Interaction of idiotype-specific T suppressor factor with the hapten-specific third-order T suppressor subset results in antigen-nonspecific suppression

The interaction between the third-order T suppressor (Ts3) cell and the idiotype (Id)-specific second-order Ts factor (TsF2) was studied in the phenyltrimethylamino (TMA) hapten system. The experimental system which we used allowed the independent analysis of induction and activation requirements of Ts3. The procedure consisted of inducing the Ts3 in vivo and activating the enriched T-cell populations containing Ts3 in vitro with TsF2. The suppressive potential was then tested in mice previously primed for delayed-type hypersensitivity responses which were also treated with cyclophosphamide to deplete Ts3 and other drug-sensitive Ts cell types. Using this experimental system, it was found that the Id-specific TsF2 was required for the in vitro activation of Ts3. Furthermore, the TsF2 activated only the homologous and not heterologous antigen-primed Ts3-containing T cells and moreover, the target of TsF2 was found to be the Ts cells bearing hapten-specific receptors. Once the TMA hapten-specific Ts3 was activated with TsF2, the ensuing suppression was antigen nonspecific. The data demonstrate that the Ts3 represents a final effector Ts cell type in the TMA system.     

Mechanism of action of a T suppressor factor (TsF) in contact sensitivity: the T cell target for TsF activity in adoptive transfer of immunity is not effector cell

The passive transfer of contact sensitivity (CS) by immune cells into normal animals requires the interaction of two distinct Ly-1+ T cells, one which is Vicia villosa lectin (VV)-nonadherent, the other which adheres to VV. Functional deletion of either cell type abrogates the adoptive transfer of CS into normal animals, whereas VV-nonadherent cells alone can transfer CS into animals pretreated with cyclophosphamide (Cy). An antigen-specific T suppressor factor, designated TNP-TsF, inhibits the transfer of CS into normal adoptive recipients. TNP-TsF mediates its suppressive activity by inducing an I-J+ subfactor (designated I-J2) from the assay population by the interaction of PC1-F (a TNP-binding subfactor of TNP-TsF) with antigen-primed Ly-2+ T cells. This I-J+ subfactor then complements TNBS-F (an antigen-nonbinding subfactor of TNP-TsF) to form an antigen-nonspecific effector molecule which suppresses DTH responses in an antigen-nonspecific fashion. We report here that TNP-TsF suppresses the adoptive transfer of CS into normal animals but not into animals pretreated with Cy. TNBS-F + I-J2, the effector complex of TNP-TsF, also suppresses the transfer of CS into normal but not Cy-treated animals. When the Ly-1 immune cells were separated into VV-adherent and -nonadherent populations, the TNBS-F + I-J2 suppressor complex suppressed the functional activity of the VV-adherent cell population, but not the VV-nonadherent cells. This suppressive activity correlates with the need for VV-adherent cells in the transfer of CS into normal but not Cy-treated recipients. When an I-J+ molecule (I-J1) from an SRBC-specific TsF was used in place of I-J2 to form a suppressor complex with TNBS-F, this TNBS-F + I-J1 TsF suppressed the transfer of CS into both normal and Cy-treated recipients. This difference in functional suppressive activity correlated with a difference in target cell specificity: TNBS-F + I-J1 suppressed the VV-nonadherent TDTH cell, whereas TNBS-F + I-J2 suppressed the VV-adherent T cell of CS. Immune cells which are transferred under conditions which do not require the VV-adherent cell for transfer are not suppressed by TNBS-F + I-J2 or TNP-TsF, but are suppressed by the TNBS-F + I-J1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibitory activity of interleukin B on the suppressor T cell hybrid T2D4

Interleukin B (IL-B), a product of unstimulated B cells, is defined by its ability to selectively prevent the differentiation of suppressor T lymphocytes from precursors into effectors. The present study was undertaken to determine whether IL-B could also be active in modulating the activity of the T cell hybrid T2D4, which produces immunoglobulin-binding suppressor factors. T2D4 cells can be selectively induced by incubation with various isotypes of antibody to express isotype-specific Fc receptors and to release soluble factors that suppress production of the corresponding isotype. The data presented here demonstrate that IL-B is greatly effective in inhibiting T2D4 activities. Either pretreatment with IL-B or continuous exposure to IL-B prevents isotype activation of T2D4. As a result, T2D4 cells do not express isotype receptors and do not produce detectable amounts of isotype-specific suppressor factors. This IL-B regulatory activity on T2D4 is temperature dependent and is inhibited by cytochalasin B. These findings provide new insights on the mechanism by which IL-B enhances antibody responses, and they offer a conceptual framework for analyzing IL-B activity on suppressor T cells.     

Murine interstitial nephritis. VII. Suppression of renal injury after treatment with soluble suppressor factor TsF1

We prepared soluble suppressor T cell factor (TsF1) from donor spleens harvested from mice primed with tubular antigen-derivatized lymphocytes to analyze both its functional interactions with a larger suppressor T cell network and its influence on the nephritogenic effector T cell response producing interstitial nephritis to a parenchymal antigen. Our findings indicate that TsF1 is antigen-specific, genetically restricted by I-J in its direct mediation of suppression, and capable of inhibiting the development of interstitial lesions. TsF1 also provides an inducing signal for the activation of effector Ts-2 suppressors following presentation by accessory cells. The induction of a Ts-2 effect, however, requires that the factor-presenting cell and the recipient of such cells share homology at I-J, and that the TsF1, the precursor Ts-2 cells, and the recipient of the Ts-2 effect share the same Igh-V allotype. Finally, the results of this current report clearly demonstrate a possible therapeutic role for soluble suppressor factors in the management of interstitial renal disease.     

Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity

We analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long-term cultured T cell growth factor (TCGF)-dependent human T lymphocytes. Seven cell lines tested produced colony-stimulating activity (CSA) as well as burst-promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven T-cell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU-GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long-term culture and this property is not related to the helper or suppressor activity of the cultured cells.     

Regulatory mechanisms in cytotoxic T lymphocyte development. III. Induction, specificity, and genetic restriction of an in vitro suppressor T cell

We addressed questions pertaining to the immunogenetics of an in vitro alloinduced suppressor T cell (Ts) previously shown to inhibit cytotoxic T-lymphocyte (CTL) development by suppressing CTL precursor proliferation. Using intra-MHC recombinant strains of B10 congenic mice, the requirements for H-2 differences to induce Ts activity, the antigen specificity of the Ts, and the genetic restriction of Ts function were studied. It was found that differences at the K, D, or I regions alone can induce strong suppressor activity. Suppression of CTL development does not appear to be genetically restricted since the Ts inhibit CTL from responder cells disparate at K, K and D, I, or K and I. The alloinduced Ts is specific for the antigen stimulating its induction, but also inhibits CTL responses against immunologically unrelated determinants, even between class I and class II antigens, provided those determinants are carried on cells expressing the original inducing antigen. Ts can be triggered by antigens present on the responder cells but absent on the stimulator cells, indicating that the suppressive signal may be exerted directly on the responder population without specific interaction with stimulator cells.     

Measles-specific T cell clones derived from a twin with multiple sclerosis: genetic restriction studies

The association between multiple sclerosis (MS) and HLA-DR2 suggests that the disease may be associated with an aberrant immune response, likely directed against an antigen of either viral or host origin. We have used measles virus-specific T cell clones derived from a patient with MS to study genetic restriction patterns of antigen presentation by macrophage-enriched (E-) populations. Twenty-two clones proliferated in response to measles-infected Vero cells but not to mumps-infected or uninfected Veros. E- cells from both the autologous subject and her healthy, measles nonresponder identical twin were capable of presenting antigen to all clones. Studies with E- cells obtained from a panel of cell donors demonstrated clones which recognized antigen in association with D2/DR2, DR4, subgroups of DR4, and SB3. Three clones recognized antigen only in association with the autologous or twin's cells, but not with other sets of HLA-matched E-cells obtained from healthy donors or from other patients with MS. These studies indicate that the differing responses to measles virus demonstrated by these two identical twins are not explained by alterations in the interactions between antigen-presenting cells and T cells. Furthermore, at the clonal level, no preferential role is seen for HLA-DR2 as the restricting element for presentation of measles virus to these clones.     

T suppressor efferent circuit which affects contact sensitivity to picryl chloride: the late-acting, second nonspecific T suppressor factor bears I-A determinants which are responsible for the I-A genetic restriction in its interaction with its target cell

The T suppressor efferent circuit in the picryl (TNP) system, which inhibits the passive transfer of contact sensitivity, involves at least two antigen-nonspecific factors. The second nonspecific T suppressor factor (ns-2) bears I-A determinants of both the alpha and the beta chain as shown by affinity chromatography on immobilized anti-I-A monoclonal antibodies. Sequential absorption shows that the determinants of the alpha and beta chain occur on the same molecular complex. No absorption was obtained with anti-I-E antibody. There are two genetic restrictions associated with ns-2--the first is in its release from the second T suppressor efferent cell (on exposure to antigen) and the second is in its inhibitory interaction with its target cell. Both are MHC restricted and matching in I-A (but not I-E, or I-J) is sufficient. The question was asked whether the I-A of the ns-2 was directly responsible for the I-A genetic restriction in its action. F1 TsF was made in (H-2k X H-2b)F1 mice by injecting picrylated parental cells intravenously and triggering the release of ns-2 with the corresponding picrylated parental cells. Both I-Ak- and I-Ab-positive ns-2 were produced and were separated by affinity chromatography on immobilized anti-I-A monoclonal antibody. The I-A phenotype of these separated ns-2 of F1 origin determines the genetic restriction in their action; i.e., I-Ak+ ns-2 only inhibits passive transfer by H-2k cells and I-Ab+ ns-2 only acts on H-2b cells. In contrast, the I-A haplotype of the picrylated cell used to induce the Ts cell which makes ns-2 is unimportant. It was concluded that the I-A on the ns-2, and not a possible recognition site for I-A, serves as a restriction element. This finding suggests that ns-2 may act directly on the I-A-restricted T cell which mediates contact sensitivity.     

Alignment of a restriction map with the genetic map of bacteriophage T4.

We reported previously that polycytidylate [poly(C)]-dependent RNA polymerase activity was a property of small spherical or triangular reovirus-specific particles which sedimented at 13 to 19S and were composed solely of the reovirus protein, sigma NS. Depending on the fraction of cellular extracts from which they were obtained, these particles exhibited marked differences in stability. Most 13 to 19S particles from a particular fraction repeatedly disaggregated into smaller 4 to 5S subunits with no enzymatic activity. Disruption of many particles could be prevented and polymerase activity retained after these particles had bound different single-stranded (ss) RNAs. Our previous results indicated that there was heterogeneity among the 13 to 19S particles in that possession of poly(C)-dependent RNA polymerase activity was a property of only some. Support for this heterogeneity was derived from the demonstration in this report that there were at least three types of binding sites present within particles in any purified preparation: (i) those binding only poly(C); (ii) those binding only reovirus ss RNAs; and (iii) those binding one or the other, but not both at the same time. It is suggested that only those particles able to bind either poly(C) or reovirus ss RNAs had poly(C)-dependent RNA polymerase activity, as reovirus ss RNAs markedly inhibited the polymerase activity. All three size classes of reovirus ss RNAs were equally effective in binding, but once bound, they were not copied. It is possible that heterogeneity in binding capacity of different particles comprised of only one protein, sigma NS, could result from the ability of subunits containing this protein to assemble into slightly different 13 to 19S particles with specificity of binding or polymerase activity conferred by the configuration of the assembled particles. The high capacity of sigma NS to bind many different nucleic acids with some specificity suggests that these particles may act during infection as condensing agents to bring together 10 reovirus ss RNA templates in preparation for double-stranded RNA synthesis.     

Analysis of mitochondrial DNA species in interspecific hybrid somatic cells using restriction endonucleases. Identification of recombinant mtDNA molecules

Interspecific hybrid cells were isolated by fusion between thymidine kinase-deficient (TK⁻) mouse B82 cells and hypoxanthine-guanine-phosphoribosyl-transferase-deficient (HGPRT⁻) rat L6TG cells, and cultivating them in selective medium with hypoxanthine-aminopterin-thymidine (HAT). Karyo-type analysis revealed that they contained both mouse and rat chromosomes. Mitochondrial DNA (mtDNA) species of the hybrid cells were identified by digesting them with three kinds of restriction endonucleases, Hae II, EcoR I and Hpa II. Their restriction endonuclease cleavage patterns indicated that a portion of the mtDNAs was of mouse parent cell origin, while the remainings were recombinant molecules, i.e., part of the rat mtDNA sequence could be detected, but not whole rat mtDNA. The molecular weights of hybrid cell mtDNAs were calculated to be almost the same as that of the parent cells (˜10⁷ D).     

Functional studies on the role of I-A molecules expressed by the antigen-specific T suppressor cell clone HF1

The antigen-specific T suppressor (Ts) cell clone HF1 (Thy-1.2+, I-Ak+, I-Ek+) was isolated from a CBA/J mouse tolerized to low doses of bovine serum albumin (BSA), and a subclone was adapted to grow in the absence of antigen-presenting cells. The functional role of the I-Ak molecules endogenously expressed by HF1 Ts cells was analyzed by using anti-I-Ak monoclonal antibodies (mAb) in attempts to block the antigen-induced proliferation of these cells. Among a panel of anti-I-Ak mAb tested, only the H118-49 mAb directed against the private determinant Ia.m1 blocked BSA-induced proliferation. The data presented here suggest a selective regulatory role of membrane-bound I-Ak molecules in the antigen-induced signal transmission.     

Cleavage of synthetic RNA-DNA hybrids with restriction endonucleases BamH1 and Sau3A

Synthetic oligodeoxyribonucleotides, containing one or two ribonucleotides in the recognition sequence, and RNA--DNA hybrids were tested for their activity in cleavage with BamH1 and Sau3A endonucleases. The replacement of dG with G in the first position of BamH1-site (GGATCC) of one of the chains does not affect the rate of the BamH1 hydrolysis. The similar heteroduplex, containing G residue in the second position, displays a decreased rate of the BamH1 hydrolysis of the modified strand and, to a lesser extent, of the unmodified complementary strand. Oligodeoxyribonucleotides in complex with oligoribonucleotides can be cleaved with the excess of BamH1 and Sau3A, oligoribonucleotides remaining intact.     

Antigen-specific suppressor T lymphocytes in man make use of the same set of surface molecules as do cytolytic T lymphocytes: roles of Leu-2/T8, Leu-4/T3, Leu-5/T11, LFA-1 molecules

When cultured with autologous antigen-primed Leu-3+ lymphoblasts, Leu-2+ cells differentiate into suppressor T cells (Ts) that specifically inhibit the responses of fresh autologous Leu-3+ cells to the priming antigen. We have shown previously that the Leu-4/T3 (CD-3) molecular complex and HLA-A,B molecules on the surface of Leu-3+ inducer blasts are recognized by Leu-2+ Ts during their differentiation. This study examines the role of various cell surface molecules expressed by Leu-2+ Ts during the inductive and effector phases of suppression. Leu-2+ cells were treated in the absence of complement with a variety of monoclonal antibodies recognizing distinct human lymphoid antigens either before or after their activation with alloantigen-primed Leu-3+ blasts. Antibodies to Leu-2/T8 (CD-8) and lymphocyte function-associated antigen-1 (LFA-1) (CDw-18) molecules inhibited not only the generation but also the effector function of Leu-2+ Ts. Although antibodies to Leu-4/T3 (CD-3) and Leu-5/T11 (CD-2) molecules caused profound inhibition of the activation of Ts, these antibodies failed to inhibit the effector function of Ts. On the contrary, anti-Leu-4 antibody consistently augmented the suppressor effect of Ts. Antibodies directed against Leu-1/T1 (CD-5), Leu-3/T4 (CD-4), LFA-3, and class I (HLA-A,B,C) and class II (HLA-DR,DQ) major histocompatibility complex molecules had no effect on either the generation or the effector function of Ts. These results suggest the involvement of Leu-2/T8 (CD-8), Leu-4/T3 (CD-3), Leu-5/T11 (CD-2), and LFA-1 (CDw-18) molecules on the surfaces of Leu-2+ cells in the activation and effector functions of Ts.     

T cell recognition of Neisseria meningitidis class 1 outer membrane proteins. Identification of T cell epitopes with selected synthetic peptides and determination of HLA restriction elements.

No vaccine is yet available against serogroup B meningococci, which are a common cause of bacterial meningitis. Some outer membrane proteins (OMP), LPS, and capsular polysaccharides have been identified as protective Ag. The amino acid sequence of the protective B cell epitopes present within the class 1 OMP has been described recently. Synthetic peptides containing OMP B cell epitopes as well as capsular polysaccharides or LPS protective B cell epitopes have to be presented to the immune system in association with T cell epitopes to achieve an optimal Ir. The use of homologous, i.e., meningococcal, T cell epitopes has many advantages. We therefore investigated recognition sites for human T cells within the meningococcal class 1 OMP. We have synthesized 16 class 1 OMP-derived peptides encompassing predicted T cell epitopes. Peptides corresponding to both surface loops and trans-membrane regions (some of which occur as amphipathic beta-sheets) of the class 1 OMP were found to be recognized by T cells. In addition, 10 of 11 peptides containing predicted amphipathic alpha-helices and four of five peptides containing T cell epitope motifs according to Rothbard and Taylor (Rothbard, J. B., and W. R. Taylor. 1988. EMBO J 7:93) were recognized by lymphocytes from one or more volunteers. Some of the T and B cell epitopes were shown to map to identical regions of the protein. At least six of the peptides that were found to contain T cell epitopes show homology to constant regions of the meningococcal class 3 OMP and the gonococcal porins PIA and PIB. Peptide-specific T cell lines and T cell clones were established to investigate peptide recognition in more detail. The use of a panel of HLA-typed APC revealed clear HLA-DR restriction patterns. It seems possible now to develop a (semi-) synthetic meningococcal vaccine with a limited number of constant T cell epitopes that cover all HLA-DR locus products.     

Cellular interactions of L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT)-specific suppressor factors. I. Inhibition of the activity of GAT-specific helper T cell clones by monoclonal GAT-specific suppressor T cell factors

Considerable information concerning the serology and biochemistry of antigen-specific, T cell-derived suppressor factors has been obtained with the use of T cell hybridomas as a source of homogeneous material. Similarly, knowledge of helper T cell products and receptors is accumulating from studies of helper T cell clones and hybridomas. Our strategy for studying the mechanisms by which suppressor factors inhibit responses was to determine whether monoclonal suppressor factors could inhibit antibody responses specific for L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) in cultures containing unprimed splenic B cells, macrophages, and GAT-specific T cell clones as a source of helper activity. The MHC-restricted, two chain suppressor factors, GAT-TsF2, inhibited these responses if the helper T cell clones and suppressor factor were derived from H-2-compatible mice. Furthermore, responses were inhibited by briefly pulsing T cell clones with GAT-TsF2 in the presence of GAT, indicating that suppressor factors need not be present continuously. In addition, helper T cell clones adsorbed syngeneic, but not allogeneic, GAT-TsF2 in the presence of GAT. Adsorption also requires a shared antigenic specificity between the H-2b-derived helper T cells and TsF2 factor. Thus, helper T cells can serve as the cellular target of antigen-specific, MHC-restricted GAT-TsF2, and cloned helper T cells can be used as a homogeneous target population for analysis of the molecular mechanisms of T cell suppression.     

Down-regulation of cytotoxic T lymphocyte development by a minor stimulating locus-induced suppressor cascade that involves Lyt-1+ suppressor T cells, IA- macrophages, and their factors

Down-regulation of the development of CTL has been studied in mice both in vivo and in vitro. To generate CTL to hapten-altered self Ag in vivo, an immunization protocol has been used in which the host's Th cells are stimulated by a minor locus histocompatibility Ag (Mlsd) and its precursor CTL are activated by trinitrophenylated syngeneic spleen cells. Injecting the H-2 compatible Mls-disparate spleen cells along with the TNP-coupled self cells into the hind paws causes TNP-self specific CTL to appear in popliteal lymph nodes within 5 days. We have previously reported that inducing Ts cells by i.v. injecting Mlsd-bearing cells prevents in vivo generation of TNP-self specific CTL after immunization in this way. Here the induced Ts cell as well as the mechanism by which it functions have been further examined. The suppression was seen to extend to allogeneic as well as TNP-self Ag, provided the Mlsd-tolerized animal was reexposed to Mlsd-bearing cells at the time of immunization for CTL. By transferring the Mlsd-induced suppression adoptively we have learned that the splenic suppressor cell bears Thy-1.2 as well as Lyt-1.1 Ag and inhibits the generation of CTL at the afferent limb. In addition, Mlsd-induced PEC of Mlsd-tolerized mice, but not of normal mice, mediated suppression of development of CTL in vivo. The active cells within the tolerized PEC have been identified as T cells and macrophages (M phi). Furthermore, PEC from mice tolerized to Mlsd suppressed generation of CTL directed toward TNP-self targets in vitro. T cells and M phi separated from PEC of Mlsd-tolerized mice achieved suppression best in culture when present together. In addition, Lyt-1+ splenic cells from tolerized but not normal mice cooperated to down-regulate CTL generation in vitro with peritoneal M phi from either tolerized or normal mice. Supernatants of 24- to 72-h cultures of PEC from tolerized mice were suppressive of CTL generation when incorporated at 40 to 50% of culture volume. Supernatants of T cells from tolerized PEC or spleen were suppressive in culture only when M phi from normal mice were also present. To achieve suppression dialyzed supernatants of M phi from tolerized mice could replace the M phi.(ABSTRACT TRUNCATED AT 400 WORDS)     

Deficiencies in suppressor T cell activity seen in patients with active systemic lupus erythematosus are due to the dilution of normally functioning suppressor T cells by nonsuppressor T cells

Concanavalin A (Con A)-activated T lymphocytes from patients with active, but not inactive, systemic lupus erythematosus (SLE) failed to express normal suppressor activity, regardless of the phenotype of CD4+ or CD8+. Con A-activated CD4+ or CD8+ T lymphocytes from the SLE patients and from normal controls were further separated into two populations, using the autologous erythrocyte rosette technique. One population very rich in cells capable of forming rosettes with autologous erythrocytes from the active patients showed the same degree of suppressor activity, as did that from normal controls; the CD4+ or CD8+ population poor in autorosetting cells derived from Con A-activated T lymphocytes from both the controls and patients did not express suppressor activity. Moreover, when autorosetting T cells from the active patients and nonrosetting cells from the same patients were mixed at a normal ratio (4:6), normal suppressor activity could be restored. It was notable that the frequency of autorosette-forming cells was markedly reduced in the Con A-activated T lymphocytes from the active, but not inactive, SLE patients, regardless of the phenotype of CD4+ or CD8+. These findings indicate the presence of a normally functioning suppressor T cell population in patients with active SLE. It seems that the lack of suppressor T cell function in patients with active SLE is due to the dilution of a few normal suppressor T cells by large numbers of nonsuppressor T lymphocytes.