Class II antigen-specific murine cytolytic T lymphocytes (CTL). I. Analysis of bulk populations and establishment of Lyt-2+L3T4- and Lyt-2-L3T4+ bulk CTL lines

Murine allogeneic cytolytic T lymphocytes (CTLs), including long-term bulk CTL lines, were induced in I-region-incompatible combinations of strains in vitro in order to study the phenotypes of class II major histocompatibility complex (MHC) antigen-specific CTLs, as well as the possible functional involvement of accessory cell interaction molecules such as Lyt-2 and L3T4. This report shows that class II-specific allogeneic CTL populations consist of two types of T cells. Lyt-2+L3T4- (2+4-) and Lyt-2-L3T4+ (2-4+), in variable proportions depending on the strain combination, that in vitro bulk CTL lines with each of these phenotypes can be established, that the killing function of 2-4+ CTL is sensitive to the blocking effect of anti-L3T4 antibodies, suggesting functional involvement of this molecule in the CTL-target interaction, that anti-Lyt-2 antibodies fail to block killing by 2+4- cells, suggesting that such CTLs do not utilize this molecule in their killing function, and that while I-A-specific CTLs of both phenotypes are detectable, 2-4+ cells could not be detected among I-E-specific CTL populations.     

Lyt-2+ suppressor T cells are resistant to anti-Lyt-2 antibody blocking

Lyt-2 molecules play a role in antigen recognition by cytotoxic T lymphocytes (CTL). In an attempt to determine whether Lyt-2 molecules play a similar role in suppressor T cell (Ts) functions, the effect of anti-Lyt-2 antibodies on Ts generation and effector activity was studied. Allospecific Ts were induced in allogeneic mixed lymphocyte cultures (MLC). Anti-Lyt-2 antibodies added to MLC in the absence of complement abolished CTL generation, but had no effect on concomitant induction of Ts. In a different experimental system, allospecific Ts were induced in cultures treated with pyrilamine, which blocks generation of CTL but allows differentiation of Ts. The addition of anti-Lyt-2 antibodies to pyrilamine-treated MLC resulted in unaffected induction of Ts. It was further demonstrated that the effector activity of Ts was as resistant to anti-Lyt-2 antibodies as their induction, in contrast to the cytolytic activity of CTL, which was inhibited by the same antibodies. Ts in the present experimental system were Lyt-2+ antigen-specific cells. It therefore appears that Lyt-2 molecules, although expressed on both CTL and Ts, are involved in CTL activity, but do not play an essential role in Ts function.     

Mouse alloantibodies capable of blocking cytotoxic T cell function. V. The majority of I region-specific CTL are Lyt-2+ but are relatively resistant to anti-Lyt-2 blocking

In an attempt to solve the conflict concerning the correlation between the Lyt-2 phenotype of T cells subsets and the type of the MHC antigens involved in the recognition by T cells, class 2 (I region) antigen-specific CTL were studied for their Lyt phenotypes and the sensitivity to the blocking effects of anti-Lyt-2,3 antibodies. To avoid contamination by CTL to class 1 antigens such as Qa antigens, A.TH anti-A. TL attackers and A.TH anti-A attackers were tested on LPS blasts of the A strain and the A.TL stain, respectively. By using these combinations, it was shown that the majority of I region-specific killers were Thy-1+, Lyt-1+23+. Specific target cell lysis by these cells were, however, found to be far less sensitive to the blocking effects of various monoclonal antibodies to the Lyt-2,3 antigens than conventional class 1-specific CTL. This conclusion was drawn by directly comparing the sensitivity of the I region-specific and K region-specific killing by identical numbers of the same attacker cells (A.TH anti-A). No significant difference was seen between the primary and the hyperimmune CTL. Lyt-2-, Thy-1+ killer cells with I region specificity could be induced when Lyt-2-depleted A.TH responder cells were stimulated in vitro. Such Lyt-2- killer cells were not induced to the H-2K alloantigen.     

Mechanism of recovery from acute virus infection: treatment of lymphocytic choriomeningitis virus-infected mice with monoclonal antibodies reveals that Lyt-2+ T lymphocytes mediate clearance of virus and regulate the antiviral antibody response.

After intravenous infection of mice, lymphocytic choriomeningitis virus multiplied in spleens and livers, attaining highest concentrations on days 4 to 6. The subsequent clearance was as rapid, and 8 to 10 days after inoculation, infectivity was usually below detectability. During the effector phase of virus elimination, both cytotoxic T-cell (CTL) activity and the number of cells producing antiviral antibodies were high. Monoclonal antibodies directed against T lymphocytes and T-lymphocyte subsets were inoculated once intravenously 5, 6, or 7 days after infection of the animals, and the effects on antiviral immune responses, as well as on elimination of virus from the organs, were determined. Treatment with anti-Thy-1 and anti-Lyt-2 antibodies blocked elimination of the virus and profoundly diminished the activity of spleen CTLs but reduced the antibody response partially (anti-Thy-1) or increased it (anti-Lyt-2). In contrast, treatment with the anti-L3T4 antibody had essentially no effect on either virus elimination or CTL response but abolished antibody production. We conclude that Lyt-2+ (cytotoxic-suppressive) T lymphocytes are needed for elimination of the virus and also regulate the humoral response but that antiviral antibodies are not essential for control of the infection.     

Immunotherapy of lymphoid and nonlymphoid tumors with monoclonal anti-Lyt-1 antibodies

Monoclonal antibodies against the T cell differentiation antigen Lyt-1 were effective in the therapy of transplanted mouse tumors. A possible mechanism whereby anti-Lyt-1 antibodies directly bind and affect the tumor cells was excluded by the following findings: a) growth of lymphoid and nonlymphoid tumors (which lack Lyt-1 antigen) was affected by anti-Lyt-1 antibodies; and b) the curative effect of passively administered anti-Lyt-1 anti-bodies was abrogated in mice depleted of T cells, supporting a mechanism whereby host Lyt-1+ cells were involved in tumor therapy. Treatment with anti-Lyt-1 antibodies was not accompanied by depletion of Lyt-1+ cells from lymphoid organs, indicating that the administered antibodies altered Lyt-1+ cell functions without affecting their frequency. In view of the in vitro enhancing effects of anti-Lyt-1 antibodies on a variety of immune responses (including lymphokine secretion and generation of cytotoxic T cell), it is suggested that the potentiation of Lyt-1+ cell activity by passively administered anti-Lyt-1 antibodies results in tumor rejection.     

The mechanism of anti-Lyt-2 inhibition of antibody-directed lysis by cytotoxic T lymphocytes

Bifunctional antibodies specific for a determinant within the T cell receptor (TcR) complex of cytotoxic T lymphocytes (CTL) and a determinant expressed on the surface of the target cell will effectively mediate cytolysis. In such a lytic system anti-Lyt-2 antibody can block cytolysis. We have observed that the amount of inhibition varies considerably from clone to clone and surprisingly correlates well with inhibition of conjugate formation as mediated by bifunctional antibody. This implies that inhibition of antibody-mediated killing occurs as the result of reduction of the avidity of the effector cell for its target, the same mechanism responsible for inhibition of receptor-mediated lysis by anti-Lyt-2. In light of the similarity between the mechanism of inhibition by anti-Lyt-2 of receptor-mediated and antibody-mediated cytolysis, we compared the ability of anti-Lyt-2 to inhibit cytolysis in these two different assay systems by using a number of different CTL clones. Whereas the majority of secondary CTL clones (presumed to have high affinity TcR) are inhibited equally in both assay systems, most primary CTL (presumed to have low affinity TcR) are more susceptible to inhibition by anti-Lyt-2 in their receptor-specific than their antibody-directed cytolysis. These results, taken together with an apparent correlation between the amount of Lyt-2 expressed on the cell surface and susceptibility to inhibition, suggest anti-Lyt-2 may block CTL function by sterically inhibiting mobility of the TcR complex.     

Antibodies to the L3T4 and Lyt-2 molecules interfere with antigen receptor-driven activation of cloned murine T cells

When L3T4+ cloned murine helper T lymphocytes (HTL) are stimulated with antigen or immobilized anti-T cell receptor (TCR) monoclonal antibodies (mAb) at concentrations which are optimal for proliferation, anti-L3T4 mAb inhibits activation as measured by proliferation and lymphokine production. Under similar conditions, IL 2-independent proliferation of Lyt-2+ cloned murine cytolytic T lymphocytes (CTL) stimulated by anti-TCR mAb is inhibited by anti-Lyt-2 antibodies. Proliferation of cloned HTL and CTL cells stimulated by IL 2 is not affected by the anti-L3T4 and anti-Lyt-2 mAb. The inhibition of TCR-induced activation of the T cell clones is not due to interference with the binding of the anti-TCR mAb. Stimulation of the TCR has been proposed to induce lymphokine secretion and proliferation by T cells through a pathway involving the activation of protein kinase C and the stimulation of an increase in the concentration of intracellular free calcium. However, proliferation of T cells stimulated by PMA (which activates protein kinase C) plus the calcium ionophore A23187 (which increases the concentration of intracellular free calcium) is not affected by mAb reactive with the Lyt-2 or L3T4 structures. If TCR stimulation does indeed activate T cells by activating protein kinase and increasing intracellular free calcium, then our data suggest that anti-L3T4 and anti-Lyt-2 mAb inhibit TCR-driven proliferation at some step before the activation of protein kinase C and the stimulation of a rise in intracellular free calcium concentration. Our results suggest that anti-L3T4 and anti-Lyt-2 mAb interfere with early biochemical processes induced by stimulation of the TCR. In HTL, which proliferate via an autocrine pathway, anti-L3T4 mAb appears to inhibit proliferation by interfering with signaling events involved in lymphokine production. Inhibition of IL 2-independent proliferation of Lyt-2+ cells by anti-Lyt-2 mAb appears to occur by a different mechanism. The precise molecular basis for the interference of each cell type has not yet been characterized.     

Bone marrow transplantation across major histocompatability barriers in mice. III. Treatment of donor grafts with monoclonal antibodies directed against Lyt determinants

We studied the effect of eliminating T cells from donor grafts of mice in a system in which bone marrow was transplanted across major histocompatibility barriers. BALB/c bone marrow (added as a source of hematopoietic stem cells) combined with equal volumes of spleen cells (added as a source of GVHD-promoting cells) was pretreated in vitro with monoclonal anti-Lyt-1.2 or Lyt-2.2 plus absorbed rabbit complement before injection into C57BL/6 total-body-irradiated recipients. Functional activity of anti-Lyt monoclonal antibodies was determined in CML assay. Treatment with anti Lyt-1.2 plus C did not have any anti-stem cell activity, as measured by CFU-S assay, and protected recipients from the onset of lethal GVHD. Treatment with Lyt-2.2 plus C also did not reduce CFU-S; however, mice receiving treated marrow did develop GVHD and were all dead by 2 mo, as were untreated control mice. Surviving "anti-Lyt-1.2 + C chimeras" demonstrated a high percentage of donor mononuclear cells in their peripheral blood. Similar results were obtained when C3H/HeN donor BMS was treated with monoclonal anti-Lyt-1.1 plus C and injected into C57BL/6 recipients. These findings show that monoclonal antibodies directed against determinants unrelated to Thy-1 can eliminate T cells in the presence of C and successfully protect transplanted mice from lethal GVHD. They also suggest that these anti-Lyt antibodies may be useful tools in determining subpopulations of T cells that contribute to the development of GVHD.     

Cytolytic T lymphocyte clones that proliferate autonomously to specific alloantigenic stimulation. II. Relationship of the Lyt-2 molecular complex to cytolytic activity, proliferation, and lymphokine secretion

Previous analyses of the inhibitory effects of anti-Lyt-2 monoclonal antibodies (mAb) on cytolytic activity suggested that Lyt-2/3 antigens expressed on the surface of murine cytolytic T lymphocytes (CTL) are involved in antigen recognition. In the present study, we investigated the effects of anti-Lyt-2 mAb (in the absence of complement) on the functional activities of H-2K/D-specific Lyt-2+ CTL clones that proliferate to antigenic stimulation in the absence of helper T cells or added interleukin 2 (IL 2) and secrete lymphokines. For those clones that were inhibited in cytolysis by anti-Lyt-2 mAb, a parallel inhibition of antigen-dependent proliferation and lymphokine secretion (interferon, macrophage-activating factor) was observed. Inhibition of proliferation or lymphokine secretion could be overcome by the addition of IL 2 or lectin, respectively. Collectively, these results would strongly suggest that anti-Lyt-2 mAb were inhibiting CTL antigen recognition. Not all CTL clones, however, were inhibited in cytolysis by anti-Lyt-2 mAb, in which case proliferation and lymphokine secretion were similarly unaffected. This heterogeneity of Lyt-2+ CTL clones in their susceptibility to inhibition of cytolytic activity, proliferation, and lymphokine secretion by anti-Lyt-2 mAb is discussed in the context of a model proposing that Lyt-2/3 molecules function to stabilize the interaction between CTL receptors and the corresponding target/stimulating cell antigens. Such a stabilization may be required by CTL possessing few and/or low affinity receptors.     

Construction and properties of new Lyt-congenic strains and anti-Lyt-2.2 and anti-Lyt-3.1 monoclonal antibodies

Hybridomas producing mouse monoclonal IgM antibodies specific for Lyt-2.2 and Lyt-3.1 T-cell surface alloantigens have been constructed. Cytotoxic titers of ascites fluids were found to be 10(-6) or greater and no lysis of thymocytes of congenic strains bearing the alternative allele was observed at the lowest dilutions tested (1:2). The anti-Lyt-2.2 monoclonal antibody (HO-2.2) specifically precipiated from extracts of Lyt-2.2-positive thymocytes molecular species indistinguishable from those precipitated by conventional anti-Lyt-2.2 sera. However, by immunoprecipitation criteria (though not by cytotoxicity), the anti-Lyt-3.1 antibody (HO-3.1) demonstrated some cross-reactivity with similar molecular species from Lyt-3.1-negative thymocytes. In addition, three new strains of mice differing from existing strains in the region of the Lyt-2 and Lyt-3 loci have been constructed. They are: C.C58-Lyt-2a, Lyt-3a and C.AKR-Lyt-2a, Lyt-3a, congenic with Balb/cAn and bearing Lyt-2a and Lyt-3a alleles of C58/J and AKR/J, respectively; and AKR.C-Lyt-2b, Lyt-3b congenic with AKR/J and bearing the Lyt-2b and Lyt-3b alleles of Balb/cJ.     

Idiotype-specific T lymphocytes. I. Regulation of antibody production by idiotype-specific H-2-restricted T lymphocytes

Cytotoxic T lymphocytes (CTL) specific for MOPC-104E myeloma cells of BALB/c origin could be induced in BALB/c, (BALB/c X BALB.B)F1, and (BALB/c X BALB.K)F1 mice. (BALB/c X BALB.B)F1 CTL activity specific for MOPC-104E was effectively inhibited by anti-H-2d but not by anti-H-2b alloantiserum. However, the activity was hardly blocked by specific anti-idiotypic antibodies to MOPC-104E. For further analysis of the recognition of idiotype on target cells by CTL, the effect of those lymphocytes on anti-dextran B1355S antibody-producing B lymphocytes, which have a cross-reactive idiotype to MOPC-104E, was investigated. Lymphocytes from the CTL population did inhibit antibody production by dextran-immune spleen cells, but those from the CTL population specific for irrelevant myeloma cells (MOPC-167) did not. The (BALB/c X BALB.K)F1 CTL population suppressed the antibody production of BALB/c but not of BALB.K. This indicates that F1 cells can preferentially see H-2 antigens of immunizing myeloma cells on target B lymphocytes. The inhibition of antibody production was antigen specific and was only restricted to the PFC that were inhibitable by anti-idiotypic antibodies. The surface phenotypes of the cells that inhibited the antibody production were Thy-1+, Lyt-1-, Lyt-2+, and I-J-. These results strongly suggest that CTL specific for MOPC-104E recognize self H-2 antigens simultaneously with idiotypic determinants on B lymphocytes. Possible immunoregulatory roles of idiotype-specific CTL on antibody production systems are also suggested.     

Conditions of anti-Lyt-2-mediated inhibition of TcR/CD3-induced IFN-gamma secretion by a CTL clone

The relationship between the T cell receptor (TcR) for antigen (Ag) and the Lyt-2/3 molecule during T cell activation was studied using the T cell clone KB5.C20, which is dependent upon Lyt-2 for target cell killing. This cytolytic T cell clone can be activated to secrete IFN-gamma by stimulation with H-2Kb expressing cells or with monoclonal antibodies directed against a clonotypic structure of the TcR or against associated CD3 molecules. IFN-gamma production induced by H-2Kb can be inhibited by anti-Lyt-2mAb. In addition, TcR-mediated activation using the anticlonotypic mAb Désiré-1 in soluble form can be inhibited by anti-Lyt-2 mAb in soluble form either as a divalent IgG or as its monovalent Fab fragment. Anti-Lyt-2 mAb immobilized on plastic wells was also inhibitory. Stimulation induced by the anti-TcR mAb or by anti-CD3 mAb immobilized on plastic can be inhibited only with plastic immobilized and not with soluble anti-Lyt-2mAb, however. These results are discussed in terms of local interactions between TcR and Lyt-2 molecules.     

Positively selected Lyt-2+ and Lyt-2- mouse T lymphocytes are comparable, after Con A stimulation, in release of IL 2 and of lymphokines acting on B cells, macrophages, and mast cells, but differ in interferon production

Purified mouse T lymphocytes were separated into Lyt-2+ and Lyt-2- populations by the procedure of panning, in which a monoclonal rat anti-Lyt-2 antibody and dishes coated with affinity-purified mouse anti-rat Ig antibodies were used. The populations obtained were 95 to 99% pure as determined by immunofluorescence. Graded doses of these T cells were cultured with optimal mitogenic doses of concanavalin A and the 0 to 24 and 24 to 48-hr culture supernatants were collected. The dose-curve assays of the supernatants of Lyt-2+ and Lyt-2- cells showed comparable activity in interleukin 2 (IL 2) and T cell-replacing factor (TRF), assayed on antigen-stimulated culture of T-depleted spleen cells. Limiting dilution assays of IL 2-secreting precursor cells stimulated by Con A showed a high frequency of precursors in both populations, slightly higher among Lyt-2- cells. The supernatants also contained comparable levels of IPA (inducer of plasminogen activator production by the macrophages), MAF (macrophage-activating factor, assayed by induction of their cytolytic function), and MCGF (mast cells growth factor, assayed on a mast cell line). IPA and MAF were not produced with the same kinetics and in the same T cell concentration conditions as IL 2 and TRF. In contrast, interferon was principally produced by the Lyt-2+ cells.     

Role of accessory molecules in signal transduction of cytolytic T lymphocyte by anti-T cell receptor and anti-Ly-6.2C monoclonal antibodies

Accessory molecules present on the cell surface of cytolytic T lymphocytes (CTL) play an important role in their activation. Antigen-specific recognition by CTL is inhibited by antibodies against Lyt-2, L3T4, or LFA-1 molecules. Presently it is not known whether these molecules function by binding a ligand such as class I or class II on the target cell or by delivering a signal that down-regulates T cell activation. In the present study we utilized anti-T cell antibodies including anti-T3 and anti-T cell receptor (alpha/beta) as well as an anti-Ly-6.2C monoclonal antibody to activate CTL clones to kill irrelevant targets or secrete BLT esterase. The redirected lysis assay system utilizes the fact that heteroconjugates between anti-T3, and anti-T cell receptor, or anti-Ly-6.2C and anti-trinitrophenyl can trigger CTL lysis of trinitrophenyl-coupled targets that did not express antigen. In this system anti-Lyt-2 antibodies as well as anti-LFA-1 antibodies inhibited triggering via T cell receptor-related molecules but not via the anti-Ly-6.2C heteroconjugate. In addition, the anti-Lyt-2 was shown to inhibit conjugate formation in the heteroaggregate assay system suggesting that the anti-Lyt-2 antibodies acted early in inhibiting CTL activity. Similar results were observed in a system in which the CTL clones were triggered to secrete a BLT-esterase-like activity in the absence of target cells. Anti-T3 coated on plastic was shown to activate BLT-esterase secretion. This secretion was inhibited by anti-Lyt-2 and anti-LFA-1. Thus, it would appear that both the Lyt-2 molecule and the LFA-1 molecule act as signal-transducing elements involved in CTL activation. In particular, the Lyt-2 molecule appears to preferentially function in receptor-mediated T cell activation.     

A role for Lyt-2+ T cells in resistance to cutaneous leishmaniasis in immunized mice

The role of Lyt-2+ T cells in immunologic resistance to cutaneous leishmaniasis was analyzed by comparing infection patterns in resistant C57BL/6 mice and susceptible BALB/c mice induced to heal their infections after sub-lethal irradiation or i.v. immunization, with similar mice treated in vivo with anti-Lyt-2 antibodies. Administration of anti-Lyt-2 mAb resulted in a dramatic reduction in the number of lymphoid cells expressing the Lyt-2+ phenotype. Such treatment led to enhanced disease in both resistant C57BL/6 and irradiated BALB/c mice, as assessed by lesion size, but did not affect the capacity of these mice to ultimately resolve their infections. In contrast, anti-Lyt-2 treatment totally blocked the induction of resistance in i.v. immunized mice. These results suggest, that Lyt-2+ T cells may play a role in immunity to a Leishmania major infection and that their relative importance to resistance may depend on how resistance is induced.     

Sequential precipitation of mouse thymocyte extracts with anti-Lyt-2 and anti-Lyt-3 sera. I. Lyt-2.1 and Lyt-3.1 antigenic determinants reside on separable molecular species.

Anti-Lyt-2.1 and anti-Lyt-3.1 sera were employed for sequential precipitation of NP-40 extracts of 125I-labeled C57BL/6-Lyt-2a, Lyt-3a thymocytes (Lyt-2.1, Lyt-3.1) to determine whether these alloantigenic determinants are present on the same or different molecular species. Treatment of extracts with anti-Lyt-3.1 serum and SaCI completely precipitated both Lyt-3.1 and Lyt-2.1-specific components, whereas treatment with anti-Lyt-2.1 serum reduced by approximately 37% the quantity of labeled species subsequently precipitable by anti-Lyt-3.1 serum. When 125I-labeled thymocytes were subjected to mild trypsinization before NP-40 extraction, the quantity of radioactive components precipitated by anti-Lyt-2.1 serum was essentially unchanged, but that of anti-Lyt-3.1-precipitable components was greatly reduced. Moreover, sequential precipitation of extracts of trypsinized thymocytes with anti-Lyt-2.1 and anti-Lyt-3.1 sera demonstrated that these molecular species were precipitated independently. Thus 1) Lyt-2.1 and Lyt-3.1 antigenic determinants appear to reside on different molecular species; 2) some Lyt-2.1- and Lyt-3.1-positive molecules appear to be complexed with each other in the NP-40 extract; and 3) this association of Lyt-2.1- and Lyt-3.1-positive species was dependent upon components that were labile to trypsinization of intact thymocytes.     

Role of L3T4+ and Lyt-2+ T cell subsets in protective immune responses of mice against infection with a low or high virulent strain of Toxoplasma gondii

In order to elucidate the role of T cell subsets in protective immunity against infection with high virulent and low virulent strains of Toxoplasma gondii, monoclonal antibodies specific for T cell subsets were injected into mice before immunization or challenge infection. Treatment of mice with monoclonal antibody to either L3T4+ or Lyt-2+ T cells before they were immunized with Toxoplasma cell homogenate prepared from high virulent RH strain tachyzoites markedly reduced survival after mice were challenged with low virulent bradyzoites of the Beverley strain. Thus, induction of protective immunity against bradyzoites of the Beverley strain requires the presence of both L3T4+ and Lyt-2+ T cells. In contrast, mice injected with living bradyzoites of the low virulent Beverley strain after immunization with Toxoplasma cell homogenate acquired protective immunity against high virulent tachyzoites of the RH strain. Lyt-2+ T cells alone appear to be final effector cells for protection against the challenge with high virulent RH strain tachyzoites, since treatment of the bradyzoite-immune mice with anti-Lyt-2 antibody, but not anti-L3T4 antibody, before challenge significantly increased mortality.     

Enterically induced regulation of systemic immune responses. II. Suppression of proliferating T cells by an Lyt-1+, 2- T effector cell

Peripheral lymph node cells from C3H mice that were fed and injected with bovine serum albumin (REG cells) demonstrate an impaired proliferative response to antigenic stimulation in vitro compared to cells from mice only injected with BSA. To determine whether suppressor cells contributed to this enterically induced impairment of systemic T cell responses, REG cells were pretreated with various monoclonal antibodies and complement (C), and were then co-cultured with antigen-reactive indicator T cells (IND) from parenterally immunized mice. Proliferation of IND cells [( 3H]thymidine uptake) was suppressed only if REG cells were treated with anti-Lyt-2 and C before co-culture. The ability of anti-Lyt-1 plus anti-Lyt-2 and C treatment to abrogate suppression suggested that the suppressor effect was due to an Lyt-1+, 2- REG cell. Suppression was independent of Lyt-2+ IND cells, and was observed at different antigen concentrations, cultivation times, and cell densities. The cells responsible for suppressor activity were radiosensitive, nylon wool nonadherent, and antigen specific. These data suggest that an Lyt-1+, 2- T cell could be an important component in mediating enterically induced regulation of systemic T cell responses.     

T helper cells in cytotoxic T lymphocyte development: role of L3T4(+)-dependent and -independent T helper cell pathways in virus-specific and alloreactive cytotoxic T lymphocyte responses

I have compared the requirements for T helper (Th) cell function during the generation of virus-specific and alloreactive cytotoxic thymus (T)-derived lymphocyte (CTL) responses. Restimulation of vesicular stomatitis virus (VSV)-immune T cells (VSV memory CTLs) with VSV-infected stimulators resulted in the generation of class I-restricted, VSV-specific CTLs. Progression of VSV memory CTLs (Lyt-1-2+) into VSV-specific CTLs required inductive signals derived from VSV-induced, Lyt-1+2- Th cells because: (i) cultures depleted by negative selection of Lyt-1+ T cells failed to generate CTLs; (ii) titration of VSV memory CTLs into a limiting dilution (LD) microculture system depleted of Th cells generated curves which were not consistent with a single limiting cell type; (iii) LD analysis of VSV memory CTLs did produce single-hit curves in the presence of Lyt-1+2- T cells sensitized against VSV; and (iv) monoclonal anti-L3T4 antibody completely abrogated CTL generation against VSV. Similar results were also obtained with Sendai virus (SV), a member of the paramyxovirus family. The notion that a class II-restricted, L3T4+ Th cell plays an obligatory role in the generation of CTLs against these viruses is also supported by the observation that purified T cell lymphoblasts (class II antigen negative) failed to function as antigen-presenting cells for CTL responses against VSV and SV. T cell lymphoblasts were efficiently lysed by class I-restricted, anti-VSV and -SV CTLs, indicating that activated T cells expressed the appropriate viral peptides for CTL recognition. Furthermore, heterogeneity in the VSV-induced Th cell population was detected by LD analysis, suggesting that at least two types of Th cells were required for the generation of an anti-VSV CTL response. VSV-induced Th cell function could not simply be replaced by exogenous IL-2 because this lymphokine induced cytotoxic cells that had the characteristics of lymphokine-activated killer (LAK) cells and not anti-viral CTLs. In contrast, CTL responses against allogeneic determinants could not be completely blocked with antibodies against L3T4 and depletion of L3T4+ cells did not prevent the generation of alloreactive CTLs in cultures stimulated with allogeneic spleen cells or activated T cell lymphoblasts. Thus, these studies demonstrate an obligatory requirement for an L3T4-dependent Th cell pathway for CTL responses against viruses such as VSV and SV; whereas, CTL responses against allogeneic determinants can utilize an L3T4-independent pathway.     

Inhibition of T cell-mediated cytolysis by monoclonal antibodies directed against Lyt-2: heterogeneity of inhibition at the clonal level

The inhibitory effect of monoclonal anti-Lyt-2 antibodies on T cell-mediated cytolysis has been investigated at the clonal level. In agreement with previous reports from several laboratories, populations of cytolytic T lymphocytes (CTL) generated in vitro in mixed leukocyte cultures (MLC) were reversibly inhibited by monoclonal anti-Lyt-2 antibodies in a dose-dependent fashion. However, when alloimmune peritoneal exudate lymphocytes (PEL) were used as a source of CTL, little or no inhibitory effect of anti-Lyt-2 antibodies on cytolysis was observed. A series of CTL clones derived from MLC or PEL populations was also tested for inhibition of cytolysis by anti-Lyt-2 antibodies. In agreement with results obtained at the population level, most MLC-derived clones (81%) were strongly inhibited by the reagent, whereas few PEL clones (15%) were inhibited. Several of these clones were expanded and maintained in culture without loss of their "inhibition phenotype." Flow cytofluorometric analysis using the same monoclonal anti-Lyt-2 antibodies further revealed that both inhibited and uninhibited clones expressed comparable amounts of Lyt-2 antigen. These results provide direct evidence that inhibition of CTL by anti-Lyt-2 antibodies is heterogeneous at the clonal level. The possibility that this heterogeneity may be related to avidity of antigen receptors is discussed.