Ts Clones Research Articles

Production of Phenylpropanoids, Naphthodianthrones and Antioxidant Status of Hypericum perforatum L. Transgenic Shoots

Fifteen transgenic shoot clones (TS A-TS O) regenerated from Hypericum perforatum hairy roots were evaluated for growth characteristics, phenylpropanoid, and naphthodianthrone production, as well as non-enzymatic and enzymatic antioxidant status. Transgenic shoots displayed higher biomass production and non-altered phenotypes compared to control shoots. Phenylpropanoid production in TS was not related to phenylalanine ammonia lyase activity indicating that alternative enzymes could be involved in the regulation of phenylpropanoid/flavonoid biosynthesis. The positive correlation between phenylpropanoids (phenolic acids, flavonoids, flavan-3-ols, tannins) and non-enzymatic antioxidant assays suggested that these compounds are the main contributors to the redox state and radical scavenging properties of TS clones. More importantly, enhanced naphthodianthrone production (hypericin, pseudohypericin and protopseudohypericin) in TS was associated with the number of leaf dark glands. Concerning the enzymatic antioxidant status of TS, guaiacol peroxidase was shown as the main enzyme that turns down H2O2 levels, while the upregulation of superoxide dismutase coincided with decreased O2•− production. The efficiency of antioxidant enzymes for O2•− and H2O2 neutralization decreased the level of MDA and consequently delayed lipid peroxidation in TS clones. Altogether, H. perforatum transgenic shoots could be proposed as a prospective biotechnological system for scale-up production of naphthodianthrones and phenylpropanoids with antioxidant properties.

Identification of Oncofetal Antigen/Immature Laminin Receptor Protein Epitopes That Activate BALB/c Mouse OFA/iLRP-Specific Effector and Regulatory T Cell Clones

During tumor development in mice and humans, oncofetal Ag/immature laminin receptor (OFA/iLRP)-specific Th1, CTL, and IL-10-secreting T (Ts) cells are induced. The presence of too many Ts or too few effector T cells appears to predict a poor prognosis. We established clones of OFA/iLRP-specific splenic Th1, CTL, and Ts cells from the OFA/iLRP+ MCA1315 fibrosarcoma-bearing BALB/c mice or from BALB/c mice vaccinated with 1 or 10 microg of rOFA/iLRP. The MCA1315 tumor cell-reactive T cell clones were characterized as to surface Ag phenotype, cytokine secretion profile, and specificity for OFA/iLRP presented by syngeneic splenic APC. OFA/iLRP-specific Th1 and Ts clones were established from all mice. OFA/iLRP-specific CTL could be established from all mice except for mice immunized with 10 microg of rOFA/iLRP. Analysis of the proliferation profile of the OFA/iLRP-specific clones to overlapping OFA/iLRP 12-mer peptides that spanned the OFA/iLRP protein sequence defined the epitopes to which the T cell clones responded. There was a similar spatial distribution of the epitopes to which the two types of CD8 T cell clones responded. The nonapeptide epitopes of the Ts clones were located between aa 36 and 147 of OFA/iLRP, while the epitopes of the CTL clones were located between aa 52 and 163. Even though the CTL and Ts epitopes shared part of the protein, all of the CD8 CTL epitopes were distinct and separable from those of CD8 Ts cells.

Regulation of FasL by NF-κB and AP-1 in Fas-dependent Thymineless Death of Human Colon Carcinoma Cells

Cell death due to thymine (dThd) deficiency, associated with the cytotoxic action of 5-fluorouracil in colon cancer, is regulated in thymidylate synthase-deficient (TS(-)) human colon carcinoma cells via the Fas (CD95, APO-1) death receptor. This was demonstrated by inhibiting the loss in clonogenicity of TS(-) cells by anti-FasL and in enhanced survival of TS(-) clones selected for resistance to Fas-mediated apoptosis, following dThd deprivation. During thymineless stress in TS(-) cells, Fas ligand (FasL) is expressed, and its promoter (hFasLPr) is activated. Transactivation of hFasLPr, dependent upon dThd deficiency, was inhibited following mutation of the binding sites for NF-kappaB or AP-1 and by preventing NF-kappaB or AP-1 activation, which inhibited expression of FasL and enhanced clonogenic survival in stable transformants expressing IkappaBalphaM or DN-MEKK, respectively. These results demonstrate the crucial roles for NF-kappaB and AP-1 in the regulation of FasL in Fas-mediated thymineless death of colon carcinoma cells.

A new immunobiological view of radiation-promoted lymphomagenesis

Whole-body irradiation produces T-cell leukaemias/ lymphomas (TCL) in some strains of inbred mice in an X-ray dose-related manner. Radiation biologists have related the rapid 'initiation' and early appearance of preleukaemic cells in these mice to unrepaired DNA damage inflicted by radiation. Following initiation, radiation-altered thymic differentiation fosters multi-step transformation changes in proto-oncogenes and suppressor gene expression in individual clones of noninvasive preleukaemia cells as they progress to malignancy. The malignant clones arising from small numbers of initiated preleukaemia thymocytes become fully transformed only after several more monthsto a year afterirradiation in those strains of mice which develop T-cell lymphomas. When the RFM mouse was subjected to sublethal whole-body X-ray, only 50% of the mice developed TCL by 6 months, yet nearly all developed preleukaemia thymocytes. The T-cell-mediated immune response of the irradiated host has never been substantiated to contribute to malignant TCL development. Until recently, X-ray-induced TCL were not known to carry common tumour rejection antigens TATA. However, several studies have revealed that both preleukaemia cells and fully malignant TCL express an immunogenic, common oncofoetal glycoprotein, termed 44 kD OFA. OFA-activated memory CD4 T H1 and CD8 TCTL T-effector cells in irradiated mice expressing OFA. As most irradiated RFM mice exhibit preleukaemia thymocytes yet only half develop tumours, this finding implicates active host T-cell effector responses in X-ray-initiated tumorigenesis. Further, the recent discovery of OFA-specific CD8 Ts clones in irradiated mice, which inhibited cytotoxicity of CD8 clones to OFA or TSTA, may explain which mice develop T-cell lymphomas.

Definition of a human suppressor T-cell epitope.

The quality of the response produced by regulatory or helper T (Th) cells presently receives much attention because of its possible implications for vaccine development and immunomodulation. Apart from cytokines and so-called costimulatory signals, antigens and the presenting major histocompatibility complex (MHC) molecules may play a role in determining the type of T-cell response generated toward antigens. To examine the role of antigen and/or HLA in control of T-cell subset activation, we have studied a special case, namely CD4+ suppressor T (Ts) cells in leprosy. Mycobacterium leprae-induced Ts cell clones have been previously isolated from peripheral blood and skin lesions of lepromatous leprosy patients and were shown to specifically down-regulate mycobacterium-specific Th cell responses. Despite considerable effort, the antigens recognized by these Ts cells have thus far not been identified. Here we report that all HLA-DR2-restricted CD4+ Ts cell clones derived from a lepromatous leprosy patient recognize an epitope that maps between the amino acid residues 439 and 448 of the mycobacterial hsp65. The peptide was presented to these Ts cells by HLA-DRB1*1503, a recently discovered HLA-DR2 variant. Non-suppressor T-cell clones derived from the same patient recognized antigens other than the hsp65 and were also stimulated by other HLA-DR2 variants. In independent cloning experiments peptide 435-449 and recombinant hsp65 induced exclusively Ts cells in this lepromatous leprosy patient. The Ts clones recognizing this particular epitope were derived from at least seven different progenitors, as they expressed different T-cell receptor alpha and beta chains. Thus, our data indicate that a specific peptide-HLA class II combination may exclusively activate Ts cells.

Analysis of cytokine production by Mycobacterium-reactive T cells. Failure to explain Mycobacterium leprae-specific nonresponsiveness of peripheral blood T cells from lepromatous leprosy patients.

Recent analyses of antimycobacterial T cells clones from a small number of individuals indicate that mycobacteria preferentially induce Th cells that produce high levels of IFN-gamma and no or little IL-4 in Mycobacterium leprae-resistant tuberculoid leprosy (TT) patients and healthy subjects, whereas in one study M. leprae-induced Ts clones from polar lepromatous leprosy (LL) patients showed a reciprocal cytokine secretion profile and mediated their suppressive activity via the release of high levels of IL-4. We have evaluated these findings in peripheral blood T cells from a larger panel of TT and LL patients as well as healthy individuals. Mycobacterium-reactive T cell lines generated from the PBMC of these individuals were tested for cytokine secretion and proliferative capacity in response to M. leprae, Mycobacterium tuberculosis, and various individual mycobacterial Ag. The lepromatous pole of the leprosy spectrum was additionally investigated by analyzing the cytokine-secretion profile of M. leprae-induced (suppressor) T cell clones as well as primary ex vivo PBMC. All T cell lines from healthy individuals and TT patients responding to M. leprae, M. tuberculosis, or individual Ag, produced high levels of IFN-gamma and TNF-alpha but little or no IL-4 and IL-6. At the lepromatous pole, T cell lines failed to proliferate upon stimulation with M. leprae but in some cases produced significant levels of IFN-gamma. No IL-4 or IL-6 secretion was observed in response to M. leprae. These lines displayed strong proliferation and Th1-like cytokine production upon stimulation with M. tuberculosis. Similarly, stimulation of primary PBMC from LL patients with M. leprae or M. tuberculosis resulted in the release of IFN-gamma but no detectable IL-4 production. Control tetanus toxoid-reactive T cell lines from the same individuals instead produced large amounts of IL-4 and low levels of IFN-gamma. The analysis of M. leprae-induced T cell clones, including those with known suppressive activity, revealed that all lepromatous T cell clones produced large amounts of IFN-gamma. Most of these clones released no or little IL-4, but some clones produced higher levels of IL-4 in addition to IFN-gamma. Most clones tested produced IL-10 as well. The suppressor activity of suppressor T cell clones could not be inhibited by a neutralizing anti-IL-4 antibody and only in one case by neutralizing anti-IL-10 antibody. Anti-IL-4 and anti-IL-10 could not overcome the M. leprae-specific unresponsiveness observed in primary PBMC from LL patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Distinction of mouse CD8+ suppressor effector T cell clones from cytotoxic T cell clones by cytokine production and CD45 isoforms.

Mouse CD8+ T cell clones could be subdivided into two subgroups by the pattern of cytokine production and CD45 isoforms that exactly corresponded with the functionally assigned cytotoxic (CTL) and suppressor (Ts) subsets. All the Ts clones invariably produced IL-10 after stimulation with immobilized anti-CD3, whereas none of CTL clones expressed mRNA of IL-10 by the same stimulation. All the CTL clones were positive for CD45RA Ag, a product of exon 4 of CD45 gene, whereas none of the Ts clones were positive for this Ag. The results are analogous to the situation within CD4+ T cell clones in which Th1 and Th2 subtypes can be distinguished by CD45 isoforms and cytokines they produce. Ts clones could suppress the proliferative responses of both Th1 and Th2 type CD4+ clones by the production of two different cytokines, IL-10 and IFN gamma, respectively, but none of the CTL clones could suppress the proliferation of CD4+ T cells. The participation of an additional nonspecific molecule in the suppression of antibody response is suggested.

Suppressor T Lymphocyte Dysfunction Is Important in the Pathogenesis of Autoimmune Thyroid Disease: A Perspective

Over the past decade, there was considerable scepticism regarding the existence, nature, and function of suppressor T (Ts) lymphocytes, particularly antigen-specific Ts lymphocytes. The receptors of putative antigen-specific murine Ts hybrids revealed that most either lacked the T cell receptor beta (TCR beta) chain or had failed to properly rearrange them and thus could not make a functional receptor. Moreover, studies of the DNA of sequences of the MHC I-J region (considered an important determinant for suppression) fail to show evidence for a unique open reading frame capable of encoding the I-J restriction element. In addition, no molecular basis for suppression had been characterized. These criticisms have now been satisfactorily resolved and the pendulum is swinging in favor of participation by antigen-specific Ts lymphocytes in immune tolerance. Ts lymphocytes have now been cloned and cell lines have been found to be idiotypic-specific. Indeed, specific Ts lymphocytes in the periphery have been shown to prevent the appearance of autoreactivity. It is, therefore, legitimate to consider their control as part of the mechanisms of maintaining the integrity of the immune system. Moreover, all human primary Ts clones and the majority of murine Ts clones have now been shown to rearrange TCR alpha beta. Ts lymphocytes are induced by antigen via antigen-presenting cells (APCs), and may do so in the context of MHC class II antigens, a concept not previously accepted. Moreover, there is now considerable evidence that such cells many well be involved in various autoimmune disease states.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of stable CD8 + suppressor T cell clones and the analysis of their suppressive function

Stable CD8 + suppressor T cell (Ts) clones were established by a relatively simple method. Keyhole limpet hemocyanin (KLH)-primed spleen cells from C3H mice were depleted of B cells and CD4 + T cells by panning and cytotoxic treatment, and the resulting CD8 + T cells were periodically stimulated with antigen and irradiated syngeneic spleen cells followed by manifestation interleukin-2 (IL-2) containing medium. T cell clones with a definite suppressor function were established by limiting dilution. They were defined as classical effector type Ts of CD8 + phenotype as they had constant and definite suppressor functions in antigen-induced T cell proliferation and specific antibody response against T cell-dependent antigens without detectable cytotoxic activity against both antigen presenting cells (APC) and helper T cells (Th). They showed no helper activity for B cells and produced no detectable helper type lymphokines such as IL-2 and IL-4. CD8 + Ts clones were able to inhibit the antigen-induced IL-2 production of normal and cloned T cells. Their suppressive activity was antigen-nonspecific and major histocompatibility complex-unrestricted. CD8 + Ts clones were also able to suppress the proliferative response of Th clones induced by immobilized anti-T cell receptor (TcR) and anti-CD3 mAbs but not the response induced by concavalin A (ConA) and IL-2. All the CD8 + T cell clones established independently utilized the TcR Vβ8 gene. Syngeneic antigen presenting cells could induce proliferation of these CD8 + clones, which was blocked by anti-CD8 and anti-I-A k monoclonal antibody (mAb) but not by anti-class I mAbs. The stimulation of CD8 + T3 clones with immobilized anti-CD3 resulted in the release of a suppressor factor(s) that potently inhibited the antigen-induced proliferation of CD4 + Th clones and the in vitro secondary antibody formation.

Analysis of suppressor T cells induced by donor-specific transfusion (DST): establishment of a human T cell hybridoma producing an antigen-nonspecific suppressor factor

Formation of suppressor T cells (Ts) induced by donor-specific transfusion (DST) is one of the most commonly suggested mechanisms for the beneficial effect of DST. In this study, we established a human T cell hybridoma derived from the peripheral blood lymphocytes (PBL) of a DST-treated patient, which produced an antigen-nonspecific suppressor factor. Post-DST PBL were fused with an azaguanine-resistant mutant of a human T cell leukemia cell line, CCRF-CEMAG. After selection and cloning, we established one clone producing the mixed lymphocyte reaction (MLR) inhibitory factor (C524: 18%–43 % suppression). Suppressive activity of the supernatant obtained from C524 after activation by PHA was highly augmented (64%–88 % MLR suppression). This factor inhibited MLR dose-dependently in an antigen-nonspecific and HLA non-restricted manner. These results indicated that Ts clones could be generated in patients receiving DST and that the immunoregulatory factors produced by activated clones may play a role in the prolongation of renal allograft survival.

Molecular Events in the T Cell‐Mediated Suppression of the Immune Response

To understand the mechanism of T cell-mediated suppression, we have established a number of suppressor T cell (Ts) clones of both CD4+ and CD8+ phenotypes that exert a definite suppressive effect on antigen-induced proliferative response of normal and cloned CD4+ helper T cells (Th). When an antigen-activated Ts clone was added to Th clones that were subsequently stimulated with antigen and APC, the increase of intracellular Ca2+ in the latter was greatly inhibited. The suppression was unidirectional where Ts suppressed Th but not vice versa. A Ts clone could not suppress other Ts clones. Exactly the same suppression of Ca2+ response could be induced by the treatment of T cells with an anti-I-J antibody. The anti-I-J suppressed the Ca2+ response of Th clones induced by antigen-pulsed APC and anti-TcR alpha beta antibody, whereas the responses to anti-CD3 and Con A were not inhibited. The difference in the effect of anti-TcR alpha beta and anti-CD3 suggests that the suppression is caused by a functional uncoupling of TcR alpha beta and CD3. The stimulation of Ts clones with anti-CD3, on the other hand, induced a unique suppressor factor that potently inhibits the antigen- and anti-TcR induced proliferation of CD4+ Th clones.

Human suppressor T cell clones lack CD28

Previously we showed that certain T cell lines and clones from a lepromatous leprosy patient displayed a dose-dependent suppression of the proliferation of autologous T cells to Mycobacterium leprae (M. leprae) but not mitogen or an unrelated antigen. The latter cells were also cloned and did not display this suppressive activity, were CD4+ and proliferated vigorously to M. leprae presented by autologous HLA-DR molecules. We shall refer to these cells as T helper (Th) cells. Most of the suppressive T cell clones (Ts) were also CD4+ and also proliferated to M. leprae presented by HLA-DR, but much less strongly than Th cells. In this study we report on our search for (a) the mechanism of this apparently antigen-specific suppression by T cells, and (b) a possible phenotypic difference between Th and Ts clones. The two main conclusions are that Ts clones possess a lytic machinery, but that M. leprae-specific suppression and cytotoxicity can be clearly dissociated, and that the only phenotypic difference between Th and Ts is the presence of the CD28 marker on Th and its absence on Ts clones.

Differences in surface phenotype and mechanism of action between alloantigen-specific CD8+ cytotoxic and suppressor T cell clones.

We have previously demonstrated that fresh CD8+ T cells proliferate in response to autologous, alloantigen-primed CD4+ T cells, and differentiate into Ts cells, which inhibit the response of fresh T cells to the primary allogeneic stimulator cell but not irrelevant stimulators. Although such Ts do not have discernible cytolytic activity, like classical cytotoxic T cells (Tc) they express CD3 and CD8 on their surface and function in a class I MHC-restricted manner. Our study was an attempt to compare the surface phenotype and mechanism of action of Ts and Tc clones derived from the same individual. Ts clones were generated from donor JK by repeated stimulation of CD8+ T cells with an autologous CD4+ T inducer line specific for an allogeneic lymphoblastoid cell line (LCL). These clones were noncytolytic for either the inducer line or the allogeneic stimulator LCL. Tc clones, generated by direct stimulation of JK CD8+ T cells with the same allogeneic LCL, mediated potent, alloantigen-specific cytolysis. All Tc clones were alpha, beta TCR+, CD3+, CD4-, CD8+, CD11b-, and CD28+. Ts clones were also alpha, beta TCR+, CD3+, and CD8+, but in contrast to Tc clones, Ts clones were CD11b+ and CD28-. When added to MLR both Ts and Tc clones inhibited the response of fresh JK CD4+ T cells to the original but not irrelevant allogeneic LCL. However, Ts inhibited the response of only those CD4+ T cells that shared class I)MHC determinants with the Ts donor, whereas Tc inhibited the response of CD4+ T cells from all responders, regardless of HLA type. Pretreatment of Ts clones with mAb to CD2, CD3, or CD8 blocked suppression, whereas similar pretreatment of Tc clones blocked cytotoxicity in 4-h 51Cr release assays but had no effect on Tc-mediated suppression of the MLR. These results suggest that both Ts and Tc clones can inhibit the MLR but they do so through different mechanisms. Moreover, the maintenance of distinct surface phenotypes on these long term clones suggests that Ts may be a distinct sublineage of CD8+ T cells rather than a variant of CD8+ Tc.

Unidirectional inhibition of early signal transduction of helper T cells by cloned suppressor T cells

Early intercellular events occurring in cloned T helper (Th) cells following interaction with cloned T suppressor (Ts) cells were studied by stopped-flow fluorometry. It was found that the increase of intracellular Ca2+ ([Ca2+]i) in major histocompatibility complex (MHC)-restricted Th clones induced by the stimulation with antigen and antigen-presenting cells (APC) is inhibited by the incubation with antigen-activated Ts clones. Optimal suppression required that the two cells recognize antigen on the same APC, although the restriction element for recognition could be different. There was an absolute requirement for recognition of the same antigen by these two cell types. The inhibitory effect was unidirectional in that Ts clones could inhibit the increase of [Ca2+]i of Th clones but not vice versa. Ts clones could not suppress the [Ca2+]i response of other Ts clones. If Th and Ts clones do not share the same MHC restriction specificity, a longer co-incubation time for activation of Ts is required for the inhibition of the [Ca2+]i response of the Th clone, suggesting the presence of a non-specific suppressive mediator that selectively acts on Th.

On the mechanism of human T cell suppression

Previous evidence from several laboratories suggests that CD8+ T suppressor cells may be important regulatory elements governing specific unresponsiveness of lepromatous lepromatous leprosy patients to M.leprae. To analyse the mechanism of suppression, CD8+ Ts clones were established from lesions and peripheral blood of lepromatous patients and tested for ability to suppress antigen-responsive CD4+. Th clones or PBL. Suppression required induction by specific M.leprae antigen, but was effected in an antigen-non-specific fashion. The Ts clones failed to exhibit cytotoxicity of four antigen-exposed MHC-matched target cells: (i) an ori-SV40 transformed macrophage line; (ii) EBV transformed B cell lines; (iii) primary macrophages; and (iv) M.leprae responsive CD4+ cells. The possibility that Ts clones induce functional inactivation of CD4+ clones in vitro was investigated. M.leprae-responsive CD4+ clones were preincubated with Ts CD8+ clones, APC, and antigen for 16 h, after which the CD8+ cells were removed. The CD4+ clones with M.leprae and APC remained unresponsive to restimulation with APC and antigen for at least 10 days, although they responded to IL-2. Addition of IL-2 to the pre- or post-incubation cultures neither prevented the induction of unresponsiveness, nor reversed it. Earlier models of tolerance have suggested that receptor occupancy in the absence of second signals induces tolerance in B and T cells. Under conditions in which antigen responses of Th clones were HLA-DR-restricted, the Ts clones were able to suppress the response of DR mismatched Th clones. Thus, the effect of the Ts cells, like mechanisms requiring antigen presentation without a second signal, appears to be induction of clonal anergy in Th cells, perhaps by a novel mechanism.

T-cell receptors of human suppressor cells.

Cells which can suppress the immune response to an antigen (TS cells) appear to be essential for regulation of the immune system. But the characterization of the TS lineage has not been extensive and many are sceptical of studies using uncloned or hybrid T-cell lines. The nature of the antigen receptor on these cells is unclear. T cells of the helper or cytotoxic lineages appear to recognize their targets using the T-cell receptor (TCR) alpha beta-CD3 complex. TCR beta-gene rearrangements are also found in some murine and human suppressor cell lines but others have been shown not to rearrange or express the beta-chain or alpha-chain genes. We previously established TS clones derived from lepromatous leprosy patients which carry the CD8 antigen and recognize antigen in the context of the major histocompatibility complex (MHC) class II molecules in vitro. We here report the characterization of additional MHC-restricted TS clones which rearrange TCR beta genes, express messenger RNA for the alpha and beta chains of the TCR and express clonally unique CD3-associated TCR alpha beta structures on their cell surface but do not express the gamma chain of the gamma delta TCR on the cell surface. We conclude that antigen recognition by at least some human CD8+ suppressor cells is likely to be mediated by TCR alpha beta heterodimers.

Characterization of the Ia antigens involved in suppressor T cell generation in the rat.

The WRC rat, an intra-class II recombinant strain (RT1.B beta nB alpha aD alpha, beta a), was used to study the relative roles of the two class II loci in mixed lymphocyte reaction (MLR) proliferation and suppressor T cell (Ts) generation. Both MLR proliferation and Ts generation were noted in cultures of WRC with DA (RT1a) stimulator cells. In contrast, cultures of WRC with BN (RT1n) stimulator cells proliferate but do not generate significant amounts of Ts. The data suggest that RT1.B beta incompatibility is important in the generation of Ts in the WRC rat. Suppressor cells generated in cultures of WF (RT1u) with WRC stimulator cells potently suppressed a WF + WRCx test MLR, with less suppression when tested against either the WF + DAx or WF + BNx MLRs. The latter experiments suggest that Ts clones may be produced to either class II subregion, and therefore that MLR proliferation and Ts induction are not necessarily linked, but vary with particular genotypes. The current lack of other rat intra-class II recombinant strains precludes assignment of suppressor induction/activation to a single locus.

T cell regulation of b cell activation. Cloned Lyt-1+2-T suppressor cells inhibit the major histocompatibility complex-restricted interaction of T helper cells with B cells and/or accessory cells.

The present studies have identified cloned Lyt-1+2- T suppressor (Ts) cells that are both antigen specific and major histocompatibility complex (MHC) restricted in their activation requirements and that function to regulate the MHC-restricted activation of B cells by T helper (Th) cells. ParentA-restricted Ts clones suppressed, in antigen-specific fashion, the responses generated by (A X B)F1 Th cells cooperating with parentA (B plus accessory) cells, but did not suppress responses by the same (A X B)F1 Th cell population cooperating with parentB (B plus accessory) cells. Moreover, responses of (A X B)F1 leads to parentA Th cells and (A X B)F1 (B plus accessory) cells were suppressed by parentA-restricted Ts clones but not by parentB-restricted Ts clones. Thus, these findings suggest that the cloned Ts cells that have been characterized here function by specifically inhibiting the MHC-restricted interaction between Th cells and B and/or accessory cells. It was further demonstrated in experiments using cloned Th and Ts populations that these Lyt-1+2-Ts cells act not simply as inducers of suppressor but rather function in a restricted fashion as effector cells in the suppressor pathway.