Synthetic Polypeptide Antigen Research Articles

From Proteins and Protein Models to Their Use in Immunology and Immunotherapy

The two leitmotifs of my scientific research were synthetic polymeric models of proteins and the structure of proteins as reflected by the three-dimensional conformation controlled by their amino acid sequence. This in turn led me to synthetic polypeptide antigens and to investigation of antibodies, ultimately resulting in the discovery of the determinant-specific genetic control of immune response and to the development of a therapeutic vaccine against the exacerbating-remitting form of multiple sclerosis. In protein chemistry I had two great teachers who became very close friends, Ephraim Katchalski-Katzir and Christian B. Anfinsen (Fig. 1), but in immunology I lacked the benefit of a great mentor, and so to some extent I was self-taught. Thus, I had to “figure out” many things myself.

Evaluation of INNO-LIA syphilis assay as a confirmatory test for syphilis.

We evaluated the sensitivity and specificity of a new confirmatory test for treponemal antibodies, INNO-LIA Syphilis (Innogenetics NV, Ghent, Belgium), on a large number of sera from a clinical laboratory. This multiparameter line immunoassay (LIA) uses recombinant and synthetic polypeptide antigens derived from Treponema pallidum proteins. In a single-blinded cross-sectional retrospective study, 289 seronegative sera, 219 seropositive sera, and 23 sera with an indeterminate serological status for syphilis were analyzed. All sera were tested by the T. pallidum hemagglutination assay (TPHA), the immunoglobulin (IgG)-fluorescent T. pallidum absorption assay (IgG-FTA-ABS), and the Venereal Disease Research Laboratory (VDRL) test. In addition, some seropositive samples were analyzed by the 19S-IgM-FTA-ABS test, an enzyme immunoassay (IgM-EIA), and the MarDx immunoblotting assay. Based on a consensus diagnosis derived from conventional serology, all of the sera were classified as positive, negative, or indeterminate, and the results were compared with the findings of the INNO-LIA Syphilis assay. The sensitivity and specificity of the LIA were 100% (219 of 219) and 99.3% (286 of 288), respectively. Compared to TPHA and IgG-FTA-ABS, the new test gave a significantly higher number (P = 0.021 and P < 0.0001, respectively) of correct results than either of the other two tests. The multiparameter INNO-LIA Syphilis assay is a useful confirmatory test for syphilis because it increases the reliability of syphilis diagnosis with respect to current conventional techniques.

Molecular tools for the diagnosis of cystic and alveolar echinococcosis.

In the past years, the diagnostic tools applied to identify alveolar (AE) and cystic echinococcosis (CE) in human patients have not only increased in number but also substantially improved in quality. The identification and characterization of species-specific parasite proteins/antigens allowed to generate subsequently recombinant or synthetic polypeptide antigens, as well as corresponding monoclonal antibodies. Some of these new tools have already demonstrated operating characteristics superior to conventional tests used for the immunodiagnosis of CE and AE, and thus may be suggested for routine laboratory application. Powerful molecular techniques, such as the polymerase chain reaction (PCR), have been developed and adapted to advance laboratory diagnosis of AE and CE. Detecting minute amounts of parasite DNA and mRNA, not only to identify but also to characterize the biological status of parasite material, thus becomes a complementary method to synergize immunodiagnostic techniques. This review focuses on recent developments of molecular tools, discussing their potential use as a primary or a supporting diagnostic element. We also outline some future developments to be undertaken in the field of molecular diagnosis, linked to clinical and laboratory problems.

Elucidation of peptide conformation involved in the recognition of (EYA) 5, EYK(EYA) 4 and EYAEAA(EYA) 3 peptides by MHC class II molecules and T-cell receptor

The role of peptide structure in the recognition of antigens by T cells remains unclear. We have analyzed the structures of two T-cell epitopes of the synthetic polypeptide antigen poly-18, poly-EYK(EYA) 5, namely EYK(EYA) 4 and (EYA) 5, and of the peptide analogue EYAEAA(EYA) 3, hereafter abbreviated as EYK, EYA and EAA, respectively. The results obtained in the theoretical simulation of their tertiary structures confirm the conclusions from 2D-NMR studies regarding their relative propensities to form α-helical structures (EYA ⪢ EYK ⪢ EAA) and extended structures (EYA < EYK < EAA). Experimental evidence has shown that the region formed by residues 7–12 constitutes the agretope for the recognition of EYK by Ia molecules, with a critical requirement for Lys 3; and the theoretical results indicate that Lys 3 has been brought to the vicinity ofthat region by the folding of the chain. The theoretical prediction for EAA, showing that this peptide has amphipathic character with a hydrophobic face lined with the methyl groups of Ala 3, Ala 6 and Ala 9, supports the experimental observation concerning the critical role of Ala 5.

Ia-antigen-T-cell interactions for a thymus-independent antigen composed of D amino acids.

Synthetic polypeptide antigens of L amino acids, although bearing repeating sequences, are thymus-dependent (L-TD), whereas the same polymers composed of D amino acids are thymus-independent (D-TI), probably due to a slower rate of metabolism. Yet we found that lymph-node cells of BALB/c mice immunized with D-TI proliferate in response to it in vitro. To follow T-cell activation by D-TI, we established T-cell hybridomas to D-TI and to its analog composed of L isomers, L-TD, for comparison. The T-cell hybridomas express membrane alpha/beta T-cell receptors and secrete interleukin 2 upon stimulation with the respective antigen. In addition, D-TI-specific hybridomas are stimulated, to a lesser extent, by the L-TD antigen, whereas only some L-TD-specific hybridomas recognize D-TI. Moreover, biotinylated analogs of D-TI and L-TD bind to splenic antigen-presenting cells (APCs) from BALB/c mice. Binding is inhibited by an excess of nonbiotinylated L-TD, and by an excess of a peptide comprising residues 259-271 of the human acetylcholine receptor alpha subunit, which binds to I-Ad and I-Ed molecules without prior processing. Analysis of APC lysates following incubation of the APCs with biotinylated D-TI and L-TD reveals that the biotinylated antigen moiety is associated with Ia molecules. D-TI and L-TD bind to Ia molecules on intact APCs with similar KD values, 5 x 10(-8) M and 3 x 10(-8) M, respectively. However, D-TI has faster kinetics of binding than L-TD, probably due to different processing requirements. Hence, we have demonstrated a major histocompatibility complex class II-mediated T-cell response to a thymus-independent antigen.

Evidence for immunodominance between closely related epitopes in the selection of T cell repertoire: Hierarchy of T cell epitopes in a repeating sequence

In a protein antigen the number of epitopes that are presented by the MHC molecules to the T cells is generally limited. This phenomenon of immunodominance determines the T cell response to a given antigen. To understand the molecular basis of epitope selection we have analysed the hierarchy of T cell epitopes in a repeating synthetic polypeptide antigen Poly 18, Poly EYK(EYA) 5 in the mice of H-2 d haplotype. Because of its repeating nature, all the potential epitopes of Poly EYK(EYA) 5 generated as a result of antigen processing will have extensive sequence overlap, therefore, providing an excellent system to investigate the molecular basis of T cell peptide epitope selection in vivo. We have synthesized a series of 12 and 15 amino acid peptides to mimic these epitopes. In H-2 d mice Poly EYK(EYA) 5 elicits T cell clones that optimally react with 15 amino acid peptides EYK(EYA) 4 and/or (EYA) 5. Similar results were obtained when related 12 amino acid peptides EYK(EYA) 3 and/or (EYA) 4 are used. (EYA) 5 reactive T cell clones appear to be very heterogenous and much larger in number than EYK(EYA) 4 reactive clones. (EYA) 5 reactive clones could be elicited by at least three short Poly-18 derived epitopes (EYA) 4, EYK(EYA) 3 and (EYA) 3EYK while EYK(EYA) 4 reactive clones elicited only by the EYK(EYA) 3 epitope. However, we observed the dominance of (EYA) 5 reactive clones even when EYK(EYA) 3 was used as an immunogen and this could be related to the degeneracy of their antigen specificity. Our earlier antigen competition studies suggest that (EYA) 5 does not compete with EYK(EYA) 4 epitope in binding to I-A d. Therefore, there is no intramolecular competition between these epitopes to activate T cells. The epitope (EYA) 3EYK appears to be subdominant since it can elicit Poly EYK(EYA) 5 specific T cells upon immunization but does not appear to be part of Poly EYK(EYA) 5 repertoire. Peptides such as (EYA) 2EYKEYA or EYAEYK(EYA) 2 with lysine substitution in the middle of the sequence were non immunogeneic. Similar results were obtained when the larger 15 amino acid peptides were used as antigen. Another level of epitope immunodominance is seen when substituted peptides of the two immunodominant epitopes are used. Some of these epitopes have potential to be part of the Poly 18 repertoire but they are greatly under represented when intact Poly 18 is used as antigen. The unusual hierarchy observed for immunodominance in these overlapping epitopes of EYK(EYA) 5 sequence suggest a bias in the selection of T cell repertoire based upon the crossreactivity between potential epitopes generated as a result of antigen processing.

Comparison of Freund's and Ribi adjuvants for inducing antibodies to the synthetic antigen (TG)-AL in rabbits

Antibody responses and health parameters were compared in rabbits immunized with a synthetic polypeptide antigen, [ l- Tyr, l- Glu, DL- Ala]- poly- L- lysine , in Freund's (FA) or RIbi (RA) adjuvants, Rabbits, 12 weeks old, of both sexes, were inoculated with 0.5 ml divided between two intramuscular (i.m.) sites. Eight received FA and antigen (50 μg); eight RA and antigen, eight PBS and antigen; four FA and PBS; four RA and PBS, and four PBS. Identical booster inoculations were made 21 days later, except that incomplete FA was substituted for complete FA. Rabbits were monitored until euthanasia and necropsy 7 weeks after the primary inoculation. Sera, obtained weekly, were analyzed for immunoglobulins using an enzyme immunoassay. Only rabbits given antigen with adjuvant produced high titered antibodies. Mean optical density values for immunoblobulin (Ig)M were greater the week after the booster in the group given FA. IgG values were similar for both adjuvant/antigen groups the week after the booster, but thereafter decreased in rabbits given RA. Antisera from rabbits given antigen with FA had greater avidity for the antigen than that from rabbits given antigen with RA, however, the difference was not significant ( p>0.05). Rabbits inoculated with FA and antigen had high serum creatinine kinase levels the day after inoculation, showed evidence of discomfort, and extensive granulomatous inflammation at the inoculation sites. Lesions were minimal to mild in rabbits given antigen with RA and PBS with either adjuvant. While RA did not result in adverse side effects, the IgG response to (TG)-AL with RA was transient compared to FA.

Variable regions of antibodies to synthetic polypeptides—III. Antibodies arising in response to administration of anti-idiotope

Antibodies elicited by the synthetic polypeptide antigen (T,G)-A-L are directed against two distinct epitopes. The majority of antibodies bind to a GT containing epitope and bear an idiotope defined by monoclonal antibodies 17 and 19. In this study, we have examined the effect of in vivo administration of the 17 and 19 antibodies to mice. Administration of anti-idiotope elicits anti-(T,G)-A-L antibodies in all strains of mice tested, including genetic non-responders to (T,G)-A-L. These antibodies bind to GT and express the idiotope. Additionally, idiotope expressing antibodies that fail to bind to antigen are also produced. Monoclonal anti-(anti-idiotope) antibodies were made. One antibody bound to (T,G)-A-L, the other did not. Sequence analysis was performed and the V-regions of (T,G)-A-L binding antibodies were compared to those of the antibody that failed to bind antigen. Both sets of antibodies are derived from the same germline V-genes as the anti-(T,G)-A-L antibodies. These results have implications for understanding the nature of network regulation of the immune system and for those attempting idiotypic vaccination.

Unusually diverse T cell response to a repeating tripeptide epitope

The immune system utilizes a diverse T cell repertoire for the recognition of foreign antigens in the context of self MHC gene products. We have examined the potential diversity of the T cell response directed to a immunodominant repeating tripeptide epitope (EYA) 5. This peptide represents one of the two T cell epitopes on the synthetic α-helical polypeptide antigen Poly 18, Poly EYK(EYA) 5 in H-2 d mice and does not require antigen processing prior to presentation to Poly 18-specific T cell hybridomas. The T cell response directed to the repeating tripeptide epitope (EYA) 5 is extremely heterogenous even though the epitope has a relatively simple amino acid sequence. We have analyzed the fine specificity of 21 randomly chosen Poly 18-reactive, (EYA) 5− specific and H-2 d-restricted T cell hybridomas derived from H-2 d, H-2 bxd, and H-2 b → H-2 bxd Poly 18-responding mice to determine the number of unique antigen reactivity patterns represented by this T cell population. We used alanine- and/or lysine-substituted (EYA) 5 peptides and a panel of haplotype-varied splenocytes and observed a great deal of microheterogeneity in response. We find that 13 of the 21 hybridomas have a distinct fine antigen specificity and T cell receptors. The binding of (EYA) 5 to the antigen-binding groove of I-A d appears to generate a highly diversified T cell response. Therefore, (EYA) 5-I-A d complex allows the activation of unrelated T cell clonotypes with the same overall antigen specificity and MHC restriction, but with distinct microheterogeneity in response and receptor usage.

Critical role of an amino acid residue in a T cell determinant is due to its interaction with a neighboring non‐critical residue

Several lines of evidence support the concept of two functionally distinct sites on antigen: the epitope, involved in interaction with the T cell receptor and the agretope, interacting with Ia. We investigated the Ia and T cell receptor interaction sites on the synthetic polypeptide antigen poly-18 [poly-EYK(EYA)5] using T cell hybridoma clones specific for this antigen in the context of I-Ad. Peptides with amino acid sequences related to poly-18 were synthesized. These were used to identify the critical residues in the minimum peptide sequence required for activation. Clone A.1.1 responds to the minimal peptide EYK(EYA)4 but not to (EYA)5. This identifies Lys3 as a critical amino acid for this hybridoma. Surprisingly, the substituted peptide EYAEAA(EYA)3 could activate A.1.1, indicating that an Ala at position 5 instead of a Tyr obviates the critical requirement for Lys3. This demonstrates that the function of critical residues may extend beyond contacting the T cell receptor or Ia, to include a third role: that of interacting with other amino acids of the T cell epitope, thus influencing the antigen's recognition by T cells.

Current status of clinical laboratory tests for the human immunodeficiency virus

The predictive values of positive and negative test results for human immunodeficiency virus (HIV) antibody are extremely high in laboratories that have good quality control and high performance standards and use licensed FDA-approved enzyme immunoassay (EIA) and Western blot standardized tests. With a carefully designed protocol, the false-positive rate of combined EIA and Western blot has been reported to be as low as 1 in 10(5). When results of HIV confirmatory antibody tests are indeterminate, other tests such as culture and nucleotide probe methods for HIV DNA or RNA may help resolve false-reactive screening EIA tests. Improvements are constantly in progress for HIV laboratory tests with the use of recombinant DNA-derived antigens and synthetic polypeptides. With the use of new-generation synthetic polypeptide antigens, specific assays to identify HIV-1 and HIV-2 have been developed. Recently, assays for the HIV regulatory gene products have been studied for their predictive potential. Antibodies to nef protein, a regulator of HIV-1 replication, may be an early indicator of HIV infection.

Specific inhibition of cell-surface T-cell receptor expression by antisense oligodeoxynucleotides and its effect on the production of an antigen-specific regulatory T-cell factor.

We have used antisense oligodeoxynucleotides corresponding to genes encoding the variable (V) region of the T-cell receptor (TCR) alpha and beta chains (V alpha and V beta) to control TCR expression in T-cell hybridomas. Two hybridomas, A1.1 and B1.1, recognize a synthetic polypeptide antigen designated poly 18 (poly[Glu-Tyr-Lys-(Glu-Tyr-Ala)5]) together with I-Ad. We have found that TCR function (production of lymphokines in response to antigen) and T3 expression were removed after protease treatment of the cells and were fully recovered 48 hr later. However, when antisense oligodeoxynucleotides corresponding to the appropriate TCR V genes were present after protease treatment, little or no recovery of TCR function or T3 expression was observed. This effect was specific for the TCR V genes utilized by the T cell: antisense oligodeoxynucleotides corresponding to the TCR V regions of A1.1 had no effect on TCR expression in B1.1 and vice versa. Thus, antisense oligodeoxynucleotides can be used to temporarily block expression of a TCR gene in a T-cell hybridoma. This technique was then applied to a paradoxical phenomenon in A1.1 cells. We had observed previously that A1.1 releases an antigen-specific immunoregulatory activity that shows the same antigenic fine specificity as is displayed by the TCR of A1.1. We now report that antisense oligodeoxynucleotides corresponding to the A1.1 V alpha gene blocked the production of this soluble antigen-specific activity by the cell. Antisense oligodeoxynucleotides corresponding to A1.1 V beta, on the other hand, had no effect on the production of this antigen-specific activity. We discuss these observations in the context of recent findings on the nature of T cell-derived antigen-specific regulatory factors.

Contribution of antigen processing to the recognition of a synthetic peptide antigen by specific T cell hybridomas.

Most antigens recognized by T cells require unfolding or partial degradation (processing) followed by association with Major Histocompatibility Complex (MHC) molecules. We examined the processing requirements for the presentation of antigen to two T cell hybridomas which recognize the alpha-helical synthetic polypeptide antigen Poly 18, Poly [EYK(EYA)5], in association with I-Ad. Hybridoma A.1.1 responds to EYK(EYA)4 as the minimum antigenic sequence while hybridoma B.1.1 recognizes (EYA)5 sequence. It was found that these hybridomas responded to Poly 18 and to minimum peptide sequences presented by glutaraldehyde and chloroquine treated antigen presenting cells (APC), suggesting that antigen processing is not a requirement for the activation of these cells. The reactivity pattern of hybridoma B.1.1 in the presence of glutaraldehyde fixed APC revealed that antigens containing lysine were presented with much less efficiency than antigens without lysine, suggesting an interaction of these residues with the antigen presenting cell surface. We discuss the possibility that alanine residues in the alpha-helical Poly 18 form a hydrophobic ridge which may be required for appropriate interaction between antigen, the T cell receptor, and MHC molecules.

Characterization and biologic activities of an anti-idiotype-specific T cell line and its derived clones.

Monoclonal anti-idiotypic antibodies were prepared against monoclonal antibodies (mAb103) specific to the synthetic polypeptide antigen (T,G)-A-L. A cell line was established by the stimulation of C3H.SW mouse T cells with one of the monoclonal anti-idiotypes (mAbA-6) that reacted with both mAb103 and conventional (T,G)-A-L-specific antibodies. The T cell line proliferated specifically in the presence of the homologous mAbA-6 and to a lesser degree when triggered with (T,G)-A-L. The line could help (T,G)-A-L primed B cells in the production of (T,G)-A-L-specific antibodies when stimulated in vitro with either (T,G)-A-L or mAbA-6. Clones obtained from the line were stimulated and maintained in culture in presence of mAbA-6 whereas others were stimulated and grown in the presence of (T,G)-A-L. Both types of clones proliferated only in the presence of mAbA-6 although (T,G)-A-L could inhibit efficiently and specifically the latter proliferation. A significant number of the (T,G)-A-L-stimulated clones could collaborate with (T,G)-A-L primed B cells in the presence of either (T,G)-A-L or mAbA-6 for the production of specific antibodies. Immunoblotting experiments indicated that mAbA-6 reacted with both the T cell receptor of the mAbA-6-specific T cell line and of a (T,G)-A-L-specific T cell line but not with that of a line specific to a nonrelated antigen.

Analysis of the antigen specific helper T cell function and HLA-DR of Israeli patients with rheumatoid arthritis (RA).

Forty-three patients with rheumatoid arthritis (RA) were studied for their ability to respond to the synthetic polypeptide antigen (T, G)-A-L as measured by the production of a T cell helper factor by their antigen activated T cells. Sixteen patients (37%) responded to (T, G)-A-L by the production of an antigen specific helper T cell factor, a percentage not significantly different from healthy donors. The production of antigen specific T cell helper factors was affected, although not significantly, by immune modulating drugs and by the presence of rheumatoid factor in sera of patients. The high incidence of HLA-DR 4 reported for RA patients was not observed in this group of RA patients.

Covalent linkage of the synthetic adjuvant MDP to the synthetic polypeptide (T,G)-A—L changes the specificity of the immune response at the T and B cell level

Is now well established that the synthetic molecule MDP ( N-acetylmuramyl- l-alanyl- d-isoglutamine) can be a good adjuvant of immunity when covalently linked to antigen. The question raised in this work is whether conjugation of antigen to the immunomodulatory molecule MDP can modify the specificity of the antibodies and T cells induced following immunization. Using the well characterized synthetic polypeptide antigen, poly( l-Tyr, l-Glu)-poly( dl-Ala)—poly( l-Lys) [(T,G)-A—L], we show that immunization of C57B1/6 (H-2 b) mice with MDP-(T,G)-A—L conjugate elicits at least two types of antibody directed against the poly( dl-Ala)—poly( l-Lys) (A—L) part of the antigen, and against new determinant(s) formed by MDP and a portion of the (T,G)-A—L molecule. Interestingly, the poly-( l-Try l-Glu) side chains thought to constitute the major antigenic determinants of the (T,G)-A—L molecule were not recognized. Lymph node cells from (T,G)-A—L immunized mice can be equally well stimulated in vitro by (T,G)-A—L or by MDP-(T,G)-A—L, whereas lymph node cells from MDP-(T,G)-A—L primed animals can be stimulated only when challenged by the conjugate used for immunization, and not by the free synthetic polypeptide (T,G)-A—L. The data presented here show that the coupling of a low mol. wt molecule such as MDP (mol. wt approx. 500) to an antigen can greatly modify the immune response directed against this antigen. Furthermore, (1) different antibody specificities are elicited depending upon whether the priming is done with free MDP and antigen or with MDP covalently linked to the antigen; (2) although still accessible on the conjugate, an epitope which represents the major antigenic determinant on the free polypeptide appears to be silent when presented on the conjugate; and (3) new determinant(s) formed by the chemical linkage of the polypeptide to the synthetic adjuvant are involved in the priming of T cells.

Characterization of monoclonal antibodies with specificity for dna and the synthetic polypeptide antigen (T,G)-A-L

Because of evidence for structural similarity of variable region genes of anti-DNA and anti-(T,G)-A-L antibodies, polyspecific interactions of monoclonal anti-DNA and anti-(T,G)-A-L antibodies were investigated. Of 20 monoclonal antibodies from C57BL/10 mice with (T,G)-A-L binding, two bound DNA as determined by ELISA. In contrast, two of five anti-DNA monoclonal antibodies from MRL- 1pr/1pr mice bound (T,G)-A-L. For both sets of antibodies, antigen binding was shown to be the activity of the same antibody by cross-inhibition studies. To determine whether such polyspecific antibodies were expressed during autoimmune disease, sera of autoimmune MRL- 1pr/1pr mice were tested for anti-(T,G)-A-L activity. This analysis demonstrated minimal elevations of anti-(T,G)-A-L in comparison to BALB/c controls. These studies thus confirm predictions about the binding activity of anti-(T,G)-A-L and anti-DNA antibodies based on structural analysis of variable region genes. They further indicate, that while anti-(T,G)-A-L and anti-DNA antibodies may have overlapping specificity, polyspecific antibodies of this kind are not preferentially expressed during autoimmunity.

Variable regions of antibodies to synthetic polypeptides. II. Analysis of variable region genes encoding antibodies specific for (T,G)-A--L.

Monoclonal antibodies specific for the synthetic polypeptide antigen (T,G)-A--L have been produced in two strains of mice, C57BL/10 and C3H.SW. The genes encoding the variable (V) regions of these antibodies have been studied by using the DNA hybridization technique of Southern, as well as by gene cloning and sequencing. Hybridization of DNA from 14 different cell lines with a kappa-chain probe revealed that the different cell lines used one of two different gene rearrangements to encode the recombined V region gene. There was a perfect correlation between light chain rearrangement, idiotype expression, and fine specificity. Hybridization analyses of the heavy chain revealed a more complex pattern. Seven hybridomas had the rearranged heavy chain V region genes on a 4.4 kb EcoRI restriction fragment. Others were found on restriction fragments that differed in length by several hundred base pairs. The recombined heavy chain V region genes were cloned from three different hybridoma cell lines secreting anti-(T,G)-A--L antibodies, all of which express the same idiotype and fine specificity pattern. Restriction mapping and sequencing indicate that all three utilize the same V gene, identified as the 186-2 germline gene. However, different D and J genes are used to encode each of the antibodies. In contrast to the results seen in other antigen systems, heavy chain D and J genes do not have a major influence on idiotype expression and fine specificity of antibodies to the synthetic polypeptide (T,G)-A--L.

Functional requirements of (Phe, G)-A--L-specific T-cell clones of (H-2b X H-2k)F1 origin.

T-cell clones specific for the synthetic polypeptide antigen poly(LPhe, LGlu)-poly(DLAla)--poly(LLys) of (C57BL/6 X C3H/HeJ)F1 origin were tested for their biological activities. One group of clones was restricted in its proliferative response to the H-2b haplotype, the second to the H-2k haplotype, and the third to the F1 unique Ia determinants. All the clones which proliferated in response to antigen secreted interleukin-2 (IL-2) following stimulation. The H-2 restriction of the IL-2 secretion was the same as that of the proliferation. Two of the clones tested, C.6 and C.10, could provide help to B cells in antibody production. However, the genetic restriction profile of the helper activity was less stringent than that for the proliferative response. Thus, C.6, which proliferated in the presence of F1 antigen-presenting cells only, could help B cells and accessory cells of C3H/HeJ. C.10, which was restricted in its proliferative response to the H-2b haplotype, could collaborate with B cells and accessory cells of the H-2k haplotype as well. The antibody response of both clones was restricted to the parental or F1 strains.

Analysis of the biological functions and fine specificity of (T,G)-A--L specific. T cell clones

Two T cell lines, TPB1 and TPB2, specific for the synthetic polypeptide antigen (T,G)-A-L, were established from (T,G)-A--L primed lymph node cells of C3H.SW(H-2 b) mice. Both lines proliferated in the presence of (T,G)-A--L, helped in antibody production in vitro, secreted IL2 upon stimulation with antigen. The lines differed in the fine specificity of their responses to antigenic stimulation. The line with the broader specificity TPB2 was cloned by limiting dilution, its derived clones were analyzed. No efficient manifestation of both proliferative activity and helper function could be detected in a single clone. Most of the clones were highly specific to (T,G)-A--L, although 2 of them cross-reacted with the closely related polypeptide (Phe,G)-A--L. Individual clones could trigger B cells for the production of antibodies of the IgM and IgG classes. All helper clones secreted (T,G)-A--L specific helper factors. No correlation was found between efficient secretion of IL2 by the clones and their other biological functions.