and suppressor T cell factors under study in different systems and laboratories.     

Clonal analysis of antigen-specific interactions between T cells and genetically engineered B cells

In order to investigate T cell-B cell interactions we constructed monoclonal, antigen-specific T- and B-cell populations. The Ia+ B-cell lymphoma A20-2J was transfected with trinitrophenyl (TNP)-specific heavy (mu) and light (kappa) chain Ig genes. A hapten-carrier complex (TNP-keyhole limpet hemocyanin (KLH)) bound to the surface Ig expressed on the transfectant and was presented to carrier-specific T-cell hybridoma clones at markedly low doses of antigen (0.01 microgram/ml) and in an Ia-restricted fashion. Two responses were elicited in the responding T-cell clones: (i) high levels of IL-2 secretion (320 units/ml), and (ii) cytotoxicity directed against the antigen-presenting B cell. This cytotoxicity was inhibited by D-mannose and was directed against innocent bystander cells, unlike cytotoxicity mediated by NK cells or alloreactive cytotoxic T lymphocyte. Helper and cytotoxic functions were often present in different T-cell hybridomas but some clones exhibited both activities. One representative T-cell hybridoma exhibited strong helper function for TNP-primed splenic B cells as detected in a plaque-forming cell assay, but was cytotoxic toward antigen-presenting B cells. Such monoclonal assay systems for studying cognate interactions of heterogeneous T cells and specific antigen-presenting cells will provide us with valuable new approaches for the study of antigen-specific T-cell regulation of B-cell activation in immune responses.     

Establishment of an antigen-specific B cell clone by somatic hybridization

Splenic B cells of A/J mice immunized with 2,4,6-trinitrophenyl (TNP)-lipopolysaccharide were fused with 2.52M, a mutant of a B cell line, in the presence of polyethylene glycol and dimethyl sulfoxide. TP67.21, a subclone of a resulting hybridoma, expresses IAk, IEk, IgM, B220, P50, and receptors for C3 fragment of complement, the Fc portion of IgG, and interleukin 2 receptor on the cell membrane; it also possesses receptor molecules for TNP on its surface, derived from TNP-reactive B cells of A/J mice primed with TNP-lipopolysaccharide used for somatic hybridization, by a rosette-forming assay with TNP-sheep erythrocytes. In contrast, parental 2.52M lacks IAk and IEk on the cell membrane and does not bind to TNP-sheep erythrocytes under the same conditions. Thus, it is likely that TP67.21 is an antigen-specific B cell clone directed against TNP. The antigen binding of cells was markedly inhibited by the specific free hapten or anti-IgM antibodies. Interestingly, TP67.21 was induced to generate a significant amount of anti-TNP antibody when treated with TNP conjugates including T cell-independent and -dependent antigens, such as TNP-lipopolysaccharide, TNP-bovine serum albumin, TNP-ovalbumin, and TNP-keyhole limpet hemocyanine in the absence of T cell help, as well as polyclonal activators; this was followed by a marked decrease in the expression of B cell surface markers on the cell membrane. This suggests that the cross-linkage of receptor molecules on TP67.21 by antigen may directly provide a differentiative signal for maturation to a lineage of B cells, and consequently results in the generation of antigen-specific antibodies without T cell involvement.     

Receptor specificity of Ia-restricted T lymphoblasts activated against trinitrobenzene sulfonate-coupled spleen cells: recognition of distinct trinitrophenyl and Ia moieties

The receptor specificity of H-2-restricted T lymphoblasts activated against trinitrobenzene sulfonate (TNBS)-coupled spleen cells was examined using an antigen binding assay. A population of Lyt-1+,2-T lymphoblasts acquired syngeneic Ia determinants during 4 days of primary culture with hapten-coupled stimulator cells. Syngeneic Ia was not reexpressed after trypsin treatment of the T cells, but was found after incubation with soluble Ia shed from lipopolysaccharide-activated blasts. Self-Ia binding was specific in that Lyt-1+,2- but not Lyt-1-,2+ cells acquired the antigen, and in that self-Ia bound more effectively than allogeneic Ia material. To determine the relationship of self-Ia binding to the recognition of foreign antigen, the binding of trinitrophenyl (TNP)-coupled plasma membrane vesicles by TNP-specific T cells was studied. TNP-vesicle binding occurred via TNP and H-2(Ia) molecules on the vesicles in that binding was inhibited with antibodies against TNP or H-2(Ia) molecules but not non-major histocompatibility complex (e.g., Ly-6.2) molecules on the vesicles. Complete inhibition of TNP-vesicle binding by an Iak-restricted TNP-specific T-cell line occurred with soluble TNP-lysine, but not an unrelated hapten, N-iodoacetyl-N-(5-sulfonic-1-naphthyl)ethylenediamine (I-AED)-cysteine. Conversely, I-AED-cysteine, but not TNP-lysine, inhibited binding of I-AED-coupled B6 vesicles by B6 anti-I-AED T cells. Significant, but weak inhibition of TNP-vesicle binding by the anti-TNP line was observed with glycoprotein preparations containing partially purified self-Ia molecules. However, inhibition was specific for I-Ak molecules, in that inhibition was lost after removal of I-Ak molecules from the glycoprotein preparation, and very little inhibition occurred with soluble glycoproteins prepared from thymocytes which contained very little Ia material or from LPS blasts of an unrelated H-2 haplotype. These results suggest a recognition model in which TNP and Ia determinants are recognized by neighboring receptor combining sites.     

Comparison between different rabbit antisera against the glucocorticoid receptor

Seven antisera against the glucocorticoid receptor (GR), raised in different rabbits immunized with highly purified (in case of five rabbits apparently homogeneous) preparation of GR from rat liver cytosol, were compared concerning titer and cross-reactivity. The titers of protein A-purified antisera (10 mg/ml) were in the range 1:100-1:320 as measured by enzyme-linked immunosorbent assay, ELISA, (defined as the dilution giving 50% of maximum absorbance). All seven antisera bound to the rat GR with a Stokes radius of 6.1 nm, but no antiserum reacted with the proteolytically induced steroid binding domain with Stokes radius 3 nm. However, the antigenic determinant(s) of the non-ligand-binding domain(s), split off from the steroid binding domain, is preserved following digestion with alpha-chymotrypsin or trypsin, respectively, since immunoactivity is still detectable by ELISA. Only two of four antisera tested cross-reacted with the GR from human lymphocytes. The same two antisera cross-reacted with chick embryo liver GR. Four out of four antisera tested cross-reacted with mouse liver GR as well as with rabbit lung GR. For these antisera, antibody binding to the GR prior to steroid- or DNA-binding did not influence the ability of the GR to interact with the ligand or DNA-cellulose, respectively. No difference regarding avidity of the antisera for activated or non-activated GR was observed. Furthermore, none of the antisera tested cross-reacted with the estrogen, progestin, androgen or mineralocorticoid receptors in rat. These findings indicate that the antisera from different rabbits raised against the same antigen all react with a certain domain of the rat GR, but show species differences as well as receptor class specificity.     

Antigen presentation by human antigen-presenting cells to antigen-specific xenogeneic murine T cells

Successful antigen presentation by xenogeneic human antigen-presenting cells (APC) to stimulate the proliferation of antigen-specific, keyhole limpet hemocyanin (KLH)-specific, ovalbumin (OVA)-specific, and purified protein derivative of Mycobacterium tuberculosis (PPD)-specific murine T cells was observed. Evidence indicating a direct cell interaction between antigen-specific murine T cells and xenogeneic human APC was given by experiments using antigen-specific murine T cell clones. The OVA-specific B10.S(9R) T cell line (9-0-A1) and PPD-specific B10.A(4R) T cell line (4-P-1) were stimulated by both xenogeneic human APC and murine APC from syngeneic or I-A compatible strains, while the PPD-specific human T cell line (Y-P-5) was stimulated by autologous human APC but not by murine APC. Anti-HLA-DR monoclonal antibodies (MoAb) blocked the xenogeneic human APC-antigen-specific murine T cell clone interaction. Thus, human xenogeneic APC can stimulate antigen-specific murine T cells through HLA-DR molecules in the same manner as syngeneic murine APC do through Ia molecules coded for by the I region of the H-2 complex, while murine APC failed to present antigen to stimulate human antigen-specific T cells.     

Antigen-binding receptors on T cells from long-term MLR. Evidence of binding sites for allogeneic and self-MHC products

Antibody inhibition of radiolabelled stimulator membrane vesicle binding by T blasts activated in the mixed lymphocyte reaction (MLR) was used to identify responder-cell determinants involved in the binding phenomenon. Antisera or monoclonal antibodies against Thy-1, Lyt-1, Lyt-2 and Ly-6 antigens were not inhibitory. However, antibodies against heavy-chain V region (V_H) determinants strongly inhibited vesicle binding by both primary and longterm MLR blasts. Anti-Ia (both alloantisera and monoclonal reagents) caused inhibition of antigen binding by primary MLR blasts only. T blasts from long-term MLR lines were neither Ia-positive, nor susceptible to blocking of antigen binding with anti-Ia. However, these cells were capable of specifically absorbing soluble syngeneic Ia material, with the concomitant appearance of vesiclebinding inhibition with anti-Ia sera. Acquisition of syngeneic Ia by T blasts was effectively blocked with the anti-V_H reagent. Passively bound self-Ia did not interfere with vesicle binding in the absence of anti-Ia. These results strongly suggest the existance of specific self-Ia acceptor sites closely linked to the receptors for stimulator alloantigens on T cells proliferating in MLR. A receptor model based on these findings is briefly discussed.Abbreviations used in this paper B10 C57BL/10 - Con A concanavalin A - FcR Fc receptor - FCS fetal calf serum - H heavy chain - Ia I-region associated antigen - Ig immunoglobulin - LPS lipopolysaccharide - Lyt T-lymphocyte differentiation antigen - MHC major histocompatibility complex - MLR mixed lymphocyte reaction - PM plasma membrane - T thymus derived - Tcr T-cell receptor - V variable region of Ig     

The partial isolation of subcellular MHC products which are recognized by alloimmune T lymphocytes

H-2 antigens from three murine tumor-cell lines (YAC, EL-4 and P815) were solubilized with triton X-100 and separated by SDS-polyacrylamide gel electrophoresis. Proteins were eluted from gel slices and assayed for inhibition by a target-effector binding (TBC) assay. Fractions inhibiting alloimmune T cells also contained serologically detectable H-2 molecules as judged by a complement-dependent microcytotoxicity-inhibition assay. Fractions containing Moloney cell-surface antigen (MCSA), gp 71, or p30 did not inhibit alloimmune TBC. H-2 antigens were selective for T cells since they failed to inhibit binding of natural killer (NK) cells to NK-sensitive targets, whereas, in the reciprocal experiment, the NK target antigens inhibited NK cells but not alloimmune T cell binding to intact targets. Cross inhibition tests in the T-cell system revealed that the H-2 molecules maintained their specific antigenic structure. These observations suggest that it will be possible to characterize further those parts of the subcellular H-2 preparations reacting with distinct killer T-cell clones.     

Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-beta-galactosidase fusion proteins.

Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.     

The partial isolation of subcellular MHC products which are recognized by alloimmune T lymphocytes

H-2 antigens from three murine tumor-cell lines (YAC, EL-4 and P815) were solubilized with triton X-100 and separated by SDS-polyacrylamide gel electrophoresis. Proteins were eluted from gel slices and assayed for inhibition by a target-effector binding (TBC) assay. Fractions inhibiting alloimmune T cells also contained serologically detectable H-2 molecules as judged by a complement-dependent microcytotoxicity-inhibition assay. Fractions containing Moloney cell-surface antigen (MCSA), gp 71, or p30 did not inhibit alloimmune TBC. H-2 antigens were selective for T cells since they failed to inhibit binding of natural killer (NK) cells to NK-sensitive targets, whereas, in the reciprocal experiment, the NK target antigens inhibited NK cells but not alloimmune T cell binding to intact targets. Cross inhibition tests in the T-cell system revealed that the H-2 molecules maintained their specific antigenic structure. These observations suggest that it will be possible to characterize further those parts of the subcellular H-2 preparations reacting with distinct killer T-cell clones.     

T-cell receptor tetramer binding or the lack there of does not necessitate antigen reactivity in T-cell receptor transduced T cells

Genetic transfer of T-cell receptor (TCR) chains provides a means of transferring tumor antigen specificity onto an alternate T-cell population. To determine which tumor reactive TCRs are best suitable for such adoptive transfer, careful evaluation of the resulting TCR modified populations need to be performed. We have previously cloned, and expressed TCRs from melanoma, EBV, HCV, and HPV reactive T-cell clones and found that several routine indicators of T-cell function do not always predict the relative strength of a TCR. Using a combination of tetramer binding assays and antigen recognition assays, we identified TCRs that fall into three classes. One class of TCR did not bind tetramers yet resulted in cells with high avidity for antigen. A second TCR class bound tetramer but did not secrete cytokines in response to antigen. Finally, the third class of TCRs bound tetramer and reacted to antigen as would be expected. We conclude that tetramer binding is not always a good indicator of the function of a cloned TCR or the avidity of a TCR gene modified T cell. Furthermore, our data indicate that the use of tetramer binding alone to identify antigen reactive TCRs may result in the exclusion of TCRs that may be highly reactive or cross reactive to the relevant tumor antigen.     

Macrophage-like suppressor cells in rats. I. Inhibition of natural macrophage-like suppressor cells by red blood cells

When trinitrobenzenesulfonic acid (TNBS), the reactive form of trinitrophenyl (TNP) hapten, is injected into a mouse, a brief intrinsic B-cell tolerance to TNP has been shown to result. Yet antigen-binding cells (ABC) with receptors for TNP persist in the TNBS-treated animal.After treatment with Pronase under conditions preserving cell recovery and viability, 80–90% of TNP-ABC failed to bind antigen. After 2 hr in vitro, Pronase-treated 4-day immune TNP-ABC displayed significant recovery of antigen binding, whereas nonimmune TNP-ABC performed the same feat by 18 hr. However, TNP-ABC tested 2 to 11 days after TNBS failed to replace digested receptors by 18 hr in vitro. Thirty days after TNBS, they had recovered this ability. This defective receptor replacement by TNP-ABC was not reversed by colchicine, and was not shared by the sheep-erythrocyte ABC of the same animals, which replaced receptors normally. When challenged with antigen (TNP-sheep erythrocytes) simultaneously with TNBS, recovery by 2 hr was evident on Day 11. When challenged with antigen 4 days after TNBS, receptor regeneration had returned to normal by the next day, and partial recovery of the anti-TNP plaque-forming cell response was evident 4 days later.Thus, the inability to replace receptors and immune unresponsiveness coincides in time, so that a causal relationship between these two defects may be hypothesized. This result contrasts with the membrane locking defect, previously described in the TNP-ABC of TNBS-treated animals, which far outlasted the unresponsive state.     

A modification of the unlabeled antibody enzyme method using heterologous antisera for the light microscopic and ultrastructural localization of insulin, glucagon and growth hormone.

The requirement of using homologous antisera (primary antiserum and peroxidase-antiperoxidase (PAP) complex raised in the same species) in the unlabeled antibody enzyme method has been investigated at the light and electron microscopic level using the localization of insulin, glucagon and growth hormone as model systems. Optimum immunocytochemical staining for all three antigens was observed when sheep or goat antirabbit gamma-globulin (S-ARgammaG or G-ARgammaG) were used to couple rabbit peroxidase-antiperoxidase complex with either guinea pig antisera to insulin (GP-AIS) or glucagon (GP-AGS), or monkey antisera to rat growth hormone (M-ARGH). The cross-reactivity between S-ARgammaG or G-ARgammaG and immunoglobulins in these primary antisera were substantiated by immunoelectrophoresis and radioimmunoassay. S-ARgammaG was shown to produce precipitation arcs with GP-AIS and M-ARGH that were similar to those seen when the latter were reacted with rabbit antiguinea pig gamma-globulin antiserum and goat antimonkey gamma-globulin antiserum, respectively. Radioimmunoassay results revealed that immunoprecipitation of 6-10% as compared to homologous antisera controls yielded excellent staining localization when S-ARgammaG was used for immunocytochemistry. Thus, heterologous antisera (primary antiserum and PAP complex raised in different species) may be used in the unlabeled antibody enzyme method as long as the coupling antiserum shows cross-reactivity with immunoglobulins of the primary antiserum and the PAP complex.     

Functional interactions of human and murine lymphoid cells. I. Analysis of primary and secondary responses

In this study human T-cell responses against murine alloantigens were analyzed. The results show that optimal primary responses are obtained from peripheral blood mononuclear cells only when murine splenic adherent cells (SAC) were used as antigen. Further analysis revealed that human T cells were able to respond directly to murine cells without the need for antigen reprocessing; however, human interleukin 1 (IL-1) was required for optimal stimulation. In contrast, secondary proliferative responses to murine cellular antigens could be induced from primed T cells even in the absence of SAC and/or IL-1. These proliferative responses, and in addition, cytotoxic T-cell responses, were specific for the priming antigen. Long-term human T-cell lines specific for murine alloantigens were found to replace the need for murine T cells in antigen-specific murine B-cell responses to sheep red blood cells. The mechanism of help delivered by the human T cells appeared to be by the release of nonspecific helper-T-cell factors. The evidence presented for this is the inability of these cells to stimulate cells from mice that express the X chromosome B-cell defect xid.     

Prostaglandin synthesis by primary cultures of mouse embryo palate mesenchyme cells

Evidence is presented for a concomitant storage of α-Neo-endorphin and dynorphin immunoreactivities in neurons of the rat brain. Antisera were raised against the structurally related opioid peptides dynorphin(1–17) and α-Neo-endorphin. Both antisera were highly specific for their respective antigen. Thus, the α-Neo-endorphin antisera did not crossreact with dynorphin and the dynorphin antisera did not crossreact with α-Neo-endorphin. Both antisera were also not cross-reactive with leu-enkephalin which is contained within the sequence of both dynorphin and α-Neo-endorphin. The antisera were used for immunofluorescent staining of frozen sections through brains from rats which had been treated with colchicine 48 hours prior to death. Both antisera revealed strong and specific immunoreactivities of magnocellular neurons in the supraoptic, retrochiasmatic supraoptic and paraventricular nuclei. Neuronal fiber systems in various areas of the brain were also labeled by the two antisera. Consecutive immunostaining of the same sections, first with dynorphin antisera and — after electrophoretic elution of the antibodies — with α-Neo-endorphin antisera or vice versa, showed that immunoreactivities for the two peptides are contained within the same hypothalamic magnocellular neurons. The neuronal fiber systems for α-Neo-endorphin and dynorphin also showed a close overlap. These studies demonstrating colocalization raise the question as to whether the two peptides have a common origin from a single precursor molecule.     

Mapping of B-neutralizing and T-helper cell epitopes on the bovine leukemia virus external glycoprotein gp51.

A battery of 19 synthetic peptides was used to characterize efficient neutralizing and helper T-cell epitopes on the bovine leukemia virus (BLV) external envelope glycoprotein gp51. Four of the antipeptide antisera raised in rabbits inhibited the formation of BLV-induced syncytia; these antisera are directed against peptides 64-73, 98-117, and 177-192. Only antisera directed against the 177-192 region also neutralized vesicular stomatitis virus-BLV pseudotypes. This study clearly demonstrates that neutralizing properties can be observed with antibodies raised to regions undescribed so far and included in both the amino-terminal and central parts of the antigen. In addition, some helper T-cell determinants were defined from gp51-immunized mice and from BLV-infected cattle. Although none of the peptides tested behaved as a universal helper T-cell epitope, peptide 98-117 stimulated T-cell proliferation from BALB/c mice and from three infected cows, while peptide 169-188 strongly stimulated T-cell proliferation from one infected cow. Further experiments performed with three peptides overlapping the 169-188 region (177-192, 179-192, 181-192) demonstrated the particular relevance of residue(s) P-177 and/or D-178 in the helper T-cell epitope. These data should assist in the design of an efficient subunit vaccine against BLV infection that contains peptides possessing both B-neutralizing and helper T-cell determinants.     

Human leukaemia-associated antigens expressed by acute myelocytic leukaemia cells and their detection by heterologous antisera.

Antisera against human acute myelocytic leukaemias were tested in complement-dependent in-vitro cytotocity tests against leukaemia cells and normal cells as targets. After absorption with erythrocytes and spleen cells from allogeneous donors the antisera reacted with leukaemia cells, but not with leukocytes from bone marrow and the peripheral blood of children in remission, lymphocytes from healthy donors, enriched B-lymphocytes, enriched T-lymphocytes, PHA-induced blasts and cord blood lymphocytes. Extensive cross reactions were obtained in the tests against leukaemia cells. The antisera reacted not only with AML cells, but also with ALL, CLL, and CML cells. It was possible to remove the cross-reactivity with ALL cells through absorption with ALL cells or with fetal tissue, and to remove the cross reactivity with CLL cells through absorption with CLL. A complete absorption of the anti-AML sera was possible with AML and CML cells. After absorption with fetal tissue and CLL cells the antisera showed exclusively specificity for myelocytic leukaemias. Thus, AML cells contain three leukaemia-associated membrane antigen components: an antigen of fetal origin, a "CLL-specific" antigen, and an antigen that occurs on myelocytic leukaemias.     

Antigen-specific and nonspecific mediatiors of T cell/B cell cooperation. II. Two helper T cells distinguished by their antigen sensitivities.

Further evidence is presented for two types of helper T cells in the mouse specific for the protein antigen, keyhole limpet hemocyanin (KLH). The first cell helps B cells respond to the trinitrophenyl hapten (TNP) coupled to KLH, is primed by relatively high doses of antigen in vivo, and yet the effector cell is stimulated by very low doses of antigen in vitro. The second cell helps B cells respond to a non-cross-reacting antigen, sheep red blood cells, presumably via production of a nonspecific factor. This cell is primed by relatively low doses of antigen in vivo, but the effector cell requires relatively high doses of antigen in vitro. Thus, the two T cell types are differently sensitive to antigen dose, both in priming and challenge. The properties of T cells responding to KLH by proliferation in vitro were also studied. These cells showed the same antigen-sensitivity in vitro, as cells producing nonspecific B cell-stimulating factors.     

Association of p60fyn with middle tumor antigen in murine polyomavirus-transformed rat cells.

Rabbit antisera raised against human FYN-specific peptides were used to evaluate the expression of the fyn gene product in normal and murine polyomavirus middle tumor antigen (MTAg)-transformed rat cells. The antisera were capable of detecting p60fyn in both normal and MTAg-transformed cells. Two different antisera directed against unique p60fyn sequences were found to detect p60fyn-MTAg complexes in cell lysates from the MTAg-transformed cells. The MTAg molecules immunoprecipitated by FYN antisera were phosphorylated on tyrosine during immune-complex kinase reactions at sites similar to those found on MTAg in complexes with pp60c-src. Whereas the abundance of p60fyn was estimated to be less in the MTAg-transformed cells than in their normal counterparts, the specific activities of p60fyn molecules in the normal and transformed cells were similar.     

Plant lectin,ATF1011, on the tumor cell surface augments tumor-specific immunity through activation of T cells specific for the lectin

Summary The possibility that a plant lectin as a carrier protein would specifically activate T cells, resulting in the augmentation of antitumor immunity was investigated. ATF1011, a nonmitogenic lectin for T cells purified from Aloe arborescens Mill, bound equally to normal and tumor cells. ATF1011 binding on the MM102 tumor cell surfaces augmented anti-trinitrophenyl (TNP) antibody production of murine splenocytes when the mice were primarily immunized with TNP-conjugated MM102 tumor cells. The alloreactive cytotoxic T cell response was also augmented by allostimulator cells binding ATF1011 on the cell surfaces. These augmented responses may be assumed to be mediated by the activation of helper T cells recognizing ATF1011 as a carrier protein. Killer T cells were induced against ATF1011 antigen in the H-2 restricted manner using syngeneic stimulator cells bearing ATF1011 on the cell surfaces. When this lectin was administered intralesionally into the tumors, induction of cytotoxic effector cells was demonstrated. These results suggest that intralesionally administered ATF1011 binds to the tumor cell membrane and activates T cells specific for this carrier lectin in situ, which results in the augmented induction of systemic antitumor immunity.