Antigen-dependent,H-2-restricted cytolytic and noncytolytic T cell lines with specificity for minor histocompatibility antigens

Several cloned T cell lines were isolated from primed mixed lymphocyte cultures immunized against minor histocompatibility antigens. These lines were selected with irradiated stimulator cells as antigen and require restimulation at intervals to keep growing. They are responsive, as measured by proliferation, to interleukin 2 (T cell growth factor) but cannot be grown in it continuously. These T cell lines have either the H-2^d or H-2^k haplotype. They all show exquisite H-2 restriction and minor histocompatibility antigen specificity. We did not observe any alloreactivity on 8 different H-2 haplotypes. For the H-2^k T cell lines, the restriction element could be mapped to either the K or D end of the H-2 complex. No I-A-restricted cell line was found. It is of interest that all these T cell lines need the presence of T cells in the irradiated stimulator cell population. This suggests a more complex interaction between irradiated stimulators and responder T cells than just H-2K- or D-restricted antigen interaction. This recognition, though necessary, does not seem sufficient to induce the T cell clones to proliferate.     

A comment on H-2-restricted T cell recognition of Mls determinants: a question of perspective

Data presented here addressed the question of whether T lymphocyte recognition of Mls determinants is MHC restricted. Using isolated T cell blasts from primary MLCs involving H-2-identical, Mls-disparate strain combinations as responding cell populations in secondary MLCs, and measuring their responses against fresh stimulating cells from different strains, has permitted the study of reactions towards Mls determinants in the presence of MHC antigenic differences. Results strongly suggest that recognition of Mlsc (Mls-2 locus) determinants is H-2 restricted. In contrast, there is still little evidence that the recognition of Mlsa (Mls-1 locus) determinants is H-2 restricted. Furthermore, data are also presented which refute the notion that different H-2 haplotypes possess different abilities to present Mlsa determinants to responding T cells.     

The molecular interactions with helper T cells which limit antigen-specific B cell differentiation

Helper T (Th) cell-dependent activation requirements for 2,4,6-trinitrophenyl (TNP)-specific resting B cells obtained from mice transgenic for Sp-6 mu, kappa genes were analyzed. Carrier-specific T cell help required linked recognition of TNP carrier and was functionally restricted by the B cell major histocompatibility complex. However, histoincompatible T cell-B cell conjugates formed by bridging surface immunoglobulin and Th cell receptor for antigen (TcR) through TNP-conjugated anti-TcR antibodies resulted in the efficient differentiation of TNP-specific B cells. Thus, Th cell-dependent cognate recognition of B cells is not obligatory. Specific conjugate formation could be obviated by using unconjugated fragments of anti-TcR antibodies. If dimeric, these fragments supported the Th cell-dependent differentiation of co-cultured histoincompatible resting B cells. Unconjugated monomeric fragments were ineffective, demonstrating the necessity for TcR cross-linking. Resting B cells from Sp-6+ mice rendered TNP-conjugated monomeric fragments of anti-TcR antibodies effectively multivalent, thereby satisfying conditions for the activation of co-cultured Th cells. The results demonstrate that Th cells do not transduce activation signals through TcR recognition of B cell membrane-associated ligand which limit the induction of B cell differentiation. Cross-linking of TcR on Th cells is required, sufficient and can be induced through interaction with the antigen-specific B cell surface.     

Antigen presentation to T cell lines and clones by peritoneal macrophages, peritoneal B cells and antigen-specific B cell hybridomas

The antigen presenting cell (APC) activity of uninduced, resident peritoneal macrophages and B cells was compared to that of antigen-specific B cell hybridomas by measuring proliferative responses of antigen-specific, MHC-restricted T cell clones. The results demonstrate that peritoneal macrophages and B cells are much more efficient APC than irradiated splenic filler cells, and that unirradiated B cells were as good as, if not better than, macrophages. Both B cells and macrophages can be pulsed with antigen, although pulsed B cells were always found to be more efficient than pulsed macrophages. However, the APC activity of B cells was exquisitely sensitive to irradiation. The relative contribution of macrophages and B cells to the APC activity of mixed populations was easily distinguished by complement dependent lysis with monoclonal antibodies specific for unique differentiation antigens expressed by these cells. Normal peritoneal macrophages and B cells present the synthetic terpolymer of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) to GAT-specific T cells clones and beef insulin to insulin-specific T cell lines nonspecifically. The APC activity of antigen-specific B cells was also examined by using novel GAT-specific, nonsecretor B cell hybridomas produced by fusing GAT-primed spleen cells to the HAT sensitive Balb/c lymphoma, M12.4.5. The hybridomas selected for these studies were GAT-specific, sIg+, Ia+ cells. These hybridomas presented GAT to GAT-specific T cells more efficiently than heterogeneous B cells suggesting that interaction with surface Ig receptors facilitated the uptake and/or processing of antigen. GAT-specific B cell hybridomas, like normal B cells, presented soluble beef insulin to an insulin-specific T cell clone nonspecifically. However, after pulsing with antigen overnight, the GAT-specific B cell hybridoma could activate only GAT-specific T cells.     

Increased production of antigen-specific B lymphocytes during in vitro immunization using carrier-specific T helper hybridomas.

An in vitro method to increase the production of hapten-specific antibody-forming B cells (AFC) using a carrier-specific T helper hybridoma and murine splenocytes is described. Naive splenocytes (6 x 10(6)/ml) are cultured in vitro in the presence of a hapten-carrier conjugate (DNP.OVA) and OVA-specific T helper hybridomas (0.5 x 10(6)/ml). After 4-5 days in vitro immunization (IVI), the maximum number of DNP-specific AFC were found using a spot-ELISA with twice the number of IgM positive cells as IgG positive AFC. The presence of antigen in the form of a hapten-carrier complex and the use of a carrier-specific Th hybridoma resulted in more hapten-specific AFC than when neither antigen nor Th hybridoma were present or when antigen alone or T help alone were used. Also when the hapten was conjugated to a carrier not recognised by the carrier-specific Th hybridoma there were considerably fewer (less than 50%) hapten specific AFC formed. When in vivo primed splenocytes (DNP) were boosted in vitro (IVB) under the same conditions as for IVI most hapten-specific AFC were found on day 4 and both anti-DNP IgM and IgG AFC were increased relative to IVI. Again most AFC were found when hapten was bound to the relevant carrier. In conclusion, carrier-specific T hybridomas can be used in an in vitro immunization procedure with naive or primed splenocytes to increase the frequency of anti-hapten AFC. This method offers an improvement over the current in vitro immunization procedures for the production of monoclonal antibodies.     

In vitro studies on H-2-linked unresponsiveness to synthetic polypeptides. III. Production of an antigen-specific T helper cell factor to (T,G)-A--L.

The in vitro production, assay and partial characterization of an extremely potent antigen-specific T helper cell factor to poly-L-(Tyr, Glu)-poly-DL-Ala--poly-L-Lys [(T,G)-A--L] which may be an important physiological mediator of T cell function in vivo is described. The factor has an antigen-binding site, Ia and Ig-like determinants on the same molecular complex. The response to (T, G)-A--L is under H-2-linked Ir gene control, some but not all low responder haplotypes will produce the factor.     

Generation and characterisation of bovine antigen-specific T cell lines.

15 antigen-specific T cell lines have been generated from eight individual cattle immunised with ovalbumin. Several sources of interleukin-2 (IL-2) were used, including a supernatant from a gibbon cell line (MLA-Sup), human recombinant IL-2 (hrIL-2) and bovine recombinant IL-2 (brIL-2). These IL-2 sources were used alternately with autologous peripheral blood mononuclear cells (PBM) together with ovalbumin to generate the lines. They grew least well in MLA-Sup and best in brIL-2. FACS analysis indicated that the lines generated with the recombinant IL-2s were extremely homogeneous in that the majority of cells were BoCD4+ (bovine CD4 equivalent) and therefore of TH phenotype. The lines were antigen specific and responded to antigen only in the presence of autologous PBM and not allogeneic (MHC class I nonidentical) PBM. However, allogeneic PBM did support their proliferation to ConA. No MLR response was observed by the cell lines to allogeneic PBM. The response to antigen was inhibited by anti bovine class II mAbs but not an anti bovine class I mAb. The subpopulation of PBM which acted as antigen presenting cells for these bovine TH cell lines had typical macrophage characteristics.     

On the diversity and heterogeneity of H-2(d)-restricted determinants and T cell epitopes from the major bee venom allergen.

One of the main limitations of using synthetic peptides for immunotherapy in allergic patients is the difficulty to delineate the immunodominant T cell epitopes which are necessarily dependent on HLA molecules. We have thus addressed the question of the role of MHC II molecules in immunodominant epitopes selection in the particular case of the major bee venom allergen (API m1). To exhaustively and easily explore it, we used BALB/c mice whose H-2 haplotype is associated with high IgE and IgG responses to API m1. By means of extensive sets of synthetic peptides, we investigated the specificity of polyclonal T cells and monoclonal hybridomas from mice immunized with API m1 and delineated four immunodominant regions, restricted to either the I-E(d) or the I-A(d) molecule. All the peptides were also tested for their capacity to bind to immunopurified MHC II molecules. Eight determinants of high affinity were identified. They clustered into three distinct regions and were largely overlapping. They included all the immunodominant epitopes, but half of them were not capable of stimulating T cells. Strikingly, interacting surfaces with either the TCR or MHC II molecule greatly differed from one determinant to another. In one case, we observed that flanking regions exerted a particular action on T cell stimulation which prevented the fine epitope localization. Our results underline the diversity and complexity of MHC II-restricted determinants and T cell epitopes from the major bee venom allergen, even in a single haplotype. These data also participate in the development of alternative approaches to conventional immunotherapy.     

Immunogenetic aspects of human thyroglobulin-reactive T cell lines and hybridomas

The in vitro proliferative response to T cells primed with human thyroglobulin (Tg) was compared in 11 independent haplotypes on B10 background. B10.K and B10.S mice were the most responsive, whereas, with the exception of B10.PL (H-2u), all other B10 congenics were intermediate responders. The two best responders to in vitro challenge with human Tg, of the k and s haplotype, are the same as those showing H-2-linked susceptibility to induction of experimental autoimmune thyroiditis (EAT) with mouse Tg. Since shared epitopes on human and mouse Tgs have been shown to be thyroiditogenic by adoptive transfer studies in CBA (H2k) mice, the findings indicate that shared epitopes may be studied in appropriate (i.e. EAT-susceptible) strains of mice. Therefore, we proceeded to develop methods to produce T-cell lines and hybridomas to human Tg in B10.K and B10.S mice, test their cross-reactivity to heterologous Tgs and their Ia restriction patterns. By using antigen-presenting cells from recombinant strains, we identified restriction elements encoded by the I-A subregion alone and a combinatorial molecule from the I-A/I-E subregions.     

The molecular basis of T cell differentiation

Our laboratory has studied the molecular basis of T helper cell differentiation. We have used reporter transgenic mice, selective hybridization techniques, and studies of cell signaling to show that a complex pattern of gene expression is reprogrammed as the decision is made to become either a Th1 or Th2 cell. Many of these components have been identified, and their mechanisms of action elucidated. Understanding these mechanisms is likely to lead in the long-term to ways to intervene in these processes and, therefore, to direct immune response in therapeutically useful directions.     

The relationship between secreted and cell surface antigen-binding molecules synthesized by T cells. I. Function-related isotypic determinants on T cells defined by antisera to antigen-specific helper and suppressor factors.

Rabbit antisera have been used to define 'constant region' markers which distinguish between mouse T cell-derived helper and suppressor factors, regardless of their antigen-specificity or strain of origin. These antisera have also been shown to bind to functional T-cell lines. After several absorption steps, rabbit anti-helper factor serum bound specifically to mouse helper cell lines, whereas rabbit anti-suppressor factor serum bound specifically to suppressor cell lines. Neither antiserum bound to cytotoxic T cell lines. The 'isotypic' determinants defined by these antisera were demonstrated to be present on distinct subpopulations of non-transformed T cell populations, such as splenic T cells, cortisone-resistant thymocytes and Con A blasts, but were not found on Thy-1- spleen cells, bone marrow, brain, heart, liver, kidney or heart tissue. The antisera did not stain significant numbers of normal thymocytes, and so expression was restricted to mature T cells. T cells reactive with rabbit anti-helper factor serum were found in the Lyt 2- population of cortisone-resistant thymocytes, and constituted a major subpopulation of in vitro induced helper cells, while rabbit anti-suppressor factor serum stained cells found in the Lyt 2+ population of cortisone-resistant thymocytes, as well as the majority of in vitro induced suppressor cells. Thus, these antisera are potentially of great value in the definition and isolation of functionally-distinct T cell subpopulations.     

T helper factor in contact sensitivity: antigen-specific I-A+ helper factor is made by an Lyt-1+2-, I-A+, I-J- T cell.

Antigen-specific T helper factor appears in the 24 hr supernatant of lymph node cells taken 4 days after immunization with contact sensitizer. The factor is assayed by its ability to augment the contact sensitivity response induced by haptenized spleen cells. In practice, picrylated or oxazolonated spleen cells are treated with the factor for 1 hr at 4 degrees and 4 x 10(6) cells are injected into the footpads of recipient mice. Contact sensitivity is assessed 5 days later. The factor first appears 3 days after immunization and its production depends on an Lyt-1+2-, I-A+, I-J- T cell. It is antigen-specific in its action in a criss-cross experiment, and can be absorbed with and eluted from haptenized beads. It bears I-A determinant(s) and the I-A determinant and the antigen binding site(s) occur on the same molecule. The molecular weight is around 60,000. The possible role of T helper factors in the activation of the antigen-presenting cell in the induction stage of the immune response is discussed.     

Direct interaction between an antigen-specific B cell clone and an MHC class II-reactive helper T cell clone.

TP67.14 established by somatic hybridization is a 2,4,6-trinitrobenzenesulfonic acid (trinitrophenyl, TNP)-specific B cell clone with a receptor molecule for TNP on the cell membrane, and MS202 is an interleukin-2 (IL-2)-dependent T helper (Th) cell clone reactive to auto-MHC class II antigens (IAk and IEk) as previously reported. In the present study it was shown that MS202 considerably induced the maturation of TP67.14 into anti-TNP plaque-forming cells (PFCs), and this response was markedly augmented by the addition of TNP-keyhole limpet hemocyanin (KLH). Recombinant cytokines and the culture supernatant of MS202 with TP67.14 did not affect the generation of anti-TNP antibodies by TP67.14. Also, neither anti-IL-4 nor anti-IL-5 monoclonal antibody (mAb) inhibited the maturation of TP67.14 mediated by MS202. The differentiative effect of MS202 on TP67.14 was completely lost when each cell was separately cultured using a semipermeable membrane. Monoclonal antibodies against LFA-1 beta molecules significantly blocked the development of anti-TNP PFCs induced by MS202, as well as anti-IAk and anti-IEk mAbs. Interestingly, the plasma membrane-enriched fraction (PM) derived from MS202 exhibited much more differentiative effects on TP67.14 treated with TNP-KLH than PM from other T cell lines and concanavalin A-induced T lymphoblasts. In addition, TNP-conjugated PM from MS202 by itself induced a great number of anti-TNP PFCs. The present findings indicate that MS202 is capable of inducing the maturation of TP67.14, which is considered to represent a population of B cells with antigen specificity in a late lineage of B cell maturation, through direct cell contact but not soluble factors. This suggests that B cells with antigen specificity, in the presence of antigen, can be induced to mature into antibody-secreting cells through direct contact with Th cells; in this process surface major histocompatibility complex class II and lymphocyte function-associated antigen 1 (LFA-1) molecules are directly involved and the cell membrane derived from Th cells provides a transductional signal for maturation of B cells with antigen specificity in the presence of antigen.     

Mechanism of antigen binding by T cells: H-2 (I-A)-restricted binding of antigen plus Ia by helper cells

The binding of a complex containing self Ia and antigen (IAC) to T cells was investigated. Poly-L (Tyr, Glu)-poly-DLAla—poly-LLys [(T, G)-A—L], bovine serum albumin, ovalbumin or fowl gamma-globulin were used as antigen. The effect of this complex was investigated in three experimental systems: autoradiographic antigen binding, in vivo immunization, and in vitro radioactive suicide of helper T cells. Autoradiographic experiments have shown that antigen binding to T cell-enriched spleen cells is a slow process with a half-life period of 20 min. It was found that during this time, antigen which can bind to Lyt-1⁺ T cells with much faster kinetics (half-life period = 2 min), is liberated from adherent cells. This “processed” antigen was retained by anti-Ia or lentil lectin affinity columns, and its binding to T cells was restricted by the I-A subregion of H-2. IAC was 100 to 1000-fold more immunogenic in vivo than the same amount of untreated antigen. This immunogenicity could be removed on anti-Ia columns, and was found to be under similar H-2 restriction as was its binding to T cells. The functional T cells, to which IAC binds, were identified by radioactive antigen suicide. It was found that the IAC killed syngeneic but not allogeneic, helper T cells. For the binding of processed antigen, helper cells required metabolic energy and a nonspecific soluble factor of adherent cells. These data are interpreted to suggest that H-2 restriction is directly determined by the interaction of the helper cell receptor with self Ia and foreign antigen. It also appears that the Ia-containing antigen may be a potent, cell type-directed immunogen.     

Selected populations of alloreactive T cells contain helper T cells but lack ThId,an antigen-specific helper T cell required for dominant production of the T15 idiotype

Isolated, alloreactive T cell populations were primed with protein carriers in vivo and tested for their ability to provide help for an anti-phosphorylcholine (PC) antibody response and for production of the T15 idiotype. It was found that alloreactive T cell populations would support anti-PC antibody responses but did not selectively activate B cells capable of producing the T15 idiotype that normally dominates such responses. This failure to help for the production of the T15 idiotype was shown to be due to the absence of an antigen-specific helper T cell that is required for dominant idiotype production (ThId). These studies suggest that ThId cells have recognition structures for antigen and for self idiotype, but lack recognition structures for major histocompatibility complex-encoded antigens.     

Patterns of lymphokine production by primary antigen-specific/MHC restricted murine helper T cell clones.

In this study we examined a panel of CD4+ antigen specific/MHC restricted T cell clones for their ability to secrete IL-2, IL-4, and IFN-gamma upon stimulation with con A, three lymphokines which are diagnostic for the TH1 and TH2 subtypes of helper T cells. Eight of the twelve clones we analyzed did not fit the classical TH1/TH2 patterns of lymphokine secretion. Seven of these clones secreted both IL-2 and IL-4 and two of these also produced IFN-gamma. The remaining non-classical clone secreted IL-4 and IFN-gamma but not IL-2. Data from the subcloning of the IL-2/IL-4/IFN-gamma triple producers were not consistent with the parental lines being a mixture of TH1 and TH2 cells. The IL-2/IL-4 double producers (IFN-gamma negative) cannot be explained by the parental lines being a mixture of the TH1 and TH2 subtypes. Nevertheless, these double producers were subcloned and the results provided convincing evidence that clones which secrete both IL-2 and IL-4 do exist. Lymphokine loss variants involving IL-2, IL-4 or IFN-gamma were observed among subclones derived from the double and triple producers as well as in several parental lines maintained in continuous culture. We also observed the appearance of inducible IFN-gamma production in some subclones derived from parental clones where production of IFN-gamma was not detectable. The phenotypes of these variants failed to indicate an obvious trend toward the TH1 and TH2 subtypes. Thus, our results suggest that more heterogeneity in the population of CD4+ helper T cells exists than can be explained by the TH1 and TH2 subtypes of these cells.     

The role of H-2 and H-2-like determinants in syngeneic and allogeneic cytostasis.

Specific sensitization against H-2 determinants was affected by immunizing allogeneic mice with spleen and lymph node cells in H-2 congenic combinations. Lymph nodes from the sensitized and non-sensitized mice were respectively cultured together with H-2 syngeneic tumour cell lines. The growth and viability of the tumour cells was subsequently measured by the amount of radiothymidine incorporation. If the tumour cells incorporated less isotope when cultured with the immune cells than with the normal cells this was termed 'cytostasis'. To identify the H-2 genes controlling the sensitization phase in the cytostasis assay, we studied the effect on different transplantable tumour target cells of lymphoid cells from mice sensitized against different congenic spleen cells. The results suggest that the cytostasis assay can can measure an in vitro specific response to H-2-incompatible sensitizing antigens, and that I--B incompatibility, together with K and/or D, is essential to produce effectors. Furthermore, H-2 allogeneic sensitization could induce cytostasis even against tumour cells syngeneic for the H-2 halotype of the responder strain. The implications of these findings are discussed.     

T-T cell interactions during in vitro cytotoxic T lymphocyte responses. III. Antigen-specific T helper cells release nonspecific mediator(s) able to help induction of H-2-restricted cytotoxic T lymphocyte responses across cell-impermeable membranes

T helper cell induction and the specificity of T cell-mediated help as generated during alloreactive and H-2-restricted, virus- or hapten-specific cytotoxic T lymphocyte (CTL) responses have been compared. With the use of a double-chamber culture system, it was possible to dissect and separately analyze the induction phase of T helper cells from the T helper cell effector function. The data obtained revealed that during alloreactive as well as H-2-restricted T cell responses, antigen-specific T helper cells are induced. Upon specific restimulation of T helper cells, helper cell function is mediated across a cell-impermeable membrane via soluble products in an apparently nonspecific and nonrestricted manner. The data suggest that similar rules govern T-T cell interactions in alloreactive and H-2-restricted CTL responses.