Somatic variation of antigen-recognition specificity in H-2b-TNP-specific cytotoxic T-cell clones.
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Biomedical subjects
K Eichmann
Publications and source records attributed to K Eichmann.
Synergism in the activation of human CD8 T cells by cross-linking the T-cell receptor complex with the CD8 differentiation antigen.
Resting human T cells can be activated and induced to proliferate by cross-linking the T-cell receptor complex (Ti/CD3) with anti-CD3 (T3) antibodies, such as OKT3, together with interleukin 2. Here we describe functional properties of another monoclonal anti-CD3 antibody (BMA 030) that, cross-linked in various ways, only weakly stimulates accessory-cell-depleted T-cell cultures. However, when cross-linked to anti-CD4 or anti-CD8 antibodies a markedly enhanced proliferation of the corresponding subpopulation is observed. We have concentrated on the analysis of CD8 cells and have found that BMA 030, when cross-linked together with anti-CD8 (T811), induced proliferation more than 100-fold greater than BMA 030 alone, whereas cross-linking with antibodies to other T-cell membrane antigens (HLA-A, B, or CD5) provided no or marginal synergistic signals. There was no synergistic effect when only one of the two antibodies, BMA 030 or T811, was cross-linked and the other was applied in soluble form. In contrast, each of the two antibodies alone, when applied in soluble form, inhibited activation induced by the cross-linked antibodies. The T-cell differentiation antigen CD8 has been implicated in the major histocompatibility complex (MHC) class I restricted specificity of CD8 T cells. In previous work from other laboratories only the negative influences of soluble anti-CD8 antibodies have been noted. In contrast, our results suggest that cross-linking between Ti/CD3 and CD8 may be a critical event in the activation of mature CD8 cells. We hypothesize that, in antigen-induced T-cell activation, CD8 and Ti/CD3 become cross-linked by their simultaneous binding to class I-associated structures. Such a mechanism, if required for proliferation in early T-cell ontogeny, could generate a selective pressure for CD8 cells to recognize class I-associated antigens.
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Limiting dilution analysis of proliferating and helper T cells in the in vivo immune response to KLH: derepression of helper T cells at moderately increased frequencies.
While it is clear that some T cells have the capacity for almost indefinite proliferation in vitro, it is a controversial issue how much of this proliferative capacity is utilized by T cells in a response to antigen in vivo. In the framework of a strict clonal selection model the functional activities of normal and immune lymphocyte populations are essentially determined by the frequencies of antigen-specific cells which are clonally expanded after recognition of antigen. In contrast, our group has proposed a network model in which the more prominent effect of immunization is a release of antigen-specific T cells from a state of suppression (= derepression) which exists in the non-immune situation and which is generated through interactions between T cells involving their antigen-specific receptors. In this model, derepression is achieved by competition of antigen with the interactions among T cells through which suppression is exerted. To test our model, we analyze in this paper how much of the immune response to keyhole limpet hemocyanin (KLH) in draining lymph nodes is accounted for by an increase in the numbers of KLH-reactive T cells or by their derepression. To this end, we immunize mice subcutaneously with KLH in CFA. For a period of 14 days after immunization draining lymph nodes are removed, and the frequencies of KLH-reactive proliferating T cells and of KLH-reactive helper T cells determined. We find that proliferating T cells increase 5 to 8 fold in frequency from day 1 to 4 after immunization (approximately 1/30,000 to approximately 1/5000) and no evidence for suppression of these T cells in the non-immune situation can be obtained. In contrast, T helper cells are strongly suppressed in non-immune lymph nodes and become derepressed suddenly between days 3 and 4 following immunization. From day 0 to day 3 T helper cell frequencies are in the order of 1/14,000-1/38,000, then increase suddenly approximately 3-6 fold within 1 day from 1/16,000-1/8,000 to 1/3,000-1/5,000 with no further change until day 14. Thus, helper T cell immunity in draining lymph nodes appears to be generated by a combination of increased frequencies of specific T cells with their release from suppression. In addition, we have reasons to suspect that we overestimate the increase in T cell frequencies. We therefore think that derepression is a major factor in the response of T helper cells to antigen.
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Cluster formation in a symmetrical network: a dynamical system for the description of the suppression among non-immune T lymphocytes and its application to the effects of immunization.
A mathematical model has been developed for the description of the suppressive regulation between polyclonally activated normal and immune T cells. The model assumes reversible cell-cell interactions to interpret results from limiting dilution experiments performed to determine the frequencies of precursor cells for antigen-specific T effector lymphocytes and to analyse mechanisms regulating the maturation of precursor into effector T cells. In particular, the model deals with the changes induced in the T lymphocytes population following immunization with antigens. In these limiting dilution experiments, T cells are placed in cultures at varying cell numbers with all other essential culture constituents kept in excess. After polyclonal activation of the T cells in culture they are supplied with growth and maturation factors so that they form daughter clones of functionally active T effector cells. The typical result observed was that effector T cells develop in cultures at low cell input but that this development is totally suppressed at high cell numbers. This result suggested that, at high cell numbers, the effector T cells are exposed to a sufficient number of other T cells of appropriate specificity to permit suppressive interactions. Whereas this is the case for non-immune T cells, T cells after immunization develop into effector cells both at high as well as at low cell concentrations, though with efficiencies less than proportional to their number of precursors. Our mathematical model is made up of a set of first order autonomous ordinary differential equations in many variables permitting the calculations of numbers of free cells and of cells engaged in cellular clusters of varying sizes. Free cells can develop into effector cells whereas cells engaged in clusters cannot. We calculate the consequences of several reasonable hypotheses concerning the effects of immunization. We consider the possibility that immunization modifies the growth behavior of the antigen-specific cells to permit an increased or accelerated clonal expansion in culture. Alternatively, we consider the possibility that immunization changes the interaction strength between cells specific for the immunizing antigen and other cells. Thirdly, we have connected both behaviors by calculating the case of an inverse relationship between growth rates and intensities of interaction between cells. Our model has been inspired by the symmetrical network model and can be interpreted in this framework. It proposes that immune regulation is a consequence of idiotype-anti-idiotype interactions.(ABSTRACT TRUNCATED AT 400 WORDS)
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Human immune response to group A streptococcal carbohydrate (A-CHO). I. Quantitative and qualitative analysis of the A-CHO-specific B cell population responding in vitro to polyclonal and specific activation.
The immune response to the group-specific carbohydrate of group A streptococci (A-CHO) provides an informative in vitro model for the investigation of several aspects of human anticarbohydrate immune responses. A-CHO-specific B cells can be polyclonally activated by pokeweed mitogen (PWM), and, specifically, by in vitro immunization with streptococcal vaccine. High levels of A-CHO-specific antibodies, mainly directed to the immunodominant side chain N-acetyl-D-glucosamine (GlcNAc), occur in healthy adult individuals. Serum antibody levels are reflected in high frequencies of precursor B cells among peripheral blood lymphocytes. In one particular case, greater than 15% of all B cells activated by PWM for IgM production were found to produce IgM anti-A-CHO antibodies, as determined in limiting dilution experiments, as well as by analyzing Ig concentrations in bulk culture experiments. The case with the lowest proportion observed had 0.3% A-CHO-specific B cells among IgM-producing B cells. Preferential PWM activation of anti-A-CHO-producing B cells could be excluded. The comparison of the proportions of anti-A-CHO IgM produced in vivo, and of B cells producing antibodies of this specificity in peripheral blood, suggests a similar distribution of specific precursor B cells in the antibody-producing lymphoid tissue compartments and in peripheral blood. However, nearly all specific antibodies produced in vitro belong to the IgM isotype, whereas IgG anti-A-CHO in high amounts, mostly exceeding the specific IgM, was found only among anti-A-CHO antibodies produced in vivo. Low anti-A-CHO IgG production was seen in polyclonally activated as well as in antigen-activated cultures, whereas, in contrast, total IgG was produced in considerable amounts after polyclonal activation. This suggests a different distribution pattern, and/or diverse differentiation requirements for anti-A-CHO-producing B cells, compared with other B cell species.
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Genetic control of B cell function. III. IgVH-controlled polymorphism in the frequencies of B cells that recognize xenogeneic red blood cells.
Previous work has shown that the primary IgM plaque-forming cell response of inbred mice to xenogeneic red blood cells (RBC) including sheep, horse and chicken RBC is under the control of two polymorphic genes or sets of genes, one linked to the Igh linkage group and the other of unknown linkage but unlinked to H-2 and a variety of other known genetic markers. Both genes together control B cell function but do not influence the function of T cells and macrophages. Thus, this system permits the study of two polymorphic loci that control B cell responsiveness. In this study we analyze the role of the Igh region in further detail. In bulk cultures and limiting dilution experiments, we confirm its exclusive influence on B cells also when analyzed separately from the background gene, i.e. in Igh-congenic strains. Moreover, we find in the majority of experiments 4-5-fold differences in sheep RBC-specific B cell precursor frequencies among lipopolysaccharide-reactive cells from 3 pairs of Igh-congenic high and low-responder strains. Similar frequency differences exist for horse RBC and chicken RBC-specific B cells but not for B cells with specificity for (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP)-gelatine. These differences are independent of the frequencies of B cells responding to lipopolysaccharide which are shown to be equal between Igh-congenic pairs of strains. Since the differences in RBC-specific B cell frequencies closely resemble the differences in bulk culture responses to the corresponding RBC, we conclude that the role of the Igh linkage group in controlling responsiveness to RBC lies in a selective influence on the B cell repertoire concerning precursor cells for RBC specificity. In addition, we find that the VH part of Igh is responsible for the observed frequency differences, suggesting that VH germ-line genes directly influence the composition of the mature B cell repertoire.
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Two human IgM myeloma proteins with unusual specificities for streptococcal carbohydrate-associated epitopes.
Five hundred and fifty human sera from patients with IgM myeloma or Waldenström's macroglobulinaemia were screened by a solid-phase enzyme-linked immunoassay for binding to the carbohydrate of group A streptococci (A-CHO). Two of them (AC8 and AC179) contained immunoglobulin, which bound specifically to A-CHO even at serum dilutions of 1:10(7). Using synthetic oligosaccharides coupled to protein for inhibition studies, the fine specificities of AC8 and AC179 were determined. AC179 is directed to alpha-linked rhamnose oligosaccharides. AC8 appears to be specific for N-acetyl-D-glucosamine (GlcNAc) side chains beta(1----2)-linked to rhamnose, whereas GlcNAc side chains in A-CHO are reported to be beta(1----3)-linked to the rhamnose backbone. Naturally occurring anti-A-CHO antibodies consist mainly of low-affinity antibodies to such beta(1----3)-linked GlcNAc. In contrast, both myeloma antibodies show more than 10 times higher relative affinities to A-CHO than antibodies prepared from normal human serum (anti-GlcNAc and anti-A-CHO, respectively) by selection for high affinity in the elution procedure. AC179 induced complement activation in the presence of A-CHO.
Acetylglucosamine↗
Vaccination against the intracellular bacterium Listeria monocytogenes with a clonotypic antiserum.
The T cell clone 26.1.1, which confers specific protection against the intracellular bacterium Listeria monocytogenes, was fused to BW 5147. The resulting T cell hybridoma, TLm1, could be stimulated to secret interleukin 2 by antigen plus accessory cells or concanavalin A. Stimulation was specific for an epitope expressed by L. monocytogenes EGD but not ATCC 19114 and was H-2I-A restricted. Antisera against TLm1 were raised in syngeneic mice and tested for their capacity to block TLm1 responses. Two antisera were identified that blocked antigen but not concanavalin A stimulation of TLm1 and did not affect antigen stimulation of similar but not identical L. monocytogenes-specific T cell hybridomas. Hence, these antisera had clonotypic activity. When these antisera were administered subcutaneously in complete Freund's adjuvant, mice were protected against a subsequent L. monocytogenes infection. Protection was antigen specific and H-2 nonrestricted. These findings suggest the feasibility of clonotypic antibodies for vaccination against intracellular bacterial infections.
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Immunoregulation by mouse T-cell clones. II. The same H-Y-specific T helper clone can provide help for the generation of cytotoxic lymphocytes and antibody-secreting cells.
Mouse H-Y-specific and I-Ab restricted T-cell clones have been established and compared for their helper effects in the differentiation of both T and B lymphocytes. The results demonstrate that three individual T-cell clones and one subclone could help in the antigen-driven induction of cytotoxic lymphocytes (CTL) from their precursor cells (CTL-P), and were able to activate B cells to develop into antibody-secreting cells (PFC) in the presence of SRBC, provided the cloned T cells were restimulated by H-Y antigen on antigen-presenting cells. In addition, antigen or lectin could induce the same H-Y-specific T-cell clones to secrete factor(s) expressing helper activities similar to that of the cloned T cells. Furthermore, it is shown that the T cell-derived soluble mediator(s) was distinct from T-cell growth factor (TCGF) and from immune interferon (IFN-gamma). The data reveal a new type of T cell with helper potential for the activation of CTL-P and B lymphocytes, and suggest the existence of distinct T helper cells which can provide help for both cytotoxic and antibody responses by virtue of different lymphokine activities.
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Idiotope mapping on the variable region of an antibody clonotype produced by normal (nonmalignant) human B cells.
Human anti-N-acetyl-D-glucosamine (GlcNAc) antibodies were prepared by affinity chromatography from serum of a healthy donor (MSS). They were heterogeneous but contained a unique antibody clonotype (1A) representing 7% of all anti-GlcNAc antibodies. Out of a series of monoclonal anti-idiotopic antibodies (anti-Id mAb), we identified five antibodies that bound to clonotype 1A as shown by isoelectric focusing and Western blotting. Two of them were specific for clonotype 1A (10F59 and 13F15), thus indicating its clonal origin. However, three anti-Id mAb (16F433, 16F539, and 16F812) bound to various additional portions of anti-GlcNAc antibodies of donor MSS. With the exception of one mAb, all anti-Id mAb have very similar relative affinities to clonotype 1A, so results from competition experiments between the different antibodies and between each antibody and antigen should reveal spatial relationships between the corresponding Id and between each Id and the antigen-combining site. The results show a consistent topography of Id on the V-region of clonotype 1A. Id 59, 812, and 433 were found to be arranged in one cluster (cluster I), whereas Id 15 and 539 belonged to a second cluster (cluster II). Cluster I resides completely in the antigen-combining site, whereas only Id 15 of cluster II weakly overlaps with the binding site. Our study demonstrates an analysis of spatial relationships of Id expressed on a human antibody clonotype. To our knowledge, this is the first demonstration of Id mapping on antibodies produced by a normal (nonmalignant) B cell clone that should be accessible to regulatory signals. Such analysis may contribute to a more detailed characterization of anti-Id mAb, and may provide additional information for a better understanding of their immunoregulatory effects.
Acetylgalactosamine↗
Limiting dilution analysis of induced unresponsiveness to trinitrophenyl: increased suppression of cytotoxic T precursor cells at unchanged frequencies.
In this paper we address the problem of tolerance in the immune system. We are discussing results of experiments that we designed to distinguish between the two major alternative hypotheses that have been invoked for immunological tolerance: clonal deletion/anergy versus suppression. These alternative hypotheses make essentially different predictions with respect to the frequencies of lymphocytes that react with antigens to which the immune system is tolerant: clonal deletion models predict reduced frequencies, whereas in suppression models precursors of effector cells are postulated to occur at frequencies similar to the nontolerant situation. We investigate these questions by limiting dilution analyses of cytotoxic T cell precursors (CTLP), using two different ways to activate them into functional cytotoxic cells (CTL): polyclonally by Concanavalin A (ConA) and specifically by antigen. Tolerance should be apparent in either protocol since neither antigen nor ConA usually activates self-reactive cytotoxic cells. Moreover, in addition to providing information on precursor frequencies, the multihit results usually obtained in limiting dilution experiments of ConA activated T cells allow the determination of quantitative and qualitative parameters of suppression. In addition, clonal anergy situations could be expected to become apparent by a differential sensitivity of precursors to activation with ConA and with antigen. As experimental system we chose the induced unresponsiveness of mice to trinitrophenyl (TNP) achieved by intravenous injection of reactive trinitrobenzenesulfonic acid (TNBS) and measured in a primary cytotoxic response to TNP-coupled syngeneic cells (TNP-SC). An equally specific but perhaps not identical form of unresponsiveness is induced by coupling the responder cell population with TNBS in vitro. Although we do not propose that this model ideally reflects all aspects of self tolerance, we think that in this particular system of induced unresponsiveness the antigen becomes associated with the surfaces of a large proportion of the cells of the body. Therefore, tolerance to widely expressed cell surface determinants might be quite adequately "simulated" by this experimental unresponsiveness. In our limiting dilution experiments we can distinguish between frequent and infrequent cytotoxic precursors which correspond to virgin and memory cells, respectively. For the frequent CTLP the situation is quite clear: they occur at essentially equal frequencies in normal and tolerant mice, as demonstrated by their reactivity to antigen at low cell densities. However, they are under the influence of a suppressive activity which is strongly increased when compared to that of normal mice.(ABSTRACT TRUNCATED AT 400 WORDS)
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Characterization of a major human antibody clonotype (1A) by monoclonal antibodies to combining site-associated idiotopes.
To identify major human antibody clonotypes with specificity to N-acetyl-D-glucosamine (GlcNAc), affinity-purified antibody preparations of different human individuals were analyzed by isoelectric focusing (IEF). A major clonotype (1A) was identified representing 7% of all anti-GlcNAc antibodies of one donor (no. 371). Anti-GlcNAc antibodies of donor 371 were used as immunogen to prepare monoclonal anti-idiotopic antibodies (mAb). Two anti-idiotopic mAb were specific for the major clonotype 1A, as shown by blotting of the anti-GlcNAc antibodies after IEF in thin-layer agarose to nitrocellulose filters and staining with either iodinated antigen or iodinated anti-idiotopic mAb. The two anti-idiotopic mAb 13F15 and 10F59 were further analyzed with respect to the antigenic determinant which they recognize. Both were directed to combining site-related determinants as shown by inhibition studies. Although 13F15 has a 500-fold higher binding capacity (relative affinity) equal amounts do not inhibit more than 50% of 10F59 binding, suggesting that the two mAb detect two closely related, but not identical, idiotopes in the antigen combining site of clone 1A. Although clonotype 1A is a unique antibody exclusively found in IEF of donor 371, the idiotope 1A.1, defined by mAb 13F15, is a recurrent determinant detectable on different anti-GlcNAc spectrotypes in various human sera.
Acetylglucosamine↗
Immunoregulation by mouse T-cell clones. I. Suppression and amplification of cytotoxic responses by cloned H-Y-specific cytolytic T lymphocytes.
H-Y-specific and H-2Db-restricted, Lyt-1-2+ T-cell clones ( CTLL ) with graded specific cytotoxic activities on male C57BL/6 (B6) target cells ( 1E3 , ; 2C5 , ++; 2A5 , +, 3E6 , +/-) were tested for their capacity to inhibit the generation of H-Y-specific cytotoxic T lymphocytes (CTL) in vitro. Addition of irradiated lymphocytes of CTLL 1E3 and CTLL 3E6 but not those of CTLL 2A5 or CTLL 2C5 abolished the generation of CTL from in vivo primed H-Y-specific precursor cells (CTLP) when added to fresh mixed-lymphocyte cultures (MLC). Exogenous sources of T-cell growth factors (TCGF) did not overcome suppression. Rather the presence of TCGF resulted in a further enhancement of suppressive activities in CTLL 1E3 and 3E6 and the induction of similar activities in cells from CTLL 2A5 and 2C5 , which by themselves were not inhibitory. Moreover when added to similar MLC on Day 1 instead of Day 0, only irradiated cells of CTLL 3E6 but not those of the other three CTLL were suppressive. Induction of suppressive activities in H-Y-specific CTLL was independent of the appropriate male stimulator cells since it was also observed in MLC induced by irrelevant antigens (H-2, trinitrophenol). Furthermore at low cell numbers, irradiated lymphocytes from any of the CTLL consistently enhanced CTL activities generated from H-Y-specific CTLP. This augmenting activity, which was not TCGF, could be transferred by soluble mediators present in antigen-sensitized CTLL cultures. Thus, these data indicate (i) that cytotoxic effector cells can function as suppressor cells in the generation of CTL, (ii) that the cytotoxic activity of cloned CTL does not correlate with their capacity to suppress CTL responses, (iii) that the inhibition of CTL responses by CTLL is not due to simple consumption of T-cell growth factors produced in MLC, and (iv) that different CTL clones may interfere with the generation of CTL at different stages of their maturation. Moreover, the experiments suggest an antigen-independent enhancement of suppression by the interaction of CTL with lymphokines. Together with the augmenting activity evoked by cloned CTL the data provide strong evidence for the expression of multiple immunological functions by one particular subset of T cells and suggest that cytotoxic effector cells can differentially regulate the maturation and/or clonal expression of their precursor cells.
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The growth of concanavalin-A activated, Lyt selected subsets in IL-2 containing supernatants.
The growth properties of Con A activated, Lyt selected splenic T lymphocytes were examined by limiting dilution analysis and clonally by single cell picking. Under the conditions used, a comparable frequency of Lyt 1+ and Lyt 2+ cells grew after Con A activation in the presence of Con A rat spleen supernatant. At a clonal level, however, the growth of these subsets differed qualitatively and quantitatively. While Lyt 2+ cells obtained clone sizes of several hundred cells, Lyt 1+ clone sizes were usually less than 100 cells, and many clones aborted their growth after a few days. Morphologically, the Lyt 1+ cell was smaller and usually showed fewer pseudopodial protusions as compared to the Lyt 2+ cell.
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Network regulation among T cells: qualitative and quantitative studies on suppression in the non-immune state.
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Quantitative studies on T cell diversity. IV. Mathematical analysis of multiple limiting populations of effector and suppressor T cells.
Limiting dilution (LD) analyses of polyclonally activated T cells yielded results suggesting the existence of multiple paired populations of effector and suppressor precursors for a number of different T cell functions and specificities analyzed. These populations occur at graded frequencies and suppression occurs within a pair but not between pairs. In this paper, we establish the mathematical basis for the interpretation of these multi-component limiting dilution results. First, we derive equations for a number of mathematical models and identify one model that both makes biological sense and can be used to reproduce experimental data. Second, within this model, we identify parameters such as the frequency of suppressive cells and the number of suppressive cells required for suppression. The results suggest that within each paired population, suppressor precursors are 20 times more frequent that effector precursors. Furthermore, a similar but variable excess of suppressor cells is required for suppression to become effective. Together with the high frequency (1/50-1/500) of most effector T cell precursors previously reported, the results suggest that up to 40% of the T cells can become involved in suppression of an antigen-specific effector T cell population. These studies may provide exact estimates for predictions to be tested in experiments on immune regulation.
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A cross-reacting human idiotype (B17) associated with antibodies to N-acetyl-D-glucosamine. Specificity, immunoglobulin class association, and distribution in the population.
This report describes the study of the expression of an idiotype in the human population which is associated with antibodies to N-acetyl-D-glucosamine (GlcNAc) present in most human sera presumably due to streptococcal infections. The idiotype is identified with antisera and monoclonal antibodies prepared against the IgM (kappa) antibody secreted by the Epstein-Barr virus-transformed human B cell line B17. At least 90% of 207 individuals tested had immunoglobulin with B17 idiotypic determinants in their sera, as demonstrated with conventional and one monoclonal anti-idiotypic antibody. Another monoclonal anti-idiotypic antibody reacted with antibodies in only a few of the sera. No correlation was found between the level of expression of different idiotopes in individual human sera, suggesting molecular heterogeneity of the B17-positive antibody population. B17-positive immunoglobulins are to a large extent specific for GlcNAc but represent only a minor population of all GlcNAc-specific antibodies in human sera. B17 determinants are on IgM (kappa) in all human sera and on IgG and IgA in some. In addition, some lambda-bearing Ig was found to react with anti-B17 antisera, suggesting the detection of VH-associated idiotypic determinants in this experimental system.
Acetylglucosamine↗
Network regulation among T cells; conclusions from limiting dilution experiments.
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