Functional heterogeneity among human inducer T cell clones

Analysis of mouse CD4+ inducer T cells at the clonal level has established that a dichotomy among CD4+ T cell clones exists with regard to types of lymphokines secreted. Mouse T cell clones designated Th1 have been shown to secrete IL-2 and IFN-gamma, whereas T cell clones designated Th2 have been shown to produce IL-4 but not IL-2 or IFN-gamma. To determine if such a dichotomy in the helper inducer T cell subset occurred in man, we examined a panel of human CD4+ helper/inducer T cell clones for patterns of lymphokine secretion and for functional activity. We identified human T cell clones which secrete IL-4 but not IL-2 or IFN-gamma, and which appeared to correspond to murine Th2 clones. In marked contrast to murine IL-2 secreting Th1 clones which do not produce IL-4 or IFN-gamma, we observed that some human T cell clones secrete IL-2, and IFN-gamma as well as IL-4. Southern blot analysis indicated that these multi-lymphokine-secreting clones represented the progeny of a single T cell. IL-4 secretion did not always correlated with enhanced ability to induce Ig synthesis. Although one T cell clone which secreted IL-2, IL-4, and IFN-gamma could efficiently induce Ig synthesis, another expressed potent cytolytic and growth inhibitory activity for B cells, and was ineffective or inhibitory in inducing Ig synthesis. These results indicate that although the equivalent of murine Th2 type cells appears to be present in man, the simple division of T cells into a Th1 and Th2 dichotomy may not hold true for human T cells.     

Activated self-MHC-reactive T cells have the cytokine phenotype of Th3/T regulatory cell 1 T cells

In the present study, we show that human self-MHC-reactive (autoreactive) T cell clones are functionally distinct from Ag-specific T cell clones. Self-MHC-reactive T cells exhibited helper function for B cell Ig production when cultured with non-T cells alone, and they exhibit suppressor function when cultured with PWM- or rCD40 ligand (rCD40L)-activated non-T cells, whereas tetanus toxoid (TT)-specific clones exhibited only helper function in the presence of TT with or without PWM or rCD40L. Addition of neutralizing Abs to the cultures showed that the suppression was mediated by TGF-beta but not by IL-10 or IFN-gamma. The self-MHC-reactive clones also inhibited proliferation of primary CD4+ T cells and TT-specific T cell clones, but in this case the inhibition was mediated by both IL-10 and TGF-beta. In further studies, the interactions between self-MHC-reactive T cell clones and non-T cells that led to suppressor cytokine production have been explored. We found that prestimulation of non-T cells for 8 h with PWM or for 48 h for rCD40L results in non-T cells capable of inducing self-MHC-reactive T cell to produce high levels of TGF-beta and IL-10. In addition, these prestimulation times coincided with peak induction of HLA-DR and costimulatory B7 molecule (especially CD86) expression on B cells. Finally, addition of CTLA-4/Fc or blocking F(ab')2 anti-CTLA-4 mAb, plus optimally stimulated non-T cells, to cultures of self-MHC-reactive clones inhibited the induction of TGF-beta but not IL-10 or IFN-gamma production. In summary, these studies show that activated self-MHC-reactive T cells have the cytokine phenotype of Th3 or T regulatory cell 1 and thus may be important regulatory cells that mediate oral and peripheral tolerance and prevent the development of autoimmunity.     

T cells in B cell chronic lymphocytic leukemia. II. Lack of antigen-specific T suppressor cells and their progenitors

Nylon wool-purified T cells (Tn) of two patients with chronic lymphocytic leukemia of the B cell type were phenotyped and tested in various assays for antigen-specific T helper (Th), T suppressor effector (Tse), T suppressor precursor (Tsp), and T suppressor inducer (Tsi) function. Antigen-specific Th as well as Tsi activity could be effectively generated. Although phenotypically CD8+ T cells, carrying the receptor for the Fc part of IgG, were present in mononuclear blood cells and Tn fractions, no antigen-specific Tse cell activity could be induced. In addition, Tsp cells were found to be functionally absent. These findings are discussed in relation to a tumor-induced limited heterogeneity within the T suppressor (Ts) cell compartment.     

Contrasuppressor cells that break oral tolerance are antigen-specific T cells distinct from T helper (L3T4+), T suppressor (Lyt-2+), and B cells

Continuous gastric intubation of mice with the T cell-dependent antigen sheep erythrocytes (SRBC) leads to a state of systemic unresponsiveness to parenteral SRBC challenge, a state termed oral tolerance. The systemic unresponsiveness of mice rendered orally tolerant to SRBC, however, is converted to humoral immune responsiveness by adoptive transfer of effector T contrasuppressor (Tcs) cells. In this study, the authors have isolated and characterized the Tcs cell subset, from the spleens of orally immunized mice, which abrogates oral tolerance. This Tcs cell is a novel cell type, which can be separated from functional T suppressor (Lyt-2+) and T helper (L3T4+) cells, and the effector Tcs cell exhibits a Lyt-1+, 2-, L3T4- phenotype. Furthermore, contrasuppression is not mediated by B cells, including those of the Lyt-1+ phenotype. Adoptive transfer of splenic Lyt-1+, 2-, L3T4- T cells from C3H/HeJ mice given oral SRBC for 21 to 28 days and splenic Lyt-1+, 2-, L3T4- T cells of C3H/HeN mice orally immunized for a shorter interval abrogated oral tolerance. Furthermore, separation of Lyt-1+ T cells into L3T4+ and L3T4- subsets by flow cytometry resulted in Lyt-1+, L3T4+ T cells with helper but not contrasuppressor function, whereas the Lyt-1+, L3T4- T cell fraction abrogated oral tolerance even though it was without helper activity. This Tcs cell subset was also effective when added to cultures of tolerized spleen cells derived from SRBC-fed mice. The effector Tcs cells are antigen-specific, because Tcs cells from SRBC-immunized mice reverse tolerance to SRBC but not to horse erythrocytes (HRBC), and Tcs cells from HRBC-immunized mice reverse tolerance to HRBC but not to SRBC. When splenic T3 (CD3)-positive T cells (Lyt-1+, 2-, and L3T4-) were separated into Vicia villosa-adherent and nonadherent subpopulations, active contrasuppression was associated with the T3-positive and Vicia villosa-adherent T cell fraction. Thus, a distinct Lyt-1+, 2-, L3T4- T cell subset that contains a T3-T cell receptor complex, which can regulate oral tolerance, is present in spleens of orally immunized mice.     

Antigen-specific, MHC-restricted B cell activation by cell-free Th2 cell products. Synergy between antigen-specific helper factors and IL-4

Ag-specific and MHC-restricted Th clones of different Ag specificities and MHC haplotypes were tested for their ability to produce soluble factors capable of providing the signals required for B cell activation and IgG antibody production. Each of five Th clones tested generated significant helper activity in supernatants derived from coculture of the T cell clone with specific Ag and syngeneic APC. The same helper activity was detected in supernatants of clones stimulated with immobilized anti-CD3 antibody in the absence of Ag or APC. The secreted helper activity resembled the activity of the intact Th cells in that it was Ag-specific, carrier-hapten-linked and MHC-restricted. These T cell products functioned to activate only those B cells expressing MHC products which corresponded to the specificity of each Th clone. Thus, the specificity of the cell-free T cell product mimicked precisely that expressed by the intact Th cell and presumably mediated by the cell surface TcR. In addition to the apparent presence of specific helper factor in Th clone supernatants, a role for nonspecific lymphokines was also identified in these preparations. Although recombinant or purified IL-4 alone was not sufficient to stimulate hapten-primed B cells to secrete hapten-specific IgG antibodies, mAb specific for IL-4 blocked the induction of antibody secretion by Th cell supernatant. These results indicate that stimulation of B cells to produce hapten-specific IgG antibody requires at least two distinct signals: an Ag-specific T cell signal which is restricted by MHC products expressed on the B cells, and a nonspecific signal mediated at least in part by the lymphokine IL-4.     

IL-10 is produced by subsets of human CD4+ T cell clones and peripheral blood T cells.

Murine IL-10 has been reported originally to be produced by the Th2 subset of CD4+ T cell clones. In this study, we demonstrate that human IL-10 is produced by Th0, Th1-, and Th2-like CD4+ T cell clones after both Ag-specific and polyclonal activation. In purified peripheral blood T cells, low, but significant, levels of IL-10 were found to be produced by the CD4+CD45RA+ population, whereas CD4+CD45RA- "memory" cells secreted 5- to 20-fold higher levels of IL-10. In addition, IL-10 was produced by activated CD8+ peripheral blood T cells. Optimal induction of IL-10 was observed after activation by specific Ag and by the combination of anti-CD3 mAb and the phorbol ester tetradecanoyl phorbol acetate, whereas the combination of calcium ionophore A23187 and 12-O-tetradecanoylphorbol-13-acetate acetate was a poor inducer of IL-10 production. Kinetic studies indicated that IL-10 was produced relatively late as compared with other cytokines. Maximal IL-10 mRNA expression in CD4+ T cell clones and purified peripheral blood T cells was obtained after 24 h, whereas maximal IL-10 protein synthesis occurred between 24 h and 48 h after activation. No differences were observed in the kinetics of IL-10 production among Th0, Th1-, and Th2-like subsets of CD4+ T cell clones. The results indicate a regulatory role for IL-10 in later phases of the immune response.     

Intrahepatic, nucleocapsid antigen-specific T cells in chronic active hepatitis B

Hepatitis B core antigen (HBcAg)-specific T cell lines were established from hepatic lymphomononuclear cells derived from five patients with chronic active hepatitis B. No hepatitis B virus envelope antigen-specific cell lines were established. Proliferation in response to recombinant and native HBcAg, but not to native hepatitis B surface antigen containing the pre-S(2) region, confirmed the specificity of the five T cell lines. All cell lines represented mixed populations of CD4+ and CD8+ T cells. The CD4+ subset provided antigen-specific help to autologous B cells with respect to anti-HBc production and to CD8+ cells with regard to HBcAg-induced proliferation and suppressor activity. The CD8+ subset contained suppressor cells that selectively inhibited the proliferative response of autologous HBcAg-specific CD4+ cells without inhibiting CD4+ cells of unrelated specificity (tetanus toxoid). Moreover, the CD8+ cells were also capable of suppressing HBcAg-stimulated antibody to HBcAg production without showing inhibition of total immunoglobulin production stimulated by pokeweed mitogen. The cytotoxic potential of the T cell lines was established in a lectin-dependent cytotoxicity system; natural killer cytotoxicity was completely absent. Our data suggest that the lesional T cells present at the site of hepatocellular injury in chronic active hepatitis B are primarily HBcAg-specific lymphocytes of the helper and suppressor/cytotoxic phenotypes and that both are functionally competent.     

Thymic stroma-derived T cell growth factor (TSTGF). I. Functional distinction of TSTGF from interleukins 2 and 4 and its preferential growth-promoting effect on helper T cell clones

A recently established thymic stroma-derived cell line (TSCL) supported the growth of the interleukin (IL) 2-dependent, antigen-specific helper T cell (Th) clone, 9-16, without requirement for IL-2 and antigen, and such growth was substituted by a factor produced into cultures by this established TSCL. This substance, thymic stroma-derived T cell-growth factor (TSTGF), was capable of inducing the proliferation of various Th clones including 9-16 Th clone, but not of cytotoxic T cell clones. TSTGF-induced growth promotion was obtained in a dose-dependent fashion and in maintaining antigen specificity of Th clones. The culture supernatant from the TSCL did not contain detectable level of IL-1, IL-2, IL-3, IL-4, or interferon activity. The proliferation of 9-16 Th clone was stimulated by recombinant IL-2 and IL-4 as well as TSTGF, but not by IL-1, IL-3, or interferons. However, the proliferation of this Th clone by IL-2 or IL-4 was almost completely inhibited by anti-IL-2 receptor or anti-IL-4 monoclonal antibody, respectively, whereas TSTGF-induced growth of 9-16 Th clones was not affected by either type of antibody, demonstrating that TSTGF is functionally distinct from IL-2 and IL-4. In addition, TSTGF activity was also obtained from the culture supernatant of the primary thymic explant, which was freshly prepared. These results indicate that the primary thymic explant as well as an established TSCL produce factors capable of promoting the growth of helper but not cytotoxic type of T cells in the absence of T cell growth factors thus far defined.     

Clonal analysis of CD4+ T helper cell subsets that induce the monocyte procoagulant response

Monocyte procoagulant inducing factor (MPIF) is a T helper cell-derived cytokine that may play a collaborative role in the expression of cell-mediated immune responses. We have attempted to elucidate whether there is a relationship between MPIF-producing T cell clones and currently proposed subsets of murine T helper cells. A large collection of murine CD4+ T cell clones, both Con A-induced and long-term alloreactive clones, was generated for this study. Four subsets were identified among these T cell clones according to their cytokine secreting profiles: Th0 producing IL-2 and IL-4, Th1 producing IL-2, Th2 producing IL-4, and Tnull, a subset producing neither cytokine. The ability to produce MPIF was found to residue within the Th0 and Th1 subsets regardless of whether the clone was Con A-induced or alloreactive. Neither Th2 clones nor Tnull exhibited significant MPIF activity. In addition, a few instances of transition from Th0 to Th2 were associated with a concomitant loss of MPIF expression. The ability to secrete MPIF after stimulation was heterogeneous among Th0 and Th1 clones and did not correlate with IL-2 production by these clones. Our results that the Th1 subset produces MPIF are consistent with findings that the Th1 subset as well as the cytokine MPIF mediates DTH. Additionally, these results suggest that MPIF-producing Th0 clones may also play a role in cell-mediated immune responses.     

Amplification of suppressor inducer pathway with monoclonal antibody, anti-2H4, identifying a novel epitope of the common leukocyte antigen/T200 antigen

The 2H4 antigen, comprised of a 200/220-kDa glycoprotein of the leukocyte common antigen (LCA) family, is expressed on a suppressor inducer, but not a helper inducer subset of T4 cells. Earlier studies have demonstrated that the T4+2H4+ subset of cells maximally responded to the AMLR and this molecule has an important role in generated suppressor signals in AMLR/Con A-activated T cell systems. In the present study, we examined the effect of a series of monoclonal antibodies including anti-2H4 antibody on the initial activation of T4 cells in response to self-Ia antigens. We found that the addition of anti-2H4 antibody resulted in an augmentation of the proliferative response of T4 cells in AMLR, whereas other antibodies reactive with LCA/T200 antigens lacked this ability. Furthermore, anti-2H4 antibody enhanced both IL-2 production and IL-2R expression in this AMLR system. This enhancing effect was inhibited by anti-T3 antibody. Moreover, the suppressor inducer function of AMLR T4 cells was enhanced with anti-2H4 antibody by increasing the number of 2H4+ cells with high antigen density. Taken together, these results suggest that the 2H4 antigen may serve as an accessory structure for enhancing the activation of the T4+2H4+ suppressor inducer subset at initiation of cell triggering.     

Regulation of hapten-specific T-cell response. II. Functional analysis of helper T cells and cytotoxic T cells in animals suppressed by azobenzenearsonate (ABA)-specific suppressor T cells

The administration of azobenzenearsonate-modified syngeneic spleen cells (ABA-SC) intravenously induces a population of first order hapten-specific inducer suppressor T cells (Ts1), which downregulate various aspects of T-cell-mediated immune responses via a well defined suppressor-T-cell pathway. In this study, we investigated the effects of these suppressor cells on the generation of ABA-specific cytolytic T lymphocytes (CTL) and helper T cells (Th) in vivo. We found evidence for functional impairment of ABA-activated Th and ABA-specific CTL precursors (CTLp) in the suppressed animals by a number of different in vitro criteria. Functional analysis of ABA-specific CTLp and ABA-activated Th in suppressed animals revealed that ABA-specific Ts inhibit the generation of CTL by impairing the antigen-specific activation of Th, which may in turn, prevent the clonal expansion of antigen-specific CTLp. The significance of these findings in relationship to our understanding of the cellular interactions necessary for the generation of CTL and the mode of action and mechanisms of suppressor T cells is discussed.     

Regulation of myelin basic protein-specific helper T cells in multiple sclerosis: generation of suppressor T cell lines.

Suppressor T cell (Ts) lines specific for myelin basic protein (MBP)-reactive helper T cell (Th) clones were generated from two patients with multiple sclerosis (MS) following a primary culture of peripheral blood mononuclear cells (PBMC) with MBP and cyclosporine A (CsA). These suppressor T cell lines were maintained in culture by alternate stimulation with MBP and antigen-presenting cells (APC). The Ts lines expressed preferentially the CD4 phenotype (5/6 Ts lines tested) and exhibited potent antigen-specific suppressor activity on the proliferation of MBP-specific Th clones and not on the T cell lines with other antigen specificity. For some Ts lines, a Ts-to-Th ratio of 1 was sufficient to inhibit the proliferation of MBP-specific T cells by 90%. The suppressor T cells obtained were weakly responsive to MBP and required the presence of the autologous PBMC for proliferation. Furthermore, proliferation of these suppressor T cell lines was restricted by HLA-DR molecules (for CD4+ Ts lines) and HLA class I (for a CD8+ Ts line). The suppressor T cell lines generated and the techniques described in this study may be helpful in our understanding of the events involved in the immune regulation in MS and other autoimmune diseases.     

Long-term CD4+ memory T cells from the spleen lack MEL-14, the lymph node homing receptor.

We have characterized the surface phenotype and function of long-lived, Ag-specific memory CD4+ T cells generated in vivo by immunization with keyhole limpet hemocyanin (KLH). CD4+ T cells from the spleens of mice primed more than 2 mo previously with KLH, produced high levels of IL-2 and IL-3, and low levels of IL-4 and IFN-gamma in response to in vitro restimulation with specific Ag. The KLH-primed T cells mediated carrier-specific helper activity for the antibody production by NIP-primed B cells in secondary in vitro responses to NIP-KLH. Subsets of CD4+ T cells from KLH-primed mice were isolated on the basis of surface CD45RB (23G2) by magnetic separation and were examined for functional capacity in several assays of Ag-specific recall. Virtually all of the secretion of IL-2, IL-3, IL-4, and IFN-gamma in response to restimulation with Ag in vitro was associated with, and considerably enriched in, the CD45RB- subset of CD4+ T cells. Similarly, carrier-specific helper function and Ag-specific proliferation in vitro were also confined to the CD45RB-, CD4+ subset of T cells, confirming the previous association of this surface phenotype with memory Th cell activity. We also examined expression of the lymphocyte homing receptor, MEL-14 (gp90MEL), which is required for lymphocyte extravasation to peripheral lymph nodes and is present in high levels on naive T cells. MEL-14 positive and negative subsets of CD4+ T cells from long term KLH-primed mice were evaluated for Ag-specific memory function in terms of lymphokine production, Ag-induced proliferation, and helper activity. Each of these functions was associated exclusively with the MEL-14- subset of CD4+ T cells, which exhibited responses comparable to the CD45RB- subset. These data indicate that memory Th cell function in the spleen is contained within the MEL-14-, CD45RB- subset of CD4+ T cells and suggest that memory helper cells may have different patterns of recirculation from naive T cells.     

Inducer T-cell-mediated killing of antigen-presenting cells

L3T4+ inducer/helper T-cell clones, once activated by antigen-presenting cells (APC) expressing the appropriate Ia allele and antigen, autonomously kill their target APC. All 13 L3T4+ inducer T-cell clones tested demonstrated this cytolytic activity. In addition, 11 different target cells representing the three major APC types, namely, macrophages, B cells, and dendritic cells, were all sensitive to this cytolytic activity. Moreover, normal macrophages which were treated with interferon-gamma to increase Ia expression were also killed. These observations convincingly demonstrate that the cytolytic activity of L3T4+ inducer T-cell clones is a general phenomenon. In contrast to other reports, lysis of target APC could not be detected following 4-6 hr of incubation. Marginal lysis was observed after 9 hr and a 20-hr incubation period was required to achieve maximal killing. The kinetics of killing paralleled other parameters of T-cell activation such as IL-2 release and cell proliferation. Activation of T cells for cytolysis of APC requires the interaction of T-cell receptors with Ia and antigen. Monoclonal antibody to Ia, L3T4 and the T-cell receptor inhibited the cytolysis of APC. The ability to mediate nonspecific bystander killing was variable depending on both the T-cell clone and the target. The implications of these findings to immune regulation and autoimmunity are discussed.     

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.     

Characterization of the accessory cells involved in suppressor T cell induction

The ability of UV-treated splenic adherent cells (SAC) to induce T cell-mediated immunity and suppressor T cells was analyzed in the 4-hydroxy-3-nitrophenyl acetyl (NP) system. UV irradiation of 0.88 KJ/m2 decreased the capacity of NP-coupled SAC to induce delayed-type hypersensitivity (DTH) responses by about 50%. The ability of uncoupled UV-treated SAC to induce allogeneic DTH response was also imparied, indicating that UV-treated SAC are inefficient at inducing DTH in these systems. TS1 induction by UV-treated NP-SAC was evaluated TS1 induction by UV-treated NP-SAC was evaluated by using adherent cells that were subjected to the same dose of UV irradiation that impaired DTH induction. Intravenous administration of 10(3) or 10(4) UV-treated NP-coupled SAC induced TS1 cells with the same efficiency as non-UV-irradiated cells. The TS1 cells induced in this fashion were antigen specific. Furthermore, to establish that the antigen was not reprocessed by the host, I-J-mismatched, UV-treated NP-SAC were unable to induce TS1 cells. The population of antigen-presenting cells responsible for TS1 induction appear to express both I-A and I-J determinants. TS2 induction by UV-treated accessory cells was also analyzed. TSF1 inducer suppressor factor was pulsed onto graded numbers of either normal or UV-treated adherent cells. The same levels of antigen-specific suppression were induced with normal and UV-treated cells. Finally, TS3 induction by UV-treated NP-SAC was analyzed. UV-treated and normal NP-SAC (3 X 10(3] induced antigen-specific suppression of NP DTH responses. I-J-mismatched, UV-treated NP-SAC failed to induce suppression, suggesting that the hapten was not reprocessed by the host under these experimental conditions. The accessory cell population responsible for TS3 induction appears to express both I-A and I-J determinants. Thus, there are at least two functional distinctions between the antigen-presenting cells that induce immunity vs those that induce suppressor cells. First, UV treatment selectively impairs the antigen-presenting cells, which activate the positive limb of the immune response. Second, I-J determinants appear to be specifically associated with the SAC, which induce suppressor T cells. Although these criteria can be used to distinguish the accessory cells involved in suppressor cell pathways from those controlling helper T cell induction, there were no discernible phenotypic differences among the accessory cells that induce the TS1, TS2, and TS3 subsets.     

An antigen-specific helper T cell hybridoma produces an antigen-specific suppressor inducer molecule with identical antigenic fine specificity. Implications for the antigen recognition and function of helper and suppressor inducer T cells

We have previously described a T cell hybridoma, A.1.1, that responds to specific Ag (P18, a synthetic polypeptide of defined sequence) in the context of I-Ad by producing lymphokines. Herein we report that this cell also releases, into culture supernatants and ascites fluid, an Ag-specific activity that functions in the induction of suppression of anti-SRBC PFC responses. This suppressive activity requires a) Ag-non-specific accessory molecules from a T suppressor inducer factor, b) Ly-2+ T cells in the assay cultures, and c) the specific Ag (P18) conjugated to the SRBC in the assay cultures. The specificity of the A.1.1-derived activity was demonstrated by the absence of suppression in cultures containing SRBC, BSA-SRBC, or conalbumin-SRBC rather than P18-SRBC. Further, the A.1.1-derived activity bound to, and could be eluted from, P18 but not conalbumin. Using a panel of synthetic variant peptides, we have mapped the critical residues in P18 required for Ag/I-Ad induced activation of A.1.1. These peptides were tested for their ability to act as targets for the A.1.1-derived suppressive activity when conjugated to SRBC and added to assay cultures. All peptides capable of stimulating the A.1.1 T cells to release lymphokines were similarly effective in the suppressor assay. Thus, the recognition of Ag by the T cells and by the T cell-derived activity appeared to be identical. The A.1.1-derived molecule was found to be capable of inducing L3T4- T cells to act as suppressor T cells following culture. These suppressor cells were active in inhibiting anti-SRBC responses in the absence of P18 and bore the Ly-2 surface marker. Thus, it is likely that the function of this Ag-specific molecule is to induce Ly-2+ suppressor T cells and thereby cause the inhibition of the response. This function is distinct from that normally associated with helper T cells and may shed new light on the possible relationship between the cell surface T cell receptor for Ag and Ag-specific T suppressor inducer molecules.     

Evaluation of Ia+ tumor cell lines and peritoneal exudate macrophages as accessory cells: differential requirements for the activation of certain T cell functions

Several Ia+ (BC3A, TA3, D1B) or Ia-inducible (WEHI-3, P388D1) tumor lines were tested for accessory cell function for the activation of antigen-specific T cell proliferation and for the induction of T helper cells that help B cells in antibody production. All lines were able to induce antigen-specific T cell proliferation in an MHC-restricted way, but none activated T helper cells to soluble antigens under all conditions tested. In comparison, starch-induced peritoneal exudate macrophages induced T cell proliferation as well as T cell help. Some of the lines tested induced nonspecific suppressor cells that were Ly-2-positive and partially or completely inhibited antibody responses. The induction of suppressor cells, however, is not the reason for the failure of the tumor lines to activate T helper cells. These data indicate that antigen-specific T cell proliferation and helper activity do not necessarily correlate.     

A single cell analysis of TCR AV24AJ18+ DN T cells.

The T-cell receptor (TCR) BV gene of human TCR AV24+ double-negative (DN) T cells, a novel subset of natural killer (NK) T cells, was investigated by single-cell sorting and single-cell polymerase chain reaction (PCR) methods. Seven of eleven TCR AV24+ DN T-cell clones utilized TCR BV8, three BV9, and one BV6. Six of seven TCR AV24/BV8+ DN T-cell clones had identical TCR beta and alpha chains, indicating that they were the same clone. All three TCR AV24/BV9+ DN T-cell clones also demonstrated the same amino acids in the CDR3 region. These findings strongly suggest that the usage of TCR beta and alpha chains on TCR AV24+ DN T cells is extremely restricted, supporting the notion that these cells recognize highly limited T-cell epitopes on antigens. All TCR AV24+ clones expressed the NKR-P1A mRNA, and so were true NK T cells. IL-2 and IL-4 mRNAs were detected in all clones, suggesting that the majority of these cells were Th0-type T cells. Six clones overexpressed Fas-ligand (Fas-L) mRNA and Fas antigen was detected on all clones at the mRNA level. In conclusion, TCR AV24+ DN T cells might recognize restricted T-cell epitopes on antigens and function as Th0-type T cells, inducer cells to Th1- or Th2-type T cells (regulatory T cells), and as Fas-L-positive cytolytic T cells.     

The isolation and characterization of the human helper inducer T cell subset

Monoclonal antibody anti-4B4 was produced by fusing NS1 myeloma with spleen cells of a mouse immunized with Saguinus oedipus lymphocyte. This anti-4B4 antibody defines a 135-KD cell surface protein that is widely distributed throughout the hematopoietic system. More importantly, anti-4B4 is reactive with functionally unique human T cell subsets. Anti-4B4 antibody was reactive with approximately 41% of unfractionated T cells, 41% of T4+ inducer cells, and approximately 43% of T8+ cytotoxic/suppressor population. This antibody subdivided peripheral blood T4+ cells into two functionally distinct populations. The T4+4B4+ subset proliferates relatively poorly upon stimulation with Con A and autologous cell antigens (AMLR) but well on exposure to soluble antigens, and it provides a good helper signal for PWM-induced Ig synthesis. The T4+4B4- subset, in contrast, proliferates well to Con A stimulation and autologous cell antigen (AMLR) but relatively poorly to soluble antigen stimulation, and provides little help to B cells for PWM-induced Ig synthesis. The T4+4B4- subset is largely 2H4+ and functions as the inducer of the T8+ suppressor cells. Thus, the present results suggest that one can divide the human T4 population into two major subsets that are phenotypically and functionally distinct, the human helper inducer subset (T4+4B4+/H.I.) and its reciprocal population defined by anti-2H4, the suppressor inducer subset (T4+2H4+/S.I.).