Idiotype expression and fine specificity of Glu60Ala30Tyr10-specific T proliferating cells

The fine specificity of anti-Glu60Ala30Tyr10 (GAT) and anti-Glu60Ala40 (GA) proliferating cells was studied. T cells primed with GAT proliferate both to GAT and GA and GA-primed T cells proliferate also to GA and GAT. This cross-reactivity was unexpected given the results previously reported on the fine specificity of anti-GAT antibodies. The effect on the proliferation of BALB/c lymph node cells (LNC) of a syngeneic anti-idiotypic serum, prepared in BALB/c against anti-GAT antibodies, was studied. Two major points are made in this paper: (i) the in vitro addition of the anti-idiotypic serum in cultures containing GAT-primed LNC and GAT enhances the proliferation of GAT-specific T cells; (ii) the anti-idiotypic serum is effective in priming in vivo LNC which then acquire the capacity to proliferate specifically with GAT in vitro. These results further confirm the existence of idiotype-like determinants on T cells.     

Dissection of the poly(Glu60 Ala30 Tyr10) (GAT)-specific T-cell repertoire in H-2I k mice

Twenty-five allospecific monoclonal antibodies (mAb), produced in the A. TH. A.BY, or B10.S (7R) anti-A.TL combinations, were shown to recognize determinants organized in four spatially distinct polymorphic regions on the same I-Ak-encoded molecule(s). These reagents were used to assess the recognition of the class II major histocompatibility complex (MHC) determinants in a series of GAT-reactive A.TL T-cell clones exhibiting various restriction specificity or alloreactivity patterns. Of the proliferative responses of 13 cloned T cells, 12 responses were found to be inhibited similarly by the same set of mAbs.A hierarchy in the blocking effects of these reagents that could be correlated with the spatial organization of their determinants was observed. (i) All the mAbs defining the epitope region I (i.e., recognizing public Ia.1- or Ia.17-like determinants, presumably expressed on the A beta subunit) and some of those identifying new public determinants in the epitope region II profoundly inhibited these T-cell responses. (ii) Intermediate blocking was observed when mAbs recognizing public determinants in the epitope region III were used. (iii) Finally, among the mAbs that identified the epitope group IV, the Ia.19-specific mAb 39.J was inhibitory, whereas mAbs directed against private Ia.2-like determinants were not. By contrast, the GAT-specific proliferative response of the T-cell clone AT-20.1, which recognized its nominal antigen in an extensively cross-reactive MHC-restricted fashion, could only be inhibited by a subset of the mAbs recognizing epitopes in groups I and II, but not by those recognizing epitopes in groups III and IV. It was also shown that the same subset of I-Ak-and I-Au-reactive mAbs displayed similar blocking effects on the proliferation of two T-cell clones exhibiting dual specificity for I-Ak- and I-Au-restricting and/or I-Ak- and I-Au-alloactivating determinants. Finally, all the cloned T-cell responses examined were found to be inhibited by rat mAbs against the LFA.1 molecule or the murine equivalent of the human OKT4 differentiation antigen. These studies suggest that class II specific mAbs can impair proliferation of cloned T-cells by a mechanism(s) other than the masking of the T-cells' restriction determinants per se.     

Acquisition of syngeneic I-A determinants by T cells proliferating in response to poly (Glu60Ala30Tyr10)

The T cell proliferative response in mice to the synthetic polymer GAT is under Ir gene control, mapping to the I-A subregion of the H-2 major histocompatibility complex (MHC). Antigen-dependent proliferation in vitro of in vivo GAT-primed lymph node cells can be inhibited by a monoclonal antibody to Ia-17, an I-A public determinant. Using this antibody for direct immunofluorescent analysis, T cells in GAT-stimulated proliferative culture are identified that express syngeneic I-A during culture. This expression is strictly antigen dependent, requires restimulation in vitro, and requires the presence of I-A-positive adherent antigen-presenting cells. T cells bearing I-A can be enriched by a simple affinity procedure, and I-A-positive cells separated on a FACS are shown to retain antigen-specific reactivity. The acquisition of I-A determinants by T cells under these culture conditions is not nonspecific. The Ia determinants borne by T cell blasts appear to be dictated by the I subregion to which the relevant Ir gene maps, and which codes for the Ia molecule involved in presentation of the antigen. Thus, (B6A)F1 (H-2b X H-2a)F1 LNC express I-Ak antigens when proliferating to GAT but not when stimulated by GLPhe, the response to which is under I-E subregion control. The relation of Ir gene function to Ia-restricted antigen presentation and self-Ia recognition is discussed.     

Idiotypic analysis of hybridoma antibodies to branched synthetic polymer (Tyr, Glu)-Ala-Lys: idiotypic relationship with antibodies to linear random polymer (Glu60, Ala30, Tyr10)

The expression of three anti-GAT idiotypes, CGAT, Gte, and GA-1, on 17 C57BL/10 and four C3H.SW hybridoma anti-(T,G)-A--L antibodies was analyzed. These hybridoma anti-(T,G)-A--L antibodies exhibited two patterns of fine antigen binding specificity. The majority of the hybridoma antibodies bound the (T,G)-A--L, GT, and GAT polymers but not the GA polymer, and were designated as GT-reactive hybridoma antibodies. A minor population of hybridoma anti(T,G)-A--L antibodies bound to (T,G)-A--L but not to GT, GAT, or GA, i.e., (T,G)-A--L-specific. A complete correlation between fine antigen binding pattern and the expression of CGAT idiotype was demonstrated. None of the 21 hybridoma anti-(T,G)-A--L antibodies expressed the GA-1 idiotype. All of the GT-reactive and none of the GT-nonreactive hybridoma anti-(%,G)-A--L antibodies expressed the CGAT idiotype. Furthermore, the Gte idiotype was found on the majority of CGAT+-bearing C57BL/10 hybridoma anti-(T,G)-A--L antibodies. These results indicate that C57BL/10 anti-(T,G)-A--L antibody repertoire can be grouped into a minimum of three families; i.e., CGAT+ Gte+, CGAT+ Gte-, and CGAT- Gte- families, with the CGAT+ Gte+ family as the major compartment. This is confirmed by the high percentage idiotype binding of serum anti-(T,G)-A--L antibodies with anti-CGAT idiotypic antisera. Finally, anti-idiotypic antisera made against CGAT+ hybridoma anti-GAT or anti-(T,G)-A--L antibodies crossreact extensively with other CGAT+ hybridoma anti-GAT and anti-(T,G)-A--L antibodies. However, additional experiments demonstrated that CGAT+ hybridoma anti-(T,G)-A--L antibodies also possess private idiotypes.     

Epitope-specific regulation in Ir gene systems. I. Conditions for the induction of epitope-specific suppressors to poly(Glu60Ala30Tyr10)

Injection of responder mice with poly(Glu60Ala30Tyr10) (GAT) followed by immunization with GAT-methylated bovine serum albumin (GATMBSA) selectively suppresses anti-MBSA plaque-forming cell (PFC) and delayed hypersensitivity (DTH) reactions. Conversely, MBSA injection followed by GATMBSA immunization suppresses anti-GAT PFC and DTH, while anti-MBSA responses remain intact. Suppression occurs for doses of antigen which are optimally immunogenic. The suppression is specific and does not act in a bystander fashion. These results demonstrate that epitope-specific regulation is reciprocal, is not limited to humoral responses, and is not limited to molecules of low molecular weight.     

The functional helper T cell repertoire specific for L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)

Experiments have been carried out to examine the potential helper T cell repertoire specific for the random terpolymer GAT on responder, nonresponder, and (responder x nonresponder)F1 murine strains. The ability of GAT-MBSA immunized T cells to collaborate with DNP-specific primary and secondary B lymphocytes of each strain in response to the antigen DNP-GAT was tested with the splenic fragment culture system. The results of these experiments show that there are GAT-specific T lymphocytes in the responder, nonresponder, and F1 strains but that these 3 GAT-specific T cell populations differ in their collaborative potential. In sum, these findings present new evidence that the nonresponder status to the terpolymer GAT is due, in part, to a functional deletion of helper T cells capable of recognizing the antigen in the context of the nonresponder haplotype. Further, a new responsive phenotype is evidenced when F1 secondary B cells are stimulated in nonresponder GAT-MBSA-primed recipients. In this case, rather than the IgG1 responses observed in such strain combinations to other antigens such as DNP-Hy or DNP-Gl phi 9, only IgM responses were obtained. This new phenotype may be the result of GAT-specific suppression of isotype switching by B cells bearing the nonresponder cell surface alloantigens.     

Identification and characterization of a suppressor T cell hybridoma specifically inducible by L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)

In vitro activation of naive spleen cells from C57BL/10 mice with GAT and the monoclonal GAT-TsF1, 372B3.5, followed by fusion with BW5147 resulted in generation of a hybridoma that fails to produce GAT-TsF constitutively, but upon reexposure to GAT and 372B3.5 is induced to secrete GAT-TsF2. The induction is GAT specific and requires de novo RNA, protein synthesis, and DNA synthesis. Although both GAT and 372B3.5 are required for induction, they may be added sequentially, provided the GAT is added first. The GAT-TsF produced by the induced cell is antigen specific and composed of two polypeptide chains: one capable of binding antigen, the other bearing determinants encoded by the I-J region of the MHC. The utility of this inducible GAT-TsF2 cell line for molecular biology and other studies is discussed.     

Recessive T cell response to poly (Glu50Tyr50) possibly caused by self tolerance

The proliferative T cell response of inbred mouse strains to the random copolymer poly(Glu50Tyr50) (GT) was found to fall into two categories. Some strains responded only marginally (delta cpm values less than 10,000 and stimulation indices less than 3), whereas other strains mounted a substantial response (delta cpm 10,000 to 80,000, SI 3 to 30). The response is controlled by the A alpha and A beta loci of the major histocompatibility complex (MHC), as well as by genes not linked to the MHC. Because the response is selectively inhibited by monoclonal antibodies specific for the A alpha A beta molecule, we assume that its control by A loci is manifested as an A-restriction of the participating T (Ly-1high, Ly-2-) cells. It is of interest that the responsiveness is recessive in F1 hybrids of responder and nonresponder strains that are H-2-identical, but differ at their genetic background. Nonresponsiveness of these F1 mice is caused neither by a defect of antigen presentation, nor the result of immune suppression on priming or at the effector phase of the response. It is most likely the consequence of clonal deletion during the establishment of self-tolerance.     

T cell subsets regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in virgin and immunized nonresponder mice

T cell subsets from virgin and immunized mice, which are Ir gene controlled nonresponders to GAT, which regulate antibody responses to GAT have been characterized. Virgin nonresponder B10.Q B cells develop GAT-specific antibody responses to GAT, B10.Q GAT-M phi, and GAT-MBSA when cultured with virgin or GAT-primed Lyt-1+, I-J-, Qa1- B10.Q helper T cells. Virgin T cells are radiosensitive, whereas immune T cells are radioresistant (750 R); qualitatively identical helper activity is obtained with T cells from mice immunized with soluble GAT, B10.Q GAT-M phi, and GAT-MBSA. Responses to GAT and GAT-M phi are not observed when virgin or GAT-primed Lyt-1+, I-J+, Qal+ T cells are added to culture of virgin or GAT-primed Lyt-1+, I-J-, Qa1- helper T cells and virgin B cells; the GAT-specific response to GAT-MBSA is intact. The Lyt-1+, I-J+, Qa1+ T cells from mice primed with GAT, GAT-M phi, and GAT-MBSA were qualitatively identical in mediating this suppression. Virgin Lyt-2+ T cells have no suppressive activity alone or with virgin Lyt-1+, I-J+, Qa1+ T cells, whereas responses to GAT, GAT-M phi, and GAT-MBSA are suppressed in cultures of GAT-primed helper T cells containing GAT-primed Lyt-2+ T cells (with or without GAT-primed Lyt-1+, I-J+, Qa1+ T cells). Suppression of responses to GAT-MBSA in cultures of GAT-M phi-primed helper T cells requires both GAT-M phi-primed Lyt-1+, I-J+, Qa1+ T cells and Lyt-2+ T cells; the Lyt-1+, I-J+, Qa1+ T cells appear to function as inducer cells in this case. In cultures containing GAT-MBSA-primed helper T cells, either GAT-MBSA-primed Lyt-1+, I-J+, Qa1+ or Lyt-2+ T cells suppress responses to GAT and GAT-M phi; under no circumstances are responses to GAT-MBSA suppressed by GAT-MBSA-primed regulatory T cells. This regulation of antibody responses to GAT by suppressor T cells is discussed in the context of the involvement of suppressor T cells in responses to antigens under Ir control, and of the evidence that nonresponsiveness to GAT is not due to a defect in the T cell repertoire, but rather is due to an imbalance in the activation of suppressor vs helper T cells.     

Dissection of the poly(glu60ala30tyr10) (GAT)-specific T-cell repertoire in H-21 k mice

We examined the antigen recognition of the class II major histocompatibility complex (MHC) of 45 poly(glu60 ala30 tyr10) (GAT)-reactive T-cell clones isolated by limiting dilution cloning of a pool of in vivo-primed and in vitro-restimulated A.TL lymph-node T cells. Each clone expressed the Thy-1.2+, Lyt-1+, Lyt-2-, LFA-1+, Ia-, and H-2Dd+ cell-surface phenotype and exhibited strict specificity for GAT on syngeneic antigen-presenting cells (APCs). The monitoring of the proliferative responses of these clones in the presence or absence of GAT, using APCs from strains with 11 independent H-2 haplotypes, revealed several distinct specificity patterns: (i) most (31 of 45, 73%) T-cell clones recognized GAT in a self-I-Ak-restricted manner; (ii) other alloreactive clones (5 of 45, 11%) were stimulated to proliferate, irrespective of the presence of GAT, in response to allodeterminants expressed on H-2s, H-2d, H-2f or H-2u spleen cells; (iii) a third T-cell clone subset (4 of 45, 9%) was activated by GAT in the context of not only self-I-Ak but also nonself restriction Ia determinants; and (iv) three clones (7%) exhibited a triple specificity, i.e., they recognized GAT in the context of self and nonself Ia determinants and were alloreactive. One of the latter clones responded to GAT in an apparently non-MHC-restricted manner and recognized an I-Ab allodeterminant. These data provide direct evidence that the antigen-specific and alloreactive T-cell repertoires overlap and that the self-MHC restriction of GAT-specific T-cell responses is not absolute in A.TL mice.     

Multigenic control of immune responses of inbred mice against the terpolymers poly(Glu57Lys38Ala5) and poly(Glu54Lys36Ala10) and linkage withH-2 haplotype

Results of immunizations of recombinant inbred and congenic strains of mice with the random polymers poly(glu⁵⁷ lys³⁸ala⁵) or GLA⁵ and poly(glu⁵⁴lys³⁶ala¹⁰) or GLA¹⁰ indicate that there is an association of the responsiveness with theH-2 haplotype. Although the C57BL/6J mice (H-2 ^b haplotype) are “non responders”, the C57BL/6By originally derived from mice of the same haplotype are responders. The immune response pattern of recombinant strains carrying haplotypes derived by crossing over within theH-2 complex indicate that the responsiveness is under control of anIr gene which maps to the left of theIB subregion. Studies with the backcross mice indicated multigenic control of the responsiveness, with one locus beingH-2 linked and another locus segregating independently ofH-2.     

The T-Cell factor specific for poly(Tyr,Glu)-Poly(Pro)-Poly(Lys) is an I-Region gene product

The nature of a T-cell factor specific for poly(Tyr,Glu)-poly(Pro)-poly(Lys) [(T,G)-pro-L] was established in the present study. The activity of the (T,G)-Pro-L-specific factor was not removed by anti-mouse immunoglobulin Sepharose columns, suggesting that it is not a classical immunoglobulin. On the other hand, the factor lost its activity after passage through immunoadsorbents prepared with anti-H-2 sera raised against theH-2 haplotypes of the mouse strains in which the factor was prepared. Furthermore, this factor was adsorbed byI region-specific antisera but not by antisera directed against theI-J andI-C subregions as well as theK andD regions of theH-2 complex. Thus, the (T,G)-Pro-L-specific T-cell factor is most probably anI-A subregion gene product.     

Activation and function of an autoreactive T cell clone with dual immunoregulatory activity

Previously it was demonstrated that the human autoreactive CD4+ T cell clone MTC-4 is bifunctional, having the capacity to augment differentiation of autologous B cells into Ig-secreting cells in the absence of PWM and the capacity to suppress such differentiation in the presence of PWM. In the present study it was shown that these two functions of MTC-4 are mediated by distinctly different mechanisms. In the presence of autologous class II MHC Ag, MTC-4 releases one or more non-MHC-restricted soluble factors which stimulate B cell differentiation. The helper factors are different from IL-2, and act on both resting (small) and activated (large) B cells. The suppressor function of MTC-4 cells is elicited when MTC-4 cells are co-cultured with autologous non-T cells preincubated with PWM for 4 h, but not with non-T cells preincubated with PWM for 24 h; thus, activated autologous non-T cells have a transient capacity to induce MTC-4 suppressor function. Induction of MTC-4 suppressor activity is not associated with increased proliferation of MTC-4 and is mediated by low numbers of these cells. Unlike helper function, MTC-4 suppression of Ig synthesis can occur late in B cell cultures, and MTC-4 suppresses Ig production by autologous B cells, but not by allogeneic B cells. Finally, in co-cultures with activated autologous non-T cells and allogeneic B cells, MTC-4 can simultaneously produce helper factors that augment Ig synthesis by allogeneic B cells and suppress Ig synthesis by autologous B cells. In summary, exposure of MTC-4 to autologous non-T cells causes release of non-MHC-restricted factors which augment Ig production by both resting and activated autologous B cells, whereas exposure of MTC-4 to recently activated B cells causes MTC-4 to express the additional function of directly suppressing Ig production by differentiated autologous B cells. Thus autoreactive T cells may be uniquely suited to regulate Ig production.     

Identification of suppressor T cells in virgin non-responder spleen cells responsible for primary unresponsiveness to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)

T cell subsets that regulate antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in mice that are Ir gene non-responders have been further characterized. We previously defined several T cell subsets in GAT-primed non-responder mice. The Lyt-2+ suppressor-effector T cells suppress responses to GAT and GAT complexed to methylated BSA (GAT-MBSA). The Lyt-1+ cell population is complex and can be separated into I-J- Th cells, which support responses to GAT and GAT-MBSA. After priming, the Lyt-1+, I-J+ cell population contains suppressor-inducer cells that activate precursors of suppressor-effector cells to suppress responses to GAT and GAT-MBSA as well as Ts cells that directly inhibit responses to GAT but not GAT-MBSA. By contrast, the Lyt-1+ cells from virgin mice contain only cells that directly suppress responses to GAT but not GAT-MBSA. The major question addressed in the present studies was whether the Lyt-1+, I-J+ Ts cells in virgin and primed mice and the suppressor-inducer cells in GAT-primed mice were functionally and serologically distinct subsets. The studies used mAb and panning procedures to separate cell populations and inhibition of PFC cell responses to functionally define the activity of the cell populations. We used the following two mAb that were raised by immunizing rats with GAT-specific suppressor factors: 1248A4.10 (known to react with suppressor-inducer cells) and 1248A4.3, another reagent from the same fusion. Lyt-1+ cells from virgin spleens contained Ts cells that were A4.10-, A4.3+ and no suppressor-inducer T cells, whereas Lyt-1+ cells from GAT-primed spleens contained Ts cells that were A4.10-, A4.3+ as well as A4.10+, A4.3- suppressor-inducer cells. Thus, the Lyt1+, I-J+ cell subset can be divided into two functionally and serologically distinct subsets, direct Ts cells (1248A4.3+), which suppress responses to GAT but not GAT-MBSA, and GAT-primed suppressor-inducer T cells (1248A4.10+).     

T cell subsets in (responder x nonresponder)F1 mice regulating antibody responses to L-glutamic acid60-L-alanine30-L-tyrosine (GAT)

Immune responses to GAT are controlled by H-2-linked Ir genes; soluble GAT stimulates antibody responses in responder mice (H-2b) but not in nonresponder mice (H-2q). In nonresponder mice, soluble GAT stimulates suppressor T cells that preempt function of helper T cells. After immunization with soluble GAT, spleen cells from (responder x nonresponder: H-2b X H-2q)F1 mice develop antibody responses to responder H-2b GAT-M phi but not to nonresponder H-2q GAT-M phi. This failure of immune F1 spleen cells to respond is due to an active suppressor T cell mechanism that is activated by H-2q, but not H-2b, GAT-M phi and involves two regulatory T cell subsets. Suppressor-inducer T cells are immune radiosensitive Lyt-1 +2-, I-A-, I-J+, Qa-1+ cells. Suppressor-effector T cells can be derived from virgin or immune spleens and are radiosensitive Lyt-1-2+, I-A-, I-J+, Qa-1+ cells. This suppressor mechanism can suppress responses of virgin or immune F1 helper T cells and B cells. Helper T cells specific for H-2b GAT-M phi are easily detected in F1 mice after immunization with soluble GAT; helper T cells specific for H-2q GAT-M phi are demonstrated after elimination of the suppressor-inducer and -effector cells. These helper T cells are radioresistant Lyt-1+2-, I-A+, I-J-, Qa-1- cells. These data indicate that the Ir gene defect in responses to GAT is not due to a failure of nonresponder M phi to present GAT and most likely is not due to a defective T cell repertoire, because the relevant helper T cells are primed in F1 mice by soluble GAT and can be demonstrated when suppressor cells are removed. These data are discussed in the context of mechanisms for expression of Ir gene function in responses to GAT, especially the balance between stimulation of helper vs suppressor T cells.