Design of T-cell receptor libraries with diverse binding properties to examine adoptive T-cell responses.

Adoptive T-cell therapies have shown significant promise in the treatment of cancer and viral diseases. One approach, which introduces antigen-specific T-cell receptors (TCRs) into ex vivo activated T cells, is designed to overcome central tolerance mechanisms that prevent responses by endogenous T-cell repertoires. Studies have suggested that use of higher-affinity TCRs against class I major histocompatibility complex antigens could drive the activity of both CD4(+) and CD8(+) T cells, but the rules that govern the TCR binding optimal for in vivo activity are unknown. Here, we describe a high-throughput platform of 'reverse biochemistry' whereby a library of TCRs with a wide range of binding properties to the same antigen is introduced into T cells and adoptively transferred into mice with antigen-positive tumors. Extraction of RNA from tumor-infiltrating lymphocytes (TILs) or lymphoid organs allowed high-throughput sequencing to determine which TCRs were selected in vivo. The results showed that CD8(+) T cells expressing the highest-affinity TCR variants were deleted in both the TIL population and in peripheral lymphoid tissues. In contrast, these same high-affinity TCR variants were preferentially expressed within CD4(+) T cells in the tumor, suggesting they had a role in antigen-specific tumor control. The findings thus revealed that the affinity of the transduced TCRs controlled the survival and tumor infiltration of the transferred T cells. Accordingly, the TCR library strategy enables rapid assessment of TCR-binding properties that promote peripheral T-cell survival and tumor elimination.

Single-chain VαVß T-cell receptors function without mispairing with endogenous TCR chains.

Transduction of exogenous T-cell receptor (TCR) genes into patients' activated peripheral blood T cells is a potent strategy to generate large numbers of specific T cells for adoptive therapy of cancer and viral diseases. However, the remarkable clinical promise of this powerful approach is still being overshadowed by a serious potential consequence: mispairing of the exogenous TCR chains with endogenous TCR chains. These 'mixed' heterodimers can generate new specificities that result in graft-versus-host reactions. Engineering TCR constant regions of the exogenous chains with a cysteine promotes proper pairing and reduces the mispairing, but, as we show here, does not eliminate the formation of mixed heterodimers. By contrast, deletion of the constant regions, through use of a stabilized Vα/Vß single-chain TCR (scTv), avoided mispairing completely. By linking a high-affinity scTv to intracellular signaling domains, such as Lck and CD28, the scTv was capable of activating functional T-cell responses in the absence of either the CD3 subunits or the co-receptors, and circumvented mispairing with endogenous TCRs. Such transduced T cells can respond to the targeted antigen independent of CD3 subunits via the introduced scTv, without the transduced T cells acquiring any new undefined and potentially dangerous specificities.

Expression of structurally diverse Qa-2-encoded molecules on the surface of cloned cytotoxic T lymphocytes.

Extracts of 125I-labeled cloned murine cytotoxic T lymphocytes (CTL) were immunoprecipitated with alloantisera to the cloned CTL and rabbit antisera to beta-2 microglobulin. Polyacrylamide gel electrophoresis (PAGE) of the specific precipitates revealed, as expected, 125I-labeled components that corresponded to products of class I genes of the major histocompatibility complex (MHC). However, additional class I gene products of relatively low apparent molecular weight (Mr) were also observed. Similar analyses of spleen cells from a variety of MHC-congenic mouse strains suggested that the class I molecules of relatively low Mr are encoded in the Qa-2 region of the MHC, and this was confirmed by immunoprecipitation with a monoclonal antibody to Qa-2. Surprisingly, however, the cell surface Qa-2 molecules of different CTL clones differed in Mr, in isoelectric focusing (IEF) pattern, and in the number of distinguishable molecules expressed per clone: some clones seemed to express only a single Qa-2-encoded molecule while others expressed two distinct ones. Treatment of the immunoprecipitated Qa-2 with endoglycosidase F (Endo F) resulted in a decrease in Mr of approximately 5,000-6,000, corresponding to the expected loss of N-linked oligosaccharides, but the decrease did not eliminate structural variability among the clones. Structural diversity of the Qa-2-encoded molecules expressed on CTL could arise because CTL clones differ (a) in the particular Qa-2 genes they express, (b) in the way they splice Qa-2 gene transcripts or, perhaps, (c) in Endo F-resistant oligosaccharides on their Qa-2 molecules.

Effects of complementarity determining region mutations on the affinity of an alpha/beta T cell receptor: measuring the energy associated with CD4/CD8 repertoire skewing.

It has been proposed that the generally low affinities of T cell receptors (TCRs) for their peptide-major histocompatibility complex (pMHC) ligands (Kd approximately 10(-4) to 10(-7) M) are the result of biological selection rather than an intrinsic affinity limitation imposed by the TCR framework. Using a soluble version of the 2C TCR, we have used complementarity determining region (CDR)-directed mutagenesis to investigate whether the affinity of this receptor for its allogeneic pMHC ligand can be improved upon. We report that several mutants at positions lying within CDR3alpha and CDR2beta showed increased affinities for pMHC compared with the wild-type receptor. Additionally, we have investigated whether Valpha mutations that have been implicated in the phenomenon of CD8(+) repertoire skewing achieve this skewing by means of generalized increases in affinity for MHC-I molecules. Two mutants (S27F and S51P), which each promote skewing toward a CD8(+) phenotype, exhibited significantly reduced affinity for pMHC-I, consistent with a quantitative-instructional model of CD4/CD8 lineage commitment. This model predicts that CD8 is downregulated on thymocytes that have TCR-ligand interactions above a minimal energy threshold. Together, the results (a) demonstrate that engineering higher affinity TCRs is feasible, and (b) provide TCR-pMHC energy values associated with CD4/CD8 repertoire skewing.

Resistance of cytotoxic T lymphocytes to the lytic effects of their toxic granules.

A cytotoxic T lymphocyte (CTL) characteristically kills target cells one after the other by releasing toxic granules that contain one or more cytolytic components. To determine how CTLs avoid destroying themselves when they release granules and lyse target cells, 7 murine CD8+ CTL cell lines were compared with 19 other cell lines for susceptibility to lysis by the isolated toxic granules. Murine CD8+ CTLs were clearly the most resistant cells: granules did not lyse them even after they were exposed to azide, cyanide, and 2-deoxyglucose, conditions that were found to enhance the susceptibility of all the other cells tested, including other T cells. Thus, resistance of CD8+ CTLs to cytotoxic granules appears to be independent of cellular ATP. To reconcile these findings with other observations that, under some circumstances, CTLs can be lysed by other CTLs, we suggest a model in which a CTL releases only a limited proportion of its toxic granules at each antigen-specific encounter with a target cell; the amount released is sufficient to kill most target cells but to leave the CTL undamaged and with enough granules to attack other target cells.

Role of 2CT cell receptor residues in the binding of self- and allo-major histocompatibility complexes.

T cell clone 2C recognizes the alloantigen L(d) and the positive selecting major histocompatibility complex (MHC), K(b). To explore the molecular basis of T cell antigen receptor (TCR) binding to different peptide/MHC (pMHC) complexes, we performed alanine scanning mutagenesis of the 2C TCR. The TCR energy maps for QL9/L(d) and SIYR/K(b) were remarkably similar, in that 16 of 41 Valpha and Vbeta alanine mutants showed reduced binding to both ligands. Several TCR residues varied in the magnitude of energy contributed to binding the two ligands, indicating that there are also unique interactions. Residues in complementarity determining region 3alpha showed the most notable differences in binding energetics among the ligands QL9/L(d), SIYR/K(b), and the clonotypic antibody 1B2. Various lines of evidence suggest that these differences relate to the mobility of this loop and point to the key role of conformational dynamics in pMHC recognition.

Mapping the energy of superantigen Staphylococcus enterotoxin C3 recognition of an alpha/beta T cell receptor using alanine scanning mutagenesis.

Binding of the T cell receptor (TCR) to a bacterial superantigen (SAG) results in stimulation of a large population of T cells and subsequent inflammatory reactions. To define the functional contribution of TCR residues to SAG recognition, binding by 24 single-site alanine substitutions in the TCR Vbeta domain to Staphylococcus aureus enterotoxin (SE) C3 was measured, producing an energy map of the TCR-SAG interaction. The results showed that complementarity determining region 2 (CDR2) of the Vbeta contributed the majority of binding energy, whereas hypervariable region 4 (HV4) and framework region 3 (FR3) contributed a minimal amount of energy. The crystal structure of the Vbeta8.2-SEC3 complex suggests that the CDR2 mutations act by disrupting Vbeta main chain interactions with SEC3, perhaps by affecting the conformation of CDR2. The finding that single Vbeta side chain substitutions had significant effects on binding and that other SEC3-reactive Vbeta are diverse at these same positions indicates that SEC3 binds to other TCRs through compensatory mechanisms. Thus, there appears to be strong selective pressure on SAGs to maintain binding to diverse T cells.

CD8(-) T cell transfectants that express a high affinity T cell receptor exhibit enhanced peptide-dependent activation.

T cells are activated by binding of the T cell receptor (TCR) to a peptide-major histocompatibility complex (MHC) complex (pMHC) expressed on the surface of antigen presenting cells. Various models have predicted that activation is limited to a narrow window of affinities (or dissociation rates) for the TCR-pMHC interaction and that above or below this window, T cells will fail to undergo activation. However, to date there have not been TCRs with sufficiently high affinities in order to test this hypothesis. In this report we examined the activity of a CD8-negative T cell line transfected with a high affinity mutant TCR (K(D) = 10 nM) derived from cytotoxic T lymphocyte clone 2C by in vitro engineering. The results show that despite a 300-fold higher affinity and a 45-fold longer off-rate compared with the wild-type TCR, T cells that expressed the mutant TCRs were activated by peptide. In fact, activation could be detected at significantly lower peptide concentrations than with T cells that expressed the wild-type TCR. Furthermore, binding and functional analyses of a panel of peptide variants suggested that pMHC stability could account for apparent discrepancies between TCR affinity and T cell activity observed in several prior studies.

Selection of variable-joining region combinations in the alpha chain of the T cell receptor.

Most T lymphocytes express an antigen-specific receptor composed of two subunits, alpha and beta, each of which can exhibit structural variability. A complex selection process operates on T cells during development in the thymus such that cells expressing only particular alpha beta-receptors migrate to the periphery. The alpha-chain repertoire was dissected at different stages of the selection process by using the polymerase chain reaction (PCR) technique to amplify only those transcripts of a particular variable region gene (V58). Sequences from these V58 cDNAs reveal the predominant expression of four joining (J) segments by T cells in the adult thymus, suggesting that molecular or cellular processes select particular V alpha J alpha combinations during development. T cells expressing one of these V58J alpha chains appear to have been negatively selected at a later stage, since these transcripts were present in the spleen at approximately one-tenth the level in the thymus. Results also indicate that residues present at the V alpha J alpha junction may be important in an early selection process.

Chromosomal locations of the murine T-cell receptor alpha-chain gene and the T-cell gamma gene.

Two independent methods were used to identify the mouse chromosomes on which are located two families of immunoglobulin (Ig)-like genes that are rearranged and expressed in T lymphocytes. The genes coding for the alpha subunit of T-cell receptors are on chromosome 14 and the gamma genes, whose function is yet to be determined, are on chromosome 13. Since genes for the T-cell receptor beta chain were previously shown to be on mouse chromosome 6, all three of the Ig-like multigene families expressed and rearranged in T cells are located on different chromosomes, just as are the B-cell multigene families for the Ig heavy chain, and the Ig kappa and lambda light chains. The findings do not support earlier contentions that genes for T-cell receptors are linked to the Ig heavy chain locus (mouse chromosome 12) or to the major histocompatibility complex (mouse chromosome 17).

Directed evolution of a stable scaffold for T-cell receptor engineering.

Here we have constructed a single-chain T-cell receptor (scTCR) scaffold with high stability and soluble expression efficiency by directed evolution and yeast surface display. We evolved scTCRs in parallel for either enhanced resistance to thermal denaturation at 46 degrees C, or improved intracellular processing at 37 degrees C, with essentially equivalent results. This indicates that the efficiency of the consecutive kinetic processes of membrane translocation, protein folding, quality control, and vesicular transport can be well predicted by the single thermodynamic parameter of thermal stability. Selected mutations were recombined to create an scTCR scaffold that was stable for over an hour at 65 degrees C, had solubility of over 4 mg ml(-1), and shake-flask expression levels of 7.5 mg l(-1), while retaining specific ligand binding to peptide-major histocompatibility complexes (pMHCs) and bacterial superantigen. These properties are comparable to those for stable single-chain antibodies, but are markedly improved over existing scTCR constructs. Availability of this scaffold allows engineering of high-affinity soluble scTCRs as antigen-specific antagonists of cell-mediated immunity. Moreover, yeast displaying the scTCR formed specific conjugates with antigen-presenting cells (APCs), which could allow development of novel cell-to-cell selection strategies for evolving scTCRs with improved binding to various pMHC ligands in situ.

Development of a novel strategy for engineering high-affinity proteins by yeast display.

Yeast display provides a system for engineering high-affinity proteins using a fluorescent-labeled ligand and fluorescence-activated cell sorting (FACS). In cases where it is difficult to obtain purified ligands, or to access FACS instrumentation, an alternative selection strategy would be useful. Here we show that yeast expressing high-affinity proteins against a mammalian cell surface ligand could be rapidly selected by density centrifugation. Yeast cell-mammalian cell conjugates were retained at the density interface, separated from unbound yeast. High-affinity T cell receptors (TCRs) displayed on yeast were isolated using antigen presenting cells that expressed TCR ligands, peptides bound to products of the major histocompatibility complex (MHC). The procedure yielded 1000-fold enrichments, in a single centrifugation, of yeast displaying high-affinity TCRs. We defined the affinity limits of the method and isolated high-affinity TCR mutants against peptide variants that differed by only a single residue. The approach was applied to TCRs specific for class I or class II MHC, an important finding since peptide-class II MHC ligands have been particularly difficult to purify. As yeast display has also been used previously to identify antigen-specific antibodies, the method should be applicable to the selection of antibodies, as well as TCRs, with high-affinity for tumor cell-surface antigens.

Generation of cytotoxic T-lymphocytes to a self-peptide/class I complex: a model for peptide-mediated tumor rejection.

Cytotoxic T-lymphocytes (CTL) typically recognize foreign peptides bound to class I products of the major histocompatibility complex. A function of CTL is to identify and eliminate tumor cells that bear inappropriately expressed peptide/class I complexes (i.e., mutated self-peptides or self-peptides that are expressed at abnormally high levels). The processes that result in tolerance to self-antigens can undermine the effectiveness of this system by deleting or inactivating T-cells that might potentially be reactive with tumor-associated antigens. To up-regulate the response to tumor antigens it will be useful to develop methods whereby CTL responses to specific self-peptides can be elicited without damage to normal tissue. In this report a CTL response was generated in BALB/c mice against the ubiquitous self-peptide p2Ca (LSPFPFDL), which binds to Ld and is derived from the mitochondrial enzyme alpha-ketoglutarate dehydrogenase. CTL derived in vitro recognize specifically the p2Ca/Ld complex and use V beta 8 regions predominantly. The cultured cells lysed target cells with lower levels of p2Ca than the levels used for induction. This result suggests that it may be possible to use peptides at high concentrations to elicit CTL react with endogenous levels of a peptide/class I complex. The in vivo potential of the response was demonstrated by the observation that BALB/c mice, coinjected with a syngeneic BALB/c myeloma and exogenous p2Ca, are able to reject the tumor. The p2Ca/Ld system may thus provide a model for evaluating the parameters for effective immunotherapy with tumor-associated peptides.

Yeast polypeptide fusion surface display levels predict thermal stability and soluble secretion efficiency.

Efficiency of yeast cell surface display can serve as a proxy screening variable for enhanced thermal stability and soluble secretion efficiency of mutant proteins. Several single-chain T cell receptor (scTCR) single-site mutants that enable yeast surface display, along with their double and triple mutant combinations, were analyzed for soluble secretion from the yeast Saccharomyces cerevisiae. While secretion of the wild-type scTCR was not detected, each of the single, double, and triple mutants were produced in yeast supernatants, with increased expression resulting from the double and triple mutants. Soluble secretion levels were strongly correlated with the quantity of active scTCR displayed as a fusion to Aga2p on the surface of yeast. Thermal stability of the scTCR mutants correlated directly with the secreted and surface levels of scTCR, with evidence suggesting that intracellular proteolysis by the endoplasmic reticulum quality control apparatus dictates display efficiency. Thus, yeast display is a directed evolution scaffold that can be used for the identification of mutant eucaryotic proteins with significantly enhanced stability and secretion properties.

Specificity and binding properties of a single-chain T cell receptor.

The specificity of a T cell is dictated by an alpha beta T cell receptor (TCR) that recognizes a complex of peptide and a product of the major histocompatibility complex (MHC). Recent studies have begun to characterize the affinities and kinetics of these interactions, but details of the alpha beta TCR structure and function are not known. To examine some of these issues we focus in this report on a TCR derived from the T cell clone 2C. This TCR binds to a complex of the nonapeptide QL9 and the class I MHC product Ld with the highest affinity of any known TCR/ligand interaction (KD approximately 10 (-7) M). Circular dichroism showed that a single-chain TCR (scTCR) containing linked V alpha and V beta regions from T cell 2C and refolded from Escherichia coli inclusion bodies exhibited the characteristic beta-sheet structure of immunoglobulins. A sensitive assay that is capable of detecting the interaction of soluble scTCR with peptide /MHC ligand on the surface of target cells was used to demonstrate that the peptide specificity of this scTCR reflects that of the TCR found on the surface of 2C. Analysis of several scTCR V alpha region mutants confirmed that the V alpha domain is critical for the specificity of scTCR binding. Finally, we identified some notable differences in the complementarity determining regions (CDR) of the 2C TCR compared to the CDR of previously characterized, cytochrome- specific TCR. These differences are discussed in the light of what is known about antibody binding sites, the high affinity of the 2C TCR, and the nature of the residues on QL9 that are predicted to interact with the TCR.

High affinity T cell receptors from yeast display libraries block T cell activation by superantigens.

The alphabeta T cell receptor (TCR) can be triggered by a class of ligands called superantigens. Enterotoxins secreted by bacteria act as superantigens by simultaneously binding to an MHC class II molecule on an antigen- presenting cell and to a TCR beta-chain, thereby causing activation of the T cell. The cross-reactivity of enterotoxins with different Vbeta regions can lead to stimulation of a large fraction of T cells. To understand the molecular details of TCR-enterotoxin interactions and to generate potential antagonists of these serious hyperimmune reactions, we engineered soluble TCR mutants with improved affinity for staphylococcal enterotoxin C3 (SEC3). A library of randomly mutated, single-chain TCRs (Vbeta-linker-Valpha) were expressed as fusions to the Aga2p protein on the surface of yeast cells. Mutants were selected by flow cytometric cell sorting with a fluorescent-labeled SEC3. Various mutations were identified, primarily in Vbeta residues that are located at the TCR:SEC3 interface. The combined mutations created a remodeled SEC3-binding surface and yielded a Vbeta domain with an affinity that was increased by 1000-fold (K(D)=7 nM). A soluble form of this Vbeta mutant was a potent inhibitor of SEC3-mediated T cell activity, suggesting that these engineered proteins may be useful as antagonists.

Idiotypic analysis of monoclonal anti-fluorescyl antibodies: localization and characterization of idiotypic determinants.

Nine monoclonal IgG anti-fluorescyl antibodies, which exhibit diverse affinities for fluorescein (Fl) (Ka values ranging from 5 X 10(6) to 10(10) M-1) were analyzed idiotypically. Each of the BALB/c hybridoma proteins (gamma, kappa) exhibited unique idiotypic determinants although two clones (6-10-6 and 20-19-1) were partially (15-20%) cross-reactive. Of two other clones (4-6-9 and 4-6-10) derived from the same cell line, 4-6-9 contained gamma 1 heavy (H) chains and 4-6-10 contained both gamma 1 and gamma 2b H-chains. In addition, 4-6-9 shared idiotypic determinants with 4-6-10 although the latter also displayed unique idiotypic specificities. Collectively, the nine clones demonstrated structural diversity analogous to previous studies which defined binding mechanism diversity. The location of determinants recognized by anti-idiotype reagents directed against each of the monoclonal antibodies was examined by binding inhibition with free Fl and fluorescein-BSA (Fl-BSA). All clones contained determinants both within the active site (Fl-inhibitable) and in close proximity to it (Fl-BSA-inhibitable), although the relative proportions of these determinants varied among the clones. Inhibitor concns required for 50% inhibition varied independently of ligand binding affinity, and therefore were more likely influenced by the heterogeneous nature and affinity of the anti-idiotype reagents toward the individual determinants. Idiotypic analysis of H- and light (L) chains derived from five monoclonal antibodies of diverse affinities was performed. Fl binding and expression of idiotypic determinants by all clones required both H- and L-chains. Restoration of the idiotype by reassociated H- and L-chains was found to be highly restricted to homologous H- and L-chain pairs, as heterologous combinations did not result in the expression of either parental idiotype. The latter was true whether the heterologous pairs were derived from clones of the same isotype or the heterologous combination associated to form an intact molecule with greater affinity than the parental H- and L-chain combination. Heterologous recombinants from the two clones (6-10-6 and 20-19-1) exhibiting partial idiotypic cross-reactivity were able to restore a fraction (approximately 25%) of their idiotypic determinants. Results demonstrated the extensive conformational requirements of ligand binding and idiotype expression and indicated that a high degree of specificity in the VH- and VL-chain interaction must exist for the expression of these idiotypes.

Sequence restrictions in T cell receptor beta-chains that have specificity for a self-peptide/Ld complex.

Cytotoxic T lymphocytes that react with a complex of Ld and a ubiquitous self-peptide derived from the enzyme alpha-ketoglutarate dehydrogenase (p2Ca, LSPFPFDL) can be readily elicited by the addition of synthetic peptide to cultures of BALB/c spleen cells. As with other Ld-restricted CTL, the p2Ca-specific cells use predominantly the V beta 8.3 region. In addition, the p2Ca-specific cells use almost exclusively one of three J beta gene segments. Selection for these J beta regions appears to be related to the presence of a glutamic acid residue that is encoded at the 5' end of the J beta and is present within the CDR3. As p2Ca does not contain a complementary charged residue, this finding may suggest that the beta-chain CDR3 from p2Ca-specific CTL contacts one of the five basic residues located on the Ld helices. Together, the results support the possibility that CDR1 and/or CDR2 (within V beta 8.3) and the CDR3 may each contact the Ld molecule. In contrast to the V beta and J beta regions, the V beta D beta J beta junctions and V alpha J alpha repertoires were diverse. The diversity could explain why p2Ca-specific CTL have relatively high precursor frequencies allowing them to be generated rapidly in primary cultures.

Binding properties and solubility of single-chain T cell receptors expressed in E. coli.

The diversity and domain structure of alpha beta T cell receptors (TCR) are similar to immunoglobulins based on sequence homologies, but the three-dimensional structure of the alpha beta-heterodimer has not been solved. To begin structure/function studies, we have compared the properties of a soluble single-chain V alpha V beta TCR (scTCR) expressed in three E. coli systems. The V alpha and V beta regions were expressed with pelB or ompA signal sequences or as a thioredoxin fusion protein. The scTCRs were detected only in the insoluble fraction of the cells and could be solubilized in guanidine and renatured to obtain properly folded scTCR from each system. Only a small fraction (1-5%) of the ompA and pelB scTCRs folded properly. In contrast, the thioredoxin fusion protein exhibited high total yields and a solubility that was ten times higher than the other scTCRs. The thioredoxin fusion protein also bound specifically to the peptide/MHC ligand with a KD of approximately 0.7 microM, as shown by a competitive inhibition assay with Fab fragments that recognize the MHC complex. Furthermore, estimates from saturation binding with antibodies that react with the native TCR indicated that up to 80% of the thioredoxin fusion protein was in the properly folded form. The improved yield, solubility, and binding activity of the thioredoxin-scTCR should make it useful for various structure/ function studies.

Expression of defined idiotypes throughout the BALB/c anti-fluorescyl antibody response: affinity and idiotype analyses of heterogeneous antibodies.

Heterogeneous BALB/c anti-fluorescyl antibodies were shown to display increases (greater than 50-fold) in binding affinity from the primary through the tertiary responses. The structural basis of such affinity maturation and the diversity exhibited by anti-fluorescyl antibodies was examined by idiotypic analysis using a panel of anti-idiotype reagents specific for seven different monoclonal antifluorescyl antibodies. Because these clones exhibited binding affinities characteristic of a secondary or hyperimmune response, it was possible to examine the mechanism of affinity maturation by determining the prevalence of the seven idiotypes (Id-4-4-20, Id-20-19-1, Id-20-20-3, Id-6-10-6, Id-20-4-4, Id-4-6-10 and Id-6-19-1) in specifically purified heterogeneous preparations with low (i.e. primary response) or high (i.e. secondary and tertiary responses) binding affinities. Four of the idiotypes were not detected in heterogeneous preparations and thus each represented less than 0.1% of the total anti-fluorescein repertoire. Although results indicated that each of three other clones expressed unique or private idiotypic determinants not present in the heterogeneous population, these idiotypes (Id-4-4-20, Id-6-10-6, Id-6-19-1) were detected and ranged from approximately 0.2 to 2.0% of the repertoire. However, results indicated that each clone expressed unique or private idiotypic determinants not present in the heterogeneous population. Determinants expressed by such high-affinity monoclonal antibodies were expressed equally in all heterogeneous preparations examined. Because those determinants which were expressed were found in either low- or high-affinity heterogeneous antibodies, it is likely that the higher affinities exhibited by monoclonal antibodies derived from a secondary response are associated with unique idiotypic determinants which were not detected in polyclonal preparations. Hence, the process of affinity maturation may find as its structural correlate a mechanism such as somatic mutation which generates individual or unique idiotypes.