Cloned, Ia-restricted T cells that do not produce interleukin 4(IL 4)/B cell stimulatory factor 1(BSF-1) fail to help antigen-specific B cells

T cells can be subdivided based on cell surface markers, MHC restriction, function, and production of soluble factors. Analysis of the ability of cloned, Ia-restricted, L3T4+ T cells to induce an in vitro anti-hapten antibody response to hapten-carrier conjugates allowed the definition of three functional subtypes. To examine whether these functional subtypes also differed in the production of soluble mediators, supernatants of the cloned lines were examined for the production of T cell growth factors and factors inducing increased expression of Ia glycoproteins on small resting B cells. All of the cloned lines produced T cell growth factors that could be further differentiated by inhibition with monoclonal antibodies. None of the Ia-restricted, L3T4+ cloned T cell lines that failed to produce IL 4/BSF-1 could provide helper function. Thus, the activation of antigen-specific B cells by helper T cells appears to require IL 4/BSF-1 as a necessary but not sufficient signal for differentiation into antibody-forming cells.     

Selective activation of helper and cytolytic T-cell functions of L3T4+ clones with either antireceptor antibody or phorbol ester and ionophore

After activation with specific antigen and antigen presenting cells (APC) L3T4+ inducer T-cell clones can lyse Ia+ APC. The present study characterizes the mechanism of activation and specificity of L3T4+ inducer cell-mediated cytolytic function. Two methods that bypass the physiological stimulus of antigen presented on Ia+ APC were used to activate L3T4+ clones. The first method utilized an antireceptor monoclonal antibody (MAb), KJ16.133, to activate KJ16.133+ clones. The activated clones expressed nonspecific cytolytic activity, killing target cells irrespective of their H-2 haplotype or their ability to express cell surface Ia molecules. The crosslinking of bound KJ16.133 antibody greatly enhanced cytolytic activity. This activation is receptor specific because KJ16.133- clones were not activated under identical conditions. The second method of activation was provided by a synergistic action of phorbol-12-myristate-13-acetate (PMA) and ionophore A23187. These agents nonspecifically activated all L3T4+ clones tested. The simultaneous presence of the two agents is required for maximal activation. Again, the activated clones expressed potent nonspecific cytolytic activity. These observations demonstrated that L3T4+ inducer T-cell-mediated killing can be separated into two stages: an activation step, which can be specifically and nonspecifically triggered and an effector phase which causes nonspecific lysis of bystander targets. The induction of nonspecific cytolytic activity by antireceptor MAb was inhibited by anti-L3T4 MAb (GK1.5). In contrast, activation of nonspecific cytolytic activity by treatment with PMA plus A23187 was not inhibited by anti-L3T4 MAb. Under the above activation conditions, antireceptor MAb selectively induced the secretion of IL-3 and expression of nonspecific cytolytic activity. However, there was little or no concomitant proliferation and production of IL-2. In contrast, activation by PMA plus A23187 coordinately induces expression of nonspecific cytolytic activity, secretion of lymphokines (IL-3 and IL-2), and cell proliferation. Thus, the anticlonotypic activation preferentially induces certain functions whereas activation with PMA plus A23187 is not selective.     

The immunobiology of T cell responses to Mls-locus-disparate stimulator cells. III. Helper and cytolytic functions of cloned, Mls-reactive T cell lines

Mls-specific T cell clones derived by limiting dilution were tested for cytotoxic activity in a lectin-dependent 51Cr-release assay. All the T cell clones tested were cytotoxic in such an assay in apparent contrast to previous reports. However, only those target cells sensitive to cytolysis by other L3T4a+ cytolytic T cells were killed by Mls-specific T cell clones in short term 51Cr-release assays, possibly explaining this discrepancy. All the T cell clones tested were L3T4a+, Lyt-2- and stimulated B cells from Mlsa,d strains of mice to proliferate and secrete immunoglobulin. Furthermore, lysis of innocent bystander targets was observed when the T cells were stimulated with Mls-disparate stimulator cells. These results are consistent with those obtained with L3T4a+ T cells specific for protein antigen:self Ia and that express cytotoxic potential.     

Interferon-beta and recombinant IL 2 can both enhance, but by different pathways, the nonspecific cytolytic potential of T3- natural killer cell-derived clones rather than that of T3+ clones

We studied the enhancement of cytolytic activity of T3- natural killer cell-derived clones, of T3+ T cell activated killer (AK) clones, and of fresh peripheral blood lymphocytes (PBL) by various crude and recombinant interferon (r-IFN) as well as IL 2 preparations. It was found that IFN-beta had the highest cytotoxicity inducing potency as compared to crude or r-IFN-alpha or -gamma preparations. This enhancement was blocked by anti-IFN-beta antibodies but not by anti-IFN-gamma antibodies. IL 2 also strongly enhances cytolytic activity in cloned T3- killer cells that express the IL 2 receptors as determined with the anti-Tac monoclonal antibody (MAb) at concentrations of IL 2 (25 U/ml) which induced one-half of the maximal proliferation capacity in human T cells and murine CTLL cells. For enhancement of cytolytic activity in fresh NK cells, a much higher concentration of IL 2 is required. In addition, the enhancement of cytolytic activity by r-IL 2 but not that by IFN-beta can be reduced by anti-Tac MAb, suggesting that the IL 2 receptor is involved in the enhancement by IL 2, but not by IFN. Both IFN-beta and IL 2 were able to enhance (over threefold) the cytolytic activity of T3- cloned killer cells against a variety of tumor target cell types. Another remarkable observation was that K562 cells, the most commonly used target cell for determining NK cell cytolytic activity, are not the most suitable targets to assess enhancement of nonspecific lytic activity as compared to Daudi or lung tumor-derived cell lines. No enhancement of anti-body-dependent cellular cytotoxicity was observed. Finally, the effects of these biological response modifiers were much more pronounced on "fresh" and cloned T3- natural killer cell-derived than on T3+-activated killer mature T cell-derived clones.     

Antigen-specific Lyt-2+ cytolytic T lymphocytes from mice infected with the intracellular bacterium Listeria monocytogenes

In vitro expanded T cell lines were used to determine whether antigen-specific cytolytic T lymphocytes are generated after infection with the intracellular bacterium, Listeria monocytogenes. Spleen cells from infected mice were cultured in the presence of syngeneic accessory cells, listerial antigen, and interleukin 2 containing supernatants. Cell lines were greater than 98% Thy-1+, L3T4-, Lyt-2+. Bone-marrow macrophages were used as target cells in two in vitro cytolytic assay systems. The Lyt-2+ T cells killed bone marrow macrophages only when infected with L. monocytogenes as assessed in a 4-hr 51Cr release assay and in an 18-hr neutral red uptake assay. Cytolysis was blocked by anti-LFA-1 and anti-Lyt-2 monoclonal antibodies. These cytolytic T cells produced interferon-gamma after co-stimulation with antigen, accessory cells, and recombinant interleukin 2. Bone marrow macrophages infected with Mycobacterium bovis were not killed by T cells from L. monocytogenes-infected mice but by T cell lines from M. bovis-infected mice, indicating that cytolysis was antigen specific. L. monocytogenes-infected target cells of different haplotype were lysed by the Lyt-2+ T cells. By using a low cell density split culture system, antigen-specific, H-2-restricted cytolytic T cells could be identified. These findings demonstrate that during infection with intracellular bacteria, Lyt-2+ T cells with cytolytic activity are generated that may be involved in antibacterial protection.     

Generation of H-2-reactive T cell lines that bear the 5936 idiotype(s)

The present experiments showed 1) that it was possible to produce mouse T cell lines against MHC determinants with a relatively high success rate by stimulation of purified T cells with allogeneic cells in the presence of irradiated syngeneic spleen cells; 2) that these lines could be led to react against selected H-2 specificities; 3) that only T cell lines established from Ig-1b allotype mice contained 5936-Id+ T cells (5936-Idiotypes are defined by an antiserum against B6 anti-CBA IgG produced in rabbit no 5936, which was tolerant to mouse gamma-globulin); and 4) that antigenic determinants coded by IAk genes induce the 5936-Idiotype(s). The latter data are in accordance with the 5936-idiotype characteristics of primary MLC T blasts. All T cell lines contained both specific MLC-responding cells and cytolytic cells. However, studies on the functional capacity of 5936-Id+ T cells from both primary MLC and the T cell lines showed that neither MLC-responding cells nor cytolytic cells directed against H-2Kk, IAk, or H-2Dk were 5936-Id+. Thus, 5936-Id+ T cells may be regulator cells induced by IAk antigens.     

Differentiation of thymocytes in fetal organ culture: analysis of phenotypic changes accompanying the appearance of cytolytic and interleukin 2-producing cells

At the 14th day of gestation, embryonic thymocytes+ are large, functionally incompetent cells with H-2K+ Thy-1+ B14- Ly-2- L3T4- phenotype, some of which express TL antigen. Differentiation of these cells in organ culture is characterized by: 1) appearance of cells expressing Ly-2 and L3T4 molecules, first among the population of large cells, after 2 days of culture; 2) appearance of small H-2K- Thy-1+TL+B14+Ly-2+L3T4+ and H-2K-Thy-1+TL+B14-Ly-2+ L3T4+ cells between days 2 and 4; 3) accumulation of small H-2K- Thy-1+ TL+ B14- Ly-2+ L3T4+ (but not H-2K- Thy-1+ TL+ B14+ Ly-2+ L3T4+) cells until day 5 of culture, and their subsequent gradual disappearance which is paralleled by an increase of the proportion of medium-sized H-2K+ Thy-1+ TL- B14- cells with various Ly-2 L3T4 phenotypes; 4) appearance and subsequent accumulation of cytolytic and IL 2-producing cells between days 4 and 6. Comparison of these results with the data from similar in vivo studies shows that differentiation of organ-cultured thymocytes rather closely follows the in vivo development only during the first week of culture and shows significant deviations thereafter. Precursors of cytolytic cells and cytolytic effector cells as well as IL 2-producing cells are found among both Ly-2+ and Ly-2- populations of thymocytes, indicating that there is no clear association between Ly-2 phenotype and the ability to kill or to secrete IL 2.     

Dissociation of alloantigen recognition from self major histocompatibility complex-restricted recognition of cytolytic T lymphocytes by monoclonal antireceptor antibodies

Two monoclonal antibodies (mAb) directed to the dual reactive cytolytic T lymphocyte clone OH8 (Db + H-Y and H-2d) were established. Analysis by cell surface staining and immunoprecipitation of radiolabeled surface molecules of OH8 followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that both mAb recognized an identical heterodimeric, clonotypic structure on OH8 cells, i.e., T cell receptor. However, although the MR3-2 mAb inhibited the lysis of either Db + H-Y or H-2d targets by OH8, the MR3-6 mAb inhibited the lysis of H-2d target cells, but not that of Db + H-Y target cells. Modulation of T cell receptor by either MR3-2 or MR3-6 mAb rendered the OH8 cytolytic T lymphocyte incapable of killing both Db + H-Y and H-2d target cells. These findings suggest that different epitopes of OH8 T cell receptor were involved for the recognition of self + antigen and alloantigen.     

Hapten-specific, class II-restricted killing by cloned T cells: direct lysis and production of a cytotoxic factor

DNP-specific, class II-restricted cloned T cells were shown to kill DNP-bearing A20.2J (A20-DNP) antigen-presenting cells. This killing was DNP-specific and was restricted by IA. Results from bystander cytotoxicity, cold-target inhibition, and protein and lymphokine inhibition experiments indicated that killing of A20-DNP targets was mediated by direct lysis. In addition to the direct lysis, antigen stimulation of the T cells also resulted in production of a soluble cytolytic factor which killed bystander L929 fibroblast cells. This killing was sensitive to inhibition of protein synthesis and lymphokine production but was not affected by the addition of cold A20-DNP target cells. Additional studies showed that other antigen-presenting cells, i.e., DNP-bearing P388D1 and splenic macrophages, were also lysed by the cloned T cells. These findings may indicate that lysis of target cells by nominal antigen-specific, class II-restricted T cells plays a role in immune regulation and/or immune protection.     

CD4+ T cell-mediated killing of MHC class II-positive antigen-presenting cells. I. Characterization of target cell recognition by in vivo or in vitro activated CD4+ killer T cells

Ag-specific as well as Ia-restricted killing of certain APC by CD4+ T cells was investigated. The CD4-mediated killing is not only a characteristic of in vitro long term cultured T cell lines or clones, but is also manifest after in vivo priming. Thus, CD4+ killer T cells are generated in vivo as well. CD4+ killer T cells are detected in the Th1, but not in the Th2 subset, and they do not appear to lyse Ia+ APC or bystander cells by a pathway mediated by secreted T cell factors. The latter observation is demonstrated by cold target inhibition experiments as well as by the failure of puromycin to inhibit killing, if applied in doses which completely block lymphokine secretion. Ia+ APC differ in their susceptibility to lysis. Transformed APC are usually better lysed than nontransformed APC. Unstimulated B cells are not killed, while LPS-stimulated B cell blasts are killed. The results of cold target inhibition and bystander killing experiments suggest that CD4+ killer T cells are activated by the common pathway, i.e., by Ag presented in the context of Ia, but killing requires the recognition of additional determinant(s) on APC. It is proposed that these killing-inducing determinants are continuously expressed on most transformed Ia+ cells and on nontransformed but stimulated APC.     

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.     

Resistance of primary CD8+ cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines

Recent evidence has shown that cloned, murine CTL cell lines are resistant to the cytotoxic components of the toxic granules they release upon specific interaction with their target cells. Inasmuch as the resistance might be due to selection in culture over many months by repeated exposure to these cytolytic components (which are released repeatedly as a result of the cultured CTL being periodically stimulated by target cells), we asked whether primary CTL are also resistant. The primary CTL were elicited in vivo by i.p. injection of allogeneic tumor cells or in vitro by 5- to 6-day MLC or by 48-h exposure to the lectin Con A. The responding cells were separated into purified CD8+ (i.e., CD4-, CD8+) and purified CD4+ (i.e., CD4+, CD8-) T cell populations that were analyzed for cytolytic activity and for resistance to lysis by toxic secretory granules derived from cloned CTL cell lines. The CD8+ T cells were highly cytolytic and relatively resistant; they retained their cytolytic activity and were lysed to a minimal extent (0 to 10%) by quantities of isolated granules that lysed 80 to 90% of the P815 tumor cell line (tested as a representative standard cell line). The CD4+ T cells, in contrast, had only minimal cytolytic activity and were far more susceptible to granule-mediated lysis. Although the resistance of primary CD8+ T cells is impressive, it is not as pronounced as the resistance of the cloned CTL cell lines, indicating that during long-term culture there is some selection for increased resistance to granule-mediated lysis. In contrast to T cells (especially CD8+ T cells), Ia+ macrophages, isolated from primary immune peritoneal exudates, were highly susceptible to granule-mediated lysis.     

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.     

Features of target cell lysis by class I and class II MHC-restricted cytolytic T lymphocytes

The lytic activity of influenza virus-specific murine cytolytic T lymphocyte (CTL) clones that are restricted by either H-2K/D (class I) or H-2I (class II) major histocompatibility (MHC) locus products was compared on an influenza virus-infected target cell expressing both K/D and I locus products. With the use of two in vitro measurements of cytotoxicity, conventional 51Cr release, and detergent-releasable radiolabeled DNA (as a measure of nuclear disintegration in the early post-lethal hit period), we found no difference between class I and class II MHC-restricted CTL in the kinetics of target cell destruction. In addition, class II MHC-restricted antiviral CTL failed to show any lysis of radiolabeled bystander cells. Killing of labeled specific targets by these class II MHC-restricted CTL was also efficiently inhibited by unlabeled specific competitor cells in a cold target inhibition assay. In sum, these data suggest that class I and class II MHC-restricted CTL mediate target cell destruction by an essentially similar direct mechanism.     

Cultured Lyt-2- L3T4- T lymphocytes from normal thymus or lpr mice express a broad spectrum of cytolytic activity

T lymphocytes expressing the surface phenotype Lyt-2- L3T4- represent a minor population of immature thymocytes that appear to be the precursors of mature T cells. Cells with the same apparent surface phenotype also accumulate in vast numbers in the lymphoid tissues of the autoimmune lpr mouse. Lyt-2- L3T4- T lymphocytes from lpr lymph node (LN) or normal thymus express low to undetectable levels, respectively, of surface antigen receptor. In addition, they produce reduced amounts of lymphokines compared with normal T cells and lack precursors of alloantigen-specific cytolytic T lymphocytes. We previously showed that after culture with phorbol esters and interleukin 2, lpr Lyt-2- L3T4- T lymphocytes proliferate and differentiate, acquiring increased levels of surface antigen receptor by most cells, as well as Lyt-2 by a portion. We now show that cultured Lyt-2- L3T4- T cells from lpr LN or normal thymus are very efficiently cytolytic toward not only allogeneic tumor targets, but also natural killer (NK)-susceptible targets and syngeneic targets. Such killing was not inhibited by antibodies to H-2 or Lyt-2. In contrast, cultured mature Lyt-2+ L3T4- T cells from normal LN, thymus, or lpr LN were cytolytic only toward allogeneic targets and were dependent on Lyt-2 expression and H-2 recognition. The similarities of cultured Lyt-2- L3T4- T cells to NK and lymphokine-activated killer cells are discussed.     

Delta 9-tetrahydrocannabinol decreases cytotoxic T lymphocyte activity to herpes simplex virus type 1-infected cells.

The purpose of this study was to examine the effect of delta 9-tetrahydrocannabinol (delta 9-THC), the major psychoactive component of marijuana, on T lymphocyte functional competence against herpes simplex virus Type 1 (HSV1) infection. Spleen cells from C3H/HeJ (H-2k) mice primed with HSV1 and exposed to delta 9-THC were examined for anti-HSV1 cytolytic T lymphocyte (CTL) activity. Flow cytometry was used to determine whether delta 9-THC altered T cytotoxic (Lyt-2+) and T helper (L3T4+) lymphocyte numbers or cell ratios. Nomarski optics microscopy was used to determine whether effector lymphocytes from drug-treated mice were able to bind to virally infected L929 (H-2k) target cells. Cytotoxicity assays demonstrated that CTL from mice exposed to delta 9-THC were deficient in anti-HSV1 cytolytic activity. delta 9-THC in vivo treatment had little effect on the number of T lymphocytes expressing the Lyt-2 or L3T4 antigens. Nomarski optics microscopy revealed that the CTL from the drug-treated mice were able to bind specifically to the HSV1-infected targets. However, delta 9-THC in vivo exposure affected CTL cytoplasmic polarization toward the virus-infected target cell. CTL granule reorientation toward the effector cell-target cell interface following cell conjugation occurred at a lower frequency in co-cultures containing CTL from drug-treated mice. These results suggest that delta 9-THC elicits dysfunction in CTL by altering effector cell-target cell postconjugation events.     

Sequential activation and lethal hit measured by [Ca2+]i in individual cytolytic T cells and targets.

Changes in cytosolic free calcium ([Ca2+]i) have been continuously imaged during the interaction of the H-2Kb specific cytotoxic T cell lymphocyte (CTL) BM 3.3, with either the H-2Kb EL4.BU or the H-2Kk RDM4 cell lines. Activation of the CTLs by EL4.BU raises [Ca2+]i to several hundred nanomolar in the CTL. Frequently [Ca2+]i is preferentially elevated in the region of the CTL furthest from the site of target contact. These responses require external Ca2+ suggesting that they are generated by the plasma membrane and not internal stores. Inappropriate targets such as RDM4 evoke no changes in [Ca2+]i. Activation of the BM 3.3 CTL is followed by increases of [Ca2+]i to several micromolar or higher in the EL4.BU targets. This massive increase can be mimicked by direct application of cytolytic granules isolated from rat natural killer cells. The increase in plasma membrane permeability is ion-specific since external Mn2+ can also readily enter target cells that have been 'hit', as evidenced by the rapid selective quenching of fura-2 in those targets. The flood of Ca2+ into the target cell is followed by a leakage of the trapped fura-2. Since both processes continue after the CTL has disengaged, they provide a useful assay for the lethal hit. Furthermore, this technique can be used to follow complete cycles of CTL activation and lethal hit delivery, which under some circumstances can be as rapid as 6 min per cycle.     

CD8+ T cell protective immunity against Chlamydia pneumoniae includes an H2-M3-restricted response that is largely CD4+ T cell-independent

CD8+ T cells are important for immunity to the intracellular bacterial pathogen Chlamydia pneumoniae (Cpn). Recently, we reported that type 1 CD8+ (Tc1) from Cpn-infected B6 mice recognize peptides from multiple Cpn Ags in a classical MHC class Ia-restricted fashion. In this study, we show that Cpn infection also induces nonclassical MHC class Ib-(H2-M3)-restricted CD8+ T cell responses. H2-M3-binding peptides representing the N-terminal formylated sequences from five Cpn Ags sensitized target cells for lysis by cytolytic effectors from the spleens of infected B6 mice. Of these, only peptides fMFFAPL (P1) and fMLYWFL (P4) stimulated IFN-gamma production by infection-primed splenic and pulmonary CD8+ T cells. Studies with Cpn-infected Kb-/-/Db-/- mice confirmed the Tc1 cytokine profile of P1- and P4-specific CD8+ T cells and revealed the capacity of these effectors to exert in vitro H2-M3-restricted lysis of Cpn-infected macrophages and in vivo pulmonary killing of P1- and P4-coated splenocytes. Furthermore, adoptive transfer of P1- and P4-specific CD8+ T cells into naive Kb-/-/Db-/- mice reduced lung Cpn loads following challenge. Finally, we show that in the absence of MHC class Ia-restricted CD8+ T cell responses, CD4+ T cells are largely expendable for the control of Cpn growth, and for the generation, memory maintenance, and secondary expansion of P1- and P4-specific CD8+ T cells. These results suggest that H2-M3-restricted CD8+ T cells contribute to protective immunity against Cpn, and that chlamydial Ags presented by MHC class Ib molecules may represent novel targets for inclusion in anti-Cpn vaccines.     

Lymphokine-mediated induction of antigen-presenting ability in thymic stromal cells

We have established and characterized long term thymic stromal cultures from BALB/c (H-2d) and CBA/J (H-2k) mice. All cultures contained multiple adherent cell types, whereas some also contained thymic macrophages (TM). Culture supernatants from all cultures tested contained macrophage colony-stimulating factor activity, whereas only cultures with TM had soluble or membrane-associated interleukin (IL)-1. However, a thymic epithelial cell line (3D . 1), cloned from one of these cultures, produced IL-1 bioactivity. Further analysis confirmed the production of IL-1 alpha mRNA by the epithelial cell. No IL-2 or IL-4 (formerly called B cell stimulatory factor 1) activity was detected in any of the cultures. Antigen-presenting (AP) ability was determined using the chicken ovalbumin (OVA)-specific, I-Ad-restricted T cell hybridoma 3DO-18.3. Harvested TM exhibited antigen-specific, Ia-restricted AP ability which was enhanced by IL-4 as well as interferon-gamma (IFN-gamma). In contrast, AP ability was detected in non-macrophage stromal cell cultures (NMSC) only after preincubation with IFN-gamma. AP by preinduced NMSC was also Ia-restricted and could be blocked by anti-I-Ad antibodies. Since the T cell receptor of 3DO-18.3 is known to recognize a peptide produced by CNBr degradation of OVA, these observations suggest that both TM and NMSC can process OVA to produce this peptide. Glutaraldehyde-fixation experiments confirmed that NMSC must process native OVA into antigenic peptides for successful AP. Assays using several cloned stromal cell lines of different lineages suggested that only epithelial cells could be induced with IFN-gamma to exhibit competent AP. Given the possible role for IFN-gamma in the maintenance of Ia in the thymus, we investigated whether IFN-gamma production could be ascribed to a subpopulation of thymocytes. Culture supernatants from calcium ionophore and phorbol ester-stimulated peanut agglutinin-negative, but not peanut agglutinin-positive, thymocytes induced AP ability in NMSC. Thus, some thymocytes can produce an Ia-inducing lymphokine (most likely IFN-gamma) which may play an important role in T cell ontogeny through its effects on both thymic macrophages and thymic epithelial cells.     

Phytohemagglutinin-induced proliferation and cytolytic activity in T3+ but not in T3- cloned T lymphocytes requires the involvement of the T3 antigen for signal transmission

Proliferation and the cloning efficiency of T3+ but not T3- T cells are increased by the addition of lectins (phytohemagglutinin; PHA) to the culture medium. In contrast to that of T3+ cloned cell lines, the cytolytic activity of T3- clones is not enhanced by PHA, as we report here. We have investigated the effects of anti-T3 monoclonal antibody (MAb) and PHA on the proliferative capacity and cytolytic activity of various T3+ and T3- clones and cells to determine the possible involvement of the T3 receptor in these processes. We found that, in addition to inhibition of allospecific cytotoxicity, anti-T3 MAb can induce and/or enhance nonspecific cytolytic activity against particular target cells in cloned allospecific cytotoxic T cells (CTL) following preincubation of the effector cells with PHA or anti-T3. This enhancement of cytolytic activity is seen in T3+ but not T3- activated killer (AK) clones or fresh T3- natural killer (NK) cells and depends on the concentrations of anti-T3 MAb or PHA used. We conclude that the T3-Ti antigen-receptor complex is involved in the transmission of the activation signals by anti-T3 and PHA.