CD4+ T cells have been shown to be crucial in the development of experimental autoimmune myasthenia gravis (EAMG). The role of CD8+ T cells in EAMG is less well established. We previously showed that antibody depletion of CD8+ T cells in rats effectively suppresses EAMG. To further study the role and relationship of CD4+ versus CD8+ T cells in induction of EAMG, CD4-/-, CD8-/-, and CD4-8- mutant C57BL/6 mice and the parent CD4+8- wild-type mice were immunized with Torpedo acetylcholine receptor (AChR) plus complete Freund's adjuvant. Clinical EAMG was nearly completely prevented in CD4-8-, CD4-/-, and CD8-/- mice. This was associated with strongly reduced AChR-specific T and B cell responses, and with reduced levels of AChR-reactive interferon gamma (IFN-gamma) and interleukin 4 (IL-4) mRNA-expressing cells in lymphoid organs when compared with CD4+8+ wild-type mice. We conclude that (a) both CD4+ and CD8+ T cells are essential for development of EAMG, and a collaboration between these cell types may be necessary; (b) CD4+ as well as CD8+ T cells secrete IFN-gamma and IL-4, and both cytokines are involved in the development of EAMG; and (c), besides T cells, other immune cells might also be responsible for help of anti-AChR antibody production.

We compared T cell receptor (TCR) V-segment frequencies in human leukocyte antigen (HLA) identical siblings to sibling pairs who differ at one or both HLA haplotypes using four V beta-specific and one V alpha-specific monoclonal antibody. In every one of nine families HLA-identical sibs had the most similar patterns of V-segment frequencies in their peripheral blood, whereas totally mismatched sibs were, in general, the most dissimilar; HLA haploidentical sibs tended to be intermediate between the two groups. The degree of similarity among HLA-identical sibs was comparable to that observed among three pairs of identical twins suggesting that HLA is the major genetic component influencing TCR V-segment frequency. Consistent with this observation, it was found that the frequency of T cells expressing particular V beta segments was skewed towards either CD4+ or CD8+ cells indicating that T cells expressing some V beta genes may be positively selected primarily by class I or class II major histocompatibility complex proteins. Finally, it was observed that individuals who express the HLA class I specificity, B38, tend to express high levels of V alpha 2.3+ cells among their CD8+ T cells. These observations represent definitive proof that human V-segment frequencies are profoundly influenced by the HLA complex.

Dimethylbenzanthracene-induced P1 sarcoma cells induce P1-specific antibodies in syngeneic DA rats. Antiidiotypic antibodies of specificity DA anti-(DA anti-P1) were induced against the tumor-specific antibodies and used to restimulate P1-primed DA T cells in vitro. Using antiidiotypic antibodies and T cell growth factor, P1-specific cytotoxic DA T cell clones were established by limiting dilution and kept in vitro. Two of these clones acquired during culture periods in addition to the P1 specificity lytic activity towards natural killer (NK) targets YAC-1 or K562. Cold target inhibition experiments showed that the very same cytotoxic T cells kill P1 and NK targets. Antiidiotypic antibodies of specificity DA anti-(DA anti-P1) inhibited cytotoxicity against P1 but not against YAC-1 or K562. We conclude that two independent receptors are located on these double-reactive T cell clones, one that is idiotypic and antigen-specific, and another displaying the binding profile of NK cells.

Out of a panel of seven monoclonal antibodies with affinity for human lymphoid cells, three were shown to prevent cytotoxic T cell activity, whereas none affected natural killer cell activity when applied without complement. Anti-OKT3 and anti-Leu-2a, with affinity for all T cells and the cytotoxic/suppressive subset, respectively were both shown to inhibit T killing by their interaction with the effector cell. For anti-OKT3, the inhibition remained after free antibody was washed away. Anti-Leu-2a, in contrast, induced a rapidly reversible inhibition. Using a single cell assay, anti-OKT3 was shown to reduce the lytic ability without affecting target cell binding, whereas anti-Leu-2a prevented the effectors from binding target cells.

Monoclonal or polyclonal IgM-anti-SRBC antibodies were used to enhance the anti-SRBC PFC response in mice. For potentiation to occur, the IgM antibodies must always be presented with the antigen for which they have specificity. No enhancement of anti-SRBC response above control levels was noted with either antibodies alone or with antibodies used together with non-cross-reacting antigens. The degree of enhancement was independent of whether only one or several different monoclonal IgM antibodies were used. Likewise, the fine specificity variation among the antibody clones failed to influence the anti- SRBC potentiation, which was shown to vary only with the amount of IgM bound to SRBC measured by hemolytic titers. The response against epitopes on the SRBC other than those the IgM recognized was also enhanced. This was determined by injecting SRBC and a monoclonal anti-SRBC IgM that did not crossreact with GRBC into mice, and measuring the response against both antigens. Normally SRBC and GRBC cross- react at the B cell level to approximately 30 percent, and in this experiment they did so both in the control group and in the IgM group. Using antigens that only cross-react significantly at the T cell level (SRBC and HRBC), IgM-antibodies would only enhance the anti-HRBC response if SRBC and HRBC were inoculated together. No anti-HRBC potentiation was noted when antibodies were injected alone or together with either SRBC or HRBC. The data indicate that the constant part of the IgM molecule is of major importance in determining its enhancing properties in acute IgM-mediated potentiation of the immune responses. No evidence was obtained for a decisive role of variable regions. Furthermore, no general B cell activating properties of either mono- or polyclonal IgM-anti-SRBC antibodies could be demonstrated.

Antisera specific for the constant part Ctau of T cell receptor molecules in mice and rats have been produced in rabbits by immunization with idiotype-positive rat T cell molecules. Such antisera could be shown to react with the majority of normal T lymphocytes from both rats and mice. Mixed leukocyte culture-activated T blasts displayed a higher percentage of positive cells than concanovalin A- or phytohemaglutinin-induced T blasts. Lipopolysaccharide-induced B blasts were not reactive with the antiserum. Lyt-1-,2+,3+ blasts displayed a significantly brighter straining than the corresponding Lyt-1+,2-,3- blasts. Isolation of the reactive molecules from T cells by immunosorption yielded a 70,000-dalton single chain polypeptide as the dominant group of molecules. Plasmin caused the chain to split into 45,000- and 25,000-dalton polypeptides, with the smaller fragment displaying antigen-binding capacity. Molecules identical in size and plasma degradation patterns were isolated from Lyt-1+,2-3- and 1-,2+,3+ blasts. Preliminary functional data supported the view that the antiserum is directed against the constant region Ctau relevant receptor structures on immunocompetent T lymphocytes.

A special class of immunologic cells can lyse or damage a variety of target cells, notably malignant cells in vitro. These cells have been called natural killer (NK) cells because lysis does not require deliberate immunization by tumor cells. Although these cells can be distinguished from conventional T cells, B cells, and phagocytic cells, they have been difficult to define. We describe a representative cloned cell line that was obtained by cloning Ig -Ly-5+ cells from spleen. This clone, Cl.Ly-1-2-NK-1+/11, displays Thy-1, Ly-5, Qat-4, Qat-5 and NK-1 cell surface antigens and lyses the NK-sensitive YAC-1 lymphoma cells, but does not lyse RL-12 cells, an NK-resistant lymphoma. In addition, this clone lysed the P815 mastocytoma, EL4 lymphoma, and lipopolysaccharide-activated B lymphocyte targets. This cloned population therefore combined information for a unique display of cell surface antigens and specialized function similar to "activated" NK cells. Because this cloned population forms conjugates with susceptible but not resistant target cells, it may prove useful to identify the structure of cell surface molecules that recognize foreign cells. Finally, cells of this clone also specificity lysed target cells coated by antibodies to determinants on the target cell surface, demonstrating that a single cloned cell population can mediate two specialized immunologic functions: antibody-dependent cellular cytotoxicity and NK cell lysis.

In this study, we tried to get information about the fine antigen-binding ability of purified, soluble, idiotype-positive T-cell receptor molecules. Lewis anti-DA T-cell receptors were purified from normal Lewis serum by the use of anti-idiotypic immunosorbent and sodium dodecyl sulfate-polyacrylamide gel, and were coupled to cyanogen bromide-activated Sepharose 4B. In parallel, Lewis anti-DA, Lewis anti-BN, and DA anti-Lewis alloantibody immunosorbents were prepared. The major Ag-B chain (44,000 daltons) and the two polypeptide chains (34,000 and 27,000 daltons) of Ia were purified from Lewis, DA, and BN lymphocytes and absorbent on the above-mentioned immunosorbents. We found that the major Ag-B chain as well as the two Ia chains were bound to the alloantibody columns if they were derived from the corresponding allogeneic strain. No retaining ability for self-major histocompatibility complex (MHC) or third-party MHC chains was noted with the alloantibody immunosorbents. When using immunosorbents made up of idiotypic T-cell receptors, only two MHC polypeptides of the relevant allo-MHC type were retained, namely, the Ag-B and the heavy Ia chains. No detectable activity was observed when testing the same column for reactivity against third-party MHC polypeptide chains. However, the Lewis anti-DA T-cell receptors could be shown to display weak, but significant, reactivity toward one Lewis MHC polypeptide chain, that is, the heavy chain of Ia type.

Killer T cells with specificity for major histocompatibility antigens have been shown in mice and rats to display idiotypic receptors allowing the lysis of such cells at the effector phase by anti-idiotypic antibodies and complement. A comparison was made between idiotypes displayed by Lyt-1-2+3+ and Lyt-1+2-3- T blasts, generated in the same mixed leucocyte culture (MLC), across an entire H-2 locus barrier. This was done by absorption of anti-idiotypic antibodies with respective T blasts, followed by estimation of the ability of the absorbed antiserum to inhibit MLC or killer T-cell function. Further, the capacity of Lyt-purified, MLC-generated T blasts to provoke specific unresponsiveness via anti-idiotypic immunity in syngeneic recipients was analyzed. Taken together, the results demonstrate that Lyt-1+2-3- T blasts responsible for the major part of MLC proliferation have distincly different idiotypes from those on the Lyt 1-2+3+ killer T cells. That the idiotypes on the killer T-cell presursors can serve as triggering sites for induction of effector T-cell function was then suggested by experiments with Lyt-1-2+3+-purified, normal T cells as precursor cells in vitro. The fact that autoanti-idiotypic antibodies may circumvent the need for helper Lyt-1+2-3- T cells in the generation of allospecific killer T cells indicates that the former cells may normally function partly via such anti-idiotypic reactions.

We have analyzed the lectin binding characteristics of cytotoxic T lymphocyte (CTL)-derived surface labeled glycoproteins by affinity chromatography of the labeled glycoproteins on a panel of immobilized lectin adsorbents. Evidence is presented for the specific interaction of the CTL-associated glycoprotein T 145 with a lectin derived from Vicia villosa seeds. Conditions are described for the preparation and use of lectin affinity adsorbents for the rapid isolation of T 145 bearing cytotoxic T lymphocytes. Direct proof is given to show that T 145-positive cells arising from a variety of T-cell activations constitute the only subpopulation of cells with ability to perform cell-mediated T-cell cytotoxicity. Specific depletion of the CTLs by adherence to V. villosa adsorbents is shown by their depletion in the nonbound cell fraction and correspondingly enriched recovery in the sugar eluted cell fraction. Specific affinity fractionation of CTLs has occurred in every strain combination tested and irrespective of the actual antigen specificity of the effector cell population.

Alpha-fetoprotein (AFP), a major component of fetal and newborn sera, was shown to exert significant immunosuppressive activity on the in vitro generation of cytotoxic T lymphocytes (CTLs). This suppression proved independent of the suppressibility of the mixed leukocyte culture activation phase, since strain combinations whose proliferative responses were refractive to AFP-induced suppression also failed to develop demonstrable CTLs in the presence of AFP. Several strain combinations were also found in which normal generation of CTLs occurred in cultures containing AFP. This refractive nature correlated with the presence of nonsuppressible lymphocyte-stimulating alloantigenic systems on the stimulating cell population. These data provide the basis for proposing several possible mechanisms for AFP-induced suppression of T-cell-mediated cytotoxicity, as well as suggesting that the primary target of this suppression is the proliferating helper T cell precommited to respond towards the major histocompatibility complex-associated lymphocyte-activating determinants.

T lymphocytes at various stages of maturation and differentiation have been isolated by cellular fractionation procedures and characterized by cell surface markers and functional assays, The cell surface glycoproteins of the various T-cell preparations have been selectively radiolabeled by the galactose oxidase-tritiated sodium borohydride technique and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Details are presented on the appearance of a new cell surface glycoprotein (T 145), present on immunocompetent T lymphocytes after activation by either major histocompatibility complex alloantigens or by concanavalin A. The intensity of T 145 expression on T lymphoblasts is shown to be directly correlated in time and extent to the levels of cytotoxicity generated in a variety of T-cell activations. Specific enrichment procedures of purified populations of mixed leukocyte culture blasts have shown Ly 1(+)2(-) blasts to be T 145(-) and Ly 1(-)2(+) blasts to be strongly T 145(+). Similar enrichment procedures on normal peripheral T cells have failed to reveal any significant expression of T 145 on a highly enriched population of Ly 1(-)2(+) T cells, Further studies on the stability of T 145 expression after induction have shown it to be a more permanent-type differentiation structure whose expression is clearly not linked to the blast stage of activation. T 145 would thus appear to represent a membrane glycoprotein whose exclusive expression on T lymphoblasts is further restricted to a defined group of cells endowed with cytolytic activity and bearing the Ly phenotype Ly 1(-)2(+).

The technique of antigen-driven, 5-bromo-deoxyuridine and light suicide has been adapted to eliminate the precursors of cytotoxic effector cells both for alloantigen and for 2,4,6-trinitrophenyl(TNP)-modified stimulator and target cells. Using this technique, the following observations have been made. Precursors of killer cells specific for alloantigen can be suicided independently of precursors of killer cells specific for TNP-modified self cells. The loss of activity during this procedure is not due to either specific or nonspecific suppressor cells, as judged by mixing experiments. With responder cells from F1 animals, it has been possible to show that precursors specific for TNP-modified cells from one parent are suicided independently of precursors specific for TNP-modified cells of the other parent, but only if the parental strains differ in the K and D regions of the H-2 complex. Cells of F1 mice derived from K and D identical, I region different, parental strains were specifically suicided by TNP-modified stimulator cells from either parent. However, the cross-reactive killing of TNP-self targets induced by stimulation with allogeneic cells is not eliminated by first suiciding with TNP-parental cells, suggesting that the precursors of these two types of TNP-self killer cells are different. This is compatible with reported differences in their specificity, as confirmed in this report. Finally, deletion of alloreactive cells by this technique reveals little or no reactivity specific for TNP-modified allogeneic stimulator cells. In summary, these results strongly suggest that recognition of self MHC antigens is preprogrammed in peripheral T cells of normal animals, and is not acquired during the immunization process. They also suggest that cells specific for modified alloantigen are relatively rare in the strains of mice studied.

Alpha-fetoprotein (AFP), a major alpha-globulin component of fetal and newborn sera, has earlier been shown to exert significant immunosuppressive activity in vitro on T-dependent-immune responses. In the present investigation we have examined the effects of AFP on the recognition and proliferation of T lymphocytes responding in mixed leukocyte culture against histocompatibility-associated alloantigens. Fetal-derived AFP could be shown to exert differential effects on both primary and secondary responses ranging from strong inhibition to occasional enhancement, depending on the stimulating antigens. Proliferative responses against major histocompatibility complex (MHC) I region determinants, mediated predominantly by Ly 1 + cells, were markedly suppressed. Suppression was also observed in responses against Mls locus products, an antigenic system whose recognition requires concomitant recognition of I region gene products on the stimulating cells. In contrast, responses against MHC K or D region determinants, mediated predominantly by Ly 2 + cells, were generally unaffected by AFP. Similarly, non-MHC loci alloantigens distinct from Mls locus products also induced T-cell proliferation which was refractive to suppression by AFP. Because neither Ly 1 + nor Ly 2 + cells responding in this latter situation could be inhibited by AFP, we concluded that the mere fact that a T cell expresses a particular Ly phenotype does not predetermine sensitivity to AFP-induced suppression. In any case, AFP exerts a highly selective suppressive activity on I region-associated immune responses. These data may help to resolve the present controversy over the possibility that AFP has an in vivo relevance as an immunosuppressive agent by pointing out the importance of selecting proper genetic situations for study.

B and T lymphocytes with reactivity against major histocompatibility antigens are known to express this immune potential via a display on the outer surface of antigen-specific, idiotypic receptors. Here, we show that anti-idiotypic antibodies directed against such receptors may serve as specific triggering agents of the idiotype-positive lymphocytes in the physical absence of foreign histocompatibility antigens. This was shown in vitro using normal or immune spleen T cells where anti-idiotypic antibodies would lead to the selective proliferation and development of antigen-specific cytolytic T cells as determined by short-time 51Cr release assays. Furthermore, purified anti-idiotypic antibodies in adjuvant administered in vivo to normal syngeneic animals could be shown to lead to production of high titers of specific alloantibodies. The present experiments were in most cases carried out using auto-anti-idiotypic antibodies as triggering agents. The present results thus lend further support to the concept that idiotype-anti-idiotype reactions may be normal parts of conventional immune processes with either stimulatory or inhibitory consequences, depending upon the prevailing conditions.

Specific immune unresponsiveness against a given set of histocompatibility antigens can be induced by immunization with autologous, antigen-specific T lymphoblasts. Such unresponsiveness can be transferred by lymphoid cells from autoblast-immunized donors to normal syngeneic recipients. The cells being most efficient in transferring the selective suppression are T lymphocytes from the spleen, especially if of Ly 1-2+3+ phenotype. By using such T lymphocytes we deem it likely that the actual underlying mechanism is one of actual transfer of autoanti-idiotypic killer T cells. In support for this view is the fact that such T cells endowed with exquisite specific, cytolytic reactivity towards autologous idiotype-positive T target cells exist in autoblast immune animals. Significant suppression may also be transferred with T cells of Ly 1+2-3- phenotype or with B cells. Here, we consider the suppressive mechanism to be one of production of autoanti-idiotypic antibodies. By using affinity fraction procedures, it was finally possible to prove that all T-cell suppressive activity resides in a population with true antigen-binding-specific receptors for the relevant idiotypes.

T lymphoblasts specific for foreign histocompatibility antigens and purified via mixed leukocyte culture (MLC) and 1 g velocity sedimentation procedures can be used as autoimmunogen to produce specific immunological unresponsiveness in adult animals. This unresponsiveness is positively correlated to the production of autoanti-idiotypic antibodies in the blast immunized animals and no evidence of coexisting alloimmunity was found. We consider this autoanti-idiotypic immunity to be the specific inducing agent of the immune tolerance. The blast immunization procedure will lead to selective reduction in T-cell reactivity against the relevant alloantigens as measured by MLC, cell-mediated lympholysis, or graft-versus-host assays. However, in individual animals, dichtomy in suppression between two T-cell assays could sometimes be observed indicating elimination of only a select group of idiotypic functionally distinct population of T cells in these blast-immunized animals. Attempts to abrogate already immune animals by the autoblast procedure were successful, in part suggesting the use of the present procedure when trying to induce in accelerated reversion of such immunity.

Normal immunocompetent T lymphocytes can be induced into specific proliferation if confronted with the relevant alloantigen in vitro. Such mixed leuko-cyteculture-activated T lymphoblasts carring idiotypic receptors on their surface can be purified using velocity sedimentation and serve as immunogen if administered in adjuvant to the autologous host. Autoblast immunization can be shown to lead to specific, long-lasting unresponsiveness against the relevant alloantigens, while leaving reactivity against third-party antigens intact. When tested as to general validity, it could be shown to function in all species analyzed (mouse, rat, and guinea pig) as well as across both major and minor histocompatibility barriers. No negative side effects have been noted so far. It would thus seem clear that autoblast immunization using the above described scheme may serve as a general tool in inducing long-lasting, specific unresponsiveness in any species and across any histocompatibility barrier.

Generation of natural killer (NK) cells in spleens from radiation chimeras produced between pairs of histocompatible 'high' and 'low' NK-reactive mouse strains has been investigated. Spleen cells of high-reactive recipients reconstituted with bone marrow from low-reactive mice were found to be low reactive. Conversely, spleen cells of low mice grafted with bone marrow or fetal liver cells from high donors were high reactive. Similarly, the age-related changes of NK activity were shown to be expressed at the bone marrow precursor cell level. These results indicate that the generation of natural killer cells is an inborn and autonomous function of the bone marrow and does not depend on the genotype or other influences of the host environment.

The role of non-H-2 alloantigens, specifically Mls locus products, in secondary in vitro T-cell-mediated cytotoxicity has been studied. Splenic T lymphocytes, activated against Mls locus alloantigens in primary-mixed cultures and isolated by velocity sedimentation gradient separation techniques, were used as responding populations in secondary mixed leukocyte cultures (MLCs) and cell-mediated lympholysis (CML). Such T-cell clones could be shown to exhibit either "self"-H-2-restricted or anti-Mls locus-specific reactivity, with this dichotomy of reactivity depending only on the primary culture conditions. Mls locus-activated T lymphocytes generated in cultures supplemented with homologous serum exhibited specific memory responses in MLC, yet remained incapable of effecting target cell destruction against Mls locus antigens or against "self"-H-2-structures in CML. In contrast, activated T-cell clones generated in the presence of heterologous serum displayed H-2-restricted reactivity in both secondary MLC and CML. H-2-restricted MLC activation was controlled by products of the H-2 serologically defined regions. Although heterologous serum was a necessary (and sufficient) entity for development of H-2-restricted responses, evidence argues against the possibility that heterologous serum acts via modification of cell surface components.