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Release of BLT esterase by type I and type II CTL clones stimulated with TL target cells. CTL clones (1 10 5 ) were incubated with TL or TL target cells (1 10 5 ) for 4 h in 96-well tissue culture plates, and then the BLT esterase activity of the supernatants was measured.
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TCRalphabeta CTL clones recognizing mouse thymus leukemia (TL) Ags were established and categorized into two groups: those killing any TL+ target cells (type I) and those killing only TL+ Con A blasts (type II). Cold target inhibition assays showed that the antigenic determinant(s) recognized by type II clones are expressed not only on TL+ Con A bl...
Context in source publication
Context 1
... we next attempted to compare the amounts of BLT esterase secretion of type I and type II CTL clones when they were stimulated by various TL target cells. The results for two clones of each type are shown in Figure 7. TL target cells stimulated type I CTL clones to secrete a large amount of BLT esterase, whereas TL target cells did not. ...
Citations
... This corroborates the observation that no peptides have been identified in elution experiments with soluble TL expressed in insect cells (8) or bacteria (10). Despite the absence of Ag presentation, TL molecules can serve as transplantation Ags and can mediate a TCR␥␦ ϩ or even TCR␣ ϩ cytotoxic T cell responses (11)(12)(13)(14). This cannot be due to Ag presentation by TL, and in some cases it has been shown that TL Ag-reactive T cells respond to a conformational epitope in the TL␣ 1 ␣ 2 domains (15). ...
The mouse thymic leukemia (TL) Ag is a nonclassical MHC class I molecule that binds with higher affinity to CD8alphaalpha than CD8alphabeta. The interaction of CD8alphaalpha with TL is important for lymphocyte regulation in the intestine. Therefore, we studied the molecular basis for TL Ag binding to CD8alphaalpha. The stronger affinity of the TL Ag for CD8alphaalpha is largely mediated by three amino acids on exposed loops of the conserved alpha3 domain. Mutant classical class I molecules substituted with TL Ag amino acids at these positions mimic the ability to interact with CD8alphaalpha and modulate lymphocyte function. These data indicate that small changes in the alpha3 domain of class I molecules potentially can have profound physiologic consequences.
... Skin graft and test bleed. Skin grafting was performed as previously described, 9,15,19) and test bleeds from the tail vain were performed weekly. Antibody activity against TL in the sera was assayed by indirect flow cytometric analysis using T3 b -TL transfected and untransfected RMA-S as indicator cells. ...
... All procedures were performed as previously described. 9,15,19) Tetramer analysis. Mixed lymphocyte culture (MLC) was performed with spleen cells from skin-grafted mice 8 weeks after transplantation as described previously, 9,15,19) and cells from MLC were stained with T3 b -TL tetramers, which were prepared as described previously. ...
... 9,15,19) Tetramer analysis. Mixed lymphocyte culture (MLC) was performed with spleen cells from skin-grafted mice 8 weeks after transplantation as described previously, 9,15,19) and cells from MLC were stained with T3 b -TL tetramers, which were prepared as described previously. 6) Briefly, spleen cells from skin-grafted mice were stimulated with γ-irradiated spleen cells from TL transgenic strains for 5 days and used for the experiments. ...
Mouse thymus-leukemia antigens (TL) are aberrantly expressed on T lymphomas in C57BL/6 (B6) and C3H/He (C3H) mice, while they are not expressed on normal T lymphocytes in these strains. When N-butyl-N-nitrosourea (NBU), a chemical carcinogen, was administered orally to B6 and C3H strains, lymphoma development was slower than in T3(b)-TL gene-transduced counterpart strains expressing TL ubiquitously as self-antigens, suggesting that anti-TL immunity may play a protective role. In addition, the development of lymphomas was slightly slower in C3H than in B6, which seems to be in accordance with the results of skin graft experiments indicating that both cellular and humoral immunities against TL were stronger in C3H than B6 mice. The interesting finding that B lymphomas derived from a T3(b)-TL transgenic strain (C3H background) expressing a very high level of TL were rejected in C3H, but not in H-2K(b) transgenic mice (C3H background), raises the possibility that TL-specific effector T cell populations are eliminated and/or energized to a certain extent by interacting with H-2K(b) molecules.
... We recently demonstrated that TL can also serve as a transplantation and tumor rejection antigen for T cells 19,20) and that both T cell receptor (TCR) αβ and γδ cytotoxic T lymphocytes (CTL) specific for TL can kill TL + target cells. 5,19,21) In this review, we summarize our recent findings on TL as both MHC class Ib and tumor-specific antigens through characterization of TL-specific CTLs. ...
Mouse thymus-leukemia antigens (TL) belong to the family of major histocompatibility complex (MHC) class Ib antigens and have a unique mode of expression, i.e., in contrast to other MHC class Ib or Ia antigens, they are found restricted to the intestines in all mouse strains, but also in the thymus of certain strains (TL(+) strains). Nevertheless, a proportion of T lymphomas/leukemias in strains that do not express TL in the thymus (TL(-) strains) feature TL as a tumor antigen. TL was originally defined serologically, but subsequently we have succeeded in generating T cell receptor (TCR) and cytotoxic T lymphocytes (CTL) recognizing TL. By use of TL tetramers free from peptides and transfectants expressing various TL/H-2 chimeric molecules, we have been able to show that TL-specific CTL recognize the alpha1/alpha2 domain of TL without any additional antigen molecules. We previously reported that one of TL's functions in the thymus is positive selection of TCR CTL. Recent studies with TL tetramers revealed that they can bind to normal intestinal intraepithelial lymphocytes (iIEL) and thymocytes in a CD8-dependent, but TCR/CD3-independent manner, while their binding to TL-specific CTL is TCR/CD3- and CD8-dependent. The possible significance of these findings in relation to the roles of TL in the intestines is discussed. We have long been interested in TL as a model tumor antigen which shares characteristics with human differentiation tumor antigens, and we have demonstrated that growth of TL(+) lymphoma cells in vivo is suppressed by immunization with TL(+) skin or dendritic cells (DC) from TL transgenic mice. In addition, anti-tumor effects against TL(+) T lymphomas were obtained by adoptive transfer of TL tetramer strongly-positive TL-specific CTLs.
... CTL mediated killing of target cells has been shown to proceed by two possible mechanisms, either by the Fas/FasL or the perforin/granzyme pathways. [35][36][37] The latter pathway is dependent on the availability of Ca ++ ions whereas the Fas/ FasL signalling pathway proceeds independently of Ca ++ . 36,37 In order to determine which pathway was involved in the CTL mediated killing responses induced by the IFNγ72/β48treated B16-F10/B7-1 hi cells in vaccinated syngeneic mice, we utilized the two agents, Concanamycin A and ethylene glycol-bis(ß-aminoethylether)-N,N,N',N'-tetra-acetic (EGTA) for their effects on the CTL killing. ...
... 36,37 In order to determine which pathway was involved in the CTL mediated killing responses induced by the IFNγ72/β48treated B16-F10/B7-1 hi cells in vaccinated syngeneic mice, we utilized the two agents, Concanamycin A and ethylene glycol-bis(ß-aminoethylether)-N,N,N',N'-tetra-acetic (EGTA) for their effects on the CTL killing. [35][36][37] The results (Fig. 7) revealed that the CTL killing was dependent on the perforin/ granzyme pathway because both agents severely reduced the level of cytotoxicity. In addition, supplementing with 5 mmol/L Ca ++ significantly overcame the inhibitory effect of the chelating agent, EGTA. ...
Sequentially treating human melanoma cell lines by priming with interferon-gamma before adding interferon-beta was previously found to be the most efficient protocol for producing concurrently increased expression of the three surface antigens B7-1, intercellular adhesion molecule-1 and human histocompatibility leucocyte antigens Class I. The present study describes similar outcomes when the same sequential intercellular adhesion molecule-based protocol is applied to murine B16-F10 melanoma cells as well as preclinical studies using the B16-F10 model as a poorly immunogenic melanoma. Thus, treating B16-F10 cells or a highly expressing B7-1 transfected subline (B16-F10/B7-1 hi) by priming with interferon-gamma for 24 h before adding interferon-beta for a further 48 h (interferon-gamma 72/beta 48) increased expression of all three surface antigens, particularly major histocompatibility complex class I whose increased expression was sustained for several days. As a whole tumour cell vaccine, interferon-gamma 72/beta 48 treated B16-F10 cells produced greater levels of cytoxic T lymphocyte response compared to vaccines prepared from cells treated with a single type of interferon. Furthermore, B16-F10 cells expressing high levels of B7-1 and treated using the interferon-gamma 72/beta 48 protocol (interferon-gamma 72/beta 48-treated B16-F10/B7-1 hi) produced substantially increased cytoxic T lymphocyte responses with a fivefold greater synergy than the combined results of either interferon treated or B7-1 expressing cells tested individually. The resulting CD8+ cytoxic T lymphocyte showed greater specificity for B16-F10 cells with tenfold higher killing than for syngeneic EL-4 lymphoma cells. Killing proceeded via the perforin-mediated pathway. CTL responses were induced independent of CD4+ T helper cells. The majority of mice receiving interferon-gamma 72/beta 48-treated B16-F10/B7-1 hi vaccine in vivo remained tumour free after challenge with 5 x 105 live B16-F10 cells expressing intermediate B7-1 levels. The novel strategy described will help enhance vaccine potency when applied clinically to prepare whole cell based cancer vaccine therapies.
... In our previous studies, we showed that TL can be recognized by both TCR␣ and TCR␥␦ CTL (7)(8)(9). Because the cytotoxic activity of these CTL is inhibited by TL, but not by anti-H-2 Abs, it was concluded that recognition of TL is direct without any requirement for Ag presentation by H-2 molecules. ...
... Rat mAb against TL (HD168; Ref. 28) and mouse mAb to TL.2 (TT213; Ref. 7) were described previously. The following mAbs were kindly provided by other investigators, as detailed earlier (7)(8)(9)(10) (35-17-2; kindly provided by N. Shinohara, Kitasato University, Sagamihara, Japan); and mouse mAb against  2 m (S19. 8;Ref. ...
... An additional five H-2K b -restricted CTL clones were tested and found to be unstained (data not shown). Staining intensity roughly correlated with the cytotoxic activity of each CTL clone against TL ϩ target cells (9). Type I CTL clones that can kill any TL ϩ target cells were brightly stained, whereas type II CTL clones that can kill TL ϩ Con A blast cells, but not leukemia cells, were very weakly stained. ...
Thymus leukemia (TL) Ags belong to the family of nonclassical MHC class I Ags and can be recognized by both TCRαβ and TCRγδ CTL with TL, but not H-2 restriction. We previously reported that the CTL epitope is TAP independent, but the antigenic molecule(s) presented by TL has yet to be determined. In the present study, TL tetramers were prepared with T3b-TL and murine β2-microglobulin, not including antigenic peptides, and binding specificity was studied. CTL clones against TL Ags were stained with the T3b-TL tetramer, and the binding shown to be CD3 and CD8 dependent. Normal lymphocytes from various origins were also studied. Surprisingly, most CD8+ intraepithelial lymphocytes derived from the small intestines (iIEL), as well as CD8+ and CD4+CD8+ thymocytes, were stained, while only very minor populations of CD8+ cells derived from other peripheral lymphoid tissues, such as spleen and lymph nodes, were positive. The binding of T3b-TL tetramers to CD8+ iIEL and thymocytes was CD8 dependent, but CD3 independent, in contrast to that to TL-restricted CTL. These results altogether showed that TL-restricted CTL can be monitored by CD3-dependent binding of T3b-TL tetramers. In addition, CD3-independent T3b-TL tetramer binding to iIEL and thymocytes may imply that TL expressed on intestinal epithelium and cortical thymocytes has a physiological function interacting with these tetramer+CD8+ T lymphocytes.
... TL is known to be a serologically defined antigen, and by grafting the skin of Tg.Con.3-1 mice onto syngeneic C3H, we demonstrated that TL can serve as a transplantation antigen, since the skin was rejected in 2-3 weeks, and mediate cytotoxic T cell (CTL) responses with TL (but not H-2) restriction (19,43,44). The major reason for successful immunization by skin grafting was assumed to be potent antigen presentation by dendritic cells (DCs) expressing a large amount of TL in the skin of Tg.Con. ...
When the skin of Tg.Con.3-1 transgenic mice expressing the TL (thymus leukemia) antigen in most tissues is grafted on syngeneic C3H mice, it is rejected, and a cytotoxic T cell (CTL) response against the TL antigen is induced. In this study, we first demonstrated that growth of TL positive lymphoma is suppressed in mice immunized by skin grafting. Immunization with bone marrow derived dendritic cells (DCs) from Tg.Con.3-1, was also found to be associated with an anti-tumor response, but less potent than skin grafting. Relative CTL precursor frequency with DC immunization was also approximately only one third that of skin grafting. The numbers of IFN-gamma producing cells in responder CD8 and CD4 T cell populations were higher with DC immunization than with skin grafting. However, DC immunization seems to induce non-specific immune responses, as re-stimulation with TL negative C3H spleen cells resulted in induction of almost half the number observed with TL positive cells. Thus, the actual number of IFN-gamma producing cells in specific responses to TL is not necessarily larger than with skin grafting immunization. The present results altogether suggest that DC immunization is capable of inducing an anti-tumor reaction, but also possibly unwanted immune responses. In vitro monitoring of specific and non-specific responses in the immune system, thus, is of particular importance for future development of cancer immunotherapy.
... Although the literature is replete with articles using murine models, including knock-out and transgenic animals, which describe the contributions of perforin and Fas-mediated lysis in the rejection of allografts [2], in the pathogenesis of viral [3,4] and autoimmune diseases [5], and in a few instances against leukaemic malignant cells [6,7], the question of which lytic pathways are taken by TIL in the destruction of solid tumours has rarely been addressed [8]. Thus, although there is much data to suggest that tumours may escape from immune recognition by a variety of pathways, there is no evaluation of the contribution of the Fas versus the perforin and granzyme pathways in the lysis of solid tumours by human TIL. ...
In order to determine the mechanism of tumour destruction by tumour-infiltrating lymphocytes (TIL), we examined the ability of both CD4+ and CD8+ effector TIL, and TIL clones, to manifest granzyme-mediated and Fas-mediated destruction of tumour targets. In many in vitro studies TIL have been shown to manifest anti-tumour reactivity, yet many tumours escape immunological destruction. To investigate the role of Fas expression and the concomitant sensitivity to the inducibility of apoptotic death, we derived TIL from four melanomas and one glioma. The glioma, and all but one of the melanomas, expressed Fas, but Fas-mediated apoptosis could only be detected if the targets were treated with cyclohexamide. The melanomas and the glioma all expressed detectable cytoplasmic Bcl-2 protein, known to exert anti-apoptotic activity. Lysis of tumours by CD8-enriched cultures and CD8+ clones was Ca2+-dependent and could not be modified by an anti-Fas MoAb. In CD4-enriched cultures or CD4+ clones with cytotoxic potential against tumour cells, cytotoxicity was also Ca2+-dependent. As Ca2+-dependent cytotoxicity is usually the result of secretion of perforin/granzyme-B, we investigated the presence of perforin in cytotoxic CD4+ clones and demonstrated the presence of granular deposits of this enzyme in some of the CD4+ clones. Although an anti-Fas MoAb did not block the lysis of melanoma targets by CD4+ clones, the examination of Fas-dependent targets demonstrated that these clones also had the potential to kill by the Fas/Fas ligand system. These data suggest that the predominant mechanism in tumour killing by TIL appears to be perforin-granzyme-dependent, and that the solid tumour cell lines we studied are less susceptible to Fas-mediated apoptosis. As non-apoptotic pathways may enhance tumour immunogenicity, exploitation of the perforin-granzyme-dependent cytotoxic T lymphocyte (CTL) pathways may be important for achieving successful anti-tumour responses.
Cytolytic CD8+ T cells fall into two subpopulations based on cytokine-secretion. Type 1 CD8+ cells (Tc1) characteristically secrete IFN-γ, whereas type 2 CD8+ cells (Tc2) secrete IL-4 and IL-5. We assessed the relative therapeutic effects of adoptively transferred OVA-specific Tc1 and Tc2 CD8+ cells in mice bearing established OVA-transfected B16 melanoma lung metastases. Both Tc1 and Tc2 subpopulations mediated a reduction in lung tumor growth that subsequently prolonged survival times in mice with both early (day 7) and more advanced (day 14) levels of tumor development. CD8+ T cell populations recovered from spleens of tumor-bearing mice receiving Tc1 or Tc2 cells showed markedly enhanced tumor Ag-specific cytolytic and cytokine-releasing activities that correlated with delays in tumor cell growth and progression. Initially, both tumor-reactive Tc1 and Tc2 effector cells accumulated at the tumor site with nearly equal frequency. Tc1 cells persisted, whereas Tc2 cell numbers progressively diminished over time. Titration of Tc1 and Tc2 effector cells showed that protection was dose dependent with the former being 5-fold more effective. Tc2 cells achieved a comparable reduction in lung tumor cell growth at higher concentrations of cell transfer. Tc1 effectors from IFN-γ-deficient mice were less therapeutically effective than wild-type mice, but there was no significant reduction in activity between corresponding Tc2 populations. We speculate that the effectiveness of Tc1 and Tc2 cells may depend on different mechanisms. These studies suggest a potential role for Tc1 and Tc2 CD8+ subpopulations in tumor regression and immunotherapy.
Cytotoxic T lymphocytes (CTL) lyse antigen-bearing target cells by two distinct pathways. Whereas granule exocytosis targets any antigen-bearing cell, fas-mediated cytotoxicity kills only fas-expressing cells and does not require antigen expression. Fas pathway activation can potentially lead to lysis of uninfected bystander cells. We examined the relative usage of the two pathways by CTL clones and cell lines directed against four different human immunodeficiency virus (HIV) proteins in lysing primary HIV-infected targets. Although fas was expressed on HIV-infected primary CD4+ T cells, their lysis by antigen-specific CD8+ CTL was only by the granule pathway. Fas ligand (fasL) was not detectable on antigen-specific CD8 clones, T-cell lines, or circulating HIV-specific CD8 T cells from HIV-infected donors, stained with a tetrameric HLA-A2-HIV-peptide complex. FasL expression by HIV-specific CTL clones was not activated by exposure to HIV-presenting cells, but was after unphysiological stimulation with phorbol myristate acetate (PMA). CTL clones did not lyse bystander Jurkat cells, but HIV-infected primary CD4+ T cells lysed uninfected bystander cells by the fas-mediated pathway. These results suggest that HIV-specific CD8+ CTL do not cause HIV immunopathology by lysing bystander cells. On the contrary, fas-mediated lysis of uninfected cells by HIV-infected cells may contribute to CD4 decline.
The nonclassical class I molecule, thymic leukemia (TL), has been shown to be expressed on intestinal epithelial cells and to interact with CD8+ intraepithelial T lymphocytes. We generated recombinant soluble TL (T18d) H chains in bacteria as inclusion bodies and refolded them with β2-microglobulin in the presence or absence of a random peptide library. Using a mAb, HD168, that recognizes a conformational epitope on native TL molecules, we observed that protein folds efficiently in the absence of peptide. Circular dichroism analysis demonstrated that TL molecules have structural features similar to classical class I molecules. Moreover, thermal denaturation experiments indicated that the melting temperature for peptide-free TL is similar to values reported previously for conventional class I-peptide complexes. Our results also show that CD8αα binding is not dependent on either TL-associated peptide or TL glycosylation.
