Cross-linking of Lyt-2 (CD8) to the T cell antigen receptor optimally activates T lymphocytes.
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Biomedical subjects
K Eichmann
Publications and source records attributed to K Eichmann.
T-cell receptors and cellular interactions.
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Animals↗
Heterologous cross-linking of Lyt-2 (CD8) to the alpha beta-T cell receptor is more effective in T cell activation than homologous alpha beta-T cell receptor cross-linking.
Ag recognition of Lyt-2 (CD8)-positive T lymphocytes requires the presentation by APC of a suitably processed Ag in association with MHC class I molecules. In previous studies we have obtained evidence that, for optimal activation, both the alpha beta-TCR and Lyt-2 have to participate in this recognition process. In the current study we investigate the functional consequences of limited cross-linking of these cell surface molecules by using soluble, dimeric hetero- and homoconjugates of mAb to Lyt-2 and to the TCR beta-chain (F23.1). Heterologous cross-linking of Lyt-2 to the TCR induced a vigorous, selective Lyt-2+ T cell proliferative response. Functionally active cytotoxic cells were generated, and a high frequency of responding cells was observed in limiting dilution analyses. In contrast, homologous TCR cross-linking initiated a less pronounced proliferation with a relatively low frequency of response, whereas Lyt-2 cross-linking resulted in no cellular proliferation. Significant T cell activation occurred with exposure to anti-Lyt-2: F23.1 mAb dimers at concentrations an order of magnitude lower than those required for stimulation by F23.1:F23.1 mAb dimers. The induction of proliferation by mAb dimers occurred in the absence of Fc components and in rigorously APC depleted, purified T cell preparations. Effective stimulation of resting T cells could be induced also by heterodimers of monovalent Fab fragments. Heterologous cross-linking of Lyt-2 to the TCR was superior to homologous TCR cross-linking primarily with respect to proliferation in IL-2 containing media and to IL-2R expression, whereas proliferation in response to other lymphokines and the production of IL-2 itself were similar under both cross-linking regimens. Thus, when linked to the TCR, Lyt-2 contributed a strong, positive signal toward IL-2-dependent growth of resting T cells. We assume that in the case of Ag-driven T cell activation, the class I MHC molecule acts as the physiologic cross-linking ligand for Lyt-2 and the TCR.
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Human immune response to group A streptococcal carbohydrate (A-CHO). II. Antigen-independent stimulation of IgM anti-A-CHO production in purified B cells by a monoclonal anti-idiotopic antibody.
The paper describes the induction by a monoclonal anti-idiotopic antibody (anti-Id mAb) of specific antibody production to group A streptococcal carbohydrate (A-CHO) in purified human B cells of several unrelated individuals. The anti-Id mAb, designated 16F498 (anti-Id498), recognizes a recurrent idiotope (Id 498) associated with the combining site of human antibodies to N-acetyl-D-glucosamine (GlcNAc), the immunodominant group of A-CHO. Id498 is expressed on IgM anti-GlcNAc antibodies but does not occur on IgG antibodies with the same specificity. It occurs also on a minor population of IgM antibodies without specificity for A-CHO. Id498 was found in 19 of 27 sera from unselected healthy donors and thus seems to be frequently expressed within the adult B cell repertoire. The in vitro induction of anti-A-CHO antibodies was analyzed in human B cells extensively depleted for T cells. Specific antibody secretion required cross-linked anti-Id which was achieved by coupling the mAb to agarose beads. No antibody secretion could be induced by soluble anti-Id (1 and 10 micrograms/ml). An optimal response required soluble T cell-derived factors which were added as a mixture of recombinant interleukin 2 with a T cell hybridoma supernatant that augments B cell growth and differentiation. Under these conditions an antigen-independent specific increase of IgM anti-A-CHO production (2.6- to 10-fold, or up to 2000 ng/ml respectively) could be induced in blood B cell populations of four of six normal individuals expressing the Id498 at serum level.
Acetylglucosamine↗
Growth-dependent variation of major histocompatibility complex (MHC) restriction and expression of Ly-2 and CD3/alpha/beta T cell receptor in cloned cytotoxic T cells.
The specificity of major histocompatibility complex (MHC)-restricted antigen recognition by cytotoxic T cells (CTL) has been clearly correlated to the alpha/beta T cell receptor (TcR) complex on the T cell surface. Occasional changes in the specificity of in vitro cultivated CTL clones, therefore, have been suspected to result from alterations of the genes coding for the TcR alpha and/or beta chain. Here we demonstrate that pronounced variations in the stringency of MHC restriction, previously reported to occur during long-term culture of 2,4,6-trinitrophenyl (TNP)-specific CTL clones, may occur rapidly in a growth-dependent, reversible manner, i.e. without structural TcR variation. Several H-2b TNP-specific CTL clones were shown to possess strong cross-reactivity for H-2k TNP target cells when seeded at low cell numbers, but exhibit reduced or undetectable cross-reaction to H-2k TNP in high-density cultures. Another clone revealed "heteroclitic" properties with significantly stronger cytotoxic activity towards allogeneic (H-2k) than syngeneic (H-2b) TNP-modified target cells. In this case dilute cultures appeared as exclusively allo-MHC restricted, whereas dense cultures were allo/self cross-restricted. In all instances these phenomena were accompanied by cell density-dependent quantitative changes in the expression of Ly-2 and T cell antigen receptor. CTL from dilute cultures had at least 2-fold higher surface concentrations of Ly-2 and CD3 antigens than cells from dense cultures while other surface markers such as Thy-1 or LFA-1 were completely identical. No such effects were observed for CTL clones exhibiting cell density-independent specificity patterns. We conclude from these findings that (a) the stringency of MHC restriction specificity may be significantly affected by the amount of expressed TcR and/or Ly-2 molecules, (b) CTL possess mechanisms to regulate Ly-2 and TcR expression and, hence, their MHC-restricted antigen recognition, and (c) the ability to regulate Ly-2 and TcR expression may be altered during prolonged culture of a CTL clone.
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Selective stimulation of human T lymphocyte subsets by heteroconjugates of antibodies to the T cell receptor and to subset-specific differentiation antigens.
Ligand binding under conditions that generate microaggregates of the T cell receptor complex (Ti/CD3) with the membrane molecules CD4 or CD8 can induce activation of small resting T lymphocytes. We demonstrate this by using soluble dimeric heteroconjugates consisting of monoclonal antibodies to CD4/CD8 and of a novel anti-CD3 monoclonal antibody (BMA030) which even in aggregated form is not stimulatory on its own under limiting conditions in the absence of accessory cells. Using combinations of BMA030 with either anti-CD4 or anti-CD8, the corresponding T cell subpopulation could be selectively expanded in vitro. Selective expansion of T cell subpopulations in vitro or in vivo might be helpful for certain therapeutical manipulations. In addition, this finding may contribute to a better understanding of major histocompatibility complex-restricted T cell activation.
Antibodies, Monoclonal↗
Human immune response to group A streptococcal carbohydrate (A-CHO). III. Comparison of the efficiencies of anti-idiotopic antibody and of nominal antigen in the induction of IgM anti-A-CHO-producing B cells.
In this study the efficiencies of a monoclonal anti-idiotopic (Id) antibody (anti-Id498) and of various preparations of nominal antigen in the induction of an antigen-specific human B cell response in vitro were compared. Anti-Id498 recognizes a recurrent, binding site-related Id present on IgM antibodies with specificity for N-acetyl-D-glucosamine, the immunodominant group of streptococcal group A carbohydrate (A-CHO). We have previously shown that anti-Id498 induces IgM anti-A-CHO secretion from B cells of donors that possess Id-498+ antibodies in their serum. A-CHO was presented to B cells either in soluble or insoluble form, i.e. coupled to beads or as intact bacteria. Purified blood B cell populations from three Id+ healthy donors with high numbers of circulating anti-A-CHO B cells were used and antibody-producing B cells are enumerated in a single-cell assay (spot ELISA). The data show that anti-Id498 was superior in the induction of IgM anti-A-CHO-secreting B cells in two donors (factor 4.6 and 13.5 as compared to the most efficient antigenic stimulation). In the third donor antigen stimulation was slightly more efficient than anti-Id but only with Sepharose-bound A-CHO and not with soluble A-CHO or intact bacteria. The increase of specific B cells induced after stimulation with anti-Id498 could be abolished after addition of autologous T cells in two donors. On the contrary, an enhancement of the specific response was observed after addition of autologous T cells in antigen-stimulated cultures. Neither suppression nor enhancement were induced by addition of irradiated T cells.
Antibodies, Anti-Idiotypic↗
Selective induction of T-cell subsets by antibodies to the T-cell antigen receptor and to the subset-specific differentiation antigens CD8 and CD4.
This report shows that small, resting T lymphocytes from mouse and man can be activated to proliferation and function by submitogenic concentrations of antibodies to T-cell antigen receptor in combination with antibodies to either CD8 or CD4. The combined antibodies can be applied either in solid phase condition or as soluble dimeric heteroconjugates. Activation is more efficient than by high concentrations of anti-T-cell-receptor antibody alone. Most importantly, activation is subset-specific such that the antibody to CD8/CD4 determines which T-cell subset will be induced.
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Suppression needs a new hypothesis.
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Role of CD8 (Lyt-2) in cytotoxic T-cell function. Analysis of variants of a cytotoxic T-cell clone with reduced Lyt-2 expression.
The role of CD8 (Lyt-2) in the function of a long-term cytotoxic T-cell (CTL) clone (Cl96) was analysed. Previous studies had shown that C196 cells utilize the alpha beta T-cell receptor and depend on Lyt-2 to recognize and lyse P815 mastocytoma target cells. Recognition is H-2Kd-restricted, presumably involving a P815 specific antigenic structure. Here we analyse a number of variants selected from Cl96 that have reduced or virtually abolished expression of Lyt-2, alone or in combination with other deficiencies. We studied the ability of these variant cells to lyse either P815, a process requiring both specific antigen recognition and triggering of cytolytic function, or to lyse the anti-CD3 hybridoma 145-2C11, a process that requires the triggering of cytolytic function only. The results shows that in the case or a permanently activated CTL effector cell such as Cl96, Lyt-2 is required for antigen recognition but is not essential for the triggering of cytotoxicity.
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Roles of CD4- and CD8-bearing T lymphocytes in the immune response to the erythrocytic stages of Plasmodium chabaudi.
The possible role of CD4- and CD8-bearing T lymphocytes in parasite clearance in vivo was investigated, using Plasmodium chabaudi in C57BL/6 mice as a model. Monoclonal antibodies specific for the CD4 and CD8 molecules were administered in vivo to deplete selectively the appropriate subset of T cells. The efficacy of depletion was ascertained by flow cytometry and functional studies. These mice were then infected with P. chabaudi, and the course of infection was followed. The control groups had maximum parasitemias of approximately 30% 10 days after infection, and the infection was cleared within 27 days. Mice without CD4+ cells had significantly higher parasitemias which they were unable to reduce below 20% for the duration of the experiment. Mice without CD8 cells had slightly higher parasitemias which were cleared after 34 days. Because of the possibility that CD8+ cells alone could not be activated in the absence of growth factors, exogenous interleukin-2 was administered to the mice depleted of CD4 cells. This did not significantly affect parasitemias, and the mice were still unable to clear their infections. The data suggest that CD4+ T cells play a crucial role in the protective immune response to the erythrocytic stages of P. chabaudi.
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Cross-linking of the T cell receptor complex with the subset-specific differentiation antigen stimulates interleukin 2 receptor expression in human CD4 and CD8 T cells.
Nonpolymorphic interactions between the T cell differentiation antigens CD4 or CD8 and major histocompatibility complex (MHC)-encoded molecules have been postulated to participate in antigen recognition of MHC-restricted T cells. This would imply simultaneous binding of CD4/8 and of the T cell receptor complex (Ti/CD3) to MHC molecules on the stimulator or target cell. In this report experimental evidence is provided that simultaneous binding by antibodies of Ti/CD3 and of CD4 or CD8 leads to the expression of interleukin 2 (IL 2) receptors in resting human T cells and to their subsequent proliferation in the presence of recombinant IL 2 (rIL 2). This could be shown by using a novel anti-CD3 monoclonal antibody (BMA 030) which alone only marginally stimulates highly purified human T cells even when applied in cross-linked form. However, human T cell subpopulations could be stimulated to grow in the presence of rIL 2 when BMA 030 was fixed to a solid support in combination with antibodies to either CD4 or CD8. In limiting dilution experiments, the frequencies of CD4 and CD8 T cells activated by the antibody combinations were similar to those activated by phytohemagglutinin in the presence of irradiated adherent cells. No stimulation was achieved if both or one antibody was applied in soluble form. In contrast, soluble antibodies inhibited activation by solid-phase antibodies. Taken together, cross-linking of Ti/CD3 with CD4/8 seems to be essential for T cell activation in cases of ligands that bind but do not activate T cells on their own--a situation that may reflect the interaction of T cell receptors with MHC-encoded molecules in association with antigen.
Antibodies, Monoclonal↗
Effective activation of resting mouse T lymphocytes by cross-linking submitogenic concentrations of the T cell antigen receptor with either Lyt-2 or L3T4.
We studied the activation of small resting mouse T lymphocytes by antibodies to the T cell antigen receptor in combination with antibodies to other T cell surface antigens. Solid-phase but not soluble antibodies KJ16-133 and F23.1, both directed to beta chains of the V beta 8 family, activate T cells to proliferate in the presence of growth factors, in a dose-dependent fashion. Antibodies to Lyt-2 and to L3T4 had no activating effect at any concentration. However, submitogenic concentrations of KJ16-133 and of F23.1 synergized with a wide range of concentrations of anti-Lyt-2 and anti-L3T4 to cause T cell proliferation similar or greater in magnitude to that caused by high concentrations of anti-T cell receptor antibody. Synergistic activation was also observed with antibodies to Lyt-1, LFA-1 and H-2 class I antigens but to a significantly lower degree. This was particularly clear in limiting dilution experiments in which the corrected frequencies of T cells proliferating in response to low amounts of anti-T cell receptor antibody together with anti-Lyt-2 were 1/4 to 1/7 for BALB/c T cells. The frequencies of BALB/c T cells responding to high concentrations of anti-T cell receptor antibody alone were between 1/14 and 1/126 and still lower frequencies of T cells proliferated in synergistic responses with anti-LFA-1 or anti-Lyt-1. Synergistic activation leads to the induction of functional cytotoxic cells. We interpret these data as suggestive that cross-linking of the T cell antigen receptor with either Lyt-2 (CD8) or L3T4 (CD4) represents an optimal activating signal for resting T cells. We think that, in physiological T cell activation, cross-linking of the T cell receptor to CD8 or CD4 is induced by their simultaneous binding to major histocompatibility complex (MHC) class I (for CD8) or MHC class II (for CD4) molecules on stimulator cells. We consider the possibility that similar cross-linking requirements may also exist during T cell repertoire selection in ontogeny, thus accounting for the strict coexpression of MHC class I and class II-restricted T cell receptors with CD8 and CD4 molecules, respectively.
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Loss of antigen recognition and impaired cytolytic function in most hybrids between cytotoxic T cells and BW5147.
In this paper we demonstrate that the generation of cytotoxic T-cell hybridomas (CTH) that retain classically H-2 restricted and antigen-specific lytic activity is not generally feasible by fusion of cytotoxic T cells (CTL) with the AKR thymoma BW5147. However, CTH that express a nonspecific lytic activity are readily generated, as revealed by lectin-dependent assays. We analyse several such hybridomas for the nature of their lytic activity and for the expression of various function-associated T-cell molecules. We show that the lytic activity retained by CTH is atypical in that only P815 mastocytoma cells, and no other tumour targets, are lysed in the presence of phytohaemagglutinin (PHA). However, in other respects this lytic activity resembles that of normal CTL: it requires cellular contact, results in a reduction of the viability of the target cells, and is sensitive to both EDTA and low temperature. Lytic CTH express low amounts of T-cell antigen receptor (TCR), whereas non-lytic CTH have no detectable TCR on their surface. Most CTH, lytic or non-lytic, fail to express Lyt 2. We conclude that the loss of antigen specificity in CTH is due to reduced expression of T-cell antigen receptor as well as of other functionally relevant molecules. We further conclude from this work that a search for a better fusion partner may be indicated to facilitate the reliable production of cytotoxic T-cell hybridomas.
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Idiotypic vaccinations: consideration towards a practical application.
We review the theoretical background as well as the available experimental data in animals and man on the possible use of anti-idiotypic antibodies as vaccines for the prevention of infectious diseases. In the first part, the basic experiments and concepts that fostered the idea of idiotypic vaccination are discussed. Although many basic aspects are still unknown, we conclude that the immune system can take antibody variable domains as representatives ("semiotypes") of foreign antigens not only in special cases, but also in a general sense. Among the major areas to be studied further are the events that regulate response to antibody in relation to those that regulate responses to antigen. Initial experiments suggest that responses to antibody may be directed intentionally towards a desired outcome. In the second part, we evaluate the actual medical need for idiotypic vaccination. We conclude that most novel vaccination regimes are likely to be developed with the help of protein chemistry and gene technology. Idiotypic vaccines may become applicable only in special, well-defined situations, such as cases of nonresponsiveness to antigen or cases of severe dysregulation of immunity by the antigen. The third part of the article deals with experimental models for idiotypic vaccination. A number of groups have performed protection experiments in various model infections of experimental animals using anti-idiotypic antibodies as vaccines. In a fair number of cases, involving infections with viral, bacterial, and parasitic microorganisms, protection has been successfully induced. In the fourth part, we summarize studies on idiotype expression in human antigen-driven immune responses. The limited data available suggest that human idiotype expression follows similar rules as in experimental animals. In particular, widely cross-relative idiotopes are readily detected using monoclonal anti-idiotopes. Antibodies to such idiotopes reacted with major proportions of the antibodies of a given specificity. Taken together, many factors point towards the feasibility of idiotypic vaccination.
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Involvement of the T cell antigen receptor and of Lyt-2 in the cytotoxic function of aged killer (AK) T cells.
Aged killer (AK) T cells are antigen-independent, IL-2-requiring variants of antigen-dependent CTL clones that have lost their original antigen specificity and have acquired, instead, specific cytotoxicity for P815 target cells. In this report we study whether AK cells use a similar or a different target cell recognition system than that of bona fide CTL. To this end, we selected from a cloned AK line variants that are partially or completely deficient in specific target recognition and/or in cytotoxic function, and analyzed these variants for expression of the T cell antigen receptor and of Lyt-2. Variants were selected from the prototype AK line (Cl 96) with specific, as well as lectin-facilitated, cytotoxicity for P815 tumor cells. Variants could be grouped into four types with increasing degrees of functional deficiency, which correlated with loss of T cell receptor and/or loss of Lyt-2. In short, loss of Lyt-2 was reflected in loss of specific target recognition, and loss of the T cell antigen receptor was reflected in loss of all cytotoxic activity. We conclude from these results that both Lyt-2 and the T cell antigen receptor are required for specific target cell recognition and the T cell antigen receptor is, in addition, required for cytotoxic function. Moreover, since AK cells express a somatically acquired specificity that differs from that of their clonal precursors, it appears that cytotoxic T cells may change their antigen receptor from one specificity to another during tissue culture.
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Isotype restriction of idiotopes associated with human anti-streptococcal A carbohydrate antibodies.
Sharing of idiotopes between IgG and IgM antibodies has been described with antibodies of various specificities in the literature. However, this does not seem to be the rule for human IgG and IgM antibodies with specificity to streptococcal A carbohydrate. Six idiotopes were described which are associated either with IgM or with IgG antibodies to one of the two major epitopes of streptococcal A carbohydrate: N-acetyl-D-glucosamine or alpha(1,2), alpha(1,3)-linked rhamnose oligomers. Some of the idiotopes are widely cross-reactive with antibodies of the corresponding specificity in other individuals. They were constantly found to be restricted to either IgM or IgG in all individuals tested so far. We discuss several alternatives to explain this finding and conclude that switching from IgM to IgG is either a very rare event in human B cells of this specificity, or leads to loss and gain of idiotopes and/or specificity due to frequent somatic mutations. The phenomenon of idiotope restriction to certain isotypes might be important if anti-idiotopic monoclonal antibodies are considered for use in therapeutical approaches aimed at the manipulation of antibody production.
Acetylglucosamine↗
Hapten-specific cytotoxic T cell clones undergo somatic variation of their antigen recognition specificity.
Two experimental systems have demonstrated somatic variation of antigen recognition specificity of long-term cytotoxic T cell (CTL) clones. System 1 used CTL clone BT7.4.1 with strict specificity for Kb/TNP, which had been continuously cultured for 15 months in the presence of H-2b/TNP stimulator cells and interleukin 2. Upon removal of the TNP antigen from the cultures, 99% of the clone cells within about 10 cell divisions lost their ability to grow in the presence of antigen and interleukin 2 (lethal variants). Of the surviving 1%, about 60% retained the ability to lyse target cells in the presence of lectins but only 12% could be considered as "wild type" BT7.4.1 cells, i.e. they still specifically lysed H-2b/TNP-bearing target cells. The majority of the growing cells, thus, had to be considered as specificity loss variants. Several specificity loss variants were established in culture and were shown to express membrane-bound T cell "receptor" heterodimer similar to their TNP-specific ancestor, BT7.4.1. Principally the same types of variants were generated in cultures growing in the presence of TNP antigen, although in quantitatively reduced numbers. Under these conditions the specific stimulator cells appeared to impose a significant selective advantage for "wild type" CTL since even after 15 months the cultures fully retained their specificity for the nominal antigen. In system 2, the development of cytolytic fine specificity of a panel of 42 individual Kb/TNP-specific CTL clones was followed over a period of 8 months of in vitro culture. At the beginning of the test, 37 of these clones exhibited significant cross-reactivity for lysis of H-2k/TNP target cells. This number of cross-reactive clones continuously diminished with time and dropped to only 4 clones after 8 months in culture. All 42 clones retained their original Kb/TNP specificity and after losing their reactivity for H-2k/TNP usually showed no decrease but rather an increase in their cytotoxic activity towards Kb/TNP target cells. Loss of H-2k/TNP cross-reactivity was not accompanied by loss of Lyt-2 or of LFA-1 surface antigens or by loss of sensitivity of the cytotoxicity to inhibition by anti-Lyt-2 or by anti-LFA-1 antibody. We conclude from these observations that in vitro cultivated CTL clones, at least those of C57BL/6 anti-TNP-C57BL/6 specificity, are not stable in terms of their antigen recognition specificity.(ABSTRACT TRUNCATED AT 400 WORDS)
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